Apical Membrane Of Proximal Tubules Research Articles

Activation of 2-oxoglutarate receptor 1 (OXGR1) by α-ketoglutarate (αKG) does not detectably stimulate Pendrin-mediated anion exchange in Xenopus oocytes.

SLC26A4/Pendrin is the major electroneutral Cl−/HCO3 − exchanger of the apical membrane of the Type B intercalated cell (IC) of the connecting segment (CNT) and cortical collecting duct (CCD). Pendrin mediates both base secretion in response to systemic base load and Cl− reabsorption in response to systemic volume depletion, manifested as decreased nephron salt and water delivery to the distal nephron. Pendrin‐mediated Cl−/HCO3 − exchange in the apical membrane is upregulated through stimulation of the β‐IC apical membrane G protein‐coupled receptor, 2‐oxoglutarate receptor 1 (OXGR1/GPR99), by its ligand α‐ketoglutarate (αKG). αKG is both filtered by the glomerulus and lumenally secreted by proximal tubule apical membrane organic anion transporters (OATs). OXGR1‐mediated regulation of Pendrin by αKG has been documented in transgenic mice and in isolated perfused CCD. However, aspects of the OXGR1 signaling pathway have remained little investigated since its original discovery in lymphocytes. Moreover, no ex vivo cellular system has been reported in which to study the OXGR1 signaling pathway of Type B‐IC, a cell type refractory to survival in culture in its differentiated state. As Xenopus oocytes express robust heterologous Pendrin activity, we investigated OXGR1 regulation of Pendrin in oocytes. Despite functional expression of OXGR1 in oocytes, co‐expression of Pendrin and OXGR1 failed to exhibit αKG‐sensitive stimulation of Pendrin‐mediated Cl−/anion exchange under a wide range of conditions. We conclude that Xenopus oocytes lack one or more essential molecular components or physical conditions required for OXGR1 to regulate Pendrin activity.

Combination therapy of cisplatin with cilastatin enables an increased dose of cisplatin, enhancing its antitumor effect by suppression of nephrotoxicity

Cisplatin, one of the most active anticancer agents, is widely used in standard chemotherapy for various cancers. Cisplatin is more poorly tolerated than other chemotherapeutic drugs, and the main dose-limiting toxicity of cisplatin is its nephrotoxicity, which is dose-dependent. Although less toxic methods of cisplatin administration have been established, cisplatin-induced nephrotoxicity remains an unsolved problem. Megalin is an endocytic receptor expressed at the apical membrane of proximal tubules. We previously demonstrated that nephrotoxic drugs, including cisplatin, are reabsorbed through megalin and cause proximal tubular cell injury. We further found that cilastatin blocked the binding of cisplatin to megalin in vitro. In this study, we investigated whether cilastatin could reduce cisplatin-induced nephrotoxicity without influencing the antitumor effects of cisplatin. Nephrotoxicity was decreased or absent in mice treated with cisplatin and cilastatin, as determined by kidney injury molecule-1 staining and the blood urea nitrogen content. Combined with cilastatin, a twofold dose of cisplatin was used to successfully treat the mice, which enhanced the antitumor effects of cisplatin but reduced its nephrotoxicity. These findings suggest that we can increase the dose of cisplatin when combined with cilastatin and improve the outcome of cancer patients.

SUN-138 Roles of Na+/HCO3- cotransporter NBCn2 in acid-base transport in the apical membrane of renal proximal tubules

Proximal tubules (PTs) reclaim approximately 80% of filtered HCO3- Acid-base transport in PTs is mainly Na dependent and conducted coordinately by apical Na+/H+ exchanger (NHE)3 and basolateral Na+/HCO3- cotransporter (NBC)e1. It is believed that the apical NHE3 and the vacuolar H+ ATPase (V-ATPase) are mainly responsible for the reabsorption in the apical membrane of PTs. However, an NHE inhibitor, S3226 or amiloride inhibited less than 50% of Na or HCO3- reabsorption in vivo. A recent study reported that devastating mutation of NHE3 did not cause proximal renal tubular acidosis in human.

