RESUMEN
The global outcome of acute aortic dissection (AD) remains poor, with a high risk of the need for urgent dialysis. This study aimed to clarify the association between sex and the requirement for urgent dialysis within 30 days after admission among patients with AD. This study included 79,998 cases who were hospitalized due to AD in Japan from 2010 to 2020 using an administrative claims database. The association between the risk of urgent dialysis and sex was investigated using the Fine and Gray model. Patients were classified into two groups based on the Stanford classification: type A AD (TAAD) and type B AD (TBAD). The lower subdistribution hazard ratio (SHR) in women was observed in both groups: TAAD (SHR: 0.58, 95% confidence interval [CI]: 0.54-0.62); TBAD (SHR: 0.49, 95% CI: 0.41-0.58). Our study revealed that women had a lower risk of requiring urgent dialysis than men in TAAD and TBAD.
RESUMEN
Coronavirus disease 2019 (COVID-19) affects both life and health. However, the differentiation from other types of pneumonia and effect of kidney disease remains uncertain. This retrospective observational study investigated the risk of in-hospital death and functional decline in ≥ 20% of Barthel Index scores after COVID-19 compared to other forms of pneumonia among Japanese adults, both with and without end-stage kidney disease (ESKD). The study enrolled 123,378 patients aged 18 years and older from a national inpatient administrative claims database in Japan that covers the first three waves of the COVID-19 pandemic in 2020. After a 1:1:1:1 propensity score matching into non-COVID-19/non-dialysis, COVID-19/non-dialysis, non-COVID-19/dialysis, and COVID-19/dialysis groups, 2136 adults were included in the analyses. The multivariable logistic regression analyses revealed greater odds ratios (ORs) of death [5.92 (95% CI 3.62-9.96)] and functional decline [1.93 (95% CI 1.26-2.99)] only in the COVID-19/dialysis group versus the non-COVID-19/non-dialysis group. The COVID-19/dialysis group had a higher risk of death directly due to pneumonia (OR 6.02, 95% CI 3.50-10.8) or death due to other diseases (OR 3.00, 95% CI 1.11-8.48; versus the non-COVID-19/non-dialysis group). COVID-19 displayed a greater impact on physical function than other types of pneumonia particularly in ESKD.
Asunto(s)
COVID-19 , Fallo Renal Crónico , Neumonía , Adulto , Humanos , Diálisis Renal , COVID-19/epidemiología , Mortalidad Hospitalaria , Japón/epidemiología , Estudios Retrospectivos , Pandemias , Fallo Renal Crónico/epidemiología , Fallo Renal Crónico/terapia , Neumonía/epidemiologíaRESUMEN
In recent times, new onset or relapse of nephrotic syndrome following the first dose of SARS-CoV-2 vaccines has been reported. Although the vaccination could trigger nephrotic syndrome, the question of whether the same vaccine should be administered as the second dose remains unanswered. A 25-year-old woman had taken the Moderna mRNA-1273 SARS-CoV-2 vaccine (mRNA-1273) and 26 days later, she noticed facial and peripheral edema. One week later she was referred and admitted to our hospital, wherein laboratory tests revealed that her serum creatinine level, serum albumin level, and urine protein-creatinine ratio were respectively 0.79 mg/dL, 2.5 g/dL, and 7.0 g/gCr. After a thorough inpatient examination including renal biopsy, she was diagnosed with minimal change disease (MCD) and treatment with steroids was initiated. She achieved complete remission the next day and did not experience a relapse upon receiving the second mRNA-1273 dose 56 days after the first, under treatment with 35 mg/day of oral prednisolone. This case report yields insight into determining whether patients who develop de novo MCD after the first mRNA-1273 dose should receive the second dose.
Asunto(s)
COVID-19 , Nefrosis Lipoidea , Síndrome Nefrótico , Vacunas , Femenino , Humanos , Adulto , Nefrosis Lipoidea/tratamiento farmacológico , Síndrome Nefrótico/tratamiento farmacológico , Vacunas contra la COVID-19 , Vacuna nCoV-2019 mRNA-1273 , SARS-CoV-2 , Recurrencia , Vacunas/uso terapéuticoRESUMEN
Encephalopathy is a rare side effect of cephalosporin treatment. We herein present a case of encephalopathy induced by ceftriaxone, a third-generation cephalosporin, in a patient with renal failure. An 86-year-old woman on maintenance hemodialysis received ceftriaxone for Helicobacter cinaedi bacteremia. Her mental status deteriorated during antibiotic treatment, and an electroencephalogram revealed triphasic waves predominantly in the frontal area. Her consciousness improved after the discontinuation of the antibiotic due to the suspicion of ceftriaxone-induced encephalopathy. This is the first reported case of encephalopathy associated with high plasma and cerebrospinal fluid ceftriaxone concentrations, and provides significant evidence for a causal relationship between the administration of ceftriaxone and the onset of encephalopathy.
