Generation Of A Novel Mouse Model Of Glomerulosclerosis Through Podocyte-specific Knockout Of Cdc50a Gene

Objective:Phospholipid turnover enzyme maintains the asymmetry of phospholipid distribution in the inner and outer layers of cell membrane.When phosphatidylserine is lost,the phosphatidylserine inside the cell membrane is exposed to the cell surface and induced apoptosis.CDC50A is an important subunit of phospholipid flipping enzyme.In this study,the deletion of Cdc50a gene in mouse podocytes showed that podocytes were deformed and damaged,resulting in the early symptom of chronic kidney disease,namely proteinuria.With the increase of age,the damage and loss of podocytes in knockout mice were aggravated,resulting in severe impairment of glomerular filtration barrier function,and a series of pathological phenotypic changes such as decreased glomerular filtration rate,proliferation of mesangial cells,accumulation of extracellular matrix,and focal segmental glomerulosclerosis.This study has provided evidence for that asymmetric distribution of phospholipids in the plasma membrane is critical for the survival of podocytes and integrity of the glomerular filtration barrier,and generated a novel mouse model of spontaneous glomerulosclerosis.Methods:1.Crossbreeding of podocyte-specific Cdc50a gene knockout mice:In this study,2.5P-Cre transgenic mice expressing podocyte specific Cre recombinase driven by human NPHS2 promoter were used.Cdc50a conditional knockout mice were generated by inserting two Lox P sites in the same direction on both sides of exon 3 of Cdc50a gene.After mating with 2.5P-Cre transgenic mice,the Cre recombinase expressed in podocytes cleaved and recombined the two Lox P sites,resulting in deletion of exon 3 of the Cdc50a gene and failure to produce functional CDC50A protein,thereby achieving tissue-specific knockout.By Crossbreeding 2.5P-Cre⁺with Cdc50a^{lox P/lox P}parent mice,two genotypes of 2.5P-Cre⁺/Cdc50a^{lox P/+}and 2.5P-Cre^-/Cdc50a^{lox P/+}were obtained.Then 2.5P-Cre⁺/Cdc50a^{lox P/+}mice were crossbred with Cdc50a^{lox P/lox P}mice.Mice with 2.5P-Cre⁺/Cdc50a^{lox P/lox P}（c KO）and 2.5P-Cre^-/Cdc50a^{lox P/lox P}（Control）genotypes were obtained and their progenies were produced for the experiment.2.Urinary biochemical assay:Urine was collected from 1.5-,2.5-and 3.5-month-old mice to detect urinary microalbumin,urinary creatinine and urinary electrolyte metabolism in c KO and Control mice.3.Glomerular filtration rate（GFR）measurement:GFR of c KO and Control mice was dynamically monitored in real time by FITC-Sinistrin injection into the tail vein of mice to assess the degree of glomerular injury.4.HE and PAS staining were used to observe the renal structure,podocyte damage and morphological changes in c KO and Control mice.5.TUNEL fluorescence staining was used to detect the apoptosis of c KO and Control glomerular podocytes.Results:1.By crossing 2.5P-Cre mice and Cdc50a mice,2.5P-Cre⁺/Cdc50a^{lox P/+}（glomerular podocyte-specific Cre and Cdc50a heterozygote）and 2.5P-Cre^-/Cdc50a^{lox P/+}（no Cre and Cdc50a heterozygote）were obtained.2.5P-Cre⁺/Cdc50a^{lox P/+}mice and Cdc50a^{lox P/lox P}mice were then crossbred to obtain genotypes 2.5P-Cre⁺/Cdc50a^{lox P/lox P}（c KO）and2.5P-Cre^-/Cdc50a^{lox P/lox P}（Control）.2.Urinary biochemical analysis and monitoring,there were no significant differences in urinary microalbumin,electrolyte metabolism and glomerular filtration rate between c KO mice and Control mice at 1.5 months,and no statistical differences in renal weight ratio.However,at 2.5 months,there were statistically significant differences in proteinuria,renal weight ratio and glomerular filtration rate between c KO mice and Control mice（P<0.05）,while there were no significant differences in urinary electrolytes.At 3.5 months,there were statistically significant differences in proteinuria,renal weight ratio and glomerular filtration rate between c KO mice and Control mice（P<0.05）,while there were still no significant differences in urinary electrolytes.3.HE and PAS staining showed no obvious pathological changes in glomerular morphology of c KO mice and Control mice at 1.5 months,while at 2.5 months,c KO mice showed thickening of glomerular basement membrane,matrix deposition,glomerular fibrosis,glomerular sclerosis and even glomerular loss.Compared with Control mice of the same age,there were statistical differences.At 3.5 months,c KO mice showed thickening of glomerular basement membrane,matrix deposition,glomerular fibrosis,glomerular sclerosis and even glomerular loss.Compared with Control mice of the same age,there were statistical differences.4.TUNEL fluorescence apoptosis assay showed that there was no obvious apoptotic signal in c KO mice and Control mice at the age of 1.5 months,while there was increased apoptotic signal in glomeruli of c KO mice than Control mice at the age of 2.5 months.Conclusion:1.CDC50A is widely expressed in podocytes and maintains the asymmetric and dynamic distribution of lipids on the podocyte plasma membrane.The deletion of podocyte specific CDC50A leads to deformation and damage of podocytes,resulting in the early symptom of chronic kidney disease,namely the production of proteinuria,and the onset of this process is relatively rapid.2.The progression of podocyte specific CDC50A loss will aggravate podocyte damage and loss,further affect podocyte survival and function,and lead to a series of pathological phenotypic changes such as decreased glomerular filtration rate,proliferation of mesangial cells,accumulation of extracellular matrix,glomerulosclerosisand and a series of pathological phenotypic changes.3.A novel mouse model of idiopathic glomerulosclerosis can be constructed by Cdc50a gene knockout in podocyte,which has a short onset time and a good phenotype.