Re-expression Of Nphp1 In Nphp1^{del2-20/del2-20} Mice Reversed Renal And Extra-renal Phenotypes

Aim:Nephronophthisis is an important genetic cause of end-stage renal disease in children and young adults.It shows a pattern of autosomal recessive inheritance.More than 25 genes have been found to be associated with this disease,with intricate mechanisms.Gene therapy can fundamentally cure some diseases that cannot be solved by existing conventional therapies.Our research group has already successfully constructed an Nphp1 knockout mouse model with both renal and extra-renal phenotypes in previous studies.In this study,we used the adeno-associated virus carrying the sequences of Nphp1 gene to transfect Nphpl knockout mice model,aiming to observe whether the renal phenotypes and extra-renal phenotypes were improved.These results contributed important basis to translational researches on Nphpl gene therapy.Methods:Mice were mainly divided into 6 groups,including WT,WT mice with empty vector,WT mice with Nphp1 carrying vector,Nphp1 knockout mice,Nphpl knockout mice with empty vector,and Nphp1 knockout mice with Nphp1 carrying vector.For mice sacrificed at 12-week-old,they were transfected with virus vector at 5-week-old.For mice sacrificed at 36-week-old,they were transfected with virus vector twice at 5-week-old and 20-week-old,respectively.Body weights were measured weekly.After mice sacrificing and kidney removal,hematoxylin-eosin staining,periodic acid Schiff staining and Masson’s trichrome staining were used to observe the histopathological morphology of the kidneys.Transmission electron microscope was also used to observe the ultrastructure of kidneys.Immunofluorescence,western blot and real-time quantitative PCR were conducted to detect indicators related to epithelial-mesenchymal transition,including α-smooth muscle protein and fibroblast-specific protein 1.Hematoxylin-eosin staining was applied for detecting the histopathological changes of retina and testis.Results:After Nphpl re-expression,the number of renal cysts at the cortical medullary junction in 12-week-old Nphpl knockout mice was decreased,the degree of tubular dilation was reduced,and the thickening or stratification of the tubular basement membrane was improved.The degree of interstitial fibrosis and inflammation infiltration in the kidneys of 36-week-old mice was also alleviated.The 24h urine volume was decreased and the urine specific gravity was increased.The cell density of the inner nuclear layer in the retina in 12-week-old Nphp1 knockout mice was increased,and the phenotype was partially improved.However,in the testis of 12-week-old Nphpl knockout mice,we cannot detect the re-expression of Nphp1 at the mRNA level,neither an improvement in the degenerated phenotype of the testis.Conclusion:Gene therapy of Nphp1 knockout mice model by adeno-associated virus carrying the sequences of Nphp1 was effective.It significantly improved the renal phenotype and partially reversed the extra-renal phenotypes of Nphp1 knockout mice.