Generation and analysis of the thiazide-sensitive Na+-Cl− cotransporter (Ncc/Slc12a3) Ser707X knockin mouse as a model of Gitelman syndrome

Yang, Sung-Sen; Lo, Yi-Fen; Yu, I‐Shing; Lin, Shu‐Wha; Chang, Tai-Hsiang; Hsu, Yu‐Juei; Chao, Tai-Kuang; Sytwu, Huey‐Kang; Uchida, Shinichi; Sasaki, Sei; Lin, Shih‐Hua

doi:10.1002/humu.21364

Cited by 61 publications

(52 citation statements)

References 49 publications

(80 reference statements)

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“…Reduced T60M-NCC expression in MDCK cells, markedly diminished Ncc abundance in the DCT cells of Ncc T58M/T58M mice, and decreased urinary NCC excretion, an index of membrane-expressed NCC, in GS patients all indicated that T60M led to reduced NCC expression primarily through post-translational modification (phosphorylation) rather than translational change. Because increased late DCT volume was found in Ncc T58M/T58M mice, similar to our previously reported NccS7073 knock-in mice, 30 the reduced total T58M-Ncc abundance may also be independent of the altered DCT cellular volume. Reminiscent of the markedly reduced total and phosphorylated Ncc expression in Wnk4 31 and Spak knockout 32 as well as kinase-dead Spak knock-in mice, 33 the constitutive inactivation of the Wnk4-Spak cascade may not only decrease Ncc phosphorylation but also reduce its accumulation in apical membranes.…”

Section: Discussionsupporting

confidence: 88%

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Phosphorylation Regulates NCC Stability and Transporter Activity In Vivo

Yang

Fang²,

Tseng

et al. 2013

Journal of the American Society of Nephrology

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A T60M mutation in the thiazide-sensitive sodium chloride cotransporter (NCC) is common in patients with Gitelman's syndrome (GS). This mutation prevents Ste20-related proline and alanine-rich kinase (SPAK)/ oxidative stress responsive kinase-1 (OSR1)-mediated phosphorylation of NCC and alters NCC transporter activity in vitro. Here, we examined the physiologic effects of NCC phosphorylation in vivo using a novel Ncc T58M (human T60M) knock-in mouse model. Ncc T58M/T58M mice exhibited typical features of GS with a blunted response to thiazide diuretics. Despite expressing normal levels of Ncc mRNA, these mice had lower levels of total Ncc and p-Ncc protein that did not change with a low-salt diet that increased p-Spak. In contrast to wild-type Ncc, which localized to the apical membrane of distal convoluted tubule cells, T58M Ncc localized primarily to the cytosolic region and caused an increase in late distal convoluted tubule volume. In MDCK cells, exogenous expression of phosphorylation-defective NCC mutants reduced total protein expression levels and membrane stability. Furthermore, our analysis found diminished total urine NCC excretion in a cohort of GS patients with homozygous NCC T60M mutations. When Wnk4 D561A/+ mice, a model of pseudohypoaldosteronism type II expressing an activated Spak/ Osr1-Ncc, were crossed with Ncc T58M/T58M mice, total Ncc and p-Ncc protein levels decreased and the GS phenotype persisted over the hypertensive phenotype. Overall, these data suggest that SPAK-mediated phosphorylation of NCC at T60 regulates NCC stability and function, and defective phosphorylation at this residue corrects the phenotype of pseudohypoaldosteronism type II.

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Section: Discussionsupporting

confidence: 88%

“…50 The BPs of restrained conscious mice were measured with a programmable tailcuff sphygmomanometer (MK-2000A; Muromachi, Tokyo, Japan). 23,30,51 Hydrochlorothiazide, Furosemide, and Amiloride Challenge Studies…”

Section: Blood and Urine Analyses And Bp Measurementmentioning

confidence: 99%

Phosphorylation Regulates NCC Stability and Transporter Activity In Vivo

Yang

Fang²,

Tseng

et al. 2013

Journal of the American Society of Nephrology

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“…The maintenance of a certain level of NCC phosphorylation may explain the rather mild phenotype of the I-1 2/2 mice. In contrast to patients with Gitelman syndrome and NCC-deficient mice as well as NCC knock-in mice bearing a Gitelman mutation (Ser707/X) 44 and SPAKdeficient mice, 45,46 the I-1 2 /2 mice have no secondary hyperaldosteronism, hypomagnesaemia, and hypocalciuria. However, I-1 2/2 mice tend to have a reduced thiazide response on dietary Na + restriction and a trend for slightly lowered plasma K + concentrations.…”

Section: Discussionmentioning

confidence: 96%

Protein Phosphatase 1 Inhibitor-1 Deficiency Reduces Phosphorylation of Renal NaCl Cotransporter and Causes Arterial Hypotension

