Evaluation of toxicological mechanisms of ochratoxin-A in human primary proximal tubule epithelial cells

Ochratoxin-A (OTA) is a fungal mycotoxin that is one of the most common food contaminants, ubiquitously found in a variety of foodstuffs worldwide. As a known nephrotoxin, hepatotoxin, and class II-b carcinogen, exposure to this compound is a major health concern. Specifically, OTA has been implicated as a potential agent in chronic kidney disease of unknown etiology (CKDu), an endemic form of chronic kidney disease that disproportionally affects rural agricultural communities and is thought to arise from environmental factors rather than diabetes and hypertension. A key pathological difference between CKDu and CKD is the degeneration of the kidney proximal tubule, where OTA has been shown to accumulate. The mechanism of OTA toxicity in the human kidney remains elusive, and this study aims to elucidate OTA-induced molecular toxicological pathways using primary human proximal tubule epithelial cells (PTECs). We demonstrated that the previously reported downregulation of NRF2 target antioxidant response elements is not due to a direct interaction between NRF2 and its cytosolic binder, KEAP1, evidenced by the lack of recovery of mRNA transcripts with co-administration of NRF2 agonists, sulforaphane or tert-butylhydroquinone. Furthermore, exposure to OTA (10 uM) induces 7192 differentially expressed genes, including key regulators of mitochondrial fission and fusion. This was confirmed at the cellular level by confocal microscopy, where a breakdown of the mitochondrial network was observed at 100 nM OTA. Furthermore, OTA was found to significantly induce reactive oxygen species (mROS) and inhibit basal mitochondrial oxidative phosphorylation as well as glycolysis through measurements of oxygen consumption rate and extracellular acidification, corroborating transcriptional findings that OTA induces hypoxic response elements. In conclusion, in this work we demonstrate that OTA causes mitochondrial dysfunction and reduced ATP production, leading to a senescent-like state in PTECs characteristic of renal disease progression. These findings provide insight into early toxicological endpoints induced by OTA which have been established as pathophysiological changes involved in chronic kidney disease.