Molecular Genetic Mechanism Of Collagen Type ? Related Nephropathy And Podocyte Injury

Alport syndrome(AS),thin basement membrane nephropathy(TBMN)and familial focal segmental glomerulosclerosis(FFSGS)are the most common hereditary glomerular diseases.It has been clarified that mutations in Type ? Collagen(COL4)are important pathogenesis of these diseases,in which AS and TBMN are clearly identified as caused by mutations of type ? collagen ?3/?4/?5 chains(COL4A3/COL4A4/COL4A5,COL4A3-5)A number of studies have also shown that COL4A3 and COL4A4 are new pathogenic genes for FFSGS.But the number of mutation sites currently identified is still not much,and the mechanism has not yet been fully elucidated.Therefore,it is of great significance to explore novel type ? collagen(COL4A3-5)mutations and molecular mechanism of podocyte injury,which provide theoretical basis for finding new possible therapeutic measures in the futureOn the basis of previous work,we performed whole exon sequencing(WES)and further Sanger sequencing in 45 FSGS families and 77 patients with FSGS(SFSGS)with basement membrane changes.We found nine novel COL4A3-5 mutations.Three COL4A5 missense mutations(p.G129V,p.G603D and p.K474N),two COL4A3 missense mutations(p.I1264T and p.G991E)and one COL4A4 missense mutation(p.G71R)were found in six FSGS families.COL4A3 p.I696M,COL4A4 p.A1577E and p.G199R mutations were found in 3 SFSGS patients.We further collected the clinical characteristics of FSGS patients with glomerular basement membrane(GBM)changes as well as TBMN patients and did analysis on their prognosis.The thinnest thickness of GBM?250nm was used as the distinguishing standard,and the FSGS group was divided into GBM local thinning group(group A-97 cases)and GBM without local thinning group(group B-51 cases).The results showed that compared with group B,there were more female than male patients in group A The systolic blood pressure,serum creatinine and the glomerular sclerosis rate in renal biopsy of group A are relative milder than group B.No significant differences were found in other clinical features.FSGS patients with local GBM thinning performed heavier manifestations compared with TBMN group(33 cases).According to our former research,we suggested classifying the diseases caused by mutations of COL4A3/A4/A5(TBMN,benign familial hematuria,AS and FSGS)into type IV collagen-associated nephropathy,combining the clinical diagnosis with the molecular diagnosisIn order to explore the possible molecular mechanism of podocyte COL4A3 mutations in glomerular diseases,in the second part of this study we constructed human podocyte cell lines with lentiviral vector,which overexpressed COL4A3 wild type(WT)and four mutants(including 3 COL4A3 missense point mutations-G619R/G801R/C1616Y and COL4A3 p Leu1528 stop mutation).The significant overexpression of COL4A3 mRNA in all cell lines was detected by RT-qPCR.Nevertheless,while Western blot(WB)showed significantly overexpression of COL4A3 in WT,G619R,G801R and C1616Y group,protein expression in p.Leu1528 stop group was significantly lower than the above 4 groups.We further analyzed the mRNA-seq expression profiles of each group.The results showed that the mRNA expression of p.Leu1528 stop group and WT group was significantly different,and the difference between WT group and the other three groups was less significant.The expression of MCP-1 mRNA in the C1616Y and p.Leu1528 stop groups was significantly higher than that in the WT group(P<0.05).The level of MCP-1 protein in cell culture medium of the C1616Y and p.Leu1528 stop group was significantly higher than the WT group according to ELISA test(P<0.05).Further RT-qPCR revealed that the mRNA expression level of ERS associated genes CHOP and sXBP-1 in the C1616Y and p.Leu1528 stop groups were significantly higher than those in the control group(P<0.05).The expression level of ERS associated proteins PERK,GRP94,GRP78 and eIF2? were significantly increased,so did the apoptosis related proteins cleaved-caspase3,CHOP and Bax,but the expression level of Bcl2 was decreased.The increased protein expression level of PERK,eIF2?,CHOP and Bax also showed in the C1616Y group.We obtain a significant increase in the expression of