Schwerdt Gerald, Freudinger Ruth, Schuster Claudia, Silbernagl Stefan, Gekle Michael
Department of Physiology, University of Wuerzburg, Germany.
Cell Physiol Biochem. 2004;14(1-2):47-56. doi: 10.1159/000076926.
Ochratoxin A (OTA) is a potent nephrotoxin and suspected to be involved in the etiology of Balkan endemic nephropathy. Nanomolar concentrations of this mycotoxin induce apoptosis in renal collecting duct-derived cells (MDCK-C7 cells, resembling principal cells). We studied the role of mitochondria in this process by inhibition of the mitochondrial respiratory chain, the F1FO-ATP synthase or by uncoupling. Also, the role of intra- and extracellular pH in apoptosis induction was investigated. Activation of caspase-3 and DNA ladder formation were used to monitor the apoptotic response. When cells were incubated with inhibitors of the mitochondrial respiratory chain or an inhibitor of the ATP-synthase, OTA-induced apoptosis was enhanced dramatically. Also, mitochondrial uncoupling potentiated the effects of OTA. OTA-induced apoptosis was not dependent on a decrease of the mitochondrial potential. Mitochondrial blockade led to medium acidification due to enhanced production of lactic acid. Artificial extracellular acidification potentiated OTA-induced caspase-3 activation. Artificial extracellular alkalization had no influence on caspase-3 activity. Intracellular pH after 24 hours exposure to inhibitors of mitochondria or acidic or alkaline media did not correlate with caspase-3 activity but correlated with caspase-3 activity when OTA was present: acidic intracellular pH (pHin) was associated with higher caspase-3 activity as compared to alkaline pHin. We conclude that extra- and intracellular pH are important factors in OTA-induced apoptosis in MDCK-C7 cells. The physiologically changing pH conditions in the collecting duct can thus alter or even aggravate the toxic effects of OTA.
赭曲霉毒素A（OTA）是一种强效肾毒素，被怀疑与巴尔干地方性肾病的病因有关。纳摩尔浓度的这种霉菌毒素可诱导肾集合管衍生细胞（MDCK-C7细胞，类似于主细胞）凋亡。我们通过抑制线粒体呼吸链、F1FO-ATP合酶或通过解偶联来研究线粒体在此过程中的作用。此外，还研究了细胞内和细胞外pH在凋亡诱导中的作用。使用半胱天冬酶-3的激活和DNA梯状条带形成来监测凋亡反应。当细胞与线粒体呼吸链抑制剂或ATP合酶抑制剂孵育时，OTA诱导的凋亡显著增强。此外，线粒体解偶联增强了OTA的作用。OTA诱导的凋亡不依赖于线粒体膜电位的降低。线粒体阻断由于乳酸产生增加导致培养基酸化。人工细胞外酸化增强了OTA诱导的半胱天冬酶-3激活。人工细胞外碱化对半胱天冬酶-3活性没有影响。暴露于线粒体抑制剂或酸性或碱性培养基24小时后的细胞内pH与半胱天冬酶-3活性无关，但在存在OTA时与半胱天冬酶-3活性相关：与碱性细胞内pH相比，酸性细胞内pH（pHin）与更高的半胱天冬酶-3活性相关。我们得出结论，细胞外和细胞内pH是OTA诱导MDCK-C7细胞凋亡的重要因素。因此，集合管中生理上不断变化的pH条件可以改变甚至加重OTA的毒性作用。
