Sara Giunti

Effect of the Monocyte Chemoattractant Protein-1/CC Chemokine Receptor 2 System on Nephrin Expression in Streptozotocin-Treated Mice and Human Cultured Podocytes

OBJECTIVE Monocyte chemoattractant protein-1 (MCP-1), a chemokine binding to the CC chemokine receptor 2 (CCR2) and promoting monocyte infiltration, has been implicated in the pathogenesis of diabetic nephropathy. To assess the potential relevance of the MCP-1/CCR2 system in the pathogenesis of diabetic proteinuria, we studied in vitro if MCP-1 binding to the CCR2 receptor modulates nephrin expression in cultured podocytes. Moreover, we investigated in vivo if glomerular CCR2 expression is altered in kidney biopsies from patients with diabetic nephropathy and whether lack of MCP-1 affects proteinuria and expression of nephrin in experimental diabetes. RESEARCH DESIGN AND METHODS Expression of nephrin was assessed in human podocytes exposed to rh-MCP-1 by immunofluorescence and real-time PCR. Glomerular CCR2 expression was studied in 10 kidney sections from patients with overt nephropathy and eight control subjects by immunohistochemistry. Both wild-type and MCP-1 knockout mice were made diabetic with streptozotocin. Ten weeks after the onset of diabetes, albuminuria and expression of nephrin, synaptopodin, and zonula occludens-1 were examined by immunofluorescence and immunoblotting. RESULTS In human podocytes, MCP-1 binding to the CCR2 receptor induced a significant reduction in nephrin both mRNA and protein expression via a Rho-dependent mechanism. The MCP-1 receptor, CCR2, was overexpressed in the glomerular podocytes of patients with overt nephropathy. In experimental diabetes, MCP-1 was overexpressed within the glomeruli and the absence of MCP-1 reduced both albuminuria and downregulation of nephrin and synaptopodin. CONCLUSIONS These findings suggest that the MCP-1/CCR2 system may be relevant in the pathogenesis of proteinuria in diabetes.

Heat shock protein expression in diabetic nephropathy.

Heat shock protein (HSP) HSP27, HSP60, HSP70, and HSP90 are induced by cellular stresses and play a key role in cytoprotection. Both hyperglycemia and glomerular hypertension are crucial determinants in the pathogenesis of diabetic nephropathy and impose cellular stresses on renal target cells. We studied both the expression and the phosphorylation state of HSP27, HSP60, HSP70, and HSP90 in vivo in rats made diabetic with streptozotocin and in vitro in mesangial cells and podocytes exposed to either high glucose or mechanical stretch. Diabetic and control animals were studied 4, 12, and 24 wk after the onset of diabetes. Immunohistochemical analysis revealed an overexpression of HSP25, HSP60, and HSP72 in the diabetic outer medulla, whereas no differences were seen in the glomeruli. Similarly, exposure neither to high glucose nor to stretch altered HSP expression in mesangial cells and podocytes. By contrast, the phosphorylated form of HSP27 was enhanced in the glomerular podocytes of diabetic animals, and in vitro exposure of podocytes to stretch induced HSP27 phosphorylation via a P38-dependent mechanism. In conclusion, diabetes and diabetes-related insults differentially modulate HSP27, HSP60, and HSP70 expression/phosphorylation in the glomeruli and in the medulla, and this may affect the ability of renal cells to mount an effective cytoprotective response.

QTc interval prolongation is independently associated with severe hypoglycemic attacks in type 1 diabetes from the EURODIAB IDDM complications study.

OBJECTIVE Our aim was to assess whether severe hypoglycemic attacks were cross-sectionally associated with abnormalities of the QTc interval in type 1 diabetic patients. RESEARCH DESIGN AND METHODS The study included 3,248 type 1 diabetic patients from the EURODIAB IDDM Complications Study. Severe hypoglycemia was defined as an attack serious enough to require the help of another person. A corrected QTc interval (QTc) >0.44 s was considered abnormally prolonged. RESULTS Nineteen percent of patients declared one to two attacks, and 13.2% of patients had three or more attacks. Prevalence of QTc prolongation was greater in patients who experienced three or more hypoglycemic attacks. Logistic regression analysis showed that the frequency of severe hypoglycemia was independently associated with QTc prolongation, even after adjustment for diabetes complications, including autonomic neuropathy (odds ratio 1.27, 95% CI 1.02–1.58). CONCLUSIONS We have provided evidence that severe hypoglycemic attacks are independently associated with a prolonged QTc interval in type 1 diabetic patients from the EURODIAB IDDM Complications Study.

