Urinary Angiotensin-converting Enzyme 2 Research Articles

Altered kidney distribution and loss of ACE2 into the urine in acute kidney injury.

There are diverse pathophysiological mechanisms involved in acute kidney injury (AKI). Among them, overactivity of the renin-angiotensin system (RAS) has been described. Angiotensin-converting enzyme 2 (ACE2) is a tissue RAS enzyme expressed in the apical border of proximal tubules. Given the important role of ACE2 in the metabolism of angiotensin II, this study aimed to characterize kidney and urinary ACE2 in a mouse model of AKI. Ischemia-reperfusion injury (IRI) was induced in C57BL/6 mice by clamping of the left renal artery followed by removal of the right kidney. In kidneys harvested 48 h after IRI, immunostaining revealed a striking maldistribution of ACE2 including spillage into the tubular lumen and the presence of ACE2-positive luminal casts in the medulla. In cortical membranes, ACE2 protein and enzymatic activity were both markedly reduced (37 ± 4 vs. 100 ± 6 ACE2/β-actin, P = 0.0004, and 96 ± 14 vs. 152 ± 6 RFU/μg protein/h, P = 0.006). In urine, full-length membrane-bound ACE2 protein (100 kDa) was markedly increased (1,120 ± 405 vs. 100 ± 46 ACE2/µg creatinine, P = 0.04), and casts stained for ACE2 were recovered in the urine sediment. In conclusion, in AKI caused by IRI, there is a marked loss of ACE2 from the apical tubular border with deposition of ACE2-positive material in the medulla and increased urinary excretion of full-length membrane-bound ACE2 protein. The deficiency of tubular ACE2 in AKI suggests that provision of this enzyme could have therapeutic applications and that its excretion in the urine may also serve as a diagnostic marker of severe proximal tubular injury.NEW & NOTEWORTHY This study provides novel insights into the distribution of kidney ACE2 in a model of AKI by IRI showing a striking detachment of apical ACE2 from proximal tubules and its loss in urine and urine sediment. The observed deficiency of kidney ACE2 protein and enzymatic activity in severe AKI suggests that administration of forms of this enzyme may mitigate AKI and that urinary ACE2 may serve as a potential biomarker for tubular injury.

Plasma angiotensin-converting enzyme 2 (ACE2) is a marker for renal outcome of diabetic kidney disease (DKD) (U-CARE study 3)

IntroductionACE cleaves angiotensin I (Ang I) to angiotensin II (Ang II) inducing vasoconstriction via Ang II type 1 (AT1) receptor, while ACE2 cleaves Ang II to Ang (1–7) causing vasodilatation...

Urinary angiotensin-converting enzyme 2 and its activity in cats with chronic kidney disease.

Angiotensin-converting enzyme 2 (ACE2) played an important role in the renin-angiotensin-aldosterone system (RAAS) and it was proved to be renoprotective in renal disease. Urinary angiotensin-converting enzyme 2 (uACE2) has been shown to reflect renal injury in human and experimental studies, but its role in feline kidney disease remains unknown. Our objectives involve comparing uACE2 concentrations and activities in cats across CKD stages with healthy controls, investigating the relationship between uACE2 concentrations, activities, and clinicopathological data in feline CKD patients, and assessing the predictive abilities of both for CKD progression. A retrospective, case-control study. The concentration and activity of uACE2 were measured by commercial ELISA and fluorometric assay kits, respectively. The concentration was adjusted to give uACE2 concentration-to-creatinine ratios (UACCRs). In total, 67 cats consisting of 24 control and 43 chronic kidney disease (CKD), including 24 early-stage CKD and 19 late-stage CKD, were enrolled in this study. UACCR values were significantly higher in both early-stage (2.100 [1.142-4.242] x 10-6) and late-stage feline CKD (4.343 [2.992-5.0.71] x 10-6) compared to healthy controls (0.894 [0.610-1.076] x 10-6; p < 0.001), and there was also significant difference between-early stage group and late-stage group (p = 0.026). Urinary ACE2 activity (UAA) was significantly lower in CKD cats (1.338 [0.644-2.755] x pmol/min/ml) compared to the healthy cats (7.989 [3.711-15.903] x pmol/min/ml; p < 0.001). UACCR demonstrated an independent, positive correlation with BUN (p < 0.001), and UAA exhibited an independent, negative correlation with plasma creatinine (p < 0.001). Both UACCR and UAA did not yield significant results in predicting CKD progression based on the ROC curve analysis. uACE2 concentration and activity exhibit varying changes as renal function declines, particularly in advanced CKD cats.

