Targeting receptor-interacting serine/threonine- protein kinase (RIPK)3 in renal tubulointerstitial
fibrosis
Ying Shi
A thesis submitted in fulfilment of the requirements for the
degree of Doctor of Philosophy
Sydney Medical School
The University of Sydney
2019
Statement of originality
This thesis is submitted to The University of Sydney in fulfilment of the requirement for the degree of Doctor of Philosophy.
This is to certify that to the best of my knowledge and belief, the content of this thesis is my own work and that all the assistance received in preparing this thesis and sources have been acknowledged. This thesis has not been submitted for any degree or other purposes.
Ying Shi
1
Abstract
Chronic kidney disease (CKD) affects almost 10% of the adult population worldwide.
Regardless of the initial cause of renal injury, renal fibrosis is the final common pathway of all forms of CKD, including diabetic kidney disease (DKD). However, current therapies to attenuate the development of progressive renal fibrosis are limited to blockade of the renin- angiotensin-aldosterone system (RAAS), achievement of blood pressure targets and in the case of DKD, blood glucose control. More recently inhibition of sodium-glucose linked transporter-2 has shown impressive renoprotective benefits in secondary analyses of major cardiovascular end-point trials. However, studies, where renal disease is a primary endpoint, are awaited. Given the personal and societal impacts of the increasing burden of CKD, it is of utmost importance to identify novel interventions for preventing the progressive renal fibrosis and thus progressive CKD.
Receptor-interacting serine/threonine-protein kinase (RIPK) 3, known as a necroptotic kinase, is recognised to be involved in various innate immune responses, including necroptosis and activation of the toll-like receptor (TLR) 2 and 4 pathways and the pyrin domain-containing protein (NLRP) 3 inflammasome. However, the role of RIPK3 in the development of renal cortical fibrosis, and in particular in the pathology observed in the renal tubule under conditions that stimulate a fibrotic response has not been elucidated.
In this thesis, mouse models of folic acid (FA)-induced nephropathy (studied at 28 days) and streptozotocin (STZ)-induced diabetic nephropathy (studied at 24 weeks) were developed.
RIPK3 knock out (KO) mice were used to determine the therapeutic effect of RIPK3 in renal fibrosis, and the RIPK3 inhibitor dabrafenib was utilized as a pharmacological inhibitor of
RIPK3. Human proximal tubular cells exposed to transforming growth factor beta-β1 (TGF-
II
β1) were used as in vitro model to determine the role of necroptosis in the development of tubular pathology under pro-fibrotic influences. Inflammatory and fibrotic markers, as well as the relevant signalling pathways, were examined.
Our results show that blockade of RIPK3 genetically or pharmacologically suppressed FA- induced activation of the TLR2 and 4 pathways, the NLRP3 inflammasome and TGF-β1 expression, associated with a reduced myofibroblast activation and interstitial extracellular matrix (ECM) deposition. RIPK3 inhibition also confers renoprotection in the STZ-induced diabetic nephropathy model. A reduction of TGF-β1, NLRP3 activation and TLR4 but not
TLR2 was observed in RIPK3 KO mice. Inhibition of necroptosis also reduced TGF-β1 and
ECM secretion in tubular cells exposed to TGF-β1. Collectively, the data shown in the thesis uniquely demonstrate that RIPK3 is involved in the development of renal fibrosis via multiple pathways, including necroptosis, TLR2 and 4 signalling and activation of the NLRP3 inflammasome. Given the two strategies to inhibit RIPK3 converge on inhibition of the TLR4 pathway and the NLRP3 inflammasome activation, we consider these are key pathways to inhibit to confer renoprotection. Dabrafenib, the RIPK3 inhibitor, deserves further exploration for repurposing as a targeted renal therapy.
III
Acknowledgement
It would not have been possible to finish this doctoral thesis without the guidance and kind support from my supervisors, help from friends, and endless amounts of love from my parents.
Here I would like to express my deepest appreciation to all those who provide me with the possibility to complete this thesis.
First and foremost, my thanks go to my supervisor Professor Carol Pollock for her guidance, absolute support throughout my studies. I have been extremely lucky to have a supervisor who was so supportive and cared so much about my work, and who responded to my questions and queries so promptly. She is truly an inspirational mentor and it has been an enormous privilege to work under the supervision of such an exceptional person.
My co-supervisor A/Professor Xinming has been a long-standing mentor and provided unfailing support during the three years of study. Thanks for always being available when I needed help and sharing a lot of science and knowledge with me.
I would like to express my sincere gratitude to my co-supervisor Dr Chunling Huang for her constant source of guidance and emotional support throughout. Thank you for all your words of advice and for sharing your knowledge.
IV
I would like to thank Dr Jason Chen for being so kind in helping me with the processing of animal tissues and subsequent analysis.
I must express my gratitude to my parents for their continued support and encouragement. I was, and still am, continually amazed by their patience and willingness to listen to the minutiae of my project and day-to-day laboratory work and for being on my journey of the ups and downs of research. I am grateful for their support in all my decisions.
I would like to thank A/Professor Usha Panchapakesan, Dr Sonia Saad and Dr Muh Geot for their technical expertise, invaluable advice and kind encouragement. To all my colleagues at the Kolling Institute within the Renal Laboratory group including Long, Hao, Sanela, Jessica,
Amgad, Ben, Sarah and Stephanie, thanks for your friendship and for always being there when
I needed help and emotional support. Especially thanks to Hao and Long for ordering reagents.
To many other colleagues and friends at the Kolling Institute, including Grace, Lauren, Jingting thanks for making the institute a very friendly place. Special thanks to Martyn for advice required during my experimental troubleshooting and kindly sharing his experience and protocols.
I would like to thank for Royal North Shore Hospital and Professor Carol Pollock and the China
Scholarship Council for Postgraduate Research Scholarship support.
V
Publications
1. Huang, C., L. Zhang, Y. Shi, H. Yi, Y. Zhao, J. Chen, C. A. Pollock and X. M. Chen (2018).
"The KCa3.1 blocker TRAM34 reverses renal damage in a mouse model of established diabetic nephropathy." PLoS One 13(2): e0192800.
(not directly the subject of my thesis but I acquired skills relevant to my project by virtue of contribution to this work)
2. Yi, H., C. Huang, Y. Shi, Q. Cao, Y. Zhao, L. Zhang, J. Chen, C. A. Pollock and X. M. Chen
(2018). "Metformin attenuates folic-acid induced renal fibrosis in mice." J Cell Physiol an 30. doi: 10.1002/jcp.26505.
(not directly the subject of my thesis but I acquired skills relevant to my project by virtue of contribution to this work)
Manuscripts in preparation
1. Ying Shi, Chunling Huang, Yongli Zhao, Jason Chen, Hao Yi, Qinghua Cao, Carol Pollock,
Xinming Chen - RIPK3 blockade attenuates kidney fibrosis in a folic acid model of renal injury.
2. Ying Shi, Chunling Huang, Yongli Zhao, Hao Yi, Qinghua Cao, Carol Pollock, Xinming Chen -
RIPK3 blockade attenuates tubulointerstitial fibrosis in a mouse model of diabetic nephropathy.
VI
Presentations arising from this work
• RIPK1: the next novel target for renal fibrosis, Oral presentation, 15th Asian Pacific
Conference of Nephrology (APCN) 2016, Perth, Australia, September 17-21, 2016.
• RIPK1: the next novel target for renal fibrosis, Oral presentation, New Horizons 2016
Conference, Sydney, Australia, November 21-22, 2016.
• Blockade of RIPK3 attenuates folic acid-induced kidney fibrosis of C57BL/6 mouse, Young
Investigator Award Oral presentation, Annual Scientific Meeting of Australian and New
Zealand Society of Nephrology, Darwin, Australia, September 4-6, 2017
• RIPK1 inhibition alleviates renal fibrogenesis, Oral presentation, Annual Scientific
Meeting of Australian and New Zealand Society of Nephrology, Darwin, Australia,
September 4-6, 2017
• RIPK3 inhibition alleviates folic acid-induced kidney fibrosis of C57BL/6 mouse, Poster
presentation, American Society of Nephrology Kidney Week, New Orleans, America,
October 31 - November 5, 2017
• RIPK3 blockade ameliorates renal fibrosis in diabetic model of eNOS knockout mice,
Poster presentation, American Society of Nephrology Kidney Week, New Orleans,
America, October 31 - November 5, 2017
• RIPK3 inhibition alleviates folic acid-induced kidney fibrosis of C57BL/6 mouse, Oral
presentation, 16th Asian Pacific Congress of Nephrology (APCN), Beijing, China, March
27-31, 2018
• Blockade of RIPK3 alleviates renal fibrogenesis trough NLRP3 inflammasome in folic acid
induced kidney fibrosis model, Oral presentation, Annual Scientific Meeting of Australian
and New Zealand Society of Nephrology, Sydney, Australia, September 10-12, 2018
VII
Awards arising from this work
• Postgraduate Research Support Scheme (PRSS) 2015
• Postgraduate Research Support Scheme (PRSS) 2016
• Annual Scientific Meeting of the Australian and New Zealand Society of Nephrology
(ANZSN) 2017 Young Investigator Award finalist
• Scientific Staff Council Australasian Travel Fellowship 2017
• ANZSN Travel Grant for ANZSN Annual Scientific Meeting 2017
• ANZSN Travel Grant for American Society of Nephrology kidney week 2017
• APCN Travel grant for 16th Asian Pacific Congress of Nephrology (APCN) 2018
• Best abstract award of 16th Asian Pacific Congress of Nephrology (APCN) 2018
• ANZSN Travel Grant for ANZSN Annual Scientific Meeting 2018
• ANZSN Travel Grant for American Society of Nephrology kidney week 2018
VIII
Table of Contents
Statement of originality ...... I
Abstract...... II
Acknowledgement ...... IV
Publications ...... VI
Manuscripts in preparation ...... VI
Presentations arising from this work ...... VII
Awards arising from this work ...... VIII
Table of Contents ...... IX
List of Figures ...... XVII
List of Tables ...... XX
List of Abbreviations ...... XXI
Chapter 1 Introduction ...... 1
Chapter 2 Literature review ...... 8
2.1 Origin of renal fibrosis ...... 8
2.1.1 Renal physiology...... 8
2.1.2 CKD ...... 9
2.1.3 ECM in renal fibrosis ...... 11
2.1.3.1 ECM and ECM components ...... 12
2.1.3.2 ECM in renal fibrosis ...... 13
IX
2.1.3.3 ECM breakdown by proteases ...... 15
2.2 Cells and signalling pathways in renal fibrosis ...... 17
2.2.1 Fibroblasts in renal fibrosis ...... 17
2.2.2 Myofibroblasts in renal fibrosis ...... 18
2.2.3 Inflammatory cells in renal fibrosis ...... 19
2.2.4 TGF-β1 ...... 20
2.2.4.1 Overview of TGF-β1 ...... 20
2.2.4.2 TGF-β-Smad pathway ...... 21
2.2.4.2.1 R-Smads ...... 23
2.2.4.2.2 Co-Smad4 ...... 24
2.2.4.2.3 I-Smads ...... 25
2.2.4.3 Non-Smad pathways ...... 25
2.2.5 Pyrin domain-containing protein 3 (NLRP3) inflammasome ...... 26
2.2.6 Toll-like receptors (TLRs) ...... 30
2.2.7 Tumour necrosis factor-α (TNF-α) ...... 34
2.3 Animal models of renal fibrosis ...... 35
2.3.1 FA nephropathy ...... 35
2.3.2 Streptozotocin (STZ)-induced diabetic nephropathy (DN) ...... 36
2.3.3 Adenine ...... 38
2.3.4 5/6 Nephrectomy ...... 39
X
2.3.5 Ureteric ureteral obstruction (UUO) ...... 40
2.4 Receptor-interacting serine/threonine-protein kinase (RIPK) 3 ...... 41
2.4.1 RIPK3 in necroptosis ...... 42
2.4.2 RIPK3 in inflammation ...... 47
2.4.3 RIPK3 in kidney disease ...... 48
2.4.4 Pharmacological blockers of RIPK3 ...... 49
Chapter 3 Methodology ...... 51
3.1 Materials ...... 51
3.1.1 General chemicals ...... 51
3.1.2 Buffers and solutions ...... 52
3.1.2.1 Buffers for genotyping ...... 52
3.1.2.2 Buffers for immunohistochemistry ...... 53
3.1.2.3 Buffers for western blot ...... 54
3.1.3 The sequence of probes and primers ...... 55
3.2 Animal models ...... 59
3.2.1 Genotyping of genetically modified mice ...... 60
3.2.2 Establishment of diabetic mice ...... 61
3.2.3 Establishment of FA-induced nephropathy ...... 62
3.2.4 Implantation of osmotic minipumps ...... 63
3.2.5 Euthanasia of animals and tissue collection ...... 63
XI
3.2.6 Tissue embedding and fixation ...... 64
3.3 Histology ...... 64
3.3.1 PAS staining ...... 64
3.3.2 Picrosirius Red stain ...... 64
3.4 Immunohistochemistry ...... 65
3.5 mRNA expression by RT-PCR analysis ...... 66
3.5.1 RNA extraction ...... 66
3.5.2 cDNA synthesis ...... 66
3.5.3 PCR ...... 67
3.6 Western blot analysis ...... 68
3.6.1 Protein extraction ...... 68
3.6.2 Bicinchoninic acid (BCA) protein assay ...... 68
3.6.3 Protein gel electrophoresis ...... 69
3.7 In vitro studies ...... 69
3.7.1 HK2 cell culture ...... 69
3.7.2 Cell proliferation assay ...... 70
3.8 Statistics ...... 70
Chapter 4 RIPK3 blockade attenuates kidney fibrosis in a folic acid model of renal injury………………………………………………………………………………………………………………………………….71
4.1 Introduction ...... 73
4.2 Methods ...... 75
XII
4.2.1 Animal study ...... 75
4.2.2 Histology and Immunohistochemistry ...... 76
4.2.3 Renal cortical injury ...... 77
4.2.4 RNA isolation and RT-PCR analysis ...... 78
4.2.5 Western blot analysis ...... 78
4.2.6 Statistical analysis...... 79
4.3 Results ...... 79
4.3.1 RIPK3 gene expression is increased in mice with FA nephropathy but no change observed in downstream pMLKL protein ...... 79
4.3.2 RIPK3 deficiency inhibits TLR2/4 signalling in mice with FA nephropathy...... 81
4.3.3 RIPK3 deficiency reduces NLRP3 inflammasome activation in mice with FA nephropathy...... 83
4.3.4 RIPK3 deficiency reduces inflammatory responses and renal injury in mice with FA nephropathy...... 84
4.3.5 RIPK3 deficiency decreases TGF-β1 signalling and myofibroblast activation in mice with FA nephropathy...... 87
4.3.6 RIPK3 deficiency reduces collagen deposition in mice with FA nephropathy...... 90
4.3.7 Dabrafenib reduces TLR2/4 activation in mice with FA nephropathy...... 92
4.3.8 Dabrafenib downregulates necroptotic NLRP3 inflammasome activation in mice with
FA nephropathy...... 94
XIII
4.3.9 Dabrafenib reduces inflammatory responses and histological evidence of renal injury
in mice with FA nephropathy...... 95
4.3.10 Dabrafenib alleviates myofibroblast activation in mice with FA nephropathy...... 97
4.3.11 Dabrafenib decreases collagen deposition in mice with FA nephropathy...... 99
4.4 Discussion ...... 101
Chapter 5 RIPK3 blockade attenuates tubulointerstitial fibrosis in a mouse model of diabetic nephropathy ...... 105
5.1 Introduction ...... 107
5.2 Methods ...... 108
5.2.1 Human kidney biopsies ...... 109
5.2.2 Animal studies ...... 109
5.2.3 RNA isolation and RT-PCR analysis ...... 110
5.2.4 Histology and Immunohistochemistry ...... 111
5.2.5 Statistical analysis...... 112
5.3 Results ...... 112
5.3.1 Upregulation of RIPK3 expression in the diabetic kidney ...... 112
5.3.2 RIPK3 gene knockout reduced fibronectin and collagen IV deposition induced by
diabetes ...... 114
5.3.3 RIPK3 gene knockout decreased transforming growth factor beta (TGF-β)1
expression and myofibroblast activation ...... 117
5.3.4 RIPK3 gene knockout reduced kidney inflammatory cell infiltration ...... 118
XIV
5.3.5 RIPK3 gene knockout reduced TLR4 signalling activation ...... 119
5.3.6 RIPK3 gene knockout reduced NLRP3 inflammasome formation...... 120
5.3.7 Dabrafenib treatment increases survival rate in mice with established diabetic
nephropathy...... 122
5.3.8 Dabrafenib treatment reduces ECM deposition in mice with established diabetic
nephropathy ...... 123
5.3.9 Dabrafenib treatment decreased myofibroblast activation and inflammatory
response ...... 126
5.3.10 Dabrafenib treatment decreased myofibroblast activation in mice with established
diabetic nephropathy ...... 128
5.4 Discussion ...... 130
Chapter 6 Blockade of necroptosis attenuates fibrotic responses in human kidney proximal tubular cells exposed to TGF-β1 ...... 133
6.1 Introduction ...... 134
6.2 Methods ...... 136
6.2.1 Cell culture ...... 136
6.2.2 Cell viability assay ...... 136
6.2.3 RNA isolation and RT-PCR analysis ...... 137
6.2.4 Western blot analysis ...... 137
6.2.5 Statistical analysis...... 137
6.3 Results ...... 138
XV
6.3.1 Inhibition of necroptosis decreased ECM secretion in HK2 cells exposed to TGF-β1.
...... 138
6.3.2 Inhibition of necroptosis downregulated TGF-β1 expression...... 142
6.3.3 Inhibition of necroptosis showed a trend to downregulate phosphor-extracellular-
signal-regulated kinase (ERK) but not p38 expression ...... 143
6.4 Discussion ...... 144
Chapter 7 Conclusions and future directions ...... 148
7.1 Conclusions ...... 149
7.2 Future directions ...... 150
Reference ...... 155
XVI
List of Figures
Chapter 2
Figure 2.1 Schematic depiction of the nephron ...... 9
Figure 2.2 TGF-β-Smad pathway ...... 22
Figure 2.3 Schematic illustration of the NLRP3 inflammasome activation ...... 28
Figure 2.4 TLRs pathway ...... 31
Figure 2.5 Role of Toll-like receptor -2 and -4 in renal fibrosis ...... 34
Figure 2.6 RIPK3 structure ...... 42
Figure 2.7 RIPK1-RIPK3 regulates and integrates necroptotic cell death from multiple receptors ...... 45
Figure 2.8 Time of death of mice mutant for genes controlling apoptosis or necroptosis ..... 47
Chapter 4
Figure 4.1 Increased receptor-interacting serine/threonine-protein kinase (RIPK) 3 in mice with folic acid nephropathy ...... 80
Figure 4.2 Increased toll-like receptor (TLR) 2/4 in mice with folic acid nephropathy ...... 82
Figure 4.3 Increased pyrin domain-containing protein 3 (NLRP3) inflammasome activation in mice with folic acid nephropathy ...... 84
Figure 4.4 Increased inflammation and kidney injury in mice with folic acid nephropathy. .. 86
Figure 4.5 Increased transforming growth factor beta (TGF-β) 1 signalling and myofibroblast activation in mice with folic acid nephropathy ...... 89
Figure 4.6 Increased collagen deposition in mice with folic acid nephropathy...... 91
Figure 4.7 Reduced RIPK3 mediated TLR2/4 signalling in mice with folic acid-induced nephropathy treated with dabrafenib...... 94
XVII
Figure 4.8 Reduced RIPK3 mediated NLRP3 inflammasome activation in mice with folic acid- induced nephropathy treated with dabrafenib...... 95
Figure 4.9 Reduced inflammatory response and kidney injury in mice with folic acid-induced nephropathy treated with dabrafenib...... 96
Figure 4.10 Reduced TGF-β1 signalling and myofibroblast activation in mice with folic acid- induced nephropathy treated with dabrafenib...... 99
Figure 4.11 Reduced collagen deposition in mice with folic acid-induced nephropathy treated with dabrafenib...... 100
Chapter 5
Figure 5.1 Timeline of DM model and dual strategies to assess the effect of limitation of
RIPK3 on the development of diabetic kidney disease...... 110
Figure 5.2 Increased receptor-interacting protein kinase (RIPK) 3 in diabetic kidney disease
...... 113
Figure 5.3 Increased fibronectin and collagen IV deposition in kidneys of diabetic mice. ... 116
Figure 5.4 Increased transforming growth factor beta (TGF-β) 1 mediated myofibroblast activation in kidneys of diabetic mice...... 118
Figure 5.5 Increased inflammatory cells infiltration in the kidneys of diabetic mice ...... 119
Figure 5.6 Increased toll-like receptor (TLR) 4 signalling in kidneys of diabetic mice...... 120
Figure 5.7 Increased pyrin domain-containing protein (NLRP) 3 formation in kidneys of diabetic mice...... 121
Figure 5.8 Decreased survival rate in diabetic mice...... 123
Figure 5.9 Increased extracellular matrix (ECM) deposition in kidneys of diabetic mice. .... 125
Figure 5.10 Dabrafenib reduced the inflammatory response and trended to a reduction in mRNA expression of TGF-β1 and the inflammasome in kidneys of diabetic mice...... 128
XVIII
Figure 5.11 Increased ECM deposition and pSmad2/3 expression in kidneys of diabetic mice
...... 129
Chapter 6
Figure 6.1 Toxicity of Nec-1s...... 138
Figure 6.2 Increased collagen IV and fibronectin secretion in HK2 cells exposed to TGF-β1.
...... 141
Figure 6.3 Increased TGF-β1 mRNA expression in HK2 cells exposed to TGF-β1...... 142
Figure 6.4 Increased ERK and p38 activation in HK2 cells exposed to TGF-β1...... 144
XIX
List of Tables
Chapter 2
Table 2.1 GFR and Albuminuria categories in CKD ...... 10
Table 2.2 Correlation of percentage of fibrosis measurements between techniques ...... 14
Table 2.3 Correlation of renal function with percentage of fibrosis measured by each technique ...... 14
Table 2.4 MMP/TIMP Mouse Models in Renal Pathophysiology ...... 16
Table 2.5 TLR2 and TLR4 expression in association with chronic kidney diseases ...... 32
Table 2.6 Comparison of CKD manifestations with different adenine doses ...... 39
Chapter 3
Table 3.1. General chemicals and compounds utilised in this thesis ...... 51
Table 3.2. The sequence of probes used for quantitative real-time PCR ...... 55
Table 3.3. Primer sets used for genotyping ...... 59
Table 3.4 Protocol of genotyping ...... 60
Table 3.5 Components of cDNA synthesis ...... 66
Table 3.6 Reaction Protocol of cDNA synthesis ...... 67
Table 3.7 Reaction Protocol of PCR ...... 68
XX
List of Abbreviations
ACL ATP citrate lyase
ACR urine albumin-to-creatinine ratio
ACR albumin-to-creatinine ratio
AKI acute kidney injury
ALK activin receptor-like kinase
AMDCC Animal Models for Diabetic Complications Consortium
ASC adapter protein apoptosis-associated speck-like protein
BMP bone morphogenetic protein
BSA bovine serum albumin
BUN blood urea nitrogen cFLIP cellular FADD-like IL-1β-converting enzyme-inhibitory protein
CKD chronic kidney disease
Co-Smads common mediator Smads
DAMPs danger-associated molecular patterns
DC dendritic cell
DD death domain
DISC death-inducing signalling complex
DKD diabetic kidney disease
DM diabetes mellitus
DMSO dimethyl sulfoxide
DN diabetic nephropathy
DRs death receptors
XXI
ECM extracellular matrix
EDTA Ethylenediaminetetraacetic acid eGFR estimated glomerular filtration rate
EMT epithelial-mesenchymal transition eNOS endothelium-derived nitric oxide synthase
ERK extracellular-signal-regulated kinase
ESRD end-stage renal disease
FA folic acid
FADD Fas-associated protein with death domain
FASL Fas ligand
FGF fibroblast growth factor
FSGS focal segmental glomerulosclerosis
FSP fibroblast-specific protein
GBM glomerular basement membrane
GFR glomerular filtration rate
GlcNAc N-acetylglucosamine
HMGB1 high-mobility-group box 1
HSPs heat shock proteins
I/R ischemia/reperfusion
IAP inhibitors of apoptosis protein
ICP RHIM-containing protein
IgA immunoglobulin A
IKK inhibitor of κB-kinase
XXII
IL interleukin
IRAK interleukin-1 receptor-associated kinase
I-Smad inhibitory Smad
JNK c-Jun N-terminal kinase
KDIGO Kidney Disease: Improving Global Outcomes
KO knock out
LAP latency associated peptide
LTBP latent TGF-β binding protein
MAPK mitogen-activated protein kinase
MCMV murine cytomegalovirus
MKK MAPK kinase
MLKL mixed-lineage kinase domain-like
MMP matrix metalloproteinase
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4sulfophenyl)-2H MTS tetrazolium
MyD88 myeloid differentiation primary response protein 88
NEMO NF-κB essential modulator
NF κB nuclear factor kappa light chain enhancer of activated B cells
NK natural killer
NLRP3 pyrin domain-containing protein 3
NLRs nucleotide-binding and oligomerization domain (NOD)-like receptors
NOD nucleotide-binding and oligomerization domain
XXIII
PAMPs pathogen-associated molecular patterns pDCs plasmacytoid dentritic cells
PDGF platelet-derived growth factor
PGs proteoglycans
PKR ripoptosome and protein kinase R
PRRs pattern recognition receptors
PTCs proximal tubular cells
RAAS renin angiotensin aldosterone system
RGRs retinoic acid-inducible gene I (RIG-I)-like receptors
RHIM homotypic interaction motif
RIG-I retinoic acid-inducible gene I
RIPA Radioimmunoprecipitation assay
RIPK receptor-interacting serine/threonine-protein kinase
ROS reactive oxygen species
R-Smads receptor-regulated Smads
SNx subtotal nephrectomy
STZ streptozotocin
TAB-1 TAK1-binding protein
TAK TGF-β activated kinase
TAZ transcriptional co-activator with PDZ-binding motif
TBE Tris-borate-EDTA
TBST Tris-buffered saline with Tween 20
TGFR TGF-β receptor
XXIV
TGF-β transforming growth factor beta
TICAM1 TIR-containing adaptor molecule 1
TIMP tissue inhibitors of metalloproteinase
TIR toll-IL-1 receptor
TIRAP TIR-domain-containing adaptor protein
TLR toll like receptor
TNFR TNF receptor
TNF-α tumour necrosis factor-α
TRAF tumour necrosis factor receptor-associated factor
TRAILR1 TNF-related apoptosis-inducing ligand receptor 1
TRAM TRIF-related adaptor molecule
TRIF TIR-domain-containing adaptor-inducing interferon-β
UUO unilateral ureteral obstruction
VEGF vascular endothelial growth factor
YAP yes-associated protein
α-SMA α-smooth muscle actin
β-ME 2-Mercaptoethanol
XXV
Chapter 1 Introduction
Chronic kidney disease (CKD) is defined as the presence of abnormal kidney function, including a loss of glomerular filtration and or proteinuria, persisting for at least 3 months. In the majority of cases CKD eventually leads to end-stage kidney disease (ESKD) requiring renal replacement therapy or death will ensue. CKD affects a large proportion of the population and considerably more than is widely appreciated by the general public. In 2014-5 1.7 million hospitalisations were associated with CKD, which accounts for 17% of all hospitalisations in
Australia. Of those hospitalisations, 80% were for regular dialysis [1]. Having CKD increases the length of stay, cost and complications of non-CKD related hospital admissions. In US, the overall prevalence of CKD in the adult general population was 14.8% in 2011-2014 [2].
Regardless of the cause of the initial renal injury, progressive renal fibrosis is common to all forms of CKD, characterized pathologically by extracellular matrix (ECM) accumulation, myofibroblast activation and inflammatory cell infiltration [3, 4].
To date, inhibition of the renin-angiotensin-aldosterone system (RAAS) is the key strategy utilised to slow deterioration of renal functional decline [5]. However, this influences intrarenal and extrarenal haemodynamics and only secondarily reduces the development of renal fibrosis [5, 6]. It is primarily beneficial in those with proteinuric renal disease and at best it delays the time to ESKD, leading to renal replacement therapy or death, by a factor of months. Thus, novel therapies directed to reducing the fibrotic response in the kidney is urgently needed to arrest the progression of CKD and improve the outcome of patients.
1
It is known that inflammatory pathways play a central role in the progression of renal fibrosis [7]. Inflammation is incited by local and systemic factors and may be differentially activated in different forms of kidney disease. However, it is now well recognised that renal diseases traditionally considered as ‘non-inflammatory’, such as diabetic nephropathy, have a significant component of inflammation underpinning progressive disease. They include tubular-derived proinflammatory cytokines (such as, but not limited to, transforming growth factor beta-β1 (TGF-β1), macrophage chemotactic protein-1 (MCP-1), tumour necrosis factor (TNF)-α, interleukin (IL)-1β) [8, 9], tubular injury associated pathways (such as Wnt/β- catenin signalling, Notch signalling) [10] and immune cells in promoting kidney injury and repair (such as macrophages, T- and B-cells) [11]. Recently, a broader recognition of inflammatory responses contributing to fibrogenesis has been recognised to be due to activation of the innate immune response. These responses are initiated by several classes of pattern recognition receptors (PRRs) in response to immune activators such as pathogen- associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs)
[12]. To date, the most recognised innate immune responses contributing to renal fibrosis are toll-like receptor (TLR)s pathways and the pyrin domain-containing protein 3 (NLRP3) inflammasome [13].
Necroptosis is a programmed cell necrosis process initiated by receptor-interacting protein kinase-3/1 (RIPK3/1) phosphorylation of mixed-lineage kinase domain-like protein (MLKL), resulting in necroptosis associated DAMP production, inflammation and tissue damage [14,
15]. Recent studies have reported that blockade of necroptosis with the inhibitor Nec-1 reduces the interstitial fibrotic response in the kidney in both the unilateral ureteral
2 obstruction (UUO) mouse model [16] and in rats subjected to subtotal nephrectomy (SNx)
[17]. Although the mechanism remains unclear, these studies highly suggest that activation of the necroptotic pathway as a consequence of renal injury contributes to renal fibrosis.
It is increasingly reported that blocking of RIPK3, RIPK1 or MLKL reduces necroptotic cell death [18-20]. However, RIPK1 has been paradoxically demonstrated to protect hepatocytes from death in the Fas-induced hepatitis model [21], suggesting that RIPK1 might serve multiple roles resulting in both mediating and conversely protecting cells from death, which is likely to be context specific. In addition, it has reported deficiency of MLKL failed to protect the kidney from fibrosis [22]. Hence targeting either RIPK1 or MLKL may not be an effective strategy to inhibit renal fibrosis. Given the inhibitor of RIPK3, dabrafenib is already used clinically in patients with non-small cell lung cancer and melanoma, if proven efficacious, the route to market in patients with CKD would be accelerated. Consequently, my thesis has focused on the effectiveness of inhibiting RIKP3 to reduce renal disease in two models of CKD. To support the mechanism, I also undertook in vitro studies to explore the role of necroptosis in the fibrotic response.
As discussed above, DAMPs are important triggers that activate the innate immune response. Recent studies in a macrophage cell culture model confirmed that activation of the NLRP3 inflammasome can be mediated by all three necroptotic kinases, i.e RIPK1, RIPK3 and MLKL acting in a coordinated manner [23-27]. However, of note, RIPK3 can directly promote NLRP3 inflammasome activation, independent of its kinase activity and without concomitant necroptosis. RIPK3 has been demonstrated to promote cell-death-independent
3 inflammation through toll-like receptor (TLR) 4 activation in macrophages [28]. These studies implicate RIPK3 in the innate immune response via multiple mechanisms including necroptosis, as well as activation of the NLRP3 inflammasome and the TLR4 pathway. Hence targeting RIPK3 may limit several mechanisms that collectively contribute to renal fibrosis. A very recent report has also demonstrated that RIPK3 promotes renal fibrosis via the AKT- dependent ATP citrate lyase (ACL), improving the survival and proliferation of fibroblasts in both the UUO-induced and adenine diet-induced renal fibrosis models [22]. However, the role of RIPK3 in a model of acute tubular necrosis leading to renal cortical fibrosis and in diabetic nephropathy has not been elucidated. Thus, the central hypothesis of this thesis is that blockade of RIPK3 will mitigate or limit the development of renal fibrosis.
The first chapter of my thesis serves as the introduction (Chapter 1), which describes the research problem explored in this thesis and addresses the overall objectives and specific aims of the thesis. Chapter 2, the literature review, provides a summary of the current literature, which provides a basic understanding of the research presented in this study.
Firstly, renal physiology and typical fibrotic processes in the kidney are described. Cellular and molecular pathways of renal fibrosis, the mechanisms driving the fibrotic processes and current research in development aimed at the treatment of renal fibrosis are then outlined.
Emphasis is placed on the TGF-β signalling pathway due to its central role in fibrotic and innate immune responses. Then, common animal models of renal fibrosis are discussed, with a focus on the models presented in this study. Lastly, RIPK3 is introduced, its function, its role in necroptosis, inflammation, and our hypothesis of the renoprotective effects in kidney disease and current RIPK3 pharmacological blockers.
4
The studies presented in this thesis were determined to examine the therapeutic potential of RIPK3 inhibition in renal fibrosis. The experimental protocols utilised in my studies are detailed in Chapter 3. Experimental details of procedures specific to certain studies are included within the respective chapters.
The subsequent chapters describe the experimental outcomes of my investigations into the pathological role of RIPK3 in the development of renal fibrosis and the efficacy of various strategies to block RIPK3. To address the aim of my thesis, mouse models of folic acid (FA)- induced nephropathy (studied at 28 days) and streptozotocin (STZ)-induced diabetic nephropathy (studied at 24 weeks) were developed (Chapter 4 and 5). My data shows that
FA nephropathy induces a significant upregulation of RIPK3 expression within the kidney. FA nephropathy developed in the RIPK3-/- mouse model or using the RIPK3 inhibitor dabrafenib attenuated the FA-induced fibrotic response, resulted in reduced renal interstitial collagen deposition. A consistent decrease of TLR2 and 4 activation, NLRP3 activation and TGF-β1 expression were observed using both strategies to inhibit RIPK3.
However, the activity of necroptosis was differentially affected in the knock out model and by the inhibitor dabrafenib. These results suggest that the activation of TLR2 and 4 signalling and NLRP3 activation is central to the fibrogenesis observed in FA-induced nephropathy
(Chapter 4).
5
To confirm our findings of RIPK3 on NLRP3 and TLRs, we determined the effect of blockade of RIPK3 in two STZ animal models of diabetic nephropathy namely RIPK3-/- and endothelium-derived nitric oxide synthase (eNOS) -/- treated with dabrafenib. Consistently,
RIPK3 inhibition with either gene knock out or the inhibitor reduced the fibrotic response and conferred renoprotection. A reduction of TGF-β1, NLRP3 activation and TLR4 but not
TLR2 was seen (Chapter 5). Therefore, we conclude that RIPK3 mediates renal fibrosis mainly via activation of the NLRP3 inflammasome and the TLR4 pathway.
Although we confirmed that the role of RIPK3 in fibrosis can be independent of necroptosis, a role for necroptosis in renal fibrosis cannot be excluded. Nec-1s inhibits necroptosis via inhibiting RIPK1 thereby the formation of the RIPK1-RIPK3 necrosome which induces downstream necroptosis [29]. I assessed the fibrotic response in human proximal tubular cells (PTCs) exposed to TGF-β1 exposed to the necroptotic inhibitor Nec-1s. Hence this strategy selectively inhibited necroptosis but did not limit the effect of RIPK3 on other pathways such as activation of TLR4 or NLRP3 inflammasome. Our data suggest that Nec-1s reduced TGF-β1 induced ECM secretion and TGF-β expression (Chapter 6), suggesting that necroptosis may also contribute to renal fibrosis. However, our in vivo models suggest that necroptosis is unlikely to be the major pathways contributing to the renal injury.
Collectively, our findings confirm:
6
1: RIPK3 is a crucial mediator in renal fibrosis. Blockade of RIPK3 attenuates renal fibrosis with an accompanying reduction in both innate and adaptive inflammatory responses, myofibroblast activation and ECM deposition.
