RESUMEN
We have previously demonstrated that KS-133 is a specific and potent antagonist of vasoactive intestinal peptide receptor 2 (VIPR2). We have also shown that vasoactive intestinal peptide-VIPR2 signaling affects the polarity and activation of tumor-associated macrophages, which is another strategy for cancer immunotherapy apart from the activation of effector T cells. In this study, we aimed to examine whether the selective blockade of VIPR2 by KS-133 changes the polarization of macrophages and induces anti-tumor effects. In the presence of KS-133, genetic markers indicative of tumor-aggressive M1-type macrophages were upregulated, and conversely, those of tumor-supportive M2-type macrophages were downregulated. Daily subcutaneous administration of KS-133 tended to suppress the growth of CT26 tumors (murine colorectal cancer-derived cells) implanted subcutaneously in Balb/c mice. To improve the pharmacological efficacy and reduce the number of doses, we examined a nanoformulation of KS-133 using the US Food and Drug Administration-approved pharmaceutical additive surfactant Cremophor® EL. KS-133 nanoparticles (NPs) were approximately 15 nm in size and stable at 4°C after preparation. Meanwhile, KS-133 was gradually released from the NPs as the temperature was increased. Subcutaneous administration of KS-133 NPs once every 3 days had stronger anti-tumor effects than daily subcutaneous administration of KS-133. Furthermore, KS-133 NPs significantly enhanced the pharmacological efficacy of an immune checkpoint-inhibiting anti-PD-1 antibody. A pharmacokinetic study suggested that the enhancement of anti-tumor activity was associated with improvement of the pharmacokinetic profile of KS-133 upon nanoformulation. Our data have revealed that specific blockade of VIPR2 by KS-133 has therapeutic potential for cancer both alone and in combination with immune checkpoint inhibitors.
Asunto(s)
Neoplasias , Receptores de Tipo II del Péptido Intestinal Vasoactivo , Animales , Ratones , Línea Celular Tumoral , Inmunoterapia , Macrófagos , Microambiente TumoralRESUMEN
Macrophages are a major population of immune cells in solid cancers, especially colorectal cancers. Tumor-associated macrophages (TAMs) are commonly divided into M1-like (tumor suppression) and M2-like (tumor promotion) phenotypes. Vasoactive intestinal peptide (VIP) is an immunoregulatory neuropeptide with a potent anti-inflammatory function. Inhibition of VIP signaling has been shown to increase CD8+ T cell proliferation and function in viral infection and lymphoma. However, the role of VIP in macrophage polarization and function in solid tumors remains unknown. Here, we demonstrated that conditioned medium from CT26 (CT26-CM) cells enhanced M2-related marker and VIP receptor (VPAC) gene expression in RAW264.7 macrophages. VIP hybrid, a VIP antagonist, enhanced M1-related genes but reduced Mrc1 gene expression and increased phagocytic ability in CT26-CM-treated RAW264.7 cells. In immunodeficient SCID mice, VIP antagonist alone or in combination with anti-PD-1 antibody attenuated CT26 tumor growth compared with the control. Analysis of tumor-infiltrating leukocytes found that VIP antagonist increased M1/M2 ratios and macrophage phagocytosis of CT26-GFP cells. Furthermore, Vipr2 gene silencing or VPAC2 activation affected the polarization of CT26-CM-treated RAW264.7 cells. In conclusion, the inhibition of VIP signaling enhanced M1 macrophage polarization and macrophage phagocytic function, resulting in tumor regression in a CT26 colon cancer model.
Asunto(s)
Neoplasias del Colon , Macrófagos , Péptido Intestinal Vasoactivo , Animales , Ratones , Neoplasias del Colon/patología , Macrófagos/metabolismo , Ratones SCID , Receptores de Péptido Intestinal Vasoactivo/metabolismo , Receptores de Tipo II del Péptido Intestinal Vasoactivo/metabolismo , Transducción de Señal , Péptido Intestinal Vasoactivo/antagonistas & inhibidores , Péptido Intestinal Vasoactivo/metabolismo , Células RAW 264.7RESUMEN
AIMS: The number of cancer survivors with cardiovascular disease is increasing. However, the effect of cancer on body fluid regulation remains to be clarified. In this study, we evaluated body osmolyte and water imbalance in rats with hepatocellular carcinoma. MAIN METHODS: Wistar rats were administered diethylnitrosamine, a carcinogenic drug, to establish liver cancer. We analyzed tissue osmolyte and water content, and their associations with aldosterone secretion. KEY FINDINGS: Hepatocellular carcinoma rats had significantly reduced body mass and the amount of total body sodium, potassium, and water. However, these rats had significantly increased relative tissue sodium, potassium, and water content per tissue dry weight. Furthermore, these changes in sodium and water balance in hepatocellular carcinoma rats were significantly associated with increased 24-h urinary aldosterone excretion. Supplementation with 0.25% salt in drinking water improved body weight reduction associated with sodium and water retention in hepatocellular carcinoma rats, which was suppressed by treatment with spironolactone, a mineralocorticoid receptor antagonist. Additionally, the urea-driven water conservation system was activated in hepatocellular carcinoma rats. SIGNIFICANCE: These findings suggest that hepatocellular carcinoma induces body mass loss in parallel with activation of the water conservation system including aldosterone secretion and urea accumulation to retain osmolyte and water. The osmolyte and water retention at the tissue level may be a causative factor for ascites and edema formation in liver failure rats.
