| Background and objective: Diabetic nephropathy(DN) is one of the mostcommon chronic complications of diabetes mellitus and the major cause of end-stage renal disease(ESRD). It widely accepted for the time being that therapeutic principles for DN are as follows: the control of hyperglycemia, blood pressure and diet as well as renal function protection. Renal function would decline quickly when clinical proteinuria appears in the diabetic patients. It has been estimated that more than 20% of diabetic patients with clinical proteinuria would progress into ESRD. Thus the expenditure on dialysis and kidney transplantation increases every year and causes heavy economical burdens on the patients and the society. An interaction of metabolic and hemodynamic factors has been considered a traditional aspect involved in the development of renal lesions in patients with type 1 or type 2 diabetes.However,the recent study in vivo and in vitro has demonstrated that inflammation is an important cause in chronic progressive renal disease in diabetic patients. The kidney inherent cells can produce inflammatory cytokines, such as TNF-α, MCP-1 and IL-1 in pathologic conditions, and these cytokines can amplify the inflammation by autocrine and paracrine and lead to an inflammation cascade.Mycophenolate mofetil(MMF), is a new immune suppressor and its active metabolic form in vivo is mycophenolic acid(MPA). Besides highly selective inhibition of lymphocyte proliferation, it could also inhibit the proliferation of mesangial cells and monocytes, reduce expression of cell adhesion molecules and reduce deposition and accumulation of ECM. MMF was initially used to suppress immunological rejection in patients after renal transplantation. In recently years, studies demonstrated that MMF had obvious therapeutic effect on non-inflammatory renal diseases including DN. It could prevent albuminuria, glomerulosclerosis and inflammatory cells infiltration, and does not interfere with blood pressure, glomerular dynamics or blood glucose levels. Up to now, no paper has been published about whether MMF has effects on preventing podocytes and mesangial cells loss at the early stage of DN. Therefore, we focused our study on the effects of MMF on cytokines, more precisely on MCP-1, TGF-βand CTGF, to elucidate the interaction between cytokines and MMF in ECM accumulation in the pathogenesis of DN. Diabetes was induced in uninephrectomized male Wistar rats by peritoneal injection of STZ. Our purposes are to compare the effect of high glucose with low glucose on the proliferation of mesangial cells(MCs), to investigate the effect of MMF on high glucose-induced proliferation, and to investigate the effect of MMF on preventing podocyte loss and renal tubulointerstitial injury in STZ induced diabetic model.The present study is to explore the mechanism of MMF's protection on DN.Methods: 1. Rat MCs were cultured with the medium which contains 5.60mmol/L glucose, or 30 mmol/L glucose and 10, 25 or 50μmol/L of MPA. Cell proliferation was tested with MTT and observed by spectrometry 6, 12, 24 and 48h later. The concentration of type IV collagen and MCP-1 in supernatant were measured by radioimmunoassay (RIA) and ELISA, respectively. The mRNA expressions of transforming growth factor (TGF)-beta, connective tissue growth factor (CTGF) and MCP-1 in cells were determined by real time RT-PCR.2. Diabetes was induced in uninephrectomized male Wistar rats by peritoneal injection of STZ(65 mg/kg). Rats were randomly divided into five groups: control group(NC), diabetic without treatment group(DM), Valsartan treated group(DM.V), MMF treated group(DM·M), and combined treatment group(DM·V·M). DM·M group were given MMF (15mg.kg-1.d-1), DM-V group Valsartan (40mg.kg-1.d-1), and DM·V·M group a combined therapy of MMF(15mg.kg-1.d-1) and Valsartan(40mg.kg-1.d-1). The medication last for 8 weeks. Serum creatinine, creatinine clearance rate, 24h urinary protein and the ratio of kidney weight/body weight were determined after 8 weeks. The renal tissue morphology was observed by light microscopy and electron microscope. Expressions of nephrin, desmin and MCP-1 were measured by semi-quantitative immunohistochemical assays. Real-time quantitative PCR was used to detect the mRNA levels of nephrin and MCP-1 in renal tissues.3. Diabetes induced in uninephrectomized male Wistar rats by peritoneal injection of STZ (65mg/kg) were randomly divided into three groups: control group (NC), diabetic group (DM) and treated group (DM+MMF) with MMF(15mg.kg-1.d-1.). This study lasted for 8 weeks. Blood glucose, 24h urinary protein and the ratio of left kidney weight/body weight were determined after 8 weeks. Morphological changes in renal tubulo-interstitium were observed. Immunohistochemical method was applied to analyze the expression of MCP-1, OPN, M-CSF and CD68. The expressions of MCP-1 and OPN mRNA in renal tissue were measured by quantitative Real-time PCR.Results:Cell culture:1. The MCs proliferation presented a time-and dose-dependent feature in different concentrations of glucose, and got to the highest point at the concentration of 30mmol/L for 24 hours. MMF could inhibit the high glucose induced proliferation in a time-and dose-dependent feature.2.The concentration of type IV collagen in supernatant with high glucose was higher than that of control group(P<0.01); High glucose plus 50umol/L MPA group had a lower concentration of type IV collagen than that of high glucosegroup(P<0.01).3.ELISA and RT-PCR suggested that hyperglycemia could stimulate the synthesisof MCP-1, low concentrations of MPA(10,25umol/L) had no obvious