Iups2009プロpok.doc

IUPS2009 Satellite Symposium
“The Kidney and Hypertension”
POSTER SESSION
ABSTRACTS
CONTENTS

P1. URETERIC BUD-DERIVED BMP7 MAINTAINS RENAL PROGENITOR CELLS: A
NOVEL REGULATORY MECHANISM THROUGH THE EXPRESSION OF EARLY NEPHROGENIC MARKERS P2. A NOVEL RENOPROTECTIVE PATHWAY MEDIATED BY AATF P3. MODULATION OF BCL-2 BY PAR-4 AND AATF IN RENAL TUBULAR EPITHELIAL P4. LOCALIZATION AND EXPRESSION OF CARBONIC ANHYDRASE ISOFORMS ALONG THE MOUSE NEPHRON DURING METABOLIC ACIDOSIS P5. NHE3 IS ACTIVATED VIA AN ELECTROSTATIC INTERACTION BETWEEN THE CYTOSOLIC -TERMINUS AND THE INNER LEAFLET OF THE PLASMA MEMBRANE P6. ALDOSTERONE STIMULATES NF-κB ACTIVITY AND TRANSCRIPTION OF ICAM-1 VIA SERUM AND GLUCOCORTICOID -INDUCIBLE PROTEIN KINASE (SGK)-1 IN RAT MESANGIAL CELLS IN VIVO AND IN VITRO P7. ACUTE SUPPRESSIVE EFFECT OF INTERLEUKIN-1β ON K+ CHANNEL P8. INHIBITION OF AN INWARDLY RECTIFYING K+ CHANNEL BY CYCLOSPORIN A INVOLVES Ca2+/CAM-KINASE II-MEDIATED PROCESSES IN HUMAN PROXIMAL TUBULE CELLS P9. THE VOLTAGE-DRIVEN ORGANIC ANION TRANSPORT OF TYPE I SODIUM-PHOSPHATE COTRANSPORTER AND OATv1 P10. VOLTAGE-DEPENDENT TRANSPORT OF URATE ON A NOVEL HUMAN URATE EFFLUX TRANSPORTER URATv1 EXPRESSED IN Xenopus OOCYTES P11. IDENTIFICATION OF THE MULTIVALENT PDZ PROTEIN PDZK1 AS A BINDING PARTNER OF SODIUM–COUPLED MONOCARBOXYLATE TRANSPORTER SMCT1 (SLC5A8) AND SMCT2 (SLC5A12) BY YEAST TWO-HYBRID ASSAY P12. INTERACTION OF A NOVEL URATE EFFLUX TRANSPORTER URATv1 P13. CALCIUM, MAGNESIUM, AND CALCIMIMETICS DECREASE PNa/PCl RATIO IN MOUSE THIN ASCENDING LIMB OF HENLE’S LOOP P14. ROMK1 KNOCKOUT MICE DO NOT PRODUCE BARTTER’S PHENOTYPE P15. CRITICAL ROLES OF V1A AND V1B VASOPRESSIN RECEPTORS IN SYSTEMIC BLOOD PRESSURE HOMEOSTASIS: STUDIES FROM GENE KNOCKOUT MICE P16. FUROSEMIDE-INDUCED nNOS OF THE MOUSE MACULA DENSA CELL LINE LACKS A C-TERMINAL REDUCTASE DOMAIN, BUT GENERATES NO Ca2+-DEPENDENTLY P17. REGULATION OF PGE2 PRODUCTION IN MOUSE MACULA DENSA CELLS P18. A ROLE OF V1A VASOPRESSIN RECEPTOR FOR URINARY SODIUM P19. DIFFERENT RESPONSES OF MACULA DENSA TO SALT LOAD BETWEEN DIABETIC AND NON-DIABETIC KIDNEY IN RATS P20. DIFFERENCE OF HIGH SALT DIET-INDUCED GLOMERULAR HYPERTENSION BETWEEN TYPE-2 DIABETES AND NON-DIABETES P21. CARBONYL STRESS MEDIATED MYOCARDIAL FIBROSIS AND RENAL P22. FUNCTIONAL CHARACTERIZATION OF CONNEXINS IN P23. THE ACTIVATION OF PGE2 RECEPTOR TYPE4 IN RENAL COLLECTING DUCTS INHIBITS THE ENAC EXPRESSION BY THE ACTIVATION OF PKA AND THE INHIBITION OF P38 VIA NON-PKA PATHWAY 1. URETERIC BUD-DERIVED BMP7 MAINTAINS RENAL
PROGENITOR CELLS: A NOVEL REGULATORY MECHANISM
THROUGH THE EXPRESSION OF EARLY NEPHROGENIC MARKERS
Itsuro KAZAMA1,2, Daniel GRAF3, Aris N ECONOMIDES4, Motoko YANAGITA 5,
Jordan A. KREIDBERG2
1Department of Nephrology, Jichi Medical University, Shimotsuke, Tochigi 329-0498,
Japan
2Division of Nephrology, Children’s Hospital, Boston, MA, USA; Department of
Pediatrics, Harvard Medical School, Boston, MA, USA
3Institute of Immunology, Biomedical Science Research Center, Hellas, Greece 4Regeneron Pharmaceuticals, Inc, Tarrytown, NY, USA 5Graduate School of Medicine, Kyoto University, Sakyo-ku, Kyoto, Japan Renal progenitor cells are the precursors of the differentiated epithelial cells that give rise to nephrons. However, we know little about the molecular regulation in the maintenance of these cells. During the initial induction of nephrons within the nephrogenic zone, Bone Morphogenetic Protein 7 (BMP7) is mainly expressed in ureteric bud derivatives that give rise to collecting ducts. Since ureteric bud derivatives are adjacent to the cap mesenchyme that contains the precursor population, ureteric bud-derived BMP7 was hypothesized to affect those precursor cells. In the present study, to determine the role of ureteric bud-derived BMP7 in the maintenance of renal progenitor cells, we generated a ureteric bud-specific conditional knock out mouse of BMP7 (BMP7 CKO). At embryonic day 18 (E18), the cap mesenchyme in BMP7 CKO kidneys looked more disorganized and less condensed than control kidneys, followed by a dramatic reduction in the progenitor cells by postnatal day 0 (P0). Decreased numbers of Ki67 positive cells and increased numbers of TUNEL positive cells in the cap mesenchyme indicated impaired cellular proliferation and increased apoptosis. Six2, Pax2 and Osr1 (Odd skipped-1) are transcription factors expressed in the renal progenitor cells in the earliest stages of nephrogenesis. In particular, Six2 has recently been shown to have a role in maintaining the progenitor cell population. In situ hybridization demonstrated decreased expression of those genes in cap mesenchyme of BMP7 CKO kidneys, while the expression of WT1 and Wnt4 remained unchanged. This work demonstrated the crucial role of BMP7 in the maintenance of renal progenitor cells, mediated through the expression of early nephrogenic markers. 2. A NOVEL RENOPROTECTIVE PATHWAY MEDIATED BY AATF *

