RESUMO
DNA is a danger signal sensed by cGAS to engage signaling through STING to activate innate immune functions. The best-studied downstream responses to STING activation include expression of type I interferon and inflammatory genes, but STING also activates other pathways, including apoptosis. Here, we report that STING-dependent induction of apoptosis in macrophages occurs through the intrinsic mitochondrial pathway and is mediated via IRF3 but acts independently of gene transcription. By intersecting four mass spectrometry datasets, we identify SAM68 as crucial for the induction of apoptosis downstream of STING activation. SAM68 is essential for the full activation of apoptosis. Still, it is not required for STING-mediated activation of IFN expression or activation of NF-κB. Mechanistic studies reveal that protein trafficking is required and involves SAM68 recruitment to STING upon activation, with the two proteins associating at the Golgi or a post-Golgi compartment. Collectively, our work identifies SAM68 as a STING-interacting protein enabling induction of apoptosis through this DNA-activated innate immune pathway.
Assuntos
Proteínas de Membrana , Transdução de Sinais , Proteínas de Membrana/metabolismo , Macrófagos/metabolismo , Proteínas de Ciclo Celular/metabolismo , DNA/metabolismo , ApoptoseRESUMO
BACKGROUND: Inhibition of prostaglandin synthesis by nonsteroidal anti-inflammatory drugs is associated with cardiovascular mortality and kidney disease. This study hypothesizes that urinary prostaglandin E2 (PGE2) and PGE2 metabolite (PGEM) excretions are markers of cardiovascular and kidney health, because they reflect both systemic and kidney-derived PGE2 production. METHODS AND RESULTS: PGE2 and PGEM were measured in spot urine samples from 2291 participants (≥55 years old) of the population-based Rotterdam Study. Urinary PGE2 and PGEM excretions were analyzed using linear regression analyses to identify cross-sectional associations with cardiovascular risk factors and baseline estimated glomerular filtration rate (eGFR). Longitudinal associations with cardiovascular mortality and kidney outcomes (eGFR <60 or <45 mL/min per 1.73 m2 and the composite outcome 40% eGFR loss or kidney failure) were assessed with Cox regression. Urinary PGE2 and PGEM excretions were higher with increasing age, lower eGFR, smoking, diabetes, and albuminuria. A 2-fold higher urinary PGE2 and PGEM excretion was associated with a higher risk of cardiovascular mortality (28 825 patient-years; 160 events; PGE2 hazard ratio [HR], 1.27, [95% CI, 1.06-1.54]; PGEM HR, 1.36 [95% CI, 1.10-1.67]). Higher PGE2 excretions were also associated with a higher risk of incident eGFR <60 mL/min per 1.73 m2 (31 530 person-years; 691 events; HR, 1.13 [95% CI, 1.02-1.25]) with similar HRs for the other kidney outcomes. CONCLUSIONS: Urinary PGE2 and PGEM excretions are novel markers for the presence and progression of cardiovascular and kidney disease. Future studies should address whether these associations are causal and can be targeted to improve cardiovascular and kidney outcomes.
Assuntos
Doenças Cardiovasculares , Nefropatias , Humanos , Pessoa de Meia-Idade , Dinoprostona , Doenças Cardiovasculares/diagnóstico , Doenças Cardiovasculares/epidemiologia , Doenças Cardiovasculares/etiologia , Estudos Transversais , Nefropatias/diagnóstico , Nefropatias/epidemiologia , Nefropatias/complicações , Rim , Taxa de Filtração Glomerular/fisiologia , Albuminúria/urina , Fatores de RiscoRESUMO
Transmembrane potassium (K) gradients are key determinants of membrane potential that can modulate action potentials, control muscle contractility, and influence ion channel and transporter activity. Daily K intake is normally equal to the amount of K in the entire extracellular fluid (ECF) creating a critical challenge - how to maintain ECF [K] and membrane potential in a narrow range during feast and famine. Adaptations to maintain ECF [K] include sensing the K intake, sensing ECF [K] vs. desired set-point and activating mediators that regulate K distribution between ECF and ICF, and regulate renal K excretion. In this focused review, we discuss the basis of these adaptions, including (1) potential mechanisms for rapid feedforward signaling to kidney and muscle after a meal (before a rise in ECF [K]), (2) how skeletal muscles sense and respond to changes in ECF [K], (3) effects of K on aldosterone biosynthesis, and (4) how the kidney responds to changes in ECF [K] to modify K excretion. The concepts of sexual dimorphisms in renal K handling adaptation are introduced, and the molecular mechanisms that can account for the benefits of a K-rich diet to maintain cardiovascular health are discussed. Although the big picture of K homeostasis is becoming more clear, we also highlight significant pieces of the puzzle that remain to be solved, including knowledge gaps in our understanding of initiating signals, sensors and their connection to homeostatic adjustments of ECF [K].
