The Role And Mechanism Of Renal Interstitial Fibroblast TP53RK In Chronic Kidney Disease

The increasing morbidity and mortality of chronic kidney disease（CKD）has become a public health threat to human health.Renal interstitial fibrosis is the main pathological manifestation of CKD,in which abnormally activated renal interstitial fibroblasts play an important role in the CKD progression.TP53RK is a serine/threonine and mutations in this gene can lead to early-onset steroid-resistant nephrotic syndrome.The pathological manifestations were focal staged glomerulosclerosis and disappearance of foot processes,suggesting that TP53RK may play an important role in the physiology and pathology of the kidney.However,the role and mechanism of TP53RK in CKD remain unexplored.Therefore,this study focuses on:（1）To explore the expression and clinical relevance of TP53RK in CKD,as well as its role and function.（2）To investigate the mechanism of TP53RK-mediated fibroblast activation and proliferation in CKD renal interstitial fibrosis.This project explores the specific role and mechanism of TP53RK in fibroblasts,and thus provide new therapeutic approach for the the prevention and treatment of CKD in clinic.Part I:The role of TP53RK in chronic kidney diseaseObjective:To investigate the role of TP53RK in renal interstitial fibrosis of CKD.Methods:（1）Immunohistochemistry（IHC）and immunofluorescence（IF）staining were used to observe the expression and localization of TP53RK in CKD patients and animal models.Western blot was used to detect the protein expression of TP53RK in animal models of CKD and TGF-β1-stimulated rat renal interstitial fibroblast（NRK-49F）cells.TP53RK^fl/flmice（named as WT）and fibroblast conditional TP53RK knockout mice(TP53RK^fl/fl;S100A4-Cre,named as c KO)were constructed and subjected to.Unilateral ureteral obstruction（UUO）and unilateral ischemia-reperfusion（UIR）injury sugeries.Kidney fibrosis was evaluated by Masson and Sirius red staining,and Western blot,and q RT-PCR analysis of fibrosis-related MAPKers.TP53RK knockdown plasmid（sg TP53RK）was transfected in TGF-β1-stimulated NRK-49F cells to assess the activation and proliferation of renal interstitial fibroblasts.（3）The TP53RK inhibitor fusidic acid（FA）was employed in CKD mice and TGF-β1-stimulated NRK-49F cells,to verify its role in improving renal interstitial fibrosis.Results:（1）The IHC results show that the expression of TP53RK was significantly increased in different degrees of renal fibrosis caused by Ig AN and FSGS,and the expression level of TP53RK was positively correlated with the degree of renal interstitial fibrosis and the concentration of renal function MAPKers（BUN and s Cr）.In UUO/UIR animal models,IHC,IF and Western blot results show that the expression of TP53RK was significantly increased compared with the Sham group,and it was colocalized with the fibroblast MAPKer S100A4.In TGF-β1-stimulated NRK-49F cells,TGF-β1 can dose-dependently promote the expression of TP53RK.（2）The results of Masson and Sirus red staining of UUO mice kidneys show that the collagen deposition area of the c KO+UUO group was reduced by 64.09%and 48.54%by compared with the WT+UUO group.Western blot and q RT-PCR results show that knocking out TP53RK could downregulate the high expression of FN,Vimentin,α-SMA,and Periostin（decreased by 42.36%&40.21%,49.78%&49.82%,47.27%&45.68%and41.64&48.24%,respectively）.Similarly,in UIR mice,the kidney fibrotic damage area of the c KO+UIR group decreased by 49.82%and 44.36%by compared with the WT+UIR group.Conditional knocking out of TP53RK also inhibited protein and m RNA expression in FN,Vimentin,α-SMA,Periostin,as well as transcription levels of Col1a1 and Col3a1.In NRK-49F,the protein levels of Periostin and FN in sg TP53RK+TGF-β1 group were downregulated by 55.89%and 35.16%,respectively,compared with the Vector+TGF-β1group.TP53RK knocking down also significantly reduced m RNA levels in Fn,Acta2,Vim,Col1a1,and Col3a1,all with p<0.01.Ed U proliferation experiments show that the proliferation rate of NRK-49F cells were decreased by 55.56%in sg