Pacheco Álvarez, Diana

Main Affiliation

Facultad de Ciencias de la Salud - CampCM

Preferred name

Official Name

Pacheco Álvarez, Diana

ORCID

0000-0001-5454-9267

Researcher ID

DVP-2845-2022

Scopus Author ID

14827257500

Now showing 1 - 10 of 15

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WNK-SPAK-NCC cascade revisited: WNK1 stimulates the activity of the Na-Cl cotransporter via SPAK, an effect antagonized by WNK4
(2014)
Chávez-Canales, María
;
Zhang, Chong
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Soukaseum, Christelle
;
Moreno, Erika
;
Pacheco Álvarez, Diana
The with-no-lysine (K) kinases, WNK1 and WNK4, are key regulators of blood pressure. Their mutations lead to familial hyperkalemic hypertension (FHHt), associated with an activation of the Na-Cl cotransporter (NCC). Although it is clear that WNK4 mutants activate NCC via Ste20 proline-alanine-rich kinase, the mechanisms responsible for WNK1-related FHHt and alterations in NCC activity are not as clear. We tested whether WNK1 modulates NCC through WNK4, as predicted by some models, by crossing our recently developed WNK1-FHHt mice (WNK1(+/FHHt)) with WNK4(-/-) mice. Surprisingly, the activated NCC, hypertension, and hyperkalemia of WNK1(+/FHHt) mice remain in the absence of WNK4. We demonstrate that WNK1 powerfully stimulates NCC in a WNK4-independent and Ste20 proline-alanine-rich kinase-dependent manner. Moreover, WNK4 decreases the WNK1 and WNK3-mediated activation of NCC. Finally, the formation of oligomers of WNK kinases through their C-terminal coiled-coil domain is essential for their activity toward NCC. In conclusion, WNK kinases form a network in which WNK4 associates with WNK1 and WNK3 to regulate NCC. © 2014 American Heart Association, Inc.
Scopus© Citations 77 1 21
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Aspectos estructurales, funcionales y patológicos del cotransportador de NaCl sensible a tiazidas
(Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, 2014-11)
Moreno, Erika
;
Pacheco Álvarez, Diana
;
Ríos Argaiz, Eduardo
NCC cotransporter is the mayor pathway for sodium chloride reabsorption in the distal nephron and the target of thiazide diuretics which, given their clinical utility in the management of arterial hypertension, are amongst the top sold drugs in the world. NCC protein is of great physiological importance given its role in the maintenance of water and salt homeostasis on the organism. Inactivating mutations in the gene that codes for NCC cause Gitelman's syndrome: an autosomal recessive disease associated with arterial hypotension, metabolic alkalosis, hipokalemia and hypocalciuria. This syndrome represents strong evidence of the relevance of the role of NCC in blood pressure regulation, electrolyte and acid base balance. In this work we review the up to date knowledge regarding this cotransporter with special attention to the molecular aspects of the protein that determine is physiological function and pathological roles. ©Revista de Investigación Clínica
Scopus© Citations 1 1 23
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Angiotensin II signaling increases activity of the renal Na-Cl cotransporter through a WNK4-SPAK-dependent pathway
(2009)
San-Cristóbal, Pedro
;
Pacheco Álvarez, Diana
;
Richardson, Ciaran
;
Ring, Aaron M.
;
Vazquez, Norma
Mutations in the kinase WNK4 cause pseudohypoaldosteronism type II (PHAII), a syndrome featuring hypertension and high serum K(+) levels (hyperkalemia). WNK4 has distinct functional states that regulate the balance between renal salt reabsorption and K(+) secretion by modulating the activities of renal transporters and channels, including the Na-Cl cotransporter NCC and the K(+) channel ROMK. WNK4's functions could enable differential responses to intravascular volume depletion (hypovolemia) and hyperkalemia. Because hypovolemia is uniquely associated with high angiotensin II (AngII) levels, AngII signaling might modulate WNK4 activity. We show that AngII signaling in Xenopus oocytes increases NCC activity by abrogating WNK4's inhibition of NCC but does not alter WNK4's inhibition of ROMK. This effect requires AngII, its receptor AT1R, and WNK4, and is prevented by the AT1R inhibitor losartan. NCC activity is also increased by WNK4 harboring mutations found in PHAII, and this activity cannot be further augmented by AngII signaling, consistent with PHAII mutations providing constitutive activation of the signaling pathway between AT1R and NCC. AngII's effect on NCC is also dependent on the kinase SPAK because dominant-negative SPAK or elimination of the SPAK binding motif in NCC prevent activation of NCC by AngII signaling. These effects extend to mammalian cells. AngII increases phosphorylation of specific sites on SPAK and NCC that are necessary for activation of each in mpkDCT cells. These findings place WNK4 in the signaling pathway between AngII and NCC, and provide a mechanism by which hypovolemia maximizes renal salt reabsoprtion without concomitantly increasing K(+) secretion. © Proceedings of the National Academy of Sciences
