RESUMEN
BACKGROUND: Chronic kidney disease (CKD) accelerates vascular calcification via phenotypic switching of vascular smooth muscle cells (VSMCs). We investigated the roles of circulating small extracellular vesicles (sEVs) between the kidneys and VSMCs and uncovered relevant sEV-propagated microRNAs (miRNAs) and their biological signaling pathways. METHODS AND RESULTS: We established CKD models in rats and mice by adenine-induced tubulointerstitial fibrosis. Cultures of A10 embryonic rat VSMCs showed increased calcification and transcription of osterix (Sp7), osteocalcin (Bglap), and osteopontin (Spp1) when treated with rat CKD serum. sEVs, but not sEV-depleted serum, accelerated calcification in VSMCs. Intraperitoneal administration of a neutral sphingomyelinase and biogenesis/release inhibitor of sEVs, GW4869 (2.5 mg/kg per 2 days), inhibited thoracic aortic calcification in CKD mice under a high-phosphorus diet. GW4869 induced a nearly full recovery of calcification and transcription of osteogenic marker genes. In CKD, the miRNA transcriptome of sEVs revealed a depletion of 4 miRNAs, miR-16-5p, miR-17~92 cluster-originated miR-17-5p/miR-20a-5p, and miR-106b-5p. Their expression decreased in sEVs from CKD patients as kidney function deteriorated. Transfection of VSMCs with each miRNA-mimic mitigated calcification. In silico analyses revealed VEGFA (vascular endothelial growth factor A) as a convergent target of these miRNAs. We found a 16-fold increase in VEGFA transcription in the thoracic aorta of CKD mice under a high-phosphorus diet, which GW4869 reversed. Inhibition of VEGFA-VEGFR2 signaling with sorafenib, fruquintinib, sunitinib, or VEGFR2-targeted siRNA mitigated calcification in VSMCs. Orally administered fruquintinib (2.5 mg/kg per day) for 4 weeks suppressed the transcription of osteogenic marker genes in the mouse aorta. The area under the curve of miR-16-5p, miR-17-5p, 20a-5p, and miR-106b-5p for the prediction of abdominal aortic calcification was 0.7630, 0.7704, 0.7407, and 0.7704, respectively. CONCLUSIONS: The miRNA transcriptomic signature of circulating sEVs uncovered their pathologic role, devoid of the calcification-protective miRNAs that target VEGFA signaling in CKD-driven vascular calcification. These sEV-propagated miRNAs are potential biomarkers and therapeutic targets for vascular calcification.
Asunto(s)
Vesículas Extracelulares , MicroARNs , Insuficiencia Renal Crónica , Calcificación Vascular , Ratas , Ratones , Animales , MicroARNs/genética , MicroARNs/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Músculo Liso Vascular/metabolismo , Calcificación Vascular/metabolismo , Insuficiencia Renal Crónica/metabolismo , Vesículas Extracelulares/metabolismo , Fósforo/metabolismo , Miocitos del Músculo Liso/metabolismoRESUMEN
Protein kinase A (PKA) directly phosphorylates aquaporin-2 (AQP2) water channels in renal collecting ducts to reabsorb water from urine for the maintenance of systemic water homeostasis. More than 50 functionally distinct PKA-anchoring proteins (AKAPs) respectively create compartmentalized PKA signaling to determine the substrate specificity of PKA. Identification of an AKAP responsible for AQP2 phosphorylation is an essential step toward elucidating the molecular mechanisms of urinary concentration. PKA activation by several compounds is a novel screening strategy to uncover PKA substrates whose phosphorylation levels were nearly perfectly correlated with that of AQP2. The leading candidate in this assay proved to be an AKAP termed lipopolysaccharide-responsive and beige-like anchor protein (LRBA). We found that LRBA colocalized with AQP2 in vivo, and Lrba knockout mice displayed a polyuric phenotype with severely impaired AQP2 phosphorylation. Most of the PKA substrates other than AQP2 were adequately phosphorylated by PKA in the absence of LRBA, demonstrating that LRBA-anchored PKA preferentially phosphorylated AQP2 in renal collecting ducts. Furthermore, the LRBA-PKA interaction, rather than other AKAP-PKA interactions, was robustly dissociated by PKA activation. AKAP-PKA interaction inhibitors have attracted attention for their ability to directly phosphorylate AQP2. Therefore, the LRBA-PKA interaction is a promising drug target for the development of anti-aquaretics.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Acuaporina 2 , Agua Corporal , Proteínas de Anclaje a la Quinasa A/genética , Proteínas de Anclaje a la Quinasa A/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Acuaporina 2/genética , Acuaporina 2/metabolismo , Agua Corporal/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Homeostasis , Ratones , FosforilaciónRESUMEN
