Low doses of ochratoxin A upregulate the protein expression of organic anion transporters Oat1, Oat2, Oat3 and Oat5 in rat kidney cortex

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Abstract

Mycotoxin ochratoxin A (OTA) is nephrotoxic in various animal species. In rodents, OTA intoxication impairs various proximal tubule (PT) functions, including secretion of p-aminohippurate (PAH), possibly via affecting the renal organic anion (OA) transporters (Oat). However, an effect of OTA on the activity/expression of specific Oats in the mammalian kidney has not been reported. In this work, male rats were gavaged various doses of OTA every 2nd day for 10 days, and in their kidneys we studied: tubule integrity by microscopy, abundance of basolateral (rOat1, rOat3) and brush-border (rOat2, rOat5) rOat proteins by immunochemical methods, and expression of rOats mRNA by RT-PCR. The OTA treatment caused: a) dose-dependent damage of the cells in S3 segments of medullary rays, b) dual effect upon rOats in PT: low doses (50–250 μg OTA/kg b.m.) upregulated the abundance of all rOats, while a high dose (500 μg OTA/kg b.m.) downregulated the abundance of rOat1, and c) unchanged mRNA expression for all rOats at low OTA doses, and its downregulation at high OTA dose. Changes in the expression of renal Oats were associated with enhanced OTA accumulation in tissue and excretion in urine, whereas the indicators of oxidative stress either remained unchanged (malondialdehyde, glutathione, 8-hydroxydeoxyguanosine) or became deranged (microtubules). While OTA accumulation and downregulation of rOats in the kidney are consistent with the previously reported impaired renal PAH secretion in rodents intoxicated with high OTA doses, the post-transcriptional upregulation of Oats at low OTA doses may contribute to OTA accumulation and development of nephrotoxicity.

Introduction

Ochratoxin A (OTA) is ubiquitous fungal metabolite and food contaminant with nephrotoxic and carcinogenic potential in animals and humans (EFSA., 2006, Kuiper-Goodman and Scott, 1989, Pfohl-Leszkowicz and Manderville, 2007). It has been shown in various animal models (rat, pig, chicken) that OTA: a) accumulates largely in the proximal tubule (PT) cells, b) induces dose- and time-dependent impairment of PT function, resulting in limited polyuria, glucosuria, proteinuria and enzymuria, and diminished secretion of organic anions (OA), c) damages the renal structure, manifested by tubule degeneration, apoptosis, necrosis, and exfoliation of PT cells, predominantly involving the pars recta segment (S3), and d) causes focal tubular proliferative effects, including cell hyperplasia and tubular cell adenoma and carcinoma (Boorman et al., 1992, Castegnaro et al., 1998, Kuiper-Goodman and Scott, 1989, Kumar et al., 2007, Lee et al., 1984, Rached et al., 2007, Sauvant et al., 2005). In some studies, usually after treating experimental animals with OTA for a few months or years, cellular mechanisms of OTA toxicity were associated with increased lipid peroxidation and formation of malondialdehyde (MDA), DNA damage, and apoptosis, and were counteracted by the scavengers of reactive oxygen species (ROS), thus indicating oxidative stress as a possible mediator of toxicity (Cavin et al., 2007, Grosse et al., 1997, Kamp et al., 2005, Petrik et al., 2003, Ringot et al., 2006, Schaaf et al., 2002).

OTA has a long plasma half life in the body, mainly due to binding to serum proteins, slow biotransformation, entero-hepatic circulation, and reabsorption in kidney (Fuchs and Hult, 1992). The renal excretion of OTA via glomerular filtration is limited, and largely proceeds via transepithelial secretion in PT (Bahnemann et al., 1997, Jung et al., 2001, Welborn et al., 1998). The filtered and/or secreted OTA is partially reabsorbed along the nephron, mostly in the PT S3 segments (Dahlmann et al., 1998, Zingerle et al., 1997). Filtration and transepithelial secretion, followed by reabsorption, represent an effective way of OTA accumulation in kidneys and may be a base for development of nephrotoxicity. OTA itself is an OA and a substrate of various renal OA transporters (Oat) located in the basolateral (BLM) or luminal membrane domain of (mainly) PT cells. In the mammalian kidney, OTA can be transported by Oat1 (Slc22a6), Oat2 (Slc22a7), Oat3 (Slc22a8), OAT4 (SLC22A11; absent in rodents), Oat5 (Slc22a19; absent in humans), Oat-K1 (Oatp1a3/Slco1a3), H+-dipeptide cotransporter (PEPT/family Slc15), and ATP-driven multidrug resistance-associated protein Mrp2 (Abcc2) (Anzai et al., 2005, Babu et al., 2002, Cha et al., 2001, Enomoto et al., 2002, Kobayashi et al., 2002, Kusuhara et al., 1999, Kwak et al., 2005, Leier et al., 2000, Takeuchi et al., 2001, Tsuda et al., 1999, Youngblood and Sweet, 2004). In the rodent PT, Oat1 and Oat3 are localized in the BLM and thus may mediate internalization of OTA at the basolateral side. Oat2, Oat5, Oat-K1, and PEPT are localized in the brush-border membrane (BBM) and thus may mediate reabsorption of OTA, whereas Mrp2 in the BBM may serve as an active extruder of OTA.