Abstract 304: Combination therapy of cisplatin with cilastatin enables to increase the dose of cisplatin for enhancing its antitumor effect by suppressing nephrotoxicity

Abstract Cisplatin is an anticancer drug widely used in the treatment of many cancers, including lung cancers. Although cisplatin causes various types of adverse events, the main dose-limiting toxicity of cisplatin is nephrotoxicity. Megalin is an endocytic receptor expressed at the apical membranes of proximal tubules. We previously demonstrated that cisplatin was reabsorbed through megalin and caused kidney injury. Cilastatin, an inhibitor of renal dehydropeptidase-I and used with imipenem, blocked the binding of cisplatin to megalin and reduced the nephrotoxicity induced by cisplatin. In the current study, we precisely evaluated the effect of cilastatin-mediated suppression of cisplatin nephrotoxicity to safely enhance the antitumor activity of cisplatin. BALB/c mice were administrated cisplatin with or without cilastatin. Tubular dilation or atrophy, brush border loss, tubular cell lysis and cast formation were observed in mice treated with cisplatin alone. However, these kidney injuries were decreased or disappeared in mice treated with cisplatin and cilastatin. Cilastatin also decreased the urinary levels of N-acetyl-β-D-glucosaminidase and neutrophil gelatinase-associated lipocalin, proximal tubular injury markers. Next, SCID mice were injected s.c. with A549, a human lung cancer cell line, and treated with cisplatin with or without cilastatin. Cilastatin did not affect the antitumor activity of cilastatin. Notably, A549 did not express megalin. Combined with cilastatin, the mice were successfully treated with 1.5 times dose of cisplatin with enhanced antitumor effects of cisplatin but without nephrotoxicity. In conclusion, cilastatin effectively suppressed nephrotoxicity of cisplatin by blocking the binding of cisplatin to megalin. These findings indicated that we could administer cisplatin into cancer patients without nephrotoxicity if we used cilastatin. Moreover, we might increase the dose of cisplatin and improve the outcome of cancer patients. Citation Format: Masashi Arita, Satoshi Watanabe, Nobumasa Aoki, Miho Takahashi, Satoshi Shoji, Koichiro Nozaki, Kosuke Ichikawa, Rie Kondo, Shoji Kuwahara, Junta Tanaka, Toshiyuki Koya, Akihiko Saito, Toshiaki Kikuchi. Combination therapy of cisplatin with cilastatin enables to increase the dose of cisplatin for enhancing its antitumor effect by suppressing nephrotoxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 304.

The potential role of myosin motor proteins in mediating the subcellular distribution of NHE3 in the renal proximal tubule.

Isoform 3 of the Na+/H+ exchanger (NHE3) is responsible for the majority of the reabsorption of NaCl, NaHCO3, and, consequently, water in the renal proximal tubule. As such, this transporter plays an essential role in acid-base balance and extracellular fluid volume homeostasis and determining systemic arterial blood pressure levels. NHE3 activity is modulated by a number of mechanisms, including the redistribution of the transporter between the body of the microvilli (where NHE3 is active) and the base of the microvilli (where NHE3 is less active). Although the physiological, pathophysiological, and pharmacological importance of the subcellular distribution of NHE3 has been well established, the exact mechanism whereby NHE3 is translocated along microvilli microdomains of the proximal tubule apical membrane is unknown. Nonmuscle myosin IIA and unconventional myosin VI move cargoes in anterograde and retrograde directions, respectively, and are known to redistribute along with NHE3 in the proximal tubule in response to a variety of natriuretic and antinatriuretic stimuli, including stimulation or inhibition of the renin-angiotensin system, high dietary Na+ intake, and high blood pressure. Therefore, this review aims to discuss the current evidence that suggests a potential role of myosin IIA and myosin VI in mediating the subcellular distribution of NHE3 along the kidney proximal tubule microvilli and their possible contribution in modifying NHE3-mediated Na+ reabsorption under both physiological and pathophysiological conditions.

Calcium-sensing receptor (CaSR) modulates vacuolar H+-ATPase activity in a cell model of proximal tubule.

The calcium-sensing receptor (CaSR) is localized in the apical membrane of proximal tubules in close proximity to the transporters responsible for proton secretion. Therefore, the aim of the present study was to analyze the effects of CaSR stimulation on the biochemical activity of the vacuolar H+-ATPase in a cellular model of proximal tubule cells, OKP cells. Biochemical activity of H+-ATPase was performed using cell homogenates, and the inorganic phosphate released was determined by a colorimetric method. Changes in cytosolic ionized calcium [Ca2+]i were also determined using Fluo-4. A significant increase of vacuolar H+-ATPase activity was observed when the CaSR was stimulated with agonists such as Gd3+ (300µM) and neomycin (200µM). This activity was also stimulated in a dose-dependent fashion by changes in extracellular Ca2+ (Ca2+o) between 10-4 and 2mM. Gd3+ and neomycin produced a sustained rise of [Ca2+]i, an effect that disappears when extracellular calcium was removed in the presence of 0.1µM thapsigargin. Inhibition of phospholipase C (PLC) activity with U73122 (5 × 10-8M) reduced the increase in [Ca2+]i induced by neomycin. CaSR stimulation induces an increase in the vacuolar H+-ATPase activity of OKP cells, an effect that involves an increase in [Ca2+]i and require phospholipase C activity. The consequent decrease in intratubular pH could lead to increase ionization of luminal calcium, potentially enhancing its reabsorption in distal tubule segments and reducing the formation of calcium phosphate stones.