AMP-activated protein kinase (AMPK) inactivation in chronic kidney disease (CKD) leads to energy status deterioration in the kidney, constituting the vicious cycle of CKD exacerbation. Unc-51-like kinase 1 (ULK1) is considered a downstream molecule of AMPK; however, it was recently reported that the activity of AMPK could be regulated by ULK1 conversely. We demonstrated that AMPK and ULK1 activities were decreased in the kidneys of CKD mice. However, whether and how ULK1 is involved in the underlying mechanism of CKD exacerbation remains unknown. In this study, we investigated the ULK1 involvement in CKD, using ULK1 knockout mice. The CKD model of Ulk1-/- mice exhibited significantly exacerbated renal function and worsening renal fibrosis. In the kidneys of the CKD model of Ulk1-/- mice, reduced AMPK and its downstream ß-oxidation could be observed, leading to an energy deficit of increased AMP/ATP ratio. In addition, AMPK signaling in the kidney was reduced in control Ulk1-/- mice with normal renal function compared to control wild-type mice, suggesting that ULK1 deficiency suppressed AMPK activity in the kidney. This study is the first to present ULK1 as a novel therapeutic target for CKD treatment, which regulates AMPK activity in the kidney.
Asunto(s)
Proteínas Quinasas Activadas por AMP , Insuficiencia Renal Crónica , Ratones , Animales , Homólogo de la Proteína 1 Relacionada con la Autofagia/genética , Homólogo de la Proteína 1 Relacionada con la Autofagia/metabolismo , Proteínas Quinasas Activadas por AMP/genética , Proteínas Quinasas Activadas por AMP/metabolismo , Riñón/metabolismo , Insuficiencia Renal Crónica/metabolismo , Fosforilación , AutofagiaRESUMEN
Fibronectin (FN) glomerulopathy (FNG), a rare autosomal hereditary renal disease, is characterized by proteinuria resulting from the massive accumulation of FN in the glomeruli. It typically affects individuals aged 10-50 years. In this report, we describe the case of a 57-year-old man who was diagnosed with FNG through genetic analysis and histological examination that revealed membranoproliferative glomerulonephritis. Despite treatment with prednisolone, the therapeutic response was unsatisfactory. Prednisolone was subsequently tapered and discontinued because the patient had pulmonary thromboembolism. Subsequent comprehensive genetic testing, which was initially not conducted because the patient's parents did not have a history of kidney disease, identified a known disease-causing variant in the FN1 gene, indicating a de novo variant. FNG was further confirmed by positive staining of glomeruli with FN using an IST-4 antibody. Although corticosteroid therapy is commonly employed as the initial treatment for MPGN, its appropriateness depends on the underlying etiology. Thus, clinicians must be aware of potential rare genetic causes underlying MPGN.
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Glomerulonefritis Membranoproliferativa , Masculino , Humanos , Persona de Mediana Edad , Glomerulonefritis Membranoproliferativa/diagnóstico , Glomerulonefritis Membranoproliferativa/tratamiento farmacológico , Glomerulonefritis Membranoproliferativa/genética , Glomérulos Renales , Riñón , Prednisolona/uso terapéuticoRESUMEN
Gitelman syndrome (GS), an autosomal recessive kidney disorder, is characterized by hypokalemia, hypomagnesemia, hypocalciuria, and metabolic alkalosis. Generally, diagnosis is made in school-aged children but multiple cases have been diagnosed in adulthood. This study examines the phenotypic differences between genetically confirmed cases and mutation-negative cases in adults. A comprehensive screening of 168 genes, including GS-related genes, was performed for 84 independent individuals who were referred to our institute with a clinical diagnosis of GS. The cases of pseudo-Bartter syndrome (BS)/GS because of diuretic abuse or other causes, which was determined based on patients' medical records, were excluded during registration. Of these 70 eligible cases for analysis, 27 (38.6%) had genetic confirmation of GS, while 37 (52.8%) had no known variants associated with GS and were considered to be unsolved cases. Note that unsolved cases comprised older, mostly female, individuals with decreased kidney function and multiple basic features of GS. The phenotype of unsolved cases is similar to that of pseudo BS/GS cases, although these cases were excluded in advance. However, the genetic and autoimmune profiles of these unsolved cases have not yet been investigated to date. Therefore, these cases may be categorized into new disease groups.
