Novel Role of AGE-R1/OST48 in the Metabolome and Proteome Promoting ER Stress in the Kidney and Liver Aowen Zhuang Bbiomsc (Hons)

Novel role of AGE-R1/OST48 in the metabolome and proteome promoting ER stress in the kidney and liver Aowen Zhuang BBiomSc (Hons)

A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2018 Faculty of Medicine

Abstract The protein oligosaccharyltransferase-48 (OST48/AGE-R1) is integral to protein N-glycosylation in the endoplasmic reticulum (ER) but is also postulated to act as a membrane localised clearance receptor for advanced glycation end-products (AGE). AGE accumulation is implicated in liver injury, diabetic kidney disease and diabetes-associated metabolic disorders. To date, however, there has been minimal investigation into the role of OST48 in kidney and liver function under either physiological or diabetic conditions. Here we have used both global (DDOST+/-) and targeted (podocyte specific; DDOST+/-Pod-Cre) knock-in mouse models to induce subtle increases in OST48. The burden of OST48 ligands, AGEs in these models, was modified by either dietary intervention or by the induction of diabetes to delineate the effects of increasing OST48 on the kidney and liver metabolome and proteome. Given previous studies, our postulate was that increasing OST48 would improve liver and kidney function in diabetes by facilitating removal of AGEs from the body.

In the first study, we examined hepatic function and structure following global knock in of OST48 in combination with a therapeutic reduction in dietary AGE intake. Global knock-in of OST48 increased protein OST48 in the gastrointestinal tract, where it was highest in the duodenum. Both increasing gastrointestinal OST48 expression and/or greater consumption of AGEs modestly impaired liver function resulting in hepatic fibrosis. However, a combination of both high AGE dietary consumption and increased OST48 expression significantly exacerbated liver injury, but this was in the absence of steatosis. Interestingly, DDOST+/- mice had increased portal vein delivery and accumulation of hepatic AGEs which were associated with central adiposity, insulin secretory defects, shifting of fuel usage to fatty and keto-acids, and hepatic glycogen accumulation. These culminated in hepatomegaly along with hepatic ER and oxidative stress. This work is significant in that it revealed a novel role of the OST48 and AGE axis as a two-hit model of hepatic injury through ER stress, changes in fuel utilisation and impaired glucose tolerance.

Our second and third studies aimed to delineate the physiological role of OST48 in the kidney through a global and podocyte-specific knock-in of OST48. Firstly, we investigated whether increased AGE excretion facilitated by modest increases in OST48, could prevent the development of diabetic kidney disease. This body of work is particularly significant, as to date there have been no studies examining whether elevating OST48 in diabetes is reno-protective, despite associative studies suggesting that a loss of OST48 is a pathological contributor to diabetes complications.

Globally increasing OST48 did not protect against the development of experimental diabetic kidney disease. However, there appeared to be modest improvements in insulin secretion seen in both non-

2 diabetic and diabetic DDOST+/- mice. This work is significant in that it revealed that globally elevating OST48 to facilitate AGE excretion does not protect against the development of diabetic kidney disease, contrary to perceptions in the current literature.

Our third study used a more selective knock in of OST48 in an important kidney cell type, podocytes. Healthy podocytes, are critical for kidney health and function, and podocyte damage is postulated to be an early event in the development of diabetic kidney disease. We site-specifically increased podocyte expression of OST48 (DDOST+/-Pod-Cre). Despite facilitating greater clearance of AGEs into the urine, increasing podocyte OST48 concentration by 2.5-fold resulted in a decline in kidney function, even in the absence of diabetes. Specifically, serum creatinine was elevated which was exacerbated by diabetes and glomerular filtration rate was decreased. As expected, diabetes increased urinary albumin excretion, but this was not improved by increasing AGE clearance via podocyte OST48. Glomerular damage including mesangial matrix expansion was evident in all diabetic mice. Non-diabetic mice with podocyte specific over-expression of OST48 also had significant glomerular damage including glomerulosclerosis, collagen IV deposition, basement membrane thickening and foot process effacement as compared with wild-type littermates. Tubulointerstitial fibrosis was also demonstrable in non-diabetic mice with increased podocyte OST48 expression and in all diabetic mice. Non-diabetic mice with increased podocyte OST48 had localized endoplasmic reticulum stress markers, evidenced by elevated sXBP1 and GRP78 expression, as seen in diabetic mice. Overall these studies suggest that facilitating AGE clearance by modestly increasing podocyte expression of OST48, results in endoplasmic reticulum stress and kidney dysfunction thereby worsening diabetic kidney disease. This work is of significance as it clarifies that therapeutic strategies which expose kidney tissues to greater AGE flux into the urine need to be better understood.

Taken together, in this thesis, we present evidence that either global or podocyte-specific increases in OST48 expression do not protect against the development of experimental diabetic kidney disease. There may be yet undiscovered roles for this protein at this site. In addition, mice with global increases in OST48 had liver injury which were compounded by consumption of a high AGE diet. Hence, our studies indicate that efforts to increase the expression of OST-48 to improve pathology in these and other diseases, as well as to prevent aging may have undesirable side-effects leading to kidney dysfunction, liver fibrosis and glucose intolerance.

Declaration by author This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis.

I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my research higher degree candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award.

I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School.

I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis.

Publications during candidature Peer-reviewed publications Zhuang A., Yap, F.Y.T., McCarthy, D., Gallo, L.A., Penfold, S.A., Sourris, K.C., Coughlan, M.T., Schulz, B.L., Forbes, J.M. The AGE receptor, OST48 contributes to podocyte dysfunction in experimental diabetic kidney disease via induction of endoplasmic reticulum stress. JASN. 2018 (Under Review).

Zhuang A., Yap, F.Y.T., McCarthy, D., Leung, C., Sourris, K.C., Penfold, S.A., Thalles-Bonke, V., Coughlan, M.T., Schulz, B.L., Forbes, J.M. Globally elevating the AGE clearance receptor, OST48 does not protect against the development of diabetic kidney disease, despite improving insulin secretion. Sci Rep. 2018 (Under Review).

Borg, J.B., Yap, F.Y.T., Keshvari, S., Simmons, D.G., Gallo, L.A., Fotheringham, A.K., Zhuang, A., Slattery, R.M., Hasnain, S.Z., Coughlan, M.T., Kantharidis, P., Forbes, J.M. Perinatal exposure to high dietary advanced glycation end products in transgenic NOD8.3 mice leads to pancreatic beta cell dysfunction. Islets. 2017 Nov 20;(00): doi: 10.1080/19382014.2017.1405189.

Ward, M., Flemming, N., Gallo, L., Fotheringham, A., McCarthy, D., Zhuang, A., Tang, P., Borg, D., Shaw, H., Harvie, B., Briskey, D., Roberts, L., Plan, M., Murphy, M., Hodson, M., Forbes, J.M. Targeted mitochondrial therapy using MitoQ shows equivalent renoprotection to angiotensin converting enzyme inhibition but no combined synergy in diabetes. Sci Rep. 2017 Nov 9;7(1): 15190. doi:10.1038/s41598-017-15589-x.

Zhuang, A., Yap, F.YT., Bruce, C., Leung, C., Plan, M.R., Sullivan, M.A., Herath, C., McCarthy, D., Sourris, K.C., Kantharidis, P., Coughlan, M.T., Febbraio, M.A., Hodson, M.P., Watt, M.J., Angus, P., Schulz, B.L., Forbes, J.M. Increased liver AGEs mediated through an OST48 pathway induces hepatic injury. Sci Rep. 2017 Sep 25;7(1):12292. doi: 10.1038/s41598-017-12548-4. (Incorporated as Chapter 3)

Gallo, L., Ward, M.S., Fotheringham, A.K., Zhuang, A., Borg, D., Flemming N.B., Harvie, B.M., Kinneally, T.L., Yeh, S.M, McCarthy, D., Koepsell, H., Vallon, V., Pollock, C., Panchapakesan, U., Forbes, J.M. Once daily administration of the SGLT2 inhibitor, empagliflozin, attenuates markers of renal fibrosis without improving albuminuria in diabetic db/db mice. Sci Rep. 2016. 6:26428, DOI: 10.1038/srep26428.

Zhuang, A., Forbes, J.M. Diabetic kidney disease: a role for advanced glycation end-product receptor 1 (AGE-R1)?. Glycoconjugate J. 2016. 33(4). doi: 10.1007/s10719-016-9693-z. (Incorporated as Chapter 2)

Foo, S.Y., Zhang, V., Lalwani, A., Lynch, J., Zhuang, A., En Lam, C., Foster, P.S., King, C., Steptoe, R., Mazzone, S., Sly, P., Phipps, S. Regulatory T cells prevent inducible BALT formation by dampening neutrophilic inflammation. J Immunol. 2015. 194(9). doi: 10.4049/jimmunol.1400909.

Zhuang, A., Forbes, J.M., Stress in the kidney is the road to pERdition: Is endoplasmic reticulum stress a pathogenic mediator of diabetic nephropathy?. J Endocrinol. 2014. 222(3). doi: 10.1530/JOE-13-0517. (Incorporated as Chapter 2)

Published conference abstracts Borg, D., Leung, S., Zhuang, A., Fotheringham, A.K., McCarthy, D., Di Trapani, J., Groop, P.H., Knip, M., Forbes, J.M., Delivery of recombinant human soluble receptor for advanced glycation end products delays autoimmune diabetes in the NOD mouse model. Diabetologia. 2015. http://dx.doi.org/10.1007/s00125-015-3687-4.

Leung, C., Fotheringham, A.K., Zhuang, A., Borg, D., Ward, M.S., Coughlan, M.T., McCarthy, D., Gallo, L.A., Harcourt, B.E., Forbes, J.M., Flux of advanced glycation end products across the kidney as a contributor to renal disease in diabetes. Nephrology, 2014. 19:62-63. http://dx.doi.org/10.1111/nep.12302.

Zhuang, A., Sourris, K.C., Harcourt, B.E., Penfold, S.A., Fotheringham, A.K., McCarthy, D., Schulz, B., Forbes, J.M., AGER1 overexpression in glomerular podocytes results in renal disease which is exacerbated by diabetes. Nephrology. 2014. 19:56-56. doi:10.1111/nep.12301.

Conference presentations during candidature Oral Presentations Zhuang, A., Yap, F.Y.T., McCarthy, D.A., Gasser, A., Plan, M.R., Hodson, M.P., Leung, C., Angus, P., Bruce, C., Febbraio, M.A., Watt, M.J., Schulz, B., Forbes, J.M., Overexpression of the AGE detoxification receptor AGER1 results in hepatic fibrosis and hepatic insulin resistance. International Society of the Maillard Reaction Symposium, 2015, Tokyo, Japan.

Zhuang, A., Sourris, K.C., Rodriguez, V.G., Harcourt, B.E., Penfold, S.A., Fotheringham, A.K., McCarthy, D.A., Bertram, J.F., Schulz, B., Forbes, J.M., AGER1 overexpression in glomerular podocytes results in renal disease which is exacerbated by diabetes. International Society of the Maillard Reaction Symposium, 2015, Tokyo, Japan.

Zhuang, A., Yap, F.Y.T., McCarthy, D.A., Gasser, A., Plan, M.R., Hodson, M.P., Leung, C., Angus, P., Bruce, C., Febbraio, M.A., Watt, M.J., Schulz, B., Forbes, J.M., Over-expression of the AGE receptor (AGE-R1) during high AGE feeding results in hepatic fibrosis and exacerbates glucose intolerance. Endocrine Society Australia - Early Career Researcher Mentor Workshop, 2015, Adelaide, Australia.

Zhuang, A., Sourris, K.C., Rodriguez, V.G., Harcourt, B.E., Penfold, S.A., Fotheringham, A.K., McCarthy, D.A., Bertram, J.F., Schulz, B., Forbes, J.M., AGER1 overexpression in glomerular podocytes results in renal disease which is exacerbated by diabetes. Endocrine Society Australia - Early Career Researcher Mentor Workshop, 2015, Adelaide, Australia.

Zhuang, A., Harcourt, B.E., Sourris, K.C., Penfold, S.A., Fotheringham, A.K., Thorn, P., Forbes, J.M., A “sweet” idea for a deadly disease. UQ School of Medicine and Biomedical Sciences 3 Minute Thesis Competition, 2015. Brisbane, Australia.

Zhuang, A., Sourris, K.C., Harcourt, B.E., Penfold, S.A., Fotheringham, A.K., McCarthy, D.A., Schulz, B., Forbes, J.M., AGER1 overexpression in glomerular podocytes results in renal disease which is exacerbated by diabetes. Australian and New Zealand Society of Nephrology, 2014, Melbourne, Australia.

Zhuang, A., Harcourt, B.E., Sourris, K.C., Penfold, S.A., Fotheringham, A.K., Thorn, P., Forbes, J.M., A “sweet” idea for a deadly disease. Mater Research 3 Minute Thesis Competition, 2014. Brisbane, Australia.

Poster Presentations Zhuang, A., Sourris, K.C., Harcourt, B.E., Penfold, S.A., Fotheringham, A.K., McCarthy, D.A., Schulz, B., Forbes, J.M., A new insight into AGER1; overexpression of AGER1 in glomerular podocytes results in kidney disease which is exacerbated by diabetes, UQ International Biomedical Science Symposium, 2014, Brisbane, Australia.

Zhuang, A., Sourris, K.C., Gallo, L.A., Rodriguez, V.G., Harcourt, B.E., Penfold, S.A., Fotheringham, A.K., McCarthy, D.A., Bertram J.F., Schulz, B., Forbes, J.M., AGE-R1 overexpression in glomerular podocytes leads to features resembling kidney disease, PanAmerican Congress of Physiology Sciences, 2014, Misiones Province, Argentina.

Zhuang, A., Harcourt, B.E., Sourris, K.C., Penfold, S.A., Fotheringham, A.K., McCarthy, D.A., Schulz, B., Forbes, J.M., AGE-R1 overexpression in glomerular podocytes leads to features resembling kidney disease, Keystone Symposia Complications of Diabetes, 2014, British Columbia, Canada.

Awards obtained during candidature 2017: Frank Claire PhD Top Up Scholarship – Mater Research: $3000 2016: Frank Claire PhD Top Up Scholarship – Mater Research: $3000 2015: Frank Claire PhD Top Up Scholarship – Mater Research: $3000 Mater Research Travel Award – Mater Research: $1000 ESA-ECR Snapshot Award – Endocrine Society: $250 2014: 1st Place, Mater Research 3MT Competition (PhD) – Mater Research: $200 Frank Claire PhD Top Up Scholarship – Mater Research: $3000. Awarded Biomedical Research PhD Scholarship – Kidney Health Australia: $27000p/a.

Society involvement during candidature 2016: Mater Mentors All Hallows’ and Somerville Student Programs. 2015: Chair of Organising Committee for the Annual TRI Poster Symposium. Organising Committee for the Weekly TRI Friday Seminar Series. 2014: Invited Participant in a Review of the Children’s Hospital Foundation’s Research Strategy and Associated Grants Program. Organising Committee for the Weekly TRI Friday Seminar Series. Secretary for the Weekly Diamantina Health Partners Metabolic Collaborative Research Meeting.

Publications included in this thesis Zhuang, A., Forbes, J.M., Stress in the kidney is the road to pERdition: Is endoplasmic reticulum stress a pathogenic mediator of diabetic nephropathy?. J Endocrinol. 2014. 222(3). doi: 10.1530/JOE-13-0517. Incorporated as Chapter 2.

Contributor Statement of contribution Author Aowen Zhuang (Candidate) Contribution to main text of manuscript (100%) Figure design (100%) Edited the paper (80%) Author Josephine M. Forbes Edited paper (20%)

Contributor Statement of contribution Author Aowen Zhuang (Candidate) Contribution to main text of manuscript (100%) Figure and table design (100%) Edited the paper (70%) Author Josephine M. Forbes Edited paper (30%)

Contributor Statement of contribution Author Aowen Zhuang (Candidate) Contribution to main text of manuscript (100%) Edited the paper (80%) Designed the experiments (70%) Conducted the experiments (80%) Figure 1-7 design (100%) Supplementary figure design (100%)

Study design (60%) Statistical analysis of data tables and figures (90%) Author Felicia YT. Yap Assisted with mouse model and collection of tissues (20%) Author Clinton Bruce Assisted with CLAMS (50%) Author Chris Leung Assisted with H&E and LFT and imaging (IR dye) (100%) Author Manuel R. Plan Assisted with AA analysis (100%) Author Mitchell A. Sullivan Assisted with glycogen analysis (100%) Author Chandana Herath Assisted with H&E and LFT (100%) Author Domenica M. McCarthy Assisted with staining and microscopy (25%) Author Karly C. Sourris Assisted with mouse model and collection of tissues (20%) Author Phillip Kantharidis Assisted with CLAMS (50%) Author Melinda T. Coughlan Assisted with mouse model and collection of tissues (20%) Author Mark A. Febbraio Assisted with mouse model design (50%) Author Mark P. Hodson Assisted with statistical analysis and AA analysis (50%) Editing (5%) Author Matthew J. Watt Assisted with ceramides and diacylglycerides (100%) Editing (5%) Author Peter Angus Assisted with study design (5%) Author Benjamin L. Schulz Assisted with study design (10%) Editing (5%) Author Josephine M. Forbes Assisted with study design (20%) Editing (5%)

Manuscripts included in this thesis Zhuang A., Yap, F.Y.T., McCarthy, D., Leung, C., Sourris, K.C., Penfold, S.A., Thalles-Bonke, V., Coughlan, M.T., Schulz, B.L., Forbes, J.M. Globally elevating the AGE clearance receptor, OST48 does not protect against the development of diabetic kidney disease, despite improving insulin secretion. Sci Rep. 2018 (Under Review). Incorporated as Chapter 4. 10

Contributor Statement of contribution Author Aowen Zhuang (Candidate) Contribution to main text of manuscript (100%) Edited the paper (85%) Designed the experiments (70%) Conducted the experiments (80%) Figure 1-5 design (100%) Study design (70%) Statistical analysis of data tables and figures (100%) Author Felicia YT. Yap Assisted with mouse model and collection of tissues (20%) Author Domenica M. McCarthy Assisted with staining and microscopy (10%) Author Chris Leung Assisted with imaging (IR dye) (100%) Author Karly C. Sourris Assisted with mouse model and collection of tissues (20%) Author Vicki Thalles-Bonke Assisted with mouse model and collection of tissues (20%) Author Melinda T. Coughlan Assisted with mouse model and collection of tissues (20%) Author Benjamin L. Schulz Assisted with study design (10%) Editing (5%) Author Josephine M. Forbes Assisted with study design (20%) Editing (15%)

Zhuang A., Yap, F.Y.T., McCarthy, D., Gallo, L.A., Penfold, S.A., Sourris, K.C., Coughlan, M.T., Schulz, B.L., Forbes, J.M. The AGE receptor, OST48 contributes to podocyte dysfunction in experimental diabetic kidney disease via induction of endoplasmic reticulum stress. JASN. 2018 (Under Review). Incorporated as Chapter 5.

Contributor Statement of contribution Author Aowen Zhuang (Candidate) Contribution to main text of manuscript (100%) Edited the paper (70%) Designed the experiments (70%) Conducted the experiments (80%)

Figure 1-6 design (100%) Study design (65%) Statistical analysis of data tables and figures (100%) Author Felicia YT. Yap Assisted with mouse model and collection of tissues (20%) Author Domenica M. McCarthy Assisted with staining and microscopy (40%) Author Linda A. Gallo Assisted with study design (5%) Author Karly C. Sourris Assisted with mouse model and collection of tissues (20%) Author Vicki Thallas-Bonke Assisted with mouse model and collection of tissues (20%) Author Melinda T. Coughlan Assisted with mouse model and collection of tissues (20%) Author Benjamin L. Schulz Assisted with study design (10%) Editing (5%) Author Josephine M. Forbes Assisted with study design (20%) Editing (25%)

Contributions by others to the thesis The publications contributing towards the thesis have involved multiple collaborations from other authors. The nature and extent of these contributions have been detailed above.

Statement of parts of the thesis submitted to qualify for the award of another degree Chapter 3, Supporting Figure 4 submitted in part for BBiomSc (Honours), University of Queensland, 2013, degree awarded December 2013.

Chapter 4, Figure 1A-B, Figure 2A, Figure 3A-B, Figure 5C submitted in part for BBiomSc (Honours), University of Queensland, 2013, degree awarded December 2013.

Chapter 5, Figure 2C-E, Figure 3A, Figure 4A submitted in part for BBiomSc (Honours), University of Queensland, 2013, degree awarded December 2013.

Research Involving Human or Animal Subjects All animal studies were performed in accordance with guidelines provided by the AMREP (Alfred

Medical Research and Education Precinct) Animal Ethics Committee, University of Queensland and the National Health and Medical Research Council of Australia (E/0846/2009/B,

E/1184/2012/B and MRI-UQ/TRI/256/15/KHA). A copy of the approval letter is provided in the appendices.

Acknowledgements Acknowledgements to personnel contributions towards this thesis and subsequent publications are detailed in the relevant chapters. We would like to acknowledge the Translational Research

Institute (TRI) and Mater Research Institute for providing the excellent research environment and core facilities that enabled this research.

Financial Support This research was supported by the NH&MRC, Kidney Health Australia (SCH17; 141516) and the

Mater Foundation, which had no role in the study design, data collection and analysis, decision to publish, or preparation or the manuscript.

Keywords diabetic kidney disease, diabetes, advanced glycation end-products (AGEs), oligosaccharyltransferase-48 (OST48), advanced glycation end-product receptor-1 (AGE-R1), liver fibrosis, endoplasmic reticulum (ER) stress, glucose homeostasis, glomerulosclerosis, tubulointerstitial fibrosis (TIF).

Australian and New Zealand Standard Research Classifications (ANZSRC) ANZSRC code: 060104, Cell Metabolism, 60% ANZSRC code: 060603, Animal Physiology - Systems, 30% ANZSRC code: 060109, Proteomics and Intermolecular Interactions (excl. Medical Proteomics), 10%

Fields of Research (FoR) Classification FoR code: 0601, Biochemistry and Cell Biology, 70% FoR code: 0606, Physiology, 30%

Table of Contents List of Abbreviations ...... 18 Chapter 1: General introduction ...... 19 Chapter 2: Literature review ...... 21 Introduction to this publication...... 22 Introduction to this publication...... 34 Chapter 3: Increased liver AGEs induce hepatic injury mediated through an OST48 pathway...... 56 Introduction to this publication...... 57 Graphical Abstract ...... 58 Abstract ...... 59 Introduction ...... 59 Results...... 61 Discussion ...... 67 Materials and Methods ...... 69 Figures and Figure Legends ...... 74 Tables...... 81 Supplementary Materials and Methods...... 82 Supporting Figures and Figure Legends...... 83 Chapter 4: Globally elevating the AGE clearance receptor, OST48 does not protect against the development of diabetic kidney disease, despite improving insulin secretion...... 88 Introduction to this chapter...... 89 Graphical Abstract ...... 90 Abstract ...... 91 Results...... 93 Discussion ...... 96 Materials and Methods ...... 99 Figures and Figure Legends ...... 103 Tables ...... 108 Chapter 5: The AGE receptor, OST48 contributes to podocyte dysfunction in experimental diabetic kidney disease via induction of endoplasmic reticulum stress...... 110 Introduction to this chapter...... 111 Graphical abstract ...... 112 Abstract ...... 113 Introduction ...... 114 Methods ...... 116 Results...... 119 Discussion ...... 123 Tables ...... 136 Supplementary Figure Legends ...... 137 Chapter 6: Conclusions ...... 139 References ...... 143 Appendices ...... 181

List of Abbreviations ACE - angiotensin converting enzyme AGE-R1 – advanced glycation end-product receptor-1 AGE-R3 – galectin-3 AGEs – advanced glycation end-products ANG – angiotensin ARB – angiotensin receptor blockers ATF6 – activating transcription factor-6 ATP – adenosine-5’-triphosphate CVD – cardiovascular disease DDOST/OST48 – Dolichyl-diphosphooligosaccharide-protein glycosyltransferase 48 kDa subunit DN – diabetic nephropathy eGFR – estimated glomerular filtration rate eIF2α – eukaryotic translation initiation factor-2α ER – endoplasmic reticulum ERAD – ER-associated degradation ESKD/ESRD – end-stage kidney/renal disease GHb – glycated haemoglobin ipGTT – intraperitoneal glucose tolerance test ipITT – intraperitoneal insulin tolerance test IRE1 – inositol-requiring protein 1 LC-MS/MS – liquid chromatography–mass spectrometry mTORC1 – mammalian target of rapamycin complex 1 NAFLD – non-alcoholic fatty liver disease NASH – non-alcoholic steatohepatitis oGTT – oral glucose tolerance test PBA – 4-phenylbutyric acid PERK – PKR-like ER kinase-mediated PTCs – proximal tubule cells RAGE – receptor for AGEs ROS – reactive oxygen species SGLT2 – sodium/glucose cotransporter 2 SIRT1 – NAD-dependent deacetylase sirtuin-1 T1D – type 1 diabetes T2D – type 2 diabetes TUDCA – taurine-conjugated ursodeoxycholic acid UPR – unfolded protein response XBP1 – X-box-binding protein 1

Chapter 1: General introduction

Diabetes, has two major forms, type 1 and type 2, both of which are characterised by uncontrolled hyperglycaemia, a predominant factor in the development of concomitant chronic complications.