TP53RK+TGF-β1group with the Vector+TGF-β1 group.The TP53RK inhibitor FA significantly reduced the fibrotic area of UUO mice（reduced by 43.69%and 54.85%,estimated by Masson and Sirius red staining respectively）.FA also inhibited the high expression of proteins and m RNA of FN,α-SMA,Vimentin.In vitro,FA significantly inhibited TGF-β1-induced NRK-49F cell activation and ECM production.Conclusion:The expression of TP53RK was significantly increased in fibrotic kidneys and localized in renal intesetial fibroblasts.Knockout,knockdown,and pharmaceutical inhibition of TP53RK can inhibit the activation and proliferation of renal interstitial fibroblasts,thereby improving renal fibrosis.Part II: The mechanism of TP53 RK in renal interstitial fibroblast in chronic kidney diseaseObjective: To investigate the upstream and downstream regulatory relationship between TP53 RK and Birc5,and to study the specific mechanism of TP53RK/Birc5 in mediating the proliferation and activation of renal interstitial fibroblast.Methods:（1）Western blot was used to detected the expression levels of Birc5 and p-Birc5 in fibroblast TP53RK-specific knockout UIR mice.TP53RK-Flag and Birc5-His plasmids were co-transfected in NRK-49 F cells and stimulated by TGF-β1,Co-inmunoprecipitation（Co-IP）was used to detect the interaction between TP53 RK and Birc5.（2）IHC staining was used to detected the expression of Birc5 in renal biopsy samples from CKD children.（3）The Birc5-specific inhibitor YM155 was employed in UUO mice and TGF-β1-stimulated NRK-49 F cells,to observe its role in improving CKD renal interstitial fibrosis.（4）Birc5 overexpression and knockdown plasmids were transfected in NRK-49 F,Western blot was ued to detect the protein expression levels of AKT,p-AKT,P38 and p-p38.（5）Mice were injected with the Birc5 targeting CRISPR/Cas9 plasmid via vein high-pressure technique and constructed for UUO model.Western blot was used for detecting the activation of PI3K/Akt and MAPK signaling pathways,as well as protein levels of cell proliferation and cell cycle associated moleculeswith.Ki-67 and S100A4 immunofluorescency was used to detect the proliferation of renal interstitial fibroblasts in UUO mice.Results:（1）In the fibroblast-specific TP53 RK knockout UIR mice,the protein levels of Birc5 and p-Birc5 were decreased by 64.45% and 61.92%,respectively.Co-IP show that TP53 RK binded and interated with Birc5,which was significantly enhanced under the stimulation of TGF-β1.（2）IHC showed that the expression of Birc5 was significantly increased in the kidney tissue of children with moderate and severe fibrosis caused by Ig AN and FSGS,which were positively correlated with the degree of fibrosis and the concentration of renal function MAPKers BUN and s Cr.（3）The results of Masson and Sirus red staining show that the Birc5-specific inhibitor YM155 significantly alleviated the pathological damage of renal fibrosis in UUO mice.The levels of protein and m RNA of FN,Vimentin and α-SMA in YM155 + UUO mice were significantly reduced,and decreased by43.18%&40.26%,60.31%&50.90%,30.21%&45.07%,respectively.Overexpressing and knocking down Birc5 in NRK-49 F cells,the protein level of AKT,p-AKT,P38 and p-p38 were consistent with the expression trend of Birc5,indicating that Birc5 posed a regulatory effect on PI3K/Akt and MAPK signaling pathways.Knocking down Birc5 in the UUO model also inhibited the overactivation of PI3K/Akt and MAPK signaling pathways,and the abnormal upregulation of proliferating nuclear antigen（PCNA）,Cyclin D1 and cyclin A2 was also reduced by 59.15%,41.78% and 33.95%.IF showed that the fluorescence intensity of S100A4 and Ki-67 in the kidney tissue of UUO mice after knocking down Birc5 was weakened by 48.76% and 45.28%,respectively,and the proportion of S100A4 and Ki-67 positive cells was reduced by 41.28%.Conclusion: TP53 RK regulates renal interstitial fibroblasts by promoting the phosphorylation of Birc5 and the down-stream MAPK and PI3K/Akt signaling pathways.Birc5-specific inhibitor YM155 can alleviate renal interstitial fibrosis and retard CKD progression.