Peptidomic Analysis of Urine from Youths with Early Type 1 Diabetes Reveals Novel Bioactivity of Uromodulin Peptides In Vitro.

Chronic hyperglycemia is known to disrupt the proteolytic milieu, initiating compensatory and maladaptive pathways in the diabetic kidney. Such changes in intrarenal proteolysis are captured by the urinary peptidome. To elucidate the early kidney response to chronic hyperglycemia, we conducted a peptidomic investigation into urines from otherwise healthy youths with type 1 diabetes and their non-diabetic peers using unbiased and targeted mass spectrometry-based techniques. This cross-sectional study included two separate cohorts for the discovery (n = 30) and internal validation (n = 30) of differential peptide excretion. Peptide bioactivity was predicted using PeptideRanker and subsequently verified in vitro Proteasix and the Nephroseq database were used to identify putative proteases responsible for peptide generation and examine their expression in diabetic nephropathy. A total of 6550 urinary peptides were identified in the discovery analysis. We further examined the subset of 162 peptides, which were quantified across all thirty samples. Of the 15 differentially excreted peptides (p < 0.05), seven derived from a C-terminal region (589SGSVIDQSRVLNLGPITRK607) of uromodulin, a kidney-specific protein. Increased excretion of five uromodulin peptides was replicated in the validation cohort using parallel reaction monitoring (p < 0.05). One of the validated peptides (SGSVIDQSRVLNLGPI) activated NFκB and AP-1 signaling, stimulated cytokine release, and enhanced neutrophil migration in vitro. In silico analyses highlighted several potential proteases such as hepsin, meprin A, and cathepsin B to be responsible for generating these peptides. In summary, we identified a urinary signature of uromodulin peptides associated with early type 1 diabetes before clinical manifestations of kidney disease and discovered novel bioactivity of uromodulin peptides in vitro Our present findings lay the groundwork for future studies to validate peptide excretion in larger and broader populations, to investigate the role of bioactive uromodulin peptides in high glucose conditions, and to examine proteases that cleave uromodulin.

Recent Approaches to Targeting Canonical NFκB Signaling in the Early Inflammatory Response to Renal IRI.

Ischemia reperfusion injury (IRI) is the most common cause of in-hospital AKI and is associated with increased morbidity and mortality. IRI is associated with an early phase of inflammation primarily regulated by the canonical NFκB signaling pathway. Despite recent advances in our understanding of the pathogenesis of IRI, few therapeutic strategies have emerged. The purpose of this manuscript is to review interventions targeting NFκB after IRI.

Follistatin-Like-1 (FSTL1) Is a Fibroblast-Derived Growth Factor That Contributes to Progression of Chronic Kidney Disease.

Our understanding of the mechanisms responsible for the progression of chronic kidney disease (CKD) is incomplete. Microarray analysis of kidneys at 4 and 7 weeks of age in Col4a3-/- mice, a model of progressive nephropathy characterized by proteinuria, interstitial fibrosis, and inflammation, revealed that Follistatin-like-1 (Fstl1) was one of only four genes significantly overexpressed at 4 weeks of age. mRNA levels for the Fstl1 receptors, Tlr4 and Dip2a, increased in both Col4a-/- mice and mice subjected to unilateral ureteral obstruction (UUO). RNAscope® (Advanced Cell Diagnostics, Newark CA, USA) localized Fstl1 to interstitial cells, and in silico analysis of single cell transcriptomic data from human kidneys showed Fstl1 confined to interstitial fibroblasts/myofibroblasts. In vitro, FSTL1 activated AP1 and NFκB, increased collagen I (COL1A1) and interleukin-6 (IL6) expression, and induced apoptosis in cultured kidney cells. FSTL1 expression in the NEPTUNE cohort of humans with focal segmental glomerulosclerosis (FSGS), membranous nephropathy (MN), and IgA nephropathy (IgAN) was positively associated with age, eGFR, and proteinuria by multiple linear regression, as well as with interstitial fibrosis and tubular atrophy. Clinical disease progression, defined as dialysis or a 40 percent reduction in eGFR, was greater in patients with high baseline FSTL1 mRNA levels. FSTL1 is a fibroblast-derived cytokine linked to the progression of experimental and clinical CKD.

