Urinary Angiotensin Converting Enzyme Research Articles

#68 Novel clinical application of urinary angiotensin-converting enzyme assay in renal sarcoidosis: a retrospective observational study

Abstract Background and Aims Renal manifestations of sarcoidosis are observed in approximately 1–5% and characterized mainly by granulomatous interstitial nephritis, which could lead to kidney failure. Angiotensin-converting enzyme (ACE) reflects the presence of granuloma; hence, serum ACE and tubular injury markers are measured in renal sarcoidosis (RS); however, they either have low sensitivity or low specificity. Although its practicality has not been established, the use of ACE levels in cerebrospinal fluid has been reported for neuro-sarcoidosis. Therefore, we hypothesized that urinary ACE could reflect granuloma presence in the kidneys and could be a disease-specific marker for RS. Method This was a single-center, retrospective observational study. We evaluated clinical characteristics and kidney biopsy findings from electronic medical record surveys and kidney biopsy specimens. The control groups included sarcoidosis without renal involvement (SAR-control) and tubulointerstitial nephritis without a sarcoidosis etiology (TIN-control). Serum and urinary ACE levels were measured, and urinary fractional excretion of ACE (FEACE) was investigated. Serum ACE assay and urinary ACE levels were measured using the colorimetric method with p-hydroxybenzoyl-glycyl-L-histidyl-L-leucine. A total of six patients (three with RS; one with sarcoidosis as controls; and two with TIN controls) with undetectable urinary ACE levels were excluded from the urinary ACE and FEACE analyses. Tubulointerstitial lesions were visually assessed for essentially normal (%), inflammatory cell infiltrates (%), tubular atrophy (%), and interstitial fibrosis (%) by several board-certified nephrologists, and analyzed the correlation of these lesions with each marker. Results This study included 18 patients with RS, 18 as SAR-control, and 10 as TIN-control. Serum ACE, urinary ACE, and FEACE levels in the RS group were significantly higher than those in the control groups. Receiver operating characteristic analysis identified FEACE and serum ACE levels as useful biomarkers for diagnosing RS among patients with both controls, respectively. In the RS group, FEACE was positively correlated with the degree of tubulointerstitial injury (r = 0.69, P = 0.0045), and the cutoff value of FEACE for diffuse tubulointerstitial injury (50.0% or more) was 0.39%, with a sensitivity and specificity of 100.0%. Furthermore, obvious positive correlations were observed among FEACE, inflammatory cell infiltrates (r = 0.53, P = 0.044), and interstitial fibrosis (r = 0.56, P = 0.029) in the RS group but not in the TIN-control. Conclusion Combination with serum and urinary ACE could help diagnose and assess disease severity among patients with RS.

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.

Pediatric dyslipidemia is associated with increased urinary ACE activity, blood pressure values, and carotidal-femoral pulse wave velocity.

This study aimed to evaluate the enzymatic activity of the angiotensin-converting enzyme (ACE) in children and adolescents to investigate their relationship with dyslipidemia and other cardiometabolic alterations. Anthropometric measurements, blood pressure (BP), and fasting lipid concentrations were taken from 360 subjects. Categorization was done according to the levels of each lipoprotein (total cholesterol, triglycerides (TG), LDL-C, HDL-C, and non-HDL-C) into three groups: normolipidemic (NL), borderline (BL), and dyslipidemic (DL). Enzymatic activity in urine was measured using the substrates Z-FHL-OH and hippuryl-HL-OH (h-HL-OH) and the ACE activity ratio (Z-FHL-OH/h-HL-OH) was calculated. Dyslipidemic levels of HDL-C, TG, and LDL-C were observed in 23%, 9%, and 3% of the participants, respectively, and were more frequent in obese children (Chi-square, p < 0.001). ACE activity ratio was augmented in BL(HDL-C) when compared to NL(HDL-C) (5.06 vs. 2.39, p < 0.01), in DL(LDL-C) in comparison to BL(LDL-C) and NL(LDL-C) (8.7 vs. 1.8 vs. 3.0, p < 0.01), and in DL(non-HDL-C) than in BL(non-HDL-C) and in NL(non-HDL-C) (6.3 vs. 2.1 vs. 2.9, p = 0.02). The groups with impaired HDL-C and TG levels presented an increased diastolic BP percentile, and a higher systolic BP percentile was observed in BL(TG) and DL(TG). The carotidal-femoral pulse wave velocity (cfPWV) was higher in the groups with DL levels of TG and LDL-C than in NL groups. Hypertriglyceridemia was associated with higher cfPWV. No direct impact of the ACE activity on BP values was observed in this cohort, however, there was an association between hyperlipidemia and ACE upregulation which can trigger mechanisms driving to early onset of hypertension and cardiovascular disease. Graphical abstract exemplifying the cohort, categorization of subjects into the groups NL normolipidemic, BL borderline, DL dyslipidemic, methods, and main findings. Pediatric dyslipidemia was consistent with dyslipidemia secondary to obesity (DSO), associated with higher urinary angiotensin-converting enzyme (ACE) activity ratio, BP blood pressure values, and carotidal-femoral pulse wave velocity (cfPWV).

