Up-regulation of Mrp4 expression in kidney of Mrp2-deficient TR− rats
Introduction
Multidrug resistance-associated proteins (Mrps) are efflux transporters for many structurally diverse amphipathic chemicals and organic anions, including glucuronide conjugates, glutathione S-conjugates, as well as a variety of anticancer drugs (i.e., anthracyclines, vinca alkaloids, and methotrexate) [1]. The human MRP family is currently composed of nine members, MRP1–9. Several Mrps participate in the hepatic efflux transport process. Mrp2 is localized to the canalicular membrane domain of hepatocytes, where it transports organic anion compounds into bile. Deficiency of Mrp2 protein in transport-deficient (TR−) rats, as well as in Dubin–Johnson syndrome patients, causes chronic conjugated hyperbilirubinemia [2], [3]. Mrp1, Mrp3, and Mrp4 are localized to the basolateral membrane of hepatocytes. However, under normal physiological conditions, these three basolateral transporters are expressed at relatively low levels in liver of rats [1], [4]. Hepatic expression of Mrp3 is increased in the Mrp2-deficient TR− rats [5], as well as in bile-duct ligated rats in which a significant decrease in Mrp2 expression is observed [6], [7]. The inducible nature of rat Mrp3 suggests that in liver, Mrp3 is important in the efflux transport of organic anions under conditions where normal biliary excretory functions are altered.
Mrps are also expressed in kidney, another organ essential for the maintenance of body homeostasis. Both rat and human MRP2 have been shown to localize to the apical (brush-border) membrane of proximal tubular cells, where it may function in the efflux transport of organic anions across the luminal membrane [8], [9]. Despite its low abundance in liver, rat Mrp4 is highly expressed in kidney [4]. Mrp4 sub-cellular localization is unique among the Mrp family of transporters. In renal proximal tubular cells, Mrp4 is found to be localized to the apical membrane domain [10], [11], whereas in hepatocytes, Mrp4 is routed to the basolateral membrane domain [11]. The substrate profile of Mrp4 is also unique in that besides transporting glucuronide- and glutathione S-conjugates [10], Mrp4 also transports cyclic nucleotides and nucleotide analogues [12], [13], [14], which have not been shown to be transported by Mrp1, Mrp2, and Mrp3.
The Mrp2-deficient TR− rats are commonly used to examine the role of Mrp2 in the efflux transport of compounds into bile. For example, the findings that organic anions such as cysteinyl leukotrienes [15], acetaminophen–glutathione conjugate [16], reduced and oxidized glutathione [17] are almost absent in bile from TR− rats strongly indicate that Mrp2 is the predominant transporter in liver that mediates hepatobiliary transport of these compounds. Interestingly, impaired biliary excretion of cysteinyl leukotrienes [15], [18] and acetaminophen–glucuronide conjugate [16] in TR− rats is associated with increased urinary excretion of these compounds. One possible explanation for these finding is that the loss of Mrp2 function may be compensated by (an)other organic anion transporter(s). In light of the fact that Mrp2 and Mrp4 have overlapping substrate profiles, and are co-localized to the apical membrane domain of proximal tubules, we hypothesize that the loss of Mrp2 in TR− rats is associated with up-regulation of renal expression of Mrp4, which explains why the capacity for the urinary excretion of some organic anions is retained in TR− rats. To test this hypothesis, we compared Mrp4 expression in kidney and liver from normal Wistar and TR− rats at both mRNA and protein levels. Mrp4 mRNA levels were quantified using the high-throughput Quantigene® branched DNA (bDNA) signal amplification assay. Mrp4 protein expression was determined by Western blot and immunohistochemical analysis. Hepatic and renal expression of Mrp3 is elevated not only in Mrp2-deficient mutant rats with chronic conjugated hyperbilirubinemia [19], but also in UDP-glucuronosyltransferase (UGT) 1A-deficient Gunn rats with unconjugated hyperbilirubinemia [7], [20]. Therefore, we also compared Mrp4 expression between normal Wistar and Gunn rats. In vitro transport studies showed that Mrp4 transports cAMP [12], [13], [14]. To determine whether changes in Mrp4 expression in renal proximal tubular cells of Mrp2-deficient TR− rats alter urinary excretion of endogenous cAMP, cAMP levels in urine and kidney tissue extracts from normal Wistar and TR− rats were compared.
Section snippets
Materials
The anti-human MRP4 polyclonal antibody was obtained from Alexis Biochemicals (Lausen, Switzerland). Vectastain Elite ABC kit for rat IgG and Avidin/Biotin blocking kit were purchased from Vector Laboratories (Burlingame, CA, USA). HistoMark Black Substrate system was purchased from Kierkegaard and Perry Laboratories (Gaithersburg, MD, USA). Donkey anti-rabbit horseradish peroxidase-linked antibodies were from Amersham Life Science (Arlington Heights, IL, USA). A rat Mrp4 peptide (20 amino acid
Mrp4 mRNA expression in Mrp2-deficient TR− rats
As shown in Fig. 1, mRNA of Mrp4 was much more abundant in kidney than in liver. Mrp4 expression in both kidney and liver of Mrp2-deficient TR− rats was about twice that of Wistar rats.
Western blot analysis of Mrp4 protein expression in Mrp2-deficient TR− rats
To determine whether the differences in Mrp4 mRNA expression translate into corresponding changes in Mrp4 protein, membrane fractions were prepared from kidney and liver of both strains of rats and Western blot analysis was performed using polyclonal anti-human MRP4 antibody as primary antibody. The peptide
Discussion
The data from the present study demonstrate that renal Mrp4 expression is significantly increased in Mrp2-deficient TR− rats compared to normal Wistar rats (Fig. 1, Fig. 2). Our immunohistochemical analysis of Mrp4 expression (Fig. 3) shows that (1) immunostaining is localized to the apical membrane domain of proximal tubules, and (2) staining intensity in kidney of TR− rats is higher than that in normal Wistar rats. These results strongly suggest that Mrp4 functions as an efflux transporter
Acknowledgement
This work was funded by grants from National Institute of Health (ES-09716 and ES-07079).
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