ABSTRACT:Different gene-silencing methods, like antisense and short interfering RNA (siRNA), are widely used as experimental tools to inhibit gene expression. In the present study, the in vivo behavior of siRNA in rats and siRNA-mediated silencing of genes in the renal proximal tubule were investigated. To study the biodistribution of siRNA, rats were injected i.v. with radiolabeled siRNA or radiolabel alone (control), and scintigraphic images were acquired at different time intervals postinjection. The siRNA preferentially accumulated in the kidneys and was excreted in the urine. One hour after injection, the amount of siRNA present in both kidneys (1.7 ؎ 0.3% of injected dose/g tissue) was on average 40 times higher than in other tissues (liver, brain, intestine, muscle, lung, spleen, and blood). Besides the biodistribution, the effect of siRNA on multidrug resistance protein isoform 2 (Mrp2/Abcc2, siRNA Mrp2 ) in renal proximal tubules was investigated. Mrp2 function was assessed by measuring the excretion of its fluorescent substrate calcein in the isolated perfused rat kidney. Four days after administration, siRNA Mrp2 reduced the urinary calcein excretion rate significantly (35% inhibition over the period 80-150 min of perfusion). This down-regulation was specific because another siRNA sequence directed against a different transporter in the proximal tubule, Mrp4 (Abcc4, siRNA Mrp4 ), did not alter the Mrp2-mediated excretion of calcein. In conclusion, siRNA accumulates spontaneously in the kidney after i.v. injection, where it selectively suppresses gene function in the proximal tubules. Therefore, i.v. administered siRNA provides a novel experimental and potential therapeutic tool for gene silencing in the kidney.
Abstract. p-Aminohippurate (PAH) is the classical substrate used in the characterization of organic anion transport in renal proximal tubular cells. Although basolateral transporters for PAH uptake from blood into the cell have been well characterized, there is still little knowledge on the apical urinary efflux transporters. The multidrug resistance protein 2 (MRP2/ ABCC2) is localized to the apical membrane and mediates ATP-dependent PAH transport, but its contribution to urinary PAH excretion is not known. In this report, we show that renal excretion of PAH in isolated perfused kidneys from wild-type and Mrp2-deficient (TR Ϫ ) rats is not significantly different. Uptake of [14 C]PAH in membrane vesicles expressing two different MRP2 clones isolated from Sf9 and MDCKII cells exhibited a low affinity for PAH (Sf9, 5 Ϯ 2 mM; MDCKII, 2.1 Ϯ 0.6 mM). Human MRP4 (ABCC4), which has recently been localized to the apical membrane, expressed in Sf9 cells had a much higher affinity for PAH (K m ϭ 160 Ϯ 50 M). Various inhibitors of MRP2-mediated PAH transport also inhibited MRP4. Probenecid stimulated MRP2 at low concentrations but had no effect on MRP4; but at high probenecid concentrations, both MRP2 and MRP4 were inhibited. Sulfinpyrazone only stimulated MRP2, but inhibited MRP4. Realtime PCR and Western blot analysis showed that renal cortical expression of MRP4 is approximately fivefold higher as compared with MRP2. MRP4 is a novel PAH transporter that has higher affinity for PAH and is expressed more highly in kidney than MRP2, and may therefore be more important in renal PAH excretion.
Nitric oxide (NO) is an important regulator of renal transport processes. In the present study, we investigated the role of NO, produced by inducible NO synthase (iNOS)
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
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