RESUMEN
Adhesion and migration of T cells are controlled by chemokines and by adhesion molecules, especially integrins, and have critical roles in the normal physiological function of T lymphocytes. Using an RNA-mediated interference screen, we identified the WNK1 kinase as a regulator of both integrin-mediated adhesion and T cell migration. We found that WNK1 is a negative regulator of integrin-mediated adhesion, whereas it acts as a positive regulator of migration via the kinases OXSR1 and STK39 and the ion co-transporter SLC12A2. WNK1-deficient T cells home less efficiently to lymphoid organs and migrate more slowly through them. Our results reveal that a pathway previously known only to regulate salt homeostasis in the kidney functions to balance T cell adhesion and migration.
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Adhesión Celular/genética , Movimiento Celular/genética , Antígenos de Histocompatibilidad Menor/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores Mensajeros de Linfocitos/metabolismo , Linfocitos T/fisiología , Animales , Células Cultivadas , Homeostasis , Transporte Iónico , Riñón/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Antígenos de Histocompatibilidad Menor/genética , Proteínas Serina-Treonina Quinasas/genética , Interferencia de ARN , Receptores Mensajeros de Linfocitos/genética , Miembro 2 de la Familia de Transportadores de Soluto 12/metabolismo , Proteína Quinasa Deficiente en Lisina WNK 1RESUMEN
Renal tubules are featured with copious mitochondria and robust transport activity. Mutations in mitochondrial genes cause congenital renal tubulopathies, and changes in transport activity affect mitochondrial morphology, suggesting mitochondrial function and transport activity are tightly coupled. Current methods of using bulk kidney tissues or cultured cells to study mitochondrial bioenergetics are limited. Here, we optimized an extracellular flux analysis (EFA) to study mitochondrial respiration and energy metabolism using microdissected mouse renal tubule segments. EFA detects mitochondrial respiration and glycolysis by measuring oxygen consumption and extracellular acidification rates, respectively. We show that both measurements positively correlate with sample sizes of a few centimeter-length renal tubules. The thick ascending limbs (TALs) and distal convoluted tubules (DCTs) critically utilize glucose/pyruvate as energy substrates, whereas proximal tubules (PTs) are significantly much less so. Acute inhibition of TALs' transport activity by ouabain treatment reduces basal and ATP-linked mitochondrial respiration. Chronic inhibition of transport activity by 2-week furosemide treatment or deletion of with-no-lysine kinase 4 (Wnk4) decreases maximal mitochondrial capacity. In addition, chronic inhibition downregulates mitochondrial DNA mass and mitochondrial length/density in TALs and DCTs. Conversely, gain-of-function Wnk4 mutation increases maximal mitochondrial capacity and mitochondrial length/density without increasing mitochondrial DNA mass. In conclusion, EFA is a sensitive and reliable method to investigate mitochondrial functions in isolated renal tubules. Transport activity tightly regulates mitochondrial bioenergetics and biogenesis to meet the energy demand in renal tubules. The system allows future investigation into whether and how mitochondria contribute to tubular remodeling adapted to changes in transport activity.
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Metabolismo Energético , Túbulos Renales , Mitocondrias , Animales , Ratones , Mitocondrias/metabolismo , Túbulos Renales/metabolismo , Masculino , Ratones Endogámicos C57BL , Consumo de Oxígeno , Biogénesis de Organelos , Transporte Biológico , Glucólisis/fisiología , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
Mutations of PKD1 coding for polycystin-1 (PC1) account for most cases of autosomal-dominant polycystic kidney disease (ADPKD). The extracellular region of PC1 contains many evolutionarily conserved domains for ligand interactions. Among these are the leucine-rich repeats (LRRs) in the far N-terminus of PC1. Using zebrafish (Danio rerio) as an in vivo model system, we explored the role of LRRs in the function of PC1. Zebrafish expresses two human PKD1 paralogs, pkd1a and pkd1b. Knockdown of both genes in zebrafish by morpholino antisense oligonucleotides produced phenotypes of dorsal-axis curvature and pronephric cyst formation. We found that overexpression of LRRs suppressed both phenotypes in pkd1-morphant zebrafish. Purified recombinant LRR domain inhibited proliferation of HEK cells in culture and interacted with the heterotrimeric basement membrane protein laminin-511 (α5ß1γ1) in vitro. Mutations of amino acid residues in LRRs structurally predicted to bind laminin-511 disrupted LRR-laminin interaction in vitro and neutralized the ability of LRRs to inhibit cell proliferation and cystogenesis. Our data support the hypothesis that the extracellular region of PC1 plays a role in modulating PC1 interaction with the extracellular matrix and contributes to cystogenesis of PC1 deficiency.