One major complication of key interest is diabetic kidney disease (DKD), which is a key contributing factor in diabetes related mortality and future risk of cardiovascular disease 1, 2.

Therefore, understanding the pathophysiology of DKD is a focus for reducing the associated mortality in diabetes. Moreover, discovery of new pathological pathways is essential in identifying potential targets for therapeutics. In this thesis, the role of the AGE receptor 1 (OST48) as a novel contributor to DKD will be thoroughly described. Advanced glycation end-products (AGEs) are a heterogeneous and complex group of non-enzymatic, post-translational modifications on proteins.

The formation of AGEs is accelerated by chronic hyperglycaemia and oxidative stress 3. The systemic reduction of AGEs using pharmacotherapies has been previously shown to improve kidney function 4 and AGE modifications on skin collagen are useful long term prognostic predictor of complications in individuals with type 1 diabetes5, as well as correlating with chronic kidney disease progression 6. Hence, the kidney is crucial in the detoxification of AGEs, through the filtration and active excretion into the urine 7, 8. Moreover, the receptor hypothesised to have a role in the clearance of AGE modified proteins from the circulation is OST48 9. However, the specific nature of OST48 and AGE ligand binding is not well understood. Whether there is specificity to certain types of AGE adducts or preferential binding to large AGE multimeric complexes, much like RAGE, is currently unknown. Clearance of circulating AGEs is an exciting avenue of interest in the improvement of diabetic complications.

Chapter 2: Literature review

Introduction to this publication. This chapter consists of two peer-reviewed review article publications, the first was published in the Glycoconjugate Journal and focuses on the role of

OST48/AGE-R1 in diabetic kidney disease.

Introduction.

Currently, the world is faced with a rising pandemic of both type 1 (T1D) and type 2 (T2D) diabetes mellitus 10, 11, defined by persistent hyperglycaemia, which is an important factor in the development of concomitant complications 2, 12. A major complication, diabetic nephropathy (DN) is a global health concern and is an important risk factor of end-stage kidney disease (ESKD), and an independent risk factor of cardiovascular disease (CVD) 1, 2. Like all other forms of chronic kidney disease, patients with DN are most likely to die of premature CVD, than to survive to the point of ESKD. Understanding the pathophysiology of diabetic nephropathy is, therefore, a key challenge to reduce the burden of diabetes. The early clinical presentation of diabetic nephropathy is characterised by hyperfiltration, progressive proteinuria, and associative glomerular injury accompanied by tubulointerstitial fibrosis and, in the later stages, a steady progressive decline in renal function 13. Moreover, low estimated glomerular filtration rate (eGFR) and albuminuria are the two most potent known risk factors for CVD. Blood pressure lowering drugs such as angiotensin-converting-enzyme (ACE) inhibitors and angiotensin receptor blockers (ARB) therapy are the only renoprotective interventions used clinically. While, ACE inhibitors are often given as first line therapy, unfortunately, they are only partially effective, achieving approximately a 20% reduction in risk of future ESRD 14. Thus, there is an urgent need for interventions that will effectively delay or reverse CKD progression in patients with DN. Furthermore, despite the best available clinical management, involving both glycaemic 15, 16 and blood pressure control 17, including inhibitors of the renin-angiotensin-aldosterone system 18, 19, it is only possible to achieve at most a 30% improvement in declining diabetic kidney function. This is the principal reason why early detection and intervention are most important. Consequently even despite early intervention, end stage disease requiring a kidney transplant or dialysis develops in a considerable number of patients. In this review, we describe the role of AGEs and how a receptor, the AGE receptor 1

(AGE-R1) provides a mechanistic and non-traditional aspect of DN. We also discuss the potential

23 targeting of AGE-R1 and whether this will improve or impair glomerular disorders and the process in understanding the clinical context of this receptor.

Diabetic nephropathy.

Although diabetes is predominately characterised as either T1D or T2D, the clinical distinction between kidney diseases in these two forms of diabetes is not easily defined. Rather, patient descriptions are often more complex and do not follow the linear trajectories often described as diabetic kidney disease. Risk of progression to end stage kidney disease is primarily predicted by low eGFR; previous rate of eGFR decline; and albuminuria. Indeed, other clinical factors add little to the predictive algorithm 20, 21. However, early predictors of progressive DN are less clear.

Interestingly, current reports from the United Kingdom, North America, and Australia suggest a gradual decline or plateau in ESKD incidence. However, this may simply be due to a combination of improved detection and intervention strategies, increased utilisation of non-dialysis supportive care (palliative care), and reversal of the trend for early-start dialysis in ESKD patients following the IDEAL trial 22, 23.

Currently, there are no cell-specific therapeutics that target the underlying pathology in the kidney, as the specific roles of individual renal cell types in disease development are not well understood.

However, given the complex cellular diversity and the various physiological roles of the kidney, it is difficult to pinpoint which functional aspects of the kidney are most affected by diabetes. Indeed, the kidney is a highly synthetic and secretory organ 24, 25 involved in the release of hormones, acute control of glycaemia through the modulation of gluconeogenesis and glucose reabsorption, as well as maintenance of fluid balance and blood pressure. Interestingly, it is thought that hyperglycaemia is the predominant mediator of pathology in resident kidney cells in diabetes through the specific induction of cellular events 26. However, this paradigm is also being challenged.

Early damage to the filtration units of the kidney, the glomeruli, is seen in DN, and the number of podocytes are drastically reduced in diabetic patients 27, 28. Podocytes are terminally-differentiated epithelial cells that, together with endothelial cells and the glomerular basement membrane, comprise the glomerular filtration barrier which functions as a barrier between the circulation and urinary filtrate. This filtration barrier selectively retains proteins in the bloodstream whilst freely filtering small molecules (such as glucose), into the urinary filtrate, the fate of which are determined through either reabsorption into the circulation, or excretion into the urine. Particularly in diabetes, podocytes are exposed to persistent long-term challenges, including glomerular hypertension 29, metabolic imbalances 30-32, and abnormal receptor stimulation 33, all of which are thought to contribute to the functional loss of podocytes, compromising the integrity of the filtration barrier. It is postulated that lost podocytes cannot be replaced by residual podocytes 34, and that residual podocytes are thought to be damaged by neighbouring damaged podocytes, in a cyclical manner 35.

It is this decrease in podocyte density which mediates a progressive loss of nephrons (the filtration and reabsorption units of the kidney), and correlates with the degree of proteinuria (increased protein excretion in urine) over time 35. However, the pathological events that ultimately precipitate in nephron loss remain to be completely delineated. Progression of nephron loss and therefore kidney disease occurs in a stereotypical manner that generally initiates with podocyte injury, leading to tuft adhesion in the Bowmans capsule, and subsequent filtration of abnormal filtrate encroaching the glomerulotubular junction and degeneration of proximal tubules. Indeed, glomerular injury, more often than not, precedes tubulointerstitial damage in diabetes 36, although this paradigm is being increasingly challenged by more recent studies. Nevertheless, it is most likely that the additional changes in the tubulointerstitium and vasculature, in combination with early glomerular changes, are the major precipitating events in the pathology of DN.

Role of advanced glycation end-products in diabetic kidney disease.

The kidney is most likely involved in the detoxification of AGEs through the filtration of circulating AGEs, and subsequently their active uptake and excretion 7, 8. AGEs are a heterogeneous and complex group of non-enzymatic, post-translational modifications on proteins, which begin with the covalent attachment of sugar moieties, which are described in other reviews in this issue.

They can be both manufactured in the body, or absorbed from the dietary sources. Indeed, states of chronic hyperglycaemia and oxidative stress are known to accelerate the formation of AGEs 3.

Consequently, AGE accumulation either through endogenous or exogenous sources 37, can have numerous detrimental effects that can be broadly characterised into three main pathways in the kidney. First, extracellular generation of AGEs have been implicated in modifications of matrix- matrix, cell-cell, or matrix-cell interactions and under pathological conditions, excessive AGEs may lead to increased stiffening of the matrix 38, 39. Indeed the accumulation of AGE modifications on skin collagen is the best long term predictor of complications in individuals with T1D 5, as well as correlating with chronic kidney disease progression 6. Second, intracellular formation of AGEs can directly affect protein trafficking, protein function and protein turnover, and can contribute to intracellular reactive oxygen species (ROS) generation the third pathway by which AGEs may exert their pathological effect is via interactions with cellular receptors. Though there are a number of

AGE receptors, the receptor hypothesised to have a role in the clearance of AGE modified proteins from the circulation is AGE-R1 9. However, the specific nature of AGE-R1 and AGE ligand binding is not well understood. Whether there is specificity to certain types of AGE adducts or preferential binding to large AGE multimeric complexes, much like RAGE, is currently unknown.

Clearance of circulating AGEs is an exciting avenue of interest in the improvement of diabetic complications. Reduced AGE absorption through the gut has been recently identified to improve glycaemic control in individuals with diabetes 40, which suggests that AGE clearance could be beneficial in the progression of diabetic nephropathy 41, 42. Therefore, understanding the role and function of AGE-R1 in the kidney in likely to provide insight into the pathogenesis of DN.

Furthermore, whether AGE excretion by the kidney is mediated by AGE-R1 expression is still not well defined, but may play an integral role in the development and progression of the chronic complications of the diabetic milieu.

AGE-R1: a simple receptor with a complex function?

AGE-R1, an evolutionarily conserved type 1 transmembrane protein 43, is encoded by the DDOST gene and has been postulated to function as a receptor for AGE turnover and clearance 44. Initially it was discovered that a p60 kDa sized protein could be involved in AGE binding and clearance. Upon further analysis it was identified that the plasma membrane component of this protein had a 95% similarity to that of OST-48 44, OST-48 is a 48kDa subunit that functions as part of the oligosaccharyltransferase complex, mediating the transfer of high-mannose oligosaccharides to asparagine residues within the lumen of the rough endoplasmic reticulum, a process known as N- glycosylation 43. It was this N-terminal protein that was exposed on the plasma membrane as the p60 protein and was postulated to interact with AGEs 44, and function as a clearance receptor 44.

Therefore, we believe that it is important to distinguish the difference between what is currently described interchangeably as OST-48 and AGE-R1. Specifically, while OST-48 may act as the active binding component of the AGE-R1 protein, OST-48 is also the key subunit in the oligosaccharyltransferase complex with disparate functions. This is an important distinction since although they are from the same gene, DDOST, OST-48 is not analogous to AGE-R1 and vice- versa, but rather the AGE-R1 protein contains OST-48, and it is this “OST-48” N-terminal component of AGE-R1 that is postulated to bind to AGEs 44.

AGE-R1 an alternative function in protein N-glycosylation.

The homology between AGE-R1 and OST-48 becomes an important aspect to consider when targeting AGE-R1 expression in an attempt to decrease the AGE burden. Indeed, it is possible that secondary effects on oligosaccharyltransferase activity, and therefore the N-glycosylation pathway,

27 could occur following changes to AGE-R1. N-glycosylation of key kidney proteins has been previously shown to have an effect on kidney health and function. Notably, N-glycosylation is essential for the localisation of important kidney proteins such as nephrin, a podocyte-specific protein that mediates actin reorganisation under mechanical stress to assist with glomerular filtration 45. Mutations in nephrin, encoded by the NPHS1, are thought to be responsible for most reported cases of hereditary nephrotic syndrome 46. Moreover, N-glycosylation has been identified as an essential modification necessary to retain proteins in the blood thereby preventing proteinuria through complex regulation of the actin cytoskeleton and downstream foot processes of podocytes

47. Hence, it is important to discern which role of AGE-R1, whether its function as a proponent of

N-glycosylation or its role as an AGE receptor, is more important to the development and progression of diabetic kidney disease, where both AGE accumulation and defects in N- glycosylation play a central role in the pathogenesis.

Interestingly, the AGE-R1 protein is present on most cells and tissues, including macrophages 48, mesangial cells 9, and mononuclear cells 49, and is also postulated to mediate the uptake and degradation of AGEs by the kidney 50. This process of AGE clearance by AGE-R1 is not well described and is thought to function primarily by sequestration and degradation of AGEs.

Subsequently this process simultaneously results in a decrease in AGE accumulation, would likely decrease ROS generation thereby reducing AGE interaction and signalling with other AGE receptors (RAGE, galactin-3) or scavenger type receptors (SR-A, SR-BI and CD36). AGE-R1 is reported to be down-regulated by both, diets abundantly rich in AGE content 51, and by diabetes 50,

52 in the kidney cortex. These changes are associated with increased intracellular AGE accumulation and decreases in urinary AGEs 53. In addition, there are associations between lower

AGE-R1 expression on circulating immune cells and a greater degree of kidney damage in a small cohort of patients with T1D 49. However, there is some contradiction within the literature where

T2D individuals with progressive diabetic nephropathy were reported to have an increased cell

28 surface and intracellular expression of AGE-R1 in their peripheral mononuclear cells 54. It is therefore difficult to ascertain whether altered AGE-R1 expression in circulating immune cells is a direct or indirect result of diabetes.

AGE-R1 an unusual receptor involved in diabetic nephropathy.

Evidence linking AGE-R1 and diabetic nephropathy in clinical studies is sparse, where the only two studies that investigated this association have contradicting conclusions. A SNP array from the

FinnDiane population study inferred that there was no association between SNPs of the AGE-R1 gene, DDOST with nephropathy development in individuals with T1D 55. This contrasts a smaller study in T1D individuals which identified low levels of AGE-R1 mRNA in circulating mononuclear cells in conjunction with elevated levels of serum AGEs, in those individuals with nephropathy 49. It is worth noting however, that despite not reaching significance, the FinnDiane SNP array identified a trend towards an association between a SNP (rs2170336) in DDOST and DN in T1D 55.

Therefore, this may be resolved by other longitudinal studies in DN and in the expansion of the sample size in future screens. In addition, the genomic profile does not directly translate into a corresponding proteomic profile. As such, these genome associative studies are not the most accurate indicator of the protein expression or function of AGE-R1 in the kidney, and further mechanistic work is required to better understand the role of AGE-R1 in the pathogenesis of DN.

The mechanistic pathways of AGE-R1.

Identifying the mechanistic function of AGE-R1 has proven difficult (Figure 1), since AGE-R1 deletion is lethal in yeast 43, and embryonically lethal in mice and other organisms 56. This is likely attributable to impaired N-glycosylation. Indeed, there is only one report of a living family with a mutation in DDOST where the children had a congenital disorder of glycosylation 57. This loss of function mutation in DDOST, did not result in diabetes or kidney disease, however, this is because

29 individuals with mutations in genes involved in glycosylation generally do not survive past childhood, due to systemic organ dysfunction 57, 58.

Initially the biological functions of AGE-R1 were identified primarily through in vitro gene transfer and gene silencing studies 9, 59. Those studies suggested that decreases in AGE-R1 expression inhibited NADPH oxidases p47phox (also known as neutrophil cytosol factor 1) activity and gp91phox expression through suppression of Tyr311 and Tyr332 phosphorylation of protein kinase

C-δ 60. Decreasing AGE-R1 altered the increased AGE-RAGE signal transduction via nuclear translocation of the p65 subunit of NFᴋB 61. Decreasing AGE-R1 expression in these studies also resulted in epidermal growth factor mediation of oxidative stress in a high AGE environment 51, 60.

Furthermore, AGE-R1 inhibits AGE-induced Ser36 phosphorylation of the p66Shc isoform of

SHC-transforming protein 1, a major oxidative stress and apoptosis-promoting adapter protein 59.

This pathway may be of importance, since increased levels of p66Shc are linked with diabetes, atherosclerosis and kidney disease 62.

Interestingly AGE-R1 has also been localised in astrocytes and retinal cells where binding studies identify that diabetes did not change the expression and/or the localisation of AGE-R1 63. However, these studies confirmed that AGEs bind to AGE-R1 and are subsequently internalised Furthermore, there has only been a single in vivo investigation of untargeted AGE-R1 over-expression in transgenic mice, which demonstrated improvements in longevity, insulin sensitivity, and resistance to balloon injury in blood vessels 64. However, this study investigated aged mice (>500 days old).

To our knowledge, there are no studies which directly compare AGE-R1 modulation in young or adult mice, to investigate kidney function. More recently, another study has shown that AGE-R1 may alter sirtuin-1 (SIRT1) activity. SIRT1 is similarly suppressed like AGE-R1 during diabetes, high fat feeding and aging 65-67. Furthermore, SIRT1 is thought to be integral in insulin signalling and secretion, insulin resistance, inflammation, and lifespan 65. Of note, Vlassara et al. suggests that

AGE-R1 overexpression blocks AGE-induced suppression of SIRT1 via NAD+/NADH, however this data has not yet been published, but was mentioned in a recent review 68. The pathway of AGE-

R1 activation and downstream effects on SIRT1 activity is of particular interest given that a recent study identified that SIRT1 overexpression in proximal tubule cells attenuated early glomerular disease in an experimental model of diabetes 69. Although the link between AGE-R1 and regulatory control of SIRT1 activity in podocytes and proximal tubule cells in the diabetic kidney remains to be defined, these studies underscore the potential importance of these two interdependent pathways in the diabetic kidney.

Another consideration is the recent and accumulating evidence demonstrating that AGE modified proteins can modulate processes such as insulin secretion 70 and insulin sensitivity 71, 72, which could also influence the progression of diabetic kidney disease via changes in glucose and insulin handling. Hence, the story of increasing “protective” AGE-R1 expression during diabetic nephropathy may not be so straightforward, and AGE-R1 expression may reflect the current metabolic environment. Moreover, AGE-R1 has been shown to prevent AGE-induced impaired insulin signalling through the insulin receptor and insulin receptor substrate 1 (IRS1) in adipocytes both in vitro and in vivo 73, 74. Although this body of work was primarily investigated in vitro in cultured adipocytes, conclusions could be drawn that there are similar mechanisms at play in other insulin sensitive cells. One insulin sensitive cell of particular importance in diabetic nephropathy is the kidney podocyte. Without insulin receptor activity, the podocytes do not functionally develop and this impairs kidney function 30.

Conclusions: AGE-R1 more than a clearance receptor?

In this review we suggest that AGE-R1 is not just a clearance receptor for AGEs. Moreover, given the signal transduction pathways activated during AGE-AGE-R1 ligation, it is highly likely that it has an influential role in kidney function. Our understanding of how AGE-R1 functions in the

31 context of diabetic nephropathy is limited, since the focus has been largely on its function as a clearance receptor for AGEs. Although this is feasible from a mechanistic perspective given the increased accumulation of AGEs seen in patients with diabetes, it largely ignores the important distinction that the component of AGE-R1 that is postulated to bind to AGEs is OST-48. Hence given the distinct role of OST-48 in N-glycosylation, it is very likely that these two distinct pathways of sugar chemistry may have some as yet undefined relationship as pathogenic mediators of nephropathy in diabetes. Further, increasing AGE-R1 levels in the kidney or in circulating lymphocytes following interventions has any beneficial effects in diabetic kidney disease remains to be elucidated by future studies. There are several important questions that remain to be answered specifically regarding the effect of AGE-R1 on kidney function and on kidney specific cells.

Understanding the physiological effect of AGE-R1 in the diabetic milieu could provide us important information in further understanding and potentially developing AGE-R1 as a mechanism to counteract the burden of a high AGE environment.

Figure 1. Excessive AGEs impair insulin sensitivity in insulin-sensitive cells. In particular the podocytes in the kidney may be a key cell of interest. AGE mediated effects on the cell are controlled predominately through receptor interaction and AGE clearance is facilitated by AGE-R1.

In a high AGE environment, prevalent in diabetic individuals, AGE-R1 expression is thought to be down-regulated by AGEs and therefore the protective effects of AGE-R1 receptor function through the upregulation of SIRT1 is thought to be impaired. Reduced AGE-R1 receptor could subsequently prevent the inhibition of AGE-induced Ser36 phosphorylation of p66Shc isoform of the SHC- transforming protein 1 resulting in increased ROS. Furthermore, reduced AGE-R1 expression could subsequently affect protein post-translation modifications through impaired N-glycosylation of key podocyte proteins such as nephrin resulting in podocyte detachment from the GBM. Alternatively, exposure to AGEs reduces insulin receptor and IRS1 tyrosine phosphorylation and increases IRS1 serine phosphorylation, resulting in impaired insulin signalling and decreased glucose uptake. This may have severe implications in an insulin sensitive cell such as the podocytes, leading to impaired metabolic activity, and consequently affecting podocyte function and health. Abbreviations: AGE, advanced glycation end productendproduct; AGE-R1, AGE receptor 1; FOXO, Forkhead box protein; FOXO3α, FOXO O3 α; IR, Insulin receptor; NADPH, nicotinamide adenine dinucleotide phosphate-oxidase; p66Shc, isoform p66 SHC-transforming protein; ROS, reactive oxygen species;

SIRT1, NAD-dependent deacetylase sirtuin-1; SOD2, mitochondrial superoxide dismutase 2 [Mn].

Introduction to this publication. The second review was published in Endocrinology and focuses on the role of ER stress in the pathophysiology of diabetic kidney disease.

Introduction.

A tightly balanced homeostasis exists between the synthesis and degradation of proteins, which is maintained in the endoplasmic reticulum (ER). When imbalance occurs in either one or both of these facets, the ER activates coordinated adaptive responses known as the unfolded protein response (UPR) and the ER-associated degradation (ERAD) pathway in an attempt to restore balance in the ER. However, when these responses are saturated, the misfolded proteins accumulate in the ER, and cells develop a detrimental condition referred to as ER stress. The principles of the

UPR are now relatively well defined 75-77, and the mechanisms of the signal transducers involved in the activation of this pathway have already been the subject of detailed reviews 75. However several key questions remain unanswered; (1) understanding how the UPR system integrates within specific resident cells of the kidney, (2) how diseases such as diabetes can promote an imbalance at these sites and (3) whether this ultimately leads to ER stress induced pathology culminating in nephropathy. This Review summarises recent developments in these areas and highlights new insights into translational applications into the clinical environment.

Function of the endoplasmic reticulum.

The ER functions as a site of synthesis, folding and maturation of secreted, luminal and transmembrane proteins. Synthesis of unfolded polypeptide chains occur in the rough ER where they undergo a maturation process, mediated by ER-resident enzymes and chaperones to form a stoichiometric stable conformation that is both energetically efficient and biologically activate 78, 79.