effects onMCP-1mRNA expression and high concentrations of MPA(50umol/L) couldinhibit MCP-1 expression significantly(P <0.01).4. Forty-eight hours later the expression of TGF-B and CTGF mRNA in highglucose group were 4.54 fold and 5.65 fold to that of normal glucose respectively;High glucose plus 50um/L MPA group had lower expressions of TGF-B andCTGF mRNA than that of high glucose group(P<0.01).Zoopery:1. At the end of the 8th week, blood glucose concentration of diabetic rats and all treatment groups were persistently higher than that of control rats. Diabetic rats showed a higher 24h urinary protein and hypertrophy index (left renal weight/body weight) than that of control group(P<0.01), but the index was lower among all treatment groups than that of diabetic non-treatment group (P<0.01). Valsartan reduced systolic blood pressure(SBP) by approximately 30mmHg when compared with untreated diabetic rats (p<0.01). There was no statistically significant difference in blood pressure between the DM-V and DM·V·M groups. MMF alone did not reduce SBP to any significant extent. Increased proteinuria tended to decrease in diabetic rats treated with MMF, Valsartan or combined therapy. There was no significant difference in the serum creatinine among all groups(P>0.05).2. At the end of the 8th week, glomerular lesions in rats were characterized by thickening of the basement membrane, mesangial expansion and sclerosis. The glomerulosclerosis score in diabetic rats increased significantly compared with that of control(P<0.01). Treatment with MMF could significantly reduce glomerulosclerosis in diabetic rats (P<0.01). Interstitial fibrosis was focal and mild during our study. The interstitial fibrosis score was higher in untreated diabetic rats than that of control rats (P<0.01). Interstitial fibrotic lesions decreased significantly in diabetic rats treated with MMF compared with that of untreated diabetic rats (P<0.05). There were significant correlations between 24h urinary protein and glomerulosclerosis score (r=0.67,P<0.01), and interstitial fibrosis score(r=0.74, P<0.01). Renal tissue section was observed through an electron microscope. Compared with that of NC group, DM group showed thickened glomerular basement membrane, vacuolization in part of the renal tubular cells, foot process denudation, exposed GBM and shedded podocytes. But in treatment groups foot process fusion is less serious, and shedded podocytes were rarely seen.3.Immunohistochemical staining for nephrin, desmin and MCP-1 Immunohistochemistry demonstrated the localization of podocyte specific nephrin in NC rats which was mainly in the central area of glomeruli. The nephrin expression decreased in diabetic rats with an altered pattern in the peripheral area of glomeruli. MMF and Valsartan treatment resulted in the restoration of nephrin expression with dominant localization along the glomerular capillary area. MCP-1 and desmin expression was almost negative in the control rats, except a weak expression occasionally seen in tubular epithelial cells and glomeruli. In the diabetic kidney, MCP-1 synthesis was mainly detected in the tubulointerstitial areas. In concert with MCP-1, desmin synthesis increased rapidly in the diabetic kidney. Different from MCP-1, desmin was mainly detected in the glomeruli. Interestingly, MMF treatment significantly suppressed the synthesis of tubulointerstitial MCP-1 and intraglomerular desmin.4. Quantitative real-time PCR Nephrin mRNA expression in diabetic rats was lower than that of control rats(P<0.05). Treatment with MMF in diabetic rats significantly restored nephrin mRNA expression. MCP-1 mRNA level in renal cortex was significantly higher in diabetic rats than that of control rats(P<0.01).Over-expression of MCP-1 mRNA in diabetic rats was significantly suppressed by MMF(P<0.01). There were significant negative correlations between nephrin and MCP-1 mRNA(r=-0.86, P<0.01), 24h urinary protein and mRNA levels of MCP-1 (r=0.56, P<0.01) or nephrin(r=-0.78, P<0.01). 5. Compared with NC group, the area of interstitial fibrosis was also significantly enlarged in DM group(P < 0.01); The expressions of CD68, MCP-1(mRNA and protein)were significantly upregulated in DM group(P <0.01); the expressions of OPN, M-CSF(protein) and OPN(mRNA)were also significantly upregulated in DM group(P <0.01); Treatment with MMF in diabetic rats can significantly reverse all those up-regulation (P <0.05or 0.01).Conclusion:1. High concentration of glucose can stimulate MCs proliferation with a time-and dose-dependent feature. MMF can inhibit such a high glucose-induced proliferation of MCs also in a time-and dose-dependent way.2. High glucose contributes to the upregulation of TGF-B, CTGF, MCP-1 and MCP-1 expression in rat MCs. MMF has some protective effect on DN by inhibiting the expression of TGF-B, CTGF and MCP-1. These findings may provide an experimental evidence for further study of the possibly protective effect of MMF against DN.3. MMF and Valsartan could suppress MCP-1 and desmin expression, increase the nephrin expression, and decrease proteinuria level in the diabetic rat. The combination of Valsartan and MMF has no superiority over monotherapies on renal protection. These results suggest that MMF may have renoprotective effects in the early stages of DN through an anti-inflammatory activity and preventing podocytes loss.4. MMF plays an obvious protective role against renal tubulointerstitial injury, and was probably associated with down-regulation of MCP-1 expression and suppression of macrophages proliferation. |
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