The Nephrotoxin Ochratoxin A Induces Key Parameters of Chronic Interstitial Nephropathy in Renal Proximal Tubular Cells

Abstract

Ochratoxin A (OTA) is a nephrotoxic and cancerogenic mycotoxin. There is epidemiological evidence that OTA exposition leads to cortical interstitial nephropathies in humans. However, virtually no data are available investigating the effect of OTA on renal cortical cells with respect to induction of nephropathy. Thus, we investigated whether OTA is able to induce changes of cellular properties potentially leading to interstitial nephropathy, using proximal tubular cell lines (OK, NRK-52E). OTA decreased cell number and cell protein time and dose dependently. Accordingly we investigated the effect of 100 nM or 1000 nM OTA. The decline of cell number after OTA exposure is due to necrosis and apoptosis, as measured by LDH release or DNA ladder formation and caspase-3 activation, respectively. OTA incubation of proximal tubular cells also resulted in a loss of epithelial tightness as determined by diffusion of FITC labeled inulin. Inflammation, fibrosis and epithelial-to-mesenchymal transition are described in chronic interstitial renal disease. Therefore, we also investigated the effect of OTA on NFκB activity, collagen secretion and generation of α smooth muscle actin. OTA alone was sufficient to induce the latter parameters in proximal tubular cells. Finally, OTA is a nephrotoxcic substance and elevated activity of mitogen activated protein kinases (MAPK) is described in nephropathies. As we investigated the effect of OTA on activity of ERK, JNK and p38 by ELISA, we found that OTA activates the MAPK measured dose dependently. In summary, OTA induced phenomena typical for chronic interstitial nephropathy, like loss of cells and epithelial tightness, necrosis and apoptosis as well as markers of inflammation, fibrosis and epithelial-to-mesenchymal transition in proximal tubular cells. Thus, we could show for the first time that OTA is able to induce key parameters of nephropathy in proximal tubular cells in culture. Moreover OTA interacts with MAPK and thus may exert its specific toxic actions.

A Journey Through Mitogen-Activated Protein Kinase and Ochratoxin A Interactions

“…Some other studies also confirmed OTAinduced MAPK phosphorylation (5,24,25,28). Phosphorylation of ERK1/2 and of their substrates Elk-1 and p90RSK was observed in kidney samples of male Fischer 344 rats fed with a carcinogenic dose of OTA over 7 days, 21 days, and 12 months (24).…”

“…According to the European survey conducted by Miraglia et al (2), mean plasma concentration of OTA in adults is 0.875 nmol L -1 , and ranges from 0.53 nmol L -1 to 2.75 nmol L -1 . OTA half-life in serum ranges from 120 h in rats to 840 h in monkeys and humans (4,5). This prolonged retention in serum is the consequence of strong binding to serum proteins, mainly to albumin.…”

“…As OTA inhibits protein synthesis at relatively high doses, which are not seen in everyday exposure, these effects have only a minor toxicological relevance. However, Gekle et al (5) have suggested that in the low-dose range (naturally occurring exposure) OTA interacts with key cell targets, changing the signalling pathways. These changes affect the cell function, and may disturb the whole-organism homeostasis.…”

“…In most cases, high OTA concentrations (>1 μmol L -1 ) cause necrosis of renal cells (both of proximal tubular and collecting duct origin), as demonstrated by lactate dehydrogenase (LDH) release (5,49). In contrast, at low nanomolar concentrations, OTA stimulates apoptosis, which has been confi rmed by chromatin condensation and fragmentation (DAPI staining, haematoxylin/eosin staining), DNA fragmentation (TUNEL assay, diphenylamine method), DNA ladder formation (DNA electrophoresis on agarose gel), and/or apoptotic enzyme caspase-3 activation (5,26,28,50). In addition, at 1 μmol L -1 , OTA was associated with both types of cell death (5,26,28).…”

“…In contrast, at low nanomolar concentrations, OTA stimulates apoptosis, which has been confi rmed by chromatin condensation and fragmentation (DAPI staining, haematoxylin/eosin staining), DNA fragmentation (TUNEL assay, diphenylamine method), DNA ladder formation (DNA electrophoresis on agarose gel), and/or apoptotic enzyme caspase-3 activation (5,26,28,50). In addition, at 1 μmol L -1 , OTA was associated with both types of cell death (5,26,28). These effects also appear to be cell-specifi c, which might explain why certain epithelial cell types along the nephron differ in susceptibility to OTA.…”