RESUMEN
Kidney-specific with-no-lysine kinase 1 (KS-WNK1) is an isoform of WNK1 kinase that is predominantly found in the distal convoluted tubule of the kidney. The precise physiological function of KS-WNK1 remains unclear. Some studies have suggested that it could play a role in regulating potassium renal excretion by modulating the activity of the Na+-Cl- cotransporter (NCC). However, changes in the potassium diet from normal to high failed to reveal a role for KS-WNK1, but under a normal-potassium diet, the expression of KS-WNK1 is negligible. It is only detectable when mice are exposed to a low-potassium diet. In this study, we investigated the role of KS-WNK1 in regulating potassium excretion under extreme changes in potassium intake. After following a zero-potassium diet (0KD) for 10 days, KS-WNK1-/- mice had lower plasma levels of K+ and Cl- while exhibiting higher urinary excretion of Na+, Cl-, and K+ compared with KS-WNK1+/+ mice. After 10 days of 0KD or normal-potassium diet (NKD), all mice were challenged with a high-potassium diet (HKD). Plasma K+ levels markedly increased after the HKD challenge only in mice previously fed with 0KD, regardless of genotype. KSWNK1+/+ mice adapt better to HKD challenge than KS-WNK1-/- mice after a potassium-retaining state. The difference in the phosphorylated NCC-to-NCC ratio between KS-WNK1+/+ and KS-WNK1-/- mice after 0KD and HKD indicates a role for KS-WNK1 in both NCC phosphorylation and dephosphorylation. These observations show that KS-WNK1 helps the distal convoluted tubule to respond to extreme changes in potassium intake, such as those occurring in wildlife.NEW & NOTEWORTHY The findings of this study demonstrate that kidney-specific with-no-lysine kinase 1 plays a role in regulating urinary electrolyte excretion during extreme changes in potassium intake, such as those occurring in wildlife. .
Asunto(s)
Ratones Noqueados , Potasio en la Dieta , Proteína Quinasa Deficiente en Lisina WNK 1 , Animales , Masculino , Ratones , Riñón/metabolismo , Túbulos Renales Distales/metabolismo , Ratones Endogámicos C57BL , Fosforilación , Potasio/orina , Potasio/metabolismo , Potasio/sangre , Potasio en la Dieta/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Eliminación Renal , Miembro 3 de la Familia de Transportadores de Soluto 12/metabolismo , Miembro 3 de la Familia de Transportadores de Soluto 12/genética , Proteína Quinasa Deficiente en Lisina WNK 1/metabolismo , Proteína Quinasa Deficiente en Lisina WNK 1/genética , FemeninoRESUMEN
BACKGROUND: The calcium-sensing receptor (CaSR) in the distal convoluted tubule (DCT) activates the NaCl cotransporter (NCC). Glucose acts as a positive allosteric modulator of the CaSR. Under physiologic conditions, no glucose is delivered to the DCT, and fructose delivery depends on consumption. We hypothesized that glucose/fructose delivery to the DCT modulates the CaSR in a positive allosteric way, activating the WNK4-SPAK-NCC pathway and thus increasing salt retention. METHODS: We evaluated the effect of glucose/fructose arrival to the distal nephron on the CaSR-WNK4-SPAK-NCC pathway using HEK-293 cells, C57BL/6 and WNK4-knockout mice, ex vivo perfused kidneys, and healthy humans. RESULTS: HEK-293 cells exposed to glucose/fructose increased SPAK phosphorylation in a WNK4- and CaSR-dependent manner. C57BL/6 mice exposed to fructose or a single dose of dapagliflozin to induce transient glycosuria showed increased activity of the WNK4-SPAK-NCC pathway. The calcilytic NPS2143 ameliorated this effect, which was not observed in WNK4-KO mice. C57BL/6 mice treated with fructose or dapagliflozin showed markedly increased natriuresis after thiazide challenge. Ex vivo rat kidney perfused with glucose above the physiologic threshold levels for proximal reabsorption showed increased NCC and SPAK phosphorylation. NPS2143 prevented this effect. In healthy volunteers, cinacalcet administration, fructose intake, or a single dose of dapagliflozin increased SPAK and NCC phosphorylation in urinary extracellular vesicles. CONCLUSIONS: Glycosuria or fructosuria was associated with increased NCC, SPAK, and WNK4 phosphorylation in a CaSR-dependent manner.