Assuntos
Rim , Potássio , Líquido Extracelular/metabolismo , Homeostase/fisiologia , Rim/metabolismo , Músculo Esquelético/metabolismo , Potássio/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
BACKGROUND: Diabetic nephropathy (DN) is a serious complication of diabetes and a common cause of end stage renal failure. Insulin-like growth factor (IGF)-signaling has been implicated in DN, but is mechanistically poorly understood. Here, we assessed the activity of the metalloproteinase PAPP-A, an activator of IGF activity, and its possible interaction with the endogenous PAPP-A inhibitors stanniocalcin (STC)-1 and -2 in the mammalian kidney under normal and hyperglycemic conditions. METHODS AND RESULTS: Immunohistochemistry demonstrated that PAPP-A, its proteolytic substrate IGF binding protein-4, STC1 and STC2 are present in the human kidney. Endogenous inhibited complexes of PAPP-A (PAPP-A:STC1 and PAPP-A:STC2) were demonstrated in media conditioned by human mesangial cells (HMCs), suggesting that PAPP-A activity is regulated by the STCs in kidney tissue. A method for the selective detection of active PAPP-A in tissue was developed and a significant increase in glomerular active PAPP-A in human diabetic kidney relative to normal was observed. In DN patients, the estimated glomerular filtration rate correlated with PAPP-A activity. In diabetic mice, glomerular growth was reduced when PAPP-A activity was antagonized by adeno-associated virus-mediated overexpression of STC2. CONCLUSION: We propose that PAPP-A activity in renal tissue is precisely balanced by STC1 and STC2. An imbalance in this equilibrium causing increased PAPP-A enzymatic activity potentially contributes to the development of DN, and thus, therapeutic targeting of PAPP-A activity may represent a novel strategy for its treatment.
Assuntos
Diabetes Mellitus Experimental , Nefropatias Diabéticas , Proteína Plasmática A Associada à Gravidez , Animais , Diabetes Mellitus Experimental/complicações , Nefropatias Diabéticas/etiologia , Humanos , Hipertrofia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Mamíferos/metabolismo , Camundongos , Proteína Plasmática A Associada à Gravidez/metabolismo , ProteóliseRESUMO
AIMS: The kidneys play a major role in maintaining Pi homeostasis. Patients in later stages of CKD develop hyperphosphatemia. One novel treatment option is tenapanor, an intestinal-specific NHE3 inhibitor. To gain mechanistic insight into the role of intestinal NHE3 in Pi homeostasis, we studied tamoxifen-inducible intestinal epithelial cell-specific NHE3 knockout (NHE3IEC-KO ) mice. METHODS: Mice underwent dietary Pi challenges, and hormones as well as urinary/plasma Pi were determined. Intestinal 33 P uptake studies were conducted in vivo to compare the effects of tenapanor and NHE3IEC-KO . Ex vivo Pi transport was measured in everted gut sacs and brush border membrane vesicles. Intestinal and renal protein expression of Pi transporters were determined. RESULTS: On the control diet, NHE3IEC-KO mice had similar Pi homeostasis, but a ~25% reduction in FGF23 compared with control mice. Everted gut sacs and brush border membrane vesicles showed enhanced Pi uptake associated with increased Npt2b expression in NHE3IEC-KO mice. Acute oral Pi loading resulted in higher plasma Pi in NHE3IEC-KO mice. Tenapanor inhibited intestinal 33 P uptake acutely but then led to hyper-absorption at later time points compared to vehicle. In response to high dietary Pi , plasma Pi and FGF23 increased to higher levels in NHE3IEC-KO mice which was associated with greater Npt2b expression. Reduced renal Npt2c and a trend for reduced Npt2a expression were unable to correct for higher plasma Pi . CONCLUSION: Intestinal NHE3 has a significant contribution to Pi homeostasis. In contrast to effects described for tenapanor on Pi homeostasis, NHE3IEC-KO mice show enhanced, rather than reduced, intestinal Pi uptake.
Assuntos
Células Epiteliais , Fosfatos , Trocador 3 de Sódio-Hidrogênio/genética , Animais , Células Epiteliais/metabolismo , Homeostase , Absorção Intestinal , Mucosa Intestinal/metabolismo , Camundongos , Camundongos Knockout , Fosfatos/metabolismo , Trocador 3 de Sódio-Hidrogênio/metabolismoRESUMO
Mutations in the Melanoma-Associated Antigen D2 (MAGED2) cause antenatal Bartter syndrome type 5 (BARTS5). This rare disease is characterized by perinatal loss of urinary concentration capability and large urine volumes. The underlying molecular mechanisms of this disease are largely unclear. Here, we study the effect of MAGED2 knockdown on kidney cell cultures using proteomic and phosphoproteomic analyses. In HEK293T cells, MAGED2 knockdown induces prominent changes in protein phosphorylation rather than changes in protein abundance. MAGED2 is expressed in mouse embryonic kidneys and its expression declines during development. MAGED2 interacts with G-protein alpha subunit (GNAS), suggesting a role in G-protein coupled receptors (GPCR) signalling. In kidney collecting duct cell lines, Maged2 knockdown subtly modulated vasopressin type 2 receptor (V2R)-induced cAMP-generation kinetics, rewired phosphorylation-dependent signalling, and phosphorylation of CREB. Maged2 knockdown resulted in a large increase in aquaporin-2 abundance during long-term V2R activation. The increase in aquaporin-2 protein was mediated transcriptionally. Taken together, we link MAGED2 function to cellular signalling as a desensitizer of V2R-induced aquaporin-2 expression. SIGNIFICANCE: In most forms of Bartter Syndrome, the underlying cause of the disease is well understood. In contrast, the role of MAGED2 mutations in a newly discovered form of Bartter Syndrome (BARTS5) is unknown. In our manuscript we could show that MAGED2 modulates vasopressin-induced protein and phosphorylation patterns in kidney cells, providing a broad basis for further studies of MAGED2 function in development and disease.