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Aldosterona/orina , Carcinoma Hepatocelular/orina , Dietilnitrosamina/toxicidad , Neoplasias Hepáticas Experimentales/orina , Equilibrio Hidroelectrolítico , Pérdida de Peso , Animales , Carcinoma Hepatocelular/inducido químicamente , Carcinoma Hepatocelular/tratamiento farmacológico , Neoplasias Hepáticas Experimentales/inducido químicamente , Neoplasias Hepáticas Experimentales/tratamiento farmacológico , Masculino , Antagonistas de Receptores de Mineralocorticoides/farmacología , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/metabolismo , Ratas , Ratas Endogámicas WKY , Receptores de Mineralocorticoides/metabolismo , Espironolactona/farmacologíaRESUMEN
Proximal tubules (PTs) take up most of the glucose in the glomerular filtrate and return it to peritubular capillary blood. Sodium-glucose cotransporter 2 (SGLT2) at the apical membrane takes up glucose into the cell. Glucose then flows across the cells and is transported to the interstitium via glucose transporter 2 (GLUT2) at the basolateral membrane. However, glucose transport under SGLT2 inhibition remains poorly understood. In this study, we evaluated the dynamics of a fluorescent glucose analog, 2-NBDG, in the PTs of live mice treated with or without the SGLT2 inhibitor, luseogliflozin. We employed real-time multiphoton microscopy, in which insulin enhanced 2-NBDG uptake in skeletal muscle. Influx and efflux of 2-NBDG in PT cells were compared under hypo-, normo-, and hyperglycemic conditions. Luseogliflozin did not exert significant effects on glucose influx parameters under any level of blood glucose. Our results suggest that blood glucose level per se does not alter glucose influx or efflux kinetics in PTs. In conclusion, neither SGLT2 inhibition nor blood glucose level affect glucose uptake kinetics in PTs. The former was because of glucose influx through basolateral GLUT2, which is an established bidirectional transporter.
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Transporte Biológico/efectos de los fármacos , Glucemia/efectos de los fármacos , Glucosa/metabolismo , Túbulos Renales Proximales , Sorbitol/análogos & derivados , Animales , Línea Celular , Túbulos Renales Proximales/efectos de los fármacos , Túbulos Renales Proximales/metabolismo , Ratones , Ratones Endogámicos C57BL , Sorbitol/farmacologíaRESUMEN
Pericytes play an important role in the recovery process after ischemic injury of many tissues. Brain pericytes in the peri-infarct area express macrophage markers in response to injury stimuli and are involved in neovascularization. In the kidney, nerve/glial antigen 2 (NG2)+ pericytes have been found to accumulate after renal injury. These accumulated NG2+ cells are not involved in scar formation. However, the role of accumulated NG2+ cells in injured kidneys remains unknown. Here, using a reversible ischemia-reperfusion (I/R) model, we found that renal NG2+ cells were increased in injured kidneys and expressed macrophage markers (CD11b or F4/80) on day 3 after reperfusion. Isolated NG2+ cells from I/R kidneys also had phagocytic activity and expressed anti-inflammatory cytokine genes, including mannose receptor and IL-10. These macrophage-like NG2+ cells did not likely differentiate into myofibroblasts because they did not increase α-smooth muscle actin expression. Intravenous transfusion of renal NG2+ cells isolated from donor mice on day 3 after reperfusion into recipient mice on day 1 after I/R surgery revealed that NG2+ cell-injected mice had lower plasma blood urea nitrogen, reduced kidney injury molecule-1 mRNA expression, ameliorated renal damage, and reduced cellular debris accumulation compared with PBS-injected mice on day 5 after reperfusion. In conclusion, these data suggest that renal NG2+ cells have an M2 macrophage-like ability and play a novel role in facilitating the recovery process after renal I/R injury.NEW & NOTEWORTHY Brain pericytes have macrophage-like activities after injury. However, such properties of pericytes in peripheral tissues have not been investigated. Here, we provide evidence that nerve/glial antigen 2-positive cells increase after renal injury. The population of nerve/glial antigen 2-positive cells, which does not increase expression of myofibroblast-associated gene, express macrophage markers and anti-inflammatory cytokine genes, have phagocytic activity, and play a role in renal recovery after kidney injury.