Picard

Trompf

Yang

et al. 2014

Journal of the American Society of Nephrology

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The thiazide-sensitive NaCl cotransporter (NCC) of the renal distal convoluted tubule (DCT) controls ion homeostasis and arterial BP. Loss-of-function mutations of NCC cause renal salt wasting with arterial hypotension (Gitelman syndrome). Conversely, mutations in the NCC-regulating WNK kinases or kelchlike 3 protein cause familial hyperkalemic hypertension. Here, we performed automated sorting of mouse DCTs and microarray analysis for comprehensive identification of novel DCT-enriched gene products, which may potentially regulate DCT and NCC function. This approach identified protein phosphatase 1 inhibitor-1 (I-1) as a DCT-enriched transcript, and immunohistochemistry revealed I-1 expression in mouse and human DCTs and thick ascending limbs. In heterologous expression systems, coexpression of NCC with I-1 increased thiazide-dependent Na + uptake, whereas RNAi-mediated knockdown of endogenous I-1 reduced NCC phosphorylation. Likewise, levels of phosphorylated NCC decreased by approximately 50% in I-1 (I-1 2/2 ) knockout mice without changes in total NCC expression. The abundance and phosphorylation of other renal sodium-transporting proteins, including NaPi-IIa, NKCC2, and ENaC, did not change, although the abundance of pendrin increased in these mice. The abundance, phosphorylation, and subcellular localization of SPAK were similar in wild-type (WT) and I-1 2/2 mice. Compared with WT mice, I-1 2/2 mice exhibited significantly lower arterial BP but did not display other metabolic features of NCC dysregulation. Thus, I-1 is a DCT-enriched gene product that controls arterial BP, possibly through regulation of NCC activity.

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“…It is difficult to determine whether upregulation of Ca-transporting molecules is due to the salt supplement alone or if there is any contribution of a direct effect from thiazides. Recently, Yang et al (24) created a mouse model of Gitelman syndrome by knocking in a gene with a known point mutation of NCC (24). The phenotype of this mouse model is identical to human Gitelman syndrome: relatively hypotensive, hypokalemic, hypomagnesemic, and hypocalciuric.…”

Section: Discussionmentioning

confidence: 99%

The role of calbindin-D28k on renal calcium and magnesium handling during treatment with loop and thiazide diuretics

Lee

et al. 2016

American Journal of Physiology-Renal Physiology

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Lee C, Ng H, Lee Y, Lai L, Lien YH. The role of calbindinD28k on renal calcium and magnesium handling during treatment with loop and thiazide diuretics. Am J Physiol Renal Physiol 310: F230 -F236, 2016. First published November 18, 2015 doi:10.1152/ajprenal.00057.2015) is a calcium binding protein located in the distal convoluted tubule (DCT) and plays an important role in active calcium transport in the kidney. Loop and thiazide diuretics affect renal Ca and Mg handling: both cause Mg wasting, but have opposite effects on Ca excretion as loop diuretics increase, but thiazides decrease, Ca excretion. To understand the role of CBD-28k in renal Ca and Mg handling in response to diuretics treatment, we investigated renal Ca and Mg excretion and gene expression of DCT Ca and Mg transport molecules in wild-type (WT) and CBD-28k knockout (KO) mice. Mice were treated with chlorothiazide (CTZ; 50 mg·kg Ϫ1 ·day Ϫ1 ) or furosemide (FSM; 30 mg·kg Ϫ1 ·day Ϫ1 ) for 3 days. To avoid volume depletion, salt was supplemented in the drinking water. Urine Ca excretion was reduced in WT, but not in KO mice, by CTZ. FSM induced similar hypercalciuria in both groups. DCT Ca transport molecules, including transient receptor potential vanilloid 5 (TRPV5), TRPV6, and CBD-9k, were upregulated by CTZ and FSM in WT, but not in KO mice. Urine Mg excretion was increased and transient receptor potential subfamily M, member 6 (TRPM6) was upregulated by both CTZ and FSM in WT and KO mice. In conclusion, CBD-28k plays an important role in gene expression of DCT Ca, but not Mg, transport molecules, which may be related to its being a Ca, but not a Mg, intracellular sensor. The lack of upregulation of DCT Ca transport molecules by thiazides in the KO mice indicates that the DCT Ca transport system is critical for Ca conservation by thiazides.furosemide; thiazides; TRPV5; TRPV6; TRPM6; calbindin-D28k; calbindin-D9k LOOP AND THIAZIDE DIURETICS are commonly used for treating hypertension, congestive heart failure, and other disorders with fluid overload (21). Loop diuretics are inhibitors of the Na-K2Cl cotransporter (NKCC) in the thick ascending limb of the loop of Henle (TALH), while thiazides inhibit the Na-Cl cotransporter (NCC) in the distal convoluted tubule (DCT). Both cause renal loss of Na, K, and Mg, but have different effects on renal Ca handling: loop diuretics cause hypercalciuria, while thiazides cause hypocalciuria (6,19). Those distinct effects on Ca excretion are also observed in patients with mutations in the NKCC (Barter syndrome) and NCC (Gitelman syndrome), respectively (22).In the kidney, Ca is reabsorbed through passive paracellular transport in the proximal tubule (PT) and TALH and active transcellular transport in the DCT (3). The gatekeeper of this active Ca transport mechanism is TRPV5. Ca enters cells across the apical TRPV5, then binds to calbindin-D28k (CBD28k), the major intracellular calcium binding proteins in DCT, and diffuses to the basolateral membrane, where Ca is extruded via the Na/Ca exchanger or Ca pumps (5)....

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Generation and analysis of the thiazide-sensitive Na+-Cl− cotransporter (Ncc/Slc12a3) Ser707X knockin mouse as a model of Gitelman syndrome

Cited by 61 publications

References 49 publications

Phosphorylation Regulates NCC Stability and Transporter Activity In Vivo

Phosphorylation Regulates NCC Stability and Transporter Activity In Vivo

Protein Phosphatase 1 Inhibitor-1 Deficiency Reduces Phosphorylation of Renal NaCl Cotransporter and Causes Arterial Hypotension

The role of calbindin-D28k on renal calcium and magnesium handling during treatment with loop and thiazide diuretics

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