Assuntos
Aquaporina 2 , Túbulos Renais Coletores , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Antígenos de Neoplasias , Aquaporina 2/genética , Aquaporina 2/metabolismo , Feminino , Células HEK293 , Humanos , Túbulos Renais Coletores/metabolismo , Camundongos , Gravidez , Proteômica , Vasopressinas/metabolismoRESUMO
Bile acid diarrhea is a chronic condition caused by increased delivery of bile acids to the colon. The underlying mechanisms remain to be elucidated. To investigate genes involved in bile acid diarrhea, systems-level analyses were used on a rat bile acid diarrhea model. Twelve male Wistar Munich rats, housed in metabolic cages, were fed either control or bile acid-mixed (1% wt/wt) diets for 10 days. Food intake, water intake, urine volume, body weight, and fecal output were monitored daily. After euthanasia, colonic epithelial cells were isolated using calcium chelation and processed for systems-level analyses, that is, RNA-sequencing transcriptomics and mass spectrometry proteomics. Bile acid-fed rats suffered diarrhea, indicated by increased drinking, feces weight, and fecal water content compared with control rats. Urine output was unchanged. With bile acid feeding, RNA-sequencing revealed 204 increased and 401 decreased mRNAs; mass spectrometry revealed 183 increased and 111 decreased proteins. Among the altered genes were genes associated with electrolyte and water transport (including Slc12a7, Clca4, and Aqp3) and genes associated with bile acid transport (Slc2b1, Abcg2, Slc51a, Slc51b, and Fabps). Correlation analysis showed a significant positive correlation (Pearson's r = 0.28) between changes in mRNA expression and changes in protein expression. However, caution must be exercised in making a direct correlation between experimentally determined transcriptomes and proteomes. Genes associated with bile acid transport responded to bile acid feeding, suggesting that colonic bile acid transport also occur by regulated protein facilitated mechanisms in addition to passive diffusion. In summary, the study provides annotated rat colonic epithelial cell transcriptome and proteome with response to bile acid feeding.NEW & NOTEWORTHY Feeding rats with a bile acid caused changes in fecal output, underlining this bile acid diarrhea model's usefulness. Colonic epithelial expression of genes associated with facilitated transport of bile acids was altered during bile acid feeding. The study raises the possibility of regulated colonic transepithelial transport of bile acids in response to luminal bile acids. In addition, this study provides annotated rat colonic epithelial cell transcriptome and proteome with response to bile acid feeding.
Assuntos
Ácidos e Sais Biliares/metabolismo , Colo/metabolismo , Neoplasias do Colo/metabolismo , Células Epiteliais/metabolismo , Animais , Diarreia/metabolismo , Dieta , Fezes/química , Masculino , Ratos WistarRESUMO
Targeting the collecting duct water channel aquaporin 2 (AQP2) to the plasma membrane is essential for the maintenance of mammalian water homeostasis. The vasopressin V2 receptor (V2R), which is a GS protein-coupled receptor that increases intracellular cAMP levels, has a major role in this targeting process. Although a rise in cAMP levels and activation of protein kinase A are involved in facilitating the actions of V2R, studies in knockout mice and cell models have suggested that cAMP signalling pathways are not an absolute requirement for V2R-mediated AQP2 trafficking to the plasma membrane. In addition, although AQP2 phosphorylation is a known prerequisite for V2R-mediated plasma membrane targeting, none of the known AQP2 phosphorylation events appears to be rate-limiting in this process, which suggests the involvement of other factors; cytoskeletal remodelling has also been implicated. Notably, several regulatory processes and signalling pathways involved in AQP2 trafficking also have a role in the pathophysiology of autosomal dominant polycystic kidney disease, although the role of AQP2 in cyst progression is unknown. Here, we highlight advances in the field of AQP2 regulation that might be exploited for the treatment of water balance disorders and provide a rationale for targeting these pathways in autosomal dominant polycystic kidney disease.
