Analysis of WNK-NCC signaling cascade, a novel blood pressure controlling system, in the kidney

2013 Fiscal Year Final Research Report

• Project/Area Number: 22390168
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Kidney internal medicine
• Research Institution: Tokyo Medical and Dental University
• Principal Investigator: RAI TATEMITSU 東京医科歯科大学, 医学部附属病院, 准教授 (80334431)
• Project Period (FY): 2010-04-01 – 2014-03-31
• Keywords: 偽性低アルドステロン血症II型 / KLHL3 / Cullin3 / ユビキチン / SPAK / Na-K-Cl共輸送体 / OSR1 / angiotensin II

Research Abstract

The purpose of this study is to further characterize the WNK-NCC signal transduction system and to propose a novel strategy for treatment of hypertension.
Transgenic mice overexpressing WNK4 showed PHAII phenotypes, indicating that wild-type WNK4 protein was a positive regulator of NCC. KLHL3 interacted with Cullin3 and WNK4, induced WNK4 ubiquitination, and reduced the WNK4 protein level. PHAII-causing mutations in either protein reduced the ubiquitination of WNK4, resulting in an increased level of WNK4 protein. Thus, we found that WNK4 was a target for KLHL3-mediated ubiquitination, and impaired ubiquitination of WNK4 was the common mechanism of human hereditary hypertension.
As a result of screening chemical compounds that disrupted WNK/SPAK binding, we discovered three novel compounds that could be promising seeds of new types of antihypertensive drugs.

Research Products

• [Journal Article] Impaired degradation of WNK1 and WNK4 kinases causes PHAII in mutant KLHL3 knock-in mice (2014)
  • Author(s): Susa K, Sohara E, Rai T, Zeniya M, Mori Y, Mori T, Chiga M, Nomura N, Nishida H, Takahashi D, Isobe K, Inoue Y, Takeishi K, Takeda N, Sasaki S, Uchida S
  • Journal Title: Hum Mol Genet Volume: (Epub ahead of print)
  • Peer Reviewed
• [Journal Article] WNK4 is the major WNK kinase positively regulating NCC in the mouse kidney (2014)
  • Author(s): Takahashi D, Mori T, Nomura N, Khan MZ, Araki Y, Zeniya M, Sohara E, Rai T, Sasaki S, Uchida S
  • Journal Title: Biosci Rep Volume: (Epub ahead of print)
  • Peer Reviewed
• [Journal Article] Regulation of with-no-lysine kinase signaling by Kelch-like proteins (2014)
  • Author(s): Uchida S, Sohara E, Rai T, Sasaki S
  • Journal Title: Biol Cell Volume: 106(2) Pages: 45-56
  • DOI: 10.1111/boc.201300069
  • Peer Reviewed
• [Journal Article] Development of enzyme-linked immunosorbent assays for urinary thiazide-sensitive Na-Cl cotransporter (NCC) measurement (2013)
  • Author(s): Isobe K, Mori T, Asano T, Kawaguchi H, Nonoyama S, Kumagai N, Kamada F, Morimoto T, Hayashi M, Sohara E, Rai T, Sasaki S, Uchida S
  • Journal Title: Am J Physiol Renal Physiol Volume: 305(9) Pages: F1374-81
  • DOI: 10.1152/ajprenal.00208.2013
  • Peer Reviewed
• [Journal Article] Dietary salt intake regulates WNK3-SPAK-NKCC1 phosphorylation cascade in mouse aorta through angiotensin II (2013)
  • Author(s): Zeniya M, Sohara E, Kita S, Iwamoto T, Susa K, Mori T, Oi K, Chiga M, Takahashi D, Yang SS, Lin SH, Rai T, Sasaki S, Uchida S
  • Journal Title: Hypertension Volume: 62(5) Pages: 872-8
  • DOI: 10.1161/HYPERTENSIONAHA.113.01543
  • Peer Reviewed
• [Journal Article] Chemical library screening for WNK signalling inhibitors using fluorescence correlation spectroscopy (2013)
  • Author(s): Mori T, Kikuchi E, Watanabe Y, Fujii S, Ishigami-Yuasa M, Kagechika H, Sohara E, Rai T, Sasaki S, Uchida S
  • Journal Title: Biochem J Volume: 455(3) Pages: 339-45
  • DOI: 10.1042/BJ20130597
  • Peer Reviewed
• [Journal Article] Decrease of WNK4 ubiquitination by disease-causing mutations of KLHL3 through different molecular mechanisms (2013)
