Inorganic anions and the renal organic cation transport system

During renal secretion, organic cations (OC) have to pass two hydrophobic membranes (basolateral and luminal) and the intervening aqueous cytoplasm. Furthermore, an uptake in intracellular endosomes may also occur. OC transport critically depends on the presence or absence of certain inorganic anions, such as Cl-, HCO3-, and others. The interaction between inorganic anions and OC may occur during the transport across the membranes or uptake by endosomes, by alterations of the transport protein or the substrate and by changes of the intracellular pH.     

Functional expression of renal organic anion transport in Xenopus laevis oocytes

Secretion of organic anions by the kidney plays a critical role in the elimination of toxic agents from the body. Recent findings in isolated membranes and intact tissue have demonstrated the participation of multiple transport proteins in this process. As a first step toward molecular characterization of these proteins through expression cloning, the studies reported below demonstrate functional expression of both fumarate- and lithium-sensitive glutarate and probenecid-sensitive p-aminohippurate transport in Xenopus oocytes injected with rat kidney poly(A)⁺RNA. Maximal increase in substrate uptake over buffer-injected controls was reached by 5 days after mRNA injection. Expression of size-fractionated mRNA indicated that the active species with respect to both transport activities were in the range of 1.8 to 3.5 kb.     

Some major transport mechanisms of insect absorptive epithelia

1. After hormonal stimulation, fluid reabsorption (JV) in locust hindgut from the KCl-rich, low-Na primary urine is driven primarily by an unusual mucosal electrogenic Cl- pump (JCl). 2. Cyclic-AMP increases JCl and also mucosal K+ and basolateral Cl- conductances, so that KCl absorption exceeds that of Na+ in rectum but not ileum. 3. Mucosal entry of Na+ occurs by exchange for NH4+ (H+), by cotransport with some neutral amino acids, and through putative channels. 4. However, transport of proline, the predominant organic substrate, is largely Na-independent and drives a sizable component of Jv in rectal but not ileal segments. 5. There is evidence for hormonal control of Na+ reabsorption in ileum but not rectum.     

Transepithelial organic anion transport by shark choroid plexus

Spiny dogfish shark (Squalus acanthias) lateral and IV choroid plexuses (CPs) are ultrastructurally similar to the corresponding tissues of rat. However, shark IV CP is proportionally larger and easily accessible. Moreover, this epithelial sheet can be halved and studied in Ussing flux chambers. We have used confocal fluorescence microscopy and radiotracer techniques to characterize transepithelial transport of the organic anions (OAs) fluorescein (FL) and 2,4-dichlorophenoxyacetic acid (2,4-D), respectively, by shark CP. Lateral and IV CP accumulated 1 microM FL, with highest levels in the underlying extracellular spaces, intermediate levels in epithelial cells, and lowest levels in the medium. 2,4-D and probenecid inhibited FL accumulation in cells and extracellular spaces, suggesting that these substrates compete for common carriers. Unidirectional absorptive [cerebrospinal fluid (CSF)-to-blood] and secretory (blood-to-CSF) fluxes of 10 microM [(14)C]2,4-D were measured under short-circuited conditions in IV CP mounted in Ussing chambers. 2,4-D underwent net absorption, with an average flux ratio of 7. Probenecid, 2,4,5-trichlorophenoxyacetic acid, and 5-hydroxyindolacetic acid reduced net absorption, reversibly inhibiting unidirectional absorption, with no effect on secretion. Ouabain irreversibly reduced net 2,4-D absorption and cellular and extracellular accumulation of FL, suggesting energetic coupling of OA absorption to Na(+) transport. Collectively, these data indicate that shark CP actively removes OAs from CSF by a process that is specific and active.     

Biochemical and ultrastructural correlates of substrate stimulation of renal organic anion transport.

Penicillin pretreatment enhanced the rate of PAH uptake into separated proximal tubules (collagenase digestion) from 2-week New Zealand white rabbits. A double reciprocal plot of these data suggests that penicillin increases the maximal velocity of PAH uptake. Na, K-ATPase was less in adult tissue but was unaffected by penicillin. No ultrastructural changes could be attributed to the treatment. Thus substrate stimulation of PAH transport does not involve Na, K-ATPase and probably involves soluble, rather than structural proteins.     

