RESUMO
Unlike human patients with mutations in the 56-kDa B1 subunit isoform of the vacuolar proton-pumping ATPase (V-ATPase), B1-deficient mice (Atp6v1b1(-/-)) do not develop metabolic acidosis under baseline conditions. This is due to the insertion of V-ATPases containing the alternative B2 subunit isoform into the apical membrane of renal medullary collecting duct intercalated cells (ICs). We previously reported that quantitative Western blots (WBs) from whole kidneys showed similar B2 protein levels in Atp6v1b1(-/-) and wild-type mice (Paunescu TG, Russo LM, Da Silva N, Kovacikova J, Mohebbi N, Van Hoek AN, McKee M, Wagner CA, Breton S, Brown D. Am J Physiol Renal Physiol 293: F1915-F1926, 2007). However, WBs from renal medulla (including outer and inner medulla) membrane and cytosol fractions reveal a decrease in the levels of the ubiquitous V-ATPase E1 subunit. To compare V-ATPase expression specifically in ICs from wild-type and Atp6v1b1(-/-) mice, we crossed mice in which EGFP expression is driven by the B1 subunit promoter (EGFP-B1(+/+) mice) with Atp6v1b1(-/-) mice to generate novel EGFP-B1(-/-) mice. We isolated pure IC populations by fluorescence-assisted cell sorting from EGFP-B1(+/+) and EGFP-B1(-/-) mice to compare their V-ATPase subunit protein levels. We report that V-ATPase A, E1, and H subunits are all significantly downregulated in EGFP-B1(-/-) mice, while the B2 protein level is considerably increased in these animals. We conclude that under baseline conditions B2 upregulation compensates for the lack of B1 and is sufficient to maintain basal acid-base homeostasis, even when other V-ATPase subunits are downregulated.
Assuntos
Células Epiteliais/metabolismo , Rim/metabolismo , ATPases Vacuolares Próton-Translocadoras/genética , Animais , Membrana Celular/metabolismo , Células Epiteliais/citologia , Citometria de Fluxo , Rim/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Regulação para Cima , ATPases Vacuolares Próton-Translocadoras/metabolismoRESUMO
The renal-specific Na-K-2Cl co-transporter, NKCC2, plays a pivotal role in regulating body salt levels and blood pressure. NKCC2 mutations lead to type I Bartter syndrome, a life-threatening kidney disease. Regulation of NKCC2 trafficking behavior serves as a major mechanism in controlling NKCC2 activity across the plasma membrane. However, the identities of the protein partners involved in cell surface targeting of NKCC2 are largely unknown. To gain insight into these processes, we used a yeast two-hybrid system to screen a kidney cDNA library for proteins that interact with the NKCC2 C terminus. One binding partner we identified was SCAMP2 (secretory carrier membrane protein 2). Microscopic confocal imaging and co-immunoprecipitation assays confirmed NKCC2-SCAMP2 interaction in renal cells. SCAMP2 associated also with the structurally related co-transporter NCC, suggesting that the interaction with SCAMP2 is a common feature of sodium-dependent chloride co-transporters. Heterologous expression of SCAMP2 specifically decreased cell surface abundance as well as transport activity of NKCC2 across the plasma membrane. Co-immunolocalization experiments revealed that intracellularly retained NKCC2 co-localizes with SCAMP2 in recycling endosomes. The rate of NKCC2 endocytic retrieval, assessed by the sodium 2-mercaptoethane sulfonate cleavage assay, was not affected by SCAMP2. The surface-biotinylatable fraction of newly inserted NKCC2 in the plasma membrane was reduced by SCAMP2, demonstrating that SCAMP2-induced decrease in surface NKCC2 is due to decreased exocytotic trafficking. Finally, a single amino acid mutation, cysteine 201 to alanine, within the conserved cytoplasmic E peptide of SCAMP2, which is believed to regulate exocytosis, abolished SCAMP2-mediated down-regulation of the co-transporter. Taken together, these data are consistent with a model whereby SCAMP2 regulates NKCC2 transit through recycling endosomes and limits the cell surface targeting of the co-transporter by interfering with its exocytotic trafficking.