Empagliflozin Inhibits NHE3 Activity in the Proximal Tubule of Normotensive and Hypertensive Rats

BackgroundA growing body of evidence suggests an interplay between extracellular volume and glucose homeostasis. NHE3 is the main pathway for Na reabsorption in the proximal tubule (PT). The sodium glucose cotransporter SGLT2 reabsorbs most of the glucose filtered by the kidneys and colocalizes with NHE3 in the apical membrane of PT. Thus, we tested the hypothesis that SGLT2 and NHE3 functionally interact. Given that SGLT2 inhibitors confer blood‐pressure lowering effects in T2DM patients, we also tested the hypothesis that the antihypertensive effects of SGLT2 inhibitors may be associated with PT NHE3 inhibition.MethodsThe acute and long‐term effects of the SGLT2 inhibitor empagliflozin on PT NHE3 activity were determined by in vivo stationary microperfusion in fourteen‐week‐old male Wistar rats and SHR‐treated rats. Additionally, twelve‐week‐old SHRs were treated with empagliflozin (10 mg/kg/day) or vehicle (control) for two weeks. Tail‐cuff blood pressure and renal function were measured before (baseline) and after treatment (post‐treatment). NHE3 expression and phosphorylation levels were evaluated by immunoblotting.ResultsEmpagliflozin acutely inhibited PT NHE3 activity in both Wistar (1.73 ± 0.07 vs. 2.55 ± 0.14 nmol/cm2.s, P < 0.001) and SHR (0.82 ± 0.07 vs. 1.21 ± 0.10 nmol/cm2.s, P < 0.01). Blood pressure (BP) of empagliflozin‐treated SHR was lower than baseline levels (176 ± 4 vs. 194 ± 4 mm Hg, P < 0.01) whereas no change in BP was observed in vehicle‐treated SHR (194 ± 6 vs. 192 ± 5 mm Hg). Compared to baseline, empagliflozin‐treatment enhanced cumulative urinary flow, calcium, sodium and glucose excretion whereas in vehicle‐treated SHR these parameters remained unchanged. Lower BP in empagliflozin‐treated SHR was associated with lower PT NHE3 activity (0.67 ± 0.10 vs. 1.18 ± 0.10 nmol/cm2.s, P < 0.001). No differences were observed on renal cortical NHE3 protein expression or NHE3 phosphorylation levels between the two groups of rats.ConclusionThese results suggest that NHE3 and SGLT2 functionally interact in the PT. Inhibition of NHE3 by SGLT2 inhibitors may underlie, at least in part, the antihypertensive effect of empagliflozin.Support or Funding InformationFAPESPThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Renal‐selective Snx5 gene silencing increases blood pressure and renal sodium transporters in mice