Connectivity mapping of a chronic kidney disease progression signature identified lysine deacetylases as novel therapeutic targets.

Tubulointerstitial injury is an important determinant of chronic kidney disease progression, yet treatment is limited. Accordingly, we derived a chronic kidney disease progression signature based on aging and disease in Col4a3-/- mice, a model associated with proteinuria and progressive loss of kidney function. Computational drug repurposing with the Connectivity Map identified vorinostat, a lysine deacetylase inhibitor, as a candidate treatment to reverse progression signature gene expression. Vorinostat administration significantly increased the lifespan of Col4a3-/- mice and attenuated tubulointerstitial fibrosis and JNK phosphorylation in the kidneys of Col4a3-/- mice. In vitro, vorinostat reduced albumin- and angiotensin II-induced activation of canonical mitogen-activated protein kinases in kidney tubular epithelial cells. Finally, a subset of murine progression signature genes was differentially expressed across kidney transcriptomic data from patients with focal segmental glomerulosclerosis, IgA nephropathy, and diabetic nephropathy. Thus, our findings suggest that lysine deacetylase inhibition may be a novel treatment to chronic kidney disease associated with proteinuria and progressive tubulointerstitial injury.

Inhibition of BRD4 Reduces Neutrophil Activation and Adhesion to the Vascular Endothelium Following Ischemia Reperfusion Injury.

Renal ischemia reperfusion injury (IRI) is associated with inflammation, including neutrophil infiltration that exacerbates the initial ischemic insult. The molecular pathways involved are poorly characterized and there is currently no treatment. We performed an in silico analysis demonstrating changes in NFκB-mediated gene expression in early renal IRI. We then evaluated NFκB-blockade with a BRD4 inhibitor on neutrophil adhesion to endothelial cells in vitro, and tested BRD4 inhibition in an in vivo IRI model. BRD4 inhibition attenuated neutrophil adhesion to activated endothelial cells. In vivo, IRI led to increased expression of cytokines and adhesion molecules at 6 h post-IRI with sustained up-regulated expression to 48 h post-IRI. These effects were attenuated, in part, with BRD4 inhibition. Absolute neutrophil counts increased significantly in the bone marrow, blood, and kidney 24 h post-IRI. Activated neutrophils increased in the blood and kidney at 6 h post-IRI and remained elevated in the kidney until 48 h post-IRI. BRD4 inhibition reduced both total and activated neutrophil counts in the kidney. IRI-induced tubular injury correlated with neutrophil accumulation and was reduced by BRD4 inhibition. In summary, BRD4 inhibition has important systemic and renal effects on neutrophils, and these effects are associated with reduced renal injury.

Stable Isotope Labeling with Amino Acids (SILAC)-Based Proteomics of Primary Human Kidney Cells Reveals a Novel Link between Male Sex Hormones and Impaired Energy Metabolism in Diabetic Kidney Disease.

Male sex predisposes to many kidney diseases. Considering that androgens exert deleterious effects in a variety of cell types within the kidney, we hypothesized that dihydrotestosterone (DHT) would alter the biology of the renal tubular cell by inducing changes in the proteome. We employed stable isotope labeling with amino acids (SILAC) in an indirect spike-in fashion to accurately quantify the proteome in DHT- and 17ß-estradiol (EST)-treated human proximal tubular epithelial cells (PTEC). Of the 5043 quantified proteins, 76 were differentially regulated. Biological processes related to energy metabolism were significantly enriched among DHT-regulated proteins. SILAC ratios of 3 candidates representing glycolysis, N-acetylglucosamine metabolism and fatty acid ß-oxidation, namely glucose-6-phosphate isomerase (GPI), glucosamine-6-phosphate-N-acetyltransferase 1 (GNPNAT1), and mitochondrial trifunctional protein subunit alpha (HADHA), were verified in vitro. In vivo, renal GPI and HADHA protein expression was significantly increased in males. Furthermore, male sex was associated with significantly higher GPI, GNPNAT1, and HADHA kidney protein expression in two different murine models of diabetes. Enrichment analysis revealed a link between our DHT-regulated proteins and oxidative stress within the diabetic kidney. This finding was validated in vivo, as we observed increased oxidative stress levels in control and diabetic male kidneys, compared with females. This in depth quantitative proteomics study of human primary PTEC response to sex hormone administration suggests that male sex hormone stimulation results in perturbed energy metabolism in kidney cells, and that this perturbation results in increased oxidative stress in the renal cortex. The proteome-level changes associated with androgens may play a crucial role in the development of structural and functional changes in the diseased kidney. With our findings, we propose a possible link between diabetic and non-diabetic kidney disease progression and male sex hormone levels. Data are available via ProteomeXchange (https://www.ebi.ac.uk/pride/archive/) with identifier PXD003811.

