STUDIES ON ANGIOTENSIN-(1-7) AND MITOCHONDRIA: THE ROLE OF AN
INTRACELLULAR RENIN-ANGIOTENSIN SYSTEM WITHIN THE KIDNEY
BY
BRYAN ANTHONY WILSON
A Thesis Submitted to the Graduate Faculty of
WAKE FOREST UNIVERSITY
GRADUATE SCHOOL OF ARTS AND SCIENCES
in Partial Fulfillment of the Requirements
for the Degree of
DOCTOR OF PHILOSOPHY
In Molecular Medicine and Translational Sciences
May 2016
Winston Salem, North Carolina
Approved By:
Mark C. Chappell, Ph.D., Advisor
TanYa M. Gwathmey-Williams, Ph.D., Co-Advisor
Robert N. Taylor, M.D., Ph.D., M.D. Advisor
James C. Rose, Ph.D., Chair
K. Bridget Brosnihan, Ph.D. ACKNOWLEDGEMENTS
My first and most important acknowledgement goes to my Lord and Savior Jesus
Christ. He has served as a comforter, best-friend, and has been the strength I needed to help me excel throughout my graduate career. Secondly, I want to acknowledge my mother Pamela A. Wilson who has had the single most important influence on my life and has served as an example of perseverance, strength and tenacity. Her constant support and reinforcement of my dreams allowed me to dream bigger and escape the confinements of my own self-limitations. I want to acknowledge my grandmother, Willie
Mae Brown who taught me how to be creative and to view the world as a canvas and to seek beauty in every moment. I want to acknowledge my father Derrick B. Pierre who taught me how to write with detail and clarity. Although use of the written word was our only means of communication during the majority of my life, it instilled in me a desire to communicate effectively and ultimately set the basis for my literary style.
I want to take this opportunity to acknowledge my PhD adviser Dr. Mark C.
Chappell. Little do you know just how much of an impact you have had on me during our initial summer working together as a summer student in the 2007 Excellence in
Cardiovascular Sciences Program. Your attention to detail, scholarly astute and vigor as an independent investigator provided me with an example of a research mentor who is dedicated to the advancement of his trainees. Working with you has literally changed my life for the better and I am grateful for all the time you have poured into my development.
I want to thank Dr. TanYa M. Gwathmey, my PhD co-adviser for her enthusiasm, guidance and dedication to forcing me to always think outside of the box. Often times in life, we are stuck in our own confusion, but being under your tutelage has caused me to have a profound awakening and mind reshaping. As a result of your support, I now understand the importance of going beyond what is required and see myself not only as
i a scientist but as a citizen of the world. You have instilled in me a spirit of outreach, service and mentorship; skills that I plan to implement in every step of my career progression.
I want to acknowledge committee member Dr. K. Bridget Brosnihan, who believed in me and supported me in ways I could never fully repay or express. Your impact on my life has been profound and I thank you for always knowing the right combination of words to say to get me through some very tough times. I want to acknowledge Dr. James C. Rose. It has been a pleasure having you serve as the chair of my committee and I thank you for all the talks we have had regarding science and the pleasant disposition you have always displayed. I want to acknowledge Dr. Robert
Taylor for serving as the M.D. chair of my committee and current graduate program director. Words cannot express the level of gratitude I have for every letter of support you have submitted on my behalf and for always stimulating in-depth discussions as it relates to my research and career goals
I want to additionally thank Dr. Debra Diz and Dr. Ann Tallant as well as the students, faculty and staff of the Wake Forest Hypertension and Vascular Research
Center. I want to thank Dr. John Parks and Kay Collare as well as the faculty and students of the Molecular Medicine and Translational Sciences Program. I want to acknowledge Dr. Dwayne Godwin for his guidance and support as well as being a dean who could think outside the box to encourage students to achieve their best. I also want to acknowledge the students, faculty and staff of the PhD/MBA program. This unique opportunity has allowed me to grow as a scientist and expanded my thinking. Moreover,
I want to thank collaborator and mentor abroad Dr. Monte S. Willis for inspiring me to write more, trust myself and to always be curious about everything.
I want to thank my siblings Carl Wilson, Yashua Wilson and Alexia Adams as well close friends and relatives Dr. Lisa Hightower-Washington, Kyle Washington, Dr.
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Tonye A. Jones, Swapnil Shewale, Brian Burkett, Christopher Boyd, Pearlina Baker, Dr.
Ollie Kelly, Dr. David Mount and Dr. Kristen Hairston for your support. In closing, I want to acknowledge fear! For a very long time, you have kept me from greeting life with love and ultimately kept me from greeting my own destiny. My acknowledgement and subsequent rejection of your strong hold has served as a catalyst to liberate me to life and unleased my spiritual trajectory.
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TABLE OF CONTENTS
LIST OF FIGURES…………………………………………………………………………vi
LIST OF ABBREVIATIONS……………………………………………………………….ix
ABSTRACT…………………………………………………………………………………xv
CHAPTER 1……………………………………………………………………………….....1
INTRODUCTION
Overview of the Renin Angiotensin System (RAS)………………………………………1
Systemic versus local organ RAS……………………………………………………….....5
RAS within the kidney……………………………………………………………………….7
Local RAS in renal proximal tubules………………………………………………………7
Proximal tubule angiotensinogen…………………………………………………10
Proximal tubule renin………………………………………………………………12
Proximal tubule ACE and ACE2………………………………………………….14
Alternative proximal tubule peptidases…………………………………………..16
Proximal tubule angiotensin peptides and receptors…………………………...17
Tubular intracellular RAS…………………………………………………………………..19
Tubule nuclear RAS………………………………………………………………..20
Tubule mitochondrial RAS…………………………………………………………22
Experimental outline and hypothesis……………………………………………………...26
Literature Cited……………………………………………………………………………….28
CHAPTER 2…………………………………………………………………………………..44
An Angiotensin-(1-7) Endopeptidase in the Kidney Cortex, Proximal Tubules and Human HK-2 Epithelial Cells that is Distinct from Insulin Degrading Enzyme
Published in American Journal of Physiology – Renal Physiology, March 2015 Vol. 308 no. 6, F594-F601
CHAPTER 3………………………………………………………………………………….75
iv
The Ins and Outs of Angiotensin Processing
Published in American Journal of Physiology - Regulatory, Integrative and Comparative Physiology, September 2014 Vol. 307 no. 5, R487-R489
CHAPTER 4………………………………………………………………………………..83
The Angiotensin-(1-7) Metallopeptidase in Human Proximal Tubule (HK-2) Cells Identified as Dipeptidyl peptidase III
Submitted to Peptides, April 2016
CHAPTER 5………………………………………………………………………………..105
Evidence for a Mitochondrial Angiotensin-(1-7) System in the Kidney
Published in American Journal of Physiology - Renal Physiology, December 2015
CHAPTER 6………………………………………………………………………………..135
Angiotensinogen Import in Renal Proximal Tubules: Evidence for Mitochondrial Trafficking
Submitted to American Journal of Physiology – Renal Physiology, April 2016
CHAPTER 7………………………………………………………………………………..162
DISCUSSION
General summary of findings…………………………………………………………….162
Angiotensin-(1-7) degrading pathways in the kidney………………………………….163
Identification of DPP3 in renal tubules and therapeutic insights…………….165
Renal intra-mitochondrial RAS …………………………………………………………..169
Renal oxidative stress and influences of the RAS…………………………….170
Angiotensin II-stimulation of ROS in the kidney……………………………….172
NOS in the kidney…………………………………………………………………173
Mitochondrial NOS……………………………………………………….174
Angiotensinogen uptake in renal mitochondria and therapeutic implications……….176
Literature Cited……………………………………………………………………………..180
CURRICULUM VITAE……………………………………………………………………..191
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LIST OF FIGURES
CHAPTER 1
Figure 1: Renin-Angiotensin System Cascade……………………………………………2
Figure 2: Cardiovascular disease events alter the balance of the Ang-(1-7) and Ang II systems…………………………………………………………………………………………4
Figure 3: Reabsorption: Proximal convoluted tubule……………………………………..9
CHAPTER 2
Figure 1: Ang-(1-7) endopeptidase activity in the sheep kidney cortex and isolated proximal tubules………………………………………………………………………………..52
Figure 2: Ang-(1-7) endopeptidase activity in human HK-2 cell supernatant and media. ……………………………………………………………………………………..53
Figure 3: Characterization of Ang-(1-7) endopeptidase activity in human HK-2 cells………………………………………………………………………………54
Table 1: Specific activiy of Ang-(1-7) endopeptidase……………………………………...56
Table 2: Purification of endopetidase from HK-2 cells………………………………….…57
Figure 4: Purified HK-2 endopeptidase preferentially hydrolyzes Ang-(1-7) to Ang-(1-4)……………………………………………………………58
Table 3: Comparison of angiotensin peptide sequences……………………………….…59
Figure 5: Comparison of the peptidase velocities for the hydrolysis of angiotensin peptides by the purified HK-2 endopeptidase………………………………………………60
Figure 6: Inhibitor profile of the purified HK-2 endopeptidase. ………………………….61
Figure 7: ANG-(1–7) peptidase from HK-2 cells is distinct from insulin-degrading enzyme (IDE)…………………………………………………………………………………..63
Figure 8: HK-2 processing of 125I-Ang I…………………………………………………….64
Figure 9: 125I-Ang I metabolism in HK-2 cells………………………………………………65
CHAPTER 3
Figure 1: Potential scheme for the extracellular and intracellular processing of angiotensin peptides within renal proximal tubules………………………………………..78
CHAPTER 4
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Figure 1: Purified Ang-(1-7) peptidase from Phenyl column reveals a single 80 kDa band on SDS-PAGE gel stained with Royal Blue…………………………………………..91
Table 1: MALDI-MS Identification……………………………………………………………92
Figure 2: Hydrolysis of Ang-(1-7) by the purified Ang-(1-7) peptidase and recombinant DPP 3……………………………………………………………………….94
Figure 3: Dose response curves for JMV-390 inhibition of 125I-Ang-(1-7) hydrolysis to 125I-Ang-(3-4) by the purified Ang-(1-7) peptidase (A7-P) and recombinant DPP 3……95
Figure 4: Kinetics of the hydrolysis of 125I-Ang-(1-7) and 125I-Ang-(3-7) by human DPP 3………………………………………………………………97
Figure 5: Influence of JMV-390 on 125I- Ang-(1-7) metabolism and intracellular Ang-(1-7) content in HK-2 cells…………………………………………………………………………..99
CHAPTER 5
Figure 1: Purification and enrichment of mitochondria from sheep renal cortex………113
Figure 2: Renin and prorenin receptor expression in isolated mitochondria from sheep renal cortex…………………………………………………………………………………….115
Figure 3: Renin activity in the mitochondria……………………………………………….116
Figure 4: Angiotensinogen (Aogen) and angiotensin peptide expression in isolated mitochondria from sheep renal cortex………………………………………………………118
Figure 5: Mitochondrial processing of 125I-Ang I…………………………………………..119
Figure 6: Immunodetection of Mas receptor expression in sheep renal mitochondria…………………………………………………………………121
CHAPTER 6
Figure 1: 125I-Aogen import in sheep proximal tubules and isolated cortical mitochondria………………………………………………………….145
Figure 2: 125I-Aogen increases in a linear fashion with higher concentrations of exogenous 125I-Aogen in sheep cortical mitochondria……………………………………146
Table 1: Comparison of angiotensinogen import in isolated sheep tubules and mitochondria from renal cortex tissue………………………………………………………147
Figure 3: 125I-Aogen import is comparable within isolated mitochondria from sheep cortex and HK-2 cells…………………………………………………………………………148
Table 2: Comparison of angiotensinogen and angiotensin peptides uptake in HK-2 mitochondria…………………………………………………………………………………..150
vii
Figure 4: Characterization of 125I-Aogen import…………………………………………..151
Figure 5: 125I-Aogen import is not dependent upon outer mitochondrial membrane… 153
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LIST OF ABBREVIATIONS
1,25(OH)2D3 = 1,25-dyhydroxyvitamin D3
A7-P = Angiotensin-(1-7) peptidase
ACE = Angiotensin I converting enzyme
ACE2 = Angiotensin II converting enzyme
AGE = Advanced glycated endproduct
AGT = Angiotensinogen
AIF = Apoptosis-inducing factor
AM = Amastatin
Ang = Angiotensin
Ang I = [Asp1-Arg2-Val3-Tyr4-Ile5-His6-Pro7-Phe8-His9-Leu10]
Ang I-Aogen = Ang I-sequence of angiotensinogen
Ang II = [Asp1-Arg2-Val3-Tyr4-Ile5-His6-Pro7-Phe8]
Ang-(1-4) = [Asp1-Arg2-Val3-Tyr4]
Ang-(1-7) = [Asp1-Arg2-Val3-Tyr4-Ile5-His6-Pro7]
ANOVA = Analysis of variance
ARBs = AT1 receptor blockers
AT1 = Angiotensin type 1 receptor
AT2 = Angiotensin type 2 receptor
AT4 = Angiotensin type 4 receptor
AT7 = Angiotensin-(1-7) receptor or Mas receptor
ATP = Adenosine triphosphate
ATP5A = ATP synthase-complex V
Bcl-2 = B-cell lymphoma 2
Bax = Bcl-2-like protein 4
Beta = Betamethasone
ix
BH4 = Bihydrobiopterin
BS = Bestatin
BSC = Benzyl succinate
CCCP = Carbonyl cyanide 3-chlorophenylhydrazone cAMP = Cyclic adenosine monophosphate cGMP = Cyclic guanosine monophosphate
CHYM = Chymostatin
CPP = c-phenylpropyl-alanine-alanine-phenylalanine-p-aminobenzoate
CSF = Cerebral spinal fluid
DALA = [7-D-ALA]-Angiotensin-(1-7)
DEAE = Diethylaminoethyl
DMEM = Dulbecco’s modified Eagle’s medium
DNA = Deoxyribonucleic acid
DPI = Diphenyleneiodonium
DPP3 = Dipetidyl peptidase 3
E2 = 17β-estradiol
E-64 = N-[N-(L-3-trans-carboxyirane-2-carbonyl)-L-leucyl]-agmatine; [1-[N-[(L-3-trans- carboxyoxirane-2-carbonyl)-L-leucyl]amino]-4-guanidinobutane]
EDTA = Ethylenediaminetetraacetic acid
EM = Electron microscopy
ENaC = Epithelial Na+ channel eNOS = Endothelial NOS
ER = Endoplasmic reticulum
ERK 1/2 = Extracellular regulated kinase 1 and 2
ETC = Electron transport chain
Ets-1 = C-ets 1
x
FAD = Flavin adenine dinucleotide
FBS = Fetal bovine serum
FMN = Flavinmononucleotide
G-1 = G protein coupled receptor 30 agonist
GC = Glucocorticoid
GPCR = G-protein coupled receptor
GPR30 = G protein-coupled receptor 30
H2O2 = Hydrogen peroxide
HFBA = Heptafluorobutyric acid
HK-2 = Human kidney 2 proximal tubule cell
HO-1 = Heme oxygenase-1
HPLC = High performance liquid chromatography
HEPES = 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
IC50 = Half maximal inhibitory concentration
IDE = Insulin degrading enzyme iNOS = Inducible NOS
IRAP = Interleukin-1 receptor antagonist protein
JG = Juxtoglomerular
JMV-390 = N-[3-[(hydroxyamino) carbonyl]-1-oxo-2(R)-benzylpropyl]-L-leucine kDa = Kilodalton
KOH = Potassium hydroxide
L-NAME = L-NG-Nitroarginine methyl ester
MAPK = Mitogen activated protein kinase
Na+ = Sodium
NaCl = Sodium chloride
Na+-K+-ATPase = Sodium-potassium-ATPase
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NADPH = Nicotinamide adenine dinucleotide phosphate
Nedd4-2 = Neuronal precursor cells expressed developmentally downregulated
NEP = Neprilysin
NHE3 = Sodium–hydrogen antiporter 3 nNOS = Neuronal NOS
NO = Nitric oxide
NO2 = Nitrogen dioxide
NOS = Nitric oxide synthase
NOX = NADPH oxidase
NQO-1 = NADPH quinine reductase 1
Nrf2 = Nuclear factor-erythroid-2-related factor 2
NRK-52E = Normal rat kidney proximal tubular cell line
NSP = Nephrectomized sheep plasma
O2 = Superoxide
OH = Hydroxyl
ONOO = Peroxynitrite
OPHEN = Ortho-phenanthroline
OXPHOS = Oxidative phophorylation p53 = Tumor protein p53
PBS = Phosphorous-based saline
PCMB = para-chloromercuribenzoic acid
PI3K = Phosphatidylinositol-4,5-bisphosphate 3-kinase
PKA = Protein kinase A
PKC = Protein kinase C
PLC = Phospholipase C
POP = Prolyl endopeptidase
xii
Pro-Ile = Isoleucine-Proline-Isoleucine
PRR = (Pro) rein receptor
PVDF = Polyvinylidene fluoride
RAP = Receptor associated protein
RAS = Renin angiotensin system
RIA = Radio-immuno assay
ROS = Reactive oxygen species
RPTs = Renal proximal tubules
SBTI = Soybean trypsin inhibitor
Serpin = Serine protease inhibitor
S1 tubule = convoluted (higher cell complexity)
S2 tubule = convoluted (higher cell complexity)
SCH = SCH39370
SDHB = Succinate dehydrogenase complex subunit B-complex II
SDS = Sodium dodecyl sulfate
SEM = Standard error of measurement
SGK1 = Serum and glucocorticoid-inducible kinase 1 siRNA = Small interfering RNA
SOD = Super oxide dismutase
TBS = Tris buffered saline
TGF-β = Transformation growth factor
TNF-α = Tumor necrosis factor-α
TIM = Translocase of inner mitochondria membrane
TOM = Translocase of outer mitochondria membrane
TOP = Thimet oligopeptidase
Total-Aogen = Internal sequence (distal to Ang I) of angiotensinogen
xiii t-PA = Tissue plasminogen activator
UCRC2 = Ubiquinol-cytochrome C reductase core protein II
VAL = Valinomycin
VDAC = Voltage-dependent anion channel
ZnCl2 = Zinc chloride
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ABSTRACT
Several therapies we have available to treat high blood pressure and the resulting kidney failure target a classic circulating hormonal system - the renin- angiotensin System (RAS), and specifically, the ACE-Ang II-AT1 receptor axis. We have focused strategically on investigating novel intracellular pathways by which Ang-(1-7) is generated and degraded since this alternative pathway within the kidney and other tissues may antagonize the Ang II-AT1 receptor mediated actions. We recently identified the presence of a novel enzyme activity in the brain that degrades Ang-(1-7) and is negatively correlated with central Ang-(1-7) levels and blood pressure. Moreover, seminal studies as a part of my graduate work identified this enzyme activity in the kidney, specifically in the proximal tubules and in a human proximal tubule cell line.
Utilizing the human tubule cells, we purified and identified the Ang-(1-7) degrading activity as Dipeptidyl-Peptidase 3 (DPP 3). Given the bioactive role of Ang-(1-7) counteracting the deleterious effects of Ang II, we further established that the beneficial intracellular actions of Ang-(1-7) may reflect the mitochondria. Thus, we demonstrated that mitochondria isolated from sheep renal cortex expressed RAS components and contained the molecular machinery to produce Ang-(1-7) intracellularly within the kidney that include the endopeptidases neprilysin (NEP) and thimet oligopeptidase (TOP) that process Ang I directly to Ang-(1-7).
We identified the presence of the RAS pathway precursor angiotensinogen
(Aogen) in isolated kidney mitochondria, however it is unclear as to the extent that tubular Aogen reflects local synthesis or internalization within the kidney. Therefore, we sought to establish the extent that Aogen is internalized by proximal tubule cells and the intracellular pattern of distribution of the precursor. We developed a sensitive method to assess the cellular uptake of Aogen using a radiolabeled form of Aogen [125I-Aogen] and
xv we determined that 125I-Aogen was internalized by proximal tubules at a rate of 0.002 ±
0.0006 fmol/min/mg protein. We further established that approximately 20% of the internalized Aogen associated with the mitochondrial fraction of the proximal tubules.
Moreover, we demonstrated that the isolated mitochondria from both sheep kidney and human proximal tubules internalize Aogen at a comparable rate suggesting that the uptake process of Aogen likely occurs in multiple species. Further characterization of
Aogen internalization by human mitohchondria revealed that inhibiton of the mitochondrial membrane potential did not attenuate the uptake of the Aogen suggesting a novel and possibly energy-independent mechanism for mitochondrial transport.
Together these studies implicate a novel pathway for the delivery of the precursor protein Aogen into the renal tubules and the subsequent uptake by the mitochondria. Moreover, the demonstration of active renin and the NEP/TOP-Ang-(1-7)-
Mas receptor axis within the renal mitochondria may portend for an important intracellular pathway to regulate sodium handling and blood pressure by Ang-(1-7). The mitochondrial Ang-(1-7) may also be regarded as a potential therapeutic target to maintain or augment Ang-(1-7) levels and preserve renal function.
xvi
CHAPTER 1 – INTRODUCTION
Overview of the Renin Angiotensin System (RAS)
Since the discovery of renin by Tigerstedt and Bergman in 1898 (156), the renin- angiotensin system (RAS) has been classically regarded as an endocrine signaling pathway governing both cardiovascular physiology and fluid homeostasis. Several cardiovascular disease pathologies have been implicated to initiate or progress as a result of dysfunctional RAS signaling. These include, but certainly are not limited to hypertension, metabolic disorders, renal pathologies, fetal origins of disease and heart disease. The RAS was initially characterized as a systemic or circulatory system whereby peptide hormones are transported through the blood stream to target organs to elicit a physiological response. The RAS (Figure 1) is initiated when the α-glycoprotein angiotensinogen (Aogen) secreted from the liver is cleaved by the aspartyl protease renin (EC: 3.4.23.15) that is released from the juxtaglomerular (JG) complex of the kidney in the circulation, to form the decapeptide angiotensin I (Ang I) (40, 63-64,
101,120, 139). Ang I is then cleaved by angiotensin-converting enzyme, (ACE, EC:
3.4.15.1), a zinc and chloride dependent member of the M2 class of metallopeptidases to form the octapeptide angiotensin II (Ang II). Regarded as the effector peptide of the
RAS, Ang II binds to its respective receptor, the angiotensin II type 1 receptor (AT1) to cause vasoconstriction and stimulate the release of aldosterone from the adrenal cortex and an increase in blood pressure (64, 70, 124). The AT1 receptor is a member of the seven-transmembrane G protein-coupled receptor (GPCR) superfamily. GPCRs arbitrate signaling cascades by ligands ranging from large proteins to peptides and are a pharmacological target for a number of drugs used within clinical settings. Ang II binding to the AT1 receptor causes various physiological actions and plays a pivotal role in cardio-renal pathologies (50, 170, 171).
1
Figure 1: Renin-Angiotensin System Cascade. Angiotensin I is converted to Angiotensin II by the enzyme ACE. The ACE homologue ACE2 generates Angiotensin- (1-7) from Angiotensin II. Ang I is also directly processed to Ang-(1-7) by endopeptidases including neprilysin. Angiotensin II and Angiotensin-(1-7) bind to their respective receptors to produce physiological actions that oppose one another.
Angiotensin II binding to the AT1 receptor produces effects that are pro-inflammatory and vasoconstrictive, while Angiotensin-(1-7) binding to the AT7/Mas receptor produces anti- inflammatory and vasodilatory effects.
2
Moreover, the actions of Ang II may encompass a wide range of systemic control mechanisms as AT1 receptors are expressed ubiquitously throughout the renal, neuronal, cardiovascular, endocrine, hepatic tissues and organs. Thus, angiotensin receptor blockers (ARBs) are considered hallmark therapeutics in the treatment and management of cardio-renal pathologies.
Since the initial characterization of a vasoconstrictor agent by by Goldblatt (55) and the subsequent identification of angiotonin by Braun-Menendez (17) and hypertensin by Page et al (116), Ang II was long considered the only bioactive peptide of the RAS. This view was primarily supported by studies demonstrating that Ang I was physiologically inert and incapable of eliciting any type of biological activity. Moreover, there are no known receptors for Ang I. However, findings over the last three decades have revealed alternative properties of the RAS which includes the discovery of novel
Ang ligand-specific peptidases, Ang peptides and receptors (49). These new findings supported the notion of an alternative axis of the RAS that was distinct and opposite that of the classic ACE-Ang II-AT1 receptor axis. Alternative components of the RAS include angiotensin converting enzyme 2 (ACE2), the heptapeptide Ang-(1-7) and its respective receptor, the AT7/Mas receptor as well as the Ang II type 2 receptor (AT2). Ang-(1-7) is derived by enzymatic hydrolysis of Ang II, removing the C-terminal phenylalanine group from the peptide by the ACE homolog ACE2. Ang-(1-7) elicits physiological responses opposite of Ang II which are cardioprotective and vasodilatory in nature. The cardiovascular benefits of Ang-(1-7) may reflect, in part, a decrease in oxidative stress, inflammatory signaling, cell injury and hypertrophy and fluid retention (8, 44, 136) (Figure
2). Similarly, the AT2 receptor counteracts the AT1 receptor to cause vasodilation, natriuresis and the release of nitric oxide (NO) (60, 160). Based on its opposing actions, activation of the ACE2-Ang-(1-7)-Mas receptor - RAS axis, represents an alternative target for novel therapeutics to improve cardiovascular function.
3
Figure 2: Cardiovascular disease events alter the balance of the Ang-(1-7) and Ang II systems, leading to higher blood pressure, inflammation, fibrosis oxidative stress and increased renal resistance and sodium reabsorption, but reduced nitric oxide.
4
Systemic versus local organ RAS
Apart from the traditional endocrine actions of hormone-dependent signaling, it is now evident that the RAS may act in a paracrine and autocrine fashion within organ subtypes. Evidence for the presence of local and fully functional RAS pathways has been demonstrated for the brain, heart, liver, adrenal gland and the kidneys (25, 26, 33).
This work was primarily supported by studies revealing the expression of RAS components in alternative sites. For example, renin which was originally thought to be a kidney enzyme, was later found to be expressed within brain tissue (52, 53). Moreover, discovery of the differential expression of other RAS components, particularly the synthesis and bioactivity of Ang II led to the conception of new functional interpretations and insights with respect to tissue-based systems. As a result, this has changed our view of the RAS and introduced new hypotheses that this pathway could have tissue- specific functionality. Although initially met with apprehension and a plethora of controversies, the evidence for local RAS pathways is quite extensive and has provided a wealth of knowledge that has not only increased our current understanding of the RAS, but has led to innovative therapeutic clinical advances.
Great technological advances in molecular biology have significantly aided in our current understanding and identification of RAS components in various tissues. The use of transgenic and gene-deletion models containing modified RAS components have been fundamental in establishing these local systems that produce Ang peptides (5, 10,
11, 25, 30, 84, 86, 118, 149). Moreover, the development of unique methodologies used to quantify the RAS has also been beneficial (25). Together these advancements in tools to characterize the RAS along with improvements in the biochemical evaluation of its constituents have verified its presence and functional regulation in multiple tissues. For instance, gene expression studies have resulted in the successful cloning of all the major components of the RAS (18, 45, 107, 134, 145, 151, 157). Subsequent analyses
5 later revealed that RAS components were differentially expressed within multiple tissue types, thus providing insight into the potential coordination between local and classic endocrine signaling to establish a basis for a RAS tone within various organs.
The quandary that has caused considerable debate with respect to local RAS pathways within various organs, centered on the concept of whether RAS components were being generated locally or by uptake from the circulation (178). To date this issue has been partially resolved, as many questions remain unanswered. However, the remaining gaps in knowledge have not threatened this view of the RAS; as a vast amount of evidence has accumulated over decades to suggest the concept of a local
RAS. Moreover, this view of the RAS has expanded tremendously to support the notion that both local and systemic endocrine mechanisms could contribute to the local synthesis and bioactivity of RAS enzymes and Ang peptides.
Although it is well known that the RAS functions distinctively within different tissues, the kidney represents a main target for Ang II, thereby regulating fluid content and blood pressure. Ang II directly stimulates the reabsorption of sodium through an increase in various transporters along the nephron and increases constriction of the renal vasculature to reduce GFR. Ang II also acts extrarenally to stimulate the release of anti-diuretic hormone (ADH or vasopressin) from the pituitary gland to cause enhanced water reabsorption in the collecting duct, and stimulates aldosterone release to increase the epithelial Na+ channel (eNAC) activity withn the principal cells of the kidney. All these actions of Ang II, will increase Na+ reabsorption, blood volume and maintain or increase blood pressure (100, 111). Moreover, the kidney expresses all of the components of the RAS and increased or activation of the ACE-Ang II-AT1 receptor axis may reduce renal function and contribute to the development of kidney pathologies.
In the following section, we discuss the body of evidence revealing various RAS components within the kidney, as well as the physiological actions of Ang peptides.
6
RAS within the kidney
It is well established that the RAS influences kidney function to regulate body fluid homeostasis and blood pressure through the stimulatory effects of the Ang II-AT1 receptor axis. However, studies suggest the concomitant signaling of Ang II along with the renal expression, synthesis and biological activity of other RAS components such as: renin, Aogen, ACE, ACE-2, AT2 receptors, Ang-(1-7) and the AT7/Mas receptor (1, 6, 8,
33, 44, 70, 80, 103, 141, 168). Due to this body of evidence, the RAS is now considered to not only assume an endocrine role, but can also function in a paracrine capacity.
Local RAS in renal proximal tubules
The nephron of the kidney is considered the primary structural and functional unit. As a whole, the main function of the nephron is to regulate fluid homeostasis, and the excretion and absorption of biological solutes such as Na+ and other electrolytes that are filtered from the blood. The kidney is normally comprised of 0.8 – 1.5 million nephrons in both kidneys of the body and collectively these units filter the blood and concentrate the tubular filtrate that ultimately is excreted as urine. The nephron is a key site of local endocrine signaling and contains all the components of the RAS. Aogen is synthesized by the proximal tubules (73, 110), while renin is produced in the JG apparatus and distal nephron. Moreover, other RAS components such as ACE, ACE2,
Ang II and Ang-(1-7) receptors are expressed throughout the nephron. Interestingly, with respect to peptide levels, renal tissue Ang II and Ang-(1-7) levels are reportedly 40-50- fold higher than those in the plasma (19, 20, 25, 39). Given the expression of RAS components within the nephron unit of renal tissue, it is of pivotal importance to acknowledge the physiological actions of a local renal RAS. Furthermore, given the complexity and diversity of cell types within the kidney and their interactions within the nephronal segment, it is equally important to focus on each independently. Thus, in
7 respect to the nephron unit we provide here a comprehensive discussion of the physiological actions of the RAS within proximal tubules.
Renal proximal tubules (RPTs) are considered to be a functional compartment of the kidney and make up a significant portion of the tissue. RPTs are the most abundant cell type in the kidney and govern a variety of regulatory and endocrine mechanisms including the active transport of and secretion of biological solutes. The proximal tubule is the nephron segment that is continuous with Bowman’s capsule to capture the filtrate from the glomerulus and ends at the thin limb of the loop of Henle. Based on morphology and functionality, the RPTs are categorized in three segments. S1 and S2 are characterized as the first two segments and contain epithelial cells of high transport capacity and complexity. The last segment, S3, consists of cells of comparatively less complexity (69, 99). RPTs under normal conditions reabsorb approximately 65% of filtered Na+ and water, more than 90% of sodium bicarbonate and essentially 100% of glucose from the glomerular filtrate (16, 108). In order to carry out this diverse regulation of endocrine function, RPTs contain numerous and diverse transporters (Figure 3). As it relates to Na+ transport, the amount of Na+ reabsorbed by RPTs may contribute to the regulation of blood volume to ultimately influence blood pressure. A classic example of this physiological phenomenon is the development of hypertension caused by a dysregulation of the RAS. Ang II is a potent regulator of blood pressure and its stimulation of RPT Na+ transport likey plays a role in the development of Ang II-mediated hypertension (32, 33). Aside from the classical endocrine RAS, RPTs of the kidney express functional RAS components intracellularly, which have been demonstrated to respond in an autocrine fashion to regulate overall kidney function (48, 58, 80).
