Numb Contributes To Renal Fibrosis By Interacting With HIF-1?

Background Most of chronic kidney diseases(CKD)patients are all inevitably progressed to end stage renal disease(ESRD).Irrespective of the initial causes,progressive CKD often results in widespread tissue scarring that leads to the complete destruction of kidney parenchyma and end-stage renal failure,a devastating condition that requires dialysis or kidney replacement.Tubulointerstitial fibrosis is the final common pathway progressing to ESRD of many kidney diseases.Therefore,it is very important to do research on the pathogenesis and therapeutic intervention of renal fibrosis.One of important factors resulting of tubulointerstitial fibrosis is renal tubular epithelial cell injury.Hypoxia has been regarded as a potential cause as well as effect of progression of chronic renal failure.Numb gene is a highly conserved gene.Numb is a multifunctional protein,which is widely expressed in many tissues of mammals,involved in diverse important cellular processes.Our previous study showed that Numb is abundantly expressed in proximal tubules,and it is mainly distributed in the brush margin and basal side of tubule epithelial cells.Importantly,targeted depletion of Numb from proximal tubular cells attenuated interstitial fibrosis in mouse model.Hence,the aim of the present study is to investigate the role and the mechanism of Numb in CKD further.Method We constructed renal fibrosis model induced by unilateral ureteral obstruction(UUO)in BALB/c mice.The mice were killed on the 7th day after surgery.Renal tissue morphology was examined by Hematoxylin and eosin staining and Masson-trichrome staining.Protein level was examed by Western blot and immunohistochemistry.We treated proximal tubular epithelial cells with indicated concentration of TGF-?1 and CoCl2 for various time periods.To unravel the signaling pathway by which TGF-?1 regulates Numb expression,we blocked Smad-dependent and Smad-independent pathways by treating NRK52E cells with siRNA and various specific chemical inhibitors before TGF-?1 treatment,respectively.The expression of Numb,HIF-1? and fibrosis molecular markers was examined by Real-time PCR and Western blot.To illustrate the relationship between Numb and HIF-1?,we overexpressed and knock down the expression of Numb and HIF-1? by Adenovirus(or plasmid)and siRNA respectively and examined by ChIP and CoIP analysis.Results In vivo,we found that the renal expression of Numb and HIF-1? was significantly upregulated in UUO model,and the distribution of Numb changed.ChIP analysis demonstrated that HIF-1? was likely to band to the promoter of Numb in renal fibrosis model.In vitro,we discovered that TGF-?1 upregulates Numb and HIF-1? expression through PKC pathway.As showed by that the PKC inhibitor Ro-31-8220 significantly inhibited TGF-?-induced Numb expression,but not the inhibitors of Smad4,Akt,p38MAPK and PKA.We also found that overexpressed the expression of HIF-1? protein could upregulated Numb protein.ChIP results showed HIF-1? may induced Numb by banding to the promoter of Numb in HK-2 cell.We also found that overexpressed the expression of Numb induced the expression of HIF-la protein but not affected the mRNA level of HIF-1?.Finally,CoIP analysis suggested that Numb could inhibit the ubiquitination of HIF-1? throught affecting the combination of MDM2 and HIF-1?,resulting of HIF-la up-regulated expression.Conclusion Our data suggest that TGF-?1 upregulates Numb and HIF-1?expression through PKC pathway in proximal tubular epitheial cell.Numb maintains HIF-1? protein by inhibiting the ubiquitination of HIF-1?.On the contrary,HIF-1?promotes Numb transcription by banding to the promoter of Numb.Overall,Numb contributes to renal fibrosis by interacting with HIF-1?.