María José Soler

Glomerular Localization and Expression of Angiotensin-Converting Enzyme 2 and Angiotensin-Converting Enzyme: Implications for Albuminuria in Diabetes

Angiotensin-converting enzyme 2 (ACE2) expression has been shown to be altered in renal tubules from diabetic mice. This study examined the localization of ACE and ACE2 within the glomerulus of kidneys from control (db/m) and diabetic (db/db) mice and the effect of chronic pharmacologic ACE2 inhibition. ACE2 co-localized with glomerular epithelial cell (podocyte) markers, and its localization within the podocyte was confirmed by immunogold labeling. ACE, by contrast, was seen only in glomerular endothelial cells. By immunohistochemistry, in glomeruli from db/db mice, strong ACE staining was found more frequently than in control mice (db/db 64.6 +/- 6.3 versus db/m 17.8 +/- 3.4%; P < 0.005). By contrast, strong ACE2 staining in glomeruli from diabetic mice was less frequently seen than in controls (db/db 4.3 +/- 2.4 versus db/m 30.6 +/- 13.6%; P < 0.05). For investigation of the significance of reduced glomerular ACE2 expression, db/db mice were treated for 16 wk with a specific ACE2 inhibitor (MLN-4760) alone or combined with telmisartan, a specific angiotensin II type 1 receptor blocker. At the end of the study, glomerular staining for fibronectin, an extracellular matrix protein, was increased in both db/db and db/m mice that were treated with MLN-4760. Urinary albumin excretion (UAE) increased significantly in MLN-4760-treated as compared with vehicle-treated db/db mice (743 +/- 200 versus 247 +/- 53.9 microg albumin/mg creatinine, respectively; P < 0.05), and the concomitant administration of telmisartan completely prevented the increase in UAE associated with the ACE2 inhibitor (161 +/- 56; P < 0.05). It is concluded that ACE2 is localized in the podocyte and that in db/db mice glomerular expression of ACE2 is reduced whereas glomerular ACE expression is increased. The finding that chronic ACE2 inhibition increases UAE suggests that ACE2, likely by modulating the levels of glomerular angiotensin II via its degradation, may be a target for therapeutic interventions that aim to reduce albuminuria and glomerular injury.

ACE and ACE2 Activity in Diabetic Mice

ACE-related carboxypeptidase (ACE2) may counterbalance the angiotensin (ANG) II–promoting effects of ACE in tissues where both enzymes are found. Alterations in renal ACE and ACE2 expression have been described in experimental models of diabetes, but ACE2 activity was not assessed in previous studies. We developed a microplate-based fluorometric method for the concurrent determination of ACE and ACE2 activity in tissue samples. Enzymatic activity (relative fluorescence unit [RFU] · μg protein−1 · h−1) was examined in ACE and ACE2 knockout mice and in two rodent models of diabetes, the db/db and streptozotocin (STZ)-induced diabetic mice. In kidney cortex, preparations consisting mainly of proximal tubules and cortical collecting tubules, ACE2 activity had a strong positive correlation with ACE2 protein expression (90-kDa band) in both knockout models and their respective wild-type littermates (r = 0.94, P < 0.01). ACE activity, likewise, had a strong positive correlation with renal cortex ACE protein expression (170-kDa band) (r = 0.838, P < 0.005). In renal cortex, ACE2 activity was increased in both models of diabetes (46.7 ± 4.4 vs. 22.0 ± 4.7 in db/db and db/m, respectively, P < 0.01, and 22.1 ± 2.8 vs. 13.1 ± 1.5 in STZ-induced diabetic versus untreated mice, respectively, P < 0.05). ACE2 mRNA levels in renal cortex from db/db and STZ-induced diabetic mice, by contrast, were not significantly different from their respective controls. In cardiac tissue, ACE2 activity was lower than in renal cortex, and there were no significant differences between diabetic and control mice (db/db 2.03 ± 0.23 vs. db/m 1.85 ± 0.10; STZ-induced diabetic 0.42 ± 0.04 vs. untreated 0.52 ± 0.07 mice). ACE2 activity in renal cortex correlated positively with ACE2 protein in db/db and db/m mice (r = 0.666, P < 0.005) as well as in STZ-induced diabetic and control mice (r = 0.621, P < 0.05) but not with ACE2 mRNA (r = −0.468 and r = −0.522, respectively). We conclude that in renal cortex from diabetic mice, ACE2 expression is increased at the posttranscriptional level. The availability of an assay for concurrent measurement of ACE and ACE2 activity should be helpful in the evaluation of kidney-specific alterations in the balance of these two carboxypeptidases, which are involved in the control of local ANG II formation and degradation.

