Mechanism Of The COL4A3 Gene P.R408H Mutation In The Development And Progression Of Diabetic Kidney Disease

ObjectiveDiabetic kidney disease（DKD）has become the leading cause of end-stage kidney disease（ESKD）worldwide,and the risk factors affecting the progression of DKD are complex and unexplored.Recent studies on the phenomenon of familial aggregation of DKD and the genetic research of cohorts have suggested that genetic mutations play an important role.This study was divided into three parts,the first part was based on Chinese Han kidney biopsy confirmed DKD families and cohorts,aiming to explore gene mutations associated with the development and progression of DKD,the second part used gene editing technology to construct point mutation mice and induced into early-stage DKD models,aiming to verify the clinical phenotype through in vivo experiments,and the third part was based on specific cells,aiming to further explore through in vitro experiments,aiming to discuss possible mechanisms between genotype and phenotype.Materials and methods:1.Clinical studyThe literature was reviewed to summarize information on genes associated with the development and progression of DKD.We took nine families with maturity onset diabetes in young（MODY）as a discovery cohort,completed whole exome sequencing of family members,identified DKD patients diagnosed by the gold standard of kidney biopsy in the family as the proband,and contrasted the genotype and renal phenotype of probands with their parents in each family to accurately establish genotype-phenotype associations.Kidney biopsy confirmed sporadic DKD patients were used as a validation cohort to observe the correlation between mutations and renal phenotype and prognosis in sporadic DKD patients.2.In vivo studyMice with heterozygous and homozygous point mutations were generated using CRISPR/Cas9 gene editing technology,type 2 diabetic mice were established by high-fat feeding combined with streptozocin（STZ）,and mice were sacrificed 10 weeks after diabetes to be an early-stage DKD model.Urine and serum of mice was collected to compare the urine albumin/creatinine（Albumin/Creatinine Ratio,ACR）,serum creatinine,urea,total cholesterol,and triglyceride levels.Kidney tissues were collected to be performed hematoxylin eosin（HE）staining,Masson,periodic acid Schiff,PAS)staining,TUNEL（terminal deoxynucleotidyl transferase mediated nick end labeling）staining and electron microscopy was performed to compare the degree of renal pathological changes in each group.Finally,molecular experiments were performed to explore the levels of endoplasmic reticulum stress（ER stress）,apoptosis and inflammatory fibrosis in each group,so as to verify the effects of mutations in this gene on renal damage in early-stage DKD models at an in vivo level.3.In vitro studyWild type and mutant plasmids of the gene were constructed,overexpressed in specific cells,cultured under normal and high glucose conditions,respectively,and the levels of ER stress,apoptosis,and fibrosis in cells of each group were observed by immunofluorescence and molecular experiments,and the effects of mutations of the gene on the cells were verified at the in vitro level to further reveal the molecular mechanisms of genotype and phenotype.Results1.Clinical study1)A total of 33 publications reported 102 genes potentially associated with DKD,focusing on genes encoding glomerular basement membrane（GBM）structural,regulation of lipid metabolism,the renin-angiotensin-aldosterone system（RASS）,and fibrosis related genes;2)Nine MODY families comprised the discovery cohort,which had more proteinuria and worse renal function despite a shorter duration of diabetes in the offspring（proband）compared with the parents in the family.A total of 196 putative DKD associated mutations were identified by cross alignment with the literature,in which the COL4A3 p.R408H mutation was associated with a more severe renal phenotype in the offspring in multiple families;3)The validation cohort confirmed that sporadic DKD patients carrying this mutation had a shorter renal median survival time than non-carrying patients,and carrying the mutation was one of the risk factors for renal prognosis,with immunohistochemical staining showing upregulated Bip protein expression in glomeruli of patients carrying the mutation.2.In vivo study-the COL4A3 p.R408H heterozygous/homozygous point mutation aggravates kidney