Study On Biomarkers For Early Diagnosis Of Acute Kidney Injury After Cardiopulmonary Bypass In Adults

BackgroundAcute kidney injury?AKI?is a serious complication of patients after cardiac surgery and critically ill patients.AKI is associated with poor prognosis including depended of dialysis,decreased quality of life,prolonged intensive care,and increased mortality,etc.Neutrophil gelatinase-associated lipocalin?NGAL?,a novel biomarker for evaluating AKI which owns good specificity and sensitivity,has been used in clinical practice.Tissue inhibitor of metalloproteinase-2?TIMP-2?and insulin-like growth factor binding protein 7?IGFBP7?are inducing factors of G1 cell cycle arrest.Study has found that[TIMP-2]×[IGFBP7]can be used as an indicator to predict the occurrence of AKI,and it's better than the two alone to evaluate the occurrence of AKI.Related studies on AKI in animal experiments and intensive care unit?ICU?patients showed Heat Shock Protein-72?HSP-72?was a good indicator for early diagnosis of AKI,in recent years.However,there is little report on HSP-72 in AKI after Cardiopulmonary Bypass?CPB?.The expression level,mechanism of HSP-72 in AKI after cardiopulmonary bypass and whether there is a predictive effect AKIaftercardiopulmonarybypassandthecomparisonofNGAL,[TIMP-2]×[IGFBP7],HSP-72 for the early diagnosis of AKI has not been studied.ObjectiveIn this study,we observed the changes of urinary NGAL,[TIMP-2]×[IGFBP7]and HSP-72 levels in patients after cardiopulmonary bypass surgery,and compared the values of them for the early diagnosis of AKI and the correlation with the severity of the disease after cardiopulmonary bypass.At the same time,human proximal tubular epithelial cells was cultured in vitro and hypoxia-reoxygenation model was established to explore molecular mechanisms.Methods1.Clinic trialIn this study,45 patients with cardiopulmonary bypass from May 2018 to July2018 in People's Hospital of Henan Provincial were selected as subjects.Blood and urine samples were collected before and after cardiopulmonary bypass at 0h,2h,4h,6h,8h,12h,24h and 48h;20 healthy subjects in the physical examination center of People's Hospital of Henan Provincial at the same time were used as the control group.According to the diagnostic criteria for AKI,patients admitted after CPB were divided into AKI group?n=24?and non-AKI group?n=21?according to the Kidney Disease:Improving Global Outcomes Guide.Serum creatinine?Scr?was determined by enzymatic method,and urinary TIMP-2,IGFBP7 and HSP-72 were detected by enzyme-linked immunosorbent Assay?ELISA?at different time points.Urine NGAL was determined by immunoturbidimetry.We compared the changes in protein expression trends at different time points.2.Cell experimentEstablish a model of hypoxia-reoxygenation in cells and observe the expression level of damaged proteins:Normally cultured human proximal tubular epithelial cells?HK-2?were inoculated to a six-well plate in their logarithmic phase for 24 hours,and cultured with a sugar-free serum-free DMEM medium containing 300?M CoCl₂.HK-2 cells were placed in a incubator with 37?and 5%CO₂ and they were treated with hypoxia for 4 hours,and then HK-2 cells were collected after reoxygenation at0h,1h,2h,4h,8h,12h,24h and 48h,respectively.Western blot?WB?was used to detect the expresse of damage proteins of TIMP-2,IGFBP7 and HSP-72 and we compaired the protein expression trends at different points of time.Results1.There was no significant difference in age,sex composition ratio,body mass index and body surface area between the experimental group and the control group?P>0.05?.The change value of Scr within 48h in the experimental group was significantly higher than that in the control group?P<0.01?.There were no significant differences in age,sex composition ratio,body mass index,body surface area,time of cardiopulmonary bypass,intraoperative urine volume,and minimum temperature of body between the AKI group and the non-AKI group?P>0.05?.The change value of Scr within 48h in the AKI group was significantly higher than that in the non-AKI group?P<0.01?.2.The levels of TIMP-2,IGFBP7,[TIMP-2]×[IGFBP7],HSP-72 and NGAL in AKI group were significantly higher than those in non-AKI group and control group?P<0.05?.The differences were statistically significant at each time point.The level of urinary TIMP-2 in AKI group peaked at 12h after CPB and then decreased.The level of urinary IGFBP7 in AKI group peaked at 12h after CPB and then decreased.The level of urinary[TIMP-2]×[IGFBP7]in AKI group peaked at 12h after CPB and then decreased.The level of urinary HSP-72 in AKI group peaked at 2h after CPB and then decreased.The level of urinary NGAL in AKI group peaked at 4h after CPB and then decreased.3.The areas under the ROC curve of urinary[TIMP-2]×[IGFBP7],HSP-72,and NGAL in AKI group and non-AKI group showed:area under the ROC curve of urine[TIMP-2]×[IGFBP7]at 2h after CPB was 0.96?P<0.05?;area under the ROC curve of urine HSP-72 at 6h after CPB was 0.996?P<0.05?;area under the ROC curve of urine NGAL at 4h after CPB was 0.95?P<0.05?,the differences were statistically significant.4.Correlation analysis between urinary levels of[TIMP-2]×[IGFBP7],HSP-72and NGAL and the severity of disease indicated that the higher the AKI stage was,the higher the expression level of the above factors was,the expression level of the above factors was positively correlated with the severity of disease?P<0.05?.5.Western blot analysis showed that the expression levels of TIMP-2,IGFBP7and HSP-72 secreted by HK-2 cells were significantly higher than those of the control group after hypoxia-reoxygenation treatment;the expression levels of protein TIMP-2,IGFBP7 and HSP-72 secreted by HK-2 cells peaked at 2h after hypoxia for 4h,and then decreased gradually.Conclusions1.TIMP-2,IGFBP7 and HSP-72 in urine can be used to predict the occurrence of AKI in patients after cardiopulmonary bypass and the degree of elevation is related to the severity of AKI in patients after cardiopulmonary bypass.2.In patients with cardiopulmonary bypass,HSP-72 can predict AKI after cardiopulmonary bypass earlier than[TIMP-2]×[IGFBP7]and NGAL.3.TIMP-2,IGFBP7 and HSP-72 can be secreted by hypoxic HK-2 cells,and the expression levels of these protein gradually increased in initial stage and then declined with the prolongation of reoxygenation time.