Study Of Vancomycin-induced Human Kidney Tubular Epithelial Cell Injury And The Expression Of KIM-1

Objective:To observe Vancomycin-induced human kidney tubular epithelial cell injury and the expression of KIM-1, the renal toxicity of vancomycin and its mechanisms and the significance of KIM-1 will be approached. Methods: HK-2 cells were cultured in DMEM/F12 medium containing 10% FBS, under 5% carbon dioxide,37 centi-degree for 2 to 3 days, then rinsed with 0.25% trypsin and subcultured into new culture flasks. HK-2 cells in logarithmic growth phase were cultured in a well-format. While cells were 80% confluent, complete medium was removed. Cells were washed with PBS for two times and new culture medium without serum was added. After 24 hours, all the cells were randomly divided into seven concentration groups. HK-2 cells were treated for 24h by 0、10、20、30、40、50、60μg/ml vancomycin. The vancomycin toxicity on HK-2 cell was detected by CCK-8. The HK-2 cells morphology were observed under inverted microscope. The contents of MDA in supernatant fluid were detected by TBA. The KIM-1mRNA and protein were detected by RT-PCR, immunofluorescence and Western Blotting, respectively. Then 20μg/ml vancomycin which the expression of KIM-1mRNA and KIM-1 protein were highest of all the concentration groups were deployed on HK-2 cells for 0、3、6、12、24 or 48h. The vancomycin toxicity was detected by CCK-8. The contents of MDA in supernatant fluid were detected by TBA. The KIM-1mRNA and protein were detected by RT-PCR and Western Blotting, respectively. Results:(1) The effects of HK-2 cells were treated with different concentration vancomycin for 24h,①vancomycin toxicity:Compared with 0μg/ml group, the persistence of HK-2 cells stepped down with the’drug concentration increased, in which the 50μg/ml and 60μg/ml groups were significantly decreased (P<0.05 or P<0.01). It suggested that the toxicity is stronger on HK-2 cells with the vancomycin concentration increased and the injury of HK-2 cells which were suffered was more serious within 24h.②the cell morphological changes:HK-2 cells were observed under the inverted phase contrast microscope. The adherent cells grew well in normal control group (0μg/ml group), multitude cells of quantity, and presented a typical "crazy paving "-like shape. Partly adherent cells were detached and necrosis, and a few cells were shrinked and became round in 20μg/ml and 40μg/ml groups. The number of cells was decreased, but they still show "cobblestone"-like shape. Lots of cells were detached and necrosis in 60μg/ml group. The number of cells were decreased more significantly and the shape of residual adherent cells changed into oval-shape or spindle shape from the polygonal.③the contents of MDA in supernatant fluid:Compared with 0μg/ml group, the contents of MDA of HK-2 cells in the supernatant is rising with the drug concentration increased, in which 30,40,50 and 60μg/ml groups were significantly increased (P<0.01). It suggested that the level of lipid peroxidation of HK-2 cells was rising with the drug concentration increased within 24h.④the expression of KIM-1mRNA:Compared with 0μg/ml group, the expression of KIM-1mRNA were significantly higher (P<0.05 or P<0.01) in other concentration groups, in which the expression of KIM-1mRNA of 20μg/ml group is the highest. It suggested that within 24h the expression of KIM-1mRNA is more in the case of less serious injury, but less expression in the case of more serious and fewer survival cells.⑤the expression of KIM-1 protein:a immunofluorescence: Under fluorescence microscopy, few KIM-1 positive cells were seen in 0μg/ml group, but the number of positive cells is more and the green fluorescence is brighter in 20μg/ml group. Compared with 0μg/ml group, there was statistical significance of the differences of 20μg/ml group (P<0.01). b Western Blotting Method:Compared with 0μg/ml group, the expression of KIM-1 protein of 10μg/ml and 20μg/ml groups were significantly higher (P<0.01), in which the expression of KIM-1 protein of 20μg/ml groups was highest. Compared with Oμg/ml group, the expression of KIM-1 protein of 60μg/ml group was significantly decreased (P<0.01). It suggested that within 24h the expression of KIM-1mRNA was more in the case of less serious injury of HK-2 cells, but less expression in the case of more serious and fewer survival cells. (2) The drug concentration which was administrated was 20μg/ml,①the vancomycin toxicity at different time points:Compared with Oh group, the survival rate of HK-2 cells is falling with the time lasting, in which the survival rate of 48h group decreased significantly (P<0.05). It suggested that the vancomycin toxicity was stronger with the exposing time longer, and the injury of HK-2 cells which were suffered was more serious.②the contents of MDA in supernatant fluid at different time points:Compared with Oh group, the contents of MDA of HK-2 cells in the supernatant were rising with the time increased, in which the contents of MDA of 24h and 48h groups were significantly higher (P<0.05 or P<0.01). It suggested that the level of lipid peroxidation of HK-2 cells was rising with the time lasting.③the expression changes of KIM-1mRNA at different time points:Compared with Oh group, the expression of KIM-1mRNA of 24h and 48h groups was significantly higher (P<0.01). It suggested that the expression of KIM-1mRNA is basical in the high expression state after HK-2 cells was mildly injuryed which induced by low concentration vancomycin within 24-48h.④the expression changes of KIM-1 protein at different time points:Compared with Oh group, the expression of KIM-1 protein of 3h、6h、12h、24h and 48h groups was significantly higher (P<0.05 or P<0.01). It suggested that the expression of KIM-1 protein is basical increasing with the time lasting after HK-2 cells was mildly injuryed which induced by low concentration vancomycin within 48h. Conclusions:①There was some cytotoxicity of vancomycin on HK-2 cells. Its mechanism may be related to oxidative stress damage.②The expression of KIM-1mRNA and protein was high in the early stage of vancomycin-induced mild acute renal injury. It suggested that KIM-1 can be used as an early acute kidney injury biomarker and a new index for early vancomycin-induced renal damage detection in clinical.