Comparison between Vitamin-D and Urinary Angiotensin Converting Enzyme Levels in Type 2 Diabetic Hypertensive Patients with Non-Diabetic Hypertensive Patients

&#x0D; Objective: To correlate urinary Angiotensin Converting Enzyme–2 (ACE-2) with serum Vitamin D levels in Type 2 Diabetic-Hypertensive and non-Diabetic-Hypertensive patients. Methodology:The Department of Physiology at the University of Health Sciences in Lahore undertook this observational, correlational study from March 2018 to February 2019. The Services Institute of Medical Services (SIMS), Lahore's medical OPD and diabetic centers were used to select 95 hypertension patients between the ages of 30 and 60, divided into 2 groups. Anthropometric parameters, as well as Blood Sugar Random (BSR) and serum Vitamin D were recorded. Urine samples for evaluating angiotensin converting enzyme 2 were collected.Results: Urinary ACE2 showed negative correlation with serum Vitamin D level (r=-0.336, p=0.034) in group A non-diabetic hypertensive patients, but not in group B diabetic hypertensive patients (r=0.07, p=0.677). Despite somewhat greater vitamin D levels in group B, this difference was not statistically significant.Conclusion: There was no link between ACE2 and blood Vitamin D levels in the diabetic hypertensive patients, despite the fact that we hypothesised that vitamin D would be considered as a viable treatment alternative for treating these individuals.&#x0D;

Urinary neprilysin - Another marker to predict postoperative acute kidney injury.

Dear Editor, Perioperative acute kidney injury (AKI) is a serious complication which is multifactorial, which leads to increased hospital and intensive care unit stay, possibility of chronic kidney disease and hemodialysis, increased cost of treatment, significant morbidity, and mortality. Neprilysin or neutral endopeptidase (NEP) is a single-pass membrane glycoprotein with zinc-dependent endopeptidase activity and a short cytosolic tail. NEP is found predominantly within the kidneys, gastrointestinal tract, liver, male genital organs, lungs, adipose tissue, brain, and heart. The renal brush border of proximal tubule on the luminal side is rich in membrane-bound NEP. NEP is involved in the degradation of both atrial and B-type natriuretic peptides, adrenomedullin, endothelin-1 and angiotensin II, and also nonvasoactive peptides, such as glucagon, glucagon-like peptide, enkephalins, and somatostatin. Circulating NEP estimation in serum has been used as a biomarker in acute and chronic heart failure patients for predicting cardiovascular death and time-to-first admission for an acute event.[1] The prospective comparison of angiotensin receptor neprilysin inhibitor with angiotensin-converting enzyme (ACE) inhibitor (ACEi) to determine impact on global mortality and morbidity in heart failure (PARADIGM-HF) study reported significant clinical benefits with the combination of an angiotensin receptor blocker valsartan with a neprilysin inhibitor sacubitril over ACEi in heart failure with reduced ejection fraction.[2] However, trends of serum NEP could never correlate NT-pro-BNP and even clinically. In AKI, NEP is flushed out from the kidneys due to shedding of the tubular brush border and thus is available for estimation in the urine as a marker. Bernardi et al.[3] conducted a prospective, observational cohort study involving 96 patients undergoing elective cardiac surgeries with cardiopulmonary bypass. They estimated urinary neprilysin levels in the immediate postoperative period and on day 1. The authors concluded that estimation of urinary neprilysin levels after cardiac surgeries has comparable discriminatory power to presently studied AKI biomarkers and has potential as a biomarker for the early detection of AKI after cardiac surgery. Pajenda et al.[4] analyzed urinary neprilysin levels of 90 critically ill patients admitted to intensive or intermediate care unit over 2–5 days. On analysis, they found that NEP levels who suffered AKI according to the KDIGO (Kidney Disease Improving Global Outcomes) criteria were significantly elevated in comparison to healthy controls. In a comparative study performed by Gutta et al.[5] between 20 nondiabetics and 40 diabetic patients, authors compared urinary ACE2, NEP, disintegrin and metalloproteinase 17 (ADAM17) between two groups. The authors concluded that urinary ACE2 and NEP can be used as early biomarkers to predict the incidence or progression of chronic kidney disease at early stages among individuals with type 2 diabetes. The normal values of urinary neprilysin derived from recent studies are in the range of 0.5–1 ng/mL. To conclude, urinary NEP needs to be validated with adequately powered randomized controlled studies in various postoperative scenarios like thoraco-abdominal aneurysm repair, major laparotomies including cytoreductive surgeries and heated intraperitoneal chemotherapy. Financial support and sponsorship Nil. Conflicts of interest There are no conflicts of interest.

