Study On The Effect Of Humanized PLA2R1 Gene Fragment On The Morphology And Function Of Rat Kidney

Objective:This study aims to explore the effects of PLA2R1 on renal morphology and function by establishing a rat model that knocks into a fragment of the humanized M-type phospholipase A2 receptor 1(PLA2R1)gene,with a view to opening up a new path for further research into the pathogenesis of membranous nephropathy and the development of new treatment options.Methods:A rat model was constructed by using CRISPR/Cas9 technology and homologous complementary pairing methods to knock in the humanized PLA2R1 gene fragment,and PCR technology was used to verify the construction of the model.The rats were divided into KI group and WT group.The KI group was homozygous(KI mice)that successfully inserted the humanized PLA2R1 gene fragment.The WT group was wild type SD rats,with 6 rats in each group and a total of 12 rats.Urine protein concentration and 24-hour urine protein content were measured using a biochemical analyzer,serum creatinine,uric acid,albumin,triglycerides,and cholesterol biochemical indicators were detected,and renal pathological manifestations were observed using HE staining,PAS staining,and Masson staining,Immunofluorescence techniques were used to observe the immune deposition of Ig G and C3 and the expression of nephrin in the glomerulus.Electron microscopy was used to observe the ultrastructural changes in the glomerulus.Statistical analysis was conducted using nonparametric tests to investigate the effect of PLA2R1 gene fragment on the morphology and function of the rat kidney.Results:The homozygotes of PLA2R1 gene were successfully screened by PCR.The median urine protein concentration of WT and KI were 51.5 mg/d L and 14.95 mg/d L respectively.The median 24 h urinary protein content of WT group was 10.637 mg,and that of KI group was 5.195 mg.The concentration and 24 h urinary protein content of KI group were significantly lower than those of WT group,and the difference was statistically significant(P < 0.05).In serological test,the median triglycerides of WT group and KI group were 2.02mmol/L and 1.46 mmol/L,the median cholesterol values of the two groups were 3.17mmol/L and 3.17 mmol/L,and the median total protein values of the two groups were 69.1g/L and 71.8 g/L,respectively.The median albumin values of the two groups were 34.8 g/L and 35.3 g/L,the median urea values of the two groups were 6.5 mmol/L and 6.53 mmol/L,and the median creatinine values of the two groups were 34 μmol/L and 37 μmol/L,respectively.The difference was not statistically significant(P > 0.05).HE,PAS and Masson staining showed no aggregation of inflammatory cells,no glycogen deposition and no fibrosis.The deposition of complement C3 and Ig G in kidney was observed by immunofluorescence,and there was no statistically significant difference between the two groups(P > 0.05).Although there was no statistically significant difference between KI group and WT group in the expression of marker protein Nephrin in podocytes(P > 0.05),the distribution pattern was more clustered.Electron microscopy showed that the morphology of foot process and fissure diaphragm was normal,and no significant difference was found between WT group and KI group.Conclusion:By inserting a partial humanized PLA2R1 gene fragment into rats,the urinary protein level in rats was lower and the expression and aggregation of podocyte marker protein Nephrin were enhanced.This gene fragment does not have harmful effects on the renal function of rats and appears to have a certain protective effect,but the specific mechanism still needs further experimental verification.