Earlier studies in various experimental models indicated that OTA, being itself a substrate for various Oats, can interact with the transport of other OA. In these studies: a) OTA directly and competitively inhibited the basolateral transport of a prototypical OA p-aminohippurate (PAH) in isolated PT segments and renal cortical membranes, b) in the presence of OTA, the accumulation of PAH in the renal cortical slices, isolated PT cells, and PT-derived established cell lines, was impaired, and c) the OTA-treated experimental animals exhibited a diminished accumulation of PAH in the renal cortical slices in vitro and a lower PAH clearance in vivo (Friis et al., 1988, Gekle and Silbernagl, 1994, Groves et al., 1998, Groves et al., 1999, Jung et al., 2001, Sauvant et al., 1998, Sokol et al., 1988, Welborn et al., 1998). These data thus indicated that OTA may affect the activity and/or expression of renal Oats, but such possibilities have not been experimentally verified. In order to test if OTA affects the cellular expression and distribution of renal Oats, here we used an in vivo model of OTA intoxication in adult male rats, and characterized the protein and mRNA expression levels of the two basolateral (Oat1, Oat3) and two brush-border (Oat2, Oat5) OA/OTA transporters in PT. The Oat data were correlated with the tissue morphology, OTA accumulation in the renal tissue and its excretion in urine, and with the tissue and/or urine indicators of oxidative stress.

Section snippets

Animals and treatment

Adult (12–14 weeks old) male Wistar rats were from the breeding colony at the Institute in Zagreb. Animals were bred and maintained according to the Guide for Care and Use of Laboratory Animals (National Institute of Health, Bethesda, USA, 1996). The studies were approved by the Institutional Ethics Committee.

OTA was dissolved in 51 mM NaHCO3, pH 7.4, and given by gastric gavage. Animals (4 rats/experimental group) were gavaged with various OTA doses (50, 125, 250 and 500 μg/kg b.m.) every 2nd

Reabsorptive functions of renal tubules in OTA-treated rats

In order to determine the OTA-induced renal functional and structural changes, specific urine parameters were analyzed and microscopic examination of the tissue cryosections was performed in rats treated with vehicle or various OTA doses for 10 days. In this initial study, in the 24-h urine from 8 vehicle-treated and 8 OTA-treated (50–500 μg/kg b.m.) animals we compared the urine volume, glucose, creatinine, phosphate, potassium, chloride, and sodium, and found that these parameters in control

Discussion

Previous studies in OTA-treated mice, rats and rabbits described alterations in renal function and structure induced with subchronic treatment with high doses (≥ 500 μg/kg b.m./day, for 5–10 days) or with chronic treatment with lower doses (100–250 μg/kg b.m./day, for a few weeks to two years) of this mycotoxin. These studies revealed only limited, OTA dose-dependent defects in renal reabsorptive and secretory functions, whereas the morphological damage was observed primarily in PT at high OTA

Conflict of interest statement

The authors declare no conflict of interest regarding this collaborative work.

Acknowledgments

The authors acknowledge the expert technical assistance by Eva Heršak, Mirjana Matašin and Jasna Mileković. This study was approved by the Ethical Committee of the Institute for Medical Research and Occupational Health in Zagreb. The funding was provided by grants No. 022-0222148-2142 (M. Peraica) and 022-0222148-2146 (I. Sabolic) from the Ministry of Science, Education and Sports, Republic of Croatia.

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