Mechanism Study Of WDR73 Deficiency In The Pathogenesis Of Nephropathy In Galloway-Mowat Syndrome

Backgroud:Galloway–Mowat syndrome(GAMOS;OMIM 251300)is a rare recessive genetic disease characterized by neurodevelopmental defects and a progressive renal glomerulopathy.Neurological symptoms of GAMOS include microcephaly,developmental delay,intellectual disability,and other variable neural symptoms.Renal manifestations range from isolated proteinuria to early nephrotic syndrome(NS),which may rapidly progress to end-stage renal disease.Although early onset nephrotic syndrome is common and most of them result in individuals’ death,there are some late onset subtypes that even no nephrotic disease in adult individuals have been reported.WDR73 is the pathogenic gene responsible for GAMOS1.At present,most of the literatures which reported about this gene were just case reports.The regulation pathway of WDR73,the precise contributions of WDR73 in physiological function,and the mechanisms of GAMOS1 caused by WDR73 deficiency,especially nephrotic syndrome,are poorly understood.Object:In this study,we focused on exploring the pathological and molecular mechanisms of GAMSO1,especially the nephrotic syndrome caused by WDR73 deficiency.By understanding of the molecular mechanisms,we hope there will be a contribution to the prevention and treatment of GAMOS1.Methods:(1)Cell morphology: HEK293-WDR73 KO cells were generated using the CRISPR/Cas9 system.Then,the Cell cycle,apoptosis and growth of cells were investigated.(2)Omics analysis for possible pathways and targets: RNA sequencing(RNA-Seq)was used to provide insights into the transcriptome of the WDR73 KO cells.Enrichment analysis was conducted to reveal the unique biological significance and key pathways associated with WDR73 in the differentially expressed genes(DEGs).We also used human protein microarray to identify potential WDR73 interacted proteins.According to the signaling pathway of interacted proteins,the possible molecular mechanism of WDR73 involved in regulating the physiological function of cells was revealed.Then,the interactions between WDR73 and these potential proteins were further confirmed by CO-IP and pull-down.(3)Investigation of molecular regulation: m RNA and protein levels of PIP4K2 C were analyzed to reveal the effect of WDR73 deficiency.The intracellular PIP2 levels were detected by ELISA.The focal adhesions(FA)of cells were detected by immunofluorescence.We further assessed the pathways affecting PIP4K2 C stability in WDR73 KO cells by treatment with the protein synthesis inhibitor cycloheximide,the ubiquitin-proteasome system inhibitor MG132 and the autophagy-lysosomal pathway inhibitor chloroquine.(4)In vivo study: To further study the pathological characteristics and development process of GAMOS1 caused by WDR73 depletion in vivo,we generated WDR73 systemic KO mice and podocyte-specific Wdr73 conditional KO(CKO)mice.We applied the Adriamycin(ADR)to induce glomerular injury both for the WT and Wdr73 CKO strains to confirm the role of WDR73 in maintaining podocyte function,and further explore the mechanism of WDR73 defect leading to nephrotic abnormalities in GAMOS1.Results:(1)WDR73 KO cells showed a relatively slow growth rate and G2/M phase arrest.The orientations and polarity axes were abnormal in metaphase spindles.And,cellular morphology changed,including the impaired cell adhesion with decreased pseudopodia.(2)RNA-seq demonstrated that the DEGs in WDR73 KO cells were enriched in focal adhesion(FA)and Extracellular Matrix(ECM)pathways.PIP4K2 C and PIP4K2 A,two phospholipid kinases also involved in FA pathway,were validated to interact with WDR73 via protein microarray.WDR73 KO led to a significant decrease in the PIP4K2 C and PIP4K2 A protein levels.(3)The decreases in PIP4K2 C and PIP4K2 A levels reduced intracellular PIP2.PIP2 plays an important role in regulating actin reorganization and FA assembly.Reduced PIP2 resulted a decrease in FA formation.WDR73 regulated PIP4K2 C protein stability through autophagy-lysosomal pathway.(4)The fact which WDR73 KO is embryonically lethal in mice indicated that WDR73 might play an important role in early development.WDR73 deletion significantly increased the susceptibility of podocytes toward injury by ADR,which was characterized by the detection of high levels of albuminuria,glomerular basement membrane(GBM)thickening,and podocyte foot process(FPs)broadening.FA formation was also impaired in primary podocytes derived from Wdr73 CKO mice.Conclusion:In this study,we demonstrated that WDR73 interacts with phosphatidylinositol kinase PIP4K2 C and PIP4K2 A.WDR73 knockout leads to the decrease of PIP4K2 C and PIP4K2 A,resulting in a significant reduction of intracellular PIP2.PIP2 is the key regulatory factor of microfilament assembly and focal adhesion forming.The significant reduction of PIP2 results in abnormal focal adhesions.As the focal adhesion plays a key role in maintaining the structure and function of foot processes(FP)in podocyte,the abnormal focal adhesion will lead to an impaired glomerular filtration barrier,which will further cause the proteinuria and other nephrotic syndromes in GAMOS1.