  • Author(s): Mori Y, Wakabayashi M, Mori T, Araki Y, Sohara E, Rai T, Sasaki S, Uchida S
  • Journal Title: Biochem Biophys Res Commun Volume: 439(1) Pages: 30-4
  • DOI: 10.1016/j.bbrc.2013.08.035
  • Peer Reviewed
• [Journal Article] KLHL2 interacts with and ubiquitinates WNK kinases (2013)
  • Author(s): Takahashi D, Mori T, Wakabayashi M, Mori Y, Susa K, Zeniya M, Sohara E, Rai T, Sasaki S, Uchida S
  • Journal Title: Biochem Biophys Res Commun Volume: 437(3) Pages: 457-62
  • DOI: 10.1016/j.bbrc.2013.06.104
  • Peer Reviewed
• [Journal Article] Impaired KLHL3-mediated ubiquitination of WNK4 causes human hypertension (2013)
  • Author(s): Wakabayashi M, Mori T, Isobe K, Sohara E, Susa K, Araki Y, Chiga M, Kikuchi E, Nomura N, Mori Y, Matsuo H, Murata T, Nomura S, Asano T, Kawaguchi H, Nonoyama S, Rai T, Sasaki S, Uchida S
  • Journal Title: Cell Rep Volume: 3(3) Pages: 858-68
  • DOI: 10.1016/j.celrep.2013.02.024
  • Peer Reviewed
• [Journal Article] A minor role of WNK3 in regulating phosphorylation of renal NKCC2 and NCC co-transporters in vivo (2013)
  • Author(s): Oi K, Sohara E, Rai T, Misawa M, Chiga M, Alessi DR, Sasaki S, Uchida S
  • Journal Title: Biol Open Volume: 1(2) Pages: 120-7
  • DOI: 10.1242/bio.2011048
  • Peer Reviewed
• [Journal Article] WNK-OSR1/SPAK-NCC signal cascade has circadian rhythm dependent on aldosterone (2012)
  • Author(s): Susa K, Sohara E, Isobe K, Chiga M, Rai T, Sasaki S, Uchida S
  • Journal Title: Biochem Biophys Res Commun Volume: 427(4) Pages: 743-7
  • DOI: 10.1016/j.bbrc.2012.09.130
  • Peer Reviewed
• [Journal Article] Does a β(2)-adrenergic receptor - WNK4 - Na-Cl co-transporter signal cascade exist in the in vivo kidney? (2012)
  • Author(s): Uchida S, Chiga M, Sohara E, Rai T, Sasaki S
  • Journal Title: Nat Med Volume: 18(9) Pages: 1324-5
  • DOI: 10.1038/nm.2809
  • Peer Reviewed
• [Journal Article] Phosphatidylinositol 3-kinase/Akt signaling pathway activates the WNK - OSR1/SPAK - NCC phosphorylation cascade in hyperinsulinemic db/db Mice (2012)
  • Author(s): Nishida H, Sohara E, Nomura N, Chiga M, Alessi DR, Rai T, Sasaki S, Uchida S
  • Journal Title: Hypertension Volume: 60(4) Pages: 981-90
  • DOI: 10.1161/HYPERTENSIONAHA
  • Peer Reviewed
• [Journal Article] Phosphorylation of Na-Cl cotransporter by OSR1 and SPAK kinases regulates its ubiquitination (2012)
  • Author(s): Hossain Khan MZ, Sohara E, Ohta A, Chiga M, Inoue Y, Isobe K, Wakabayashi M, Oi K, Rai T, Sasaki S, Uchida S
  • Journal Title: Biochem Biophys Res Commun Volume: 425(2) Pages: 456-61
  • DOI: 10.1016/j.bbrc.2012.07.124
  • Peer Reviewed
• [Journal Article] Effect of heterozygous deletion of WNK1 on the WNK-OSR1 / SPAK-NCC / NKCC1 / NKCC2 signal cascade in the kidney and blood vessels (2012)
  • Author(s): Susa K, Kita S, Iwamoto T, Yang SS, Lin SH, Ohta A, Sohara E, Rai T, Sasaki S, Alessi DR, Uchida S
  • Journal Title: Clin Exp Nephrol Volume: 16(4) Pages: 530-8
  • Peer Reviewed
• [Journal Article] Acute insulin stimulation induces phosphorylation of the Na-Cl cotransporter in cultured distal mpkDCT cells and mouse kidney (2011)
  • Author(s): Sohara E, Rai T, Yang SS, Ohta A, Naito S, Chiga M, Nomura N, Lin SH, Vandewalle A, Ohta E, Sasaki S, Uchida S
  • Journal Title: PLos One Volume: 6(8) Pages: 6(8)
  • DOI: 10.1371/journal.pone.0024277
  • Peer Reviewed
• [Journal Article] Phenotypes of pseudohypoaldosteronism type II caused by the WNK4 D561A missense mutation are dependent on the WNK-OSR1/ SPAK kinase cascade (2011)
  • Author(s): Chiga M, Rafiqi FH, Alessi DR, Sohara E, Ohta A, Rai T, Sasaki S, Uchida S
  • Journal Title: J Cell Sci Volume: 124(Pt9) Pages: 1391-5
  • DOI: 10.1242/jcs.084111
  • Peer Reviewed
