FK506 Attenuated Proteinuria By Inhibitingendothelial-to-mesenchymal Transition In Rats With Diabetic Nephropathy

Background: Mounting evidence has shown that damage of endothelial cells plays an important role in the early diabetic nephropathy(DN)with proteinuria.Although FK506 has been found to attenuate proteinuria in DN rats,the underlying mechanism remains unknown.Previous studies have shown that endothelial-to-mesenchymal transition(End MT)is involved in the initiation and development of proteinuria in DN.In this study,we aim to explore whether End MT could be attenuated by FK506 treatment in DN.Methods: Thirty-six male Sprague-Dawley(SD)rats were randomly divided into three groups(n=12/group): normal control(NC)group,DN group and DN+FK506group.The DN model of rats was established by intraperitoneal injection of strephozotocin(58mg/kg).The DN rats were then administered with FK506 by gavage(0.5mg/kg/d)for 24 weeks.Biochemical parameters,urinary protein(UP),and kidney weight/body weight(KW/BW)were measured.Renal pathological changes were observed using light microscopes and electron microscopes.Endothelial marker Platelet endothelial cell adhesion molecule-1(CD31)and mesenchymal marker [α-smooth muscle actin(α-SMA)] were detected by double immunofluorescence staining and immunohistochemistry.Real time-polymerase chain reaction(RT-PCR)and Western blotting(WB)detected CD31,α-SMA and fibroblast-specificprotein1(FSP1).Results: Compared with NC rats,the levels of serum creatintine(SCr),blood urea nitrogen(BUN),KW/BW and UP were increased significantly in DN(P<0.05).Meanwhile,the increased glomerular volume,thickness of glomerular basement membrane(GBM),and area of glomerular mesangial matrix were aslo observed in DN(P<0.05),which was attenuated in DN rats with FK506 treatment(the trough concentration of FK506 was 0.5±0.023ng/ml).Swelling of endothelial cells and abnormal arrangement of fenestra were observed in DN rats.FK506 could attenuate the changes markedly.Double immunofluorescence staining results showed a co-localization of CD31 and α-SMA in renal tissue of DN rats,while FK506 treatment attenuated these phenomena.Immunohistochemistry,RT-PCR and WB showed that the CD31 expression was downregulated in DN,whereas the expressions of FSP1 and α-SMA were markedly upregulated.More interestingly,these changes were inhibited by FK506 treatment.Conclusion: The middle dose of FK506 can attenuate the proteinuria by inhibiting End MT in DN.