Asunto(s)
Antibacterianos/efectos adversos , Encefalopatías/inducido químicamente , Ceftriaxona/efectos adversos , Diálisis Renal/efectos adversos , Anciano de 80 o más Años , Antibacterianos/sangre , Antibacterianos/líquido cefalorraquídeo , Bacteriemia/tratamiento farmacológico , Ceftriaxona/sangre , Ceftriaxona/líquido cefalorraquídeo , Electroencefalografía , Femenino , Infecciones por Helicobacter/tratamiento farmacológico , HumanosRESUMEN
INTRODUCTION: Accumulating evidence suggests that a large hospital volume (HV) is associated with favorable outcomes in various diseases or surgical procedures. The aim of this study is to clarify the correlation of HV and dialysis case volume (DCV) with in-hospital death in patients on maintenance dialysis. METHODS: The study cohort was derived from the Diagnosis Procedure Combination database, a national inpatient database in Japan, from 2012 to 2014. We included 382,689 admissions of maintenance dialysis patients over the age of 20 years in the analysis. HV was defined as the mean number of daily hospitalized patients, and DCV was defined as the mean number of annually hospitalized patients on maintenance dialysis. The primary outcome was in-hospital all-cause mortality, evaluated using multivariable logistic regression models across the respective quartiles of HV and DCV. RESULTS: The mean age of participants was 69 ± 12 years; 94% were receiving hemodialysis, and 21,182 patients (5.5%) died after hospitalization. In unadjusted models, larger HV and DCV were both associated with lower in-hospital mortality. However, this association remained significant only for DCV after adjustment for potential confounders, with multivariable-adjusted odds ratios of 0.82 (95% confidence interval [CI], 0.79-0.85), 0.76 (95% CI, 0.73-0.80), and 0.68 (95% CI, 0.65-0.72) for DCV 249 to 432, 433 to 713, and ≥714 (vs. ≤ 248) admissions per year, respectively. Multivariable subgroup analyses determined that this association was independent of age, sex, dialysis modality, Charlson Comorbidity Index, and emergency admission. CONCLUSION: Selective admission to hospitals with a large DCV may improve outcomes of dialysis patients.
RESUMEN
INTRODUCTION: Sarcopenia, the age-related loss of muscle mass and function, frequently accompanies chronic kidney disease. The aim of this study was to clarify the prevalence and the risk factors for sarcopenia among patients with non-dialysis-dependent chronic kidney disease (NDD-CKD), focusing on the use of drugs. METHODS: We conducted a cross-sectional analysis on a cohort of 260 patients with NDD-CKD in a university hospital, recruited between June 2016 and March 2017. We extracted data on patient gender, age, cause of chronic kidney disease, use of drugs, and comorbidities that could potentially affect the prevalence of sarcopenia. Sarcopenia was diagnosed using the criteria of the Asian Working Group for Sarcopenia. Logistic regression analysis was performed to analyze the association of each factor on the prevalence of sarcopenia. RESULTS: 25.0% of our study subjects had sarcopenia. Multivariable analysis revealed that an increased risk of sarcopenia was significantly associated with age, male gender, body mass index, diabetes mellitus, and loop diuretic use (odds ratio, 4.59: 95% confidence interval, 1.81-11.61: P-value 0.001). CONCLUSIONS: In our cohort, the prevalence of sarcopenia in patients with NDD-CKD was high, and diuretics use, particularly loop diuretic use, was suggested to be a risk factor of sarcopenia. Although loop diuretics are commonly used in patients with CKD, careful consideration of the risk of sarcopenia may be necessary.
