ࡱ> =?<5@b2bjbj22 86XXbjjjjjjj~DF ~& ^QSSSSSS$ R. ,wijgggwjj333gjjQ3gQ33MjjMR >3 M=0& MZ  Z M~~jjjjZ jM>0,3\$ww~~) ~~ORGANIC ANION TRANSPORTERS IN EXPERIMENTAL OCHRATOXIN A NEPHROPTOXICITY Vilim Zlender1, Davorka Breljak2, Marija Ljubojevic2, Daniela Balen2, Hrvoje Brzica2, Naohiko Anzai3, Radovan Fuchs1, Ivan Saboli2 1 Unit of Toxicology, Institute for Medical Research and Occupational Health, 10001 Zagreb, Croatia,  HYPERLINK "mailto:vzlender@imi.hr" vzlender@imi.hr 2Molecular Toxicology, Institute for Medical Research and Occupational Health, 10001 Zagreb, Croatia,  HYPERLINK "mailto:dbreljak@imi.hr" dbreljak@imi.hr,  HYPERLINK "mailto:mljubo@imi.hr" mljubo@imi.hr,  HYPERLINK "mailto:dbalen@imi.hr" dbalen@imi.hr,  HYPERLINK "mailto:hbrzica@imi.hr" hbrzica@imi.hr,  HYPERLINK "mailto:rfuchs@imi.hr" rfuchs@imi.hr,  HYPERLINK "mailto:sabolic@imi.hr" sabolic@imi.hr 3Pharmacology and Toxicology, Kyorin University School of Medicine, Tokyo 181-8611, Japan,  HYPERLINK "mailto:anzai@kyorin-u.ac.jp" anzai@kyorin-u.ac.jp Ochratoxin A (OTA) is a ubiquitous fungal metabolite with nephrotoxic and carcinogenic potential. Various animal models have demonstrated that OTA mainly impairs proximal tubule (PT) function and causes glucosuria, enzymuria, and diminished secretion of organic anion p-aminohippurate (PAH). The last indicates that OTA toxicity may target renal organic anion transporters (Oats; subfamily of Slc22a drug transporters). Oat1 (Slc22a6) and Oat3 (Slc22a8) proteins reside in the basolateral membrane (BLM) of PT epithelial cells and mediate both OTA and PAH transport in a noncompetitive mode. Previous studies have shown that subchronic intoxication of rats with OTA leads to a dramatic reduction of PAH clearance/excretion, indicating that Oat1 and Oat3 may be involved in OTA nephrotoxicity, possibly via inhibiting their transport activity and/or expression in the cell membrane. However, these possibilities have not been clarified. In order to study effects of OTA on cell/tissue morphology and on cellular expression of renal Oat1 and Oat3, especially in view of male-dominant expression of these transporters in renal proximal tubules in rats and mice, we used an established subchronic in vivo model of OTA intoxication in adult male (M) and female (F) Wistar rats. The animals of both sexes were treated with various doses of OTA (0, 50, 125, 250 & 500 g/kg b.w., p.o., every 2nd day for 10 days). Using peptide-specific polyclonal antibodies, Oats were studied by Western blotting (WB) in total cell membranes (TCM) isolated from the cortex (C) and outer stripe (OS) tissues, and by immunofluorescence cytochemistry (IC) in tissue cryosections, whereas the expression of their mRNA was analyzed by RT-PCR using RNA from the renal C and OS. The study of tissue morphology revealed that OTA treatment resulted in damaged PT S3 segments in medullary rays, manifested by the loss of regular tubular structure and cell membranes, cell desquamation and other necrotic events. These phenomena were more severe at higher OTA doses, and were much more pronounced in M. As found in WB experiments, in spite of different levels of the Oat1 and Oat3 protein expression in isolated membranes (M>F), their expression in OTA-treated rats in both sexes followed a similar and dual, dose-dependent pattern. At low OTA doses (50-125 g/kg b.w.) the expression of Oat1 and Oat3 protein increased ~50% and ~300%, respectively, whereas high OTA doses (250-500 g/kg