Assuntos
Proteínas de Transporte/metabolismo , Membrana Celular/metabolismo , Exocitose/fisiologia , Simportadores de Cloreto de Sódio-Potássio/metabolismo , Animais , Proteínas de Transporte/genética , Membrana Celular/genética , Células HEK293 , Humanos , Masculino , Camundongos , Gambás , Transporte Proteico/fisiologia , Simportadores de Cloreto de Sódio-Potássio/genética , Membro 1 da Família 12 de Carreador de SolutoRESUMO
The sodium-independent anion exchanger pendrin is expressed in several tissues including the kidney cortical collecting duct (CCD), where it acts as a chloride/bicarbonate exchanger and has been shown to participate in the regulation of acid-base homeostasis and blood pressure. The renal sympathetic nervous system is known to play a key role in the development of salt-induced hypertension. This study aimed to determine whether pendrin may partly mediate the effects of ß adrenergic receptors (ß-AR) on renal salt handling. We investigated the regulation of pendrin activity by the cAMP/protein kinase A (PKA) signaling pathway, both in vitro in opossum kidney proximal (OKP) cells stably transfected with pendrin cDNA and ex vivo in isolated microperfused CCDs stimulated by isoproterenol, a ß-AR agonist. We found that stimulation of the cAMP/PKA pathway in OKP cells increased the amount of pendrin at the cell surface as well as its transport activity. These effects stemmed from increased exocytosis of pendrin and were associated with its phosphorylation. Furthermore, cAMP effects on the membrane expression and activity of pendrin were abolished by mutating the serine 49 located in the intracellular N-terminal domain of pendrin. Finally, we showed that isoproterenol increases pendrin trafficking to the apical membrane as well as the reabsorption of both Cl(-) and Na(+) in microperfused CCDs. All together, our results strongly suggest that pendrin activation by the cAMP/PKA pathway underlies isoproterenol-induced stimulation of NaCl reabsorption in the kidney collecting duct, a mechanism likely involved in the sodium-retaining effect of ß-adrenergic agonists.
Assuntos
Proteínas de Transporte de Ânions/metabolismo , Antiportadores de Cloreto-Bicarbonato/metabolismo , AMP Cíclico/farmacologia , Túbulos Renais Coletores/metabolismo , Receptores Adrenérgicos beta/metabolismo , Cloreto de Sódio/metabolismo , Agonistas Adrenérgicos beta/farmacologia , Animais , Membrana Celular/metabolismo , Células Cultivadas , AMP Cíclico/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Isoproterenol/farmacologia , Túbulos Renais Coletores/citologia , Túbulos Renais Coletores/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos , Modelos Animais , Gambás , Fosforilação/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Receptores Adrenérgicos beta/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Transportadores de SulfatoRESUMO
Mutations in the anion exchanger pendrin are responsible for Pendred syndrome, an autosomal recessive disease characterized by deafness and goitre. Pendrin is highly expressed in kidney collecting ducts, where it acts as a chloride/bicarbonate exchanger and thereby contributes to the regulation of acid-base homoeostasis and blood pressure. The present study aimed to characterize the intrinsic properties of pendrin. Mouse pendrin was transfected in HEK (human embryonic kidney) 293 and OKP (opossum kidney proximal tubule) cells and its activity was determined by monitoring changes in the intracellular pH induced by variations of transmembrane anion gradients. Combining measurements of pendrin activity with mathematical modelling we found that its affinity for Cl-, HCO3- and OH- varies with intracellular pH, with increased activity at low intracellular pH. Maximal pendrin activity was also stimulated at low extracellular pH, suggesting the presence of both intracellular and extracellular proton regulatory sites. We identified five putative pendrin glycosylation sites, only two of which are used. Mutagenesis-induced disruption of pendrin glycosylation did not alter its cell-surface expression or polarized targeting to the apical membrane and basal activity, but fully abrogated its sensitivity to extracellular pH. The hither to unknown regulation of pendrin by external pH may constitute a key mechanism in controlling ionic exchanges across the collecting duct and inner ear.
Assuntos
Proteínas de Transporte de Ânions/metabolismo , Cloretos/metabolismo , Radical Hidroxila/metabolismo , Animais , Proteínas de Transporte de Ânions/genética , Linhagem Celular , Clonagem Molecular , Glicosilação , Humanos , Concentração de Íons de Hidrogênio , Cinética , Camundongos , Mutagênese Sítio-Dirigida , Gambás , Transportadores de SulfatoRESUMO
Pharmaceutical companies with a medical mindset and an empowered Medical Affairs function are well equipped to meet the needs and expectations of patients and society. Yet, as capacity to understand and serve those needs accelerates, so too do expectations. Evidence-based practice, without delay, is expected throughout the development and delivery of medicine, healthcare, and information, and potential sources of evidence are legion. To keep pace and go beyond, to innovate, requires efficiencies. Not the disguised cutbacks of political language, but the collaborative constructive efficiencies of shared learning, forming new evidence bases for further progress. Here, we describe the first year of a medical transformation process at a global mid-sized pharmaceutical company. Beginning with a broad review designed to leverage collective intelligence and focus on meaningful outcomes for patients, this process examined and reshaped the structure, culture, and tools of the medical organization and its interactions within and outside the company. We report the findings of the diagnostic phase, outline the solutions implemented to date, and anticipate the next steps in this dynamic evolving journey.
