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.

The reabsorptive and excretory capacity of the kidney has an important influence on the systemic concentration of drugs. Multidrug resistance proteins (MRP/ABCC) expressed in the kidney play a critical role in the tubular efflux of a wide variety of drugs and toxicants, and, in particular, of their negatively charged phase II metabolites. Nine structurally and functionally related MRP family members have been identified (MRP1-9), which differ from each other by their localization, expression levels, and substrate specificity. During altered physiological circumstances, adaptations in these transporters are required to avoid systemic toxicity as well as renal tubular damage. Key players in these events are hormones, protein kinases, nuclear receptors, and disease conditions, which all may affect transporter protein expression levels. This review discusses current knowledge on the renal characteristics of MRP1-9, with specific focus on their regulation.

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In renal proximal tubule, multidrug resistance protein 2 (Mrp2) actively transports many organic anions into urine, including drugs and metabolic wastes. Upon exposure to nephrotoxicants or during endotoxemia, both Mrp2 activity and expression are up-regulated. This may result from induced de novo synthesis of Mrp2 or post-transcriptional events involving specific signaling pathways. Here, we investigated glucocorticoid signaling to Mrp2 in killifish renal proximal tubules, a model system in which transport activity can be measured using a fluorescent substrate and confocal imaging. Exposure of tubules to dexamethasone rapidly increased Mrp2-mediated fluorescein methotrexate transport. Other glucocorticoid receptor (GR) ligands, cortisol and triamcinolone acetonide, also stimulated Mrp2-mediated transport. The GR antagonist, mifepristone 17␤-hydroxy-11␤-[4-dimethylamino phenyl]-17␣-[1-propynyl] estra-4,9-dien-3-one (RU486), abolished stimulation by all three ligands, whereas the mineralocorticoid receptor antagonist, spironolactone, was ineffective. Consistent with action through a nongenomic mechanism, dexamethasone stimulation of Mrp2-mediated transport was insensitive to cycloheximide and actinomycin D, and immunohistochemistry revealed no alterations in Mrp2 expression at the luminal membrane. (9S-(9␣,10␤,12␣))-2,3,9,10,11,12-hexahydro-10-hydroxy-10-(methoxycarbonyl)-9-methyl-9,12-epoxy-1H-diindolo[1,2,3-fg:3Ј,2Ј,1Ј-kl]pyrrolo[3,4-i][1,6]benzodiazocin-1-one (K252a), an inhibitor of the tyrosine receptor kinase subfamily, reduced the dexamethasone effect, as did the specific hepatocyte growth factor receptor (c-Met) tyrosine kinase inhibitor, -665752). Hepatocyte growth factor (HGF), an endogenous ligand for c-Met, stimulated Mrp2-mediated transport. This effect was reversed by PHA-665752 but not by RU486. Inhibition of mitogenactivated protein kinase/extracellular signal-regulated kinase kinase (MEK 1/2) also abolished the effects of dexamethasone and HGF. Our results disclose a novel mechanism by which glucocorticoids acting through GR, c-Met, and MEK1/2 cause rapid, nongenomic stimulation of Mrp2-mediated transport in renal proximal tubules. This up-regulation may be nephroprotective, enhancing efflux of metabolic wastes and toxicants during cell and tissue stress.