Correlation between Urinary Angiotensin Converting Enzyme-2 &amp; Micro Albuminuria in Non-Diabetic-Hypertensive and Type 2 Diabetic-Hypertensive patients

Background: The Renin Angiotensin System (RAS) is one of the main contributors to insulin resistance which leads to T2DM and its complications. The ACE2/Angiotensin-(1-7)/Mas axis has lately been anticipated to function as a negative regulator of the RAS, consequently, rendering a shielding task against progress of type 2 diabetes, as well as, lowering blood pressure. Aim: To correlate urinary angiotensin 2 levels and microalbuminuria in Type 2 Diabetic and non-Diabetic-hypertensive patients. Methods: A particular population of selected subjects was categorized into 2 groups, with 49 subjects in each group. Diabetic and hypertensive patients were selected from the SIMS diabetic Clinic and medical wards, Lahore. Anthrometric parameters, as well as, BSR were recorded in the clinics. Blood and urine samples were collected and stored for evaluating the biochemical parameters in the Physiology Laboratory, UHS. Results: Microalbuminuria, as well as urinary ACE2 levels in group A (non-diabetic-hypertensive subjects), were greater than in group B (diabetic-hypertensive subjects). There was a significant positive correlation between uACE2 levels and microalbuminuria in group A patients. Conclusion: Although the current study reinforced the fact that both T2DM and hypertension are risk factors for CKD, contrary to our prediction based on evidence gained from various research studies, we were unable to prove that urinary ACE 2 levels are higher in patients with hypertension compounded with diabetes mellitus. Keywords: The Renin Angiotensin System (RAS), angiotensin converting enzyme 2, microalbuminuria levels.

Cardiac and Renal SARS-CoV-2 Viral Entry Protein Regulation by Androgens and Diet: Implications for Polycystic Ovary Syndrome and COVID-19.

The susceptibility and the severity of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are associated with hyperandrogenism, obesity, and preexisting pulmonary, metabolic, renal, and cardiac conditions. Polycystic ovary syndrome (PCOS), the most common endocrine disorder in premenopausal women, is associated with obesity, hyperandrogenism, and cardiometabolic dysregulations. We analyzed cardiac, renal, circulatory, and urinary SARS-CoV-2 viral entry proteins (ACE2, TMPRSS2, TMPRSS4, furin, cathepsin L, and ADAM17) and androgen receptor (AR) expression, in a peripubertal androgen exposure model of PCOS. Peripubertal female mice were treated with dihydrotestosterone (DHT) and low (LFD) or high (HFD) fat diet for 90 days. HFD exacerbated DHT-induced increase in body weight, fat mass, and cardiac and renal hypertrophy. In the heart, DHT upregulated AR protein in both LFD and HFD, ACE2 in HFD, and ADAM17 in LFD. In the kidney, AR protein expression was upregulated by both DHT and HFD. Moreover, ACE2 and ADAM17 were upregulated by DHT in both diets. Renal TMPRSS2, furin, and cathepsin L were upregulated by DHT and differentially modulated by the diet. DHT upregulated urinary ACE2 in both diets, while neither treatment modified serum ACE2. Renal AR mRNA expression positively correlated with Ace2, Tmprss2, furin, cathepsin L, and ADAM17. Our findings suggest that women with PCOS could be a population with a high risk of COVID-19-associated cardiac and renal complications. Furthermore, our study suggests that weight loss by lifestyle modifications (i.e., diet) could potentially mitigate COVID-19-associated deleterious cardiorenal outcomes in women with PCOS.

Distinct Regulation of U-ACE2 and P-ACE2 (Urinary and Plasma Angiotensin-Converting Enzyme 2) in a Japanese General Population.

