Minimal Change Disease: Pathogenetic Insights from Glomerular Proteomics.

The mechanism underlying podocyte dysfunction in minimal change disease (MCD) remains unknown. This study aimed to shed light on the potential pathophysiology of MCD using glomerular proteomic analysis. Shotgun proteomics using label-free quantitative mass spectrometry was performed on formalin-fixed, paraffin-embedded (FFPE) renal biopsies from two groups of samples: control (CTR) and MCD. Glomeruli were excised from FFPE renal biopsies using laser capture microdissection (LCM), and a single-pot solid-phase-enhanced sample preparation (SP3) digestion method was used to improve yield and protein identifications. Principal component analysis (PCA) revealed a distinct separation between the CTR and MCD groups. Forty-eight proteins with different abundance between the two groups (p-value ≤ 0.05 and |FC| ≥ 1.5) were identified. These may represent differences in podocyte structure, as well as changes in endothelial or mesangial cells and extracellular matrix, and some were indeed found in several of these structures. However, most differentially expressed proteins were linked to the podocyte cytoskeleton and its dynamics. Some of these proteins are known to be involved in focal adhesion (NID1 and ITGA3) or slit diaphragm signaling (ANXA2, TJP1 and MYO1C), while others are structural components of the actin and microtubule cytoskeleton of podocytes (ACTR3 and NES). This study suggests the potential of mass spectrometry-based shotgun proteomic analysis with LCM glomeruli to yield valuable insights into the pathogenesis of podocytopathies like MCD. The most significantly dysregulated proteins in MCD could be attributable to cytoskeleton dysfunction or may be a compensatory response to cytoskeleton malfunction caused by various triggers.

Relationship between vascular cell adhesion molecule-1 (VCAM-1), soluble receptor for advanced glycation end products (sRAGE) and functional hemodynamic parameters of arteriovenous fistula.

BACKGROUND: The preferred vascular access for hemodialysis is represented by arteriovenous fistula (AVF) due to fewer complications and more prolonged survival. Considerable efforts have been made to identify biomarkers associated with AVF dysfunction, but results are conflicting. Vascular cell adhesion molecule (VCAM-1) and advanced glycation end products are involved in atherogenesis, vascular calcification, peripheral artery disease, and neointimal hyperplasia in renal and non-renal patients. The objective of this study was to evaluate whether there is an association between VCAM-1, soluble receptor for advanced glycation end products (sRAGE), NcarboxymethylLysine (CML), and arteriovenous fistula dysfunction (AVF). METHODS: VCAM-1, sRAGE, and CML were performed using the ELISA technique in 88 HD patients. Ultrasound assessment of AVF reports brachial artery blood flow (Qa), brachial resistivity index (RI), presence of calcification, and the diameter. AVF dysfunction was defined as a brachial artery Qa ⩽ 500 ml/min or RI ⩾ 0.5. RESULTS: The median level of VCAM-1 [2676.5(2206.8-4203.9) versus 2613.2(1885.7-3161.8), p 0.026] was significantly higher in patients with AVF dysfunction compared to the rest of the patients. sRAGE and CML were higher in this group but without statistical significance. In patients with AVF dysfunction, significant positive correlations were found between VCAM-1and sRAGE (r = 0.417, p = 0.001), RI (r = 0.313, p = 0.046), dialysis vintage (r = 0.540, p < 0.001), AVF vintage (r = 0.336, p = 0.032), intima-media thickness (r = 0.423, p = 0.006) and C-reactive protein (r = 0.315, p = 0.045). VCAM-1 correlated inversely with cholesterol (r = -0.312, p = 0.047), triglycerides (r = -0.358, p = 0.021), body mass index (r = -0.388, p = 0.012). In multivariate regression analysis, VCAM-1 (p = 0.013) and sRAGE (p = 0.01) remained significant predictors of RI and Qa. Logistic regression disclosed calcification, VCAM-1, as risks factors for AVF dysfunction. CONCLUSION: The results we obtained showed that patients with AVF dysfunction had a significantly higher level of VCAM-l. A positive correlation between VCAM-1 and sRAGE was identified in this group.

Hemodialysis Patients with Higher Serum Levels of Soluble Receptor for Advanced Glycation End Products Have an Increased Risk for Arteriovenous Fistula Failure.