Therefore, it is plausible to suggest that the development of RPT injury is closely related to the dysregulation of the RAS and a variety of associated renal pathologies.
8
Figure 3: Reabsorption: Proximal convoluted tubule. Luminal membrane transporters include the Na+-glucose cotransporters (SGLT1 and SGLT2) and the Na+/H+ exchanger (NHE-3). Additional transporters include the Na+-3HCO3− cotransporter (NBC1), as well as K+ and Cl− channels and the peritubular membrane transporter Na+- K+-ATPase. High water permeability is mediated by AQP1 channels, which after cGMP stimulation may also increase Na+ entry. Moreover, the tight junctions of proximal tubules allow substantial and relatively nonselective fluxes of cations and anions.
9
Proximal tubule angiotensinogen
Aogen is 53 kDa member of the serpin (serine protease inhibitor) superfamily and serves as the sole precursor of all Ang peptides. Additional members of the serpin class include proteins such as antitrypsin, antithrombin II and α1-antichymotrypsin. As the sole substrate for the formation of Ang peptides, the unique N-terminal Ang I sequence of Aogen distinguishes it from other serpins. Aogen’s cleavage by renin is the rate-limiting step to generate Ang I. Although it is only the first ten N-terminal amino acids of Aogen that yields Ang I, renin is considered to be an Aogen-exclusive protease
(147). Renin’s substrate affinity for Aogen (Km: 2.6 µM) is higher compared to similar serpins (alpha1 antitrypsin) or a modified Aogen substrate consisting of the 17 N- terminal amino acids that interact with the active site (Km: 47.5 µM) (147). Moreover, renin cleavage of Aogen confers species-specific activity. This was demonstrated in studies showing that purified human Aogen was not efficiently cleaved by mouse or rat renin (51, 54, 68, 172).
Apart from Aogen’s role as a peptide precursor, the protein itself is believed to exert physiological effects independent of the bioactive Ang peptides, Ang II and Ang-(1-
7) (94). With respect to the kidney, local Aogen synthesis and uptake in RPTs may play a significant role in regulating blood pressure, Na+ retention and the extent of tubular injury. In the context of renal glomerular pathogenesis, local Ang II and Aogen content is increased along with the accumulation of albumin to cause proteinuria (81, 82, 85, 150).
Albumin and Aogen are of comparable size and normally not filtered by the glomerulus.
However, under diseased conditions and nephron injury, these filtering junctions may become disrupted, thus leading to an accumulation of proteins in the tubular fluid. As a result, RPTs are exposed to an excessive amount of protein and increase protein uptake mechanisms as a compensatory measure. The accumulation of Aogen in the diseased
10 kidney may play a key role in the progression of tubular injury and serve as a substrate for the increased production of Ang II.
Several groups have now reported that the tubular content of Aogen arises from both active uptake and local synthesis (72, 78, 112, 122). Tubular clearance of proteins filtered by the glomerulus is believed to be mediated by the endocytic multi-ligand receptors megalin and cubilin (28, 106). Administration of recombinant human Aogen via jugular vein catheter in rats resulted in tubular uptake and localization; and this uptake of
Aogen was exacerbated in diabetic animals that may reflect diabetic glomerular damage and increased protein filtration (158). Moreover, gene deletion of megalin in mice attenuated proximal tubular accumulation of Aogen (87). Additional studies supporting uptake of Aogen in RPTs was demonstrated in kidney-specific Aogen knockout mice who exhibited renal Aogen levels that were comparable to that of control animals (98).
Moreover, liver specific Aogen knockout mice exhibited a significant reduction in the renal concentration of the protein as well as the bioactive peptide Ang II, thus indicating that liver Aogen may serve as a primary source of renal Aogen and Ang II (98). The mechanisms by which Aogen is internalized within RPTs are still not fully understood, although evidence supports the role of the protein transporter megalin in the process.
However, the complexity and promiscuous nature of megalin in regards to the internalization of multiple protein ligands eludes to the plausibility that alternative and more specific transport mechanisms may be involved in the internalization of Aogen.
This is partly supported by studies indicating partial inhibition of Aogen uptake by the megalin competitor, receptor-associated protein (RAP) (122). Moreover, megalin- dependent binding of Aogen was not attenuated by the addition of exogenous Ang II, which has been shown to minimize megalin’s interactions with the Aogen protein (57,
117). Collectively, these studies provide support that RPT Aogen and its interplay with an impaired systemic RAS could confer a phenotype leading to a dysregulation of renal
11 function and the development of cardiovascular pathologies such as hypertension and nephron injury.
Proximal tubule renin
Evidence also supports the presence of renin and its enzymatic activity within
RPTs (27, 74, 105). However, studies regarding the synthetic potential of RPTs to produce renin locally remain to be fully elucidated. Past studies demonstrated positive immunoreactivity for renin that was detected in apical cytoplasmic vesicles of RPTs (41,
102, 155), but the presence of renin mRNA was not confirmed (41, 47, 56, 138).
Moreover, researchers found no evidence of renin cDNA in microdissected proximal tubule segments of control rats (105). This discrepancy in regard to the presence of the renin protein, but lack of genetic coding material was further addressed by Chen at colleagues (27). This same group discovered that the presence of mRNA for renin was differentially expressed within tubule subtypes. For instance, renin mRNA was only detected in proximal convoluted tubules and proximal straight tubules and not in the outer medullary collecting ducts (27). Moreover, renin activity was only detected in proximal convoluted tubules and not in proximal straight tubules or outer medullary collecting ducts. Interestingly, although renin cDNA was detected in both proximal convoluted tubules and proximal straight tubules, these values were 1/500th less of that found in the juxtaglomerular (JG) cells of the glomerulus (27). These studies are consistent with recent evidence demonstrating alternatively spliced renin transcripts that are expressed along cortical tubules (74). Ishigami et al recently demonstrated an additional renin transcript that lacks a portion of the pre-pro segment such that the enzyme is functionally active; the isoform constitutes ~10% of total renin and was expressed predominantly along the tubular elements of the mouse kidney (74).
Moreover, forced overexpression of this renin isoform was associated with higher blood pressure in the transgenic mice (74). Although the Ishigami study found no change in
12 circulating renin suggesting that the renin isoform is not secreted, the intracellular localization of the renin isoform within the tubules was not established.
The common theme as it relates to the uptake versus local synthesis of RAS components is clearly apparent in the scientific literature that pertains to tubular renin.
This controversy is perpetuated by the lack of studies which focused solely on proximal tubules. Therefore, it is important to take into account artifacts due to the potential contamination of renin from JG cells or uptake from the circulation. This may in turn, falsely confer renin activity within tubules during the isolation process from renal tissue.
JG cells are positioned near the renal afferent arterioles at the front axis of the glomerulus and are the body’s main source of renin. Renin is synthesized as the inactive preprorenin, which is converted to the zymogen prorenin upon trafficking to the endoplasmic reticulum (ER). The majority of prorenin (~80%) is secreted, however the remaining active pool is stored in secretory vesicles (renin granules). Well-characterized stimulants of renin release include a decrease in renal perfusion pressure, Na+ and water depletion, as well as sympathetic activation. Upon stimulation, renin is released from JG cells into the circulation in both active renin and inactive prorenin forms.
Prorenin can be rendered active by proteolytic cleavage or by binding to the prorenin receptor (PRR) that induces a conformational change in prorenin that removes the inhibitory peptide from the active site of prorenin (83). In a similar fashion to Aogen, renin may also be internalized by RPTs by a megalin-mediated process (158). Moreover, immunoprecipitation studies of human prorenin administration in rats demonstrated that human prorenin was bound to megalin and the PRR, indicating that the uptake of prorenin may be mediated by both of these receptors (158). Although there are many unanswered questions regarding the mechanisms governing tubular expression of renin, one study in particular provided a strong case to support the theory of local renin synthesis in RPTs (105). In this regard, Moe et al demonstrated renin activity in the
13 media and lysates from rabbit proximal tubule cells in culture that was blocked by the nonpeptide renin inhibitor A-74273. Given the diversity of cell types within the kidney, and potential contamination of JG cells, this data provides solid evidence of “self- contained” renin within RPTs irrespective of the whole organ/whole animal experiments.
Proximal tubule ACE and ACE2
Studies within our lab as well as those of other groups have extensively characterized ACE and ACE2 activity in RPTs from multiple animal species (21, 67, 103,
109, 141, 153). The presence of ACE and ACE2 in RPTs ensures the local production of
Ang II and Ang (1-7) independent of that synthesized by the classic circulating RAS, thus providing a cellular source of the bioactive peptides. ACE is a zinc metallopeptidase that primarily acts as a dipeptidylpeptidase to hydrolyze a variety of peptides. ACE is anchored to the plasma membrane by a C-terminal hydrophobic segment. The enzyme is expressed as two isoforms (germinal or somatic) that reflect the number of catalytic domains of each isoform. Expression of germinal ACE is restricted solely to the testis, specifically in germinal cells to facilitate the maturation of spermatogenesis (142).
Germinal ACE contains only one cataylic site that corresponds to the carboxy or C- terminal active site. Somatic ACE on the other hand, contains two active sites (the N and C terminal domains) and is expressed on the vascular endothelium and brush border of RPTs, however the level of expression of ACE may be contingent upon tubule subtype. With respect to the kidney and RPTs, ACE’s most relevant substrate is Ang I to form Ang II. Indeed, Gonzalez-Villalobos et al show that tubular deletion of ACE prevents both Ang II dependent and L-NAME dependent hypertension (179). In the mouse kidney, Reinhold and colleagues found that, ACE mRNA was not detected in microdissected nephron segments, however it was demonstrated in the whole kidney homogenate (127). This is somewhat contradictory to previous studies demonstrating
14
ACE expression in human RPTs and in immortalized human kidney proximal tubule cells
(6, 140). Moreover, ACE was detected in the tubular fluid from RPTs in rats, but its expression is decreased in late proximal tubules and early distal tubules, and is absent in the distal tubules (23). These observations coincide with an earlier study demonstrating the lack of ACE immunostaining in proximal convoluted tubules (S1 segment), or in other tubular structures such as distal convoluted tubules, connecting tubules and cortical collecting ducts (159). Based on these results, it is evident that tubule ACE expression may vary depending on the tubule subtype or species examined.
Similar to ACE, ACE2 is also expressed along the plasma membrane of RPTs.
As a homologue of ACE, ACE2 is one of the most recently discovered components of the RAS. Since its initial characterization in 2000, ACE2 has signified that the RAS is far more complex than previously proposed (45, 157). With respect to the kidney expression of ACE2, this enzyme has generated new hypotheses and added an additional level of physiological control with respect to the concept of an intra-renal RAS. The enzyme is expressed abundantly in the proximal tubule and metabolizes Ang II directly to Ang-(1-
7). Thus, ACE2 may play a pivotal role as an endogenous regulator of the RAS. Aside from actions in metabolizing Ang II, ACE2 also catalyzes conversion of Ang I to Ang (1-
9), which is further cleaved by ACE to produce Ang-(1-7). However, it is of note, that the efficiency of ACE2 to convert Ang I to Ang-(1-9) is roughly 400-fold less than the metabolism of Ang II (128).
The ACE2 gene is coded on the human X chromosome and is translated as an
805-amino acid membrane-bound glycoprotein. The N-terminal domain for ACE2 shares
~40% sequence homology with ACE. In contrast to ACE, ACE2 is a monocarboxypeptidase which removes the C-terminal 8th position phenylalanine from
Ang II to produce Ang-(1-7). Moreover, ACE2 activity is not blocked by conventional
ACE inhibitors. However, ACE inhibition increased Ang I levels which may be converted
15 by an alternative pathway that is independent from Ang II. In this case, the neutral endopeptidase neprilysin (NEP) may process Ang I directly to yield Ang-(1-7) (128, 141).
Given its ability to play a critical role in mediating the balance between intra-renal Ang II and Ang-(1-7), ACE2 represents a target for the action of novel therapeutics to treat renal pathologies while also contributing as a member of the local tubular RAS.
Alternative proximal tubule peptidases
Ang II appears to be alternatively generated from the precursor Aogen by non- renin dependent enzyme activity (77). This process can be mediated by tonin, tissue plasminogen activator (t-PA, EC: 3.4.21.68) or cathepsin. Moreover, non-ACE dependent mechanisms also exist that generate Ang II from its precursor Ang I. These pathways include chymostatin, elastase-2 and cathepsin G (EC: 3.4.21.20) (71, 165). In
RPTs, Ang-(1-7) can also be generated by alternative peptidases from the precursor
Ang I that include neprilysin (NEP, EC: 3.4.24.11), thimet oligopeptidase (TOP, EC:
3.4.24.15), and prolyl oligopeptidase (POP, EC: 3.4.21.26) (44, 146); the former is located on the brush-border membranes of RPTs and the latter enyzmes are found in the cytoplasm (163, 164). Recently, we have identified a peptidase activity (A7-P) that metabolizes Ang-(1-7) within the cerebral spinal fluid (CSF), brain medulla, renal cortex and isolated proximal tubules of sheep as well as HK-2 cells (96, 166). This peptidase may be potentially important as Ang-(1-7) serves to counter-balance the deleterious cardiovascular effects of Ang II. The peptidase activity was markedly higher in animals exposed to betamethasone (Beta); a model of glucocorticoid-induced (GC) cardiovascular fetal programming. Moreover, A7-P activity was inversely correlated to
Ang-(1-7) content in CSF, but positively correlated to blood pressure (97). However, it was not known whether the Ang-(1-7) peptidase was expressed within the proximal tubules of the kidney. Therefore, further investigation was needed to characterize and
16 establish the role of the A7-P as a novel component of the RAS and in Ang peptide processing, as well as its capacity for potentiating functional kidney responses under diseased conditions.
Proximal tubule Ang peptides and receptors
Evidence that renal concentrations of both Ang II and Ang-(1-7) are much higher in the kidney than that in the circulation supports the notion of a local RAS and its contributions to intra-renal Ang peptide content (25). The presence of endogenous Ang peptides in the kidney sets the basis for their induction of biological and physiological activity through the binding to Ang peptide-specific receptors. Four classes of Ang receptors have been characterized in RPTs of the kidney. These include the AT1 and
AT2 receptors which bind Ang II (22, 60, 114, 144, 173-175, 177), the Mas receptor (62,
135) that recognizes Ang-(1-7) and the AT4 receptor (IRAP, insulin-regulated aminopeptidase) that binds Ang-(3-8) or Ang IV (4, 66, 90). AT1 and AT2 receptors are expressed on both the basolateral and brush border membranes of RPTs. However, the specific roles of the AT1 receptor are better characterized. Ang II binding to the AT1 receptor stimulates Na+ retention in tubules and is a main culprit in the development of tubule-interstitial injury or fibrosis (95). In rodent models, the AT1 receptor may exist as two isoforms, AT1a and AT1b, although Ang II binding to the AT1a receptor is similar to the
AT1 receptor signaling in humans and the preferred pathway leading to physiological effects in rodent RPTs (38). In contrast, the AT2 receptor signaling in tubules leads to the induction of signaling cascades that are beneficial. This may encompass the stimulation of tubule mechanisms to induce natriuresis through the release of NO by cyclic guanosine monophosphate (cGMP) signaling (115, 143).
Similar to AT2 receptor signaling, the role of the Ang-(1-7)-Mas receptor axis opposes the effects of the Ang II-AT1 receptor axis and numerous reports support its role
17 in renal function. The levels of Ang-(1-7) with respect to Ang II within the kidney are comparable (25, 76), and it is plausible to infer that maintaining tubular levels of Ang-(1-
7) may be beneficial to offset Ang II-AT1 receptor tone. Ang-(1-7) through binding to the
Mas receptor stimulates beneficial vascular effects (135-137). These actions may include a reduction in proliferation, hypertrophy, fibrosis and thrombosis. The Mas receptor is expressed throughout the kidney and studies have reported the predominant localization of Mas mRNA in the renal cortex (5). Moreover, studies from our lab determined that within the renal cortex, Mas immunofluorescence staining was predominantly associated with the proximal tubule segment (62). Mas receptor expression was also detected in the thick ascending limb of Henle, the collecting ducts and vasa recta, but not in the glomerulus of the sheep kidney (62).
The effects of the Ang-(1-7)/Mas axis in the kidney is complex and may include renal protective effects through its inhibition of Ang II-induced MAP kinase signaling or by the amelioration of oxidative stress. Ang II stimulation of ERK 1/2 in the kidney may include the activation of NADPH oxidase isoforms (NOXs) and the subsequent production of reactive oxygen species (ROS) (79). However, evidence suggests that
Ang-(1-7) signaling through the Mas receptor may lead to a de-phosphorylation of ERK
1/2 within rat proximal tubular cells and an attenuation of ROS (7, 79). Moreover, our group has demonstrated that Ang-(1-7) abolished advance glycated end product (AGE)- induced cellular hypertrophy and myofibroblast transformation via inhibition of ERK1/2
(7). Indeed, the benefits of Ang-(1-7) signaling in tubules could reflect alterations in oxidative stress as studies by Rakusan et al found that deletion of the Mas receptor enhanced renal hypertension in mice that was reversed by the the antioxidants apocynin or tempol (125). Given the numerous reports highlighting both the in vivo and in vitro tubular effects of Ang-(1-7) and Ang II, there is the need to further understand their interaction in renal disease and tubular function.
18
Tubular intracellular RAS
Since the 1970s a substantial body of work has changed our view of the RAS from a classic circulating pathway, to that of a cellular or intracellular system. This concept was derived from the theory that RAS components may be internalized or synthesized locally within individual cells of key vascularized organs such as the heart, brain and kidney. The presence of intracellular RAS components were believed to play a role in exerting a number of physiological effects including cell growth and differentiation to influence both nuclear and mitochondrial function (1, 2, 9, 59, 60, 62, 119, 126, 168,
169). Moreover, there is compelling evidence for functional actions of an intracellular
RAS within the kidney including the endoplasmic reticulum, nucleus and the mitochondria. Seminal studies by Robertson et al were the first to demonstrate evidence for intracrine actions of the RAS; demonstrating rapid internalization of radio-labeled Ang
II by cardiac cells that trafficked to the nucleus and mitochondria (129). Moreover, receptors for Ang II and Ang-(1-7) were subsequenlty identified on nuclei and mitochondria (1, 58, 62, 92, 119, 169). Interestingly, subcellular Ang receptors were found to have similar characteristics as plasma membrane Ang receptors. For instance,
Ang II binding to receptors on isolated renal nuclei was associated with ROS production that was attenuated by the AT1 antagonist losartan or the NOX inhibitor diphenyliodonium (DPI) (59, 119). Indeed, distinct and opposing actions of Ang-(1-7) through the binding of the Mas receptor were also identified at the nuclear level, as studies by Gwathmey et al demonstrated a stimulation of NO in isolated renal nuclei that was blocked by addition of the Mas receptor antagonist DALA or the NOS inhibitor L-
NAME (62).
Intracellularly formed Ang II and Ang-(1-7) have been well documented in renal tissue, isolated RPTs and immortalized proximal tubule cell lines (7, 8, 76, 93, 109, 111,
19
169). Additionally, the intra-tubular expression of other RAS components including:
Aogen, renin, ACE and ACE2 have been established and supports the characterization of an intracellular RAS pathway leading to the production of local peptides. Collectively, these findings suggest that Ang peptide binding to subcellular receptors regulate redox homeostasis within renal tissue.
Tubule nuclear RAS
We have established specific intra-nuclear RAS pathways linked to Ang II and
Ang-(1-7) regulation of ROS and NO within the renal cortex. The AT1, AT2 and Mas receptors have all been identified in the nuclear compartment of renal tissue (58). Ang II stimulates RPT Na+ and fluid transport, ultimately influencing blood pressure homeostasis (91). Aside from it’s physiological regulation of renal transport, Ang II can also stimulate cytokines and chemokines to influence the development and progression of tubular and renal oxidative stress and inflammation (3, 131). At a functional level, nuclear AT1 receptor production of ROS is coupled with phosphoinositol-3 kinase (PI3K) and protein kinase C (PKC) activation (58, 60, 119, 176). In vitro studies demonstrate
+ that chronic Ang II stimulation of nuclear AT1 receptors increases Na /H exchanger 3
(NHE3) promoter expression in a similar fashion to those found on the plasma membrane of renal tubules (123). Additionally, studies from our lab as well as those from others have demonstrated that renal cortical nuclei signal through the AT1 receptor to increase production of hydrogen peroxide (58, 119). In particular, our group has focused on the alterations of the Ang II-ACE-AT1 receptor axis in GC-induced fetal programming.
With respect to tubular sodium handling, GC exposure enhances Ang II tone in isolated sheep tubules resulting in an enhanced uptake of Na+ and associated oxidative stress
(14, 148). Nuclei isolated from renal cortex of GC-exposed sheep exhibit a similar
“programmed” phenotype as cortical tubules. Renal nuclei from GC-exposed animals
20 have a higher density of AT1 receptors and respond to Ang II to stimulate ROS in an exacerbated fashion (61). The synergistic stimulation of both oxidative stress and tubular
Na+ transport by Ang II, highlights the need to further investigate the tubule mechanisms that mediate these processes. To date, it is still unclear whether the Ang II-dependent increase in tubular Na+ transport is mediated by the stimulation of oxidative stress or vice versa. However, given the conservation of a diseased phenotype in isolated organelles such as the nucleus, one could infer that substantial modifications at the genetic level may be involved. Thus, further investigations regarding an in-depth genetic analysis with respect to the changes that occur in diseased or injured RPTs should be warranted.
In contrast, Ang-(1-7) stimulates nuclear NO that is mediated by eNOS activation. The regulatory role of Ang-(1-7)’s stimulation of NO is by causing physiological changes in the renal vasculature to lower blood pressure. This was shown by Benter et al who demonstrated that infusion of Ang-(1-7) into spontaneously hypertensive rats (SHR) caused a reduction in systolic blood pressure (12). Moreover,
Ang-(1-7) has also been shown to increase renal blood flow and inhibits the pressor responses of Ang II in Wistar Kyoto and SHR rats (42, 133). These beneficial effects were blocked by antagonism of the Mas receptor, or NOS inhibition, and suggest that the vasodilatory response of Ang-(1-7) binding to the Mas receptor is mediated by the release of NO.
Renal tubules play a critical role in regulating blood pressure and fluid homeostasis and the role of Ang-(1-7) in the regulation of salt and water excretion has been the focus of several studies. In contrast to Ang II, renal tubules respond to Ang-(1-
7) in a manner that elicits a natriuretic effect to influence the regulation of tubular transport. Ang-(1-7) inhibits Na+ uptake in RPTs, which is mediated by the Mas receptor that impacts the activation of Na+ /H exchanger 3 (NHE3) (65, 104). NHE3 mediates the
21 majority of Na+ and fluid reabsorption by RPTs, and this transporter plays a major role in the increase in blood pressure within essential hypertension (161). Blood pressure has been shown to be lower in renal NHE3-deficient mice than in wild-type mice on a normal
Na+ diet (113). Moreover, antenatal GC administration alters renal function and is associated with hypertension in sheep (26). Specifically, we find that isolated renal proximal tubules from sheep in culture maintain the same “programming phenotype” cellularly and intracellularly as seen in the whole kidney (13, 152). This is evident in respect to sodium handling, as tubules isolated from GC-exposed animals displayed an attenuated effect of Ang-(1-7) to inhibit sodium uptake (152). Moreover, Bi et al finds that renal proximal tubules from Beta-exposed sheep exhibited an enhanced susceptibility to oxidative stress (14). Additionally, studies by Gwathmey et al., demonstrated that Beta- exposed animals exhibited a reduced ability to produce nuclear NO by Ang-(1-7) (61).
Thus, the reduced bioavailability of NO in combination with abnormal sodium handling may contribute to a sustained increase in blood pressure following antenatal GC exposure. Indeed, Ang-(1-7) stimulates NO that may inhibit sodium reabsorption in renal tubules and induce natriuresis. Alterations in Ang-(1-7) and NO tone within the proximal tubules of the kidney may contribute to increased sodium reabsorption and higher blood pressure.
Tubule mitochondrial RAS
Since the discovery that radiolabeled Ang II administered to rats traffics to the nucleus and mitochondria, increasing interest has focused on the dysregulation of the
RAS and mitochondrial dysfunction. Various laboratories have demonstrated that dysfunctional mitochondria may contribute to the pathophysiology of hypertension, cardiac failure, metabolic syndrome, diabetes, renal disease and aging (1, 2, 35, 46).
The elements of functional RAS signaling have been described in the mitochondria,
22 however the specific roles of Ang receptors and other components remains to be fully understood. Treatment of rats with ACE inhibitors or ARBs lengthens rodent life span and maintains mitochondrial function and integrity over time (24, 36). Moreover, Ang II stimulates mitochondrial ROS in a variety of renal proximal tubule cell models as well as decreases both mitochondrial membrane potential and NO bioavailability (43). Together these observations suggest that the deleterious actions of Ang II may reflect altered mitochondrial function.
To date, only three research manuscripts have addressed the characterization of
RAS components in mitochondria (1, 9, 169). Studies by Abadir et al recently described a functional liver mitochondrial RAS consisting of Ang II coupled to the AT2 receptor that stimulaties NO and mitochondrial respiration (1). The effect of the mitochondrial AT2 receptor was comparable in respect to receptor signaling on the plasma membrane surface. Activation of AT2 receptors may include the stimulation of phospholipase C
(PLC), protein tyrosine phosphatases or protein kinase phosphatases (160). Moreover, the production of NO mediated by AT2 stimulation could subsequently activate cGMP synthesis, which, in turn, may directly modify the post-translation of mitochondrial targets and ultimately influence respiration. An effect of aging was also observed in the Abadir studies as there was an imbalance between mitochondrial Ang II receptor subtypes (1).
Mitochondrial AT1 receptors increased with aging while AT2 receptors decreased. Given the counterbalance between AT1 and AT2 receptor signaling, a higher density of AT1 receptors in aged mitochondria could play a possible role in the deleterious effects mediated by the Ang II-AT1 receptor axis in cardiovascular disease. In this regard, Ang II stimulation of AT1 receptors in cell culture has been associated with p53 activation, cellular hypertrophy, mitochondrial dysfunction and apoptosis (79, 88, 89, 132). AT1 activation of p53 caused a decrease in the ratio of Bcl-2 to Bax two key mediators of mitochondrial-induced apoptosis in cells. With respect to renal tubules, this effect was
23 also observed in studies by Kim et al who demonstrated that Ang II also induced depolarization of the mitochondrial membrane potential, and cytosolic secretion of cytochrome C and apoptosis-inducing factor (AIF) in cultured rat proximal tubule cells. In contrast, Ang-(1-7) was shown to attenuate the Ang II-induced mitochondrial production of ROS via reduced NOX4 expression and apoptosis.
Ang II may induce mitochondrial depolarization through an increase in the generation of ROS (79). Although the role of the mitochondrial RAS and its potential to influence oxidative stress is still under investigation, evidence suggests that tubular ROS injury mediated by Ang II-induced mitochondrial NOX4 may play a pivotal role in mitochondrial dysfunction in tubular cells and ultimately cell survival (79).
Mitochondrial protection from apoptosis could involve the release of NO and downstream activation of cGMP (15, 31, 75). However, further investigations are needed to fully characterize the extent the intrinsic mitochondrial RAS plays in this process. The identification of AT2 receptors on mitochondrial membranes offers promising insight and highlights the probability that mitochondria have counter-regulatory mechanisms in place to respond to Ang peptides.
Most work with respect to the characterization of a mitochondrial RAS has focused heavily on the Ang II axis of the pathway. Moreover, the extent of expression and functionality of other RAS components in renal cortical mitochondria is not fully understood. Recent studies from our lab have taken a comprehensive biochemical approach to characterize the expression of RAS components in renal cortical mitochondria, as well as examining the Ang-(1-7) axis (169). These studies provided evidence for several RAS components including Aogen, active renin, Ang I and the bioactive peptides Ang II and Ang-(1-7) in purified mitochondria from the sheep renal cortex. Moreover, we demonstrated that purified mitochondria processed Ang I to Ang-
(1-7) by the endopeptidases NEP and TOP, suggesting the possibility for an intra-
24 mitochondrial RAS pathway that forms Ang-(1-7) (169). Studies to determine the direct effects of Ang-(1-7) on mitochondrial function are still needed, although the balance between Ang II receptors in aging may concomitantly allude to impaired Mas signaling in the progression of mitochondrial dysfunction, oxidative stress and apoptosis.
Additional mitochondrial-specific RAS components have also been identified.
With respect to renin, Peters et al originally demonstrated an alternative transcript of renin that lacked exon 1 which precluded entry of the transcribed protein into the secretory pathway and this isoform was predicted to reside within the cell (121). These investigators subsequently showed that the truncated form of renin was active and preferentially localized to the mitochondria (27). Overexpression of this renin isoform in a human cardiomyocyte cell line increased the extent of apoptosis; however, it remains unclear whether this was a direct effect of renin or reflected renin-dependent processing of Aogen (162). Of particular interest, Clausmeyer et al showed that a truncated form of renin was actively taken up and internalized by isolated mitochondria, but that the full length prorenin form did not undergo internalization (29). It is not currently known whether different renin transcripts are expressed within the sheep kidney or the extent that renin isoforms traffic to the mitochondria. Based on studies from our lab, it is plausible to hypothesize that the majority of the renin that resides in the mitochondria is constitutively active. This was primarily demonstrated by the lack of trypsin to cause a further increase in renin activity (169). A predominantly active form of renin in this compartment would support the absence of intact Aogen and the presence of the des-
Ang I form of the precursor in the renal mitochondria. Moreover, we found no immunological evidence for the prorenin receptor in mitochondria which is consistent with predominant intracellular localization of this receptor on the endoplasmic reticulum, as well as the plasma membrane (8, 62, 74, 162).
25
Experimental outline and hypothesis
Given the complexity of the intracellular renal RAS and the need for further studies to characterized the pathway, the following experiments in this thesis are designed to better understand the mechanisms involving the RAS components and particularly Ang-
(1-7) formation and degradation, as these pathways that may play a role for initiation and progression of mitochondrial and tubular dysfunction.
Chapter 2 describes how the identification of a novel peptidase activity in the brain that degrades Ang-(1-7) and is negatively correlated with Ang-(1-7) levels (96, 97), was identified in the sheep kidney (166). Moreover, we identified the enzyme activity in the sheep renal cortex and isolated proximal tubules, as well as in the human HK-2 proximal tubule cell line.
Chapter 3 consists of a review detailing both extracellular and intracellular pathways that potentially govern the formation and metabolism of angiotensin peptides within the renal proximal tubules and provides a rationale towards the role of the Ang-(1-7) peptidase in the kidney.
Chapter 4 describes the subsequent purification and identification of the renal Ang-(1-7) peptidase as Dipeptidyl-Peptidase 3 (DPP3) (34). From these studies we demonstrated that DPP3 could serve as an Ang-(1-7) peptidase and may constitute a novel component of the intra-kidney RAS.
Chapter 5 establishes an Ang-(1-7) axis within the renal mitochondria. The rationale for doing this is based on evidence revealing the benefits of ACE inhibitors towards improving mitochondrial function and oxidative stress (37). Thus, we further
26 demonstrated that mitochondria isolated from sheep renal cortex actively expressed
RAS components and contained molecular machinery to produce Ang-(1-7) intracellularly within the kidney (169). Together, this work further established the presence of a mitochondrial RAS tone and its potential contribution to kidney mitochondrial function.