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Antígenos/metabolismo , Isquemia/metabolismo , Riñón/metabolismo , Macrófagos/metabolismo , Proteoglicanos/metabolismo , Daño por Reperfusión/metabolismo , Animales , Células Epiteliales/metabolismo , Células Epiteliales/patología , Isquemia/patología , Riñón/patología , Macrófagos/patología , Masculino , Ratones , Miofibroblastos/metabolismo , Miofibroblastos/patología , Fagocitosis/fisiología , Fenotipo , Daño por Reperfusión/patologíaRESUMEN
Hypoxia-inducible factor prolyl hydroxylase (HIF-PHD) inhibitors were developed for treatment of renal anemia. Patients applicable for HIF-PHD inhibitor treatment experience complications such as chronic kidney disease, whereby water and electrolyte homeostasis is disrupted. The effects of hypoxia-inducible factor stabilization on salt accumulation in the setting of reduced renal function remain unclear. In the present study, we investigated the effect of a HIF-PHD inhibitor, molidustat, on salt distribution and excretion in rats with subtotal nephrectomy-induced chronic kidney disease. Male Wistar rats were subjected to 5/6 nephrectomy. After confirming blood pressure elevation (>150 mmHg, at 4 weeks after surgery), rats were treated with molidustat. After 1 week of treatment, molidustat did not significantly improve blood cell volume or blood pressure. Distribution of sodium, potassium, and water in skin, carcass, and bone samples was not affected by molidustat. Furthermore, molidustat had no significant effect on urinary sodium excretion or concentration in response to acute oral salt loading (1 g/kg). In conclusion, molidustat did not affect distribution or excretion of salt in rats subjected to a model of nephron loss.
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Hipertensión/metabolismo , Nefrectomía/efectos adversos , Inhibidores de Prolil-Hidroxilasa/farmacología , Pirazoles/farmacología , Insuficiencia Renal Crónica/metabolismo , Sodio/metabolismo , Triazoles/farmacología , Animales , Presión Sanguínea , Modelos Animales de Enfermedad , Masculino , Ratas Wistar , Insuficiencia Renal Crónica/etiología , Insuficiencia Renal Crónica/fisiopatología , Sodio/orinaRESUMEN
The kidneys are the major organs for erythropoietin (EPO) production in adults, and thus, kidney damage results in reduced EPO levels and anemia. Inhibitors of Hypoxia-inducible factor-prolyl hydroxylase domain-containing protein (HIF-PHD) are awaited as new therapeutic options for renal anemia. It can be predicted that most patients who receive HIF-PHD inhibitors have renal dysfunction as a cause of anemia. Therefore, in the present study, we investigated the effects of the HIF-PHD inhibitor molidustat on anemia and renal dysfunction when initiated after the onset of renal anemia. Male C57BL/6J mice received adenine orally to induce nephropathy. After the onset of nephropathy, the mice were treated with either vehicle or molidustat. After 4 weeks of administration, vehicle-treated mice displayed significant anemia, and molidustat ameliorated this anemia. Vehicle-treated mice exhibited reduced creatinine clearance and body weight, increased blood urea nitrogen levels, histopathological changes, immune cell infiltration, and dehydration. Molidustat reversed immune cell infiltration, dehydration, and renal fibrosis without improving renal functional parameters. In conclusion, molidustat treatment initiated after the onset of nephropathy and renal anemia reversed anemia in mice. Molidustat improved some parameters of renal abnormality, but it did not restore renal function.
Asunto(s)
Lesión Renal Aguda/inducido químicamente , Lesión Renal Aguda/complicaciones , Adenina/efectos adversos , Anemia/tratamiento farmacológico , Anemia/etiología , Factor 1 Inducible por Hipoxia/antagonistas & inhibidores , Inhibidores de Prolil-Hidroxilasa , Pirazoles/uso terapéutico , Triazoles/uso terapéutico , Lesión Renal Aguda/metabolismo , Animales , Modelos Animales de Enfermedad , Eritropoyetina/metabolismo , Masculino , Ratones Endogámicos C57BL , Pirazoles/administración & dosificación , Pirazoles/farmacología , Triazoles/administración & dosificación , Triazoles/farmacologíaRESUMEN
AIMS/INTRODUCTION: Clinical studies have shown that treatment with inhibitors of sodium-glucose cotransporter 2 (SGLT2) significantly increases the hematocrit in patients with type 2 diabetes. To investigate whether SGLT2 inhibitors directly promote erythropoietin production independently on blood glucose reduction, the hematopoietic effect of the specific SGLT2 inhibitor, luseogliflozin, was examined in non-diabetic rats with renal anemia. MATERIALS AND METHODS: Renal anemia was induced by treatment with adenine (200 or 600 mg/kg/day, orally for 10 days) in non-diabetic Wistar-Kyoto or Wistar rats, respectively. Luseogliflozin (10 mg/kg bodyweight) or vehicle (0.5% carboxymethyl cellulose) was then administered for 6 weeks. The hematocrit and the hemoglobin (Hb), blood urea nitrogen, plasma creatinine, and plasma erythropoietin levels were monitored. RESULTS: Treatment with adenine decreased the hematocrit and the Hb level, which were associated with increases in the blood urea nitrogen and plasma creatinine levels. In Wistar-Kyoto rats treated with 200 mg/kg/day adenine, administration of luseogliflozin induced glycosuria, but did not change the blood urea nitrogen, plasma creatinine levels, hematocrit, Hb or plasma erythropoietin levels. Similarly, luseogliflozin treatment failed to change the hematocrit or the Hb levels in Wistar rats with renal anemia induced by 600 mg/kg/day of adenine. Plasma erythropoietin concentrations were also not different between luseogliflozin- and vehicle-treated rats. Similarly, in human erythropoietin-producing cells derived from pluripotent stem cells, luseogliflozin treatment did not change the erythropoietin level in the medium. CONCLUSIONS: These data suggest that SGLT2 inhibitor fails to exert hematopoietic effects in non-diabetic conditions.