We have reported that sorting nexin 5 (SNX5) plays an important role in the positive regulation of the renal dopamine receptor subtype 1 (D1R). siRNA‐mediated gene silencing of renal Snx5 results in anti‐natriuresis and increases further the elevated blood pressure (BP) of spontaneously hypertensive rats. We tested the hypothesis that the increased renal sodium transport and increased BP following Snx5 gene silencing is accompanied by changes in the expression of renal transporters in C57BL6/J mice. We infused subcapsularly the Snx5‐specific siRNA (3 μg/kg/day), or non‐silencing “mock” siRNA as control, for 7 days in 8 1‐year old, previously uninephrectomized male C57BL6 mice. A 24‐hour urine was collected from the mice housed in metabolic cages a day prior to the end of the siRNA infusion. Renal SNX5 protein expression was decreased by 60% with Snx5‐siRNA relative to control group. Renal SNX5‐depleted mice had elevated systolic (119±5.2 mm Hg, measured under anesthesia) and diastolic BPs (91.8±7.3 mm Hg) relative to control mice (systolic BP=101.5±0.5 mm Hg, diastolic BP=72±2.3 mm Hg). Urine sodium excretion tended to decrease in SNX5‐depleted mice (0.68±0.22 mmol/Cr mg) relative to controls (1.08±0.30 mmol/ Cr mg). Moreover, renal SNX5 depletion shifted the pressure natriuresis curve to the right. Food and water intake, body weight, urine volume, and heart rate were similar in the two groups. SNX5 is mainly located at the apical membrane of proximal tubules (PT), thick ascending limbs of loop of Henle (TAL) and distal convoluted tubules (DCT). SNX5 was observed via confocal microscopy to colocalize with NHE3, NKCC2 and NCC in C57Bl/6J mice under basal conditions. However, the co‐staining is strongest with NHE3 in the PT, relative to NKCC 2 and NCC which are expressed in TAL and DCT, respectively. The expression of renal NHE3 (172±29 % of control), NaPi2 (223±12%), NKCC2 (440±12%) and NCC (177±23%) were greater in SNX5‐depleted mice than in control mice. Renal protein expressions of α,β,γ‐ENaCs, Na+K+ATPase and actin were similar in the 2 groups. These findings suggest that inhibition of SNX5 expression with siRNA increases sodium transporters in proximal and distal nephron segments. This may lead to the inability of renal D1R to inhibit renal sodium transport and may be responsible for the increased BP in Snx5‐siRNA‐ treated mice.Support or Funding InformationThis work was supported in part by grants from the National Institutes of Health, R37HL023081, R01DK039308, P01HL068686, P01HL074940, and R01HL092196. Dr. Xiaoyan Wang was supported, in part, by National Kidney Foundation of Maryland (Professional Development Award, 2014).

Na+/HCO3- Cotransporter NBCn2 Mediates HCO3- Reclamation in the Apical Membrane of Renal Proximal Tubules.

The kidney maintains systemic acid-base balance by reclaiming from the renal tubule lumen virtually all HCO3- filtered in glomeruli and by secreting additional H+ to titrate luminal buffers. For proximal tubules, which are responsible for about 80% of this activity, it is believed that HCO3- reclamation depends solely on H+ secretion, mediated by the apical Na+/H+ exchanger NHE3 and the vacuolar proton pump. However, NHE3 and the proton pump cannot account for all HCO3- reclamation. Here, we investigated the potential contribution of two variants of the electroneutral Na+/HCO3- cotransporter NBCn2, the amino termini of which start with the amino acids MCDL (MCDL-NBCn2) and MEIK (MEIK-NBCn2). Western blot analysis and immunocytochemistry revealed that MEIK-NBCn2 predominantly localizes at the basolateral membrane of medullary thick ascending limbs in the rat kidney, whereas MCDL-NBCn2 localizes at the apical membrane of proximal tubules. Notably, NH4Cl-induced systemic metabolic acidosis or hypokalemic alkalosis downregulated the abundance of MCDL-NBCn2 and reciprocally upregulated NHE3 Conversely, NaHCO3-induced metabolic alkalosis upregulated MCDL-NBCn2 and reciprocally downregulated NHE3 We propose that the apical membrane of the proximal tubules has two distinct strategies for HCO3- reclamation: the conventional indirect pathway, in which NHE3 and the proton pump secrete H+ to titrate luminal HCO3-, and the novel direct pathway, in which NBCn2 removes HCO3- from the lumen. The reciprocal regulation of NBCn2 and NHE3 under different physiologic conditions is consistent with our mathematical simulations, which suggest that HCO3- uptake and H+ secretion have reciprocal efficiencies for HCO3- reclamation versus titration of luminal buffers.

Megalin Blockade with Cilastatin Suppresses Drug-Induced Nephrotoxicity.

