Effect Of SARA On Renal Tubular Epithelial To Mesenchymal Transition In Diabetic Nephropathy

Background Diabetic nephropathy (DN) is one of leading cause of end stage renal disease (ESRD). Tubulointerstitial fibrosis (TIF) is the most common pathological changes in DN. Epithelial to mesenchymal transition (EMT) is the early and reversible stage of TIF and extracellular matrix (ECM) accumulation is the key histological change of TIF. It is established that high glucose can result in EMT and ECM accumulation in renal tubular epithelial cells and Transforming growth factor-β1 (TGF-β1) plays a key role in these process. TGF-β1/Smads is the classical pathway which is involved in TGF-β1 mediated EMT and ECM accumulation. It is reported that TGF-β1/Smad is regulated by many factors such as adaptor protein.Smad anchor for receptor activation (SARA) is one adaptor protein which can recruit Smad2 and Smad3 to the activated TGF-βreceptorⅠ(TβⅠR) and promote their phosphorylation. SARA plays an essential role in TGF-βinduced Smad2 activation and Smad3 can be phosphorylated in a SARA-independent way. It is showed that SARA is negatively related to EMT and fibrosis and modulation of SARA can reverse the development of EMT.Therefore, SARA may play a key role in the process of EMT of tubular epithelial cells and ECM accumulation in diabetic nephropathy. To this end, experimental research is carried out as follow. Objective To investigate the expression of SARA in the kidney and observe the relationship of SARA and TIF in diabetic nephropathy patients.Methods 12 patients were enrolled into the study, they were diagnosed as DN (n=6) or minimal changes disease (primary kidney disease, control group, n=6) based on clinical manifestations and renal pathology. Renal pathological changes were observed by HE, Masson and PASM staining. Interstitial damage score and collagen deposition score were evaluated. The expression of SARA was tested by immunohistochemistry and the relationship of SARA and tubular interstitial damage in DN was analyzed.Results HE, Masson and PASM staining showed that there were many lesions in the kidneys of DN patients such as moderate to severe proliferation of mesangial matrix, nodular sclerosis, diffuse or segmental thickening of the basement membrane, tubular atrophy and compensatory expansion, local interstitial fibrosis. Meanwhile, the lesions in the kidneys of minimal change disease patients were less severe. There were mild mesangial cell and matrix proliferation, basement membrane is not thick, mild granular degeneration and vacuolar degeneration in renal tubular epithelial cells and no significant abnormalities in interstitial and renal vascular. Interstitial damage score and collagen deposition score in the kidneys of DN patients were higher than that in control group. Immunohistochemistry showed that the expression of SARA was significantly down-regulated in DN patients compared with that in control group.Conclusion The expression of SARA is significantly down-regulated in the kidney of DN patients and SARA is negatively related with renal interstitial fibrosis. Objective To detect the expression of SARA in human renal proximal epithelial cell lines (HK-2) and investigate the role of SARA in the development of high glucose induced EMT and ECM in HK-2.Methods HK-2 cells were exposed to high glucose (30mM D-glucose). The expressions of Vimentin, ZO-1, FN, ColⅠ, SARA were examined by Western blot and Realtime PCR. Furth more, HK-2 cells were transfected with the plasmids of wild-type SARA (SARA (WT)) and SARA mutant (SARA with SBD deletion, called SARA (dSBD)). Then the expressions of Vimentin, ZO-1, FN, ColⅠwere detected.Results Stimulation of HK-2 with high glucose resulted in a significant reduce in the expression of ZO-1 and increase in the expressions of Vimentin, FN, ColⅠ. Meanwhile, high glucose reduced the protein and mRNA expression of SARA. Compared with high glucose group, overexpression of SARA in HK-2 unregulated the expression of ZO-1 and downregulated the expression of Vimentin, FN, ColⅠHowever, SARA (dSBD) had no significant effect on the expression ZO-1, Vimentin, FN, ColⅠConclusion High glucose leads to EMT and ECM accumulation and reduces the expression of SARA in HK-2. Overexpression of SARA can reverse the EMT of HK-2 and inhibit the ECM accumulation, while this effect is depend on the SBD domain of SARA. Objective To investigate the mechanism by which SARA modulates the process of high glucose induced EMT and ECM accumulation in renal tubular epithelial cells.Methods HK-2 cells was exposed to high glucose (30mM D-glucose) and then the expression of TGF-β1, Smad2, Smad3, p-Smad2, p-Smad3 were detected. Furthermore, HK-2 cells were transfected with the plasmids of wild-type SARA and SARA (dSBD). Then the expressions of Smad2, Smad3, p-Smad2, p-Smad3 were examined.Results The expression of TGF-β1 and Smad3 increased after stimulation of high glucose in HK-2. However, the Smad2 mRNA expression level increased while its protein expression was downregulated in a time dependent manner. Smad2 and Smad3 were activated by glucose stimulation and Smad3 kept activation for a longer time than Smad2.Overexpression of SARA increased the protein expression of Smad2 and had no effect on its mRNA expression. Meanwhile, Overexpression of SARA didn't change the protein and mRNA expression of Smad3. SARA (dSBD) had no significant effect on both Smad2 and Smad3 expression.Overexpression of SARA prolonged the activation time of Smad2 and shortened the activation time of Smad3 and then changed the balance between Smad2 and Smad3. However, SARA (dSBD) had no significant effect on the activation of Smad2 and Smad3.Conclusion High glucose induces increase of TGF-β1 and Smad3 and decrease of Smad2 protein expression. SARA can modulate the TGF-β1 pathway by regulating the balance between Smad2 and Smad3.