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Enfermedades Renales Poliquísticas , Riñón Poliquístico Autosómico Dominante , Animales , Humanos , Riñón Poliquístico Autosómico Dominante/genética , Pez Cebra/genética , Leucina/metabolismo , Canales Catiónicos TRPP/metabolismo , Enfermedades Renales Poliquísticas/metabolismo , Laminina/metabolismo , Riñón/metabolismoRESUMEN
BACKGROUND: Mutations of PKD2, which encodes polycystin-2, cause autosomal dominant polycystic kidney disease (ADPKD). The prevailing view is that defects in polycystin-2-mediated calcium ion influx in the primary cilia play a central role in the pathogenesis of cyst growth. However, polycystin-2 is predominantly expressed in the endoplasmic reticulum (ER) and more permeable to potassium ions than to calcium ions. METHODS: The trimeric intracellular cation (TRIC) channel TRIC-B is an ER-resident potassium channel that mediates potassium-calcium counterion exchange for inositol trisphosphate-mediated calcium ion release. Using TRIC-B as a tool, we examined the function of ER-localized polycystin-2 and its role in ADPKD pathogenesis in cultured cells, zebrafish, and mouse models. RESULTS: Agonist-induced ER calcium ion release was defective in cells lacking polycystin-2 and reversed by exogenous expression of TRIC-B. Vice versa, exogenous polycystin-2 reversed an ER calcium-release defect in cells lacking TRIC-B. In a zebrafish model, expression of wild-type but not nonfunctional TRIC-B suppressed polycystin-2-deficient phenotypes. Similarly, these phenotypes were suppressed by targeting the ROMK potassium channel (normally expressed on the cell surface) to the ER. In cultured cells and polycystin-2-deficient zebrafish phenotypes, polycystin-2 remained capable of reversing the ER calcium release defect even when it was not present in the cilia. Transgenic expression of Tric-b ameliorated cystogenesis in the kidneys of conditional Pkd2-inactivated mice, whereas Tric-b deletion enhanced cystogenesis in Pkd2-heterozygous kidneys. CONCLUSIONS: Polycystin-2 in the ER appears to be critical for anticystogenesis and likely functions as a potassium ion channel to facilitate potassium-calcium counterion exchange for inositol trisphosphate-mediated calcium release. The results advance the understanding of ADPKD pathogenesis and provides proof of principle for pharmacotherapy by TRIC-B activators.
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Riñón Poliquístico Autosómico Dominante , Canales Catiónicos TRPP , Animales , Calcio/metabolismo , Retículo Endoplásmico/metabolismo , Inositol/metabolismo , Canales Iónicos/genética , Ratones , Riñón Poliquístico Autosómico Dominante/genética , Riñón Poliquístico Autosómico Dominante/metabolismo , Riñón Poliquístico Autosómico Dominante/prevención & control , Potasio/metabolismo , Canales de Potasio , Canales Catiónicos TRPP/genética , Canales Catiónicos TRPP/metabolismo , Pez Cebra/metabolismoRESUMEN
Serious loss of organic substances and notable release of refractory intracellular organics and cell-free antibiotic resistance genes (ARGs) caused by cell lysis are found when quick lime, FeCl3, and cationic polyacrylamide (CPAM) were used as sludge conditioners, which is not feasible to sludge separate incineration and increases ecological risks. Therefore, persulfate oxidation through ferrous (Fe2+-Na2S2O8) activation was applied for the upgradation of sludge conditioner in China, the specific resistance to filtration (SRF) and capillary suction time (CST) significantly decreased and the removed water increased from 40% to 54%, implying that the persulfate activated by ferrous (PAF) conditioner presents good performance in sludge dewatering. Organic matter content and heating value of sludge merely decreased, and Cl- content in sludge simultaneously decreased with the use of the PAF conditioner, thereby effectively reducing the corrosion risk to the incinerator and showing good compatibility with sludge separate incineration. In accordance with ferrous activation, sulfate radical plays an important role in sludge dewatering process because remarkable decrease in polysaccharides and protein contents from tightly bound extracellular polymeric substances (TB-EPS) was discovered. Based on flow cytometry analysis, slight cell lysis presented better filtrate quality by the use of PAF conditioner, 49.3% of refractory intracellular organics was removed and the respective ermB, tetW and blaTEM decreased by factors of 37.3%, 54.5% and 63.6% due to the strong oxidizing property of sulfate radical. The intensive decrease in refractory intracellular organics and cell-free ARGs will reduce the ecological risks. The total carbon emission significantly decreases to 1771.1 kgCO2/tDS when PAF conditioner was employed, which is beneficial to the upgradation of sludge deep dewatering conditioners.