Clinical Practice Guideline on management of patients with diabetes and chronic kidney disease stage 3b or higher (eGFR <45 mL/min)

Diabetes mellitus is becoming increasingly prevalent and is considered a rapidly growing concern for healthcare systems. Besides the cardiovascular complications, diabetes mellitus is associated with chronic kidney disease (CKD). CKD in patients with diabetes can be caused by true diabetic nephropathy, but can also be caused indirectly by diabetes, e.g. due to polyneuropathic bladder dysfunction, increased incidence of relapsing urinary tract infections or macrovascular angiopathy. However, many patients who develop CKD due to a cause other than diabetes will develop ormay already have diabetes mellitus. Finally, many drugs that are used for management of CKDs, e.g. corticosteroids or calcineurin inhibitors, can cause diabetes. Despite the strong interplay between diabetes and CKD, the management of patients with diabetes and CKD stage 3b or higher (eGFR <45 mL/min) remains problematic. Many guidance-providing documents have been produced on the management of patients with diabetes to prevent or delay the progression to CKD, mostly defined as the presence of microand macro-albuminuria. However, none of these documents specifically deal with the management of patients with CKD stage 3b or higher (eGFR <45 mL/min). There is a paucity of well-designed, prospective studies in this population, as many studies exclude either patients with diabetes, or with CKD stage 3b or higher (eGFR <45 mL/min), or both. This limits the evidence base to these approaches. In addition, due to some new developments in this area, the advisory board of ERBP decided that a guideline on the management of patients with diabetes and CKD stage 3b or higher (eGFR <45 mL/min) was needed and timely:

Angiotensin-converting enzyme 2: enhancing the degradation of angiotensin II as a potential therapy for diabetic nephropathy

Angiotensin-converting enzyme 2 (ACE2) is a monocarboxypeptidase that degrades angiotensin II with high efficiency leading to the formation of angiotensin-(1-7). ACE2 within the kidneys is largely localized in tubular epithelial cells and in glomerular epithelial cells. Decreased glomerular expression of this enzyme coupled with increased expression of ACE has been described in diabetic kidney disease, both in mice and humans with type 2 diabetes. Moreover, both ACE2 genetic ablation and pharmacological ACE2 inhibition have been shown to increase albuminuria and promote glomerular injury. Studies using recombinant ACE2 have shown the ability of ACE2 to rapidly metabolize Ang II in vivo and form the basis for future studies to examine the potential of ACE2 amplification in the therapy of diabetic kidney disease and cardiovascular disease.

Localization of ACE2 in the renal vasculature: amplification by angiotensin II type 1 receptor blockade using telmisartan

Angiotensin-converting enzyme (ACE)2 is a carboxypeptidase that degrades angiotensin II and other peptides. In the kidney, ACE2 localization within the glomerulus and tubules is cell specific. This study was aimed to investigate the localization of ACE2 within the renal vasculature. We also studied the effect of the administration of a specific angiotensin II type 1 receptor blocker, telmisartan, on ACE2 expression in the renal vasculature. ACE2 and ACE were localized in renal arterioles using confocal microscopy and specific cell markers. Quantitative measurements of ACE2 and ACE mRNA were estimated in kidney arterioles isolated by laser capture microdissection using real-time PCR. In kidney arterioles, ACE was localized in the endothelial layer, whereas ACE2 was localized in the tunica media. In mice treated with telmisartan (2 mg.kg(-1).day(-1)) for 2 wk, ACE2 expression was increased by immunostaining, whereas ACE expression was decreased. This was reflected in a decrease in the ACE/ACE2 ratio compared with vehicle-treated controls (0.53 +/- 0.14 vs. 7.59 +/- 2.72, P = 0.027, respectively). In kidney arterioles isolated by laser capture microdissection, the ACE/ACE2 mRNA ratio was also decreased compared with control mice (1.21 +/- 0.31 vs. 4.63 +/- 0.86, P = 0.044, respectively). In conclusion, in kidney arterioles ACE2 is preferentially localized in the tunica media, and its expression is increased after administration of the angiotensin II type 1 receptor blocker, telmisartan. Amplification of ACE2 in the renal vasculature may contribute to the therapeutic action of telmisartan by increasing angiotensin II degradation.

