Smad7 alleviates glomerular mesangial cell proliferation via the ROS-NF-κB pathway.

OBJECTIVE: The aim of this study was to demonstrate that altered gene expression of Smad7regulated NF-κB expression and ROS production on Ang II (Angiotensin II)-induced rat glomerular mesangial cell (GMC) proliferation. METHODS: pAdTrack-CMV-Smad7 was transduced into rat GMC by adeno-transduction using an ADV (adenovirus)-mediated vector in vivo. Diphenylene iodonium chloride (DPI) pre-treated GMC, and blocked ROS generation as determined by DCFH-DA method. Altered expressions of IκBα and p65 were monitored by Western blot analysis and immunofluorescence. GMC proliferation was tested by the Cell Counting Kit-8 assay. Apoptosis of GMC was detected by flow cytometric analysis. RESULTS: Over-expression of Smad7 dampened the ability of Ang II to promote ROS synthesis and inhibited the ability of Ang II to decrease functional expression of IκBα. Moreover, Smad7 increased nuclear IκBα expression. Smad7 did not significantly influence the capacity of Ang II to increase protein expression of NF-κB p65. However, immunofluorescence analysis showed that Smad7 reduced nuclear NF-κB p65 level. Further, over-expression of Smad7 promoted GMC apoptosis by inhibiting NF-κB activation, which alleviated the Ang II-promoted proliferation of GMC. CONCLUSIONS: Smad7 influenced NF-κB expression by regulating ROS generation, and induced GMC apoptosis to counter the Ang II-promoted proliferation.

Effects of caveolin-1 and P-ERK1/2 on Ang II-induced glomerular mesangial cell proliferation.

This study explored the effects of caveolin-1, p-ERK1/2 and transient receptor potential channel 6 (TRPC6) on angiotensin II (Ang II)-induced glomerular mesangial cell (GMC) proliferation, and investigated the role of Ang II on GMC proliferation. GMC cultures were divided into Control, Ang II (Ang II 10(-7 )mol/L), PD98059 (Ang II 10(-7 )mol/L + PD98059 5 × 10(-5 )mol/L) and MßCD groups (Ang II 10(-7 )mol/L + MßCD 10(-2 )mol/L). GMCs proliferation was measured by the methyl thiazolil tetracolium and trypan blue assays. The distribution of caveolin-1, p-ERK1/2 and TRPC6 was monitored by immunocytochemistry. Real time polymerase chain reaction (PCR) was used to assess mRNA expression of caveolin-1 and TRPC6. Western blot analysis was used to assess protein expression of caveolin-1, p-ERK1/2 and TRPC6. The results showed that Ang II promoted GMC proliferation. PD98059 and MßCD blocked Ang II-induced GMC proliferation, by 31.06% and 48.96%, respectively. In comparison with the control group, the expression of p-ERK1/2 and TRPC6 was significantly higher and caveolin-1 expression was significantly lower in the Ang II group. PD98059 markedly decreased p-ERK1/2 and TRPC6 expression and increased caveolin-1 expression. MßCD decreased the expression of p-ERK1/2 and TRPC6, but had no significant effect on caveolin-1 protein expression. These findings suggested that the intact caveolae structure was associated with Ang II-induced GMC proliferation, ERK1/2 activation and TRPC6 expression. And p-ERK1/2 acted as an upstream signal molecule for TRPC6. Moreover, p-ERK1/2 and caveolin-1 appeared to be inhibited reciprocally, thus regulated GMC proliferation by regulating TRPC6 expression.

Protective effects of blocking renin-angiotensin system on the progression of renal injury in glomerulosclerosis.