During the synthesis of new proteins, the stoichiometry of the amino acid chains can potentially be altered by post-translational modifications, thereby profoundly affecting the energy signature and conformation of the folded protein. The role of post-translational modifications are becoming more apparent in the development of disease, particularly in chronic conditions such as diabetes 80, due to their impact on the generation of inappropriately folded proteins, seen most vividly in congenital

35 disorders of glycosylation 81. Subsequently the maturation of these nascent proteins are further dependent on the appropriate intracellular concentrations of calcium, glucose and adenosine-5’- triphosphate (ATP), as well as the redox environment 82; all of which function as ER quality control factors. When proteins are both correctly folded and carry the appropriate post-translational modifications, the protein exits the ER and progresses through the secretory pathway to the Golgi body. When folded inappropriately, proteins accumulate within the ER, where UPR pathways are activated or terminally misfolded proteins are retro-translocated from the ER into the cytoplasm and undergo proteasome degradation in the ERAD pathway.

Protein homeostasis within cells is particularly complex in nature as a balance is forged between constant turnover, the synthesis of new and the degradation of old proteins, as well as the influx of new proteins and precursors from the extracellular environment. Unfortunately protein synthesis is inherently an error-prone process. Nevertheless, the dynamic nature of the ER allows cells to adjust their protein-folding capacity in response to fluctuations in the environment through transmembrane sensors that face the ER lumen and effectors that signal to other compartments of the cell, ensuring that secreted proteins are maintained at a high fidelity and at the correct concentrations to maintain protein homeostasis.

Triggering the unfolded protein response (UPR).

In mammalian cells, there are three major UPR pathways, activated by transmembrane sensors located in the ER membrane. These three major pathways depicted in Figure 1 are the inositol- requiring protein 1 (IRE1), activating transcription factor-6 (ATF6) and the PKR-like ER kinase- mediated (PERK) response. When protein synthesis and degradation in the ER is under homeostatic balance these three sensors are inactive and are hypothesised to be either; inherently inactive until unfolded proteins activate the sensors, maintained in an inactive state by the ER chaperone BiP, or a hybrid of both of these mechanisms 75. When activated, these pathways elicit several physiological

36 responses in order to counteract the increased accumulation of inappropriately folded proteins.

Ultimately the purpose of the UPR pathway is to prevent an overabundance of misfolded proteins in the ER, which would consequently result in ER stress and potentially cell death.

As misfolded proteins begin to accumulate in the ER, pathways of the UPR are activated. The first identified stress transducer IRE1 83, 84, undergoes oligomerisation following the accumulation of misfolded proteins and trans-autophosphorylates, which allosterically activates IRE1 endoribonuclease activity via a conformational change 85. IRE1 mediated sequence-specific cleavage is targeted at X-box-binding protein 1 (XBP1) mRNA causing a frame shift in the coding sequence. A frame shift in XBP1 mRNA leads to the manufacture of the potent transcriptional activator (XBP1s), which activates genes encoding ER chaperones, enzymes that promote protein folding, maturation, secretion and ERAD components in an attempt to decrease ER load and relieve

ER stress 86, 87. In parallel, ATF6 traffics to the Golgi in the presence of misfolded proteins where it is cleaved by proteases S1P and S2P 88 revealing a cytosolic fragment (ATF6n) which has a DNA binding domain. ATF6n subsequently migrates to the nucleus and in a similar manner to XBP1s, functions as a transcriptional activator for proteins which assist in resolution of ER stress by increasing ER capacity 89.

Unlike the other two signal transducers, PERK phosphorylation activates a substrate known as eukaryotic translation initiation factor-2α (eIF2α). Activation of eIF2α reduces ER stress not by increasing the ER potential, rather eIF2α reduces the load on the load on the ER by inhibiting translation of new proteins from mRNA, leading to a reduction in overall protein synthesis through the inhibition of AUG codon recognition at the ribosomal level 90.

The pathways of the UPR are essential in maintaining normal physiological functions, particularly in cells with a high rate of protein synthesis (kidney tubule cells 24, 25) or secretory function

(pancreatic β-cells and liver cells 91). Additionally since the UPR is conserved through many species, perhaps the design of this pathway was intended to be acutely beneficial dependent on the disease conditions, ameliorating damage. Unfortunately when the UPR is consistently activated such as in the presence of a chronic condition (such as diabetes) or during viral infection where cellular protein machinery is a means for viral replication, this adaptive response of the UPR system develops into a cytotoxic response promoting increased autophagy and apoptosis in an attempt to halt disease progression.

ER stress in diabetes.

The ER stress response is thought to be involved in several pathophysiological developments throughout the progression of diabetes and the associated chronic complications. The relationship between ER stress and the regulatory metabolic pathways in the ER are incredibly diverse.

However as the current literature stands there appears to be a greater focus on ER stress and the downstream response in Type 2 diabetes (T2D) when compared to Type 1 diabetes (T1D), although this does not reflect the lack of relevance to this disorder as well as its role, in the development of complications associated with diabetes.

ER stress in type 2 diabetes.

Obesity is a common comorbidity in individuals with T2D characterised by a dysregulation in hepatic glucose production in combination with declining peripheral insulin sensitivity in the face of inadequate insulin secretion by the pancreatic β-cells. During the progression of T2D, there is an increased demand on the β-cells for insulin production in order to compensate for the ongoing intolerance. Moreover an unfortunate eventuality for the majority of obese type 2 diabetics is the advent of both glucotoxicity and lipotoxicity, where the imbalance between glucose and lipid homeostasis is a confounding result of nutrient excess as well as other comorbidities such as renal or hepatic disease.

Initial studies implicating ER stress in the pathogenesis of T2D identified that genetically obese mice (ob/ob) or mice fed a high fat diet inducing obesity had increased parameters of the UPR in the liver and adipose tissues 92. Similarly in insulin-resistant, non-diabetic humans there are marked increases in the presence of the IRE1 protein, an indicator of the adaptive response of the UPR 93.

Despite only detecting changes in one arm of the UPR in the aforementioned study, another study cohort of severely obese patients whom underwent surgery assisted weight loss (BMI of 51.3 compared to a BMI of 33.5) exhibited significant decreases in all aspects of the UPR arms in the majority of patients, suggesting a significant reduction of ER stress after gastric-bypass-induced weight loss 94. Noteworthy were the concomitant improvements in overall metabolic health afforded to these patients with the associative surgery. Evidently certain polymorphisms in the ATF6 gene were identified to be associated with T2D and modulation of plasma glucose in certain populations of the Dutch 95 and Pima Indians 96. Unfortunately amongst indigenous populations, specifically the

Pima Indians 97, 98 and Australian Aborigines 99, 100, there is an inherent increased prevalence of diabetic comorbidities of both cardiovascular and renal disease. Whether genetic polymorphisms resulting in dysfunctional responses to ER stress could provide a basis accompanied by other factors for the accelerated progression of renal disease requires to be further established in these populations. These genetic associations are not constitutive amongst all ethnic backgrounds, with very little association noted amongst the certain population groups of the Chinese 101, 102, Caucasian

101 and African-American 101 ethnicities.

Prolonged exposure to nutrient excess in obese individuals promoting an environment of systemic

ER stress could synergistically contribute to the pathophysiology of both insulin resistance and hyperglycaemia in obesity and T2D. Each of these would clearly impact diabetic kidney disease where there are clear benefits of strict glycaemic 103, 104 and lipid control 105, 106, as well as weight

39 loss and caloric restriction 107, 108. However it is uncertain whether ER stress is a consequence of diabetes or the developed comorbidity of obesity.

ER stress in type 1 diabetes.

The traditional notion of T1D as primarily an autoimmune disease in humans is currently being challenged, and suggestively labelled as an inflammatory pancreatic-wide disease 109. Therefore this hypothesis raises interesting possibilities for other damage mechanisms such as ER stress as pathogenic mediators of T1D, given that islets isolated from individuals with T1D have increased levels of ER stress markers 110. A particularly interesting notion is the integral role of PERK, an ER stress signalling molecule in maintaining normal pancreatic health and function. Murine models with null mutations in Perk and mutations at the Ser51 phosphorylation site in eIF2α exhibit β-cell dysfunction and diabetes 111-113. Additional work has described the necessity for PERK function during the neonatal stage in pancreas development 114. Not only does PERK appear to be essential during the development of the pancreas, there is evidence that maintenance of normal β-cell function in the adult pancreas requires PERK 115. Furthermore, a mutation in the EIF2AK3 gene causes an autosomal recessive disease in humans known as Walcott-Rallison syndrome (WRS), whom often exhibit presentation of type 1 diabetes and renal insufficiencies during the neonatal period 116. Interestingly, within the South Indian population, polymorphisms between the regions around the EIF2AK3 locus show an association with T1D susceptibility 117. Evidently when compared to the neighbouring regions, the Southern Indian population has the highest prevalence of kidney disease 118. It has also been observed that South Indian/Asian migrants have greater prevalence and faster progression of diabetic nephropathy when compared to their European counterparts 119, 120. This is particularly noteworthy as it may suggest that in the advent of ER stress a deficiency in PERK function could possibly play a contributing role in the progression of comorbidities, such as diabetic nephropathy.

ER stress and diabetic nephropathy.

The role of ER stress in diabetic injury is not a new concept, with several indications that the presence of high glucose and free fatty acids in diabetic patients can induce ER stress. Implicating

ER stress in the development of complications such as retinopathy 121, 122 and cardiovascular disease

123, 124. However it has only been recently that there has been a focus on the potential role for ER stress in the pathogenesis of diabetic nephropathy (DN), with reports identifying the activity of the

UPR in podocytes and other kidney cells 125-127. Lindenmeyer and colleagues 128 identified the induction of UPR genes in humans with DN in a manner that is dependent on the degree of kidney injury. Where biopsies of patients with established DN exhibited increased levels of XBP1 and ER chaperones (HSPA5/GRP78, ORP150/HYOU1), when compared to those with only mild diabetes.

Moreover, much like the pancreatic β-cells, the kidneys could constitutively be a site sensitised for the induction of ER stress, due to the high rates of protein synthesis. Although surprising, in humans it is estimated that the fractional rates of protein synthesis by the kidneys is approximately

42% of the total body load daily 24 which is the highest in the body, further suggesting that kidney cells could be highly susceptible to ER stress, due to fluctuations in ER load. It is important to identify the disparate functions of the different resident kidney cells, and understanding how ER stress contributes to DN pathophysiology in the unique cell populations may elucidate novel pharmacological targets to remediate disease.

ER stress mediate glomerular injury.

Glomerular epithelial cells (podocytes) play an essential role in the health and function of the glomeruli. The podocytes are insulin sensitive cells 30, 129 that contract to limit diuresis, fluxing nutrients into the urine for the primary purpose of nutrient retention and post-prandial utilisation.

Unfortunately the podocyte is a terminally differentiated cell with limited capabilities to regenerate and proliferate following injury, therefore reduced podocyte density is argued to be a crucial determinant in the development and progression of DN 130, 131. The induction of ER stress has been

41 elegantly reviewed in primary glomerular diseases 132. However developing a greater understanding as to how ER homeostasis is perturbed in these cells during diabetes and the subsequent adaptive responses is warranted.

Mechanisms culminating in a stressed diabetic podocyte ER.

Currently there are no direct studies investigating the specific role that ER stress plays through the targeted deletion of the ATF6/IRE1/PERK pathways in the podocytes, irrespective of diabetic conditions. Common insults such as the accumulation of advanced glycation end products (AGEs) and the activation of their receptors have been implicated in not only the pathogenesis of DN 133-136 but also the induction of ER stress 126, 137. Moreover the accumulation of AGEs within the skin

(representing tissue accumulation), also predicts those T1D individuals at greatest risk of developing nephropathy 138. Additionally albumin, a major component in the proteinuria observed in DN, is often reflective of progressive disease associated with a declining glomerular filtration barrier and increased leaching into the urine. Not only could excessive exposure of albumin to podocyte induce ER stress pathways leading to caspase-12 mediated podocyte apoptosis independent of mitochondrial input 139, it appears that the modification of albumin by AGEs enhances pro-apoptotic pathways in podocytes in a dose- and time-dependent manner 140. Moreover

Chuang and colleagues 141 observed that both soluble and matrix bound AGEs induced apoptosis in podocytes in an additive manner and they further observed an increase in podocyte detachment when cultured in the presence of AGE-modified collagen IV. This is of particular interest as this may implicate not only the number of podocytes as a major determinant in the development and progression of DN but also their integrity, which has been well described 142. Moreover parallels can be drawn from this postulation, with the identification that the ability for the podocyte to effectively adhere to the extracellular matrix is a major determinant of ER stress induction and the subsequent development of glomerular injury 143. This may suggest that with the onset of podocyte dysfunction, ER stress could potentially exacerbate damage to the cell.

Moreover it is suggested that during the advent of hyperglycaemia in diabetic patients, the balance of the ER load in the podocyte is perturbed through a number of systemic pathways leading to the induction of ER stress and subsequently podocyte injury. Indeed, ER stress is induced in podocytes when acutely stimulated with both high 144 and low 47 glucose conditions, and it is apparent that there are regulatory mechanisms to alleviate this stress when podocytes are chronically exposed to hyperglycaemic conditions 137. Given that cell survival is promoted through the activation of autophagy in the advent of ER stress induced in mammalian cells under a high glucose environment

145, it is likely that compensatory modifications to the ER could be directly linked with autophagy.

Adaptive responses to a perturbed ER in the diabetic podocyte.

Autophagy, a highly regulated lysosomal pathway, selectively targets and removes damaged organelles and is suspected to further function in tandem with the ER as a compensatory mechanism for proteosomal degradation 146. Not only is autophagy implicated to function in the adaptation of stress and survival mechanisms, autophagy may also play a distinctive role in the metabolic regulation of a cell. It appears that autophagy intricately regulates lipid metabolism 147, 148 and is involved in the degradation of large glycogen stores in the hepatocytes of neonates 149. Interestingly podocytes maintain a constitutively higher level of autophagy when compared to other intrinsic renal cells 150, 151. This may simply be due to the limited capacity for regeneration of the podocyte and hence requirement to constantly recycle and renew cellular components. However there may be an underlying involvement of autophagy in the metabolic capacity of the podocyte.

Recent developments in the understanding of the function of the protein kinase mammalian target of rapamycin complex 1 (mTORC1) in podocytes, has identified a relationship between autophagy and

ER function. mTORC1 is a kinase complex involved in a myriad of cellular processes including a modulatory role of autophagy in response to nutrients such as glucose, amino acids and growth

43 factors 152. Interestingly, there is evidence to suggest that much like mTORC1, the UPR can be activated through various physiological stimuli (circulating FFAs 153, hypoxia 154 and growth stimuli 155, 156) that do not necessarily act through the accumulation of unfolded proteins in the ER.

Moreover, a recent review 157 elegantly summarises how mTORC1 operates both upstream and downstream of ER stress signals, essentially inferring an intricate relationship between ER stress, autophagy and mTORC1. The speculation that mTORC1 may be involved in the pathogenesis of

DN, came from the observation that an inhibitor of mTORC1 (rapamycin) attenuates renal hypertrophy in experimental models of DN 158. Rapamycin selectively restores integrity of the podocyte foot processes through the stabilisation of the protein nephrin to the filtration barrier and reduces subsequent tubular and glomerular damage 159, 160. Nephrin is an integral podocyte protein involved in the retention of larger macromolecules in the blood as well as playing a role in the membrane docking of the insulin sensitive glucose transporter, GLUT4 161. However, the changes to the role and function of mTORC1 in podocytes, in response to the metabolic alterations that occurs during diabetes remain to be completely delineated.

It has been suggested however that mTORC1 activation in podocytes may play an essential role in the development of DN, as podocyte-specific mTORC1 activation in the absence of diabetes recapitulated several features of DN 160. Coupled with the chronic activation of mTORC1 in the podocytes, ER stress was enhanced in the glomeruli and the pathological changes in the podocyte could partially be attributed to ER stress 160. When mTORC1 activity was genetically reduced in diabetic mice, there was a significant reduction in the development of DN 160. Consistent with the ideology that the activation of the UPR can occur both downstream and upstream of mTORC1 infers a particularly interesting notion, that in certain contexts, mTORC1 activation is a component in the process of ER stress induced cell toxicity, and if perturbed in the podocyte, could be a crucial to the development of DN. Indeed, despite the fact that hyper-activation of mTORC1 in mouse podocytes led to a phenotype akin to that of DN, reducing mTORC1 activity similarly promoted

44 podocyte dysfunction 162, suggesting that mTORC1 activity is essential for the development of the podocyte. Interestingly Inoki and colleagues identified that mTORC1 was causing podocyte dysfunction by retention of nephrin in the cytoplasm, where it could not localise to the renal filtration barrier during hyper-activation of mTORC1 in podocytes 160. Not only would impairments and the subsequent accumulation of dysfunctional nephrin promote ER stress 163, a decline in nephrin is known to lead to the development of proteinuria 164 and glucose homeostatic defects in podocytes. As alluded to above, a loss of nephrin also impairs GLUT4 trafficking to the plasma membrane and therefore insulin dependent glucose uptake 161. However the mechanism by which mTORC1 activity alters the capacity of nephrin to localise to the glomerular filtration barrier needs to be further explored. One possible avenue would be to identify the effects of post translational modifications of nephrin in the ER and golgi which could alter nephrin localisation and function.

One particular modification of interest is N-glycosylation which plays a critical role in mediating membrane localisation of nephrin 45 and occurs within the ER. Hence, impaired protein modification in the ER, either through mechanisms directed or indirectly associated with ER stress, could also promote podocyte dysfunction and hence influence the progression of DN.

These data illustrate a rather intricate relationship between ER stress and mTORC1 activity (Figure

1). Although there is growing body of evidence indicating the crucial role for the mTORC1 pathway in podocyte health, unfortunately as of current there is a limited understanding of the intersecting relationship between ER stress, mTOR and the UPR in the development of human DN.

ER stress mediated tubulointerstitial injury.

Tubulointerstitial damage is considered a final common pathway to end-stage kidney disease where the deposition of extracellular matrix, oedema and infiltrating cells separate the proximal tubules from their intimate contact with the renal tubular capillaries. Since one of the primary functions of the proximal tubule is the reabsorption of molecules from the urinary filtrate such as glucose and

45 sodium and their return into the bloodstream, tubulointersitital fibrosis is the best prognostic indicator of progression to end stage renal disease requiring transplantation or dialysis. However, in patients with diabetes, renal biopsies are no longer performed routinely due to an increased risk of bleeding and poor wound healing within this population. Proximal tubular epithelial cells (PTCs) are another particular cell of interest in the pathophysiology of kidney tubular injury in DN 165, particularly as they are highly susceptible to ER stressors 166 and are highly synthetic and metabolically active cells.

Mechanisms culminating a stressed diabetic PTC ER.

A common hallmark of DN is proteinuria, specifically the loss of albumin (albuminuria) and one of the central roles of the PTCs is the ability to reabsorb a proportion of proteins that may be filtered by the glomerulus and consequently end up within the urinary filtrate, which is exacerbated in the context of diabetes. Moreover several studies show a direct association between proteinuria and pathogenesis of tubular injury, with levels of albumin and modified-albumin as the determinant factor 167, 168. The effects of albumin on PTCs are biphasic, where on one hand albumin stimulates the growth of PTCs 169, 170 while on the other hand, excessive exposure to albumin is implicated in tubular atrophy in a pro-apoptotic and pro-inflammatory environment 171. Moreover albumin induced ER stress is implicated to cause tubular damage through the activation of caspase-12 subsequently resulting in apoptosis of PTCs 171. Excessive reabsorption of albumin is also suggested to promote reactive oxygen species production and activation of PPARγ, stimulating

GRP78 and eIF2α phosphorylation, both of which are markers of ER stress. ER stress subsequently stimulated downstream phosphorylation of JNK and NF-κB, resulting in increased expression of sodium dependent glucose transporter-2 (SGLT2) expression in cultured rabbit PTCs 172. Increased

SGLT2 expression has previously been identified in PTCs isolated from the urine in T2D patients

173. However mice which have the expression of SGLT2 ablated in the PTCs are not protected against the development or progression of kidney injury 174. Whether increased expression of

SGLT2 correlates with an increased risk for the development of DN is yet to be identified.

Nevertheless, given the important role of SGLT2 in glucose reabsorption in the proximal tubule and the advent of new therapies in diabetic treatment regimens that target this protein to control hyperglycaemia by inducing glycosuria 175, 176, these pathways involving ER stress warrant further investigation and understanding.

Adaptive responses to a perturbed ER in the diabetic PTC.

What is particularly interesting to note is the identification of mTORC1 as an upstream mediator in

PTCs, prior to the induction of ER stress as a result of excess albumin exposure 177, although whether albumin derived from human sera functions in a likewise manner is yet to be determined.

Similar to podocytes, mTORC1 may also play a crucial role in the pathophysiology of kidney tubular injury through the regulation of ER stress. Interestingly it has been further elucidated in tubular cells that activation of pro-apoptotic pathways can be mediated by the induction of ER stress. Not only was mTORC1 identified to function downstream of ER stress in these cells, but subsequent mTORC1 activity involved pathways culminating in IRE1-JNK activation 159.

Interestingly inhibition of mTORC1 with rapamycin under the same physiological conditions selectively inhibited the IRE1 pathway. This further implies mTORC1 activity both upstream and downstream of ER stress, however the reason that mTORC1 selectively activates the IRE1 branch and not the other branches of the UPR need to be further elucidated. One interpretation (Figure 2) suggests that albumin may direct ER stress in the proximal tubule of diabetic patients with albuminuria, mediating reduced insulin responsiveness in the PTCs through IRE-1JNK activation driven by mTORC1. Which the IRE1-JNK pathway has been shown to reduce insulin sensitivity in adipocytes, although mTOR activity was decreased in the adipose tissue of the T2D patients 178.

Reduced mTOR activity could simply be a consequence of developed insulin resistance, as sustained activation of mTORC1 renders insulin receptor signalling pathways irresponsive to insulin 179. Ultimately whether mTORC1 activity would reduce insulin sensitivity in PTCs would

47 need to be confirmed. How insulin is relevant to the PTCs however is an evolving concept given that these cells are not physiologically insulin sensitive cells with respect to glucose uptake, nor do they manufacture the enzymes to perform glycolysis under normal conditions 180. A recent paper identified that indeed PTCs require insulin receptor mediated signalling for their normal function, and when the insulin receptor was specifically deleted in the PTCs, mice exhibited increased renal gluconeogenesis independent of glucose clearance, as well as enhanced insulin secretion and action

181. It is postulated that the down regulation of insulin receptor and/or insulin-resistance in the PTCs could further contribute to hyperglycaemia, which could effectively enhance the risk for the development of DN. Although the mechanisms and actions of mTORC1 remain to be further explored in these cells, mTORC1 activity may play a central role not only in tubular injury via changes in insulin receptor signalling but also the development of DN.

Therapeutic implications of ER stress modulators.

Current approaches to treat DN largely target systemic blood pressure and/or intraglomerular hypertension. More often than not the first line of therapies for the treatment of DN are those which influence the renin-angiotensin system, including; angiotensin converting enzyme inhibitors 182 and angiotensin II (ANG II) receptor antagonists 18, with concurrent glycaemic control agents and often therapies for hyperlipidaemia. Despite these interventions, progression of DN to end stage renal disease can only be slowed but not cured. Although these interventions are currently the most effective clinical management of microvascular complications such as nephropathy, there is also evidence to suggest that strict glycaemic control does not necessarily reduce the risk of cardiovascular disease 183 and may potentially elevate the risk of a cardiovascular event 184.