Olmesartan Attenuates Kidney Fibrosis in a Murine Model of Alport Syndrome by Suppressing Tubular Expression of TGFß.

Despite the wide use of angiotensin II receptor blockers in the treatment of Alport syndrome (AS), the mechanism as to how angiotensin II receptor blockers prevent interstitial fibrosis remains unclear. Here, we report that treatment of olmesartan effectively targets the feedback loop between the renin-angiotensin system (RAS) and transforming growth factor ß (TGFß) signals in tubular epithelial cells and preserves renal angiotensin-converting enzyme 2 (ACE2) expression in the kidney of Col4a3-/- mice, a murine model of experimental AS. Morphology analyses revealed amelioration of kidney fibrosis in Col4a3-/- mice by olmesartan treatment. Upregulation of TGFß and activation of its downstream in Col4a3-/- mice were attenuated by olmesartan in Col4a3-/- mice. Intriguingly, TGFß expression was preferentially upregulated in damaged tubular epithelial cells in Col4a3-/- mice. Concurrent upregulation of TNFα-converting enzyme and downregulation of ACE2 suggested RAS activation in Col4a3-/- mice, which was prevented by olmesartan. Mechanistically, olmesartan suppressed TGFß-induced RAS activation in tubular epithelial cells in vitro. Collectively, we concluded that olmesartan effectively suppresses the progression of tubulointerstitial fibrosis in AS by interrupting RAS-TGFß feedback loop to counterbalance intrarenal RAS activation.

Angiotensin-converting enzyme 2 and renal disease.

PURPOSE OF REVIEW: The renin-angiotensin system (RAS) is a pivotal player in the physiology and pathophysiology of cardiovascular and renal systems. Discovery of angiotensin-converting enzyme 2 (ACE2), capable of cleaving RAS effector peptide angiotensin (Ang) II into biologically active Ang-(1-7), has increased the complexity of our knowledge of the RAS. ACE2 expression is abundant in the kidney and is thought to provide protection against injury. This review emphasizes current experimental and clinical findings that examine ACE2 in the context of kidney injury and its potential therapeutic impact for treatment of kidney disease. RECENT FINDINGS: Clinical studies have reported upregulation of ACE2 in urine from diabetic patients, which may be reflective of pathological shedding of renal ACE2 as suggested by mechanistic experiments. Studies in experimental models have investigated the feasibility of pharmacological induction of ACE2 for improvement of renal function, inflammation, and fibrosis. SUMMARY: Emerging concepts about the RAS indicate that ACE2 is a critical regulator of angiotensin peptide metabolism and the pathogenesis of renal disease. Human recombinant ACE2 is available and may be a practical clinical approach to enzyme replacement. Elucidating precise roles of ACE2 throughout disease progression will enrich our view of the RAS and help identify novel targets and appropriate strategies for intervention.

Social Determinants of Health Are Associated with Markers of Renal Injury in Adolescents with Type 1 Diabetes.

OBJECTIVE: To examine the relationship between the social determinants of health and markers of early renal injury in adolescent patients with type 1 diabetes (T1D). STUDY DESIGN: Renal outcomes included estimated glomerular filtration rate (eGFR) and albumin-creatinine excretion ratio (ACR). Differences in urinary and serum inflammatory markers also were assessed in relation to social determinants of health. Regression analysis was used to evaluate the association between the Ontario Marginalization Index (ON-Marg) as a measure of the social determinants of health, patient characteristics, ACR, eGFR, and renal filtration status (hyperfiltration vs normofiltration). RESULTS: Participants with T1D (n = 199) with a mean age of 14.4 ± 1.7 years and diabetes duration of 7.2 ± 3.1 years were studied. Mean eGFR was 122.0 ± 19.4 mL/min/1.73 m2. Increasing marginalization was positively associated with eGFR (P < .0001) but not with ACR (P = .605). Greater marginalization was associated with greater median levels of urinary interleukin (IL)-2, IL-12 (p40), macrophage-derived chemokine, monocyte chemoattractant protein-3, and tumor necrosis factor-ß and serum IL-2. ON-Marg was significantly associated with eGFR after we controlled for age, sex, body mass index z score, ethnicity, serum glucose, and hemoglobin A1c in linear regression. A similar association between hyperfiltration and ON-Marg score was observed in multivariable logistic regression. CONCLUSION: Increasing marginalization is significantly associated with both eGFR and hyperfiltration in adolescents with T1D and is associated with significant changes in urinary inflammatory biomarkers. These findings highlight a potentially important interaction between social and biological determinants of health in adolescents with T1D.