Targeting the MCP-1/CCR2 System in Diabetic Kidney Disease

Diabetic nephropathy is the leading cause of end-stage renal failure in the Western World and accounts for significant morbidity and mortality in patients with diabetes. Although hyperglycaemia and hypertension are established key determinants in the development of the complication, recent studies suggest that a low-grade inflammatory response may also play a role. Monocyte Chemoattractant Protein 1 (MCP-1), a potent chemokine produced by renal cells, has emerged as a very important player in this process. Specifically, it has been shown that MCP-1 is overexpressed in the kidneys from diabetic animals. Furthermore, there is amelioration of both functional and structural abnormalities in MCP-1- knockout mice in the setting of concomitant diabetes. Over recent years the cellular mechanisms linking MCP-1 to kidney injury have been increasingly delineated and, in particular, it has become evident that MCP-1 contributes to the kidney damage not only by inducing mononuclear cell recruitment, but also by direct activation of resident renal cells. The present review focuses on the most significant advances in understanding the role of MCP-1 in diabetic kidney disease and future potential therapeutic implications.

The pleiotropic actions of rosuvastatin confer renal benefits in the diabetic Apo-E knockout mouse

Diabetic nephropathy is a leading cause of end-stage renal disease. Statins may exert renoprotective effects independently of lipid-lowering properties. We investigated the pleiotropic effects of rosuvastatin on renal structure and function in streptozotocin diabetic apolipoprotein-E knockout (Apo-E(-/-)) mice, a model of progressive nephropathy in which dyslipidemia is resistant to statin treatment. These effects were compared with those observed with conventional renin-angiotensin system blockade (candesartan) or combined treatment. Nondiabetic and diabetic Apo-E(-/-) mice were randomized to no treatment or treatment with candesartan (2.5 mg/kg), rosuvastatin (5 mg/kg), or their combination per gavage for 20 wk. Urine and blood samples were collected for assessment of albuminuria, creatinine clearance, plasma lipids, glucose, and glycated hemoglobin. Renal sclerosis was analyzed on paraffin-embedded kidney sections stained with periodic acid-Schiff. Renal expression of collagen IV, fibronectin and advanced glycation end products (AGEs), receptor for advanced glycation and products (RAGE), NADPH oxidase 4 (NOX4), and nitrotyrosine was assessed by real-time PCR and/or immunohistochemistry. Diabetes-induced albuminuria was not affected by rosuvastatin and combination treatment but was prevented by candesartan. Diabetes resulted in increased creatinine clearance, which was not modified by the treatments. Rosuvastatin and/or candesartan prevented diabetes-associated renal extracellular matrix accumulation. Rosuvastatin reduced accumulation of AGEs and expression of RAGE, NOX4, and nitrotyrosine. In conclusion, in the diabetic Apo-E(-/-) mouse, rosuvastatin confers renal benefits that are independent of lipid lowering and equivalent or greater to those observed with candesartan. The combination treatment is not superior to monotherapies.

INCIDENCE AND RISK FACTORS OF PROLONGED QTc INTERVAL IN TYPE 1 DIABETES: THE EURODIAB PROSPECTIVE COMPLICATIONS STUDY.

OBJECTIVE—Corrected QT (QTc) prolongation is predictive of cardiovascular mortality in both the general and diabetic populations. As part of the EURODIAB Prospective Complication Study, we have assessed the 7-year incidence and risk factors of prolonged QTc in people with type 1 diabetes. RESEARCH DESIGN AND METHODS—A total of 1,415 type 1 diabetic subjects, who had normal QTc at baseline, were reanalyzed after the 7-year follow-up period. QTc >0.44 s was considered abnormally prolonged. RESULTS—Cumulative incidence of prolonged QTc was 18.7%, which is twofold higher in women than in men (24.5 vs. 13.9%, P < 0.0001). At the baseline examination, incident cases were older and less physically active than nonincident cases, had higher mean values of systolic blood pressure and HDL cholesterol, and had higher frequencies of hypertension, coronary heart disease, and distal symmetrical polyneuropathy. In multivariate logistic regression analyses, female sex and higher values of A1C and systolic blood pressure were associated with the risk of prolonged QTc, whereas physical activity and BMI within the range of 21.5–23.2 kg/m2 were protective factors. In women, association with modifiable factors, particularly BMI, was stronger than in men. CONCLUSIONS—In type 1 diabetic subjects from the EURODIAB cohort, female sex, A1C, and systolic blood pressure are predictive of prolonged QTc, whereas physical activity and BMI within the range of 21.5–23.2 kg/m2 play a protective role. These findings are clinically relevant, as they may help to identify subjects at higher risk for prolonged QTc, as well as provide potential targets for risk-lowering strategies.