Asunto(s)
Glucosuria , Simportadores del Cloruro de Sodio , Humanos , Ratones , Animales , Simportadores del Cloruro de Sodio/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores Sensibles al Calcio/metabolismo , Glucosa/metabolismo , Células HEK293 , Ratones Endogámicos C57BL , Fosforilación , Miembro 3 de la Familia de Transportadores de Soluto 12/metabolismo , Túbulos Renales Distales/metabolismo , Ratones Noqueados , Glucosuria/metabolismoRESUMEN
PURPOSE OF REVIEW: Regulation of the sodium chloride cotransporter (NCC) in the distal convoluted tubule (DCT) plays a crucial role in renal salt handling. The calcium-sensing receptor (CaSR) has been shown to activate NCC through the WNK4-SPAK pathway, which is independent of the Renin-Angiotensin-Aldosterone system. In this review, we examine new information about the mechanism of how the CaSR regulates NCC through the WNK4-SPAK pathway and its physiological and therapeutic implications. RECENT FINDINGS: The activation of CaSR in TALH cells during hypercalcemia inhibits NKCC2 and ROMK activity, reducing paracellular Ca2+ reabsorption but decreasing salt reabsorption. This pathway enables NaCl reabsorption in the DCT while promoting Ca2+ excretion. CaSR activation in the apical DCT stimulates a signaling pathway involving PKC, WNK4, and SPAK, which increases NCC activation to recover the NaCl not reabsorbed in TAHL. Glucose or fructose acting as calcimimetics enhance apical CaSR sensitivity, increasing NCC activity, which contribute to the mechanism of hypertension prevalence in diabetic patients or in those with high fructose consumption. SUMMARY: These findings reveal the importance of the CaSR-mediated activation of the WNK4-SPAK pathway in regulating salt and calcium homeostasis and its potential as a therapeutic target for hypertension and related diseases.
Asunto(s)
Hipertensión , Proteínas Serina-Treonina Quinasas , Humanos , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores Sensibles al Calcio/metabolismo , Fosforilación , Cloruro de Sodio/metabolismo , Calcio/metabolismo , Túbulos Renales Distales/metabolismo , Hipertensión/metabolismo , Cloruro de Sodio Dietético/metabolismo , Miembro 3 de la Familia de Transportadores de Soluto 12/metabolismoRESUMEN
The physiological role of the shorter isoform of with no lysine kinase (WNK)1 that is exclusively expressed in the kidney (KS-WNK1), with particular abundance in the distal convoluted tubule, remains elusive. KS-WNK1, despite lacking the kinase domain, is nevertheless capable of stimulating the NaCl cotransporter, apparently through activation of WNK4. It has recently been shown that a less severe form of familial hyperkalemic hypertension featuring only hyperkalemia is caused by missense mutations in the WNK1 acidic domain that preferentially affect cullin 3 (CUL3)-Kelch-like protein 3 (KLHL3) E3-induced degradation of KS-WNK1 rather than that of full-length WNK1. Here, we show that full-length WNK1 is indeed less impacted by the CUL3-KLHL3 E3 ligase complex compared with KS-WNK1. We demonstrated that the unique 30-amino acid NH2-terminal fragment of KS-WNK1 is essential for its activating effect on the NaCl cotransporter and recognition by KLHL3. We identified specific amino acid residues in this region critical for the functional effect of KS-WNK1 and KLHL3 sensitivity. To further explore this, we generated KLHL3-R528H knockin mice that mimic human mutations causing familial hyperkalemic hypertension. These mice revealed that the KLHL3 mutation specifically increased expression of KS-WNK1 in the kidney. We also observed that in wild-type mice, the expression of KS-WNK1 was only detectable after exposure to a low-K+ diet. These findings provide new insights into the regulation and function of KS-WNK1 by the CUL3-KLHL3 complex in the distal convoluted tubule and indicate that this pathway is regulated by dietary K+ levels.NEW & NOTEWORTHY In this work, we demonstrated that the kidney-specific isoform of with no lysine kinase 1 (KS-WNK1) in the kidney is modulated by dietary K+ and activity of the ubiquitin ligase protein Kelch-like protein 3. We analyzed the role of different amino acid residues of KS-WNK1 in its activity against the NaCl cotransporter and sensitivity to Kelch-like protein 3.