Regulation of renal organic anion transporter 3 (SLC22A8) expression and function by the integrity of lipid raft domains and their associated cytoskeleton.

BACKGROUND/AIMS: In humans and rodents, organic anion transporter 3 (Oat3) is highly expressed on the basolateral membrane of renal proximal tubules and mediates the secretion of exogenous and endogenous anions. Regulation of Oat3 expression and function has been observed in both expression system and intact renal epithelia. However, information on the local membrane environment of Oat3 and its role is limited. Lipid raft domains (LRD; cholesterol-rich domains of the plasma membrane) play important roles in membrane protein expression, function and targeting. In the present study, we have examined the role of LRD-rich membranes and their associated cytoskeletal proteins on Oat3 expression and function. METHODS: LRD-rich membranes were isolated from rat renal cortical tissues and from HEK-293 cells stably expressing human OAT3 (hOAT3) by differential centrifugation with triton X-100 extraction. Western blots were subsequently analyzed to determine protein expression. In addition, the effect of disruption of LRD-rich membranes was examined on functional Oat3 mediated estrone sulfate (ES) transport in rat renal cortical slices. Cytoskeleton disruptors were investigated in both hOAT3 expressing HEK-293 cells and rat renal cortical slices. RESULTS: Lipid-enriched membranes from rat renal cortical tissues and hOAT3-expressing HEK-293 cells showed co-expression of rOat3/hOAT3 and several lipid raft-associated proteins, specifically caveolin 1 (Cav1), ß-actin and myosin. Moreover, immunohistochemistry in hOAT3-expressing HEK-293 cells demonstrated that these LRD-rich proteins co-localized with hOAT3. Potassium iodide (KI), an inhibitor of protein-cytoskeletal interaction, effectively detached cytoskeleton proteins and hOAT3 from plasma membrane, leading to redistribution of hOAT3 into non-LRD-rich compartments. In addition, inhibition of cytoskeleton integrity and membrane trafficking processes significantly reduced ES uptake mediated by both human and rat Oat3. Cholesterol depletion by methyl-ß-cyclodextrin (MßCD) also led to a dose dependent reduction Oat3 expression and ES transport by rat renal cortical slices. Moreover, the up-regulation of rOat3-mediated transport seen following insulin stimulation was completely prevented by MßCD. CONCLUSION: We have demonstrated that renal Oat3 resides in LRD-rich membranes in proximity to cytoskeletal and signaling proteins. Disruption of LRD-rich membranes by cholesterol-binding agents or protein trafficking inhibitors altered Oat3 expression and regulation. These findings indicate that the integrity of LRD-rich membranes and their associated proteins are essential for Oat3 expression and function.

Exposure to hexavalent chromium resulted in significantly higher tissue chromium burden compared with trivalent chromium following similar oral doses to male F344/N rats and female B6C3F1 mice.

In National Toxicology Program 2-year studies, hexavalent chromium [Cr(VI)] administered in drinking water was clearly carcinogenic in male and female rats and mice, resulting in small intestine epithelial neoplasms in mice at a dose equivalent to or within an order of magnitude of human doses that could result from consumption of chromium-contaminated drinking water, assuming that dose scales by body weight(3/4) (body weight raised to the 3/4 power). In contrast, exposure to trivalent chromium [Cr(III)] at much higher concentrations may have been carcinogenic in male rats but was not carcinogenic in mice or female rats. As part of these studies, total chromium was measured in tissues and excreta of additional groups of male rats and female mice. These data were used to infer the uptake and distribution of Cr(VI) because Cr(VI) is reduced to Cr(III) in vivo, and no methods are available to speciate tissue chromium. Comparable external doses resulted in much higher tissue chromium concentrations following exposure to Cr(VI) compared with Cr(III), indicating that a portion of the Cr(VI) escaped gastric reduction and was distributed systemically. Linear or supralinear dose responses of total chromium in tissues were observed following exposure to Cr(VI), indicating that these exposures did not saturate gastric reduction capacity. When Cr(VI) exposure was normalized to ingested dose, chromium concentrations in the liver and glandular stomach were higher in mice, whereas kidney concentrations were higher in rats. In vitro studies demonstrated that Cr(VI), but not Cr(III), is a substrate of the sodium/sulfate cotransporter, providing a partial explanation for the greater absorption of Cr(VI).

Activation of protein kinase Czeta increases OAT1 (SLC22A6)- and OAT3 (SLC22A8)-mediated transport.

