<i>In Vitro</i> Study of the Nephrotoxicity of Tripterygium Tablet Extract and Triptolide in Monolayer HK-2 Cells Cultured in a Transwell Chamber

Abstract

We established a monolayer polarized cell model using human kidney 2 (HK-2) cells cultured in a transwell chamber to examine the changes in the morphology and physiological functions of human-derived renal proximal tubular epithelial cells caused by tripterygium tablet extract (TTE) and triptolide. HK-2 cells were cultured on PCF membranes to form a complete monolayer of cells. A MTT assay was used to select 10, 40, 160, 640 μg·ml-1 TTE or 4, 16, 64, 256 ng·ml-1 triptolide to treat HK-2 monolayer cells. After 24 hours, a FITC permeability assay was performed; GGT, LDH and NAG secretion on the apical (AP) and basolateral (BL) sides of the cells by HK-2 cells were examined. The morphology and the monolayer structure of HK-2 cells was observed via optical microscope and scanning electron microscope, respectively. The effect on the cytoskeleton of HK-2 cells was observed under a fluorescence microscope. The IC50 of TTE was 277.122 μg·ml-1, and the IC50 of triptolide was 148.035 ng·ml-1. Compared with the DMSO group, the FITC leakage rate with TTE 160, 640 μg·ml-1 treated group and 4 - 256 ng·ml-1 triptolide dose group exhibited statistically significant increase. TTE significantly increased secretion of GGT and LDH at 160, 640 μg·ml-1, meanwhile, dramatically increased the AP/BL ratio of LDH at 160 μg·ml-1; triptolide significantly increased secretion and AP/BL ratio of GGT and LDH at 256 ng·ml-1. The morphological observations via optical and electron microscope indicated various degrees of damage to HK-2 cells by TTE and triptolide, and the degree of damage correlated positively with the dosage of the tested articles. Compared with DMSO group, the cellular damage degrees at TTE dosages of 40 - 640 μg·ml-1 and triptolide dose group at 16, 256 ng·ml-1 exhibited statistically significant differences via observation under optical microscope. Both TTE and triptolide caused various degrees of shortening and thickening of intracellular F-actin bundles of HK-2 cells; aggravation of these changes was observed with increasing drug dosage. Thus, we conclude both TTE and triptolide caused damage to human renal proximal tubular epithelial cells at certain dosages; TTE dosages of 40 μg·ml-1 and above and triptolide dose group at 16 ng·ml-1 and above exhibited the changes in the morphology, meanwhile, TTE dosages of 160 μg·ml-1 and above and triptolide dose group at 256 ng·ml-1 exhibited the changes in the physiological functions such as secretion of HK-2 cell.