Lack of interaction between atrial natriuretic factor and renal organic anion transport system

The aim of the present investigation was to test the hypothesis that atrial natriuretic factor (ANF) is secreted into the proximal tubule lumen by the organic anion transport mechanism. The rationale for this hypothesis was the reported probenecid attenuation of the natriuretic effect of ANF. Probenecid is widely regarded as an inhibitor of organic acid transport in the proximal tubule. ANF was prepared in varying degrees of purity ranging from a relatively crude extract to a highly purified form. A commercially available form was also used. All forms were bioassayed using the anesthetized rat and a diuresis and natriuresis was observed in each case which was comparable to literature reports. Interaction of ANF with the organic acid transport system was evaluated using the renal cortical slice technique. Over a wide range of concentrations, there was no effect of ANF on cortical slice accumulation of either p-aminohippurate (PAH), the classical substrate of the organic anion transport system or tetraethylammonium (TEA), a typical organic cation. It is concluded that although ANF may indeed exert its effect at the luminal membranes of the nephron, access to the lumen is not mediated by the organic cation or anion transport system in the proximal tubule.     

The molecular and cellular physiology of basolateral organic anion transport in mammalian renal tubules

Basolateral transport of organic anions (OAs) into mammalian renal proximal tubule cells is a tertiary active transport process. The final step in this process involves movement of OA into the cells against its electrochemical gradient in exchange for alpha-ketoglutarate (alphaKG) moving down its electrochemical gradient. Two homologous transport proteins (OAT1 and OAT3) that function as basolateral OA/alphaKG exchangers have been cloned and sequenced. We are in the process of determining the functional distribution and regulation of OAT1 and OAT3 in renal tubules. We are using rabbit OAT1 (rbOAT1) and OAT3 (rbOAT3) expressed in heterologous cell systems to determine substrate specificity and putative regulatory steps and isolated rabbit proximal renal tubule segments to determine functional distribution and physiological regulation of these transporters within their native epithelium. Rabbit OAT1 and OAT3 differ distinctly in substrate specificity. For example, rbOAT1 has a high affinity for the classical renal OA transport substrate, p-aminohippurate (PAH), whereas rbOAT3 has no affinity for PAH. In contrast, rbOAT3 has a high affinity for estrone sulfate (ES), whereas rbOAT1 has only a very slight affinity for ES. Both rbOAT1 and rbOAT3 appear to have about the same affinity for fluorescein (FL). These differences and similarities in substrate affinities make it possible to functionally map transporters along the renal tubules. Initial data indicate that OAT1 predominates in S2 segments of the rabbit proximal tubules, but studies of other segments are just beginning. Transport of a given substrate in any tubule segment depends on both the affinity of each transporter which can accept that substrate as well as the level of expression of each of those processes in that particular tubule segment. Basolateral PAH transport (presumably OAT1 activity) appears to be down-regulated by activation of protein kinase C (PKC) and up-regulated via mitogen-activated protein kinase (MAPK) through phospholipase A(2) (PLA(2)), prostaglandin E(2) (PGE(2)), cyclic AMP, and protein kinase A (PKA) activation.     

Regulation by protein kinase C of organic anion transport driven by rat organic anion transporter 3 (rOAT3)

The organic anion transporter 3 (rOAT3) is a multispecific OAT localized at the basolateral membrane of the proximal tubule. The purpose of this study was to elucidate the role of protein kinase C (PKC) in the regulation of organic anion transport driven by rOAT3 and its mechanism of action. For this purpose, we established and utilized cells derived from the second segment of proximal tubule from mice stably expressing rOAT3 (S2 rOAT3). Phorbol 12-myristate 13-acetate (PMA), a PKC stimulator, attenuated the cellular uptake of estrone sulfate (ES), a prototype organic anion for rOAT3, in a dose- and time-dependent manner. PMA treatment resulted in a decrease in the Vmax, but not the Km of uptake of ES in S2 rOAT3. Treatment of S2 rOAT3 with other PKC stimulators or diacylglycerols also inhibited the uptake of ES, whereas that with an inactive phorbol ester did not. Chelerythrine chloride, a PKC inhibitor, reversed the PMA-induced decrease in uptake of ES in S2 rOAT3. These results suggest that PKC activation downregulates rOAT3-mediated organic anion transport. This down-regulation may be due to the inhibition of translocation or internalization of the rOAT3 protein, resulting in the decrease in the Vmax of rOAT3-mediated organic anion transport.     