COL4A3 in the p.Leu1528 stop group by using the proteasome pathway inhibitor(MG132),but no significant change in the WT group was appeared,suggesting that the intervention of the proteasome pathway may be a new treatment of serious mutation in COL4A3(e.g.protein translation prematurely termination).In the next step,we established the Col4a3 p.Gly95stop and Col4a3 p.Thr1621Stop mus-Col4a3 knockout mouse monoclonal cell lines by using the CRISPR/Cas9 system.Compared with WT group,WB showed lower expression level of Col4a3 while RT-qPCR revealed a higher expression level of CHOP,sXBP-1 and MCP-1 in above two cell lines.Flow cytometry test showed that there are more apoptotic cells in the p.Thr1621 Stop group than the WT group The protein expression levels of PERK?GRP94 and eIF2? were increased in both Col4a3p.Gly95stop and p.Thr1621Stop cells,and p.Gly95stop cells also showed increased expression of leaved-Caspase3,CHOP,Bax and Bcl-2,suggesting a high level of apoptosis This part of the study demonstrated in vitro that ERS and apoptosis induced by ERS were involved in the molecular mechanism of podocyte injury caused by COL4A3 pathogenicity mutations(especially severe mutations such as protein termination),and suggested that the intervention of the proteasome pathway may be a new treatment of serious COL4A3 mutationsTo evaluate the pathogenic effect of COL4A3 mutants in vivo,we further constructed a new Co14a3 p.Arg1631 Stop transgenic mouse model.In the homozygous mice,significant proteinuria and hematuria were observed from the sixth week,and serum creatinine was significantly increased at 12th week.The typical pathological changes of FSGS and glomerularsclerosis were observed by light microscopy,and local GBM thickening was showed by electron microscopy.20-week-old homozygous mice were more aggravated in renal function and renal pathology than the 12-week-old,and autophagosomes were observed by electron microscopy.We did not observe hematuria and proteinuria in 20-week-old heterozygous(Het)mice,but renal biopsy in 32-week-old Het mice(2 males and 2 females)also showed changes of FSGS,and electron microscopy showed localized thickening or thinning of GBM,as well as effacement of podocyte foot processes.We analyzed the mRNA-seq data of the renal cortex in 12-week-old WT/Het/Hom mice.The results showed changes in the ERS-related genes and signal pathways,which is consistent with the previous cell research.RT-qPCR showed that the expression level of MCP-1 and Collal was significantly higher in Hom mice than WT/Het mice(12/20-week),but there was no difference in mRNA level when it comes to ERS-related genes such as Chop,Bip and sXbp1.According to the WB results,compared with WT/Het group,Hom group(12/20-week)showed higher expression level of ERS-related proteins GRP78,PERK and IRE1?,apoptosis-related proteins Chop and Bax,autophagy-related protein LC3-?,inflammatory-related chemokine MCP-1.As well as the expression of Atg16Ll and p-eIF2? in 12 weeks Hom mice was significantly increased.However,no such changes mentioned above were observed in the Het group,which is agreed with the absence of renal phenotype.This part of the study demonstrated in vivo that excessive ERS,apoptotic and autophagy were involved in renal disease phenotype caused by COL4A3 mutationsIn summary,we identified nine new COL4A3-5 mutation sites by expanding COL4 mutation detection in FFSGS and SFSGS patients.We are the first to propose to define several renal diseases caused by COL4A3/A4/A5 mutations as type ? collagen-related nephropathy.We constructed different COL4A3 mutant podocyte model by lentivirus and CRISPR/Cas9 system,and found that excessive ERS and apoptosis could be involved in the molecular mechanism of podocyte injury.Moreover,the proteasome pathway intervention may be a new treatment for severe COL4A3 mutation(such as protein translation prematurely termination).In addition,we established a new Col4a3 p.Arg1631Stop transgenic mouse model and observed significant renal disease in the Hom group.Thus we further demonstrated that the display of excessive ERS,apoptosis and autophagy were involved in the pathogenic effect of COL4A3 mutation,which laid a good model and working basis for future research.