Possible role of mtDNA depletion and respiratory chain defects in aristolochic acid I-induced acute nephrotoxicity.

This report describes an investigation of the pathological mechanism of acute renal failure caused by toxic tubular necrosis after treatment with aristolochic acid I (AAI) in Sprague-Dawley (SD) rats. The rats were gavaged with AAI at 0, 5, 20, or 80 mg/kg/day for 7 days. The pathologic examination of the kidneys showed severe acute tubular degenerative changes primarily affecting the proximal tubules. Supporting these results, we detected significantly increased concentrations of blood urea nitrogen (BUN) and creatinine (Cr) in the rats treated with AAI, indicating damage to the kidneys. Ultrastructural examination showed that proximal tubular mitochondria were extremely enlarged and dysmorphic with loss and disorientation of their cristae. Mitochondrial function analysis revealed that the two indicators for mitochondrial energy metabolism, the respiratory control ratio (RCR) and ATP content, were reduced in a dose-dependent manner after AAI treatment. The RCR in the presence of substrates for complex I was reduced more significantly than in the presence of substrates for complex II. In additional experiments, the activity of respiratory complex I, which is partly encoded by mitochondrial DNA (mtDNA), was more significantly impaired than that of respiratory complex II, which is completely encoded by nuclear DNA (nDNA). A real-time PCR assay revealed a marked reduction of mtDNA in the kidneys treated with AAI. Taken together, these results suggested that mtDNA depletion and respiratory chain defects play critical roles in the pathogenesis of kidney injury induced by AAI, and that the same processes might contribute to aristolochic acid-induced nephrotoxicity in humans.

Disappearance of sexual dimorphism in triptolide metabolism in monosodium glutamate treated neonatal rats.

Triptolide (CAS 38748-32-2), a major active component of Tripterygium wilfordii Hook F (TWHF), was reported to be sex-dependently metabolized mainly due to sex-related expression of CYP3A2. Sexual dimorphism in the expression of CYP isoforms is affected by sex difference in daily rhythm of growth hormone (GH) secretion. Neonatal administration of monosodium glutamate (MSG) can produce latent developmental defects in GH secretion and associated sex-dependent hepatic enzymes. In the present study, the triptolide metabolism, CYP3A2 expression and CYP3A-dependent activity were evaluated in Sprague-Dawley rats treated neonatally with MSG (4 mg/g) or saline (control) on postnatal days 1, 3, 5, 7 and 9. Treatment with MSG during the neonatal period in both sexes caused a number of disorders characterized by stunted body growth, notable obesity and suppression of GH secretion to barely detectable levels. In addition, neonatal treatment with MSG nearly eliminated the male-specific CYP3A2 expression and significantly reduced the microsomal erythromycin N-demethylation activity in males, while having no effects on CYP3A2 protein in females. Consistent with the P450 findings, the sexual dimorphism of triptolide metabolism completely disappeared in MSG-treated rats. This suggested that neonatal MSG treatment could eliminate the sex-dependent difference in metabolism of triptolide by suppressing CYP3A2 expression and activity in males to the same extent as females.

Toxicities of aristolochic acid I and aristololactam I in cultured renal epithelial cells.

Aristolochic acid nephropathy, a progressive tubulointerstitial renal disease, is primarily caused by aristolochic acid I (AA-I) intoxication. Aristololactam I (AL-I), the main metabolite of AA-I, may also participate in the processes that lead to renal damage. To investigate the role and mechanism of the AL-I-mediated cytotoxicity, we determined and compared the cytotoxic effects of AA-I and AL-I on cells of the human proximal tubular epithelial (HK-2) cell line. To this end, we treated HK-2 cells with AA-I and AL-I and assessed the cytotoxicity of these agents by using the 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay, flow cytometry, and an assay to determine the activity of caspase 3. The proliferation of HK-2 cells was inhibited in a concentration- and time-dependent manner. Cell-cycle analysis revealed that the cells were arrested in the S-phase. Apoptosis was evidenced by the results of the annexin V/propidium iodide (PI) assay and the occurrence of a sub-G1 peak. In addition, AA-I and AL-I increased caspase 3-like activity in a concentration-dependent manner. These results also suggested that the cytotoxic potency of AL-I is higher than that of AA-I and that the cytotoxic effects of these molecules are mediated through the induction of apoptosis in a caspase 3-dependent pathway.