Chapter 6 investigates the extent by which the RAS precursor Aogen is internalized by proximal tubule cells and the intracellular pattern of distribution (167). My hypothesis was based on previous studies identifying that RAS components within intracellular locations such as mitochondria may reflect either local synthesis or cellular uptake from the circulation. My previous studies identified the presence of Aogen in isolated kidney mitochondria (169), however it is unclear the extent that tubular Aogen reflects local synthesis or internalization. Therefore, we established intenalization assays for Aogen to assess Aogen import and subsequent trafficking to the mitochondria in proximal tubules.
27
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CHAPTER 2
An Angiotensin-(1-7) Endopeptidase in the Kidney Cortex, Proximal Tubules and Human HK-2 Epithelial Cells that is Distinct from Insulin Degrading Enzyme
Bryan A. Wilson1, Nildris Cruz-Diaz1, Allyson C. Marshall1, Nancy T. Pirro1, Yixin Su2, TanYa M. Gwathmey1, James C. Rose2, and Mark C. Chappell1
1Hypertension & Vascular Research Center, 2Department of Obstetrics and Gynecology, Wake Forest University School of Medicine,
Winston-Salem, NC USA.
This paper is published in American Journal of Physiology – Renal Physiology, March 2015 Vol. 308 no. 6, F594-F601, and and represents the efforts of the first author. Differences in organization and formatting may reflect requirements of the journal.
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ABSTRACT
Angiotensin 1-7 (Ang-(1-7)) is expressed within the kidney and exhibits renoprotective actions that antagonizes the inflammatory, fibrotic and pro-oxidant effects of Ang II. We previously identified an endopeptidase that preferentially metabolized Ang-(1-7) to Ang-
(1-4) in the brain medulla and cerebrospinal fluid (CSF) of sheep; thus, the present study established the expression of the peptidase in the kidney. Utilizing a sensitive HPLC- based approach, we demonstrate an endopeptidase activity that hydrolyzed Ang-(1-7) to
Ang-(1-4) in the sheep cortex, isolated tubules and human HK-2 renal epithelial cells.
The endopeptidase was markedly sensitive to the metallopeptidase inhibitor JMV-390; human HK-2 cells expressed subnanomolar sensitivity (IC50 = 0.5 nM) and the highest specific activity (123 ± 5 fmol/min/mg) in comparison to the tubules (96 ± 12 fmol/min/mg) and cortex (107 ± 9 fmol/min/mg). The endopeptidase was purified 41-fold from HK-2 cells; the activity was sensitive to JMV-390, the chelator o-phenanthroline and the mercury-containing compound p-chloromercuribenzoic acid (PCMB), but not to selective inhibitors against neprilysin, neurolysin and thimet oligopeptidase. Both Ang-
(1-7) and its endogenous analog [Ala1]-Ang-(1-7) (alamandine) were preferentially hydrolyzed by the endopeptidase as compared to Ang II, [Asp1]-Ang II, Ang I and Ang-
(1-12). Although the Ang-(1-7) endopeptidase and insulin degrading enzyme (IDE) share similar inhibitor characteristics of a metallothiolendopeptidase, we demonstrate marked differences in substrate specificity which suggests these peptidases are distinct.
We conclude that an Ang-(1-7) endopeptidase is expressed within the renal proximal tubule and may play a potential role in the renal renin angiotensin system to regulate
Ang-(1-7) tone.
45
INTRODUCTION
We recently identified an endopeptidase that metabolized angiotensin-(1-7) [Ang-
(1-7)] to Ang-(1-4) in the brain medulla and cerebrospinal fluid (CSF) of sheep (20-22).
The purified endopeptidase exhibited a very high affinity (IC50 < 1 nM) to the metallopeptidase agent JMV-390, but was insensitive to other metalloendopeptidase inhibitors including neprilysin (NEP, EC. 3.4.24.11), neurolysin (EC 3.4.24.16) and thimet oligopeptidase (TOP, EC 3.4.24.15) (20). The endopeptidase hydrolyzed Ang-(1-7) at a
12-fold higher rate than Ang II, but failed to metabolize other biologically active peptides apelin, bradykinin and neurotensin (20). Interestingly, Ang-(1-7) endopeptidase activity was 3-fold higher in the CSF of glucocorticoid-exposed sheep, an experimental model of in utero fetal programing that expresses higher blood pressure with associated baroreceptor dysfunction (21). Moreover, endopeptidase activity significantly correlated with the elevated blood pressure in glucocorticoid-exposed sheep, but was negatively associated with endogenous levels of Ang-(1-7) in the CSF (9). The central expression of an Ang-(1-7) endopeptidase may regulate Ang-(1-7) tone within key cardiovascular centers of the brain to influence blood pressure and baroreflex function (9).
Although Ang-(1-7) was originally identified as an endogenous component of the brain renin-angiotensin system (RAS) over 25 years ago (8), the peptide and its receptor
Mas were subsequently identified within various cell types of the kidney, as well as other peripheral tissues (3; 14; 28). In contrast to the Ang II-AT1 receptor pathway within the kidney, Ang-(1-7) increases renal blood flow and acts directly as a natriuretic to enhance sodium excretion likely through the regulation of nitric oxide (7). Ang-(1-7) also exhibits renoprotective actions and antagonizes the inflammatory, fibrotic and pro-oxidant actions of Ang II (2; 3; 9; 24; 28; 34). Since the proximal tubule is considered a key site for an intrarenal RAS, the present study sought to establish the Ang-(1-7) endopeptidase
46 activity in the renal cortex and isolated proximal tubules from adult sheep, as well as in the HK-2 human epithelial cell line, a well-characterized model of the human proximal tubule that expresses a complete RAS (1; 24; 29; 32; 33), Using a sensitive HPLC- based assay, we report that the sheep cortex, isolated tubules and the human HK-2 cells express an Ang-(1-7) endopeptidase activity that metabolizes the peptide to Ang-(1-4).
Similar to the brain peptidase, the HK-2 Ang-(1-7) endopeptidase was potently blocked by the inhibitor JMV-390 (IC50 of 0.5 nM), as well as by mercuri-containing agents and metallochelators that is characteristic of the M16A family of metallothiolpeptidases and include insulin degrading enzyme (IDE, 3.4.24.56). The endopeptidase activity was subsequently enriched approximately 40-fold from the human HK-2 cells; the purified preparation hydrolyzed Ang-(1-7) and [Ala1]-Ang-(1-7) at a 9-10 fold greater rate than
Ang II, [Ala1]-Ang II or Ang-(1-9), but did not metabolize Ang I or Ang-(1-12). A comparative analysis between human IDE and the HK-2 Ang-(1-7) endopeptidase revealed marked differences to hydrolyze 125I-Ang-(1-7) and the fluorescently quenched
7 peptide Abz-Ang-(1-7)-[Tyr (NO2)] suggesting the peptidases are distinct. From the current results, we conclude that an Ang-(1-7) endopeptidase is expressed within the proximal tubules of the kidney and may play a potential role in the renal RAS to regulate
Ang-(1-7) tone.
METHODS
Animals
Mixed breed sheep (obtained from a private local vendor) were delivered at term, farm-raised, and weaned at 3 months of age. Sheep (10-12 months of age) were anesthetized with ketamine and isoflurane and euthanized by exsanguination. Kidneys were removed immediately and renal cortex was dissected out on ice for immediate
47 isolation of proximal tubules as previously described (30). Cortical tissue or isolated tubules were stored at -80°C. All procedures were approved by the Wake Forest
University School of Medicine IACUC for animal care.
HK-2 Cells
HK-2 cells derived from human proximal tubular cells were obtained from ATCC
(Manassas, VA, USA). The cells were incubated at 37°C under 5% CO2 humidified atmosphere, and were routinely maintained in DMEM/F12 supplemented with 10% FBS, insulin-transferrin-selenium-cortisol, 100 µg/mL penicillin and 100 µg/mL streptomycin.
HK-2 cells were placed in serum-free DMEM/F12 media for 24 hours prior to the metabolism and purification studies.
Ang-(1-7) endopeptidase activity was purified from HK-2 cells using a similar approach to that of the brain peptidase (20). Sensitivity of the purified HK-2 peptidase was assessed to various inhibitors including para-chloromercuribenzoic acid (PCMB 10
µM), E-64 (10 µM), ortho-phenanthroline (10 µM), N-[N-[1-(S)-carboxyl-3-phenylpropyl]-
(S)-phenyl-alanyl]-(S)-isoserine (SCH 10 µM), N-[1-(R,S)-carboxy-3-phenylpropyl]-Ala-
Ala-Phe-p-aminobenzoate (CPP 10 µM), the dipeptide proline-isoleucine (Pro-Ile, 1 mM), and the metallopeptidase inhibitor N-[3-[(hydroxyamino)carbonyl]-1-oxo-2(R)- benzylpropyl]-L-leucine (JMV-390, 5 and 50 nM) as described for the brain and CSF endopeptidase (20). All inhibitors were obtained from Sigma (St. Louis, MO, USA) except SCH (gift from Schering Plow), CPP (Bachem, King of Prussia, PA, USA), and
JMV-390 (Tocris bioscience, Bristol, UK).
Tissue and cell preparation
Kidney cortex (1 g) was homogenized in HEPES buffer (25 mM Na+ free HEPES,
10 μM ZnCl2, 125 mM NaCl, 0.01% Triton, pH 7.4) using a Power Gen 1000 tissue
48 grinder (Fisher Scientific, Pittsburgh, PA, USA) on setting 5 for 60 s and centrifuged at
100,000 xg for 30 minutes at 4°C. Frozen proximal tubules and HK-2 cell pellets were resuspended in the HEPES buffer and sonicated briefly utilizing an ultrasonic processor
(Model W-380 Heat Systems-Ultrasonics, Inc.) and centrifuged at 100,000 xg for 30 minutes at 4°C. Supernatants were stored on ice and utilized for peptide metabolism experiments or the enzyme purification (HK-2 supernatant).
Endopeptidase Assays
Metabolism reactions were conducted at 37°C in the HEPES assay buffer
(25 mM HEPES, 125 mM NaCl, 10 μM ZnCl2, 0.01% Triton pH 7.4) using supernatants from sheep kidney cortex (10 µg), isolated proximal tubules (10 µg), HK-2 cells (10 µg), concentrated HK-2 cell media (50 µL) or the purified Q-Sepharose fraction (0.3 µg) as previously described (20). Activity was expressed as fmol of 125I-Ang-(1-4) formed per minute per mg protein (fmol/min/mg).
Recombinant human insulin degrading enzyme activity (IDE, R&D Systems,
Minneapolis, MN USA) was assayed with 2 µg of IDE and 100 µM Abz-Ang-(1-7)-
7 125 [Tyr (NO2)] for 20 hours or 0.5 nM I-(Ang-1-7) for 60 minutes at 37°C. The reactions were stopped by addition of 1.0% phosphoric acid and analyzed by HPLC-UV for Abz-
7 125 Ang-(1-7)-[Tyr (NO2)] or HPLC- γ detector for I-(Ang-1-7).
Metabolism assays with unlabeled peptides were performed in the HEPES assay buffer with the HK-2 purified peptidase (0.3 ug) and 100 µM of unlabeled Ang I, Ang-(1-
12), Ang-(1-9), Ang II, [Ala1]-Ang II, Ang-(1-7), [Ala1]-Ang-(1-7), and Abz-Ang-(1-7)-
7 [Tyr (NO2)], an internally quenched fluorescent peptide (synthesized by LifeTein, South
Plainfield, NJ, USA). The higher peptide concentration was necessary to detect the
49
Ang-(1-4) product by UV analysis as opposed to the 125I-labelled product (20).
Reactions were stopped after 20 hours at 37°C with 1.0% phosphoric acid and separated on a Shimadzu Prominence HPLC using a NovaPak C18 column (2.1 x 150 mm, Waters, Milford MA, USA) under gradient conditions (20). Peptides were monitored at 220 nm and the products identified by the retention time of standard peptides.
125I-Ang I metabolism was performed at 37°C in the HEPES reaction buffer using the concentrated HK-2 supernatant (10 µg) in a final volume of 250 μL. Each reaction contained a final concentration of 0.5 nM 125I-Ang I and 100 nM Ang I with or without the inhibitors JMV-390 (1 nM) and JMV-390 (1 nM)/CPP (10 µM) and PCMB (10 µM). The reactions were stopped after 60 minutes by addition of ice-cold 1.0% phosphoric acid and centrifuged at 16,000 xg. The supernatants were immediately filtered and the products detected by HPLC-γ detection.
Statistics
Data are expressed as mean ± SEM. One-way repeated measures ANOVA with
Bonferroni post-tests were used for the statistical analysis of data (GraphPad Prism 5,
San Diego, CA USA). The inhibitory constants (IC50) for the peptidase activity were determined by non-linear regression one-site competition with no constraints (GraphPad
Prism 5 statistical program). The criterion for statistical significance was set at p < 0.05.
RESULTS
To determine whether Ang-(1-7) endopeptidase activity in the sheep CSF and brain medulla was evident in peripheral tissues, peptidase activity was assessed in the
100,000 xg cytosolic fraction of the sheep cortex by HPLC-based detection of 125I-Ang-
(1-4) (19). As shown in the chromatograph of Figure 1A, the cortical activity hydrolyzed
125I-Ang-(1-7) to 125I-Ang-(1-4) that was reduced by the inhibitor JMV-390 (Figure 1B).
50
The cytosolic fraction from the sheep isolated proximal tubules also hydrolyzed 125I-Ang-
(1-7) to 125I-Ang-(1-4) and was sensitive to the inhibitor JMV-390 (Figures 1C and 1D, respectively). We next assessed the activity in the human HK-2 proximal tubule cell line, a well-characterized tubule model that contains a complete RAS (1; 29; 31). As shown in Figure 2, the Ang-(1-7) endopeptidase activity was expressed in the cell cytosol (Figure 2A) and the collected cell media (Figure 2C) of the HK-2 cells. The addition of JMV-390 essentially abolished the peptidase activity in both fractions (Figure
2B and 2D, respectively). Competition studies in the HK-2 cytosol revealed IC50’s of 5
µM and 2 µM for unlabeled Ang-(1-7) and [Ala1]-Ang-(1-7) (Figure 3A and 3B, respectively) to block the hydrolysis of 125I-Ang-(1-7) to 125I-Ang-(1-4). [Ala1]-Ang-(1-7) is an endogenous analog of Ang-(1-7) that may arise from the decarboxylation of aspartic acid to alanine or conversion from [Ala1]-Ang II by ACE2; however, the metabolism pathways for the peptide are not currently known (17). In comparison, the inhibitor
125 JMV-390 exhibited IC50 values of 0.5 nM and 0.2 nM for I-Ang-(1-7) hydrolysis in the
HK-2 cytosol and the cell media (Figures 3C and 3D, respectively).
51
Figure 1: Ang-(1-7) endopeptidase activity in the sheep kidney cortex and isolated proximal tubules. 125I-Ang-(1-7) [125I-A7] was hydrolyzed to 125I-Ang-(1-4) [125I-A4] in the supernatant from kidney cortex (A) and was reduced by JMV-390 (1 μM) (B); 125I-A7 was hydrolyzed to 125I-A4 in the supernatant from isolated proximal tubules (C); and was reduced by JMV-390 (1 μM) (D). Peptidase assays were conducted in the presence of an inhibitor cocktail for 60 min at 37°C and products detected by HPLC-γ detector under isocratic conditions. Data are representative of three separate experiments from different animals (see Table 1).
52
Figure 2: Ang-(1-7) endopeptidase activity in human HK-2 cell supernatant and media. 125I-Ang-(1-7) [125I-A7] was hydrolyzed to 125I-Ang-(1-4) [125I-A4] and 125I-Ang-(3- 4), the small peak eluting before 125I-A4 in the HK-2 100,000 xg cytosol fraction (A) and was abolished by JMV-390 (1 µM) (B). 125I-A7 was hydrolyzed to 125I-A4 in the media from HK-2 cells (C) and was abolished by JMV-390 (1 µM) (D). Peptidase assays were conducted without additional inhibitors for 60 min at 37°C and products detected by HPLC-γ detector under isocratic conditions. Data are representative of three separate experiments from three distinct cell passages (see Table 1).
53
Figure 3: Characterization of Ang-(1-7) endopeptidase activity in human HK-2 cells. Dose response curves for inhibition of 125I-Ang-(1-4) production by unlabeled Ang- (1-7) (A) and unlabeled [Ala1]-Ang-(1-7) (B) in HK-2 100,000 xg cytosol fraction. Dose response curves for inhibition of 125I-Ang-(1-4) production by the JMV-390 inhibitor in HK-2 cell supernatant (C) and media (D). Data are mean ± SEM; n = 3. Monophasic curve fit and IC50 values derived with GraphPad Prism 5.
54
Comparison of the specific activity of the three preparations revealed that the
HK-2 cells expressed slightly higher activity than the cortical tissue or isolated tubules
(Table 1). The majority of activity in all three preparations was sensitive to the inhibitor
JMV-390 (Table 1). Moreover, we did not detect activity in the membrane fraction of the sheep tissue or HK-2 cells (data not shown). We enriched the endopeptidase activity from the HK-2 cell cytosol using dye absorption (Cibacron Blue 3AG) and ion exchange chromatography (DEAE and Sepharose Q), an approach previously utilized to purify the brain endopeptidase (20). The HK-2 endopeptidase activity was purified approximately
40-fold with an overall yield of 29% (Table 2). The purified activity that eluted from the
Sepharose Q column in 250 mM NaCl (“Q fraction”) was subsequently used to characterize the enzyme regarding the hydrolysis of unlabeled angiotensins known to be expressed endogenously, as well as the sensitivity to various inhibitors. As shown in
Figure 4A, incubation of 100 µM unlabeled Ang-(1-7) with the Q fraction yielded a single peak that was reduced by JMV-390 (Figure 4B). [Ala1]-Ang-(1-7) was hydrolyzed to a similar extent as Ang-(1-7) (28 versus 30 nmol/min/mg; Figure 4A and 4C, Figure 5); however, the C-terminally extended peptides Ang II, Ang I and Ang-(1-12) were hydrolyzed to a lesser extent or not at all by the HK-2 peptidase (Figures 4D-4F). These and other angiotensin metabolism data are summarized in Figure 5 and the sequence for each peptide shown in Table 3. Ang-(1-7) was hydrolyzed at approximately a 10-fold higher rate than Ang-(1-9) (3 nmol/min/mg) or Ang II (2 nmol/min/mg). In regards to the sensitivity towards various enzyme inhibitors, the activity of the enriched Q fraction was markedly inhibited by 5 nM JMV-390 (>90%) and abolished with a 10-fold higher concentration (Figure 6A). The thiol inhibitor PCMB and the metal chelator o- phenanthroline inhibited 92% and 94% of the endopeptidase activity, respectively
(Figure 6A).
55
56
57
Figure 4: Purified HK-2 endopeptidase preferentially hydrolyzes Ang-(1-7) to Ang- (1-4). Purified endopeptidase (0.3 ug) from the HK-2 100,000 xg cytosol fraction was incubated with 100 μM of Ang-(1-7) [A7] (A); A7 + 1 μM JMV-390 (B); [Ala1]-Ang-(1-7) [[Ala1]-A7] (C); Ang II [AII] (D); Ang I [AI] (E) and Ang-(1-12) [A12] (F). Reactions occurred for 20 hours at 37°C and the products detected by HPLC-UV (220 nM) under identical gradient conditions.
58
59
Figure 5: Comparison of the peptidase velocities for the hydrolysis of angiotensin peptides by the purified HK-2 endopeptidase. Purified peptidase (0.3 µg) was incubated with angiotensin peptides (100 µM) and the metabolism detected by HPLC-UV (220 nM) under gradient conditions. Data are from a single experiment.
60
Figure 6: Inhibitor profile of the purified HK-2 endopeptidase. Peptidase activity was blocked by JMV-390 (JMV, 5 nM and 50 nM) and the thiol inhibitor para- chloromercuribenzoic acetate (PCMB) (10 μM), and metallochelator ortho- phenanthroline (O-Phen, 10 µM) (A). Other inhibitors including CPP (10 μM), Pro-Ile (10 µM), SCH (10 μM), and E-64 (10 μM) did not significantly inhibit activity (B). Peptidase reactions were performed with 125I-Ang-(1-7) for 60 min at 37°C. Data are mean ± SEM, n = 3; *P<0.05 vs. control conditions. Other selective inhibitors against TOP (CPP, 10 µM), neurolysin (Pro-Ile, 1 mM) and NEP (SCH, 10 µM), as well as the thiol epoxide agent E64 (10 µM) did not significantly attenuate activity (Figure 6B).
61
Based on the sensitivity of the Ang-(1-7) endopeptidase to the metallochelator o- phenanthroline and the mercuri-based inhibitor PCMB, as well as the lack of inhibition by the thiol agent E64 and other endopeptidase inhibitors, we noted that the insulin degrading enzyme (IDE) shared similar inhibitor characteristics (4). We obtained recombinant human IDE and compared its activity to the Ang-(1-7) endopeptidase. As shown in Figure 7A, the purified HK-2 Q fraction (0.3 µg) hydrolyzed 125I-Ang-(1-7) to
125I-Ang-(1-4); however, human IDE (2 µg) failed to metabolize 125I-Ang-(1-7) (Figure
7C). In contrast, the Ang-(1-7) endopeptidase did not metabolize the fluorescent
7 substrate [Abz]-Ang-(1-7)-[Tyr -(NO2)] while human IDE hydrolyzed the fluorescent peptide (Figures 7B and 7D). The large void peak in the UV chromatographs (7B and
7D) is the DMSO solvent for the fluorescent peptide.
Finally, we examined the processing of 125I-Ang I in the 100,000 xg cytosol fraction of the human HK-2 cells. As shown in the chromatographs for Figure 8, 125I-Ang I was metabolized primarily to 125I-Ang-(1-7) and 125I-Ang-(1-4). Addition of JMV-390 reduced
Ang-(1-4) and enhanced the 125I-Ang-(1-7) peak (Figure 8B, Figure 9). The co-addition of JMV-390 and the TOP inhibitor CPP reduced the 125I-Ang-(1-7) peak and preserved the 125I-Ang I peak (Figure 8C, Figure 9), while the thiol inhibitor PCMB abolished metabolism of 125I-Ang I (Figure 8D, Figure 9). In Figure 9, the 125I-Ang I metabolism studies were quantified as the extent of 125I-Ang-(1-7) or 125I-Ang-(1-4) formation in the absence or presence of JMV-390, JMV-390/CPP and PCMB (Figures 9A & 9B, respectively). We did not attempt higher concentrations of JMV-390 to completely block
125 I-Ang-(1-4) as this inhibitor may attenuate TOP activity (IC50 = 30 nM) and subsequently reduce the conversion of 125I-Ang I to 125I-Ang-(1-7) (15 ).
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Figure 7: ANG-(1–7) peptidase from HK-2 cells is distinct from insulin-degrading enzyme (IDE). Purified HK-2 peptidase (0.3 μg) hydrolyzed 125I-A7 to 125I-A4 (A) but did not hydrolyze Abz-ANG-(1–7)-[Tyr7 (NO2)] (Abz-A7; B). Human recombinant IDE (2 μg) did not hydrolyze 125I-A7 (C) but hydrolyzed Abz-A7 to Abz-ANG-(1–4) (Abz-A4; D). Products were separated by HPLC under isocratic (A and C) or gradient (B and D) conditions.
63
Figure 8: HK-2 processing of 125I-Ang I. Chromatograph reveals 125I-Ang I [AI] is metabolized to 125I-Ang-(1-7) [A7] and 125I-Ang-(1-4) [A4] in the supernatant from HK-2 100,000 xg cytosol fraction (A). A7 metabolism to A4 is reduced by 1 nM JMV-390 (B), and AI metabolism to A7 is reduced by 10 µM CPP in combination with 1 nM JMV-390 (C). AI metabolism is abolished by 10 µM PCMB (D). 125I-labelled products were separated by HPLC under gradient conditions.
64
Figure 9: 125I-Ang I metabolism in HK-2 cells. JMV-390 (JMV, 1 nM) increased 125I- Ang-(1-7), while CPP (10 µM) and JMV reduced 125I-Ang-(1-7) (A). JMV reduced 125I- Ang-(1-4) levels while addition of JMV and CPP did not further influence 125I-Ang-(1-4) (B). The mercuri-containing inhibitor PCMB (10 µM) abolished the metabolism of 125I- Ang I to 125I-Ang-(1-7) and 125I-Ang-(1-4). Data are mean ± SEM, n = 3; *P < 0.05 vs. control (CON); #P < 0.05 vs. JMV; AP<0.05 vs. JMV/CPP.
65
DISCUSSION
Abundant evidence strongly supports a local or tissue based RAS in the kidney that contributes to renal function, as well as the regulation of blood pressure and water and sodium balance (24; 25). The proximal tubule is one site within the kidney that contains many, if not all elements of a complete RAS capable of generating and responding to the bioactive peptide Ang II (24; (33). The present study provides evidence for an enzymatic pathway that specifically metabolizes the Tyr4-Ile5 bond of Ang-(1-7) to form Ang-(1-4) within the proximal tubules of the sheep kidney and the human HK-2 tubule cells. The isolation and characterization of the peptidase from the HK-2 tubule cells revealed essentially identical characteristics to the Ang-(1-7) endopeptidase isolated from the sheep brain and CSF (20). Similar to the brain peptidase, the HK-2 cellular activity and that isolated from the cell media exhibited a very low IC50 (0.2 to 0.5 nM) for the metallopeptidase inhibitor JMV-390. Moreover, the purified HK-2 endopeptidase preferentially hydrolyzed Ang-(1-7) in comparison to Ang II, Ang-(1-9), Ang I or Ang-(1-
12). Finally, although human IDE and the Ang-(1-7) endopeptidase share similar inhibitor characteristics and IDE is expressed within the renal tubules, we demonstrate marked differences in substrate specificity to suggest that the two enzymes are distinct.
An Ang-(1-7) endopeptidase or neuropeptidase was previously identified in the brain medulla and CSF of sheep (20). The current study identified the Ang-(1-7) endopeptidase in the sheep cortex, isolated tubules and human HK-2 cells that was also sensitive to the inhibitor JMV-390. The peptidase was purified from the HK-2 cells using a similar chromatographic approach to that of the brain peptidase; we achieved a 40-fold increase in specific activity that likely reflects the lower initial protein content of the cells as compared to the brain tissue (20). Nevertheless, the purified peptidase revealed essentially an identical specificity as the brain peptidase regarding the hydrolysis of
66 angiotensins. Ang-(1-7) was hydrolyzed at a greater rate than Ang II and Ang-(1-9) while the hydrolysis of Ang I or Ang-(1-12) was not detectable under the present conditions. Although the endogenous Ang-(1-7) analog [Ala1]-Ang-(1-7) exhibited a
125 lower IC50 than Ang-(1-7) for hydrolysis of I-Ang-(1-7) (2 µM vs. 5 µM), we noted little difference in the metabolism of these two peptides by the purified HK-2 endopeptidase.
The endopeptidase also hydrolyzed Ang II and [Ala1]-Ang II in a similar manner, albeit at a 10-fold lower rate than that of Ang-(1-7) or [Ala1]-Ang-(1-7). Importantly, these data suggest that substitution at the N-terminus of the peptide (alanine to aspartic acid) does not have a major influence the hydrolysis of the peptide; however, additions to the C- terminal end appear to markedly reduce metabolism. The quenched fluorescent Ang-(1-
7) peptide with a substituted tyrosine (NO2) group was not hydrolyzed suggesting that the C-terminal proline may be a critical residue in the recognition and catalysis of Ang-
(1-7) by the endopeptidase.
The purified HK-2 peptidase activity was sensitive to both the metallochelator o- phenanthroline and the thiol inhibitor PCMB, but not the thiol epoxide inhibitor E-64. The brain endopeptidase was also quite sensitive to o-phenanthroline, PCMB and another mercurial agent APMA, but not E-64 or leupeptin (20). Moreover, the chelating agent
EDTA was significantly less potent than o-phenanthroline to inhibit the brain endopeptidase (20). Interestingly, the metallothiolendopeptidase IDE exhibited essentially an identical pattern of sensitivity to this group of inhibitors (4; 31). Originally identified as the primary peptidase that metabolizes insulin, IDE contributes to the in vivo metabolism of amylin and glucagon (18). IDE also hydrolyses a number of other peptides in vitro including ANP, γ-endorphin, bradykinin and relaxin (5; 19; 23; 27); however, human IDE failed to metabolize radiolabelled Ang-(1-7). IDE hydrolyzed the quenched Abz-Ang-(1-7) fluorescent peptide to Abz-Ang-(1-4), but this substrate was not
67 metabolized by the HK-2 endopeptidase. Replacement of tyrosine (NO2) for proline in the 7th position of the fluorescent peptide may obviate recognition of this substrate by the
HK-2 endopeptidase, while the tyrosine (NO2) substitution does not appear critical for
IDE hydrolysis. IDE belongs to the M16 family of soluble metallothiolpeptidases or inverzincins; other members include the bacterial enzyme pitrilysin (EC.3.4.24.55), nardilysin (EC 3.4.24.61) and mammalian metallopeptidase I that is predominantly localized to the mitochondria (10). We have not obtained sequence data on the enriched Ang-(1-7) endopeptidase from brain or the HK-2 cells to unequivocally identify the protein. The Ang-(1-7) endopeptidase may belong to the M16 class of metallothiolendopetidases based on the inhibitor profile; however, complete purification and subsequent identification of the peptidase is necessary to address this issue. In addition, it remains to be determined whether the Ang-(1-7) peptidase is expressed in the kidney in other species such as the rat or mouse.
The Ang-(1-7) endopeptidase was prominent in the 100,000 xg cytosolic fraction of the sheep cortex, proximal tubules and HK-2 cells. The kidney exhibits a high content of
Ang-(1-7) comparable to Ang II (26), and immunocytochemical studies localized Ang-(1-
7) predominantly to the proximal tubules (13). In the NRK52-E rat epithelial cell line, the advanced glycation end product (AGE) methylglyoxal-albumin reduced intracellular levels of Ang-(1-7) which were associated with an enhanced metabolism of the peptide to Ang-(1-4), myofibroblast transition and chronic MAPK activation (2). Moreover, the intracellular expression of functional Ang-(1-7) and Ang II receptors is evident within the kidney, proximal tubules and other tissues and the endopeptidase may be a novel component of an intracellular RAS to regulate the cellular levels of Ang-(1-7) (6; 11; 12;
14; 16; 32).
68
Perspectives and Significance
Ang-(1-7) constitutes a key bioactive component of the non-classical or alternative
RAS in the kidney, circulation and other tissues (7). In general, the ACE2/NEP/TOP-
Ang-(1-7)-AT7/Mas axis opposes or functionally antagonizes a stimulated ACE-Ang II-
AT1R pathway. The renal actions of Ang-(1-7) include natriuresis, diuresis and reduced vascular resistance, as well as anti-inflammatory and anti-fibrotic effects that may encompass the release of nitric oxide, activation of cellular phosphatases and the reduction of oxidative stress (9; 28). The presence of an Ang-(1-7) endopeptidase within the proximal tubule of the kidney may constitute a novel therapeutic target to maintain the intracellular levels of the peptide and potentially provide renoprotective effects.
CONFLICT OF INTEREST
The authors declare that there are no competing financial interests in the work described.
ACKNOWLEDGEMENTS
This work represents partial fulfillment of the requirements for the degree of Doctorate of
Philosophy in the Department of Molecular Medicine and Translational Sciences at
Wake Forest University School of Medicine for Bryan A. Wilson. Mr. Wilson is supported by a National Institutes of Health (NIH) T32 grant (HL091797). Dr. Cruz-Diaz is supported by a fellowship from the PRIME Institutional Research and Academic Career
Development Award K12-GM102773. Additional support for this study was provided by
NIH grants HD-047584, HD-017644, and HL-51952; the Groskert Heart Fund, the Wake
Forest Venture Fund the Farley‐Hudson Foundation (Jacksonville, NC). The authors gratefully acknowledge Eric LeSaine for his technical and surgical support.