BACKGROUND: Arteriovenous fistula (AVF) failure due to thrombosis is a major cause of morbidity in patients undergoing regular hemodialysis (HD). Advanced glycation end products (AGEs) and their receptor (RAGE) might contribute to inflammation, neointimal hyperplasia, and thrombosis. RAGE has a C-truncated secretory receptor form, called soluble RAGE (sRAGE). In this study, we aimed to evaluate the association of serum sRAGE with AVF failure due to thrombosis in HD patients. METHODS: Eighty-eight prevalent HD patients with functional AVF were included in the study. The presence of stenosis, clinical and laboratory data, and serum sRAGE was evaluated at inclusion. sRAGE concentration was measured by a competitive enzyme-linked immunosorbent assay, and stenosis was detected by ultrasound. Patients were prospectively followed up for 36 months. During this period, AVF failure (defined as the absence of blast or palpable thrill and impossible cannulation with 2 needles because of complete thrombosis) was noted and thrombosis was certified by ultrasound examination. RESULTS: During follow-up, 16 (18.18%) patients lost their vascular access due to thrombosis. In multivariate Cox regression analysis, sRAGE was a significant predictor of vascular access thrombosis (hazard ratio = 1.15, 95% confidence interval: 1.03-1.25, p = 0.012). Kaplan-Meier analysis showed a significantly lower AVF patency time in patients with sRAGE >16.78 ng/mL than those with sRAGE <16.78 ng/mL (p = 0.02). In the subgroup of patients with stenosis at baseline, sRAGE, serum albumin, obesity, and ischemic heart disease were associated with thrombosis. CONCLUSION: In our study, baseline, systemic sRAGE is associated with the occurrence of thrombosis of AVF, and this marker has a significant impact on AVF survival.

Pentosidine in chronic hemodialysis patients: relation with arteriovenous fistula morphology and function.

PURPOSE: It has been suggested that advanced glycation end products (AGEs) are involved in atherogenesis, vascular calcification and remodeling, including neointimal hyperplasia, in renal and non-renal patients. Their relevance for arteriovenous fistula (AVF) function has been poorly studied to date, with only one clinical study addressing the issue of thrombosis of vascular access in relation to AGEs in dialysis patients. We aimed to evaluate the relationship between serum pentosidine and AVF morphology and function. METHODS: Eighty-eighth hemodialysis patients with patent native AVF were included. Ultrasound examination of AVF evaluated blood flow in the brachial artery, resistivity index (RI), the diameter of the vessels and the presence of stenosis. AVF and cardiovascular history were recorded, routine clinical and laboratory evaluation was performed and serum pentosidine was assessed. RESULTS: Forty-eight patients (54.54%) had AVF stenosis. Pentosidine correlated in univariate analysis with cholesterol (r = 0.270, p = 0.01), triglycerides (r = 0.309, p = 0.003), calcium (r = 0.040, p < 0.001) and inversely to dialysis vintage (r = - 0.453, p < 0.001), access vintage (r = - 0.432, p = 0.001), phosphate (r = - 0.211, p = 0.04), parathyroid hormone (r = - 0.211, p = 0.04), urea (r = - 0.230, p = 0.03), residual diameter of AVF (r = - 0.023, p = 0.03). In multivariate regression calcium (p = 0.006), access vintage (p = 0.03), and residual diameter of AVF vein (p = 0.02) remain significantly linked to pentosidine. Patients with pentosidine above median had higher cholesterol (179.91 vs. 160.97, p = 0.04), triglycerides (187.18 vs. 129.31, p = 0.002) and higher prevalence of hypertension (93.70% vs. 84.10%, p = 0.02). CONCLUSIONS: Our study suggests that pentosidine could be associated to vascular access morphology and function in dialysis patients.

Adiponectin is related to markers of endothelial dysfunction and neoangiogenesis in diabetic patients.

PURPOSE: Adiponectin an adipokine, produced by mature adipocyte, has an important effect on several aspects of endothelial function, including leukocyte adhesion (mediated by adhesion molecules like intercellular adhesion molecule 1 (ICAM1) endothelial cell selective adhesion molecule ESAM). Recently, it has been linked to vascular endothelial growth factor (VEGF)-modulated angiogenesis. ESAM might also be involved in modulating VEGF-dependent actions. We studied relationship of adiponectin to ESAM, ICAM1, and VEGF in type 2 diabetic patients (T2DP) with or without microvascular complications. METHODS: Incident T2DP referred for nephrologic evaluation were included (patients with no nephropathy or stage 1-4 nephropathy). T2DP with stage 5 chronic kidney disease (CKD) were selected from a dialysis center. Clinical, standard laboratory assessment and adiponectin, ESAM, ICAM1, and VEGF (ELISA) were recorded. RESULTS: Eighty-seven patients were included, 15 had no CKD, 30 with stage 1 or 2 CKD, 20 with stage 3 or 4 CKD and 22 patients on dialysis. ESAM was higher in patients with CKD than in those without CKD (p = 0.02), adiponectin, ICAM1, and VEGF were similar. Adiponectin correlated in univariate analysis to ESAM (r = 0.32, p = 0.002), ICAM1 (r = 0.23, p = 0.038), and CRP (r = 0.31, p = 0.012), and inversely to serum albumin (r = - 0.57, < 0.0001). In predialysis patients, adiponectin also correlated to albuminuria (r = 0.54, p < 0.0001) and glomerular filtration rate (r = - 0.46, p = 0.0001). In multivariate regression ESAM (p = 0.04), VEGF (p = 0.03), and albumin (p < 0.0001) are significant predictors of adiponectin. None of these cytokines were different when comparing patients with and without retinopathy. CONCLUSION: Adiponectin is directly linked to adhesion molecules and VEGF in T2DP.