Moreover an unfortunate phenomenon often observed in the treatment of diabetes and its complications is poor adherence to treatment regimens, particularly with rates of adherence inversely proportional to the number of diabetic medications prescribed 185. Not only does this provide a rationale for the necessity of developing novel therapeutics which can target multiple

48 pathways, but also therapeutics that assists in improving patient compliance and as well as addressing other pathogenic mediators. A potential avenue to assist in achieving this therapeutic goal may be to target ER stress pathways.

There are a myriad of compounds (Table 1) that improve ER folding capacity such as the chemical chaperones 4-phenylbutyric acid (PBA), taurine-conjugated ursodeoxycholic acid (TUDCA) and

ER chaperones which reduce ER stress and improve insulin action and sensitivity 186, 187. Small- molecule chemical chaperones such as TUDCA and 4-PBA are suspected to either enhance protein secretion or the folding capacity of the ER 188. Indeed, recent work in experimental models of DN show that 4-PBA 189 and TUDCA 140, 190 could potentially slow the progression of DN through the attenuation of ER stress induced apoptosis via the reduction of GRP78 and PERK expression, and the restoration of defective autophagy. However, it is not yet understood whether chemical chaperones could directly improve kidney function or these results are simply confounded by an improvement in glycaemic control seen with this class of agents. Despite a recent clinical investigation identifying that oral TUDCA administration can increase both hepatic and muscular insulin sensitivity in obese non-diabetic patients 191, markers of ER stress were not improved and therefore it is evident that there is still relatively little known on the long term efficacy and target specificity of these ER stress modulators in humans.

Rapamycin (Sirolimus®) an mTOR inhibitor is currently approved by the FDA as an anti-restenosis agent and immunosuppressant. Clinical studies of rapamycin have shown significant efficacy in improving glycaemic control in T1D patients following pancreatic islet transplantation 192.

Moreover Sirolimus-based trials of kidney transplantation also improved acute rejection and minimised nephrotoxicity often seen with other immunomodulators 193, 194. However it is difficult to determine if this class of agent or rapamycin-based therapeutics could be beneficial in the treatment of DN. Although mouse based experimental models suggest that a reduction in mTORC1 activity

49 could reduce the development of DN 160. Pharmacological inhibition of the mTOR pathway by rapamycin should be cautiously approached given the evident off-target effects described with longstanding application of rapamycin 195.

Chemical chaperones that enhance protein folding are not the only avenue for ER modulation.

Indeed, agents targeting other pathways such as dietary intervention through the reduction of palmitic acid could reduce ER stress mediated CHOP upregulation in podocytes and thereby attenuating pro-apoptotic pathways mediated by ER stress 196. Moreover antioxidant therapy using

α-lipoic acid has been identified to reduce hepatic lipid accumulation, ER stress biomarkers and glomerular mesangial matrix expansion 197, 198 in other contexts. There is evidence that increasing consumption of polyunsaturated fatty acids (PUFAs) could attenuate DN both in animal models and in humans 199, although it remains to be determined whether this is modulated through an improvement in ER stability. These results suggest that perhaps dietary intervention involving a shift to increased unsaturated FFAs and reduced saturated fats could reduce ER stress-related renal cellular toxicity, particularly in T2D where lipid accumulation plays a more integral role in disease pathology.

Concluding remarks.

Whether ER stress directly contributes to the pathogenesis of nephropathy in the context of diabetes is still an unanswered question, but it is apparent that there is ER stress within specific renal cells.

Furthermore there are certainly lessons to be learnt from other sites within the body, as it is evident that ER stress is not a uniform phenomenon particularly affecting highly synthetic sites. Hence drawing similarities between seemingly disparate sites which commonly present with ER stress in disease states may assist us to further understand this mystery. However, whether ER stress plays a causative role in the pathogenesis of DN is not yet defined, nor is the regulatory role of the UPR in highly synthetic organs such as the kidney, well understood. Certainly using modulators of ER

50 function and stress in experimental models of DN may provide some answers in this context.

However the efficacy of these modulators to specifically target the renal cells of interest and there mechanism of action, needs to be further explored particularly given the complexity of the kidney which contains more than eight resident cell types. There should also be an emphasis placed on the investigation of societal and psychological implications as to how to best integrate these novel therapeutics in conjunction with proven treatment regimens to assist in improving government regulatory authorities, patient compliance and ultimately patient health and well-being.

Figure 1. A summary of the proposed mechanisms of ER stress induced podocyte dysfunction.

(a) Glomerular stress events, in this case mediated by diabetes and the subsequent metabolic and catabolic changes perturb the balance between mTORC1, the ER and UPR. (b) AGE-BSA induces podocyte apoptosis mediated by caspase-12 activation 139, 140. (c) AGE-modified collagen IV contributes to podocyte detachment or dysfunction 141 and ER stress leading to the development of podocyte dysfunction. Obesity is likely a factor contributing to dysfunctional mTORC1 activity, given that mTORC1 activity is increased in the glomerular in obese db/db mice with a targeted genetic insertion of mTORC1 activity when compared to db/m mice 160. (d) Chronic changes in mTORC1 activity could be a determinant pathway leading to the development of diabetic nephropathy through various pathological changes to the podocyte 160, 161.

Figure 2. A summary of the proposed mechanisms of ER stress induced tubular dysfunction.

Metabolic imbalances in diabetes (hyperglycaemia, inflammation and obesity) contribute to the interacting pathways of ER stress contributing to tubular dysfunction. Enhanced glycation is directly associated with pathways implicated in the development of diabetic nephropathy through the modification albumin generating large macromolecular complexes 135. (a) Excessive reabsorption of albumin is thought to overload the PTCs leading to ER stress-induced apoptosis mediated by caspase-12 activation 171. (b) Moreover albumin appears to have an intricate signaling pathway both upstream and downstream of ER stress mediated by mTORC1. Enhanced mTORC1 activity in the PTCs not only contributes to pro-apoptotic pathways, but through the selective activation of the IRE1-JNK pathway could lead to decreased responsiveness to insulin in the PTC.

53 resistance was only identified in adipocytes from T2D patients 178 and has not been substantiated in

PTCs. (d) Furthermore JNK activation could further promote metabolic imbalances in the PTCs through ER stress induced NF-κB stimulation of SGLT2 expression 172. Understanding how ER stress might perturb the metabolic integrity of the proximal tubule could be a crucial pathway contributing to the pathogenesis of DN.

Table 1. ER stress modulators of particular interest ER stress modulator Mechanism of action Cell Disease model Outcomes Reference investigated 4 Phenyl-butyric acid (4- ER chaperone STZ-induced Attenuates proteinuria, oxidative stress 189 PBA) diabetes markers p-JNK, MCP-1 and TGF-β1, ER stress markers GRP78 and PERK Taurine-conjugated ER chaperone Podocytes Suppressed AGE-induced elevation of 140 ursodeoxycholic acid GRP78 and dose-dependently inhibited (TUDCA) podocyte apoptosis ER chaperone Clinical trial: Increased hepatic and muscle insulin 191 obese non- sensitivity. Markers of ER stress did not diabetic change Palmitoleic/oleic acid Attenuated palmitic acid- Podocytes Reduced palmitic acid-induced apoptosis of 196 induced upregulation of podocytes CHOP Rapamycin Specific suppression of PTCs TM-treated mice Suppressed renal tubular injury 159 IRE1-JNK signalling Reversed mTORC1- Podocytes PcKOTsc1 mice Early intervention regenerated podocyte 160 dependent podocyte foot processes injury Abbreviations previously not mentioned: STZ, streptozotocin; TM, tunicamycin; ATN, acute tubular necrosis; ACR, albumin:creatinine ratio

Chapter 3: Increased liver AGEs induce hepatic injury mediated through an OST48 pathway.

Introduction to this publication. This chapter was published in Scientific Reports as an original article. In this report, we over-expressed the gene encoding for oligosaccharyltransferase-48

(OST48), a protein of the N-glycosylation machinery. OST48 is also postulated to act as a membrane localised clearance receptor for advanced glycation end-products (AGE). In this novel study, over-expression of OST48 resulted in hepatic fibrosis, which proceeded without steatosis, obesity or a lack of physical activity.

Specifically, we demonstrated the following; 1) Mice over-expressing OST48 (DDOST+/-) and wild-type mice exposed to AGEs had modest liver injury, but DDOST+/- mice exposed to AGEs had significant hepatic fibrosis and elevation in plasma concentrations of liver enzymes. 2) Fibrotic, dysfunctional livers demonstrated ER stress (SWATH proteomics) however there were no changes in N-glycosylation of secreted proteins. 3) DDOST+/- mice had increases in OST48 localisation to hepatic cell membranes and the small intestine which increased delivery and accumulation of hepatic AGEs ascertained by Near IR imaging studies. 4) DDOST+/- mice with hepatic fibrosis had specific increases in central adiposity but were not overweight (using EchoMRI and adipose tissue weight) and were more physically active (CLAMS). 5) Studies using OGTT and ITT showed impaired glucose tolerance and abnormalities in insulin secretion in DDOST+/- mice which exacerbated liver fibrosis. 6) Indirect calorimetry and hepatic SWATH proteomics/qPCR showed a shift towards fatty and keto acid utilisation in DDOST+/- mice which were more pronounced with

AGE exposure. 7) High AGE fed DDOST+/- mice also had hepatic glycogen accumulation and hepatomegaly.

In summary, in this group of studies we show a novel OST48/AGE pathway to liver fibrosis that progressed despite ample physical activity and in the absence of steatosis. The data also suggests a previously unappreciated physiological trafficking role of OST48 in the gastrointestinal tract and liver, which when dysregulated result in hepatic fibrosis.

Graphical Abstract

Published Abstract

Abstract

The protein oligosaccharyltransferase-48 (OST48) is integral to protein N-glycosylation in the endoplasmic reticulum (ER) but is also postulated to act as a membrane localised clearance receptor for advanced glycation end-products (AGE). Hepatic ER stress and AGE accumulation are each implicated in liver injury. Hence the objective of this study was to increase the expression of

OST48 and examine the effects on hepatic function and structure. Groups of 8 week old male mice

(n=10-12/group) over-expressing the gene for OST48, dolichyl-diphosphooligosaccharide-protein glycosyltransferase (DDOST+/-), were followed for 24 weeks, while randomised to diets either low or high in AGE content. By week 24 of the study, either increasing OST48 expression or consumption of high AGE diet impaired liver function and modestly increased hepatic fibrosis, but their combination significantly exacerbated liver injury in the absence of steatosis. DDOST+/- mice had increased both portal delivery and accumulation of hepatic AGEs leading to central adiposity, insulin secretory defects, shifted fuel usage to fatty and ketoacids, as well as hepatic glycogen accumulation causing hepatomegaly along with hepatic ER and oxidative stress. This study revealed a novel role of the OST48 and AGE axis in hepatic injury through ER stress, changes in fuel utilisation and glucose intolerance.

Introduction

Chronic liver disease is increasing globally as a result of obesity and diabetes, with non-alcoholic fatty liver disease (NAFLD), the most common liver disorder 200. However, NAFLD is asymptomatic and most individuals do not progress to non-alcoholic steatohepatitis (NASH). The development of NASH is postulated to be the result of a ‘second hit’ environmental factor 201.

Glucose intolerance and dyslipidaemia which are often characteristic of NAFLD 202, facilitate the non-enzymatic post-translational modification of proteins, forming advanced glycation end products

(AGEs) 203, which accumulate in tissues and contribute to liver fibrogenesis 203. Excessive exposure

59 to exogenous sources of AGEs, such as from food, can also exacerbate liver injury in the absence of glucose intolerance 204. This is likely via simple diffusion through the epithelium if cleaved 7, although AGE specific receptors may facilitate receptor-mediated gastro-intestinal trafficking. Once absorbed, AGEs are directed to the liver through the portal vein and excreted via the gall bladder by hepatic sinusoidal Kupffer and endothelial cells 205, and by renal clearance 206.

The liver, however, is not only a site for the clearance of AGEs, but also a target organ and expresses various AGE receptors including the receptor for advanced glycation end-products

(RAGE) 207, advanced glycation end product-receptor 1 (OST48 also known as AGE-R1) 44 and galectin-3 (AGE-R3) 208. There is accumulating evidence which suggests that in NAFLD, AGEs are a ‘second hit’ that triggers progression from steatosis to NASH 209. Galectin-3 (AGE-R3) has also been associated with inflammation and liver fibrosis 208 and a Phase 2a multi-centre trial is underway in individuals with portal hypertension and NASH cirrhosis (NCT02462967), using galectin-3 inhibitors. Although the studies to date in galectin-3 have focused on its immunomodulatory roles, it is feasible that AGE binding and signalling via galectin-3 is also prevented by galectin-3 inhibition contributing to the efficacy of these agents in preventing liver pathology.

The effects of AGEs are mediated by their receptors, which the effects can be broadly contrasting depending on the type of receptor. For example RAGE facilitates oxidative stress, cell growth and inflammation 210. Specifically, in chronic liver injury, hepatic expression of RAGE is significantly increased 211 and several studies in acute liver injury have identified that blockade of RAGE can ameliorate toxic, ischemic and cholestatic liver damage 212. Alternatively, another AGE receptor,

OST48, is thought to be responsible for the detoxification and clearance of AGEs and negative regulation of AGE pro-inflammatory signalling 9, 49, and cellular oxidative stress 59. A decline in the expression of OST48 associated with increases in AGEs has been demonstrated both in murine

60 models 52 and in individuals with diabetes 49. Although OST48 is postulated as an AGE clearance receptor, its primary role is within the endoplasmic reticulum (ER) lumen 43 where as a subunit of the multiprotein oligosaccharyltransferase complex it facilitates enzymatic N-linked glycosylation of selected asparagine residues during protein translation 213.

The present studies have demonstrated that OST48, a protein that was previously thought to mediate its effects via N-glycosylation or detoxification and clearance of AGEs, is rather a facilitator of increased AGE deposition in the liver. Specifically, a novel OST48-AGE pathway that leads to the onset of liver injury in combination with central adiposity and glucose intolerance, despite ample physical activity. These results establish a previously unappreciated physiological trafficking role of OST48 in the gastrointestinal tract and liver, which when dysregulated result in liver injury.

Results

Generation of a ubiquitous OST48 knock-in mouse model. OST48 knock-in mice

(ROSA26tm1(DDOST)Jfo hereon termed as DDOST+/-) were heterozygous in their expression of human

DDOST at the ROSA26 locus under the control of the ubiquitin promoter (Fig. S1A). There was a significant increase in hepatic OST48 gene (DDOST) expression (Fig. S1B), whilst endogenous gene expression (Ddost) was unaffected (Fig. S1C). Targeted proteomics identified that 32 week old DDOST +/- mice did not show a significant change in total OST48 protein in the gut using the major peptides detected (Fig. S1D). However, there is a substantial increase in OST48 protein in each major section of the gut than compared to an average of 5 differently tissue locations (Fig.

S1D). Moreover, in fractionated liver tissue taken from DDOST+/- mice at the same time point, there was a significant increase in plasma membrane localisation of DDOST+/- fed on a high AGE diet (Fig. S1E). This increase in plasma membrane OST48 protein localisation and content in liver taken from DDOST+/- high AGE fed mice was not seen in the cytosol (data not shown). The small

61 intestine (duodenum, jejunum and ileum) had a high relative abundance of OST48 protein when compared with other tissues (Fig. S1D).

In the absence of steatosis, hepatic injury was evident in DDOST+/- mice and this was exacerbated by a high AGE diet. Hepatomegaly was evident in DDOST+/- mice irrespective of the diet (Fig. 1A). H&E staining indicated that wild-type mice on a high AGE content diet had some hepatocellular ballooning, with modest rarefication of the cytoplasm (Fig. 1B). All

DDOST+/- mice exhibited hepatocellular enlargement and ballooning, clusters of lobular plasma cells, increased inflammatory infiltration and an abundance of rarefied cytoplasm in hepatocytes

(Fig. 1B), as well as increases in hepatic fibrosis exhibited by increased Sirius Red staining and positive α-SMA staining, respectively, (Fig. 1C & D). Although there was no substantial differences in Sirius Red staining between high AGE fed WT mice and DDOST+/- mice (Fig. 1C).

We could however observe a gradual exacerbation of α-SMA staining in high AGE fed DDOST+/- mice (Fig. 1D). Plasma concentrations of the liver enzymes ALT and AST were also increased in

DDOST+/- mice (Fig. 1E), and plasma ALP concentrations were the highest in DDOST+/- mice fed a high AGE diet, suggesting the presence of hepatocellular damage.

Hepatocellular ballooning is often indicative of lipid droplet accumulation in the liver. Despite the presence of vascular fibrosis, there were no differences among groups in hepatic Oil Red O staining for lipid droplets (Fig. 1F; right). Further, there was a significant reduction in both hepatic DAGs

(Fig. 1F; top; P = 0.0101) and ceramides (Fig. 1F; bottom; P = 0.0246) associated with high AGE dietary intake. There was also a modest decrease (-30.9% low AGE diet and -15.9% high AGE diet,

P = 0.0516, 2-way ANOVA) in hepatic ceramide content when comparing the DDOST+/- genotype with the WT mice however, this did not reach statistical significance.

DDOST+/- mice exhibited increased absorption of AGEs in the liver. The two-hit model of a high AGE diet and DDOST+/- mice exhibited increased deposition of both CML and OST48 in hepatic tissue sections (Fig. 2A). Oral administration of near-infrared labelled AGEs using

IVIS/MR imaging, suggested prolonged exposure of AGEs in the liver of DDOST+/- mice (Fig.

2B). We further identified that hepatic AGE deposition was greater in mice fed a high AGE diet and in DDOST+/- mice irrespective of dietary alterations (Fig. 2C). Although WT mice fed a high AGE diet had similar deposition of AGEs in the liver, it was identified that circulating AGE concentrations were reduced in all DDOST+/- mice as compared with high AGE fed littermate WT mice (Fig. 2D) indicating an increase in trafficking and exposure of AGEs from the circulation in the liver.

Despite the absence of steatosis DDOST+/- mice had increased adiposity in addition to increased physical activity. There were no differences in body weight or lean body mass at either the beginning (Fig. S2A and B) or the end of the study (Fig. S2D and E) among mouse groups.

Body fat mass was increased in DDOST+/- mice as compared to their respective WT group (Fig.

3A; left), and this was more pronounced with high AGE feeding at the study end. Indeed, adiposity, measured as the intra-abdominal fat pads (mesenteric and omental fat pads), was increased in DDOST+/- mice as compared to WT littermates (Fig. 3A; right). At the beginning of the study, there were no significant differences in adiposity between WT and DDOST+/- mice (Fig.

S2C). Increased adiposity in DDOST+/- mice was not a consequence of reduced physical activity

(Fig. 3B; left), nor of increased food intake (Fig. S2F). In fact, DDOST+/- mice exhibited increased locomotor activity during both dark/awake and light/sleep phases (Fig. 3C; right), irrespective of diet.

Hepatic injury associates with ER stress, oxidative stress and activated inflammasome.

Confocal microscopy, showed co-localisation of GRP-78 with OST48 (Fig. 4A). Unbiased

63 proteomic profiling of hepatic tissue using SWATH-MS, identified increases in key proteins involved in ER stress, specifically GRP78 (Fig. 4B; top) and EIF3A (Fig. 4B; bottom) in our two- hit model of OST48 over-expression and high AGE feeding. The anti-oxidant enzymes

SOD1/SODC (Fig. 4C; top) and SOD2/SODM (Fig. 4C; bottom) were increased in DDOST+/- mice irrespective of the diet consumed. DDOST+/- mice fed a low AGE diet also had increases in the redox responsive GPX1 (Fig. S3A; left) and aconitase/ACON (Fig. S3A; right) when compared to low AGE fed WT mice. Furthermore, we also observed a significant (P = 0.034) effect of a high AGE diet on increased 8-isoprostane levels in the urine (Fig. 4D). DDOST+/- mice fed a high AGE diet also had significant increases in the inflammatory proteins CD47 (Fig. S3B; left) and HA1D (Fig. S3B; right).

Defects in N-glycosylation do not explain the hepatic injury seen in DDOST+/- mice fed on a high AGE diet. Prolonged ER stress culminating in liver injury is often associated with impaired

ER function leading to changes in glycosylation occupancy on proteins. We therefore investigated whether DDOST+/- mice altered hepatic protein synthesis N-glycosylation. Fifty-six glycosylation sites were identified on mouse plasma proteins (Fig. S4A) with six indicating partial modification by N-glycosylation (Fig. S4B), but there were no differences among groups in glycosylation occupancy on plasma proteins (Fig. S4A), nor in plasma proteins specifically glycosylated and secreted by the liver (Fig. S4C).

Hepatic injury was associated with changes in fatty acid metabolism.

Using continuous indirect calorimetry, we determined that DDOST+/- mice had decreased respiratory exchange ratios during both the sleep/light (Fig. 5A; left) and active/dark (Fig. 5A; right) phases. This downward shift in RER suggested a proportional shift towards the use of free fatty acids and/or ketones for ATP generation as compared to wild-type littermates consuming either a low/high AGE diet. In support of this, an RER of 0.9, seen in DDOST+/- mice on a high

AGE diet during their active cycle, demonstrates a ~65:35 of carbohydrate:fat/ketone oxidation as compared with WT mice fed a low AGE diet, (RER of 0.98; ~93:7 carbohydrate:fat/ketone oxidation) 214. During the light cycle, DDOST+/- mice fed a high AGE diet had an RER of 0.88, demonstrating a large shift towards fatty acid and ketone oxidation (~59:41 carbohydrate:fat/ketone oxidation) as compared with low AGE fed WT mice (RER of 0.95; ~83:17 carbohydrate:fat/ketone oxidation). Moreover, we observed significant decreases in heat generation during both the sleep/light (Fig. 5B; left) and active/dark phases (Fig. 5B; right) specifically associated with a high

AGE diet (P < 0.0001) in both WT and DDOST+/- mice.

The gene expression of key enzymes involved in fatty acid utilisation by the liver were altered in

DDOST+/- mice fed a high AGE diet (Fig. 5C). Specifically, there was a significant increase in

Pparα gene expression in DDOST+/- mice fed a high AGE diet (Fig. 5C; top) as compared with other groups. The gene expression of Lepr (Fig. 5C; bottom) was decreased by both the high AGE diet and in all DDOST+/- mice. DDOST+/- high AGE fed mice, also showed a decrease in the gene expression of both Acadvl (Fig. S5; left) and Acadm (Fig. S5; right), encoding enzymes involved in the oxidation of medium and long chain fatty acids.

Using SWATH-MS proteomics, significant increases in proteins associated with fatty acid oxidation, peroxisome proliferator-activated receptor (PPAR) protein signaling and fatty acid transport/export were demonstrated in DDOST+/- mice fed a high AGE diet when compared with

WT low AGE fed mice (Fig. 5D). Specifically, the fatty acid oxidation enzymes ACOX1 and

ACBP were increased, as well as the transport proteins FABP5 and FABPL, and the HDL core protein APOA1. The other proteins associated with lipid metabolism examined were not significantly altered.

DDOST+/- mice exhibit impaired glucose tolerance at a young age. Young (8 week old)

DDOST+/- mice had increases in fasted blood glucose concentrations (Table 1 and Fig. S6A), lower fasting insulin concentrations and decreases in first phase insulin secretion (Table 1 and Fig.

S6B) during ipGTT as compared with WT littermates. There were no differences in fed blood glucose concentrations (Table 1), or in insulin tolerance tests (Fig. S6C) among young mice.

Adult DDOST+/- mice exhibited glucose intolerance which is augmented by high AGE dietary intake. By the end of the study, all DDOST+/- mice had elevated glycated haemoglobin (Fig. 6A) and lower fasted plasma insulin concentrations (Fig. 6B), without differences in fasted or fed plasma glucose concentrations as compared with WT mice (Table 1). However, high AGE fed

DDOST+/- mice also had increased 2 hour plasma glucose concentrations following a 2 g/kg glucose bolus (ipGTT), as compared with other groups (Fig. S7A and B). While on a high AGE diet, WT mice were less glucose tolerant as compared with low AGE fed mice (Fig. S7C).