Qing Guo, Chelsea Baker and Jun Xie
Department of Physiology, The University of Oklahoma Health Sciences Center, 940
Stanton L. Young Blvd., Oklahoma City, OK 73104 U.S.A.
Ischemia/reperfusion (I/R)-induced renal injury is the most common cause of acute renal failure (ARF). AATF (apoptosis antagonizing transcription factor) is a leucine zipper domain containing protein with anti-apoptotic properties. Human AATF has an open reading frame of 560 amino acids, and is expressed in several organs and tissues including the kidney. We found that levels of AATF expression in renal tubular epithelial cells were significantly altered in an in vitro model of renal injury induced by I/R. In renal tubule epithelial cells, RNAi-mediated silencing of AATF exacerbated, while overexpression of AATF ameliorated mitochondrial dysfunction, oxidative damage, and apoptotic death induced by I/R. These results identify AATF as a novel cytoprotective factor in renal tubular cells. Par-4 (prostate apoptosis response-4) is a leucine zipper protein linked to apoptotic cell death in prostate cancer and neuronal tissues. The leucine zipper domain of Par-4 (Leu.zip) mediates protein-protein interactions that are essential for sensitization of cells to apoptosis, and overexpression of Leu.zip blocks Par-4 activity in a dominant negative fashion. We found that AATF is an endogenous interaction partner and blocker of Par-4 activity. We also provide evidence that AATF is a novel phosphorylation target of the serine/threonine protein kinase Akt1/PKBa, and that phosphorylation of AATF by Akt1 may represent a critical mechanism of signal transduction mediated by Akt1 in 3. MODULATION OF BCL-2 BY PAR-4 AND AATF IN RENAL
TUBULAR EPITHELIAL CELLS *

Jun Xie, Chelsea Baker and Qing Guo
Department of Physiology, The University of Oklahoma Health Sciences Center, 940
Stanton L. Young Blvd., Oklahoma City, OK 73104 U.S.A.
Par-4 (prostate apoptosis response-4) is a leucine zipper protein initially linked to apoptotic cell death in prostate cancer and neuronal tissues. We have identified Par-4 as an early and critical link in the chain of events leading to the initiation of apoptosis in human kidney proximal tubular cells in well-established models of renal injury induced by ischemia/reperfusion (I/R). Targeted knockdown of Par-4 expression by RNAi provides a novel cytoprotective mechanism. In pull down assays using specific sequence on the bcl-2 P1 promoter as bait, we found a significantly increased Par-4 binding to the bcl-2 promoter in proximal tubular cells following I/R. Overexpression of Par-4 effectively reduced the adaptive increase in Bcl-2 levels in the early stages of I/R injury in renal tubule cells, indicating Par-4-dependent cell-death was mediated by decreased bcl-2 transcription. AATF (apoptosis antagonizing transcription factor) is a leucine zipper protein which interacts with Par-4 via the leucine zipper domain. Binding of Par-4 to the bcl-2 promoter was significantly reduced in cells co-transfected with AATF. These results suggest that Par-4/AATF complex formation plays a critical role in renal cell survival by regulating nuclear transcription of bcl-2. 4. LOCALIZATION AND EXPRESSION OF CARBONIC ANHYDRASE
ISOFORMS ALONG THE MOUSE NEPHRON DURING METABOLIC
ACIDOSIS

Yukiko YASUOKA1, Mizuka KOBAYASHI2, Yuichi SATO3, and Katsumasa
KAWAHARA1.
1, 2. Departments of Physiology and Anesthesiology, Kitasato University School of
Medicine, Kitasato 1-15-1, Sagamihara 228-8555, Japan
3. Department of Molecular Diagnostics, Kitasato University School of Allied Health
Sciences, Kitasato 1-15-1, Sagamihara 228-8555, Japan
The kidney plays a key role in extracellular fluid pH homeostasis by reclaiming 3 ) at the proximal tubules and generating the consumed HCO3 at the intercalated cells of collecting ducts. Further, MTAL, medullary thick ascending limb of Henle’s loop, is thought to be important for H+ and ammonium excretion. We investigated localization and expression of carbonic anhydrase (CA) isoforms along the nephron to examine whether CAs are really involved in urine acidification during metabolic acidosis (MA). Methods. The CA (CAII, CAIV, CAXII, CAXIV) mRNAs
expression was examined by a quantitative In situ hybridization method along the mouse (C57BL/6J, male, 10 wks) nephron, such as PCT, PST, DTL, MTAL, CTAL, DCT, and CDs. Dots of CA mRNAs (stainings) were counted by eye under the microscope during the conditions of control and metabolic acidosis (0.28 M NH4Cl solution drinking). Results. After administration of NH4Cl, average urine pH (± SEM)
decreased significantly (P < 0.001) from 6.52 ± 0.04 (n=14) to 5.8 ± 0.03 (day 1, n=5) and was unchanged until day 6. Plasma pH instantaneously decreased from 7.32 ± 0.02 (n=6) to 7.17 ± 0.01 (n=5) on day 1 (P < 0.001), but completely recovered to 7.35 ± 0.02 (n=9) on day 6. In control mice, CAII MTAL, CTAL, DCT, and CDs. During NH4Cl load, the levels of CAII CAXIV mRNAs increased specifically at MTAL (CAII), at DTL and MTAL (CAIV), and at PCT and CD-IC (CAXII) . Interestingly, a level of the CAXIV expression was unchanged. Conclusion.
CAII and CAIV in MTAL may play an important role for maintaining the systemic acid-base balance, especially during MA. 5. NHE3 IS ACTIVATED VIA AN ELECTROSTATIC INTERACTION
BETWEEN THE CYTOSOLIC-TERMINUS AND THE INNER LEAFLET
OF THE PLASMA MEMBRANE
R. Todd A
, Tony YEUNG , Wendy FURUYA , Iskra PELTEKOVA , John 1Cell Biology Program, The Hospital for Sick Children, Toronto, M5G 1X8, Canada, 2Department of Pediatrics, The University of Alberta, Edmonton, T6J 2R7, Canada 3Department of Physiology, McGill University, Montreal, H3G 1Y6, Canada 4Department of Biochemistry, University of Toronto, M5S 1X8, Canada Implicit to the maintenance of intravascular volume and consequently blood pressure is the absorption of filtered NaCl and water by the renal tubule. The proximal tubule absorbs the bulk (∼110 L of water and 1.2 Kg of NaCl daily). Apical entry of Na + via exchange for a proton by NHE3 is the rate -limiting and regulated step in this process. The cytosolic C -terminus is known to be necessary for the regulation of NHE3 by many effectors, however the precise mechanism(s) are poorly understood. In an attempt to explain NHE3 regulation we examined its cytosolic domain and observed several regions proximal to the transmembrane domain that contained multiple cationic residues (region 1: residues 456-480 +4; region 2:503-527, +7; and region 3:645-688, +13). Given that the inner leaflet of the plasma membrane contains a high concentration of negati vely charged lipids we predicted that the tail of NHE3 would bind to the plasma membrane and that this interaction could be regulated via alterations in the juxta-membrane ion concentration and/or surface charge. To test our hypothesis we designed peptides corresponding to the three regions and conjugated a fluorescent probe (bimane) to them. The interaction of bimane with a hydrophobic environment increases its fluorescence. Consistent with our hypothesis, the addition of these peptides to liposomes containing anionic lipids increased fluorescence, a phenomenon not observed in the presence of liposomes composed of zwitterionic lipids. Moreover, fluorescence was quenched by increasing the ionic strength of the solution bathing the anionic liposomes, or by introducing cationic sterols into the anionic liposomes. Next we generated expression constructs of the three regions conjugated to GFP. In order to mimic the in vivo situation we introduced a farnesylation motif at the opposite end to the GFP. Expression of these constructs revealed plasma membrane localization, which could be prevented by increasing intracellular Ca2+, depleting the cells of ATP (which would deplete the inner membrane surface charge) or incorporating cationic sterols into the plasma membrane. Further, mutating the cationic residues to alanines, despite the presence of the farnesyl group, prevented plasma membrane localization. Next we measured the Na+-dependent recovery of pH in OK cells (which express NHE3 endogenously) after increasing intracellular Ca2+, depleting ATP or in the presence of a cationic sterol. All of these perturbations inhibited NHE3 activity. Finally, we generated full-length NHE3 constructs that had the cationic residues from each region mutated to alanines. The region 1 (residues 456-480) and 2 (residues 503-527) mutants completely lacked NHE activity, while the third mutant (residues 642-678) had similar activity to wild-type NHE3. Together these findings strongly suggest that NHE3 activity is regulated by an electrostatic interaction between cationic residues in the cytosolic C-terminus of NHE3 and anionic lipids in the inner leaflet of the plasma membrane and provides a putative mechanism for many of the processes known to regulate NHE3 activity. 6. ALDOSTERONE STIMULATES NF-κB ACTIVITY AND
TRANSCRIPTION OF ICAM -1 VIA SERUM AND
GLUCOCORTICOID-INDUCIBLE PROTEIN KINASE (SGK)-1 IN RAT
MESANGIAL CELLS IN VIVO AND IN VITRO