Insights into Diabetic Kidney Disease Using Urinary Proteomics and Bioinformatics.

A number of proteomic and peptidomic analyses of urine from diabetic subjects have been published in the quest for a biomarker that predicts progression of nephropathy. Less attention has been paid to the relationships between urinary proteins and the underlying biological processes revealed by the analyses. In this review, we focus on the biological processes identified by studying urinary proteins and protein-protein interactions at each stage of diabetic nephropathy to provide an overview of the events underlying progression of kidney disease reflected in the urine. In uncomplicated diabetes, proteomic/peptidomic analyses indicate that early activation of fibrotic pathways in the kidney occurs before the onset of microalbuminuria. In incipient nephropathy, when albumin excretion rates are abnormal, proteomic/peptidomic analyses suggest that changes in glomerular permselectivity and tubular reabsorption account, at least in part, for the proteins and peptides that appear in the urine. Finally, overt nephropathy is characterized by proteins involved in wound healing, ongoing fibrosis, and inflammation. These findings suggest that there is a spectrum of biological processes in the diabetic kidney and that assessing protein networks may be more informative than individual markers with respect to the stage of disease and the risk of progression.

Assessment of urinary microparticles in normotensive patients with type 1 diabetes.

AIMS/HYPOTHESIS: Assessment of urinary extracellular vesicles including exosomes and microparticles (MPs) is an emerging approach for non-invasive detection of renal injury. We have previously reported that podocyte-derived MPs are increased in diabetic mice in advance of albuminuria. Here, we hypothesised that type 1 diabetes and acute hyperglycaemia would increase urinary podocyte MP levels in uncomplicated diabetes. METHODS: In this post hoc exploratory analysis, we examined archived urine samples from normoalbuminuric patients with uncomplicated type 1 diabetes studied under clamped euglycaemia and hyperglycaemia and compared with healthy controls. Urinary vesicles were assessed by electron microscopy and nanoparticle tracking while podocyte MPs were assessed by flow cytometry. RESULTS: Neither vesicle size nor total number were significantly altered in type 1 diabetes or acute hyperglycaemia. By contrast, urinary podocyte MP levels were higher in type 1 diabetes (0.47 [0.00-3.42] MPs/µmol creatinine [Cr]) compared with healthy controls (0.00 [0.00-0.00] MPs/µmol Cr, p < 0.05) and increased under hyperglycaemic clamp (0.36 [0.00-4.15] MPs/µmol Cr during euglycaemia vs 2.70 [0.00-15.91] MPs/µmol Cr during hyperglycaemia, p < 0.05). Levels of urinary albumin to creatinine ratio and nephrin (surrogates of podocyte injury) were unchanged by type 1 diabetes or acute hyperglycaemia. CONCLUSION/INTERPRETATION: Taken together, our data show that urinary podocyte MP levels are higher in patients with type 1 diabetes in advance of changes in other biomarkers (albuminuria, nephrin). Examination of podocyte MPs may serve as an early biomarker of glomerular injury in uncomplicated type 1 diabetes.

Influence of sex on hyperfiltration in patients with uncomplicated type 1 diabetes.