Asunto(s)
Insuficiencia Renal Crónica/tratamiento farmacológico , Insuficiencia Renal Crónica/epidemiología , Sarcopenia/epidemiología , Inhibidores del Simportador de Cloruro Sódico y Cloruro Potásico/uso terapéutico , Factores de Edad , Anciano , Anciano de 80 o más Años , Índice de Masa Corporal , Estudios de Cohortes , Comorbilidad , Estudios Transversales , Complicaciones de la Diabetes/epidemiología , Complicaciones de la Diabetes/fisiopatología , Femenino , Humanos , Modelos Logísticos , Masculino , Análisis Multivariante , Oportunidad Relativa , Prevalencia , Insuficiencia Renal Crónica/fisiopatología , Factores de Riesgo , Sarcopenia/fisiopatología , Factores Sexuales , Inhibidores del Simportador de Cloruro Sódico y Cloruro Potásico/efectos adversosRESUMEN
The sodium chloride cotransporter (NCC) has been identified as a key molecule regulating potassium balance. The mechanisms of NCC regulation during low extracellular potassium concentrations have been studied in vitro. These studies have shown that hyperpolarization increased chloride efflux, leading to the activation of chloride-sensitive with-no-lysine kinase (WNK) kinases and their downstream molecules, including STE20/SPS1-related proline/alanine-rich kinase (SPAK) and NCC. However, this mechanism was not studied in vivo Previously, we developed the barttin hypomorphic mouse (Bsndneo/neo mice), expressing very low levels of barttin and ClC-K channels, because barttin is an essential ß-subunit of ClC-K. In contrast with Bsnd-/- mice, Bsndneo/neo mice survived to adulthood. In Bsndneo/neo mice, SPAK and NCC activation after consuming a low-potassium diet was clearly impaired compared with that in wild-type (WT) mice. In ex vivo kidney slice experiment, the increase in pNCC and SPAK in low-potassium medium was also impaired in Bsndneo/neo mice. Furthermore, increased blood pressure was observed in WT mice fed a high-salt and low-potassium diet, which was not evident in Bsndneo/neo mice. Thus, our study provides in vivo evidence that, in response to a low-potassium diet, ClC-K and barttin play important roles in the activation of the WNK4-SPAK-NCC cascade and blood pressure regulation.
Asunto(s)
Presión Sanguínea/genética , Canales de Cloruro/genética , Hipertensión/genética , Proteínas de la Membrana/genética , Simportadores de Cloruro de Sodio-Potasio/genética , Animales , Modelos Animales de Enfermedad , Humanos , Hipertensión/patología , Riñón/metabolismo , Ratones , Fosforilación , Potasio/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Transducción de Señal/genética , Miembro 3 de la Familia de Transportadores de Soluto 12/genéticaRESUMEN
WNK-OSR1/SPAK-NCC signaling cascade is important for regulating salt balance and blood pressure. Activation of WNK-OSR1/SPAK-NaCl cotransporter (NCC) cascade increases sodium reabsorption in the kidney, leading to pseudohypoaldosteronism type II (PHA II) characterized by salt-sensitive hypertension and hyperkalemia. It has been previously demonstrated that the amount of phosphorylated and total NCC markedly decreased in WNK4-/- mice, indicating that WNK4 plays a major role for activation of OSR1/SPAK-NCC signaling. However, it is unclear whether absence of WNK4 can be compensated by other WNK kinases. We recently reported that KLHL3R528H/+ knock-in mice, a PHAII model, exhibited augmented activation of OSR1/SPAK-NCC signaling by increased protein levels of both WNK1 and WNK4 due to impaired protein degradation by the mutant KLHL3. In this study, we sought to determine the contribution of WNK4 to OSR1/SPAK-NCC signaling using an in vivo model which shows extremely increased WNK1 with absence of WNK4. We generated WNK4-/-KLHL3R528H/+ mice and WNK4-/-KLHL3R528H/R528H mice by crossing WNK4-/- mice with KLHL3R528H/+ mice. Thereafter, WNK-OSR1/SPAK-NCC phosphorylation signal cascade was examined in kidneys from these mice. As expected, both WNK4-/-KLHL3R528H/+ mice and WNK4-/-KLHL3R528H/R528H mice demonstrated increased WNK1 in the kidney, due to the KLHL3 mutation, and WNK4 deficiency. However, phosphorylation of SPAK and NCC at distal convoluted tubules were almost completely absent even in WNK4-/-KLHL3R528H/R528H mice. In conclusion, increased WNK1 was unable to compensate for WNK4 deficiency and phosphorylate the NCC, indicating that WNK4 is indispensable for the onset of PHAII.
Asunto(s)
Riñón/metabolismo , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Antígenos de Histocompatibilidad Menor/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Seudohipoaldosteronismo/genética , Seudohipoaldosteronismo/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Mutación/genética , Relación Estructura-Actividad , Proteína Quinasa Deficiente en Lisina WNK 1RESUMEN
The Kelch-like ECH-associating protein 1 (Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) signaling pathway is the major regulator of cytoprotective responses to oxidative and electrophilic stress. The Cul3/Keap1 E3 ubiquitin ligase complex interacts with Nrf2, leading to Nrf2 ubiquitination and degradation. In this study, we focused on the disruption of the Keap1-Nrf2 interaction to upregulate Nrf2 expression and the transcription of ARE-controlled cytoprotective oxidative stress response enzymes, such as HO-1. We completed a drug-repositioning screening for inhibitors of Keap1-Nrf2 protein-protein interactions using a newly established fluorescence correlation spectroscopy (FCS) screening system. The binding reaction between Nrf2 and Keap1 was successfully detected with a KD of 2.6 µM using our FCS system. The initial screening of 1,633 drugs resulted in 12 candidate drugs. Among them, 2 drugs significantly increased Nrf2 protein levels in HepG2 cells. These two promising drugs also upregulated ARE gene promoter activity and increased HO-1 mRNA expression, which confirms their ability to dissociate Nrf2 and Keap1. Thus, drug-repositioning screening for Keap1-Nrf2 binding inhibitors using FCS enabled us to find two promising known drugs that can induce the activation of the Nrf2-ARE pathway.