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Aguas del Alcantarillado , Eliminación de Residuos Líquidos , Carbono , Matriz Extracelular de Sustancias Poliméricas , Incineración , AguaRESUMEN
With-no-lysine (WNK) kinases regulate renal sodium-chloride cotransporter (NCC) to maintain body sodium and potassium homeostasis. Gain-of-function mutations of WNK1 and WNK4 in humans lead to a Mendelian hypertensive and hyperkalemic disease pseudohypoaldosteronism type II (PHAII). X-ray crystal structure and in vitro studies reveal chloride ion (Cl-) binds to a hydrophobic pocket within the kinase domain of WNKs to inhibit its activity. The mechanism is thought to be important for physiological regulation of NCC by extracellular potassium. To test the hypothesis that WNK4 senses the intracellular concentration of Cl- physiologically, we generated knockin mice carrying Cl--insensitive mutant WNK4. These mice displayed hypertension, hyperkalemia, hyperactive NCC, and other features fully recapitulating human and mouse models of PHAII caused by gain-of-function WNK4. Lowering plasma potassium levels by dietary potassium restriction increased NCC activity in wild-type, but not in knockin, mice. NCC activity in knockin mice can be further enhanced by the administration of norepinephrine, a known activator of NCC. Raising plasma potassium by oral gavage of potassium inactivated NCC within 1 hour in wild-type mice, but had no effect in knockin mice. The results provide compelling support for the notion that WNK4 is a bona fide physiological intracellular Cl- sensor and that Cl- regulation of WNK4 underlies the mechanism of regulation of NCC by extracellular potassium.
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Cloruros/metabolismo , Proteínas Serina-Treonina Quinasas/fisiología , Animales , Ratones , Ratones Transgénicos , Potasio/administración & dosificación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Seudohipoaldosteronismo/genéticaRESUMEN
Lysine-deficient protein kinase-1 (WNK1) is critical for both embryonic angiogenesis and tumor-induced angiogenesis. However, the downstream effectors of WNK1 during these processes remain ambiguous. In this study, we identified that oxidative stress responsive 1b (osr1b) is upregulated in endothelial cells in both embryonic and tumor-induced angiogenesis in zebrafish, accompanied by downregulation of protein phosphatase 2A (pp2a) subunit ppp2r1bb. In addition, wnk1a and osr1b are upregulated in two liver cancer transgenic fish models: [tert x p53-/-] and [HBx,src,p53-/-,RPIA], while ppp2r1bb is downregulated in [tert x p53-/-]. Furthermore, using HUVEC endothelial cells co-cultured with HepG2 hepatoma cells, we confirmed that WNK1 plays a critical role in the induction of hepatoma cell migration in both endothelial cells and hepatoma cells. Moreover, overexpression of OSR1 can rescue the reduced cell migration caused by shWNK1 knockdown in HUVEC cells, indicating OSR1 is downstream of WNK1 in endothelial cells promoting hepatoma cell migration. Overexpression of PPP2R1A can rescue the increased cell migration caused by WNK1 overexpression in HepG2, indicating that PPP2R1A is a downstream effector in hepatoma. The combinatorial treatment with WNK1 inhibitor (WNK463) and OSR1 inhibitor (Rafoxanide) plus oligo-fucoidan via oral gavage to feed [HBx,src,p53-/-,RPIA] transgenic fish exhibits much more significant anticancer efficacy than Regorafenib for advanced HCC. Importantly, oligo-fucoidan can reduce the cell senescence marker-IL-1ß expression. Furthermore, oligo-fucoidan reduces the increased cell senescence-associated ß-galactosidase activity in tert transgenic fish treated with WNK1-OSR1 inhibitors. Our results reveal the WNK1-OSR1-PPP2R1A axis plays a critical role in both endothelial and hepatoma cells during tumor-induced angiogenesis promoting cancer cell migration. By in vitro and in vivo experiments, we further uncover the molecular mechanisms of WNK1 and its downstream effectors during tumor-induced angiogenesis. Targeting WNK1-OSR1-mediated anti-angiogenesis and anti-cancer activity, the undesired inflammation response caused by inhibiting WNK1-OSR1 can be attenuated by the combination therapy with oligo-fucoidan and may improve the efficacy.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Animales , Humanos , Proteína Quinasa Deficiente en Lisina WNK 1/genética , Proteína Quinasa Deficiente en Lisina WNK 1/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/genética , Pez Cebra/metabolismo , Rafoxanida , Proteína Fosfatasa 2/metabolismo , Lisina , Proteína p53 Supresora de Tumor , Antígenos de Histocompatibilidad Menor , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/genética , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/genética , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Factores de Transcripción/metabolismo , beta-Galactosidasa/metabolismoRESUMEN