Urinary ACE Phenotyping as a Research and Diagnostic Tool: Identification of Sex-Dependent ACE Immunoreactivity.

Angiotensin-converting enzyme (ACE) is highly expressed in renal proximal tubules, but ACE activity/levels in the urine are at least 100-fold lower than in the blood. Decreased proximal tubular ACE has been associated with renal tubular damage in both animal models and clinical studies. Because ACE is shed into urine primarily from proximal tubule epithelial cells, its urinary ACE measurement may be useful as an index of tubular damage. We applied our novel approach-ACE phenotyping-to characterize urinary ACE in volunteer subjects. ACE phenotyping includes (1) determination of ACE activity using two substrates (ZPHL and HHL); (2) calculation of the ratio of hydrolysis of the two substrates (ZPHL/HHL ratio); (3) quantification of ACE immunoreactive protein levels; and (4) fine mapping of local ACE conformation with mAbs to ACE. In normal volunteers, urinary ACE activity was 140-fold less than in corresponding plasma/serum samples and did not differ between males and females. However, urinary ACE immunoreactivity (normalized binding of 25 mAbs to different epitopes) was strongly sex-dependent for the several mAbs tested, an observation likely explained by differences in tissue ACE glycosylation/sialylation between males and females. Urinary ACE phenotyping also allowed the identification of ACE outliers. In addition, daily variability of urinary ACE has potential utility as a feedback marker for dieting individuals pursuing weight loss. Urinary ACE phenotyping is a promising new approach with potential clinical significance to advance precision medicine screening techniques.

Urinary angiotensin-converting enzyme 2 and metabolomics in COVID-19-mediated kidney injury.

BackgroundAngiotensin-converting enzyme 2 (ACE2), the receptor for SARS-CoV-2, is highly expressed in the kidneys. Beyond serving as a crucial endogenous regulator of the renin-angiotensin system, ACE2 also possess a unique function to facilitate amino acid absorption. Our observational study sought to explore the relationship between urine ACE2 (uACE2) and renal outcomes in COVID-19.MethodsIn a cohort of 104 patients with COVID-19 without acute kidney injury (AKI), 43 patients with COVID-19-mediated AKI, and 36 non-COVID-19 controls, we measured uACE2, urine tumor necrosis factor receptors I and II (uTNF-RI and uTNF-RII), neutrophil gelatinase-associated lipocalin (uNGAL). We also assessed ACE2 staining in autopsy kidney samples and generated a propensity-score matched subgroup of patients to perform a targeted urine metabolomic study to describe the characteristic signature of COVID-19.ResultsuACE2 is increased in patients with COVID-19, and further increased in those that developed AKI. After adjusting uACE2 levels for age, sex and previous comorbidities, increased uACE2 was independently associated with over 3-fold higher risk of developing AKI (OR 3.05, 95%CI:1.23‒7.58,p = 0.017). Increased uACE2 corresponded to a tubular loss of ACE2 in kidney sections and strongly correlated with uTNF-RI and uTNF-RII. Urine quantitative metabolome analysis revealed an increased excretion of essential amino acids in patients with COVID-19, including leucine, isoleucine, tryptophan and phenylalanine. Additionally, a strong correlation was observed between urine amino acids and uACE2.ConclusionsElevated uACE2 is related to AKI in patients with COVID-19. The loss of tubular ACE2 during SARS-CoV-2 infection demonstrates a potential link between aminoaciduria and proximal tubular injury.