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Síndrome de Bartter , Síndrome de Gitelman , Hipopotasemia , Adulto , Síndrome de Bartter/genética , Femenino , Síndrome de Gitelman/complicaciones , Síndrome de Gitelman/diagnóstico , Síndrome de Gitelman/genética , Humanos , Hipopotasemia/complicaciones , Hipopotasemia/diagnóstico , Hipopotasemia/genética , Masculino , Mutación , FenotipoRESUMEN
BACKGROUND: Lipid-metabolizing enzymes and their metabolites affect inflammation and fibrosis, but their roles in chronic kidney disease (CKD) have not been completely understood. METHODS: To clarify their role in CKD, we measured the mRNA levels of major lipid-metabolizing enzymes in 5/6 nephrectomized (Nx) kidneys of C57BL/6 J mice. Mediator lipidomics was performed to reveal lipid profiles of CKD kidneys. RESULTS: In 5/6 Nx kidneys, both mRNA and protein levels of Alox15 were higher when compared with those in sham kidneys. With respect to in situ hybridization, the mRNA level of Alox15 was higher in renal tubules of 5/6 Nx kidneys. To examine the role of Alox15 in CKD pathogenesis, we performed 5/6 Nx on Alox15-/- mice. Alox15-/- CKD mice exhibited better renal functions than wild-type mice. Interstitial fibrosis was also inhibited in Alox15-/- CKD mice. Mediator lipidomics revealed that Alox15-/- CKD mouse kidneys had significantly higher levels of PGD2 than the control. To investigate the effects of PGD2 on renal fibrosis, we administered PGD2 to TGF-ß1-stimulated NRK-52E cells and HK-2 cells, which lead to a dose-dependent suppression of type I collagen and αSMA in both cell lines. CONCLUSION: Increased PGD2 in Alox15-/- CKD mouse kidneys could inhibit fibrosis, thereby resulting in CKD improvement. Thus, Alox15 inhibition and PGD2 administration may be novel therapeutic targets for CKD.
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Araquidonato 12-Lipooxigenasa/genética , Araquidonato 15-Lipooxigenasa/genética , Riñón/patología , Metabolismo de los Lípidos/genética , Prostaglandina D2/genética , Insuficiencia Renal Crónica/genética , Insuficiencia Renal Crónica/fisiopatología , Actinas/genética , Actinas/metabolismo , Animales , Araquidonato 12-Lipooxigenasa/metabolismo , Araquidonato 15-Lipooxigenasa/metabolismo , Línea Celular , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Cadena alfa 1 del Colágeno Tipo I , Fibrosis , Humanos , Oxidorreductasas Intramoleculares/genética , Riñón/metabolismo , Túbulos Renales Proximales/metabolismo , Lipocalinas/genética , Masculino , Ratones Endogámicos C57BL , Nefrectomía , Prostaglandina D2/farmacología , ARN Mensajero/metabolismo , Insuficiencia Renal Crónica/patologíaRESUMEN
The inappropriate over-activation of the with-no-lysine kinase (WNK)-STE20/SPS1-related proline/alanine-rich kinase (SPAK)-sodium chloride cotransporter (NCC) phosphorylation cascade increases sodium reabsorption in distal kidney nephrons, resulting in salt-sensitive hypertension. Although chronic kidney disease (CKD) is a common cause of salt-sensitive hypertension, the involvement of the WNK phosphorylation cascade is unknown. Moreover, the effect of immune systems on WNK kinases has not been investigated despite the fact that immune systems are important for salt sensitivity. Here we demonstrate that the protein abundance of WNK1, but not of WNK4, was increased at the distal convoluted tubules in the aristolochic acid nephropathy mouse model of CKD. Accordingly, the phosphorylation of both SPAK and NCC was also increased. Moreover, a high-salt diet did not adequately suppress activation of the WNK1-SPAK-NCC phosphorylation cascade in this model, leading to salt-sensitive hypertension. WNK1 also was increased in adenine nephropathy, but not in subtotal nephrectomy, models of CKD. By comparing the transcripts of these three models focusing on immune systems, we hypothesized that tumor necrosis factor (TNF)-α regulates WNK1 protein expression. In fact, TNF-α increased WNK1 protein expression in cultured renal tubular cells by reducing the transcription and protein levels of NEDD4-2 E3-ligase, which degrades WNK1 protein. Furthermore, the TNF-α inhibitor etanercept reversed the reduction of NEDD4-2 expression and upregulation of the WNK1-SPAK-NCC phosphorylation cascade in distal convoluted tubules in vivo in the aristolochic acid nephropathy model. Thus, salt-sensitive hypertension is induced in CKD via activation of the renal WNK1- SPAK-NCC phosphorylation cascade by TNF-α, reflecting a link with the immune system.