Asunto(s)
Reposicionamiento de Medicamentos , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Elementos de Respuesta Antioxidante , Evaluación Preclínica de Medicamentos , Células Hep G2 , Humanos , Estrés Oxidativo , Unión Proteica , Espectrometría de Fluorescencia , Regulación hacia ArribaRESUMEN
Mutations in the with-no-lysine kinase 1 (WNK1), WNK4, Kelch-like 3 (KLHL3), and Cullin3 (CUL3) genes were identified as being responsible for hereditary hypertensive disease pseudohypoaldosteronism type II (PHAII). Normally, the KLHL3/CUL3 ubiquitin ligase complex degrades WNKs. In PHAII, the loss of interaction between KLHL3 and WNK4 increases levels of WNKs because of impaired ubiquitination, leading to abnormal over-activation of the WNK-OSR1/SPAK-NCC cascade in the kidney's distal convoluted tubules (DCT). KLHL2, which is highly homologous to KLHL3, was reported to ubiquitinate and degrade WNKs in vitro. Mutations in KLHL2 have not been reported in patients with PHAII, suggesting that KLHL2 plays a different physiological role than that played by KLHL3 in the kidney. To investigate the physiological roles of KLHL2 in the kidney, we generated KLHL2-/- mice. KLHL2-/- mice did not exhibit increased phosphorylation of the OSR1/SPAK-NCC cascade and PHAII-like phenotype. KLHL2 was predominantly expressed in the medulla compared with the cortex. Accordingly, medullary WNK4 protein levels were significantly increased in the kidneys of KLHL2-/- mice. KLHL2 is indeed a physiological regulator of WNK4 in vivo; however, its function might be different from that of KLHL3 because KLHL2 mainly localized in medulla.
Asunto(s)
Riñón/metabolismo , Proteínas de Microfilamentos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Ubiquitinación/fisiología , Proteínas Adaptadoras Transductoras de Señales , Animales , Regulación hacia Abajo/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Distribución TisularRESUMEN
The mechanisms of immunoactivation by salt are now becoming clearer. However, those of immunosuppression remain unknown. Since clinical evidence indicates that salt protects proximal tubules from injury, we investigated mechanisms responsible for salt causing immunosuppression in proximal tubules. We focused on cytokine-related gene expression profiles in kidneys of mice fed a high salt diet using microarray analysis and found that both an interferon gamma (IFNγ) inducible chemokine, chemokine (C-X-C motif) ligand 9 (CXCL9), and receptor, CXCR3, were suppressed. We further revealed that a high salt concentration suppressed IFNγ inducible chemokines in HK2 proximal tubular cells. Finally, we demonstrated that a high salt concentration decreased IFNGR1 expression in the basolateral membrane of HK2 cells, leading to decreased phosphorylation of activation sites of Janus kinase 1 (JAK1) and Signal Transducers and Activator of Transcription 1 (STAT1), activators of chemokines. JAK inhibitor canceled the effect of a high salt concentration on STAT1 and chemokines, indicating that the JAK1-STAT1 signaling pathway is essential for this mechanism. In conclusion, a high salt concentration suppresses IFNγ-JAK1-STAT1 signaling pathways and chemokine expressions in proximal tubules. This finding may explain how salt ameliorates proximal tubular injury and offer a new insight into the linkage between salt and immunity.
