The Functional Study Of C/EBPα In Renal Interstitial Fibrosis

Chronic kidney disease(CKD)was defined as estimated glomerular filtration rate(e GFR)less than 60 m L/min per 1·73 m2 or the presence of renal injury,which lasted more than three months.CKD is one of the most common diseases on the earth with high incidence but low patients’ awareness rate.The morbidity of CKD around the world is about 10.5% to 13.1%.The overall prevalence of chronic kidney disease in China was 10.8% therefore the number of patients is estimated to be approximately 120 million.According to the data published from Our department(Department of Nephrology,Ruijin Hospital,Shanghai Jiao Tong University),the prevalence of CKD in the community population in Shanghai is 11.8%,and the rate of awareness of CKD is 8.2%.Due to the insidious onset of CKD,it is usually ignored at early stage.Without timely diagnosis and effective treatment,it will finally progress to end stage renal disease(ESRD)which is needed renal replacement therapy with expected shorter survival time.Renal fibrosis is the final common pathway during the progression from CKD to ESRD,which is characterized by glomerulosclerosis and renal tubulointerstitial fibrosis.The manifestation of renal tubulointerstitial fibrosis include filtration of inflammatory cells,activation of myofibroblasts,increased deposition of extracellular matrix(ECM),renal tubular atrophy and microvascular rarefaction.However,according to the existing research,the mechanism of renal fibrosis has not been fully clarified,and further exploration of its molecular mechanism is of great significance for delaying the progression of CKD.Our previous research have screened and found the target transcription factor,CCAAT/enhancer binding proteinα(C/EBPα),by using proteomics technical platform of i TRAQ-2D-nano-HPLC-MS/MS technique and bioinformatics.It was showed that C/EBPα plays an important role in the occurrence and development process of renal fibrosis.In this study we made further study to explore the effect and mechanism of this transcription factor C/EBPα during renal tubulointerstitial fibrosis,which would probably provide a new therapeutic target.In the first part of this study,by using the cre-loxp system,C57BL/6J mice of C/EBPα-loxp were mated with pepck-cre mice,therefore renal tubular epithelial cells conditional knockout C/EBPα(C/EBPα-/-)mice model were successfully established.We extracted DNA from the mouse tail,amplified cre enzyme and loxp fragment by Polymerase Chain Reaction(PCR)and applied DNA gel electrophoresis for genotype identification.Total RNA from mice kidneys was extracted,reversible transcription PCR and real-time quantitative PCR(RT-q PCR)were applied to determinate the expression level of C/EBPα m RNA.Compared to Wild type(WT)mice,m RNA expression of C/EBPα in C/EBPα-/-mice decreased by 68.66%.What’s more,we took the approach of immunohistochemistry stain to detect the expression of C/EBPα in the renal tissue.In addition,further validation of C/EBPα protein expression by immunohistochemical method in kidney tissue showed that C/EBPα expression was discovered in the nuclei of renal tubular epithelial cells of WT mice,versus no positive C/EBPα expression at renal tubular epithelial cells of C/EBPα-/-mice.So we confirmed that conditional knockout C/EBPα mice were successfully established depending on the multiple validation methods of DNA,m RNA and protein expression level.In the second part of this study,Agilent Whole Mouse Genome Oligo Microarray was performed to identify the differences in gene expression profiles between C/EBPα-/-and WT mice in a sensitive and effective way.The results showed that lots of the genes expression had been changed in C/EBPα knockout mice group.1754 significantly differential expression genes were identified: 606 up-regulated genes,which included tgfb1,tgfbr1,smad1,smad3,wnt4,wnt9 a and wnt16 etc.1148 genes were down-regulated which contained Olfr1000,Olfr1180,Olfr1195,Olfr1201 and Olfr1301 ect.Most of the differential genes were distributed on cell membrane,mainly participating in cell regulation,cell communication,signal pathway and ect.Finally,the partial results of microarray were validated in next part.We further chose part of interested differential genes for next validation.The third part we chose unilateral ureteral obstruction(UUO)mice as the renal interstitial fibrosis model,it is divided into four groups: WT-control group,C/EBPα-/--control group,WT-UUO group and C/EBPα-/--UUO group.Serum creatinine,urea nitrogen,albumin,triglyceride,cholesterol,urine albumin to creatinine ratio were detected in all four groups.however,no significantly difference were discovered.The expression of C/EBPα was detected by immnuohistochemical stain,and renal histopathological staining(HE,PAS,Masson methods)were also performed.C/EBPα were expressed in nuclei of renal tubular epithelial cells at both WT-control and WT-UUO group,whereas negative C/EBPα expression were detected at C/EBPα-/-control or UUO group.Nevertheless,renal pathology manifestation of both renal tubule injury and interstitial fibrosis were more severe in C/EBPα-/-UUO group versus WT UUO group,it is demonstrated that C/EBPα knockout would aggravate kidney injury,this transcription factor C/EBPα played a protective role during renal interstitial fibrosis.To verify the results of previous m RNA microarray,q PCR were introduced to detect differential expression of several important factors.As it is expected,the expression of colony stimulating factor 1(csf-1),colony stimulating factor-1r(csf-1r),colony stimulating factor-3r(csf-3r)m RNA were significantly increased while colony stimulating factor-3(csf-3)were significantly decreased.It is implied that renal protective effect of C/EBPα may be contributed by regulating the local inflammatory reaction of renal impaired tissue.In summary,our study successfully constructed renal tubular epithelial cell conditional C/EBPα knockout mice via cre-loxp system,we found that C/EBPα played an important protective role in renal interstitial fibrosis by reducing local inflammation.It may provide a new therapeutic target for the treatment of renal fibrosis and delaying the progression of CKD.