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Hipertensión , Insuficiencia Renal Crónica , Animales , Hipertensión/inducido químicamente , Ratones , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Insuficiencia Renal Crónica/inducido químicamente , Factor de Necrosis Tumoral alfa , Proteína Quinasa Deficiente en Lisina WNK 1RESUMEN
With-no-lysine kinase (WNK) plays important roles in regulating electrolyte homeostasis, cell signaling, survival, and proliferation. It has been recently demonstrated that WNK1, a member of the WNK family, modifies the function of immune cells. Here we report that in macrophages, WNK1 has suppressive effects on lipopolysaccharide (LPS)-induced inflammatory responses via TGFß-activated kinase 1 (TAK1)-mediated activation of nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathway. We found that WNK1 heterozygous (WNK1+/-) mice produced excessive proinflammatory cytokines in an experimental LPS-induced sepsis model, and peritoneal macrophages isolated from WNK1+/- mice produced higher levels of LPS-induced cytokines and NOS2 expression as canonical proinflammatory M1 macrophage markers. We confirmed that small hairpin RNA (shRNA)-mediated knockdown of WNK1 activated LPS-induced cytokine production and NOS2 expression in RAW 264.7 macrophages. Moreover, we demonstrated that WNK1 knockdown increased the nuclear translocation of NF-κB and activated the p38 and Jun N-terminal kinase (JNK) MAPK signaling pathway and that a TAK1 inhibitor diminished these effects of WNK1 knockdown. These results suggest that WNK1 acts as a physiologic immune modulator via interactions with TAK1. WNK1 may be a therapeutic target against the cytokine storm caused by sepsis.
Asunto(s)
Citocinas/biosíntesis , Quinasas Quinasa Quinasa PAM/metabolismo , Activación de Macrófagos , Macrófagos/inmunología , Sepsis/inmunología , Proteína Quinasa Deficiente en Lisina WNK 1/metabolismo , Animales , Células Cultivadas , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Lipopolisacáridos , Quinasas Quinasa Quinasa PAM/fisiología , Sistema de Señalización de MAP Quinasas , Macrófagos/efectos de los fármacos , Macrófagos/enzimología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , FN-kappa B/metabolismo , Células RAW 264.7 , Sepsis/inducido químicamente , Sepsis/enzimología , Proteína Quinasa Deficiente en Lisina WNK 1/genética , Proteína Quinasa Deficiente en Lisina WNK 1/fisiología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
The kidneys consume a large amount of energy to regulate volume status and blood pressure and to excrete uremic toxins. The identification of factors that cause energy mismatch in the setting of chronic kidney disease (CKD) and the development of interventions aimed at improving this mismatch are key research imperatives. Although the critical cellular energy sensor 5'-adenosine monophosphate (AMP)-activated protein kinase (AMPK) is known to be inactivated in CKD, the mechanism of AMPK dysregulation is unknown. In a mouse model of CKD, metabolome analysis confirmed a decrease in AMPK activation in the kidneys despite a high AMP: ATP ratio, suggesting that AMPK did not sense energy depletion. Similar AMPK inactivation was found in heart and skeletal muscle in CKD mice. Several uremic factors were shown to inactivate AMPK in vitro and in ex vivo preparations of kidney tissue. The specific AMPK activator A-769662, which bypasses the AMP sensing mechanism, ameliorated fibrosis and improved energy status in the kidneys of CKD mice, whereas an AMP analog did not. We further demonstrated that a low-protein diet activated AMPK independent of the AMP sensing mechanism, leading to improvement in energy metabolism and kidney fibrosis. These results suggest that a failure to sense AMP is the key mechanism underlying the vicious cycle of energy depletion and CKD progression and direct AMPK activation may be a novel therapeutic approach in CKD.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Dieta con Restricción de Proteínas , Metabolismo Energético/fisiología , Riñón/patología , Insuficiencia Renal Crónica/patología , Adenosina Monofosfato/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Compuestos de Bifenilo , Modelos Animales de Enfermedad , Metabolismo Energético/efectos de los fármacos , Fibrosis/metabolismo , Humanos , Riñón/metabolismo , Masculino , Metabolómica , Ratones , Ratones Endogámicos C57BL , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Miocardio/metabolismo , Miocardio/patología , Pironas/farmacología , Insuficiencia Renal Crónica/dietoterapia , Insuficiencia Renal Crónica/etiología , Insuficiencia Renal Crónica/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Tiofenos/farmacologíaRESUMEN
Type 4 Bartter syndrome (BS) is caused by genetic mutations in barttin, which is coded for by BSND. Barttin serves as the ß-subunit of the ClC-K chloride (Cl-) channel, which is widely expressed in distal nephrons. Type 4 BS is characterized by severely impaired reabsorption of salt, which may cause polyuria, hypokalemia, and metabolic alkalosis. Calcineurin inhibitors reportedly induce renal salt retention and hyperkalemia by enhancing the phosphorylation of the sodium (Na+)-potassium (K+)-2Cl- cotransporter (NKCC2) and Na+-Cl- cotransporter (NCC). In addition, we have previously reported that tacrolimus, a calcineurin inhibitor, increases the levels of phosphorylated NCC. In this study, we administered tacrolimus to barttin hypomorphic (Bsndneo/neo) mice, a murine model of type 4 BS that exhibits polyuria, hypokalemia, and metabolic alkalosis. Administration of tacrolimus increased the serum K+ level and suppressed urinary K+ excretion. Furthermore, after treatment with tacrolimus, Bsndneo/neo mice increased levels of phosphorylated NCC and NKCC2. We conclude that tacrolimus partially improves clinical phenotypes of Bsndneo/neo mice, and that calcineurin inhibitors might be effective for treating type 4 BS.