2: The renoprotective effects of RIPK3 is primarily due to inhibition of the TLR4 pathway and
NLRP3 inflammasome activation. However, a reduction in necroptosis, TGF-β1 activation and inhibition of downstream signalling may also contribute to a reduction in renal fibrosis.
3: The pharmacological inhibitor of RIPK3, dabrafenib, afforded renoprotection and may be considered for repurposing as a targeted renal therapy in patients with CKD.
7
Chapter 2 Literature review
2.1 Origin of renal fibrosis
2.1.1 Renal physiology
Kidneys are essential organs that maintain fluid, electrolyte and acid /base homeostasis as well as regulating blood pressure, vitamin D homeostasis and erythropoesis. The essential structural and functional unit of the kidney is known as nephron, consisting of a renal corpuscle including the glomerular tuft and a tubule unit (Figure 2.1). In the physiological context, a nephron serves as a functional unit to filter blood, reabsorb the filtered electrolytes, solutes and fluid, and excrete wastes and excessive electrolytes and water. In this process, the glomerulus is responsible for filtering blood, whilst tubules are responsible for reabsorbing 99% of the filtered electrolytes and water and returning them to the circulation [30, 31].
8
Figure 2.1 Schematic depiction of the nephron. Water and small molecular solute pass from the glomerular capillaries into the urinary space, whereas protein and red blood cells are normally restricted from passing through the glomerular filter due to size or charge sensitive exclusion. Ninety-nine percent of the filtrate is reabsorbed in the renal tubule [32].
2.1.2 CKD
CKD is characterized by the loss of renal cells and their replacement by ECM, known as the pathogenesis of fibrosis, leading to the renal dysfunction [33]. CKD is defined as an estimated glomerular filtration rate (eGFR) less than 60 ml/min/1.73 m2 or a urine albumin- to-creatinine ratio (ACR) more than 3mg/mmol persisting for more than 3 months. The
Kidney Disease: Improving Global Outcomes (KDIGO) Clinical Practice Guideline divides CKD stages by proposing six categories of kidney function (eGFR) and three categories of albuminuria (ACR, A1–A3) (table 2.1). In the late-stage of CKD (G 4 and 5), some diseases specific therapies are available. However, when interstitial fibrosis and tubular atrophy occur generic supportive therapies directed to blood pressure control, reduction in albuminuria and reducing cardiovascular risk are the mainstays of therapy.
9
Table 2.1 GFR and Albuminuria categories in CKD [34]. *Relative to young adult level.
**Including nephrotic syndrome.
Abbreviations: CKD, chronic kidney disease; GFR, glomerular filtration rate; AER, albumin excretion rate; ACR, albumin-to-creatinine ratio.
According to the 2013 global burden of disease study, CKD increased from 28th in 1990 to
20th in 2013 in the global ranking of the number of death and disability-adjusted life years
[35]. In Central Latin America this change moved from 18th in 1990 to 7th in 2013, whilst it changed from 15th in 1990 to 12th in 2013 for Western Europe [36]. To date, in all developed and many developing countries, diabetes and hypertension are the leading causes of CKD, while glomerulonephritis and unknown causes are more common in Asian and sub-Saharan African countries. This difference may due to the decreased burden of chronic infectious diseases and environmentally induced nephrotoxins in developed countries [37]. Infectious diseases remain prevalent in low-income countries, which are associated with poor sanitation, high water fluorides, and high concentrations of disease- transmitting vectors [38]. In addition, the burden of CKD in developing countries correlates with environmental pollution, pesticides, analgesic abuse, herbal medications, and the use of unregulated food additives [39, 40].
Fibrosis of the kidney is the end result of all forms of progressive kidney disease, leading to an inexorable loss of renal function and eventually death or renal replacement therapy. The kidney is a complex organ with highly specialized structures which can differentially undergo
10 fibrosis depending on the underlying disease. For example, the main function of glomerulus is filtration. Trapped immune complexes in the glomerular basement membrane (GBM) or in situ from circulating and then filtered antibodies and glomerular antigens can result in a cascade of inflammation and development of glomerulonephritis. Chronic glomerulonephritis may then stimulate scar formation, leading to glomerulosclerosis. In parallel, antigens or molecules causing injury and inflammatory responses in renal tubular cells, initiate tubulointerstitial nephritis, ultimately resulting in tubulointerstitial fibrosis
[32].
As previously stated, any disease that causes progressive kidney injury may lead to renal fibrosis. The histopathology of tubulointerstitial fibrosis is characterized by the deposition of
ECM in the interstitium associated with inflammatory cell infiltration, tubular cell damage, fibroblast activation, and rarefaction of the peritubular microvasculature [41]. Collectively, the fibrotic pattern is determined by the cells that are involved. However, glomerular and tubular fibrotic processes can’t be entirely separated. Both processes can result in progressive kidney disease. Regardless of the nature of the initial renal injury, progressive tubulointerstitial fibrosis is the final common mechanism of all chronic kidney disease [42].
In primary glomerulopathies, where the injury by definition is glomerular, histological evidence of tubulointerstitial fibrosis has more prognostic value than does glomerular pathology [43]. Once renal fibrosis supervenes, progressive functional decline occurs, which relentlessly progresses leading to dialysis or renal transplantation.
2.1.3 ECM in renal fibrosis
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2.1.3.1 ECM and ECM components
The ECM is a three-dimensional, non-cellular structure that provides structural support for organs and tissues and for all cell layers in the form of basement membranes and for individual cells as substrates for cell motility [44]. In mammals, the ECM consists of around
300 proteins, known as the core matrisome, which consists of 43 collagen subunits, 36 proteoglycans (PGs) and ~200 complex glycoproteins [45]. Those components can be divided into two main types with regard to their location and components. One of the types is known as the interstitial connective tissue matrix, providing structural scaffolding for tissues. The other type is the basement membrane which separates the epithelium from the surrounding stroma [46].
Collagens are the main structural proteins of the ECM and are classified into fibrillar (collagens
I, III, V and XI) and non-fibrillar forms. Those collagen fibrils provide tensile strength to the
ECM. PGs, such as aggrecan, versican, perlecan and decorin, fill the extracellular interstitial space and promote hydration and therefore regulates stiffness of the ECM by sequestering water within the tissue [46]. Glycoproteins, such as laminins, elastin, fibronectins, thrombospondins, tenascins and nidogen, have diverse functions, including acting as ligands for cell surface receptors and small protein fragments released after proteolysis [46]. ECM remodelling occurs in response to signals transmitted by known ECM receptors (such as integrins), ECM-modifying proteins (such as matrix metalloproteinases (MMPs)) or extracellular/cellular tension. The latter is also regulated by ECM remodelling. Cells which interact with the ECM not only detect the composition and stiffness of their substrates but
12 their geometry, which trigger different signalling pathways. Imbalance between ECM degradation and assembly leads to organ fibrosis, which to date is not reversible.
2.1.3.2 ECM in renal fibrosis
Accumulation of ECM is considered as the fundamental to the process of fibrogenesis. The
ECM is known to regulate cell behaviour. The ECM, which was previously thought to have a static structure, is now recognised to be constantly undergoing remodelling. However, recent data has proposed that ECM is not only the end consequence of injury but also contributes to promoting the fibrotic response. Tissue stiffness induced by over-production of the ECM and impaired hydration activates profibrotic growth factor-like TGF-β in a yes- associated protein (YAP) / transcriptional co-activator with PDZ-binding motif (TAZ)- dependent pathway, which, in turn, promotes further tubular injury by increasing the diffusion distance of oxygen, resulting in increased hypoxia [47, 48].
By definition, fibrosis correlates with changes in the matrix in both quantitative and qualitative manners [49]. In renal fibrosis, the interstitium of the fibrotic kidneys fills with fibrillar type I and III collagens [50]. Therefore, type I and III collagens are wildly used as hallmarks of renal fibrosis, which can be measured by the techniques including immunohistochemistry, trichrome and periodic acid–Schiff and Sirius Red with and without polarization[51-53]. Type I collagen is well recognised to be a cause of pathological fibrosis but usually, deposits later in the fibrotic process in relatively low amounts. In contrast, type
III collagen has much more abundant expression [54]. Of note, collagen III morphometry and
13 a visual assessment of trichrome-stained slides have been shown to be efficient and reproducible in the assessment of kidney fibrosis and demonstrate correlation with kidney function [55] (as shown in Table 2.2 and Table 2.3).
Table 2.2 Correlation of percentage of fibrosis measurements between techniques [55].
Table 2.3 Correlation of renal function with percentage of fibrosis measured by each technique [55].
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Increased thickness of the glomerular basement membrane (GBM) has also been observed early in the development of diabetic nephropathy [56]. The specialized GBM is comprised primarily of non-fibrillar collagen ie. collagen IV [57-59]and laminin [60] which is the most abundant non-collagenous glycoprotein of basement membranes [61]. Increased accumulation of glomerular matrix components leads to glomerulosclerosis and albuminuria
[62], but as discussed above, tubulointerstitial fibrosis correlates more with functional renal decline [43].
2.1.3.3 ECM breakdown by proteases
The appropriate and regulated balance of synthesis, degradation and remodelling of ECM in the kidney is key to avoiding renal fibrosis as a consequence of injury. A variety of enzymes can remodel the matrix, of which the most important are the matrix metalloproteinases
(MMPs) [63]. MMPs are a large family of proteinases, which play a fundamental role in degrading ECM and multiple cellular processes, such as releasing tissue-bound fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) and regulating inflammation [64]. To date, there are 28 MMP genes having been identified in humans, and most of them are multidomain proteins [65].
In turn, MMP activity is regulated in various ways, of which the most investigated is tissue inhibitors of metalloproteinases (TIMPs). TIMPs bind with MMPs and inhibit or modify their function [66]. Of note, in addition to the findings shown in Table 2.4, an early increase in
15
TIMP 1 and -3 expression and activity and a decrease in MMP-1 and -9 activity have been associated with the development of tubulointerstitial fibrosis in rats and humans [67-70].
These observations indicate upregulated ECM deposition following injury, but decreased degradation, accelerating the progression of tubulointerstitial fibrosis at a later period [71].
Table 2.4 MMP/TIMP Mouse Models in Renal Pathophysiology. The generation of
MMP/TIMP knockout mice knockout mice has advanced the understanding of the role of
MMPs/TIMPs in AKI/CKD mouse models [72].
Abbreviations: MMP, matrix metalloproteinase; TIMP, tissue inhibitors of metalloproteinase.
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2.2 Cells and signalling pathways in renal fibrosis
2.2.1 Fibroblasts in renal fibrosis
Fibroblasts are mesenchymal cells, phenotypically showing a spindle-shaped morphology and present in all tissues. They are the key to the structure and function of the ECM. They maintain tissue architecture by synthesising ECM proteins, as well as proteolytic enzymes and inhibitors [73]. Depending on the anatomical site and the degree of activation, fibroblasts exhibit high heterogeneity. Compared with other tissues, the normal renal cortex has a lower number of fibroblasts, which are concentrated in perivascular or peritubular areas. Unlike myofibroblasts, these cells are vimentin positive but not positive for α-smooth muscle actin (α-SMA) staining [73-75], suggesting that in a resting state they do not acquire a myofibroblastic phenotype.
It is clear that the number of fibroblasts increase in disease states and differentiate into myofibroblasts, which may be directed by various cytokines, such as TGF-β1 [73]. However, the origin of resident fibroblasts in renal cortex remains largely unknown. Some evidence suggests that bone marrow-derived fibroblasts make up 12% of the population in the normal kidney interstitium [76] but increases to 30% in the context of disease [77]. It is also proposed that resident interstitial fibroblasts can originate from uninduced mesenchyme in the embryonic kidney [78]. The source of both resident and accumulating renal fibroblasts has been the subject of considerable debate, which to date has not been definitively resolved.
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2.2.2 Myofibroblasts in renal fibrosis
Myofibroblasts are terminally differentiated cells with an increased capability of producing matrix production, which in turn promotes ECM accumulation in fibrosis. Myofibroblasts are rarely found in normal tissue. They are contractile cells displaying many of the morphological features of smooth muscle cells with an abundant expression of α-SMA, which is integrated into stress fibres and elevate the contractile activity of cells [73, 79-81].
In the normal tissue repair process, myofibroblasts appear but shortly thereafter undergo apoptosis. Under pathological situations, myofibroblasts can be induced and sustained by a number of growth factors including TGF-β and platelet-derived growth factor and enhanced signalling through the Wnt pathway (PDGF) β [82-85].
Given that the myofibroblast is well recognized as a key producer of ECM, the mechanism behind myofibroblast persistence in the kidneys of patients with CKD may be of key importance in studying the fibrotic response. Numerous investigations have been conducted on the origin of renal myofibroblasts. Previous work proposed that the proximal tubular cells (PTCs) can transform into interstitial myofibroblasts due to a process of epithelial- mesenchymal transition (EMT) [76]. Alternatively, or in addition, resident fibroblasts and/or pericytes may serve as the likely precursor to the myofibroblasts [86]. Further studies have also demonstrated inflammatory cells and bone-marrow derived fibrocytes may also transdifferentiate into myofibroblasts [7, 87, 88]. As alluded to above, the concept of EMT and endothelial to mesenchymal transition (Endo MT), has waxed and waned for over 20
18 years [89, 90]. By definition, EMT and endoMT refer to epithelial or endothelial cells undergoing morphological changes following injury. In this process there is a loss of epithelial or endothelial characteristics and acquisition of fibroblast markers (fibroblast- specific protein (FSP), vimentin and α-SMA) [76, 91-93]. Consequently, these cells start to function as (myo)fibroblasts in the tubulointerstitium with the secretion of stress fibres and unregulated ECM production.
To date, it is well accepted that α-SMA is the best available marker to identify activated myofibroblasts. However, recent literature suggested many matrix-producing cells do not always express α-SMA [94, 95]. Hence, the origin of renal myofibroblasts during ECM expansion in CKD is still under debate.
2.2.3 Inflammatory cells in renal fibrosis
Cells infiltrating the renal interstitium have long been believed to play a major role in the initiation and progression of tubulointerstitial fibrosis. This is because the degree to which a number of cell types, including macrophages, dendritic cells (DCs), T cells and natural killer
(NK) cells accumulate in the renal interstitium parallels the extent of fibrosis. Among all the cell types, the macrophage is the cell type in most models of CKD that is highly associated with tubulointerstitial fibrosis and poor renal outcomes [96, 97]. Blocking the function of macrophages either genetically or pharmacologically attenuates renal fibrosis in rodent models of unilateral ureteral obstruction (UUO) and diabetic nephropathy [98-100], which further suggests macrophages have a key role in the development of renal fibrosis.
19
Macrophages have been demonstrated to be a crucial source of MMP-9 in a late-stage UUO model [101], which indicates a controversial or perhaps a dual function of macrophages that may be dependent on the activation into an M1 or M2 phenotype, which is often context dependent [102, 103]. Thus, the macrophage-derived MMPs in CKD may need further investigation since the function of some MMPs differs in different CKD model and depending on the phase of injury. Furthermore, the macrophage has a key role in independent innate and adaptive immune pathways that further direct the injury response to repair or fibrosis.
2.2.4 TGF-β1
2.2.4.1 Overview of TGF-β1
TGF-β is the prototype of a family of secreted polypeptide growth factors. Three isoforms of
TGF-β have been identified in mammals, including TGF-β1, TGF-β2 and TGF-β3 [104]. All three TGF-β isoforms firstly bind with TGF-β receptor (TGFR) 2 as an active form
(homodimers) and then activates TGFR1 to initial receptor signalling [105]. After synthesizing in a precursor form, TGF-β is cleaved to release the latency associated peptide
(LAP), which binds to the TGF-β homodimer to promote the formation of the latent TGF-β binding protein (LTBP) [106]. However, at this stage, the TGF-β/LAP/LTBP complex remains inactive. To release active TGF-β in the ECM, one or more of a wide range of proteases including plasmin, matrix metalloproteinase (MMP) 2 and MMP9, thrombospondin,
20 integrins and the cationic independent mannose 6 phosphate receptor are needed to cleave the TGF-β complex into the active component [107].
It is well accepted that overexpression of active TGF-β1 induces a fibrotic response in multiple organs, including the kidney [108]. TGF-β1 is a well-characterised key mediator in the pathogenesis of tubulointerstitial fibrosis, due to its direct and indirect effect on various cells types [109-111]. The direct action of TGF-β1 transition of cells to a fibroblastic phenotype as described above and proliferation, migration and synthesis of profibrotic proteins, such as collagens and fibronectin [112-114]. TGF-β1 can also induce an indirect fibrotic response, via accelerating apoptosis of resident healthy cells and promoting resident and infiltrating cells to increase ECM deposition [115-117]. Inhibiting TGF-β1 in animal models of kidney disease attenuates fibroblast activation and ECM accumulation [118-121].
However, targeting the entire spectrum of actions of TGF- β1 in preventing renal disease has not been fruitful in that TGF-β1-specific, humanized, neutralizing monoclonal antibody added to RAAS inhibitors failed to slow progression of diabetic nephropathy [122].
2.2.4.2 TGF-β-Smad pathway
Smads are identified as the intracellular mediator of TGF-β1. Smads separate into different classes with regards to their functions: two TGF-β receptor-regulated Smads (R-Smad)
(Smad2 and Smad3), three bone morphogenetic protein (BMP) R-Smads (Smad1, Smad5, and Smad8) , one common mediator Smad (Co-Smad) (Smad 4) and two inhibitory Smads (I-
Smad) (Smad 6 and Smad 7) [123, 124]. TGF-β signalling is initiated with activation of the
21 receptor and R-Smad phosphorylation, followed by translocation of R-Smad/Co-Smad complex into the nucleus to regulate gene transcription [125]. By contrast, I-Smads can antagonize the activity of the R-Smads and prevent the phosphorylation of R-Smads [123]
(Figure 2.2).
Figure 2.2 TGF-β-Smad pathway. TGF-β binds to the primary TGF-β receptor type II (TGFR2), which is a constitutively active serine/threonine kinase that recruits receptor type I (TGFR1) to the complex. Subsequent transphosphorylation and activation of TGFR 1 by TGFR2 allows the TGFR1 kinase to phosphorylate selected Smads, which are inhibited by the so-called inhibitory Smads (Smad6 or Smad7). These receptor-activated Smads (R-Smads, e.g.
Smad2/3) then form active complexes with the common Smad4, which translocate into the nucleus, where they regulate transcription of certain target genes. In addition, the activated
22 receptor complex activates non-Smad signalling pathways, such as Ras, ERK, JNK, p38, RhoA and PI3K [9].
Abbreviations: ERK, extracellular-signal-regulated kinase; JNK, c-Jun N-terminal kinase; PI3K, phosphoinositide 3-kinase.
2.2.4.2.1 R-Smads
Among R-Smads, it is increasingly reported that BMP R-Smads (1, 5, 8) are involved in the development of kidney and renal cell cancer [126-128]. Some studies have reported that the
BMP-7-Smad1/5/8 pathway contributes to ECM deposition in the kidneys of UUO rats [129] and activin receptor-like kinase (ALK)-1 /Smad1/5 pathway may influence ECM protein expression in several cell types, such as rat myoblasts, hepatocytes and human chondrocytes [130]. However, the role of BMP R-Smads in fibrotic disorders is still unknown.
It is well demonstrated that Smad2 and Smad3 are extensively activated in the fibrotic response induced by TGF-β1 in various animal models and human kidney disease, including diabetic [131-135] and obstructive nephropathy [136-140], remnant kidney disease [141,
142], hypertensive nephropathy [143], drug-associated nephropathy, and immunologically- mediated glomerulonephritis [144, 145]. The binding of TGF-β1 to TGFR2 activates the
TGFR2 kinase and phosphorylates Smad2 and Smad3 [146]. TGF-β/Smad3 signalling mediates transcription of multiple downstream genes, such as the collagen chains ColIa1,
ColIa2, ColIIIa1, ColaVa2, ColVIa1 and ColVIa3, and TIMP-1 [147], suggesting a critical role of
23
Smad3 in fibrosis. In addition, it is increasingly reported that deletion of Smad3 in mice suppresses fibrosis in rodent models of kidney disease [133, 137, 148].
Relative to Smad3, the function of Smad2 in renal fibrosis is not fully elucidated. Because of the unavailability of Smad2 knock out (KO) mice, conditional kidney tubular epithelial cells
Smad2 KO mice have been generated by crossing the Smad2 floxed mouse with the kidney- specific promoter (Cadherin 16)-driven Cre transgenic mouse [149]. Unexpectedly, deletion of Smad2 in tubular cells significantly enhances fibrosis, with an associated elevated Smad3 signalling in the UUO mouse model [150]. It is commonly believed that Smad2 may inhibit the phosphorylation of Smad3 when exposed to TGF-β1. It is also possible that Smad2 and
Smad4 may reduce phosphor-Smad3 nuclear translocation and then binding to its target genes. Consistent with this view, a reduction in Smad 2 has been shown to increase activation of Smad3 and promote collagen expression in response to fibrotic mediators
[132, 141, 143, 148, 151]. Therefore, Smad2 and Smad3 may have distinct roles in mediating the fibrosis upon exposure to TGF-β1.
2.2.4.2.2 Co-Smad4
Smad4 plays a key role in nucleocytoplasmic shuttling of Smad2/3 and Smad1/5/8 into the nucleus [152]. Loss of Smad4 has been demonstrated to inhibit TGF-β1 induced ECM accumulation in mesangial cells. In addition, specific deletion of Smad4 from renal tubular cells alleviates renal fibrosis in a mouse model of UUO by suppressing Smad3 function [153].
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2.2.4.2.3 I-Smads
Smad 6 and Smad7 act as inhibitory mediators in the TGF-β/Smad pathway via a negative feedback loop that prevents the phosphorylation of Smad2 and 3 [154-157]. However, the phosphorylation of I-Smads remains largely unknown. Despite the phosphorylation sites, the importance of Smad6 for cellular signalling remains unclear [158-160].
TGF-β induces Smad7 transcription but then promotes its degradation by initiating the
Smad3-dependent Smurfs/Arkadia-mediated ubiquitin-proteasome degradation pathway that degrades Smad7 protein [156, 157, 161, 162]. Furthermore, deletion of Smad7 promotes fibrogenesis in a number of mouse models, including UUO [140], diabetic [135] and hypertensive nephropathy [163]. Collectively these studies suggest that activation of
Smad3 is tightly associated with the degradation of Smad7. An imbalance in the activity of
Smad3 and Smad7 has been reported to be a major mechanism in fibrogenesis.
2.2.4.3 Non-Smad pathways
In addition to Smad mediated signalling cascades, TGF-β can independently and directly activate other pathways, such as Ras/Raf/extracellular-signal-regulated kinase (ERK)/ mitogen-activated protein kinase (MAPK) pathways, c-Jun N-terminal kinase (JNK), p38
MAPK signalling and Rho-like GTPase signalling pathways [9]. TGF-β can induce phosphorylation of tyrosine residues on TGFRs (I and II) and recruit ERK through Ras, Raf and their downstream MAPK cascades. In this thesis, I specifically focussed on ERK as it regulates target gene transcription through its downstream transcription factors in
25 conjunction with Smads to control EMT [164]. ERK can also regulate the activity of R-Smads, including Smad1, Smad2 and Smad3 [165-168]. Moreover, ERK is involved in the autoinduction of TGF-β1 via distinct transcriptional and translational mechanisms in tubular epithelial cells [169]. These studies suggest a dominant role of ERK in the non-Smad mediated transduction of TGF-β1.
The Rho-like GTPases, including RhoA, Rac and Cdc42, play crucial roles in controlling dynamic cytoskeletal organization, cell motility, and gene expression through a variety of effectors [170]. In addition to MAPK pathways, RhoA is an important regulator, which can be activated by TGF-β via either Smad-dependent or independent pathways to induce stress fibre formation during EMT [171, 172].
JNK and p38 MAPK pathways are the best characterized non-Smad pathways involved in renal fibrosis. TGF-β can rapidly activate JNK and p38 MAPK via MAPK kinase (MKK) 4 and
MKK 3/6, respectively [173-179]. Activated JNK/p38 acts in conjunction with Smad2/3 to regulate apoptosis and EMT by controlling the activities of downstream transcription factors
[179-185]. JNK can also regulate Smad 3 activity directly by phosphorylation [185, 186].
2.2.5 Pyrin domain-containing protein 3 (NLRP3) inflammasome
Inflammation is integral to most kidney diseases. Adaptive immunity is associated with kidney disease where foreign antigens incite injury (e.g. in post-infectious glomerulonephritis) or autoantigens (e.g. in anti-glomerular basement membrane
26 glomerulonephritis, renal transplantation, IgA nephropathy). Innate immunity is responsible for antigen-independent inflammation [187]. Increasing research interest has focused on innate immune responses during renal infection, toxic, ischemic, and traumatic kidney injury[187]. However, evidence also exists that innate immunity contributes to CKD including diabetic nephropathy [188] and polycystic kidney disease [189]. Hence the importance of innate immune response in the progression of CKD is increasingly being investigated.
Innate immune response is initiated by several classes of pattern recognition receptors
(PRRs), such as TLRs and nucleotide-binding and oligomerization domain (NOD)-like receptors (NLRs), along with inflammasomes leading to the early innate immune response
[12]. The NLRP3 inflammasome consists of NLRP3 protein, adapter protein apoptosis- associated speck-like protein (ASC) and procaspase-1[190]. It is well established that the
NLRP3 Inflammasome plays a vital role in innate immunity. NLRP3 inflammasome activity can be induced in response to immune activators such as pathogen-associated molecular patterns (PAMPs), danger-associated molecular patterns (DAMPs), other exogenous invaders or environmental stress. Induced inflammasome complex triggers the transformation of procaspase-1 to caspase-1, as well as the conversion of interleukin (IL) -1β and IL-18 into a mature form that facilitates the biological function of inflammasome (Figure
2.3) [191, 192].
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Figure 2.3 Schematic illustration of the NLRP3 inflammasome activation. Upon exposure to
PAMPs or DAMPs, TLRs are phosphorylated and subsequently activate NF-κB. In the nucleus,
NF-κB promotes the transcription of NLRP3, pro-IL-1β, and pro-IL-18, which, after translation, remain in the cytoplasm in inactive forms. Thus, this signal (depicted in red as
“Signal 1”) is a priming event. A subsequent stimulus (shown as “Signal 2” in black) activates the NLRP3 inflammasome by facilitating the oligomerization of inactive NLRP3, ASC, and procaspase-1. This complex, in turn, catalyzes the conversion of procaspase-1 to caspase-1, which contributes to the production and secretion of the mature IL-1β and IL-18 [192].
Abbreviations: NLRP3, pyrin domain-containing protein 3; PAMPs, pathogen-associated molecular patterns; DAMPs, danger-associated molecular patterns; TLRs, Toll-like receptors;
NF-κB, nuclear factor kappa light chain enhancer of activated B cells; IL, interleukin; ASC, apoptosis-associated speck-like protein; ROS, reactive oxygen species.
28
To date, the cell-specific expression of the components of NLRP3 inflammasome within the kidney remains only partially elucidated. Renal immune cells, such as DCs and macrophages, express all NLRP3 components. Some non-immune renal parenchymal cells don’t express all the components of the inflammasome. For example, mesangial cells can express and release
IL-18 but not IL-1β. As such, the canonical effect of the NLRP3 inflammasome in renal non- immune cells remains uncertain. Of note, expressing and releasing of IL-18 and IL-1β has been observed in tubular epithelial cells, indicating the potential function of NLRP3 in tubular injury [193].
Given that the NLRP3 inflammasome is a key mediator in the inflammatory response,
NLRP3-deficiency has been reported to protect mice from ischemic acute tubular necrosis
[194, 195]. However, consistent results have not observed in ASC deficiency animals [195].
Similarly, blockade of IL-1 or IL-18 has had no effect on ischemic kidney injury [195, 196].
Thus, renoprotection dependent on IL-1 or IL-18 deficiency has not been consistently reported. Conversely, NLRP3 deficiency has been reported to reduce renal ischemia- reperfusion injury in all studies [195-199], suggesting the NLRP3 inflammasome has non- canonical effects independent of IL-1β or IL-18. These non-canonical effects of NLRP3 remain under debate but some studies have suggested it could be related to TGF-β signalling within tubular epithelial cells[193]. The NLRP3 expression can be induced by TGF-β in a Smad3-dependent manner and is required for TGF-β-mediated Smad2/3 phosphorylation. This function of NLRP3 seems to correlate with EMT, which is highly associated with interstitial fibrosis in CKD [200, 201].
29
2.2.6 Toll-like receptors (TLRs)
TLRs are a conserved family of pattern recognition receptors which share the same cytoplasmic domain as the toll-IL-1 receptor (TIR) and similar activation pathways, including identified 11 human and 13 mouse TLRs [202]. In response to PAMPs or DAMPs, TLRs activate host cell inflammatory response. After activation, TLRs recruit differential adapter molecules and catalyze the downstream signalling pathway including myeloid differentiation primary response protein 88 (MyD88)-dependent and independent, and thus result in the release of cytokines and chemokines (Figure 2.4) [203, 204].
30
Figure 2.4 TLRs pathway. All TLRs (except for TLR3) signal through the adaptor protein
MyD88 to activate nuclear factor (NF)-κB and mitogen-activated protein kinases for the induction of proinflammatory cytokines. TLR3 signals through TRIF /TICAM1. Other TLR adaptors include TRIF, TIRAP, and TRAM [205, 206].
Abbreviations: MyD88, myeloid differentiation primary response protein 88; TRIF, TIR- domain-containing adaptor-inducing interferon-β; TICAM1, TIR-containing adaptor molecule
1; TIRAP, TIR-domain-containing adaptor protein; TRAM, TRIF-related adaptor molecule; IKK, inhibitor of κB-kinase; IRAK, interleukin-1 receptor-associated kinase; NEMO, NF-κB essential modulator; pDCs, plasmacytoid dendritic cells; RIP1, receptor-interacting protein-
1; TAB-1, TAK1-binding protein; TAK, TGF-β activated kinase; TRAF, tumour necrosis factor receptor-associated factor.
TLRs are expressed in a number of cells within the kidney, such as DCs [207, 208], lymphocytes [209], macrophages [210-212], intrinsic renal cells [213], podocytes [213], mesangial [214, 215], endothelial [216-218] and tubular epithelial cells [219]. Specifically, interstitial and glomerular macrophages express TLR1, 2, 4, and 6, and DCs express TLR4, 7,
8, and 9. TLR2 and TLR4 are upregulated in monocytes as well as in tubular cells and myofibroblasts in models of renal injury stage [206]. To date, most of the TLRs studies on
CKD focus on TLR2 and TLR4 (Table 2.5).
31
Table 2.5 TLR2 and TLR4 expression in association with chronic kidney diseases [206].
Abbreviations: CKD, chronic kidney disease; DN, diabetic nephropathy; ESRD, end-stage renal disease; IgA, immunoglobulin A; TLR, Toll-like receptor; UUO, unilateral ureteral obstruction.
TLR signalling, mainly TLR2 and TLR4, is implicated in inflammatory and autoimmune kidney disease as well as in non-immune kidney disease, including ischemia/reperfusion (I/R) injury
[220, 221], UUO [222-224], tubulointerstitial nephritis [225], nephrotoxicity [226-228] and diabetic nephropathy [229, 230]. There is strong evidence implicating TLR2 and 4 as mediators of inflammation in the non-immune UUO model of renal injury. TLR2 was markedly upregulated in the kidney subjected to UUO, as early as 2 days after surgery [231,
232]. Evidence from TLR2-/- mice studies supports that TLR2 initiates renal inflammation during the early stage of UUO [231]. However, deletion of TLR2 did not change the ECM accumulation in this model [231], which is supported by independent reports that the development of renal fibrosis in the UUO model is independent of TLR2-MyD88 signalling
32
[232]. Similarly, renal TLR4 expression was found to be progressively elevated in the UUO model [224]. In contrast to the TLR2-/- mice, TLR4 -/- mice had less renal fibrosis with less α-
SMA and fibroblast accumulation [224], suggesting that TLR2 and TLR4 may have distinct roles in the development and progression of renal pathology.
Renal biopsies of patients with diabetic nephropathy show a significant up-regulation of
TLR2 [230] and TLR4 [229] in the tubules compared to those from normal kidneys. In mouse models of diabetic nephropathy, TLR2 plays a pro-inflammatory role, which is consistent with the role demonstrated in the UUO model. Deletion of TLR2 reduced macrophage- mediated inflammation, albuminuria and podocyte loss in STZ-induced type 1 DM [233].
Conversely, TLR4 induces tubulointerstitial fibrosis via increasing the susceptibility of tubular cells and myofibroblasts upon TGF-β independent of TGF-β/Smad pathway [222].
Additionally, fibrinogen, an endogenous ligand for TLRs, is known to initiate resident fibroblast proliferation through the TLR2/TLR4 MyD88/ nuclear factor kappa light chain enhancer of activated B cells (NF-κB) pathway in the kidney [223, 234].
Therefore, although TLR2 and TLR4 are often contemporaneously upregulated in multiple models of renal disease, they have distinct roles in the development of renal fibrosis. TLR2 triggers pro-inflammatory responses while TLR4 regulates both pro-inflammatory and pro- fibrotic pathways (Figure 2.5).
33
Figure 2.5 Role of Toll-like receptor -2 and -4 in renal fibrosis [206].
Abbreviations: ECM, extracellular matrix; HMGB1, high-mobility-group box 1; HSPs, heat shock proteins; TGF-β, transforming growth factor-β.
2.2.7 Tumour necrosis factor-α (TNF-α)
TNF-α, produced by various cells including macrophages, mesangial cells and renal tubular epithelial cells, stimulates the release of multiple cytokines and contributes to renal fibrosis
[235, 236]. As the first cytokine to appear in the blood after injury or stress, TNF-α is well recognized as an important host defence molecule. The release of other pro-inflammatory mediators, such as IL-1 or IL-6, is dependent on the prior release of TNF-α. TNF-α ‘s biological function is mediated by binding with two TNF receptors (TNFR)1 and TNFR2, which then promotes the downstream generation of inflammatory cytokines [237].
Elevated TNF-α levels have been observed in various rodent models of kidney disease, such as cyclosporine A-induced nephropathy [238], diabetic nephropathy [239, 240], adenine- induced nephropathy [241]. Moreover, inhibition of TNF-α slows the progression of renal
34 disease, including in diabetic nephropathy [242]. An anti-TNF-α monoclonal antibody has been evaluated in Phase II clinical trial (NCT00814255) as an anti-fibrotic agent in patients with primary focal segmental glomerulosclerosis (FSGS). Unfortunately, the trial failed to recruit sufficient patients to reach a definitive conclusion [235]. Furthermore, in patients with type 1 diabetes, the renal functional decline was associated only modestly with total
TNFα concentration in serum and was unrelated to free TNFα, whilst circulating concentrations of TNFR1 and TNFR2 were strongly associated with a risk for the early renal decline and remained stable over long periods [243]. Similar results have also been found in patients with type 2 diabetes. Elevated TNFR1 and TNFR2 were associated with an increased risk for progression of functional renal decline in patients with diabetic kidney disease [244].
2.3 Animal models of renal fibrosis
To study the mechanisms and pathogenesis of renal fibrosis and provide an avenue for future treatment, many animal models have been developed, each of those mirrors various elements of human CKD. However, each model has strengths and disadvantages. There is no perfect model which can exactly mimic all characteristics of human renal fibrosis.
2.3.1 FA nephropathy
It is well known that high dosages of folic acid given i.p. in mice induces tubular necrosis by direct tubular toxicity, deposition of FA in the tubular lumen, interstitial oedema, oliguria and proliferation of interstitial connective tissue [245]. Thus, FA induces renal injury via two mechanisms: direct nephrotoxicity and the deposition of intratubular FA crystals, resulting
35 in intrarenal obstruction. To reduce the deposition of FA and separate the nephrotoxic and obstructive effects of FA, the alkalinity of the urine is increased with co-administration of sodium bicarbonate (FA in 0.3 M NaHCO3 vehicle) to decrease intratubular crystal formation when using this model [246-248].