Asunto(s)
Anemia/tratamiento farmacológico , Células Madre Hematopoyéticas/efectos de los fármacos , Insuficiencia Renal/tratamiento farmacológico , Inhibidores del Cotransportador de Sodio-Glucosa 2/farmacología , Sorbitol/análogos & derivados , Adenina , Anemia/sangre , Anemia/inducido químicamente , Animales , Modelos Animales de Enfermedad , Eritropoyetina/sangre , Hematócrito , Hemoglobinas/efectos de los fármacos , Humanos , Riñón/efectos de los fármacos , Masculino , Ratas , Ratas Endogámicas WKY , Ratas Wistar , Insuficiencia Renal/sangre , Insuficiencia Renal/inducido químicamente , Sorbitol/farmacologíaRESUMEN
BACKGROUND: The distal convoluted tubule (DCT) is an important nephron site for parathyroid hormone (PTH) and calcitonin regulation of urinary divalent cation excretion. These hormones exert their effects on the DCT in substantial part through activation of adenylyl cyclase (AC); however, it is unknown which AC isoforms are involved. METHODS: To examine this, two mouse DCT cell lines were studied: 209 and D1 cells. AC isoform mRNA expression was analyzed by real-time PCR. Cyclic AMP was measured using enzyme immunoassay. RESULTS: Calcitonin, but not PTH, stimulated cAMP accumulation in 209 cells, while PTH, but not calcitonin, increased cAMP content in D1 cells. Both cell types expressed AC3, AC4, AC6, AC7, and AC9 mRNA; in both cell types, AC6 mRNA was most abundant, followed by AC9, then AC3 and AC7, with relatively very small amounts of AC4 mRNA. Microdissected mouse DCT had a similar pattern of AC isoform mRNA expression although AC5 mRNA was detected. Individual siRNA knockdown of AC6 and AC9 reduced calcitonin-stimulated cAMP accumulation in 209 cells and PTH-induced cAMP levels in D1 cells. Knockdown of AC3 had no effect on hormonal augmentation of cAMP in either cell line. Surprisingly, knockdown of AC7 increased calcitonin-induced cAMP accumulation in 209 cells as well as PTH-stimulated cAMP content in D1 cells. CONCLUSIONS: Taken together, these findings indicate that AC6 and AC9 mediate calcitonin- and PTH-stimulated cAMP accumulation in DCT cells, while activation of AC7 may paradoxically reduce the stimulatory effects of PTH and calcitonin on cultured DCT cAMP levels.
Asunto(s)
Adenilil Ciclasas/fisiología , Calcitonina/farmacología , AMP Cíclico/metabolismo , Túbulos Renales Distales/metabolismo , Hormona Paratiroidea/farmacología , Animales , Línea Celular , Isoenzimas/fisiología , Túbulos Renales Distales/efectos de los fármacos , RatonesRESUMEN
Hypoxia predisposes renal fibrosis. This study was conducted to identify novel approaches to ameliorate the pathogenic effect of hypoxia. Using human proximal tubular epithelial cells we showed that a pan-phosphodiesterase (PDE) inhibitor, 3-isobutyl-1-methylxanthine (IBMX) dose and time dependently downregulated hypoxia-inducible factor 1α (HIF-1α) mRNA expression, which was further augmented by addition of a transcriptional inhibitor, actinomycin D. IBMX also increased the cellular cyclic adenosine monophosphate (cAMP) level. Luciferase assay showed that blocking of protein kinase A (PKA) using H89 reduced, while 8-Br-cAMP agonized the repression of HIF-1α promoter activity in hypoxic condition. Deletion of cAMP response element binding sites from the HIF-1α promoter abrogated the effect of IBMX. Western blot and immunofluorescent study confirmed that the CoCl2 induced increased HIF-1α protein in whole cell lysate and in nucleus was reduced by the IBMX. Through this process, IBMX attenuated both CoCl2 and hypoxia induced mRNA expressions of two pro-fibrogenic factors, platelet-derived growth factor B and lysyl oxidase. Moreover, IBMX reduced production of a mesenchymal transformation factor, ß-catenin; as well as protected against hypoxia induced cell-death. Taken together, our study showed novel evidence that the PDE inhibitor IBMX can downregulate the transcription of HIF-1α, and thus may attenuate hypoxia induced renal fibrosis.