In response to an insulin bolus (ipITT), DDOST+/- mice fed a high AGE diet had higher plasma glucose concentrations than other groups (Fig. 6C). Fasting plasma glucagon concentrations were not increased (Table 1), but hepatic glycogen storage was increased by 30-40% in DDOST+/- mice

(Fig. 6D). When compared with the WT low AGE fed group, all mice also had lower protein intensities of 1,4-alpha-glucan-branching enzyme (GLGB), a facilitator of glycogenolysis (Fig. 6E), consistent with greater levels of liver glycogen described above.

The gene expression of the gluconeogenic enzyme, pyruvate carboxylase kinase (Pck2; Fig. 6F) was increased by high AGE dietary feeding. Pathway analysis of proteins involved in GNG showed that hepatic proteins involved in oxidative phosphorylation were elevated in all mouse groups when compared with WT low AGE fed mice (Fig. 6G).

DDOST+/- mice on a high AGE diet exhibit increased amino acid metabolism.

Increased gluconeogenesis and hepatic glucose release to potentially sustain an associated increased physical activity can be further substantiated by the evident increased circulating concentrations of the amino acids alanine (Fig. 7A) and serine (Fig. 7B) and the loss of glucogenic glycine in

DDOST+/- mice (Fig. 7A). Plasma concentrations of the essential amino acids lysine and histidine

(Fig. 7C) were also increased in DDOST+/- mice. Plasma concentrations of both tyrosine and tryptophan were also increased in the high AGE fed DDOST+/- mice when compared with other mouse groups (Fig. 7D). Pathway analyses showed an increase in protein associated with amino acid metabolism in high AGE fed DDOST+/- mice (Fig. 7E). There were no other changes observed in circulating concentration of other amino acids (Fig. S8).

Discussion

Our discovery of the accumulation of AGEs in the liver through an OST48 mediated pathway of uptake and clearance is novel. Despite the unconventional pathway of liver injury (normally the presence of steatosis leading to the onset of fibrosis) in our DDOST+/- mice, this allowed us to identify other metabolic factors that may contribute an important role in the development of liver injury 215. A recent study in children with NAFLD demonstrated that the presence of early portal inflammation on biopsy was independently associated with severity of liver fibrosis and metabolic risk factors for type 2 diabetes, in particular waist circumference 216. Indeed in that study, central adiposity was the only non-invasive variable associated with biopsy proven portal inflammation.

These findings are consistent with the present study, where central adiposity was seen in concert with early liver injury and portal fibrosis in DDOST+/- mice fed a high AGE diet. Whether

DDOST+/- mice bypass the development of steatosis and proceed directly into fibrogenic pathways would require further investigation into a high fat diet model. Despite this caveat what was particularly interesting was that central adiposity and liver injury persisted in DDOST+/- mice fed a high AGE diet, despite them partaking in greater levels of physical activity. This interesting finding

67 reiterates the complexity of the relationship between metabolic factors and liver injury, given that exercise is a feasible intervention to improve liver fibrosis in patients with NAFLD 217 and relates to adiposity in NAFLD 218.

The liver is a node which controls systemic glucose and lipid fluxes in the body, balancing these against its own energy requirements 219. These processes are influenced heavily by pancreatic islet function via the endocrine hormones insulin and glucagon 220 as well as other hormones. In the present study, abnormalities in insulin secretion and elevations in glycated haemoglobin were seen in the context of hepatic injury and increased fuel oxidation, particularly of fatty acids in high AGE fed DDOST+/- mice. This is in agreement with previous evidence that a shift in hepatic fuel utilisation towards fatty acids results in liver damage 221. The increased utilisation of fatty acids for

-oxidation, and likely increased fatty acid export/transport proteins, may also explain the lack of hepatic fat accumulation seen in DDOST+/- mice fed a high AGE diet. Surprisingly, this led to an increase in central adiposity, despite increased physical activity and the absence of changes in caloric intake and lean body mass. There also appeared to be an excess supply of circulating glucose generated from hepatic gluconeogenesis which may be required by the skeletal muscle to facilitate the increases in physical activity seen in DDOST+/- mice, rather than for thermogenesis.

This is also supported by the decreases in heat generation seen in AGE-R1 mice fed a high AGE diet. A futile cycle of energy generation in adipocytes has been linked to reduced body fat storage

222, however it has not been previously linked to the development of liver fibrosis.

In summary, this group of studies revealed a previously unappreciated role of the OST48-AGE axis within the body. Indeed, the data suggest that the “trafficking role” of OST48 in normal hepatic physiology is not solely involved in protein N-glycosylation or elimination

(detoxification/clearance) of AGEs but could also alter gastrointestinal uptake through the small intestine of AGEs leading to changes in glucose homeostasis, hepatic function and fuel utilisation.

The physiological significance of this should form the basis of future studies. In the context of a high AGE diet, increases in OST48 expression exacerbated liver injury leading to fibrosis, likely via known pathways of injury including oxidative and ER stress. Further studies into the role of hepatic and gastrointestinal OST48 mediated AGE uptake could lead to a novel approach to minimise liver injury.

Materials and Methods

Animal Model. C57BL/6J mice (The Jackson Laboratory, United States) were genetically modified via the Cre-loxP recombination system (Ozgene, WA, Australia) and ubiquitous genetic knock-in of the human gene encoding OST48 (DDOST) at the ROSA26 locus with removal of the delivery neomycin cassette, as described in Supporting Experimental Procedure 1 and Supporting Fig. 1.

DDOSTflox/flox mice are referred to as the wild-type (WT) background and DDOSTflox•Cre/flox are labelled as the genetic OST48 heterozygous knock-in mouse model (DDOST+/-). Eight week old male DDOST+/- mice and littermate controls (WT) were randomised to be fed either AIN-93G

(low AGE diet; Specialty Feeds, Perth, Australia) 223 or baked AIN-93G (1 hour at 1000C; high

AGE diet), which contained a 5-fold higher content of the AGEs, N(ε)-(carboxymethyl)lysine

(CML), N(ε)-(carboxyethyl)lysine and methylglyoxal 72 for 24 weeks. Heat labile vitamin contents

(vitamin A and thiamine) were not decreased by the heating protocol 72. Mice were allowed access to food and water ad libitum and were maintained on a 12 hour light:dark cycle at 220C. All mouse experiments were performed following approval from the AMREP Animal Ethics Committee and as per guidelines from the National Health and Medical Research Council of Australia.

Plasma AGE measurement. CML concentrations in plasma were measured by an in-house indirect

ELISA as previously described 50.

Human and mouse OST48 ELISAs. Mouse and human OST48 concentrations were determined in hepatic cortical membrane and cytosolic fractions by commercial sandwich ELISAs (Cloud-Clone

Corp., Houston, United States), according to the manufacturer’s specifications.

Liquid Chromatography-Mass Spectrometry (LC-MS/MS). As previously described 224, proteins were extracted from whole liver tissue samples using guanidine denaturing buffer (6 M guanidinium, 10 mM DTT and 50 mM Tris-HCl). Reduced cysteines were alkylated with acrylamide, and quenched with excess DTT. Proteins were precipitated in 4 volumes of 1:1 methanol:acetone and digested with trypsin. Peptides were desalted and analysed by Information

Dependent Acquisition LC-MS/MS as described 225 using a Prominence nanoLC system

(Shimadzu, NSW, Australia) and Triple TOF 5600 mass spectrometer with a Nanospray III interface (SCIEX). SWATH-MS analysis was performed 226 and analysed with MSstats as previously described. Differentially abundant proteins were analysed using DAVID 227.

Histology and Immunofluorescence. Haemotoxylin and Eosin, Masson’s Trichrome and Sirius

Red (Sigma-Aldrich, United States) stains were completed on 10% Buffered formalin fixed paraffin sections. Collagen content of the liver was quantified histologically using computerised quantification of picrosirius red staining, as described previously 212. Oil-Red-O (Sigma-Aldrich,

USA) staining was performed on frozen OCT embedded cryosections as previously described 228.

All sections were visualized on a slide scanner (Virtual Slide System VS120, Olympus, Tokyo,

Japan) and viewed in the supplied program (OlyVIA Build 10555, Olympus, Tokyo, Japan).

Briefly, for immunofluorescence staining, 4% PFA fixed frozen liver sections were dual stained with both anti-CML (1:125 dilution; ab30917; Abcam, United Kingdom) and OST48 (1:100 dilution; sc25558; Santa Cruz biotechnologies, United States). Dual staining on paraffin buffered formalin fixed sections was with GRP78 (1:50 dilution; sc1050; Santa Cruz biotechnologies, United

States) and OST-48 (1:100 dilution; sc74407; Santa Cruz biotechnologies, United States).

Serum and urine biochemistry and analysis of hepatic lipids. The plasma concentrations of alanine transaminase (ALT), aspartate transaminase (AST) and alkaline phosphatase (ALP) were measured by an auto-analyzer (Beckman Instruments, USA). Hepatic diacylglycerols (DAGs) and ceramides were extracted and quantified by thin layer chromatography and a liquid scintillation analyzer (LS6500; Beckmann Coulter Inc., CA, USA) as previously described 229. Lipid peroxidation was examined by urinary 8-isoprostane in a competitive ELISA (Oxford Biomedical

Research, United States), according to the manufacturer’s specifications.

Glucose and insulin tolerance tests. Intraperitoneal glucose tolerance tests (ipGTT) were performed following a 2 g/kg D-glucose bolus as previously described 230. Insulin was determined by rat/mouse insulin ELISA (RnD systems, MN, United States). Intraperitoneal insulin tolerance test (ipITT), to determine glucose output was performed using a 1 IU/kg bolus of Humulog fast- acting insulin (Eli Lilly, IN, United States). Area under the curve was calculated using the trapezoidal rule (GraphPad Software, CA, United States).

Indirect calorimetry and assessment of body composition. Whole body composition was measured at 32-weeks of age in conscious, but physically restrained mice using an EchoMRI™ 3- in-1 body composition analyzer (EchoMRI, TX, USA). A cohort of mice were allocated for repeated measures of energy expenditure by indirect calorimetry (VO2, VCO2) normalised to lean body mass, locomotor/physical activity and rate of energy expenditure (heat generation) using a

Comprehensive Lab Animal Monitoring System (CLAMS; Columbus Instruments, OH, United

States). Data was collected over a 24-hour cycle period, post-acclimatisation to the cages for a 12- hour period.

Liver glycogen content. As previously described 231, total liver glycogen content was determined using a glucose oxidase/peroxidase assay procedure and absorbance was analysed on a UV-1700

PharmaSpec UV-vis spectrophotometer (Shimadzu, NSW, Australia).

Quantitative real-time PCR. Messenger RNA was purified and was used (1 µg) to synthesize cDNA using SuperScript first-strand synthesis system (Invitrogen). Quantitative real-time PCR was performed using pre-designed TaqMan Gene Expression Assays® for Acadm, Acadvl, Acox1,

ADRB2, Ccl2, Col1a1, Col3a1, Ddost, DDOST, G6pc, Gcgr, Got1, Gyk, Kcnma1, Lepr, Pck1,

Pck2, Ppara, Slc27a4 and Slc37a4 (Supporting Experimental Procedure 2; Life Technologies,

Mulgrave, VIC, Australia) in ViiA™ 7 real-time PCR system (Applied Biosystem, Darmstadt,

Germany). Gene expression levels were calculated after normalisation to an endogenous multiplexed control (18S) using the ΔΔCT method as previously described 72 and expressed as relative fold change compared to the wild-type mice fed a high AGE content diet. The results are represented as mean ± %CV.

Amino acid measurements. Approximately 40 µl of serum was mixed with 160 µl of an extraction solution (3:1 ratio of analytical grade methanol to ddH2O), centrifuged at 16,000 × g for 10 minutes and the supernatant removed for processing. Amino acids were measured by HPLC as described in

Chacko et al. 232.

Statistical analysis. Results are expressed as mean ± SD (standard deviation), and analyzed by 2- way ANOVA followed by post hoc testing for multiple comparisons using the Bonferroni method unless otherwise specified. α (genotype effect) P < 0.05, β (diet effect) P < 0.05, δ (interaction effect) P < 0.05 were reported. For comparison between groups as required, a two-tailed unpaired

Student’s t-test was used. For SWATH-MS, MSstatsV3.5.1 was used to detect differentially abundant proteins estimating the log-fold changes between compared conditions of the chosen 72 experimental group and with the WT mice fed a low AGE diet group. For all calculations a P < 0.05 was considered as statistically significant.

Figures and Figure Legends

Figure 1. A diet high in AGE content is a promoter of portal vein fibrosis following upregulation of DDOST+/- in the absence of steatosis. (A) Cull liver weight. (B) Representative H

& E stained paraffin-embedded liver tissue sections. (C) Hepatic fibrillar collagen quantification of

Sirius Red stained images (left). Representative images of fibrosis stained images of Sirius Red localised around fibrillar fibrosis extending between hepatocytes (right). (D) Hepatic α-SMA positive quantification of immunofluorescent stained images (left). Representative images of α-

SMA positive staining extending between hepatocytes (right). (E) Liver function tests for ALT

(left), AST (middle) and ALP (right). (F) Hepatic diacylglyceride (top) and ceramide (bottom) accumulation. Representative images of Oil Red O staining for lipid droplets in liver tissue (right).

Data represented as means ± SD (n = 4-9/group). *P < 0.05, student’s t-test. Genotype effect P <

0.05, (diet effect) P < 0.05, 2-way ANOVA and multiple comparison of genotype, diet and interaction by Bonferroni’s post hoc test. Representative images scale bar = 50µm (outside box) and 20µm (inside box).

Figure 2. DDOST+/- mice have increased hepatic uptake of AGEs. Wild-type and DDOST+/- mice were fed either a high AGE (baked AIN-93G diet) or a low AGE (unbaked AIN-93G) diet for

24 weeks. (A) Immunofluorescence for OST48 (red) and the AGE, CML (green) in OCT liver sections. Representative images; scale bar = 20µm (B) Near-infrared imaging of AGE uptake in the liver following oral gavage using IVIS/MRI. (C) AGE concentrations in hepatic tissue by immunofluorescence quantification. (D) Plasma AGE concentrations by ELISA. Data represented 75 as means ± SD (n = 4-9/group). Genotype effect P < 0.05, 2-way ANOVA and multiple comparison of genotype, diet and interaction by Bonferroni’s post hoc test.

Figure 3. DDOST+/- mice have increased central adiposity despite greater physical activity.

(A) Fat percentage measured by EchoMRI (left) and weight of omental fat deposits (right). (B-C)

CLAMS apparatus measured 24 hour physical activity and caloric intake at 24 weeks post-dietary intervention in WT (●) and DDOST+/- (●) mice fed on either a high AGE diet (closed circles) or a low AGE diet (open circles). (B) Total locomotor activity in the horizontal plane measured by infrared beam breaks. (C) The line graphs show average hourly heat generation over the 24-hour period. The bar graphs on the right show the average physical activity over the entire 12-hour light/sleep (left) and dark/active (right) period. Data represented as means ± SD (n = 4-9/group).

*P < 0.05, student’s t-test, α (genotype effect) P < 0.05, β (diet effect) P < 0.05, as per figure above.

2-way ANOVA and multiple comparison of genotype, diet and interaction by Bonferroni’s post hoc test.

Figure 4. A high AGE diet promotes liver fibrosis and correlates with increased ER stress markers following increases in DDOST+/- mice. (A) Immunofluorescence for OST48 (red), the

ER stress marker GRP-78 (green) on paraffin liver sections (hepatocytes). (B) SWATH-MS protein intensities of ER stress pathway related proteins. GRP78 (top) and EIF3A (bottom). (C) SWATH-

MS protein intensities of oxidative stress pathway related proteins. SODC (top) and SODM

(bottom). (D) ELISA identifying content of urinary 8-isoprostane. Data represented as means ± SD

(n = 4-9/group). *P < 0.05, MSstatsV3.5.1 determined significant log fold changes in the protein intensities between the selected experimental group and the WT low AGE diet group.

Representative images scale bar = 20µm.

Figure 5. DDOST+/- mice have a shift towards increased fatty acid utilization. (A) CLAMS apparatus measured respiratory exchange rate at 24 weeks post-diet modification. Average difference in respiratory quotient corrected to lean body mass measured by indirect calorimetry during the entire 12-hour light/sleep (left) and dark/active (right) period. (B) Average difference in heat production measured by indirect calorimetry during the entire 12-hour light/sleep (left) and dark/active (right) period. (C) Real-time PCR of liver tissue targeting genes of interest Pparα (top) and Lepr (bottom) (n = 4-8 per group in triplicate). (D) Heat map representation of SWATH-MS proteomics data for enzymatic pathways involved in fatty acid oxidation. Significant proteins are represented as the Log2 fold change where red indicates a decreased and blue indicates an increase in protein concentrations. Data represented as means ± SD (n = 4-9/group). For proteomics,

MSstatsV3.5.1 determined significant (P < 0.05) log fold changes in the protein intensities between the selected experimental group and the WT low AGE diet group. *P < 0.05, student’s t-test.

Genotype effect P < 0.05, (diet effect) P < 0.05, 2-way ANOVA and multiple comparison of genotype, diet and interaction by Bonferroni’s post hoc test.

Figure 6. Hepatic fibrosis associated with glucose intolerance, increased glucose synthesis and storage in high AGE fed DDOST+/- mice. (A) Glycated haemoglobin. (B) Fasting plasma insulin concentrations. (C) Plasma glucose curve over 120 mins following a 1IU/kg insulin bolus (ipITT) and area-under-the-curve (AUC) analysis. (D) Hepatic glycogen measured in liver tissue. (E)

SWATH-MS protein intensities for the glycogen storage pathway protein (GLGB). (F) qPCR of

Pck2, the enzyme involved in gluconeogenesis. (G) Heat map representation of SWATH-MS proteomics data for enzymatic pathways involved in GNG. Significant proteins are represented as the Log2 fold change where red indicates a decreased and blue indicates an increase in protein concentrations. Data represented as means ± SD (n = 4-9/group). For proteomics, MSstatsV3.5.1 determined significant (P < 0.05) log fold changes in the protein intensities between the selected experimental group the WT low AGE diet group. *P < 0.05, student’s t-test. Genotype effect P <

0.05, (diet effect) P < 0.05, 2-way ANOVA and multiple comparison of genotype, diet and interaction by Bonferroni’s post hoc test.

Figure 7. DDOST+/- mice exhibit increased ketogenesis. (A) Concentrations of serum amino acids. (B) Heat map representation of SWATH-MS proteomics data for enzymatic pathways involved in ketogenesis. Significant proteins are represented as the Log2 fold change where red indicates a decreased and blue indicates an increase in protein concentrations. Data represented as means ± SD (n = 4-9/group). For proteomics, MSstatsV3.5.1 determined significant (P < 0.05) log fold changes in the protein intensities between the selected experimental group the WT low AGE diet group.

Tables.

Table 1. Biochemical measurements in mice post-diet modifications (32 weeks of age). Mean and standard deviation for biochemical measurements of each genotype during pre- and post-diet modification. Post-diet modification significance levels were determined by two-way ANOVA, testing the effect of genotype and diet. Differences between variables identified by Bonferroni’s post hoc test. Bold P values indicates significant effect of at least < 0.05. G: Genotype; D: Diet;

G∙D: Interaction.

Post-diabetes modification

WT DDOST+/- Two-Way ANOVA

AGELow AGEHigh AGELow AGEHigh G D G∙D

Fed

Glucose 15.70 ± 1.80 13.77 ± 1.53 15.66 ± 1.38 13.95 ± 1.18 0.96 0.23 0.94

(mmol/L)

Fasting

Glucose 10.65 ± 0.46 9.417 ± 1.13 9.913 ± 0.76 10.03 ± 0.47 0.94 0.49 0.96

(mmol/L)

Fasting 0.9438 ± Insulin 1.255 ± 0.41 1.837 ± 0.53 0.7999 ± 0.14 0.05 0.51 0.28 0.27 (ng/ml)

Fasting

Glucagon 1366 ± 187.8 2087 ± 287.6 1552 ± 80.13 1677 ± 279.8 0.61 0.09 0.93

(pg/ml)

Supplementary Materials and Methods.

Genotyping of genetic knock-in mutation for DDOST+/- mice. All genotyping was confirmed with genomic Southern blot hybridization using the ScaI and the 5’ probe. Mice were then re-screened with the same filter with a generic Cre probe to determine whether they contain the ubiquitous promoter (Supporting Fig. 1).

Pre-designed TaqMan Gene Expression Assays® IDs

Gene name Life Technologies Assay ID Acadm Mm01323360_g1 Acadvl Mm00444293_m1 Acox1 Mm01246834_m1 ADRB2 Hs00240532_s1 Col1a1 Mm00801666_g1 Col3a1 Mm01254476_m1 Ccl2 Mm00441242_m1 Ddost Mm00492100_m1 DDOST Hs00193263_m1 G6pc Mm00839363_m1 Gcgr Mm00433546_m1 Got1 Mm01195792_g1 Gyk Mm00433896_m1 Kcnma1 Mm01268569_m1 Lepr Mm00440181_m1 Pck1 Mm01247058_m1 Pck2 Mm00551411_m1 Ppara Mm00440939_m1 Slc27a4 Mm01327405_m1 Slc37a4 Mm00484574_m1

Supporting Figures and Figure Legends.

Supporting Figure 1. Generation of a ubiquitous DDOST heterozygous knock-in mutant. (A)

Genomic clone of DDOST bearing exon 1 used for the construction of the floxed targeting vector as indicated (top). Heterozygous mice were crossed with mice expressing Cre and subsequent progeny contained a ubiquitous over-expression allele. Genomic Southern blotting confirmed the predicted

DDOST allelic structures in the ubiquitous knock-in strain (bottom). Left and middle panels: ES cell clones bearing indicated mutant DDOST allele in comparison to the parental wild-type ES cells; Right panels: adult mouse genotypes with either DDOST germline modifications or wild- type, successfully crossed with the Ubi-Cre (UbiC) strain. (B) Hepatic gene expression of DDOST, encoding for human DDOST by real-time-PCR. (C) Hepatic gene expression of Ddost and DDOST, encoding for mouse AGE-R1 and human AGE-R1 respectively by real-time-PCR. (D) Targeted

SWATH proteomics for OST48 protein content in gut tissue and an average of 5 different tissues

(left kidney, liver, skeletal muscle, left ventricle and epididymal fat). (E) Endogenous mouse

DDOST and human DDOST (striped) protein content measured in hepatic plasma membrane by

ELISA

Supporting Figure 2. DDOST+/- mice do not have differences in body weight or body composition pre-diet modification. (A) Body weight. (B) Lean body mass percentage measured by EchoMRI.

Supporting Figure 3. SWATH-MS protein intensities of oxidative stress pathway related proteins and inflammatory pathway related proteins. (A) GPX1 (left) and ACON (right). (B) CD47 (left) and HA1D (right). Data represented as means ± SD (n = 4-9/group). *P < 0.05, MSstatsV3.5.1 determined significant log fold changes in the protein intensities between the selected experimental group and the WT low AGE diet group.

Supporting Figure 4. Glycosylation occupancy in serum proteins. ((A) Peptide-centric glycosylation occupancy at asparagine residues in mouse serum proteins measured after trypsin digest.

Glycosylation occupancy was measured at different glycosylation sites (e.g. THHY N188, Asn118 in TTHY) in wild-type and AGE-R1 mice. Color is mapped to the extent of glycosylation occupancy from 100% (blue) to 0% (red). Data is mean (n = 3/group of 3 replicates). (B) Heat map representing several proteins secreted from the liver and their degree of glycosylation occupancy.