Organic anion transporters (OATs) play a pivotal role in the clearance of small organic anions by the kidney, yet little is known about how their activity is regulated. A yeast two-hybrid assay was used to identify putative OAT3-associated proteins in the kidney. Atypical protein kinase Czeta (PKCzeta) was shown to bind to OAT3. Binding was confirmed in immunoprecipitation assays. The OAT3/PKCzeta interaction was investigated in rodent renal cortical slices from fasted animals. Insulin, an upstream activator of PKCzeta, increased both OAT3-mediated uptake of estrone sulfate (ES) and PKCzeta activity. Both effects were abolished by a PKCzeta-specific pseudosubstrate inhibitor. Increased ES transport was not observed in renal slices from OAT3-null mice. Transport of the shared OAT1/OAT3 substrate, rho-aminohippurate, behaved similarly, except that stimulation was reduced, not abolished, in the OAT3-null mice. This suggested that OAT1 activity was also modified by PKCzeta, subsequently confirmed using an OAT1-specific substrate, adefovir. Inhibition of PKCzeta also blocked the increase in ES uptake seen in response to epidermal growth factor and to activation of protein kinase A. Thus, PKCzeta acted downstream of the epidermal growth factor to protein kinase A signaling pathway. Activation of transport was accompanied by an increase in V(max) and was blocked by microtubule disruption, indicating that activation may result from trafficking of OAT3 into the plasma membrane. These data demonstrate that PKCzeta activation up-regulates OAT1 and OAT3 function, and that protein-protein interactions play a central role controlling these two important renal drug transporters.

Stoichiometry of organic anion/dicarboxylate exchange in membrane vesicles from rat renal cortex and hOAT1-expressing cells.

Although membrane vesicle studies have established the driving forces that mediate renal organic anion secretion and the organic anion transporter Oat1 has now been cloned in several species, its stoichiometry has remained uncertain. In this study, we used electrophysiology, kinetic measurements, and static head experiments to determine the coupling ratio for Oat1-mediated organic anion/dicarboxylate exchange. Initial experiments demonstrated that uptake of PAH by voltage-clamped Xenopus laevis oocytes expressing rOat1 led to net entry of positive charge, suggesting that coupling was one-to-one. This conclusion was confirmed by kinetic analysis of PAH and glutarate fluxes in native basolateral membrane vesicles from the rat renal cortex, which showed a Hill coefficient of 1. Similarly, static head experiments on the rat vesicles also showed a 1:1 coupling ratio. To confirm these conclusions in a system expressing a single cloned transporter, Madin-Darby canine kidney cells were stably transfected with the human exchanger hOAT1. The hOAT1-expressing cell line showed extensive PAH transport, which was very similar in all respects to transport expressed by hOAT1 in Xenopus oocytes. Its Km for PAH was 8 microM and glutarate effectively trans-stimulated PAH transport. When stoichiometry was assessed using plasma membranes isolated from the hOAT1-expressing cells, both kinetic and static head data indicated that hOAT1 also demonstrated a 1:1 coupling between organic anion and dicarboxylate.

Organic anion transporter 3 (Slc22a8) is a dicarboxylate exchanger indirectly coupled to the Na+ gradient.

Basolateral uptake of organic anions in renal proximal tubule cells is indirectly coupled to the Na(+) gradient through Na(+)-dicarboxylate cotransport and organic anion/dicarboxylate exchange. One member of the organic anion transporter (OAT) family, Oat1, is expressed in the proximal tubule and is an organic anion/dicarboxylate exchanger. However, a second organic anion carrier, Oat3, is also highly expressed in the renal proximal tubule, but its mechanism is unclear. Thus we have assessed Oat3 function in Xenopus laevis oocytes and rat renal cortical slices. Probenecid-sensitive uptake of p-aminohippurate (PAH, an Oat1 and Oat3 substrate) and estrone sulfate (ES, an Oat3 substrate) in rat Oat3-expressing oocytes was significantly trans-stimulated by preloading the oocytes with the dicarboxylate glutarate (GA). GA stimulation of ES transport by oocytes coexpressing rabbit Na(+)-dicarboxylate cotransporter 1 and rat Oat3 was significantly inhibited when the preloading medium contained Li(+) or methylsuccinate (MS) or when Na(+) was absent. All these treatments inhibit the Na(+)-dicarboxylate cotransporter, but not rat Oat3. Li(+), MS, and Na(+) removal had no effect when applied during the ES uptake step, rather than during the GA preloading step. Concentrative ES uptake in rat renal cortical slices was also demonstrated to be probenecid and Na(+) sensitive. Accumulation of ES was stimulated by GA, and this stimulation was completely blocked by probenecid, Li(+), MS, taurocholate, and removal of Na(+). Thus Oat3 functions as an organic anion/dicarboxylate exchanger that couples organic anion uptake indirectly to the Na(+) gradient.