FA leads to acute injury (1-14 days) [249] and patchy interstitial fibrosis in the chronic phase
(28-42 days) [250]. Because of the varying pathological change in the kidney at different time points, the FA-induced kidney injury model is widely used for studying both acute kidney injury [251, 252] and chronic kidney fibrosis [248]. In addition, compared with the
UUO model, which by virtue of it being a unilateral renal injury doesn’t result in significant renal impairment, renal function correlates with the development of CKD [253]. Having said this, it is reported that after FA injection in rats, plasma creatinine and blood urea nitrogen
(BUN) peak at 24 hours and plateau up to day 6 and decrease thereafter [254]. Hence assessment of kidney function in the acute phase is indicative of acute kidney injury but is less reflective of renal pathology in the chronic phase. This is consistent with serum markers of renal function in humans where considerable renal pathology can exist before serum markers become abnormal [255].
2.3.2 Streptozotocin (STZ)-induced diabetic nephropathy (DN)
A reliable animal model of diabetic renal injury is a useful tool to investigate the underlying mechanism of diabetic nephropathy. It is well studied as diabetic kidney disease is the commonest cause of CKD and hence a rodent model of DN is highly relevant to human
36 disease. STZ is an analogue of N-acetylglucosamine (GlcNAc), which is transported into pancreatic β cells. STZ selectively inhibits the activity O- GlcNAcase in β cells, causing O- glycosylation of intracellular proteins, β -cell apoptosis and ensuing insulin deficiency [256].
Using STZ to establish type 1 diabetes has been shown to develop modest elevations in albuminuria and serum creatinine and histological lesions reflective of diabetic nephropathy
18-24 weeks following the induction of diabetes [257, 258]. There are two approaches to induce diabetic nephropathy using STZ in mice. One is a low-dose injection of STZ (55 mg/kg) IP for five consecutive days. One week after the final STZ injection, over 90% of STZ- treated mice have a non-fasting blood glucose of more than 15 mmol/L. The second model uses a single high dosage of STZ (≥200 mg/kg) or a two-dose regimen of STZ (2 x 100–125 mg/kg) given on consecutive days. STZ-induced diabetic mice may have significant weight loss, which may due to the catabolic effects of insulin deficiency, severe hyperglycaemia and the volume depletion associated with osmotic diuresis. Low dose insulin treatment may be required to stabilise the catabolic effects without reversing hyperglycaemia [259]. Of note, at high doses, STZ has non-specific cytotoxicity to kidney, which can result in acute kidney injury in mice and rats [260, 261], thus makes using this model less reflective of pure diabetic nephropathy. Hence the low dose model is preferred.
The use of STZ is a robust method for inducing diabetes in rodents. However, all animal models of diabetic nephropathy vary in the degree to which they resemble human disease
[262]. Furthermore, the susceptibility to induce diabetes and diabetic nephropathy differs in different strains of mice with different genetic backgrounds [263]. For example, C57BL/6
37 mice are the most commonly used strain of mice used experimentally for a number of different diseases. Compared with other strains, C57BL/6 mice are resistant to the development of renal injury induced by diabetes. C57BL/6 diabetic mice show mild to moderate albuminuria and display early nephropathic changes at 6 months after the induction of diabetes [264].
Endothelial dysfunction has been demonstrated as a crucial part in the progression of DN
[265, 266]. Nitric oxide (NO) produced by NO synthase (NOS), mainly endothelial nitric oxide synthase (eNOS) in renal vasculature, contributes to the endothelial dysfunction in diabetes
[267, 268]. Fundamentally, DN is a microvascular disease. Multiple studies have reported that the eNOS-/- diabetic mice display advanced nephropathic changes the features similar to human DN compared to its wild-type mouse model [269, 270]. These studies clearly defined eNOS-/- diabetic mouse model a robust animal model of DN. Thus, the genetic background and strain of mice are necessary to consider when using an insulin-deficient
STZ-induced model.
2.3.3 Adenine
CKD induced by adenine has been shown to be a reliable model of CKD in mice and rats.
Adenine is metabolized to 2,8-dihydroxyadenine, which precipitates and produces tubular crystals within the kidney [271-273]. Adenine can be administrated by oral gavage or diet. It is reported that oral dosing with adenine at 50 mg/kg for 28 days is suitable to induce a stable anemia associated with CKD in mice [274]. Furthermore, the administration of a
38
0.75% adenine diet in rats for 4 weeks results in irreversible chronic renal failure, which reflects CKD seen clinically [275]. Of interest, different manifestations of CKD can be observed using different dietary concentrations of adenine (Table 2.6) [276].
Table 2.6 Comparison of CKD manifestations with different adenine doses [276].
2.3.4 5/6 Nephrectomy
5/6 nephrectomy mimics progressive renal failure after the loss of renal mass in human. It is essentially a hyperfiltration model of the renal disease. To induce this model, there are different ways to ablate 2/3 of the remaining kidney after removing one kidney [277]. One approach often used in rats is ligation of the branches of the renal artery after contralateral uninephrectomy. However, this approach is not usually suitable in mice due to limited renal artery branching. Hence an alternative method is to remove approximately 50% of the remaining kidney by polar excision 1-2 weeks after uninephrectomy. The final approach is to ligate one or more branches of the mouse renal artery, with cauterisation performed as needed to remove additional renal mass to establish the model.
39
The ligation results in more severe proteinuria and hypertension than those with excision, which may be due to the upregulated RAAS observed with regional ischemia. Proteinuria develops from week 2 and reaches 200-600mg/24h. Glomerular hypertrophy occurs in the acute phase (0-4 weeks). After 8 weeks, about up to 20% glomeruli develop mesangial expansion and focal and segmental glomerular sclerosis, associated with early interstitial fibrosis and tubular atrophy. By 12 weeks, universal glomerulosclerosis and tubulointerstitial fibrosis appear [253, 278].
2.3.5 Ureteric ureteral obstruction (UUO)
The UUO model is widely used to study mechanisms of tubulointerstitial fibrosis [279-281].
UUO induces severe renal injury, characterised by reduced renal blood flow and glomerular filtration rate within 24 h, followed by interstitial inflammation (peak at 2-3 days), tubular dilation, tubular atrophy and fibrosis within a week. By 2 weeks, the obstructed kidney has no function [282]. The model develops interstitial infiltration of macrophages, tubular cell death, the phenotypic transition of resident renal cells and severe interstitial renal fibrosis with the associated expansion of the ECM.
Complete ureteral obstruction is not a usual cause of human renal disease. However, partial obstructive nephropathy is not uncommon. Therefore, models of partial or reversible UUO have been developed in the rat and mouse. However, compared to the complete obstruction model, renal pathology in the partial or reversible models is variable and unpredictable [283]. Thus, the advantage of the complete obstruction model is that it is
40 reproducible, and injury develops within a short time of obstruction. The disadvantages include the lack of serum biochemical markers of CKD and given the mechanical nature of the injury, pharmacological interventions are unlikely to positively impact on the pathology observed.
Compared to other models of progressive renal injury, the chronic phase of the FA model of
CKD provides a useful tool to study the mechanism of the fibrotic response as well as the impact of pharmacological intervention on fibrosis. The low-dose STZ induced diabetic model is a reliable and robust model for diabetic study, which is approved by the Animal
Models for Diabetic Complications Consortium (AMDCC) [284]. Therefore, I chose FA nephropathy and the low-dose STZ induced diabetic models for my studies.
2.4 Receptor-interacting serine/threonine-protein kinase (RIPK) 3
The RIP kinase family contains seven members, each of which contains a homologous kinase domain. To date, the functions of RIPKs 4–7 are poorly understood [285]. RIPK2 is a mediator of mucosal immunity. Extensive studies have clarified the important molecular and physiological roles of RIPK1 and RIPK3 in inflammation and cell death [286, 287].
The RIPK3 gene is located on chromosome 14 in both human and mouse. This gene encodes a 518 amino acid (aa) protein with a molecular mass of 57kDa in humans, whereas it encodes a 486 aa protein of 53kDa in mice 1-3 (as shown in Figure 2.6). RIPK3 is a threonine/serine protein kinase which shares almost 30% identity and 60% with the other
41 two RIP members, RIPK1 and RIPK2 [288, 289]. Compared with RIPK2, RIPK3 and RIPK1 have a unique C-terminal RIP homotypic interaction motif (RHIM) [288], which enables homotypic protein interactions [290].
To date, several phosphorylation sites of RIPK3 have been identified. Ser227 (Thr231/Ser232 for mouse RIP3) and Ser199 (Ser204 in mouse) are particularly crucial for the activation of its downstream substrate in the necroptosis pathway, mixed-lineage kinase domain-like
(MLKL) [291-294].
Figure 2.6 RIPK3 structure. (a) RIPK3 structure of human RIPK3 (hRIPK3) and (b) mouse RIPK3
(mRIPK3) [295]. Abbreviations: RIPK3, receptor-interacting serine/threonine-protein kinase
3; RHIM, RIP homotypic interaction motif.
2.4.1 RIPK3 in necroptosis
In response to physiological signals and pathological stimuli, cell death is crucial to maintaining homeostasis. To date, several types of cell death have been identified. Among them, necrosis is a type of cell death characterized by loss of intracellular contents and the
42 triggering of a subsequent inflammatory response. For many years, necrosis was considered to be accidental and therefore unregulated cell death [296-299]. The recognition that necroptosis is programmed necrosis has provided new insights into necrosis-induced cell death. Necroptosis has been well demonstrated to be mediated by the dimerization of
RIPK1-RIPK3 to form the necrosome with downstream expression of MLKL [300]. RIPK1 and
RIPK3 both possess RHIM, with bilateral interaction between RIPK1 and RIPK3.
Subsequently, the necrosome facilitates the aggregation of phosphorylated RIPK3 and phosphorylation of MLKL by RIPK3. Phosphorylated MLKL translocates to the cell membrane and thus induces necroptosis (Figure 2.7) [301]. Along with the activity of the inhibitors of apoptosis protein (IAP) ubiquitin ligases, a death domain (DD) on RIPK1 can also induce the adapter Fas-associated protein with death domain (FADD), caspase 8 and the caspase paralog cellular FADD-like IL-1β-converting enzyme-inhibitory protein (cFLIP) to the necrosome [302-305]. Consistently, in the absence of cIAPs and/or blockade of caspase 8, necroptosis will occur [304].
The cytokine TNF has a key effect on cell death and tissue injury mainly via TNFR1 associated TNF signalling [297]. TNF is known as a potent inducer of necrosis and apoptosis and hence much of the knowledge on necroptosis comes from the studying of TNF [294,
306, 307]. TNFR1 stimulation induces a different response in cells depending on the cell type and pervading inflammatory conditions. For example, for most cells, TNFR1 stimulation activates inflammatory pathways via the formation of a membrane-associated protein complex (complex I) [297]. In the presence of specific conditions or in certain cells, TNFR1
43 activation can recruit and trigger the necrosome formation and thus induce necroptosis
(Figure 2.7) [25].
In addition to TNFR1, there are other death receptors (DRs) of the TNF superfamily, such as
Fas (also known as CD95 or apoptosis antigen 1), DR3 (also known as apoptosis antigen 3),
TNF-related apoptosis-inducing ligand receptor 1 (TRAILR1, also known as DR4), TRAILR2
(also known as DR5), and DR6 [308]. Binding of Fas ligand (FASL) to Fas or TRAIL to TRAILR1 or TRAILR 2 stimulates the formation of a membrane-associated death-inducing signalling complex (DISC) via FADD, resulting in an activation of caspase 8 and subsequent apoptosis.
In the absence of cIAPs and blockade of caspase 8, these ligands can also induce necroptosis
[303]. In addition, other receptors including TLRs, NLRs, retinoic acid-inducible gene I (RIG-I)- like receptors (RGRs), ripoptosome and protein kinase R (PKR) complexes have also been demonstrated to initiate necroptosis [14].
44
Figure 2.7 RIPK1-RIPK3 regulates and integrates necroptotic cell death from multiple receptors. Necroptosis can be induced by death receptors including TNFR1 (top left); TLR4, via a TRIF-RIPK1-RIPK3 complex (top middle); TLR3, via a TRIF–RIPK1–FADD–cFLIP–caspase-8 complex (top right); or IFNRs, via a RIPK1-RIPK3 complex (top, middle). These pathways can induce the association of RIPK1 with RIPK3 via RHIM-RHIM domain interactions (light blue) and phosphorylation of RIPK3, which leads to aggregation of phosphorylated RIPK3 (pRIPK3) and phosphorylation of MLKL by RIPK3. Translocation of phosphorylated MLKL (pMLKL) to the cell membrane leads to necroptosis, with the release of DAMPs [25].
Abbreviations: RIPK, receptor-interacting serine/threonine-protein kinase; TNFR, tumour necrosis factor receptor; FADD, Fas-associated protein with death domain; cFLIP, cellular
FADD-like IL-1β-converting enzyme-inhibitory protein; MLKL, mixed-lineage kinase domain-
45 like; IFN, interferon; LPS, lipopolysaccharide; mtDNA, mitochondrial DNA; MCMV, murine cytomegalovirus; dsDNA, double-stranded DNA.
As discussed above, necroptosis is a type of regulated necrosis which is mediated by RIPK3,
RIPK1 and the executor MLKL. However, in contrast to the obligatory involvement of RIPK3,
RIPK1 is not always required. The M45-mutant strain of murine cytomegalovirus (MCMV) infection induces RIPK3 activation in the absence of activation of RIPK1 [309, 310]. There is also evidence that the RHIM-containing protein (ICP) 6 protein of herpes simplex virus 1 recruits RIPK3 directly and independent of RIPK1 [311]. In addition, as shown in Figure 2.8,
RIPK1 may have dual influences on cell death by both promoting necroptosis and protecting cells from death under certain conditions. However, the necroptosis inhibitor Nec-1s, known as the RIPK1 inhibitor, has been consistently proven to abolish the RIPK1/RIPK3 necrosome formation and following necroptosis in various models and cells [312-314].
46
Figure 2.8 Time of death of mice mutant for genes controlling apoptosis or necroptosis.
Mouse genotypes are indicated on the left and the length of the line on the right indicates how long each genotype survives. Blue lines indicate where death is due to inappropriate necroptosis, red lines indicate where death is due to inappropriate apoptosis, and purple lines indicate where death is due to apoptosis and necroptosis. Embryos are designated E0 when a vaginal plug is detected. P0 refers to newborn mice [315].
Abbreviations: kd, D161N kinase dead mutant. Cflar encodes c-FLIP (cellular FADD-like IL-
1β-converting enzyme-inhibitory protein).
2.4.2 RIPK3 in inflammation
47
In addition to its functional involvement in necroptosis, under specific conditions, RIPK3 can also serve as a pro-apoptosis adaptor, which recruits RIPK1 and FADD to activate caspase 8 and thus apoptosis. This effect relies on the involvement of caspase 8 when RIPK3 is inactive or MLKL is absent [316, 317]. RIPK3 deficient animals develop normally whereas mice expressing catalytically inactive RIPK3 D161N died around embryonic day 11.5 from unregulated apoptotic injury [316]. Similar effects are observed in a study using compounds that selectively inhibit RIPK3 interaction with caspase 8 independent of pro-necrotic kinase activity and MLKL [317].
RIPK3 is mainly reported as a regulator of cell death, but recent studies also identify that
RIPK3 is an important mediator in NLRP3 inflammasome formation [23]. It is well documented that elevation of RIPK3 activity induces an increase of phosphorylation of MLKL activity, which leads to the NLRP3 activation. Furthermore, increased RIPK3 expression can directly promote NLRP3 inflammasome and the associated inflammatory responses independent of MLKL [23-27, 318].
2.4.3 RIPK3 in kidney disease
Given the importance of RIPK3 in inflammation is increasingly recognised, recent studies have investigated the role of RIPK3 in kidney disease. The majority of these studies have used an acute kidney injury (AKI) animal model [319, 320] or allograft survival after transplantation [321]. Lack of RIPK3 protects kidney tubular injury in both sepsis-induced
AKI [319] and FA-induced AKI [320]. Interestingly, MLKL deficiency aggravates kidney
48 function associated with amplified histologic damage in the model of FA-induced AKI [320], suggesting that the effect of RIPK3 may be independent of MLKL. However, no study has assessed the role of RIPK3 in the chronic phase of kidney disease.
A recent report has demonstrated that RIPK3 promotes kidney fibrosis, which is correlated with AKT-dependent ATP citrate lyase (ACL) in both the UUO-induced and adenine diet- induced renal fibrosis. This study, contemporaneously undertaken at the time of my PhD, is the first to address the role of RIPK3 in renal fibrosis by mediating survive and proliferation of fibroblasts [22]. It needs to be noted that deficiency of MLKL also failed to protect the kidney from fibrosis in this study [22].
2.4.4 Pharmacological blockers of RIPK3
To date, several selective small-molecule compounds have been shown to inhibit RIPK3- dependent necroptosis [317], providing an impressive toolbox for the investigation of the role of RIPK3 in diverse tissues. GSK’872 is the most widely-used cell-permeable inhibitor of the
RIPK3-selective kinase, with >1,000-fold selectivity over a vast majority of more than 300 other kinases [322]. GSK’872 binds the kinase domain and inhibits kinase activity with high specificity, targeting a broad range of pro-necrotic stimuli [317] and has been used to specifically inhibit RIPK3 [22, 322-325].
Compared with GSK’872, dabrafenib is not a selective RIPK3 inhibitor. Previous studies have reported that dabrafenib is a RIPK3 inhibitor in various models, including human hepatocytes
49
[326], mouse models of acetaminophen-induced liver injury [326] and ischemic brain injury
[327]. In addition, dabrafenib is a well-known inhibitor of B-Raf, which suppresses the downstream Ras/Raf/ERK/MAPK pathway [328] which has been approved for clinical use for the treatment of non-small cell lung cancer expressing B-Raf V600E mutations and in melanoma
[329]. Inhibition of Raf kinase is known to attenuate renal fibrosis in a rat model of autosomal dominant polycystic kidney disease [330]. It is also recognised that transforming growth factor beta (TGF-β) 1 can induce Ras/Raf/ERK/MAPK pathways [331], resulting in renal fibrosis.
Therefore, dabrafenib may show renoprotection in the context of diverse models of renal fibrosis.
Collectively, RIPK3 might be a crucial regulator in renal fibrosis via its mediated-necroptosis or -inflammation. By using the RIPK3 -/- mouse model in 2 diverse forms of renal disease and comparing to the results observed in the dabrafenib treated mice, we considered it would be possible to dissect mechanisms of benefit of RIPK3 inhibition and conversely benefit of dabrafenib independently of RIPK3.
50
Chapter 3 Methodology
3.1 Materials
3.1.1 General chemicals
Table 3.1. General chemicals and compounds utilised in this thesis
Name Supplier Cat. No
2-Mercaptoethanol Sigma-Aldrich M3148
Agarose Bio-Rad 161-3102
Aluminium ammonium sulphate Sigma-Aldrich 202568
Ammonium hydroxide Sigma-Aldrich 320145
Boric acid Sigma-Aldrich 185094
Bovine serum albumin Sigma-Aldrich A9647
Citric acid UNIVAR B/NO.1203045
Dabrafenib CAYMAN CHEMICAL 1195765-45-7
Ethanol POCD ETHABS5P
Ethylenediaminetetraacetic acid Sigma-Aldrich E6758
Folic acid Sigma-Aldrich F7876
Formalin Sigma-Aldrich HT501128
Glacial Acetic Acid Sigma-Aldrich PHR1748
51
Glycerol Sigma-Aldrich G9012
Glycine ANALAR NORMAPUR 10119CU
Haematoxylin Sigma-Aldrich H9627
Hydrogen peroxide UNIVAR B/NO.1611231569
Methanol POCD METH5P
Periodic acid POCD PERIO50%/100
Schiff's reagent POCD SCHIFFS500ML
Sodium chloride MERCK K40428104 939
Sodium hydrogen carbonate ANALAR NORMAPUR 10247
Sodium hydroxide UNIVAR B/NO.1301133957
Sodium iodate Sigma-Aldrich S4007
Streptozotocin Sigma-Aldrich S0130
TRIS ASTRAL SCIENTIFIC 497
TRIS hydrochloride Sigma-Aldrich T3253
Tween 20 Sigma-Aldrich P1379
Xylene POCD XYLENEPL5LT
3.1.2 Buffers and solutions
3.1.2.1 Buffers for genotyping
52
0.2M NaOH/ 1 mM Ethylenediaminetetraacetic acid (EDTA) solution
Tris-HCl/Citric acid solution:
0.33M Tris-HCl pH 8.5
0.1M citric acid
Tris-borate-EDTA (TBE) electrophoresis buffer (10X)
1 M Tris base
1 M Boric acid
0.02 M EDTA
3.1.2.2 Buffers for immunohistochemistry
Heat retrieval solution, pH 6.0
0.01M Citrate buffer
Tris-buffered saline with Tween 20 (TBST) wash buffer, pH 7.6
0.05M Tris
0.15M NaCl
0.05% Tween 20
53
Mayer’s Haematoxylin
0.5% (w/v) Haematoxylin
5% (w/v) Aluminium ammonium sulphate
0.03% (w/v) Sodium iodate
2% (v/v) Glacial acetic acid
30% (v/v) Glycerol
Scott's bluing solution
0.5% (v/v) Ammonium hydroxide
3.1.2.3 Buffers for western blot
Lysis buffer
1× Radioimmunoprecipitation assay (RIPA) buffer (Thermo Scientific)
1× Protease and Phosphatase Inhibitor Cocktail, EDTA-free (Roche)
Transfer buffer
25mM Tris
190mM Glycine
54
20% Methanol
TBST buffer
20mM Tris, pH7.5
150mM NaCl
0.1% Tween 20
Blocking buffer
5% BSA in TBST
3.1.3 The sequence of probes and primers
Table 3.2. The sequence of probes used for quantitative real-time PCR
Target (Assay ID) Sequence (FAM labelled Probe/ Primer 2 /Primer 1 Sequence 5' -
-> 3')
ASC /56-FAM/CAG TGT CCC /ZEN/TGC TCA GAG TAC AGC /3IABkFQ/
Mm.PT.56a.42872867 GCT TAG AGA CAT GGG CTT ACA G
GGT CCA CAA AGT GTC CTG TT
F4/80 /56-FAM/AGT CTG GGA /ZEN/ATG GGA GCT AAG GTC
A/3IABkFQ/
55
Mm.PT.58.11087779 ATT CAC TGT CTG CTC AAC CG
GGA AGT GGA TGG CAT AGA TGA hCollagen IV /56-FAM/TCA TAC AGA /ZEN/CTT GGC AGC GGC T/3IABkFQ/
Hs.PT.58.15679435 AGA GAG GAG CGA GAT GTT CA
TGA GTC AGG CTT CAT TAT GTT CT hFibronectin /56-FAM/TAC AGC TTA /ZEN/TTC TCC CTC GCC CAG /3IABkFQ/
Hs.PT.58.40005963 CGT CCT AAA GAC TCC ATG ATC TG
ACC AAT CTT GTA GGA CTG ACC hTGF-β1 /56-FAM/ACC CGC GTG /ZEN/CTA ATG GTG GAA /3IABkFQ/
Hs.PT.58.39813975 CCG ACT ACT ACG CCA AGG A
GTT CAG GTA CCG CTT CTC G hTubulin /56-FAM/ACT CAC GCA /ZEN/TGG TTG CTG CTT C/3IABkFQ/
Hs.PT.58.27209704 TCT CTT ACA TCG ACC GCC TA
CAT TGC CAA TCT GGA CAC CA
IL-1β /56-FAM/TTC CAA ACC /ZEN/TTT GAC CTG GGC TGT /3IABkFQ/
Mm.PT.58.41616450 GAC CTG TTC TTT GAA GTT GAC G
CTC TTG TTG ATG TGC TGC TG mCollagen IV /56-FAM/TCT TTC TCC /ZEN/CTT TTG TCC CTT CAC GC/3IABkFQ/
56
Mm.PT.58.31838522 TCT GGC TGT GGA AAA TGT GA
AAT CCA ATG ACA CCT TGC AAC
MCP-1 /56-FAM/TCA ATG CCC /ZEN/CAG TCA CCT GCT /3IABkFQ/
Hs.PT.58.45467977 AGC AGC CAC CTT CAT TCC
GCC TCT GCA CTG AGA TCT TC mFibronectin /56-FAM/CAG GAG ATT /ZEN/TGT TAG GAC CAC GGC
A/3IABkFQ/ Mm.PT.58.8135568
GAG CTA TCC ATT TCA CCT TCA GA
TTG TTC GTA GAC ACT GGA GAC mGAPDH /56-FAM/TGC AAA TGG /ZEN/CAG CCC TGG TG/3IABkFQ/
Mm.PT.39a.1 AAT GGT GAA GGT CGG TGT G
GTG GAG TCA TAC TGG AAC ATG TAG mTGF-β1 /56-FAM/ATA GAT GGC /ZEN/GTT GTT GCG GTC CA/3IABkFQ/
Mm.PT.58.11254750 GCG GAC TAC TAT GCT AAA GAG G
CCG AAT GTC TGA CGT ATT GAA GA mTubulin /56-FAM/CAT GTT CCA /ZEN/GGC AGT AGA GCT CCC /3IABkFQ/
Mm.PT.58.13069923 CGC GAA GCA GCA ACC AT
TCT TGT CAC TTG GCA TCT GG
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NLRP3 /56-FAM/TGC CTC ACT /ZEN/TCT AGC TTC TGC CG/3IABkFQ/
Mm.PT.58.13974318 CAC TCA TGT TGC CTG TTC TTC
CGG TTG GTG CTT AGA CTT GA
RIPK3 /56-FAM/TCC CAG GAT /ZEN/ATC TTC TTC GAG TTC ACG
AT/3IABkFQ/ Mm.PT.58.12712973.g s GGA ACC ATG ATG TAG CAG TCA
CTC ATT ACG AAG ATT AAC CAT AGC C
TLR2 /56-FAM/CCA AAG AGC /ZEN/TCG TAG CAT CCT CTG A/3IABkFQ/
Mm.PT.58.45820113 CAA CTT ACC GAA ACC TCA GAC A
CCA GAA GCA TCA CAT GAC AGA
TLR4 /56-FAM/ACT CAG CAA /ZEN/AGT CCC TGA TGA CAT TCC
/3IABkFQ/ Mm.PT.58.41643680
GAA GCT TGA ATC CCT GCA TAG
AGC TCA GAT CTA TGT TCT TGG TTG
TNFα /56-FAM/CCA CGT CGT /ZEN/AGC AAA CCA CCA AGT /3IABkFQ/
Mm.PT.58.12575861 AGA CCC TCA CAC TCA GAT CA
TCT TTG AGA TCC ATG CCG TTG
α-SMA /56-FAM/CCG CTG ACT /ZEN/CCA TCC CAA TGA AAG A/3IABkFQ/
Mm.PT.58.16320644 GAG CTA CGA ACT GCC TGA C
58
CTG TTA TAG GTG GTT TCG TGG A
Table 3.3. Primer sets used for genotyping
Sequence 5' --> 3'
eNOS primers AAT TCG CCA ATG ACA AGA CG
AGG GGA ACA AGC CCA GTA GT
CTT GTC CCC TAG GCA CCT CT
RIPK3 primers CGC TTT AGA AGC CTT CAG GTT GAC
GCC TGC CCA TCA GCA ACT C
CCA GAG GCC ACT TGT GTA GCG
3.2 Animal models
All animals were housed in the Kearns Animal Facility of the Kolling Institute of Medical
Research, with a stable environment maintained at 22 ± 1°C with a 12/12h light-dark cycle.
Experiments were done in accordance with the National Health and Medical Research
Council of Australia’s Code for the Care and Use of Animals for Scientific Purposes and were approved by the Animal Research Ethics Committee of the Royal North Shore Hospital
(NSLHD reference RESP/15/206).
59
Wild-type (WT) mice were obtained from Kearns Animal Facility of the Kolling Institute of
Medical Research, RIPK3-/- mice were a gift from Genetech, San Francisco, CA 94080; eNOS-
/- mice were purchased Jacksons Laboratory, USA. 7-9 weeks old male mice weighing 20-25g were used for this study. All mice had a C57BL/6 background.
3.2.1 Genotyping of genetically modified mice
Mouse genomic DNA was isolated from mouse tail tips (1-2 mm) by incubating tips with
40μl 0.2M NaOH/ 1 mM EDTA solution and heated at 95°C for 10 minutes. After incubation, the tubes were vortexed for 2 minutes while still hot. 40μl of Tris-HCl/Citric acid solution was then added to each tube and further vortexed for 2 minutes. The tubes were then centrifuged for 2 minutes at 20200 rpm. 1μl solution from the tube was added to the
HotFirePol master mix for the PCR. The final concentration of primers in the PCR reaction was 0.4μM (as showed in Table 3.4).
Table 3.4 Protocol of genotyping
Reagent For 1 reaction For 10 reactions
5x HotFirePol 4 μl 40 μl
Primers from 4μM stock 2 μl 20 μl water 13 μl 130 μl
Add DNA (1μl/tube)
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PCR conditions were as follows:
95°C - 8 minutes
95°C – 30 seconds
60°C – 30 seconds (for mRIP3= 60°C, for eNOS=65°C)
72°C – 60 seconds
42 cycles
PCR products were measured with 2% agarose DNA electrophoresis. Agarose gels were prepared at 2% (w/v) using TBE buffer, with SafeView DNA Stains (abm) being added to the molten agarose, to allow for visualisation of DNA bands under UV light, and allowed to cool before PCR samples were loaded into the gel. Electrophoresis was run at 100-120V for 45-60 min, or until the dye reached the end of the gel. DNA bands were visualised under UV light and recorded using the BioRad Chemidoc system.
3.2.2 Establishment of diabetic mice
Mice were fasted for 4 hours and then received either 55 mg/kg STZ diluted in 0.1 M citrate buffer, pH 4.5, or citrate buffer alone by intraperitoneal injection for five consecutive days.
Animals serving as non-diabetic controls were injected with citrate buffer alone. Blood glucose levels were monitored weekly to confirm the diabetic status of these mice. Only
STZ-treated animals with blood glucose >16 mmol/L were considered diabetic for the purpose of inclusion in subsequent studies as detailed in this thesis.
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Post STZ induction and experimental procedures, blood glucose monitoring was done twice a week until diabetes was established, then it was done weekly. To do so, the tip of the tail was wiped with 70% ethanol prior to blood collection and a blood sample (approximately
5µl) was obtained via tail vein prick near the tip of the tail using a sterile scalpel blade, and blood glucose was measured using a Nano Accu-Chek Blood Glucose Monitor. Insulin
(Lantus 2U/ day) was given subcutaneously if glucose exceeded 28 mmol or if they showed significant weight loss after induction of diabetes (10-15% body weight loss of mouse with blood glucose >16 mmol/L). For animals that required insulin, blood sugar measurement was performed twice a week. Mice were monitored by clinical observation for signs of dehydration and signs of distress (e.g. inactive, matted hair, etc). Weight was measured every week to ensure animal wellbeing. Urine collection from the metabolic cage was performed at week 24 after the last STZ injection.
3.2.3 Establishment of FA-induced nephropathy
Mice were fasted for 4 hours and the folic acid was administrated to mice by intraperitoneal injection (i.p.) at a dose of 250 mg/kg in the vehicle (0.5 ml of 0.3 M NaHCO3) to induce nephropathy. After injection, mice were carefully observed for any sign of distress and lesions at injection site. A 28-day model was used in our study. Hence animals were culled at week 4 after folic acid administration. Mice were also carefully observed for general well- being throughout the duration of the experiments.
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3.2.4 Implantation of osmotic minipumps
Osmotic minipumps were implanted in the interscapular region subcutaneously under short inhalational anaesthesia with 2% isoflurane, nitrous oxide (2L/minute) and oxygen (1L/min).
The anaesthetised mice were immobilised on a surgical pad. A mid-scapular incision was made on the back of the animal. By inserting a haemostat into the incision, and opening and closing the jaws of the haemostat, a pocket for the pump was created by spreading the subcutaneous tissue, which was used to insert the filled pump. The wound was sutured using surgical silk. The mice were recovered on a heating pad and once recovered, they were moved to their original cages. Mice were given an analgesic drug (Buprenorphine,
Temgesic 0.03mg/kg IP) for pain induced by surgery immediately before the procedure and
4 hours after the procedure. Potential infection at the incision point, and the wellbeing of mice were observed closely daily. For the diabetic kidney disease model, the osmotic minipumps were replaced every 6 weeks, while implantation surgery was done only once on folic acid mice model (implantation of minipump was performed as described in methods of chapter 4 and 5).
3.2.5 Euthanasia of animals and tissue collection
At the designated experimental endpoint, mice were humanely euthanised and sacrificed under anaesthetic by 2% Isoflurane, nitrous oxide (2L/min) and oxygen (1L/min).
Thoracotomy was performed when the animal was fully anaesthetised and then tissues were harvested. The kidney, in particular, was sliced longitudinally, with one half being fixated in 10% neutral-buffered formalin for histological analysis, while the other half was snap-frozen in liquid nitrogen and stored at -80°C.
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3.2.6 Tissue embedding and fixation
Tissue samples for fixing were immediately placed in cassettes and placed in formalin for 12-
24 hours depending on the size of the tissue, then dehydrated in ethanol (70%, 95% then
100%) and processed using a tissue embedding processor (Excelsior ES, Thermo Scientific).
The samples were then embedded in paraffin using a paraffin dispenser before sectioning.
The kidneys were sectioned at the thickness of 4 µM.
3.3 Histology
3.3.1 PAS staining
After being deparaffinised using xylene and hydrated in distilled water, slides were immersed in 1% periodic acid solution for 15 minutes at room temperature. The slides were washed and immersed in Schiff’s solution for 15 minutes. Once washed, slides were incubated in Mayer’s Haematoxylin for 3 minutes and washed in running water for 3 minutes, followed by incubation in Scott’s bluing solution for 1 minute to stain the nuclear.
Then slides were washed, dehydrated and mounted (HEUCKITT, ThermoFisher).
3.3.2 Picrosirius Red stain
According to manufacturer’s instructions (Picrosirius Red Stain Kit, 24901-500,
Polysciences), paraffin sections on slides were firstly deparaffinized and hydrated in distilled water and then placed in phosphomolybdic acid (solution A) for 2 minutes. Once washed,
64 slides were incubated in Picrosirius Red F3BA Stain (solution B) for 60 minutes, followed by incubation in 0.1N Hydrochloride Acid solution (solution C) for 2 minutes. After staining, slides were placed in 70% ethanol for 45 seconds dehydrated and mounted.
3.4 Immunohistochemistry
Deparaffinisation and hydration of slides are as described above. Heat retrieval was processed using a boiling water bath. The slides were heated in hot retrieval buffer at 99 ℃ for 20 minutes and cooled for another 20 minutes at room temperature within the hot retrieval buffer. Slides were then washed in running tap water for 3 minutes and immersed in 0.3% H2O2 for 5 minutes. Once washed, slides were ready for antibody incubation using
Sequenza system (Thermo Scientific Shandon Coverplate Technology). Firstly, blocking buffer (Protein Block, Serum-Free, Dako X0909) was used and slides were blocked for 10 minutes. The diluted primary antibody (Antibody Diluent, Dako S0809) was added to each chamber along with the negative control (mouse negative control Dako X0931, rabbit negative control Dako X0930) and incubated at 4℃ overnight. The next day, the secondary antibody (EnVision + Rabbit Dako K4003, EnVision + Mouse Dako K4001) was added to slides after washing with TBST and incubation for 30 minutes. After washing, slides were incubated with substrate chromogen to cover the kidney sections (DAB+ Peroxidase
Substrate Dako K3468) for 10 minutes at room temperature. The reaction was stopped and slides washed in running tap water, nuclear staining, dehydration and mounting were processed as previously described.
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3.5 mRNA expression by RT-PCR analysis
3.5.1 RNA extraction
RNA was extracted from cells using the isolate RNA kit (Isolate II RNA Mini Kit, BIO-52073,
Bioline). Cell lysis was generated by adding lysis butter and β-ME to the cell pellet or to the ground tissue and vortexed vigorously. The lysate was filtered with the supplied collection tubes and mixed with 70% ethanol to adjust the RNA binding conditions. After binding RNA to the silica membrane of the RNA minicolumns, membrane desalting buffer was added to dry the membrane and DNase was added to digest DNA. Once the membrane was washed and dried, RNA was eluted with RNase-free water and measured by Nano-drop (ND-1000
Spectrophotometer, ThermoFisher Scientific).