Yoshio TERADA, Department of Endocrinology, Metabolism, and Nephrology, Kochi
University, Kochi 785-8505, Japan
Recently, attention has focused on the role of aldosterone (Ald) in the pathophysiology of hypertension and cardiovascular disease. Several clinical and experimental data support a contribution of Ald to the inflammation of several organs. However, the molecular mechanisms of Ald on glomerular inflammation are not known We at first showed that mineralcorticoid receptor (MR) was expressed in rat mesangial cells and glomeruli, and that Ald translocated MR from cytosolic lesions to the nucleus. To determine the signaling pathway of Ald concerning inflammation in cultured mesangial cells, we investigated the effects of Ald on serum and glicocorticoid-inducible protein kinase (SGK)-1 expression and activation, NF-κB activation, and ICAM-1 expression. Ald (1-100 nM) stimulated SGK-1 expression and phosphorylation (Ser-422) dose-dependently. Ald (10 nM) stimulated phosphorylation of SGK-1 from 1 h, and increased protein expression of SGK-1 from 3h. The increments of phosphorylation and expression of SGK-1 by Ald were inhibited by MR inhibitor (Eplerenone). Ald (10 nM) stimulated NF-κB activity measured by NF-κB responsive elements-luciferase assay and phosphorylation of Ik -B from 3 h. This activation of NF-κB by Ald was inhibited to 30% by transfection of dominant-negative SGK-1, and enhanced by wild-type SGK-1. Furthermore, ICAM-1 promoter activity and protein expression increased by Ald (10 nM) from 3 h and 6 h, respectively. The effects of Ald on ICAM-1 promoter activity and protein expression were inhibited by transfection of dominant-negative SGK-1 and dominant-negative Ik-B in rat mesangial cells. We also demonstrated that Ald (10 nM) activated Ki-Ras-A, c-Raf kinase, MEK1/2, MAPK1/2, and induced cyclin D1, cyclin A expression, and cell cycle progression in rat mesangial cells. Moreover, we found that the MR inhibitor significantly ameliorated glomerular sclerosis and inflammation induced by aldosterone-infusion (five weeks in 21 rats) in vivo. The expression of SGK-1 and ICAM-1 in glomeruli was markedly increased by Ald treatment. Increments of SGK-1 and ICAM-1 expression induced by Ald in glemeruli were markedly reduced by MR In conclusion, our findings suggest that Ald stimulates SGK phosphorylation and protein expression in mesangial cells via MR. Ald stimulates NF -kB at least in part by activation of SGK-1. Ald also stimulates cell cycle progression in mesangial cells. Furthermore, Ald stimulates ICAM-1 transcription via NF-κB and SGK-1. Ald may have key roles in NF-κB- and ICAM-1-related inflammation and cell proliferation in mesangial cells in vivo and in vitro. MR antagonist may serves as a potential therapeutic approach to glomerular inflammatory diseases. 7. ACUTE SUPPRESSIVE EFFECT OF INTERLEUKIN-1β ON K+
CHANNEL ACTIVITY IN HUMAN PROXIMAL TUBULE CELLS

Kazuyoshi NAKAMURA, You KOMAGIRI, Toshiyuki KOJO, Manabu KUBOKAWA
Department of Physiology, Iwate Medical University School of Medicine, Morioka
020-8505, Japan

Interleukin-1ß (IL-1ß) is one of the cytokines which augment cell injury in various organs during inflammatory diseases. Although some investigators reported that changes in K+ channel activity were involved in the renal tubular cell injury induced by endotoxemia or ischemia, little information is available regarding effects of cytokines on activity of renal K+ channels. In this study, we investigated the effect of IL-1ß on activity of an inwardly rectifying K+ channel (Gi: 40 pS, Go: 7 pS) in cultured human proximal tubule cells, using the patch-clamp technique. In cell-attached patches, IL-1ß (15 pg/ml) suppressed the K+ channel activity in a few minutes after its addition to the bath. This acute suppressive effect was blocked by IL -1 receptor antagonist (20 ng/ml). As we reported previously, the activity of this K+ channel was increased by 8Br-cAMP (100 µM) and 8Br-cGMP (100 µM) through activation of protein kinases A (PKA) and G (PKG), respectively. However, these cyclic nucleotides did not stimulate channel activity in the presence of IL-1ß, suggesting that IL -1β might interfere with the effects of cAMP/PKA and cGMP/PKG pathways on channel activity. Since it was reported that protein kinase C (PKC) suppressed K+ channel activity in opossum kidney proximal tubule cells or rat cortical collecting duct, we next examined whether PKC would mediate the suppressive effect of IL -1β. When the cultured human proximal tubule cells were pretreated with a PKC inhibitor, GF109203X (500 nM), IL-1β did not suppress channel activity. Furthermore, GF109203X reactivated the channel which was suppressed by IL-1β. In inside-out patches, PKC- α (1 U/ml) reduced channel activity in the presence of phorbol 12-myristate 13-acetate (PMA, 500 nM) and 10-6 M Ca2+. PMA alone had no appreciable effect on channel activity. In addition, IL-1β itself did not affect channel activity in inside-out patches. These results suggested that IL-1β acutely suppressed K+ channel activity through its specific receptor in cultured human proximal tubule cells, and that the suppressive effect would be dependent on activation of the PKC pathway which overcame the PKA/PKG-dependent processes. 8. INHIBITION OF AN INWARDLY RECTIFYING K + CHANNEL BY
CYCLOSPORIN A INVOLVES Ca2+/CAM -KINASE II-MEDIATED
PROCESSES IN HUMAN PROXIMAL TUBULE CELLS