[Figure: see text].

Urinary Levels of SARS-CoV-2 Nucleocapsid Protein Associate With Risk of AKI and COVID-19 Severity: A Single-Center Observational Study.

Background: Acute kidney injury (AKI) is very common in severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) disease 2019 (COVID-19) and considered as a risk factor for COVID-19 severity. SARS-CoV-2 renal tropism has been observed in COVID-19 patients, suggesting that direct viral injury of the kidneys may contribute to AKI. We examined 20 adult cases with confirmed SARS-CoV-2 infection requiring ICU supportive care in a single-center prospective observational study and investigated whether urinary markers for viral infection (SARS-CoV-2 N) and shedded cellular membrane proteins (ACE2, TMPRSS2) allow identification of patients at risk for AKI and outcome of COVID-19.Objectives: The objective of the study was to evaluate whether urinary markers for viral infection (SARS-CoV-2 N) and shedded cellular membrane proteins (ACE2, TMPRSS2) allow identification of patients at risk for AKI and outcome of COVID-19.Results: Urinary SARS-CoV-2 N measured at ICU admission identified patients at risk for AKI in COVID-19 (HR 5.9, 95% CI 1.4–26, p = 0.0095). In addition, the combination of urinary SARS-CoV-2 N and plasma albumin measurements further improved the association with AKI (HR 11.4, 95% CI 2.7–48, p = 0.0016). Finally, combining urinary SARS-CoV-2 N and plasma albumin measurements associated with the length of ICU supportive care (HR 3.3, 95% CI 1.1–9.9, p = 0.0273) and premature death (HR 7.6, 95% CI 1.3–44, p = 0.0240). In contrast, urinary ACE2 and TMPRSS2 did not correlate with AKI in COVID-19.Conclusions: In conclusion, urinary SARS-CoV-2 N levels associate with risk for AKI and correlate with COVID-19 severity.

Androgens and Diet Regulation of SARS‐CoV‐2 Viral Entry Proteins: Implications for COVID‐19 Cardiorenal Outcomes Severity in Polycystic Ovary Syndrome

BackgroundThe susceptibility and the severity of coronavirus disease 2019 (COVID‐19) caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) are associated with hyperandrogenism, obesity, and pre‐existing pulmonary, metabolic, renal or cardiac conditions. Hyperandrogenemia and ovarian dysfunction characterize polycystic ovary syndrome (PCOS), the most common endocrine disorder in premenopausal women. Women with PCOS have higher prevalence of obesity and higher incidence of cardiovascular diseases and renal injury, making them a population at risk for severe COVID‐19 outcomes.MethodsThree‐week‐old female mice were implanted with Silastic tubes filled with the nonaromatizable androgen dihydrotestosterone (DHT, 8 mg) or vehicle (empty tubes). Animals were maintained on high‐fat diet (HFD; 60% kcal fat) or low‐fat diet (LFD; 10% kcal fat, sucrose‐matched) for 90 days. Fat mass (EchoMRI), kidney and left ventricle (LV) weights as well as the cardiac and renal expression of the inflammatory markers interleukin‐6 (IL‐6), tumor necrosis factor‐α (TNF‐α), and interleukin‐1β (IL‐1β) were assessed. Moreover, circulatory, urinary, renal and cardiac levels of SARS‐CoV‐2 host cell receptor angiotensin converting enzyme 2 (ACE2) were quantified. Additionally, renal and cardiac mRNA and protein expression of the multiple cellular proteases required for viral entry (TMPRSS2, TMPRSS4, furin, cathepsin L, and ADAM17) were assessed.ResultsHFD exacerbated DHT‐mediated increases in fat mass (20.28 ± 1.10 vs. 6.78 ± 0.43 g, p<0.05), kidney weight (33.79 ± 1.35 vs. 26.52 ± 1.08 g, p<0.05), LV hypertrophy (7.48 ± 0.21 vs. 6.41 ± 0.28 g, p<0.05) and renal and cardiac expression of the inflammatory marker IL‐6 (1.5 and 2.5‐fold, respectively). PCOS mice on both diets showed higher urinary ACE2 (LFD: 18‐fold, HFD: 10‐fold) and altered renal mRNA and/or protein expression of all the measured targets. DHT upregulated renal TMPRSS2 (1.19 ± 0.04 vs. 0.98 ± 0.03 AU, p<0.05) and cathepsin L (2.64 ± 0.25 vs. 0.62 ± 0.25 AU, p<0.05) proteins in HFD‐fed DHT‐treated mice compared to LFD‐fed DHT‐treated mice. In the heart, LFD‐fed DHT‐treated mice showed no change in ACE2 and the cellular proteases on either the mRNA or protein levels except for ADAM17 protein that was significantly upregulated (1.63‐fold) compared to the vehicle‐treated group. On the other hand, while HFD‐fed DHT‐treated mice showed similar increase in cardiac ADAM17 protein (1.64‐fold vs. LFD‐fed vehicle‐treated), they showed higher mRNA expression of ACE2 (1.83‐fold) and furin (1.32‐fold) compared to the HFD‐fed vehicle‐treated mice.Conclusion and significanceOur findings highlight women with PCOS as a population with a high risk of COVID‐19 associated cardiac and renal complications. Further, our study suggests dietary control recommendations for optimal management of women with PCOS that can ultimately attenuate COVID‐19 cardiorenal outcomes.