Abstract 085: Pediatric Dyslipidemia Is Associated With Higher Urinary Angiotensin Converting Enzyme Activity And Blood Pressure Values In A Brazilian Cohort

Angiotensin converting enzyme (ACE) upregulation in adults contributes to cardiometabolic diseases including dyslipidemia and atherosclerosis. Objective: Investigate whether ACE activity is modulated in pediatric dyslipidemia. Methods: We evaluated anthropometric measurements, blood pressure and fasting lipid concentrations of 360 individuals (190 boys and 170 girls) aged from 6 to 19 year (mean 11.6). Categorization was done according to the levels of each lipoprotein (Total cholesterol (TC), triglycerides (TG), LDL-C, HDL-C, and non-HDL) into three groups: normolipidemic, borderline and dyslipidemic; reference values were according to American Academy of Pediatrics and Brazilian Pediatric Society. ACE activity in urine was measured with the substrates Z-FHL-OH and hippuryl-HL-OH. ACE activity ratio to infer N-domain activity was calculated (Z-FHL/h-HL). Results: Dyslipidemic levels of HDL-C, TG and LDL-C were observed in 23%, 9% and 3% of the participants, respectively. These clinical alterations were more frequent in obese children (Chi-square, p &lt;0.001). ACE activity (Z-FHL) was higher in the groups with borderline and dyslipidemic values of HDL-C than in normolipidemic group (0.077 vs 0.070 vs 0.037 nmol/min/mg of creatinine, p = 0.01). The ratio of ACE activity was augmented in HDL-C borderline group when compared to HDL-C normolipidemic group (5.06 vs 2.39, p &lt;0.01) and in LDL-C dyslipidemic group than in LDL-C borderline and LDL-C normolipidemic groups (8.66 vs 1.84 vs 2.88, p = 0.02). Volunteers with normal levels of TG presented lower diastolic blood pressure (DBP) mmHg (p = 0.02), percentile of DBP (p &lt; 0.01), and percentile of systolic blood pressure (SBP) (p &lt;0.01) than volunteers with borderline and dyslipidemic levels of TG. Also, increased DBP mmHg was observed in borderline and dyslipidemic HDL-C groups when compared to normolipidic HDL-C group. Conclusion: Pediatric hypertriglyceridemia, HDL-C deficiency and high LDL-C were related to higher urinary ACE activity (mainly N-domain). ACE modulation can contribute to higher risk of hypertension and other cardiometabolic alterations still in childhood and adolescence. Also, impairment in lipoprotein levels was associated with higher blood pressure values.

Levels of angiotensin-converting enzyme 1 and 2 in serum and urine of children with Sickle Cell Disease

Introduction:Sickle cell nephropathy begins in childhood and presents early increases in glomerular filtration, which, over the long term, can lead to chronic renal failure. Several diseases have increased circulating and urinary angiotensin-converting enzyme (ACE) activity, but there is little information about changes in ACEs activity in children with sickle cell disease (SCD).Objective:We examined circulating and urinary ACE 1 activity in children with SCD.Methods:This cross-sectional study compared children who were carriers of SCD with children who comprised a control group (CG). Serum and urinary activities of ACE were evaluated, as were biochemical factors, urinary album/creatinine rates, and estimated glomerular filtration rate.Results:Urinary ACE activity was significantly higher in patients with SCD than in healthy children (median 0.01; range 0.00-0.07 vs median 0.00; range 0.00-0.01 mU/mL·creatinine, p < 0.001. No significant difference in serum ACE activities between the SCD and CG groups was observed (median 32.25; range 16.2-59.3 vs median 40.9; range 18.0-53.4) mU/m`L·creatinine, p < 0.05.Conclusion:Our data revealed a high urinary ACE 1 activity, different than plasmatic level, in SCD patients suggesting a dissociation between the intrarenal and systemic RAAS. The increase of urinary ACE 1 activity in SCD patients suggests higher levels of Ang II with a predominance of classical RAAS axis, that can induce kidney damage.