Regulation of renal peripheral benzodiazepine receptors by anion transport inhibitors

The in vitro and in vivo regulation of [3H]Ro 5-4864 binding to peripheral benzodiazepine receptors (PBR) by ion transport/exchange inhibitors was studied in the kidney. The potencies of 9-anthroic acid, furosemide, bumetanide, hydrochlorothiazide and SITS as inhibitors of [3H]Ro 5-4864 binding to renal membranes were consistent with their actions as anion transport inhibitors (Ki approximately equal to 30 - 130 microM). In contrast, spironolactone, amiloride, acetazolamide, and ouabain were less potent (Ki = 100-1000 microM). Administration of furosemide to rats for five days resulted in a profound diuresis (approximately equal to 350% increase in urine volume) accompanied by a significant increase in PBR density (43%) that was apparent by the fifth day of treatment. Administration of hydrochlorothiazide or Ro 5-4864 for five days also caused diuresis and increased renal PBR density. Both the diuresis and increased density of PBR produced by Ro 5-4864 were blocked by coadministration of PK 11195, which alone had no effect on either PBR density or urine volume. The equilibrium binding constants of [3H]Ro 5-4864 to cardiac membranes were unaffected by administration of any of these drugs. These findings suggest that renal PBR may be selectively modulated in vivo and in vitro by administration of ion transport/exchange inhibitors.     

Fluid transport by airway epithelia

Airway epithelia possess transepithelial ion transport processes which may help to regulate the fluid content of airway secretions. Chloride secretion promotes fluid movement from blood to airway lumen. Active absorption of Na favours fluid movement in the opposite direction. The balance between these two processes can be altered by a number of agents which stimulate Cl secretion. The importance of ion transport for normal mucociliary clearance is suggested by the finding that airway epithelia in patients with cystic fibrosis are unable to secrete chloride. This defect may cause the characteristically sticky and tenacious mucous secretions which are the major cause of death in this disease.     

Effects of metabolic substrates and inhibitors on weak organic anion transport in rat renal proximal tubules

Stimulatory effects of intermediates of the tricarboxylic acid cycle on renal uptake of a weak organic anion, fluorescein, were studied with the aid of the method of contact microfluorimetry of individual convoluted proximal tubules ascending to the surface of the rat renal cortex slices. The study was undertaken for verifying the hypothesis that energization of renal excretion of anionic exenobiotics is mediated through their transport across the basolateral membrane in exchange for cytoplasmic alpha-ketoglutarate serving as a counter-anion. Effects of inhibitors of the tricarboxylic acid cycle such as fluoroacetate, malonate and 5-methoxyindole-2-carboxylate on the fluorescein uptake and renal gluconeogenesis in the presence of the metabolic substrates were investigated in order to outline metabolic pathways that could be responsible for elevation of the cytoplasmic alpha-ketoglutarate. Obtained data evidence that the stimulatory effects of the tricarboxylic acid cycle intermediates on the transport process under study depend on the metabolic state of the mitochondria and involve an activation of certain reactions but not the cycle as a whole. It has been suggested that an elevation of the cytoplasmic alpha-ketoglutarate resulting from this activation can be conditioned by export of isocitrate from the mitochondria with its subsequent transformation into alpha-ketoglutarate in the cytoplasm in the isocitrate dehydrogenase reaction.     

Characterization of ochratoxin A transport by human organic anion transporters.

The purpose of this study was to investigate the characteristics of ochratoxin A (OTA) transport by multispecific human organic anion transporters (hOAT1 and hOAT3, respectively) using the second segment of proximal tubule (S2) cells from mice stably expressing hOAT1 and hOAT3 (S2 hOAT1 and S2 hOAT3). S2 hOAT1 and S2 hOAT3 exhibited a time- and dose-dependent, and a saturable increase in uptake of [3H]-OTA, with apparent Km values of 0.42 microM (hOAT1) and 0.75 microM (hOAT3). These OTA uptakes were inhibited by several substrates for the OATs. Para-aminohippuric acid (PAH), probenecid, piroxicam, octanoate and citrinin inhibited [3H]-OTA uptake by hOAT1 and hOAT3 in a competitive manner (Ki = 4.29-3080 microM), with the following order of potency: probenecid > octanoate > PAH > piroxicam > citrinin for hOAT1; probenecid > piroxicam > octanoate> citrinin > PAH for hOAT3. These results indicate that hOAT1, as well as hOAT3, mediates a high-affinity transport of OTA on the basolateral side of the proximal tubule, but hOAT1- and hOAT3-mediated OTA transport are differently influenced by the substrates for the OATs. These pharmacological characteristics of hOAT1 and hOAT3 may be significantly related with the events in the development of OTA-induced nephrotoxicity in the human kidney.     

Organic acid (or anion) and organic base (or cation) transport by renal tubules of nonmammalian vertebrates

Organic acids (or anions) and organic bases (or cations) are transported by the renal tubules of nonmammalian vertebrates, but until recently the details of the transport processes have been poorly studied. Work with isolated perfused and nonperfused renal tubules and with membrane vesicles has now begun to supply information on the transepithelial transport processes and the transport steps at the individual cell membranes. The current information is reviewed for organic acids (or anions) as a general group, for urate (which generally appears to be transported by a separate system from that for other organic anions), and for organic bases (or cations) as a general group. Tentative cellular models for the transepithelial transport of each of these general categories of compounds are suggested.     