Asunto(s)
1-Metil-3-Isobutilxantina/farmacología , Células Epiteliales/efectos de los fármacos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Hipoxia de la Célula/efectos de los fármacos , Células Cultivadas , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Células Epiteliales/metabolismo , Humanos , Proteínas Proto-Oncogénicas c-sis/metabolismo , Transducción de Señal/efectos de los fármacos , Xantinas/farmacología , beta Catenina/metabolismoRESUMEN
OBJECTIVE: Experiments were carried out to investigate whether diuretics (hydrochlorothiazideâ+âfurosemide) impact on the effects of a sodium-dependent glucose cotransporter 2 (SGLT2) inhibitor on glucose metabolism and blood pressure (BP) in metabolic syndrome SHR/NDmcr-cp(+/+) rats (SHRcp). METHODS: Male 13-week-old SHRcp were treated with: vehicle; the SGLT2-inhibitor luseogliflozin (10âmg/kg per day); diuretics (hydrochlorothiazide; 10âmg/kg/dayâ+âfurosemide; 5âmg/kg per day); or luseogliflozinâ+âdiuretics (n = 5-8 for each group) daily by oral gavage for 5 weeks. BP and glucose metabolism were evaluated by a telemetry system and oral glucose tolerance test, respectively. RESULTS: Vehicle-treated SHRcp developed nondipper type hypertension (dark vs. light-period mean arterial pressure: 148.6â±â0.7 and 148.0â±â0.7âmmHg, respectively, Pâ=â0.2) and insulin resistance. Compared with vehicle-treated animals, luseogliflozin-treated rats showed an approximately 4000-fold increase in urinary excretion of glucose and improved glucose metabolism. Luseogliflozin also significantly decreased BP and turned the circadian rhythm of BP from a nondipper to dipper pattern (dark vs. light-period mean arterial pressure: 138.0â±â1.6 and 132.0â±â1.3âmmHg, respectively, Pâ<â0.01), which were associated with a significant increase in urinary excretion of sodium. Addition of diuretics did not influence luseogliflozin-induced improvement of glucose metabolism and circadian rhythm of BP in SHRcp. CONCLUSION: These data suggest that a SGLT2 inhibitor elicits its beneficial effects on glucose metabolism and hypertension in study participants with metabolic syndrome undergoing treatment with diuretics.
Asunto(s)
Diuréticos/uso terapéutico , Hipertensión/tratamiento farmacológico , Síndrome Metabólico/complicaciones , Obesidad , Inhibidores del Cotransportador de Sodio-Glucosa 2 , Administración Oral , Animales , Presión Sanguínea , Diuréticos/administración & dosificación , Furosemida/administración & dosificación , Furosemida/uso terapéutico , Hidroclorotiazida/administración & dosificación , Hidroclorotiazida/uso terapéutico , Hipertensión/sangre , Hipertensión/complicaciones , Hipertensión/fisiopatología , Masculino , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Transportador 2 de Sodio-Glucosa , Sorbitol/administración & dosificación , Sorbitol/análogos & derivados , Sorbitol/uso terapéuticoRESUMEN
Studies were performed to examine the effects of the selective sodium-glucose co-transporter 2 (SGLT2) inhibitor empagliflozin on urinary sodium excretion and circadian blood pressure in salt-treated obese Otsuka Long Evans Tokushima Fatty (OLETF) rats. Fifteen-week-old obese OLETF rats were treated with 1% NaCl (in drinking water), and vehicle (0.5% carboxymethylcellulose, n=10) or empagliflozin (10 mg kg(-1)per day, p.o., n=11) for 5 weeks. Blood pressure was continuously measured by telemetry system. Glucose metabolism and urinary sodium excretion were evaluated by oral glucose tolerance test and high salt challenge test, respectively. Vehicle-treated OLETF rats developed non-dipper type blood pressure elevation with glucose intolerance and insulin resistance. Compared with vehicle-treated animals, empagliflozin-treated OLETF rats showed an approximately 1000-fold increase in urinary glucose excretion and improved glucose metabolism and insulin resistance. Furthermore, empagliflozin prevented the development of blood pressure elevation with normalization of its circadian rhythm to a dipper profile, which was associated with increased urinary sodium excretion. These data suggest that empagliflozin elicits beneficial effects on both glucose homeostasis and hypertension in salt-replete obese states.
