Membrane Of Renal Proximal Tubules Research Articles

Sodium‐glucose cotransporter 2‐mediated oxidative stress augments advanced glycation end products‐induced tubular cell apoptosis

Ninety percent of glucose filtered by the glomerulus is reabsorbed by a sodium-glucose cotransporter 2 (SGLT2), which is expressed mainly on the apical membrane of renal proximal tubules. Because blockade of SGLT2 promotes urinary glucose excretion and thereby improves hyperglycaemia, selective inhibition of SGLT2 has been proposed as a potential therapeutic target for the treatment of patients with diabetes. Moreover, advanced glycation end products (AGEs)-receptor (RAGE) system induces apoptosis of tubular cells, thereby playing a role in diabetic nephropathy as well. However, the pathophysiological crosstalk of SGLT2 with AGEs-RAGE axis and its role in diabetic nephropathy remains unknown. This study investigated whether and how blockade of SGLT2 could prevent AGEs-elicited apoptosis of high glucose-exposed proximal tubular cells in vitro. SGLT2 was expressed in tubular cells. Tubular SGLT2 expression and glucose entry into the cells were completely blocked by the treatment with small interfering RNAs (siRNAs) raised against SGLT2. High glucose increased reactive oxygen species generation and RAGE expression levels in tubular cells, both of which were partly suppressed by SGLT2 siRNAs or an antioxidant, N-acetylcysteine. Further, high glucose was found to augment the AGEs-induced tubular cell apoptosis, which was also inhibited by SGLT2 siRNAs. Our present data suggest that SGLT2-mediated, high glucose-induced reactive oxygen species generation could augment the AGEs-induced apoptotic cell death of tubular cells via RAGE induction. SGLT2 may play some role in tubular apoptosis in diabetic nephropathy.

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), β-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-β-cyclodextrin (MβCD) 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 MβCD. 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.

Renal Expression and Function of Oat1 and Oat3 in Rats with Vascular Calcification

Background/Aims: Calcium overload in vascular smooth muscle is a highly pathogenic event, which progresses with advancing age. Old patients are polymedicated, and several pharmacotherapeutic agents circulate in the plasma as organic anions. The organic anion transporters 1 and 3 (Oat1 and Oat3) are present in renal basolateral membranes, which transport organic anions of pharmacological and physiological interest. This study was designed to evaluate the renal expression and function of Oat1 and Oat3 in rats with vascular calcification. Methods: Vascular calcification was induced by administration of a single dose of vitamin D₃ (300,000 UI/ kg b.w., i.m.) to male Wistar rats 10 days before the experiments. Oat1 and Oat3 expression was assessed by immunoblotting, immunohistochemistry and reverse-transcriptase polymerase chain reaction. The renal clearance of p-aminohippurate (PAH, a prototypical organic anion, substrate of Oat1 and Oat3) was measured by conventional clearance techniques. Results: Oat1 and Oat3 protein levels showed an increase in plasma membranes of renal proximal tubules of treated animals, where both transporters are functional. This could explain the increase observed in the renal clearance of PAH in treated rats. Conclusions: These results suggest the relevance of considering the existence of vascular calcification, which is common in ageing, when organic anion drugs are prescribed.

Interaction of human MATE1 and MATE2‐K with N‐Butylpyridinium chloride and other cationic Ionic liquids

‘Ionic Liquids’ (ILs; salts in the liquid state) are seeing increasing use in industrial applications and, consequently, are of increasing interest with respect to their potential toxicity. Three cationic ILs (NBuPy‐Cl, Bmim‐Cl and BmPy‐Cl) have been shown to be excreted in the urine of rats and mice and are inhibitors of the human ortholog of the Organic Cation Transporter, hOCT2, which is the entry step for secretion of type I OCs across the basolateral membrane of renal proximal tubule (RPT) cells. In the present study we assessed the interaction of these model ILs with the human Multidrug and Toxin Extrusion transporters, hMATE1 and hMATE2‐K, which mediate the apical exit of OCs from RPT cells into the filtrate. The uptake of [3H]1‐methyl‐4‐phenylpyridinium ([3H]MPP) and [3H]triethylmethylammonium ([3H]TEMA) into Chinese hamster ovary CHO cells that stably expressed hMATE1 or hMATE2‐K was inhibited by the model ILs with comparatively high affinity (IC50 values from 3 to 70 μM). In addition, all three ILs trans‐stimulated MATE's mediated [3H]MPP efflux, supporting the conclusion that these model ILs are transported substrates as well as inhibitors of the human MATE homologs. Together, these data suggest that cationic ILs are secreted by the human kidney through the OCT2‐MATE1/2‐K pathway, and that inhibition of MATE transporters by ILs may influence the pharmacokinetics of (at least) some cationic drugs.