Total RNA was extracted from mouse kidney cortex tissues using the RNeasy plus mini kit
(Qiagen) by QIAcube (Qiagen). The obtained RNA was eluted with RNase-free water and measured with Nano-drop (ND-1000 Spectrophotometer, ThermoFisher Scientific).
3.5.2 cDNA synthesis iScript cDNA Synthesis Kit (1708891, BioRad) was used in our study. As per the manufacturer’s instructions, 1µg total RNA from each sample was used as the RNA template. The reaction was undertaken as per the following tables (Table 3.5 and 3.6).
Table 3.5 Components of cDNA synthesis
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Components Volume per reaction (µl)
5×iScript Reaction Mix 4 iScript Reverse Transcriptase 1
Nuclease-free water + RNA template (1µg) 15
Total volume 20
Table 3.6 Reaction Protocol of cDNA synthesis
Priming 5 minutes at 25 ℃
Reverse transcription 20 minutes at 46℃
RT inactivation 1 minute at 95℃
Holding step 4℃
3.5.3 PCR
Quantitative real-time PCR was performed using the master mix (BIO-82005, Bioline) with the probes as shown in Table 3.2 on the ABIPrism-7900 Sequence Detection System (Applied
Biosystems). The reaction was per the following table (Table 3.7). The relative mRNA expression levels were calculated according to the 2_∆∆Ct method using Expression Suite software (ThermoFisher Scientific).
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Table 3.7 Reaction Protocol of PCR
Polymerase activation 2 minutes at 95 ℃
Denaturation 10 seconds at 95℃ 40 cycles Annealing/extension 1 minute at 60℃
3.6 Western blot analysis
3.6.1 Protein extraction
For protein extraction, the supernatant of cell culture medium was collected and stored at -
80℃. Cell culture dishes or plates were placed on ice and washed with ice-cold TBS. After aspiration of TBS, ice-cold RIPA buffer was added to the dish or plate and a cold plastic cell scraper was used to scrape adherent cells off the dish/plate and then the cell suspension was transferred into a precooled microcentrifuge tube and maintained with constant agitation for 30 minutes at 4 ℃. The tube was spun at 16,000×g for 20 minutes in a 4℃- precooled centrifuge and the supernatant was kept at -20℃ and the pellet discarded.
Protein extraction from tissue was similar. After the kidney was cut, TissueRuptor II
(QIAGEN) was used to homogenize the tissue together with the RIPA buffer. The lysate was centrifuged, and the supernatant collected and stored at -20℃.
3.6.2 Bicinchoninic acid (BCA) protein assay
The protein concentration was quantified using the Pierce BCA Protein Assay Kit (Thermo
Scientific). According to the manufacturer’s instructions, samples were applied in duplicate
68 to the plate, along with a reference BSA standard dilution series, and incubated for 30 minutes at 37℃. The absorbance at 560nm was determined using a microplate reader.
Using the microplate reader software, a standard curve was produced to calculate the protein concentration of each sample.
3.6.3 Protein gel electrophoresis
Samples were mixed with the sample buffer and the reducing buffer (Novex Bolt TM,
Invitrogen) and heated at 70°C for 10 minutes followed by 5 minutes cooling to 4 °C.
Approximately 20 μg/lane of the sample was loaded into Bolt 4-12% Bis-Tris Plus Gels
(Thermo Fisher Scientific). The gel was run at 165 V for 1.5 hr or until the dye front reached the end of the gel (BlotTM MES SDS Running buffer, Invitrogen). Electrophoresed proteins were then transferred from the gel onto nitrocellulose membranes (A10235589, Amersham) using the Wet/Tank Blotting System (Bio-Rad). Membranes were blocked with 5% (w/v) BSA in TBST for 1 hour at room temperature, before being incubated with the primary antibody overnight at 4 °C. After several washes with TBST, membranes were probed with an appropriate secondary antibody for 1 hour at room temperature. The membranes were then detected using ECL (Immobilon Crescendo Western HRP substrate, Millipore) with LAS-
4000 Imaging System (Fujifilm). Gel-pro software was used to analyse the densitometry of immunoblot images.
3.7 In vitro studies
3.7.1 HK2 cell culture
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HK2 cells (human renal proximal tubular epithelial cells immortalized by transduction with human papilloma virus16 E6/E7 genes) purchased from ATCC were grown in keratinocyte serum-free media medium (Invitrogen, CA). Cells were grown at 37°C in 5% CO2 and 95% air.
Fresh growth medium was added to cells every other day until confluent. The cells used in this experiment were at passages 10-15.
3.7.2 Cell proliferation assay
Cell proliferation assays were measured with Cell Proliferation Assay kit (TB245, Promega).
20 µl of reagent was added into each well of the 96-well plate containing the samples in
100µl of culture medium. The plate was incubated at 37°C for 4 hours in a humidified, 5%
CO2 atmosphere and absorbance was recorded at 490nm using a plate reader.
3.8 Statistics
Unless otherwise stated all data are presented as mean ± S.E.M. The differences between two groups were analysed by the two-tailed t-test, and Comparison of multiple groups was analysed by one-way analysis of variance ANOVA followed by Tukey's multiple comparisons test. A two-sided p-value < 0.05 was considered statistically significant.
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Chapter 4 RIPK3 blockade attenuates kidney fibrosis in a
folic acid model of renal injury
71
Abstract
Renal fibrosis is common to all forms of progressive kidney disease. However, current therapies to limit renal fibrosis are largely ineffective. Phosphorylation of receptor-interacting serine/threonine-protein kinase (RIPK) 3 has been recently suggested to be a key regulator of the pyrin domain containing 3 (NLRP3) inflammasome and the common pathway of toll-like receptor (TLR) 2 and 4, which provides new insights into mechanisms of chronic kidney disease (CKD). However, the specific effect of RIPK3 on renal cortical fibrosis has not been fully understood. To study the function of RIPK3, both genetic ablation and pharmacological inhibition of RIPK3 (dabrafenib) were used in the study. Our studies identify that RIPK3 promotes renal fibrosis via the common pathways of activation of TLR2 and 4 and the NLRP3 inflammasome in a mouse model of folic acid-induced nephropathy. Both interventional strategies decreased the renal fibrotic response. In dissecting the relevant pathways, differential inhibition was observed in the transforming growth factor beta (TGF-β) 1 and mitogen-activated protein kinase (MAPK) pathways, but beneficial effects converged on the
TLR2/4 and NLRP3 pathways. This study demonstrates a role for RIPK3 as the mediator of renal fibrosis via upregulation of TLR2/4 pathways and inflammasome activation. Dabrafenib, as an inhibitor of RIPK3, may be an effective treatment to limit the progression of the tubulointerstitial fibrosis.
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4.1 Introduction
Chronic kidney disease (CKD) is a worldwide public health issue. Regardless of the cause of initial renal injury, CKD leads to renal fibrosis, characterized by renal dysfunction, extracellular matrix (ECM) accumulation, myofibroblast activation and inflammatory responses [3, 4]. To date, the most widely used strategy to limit the progression of CKD is blockade of the renin- angiotensin system, an approach that was developed more than 20 years ago with subsequently little therapeutic progress [332]. Therefore, identifying a novel therapeutic target to limit renal fibrogenesis is crucial to improve the outcomes of patients with CKD.
As a key kinase in inducing cell death, receptor-interacting protein kinase (RIPK) 3 has been increasingly reported to promote inflammatory responses and tissue damage [25]. However, the role of RIPK3 on renal tubules in renal cortical fibrosis has not been elucidated. Our pilot studies on human kidney biopsies from patients with diabetic nephropathy demonstrated a higher RIPK3 protein expression in proximal tubules, suggesting a possible functional role for
RIPK3 in tubular epithelial cells subjected to injury resulting in progressive renal fibrosis.
Recent studies have identified that RIPK3 is an important mediator in the pyrin domain containing 3 (NLRP3) inflammasome formation [23]. It is well documented that elevation of
RIPK3 activity induces an increase of phosphorylation of mixed-lineage kinase domain-like protein (MLKL) activity [25, 311], which leads to the NLRP3 activation [26]. Additionally, increased RIPK3 expression can directly promote expression of the NLRP3 inflammasome and associated inflammatory responses independent of MLKL [23-27, 318]. Furthermore, RIPK3 has
73 been demonstrated to promote cell-death-independent inflammation through toll like receptor (TLR) 4 activation in macrophages [28], indicating that RIPK3 is a crucial mediator of the TLR4 pathway. Importantly, it is well documented that activation of both NLRP3 and the
TLR4 pathway contribute to tubulointerstitial fibrosis in diverse rodent models of kidney disease, such as unilateral ureteral obstruction and diabetic nephropathy [232, 258, 333-340].
Together, these studies indicate that RIPK3 may promote renal fibrosis through activation of the NLRP3 inflammasome and the TLR4 pathway.
Dabrafenib is a well-known inhibitor of B-Raf, which suppresses the downstream
Ras/Raf/extracellular-signal-regulated kinase (ERK)/ mitogen-activated protein kinase (MAPK) pathway [328] which has been approval for clinical use for the treatment of melanoma and non-small cell lung cancer expressing B-Raf V600E mutations [329]. Inhibition of Raf kinase is known to attenuate renal fibrosis in a rat model of autosomal dominant polycystic kidney disease (ADPKD) [330]. It is also recognised that transforming growth factor beta (TGF-β) 1 can induce Ras/Raf/ERK/MAPK pathways [331], resulting in renal fibrosis. These studies indicate a potential renoprotective role of dabrafenib. Importantly, previous studies reported that dabrafenib is also a RIPK3 inhibitor which have been demonstrated in various models including human hepatocytes [326], mouse models of acetaminophen-induced liver injury
[326] and ischemic brain injury [327]. Therefore, although GSK’872 is a more selective RIPK3- inhibitor [322], dabrafenib may have several mechanisms of benefit in the context of renal fibrosis.
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Folic acid (FA) nephropathy is a widely used animal model to study interstitial fibrosis. FA causes renal injury as it is directly toxic to tubular epithelial cells. Hence it is a useful model to study tubular injury and subsequent renal pathology. FA initiates tubular dysfunction in the acute phase (1-14 days) and interstitial fibrosis in chronic phase (28-42 days) [253], thus a 28- day FA nephropathy model of tubulointerstitial fibrosis was used in this study.
In the present study, we aimed to test the hypothesis that RIPK3 inhibition limits FA induced renal fibrosis. By developing the model in RIPK3 deficient mice and in wild-type mice treated with dabrafenib we were able to dissect the downstream pathways responsible for renal fibrosis.
4.2 Methods
4.2.1 Animal study
RIPK3-/- mice were a kind gift from K. N [341]. Male RIPK3-/-(c57BL/6), RIPK3+/+ (c57BL/6) mice of 7-9 weeks old with weighing 20-25g were studied. All animals were housed in the Kearns
Animal Facility of the Kolling Institute of Medical Research, with a stable environment maintained at 22 ± 1°C with a 2/12h light-dark cycle. FA nephropathy model was induced by a single intraperitoneal injection of FA (250 mg/kg in the vehicle of 0.3 M NaHCO3, pH 7.4).
Control mice were injected with vehicle.
Dabrafenib was used as the RIPK3 inhibitor in our study. Given that dabrafenib has very different oral bioavailability in humans (t1/2 4.8 hours for oral administration) [342, 343]
75 compared to mice (t1/2 23.7 min for oral administration) [344], Alzet model 2006 osmotic pumps (0.15 μl/hr, 42-day-pump model) were used to deliver dabrafenib. Before implantation surgery, pumps were incubated in sterile saline at 37 ℃ for 60 hours. Dabrafenib was dissolved in DMSO at a concentration of 16.7 mg/ml [344, 345]. Dabrafenib solution was delivered at a rate of 2.5 μg/hr. 24 hours following the FA injection, osmotic pumps were implanted on the back of mice to deliver dabrafenib or its vehicle DMSO. Mice were sacrificed
28 days after the surgery [253].
Experiments were done in accordance with the National Health and Medical Research Council of Australia’s Code for the Care and Use of Animals for Scientific Purposes and were approved by the Animal Research Ethics Committee of the Royal North Shore Hospital (NSLHD reference
RESP/15/206).
4.2.2 Histology and Immunohistochemistry
Kidneys were fixed in 4% paraformaldehyde and embedded with paraffin. 4 μm kidney sections were cut and stained as follows. After deparaffinisation with xylene, slides were immersed in decreasing concentration of ethanol (100%, 100%, 95%, 70%) and rinsed in a container of running water. 1% periodic acid solution (PER50%/100, POCD Scientific) and
Schiff’s reagent (SCH500, POCD Scientific) was used for Periodic Acid-Schiff (PAS) staining.
Picrosirius Red stain (24901, Polysciences) was undertaken according to the manufacturer's protocol.
76
Citrate buffer in pH 6 was heated to 99 ℃ for epitope retrieval. Slides were placed in hot retrieval buffer for 20 minutes’ incubation followed by 20 minutes’ cooling to room temperature. Slides were blocked for 15 minutes (protein block serum-free ready to use x0909, Dako). Immunohistochemical staining was processed in Thermo Scientific Sequenza system. Slides were incubated overnight at 4℃ with primary antibodies against type I collagen
(Abcam, ab34710, 1:750), a-SMA (Sigma-Aldrich, A2547, 1:2000), phospho-Smad2/3 (Santa
Cruz, sc-11769, 1:75). Slides were then washed with TBS-T (50mM Tris, 150mM NaCl, 0.05%
Tween 20, pH 7.6), and then incubated with associated secondary antibodies
(EnVision+system-HRP labelled polymer anti-rabbit k4003, EnVision+system-HRP labelled polymer anti-mouse k4001, Dako) for 30 minutes at room temperature. After washing with
TBS-T, kidney sections were covered with the DAB (liquid DAB+substrate chromogen system k3468, Dako) for 10 minutes. All experiments included controls without primary antibody.
Staining was scored by multiplying the percentage of positive signals by the intensity. Score of intensity was from 1 to 5. Positive signals in the renal cortex regions were identified and quantified using Image J software.
4.2.3 Renal cortical injury
Renal cortical injury was blindly assessed in PAS-stained kidney sections. Abnormal cortical tissues containing tubular dilation, atrophy, cast formation or inflammatory cell infiltration were considered as being indicative of interstitial fibrosis/ tubular atrophy [55, 320].
Abnormal cortical area was identified and marked and then expressed as a percentage compared to the total cortical area using Photoshop software.
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4.2.4 RNA isolation and RT-PCR analysis
Total RNA was extracted from mouse kidney cortex tissues using RNeasy plus mini kit (Qiagen) by QIAcube (Qiagen). The cDNA was synthesized with a cDNA synthesis kit (Roche).
Predesigned probes (PrimeTime® Assay Std Probe 5' 6-FAM™/ZEN™/3' IB®FQ, Integrated DNA
Technologies), including RIPK3 (Assay ID: Mm.PT.58.12712973.gs), α-SMA (Assay ID:
Mm.PT.58.16320644), TLR4 (Assay ID: Mm.PT.58.41643680), TLR2 (Assay ID:
Mm.PT.58.45820113), NLRP3 (Assay ID: Mm.PT.58.13974318), ASC (Assay ID:
Mm.PT.56a.42872867), IL-1β (Assay ID: Mm.PT.58.41616450), TNF a (Assay ID:
Mm.PT.58.12575861), TGF-β1 (Assay ID: Mm.PT.58.11254750), MCP-1 (Assay ID:
Mm.PT.58.42151692), F4/80 (Assay ID: Mm.PT.58.11087779), Glyceraldehyde-3-Phosphate dehydrogenase (GAPDH) (Assay ID: Mm.PT.39a.1) were used. Quantitative real-time PCR was performed using the Bioline SensiFast Probe Hi-Rox kit (BIO-82020) with probes on the ABI-
Prism-7900 Sequence Detection System (Applied Biosystems). The PCR reactions were run for
40 cycles. Each amplification cycle included denaturation at 95℃ for 10 seconds and annealing/extension at 60℃ for 50 seconds. All reactions were done in duplicate. The relative mRNA expression level was calculated by ExpressionSuite software and GAPDH was used as an endogenous reference gene.
4.2.5 Western blot analysis
Mouse kidney cortex tissues were homogenized and lysed in RIPA buffer with protease inhibitors (Thermofisher). Tissue lysates were separated on 4-12% gels (Invitrogen) and then transferred to PVDF membrane (PALL) for 2 hours at 4 ℃. Membranes were blocked with 5%
BSA in TBS-T (20 mM Tris, 150 mM NaCl, 0.1% Tween 20, pH 7.6) for 1 hour and then incubated
78 with primary antibodies (GAPDH, ab8245, Abcam; phospho-p65, sc-101749, Santa Cruz; p65, sc-372, Santa Cruz; phospho-ERK1/2, 9101s, Cell Signalling; ERK1/2, 4695s, Cell Signalling;
MLKL, #37705, Cell Signalling; phospho-MLKL, #62233, Cell Signalling) at 4 ℃ overnight followed by relevant horseradish peroxidase-conjugated secondary antibody (anti-Rabbit antibody, Cell Signalling; anti-Mouse antibody, Santa Cruz). The membranes were then detected using ECL (Millipore) with LAS-4000 Imaging System (Fujifilm). Gel-pro software was used to analyse the densitometry of immunoblot images.
4.2.6 Statistical analysis
The results are presented as mean ± S.E.M. All statistical analyses were performed with
GraphPad software. Statistical analysis of two groups was performed with the two-tailed t- test. Comparison of multiple groups was analysed by one-way analysis of variance ANOVA followed by Tukey's multiple comparisons test. A two-sided p-value < 0.05 was considered statistically significant.
4.3 Results
4.3.1 RIPK3 gene expression is increased in mice with FA nephropathy but no change observed in downstream pMLKL protein
RIPK3 mRNA expression in the kidney was significantly increased in wild-type mice with FA nephropathy compared with control mice (Fig 4.1a P<0.0001). Despite the upregulation of
RIPK3, FA nephropathy did not induce a significant downstream effect on phosphorylated
MLKL protein, suggesting renal injury and the subsequent fibrotic response is independent of
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MLKL induced necroinflammation (Fig. 4.1 b, c). As expected, no mRNA expression of RIPK3 was found in RIPK3-/- mice.
Figure 4.1 Increased receptor-interacting serine/threonine-protein kinase (RIPK) 3 in mice with folic acid nephropathy. (a) mRNA expression of RIPK3 was measured with q-PCR in mice
(wild-type vs RIPK3-/-) with/without folic acid nephropathy. Glyceraldehyde-3-Phosphate dehydrogenase (GAPDH) was used as the reference gene. Statistical analysis of RIPK3 mRNA expression was performed using the two-tailed t-test (t=5.304; df=20). ****, P < 0.0001. n =
10-12 animals per group. (b, c) Immunoblot analyses of kidneys from the same groups as in
(a) using antibodies for phospho-mixed-lineage kinase domain-like (MLKL), total MLKL and
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GAPDH as loading control. Statistical analysis was performed using one-way ANOVA followed by Tukey's multiple comparisons test. Results are presented as mean ± S.E.M. n = 4 animals per group.
4.3.2 RIPK3 deficiency inhibits TLR2/4 signalling in mice with FA nephropathy.
To determine whether RIPK3 mediated TLR2/4 signalling is elevated in FA-induced renal fibrosis, we compared wild-type and RIPK3 -/- mice, with or without FA-induced nephropathy.
RT-PCR was performed to quantify TLR2 and TLR4 mRNA expression. Both TLR2 and TLR4 mRNA expression levels were significantly upregulated in wild-type FA nephropathy mice compared to control mice (P<0.0001, Fig. 4.2 a, b). RIPK3 deletion significantly reduced the
TLR2 and TLR4 mRNA expression (all P<0.05, Fig. 4.2 a, b). We then measured downstream markers of TLR2/4 common signalling, including nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) signalling pathways and proinflammatory cytokines interleukin-1 (IL-
1) β and tumor necrosis factor (TNF) α. Phosphorylated p65 protein level was assessed as a downstream marker of the NFκB signalling pathway. FA nephropathy was associated with significantly elevated mRNA expression levels of IL-1β and TNF α (both P<0.05, Fig. 4.2 c, d).
However, there were no significant differences in phosphorylated p65 levels in the kidneys between these groups. Deletion of RIPK3 decreased mRNA expression of IL-1β and TNF α (Fig.
4.2 c, d). These results indicate that ablation of RIPK3 suppresses FA-induced TLR2/4 pathway activation with associated reduction in downstream IL-1β and TNF α.
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Figure 4.2 Increased toll-like receptor (TLR) 2/4 in mice with folic acid nephropathy. (a-d) mRNA expression of TLR2, TLR4, interleukin (IL) -1β, tumour necrosis factor-α (TNFα), were
82 measured with q-PCR in mice (wild-type vs RIPK3-/-) with/without folic acid nephropathy.
GAPDH was used as the reference gene. Statistical analysis was performed using one-way
ANOVA followed by Tukey's multiple comparisons test. Results are presented as mean ± S.E.M.
*, P < 0.05. ***, P < 0.001. ****, P < 0.0001. n = 10-12 animals per group. (e, f) Immunoblot analyses of kidneys from the same groups as in (a) using antibodies for phospho-p65, total p65 and GAPDH as loading control. Statistical analysis was performed using one-way ANOVA followed by Tukey's multiple comparisons test. Results are presented as mean ± S.E.M. n = 4 animals per group.
4.3.3 RIPK3 deficiency reduces NLRP3 inflammasome activation in mice with FA nephropathy.
To assess whether RIPK3 mediated NLRP3 activation is increased in FA-induced nephropathy, the inflammasome components were determined by RT-PCR. FA-induced nephropathy significantly elevated the mRNA expression levels of NLRP3 and adapter protein apoptosis- associated speck-like protein (ASC), which is known to facilitate recruitment of inflammasome complex [191] (P<0.0001, Fig. 4.3 a, b). These effects were down-regulated in RIPK3-/- mice exposed to FA (Fig. 4.3a, b). These data demonstrate that RIPK3 deletion inhibits NLRP3 activation in the context of a FA nephropathy model.
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Figure 4.3 Increased pyrin domain-containing protein 3 (NLRP3) inflammasome activation in mice with folic acid nephropathy. (a, b) mRNA expression of NLRP3, adapter protein apoptosis-associated speck-like protein (ASC) were measured with q-PCR in mice (wild-type vs RIPK3-/-) with/without folic acid nephropathy. GAPDH was used as the reference gene.
Statistical analysis was performed using one-way ANOVA followed by Tukey's multiple comparisons test. Results are presented as mean ± S.E.M. *, P < 0.05. ****, P < 0.0001. n =
10-12 animals per group.
4.3.4 RIPK3 deficiency reduces inflammatory responses and renal injury in mice with FA nephropathy.
Monocyte chemoattractant protein (MCP)-1 and the marker of macrophage infiltration F4/80 are well-recognized components of the inflammatory response, which can be regulated by multiple signalling pathways including NF κB [346], MAPK [347] and AKT [348]. To investigate whether RIPK3 deletion reduces FA-induced inflammatory responses and associated renal injury, RT-PCR was undertaken to assess MCP-1 and F4/80 mRNA expression levels within
84 kidneys of control and FA models in wild-type and RIPK3 -/- mice. As shown in Fig. 4.4 a, MCP-
1 mRNA expression was significantly increased in wild-type mice with FA nephropathy versus control (P<0.0001, Fig. 4.4 a). In contrast, RIPK3-/-mice with FA nephropathy exhibited lower
MCP-1 gene expression than their wild-type counterparts (P<0.001, Fig. 4.4 a). In addition, we observed wild-type mice with FA nephropathy had significantly higher mRNA expression of
F4/80 compared to control (P<0.0001, Fig. 4.4 b). In RIPK3-/- mice this increase was largely abrogated (P<0.01, Fig. 4.4 b). Together, these data suggest that the absence of RIPK3 attenuates FA-induced inflammatory responses within the kidney.
Blinded assessment of renal cortical injury consistently demonstrated increased renal injury within the kidney cortex in the FA-exposed wild-type animals, characterized by tubular dilation, tubule atrophy, cast formation or inflammatory cell infiltration (P<0.0001, Fig. 4.4 c, d). RIPK3 deletion was associated with less histological evidence of renal cortical injury
(P<0.01, Fig. 4.4 c, d).
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Figure 4.4 Increased inflammation and kidney injury in mice with folic acid nephropathy. (a, b) mRNA expressions of monocyte chemoattractant protein (MCP-1) and F4/80 were measured with q-PCR in mice (wild-type vs RIPK3-/-) with/without folic acid nephropathy.
GAPDH was used as the reference gene. (c) Representative images of Periodic Acid-Schiff
(PAS)-stained kidney sections. Magnification: 1× (left column); 20× (right column). (d)
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Quantitation of renal cortical injury in the same groups as in (a). Statistical analysis was performed using one-way ANOVA followed by Tukey's multiple comparisons test. Results are presented as mean ± S.E.M. **, P < 0.01. ***, P < 0.001. ****, P < 0.0001. n = 10-12 animals per group.
4.3.5 RIPK3 deficiency decreases TGF-β1 signalling and myofibroblast activation in mice with FA nephropathy.
To investigate the association between RIPK3 and TGF-β1 in renal fibrogenesis, components of the TGF-β1 signalling pathway were determined using RT-PCR and immunohistochemistry.
FA resulted in significant up-regulation of TGF-β1 mRNA expression, which was reduced by
RIPK3 deletion (all P<0.05, Fig. 4.5 a). It is well documented that Smad 2/3 and ERK1/2 are crucial downstream proteins of TGF-β1 fibrogenic signalling. As shown in Fig. 4.5 b and c, FA- induced nephropathy resulted in a significant elevation of p-Smad2/3 protein expression within the kidney cortex (P<0.01), whereas this effect was reversed in RIPK3-/- mice exposed to FA (P<0.001). In addition, FA nephropathy was associated with a significant increase in phosphorylated ERK1/2 (P<0.05, Fig. 4.5 d, e), which was partially reduced by RIPK3 deletion
(P=0.0585, Fig. 4.5 d, e).
It is well known that myofibroblast activation is a dominant feature of renal fibrosis. To assess if RIPK3 contributes to myofibroblast activation, α-smooth muscle actin (α-SMA) was measured. As shown in Fig. 4.5 f, α-SMA mRNA expression was significantly higher in wild- type mice with FA nephropathy compared to control mice (P<0.0001), which was significantly
87 decreased in RIPK3 deficient mice (P<0.0001). To ascertain myofibroblast activation in the kidney cortex, α-SMA protein expression was evaluated by immunohistochemistry. α-SMA protein expression was consistently upregulated in FA-induced nephropathy compared to control kidneys (P<0.0001, Fig. 4.5 g, h), which was reversed in RIPK3-/- mice (P<0.0001, Fig.
4.5 g, h). These data demonstrate that RIPK3 gene deletion results in suppressed TGF-β1 signalling and myofibroblast activation within the kidney cortex in the FA nephropathy model.
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Figure 4.5 Increased transforming growth factor beta (TGF-β) 1 signalling and myofibroblast activation in mice with folic acid nephropathy. (a, f) mRNA expression of α-smooth muscle actin (α-SMA) and TGF-β1 were measured with q-PCR in mice (wild-type vs RIPK3-/-) with/without folic acid nephropathy. GAPDH was used as reference gene. (b, g)
Representative images of p-Smad2/3 and α-SMA stained kidney sections from the same groups as in (a). Scale bar: 200 μm. Magnification: 10× (left column); 40× (right column). (c, h)
Quantitation of p-Smad2/3 and α-SMA immunohistochemical staining in the same groups as in (a). Statistical analysis was performed using one-way ANOVA followed by Tukey's multiple comparisons test. Results are presented as mean ± S.E.M. *, P < 0.05. **, P < 0.01, ***, P <
0.001. ****, P < 0.0001. n = 6-12 animals per group. (d, e) Immunoblot analyses of kidneys
89 from the same groups as in (a) using antibodies for phospho-extracellular signal-regulated kinase (ERK) 1/2, total ERK1/2, and GAPDH as the loading control. Statistical analysis was performed using one-way ANOVA followed by Tukey's multiple comparisons test. Results are presented as mean ± S.E.M. *, P < 0.05. n = 4 animals per group.
4.3.6 RIPK3 deficiency reduces collagen deposition in mice with FA nephropathy.
To assess collagen deposition within the kidney cortex, we undertook picrosirius red staining
(PSR) and immunohistochemistry to measure type I collagen protein expression. As shown in
Fig. 4.6 a and b, FA nephropathy stimulated significant collagen deposition within the kidney cortex, which was decreased in RIPK3 deficient mice (all P<0.05). Collagen I was non- significantly increased in the mice with FA nephropathy (P=0.0609, Fig. 4.6 a, c), which was decreased in RIPK3-/- mice (P<0.0001, Fig. 4.6 a, c). Thus, RIPK3 deletion reduced the increase in collagen accumulation observed in FA-induced nephropathy.
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Figure 4.6 Increased collagen deposition in mice with folic acid nephropathy. (a)
Representative images of picrosirius red staining (PSR) and collagen I stained kidney sections in mice (wild-type vs RIPK3-/-) with/without folic acid nephropathy. Scale bar: 200 μm.
Magnification: 10× (left column); 40× (right column). (b) Quantitation of PSR staining in the same groups as in (a). (c) Quantitation of Collagen I immunohistochemical staining in the same groups as in (a). Statistical analysis was performed using one-way ANOVA followed by
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Tukey's multiple comparisons test. Results are presented as mean ± S.E.M. *, P < 0.05. ***,
P < 0.001. ****, P < 0.0001. n = 6-12 animals per group.
4.3.7 Dabrafenib reduces TLR2/4 activation in mice with FA nephropathy.
Given genetic deletion of RIPK3 does not reflect pharmacological inhibition of RIPK3 we assessed TLR2/4 signalling in mice with FA nephropathy in the presence or absence of dabrafenib. Dabrafenib showed a trend to downregulate the FA-induced elevation of RIPK3 mRNA expression (P=0.0758, Fig. 4.7 a), although paradoxically, decreased phosphorylation of MLKL protein was observed (P<0.05, Fig. 4.7 f, g). Taken together with the lack of inhibition of MLKL observed in the RIPK3 knockout mice this suggests that MLKL expression may be regulated independent of RIPK3. Decreased TLR2 and TLR4 gene expression, as well as inhibition of downstream phosphorylated p65 and cytokines IL-1β and TNFα (Fig. 4.7 b, c, d, e, f, h), were also observed with dabrafenib treatment. These results indicate that dabrafenib suppresses the FA-induced TLR2/4 pathway activation and downstream inflammatory responses.
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Figure 4.7 Reduced RIPK3 mediated TLR2/4 signalling in mice with folic acid-induced nephropathy treated with dabrafenib. (a-e) mRNA expression of RIPK3, TLR4, TLR2, IL-1β,
TNFα were measured with q-PCR in mice (with or without treatment) with/without folic acid nephropathy. GAPDH was used as reference gene. Statistical analysis of genes was performed using one-way ANOVA followed by Tukey's multiple comparisons test. Results are presented as mean ± S.E.M. *, P < 0.05. **, P < 0.01. ***, P < 0.001. n = 6-12 animals per group. (f-h) Immunoblot analyses of kidneys from the same groups as in (a) using antibodies for phospho-MLKL, total MLKL, phospho-p65, total p65 and GAPDH as the loading control.
Statistical analysis was performed using one-way ANOVA followed by Tukey's multiple comparisons test. Results are presented as mean ± S.E.M. *, P < 0.05. **, P < 0.01. ***, P <
0.001. n = 5 animals per group.
4.3.8 Dabrafenib downregulates necroptotic NLRP3 inflammasome activation in mice with
FA nephropathy.
To understand if RIPK3 inhibition with dabrafenib blocks necroptotic NLRP3 inflammasome activation, inflammasome components were measured by RT-PCR. Similar to the results observed in RIPK3 deficient animals, dabrafenib reduced NLRP3 inflammasome formation in
FA-induced nephropathy by downregulating NLRP3 and ASC mRNA expression (Fig. 4.8 a, b).
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Figure 4.8 Reduced RIPK3 mediated NLRP3 inflammasome activation in mice with folic acid-induced nephropathy treated with dabrafenib. (a, b) mRNA expression of NLRP3, ASC were measured with q-PCR in mice (with or without treatment) with/without folic acid nephropathy. GAPDH was used as reference gene. Statistical analysis of genes was performed using one-way ANOVA followed by Tukey's multiple comparisons test. Results are presented as mean ± S.E.M. *, P < 0.05. ***, P < 0.001. n = 6-12 animals per group.
4.3.9 Dabrafenib reduces inflammatory responses and histological evidence of renal injury in mice with FA nephropathy.
To further study if dabrafenib protects the kidney against FA-induced nephropathy, we measured inflammatory responses and objective markers of renal injury. Consistently, MCP-
1 and F4/80 mRNA expression were downregulated by dabrafenib (P<0.05 for all, Fig. 4.9 a, b). Furthermore, dabrafenib significantly reduced FA-induced histological markers of injury as described above in the kidney cortex (P<0.0001, Fig. 4.9 c, d).
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Figure 4.9 Reduced inflammatory response and kidney injury in mice with folic acid- induced nephropathy treated with dabrafenib. (a, b) mRNA expression of MCP-1 and F4/80 were measured with q-PCR in mice (with or without treatment) with/without folic acid nephropathy. GAPDH was used as reference gene. (c) Representative images of PAS-stained kidney sections from the same groups as in (a). Magnification: 1× (left column); 20× (right column). (d) Quantitation of renal cortical injury in the same groups as in (a). Statistical analysis was performed using one-way ANOVA followed by Tukey's multiple comparisons test. Results are presented as mean ± S.E.M. *, P < 0.05. **, P < 0.01. ***, P < 0.001. ****, P
< 0.0001. n = 6-12 animals per group.
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4.3.10 Dabrafenib alleviates myofibroblast activation in mice with FA nephropathy.
Having demonstrated FA nephropathy is associated with elevated TGF-β1 signalling and myofibroblast activation, we sought to determine whether dabrafenib treatment limited renal injury by attenuation of the activation of these pathways in FA nephropathy. RT-PCR and immunohistochemistry indicated that dabrafenib treatment decreased TGF-β1 mRNA expression and p-ERK protein level (Fig. 4.10 a, d, e) without changing p-Smad2/3 (Fig. 4.10 b, c). Furthermore, dabrafenib significantly lowered α-SMA mRNA and protein expression, suggesting that dabrafenib effectively inhibits myofibroblast activation within the kidney cortex in FA nephropathy (P<0.05 for all, Fig. 4.10 f-h) but the lack of change in p-Smad2/3 expression suggests this occurs independently of TGF-β1 signalling.
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Figure 4.10 Reduced TGF-β1 signalling and myofibroblast activation in mice with folic acid- induced nephropathy treated with dabrafenib. (a, f) mRNA expression of TGF-β1 and α-SMA were measured with q-PCR in mice (with or without treatment) with/without folic acid nephropathy. GAPDH was used as the reference gene. (b, g) Representative images of p-
Smad2/3 and α-SMA stained kidney sections from the same groups as in (a). Scale bar: 200
μm. Magnification: 10× (left column); 40× (right column). (c, h) Quantitation of p-Smad2/3 and α-SMA immunohistochemical staining in the same groups as in (a). Statistical analysis was performed using one-way ANOVA followed by Tukey's multiple comparisons test. Results are presented as mean ± S.E.M. *, P < 0.05. ***, P < 0.001. ****, P < 0.0001. n = 5-12 animals per group. (d, e) Immunoblot analyses of kidneys from the same groups as in (a) using antibodies for phospho-ERK1/2, total ERK1/2, and GAPDH as loading control. Statistical analysis was performed using one-way ANOVA followed by Tukey's multiple comparisons test. Results are presented as mean ± S.E.M. ***, P < 0.001. n = 5 animals per group.
4.3.11 Dabrafenib decreases collagen deposition in mice with FA nephropathy.
To examine total collagen deposition and specifically collagen I expression, kidney tissues were analyzed by picrosirius red staining and immunohistochemistry. Picrosirius red staining demonstrated increased collagen deposition in the kidneys of FA-induced nephropathy compared to control mice and significantly reduced collagen deposition in kidneys of mice treated with dabrafenib (Fig 4.11 a and b, P<0.01). Immunohistochemical analyses demonstrated increased collagen I in the kidney cortex of FA-induced nephropathy compared to control mice, which was reversed by dabrafenib treatment (Fig 4.11 a and c, P<0.01). This
99 data confirms that dabrafenib suppresses collagen deposition and specifically collagen I expression in FA nephropathy.