Angiopoietin 2 induces cell cycle arrest in endothelial cells: a possible mechanism involved in advanced plaque neovascularization

Objective—To characterize the molecules and the mechanisms regulating the neoangiogenetic process in advanced atherosclerotic plaques. Methods and Results—Western blot and immunofluorescence analysis of atherosclerotic specimens demonstrated that unlike neovessels from early lesions that expressed vascular endothelial growth factor (VEGF) and angiopoietin1 (Angio1), vessels from advanced lesions expressed VEGF and angiopoietin 2 (Angio2). Moreover, only few neovessels from advanced lesions showed a positive immunostaining for proliferating cell nuclear antigen. Angio1-elicited and Angio2-elicited intracellular events in endothelial cells (EC) demonstrated that while Angio1 triggered Erk1/Erk2 mitogen activated protein kinases (MAPK) and Akt activation, Angio2 (50 ng/mL) induced STAT5 activation and p21waf expression and increased the fraction of cells in G1. Both Angio2-mediated events were abrogated by expressing a dominant negative STAT5 construct (&Dgr;STAT5). Consistent with the expression of Angio2 in neovessels of advanced lesions a transcriptionally active STAT5 was detected. Moreover, co-immunoprecipitation experiments revealed the presence of a STAT5/Tie2 molecular complex in neointima vessels from advanced, but not from early, lesions. Conclusions—In advanced lesions, the activation of the Tie2-mediated STAT5 signaling pathway may negatively regulate vessel growth.

Sex-differences in prevalence of electrocardiographic left ventricular hypertrophy in Type 2 diabetes: The Casale Monferrato Study

Aims  Although left ventricular hypertrophy (LVH) defined by either standard 12‐lead ECG or echocardiography strongly predicts cardiovascular mortality, its prevalence in Type 2 diabetes is largely unknown. We have assessed prevalence of ECG‐LVH and its relationship with clinical and metabolic variables in an Italian population‐based cohort of subjects with Type 2 diabetes.

Increased QT Interval Dispersion Predicts 15-Year Cardiovascular Mortality in Type 2 Diabetic Subjects The population-based Casale Monferrato Study

OBJECTIVE To evaluate the predictive role of increased corrected QT (QTc) and QT interval dispersion (QTd) on all-cause and cardiovascular mortality in a large, unselected type 2 diabetic population. RESEARCH DESIGN AND METHODS The prospective study included 1,357 type 2 diabetic patients from the Casale Monferrato Study. At baseline, QTc intervals >0.44 s and QTd intervals >0.08 s were considered abnormally prolonged. Both all-cause and cardiovascular mortality were assessed 15 years after the baseline examination. RESULTS During the follow-up period, 862 subjects per 12,450 person-years died. Multivariate analysis showed that the hazard ratio (HR) of cardiovascular mortality was significantly increased in subjects with prolonged QTd (1.26 [95% CI 1.02–1.55]) and was only slightly reduced after multiple adjustments. Conversely, prolonged QTc did not increase the HRs for all-cause or cardiovascular mortality. CONCLUSIONS Increased QTd predicts cardiovascular mortality after a long-term follow-up period in a large, unselected population of type 2 diabetic subjects.

Comparison between 24‐h proteinuria, urinary protein/creatinine ratio and dipstick test in patients with nephropathy: Patterns of proteinuria in dipstick‐negative patients

Objective. Three main tests are commonly employed for the measurement of proteinuria: the dipstick test, the urinary protein/creatinine ratio (P/C) and the 24‐h urine collection. The aim of this study was to evaluate the correlation between these methods, comparing linear regression and ROC curve data. Material and methods. A total of 297 consecutive outpatients with different renal diseases were included in the study. Twenty‐four‐hour proteinuria was considered the reference test. Results. A high degree of correlation was observed between all the tests (p<0.0001), the highest regression coefficient being between 24‐h proteinuria and P/C (R = 0.82), and the lowest between P/C and the dipstick test (R = 0.72). The dipstick test failed to detect pathological proteinuria in 94 patients (31.6 %). Therefore, in these subjects, the patterns of proteinuria were assessed by immunofixation and sodium dodecyl sulphate (SDS) electrophoresis. Conclusions. Our data strongly support the use of urinary P/C for the detection of proteinuria, at least in nephrology units, where the prevalence of proteinuria is likely to be high.