Nephrotoxicity induced by antimicrobial or anticancer drugs is a serious clinical problem. Megalin, an endocytic receptor expressed at the apical membranes of proximal tubules, mediates the nephrotoxicity of aminoglycosides and colistin, key antimicrobials for multidrug-resistant organisms. The mechanisms underlying the nephrotoxicity induced by vancomycin, an antimicrobial for methicillin-resistant Staphylococcus aureus, and cisplatin, an important anticancer drug, are unknown, although the nephrotoxicity of these drugs and gentamicin, an aminoglycoside, is suppressed experimentally with cilastatin. In the clinical setting, cilastatin has been used safely to suppress dehydropeptidase-I-mediated renal metabolism of imipenem, a carbapenem antimicrobial, and thereby limit tubular injury. Here, we tested the hypothesis that cilastatin also blocks megalin-mediated uptake of vancomycin, cisplatin, colistin, and aminoglycosides, thereby limiting the nephrotoxicity of these drugs. Quartz crystal microbalance analysis showed that megalin also binds vancomycin and cisplatin and that cilastatin competes with megalin for binding to gentamicin, colistin, vancomycin, and cisplatin. In kidney-specific mosaic megalin knockout mice treated with colistin, vancomycin, or cisplatin, the megalin-replete proximal tubule epithelial cells exhibited signs of injury, whereas the megalin-deficient cells did not. Furthermore, concomitant cilastatin administration suppressed colistin-induced nephrotoxicity in C57BL/6J mice. Notably, cilastatin did not inhibit the antibacterial activity of gentamicin, colistin, or vancomycin in vitro, just as cilastatin did not affect the anticancer activity of cisplatin in previous studies. In conclusion, megalin blockade with cilastatin efficiently suppresses the nephrotoxicity induced by gentamicin, colistin, vancomycin, or cisplatin. Cilastatin may be a promising agent for inhibiting various forms of drug-induced nephrotoxicity mediated via megalin in the clinical setting.

Roles of organic anion transporters (OATs) in renal proximal tubules and their localization.

Organic anions (OAs) are secreted in renal proximal tubules in two steps. In the first step, OAs are transported from the blood through basolateral membranes into proximal tubular cells. The prototypical substrate for renal organic anion transport systems, para-aminohippurate (PAH), is transported across basolateral membranes of proximal tubular cells via OAT1 (SLC22A6) and OAT3 (SLC22A8) against an electrochemical gradient in exchange for intracellular dicarboxylates. In the second step, OAs exit into urine through apical membranes of proximal tubules. This step is thought to be performed by multidrug efflux transporters and a voltage-driven organic anion transporter. However, the molecular nature and precise functional properties of these efflux systems are largely unknown. Recently, we characterized an orphan transporter known as human type I sodium-phosphate transporter 4, hNPT4 (SLC17A3), using the Xenopus oocyte expression system. hNPT4 acts as a voltage-driven efflux transporter ("human OATv1") for several OAs such as PAH, estrone sulfate, diuretic drugs, and urate. Here, we describe a model for an OA secretory pathway in renal tubular cells in which OAs exit cells and enter the tubular lumen via hOATv1 (hNPT4). Additionally, hOATv1 functions as a common renal secretory pathway for both urate and drugs, indicating that hOATv1 may be a leak pathway for excess urate that is reabsorbed via apical URAT1 to control the intracellular urate levels. Therefore, we propose a molecular mechanism for the induction of hyperuricemia by diuretics: the diuretics enter proximal tubular cells via basolateral OAT1 and/or OAT3 and may then interfere with the NPT4-mediated apical urate efflux in the renal proximal tubule.

Kidney Injury Molecule-1 Enhances Endocytosis of Albumin in Renal Proximal Tubular Cells.

Receptor-mediated endocytosis plays an important role in albumin reabsorption by renal proximal tubule epithelial cells. Kidney injury molecule-1 (KIM-1) is a scavenger receptor that is upregulated on the apical membrane of proximal tubules in proteinuric kidney disease. In this study, we examined the cellular localization and functional role of KIM-1 in cultured renal tubule epithelial cells (TECs). Confocal immunofluorescence microscopy reveals intracellular and cell surface localization of KIM-1 in primary renal TECs. Albumin stimulation resulted in a redistribution of KIM-1 and tight junction protein zonula occludens-1 in primary TEC monolayer. An increase in albumin internalization was observed in both primary TECs expressing endogenous KIM-1 and rat kidney cell line (NRK-52E) overexpressing exogenous KIM-1. KIM-1-induced albumin accumulation was abolished by its specific antibody. Moreover, endocytosed KIM-1 and its cargo proteins were delivered from endosomes to lysosomes for degradation in a clathrin-dependent pathway. Supportive evidence includes (1) detection of KIM-1 in Rab5-positive early endosomes, Rab7-positive late endosomes/multivesicular bodies, and LAMP1-positive lysosomes, (2) colocalization of KIM-1 and clathrin in the intracellular vesicles, and (3) blockade of KIM-1-mediated albumin internalization by chlorpromazine, an inhibitor of clathrin-dependent endocytosis. KIM-1 expression was upregulated by albumin but downregulated by transforming growth factor-β1. Taken together, our data indicate that KIM-1 increases albumin endocytosis in renal tubule epithelial cells, at least partially via a clathrin-dependent mechanism. J. Cell. Physiol. 231: 896-907, 2016. © 2015 Wiley Periodicals, Inc.