Assuntos
Aquaporina 2/metabolismo , Túbulos Renais Coletores/metabolismo , Rim Policístico Autossômico Dominante/metabolismo , Equilíbrio Hidroeletrolítico/fisiologia , Água/metabolismo , Proteínas de Ancoragem à Quinase A/metabolismo , Antagonistas dos Receptores de Hormônios Antidiuréticos/uso terapêutico , Membrana Celular/metabolismo , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Fluconazol , Humanos , Rim Policístico Autossômico Dominante/tratamento farmacológico , Transporte Proteico , Proteínas Proto-Oncogênicas pp60(c-src)/antagonistas & inibidores , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Receptores de Vasopressinas/metabolismo , Transdução de Sinais , Tolvaptan/uso terapêutico , Proteína Wnt-5a/metabolismoRESUMO
(1) Background: The unusual accumulation of Na,K-ATPase complexes in the brush border membrane of choroid plexus epithelial cells have intrigued researchers for decades. However, the full range of the expressed Na,K-ATPase subunits and their relation to the microvillus cytoskeleton remains unknown. (2) Methods: RT-PCR analysis, co-immunoprecipitation, native PAGE, mass spectrometry, and differential centrifugation were combined with high-resolution immunofluorescence histochemistry, proximity ligase assays, and stimulated emission depletion (STED) microscopy on mouse choroid plexus cells or tissues in order to resolve these issues. (3) Results: The choroid plexus epithelium expresses Na,K-ATPase subunits α1, α2, ß1, ß2, ß3, and phospholemman. The α1, α2, ß1, and ß2, subunits are all localized to the brush border membrane, where they appear to form a complex. The ATPase complexes may stabilize in the brush border membrane via anchoring to microvillar actin indirectly through ankyrin-3 or directly via other co-precipitated proteins. Aquaporin 1 (AQP1) may form part of the proposed multi-protein complexes in contrast to another membrane protein, the Na-K-2Cl cotransporter 1 (NKCC1). NKCC1 expression seems necessary for full brush border membrane accumulation of the Na,K-ATPase in the choroid plexus. (4) Conclusion: A multitude of Na,K-ATPase subunits form molecular complexes in the choroid plexus brush border, which may bind to the cytoskeleton by various alternative actin binding proteins.
Assuntos
Aquaporina 1/fisiologia , Plexo Corióideo/metabolismo , Células Epiteliais/metabolismo , Microvilosidades/metabolismo , Membro 2 da Família 12 de Carreador de Soluto/fisiologia , Actinas/metabolismo , Animais , Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Arginine vasopressin (AVP) stimulates the concentration of renal urine by increasing the principal cell expression of aquaporin-2 (AQP2) water channels. Prostaglandin E2 (PGE2) and prostaglandin2α (PGF2α) increase the water absorption of the principal cell without AVP, but PGE2 decreases it in the presence of AVP. The underlying mechanism of this paradoxical response was investigated here. Mouse cortical collecting duct (mkpCCDc14) cells mimic principal cells as they endogenously express AQP2 in response to AVP. PGE2 increased AQP2 abundance without desmopressin (dDAVP), while in the presence of dDAVP, PGE2, and PGF2α reduced AQP2 abundance. dDAVP increased the cellular PGD2 and PGE2 release and decreased the PGF2α release. MpkCCD cells expressed mRNAs for the receptors of PGE2 (EP1/EP4), PGF2 (FP), and TxB2 (TP). Incubation with dDAVP increased the expression of EP1 and FP but decreased the expression of EP4. In the absence of dDAVP, incubation of mpkCCD cells with an EP4, but not EP1/3, agonist increased AQP2 abundance, and the PGE2-induced increase in AQP2 was blocked with an EP4 antagonist. Moreover, in the presence of dDAVP, an EP1/3, but not EP4, agonist decreased the AQP2 abundance, and the addition of EP1 antagonists prevented the PGE2-mediated downregulation of AQP2. Our study shows that in mpkCCDc14 cells, reduced EP4 receptor and increased EP1/FP receptor expression by dDAVP explains the differential effects of PGE2 and PGF2α on AQP2 abundance with or without dDAVP. As the V2R and EP4 receptor, but not the EP1 and FP receptor, can couple to Gs and stimulate the cyclic adenosine monophosphate (cAMP) pathway, our data support a view that cells can desensitize themselves for receptors activating the same pathway and sensitize themselves for receptors of alternative pathways.
RESUMO
The connexin 37 (Cx37) channel is clustered at gap junctions between cells in the renal vasculature or the renal tubule where it is abundant in basolateral cell interdigitations and infoldings of epithelial cells in the proximal tubule, thick ascending limb, distal convoluted tubule and collecting duct; however, physiological data regarding its role are limited. In this study, we investigated the role of Cx37 in fluid homeostasis using mice with a global deletion of Cx37 (Cx37-/- mice). Under baseline conditions, Cx37-/- had ~40% higher fluid intake associated with ~40% lower urine osmolality compared to wild-type (WT) mice. No differences were observed between genotypes in urinary adenosine triphosphate or prostaglandin E2, paracrine factors that alter renal water handling. After 18-hours of water deprivation, plasma aldosterone and urine osmolality increased significantly in Cx37-/- and WT mice; however, the latter remained ~375 mmol/kg lower in Cx37-/- mice, an effect associated with a more pronounced body weight loss despite higher urinary AVP/creatinine ratios compared to WT mice. Consistent with this, fluid intake in the first 3 hours after water deprivation was 37% greater in Cx37-/- vs WT mice. Cx37-/- mice showed significantly lower renal AQP2 abundance and AQP2 phosphorylation at serine 256 than WT mice in response to vehicle or dDAVP, suggesting a partial contribution of the kidney to the lower urine osmolality. The abundance and responses of the vasopressin V2 receptor, AQP3, NHE3, NKCC2, NCC, H+-ATPase, αENaC, γENaC or Na+/K+-ATPase were not significantly different between genotypes. In summary, these results demonstrate that Cx37 is important for body water handling.