Large-conductance K+ (BK) channels expressed in intercalated cells (ICs) in the aldosterone-sensitive distal nephron (ASDN) mediate flow-induced K+ secretion. In the ASDN of mice and rabbits, IC BK channel expression and activity increase with a high-K+ diet. In cell culture, the long isoform of with-no-lysine kinase 1 (L-WNK1) increases BK channel expression and activity. Apical L-WNK1 expression is selectively enhanced in ICs in the ASDN of rabbits on a high-K+ diet, suggesting that L-WNK1 contributes to BK channel regulation by dietary K+. We examined the role of IC L-WNK1 expression in enhancing BK channel activity in response to a high-K+ diet. Mice with IC-selective deletion of L-WNK1 (IC-L-WNK1-KO) and littermate control mice were placed on a high-K+ (5% K+, as KCl) diet for 10 or more days. IC-L-WNK1-KO mice exhibited reduced IC apical + subapical α-subunit expression and BK channel-dependent whole cell currents compared with controls. Six-hour urinary K+ excretion in response a saline load was similar in IC-L-WNK1-KO mice and controls. The observations that IC-L-WNK1-KO mice on a high-K+ diet have higher blood K+ concentration and reduced IC BK channel activity are consistent with impaired urinary K+ secretion, demonstrating that IC L-WNK1 has a role in the renal adaptation to a high-K+ diet.NEW & NOTEWORTHY When mice are placed on a high-K+ diet, genetic disruption of the long form of with no lysine kinase 1 (L-WNK1) in intercalated cells reduced relative apical + subapical localization of the large-conductance K+ channel, blunted large-conductance K+ channel currents in intercalated cells, and increased blood K+ concentration. These data confirm an in vivo role of L-WNK1 in intercalated cells in adaptation to a high-K+ diet.
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Riñón/metabolismo , Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Nefronas/metabolismo , Potasio/metabolismo , Proteína Quinasa Deficiente en Lisina WNK 1/metabolismo , Animales , Transporte Iónico , Riñón/citología , Ratones , Proteína Quinasa Deficiente en Lisina WNK 1/genéticaRESUMEN
The with-no-lysine (K) (WNK) signaling pathway to STE20/SPS1-related proline- and alanine-rich kinase (SPAK) and oxidative stress-responsive 1 (OSR1) kinase is an important mediator of cell volume and ion transport. SPAK and OSR1 associate with upstream kinases WNK 1-4, substrates, and other proteins through their C-terminal domains which interact with linear R-F-x-V/I sequence motifs. In this study we find that SPAK and OSR1 also interact with similar affinity with a motif variant, R-x-F-x-V/I. Eight of 16 human inward rectifier K+ channels have an R-x-F-x-V motif. We demonstrate that two of these channels, Kir2.1 and Kir2.3, are activated by OSR1, while Kir4.1, which does not contain the motif, is not sensitive to changes in OSR1 or WNK activity. Mutation of the motif prevents activation of Kir2.3 by OSR1. Both siRNA knockdown of OSR1 and chemical inhibition of WNK activity disrupt NaCl-induced plasma membrane localization of Kir2.3. Our results suggest a mechanism by which WNK-OSR1 enhance Kir2.1 and Kir2.3 channel activity by increasing their plasma membrane localization. Regulation of members of the inward rectifier K+ channel family adds functional and mechanistic insight into the physiological impact of the WNK pathway.
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Canales de Potasio de Rectificación Interna/química , Canales de Potasio de Rectificación Interna/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Humanos , Datos de Secuencia Molecular , Familia de Multigenes , Mutación , Canales de Potasio de Rectificación Interna/genética , Dominios Proteicos , Proteínas Serina-Treonina Quinasas/genética , Alineación de Secuencia , Transducción de SeñalRESUMEN
Soluble klotho (sKlotho), the shed ectodomain of α-klotho, protects the heart by down-regulating transient receptor potential canonical isoform 6 (TRPC6)-mediated calcium signaling. Binding to α2-3-sialyllactose moiety of gangliosides in lipid rafts and inhibition of raft-dependent signaling underlies the mechanism. A recent 3-Å X-ray structure of sKlotho in complex with fibroblast growth factor receptor (FGFR) and fibroblast growth factor 23 (FGF23) indicates that its ß6α6 loop might block access to the proposed binding site for α2-3-sialyllactose. It was concluded that sKlotho only functions in complex with FGFR and FGF23 and that sKlotho's pleiotropic effects all depend on FGF23. Here, we report that sKlotho can inhibit TRPC6 channels expressed in cells lacking endogenous FGFRs. Structural modeling and molecular docking show that a repositioned ß6α6 loop allows sKlotho to bind α2-3-sialyllactose. Molecular dynamic simulations further show the α2-3-sialyllactose-bound sKlotho complex to be stable. Domains mimicking sKlotho's sialic acid-recognizing activity inhibit TRPC6. The results strongly support the hypothesis that sKlotho can exert effects independent of FGF23 and FGFR.-Wright, J. D., An, S.-W., Xie, J., Lim, C., Huang, C.-L. Soluble klotho regulates TRPC6 calcium signaling via lipid rafts, independent of the FGFR-FGF23 pathway.