Manabu KUBOKAWA, Toshiyuki KOJO, You KOMAGIRI, Kazuyoshi NAKAMURA
Department of Physiology, Iwate Medical University School of Medicine, Morioka,
020-8505 Japan
Cyclosporin A (CsA) is an inhibitor of a Ca2+/CaM-depdent phosphatase (PP-2B), and a well-known immunosuppressive agent. CsA is clinically used after organ transplantation or for the therapy of autoimmune diseases. However, several complications, such as hypertension or decrease in the renal function, are observed during use of CsA. One of the significant findings of CsA-induced changes in renal cells is suppression of K+ channel activity in the apical or basolateral membrane of several nephron segments. In cultured human proximal tubule cells (RPTECs), an inwardly rectifying K+ channel with inward conductance of about 40 pS is the K+ channel which is most frequently observed in RPTECs, and its activity was inhibited by application of CsA (5 µM). In this study, we examined the mechanism for the CsA-induced suppression of this K+ channel in RPTECs using the patch-clamp technique and fluorescence imaging with fura-2. Application of CsA (5 µM) to RPTECs significantly suppressed the channel activity in cell-attached patches with a moderate elevation of the intracellular Ca2+ concentration ([Ca2+]i) which was evaluated by using fura-2 imaging. Since CsA attenuates the PP-2B-induced dephosphorylation, it is conceivable that the CsA-induced channel suppression may involve Ca2+-dependent protein phosphorylation. Thus we examined the effects of inhibitors of protein kinase C (PKC) and Ca2+/CaM kinase II (CaMKII) on CsA-induced channel suppression. In the presence of a PKC inhibitor, GF109203X (500 nM), CsA-induced channel suppression occurred, but it was prevented in the presence of an inhibitor of CaMKII, KN-62 (20 nM), suggesting that the CsA-induced channel suppression was CaMKII-dependent. Furthermore, we examined the direct effect of CaMKII on channel activity in inside-out patches in the absence and presence of PP-2B. In inside-out experiments, bath solution contained 1 mM ATP to maintain channel activity and 10-6 M Ca2+ and 0.6 µM CaM to stimulate CaMKII or PP-2B. Application of CaMKII (0.15 U/ml) induced channel suppression in inside-out patches, indicating that CaMKII has the inhibitory effect on channel activity. Moreover, the following addition of PP-2B (800 U/ml) re-activated the suppressed channel activity even in the presence of CaMKII. Thus, it is strongly suggested that inhibition of PP-2B by CsA elevates [Ca2+]i and evoked CaMKII-mediated channel suppression. We conclude that CsA-induced channel suppression is mainly caused by CaMKII-mediated phosphorylation processes in 9. THE VOLTAGE-DRIVEN ORGANIC ANION TRANSPORT OF TYPE I
SODIUM-PHOSPHATE COTRANSPORTER AND OATv1

Promsuk JUTABHA, Naohiko ANZAI, Hitoshi ENDOU, Hiroyuki SAKURAI
Department of Pharmacology and Toxicology, Kyorin University School of Medicine,
Mitaka-shi, Tokyo 181-8611, Japan
Type I sodium -phosphate cotransporter (NPT1), a protein initially characterized as phosphate transporter, is expressed at the apical membrane of renal proximal tubular cells and mediated the transport of organic anions (Uchino et al. Biochem Biophys Res Commun, 2000). The predicted amino acid sequence of NPT1 exhibited 60-65% identity to that of OATv1, a voltage-driven organic anion transporter identified in pig kidney. NPT1 is suggested to be the human orthologue of OATv1 (Jutabha et al. J Biol Chem, 2003). To clarify the functional properties of NPT1 in comparison to OATv1, we used HEK293 expressing either OATv1 or NPT1 as an expression system. NPT1 showed high affinity transport of estrone sulfate, as shown in that of OATv1. The transport was Na+-independent and enhanced at high concentrations of extracellular potassium and chloride-free condition. Using oocyte expression system, the transport of PAH and estrone sulfate by NPT1 was enhanced by chloride-free condition not by high potassium concentration, different from that observed in OATv1-expressed oocytes. This finding is the first report supporting the functional property of NPT1 as 10. VOLTAGE-DEPENDENT TRANSPORT OF URATE ON A NOVEL
HUMAN URATE EFFLUX TRANSPORTER URATv1 EXPRESSED IN
Xenopus OOCYTES

Promsuk JUTABHA1, Naohiko ANZAI1, Shuji KANEKO2, Hiroyuki SAKURAI1
1Department of Pharmacology and Toxicology, Kyorin University School of Medicine, Mitaka-shi, Tokyo 181-8611, Japan 2Department of Molecular Pharmacology, Graduate School of Pharmaceutical Sciences, Kyoto University Voltage-driven urate efflux transporter URATv1 (SLC2A9) is a member of the facilitative glucose transporter (GLUT) family that regulates blood urate level in humans and its function is inhibited by a uricosuric agent benzbromarone (Anzai et al., J Biol Chem, 2008). Although depolarization induced by the elevation of extracellular K+ concentration enhanced URATv1-mediated urate transport in Xenopus oocytes, it is still unclear whether this effect is due to change in membrane potential or extracellular K+ concentration. In the present study, we used two-electrode voltage-clamp to demonstrate the voltage-dependent urate transport of URATv1 heterologously expressed in Xenopus oocytes. The urate-evoked, outward current was linearly increased with depolarization when the oocytes were perfused with 0.5 mM urate, indicating that urate transport through URATv1 is affected by membrane potential. The urate-evoked current was completely inhibited in the presence of benzbromarone (0.1 mM), confirming the involvement of URATv1. Moreover, when external Na+ was fully replaced with K+, the urate -evoked current was not affected, indicating that the URATv1-mediated conductance is not dependent on external Na+ or K + concentrations. These results suggest that urate is transported into oocytes through URATv1 in a membrane potential, but not Na+ or K+, dependent manner favoring depolarization, where urate moves from negative to positive direction. In conclusion, we provide electrophysiological evidences that URATv1 is a voltage dependent urate transporter. In a physiological state in the kidney proximal tubular cell, urate is likely to be transported out of the cell through URATv1 which located in the basolateral 11. IDENTIFICATION OF THE MULTIVALENT PDZ PROTEIN PDZK1
AS A BINDING PARTNER OF SODIUM –COUPLED
MONOCARBOXYLATE TRANSPORTER SMCT1 (SLC5A8) AND
SMCT2 (SLC5A12) BY YEAST TWO-HYBRID ASSAY

Sunena SRIVASTAVA1, Naohiko ANZAI1, Seiji MIYAUCHI2, Daisaku MIURA1,
Toshiyuki FUKUTOMI1, Vadivel GANAPATHY, Hiroyuki SAKURAI1
1Department of Pharmacology and Toxicology, Kyorin University School of Medicine,
Mitaka-shi, Tokyo 181-8611, Japan
2College of Pharmaceutical Sciences, Matsuyama University, Matsuyama-shi, Ehime, Japan 3Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA 30912, USA SMCT1 (SLC5A8) and SMCT2 (SLC5A12) are transporters that mediate the transport of lactate in the apical membrane of proximal tubular cells. Since URAT1 is also expressed in the apical membrane of renal proximal tubular cells and transports urate in exchange for intracellular organic anions such as lactate and nicotinate, SMC T1 and SMCT2 seem important for the transport function of URAT1 (Anzai et al., Curr Opin Rheumatol, 2007). In addition, SMCT1 & SMCT2 C-terminal domain is in the cytoplasm and contains PDZ motif, suggesting that it interacts with PDZ proteins. In this study, we used the yeast two-hybrid screening to investigate the putative SMCT1- and SMCT2 -associated proteins in the kidney. Using the SMCT1 and SMCT2 C-terminus as bait, we performed yeast two -hybrid screenings of a human adult kidney cDNA library. In both cases, an identical sequence encoding the gene for the multivalent PDZ domain-containing protein PDZK1 was obtained (13 out of 22 for SMCT1, 8 out of 34 for SMCT2). The elucidation of these interactions may lead to the further understanding of functional regulation of urate transport via monocarboxylate 12. INTERACTION OF A NOVEL URATE EFFLUX TRANSPORTER
URATv1 (SLC2A9) WITH URICOSURIC AGENTS