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Riñón/enzimología , Proteínas de Microfilamentos/metabolismo , Potasio en la Dieta/metabolismo , Seudohipoaldosteronismo/enzimología , Proteína Quinasa Deficiente en Lisina WNK 1/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Proteínas Cullin/metabolismo , Estabilidad de Enzimas , Femenino , Riñón/fisiopatología , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas de Microfilamentos/genética , Mutación , Dominios y Motivos de Interacción de Proteínas , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteolisis , Seudohipoaldosteronismo/genética , Seudohipoaldosteronismo/fisiopatología , Miembro 3 de la Familia de Transportadores de Soluto 12/genética , Miembro 3 de la Familia de Transportadores de Soluto 12/metabolismo , Proteína Quinasa Deficiente en Lisina WNK 1/deficiencia , Proteína Quinasa Deficiente en Lisina WNK 1/genética , Xenopus laevisRESUMEN
Familial hyperkalemic hypertension (FHHt) can be mainly attributed to increased activity of the renal Na+:Cl- cotransporter (NCC), which is caused by altered expression and regulation of the with-no-lysine (K) 1 (WNK1) or WNK4 kinases. The WNK1 gene gives rise to a kidney-specific isoform that lacks the kinase domain (KS-WNK1), the expression of which occurs primarily in the distal convoluted tubule. The role played by KS-WNK1 in the modulation of the WNK/STE20-proline-alanine rich kinase (SPAK)/NCC pathway remains elusive. In the present study, we assessed the effect of human KS-WNK1 on NCC activity and on the WNK4-SPAK pathway. Microinjection of oocytes with human KS-WNK1 cRNA induces remarkable activation and phosphorylation of SPAK and NCC. The effect of KS-WNK1 was abrogated by eliminating a WNK-WNK-interacting domain and by a specific WNK inhibitor, WNK463, indicating that the activation of SPAK/NCC by KS-WNK1 is due to interaction with another WNK kinase. Under control conditions in oocytes, the activating serine 335 of the WNK4 T loop is not phosphorylated. In contrast, this serine becomes phosphorylated when the intracellular chloride concentration ([Cl-]i) is reduced or when KS-WNK1 is coexpressed with WNK4. KS-WNK1-mediated activation of WNK4 is not due to a decrease of the [Cl-]i. Coimmunoprecipitation analysis revealed that KS-WNK1 and WNK4 interact with each other and that WNK4 becomes autophosphorylated at serine 335 when it is associated with KS-WNK1. Together, these observations suggest that WNK4 becomes active in the presence of KS-WNK1, despite a constant [Cl-]i.
Asunto(s)
Cloruros/metabolismo , Riñón/enzimología , Proteínas Serina-Treonina Quinasas/metabolismo , Sodio/metabolismo , Proteína Quinasa Deficiente en Lisina WNK 1/metabolismo , Animales , Activación Enzimática , Femenino , Humanos , Oocitos , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Ratas , Miembro 3 de la Familia de Transportadores de Soluto 12/genética , Miembro 3 de la Familia de Transportadores de Soluto 12/metabolismo , Proteína Quinasa Deficiente en Lisina WNK 1/genética , Proteínas de Xenopus/metabolismo , Xenopus laevisRESUMEN
The STE20/SPS1-related proline-alanine-rich protein kinase (SPAK) controls the activity of the electroneutral cation-chloride cotransporters (SLC12 family) and thus physiological processes such as modulation of cell volume, intracellular chloride concentration [Cl-]i, and transepithelial salt transport. Modulation of SPAK kinase activity may have an impact on hypertension and obesity, as STK39, the gene encoding SPAK, has been suggested as a hypertension and obesity susceptibility gene. In fact, the absence of SPAK activity in mice in which the activating threonine in the T loop was substituted by alanine (SPAK-KI mice) is associated with decreased blood pressure; however its consequences in metabolism have not been explored. Here, we fed wild-type and homozygous SPAK-KI mice a high-fat diet for 17 wk to evaluate weight gain, circulating substrates and hormones, energy expenditure, glucose tolerance, and insulin sensitivity. SPAK-KI mice exhibit resistance to HFD-induced obesity and hepatic steatosis associated with increased energy expenditure, higher thermogenic activity in brown adipose tissue, increased mitochondrial activity in skeletal muscle, and reduced white adipose tissue hypertrophy mediated by augmented whole body insulin sensitivity and glucose tolerance. Our data reveal a previously unrecognized role for the SPAK kinase in the regulation of energy balance, thermogenesis, and insulin sensitivity, suggesting that this kinase could be a new drug target for the treatment of obesity and the metabolic syndrome.