Assuntos
Medicina , Atenção à Saúde , Humanos , Idioma , Preparações FarmacêuticasRESUMO
Modern dietary habits are characterized by high-sodium and low-potassium intakes, each of which was correlated with a higher risk for hypertension. In this study, we examined whether long-term variations in the intake of sodium and potassium induce lasting changes in the plasma concentration of circulating steroids by developing a mathematical model of steroidogenesis in mice. One finding of this model was that mice increase their plasma progesterone levels specifically in response to potassium depletion. This prediction was confirmed by measurements in both male mice and men. Further investigation showed that progesterone regulates renal potassium handling both in males and females under potassium restriction, independent of its role in reproduction. The increase in progesterone production by male mice was time dependent and correlated with decreased urinary potassium content. The progesterone-dependent ability to efficiently retain potassium was because of an RU486 (a progesterone receptor antagonist)-sensitive stimulation of the colonic hydrogen, potassium-ATPase (known as the non-gastric or hydrogen, potassium-ATPase type 2) in the kidney. Thus, in males, a specific progesterone concentration profile induced by chronic potassium restriction regulates potassium balance.
Assuntos
Glândulas Suprarrenais/metabolismo , Hipopotassemia/metabolismo , Rim/metabolismo , Potássio na Dieta/metabolismo , Progesterona/biossíntese , Glândulas Suprarrenais/efeitos dos fármacos , Glândulas Suprarrenais/enzimologia , Aldosterona/biossíntese , Análise de Variância , Animais , Linhagem Celular , Doença Crônica , Corticosterona/biossíntese , Modelos Animais de Doenças , Feminino , Regulação Enzimológica da Expressão Gênica , ATPase Trocadora de Hidrogênio-Potássio/genética , ATPase Trocadora de Hidrogênio-Potássio/metabolismo , Antagonistas de Hormônios/farmacologia , Humanos , Hipopotassemia/enzimologia , Hipopotassemia/genética , Rim/efeitos dos fármacos , Rim/enzimologia , Masculino , Camundongos , Camundongos Knockout , Mifepristona/farmacologia , Modelos Biológicos , Potássio na Dieta/administração & dosagem , Potássio na Dieta/urina , Progesterona/sangue , Receptores de Progesterona/antagonistas & inibidores , Receptores de Progesterona/metabolismo , Sódio na Dieta/metabolismo , Fatores de Tempo , Regulação para CimaRESUMO
AIM: Type A intercalated cells of the renal collecting duct participate in the maintenance of the acid/base balance through their capacity to adapt proton secretion to homeostatic requirements. We previously showed that increased proton secretion stems in part from the enlargement of the population of proton secreting cells in the outer medullary collecting duct through division of fully differentiated cells, and that this response is triggered by growth/differentiation factor 15. This study aimed at deciphering the mechanism of acid load-induced secretion of Gdf15 and its mechanism of action. METHODS: We developed an original method to evaluate the proliferation of intercalated cells and applied it to genetically modified or pharmacologically treated mice under basal and acid-loaded conditions. RESULTS: Gdf15 is secreted by principal cells of the collecting duct in response to the stimulation of vasopressin receptors. Vasopressin-induced production of cAMP triggers activation of AMP-stimulated kinases and of Na,K-ATPase, and induction of p53 and Gdf15. Gdf15 action on intercalated cells is mediated by ErbB2 receptors, the activation of which triggers the expression of cyclin d1, of p53 and anti-proliferative genes, and of Egr1. CONCLUSION: Acidosis-induced proliferation of intercalated cells results from a cross talk with principal cells which secrete Gdf15 in response to their stimulation by vasopressin. Thus, vasopressin is a major determinant of the collecting duct cellular homeostasis as it promotes proliferation of intercalated cells under acidosis conditions and of principal cells under normal acid-base status.