The aim of this analysis was to examine sex-based differences in renal segmental resistances in healthy controls (HCs) and patients with type 1 diabetes (T1D). We hypothesized that hyperfiltration-an early hemodynamic abnormality associated with diabetic nephropathy-would disproportionately affect women with T1D, thereby attenuating protection against the development of renal complications. Glomerular hemodynamic parameters were evaluated in HC (n = 30) and in normotensive, normoalbuminuric patients with T1D and either baseline normofiltration [n = 36, T1D-N, glomerular filtration rate (GFR) 90-134 ml·min-1·1.73 m2] or hyperfiltration (n = 32, T1D-H, GFR ≥ 135 ml·min-1·1.73 m2) during euglycemic conditions (4-6 mmol/l). Gomez's equations were used to derive efferent (RE) and afferent (RA) arteriolar resistances, glomerular hydrostatic pressure (PGLO) from inulin (GFR) and paraaminohippurate [effective renal plasma flow (ERPF)] clearances, plasma protein and estimated ultrafiltration coefficients (KFG). Female patients with T1D with hyperfiltration (T1D-H) had higher RE (1,985 ± 487 vs. 1,381 ± 296 dyne·sec-1·cm-5, P < 0.001) and filtration fraction (FF, 0.20 ± 0.047 vs. 0.16 ± 0.03 P < 0.05) and lower ERPF (876 ± 245 vs. 1,111 ± 298 134 ml·min-1·1.73 m2P < 0.05) compared with male T1D-H patients. Overall, T1D-H patients had higher PGLO and lower RA vs. HC subjects, although there were no sex-based differences. In conclusion, female T1D-H patients had higher RE and FF and lower ERPF than their male counterparts with no associated sex differences in RA Prospective intervention studies should consider sex as a modifier of renal hemodynamic responses to renal protective therapies.

The relationship between urinary renin-angiotensin system markers, renal function, and blood pressure in adolescents with type 1 diabetes.

The relationship between the renal renin-angiotensin aldosterone system (RAAS) and cardiorenal pathophysiology is unclear. Our aims were to assess 1) levels of urinary RAAS components and 2) the association between RAAS components and HbA1c, the urine albumin/creatinine ratio (ACR), estimated glomerular filtration rate (eGFR), and blood pressure (BP) in otherwise healthy adolescents with type 1 diabetes mellitus (TID) vs. healthy controls (HC). Urinary angiotensinogen and angtionsin-converting enzyme (ACE) 2 levels, activity of ACE and ACE2, BP, HbA1c, ACR, and eGFR were measured in 65 HC and 194 T1D from the Adolescent Type 1 Diabetes Cardio-Renal Intervention Trial (AdDIT). Urinary levels of all RAAS components were higher in T1D vs. HC (P < 0.0001). Higher HbA1c was associated with higher urinary angiotensinogen, ACE2, and higher activity of ACE and ACE2 (P < 0.0001, P = 0.0003, P = 0.003, and P = 0.007 respectively) in T1D. Higher ACR (within the normal range) was associated with higher urinary angiotensinogen (P < 0.0001) and ACE activity (P = 0.007), but not with urinary ACE2 activity or ACE2 levels. These observations were absent in HC. Urinary RAAS components were not associated with BP or eGFR in T1D or HC. Otherwise healthy adolescents with T1D exhibit higher levels of urinary RAAS components compared with HC. While levels of all urinary RAAS components correlate with HbA1c in T1D, only urinary angiotensinogen and ACE activity correlate with ACR, suggesting that these factors reflect an intermediary pathogenic link between hyperglycemia and albuminuria within the normal range.

Murine recombinant angiotensin-converting enzyme 2 attenuates kidney injury in experimental Alport syndrome.

Angiotensin-converting enzyme 2 (ACE2) is a monocarboxypeptidase in the renin-angiotensin system that catalyzes the breakdown of angiotensin II to angiotensin 1-7. We have reported that ACE2 expression in the kidney is reduced in experimental Alport syndrome but the impact of this finding on disease progression has not been studied. Accordingly, we evaluated effects of murine recombinant ACE2 treatment in Col4a3 knockout mice, a model of Alport syndrome characterized by proteinuria and progressive renal injury. Murine recombinant ACE2 (0.5 mg/kg/day) was administered from four to seven weeks of age via osmotic mini-pump. Pathological changes were attenuated by murine recombinant ACE2 treatment which ameliorated kidney fibrosis as shown by decreased expression of COL1α1 mRNA, less accumulation of extracellular matrix proteins, and inhibition of transforming growth factor-ß signaling. Further, increases in proinflammatory cytokine expression, macrophage infiltration, inflammatory signaling pathway activation, and heme oxygenase-1 levels in Col4a3 knockout mice were also reduced by murine recombinant ACE2 treatment. Lastly, murine recombinant ACE2 influenced the turnover of renal ACE2, as it suppressed the expression of tumor necrosis factor-α converting enzyme, a negative regulator of ACE2. Thus, treatment with exogenous ACE2 alters angiotensin peptide metabolism in the kidneys of Col4a3 knockout mice and attenuates the progression of Alport syndrome nephropathy.