Asunto(s)
Dieta Alta en Grasa/efectos adversos , Metabolismo Energético/genética , Resistencia a la Insulina/genética , Proteínas Serina-Treonina Quinasas/genética , Aumento de Peso/genética , Animales , Células Cultivadas , Grasas de la Dieta/farmacología , Metabolismo Energético/efectos de los fármacos , Técnicas de Sustitución del Gen , Silenciador del Gen , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Serina-Treonina Quinasas/fisiología , Aumento de Peso/efectos de los fármacosRESUMEN
Pseudohypoaldosteronism type II (PHAII) is a genetic disease characterized by association of hyperkalemia, hyperchloremic metabolic acidosis, hypertension, low renin, and high sensitivity to thiazide diuretics. It is caused by mutations in the WNK1, WNK4, KLHL3 or CUL3 gene. There is strong evidence that excessive sodium chloride reabsorption by the sodium chloride cotransporter NCC in the distal convoluted tubule is involved. WNK4 is expressed not only in distal convoluted tubule cells but also in ß-intercalated cells of the cortical collecting duct. These latter cells exchange intracellular bicarbonate for external chloride through pendrin, and therefore, account for renal base excretion. However, these cells can also mediate thiazide-sensitive sodium chloride absorption when the pendrin-dependent apical chloride influx is coupled to apical sodium influx by the sodium-driven chloride/bicarbonate exchanger. Here we determine whether this system is involved in the pathogenesis of PHAII. Renal pendrin activity was markedly increased in a mouse model carrying a WNK4 missense mutation (Q562E) previously identified in patients with PHAII. The upregulation of pendrin led to an increase in thiazide-sensitive sodium chloride absorption by the cortical collecting duct, and it caused metabolic acidosis. The function of apical potassium channels was altered in this model, and hyperkalemia was fully corrected by pendrin genetic ablation. Thus, we demonstrate an important contribution of pendrin in renal regulation of sodium chloride, potassium and acid-base homeostasis and in the pathophysiology of PHAII. Furthermore, we identify renal distal bicarbonate secretion as a novel mechanism of renal tubular acidosis.
Asunto(s)
Acidosis Tubular Renal/fisiopatología , Túbulos Renales Colectores/fisiopatología , Proteínas Serina-Treonina Quinasas/genética , Seudohipoaldosteronismo/complicaciones , Transportadores de Sulfato/metabolismo , Acidosis Tubular Renal/sangre , Acidosis Tubular Renal/etiología , Animales , Modelos Animales de Enfermedad , Técnicas de Inactivación de Genes , Humanos , Túbulos Renales Colectores/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación Missense , Potasio/sangre , Potasio/metabolismo , Seudohipoaldosteronismo/genética , Seudohipoaldosteronismo/fisiopatología , Eliminación Renal , Cloruro de Sodio/metabolismo , Simportadores de Sodio-Bicarbonato/metabolismo , Transportadores de Sulfato/genética , Regulación hacia ArribaRESUMEN
The K(+)-Cl(-) cotransporters (KCC1-KCC4) encompass a branch of the SLC12 family of electroneutral cation-coupled chloride cotransporters that translocate ions out of the cell to regulate various factors, including cell volume and intracellular chloride concentration, among others. L-WNK1 is an ubiquitously expressed kinase that is activated in response to osmotic stress and intracellular chloride depletion, and it is implicated in two distinct hereditary syndromes: the renal disease pseudohypoaldosteronism type II (PHAII) and the neurological disease hereditary sensory neuropathy 2 (HSN2). The effect of L-WNK1 on KCC activity is unknown. Using Xenopus laevis oocytes and HEK-293 cells, we show that the activation of KCCs by cell swelling was prevented by L-WNK1 coexpression. In contrast, the activity of the Na(+)-K(+)-2Cl(-) cotransporter NKCC1 was remarkably increased with L-WNK1 coexpression. The negative effect of L-WNK1 on the KCCs is kinase dependent. Elimination of the STE20 proline-alanine rich kinase (SPAK)/oxidative stress-responsive kinase (OSR1) binding site or the HQ motif required for the WNK-WNK interaction prevented the effect of L-WNK1 on KCCs, suggesting a required interaction between L-WNK1 molecules and SPAK. Together, our data support that NKCC1 and KCCs are coordinately regulated by L-WNK1 isoforms.