The different mode of organic anion transport in NPT1/SLC17A1 and NPT4/SLC17A3

Voltage‐driven organic anion transporter has been hypothesized at the apical side of renal proximal tubule based on the classical membrane vesicle study. We first clarified its molecular identity (OATv1) in pig kidney, and searched its human ortholog. Human NPT1 (SLC17A1) has the highest identity (63%) to amino acid sequence of pig OATv1, but does not mediate voltage‐driven transport. Recently, we identified NPT4 (SLC17A3) as a human voltage‐driven organic anion transporter in kidney. To further clarify the difference of NPT1 and NPT4, we injected their cRNAs into Xenopus oocytes and compared transport properties. NPT1 showed broader substrate recognition for organic anions than NPT4. Estrone sulfate is a new substrate of NPT1 similar to NPT4. The major transport modes are different: Cl− exchange for NPT1 and voltage‐driven for NPT4. Both modes are observed in pig OATv1‐mediated transport. Deletion of PDZ‐motif at C‐terminus of NPT1, not in NPT4, abolished its transport function, suggesting that PDZ‐domain containing protein may regulate NPT1 function. The co‐localization of NPT1 and NPT4 at apical membrane of renal proximal tubules may be useful for the efficient organic anion secretion in kidney.

Renal Tubular Secretion of Varenicline by Multidrug and Toxin Extrusion (MATE) Transporters

Multidrug and toxin extrusion (MATE) 1 and MATE2-K, H(+)/organic cation antiporters, are located at the brush-border membrane of renal proximal tubules. The present study aimed to clarify the role of MATE transporters in tubular secretion of varenicline. Varenicline at a dose of 5 mg/kg was administered to wild-type and Mate1-knockout mice via the jugular vein, and its uptake was measured by high-performance liquid chromatography. The renal secretory clearance of and systemic exposure to varenicline were significantly decreased (54.6%, p < 0.05) and increased (116%, p < 0.05) respectively, by the genetic disruption of Mate1 in mice. Uptake of varenicline and [(14)C]tetraethylammonium (TEA) was examined in HEK293 cells transiently expressing the human (h) MATE1, hMATE2-K, mouse (m) MATE1, and hOCT2 basolateral organic cation transporter. [(14)C]TEA uptake in HEK293 cells expressing MATE transporters and hOCT2 was decreased in the presence of varenicline. The calculated IC(50) values for hMATE1, hMATE2-K, mMATE1, and hOCT2 were 62.2 ± 6.5, 122.3 ± 67.6, 255.0 ± 37.9, and 1,003.9 ± 135.8 (µM; mean ± S.E. for three separate experiments), respectively. Varenicline uptake was significantly increased in HEK293 cells expressing mMATE1, hMATE1, or hMATE2-K cDNA as well as hOCT2 compared to empty vector-transfected cells. In conclusion, renal MATE transporters were found to be responsible for renal tubular secretion of varenicline.

Identification and apical membrane localization of an electrogenic Na+/Ca2+exchanger NCX2a likely to be involved in renal Ca2+excretion by seawater fish

