The Effect Of Active Vitamin D₃ On Podocyte In Adriamycin-induced Nephropathy Rats

Object:To detect the effect of active vitamin D₃[1,25-?OH?₂D₃]on the pathological changes of adriamycin?ADR?nephropathy,and analyze the degree of podocyte nephrin,CD2AP,podocin and p-AKT injury at different time points.Meanwhile,detect the changes of protein levels in rats and the correlations between them,ensure the effects of active vitamin D₃ on podocyte injury in rats with adriamycin-induced nephropathy.Methods:1.Preparation of animal model:6-week-old male SD rats were randomly divided into three groups,normal control group?Control?,model group with Adriamycin?ADR?and vitamin D₃treatment group[ADR+1,25-?OH?₂D₃].A adriamycin-induced nephropathies model was prepared by a single tail vein injection of adriamycin solution.One week later,collected 24h urine from metabolic cages to measure 24-hour urinary protein.2.Active Vitamin D₃ treatment:In the treatment group,active vitamin D₃ was given daily at a dose of 0.25?g/kg,and the control and model groups were given equal amounts of peanut oil daily.3.Measurement of body weight,blood,and urine biochemical indicators:Rats were weighed weekly and sacrificed on the 2th,4th,6th,8th,and 10th weekends after the injection of adriamycin.collected blood and weighted the kidneys.One day prior to sacrifice using metabolic cages to collect urine 24h rats,using automatic biochemical analyzer rat urine specimens?24h urinary protein,serum total protein,albumin,creatinine,blood urea nitrogen and total cholesterol?.4.Kidney pathological examination:Microscope were used to observe glomerular sclerosis,cell proliferation,hypertrophy and apoptosis after HE,Masson staining.Transmission electron microscope?TEM?was used to observe the disappearance and fusion of the podocyte foot process,whether there were abnormal granules in the cell body,and whether there were chromatin condensation,nuclear chromatin apical phenomenon and apoptotic bodies in the apoptotic cells.5.Immunohistochemical observe of the degree of podocyte injury,the expression and distribution of nephrin,CD2AP,podocin and p-AKT protein expression.6 Real-time fluorescence quantitative PCR was used to detect the expression of nephrin,CD2AP,podocin and AKT at the mRNA level.7.Western Blot was used to detect the changes of protein expression of nephrin,CD2AP,podocin and p-AKT.Results:1.Successful preparation of adriamycin-induced rat nephropathy animal model:Compared with the control group,rats in the adriamycin model group had lighter weight after a week?P<0.05?,and 24-hour urine protein quantification was significantly higher than normal?P<0.05?.2.Serum cholesterol,serum creatinine,and urea nitrogen in rats with adriamycin-induced nephropathy were significantly higher than those of the control group In the same period?P<0.05?.Serum albumin and total protein were significantly lower?P<0.05?,Compared with the model group rats,the indicators improved significantly in the treatment group by the end of the 6th week,and the difference was statistically significant.?P<0.05?.3.Kidney histopathological results showed that the kidney of the control group was reddish-brown in appearance,and the touch was smooth and soft.In the model group,the appearance of the kidney was pale and the capsule was tense;compared with the model group,the above-mentioned pathological changes in the treatment group all improved,the texture was ruddy,and the degree of edema was reduced.HE and Masson staining of rat renal tissue showed normal glomerular morphous structure in the control group;vacuolar degeneration of the glomerular epithelial cells in the model group,hyperplasia of the extracellular matrix,and segmental sclerosis;the above pathology in the treatment group,the change is not obvious.TEM observation showed that the podocyte morphology of the control group was normal and the foot process was clear and complete.In the model group,the peduncles became flat and the foot processes were extensively fused,and in some areas,the foot processes were seen to fall off from the basement membrane.Compared with the model group,the above-mentioned lesions in the treatment group were relieved to varying degrees,a nd it was seen that the number of podocytes recovered,the fusion of the foot processes was slight,and the basement membrane was not significantly thickened.4.Immunohistochemistry results showed that nephrin,podocin,CD2AP,and p-AKT positive substances were brownish yellow particles.In the control group,the expression of the above proteins was high in the glomerulus,and appeared continuous in capillary loops and thick line distribution.In the model group,only a small amount of protein was expressed,and the yellow deposition gradually faded and disappeared.The treatment group was improved compared with the model group.The positive expression rate of the treatment group was significantly higher than that of the model group at the end of the 10th week?P<0.05?.5.The results of real-time fluorescence quantitative PCR showed that compared with the control group,the expression of nephrin,podocin,CD2AP,and AKT mRNA in the model group was decreased?P<0.05?.Compared with the model group,the expression of mRNA in the treatment group was increased?P<0.05?.6.Western blot results showed that compared with the control group,the expression of nephrin,podocin,CD2AP,and p-AKT protein in the model group decreased?P<0.05?.Compared with the model group,the protein expression of the treatment group increased?P<0.05?.Conclusions:1.Active vitamin D₃ can reduce proteinuria in rats with adriamycin nephropathy and improve their renal function.2.The decreased expression of nephrin,podocin and CD2AP protein is closely related to the podocyte injury and proteinuria formation in ADR rats.3.Active vitamin D₃ can promote the recovery of podocytes in rats with adriamycin-induced nephropathy,and may regulate the survival of podocytes through the PI3K/p-AKT signaling pathway.