Assuntos
Acidose , Túbulos Renais Coletores , Animais , Proliferação de Células , Camundongos , Néfrons , ATPase Trocadora de Sódio-PotássioRESUMO
V-ATPases (H(+) ATPases) are multisubunit, ATP-dependent proton pumps that regulate pH homeostasis in virtually all eukaryotes. They are involved in key cell biological processes including vesicle trafficking, endosomal pH sensing, membrane fusion and intracellular signaling. They also have critical systemic roles in renal acid excretion and blood pH balance, male fertility, bone remodeling, synaptic transmission, olfaction and hearing. Furthermore, V-ATPase dysfunction either results in or aggravates various other diseases, but little is known about the complex protein interactions that regulate these varied V-ATPase functions. Therefore, we performed a proteomic analysis to identify V-ATPase associated proteins and construct a V-ATPase interactome. Our analysis using kidney tissue revealed V-ATPase-associated protein clusters involved in protein quality control, complex assembly and intracellular trafficking. ARHGEF7, DMXL1, EZR, NCOA7, OXR1, RPS6KA3, SNX27 and 9 subunits of the chaperonin containing TCP1 complex (CCT) were found to interact with V-ATPase for the first time in this study. Knockdown of two interacting proteins, DMXL1 and WDR7, inhibited V-ATPase-mediated intracellular vesicle acidification in a kidney cell line, providing validation for the utility of our interactome as a screen for functionally important novel V-ATPase-regulating proteins. Our data, therefore, provide new insights and directions for the analysis of V-ATPase cell biology and (patho)physiology.
Assuntos
Mapas de Interação de Proteínas , ATPases Vacuolares Próton-Translocadoras/metabolismo , Animais , Imunoprecipitação , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Fosforilação , Dobramento de Proteína , Mapeamento de Interação de Proteínas/métodos , Transporte Proteico , ATPases Vacuolares Próton-Translocadoras/genéticaRESUMO
Uncontrolled inflammation is one of the leading causes of kidney failure. Pro-inflammatory responses can occur in the absence of infection, a process called sterile inflammation. Here we show that the purinergic receptor P2Y14 (GPR105) is specifically and highly expressed in collecting duct intercalated cells (ICs) and mediates sterile inflammation in the kidney. P2Y14 is activated by UDP-glucose, a damage-associated molecular pattern molecule (DAMP) released by injured cells. We found that UDP-glucose increases pro-inflammatory chemokine expression in ICs as well as MDCK-C11 cells, and UDP-glucose activates the MEK1/2-ERK1/2 pathway in MDCK-C11 cells. These effects were prevented following inhibition of P2Y14 with the small molecule PPTN. Tail vein injection of mice with UDP-glucose induced the recruitment of neutrophils to the renal medulla. This study identifies ICs as novel sensors, mediators and effectors of inflammation in the kidney via P2Y14.
Assuntos
Inflamação/metabolismo , Túbulos Renais Coletores/patologia , Receptores Purinérgicos P2Y/metabolismo , Uridina Difosfato Glucose/farmacologia , Animais , Células Cultivadas , Cães , Inflamação/imunologia , Inflamação/patologia , Túbulos Renais Coletores/citologia , Túbulos Renais Coletores/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Células Madin Darby de Rim Canino , Masculino , Camundongos , Neutrófilos/metabolismoRESUMO
Tumor necrosis factor alpha (TNFalpha) plays a major role in chronic heart failure, signaling through two different receptor subtypes, TNFR1 and TNFR2. Our aim was to further delineate the functional role and signaling pathways related to TNFR1 and TNFR2 in cardiac myocytes. In cardiac myocytes isolated from control rats, TNFalpha induced ROS production, exerted a dual positive and negative action on [Ca(2+)] transient and cell fractional shortening, and altered cell survival. Neutralizing anti-TNFR2 antibodies exacerbated TNFalpha responses on ROS production and cell death, arguing for a major protective role of the TNFR2 pathway. Treatment with either neutralizing anti-TNFR1 antibodies or the glutathione precursor, N-acetylcysteine (NAC), favored the emergence of TNFR2 signaling that mediated a positive effect of TNFalpha on [Ca(2+)] transient and cell fractional shortening. The positive effect of TNFalpha relied on TNFR2-dependent activation of the cPLA(2) activity, independently of serine 505 phosphorylation of the enzyme. Together with cPLA(2) redistribution and AA release, TNFalpha induced a time-dependent phosphorylation of ERK, MSK1, PKCzeta, CaMKII, and phospholamban on the threonine 17 residue. Taken together, our results characterized a TNFR2-dependent signaling and illustrated the close interplay between TNFR1 and TNFR2 pathways in cardiac myocytes. Although apparently predominant, TNFR1-dependent responses were under the yoke of TNFR2, acting as a critical limiting factor. In vivo NAC treatment proved to be a unique tool to selectively neutralize TNFR1-mediated effects of TNFalpha while releasing TNFR2 pathways.