Establishment Of X-linked Alport Syndrome Modelmce And The Mechanism Of CD44 In Disease Progression

Background&Objectives:X-linked Alport syndrome（XLAS）is a hereditary kidney disease caused by mutation of COL4A5 gene encoding the α5 chain of collagen type Ⅳ,with typical manifestations of hematuria and proteinuria.As disease progress,kidney tissues undergo progressive glomerulosclerosis and interstitial fibrosis,and could eventually develop into end-stage renal disease（ESRD）.The severity of the phenotype varies depending on the genotype of COL4A5 mutation,the precise mechanisms linking the gene mutation to progressive kidney tissue damage in AS disease are still unclear,and the differential expression profiles of genes and proteins in AS renal tissue have not been elucidated.Mouse models are essential tools for studying involved pathways and identifying novel therapeutic targets for AS,however,till today only two nonsense mutant XLAS mouse models have been reported.In this study,we created a frameshift mutant XLAS mouse model based on a mutation identified in our previous research,and revealed the differential gene and protein expression profiles of kidney tissues XLAS mice by using multi-omics technology.At last,we further explored the role of CD44 and other molecules in the pathogenesis in XLAS.Methods:CRISPR/Cas9 was used to knock in a COL4A5 frameshift mutation c.980₉83delATGG,which was previously discovered in a typical XLAS family,to create a XL AS mouse model.DNA sequencing,urine protein and urine creatinine quantification,immunofluorescence of Ⅳ collagen α5 chain immunostaining,histological analyses and transmission electron microscopy（TEM）were performed to validate the phenotype of the XLAS mouse model.RNA sequencing and Proteomic Mass spectrum were performed to draw the profile of the differently expressed genes and proteins in AS renal tissue.Subsequently,the differential expressions of CD44 and related factors in XLAS mice kidney tissues were verified by qPCR,and the roles of CD44 HAS and TGF-β to promote XLAS pathogenesis were studied using WB,qPCR and other methods.Results:The DNA sequencing results confirmed that we successfully generated the Col4a5 frameshift c.980₉83delATGG mutant XLAS mice.The XLAS mice showed typical symptoms at 16w with proteinuria,absence of Ⅳ collagen α5 chain in immunostaining,and thinned,thickened or splintered multi-laminated glomerular basement membrane in TEM.Besides,the expression of Col4a5 in XLAS mice was significantly decreased.In addition,we identified 16.67%（4042/24003）differential genes and 4.3%（264/6089）differential proteins out of total genes and proteins respectively in XLAS versus healthy control mice kidney tissues.Enrichment analysis based on transcriptome and proteome yielded consistent pathways including collagen metabolism,extracellular matrix receptor interactions and local adhesion.Moreover,qPCR and WB results showed significantly increased levels of CD44,HAS and TGF-β expression in XLAS mice.Furthermore,COL4A5 knockdown at cellular level induced an increased expression of HAS,resulting in increased HA levels by which activated CD44 and up-regulated TGF-β to promote renal fibrosis.Conclusions:This study successfully generated a novel frameshift mutant XLAS mouse model and for the first time comprehensively identified the profiles of differential genes and proteins in XLAS kidney tissues based on this new mouse model.By screening the profiles,we discovered that CD44 plays an important role in XLAS pathogenesis,and further clarified a critical mechanism that Col4a5 deficiency can activate the expression of HA synthase,leading to the activation of HA-CD44-TGF-β pathway and thereby promoting renal fibrosis.