Observation of penetration, distribution and accumulation in human renal proximal tubular epithelial cells by aristololactam-I / 中国中药杂志

<p><b>OBJECTIVE</b>To study whether aristololactam I (AL-I) can enter renal proximal tubular epithelial cells and the situation of intracellular distribution and accumulation.</p><p><b>METHOD</b>Cultured human renal proximal tubular epithelial cell line (HK-2) was used as the subject. Intracellular fluorescence from AL-I and its distribution are examined by fluorescence microscopy after a treatment with different concentration of AL-I, the intracellular accumulation of AL-I was also investigated by incubated cells in AL-I -free medium for 48 h after washing-out the media containing AL-I.</p><p><b>RESULT</b>After treatment of AL-I (concentration from 5 microg x mL(-1) to 20 microg x mL(-1)), glaucous fluorescence could be observed inside renal proximal tubular epithelial cells at 0.5 h, and the fluorescence distributed only in cytoplasm while not be observed in nuclei. Moreover, the fluorescence of AL-I could be kept in cytoplasm for more than 48 h after washing out the media containing AL-I .</p><p><b>CONCLUSION</b>AL-I is able to enter renal proximal tubular epithelial cells in short time and accumulate in cytoplasm, but not enter nuclei. This property may contribute to the cytotoxic mechanism of renal injury induced by AL-I, which may partially explain the persistent renal toxicity of AAs and its metabolites in the development of aristolochic acid nephropathy.</p>

Effects of exogenous epidermal growth factor (EGF) on growth inhibition induced by aristolochic acid I (AA-I) in renal proximal tubular epithelial cells / 中国中药杂志

<p><b>OBJECTIVE</b>The purpose of this study was to investigate the effects of exogenous epidermal growth factor (EGF) on the growth inhibition of renal proximal tubular epithelial cell induced by AA-I.</p><p><b>METHOD</b>Cultured human renal proximal tubular epithelial cell line HK-2 was used as the subject. The changes of the survived HK-2 cells were observed and compared among control, AA-I stimulation, pre-EGF, together-EGF and post-EGF groups. In the study, cellular morphologic assessments were performed with a phase-contrast inverted microscope. Cell counting after stained with 0.04% trypan blue was adopted to analyze cell proliferation. Cell cycle was assessed by flow cytometry.</p><p><b>RESULT</b>Number of the survived cells stimulated by AA-I for 12, 24, 48 hours decreased gradually in a dose and time dependent manner. At 24, 48 hours, the survived cells showed a significant disturbance in the cell cycle procedure, which was characterized as decreased percentage of cells in G0/G1 phase, significant increased percentage of cells in G2/M phases. Exogenous EGF (20 microg L(-1)) could significantly promote the proliferation of HK-2 cells, which shown a increased cell number, accompanied down-regulated cells in G0/G1 phase, increased cells in S and G2/M phase. Compared with AA-I groups, it failed to improve the inhibitory effect on cell proliferation and abnormal cell cycle procedure by AA-I, no matter EGF was added in before, at same time or after AA-I stimulation.</p><p><b>CONCLUSION</b>AA-I (10 g L(-1)) has remarkable growth inhibition effects on survived RTEC, and can induce a blockage of G2/M phase in the cell cycle procedure. Exogenous EGF (20 microg L(-1)) promote the proliferation in normal cultured HK-2 cell. EGF treatment could not improve the proliferation inhibitory effect induced by AA-I, no matter adding EGF to cultures before, together with AA-I or after AA-I stimulation.</p>