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.

Reduced urinary levels of angiotensin-converting enzyme 2 activity predict acute kidney injury in critically ill patients

Objective: Angiotensin-converting enzyme 2 activity reflects non-classical renin-angiotensin system upregulation. We assessed the association of urinary angiotensin-converting enzyme 2 (uACE2) activity with acute kidney injury (AKI). Design, setting and participants: A prospective observational study in which we measured uACE2 activity in 105 critically ill patients at risk of AKI. We report AKI stage 2 or 3 at 12 hours of urine collection (AKI12h) and AKI stage 2 or 3 at any time during intensive care unit stay in patients free from any stage of AKI at inclusion (AKIICU). AKI prediction was assessed using area under the receiver-operating characteristics curve (AUROC) and net reclassification indices (NRIs). Main outcome measure: AKI stage 2 or 3 at 12 hours of urine collection. Results: Within 12 hours of inclusion, 32 of 105 patients (30%) had developed AKI12h. Corrected uACE2 activity was significantly higher in patients without AKI12h compared with those with AKI12h (median [interquartile range], 13 [6-24] v 7 [4-10] pmol/min/mL per mmol/L of urine creatinine; P < 0.01). A 10-unit increase in uACE2 was associated with a 28% decrease in AKI12h risk (odds ratio [95% CI], 0.72 [0.46-0.97]). During intensive care unit admission, 39 of 76 patients (51%) developed AKIICU. uACE2 had an AUROC for the prediction of AKI12h of 0.68 (95% CI, 0.57-0.79), and correctly reclassified 28% of patients (positive NRI) to AKI12h. Patients with uACE2 > 8.7 pmol/min/mL per mmol/L of urine creatinine had a significantly lower risk of AKIICU on log-rank analysis (52% v 84%; P < 0.01). Conclusions: Higher uACE2 activity was associated with a decreased risk of AKI stage 2 or 3. Our findings support future evaluations of the role of the non-classical renin-angiotensin system during AKI.

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.

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.

Abstract P394: Ace Activity and Its Expression as a Hypertension Biomarker in the Urine of Pediatric Renal Transplant Recipients

Pediatric hypertension is a risk factor for hypertension in adults. Primary hypertension results from genetic, environmental, and behavioral factors. Renin Angiotensin System is important to control cardiovascular and renal functions. Angiotensin Converting Enzyme (ACE) has isoforms: somatic (130-190 kDa) and testicular (90-110 kDa). Casarini et al. described the N-domain isoform of 90kDa, in urine of hypertensive humans as a possible hypertension biomarker. Objective: Analyze ACE isoform in urine of pediatric renal transplant recipients before and after transplantation and urine of donor, to verify the 90 kDa isoform transmission and correlating with hypertension. Methods: 30 pediatric recipients with living donors; ACE activity was performed before, 1, 3 and 6 months post transplantation using ZPhe and HHL as substrates and its ratio. Results: 69% had pre-transplant hypertension and 85.7% family history. After 6 months 25% remained. From the pre-transplant until the sixth month after, ACE activity was reduced. In the model of linear regression between ACE activity and variables, including pre-transplantation for the substrates ZPhe-HL remained significant, time and uropathy (0.023 more); with HHL, time and uropathy with higher activity of ACE (0.019 more) and for weight, reduction of ACE activity ( 0.00038 at 1 kg increase in weight). At post-transplant only time and weight remained significant with ZPhe-HL, and for HHL uropathy, proteinuria (0.010 more ACE activity) and weight. Recipients before transplantation had higher urinary ACE activity when compared to controls. Western blotting data showed that all recipients without ACE 90KDa when received a kidney from the donor that had this isoform, started to express it. Conclusions: Increased ACE activity in uropathies may be associated with renal injury or intra-renal production. Proteinuria, a marker of renal injury, can be associated with increase on ACE activity. With nutritional recovery and a healthy kidney after transplantation, patients can reverse the degree of renal damage and decrease the production of ACE.

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.

Urinary angiotensin converting enzyme 2 is strongly related to urinary nephrin in type 2 diabetes patients.