Assuntos
Aquaporina 2/metabolismo , Conexinas/fisiologia , Deleção de Genes , Polidipsia/etiologia , Poliúria/etiologia , Animais , Aquaporina 2/genética , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , Polidipsia/patologia , Poliúria/patologia , Proteína alfa-4 de Junções ComunicantesRESUMO
The sodium-hydrogen exchanger isoform 3 (NHE3, SLC9A3) is abundantly expressed in the gastrointestinal tract and is proposed to play essential roles in Na+ and fluid absorption as well as acid-base homeostasis. Mutations in the SLC9A3 gene can cause congenital sodium diarrhea (CSD). However, understanding the precise role of intestinal NHE3 has been severely hampered due to the lack of a suitable animal model. To navigate this problem and better understand the role of intestinal NHE3, we generated a tamoxifen-inducible intestinal epithelial cell-specific NHE3 knockout mouse model (NHE3IEC-KO). Before tamoxifen administration, the phenotype and blood parameters of NHE3IEC-KO were unremarkable compared with control mice. After tamoxifen administration, NHE3IEC-KO mice have undetectable levels of NHE3 in the intestine. NHE3IEC-KO mice develop watery, alkaline diarrhea in combination with a swollen small intestine, cecum and colon. The persistent diarrhea results in higher fluid intake. After 3 weeks, NHE3IEC-KO mice show a â¼25% mortality rate. The contribution of intestinal NHE3 to acid-base and Na+ homeostasis under normal conditions becomes evident in NHE3IEC-KO mice that have metabolic acidosis, lower blood bicarbonate levels, hyponatremia and hyperkalemia associated with drastically elevated plasma aldosterone levels. These results demonstrate that intestinal NHE3 has a significant contribution to acid-base, Na+ and volume homeostasis, and lack of intestinal NHE3 has consequences on intestinal structural integrity. This mouse model mimics and explains the phenotype of individuals with CSD carrying SLC9A3 mutations.
Assuntos
Anormalidades Múltiplas/genética , Diarreia/congênito , Células Epiteliais/metabolismo , Erros Inatos do Metabolismo/genética , Trocador 3 de Sódio-Hidrogênio/genética , Anormalidades Múltiplas/metabolismo , Anormalidades Múltiplas/mortalidade , Anormalidades Múltiplas/patologia , Animais , Diarreia/genética , Diarreia/metabolismo , Diarreia/mortalidade , Diarreia/patologia , Modelos Animais de Doenças , Feminino , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Masculino , Erros Inatos do Metabolismo/metabolismo , Erros Inatos do Metabolismo/mortalidade , Erros Inatos do Metabolismo/patologia , Camundongos , Camundongos Knockout , Mutação , Trocador 3 de Sódio-Hidrogênio/metabolismoRESUMO
The metabolic sensor AMP-activated protein kinase (AMPK) inhibits the epithelial Na+ channel (ENaC), a key regulator of salt reabsorption by the kidney and thus total body volume and blood pressure. Recent studies have suggested that AMPK promotes the association of p21-activated kinase-interacting exchange factor-ß1 ß1Pix, 14-3-3 proteins, and the ubiquitin ligase neural precursor cell expressed developmentally downregulated protein (Nedd)4-2 into a complex that inhibits ENaC by enhancing Nedd4-2 binding to ENaC and ENaC degradation. Functional ß1Pix is required for ENaC inhibition by AMPK and promotes Nedd4-2 phosphorylation and stability in mouse kidney cortical collecting duct cells. Here, we report that AMPK directly phosphorylates ß1Pix in vitro. Among several AMPK phosphorylation sites on ß1Pix detected by mass spectrometry, Ser71 was validated as functionally significant. Compared with wild-type ß1Pix, overexpression of a phosphorylation-deficient ß1Pix-S71A mutant attenuated ENaC inhibition and the AMPK-activated interaction of both ß1Pix and Nedd4-2 to 14-3-3 proteins in cortical collecting duct cells. Similarly, overexpression of a ß1Pix-Δ602-611 deletion tract mutant unable to bind 14-3-3 proteins decreased the interaction between Nedd4-2 and 14-3-3 proteins, suggesting that 14-3-3 binding to ß1Pix is critical for the formation of a ß1Pix/Nedd4-2/14-3-3 complex. With expression of a general peptide inhibitor of 14-3-3-target protein interactions (R18), binding of both ß1Pix and Nedd4-2 to 14-3-3 proteins was reduced, and AMPK-dependent ENaC inhibition was also attenuated. Altogether, our results demonstrate the importance of AMPK-mediated phosphorylation of ß1Pix at Ser71, which promotes 14-3-3 interactions with ß1Pix and Nedd4-2 to form a tripartite ENaC inhibitory complex, in the mechanism of ENaC regulation by AMPK.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Células Epiteliais/metabolismo , Canais Epiteliais de Sódio/metabolismo , Rim/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Animais , Regulação Enzimológica da Expressão Gênica/genética , Células HEK293 , Humanos , Túbulos Renais Coletores/metabolismo , Camundongos , Mutação/genética , Ubiquitina-Proteína Ligases Nedd4/genética , Ubiquitina-Proteína Ligases Nedd4/metabolismo , Fosforilação , Fatores de Troca de Nucleotídeo Guanina Rho/genéticaRESUMO