Asunto(s)
Síndrome de Bartter/tratamiento farmacológico , Inhibidores de la Calcineurina/uso terapéutico , Pérdida Auditiva Sensorineural/tratamiento farmacológico , Simportadores de Cloruro de Sodio-Potasio/agonistas , Miembro 3 de la Familia de Transportadores de Soluto 12/agonistas , Tacrolimus/uso terapéutico , Animales , Síndrome de Bartter/metabolismo , Modelos Animales de Enfermedad , Pérdida Auditiva Sensorineural/metabolismo , Hipopotasemia/tratamiento farmacológico , Hipopotasemia/metabolismo , Ratones , Ratones Endogámicos C57BL , Fosforilación/efectos de los fármacos , Simportadores de Cloruro de Sodio-Potasio/metabolismo , Miembro 3 de la Familia de Transportadores de Soluto 12/metabolismoRESUMEN
BACKGROUND: Elevated white blood cell (WBC) count is a well-known predictor of chronic kidney disease (CKD) progression. However, elderly patients commonly fail to develop a high WBC count in response to several diseased states and may instead present a low WBC count. Therefore, we hypothesized that low WBC count, in addition to high WBC count, is associated with CKD progression in the elderly. METHODS: We conducted a prospective cohort study using 3-year follow-up data from the CKD Research of Outcomes in Treatment and Epidemiology study. In the present study, participants aged over 60 years with pre-dialysis CKD stages G2-G5 were eligible. Patients were stratified into three groups according to WBC count using tertiles (T). The primary outcome was a composite of end-stage renal disease and a 50% reduction in estimated glomerular filtration rate. Data were analyzed using Cox proportional hazard models with adjustments for covariates. RESULTS: We enrolled 697 patients (males, 69%). The median WBC count was 6100 cells/µl (T1, <5400, n = 222; T2, 5400-6900, n = 235; T3, ≥6900, n = 240). During a median follow-up of 868 days, the primary outcome was observed in 170 patients, whereas 54 patients died. T1 and T3 had significantly higher hazard ratios (HR) than T2 (T1, HR 1.69, 95% confidence interval 1.14-2.51; T3, HR 1.63, 95% confidence interval 1.10-2.41). Moreover, T1 had a significantly higher adjusted HR (1.54, 95% confidence interval 1.00-2.37). CONCLUSION: Low WBC count is independently associated with CKD progression in the elderly.