Seawater (SW) contains ∼10 mM Ca(2+), yet marine fish must drink seawater as their major water source. Thus marine teleosts fish need to excrete Ca(2+) to maintain whole body Ca(2+) homeostasis. In the intestine, seawater Ca(2+) interreacts with epithelial-secreted HCO(3)(-) by the intestinal epithelium, and the resulting CaCO(3) precipitates, which is rectally excreted. Recently the transporters involved in intestinal HCO(3)(-) secretion were identified. Ca(2+) is also excreted by the kidney, but the protein(s) involved in renal Ca(2+) excretion have not been identified. Here we identified a candidate transporter by using SW pufferfish torafugu (Takifugu rubripes) and its closely related euryhaline species mefugu (Takifugu obscurus), which are becoming useful animal models for studying molecular mechanisms of seawater adaptation. RT-PCR analyses of Na(+)/Ca(2+) exchanger (NCX) family members in various torafugu tissues demonstrated that only NCX2a is highly expressed in the kidney. Renal expression of NCX2a was markedly elevated when mefugu were transferred from freshwater to seawater. In situ hybridization and immunohistochemical analyses indicated that NCX2a is expressed in the proximal tubule at the apical membrane. NCX2a, expressed in Xenopus oocytes, conferred [Ca(2+)](out)- and Na(+)-dependent currents. These results suggest that NCX2a mediates renal Ca(2+) secretion at the apical membrane of renal proximal tubules and has an important role in whole body Ca(2+) homeostasis of marine teleosts.

Interaction of H+with the extracellular and intracellular aspects of hMATE1

Human multidrug and toxin extrusion 1 (hMATE1, SLC47A1) is a major candidate for being the molecular identity of organic cation/proton (OC/H(+)) exchange activity in the luminal membrane of renal proximal tubules. Although physiological function of hMATE1 supports luminal OC efflux, the kinetics of hMATE1-mediated OC transport have typically been characterized through measurement of uptake, i.e., the interaction between outward-facing hMATE1 and OCs. To examine kinetics of hMATE1-mediated transport in a more physiologically relevant direction, i.e., an interaction between inward-facing hMATE1 and cytoplasmic substrates, we measured the time course of hMATE1-mediated efflux of the prototypic MATE1 substrate, [(3)H]1-methyl-4-phenylpyridinium, under a variety of intra- and extracellular pH conditions, from Chinese hamster ovary cells that stably expressed the transporter. In this study, we showed that an IC(50)/K(i) for interaction between extracellular H(+) and outward-facing hMATE1 determined from conventional uptake experiments [12.9 ± 1.23 nM (pH 7.89); n = 9] and from the efflux protocol [14.7 ± 3.45 nM (pH 7.83); n = 3] was not significantly different (P = 0.6). Furthermore, kinetics of interaction between intracellular H(+) and inward-facing hMATE1 determined using the efflux protocol revealed an IC(50) for H(+) of 11.5 nM (pH 7.91), consistent with symmetrical interactions of H(+) with the inward-facing and outward-facing aspects of hMATE1.

Near Infrared Fluorescence Imaging With Robotic Assisted Laparoscopic Partial Nephrectomy: Initial Clinical Experience for Renal Cortical Tumors

We evaluated the utility of near infrared fluorescence of intravenously injected indocyanine green in performing robotic assisted laparoscopic partial nephrectomy. In addition, we evaluated the initial performance of a novel near infrared fluorescence imaging system integrated into the da Vinci® Si Surgical System during robotic assisted laparoscopic nephrectomy. Fluorescence imaging for the da Vinci Si Surgical System was used for all cases. Indocyanine green was injected before near infrared imaging. Immediate imaging assessed the renal vasculature while delayed imaging differentiated renal cortical tumors from normal parenchyma. The intraoperative performance of near infrared fluorescence of intravenous indocyanine green was evaluated for tumor appearance relative to surrounding renal parenchyma as well as identification of the renal vasculature. A total of 11 patients underwent robotic assisted laparoscopic nephrectomy with 2 converted to robotic assisted laparoscopic radical nephrectomy. Indocyanine green injections were repeated up to a total of 5 times depending on the goal of visualization. Of the 11 patients 10 demonstrated malignancy on final pathology. Of the malignant tumors 7 were hypofluorescent and 3 were isofluorescent compared to the surrounding renal parenchyma. Near infrared fluorescence imaging delineated the vascular anatomy in all cases. All surgical margins were negative on final pathology. Intraoperative imaging of indocyanine green with near infrared fluorescence is a safe and effective method to accurately identify the renal vasculature and to differentiate renal tumors from surrounding normal parenchyma. The capacity for multimodal imaging within the surgical console further facilitates this imaging. Further study is needed to determine if this technique will help improve outcomes of robotic assisted laparoscopic nephrectomy.