Recombinant N-Terminal Slit2 Inhibits TGF-ß-Induced Fibroblast Activation and Renal Fibrosis.

Fibrosis and inflammation are closely intertwined injury pathways present in nearly all forms of CKD for which few safe and effective therapies exist. Slit glycoproteins signaling through Roundabout (Robo) receptors have been described to have anti-inflammatory effects through regulation of leukocyte cytoskeletal organization. Notably, cytoskeletal reorganization is also required for fibroblast responses to TGF-ß Here, we examined whether Slit2 also controls TGF-ß-induced renal fibrosis. In cultured renal fibroblasts, which we found to express Slit2 and Robo-1, the bioactive N-terminal fragment of Slit2 inhibited TGF-ß-induced collagen synthesis, actin cytoskeletal reorganization, and Smad2/3 transcriptional activity, but the inactive C-terminal fragment of Slit2 did not. In mouse models of postischemic renal fibrosis and obstructive uropathy, treatment with N-terminal Slit2 before or after injury inhibited the development of renal fibrosis and preserved renal function, whereas the C-terminal Slit2 had no effect. Our data suggest that administration of recombinant Slit2 may be a new treatment strategy to arrest chronic injury progression after ischemic and obstructive renal insults by not only attenuating inflammation but also, directly inhibiting renal fibrosis.

Ischemia reperfusion injury, ischemic conditioning and diabetes mellitus.

Ischemia/reperfusion, which is characterized by deficient oxygen supply and subsequent restoration of blood flow, can cause irreversible damages to tissue. Mechanisms contributing to the pathogenesis of ischemia reperfusion injury are complex, multifactorial and highly integrated. Extensive research has focused on increasing organ tolerance to ischemia reperfusion injury, especially through the use of ischemic conditioning strategies. Of morbidities that potentially compromise the protective mechanisms of the heart, diabetes mellitus appears primarily important to study. Diabetes mellitus increases myocardial susceptibility to ischemia reperfusion injury and also modifies myocardial responses to ischemic conditioning strategies by disruption of intracellular signaling responsible for enhancement of resistance to cell death. The purpose of this review is twofold: first, to summarize mechanisms underlying ischemia reperfusion injury and the signal transduction pathways underlying ischemic conditioning cardioprotection; and second, to focus on diabetes mellitus and mechanisms that may be responsible for the lack of effect of ischemic conditioning strategies in diabetes.

Chronology of mitochondrial and cellular events during skeletal muscle ischemia-reperfusion.

Peripheral artery disease (PAD) is a common circulatory disorder of the lower limb arteries that reduces functional capacity and quality of life of patients. Despite relatively effective available treatments, PAD is a serious public health issue associated with significant morbidity and mortality. Ischemia-reperfusion (I/R) cycles during PAD are responsible for insufficient oxygen supply, mitochondriopathy, free radical production, and inflammation and lead to events that contribute to myocyte death and remote organ failure. However, the chronology of mitochondrial and cellular events during the ischemic period and at the moment of reperfusion in skeletal muscle fibers has been poorly reviewed. Thus, after a review of the basal myocyte state and normal mitochondrial biology, we discuss the physiopathology of ischemia and reperfusion at the mitochondrial and cellular levels. First we describe the chronology of the deleterious biochemical and mitochondrial mechanisms activated by I/R. Then we discuss skeletal muscle I/R injury in the muscle environment, mitochondrial dynamics, and inflammation. A better understanding of the chronology of the events underlying I/R will allow us to identify key factors in the development of this pathology and point to suitable new therapies. Emerging data on mitochondrial dynamics should help identify new molecular and therapeutic targets and develop protective strategies against PAD.

Characterization of the intrarenal renin-angiotensin system in experimental alport syndrome.

