The Regulation Mechanism Of PAX2 To ADAM10 In Promoting Renal Tubular Epithelial Mesenchymal Transition And Renal Fibrosis

Objective: Renal fibrosis is the final comman pathway leading to chronic kidney disease.Proper kidney function depends on the correct cellular ineraction of over 20 different cell types.In fibrosis this complex architectural is changed and the cell-cell interaction balance is altered.It is characterized by accumulation of collagen,activated myofibroblasts,inflammatory cells as well as loss of microvasculature and epithelial cells.The deposition of extracellular matrix in the interstitial is the main feature,and myofibroblasts are the main sources to secret the extracellular matrix.The present studies reveal that renal tubular epithelial mesenchmal transition is the important sourse for myofibroblasts.Paired box gene 2(PAX2)is a nuclear transcription factor and regulates the development of the embryonic kidney,with no expression in the mature kidney.Both our research group and recent studies suggest that PAX2 promotes renal fibrosis,and induces renal tubular epithelial mesenchymal transition.A disintegrin and metalloprotease 10(ADAM10)is a transmembrane protein with the activity of metal protease,which promotes the maturity of a variety of cell surface molecules and has more than forty substrates including epithelial marker E-cadherin and Notch.ADAM10 is regulated by PAX2 in renal cell carcinoma and melanoma cells.According to the above background,we detect the transcription and expression of ADAM10 in the renal tubular epithelial cells when it is overexpressed with PAX2,and explore the mechanism of PAX2 to regulate ADAM10 by the methods of chromatin immunoprecipitation and dual luciferase reporter gene assay.By interventing the expression of ADAM10 to investigate if ADAM10 mediates the process of the renal tubular epithelial mesenchymal transition induced by PAX2.Finally,we validate ADAM10 involved in the process of renal interstitial fibrosis by the unilateral ureteral obstruction model of rats and the renal biopay samples of chronic kidney disease patients and provide a new choice for the treatment of renal fibrosis.Methods: A rat renal tubular epithelial cell line,NRK52 E,was transfected with lentivirus carrying PAX2,and E-cadherin and ?-SMA expressions were measured.The influence of PAX2 on ADAM10 promoter activity was evaluated using chromatin immunoprecipitation(CHIP)and dual-luciferase reporter assay.We also treated NRK52 E with ADAM10-specific overexpression vector and inhibitors and measured E-cadherin and ?-SMA expression.In vivo,Wistar rats(n=60)were subjected to unilateral ureteral obstruction(UUO)(n=30)or sham surgery(n=30),with tissues from post-operative day 3,7,14,21 and 28 days examined,and PAX2/ADAM10 activity measured.ADAM10 expression was also assessed in kidneys from patients with chronic kidney disease(CKD).Results: 1.NRK52 E cells were transfected with PAX2 lentivirus expression vector,the results show that E-cadherin m RNA and protein expression were downregulated(P<0.05)and ?-SMA m RNA and protein expression were upregulated(P<0.05)in the PAX2 overexpression group compared to the empty vector group,which indicated renal tubular epithelial mesenchymal transition.ADAM10 m RNA and protein expression was also upregulated in the PAX2 overexpression group by real-time q PCR,Western blot and immunofluorescence(P<0.05),which suggested that the overexpression of PAX2 induced the transcriptional activation and increased expression of ADAM10.2.It was identified a putative PAX2 binding site(-281bp)in the ADAM10 promoter in renal tubular epithelial cells by bioinformatic software Jaspar and PROMO.Our data demonstrated that PAX2 can directly interact with the ADAM10 promoter in renal tubular epithelial cells(P<0.01)by chromatin immunoprecipitation assay,to regulate the expression of ADAM10.3.We assessed the influence of PAX2 to ADAM10 promoter transcriptional activity in renal tubular epithelial cells by dual luciferase reporter gene assay.The results indicated that ADAM10 promoter transcriptional activity significantly increased,it was 3.2±0.26 compared to the control group(P<0.001).4.HK-2 cells were transfected with ADAM10 plasmid,and compared to the control group the results indicated that E-cadherin m RNA and protein expression were decreased(P<0.05),and ?-SMA m RNA and protein expression were increased(P<0.05)by the detection of real-time q PCR,Western blot and immunofluorescence.It suggested that overexpression of ADAM10 can induce human proximal epithelial mesenchymal transition.5.A specific inhibitor GI254023 X of ADAM10 was added into NRK52 E cells which were overexpressed with PAX2.The results show that E-cadherin protein was increased,and ?-SMA protein was decreased in the GI254023 X group compared to the control DMSO group by Western blot(P<0.05),but not completely reversed the EMT induced by PAX2.It suggested that ADAM10 partly mediated EMT induced by PAX2 in rat renal tubular epithelial cells.6.The expression of E-cadherin protein became decreased compared to sham group on 3d after operation,and gradually diminished in a time-dependent manner(P<0.05),and ?-SMA protein was increased in a time-dependent manner in UUO rats(P<0.05)by Western blot and immunohistochemical staining,which indicated that the renal tubular epithelial mesenchymal transition existed in UUO rats.7.Real-time q PCR,Western blot and immunohistochemical detection show that ADAM10 m RNA and protein were increased in UUO rats compared to sham rats(P<0.05),and was a time-dependent manner in UUO rats.PAX2 m RNA was increased(P<0.05),and the expression of protein was increased too(P<0.05)in UUO rats,which also increased in a time-dependent manner.The results suggested that the expression of ADAM10 was increased in the obstructive kidney in UUO rats,and the expression was consistent with the process of renal interstitial fibrosis and the expression of PAX2.8.Paraffin-embeded renal tissues from 5 patients with chronic kidney disease and 1 with mild mesangial proliferation were examined for ADAM10 expression.In 5 CKD patients 2 were CKD stage ?,3 were CKD stage ?.Focal segmental glomerular sclerosis were 3,and chronic interstitial nephritis were 2.The histochemical staining show that ADAM10 was overexpressed in renal biopsy samples in 5 patients with chronic kidney disease compared to the patient with mild mesangial proliferation.Conclusion: 1.PAX2 can directly induce the transcriptional activity and increased expression of ADAM10 associated with epithelial mesenchymal transition in rat renal tubular epithelial cells.2.ADAM10 can induce epithelial mesenchymal transition in human proximal tubular epithelial cells.3.The epithelial mesenchymal transition induced by PAX2 was partly mediated by ADAM10 in rat renal tubular epithelial cells.4.ADAM10 was involved in the process of renal interstitial fibrosis in vivo.