Scopus© Citations 216 1 3
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Rare mutations in SLC12A1 and SLC12A3 protect against hypertension by reducing the activity of renal salt cotransporters
(2011)
Acuña, Rocío
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Martínez-de-la-Maza, Lilia
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Ponce-Coria, José
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Vázquez, Norma
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Ortal-Vite, Penélope
Objectives: Screening for variants in SLC12A1 and SLC12A3 genes, encoding the renal Na:Cl (NCC) and Na:K:2Cl (NKCC2) cotransporters, respectively, in 3125 members of the Framingham Heart Study (FHS) revealed that carrying a rare mutation in one of these genes was associated with a significant reduction in blood pressure, in the risk of arterial hypertension, and of death due to cardiovascular disease. Because near 60% of the rare mutations identified have not been related to Bartter's or Gitelman's disease, the consequence of such mutations on cotransporter activity is unknown. Methods: We used the heterologous expression system of Xenopus laevis oocytes, microinjected with wild-type or mutant NCC or NKCC2 cRNAs, to examine the effect of these inferred NCC and NKCC2 mutations on the cotransporters' functional properties. Cotransporter activity was defined as the diuretic-sensitive radioactive tracer uptake and response to known modulators was assessed. © Journal of Hypertension
2 13Scopus© Citations 56
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The European Eel NCCβ Gene Encodes a Thiazide-resistant Na-Cl Cotransporter
(2016)
Moreno, Erika
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Plata, Consuelo
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Rodríguez-Gama, Alejandro
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Argaiz, Eduardo R.
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Vázquez, Norma
The thiazide-sensitive Na-Cl cotransporter (NCC) is the major pathway for salt reabsorption in the mammalian distal convoluted tubule. NCC plays a key role in the regulation of blood pressure. Its inhibition with thiazides constitutes the primary baseline therapy for arterial hypertension. However, the thiazide-binding site in NCC is unknown. Mammals have only one gene encoding for NCC. The eel, however, contains a duplicate gene. NCCα is an ortholog of mammalian NCC and is expressed in the kidney. NCCβ is present in the apical membrane of the rectum. Here we cloned and functionally characterized NCCβ from the European eel. The cRNA encodes a 1043-amino acid membrane protein that, when expressed in Xenopus oocytes, functions as an Na-Cl cotransporter with two major characteristics, making it different from other known NCCs. First, eel NCCβ is resistant to thiazides. Single-point mutagenesis supports that the absence of thiazide inhibition is, at least in part, due to the substitution of a conserved serine for a cysteine at position 379. Second, NCCβ is not activated by low-chloride hypotonic stress, although the unique Ste20-related proline alanine-rich kinase (SPAK) binding site in the amino-terminal domain is conserved. Thus, NCCβ exhibits significant functional differences from NCCs that could be helpful in defining several aspects of the structure-function relationship of this important cotransporter. © 2016 ASBMB. Currently published by Elsevier Inc; originally published by American Society for Biochemistry and Molecular Biology.
1 19Scopus© Citations 13
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WNK3 is a Putative Chloride-sensing Kinase
(2011)
Pacheco Álvarez, Diana
;
Gamba, Gerardo