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Señalización del Calcio , Glucuronidasa/metabolismo , Microdominios de Membrana/metabolismo , Canal Catiónico TRPC6/metabolismo , Animales , Sitios de Unión , Unión Competitiva , Línea Celular , Factor-23 de Crecimiento de Fibroblastos , Factores de Crecimiento de Fibroblastos/química , Factores de Crecimiento de Fibroblastos/metabolismo , Glucuronidasa/química , Células HEK293 , Humanos , Proteínas Klotho , Lactosa/análogos & derivados , Lactosa/química , Lactosa/metabolismo , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Dominios y Motivos de Interacción de Proteínas , Ratas , Receptores de Factores de Crecimiento de Fibroblastos/química , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Ácidos Siálicos/química , Ácidos Siálicos/metabolismo , Solubilidad , Canal Catiónico TRPC6/antagonistas & inhibidores , Canal Catiónico TRPC6/químicaRESUMEN
WNK [with-no-lysine (K)] kinases are a family of four members of serine and threonine kinases that regulate renal Na+ and K+ transport. Mutations of WNK1 and WNK4 cause a hereditary hypertensive and hyperkalemic disease known as pseudohypoaldosteronism type II (PHA2). Unlike other WNK isoforms, WNK1 is ubiquitously expressed and regulates many other cellular processes outside the kidney. Oxidative stress response kinase (OSR1) and related STE 20/SPS1-related proline alanine-rich kinase (SPAK) are downstream kinases of WNK kinases. To examine the role of WNK kinase cascade in vivo, we generated global Wnk1-deleted mice and found that Wnk1-ablated mice die in utero from embryonic angiogenesis and cardiac developmental defects. Endothelial-specific Wnk1 deletion reveals that angiogenesis defect is due to WNK1 requirement in endothelium. We further showed that global and endothelial-deletion of Osr1 phenocopies Wnk1 deletion. Furthermore, expression of a catalytic constitutively active Osr1 transgene rescues angiogenesis defects and embryonic lethality of Wnk1-ablated mice. In zebrafish, Wnk1 knockdown causes similar angiogenesis defects to Vegf2 (Flk1) knockdown and that expression of WNK1 partially rescues Flk1 angiogenesis defects. The results indicate that WNK1 is downstream of VEGF signaling cascade. T-lymphocytes isolated from Wnk1-null mice exhibit migration defects. Inhibition of WNK1-OSR1 downstream target Na-K-2Cl cotransporter NKCC1 mimics migration defect of WNK1-deficient T-lymphocytes. Thus, WNK1-OSR1/SPAK cascade is important for angiogenesis. Regulation of ion homeostasis and cell volume may underlie the mechanism for WNK1 regulation of endothelial cell migration and angiogenesis.
RESUMEN
Soluble klotho, the shed ectodomain of the antiaging membrane protein α-klotho, is a pleiotropic endocrine/paracrine factor with no known receptors and poorly understood mechanism of action. Soluble klotho down-regulates growth factor-driven PI3K signaling, contributing to extension of lifespan, cardioprotection, and tumor inhibition. Here we show that soluble klotho binds membrane lipid rafts. Klotho binding to rafts alters lipid organization, decreases membrane's propensity to form large ordered domains for endocytosis, and down-regulates raft-dependent PI3K/Akt signaling. We identify α2-3-sialyllactose present in the glycan of monosialogangliosides as targets of soluble klotho. α2-3-Sialyllactose is a common motif of glycans. To explain why klotho preferentially targets lipid rafts we show that clustering of gangliosides in lipid rafts is important. In vivo, raft-dependent PI3K signaling is up-regulated in klotho-deficient mouse hearts vs. wild-type hearts. Our results identify ganglioside-enriched lipid rafts to be receptors that mediate soluble klotho regulation of PI3K signaling. Targeting sialic acids may be a general mechanism for pleiotropic actions of soluble klotho.