69
Portions of this work were originally presented at the 2014 Experimental Biology meeting in San Diego CA.
70
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5. Bennett RG, Heimann DG and Hamel FG. Degradation of relaxin family peptides by insulin-degrading enzyme. Ann N Y Acad Sci 1160: 38-41, 2009.
6. Bkaily G, Nader M, Avedanian L, Choufani S, Jacques D, Orleans-Juste P, Gobeil F, Chemtob S and Al-Khoury J. G-protein-coupled receptors, channels, and Na+-H+ exchanger in nuclear membranes of heart, hepatic, vascular endothelial, and smooth muscle cells. Can J Physiol Pharmacol 84: 431-441, 2006.
7. Chappell MC. Nonclassical Renin-Angiotensin system and renal function. Compr Physiol 2: 2733-2752, 2012.
8. Chappell MC, Brosnihan KB, Diz DI and Ferrario CM. Identification of angiotensin-(1-7) in rat brain: evidence for differential processing of angiotensin peptides. J Biol Chem 264: 16518-16523, 1989.
9. Chappell MC, Marshall AC, Alzayadneh EM, Shaltout HA and Diz DI. Update on the Angiotensin converting enzyme 2-Angiotensin (1-7)-MAS receptor axis: fetal programing, sex differences, and intracellular pathways. Front Endocrinol (Lausanne) 4: 201-214, 2014.
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10. Chow KM, Gakh O, Payne IC, Juliano MA, Juliano L, Isaya G and Hersh LB. Mammalian pitrilysin: substrate specificity and mitochondrial targeting. Biochemistry 48: 2868-2877, 2009.
11. Cook JL and Re RN. Invited Review: Lessons from in vitro studies and a related intracellular angiotensin II transgenic mouse model. Am J Physiol Regul Integr Comp Physiol 302: R482-93, 2011.
12. Ellis B, Li XC, Miguel-Qin E, Gu V and Zhuo JL. Evidence for a functional intracellular angiotensin system in the proximal tubule of the kidney. Am J Physiol Regul Integr Comp Physiol 302: R494-R509, 2012.
13. Ferrario CM, Averill DB, Brosnihan KB, Chappell MC, Iskandar SS, Dean RH and Diz DI. Vasopeptidase inhibition and Ang-(1-7) in the spontaneously hypertensive rat. Kid Int 62: 1349-1357, 2002.
14. Gwathmey TM, Alzayadneh EM, Pendergrass KD and Chappell MC. Novel roles of nuclear angiotensin receptors and signaling mechanisms. Am J Physiol Regul Integr Comp Physiol 302: R518-R530, 2012.
15. Kitabgi P, Dubuc I, Nouel D, Costentin J, Cuber JC, Fulcradn H, Doulut S, Rodriguez M, Martinez J. Effects of thiophan estatin and a novel metallopeptidase inhibitor JMV 390-1 on the reovery of neurotensio andneuromedian N released from mouse hypothalmus. Neursoci Lett 14: 200-204, 1993.
16. Kumar R, Thomas CM, Yong QC, Chen W and Baker KM. The intracrine renin- angiotensin system. Clin Sci (Lond) 123: 273-284, 2012.
17. Lautner RQ, Villela DC, Fraga-Silva RA, Silva N, Verano-Braga T, Costa-Fraga F, Jankowski J, Jankowski V, Sousa F, Alzamora A, Soares E, Barbosa C, Kjeldsen F, Oliveira A, Braga J, Savergnini S, Maia G, Peluso AB, Passos- Silva D, Ferreira A, Alves F, Martins A, Raizada M, Paula R, Motta-Santos D, Klempin F, Pimenta A, Alenina N, Sinisterra R, Bader M, Campagnole-Santos MJ and Santos RA. Discovery and characterization of alamandine: a novel component of the renin-angiotensin system. Circ Res 112: 1104-1111, 2013.
18. Maianti JP, McFedries A, Foda ZH, Kleiner RE, Du XQ, Leissring MA, Tang WJ, Charron MJ, Seeliger MA, Saghatelian A and Liu DR. Anti-diabetic activity
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20. Marshall AC, Pirro NT, Rose JC, Diz DI and Chappell MC. Evidence for an angiotensin-(1-7) neuropeptidase expressed in the brain medulla and CSF of sheep. J Neurochem 130: 313-323, 2014.
21. Marshall AC, Shaltout HA, Pirro NT, Rose JC, Diz DI and Chappell MC. Antenatal betamethasone exposure is associated with lower ANG-(1-7) and increased ACE in the CSF of adult sheep. Am J Physiol Regul Integr Comp Physiol 305: R679-R688, 2013.
22. Marshall AC, Shaltout HA, Pirro NT, Rose JC, Diz DI and Chappell MC. Enhanced activity of an angiotensin-(1-7) neuropeptidase in glucocorticoid-induced fetal programming. Peptides 52: 74-81, 2014.
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30. Shaltout HA, Westwood B, Averill DB, Ferrario CM, Figueroa J, Diz DI, Rose JC and Chappell MC. Angiotensin metabolism in renal proximal tubules, urine and serum of sheep: Evidence for ACE2-dependent processing of Angiotensin II. Am J Physiol Renal Physiol 292: F82-F91, 2006.
31. Shi K, Yokono K, Baba S, Roth RA. Purification and characterization of an insulin degrading enzyme fom human erythrocytes. Diabetes 35: 675-683.
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CHAPTER 3 The Ins and Outs of Angiotensin Processing
Bryan A. Wilson, Allyson C. Marshall, Ebaa M. Alzayadneh and Mark C. Chappell
Hypertension & Vascular Research Center, Wake Forest University School of Medicine, Winston-Salem, NC,
This paper is published online in the American Journal of Physiology Regulatory Integrative Comparative Physiology 2014; 307(5):R487-R489, and represents the efforts of the first author. Differences in organization and formatting may reflect requirements of the journal.
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ABSTRACT
The kidney is key target organ for bioactive components of the renin-angiotensin system (RAS); however, various renal cells such as the tubular epithelium contain an intrinsic RAS. The renal RAS can be functionally divided into Ang II-AT1 receptor and
Ang-(1-7)-AT7/Mas receptor arms that functionally oppose one another. The current review considers both extracellular and intracellular pathways that potentially govern the formation and metabolism of angiotensin peptides within the renal proximal tubules.
REVIEW
The kidney is a key target of the diverse components of the renin-angiotensin- aldosterone system that include prorenin/renin, angiotensin II (Ang II), Ang-(2-8) (Ang
III), Ang-(1-7), Ang-(3-8), Ang-(1-9) and aldosterone (2; 3; 6; 10). The kidney also comprises an intrinsic renin-angiotensin system (RAS) particularly within the proximal tubule epithelium capable of producing bioactive peptides to activate their respective receptors (R) in a paracrine or autocrine manner (2; 6; 10). Currently, the renal RAS can be functionally partitioned into at least two arms based on the distinct processing enzymes and receptors that comprise the Ang II-AT1R and the Ang-(1-7)-AT7/MasR axis (2; 3; 7; 10; 14) In general, these two pathways exhibit opposing effects in the kidney and may antagonize the actions of one another (2; 3). Within the tubular system of the kidney, Ang II stimulates the AT1 receptor to enhance the activity of various transport mechanisms to maintain the efficient reabsorption of sodium (10). In contrast,
Ang-(1-7) evokes natriuretic and diuretic effects, stimulates nitric oxide release, induces scavenging enzymes to attenuate oxidative stress and stimulates cellular phosphatases to inhibit MAP kinase pathways (2; 3). Apart from the distinct Ang peptide receptors, evidence to date suggests a complex array of peptidases involved in the synthesis and metabolism of Ang II and Ang-(1-7) (14). Both peptides ultimately arise from the same precursor protein angiotensinogen that may be internalized by the proximal tubular cells
76 and/or locally synthesized within the kidney (2) (Figure 1). ACE is well-recognized as the key Ang II-forming enzyme in the kidney, circulation and other peripheral and central tissues. In contrast, the ACE homolog ACE2 efficiently metabolizes Ang II to Ang-(1-7) and may markedly alter the functional signature of the RAS (2). ACE2 is a mono- carboxypeptidase that does not continue to metabolize Ang-(1-7) due to the C-terminal proline; however, ACE hydrolyzes the Ile5-His6 bond of Ang-(1-7) to form Ang-(1-5) (2).
ACE inhibitors increase circulating levels of Ang-(1-7) by preventing the rapid metabolism of the peptide, as well as shifting the processing of Ang I to Ang-(1-7) by the endopeptidase neprilysin (NEP) (2; 13; 14). All three enzymes are classified as metallopeptidases with membrane-anchoring domains that orient their active sites on the extracellular cell surface to process substrates within the glomerular filtrate, interstitial fluid, cerebrospinal fluid (CSF) or the blood (Figure 1). These peptidases comprise the extracellular processing paths for the formation of Ang II and Ang-(1-7) to subsequently bind to AT1R or AT7R on the cell surface and activate various signaling pathways
(Figure 1). ACE, ACE2 and NEP also contribute to the metabolism of both peptides to either inactive forms or, in the case of Ang-(1-7), a metabolite that functionally opposes the Ang II-AT1R axis (2; 13).
In addition to the extracellular processing of angiotensin peptides, there is compelling evidence for the intracellular expression of both Ang II and Ang-(1-7) in the kidney and other tissues (1--8). The tissue expression of angiotensins may indeed lead to their subsequent release into the extracellular space; however, evidence of intracellular AT1R, AT2R and AT7R may portend for processing pathways that provide an intracellular source of Ang II or Ang-(1-7) as potential receptor ligands (1-8). In glycemic or diabetic conditions, Ang II content is increased in various cell types and appears to reflect the contribution of non-ACE dependent pathways such as chymase or cathepsins (5; 8; 11).
77
Figure 1. Potential scheme for the extracellular and intracellular processing of angiotensin peptides within renal proximal tubules. Extracellular metallopeptidases, angiotensin converting enzyme (ACE), ACE2 and neprilysin (NEP) process Ang I to Ang II or Ang-(1-7) (Ang 7) that subsequently bind to receptors on the cell surface. Intracellular peptidases include thimet oligopeptidase (TOP) to process Ang I to Ang-(1- 7) or ACE-independent (non-ACE) pathways such as chymase to form Ang II. An Ang- (1-7) endopeptidase (A7-EP) hydrolyzes the peptide to Ang-(1-4) (Ang 4) and the peptidase may be secreted for extracellular metabolism of Ang-(1-7) in addition to that by ACE. The precursor angiotensinogen (Aogen) may be internalized by the tubules from extracellular sources or arise from intracellular synthesis.
78
Elucidation of the intracellular pathways for Ang-(1-7) formation and metabolism within the intracellular compartments of the kidney and other tissues is less clear. Costa-Neto and colleagues reported that the soluble enzyme thimet oligopeptidase (TOP) processed
Ang I predominantly to Ang-(1-7) in the rat hippocampus independent of Ang II (12).
TOP was also identified as the primary activity that directly converted Ang I to Ang-(1-7) without the prerequisite formation of Ang II in nuclei isolated from the NRK-52E renal epithelial cells (1). In regards to Ang-(1-7) metabolism, our recent studies reveal a soluble endopeptidase that degrades Ang-(1-7) to the inactive metabolite Ang-(1-4) in the CSF and brain medulla of sheep (3; 9). The purified peptidase exhibits a preferred specificity for Ang-(1-7) in comparison to other angiotensins; the rate of Ang-(1-7) processing to Ang-(1-4) was 10-20 fold higher than for Ang II and Ang I, while other peptides including bradykinin, neurotensin and apelin were not hydrolyzed by the enzyme following a 24 hour incubation (9). The Ang-(1-7) endopeptidase also exhibits subnanomolar affinity (IC=0.8 nM) against the inhibitor JMV-390 that is markedly lower than that reported for other metallopeptidases such as neprilysin, neurolysin, TOP and
ACE (9). Preliminary studies now identify the Ang-(1-7) endopeptidase in the soluble fraction of isolated proximal tubules of the sheep kidney, as well as in the human proximal tubule HK-2 cell line (15). Both peptidase activities in the tubules and HK-2 cells exhibit similar high affinities for the JMV inhibitor to block the conversion of Ang-(1-
7) to Ang-(1-4) (15). Moreover, endopeptidase activity was evident in the serum-free media of the HK-2 cells suggesting that the enzyme is released and may potentially participate in the extracellular metabolism of Ang-(1-7) within the filtrate or the interstitial compartment of the kidney (15). Although additional characterization of the renal endopeptidase regarding the enzyme’s specificity and regulation is warranted, these studies suggest both intracellular and extracellular processing pathways may contribute
79 to the formation and metabolism of the bioactive peptides Ang II and Ang-(1-7) within the kidney to influence renal function and pathology (Figure 1).
ACKNOWLEDGMENTS
This work was supported by NIH grants HL56973, HL52972, HL112237, and HD047584.
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12. Pereira MG, Souza L L, Becari C, Duarte DA, Camacho FR, Oliveira JA, Gomes MD, Oliveira EB, Salgado MC, Garcia-Cairasco N, Costa-Neto CM. Angiotensin II-independent angiotensin-(1-7) formation in rat hippocampus: involvement of thimet oligopeptidase. Hypertension 62,879–885, 2013.
13. Schwacke JH, Spainhour JC, Ierardi JL, Chaves JM, Arthur JM, Janech MG, Velez JC. Network modeling reveals steps in angiotensin peptide processing. Hypertension.61:690-700, 2013.
14. Shaltout HA, Westwood B, Averill DB, Ferrario CM, Figueroa J, Diz DI, Rose JC and Chappell MC. Angiotensin metabolism in renal proximal tubules, urine and serum of sheep: ACE2-dependent processing of angiotensin II. Am J Physiol Renal Physiol 292: F82-F91, 2007.
15. Wilson BA, Marsall AC, Pirro NT, Su Y, Rose JC, Chappell MC. [Abstract] Expression of an Angiotensin-(1-7) endopeptidase in proximal tubules of sheep and human kidney. FASEB J 28:1088.1014, 2014.
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CHAPTER 4
Identification of Dipeptidyl Peptidase 3 as the Angiotensin-(1-7)-Degrading Peptidase in Human HK-2 Epithelial Cells
Bryan A. Wilson*, Nildris Cruz-Diaz*, Allyson C. Marshall, Nancy T. Pirro, K. Bridget Brosnihan and Mark C. Chappell
Hypertension & Vascular Research Center, Wake Forest University School of Medicine, Winston-Salem, NC USA.
Submitted to Peptides, April 2016
Note: (*) indicates that both authors contributed to this work equally.
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ABSTRACT
Angiotensin 1-7 (Ang-(1-7)) is expressed within the kidney and exhibits renoprotective actions that antagonizes the inflammatory, fibrotic and pro-oxidant effects of the Ang II-
AT1 receptor axis. We previously identified a peptidase activity that metabolized Ang-(1-
7) in sheep isolated proximal tubules and in human HK-2 proximal tubule cells; thus, the present study isolated and identified the Ang-(1-7) peptidase from the 100,000 x g cytosolic fraction of the HK-2 cells. Following several chromatographic steps, a single protein band at 80 kDa was excised from SDS-PAGE and analyzed by LC-MS. The protein band was identified as the enzyme dipeptidylpeptidase III (DPP 3, EC: 3.4.14.4).
A DPP 3 antibody identified a single 80 kDa band from the purified enzyme, as well as a recombinant human DPP 3. Both the purified Ang-(1-7) peptidase and recombinant
DPP 3 were potently inhibited by the metallopeptidase agent JMV-390 and exhibited
IC50’s of 0.2 nM and 1.4 nM, respectively. DPP 3 sequentially hydrolyzed Ang-(1-7) to
Ang-(3-7) and rapidly converted Ang-(3-7) to Ang-(5-7). Kinetic analysis revealed that
Ang-(3-7) was hydrolyzed at a greater rate than Ang-(1-7) [14.8 vs. 5.5 nmol/min/µg protein]; the Km for Ang-(3-7) was lower than Ang-(1-7) [2.0 vs. 12 µM]. Finally, chronic treatment of the HK-2 cells with the JMV-390 inhibitor significantly reduced intracellular DPP 3 activity and tended to augment the cellular levels of Ang-(1-7). We conclude that DPP 3 may contribute to the intracellular metabolism of Ang-(1-7) and potentially influence the cellular expression and function of Ang-(1-7).
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INTRODUCTION
We recently identified a peptidase activity in the brain medulla and cerebrospinal fluid (CSF) of sheep that metabolized low concentrations (pM) of angiotensin-(1-7) [Ang-
(1-7)] (8; 10; 11). The peptidase was purified approximately 2000-fold from brain medulla and the enzyme activity exhibited a very high affinity (IC50<1 nM) to the metallopeptidase agent JMV-390, but was insensitive to other metallopeptidase inhibitors including neprilysin (NEP, EC. 3.4.24.11), neurolysin (EC 3.4.24.16) and thimet oligopeptidase (TOP, EC 3.4.24.15) (8). The purified peptidase hydrolyzed Ang-(1-7) at a 12-fold higher rate than Ang II, but failed to metabolize the larger biologically active peptides apelin 13, bradykinin and neurotensin (8). Interestingly, the Ang-(1-7) peptidase activity was 3-fold higher in the CSF of glucocorticoid-exposed sheep, an experimental model of in utero fetal programing that exhibits higher blood pressure associated with baroreceptor dysfunction (11). In this regard, the peptidase activity significantly correlated with the higher blood pressure in glucocorticoid-exposed sheep, but was negatively associated with endogenous levels of Ang-(1-7) in the CSF (6).
Based on this evidence, the central expression of an Ang-(1-7) peptidase may potentially regulate Ang-(1-7) tone within key cardiovascular centers of the brain to influence blood pressure and baroreflex function (6).
Although Ang-(1-7) was originally identified as an endogenous component of the brain renin-angiotensin system (RAS) over 25 years ago (5), the peptide and its receptor
Mas were subsequently identified within various cell types of the kidney, as well as other peripheral tissues (1; 13). In contrast to the Ang II-AT1 receptor pathway within the kidney, Ang-(1-7) increases renal blood flow and acts directly as a natriuretic to enhance sodium excretion likely through the regulation of nitric oxide (3). Ang-(1-7) also exhibits renoprotective actions and antagonizes the inflammatory, fibrotic and pro-oxidant actions
85 of Ang II (1; 6; 13). Indeed, Ang-(1-7) treatment was recently shown to be superior to
AT1 receptor antagonists to attenuate the progression of diabetic renal damage and advanced glycation end product (AGE)-induced myofibroblast transition and cellular hypertrophy (2; 32). The proximal tubule is considered a key site for an intrarenal RAS expressing the biosynthetic components angiotensinogen, renin, and ACE that participate in the local formation of Ang II (12). The tubules also express the ACE homolog ACE2 that efficiently converts Ang II to Ang-(1-7), as well as the endopeptidases NEP and TOP that process Ang I directly to Ang-(1-7) (16). We recently showed that the Ang-(1-7) peptidase activity was expressed in the sheep renal cortex, isolated proximal tubules and in human HK-2 proximal tubule cells. Interestingly, the
Ang-(1-7) peptidase constituted the sole activity in the HK-2 cells that metabolized Ang-
(1-7) (15). Therefore, we utilized the HK-2 cells as a source of the Ang-(1-7) degrading activity to purify, identify and characterize the peptidase. Following extensive purification, a single protein band revealed on SDS-gel was analyzed by LC-MS and the enzyme dipeptidyl peptidase III (DPP 3, EC: 3.4.14.4) was identified.
METHODS
Cell culture
HK-2 cells derived from human proximal tubular cells were obtained from ATCC
(Manassas, VA, USA). The cells were incubated at 37°C under 5% CO2 humidified atmosphere, and were routinely maintained in DMEM/F12 supplemented with 10% FBS, insulin-transferrin-selenium-cortisol, 100 µg/mL penicillin and 100 µg/mL streptomycin.
HK-2 cells were placed in serum-free DMEM/F12 media for 24 hours prior to the metabolism and purification studies.
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Cell preparation
Frozen cell pellets were resuspended in HEPES buffer (25 mM Na+ free HEPES, 10 μM
ZnCl2, 125 mM NaCl, 0.01% Triton, pH 7.4) and sonicated briefly utilizing an ultrasonic processor (Model W-380 Heat Systems-Ultrasonics, Inc.) and centrifuged at 100,000 x g for 30 minutes at 4°C. Supernatants were stored on ice and utilized for peptide metabolism experiments or the enzyme purification (HK-2 supernatant).
HK-2 endopeptidase purification
Ang-(1-7) was purified from HK-2 cells using an identical approach to that of the brain peptidase (8). HK-2 supernatant was pooled and concentrated on a 30 kDa cut-off filter
(Millipore, Bedford, MA, USA). Concentrated HK-2 supernatant was resuspended in a final volume of 5 mL HEPES buffer (25 mM HEPES, 50 mM NaCl, 10 μM ZnCl2, 0.01%
Triton pH = 8.0) and applied to a Cibacron Blue Sepharose Fast Flow (GE Healthcare
Bio-Sciences) column (1 x 5 cm) equilibrated in the same HEPES buffer. The flow- through fraction was applied directly to a 1 x 10 cm diethylaminoethyl Sepharose column
(DEAE, Sigma-Aldrich, St. Louis, MO, USA). The column was subsequently washed in
HEPES buffer with an increasing step gradient of NaCl (125, 250, 500 mM, 1M). The eluted activity in the 125 mM NaCl fraction was concentrated in 50 mM NaCl of the
HEPES buffer and applied to a 1 × 10 cm Q-Sepharose Fast Flow column (Sigma-
Aldrich) with a step gradient of increasing NaCl (50, 125, 250, 500 mM and 1 M). The endopeptidase activity eluted in the 250 mM NaCl and the fraction was concentrated, diluted in the HEPES buffer containing 1 M NH4SO4 and applied to 2 x 10 cm Phenyl-
Sepharose column. This column was washed with 1 M NH4SO4 and a decreasing step gradient was applied. The enzyme activity primarily eluted in 250 mM NH4SO4 and this fraction was concentrated on a 30 kDa filter in the HEPES buffer to remove the NH4SO4.
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Native and SDS Polyacrylamide Electrophoresis
The purified peptidase from the phenyl column was fractionated by both native and SDS 10% polyacrylamide gels for 1 hour at 120V in Tris-glycine with or without
SDS. Gels were stained with the protein dye Royal Blue and the sole band at 80 kDa excised for peptidase activity (native gel) or for MS analysis (SDS gel, MS Bioworks,
Ann Arbor MI). For western blot analysis of the purified enzyme and the DPP 3 standard, the SDS gels were transferred to a polyvinylidene difluoride (PVDF) membrane. Blots were blocked with 5% Bio-Rad Dry Milk (Bio-Rad, Hercules, CA, USA) and Tris buffered saline (TBS) with Tween (0.05%) and probed overnight at 4°C with a human DPP 3 primary antibody (Abcam, Cambridge MA).
Peptidase Assay
Metabolism reactions were conducted at 37°C in the HEPES assay buffer (25 mM
HEPES, 125 mM NaCl, 10 μM ZnCl2, 0.01% Triton pH 7.5) using supernatants from HK-
2 cells (10 µg), the purified Phenyl column fraction (0.3 µg) or the supernatant from the excised band of the native gel. The assay contained a final concentration 0.5 nM 125I-
Ang-(1-7) and 100 nM Ang-(1-7) in a volume of 250 μL. The reactions were stopped after 60 minutes by addition of ice-cold 1.0% phosphoric acid and centrifuged at
16,000 x g. The supernatants were filtered for separation on a Shimadzu Prominence
HPLC equipped with an Aeris Peptide XB-C18 (2.1 × 100 mm, Phenomenex, Torrance,
CA, USA) and a Bioscan flow-through γ detector using isocratic elution conditions (8).
Activity was expressed as fmol of 125I-Ang-(3-4) formed per minute per mg protein
(fmol/min/mg).
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HPLC-UV Analysis
Metabolism assays with unlabeled peptides were performed in the HEPES assay buffer with the HK-2 purified peptidase or the recombinant DPP 3 (300 ng) and 100 µM of unlabeled Ang-(1-7). Reactions were stopped after 20 hours at 37°C with 1.0% heptafluorobutyric acid (HFBA) and separated on a Shimadzu Prominence HPLC using a NovaPak C18 column (2.1 x 150 mm, Waters, Milford MA, USA) using HFBA as the counter ion (7). Peptides were monitored at 220 nm and the products identified by the retention time of standard peptides. All inhibitors were obtained from Sigma (St. Louis,
MO, USA) except SCH (gift from Schering Plow), CPP (Bachem, King of Prussia, PA,
USA), and JMV-390 (Tocris Bioscience, Bristol, UK).
HK-2 Cell Inhibitor Studies
To establish the effect of peptidase inhibition on the cellular expression of Ang-(1-7), the
HK-2 cells were placed in 1% FBS DMEM/F12 media for 24 hours and then treated with
0, 20 or 200 nM JVM-390 every 24 hours for 3 days. Cells were rinsed in cold PBS on ice, scraped and stored at -80°C. The cell pellet was brought up to 5 ml of Milli-Q water and immediately placed in a boiling water bath for 15 minutes. The solution was acidified with 1% HFBA, sonicated and kept on ice overnight in a refrigerator. Following a 25,000 x g centrifugation step, the supernatant was applied to an Oasis mixed mode extraction column. The column was washed with 0.1% HFBA and the peptide fraction eluted in 80% methanol. The eluent was evaporated in a Savant vacuum centrifuge, reconstituted in the Ang-(1-7) RIA buffer and peptide content determined. As previously described, the Ang-(1-7) RIA recognizes Ang-(2-7) and Ang-(3-7), but does not recognize (<0.01%) other angiotensin peptides including Ang II and Ang I (17). The
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Ang-(1-7) RIA exhibits a sensitivity of 2.5 fmol per tube. The peptide content of Ang-(1-
7) in the HK-2 cells was expressed as fmol per mg protein of the cell extract.
Statistics
Data are expressed as mean ± SEM. One-way repeated measures ANOVA with
Bonferroni post-tests were used for the statistical analysis of data (GraphPad Prism 6,
San Diego, CA USA). The inhibitory constants (IC50) for the peptidase activity were determined by non-linear regression one-site competition with no constraints (GraphPad
Prism 6 statistical program). The criterion for statistical significance was set at p < 0.05.
RESULTS
The 100,000 x g cytosolic fraction from the HK-2 cells was initially concentrated on a
30 kDa filter and applied to a Ciba Chron 3AG column. The flow through was immediately applied to DEAE Sepharose and the Ang-(1-7) degrading activity eluted in
185 mM NaCl. This fraction was then applied to a Mono Q ion exchange column, washed and peptidase activity eluted in 210 mM NaCl. The Q fraction was adjusted to
1M NH4SO4 and applied to a phenyl Sepharose column. The peptidase was retained on the column under the 1M NH4SO4 loading conditions and the majority of activity eluted in
0.25 M NH4SO4 and was concentrated on a 30 kDa filter. The purified material from the
Phenyl column was fractionated on both a reducing SDS and a native or non-reducing polyacrylamide gel (10%), both gels were stained with Royal Blue. As shown in Figure
1A, the SDS gel reveals a single protein band at ~80 kDa using 2 different concentrations of the purified peptidase. The band from the SDS gel was excised and submitted for MALDI-MS analysis (MS Bioworks).
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125I-A3-4
Figure 1. Purified Ang-(1-7) peptidase from Phenyl column reveals a single 80 kDa band (arrow) on SDS-PAGE gel stained with Royal Blue (panel A). Western blot of DPP 3 standards (lanes 1-2) and purified Ang-(1-7) peptidase with DPP 3 antibody reveals a single 80 kDa band (arrow). Excised 80 kDa band from native PAGE gel of purified Ang-(1-7) peptidase from Phenyl column reveals activity that hydrolyzed 125I- Ang-(1-7) (A7) to 125I-Ang-(3-4) (panel C). Molecular weight
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Table 1: MALDI-MS Identification
Identified Proteins Molecular Weight (kDa)
Bovine Albumin 69
Keratin 62
Lysozyme C 17
Heat Shock Protein 71
IgGa-1 38
DPP III 83
α-trypsin inhibitor 1 103
Peptidyl prolyl isomerase 64
A-2 Glycoprotein 39
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The MS analysis revealed 7 human proteins that included keratin, heat shock protein
70, and DPP 3, as well as bovine serum albumin (BSA) that is likely internalized from fetal bovine serum by the tubule cells (Table 1). Of the proteins identified, only DPP 3 exhibited the expected molecular size and proteolytic activity that would hydrolyze Ang-
(1-7). We obtained a human DPP 3 antibody and recombinant human DPP 3 and compared the immunostaining by Western blot of DPP 3 and the purified Ang-(1-7) peptidase. As shown in Figure 1B, the immunoblot reveals an identical band at 80 kDa for human DPP 3 (lanes 1-2) and the purified Ang-(1-7) peptidase (lanes 3-4) from the
HK-2 cells. Although not shown, fractionation of the Ang-(1-7) peptidase by a non- reducing or native PAGE gel also revealed a single 80 kDa band. This band was excised from the native gel, incubated overnight in the peptidase assay buffer and the supernatant exhibited significant peptidase activity (Figure 1C).
We then compared the hydrolysis of unlabeled Ang-(1-7) by the purified Ang-(1-7) peptidase. Separation of peptide standards is shown in Figure 2A and the products of
Ang-(1-7) metabolism are shown in Figure 2B. For the unlabeled peptides, we utilized the HFBA solvent system as the counter-ion since the phosphoric system does not distinguish Ang-(1-7) and Ang-(3-7). Figure 3C is the metabolism of 125I-Ang-(1-7) by the purified Ang-(1-7) peptidase that reveals the metabolites 125I-Ang-(3-4) and 125I-Ang-
(3-7). A similar profile of metabolism for 125I-Ang-(1-7) by rH-DPP 3 is revealed by the chromatograph in Figure 2D.
We then compared the sensitivity of the JMV-3909 inhibitor to attenuate 125I-Ang-(1-
7) metabolism by the purified Ang-(1-7) peptidase and rH-DPP 3. As shown in the chromatographs, both the Ang-(1-7) peptidase and rH-DPP 3 cleaved 125I-Ang-(1-7) to
125I-Ang-(3-7) and 125I-Ang-(3-4) (Figure 3A and 3D).
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Figure 2. Hydrolysis of Ang-(1-7) by the purified Ang-(1-7) peptidase and recombinant DPP 3. Panel A: Chromatograph of Ang-(5-7) (A5-7), Ang-(3-7) (A3-7) and Ang-(1-7) standards (3 nmols each) by HPLC-UV detected at 220 nM. Panel B: Purified Ang-(1-7) peptidase from HK-2 cells was incubated with Ang-(1-7) (200 µM) and the metabolism detected by HPLC-UV using the HFBA solvent system (220 nM). Conversion of 125I-Ang-(1-7) (A7) to 125I-Ang-(3-4) and 125I-Ang-(3-7) by purified Ang-(1- 7) peptidase (C) and recombinant DPP 3 (D) by HPLC using the phosphoric acid solvent system.
94
Figure 3. Dose response curves for JMV-390 inhibition of 125I-Ang-(1-7) hydrolysis to 125I-Ang-(3-4) by the purified Ang-(1-7) peptidase (A7-P) and recombinant DPP 3. Chromatographs show A7-P and DPP 3 hydrolysis of 125I-Ang-(1-7) alone (A and D) or with 0.5 nM JMV-390 (B and E) or with 10 nM JMV-390 (C and F). The IC50 for Ang-(1-
7) peptidase (A7-P) was of 0.2 nM (-ο-) and the IC50 for DPP 3 was of 1.4 nM (-●-) (G).