Identifying and Validating Plasma Circulating Renin‐Angiotensin System Components as Reliable Biomarkers of Rheumatoid Arthritis

Objective The renin-angiotensin system (RAS) is an enzymatic cascade that controls the cardiovascular, renal, and adrenal functions responsible for maintaining arterial blood pressure and balancing body fluids and electrolytes. The RAS comprised of two opposing arms: i) classical arm, consists of Angiotensin (Ang) Converting Enzyme (ACE) /Ang II/ Ang II type 1 receptor (AT1R), which modulates a diverse range of biological effects, including vasoconstriction, vascular smooth muscle cell proliferation, and hypertrophy of heart vessel wall, ii) protective arm, consists of ACE2/Ang-(1-7)/Mas receptor (MasR), that acts as the counter regulatory within the RAS. Chronic imbalances of these two arms of the RAS leads to different pathophysiological conditions in renal, cardiovascular (CV), and central nervous systems. The urinary or plasma circulating level of ACE2 has been associated with different disease conditions such as renal, CV, and metabolic disorders. The plasma circulating level of ACE2 has been associated with different diseases. As a body's defense mechanism, the increased level of circulating ACE2 degrades Ang II and keeps its level within the normal range. Several studies links plasma or urinary ACE2 levels in patients with various disease. These patients exhibited reduced ACE2 activity and considerable high levels of circulating anti-ACE2 autoantibody, suggesting that an assessment of circulating ACE2, anti-ACE2, Ang II and Ang1-7 could provide a useful diagnostic biomarkers panel for patients with different inflammatory diseases. Methods This study was conducted based on an approved IRB and using twelve unidentifiable rheumatoid arthritis (RA) patients (five active and seven remission patients) samples, which were provided by the Institute of Arthritis Research. Diagnosis of RA was made using a RAPID3 questionnaire in tandem with comparing the measured amount of C-reactive protein and Vectra DA score. Levels of Ang-(1-7) and Ang II peptides were measured using liquid chromatography in tandem with mass spectrometry. Anti-ACE2 autoantibodies were measured using a previously-established ELISA method. Results The Mean ± SD results of the levels of Ang-(1-7) and Ang II peptides show that Ang-(1-7) level in the active RA group (1.29 ± 1.79) was significantly lower than in the remission RA group (7.62 ± 7.035). In contrast to Ang-(1-7), Ang II levels were significantly higher in the active RA patients (5.43 ± 4.05) when compared with the remission group (0.86 ± 0.43) (Fig.1). The mean ELISA score was significantly higher in the active RA patients (Fig.1) and was also correlated with Ang II levels in the active but not in the remission group (Fig. 2). These results suggest that anti-ACE2 antibody is associated with RA activity and intensity. Conclusion Our study suggests that estimation of the level of Ang-(1-7) and Ang II peptides in addition to assessing anti-ACE2 antibodies in patient's plasma can be considered as effective means of RA stage diagnosis. This approach also could be applied to other inflammatory conditions such as cardiovascular, renal and pulmonary diseases, diabetes and cancer.

Effects of Angiotensin II Type 1A Receptor on ACE2, Neprilysin and KIM-1 in Two Kidney One Clip (2K1C) Model of Renovascular Hypertension.