Asunto(s)
Compuestos de Bencidrilo/farmacología , Presión Sanguínea/efectos de los fármacos , Ritmo Circadiano/efectos de los fármacos , Glucósidos/farmacología , Hipoglucemiantes/farmacología , Obesidad/fisiopatología , Inhibidores del Cotransportador de Sodio-Glucosa 2 , Animales , Compuestos de Bencidrilo/uso terapéutico , Glucemia , Prueba de Tolerancia a la Glucosa , Glucósidos/uso terapéutico , Homeostasis/efectos de los fármacos , Hipertensión/tratamiento farmacológico , Hipertensión/fisiopatología , Hipoglucemiantes/uso terapéutico , Resistencia a la Insulina , Ratas , Ratas Endogámicas OLETF , Cloruro de Sodio Dietético/farmacología , Transportador 2 de Sodio-GlucosaRESUMEN
The transient receptor potential melastatin type 6 (TRPM6) epithelial Mg(2+) channels participate in transcellular Mg(2+) transport in the kidney and intestine. Previous reports suggested a hormonal cAMP-dependent regulation of Mg(2+) reabsorption in the kidney. The molecular details of this process are, however, unknown. Adenylate cyclase 3 (Adcy3) has been shown to colocalize with the Na(+)/Cl(-) cotransporter, a marker of the distal convoluted segment of the kidney, the principal site of TRPM6 expression. Given the critical role of TRPM6 in Mg(2+) reabsorption, an inducible kidney-specific Adcy3 deletion mouse model was characterized for blood and urinary electrolyte disturbances under a normal--and low--Mg(2+) diet. Increased urinary Mg(2+) wasting and Trpm6 mRNA levels were observed in the urine and kidney of Adcy3-deleted animals compared with wild-type controls. Serum Mg(2+) concentration was significantly lower in Adcy3-deleted animals at day 7 on the low Mg(2+) diet. Using patch clamp electrophysiology, cell surface biotinylation, and total internal reflection fluorescence live cell imaging of transfected HEK293 cells, we demonstrated that cAMP signaling rapidly potentiates TRPM6 activity by promoting TRPM6 accumulation at the plasma membrane and increasing its single-channel conductance. Comparison of electrophysiological data from cells expressing the phosphorylation-deficient S1252A or phosphomimetic S1252D TRPM6 mutants suggests that phosphorylation at this intracellular residue participates in the observed stimulation of channel activity. Altogether, these data support a physiologically relevant magnesiotropic role of cAMP signaling in the kidney by a direct stimulatory action of protein kinase A on the plasma membrane trafficking and function of TRPM6 ion channels.
Asunto(s)
Adenilil Ciclasas/metabolismo , AMP Cíclico/metabolismo , Riñón/metabolismo , Magnesio/metabolismo , Reabsorción Renal , Canales Catiónicos TRPM/metabolismo , Adenilil Ciclasas/genética , Animales , Biotinilación , Membrana Celular/metabolismo , Colforsina/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Fenómenos Electrofisiológicos , Células HEK293 , Humanos , Magnesio/administración & dosificación , Masculino , Ratones , Ratones Noqueados , Microscopía Fluorescente , Fosforilación , ARN Mensajero/orina , Transducción de Señal , Canales Catiónicos TRPM/genética , Transfección , Vasodilatadores/farmacologíaRESUMEN
Adenylyl cyclase (AC)-stimulated cAMP plays a key role in modulating transport and channel activity along the nephron. However, the role of individual adenylyl cyclase isoforms in such regulation is largely unknown. Since adenylyl cyclase 3 (AC3) is expressed throughout nephron, we investigated its role in the physiologic regulation of renal Na(+) and water transport. Mice were generated with inducible nephron knockout of AC3 (AC3 KO) by breeding mice with loxP-flanked critical exons in the Adcy3 gene with mice expressing Pax8-rtTA and LC-1 transgenes. After doxycycline treatment at 1 month of age, nephron AC3 KO mice had 100% Adcy3 gene recombination in all renal tubule segments, but not in glomeruli. Sodium intake, urinary Na(+) excretion, glomerular filtration rate, and blood pressure were similar between nephron KO mice and the controls during normal, high, and low Na(+) diets. Plasma renin concentration was not different between the two groups during varied Na(+) intake. Moreover, there were no differences in urine volume and urine osmolality between the two genotypes during normal or restricted water intake. In conclusion, these data suggested that AC3 is not involved in the physiological regulation of nephron Na(+) and water handling.
RESUMEN
Abstract Adenylyl cyclase (AC)-stimulated cAMP is a key mediator of collecting duct (CD) Na and water transport. AC isoforms 3, 4, and 6 are expressed in the CD. Our group demonstrated that AC6, but not AC3, is involved in regulating CD Na and water transport. However, the role of AC4 in such regulation remains unknown. Therefore, we generated mice with loxP-flanked critical exons in the Adcy4 gene and bred with mice expressing the aquaporin-2/Cre recombinase transgene to yield CD principal cell-specific knockout of AC4 (CD AC4 KO). Isolated inner medullary CD showed 100% genomic target gene recombination in CD AC4 KO mice, while microdissected cortical CD and renal papillary AC4 mRNA was significantly reduced in CD AC4 KO mice. CD AC4 KO had no effect on vasopressin (AVP)-stimulated cAMP generation in the inner medulla. Water intake, urine volume, and urine osmolality were similar between CD AC4 KO and control mice during normal or restricted water intake. Sodium intake, urinary Na excretion, and blood pressure on a normal-, high-, or low-Na diet were not affected by CD AC4 KO. Moreover, there were no differences in plasma AVP or plasma renin concentration between CD AC4 KO and control mice. In summary, these data suggest that CD AC4 does not play a role in the physiologic regulation of CD Na and water handling.
