The Role Of PHF14 In The Renal Progressive Tubulointerstitial Fibrosis After Kidney Injury

Objective:The primary objectives of our study were to determine the expression profile of PHF14 in the scenario of tubulointerstitial fibrosis following folic acid induced renal injury and to illuminate that whether PHF14 protein plays a role in renal fibrosis deterioration.The interaction between PHF14 and TGF-beta signaling was also investigated.Methods:1.With the folic acid nephropathy mouse model,we figured out the expression profile of PHF14 using real-time PCR,western blot.Morphologic and immunogistochemical(collagen 1 and alpha-SMA)analyses were used to validate the animal model,while the immunohistochemical analysis with PHF14 antibody were used to reveal the expression level and location of PHF14.2.Mice with genotype phf14 lox+/+ and CAGGCre-ERTM+ were used to generate created phf14 lox+/+;Cre+ mice.High recombination efficiency of Cre+;phf14 lox+/+ adult mice was validated with real-time PCR and wstern blot.3.PHF14 knockout mice and control mice were administrated folic acid.At the predesigned time points,we used real-time PCR,western blot and morphologic analyses to evaluate the severity of fibrosis.4.Real-time PCR and western blot were used to examine the PHF14 expression profile in NRK-49 F cells at the existence of TGF-beta.5.We explored whether TGF-beta regulated PHF14 expression directly through TGF-beta/smad pathway using chromatin immunoprecipitation analysis.6.We tested the role of PHF14 in collagen 1 and alpha-SMA synthesis using western blot and immunofluorescence in the condition of PHF14 knockout in NRK-49 F cells.7.We further explored whether PHF14 directly represses the transcription of PDGFR-alpha with real-time PCR,western blot and chromatin immunoprecipitation analysis.Results:1.Compared with sham controls,PHF14 expression was upregulated persistently in the kidneys in acute injury phase and chronic fibrosis phase for mice underwent FA injection.PHF14 conditional knockout mice under the same pro-fibrotic insult may experienced more severe renal fibrosis.2.NRK-49 F were incubated in the serum-free medium or in the presence of TGF-beta.After TGF-beta treatment,we detected the expected increased levels of p-smad3,smad2/3 and alpha-SMA.PHF14 mRNA and protein expression was also remarkably elevated.We detected that endogenous p-smad2/3 bound to the proximal upstream region of the phf14 transcription start site and stimulates the transcription of PHF14.And PHF14 may functions in a complex and binds to the proximal region of the PDGFR-alpha transcription start site and downregulates PDGFR-alpha transcription.Conclusions:In the situation of folic acid nephropathy,PHF14 protein expression unregulates persistently as the progression of renal fibrosis.The overexpression of PHF14 is induced by TGF-beta/p-smad3 signaling.And the elevated PHF14 may hinder the process of renal fibrogenesis by supressing PDGFR-alpha expression.The TGF-beta/ smad/ PHF14 pathway could be regarded as a self-limiting mechanism of TGF-beta dominated renal pro-fibrotic signaling and a re-balancing biological route for pro-/anti-fibrotic regulators.