Increased Apical Insertion of the Multidrug Resistance Protein 2 (MRP2/ABCC2) in Renal Proximal Tubules following Gentamicin Exposure Sylvia Notenboom, Alfons C. Wouterse, Bram Peters, Leon H. Kuik, Suzanne Heemskerk, Frans G. M. Russel, and Rosalinde Masereeuw Department of Pharmacology and Toxicology, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands Received March 14, 2006; accepted June 2, 2006 ABSTRACT Multidrug resistance protein (MRP) 2 (MRP2; ABCC2), an or- ganic anion transporter apically expressed in liver, kidney, and intestine, plays an important protective role through facilitating the efflux of potentially toxic compounds. We hypothesized that upon a toxic insult, MRP2 is up-regulated in mammalian kidney, thereby protecting the tissue from damage. We studied the effects of the nephrotoxicant gentamicin on the functional ex- pression of MRP2 in transfected Madin-Darby canine kidney type II (MDCKII) cells and rat kidney. Transport of glutathione- methyl fluorescein by cells or calcein by isolated perfused rat kidney was measured to monitor MRP2 activity. MDCKII cells were exposed to gentamicin (0 –1000 M) for either 1 h, 24 h, or for 1 h followed by 24-h recovery. No effect was observed on MRP2 after 1-h exposure. After 24-h gentamicin exposure or after a 24-h recovery period following 1-h exposure, an in- crease in MRP2-mediated transport was seen. This up-regula- tion was accompanied by a 2-fold increase in MRP2 protein expression in the apical membrane, whereas the expression in total cell lysates remained unchanged. In perfused kidneys of rats exposed to gentamicin (100 mg/kg) for seven consecutive days, an increase in Mrp2 function and expression was found, which was prevented by addition of a dual endothelin-receptor antagonist, bosentan. We conclude that an increased shuttling of the transporter to the apical membrane takes place in re- sponse to gentamicin exposure, which is triggered by endothe- lin. Up-regulation of MRP2 in the kidney may be interpreted as part of a protective mechanism. The renal proximal tubule fulfills an important role in the elimination of anionic, cationic, and neutral waste products, varying from endogenous (metabolic) waste products to xe- nobiotics. One of the transporters involved in the active se- cretion of organic anions in the preurine is multidrug resis- tance protein (MRP) 2 (MRP2; ABCC2). MRP2 is located at the brush-border membrane of the proximal tubule (Schaub et al., 1997) and transports a variety of organic anionic con- jugates, amphiphilic anions, and neutral substrates (for re- view, see Van de Water et al., 2005). Low expression or absence of MRP2 causes conjugated hyperbilirubinemia and pigment disposition in the liver, as observed in patients with the autosomal recessively inherited Dubin-Johnson syn- drome, partly due to an impaired canalicular secretion of glutathione, glutathione conjugates, and bilirubin glucu- ronides (Paulusma et al., 1997; Smitherman et al., 2004). We found previously that the renal excretion capacity for a num- ber of known Mrp2 substrates was decreased as well in an Mrp2-deficient rat (Masereeuw et al., 2003). The functional expression of MRP2 may be influenced by exogenous factors, such as exposure to toxicants, cellular stress, and disease conditions. For example, cholestasis re- sults in a decreased expression of Mrp2 in the liver, whereas the expression of the transporter protein in the kidney is up-regulated (Tanaka et al., 2002). Less dramatic changes in Mrp2 were observed after exposure to the nephrotoxic anti- biotic agent gentamicin using a killfish renal model. Expo- sure to gentamicin results in a rapid reduction in Mrp2, This study was supported by the Dutch Kidney Foundation. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.106.104547. ABBREVIATIONS: MRP/Mrp, multidrug resistance protein; ABC, ATP-binding cassette; DMEM, Dulbecco’s modified Eagle’s medium; HBSS, Hanks’ balanced salt solution; AM, acetoxymethylester; CMFDA, 5-chloromethylfluorescein diacetate; MK-571, (3-([{3-(2-[7-chloro-2-quinoli- nyl]ethenyl)phenyl}-{(3-dimethyl-amino-3-oxopropyl)-thio}-methyl]thio)propanoic acid); CDNB, 1-chloro-3,4-nitrobenzene; ET, endothelin; MD- CKII, Madin-Darby canine kidney type II; wt, wild type/wild-type; OK, opossum kidney; GS-MF, glutathione-methylfluorescein; LDH, lactate dehydrogenase; WH, Wistar-Hannover; PBS, phosphate-buffered saline; NFDM, nonfat dried milk; E-64, N-(trans-epoxysuccinyl)-L-leucine 4-guanidinobutylamide; PCR, polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Gent, gentamicin; WT, wild type. 0022-3565/06/3183-1194–1202$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 318, No. 3 Copyright © 2006 by The American Society for Pharmacology and Experimental Therapeutics 104547/3132674 JPET 318:1194–1202, 2006 Printed in U.S.A. 1194 at ASPET Journals on April 30, 2016 jpet.aspetjournals.org Downloaded from