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Leucocitos , Insuficiencia Renal Crónica/sangre , Factores de Edad , Anciano , Anciano de 80 o más Años , Progresión de la Enfermedad , Femenino , Tasa de Filtración Glomerular , Humanos , Japón/epidemiología , Estimación de Kaplan-Meier , Riñón/fisiopatología , Fallo Renal Crónico/sangre , Fallo Renal Crónico/mortalidad , Fallo Renal Crónico/fisiopatología , Recuento de Leucocitos , Modelos Logísticos , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Pronóstico , Modelos de Riesgos Proporcionales , Estudios Prospectivos , Insuficiencia Renal Crónica/diagnóstico , Insuficiencia Renal Crónica/mortalidad , Insuficiencia Renal Crónica/fisiopatología , Factores de Riesgo , Factores de TiempoRESUMEN
BACKGROUND: Pseudohypoaldosteronism type II (PHAII) is a hereditary hypertensive disease caused by mutations in four genes: WNK1, WNK4, Kelch-like3 (KLHL3), and cullin3 (CUL3). Recently, it was revealed that CUL3-KLHL3 E3 ligase complex ubiquitinates WNK1 and WNK4, leading to their degradation, and that a common pathogenesis of PHAII is defective WNK degradation due to CUL3-KLHL3 E3 ligase complex impairment. PHAII-causing CUL3 mutations mediate exon9 skipping, producing a CUL3 protein with a 57-amino acid deletion (Δ403-459). However, the pathogenic effects of KLHL3, an adaptor protein that links WNKs with CUL3, in PHAII caused by CUL3 mutation remain unclear. METHODS: To clarify detailed pathophysiological mechanisms underlying PHAII caused by CUL3 mutation in vivo, we generated and analyzed knock-in mice carrying the same CUL3 exon9 deletion (CUL3WT/Δex9) as that reported in PHAII patients. RESULTS: CUL3WT/Δex9 mice exhibited a PHAII-like phenotype. Interestingly, we confirmed markedly decreased KLHL3 expression in CUL3WT/Δex9 mice by confirming the true KLHL3 band in vivo. However, the expression of other KLHL family proteins, such as KLHL2, was comparable between WT and mutant mice. CONCLUSION: KLHL3 expression was decreased in CUL3WT/Δex9 mice. However, expression levels of other KLHL family proteins were comparable between the wild-type and mutant mice. These findings indicate that the decreased abundance of KLHL3 is a specific phenomenon caused by mutant CUL3 (Δexon9). Our findings would improve our understanding of the pathogenesis of PHAII caused by CUL3 mutation in vivo.
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Proteínas Portadoras/fisiología , Proteínas Cullin/genética , Mutación , Seudohipoaldosteronismo/etiología , Proteínas Adaptadoras Transductoras de Señales , Animales , Proteínas Portadoras/análisis , Humanos , Ratones , Proteínas de Microfilamentos , Seudohipoaldosteronismo/genéticaRESUMEN
Dietary potassium intake is inversely related to blood pressure and mortality. Moreover, the sodium-chloride cotransporter (NCC) plays an important role in blood pressure regulation and urinary potassium excretion in response to potassium intake. Previously, it was shown that NCC is activated by the WNK4-SPAK cascade and dephosphorylated by protein phosphatase. However, the mechanism of NCC regulation with acute potassium intake is still unclear. To identify the molecular mechanism of NCC regulation in response to potassium intake, we used adult C57BL/6 mice fed a 1.7% potassium solution by oral gavage. We confirmed that acute potassium load rapidly dephosphorylated NCC, which was not dependent on the accompanying anions. Mice were treated with tacrolimus (calcineurin inhibitor) and W7 (calmodulin inhibitor) before the oral potassium loads. Dephosphorylation of NCC induced by potassium was significantly inhibited by both tacrolimus and W7 treatment. There was no significant difference in WNK4, OSR1, and SPAK expression after high potassium intake, even after tacrolimus and W7 treatment. Another phosphatase, protein phosphatase 1, and its endogenous inhibitor I-1 did not show a significant change after potassium intake. Hyperkaliuria, induced by high potassium intake, was significantly suppressed by tacrolimus treatment. Thus, calcineurin is activated by an acute potassium load, which rapidly dephosphorylates NCC, leading to increased urinary potassium excretion.
Asunto(s)
Inhibidores de la Calcineurina/farmacología , Calcineurina/metabolismo , Riñón/efectos de los fármacos , Potasio en la Dieta/metabolismo , Eliminación Renal/efectos de los fármacos , Tacrolimus/farmacología , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Concentración de Iones de Hidrógeno , Riñón/metabolismo , Masculino , Ratones Endogámicos C57BL , Fosforilación , Potasio en la Dieta/sangre , Potasio en la Dieta/orina , Inhibidores de Proteínas Quinasas/farmacología , Proteína Fosfatasa 1/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal/efectos de los fármacos , Miembro 3 de la Familia de Transportadores de Soluto 12/efectos de los fármacos , Miembro 3 de la Familia de Transportadores de Soluto 12/metabolismo , Sulfonamidas/farmacología , Factores de Tiempo , Factores de Transcripción/metabolismoRESUMEN
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.
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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
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.
