| Objective:To detect the expression of p53 and p21 in adriamycin nephropathy mice.To investigate whether the cell senescence was involved in the pathogenesis and development of adriamycin nephropathy.Method:60 male balb/c mices aged 6 weeks were randomly divided into two groups: the normal group and the adriamycin nephropathy model group(model group),30 mices in each group.The model group was disposable injected with adriamycin(10mg/kg body weight)into the tail vein.The normal group was injected with equal amount of saline.Seven days after adriamycin injection,the mices which 24-h urinary protein excretion was more than 50mg/kg were included in this experiment.At the end of the oth,2nd,4th,6th weeks,six mices which were randomly selected in each group were sacrificed,with the blood,24 h and kidneys collected.To detect the 24-h urinary protein excretion and blood biochemical indexes.The degree of glomerular sclerosis and renal tubular injury was observed by HE and PAS staining in renal tissue.The expression of p53 and p21 protein was detected by immunohistochemistry.The mRNA expression of p53 and p21 was detected by Real-time PCR.The correlations between the expression of p53,p21 protein and p53,p21 mRNA and other indexes were analyzed.Results:1 24-h urinary protein changes of the adriamycin nephropathy model group and matched group in different periodsIn matched group,there was no significant difference in the urinary protein at the end of the oth,2nd,4th,6th weeks(P>0.05).The urinary protein of model group at the end of the 2nd[(16.19±1.13)g/24h],4th[(14.50±1.46)g/24h],6th[(12.52±1.2)g/24h] weeks was significantly higher than that of matched group at the end of the 2nd[(2.02±0.47)g/24h],4th[(2.25±0.55)g/24h],6th[(2.09±0.56)g/24h] weeks,and the differences were statistically significant(P < 0.05).In model group,urinary protein increased from the end of 2nd week,and resched the peak at the end of 2nd week.And compare with urinary protein at the end of2 nd week,the urinary protein decreased slightly at the end of the 4th,6th weeks.2 The biochemical index changes of the adriamycin nephropathy model group and matched group in different periodsIn matched group,there was no significant difference in serum albumin,total cholesterol and serum creatinine at the end of the oth,2nd,4th,6th weeks(P>0.05).The serum albumin of model group at the end of the 2nd[(19.63±1.28)g/L],4th[(23.09±1.91)g/L],6th[(36.74±1.49)g/L]weeks was significantly lower than that of matched group at the end of the 2nd[(36.77±1.81)g/L],4th[(37.04±1.79)g/L],6th[(2.09±0.56)g/L]weeks,and the differences were statistically significant(P < 0.05).In model group,serum albumin decreased from the end of 2nd week,and resched the lowest level at the end of 2nd week.And compare with urinary protein at the end of 2nd week,the serum albumin increased slightly at the end of the 4th,6th weeks.The total cholesterol of model group at the end of the 2nd[(7.56±1.10)mmol/L],4th[(9.07±1.02)mmol/L],6th[(7.18±0.62)mmol/L] weeks was significantly higher than that of matched group at the end of the 2nd[(2.17±0.15)mmol/L],4th[(2.16±0.24)mmol/L],6th[(2.25±0.25)mmol/L] weeks,and the differences were statistically significant(P<0.05).In model group,total cholesterol increased from the end of 2nd week,and resched the peak at the end of 4th week.And compare with total cholesterol at the end of 4th week,the total cholesterol decreased slightly at the end of the 6th weeks.There was no significant difference in serum creatinine between normal group and model group at the end of the oth,2nd,4th,6th weeks(P>0.05).(Table 3)3 Renal histopathological findings of the adriamycin nephropathy model group and matched group in different periodsUnder light microscope study,in normal group,morphological structure of glomerulus and tubule was normal at the end of the oth,2nd,4th,6th weeks.In model group,morphological structure of glomerulus and tubule was normal at the end of the oth week.Adhesion between glomeruli and Bowman's capsule,protein cast in the nephric tubule and inflammatory cell infiltration in renal interstitium were observed at the end of the 2nd week.At the end of the 4th week,mesangial cells hyperplasia of mild or medium degree was observed accompanied with partial glomerulosclerosis as well as segmental tubular expansion,and inflammatory cell infiltration increased in renal interstitium.At the end of the 6th week,mesangial cells