(C) Extracted ion chromatograms for the peptide containing ESTN N377 in liver extracts; corresponding to de-glycosylated (dashed) and non-glycosylated (solid) forms of tryptic peptide

(m/z of 826.4297 and m/z of 826.1017, respectively), peak corresponds to the monoisotopic peak from each peptide.

Supporting Figure 5. Real-time PCR of liver tissue targeting genes of interest. Acadm (left) and

Acadvl (right). Data represented as means ± SD (n = 4-8/group in triplicate).

Supporting Figure 6. Young DDOST+/- mice have impaired glucose tolerance. Young (8 week old)

WT (●) and AGE-R1 (●) mice serum glucose and insulin levels in mice fasted for 6 hours following intraperitoneal insulin or glucose injection. (A) ipGTT measuring glucose homeostasis (top) with subsequent area-under-the-curve (AUC) analysis (bottom). (B) ipITT assessing glucose effectiveness (top) and ratio of AUIC:AUGC of early-phase insulin response (bottom). (C) ipGTT measuring insulin effectiveness (top) with subsequent area-under-the-curve (AUC) analysis

(bottom). Data represented as means ± SD (n = 4-9/group). *P < 0.05, student’s t-test.

Supporting Figure 7. DDOST+/- mice exhibit impaired first phase insulin secretion. (A-B)

Intraperitoneal glucose and insulin tolerance testing in wild-type (●) and DDOST+/- (●) mice fasted for 6 hours after 24 weeks of high or low AGE diet feeding. (A) ipITT (left) and ipGTT (right) measuring glucose homeostasis and (B) ipGTT measuring insulin effectiveness with subsequent area-under-the-curve (AUC) analysis of serum glucose (AUGC) and insulin (AUIC). (C) Fasted insulin concentrations in plasma serum (top). Ratio of AUIC:AUGC to determine insulin effectiveness during the first phase insulin secretion response (bottom). Data represented as means

± SD (n = 4-9/group). #P < 0.1, *P < 0.05, **P < 0.01, α (genotype effect) P < 0.05, β (diet effect)

P < 0.05, δ (interaction effect) P < 0.05, 2-way ANOVA and multiple comparison of genotype, diet and interaction by Bonferroni’s post hoc test.

Supporting Figure 8. Serum concentrations of amino acids. Amino acid metabolite concentration measured in plasma serum. A (genotype effect) P < 0.05, B (diet effect) P < 0.05, C (interaction effect) P < 0.05, 2-way ANOVA and multiple comparison of genotype, diet and interaction by

Bonferroni’s post hoc test. 87

Chapter 4: Globally elevating the AGE clearance receptor, OST48 does not protect against the development of diabetic kidney disease, despite improving insulin secretion.

Introduction to this chapter. This chapter is submitted for publication in Scientific Reports as an original article. It has currently been peer-reviewed and is under revisions prior to publication. In this report, we over-expressed the gene encoding for oligosaccharyltrasnferase-48 (OST48).

Although the current literature has suggested that increasing OST48 could be a potential mechanism to decrease systemic AGE toxicity by facilitating AGE clearance from the body, in diabetic complications, there has been little evidence published to directly support this claim. Therefore, the focus of this chapter is to elucidate whether increased OST48 could prevent the development of diabetic kidney disease. Furthermore, beyond the scope of this chapter, this work provides crucial information in elucidating the role of OST48 in the kidney. In this novel study, over-expression of

OST48 did not protect against the development of diabetic kidney disease and diabetic mice proceeded to develop glomerulosclerosis, tubulointerstitial fibrosis and kidney function decline.

Specifically, we demonstrated the following; 1) Mice over-expressing OST48 (DDOST+/-) and wild-type mice induced with experimental diabetes both exhibited kidney injury and declined kidney function. 2) Increased urinary excretion of AGEs and reduced kidney deposition in

DDOST+/- diabetic mice did not translate into protection against kidney damage and dysfunction.

3) Diabetic DDOST+/- mice exhibited significant improvements in fasting plasma insulin, insulin sensitivity and short-term glucose homeostasis. 4) However, this did not improve long-term glycaemic control.

In summary, in this group of experiments we showed conclusively that increased OST48 in the kidney does not protect against the development of diabetic kidney disease. The data however suggests that OST48 may influence insulin sensitivity and insulin secretion and that further studies are required to elucidate the role OST48 has in the kidney.

Graphical Abstract

Abstract

The accumulation of advanced glycation end products (AGEs) have been implicated in the development and progression of diabetic kidney disease (DKD). There has been interest in investigating the potential of AGE clearance receptors, such as oligosaccharyltransferase-48kDa subunit (OST48) to prevent the detrimental effects of excess AGE accumulation seen in the diabetic kidney. Here the objective of the study was to increase the expression of OST48 to examine if this slowed the development of DKD by facilitating the clearance of AGEs. Groups of 8-week-old heterozygous knock-in male mice (n=9-12/group) over-expressing the gene encoding for OST48, dolichyl-diphosphooligosaccharide-protein glycosyltransferase (DDOST+/-) and litter mate controls were randomised to either (i) no diabetes or (ii) diabetes induced via multiple low-dose streptozotocin and followed for 24 weeks. By the study end, global over expression of OST48 increased glomerular OST48. This facilitated greater renal excretion of AGEs but did not affect circulating or renal AGE concentrations. Diabetes resulted in kidney damage including lower glomerular filtration rate, albuminuria, glomerulosclerosis and tubulointerstitial fibrosis. In diabetic mice, tubulointerstitial fibrosis was further exacerbated by global increases in OST48. There was significantly insulin effectiveness, increased acute insulin secretion, fasting insulin concentrations and AUCinsulin observed during glucose tolerance testing in diabetic mice with global elevations in

OST48 when compared to diabetic wild-type littermates. Overall, this study suggested that despite facilitating urinary-renal AGE clearance, there were no benefits observed on kidney functional and structural parameters in diabetes afforded by globally increasing OST48 expression. However, the improvements in insulin secretion seen in diabetic mice with global over-expression of OST48 and their dissociation from effects on kidney function warrant future investigation.

Introduction

Currently, the world is faced with a pandemic of both type 1 and type 2 diabetes (T1D and T2D)10,

233, defined by persistent hyperglycaemia, which is a predominant factor in the development of concomitant complications12, 234. A major complication, diabetic kidney disease (DKD) is a worldwide health concern and is an important risk factor for end-stage renal disease (ESRD)235 and cardiovascular disease234, 236. Understanding the pathophysiology of DKD development and progression is therefore, a key challenge to reduce the burden of diabetic complications. The early clinical presentation of DKD is characterised by hyperfiltration, progressive proteinuria and associative glomerular injury accompanied by tubulointerstitial fibrosis and in the later stages, a steady progressive decline in renal function13. Unfortunately, despite optimal clinical management, involving both glycaemic16, 237 and blood pressure control17, including inhibitors of the renin- angiotensin-aldosterone system238, 239, it is only possible to achieve a 30% improvement in declining kidney function in DKD. Despite intervention, many individuals reach end stage disease requiring renal replacement therapy or die prematurely from a cardiovascular event240. Hence new therapies to combat DKD are urgently required.

AGE accumulation within tissues is thought to one pathological mediator in DKD241. It is postulated that under physiological conditions, OST48 may facilitate AGE clearance into the urine53 and that this function is impaired during the development and progression of DKD241 resulting in pathological accumulation of AGE at sites such as the kidney. OST48 (also known as AGER1) dually functions as an extracellular AGE binding protein242 and as a 48kDa subunit that functions as part of the oligosaccharyltransferase complex, which mediates the transfer of high-mannose oligosaccharides to asparagine residues within the lumen of the rough ER. OST48 gene and protein expression are decreased by diets abundantly rich in AGEs51 and by diabetes50, 52. In addition, there are associations between lower OST48 levels in circulating immune cells and progressive diabetic

92 nephropathy in a small cohort of patients with type 1 diabetes49, 243 and with impaired insulin sensitivity patients with in type 2 diabetes54, 74. To date, there has been one, in vivo investigation using untargeted over-expression of OST48. This study in elderly mice (>500 days old) demonstrated improvements in longevity, insulin sensitivity and resistance to balloon injury in blood vessels64. However, there are no studies where the efficacy of increasing OST48 expression to facilitate AGE clearance has been tested in the development of kidney disease including DKD.

A SNP array from the FinnDiane population study (n = 2719) inferred that there was an association of the OST48 gene, DDOST (rs2170336), with nephropathy development in individuals with T1D55.

Additionally, a study in T1D patients identified that elevated serum AGEs were associated with increased OST48 mRNA in circulating mononuclear cells49. Moreover, the functional loss of

DDOST in humans, demonstrates a phenotype with characteristics known as congenital disorders of glycosylation (CDG)57. A sole case was described in a seven-year-old male child with CDG, whose condition arose from the inheritance of a maternal missense mutation and a paternal point mutation resulting in a premature stop codon and as a result the patient exhibited severe hypoglycosylation57.

It was shown however that restored wild-type DDOST cDNA in the patients’ fibroblasts rescued glycosylation, thus indicating the importance of functional DDOST in a pathological environment57.

For the present study, we hypothesised that mice with a global OST48 overexpression would be protected from increases in circulating AGEs and impaired kidney function in the context of diabetes. Specifically, we aimed to identify whether increased OST48 in the presence of hyperglycaemia could drive AGE lowering to protect against AGE-mediated microvascular damage typically seen in DKD, such as glomerular pathology and greater tubulointerstitial fibrosis.

Results

Characterisation of diabetes in a site directed global OST48 knock-in mouse model. Mice were generated with a global over-expression of OST48 (DDOST+/-)244. These mice exhibited variation 93 in food and water consumption, urine output and a significant reduction in kidney weight compared with wild-type mice (Table 1). Diabetes was characterised by elevated GHb, fed/fasting blood glucose and increased fasting insulin (Table 1). Diabetic mice had significantly lower mean total body weight, increased urine output as well as increased food and water consumption and renal hypertrophy (Table 1).

Increased glomerular OST48 facilitated AGE urinary excretion in DDOST+/- mice. SWATH proteomics in renal cortices enriched for glomerular proteins identified that non-diabetic DDOST+/- mice had a significant increase in OST48 protein abundancy (48.39-fold increase) over non-diabetic wild-type mice (Figure 1A). There was also a tendency toward increased OST48 protein in glomerular fractions from diabetic DDOST+/- mice (P=0.07; 14.44-fold increase) compared to diabetic wild-type mice (Figure 1A). Surprisingly, there was no significant increase in OST48 seen in tubular enriched protein fractions (Figure 1B) from DDOST+/- mice (Figure 1C). Glomerular

OST48 appeared to be predominately expressed by podocytes as indicated by the co-localisation of nephrin (Figure 1D) as well as within proximal tubule cells expressing SGLT2 (Figure 1E).

Subsequently, there was a significant increase in renal AGE excretion by all diabetic mice as well as the non-diabetic DDOST+/- mice (Figure 1F). Specifically, urinary AGE excretion increased by

~120% in non-diabetic DDOST+/- mice compared to wild-type mice (Figure 1F; P=0.033).

However, DDOST+/- mice showed no increases in AGE accumulation in either kidney tissue

(Figure 1G) or within the circulation (Figure 1H).

Diabetic DDOST+/- mice had no improvements in kidney function despite increased urinary

AGE excretion. All diabetic mice had impaired kidney function as ascertained by a decrease in creatinine clearance (Figure 2A), which was not affected by OST48 overexpression. Diabetic mice also had albuminuria when assessed by either albumin:creatinine ratio (Figure 2B) or 24 hour

94 urinary albumin excretion (Figure 2C). Therefore, increased OST48 expression did not prevent the decline in kidney function which is characteristic of diabetic kidney disease.

Globally increasing OST48 expression did not protect against diabetes-induced kidney structural damage. Diabetes resulted in glomerulosclerosis in both genotypes to the same degree by the study end (Figure 3A). This was supported by SWATH proteomics data from kidney cortices enriched for glomerular proteins which demonstrated significant increases in the abundance of collagen proteins in diabetic mice (Figure 3B). Increasing the expression of OST48 also did not prevent tubulointerstitial fibrosis (TIF) in the kidneys of diabetic mice, as seen with Masson’s trichrome (Figure 3C) and Sirius red (Figure 3D) staining. Indeed, there was exacerbation of TIF in diabetic DDOST+/- by comparison to diabetic wild-type littermates (Figure 3D; 32.0% increase,

P=0.043).

Diabetic mice with globally increased OST48 expression have greater insulin secretory capacity and increased P13K-AKT activity in glomeruli, which is dissociated from deterioration in kidney function. In the absence of diabetes, globally increasing the expression of

OST48 had no significant effect on insulin sensitivity or glucose tolerance (Figure 4A-G).

However, when diabetes was induced, mice with a global over-expression of OST48 had significant reduction in fractional glucose excretion (Figure 4A), increases in fasting plasma insulin (Figure

4B) and lower blood glucose concentrations during an ipGTT, both acutely at 15 minutes (11.9% decrease, P = 0.097) and 30 minutes (14.2% decrease, P = 0.017) post-bolus of D-glucose (Figure

4C). Consistent with these changes, overall insulin secretion (Figure 4D; average 77.4% increase,

P < 0.05 – 0.001), first phase insulin secretion (Figure 4E, 99.5% increase, P = 0.0005) and the

AUC insulin (Figure 4F) during the ipGTT were all greater in diabetic mice over expressing

OST48, when compared to littermate wild type diabetic mice. Insulin effectiveness (AUIC:AUGC) was also greater in diabetic DDOST+/- mice compared to diabetic wild-type mice (Figure 4E-G;

99.5% increase, P=0.0005). During fed conditions an insulin tolerance test (ipITT), all diabetic

DDOST+/- mice had decreased responsiveness to insulin with persistently elevated plasma glucose concentrations, when compared to non-diabetic mice (Figure 4H) but did not differ between genotypes. This was particularly evident during the first 60-minutes of the ipITT, where there was a rapid decline in plasma glucose concentrations in non-diabetic mice (Figure 4H-I; 490% decline compared to diabetic mice), with rapid recovery of blood glucose concentrations by 120 mins. This increase in insulin secretion and effectiveness was also confirmed by increased PI3K-AKT activation in the glomerular enriched fractions from mice over-expressing OST48 Figure 4J).

Discussion

There has been some speculation that increasing AGE clearance via increases in OST48 could alleviate the development and progression of diabetic kidney disease49, 51, 52, 59, 60. We have shown for the first time that globally increasing OST48 through the over-expression of DDOST in a mouse model of diabetes, did not protect against kidney disease. Indeed, there was an exacerbation of tubulointerstitial fibrosis seen in diabetic mice with a global over-expression of OST48. Quite surprisingly, however, this was despite significant improvements in insulin secretory capacity and action in these diabetic DDOST+/- mice. Therefore, this study dissociated improvements in insulin secretion from slowing the onset and progression of DKD, in mice over-expressing

OST48.

Previous studies have suggested that increasing AGE clearance from the body through the OST48 pathway prevents oxidative stress51 and inflammatory responses9, as well as improving healing and hyperglycaemia64, concluding that increased OST48 activity could have potential benefits to alleviate vascular complications in diabetes51, 60 including DKD9, 51. Moreover, previous studies have alluded at the potential benefits of AGE restriction and reducing serum AGE levels in diabetic patients4, 74, 245, 246, and suggested that AGE excretion facilitated through increased OST48 could

96 reduce the AGE burden53, 64, 71, 74, 247. Hence, we were surprised that increasing AGE clearance by the kidneys, via globally increasing OST48 expression had no effect on kidney function and structural parameters in this mouse model of diabetes and worsened renal pathology.

In our mouse model the lack of effect of OST48 to alleviate DKD was despite modest improvements to insulin secretory capacity, although this was not sufficient to improve the long- term markers of glycaemic control (GHb), nor fasting and fed plasma glucose concentrations.

However, our data is in agreement with the DCCT/EDIC clinical trials, which indicate that without effective reduction in blood glucose early in disease development, there was no reduction in the risk in development of diabetic vascular complications including DKD248, 249. There have been previous studies where AGEs have been shown to impair insulin secretion70, 250 although it is unclear as to the role that OST48 plays in beta cells. However, we have identified that OST48 is expressed in beta cells251. In addition, further investigation would be required to elucidate why these apparent improvements in insulin secretion seen in diabetic OST48 mice did not translate to improved long- term clinical markers of glycaemic control. However, this may just indicate that modest improvements in insulin secretion later in disease are not sufficient to alter the progression of diabetic kidney disease.

There was also a decline in renal AGE content seen with diabetes, which was consistent with the increases in AGE excretion into the urine, but this was not affected by OST48. In agreement with our findings, another study showed that increasing OST48 expression in mice also facilitated urinary AGE clearance, without affecting renal AGE concentrations64. This would suggest that rather than specific AGE uptake into the kidney the flux of AGEs through the kidney into the urine, may trip signalling cascades for other AGE receptors such as RAGE252. This could be feasibly responsible for the observed glomerulosclerosis and tubulointerstitial fibrosis253 with diabetes and with OST48 overexpression where chronic binding of circulating AGEs to RAGE results in diabetic

97 kidney and cardiovascular disease136, 246, 253. Increased AGE flux from the circulation to the urine, is also postulated in other studies to have detrimental effects on proximal tubules contributing to tubulointerstitial fibrosis254, which is consistent with our model. However, we do not suspect defects in protein N-glycosylation contributed to the kidney pathology seen with OST48 overexpression 255. This is because, we have previously shown that ubiquitous global overexpression of OST48 does not impair nor alter protein N-glycosylation244.

Despite the ineffectiveness of increased OST48 to protect against the development of diabetic kidney disease, we did however observe benefits on insulin secretion, fasting insulin and insulin sensitivity by globally elevating OST48 in diabetic mice. Our data suggests that downstream insulin receptor activity via P13K and diverging into increased lipogenesis, glycolysis and glycogenesis

(starch and sucrose metabolism) occurs in the kidney when OST48 is increased. Our data is in concordance with previous human studies which suggest that AGE restriction whether through dietary74 or therapeutic245 methods improve insulin sensitivity and decrease in serum OST48 concentrations74. We have also consistently shown that AGE lowering therapies which decrease the burden of AGEs improve both insulin secretion70 and improve insulin sensitivity72, 256.

In summary, these studies revealed that increasing OST48 expression globally does not prevent the development of DKD. Specifically, in diabetic mice, increasing OST48 expression did not prevent the decline in kidney function, glomerulosclerosis and exacerbated tubulointerstitial fibrosis.

Therefore, we would suggest that increasing the flux of AGEs into the urine is not a strategy worth pursuing in DKD. Similarly, it appears that increasing AGE signalling in the kidney via receptors such as RAGE without increasing kidney AGE content, is responsible for this lack of protective effect. Therefore, this study suggests that globally increasing the expression of OST48 thereby increasing urinary AGE excretion does not improve kidney function in the context of diabetes.

However, further studies are required to identify whether kidney cell-specific modulation of OST48 may be superior to a global approach.

Materials and Methods

Animal husbandry. Male C57BL/6J wild-type mice and littermate heterozygotes with a ubiquitous genetic insertion of the human gene encoding AGE-R1/OST48 (DDOST+/-) at the ROSA26 locus

(ROSA26tm1(DDOST)Jfo; termed as DDOST+/-) were generated (Ozgene, Australia). Between 6-8 weeks of age (Week 0), diabetes was induced in male wildtype (n = 9) and DDOST+/- (n = 9) mice by multiple low dose intraperitoneal injections of streptozotocin (STZ)257, at a dosage of

55mg/kg/day (dissolved in sodium citrate buffer, pH 4.5) for 5 consecutive days. Control or non- diabetic male wild-type (n = 11) or DDOST+/- (n = 12) mice received equivalent injections of sodium citrate buffer alone. After 10 days of recovery, blood glucose concentrations were determined and then repeated weekly to ensure mice included were diabetic (blood glucose concentrations > 15mmol/L). Mice were housed in specific pathogen free housing conditions and allowed access to food and water ad libitum and were maintained on a 12 hour light:dark cycle at

22°C. All mice received a diet of standard mouse chow low in AGE content (AIN-93G70; Specialty

Feeds, Australia) ad libitum. At 0 and 12 weeks of the study, mice were housed in metabolic cages

(Iffa Credo, l’Arbresle, France) for 24 hours to determine food and water consumption. Urine output was also measured during caging and urinary glucose determined by a glucometer

(SensoCard Plus, POCD, Australia).

Intraperitoneal glucose and insulin tolerance testing (IPGTT/IPITT). Intraperitoneal glucose

(ipGTT) and insulin (ipITT) tolerance tests were performed as outlined previously230. Briefly, for ipGTT experiments, mice were fasted for 6 hours and a 1g/kg bolus of glucose was injected intraperitoneally (ip) and 50µl of blood was sampled at 0, 15, 30, 60, and 120 mins for plasma glucose and insulin analysis. During ipITT experiments, mice were injected with 1.0U of fast acting

99 insulin/kg (Humulin) diluted in 0.9% saline and 50µl of blood was sampled at 0, 30, 60, and 120 mins for plasma glucose analysis.

Liquid Chromatography-Mass Spectrometry (LC-MS/MS). As previously described224, proteins were extracted from whole liver tissue samples using guanidine denaturing buffer (6 M guanidinium, 10 mM DTT and 50 mM Tris-HCl). Reduced cysteines were alkylated with acrylamide, and quenched with excess DTT. Proteins were precipitated in 4 volumes of 1:1 methanol:acetone and digested with trypsin. Peptides were desalted and analysed by Information

Dependent Acquisition LC-MS/MS as described225 using a Prominence nanoLC system (Shimadzu,

NSW, Australia) and Triple TOF 5600 mass spectrometer with a Nanospray III interface (SCIEX).

SWATH-MS analysis was performed226 and analysed with MSstats as previously described.

Differentially abundant proteins were analysed using DAVID227.

Serum/urine creatinine and clearance. Serum and urinary creatinine were measured spectrophotometrically at 550nm (Cobas Mira, Roche Diagnostics, Australia), and the ratio of creatinine clearance was determined as specific measure of renal function.

Albumin excretion rate. Urine albumin was measured spectrophotometrically at 620nm

(FLUOstar Optima, BMG Labtech, Australia), and 24-hour albumin excretion rate was calculated by normalizing the levels of albumin to the flow rate of urine.

Histology and imaging. Paraffin-embedded sections were stained with either a Periodic acid Schiff

(PAS) staining kit (Sigma-Aldrich, United States), a Trichrome (Masson) staining kit (Sigma-

Aldrich, St. Louis, Missouri, USA) or Sirius Red (Sigma-Aldrich, United States). All sections were visualized on an Olympus Slide scanner VS120 (Olympus, Japan) and viewed in the supplied program (OlyVIA Build 10555, Olympus, Japan). Slides were quantified based on threshold

100 analysis in Fiji258. Briefly, for immunofluorescence staining, paraffin-embedded sections were stained with a combination of either anti-CML (1:200 dilution; ab27684; Abcam, United Kingdom),

OST48 (H-1; 1:100 dilution; SC-74408; Santa Cruz biotechnologies, United States), nephrin (1:100 dilution; ab27684; Abcam, United Kingdom) and SGLT2 (M-17; 1:100 dilution; sc-47403; Santa

Cruz biotechnologies, United States). Confocal images were visualized on an Olympus FV1200 confocal microscope (Olympus, Japan) and viewed in the supplied program (FV10, Olympus,

Japan).

Glomerulosclerotic index (GSI). GSI as a measure of glomerular fibrosis was evaluated in a blinded manner by a semi-quantitative method259. Severity of glomerular damage was assessed on the following parameters; mesangial matrix expansion and/or hyalinosis of focal adhesions, true glomerular tuft occlusion, sclerosis and capillary dilation. Specifically, grade 0 indicates a normal glomerulus; 1, <25% glomerular injury; grade 2, 26-50%; grade 3, 51-75%; and grade 4, >75%.