Analysis of different complexes of type IIa sodium-dependent phosphate transporter in rat renal cortex using blue-native polyacrylamide gel electrophoresis

Type IIa sodium-dependent phosphate transporter (NaPi-IIa) can be localized in the apical plasma membrane of renal proximal tubule to carry out a rate-limiting step of phosphate reabsorption. For the apical localization, NaPi-IIa is required to form a macromolecular complex with some adaptor proteins such as Na(+)/H(+) exchanger regulatory factor 1 (NHERF-1) and ezrin. However, the detail of macromolecular complex containing NaPi-IIa in the apical membrane of the renal proximal tubular cells has not been clarified. In this study, we identified at least four different complexes (220, 480, 920, 1,100 kDa) containing NaPi-IIa by using blue-native polyacrylamide gel electrophoresis. Interestingly, LC-MS/MS analysis and immunoprecipitation analysis reveal that megalin is a component of larger complexes (920 and 1,100 kDa). In addition, NaPi-IIa can be heterogeneously co-localized with ezrin and megalin on the apical membrane of renal proximal tubuler cells by fluorescence microscopy analysis. These results suggest that NaPi-IIa can form some different complexes on the apical plasma membrane of renal proximal tubular cells.

Disruption of multidrug and toxin extrusion MATE1 potentiates cisplatin-induced nephrotoxicity

Multidrug and toxin extrusion 1 (MATE1/SLC47A1) is expressed in the brush-border membrane of renal proximal tubules and mediates the efflux of cationic drugs. In the present study, the role of MATE1 in the nephrotoxicity of cisplatin was investigated in vivo and in vitro. Cisplatin (15 mg/kg) was administered intraperitoneally to wild-type (Mate1(+/+)) and Mate1 knockout (Mate1(−/−)) mice. Lifespan was significantly shorter in Mate1(−/−) mice than Mate1(+/+) mice. Three days after the administration of cisplatin, plasma creatinine and blood urea nitrogen (BUN) levels were increased in both Mate1(+/+) and Mate1(−/−) mice compared with vehicle-treated controls, and creatinine clearance was decreased. Moreover, a significant rise in creatinine and BUN levels was observed in cisplatin-treated Mate1(−/−) mice in comparison to Mate1(+/+) mice. A pharmacokinetic analysis revealed the plasma concentration and renal accumulation of cisplatin to be higher in Mate1(−/−) mice than Mate1(+/+) mice 1 h after a single intravenous administration of cisplatin (0.5 mg/kg). Furthermore, the combination of a selective MATE inhibitor, pyrimethamine, with cisplatin also elevated creatinine and BUN levels compared to cisplatin alone. In experiments in vitro, the cellular uptake of cisplatin was stimulated by the expression of mouse MATE1 as well as organic cation transporters OCT1 and OCT2. In conclusion, MATE1 mediates the efflux of cisplatin and is involved in cisplatin-induced nephrotoxicity.

NaPi-IIa interacting proteins and regulation of renal reabsorption of phosphate

Control of phosphate (P(i)) homeostasis is essential for many biologic functions and inappropriate low levels of P(i) in plasma have been suggested to associate with several pathological states, including renal stone formation and stone recurrence. P(i) homeostasis is achieved mainly by adjusting the renal reabsorption of P(i) to the body's requirements. This task is performed to a major extent by the Na/Pi cotransporter NaPi-IIa that is specifically expressed in the brush border membrane of renal proximal tubules. While the presence of tight junctions in epithelial cells prevents the diffusion and mixing of the apical and basolateral components, the location of a protein within a particular membrane subdomain (i.e., the presence of NaPi-IIa at the tip of the apical microvilli) often requires its association with scaffolding elements which directly or indirectly connect the protein with the underlying cellular cytoskeleton. NaPi-IIa interacts with the four members of the Na(+)/H(+) exchanger regulatory factor family as well as with the GABA(A)-receptor associated protein . Here we will discuss the most relevant findings regarding the role of these proteins on the expression and regulation of the cotransporter, as well as the impact that their absence has in P(i) homeostasis.