• [Journal Article] Regulation of WNK1 kinase by extracellular potassium (2011)
  • Author(s): Naito S, Ohta A, Sohara E, Ohta E, Rai T, Sasaki S, Shinichi Uchida
  • Journal Title: Clin Exp Nephrol Volume: 15(2) Pages: 195-202
  • DOI: 10.1007/s10157-010-0378-9
  • Peer Reviewed
• [Journal Article] Mechanisms for hypercalciuria in pseudohypoaldosteronism type II-causing WNK4 knock-in mice (2010)
  • Author(s): Yang SS, Hsu YJ, Chiga M, Rai T, Sasaki S, Uchida S, Lin SH
  • Journal Title: Endocrinology Volume: 151(4) Pages: 1829-36
  • DOI: 10.1210/en.2009-0951
  • Peer Reviewed
• [Journal Article] Effect of angiotensin II on the WNK-OSR1/SPAK-NCC phosphorylation cascade in cultured mpkDCT cells and in vivo mouse kidney (2010)
  • Author(s): Talati G, Ohta A, Rai T, Sohara E, Naito S, Vandewalle A, Sasaki S, Uchida S
  • Journal Title: Biochem Biophys Res Commun Volume: 393(4) Pages: 844-8
  • DOI: 10.1016/j.bbrc.2010.02.096
  • Peer Reviewed
• [Presentation] Clinical significance of urinary thiazide-sensitive Na-Cl cotransporter (NCC) measurement by newly developed enzyme-linked immunosorbent assays (2013)
  • Author(s): Isobe K, Mori T, Sohara E, Rai T, Sasaki S, Uchida S
  • Organizer: American Society of Nephrology Kidney Week 2013
  • Place of Presentation: Atlanta, U.S.A.
  • Year and Date: 20131105-10
• [Presentation] Low salt intake decreased transcription and protein level of KLHL3 in mouse kidney (2013)
  • Author(s): Susa K, Sohara E, Zeniya M, Rai T, Sasaki S, Uchida S
  • Organizer: American Society of Nephrology Kidney Week 2013
  • Place of Presentation: Atlanta, U.S.A.
  • Year and Date: 20131105-10
• [Presentation] Chemical library screening for WNK signaling inhibitors by using fluorescent correlation spectroscopy (2013)
  • Author(s): Mori T, Kikuchi E, Sohara E, Rai T, Sasaki S, Uchida S
  • Organizer: American Society of Nephrology Kidney Week 2013
  • Place of Presentation: Atlanta, U.S.A.
  • Year and Date: 20131105-10
• [Presentation] Impaired KHLH3-mediated ubiquitination of WNK4 activates OSR1 and SPAK kinases - NaCl cotransporter (NCC) signaling and causes hypertension (2013)
  • Author(s): Wakabayashi M, Mori T, Isobe K, Sohara E, Susa K, Araki Y, Chiga M, Kikuchi E, Nomura N, Mori Y, Rai T, Sasaki S, Uchida S
  • Organizer: American Society of Nephrology Kidney Week 2013
  • Place of Presentation: Atlanta, U.S.A.
  • Year and Date: 20131105-10
• [Presentation] Analyses of KHLH3 mutants that cause Pseudohypoaldosteronism type II (2013)
  • Author(s): Mori Y, Wakabayashi M, Mori T, Araki Y, Sohara E, Rai T, Sasaki S, Uchida S
  • Organizer: American Society of Nephrology Kidney Week 2013
  • Place of Presentation: Atlanta, U.S.A.
  • Year and Date: 20131105-10
• [Presentation] KHLH2 interacts with and ubiquitinates WNK4 kinases (2013)
  • Author(s): Takahashi D, Mori T, Wakabayashi M, Mori Y, Susa K, Zeniya M, Rai T, Sohara E, Sasaki S, Uchida S
  • Organizer: American Society of Nephrology Kidney Week 2013
  • Place of Presentation: Atlanta, U.S.A.
  • Year and Date: 20131105-10
• [Presentation] WNK3 regulates blood pressure through the regulation of vascular OSR1 / SPAK-NKCC1 phosphorylation cascade (2013)
  • Author(s): Zeniya M, Sohara E, Oi K, Chiga M, Susa K, Mori T, Takahashi D, Rai T, Sasaki S, Uchida S
  • Organizer: American Society of Nephrology Kidney Week 2013
  • Place of Presentation: Atlanta, U.S.A.
  • Year and Date: 20131105-10
• [Presentation] Chemical library screening for direct SPAK inhibitors by a newly devoloped ELISA (2012)
  • Author(s): Kikuchi E, Mori T, Isobe K, Sohara E, Rai T, Sasaki S, Uchida S
  • Organizer: American Society of Nephrology Kidney Week 2012
  • Place of Presentation: San Diego, U.S.A.