Podocyte lesion is recently recognized as an early event in diabetic kidney disease (DKD) and is reflected by urinary (u) nephrin (Neph) shedding. Angiotensin II plays an important role in podocyte dysfunction of diabetes. Angiotensin converting enzyme 2 (ACE2) is the main ACE variant in podocytes and counteracts deleterious angiotensin II effects. We assessed for the first time the relation of uACE2 and uNeph in type 2 diabetes subjects. Seventy-five type 2 diabetes patients were included in a transversal study. History, clinical and laboratory data, urinary albumin-to-creatinine ratio (uACR), and ELISA determination of uNeph and uACE2 were obtained. uNeph was 349.00±133.42pg/ml, and uACE2 was 45.50 (36.35-62.60) pg/ml. uNeph correlated to uACE2 (r=0.44, p<0.001) and to uACR (r=0.25, p=0.032). In multivariate regression, introducing parameters that are known to be related to DKD, uACE2 (p<0.0001), LDL cholesterol (p=0.02) and glycated hemoglobin (p=0.03) remained significant predictors of uNeph. Normoalbuminuric patients had lower uNeph than patients with uACR>30mg/g (325.50±135.45 vs 391.03±121.40pg/ml, p=0.04); they also had a tendency versus lower uACE2 [41.40 (34.30-60.65) vs 52.57 (37.95-69.85) pg/ml, p=0.06]. A cutoff for uNeph of 451.6pg/ml was derived from the ROC curve analysis; uACE2 was the main determinant for uNeph being above or below this cutoff-OR=1.09; 95%CI (1.04-1.15), p=0.001. Patients taking blockers of the renin angiotensin system had similar uNeph and uACE2. uNeph and uACE2 were not influenced by renal function. uNeph is significantly correlated to uACE2 and uACR in type 2 diabetes patients.

Urinary angiotensin-converting enzyme 2 increases in diabetic nephropathy by angiotensin II type 1 receptor blocker olmesartan.

Angiotensin-converting enzyme 2 (ACE2) is a member of the renin-angiotensin system that degrades angiotensin (Ang) II to the seven-amino acid peptide fragment Ang-(1-7). We evaluated the changes in urinary ACE2 levels in response to treatment with the angiotensin II type 1 receptor blocker olmesartan in diabetes patients with nephropathy. This prospective, open-label, interventional study was conducted with 31 type 2 diabetes patients with nephropathy. After initial evaluation, patients received 20 mg/day olmesartan, which was increased to 40 mg/day over a 24-week period. In diabetes patients with chronic kidney disease, olmesartan significantly increased urinary ACE2 levels independently of blood pressure and plasma aldosterone levels and reduced albuminuria, urinary liver-type fatty acid binding protein (L-FABP), and plasma aldosterone levels. Multivariable regression analysis revealed that the change in urinary L-FABP levels was an independent predictor of increased urinary ACE2 levels. Olmesartan may have the unique effect of increasing urinary ACE2 levels. However, whether this contributes to olmesartan's renoprotective effect must be examined further.

Increased Urinary Angiotensin-Converting Enzyme 2 in Renal Transplant Patients with Diabetes

Angiotensin-converting enzyme 2 (ACE2) is expressed in the kidney and may be a renoprotective enzyme, since it converts angiotensin (Ang) II to Ang-(1-7). ACE2 has been detected in urine from patients with chronic kidney disease. We measured urinary ACE2 activity and protein levels in renal transplant patients (age 54 yrs, 65% male, 38% diabetes, n = 100) and healthy controls (age 45 yrs, 26% male, n = 50), and determined factors associated with elevated urinary ACE2 in the patients. Urine from transplant subjects was also assayed for ACE mRNA and protein. No subjects were taking inhibitors of the renin-angiotensin system. Urinary ACE2 levels were significantly higher in transplant patients compared to controls (p = 0.003 for ACE2 activity, and p≤0.001 for ACE2 protein by ELISA or western analysis). Transplant patients with diabetes mellitus had significantly increased urinary ACE2 activity and protein levels compared to non-diabetics (p<0.001), while ACE2 mRNA levels did not differ. Urinary ACE activity and protein were significantly increased in diabetic transplant subjects, while ACE mRNA levels did not differ from non-diabetic subjects. After adjusting for confounding variables, diabetes was significantly associated with urinary ACE2 activity (p = 0.003) and protein levels (p<0.001), while female gender was associated with urinary mRNA levels for both ACE2 and ACE. These data indicate that urinary ACE2 is increased in renal transplant recipients with diabetes, possibly due to increased shedding from tubular cells. Urinary ACE2 could be a marker of renal renin-angiotensin system activation in these patients.