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Antígenos de Histocompatibilidad Menor/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Miembro 2 de la Familia de Transportadores de Soluto 12/metabolismo , Simportadores/metabolismo , Animales , Tamaño de la Célula , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Lisina , Antígenos de Histocompatibilidad Menor/genética , Mutación , Osmorregulación , Fosforilación , Dominios y Motivos de Interacción de Proteínas , Isoformas de Proteínas , Proteínas Serina-Treonina Quinasas/genética , Transducción de Señal , Transfección , Proteína Quinasa Deficiente en Lisina WNK 1 , Xenopus laevis , Cotransportadores de K ClRESUMEN
It is widely recognized that the phenotype of familial hyperkalemic hypertension is mainly a consequence of increased activity of the renal Na(+)-Cl(-) cotransporter (NCC) because of altered regulation by with no-lysine-kinase 1 (WNK1) or WNK4. The effect of WNK4 on NCC, however, has been controversial because both inhibition and activation have been reported. It has been recently shown that the long isoform of WNK1 (L-WNK1) is a chloride-sensitive kinase activated by a low Cl(-) concentration. Therefore, we hypothesized that WNK4 effects on NCC could be modulated by intracellular chloride concentration ([Cl(-)]i), and we tested this hypothesis in oocytes injected with NCC cRNA with or without WNK4 cRNA. At baseline in oocytes, [Cl(-)]i was near 50 mM, autophosphorylation of WNK4 was undetectable, and NCC activity was either decreased or unaffected by WNK4. A reduction of [Cl(-)]i, either by low chloride hypotonic stress or coinjection of oocytes with the solute carrier family 26 (anion exchanger)-member 9 (SLC26A9) cRNA, promoted WNK4 autophosphorylation and increased NCC-dependent Na(+) transport in a WNK4-dependent manner. Substitution of the leucine with phenylalanine at residue 322 of WNK4, homologous to the chloride-binding pocket in L-WNK1, converted WNK4 into a constitutively autophosphorylated kinase that activated NCC, even without chloride depletion. Elimination of the catalytic activity (D321A or D321K-K186D) or the autophosphorylation site (S335A) in mutant WNK4-L322F abrogated the positive effect on NCC. These observations suggest that WNK4 can exert differential effects on NCC, depending on the intracellular chloride concentration.
Asunto(s)
Cloruros/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Simportadores del Cloruro de Sodio/metabolismo , Proteínas de Xenopus/metabolismo , Animales , Humanos , Ratones , Xenopus laevisRESUMEN
The serine/threonine kinase WNK3 and the ubiquitin-protein ligase NEDD4-2 are key regulators of the thiazide-sensitive Na+-Cl- cotransporter (NCC), WNK3 as an activator and NEDD2-4 as an inhibitor. Nedd4-2 was identified as an interacting partner of WNK3 through a glutathione-S-transferase pull-down assay using the N-terminal domain of WNK3, combined with LC-MS/MS analysis. This was validated by coimmunoprecipitation of WNK3 and NEDD4-2 expressed in HEK293 cells. Our data also revealed that the interaction between Nedd4-2 and WNK3 does not involve the PY-like motif found in WNK3. The level of WNK3 ubiquitylation did not change when NEDD4-2 was expressed in HEK293 cells. Moreover, in contrast to SGK1, WNK3 did not phosphorylate NEDD4-2 on S222 or S328. Coimmunoprecipitation assays showed that WNK3 does not regulate the interaction between NCC and NEDD4-2. Interestingly, in Xenopus laevis oocytes, WNK3 was able to recover the SGK1-resistant NEDD4-2 S222A/S328A-mediated inhibition of NCC and further activate NCC. Furthermore, elimination of the SPAK binding site in the kinase domain of WNK3 (WNK3-F242A, which lacks the capacity to bind the serine/threonine kinase SPAK) prevented the WNK3 NCC-activating effect, but not the Nedd4-2-inhibitory effect. Together, these results suggest that a novel role for WNK3 on NCC expression at the plasma membrane, an effect apparently independent of the SPAK kinase and the aldosterone-SGK1 pathway.
