Nrf2 Inhibits Tubularinterstitial Disease Progression Via Regulating Wnt Pathway Dependent On Downregulating CTGF Secreation In Epithelial Cell

Objective:Acute kidney injury?AKI?is an independent risk factor for chronic kidney disease?CKD?.If the injury is mild,a repair process can be adaptive and lead to complete renal recovery.However,severe injury will be accompanied by a maladaptive repair which usually leads to nephron loss,fibrosis,vascular rarefaction,and chronic inflammation.Nuclear factor erythroid 2-related factor 2?Nrf2?is a redoxsensitive transcription factor that regulates antioxidant proteins,cell cycle-related regulators and detoxification enzymes.Nrf2 has shown a renal protective role in tubularinerstial disease including acute kidney injury?AKI?models induced by rhabdomyolysis,ischemia/reperfusion and cisplatin,as well as,chronic injury models including pristine-induced lupus nephritis and diabetic nephropathy.The unilateral ureteral obstruction?UUO?mouse model is characterized by progressive tubulointerstitial fibrosis and reflects the biological processes present in clinical obstructive nephropathy.Nrf2 activators,such as CDDO-imidazolide,Oleanolic acid,DMF and Fimasartan,can attenuate UUO-induced kidney injury.These results suggest that Nrf2 plays a protective role against tubulointerstitial fibrosis.However,the mechanisms by which Nrf2 prevented progression from acute tubular damage to chronic tubulointerstitial fibrosis are remained to be clarified.In this study,we aim to explore the Nrf2 expression in the course of tubulointerstitial disease in UUO model and human samples.Then we will verify whether Nrf2 deletion aggravates the progression of renal injury at different stages of UUO.Finally,in tubular epithelia cells the regulation role of Nrf2 on CTGF expression which promotes epithelia cell transdifferentiation will be verified.Methods:Nrf2 expression in wildtype mice at 2,5,14 and 21 days after UUO were measured.Nrf2 expression in renal biopsies samples of patients with acute,subacute and chronic tubulointerstitialnephritis?TIN?were also studied.We compared Nrf2^-/-and Nrf2^+/+mice following UUO in tubular damage on day 2 after UUO;transdifferentiation?Vimentin?and proliferation?PCNA?on day 5 after UUO;fibrosis?Fibronectin and a-smooth muscle actin?and macrophage infiltration?F4/80?on day 14 after UUO.Antioxidative and inflammatory responses gene expression were studied in day 2,5 and14 days after uuo.CTGF expression level and Wnt signaling were evaluated in kidney tissue of day 5 after UUO and in hypoxia treated NRK52E cells by silencing Nrf2.Then we silenced CTGF and Nrf2 simulately and measured Wnt signaling and transdifferentiation markers.To find how Nrf2 regulates CTGF expression,CTGF transcriptors and post transcript regulated micro-RNAs were also studied.Results:1.In wildtype mice,the expression of Nrf2 and Nrf2 downstream gene,Gclc and Ho-1,were elevated after UUO.On day 2 after UUO,the expression Nrf2 began to increase and reached its peak on day 14.In IHC staining,Nrf2-positive staining can be detected as early as day 2,which mainly located in nuclei and less in cytoplasm of healthy-looking or slightly injured tubules.By day14,positive Nrf2 was limited in the tubules with nuclear detachment into the tubular lumen.Nrf2 expression was upregulated in renal biopsies of patients with TIN and acute TIN group had the highest expression with nuclear positive staining in immunochemistry stain.2.On day 2 following UUO,in Nrf2^-/-mice compared with Nrf2^+/+mice,tubular damage,apoptotic cell numbers,cleaved caspase3 and cleaved-PARP expression were increased.On day 5,protein levels of vimentin and PCNA and the co-expressed cells of both proteins were increased in Nrf2^-/-mice.On day 14,Fbronectin,a-SMA and F4/80 protein levels were increased in Nrf2^-/-mice.Antioxidative response genes Gclc and Ho-1 were up-regulated in since day 2 after UUO.Nrf2 deficiency reduced Gclc and Ho-1 mRNA levels.The inflammatory cytokines Tgf?1 and Tnf mRNA gradually increased from day 2 to 14.IL-6 and IL-1?mRNA were remarkably elevated on day 14.Nrf2 gene deletion upregulated Tgf?1,Tnf,IL-6 and IL-1?further.3.In vitro,we found that Nrf2 silencing significantly increased CTGF expression in response to hypoxia in NRK52E cell line.In hypoxia treated NRK52E cell line,Nrf2silencing promoted p-Lrp6 and?-catenine expression and epithelia cell Transdifferentiation.By silencing Nrf2 and CTGF,the exaberation functions in p-Lrp6,?-catenine and Vimentin by Nrf2 silencing have been attenuated.The similar change has been confirmed in day 5 of UUO mice model.We also analysed how Nrf2 regulates CTGF expression in two aspects including transcription and post-transcription regulation.Nrf2knockdown increased c-fos,a CTGF transcription regulators,expression in epithelial cells.But Nrf2 had no regulation role on post transcript regulated micro-RNAs?mir-26a,mir-26b,mir-30a and mir-133a?.Conclusion:1.Nrf2 expression were up-regulated in UUO model and renal biopsies from TIN patients;2.Nrf2 deficiency aggravated tubular injury in early phase,promoted maladaptive proliferation and dedifferentiation in transition phase and increased fibrogenesis in late phase in UUO model;3.Nrf2 deletion decreased antioxidative genes and increased profibrotic inflammatory cytokines expression in UUO;4.In tubular epithelia cell Nrf2 downregulated CTGF expression to inhibit tubularinterstitial disease progression.