Asunto(s)
Gangliósidos/metabolismo , Glucuronidasa/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Microdominios de Membrana/metabolismo , Transducción de Señal/fisiología , Animales , Fenómenos Biofísicos/fisiología , Línea Celular , Línea Celular Tumoral , Células HEK293 , Células HeLa , Humanos , Proteínas Klotho , Ratones , Fosfatidilinositol 3-Quinasas/metabolismoRESUMEN
The ubiquitin-proteasome system degrades viral oncoproteins and other microbial virulence factors; however, the role of endolysosomal degradation pathways in these processes is unclear. Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma, and a constitutively active viral G protein-coupled receptor (vGPCR) contributes to the pathogenesis of KSHV-induced tumors. We report that a recently discovered autophagy-related protein, Beclin 2, interacts with KSHV GPCR, facilitates its endolysosomal degradation, and inhibits vGPCR-driven oncogenic signaling. Furthermore, monoallelic loss of Becn2 in mice accelerates the progression of vGPCR-induced lesions that resemble human Kaposi's sarcoma. Taken together, these findings indicate that Beclin 2 is a host antiviral molecule that protects against the pathogenic effects of KSHV GPCR by facilitating its endolysosomal degradation. More broadly, our data suggest a role for host endolysosomal trafficking pathways in regulating viral pathogenesis and oncogenic signaling.
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Proteínas Reguladoras de la Apoptosis/fisiología , Herpesvirus Humano 8/fisiología , Lisosomas/fisiología , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Virales/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/genética , Autofagia/fisiología , Beclina-1 , Transformación Celular Viral , Cruzamientos Genéticos , Modelos Animales de Enfermedad , Endocitosis/fisiología , Predisposición Genética a la Enfermedad , Células HEK293 , Herpesvirus Humano 8/inmunología , Herpesvirus Humano 8/patogenicidad , Heterocigoto , Humanos , Inmunidad Innata , Péptidos y Proteínas de Señalización Intercelular , Interleucina-6/biosíntesis , Interleucina-6/genética , Lisosomas/virología , Ratones , FN-kappa B/metabolismo , Proteínas/fisiología , Proteolisis , ARN Interferente Pequeño , Receptores de Quimiocina/metabolismo , Sarcoma de Kaposi/genética , Sarcoma de Kaposi/patología , Sarcoma de Kaposi/virología , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Neoplasias Cutáneas/virologíaRESUMEN
BACKGROUND: This study aimed to investigate whether cervical disc arthroplasty (CDA) would be equally effective in elderly patients as in the young. The inclusion criteria of published clinical trials for CDA-enrolled patients covered the ages from 18 to 78 years. However, there was a paucity of data addressing the differences of outcomes between older and the younger patients. METHODS: A series of consecutive patients who underwent one- or two-level CDA were retrospectively reviewed. Patients at the two extreme ends of the age distribution (≥65 and ≤ 40 years) were selected for comparison. Clinical outcome parameters included visual analog scale (VAS) of neck and arm pain, neck disability index (NDI), and Japanese Orthopaedic Association (JOA) scores. Radiographic outcomes included range of motion (ROM) at the indexed level and evaluation of heterotopic ossification (HO) by computed tomography (CT). Complication profiles were also investigated. RESULTS: There were 24 patients in the elderly group (≥65 years old) and 47 patients in the young group (≤40 years old) with an overall mean follow-up of 28.0 ± 21.97 months. The elderly group had more two-level CDA, and thus the mean operative time was longer (239 vs. 179 min, p < 0.05) than the young group. Both groups had similarly significant improvement in clinical outcomes at the final follow-up. All the replaced disc segments remained mobile on post-operative lateral flexion and extension radiographs. However, the elderly group had a slight decrease in mean ROM (- 0.32° ± 3.93°) at the index level after CDA when compared to that of pre-operation. In contrast, the young group had an increase in mean ROM (+ 0.68° ± 3.60°). The complication profiles were not different, though a trend toward dysphagia was noted in the elderly group (p = 0.073). The incidence or severity (grading) of HO was similar between the two groups. CONCLUSIONS: During the follow-up of two years, CDA was equally effective for patients over 65 years old and those under 40 years in clinical improvement. Although the elderly group demonstrated a small reduction of mean ROM after CDA, in contrast to the young group which had a small increase, the segmental mobility was well preserved at every indexed level for each group.