Statistical Analyses. Results are expressed as mean ± SD (standard deviation), and assessed in

GraphPad Prism V7.01 for Windows (GraphPad Software, United States). Normally distributed parameters (tested with D’Agostino & Pearson omnibus normality test) were tested for statistical significance by 2-way ANOVA followed by post hoc testing for multiple comparisons using the

Bonferroni method unless otherwise specified. For comparison between groups as required, a two- tailed unpaired Student’s t-test was used where specified. For SWATH-MS, MSstatsV3.10.0260 was used to detect differentially abundant proteins estimating the log-fold changes between compared conditions of the chosen experimental group and with the wild-type non-diabetic mice. For all calculations, a P < 0.05 was considered as statistically significant.

Experimental animal ethics statement. All animal studies and experiments were performed in accordance with guidelines provided and approved by the AMREP (Alfred Medical Research and

101

Education Precinct) Animal Ethics Committee and the National Health and Medical Research

Council of Australia (E/0846/2009B).

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Figures and Figure Legends

Figure 1. DDOST+/- mice have increased glomerular expression of OST48, increased CML excretion, however no changes in CML deposition in the kidney or plasma CML levels.

OST48 expression and localization in the kidney. SWATH proteomics (A-C) quantification of the protein intensities of OST48 in processed kidney tissue enriched for either (A) glomeruli or (B) tubules and in the kidney cortex (C). Confocal photomicrographs (D-E) of OST48 (green), CML

(orange) and either a podocyte foot process marker, nephrin (red) or a proximal tubule marker,

SGLT2 (red) on kidney sections imaged at either a (D) glomerulus or (E) proximal tubule. CML

ELISA (F-H) measuring the total content of CML in (F) 24-hour urine collections, (G) whole kidney and (H) plasma. Results are expressed as mean ± SD with either two-way ANOVA or unpaired t-test analysis (n = 4-9) *P < 0.05. Scale bars from representative images for confocal

103 microscopy are representative of 5µm. For proteomics, MSstatsV3.10.0 determined significant (P <

0.05) log fold changes in the protein intensities between the selected experimental group the wild- type non-diabetic group (n = 4-5).

Figure 2. Diabetic mice with global increases in OST48 are not protected against the development of diabetic kidney disease. Serum and 24-hour urine was collected from mice in metabolic cages at week 0 and week 24 of the study. Creatinine was measured spectrophotometrically and the (A) change in creatinine clearance over the study period was determined from matched creatinine clearance values from week 0 of the study (6-8 weeks of age) to week 24 of the study. (B) Urinary albumin:creatinine ratio measured at week 24 of the study. (C)

Albumin was measured spectrophotometrically at 620nm in a biochemical analyzer and the albumin excretion rate (AER) was determined based on the 24-hour urine flow rate. Results are expressed as mean ± SD with either two-way ANOVA or paired t-test analysis (n = 3-8) *P < 0.05.

104

Figure 3. DDOST+/- mice were not protected from renal structural damage and exhibited similar patterns of collagen protein content exhibited in mice with diabetes. (A) Assessment of renal glomerular damage in the kidney with Periodic-acid Schiff staining (PAS), which was then quantified based on a positive threshold protocol. DDOST+/- mice and mice with a diabetic phenotype exhibited moderate glomerulosclerosis, indicated by an increase in mesangial matrix expansion (black arrow). (B) Heat map representation of SWATH-MS proteomics of glomeruli enriched proteins for enzymatic pathways involved in collagen fibril organization. Significant proteins are represented as bolded cells, where red indicates an increase and blue indicates a decrease in protein concentrations. (C) Presence of collagen in Masson’s trichrome (blue staining) and (D) Sirius red (red staining) in the interstitium of the tubules (black arrow) is an indicator of progressive kidney damage. The severity of these changes was more pronounced in mice with a diabetic phenotype. Scale bars from representative images of (A) glomeruli stained with PAS or (C- 105

D) tubule sections stained with either Masson’s trichrome or Sirius red were 20µm and 100µm, respectively. Results are expressed as mean ± SD with either two-way ANOVA or unpaired t-test analysis (n = 5-9) *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. For proteomics,

MSstatsV3.10.0 determined significant (P < 0.05) log fold changes in the protein intensities between the selected experimental group the wild-type non-diabetic group (n = 3-5).

Figure 4. Diabetic DDOST+/- mice exhibit increased first phase insulin secretion and activation of the P13K-AKT pathway akin to a diabetic phenotype. (A) 24-hour fractional excretion of glucose. (B) Fasting plasma insulin concentrations. (C) Plasma glucose and (D) insulin

106 curve over 120-minutes following an intraperitoneal glucose injection and (E) first phase area- under-the-curve (AUC) analysis and (F) 120-minute AUC analysis. (G) Ratio of AUIC:AUGC of early-phase insulin response. (H) Plasma glucose over 120-minutes following an intraperitoneal insulin injection and (I) 120-minute AUC analysis. (J) Heat map representation of SWATH-MS proteomics data for enzymatic pathways in glomerular proteins involved the P13K-AKT signalling pathway. Significant proteins are represented as the Log2 fold change where red indicates a decreased and blue indicates an increase in protein concentrations. Results are expressed as mean ±

SD with either two-way ANOVA or unpaired t-test analysis (n = 5-9) *P < 0.05, **P<0.01,

***P<0.001. For IPGTT and IPITT curves results are represented as *P<0.05, **P<0.01,

***P<0.001 for DDOST+/- diabetic compared to wild-type diabetic, α < 0.05 effect due to genotype, β < 0.05 effect due to diabetes. For proteomics, MSstatsV3.5.1 determined significant (P

< 0.05) log fold changes in the protein intensities between the selected experimental group the wild- type non-diabetic group (n = 3-5).

107

Tables

Table 1. Biochemical/anthropometric measurements post-diabetes (Week 24 of study/32 weeks of age). Mean and standard deviation for biochemical/anthropometric measurements (n = 5-

11). Significance levels were determined by two-way ANOVA, testing the effect of genotype and diabetes. Differences between variables identified by Bonferroni’s post hoc test. Bold P values indicates significant effect of at least < 0.05.

WT DDOST+/- Two-Way ANOVA

Control Diabetic Control Diabetic Genotype Diabetes Interaction

GHb 5.58 ± 11.75 ± 6.54 ± 10.39 ± 0.36 <0.0001 0.17 (mmol/mol) 0.36 2.41 1.21 2.46

Food 0.067 ± 0.31 ± 0.078 ± 0.20 ± consumption 0.014 <0.0001 0.0033 0.016 0.058 0.026 0.082 (g/24 h)

Fed Glucose 16.26 ± 25.30 ± 16.26 ± 27.25 ± 0.52 <0.0001 0.52 (mmol/L) 2.43 6.22 3.18 1.50

Fasting 10.79 ± 12.98 ± 9.70 ± 12.35 ± Glucose 0.36 0.015 0.81 3.02 2.51 0.98 1.91 (mmol/L)

Fasting Insulin 0.98 ± 0.33 ± 0.97 ± 0.71 ± 0.22 0.0056 0.21 (ng/ml) 0.61 0.07 0.32 0.15

Water 0.13 ± 1.73 ± 0.15 ± 0.91 ± consumption 0.00030 <0.0001 0.0002 0.010 0.43 0.052 0.39 (ml/24 h)

Urine 0.025 ± 1.37 ± 0.019 ± 0.77 ± production 0.0030 <0.0001 0.0035 0.012 0.43 0.012 0.33 (ml/24 h)

Kidney 11.92 ± 19.68 ± 10.71 ± 16.36 ± Weight/BW 0.020 <0.0001 0.25 2.99 0.71 1.94 1.23 ratio (g)

108

Acknowledgements

The authors would like to acknowledge Maryann Arnstein and Anna Gasser at Baker IDI Heart and

Diabetes Institute, Australia, and David Briskey at the School of Human Movement and Nutrition

Sciences, University Queensland, Australia, for technical assistance.

Duality of Interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Author Contributions

The following authors’ contributions are listed below. AZ, JMF and BS wrote the main manuscript text, AZ prepared figures 1-5 and supplementary figures, in addition to the accompanied figure legends and supplementary figure legends. FYTY, DM, CL, SSL, KCS, BEH, SAP, VTB and MTC assisted with experiments and/or revised the manuscript.

Financial Support:

JMF is supported by a Senior Research Fellowship (APP1004503;1102935) from the National

Health and Medical Research Council of Australia (NH&MRC). BLS is supported by a Career

Development Fellowship (APP1087975) from the NH&MRC. AZ received a scholarship from

Kidney Health Australia (SCH17; 141516) and the Mater Research Foundation. MTC is supported by a Career Development Fellowship from the Australian Type 1 Diabetes Clinical Research

Network, a special initiative of the Australian Research Council. This research was supported by the

NH&MRC, Kidney Health Australia and the Mater Foundation, which had no role in the study design, data collection and analysis, decision to publish, or preparation or the manuscript.

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Chapter 5: The AGE receptor, OST48 contributes to podocyte dysfunction in experimental diabetic kidney disease via induction of endoplasmic reticulum stress.

110

Introduction to this chapter. This chapter is submitted for publication in the Journal of the

American Society of Nephrology as an original article. In this report, we over-expressed the gene encoding for oligosaccharyltransferase-48 (OST48) specifically in the podocytes. It is well known that podocytes are significantly reduced in diabetic kidney disease (DKD). In diabetes, podocytes are exposed to persistent long-term challenges, including; glomerular hypertension, metabolic imbalances and abnormal receptor stimulation, all of which are thought to contribute to functional loss of podocytes, compromising the integrity of the filtration barrier. The pathological events that culminate in diabetic kidney disease is thought to occur in a stereotypical manner that initiates with podocyte injury, leading tuft adhesions in the Bowmans capsule and subsequent filtration of abnormal filtrate encroaching the glomerulo-tubular junction and subsequent degradation of proximal tubules. There have been no studies examining whether specifically increasing AGE clearance via receptor manipulation in the podocytes is protective of the kidneys in diabetes. As such, we site-specifically increased podocyte expression of OST48.

We demonstrated the following; 1) Mice over-expressing OST48 specifically at the podocytes

(DDOST+/-Pod-Cre) exhibited elevated serum creatinine, reduced eGFR and increased albumin excretion, all of which at levels worse than wild-type diabetic mice. 2) This was not improved by increased AGE clearance via podocyte OST48. 3) Glomerular damage occurred including mesangial matrix expansion, increased collagen IV depo and substantial tubulointerstitial fibrosis.

4) DDOST+/-Pod-Cre podocytes exhibited increased endoplasmic reticulum stress markers, specifically elevated sXBP1 and GRP78 expression.

In summary, we demonstrated a novel OST48 pathway that leads to increased AGE excretion, however this did not prevent podocyte damage from occurring in the forms of endoplasmic reticulum stress markers GRP78 and sXBP1. All of which, culminating in the development of kidney disease.

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Graphical abstract

112

Abstract

The accumulation of advanced glycation end products (AGEs) is implicated in the development and progression of diabetic kidney disease (DKD). There has been considerable interest in targeting the

AGE pathway by increasing “clearance” via modulating receptors such as advanced glycation end product receptor 1 (OST48). However, to date, no studies have examined whether stimulating AGE clearance via receptor manipulation is reno-protective in diabetes. Podocytes, which are involved in

AGE clearance, are early contributors to DKD and could be a target for reno-protection. As such, we generated a mouse with targeted elevation of OST48 in podocytes driven by the podocin promoter (DDOST+/-Pod-Cre). Despite promoting increased urinary clearance of AGEs, increasing

OST48 resulted in a decline in renal function, which was further exacerbated by diabetes. The resulting phenotype showed increased serum creatinine and decreased FITC-sinistrin glomerular filtration rates. DDOST+/-Pod-Cre mice had significant glomerular damage including glomerulosclerosis, collagen IV deposition, glomerular basement membrane (GBM) thickening and foot process effacement and tubulointerstitial fibrosis. SWATH-MS proteomic analysis of isolated glomeruli identified enrichment in collagen deposition, endoplasmic reticulum and oxidative stress proteins. Furthermore, ultra-resolution microscopy of podocytes revealed denudation of foot processes where there was co-localization of OST48 and AGEs. Electron microscopy confirmed that increasing OST48 expression led to podocyte foot process effacement and GBM expansion.

These studies indicate that facilitating AGE clearance by increasing podocyte expression of OST48, results in glomerular endoplasmic reticulum stress and a decline in kidney function which worsened diabetic kidney disease.

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Introduction

Globally there is a rising pandemic of diabetes10, 11, defined by persistent hyperglycemia, a principal risk factor for the development of concomitant chronic complications2, 12. Hence, the numbers of individuals with diabetic kidney disease (DKD), a major complication, are burgeoning. DKD is an important risk factor for both end-stage kidney disease and cardiovascular disease1, 2. While renin- angiotensin system blockade including angiotensin-converting-enzyme inhibition is first line therapy for DKD, in general these agents only achieve an ~ 20% reduction in end-stage kidney disease14.

Advanced glycation end products (AGEs) are a heterogeneous and complex group of non- enzymatic, post-translational modifications to amino acids and proteins, which include hemoglobin

A1C (HbA1C) a clinical marker used for the diagnosis of diabetes. Their endogenous formation can be exacerbated by chronic hyperglycemia and oxidative stress 3 and therefore the accumulation of

AGEs occurs at an accelerated rate in diabetes261, 262. Increases in AGE formation on skin collagen5,

263, 264 and within the circulation265, 266 predict poor prognosis for patients with diabetes including increased risk for kidney and cardiovascular disease. The kidneys are a major site of AGE detoxification through the filtration of circulating AGEs, and subsequently their active uptake and excretion7, 8 and hence AGE accumulation is seen in diabetic patients with chronic kidney disease267, 268. Therapies which lower AGE accumulation have shown benefit in Phase II clinical trials for the treatment DKD in individuals with Type 2 diabetes42.

OST48, an evolutionarily conserved type 1 transmembrane protein43, is encoded by the DDOST

(Dolichyl-diphosphooligosaccharide—protein glycosyltransferase 48 kDa subunit) gene and has been postulated to function as a receptor for AGE turnover and clearance44, 269. OST48 is expressed in most cells and tissues270, including macrophages48 and mononuclear cells49. In the kidney, previous studies have shown OST48 expression in glomerular cells including podocytes270, 271,

114 mesangial cells48 and proximal tubules270, 271, where it is postulated to mediate the uptake and degradation of AGEs51. The process whereby AGEs are cleared by OST48 in the kidney is not well understood50, but is thought to involve caveolin dependent sequestration and degradation of AGEs and then excretion via the urine 242. Based on previous studies, a strong link exists between impaired podocyte function and albuminuria, increased urinary AGE excretion and glomerulosclerosis136, 167, 272-275.

Early damage to the glomeruli, specifically podocyte structural damage, is characteristic of DKD

130, 276, 277. AGEs can induce podocyte cell-cycle arrest278, hypertrophy278 and apoptosis279, whilst a systemic reduction in AGEs has been shown to improve podocyte and kidney function4. Hence, increased facilitation of AGE excretion underpinned by an increase in OST48 expression could potentially improve podocyte health and alleviate DKD. Currently there are no reported studies identifying whether podocyte OST48 facilitates urinary AGE excretion and subsequently if targeting podocyte OST48 could influence kidney health.

Here, we examined if facilitating greater AGE clearance via modestly increasing podocyte specific

OST48 expression could attenuate the development and progression of DKD.

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Methods

Animal husbandry. All animal studies were performed in accordance with guidelines provided by the AMREP (Alfred Medical Research and Education Precinct) Animal Ethics Committee,

University of Queensland and the National Health and Medical Research Council of Australia

(E/1184/2012/B and MRI-UQ/TRI/256/15/KHA). Male C57BL/6J wild-type mice with a genetic insertion of the human gene encoding AGE-R1/OST-48 (DDOST) in the ROSA26 locus

(ROSA26tm1(DDOST)Jfo) and were crossed with mice only expressing Cre under the control of the

Podocin promoter (termed DDOST+/-Pod-Cre) were generated by Ozgene Australia (Ozgene, Perth,

WA, Australia). Between 6-8 weeks of age (Week 0), experimental diabetes (DIAB) was induced in male wild-type (n = 9) and DDOST+/-Pod-Cre (n = 10) mice by low dose intraperitoneal injection of streptozotocin 257. Non-diabetic male wild-type (n = 11) and DDOST+/-Pod-Cre (n = 7) mice received equivalent injections of sodium citrate buffer alone. After 10 days of recovery, blood glucose concentrations were determined and then repeated weekly to ensure mice had diabetes ([blood glucose]>15mmol/L). Mice were allowed access to food and water ad libitum and were maintained on a 12-hour light:dark cycle at 22°C. All mice received a diet of standard mouse chow (AIN-93G)

(Specialty Feeds, Glen Forrest, Perth, Australia). At 0 and 12 weeks of the study, mice were housed in metabolic cages (Iffa Credo, France) for 24 hours to determine food and water consumption and collect urine. Urine output was also measured during caging and urinary glucose determined by a glucometer (SensoCard Plus, POCD, Australia).

Biochemical analyses. CML concentrations in plasma and urine were measured by an in-house indirect ELISA as previously described50. Urinary albumin (Bethyl Laboratories, United States) was normalized to the 24-hour flow rate of urine. Serum and urinary creatinine were measured spectrophotometrically at 505nm (Roche/Hitachi 902 Analyzer, Roche Diagnostics GmBH,

Germany) using the Jaffe method.

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Glomerular Filtration Rate. At 0 and 12 weeks of the study, GFR was estimated in conscious mice using the transcutaneous decay of retro-orbitally injected FITC-sinistrin (10mg/100g body weight dissolved in 0.9% NaCl), as previously described280. GFR was calculated from the rate of constant (α2) of the single exponential excretion phase of the curve and a semi-empirical factor.

Liquid chromatography-selected reaction monitoring mass spectrometry (LC-SRM/MS-MS).

Kidney cortices were sequentially sieved on ice to obtain fractions enriched for either glomeruli or tubules, which were then processed as previously described224. Peptides were desalted and analyzed by Information Dependent Acquisition LC-MS/MS as described225 using a Prominence nanoLC system (Shimadzu, Australia) and Triple TOF 5600 mass spectrometer with a Nanospray III interface (AB SCIEX, United States). SWATH-MS analysis was performed226 and differentially abundant proteins were analyzed using DAVID227.

Histology and imaging. Paraffin-embedded sections were stained with either a Periodic acid Schiff

(PAS) staining kit (Sigma-Aldrich, United States), a Trichrome (Masson) staining kit (Sigma-

Aldrich, St. Louis, Missouri, USA) or Sirius Red (Sigma-Aldrich, United States). Collagen IV was determined by immunohistochemistry using an anti-collagen IV antibody (1:100 dilution; ab6586;

Abcam, United Kingdom). All sections were visualized on an Olympus Slide scanner VS120

(Olympus, Japan) and viewed in the supplied program (OlyVIA Build 10555, Olympus, Japan).

Slides were quantified based on threshold analysis in Fiji258. Briefly, for immunofluorescence staining, frozen kidney sections were stained with a combination of either anti-CML (1:200 dilution; ab27684; Abcam, United Kingdom), OST48 (H-1; 1:100 dilution; SC-74408; Santa Cruz biotechnologies, United States), GRP78 (A-10; 1:50 dilution; SC-376768; Santa Cruz biotechnologies, United States), WT-1 (c-terminus; 1:100 dilution; PA5-16879; ThermoFisher

Scientific, United States), synaptopodin (P-19; 1:100 dilution; SC-21537; Santa Cruz biotechnologies, United States), nephrin (1:100 dilution; ab27684; Abcam, United Kingdom).

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Confocal images were visualized on an Olympus FV1200 confocal microscope (Olympus, Japan) and viewed in the supplied program (FV10, Olympus, Japan). For ultra-resolution 3D microscopy, slides were visualized on an OMX Blaze deconvolution structured illumination (SIM) super- resolution microscope (GE Healthcare Life Science, United Kingdom).

Fixation, tissue processing and acquisition of data from electron microscopy. Renal cortex was processed as previously described281. Sections were cut at 60nm on a Leica UC6 ultramicrotome

(Leica, Germany) and were imaged at 80kV on a Jeol JSM 1011 transmission electron microscope

(Jeol, United States) equipped with an Olympus Morada (Olympus, Japan) digital camera.

Quantification of foot process width and GBM expansion was assessed as previously described282.

Statistical analyses. Results are expressed as mean ± SD (standard deviation), and assessed in

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Data availability statement. The datasets generated and analyzed during the current study are available from the corresponding author upon reasonable request.

Results

Generation of a podocyte-specific OST48 knock-in mouse model. Mice were generated with a podocyte specific knock-in of human OST48 (DDOST+/-Pod-Cre) driven by the podocin promoter

(Supplementary Information 1A-B) inserted at the ROSA26 locus and showed no differences in any anthropometric parameters or long term glycemic control (Table 1). Mice with experimentally induced diabetes were characterized by elevated fasting blood glucose and glycated hemoglobin

(GHb) (Table 1). Irrespective of genotype, diabetic mice exhibited a lower body weight, renal hypertrophy, increased consumption of food and water and a greater urinary output when compared with non-diabetic mice (Table 1).

DDOST+/-Pod-Cre mice had podocyte specific increases in OST48 protein content. SWATH proteomics measuring the relative abundancy of OST48 in renal cortices (Figure 1A) identified that

DDOST+/-Pod-Cre mice had a subtle and significant increase in OST48 protein abundancy (2.06- fold). Moreover, SWATH proteomics identified in fractions enriched for either glomeruli or tubular specific proteins (Figure 1B) subtle and significant increases in OST48 abundancy (2.59-fold and

1.79-fold, respectively). Interestingly, it appeared that there was also a significant interactive effect of impaired glycemic control on the abundancy of OST48 in only the glomeruli proteins (Figure

1B; left). Localized increases in OST48 expression in WT-1 positive cells were demonstrated in

DDOST+/-Pod-Cre mice by confocal microscopy (Figure 1C). A podocyte-specific increase in

OST48 expression was confirmed using ultra-resolution 3-dimensional structured illumination microscopy (3D-SIM) (Figure 1D). This included increased localization of OST48 to damaged podocyte foot processes, as indicated by nephrin loss (Figure 1D) and this was particularly evident in 3D reconstruction videos (Supplementary Figure 1C and Supplementary Video 1). This

119 localization of OST48 to areas of damaged foot processes was unexpected, since the current thinking is that an increase in OST48 could improve declining kidney function through the sequestering of AGEs52. These initial findings of damage to podocyte foot processes warranted further investigation into kidney function in DDOST+/-Pod-Cre mice and whether increased podocyte expression of OST48 affected glomerular filtration.

Subtle increases in podocyte OST48 expression decreases GFR, exacerbating DKD. Conscious glomerular filtration rate (GFR) determined as a ratio of subcutaneous excretion of FITC-sinistrin, identified a significant decline in kidney function in DDOST+/-Pod-Cre mice (Figure 2A – 49% decrease, P < 0.0001). Compared to their baseline kidney function, DDOST+/-Pod-Cre mice lost 59% of GFR over the study period (Figure 2B). Non-diabetic DDOST+/-Pod-Cre mice also had elevated serum creatinine compared to their wild-type counterparts (Figure 2C – average 88% increase; P =

0.0034), which was further increased by diabetes (Figure 2C – 58% increase; P = 0.043 for

DDOST+/-Pod-Cre and 123% increase; P = 0.0047 for wild-type). Diabetes increased serum creatinine in wild type mice (Figure 2C) and decreased creatinine clearance (Figure 2D), which tended to further decrease in DDOST+/-Pod-Cre mice, although this did not reach statistical significance (Figure 2D). Compared to wild-type mice, DDOST+/-Pod-Cre mice averaged a 72% reduction in creatinine clearance following simultaneous blood and 24-hour urine collection

(Figure 2E; P = 0.0052) in agreement with FITC-sinistrin based GFR assessment (Figure 2A).