Influence of [H+] on MPP efflux from CHO cells expressing hMATE1

The human multidrug and toxin extrusion protein, hMATE1, is a key transporter mediating organic cation (OC)/hydrogen ion (H+) exchange at the luminal membrane of renal proximal tubule (RPT) cells. Physiologically, hMATE1 secretes OCs from RPT cells into the filtrate. However, kinetic studies of hMATE1 have largely been confined to measurement of OC uptake into hMATE1‐expressing cells. To characterize transport activity of hMATE1 in its native mode, we developed a method to study efflux of 1‐methy‐4‐phenylpyrinidium (MPP) in CHO cells expressing hMATE1. After loading cells with [3H]MPP in pH 8.5 buffer, time course of washout was determined as a function of [H+] in the washout buffer. Efflux involved at least two phases, a fast phase representing ~87% of the accumulated MPP, and a very slow phase that could be subtracted from the first for analysis. Rate constant for MPP efflux of the fast phase (presumably from the cytoplasm) increased significantly (p &lt; 0.0001) when external [H+] was raised from 3.16 nM (pH 8.5; K=0.22 min−1) to 40 nM (pH 7.4; K=0.51 min−1). Half‐maximal MPP efflux occurred at an extracellular [H+] of 4.42 nM (pH 8.35), a concentration not significantly different from the IC50 for inhibition of MPP uptake produced by extracellular [H+] (7.26 nM; pH 8.14). This method offers the promise of measuring the kinetics and selectivity of substrate interaction at the cytoplasmic face of hMATE1. (NIH R56DK080801)

Urate Transport Across the Apical Membrane of Renal Proximal Tubules

Since the molecular cloning of the renal apical urate/anion exchanger URAT1 (SLC22A12), several membrane proteins relevant to urate transport have been identified. In addition, the identification of PDZ (PSD-95, DglA, and ZO-1) domain protein PDZK1 as a binding partner of URAT1, and the emerging role of PDZ scaffold for renal apical transporters have led to a new concept of renal urate transport: urate-transporting multimolecular complex, or “urate transportsome,” that may form an ultimate functional unit at the apical membrane of renal proximal tubules. Elucidation of urate transportsome will lead to the new drug development for hyperuricemia.

A humanized mouse model for the reduced folate carrier

The ubiquitously expressed reduced folate carrier (RFC) or SLC19A1 is recognized to be an essential transport system for folates in mammalian cells and tissues. In addition to its generalized role as a folate transporter, RFC provides specialized tissue functions including absorption across intestinal/colonic epithelia, transport across the basolateral membrane of renal proximal tubules, transplacental transport of folates, and folate transport across the blood–brain barrier. The human RFC (hRFC) gene is regulated by five major upstream non-coding regions (designated A1/A2, A, B, C, and D), each transcribed from a unique promoter. Altogether, at least 14 distinct hRFC transcripts can be envisaged in which different 5′ untranslated regions (UTRs) are fused to a common splice acceptor region (positions −1 to −49) within the first coding exon with a common 1776 bp coding sequence. The 5′ non-coding regions are characterized by alternate transcription start sites, multiple splice forms, and selective tissue distributions. Alternate 5′ UTRs impact mRNA stabilities and translation efficiencies, and result in synthesis of modified hRFC proteins translated from upstream AUGs. In this report, we describe production and characterization of transgenic mice (TghRFC1) containing a functional hRFC gene and of humanized mice in which the mRFC gene is inactivated and an active hRFC gene has been introduced. The mice appear to be healthy and to breed well. Analysis of tissue specificity of expression in both the TghRFC1 and humanized hRFC mice by real-time RT-PCR demonstrates that the hRFC gene is expressed with a specificity closely resembling that seen in human tissues. For the humanized hRFC mice, levels of B and A1/A2 5′ UTRs predominated in all mice/tissues, thus resembling results in normal human tissues. Lower levels of A and C 5′ UTRs were also detected. The availability of humanized mouse models for hRFC will permit investigators to address critical unanswered questions pertinent to human health and disease. These include the ability to analyze the hRFC gene in vivo, to control dietary and other environmental conditions that may impact levels of gene expression, and to control the genetics of the mice in order to assess the effects of hRFC gene alterations on tissue folate uptake and distribution, none of which can be easily achieved in human populations.