RESUMEN
cAMP is a key mediator of connecting tubule and collecting duct (CD) Na(+) and water reabsorption. Studies performed in vitro have suggested that CD adenylyl cyclase (AC)3 partly mediates the actions of vasopressin; however, the physiological role of CD AC3 has not been determined. To assess this, mice were developed with CD-specific disruption of AC3 [CD AC3 knockout (KO)]. Inner medullary CDs from these mice exhibited 100% target gene recombination and had reduced ANG II- but not vasopressin-induced cAMP accumulation. However, there were no differences in urine volume, urinary urea excretion, or urine osmolality between KO and control mice during normal water intake or varying degrees of water restriction in the presence or absence of chronic vasopressin administration. There were no differences between CD AC3 KO and control mice in arterial pressure or urinary Na(+) or K(+) excretion during a normal or high-salt diet, whereas plasma renin and vasopressin concentrations were similar between the two genotypes. Patch-clamp analysis of split-open cortical CDs revealed no difference in epithelial Na(+) channel activity in the presence or absence of vasopressin. Compensatory changes in AC6 were not responsible for the lack of a renal phenotype in CD AC3 KO mice since combined CD AC3/AC6 KO mice had similar arterial pressure and renal Na(+) and water handling compared with CD AC6 KO mice. In summary, these data do not support a significant role for CD AC3 in the regulation of renal Na(+) and water excretion in general or vasopressin regulation of CD function in particular.
Asunto(s)
Adenilil Ciclasas/deficiencia , Túbulos Renales Colectores/fisiología , Sodio/orina , Adenilil Ciclasas/metabolismo , Animales , Presión Sanguínea/efectos de los fármacos , Diuresis , Femenino , Masculino , Ratones , Ratones Noqueados , Cloruro de Sodio Dietético/farmacologíaRESUMEN
cAMP is an important mediator of cystogenesis in polycystic kidney disease (PKD). Several adenylyl cyclase (AC) isoforms could mediate cAMP accumulation in PKD, and identification of a specific pathogenic AC isoform is of therapeutic interest. We investigated the role of AC6 in a mouse model of PKD that is homozygous for the loxP-flanked PKD1 gene and heterozygous for an aquaporin-2-Cre recombinase transgene to achieve collecting duct-specific gene targeting. Collecting duct-specific knockout of polycystin-1 caused massive renal cyst formation, kidney enlargement, and severe kidney failure, with a mean survival time of 2 months. In contrast, coincident collecting duct-specific knockout of polycystin-1 and AC6 (also homozygous for the floxed ADCY6 gene) markedly decreased kidney size and cystogenesis, improved renal function, reduced activation of the B-Raf/ERK/MEK pathway, and greatly increased survival. Absence of collecting duct AC6 did not alter urinary cAMP excretion or kidney cAMP concentration. In conclusion, AC6 is a key mediator of cyst formation and renal injury in a model of PKD.
Asunto(s)
Adenilil Ciclasas/deficiencia , Riñón Poliquístico Autosómico Dominante/enzimología , Adenilil Ciclasas/genética , Adenilil Ciclasas/fisiología , Animales , Acuaporina 2/genética , AMP Cíclico/fisiología , Modelos Animales de Enfermedad , Femenino , Genotipo , Integrasas/genética , Riñón/patología , Fallo Renal Crónico/etiología , Fallo Renal Crónico/prevención & control , Túbulos Renales Colectores/fisiopatología , Sistema de Señalización de MAP Quinasas , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Riñón Poliquístico Autosómico Dominante/genética , Riñón Poliquístico Autosómico Dominante/metabolismo , Riñón Poliquístico Autosómico Dominante/patología , Canales Catiónicos TRPP/deficiencia , Canales Catiónicos TRPP/genética , TransgenesRESUMEN
Female rats are more resistant to blood pressure increases induced by high salt (HS) intake or angiotensin (Ang) II infusion. Because endothelin ET(B) receptors on endothelial and epithelial cells mediate tonic vasodilation and sodium excretion, we hypothesized that ET(B) receptors limit the hypertensive response and renal injury induced by HS diet alone or with chronic AngII infusion (AngII/HS) in female compared with male rats. A 4 week HS diet (4% NaCl) did not significantly change blood pressure (measured by telemetry) in either male or female Sprague-Dawley rats. Administration of the ET(B) receptor antagonist A-192621 (1, 3 and 10 mg/kg per day in food) during HS feeding caused a dose-dependent increase in blood pressure in both sexes. In AngII/HS rats, males had a larger increase in blood pressure than females. The increase in blood pressure produced by ET(B) receptor blockade in male AngII/HS rats was not significant. However, A-192621 treatment resulted in a significant further increase in blood pressure in female AngII/HS rats. Male rats had consistently higher protein excretion rates before and during AngII/HS, but this was not significantly affected by ET(B) receptor blockade in either sex. In conclusion, ET(B) receptors play a significantly greater beneficial role in protecting female compared with male rats against AngII-induced hypertension and may contribute to the sex differences in AngII-induced hypertension.