Asunto(s)
Quimiocinas/genética , Células Epiteliales/efectos de los fármacos , Interferón gamma/farmacología , Transducción de Señal/efectos de los fármacos , Cloruro de Sodio/farmacología , Animales , Antivirales/farmacología , Línea Celular , Quimiocinas/metabolismo , Células Epiteliales/metabolismo , Humanos , Interferón gamma/metabolismo , Janus Quinasa 1/metabolismo , Túbulos Renales Proximales/citología , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de Interferón/genética , Receptores de Interferón/metabolismo , Factor de Transcripción STAT1/metabolismo , Transducción de Señal/genética , Transcriptoma/efectos de los fármacos , Transcriptoma/genética , Receptor de Interferón gammaRESUMEN
Muscle wasting or sarcopenia contributes to morbidity and mortality in patients with cancer, renal failure, or heart failure, and in elderly individuals. Na+-K+-2Cl- cotransporter 1 (NKCC1) is highly expressed in mammalian skeletal muscle, where it contributes to the generation of membrane ion currents and potential. However, the physiologic function of NKCC1 in myogenesis is unclear. We investigated this issue using the NKCC1 inhibitors bumetanide and furosemide, which are commonly used loop diuretics. NKCC1 protein levels increased during C2C12 murine skeletal myoblast differentiation, similarly to those of the myogenic markers myogenin and myosin heavy chain (MHC). NKCC1 inhibitors markedly suppressed myoblast fusion into myotubes and the expression of myogenin and MHC. Furthermore, phosphorylated and total NKCC1 levels were elevated in mouse skeletal muscles after 6 weeks' voluntary wheel running. Immunofluorescence analyses of myofiber cross-sections revealed more large myofibers after exercise, but this was impaired by daily intraperitoneal bumetanide injections (0.2 or 10 mg/kg/day). NKCC1 plays an essential role in myogenesis and exercise-induced skeletal muscle hypertrophy, and sarcopenia in patients with renal or heart failure may be attributable to treatment with loop diuretics.
Asunto(s)
Diuréticos/administración & dosificación , Mioblastos/citología , Sarcopenia/etiología , Miembro 2 de la Familia de Transportadores de Soluto 12/metabolismo , Regulación hacia Arriba , Animales , Bumetanida/administración & dosificación , Bumetanida/farmacología , Diferenciación Celular/efectos de los fármacos , Línea Celular , Modelos Animales de Enfermedad , Diuréticos/farmacología , Furosemida/administración & dosificación , Furosemida/farmacología , Inyecciones Intraperitoneales , Ratones , Mioblastos/efectos de los fármacos , Mioblastos/metabolismo , Fosforilación , Carrera , Sarcopenia/metabolismoRESUMEN
The with-no-lysine kinase (WNK) 4 gene is a causative gene in pseudohypoaldosteronism type II. Although WNKs are widely expressed in the body, neither their metabolic functions nor their extrarenal role is clear. In this study, we found that WNK4 was expressed in mouse adipose tissue and 3T3-L1 adipocytes. In mouse primary preadipocytes and in 3T3-L1 adipocytes, WNK4 was markedly induced in the early phase of adipocyte differentiation. WNK4 expression preceded the expression of key transcriptional factors PPARγ and C/EBPα. WNK4-siRNA-transfected 3T3-L1 cells and human mesenchymal stem cells showed reduced expression of PPARγ and C/EBPα and lipid accumulation. WNK4 protein affected the DNA-binding ability of C/EBPß and thereby reduced PPARγ expression. In the WNK4-/- mice, PPARγ and C/EBPα expression were decreased in adipose tissues, and the mice exhibited partial resistance to high-fat diet-induced adiposity. These data suggest that WNK4 may be a proadipogenic factor, and offer insights into the relationship between WNKs and energy metabolism.
Asunto(s)
Dieta Alta en Grasa , Obesidad/etiología , Proteínas Serina-Treonina Quinasas/metabolismo , Células 3T3-L1 , Adipocitos/citología , Adipocitos/metabolismo , Tejido Adiposo Blanco/metabolismo , Tejido Adiposo Blanco/patología , Animales , Glucemia/análisis , Proteína alfa Potenciadora de Unión a CCAAT/genética , Proteína alfa Potenciadora de Unión a CCAAT/metabolismo , Células Cultivadas , Humanos , Insulina/sangre , Leptina/sangre , Masculino , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Obesidad/patología , Obesidad/prevención & control , PPAR gamma/genética , PPAR gamma/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
Mutations in the with-no-lysine kinase 1 (WNK1), WNK4, kelch-like 3 (KLHL3), and cullin3 (CUL3) genes are known to cause the hereditary disease pseudohypoaldosteronism type II (PHAII). It was recently demonstrated that this results from the defective degradation of WNK1 and WNK4 by the KLHL3/CUL3 ubiquitin ligase complex. However, the other physiological in vivo roles of KLHL3 remain unclear. Therefore, here we generated KLHL3-/- mice that expressed ß-galactosidase (ß-Gal) under the control of the endogenous KLHL3 promoter. Immunoblots of ß-Gal and LacZ staining revealed that KLHL3 was expressed in some organs, such as brain. However, the expression levels of WNK kinases were not increased in any of these organs other than the kidney, where WNK1 and WNK4 increased in KLHL3-/- mice but not in KLHL3+/- mice. KLHL3-/- mice also showed PHAII-like phenotypes, whereas KLHL3+/- mice did not. This clearly demonstrates that the heterozygous deletion of KLHL3 was not sufficient to cause PHAII, indicating that autosomal dominant type PHAII is caused by the dominant negative effect of mutant KLHL3. We further demonstrated that the dimerization of KLHL3 can explain this dominant negative effect. These findings could help us to further understand the physiological roles of KLHL3 and the pathophysiology of PHAII caused by mutant KLHL3.