Naohiko ANZAI, Promsuk JUTABHA, Chun Ji JIN, Toru KIMURA, Ellappan BABU,
Sunena SRIVASTAVA, Hitoshi ENDOU, Hiroyuki SAKURAI
Department of Pharmacology and Toxicology, Kyorin University School of Medicine,
Mitaka-shi, Tokyo 181-8611, Japan
Hyperuricemia is a significant factor in a variety of diseases, including gout and cardiovascular diseases. And renal excretion largely determines blood urate level. in 2002, we identified a major urate reabsorptive transporter, URAT1 (SLC22A12), on the apical side of the renal proximal tubular cells (Enomoto et al., Nature, 2002). Recently, we identified that URATv1 (SLC2A9) functions as a urate efflux transporter from renal tubular cells into the blood circulation (Anzai et al., J Biol Chem, 2008). URATv1-expressed Xenopus oocytes transported urate favoring negative to positive potential direction. In vivo role of URATv1 is supported by the fact that a renal hypouricemia patient without any mutations in SLC22A12 was found to have a missense mutation P412R in SLC2A9, which reduced urate transport activity in vitro. Considering its basolateral localization, SLC2A9 is likely to mediate the second step of urate reabsorption. To further characterize the substrate selectivity of URATv1, we performed the urate uptake inhibition study in the URATv1 expressed oocytes using several organic anions that are known to interact with URAT1. intracellularly-injected glucose, fructose, monocarboxylates such as lactate, nicotinate or pyrazinoate (PZA) for urate. In addition, urate efflux via URATv1 was not enhanced by extracellular glucose or fructose. Urate transport via URATv1 was affected by uricosuric agents probenecid, benzbromarone, phenylbutazone and losartan, but not by monocarboxylates such as lactate, nicotinate, orotate, PZA, or -hydroxybutyrate. In summary, different substrate selectivity between URATv1 and URAT1 suggests that URATv1 may be a novel therapeutic target for hyperuricemia. 13. CALCIUM , MAGNESIUM , AND CALCIMIMETICS DECREASE
PNa/PCl RATIO IN MOUSE THIN ASCENDING LIMB OF HENLE’S
LOOP

Tetsuji MORIMOTO1), Noriko SUGAWARA1), Elnur I. FARAJOV2), Fumiaki KAMADA1),
Naonori KUMAGAI1), Shigeru TSUCHIYA 1), Yoshiaki KONDO2)
1) Department of Pediatrics, Tohoku University Graduate School of Medicine,
1-1 Seiryo-machi, Aoba-ku, Sendai, 980-8574, Japan
2) Department of Medical Informatics, Tohoku University Graduate School of
Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai, 980-8574, Japan
It is well known that Randall’s plaque, the initial lesion in calcium-oxalate nephrolithiasis, is formed in the basement membranes of the thin ascending limbs (ATLs) of Henle’s loop. We demonstrated in 1995 that Ca2+ pump and Ca2+ channel are present in the ATLs (Takahashi et al. Am J Physiol). While it is known that NaCl transport is stimulated by ambient Ca2+ (Kondo et al. Am J Physiol 1988), the contribution of calcium-sensing receptor (CaSR) to NaCl transport in the ATLs has not In this study, the effect of Ca2+, Mg2+, and calcimimetics on NaCl transport was investigated in the in vitro isolated microperfused mouse thin ascending limb (ATL) of Henle’s loop by measuring transepithelial voltage (Vt). In the presence of a transmural NaCl concentration gradient (100 mM higher in the lumen), the transepithelial diffusional potential (Vd) was 13.7 + 0.4 mV (n=17). When Ca2+ concentration in the bathing fluid was increased from 1.5 mM to 4.5 mM at 37 °C, the relative permeability of Na+ to Cl- (PNa/PCl) estimated from the NaCl diffusion voltage changed from 0.371 + 0.017 to 0.341 + 0.015 (n=10, P<0.0001). Increasing Ca2+ concentration in the luminal fluid from 1.5 mM to 4.5 mM decreased PNa/PCl from 0.349 + 0.013 to 0.330 + 0.013 (n=5, P=0.001). When Mg2+ concentration in the bathing fluid was increased from 1 mM to 5 mM at 37°C, the PNa/PCl decreased from 0.339 + 0.020 to 0.319 + 0.020 (n=5, P<0.0001). Increasing Mg 2+ concentration in the luminal fluid from 1 mM to 5 mM decreased PNa/PCl from 0.347 + 0.033 to 0.333 + 0.033 (n=5, P<0.0001). The addition of 0.1 mM Neomycin (Neo) and 0.2 mM Gentamicin (Gen) to the basolateral or luminal solution also decreased P Na/PCl. Besides, the addition of 30uM Gadolinium (Gd) to the basolateral solution also decreased PNa/PCl. These data indicate that ambient Ca2+, Mg 2+, and calcimimetics inhibit Na+ transport in the ATLs, which is known to occur via Our observations strongly suggest that Ca2+ is a potent regulator of paracellular Na+ permeability, leading to prevention of calcium stone formation via inhibition of urine 14. ROMK1 KNOCKOUT MICE DO NOT PRODUCE BARTTER’S
PHENOTYPE BUT EXHIBIT HYPERTENSION
Ke Dong, Qingshang Yan, Gerhard Giebisch, Steven Hebert and Tong Wang. Department of Cellular & Molecular Physiology, Yale University School of Medicine, 333 Cedar Street, New Have n, CT, 06520, USA We have previously reported that ROMK knockout mice show a similar phenotype to Bartter’s syndrome, which is salt wasting and dehydration due to reduced Na-2Cl-K-cotransporter activity (Lu et al, JBC 2002, Cantone et al, AJP 2008). The salt and water wasting in ROMK Bartter’s mouse is compensated by increased thiazide-sensitive NaCl transport activity, distal tubule hypertrophy, elevated renin-Ang II and aldosterone levels (Cantone et al, AJP 2008, Wagner et al, AJP, 2008). At least three ROMK isoforms have been identified in the kidney; however, unique functions of any of the isoforms in nephron segments are still poorly understood. To establish the roles of ROMK1 in distal tubules and collecting ducts, we have generated a mouse deficient only in ROMK1 by selective deletion of the ROMK1 exon, using an ES cell Cre-LoxP strategy. We examined general and renal phenotypes in ROMK1 null mice by metabolic and renal clearance experiments. Our data show no difference in plasma Na+, K+ and acid-base parameters between littermate WT and ROMK1 null mice. Unlike ROMK null mice with a 3-fold increase in urine volume, 60% increase in Na+ and K+ excretion, and a 3-fold and 60% increase in water and food intake, the re was no significant difference in urine volume, Na+, K+ excretion, and water and food intake between WT and ROMK1 null mice. Renal clearance data show no difference in urine output (1.39 vs. 1.48ml/min) and GFR (0.9 ±0.03 vs. 0.75±0.09) between WT and ROMK1 null mice. However, the fractional Na+ excretion was significantly reduced (0.32±0.05 vs. 0.56±0.08%) in ROMK null mice compared with the WT. The mean blood pressure, measured via the carotid artery, was higher in ROMK1 null mice compared with background-matched wild-type controls. Mean blood pressure was 112.3±6.48mmHg and 133.9±5.58mmHg (n=16 and n=15, P<0.05) in WT and ROMK1 null mice respectively. To investigate the mechanism of the hypertension, we examined kidney Na+ channel activities by testing the effect of the ENaC inhibitor benzamil on renal clearance. Experimental data show that benzamil produces a higher natriuretic effect in ROMK1 null mice than in WT control. Benzamil increased ENa and FENa by 6.5-fold and 3.2-fold in ROMK1 null mice, and 2.9-fold and 1.8-fold in WT control, respectively. The increased sensitivity to benzamil suggests elevated ENaC activity in ROMK1 KO mice. We conclude that the ROMK1 knockout mouse does not produce Bartter’s phenotype of lost salt and water, but rather increased ENaC activity leading to 15. CRITICAL ROLES OF V1A AND V1B VASOPRESSIN RECEPTORS
IN SYSTEMIC BLOOD PRESSURE HOMEOSTASIS: STUDIES FROM
GENE KNOCKOUT MICE
Yoko FUJIWARA1, Taka-aki KOSHIMIZU1, Hiroyoshi TSUCHIYA 1, Gozoh TSUJIMOTO2, and Akito TANOUE3 1Div. of Molecular Pharmacol., Dep. of Pharmacol., Jichi Medical University, Yakushiji 329-0498, Tochigi, Japan. 2Dep. of Genomic Drug Discovery Science, Graduate School of Pharmaceutical Sciences Kyoto University Faculty of Pharmaceutical Sciences, Kyoto University, Yoshida shimoadachi 606-8501, Kyoto, Japan 3Dep. of Molecular and Cellular Pharmacol., National Research Institute for Child Health and Development, Setagaya 157-8535, Tokyo, Japan Three receptor subtypes, V1a, V1b, and V2, are activated by arginine -vasopressin (AVP) and modulate both osmotic and cardiovascular homeostasis. Although a potent vasoconstrictor effect of intravenously administered AVP is mediated by V1a, the physiological contribution of V1a and V1b to basal blood pressure (BP) is ill-defined. We investigated the functional roles of the V1a and V1b in cardiovascular homeostasis by generating mice lacking each receptor. The basal BP of conscious mutant mice lacking the V1a receptor gene (V1a-/-) was significantly lower compared to the wild-type mice without a notable change in heart rate. AVP-induced vasopressor responses were abolished in the V1a-/- mice; rather, AVP caused a decrease in BP, which occurred in part, through V2 receptor-mediated release of nitric oxide from the vascular endothelium. In V1a-/-, arterial baroreceptor reflexes were markedly impaired and a significant 9 % reduction in circulating blood volume was noted. The plasma renin activity and aldosterone level, as well as AVP-stimulated aldosterone release, are reduced in V1a-/- mice. The V1a was found to specifically co-express at the macula densa cells with cyclooxygenase-2 and neuronal nitric oxide synthase. In contrast to V1a-/-, systolic BP of V1b knockout mice (V1b-/-) was elevated. Cardiac contractile functions assessed by echocardiography were enhanced and heart weight/body weight ratio was increased in V1b-/-. Taken together, these results indicate that both V1a and V1b receptors are critically involved in the maintenance of resting arterial BP 16. FUROSEMIDE-INDUCED nNOS OF THE MOUSE MACULA DENSA
CELL LINE LACKS A C-TERMINAL REDUCTASE DOMAIN, BUT
GENERATES NO Ca2+-DEPENDENTLY
Hideaki KAWADA1,3), Yukiko YASUOKA1), Mizuka KOBAYASHI2), Hidekazu FUKUDA1), Yukito ISHIZAKA3), and Katsumasa KAWAHARA1) Departments of Physiology1) and anesthesiology2), Kitasato University School of Medicine, Sagamihara 228-8555, Japan Department of Intractable Diseases3), International Medical Center of Japan, Tokyo 162-8655, Japan Macula densa (MD) modulates tubuloglomerular feedback (TGF) system by generating nitric oxide (NO) in response to changes in luminal NaCl concentration. An established cell line of mouse MD (NE-MD) shows upregulation of neuronal NO synthase (nNOS) protein and generates L-arginine-induced NO in the presence of furosemide, an inhibitor of Na+-K+-2Cl- transporter. Estimated molecular weight of furosemide-induced nNOS protein (65 kD) was much shorter than brain or cardiac type of nNOS (160 kD), suggesting that nNOS protein of NE-MD may be largely truncated. To further investigate the whole structure of nNOS and the deleted parts of the functional domains, we have examined the furosemide (12 µM)-induced proteomes of NE-MD by two-dimensional gel electrophoresis (2-DE) combined with mass spectrometry (MALDI-TOF-MS). Eighteen unique proteins have been identified. These proteins include mainly cytoplasmic proteins (61%) and some mitochondrial (11%), endoplasmic reticulum (17%), and membrane proteins (11%). Although 6 protein spots were increased approximately by 1.5 times in the presence of furosemide, one unique protein spot, finally determined as nNOS, increased by more than 5 times. According to the results of MS fit, N-terminal heme binding domain was highly conserved, but, a C-terminal reductase domain, such as the flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), and nicotinamide adenine dinucleotide phosphate (NADPH) binding regions, was missing. Further, by using an NO sensing electrode, L-arginine -induced NO production was Ca2+-dependent and pH-sensitive. These findings suggest that C-terminal truncated nNOS of NE-MD cells may explain unique regulation of gene expression and activity in mouse macula densa. 17. REGULATION OF PGE2 PRODUCTION IN MOUSE MACULA DENSA

