TITLE:
In Vitro Study of the Nephrotoxicity of Tripterygium Tablet Extract and Triptolide in Monolayer HK-2 Cells Cultured in a Transwell Chamber
AUTHORS:
Ran Hao, Lianqiang Hui, Chun Li, Chunyu Cao, Yifei Yang, Jiyuan Zhang, Ting Liu, Yi Zhang
KEYWORDS:
Nephrotoxicity, Tripterygium Tablet Extract, Triptolide, HK-2 Cell, Transwell Chamber
JOURNAL NAME:
Chinese Medicine,
Vol.9 No.1,
March
14,
2018
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.