Diabetes increased 24-hour urinary albumin excretion rate, but this did not differ between wild-type and DDOST+/-Pod-Cre mice (Figure 2F and 2G).

DDOST+/-Pod-Cre mice have damaged podocyte architecture and tubulointerstitial damage.

Diabetes resulted in glomerulosclerosis in both genotypes (Figure 3A). In the absence of diabetes,

DDOST+/-Pod-Cre mice also had glomerulosclerosis (Figure 3A) and greater glomerular collagen IV accumulation (Figure 3B) than wild-type mice which was not further elevated by diabetes.

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Glomerular collagen IV accumulation was also present in all diabetic mice (Figure 3B). By electron microscopy, glomerular basement membrane thickening and podocyte foot process effacement were seen in DDOST+/-Pod-Cre, but not in wild-type mice (Figure 3C). In glomerular fractions from mouse kidney cortices, SWATH-MS proteomics identified significant increases in the abundance of collagen proteins (Figure 3D). Specifically, both diabetes and increased podocyte

OST48 expression caused an increased abundance of collagen 1, collagen 6 and other structural collagen proteins (Figure 3D). Given that all DDOST+/-Pod-Cre mice exhibited a decline in kidney function, we suspected that in addition to damaged glomeruli that there would be other structural evidence of progressive kidney damage in the tubules of these mice. As expected, diabetes increased tubulointerstitial fibrosis in WT mice (Figure 4A-C). In addition, there was significant tubulointerstitial fibrosis in all DDOST+/-Pod-Cre mice as compared to wild-type non-diabetic mice, evidenced by an increase in positive connective tissue quantified from Masson’s trichrome (Figure

4A), Sirius red (Figure 4B) and collagen IV staining in this compartment (Figure 4C). In tubule- enriched cortical fractions, SWATH-MS proteomics identified modest increases in proteins associated with collagen fibril organization (CO1A1-2) (Figure 4D) in non-diabetic DDOST+/-Pod-

Cre mice as compared with WT littermates. However, with diabetes, DDOST+/-Pod-Cre mice showed significant accumulation of collagen IV in tubule-enriched fractions which were not seen in WT diabetic or non-diabetic DDOST+/-Pod-Cre mice. There were further modest increases in markers of tubulointerstitial fibrosis in diabetic DDOST+/-Pod-Cre but these were not different to WT mice with diabetes (Figure 4A-D)

Tissue AGEs were localized to damaged podocytes. Podocyte AGE accumulation and OST48 appeared to be co-localized (Figure 5A), and were prominent in areas where concomitant deterioration of podocyte foot processes was seen, as defined by loss of both nephrin (Figure 5A) and synaptopodin (Supplementary Figure 2A). 3D-SIM confirmed the appearance of nodules/vacuoles containing both OST48 and AGEs in DDOST+/-Pod-Cre mice in areas of podocyte

121 foot process deterioration (Figure 5B, Figure 5C and Supplementary Video 2). These changes were not observed in wild-type diabetic mice. Overall, there was a significant reduction in AGE content in kidney cortical extracts taken from DDOST+/-Pod-Cre mice (Figure 5D - genotype effect P

< 0.05). Diabetes also increased urinary AGE excretion (P = 0.007; ~10-fold increase) when compared to non-diabetic mice (Figure 5E). Although not significant (P < 0.2) there was also a trend towards increased urinary AGE excretion in non-diabetic DDOST+/-Pod-Cre mice compared to wild-type non-diabetic mice. Plasma AGE concentrations at the end of the study were not different among groups (Supplementary Figure 2B).

Podocyte OST48-mediated AGE accumulation resulted in ER stress. ER stress at sites of AGE accumulation within damaged podocytes was examined. DDOST+/-Pod-Cre mice exhibited an increase in the ER stress markers GRP-78 and spliced XBP-1 when compared to wild-type mice

(Figure 6A). Furthermore, these ER stress markers were further increased by diabetes (Figure 6A).

This was confirmed using glomerular enriched fractions and SWATH proteomics where diabetic

DDOST+/-Pod-Cre mice, showed significant enrichments in proteins associated with ER stress

(GRP75, GRP78 and TERA) compared to non-diabetic wild-type mice (Figure 6B). Antioxidant enzymes SOD1/SODC and SOD2/SODM were also increased by diabetes in wild-type mice

(Figure 6B). Glomerular fractions from diabetic wild-type and DDOST+/-Pod-Cre mice were also significantly enriched for proteins associated with mitochondrial ATP synthesis, particularly biosynthesis of complex I components (NADH dehydrogenase:ubiquinone) when compared with non-diabetic mice (Figure 6C).

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Discussion

Lowering AGE burden, including facilitating renal AGE clearance has been suggested as a possible treatment for DKD. However, the assertion that increasing renal AGE clearance receptors, including OST48, may protect against DKD has not been previously investigated. Here, we have shown for the first time that modestly increasing OST48 in a podocyte-specific manner decreased glomerular filtration and caused podocyte structural damage including foot process effacement, leading to glomerulosclerosis and tubulointerstitial fibrosis, which were further exacerbated by diabetes. Surprisingly, in non-diabetic mice the decline in GFR and increased podocyte and glomerular damage were seen in the absence of elevations in albuminuria. Mechanistically, increases in OST48 in podocytes were marked by increased podocyte specific AGE uptake resulting in oxidative and endoplasmic reticulum stress and subsequent kidney functional and structural decline, where previously it has been shown that AGE uptake into cultured podocytes induces hypertrophy278 and apoptosis279.

It was particularly interesting that albuminuria was not seen concomitantly with the loss of GFR in

DDOST+/-Pod-Cre mice despite podocyte foot effacement, thickening of the glomerular basement membrane and glomerulosclerosis. Although surprising, this is consistent with clinical observations.

For example, the prospective longitudinal Joslin Proteinuria Cohort studies283, 284 identified that the overall prevalence of normo-albuminuria in patients with progressive kidney disease was around

10% and that regression from micro- and macro-albuminuria to normo-albuminuria was repeatedly observed. Puzzlingly, however, we observed podocyte foot effacement and glomerular basement membrane thickening, which are commonly attributed as causes of micro- and macro-albuminuria.

This finding is consistent with other postulated models of the origins of albuminuria, such as the high glomerular sieving coefficient (GSC) hypothesis285, 286. This hypothesis suggests that albuminuria is driven by proximal tubule damage which prevents the reabsorption/processing of albumin by these cells. In support of this, tubulointerstitial fibrosis was more pronounced in

123 diabetic mice regardless of their genotype and they also had concomitant increases in urinary albumin excretion which not seen in non-diabetic mice. Alternatively, the absence of macroalbuminuria seen in mice with increased podocyte expression of OST48, could also be attributed to the significant decline in GFR as this would limit the flux of albumin into the urine, however this warrants further investigation.

Although we showed that increases in podocyte OST48 expression decreased renal AGE accumulation and increased urinary AGE excretion, this was not associated with reno-protection. In fact, this resulted in significant renal disease. We have shown for the first time that OST48 and

AGEs colocalize within podocytes in areas of foot process denudation and damage. We observed that OST48 facilitation of podocyte AGE accumulation, resulted in a cascade of ER stress culminating in podocyte foot process effacement, GBM expansion, and renal functional decline.

Increasing the urinary flux of AGEs has however been previously shown to impair renal function in both healthy humans4, 287 and early in the development of diabetic kidney disease in rodent models136, 288.

Taken together, these results suggest that specifically increasing podocyte exposure and uptake of

AGEs by facilitating their flux into the urine is sufficient to induce significant kidney damage.

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Acknowledgements

The authors acknowledge the Translational Research Institute (TRI) for providing the excellent research environment and core facilities that enabled this research. We particularly thank Sandrine

Roy and Ali Ju from the TRI Microscopy Core Facility. Furthermore, the authors acknowledge the assistance in electron microscopy provided by Jennifer Brown from the Centre for Microscopy and

Microanalysis at the University of Queensland.

Competing Financial Interests

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Author Contributions

The following authors’ contributions are listed below. AZ wrote the main manuscript text, AZ prepared figures 1-5 and supplementary figures, in addition to the accompanied figure legends and supplementary figure legends. JMF and BS, conceived the project, raised the funding and supervised completion of the experiments, as well as assisting with statistical tests and drafting of the manuscript. FYT, DM, LAG and SAP provided technical assistance. DM, LAG, KCS and MTC assisted with manuscript revision. All authors have approved the final manuscript.

Financial Support

JMF is supported by a Senior Research Fellowship (APP1004503;1102935) from the National

Health and Medical Research Council of Australia (NH&MRC). BLS is supported by a Career

Development Fellowship (APP1087975) from the NH&MRC. AZ received a scholarship from

Kidney Health Australia (SCH17; 141516) and the Mater Research Foundation. MTC is supported by a Career Development Fellowship from the Australian Type 1 Diabetes Clinical Research

Network, a special initiative of the Australian Research Council. This research was supported by the

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NH&MRC, Kidney Health Australia and the Mater Foundation, which had no role in the study design, data collection and analysis, decision to publish, or preparation or the manuscript.

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Figures and Figure Legends

Figure 1. OST48 expression and localization in the kidney. (A-B) SWATH proteomics quantification of total OST48 in (A) renal cortex and (B) enriched for either (left) glomeruli or

(right) tubules in both the DDOST+/-Pod-Cre mice and the wild-type littermates. (C) Confocal photomicrographs of OST48 (green) and a podocyte specific marker WT-1 (red) on kidney sections imaged at the glomerulus. (D) 3D-SIM of podocyte foot processes stained with nephrin (red) and

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OST48 localization (green). Scale bars from representative images for (C) confocal microscopy and

(D) 3D-SIM were 30µm and 5µm, respectively. Results are expressed as mean ± SD with unpaired t-test analysis (n = 6-12) **P < 0.01, ***P < 0.001 and for proteomics (n = 4-6), MSstatsV3.10.0 determined significant changes in the protein intensities * P < 0.05, **P < 0.01, ****P < 0.0001.

Figure 2. DDOST+/-Pod-Cre mice exhibited a rapid and progressive decline in kidney function independent of macroalbuminuria. Serum and 24-hour urine was collected from mice in metabolic cages at week 0 and week 12 of the study. (A-C) Creatinine was measured spectrophotometrically at 550nm in a biochemical analyzer. (A) Serum creatinine measured at week 12 of the study. (B)

Ratio of creatinine clearance following correction for kidney weight measured at week 12 of the study. (C) Change in creatinine clearance over the study, which was determined from matched creatinine clearance values from week 0 of the study (6-8 weeks of age) to week 12 of the study.

(D-E) Albumin was measured spectrophotometrically at 620nm in a biochemical analyzer and the albumin excretion rate (AER) was determined based on the 24-hour urine flow rate. (D) AER 128 measured at week 12 of the study. (E) Change in albuminuria over the study, which was determined from matched albuminuria values from week 0 (6-8 weeks of age) of the study to week 12 of the study. Results are expressed as mean ± SD with either two-way ANOVA or paired t-test analysis (n

= 3-8) *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3. Podocyte OST48 was associated with a deterioration of podocyte architecture. (A-C)

Assessment of renal glomerular damage in the kidney with (A) Periodic-acid Schiff staining (PAS) and (B) collagen IV, which was then quantified based on a positive threshold protocol. DDOST+/-

Pod-Cre mice and mice with a diabetic phenotype show moderate glomerulosclerosis, indicated by an increase in mesangial matrix expansion (black arrow) and accumulation of the extracellular matrix

(white arrow head). (C) Electron microscopy determined the degree of hypertrophy in the foot 129 processes (black arrow) and the expansion of the GBM (white arrow head). Scale bars from representative images of (A-B) glomeruli stained with either PAS or collagen IV were 20µm. (C)

Representative images from electron microscopy were imaged at either X3000 magnification or

X15000 magnification were 20µm and 4µm, respectively. (D) Heat map representation of SWATH-

MS proteomics data for enzymatic pathways involved in collagen fibril organization. Significant proteins are represented as bolded cells, where red indicates an increase and blue indicates a decrease in protein concentrations. Data represented as means ± SD (n = 4-7/group). Results are expressed as mean ± SD with either two-way ANOVA or unpaired t-test analysis (n = 5-9) *P <

0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. For proteomics, MSstatsV3.10.0 determined significant (P < 0.05) log fold changes in the protein intensities between the selected experimental group the wild-type non-diabetic group.

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Figure 4. Podocyte OST48 increased tubulointerstitial fibrosis. (A-C) Moreover, the presence of collagen in (A) Masson’s trichrome (blue staining), (B) Sirius red (red) and (C) collagen IV

(brown), was quantified based on a positive threshold protocol. Fibrosis in the interstitium of the proximal tubules (black arrow) is an indicator of progressive kidney damage. The severity of these changes was more pronounced in the DDOST+/-Pod-Cre mice and the mice with a diabetic phenotype.

(D) Heat map representation of SWATH-MS proteomics data for enzymatic pathways involved in collagen fibril organization. Significant proteins are represented as bolded cells, where red indicates an increase and blue indicates a decrease in protein concentrations. Data represented as means ± SD

(n = 4-7/group). Scale bars from representative images of (A-C) proximal tubule sections stained with either Masson’s trichrome, Sirius red or collagen IV were 100µm. Results are expressed as mean ± SD with either two-way ANOVA or unpaired t-test analysis (n = 5-9) *P < 0.05, **P <

0.01. For proteomics, MSstatsV3.10.0 determined significant (P < 0.05) log fold changes in the protein intensities between the selected experimental group the wild-type non-diabetic group.

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Figure 5. Podocyte OST48 increased AGE accumulation in podocytes. (A) Confocal photomicrographs of OST48 (green), CML (orange) and a podocyte foot process marker, nephrin 132

(red) on kidney sections imaged at a glomerulus. (B) 3D-SIM of podocyte foot processes staining for nephrin (red), OST48 (green) and CML (blue) localization. (C) 3D-SIM reconstruction of podocyte foot processes staining for nephrin (red), OST48 (green) and CML (blue) localization. (D-

E) CML ELISA measuring the total content of CML detected in (D) whole kidney cortex homogenates and in (E) 24-hour urine collections. Scale bars from representative images for (A) confocal microscopy and (B-C) 3D-SIM were 30µm and 5µm, respectively. Results are expressed as mean ± SD with either two-way ANOVA or unpaired t-test analysis (n = 5-9) *P < 0.05.

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Figure 6. Podocyte OST48 increased ER stress markers. (A) Confocal photomicrographs of either

ER stress marker GRP-78 (green), or XBP-1 (green) and podocyte foot process marker, synaptopodin (red). (B-C) Heat map representation of SWATH-MS proteomics data for enzymatic

134 pathways involved in (B) endoplasmic reticulum (ER) stress and oxidative stress (OS) enzymatic pathways, and (C) ubiquinone biosynthesis pathways. Significant proteins are represented as bolded cells, where red indicates an increase and blue indicates a decrease in protein concentrations. Data represented as means ± SD (n = 4-7/group). Scale bars from representative images of confocal microscopy were 30µm. Data represented as means ± SD (n = 5/group). For proteomics,

MSstatsV3.10.0 determined significant (P < 0.05) log fold changes in the protein intensities between the selected experimental group the wild-type non-diabetic group.

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Tables

Table 1. Anthropometric and metabolic parameters. Treatment wild-type wild-type DDOST+/-Pod-Cre DDOST+/-Pod-Cre Two-way ANOVA (STZ induction) non-diabetic diabetic non-diabetic diabetic G D G•D Body weight (g) 27.14 ± 2.48 32.00 ± 4.62 0.93 0.0001 0.59 12 weeks 32.82 ± 3.12 26.56 ± 4.16 0.74 0.0001 0.33 Kidney weight (g × body weight 103) 12 weeks 11.62 ± 1.44 14.74 ± 2.27 11.03 ± 1.21 15.93 ± 3.91 Food consumption (g/24 h) 3.62 ± 0.49 3.63 ± 1.12 0.46 0.44 0.086 2 weeks 3.26 ± 0.80 2.70 ± 1.01 4.79 ± 1.13 2.50 ± 0.95 0.28 <0.0001 0.55 12 weeks 2.68 ± 0.58 4.13 ± 1.55 Water consumption (ml/24 h) 5.70 ± 2.91 4.74 ± 1.24 0.12 0.58 0.29 2 weeks 5.22 ± 2.91 3.24 ± 1.49 17.70 ± 9.77 2.94 ± 1.80 0.35 0.0004 0.79 12 weeks 4.97 ± 8.49 14.06 ± 10.28 Urine production (ml/24 h) 1.19 ± 0.69 1.34 ± 0.83 0.40 0.72 0.081 2 weeks 0.52 ± 0.34 0.78 ± 0.91 15.83 ± 8.05 2.03 ± 1.77 0.60 <0.0001 0.39 12 weeks 1.20 ± 0.76 12.53 ± 10.84 Fasting blood glucose (mmol/L) 23.53 ± 3.80 10.10 ± 2.95 0.35 <0.0001 0.14 12 weeks 9.22 ± 2.72 19.66 ± 6.94 GHb (mmol/mol) 7.72 ± 3.79 3.35 ± 0.28 0.45 0.0048 0.99 12 weeks 4.22 ± 1.68 6.86 ± 3.83 Values are mean ± SD (n = 5-11), G: genotype, D: diabetes, G•D: interaction, STZ: streptozotocin, GHb: glycated hemoglobin.

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Supplementary Figure Legends

Supplementary Figure 1. Generation of a podocyte specific DDOST heterozygous knock-in mutant. (A) Genomic clone of DDOST bearing exon 1 used for the construction of the floxed targeting vector as indicated. Heterozygous mice were crossed with mice expressing podocin-Cre and subsequent progeny contained a ubiquitous over-expression allele. (B) Genomic Southern blotting confirmed the predicted DDOST allelic structures in the podocin-targeted knock-in strain.

(C) Reconstruction of 3D-SIM of podocyte foot processes stained with nephrin (red) and OST48 localization (green). Scale bars from representative images for 3D-SIM were 5µm.

Supplementary Figure 2. Podocyte OST48 increased AGE accumulation in podocytes. (A)

Confocal photomicrographs of OST48 (green), CML (orange) and a podocyte foot process marker, synaptopodin (red) on kidney sections imaged at a glomerulus. Scale bars from representative images for confocal microscopy were 30µm. (B) CML ELISA measuring the total content of CML detected in plasma.

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Supplementary Video 1. Reconstruction of 7µm thick renal cortex section imaged in a 3D-SIM microscope. OST48 (green), nephrin (red). Scale bars from representative videos for 3D-SIM were

5µm. Attached as supplementary video (s4238045_phd_chapter5supplementaryvideo1).

Supplementary Video 2. Reconstruction of 7µm thick renal cortex section imaged in a 3D-SIM microscope. OST48 (green), CML(blue) and nephrin (red). Scale bars from representative videos for 3D-SIM were 5µm. Videos can be supplied upon request. Attached as supplementary video

(s4238045_phd_chapter5supplementaryvideo2).

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Chapter 6: Conclusions

139

Overview DKD is an important risk factor for cardiovascular disease. Understanding the development of DKD is crucial in reducing the morbidity and mortality associated with diabetes. AGEs have long been considered an important pathological factor contributing to DKD. This thesis investigates the currently lacking knowledge surrounding the lesser known AGE receptor OST48, its signalling pathways and the impact of changing its expression patterns on kidney/liver function in health and diabetes. We aimed to provide key information on which further studies to develop new therapies or re-purposing of other compounds, could be based to enhance treatment options targeting AGEs in DKD. This thesis investigated the physiological role of OST48 in the metabolome and proteome in the kidney and liver. Key findings are outlined below;

1. Initial findings examining protein synthesis and folding identified that two-fold over-

expression of OST48 does not impact N-glycosylation by the ER in the liver.

2. Increased OST48 protein resulted in hepatic fibrosis in the absence of steatosis, obesity and

reduced physical activity. Otherwise increasing OST48 activated other common pathways

implicated in NASH disease pathogenesis.

3. Hepatic fibrosis was further exacerbated when challenged with a “second hit” injury of a

high AGE diet, highlighting increased OST48 as a risk factor for liver damage in situations

where AGEs are plentiful such as in type 2 diabetes.

4. Adding a high AGE diet, reminiscent of a western style diet, increased absorption of dietary

AGEs by OST48 via both the gastrointestinal tract and the liver.

5. Mechanisms driving hepatic fibrosis centred around increased hepatic ER stress and the

preferential fuel utilisation of ketones and fatty acids.

6. Contrary to current perceptions, over-expression of OST48 in mice did not delay the onset

and/or progression of DKD.

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7. Global increases in OST48 facilitated excretion of AGEs in the urine, however, this did not

improve kidney function and/or health.

8. Ultra-resolution microscopy of mice with selective over-expression of OST48 in podocytes

imaged for the first time the selective binding of AGEs and OST48 within the podocyte.

9. Knock-in of podocyte OST48 leads to a decline in kidney function and significant

glomerular damage, which are exacerbated by diabetes.

10. Glomerular and tubular enriched protein fractions are both affected by increases in podocyte

OST48 expression.

11. Overall, we identified new insights into the role the OST48 plays in kidney health as well as

identifying a novel podocyte specific pathway relevant to diabetic kidney injury.

General discussion and future directions We completely understand that with the advent of technologies developed in recent years (e.g.

CRISPR/Cas9) that there are limitations surrounding the use of the Cre-LoxP technology in our mouse model knock-in system. However, at the time of the study design, the Cre-LoxP technology was the most advanced knock-in technology available for specific insertion at a defined genetic locus, ROSA26. All knock-out attempts for OST48 in mice were embryonically lethal. As such, a heterozygous knock-in design was utilised where OST48 expressing lines were chosen with modest over-expression (2-3 fold) and applied to better understand the physiological role of OST48 in DKD and liver health. Even the advent of CRISPR technology however, it would be difficult to pursue an

OST48 knock-out model without detrimentally affecting the N-glycosylation machinery. Our mouse lines were chosen with subtle increases in OST48 to provide the best chance of understanding if changes in OST48 would be of benefit in conditions where AGEs are plentiful and pathogenic such as diabetes. As such we feel that with the current technologies available, that our heterozygous

141

OST48 global and podocyte-specific knock-ins are currently the best possible animal models to evaluate the physiological role of OST48.

Another avenue we would like to further pursue is the establishment of a primary cell culture model of the podocyte grown from a population of diabetic patients with and without kidney disease.

Ideally, with the primary cell culture studies we would like to establish site-specific mutagenesis of

OST48 over-expression and knock-down. Optimally we would hope to establish this mutagenesis without affecting N-glycosylation pathway machinery, which would then allow us to examine the effects of OST48 specifically on isolated podocyte health and function. Moreover, a pilot study on a clinical cohort of diabetic patients with the focus on the role of OST48 in podocyte function would provide greater insight into the physiological role of OST48 in humans.

Alternative directions that could be pursued would be to establish a liver cell specific (for example hepatocyte-specific or hepatic stellate cell-specific) over-expression of OST48 in a mouse model.

This would allow us to further understand the effective role of OST48 specifically in the liver and how it could affect hepatic fibrosis and fuel utilisation in the liver. We are also currently completing studies to better understand how changes in podocyte OST48 have influenced systemic glycaemic control which was discovered but not followed up in this thesis. Therefore, a pancreatic specific over-expression of OST48 could also provide key insight into how OST48 may impact glycaemic control in conditions such as diabetes.

Taken together, in this thesis we present evidence that there are unappreciated roles for OST48 in both kidney and liver health/function. Our studies indicate that efforts to increase the expression of

OST48 to improve pathology in diabetes, and other metabolic diseases may have undesirable side- effects leading to kidney dysfunction, liver fibrosis and glucose intolerance. 142

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