Activation of the renin angiotensin system plays a pivotal role in the regulation of blood pressure, which is mainly attributed to the formation of angiotensin-II (Ang II). The actions of Ang II are mediated through binding to the Ang-II type 1 receptor (AT1R) which leads to increased blood pressure, fluid retention, and aldosterone secretion. In addition, Ang II is also involved in cell injury, vascular remodeling, and inflammation. The actions of Ang II could be antagonized by its conversion to the vasodilator peptide Ang (1–7), partly generated by the action of angiotensin converting enzyme 2 (ACE2) and/or neprilysin (NEP). Previous studies demonstrated increased urinary ACE2 shedding in the db/db mouse model of diabetic kidney disease. The aim of the study was to investigate whether renal and urinary ACE2 and NEP are altered in the 2K1C Goldblatt hypertensive mice. Since AT1R is highly expressed in the kidney, we also researched the effect of global deletion of AT1R on renal and urinary ACE2, NEP, and kidney injury marker (KIM-1). Hypertension and albuminuria were induced in AT1R knock out (AT1RKO) and WT mice by unilateral constriction of the renal artery of one kidney. The 24 h mean arterial blood pressure (MAP) was measured using radio-telemetry. Two weeks after 2K1C surgery, MAP and albuminuria were significantly increased in WT mice compared to AT1RKO mice. Results demonstrated a correlation between MAP and albuminuria. Unlike db/db diabetic mice, ACE2 and NEP expression and activities were significantly decreased in the clipped kidney of WT and AT1RKO compared with the contralateral kidney and sham control (p < 0.05). There was no detectable urinary ACE2 and NEP expression and activity in 2K1C mice. KIM-1 was significantly increased in the clipped kidney of WT and AT1KO (p < 0.05). Deletion of AT1R has no effect on the increased urinary KIM-1 excretion detected in 2K1C mice. In conclusion, renal injury in 2K1C Goldblatt mouse model is associated with loss of renal ACE2 and NEP expression and activity. Urinary KIM-1 could serve as an early indicator of acute kidney injury. Deletion of AT1R attenuates albuminuria and hypertension without affecting renal ACE2, NEP, and KIM-1 expression.

Urinary angiotensin converting enzyme 2 and disease activity in pediatric IgA nephropathy.

Background : Our previous studies demonstrated that the intrarenal renin-angiotensin system (RAS) status was activated in pediatric patients with chronic glomerulonephritis. In the present study, we tested the hypothesis that angiotensin-converting enzyme 2 (ACE2) expression in the kidney is associated with the development of pediatric IgA nephropathy. Methods : We analyzed urinary ACE2 levels and ACE2 expression in the kidney tissues of pediatric patients with IgA nephropathy treated with RAS blockade. Paired tests were used to analyze changes from the first to the second biopsy. Results : Urinary ACE2 levels were significantly decreased after RAS blockade treatment, accompanied by decreased ACE2 expression levels in kidney tissues, urinary protein levels and mesangial hypercellularity scores. Urinary ACE2 levels at the first biopsy were positively correlated with the ACE2 expression levels. Conclusions : These data suggest that urinary ACE2 is associated with ACE2 expression in the diseased kidney, which correlates with the pathogenesis of IgA nephropathy in pediatric patients. J. Med. Invest. 68 : 292-296, August, 2021.

Could urinary ACE2 protein level help identify individuals susceptible to SARS-CoV-2 infection and complication?

Could urinary ACE2 protein level help identify individuals susceptible to SARS-CoV-2 infection and complication?