New experimental models of diabetic nephropathy in mice models of type 2 diabetes: efforts to replicate human nephropathy.

Diabetic nephropathy (DN) is the leading cause of end-stage renal disease. The use of experimental models of DN has provided valuable information regarding many aspects of DN, including pathophysiology, progression, implicated genes, and new therapeutic strategies. A large number of mouse models of diabetes have been identified and their kidney disease was characterized to various degrees. Most experimental models of type 2 DN are helpful in studying early stages of DN, but these models have not been able to reproduce the characteristic features of more advanced DN in humans such as nodules in the glomerular tuft or glomerulosclerosis. The generation of new experimental models of DN created by crossing, knockdown, or knockin of genes continues to provide improved tools for studying DN. These models provide an opportunity to search for new mechanisms involving the development of DN, but their shortcomings should be recognized as well. Moreover, it is important to recognize that the genetic background has a substantial effect on the susceptibility to diabetes and kidney disease development in the various models of diabetes.

New aspects of the renin-angiotensin system: Angiotensin-converting enzyme 2 - A potential target for treatment of hypertension and diabetic nephropathy

Purpose of reviewWhereas angiotensin-converting enzyme promotes the formation of angiotensin II, angiotensin-converting enzyme 2 promotes the degradation of angiotensin II to angiotensin-(1–7). We review recent studies dealing with angiotensin-converting enzyme 2 in kidney disease and hypertension, and discuss the potential therapeutic benefit of increasing angiotensin-converting enzyme 2 activity in the treatment of these diseases. Recent findingsIn glomeruli from diabetic mice, angiotensin-converting enzyme 2 expression is downregulated, and pharmacological inhibition of angiotensin-converting enzyme 2 leads to worsening of albuminuria, increased mesangial matrix deposition and fibronectin expression. The deletion of the angiotensin-converting enzyme 2 gene in mice leads to worsening of angiotensin II-induced hypertension and has also been shown to cause glomerulosclerosis in aging male mice. SummaryAngiotensin-converting enzyme 2 is a key enzyme in the renin–angiotensin system that favors the degradation of angiotensin I and angiotensin II. Angiotensin-converting enzyme 2 inhibition by pharmacological means and by genetic deletion worsens kidney disease in diabetic mice. Strategies geared to increasing angiotensin-converting enzyme 2 activity may provide a novel therapeutic target within the renin–angiotensin system by enhancing angiotensin II degradation that may complement the current approach of inhibiting angiotensin II formation and action. Amplifying angiotensin-converting enzyme 2 activity may have a potential therapeutic role for kidney disease and hypertension.

Validity of the hospital discharge diagnosis in epidemiologic studies of biliopancreatic pathology

Background: The aim was to analyse the magnitude, direction and predictors of change in the main hospital discharge diagnosis (HDD) after a clinical expert review, among patients included in a multicentre molecular epidemiologic study of biliopancreatic diseases. Methods: A total of 602 patients with a suspicion diagnosis of pancreas cancer (PC), cancer of the extrahepatic biliary system (CEBS) or benign biliopancreatic pathologies (BPP) were prospectively recruited at five general hospitals. A structured form was used to collect information from medical records. A panel of experts revised all diagnostic information and established the main clinico-pathological diagnosis (CPD) by consensus. Results: Of the 204 cases with a HDD of PC, 176 (86%) were deemed to have a CPD of PC, eight of CEBS, twelve a neoplasm of different origin, four BPP and four syndromic diagnoses. Thus, 28 cases (14%) were false positives. Of the 129 patients with a HDD of CEBS, 15 (12%) were false positives. Nine of the 396 cases with a HDD of non-PC (2%) had a CPD of PC (false negatives), whilst 14 of 471 patients with a HDD of non-CEBS (3%) were deemed to have CEBS. Overall, sensitivity and specificity of HDD for PC were, respectively, 95 and 93%, and for CEBS, 89 and 97%. Cytohistological confirmation and laparotomy were independent predictors of diagnostic change. Conclusions: Validity of the HDD was high, but its association with some clinical variables suggests that sole reliance on HDD can significantly bias results, and highlights the need to review all HDDs. Alternatively, only patients at high risk of misdiagnosis could be reviewed: primarily, those lacking a cytohistological diagnosis or a laparotomy. No exclusions appear warranted solely on the basis of age, gender or tumour spread.