hyperplasia of medium or severe degree was observed accompanied with focal segmental glomerulonephritis as well as renal tubular atrophy,and inflammatory cell infiltration increased in renal interstitium.In matched group,there was no significant difference in glomerular sclerosis and tubulointerstitial injury at the end of the oth,2nd,4th,6th weeks(P>0.05).In model group,glomerular sclerosis and tubulointerstitial injury were significantly increased from the end of 2nd week.Compare with the end of 2nd week,the severity of glomerular sclerosis and tubulointerstitial injury gradually increased at the end of the 4th,6th weeks.And the differences comparing with the end of 0th week were statistically significant(P < 0.05).The glomerular sclerosis and tubulointerstitial injury of model group at the end of the 2nd[(0.535±0.116)(1.133±0.136)],4th[(0.738±0.112)(1.417±0.172)],6th[(0.847±0.121)(1.633±0.136)] weeks were significantly higher than that of matched group at the end of the 2nd[(0.020±0.020)(0.067±0.061)],4th[(0.022±0.017)(0.083±0.075)],6th[(0.018±0.015)(0.067±0.052)] weeks,and the differences were statistically significant(P<0.05).(Fig 1)4 The changes of p53 and p21 protein expression of adriamycin nephropathy model group and matched group in different periodsIn matched group,there was no significant difference in p53 and p21 protein expression at the end of the oth,2nd,4th,6th weeks(P>0.05).p53 and p21 protein expression of model group at the end of the2nd(51.944±15.081,59.674±15.234),4th[(164.902±12.852,202.428±24.098),6th(254.673±24.703,348.115±25.180)weeks was significantly higher than that of matched group at the end of the 2nd(8.111±1.744,11.883±1.863),4th(7.869±1.940,12.652±2.174),6th(8.537±1.262,12.422±2.848)weeks,and the differe-nces were statistically significant(P<0.05).In model group,p53 and p21 protein expression gradually increased with time.(Fig 2.Table 4)5 The changes of p53 and p21 mRNA expression of adriamycin nephropathy model group and matched group in different periodsIn matched group,there was no significant difference in p53 and p21 mRNA expression at the end of the oth,2nd,4th,6th weeks(P>0.05).p53 and p21 mRNA expression of model group at the end of the2nd(4.52±0.44,6.99±0.80),4th(6.43±0.68,9.36±0.69),6th(8.55±0.84,11.96±0.79)weeks was significantly higher than that of matched group at the end of the 2nd(0.96±0.10,1.04±0.09),4th(0.97±0.08,1.04±0.08),6th(0.97±0.11,1.05±0.08)weeks,and the differences were statistically significant(P < 0.05).In model group,p53 and p21 mRNA expression gradually increased with time.(Table 5)6 The correlation between the expression of p53 and p21 and different indexes in model groupIn model group,expression of p53 protein was positively correlated with glomerular sclerosis,tubulointerstitial injury,proteinuria and total cholesterol(r=0.877,r=0.830,r=0.456,r=0.576,P < 0.05),negatively correlated with serum albumin(r=-0.343,P < 0.05),and uncorrelated with serum creatinine(P>0.05).Expression of p53 mRNA was positively correlated with glomerular sclerosis,tubulointerstitial injury,proteinuriaand total cholesterol(r=0.940,r=0.926,r=0.473,r=0.524,P < 0.05),negatively correlated with serum albumin(r=-0.379,P < 0.05),and uncorrelated with serum creatinine(P>0.05).Expression of p21 protein was positively correlated with glomerular sclerosis,tubulointerstitial injury,proteinuria and total cholesterol(r=0.838,r=0.804,r=0.410,r=0.525,P<0.05),negatively correlated with serum albumin(r=-0.396,P<0.05),and uncorrelated with serum creatinine(P>0.05).Expression of p53 protein was positively correlated with glomerular sclerosis,tubulointerstitial injury,proteinuria and total cholesterol(r=0.948,r=0.940,r=0.520,r=0.559,P < 0.05),negatively correlated with serum albumin(r=-0.377,P<0.05),and uncorrelated with serum creatinine(P>0.05).(Table 6)Conclusions: Adriamycin nephropathy mice model which characterized by massive proteinuria,hypoalbuminemia and hypercholesterolemia could be used to simulate the minimal change nephrosis and focal segmental glomerulosclerosis in human.The expression of senescence gene p53 and p21 in adriamycin nephropathy mice increased from 2 weeks,which was correlated with the severity of adriamycin nephropathy,suggesting that cell senescence existed in early stage of renal disease and participated in the occurrence and development of kidney disease. |
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