injury in an early-stage DKD modelWe used CRISPR/Cas9 gene editing to construct COL4A3 c.1223G>A p.P408H（COL4A3^Δ）heterozygous and homozygous point mutant mice,and induced them into an early-stage DKD model,which was divided into the following 6 groups:（1）wild type group(COL4A3^+/+),（2）wild type and DKD group(COL4A3^+/++DKD),（3）heterozygous mutation group(COL4A3^+/Δ),（4）heterozygous mutation and DKD group(COL4A3^+/Δ+DKD),（5）homozygous mutation group(COL4A3^Δ/Δ),（6）homozygous mutation and DKD group(COL4A3^Δ/Δ+DKD).Comparison kidney function of mice:1)compared with the COL4A3^+/++DKD group,the urinary ACR level was increased in the COL4A3^+/Δ+DKD group[70.37±7.70μg/mg vs.90.17±10.84μg/mg,P<0.05],and COL4A3^Δ/Δ+DKD group[70.37±7.70μg/mg vs.86.03±6.52μg/mg,P<0.05].2)Similarly,compared with the COL4A3^+/++DKD group,the serum creatinine level was increased in the COL4A3^+/Δ+DKD group[10.0±1.58μmol/L vs.16.8±4.85μmol/L,P<0.05],and COL4A3^ΔΔ+DKD group[10.0±1.58μg/mg vs.24.5±3.14μg/mg,P<0.05].3)compared with the COL4A3^+/++DKD group,COL4A3^+/Δ+DKD group and COL4A3^Δ/Δ+DKD group presented with worse renal pathomorphology,larger glomerular volume,more severe renal fibrosis and glycogen deposition,and electron microscopy showed uneven thickness and thickness of the GBM.Molecular experiments indicated that:1)type Ⅳ collagenα3 chain protein expression was unchanged in the kidneys of mice in the COL4A3^+/Δand COL4A3^Δ/Δgroups.2)COL4A3^+/Δ+DKD and COL4A3^Δ/Δ+DKD groups showed increased ER stress as indicated by higher expression of Bip,ATF6,x BP1,ATF4,p-e IF2αthan COL4A3^+/++DKD group.3)COL4A3^+/Δ+DKD and COL4A3^Δ/Δ+DKD groups showed increased levels of apoptosis,as indicated by higher expression of Cleaved-caspase3,Bax/Bcl2,and Chop than COL4A3^+/++DKD group.4)COL4A3^+/Δ+DKD and COL4A3^Δ/Δ+DKD groups showed increased levels of fibrosis and inflammation,as indicated by higher expression of TGFβ,MMP9,TNFR,TNFα,and IL-6.5)COL4A3^+/Δ+DKD and COL4A3^Δ/Δ+DKD groups showed increased expression of integrinβ3 and p-AKT,suggesting that the kidney injury caused by this mutation may be associated with activation of the integrinβ3/Akt pathway in the kidneys of mice with early stage of DKD.3.In vitro study-overexpression of COL4A3 p.P408H plasmid aggravates podocyte cell line injuryCOL4A3 gene is expressed exclusively in glomerular podocytes in the kidney,we overexpressed the empty vector,COL4A3 wild-type（wild type-COL4A3,wt-COL4A3）and COL4A3 c.1223G>A p.P408H mutant（mutation-COL4A3,mut-COL4A3）plasmids in a mouse podocyte cell line,cultured with normal glucose（NG）and high glucose（HG）.Cells were divided into eight groups:（1）untransfected plasmid and normal glucose group（control+NG）,（2）transfected with empty plasmid and normal glucose group（vector+NG）,（3）transfected with COL4A3 wild type plasmid and normal glucose group（wt-COL4A3+NG）,（4）transfected with COL4A3 mutant plasmid and normal glucose group（mut-COL4A3+NG）,（5）untransfected plasmid and high glucose group（control+HG）,（6）transfected with empty plasmid and high glucose group（vector+HG）,（7）transfected with COL4A3 wild type plasmid and high glucose group（wt-COL4A3+HG）,（8）transfected with COL4A3 mutant plasmid and high glucose group（mut-COL4A3+HG）.Molecular experiments indicated that:1)type Ⅳ collagenα3 chain protein expression was higher in the mut-COL4A3+HG group compared with wt-COL4A3+HG.2)Compared with wt-COL4A3+HG group,mut-COL4A3+HG group caused increased ER stress levels as indicated by higher expression of Bip,ATF6,x BP1,and p-PERK proteins.3)Compared with wt-COL4A3+HG group,flow cytometry detected increased apoptosis of podocytes in mut-COL4A3+HG group,Western Blot also showed the higher expression of Bax/Bcl2 and Chop.4)Compared with wt-COL4A3+HG group,mut-COL4A3+HG group had the higher levels of fibrosis,as indicated by increased TGFβ,MMP-9,andα-SMA protein expression.5)Compared with wt-COL4A3+HG group,mut-COL4A3+HG group had the higher levels of Integrinβ3、p-AKT/AKT protein expression.ConclusionClinical study suggested that the COL4A3 gene p.R408H point mutation might be associated with DKD progression.The in vivo experiments confirmed that kidney injury was more severe in the DKD model with heterozygous and homozygous point mutation of COL4A3 gene p.P408H than in wild type DKD mice.In vitro experiments suggested that this mutation may cause ER stress in mouse podocytes,which eventually causes podocyte apoptosis.