Figure 4.11 Reduced collagen deposition in mice with folic acid-induced nephropathy treated with dabrafenib. (a) Representative images of PSR and collagen I stained kidney sections in mice (with or without treatment) with/without folic acid nephropathy. Scale bar:
200 μm. Magnification: 10× (left column); 40× (right column). (b) Quantitation of PSR staining in the same groups as in (a). (c) Quantitation of Collagen I immunohistochemical staining in the same groups as in (a). Statistical analysis was performed using one-way
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ANOVA followed by Tukey's multiple comparisons test. Results are presented as mean ±
S.E.M. *, P < 0.05. **, P < 0.01. ****, P < 0.0001. n = 6-8 animals per group.
4.4 Discussion
In the present study, we defined the role of RIPK3 in renal fibrogenesis using a mouse model of FA-induced nephropathy. Our results demonstrate that RIPK3 is activated in FA-induced renal fibrosis. The fibrotic response was mitigated in the RIPK3-/- mice and in the wild-type mice treated with dabrafenib. Differential effects were observed on downstream TGF-β1 and
MAPK activation in the 2 strategies used to limit RIPK3 activation. However, a reduction in
TLR2 and 4 and NLRP3 activation was seen in both models concurrent with renoprotection, suggesting it is this pathway dominantly contributes to renal fibrosis.
It is known the FA is directly toxic to renal tubular cells which is the primary mechanism of injury in this model with subsequent development of renal fibrosis. Previous reports have shown TLR2/4 activation induces tubular epithelial cell injury, resulting in upregulation of
NFκB activation and consequent inflammatory and fibrogenic responses [349]. Our studies demonstrate that FA-induced nephropathy increases TNFα, which is known to be a key upstream regulator of RIPK3 [25], which in turn, acts upstream of the TLR2/4 pathway in tubular cells. We also report here that blocking of RIPK3 is associated with lower macrophage infiltration, inflammatory and fibrotic responses.
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In support of our current findings, TLR2 and TLR4 have been previously demonstrated to be involved in renal inflammatory responses, leukocyte infiltration and fibrogenesis in various kidney diseases, such as ischemia/reperfusion injury, obstructive uropathy, tubulointerstitial nephritis and diabetic kidney disease [225, 234, 333, 350]. We have previously shown that blocking either TLR2 or 4 protects the kidney against tubular injury in diabetic kidney disease
[258, 351]. Moreover, activation of TLR2/4 triggers NFκB nuclear translocation and transcription of proinflammatory cytokines, such as TNF α, IL-1 β [234, 352], which may amplify the necroptotic response. Our data have indicated that dabrafenib treatment only partially reduces RIPK3 mRNA expression, which is likely to be due to the differential inhibition of RIPK3 with the pharmacological inhibitor vs the knockout model. However, despite the modest reduction in RIPK3 TNF α was significantly reduced, which is likely to limit the amplification response.
Previous studies have already postulated the deleterious role of NLRP3 inflammasome activation in the progression of renal inflammation and fibrosis [201]. NLRP3 deletion induces an increased repair response in tubular epithelial cells [353] and protects tubular cells from
Angiotensin II-triggered cell injury [354]. Our study has revealed that blockade of RIPK3 in FA- induced nephropathy decreases the expression of NLRP3 inflammasome components NLRP3 and ASC, suggesting that RIPK3 may also promote tubular injury via activation of the inflammasome.
It is well recognised that TGF-β1 regulates the activation of myofibroblasts and renal fibrotic responses at least partially through activation of TLR pathways [355]. mRNA levels of TGF-β1
102 are reduced in the kidneys of TLR4-/- mice [258]. TLR4 induces tubular cells and myofibroblasts to secrete extracellular matrix independent of the TGF-β/Smad pathway [222].
In addition, a novel and direct role of NLRP3 in promoting TGF-β1 mediated EMT in renal tubular epithelial cells has been confirmed [193]. TGF-β1 initiates canonical (Smad pathway) and non-canonical (non-Smad-based, p38, c-Jun N-terminal kinase (JNK) and MAPK) signalling pathways to exert multiple biological effects [355]. Our data elucidate that deletion of RIPK3 reduces TGF-β1 mRNA expression and its myofibroblast activation, collagen accumulation as well as TGF-β1/Smad2/3 and ERK signalling pathways. However, renoprotection was observed in the dabrafenib treated FA-exposed mice with no significant change in the
Smad2/3 or ERK signalling pathways. Hence, although we conclude that the benefits of RIPK3 inhibition on TGF-β1 converge on the suppression of the TLR4 and inflammasome pathways it is possible that a reduction in TGF-β reduces a proinflammatory stimulus independent of the Smad pathway.
It has been recently being independently reported that RIPK3 is implicated in renal fibrogenesis in two alternative models of renal fibrosis ie the unilateral ureteral obstruction
(UUO) and adenine-induced models of CKD [22]. These studies confirmed our findings that inhibition of RIPK3 conferred renoprotection independent of downstream MLKL. Indeed, they found that RIPK3 induced fibrogenesis through the protein kinase B (PKB, also known as AKT)
-dependent activation of ATP citrate lyase (ACL). Inhibition of RIPK3 suppressed TGF-β1 phosphorylation of AKT and ACL in fibroblasts. With a reduction in extracellular matrix production and myofibroblast differentiation. However, RIPK3 did not alter the TGF-β1 mRNA expression at 7 days in the UUO mice [22] when renal fibrogenesis is likely to have been
103 instigated in the model. Hence it is likely that RIPK3 can upregulate multiple pathways involved in renal fibrogenesis, which may differ between cell types and depend on the strength of the fibrogenic stimuli. In our study, limitation of fibrogenesis in the FA model was driven by inhibition of the TLR2/4 and inflammasome pathways.
These results suggest that the downstream activation of TLR2/4 signalling and NLRP3 activation is an unrecognised mechanism in perpetuating tubular injury and the development of renal fibrosis in FA-induced nephropathy. Genetic deletion or pharmacological blockade of
RIPK3 with dabrafenib attenuates kidney injury and renal interstitial collagen deposition via inhibition of the TLR2/4 pathway and NLRP3 activation in the mouse model of FA nephropathy.
Therefore, targeting RIPK3 may be a therapeutic approach for the prevention of progressive kidney disease. Dabrafenib is currently available for human use and here we have shown that it can be repurposed for use in patients with renal fibrosis and likely progressive kidney disease.
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Chapter 5 RIPK3 blockade attenuates tubulointerstitial
fibrosis in a mouse model of diabetic nephropathy
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Abstract
Receptor-interacting protein kinase-3 (RIPK3) is a multifunctional regulator of cell death and inflammation. RIPK3 controls cellular signalling through the toll-like receptor (TLR) pathway and the formation of the domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome, which have been well recognised to mediate renal fibrogenesis. The role of
RIPK3 in diabetic kidney disease (DKD) induced renal fibrosis has not been previously determined. To define the action of RIPK3 in the development of DKD, wild-type (WT), RIPK3
-/-, endothelium-derived nitric oxide synthase (eNOS)-/- mice were induced to develop diabetes mellitus using multiple low doses of streptozotocin and maintained for 24 weeks.
RIPK3 expression was upregulated and fibrotic responses were increased in the kidney cortex of mice with established diabetic nephropathy compared to control mice. Consistently, mRNA expression of inflammasome components, TLR4 and interleukin (IL)-1β as well as TGF-β1 and
α smooth muscle actin (α-SMA) were increased in diabetic kidneys of WT mice compared to control mice. However, these markers were normalised or significantly reversed in kidneys of diabetic RIPK3 -/- mice. Renoprotection was also observed using the RIPK3 inhibitor dabrafenib in eNOS-/- diabetic mice as demonstrated by reduced collagen and fibronectin deposition and myofibroblast activation. These results suggest that RIPK3 is associated with the development of renal fibrosis in DKD due to downstream activation of the NLRP3 inflammasome and TLR4 and TGF1 signalling. Inhibition of RIPK3 results in renoprotection.
Thus, RIPK3 may be a potential target for therapeutic intervention in patients with diabetic kidney disease.
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5.1 Introduction
End-stage kidney disease (ESKD) is a major cause of morbidity and mortality in patients with diabetes mellitus. Renal fibrosis indicates progressive renal disease will ensue in most forms of chronic kidney disease (CKD). In recent years, the increasing prevalence of diabetes mellitus accounts for the majority of chronic kidney disease worldwide [356]. The mainstay of therapy for diabetic kidney disease (DKD) is currently limited to controlling blood glucose and blood pressure, generally with an agent that blocks the renin-angiotensin system [357]. To date, no specific therapy for preventing diabetic kidney disease is available. A successful continuum between innovative discovery science and rigorous translation of research findings is required to improve the outcomes of patients with diabetic kidney disease.
The receptor-interacting protein kinase (RIPK)3, a crucial kinase mediating necroptosis, has been implicated as a potential regulator of multiple inflammatory pathways, including toll- like receptor (TLR)4 [358] and domain-like receptor family pyrin domain-containing (NLRP) 3 inflammasome [23], which have been well established to promote tubular epithelial cell injury and interstitial fibrosis in various models of kidney disease, including DKD [13, 206, 258, 336,
350, 359]. These studies suggest that RIPK3 may be a potential target to ameliorate DKD.
Limiting the activation of the NLRP3 inflammasome and TLR4 by inhibiting RIPK3 has theoretical benefits in limiting renal fibrosis, including downstream effects on TGF1 expression and signalling. However, the function of RIPK3 in the development of DKD has not been elucidated.
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Dabrafenib, known as a B-Raf inhibitor, suppresses the downstream Ras/Raf/extracellular- signal-regulated kinase (ERK)/ mitogen-activated protein kinase (MAPK) pathway [328] and has been approved by the U.S. Food and Drug Administration for the treatment of non-small cell lung cancer and melanoma expressing BRAFV600E mutations [329]. It is well documented that Ras/Raf/ERK/MAPK pathways are involved in renal fibrosis [331], suggesting dabrafenib may have renoprotective effects. Dabrafenib has also been reported to inhibit RIPK3 in mouse models of ischemic brain injury [327] and liver injury in human hepatocytes [326]. Compared to the other widely-used RIPK3 inhibitor GSK'872 [22, 323], dabrafenib shows more therapeutic potential for renal fibrosis because it has broader kinase inhibition. Given it is already in use in humans, repurposing for use in DKD is likely to have a facilitated route to market.
In this study, we examined the role of RIPK3 in DKD induced renal fibrosis using a streptozotocin (STZ)-induced diabetic mouse model. We found that RIPK3 deficiency attenuated diabetes-induced renal fibrosis, associated with reduced activation of the NLRP3 inflammasome, TLR4 and TGF1 signalling. Dabrafenib treatment also attenuated diabetes- induced renal interstitial extracellular matrix (ECM) deposition and myofibroblast activation.
Our data support the tenet that RIPK3 may mediate diabetes-induced fibrosis through the
NLRP3 inflammasome and TLR4 pathway. Hence strategies that include inhibition of RIPK3 may limit the development of diabetic kidney disease.
5.2 Methods
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5.2.1 Human kidney biopsies
Human kidney biopsy specimens were collected from patients with clinically confirmed diabetic nephropathy by the Department of Anatomical Pathology of the Royal North Shore
Hospital. Control kidney tissues were obtained from removed kidneys of patients, generally due to peripheral tumour but without known kidney disease. This study was approved by the
Human Research Ethics Committee of NSLHD and Kolling Institute of Medical Research (HREC reference LNR/17/HAWKE/497).
5.2.2 Animal studies
7-9-week-old male wild-type (WT), RIPK3-/-, endothelium-derived nitric oxide synthase
(eNOS)-/- mice (all C57BL/6 background) weighing 20-25g were used for this study. Mice were assigned to receive either 55 mg/kg STZ (Sigma-Aldrich) diluted in 0.1 M citrate buffer, pH 4.5, or citrate buffer alone by intraperitoneal injection for five consecutive days as described previously [257]. STZ-treated animals with blood glucose >16 mmol/L were considered as diabetic. eNOS-/- mice were used to study the effect of dabrafenib on the development of diabetic nephropathy. Alzet model 2006 osmotic pumps (0.15µl/hr of dabrafenib dissolved in vehicle dimethyl sulfoxide (DMSO) at a concentration of 16.7 mg/ml) were implanted in the subcutaneous tissues in the interscapular region of eNOS-/- diabetic mice to deliver dabrafenib or DMSO. Mice were weighed, and the blood glucose was monitored weekly with an ACCU-CHEK glucometer (Roche Diagnostics). Insulin (Lantus 2U/ day) was given subcutaneously if the blood glucose of mice exceeded 28 mmol or mice had significant weight loss (10-15% body weight loss in mouse with blood glucose >16 mmol/L).
Mice were sacrificed 24 weeks after the induction of diabetes (as shown in Fig 5.1). All
109 animals were housed in the Kearns Animal Facility of the Kolling Institute of Medical
Research, with a stable environment maintained at 22 ± 1°C with a 2/12h light-dark cycle.
Experiments were done in accordance with the National Health and Medical Research
Council of Australia’s Code for the Care and Use of Animals for Scientific Purposes and were approved by the Animal Research Ethics Committee of the Royal North Shore Hospital
(NSLHD reference RESP/15/206).
Figure 5.1 Timeline of DM model and dual strategies to assess the effect of limitation of
RIPK3 on the development of diabetic kidney disease. Male mice at 7-9 weeks of age were induced to develop diabetes using a regimen of 5 doses of STZ as described above. After a week, eNOS-/- diabetic mice underwent surgery to implant the pump to deliver dabrafenib or vehicle. Mice were sacrificed 24 weeks after inducing diabetes.
5.2.3 RNA isolation and RT-PCR analysis
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Total RNA was extracted from mouse kidney cortex using the RNeasy plus mini kit (Qiagen) by QIAcube (Qiagen). cDNA was synthesized with a cDNA synthesis kit (Roche). Predesigned probes (PrimeTime® Assay Std Probe 5' 6-FAM™/ZEN™/3' IB®FQ, Integrated DNA
Technologies), including RIPK3 (Assay ID: Mm.PT.58.12712973.gs), Collagen IV ((Assay ID:
Mm.PT.58.31838522), Fibronectin (Assay ID: Mm.PT.58.8135568), TGF-β1 (Assay ID:
Mm.PT.58.11254750), α-SMA (Assay ID: Mm.PT.58.16320644), MCP-1 (Assay ID:
Mm.PT.58.42151692), F4/80 (Assay ID: Mm.PT.58.11087779), TLR4 (Assay ID:
Mm.PT.58.41643680), NLRP3 (Assay ID: Mm.PT.58.13974318), ASC (Assay ID:
Mm.PT.56a.42872867), IL-1 β (Assay ID: Mm.PT.58.41616450), Tubulin (Assay ID:
Mm.PT.58.13069923) were measured. Tubulin was used as endogenous reference gene.
5.2.4 Histology and Immunohistochemistry
Kidney tissues were fixed in 4% paraformaldehyde and embedded with paraffin. 4 μm kidney sections were cut and stained as follows: After deparaffinisation with xylene, slides were immersed in decreasing concentration of ethanol (100%, 100%, 95%, 70%) and rinsed in a container of running water. Citrate buffer at pH 6 was heated to 99 ℃ for epitope retrieval. Slides were placed in hot retrieval buffer for 20 minutes incubation followed by 20 minutes cooling to room temperature. Slides were blocked for 15 minutes (protein block serum-free ready to use x0909, Dako). Immunohistochemical staining was processed in
Thermo Scientific Sequenza system. Slide were incubated overnight at 4℃ with primary antibodies as follows: RIPK3 (Abcam: ab56164), type I collagen (Abcam, ab34710, 1:750), type III collagen (Abcam, ab7778, 1:500), type IV collagen (Abcam, ab6586, 1:1000),
Fibronectin (Abcam, ab 45688, 1:750), a-SMA (Sigma-Aldrich, A2547, 1:10000), p-Smad2/3
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(Santa Cruz Biotechnology, sc-11769R, 1:150). Slides were then washed with TBS-T (50mM
Tris, 150mM NaCl, 0.05% Tween 20, pH 7.6), and incubated with associated secondary antibodies (EnVision+system-HRP labelled polymer anti-rabbit k4003, Dako) for 30 minutes at room temperature. After washing with TBS-T, kidney sections were covered with DAB
(liquid DAB+substrate chromogen system k3468, Dako) for 10 minutes. All experiments included controls without primary antibody. Staining was scored by multiplying the percentage of positive signals by the intensity. Score of intensity was from 1 to 5. Positive signals in the renal cortex regions were identified and quantified using Image J software.
5.2.5 Statistical analysis
The results are presented as mean ± S.E.M. The differences between two groups were analysed by two-tailed t-tests, and comparison of multiple groups was analysed by one-way analysis of variance ANOVA followed by Tukey's multiple comparisons test. The survival curves were analysed by Log-rank (Mantel-Cox) test. A two-sided p-value < 0.05 was considered statistically significant.
5.3 Results
5.3.1 Upregulation of RIPK3 expression in the diabetic kidney
To determine whether RIPK3 is upregulated in diabetic kidney, RIPK3 protein expression was measured in kidney biopsies collected from normal nephrectomy specimens (Fig. 5.2a: Ctrl) and patients with established diabetic nephropathy (Fig. 5.2a: DN) using
112 immunohistochemistry. RIPK3 protein was clearly upregulated in proximal tubules (black arrows) of diabetic kidneys compared to normal kidneys.
RIPK3 protein expression was examined using immunohistochemistry in kidneys of mice with or without diabetic nephropathy. Consistent upregulation of RIPK3 protein (black arrows) was observed in the kidneys of diabetic mice compared to control kidneys (Fig. 5.2b). mRNA expression of RIPK3 was assessed using RT-PCR. As shown in Fig. 5.2c, RIPK3 mRNA expression was significantly increased in diabetic mice compared to non-diabetic controls (P< 0.01). Our data indicate that RIPK3 is upregulated within the kidney in the context of diabetic kidney disease.
Figure 5.2 Increased receptor-interacting protein kinase (RIPK) 3 in diabetic kidney disease.
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(a) Representative images of RIPK3 stained kidney biopsies collected from normal nephrectomy specimens and patients with established DKD. Magnification: 20×. (b)
Representative images of RIPK3 stained kidney sections in mice (wild-type WT or RIPK3-/-) +/- diabetes mellitus. Magnification: 20×. (c) RIPK3 mRNA expression fold change was measured by RT-PCR in the same groups as in (b). Tubulin was used as reference gene. Statistical analysis was performed using two-tailed t-tests. Results are presented as mean ± S.E.M. **P < 0.01. n
= 14 animals for control group, n = 6 animals for DN group.
5.3.2 RIPK3 gene knockout reduced fibronectin and collagen IV deposition induced by diabetes
To investigate whether RIPK3 deficiency alleviates renal fibrogenesis, we compared WT and
RIPK3 -/- mice, each with or without diabetes. The expression of the extracellular matrix (ECM) proteins fibronectin and type IV collagen were measured in the kidney cortex of mice by RT-
PCR and immunohistochemical analysis. As shown in Fig. 5.3 a, elevated fibronectin mRNA expression was found in diabetic WT mice compared to non-diabetic controls (P< 0.05).
However, fibronectin mRNA expression levels in diabetic RIPK3-/- mice were significantly lower than that in the diabetic WT mice (P<0.05, Fig. 5.3 a). Similarly, type IV collagen mRNA expression was up-regulated in kidneys from diabetic mice compared to non-diabetic WT control (P< 0.05, Fig. 5.3 b). RIPK3 gene knockout reversed diabetes-induced type IV collagen mRNA expression in the kidney cortex compared to diabetic WT mice (P< 0.05, Fig. 5.3 b).
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Consistent with the RT-PCR results, immunohistochemical analysis revealed increased staining of fibronectin and type IV collagen protein in the diabetic mice (all P< 0.05, Fig. 5.3 c, d, e), and reduced accumulation of both fibrotic markers in the kidney cortex of RIPK3 knockout mice with diabetes (all P< 0.05, Fig. 5.3 c, d, e).
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Figure 5.3 Increased fibronectin and collagen IV deposition in kidneys of diabetic mice.
(a, b) Fibronectin and collagen IV mRNA expression was measured by RT-PCR in mice (WT or
RIPK3-/-) +/- diabetes mellitus. Tubulin was used as reference gene. (c) Representative images of fibronectin and collagen IV stained kidney sections are from the same groups as in (a).
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Magnification: 20×. (d, e) Positive area percentage quantitation of fibronectin and collagen IV immunohistochemical stains are from the same groups as in (a). Statistical analysis was performed by one-way analysis of variance ANOVA followed by Tukey's multiple comparisons test. Results are presented as mean ± S.E.M. *P < 0.05, **P < 0.01, ****P < 0.0001. n = 6-14 animals per group.
5.3.3 RIPK3 gene knockout decreased transforming growth factor beta (TGF-β)1 expression and myofibroblast activation
To understand the association between RIPK3 and ECM deposition we examined renal TGF-
β1 mRNA expression levels in diabetic WT and RIPK3 -/- mice using RT-PCR. Induction of diabetes in WT mice led to significantly increased TGF-β1 mRNA expression (P< 0.001, Fig. 5.4 a), while TGF-β1 gene expression was reduced in RIPK3 -/- mice (P< 0.01, Fig. 5.4 a). α-smooth muscle actin (α-SMA) gene expression is commonly regarded as a specific marker of myofibroblast differentiation downstream of TGF-β1 activation. Hence α-SMA mRNA was examined using RT-PCR. The data showed that α-SMA mRNA expression levels in kidneys of
WT mice with diabetes were upregulated compared to non-diabetic mice (P< 0.01, Fig. 5.4 b).
However, its expression levels were normalized in kidneys of diabetic RIPK3 -/- mice (P< 0.05,
Fig. 5.4 b). These data suggest that disruption of RIPK3 suppresses TGF-β1 expression and myofibroblast differentiation in kidneys.
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Figure 5.4 Increased transforming growth factor beta (TGF-β) 1 mediated myofibroblast activation in kidneys of diabetic mice.
(a, b) TGF-β1 and α-smooth muscle actin (α-SMA) mRNA expression were measured by RT-
PCR in mice (WT or RIPK3-/-) +/- diabetic nephropathy. Tubulin was used as the reference gene. Statistical analysis was performed by one-way analysis of variance ANOVA followed by
Tukey's multiple comparisons test. Results are presented as mean ± S.E.M. **P < 0.01, ***P
< 0.001. n = 6-14 animals per group.
5.3.4 RIPK3 gene knockout reduced kidney inflammatory cell infiltration
It is well known that inflammatory cell infiltration plays a crucial role in progressive diabetic kidney disease. Thus, we measured monocyte chemoattractant protein (MCP)-1 and the mRNA expression of the macrophage marker F4/80. As shown in Fig. 5.5 a, MCP-1 mRNA expression was up-regulated in kidneys of diabetic WT mice compared to non-diabetic controls. However, this upregulation was partially reversed in diabetic RIPK3 -/- mice (all P<
0.05). Similarly, F4/80 mRNA expression was increased significantly in the kidney cortex of diabetic WT mice compared to non-diabetic controls, whereas F4/80 mRNA expression in
118 kidneys of diabetic RIPK3-/- mice was much lower compared to diabetic WT mice (all P< 0.05,
Fig. 5.5 b). Collectively, these data indicate that loss of RIPK3 led to suppressed inflammatory cell infiltration in the diabetic kidney cortex.
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Figure 5.5 Increased inflammatory cells infiltration in the kidneys of diabetic mice.
(a, b) Monocyte chemoattractant protein (MCP)-1 and F4/80 mRNA expression were measured by RT-PCR in mice (WT or RIPK3-/-) +/- diabetes mellitus. Tubulin was used as the reference gene. Statistical analysis was performed by one-way analysis of variance ANOVA followed by Tukey's multiple comparisons test. Results are presented as mean ± S.E.M. *P <
0.05, ***P < 0.001, ****P < 0.0001. n = 6-14 animals per group.
5.3.5 RIPK3 gene knockout reduced TLR4 signalling activation
To understand the role of RIPK3 in renal fibrosis and to identify the mechanism whereby RIPK3 participates in TLR4 signalling, mRNA expression of TLR4 and its downstream interleukin (IL)-
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1β were measured using RT-PCR. As shown in Fig. 5.6 a, the expression of TLR4 was significantly up-regulated in diabetic WT mice compared to non-diabetic controls, while RIPK3 gene knockout alleviated this up-regulation. mRNA expression of TLR2 only showed a trend of inhibition by the blockade of RIPK3 (data not shown). IL-1β mRNA was elevated in kidneys of diabetic WT mice and gene ablation of RIPK3 reversed this effect (all P< 0.05, Fig. 5.6 b).
These data suggest that RIPK3 regulates activation of TLR4 signalling.
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Figure 5.6 Increased toll-like receptor (TLR) 4 signalling in kidneys of diabetic mice.
(a, b) TLR4 and interleukin (IL)-1 β mRNA expression were measured by RT-PCR in mice (WT or RIPK3-/-) +/- diabetes mellitus. Tubulin was used as the reference gene. Statistical analysis was performed by one-way analysis of variance ANOVA followed by Tukey's multiple comparisons test. Results are presented as mean ± S.E.M. *P < 0.05, **P < 0.01, ***P < 0.001. n = 6-14 animals per group.
5.3.6d RIPK3 gene knockout reduced NLRP3 inflammasomee formation
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It is well documented that IL-1β serves as the active downstream cytokine of NLRP3 inflammasome. To examine if RIPK3 regulates NLRP3 activation in the kidneys of diabetic mice, mRNA expression of the NLRP3 inflammasome components NLRP3 and the adapter protein apoptosis-related speck-like protein (ASC) were examined using RT-PCR. As shown in Fig. 5.7 a, mRNA expression of NLRP3 was upregulated in kidneys of diabetic WT mice compared to non-diabetic controls (P< 0.001), and RIPK3 gene knockout completely reversed this effect
(Fig. 5.7 a, P< 0.01). Consistently, ASC mRNA expression was significantly elevated in the kidney of diabetic WT mice compared to non-diabetic controls, whereas RIPK3 gene knockout also normalised ASC mRNA expression in diabetic kidneys (all P< 0.05, Fig. 5.7 b). Together, these data indicate that loss of RIPK3 reduces NLRP3 formation and activation within the diabetic kidney cortex.
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Figure 5.7 Increased pyrin domain-containing protein (NLRP) 3 formation in kidneys of diabetic mice.
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(a, b) NLRP3 and adapter protein apoptosis-associated speck-like protein (ASC) mRNA expressions were measured by RT-PCR in mice (WT or RIPK3-/-) +/- diabetes mellitus. Tubulin was used as reference gene. Statistical analysis was performed by one-way analysis of variance ANOVA followed by Tukey's multiple comparisons test. Results are presented as mean ± S.E.M. **P < 0.01, ***P < 0.001. n = 6-14 animals per group.
5.3.7 Dabrafenib treatment increases survival rate in mice with established diabetic nephropathy.
To study whether the RIPK3 inhibitor dabrafenib suppresses the progression of diabetic nephropathy and associated mortality, survival rates of mice after induction of diabetes were assessed over a period of 24 weeks. As shown in Fig.5.8, all control mice survived throughout the experimental period. Diabetic mice, without dabrafenib treatment, had a significantly decreased survival compared with control group (P=0.0015), reflecting a survival rate of 84.6% at day 10, decreasing to 76.9% at day 14. Further attrition occurred at the 134th, 135th day,
166th day and 167th day) resulting in a 46.2% survival at the time of culling. However, dabrafenib treatment conferred an increased survival rate at each time point compared to untreated mice reflected in a survival rate of 75% after 24 weeks of diabetes. This data demonstrates that dabrafenib confers a survival benefit in the animals with diabetic kidney disease.
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Figure 5.8 Decreased survival rate in diabetic mice. Survival rate eNOS-/- mice (with or without treatment) +/- diabetic nephropathy. The survival of eNOS-/- diabetic mice was monitored and compared to control mice (n = 14 for control group, n=13 for eNOS-/- diabetic group without treatment, n=12 for dabrafenib treated eNOS-/- diabetic group). After 24 weeks, number of surviving mice in eNOS-/- diabetic group without treatment was 6, while dabrafenib treated eNOS-/- diabetic group had 9 mice.
5.3.8 Dabrafenib treatment reduces ECM deposition in mice with established diabetic nephropathy
To determine if dabrafenib alleviates diabetes-induced renal fibrosis, we performed immunohistochemistry to measure ECM deposition in the kidney. As shown in Fig.5.9 a and b, diabetic mice had significantly higher renal cortical collagen I expression, whereas this increase was reversed by dabrafenib treatment (P<0.0001). Similarly, diabetes-induced an up- regulation of renal cortical collagen III, which was largely abrogated in dabrafenib treated mice (P<0.0001, Fig. 5.9 a, c). A similar upregulation was found in collagen IV expression that
123 was abrogated by dabrafenib (P<0.01, Fig.5.9 a, d). Furthermore, dabrafenib decreased diabetes-induced fibronectin protein expression (P<0.0001, Fig. 5.9 a, e). Thus, diabetes- induced ECM deposition in the kidney was attenuated by dabrafenib treatment.
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Figure 5.9 Increased extracellular matrix (ECM) deposition in kidneys of diabetic mice.
(a) Representative immunohistochemical staining images of collagen I, collagen III, collagen
IV and fibronectin in the renal cortex from eNOS-/- mice (with or without treatment) +/- diabetic nephropathy. Magnification: 20×. (b-e) The quantitation of collagen I, collagen III, collagen IV and fibronectin expression in mice kidneys. Statistical analysis was performed with
ANOVA followed by Tukey's multiple comparisons test. Results are presented as mean ± S.E.M.
*, P < 0.05. ***, P < 0.001. ****, P < 0.0001. n = 6-12 animals per group.
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5.3.9 Dabrafenib treatment decreased myofibroblast activation and inflammatory response
Given having demonstrated the upregulation of ECM and inflammasome markers in diabetic animal, we investigated whether dabrafenib reduced these markers predictive of CKD in diabetic mice. As shown in Fig 5.10 a and b, dabrafenib didn’t change the TGF-β expression but show a trend to reducing α-SMA (P=0.0643). Dabrafenib also suppressed the inflammatory response with a reduced MCP-1 (Fig. 5.10 c, P<0.05) and less F4/80 expression
(Fig. 5.10 d, P<0.05). Although no statistical difference was found in expression of inflammasome components, dabrafenib displayed a trend to inhibit the NLRP3 inflammasome
(Fig. 5.10 e, f).
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Figure 5.10 Dabrafenib reduced the inflammatory response and trended to a reduction in mRNA expression of TGF-β1 and the inflammasome in kidneys of diabetic mice.
(a-f) mRNA expressions of TGF-β, α-SMA, MCP-1. F4/80, NLRP3 and ASC were measured by
RT-PCR in the renal cortex from eNOS-/- diabetic mice (with or without treatment). Statistical analysis was performed using two-tailed t-tests. Results are presented as mean ± S.E.M. *, P
< 0.05. n = 6-12 animals per group.
5.3.10 Dabrafenib treatment decreased myofibroblast activation in mice with established diabetic nephropathy
Given no significant difference was observed in TGF1 mRNA but trends were observed to a reduction in α-SMA mRNA expression, we measured the α-SMA protein expression within the kidney cortex to investigate if dabrafenib reduces myofibroblast activation. As shown in
Fig.5.11 a and b, diabetic mice showed significantly elevated α-SMA protein expression, whereas this effect was significantly reduced in mice exposed to dabrafenib (P<0.0001).
To assess whether dabrafenib RIPK3 reduced TGF-β downstream Smad signalling independent of mRNA expression, we measured the p-Smad2/3 within the kidney cortex. As shown in Fig. 5.11 a and c, diabetes stimulated a significant up-regulation of p-Smad2/3 within the kidney cortex, which was decreased in dabrafenib treated mice similarly exposed to STZ
(P<0.05). These data suggest that dabrafenib results in suppressed myofibroblast activation within the kidney cortex through inhibition of the Smad2/3 pathway, independent of a change in TGF-β mRNA expression, in the context of a diabetic nephropathy model.
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Figure 5.11 Increased ECM deposition and pSmad2/3 expression in kidneys of diabetic mice.
(a) Representative immunohistochemical staining images of α-SMA and p-Smad2/3 in the renal cortex from eNOS-/- mice (with or without treatment) +/- diabetic nephropathy.
Magnification: 20×. (b-c) The quantitation of α-SMA and p-Smad2/3 expression in mice kidneys. Statistical analysis was performed with ANOVA followed by Tukey's multiple comparisons test. Results are presented as mean ± S.E.M. *, P < 0.05. ***, P < 0.001. ****, P
< 0.0001. n = 6-10 animals per group.
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5.4 Discussion
This study was undertaken to define the role of RIPK3 in regulating tubulointerstitial fibrosis in diabetic nephropathy. Significant up-regulation of RIPK3 was evident in the kidney cortex of animals and humans with diabetic kidney disease. RIPK3 deficiency downregulated TLR4 signalling, NLRP3 inflammasome activation, and TGF-β1 expression and signalling, associated with a less inflammatory cell infiltration, myofibroblast activation and less ECM deposition.
The RIPK3 inhibitor dabrafenib also replicates these renoprotective effects.
Evolving data suggest that TLR4 signalling and the associated downstream cytokine IL-1β drives renal inflammation and fibrosis [206, 360-362]. Our group has previously shown that blocking TLR4 protects the kidney against tubular injury in in vivo and in vitro models of diabetic kidney disease [258, 349]. The NLRP3 inflammasome, the alternate source of IL-1β, also contributes to the progression of renal fibrosis [201]. It has been reported that NLRP3 deletion protects against renal fibrosis and attenuates inflammation in diabetic mice [363] and mice with 5/6 nephrectomy [364]. In the present study, we found genetic deletion of
RIPK3 reduced TLR4 signalling as well as NLRP3 inflammasome activation, associated with reduced ECM deposition and inflammatory cell filtration. Thus, we conclude the benefit of
RIPK3 inhibition on inflammatory response may arise from inhibition of TLR4 and NLRP3 activation, in addition to reduced expression and/or downstream signalling of TGF1.
In our study, TGF-β1 was reduced by RIPK3 deficiency. Although no change in expression of
TGF1 was observed in diabetic animals treated with dabrafenib, decreased phosphorylation
130 of p-Smad2/3was observed. A recent study also reported that TGF-β1 expression was not altered by RIPK3 deficiency in a 7-days unilateral ureteral obstruction (UUO) mouse model which is well recognised as a fibrogenic model. In this model, RIPK3 contributes to fibrosis via regulating proliferation of fibroblasts [22]. Hence RIPK3 may contribute to fibrosis through multiple pathways, depending on the cell type and the context of the fibrotic stimuli. However, the downregulation of TGF-β1 expression and/or downstream signalling by RIPK3 inhibition may still contribute to the reduction in renal fibrosis.
It is well documented that activation of TLR4 and the NLRP3 inflammasome triggers downstream profibrotic pathways [365], including stimulation of TGF1 expression and signalling [363, 366], which serves to amplify the fibrotic response [367, 368]. Hence it is possible that inhibition of RIPK3 may directly inhibit TGF-β responses, or indirectly as a consequence of suppression of TLR4 and the NLRP3 inflammasome.
There are several potential reasons why dabrafenib was not as potent in reducing inflammasome activation as was demonstrated in the RIPK3-/- animal. It is inevitable that the animals with genetic deletion of RIPK3 have greater inhibition of RIPK3 compared with a pharmacological inhibitor. Serum assays to determine the level of RIPK3 inhibition were not undertaken throughout the study. Furthermore, DMSO was used as the vehicle for dabrafenib delivery in our study. Despite the demonstrated toxicity of DMSO [369], it is recognised that
DMSO administration may also lead to reduced transcription of inflammatory pathways
[34,35]. Low concentrations of DMSO have been found to suppress CD4+ T cell activation, function, and metabolism [370]. A study has also demonstrated that DMSO attenuates NLRP3
131 inflammasome activation in macrophages and LPS-induced mice [371]. Hence, the magnitude of inflammation observed in the diabetic animals treated with DMSO may have been diminished. Additionally, the eNOS-/- mice may have different response towards the treatment of dabrafenib compared to wild-type mice. Moreover, the most severely affected mice from the eNOS-/- experiment had died which is very likely to cause bias to the conclusion.
