Impaired Renal Secretion of Substrates for the Multidrug Resistance Protein 2 in Mutant Transport–Deficient (TR−) Rats

Abstract

Abstract. Previous studies with mutant transport-deficient rats (TR Ϫ ), in which the multidrug resistance protein 2 (Mrp2) is lacking, have emphasized the importance of this transport protein in the biliary excretion of a wide variety of glutathione conjugates, glucuronides, and other organic anions. Mrp2 is also present in the luminal membrane of proximal tubule cells of the kidney, but little information is available on its role in the renal excretion of xenobiotics. The authors compared renal transport of the fluorescent Mrp2 substrates calcein, fluo-3, and lucifer yellow (LY) between perfused kidneys isolated from Wistar Hannover (WH) and TR Ϫ rats. Isolated rat kidneys were perfused with 100 nM of the nonfluorescent calcein-AM or 500 nM fluo3-AM, which enter the tubular cells by diffusion and are hydrolyzed intracellularly into the fluorescent anion. The urinary excretion rates of calcein and fluo-3 were 3 to 4 times lower in perfused kidneys from TR Ϫ rats compared with WH rats. In contrast, the renal excretion of LY (10 M, free anion) was somewhat delayed but appeared unimpaired in TR Ϫ rats. Membrane vesicles from Sf9 cells expressing human MRP2 or human MRP4 indicated that MRP2 exhibits a preferential affinity for calcein and fluo-3, whereas LY is a better substrate for MRP4. We conclude that the renal clearance of the Mrp2 substrates calcein and fluo-3 is significantly reduced in TR Ϫ rat; for LY, the absence of the transporter may be compensated for by (an)other organic anion transporter(s).ATP-binding cassette (ABC) proteins play an important role in the cellular defense against xenobiotics and their metabolites by active secretion of these compounds into bile, urine, and intestinal lumen. Overexpression of the ATP-driven transporters in tumor cells is associated with multidrug resistance, which is a major complication for successful cancer chemotherapy. The proteins couple ATP hydrolysis to the transport of specific substrates and include P-glycoprotein, which transports uncharged compounds and organic cations, and the multidrug resistance proteins (MRP), with a preference for organic anions (for reviews, see references 1-3). The canalicular membrane of hepatocytes contains an isoform of MRP called MRP2 or canalicular multispecific organic anion transporter (cMOAT) (4). In mutant transport-deficient (TR Ϫ

Increased Apical Insertion of the Multidrug Resistance Protein 2 (MRP2/ABCC2) in Renal Proximal Tubules following Gentamicin Exposure

“…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 syndrome, partly due to an impaired canalicular secretion of glutathione, glutathione conjugates, and bilirubin glucuronides (Paulusma et al, 1997;Smitherman et al, 2004). We found previously that the renal excretion capacity for a number of known Mrp2 substrates was decreased as well in an Mrp2-deficient rat (Masereeuw et al, 2003).…”

“…These injections took place under anesthesia via a nose cone (5% isoflurane; Isoflo, Abbott Laboratories, Abbott Park, IL). Subsequently, rat kidneys were isolated and perfused as described in detail previously (Masereeuw et al, 2003). Based on the findings in our previous study, calcein-AM was used as a source for the fluorescent substrate calcein.…”

Contribution of Multidrug Resistance Protein 2 (MRP2/ABCC2) to the Renal Excretion of p-aminohippurate (PAH) and Identification of MRP4 (ABCC4) as a Novel PAH Transporter

“…However, renal function seems unimpaired in TR Ϫ rats, which may be explained by a relatively minor role for Mrp2 in renal physiology as a result of the presence of various other compensatory organic anion transporters. On the other hand, we recently found that renal excretion of certain exogenous bulky organic anions in TR Ϫ kidney is either reduced (calcein, fluo-3) or delayed (lucifer yellow) (26). Furthermore, under the influence of drug treatment, MRP2 function may be altered.…”

“…Rat kidneys of Wistar-Hannover (WH) and TR Ϫ rats were isolated and perfused as described in detail previously (25,26). Research was undertaken under the auspices of a protocol approved by the local university animal care and usage committee.…”

Multidrug Resistance–Associated Protein 4 Is Involved in the Urinary Excretion of Hydrochlorothiazide and Furosemide

“…Both transporters accept a variety of organic anions as substrates, such as p-aminohippurate (PAH), 17␤-estradiol-17␤-d-glucuronide, dehydroepiandrosterone sulfate (DHEAS), and methotrexate (18,(25)(26)(27). van Aubel et al (18) initially found that Mrp4 is localized on the BBM, and, subsequently, Mrp4 was suggested to play important roles in tubular secretion (18,25,28), although direct evidence of this still is lacking. For BCRP, an in vivo study using Bcrp knockout mice has demonstrated that BCRP is responsible for the urinary excretion of some organic anions, such as methotrexate and 6-hydroxy-5,7-dimethyl-2-methylamino-4-(3-pyridylmethyl) benzothiazole (E3040) sulfate (29,30).…”