ESAM predicts cardiovascular mortality in diabetic hemodialysis patients.

AIM: To assess endothelial cell selective adhesion molecule (ESAM) as predictor of cardiovascular mortality in diabetic dialysis patients (DDPs). METHODS: ESAM, clinical and laboratory parameters were assessed in 73 DDP. Cardiovascular mortality was recorded in a 2 years' prospective observational study. RESULTS: Baseline ESAM was 17.1 (10.05-24.8) ng/ml and was correlated to phosphate (r = -0.42, p = 0.008), parathormone (r = -0.36, p = 0.048), albumin (r = -0.24, p = 0.048). ESAM significantly predicted cardiovascular death in univariate [HR = 1.03, 95% CI (1.006-1.054), p = 0.01] and multivariate [HR = 1.034, 95% CI (1.003-1.066), p = 0.03] Cox analysis. Time to cardiovascular death was shorter for patients with ESAM >12.44 ng/ml, p = 0.0045. CONCLUSION: ESAM is an independent predictor of cardiovascular mortality in DDP.

ESAM predicts cardiovascular mortality in diabetic hemodialysis patients.

AIM: To assess endothelial cell selective adhesion molecule (ESAM) as predictor of cardiovascular mortality in diabetic dialysis patients (DDPs). METHODS: ESAM, clinical and laboratory parameters were assessed in 73 DDP. Cardiovascular mortality was recorded in a 2 years' prospective observational study. RESULTS: Baseline ESAM was 17.1 (10.05-24.8) ng/ml and was correlated to phosphate (r = -0.42, p = 0.008), parathormone (r = -0.36, p = 0.048), albumin (r = -0.24, p = 0.048). ESAM significantly predicted cardiovascular death in univariate [HR = 1.03, 95% CI (1.006-1.054), p = 0.01] and multivariate [HR = 1.034, 95% CI (1.003-1.066), p = 0.03] Cox analysis. Time to cardiovascular death was shorter for patients with ESAM >12.44 ng/ml, p = 0.0045. CONCLUSION: ESAM is an independent predictor of cardiovascular mortality in DDP.

Relationship of adiponectin to markers of oxidative stress in type 2 diabetic patients: influence of incipient diabetes-associated kidney disease.

PURPOSE: Adiponectin may be beneficial in incipient chronic kidney disease by antagonizing oxidative stress. We evaluated adiponectin, malondialdehyde (MDA), and superoxide dismutase (SOD), in type 2 diabetes mellitus patients (T2DP) with and without incipient nephropathy. METHODS: T2DP with glomerular filtration rate (GFR) >30 ml/min were compared with 20 healthy controls. Clinical and laboratory evaluations, levels of MDA (fluorimetric thiobarbituric test), SOD (cytochrome reduction method) and adiponectin (ELISA) were obtained. RESULTS: Sixty-four patients (GFR 91.44 ± 38.50 ml/min, urinary albumin-to-creatinine ratio [UACR] 20.81 [4.64-72.88 mg/g]) were included. MDA was higher in T2DP than in controls: 3.97 (2.43-4.59) versus 1.35 (1.16-1.81) nmol/ml, p < 0.0001. MDA correlated with glycated hemoglobin (r = 0.40, p = 0.001), adiponectin (r = -0.28, p = 0.03), systolic blood pressure (r = -0.28, p = 0.03) and SOD (r = -0.35, p = 0.005); adiponectin (p = 0.01) and glycated hemoglobin (p = 0.02) remained significant predictors of MDA in multiple regression analysis. SOD was negatively correlated with glycemia (r = -0.71, p < 0.0001) and glycated hemoglobin (r = -0.5, p < 0.0001). When patients were divided according to a ROC-derived adiponectin cutoff of 8.9 µg/ml, patients with higher adiponectin had lower MDA, [2.55 (2.35-3.60) vs. 4.10 (2.89-5.31) nmol/ml, p = 0.005] but similar SOD levels. In T2DP with nephropathy (GFR < 60 ml/min or UACR > 30 mg/g), the correlation of adiponectin with MDA was stronger, (r = -0.51, p = 0.004) confirmed in multiple regression analysis (p = 0.03). Adiponectin was not correlated with MDA, and SOD was inversely related to MDA in patients without nephropathy. CONCLUSION: Adiponectin is a significant predictor of MDA in early diabetic nephropathy, whereas SOD strongly depends only on glycemic control and is not directly related to adiponectin.