IC50 values were derived from 3 separate experiments using Prism 6.
95
Addition of increasing concentrations of JMV-390 attenuated the metabolism of 125I-Ang-
(1-7) by the Ang-(1-7) peptidase (Figure 3B and 3C) and by rH-DPP 3 (Figure 3E and
125 3F). These competition studies for I-Ang-(1-7) hydrolysis revealed an IC50 of 0.18 ± nM for the Ang-(1-7) peptidase and 1.20 ± nM for rH-DPP 3 (Figure 3G). DPP 3 is a metallothiolpeptidase that typically cleaves two amino acid residues from the N-terminus of peptide substrates 4 to 8 residues in length. The predicted pattern of Ang-(1-7) metabolism by DPP 3 should be the immediate formation of the pentapeptide Ang-(3-7) and the subsequent formation of tripeptide Ang-(5-7); there should be no further metabolism as DPP 3 does not cleave peptides of 3 residues or less. However, based on the predicted metabolism, we could not explain why we failed to routinely observe a predominant 125I-Ang-(3-7) peak in the metabolism studies. Therefore, we performed a kinetic analysis of both 125I-Ang-(1-7) and 125I-Ang-(3-7) metabolism with rH-DPP 3.
For these studies, we used a fixed concentration of the 125I-substrate and increasing concentrations of the unlabeled substrate, as well as assumed that the peptidase equally recognizes both labeled and unlabeled peptides. As shown in Figure 4, the saturation curves revealed a higher Km value for Ang-(1-7) [12 µM] as compared to Ang-(3-7) [2
µM]. Moreover, the Vmax value for Ang-(3-7) was 3-fold higher than that for Ang-(1-7)
[14.8 vs 5.5 nmol/min/µg]. These data likely explain the absence of a substantial peak for 125I-Ang-(3-7) as the pentapeptide does not sufficiently accumulate once DPP 3 initially cleaves 125I-Ang-(1-7).
Finally, we addressed whether DPP 3 influences the cellular expression of Ang-(1-7) in the HK-2 cells by chronically treating the cells with JMV-390. Cells were placed in 1% serum-containing media for 24 hours and then treated with or without 20 nM JMV-390 for 72 hours; the inhibitor was replenished every 24 hours.
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Figure 4. Kinetics of the hydrolysis of 125I-Ang-(1-7) and 125I-Ang-(3-7) by human DPP 3. Kinetic values were determined by the conversion of either 125I-Ang-(1-7) or 125I- Ang-(3-7) to 125I-Ang-(3-4) in the presence of increasing concentrations of unlabeled peptide substrates. Kinetic constants for each peptide were derived from 3 separate determinations and analyzed by Prism 6 software.
97
The dose of JMV-390 is approximately 100-fold higher than that of the IC50 (0.2 nM) for the inhibition of the purified Ang-(1-7) peptidase. As shown in Figure 5A, 20 nM
JMV-390 significantly reduced the metabolism of 125I-Ang-(1-7) in the 100,000 x g cell cytosolic fraction. Basal expression of Ang-(1-7) in the HK-2 cells was 22 fmol/mg protein (Figure 5B). Following a 72 hour exposure to 20 nM JMV-390, the cellular levels of Ang-(1-7) increased 2-fold to 38 fmol/mg protein, but the increase did not achieve statistical significance (Figure 5B).
DISCUSSION
Abundant evidence strongly supports an alternative or non-classical Ang-(1-7) axis of the RAS in the circulation, kidney and other tissues that may buffer or antagonize the actions of the Ang II-AT1 receptor axis (2; 3; 6). Enzymes the form Ang-(1-7) directly from Ang I include the endopeptidases neprilysin, thimet oligopeptidase and prolyl oligopeptidase, while ACE2 and prolyl carboxypeptidase hydrolyze Ang II to Ang-(1-7)
(4; 16).
In regards to the mechanisms that metabolize Ang-(1-7), we identified ACE as a primary pathway in the circulation that degrades Ang-(1-7) (4). Indeed, circulating levels of Ang-(1-7) markedly increase following administration of ACE inhibitors that reflect the reduced metabolism of Ang-(1-7), as well as the shunting of Ang I away from Ang II and towards Ang-(1-7)-forming pathways (4). Although Marshall et al identified ACE activity in the sheep CSF that degrades Ang-(1-7), they identified another activity in the CSF that contributed to a greater extent in Ang-(1-7) metabolism (6; 11). Moreover, the Ang-
(1-7)-degrading activity independent from ACE was approximately 3-fold higher in sheep exposed to antenatal betamethasone, an experimental model of fetal programming associated with hypertension and reduced baroreflex sensitivity (6).
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Figure 5. Influence of JMV-390 on 125I- Ang-(1-7) metabolism and intracellular Ang- (1-7) content in HK-2 cells. Cells were treated with 20 nM or 200 nM JMV-390 for 72 hours. Panel A: Both doses of JMV-390 significantly reduced the metabolism of 125I- Ang-(1-7) to 125I-Ang-(3-4) in the 100,000 x g HK-2 cell cytosolic fraction. Metabolism data are mean ± SEM, n=3; *p<0.05. Panel B: The 20 nM dose of JMV-390 increased the intracellular content of Ang-(1-7) in the HK-2 cell extracts.
99
Wilson et al further characterized a similar if not identical activity that degraded Ang-
(1-7) in the sheep proximal tubules and the human HK-2 cell line (15). The present study isolated the Ang-(1-7) peptidase activity by multiple chromatographic steps and
SDS-PAGE from the HK-2 cells and identified the enzyme as DPP 3. DPP 3 is a soluble thiol metallopeptidase that is widely distributed in both central and peripheral tissues.
The enzyme cleaves 2 residues at a time from the N-terminus of various peptides with an optimal peptide length of 4 to 8 amino acids. The fact that DPP 3 is a thiol metallopeptidase explains the unusual sensitivity of the enzyme to the thiol inhibitors
PCMB and APSM, as well as the chelating agents o-phenanthroline and EDTA (9). We originally reported that the Ang-(1-7) peptidase exhibited a very low IC50 (<1nM) to the metallopeptidase inhibitor JMV-390, and the recombinant form of DPP 3 was also potently inhibited by the JMV compound (IC50 of ~1 nM).
Although we initially considered the Ang-(1-7) activity to be an endopeptidase, the present studies clearly confirm that the DPP 3 hydrolyzes Ang-(1-7) to the pentapeptide
Ang-(3-7) and then undergoes very rapid conversion to Ang-(5-7). Indeed, using 125I-
Ang-(1-7) as the peptide substrate, it is very difficult to detect the accumulation of 125I-
Ang-(3-7). Moreover, kinetic studies on the hydrolysis of Ang-(1-7) and Ang-(3-7) by
DPP 3 revealed that Ang-(3-7) was a superior substrate. The Vmax for the hydrolysis of
Ang-(3-7) by DPP 3 was 3-fold higher than Ang-(1-7) while the Km for Ang-(3-7) was 6- fold lower than that for Ang-(1-7); these kinetic data may explain the apparent absence of an Ang-(3-7) peak following the initial hydrolysis of Ang-(1-7). Moreover, the current data are also consistent with our previous study in which the fluorescently quenched
1 7 Ang-(1-7) analog [Abz-Asp , Tyr(NO2) ]-Ang-(1-7)] was resistant to the HK-2 peptidase
(15). Peptide substrates that contain a N-terminally blocked group are likely resistant to hydrolysis by DPP 3.
100
DPP 3 is primarily an intracellular peptidase, although we detected activity in the cell media of the HK-2 cells suggesting the possible secretion or release of the enzyme (15).
The HK-cells are reported to express a complete RAS including renin, angiotensinogen,
ACE and ACE2; therefore, we assessed whether chronic treatment of the HK-2 cells with the JMV-390 inhibitor would influence the intracellular levels of Ang-(1-7) (14). We find that 20 nM JMV-390 reduced the activity of DPP 3 by 50% in the cytosol of the HK-2 cells and tended to increase the cellular levels of Ang-(1-7) approximately 2-fold. It is possible that a 50% reduction in DPP 3 activity is not sufficient to significantly influence the intracellular metabolism of Ang-(1-7). However, higher concentrations of JMV-390 may spill over to block other endopeptidases such as thimet oligopeptidase that may contribute to Ang-(1-7) formation (15). Therefore, additional studies are necessary to determine whether more selective approaches to knockdown DPP 3 would yield a more robust increase in the cellular content of Ang-(1-7) that does not impact the formation of the peptide.
In conclusion, the present studies confirm that the Ang-(1-7) degrading activity in the
HK-2 proximal tubule cells is DPP 3 and that the peptidase activity present in the sheep
CSF, brain medulla and proximal tubules is also likely DPP 3. The extent that DPP 3 participates in the intracellular expression of Ang-(1-7) in central and peripheral compartments may reflect the co-localization of the peptidase and Ang-(1-7), as well as the presence of other competing peptide substrates for DPP 3 in these tissues. The intracellular pathways involved in the expression of angiotensin peptides are not clearly established; however, elucidation of the role of DPP 3 to regulate Ang-(1-7) may be important to establish the cellular actions of the alternative axis of the RAS.
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CONFLICT OF INTEREST
The authors declare that there are no competing financial interests in the work described.
ACKNOWLEDGEMENTS
Dr. Cruz-Diaz is supported by a fellowship from the PRIME Institutional Research and
Academic Career Development Award K12-GM102773. Mr. Wilson is supported by an
American Heart Association (AHA) predoctoral fellowship grant (15PRE25120007).
Additional support for this study was provided by NIH grant HD-; the Groskert Heart
Fund, the Wake Forest Venture Fund the Farley‐Hudson Foundation (Jacksonville, NC).
These studies constitute the partial fulfillment of the requirements for the degree of
Doctorate of Philosophy in the Department of Molecular Medicine and Translational
Sciences at Wake Forest University School of Medicine for Mr. Wilson. Portions of this work were originally presented at the 2016 Experimental Biology meeting in San Diego
CA.
102
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14. Shalamanova L, Wilkinson MC, McArdle F, Jackson MJ and Rustom R. Characterisation of the expression of the Renin-Angiotensin system in primary and immortalised human renal proximal tubular cells. Nephron Exp Nephrol 116: e53- e61, 2010.
15. Wilson BA, Cruz-Diaz N, Marshall AC, Pirro NT, Su Y, Gwathmey TM, Rose JC and Chappell MC. An angiotensin-(1-7) peptidase in the kidney cortex, proximal tubules, and human HK-2 epithelial cells that is distinct from insulin-degrading enzyme. Am J Physiol Renal Physiol 308: F594-F601, 2015.
16. Wilson BA, Marshall AC, Alzayadneh EM and Chappell MC. The ins and outs of angiotensin processing within the kidney. Am J Physiol Regul Integr Comp Physiol 307: R487-R489, 2014.
17. Wilson BA, Nautiyal M, Gwathmey TM, Rose JC and Chappell MC. Evidence for a Mitochondrial Angiotensin-(1-7) System in the Kidney. Am J Physiol Renal Physiol ajprenal, 2015.
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CHAPTER 5
Evidence for a Mitochondrial Angiotensin-(1-7) System in the Kidney
Bryan A. Wilson1, Manisha Nautiyal3, TanYa M. Gwathmey1, James C. Rose2, and Mark C. Chappell1
1Hypertension & Vascular Research Center, 2Department of Obstetrics and Gynecology, Wake Forest University School of Medicine, Winston-Salem, NC, 3Division of Endocrinology, Diabetes & Metabolism, University of Florida, Gainesville, FL USA.
This paper is published online in American Journal of Physiology - Renal Physiology, 310: F637–F645, 2016, and represents the efforts of the first author. Differences in organization and formatting may reflect requirements of the journal.
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ABSTRACT
Evidence for an intracellular renin-angiotensin system (RAS) in various cell organelles now includes the endoplasmic reticulum, nucleus and mitochondria (Mito). Indeed, angiotensin (Ang) AT1 and AT2 receptor subtypes were functionally linked to Mito respiration and nitric oxide production, respectively in previous studies. We undertook a biochemical analysis of the Mito RAS from male and female sheep kidney cortex. Mito - were isolated by differential centrifugation followed by a discontinuous Percoll gradient and were co-enriched in Mito membrane markers VDAC and ATP synthase, but not β- actin or cathepsin B. Two distinct renin antibodies identified a 37 kDa protein band in
Mito; angiotensinogen (Aogen) conversion was abolished by the inhibitor aliskerin. Mito
Aogen was detected by an Aogen antibody to an internal sequence of the protein, but not with an antibody directed against the Ang I N-terminus. Angiotensin peptides were quantified by three direct RIAs; mitochondrial Ang II and Ang-(1-7) contents were higher compared to Ang I [23 ± 8 and 58 ± 17 vs. 2 ± 1 fmol/mg protein; p<0.01, n=3]. 125I-Ang
I metabolism primarily revealed the formation of 125I-Ang-(1-7) in Mito that reflects the endopeptidases neprilysin and thimet oligopeptidase. Lastly, immunoblot studies utilizing the Ang-(1–7)/Mas receptor antibody revealed the protein in isolated Mito from sheep renal cortex. Collectively, the current data demonstrate that Mito actively metabolize the
RAS precursor protein Aogen, indicating that RAS peptides Ang II and Ang-(1-7) may be generated within Mito to establish an intra-mitochondrial RAS tone and contribute to renal mitochondrial function.
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INTRODUCTION
The renin-angiotensin system (RAS) plays a pivotal role in regulating cardiovascular and renal function. Historically, the RAS has been characterized as a circulating system that contributes to the regulation of blood pressure and fluid homeostasis. In a paracrine manner, the RAS influences local tissue systems including the brain, adrenal cortex, heart and kidney. These organ systems were subsequently found to contain a functional RAS capable of responding in an autocrine fashion. Indeed, there is compelling evidence for functional actions of intracellular renin-angiotensin systems in various cell organelles including the endoplasmic reticulum, nucleus and the mitochondria within various tissues (1, 2, 4, 5, 25, 26, 42).
Mitochondria are the major energy producing organelles within a cellular system.
These organelles are a primary source of reactive oxygen/nitrogen species and may regulate intracellular redox status and influence cellular signaling. Mitochondrial function and oxidant production are altered in cardiovascular pathologies that may reflect, in part, a role of the Ang II axis of the RAS. Ang II, a potent vasoconstrictor and inflammatory peptide hormone, stimulates the production of mitochondrial reactive oxygen species
(ROS) (15, 29). In this regard, recent studies demonstrate an intra-mitochondrial RAS may exist within a variety of tissue types (1, 5). Abadir et al demonstrated that Ang II type 2 (AT2) receptors are expressed on the inner mitochondrial membrane and are coupled to nitric oxide (NO) production in liver mitochondria (1). Moreover, an increased ratio of mitochondrial Ang II type 1 (AT1) to AT2 receptors was associated with aging; chronic treatment of the aged mice with the AT1 receptor antagonist losartan abrogated the higher AT1 receptor expression (1). These results suggest that one target of chronic
RAS blockade to blunt the effects of cellular aging is the mitochondria. Indeed, Ferder,
Inversa and colleagues demonstrate that either angiotensin converting enzyme (ACE)
107 inhibition or angiotensin receptor blockers (ARBs) attenuated the decline in mitochondrial function (11-13). However, a more recent study has raised concerns on the biochemical evidence for an intra-mitochondrial RAS (5). Astin et al failed to detect
RAS components in purified liver mitochondria and reported marginal inhibition of mitochondrial respiration by Ang II at a supraphysiological dose (1 µM), suggesting a non-specific rather than a direct effect of Ang II (5).
Since the Abadir and Astin studies focused primarily on the Ang II-AT1/AT2 receptor axis in rodents, we undertook a biochemical analysis of the mitochondrial RAS that examined the Ang-(1-7) axis. The current study provides evidence for several RAS components including angiotensinogen, active renin, Ang I and the bioactive peptides
Ang II and Ang-(1-7) in purified mitochondria from the sheep renal cortex. Moreover, we demonstrate that purified mitochondria process Ang I to Ang-(1-7) by the endopeptidases neprilysin and thimet oligopeptidase, suggesting the possibility for an intra-mitochondrial RAS pathway that forms Ang-(1-7).
METHODS
Animals
Mixed breed sheep (obtained from a private local vendor) were delivered at term, farm- raised, and weaned at 3 months of age. Adult male and female sheep (10-12 months of age) were anesthetized with ketamine and isoflurane, and euthanized by exsanguination. Kidneys were removed immediately and renal cortex was dissected out on ice for immediate isolation of mitochondria. Cortical tissue or isolated mitochondria were stored at -80°C. All procedures in the current study were approved by the Wake
Forest University School of Medicine IACUC for animal care.
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Isolation of Sheep Renal Cortex Mitochondria
Mitochondria were isolated from fresh sheep renal cortex by a discontinuous Percoll gradient, as described previously (20, 39, 46). Tissue was homogenized in mitochondrial homogenization buffer [75mM Sucrose, 225 mM Mannitol, 10 mM Na+ Free HEPES and
0.1 mM EDTA, pH 7.4 (KOH only)] using a Polytron Ultra-Turrax T25 Basic (setting 5), followed by a 50-ml all-glass Dounce homogenizer (Kontes Glass, Vineland, NJ). The homogenate was centrifuged for 5 min at 1,000 g, and then the supernatant was collected and filtered through a 70 µm metal strainer and centrifuged for 10 min at
10,000 g. The resulting pellet, representing the crude mitochondrial fraction was resuspended in equal amounts of 12% Percoll (GE Healthcare, Buckinghamshire, U.K.) in Percoll buffer [250mM Sucrose, 0.4mM EDTA and 10mM Na+ Free HEPES]. This suspension was layered onto a discontinuous Percoll gradient (24% and 40% in Percoll
Buffer). The gradient was centrifuged for 5 min at 15,000 g, and the layer between the
24% and 40% Percoll suspensions containing the purified mitochondria was collected.
The preparation was washed in Percoll buffer and centrifuged for 10 min at 15,000 g.
Purity of the isolated mitochondria was determined by immunoblot analysis (Figure 1A-
1C). Antibodies were obtained from the following sources: voltage-dependent anion channel (VDAC, 1:1000, Cell Signaling), an outer mitochondrial membrane marker and several inner mitochondrial markers of the electron transport chain: ATP synthase - complex V (CV-ATP5A), ubiquinol-cytochrome c reductase core protein II – complex III
(CIII-UCRC2) and succinate dehydrogenase complex subunit B – complex II (CII-SDHB) obtained from Mito Sciences (1:1000, Total OXPHOS Antibody Cocktail). A Cathepsin B antibody was utilized as a marker for lysosomes and renin granules (50) (1:400, Abcam ab58802). Membranes were probed with mouse monoclonal anti-β-actin (Sigma, St.
Louis, Missouri, USA, 1:5000) antibody to assess the degree of purity.
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Immunoblot Analysis of RAS Components
Purified mitochondrial homogenates (~100 μg) were boiled in PBS (pH 7.4), diluted in Laemmli buffer with β-mercaptoethanol, separated on 10% Sodium dodecyl sulfate (SDS) polyacrylamide gels for 1 hour at 120V in Tris-glycine SDS and transferred to a polyvinylidene difluoride (PVDF) membrane. Blots were blocked with 5% Bio-Rad
Dry Milk (Bio-Rad, Hercules, CA, USA) and Tris buffered saline (TBS) with Tween
(0.05%) and probed overnight at 4°C with primary antibodies against the following: rat angiotensinogen (internal sequence of angiotensinogen (Total- angiotensinogen), residues 42–57, 1:1000), Ang I sequence of angiotensinogen (Ang I- angiotensinogen, residues 25–34, 1:1000), renin (1:3000, Inagami antibody no. 826), renin (1:100, Aviva), prorenin receptor (ATP6IP2, 1:500, Abcam), neprilysin (NEP, 1:2000, Cell Signaling), thimet oligopeptidase (TOP, 1:800, Epitomics) and the Ang-(1-7)/Mas receptor protein
(MAS, 1:250, Alomone AAR-013). Membranes were treated with horseradish peroxidase
(HRP)-labeled polyclonal anti-rabbit secondary antibodies (1:5000) for 1 hour and detected with ECL chemiluminescent substrates (Advansta, Menlo Park CA, USA).
Renin Assay
Purified mitochondria were isolated as described above and stored at −80°C.
The mitochondrial pellet was reconstituted in 1 ml of metabolism buffer (25 mM HEPES,
125 mM NaCl, 10µM ZnCl2) at pH 7.4, sonicated and left for 60 min on ice. A sample from the homogenate was taken for protein content. For basal renin activity assay, 400
µg of mitochondrial homogenate was added to 10 µl of nephrectomized sheep plasma
(NSP) as the source of exogenous angiotensinogen substrate. The assay was performed at 37ºC for 4 hours in the presence or absence of the renin inhibitor aliskiren
(1 μM, final concentration). Aliquots were removed at 30 min, 1, 2 and 4 hours and
110 analyzed by Western blot using the Ang I-angiotensinogen antibody. Activation of prorenin was performed by addition of 5 mg/ml trypsin (Sigma) to the mitochondrial sample for 60 min on ice. Trypsin was inactivated by incubation with an excess of soybean trypsin inhibitor (SBTI; 1 mg/ml, Sigma) for 15 min at room temperature. SBTI is a potent serine protease inhibitor that does not attenuate renin, an aspartyl protease.
Following SBTI, the renin assay was performed as described above.
Angiotensin Peptides
Sheep renal cortex mitochondria were isolated as described above and stored at
−80°C. The mitochondrial pellet was reconstituted in Milli-Q water and placed in a boiling water bath for 15 min. The mitochondrial fraction was acidified with heptafluorobutyric acid (HFBA) to a final concentration of 0.1%, sonicated and centrifuged at 20,000 g for
20 min at 4°C. The resultant supernatant was applied to an activated Sep-Pak C18 extraction column, washed with 0.1% HFBA, and the peptide fraction eluted with 3 ml of
100% methanol in 0.1% HFBA. Measurement of immunoreactive Ang I, Ang II and Ang-
(1-7) in the extracted mitochondria was performed using three distinct radioimmunoassays (RIAs) (4). The Ang-(1-7) RIA fully recognizes Ang-(1-7) and Ang-
(2-7), but cross-reacts less than 0.01% with Ang-(3–7), Ang II, Ang I, and their fragments. The Ang II RIA equally recognizes Ang III, Ang-(3-8), and Ang-(4-8), but cross-reacts less than 0.01% with Ang I and Ang-(1-7). The limit of detection was 1 fmol/tube for Ang I, 0.5 fmol/ tube for Ang II and 4 fmol/ tube for Ang-(1-7) (4, 35, 36).
125I-Ang I Metabolism
125I-Ang I metabolism was measured at 37°C in the metabolism buffer using the mitochondrial lysate (50 µg) in a final volume of 250 μl. Each reaction contained a final concentration of 0.5 nM 125I-Ang I and 100 nM Ang I with or without the inhibitors N-[N-
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[1-(S)-carboxyl-3-phenylpropyl]-(S)-phenyl-alanyl]-(S)-isoserine (SCH, 10 µM) and SCH
(10 µM)/ N-[1-(R,S)-carboxy-3-phenylpropyl]-Ala-Ala-Phe-p-aminobenzoate (CPP, 10
μM). The reactions were stopped after 60 min by addition of ice-cold 1.0% phosphoric acid and centrifuged at 16,000 g. The supernatants were immediately filtered, and the products were detected by HPLC-γ detection as previously described (34, 53).
Preliminary studies also assessed 125I-Ang II metabolism using an identical approach as that for the mitochondrial metabolism of Ang I.
Statistical analysis
All measurements are expressed as mean ± standard error (SEM). Differences between the groups were analyzed by one-way analysis of variance (ANOVA) and Newman-
Keuls multiple comparison analysis. Statistical analyses were performed and figures constructed with GraphPad Prism V (GraphPad Software, San Diego, California, USA).
A probability value of <0.05 was required for statistical significance.
RESULTS
Angiotensinogen and Renin
We verified mitochondrial enrichment and purity utilizing immunoreactive probes against proteins to outer mitochondrial (VDAC) and inner mitochondrial (ATP5A, UCRC2 and SDHB) membranes. Immunoreactivity for VDAC (Figure 1A), ATP5A, UCRC2 and
SDHB (Figure 1B) was enriched as mitochondria were purified through differential centrifugation and a discontinuous Percoll gradient. In contrast, expression of the lysosomal and renin granule marker Cathepsin B (Figure 1C) in addition to the cellular protein β-actin was absent in the final Percoll fraction (Figures 1A-1C). Based on the mitochondrial purity obtained by the Percoll gradient, this preparation was utilized in subsequent experiments to characterize the RAS components.
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Figure 1: Purification and enrichment of mitochondria from sheep renal cortex. Whole renal cortex homogenate (lane 1), crude mitochondrial fraction (lane 2) and Percoll-purified mitochondrial fraction (lane 3) were subjected to 10% SDS gel and immunoblotting with anti-VDAC (A – 50 ug total protein) as well as ATP5A, UQCRC2, SDHB (B – 5 ug total protein) and Cathepsin B (C – 50 µg total protein), as well as β- actin. Purified mitochondria exhibited greater expression of outer mitochondria marker (VDAC) and inner mitochondrial markers (ATP5A, UQCRC2 and SDHB), while Cathepsin B and β-actin expression were diminished.
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We initially assessed renin and the pro-renin receptor expression in three mitochondrial homogenates and a renal cortex homogenate. Two distinct protein bands of 37 kDa and 55 kDa were identified utilizing both the AVIVA renin antibody (Figure 2A) and the Inagami renin antibody (Figure 2B). Despite the presence of renin, there was no immunological evidence of the prorenin receptor (ATP6IP2) using the Abcam antibody in three separate mitochondrial preparations, although we noted a protein band of 35 kDa in the cortical homogenate (Figure 2C). We then assessed renin activity in isolated mitochondria from male (Figure 3 A,C,E,G) and female (Figure 3 B,D,F,H) sheep by measuring the disappearance of intact angiotensinogen from NSP in the absence or presence of the renin inhibitor aliskiren (Figure 3). The renin assay was facilitated by the specificity of the Ang I-angiotensinogen antibody to recognize the Ang I sequence of angiotensinogen. As shown in the immunoblot in Figure 3 (A-D), renin activity in isolated mitochondria was evident by the time-dependent disappearance of intact angiotensinogen at 37ºC and that Ang I-angiotensinogen processing was abolished by aliskiren (Figure 3 G-H). We did not attempt to quantify renin activity between the male and female mitochondria isolated from the renal cortex. To determine prorenin levels, mitochondria were pretreated with trypsin to process prorenin to active renin and angiotensinogen processing was determined in the presence of the trypsin inhibitor
SBTI. Trypsin activation of mitochondrial prorenin did not appear to markedly increase renin activity as the disappearance rate of Ang I-angiotensinogen was comparable for the trypsin and non-trypsin treated mitochondria samples in males and females (Figures
3 C-D and E-F).
The protein precursor angiotensinogen was assessed by immunoblot using two distinct antibodies – a “total” angiotensinogen antibody that targeted an internal amino acid sequence distal to Ang I and the Ang I-angiotensinogen antibody directed against
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Figure 2: Renin and prorenin receptor expression in isolated mitochondria from sheep renal cortex. (A) Immunoblot of rat renal proximal tubular (NRK-52E) cells (lane 2) and sheep renal mitochondria preparations (lanes 3-5) with the Inagami renin antibody revealed two distinct protein bands (37 kDa and 55 kDa). Lane 3-4 represent female mitochondrial preparations while Lane 5 represents a male preparation. (B) Similarly, immunoblot of renal cortex homogenate (lane 2) and sheep renal mitochondria preparations from three male sheep (lanes 3-5) with the Aviva renin antibody revealed two distinct protein bands (37 kDa and 55 kDa). (C) Immunoblot of prorenin receptor (ATP6IP2) revealed a predominant protein band at 35 kDa in renal cortex homogenate (lane 2), but not the mitochondrial preparations from three male control sheep (lane 3-5). Lane 1 (A-C) represents the protein molecular weight marker.
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Figure 3: Renin activity in the mitochondria. Renin activity in the mitochondria of a male (A,C,E,G) and female (B,D,F,H) sheep. Non-trypsin (A-B) or trypsin (C-D) treated mitochondrial homogenates with (+) or without (-) the renin inhibitor aliskiren was detected by immunoblot analysis using an AI-Aogen directed antibody. Relative quantification of the Ang I-Aogen band without (E-F) or containing the renin inhibitor aliskiren (G-H) was performed. The disappearance or half-life (t½) of AI-Aogen was determined from single experiments.
116 the Ang I sequence (4). Thus, the Ang I-angiotensinogen antibody detects only intact
Ang I-containing angiotensinogen, and the total angiotensinogen antibody detects both intact and des-Ang I angiotensinogen (i.e., renin-processed angiotensinogen). The total angiotensinogen antibody recognized a 55 kDa protein band in three different male mitochondrial preparations and in nephrectomized sheep plasma (NSP); however, the
Ang I-angiotensinogen antibody failed to detect a protein band in the mitochondrial homogenates (Figure 4A). The absence of Ang I-angiotensinogen may suggest active processing of the precursor, therefore, we determined the expression of angiotensin peptides. Mitochondrial Ang I, Ang II and Ang-(1-7) content was then quantified using three direct RIAs targeted to each peptide in isolated mitochondria from three separate preparations (Figure 4B). The mitochondrial content of Ang II and Ang-(1-7) were both significantly higher than Ang I [23 ± 8 and 58 ± 17 vs. 2 ± 1 fmol/mg protein; p<0.01, n=3].
Angiotensin I Processing
We examined the potential processing pathway of Ang I in isolated mitochondrial homogenates from sheep renal cortex using the 125I-labeled peptide as substrate. As shown in the chromatographs for Figure 5, 125I-Ang I was metabolized primarily to 125I-
Ang-(1-7). Addition of the neprilysin inhibitor SCH reduced the 125I-Ang-(1-7) peak
(Figure 5B). The co-addition of SCH and the thimet oligopeptidase inhibitor CPP further reduced the 125I-Ang-(1-7) peak and preserved the 125I-Ang I peak (Figure 5C).
Immunoblot analysis of the 100,000 x g mitochondrial membranes revealed NEP expression while the corresponding 100,000 x g soluble fraction expressed thimet oligopeptidase (Figure 5D). Quantification of Ang I metabolism revealed that the neprilysin inhibitor significantly increased Ang I and reduced Ang-(1-7) (Figure 5E-F, respectively). The addition of the thimet oligopeptidase inhibitor further reduced Ang-(1-
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Figure 4: Angiotensinogen (Aogen) and angiotensin peptide expression in isolated mitochondria from sheep renal cortex. (A) Immunoblot of Aogen in nephrectomized sheep plasma (NSP, lane 1) and isolated mitochondria from the renal cortex of three male sheep (lanes 2-4). A protein band of 55 kDa was detected for total Aogen in all lanes (upper blot), but AI-Aogen was not detected in the mitochondria preparations (lower blot) (B) Angiotensin peptide expression was quantified in isolated mitochondria from sheep renal cortex using distinct radioimmunoassays. Ang I, Ang II and Ang-(1-7) were detected; however there was a significant difference between the levels of Ang I compared to Ang II and Ang-(1-7). Data are mean ± SEM, n = 3; *p < 0.01 (Ang I vs. Ang II); (Ang I vs. Ang-(1-7)).