Asunto(s)
Membrana Celular/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Riñón/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Simportadores del Cloruro de Sodio/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Aldosterona/metabolismo , Animales , Membrana Celular/patología , Células Cultivadas , Femenino , Células HEK293 , Humanos , Proteínas Inmediatas-Precoces/metabolismo , Técnicas In Vitro , Riñón/patología , Modelos Animales , Ubiquitina-Proteína Ligasas Nedd4 , Oocitos/metabolismo , Oocitos/patología , Fosforilación , Transducción de Señal/fisiología , Proteínas de Xenopus/metabolismo , Xenopus laevisRESUMEN
The following is a narrative review of the fundamentals of optogenetics. It focuses on the advantages and constraints of manipulating the autonomic nervous system by modifying the pathophysiological characteristics that arise in different diseases. Although the use of this technique is currently experimental, we will discuss improvements that have been implemented and identify the necessary measures for potential preclinical translation in the control of the cardiac autonomic nervous system.
RESUMEN
BACKGROUND: SARS-CoV2 induces flu-like symptoms that can rapidly progress to severe acute lung injury and even death. The virus also invades the central nervous system (CNS), causing neuroinflammation and death from central failure. Intravenous (IV) or oral dexamethasone (DXM) reduced 28 d mortality in patients who required supplemental oxygen compared to those who received conventional care alone. Through these routes, DMX fails to reach therapeutic levels in the CNS. In contrast, the intranasal (IN) route produces therapeutic levels of DXM in the CNS, even at low doses, with similar systemic bioavailability. AIMS: To compare IN vs. IV DXM treatment in hospitalized patients with COVID-19. METHODS: A controlled, multicenter, open-label trial. Patients with COVID-19 (69) were randomly assigned to receive IN-DXM (0.12 mg/kg for three days, followed by 0.6 mg/kg for up to seven days) or IV-DXM (6 mg/d for 10 d). The primary outcome was clinical improvement, as defined by the National Early Warning Score (NEWS) ordinal scale. The secondary outcome was death at 28 d between IV and IN patients. Effects of both treatments on biochemical and immunoinflammatory profiles were also recorded. RESULTS: Initially, no significant differences in clinical severity, biometrics, and immunoinflammatory parameters were found between both groups. The NEWS-2 score was reduced, in 23 IN-DXM treated patients, with no significant variations in the 46 IV-DXM treated ones. Ten IV-DXM-treated patients and only one IN-DXM patient died. CONCLUSIONS: IN-DMX reduced NEWS-2 and mortality more efficiently than IV-DXM, suggesting that IN is a more efficient route of DXM administration.
Asunto(s)
COVID-19 , Humanos , SARS-CoV-2 , ARN Viral , Tratamiento Farmacológico de COVID-19 , Dexametasona/uso terapéuticoRESUMEN
In the renal collecting ducts, chloride reabsorption occurs through both transcellular and paracellular pathways. Recent literature highlights a functional interplay between both pathways. We recently showed that in polarized inner medullary collecting duct cells, expression of the basolateral kidney anion exchanger 1 (kAE1) results in a decreased transepithelial electrical resistance (TEER), in a claudin-4 dependent pathway. Claudin-4 is a paracellular sodium blocker and chloride pore. Here, we show that kAE1 expression in mouse inner medullary collecting duct cells triggers WNK4, SPAK and claudin-4 phosphorylation. Expression of a functionally dead kAE1 E681Q mutant has no effect on phosphorylation of these proteins. Expression of a catalytically inactive WNK4 D321A or chloride-insensitive WNK4 L319F mutant abolishes kAE1 effect on TEER, supporting a contribution of WNK4 to the process. We propose that variations of the cytosolic pH and chloride concentration upon kAE1 expression alter WNK4 kinase activity and tight junction properties.