Therefore, the benefit of dabrafenib may have been attenuated.
In summary, the results from these animal studies have demonstrated that RIPK3 is an important mediator of tubulointerstitial fibrosis in diabetic kidney disease. Furthermore, we have shown that blockade of RIPK3 exerts antifibrotic effects in the kidney that may arise due to a combination of inhibition of TLR4 signalling, NLRP3 inflammasome activation and TGF1 expression and downstream signalling.
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Chapter 6 Blockade of necroptosis attenuates fibrotic responses in human kidney proximal tubular cells exposed
to TGF-β1
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Abstract Necroptosis is increasingly recognised as a major regulator of inflammatory-related diseases, and in several conditions, necroptotic-blockade has been demonstrated to exert tissue protective effects. The inhibitor Nec-1s is a commonly used blocker of necroptosis. However, whether Nec-1s limits renal fibrosis is unclear. We utilised a human in vitro model, whereby human proximal tubular (HK2) cells were incubated with or without transforming growth factor beta (TGF-β) 1 (2ng/ml) for 48h in the presence or absence of the inhibitor Nec-1s (80
µM). We found Nec-1s treatment reduced the amplification of mRNA expression of TGF-β1, as well as the extracellular matrix (ECM) proteins (collagen I, collagen III, collagen IV and fibronectin). Our results indicate that Nec-1s may limit renal fibrosis via inhibiting TGF-β1 mediated ECM secretion in kidney proximal tubular cells.
6.1 Introduction
Renal fibrosis is common to all forms of progressive kidney disease. It is characterized by extracellular matrix (ECM) accumulation, myofibroblast activation and increased inflammation, causing renal dysfunction and ultimately end-stage kidney disease (ESKD) [372].
However, to date, specific therapies for preventing progression of renal fibrosis are lacking.
Necroptosis is a type of regulated necrosis activated by ligands of death receptors and stimuli that induce the expression of death receptor ligands under apoptotic deficient conditions. It serves essential functions in both development and tissue homeostasis [373]. The classic necroptosis pathway includes three key interacting kinases,
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receptor-interacting serine/threonine-protein kinase (RIPK) 3, RIPK1 and mixed lineage kinase domain-like protein (MLKL). The necrosome formed by RIPK3 and RIPK1 is the trigger to activate the necroptosis executioner MLKL and then induce necroptosis [15]. Conversely, deletion or inhibition of RIPK3, RIPK1 or MLKL prevents cells from undergoing necroptosis [18-
20, 374-376]. Necrostatin-1/1s (Nec-1/Nec-1s), is a widely used inhibitor of necroptosis [16,
17, 377, 378], effectively reducing necroptosis via inhibiting the kinase RIPK1 activity with an
IC50 of 0.367 µM in the induced necroptosis L929sAhFas cell model [379].
Recent studies have reported that inhibition of necroptosis with the inhibitor Nec-1 shows a protective effect against the interstitial fibrotic response in the kidney in both the unilateral ureteral obstruction (UUO) mouse model [16] and in rats subjected to subtotal nephrectomy
(SNx) [17]. In the UUO model, Nec-1 treatment reduced TGF-β gene expression [16]. Given that UUO is a classic animal model of studying tubulointerstitial fibrosis, these results indicate the antifibrotic function of Nec-1 may relate to inhibition of TGF-β effects on tubular cells.
It is well recognized that TGF-β1 is a principal mediator of renal fibrosis in all forms of kidney disease. There is now a large amount of literature to support that TGF-β1 is responsible for promoting tubulointerstitial fibrosis through tubular cell injury [380, 381]. The renal tubular epithelium is also considered as the effector of tubulointerstitial fibrosis, secreting profibrogenic cytokines and chemoattractants to enhance the inflammatory response [381].
Ultimately, tubular cells may also undergo epithelial to mesenchymal transition and acquire a myofibroblastic phenotype. Human proximal tubular cells (HK2 cell line) are widely used for studying renal fibrosis in cell culture [382]. Therefore, in this study, we studied the
135 antifibrotic potential of blockade of necroptosis and the mechanisms underpinning renoprotection in the HK2 cell line exposed to TGF-β1. Nec-1s, which is more potent in inhibiting necroptosis than is Nec-1 [383], was used as a necroptotic inhibitor in our study.
Our results demonstrate that Nec-1s attenuates ECM secretion induced via downregulation of TGF-β1.
6.2 Methods
6.2.1 Cell culture
HK2 cells were grown in keratinocyte serum-free medium (Invitrogen, CA). After reaching
60% confluence, cells were exposed to TGF-β1 (2 ng/ml) (R&D) plus the vehicle dimethyl sulfoxide (DMSO) (final concentration in culture medium was less than 0.1%) or Nec-1s (80
µM) (Biovision) in DMSO and incubated for 48 hours. Culture medium supernatant, cell lysates and total RNA were collected for further analyses.
6.2.2 Cell viability assay
For the cell proliferation assays, cells were seeded into each well of a 96-well plate (5 × 103 per well). After 24 hours, cells attached to the plate and then were incubated with Nec-1s in vehicle DMSO (40, 60, 80, 100, 160, 320, 400 µM) for 24 hours. Cell proliferation was determined with a CellTiter 96® AQueous Non-Radioactive Cell Proliferation. This assay uses
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4sulfophenyl)-2H tetrazolium
(MTS) that changes to formazan in cells with mitochondrial activity (viable cells). Formazan was measured at 490 nm with a microplate reader.
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6.2.3 RNA isolation and RT-PCR analysis
Total RNA was extracted from cells using ISOLATE II RNA Mini Kit (Bioline). The cDNA was synthesized with a cDNA synthesis kit (Roche). Predesigned probes, including tubulin (Assay
ID: Hs.PT.58.27209704), collagen IV (Assay ID: Hs.PT.58.15679435), fibronectin (Assay ID:
Hs.PT.58.40005963), TGF-β1 (Assay ID: Hs.PT.58.39813975) were measured. Tubulin was used as the endogenous reference gene.
6.2.4 Western blot analysis
Proteins in the supernatant were separated on 4-12% gels (Invitrogen) and then transferred to PVDF membrane (PALL) for 2 hours at 4 ℃. Membranes were blocked with 5% BSA in
TBS-T (20 mM Tris, 150 mM NaCl, 0.1% Tween 20, pH 7.6) for 1 hour and then incubated with primary antibodies (collagen I, Abcam, ab34710; collagen III, Abcam, ab7778; collagen
IV, Abcam, ab6586; fibronectin, Abcam, ab45688) and relevant horseradish peroxidase- conjugated secondary antibody (anti-Rabbit antibody, Cell Signalling).
6.2.5 Statistical analysis
The results are presented as mean ± S.E.M. All statistical analyses were performed with
GraphPad software. Comparison of multiple groups was analysed by one-way analysis of variance ANOVA followed by Tukey's multiple comparisons test. A two-sided p-value < 0.05 was considered statistically significant.
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6.3 Results
6.3.1 Inhibition of necroptosis decreased ECM secretion in HK2 cells exposed to TGF-β1.
In the present study, we used the MTS to measure the toxicity of Nec-1s. As shown in Fig
6.1a, Nec-1s inhibited cell proliferation in a concentration-dependent manner. However, even with a high concentration of 400 µM, the mean of the proliferation rate was 0.7729, suggesting a low toxicity of Nec-1s.
Figure 6.1 Toxicity of Nec-1s.
Proliferation rate was measured with MTS in HK2 cells exposed to the inhibitor Nec-1s at the above concentrations. Statistical analysis was performed by one-way analysis of variance
ANOVA followed by Tukey's multiple comparisons test. Results are presented as mean ± S.E.M.
****P < 0.0001 vs control. n = 6.
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To explore the function of Nec-1s, fibronectin and type IV collagen gene expression were measured in HK2 cells exposed to TGF-β1 with or without the necroptosis inhibitor Nec-1s at a concentration of 80 µM using RT-PCR. TGF-β1 treatment stimulated fibronectin gene expression (P< 0.001, Fig.6.2a), as well as type IV collagen gene expression (P< 0.05,
Fig.6.2b). Nec-1s at a concentration of 80 µM showed strong inhibition of both genes (Fig
6.2 a, b, both P<0.05). Western blotting experiments also confirmed that TGF-β1 increased collagen I, III, IV and fibronectin protein expression (Fig. 6.2c, d, e, f, g, all P<0.05), while
Nec-1s reversed this effect (6.2c, d, e, f, g, all P<0.05). These data suggest that Nec-1s treatment significantly reduces TGF-β1-induced ECM secretion in tubular cells.
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(a) Fibronectin mRNA expression fold change was measured by RT-PCR in HK2 cells exposed to TGF-β1 with or without the inhibitor Nec-1s. (b) Collagen IV mRNA expression fold change was measured by RT-PCR in the same group as in (a). Tubulin was used as the reference gene for RT-PCR. (c-g) Immunoblot analysed cells from the same groups as in (a) using antibodies for collagen I, collagen III, collagen IV, fibronectin. Statistical analysis was performed by one- way analysis of variance ANOVA followed by Tukey's multiple comparisons test. Results are presented as mean ± S.E.M. *P < 0.05, ***P < 0.001. n = 4.
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6.3.2 Inhibition of necroptosis downregulated TGF-β1 expression
Having established that TGF-β1 treated HK2 cells secrete elevated levels ECM, we sought to determine whether these stimulated cells themselves exhibited increased TGF-β1 expression. RT-PCR results demonstrated that TGF-β1 treatment significantly increased TGF-
β1 gene expression (P< 0.01, Fig.6.3a), thus amplifying the fibrotic response. We also observed that this upregulation was partially reversed by Nec-1s treatment (P< 0.05,
Fig.6.3a), suggesting that Nec-1s treatment may limit tubulointerstitial fibrosis by reducing endogenous TGF-β1 mRNA expression.
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(a) TGF-β1 mRNA expression fold change was measured by RT-PCR in HK2 cells exposed to
TGF-β1 with or without the inhibitor Nec-1s. Tubulin was used as the reference gene for RT-
PCR. Statistical analysis was performed by one-way analysis of variance ANOVA followed by
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Tukey's multiple comparisons test. Results are presented as mean ± S.E.M. *P < 0.05, **P <
0.01. n = 4.
6.3.3 Inhibition of necroptosis showed a trend to downregulate phosphor-extracellular- signal-regulated kinase (ERK) but not p38 expression
To further study the mechanism, we measured the phosphorylation level of ERK and p38 using western blot. HK2 cells showed higher phosphorylation levels of ERK in the presence of TGF-β1 (P< 0.05, Fig.6.4a, b). However, this effect showed a trend to be reduced by Nec-
1s treatment (P=0.0721, Fig. 6.4a, b), indicating that Nec-1s treatment may decrease the fibrotic response partially via regulating ERK activation. However, no trend was seen for
Nec-1s to reduce the TGF-β1-induced phosphorylation of p38. (Fig. 6.4 a, c).
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Figure 6.4 Increased ERK and p38 activation in HK2 cells exposed to TGF-β1.
(a, b, c) Immunoblot analysis of HK2 cells exposed to TGF-β1 with or without the inhibitor
Nec-1s. using antibodies for phosphor-ERK, total ERK, phosphor-p38, total p38. Statistical analysis was performed by one-way analysis of variance ANOVA followed by Tukey's multiple comparisons test. Results are presented as mean ± S.E.M. *P < 0.05, n = 4.
6.4 Discussion
In the current study, we investigated whether inhibition of necroptosis blocks the fibrotic response in HK2 cells exposed to TGF-β1. Our results showed that exogenous TGF-β1 amplified endogenous tubular cell TGF-β1 mRNA expression, which was accompanied by increasing ECM secretion and ERK activation. Moreover, the findings suggest that Nec-1s
144 inhibition of necroptosis leads to a reduction of ECM secretion and TGF-β1 expression.
Taken together, these findings suggest that necroptosis may be involved in fibrogenesis via mediating downstream activation of TGF-β1 as well as auto amplification of the TGF-β1 response.
ECM deposition is the main histological feature of renal fibrosis. It is well established that
TGF-β1 is one of the most potent cytokines regulating ECM synthesis, with tubular cells and myofibroblasts, which in turn, are often derived from tubular cells, being a significant source of TGF-β1. Downstream TGF-β1 signalling through stimulates the synthesis and overproduction of various ECM proteins, including collagens and fibronectin, accounting for the interstitial fibrosis [384]. Of note, in this study, protein levels of Collagens -1, -3 and IV as well as fibronectin were increased by TGF-β1 and reversed by Nec-1s, which supports our hypothesis that blockade of necroptosis suppresses TGF-β1 induced ECM secretion in tubular cells. These results are also consistent with our previous findings in in vivo studies, that blockade of RIPK3 reduces TGF-β1 within the kidney cortex, indicating the benefit of
RIPK3 inhibition may partially arise from abolishing necroptosis in tubular cells.
TGF-β1 induces a fibrotic response by activating Smad mediated and non-Smad signalling cascades, such as Ras/Raf/ERK/ mitogen-activated protein kinase (MAPK) pathways, p38/MAPK pathways [9]. However, the activation of p38/MAPK in renal fibrosis occurs in conjunction with Smad2/3 [179, 181-184]. The present study also shows inhibition of necroptosis failed to regulate p38, and hence unlikely to also involve Smad 2/3.
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In non-Smad signalling, only ERK contributes to the autoinduction of TGF-β1 [169]. It is also reported that activation of the ERK signalling Is required for TGF-β1-Induced epithelial-to- mesenchymal transition (EMT) in NMuMG and MCT (mouse proximal tubule epithelial cells)
[385]. Given that we obtained consistent suppression of myofibroblast activation and showed a trend to inhibition of ERK phosphorylation with inhibition of RIPK3 in in vivo studies, ERK activation may play a role in renal fibrosis. Similar to the effect of RIPK3 deficiency or dabrafenib, Nec-1s treatment only exhibited a partial suppression of the ERK pathway. Thus, we conclude ERK signalling is not a major pathway in necroptosis mediated TGF-β1 cascades.
However, it remains possible the beneficial effect of dabrafenib may be partially driven by inhibition of ERK in conjunction with inhibition of RIPK3.
Nec-1s, a widely used to block necroptosis as it abolishes the RIPK1/RIPK3 necrosome formation and ensuing necroptosis [312-314, 386]. Hence it is a useful pharmacological tool to study necroptosis but not necessarily to investigate the individual contribution of RIPK1 or RIPK3 to the necroptotic response. Given that RIPK1 shows dual influences on necroptosis in other models of fibrosis [315], the role of RIPK1 in tissue and organ-specific models of renal fibrosis needs to be further explored.
In summary, the present study demonstrated that ablation of necroptosis significantly suppressed TGF-β1 expression, as well as TGF-β1-induced ECM secretion in proximal tubular cells. Nec-1s showed a trend to be associated with suppression of ERK activation. However,
146 it is possible that other downstream signalling pathways of TGF-β1 may also be involved.
This study provides a basis for further investigating the therapeutic potential of necroptosis inhibition in the prevention of renal interstitial fibrosis.
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Chapter 7 Conclusions and future directions
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7.1 Conclusions
CKD represents an increasing component of the health burden and economic cost of non- communicable disease in the developed and developing world [3, 4]. CKD is characterized functionally by a drop in glomerular filtration rate and development of albuminuria and histologically by ECM accumulation, myofibroblast activation and inflammatory responses, which collectively comprise the features of renal fibrosis. Renal fibrosis is the pathological endpoint of all renal disease across the spectrum of causes of CKD and ESKD. CKD due to multiple causes, but primarily due to repeated episodes of acute or toxic kidney injury or diabetic kidney disease affects a large number of the population worldwide. Hence it is appropriate to focus on treatment strategies that interrupt the development of fibrosis in addition to upstream factors that may be specific to certain diseases.
Given that clinical strategies based on current findings for the prevention or management of renal fibrosis remain unsatisfactory [122, 235], and the rising importance of innate immunity in fibrotic response has been reported, we hypothesised RIPK3 as a novel target and utilised the toxin-induced FA model of acute toxic nephropathy followed by the development of CKD and a STZ induced diabetic model to test our hypothesis. An in vitro study was also done to explore the role of necroptosis in the fibrotic response. Our results are described in detail in each chapter and in summary form in Chapter 1.
Collectively, the principal finding in the thesis is the identification of RIPK3 as a crucial regulator of renal fibrosis. I have identified RIPK3 mediated TLR4 signalling and NLRP3
149 inflammasome activation, key contributors to the development of renal fibrosis, in addition to known TGF1 dependent signalling pathways that may be downstream of activation of
RIPK3 activation of TLR4 signalling and the NLRP3 inflammasome and dependent on the model of CKD. My data provide evidence of the powerful effect of RIPK3 on fibrogenesis and strongly suggests that dabrafenib should be considered for repurposing to target RIPK3 to limit the development of CKD.
7.2 Future directions
The results of this thesis showed blocking RIPK3 reduced IL-1β mRNA expression in both the
FA and diabetic CKD models. IL-1β serves as the downstream cytokine of both NLRP3 inflammasome and TLR2/4-NFκB cascades. However, the data generated from the FA model suggests NFκB is not primarily activated, suggesting IL-1β may mainly originate from inflammasome activation, thus confirming our conclusion NLRP3 activation is a major downstream mediator of RIPK3. Although not the focus of this thesis, targeting of IL1β has received very recent attention for the treatment of cardiovascular disease [387] and more recently interest to prevent the development and progression of CKD [388]. My data would support further targeted studies into using monoclonal blockers of IL-1β to assess the effect on progressive kidney disease.
As I discussed in Chapter 2, the NLRP3 inflammasome activation contributes to tubular injury and renal fibrosis. NLRP3 is the crucial component of inflammasome complex, deletion of which protected PTCs from NLRP3 inflammasome activation in Ang II-infused mice [354]. Similarly, NLRP3 deficiency attenuated tubulointerstitial fibrosis as
150 demonstrated by reduced ECM deposition and decreased cell apoptosis in a mouse UUO model [340]. In vitro studies further confirmed suppression of EMT as well as α-SMA in renal
PTCs exposed to TGF-β [193]. An inflammasome-independent role of NLRP3 has been proposed, given that the effect of NLRP3 on α-SMA expression was shown to be independent of the inflammasome and associated IL-1β expression [193]. A similar result was observed in the mouse model of renal ischemia-reperfusion injury, where loss of NLRP3 but not ASC protected tubular cells from necrosis and apoptosis. In our FA model, blocking
RIPK3 showed a trend to reduce ASC, which was more convincingly demonstrated in the DN model. Compared to ASC, ablation of RIPK3 reduced NLRP3 expression significantly in both the FA and DN models, suggesting the benefit of blocking RIPK3 may arise from independent effects on both the inflammasome and on NLRP3. Thus, dissection of these individual components and their contribution to benefits derived from interruption or inhibition of the
RIPK3 mediated pathway would be worthy of exploration in the future.
Several recent studies have established the significant roles of TLR2 and 4 in the development of kidney injury and fibrosis. Although TLR2 and TLR4 are often observed to be contemporaneously increased in various models of renal disease, TLR2 induces pro- inflammatory responses while TLR4 mediates both pro-inflammatory and pro-fibrotic pathways [206]. In my studies, both in vivo models upregulated TLR2 and TLR4 expression.
In the FA model, lack of RIPK3 reduced expression of both receptors. However, only TLR4 was downregulated by RIPK3 deficiency in the DN model. In considering these differences, the FA model is studied at 28-day i.e in the chronic phase after FA-induced tubular injury, at which stage persistent inflammatory stimulation is reduced. In contrast, hyperglycaemia is a persistent stimulus in the DN model. Hence our data may suggest a positive feedback loop
151 regulation between RIPK3 and TLR2 exists and differ dependent on the strength of the inflammatory stimuli. Experiments with graded inflammation and concomitant TLR2 blockade are required to dissect the two pathways.
We have demonstrated that TGF-β is regulated by RIPK3 in both the FA model and diabetic model of CKD. A reduction of TGF-β expression was also observed in Nec-1s treated PTCs.
Thus, inhibition of both RIPK3 and necroptosis downregulate TGF-β expression +/- activity.
Given the role of RIPK3 in necroptosis, these results support the basis of our hypothesis that targeting RIPK3 contributes to renal fibrosis via multiple mechanisms occurring in parallel.
It is well recognized that downstream signalling of TGF-β includes Smad and non-Smad mediated signalling cascades such as the Ras/Raf/ERK/MAPK pathways. To further investigate the RIPK3-sensitive downstream mediators of TGF-β, we measured these downstream mediators in our studies. Smad2/3 was downregulated in RIPK3-/- mice with
FAN as well as in diabetic mice treated with dabrafenib. Therefore, we conclude Smad2/3 signalling is an important mechanism in mediating downstream effects of RIPK3 activation.
The regulation of Smad2/3 signalling by RIPK3 may differ depending on the context of injury and needs validation in differing models of renal disease. Expression of pERK showed a consistent trend to reduction in both in vivo and in vitro studies. Although no significant changes were found, the function of this pathway in RIPK3/TGF-β mediated inhibition of fibrosis cannot be excluded given the trend to significance was high. Non-Smad pathways, such as the JNK, and Rho-like GTPase signalling pathways deserve further exploration in future studies.
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Using a universal knock out animal model resulting in RIPK3 deficiency is a potential limitation of our study in defining mechanistic effects. Investigating mechanistic aspects underpinning any therapeutic potential is limited by this approach. A tubular-specific knock out model would be required to assess tubular specific effects of RIPK3 in the contribution of the tubular cell to renal fibrosis. Such a model could be the focus of future studies to develop specific therapies. Having said this, organ and cell-specific knockout and overexpression strategies have limited relevance to therapeutic interventions which are generally unable to be targeted to organs or tissues but have global effects.
Dabrafenib was used as the RIPK3 inhibitor in our study and deliberately chosen to allow differentiation of the RIPK3 dependent pathways (as demonstrated in the RIPK3-/- animals) from other kinase-dependent pathways also inhibited by dabrafenib. As I discussed in
Chapter 2, dabrafenib can inhibit Ras/Raf/ERK/MAPK activation [328], and hence is used in targeted therapy for melanoma and non-small cell lung cancer expressing B-Raf V600E mutations [329]. Hence it has broader therapeutic potential beyond its actions as a RIPK3 inhibitor. As discussed in Chapter 2, Ras/Raf/ERK/MAPK serves as a downstream pathway of
TGF-β and contributes to renal fibrosis. However, only a trend for ERK inhibition by dabrafenib was observed in our study. These off-target effects of dabrafenib on
Ras/Raf/ERK/MAPK may contribute to its renoprotection effects. A higher dose of dabrafenib is worthy of investigation in an animal model of renal fibrosis.
The toxicity of dabrafenib has been fully investigated as required prior to approval for clinical use. A single patient has been reported to develop nephrotic syndrome during treatment with dabrafenib in combination with another BRAF inhibitor trametinib [389].
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However, kidney function and glomerular ultrastructural changes fully recovered after drug withdrawal. In patients receiving dabrafenib therapy in the Food and Drug Administration
Adverse Event Reporting System, quarterly legacy data file from the second quarter of 2013 through second quarter of 2014, thirteen cases were reported as having acute kidney injury due to tubulointerstitial injury [390]. The reasons for acute kidney injury are unclear, but given the drug was administered in patients receiving concomitant therapies for malignancy there are likely to be multiple mechanisms predisposing to acute kidney injury. A study also indicated that the dabrafenib associated nephrotoxicity may be age-dependent [391].
Although very few reported have been published on nephrotoxicity of dabrafenib, dedicated clinical studies in patients with would be demanded before repurposing this drug for kidney disease.
Compared to the enormous costs and money for the approval of a new drug, this drug may be easier to repurpose for use in patients with CKD with less time and cost. Clinical trials using dabrafenib to slow the development of CKD should be supported, with or without pharmaceutical support.
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References
1. AIHW. Chronic kidney disease. 2018 18/01/2018; Available from: https://www.aihw.gov.au/reports-statistics/health-conditions-disability-deaths/chronic- kidney-disease/overview. 2. SYSTEM, U.S.R.D. CKD in the General Population. [cited 2018 29/9]; Available from: https://www.usrds.org/2017/view/v1_01.aspx. 3. Lee, S.B. and R. Kalluri, Mechanistic connection between inflammation and fibrosis. Kidney Int Suppl, 2010(119): p. S22-6. 4. Carew, R.M., B. Wang and P. Kantharidis, The role of EMT in renal fibrosis. Cell Tissue Res, 2012. 347(1): p. 103-16. 5. Zhang, F., H. Liu, D. Liu, et al., Effects of RAAS Inhibitors in Patients with Kidney Disease. Curr Hypertens Rep, 2017. 19(9): p. 72. 6. Ruilope, L.M., Angiotensin receptor blockers: RAAS blockade and renoprotection. Curr Med Res Opin, 2008. 24(5): p. 1285-93. 7. Mack, M., Inflammation and fibrosis. Matrix Biol, 2018. 68-69: p. 106-121. 8. Liu, B.C., T.T. Tang, L.L. Lv, et al., Renal tubule injury: a driving force toward chronic kidney disease. Kidney Int, 2018. 93(3): p. 568-579. 9. Loeffler, I. and G. Wolf, Transforming growth factor-β and the progression of renal disease. Nephrology Dialysis Transplantation, 2014. 29(suppl_1): p. i37-i45. 10. Edeling, M., G. Ragi, S. Huang, et al., Developmental signalling pathways in renal fibrosis: the roles of Notch, Wnt and Hedgehog. Nat Rev Nephrol, 2016. 12(7): p. 426-39. 11. Kumar, S., Cellular and molecular pathways of renal repair after acute kidney injury. Kidney Int, 2018. 93(1): p. 27-40. 12. Wang, Y.H. and Y.G. Zhang, Kidney and innate immunity. Immunol Lett, 2017. 183: p. 73-78. 13. Wada, J. and H. Makino, Innate immunity in diabetes and diabetic nephropathy. Nat Rev Nephrol, 2016. 12(1): p. 13-26. 14. Pasparakis, M. and P. Vandenabeele, Necroptosis and its role in inflammation. Nature, 2015. 517(7534): p. 311-20. 15. Moriwaki, K. and F.K. Chan, Necroptosis-independent signaling by the RIP kinases in inflammation. Cell Mol Life Sci, 2016. 16. Xiao, X., C. Du, Z. Yan, et al., Inhibition of Necroptosis Attenuates Kidney Inflammation and Interstitial Fibrosis Induced By Unilateral Ureteral Obstruction. Am J Nephrol, 2017. 46(2): p. 131-138.
155
17. Zhu, Y., H. Cui, Y. Xia, et al., RIPK3-Mediated Necroptosis and Apoptosis Contributes to Renal Tubular Cell Progressive Loss and Chronic Kidney Disease Progression in Rats. PLoS One, 2016. 11(6): p. e0156729. 18. Roychowdhury, S., M.R. McMullen, S.G. Pisano, et al., Absence of receptor interacting protein kinase 3 prevents ethanol-induced liver injury. Hepatology, 2013. 57(5): p. 1773-83. 19. Liu, W., B. Chen, Y. Wang, et al., RGMb protects against acute kidney injury by inhibiting tubular cell necroptosis via an MLKL-dependent mechanism. Proc Natl Acad Sci U S A, 2018. 115(7): p. E1475-E1484. 20. Zhang, Y.F., W. He, C. Zhang, et al., Role of receptor interacting protein (RIP)1 on apoptosis- inducing factor-mediated necroptosis during acetaminophen-evoked acute liver failure in mice. Toxicol Lett, 2014. 225(3): p. 445-53. 21. Filliol, A., M. Farooq, C. Piquet-Pellorce, et al., RIPK1 protects hepatocytes from death in Fas- induced hepatitis. Sci Rep, 2017. 7(1): p. 9205. 22. Imamura, M., J.S. Moon, K.P. Chung, et al., RIPK3 promotes kidney fibrosis via AKT- dependent ATP citrate lyase. JCI Insight, 2018. 3(3). 23. Lawlor, K.E., N. Khan, A. Mildenhall, et al., RIPK3 promotes cell death and NLRP3 inflammasome activation in the absence of MLKL. Nat Commun, 2015. 6: p. 6282. 24. Vince, J.E. and J. Silke, The intersection of cell death and inflammasome activation. Cell Mol Life Sci, 2016. 73(11-12): p. 2349-67. 25. Silke, J., J.A. Rickard and M. Gerlic, The diverse role of RIP kinases in necroptosis and inflammation. Nat Immunol, 2015. 16(7): p. 689-97. 26. Conos, S.A., K.W. Chen, D. De Nardo, et al., Active MLKL triggers the NLRP3 inflammasome in a cell-intrinsic manner. Proc Natl Acad Sci U S A, 2017. 114(6): p. E961-E969. 27. Wang, X., W. Jiang, Y. Yan, et al., RNA viruses promote activation of the NLRP3 inflammasome through a RIP1-RIP3-DRP1 signaling pathway. Nat Immunol, 2014. 15(12): p. 1126-33. 28. Najjar, M., D. Saleh, M. Zelic, et al., RIPK1 and RIPK3 Kinases Promote Cell-Death- Independent Inflammation by Toll-like Receptor 4. Immunity, 2016. 45(1): p. 46-59. 29. Zhu, Y., H. Cui, H. Gan, et al., Necroptosis mediated by receptor interaction protein kinase 1 and 3 aggravates chronic kidney injury of subtotal nephrectomised rats. Biochem Biophys Res Commun, 2015. 461(4): p. 575-81. 30. Haberle, D.A. and H. von Baeyer, Characteristics of glomerulotubular balance. Am J Physiol, 1983. 244(4): p. F355-66.
156
31. Thomson, S.C. and R.C. Blantz, Glomerulotubular balance, tubuloglomerular feedback, and salt homeostasis. J Am Soc Nephrol, 2008. 19(12): p. 2272-5. 32. Schnaper, H.W., Renal fibrosis. Methods Mol Med, 2005. 117: p. 45-68. 33. Nogueira, A., M.J. Pires and P.A. Oliveira, Pathophysiological Mechanisms of Renal Fibrosis: A Review of Animal Models and Therapeutic Strategies. In Vivo, 2017. 31(1): p. 1-22. 34. Chapter 1: Definition and classification of CKD. Kidney Int Suppl (2011), 2013. 3(1): p. 19-62. 35. Global Burden of Disease Study, C., Global, regional, and national incidence, prevalence, and years lived with disability for 301 acute and chronic diseases and injuries in 188 countries, 1990-2013: a systematic analysis for the Global Burden of Disease Study 2013. Lancet, 2015. 386(9995): p. 743-800. 36. (IHME), I.f.H.M.a.E., GBD Compare. Seattle, WA: IHME, University of Washington, 2015. 37. Engelgau, M.M., S. El-Saharty, P. Kudesia, et al., Regional Aging and Disease Burden, in Capitalizing on the Demographic Transition. p. 15-40. 38. Ayodele, O.E. and C.O. Alebiosu, Burden of chronic kidney disease: an international perspective. Adv Chronic Kidney Dis, 2010. 17(3): p. 215-24. 39. Jha, V., End-stage renal care in developing countries: the India experience. Ren Fail, 2004. 26(3): p. 201-8. 40. Jha, V., G. Garcia-Garcia, K. Iseki, et al., Chronic kidney disease: global dimension and perspectives. Lancet, 2013. 382(9888): p. 260-72. 41. Bohle, A., H. Christ, K.E. Grund, et al., The role of the interstitium of the renal cortex in renal disease. Contrib Nephrol, 1979. 16: p. 109-14. 42. Harris, R.C. and E.G. Neilson, Toward a unified theory of renal progression. Annu Rev Med, 2006. 57: p. 365-80. 43. Hruby, Z., D. Smolska, H. Filipowski, et al., The importance of tubulointerstitial injury in the early phase of primary glomerular disease. J Intern Med, 1998. 243(3): p. 215-22. 44. Hynes, R.O., Extracellular matrix: not just pretty fibrils. Science (New York, N.Y.), 2009. 326(5957): p. 1216-1219. 45. Hynes, R.O. and A. Naba, Overview of the matrisome--an inventory of extracellular matrix constituents and functions. Cold Spring Harb Perspect Biol, 2012. 4(1): p. a004903. 46. Bonnans, C., J. Chou and Z. Werb, Remodelling the extracellular matrix in development and disease. Nat Rev Mol Cell Biol, 2014. 15(12): p. 786-801. 47. Gewin, L.S., Renal fibrosis: Primacy of the proximal tubule. Matrix Biol, 2018. 68-69: p. 248- 262.
157
48. Szeto, S.G., M. Narimatsu, M. Lu, et al., YAP/TAZ Are Mechanoregulators of TGF-beta-Smad Signaling and Renal Fibrogenesis. J Am Soc Nephrol, 2016. 27(10): p. 3117-3128. 49. Kuncio, G.S., E.G. Neilson and T. Haverty, Mechanisms of tubulointerstitial fibrosis. Kidney Int, 1991. 39(3): p. 550-6. 50. Zeisberg, M. and E.G. Neilson, Mechanisms of tubulointerstitial fibrosis. J Am Soc Nephrol, 2010. 21(11): p. 1819-34. 51. Liu, L., P. Zhang, M. Bai, et al., p53 Upregulated by HIF-1alpha Promotes Hypoxia-induced G2/M Arrest and Renal Fibrosis in vitro and in vivo. J Mol Cell Biol, 2018. 52. Wang, W., P.H. Zhou, W. Hu, et al., Cryptotanshinone hinders renal fibrosis and epithelial transdifferentiation in obstructive nephropathy by inhibiting TGF-beta1/Smad3/integrin beta1 signal. Oncotarget, 2018. 9(42): p. 26625-26637. 53. Wang, D., M. Xiong, C. Chen, et al., Legumain, an asparaginyl endopeptidase, mediates the effect of M2 macrophages on attenuating renal interstitial fibrosis in obstructive nephropathy. Kidney Int, 2018. 94(1): p. 91-101. 54. Furness, P.N., Extracellular matrix and the kidney. J Clin Pathol, 1996. 49(5): p. 355-9. 55. Farris, A.B., C.D. Adams, N. Brousaides, et al., Morphometric and visual evaluation of fibrosis in renal biopsies. J Am Soc Nephrol, 2011. 22(1): p. 176-86. 56. Tomino, Y., K. Funabiki, I. Shirato, et al., Computer imaging analysis of glomerular extracellular components in patients with IgA nephropathy. Acta Pathol Jpn, 1989. 39(5): p. 296-305. 57. Chen, X., G. Moeckel, J.D. Morrow, et al., Lack of integrin alpha1beta1 leads to severe glomerulosclerosis after glomerular injury. Am J Pathol, 2004. 165(2): p. 617-30. 58. Haverty, T.P., C.J. Kelly, W.H. Hines, et al., Characterization of a renal tubular epithelial cell line which secretes the autologous target antigen of autoimmune experimental interstitial nephritis. J Cell Biol, 1988. 107(4): p. 1359-68. 59. Howard, B.V., E.J. Macarak, D. Gunson, et al., Characterization of the collagen synthesized by endothelial cells in culture. Proc Natl Acad Sci U S A, 1976. 73(7): p. 2361-4. 60. Abrahamson, D.R., P.L. St John, L. Stroganova, et al., Laminin and type IV collagen isoform substitutions occur in temporally and spatially distinct patterns in developing kidney glomerular basement membranes. J Histochem Cytochem, 2013. 61(10): p. 706-18. 61. Suh, J.H. and J.H. Miner, The glomerular basement membrane as a barrier to albumin. Nat Rev Nephrol, 2013. 9(8): p. 470-7. 62. Wolf, G. and F.N. Ziyadeh, Cellular and molecular mechanisms of proteinuria in diabetic nephropathy. Nephron Physiol, 2007. 106(2): p. p26-31.