BACKGROUND: The NaCl cotransporter NCC in the kidney distal convoluted tubule (DCT) regulates urinary NaCl excretion and BP. Aldosterone increases NaCl reabsorption via NCC over the long-term by altering gene expression. But the acute effects of aldosterone in the DCT are less well understood. METHODS: Proteomics, bioinformatics, and cell biology approaches were combined with animal models and gene-targeted mice. RESULTS: Aldosterone significantly increases NCC activity within minutes in vivo or ex vivo. These effects were independent of transcription and translation, but were absent in the presence of high potassium. In vitro, aldosterone rapidly increased intracellular cAMP and inositol phosphate accumulation, and altered phosphorylation of various kinases/kinase substrates within the MAPK/ERK, PI3K/AKT, and cAMP/PKA pathways. Inhibiting GPR30, a membrane-associated receptor, limited aldosterone's effects on NCC activity ex vivo, and NCC phosphorylation was reduced in GPR30 knockout mice. Phosphoproteomics, network analysis, and in vitro studies determined that aldosterone activates EGFR-dependent signaling. The EGFR immunolocalized to the DCT and EGFR tyrosine kinase inhibition decreased NCC activity ex vivo and in vivo. CONCLUSIONS: Aldosterone acutely activates NCC to modulate renal NaCl excretion.
Assuntos
Aldosterona/farmacologia , Túbulos Renais Distais/metabolismo , Transdução de Sinais , Tiazidas/farmacologia , Aldosterona/metabolismo , Animais , Pressão Sanguínea , Cálcio/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Biologia Computacional , AMP Cíclico/metabolismo , Receptores ErbB/metabolismo , Síndrome de Gitelman/metabolismo , Rim/metabolismo , Masculino , Camundongos , Mineralocorticoides/metabolismo , Fosforilação , Proteômica , Receptores de Estrogênio/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Cloreto de Sódio/metabolismo , Membro 3 da Família 12 de Carreador de Soluto/metabolismoRESUMO
Cholinergic receptor activation leads to premature development of hypertension and infiltration of proinflammatory CD161a+/CD68+ M1 macrophages into the renal medulla. Renal inflammation is implicated in renal sodium retention and the development of hypertension. Renal denervation is known to decrease renal inflammation. The objective of this study was to determine the role of CD161a+/CD68+ macrophages and renal sympathetic nerves in cholinergic-hypertension and renal sodium retention. Bilateral renal nerve denervation (RND) and immune ablation of CD161a+ immune cells were performed in young prehypertensive spontaneously hypertensive rat (SHR) followed by infusion of either saline or nicotine (15 mg·kg-1·day-1) for 2 wk. Immune ablation was conducted by injection of unconjugated azide-free antibody targeting rat CD161a+. Blood pressure was monitored by tail cuff plethysmography. Tissues were harvested at the end of infusion. Nicotine induced premature hypertension, renal expression of the sodium-potassium chloride cotransporter (NKCC2), increases in renal sodium retention, and infiltration of CD161a+/CD68+ macrophages into the renal medulla. All of these effects were abrogated by RND and ablation of CD161a+ immune cells. Cholinergic activation of CD161a+ immune cells with nicotine leads to the premature development of hypertension in SHR. The effects of renal sympathetic nerves on chemotaxis of CD161a+ macrophages to the renal medulla, increased renal expression of NKCC2, and renal sodium retention contribute to cholinergic hypertension. The CD161a+ immune cells are necessary and essential for this prohypertensive nicotine-mediated inflammatory response.NEW & NOTEWORTHY This is the first study that describes a novel integrative physiological interaction between the adrenergic, cholinergic, and renal systems in the development of hypertension, describing data for the role of each in a genetic model of essential hypertension. Noteworthy findings include the prevention of nicotine-mediated hypertension following successful immune ablation of CD161a+ immune cells and the necessary role these cells play in the overexpression of the sodium-potassium-chloride cotransporter (NKCC2) in the renal medulla and renal sodium retention. Renal infiltration of these cells is demonstrated to be dependent on the presence of renal adrenergic innervation. These data offer a fertile ground of therapeutic potential for the treatment of hypertension as well as open the door for further investigation into the mechanism involved in inflammation-mediated renal sodium transporter expression. Taken together, these findings suggest immune therapy, renal denervation, and, possibly, other new molecular targets as having a potential role in the development and maintenance of essential hypertension.