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Artroplastia/tendencias , Vértebras Cervicales/diagnóstico por imagen , Vértebras Cervicales/cirugía , Adulto , Factores de Edad , Anciano , Anciano de 80 o más Años , Artroplastia/métodos , Vértebras Cervicales/fisiología , Femenino , Estudios de Seguimiento , Humanos , Masculino , Rango del Movimiento Articular/fisiología , Estudios Retrospectivos , Resultado del Tratamiento , Adulto JovenRESUMEN
The Na+-Cl- cotransporter (NCC) in distal convoluted tubule (DCT) plays important roles in renal NaCl reabsorption. The current hypothesis for the mechanism of regulation of NCC focuses on WNK4 and intracellular Cl- concentration ([Cl-]i). WNK kinases bind Cl-, and Cl- binding decreases the catalytic activity. It is believed that hypokalemia under low K+ intake decreases [Cl-]i to activate WNK4, which thereby phosphorylates and stimulates NCC through activation of SPAK. However, increased NCC activity and apical NaCl entry would mitigate the fall in [Cl-]i. Whether [Cl-]i in DCT under low-K+ diet is sufficiently low to activate WNK4 is unknown. Furthermore, increased luminal NaCl delivery also stimulates NCC and causes upregulation of the transporter. Unlike low K+ intake, increased luminal NaCl delivery would tend to increase [Cl-]i. Thus we investigated the role of WNK4 and [Cl-]i in regulating NCC. We generated Wnk4-knockout mice and examined regulation of NCC by low K+ intake and by increased luminal NaCl delivery in knockout (KO) and wild-type mice. Wnk4-KO mice have marked reduction in the abundance, phosphorylation, and functional activity of NCC vs. wild type. Low K+ intake increases NCC phosphorylation and functional activity in wild-type mice, but not in Wnk4-KO mice. Increased luminal NaCl delivery similarly upregulates NCC, which, contrary to low K+ intake, is not abolished in Wnk4-KO mice. The results reveal that modulation of WNK4 activity by [Cl-]i is not the sole mechanism for regulating NCC. Increased luminal NaCl delivery upregulates NCC via yet unknown mechanism(s) that may override inhibition of WNK4 by high [Cl-]i.
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Túbulos Renales Distales/enzimología , Potasio en la Dieta/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Cloruro de Sodio/metabolismo , Animales , Transporte Biológico , Regulación Enzimológica de la Expresión Génica , Inyecciones Subcutáneas , Túbulos Renales Distales/efectos de los fármacos , Ratones Endogámicos C57BL , Ratones Noqueados , Fosforilación , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas Serina-Treonina Quinasas/genética , Eliminación Renal , Reabsorción Renal , Cloruro de Sodio/administración & dosificación , Inhibidores de los Simportadores del Cloruro de Sodio/farmacología , Miembro 3 de la Familia de Transportadores de Soluto 12/deficiencia , Miembro 3 de la Familia de Transportadores de Soluto 12/genética , Miembro 3 de la Familia de Transportadores de Soluto 12/metabolismoRESUMEN
Soluble Klotho (sKlotho) is the shed ectodomain of antiaging membrane Klotho that contains 2 extracellular domains KL1 and KL2, each of which shares sequence homology to glycosyl hydrolases. sKlotho elicits pleiotropic cellular responses with a poorly understood mechanism of action. Notably, in injury settings, sKlotho confers cardiac and renal protection by down-regulating calcium-permeable transient receptor potential canonical type isoform 6 (TRPC6) channels in cardiomyocytes and glomerular podocytes. Inhibition of PI3K-dependent exocytosis of TRPC6 is thought to be the underlying mechanism, and recent studies showed that sKlotho interacts with α2-3-sialyllactose-containing gangliosides enriched in lipid rafts to inhibit raft-dependent PI3K signaling. However, the structural basis for binding and recognition of α2-3-sialyllactose by sKlotho is unknown. Using homology modeling followed by docking, we identified key protein residues in the KL1 domain that are likely involved in binding sialyllactose. Functional experiments based on the ability of Klotho to down-regulate TRPC6 channel activity confirm the importance of these residues. Furthermore, KL1 domain binds α2-3-sialyllactose, down-regulates TRPC6 channels, and exerts protection against stress-induced cardiac hypertrophy in mice. Our results support the notion that sialogangliosides and lipid rafts are membrane receptors for sKlotho and that the KL1 domain is sufficient for the tested biologic activities. These findings can help guide the design of a simpler Klotho mimetic.-Wright, J. D., An, S.-W., Xie, J., Yoon, J., Nischan, N., Kohler, J. J., Oliver, N., Lim, C., Huang, C.-L. Modeled structural basis for the recognition of α2-3-sialyllactose by soluble Klotho.