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Figure 5: Mitochondrial processing of 125I-Ang I. Chromatograph reveals 125I-Ang I [125I-AI] was metabolized to 125I-Ang-(1-7) [125I-A7] in renal mitochondrial homogenate fractions (A). 125I-AI metabolism to 125I-A7 was reduced by 10 µM SCH (B), and 125I-AI metabolism to 125I-A7 was reduced by 10 µM SCH in combination with 10 µM CPP (C). Immunoblot analysis revealed evidence for protein expression of neprilysin (NEP) and thimet oligopeptidase (TOP) in mitochondrial homogenates (lane 2-4, n=3) along with renal cortex homogenate as a positive control (lane 1) (D). 125I-labelled products were separated by HPLC under gradient conditions. Statistical quantification of 125I-AI metabolism demonstrated SCH (10 µM) and SCH in combination with CPP (10 µM) preserved 125I-Ang I, while SCH (10 µM) and CPP (10 µM) reduced 125I-Ang-(1-7) formation (E-F). The addition of SCH and CPP did not significantly further influence 125I- Ang-(1-7) formation or preservation of 125I-Ang I (E-F). Data are mean ± SEM, n = 3; *p < 0.05 vs. control (CON).
119
7), but this did not attain significance in comparison to the effect of the neprilysin inhibitor alone. We did not observe Ang I to Ang II processing in the mitochondrial preparation with or without the endopeptidase inhibitors (Figure 5A-C).
Expression of the Mas receptor
In lieu of the mitochondrial content of Ang-(1-7), we further assessed whether these organelles express the protein for the Ang-(1-7)/Mas receptor. For these studies, we utilized the Alomone antibody against the Mas receptor. We previously demonstrated that the antibody identified the receptor protein within the cortical tubules of the sheep kidney by immunofluorescent staining (26). Using a new batch of the antibody, the immunoblot revealed a single band of 40 kDa in the cortical homogenate, crude mitochondrial pellet and the purified Percoll fraction from the sheep renal cortex
(Figure 6A). A single immunoreactive band for the Mas protein was also evident in the
Percoll mitochondrial fraction from three male sheep (Figure 6B).
DISCUSSION
Compelling evidence strongly supports an intracellularly-based RAS in the kidney that contributes to renal function, as well as the regulation of oxidative stress (3, 25, 26,
29, 42). Mitochondria are now considered a key organelle regarding their role in chronic cardiovascular diseases and the mechanisms that underlie hypertension and renal dysfunction (10, 14, 16, 38, 45). Mitochondria are one of several intracellular organelles that contain elements of a RAS and Ang II may directly influence oxidative stress and mitochondrial respiration (1, 5, 21). The present study provides further support for an intra-mitochondrial RAS pathway that is distinct from the peripheral RAS. Although previous reports have indicated associations between RAS components and mitochondrial function (1, 5, 21, 45, 47), the biochemical approach in this study allowed
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Figure 6: Immunodetection of Mas receptor expression in sheep renal mitochondria. (A) Whole renal cortex homogenate (lane 2), crude mitochondrial fraction (lane 2) and Percoll-purified mitochondrial fraction (lane 3) were subjected to 10% SDS gel and immunoblotting with an antibody against the MAS receptor protein. (B) Whole renal cortex homogenate and purified mitochondria from three distinct male sheep exhibited protein expression of the MAS receptor. Lane 1 (A-B) represents the protein molecular weight marker.
121 for a more complete characterization of a mitochondrial RAS regarding the alternative
Ang-(1-7) axis. Our studies revealed that mitochondrial renin was active and cleaved exogenous angiotensinogen. Mitochondria expressed the des-Ang I form of angiotensinogen, but not intact angiotensinogen, suggesting that renin may process endogenous angiotensinogen within the mitochondria. We also show by direct RIA the presence of the angiotensin peptides Ang I, Ang II and Ang-(1-7) within mitochondria.
Moreover, the mitochondrial preparation processed Ang I to Ang-(1-7) by the endopeptidases neprilysin and thimet oligopeptidase. Finally, the current study revealed that purified mitochondria expressed a single protein band for the AT7/Mas receptor.
Peters and colleagues originally identified an alternative transcript of renin that lacked exon 1 which precluded entry of the transcribed protein into the secretory pathway; this isoform was predicted to reside within the cell (44). These investigators subsequently showed that the truncated form of renin was catalytically active and preferentially localized to the mitochondria (9). Overexpression of this renin isoform in a human cardiomyocyte cell line was recently shown to protect the cells from necrosis in response to reduced glucose conditions; this protective effect was not reversed by a renin inhibitor suggesting a direct effect of renin rather than renin-dependent processing of angiotensinogen (52). Clausmeyer et al reported that truncated renin was actively taken up and internalized by isolated mitochondria, but that the full length pro-renin form did not undergo internalization (9). Ishigami et al have recently demonstrated an additional renin transcript that lacks a portion of the pre-pro segment such that the enzyme is constitutively active; the isoform constitutes ~10% to total renin and was expressed predominantly along the tubular elements of the mouse kidney (27). The forced overexpression of this renin isoform was associated with higher blood pressure in the transgenic mice (27). Although the Ishigami study found no change in circulating
122 renin suggesting that the renin isoform is not secreted, the intracellular localization of the renin isoform within the tubules was not established (27). It is not currently known whether different renin transcripts are expressed within the sheep kidney or the extent that renin isoforms traffic to the mitochondria.
The renal cortex consists of multiple cell populations, and although tubules constitute the major cell type, we cannot definitively exclude juxtaglomerular or other cell contamination. Juxtaglomerular cells are the major source of renin and contain both pro- renin and active forms of renin (28, 37). Renin-containing granules sediment at a higher density than purified mitochondria; however, the possibility of renin contamination from granules is plausible and isolated mitochondria were probed with a cathepsin B antibody. Renin and cathepsin B coexist within renin granules, and cathepsin B is a major component of lysosomes (50). Both cathepsin B and β-actin protein expression were diminished as mitochondria were purified from sheep renal cortex suggesting a negligible contamination of renin granules or lysosomes in the mitochondrial preparation.
Moreover, trypsin-treatment did not result in a marked increase in renin activity. The inability of trypsin to enhance renin activity further argues for the lack of renin granule contamination in the isolated mitochondria. We found also no immunological evidence for the prorenin receptor in mitochondria which is consistent with predominant intracellular localization of this receptor on the endoplasmic reticulum, as well as the plasma membrane (4, 26, 27, 52).
Evidence of catalytically active renin in the mitochondria would support the predominant des-Ang I form of angiotensinogen in this organelle. Peters et al originally identified angiotensinogen by electron microscopy (EM) in mitochondria from the adrenal cortex along with other RAS components (44). Moreover, our preliminary data suggest that angiotensinogen is internalized by isolated mitochondria from both the sheep renal
123 cortex and human HK-2 proximal tubule cells (54). Evidence for the angiotensin peptides Ang I and Ang-(1-7) within the mitochondrial may also reflect the renin- dependent processing of angiotensinogen. Ang I metabolism studies revealed the generation of Ang-(1-7) that was attributed to the endopeptidases neprilysin (EC
3.4.24.11) and thimet oligopeptidase (EC 3.4.24.15). Neprilysin contributes to the generation of circulating Ang-(1-7), particularly following ACE inhibition; however, to our knowledge, these are the first studies to indicate neprilysin activity within the renal mitochondria (4, 19, 40, 53). Although current understanding of a mitochondrial role for neprilysin is limited, there is evidence for the peptidase in mitochondrial-mediated amyloid β degradation (33). Transgenic mice engineered to produce amyloid β that contained the human mitochondria-targeted antioxidant catalase were found to upregulate neprilysin expression that was associated with reduced amyloid β and oxidative DNA damage (33). A role for thimet oligopeptidase in the intracellular processing of Ang I to Ang-(1-7) was evident in rat vascular smooth muscle cells, the
NRK-52E renal epithelial cell line and rat brain (4, 7, 43). The enzyme is primarily an intracellular metallopeptidase of the M3 family of metallopeptidases and hydrolyzes peptides with an optimal length of 9-17 amino acids. Thimet oligopeptidase activity was originally localized to the mitochondria from rat liver; however, Barrett and colleagues subsequently identified the mitochondrial peptidase activity to be neurolysin (EC
3.4.24.16), a metallopeptidase that shares 60% homology with thimet oligopeptidase and is sensitive to higher concentrations of the CPP inhibitor (30, 31). Although our immunoblot studies identified a single protein band for thimet oligopeptidase in purified mitochondria, additional studies may be required to definitively confirm that the sheep renal mitochondrial activity is indeed thimet oligopeptidase or its homolog neurolysin.
We note that both neprilysin and thimet oligopeptidase are ubiquitous peptidases that are found in the kidney of various species (8). Finally, Ang II metabolism studies did not
124 reveal conversion to Ang-(1-7) in the isolated mitochondria and this would further support an ACE2/Ang II-independent pathway for Ang-(1-7) formation (Wilson and
Chappell, unpublished data).
The biochemical detection of Ang II corroborates several studies that have localized Ang II to the mitochondrial by immuno-gold EM (1, 18, 44). We did not detect
ACE activity in isolated mitochondria from the renal cortex consistent with the findings of
Astin et al in liver mitochondria (5). Moreover, we found no evidence of other Ang II- forming activities (cathepsin G, elastase-2, chymase) from Ang I in the absence or presence of the combined neprilysin and thimet oligopeptidase inhibitors that abolished
Ang-(1-7) formation, and these data do not support the conversion of Ang I to Ang II within mitochondria (8). However, Peters and colleagues detected immunoreactive ACE in mitochondria isolated from rat adrenal cortex and it is possible that species or tissue differences may underlie apparent discrepancies in the expression of RAS components in this organelle (44). Alternatively, both the Abadir and Astin studies reported AT1 receptors in isolated mitochondria and mitochondrial Ang II may derive from trafficking of the Ang II-AT1 receptor complex from the cell surface (1, 5). Early studies by Sirett et al described the highest density of Ang II binding sites on the purified mitochondrial fraction from rat brain (48). Moreover, Goodfriend and colleagues originally reported direct effects of Ang II on mitochondrial phosphorylation in isolated preparations (21). Danser and colleagues found that uptake of circulating 125I-Ang II by the kidney was also associated with the mitochondrial fraction (51), and Li et al recently demonstrated the trafficking of the Ang II-AT1 receptor complex to the mitochondria in proximal tubule cells
(30). We also identified the AT7/Mas receptor on purified mitochondria based on
Western blot analysis using the Alomone antibody and had previously demonstrated
Mas expression primarily on the tubular elements of the sheep kidney with the same
125 antibody (26). AT1, AT2 and Mas receptors do not express a canonical N-terminal mitochondrial targeting sequence, nor are these receptors predicted to localize to the mitochondria based on subcellular localization prediction software (TargetP 1.1 Server)
(17). Moreover, to our knowledge, there is no evidence that mammalian mitochondrial
DNA genes code for these receptors based on the MitoCarta inventory (6). However,
AT1, AT2 and Mas receptor subtypes are evident on other intracellular organelle including the nucleus and endoplasmic reticulum; thus, there may exist alternative mechanisms for the intracellular expression of angiotensin receptors (1, 4, 22-24, 26, 31,
32, 41, 42, 47, 49).
The present study isolated mitochondria from the renal cortex, and although the tubular epithelial cells are the most predominant cell type in this tissue distinguished by a high density of mitochondria, we cannot definitely localize all the RAS elements to one particular cell type at this time. However, it should be noted that Peters et al localized multiple components of the RAS including immunoreactive renin, angiotensinogen, ACE and Ang II to the mitochondria of rat adrenal cortex (44). Moreover, we did not evaluate any functional properties of Ang-(1-7) or Ang II within the sheep mitochondria. The Ang-
(1-7)-AT7/Mas receptor axis may act as a physiological antagonist of Ang II that counter- balances the deleterious effects of Ang II-mediated intracellular signaling (7, 38). In this regard, Ang-(1-7) attenuated mitochondrial ROS production and cellular apoptosis induced by Ang II in renal NRK-52E epithelial cells (29). Since the Ang-(1-7)-AT7/Mas receptor axis is typically linked to NO generation in the kidney and other tissues, investigation of this pathway to influence mitochondrial function under normal and pathological conditions may be warranted, particularly as Ang-(1-7) was recently shown to be more effective than the AT1 receptor antagonist to attenuate renal injury in a model of diabetic nephropathy (55).
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Perspectives and Significance
The RAS may influence kidney dysfunction and oxidative stress maintenance within a variety of intracellular organelles (2). In general, the Ang-(1-7)-AT7/Mas axis opposes or functionally antagonizes a stimulated ACE-Ang II-AT1R pathway. The renal actions of an intra-mitochondrial Ang-(1-7) system may encompass the release of NO, activation of anti-apoptotic pathways and/or the reduction of oxidative stress. Moreover, the loss of Ang-(1-7) tone within the kidney may accelerate deleterious mitochondrial pathways that increase oxidative stress and enhance apoptosis under pathological conditions. Therefore, the presence of a mitochondrial Ang-(1-7)-AT7/Mas axis within the kidney could constitute a novel therapeutic target to influence intracellular levels of oxidative stress and respiration; elucidation of the peptide’s functional role may identify specific intracellular targets to attenuate renal disease.
CONFLICT OF INTEREST
The authors declare that there are no competing financial interests in the work described.
ACKNOWLEDGEMENTS
This work represents partial fulfillment of the requirements for the degree of Doctorate of
Philosophy in the Department of Molecular Medicine and Translational Sciences at
Wake Forest University School of Medicine for Bryan A. Wilson. Mr. Wilson is supported by an American Heart Association (AHA) predoctoral fellowship grant
(15PRE25120007). Additional support for this study was provided by NIH grants HD-
047584, HD-017644, HD-084227, 0HL-51952, T32 grant (HL091797); the Groskert
Heart Fund, the Wake Forest Venture Fund and the Farley‐Hudson Foundation
(Jacksonville, NC). The authors gratefully acknowledge Eric LeSaine for his technical
127 and surgical support. Portions of this research were originally presented at the American
Heart Association’s Council on Hypertension 2015 Scientific Sessions meeting in
Washington, DC.
128
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CHAPTER 6
Angiotensinogen Import in Renal Proximal Tubules and Subsequent Trafficking to the Mitochondrial
Bryan A. Wilson1, Nildris Cruz-Diaz1, Yixin Su2, James C. Rose2, TanYa M. Gwathmey1, and Mark C. Chappell1
1Hypertension & Vascular Research Center, 2Department of Obstetrics and Gynecology, Wake Forest University School of Medicine, Winston-Salem, NC USA.
Submitted to American Journal of Physiology – Renal Physiology, April 2016
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ABSTRACT
Evidence for an intrarenal renin-angiotensin system (RAS) is well established. The renal proximal tubules, which constitute a key functional component of the kidney, express the
RAS precursor angiotensinogen (Aogen); however, it is unclear the extent that tubular
Aogen reflects local synthesis or internalization. Therefore, the current study established the extent that Aogen is internalized by ovine proximal tubules and the intracellular pattern of distribution. Proximal tubules were isolated from the kidney cortex of male sheep by enzymatic digestion and a discontinuous Percoll gradient. Tubules were incubated with 125I-Aogen for 2 hrs at 37°C in serum/phenol-free DMEM/F12 media.
Approximately 10% of exogenous 125I-Aogen was internalized by sheep tubules.
Subcellular fractionation revealed that 21 ± 4% of the internalized 125I-Aogen associated with the mitochondrial (Mito) fraction; additional labeling was evident in the nucleus [60 ±
7%], endoplasmic reticulum [4 ± 0.5%] and cytosol [15 ± 4%; n=4]. Subsequent studies determined whether Mito directly internalized 125I-Aogen using isolated Mito from renal cortex and human HK-2 cells. Sheep cortical Mito and the HK-2 Mito internalized 125I-
Aogen at a comparable rate of [33 ± 9 vs. 21 ± 10 pmol/min/mg protein; n=3]. Lastly, unlabeled Aogen (100 nM) competed for 125I-Aogen uptake to a greater extent than human albumin in HK-2 Mito [60 ± 2% vs. 16 ± 13%; n=3]. Collectively, our data demonstrate Aogen import and subsequent trafficking to the Mito in proximal tubules.
We conclude that this pathway constitutes a source of the Aogen precursor for the intracellular expression of angiotensin peptides in the tubules.
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INTRODUCTION
The renin-angiotensin system (RAS) plays a pivotal role to influence cardiovascular and renal function. Historically, the RAS has been characterized as a classic endocrine or circulating system that targets both peripheral and central receptors in the regulation of blood pressure and fluid homeostasis. Indeed, the RAS influences local tissue systems including the brain, pituitary, adrenal cortex, vasculature, heart and kidney. However, these organ systems were subsequently found to contain a functional
RAS capable of responding in an autocrine fashion. The proximal tubule epithelium of the renal cortex constitutes a functional system within the kidney and contains RAS components that are fully capable of regulating fluid and solute transport. Moreover, there is compelling evidence for functional actions of an intracellular RAS within the kidney including the endoplasmic reticulum, nucleus and the mitochondria (1-4, 15, 16,
26, 35).
Given the high energy demands of active reabsorption and secretion governed by this nephron component, proximal tubules are believed to contain a high density of mitochondria. Mitochondria are the major energy producing organelles within a cellular system. These organelles are a primary source of reactive oxygen/nitrogen species and may regulate intracellular redox status and influence cellular signaling. Mitochondrial function and oxidant production are altered in cardiovascular pathologies that may reflect, in part, a role of the angiotensin II (Ang II) axis of the RAS. Ang II, a potent vasoconstrictor and inflammatory peptide hormone, stimulates the production of mitochondrial reactive oxygen species (ROS) (11, 18). In this regard, recent studies demonstrate an intra-mitochondrial RAS within a variety of tissues (1, 4). Abadir et al demonstrated that Ang II type 2 (AT2) receptors are expressed on the inner mitochondrial membrane and are coupled to nitric oxide (NO) production in liver mitochondria (1). Moreover, an increased ratio of mitochondrial Ang II type 1 (AT1) to
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AT2 receptors was associated with aging, and chronic treatment of the aged mice with the AT1 receptor antagonist losartan abrogated the higher AT1 receptor expression (1).
These results suggest that one target of chronic RAS blockade to blunt the effects of cellular aging is the mitochondria. Indeed, Ferder, Inversa and colleagues demonstrate that either angiotensin converting enzyme (ACE) inhibition or angiotensin receptor blockers (ARBs) attenuated the decline in mitochondrial function (8-10). However, a more recent study has raised concerns on the biochemical evidence for an intra- mitochondrial RAS (4). Astin et al failed to detect RAS components in purified liver mitochondria and reported marginal inhibition of mitochondrial respiration by Ang II at a supraphysiological dose (1 µM), suggesting a non-specific rather than a direct effect of
Ang II (4).
In lieu of the intra-mitochondrial RAS studies demonstrated by the Abadir (1) and
Astin (4) groups who focused primarily on the Ang II-AT1/AT2 receptor axis in rodents, we also undertook a biochemical analysis of the mitochondrial RAS, with a focus on examining the Ang-(1-7) axis. The ACE2-Ang-(1-7)-AT7/Mas receptor axis generally opposes or functionally antagonizes a stimulated ACE-Ang II-AT1 receptor pathway.
Moreover, the renal mechanisms of an intra-mitochondrial Ang-(1-7) system may include activation of anti-apoptotic pathways and/or the reduction of oxidative stress (18).
Indeed, we demonstrated several RAS components including angiotensinogen
(Aogen), active renin, Ang I and the bioactive peptides Ang II and Ang-(1-7) in purified mitochondria from the sheep renal cortex (35). Moreover, purified mitochondria processed Ang I to Ang-(1-7) by the endopeptidases neprilysin and thimet oligopeptidase, suggesting the possibility for an intra-mitochondrial RAS that generates
Ang-(1-7). As previously described, cardiovascular therapeutics in the form of RAS blockade agents concomitantly improve cardiovascular outcomes with beneficial effects on mitochondrial function. Indeed, in the kidney, Ang II has been shown to produce ROS
138 via mitochondrial NADPH oxidase (18), and this effect was blunted by the addition of
Ang-(1-7). Based on this evidence, it is plausible to hypothesize that potential strategies to interrupt negative RAS signaling may involve alterations in the balance of local concentrations of bioactive Ang peptides within intracellular compartments such as the mitochondria. However, the mechanisms by which RAS components may be targeted to the mitochondria remains unknown.
Recent studies by Li and colleagues have demonstrated that Ang II is primarily taken up by the proximal tubules of the kidney via an AT1a receptor-mediated mechanism that is further transported to the mitochondria (19). Moreover, our studies additionally localized RAS components to the mitochondrial compartment of renal cortex which is primarily comprised of tubules (35). Although characterization and functional studies have documented the relevance of an intra-mitochondrial RAS, further research developments are needed to fully understand the transport and trafficking of RAS components to mitochondria or the biosynthetic processes that govern Ang peptide formation.
Due to our evidence revealing the processing of Aogen by active renin within mitochondria (35), we hypothesized that there may be intracellular pathways by which
Aogen translocates to intracellular sites to aid as a source for the generation of active
Ang peptides. The current study provides evidence for the active import of Aogen into renal tubules that subsequently traffics to the mitochondria. Moreover, we demonstrate that this process is conserved amongst sheep and human renal tissue and is not impaired by removal of outer mitochondrial membrane components or addition of mitochondrial uncoupling agents, suggesting that the import of Aogen into the mitochondria may be mediated by passive transport mechanisms.
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METHODS
Animals
Mixed breed sheep (obtained from a private local vendor) were delivered at term, farm-raised, and weaned at 3 months of age. Adult male and female sheep (10-12 months of age) were anesthetized with ketamine and isoflurane, and euthanized by exsanguination. The kidneys were removed immediately, and the renal cortex was dissected out on ice for immediate isolation of proximal tubules as previously described
(34). All procedures were approved by the Wake Forest University School of Medicine
Institutional Animal Care and Use Committee.
HK-2 Cells
HK-2 cells derived from human proximal tubular cells were obtained from
American Type Culture Collection (Manassas, VA). The cells were incubated at 37°C under 5% CO2 humidified atmosphere and were routinely maintained in Dulbecco's
Modified Eagle Medium (DMEM/F12) supplemented with 10% FBS, insulin-transferrin- selenium-cortisol, 100 μg/ml penicillin, and 100 μg/ml streptomycin. HK-2 cells were placed in serum-free DMEM/F12 media for 24 hr before mitochondria isolation.
125I-Angiotensinogen Import
Internalization of 125I-angiotensinogen (125I-Aogen) was studied in intact isolated sheep renal cortex tubules and mitochondria. Additionally, mitochondria isolated from
HK-2 cells in culture were also assessed. Purified recombinant human angiotensinogen was purchased from Sino Biolgoical Inc (Beijing, China) and iodinated utilizing the chloramine T method as previously described (7) and purified by molecular weight filtration using a 30 kDa centrifugal filter (MicrosepTM Advance Centrifugal Device, Pall
Corporation). Internalization of 125I-Aogen (20 nM) was determined in purified tubules
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(500 µg) for 2 hours at 37°C in serum/phenol-free DMEM (DMEM media; phenol red/serum-free, 4.5 g/l D-Glucose, 25mM HEPES, L-Glutamine). The import reaction was stopped by addition of 0.01% phosphoric acid and tubules were centrifuged at
16,000 x g for 1 min. Supernatant was discarded and resulting pellet was washed twice with DMEM media (0.01% triton; 0.01% bovine serum albumin (BSA)) and subsequently treated with 50mM glycine HCL to remove any surface-bound or membrane associated radioligand for 5 min at 4°C. Following glycine treatment, tubules were gently vortexed and centrifuged at 16,000 x g for 1 min. Supernatant was discarded and the pellet containing the residual radioactivity was considered the intra-tubular ligand that was derived from binding and internalization of 125I-Aogen. The incorporation of 125I-Aogen was determined by quantitatively measuring the radioactivity retained by the tubule pellet or supernatant by liquid-scintillation counting.
Subcellular Fractionation
All steps were carried out on ice or at 4°C. Following the 125I-Aogen import assay as described above, sheep tubules were centrifuged for 5 min at 1,500 x g to wash and collect cells. The supernatant was removed and tubule pellet was re-suspended in
DMEM media. Samples were then homogenized with a 20-ml all-glass Dounce homogenizer (Kontes Glass, Vineland, NJ) for 1 min and centrifuged at 1,500 x g for 5 min. The supernatant was saved and the pellet (nuclear fraction) was stored on ice. The resulting supernatant was re-centrifuged at 10,000 x g for 10 min. The pellet
(mitochondrial fraction) was stored on ice and the supernatant was further subjected to centrifugation at 100,000 x g for 30 min. The final pellet (endoplasmic reticulum fraction) and supernatant (cytosolic fraction) were stored on ice. The resulting sub-cellular fractions were quantified for 125I-Aogen radioactivity.
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Isolation of Mitochondria
Mitochondria were isolated from fresh sheep renal cortex by a discontinuous
Percoll gradient, as described previously (12, 23, 28, 35). For isolation of mitochondria from tissue culture, HK-2 cells in 100 mm cultured dishes were washed with PBS and collected by cell scraping in 1 ml DMEM media and centrifugation (1500 x g , 5 min,
4°C). The collected cells were re-suspended in DMEM media and were homogenized using a 20-ml all-glass Dounce homogenizer. The cell homogenate was then centrifuged
(1500 g, 5 min, 4°C); and the supernatant fraction was removed and centrifuged again
(10,000 g, 10 min, 4°C) to obtain the mitochondrial enriched fraction in the pellet. This
HK-2 mitochondrial pellet was used for the 125I-Aogen import assay. Internalization of
125I-Aogen (20 nM) was determined in purified mitochondrial homogenates (50 - 200 µg) for 1 hour at 37°C in DMEM media. 125I-Aogen import was qualitatively determined by autoradiography after SDS-PAGE separation of the radioactively labeled HK-2 and sheep kidney cortex mitochondria (100 µg) and exposure to GeneMate Blue
Autoradiography Flim for 18 hrs at room temperature. 125I-Aogen import rate was also determined either in the absence or presence of exogenous inhibitors and mitochondrial uncoupling agents. The exogenous inhibitors/uncoupling agents were added to the mixtures of purified mitochondria to determine their influence on 125I-Aogen specific import. The inhibitors employed were: metallopeptidase inhibitor, O-Phenantroline (O-
PHEN, 10 µM), mitochondrial uncoupling agents carbonyl cyanide 3- chlorophenylhydrazone (CCCP, 10 µM) and Valinomycin (Val, 10 µM).
Sub-mitochondrial Membrane Fractionation
In an effort to characterize the import mechanism of 125I-Aogen into isolated mitochondria, we utilized the non-ionic detergent digitonin to fractionate sub- mitochondrial compartments (30). To remove the outer mitochondrial membrane,
142 purified sheep and HK-2 mitochondria were solubilized with 5mg/ml digitonin (Sigma, high purity) in DMEM media and were subjected to constant shaking at room temperature over a time course of 15 min. Mitochondrial aliquots were removed at 1 min,
5 min and 15 min. The reaction was terminated by the addition and subsequent washing
(2x) with cold DMEM media to remove residual detergent and centrifuged at 16,000 rpm for 1 min. The resulting HK-2 and sheep mitochondrial protein content was examined by
SDS-PAGE (20 μg) for immunoblot analysis. Treated mitochondria were fractionated using 10% polyacrylamide gels for 1 hour at 120V in Tris-glycine SDS and transferred to a polyvinylidene difluoride (PVDF) membrane. Blots were blocked with 5% Bio-Rad Dry
Milk (Bio-Rad, Hercules, CA, USA) and Tris buffered saline (TBS) with Tween (0.05%) and probed overnight at 4°C with primary antibodies against the following: voltage- dependent anion channel (VDAC, 1:1000, Cell Signaling), an outer mitochondrial membrane marker.
Statistical analysis
All measurements are expressed as mean ± standard error (SEM). Differences between two individual sample means were compared using student t test analysis while differences between the groups were analyzed by one-way or two-way analysis of variance (ANOVA) and Newman-Keuls multiple comparison analysis. Statistical analyses were performed and figures constructed with GraphPad Prism V (GraphPad
Software, San Diego, California, USA). A probability value of <0.05 was required for statistical significance.
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RESULTS
Angiotensinogen internalization by renal tubules
To quantify the extent of Aogen internalization, 125I-Aogen was incubated with freshly isolated proximal tubules at 37° C in defined DMEM/F12 media (Figure 1A).
Following a 2 hr incubation period, approximately 10% of exogenous 125I-Aogen was internalized (Figure 1B). Subcellular fractionation of the proximal tubules revealed that
22% of the internalized 125I-Aogen associated with the mitochondrial fraction, with additional labeling in the nucleus (59%), endoplasmic reticulum (4%), and cytosol (15%)
(Figure 1C).
Angiotensinogen import in sheep and human renal mitochondria
We previously reported that mitochondria isolated from the sheep cortex exhibited components of the RAS including renin and the [des-Ang I]-Aogen (35). Based on the presence of 125I-Aogen within the mitochondrial fraction in the tubule studies, we then determined whether isolated mitochondria directly internalize Aogen. For these studies, mitochondria obtained from a Percoll density fraction of crude mitochondria were incubated with increasing concentrations of 125I-Aogen at 37°C. The mitochondria were then washed in twice in uptake buffer, once in the low pH glycine-HCl buffer and the resultant mitochondrial pellet counted (Figure 2A). As shown in Figure 2B, mitochondrial 125I-Aogen increased in a linear fashion with higher concentrations of exogenous 125I-Aogen. In contrast, the amount of 125I-Aogen in the pellet was negligible when the same protein content of BSA was substituted for the purified mitochondria.
Moreover, the uptake rate of 125I-Aogen in tubule cells was significantly lower as compared to isolated mitochondria in sheep (Table 1).
In three separate mitochondrial preparations from different sheep kidneys, the rate of 125I-Aogen uptake was 33 ± 5 pmol/min/mg protein (Figure 3A).
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Figure 1: 125I-Aogen import in sheep proximal tubules and isolated cortical mitochondria. Schematic presentation of sheep tubule 125I-Aogen import assay and subcellular fractionation (A). 125I-Aogen (20 nM) was incubated with isolated sheep renal proximal tubules (500 µg) for 2hrs at 37°C. 10% of exogenous 125I-Aogen was internalized by sheep tubules (B). Subcellular fractionation revealed that 21 ± 4% of the internalized 125I-Aogen associated with the mitochondrial (Mito) fraction, with additional labeling in the nucleus [60 ± 7%], endoplasmic reticulum [4 ± 0.5%] and cytosol [15 ± 4%; n=4] (C).
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Figure 2: 125I-Aogen increases in a linear fashion with higher concentrations of exogenous 125I-Aogen in sheep cortical mitochondria. Schematic presentation of sheep cortical mitochondria 125I-Aogen import assay (A). Increasing concentrations of 125I-Aogen was added to isolated cortical mitochondria (200 µg) and bovine serum albumin (200 µg). Sheep mitochondria retention of 125I-Aogen increased in a linear fashion with respect to the amount of radiolabeled loaded. In contrast, there was no retainment of 125I-Aogen in the presence of BSA (B).
146
147
Figure 3: 125I-Aogen import is comparable within isolated mitochondria from sheep cortex and HK-2 cells. Isolated mitochondria from sheep cortex (250 µg) and HK-2 cells (250 µg) were incubated with 125I-Aogen for 2hrs at 37°C. Sheep cortical mitochondria and the HK-2 mitochondria internalized 125I-Aogen at a comparable rate of [33 ± 9 vs. 21 ± 10 pmol/min/mg protein; n=3] (A). Purified sheep cortical (lane 2-5) and HK-2 (lane 6-9) mitochondria were fractionated by 10% SDS gel separation and exposed to autoradiography film for 18 hrs. A single protein band corresponding to the molecular weight of Aogen was detected at 50 kDa compared to the 125I-Aogen standard (Std. (0.4 pmol), lane 1) (inset). Unlabeled Aogen (100nM) significantly competed for 125I-Aogen uptake to a greater extent than human serum albumin (HSA, 100nM) in HK-2 mitochondria [60 ± 2% vs. 16 ± 13%; n=3; p < 0.05] (B).
