1. Signal Transduction Pathways Involved In MCP-1 Induced Tubular Epithelial-Myofibroblast Transition Of HK-2 Cells In Vitro 2. Effect Of Rapamycin On Tubular Epithelial-Myofibroblast Transition And Renal Interstitial Fibrosis

Signal Transduction Pathways Involved in MCP-1 Induced Tubular Epithelial-Myofibroblast Transition of HK-2 Cells in VitroObjectiveIt is considered that Tubular Epithelial-myofibroblast transition (EMT) is one of the primary pathomechanisms in renal interstitial fibrosis. Monocyte chemoattractant protein-1 (MCP-1) plays an important role in progressive glomerular and interstitial damage in renal diseases. We recently found that MCP-1 may induce EMT, and the mechanism of the effect involves TGFβ-1-Smad3 signal transduction pathway. However, the exact mechanism of the effect is still unclear. In the present study, the effect of MCP-1 on EMT of proximal tubular cells (HK-2) and possible mechanism of the effect were examined.MethodsThis study includes three experiments. 1. To examine the effect of MCP-1 on EMT of HK-2 cells, the cultured HK-2 cells were divided into three groups: negative control, treated with TGFβ-1(5 ng/m]), treated with different concentration (0.1, 1, 10, 100ng/ml) recombinant human MCP-1 at different time point(24h, 36h, 48h, 72h),respectively. The expressions ofα-smooth muscle actin (α-SMA) mRNA and protein of cells were detected by Western blot and RT-PCR method. 2 To determine possible mechanism of EMT induced by MCP-1, cultured HK-2 cells were divided into negative control, MCP-1 treated at different concentration(0. 1, 1, 10, 100 ng/ml). The expressions of P-Erk1/2, Erk1/2, P-P38MAPK, P38MAPK, RhoA protein levels of HK-2 cells were measured with Western blot method, and expressions of RhoC mRNA levels were detected by RT-PCR method. Then the effects of inhibitors of MEK (PD98059), P38 MAPK (SB203580) and Rho kinase (Y27632) on MCP-1 (1 ng/ml) induced EMT were examined, respectively. The levels ofα-SMA protein in each group were examined by Western blot. 3. HK-2 cells were divided into negative control, treated with TGFβ-1(5 ng/ml), treated with different concentration MCP-1 (0.1,1, 10, 100 ng/ml). The expressions of Snail mRNA levels were detected by RT-PCR method.Results1. The expressions ofα-SMAprotein and mRNA significantly increased in MCP-1 treated HK-2 cells in dose and time dependent manner. The levels ofα-SMA protein and mRNA expressions of HK-2 cells treated with MCP-1(1 ng/ml) at 48h were significantly higher in cells treated with MCP-1 at different concentrations (P＜0.05). 2. The ratio of P-Erkl/2 and Erkl/2 was significantly increased in MCP-1 treated cells and the highest ratio was at 100 ng/ml of MCP-1. MCP-1 induced phosphorylation of Erk1/2 was completely inhibited by PD98059 (P＜0.05), but MCP-1 induced expressions ofα-SMA was only partly inhibited by PD98059 (P＜0.05). The ratio of P-P38MAPK and P38MAPK was significantly increased in MCP-1 treated cells at different concentration when compared with the negative control. MCP-1 could phosphorylate P38MAPK in HK-2 cells in dose dependent manner. SB203580 blocked the phosphorylation of P38MAPK induced by MCP-1, while MCP-1 induced expressions ofα-SMA were not affected by SB203580. The level of RhoA Protein and RhoC mRNA expressions in HK-2 cell stimulated with MCP-1 were not significantly changed in MCP-1 treated cells at different concentration (P＞0.05). The expressions ofα-SMA did not changed significantly in HK-2 cells treated with MCP-1 and Rho kinase inhibitor: Y27632 (P＞0.05). 