Hidekazu FUKUDA, Katsumasa KAWAHARA
Department of Physiology, Kitasato University School of Medicine. Kitasato 1-15-1,
Sagamihara, 228-8555, Japan
Prostaglandin E2 (PGE2), derived from cyclooxgenase-2 (COX-2) expressed in the macula densa (MD), is known as a signal molecule stimulating the release of renin from juxtaglomerular cells of the afferent arterioles in the kidney, especially during volume depletion. The E prostanoid (EP) receptors comprise four isoforms (EP1-EP4) coupled with subcellular signal transduction mechanisms. Although EP receptors expressed in MD may control renin release through the tubuloglomerular feedback (TGF) system, its role and cellular mechanisms remain uncertain. We have recently established a novel mouse macula densa cell line (NE-MD). In NE-MD cells, we examined the COX-2 gene expression, cAMP formation, and the PGE2 production to determine whether EP isoforms are involved in the PGE2 production. Real-time PCR revealed that the COX-2 gene expression was increased to 3.5 times in the presence of 10 µM of forskolin, an adenylyl cyclase activator, and that the low [chloride]-induced COX-2 expression was completely abolished by the addition of 10 µM of H-89, a PKA inhibitor. Further, by using RT-PCR technique, we found an expression o f EP4 isoform, a Gs protein-coupled receptor, but no expression of EP1-EP3 isoforms. More over, the addition of PGE2 to the bathing medium stimulated significantly cAMP formation in a dose-dependent manner (radioimmunoassay). Finally, the COX-2 gene expression was increased to 1.5 times in the presence of 5 µM of PGE2. In summary, these results indicate that the COX-2-derived PGE2 elevates the levels of the COX-2 expression in MD in a positive feedback mechanism via EP4 receptor. This suggests that orchestrated signals of PGE2 may be essential for suitable renin release through TGF 18. A ROLE OF V1A VASOPRESSIN RECEPTOR FOR URINARY
SODIUM EXCRETION IN DIABETIC KIDNEY OF MICE