148
We then assessed the rate of 125I-Aogen internalization in isolated mitochondria from HK-2 cells, a well-characterized human proximal tubule cell line. The HK-2 mitochondria internalized 125I-Aogen at a comparable rate to the sheep mitochondria
(Figure 3A). The mitochondrial 125I-Aogen was subsequently analyzed by SDS-gel autoradiography in both sheep cortical and HK-2 mitochondria homogenates. The autoradiograph demonstrates a single protein band corresponding to the molecular weight of Aogen at 55 kDa (inset, Figure 3). Moreover, unlabeled Aogen (100 nM) competed for 125I-Aogen uptake to a significantly greater extent than human serum albumin (HSA) in HK-2 mitochondria (Figure 3B).
Next, we investigated whether Ang peptides are also imported within mitochondria and whether the uptake rate is comparable to that of 125I-Aogen. Ang I, Ang
II and Ang-(1-7) were iodinated and purified as previously described (7), and then incubated with the isolated HK-2 mitochondria. In these studies, HK-2 mitochondria internalized 125I-Aogen at a rate of 6 ± 1 pmol/min/mg protein, however, there was minimal uptake of 125I-Ang I, 125I-Ang II or 125I-Ang-(1-7) (Table 2).
Characterization of Angiotensinogen import
The efficiency of protein transport across mitochondrial membranes is often mediated by membrane potential. Additionally, mitochondrial processing peptidases
(MPPs) may actively cleave mitochondrial targeting signals to direct import and sorting of proteins into the mitochondrial sub-compartments (5, 14, 32). Therefore, we assessed whether agents known to inhibit the mitochondrial membrane potential or block MMP activity would influence 125I-Aogen import in both sheep cortical and HK-2 mitochondria.
As shown in Figure 4, preincubation of the mitochondria with the uncoupling agents
CCCP and Valinomycin did not inhibit 125I-Aogen uptake. Moreover, the addition of the
MPP inhibitor did not attenuate mitochondrial uptake of Aogen (Figure 4).
149
150
Figure 4: Characterization of 125I-Aogen import. Isolated HK-2 and sheep cortical mitochondria were exposed to the mitochondrial processing peptidase inhibitor o- phenanthroline (OPHEN, 10µM) along with mitochondrial membrane uncoupling agents: carbonyl cyanide 3-chlorophenylhydrazone (CCCP, 10µM) and Valinomycin (Val, 10µM). OPHEN, CCCP and Val had no significant impact on inhibiting 125I-Aogen import compared to non-treated mitochondria (control) [n=3; ns; p > 0.05].
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Angiotensinogen import is not dependent on the outer mitochondrial membrane
The outer mitochondrial membrane harbors numerous protein transporters that facilitate import of cytosolic proteins into mitochondrial compartments. The most notable of these proteins are the translocases of the outer membrane (TOM complexes). The receptor domain of TOM complexes faces the cytosolic compartment and initiates the recognition and subsequent transport of mitochondrial protein precursors (36-38).
Isolated sheep cortical and HK-2 mitochondria were treated with the non-ionic detergent digitonin to remove the TOM complexes and the uptake of 125I-Aogen was determined.
We initially confirmed that digitonin (5mg/ml) removed the outer mitochondrial membrane (OM); as evident by the disappearance of the immunoreactive band for the
OM protein VDAC for HK-2 cells (Figure 5A) and sheep cortex (Figure 5B). Digitonin treatment significantly reduced but did not abolish 125I-Aogen uptake in both sheep cortical and HK-2 mitochondria (Figure 5C).
DISCUSSION
We previously reported that mitochondria isolated from sheep renal cortex express elements of a local RAS and demonstrated the expression of renin, [des-Ang I]-
Aogen, the bioactive peptides Ang II and Ang-(1-7), as well as the AT7/Mas receptor
(35). Although these data support earlier studies by Abadir and colleagues on the evidence of a mitochondrial RAS, the mechanisms for the intracellular expression of
RAS components in the mitochondria and other compartments are unclear (3, 29, 35).
Moreover, there is some controversy regarding the extent that local Aogen synthesis versus the uptake and internalization of the protein contributes to Ang II content in the proximal tubules (20, 21). Therefore, the present studies assessed the extent that proximal tubules take up Aogen and the intracellular distribution of the internalized protein.
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Figure 5: 125I-Aogen import is not dependent upon outer mitochondrial membrane. Purified HK-2 (A) and sheep cortical (B) mitochondria were exposed to the non-ionic detergent digitonin (5mg/ml) for 15 min to remove the outer mitochondrial membrane (A- C). Following the digitonin treatment, mitochondrial preparations were subjected to 10% SDS gel fractionation and immunoblotting with VDAC (A-B - 20µg total protein). Purified mitochondria treated with digitonin exhibited a complete abolishment of protein expression for the outer mitochondrial membrane marker VDAC (A-B) in a time dependent manner. Purified sheep and HK-2 mitochondria (100 µg) from the 15 min digitonin time point were incubated with 125I-Aogen (20 nM) for 1 hr at 37°C. 125I-Aogen import was maintained in treated mitochondria but was significantly reduced compared to control [n=3 per group, *p < 0.05 compared to control] (C)
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We find that Aogen import actively occurs within isolated sheep proximal tubules and the internalized protein traffics predominantly to the nuclear and mitochondrial fractions. Moreover, we show that both sheep cortical and human mitochondria directly internalize Aogen. The uncoupling of mitochondria along with the removal of the outer membrane failed to abolish Aogen uptake, indicating that the translocation of Aogen may be facilitated via a non-motor or energy-independent mechanism (32).
The existence of a local RAS within kidney tubule cells has been well established, although the mechanisms by which RAS components are transported within tubules and traffic to intracellular sites are still equivocal. Due to its high molecular size
(50-60kDa), Aogen should not undergo glomerular filtration, however several reports suggest some degree of Aogen filtration that may provide a source of the protein precursor for the generation of Ang peptides within the kidney (21, 27, 31). Pohl et al showed that Aogen is internalized by the protein transporter megalin and that megalin knockout mice exhibit reduced tubular Aogen levels (27, 31). Indeed, Matsusaka and colleagues demonstrated that renal-specific Aogen knockout mice expressed renal
Aogen content similar to to the wildtype mice (21). However, liver-specific Aogen knockout caused a significant reduction in the renal concentration of both the protein and
Ang II, thus implicating that liver Aogen may serve as a primary source of renal Aogen and Ang II.
Although megalin is likely the predominant protein that binds and internalizes
Aogen , Pan et al demonstrated a specific Aogen binding site on the extracellular surface of human proximal tubules (25). These studies revealed a high affinity binding
6 site of Aogen [Kd = 1.73 nmol, Bmax = 23.39 pmol/10 cells] that was displaced by Aogen, but not Ang II or the AT1 receptor antagonist losartan as previously shown (13).
Moreover, these investigators demonstrated that the cell surface Aogen-receptor
154 complex underwent internalization, although the intracellular distribution of this complex within the tubules was not established. We did not assess the presence of a high affinity
Aogen receptor on the cell surface of the sheep proximal tubules or the HK-2 cells.
Moreover, to our knowledge, megalin expression has not been reported to be associated with mitochondrial membranes, nor does the mitochondrial genome contain the megalin sequence. Moreover, the affinity of the tubular Aogen receptor was reported to be ~5 nM, while we find that 100 nM Aogen reduced mitochondrial uptake by 60% which suggests distinct sites with quite different affinities for Aogen. The identification of the internalization process or receptor that mediates transport of Aogen into isolated tubule mitochondria requires further investigation.
The mitochondrial outer membrane contains a multi-subunit complex responsible for the specific recognition and translocation of precursor proteins within mitochondria.
The TOM complex consists of three receptor proteins, Tom20, Tom22 and Tom70, the channel protein Tom40, and several small Tom proteins (22). Digitonin treatment is a standard approach to remove the outer membrane components of the mitochondria (30).
Digitonin pretreatment of mitochondria revealed that Aogen import was not entirely dependent on transport mechanisms found on the outer mitochondrial membrane and suggests that other intra-mitochondrial transport proteins may be involved in Aogen’s uptake. Once precursor proteins cross the outer mitochondria membrane they can further traffic to the mitochondrial matrix by translocases of the inner membrane of mitochondria (TIMs) (14), although this process is believed to be dependent upon mitochondrial membrane potential (17). The uncoupling agents CCCP and Valinomycin also failed to inhibit the retainment of imported Aogen in renal mitochondria. Based on this, the mechanism of Aogen translocation across the outer membrane and retainment by the mitochondrial matrix is still unclear at this time, although the process may include
155 non-energy dependent mechanisms (32). The mitochondrial inner membrane is considered to be the energy-producing membrane within the cell. The electrochemical proton gradient generated by the electron transport chain of the mitochondrial matrix drives ATP synthase and promotes the proton-motive force needed by translocases to control the import of ions and proteins (24). However, van der Laan and colleagues presented evidence for a motor-free mitochondrial translocase that is capable of facilitating the integration of preproteins into sub-mitochondrial compartments (32).
Perspectives and Significance
The mechanisms that contribute to the intracellular expression of the biologically active angiotensin peptides Ang II and Ang-(1-7) within the kidney remain unclear. The present study demonstrates that the precursor protein Aogen is internalized by the proximal tubules and traffics to the mitochondria and nucleus. Previous studies suggest that functional Ang receptors are expressed on these organelles and may be coupled to the stimulation of nitric oxide and reactive oxygen species (1, 2, 18). The uptake and trafficking of Aogen in the renal epithelium may provide an intracellular source for the receptor ligands within this organelle. Alternatively, the demonstration of direct uptake of
Aogen by the mitochondria may portend for biological actions of the precursor protein that may be distinct from Ang II or Ang-(1-7) (20). Indeed, Corvol and colleagues previously showed that both Aogen and the des-Ang I isoform exhibited anti-angiogenic properties that were not blocked by AT1 receptor antagonists (6). Moreover, Aogen delays angiogenesis and tumor growth of hepatocarcinoma in transgenic mice
(33).Thus, additional studies are required to distinguish the potential effects of Aogen versus those of Ang II or Ang-(1-7) on mitochondrial function and whether this uptake pathway represents a novel therapeutic target.
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CONFLICT OF INTEREST
The authors declare that there are no competing financial interests in the work described.
ACKNOWLEDGEMENTS
This work represents partial fulfillment of the requirements for the degree of Doctorate of
Philosophy in the Department of Molecular Medicine and Translational Sciences at
Wake Forest University School of Medicine for Bryan A. Wilson. Mr. Wilson is supported by an American Heart Association (AHA) predoctoral fellowship grant
(15PRE25120007). Additional support for this study was provided by NIH grants HD-
047584, HD-017644, HL-51952, T32 grant (HL091797); the Groskert Heart Fund, the
Wake Forest Venture Fund and the Farley‐Hudson Foundation (Jacksonville, NC). The authors gratefully acknowledge Eric LeSaine for his technical and surgical support.
157
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CHAPTER 7 - DISCUSSION
General Summary of Findings
The RAS is activated and involved in the pathogenic mechanisms that result in kidney and tubular damage. These effects may reflect an active ACE-AngII-AT1 receptor axis, as well as the attenuated expression of an Ang-(1-7)-Mas receptor pathway.
Indeed, Ang II is reported to be increased in cardiovascular disease and particularly in the diseased kidney (22, 29, 65). The cellular actions of Ang II include cellular dedifferentiation and proliferation, tubuloglomerular fibrosis, the generation of ROS, inflammation and apoptosis (117). As a consequence of stimulating these deleterious pathways, increased Ang II signaling and its ramifications may ultimately contribute to the progression of hypertension and renal damage. Accordingly, research efforts are geared towards further understanding the intra-renal RAS and designing therapeutics to prevent its activation and the development of renal pathologies.
At present, the identification of local RAS systems in organs such as the kidney, has aided in our current understanding towards improving patient clinical outcomes.
However, there is controversy over the effectiveness of therapies that block the RAS
(28, 109). Upon discovery that many kidney pathologies were associated with increased renal pressure, resulting in enhanced glomerular pressure and hyperfiltration by tubules, the rationale for using therapeutics to lower pressure followed suit. Clearly, RAS inhibition in the form of ACE inhibitors or ARBs were hypothesized to be provide benefit and indeed numerous studies worldwide have shown the benefit of using ACE inhibitors and ARBs in the treatment of hypertension and renal disease (16, 72, 90, 91). Although the administration of RAS blockade therapeutics are a primary method used in treating cardiovascular disease, the efficacy of these agents are variable. This indicates that the
162 current treatments available may have limitations that could in part, reflect their inability to target intracellular signaling pathways. The concept of treating patients with drugs that act on enzymes or bind to GPCRs embedded in the plasma membrane is well-known and constitutes one of the largest classes of orally active compounds. However, research is lacking towards understanding questions that address the effectiveness of these compounds to successfully enter the cell and target intracellular receptors or enzymes. Research findings regarding the efficiency of drug compounds such as ACE inhibitors, to undergo transepithelial transport, indicate that increased lipophilicity can lead to enhanced interactions with cellular uptake processes and accumulation (106,
126). Moreover, additional parameters such as the polarity and the molecular size of drug therapeutics may present additional barriers.
The evidence of RAS components within intracellular locations such as the mitochondria and nucleus support the rationale for the need of improved therapies that target within the cell. The current work presented here within this thesis builds upon the concept of an intracellular RAS and presents support for the constant investigation of novel aspects that may impact the development of renal pathologies.
Angiotensin (1-7) degrading pathways in the kidney
In chapter two of the dissertation, I began my exploration into intracellular renal
RAS signaling by characterizing a novel peptidase activity that degraded Ang-(1-7). This peptidase activity was first described in our laboratory in the brain medulla and CSF of sheep by Marshall et al (79, 81). Moreover, these studies importantly revealed that the peptidase activity was negatively correlated with Ang-(1-7) peptide levels and was increased in a model of GC-induced fetal programming (79).
Fetal GC exposure is a well-characterized initiator of programming events in utero, and inappropriate elevations in GCs, such as Beta, during fetal development could
163 lead to the development of cardiovascular disease in adult life (85). GCs can have potent effects on the development of a variety of tissues including kidney function and there is compelling evidence that this may encompass dysregulation of the renal RAS
(13, 14, 53, 57, 110, 116). Consequently, this intrauterine insult by GCs reduces nephron number and is associated with the development of hypertension in adulthood
(46, 83). However, the sole reduction in nephron number may not fully explain the impact of fetal programming towards the development of cardiovascular/metabolic pathologies later in life. New evidence suggests that additional renal mal-adaptive changes at the molecular level may reflect the improper processing of Ang peptides, which can compromise nephrogenesis and impact molecular signaling within the kidney
(22).
The identification of a novel Ang-(1-7) peptidase may provide insight into the decreased Ang-(1-7) levels observed in GC-exposed animals (22). Moreover, it could provide insight to the physiological ramifications of these animals having reduced expression of the Mas receptor (22). In this regard, endogenous Ang-(1-7) levels may regulate the expression of the Mas receptor in a positive feedback manner (80). With respect to renal alterations, Beta exposure alters the functional complement of Ang receptor subtypes (53). We previously showed altered nuclear Ang receptor isotypes in
Beta-exposed sheep that were assocated with increased stimulation of ROS via AT1 receptors and an attenuation of NO production via both AT2 and Mas receptors (53).
Sex differences also appear to play a significant role in the responsiveness to
Ang-(1-7). Beta exposure in sheep reduces Na+ excretion in response to systemic infusions of Ang-(1-7) (13). In females, this natriuretic effect was significantly higher compared to males. The mechanism by which Beta exposure induces changes within the kidney is not clear, but our studies reveal evidence for tubular dysfunction (15) and changes in Na+ uptake within cultured RPTs isolated from sheep kidney (116). RPTs
164 isolated from Beta-exposed animals exhibited increased Na+ uptake compared to RPTs from unexposed animals (116). Based on these findings, the isolated RPTs in culture express the similar “programmed” characteristics as seen in the whole kidney. Although it is not known whether Beta exposure or sex differences influence expression of the peptidase within the proximal tubules of the kidney, the future goals will be to establish the role of the RAS and the A7-P, and their capacity for potentiating functional kidney responses that contribute to the inappropriate or accelerated metabolism of Ang-(1-7) as a result of GC-induced fetal programming and cardiovascular disease.
Identification of DPP3 in renal tubules and insights into therapeutic blockade
In chapter four we applied an extensive protein purification process coupled to the mass spectrometric identification of the Ang-(1-7) peptidase activity in HK-2 cells as
DPP3 (31). DPP3 (EC: 3.4.14.4) is a zinc-dependent aminopeptidase and member of the M49 family of metallopeptidases. It preferentially facilitates the hydrolysis of dipeptides from the N-terminus of oligopeptides ranging from three to ten amino acid residues (95). A number of studies have implicated DPP3’s role in pathophysiological signaling processes that include the degradation of bioactive peptides, hormone processing, cellular growth and homeostasis, pain and oxidative stress (26, 56, 77, 79,
81, 101, 118).
DPP3 is considered to be predominantly a cytosolic peptidase. The enzyme has been extensively purified and characterized from the soluble fraction of various mammalian tissues (4, 104) and is expressed ubiquitously in a wide range of organisms and tissues (95). DPP3 hydrolyzes an array of physiological peptide substrates; however, its ability to degrade the bioactive peptide Ang-(1-7) reinforces its presence as a novel member of the RAS. The interactions of DPP3 and the RAS are an area of research that is not well-understood. Moreover, it’s role in the development and/or
165 progression of cardiovascular disease, specifically as it pertains to renal pathologies will be the focus of future research pursuits. However, there are implications of the enzyme’s role in many pathophysiological processes that could provide valuable insight.
DPP3 is believed to participate in cytosolic protein turnover by degrading targeting peptide sequences of cellular proteins destined for elimination by the ubiquitin proteasome pathway. Zhan et al demonstrated that DPP3 activity is higher in cataractous lens compared with the normal lens, resulting in a higher turnover rate of peptides (113, 114). Moreover, DPP3 activity and age of cataract were found to positively correlate which may result in an increase in oxidative stress in the aging lens
(51, 125).
DPP3 degradation of bioactive peptides may influence other physiological parameters such as blood pressure regulation, inflammation and pain. Enkephalins and endomorphins are naturally occurring morphine-like peptides that elicit pain-attenuating, analgesic or opioid-like activity within the neuroendocrine system (44, 45). Turnover of enkephalins and endomorphins by DPP3 was shown to modulate perception of pain, stress and the reward/arousal system in the brain (45), and one report suggests the synaptic membrane localization of mammalian DPP3 in mice that would facilitate interaction of the peptidase with these substrates in the extracellular space (21).
Interestingly, the use of the JMV-390 inhibitor is associated with analgesic effects in mice that were thought to reflect the inhibition of degradation of the neuronal peptides neuromedin and neurotensin (42). The molecular design of JMV-390 was based on the recognition of the leucine-leucine sequence that corresponds to neurotensin, and the amino-terminal hydroxy amino group for an interaction with the zinc cofactor in the active site. Given the evidence that DPP3 is a zinc-dependent dipeptidylpeptidase and the vicinity of the zinc binding site has been reported to be in the substrate binding region
166 and catalytic center, JMV-390 may serve as an additional and potent inhibitor of the enzyme (8).
Furthermore, given DPP3’s high affinity for Ang-(1-7), it is also plausible that tissue levels of DPP3 may influence blood pressure regulation. Moreover, hydrolysis of
Ang-(1-7) by DPP3 may potentially contribute to a decrease in the intracellular peptide levels and the subsequent elevation in blood pressure. The exact physiological effects of
DPP3 and its role in pressure homeostasis are unclear, although oxidative stress is hypothesized to be a causative factor. In contrast, there are studies that support the beneficial effects of DPP3 activation. Cellular oxidative stress in cancer is associated with an upregulation in the nuclear translocation of Nuclear factor-erythroid-2-related factor 2 (Nrf2) (111). Studies by Liu et al reported that Nrf2 expression was enhanced in neuroblastoma cells and activates detoxifying enzymes (74) via an anti-oxidant response element. Similarly, protein C-ets-1 (Ets-1), a critical regulator of DPP3 expression is transcriptionally upregulated by increased levels of H2O2, which also activates detoxifying enzymes such as NADPH quinine reductase 1 (NQO 1). Therefore, the enhanced expression of DPP3 is believed to act as a scavenger of oxidized or damaged proteins. (107,121)
It is likely the influence of DPP3 signaling in cancer is quite different than the pathophysiology associated with cardiovascular disease and renal function. The rationale for this argument is based on studies that ovariectomized mice displayed a diminished expression in DPP3 and Heme Oxygenase-1 (HO-1) proteins that was reversed by 17β-estradiol (E2) supplementation (97). Male mice supplemented with E2 exhibited a significant increase in DPP3 expression but no change in HO-1 protein levels. Overall the authors of this study infer that E2 is generally beneficial and may influence DPP3’s activation of detoxifying enzymes such as HO-1. Similarly, studies in our lab demonstrated the beneficial effects of estradiol signaling, as ovariectomy in
167 hypertensive rats [mRen(2).Lewis] was associated with an increase in blood pressure that was attenuated by the estrogen receptor isotype, G protein-coupled receptor 30
(GPR30) agonist, G-1 (73). Moreover, these studies also demonstrated that G-1 prevented the reduction in ACE2 in ovariectomized rats, which could reflect increased renal levels of Ang-(1-7).
The increase in DPP3 by estradiol in ovariectomized mice studies does not support our hypothesis regarding the hydrolysis of Ang-(1-7) by DPP3 and the beneficial cardiovascular effects of the peptide. However, an increase in HO-1 in mice could reflect the induction of compensatory mechanisms whereby HO-1 activates the release of NO to reduce blood pressure. Indeed, a key mechanism by which Ang-(1-7) exerts its vasodilatory effects may encompass the release of NO (99).
Considering its acute activation by biological substances and ROS, DPP3 may act as ubiquitin-specific peptidase, facilitating the removal of ubiquitin sequences from proteins that would normally undergo degradation. As a result, detoxifying enzymes may remain active within the cell; however the effects of chronic inhibition of protein turnover could have deleterious effects on tissue function. The majority of intracellular proteins appear to be degraded via the ubiquitin/proteasome pathway. Therefore, inhibiting this process is likely to have broad implications on the regulation of cellular functions. Under chronic cardiovascular phenotypes similar to those induced by fetal programming, alterations in intracellular signaling pathways become activated.
One example of this is with respect to serum and glucocorticoid-inducible kinase
1 (SGK1). SGK1 is expressed on renal epithelial cells and is transcriptionally activated by a variety of factors including glucocorticoids, mineralocorticoids, 1,25- dyhydroxyvitamin D3 (1,25(OH)2D3), transforming growth factor β (TGFβ), interleukin 6 fibroblast and platelet-derived growth factor, thrombin, endothelin and AGEs (69).
Intracellular clearance of SGK1 is mediated by the ubiquitin ligase Nedd4-2 (neuronal
168 precursor cells expressed developmentally downregulated) (70). SGK1 regulates a wide variety of tubular channels, including the renal epithelial Na+ channel (ENaC) and NHE3
(69, 70). Under pathological conditions, SGK1 levels increase, and this stimulates SGK1 phosphorylation of target proteins that further phosphorylates the ubiquitin ligase Nedd4-
2, to inhibit its ability to ubiquinate SGK1. Thus, SGK1 cellular clearance is attenuated.
Under normal conditions proximal tubular SGK1 expression is low; however, elevated
SGK1 may participate in the pathophysiological progression of renal injury to promote hypertension, metabolic syndrome cell hypertrophy, inflammation and fibrosis (69, 70).
Although the regulation of DPP3 is quite complex and can include a variety of intracellular mechanisms, its substrate specificity and hydrolysis of Ang-(1-7) warrants further investigation. At this point, is not known whether or not DPP3 is upregulated in renal pathologies or GC-induced fetal programming events in the kidney or other tissues.
Moreover, it is also unclear in regards to whether DPP3 participates as an ubiquitin- specific peptidase. However, the enzyme is predicted to contain several ubiquitination sites (32). Given the cardiovascular benefits of maintaining adequate tissue levels of
Ang-(1-7), DPP3 signaling confers novel insights into potential mechanisms regulating the levels and the protective actions of this bioactive peptide. Thus, DPP3 constitutes a novel component of the intra-renal RAS and its therapeutic inhibition may potentially aid in the prevention and treatment of renal pathologies.
Renal mitochondria and therapeutic implications of an intra-mitochondrial RAS
In chapter five we continued our investigations into understanding the intracellular RAS and its role in the progression of renal/tubular pathologies. For these studies we focused on the mitochondria within the kidney. Renal proximal tubules cells are a highly specialized cell type that governs the active transport of biological solutes across the apical and basolateral membranes. To facilitate the energy demands
169 essential to the efficiency of these processes, the tubule epithelium expresses high levels of mitochondria. In the context of renal cardiovascular pathologies such as hypertension and diabetic nephropathy, compensatory renal hypertrophy may become activated. This includes the induction of a hypermetabolic state whereby nephron oxygen consumption increases to accommodate the enhancement in transcellular transport of biological solutes such as Na+ or glucose (103). Moreover, histomorphometric studies of the kidney also show that tubular compensatory mechanisms are shown to increase mitochondrial volume with no change in density in proportion to the overall increase in cell volume (98). Therefore, the intracellular density of mitochondria remains constant and undergos hypertrophy as a part of the compensatory growth response (59).
The maintenance of mitochondrial volume homeostasis is an intracellular function that regulates oxidative capacity, apoptosis and molecular signaling (54, 61).
Under normal conditions, mitochondrial volume is mainly regulated by potassium influx within the electron transport chain of the inner mitochondrial membrane. However, under cellular stress, mitochondrial swelling is one of the first events to occur to initiate cell death as volume changes have been shown to stimulate cytochrome C release (50).
Moreover, studies suggest that mitochondrial swelling stimulate mechanical signals that may affect the overall tissue function (62). Kaasik et al, demonstrated that swollen mitochondria induced impairments in cardiac muscle contractility (62). This suggests that mitochondrial volume modifications could have an important impact in mitochondria-rich cell types, like the proximal tubule cells of the kidney.
Renal oxidative stress and influences of the RAS
Increases in intracellular oxidant production results in tubular injury and is linked to decreases in mitochondrial function, sodium-potassium-ATPase (Na+-K+-
170
ATPase) activity and active Na+ transport. Intracellular redox status is a highly coordinated process. Under homeostatic conditions, free radical production facilitates normal cell signaling and functioning. However, under diseased conditions or cellular injury, oxidative stress occurs and the production of ROS exceeds local antioxidant capacity. As a result, the oxidation of macromolecules may occur including various proteins, lipids, carbohydrates, and DNA. The importance of regulating ROS levels during kidney injury has been the target of numerous experimental therapeutics. For instance, rodent studies have demonstrated that the overexpression of ROS scavenging enzymes such as superoxide dismutase (SOD) or catalase protected diabetic mice from hyperglycemia-induced renal injury (17, 18, 27, 40). Moreover, deficiency of SOD accelerated diabetic kidney injury in mice (39).
There are many intracellular pathways that initiate the production of ROS in the diseased kidney. Some of these include enzymatic and non-enzymatic sources of ROS such as: the auto-oxidation of glucose, NOS and NADPH oxidase, advanced glycation end products (AGEs) and mitochondrial respiratory chain abnormalities (47). The main sources of ROS that can be generated in the cells include free radicals such as superoxide (O2-), hydroxyl (OH), and hydrogen peroxide (H2O2) (47). Moreover, reactive nitrogen species may also be produced by similar mechanisms including NO and nitrogen dioxide (NO2-), as well as peroxynitrite (ONOO-). Together these various types of free radicals have been extensively studied within the kidney and their over accumulation may result in the development of renal damage.
Furthermore, In vivo and in vitro animal studies have demonstrated the potent regulation of oxidative stress by the RAS. In particular, Ang peptides have been shown to either stimulate or inhibit ROS production and renal tubular injury (5, 12, 29, 30, 41,
64, 100).
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Angiotensin II-stimulation of ROS in the kidney
The RAS via stimulation of the Ang II-AT1 receptor axis plays a pivotal role in the increased ROS production observed within kidney disease. Ang II stimulation of cellular
ROS induces the opening of mitochondrial potassium-ATP channels and depolarizes mitochondrial membrane potential which, in turn, further enhances ROS generation by mitochondria, resulting in the activation of deleterious cellular signaling cascades such as the MAPK and protein kinase C (PKC) pathways (33, 64).
The mechanism by which Ang II stimulates ROS in the kidney is not fully understood, although NOX isoforms are believed to play a pivotal role. NOX1, NOX2 and NOX4 are expressed in the kidney (20, 23). In renal diseases such as diabetic nephropathy, NOX2 and NOX4 are upregulated (6, 102). This was primarily supported by NOX2 studies within cultured rodent mesangial cells that showed an increase in the production of ROS under high glucose conditions; this glucose-induced increase in superoxide was abolished by addition of a siRNA directed to the p47phox subunit of the protein (58). Moreover, in vivo studies utilizing the NOX inhibitor apocynin reduced diabetic renal injury (6, 112). Although other NOX isoforms are located in the kidney,
Nox4 is believed to be the predominant protein in the proximal tubules (48, 82, 105).
Intracellularly, there is evidence that demonstrates Ang II stimulation of mitochondrial NOX4-mediated oxidative stress, depolarization of mitochondrial membrane potential and the release of cytochrome C and apoptosis inducing factor
(AIF). Furthermore, in the same study, the co-administration of Ang-(1-7) abrogated the response to Ang II, thereby indicating the dual regulation of mitochondrial function by
Ang peptides (64).
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Despite the expression of different renal isoforms in the kidney, it is still not clear which of the NOX proteins is the major determinant of initiation and progression of renal injury. The duration of renal disease, type of pathology and subcellular location of ROS production may play a role. Therefore, additional pharmacological studies are needed using more specific inhibitors of NOX isoforms and their characterization within different intracellular organelles and renal injury phenotypes could prove beneficial.
Irrespective of the health compromising insults that lead to the onset of cardiovascular disease, the RAS appears to play a predominate role. As a result, continued investigations to examine the relationship of the RAS and the risk of compromises to cardiovascular health remains the focus of standardized clinical trial cohorts (88).
NOS in the kidney
Studies supporting the presence of mitochondrial-specific NOS isoforms are equivocal. Moreover, evidence for the presence of a RAS to influence NO production within this organelle is few. Certainly, the work by Abadir et al provides a strong framework towards the functional implications of RAS components within liver mitochondria and prompts the need of further studying these mechanisms in other vascularized tissues such as the kidney.
There are three major isoforms of NOS, including inducible (iNOS), neuronal
(nNOS), and endothelial (eNOS). Each of these isoforms requires several cofactors to produce NO, including: flavinmononucleotide (FMN), tetrahydrobiopterin (BH4), calmodulin, and flavin adenine dinucleotide (FAD). In renal injury, concentrations of the
NO substrate precursor, l-arginine may become low coupled with the downregulation of cofactors leading to the uncoupling of NOS. The uncoupled NOS may contribute to the production of superoxide instead of NO. Indeed, in a model of experimental diabetic
173 nephropathy, both uncoupled NOS and NOX were shown to provide intracellular sources of glomerular superoxide (102). However, in the same study, restoration of physiological levels of BH4 attenuated ROS production and improved renal function (102).
The status of NO and its role in renal pathology is debatable. Current findings suggest that early diabetic nephropathy is associated with increased intra-renal NO production (68) mediated primarily by eNOS and nNOS (102). Moreover, enhanced production of NO may contribute to the hyperfiltration and other hemodynamic alterations that are associated with early diabetic nephropathy. This mechanism is also supported by studies in early diabetic nephropathy where the NOS inhibitor L-NAME reversed hemodynamic changes and kidney injury (24).