Abstract 141: G-Protein Coupled Receptors GPR37 and GPR37L1 Regulate Sodium Reabsorption in Renal Proximal Tubule Cells

GPR37 and GPR37L1 are closely related G-protein coupled receptors that are expressed mainly in brain glial cells and muscle-myenteric nerve layers in the GI tract. GPR37L1 transgenic mice have decreased systolic blood pressure (SBP) whereas GPR37L1 KO mice have increased SBP. However, there are no studies reporting kidney expression of GPR37 or GPR37L1 or their role in renal blood pressure regulation. Immunostaining and immunoblotting showed that GPR37 and GPR37L1 are expressed in the apical membrane of proximal tubules of the mouse kidney; RT-PCR on proximal tubule and collecting duct cells obtained by laser capture micro-dissection of mouse kidney sections, confirmed these findings. In addition, chronic high salt diet increased the renal expression of prosaposin, a precursor for saposin C, a natural ligand for GPR37 and GPR37L1. Infusion of prosaptide, a synthetic ligand for GPR37 and GPR37L1, decreased SBP in mice by 10 mm Hg. To determine the roles of GPR37 and GPR37L1 in renal Na+ transport, we over-expressed separately the two proteins in human renal proximal tubule (RPT) cells (n=3). Intracellular Na+ was increased in GPR37- or GPR37L1-transfected RPT cells (3.1 ± 0.8 and 3.2 ± 0.6 fold respectively, P<0.001) compared with mock-transfected cells. Immunoblot analyses showed increased phosphorylation of Erk1/2 (1.33 ± 0.03 and 1.52 ± 0.06 fold, P<0.05), and ribosomal S6 protein (1.29 ± 0.03.5 and 1.39 ± 0.08 fold, P<0.01) in RPT cells over-expressing GPR37 or GPR37L1, respectively. Na+,K+-ATPase expression was decreased by 29% ± 3.5 (P<0.05) in GPR37L1 transfected RPT cells, but was unaltered in GPR37 transfected RPT cells. Taken together, these results show that both GPR37 and GPR37L1 are expressed in RPT cells and signal via the Erk1/2 pathway. GPR37L1 increases intracellular Na+ in RPT cells by decreasing the exit of Na+ due to a decrease in Na+,K+-ATPase expression and activity at the basolateral membrane while GPR37 increases intracellular Na+, by a mechanism independent of Na+,K+-ATPase. These results indicate that GPR37 and GPR37L1 may play a role in Na+ reabsorption in RPT cells and may be novel targets to designing drugs to treat patients with hypertension.

Expression of basolateral organic anion and cation transporters in experimental cadmium nephrotoxicity in rat kidney.

Cadmium (Cd)-intoxicated experimental animals exhibit impaired renal secretion of organic anions (OA) and cations (OC), indicating their transporters (Oats and Octs) in the proximal tubule (PT) basolateral membrane as possible targets of Cd. To correlate transport data from the literature with the expression of relevant transporters, we performed immunochemical and RT-PCR studies of renal Oats and Octs in the subchronic (treatment with CdCl2; 2 mg Cd/kg b.m./day, for 2 weeks) and acute (treatment with Cd-metallothionein (CdMT); 0.4 mg Cd/kg b.m., 6 or 12 h before killing) models of Cd nephrotoxicity. In the subchronic model, PT exhibited a minor loss of basolateral invaginations and overall unchanged expression of Na(+)/K(+)-ATPase and GAPDH proteins and mRNAs, while the expression of Oat and Oct proteins and their mRNAs was strongly downregulated. In the acute model, a time-related redistribution of basolateral transporters to the intracellular vesicular compartment was a major finding. However, 6 h following CdMT treatment, the total abundance of Oat and Oct proteins in the renal tissue remained unchanged, the expression of mRNAs decreased only for Oats, while a limited Oat1 and Na(+)/K(+)-ATPase immunoreactivity in the PT apical membrane indicated loss of cell polarity. As tested in rats treated with colchicine, the observed loss/redistribution of basolateral transporters in both models may be independent on microtubules. Therefore, the diminished renal secretion of OA and OC via PT in Cd nephrotoxicity may result from (a) limited loss of secretory surface (basolateral invaginations), (b) selective loss of Oats and Octs, and