Abstract P2044: Renoprotective Effects of Canagliflozin in Db/db Diabetic Mice

Diabetes is one of the major causes of chronic kidney disease which could lead to end-stage renal disease. Previously, we have shown increased urinary angiotensin converting enzyme 2 (ACE2) and neprilysin (NEP) in db/db mouse model of type 2 diabetes, which was attenuated by normalizing hyperglycemia and glycosuria with the PPAR-γ agonist, rosiglitazone and pioglitazone. Sodium glucose co-transporter 2 (SGLT-2) inhibitor controls hyperglycemia by inhibiting renal glucose reabsorption and cause glycosuria. The aim of this study is to investigate the effect of SGLT-2 inhibitor, Canagliflozin on the urinary ACE2 and NEP and also to determine its effects on albuminuria and some of the renal injury biomarkers, neutrophil-gelatinase associated lipocalin (NGAL), sirtuins-1 (SIRT1) and arginase-II (Arg-II). Six- weeks old db/db and lean control mice were fed either normal chow or canagliflozin diet (20mg/kg/day) for 15 weeks. Metabolic and renal parameters were measured weekly and urine was collected for evaluating glycosuria, protein expression and enzyme activity. At 5 weeks, db/db mice showed significant increase in urinary ACE2, NEP and NGAL expression compared to lean mice (p&lt;0.001). Treatment with canagliflozin for 15 weeks significantly decreased blood glucose in db/db compared to untreated mice (206.58 ± 38.35 vs. 461.82 ± 40.63 mg/dL, p &lt;0.0001), with no effect in lean mice. Unlike PPAR-γ agonist, management of hyperglycemia with canagliflozin was not associated with attenuation of urinary albumin and ACE2. However, it attenuated urinary NGAL expression. As expected canagliflozin increased renal glycosuria in db/db and lean mice ( p &lt;0.0001) which was associated with significant increase in urinary NEP expression and activity. Western blot showed significant downregulation of SIRT1 in the kidney of 21 weeks old db/db mice which was upregulated by canagliflozin treatment. Further, there was upregulation of renal Arg-II in the db/db mice which was downregulated by canagliflozin. In conclusion, the renoprotective effects of canagliflozin could be mediated by upregulation of renal SIRT1 expression and downregulation of the renal injury markers, NGAL and Arg-II.

Abstract P2036: Effects of Angiotensin II Type 1 A Receptor (AT1aR) on Renal and Urinary Biomarkers of Acute Kidney Injury in Two Kidney One Clip (2K1C) Model of Renovascular Hypertension

Renin angiotensin system (RAS) plays a key role in the pathogenesis and progression of hypertension and renal injury. Angiotensin-II (Ang-II) type 1 receptor (AT1R) mediates the actions of Ang II to increase blood pressure, fluid retention, and aldosterone secretion. Ang II is involved in cell injury, vascular remodeling, and inflammation by releasing inflammatory and pro-inflammatory mediators. The effects of Ang II could be counteracted through its catabolism into the vasodilator peptide Ang (1-7) by angiotensin converting enzyme 2 (ACE2) and/or neprilysin (NEP). Diabetes and high blood pressure are the major causes of chronic kidney disease which could lead to end stage renal disease. Our previous studies demonstrated that shedding of renal ACE2 and NEP in the urine of db / db diabetic mice could act as biomarkers for diabetic kidney disease. The aim of the study is to test the hypothesis that there is increased renal ACE2 and NEP shedding in the urine of two-kidney one clip (2K1C) model of renovascular hypertension. In addition, we investigated the effects of deleting AT1aR on these RAS components and on kidney injury molecule-1 (KIM-1). Renovascular hypertension was induced in AT1aR knock out (KO) and WT mice by 2K1C surgery. Blood pressure (BP) measurement by radio-telemetry revealed significant increase of 46.1± 3.6 mmHg in 2K1C mice compared to control mice (p&lt;0.001). Deleting AT1aR significantly decreased the BP in 2K1C mice compared to 2K1C WT mice (69.86 ± 0.19 vs. 151.69 ± 12.22 mmHg, p&lt;0.001). Renal expression and activity of ACE2 and NEP were significantly decreased in the clipped kidney of WT and AT1KO group (p&lt;0.05). In contrast to diabetes, there was no detectable urinary ACE2 and NEP expression and activity in 2KIC mice. In addition, urinary expression of KIM-1 was increased in the clipped kidney. Although deleting AT1R attenuated albuminuria and hypertension, it did not altered urinary expression of KIM-1. These results suggest that the decreased renal NEP and ACE2 in the clipped kidney of 2K1C model may thus worsen kidney injury via impairment of Ang (1-7) formation. In conclusion, urinary KIM-1 could serve as an early indicator of non-diabetic acute kidney injury. Urinary ACE2 and NEP could be used as biomarker of diabetic kidney disease.

A review of urinary angiotensin converting enzyme 2 in diabetes and diabetic nephropathy.