Associations between renal sodium‐citrate cotransporter (hNaDC‐1) gene polymorphism and urinary citrate excretion in recurrent renal calcium stone formers and normal controls

Urinary citrate is a potent inhibitor of renal stone formation. Its excretion is regulated by Na(+)/dicarboxylate cotransporter-1 (NaDC-1), which is expressed on the apical membrane of renal proximal tubules. Many patients with calcium urolithiasis exhibit hypocitraturia, however, the mechanisms are not perfectly understood. We examined whether or not the I550V polymorphism in human NaDC-1 gene (hNaDC-1) influenced urinary citrate excretion. I550V polymorphism was investigated in 105 patients with recurrent renal calcium stone formation (RSF) and 107 age-matched healthy volunteers with non-renal stone formation (NSF), using polymerase chain reaction (PCR) restriction fragment length polymorphism analysis and two 24-h urine samples. Overall and in the RSF groups, subjects with a BB (homozygous for the digested Bcl-I allele) genotype exhibited a significantly lower urinary citrate excretion level than subjects with a bb (homozygous for the undigested allele) genotype. Genotype distributions between subjects with hypocitraturia and normocitraturia were significantly different, with the BB genotype being more frequently observed in subjects with hypocitraturia - both overall and in each of the RSF and NSF groups. Although the BB genotype was observed more frequently in the RSF group than in the NSF group, no statistical differences among the distributions of the three genotypes (BB, Bb [heterozygous] and bb) were observed between the RSF and NSF groups. These results suggest that the B allele of I550V polymorphism of hNaDC-1 may be associated with a reduction in urinary citrate excretion and contribute to hypocitraturia in recurrent renal stone formers.

Regulation of organic anion transporter 3 (OAT3) expression and function by membrane lipid raft‐associated proteins and cytoskeletons

In humans and rodents, two organic anion transporters (OATs), OATs 1 and 3, are expressed on the basolateral membrane of renal proximal tubules and mediate the secretion of exogenous or endogenous anions. Regulation of OAT1 and 3 has been reported in intact renal epithelia. For example, PKC activation inhibits organic anion uptake and EGF stimulates organic anion uptake mediated by OAT1 or OAT3. However, molecular mechanisms that regulate OAT expression and function are poorly understood. In the present study, plasma membrane lipid raft domains (LRDs) involved in protein trafficking and control of membrane processes were isolated from rat renal cortex and from HEK-293, human kidney epithelial cell line. Results indicate LRDs from rat renal cortical tissues contain caveolin 1, lipid-associated protein, and myosin – components previously implicated in protein sorting, trafficking, and cell signaling cascades. Similarly, LRDs from HEK-293 cells stably expressing human OAT3 (hOAT3) show co-localization of myosin and caveolin 1 with hOAT3. Cytoskeletal disruptors also alter hOAT3 expression and transport in HEK-293 cells, while a cholesterol depleting agent reduces OAT3 transport in rat renal slices. Thus, OAT3 expression and function may be regulated by LRDs and cytoskeletons. This research was supported by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences.

Modulation of Renal Apical Organic Anion Transporter 4 Function by Two PDZ Domain–Containing Proteins

Human organic anion transporter 4 (OAT4) is an apical organic anion/dicarboxylate exchanger in the renal proximal tubules and mediates high-affinity transport of steroid sulfates such as estrone-3-sulfate (E1S) and dehydroepiandrosterone sulfate. Here, two multivalent PDZ (PSD-95/Discs Large/ZO-1) proteins PDZK1 and NHERF1 were examined as interactors of OAT4 by a yeast two-hybrid assay. These interactions require the extreme C-terminal region of OAT4 and the first and fourth PDZ domains of PDZK1 and the first PDZ domain of NHERF1. These interactions were confirmed by surface plasmon resonance assays (K(D): 36 nM, 1.2 microM, and 41.7 microM, respectively). In vitro binding assays and co-immunoprecipitation studies revealed that the OAT4 wild-type but not a mutant lacking the PDZ motif interacted directly with both PDZK1 and NHERF1. OAT4, PDZK1, and NHERF1 proteins were shown to be localized at the apical membrane of renal proximal tubules. The association with PDZK1 or NHERF1 enhanced OAT4-mediated E1S transport activities in HEK293 cells (1.2- to 1.4-fold), and the deletion of the OAT4 C-terminal PDZ motif abolished this effect. The augmentation of the transport activity was accompanied by alteration in V(max) of E(1)S transport via OAT4 and was associated with the increased surface expression level of OAT4 protein. This study indicates that the functional activity of OAT4 is modulated through the PDZ interaction with the network of PDZK1 and NHERF1 and suggests that OAT4 is involved in the regulated apical organic anion handling in the renal proximal tubules, provided by the PDZ scaffold.