Asunto(s)
Proteínas de Microfilamentos/genética , Mutación/genética , Seudohipoaldosteronismo/genética , Seudohipoaldosteronismo/fisiopatología , Proteínas Adaptadoras Transductoras de Señales , Animales , Técnicas de Sustitución del Gen , Genes Dominantes , Riñón/enzimología , Riñón/patología , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Microfilamentos/deficiencia , Modelos Biológicos , Proteínas Mutantes/metabolismo , Fenotipo , Fosforilación , Multimerización de Proteína , Proteínas Serina-Treonina Quinasas/metabolismo , Seudohipoaldosteronismo/sangre , Distribución TisularRESUMEN
Heritable nephrogenic diabetes insipidus (NDI) is characterized by defective urine concentration mechanisms in the kidney, which are mainly caused by loss-of-function mutations in the vasopressin type 2 receptor. For the treatment of heritable NDI, novel strategies that bypass the defective vasopressin type 2 receptor are required to activate the aquaporin-2 (AQP2) water channel. Here we show that Wnt5a regulates AQP2 protein expression, phosphorylation and trafficking, suggesting that Wnt5a is an endogenous ligand that can regulate AQP2 without the activation of the classic vasopressin/cAMP signalling pathway. Wnt5a successfully increases the apical membrane localization of AQP2 and urine osmolality in an NDI mouse model. We also demonstrate that calcineurin is a key regulator of Wnt5a-induced AQP2 activation without affecting intracellular cAMP level and PKA activity. The importance of calcineurin is further confirmed with its activator, arachidonic acid, which shows vasopressin-like effects underlining that calcineurin activators may be potential therapeutic targets for heritable NDI.
Asunto(s)
Acuaporina 2/genética , Calcineurina/metabolismo , Regulación de la Expresión Génica , Riñón/metabolismo , Transducción de Señal , Proteína Wnt-5a/metabolismo , Animales , Acuaporina 2/metabolismo , Ácido Araquidónico/farmacología , Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Diabetes Insípida Nefrogénica/metabolismo , Diabetes Insípida Nefrogénica/patología , Diabetes Insípida Nefrogénica/orina , Modelos Animales de Enfermedad , Proteínas Dishevelled/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Masculino , Ratones Endogámicos C57BL , Modelos Biológicos , Concentración Osmolar , Permeabilidad , Fosforilación/efectos de los fármacos , Fosfoserina/metabolismo , Transporte de Proteínas/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal/efectos de los fármacos , Agua , beta Catenina/metabolismoRESUMEN
Mutations in with-no-lysine kinase (WNK) 1, WNK4, Kelch-like 3 (KLHL3), and Cullin3 result in an inherited hypertensive disease, pseudohypoaldosteronism type II. WNK activates the Na-Cl cotransporter (NCC), increasing sodium reabsorption in the kidney. Further, KLHL3, an adapter protein of Cullin3-based E3 ubiquitin ligase, has been recently found to bind to WNK, thereby degrading them. Insulin and vasopressin have been identified as powerful activators of WNK signaling. In this study, we investigated effects of Akt and PKA, key downstream substrates of insulin and vasopressin signaling, respectively, on KLHL3. Mass spectrometry analysis revealed that KLHL3 phosphorylation at S433. Phospho-specific antibody demonstrated defective binding between phosphorylated KLHL3 and WNK4. Consistent with the fact that S433 is a component of Akt and PKA phosphorylation motifs, in vitro kinase assay demonstrated that Akt and PKA can phosphorylate KLHL3 at S433, that was previously reported to be phosphorylated by PKC. Further, forskolin, a representative PKA stimulator, increased phosphorylation of KLHL3 at S433 and WNK4 protein expression in HEK293 cells by inhibiting the KLHL3 effect that leads to WNK4 degradation. Insulin also increased phosphorylation of KLHL3 at S433 in cultured cells. In conclusion, we found that Akt and PKA phosphorylated KLHL3 at S433, and phosphorylation of KLHL3 by PKA inhibited WNK4 degradation. This could be a novel mechanism on how insulin and vasopressin physiologically activate the WNK signal.
