Involvement of Elf3 on Smad3 activation-dependent injuries in podocytes and excretion of urinary exosome in diabetic nephropathy.

Diabetic nephropathy (DN) is among the most serious complications of diabetes mellitus, and often leads to end-stage renal disease ultimately requiring dialysis or renal transplantation. The loss of podocytes has been reported to have a role in the onset and progression of DN. Here, we addressed the activation mechanism of Smad3 signaling in podocytes. Expression of RII and activation of Smad3 were induced by AGE exposure (P<0.05). Reduction of the activation of RII-Smad3 signaling ameliorated podocyte injuries in Smad3-knockout diabetic mice. The bone morphogenetic protein 4 (BMP4) significantly regulated activation of RII-Smad3 signalings (P<0.05). Moreover, the epithelium-specific transcription factor, Elf3was induced by AGE stimulation and, subsequently, upregulated RII expression in cultured podocytes. Induction of Elf3 and activation of RII-Smad3 signaling, leading to a decrease in WT1 expression, were observed in podocytes in diabetic human kidneys. Moreover, AGE treatment induced the secretion of Elf3-containing exosomes from cultured podocytes, which was dependent on the activation of the TGF-ß-Smad3 signaling pathway. In addition, exosomal Elf3 protein in urine could be measured only in urinary exosomes from patients with DN. The appearance of urinary exosomal Elf3 protein in patients with DN suggested the existence of irreversible injuries in podocytes. The rate of decline in the estimated Glomerular Filtration Rate (eGFR) after measurement of urinary exosomal Elf3 protein levels in patients with DN (R2 = 0.7259) might be useful as an early non-invasive marker for podocyte injuries in DN.

Increased urinary Smad3 is significantly correlated with glomerular hyperfiltration and a reduced glomerular filtration rate and is a new urinary biomarker for diabetic nephropathy.

BACKGROUND: Diabetic nephropathy is one of the major microvascular complications of diabetes. We investigated the association between urinary Smad3 (usmad3) levels, glomerular hyperfiltration, and the development of nephropathy in patients with type 2 diabetes mellitus (T2DM). METHODS: The usmad3 level was determined by enzyme-linked immunosorbent assay in 245 well-characterised patients with T2DM and 82 healthy control subjects. The associations of the usmad3 level with glomerular hyperfiltration, glucose and lipid profiles, and renal function were evaluated. RESULTS: The usmad3 level was significantly higher in patients with diabetes than in the control group. The level in the hyperfiltration group was higher than that in the normofiltering group, regardless of whether patients were in the normoalbuminuric or the proteinuria groups. Pearson's correlation analysis suggested that the usmad3 level was significantly correlated with age, systolic blood pressure, fasting plasma glucose, insulin, C-peptide, glycated haemoglobin, and estimated glomerular filtration rate (eGFR). A multiple linear stepwise regression analysis revealed that usmad3 levels in patients with T2DM and an eGFR ≥ 90 ml/min/1.73 m(2) were independently and positively correlated with eGFR, whereas in patients with T2DM and eGFR <90 ml/min/1.73 m(2), the levels were independently and negatively correlated with eGFR. CONCLUSIONS: The usmad3 level was significantly correlated with biphasic changes in the GFR (both glomerular hyperfiltration and reduced eGFR) in patients with T2DM. Usmad3 may serve as a novel marker for hyperfiltration and for screening patients with T2DM for nephropathy.

[Change and significance of urinary smad3 in type 2 diabetic nephropathy].

OBJECTIVE: To explore the significance of urinary smad3(usmad3) protein level change in diabetic nephropathy (DN) in type 2 diabetes mellitus and examine its relationship with the progression of DN. METHODS: From May 2010 to August 2011, a total of 282 patients with type 2 diabetes were selected for the experimental group according to instant urine specimen albumin-creatinine ratio (UACR). Another 100 healthy subjects were taken as the control group. Then the diabetics were divided into 3 groups, including 110 with normal albuminuria (NA group), 114 with microalbuminuria (MA group) and 58 with macroalbuminuria (DN group). Enzyme-linked immunosorbent assay (ELISA) was used to detect the content of usmad3. The parameters of age, systolic blood pressure, body mass index (BMI), blood glucose, blood lipids, urinary albumin/creatinine (ACR), estimated glomerular filtration rate (eGFR) and glycosylated hemoglobin were measured. And non-mydriatic fundus camera was used to evaluate retinopathy, the DN group (n = 58) was then divided into two groups of those without retinopathy (n = 25) and with retinopathy (n = 33). RESULTS: (1) The usmad3 level in type 2 diabetes group was significantly higher than that in the control group (489(273,1193) vs 311 (179, 497) ng/mmol Cr (P < 0.01). (2) According to UACR, type 2 diabetes group was divided into 3 different groups to compare the relative level differences of usmad3 in different groups: MA group versus NA group, 552 (316,1338) vs 317 (200,594), DN group versus NA group, 1035 (503,3035)vs 317 (200,594), DN group versus MA group, 1035(503,3035)vs 552(316,1338), all P < 0.01. (3) Pearson correlation test showed the level of usmad3 was significantly correlated with age, SBP, HbA1c, blood urea nitrogen, creatinine, total cholesterol, low density lipoprotein, eGFR and UACR (r = 0.57, P < 0.01). And multiple linear regression analysis showed that usmad3 and UACR were independently correlated (ß = 0.754, P < 0.01). (4) The usmad3 level in DN with retinopathy were significantly higher than that in DN without retinopathy (1905(806,4303) vs 595 (331,1183), P < 0.01). No significant difference existed in uACR level between DN with retinopathy and DN without retinopathy(P > 0.05). CONCLUSIONS: Urinary level of smad3 is significantly elevated in type 2 diabetics and it is significantly associated with ACR. It suggests that usmad3 is a potential marker in the diagnosis of DN and may be used to predict the severity of DN.