158
63. Cui, N., M. Hu and R.A. Khalil, Biochemical and Biological Attributes of Matrix Metalloproteinases. Prog Mol Biol Transl Sci, 2017. 147: p. 1-73. 64. Malemud, C.J., Matrix metalloproteinases (MMPs) in health and disease: an overview. Front Biosci, 2006. 11: p. 1696-701. 65. Catania, J.M., G. Chen and A.R. Parrish, Role of matrix metalloproteinases in renal pathophysiologies. Am J Physiol Renal Physiol, 2007. 292(3): p. F905-11. 66. Keeling, J. and G.A. Herrera, Human matrix metalloproteinases: characteristics and pathologic role in altering mesangial homeostasis. Microsc Res Tech, 2008. 71(5): p. 371-9. 67. Bauvois, B., N. Mothu, J. Nguyen, et al., Specific changes in plasma concentrations of matrix metalloproteinase-2 and -9, TIMP-1 and TGF-beta1 in patients with distinct types of primary glomerulonephritis. Nephrol Dial Transplant, 2007. 22(4): p. 1115-22. 68. Gonzalez-Avila, G., C. Iturria, F. Vadillo-Ortega, et al., Changes in matrix metalloproteinases during the evolution of interstitial renal fibrosis in a rat experimental model. Pathobiology, 1998. 66(5): p. 196-204. 69. Ewens, K.G., R.A. George, K. Sharma, et al., Assessment of 115 candidate genes for diabetic nephropathy by transmission/disequilibrium test. Diabetes, 2005. 54(11): p. 3305-18. 70. Kanauchi, M., H. Nishioka, Y. Nakashima, et al., Role of tissue inhibitors of metalloproteinase in diabetic nephropathy. Nihon Jinzo Gakkai Shi, 1996. 38(3): p. 124-8. 71. Dimas, G., F. Iliadis and D. Grekas, Matrix metalloproteinases, atherosclerosis, proteinuria and kidney disease: Linkage-based approaches. Hippokratia, 2013. 17(4): p. 292-7. 72. Parrish, A.R., Chapter Two - Matrix Metalloproteinases in Kidney Disease: Role in Pathogenesis and Potential as a Therapeutic Target, in Progress in Molecular Biology and Translational Science, R.A. Khalil, Editor. 2017, Academic Press. p. 31-65. 73. Meran, S. and R. Steadman, Fibroblasts and myofibroblasts in renal fibrosis. Int J Exp Pathol, 2011. 92(3): p. 158-67. 74. Alpers, C.E., K.L. Hudkins, J. Floege, et al., Human renal cortical interstitial cells with some features of smooth muscle cells participate in tubulointerstitial and crescentic glomerular injury. J Am Soc Nephrol, 1994. 5(2): p. 201-9. 75. Clayton, A., R. Steadman and J.D. Williams, Cells isolated from the human cortical interstitium resemble myofibroblasts and bind neutrophils in an ICAM-1--dependent manner. J Am Soc Nephrol, 1997. 8(4): p. 604-15. 76. Iwano, M., D. Plieth, T.M. Danoff, et al., Evidence that fibroblasts derive from epithelium during tissue fibrosis. J Clin Invest, 2002. 110(3): p. 341-50.
159
77. Grimm, P.C., P. Nickerson, J. Jeffery, et al., Neointimal and tubulointerstitial infiltration by recipient mesenchymal cells in chronic renal-allograft rejection. N Engl J Med, 2001. 345(2): p. 93-7. 78. Ekblom, P. and A. Weller, Ontogeny of tubulointerstitial cells. Kidney Int, 1991. 39(3): p. 394- 400. 79. Dugina, V., L. Fontao, C. Chaponnier, et al., Focal adhesion features during myofibroblastic differentiation are controlled by intracellular and extracellular factors. J Cell Sci, 2001. 114(Pt 18): p. 3285-96. 80. Hinz, B., Tissue stiffness, latent TGF-beta1 activation, and mechanical signal transduction: implications for the pathogenesis and treatment of fibrosis. Curr Rheumatol Rep, 2009. 11(2): p. 120-6. 81. Hinz, B., V. Dugina, C. Ballestrem, et al., Alpha-smooth muscle actin is crucial for focal adhesion maturation in myofibroblasts. Mol Biol Cell, 2003. 14(6): p. 2508-19. 82. Koesters, R., B. Kaissling, M. Lehir, et al., Tubular overexpression of transforming growth factor-beta1 induces autophagy and fibrosis but not mesenchymal transition of renal epithelial cells. Am J Pathol, 2010. 177(2): p. 632-43. 83. Borges, F.T., S.A. Melo, B.C. Ozdemir, et al., TGF-beta1-containing exosomes from injured epithelial cells activate fibroblasts to initiate tissue regenerative responses and fibrosis. J Am Soc Nephrol, 2013. 24(3): p. 385-92. 84. Chen, Y.T., F.C. Chang, C.F. Wu, et al., Platelet-derived growth factor receptor signaling activates pericyte-myofibroblast transition in obstructive and post-ischemic kidney fibrosis. Kidney Int, 2011. 80(11): p. 1170-81. 85. Maarouf, O.H., A. Aravamudhan, D. Rangarajan, et al., Paracrine Wnt1 Drives Interstitial Fibrosis without Inflammation by Tubulointerstitial Cross-Talk. J Am Soc Nephrol, 2016. 27(3): p. 781-90. 86. Hung, C., G. Linn, Y.H. Chow, et al., Role of lung pericytes and resident fibroblasts in the pathogenesis of pulmonary fibrosis. Am J Respir Crit Care Med, 2013. 188(7): p. 820-30. 87. LeBleu, V.S., G. Taduri, J. O'Connell, et al., Origin and function of myofibroblasts in kidney fibrosis. Nat Med, 2013. 19(8): p. 1047-53. 88. Boor, P., T. Ostendorf and J. Floege, Renal fibrosis: novel insights into mechanisms and therapeutic targets. Nat Rev Nephrol, 2010. 6(11): p. 643-56. 89. Strutz, F. and E.G. Neilson, New insights into mechanisms of fibrosis in immune renal injury. Springer Semin Immunopathol, 2003. 24(4): p. 459-76.
160
90. Strutz, F., Pathogenesis of tubulointerstitial fibrosis in chronic allograft dysfunction. Clin Transplant, 2009. 23 Suppl 21: p. 26-32. 91. Strutz, F., H. Okada, C.W. Lo, et al., Identification and characterization of a fibroblast marker: FSP1. J Cell Biol, 1995. 130(2): p. 393-405. 92. Ng, Y.Y., T.P. Huang, W.C. Yang, et al., Tubular epithelial-myofibroblast transdifferentiation in progressive tubulointerstitial fibrosis in 5/6 nephrectomized rats. Kidney Int, 1998. 54(3): p. 864-76. 93. Zeisberg, M., F. Strutz and G.A. Muller, Renal fibrosis: an update. Curr Opin Nephrol Hypertens, 2001. 10(3): p. 315-20. 94. Sun, K.H., Y. Chang, N.I. Reed, et al., alpha-Smooth muscle actin is an inconsistent marker of fibroblasts responsible for force-dependent TGFbeta activation or collagen production across multiple models of organ fibrosis. Am J Physiol Lung Cell Mol Physiol, 2016. 310(9): p. L824- 36. 95. Neelisetty, S., C. Alford, K. Reynolds, et al., Renal fibrosis is not reduced by blocking transforming growth factor-beta signaling in matrix-producing interstitial cells. Kidney Int, 2015. 88(3): p. 503-14. 96. Eardley, K.S., C. Kubal, D. Zehnder, et al., The role of capillary density, macrophage infiltration and interstitial scarring in the pathogenesis of human chronic kidney disease. Kidney Int, 2008. 74(4): p. 495-504. 97. Bohle, A., G.A. Muller, M. Wehrmann, et al., Pathogenesis of chronic renal failure in the primary glomerulopathies, renal vasculopathies, and chronic interstitial nephritides. Kidney Int Suppl, 1996. 54: p. S2-9. 98. Duffield, J.S., Macrophages and immunologic inflammation of the kidney. Semin Nephrol, 2010. 30(3): p. 234-54. 99. Henderson, N.C., A.C. Mackinnon, S.L. Farnworth, et al., Galectin-3 expression and secretion links macrophages to the promotion of renal fibrosis. Am J Pathol, 2008. 172(2): p. 288-98. 100. Ninichuk, V., A.G. Khandoga, S. Segerer, et al., The role of interstitial macrophages in nephropathy of type 2 diabetic db/db mice. Am J Pathol, 2007. 170(4): p. 1267-76. 101. Zeisberg, M., M. Khurana, V.H. Rao, et al., Stage-specific action of matrix metalloproteinases influences progressive hereditary kidney disease. PLoS Med, 2006. 3(4): p. e100. 102. Roma-Lavisse, C., M. Tagzirt, C. Zawadzki, et al., M1 and M2 macrophage proteolytic and angiogenic profile analysis in atherosclerotic patients reveals a distinctive profile in type 2 diabetes. Diab Vasc Dis Res, 2015. 12(4): p. 279-89.
161
103. Zajac, E., B. Schweighofer, T.A. Kupriyanova, et al., Angiogenic capacity of M1- and M2- polarized macrophages is determined by the levels of TIMP-1 complexed with their secreted proMMP-9. Blood, 2013. 122(25): p. 4054-67. 104. Yu, L., W.A. Border, Y. Huang, et al., TGF-beta isoforms in renal fibrogenesis. Kidney Int, 2003. 64(3): p. 844-56. 105. Xu, P., J. Liu and R. Derynck, Post-translational regulation of TGF-beta receptor and Smad signaling. FEBS Lett, 2012. 586(14): p. 1871-84. 106. Robertson, I.B., M. Horiguchi, L. Zilberberg, et al., Latent TGF-beta-binding proteins. Matrix Biol, 2015. 47: p. 44-53. 107. Annes, J.P., J.S. Munger and D.B. Rifkin, Making sense of latent TGFbeta activation. J Cell Sci, 2003. 116(Pt 2): p. 217-24. 108. Sanderson, N., V. Factor, P. Nagy, et al., Hepatic expression of mature transforming growth factor beta 1 in transgenic mice results in multiple tissue lesions. Proc Natl Acad Sci U S A, 1995. 92(7): p. 2572-6. 109. Wang, W., V. Koka and H.Y. Lan, Transforming growth factor-beta and Smad signalling in kidney diseases. Nephrology (Carlton), 2005. 10(1): p. 48-56. 110. Bottinger, E.P., TGF-beta in renal injury and disease. Semin Nephrol, 2007. 27(3): p. 309-20. 111. Roberts, A.B., Molecular and cell biology of TGF-beta. Miner Electrolyte Metab, 1998. 24(2- 3): p. 111-9. 112. Border, W.A., S. Okuda, L.R. Languino, et al., Transforming growth factor-beta regulates production of proteoglycans by mesangial cells. Kidney Int, 1990. 37(2): p. 689-95. 113. Haberstroh, U., G. Zahner, M. Disser, et al., TGF-beta stimulates rat mesangial cell proliferation in culture: role of PDGF beta-receptor expression. Am J Physiol, 1993. 264(2 Pt 2): p. F199-205. 114. Wilson, H.M., F.J. Reid, P.A. Brown, et al., Effect of transforming growth factor-beta 1 on plasminogen activators and plasminogen activator inhibitor-1 in renal glomerular cells. Exp Nephrol, 1993. 1(6): p. 343-50. 115. Lebrin, F., M. Deckers, P. Bertolino, et al., TGF-beta receptor function in the endothelium. Cardiovasc Res, 2005. 65(3): p. 599-608. 116. Das, R., S. Xu, X. Quan, et al., Upregulation of mitochondrial Nox4 mediates TGF-beta- induced apoptosis in cultured mouse podocytes. Am J Physiol Renal Physiol, 2014. 306(2): p. F155-67. 117. Mack, M. and M. Yanagita, Origin of myofibroblasts and cellular events triggering fibrosis. Kidney Int, 2015. 87(2): p. 297-307.
162
118. McGaraughty, S., R.A. Davis-Taber, C.Z. Zhu, et al., Targeting Anti-TGF-beta Therapy to Fibrotic Kidneys with a Dual Specificity Antibody Approach. J Am Soc Nephrol, 2017. 28(12): p. 3616-3626. 119. Murphy, S.R., A.J. Dahly-Vernon, K.M. Dunn, et al., Renoprotective effects of anti-TGF-beta antibody and antihypertensive therapies in Dahl S rats. Am J Physiol Regul Integr Comp Physiol, 2012. 303(1): p. R57-69. 120. Russo, L.M., E. del Re, D. Brown, et al., Evidence for a role of transforming growth factor (TGF)-beta1 in the induction of postglomerular albuminuria in diabetic nephropathy: amelioration by soluble TGF-beta type II receptor. Diabetes, 2007. 56(2): p. 380-8. 121. Moon, J.A., H.T. Kim, I.S. Cho, et al., IN-1130, a novel transforming growth factor-beta type I receptor kinase (ALK5) inhibitor, suppresses renal fibrosis in obstructive nephropathy. Kidney Int, 2006. 70(7): p. 1234-43. 122. Voelker, J., P.H. Berg, M. Sheetz, et al., Anti-TGF-beta1 Antibody Therapy in Patients with Diabetic Nephropathy. J Am Soc Nephrol, 2017. 28(3): p. 953-962. 123. Hill, C.S., The Smads. Int J Biochem Cell Biol, 1999. 31(11): p. 1249-54. 124. Heldin, C.H. and A. Moustakas, Role of Smads in TGFbeta signaling. Cell Tissue Res, 2012. 347(1): p. 21-36. 125. Moustakas, A., S. Souchelnytskyi and C.H. Heldin, Smad regulation in TGF-beta signal transduction. J Cell Sci, 2001. 114(Pt 24): p. 4359-69. 126. Markic, D., T. Celic, J. Spanjol, et al., Expression of bone morphogenetic protein-7, its receptors and Smad1/5/8 in normal human kidney and renal cell cancer. Coll Antropol, 2010. 34 Suppl 2: p. 149-53. 127. Blank, U., M.L. Seto, D.C. Adams, et al., An in vivo reporter of BMP signaling in organogenesis reveals targets in the developing kidney. BMC Dev Biol, 2008. 8: p. 86. 128. Oxburgh, L. and E.J. Robertson, Dynamic regulation of Smad expression during mesenchyme to epithelium transition in the metanephric kidney. Mech Dev, 2002. 112(1-2): p. 207-11. 129. Cao, J., Y. Li, Y. Peng, et al., Febuxostat Prevents Renal Interstitial Fibrosis by the Activation of BMP-7 Signaling and Inhibition of USAG-1 Expression in Rats. Am J Nephrol, 2015. 42(5): p. 369-78. 130. Munoz-Felix, J.M., M. Gonzalez-Nunez and J.M. Lopez-Novoa, ALK1-Smad1/5 signaling pathway in fibrosis development: friend or foe? Cytokine Growth Factor Rev, 2013. 24(6): p. 523-37.
163
131. Li, J.H., X.R. Huang, H.J. Zhu, et al., Advanced glycation end products activate Smad signaling via TGF-beta-dependent and independent mechanisms: implications for diabetic renal and vascular disease. FASEB J, 2004. 18(1): p. 176-8. 132. Chung, A.C., H. Zhang, Y.Z. Kong, et al., Advanced glycation end-products induce tubular CTGF via TGF-beta-independent Smad3 signaling. J Am Soc Nephrol, 2010. 21(2): p. 249-60. 133. Fujimoto, M., Y. Maezawa, K. Yokote, et al., Mice lacking Smad3 are protected against streptozotocin-induced diabetic glomerulopathy. Biochem Biophys Res Commun, 2003. 305(4): p. 1002-7. 134. Isono, M., S. Chen, S. Won Hong, et al., Smad pathway is activated in the diabetic mouse kidney and Smad3 mediates TGF-β-induced fibronectin in mesangial cells. Biochemical and Biophysical Research Communications, 2002. 296(5): p. 1356-1365. 135. Chen, H.Y., X.R. Huang, W. Wang, et al., The protective role of Smad7 in diabetic kidney disease: mechanism and therapeutic potential. Diabetes, 2011. 60(2): p. 590-601. 136. Huang, X.R., A.C. Chung, X.J. Wang, et al., Mice overexpressing latent TGF-beta1 are protected against renal fibrosis in obstructive kidney disease. Am J Physiol Renal Physiol, 2008. 295(1): p. F118-27. 137. Sato, M., Y. Muragaki, S. Saika, et al., Targeted disruption of TGF-beta1/Smad3 signaling protects against renal tubulointerstitial fibrosis induced by unilateral ureteral obstruction. J Clin Invest, 2003. 112(10): p. 1486-94. 138. Lan, H.Y., W. Mu, N. Tomita, et al., Inhibition of renal fibrosis by gene transfer of inducible Smad7 using ultrasound-microbubble system in rat UUO model. J Am Soc Nephrol, 2003. 14(6): p. 1535-48. 139. Terada, Y., S. Hanada, A. Nakao, et al., Gene transfer of Smad7 using electroporation of adenovirus prevents renal fibrosis in post-obstructed kidney. Kidney Int, 2002. 61(1 Suppl): p. S94-8. 140. Chung, A.C., X.R. Huang, L. Zhou, et al., Disruption of the Smad7 gene promotes renal fibrosis and inflammation in unilateral ureteral obstruction (UUO) in mice. Nephrol Dial Transplant, 2009. 24(5): p. 1443-54. 141. Yang, F., X.R. Huang, A.C. Chung, et al., Essential role for Smad3 in angiotensin II-induced tubular epithelial-mesenchymal transition. J Pathol, 2010. 221(4): p. 390-401. 142. Hou, C.C., W. Wang, X.R. Huang, et al., Ultrasound-microbubble-mediated gene transfer of inducible Smad7 blocks transforming growth factor-beta signaling and fibrosis in rat remnant kidney. Am J Pathol, 2005. 166(3): p. 761-71.
164
143. Wang, W., X.R. Huang, E. Canlas, et al., Essential role of Smad3 in angiotensin II-induced vascular fibrosis. Circ Res, 2006. 98(8): p. 1032-9. 144. Huang, X.R., A.C. Chung, L. Zhou, et al., Latent TGF-beta1 protects against crescentic glomerulonephritis. J Am Soc Nephrol, 2008. 19(2): p. 233-42. 145. Ka, S.M., X.R. Huang, H.Y. Lan, et al., Smad7 gene therapy ameliorates an autoimmune crescentic glomerulonephritis in mice. J Am Soc Nephrol, 2007. 18(6): p. 1777-88. 146. Lan, H.Y. and A.C. Chung, TGF-beta/Smad signaling in kidney disease. Semin Nephrol, 2012. 32(3): p. 236-43. 147. Verrecchia, F., M.L. Chu and A. Mauviel, Identification of novel TGF-beta /Smad gene targets in dermal fibroblasts using a combined cDNA microarray/promoter transactivation approach. J Biol Chem, 2001. 276(20): p. 17058-62. 148. Zhou, L., P. Fu, X.R. Huang, et al., Mechanism of chronic aristolochic acid nephropathy: role of Smad3. Am J Physiol Renal Physiol, 2010. 298(4): p. F1006-17. 149. Shao, X., S. Somlo and P. Igarashi, Epithelial-specific Cre/lox recombination in the developing kidney and genitourinary tract. J Am Soc Nephrol, 2002. 13(7): p. 1837-46. 150. Meng, X.M., X.R. Huang, A.C. Chung, et al., Smad2 protects against TGF-beta/Smad3- mediated renal fibrosis. J Am Soc Nephrol, 2010. 21(9): p. 1477-87. 151. Yang, F., A.C. Chung, X.R. Huang, et al., Angiotensin II induces connective tissue growth factor and collagen I expression via transforming growth factor-beta-dependent and -independent Smad pathways: the role of Smad3. Hypertension, 2009. 54(4): p. 877-84. 152. Massague, J. and D. Wotton, Transcriptional control by the TGF-beta/Smad signaling system. EMBO J, 2000. 19(8): p. 1745-54. 153. Meng, X.M., X.R. Huang, J. Xiao, et al., Disruption of Smad4 impairs TGF-beta/Smad3 and Smad7 transcriptional regulation during renal inflammation and fibrosis in vivo and in vitro. Kidney Int, 2012. 81(3): p. 266-79. 154. Afrakhte, M., A. Moren, S. Jossan, et al., Induction of inhibitory Smad6 and Smad7 mRNA by TGF-beta family members. Biochem Biophys Res Commun, 1998. 249(2): p. 505-11. 155. Zhu, H.J., J. Iaria and A.M. Sizeland, Smad7 differentially regulates transforming growth factor beta-mediated signaling pathways. J Biol Chem, 1999. 274(45): p. 32258-64. 156. Kavsak, P., R.K. Rasmussen, C.G. Causing, et al., Smad7 binds to Smurf2 to form an E3 ubiquitin ligase that targets the TGF beta receptor for degradation. Mol Cell, 2000. 6(6): p. 1365-75.
165
157. Ebisawa, T., M. Fukuchi, G. Murakami, et al., Smurf1 interacts with transforming growth factor-beta type I receptor through Smad7 and induces receptor degradation. J Biol Chem, 2001. 276(16): p. 12477-80. 158. Imamura, T., M. Takase, A. Nishihara, et al., Smad6 inhibits signalling by the TGF-beta superfamily. Nature, 1997. 389(6651): p. 622-6. 159. Zhao, J., W. Shi, H. Chen, et al., Smad7 and Smad6 differentially modulate transforming growth factor beta -induced inhibition of embryonic lung morphogenesis. J Biol Chem, 2000. 275(31): p. 23992-7. 160. Liu, X., J. Lee, M. Cooley, et al., Smad7 but not Smad6 cooperates with oncogenic ras to cause malignant conversion in a mouse model for squamous cell carcinoma. Cancer Res, 2003. 63(22): p. 7760-8. 161. Fukasawa, H., T. Yamamoto, A. Togawa, et al., Down-regulation of Smad7 expression by ubiquitin-dependent degradation contributes to renal fibrosis in obstructive nephropathy in mice. Proc Natl Acad Sci U S A, 2004. 101(23): p. 8687-92. 162. Liu, F.Y., X.Z. Li, Y.M. Peng, et al., Arkadia regulates TGF-beta signaling during renal tubular epithelial to mesenchymal cell transition. Kidney Int, 2008. 73(5): p. 588-94. 163. Liu, G.X., Y.Q. Li, X.R. Huang, et al., Disruption of Smad7 promotes ANG II-mediated renal inflammation and fibrosis via Sp1-TGF-beta/Smad3-NF.kappaB-dependent mechanisms in mice. PLoS One, 2013. 8(1): p. e53573. 164. Lee, M.K., C. Pardoux, M.C. Hall, et al., TGF‐β activates Erk MAP kinase signalling through
direct phosphorylation of ShcA. The EMBO Journal, 2007. 26(17): p. 3957-3967. 165. Kretzschmar, M., J. Doody and J. Massague, Opposing BMP and EGF signalling pathways converge on the TGF-beta family mediator Smad1. Nature, 1997. 389(6651): p. 618-22. 166. Kretzschmar, M., J. Doody, I. Timokhina, et al., A mechanism of repression of TGFbeta/ Smad signaling by oncogenic Ras. Genes Dev, 1999. 13(7): p. 804-16. 167. Funaba, M., C.M. Zimmerman and L.S. Mathews, Modulation of Smad2-mediated signaling by extracellular signal-regulated kinase. J Biol Chem, 2002. 277(44): p. 41361-8. 168. Matsuura, I., G. Wang, D. He, et al., Identification and characterization of ERK MAP kinase phosphorylation sites in Smad3. Biochemistry, 2005. 44(37): p. 12546-53. 169. Zhang, M., D. Fraser and A. Phillips, ERK, p38, and Smad signaling pathways differentially regulate transforming growth factor-beta1 autoinduction in proximal tubular epithelial cells. Am J Pathol, 2006. 169(4): p. 1282-93. 170. Jaffe, A.B. and A. Hall, Rho GTPases: biochemistry and biology. Annu Rev Cell Dev Biol, 2005. 21: p. 247-69.
166
171. Bhowmick, N.A., M. Ghiassi, A. Bakin, et al., Transforming growth factor-beta1 mediates epithelial to mesenchymal transdifferentiation through a RhoA-dependent mechanism. Mol Biol Cell, 2001. 12(1): p. 27-36. 172. Edlund, S., M. Landstrom, C.H. Heldin, et al., Transforming growth factor-beta-induced mobilization of actin cytoskeleton requires signaling by small GTPases Cdc42 and RhoA. Mol Biol Cell, 2002. 13(3): p. 902-14. 173. Frey, R.S. and K.M. Mulder, Involvement of extracellular signal-regulated kinase 2 and stress- activated protein kinase/Jun N-terminal kinase activation by transforming growth factor beta in the negative growth control of breast cancer cells. Cancer Res, 1997. 57(4): p. 628-33. 174. Engel, M.E., M.A. McDonnell, B.K. Law, et al., Interdependent SMAD and JNK signaling in transforming growth factor-beta-mediated transcription. J Biol Chem, 1999. 274(52): p. 37413-20. 175. Hocevar, B.A., T.L. Brown and P.H. Howe, TGF-beta induces fibronectin synthesis through a c- Jun N-terminal kinase-dependent, Smad4-independent pathway. EMBO J, 1999. 18(5): p. 1345-56. 176. Hanafusa, H., J. Ninomiya-Tsuji, N. Masuyama, et al., Involvement of the p38 mitogen- activated protein kinase pathway in transforming growth factor-beta-induced gene expression. J Biol Chem, 1999. 274(38): p. 27161-7. 177. Sano, Y., J. Harada, S. Tashiro, et al., ATF-2 is a common nuclear target of Smad and TAK1 pathways in transforming growth factor-beta signaling. J Biol Chem, 1999. 274(13): p. 8949- 57. 178. Bhowmick, N.A., R. Zent, M. Ghiassi, et al., Integrin beta 1 signaling is necessary for transforming growth factor-beta activation of p38MAPK and epithelial plasticity. J Biol Chem, 2001. 276(50): p. 46707-13. 179. Yu, L., M.C. Hebert and Y.E. Zhang, TGF-beta receptor-activated p38 MAP kinase mediates Smad-independent TGF-beta responses. EMBO J, 2002. 21(14): p. 3749-59. 180. Shibuya, H., H. Iwata, N. Masuyama, et al., Role of TAK1 and TAB1 in BMP signaling in early Xenopus development. EMBO J, 1998. 17(4): p. 1019-28. 181. Yamashita, M., K. Fatyol, C. Jin, et al., TRAF6 mediates Smad-independent activation of JNK and p38 by TGF-beta. Mol Cell, 2008. 31(6): p. 918-24. 182. Edlund, S., S. Bu, N. Schuster, et al., Transforming growth factor-beta1 (TGF-beta)-induced apoptosis of prostate cancer cells involves Smad7-dependent activation of p38 by TGF-beta- activated kinase 1 and mitogen-activated protein kinase kinase 3. Mol Biol Cell, 2003. 14(2): p. 529-44.
167
183. Liao, J.H., J.S. Chen, M.Q. Chai, et al., The involvement of p38 MAPK in transforming growth factor beta1-induced apoptosis in murine hepatocytes. Cell Res, 2001. 11(2): p. 89-94. 184. Bakin, A.V., C. Rinehart, A.K. Tomlinson, et al., p38 mitogen-activated protein kinase is required for TGFbeta-mediated fibroblastic transdifferentiation and cell migration. J Cell Sci, 2002. 115(Pt 15): p. 3193-206. 185. Zhang, Y.E., Non-Smad pathways in TGF-beta signaling. Cell Res, 2009. 19(1): p. 128-39. 186. Grynberg, K., F.Y. Ma and D.J. Nikolic-Paterson, The JNK Signaling Pathway in Renal Fibrosis. Front Physiol, 2017. 8: p. 829. 187. Anders, H.J., Innate versus adaptive immunity in kidney immunopathology. BMC Nephrol, 2013. 14: p. 138. 188. Navarro-Gonzalez, J.F., C. Mora-Fernandez, M. Muros de Fuentes, et al., Inflammatory molecules and pathways in the pathogenesis of diabetic nephropathy. Nat Rev Nephrol, 2011. 7(6): p. 327-40. 189. Swenson-Fields, K.I., C.J. Vivian, S.M. Salah, et al., Macrophages promote polycystic kidney disease progression. Kidney Int, 2013. 83(5): p. 855-64. 190. Inoue, M. and M.L. Shinohara, NLRP3 Inflammasome and MS/EAE. Autoimmune Dis, 2013. 2013: p. 859145. 191. Schroder, K. and J. Tschopp, The inflammasomes. Cell, 2010. 140(6): p. 821-32. 192. Shao, B.Z., Z.Q. Xu, B.Z. Han, et al., NLRP3 inflammasome and its inhibitors: a review. Front Pharmacol, 2015. 6: p. 262. 193. Wang, W., X. Wang, J. Chun, et al., Inflammasome-independent NLRP3 augments TGF-beta signaling in kidney epithelium. J Immunol, 2013. 190(3): p. 1239-49. 194. Iyer, S.S., W.P. Pulskens, J.J. Sadler, et al., Necrotic cells trigger a sterile inflammatory response through the Nlrp3 inflammasome. Proc Natl Acad Sci U S A, 2009. 106(48): p. 20388-93. 195. Shigeoka, A.A., J.L. Mueller, A. Kambo, et al., An inflammasome-independent role for epithelial-expressed Nlrp3 in renal ischemia-reperfusion injury. J Immunol, 2010. 185(10): p. 6277-85. 196. Haq, M., J. Norman, S.R. Saba, et al., Role of IL-1 in renal ischemic reperfusion injury. J Am Soc Nephrol, 1998. 9(4): p. 614-9. 197. Anders, H.J. and D.A. Muruve, The inflammasomes in kidney disease. J Am Soc Nephrol, 2011. 22(6): p. 1007-18. 198. Wu, H., M.L. Craft, P. Wang, et al., IL-18 contributes to renal damage after ischemia- reperfusion. J Am Soc Nephrol, 2008. 19(12): p. 2331-41.
168
199. Daemen, M.A., C. van't Veer, T.G. Wolfs, et al., Ischemia/reperfusion-induced IFN-gamma up- regulation: involvement of IL-12 and IL-18. J Immunol, 1999. 162(9): p. 5506-10. 200. Kalluri, R. and R.A. Weinberg, The basics of epithelial-mesenchymal transition. J Clin Invest, 2009. 119(6): p. 1420-8. 201. Lorenz, G., M.N. Darisipudi and H.J. Anders, Canonical and non-canonical effects of the NLRP3 inflammasome in kidney inflammation and fibrosis. Nephrol Dial Transplant, 2014. 29(1): p. 41-8. 202. Sheedy, F.J. and L.A. O'Neill, The Troll in Toll: Mal and Tram as bridges for TLR2 and TLR4 signaling. J Leukoc Biol, 2007. 82(2): p. 196-203. 203. Beutler, B., Inferences, questions and possibilities in Toll-like receptor signalling. Nature, 2004. 430(6996): p. 257-63. 204. Kawai, T. and S. Akira, The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors. Nat Immunol, 2010. 11(5): p. 373-84. 205. Moresco, E.M.Y., D. LaVine and B. Beutler, Toll-like receptors. Current Biology, 2011. 21(13): p. R488-R493. 206. Yiu, W.H., M. Lin and S.C. Tang, Toll-like receptor activation: from renal inflammation to fibrosis. Kidney Int Suppl (2011), 2014. 4(1): p. 20-25. 207. Watts, C., M.A. West and R. Zaru, TLR signalling regulated antigen presentation in dendritic cells. Curr Opin Immunol, 2010. 22(1): p. 124-30. 208. Kokkinopoulos, I., W.J. Jordan and M.A. Ritter, Toll-like receptor mRNA expression patterns in human dendritic cells and monocytes. Mol Immunol, 2005. 42(8): p. 957-68. 209. Anders, H.J., B. Banas and D. Schlondorff, Signaling danger: toll-like receptors and their potential roles in kidney disease. J Am Soc Nephrol, 2004. 15(4): p. 854-67. 210. Devaraj, S., M.R. Dasu, J. Rockwood, et al., Increased toll-like receptor (TLR) 2 and TLR4 expression in monocytes from patients with type 1 diabetes: further evidence of a proinflammatory state. J Clin Endocrinol Metab, 2008. 93(2): p. 578-83. 211. Devaraj, S., M.R. Dasu, S.H. Park, et al., Increased levels of ligands of Toll-like receptors 2 and 4 in type 1 diabetes. Diabetologia, 2009. 52(8): p. 1665-8. 212. Dasu, M.R., S. Devaraj, S. Park, et al., Increased toll-like receptor (TLR) activation and TLR ligands in recently diagnosed type 2 diabetic subjects. Diabetes Care, 2010. 33(4): p. 861-8. 213. Banas, M.C., B. Banas, K.L. Hudkins, et al., TLR4 links podocytes with the innate immune system to mediate glomerular injury. J Am Soc Nephrol, 2008. 19(4): p. 704-13. 214. Wolf, G., J. Bohlender, T. Bondeva, et al., Angiotensin II upregulates toll-like receptor 4 on mesangial cells. J Am Soc Nephrol, 2006. 17(6): p. 1585-93.
169
215. Kaur, H., A. Chien and I. Jialal, Hyperglycemia induces Toll like receptor 4 expression and activity in mouse mesangial cells: relevance to diabetic nephropathy. Am J Physiol Renal Physiol, 2012. 303(8): p. F1145-50. 216. Chen, J., R. John, J.A. Richardson, et al., Toll-like receptor 4 regulates early endothelial activation during ischemic acute kidney injury. Kidney Int, 2011. 79(3): p. 288-99. 217. Takata, S., Y. Sawa, T. Uchiyama, et al., Expression of Toll-Like Receptor 4 in Glomerular Endothelial Cells under Diabetic Conditions. Acta Histochem Cytochem, 2013. 46(1): p. 35-42. 218. Summers, S.A., B.S. van der Veen, K.M. O'Sullivan, et al., Intrinsic renal cell and leukocyte- derived TLR4 aggravate experimental anti-MPO glomerulonephritis. Kidney Int, 2010. 78(12): p. 1263-74. 219. Kruger, B., S. Krick, N. Dhillon, et al., Donor Toll-like receptor 4 contributes to ischemia and reperfusion injury following human kidney transplantation. Proc Natl Acad Sci U S A, 2009. 106(9): p. 3390-5. 220. Wu, H., G. Chen, K.R. Wyburn, et al., TLR4 activation mediates kidney ischemia/reperfusion injury. J Clin Invest, 2007. 117(10): p. 2847-59. 221. Pulskens, W.P., G.J. Teske, L.M. Butter, et al., Toll-like receptor-4 coordinates the innate immune response of the kidney to renal ischemia/reperfusion injury. PLoS One, 2008. 3(10): p. e3596. 222. Pulskens, W.P., E. Rampanelli, G.J. Teske, et al., TLR4 promotes fibrosis but attenuates tubular damage in progressive renal injury. J Am Soc Nephrol, 2010. 21(8): p. 1299-308. 223. Sorensen, I., N. Susnik, T. Inhester, et al., Fibrinogen, acting as a mitogen for tubulointerstitial fibroblasts, promotes renal fibrosis. Kidney Int, 2011. 80(10): p. 1035-44. 224. Campbell, M.T., K.L. Hile, H. Zhang, et al., Toll-like receptor 4: a novel signaling pathway during renal fibrogenesis. J Surg Res, 2011. 168(1): p. e61-9. 225. Correa-Costa, M., T.T. Braga, P. Semedo, et al., Pivotal role of Toll-like receptors 2 and 4, its adaptor molecule MyD88, and inflammasome complex in experimental tubule-interstitial nephritis. PLoS One, 2011. 6(12): p. e29004. 226. Zhang, B., G. Ramesh, S. Uematsu, et al., TLR4 signaling mediates inflammation and tissue injury in nephrotoxicity. J Am Soc Nephrol, 2008. 19(5): p. 923-32. 227. Lim, B.J., S.W. Hong and H.J. Jeong, Renal tubular expression of Toll-like receptor 4 in cyclosporine nephrotoxicity. APMIS, 2009. 117(8): p. 583-91. 228. Kim, J., K.E. Long, K. Tang, et al., Poly(ADP-ribose) polymerase 1 activation is required for cisplatin nephrotoxicity. Kidney Int, 2012. 82(2): p. 193-203.