Asunto(s)
Proteínas Portadoras/genética , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos , Proteínas Portadoras/metabolismo , Colforsina/farmacología , Proteínas Cullin/genética , Proteínas Cullin/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Regulación de la Expresión Génica , Células HEK293 , Humanos , Insulina/farmacología , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de Microfilamentos , Antígenos de Histocompatibilidad Menor , Datos de Secuencia Molecular , Fosforilación/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Vasopresinas/farmacología , Proteína Quinasa Deficiente en Lisina WNK 1RESUMEN
Recently, the kelch-like protein 3 (KLHL3)-Cullin3 complex was identified as an E3 ubiquitin ligase for with no lysine (WNK) kinases, and the impaired ubiquitination of WNK4 causes pseudohypoaldosteronism type II (PHAII), a hereditary hypertensive disease. However, the involvement of WNK kinase regulation by ubiquitination in situations other than PHAII has not been identified. Previously, we identified the WNK3-STE20/SPS1-related proline/alanine-rich kinase-Na/K/Cl cotransporter isoform 1 phosphorylation cascade in vascular smooth muscle cells and found that it constitutes an important mechanism of vascular constriction by angiotensin II (AngII). In this study, we investigated the involvement of KLHL proteins in AngII-induced WNK3 activation of vascular smooth muscle cells. In the mouse aorta and mouse vascular smooth muscle (MOVAS) cells, KLHL3 was not expressed, but KLHL2, the closest homolog of KLHL3, was expressed. Salt depletion and acute infusion of AngII decreased KLHL2 and increased WNK3 levels in the mouse aorta. Notably, the AngII-induced changes in KLHL2 and WNK3 expression occurred within minutes in MOVAS cells. Results of KLHL2 overexpression and knockdown experiments in MOVAS cells confirmed that KLHL2 is the major regulator of WNK3 protein abundance. The AngII-induced decrease in KLHL2 was not caused by decreased transcription but increased autophagy-mediated degradation. Furthermore, knockdown of sequestosome 1/p62 prevented the decrease in KLHL2, suggesting that the mechanism of KLHL2 autophagy could be selective autophagy mediated by sequestosome 1/p62. Thus, we identified a novel component of signal transduction in AngII-induced vascular contraction that could be a promising drug target.
Asunto(s)
Angiotensina II/farmacocinética , Proteínas de Microfilamentos/metabolismo , Tono Muscular/fisiología , Proteínas del Tejido Nervioso/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal/efectos de los fármacos , Proteínas Adaptadoras Transductoras de Señales/genética , Angiotensina II/farmacología , Animales , Aorta , Autofagia/efectos de los fármacos , Células Cultivadas , Técnicas de Silenciamiento del Gen , Proteínas de Choque Térmico/genética , Ratones , Proteínas de Microfilamentos/efectos de los fármacos , Proteínas de Microfilamentos/genética , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Proteínas del Tejido Nervioso/efectos de los fármacos , Proteínas del Tejido Nervioso/genética , Proteínas Serina-Treonina Quinasas/efectos de los fármacos , Proteína Sequestosoma-1 , Sodio en la Dieta/farmacología , Miembro 2 de la Familia de Transportadores de Soluto 12/metabolismo , VasoconstricciónRESUMEN
Pseudohypoaldosteronism type II (PHAII) is a hereditary disease characterized by salt-sensitive hypertension, hyperkalemia and metabolic acidosis, and genes encoding with-no-lysine kinase 1 (WNK1) and WNK4 kinases are known to be responsible. Recently, Kelch-like 3 (KLHL3) and Cullin3, components of KLHL3-Cullin3 E3 ligase, were newly identified as responsible for PHAII. We have reported that WNK4 is the substrate of KLHL3-Cullin3 E3 ligase-mediated ubiquitination. However, WNK1 and Na-Cl cotransporter (NCC) were also reported to be a substrate of KLHL3-Cullin3 E3 ligase by other groups. Therefore, it remains unclear which molecule is the target(s) of KLHL3. To investigate the pathogenesis of PHAII caused by KLHL3 mutation, we generated and analyzed KLHL3(R528H/+) knock-in mice. KLHL3(R528H/+) knock-in mice exhibited salt-sensitive hypertension, hyperkalemia and metabolic acidosis. Moreover, the phosphorylation of NCC was increased in the KLHL3(R528H/+) mouse kidney, indicating that the KLHL3(R528H/+) knock-in mouse is an ideal mouse model of PHAII. Interestingly, the protein expression of both WNK1 and WNK4 was significantly increased in the KLHL3(R528H/+) mouse kidney, confirming that increases in these WNK kinases activated the WNK-OSR1/SPAK-NCC phosphorylation cascade in KLHL3(R528H/+) knock-in mice. To examine whether mutant KLHL3 R528H can interact with WNK kinases, we measured the binding of TAMRA-labeled WNK1 and WNK4 peptides to full-length KLHL3 using fluorescence correlation spectroscopy, and found that neither WNK1 nor WNK4 bound to mutant KLHL3 R528H. Thus, we found that increased protein expression levels of WNK1 and WNK4 kinases cause PHAII by KLHL3 R528H mutation due to impaired KLHL3-Cullin3-mediated ubiquitination.