To investigate the protective effects of blocking rennin-angiotensin system (RAS) on the progression of renal injury in glomerulosclerosis, a glomerulosclerosis model was made for SD rats by unilateral nephrectomy and being injected with Adriamycin into caudal vein. The rats with glomerulosclerosis were randomly divided as ten per group into those without further treatment (group D) and those treated with Benazepril (group DB), Losartan (group DL), or sham-operation (group C), respectively. After 6 weeks of administration of Benazepril or Losartan, the mRNA expressions of TGF-beta1, Col IV, Fn, ET-1 and iNOS in renal cortex were measured by RT-PCR. Besides, the expressions of TGF-beta1, ET-1 and iNOS at protein level were detected by Western blotting and the concentrations of Col IV and Fn were analyzed with immunohistochemistry respectively. Results showed that the rats in group D appeared as obvious proteinuria, hypoalbuminemia and hypercholesterolemia, which had a significant difference compared with group C (p < 0.05), and most of their mesangiums were detected with cellular proliferation and significant increasing for extracellular matrix. Renal cortex TGF-beta1, Col IV, Fn, ET-1 and iNOS in rats of group D were increased by 3.59, 2.57, 2.21, 2.58 and 3.28 times at mRNA level, and by 2.60, 1.40, 0.75, 1.83 and 2.15 times at protein level, respectively, compared with group C. When the animals were treated with Benazepril (group DB) or Losartan (group DL), however, the biochemical and pathological damages were significantly recovered, and protein expressions of TGF-beta1, Col IV, Fn, ET-1 and iNOS were also significantly diminished (p < 0.05). This study suggested that blocking RAS using Benazepril or Losartan can have protective effects on the renal injury in glomerulosclerosis by down-regulating the expressions of TGF-beta1, Col IV, Fn, ET-1 and iNOS.

[Effects of rhein on activity of caspase-3 in kidney and cell apoptosis on the progression of renal injury in glomerulosclerosis].

OBJECTIVE: To investigate the effects of rhein on the progression of renal injury and cell apoptosis in glomerulosclerosis, and further explore the protective mechanism of rhein on glomerulosclerosis. METHODS: Glomerulosclerosis models were made for SD rats by unilateral nephrectomy and being injected with Adriamycin into caudal vein, and randomly divided into control group, renal disease group, Rhein treatment group and Benazepril treatment group, and 6 rats in each group were killed at the 6th, 8th, 10th, 12th week respectively. The apoptosis protease-3 (caspase-3) in renal cortex was determined by immunohistochemistry stain method, and the activity of caspase-3 was measured by colorimetry, and the activity of nuclear factor-kappa B (NF-kappaB) was analyzed by gel electrophoretic mobility shift assay (EMSA), and renal tissue cell apoptosis was tested by terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) in order to observe expressions of caspase-3 and NF-kappaB and cell apoptosis of renal tissue. RESULTS: Renal disease group presented with distinct proteinuria, decreasing of blood albumin content and increasing of cholesterol concentration. Glomerulosclerosis index, apoptosis index, activity of NF-kappaB and expression of caspase-3 in renal disease group were more significantly higher than those in control group (P < 0.05 or P < 0.01) as time passed. Compared with the other time points in renal disease group, there were a great number of TUNEL-positive cells observed at the 10th week, slightly higher than that at the 12th week (9.3 +/- 2.3 vs 8.4 +/- 1.2, P > 0.05), the expression of Caspase-3 was also most obvious at the 10th week, significantly higher than that at the 12th week (11.4 +/- 2.5 vs 8.2 +/- 1.7, P < 0.05), which mainly located around capillary vessel in renal cortex, tending to be consistent with apoptosis cells expression. After the 8 weeks treatment of rhein or Benazepril, the number of TUNEL-positive cells significantly decreased and maintained at a certain level, and the activity of NF-kappaB and expression of caspase-3 decreased (P < 0.05), and renal pathological changes and biochemical changes improved magnificently, moreover, the expression of caspase-3 showed positive correlation with apoptosis index (r = 0.836, P < 0.01). CONCLUSION: Rhein could have significant protective effects on the progression of renal injury, and might regulate pathological changes by influencing the activities of NF-kappaB and caspase-3 in the early phase of glomerulosclerosis. Therefore, down-regulating caspase-3 expression in kidney might be one of the molecular mechanisms in the way that rhein could alleviate renal tissue cell apoptosis in glomerulosclerosis.

Detection of urinary podocytes and nephrin as markers for children with glomerular diseases.

The purpose of this study was to detect the urinary podocytes and its related protein, nephrin, in the urine of the children with glomerular disease in order to analyze the relationship of the clinical testing with the significance of the glomerular disease. A total of 65 children with nephrotic syndrome were selected for this study. The podocytes and nephrin were detected in the urinary sediment by indirect immunofluorescence, enzyme-linked immunosorbent assay, and Western blotting. The urinary podocytes and nephrin positive rates were 53.8% and 50.8%, respectively, in the children with glomerular disease. The serum total protein and albumin decreased in the podocyte-positive children, while the urine total protein at 24 h, urinary albumin/creatinine ratio, blood urea nitrogen, and serum creatinine were significantly elevated as compared to those of the podocyte-negative patients. Furthermore, the results were the same in the patients with positive nephrin as compared to that of the patients with negative nephrin. The podocyte number and nephrin level were significantly higher in the lupus nephritis group as compared to those of the other groups. Likewise, the podocyte number and nephrin level dramatically increased in the focal segmental glomerulosclerosis group as compared to those of the mesangial proliferative glomerulonephritis and minimal change disease groups. In addition, the podocyte numbers and nephrin expression were significantly higher in severe proteinuria group as compared to those of the mild proteinuria group. The urinary nephrin expression was positively related to podocyte and urinary albumin/creatinine ratio. We concluded that the detection of the urinary podocytes and nephrin could be taken as markers for children with glomerular disease, reflecting the type of the disease. Therefore, this can be used as a noninvasive method to evaluate the severity of the kidney disease in children.