Cloning, localization and characterization of a Zn transporter (Zip10) from fly, dog, and human (893.39)

Background: Zn is implicated as having a role in the formation of calcium oxalate (CaOx) kidney stones. Our studies indicate that Slc39a10 (Zip10) may be important in this process. Zip10 has identifiable sequences in human, dog and fly (Drosophila). As we are interested in a translational model of kidney stones, we examined Zip10 from all 3 species.Methods: Zip10 cDNAs were cloned from human & dog (kidney) and fly (whole fly) using gene specific primers and RT‐PCR. Clones were put into a Xenopus oocyte expression vector so that Zip10 function could be measured. Three days post‐injection, oocytes were pre‐incubated in ND96, followed by 30 min in ND96 + 10 μM ZnCl + the PET isotope 63Zn, with varying pHs and ion concentrations. 63Zn uptake was measured in each oocyte using a gamma counter. Zip10 oocytes were also checked using electrophysiology (ion selective electrodes & voltage clamping).Results: Zip10 mRNA & protein are found in gut and kidney. In mouse kidney, Zip10 antibodies stained the apical membrane of proximal tubules. All clones transported 63Zn (nmol/oocyte/hr): hZip10 = 70±8; cZip10= 44 ±7; dZip10= 64 ±8; water = 0.4 ±0.1. These clones seem to be most active at pH 7.5 as uptakes decreased at 5.5 and 8.5. Replacement of either all Cl‐ (with HCO3舉) or all Na+ (with choline) reduced Zn‐uptake. Electrophysiology revealed no evidence of I or V changes with addition of Zn (electroneutral).Conclusions: Our results indicated that all three Zip10 clones transport Zn2+. Understanding the detailed mechanisms of these Zn transporters in oocytes presents the opportunity to better use our Drosophila model in the study of CaOx stone formation and absorption.Grant Funding Source: Supported by DK83007, DK92408

The membrane cytoskeletal crosslinker ezrin is essential for the regulation of phosphate and calcium homeostasis

Ezrin is a cross‐linker between the plasma membrane proteins and actin cytoskeleton. In the kidney, ezrin is mainly localized at brush border membrane of proximal tubules with a scaffolding protein, Na+/H+ exchanger regulatory factor (NHERF) 1. NHERF1 interacts with Na+/phosphate (Pi) cotransporter Npt2a. We focused on the physiological role of ezrin in the renal Pi reabsorption by using ezrin knockdown mice (Vil2kd/kd). The Vil2kd/kd mice exhibited hypophosphatemia, hypocalcemia, and osteomalacia. The reduced plasma concentration of Pi was attributed to the defective urinary Pi reabsorption. Immunofluorescence staining and immunoblotting revealed that the Npt2a and NHERF1 expressions at apical membrane of proximal tubules were markedly reduced in the kidney of Vil2kd/kd mice. On the other hand, urinary loss of Ca2+ was not observed in Vil2kd/kd mice. Plasma concentration of 1,25‐dihydroxyvitamin D was elevated following reduced plasma Pi level. Furthermore, bone mineral density in tibiae was significantly lowered in the adult and young Vil2kd/kd mice. These results suggest that ezrin is required for the regulation of systemic Pi homeostasis in vivo.

Altered expression and localization of insulin receptor in proximal tubule cells from human and rat diabetic kidney

Diabetes is the major cause of end stage renal disease, and tubular alterations are now considered to participate in the development and progression of diabetic nephropathy (DN). Here, we report for the first time that expression of the insulin receptor (IR) in human kidney is altered during diabetes. We detected a strong expression in proximal and distal tubules from human renal cortex, and a significant reduction in type 2 diabetic patients. Moreover, isolated proximal tubules from type 1 diabetic rat kidney showed a similar response, supporting its use as an excellent model for in vitro study of human DN. IR protein down-regulation was paralleled in proximal and distal tubules from diabetic rats, but prominent in proximal tubules from diabetic patients. A target of renal insulin signaling, the gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK), showed increased expression and activity, and localization in compartments near the apical membrane of proximal tubules, which was correlated with activation of the GSK3β kinase in this specific renal structure in the diabetic condition. Thus, expression of IR protein in proximal tubules from type 1 and type 2 diabetic kidney indicates that this is a common regulatory mechanism which is altered in DN, triggering enhanced gluconeogenesis regardless the etiology of the disease.