  • Year and Date: 20121030-1104
• [Presentation] Development of sandwich enzyme linked immunosorbent assay for measurement of urinary total and phosphorylated Na-Cl cotransporter protein (2012)
  • Author(s): Isobe K, Sohara E, Rai T, Sasaki S, Uchida S
  • Organizer: American Society of Nephrology Kidney Week 2012
  • Place of Presentation: San Diego, U.S.A.
  • Year and Date: 20121030-1104
• [Presentation] Dietary salt intake and angiotensin II regulates WNK - SPAK - NKCC1 phosphorylation cascade in mouse aorta (2012)
  • Author(s): Zeniya M, Sohara E, Oi K, Chiga M, Susa K, Mori T, Rai T, Sasaki S, Uchida S
  • Organizer: American Society of Nephrology Kidney Week 2012
  • Place of Presentation: San Diego, U.S.A.
  • Year and Date: 20121030-1104
• [Presentation] Phosphorylation of Na-Cl cotransporter by OSR1 and SPAK kinases regulates its ubiquitination (2012)
  • Author(s): Kahn MZH, Sohara E, Rai T, Sasaki S, Uchida S
  • Organizer: American Society of Nephrology Kidney Week 2012
  • Place of Presentation: San Diego, U.S.A.
  • Year and Date: 20121030-1104
• [Presentation] Generation and analysis of WT-WNK4 transgenic mice reveal the physiological role of WNK4 (2012)
  • Author(s): Mori T, Sohara E, Rai T, Sasaki S, Uchida S
  • Organizer: American Society of Nephrology Kidney Week 2012
  • Place of Presentation: San Diego, U.S.A.
  • Year and Date: 20121030-1104
• [Presentation] WNK4 - OSR1/SPAK - NCC signal cascade has circadian rhythm dependent on aldosterone (2012)
  • Author(s): Susa K, Sohara E, Isobe K, Chiga M, Rai T, Sasaki S, Uchida S
  • Organizer: American Society of Nephrology Kidney Week 2012
  • Place of Presentation: San Diego, U.S.A.
  • Year and Date: 20121030-1104
• [Presentation] Increased Na-Cl cotransporter phosphorylation in hyperinsulinemic db/db mice is regulated by insulin/PI3K pathway (2011)
  • Author(s): Nishida H, Sohara E, Alessi D, Nomura N, Rai T, Sasaki S, Uchida S
  • Organizer: American Society of Nephrology Kidney Week 2011
  • Place of Presentation: Philadelphia, U.S.A
  • Year and Date: 20111108-13
• [Presentation] Increased protein abundance of the mutant WNK4 may be a cause of the increased WNK4 kinase activity in the mouse model of pseudohypoaldosteronism type II(PHA II). (2011)
  • Author(s): Wakabayashi M, Naito S, Sohara E, Rai T, Sasaki S, Uchida S
  • Organizer: American Society of Nephrology Kidney Week 2011
  • Place of Presentation: Philadelphia, U.S.A.
  • Year and Date: 20111108-13
• [Presentation] Acute insulin stimulation induces phosphorylation of the Na-Cl cotransporter in cultured distal mpkDCT cells and mouse kidney (2011)
  • Author(s): Sohara E, Rai T, Yang SS, Ohta A, Lin SH, Vandewalle A, Sasaki S, Uchida S
  • Organizer: American Society of Nephrology Kidney Week 2011
  • Place of Presentation: Philadelphia, U.S.A.
  • Year and Date: 20111108-13
• [Presentation] Development of new systems to measure total and phosphorylated Na-Cl cotransporter (NCC) protein in human urine (2011)
  • Author(s): Isobe K, Sohara E, Rai T, Sasaki S, Uchida S
  • Organizer: American Society of Nephrology Kidney Week 2011
  • Place of Presentation: Philadelphia, U.S.A.
  • Year and Date: 20111108-13
• [Presentation] Urinary excretion of Na-Cl cotransporter in exosomes is increased by high salt diet as well as low salt diet (2011)
  • Author(s): Kahn MZH, Sohara E, Ohta A, Naito S, Chiga M, Rai T, Sasaki S, Uchida S
  • Organizer: American Society of Nephrology Kidney Week 2011
  • Place of Presentation: Philadelphia, U.S.A.
  • Year and Date: 20111108-13
• [Presentation] WNK - OSR1/SPAK - SLC12A phosphorylation cascade in the WNK1 (+/-) mice (2011)
  • Author(s): Susa K, Sohara E, Rai T, Kita S, Iwamoto T, Alessi D, Sasaki S, Uchida S
  • Organizer: American Society of Nephrology Kidney Week 2011
  • Place of Presentation: Philadelphia, U.S.A
  • Year and Date: 20111108-13