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Pressão Sanguínea , Hipertensão/prevenção & controle , Rim , Macrófagos/efeitos dos fármacos , Subfamília B de Receptores Semelhantes a Lectina de Células NK/antagonistas & inibidores , Nicotina , Agonistas Nicotínicos , Fenol/administração & dosagem , Artéria Renal/inervação , Reabsorção Renal , Sódio/urina , Simpatectomia Química , Animais , Modelos Animais de Doenças , Hipertensão/induzido quimicamente , Hipertensão/imunologia , Hipertensão/fisiopatologia , Mediadores da Inflamação/imunologia , Rim/irrigação sanguínea , Rim/efeitos dos fármacos , Rim/imunologia , Macrófagos/imunologia , Masculino , Subfamília B de Receptores Semelhantes a Lectina de Células NK/imunologia , Ratos Endogâmicos SHR , Membro 1 da Família 12 de Carreador de Soluto/metabolismoRESUMO
Aquaporin 2 (AQP2) mediates the osmotic water permeability of the kidney collecting duct in response to arginine vasopressin (VP) and is essential for body water homeostasis. VP effects on AQP2 occur via long-term alterations in AQP2 abundance and short-term changes in AQP2 localization. Several of the effects of VP on AQP2 are dependent on AQP2 phosphorylation and ubiquitylation; post-translational modifications (PTM) that modulate AQP2 subcellular distribution and function. Although several protein kinases, phosphatases, and ubiquitin E3 ligases have been implicated in AQP2 PTM, how AQP2 is deubiquitylated or the role of deubiquitylases (DUBS) in AQP2 function is unknown. Here, we report a novel role of the ubiquitin-specific protease USP4 in modulating AQP2 function. USP4 co-localized with AQP2 in the mouse kidney, and in mpkCCD14 cells USP4 and AQP2 abundance are increased by VP. AQP2 and USP4 co-immunoprecipitated from mpkCCD14 cells and mouse kidney, and in vitro, USP4 can deubiquitylate AQP2. In mpkCCD14 cells, shRNA mediated knockdown of USP4 decreased AQP2 protein abundance, whereas no changes in AQP2 mRNA levels or VP-induced cAMP production were detected. VP-induced AQP2 membrane accumulation in knockdown cells was significantly reduced, which was associated with higher levels of ubiquitylated AQP2. AQP2 protein half-life was also significantly reduced in USP4 knockdown cells. Taken together, the data suggest that USP4 is a key regulator of AQP2 deubiquitylation and that loss of USP4 leads to increased AQP2 ubiquitylation, decreased AQP2 levels, and decreased cell surface AQP2 accumulation upon VP treatment. These studies have implications for understanding body water homeostasis.
Assuntos
Aquaporina 2/metabolismo , Membrana Celular/metabolismo , Proteases Específicas de Ubiquitina/metabolismo , Animais , Linhagem Celular , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Meia-Vida , Rim/citologia , Camundongos Endogâmicos C57BL , Modelos Biológicos , Ligação Proteica , Ubiquitinação/efeitos dos fármacos , Vasopressinas/farmacologiaRESUMO
Receptor-mediated endocytosis is responsible for reabsorption of transferrin (Tf) in renal proximal tubules (PTs). Although the role of the megalin-cubilin receptor complex (MCRC) in this process is unequivocal, modalities independent of this complex are evident but as yet undefined. Here, using immunostaining and Tf-flux assays, FACS analysis, and fluorescence imaging, we report localization of Tf receptor 1 (TfR1), the cognate Tf receptor mediating cellular holo-Tf (hTf) acquisition, to the apical brush border of the PT, with expression gradually declining along the PT in mouse and rat kidneys. In functional studies, hTf uptake across the apical membrane of cultured PT epithelial cell (PTEC) monolayers increased in response to decreased cellular iron after desferrioxamine (DFO) treatment. We also found that apical hTf uptake under basal conditions is receptor-associated protein (RAP)-sensitive and therefore mediated by the MCRC but becomes RAP-insensitive under DFO treatment, with concomitantly decreased megalin and cubilin expression levels and increased TfR1 expression. Thus, as well as the MCRC, TfR1 mediates hTf uptake across the PT apical brush border, but in conditions of decreased cellular iron, hTf uptake is predominated by augmented apical TfR1. In conclusion, both the MCRC and TfR1 mediate hTf uptake across apical brush border membranes of PTECs and reciprocally respond to decreased cellular iron. Our findings have implications for renal health, whole-body iron homeostasis, and pathologies arising from disrupted iron balance.