RESUMEN
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.
RESUMEN
Caveolae-associated signaling toward mitochondria contributes to the cardioprotective mechanisms against ischemia-reperfusion (I/R) injury induced by ischemic postconditioning. In this work, we evaluated the role that the actin-cytoskeleton network exerts on caveolae-mitochondria communication during postconditioning. Isolated rat hearts subjected to I/R and to postconditioning were treated with latrunculin A, a cytoskeleton disruptor. Cardiac function was compared between these hearts and those exposed only to I/R and to the cardioprotective maneuver. Caveolae and mitochondria structures were determined by electron microscopy and maintenance of the actin-cytoskeleton was evaluated by phalloidin staining. Caveolin-3 and other putative caveolae-conforming proteins were detected by immunoblot analysis. Co-expression of caveolin-3 and actin was evaluated both in lipid raft fractions and in heart tissue from the different groups. Mitochondrial function was assessed by respirometry and correlated with cholesterol levels. Treatment with latrunculin A abolishes the cardioprotective postconditioning effect, inducing morphological and structural changes in cardiac tissue, reducing F-actin staining and diminishing caveolae formation. Latrunculin A administration to post-conditioned hearts decreases the interaction between caveolae-forming proteins, the co-localization of caveolin with actin and inhibits oxygen consumption rates in both subsarcolemmal and interfibrillar mitochondria. We conclude that actin-cytoskeleton drives caveolae signaling to mitochondria during postconditioning, supporting their functional integrity and contributing to cardiac adaption against reperfusion injury.
Asunto(s)
Caveolas , Daño por Reperfusión , Ratas , Animales , Caveolas/metabolismo , Actinas/metabolismo , Caveolina 3/metabolismo , Citoesqueleto/metabolismo , Caveolina 1/metabolismo , Daño por Reperfusión/metabolismo , Mitocondrias/metabolismoRESUMEN
BACKGROUND: By end December of 2021, COVID-19 has infected around 276 million individuals and caused over 5 million deaths worldwide. Infection results in dysregulated systemic inflammation, multi-organ dysfunction, and critical illness. Cells of the central nervous system are also affected, triggering an uncontrolled neuroinflammatory response. Low doses of glucocorticoids, administered orally or intravenously, reduce mortality among moderate and severe COVID-19 patients. However, low doses administered by these routes do not reach therapeutic levels in the CNS. In contrast, intranasally administered dexamethasone can result in therapeutic doses in the CNS even at low doses. METHODS: This is an approved open-label, multicenter, randomized controlled trial to compare the effectiveness of intranasal versus intravenous dexamethasone administered in low doses to moderate and severe COVID-19 adult patients. The protocol is conducted in five health institutions in Mexico City. A total of 120 patients will be randomized into two groups (intravenous vs. intranasal) at a 1:1 ratio. Both groups will be treated with the corresponding dexamethasone scheme for 10 days. The primary outcome of the study will be clinical improvement, defined as a statistically significant reduction in the NEWS-2 score of patients with intranasal versus intravenous dexamethasone administration. The secondary outcome will be the reduction in mortality during hospitalization. CONCLUSIONS: This protocol is currently in progress to improve the efficacy of the standard therapeutic dexamethasone regimen for moderate and severe COVID-19 patients. TRIAL REGISTRATION: ClinicalTrials.gov NCT04513184 . Registered November 12, 2020. Approved by La Comisión Federal para la Protección contra Riesgos Sanitarios (COFEPRIS) with identification number DI/20/407/04/36. People are currently being recruited.