Contrastingly, most studies in renal disease demonstrate that proteinuria, negative alterations in renal function, and hypertension are associated with progressive loss of NO (94). The alterations in renal function as a result of NO deficiency are believed to be mediated through multiple mechanisms, including high glucose, AGEs and the attenuation of posttranslational modification of NOS isoforms, which can alter the activity of both endothelial and inducible isoforms. Indeed, studies report no effect
(108) or stimulation of renal damage by chronic NO inhibition in models of type 1 and type 2 diabetic nephropathy (63). Evidence of the contrasting roles of NO in the kidney, indicates that further research is needed to fully understand its signaling within renal pathologies.
Mitochondrial NOS
Recent findings suggest that mitochondria may contain a specific NOS isoform that produces significant amounts of NO to regulate respiration and function. However, definitive evidence for mitochondria specific NOS and its effects in vivo are still lacking,
174 and interpretations are complicated. The first evidence of a mitochondrial NOS was described by Loesch et al in perivascular nerves of the basilar artery that was localized to mitochondria in the vessel wall (75). Additional studies followed using immunogold electron microscopy that revealed eNOS immunoreactivity in mitochondria from various rat tissues including liver, heart and brain (9, 10). Indeed, numerous studies have quantified mitochondrial NO production in different tissue types by various biochemical techniques including: fluorescent assays, spectrophotometry and immunocytochemistry
(2, 19, 49, 76, 92). Together these observations support the role that NO may be an intrinsic regulator of mitochondrial function.
There is considerable synergism with respect to RAS signaling and its influence on NO production within the mitochondria. Studies by Abadir et al effectively measured mitochondrial NO production that was coupled to the AT2 receptor (2). Furthermore, this same group demonstrated an effect of aging on the overall expression of Ang II receptor subtypes revealing a higher ratio of the AT1 versus the AT2 in aged mice (2, 3).
Interestingly, this aged-related decline in AT2 expression was attenuated by administration of the AT1 antagonist losartan (2). Interestingly, studies by Kim et al broadened the view of RAS components and their influence on mitochondrial function
(64). Although their studies did not focus on NO per se, this group demonstrated that
Ang-(1-7) attenuated Ang II-induced mitochondrial oxidative stress, NOX4 expression and apoptosis that was comparable to that of a NOX inhibitor (64). These findings suggest that the ACE2-Ang-(1-7)-Mas receptor axis of the RAS may constitute a novel target in the attenuation of mitochondrial injury.
Indeed, studies from our lab have further characterized an intra-mitochondrial
RAS by revealing elements of the Ang-(1-7) axis. We determined that isolated renal mitochondrial expressed endogenous Ang-(1-7) and the Mas receptor (120). Although we did not characterize a functional role of Ang-(1-7) in mitochondria, the regulation of
175
Ang-(1-7) peptide levels and bioactivity through the Mas may play a pivotal role in mitochondrial homeostasis. One of the key molecular roles of Ang-(1-7) activity through the Mas receptor is the stimulation of NO (99). Given the established role of NO in cardio-renal protection (60), it is plausible to speculate that the Ang-(1-7)-Mas receptor axis could be involved in the mitigation of renal mitochondrial injury.
Angiotensinogen uptake in renal mitochondria and therapeutic implications
In chapter six we examined import of the RAS protein precursor Aogen within
RPTs and its intracellular trafficking to the mitochondria. Aogen’s expression in RPTs may reflect local synthesis or internalization of the extracellular protein from the circulation (65-67, 78, 84, 89). Therefore, it is important to understand the mechanisms by which internalized Aogen serves as a precursor for the generation of Ang peptides and the intracellular establishment of a RAS tone within the kidney.
Emerging reports suggest that once Aogen is internalized by RPTs, it further traffics to intracellular sites including the nucleus, ER and mitochondria (119).
Specifically, the des-Ang I form of Aogen was identified within isolated renal mitochondria and this may suggest that intact Aogen is cleaved by active renin (120).
We did not find evidence for the prorenin receptor on the isolated mitochondria nor the presence of inactive or prorenin (120). Moreover, the mitochondrial expression of the bioactive Ang peptides, Ang II and Ang-(1-7) have been quantified (120). Given this information, it is plausible to hypothesize that Aogen’s import into mitochondria adds an additional level of intracrine RAS signaling and expands the functional ramifications the system may have on this organelle.
The transport of proteins into mitochondria is a complex and highly coordinated process involving numerous protein transporters. The most notable of these proteins are the translocases of the outer membrane (TOM complexes) and the translocases of the
176 inner membrane (TIM complexes). The receptor domain of TOM complexes faces the cytosolic compartment and initiates the recognition and subsequent transport of mitochondrial protein precursors (122-124). TIM complexes are embedded in the inner mitochondrial membrane and direct internalized proteins from the outer mitochondrial membrane envelope into the mitochondrial matrix (43). The efficiency of mitochondrial protein transport is contingent upon the internal mitochondrial membrane potential generated by the energy produced by the electron transport chain within the mitochondrial matrix (43, 87, 96).
Out of the 1000-2000 proteins that are integral to the mammalian mitochondria, only a small fraction are encoded by the mitochondrial genome (37 proteins in humans)
(1, 87). Therefore, numerous proteins are transported into the mitochondria where they may play various roles in regulating mitochondrial function. Despite the wealth of research describing the diverse pathways by which proteins are imported into mitochondria, an extensive number of questions regarding mitochondrial transport still remain unanswered. This is partly contributed to the limited expression of protein transport mechanisms normally associated with the plasma membrane that are not identified on mitochondrial membranes (43, 87).
The transport of Aogen within RPTs occurs along the plasma membrane and is mediated by the megalin receptor (25, 52, 86, 93). However, there are no reports of this receptor’s localization on mitochondria nor does mitochondrial DNA code for the megalin gene. Together, this reveals that Aogen transport into renal mitochondria occurs via alternative mechanisms that may be mitochondrial specific. The electrochemical potential of mitochondria is known to influence the transport of proteins into the mitochondrial matrix (43, 87). However our use of the mitochondrial uncoupling agents
CCCP and Valinomycin failed to cause a reduction in the mitochondrial import of Aogen.
Interestingly, removal of the outer mitochondrial membrane and TOM complexes by the
177 non-ionic detergent digitonin significantly reduced Aogen mitochondrial import, but did not completely inhibit it. Therefore, this reveals that transport mechanisms for Aogen’s import into mitochondria are conserved on both the inner and outer membranes and provides insight into the complexity of an intra-mitochondrial RAS.
A key question prompted from these studies is in regards to the exact influence mitochondrial Aogen could be having with respect to mitochondrial function. GPCR Ang peptide receptor signaling for many years was believed to function at the plasma membrane. However, recently did it become evident that Ang peptide GPCRs can be localized at and signal to alternative endomembranes, including those of the mitochondria (2, 3, 7). Moreover, recent reports confirm that these Ang peptide GPCRs stimulated functional pathways such as NO (2). To date, the G protein coupled signaling cascade of Aogen binding to its respective receptor has not been fully elucidated (78), although no subsequent reports have confirmed the presence of an Aogen receptor within the kidney or other tissues. Furthermore, the megalin receptor does not constitute the sole binder of the Aogen protein, as megalin has been shown to bind and mediate the transcytosis of other protein substrates (25). Despite the absence of evidence for a canonical G protein coupled Aogen receptor, within the mitochondria, the glycoprotein may contribute to the production of the bioactive peptides Ang II and Ang-(1-7). Indeed, evidence of the AT1, AT2 and Mas receptors have been located on the membranes of isolated mitochondria (2, 3, 7, 120), and may play a role in the highly dynamic and physiological signaling cascades that influence mitochondrial function to impact end- organ damage.
Mitochondrial dysfunction may be a principal underlying event in cardiovascular disease that impacts the kidney. Mitochondria provide energy for basic metabolic processes, and altered miochondrial function in the context of renal disease impairs cellular metabolism and leads to tubular injury (11, 59). In conditions of renal
178 hypertrophy, nephron mitochondrial energy demands increase leading to enhanced oxygen consumption and the induction of a hypermetabolic state (55, 71). In addition to these modifications in mitochondrial bioenergetics, renal injury is associated with enhanced intracellular RAS signaling (5, 64, 115). Together these data suggest the possibility that an increase in intracellular RAS signaling may be coupled to impairment in renal mitochondrial energy flux. Indeed, studies by De Cavanagh et al have demonstrated that blockade of the RAS were associated with improved mitochondrial function (33-38). Thus, distinguishing the potential effects of canonical Aogen versus
Ang II or Ang-(1-7) pathways within mitochondria may reveal novel therapeutic approaches to counteract the increased potential for renal injury.
The current clinical therapies available to treat cardiovascular disease and the resulting renal failure primarily target: the ACE-Ang II-AT1 receptor axis. For example,
ACE inhibitors and AT1 receptor antagonists are used ubiquitously to reduce high blood pressure and renal damage. However, they fail to treat a primary intracellular defect in renal failure; consisting of a lack of energy for which no current treatments are available.
Therefore, further research into understanding the role of an intracellular RAS within the kidney and the bioactivity of its components within organelles such as the mitochondria may lead to the next generation of protective therapeutics to optimize cellular energy and improve overall kidney function.
179
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Bryan A. Wilson, Ph.D., MBA
Medical Center Blvd ● Molecular Medicine and Translational Sciences ● Winston Salem, NC
PROFESSIONAL INTERESTS
Understanding the molecular mechanisms of hypertension and cardio-renal physiology and medicine ● Intracellular signaling and mitochondrial function ● Science business applications, media, marketing and outreach ● Mentoring, educating and participating in the professional development of future science trainees
EDUCATION
Ph.D./MBA Doctoral Candidate – Molecular Medicine and Translational Sciences, Wake Forest School of Medicine, Winston Salem, NC Dissertation Topic: “Studies of Angiotensin-(1-7) and Mitochondria: The Role of an Intracellular Renin Angiotensin System within the Kidney” Advisor: Dr. Mark C. Chappell and Dr. TanYa M. Gwathmey-Williams
MBA (Masters of Business Administration), April 2015, Wake Forest University School of Business, Winston Salem, NC
NIH PREP Scholar (National Institutes of Health Post Baccalaureate Research Education Program), July 2010, Wake Forest School of Medicine, Winston Salem, NC
B.S., Biological Sciences, minor in Nutritional Sciences , December 2008, Louisiana State University, Baton Rouge, LA LSU Distinguished Communicator in Biological Sciences Certification
AWARDS AND HONORS
2016 American Heart Association (Mid-Atlantic Affiliate) – Emerging Leader 2016 Mid-Atlantic PREP/IMSD Research Symposium Distinguished PREP Alumni Award 2016 February 2016 APSselect featured article, “Evidence for a Mitochondrial Angiotensin-(1-7) System in the Kidney” was acknowledged with a certificate for distinction in scholarship in the American Journal of Physiological-Renal Physiology 2015 American Physiological Society – Minority Travel Fellowship Award – 2016 Experimental Biology, San Diego, CA 2015 Winston Under 40 Leadership Award – City of Winston Salem, NC – Chamber of Commerce 2015 American Heart Association - Predoctoral Fellowship 2014 MARC FASEB Exhibitor Award – 2014 Annual Biomedical Research Conference for Minority Students (ABRCMS) – San Antonio, TX 2014 Nominee - Forbes Top 30 Under 30 in Science and Medicine 2014 Wake Forest School of Medicine - Molecular Medicine and Translational Science Travel Award to attend the 2014 Endocrine Society Meeting – Chicago, Illinois 2014-2015 Internship/Fellowship -The Future Leaders Advancing Research in Endocrinology (FLARE) – The Endocrine Society 2014 Invitation to submit a mini-review and participate in the American Physiological Society Section on Water and Electrolyte Homeostasis Data Diuresis Oral Session at the 2014 Experimental Biology Meeting in San Diego, California
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2014 American Society for Investigative Pathology – Executive Award for Media Outreach 2014 American Society for Nutrition – MARC FASEB Travel Award Winner – 2014 Experimental Biology Meeting – San Diego, California 2014 Travel Award Winner - The Future Leaders Advancing Research in Endocrinology (FLARE) Workshop – The Endocrine Society – New Orleans, Louisiana 2013 MARC FASEB Travel Award Winner – Endocrine Society Meeting (ENDO 2013), San Francisco, California 2013 Endocrine Society – Presidential Poster Competition Award Winner – ENDO 2013 Meeting, San Francisco, California 2013 Intersociety Council for Pathology Information Inc. Travel Award Winner – 2013 Experimental Biology Conference Boston, Massachusetts 2013 1st Place - 2013 Maya Angelou Center for Health Equity Bowl – Wake Forest School of Medicine, Winston Salem, North Carolina 2013 FASEB Stand Up For Science Competition – Recognition of Community Hunger Campaign donation of $500 towards service efforts 2012 Wells Fargo PhD/MBA Student Scholarship 2012 MARC FASEB Travel Award Winner – 2012 APS Writing and Reviewing for Science Journals Workshop; Orlando, Florida 2012 3rd Place Winner (Poster Competition) – 2012 Charlotte Life Sciences Conference. Charlotte, North Carolina 2012-13 MARC FASEB Travel Award Winner – 2012, 2013 Experimental Biology Conference San Diego, CA; Boston, MA 2012 1st Place Winner (Oral Competition) – Georgia Tech 9th Annual Graduate Technical Symposium 2006 – 2008 LSU Howard Hughes Medical Institute Professor Program Scholar 2007 – 2008 LSU Scholarships for Science, Technology, Engineering and Math Scholar 2008 LSU Communication Across the Curriculum – Distinguished Communicator Certification 2008 Louis Stokes – Louisiana Alliance for Minority Participation Award 2004 – 2007 Louisiana Tuition Opportunity Program Scholarship (TOPS) 2007 South Western Association of Student Assistance Programs (SWASAP) Leadership Scholarship 2004 Louisiana State University Black Faculty and Staff Caucus Scholar 2004 LSU Center for Community Engagement and Learning and Leadership Service Learning Award 2004 Highland Elementary Reading Friends Star Reader Award
PROFESSIONAL EXPERIENCE
Doctoral Research
PhD/MBA Student – Molecular Medicine and Translational Sciences - Wake Forest School of Medicine, Winston Salem, NC August 2010 – present Currently investigating intracellular signaling mechanisms of the renin-angiotensin system (RAS) and the role it plays in the development of cardiovascular disease and renal pathologies. Specifically, I am examining changes in cell signaling cascades and mitochondrial function with respect to the production of oxidative stress utilizing in-vitro/animal models.
Industrial Experience
Intern – Marketing and Communications, Wake Forest Innovations, Winston Salem, NC
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August 2015 – present Developing expertise in the hands-on creation, distribution and measurement of science/medical digital marketing engines. I am learning how to code, create and connect science/medical digital marketing initiatives worldwide, website design and management, content development and digital strategy.
Intern – The Endocrine Society, Meetings and Education Office, Washington, DC July 2014 – July 2015 Served on the Endocrine Society's Minority Affairs Committee which includes attending year-round committee meetings and mentoring and recruiting undergraduates for the society’s Minority Access Program (MAP). The MAP is a career development and summer research training program for undergraduate students from underrepresented minority communities. Assisted with the development of innovative strategies to enhance trainee participation and experience at the annual meeting as a member of the ENDO Task Force.
Co-Chair – Head of Marketing – Wake Forest Healthcare Strategy Conference and Case Competition, Winston Salem, NC March 2015 I led marketing efforts for the annual Wake Forest University Healthcare Strategy Conference and Case Competition, which is an international meeting focused on biotech innovation. Along with my team, we raised over $55,000 in funding from various leaders in biotechnology, with leading sponsor being Boston Scientific. Independently, I raised $6,000 by writing and submitting a meeting grant proposal that was funded by the North Carolina Biotechnology Center. Other duties included me managing and updating the conference websites, social media accounts, generating marketing materials as well as maintaining relationships between our sponsors and academic business school partners.
Official Meeting Blogger and Media Outreach – American Society for Investigative Pathology, Bethesda, MD April 2012 – present I represent the American Society for Investigative Pathology by compiling articles and promoting the science accomplishments of researchers and investigators in this society at their annual international meeting.
STEM Education
Instructor/Tutor - LEAP Intervention Instructor/Tutor STEM Scholars Program, Glen Oaks Middle School, Baton Rouge, LA 2008 - 2009 Worked as an in-school fulltime instructor/tutor and assistant to the Math and Science coordinator. Extensively tutored students in math and science preparation for the Louisiana LEAP exam. Mentored and advised students for high school and college preparation. Assisted with the planning of Math and Science night and the Glenn Oaks Science Fair and Leadership Conference. Mentored students with future plans of pursuing careers in the STEM (Science, Technology, Engineering and Math) disciplines.
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Biology Tutor/Peer Mentor – Student Support Service, Louisiana State University, Baton Rouge, LA 2005 - 2008 Worked as a Biology tutor and peer mentor for the comprehensive, federally funded TRIO program at LSU. This program was designed to provide educational opportunities and outreach programs to support students from disadvantaged backgrounds in motivating them to overcome deficiencies in their course work and achieve academic excellence.
FUNDING
2015-2017 15PRE25120007 - American Heart Association – Mid-Atlantic Affiliate Winter 2015 Predoctoral Fellowship – PI
2015 2015-BMG-3005 – North Carolina Biotechnology Center Meeting Grant – 2015 Wake Forest Healthcare Strategy Conference and Case Competition – co-PI (completed)
2014-2015 5T32HL091797-08 - NIH-NHLBI T-32 Grant Trainee - Integrative Lipid Sciences, Inflammation, and Chronic Diseases Training Program – Wake Forest School of Medicine (completed)
2011-2013 3P50AT002782-07S1 – NIH-NCCAM - Predoctoral Minority Supplement (completed)
ABSTRACTS
1. Wilson, Bryan A., Cruz-Diaz, Nildris, Su, Yixin, Rose, James C., Gwathmey, TanYa M. Chappell, Mark C. Internalization of Angiotensinogen in Renal Proximal Tubules: Evidence for Mitochondrial Trafficking FASEB J April 2016 (Oral Talk/Poster – 2016 Experimental Biology Meeting – San Diego, CA) 2. Cruz-Diaz, Nildris, Wilson, Bryan A., Pirro, Nancy T., Chappell, Mark C. The Angiotensin-(1-7) Metallopeptidase in Human Proximal Tubule (HK-2) Cells Identified as Dipeptidyl peptidase III FASEB J April 2016 (Poster – 2016 Experimental Biology Meeting – San Diego, CA) 3. Wilson, Bryan A., Pirro, Nancy T., Gwathmey, TanYa M., Rose, James C., Chappell, Mark C. A Mitochondrial Renin-Angiotensin System: Internalization of Angiotensinogen. Hypertension 66.Suppl 1 (2015): A020-A020. 2015 4. Alzayadneh, Ebaa M., Wilson, Bryan A., Marshall, Allyson C., Chappell, Mark C. AGE’s Enhance the Metabolism of Angiotensin-(1-7) and Lower Intracellular Peptide Expression. Hypertension 2014 64:A234 5. Wilson, Bryan A., Rose, James C., Chappell, Mark C., Functional Neprilysin- Angiotensin-(1-7) System within Mitochondria of the Sheep Kidney. FASEB J April 2014 28:1088.9 6. Wilson, Bryan A., Marshall, Allyson C., Pirro, Nancy T., Su, Yixin, Rose, James C., Chappell, Mark C., Expression of an Angiotensin-(1-7) Endopeptidase in Proximal Tubules of the Sheep and Human Kidney FASEB J April 2014 28:1088.10 7. Chilton Floyd, Howard Timothy, Sergeant Susan, Manichaikul Ani, Rich Stephen, Wilson Bryan, Seeds Michael. Differences in n-3 and n-6 PUFA biosynthesis in African and European Americans. FASEB J April 2014 28:806.16
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8. Wilson Bryan A., Ivester, Priscilla, Sergeant, Susan, Murphy, Karin, Hundley, William G, Chilton, Floyd H. Differences in the Biosynthesis of Long Chain Omega-3 and Omega-6 Polyunsaturated fatty acids (PUFAs) between African and European American patients with Coronary Artery Disease FASEB J April 9, 2013 27:1192.16 9. Wilson, Bryan A, Sergeant, Susan, Ivester, Priscilla, Hundley, William, Chilton, Floyd H. Racial Differences in Serum Omega-9 (ω9) Lipid Fatty Acid Composition and Desaturase Activity in Patients with Diabetes Type-II. Endocr Rev June 2013; 34(03_MeetingAbstracts): SAT-745 10. Wilson, Bryan A., Sergeant, Susan, Ainsworth, Hannah, Mathias, Rasika, Chilton, Floyd H., III. Racial Differences in Plasma Omega-3 Long Chain Fatty Acid Levels in a Cohort of African Americans and European Americans with Diabetes and Metabolic Syndrome. FASEB J March 29, 2012 26:266.4 11. Wilson , Bryan A, Gwathmey T, Pirro N, Chappell MC. Evidence for protein kinase C dependent stimulation of reactive oxygen species in isolated nuclei of renal epithelial cells. FASEB J April 6, 2010 24:1059.3 12. Yamaleyeva, Liliya, Wilson, Bryan A., Westwood Brian M, Pirro, Nancy T., Chappell, Mark C. Estrogen-Depleted mRen2.Lewis Rats Exhibit COX-2 Dependent Responses Distinct From Either the Intact Female or Male Strain. Hypertension 2007 50:4
RESEARCH PUBLICATIONS
1. Wilson, Bryan A., Cruz-Diaz, Nildris, Marshall, Allyson C., Pirro, Nancy T., Su, Yixin, Gwathmey TanYa M., Rose, James C., and Chappell, Mark C. An Angiotensin-(1-7) Endopeptidase in the Kidney Cortex, Proximal Tubules and Human HK-2 Epithelial Cells that is Distinct from Insulin Degrading Enzyme. Am J Physiol Renal Physiol. 2015 Mar 15;308(6):F594-601 2. Wilson, Bryan A., Nautiyal, Manisha, Gwathmey TanYa M., Rose, James C., and Chappell, Mark C. Evidence for a Mitochondrial Angiotensin-(1-7) System in the Kidney. Am J Physiol Renal Physiol. 2015, E-pub ajprenal-00479 3. Wilson, Bryan A., Cruz-Diaz, Nildris, Su, Yixin, Rose, James C., Gwathmey, TanYa M. Chappell, Mark C. Angiotensinogen Import in Renal Proximal Tubules: Evidence for Mitochondrial Trafficking (in-preparation) 4. Cruz-Diaz, Nildris, Wilson, Bryan A., Pirro, Nancy T., Chappell, Mark C. The Angiotensin-(1-7) Metallopeptidase in Human Proximal Tubule (HK-2) Cells Identified as Dipeptidyl peptidase III (in-preparation)
REVIEW ARTICLES
1. Wilson, Bryan A., Marshall, Allyson C., Alzayadneh, Ebaa M., Chappell, Mark C. The Ins and Outs of Angiotensin Processing. Am J Physiol Regul Integr Comp Physiol 2014 Sep 1;307(5):R487-9 2. Chilton, Floyd H., Murphy, Robert C., Wilson, Bryan A., Sergeant, Susan, Ainsworth, Hannah, Seeds, Michael C., Mathias, Rasika M. Diet-Gene Interactions and PUFA Metabolism: A Potential Contributor to Health Disparities and Human Diseases. Nutrients 6 (2014): 1993-2022 3. Wilson, Bryan A., Pollard, Ricquita D., Ferguson, Daniel S., Nutriential Hazards: Macronutrients: Essential Fatty Acids, Encyclopedia of Food Safety, edited by Yasmine Motarjemi, Academic Press, Waltham, 2014, Pages 95-102 4. Wilson, Bryan A., “Video Education America (VEA). Nutrition and Hydration in Sports (Review).” Science, Books and Film 49.4 (2013): 98. 5. Williams Katelyn, Wilson, Bryan A., O’Connor, Wendi G., Monte S. Willis “Ernest Everett Just, PhD: Pioneer in Ecological Developmental (Eco-Devo) Biology. Journal of the South Carolina Academy of Science. 2013
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6. Wilson, Bryan A. "Kitcher, Philip. Science in a Democratic Society (Book Review)." Science, Books and Film 48.1 (2012): 19. 7. Wilson, Bryan A. “Hazen, Robert. The Story of Earth: The First 4.5 Billion Years, from Stardust to Living Planet (Book Review).” Science, Books and Film 48.8 (2012): 209. 8. Wilson, Bryan A., O’Connor, Wendi G., Monte S. Willis. “The Legacy of Charles R. Drew MD, CM, MDSci.” Immunohematology 27 (3) 2011. 9. Wilson, Bryan A., Monte S. Willis, and Jonathan C. Schisler. "Sir Hans Adolf Krebs: Architect of Metabolic Cycles." Lab Medicine 41 (2010): 377-80. Print. 10. Wilson, Bryan A. and Monte S. Willis. “Percy Lavon Julian: Pioneer of Medicinal Chemistry Synthesis.” Lab Medicine 41 (2010): 688-692. Print.
PROFESSIONAL ACTIVITIES/MEMBERSHIP
American Heart Association (Trainee Member) – 2014-present o Corporate Relations Liason Endocrine Society (2013 – present) o Minority Affairs Committee Intern (2014-2015) o Media Blogger (2014) o Endo Task Force (2015-2016) o Trainee and Career Development Core Committee (2015-2017) American Society for Investigative Pathology (2012-present) o Media Blogger/Outreach (2012-present) American Society for Nutrition (2011-2014) o Student Chair – Dietary bioactive components of medicinal, functional and whole foods minisymposium – Experimental Biology Meeting 2012, San Diego, California American Physiological Society (2011-present) o Instructor/Presenter - Physiology Understanding Week (PhUn Week) 2009- present Wake Forest Graduate Student Association: (2009-present) o Social Chair (2013-2014) o Executive Co-Chair (2012-2013) o Community Service Co-Chair (2011-2012) o Molecular Medicine and Translational Sciences Department Representative (2011-present) o Wake Forest University Graduate Newsletter – “The Graduate” – Editor (2011- present) Wake Forest Black Graduate Student Association (2009-present) o Vice President (2011-2012)
SYNERGISTIC ACTIVITIES – SCIENCE OUTREACH AND VOLUNTEERISM
Mentor – City of Winston Salem Chamber of Commerce – Senior Academy (2015- present) Recruiter/Exhibitor – Annual Biomedical Research Conference for Minority Students – Fall 2014-2015 Panelist- Minority Affairs Committee Luncheon – 2014 Endocrine Society Meeting – Chicago, IL. For Inspiration and Recognition of Science and Technology (FIRST®) Lego League Competition Judge (2014) Maya Angelou Center for Health Equity – Wake Forest School of Medicine – Diabetes Support Group – Instructor (2014) Calvary Baptist Day School College Career Day 2014 – College Essay Writing Workshop Instructor – Winston Salem, North Carolina
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Tri-Beta Biology Honor Society – Winston Salem State University – Career Panel Participant – Spring 2012-2015 Judge/Finalist Mentor – Spirit of Innovation Challenge – Conrad Foundation (2011-2012) Reviewer (Nutrition) – AAAS – Science Books and Film - (2011-present) Wake Forest School of Medicine Translational Science Institute - Program in Community Engagement and Implementation – Elementary Science Fair Workshop Facilitator/Buddy – (2011-present) o 3rd Grade Student – Daquan Thompson – 2nd Place School Winner Carver Road Church of Christ – Career Day – Becoming a Scientist Booth – (2010-2011) Chemical Education Foundation – “You Be The Chemist Challenge” team coach – Glen Oaks Middle School (2009) Organized the First Annual Glen Oaks Middle School STEM Leadership Conference (2008) Organized the First Minority Science and Pre-Professional Society’s (LSU-MSPPS) Health Week (2008) Glen Oaks Middle School – Math and Science Night Volunteer (2008) Service Learning Student Advisory Council Hurricane Katrina City Block Clean Up (2006) Highland Elementary (Baton Rouge, LA) – Reading Friend and Wacky Science Demonstrator (2005) Alternative Service Break Trip with La Union De Pueblo Entero (LUPE) – An organization committed to responding to the injustices facing migrant farm workers and immigrant communities. (2004)
TEACHING ACTIVITIES
Summer 2013 – Winston Salem State University - Gastrointestinal Physiology - DPT6403 - Applied Physiology. Dr. Teresa Conner-Kerr (Chair Physical Therapy), and Dr. Allyn Howlett (Course Director)
SELECTED PRESENTATIONS
A Mitochondrial Renin-Angiotensin System: Internalization of Angiotensinogen. Wilson, Bryan A., Pirro, Nancy T., Gwathmey, TanYa M., Rose, James C., Chappell, Mark C. (Poster) Wake Forest University Department of Surgical Sciences Research Day, November 2015
Expression Of An Angiotensin-(1-7) Endopeptidase In Proximal Tubules Of The Sheep And Human Kidney Bryan A. Wilson, Allyson Marshall, Nancy Pirro, Yixin Su, James C. Rose, Mark C. Chappell (Oral) Wake Forest University Department of Lipid Sciences Minisymposium, September 2014
Expression Of An Angiotensin-(1-7) Endopeptidase In Proximal Tubules Of The Sheep And Human Kidney Bryan A. Wilson, Allyson Marshall, Nancy Pirro, Yixin Su, James C. Rose, Mark C. Chappell (Poster) Wake Forest University Department of Surgical Sciences Research Day, November 2014
Racial Differences in Plasma Omega-3 Long Chain Fatty Acid Levels in a Cohort of African Americans and European Americans with Diabetes and Metabolic Syndrome: Insight to Personalized Nutrition Bryan A. Wilson, Susan S. Sergeant, Rasika Mathias, Hannah Ainsworth, Priscilla Ivester, Floyd H. Chilton (Poster) Charlotte Life Sciences Conference 2012, Charlotte, North Carolina
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Racial Differences in Plasma Omega-3 Long Chain Fatty Acid Levels in a Cohort of African Americans and European Americans with Diabetes and Metabolic Syndrome Bryan A. Wilson, Susan S. Sergeant, Rasika Mathias, Hannah Ainsworth, Priscilla Ivester, Floyd H. Chilton (Oral) Georgia Tech 9th Annual Graduate Technical Symposium 2012, Atlanta, Georgia
The Role of Fatty Acid Desaturase (FADS) and Elongase (ELOVL) Gene Variants on Omega3/Omega6 PUFA metabolism in African Americans and European Americans. Bryan A. Wilson, Susan S. Sergeant, Rasika Mathias, Hannah Ainsworth, Priscilla Ivester, Floyd H. Chilton (Poster) Southeast Lipid Conference 2011, Pine Mountain, Georgia
Fostering Your Subconscious Creativity, by Running Your Own Science Race (Oral Talk) Winston Salem State University, Tri-Beta Honor Society Induction Ceremony, April 19, 2011
Evidence for Protein Kinase C Dependent Stimulation of Reactive Oxygen Species in Isolated Nuclei of Renal Epithelial Cells Bryan A. Wilson, TanYa Gwathmey, Nancy Pirro, Mark C. Chappell (Poster) Wake Forest University Post Baccalaureate Research Education Symposium 2010
PMA Stimulates Reactive Oxygen Species in Nuclei from NRK52E Proximal Tubule Cells Bryan A Wilson, TanYa Gwathmey, Mike Robbins, Mark C. Chappell (Poster) Wake Forest University Department of Surgical Sciences Research Day, November 2009
Sex Differences in Response to Cyclooxygenase 2 inhibition in the mRen2.Lewis Rat Bryan Wilson, Liliya Yamaleyeva, Brian M. Westwood, Nancy T. Pirro, Mark C. Chappell (Oral Talk) Presented at the Annual Biomedical Research Conference for Minority Students 2008, Orlando, Florida.
RESEARCH SUPERVISION
Undergraduate Students Kijana George (Summer 2015) – Excellence in Cardiovascular Sciences Summer Research Program Lily Zerihun (Summer 2012) – Excellence in Cardiovascular Sciences Summer Research Program
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