3. The expression of Snail mRNA was significantly increased in dose dependent manner in MCP-1 treated cells at different concentration when compared with negative control. The level of Snail mRNA was highest at 100ng/ml of MCP-1.ConclusionsMCP-1 may induce EMT of HK-2 cells in vitro, and this effect may involve upregulation of Erk1/2 Map kinase and Snail signal pathways. P38MAPK or Rho kinase signal pathways were not proven to be involved in MCP-1 induced EMT. Effect of Rapamycin on Tubular Epithelial-Myofibroblast Transition and Renal Interstitial fibrosisBackground:Renal interstitial fibrosis is closely correlated to progression of chronic renal disease and Epithelial-Myofibroblast Transition (EMT). EMT is a key event in the progression of chronic renal diseases.Rapamycin (RAPA) is a macrocyclic lactone product of Streptomyces hygroscopicus. It's a potent anti-proliferative immunosuppressant and has been used clinically to suppress rejection of transplanted organs. Rapamycin has also been shown to reduce renal injury, including renal fibrosis, in a variety of animal models of chronic renal disease. However, the effect of rapamycin on EMT and renal fibrosis has hardly been investigated.Objective: In this present study, we observed the effect of rapamycin on EMT of cultured HKC cells in vitro and examined the effect of rapamycin on renal fibrosis and EMT in rats of UUO model.Methods: In Vitro study, Cultured human proximal tubular epithelial cells (HKC) were divided into three groups: negative control, treated with TGFβ-1 (lng/ml) and treated with TGFβ-1 (1 ng/ml) andrapamycin (0. 1, 1, 10, 100 ng/ml) at different time point (12h, 24h, 48h, 72h). The protein and mRNA forα-SMA and E-cadherin in HKC cells were determined by Western Blot and RT-PCR. The mRNA level of Snail in HKC was detected by RT-PCR. In Vivo study, The Wistar rats were divided into sham-operation, UUO and UUO treated with rapamycin groups, and sacrificed at day 3, 7, 14 and 21, respectively. The histopathologic degrees of interstitial fibrosis were scored in Masson-stained sections. The protein and mRNA forα-SMA and E-cadherin in kidney tissue were semiquantitatively measured with immunohistochemistry, Western Blot and RT-PCR. The expressions for TGFβ-1 and MCP-1 were determined by immunohistochemistry, Western blot and RT-PCR, respectively. dramatically abrogated TGFβ-1 inducedα-SMA expression and restored the E-cadherin expression in HKC cells in a dose-dependent manner. At a concentration of 100 ng/ml, Rapamycin almost completely blockedα-SMA mRNA and Protein expression induced by TGFβ-1(lng/ml). Rapamycin also suppressed expression ofα-SMA in HKC cells at both mRNA and protein levels in a time dependent manner. We also found rapamycin dramatically abrogated TGFβ-1 induced snail mRNA expression in HKC cells in a dose-dependent manner. In Vivo study, The semiquantitative renal fibrosis scores were reduced in RAPA-treated group compared with the UUO group at day 3 and 7 (P＜0.05), respectively. The expressions ofα-SMA protein and mRNA were also reduced in RAPAtreated group, when compared with the UUO group at day 3 and 7 (P＜0.05), respectively. Meanwhile, the E-cadherin protein and mRNA expressions were upregulated in RAPA-treated group when compared with the UUO group at day 3 and 7, respectively (P＜0.05). The MCP-1 and TGFβ-1 expressions were decreased in RAPA-treated group when compared with the UUO group at day 3, 7, and 14 (P＜0.05), respectively. No differences in fibrosis score, expression ofα-SMA, MCP-1 and TGFβ-1 were found between the RAPA-treated and UUO group at day 21.Conclusion:Rapamycin may inhibit EMT of tubular cells both in vitro and in vivo. The upregulation of Snail expression might be one of the mechanisms of Rapamycin blocking EMT. Rapamycin could attenuate the interstitial fibrosis in rat model of obstructive nephropathy. The inhibition of the EMT and suppression of MCP-1 and TGFβ-1 expressions might be two important mechanisms that account for it beneficial effects.