The with-no-lysine kinase 3 (WNK3) is a serine/threonine kinase that modulates the activity of the electroneutral cation-coupled chloride cotransporters (CCC). Using the Xenopus laevis oocyte heterologous expression system, it has been shown that WNK3 activates the Na(+)-coupled chloride cotransporters NKCC1, NKCC2, and NCC and inhibits the K(+)-coupled chloride cotransporters KCC1 through KCC4. Interestingly, the effect of catalytically inactive WNK3 is opposite to that of wild type WNK3: inactive WNK3 inhibits NKCCs and activates KCCs. In doing so, wild type and catalytically inactive WNK3 bypass the tonicity requirement for activation/inhibition of the cotransporter. Thus, WNK3 modulation of the electroneutral cotransporters promotes Cl(-) influx and prevents Cl(-) efflux, thus fitting the profile for a putative "Cl(-)-sensing kinase". Other kinases that potentially have these properties are the Ste20-type kinases, SPAK/OSR1, which become phosphorylated in response to reductions in intracellular chloride concentration and regulate the activity of NKCC1. It has been demonstrated that WNKs lie upstream of SPAK/OSR1 and that the activity of these kinases is activated by phosphorylation of threonines in the T-loop by WNKs. It is possible that a protein phosphatase is also involved in the WNK3 effects on its associated cotransporters because activation of KCCs and inhibition of NKCCs by inactive WNK3 can be prevented by known inhibitors of protein phosphatases, such as calyculin A and cyclosporine, suggesting that a protein phosphatase is also involved in the protein complex. Copyright © 2011 S. Karger AG, Basel. © Cellular Physiology and Biochemistry
1 9Scopus© Citations 37
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Role of WNK Kinases in the Modulation of Cell Volume
(2018)
Pacheco Álvarez, Diana
;
Gamba, Gerardo
Ion Transport across the cell membrane is required to maintain cell volume homeostasis. In response to changes in extracellular osmolarity, most cells activate specific metabolic or membrane-transport pathways to respond to cell swelling or shrinkage and return their volume to its normal resting state. This process involves the rapid adjustment of the activities of channels and transporters that mediate flux of K+, Na+, Cl-, and small organic osmolytes. Cation chloride cotransporters (CCCs) NKCCs and KCCs are a family of membrane proteins modulated by changes in cell volume and/or in the intracellular chloride concentration ([Cl-]i). Cell swelling triggers regulatory volume decrease (RVD), promoting solute and water efflux to restore normal cell volume. Swelling-activated KCCs mediate RVD in most cell types. In contrast, cell shrinkage triggers regulatory volume increase (RVI), which involves the activation of the NKCC1 cotransporter of the CCC family. Regulation of the CCCs during RVI and RVD by protein phosphorylation is a well-characterized mechanism, where WNK kinases and their downstream kinase substrates, SPAK and OSR1 constitute the essential phospho-regulators. WNKs-SPAK/OSR1-CCCs complex is required to regulate cell shrinkage-induced RVI or cell swelling-induced RVD via activating or inhibitory phosphorylation of NKCCs or KCCs, respectively. WNK1 and WNK4 kinases have been established as [Cl-]i sensors/regulators, while a role for WNK3 kinase as a cell volume-sensing kinase has emerged and is proposed in this chapter.
2 6Scopus© Citations 40
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Regulation of NKCC2 by a chloride-sensing mechanism involving the WNK3 and SPAK kinases
(2008)
Ponce-Coria, José
;
San-Cristóbal, Pedro
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Kahle, Kristopher T.
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Vázquez, Norma
;
Pacheco Álvarez, Diana
The Na+:K+:2Cl− cotransporter (NKCC2) is the target of loop diuretics and is mutated in Bartter's syndrome, a heterogeneous autosomal recessive disease that impairs salt reabsorption in the kidney's thick ascending limb (TAL). Despite the importance of this cation/chloride cotransporter (CCC), the mechanisms that underlie its regulation are largely unknown. Here, we show that intracellular chloride depletion in Xenopus laevis oocytes, achieved by either coexpression of the K-Cl cotransporter KCC2 or low-chloride hypotonic stress, activates NKCC2 by promoting the phosphorylation of three highly conserved threonines (96, 101, and 111) in the amino terminus. Elimination of these residues renders NKCC2 unresponsive to reductions of [Cl−]i. The chloride-sensitive activation of NKCC2 requires the interaction of two serine-threonine kinases, WNK3 (related to WNK1 and WNK4, genes mutated in a Mendelian form of hypertension) and SPAK (a Ste20-type kinase known to interact with and phosphorylate other CCCs). WNK3 is positioned upstream of SPAK and appears to be the chloride-sensitive kinase. Elimination of WNK3's unique SPAK-binding motif prevents its activation of NKCC2, as does the mutation of threonines 96, 101, and 111. A catalytically inactive WNK3 mutant also completely prevents NKCC2 activation by intracellular chloride depletion. Together these data reveal a chloride-sensing mechanism that regulates NKCC2 and provide insight into how increases in the level of intracellular chloride in TAL cells, as seen in certain pathological states, could drastically impair renal salt reabsorption.