AngII-induced glomerular mesangial cell proliferation inhibited by losartan via changes in intracellular calcium ion concentration.

This study investigated the changes in intracellular [Ca(2+)](i) (intracellular calcium ion concentration) and TRPC6 (transient receptor potential channel 6) expression during angiotensin II (AngII)-induced glomerular mesangial cell (GMC) proliferation, as well as the inhibitory effect of losartan. GMC cultures were split into four groups treated for 24 h: Group N (blank control group), Group A (10(-7 )mol/L AngII), Group LT (10(-7 )mol/L AngII and 10(-5 )mol/L losartan), and Group Pred (10(-7 )mol/L AngII and 10(-5 )mol/L prednisone). GMCs proliferation was measured by the MTT and trypan blue assays. The distribution of TRPC6 was monitored by immunofluorescence, the expression of TRPC6 was detected by RT-PCR and Western blotting, and [Ca(2+)](i) was measured by laser scanning confocal microscopy. The results showed that the maximal proliferation of GMCs was induced by treatment with 10(-7 )mol/L AngII for 24 h. In Group A, the distribution of TRPC6 was not uniform in the cell membrane, there was increased accumulation of this protein within the cytoplasm, and the increased expression of TRPC6 and [Ca(2+)](i) was consistent with the proliferation of cells. In Group LT, losartan inhibited the proliferation of GMCs significantly, the levels of TRPC6 and [Ca(2+)](i) were diminished, and the distribution of TRPC6 was improved. Prednisone also significantly inhibited the proliferation of GMCs and had no effects on the expression of TRPC6 and [Ca(2+)](i) in Group Pred. These findings suggested that AngII could enhance the expression of TRPC6, increase [Ca(2+)](i,) and demonstrate a time-dose-response relationship with the proliferation of GMCs, while losartan reversed the effect of AngII on GMC proliferation.

FK506 alleviates proteinuria in rats with adriamycin-induced nephropathy by down-regulating TRPC6 and CaN expression.

BACKGROUND: To investigate the roles of transient receptor potential cation channel 6 (TRPC6) and calcineurin (CaN) in proteinuria pathogenesis and the mechanism of action of the calcineurin inhibitor tacrolimus (FK506) in adriamycin-induced nephropathy. METHODS: The adriamycin-induced nephropathy rats were established and randomly divided into adriamycin nephropathy (ADR), low-dose FK506 treated (ADR + FK0.5), high-dose FK506 treated (ADR + FK1.0) and Control groups. Twenty-four hour urinary protein and blood biochemistry were measured on weeks 2, 3, 5 and 7, and the distributions and expressions of TRPC6 and CaN in the renal tissue were detected by immunohistochemistry, real-time PCR and Western blot. RESULTS: The study showed the 24-hour urinary protein increased significantly in ADR rats compared with Controls whilst for ADR rats hypoalbuminemia, hypercholesterolemia, renal functional lesion and renal pathologic changes appeared. The areas and intensities of TRPC6 and CaN expressed in the glomerulus and tubulointerstitium of ADR rats increased significantly. The expression of TRPC6 mRNA in ADR rats began to increase on the 3rd week and persisted to the 7th week, and CaN mRNA increased throughout the period. Protein expressions of TRPC6 and CaN in ADR rats were significantly higher than those of Controls. FK506 can inhibit CaN activity in the renal tissues and further decrease TRPC6 expression and therefore reduce renal damage and proteinuria in a dose-dependent manner. CONCLUSIONS: These findings suggested that TRPC6 and CaN were up-regulated on mRNA and protein levels in adriamycin-induced rats. FK506 had a therapeutic effect on the progression of proteinuria and renal damage by down-regulating of TRPC6 and CaN in the renal tissues.