b.w.) decreased the expression of Oat1 (~70%) but not of Oat3 in respect to controls (vehicle-treated rats). By IC, the staining intensity of both transporters in proximal tubules completely matched the WB data. However, the RT-PCR data showed that the Oat1 mRNA expression was unaffected by low OTA doses, whereas high OTA doses downregulated it, and the expression of Oat3 mRNA was unaffected by any OTA dose. Also the expression of housekeeping gene GAPDH mRNA remained unchanged in all OTA-treated animals. Our data indicate that in rats: 1) the OTA treatment causes dose-related and M-dominant damage to cell morphology in proximal tubule S3 segments, 2) Oat1 and Oat3 have a sim0p   : < @ Z \ ^ ` z | ymdXh,[*h,[*H*mH sH h~^H*mH sH hSxhSNH*mH sH hSxhfmH sH hSxhfH*mH sH hfmH sH ht9H*mH sH h2\mH sH hSxhSNmH sH ht9hSNH*mH sH ht9h2\mH sH hSxhSx5mH sH ht9hSNmH sH ht9mH sH ht9ht9mH sH ht9hSxmH sH " m n   a2b2$a$gd gdSN$a$gdt9 b2  # 2 3 4 5 Y Z [ j k m n o  0 1 2 ? @ B C e ɪ࢖yɪgɪ#jh&ht9UmH sH #jh&ht9UmH sH ht9ht9mH sH ht9ht9H*mH sH h,[*mH sH h&ht90JmH sH #jh&ht9UmH sH jht9UmH sH ht9h"mH sH ht9mH sH ht9hSNmH sH ht9hSNH*mH sH &e f g t u w x        d e m t v w ̨̺̖̎wohw]ht9ht9mH sH  ha *h-Rh-RmH sH ht9hSNmH sH ht9ht9H*mH sH h,[*mH sH #jh&ht9UmH sH #j3h&ht9UmH sH #j\h&ht9UmH sH ht9mH sH h&ht90JmH sH jht9UmH sH #jh&ht9UmH sH $   4 E K -056]_u¶tiiiiihSxh-mH sH h)h)6mH sH h)mH sH hSxh}mH sH h~%mH sH hWaZmH sH hSxh.mH sH h mH sH ht9h.5mH sH ht9hSNmH sH h&ht90JmH sH jht9UmH sH #jh&ht9UmH sH ht9mH sH $ DEMQYZ[iOcu|}1BDEHfmļļĴפ״ללhSxh mH sH hPc*hk6mH sH hmH sH hWaZmH sH h~%mH sH hkmH sH h*mH sH hSxhOmH sH h)mH sH hPc*mH sH hSxh-mH sH hSxhpmH sH hSxhN~(eij$Riqu01070׿⿯UhmH sH h!#mH sH hZj&mH sH hBmH sH hPtmH sH hPthPtmH sH hWaZmH sH hPthwmH sH hPthOmH sH Dilar pattern of protein expression for both sexes, with upregulation of both transporters at low but downregulation of only Oat1 at high OTA doses, 3) the upregulation of Oat1 and Oat3 proteins seems to be mRNA-unrelated and post-transcriptional, 4) previously observed inhibition of the renal PAH transport/secretion in rodents treated with high OTA doses may be explained by the loss of cell plasma membrane (transporting surface) and Oat1 protein in this membrane, and 5) upregulated Oat1 and Oat3 proteins at low OTA doses may possibly enhance OTA uptake and accelerate the development of nephrotoxicity. 70A0`000000*141H1111111222@2`2a2b2hSxh HmH sH h~mH sH h HmH sH hSxh~mH sH h!#mH sH hWaZmH sH hmH sH 6&P 1h:p8,. A!"#$% DyK vzlender@imi.hryK Fmailto:vzlender@imi.hryX;H,]ą'cDyK dbreljak@imi.hryK Fmailto:dbreljak@imi.hryX;H,]ą'cDyK mljubo@imi.hryK Bmailto:mljubo@imi.hryX;H,]ą'cDyK dbalen@imi.hryK Bmailto:dbalen@imi.hryX;H,]ą'cDyK hbrzica@imi.hryK Dmailto:hbrzica@imi.hryX;H,]ą'cDyK rfuchs@imi.hryK Bmailto:rfuchs@imi.hryX;H,]ą'cDyK sabolic@imi.hryK Dmailto:sabolic@imi.hryX;H,]ą'cDyK anzai@kyorin-u.ac.jpyK Pmailto:anzai@kyorin-u.ac.jpyX;H,]ą'c@@@ NormalCJ_HaJmHsHtHDA@D Default Paragraph FontRiR  Table Normal4 l4a (k(No List6U@6 t9 Hyperlink >*B*phb6IJmnad00000000 000M90 e 70b2b2b24Zj  1?BftwvbXXXXXXXXK:"L,8#Md?#ND#__oodddttd9*urn:schemas-microsoft-com:office:smarttagsplace8*urn:schemas-microsoft-com:office:smarttagsCityB*urn:schemas-microsoft-com:office:smarttagscountry-region JOZalr9?em-7[e        & 2 N P T W 8DesQ_dd3ddKorisnik65BPtSx lL!!#Zj&E)*,[*Pc*8,+/OARoot Entry FbV3@Data 1Table$WordDocument86SummaryInformation(,DocumentSummaryInformation84CompObjj  FMicrosoft Word Document MSWordDocWord.Document.89q