Functional Characterization of Rat Organic Anion Transporter 5 (Slc22a19) at the Apical Membrane of Renal Proximal Tubules

A novel member of the organic anion transporter (OAT) family, Oat5 (Slc22a19), has been reported to transport a naturally occurring mycotoxin, ochratoxin A (OTA). However, neither its endogenous substrate and driving force nor physiological functions have been determined. Herein, we report the functional characterization of rat Oat5 (rOat5), as well as its intrarenal distribution and membrane localization. When expressed in Xenopus laevis oocytes, rOat5 mediated the transport of sulfate conjugates of steroids such as estrone-3-sulfate (E(1)S; K(m) = 18.9 +/- 3.9 microM) and dehydroepiandrosterone sulfate (K(m) = 2.3 +/- 0.2 microM) in a sodium-independent manner, in addition to OTA. The rOat5-mediated E(1)S transport was strongly inhibited by four-carbon (C4) dicarboxylate succinate and longer dicarboxylates (C7-C9). The uptake of [(3)H]E(1)S via rOat5 was significantly trans-stimulated by succinate, and the efflux of [(14)C]succinate was significantly trans-stimulated by E(1)S. A similar trans-stimulatory effect of preloaded succinate on E(1)S uptake was also detected in cells stably expressing rOat5 (S(2) rOat5). rOat5 interacted with chemically heterogenous anionic compounds. The rOat5-mediated E(1)S transport was inhibited by several sulfate conjugates, such as 4-methylumbelliferyl sulfate and beta-estradiol sulfate, but not by glucuronide conjugates. An immunohistochemical study showed that rOat5 was localized at the apical membrane of renal proximal tubules in the corticomedullary region. rOat5 mRNA was expressed in the late segments (S(2) and S(3)) of proximal tubules. These results indicate that rOat5 is renal organic anion/dicarboxylates exchanger and, under physiological conditions, may function as an apical reabsorptive pathway for organic anions in proximal tubules driven by an outward gradient of dicarboxylates.

Shank2E binds NaPi cotransporter at the apical membrane of proximal tubule cells

Proteins expressing postsynaptic density (PSD)-95/Drosophila disk large (Dlg)/zonula occludens-1 (ZO-1) (PDZ) domains are commonly involved in moderating receptor, channel, and transporter activities at the plasma membrane in a variety of cell types. At the apical membrane of renal proximal tubules (PT), the type IIa NaP(i) cotransporter (NaP(i)-IIa) binds specific PDZ domain proteins. Shank2E is a spliceoform of a family of PDZ proteins that is concentrated at the apical domain of liver and pancreatic epithelial cell types and is expressed in kidney. In the present study, immunoblotting of enriched plasma membrane fractions and immunohistology found Shank2E concentrated at the brush border membrane of rat PT cells. Confocal localization of Flag-Shank2E and enhanced green fluorescent protein-NaP(i)-IIa in cotransfected OK cells showed these proteins colocalized in the apical microvilli of this PT cell model. Shank2E co-immunoprecipitated with NaP(i)-IIa from rat renal cortex tissue and HA-NaP(i)-IIa coprecipitated with Flag-Shank2E in cotransfected human embryonic kidney HEK cells. Domain analysis showed that the PDZ domain of Shank2E specifically bound NaP(i)-IIa and truncation of the COOH-terminal TRL motif from NaP(i)-IIa abolished this binding, and Far Western blotting showed that the Shank2E- NaP(i)-IIa interaction occurred directly between the two proteins. NaP(i)-IIa activity is regulated by moderating its abundance in the apical membrane. High-P(i) conditions induce NaP(i)-IIa internalization and degradation. In both rat kidney PT cells and OK cells, shifting to high-P(i) conditions induced an acute internal redistribution of Shank2E and, in OK cells, a significant degree of degradation. In sum, Shank2E is concentrated in the apical domain of renal PT cells, specifically binds NaP(i)-IIa via PDZ interactions, and undergoes P(i)-induced internalization.