Urinary angiotensin converting enzyme (ACE) activity in normotensive hyperoxaluric rats.

Urinary angiotensin converting enzyme (ACE) activity in normotensive hyperoxaluric rats.

Urinary angiotensin-converting enzyme activity in type 2 diabetes mellitus: its relationship to diabetic nephropathy.

Urinary angiotensin-converting enzyme (ACE) and urinary N-acetyl-beta-D-glucosaminidase (NAG) were measured after a 5-year interval in 38 non-azotemic type 2 diabetic patients. Of these patients at baseline, 16 had nil nephropathy, 15 had incipient nephropathy, and 7 had overt nephropathy. During the follow-up, 6 and 1 of the 16 patients with nil nephropathy developed incipient and overt nephropathy, respectively. Four of the 15 patients with incipient nephropathy progressed to overt nephropathy. The 7 patients with overt nephropathy continued to have overt nephropathy, with slight azotemia in one. Urinary ACE and NAG levels were normal at baseline and showed no significant elevations at follow-up in the patients with nil nephropathy, no significant changes in baseline and modest elevations at follow-up in the patients with incipient nephropathy, and high at baseline and marked elevations at follow-up in the patients with overt nephropathy. In all patients, urinary ACE during the follow-up was positively correlated with urinary albumin or NAG, but not with glomerular filtration rate. Urinary ACE may be of poor prognostic value for the follow-up of diabetic patients, which is at variance with urinary albumin.

Angiotensin I converting enzyme activity in adriamycin induced nephrosis in rats

Activity of the dipeptidyl hydrolase angiotensin converting enzyme (ACE) has been observed to be altered by treatment with adriamycin (ADR). We used an animal model of ADR nephrotoxicity to study the effects on ACE in serum, urine and tissues on days 5, 10, 15, 20, 25 and 30 after ADR administration. Both glomerular and tubular injury occurred as evidenced by heavy proteinuria, albuminuria and increased urine N-acetyl glucosaminidase (NAG) excretion. Serum ACE was signifcaantly elevated on days 20, 25 and 30. Of great interest was the excretion of ACE in urine of treated rats which ran parallel with the total protein excretion above the barely detectable levels found in controls. ACE activity increased in kidney, adrenal gland and liver on days 15, 20, 25 and 30. Heart and brain ACE levels increased on days 25 and 30. Increased ACE activity in aorta and lungs occurred on days 20, 25 and 30. ACE activity decreased in kidney, aorta, heart and brain on days 5 and 10. These observations strongly suggest a contribution of various tissues to elevate the serum ACE level. Urinary ACE may be of potential use as an index for renal glomerular and tubular damage.

A Reliable Radiometric Assay for the Determination of Angiotensin I-Converting Enzyme Activity in Urine

We present a radiometric assay for the determination of urinary angiotensin-converting enzyme activity, using benzoyl-[1-14C]glycyl-L-histidyl-L-leucine as the substrate. An optimal pH of 8.3, an optimal chloride concentration of 0.375 mol/l and complete inhibition by EDTA-Na2, captopril and enalaprilat confirm the specificity of the assay. Comparison of dialysis and ultrafiltration for concentration of urine showed the existence of angiotensin-converting enzyme inhibitors in human urine. Dialysis against water was the more effective method for avoiding enzyme inhibition. After dialysis of urine, the assay was linear with time and with enzyme concentration; it was highly sensitive (60 mU/l) and showed good reproducibility. Under our technical conditions, we found angiotensin-converting enzyme activity in urine samples with quantitatively abnormal protein contents, but not in normal urine. Urinary angiotensin-converting enzyme did not correlate with proteinuria nor with water-salt parameters or creatinine. We confirm the kidney tubular epithelial origin of the enzyme, and propose the use of our assay to study urinary angiotensin-converting enzyme as a marker of renal tubular damage.