Assuntos
Ferro/metabolismo , Túbulos Renais Proximais/metabolismo , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores da Transferrina/metabolismo , Transferrina/metabolismo , Animais , Linhagem Celular Transformada , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Endogâmicos WKYRESUMO
BACKGROUND: Cholera toxin (CT)-induced diarrhea is mediated by cyclic adenosine monophosphate (cAMP)-mediated active Cl- secretion via the cystic fibrosis transmembrane conductance regulator (CFTR). Although the constitutive activation of adenylyl cyclase (AC) in response to CT is due to adenosine diphosphate ribosylation of the small G protein α-subunit activating CFTR with consequent secretory diarrhea, the AC isoform(s) involved remain unknown. METHODS: We generated intestinal epithelial cell-specific adenylyl cyclase 6 (AC6) knockout mice to study its role in CT-induced diarrhea. RESULTS: AC6 messenger RNA levels were the highest of all 9 membrane-bound AC isoforms in mouse intestinal epithelial cells. Intestinal epithelial-specific AC6 knockout mice (AC6loxloxVillinCre) had undetectable AC6 levels in small intestinal and colonic epithelial cells. No significant differences in fluid and food intake, plasma electrolytes, intestinal/colon anatomy and morphology, or fecal water content were observed between genotypes. Nevertheless, CT-induced fluid accumulation in vivo was completely absent in AC6loxloxVillinCre mice, associated with a lack of forskolin- and CT-induced changes in the short-circuit current (ISC) of the intestinal mucosa, impaired cAMP generation in acutely isolated small intestinal epithelial cells, and significantly impaired apical CFTR levels in response to forskolin. CONCLUSIONS: AC6 is a novel target for the treatment of CT-induced diarrhea.
Assuntos
Adenilil Ciclases/metabolismo , Toxina da Cólera/toxicidade , Cólera/fisiopatologia , Diarreia/fisiopatologia , Células Epiteliais/enzimologia , Células Epiteliais/metabolismo , Adenilil Ciclases/deficiência , Animais , Colforsina/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células Epiteliais/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
The choroid plexus epithelium within the brain ventricles secretes the majority of the cerebrospinal fluid (CSF). The luminal Na+-K+-ATPase acts in concert with a host of other transport proteins to mediate efficient fluid secretion across the epithelium. The CSF contains little protein buffer, but the pH value seems nonetheless maintained within narrow limits, even when faced with acid-base challenges. The involvement of choroid plexus acid-base transporters in CSF pH regulation is highlighted by the expression of several acid-base transporters in the epithelium. The aim of the present study was to identify novel acid-base transporters expressed in the luminal membrane of the choroid plexus epithelium to pave the way for systematic investigations of each candidate transporter in the regulation of CSF pH. Mass spectrometry analysis of proteins from epithelial cells isolated by fluorescence-activated cell sorting identified the Cl-/H+ exchangers ClC-3, -4, -5, and -7 in addition to known choroid plexus acid-base transporters. RT-PCR on FACS isolated epithelial cells confirmed the expression of the corresponding mRNAs, as well as Na+/H+ exchanger NHE6 mRNA. Both NHE6 and ClC-7 were immunolocalized to the luminal plasma membrane domain of the choroid plexus epithelial cells. Dynamic imaging of intracellular pH and membrane potential changes in isolated choroid plexus epithelial cells demonstrated Cl- gradient-driven changes in intracellular pH and membrane potential that are consistent with Cl-/H+ exchange. In conclusion, we have detected for the first time NHE6 and ClC-7 in the choroid plexus, which are potentially involved in pH regulation of the CSF.
Assuntos
Membrana Celular/metabolismo , Líquido Cefalorraquidiano/metabolismo , Canais de Cloreto/metabolismo , Plexo Corióideo/metabolismo , Células Epiteliais/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Animais , Separação Celular/métodos , Canais de Cloreto/genética , Plexo Corióideo/citologia , Citometria de Fluxo , Concentração de Íons de Hidrogênio , Masculino , Potenciais da Membrana , Camundongos Endogâmicos C57BL , Proteômica/métodos , Trocadores de Sódio-Hidrogênio/genética , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
The thiazide-sensitive sodium chloride cotransporter NCC is important for maintaining serum sodium (Na+) and, indirectly, serum potassium (K+) levels. Functional studies on NCC have used cell lines with native NCC expression, transiently transfected nonpolarized cell lines, or Xenopus laevis oocytes. Here, we developed the use of polarized Madin-Darby canine kidney type I (MDCKI) mammalian epithelial cell lines with tetracycline-inducible human NCC expression to study NCC activity and membrane abundance in the same system. In radiotracer assays, induced cells grown on filters had robust thiazide-sensitive and chloride dependent sodium-22 (22Na) uptake from the apical side. To minimize cost and maximize throughput, assays were modified to use cells grown on plastic. On plastic, cells had similar thiazide-sensitive 22Na uptakes that increased following preincubation of cells in chloride-free solutions. NCC was detected in the plasma membrane, and both membrane abundance and phosphorylation of NCC were increased by incubation in chloride-free solutions. Furthermore, in cells exposed for 15 min to low or high extracellular K+, the levels of phosphorylated NCC increased and decreased, respectively. To demonstrate that the system allows rapid and systematic assessment of mutated NCC, three phosphorylation sites in NCC were mutated, and NCC activity was examined. 22Na fluxes in phosphorylation-deficient mutants were reduced to baseline levels, whereas phosphorylation-mimicking mutants were constitutively active, even without chloride-free stimulation. In conclusion, this system allows the activity, cellular localization, and abundance of wild-type or mutant NCC to be examined in the same polarized mammalian expression system in a rapid, easy, and low-cost fashion.