Asunto(s)
Angiotensina II/farmacología , Hipertensión/fisiopatología , Receptor de Endotelina B/fisiología , Caracteres Sexuales , Cloruro de Sodio Dietético/farmacología , Animales , Presión Sanguínea/efectos de los fármacos , Presión Sanguínea/fisiología , Monitoreo Ambulatorio de la Presión Arterial , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Antagonistas de los Receptores de la Endotelina B , Femenino , Hipertensión/inducido químicamente , Masculino , Proteinuria/inducido químicamente , Proteinuria/fisiopatología , Pirrolidinas/farmacología , RatasRESUMEN
We have recently demonstrated that chronic infusion of exogenous ANG II, which induces blood pressure elevation, attenuates renal medullary endothelin B (ET(B)) receptor function in rats. Moreover, this was associated with a reduction of ET(B) receptor expression in the renal inner medulla. The aim of this present work was to investigate the effect of a physiological increase in endogenous ANG II (low-salt diet) on the renal ET system, including ET(B) receptor function. We hypothesized that endogenous ANG II reduces renal medullary ET(B) receptor function during low-salt intake. Rats were placed on a low-salt diet (0.01-0.02% NaCl) for 2 wk to allow an increase in endogenous ANG II. In rats on normal-salt chow, the stimulation of renal medullary ET(B) receptor by ET(B) receptor agonist sarafotoxin 6c (S6c) causes an increase in water (3.6 ± 0.4 from baseline vs. 10.5 ± 1.3 µl/min following S6c infusion; P < 0.05) and sodium excretion (0.38 ± 0.06 vs. 1.23 ± 0.17 µmol/min; P < 0.05). The low-salt diet reduced the ET(B)-dependent diuresis (4.5 ± 0.5 vs. 6.1 ± 0.9 µl/min) and natriuresis (0.40 ± 0.11 vs. 0.46 ± 0.12 µmol/min) in response to acute intramedullary infusion of S6c. Chronic treatment with candesartan restored renal medullary ET(B) receptor function; urine flow was 7.1 ± 0.9 vs. 15.9 ± 1.7 µl/min (P < 0.05), and sodium excretion was 0.4 ± 0.1 vs. 1.1 ± 0.1 µmol/min (P < 0.05) before and after intramedullary S6c infusion, respectively. Receptor binding assays determined that the sodium-depleted diet resulted in a similar level of ET(B) receptor binding in renal inner medulla compared with rats on a normal-salt diet. Candesartan reduced renal inner medullary ET(B) receptor binding (1,414 ± 95 vs. 862 ± 50 fmol/mg; P < 0.05). We conclude that endogenous ANG II attenuates renal medullary ET(B) receptor function to conserve sodium during salt deprivation independently of receptor expression.
Asunto(s)
Angiotensina II/fisiología , Dieta Hiposódica , Médula Renal/metabolismo , Receptor de Endotelina B/fisiología , Animales , Bencimidazoles/farmacología , Compuestos de Bifenilo , Diuresis/efectos de los fármacos , Endotelina-1/metabolismo , Endotelina-2/metabolismo , Masculino , Natriuresis/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Receptor de Endotelina B/agonistas , Cloruro de Sodio Dietético/metabolismo , Sodio en la Dieta/farmacología , Tetrazoles/farmacología , Venenos de Víboras/farmacologíaRESUMEN
We hypothesized that angiotensin (Ang) II hypertensive rats have impaired natriuresis after renal medullary endothelin (ET) B receptor stimulation that would be more evident in male versus female rats. Acute intramedullary infusion of the ET(B) agonist sarafotoxin 6c in normotensive male rats increased sodium excretion from 0.51±0.11 µmol/min during baseline to 1.64±0.19 µmol/min (P<0.05) after S6c. After 2 weeks of Ang II infusion (260 ng/kg per minute SC), male rats had an attenuated natriuretic response to S6c of 0.62±0.16 µmol/min during baseline versus 0.95±0.07 µmol/min after S6c. In contrast, ET(B)-dependent natriuresis was similar in female hypertensive rats (0.48±0.07 versus 1.5±0.18 µmol/min; P<0.05) compared with normotensive controls (1.05±0.07 versus 2.14±0.24 µmol/min; P<0.05). Because ET(A) receptors also mediate natriuresis in normotensive female rats, we examined ET(A) receptor function in female Ang II hypertensive rats. Intramedullary infusion of ET-1 increased sodium excretion in both hypertensive and normotensive female rats, which was partially blocked by the ET(A) antagonist BQ-123. Maximum ET(B) receptor binding in inner medullary membrane preparations was comparable between vehicle and Ang II hypertensive females; however, maximum ET(B) binding was significantly lower in male hypertensive rats (1952±251 versus 985±176 fmol/mg; P<0.05). These results indicate that renal ET(B) function is impaired in male Ang II hypertension attributed, at least in part, to a reduced number of ET(B) binding sites. Furthermore, renal ET receptor function is preserved in female rats during chronic Ang II infusion, suggesting that renal ET receptor function could serve to limit hypertension in females compared with males.