2 5Scopus© Citations 208
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Structure-function relationships in the sodium chloride cotransporter
(2023)
Moreno, Erika
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Pacheco Álvarez, Diana
;
Chávez-Canales, María
;
Elizalde, Stephanie
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Leyva Ríos, Karla
The thiazide sensitive Na+:Cl− cotransporter (NCC) is the principal via for salt reabsorption in the apical membrane of the distal convoluted tubule (DCT) in mammals and plays a fundamental role in managing blood pressure. The cotransporter is targeted by thiazide diuretics, a highly prescribed medication that is effective in treating arterial hypertension and edema. NCC was the first member of the electroneutral cation-coupled chloride cotransporter family to be identified at a molecular level. It was cloned from the urinary bladder of the Pseudopleuronectes americanus (winter flounder) 30 years ago. The structural topology, kinetic and pharmacology properties of NCC have been extensively studied, determining that the transmembrane domain (TM) coordinates ion and thiazide binding. Functional and mutational studies have discovered residues involved in the phosphorylation and glycosylation of NCC, particularly on the N-terminal domain, as well as the extracellular loop connected to TM7-8 (EL7-8). In the last decade, single-particle cryogenic electron microscopy (cryo-EM) has permitted the visualization of structures at high atomic resolution for six members of the SLC12 family (NCC, NKCC1, KCC1-KCC4). Cryo-EM insights of NCC confirm an inverted conformation of the TM1-5 and TM6-10 regions, a characteristic also found in the amino acid-polyamine-organocation (APC) superfamily, in which TM1 and TM6 clearly coordinate ion binding. The high-resolution structure also displays two glycosylation sites (N-406 and N-426) in EL7-8 that are essential for NCC expression and function. In this review, we briefly describe the studies related to the structure-function relationship of NCC, beginning with the first biochemical/functional studies up to the recent cryo-EM structure obtained, to acquire an overall view enriched with the structural and functional aspects of the cotransporter. Copyright © 2023 Moreno, Pacheco-Alvarez, Chávez-Canales, Elizalde, Leyva-Ríos and Gamba.
Scopus© Citations 3 1 13
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C-terminally truncated, kidney-specific variants of the WNK4 kinase lack several sites that regulate its activity
(2018)
Murillo-de-Ozores, Adrián Rafael
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Rodríguez Moya, Alejandro
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Bazúa-Valenti, Silvana
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Leyva Ríos, Karla
;
Vázquez, Norma
WNK lysine-deficient protein kinase 4 (WNK4) is an important regulator of renal salt handling. Mutations in its gene cause pseudohypoaldosteronism type II, mainly arising from overac-tivation of the renal Na/Cl cotransporter (NCC). In addition to full-length WNK4, we have observed faster migrating bands (between 95 and 130 kDa) in Western blots of kidney lysates. Therefore, we hypothesized that these could correspond to uncharacterized WNK4 variants. Here, using several WNK4 antibodies and WNK4/ mice as controls, we showed that these bands indeed correspond to short WNK4 variants that are not observed in other tissue lysates. LC-MS/MS confirmed these bands as WNK4 variants that lack C-terminal segments. In HEK293 cells, truncation of WNK4’s C terminus at several positions increased its kinase activity toward Ste20-related proline/ alanine-rich kinase (SPAK), unless the truncated segment included the SPAK-binding site. Of note, this gain-of-function effect was due to the loss of a protein phosphatase 1 (PP1)-bind-ing site in WNK4. Cotransfection with PP1 resulted in WNK4 dephosphorylation, an activity that was abrogated in the PP1-binding site WNK4 mutant. The electrophoretic mobility of the in vivo short variants of renal WNK4 suggested that they lack the SPAK-binding site and thus may not behave as constitutively active kinases toward SPAK. Finally, we show that at least one of the WNK4 short variants may be produced by proteolysis involving a Zn2-dependent metalloprotease, as recombinant full-length WNK4 was cleaved when incubated with kidney lysate. © 2018 American Society for Biochemistry and Molecular Biology Inc. All rights reserved.
1 9Scopus© Citations 16

Pacheco Álvarez, Diana

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