Murine renal organic anion transporters mOAT1 and mOAT3 facilitate the transport of neuroactive tryptophan metabolites

Tryptophan metabolites such as kynurenate (KYNA), xanthurenate (XA), and quinolinate are considered to have an important impact on many physiological processes, especially brain function. Many of these metabolites are secreted with the urine. Because organic anion transporters (OATs) facilitate the renal secretion of weak organic acids, we investigated whether the secretion of bioactive tryptophan metabolites is mediated by OAT1 and OAT3, two prominent members of the OAT family. Immunohistochemical analyses of the mouse kidneys revealed the expression of OAT1 to be restricted to the proximal convoluted tubule (representing S1 and S2 segments), whereas OAT3 was detected in almost all parts of the nephron, including macula densa cells. In the mouse brain, OAT1 was found to be expressed in neurons of the cortex cerebri and hippocampus as well as in the ependymal cell layer of the choroid plexus. Six tryptophan metabolites, including the bioactive substances KYNA, XA, and the serotonin metabolite 5-hydroxyindol acetate inhibited [³H]p-aminohippurate (PAH) or 6-carboxyfluorescein (6-CF) uptake by 50–85%, demonstrating that these compounds interact with OAT1 as well as with OAT3. Half-maximal inhibition of mOAT1 occurred at 34 µM KYNA and 15 µM XA, and it occurred at 8 µM KYNA and 11.5 µM XA for mOAT3. Quinolinate showed a slight but significant inhibition of [³H]PAH uptake by mOAT1 and no alteration of 6-CF uptake by mOAT3. [¹⁴C]-Glutarate (GA) uptake was examined for both transporters and demonstrated differences in the transport rate for this substrate by a factor of 4. Trans-stimulation experiments with GA revealed that KYNA and XA are substrates for mOAT1. Our results support the idea that OAT1 and OAT3 are involved in the secretion of bioactive tryptophan metabolites from the body. Consequently, they are crucial for the regulation of central nervous system tryptophan metabolite concentration. kidneys; brain; macula densa; transforming growth factor; N-methyl-D-aspartate receptor     

Inhibition of organic anion transport in endothelial cells by hydrogen peroxide.

ATP loss is a prominent feature of cellular injury induced by oxidants or ischemia. How reduction of cellular ATP levels contributes to lethal injury is still poorly understood. In this study we examined the ability of H2O2 to inhibit in a dose-dependent manner the extrusion of fluorescent organic anions from bovine pulmonary artery endothelial cells. Extrusion of fluorescent organic anions was inhibited by probenecid, suggesting an organic anion transporter was involved. In experiments in which ATP levels in endothelial cells were varied by treatment with different degrees of metabolic inhibition, it was determined that organic anion transport was ATP-dependent. H2O2-induced inhibition of organic anion transport correlated well with the oxidant's effect on cellular ATP levels. Thus H2O2-mediated inhibition of organic anion transport appears to be via depletion of ATP, a required substrate for the transport reaction. Inhibition of organic anion transport directly by probenecid or indirectly by metabolic inhibition with reduction of cellular ATP levels was correlated with similar reductions of short term viability. This supports the hypothesis that inhibition of organic anion transport after oxidant exposure or during ischemia results from depletion of ATP and may significantly contribute to cytotoxicity.     

Renal organic anion transport: a comparative and cellular perspective

A major system for net transepithelial secretion of a wide range of hydrophobic organic anions (OAs) exists in the proximal renal tubules of almost all vertebrates. This process involves transport into the cells against an electrochemical gradient at the basolateral membrane and movement from the cells into the lumen down an electrochemical gradient. Transport into the cells at the basolateral membrane, which is the dominant, rate-limiting step, is a tertiary active transport process, the final step which involves countertransport of the OA into the cells against its electrochemical gradient in exchange for alpha-ketoglutarate moving out of the cells down its electrochemical gradient. The outwardly directed gradient for alpha-ketoglutarate is maintained by metabolism ( approximately 40%) and by transport into the cells across both the basolateral and luminal membranes by separate sodium-dicarboxylate cotransporters ( approximately 60%). The inwardly directed sodium gradient driving alpha-ketoglutarate uptake is maintained by the basolateral Na(+)-K(+)-ATPase, the primary energy-requiring transport step in the total tertiary process. The basolateral OA/alpha-ketoglutarate exchange process now appears to be physiologically regulated by several factors in mammalian tubules, including peptide hormones (e.g., bradykinin) and the autonomic nervous system acting via protein kinase C (PKC) pathways and epidermal growth factor (EGF) working via the mitogen-activated protein kinase (MAPK) pathway.