Study On The Expression And Role Of Transcription Factor C/EBP? In Renal Interstitial Fibrosis

All chronic kidney diseases(CKD)may progress to end renal stage disease(ESRD). Prevalence of CKD is high and the rate of awareness is low. A study published by Ruijin hospital shows that the morbidity rate of CKD is 11.8% in Shanghai District,however the awareness rate was only 8.2%.The process of CKD is characterized by progressive loss of renal function which is caused by cell necrosis and apoptosis and deposition of extracellular matrix in interstitium. The process includes glomerular sclerosis and renal tubulointerstitial fibrosis. NF-kapaB is a multi-transcripted-regulated nuclear factor which was involved in multiple pathways in regulation of renal tubular function and secretion of inflammable factors. So it plays an important role in renal interstitial fibrosis. Peroxisome proliferator-activated receptor, a transcription factor also involves in many pathways in renal interstitial fibrosis including inhibition of epithelial-mesenchymal transistion(EMT), extracellular matrix(ECM) deposition and secretion of inflammation factor. Our previous study had screened a protein called C/EBP? by proteomics through iTRAQ-2D-nano-HPLC-MS/MS technology,and the preliminary investigation reveals that C/EBP? is involved in the process of CKD and can inhibit the process to some extent. So the current study is focused on C/EBP?'s mechanism in interstitial renal fibrosis and the interaction among the three nuclear factors.The study includes two parts.The first part is about in vitro study. TGF?1 was used as the main stimulus for HK-2 to investigate whether C/EBP? was involved in the process of inflammation and cell apoptosis by the method of gain and loss of function. First of all,C/EBP? plasmid was transfected into HK-2 cells, then the expression of PPAR? and NF-?B were detected. Then 10ng/ml TGF?1 was used as a stimulus for 12 h after HK-2 cell were transfected for 24 h. The mRNA of Col?a1, PPAR? and C/EBP? were detected. SiRNA of C/EBP? was transfected into HK-2 cells with a concentration of 5ng/ml of TGF? for 0h, 4h, 12 h and 24 h. The supernatant was collected and protein expression of MCP-1 in supernatant was detected with ELISA.The results showed that the transfection is successful and the transfection rate is over 70-80%. In normal condition, the expression of PPAR? and NF-?B were not influenced by overexpression of C/EBP?. Expression of MCP-1 in siRNA-C/EBP? group SiRNA-control group and control group showed the same trend. There is no difference among the four time points. Apoptosis could be induced by TGF?1 in HK-2. And the apoptosis between p EGFP-N1-Control group and pEGFP-N1- C/EBP? group is of no difference in HK-2. The expression of C/EBP? was decreaseed by TGF?1. And the expression of C/EBP? in control group, pEGFP-N1-Control group and pEGFP-N1-C/EBP? group had a declination of 65%, 74% and 38% compared with basal state respectively. And the PPAR? had a declination of 66%, 50% and 50%,respectively. However, the expression of Col ? was increased by TGF ? 1 in Control group, pEGFP-N1-Control group and pEGFP-N1- C/EBP? group by 10 times, 7 times and 4.5 times,respectively.In the second part, we brought C/EBP?-floxp heterozygous mice and pepck-cre mice from Mt. Sinai School of Medicine to acquire the tubular-conditional C/EBP?-knockout mice. The genotype identification was done as well. Wild type and heterozygous mice were used to construct the UUO model. The time end point is 7 days. We used RT-qPCR to detect if the expression of C/EBP? is changed in WT-control group, WT-UUO group, KO-operated group and KO-non-operated group. The expression of C/EBP? was decreased in UUO model and in heterozygous mutant mice was lowered by 65%. The expression of NF-?B in KO-operated group increased 1.5 times compared with WT-UUO group. Resveratrol was also used to explore its effect on renal fibrosis. The expression of C/EBP? was increaseed by resveratrol in Resv-UUO group compared with UUO group. The expression of C/EBP? was increased twice by ResV in ResV-UUO group compared with UUO group. While the expression of NF-?B in Res V-UUO was decreased to 25% compared with UUO group.The expression of ?-SMA in Res V-UUO group had a decrease of 60%, and E-cadherin increased 3 times compared with UUO group. The results showed that resveratrol could inhibit renal interstitial fibrosis by inhibiting EMT and inhibiting the activation of NF-?B, and the C/EBP? may be involved in the mechanism of the effects.In a word, the expression of C/EBP? was decreased in the process of renal interstitial fibrosis. And C/EBP? may have a inhibitory function with NF-?B. Resveratrol can inhibit renal interstitial fibrosis by inhibiting EMT process and NF-?B expression. C/EBP? may be involved in the pathway of resveratrol inhibition of renal interstitial fibrosis.