Experimental Study Of Early Diagnosis Of Acute Renal Injury In Sepsis Rats By NGAL, KIM-1 And TIMP-2

Part Ⅰ Prepare Septic AKI Rat ModelObjective:To establish an easy an steady relevant septic AKI rat model.Methods:We made the sepstic AKI rat model by cecal ligation and puncture(CLP).50 healthy male Sprague-Dawley rats were randomly assigned to three groups:Normal control group(n=10),don’t do any treatment.CLP group(n=20),according to Rittirsch,perform CLP surgery. Sham operation group(sham group,n=20),the cecum was isolated but neither ligated nor punctured,the rest of treatment equally to CLP group.Rat blood were collected by cardiac puncture after 24 hours,for serum creatinine(SCr),blood urea nitrogen(BUN),tumor necrosis factor-a(TNF-a) and Interleukin-6(IL-6)detecting.The biological behavivour changes and the 24 hours and 48 hours mortality rats of sham group and CLP group were observed(n=10). The rest of survival rats were killed after 24h,the right renal tissue was taken for pathological section.Renal pathology was obsered with light microscopy, using hematoxy-eosin staining.We determined that whether sepstic acute kidney injury accured in the rats according to creatinine and renal pathological changes in rats with sepsis.Data were presented as mean± SD.One-way ANOVA was used to analyze changes among multiple groups,and followed by LSD-test, it was considered statistically significant( P<0.05).Results:The sham group rats were all survived,the 24 hours and 48 hours mortality rates of CLP group were 20%and 60%.There was no great difference in renal function,inflammatory factors and histological changes between sham group and control group(P>0.05).Compared with sham group,the levels of SCr, BUN,TNF-a and IL-6 increased significantiy at 24h(P<0.05) in CLP group,(Cr(μmol/L):17.74±1.80 VS.40.96 ± 3.03,BUN(nmol/L):1.35 ± 0.23 VS. 2.59 ± 0.28,TNF-a (ng/L):100.81+7.97 VS.239.30±17.57,IL-6(ng/L):76.35±8.94 VS. 161.43 ± 14.76,all P<0.05).There were no significant histological changes in control and sham group.The kidney tissue structure was clear, no obvious pathological changes were detacted in control and sham group, there were also on difference in kidney tissues injury scores(P>0.05).Compared with the sham group, the renal tissue structure disarranged, tubular epithelial cell swelling, vacuolar degeneration and cell shedding changes in CLP group, the renal injury scores increased, the difference was statistically significant(0.48 ± 0.25VS. 2.83±0.27,P<0.05).Conclusion:1.Our research successfully create septic AKI rat model induced by CLP.2. The septic AKI rat model induced by CLP is easy and steady, via controlling the variety,age of rats and the standarization of the CLP.Part Ⅱ the Early Prediction Value of NGAL, KIM-1 and TIMP-2 on Septic AKI RatsObjective:To investigate the early prediction value of NGAL, KIM-1 and TIMP-2 on septic AKI rats.Methods:108 healthy male Sprague-Dawley rats were randomly assigned to three groups: normal control group(w=12),septic AKI group(SAKI group,n=48),perform cecal ligation and puncture;Sham operation group(sham group,n=48),the cecum was isolated but neither ligated nor punctured.Sham group and SAKI group were devided into four time point(6h,12h,18h and 24h after operation).At four time points 12 rats were in each group. Blood samples were collected by cardiac puncture for detecting the serum Cr and BUN.Urine was collected through bladder puncture to detect NGAL,KIM-1 and TIMP-2.The right renal tissue was taken for pathological section.Renal pathology was observed with light microscopy, using hematoxy-eosin staining.Data were presented as mean ±SD.One-way ANOVA was used to analyze changes among multiple groups,and followed by LSD-test,there was statistically significant(P<0.05).Correlation analysis was detected using Pearson method.Results:1.Comparsion of renal function among three groupsCompared with normal control group,there was no difference in serum Cr and BUN in sham group (P>0.05). Compared with sham group, the level of serum Cr and BUN in the SAKI group were increased significantly at 12h (P<0.05),(Cr(μmol/L):16.34±2.42 VS. 23.53 ±2.88,BUN(nmol/L):1.27± 0.17 VS.1.69 ± 0.26, all P<0.05), and the concentration continued to rise at 18h and 24h. Compared with 24h after operation,the level of serum Cr and BUN were significantly different in the SAKI group at 12h and 18h(P<0.05).2.Comparsion of urine renal injury biomarkers among three groupsCompared with normal control group,there was no significance in the concentration of urine NGAL,KIM-1 and TIMP-2 in sham group(P>0.05).In SAKI group,the levels of urine injury biomarkers increased significantly at 6h,compared with sham group(P<0.05). Urine KIM-1 reached a peak at 12h(153.80± 20.08 (ng/L)),with declining at 18h and 24h,still higher compared with sham group(P<0.05);Compared with 12h after operation,the level of urine KIM-1 at 6h,18h and 24h in the SAKI group were significantly different(P<0.05).The level of urine NGAL reached a peak at 18h after operation(3166.74±337.64(ng/L)),with a trend of decreasing at 24h,it was still higher than sham group(P<0.05);compared with 18h,there were significant difference in the SAKI group at 6h and 12h(P<0.05),while there was no difference at 24h(P>0.05).The level of urine TIMP-2 continued to increase at 12h,18h and 24h,and reached the peak at 24h in the SAKI group(2388.26±353.74(ng/L))(P<0.05),compared with 24h,the levels of urine TIMP-2 were significantly lower at 6h,12h and 18h(P<0.05).3.Histological changes and comparsion of renal injury scores among three groupsThe rat kidney tissues showed no obvious abnormalities in sham group and normal control group,there wre no cell edema, vacuolar degeneration,necrosis,shedding and cell tube formation in renal tubular epithelial cells; Tissue structure changes were observed in rat kidney in SAKI group,with different degreen of swelling, vacuolar degeneration,necrosisin in renal tubular epithelial cells, or cell tube formation.in renal tubule.In septic AKI group,the renal injury scores increased gradually at all the time points,and there were significant differences,compared with the sham group(P<0.05).4.Correlation analysis between urine biomarkers and renal pathology scoresCorrelation analysis showed that the renal pathology scores had a positive corrlation with urine KIM-1, urine NGAL, urine TIMP-2, the correlation coefficient were 0.638,0.897,0.810(P <0.01).Conclusion:The urinary biomarkers are significantly increased in septic AKI rats at 6h after CLP, and earlier than serum creatinine(Cr), blood urea nitrogen (BUN).Urine KIM-1, urine NGAL, urine TIMP-2 with renal pathology scores have a positive correlation,the urinary biomarkers have an early predictive value on septic AKI.