Urinary angiotensin converting enzyme 2 (ACE2) is significantly increased in diabetes and diabetic nephropathy. While studies on its clinical significance are still underway, its urinary expression, association with metabolic and renal parameters has been in the recent past considerably studied. The recent studies have demystified urine ACE2 in many ways and suggested the roles it could play in the management of diabetic nephropathy. In all studies the expression of urinary ACE2 was determined by enzyme activity assay and/with the quantification of ACE2 protein and mRNA by methods whose reliability are yet to be evaluated. This review summarizes recent findings on expression of urinary ACE2, examines its relationship with clinical parameters and highlights possible applications in management of diabetic nephropathy.

Increased urinary angiotensin converting enzyme 2 and neprilysin in patients with type 2 diabetes.

Angiotensin converting enzyme 2 (ACE2) and neprilysin (NEP) are metalloproteases that are highly expressed in the renal proximal tubules. ACE2 and NEP generate renoprotective angiotensin (1-7) from angiotensin II and angiotensin I, respectively, and therefore could have a major role in chronic kidney disease (CKD). Recent data demonstrated increased urinary ACE2 in patients with diabetes with CKD and kidney transplants. We tested the hypothesis that urinary ACE2, NEP, and a disintegrin and metalloproteinase 17 (ADAM17) are increased and could be risk predictors of CKD in patients with diabetes. ACE2, NEP, and ADAM17 were investigated in 20 nondiabetics (ND) and 40 patients with diabetes with normoalbuminuria (Dnormo), microalbuminuria (Dmicro), and macroalbuminuria (Dmacro) using ELISA, Western blot, and fluorogenic and mass spectrometric-based enzyme assays. Logistic regression model was applied to predict the risk prediction. Receiver operating characteristic curves were drawn, and prediction accuracies were calculated to explore the effectiveness of ACE2 and NEP in predicting diabetes and CKD. Results demonstrated that there is no evidence of urinary ACE2 and ADAM17 in ND subjects, but both enzymes were increased in patients with diabetes, including Dnormo. Although there was no detectable plasma ACE2 activity, there was evidence of urinary and plasma NEP in all the subjects, and urinary NEP was significantly increased in Dmicro patients. NEP and ACE2 showed significant correlations with metabolic and renal characteristics. In summary, urinary ACE2, NEP, and ADAM17 are increased in patients with diabetes and could be used as early biomarkers to predict the incidence or progression of CKD at early stages among individuals with type 2 diabetes.

Angiotensin-converting enzyme 2 amplification limited to the circulation does not protect mice from development of diabetic nephropathy

Blockers of the renin-angiotensin system are effective inthe treatment of experimental and clinical diabetic nephropathy. An approach different from blocking the formation or action of angiotensin II (1-8) that could also be effective involves fostering its degradation. Angiotensin-converting enzyme 2 (ACE2) is a monocarboxypeptidase that cleaves angiotensin II (1-8) to form angiotensin (1-7). Therefore, we examined the renal effects of murine recombinant ACE2 in mice with streptozotocin-induced diabetic nephropathy as well as that of amplification of circulating ACE2 using minicircle DNA delivery prior to induction of experimental diabetes. This delivery resulted in a long-term sustained and profound increase in serum ACE2 activity and enhanced ability to metabolize an acute angiotensin II (1-8) load. In mice with streptozotocin-induced diabetes pretreated with minicircle ACE2, ACE2 protein in plasma increased markedly and this was associated with a more than 100-fold increase in serum ACE2 activity. However, minicircle ACE2 did not result in changes in urinary ACE2 activity as compared to untreated diabetic mice. In both diabetic groups, glomerular filtration rate increased significantly and to the same extent ascompared to non-diabetic controls. Albuminuria, glomerular mesangial expansion, glomerular cellularity, and glomerular size were all increased to a similar extent in minicircle ACE2-treated and untreated diabetic mice, as compared to non-diabetic controls. Recombinant mouse ACE2 given for 4 weeks by intraperitoneal daily injections in mice with streptozotocin-induced diabetic nephropathy also failed to improve albuminuria or kidney pathology. Thus, a profound augmentation of ACE2 confined to the circulation failed to ameliorate the glomerular lesions and hyperfiltration characteristic of early diabetic nephropathy. These findings emphasize the importance of targeting thekidney rather than the circulatory renin angiotensin system to combat diabetic nephropathy.

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.