Expression of TLR4 protein is reduced in chronic renal failure: evidence from an experimental model of nephron reduction.

Toll-like receptor 4 (TLR4) signaling is involved in various acute and chronic renal lesions and contributes to inflammation and fibrosis in several organs; the latter are important determinants to the progression of chronic kidney disease (CKD). We aimed to assess TLR4 expression in progressive CKD and relate it to severity of kidney damage, using an experimental nephron reduction model. Male Wistar rats were subjected to subtotal nephrectomy using the ligation technique, after 12 weeks of observation, serum creatinine and proteinuria were determined, animals were sacrificed, glomerulosclerosis and interstitial scarring were quantified histologically, and TLR4 expression was assessed by immunohistochemistry. Sham-operated rats served as controls. Case animals had significantly higher creatinine, proteinuria, glomerulosclerosis and tubulointerstitial involvement. TLR4 expression was prominent in proximal tubes, less staining was observed on infiltrating inflammatory cells. Percentage of TLR4-positive tubes was reduced in the subtotal nephrectomy animals, when compared to controls (0.67±0.09 versus 0.79±0.07, p=0.003). Percentage of TLR4-positive tubes correlated inversely to markers of kidney damage: to proteinuria (r=-0.55, p=0.02), serum creatinine (r=-0.53, p=0.01); percentage of glomeruli with glomerulosclerosis (r=-0.54, p=0.01) and tubulointerstitial score (r=-0.36, p=0.01). As TLR4 staining appears in tubular casts only in nephrectomy animals, shedding from damaged tubular cells is a very likely explanation for the reduced TLR4 expression in the kidneys of subjects with experimental nephron reduction.

276G>T Polymorphism of the ADIPOQ Gene Influences Plasma Adiponectin in Type 2 Diabetes Patients but Is Not Predictive for Presence of Type 2 Diabetes in a Caucasian Cohort from Romania.

BACKGROUND: Previous reports associated ADIPOQ 276G>T polymorphism with plasma adiponectin levels and diabetes. Our objective was to study this polymorphism in type 2 diabetes (T2D) Romanian patients and to assess its influence on plasma adiponectin levels; possible link to prevalence of T2D was also addressed. DESIGN: Case control studyMaterial and Methods: Consecutive T2D patients, age and sex matched controls were genotyped for the 276 ADIPOQ locus. Medical history, laboratory evaluation, plasma adiponectin were assessed. OUTCOMES: 105 T2D patients and 48 controls were included. Adiponectin was higher in controls (17.04±3.02 µg/ml) than in T2D patients (10.32±1.16 µg/ml), difference failed to reach significance (p=0.06). Genotype distribution wasn't different between T2D patients and controls. 44 (41.90%) of T2D patients had GG genotype, 51 (48.57%) GT and 10 (9.52%) TT genotype. Adiponectin was higher (19.03±3.46 µg/ml) in diabetic TT allele carriers than in GT (9.96±1.76 µg/ml) or GG patients (8.71±1.60 µg/ml) p=0.003. In controls, 28 (58.33 %) subjects were carriers of the GG genotype, 16 (33.33%) had GT genotype and 4 (8.33%) had TT genotype. There weren't significant differences in the studied parameters between different genotypes in the control group. Logistic regression disclosed age p=0.0001 (OR 1.086; CI 1.041/1.133), waist circumference p=0.00049 (OR 1.084; CI 1.036/1.135), adiponectinemia p=0.036 (OR 0.963; CI 0.929/0.998) but not genotype as predictors for the presence of diabetes. CONCLUSION: Presence of the TT allele at the 276 locus of the ADIPOQ gene is associated with higher plasma adiponectin levels in type 2 diabetes patients. Plasma adiponectin, and not genotype at the 276 locus is predictive for the presence of T2D.