170
229. Lin, M., W.H. Yiu, H.J. Wu, et al., Toll-like receptor 4 promotes tubular inflammation in diabetic nephropathy. J Am Soc Nephrol, 2012. 23(1): p. 86-102. 230. Li, F., N. Yang, L. Zhang, et al., Increased expression of toll-like receptor 2 in rat diabetic nephropathy. Am J Nephrol, 2010. 32(2): p. 179-86. 231. Leemans, J.C., L.M. Butter, W.P. Pulskens, et al., The role of Toll-like receptor 2 in inflammation and fibrosis during progressive renal injury. PLoS One, 2009. 4(5): p. e5704. 232. Skuginna, V., M. Lech, R. Allam, et al., Toll-like receptor signaling and SIGIRR in renal fibrosis upon unilateral ureteral obstruction. PLoS One, 2011. 6(4): p. e19204. 233. Devaraj, S., P. Tobias, B.S. Kasinath, et al., Knockout of toll-like receptor-2 attenuates both the proinflammatory state of diabetes and incipient diabetic nephropathy. Arterioscler Thromb Vasc Biol, 2011. 31(8): p. 1796-804. 234. Lin, M. and S.C. Tang, Toll-like receptors: sensing and reacting to diabetic injury in the kidney. Nephrol Dial Transplant, 2014. 29(4): p. 746-54. 235. Lv, W., G.W. Booz, Y. Wang, et al., Inflammation and renal fibrosis: Recent developments on key signaling molecules as potential therapeutic targets. Eur J Pharmacol, 2018. 820: p. 65- 76. 236. Idasiak-Piechocka, I., A. Oko, E. Pawliczak, et al., Urinary excretion of soluble tumour necrosis factor receptor 1 as a marker of increased risk of progressive kidney function deterioration in patients with primary chronic glomerulonephritis. Nephrol Dial Transplant, 2010. 25(12): p. 3948-56. 237. Monaco, C., J. Nanchahal, P. Taylor, et al., Anti-TNF therapy: past, present and future. Int Immunol, 2015. 27(1): p. 55-62. 238. Carlos, C.P., N.M. Sonehara, S.M. Oliani, et al., Predictive usefulness of urinary biomarkers for the identification of cyclosporine A-induced nephrotoxicity in a rat model. PLoS One, 2014. 9(7): p. e103660. 239. Moriwaki, Y., T. Inokuchi, A. Yamamoto, et al., Effect of TNF-alpha inhibition on urinary albumin excretion in experimental diabetic rats. Acta Diabetol, 2007. 44(4): p. 215-8. 240. Omote, K., T. Gohda, M. Murakoshi, et al., Role of the TNF pathway in the progression of diabetic nephropathy in KK-A(y) mice. Am J Physiol Renal Physiol, 2014. 306(11): p. F1335-47. 241. Santana, A.C., S. Degaspari, S. Catanozi, et al., Thalidomide suppresses inflammation in adenine-induced CKD with uraemia in mice. Nephrol Dial Transplant, 2013. 28(5): p. 1140-9. 242. Quiroga, B., D. Arroyo and G. de Arriba, Present and future in the treatment of diabetic kidney disease. J Diabetes Res, 2015. 2015: p. 801348.
171
243. Gohda, T., M.A. Niewczas, L.H. Ficociello, et al., Circulating TNF receptors 1 and 2 predict stage 3 CKD in type 1 diabetes. J Am Soc Nephrol, 2012. 23(3): p. 516-24. 244. Niewczas, M.A., T. Gohda, J. Skupien, et al., Circulating TNF receptors 1 and 2 predict ESRD in type 2 diabetes. J Am Soc Nephrol, 2012. 23(3): p. 507-15. 245. Fink, M., M. Henry and J.D. Tange, Experimental folic acid nephropathy. Pathology, 1987. 19(2): p. 143-9. 246. Soofi, A., P. Zhang and G.R. Dressler, Kielin/chordin-like protein attenuates both acute and chronic renal injury. J Am Soc Nephrol, 2013. 24(6): p. 897-905. 247. Li, X., J. Dai, Y. Qiu, et al., 4-1BB-mediated signals confer protection against folic acid-induced nephrotoxicity. Kidney Blood Press Res, 2012. 36(1): p. 10-7. 248. Vasko, R., S. Xavier, J. Chen, et al., Endothelial sirtuin 1 deficiency perpetrates nephrosclerosis through downregulation of matrix metalloproteinase-14: relevance to fibrosis of vascular senescence. J Am Soc Nephrol, 2014. 25(2): p. 276-91. 249. Jiang, C., Q. Shao, B. Jin, et al., Tanshinone IIA Attenuates Renal Fibrosis after Acute Kidney Injury in a Mouse Model through Inhibition of Fibrocytes Recruitment. Biomed Res Int, 2015. 2015: p. 867140. 250. Grande, M.T., B. Sanchez-Laorden, C. Lopez-Blau, et al., Snail1-induced partial epithelial-to- mesenchymal transition drives renal fibrosis in mice and can be targeted to reverse established disease. Nat Med, 2015. 21(9): p. 989-97. 251. He, S., N. Liu, G. Bayliss, et al., EGFR activity is required for renal tubular cell dedifferentiation and proliferation in a murine model of folic acid-induced acute kidney injury. Am J Physiol Renal Physiol, 2013. 304(4): p. F356-66. 252. Stallons, L.J., R.M. Whitaker and R.G. Schnellmann, Suppressed mitochondrial biogenesis in folic acid-induced acute kidney injury and early fibrosis. Toxicol Lett, 2014. 224(3): p. 326-32. 253. Yang, H.C., Y. Zuo and A.B. Fogo, Models of chronic kidney disease. Drug Discov Today Dis Models, 2010. 7(1-2): p. 13-19. 254. Santos, S., R.J. Bosch, A. Ortega, et al., Up-regulation of parathyroid hormone-related protein in folic acid-induced acute renal failure. Kidney Int, 2001. 60(3): p. 982-95. 255. Rysz, J., A. Gluba-Brzozka, B. Franczyk, et al., Novel Biomarkers in the Diagnosis of Chronic Kidney Disease and the Prediction of Its Outcome. Int J Mol Sci, 2017. 18(8). 256. Lee, T.N., W.E. Alborn, M.D. Knierman, et al., The diabetogenic antibiotic streptozotocin modifies the tryptic digest pattern for peptides of the enzyme O-GlcNAc-selective N-acetyl- beta-d-glucosaminidase that contain amino acid residues essential for enzymatic activity. Biochem Pharmacol, 2006. 72(6): p. 710-8.
172
257. Huang, C., S. Shen, Q. Ma, et al., Blockade of KCa3.1 ameliorates renal fibrosis through the TGF-beta1/Smad pathway in diabetic mice. Diabetes, 2013. 62(8): p. 2923-34. 258. Ma, J., S.J. Chadban, C.Y. Zhao, et al., TLR4 activation promotes podocyte injury and interstitial fibrosis in diabetic nephropathy. PLoS One, 2014. 9(5): p. e97985. 259. Kitada, M., Y. Ogura and D. Koya, Rodent models of diabetic nephropathy: their utility and limitations. Int J Nephrol Renovasc Dis, 2016. 9: p. 279-290. 260. Tay, Y.C., Y. Wang, L. Kairaitis, et al., Can murine diabetic nephropathy be separated from superimposed acute renal failure? Kidney Int, 2005. 68(1): p. 391-8. 261. Kraynak, A.R., R.D. Storer, R.D. Jensen, et al., Extent and persistence of streptozotocin- induced DNA damage and cell proliferation in rat kidney as determined by in vivo alkaline elution and BrdUrd labeling assays. Toxicol Appl Pharmacol, 1995. 135(2): p. 279-86. 262. Tesch, G.H. and T.J. Allen, Rodent models of streptozotocin-induced diabetic nephropathy. Nephrology (Carlton), 2007. 12(3): p. 261-6. 263. Breyer, M.D., E. Bottinger, F.C. Brosius, 3rd, et al., Mouse models of diabetic nephropathy. J Am Soc Nephrol, 2005. 16(1): p. 27-45. 264. Sugimoto, H., G. Grahovac, M. Zeisberg, et al., Renal fibrosis and glomerulosclerosis in a new mouse model of diabetic nephropathy and its regression by bone morphogenic protein-7 and advanced glycation end product inhibitors. Diabetes, 2007. 56(7): p. 1825-33. 265. Xu, J. and M.H. Zou, Molecular insights and therapeutic targets for diabetic endothelial dysfunction. Circulation, 2009. 120(13): p. 1266-86. 266. Rask-Madsen, C. and G.L. King, Mechanisms of Disease: endothelial dysfunction in insulin resistance and diabetes. Nat Clin Pract Endocrinol Metab, 2007. 3(1): p. 46-56. 267. Forstermann, U. and W.C. Sessa, Nitric oxide synthases: regulation and function. Eur Heart J, 2012. 33(7): p. 829-37, 837a-837d. 268. Massion, P.B. and J.L. Balligand, Modulation of cardiac contraction, relaxation and rate by the endothelial nitric oxide synthase (eNOS): lessons from genetically modified mice. J Physiol, 2003. 546(Pt 1): p. 63-75. 269. Takahashi, T. and R.C. Harris, Role of endothelial nitric oxide synthase in diabetic nephropathy: lessons from diabetic eNOS knockout mice. J Diabetes Res, 2014. 2014: p. 590541. 270. Mohan, S., R.L. Reddick, N. Musi, et al., Diabetic eNOS knockout mice develop distinct macro- and microvascular complications. Lab Invest, 2008. 88(5): p. 515-28. 271. Yokozawa, T., P.D. Zheng, H. Oura, et al., Animal model of adenine-induced chronic renal failure in rats. Nephron, 1986. 44(3): p. 230-4.
173
272. Ali, B.H., S. Al-Salam, I. Al Husseni, et al., Effects of Gum Arabic in rats with adenine-induced chronic renal failure. Exp Biol Med (Maywood), 2010. 235(3): p. 373-82. 273. Ali, B.H., S. Al-Salam, M. Al Za'abi, et al., New model for adenine-induced chronic renal failure in mice, and the effect of gum acacia treatment thereon: comparison with rats. J Pharmacol Toxicol Methods, 2013. 68(3): p. 384-93. 274. Rahman, A., D. Yamazaki, A. Sufiun, et al., A novel approach to adenine-induced chronic kidney disease associated anemia in rodents. PLoS One, 2018. 13(2): p. e0192531. 275. Okada, H., Y. Kaneko, T. Yawata, et al., Reversibility of adenine-induced renal failure in rats. Clinical and Experimental Nephrology, 1999. 3(2): p. 82-88. 276. Diwan, V., L. Brown and G.C. Gobe, Adenine-induced chronic kidney disease in rats. Nephrology (Carlton), 2018. 23(1): p. 5-11. 277. Fogo, A.B., Hypertensive risk factors in kidney disease in African Americans. Kidney Int Suppl, 2003(83): p. S17-21. 278. Ma, L.J., S. Nakamura, J.C. Aldigier, et al., Regression of glomerulosclerosis with high-dose angiotensin inhibition is linked to decreased plasminogen activator inhibitor-1. J Am Soc Nephrol, 2005. 16(4): p. 966-76. 279. Chevalier, R.L., M.S. Forbes and B.A. Thornhill, Ureteral obstruction as a model of renal interstitial fibrosis and obstructive nephropathy. Kidney Int, 2009. 75(11): p. 1145-1152. 280. Oliveira-Silva, G.L., I.B.M. Morais, J. Fortunato-Silva, et al., Testosterone and Mast Cell Interactions in the Development of Kidney Fibrosis after Unilateral Ureteral Obstruction in Rats. Biol Pharm Bull, 2018. 41(8): p. 1164-1169. 281. Xi, W., X. Zhao, M. Wu, et al., Lack of microRNA-155 ameliorates renal fibrosis by targeting PDE3A/TGF-beta1/Smad signaling in mice with obstructive nephropathy. Cell Biol Int, 2018. 282. Chevalier, R.L., Obstructive nephropathy: towards biomarker discovery and gene therapy. Nat Clin Pract Nephrol, 2006. 2(3): p. 157-68. 283. Josephson, S., B. Robertson, G. Claesson, et al., Experimental obstructive hydronephrosis in newborn rats. I. Surgical technique and long-term morphologic effects. Invest Urol, 1980. 17(6): p. 478-83. 284. Brosius, F.C., 3rd, C.E. Alpers, E.P. Bottinger, et al., Mouse models of diabetic nephropathy. J Am Soc Nephrol, 2009. 20(12): p. 2503-12. 285. Zhang, D., J. Lin and J. Han, Receptor-interacting protein (RIP) kinase family. Cell Mol Immunol, 2010. 7(4): p. 243-9. 286. Christofferson, D.E., Y. Li and J. Yuan, Control of life-or-death decisions by RIP1 kinase. Annu Rev Physiol, 2014. 76: p. 129-50.
174
287. Newton, K., RIPK1 and RIPK3: critical regulators of inflammation and cell death. Trends Cell Biol, 2015. 288. Sun, X., J. Lee, T. Navas, et al., RIP3, a novel apoptosis-inducing kinase. J Biol Chem, 1999. 274(24): p. 16871-5. 289. Yu, P.W., B.C. Huang, M. Shen, et al., Identification of RIP3, a RIP-like kinase that activates apoptosis and NFkappaB. Curr Biol, 1999. 9(10): p. 539-42. 290. Sun, X., J. Yin, M.A. Starovasnik, et al., Identification of a novel homotypic interaction motif required for the phosphorylation of receptor-interacting protein (RIP) by RIP3. J Biol Chem, 2002. 277(11): p. 9505-11. 291. Sun, L., H. Wang, Z. Wang, et al., Mixed lineage kinase domain-like protein mediates necrosis signaling downstream of RIP3 kinase. Cell, 2012. 148(1-2): p. 213-27. 292. Chen, W., Z. Zhou, L. Li, et al., Diverse sequence determinants control human and mouse receptor interacting protein 3 (RIP3) and mixed lineage kinase domain-like (MLKL) interaction in necroptotic signaling. J Biol Chem, 2013. 288(23): p. 16247-61. 293. McQuade, T., Y. Cho and F.K. Chan, Positive and negative phosphorylation regulates RIP1- and RIP3-induced programmed necrosis. Biochem J, 2013. 456(3): p. 409-15. 294. He, S., L. Wang, L. Miao, et al., Receptor interacting protein kinase-3 determines cellular necrotic response to TNF-alpha. Cell, 2009. 137(6): p. 1100-11. 295. Shlomovitz, I., S. Zargrian and M. Gerlic, Mechanisms of RIPK3-induced inflammation. Immunol Cell Biol, 2017. 95(2): p. 166-172. 296. Zong, W.X. and C.B. Thompson, Necrotic death as a cell fate. Genes Dev, 2006. 20(1): p. 1-15. 297. Vandenabeele, P., L. Galluzzi, T. Vanden Berghe, et al., Molecular mechanisms of necroptosis: an ordered cellular explosion. Nat Rev Mol Cell Biol, 2010. 11(10): p. 700-14. 298. Festjens, N., T. Vanden Berghe and P. Vandenabeele, Necrosis, a well-orchestrated form of cell demise: signalling cascades, important mediators and concomitant immune response. Biochim Biophys Acta, 2006. 1757(9-10): p. 1371-87. 299. Proskuryakov, S.Y., A.G. Konoplyannikov and V.L. Gabai, Necrosis: a specific form of programmed cell death? Exp Cell Res, 2003. 283(1): p. 1-16. 300. Vandenabeele, P., W. Declercq, F. Van Herreweghe, et al., The role of the kinases RIP1 and RIP3 in TNF-induced necrosis. Sci Signal, 2010. 3(115): p. re4. 301. Li, J., T. McQuade, A.B. Siemer, et al., The RIP1/RIP3 necrosome forms a functional amyloid signaling complex required for programmed necrosis. Cell, 2012. 150(2): p. 339-50.
175
302. Feoktistova, M., P. Geserick, B. Kellert, et al., cIAPs block Ripoptosome formation, a RIP1/caspase-8 containing intracellular cell death complex differentially regulated by cFLIP isoforms. Mol Cell, 2011. 43(3): p. 449-63. 303. Geserick, P., M. Hupe, M. Moulin, et al., Cellular IAPs inhibit a cryptic CD95-induced cell death by limiting RIP1 kinase recruitment. J Cell Biol, 2009. 187(7): p. 1037-54. 304. Oberst, A., C.P. Dillon, R. Weinlich, et al., Catalytic activity of the caspase-8-FLIP(L) complex inhibits RIPK3-dependent necrosis. Nature, 2011. 471(7338): p. 363-7. 305. Orozco, S., N. Yatim, M.R. Werner, et al., RIPK1 both positively and negatively regulates RIPK3 oligomerization and necroptosis. Cell Death Differ, 2014. 21(10): p. 1511-21. 306. Cho, Y.S., S. Challa, D. Moquin, et al., Phosphorylation-driven assembly of the RIP1-RIP3 complex regulates programmed necrosis and virus-induced inflammation. Cell, 2009. 137(6): p. 1112-23. 307. Zhang, D.W., J. Shao, J. Lin, et al., RIP3, an energy metabolism regulator that switches TNF- induced cell death from apoptosis to necrosis. Science, 2009. 325(5938): p. 332-6. 308. Wilson, N.S., V. Dixit and A. Ashkenazi, Death receptor signal transducers: nodes of coordination in immune signaling networks. Nat Immunol, 2009. 10(4): p. 348-55. 309. Upton, J.W., W.J. Kaiser and E.S. Mocarski, Virus inhibition of RIP3-dependent necrosis. Cell Host Microbe, 2010. 7(4): p. 302-13. 310. Upton, J.W., W.J. Kaiser and E.S. Mocarski, DAI/ZBP1/DLM-1 complexes with RIP3 to mediate virus-induced programmed necrosis that is targeted by murine cytomegalovirus vIRA. Cell Host Microbe, 2012. 11(3): p. 290-7. 311. Wang, X., Y. Li, S. Liu, et al., Direct activation of RIP3/MLKL-dependent necrosis by herpes simplex virus 1 (HSV-1) protein ICP6 triggers host antiviral defense. Proc Natl Acad Sci U S A, 2014. 111(43): p. 15438-43. 312. Lee, Y.S., K.M. Park, L. Yu, et al., Necroptosis Is a Mechanism of Death in Mouse Induced Hepatocyte-Like Cells Reprogrammed from Mouse Embryonic Fibroblasts. Mol Cells, 2018. 41(7): p. 639-645. 313. Dionisio, P.E.A., S.R. Oliveira, J. Amaral, et al., Loss of Microglial Parkin Inhibits Necroptosis and Contributes to Neuroinflammation. Mol Neurobiol, 2018. 314. Hong, J.M., S.J. Kim and S.M. Lee, Role of necroptosis in autophagy signaling during hepatic ischemia and reperfusion. Toxicol Appl Pharmacol, 2016. 308: p. 1-10. 315. Newton, K., RIPK1 and RIPK3: critical regulators of inflammation and cell death. Trends Cell Biol, 2015. 25(6): p. 347-53.
176
316. Newton, K., D.L. Dugger, K.E. Wickliffe, et al., Activity of protein kinase RIPK3 determines whether cells die by necroptosis or apoptosis. Science, 2014. 343(6177): p. 1357-60. 317. Mandal, P., S.B. Berger, S. Pillay, et al., RIP3 induces apoptosis independent of pronecrotic kinase activity. Mol Cell, 2014. 56(4): p. 481-95. 318. Kang, T.B., S.H. Yang, B. Toth, et al., Caspase-8 blocks kinase RIPK3-mediated activation of the NLRP3 inflammasome. Immunity, 2013. 38(1): p. 27-40. 319. Sureshbabu, A., E. Patino, K.C. Ma, et al., RIPK3 promotes sepsis-induced acute kidney injury via mitochondrial dysfunction. JCI Insight, 2018. 3(11). 320. Martin-Sanchez, D., O. Ruiz-Andres, J. Poveda, et al., Ferroptosis, but Not Necroptosis, Is Important in Nephrotoxic Folic Acid-Induced AKI. J Am Soc Nephrol, 2017. 28(1): p. 218-229. 321. Lau, A., S. Wang, J. Jiang, et al., RIPK3-mediated necroptosis promotes donor kidney inflammatory injury and reduces allograft survival. Am J Transplant, 2013. 13(11): p. 2805- 18. 322. Kaiser, W.J., H. Sridharan, C. Huang, et al., Toll-like receptor 3-mediated necrosis via TRIF, RIP3, and MLKL. J Biol Chem, 2013. 288(43): p. 31268-79. 323. Yang, X.S., T.L. Yi, S. Zhang, et al., Hypoxia-inducible factor-1 alpha is involved in RIP-induced necroptosis caused by in vitro and in vivo ischemic brain injury. Sci Rep, 2017. 7(1): p. 5818. 324. Weng, D., R. Marty-Roix, S. Ganesan, et al., Caspase-8 and RIP kinases regulate bacteria- induced innate immune responses and cell death. Proc Natl Acad Sci U S A, 2014. 111(20): p. 7391-6. 325. Kaiser, W.J., L.P. Daley-Bauer, R.J. Thapa, et al., RIP1 suppresses innate immune necrotic as well as apoptotic cell death during mammalian parturition. Proc Natl Acad Sci U S A, 2014. 111(21): p. 7753-8. 326. Li, J.X., J.M. Feng, Y. Wang, et al., The B-Raf(V600E) inhibitor dabrafenib selectively inhibits RIP3 and alleviates acetaminophen-induced liver injury. Cell Death Dis, 2014. 5: p. e1278. 327. Cruz, S.A., Z. Qin, A.F.R. Stewart, et al., Dabrafenib, an inhibitor of RIP3 kinase-dependent necroptosis, reduces ischemic brain injury. Neural Regen Res, 2018. 13(2): p. 252-256. 328. Spagnolo, F., P. Ghiorzo and P. Queirolo, Overcoming resistance to BRAF inhibition in BRAF- mutated metastatic melanoma. Oncotarget, 2014. 5(21): p. 10206-21. 329. Odogwu, L., L. Mathieu, G. Blumenthal, et al., FDA Approval Summary: Dabrafenib and Trametinib for the Treatment of Metastatic Non-Small Cell Lung Cancers Harboring BRAF V600E Mutations. Oncologist, 2018.
177
330. Buchholz, B., B. Klanke, G. Schley, et al., The Raf kinase inhibitor PLX5568 slows cyst proliferation in rat polycystic kidney disease but promotes renal and hepatic fibrosis. Nephrol Dial Transplant, 2011. 26(11): p. 3458-65. 331. Gerarduzzi, C., Q. He, B. Zhai, et al., Prostaglandin E2-Dependent Phosphorylation of RAS Inhibition 1 (RIN1) at Ser 291 and 292 Inhibits Transforming Growth Factor-beta-Induced RAS Activation Pathway in Human Synovial Fibroblasts: Role in Cell Migration. J Cell Physiol, 2017. 232(1): p. 202-15. 332. Francois, H. and C. Chatziantoniou, Renal fibrosis: Recent translational aspects. Matrix Biol, 2017. 333. Panchapakesan, U. and C. Pollock, The role of toll-like receptors in diabetic kidney disease. Curr Opin Nephrol Hypertens, 2018. 27(1): p. 30-34. 334. Garibotto, G., A. Carta, D. Picciotto, et al., Toll-like receptor-4 signaling mediates inflammation and tissue injury in diabetic nephropathy. J Nephrol, 2017. 30(6): p. 719-727. 335. Yang, S., J. Zhang, S. Wang, et al., SOCS2 overexpression alleviates diabetic nephropathy in rats by inhibiting the TLR4/NF-kappaB pathway. Oncotarget, 2017. 8(53): p. 91185-91198. 336. Wang, S., Y. Li, J. Fan, et al., Interleukin-22 ameliorated renal injury and fibrosis in diabetic nephropathy through inhibition of NLRP3 inflammasome activation. Cell Death Dis, 2017. 8(7): p. e2937. 337. Anders, H.J., B. Suarez-Alvarez, M. Grigorescu, et al., The macrophage phenotype and inflammasome component NLRP3 contributes to nephrocalcinosis-related chronic kidney disease independent from IL-1-mediated tissue injury. Kidney Int, 2018. 93(3): p. 656-669. 338. Zheng, L., J. Zhang, X. Yuan, et al., Fluorofenidone Attenuates IL-1beta Production by Interacting with NLRP3 Inflammasome in Unilateral Ureteral Obstruction. Nephrology (Carlton), 2017. 339. Pulskens, W.P., L.M. Butter, G.J. Teske, et al., Nlrp3 prevents early renal interstitial edema and vascular permeability in unilateral ureteral obstruction. PLoS One, 2014. 9(1): p. e85775. 340. Guo, H., X. Bi, P. Zhou, et al., NLRP3 Deficiency Attenuates Renal Fibrosis and Ameliorates Mitochondrial Dysfunction in a Mouse Unilateral Ureteral Obstruction Model of Chronic Kidney Disease. Mediators Inflamm, 2017. 2017: p. 8316560. 341. Newton, K., X. Sun and V.M. Dixit, Kinase RIP3 is dispensable for normal NF-kappa Bs, signaling by the B-cell and T-cell receptors, tumor necrosis factor receptor 1, and Toll-like receptors 2 and 4. Mol Cell Biol, 2004. 24(4): p. 1464-9.
178
342. Ouellet, D., E. Gibiansky, C. Leonowens, et al., Population pharmacokinetics of dabrafenib, a BRAF inhibitor: effect of dose, time, covariates, and relationship with its metabolites. J Clin Pharmacol, 2014. 54(6): p. 696-706. 343. Denton, C.L., E. Minthorn, S.W. Carson, et al., Concomitant oral and intravenous pharmacokinetics of dabrafenib, a BRAF inhibitor, in patients with BRAF V600 mutation- positive solid tumors. J Clin Pharmacol, 2013. 53(9): p. 955-61. 344. Mittapalli, R.K., S. Vaidhyanathan, A.Z. Dudek, et al., Mechanisms limiting distribution of the threonine-protein kinase B-RaF(V600E) inhibitor dabrafenib to the brain: implications for the treatment of melanoma brain metastases. J Pharmacol Exp Ther, 2013. 344(3): p. 655-64. 345. Vaidhyanathan, S., B. Wilken-Resman, D.J. Ma, et al., Factors Influencing the Central Nervous System Distribution of a Novel Phosphoinositide 3-Kinase/Mammalian Target of Rapamycin Inhibitor GSK2126458: Implications for Overcoming Resistance with Combination Therapy for Melanoma Brain Metastases. J Pharmacol Exp Ther, 2016. 356(2): p. 251-9. 346. Feng, Y., Z.R. Cai, Y. Tang, et al., TLR4/NF-kappaB signaling pathway-mediated and oxLDL- induced up-regulation of LOX-1, MCP-1, and VCAM-1 expressions in human umbilical vein endothelial cells. Genet Mol Res, 2014. 13(1): p. 680-95. 347. Xie, J., L. Yang, L. Tian, et al., Macrophage Migration Inhibitor Factor Upregulates MCP-1 Expression in an Autocrine Manner in Hepatocytes during Acute Mouse Liver Injury. Sci Rep, 2016. 6: p. 27665. 348. Bian, Z.M., S.G. Elner, A. Yoshida, et al., Differential involvement of phosphoinositide 3- kinase/Akt in human RPE MCP-1 and IL-8 expression. Invest Ophthalmol Vis Sci, 2004. 45(6): p. 1887-96. 349. Mudaliar, H., C. Pollock, M.G. Komala, et al., The role of Toll-like receptor proteins (TLR) 2 and 4 in mediating inflammation in proximal tubules. Am J Physiol Renal Physiol, 2013. 305(2): p. F143-54. 350. Chen, L., M.L. Sha, D. Li, et al., Relaxin abrogates renal interstitial fibrosis by regulating macrophage polarization via inhibition of Toll-like receptor 4 signaling. Oncotarget, 2017. 8(13): p. 21044-21053. 351. Ma, J., H. Wu, C.Y. Zhao, et al., Requirement for TLR2 in the development of albuminuria, inflammation and fibrosis in experimental diabetic nephropathy. Int J Clin Exp Pathol, 2014. 7(2): p. 481-95. 352. Li, X., S. Jiang and R.I. Tapping, Toll-like receptor signaling in cell proliferation and survival. Cytokine, 2010. 49(1): p. 1-9.
179
353. Bakker, P.J., L.M. Butter, N. Claessen, et al., A tissue-specific role for Nlrp3 in tubular epithelial repair after renal ischemia/reperfusion. Am J Pathol, 2014. 184(7): p. 2013-22. 354. Wen, Y., Y. Liu, T. Tang, et al., NLRP3 inflammasome activation is involved in Ang II-induced kidney damage via mitochondrial dysfunction. Oncotarget, 2016. 7(34): p. 54290-54302. 355. Meng, X.M., D.J. Nikolic-Paterson and H.Y. Lan, TGF-beta: the master regulator of fibrosis. Nat Rev Nephrol, 2016. 12(6): p. 325-38. 356. Li, P.K. and T.K. Ma, Global impact of nephropathies. Nephrology (Carlton), 2017. 22 Suppl 4: p. 9-13. 357. Kim, M.K., Treatment of diabetic kidney disease: current and future targets. Korean J Intern Med, 2017. 32(4): p. 622-630. 358. He, S., Y. Liang, F. Shao, et al., Toll-like receptors activate programmed necrosis in macrophages through a receptor-interacting kinase-3-mediated pathway. Proc Natl Acad Sci U S A, 2011. 108(50): p. 20054-9. 359. Pushpakumar, S., L. Ren, S. Kundu, et al., Toll-like Receptor 4 Deficiency Reduces Oxidative Stress and Macrophage Mediated Inflammation in Hypertensive Kidney. Sci Rep, 2017. 7(1): p. 6349. 360. Souza, A.C., T. Tsuji, I.N. Baranova, et al., TLR4 mutant mice are protected from renal fibrosis and chronic kidney disease progression. Physiol Rep, 2015. 3(9). 361. Hughes, M.M. and L.A.J. O'Neill, Metabolic regulation of NLRP3. Immunol Rev, 2018. 281(1): p. 88-98. 362. Zhang, M., Y. Guo, H. Fu, et al., Chop deficiency prevents UUO-induced renal fibrosis by attenuating fibrotic signals originated from Hmgb1/TLR4/NFkappaB/IL-1beta signaling. Cell Death Dis, 2015. 6: p. e1847. 363. Wu, M., W. Han, S. Song, et al., NLRP3 deficiency ameliorates renal inflammation and fibrosis in diabetic mice. Mol Cell Endocrinol, 2018. 364. Gong, W., S. Mao, J. Yu, et al., NLRP3 deletion protects against renal fibrosis and attenuates mitochondrial abnormality in mouse with 5/6 nephrectomy. Am J Physiol Renal Physiol, 2016. 310(10): p. F1081-8. 365. Lemos, D.R., M. McMurdo, G. Karaca, et al., Interleukin-1beta Activates a MYC-Dependent Metabolic Switch in Kidney Stromal Cells Necessary for Progressive Tubulointerstitial Fibrosis. J Am Soc Nephrol, 2018. 29(6): p. 1690-1705. 366. Lin, M., W.H. Yiu, R.X. Li, et al., The TLR4 antagonist CRX-526 protects against advanced diabetic nephropathy. Kidney Int, 2013. 83(5): p. 887-900. 367. Otto, G., IL-1beta switches on kidney fibrosis. Nat Rev Nephrol, 2018. 14(8): p. 475.
180
368. Nee, L.E., T. McMorrow, E. Campbell, et al., TNF-alpha and IL-1beta-mediated regulation of MMP-9 and TIMP-1 in renal proximal tubular cells. Kidney Int, 2004. 66(4): p. 1376-86. 369. Noel, P.R., K.C. Barnett, R.E. Davies, et al., The toxicity of dimethyl sulphoxide (DMSO) for the dog, pig, rat and rabbit. Toxicology, 1975. 3(2): p. 143-69. 370. Holthaus, L., D. Lamp, A. Gavrisan, et al., CD4(+) T cell activation, function, and metabolism are inhibited by low concentrations of DMSO. J Immunol Methods, 2018. 371. Ahn, H., J. Kim, E.B. Jeung, et al., Dimethyl sulfoxide inhibits NLRP3 inflammasome activation. Immunobiology, 2014. 219(4): p. 315-22. 372. Humphreys, B.D., Mechanisms of Renal Fibrosis. Annu Rev Physiol, 2017. 373. Zhou, W. and J. Yuan, Necroptosis in health and diseases. Semin Cell Dev Biol, 2014. 35: p. 14-23. 374. Geng, J., Y. Ito, L. Shi, et al., Regulation of RIPK1 activation by TAK1-mediated phosphorylation dictates apoptosis and necroptosis. Nat Commun, 2017. 8(1): p. 359. 375. Moriwaki, K., S. Balaji, T. McQuade, et al., The necroptosis adaptor RIPK3 promotes injury- induced cytokine expression and tissue repair. Immunity, 2014. 41(4): p. 567-78. 376. Zhou, Y., B. Zhou, H. Tu, et al., The degradation of mixed lineage kinase domain-like protein promotes neuroprotection after ischemic brain injury. Oncotarget, 2017. 8(40): p. 68393- 68401. 377. Martin-Sanchez, D., M. Fontecha-Barriuso, S. Carrasco, et al., TWEAK and RIPK1 mediate a second wave of cell death during AKI. Proc Natl Acad Sci U S A, 2018. 378. Wang, Q., T. Zhou, Z. Liu, et al., Inhibition of Receptor-Interacting Protein Kinase 1 with Necrostatin-1s ameliorates disease progression in elastase-induced mouse abdominal aortic aneurysm model. Sci Rep, 2017. 7: p. 42159. 379. Martens, S., V. Goossens, L. Devisscher, et al., RIPK1-dependent cell death: a novel target of the Aurora kinase inhibitor Tozasertib (VX-680). Cell Death Dis, 2018. 9(2): p. 211. 380. Lopez-Hernandez, F.J. and J.M. Lopez-Novoa, Role of TGF-beta in chronic kidney disease: an integration of tubular, glomerular and vascular effects. Cell Tissue Res, 2012. 347(1): p. 141- 54. 381. Gewin, L. and R. Zent, How does TGF-beta mediate tubulointerstitial fibrosis? Semin Nephrol, 2012. 32(3): p. 228-35. 382. Chen, P., Y. Yuan, T. Zhang, et al., Pentosan polysulfate ameliorates apoptosis and inflammation by suppressing activation of the p38 MAPK pathway in high glucosetreated HK2 cells. Int J Mol Med, 2018. 41(2): p. 908-914.
181
383. Greenlee-Wacker, M.C., S. Kremserova and W.M. Nauseef, Lysis of human neutrophils by community-associated methicillin-resistant Staphylococcus aureus. Blood, 2017. 129(24): p. 3237-3244. 384. Eikmans, M., J.J. Baelde, E. de Heer, et al., ECM homeostasis in renal diseases: a genomic approach. J Pathol, 2003. 200(4): p. 526-36. 385. Xie, L., B.K. Law, A.M. Chytil, et al., Activation of the Erk pathway is required for TGF-beta1- induced EMT in vitro. Neoplasia, 2004. 6(5): p. 603-10. 386. Zhu, C., Y. Liu, Z. Guan, et al., Hypoxia-reoxygenation induced necroptosis in cultured rat renal tubular epithelial cell line. Iran J Basic Med Sci, 2018. 21(8): p. 863-868. 387. Ridker, P.M., J.G. MacFadyen, R.J. Glynn, et al., Inhibition of Interleukin-1beta by Canakinumab and Cardiovascular Outcomes in Patients With Chronic Kidney Disease. J Am Coll Cardiol, 2018. 71(21): p. 2405-2414. 388. Afsar, B., A. Covic, A. Ortiz, et al., The Future of IL-1 Targeting in Kidney Disease. Drugs, 2018. 78(11): p. 1073-1083. 389. Perico, L., M. Mandala, A. Schieppati, et al., BRAF Signaling Pathway Inhibition, Podocyte Injury, and Nephrotic Syndrome. Am J Kidney Dis, 2017. 70(1): p. 145-150. 390. Jhaveri, K.D., V. Sakhiya and S. Fishbane, Nephrotoxicity of the BRAF Inhibitors Vemurafenib and Dabrafenib. JAMA Oncol, 2015. 1(8): p. 1133-4. 391. Groseclose, M.R., S.B. Laffan, K.S. Frazier, et al., Imaging MS in Toxicology: An Investigation of Juvenile Rat Nephrotoxicity Associated with Dabrafenib Administration. J Am Soc Mass Spectrom, 2015. 26(6): p. 887-98.
182