Asunto(s)
Proteínas de Microfilamentos/genética , Mutación , Proteínas Serina-Treonina Quinasas/metabolismo , Seudohipoaldosteronismo/genética , Seudohipoaldosteronismo/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Modelos Animales de Enfermedad , Células Epiteliales/metabolismo , Expresión Génica , Orden Génico , Marcación de Gen , Vectores Genéticos/genética , Genotipo , Riñón/metabolismo , Ratones , Ratones Transgénicos , Antígenos de Histocompatibilidad Menor , Fenotipo , Canales de Potasio de Rectificación Interna/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas Serina-Treonina Quinasas/genética , Proteolisis , Canales de Sodio/metabolismo , Ubiquitinación , Proteína Quinasa Deficiente en Lisina WNK 1RESUMEN
By analysing the pathogenesis of a hereditary hypertensive disease, PHAII (pseudohypoaldosteronism type II), we previously discovered that WNK (with-no-lysine kinase)-OSR1/SPAK (oxidative stress-responsive 1/Ste20-like proline/alanine-rich kinase) cascade regulates NCC (Na-Cl co-transporter) in the DCT (distal convoluted tubules) of the kidney. However, the role of WNK4 in the regulation of NCC remains controversial. To address this, we generated and analysed WNK4-/- mice. Although a moderate decrease in SPAK phosphorylation and a marked increase in WNK1 expression were evident in the kidneys of WNK4-/- mice, the amount of phosphorylated and total NCC decreased to almost undetectable levels, indicating that WNK4 is the major WNK positively regulating NCC, and that WNK1 cannot compensate for WNK4 deficiency in the DCT. Insulin- and low-potassium diet-induced NCC phosphorylation were abolished in WNK4-/- mice, establishing that both signals to NCC were mediated by WNK4. As shown previously, a high-salt diet decreases phosphorylated and total NCC in WNK4+/+ mice via AngII (angiotensin II) and aldosterone suppression. This was not ameliorated by WNK4 knock out, excluding the negative regulation of WNK4 on NCC postulated to be active in the absence of AngII stimulation. Thus, WNK4 is the major positive regulator of NCC in the kidneys.
Asunto(s)
Túbulos Renales Distales/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Angiotensina II/genética , Angiotensina II/metabolismo , Animales , Transporte Iónico/fisiología , Túbulos Renales Distales/citología , Ratones , Ratones Noqueados , Fosforilación/fisiología , Proteínas Serina-Treonina Quinasas/genética , Transducción de Señal/fisiología , Miembro 3 de la Familia de Transportadores de Soluto 12/genética , Miembro 3 de la Familia de Transportadores de Soluto 12/metabolismoRESUMEN
Na-K-Cl cotransporter isoform 1 (NKCC1) is involved in the regulation of vascular smooth muscle cell contraction. Recently, the with-no-lysine kinase (WNK)-STE20/SPS1-related proline/alanine-rich kinase (SPAK)-NKCC1 phosphorylation cascade in vascular smooth muscle cells was found to be important in the regulation of vascular tone. In this study, we investigated whether the WNK-SPAK-NKCC1 cascade in mouse aortic tissue is regulated by dietary salt intake and the mechanisms responsible. Phosphorylation of SPAK and NKCC1 was significantly reduced in the aorta in high-salt-fed mice and was increased in the aorta in low-salt-fed mice, indicating that the WNK-SPAK-NKCC1 phosphorylation cascade in the aorta was indeed regulated by dietary salt intake. Acute and chronic angiotensin II infusion increased phosphorylation of SPAK and NKCC1 in the mouse aorta. In addition, valsartan, an antagonist of angiotensin II type 1 receptor, inhibited low-salt diet-induced phosphorylation of SPAK and NKCC1, demonstrating that angiotensin II activates the WNK-SPAK-NKCC1 phosphorylation cascade through the angiotensin II type 1 receptor. However, a low-salt diet and angiotensin II together did not increase phosphorylation of SPAK and NKCC1 in the aorta in WNK3 knockout mice, indicating that activation of the WNK-SPAK-NKCC1 phosphorylation cascade induced by a low-salt diet and angiotensin II is dependent on WNK3. Indeed, angiotensin II-induced increases in blood pressure were diminished in WNK3 knockout mice. In addition, decreased response to angiotensin II in the mesenteric arteries was observed in WNK3 knockout mice. Our data also clarified a novel mechanism for regulation of vascular tonus by angiotensin II. Inhibition of this cascade could, therefore, be a novel therapeutic target in hypertension.