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Glucuronidasa/metabolismo , Lactosa/análogos & derivados , Ácidos Siálicos/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Conformación de Carbohidratos , Cardiomegalia/metabolismo , Simulación por Computador , Gangliósidos/química , Gangliósidos/metabolismo , Regulación de la Expresión Génica/fisiología , Glucuronidasa/genética , Células HEK293 , Humanos , Proteínas Klotho , Lactosa/química , Lactosa/metabolismo , Microdominios de Membrana , Ratones , Modelos Moleculares , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Conformación Proteica , Ácidos Siálicos/química , Transducción de Señal/fisiologíaRESUMEN
Klotho is a type-1 membrane protein predominantly produced in the kidney, the extracellular domain of which is secreted into the systemic circulation. Membranous and secreted Klotho protect organs, including the kidney, but whether and how Klotho directly protects the glomerular filter is unknown. Here, we report that secreted Klotho suppressed transient receptor potential channel 6 (TRPC6)-mediated Ca2+ influx in cultured mouse podocytes by inhibiting phosphoinositide 3-kinase-dependent exocytosis of the channel. Furthermore, soluble Klotho reduced ATP-stimulated actin cytoskeletal remodeling and transepithelial albumin leakage in these cells. Overexpression of TRPC6 by gene delivery in mice induced albuminuria, and exogenous administration of Klotho ameliorated the albuminuria. Notably, immunofluorescence and in situ hybridization revealed Klotho expression in podocytes of mouse and human kidney. Heterozygous Klotho-deficient CKD mice had aggravated albuminuria compared with that in wild-type CKD mice with a similar degree of hypertension and reduced clearance function. Finally, disrupting the integrity of glomerular filter by saline infusion-mediated extracellular fluid volume expansion increased urinary Klotho excretion. These results reveal a potential novel function of Klotho in protecting the glomerular filter, and may offer a new therapeutic strategy for treatment of proteinuria.
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Glucuronidasa/fisiología , Podocitos , Proteinuria/etiología , Canales Catiónicos TRPC/fisiología , Albuminuria/etiología , Animales , Células Cultivadas , Humanos , Proteínas Klotho , Ratones , Insuficiencia Renal Crónica/complicaciones , Canal Catiónico TRPC6RESUMEN
KEY POINTS: The highly variable phenotypes observed in patients with classic Bartter's syndrome (BS) remain unsatisfactorily explained. The wide spectrum of functional severity of CLCNKB mutations may contribute to the phenotypic variability, and the genotype-phenotype association has not been established. Low-level expression of the human ClC-Kb channel in mammalian cells impedes the functional study of CLCNKB mutations, and the underlying cause is still unclear. The human ClC-Kb channel is highly degraded by proteasome in human embryonic kidney cells. The C-terminal in-frame green fluorescent protein fusion may slow down the proteasome-mediated proteolysis. Barttin co-expression necessarily improves the stability, membrane trafficking and gating of ClC-Kb. CLCNKB mutations in barttin-binding sites, dimer interface or selectivity filter often have severe functional consequences. The remaining chloride conductance of the ClC-Kb mutant channel significantly correlates with the phenotypes, such as age at diagnosis, plasma chloride concentration, and the degree of calciuria in patients with classic BS. ABSTRACT: Mutations in the CLCNKB gene encoding the human voltage-gated chloride ClC-Kb (hClC-Kb) channel cause classic Bartter's syndrome (BS). In contrast to antenatal BS, classic BS manifests with highly variable phenotypes. The functional severity of the mutant channel has been proposed to explain this phenomenon. Due to difficulties in the expression of hClC-Kb in heterologous expression systems, the functional consequences of mutant channels have not been thoroughly examined, and the genotype-phenotype association has not been established. In this study, we found that hClC-Kb, when expressed in human embryonic kidney (HEK) cells, was unstable due to degradation by proteasome. In-frame fusion of green fluorescent protein (GFP) to the C-terminus of the channel may ameliorate proteasome degradation. Co-expression of barttin increased protein abundance and membrane trafficking of hClC-Kb and markedly increased functional chloride current. We then functionally characterized 18 missense mutations identified in our classic BS cohort and others using HEK cells expressing hClC-Kb-GFP. Most CLCNKB mutations resulted in marked reduction in protein abundance and chloride current, especially those residing at barttin binding sites, dimer interface and selectivity filter. We enrolled classic BS patients carrying homozygous missense mutations with well-described functional consequences and clinical presentations for genotype-phenotype analysis. We found significant correlations of mutant chloride current with the age at diagnosis, plasma chloride concentration and urine calcium excretion rate. In conclusion, hClC-Kb expression in HEK cells is susceptible to proteasome degradation, and fusion of GFP to the C-terminus of hClC-Kb improves protein expression. The functional severity of the CLCNKB mutation is an important determinant of the phenotype in classic BS.