Risk Factors for Acute Kidney Injury in Severe Rhabdomyolysis

Background Acute kidney injury (AKI) is a life-threatening complication of severe rhabdomyolysis. This study was conducted to assess risk factors for AKI and to develop a risk score for early prediction. Methods Retrospective observational cohort study with a 9-year follow-up, carried out in an acute-care teaching-affiliated hospital. A total of 126 patients with severe rhabdomyolysis defined as serum creatine kinase (CK) > 5,000 IU/L fulfilled the inclusion criteria. Univariate and logistic regression analyses were performed to determine risk factors for AKI. Based on the values obtained for each variable, a risk score and prognostic probabilities were estimated to establish the risk for developing AKI. Results The incidence of AKI was 58%. Death during hospitalization was significantly higher among patients with AKI, compared to patients without AKI (19.2% vs 3.6%, p = 0.008). The following variables were independently associated with AKI: peak CK (odds ratio [OR] 4.9, 95%CI 1.4-16.8), hypoalbuminemia (< 33 mg/dL, [OR 5.1, 95%CI 1.4-17-7]), metabolic acidosis (OR 5.3, 95%CI 1.4-20.3), and decreased prothrombin time (OR 4.4, 95% CI 1.3-14.5). A risk score for AKI was calculated for each patient, with an OR of 1.72 (95%CI 1.45-2.04). The discrimination value of the predictive model was established by means of a ROC curve, with the area under the curve of 0.871 (p<0.001). Conclusions The identification of independent factors associated with AKI and a risk score for early prediction of this complication in patients with severe rhabdomyolysis may be useful in clinical practice, particularly to implement early preventive measures.

Role of Circulating Angiotensin Converting Enzyme 2 in Left Ventricular Remodeling following Myocardial Infarction: A Prospective Controlled Study

Angiotensin-converting enzyme 2 (ACE2) cleaves Angiotensin-II to Angiotensin-(1–7), a cardioprotective peptide. Serum soluble ACE2 (sACE2) activity is raised in chronic heart failure, suggesting a compensatory role in left ventricular dysfunction. Our aim was to study the relationship between sACE2 activity, infarct size, left ventricular systolic function and remodeling following ST-elevation myocardial infarction (STEMI). A contrast-enhanced cardiac magnetic resonance study was performed acutely in 95 patients with first STEMI and repeated at 6 months to measure LV end-diastolic volume index, ejection fraction and infarct size. Baseline sACE2 activities, measured by fluorescent enzymatic assay 24 to 48 hours and at 7 days from admission, were compared to that obtained in 22 matched controls. Patients showed higher sACE2 at baseline than controls (104.4 [87.4–134.8] vs 74.9 [62.8–87.5] RFU/µl/hr, p<0.001). At seven days, sACE2 activity significantly increased from baseline (115.5 [92.9–168.6] RFU/µl/hr, p<0.01). An inverse correlation between sACE2 activity with acute and follow-up ejection fraction was observed (r = −0.519, p<0.001; r = −0.453, p = 0.001, respectively). Additionally, sACE2 directly correlated with infarct size (r = 0.373, p<0.001). Both, infarct size (β = −0.470 [95%CI:−0.691:−0.248], p<0.001) and sACE2 at 7 days (β = −0.025 [95%CI:−0.048:−0.002], p = 0.030) were independent predictors of follow-up ejection fraction. Patients with sACE2 in the upper tertile had a 4.4 fold increase in the incidence of adverse left ventricular remodeling (95% confidence interval: 1.3 to 15.2, p = 0.027). In conclusion, serum sACE2 activity rises in relation to infarct size, left ventricular systolic dysfunction and is associated with the occurrence of left ventricular remodeling.