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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
BACKGROUND: Electrolyte abnormalities, particularly dysnatremia, are independent predictors of adverse outcome in individuals with and without renal failure. However, the association of serum chloride level (Cl-) with mortality or risk of cardiovascular (CV) events in chronic kidney disease (CKD) remains unclear. METHODS: This prospective cohort study included 923 pre-dialysis CKD G2-G5 patients among the participants of the CKD Research of Outcomes in Treatment and Epidemiology (CKD-ROUTE) study, who newly visited 16 nephrology centers. The primary outcome was a composite of overall death and CV events, and the secondary outcome was overall death. Data were analyzed using the Cox hazards model with adjustment for potential confounders. RESULTS: Median Cl- was 106.0 mEq/L at enrollment [quartile (Q) 1: ≤103.9, n = 207; Q2: 104.0-105.9, n = 207; Q3: 106.0-108.0, n = 289; Q4: ≥108.1, n = 220]. During a median follow-up of 33 months, there were 98 CV events, 66 deaths, and 154 composite outcomes. The hazard ratio (HR) for the composite outcome was higher for Q1 than Q3 [HR 1.72; 95 % confidence interval (CI) 1.08-2.72; P = 0.022]. As a continuous variable in a subset of patients whose Cl- was ≤106.0 mEq/L, higher Cl- was associated with lower risk of the composite outcome (HR 0.93; 95 % CI 0.87-0.99; P = 0.023). HR for all-cause mortality was also higher for Q1 than Q3 (HR 2.48; 95 % CI 1.22-5.03; P = 0.012). CONCLUSION: Low Cl- was associated with increased mortality and risk of CV events in pre-dialysis CKD patients. Cl- may be an additional prognostic indicator in CKD.
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Enfermedades Cardiovasculares/sangre , Enfermedades Cardiovasculares/mortalidad , Cloruros/sangre , Insuficiencia Renal Crónica/sangre , Insuficiencia Renal Crónica/mortalidad , Anciano , Anciano de 80 o más Años , Biomarcadores/sangre , Enfermedades Cardiovasculares/diagnóstico , Causas de Muerte , Distribución de Chi-Cuadrado , Regulación hacia Abajo , Femenino , Humanos , Japón/epidemiología , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Análisis Multivariante , Pronóstico , Modelos de Riesgos Proporcionales , Estudios Prospectivos , Insuficiencia Renal Crónica/diagnóstico , Factores de Riesgo , Factores de TiempoRESUMEN
BACKGROUND: Gene identification of hereditary kidney diseases by DNA sequencing is important for precise diagnosis, treatment, and genetic consultations. However, the conventional Sanger sequencing is now practically powerless in the face of ever increasing numbers of reported causative genes of various hereditary diseases. The advent of next-generation sequencing technology has enabled large-scale, genome-wide, simultaneous sequence analyses of multiple candidate genes. METHODS: We designed and verified a comprehensive diagnosis panel for approximately 100 major inherited kidney diseases, including 127 known genes. The panel was named Simple, sPEedy and Efficient Diagnosis of Inherited KIdney Diseases (SPEEDI-KID). We applied the panel to 73 individuals, clinically diagnosed with an inherited kidney disease, from 56 families. RESULTS: The panel efficiently covered the candidate genes and allowed a prompt and accurate genetic diagnosis. Moreover, 18 unreported mutations suspected as the disease causes were detected. All these mutations were validated by Sanger sequencing, with 100 % concordance. CONCLUSION: In conclusion, we developed a powerful diagnostic method, focusing on inherited kidney diseases, using a custom panel, SPEEDI-KID, allowing a fast, easy, and comprehensive diagnosis regardless of the disease type.
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Análisis Mutacional de ADN , Perfilación de la Expresión Génica , Pruebas Genéticas/métodos , Secuenciación de Nucleótidos de Alto Rendimiento , Enfermedades Renales/diagnóstico , Enfermedades Renales/genética , Mutación , Adolescente , Adulto , Anciano , Niño , Preescolar , Femenino , Estudios de Asociación Genética , Marcadores Genéticos , Predisposición Genética a la Enfermedad , Herencia , Humanos , Lactante , Masculino , Persona de Mediana Edad , Fenotipo , Valor Predictivo de las Pruebas , Reproducibilidad de los ResultadosRESUMEN
BACKGROUND: Vitamin D analogs have generally been recommended for treatment of mineral bone disease in chronic kidney disease (CKD). However, the association between this treatment and CKD progression has not yet been established. METHODS: We designed a post hoc propensity score-matched cohort analysis derived from 3-year follow-up data of a prospective cohort. Adult participants with pre-dialysis CKD stages 4-5 who had newly been prescribed active vitamin D analogs during the observation period were eligible as matched cases. Then, matched controls were extracted from participants who had never been prescribed active vitamin D analogs. The primary outcome was a composite of end-stage renal disease or a 50 % reduction in estimated glomerular filtration rate (eGFR). A Cox proportional hazards model evaluated the association between the use of vitamin D analogs and the primary outcome. RESULTS: We enrolled 240 patients (males, 65 %). The number of matched cases and controls was 30 and 210, respectively. The primary outcome was observed in 94 patients, whereas 25 patients died. The mean ± standard deviation age and eGFR were 69 ± 12 years and 17 ± 5.7 ml/min/1.73 m2, respectively. In a Cox proportional hazard model, the use of vitamin D analogs was independently associated with a lower risk of the primary outcome (crude hazard ratio 0.41; 95 % confidence interval 0.19, 0.89; adjusted hazard ratio 0.38; 95 % confidence interval 0.17, 0.88). CONCLUSION: The use of vitamin D analogs is independently associated with the preservation of renal function in patients with pre-dialysis CKD stages 4-5.
