| BackgroundCyclosporine(Cs A)is the first-line drug for the treatment of refractory nephrotic syndrome and to achieve longer-lasting renal disease remission while prolonging treatment time,but Cs A-associated nephrotoxicity(Cs AN)limits its long-term use.Therefore,finding biomarkers that are sensitive,and specific to monitor Cs AN is the key to solve the Cs A treatment problem.In our previous study,we found humoral(blood or urine)renal injury markers NGAL and TGF-β,in children with nephrotic syndrome or in the doxorubicin rat model simulating human minimal lesion nephrotic syndrome,they all have potential guiding value.However,the changes of body fluid markers are always affected by many factors in the system.Therefore,further understanding of changes of biomarkers in situ renal tissues expression has a significant predictive value for screening Cs A-associated nephrotoxicity.ObjectiveIn rats with nephrotic syndrome induced by doxorubicin,immunohistochemistry was used to detect neutrophil gelatinase-associated lipocalin(NGAL),renal injury molecule-1(KIM-1),and transformation growth factor-β(TGF-β)in renal tissues and the dynamic changes of Cs A treatment at different stages(7d,14 d,21d),combined with the degree of pathological damage of renal tissues at the same time,explored that these biomarkers are different for Cs A treatment,and the significance of the evaluation of kidney damage at different time.Methods1.Animal model group: male SD rats,120~150g,a total of 80,8 were left in the normal group,no drugs were applied,and the other 72 were randomly divided into the control group and the experimental group.The control group include blank control and the hormone control group,experimental group was the hormone + Cs A group.The control group and the experimental group were given a single tail vein injection of doxorubicin hydrochloride 7.5 mg/kg to make a rat model of adriamycin nephropathy to simulate children with minimal lesion nephrotic syndrome;2.The blank control group,the hormone control group and the hormone + Cs A group were treated with normal saline,prednisone,prednisone + Cs A for 7 days,14 days,21 days as the early,middle and late stages of treatment.The kidneys were collected at three time points;3.Preparation of renal tissue paraffin sections for HE and PAS staining,light pathological observation of renal histopathological changes including renal tubular injury,interstitial endothelium cell infiltration and vascular lesions;immunohistochemical detection of NGAL,KIM-1 and TGF-β expression in kidney tissue;4.Calculate the integrated optical density(IOD)of positive staining in each field of view using Image-Pro Plus image analysis software.5.Statistical analysis of the data using SPSS 22.0 software.The measurement data was first tested for normality,and the data of the normal distribution adopts the mean ±standard deviation(x ±S),the non-normal distribution was expressed as the median(upper and lower quartiles)[M(P25,P75)],and Kruskal-Wallis single factor analysis was used to analyze the difference between groups.<0.05 was considered statistically significant.ResultsThere was no obvious damage in the renal pathology of the rats in the early stage of treatment(7d),the difference was not statistically significant(P>0.05),while the NGAL and KIM-1 expression sites were mainly distributed in the glomerular mesangial area.The distribution of renal tubular epithelial cells was mainly changed in the control group and the experimental group.Compared with the normal control group,the expression of NGAL and KIM-1 in the renal tubules of the blank control group was significantly higher than that of the hormone group and the hormone + Cs A group(P<0.05).Kidney injury began to appear in the middle of treatment(14d),mainly in the vacuolar degeneration of renal tubular epithelial cells;NGAL was rarely expressed in the glomeruli of each group,mainly distributed in the renal tubular epithelial cells in the cortex,compared with the blank control group at 7d The expression of NGAL in renal tubules decreased(P<0.05),while the hormone + Cs A group showed a trend of increase,especially at 21 days,which was significantly higher than that of the control group and the hormone group at the same time(P<0.05);KIM-1 and NGAL expression sites and The trend of change is similar.In the late stage of treatment,the renal injury was significantly aggravated.In addition to the vacuolar degeneration of renal tubular epithelial cells,brush-like detachment or high-column dwarf column appeared,and interstitial inflammatory cell infiltration and small arterial hyaline degeneration appeared in the hormone+Cs A group.The injury site gradually spread from the outer cortex to the inner zone close to the medulla;NGAL and KIM-1 showed no significant changes in the expression of the blank control group,but with the prolongation of time,the distribution of renal tubules gradually decreased,and the expression of the hormone group and There was no significant change in the expression level.The distribution of NGAL and KIM-1 in the hormone+Cs A group increased in the renal tubules(P<0.05),and multifocal distribution and expansion to the proximal medulla.In addition,KIM-1 was also released during this period.It shows a trend of increased glomerular distribution.TGF-β was not obviously expressed in glomeruli at each time,and only in the14 d and 21 d hormone groups and hormone + Cs A group,some specimens were slightly expressed in tubulointerstitial and peribulbar.ConclusionsThe in situ expression of NGAL and KIM-1 in the middle and late stage of renal tubular epithelial cells treated with Cs A was significantly increased,and it was consistent with the degree of tubulointerstitial injury.It can be used as a biomarker for evaluating the late stage of Cs A-associated nephrotoxicity. |
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