Ezrin, a membrane cytoskeletal cross-linker, is essential for the regulation of phosphate and calcium homeostasis

Ezrin cross-links plasma membrane proteins with the actin cytoskeleton. In the kidney, ezrin mainly localizes at the brush border membrane of proximal tubules with the scaffolding protein, Na(+)/H(+) exchanger regulatory factor (NHERF) 1. NHERF1 interacts with the sodium/phosphate cotransporter, Npt2a. Defects in NHERF1 or Npt2a in mice cause hypophosphatemia. Here we studied the physiological role of ezrin in renal phosphate reabsorption using ezrin knockdown mice (Vil2). These mice exhibit hypophosphatemia, hypocalcemia, and osteomalacia. The reduced plasma phosphate concentrations were ascribed to defects in urinary phosphate reabsorption. Immunofluorescence and immunoblotting indicated a marked reduction in renal Npt2a and NHERF1 expression at the apical membrane of proximal tubules in the knockdown mice. On the other hand, urinary loss of calcium was not found in Vil2 mice. Plasma concentrations of 1,25-dihydroxyvitamin D were elevated following reduced plasma phosphate levels, and mRNA of the vitamin D-dependent TRPV6 calcium channel were significantly increased in the duodenum of knockdown mice. Expression of TRPV6 at the apical membrane, however, was significantly decreased. Furthermore, tibial bone mineral density was significantly lower in both the adult and young Vil2 mice. These results suggest that ezrin is required for the regulation of systemic phosphate and calcium homeostasis in vivo.

Alkaline Phosphatase as a Treatment of Sepsis-Associated Acute Kidney Injury

Currently there are no pharmacological therapies licensed to treat sepsis-associated acute kidney injury (AKI). Considering the high incidence and mortality of sepsis-associated AKI, there is an urgent medical need to develop effective pharmacological interventions. Two phase II clinical trials recently demonstrated beneficial effects of the enzyme alkaline phosphatase (AP). In critically ill patients with sepsis-associated AKI, treatment with AP reduced the urinary excretion of tubular injury biomarkers and plasma markers of inflammation, which was associated with improvement of renal function. The dephosphorylating enzyme, AP, is endogenously present in the renal proximal tubule apical membrane but becomes depleted during ischemia-induced AKI, thereby possibly contributing to further renal damage. The exact mechanism of action of AP in AKI is unknown, but might be related to detoxification of circulating lipopolysaccharide and other proinflammatory mediators that lose their proinflammatory effects after dephosphorylation. Alternatively, tissue damage associated with systemic inflammation might be attenuated by an AP-mediated effect on adenosine metabolism. Adenosine is a signaling molecule that has been shown to protect the body from inflammation-induced tissue injury, which is derived through dephosphorylation of ATP. In this Perspectives article, we discuss the clinical activity of AP and its putative molecular modes of action, and we speculate on its use to treat and possibly prevent sepsis-associated AKI.

Abstract 633: Lack of ACE2 Abolishes High Fat Diet-Induced Upregulation of Prolyl Carboxypeptidase

Emerging evidence suggests that activation of the proteases, Ang converting enzyme 2 (ACE2) and prolyl carboxypeptidase (PCP), are associated with metabolic pathologies. We hypothesized that global loss of ACE2 affects high fat diet induced renal expression of PCP. ACE2 knockout (ACE2KO) and wild type (WT) male mice were fed a 60% high fat (HF) or normal chow (NC) diet for 12 weeks. Using dual immunofluorescence microscopy and western blotting, we determined the expression pattern and abundance of ACE2 and PCP in kidney. PCP was co-localized with ACE2 in the Bowman’s capsule of the glomerulus. PCP was also localized along the apical membrane of proximal tubules along with ACE2. There was no evidence for protease co-localization in the tubules. ACE2 was absent in kidney from ACE2KO (immunofluorescence or western). PCP localization was unchanged in ACE2KO and the localization of both proteases did not change in response to HF diet. Protein intensity, normalized to ß-actin, showed no difference in PCP between untreated WT and KO (0.76±0.18 vs 0.57±0.07). PCP in HF WT was significantly elevated as compared to HF KO (0.94±0.02 vs 0.57±0.03, p≤0.05) while HF WT ACE2 levels remained unchanged as compared to untreated WT (0.90±0.24 vs 0.94±0.16). Results indicate loss of ACE2 negatively affects HF diet-induced PCP expression. Neither the absence of ACE2 nor HF diet changed the localization of PCP. These results suggest interactions between ACE2 and PCP in metabolic pathologies.