Blockade of the renin-angiotensin system attenuates the progression of experimental and clinical Alport syndrome (AS); however, the underlying mechanism(s) remains largely unknown. We evaluated the renin-angiotensin system in 4- and 7-week-old homozygous for collagen, type IV, α3 gene (Col4A3(-/-)) and wild-type mice, a model of AS characterized by proteinuria and progressive renal injury. Renal angiotensin (Ang) II levels increased, whereas renal Ang-(1-7) levels decreased in 7-week-old Col4a3(-/-) mice compared with age-matched controls; these changes were partially reversed by recombinant angiotensin-converting enzyme 2 (ACE2) treatment. The expression of both the angiotensinogen and renin protein increased in Col4a3(-/-) compared with wild-type mice. Consistent with the Ang-(1-7) levels, the expression and activity of kidney ACE2 decreased in 7-week-old Col4a3(-/-) mice. The urinary excretion rate of ACE2 paralleled the decline in tissue expression. Expression of an Ang II-induced gene, heme oxygenase-1, was up-regulated in the kidneys of 7-week-old Col4a3(-/-) mice compared with wild-type mice by microarray analysis. Heme oxygenase-1 (HO-1) protein expression was increased in kidneys of Col4a3(-/-) mice and normalized by treatment with ACE inhibitor. Urinary HO-1 excretion paralleled renal HO-1 expression. In conclusion, progressive kidney injury in AS is associated with changes in expression of intrarenal renin Ang system components and Ang peptides. HO-1 and ACE2 may represent novel markers of AS-associated kidney injury, whereas administration of recombinant ACE2 and/or Ang-(1-7) may represent novel therapeutic approaches in AS.

Quantification of angiotensin II-regulated proteins in urine of patients with polycystic and other chronic kidney diseases by selected reaction monitoring.

BACKGROUND: Angiotensin-II (Ang II) mediates progression of autosomal-dominant polycystic kidney disease (ADPKD) and other chronic kidney diseases (CKD). However, markers of kidney Ang II activity are lacking. We previously defined 83 Ang II-regulated proteins in vitro, which reflected kidney Ang II activity in vivo. METHODS: In this study, we developed selected reaction monitoring (SRM) assays for quantification of Ang II-regulated proteins in urine of ADPKD and CKD patients. We demonstrated that 47 of 83 Ang II-regulated transcripts were differentially expressed in cystic compared to normal kidney tissue. We then developed SRM assays for 18 Ang II-regulated proteins overexpressed in cysts and/or secreted in urine. Methods that yielded CV ≤ 6 % for control proteins, and recovery ~100 % were selected. Heavy-labeled peptides corresponding to 13 identified Ang II-regulated peptides were spiked into urine samples of 17 ADPKD patients, 9 patients with CKD predicted to have high kidney Ang II activity and 11 healthy subjects. Samples were then digested and analyzed on triple-quadrupole mass spectrometer in duplicates. RESLUTS: Calibration curves demonstrated linearity (R(2) > 0.99) and within-run CVs < 9 % in the concentration range of 7/13 peptides. Peptide concentrations were normalized by urine creatinine. Deamidated peptide forms were monitored, and accounted for <15 % of the final concentrations. Urine excretion rates of proteins BST1, LAMB2, LYPA1, RHOB and TSP1 were significantly different (p < 0.05, one-way ANOVA) between patients with CKD, those with ADPKD and healthy controls. Urine protein excretion rates were highest in CKD patients and lowest in ADPKD patients. Univariate analysis demonstrated significant association between urine protein excretion rates of most proteins and disease group (p < 0.05, ANOVA) as well as sex (p < 0.05, unpaired t test). Multivariate analysis across protein concentration, age and sex demonstrated good separation between ADPKD and CKD patients. CONCLUSIONS: We have optimized methods for quantification of Ang II-regulated proteins, and we demonstrated that they reflected differences in underlying kidney disease in this pilot study. High urine excretion of Ang II-regulated proteins in CKD patients likely reflects high kidney Ang II activity. Low excretion in ADPKD appears related to lack of communication between cysts and tubules. Future studies will determine whether urine excretion rate of Ang II-regulated proteins correlates with kidney Ang II activity in larger cohorts of chronic kidney disease patients.