Mizuka KOBAYASHI1, Yukiko YASUOKA2, Akito TANOUE3, Katsumasa KAWAHARA2,
and Hirotsugu OKAMOTO1
1,2. Departments of Anesthesiology and Cellular & Molecular Physiology, Kitasato
University Grad. Sch. of Med. Sci., Kitasato 1 -15-1, Sagamihara 228-8555, Japan
3. Department of Pharmacology, National Research Institute for Child Health and
Development, Okura 2-10-1 Tokyo 157-8535, Japan
V1a vasopressin receptor (V1aR) plays a key role not only in body water/electrolytes balance (Koshimizu et al, 2006), but also in plasma glucose homeostasis (Aoyagi et al, 2007). Here we report that V1aR-deficient (V1aR-/-) mice may lose a regulatory function of auto -regulation systems in kidney, such as tubuloglomerular feedback (TGF) and urinary Na+ excretion, especially during diabetic nephropathy. Streptozotocin-induced diabetic mice of control (WT) and V1aR-/- (KO) were fed under conditions of standard animal diet for 14-16 wks. There were no significant differences in body weight increases and blood glucose levels between WT and KO mice. In contrast, although water intake and urine volume was regulated within the same levels, plasma renin activity significantly (P < 0.05) increased from 20.8 to 42.3 ng/ml/hr in control (n=4), but was unchanged in KO mice (24.0 to 28.6 ng/ml/hr, n=4) after diabetes. Interestingly, urinary Na+ excretion remained high in KO mice after diabetes (22.3 to 20.5 µmol/d/Cr, n=4), whereas it was significantly (P < 0.05) decreased from 23.9 to 18.7 µmol/d/Cr in control (n=4). Further, immunohistochemistry revealed that a population of nNOS (neuronal nitric oxide synthase)-immunoreactive cells (macula densa: MD) was unchanged, but a level of the staining in MD cells markedly decreased in diabetic KO mice, compared with control (diabetic WT mice). Taken together, these results suggest that V1aR-deficient (V1aR-/-) mice may lose a normal function of TGF system against urinary Na+ loss. This may explain a mechanism of unusual loss of sodium in diabetic nephropathy. 19. DIFFERENT RESPONSES OF MACULA DENSA TO SALT LOAD
BETWEEN DIABETIC AND NON-DIABETIC KIDNEY IN RATS

Hajime HASEGAWA, Kaori TAKAYANAGI, Taisuke SHIMIZU, Takatsugu IWASHITA ,
Yosuke TAYAMA, Juko ASAKURA, Koichi KANOZAWA, Hitoshi KATO, Tetsuya
MITARAI
Dept of Nephrology and Hypertension, Saitama Medical Center, Saitama Medical
University, Kamoda 1981, Kawagoe, Saitama 350-8550, Japan
Distinctive tubuloglomerular (TG) feedback response in diabetic kidney is considered as a one of possible cause for the development of glomerular hypertension (GHT) which is a key step of progressive renal damage in early diabetes. The aim o f this study is to examine the contrasts of macula densa responses to salt load between METHOD: Streptozotocin (60 mg i.p.) induced diabetic rats were fed by low (LS: 0.1% NaCl) and high salt (HS: 1.2% NaCl) diet for 4 weeks. Evaluation of GHT was studied by histological analysis. Gene expression and immunoreactivity of molecular markers for macula densa function were studied by RT-PCR and RESULTS: Diabetic kidney showed more significant glomerular hypertrophy and the increase in creatinine clearance and urine albumine excretion by HS diet than non-diabetic kidney. nNOS expression was increased by HS diet in non-diabetic kidney (109.0±19.5% vs 161.3±46.1%), but decreased in diabetic kidney (77.4±8.3 vs 51.0±13.9%). Similarly, COX-2 expression was decreased by HS in non-DM (102.4±13.0% vs 73.0±18.8%), but not in DM (50.7±7.4% vs 49.0±13.7%). Difference of immunoreactivity of nNOS and COX-2 in macula densa between LS and HS diet DISCUSSION: Lack or attenuation of macula densa response to high salt load in diabetic kidney might indicate that HS diet-induced increase in sodium delivery to macula densa was blunted in diabetic kidney by increased sodium reabsorption in proximal tubules. The changes in macula densa response might cause the attenuation of TG feedback and sustained GHT in diabetic kidney. 20. DIFFERENCE OF HIGH SALT DIET-INDUCED GLOMERULAR
HYPERTENSION BETWEEN TYPE-2 DIABETES AND
NON-DIABETES
Kaori TAKAYANAGI, Hajime HASEGAWA, Taisuke SHIMIZU, Takatsugu IWASHITA, Yosuke TAYAMA, Juko ASAKURA, Koichi KANOZAWA, Akihiko MATSUDA, Tetsuya MITARAI Dept of Nephrology and Hypertension, Saitama Medical Center, Saitama Medical University, Kamoda 1981, Kawagoe, Saitama 350-8550, Japan Although glomerular hypertension (GHT) is considered as a key step for the progressive renal damage in early diabetes, the mechanisms of its development are not well understood. The aim of this study is to examine the different responses to salt load in the severity of glomerular damages between diabetic and non-diabetic rats for the evaluation of significance of salt diet as an accelerating factor of diabetic METHOD: Goto-Kakizaki rats (GK), model of non-obese type 2 diabetes, and normal Wistar rats (W) were grown by high salt (HS, 6.0% NaCl) or low salt (LS, 0.03% NaCl) diet for 12 weeks. Severity of glomerular damages were evaluated by histological analysis. Gene expression of tubular salt transporting molecules were studied by RT-PCR. For evaluation of tubular function, furosemide (5 mg/kg, ip) or hydrochlorothiazide (10 mg/kg, ip) were administered as diuretics challenge tests. Primary cultured cells originated from renal cortex and outer medulla were used for in RESULTS: GK with HS diet showed more significant glomerular hypertrophy and an increase in the expression of TGF beta and desmin in glomerulus that W with HS diet. number of TGF beta positive glomerular cells than W-HS. Gene expression of salt transporting molecules in pre-macula densa segments was enhanced by HS diet in GK but not in W (NHE3: 101.8±6.1% in W-LS, 84.6±4.9% in W-HS, 99.2±5.5% in GK-LS, 162.7±11.3% in GK-HS, NKCC2: 103.1±9.7% in W-LS, 106.2±12.5% in W-HS, 101.4±6.5% in GK-LS, 144.3±10.1% in GK-HS). However, the expression of the molecules in post-macula densa segments was not different between GK and W. Increase in FENa by furosemide, but not thiazide, was significantly high in GK with HS diet (13.8±1.9-times in W-LS, 12.8±2.7 -times in W-HS, 19.3±3.1-times in GK-LS, 43.2±2.3-times in GK-HS). In vitro study of gene expression of the molecules showed that high salt condition increased gene expression of pre-macula densa molecules but not post-macula densa molecules (NHE3: 184±41.8% in high glucose, 1791.2±202.9% in high glucose+HS, NKCC2: 162.7±38.0% in high glucose, 1292.4±192.9% in high glucose+HS), although high glucose or mannitol-induced high osmolality did not DISCUSSION: Present study showed that high salt load caused more serious GHT in diabetic kidney than non-diabetic kidney. These results might indicate that diabetic kidney showed different response to salt load comparing to non-diabetic kidney, resulting the increase in salt reabsorption in pre-macula densa segments, possible attenuation of tubuloglomerular feedback and development of GHT. 21. CARBONYL STRESS MEDIATED MYOCARDIAL FIBROSIS AND
RENAL INJURY IN DAHL SALT SENSITIVE RATS