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Tasa de Filtración Glomerular/efectos de los fármacos , Fallo Renal Crónico/prevención & control , Riñón/efectos de los fármacos , Insuficiencia Renal Crónica/tratamiento farmacológico , Vitamina D/uso terapéutico , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Progresión de la Enfermedad , Femenino , Humanos , Japón , Riñón/fisiopatología , Fallo Renal Crónico/fisiopatología , Masculino , Persona de Mediana Edad , Puntaje de Propensión , Modelos de Riesgos Proporcionales , Estudios Prospectivos , Factores Protectores , Insuficiencia Renal Crónica/diagnóstico , Insuficiencia Renal Crónica/fisiopatología , Factores de Riesgo , Factores de Tiempo , Resultado del Tratamiento , Vitamina D/efectos adversos , Vitamina D/análogos & derivadosRESUMEN
BACKGROUND: The relationship between protein-energy wasting and chronic kidney disease (CKD) progression is unknown. In the present prospective cohort study, we evaluated the hypothesis that a combination of low body mass index (BMI) and serum albumin level is associated with rapid CKD progression. METHODS: The study cohort comprised 728 predialysis Japanese patients with CKD (stages 2-5) enrolled from 2010 to 2011. Patients were categorized into four groups according to their serum albumin levels and BMI: group 1, low serum albumin level (<4 g/dL) and low BMI (<23.5 kg/m2); group 2, high serum albumin level (≥4 g/dL) and low BMI; group 3, low serum albumin level and high BMI (≥23.5 kg/m2); and group 4, high serum albumin level and high BMI. The primary outcome was a 30 % decline in estimated glomerular filtration rate (eGFR) or start of dialysis within 2 years. The secondary outcome was an annual GFR decline (mL/min/1.73 m2/year). RESULTS: Logistic regression analysis adjusted for baseline characteristics (reference, group 4) showed that only group 1 was associated with a significant risk of CKD progression, with adjusted odds ratio of 3.51 [95 % confidence interval (CI) (1.63, 7.56)]. A multivariate linear regression analysis adjusted for baseline characteristics showed a significant difference in annual eGFR decline between groups 1 and 4 [coefficients ß (standard error) -2.62 (0.75), p = 0.001]. CONCLUSION: This study suggests that combined effects of low BMI (<23.5 kg/m2) and serum albumin level (<4 g/dL) are associated with CKD progression.
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Índice de Masa Corporal , Hipoalbuminemia/complicaciones , Desnutrición Proteico-Calórica/complicaciones , Insuficiencia Renal Crónica/complicaciones , Albúmina Sérica/análisis , Delgadez/complicaciones , Anciano , Anciano de 80 o más Años , Biomarcadores/sangre , Distribución de Chi-Cuadrado , Progresión de la Enfermedad , Regulación hacia Abajo , Femenino , Tasa de Filtración Glomerular , Humanos , Hipoalbuminemia/sangre , Hipoalbuminemia/diagnóstico , Riñón/fisiopatología , Modelos Lineales , Modelos Logísticos , Masculino , Persona de Mediana Edad , Análisis Multivariante , Estado Nutricional , Oportunidad Relativa , Estudios Prospectivos , Desnutrición Proteico-Calórica/sangre , Desnutrición Proteico-Calórica/diagnóstico , Desnutrición Proteico-Calórica/fisiopatología , Diálisis Renal , Insuficiencia Renal Crónica/sangre , Insuficiencia Renal Crónica/diagnóstico , Insuficiencia Renal Crónica/fisiopatología , Factores de Riesgo , Albúmina Sérica Humana , Delgadez/diagnóstico , Delgadez/fisiopatología , Factores de Tiempo , TokioRESUMEN
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.