Asunto(s)
Tratamiento Farmacológico de COVID-19 , Dexametasona/efectos adversos , Humanos , Inflamación , Enfermedades Neuroinflamatorias , SARS-CoV-2 , Resultado del TratamientoRESUMEN
The role of Cl- as an intracellular signaling ion has been increasingly recognized in recent years. One of the currently best described roles of Cl- in signaling is the modulation of the With-No-Lysine (K) (WNK) - STE20-Proline Alanine rich Kinase (SPAK)/Oxidative Stress Responsive Kinase 1 (OSR1) - Cation-Coupled Cl- Cotransporters (CCCs) cascade. Binding of a Cl- anion to the active site of WNK kinases directly modulates their activity, promoting their inhibition. WNK activation due to Cl- release from the binding site leads to phosphorylation and activation of SPAK/OSR1, which in turn phosphorylate the CCCs. Phosphorylation by WNKs-SPAK/OSR1 of the Na+-driven CCCs (mediating ions influx) promote their activation, whereas that of the K+-driven CCCs (mediating ions efflux) promote their inhibition. This results in net Cl- influx and feedback inhibition of WNK kinases. A wide variety of alterations to this pathway have been recognized as the cause of several human diseases, with manifestations in different systems. The understanding of WNK kinases as Cl- sensitive proteins has allowed us to better understand the mechanistic details of regulatory processes involved in diverse physiological phenomena that are reviewed here. These include cell volume regulation, potassium sensing and intracellular signaling in the renal distal convoluted tubule, and regulation of the neuronal response to the neurotransmitter GABA.
RESUMEN
Gain-of-function mutations in with no lysine (K) 1 (WNK1) and WNK4 genes are responsible for familial hyperkalemic hypertension (FHHt), a rare, inherited disorder characterized by arterial hypertension and hyperkalemia with metabolic acidosis. More recently, FHHt-causing mutations in the Kelch-like 3-Cullin 3 (KLHL3-CUL3) E3 ubiquitin ligase complex have shed light on the importance of WNK's cellular degradation on renal ion transport. Using full exome sequencing for a 4-generation family and then targeted sequencing in other suspected cases, we have identified new missense variants in the WNK1 gene clustering in the short conserved acidic motif known to interact with the KLHL3-CUL3 ubiquitin complex. Affected subjects had an early onset of a hyperkalemic hyperchloremic phenotype, but normal blood pressure values"Functional experiments in Xenopus laevis oocytes and HEK293T cells demonstrated that these mutations strongly decrease the ubiquitination of the kidney-specific isoform KS-WNK1 by the KLHL3-CUL3 complex rather than the long ubiquitous catalytically active L-WNK1 isoform. A corresponding CRISPR/Cas9 engineered mouse model recapitulated both the clinical and biological phenotypes. Renal investigations showed increased activation of the Ste20 proline alanine-rich kinase-Na+-Cl- cotransporter (SPAK-NCC) phosphorylation cascade, associated with impaired ROMK apical expression in the distal part of the renal tubule. Together, these new WNK1 genetic variants highlight the importance of the KS-WNK1 isoform abundance on potassium homeostasis.
Asunto(s)
Acidosis/metabolismo , Túbulos Renales Distales/metabolismo , Mutación , Seudohipoaldosteronismo/metabolismo , Proteína Quinasa Deficiente en Lisina WNK 1/metabolismo , Acidosis/genética , Acidosis/patología , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Secuencias de Aminoácidos , Animales , Proteínas Cullin/genética , Proteínas Cullin/metabolismo , Células HEK293 , Humanos , Túbulos Renales Distales/patología , Ratones , Ratones Mutantes , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Seudohipoaldosteronismo/genética , Seudohipoaldosteronismo/patología , Proteína Quinasa Deficiente en Lisina WNK 1/genética , Xenopus laevisRESUMEN
The ArcB/A two-component signal transduction system of Escherichia coli modulates the expression of numerous operons in response to redox conditions of growth. We demonstrate that the putative arcA and arcB genes of Mannheimia succiniciproducens MBEL55E, a capnophilic (CO2-loving) rumen bacterium, encode functional proteins that specify a two-component system. The Arc proteins of the two bacterial species sufficiently resemble each other that they can participate in heterologous transphosphorylation in vitro, and the arcA and arcB genes of M. succiniciproducens confer toluidine blue resistance to E. coli arcA and arcB mutants. However, neither the quinone analogs (ubiquinone 0 and menadione) nor the cytosolic effectors (d-lactate, acetate, and pyruvate) affect the net phosphorylation of M. succiniciproducens ArcB. Our results indicate that different types of signaling molecules and distinct modes of kinase regulation are used by the ArcB proteins of E. coli and M. succiniciproducens.