Qi GUO1, Takefumi MORI1,2, Xianguang CHEN1, Chunyan HU1, Yusuke OHSAKI1,
Yoshimi YONEKI1, Toshio MIYATA4, Masayuki KANAZAWA3, Masahiro KOHZUKI3,
Sadayoshi ITO1.
1 Division of Nephrology, Endocrinology and Vascular Medicine, Tohoku University
Graduate School of Medicine, Sendai, 980-8575, Japan.
2 Health Administration Center, Tohoku University, Sendai, 980-8575, Japan.
3 Department of internal Medicine and Rehabilitation Science, Tohoku University
Graduate School of Medicine, Sendai, 980-8575, Japan
4 Center for Translational and Advanced Research, Tohoku University Graduate
School of Medicine, Sendai, 980-8575, Japan.
Objectives Carbonyl stress is enhanced in subjects with chronic kidney disease, but
whether it involves in the cardio-renal connection is not fully known. The present study determined the role of carbonyl stress in the pathogenesis of cardio-renal connection Methods Rats were given either 1% methylglyoxal in tap drinking water or tap water
alone for 12 weeks. In another set of rats, either candesartan (an angiotensin II receptor blocker, 10 mg/kg/day) alone, standard rat chow with 1% methylglyoxal in tap drinking water or candesartan plus methylglyoxal was given for 4 weeks. Results Increase in blood pressure was observed from a baseline level of 123±1
mmHg to a level of 148±5 mmHg after 12 weeks administration of methylglyoxal, a precursor of carbonyl stress. This was followed by increased urinary albumin excretion, glomerular sclerosis and tubular injury. Enhanced expression of renal advanced glycation endproducts (Ne-carboxyethyl-lysine, CEL), 8-hydroxydeoxyguanosine (8-OHdG, a marker of oxidative stress), and NAD(P)H oxidase activity was observed in the kidney of rats treated methylglyoxal for 4 weeks. Treatment of candesartan fo r 4 weeks completely attenuated the hypertensive response to methylglyoxal and reversed the tubulo-glomerular injury. Co-administration of cadesartan and methylglyoxal significantly attenuated renal expression of CEL, 8 -OHdG and NAD(P)H oxidase activity compared with methylglyoxal alone. Methylglyoxal significantly increased myocardial total collagen content (a percent rate of Sirius -red stained collagen area to total myocardial area), and candesartan significantly attenuated this in Conclusion These results indicate that carbonyl stress play a role in the pathogenesis
of hypertension and cardio-renal connection in chronic kidney disease, which is at least in part mediated through oxidative stress in Dahl S rats. 22. FUNCTIONAL CHARACTERIZATION OF CONNEXINS IN
JUXTAGLOMERULAR APPARATUS
Tsuneo TAKENAKA, Tsutomu INOUE, Yoshihiko KANNO, Hiromichi SUZUKI. Department of Nephrology, Saitama Medical University Faculty of Medicine, Iruma 350-0495 JAPAN Gap junctions are composed of connexins, and present in the juxtaglomerular apparatus enabling intercellular communication. Our study determined the location of different connexin subtypes within the juxtaglomerular apparatus of the rat, and the role of these subtypes in renal hemodynamics through the use of specific mimetic peptides. Immunohistochemical analysis showed connexin 37 and 40 expression in the endothelial and renin-secreting cells of the afferent arteriole while connexin 40 was also found in extra- and intraglomerular mesangial cells. In contrast, connexin 43 was weakly expressed in endothelial cells of the afferent arteriole and within the glomerulus. Intra-renal infusion of the peptides (GAP) reported to block specific Gap junctions ( Cx37,43GAP27 or Cx40GAP27) elevated blood pressure, plasma renin activity and angiotensin II levels while decreasing renal plasma flow without a significant change in the glomerular filtration rate. Subsequent restoration of blood pressure reduced both renal plasma flow and glomerular filtration rate. In contrast, Cx43GAP26 reduced glomerular filtration rate without alterations in blood pressure, renal plasma flow, plasma renin activity or angiotensin II levels. Hence, connexins 37 and 40 are expressed in the rat juxtaglomerular apparatus and these proteins control, in part, the renin-angiotensin system and renal autoregulation. 23. THE ACTIVATION OF PGE2 RECEPTOR TYPE4 IN RENAL
COLLECTING DUCTS INHIBITS THE ENAC EXPRESSION BY THE
ACTIVATION OF PKA AND THE INHIBITION OF P38 VIA NON-PKA
PATHWAY
Noritaka KAWADA1, Toshiki MORIYAMA1, Harumi K ITAMURA1, Carolyn M.
ECELBARGER2, William J. WELCH3, Christopher S. W ILCOX3, Enyu IMAI1, Yoshitaka
ISAKA1, and Hiromi RAKUGI1.
1Division of Nephrology, Osaka Univ Graduate School of Medicine, Osaka, JAPAN.
2Division of Endocrinology and Metabolism, Georgetown Univ, Washington, DC, USA. 3Center for Hypertension and Kidney, Diabetic and Vascular Diseases and Division of Nephrology and Hypertension, Georgetown University, Washington, DC, USA. Cyclooxygenase-1 (COX-1) deficient mice are salt-sensitive and have blunted production of prostaglandin E 2 (PGE2) and thromboxane A2 (TxA2). We hypothesized that the PGE2 deficiency in the kidney of COX-1 deficient mice causes sodium retention via the abnormal activation of sodium transporters. We have reported that the activation of PGE2 receptor type 4 (EP4) by its selective agonist (EP4AG; ONO-AE1-329, 10-6M) in cultured mice collecting duct (M-1) cells reduces the expression of alpha-, beta-, and gamma- amiloride-sensitive epithelial sodium transporter (ENaC) mRNAs and the protein phosphorylation of Akt and Sgk1. Established role of EP4 is the activation of adenylate cyclase, which leads to activate protein kinase A (PKA) via cAMP production. Thus the aim of this study was to dissect the role of PKA and non-PKA pathways on the expression of ENaCs in M-1 cells exposed to EP4AG. The inhibition of beta- and gamma-ENaC mRNAs and phosphorylated Akt by EP4AG were blunted by selective protein kinase A (PKA) inhibitor (H89, 10-6M), but H89 had no effect on the alpha-ENaC mRNA and the phosphorylated Sgk1 (ß:+41%, ?:+50%, p<0.01 vs EP4AG; n=5). These indicate the involvement of PKA pathway on the expression of beta-, and gamma-ENaC mRNAs and on the phosphorylation of Akt. In M-1 cell co-incubated with H89, EP4AG reduced the expression of alpha-, beta-, and gamma-ENaC mRNAs and the phosphorylation of Akt and increased the expression of MKP-1 mRNA (a:-24%, ß:-27%, ?:-42%, MKP-1:+3100%, p<0.01 vs Veh; n=5). These indicate the involvement of non-PKA pathway in the expression of alpha-, beta-, and gamma-ENaC and MKP-1 mRNAs and the protein phosphorylation of Akt. p38 is the targets of MKP-1 and, as anticipated, EP4AG reduced the protein phospholylation of p38. Selective inhibitor for p38 (SB203580) reduced the expression of alpha -, beta-, and gamma-ENaC mRNAs (SB203580, 10-5M for 6hrs; a:-14%, ß:-16%, ?:-40%, p<0.01 vs Veh; n=4). In conclusion; EP4AG inhibits the ENaC subunits mRNA and the protein phosphorylation of Akt and Sgk1 via the activation of PKA and non-PKA pathway. Inhibition of p38 signal via the induction of MKP-1 is a possible explanation for the reduced ENaC mRNAs by EP4-nonPKA pathway in collecting duct cells. Abnormal ENaC regulation due to the PGE2 deficiency in the kidney may contribute to the salt-sensitivity in COX-1

Source: http://www.med.kitasato-u.ac.jp/IUPS2009/image/poster.pdf