RESUMEN
Exchange proteins directly activated by cAMP (Epacs) are abundantly expressed in the renal tubules. We used genetic and pharmacological tools in combination with balance, electrophysiological, and biochemical approaches to examine the role of Epac1 and Epac2 in renal sodium handling. We demonstrate that Epac1-/- and Epac2-/- mice exhibit a delayed anti-natriuresis to dietary sodium restriction despite augmented aldosterone levels. This was associated with a significantly lower response to the epithelial Na+ channel (ENaC) blocker amiloride, reduced ENaC activity in split-opened collecting ducts, and defective posttranslational processing of α and γENaC subunits in the KO mice fed with a Na+-deficient diet. Concomitant deletion of both isoforms led to a marginally greater natriuresis but further increased aldosterone levels. Epac2 blocker ESI-05 and Epac1&2 blocker ESI-09 decreased ENaC activity in Epac WT mice kept on the Na+-deficient diet but not on the regular diet. ESI-09 injections led to natriuresis in Epac WT mice on the Na+-deficient diet, which was caused by ENaC inhibition. In summary, our results demonstrate similar but nonredundant actions of Epac1 and Epac2 in stimulation of ENaC activity during variations in dietary salt intake. We speculate that inhibition of Epac signaling could be instrumental in treatment of hypertensive states associated with ENaC overactivation.
Asunto(s)
Canales de Calcio/genética , Regulación de la Expresión Génica , Factores de Intercambio de Guanina Nucleótido/genética , Enfermedades Renales/genética , Natriuresis/genética , Sodio/orina , Canales Catiónicos TRPV/genética , Animales , Biomarcadores/orina , Canales de Calcio/biosíntesis , Células Cultivadas , Modelos Animales de Enfermedad , Factores de Intercambio de Guanina Nucleótido/biosíntesis , Enfermedades Renales/metabolismo , Enfermedades Renales/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , ARN/genética , Canales Catiónicos TRPV/biosíntesisRESUMEN
BACKGROUND: Over the last decade, advances in genetic techniques have resulted in the identification of rare hereditary disorders of renal magnesium and salt handling. Nevertheless, approximately 20% of all patients with tubulopathy lack a genetic diagnosis. METHODS: We performed whole-exome and -genome sequencing of a patient cohort with a novel, inherited, salt-losing tubulopathy; hypomagnesemia; and dilated cardiomyopathy. We also conducted subsequent in vitro functional analyses of identified variants of RRAGD, a gene that encodes a small Rag guanosine triphosphatase (GTPase). RESULTS: In eight children from unrelated families with a tubulopathy characterized by hypomagnesemia, hypokalemia, salt wasting, and nephrocalcinosis, we identified heterozygous missense variants in RRAGD that mostly occurred de novo. Six of these patients also had dilated cardiomyopathy and three underwent heart transplantation. We identified a heterozygous variant in RRAGD that segregated with the phenotype in eight members of a large family with similar kidney manifestations. The GTPase RagD, encoded by RRAGD, plays a role in mediating amino acid signaling to the mechanistic target of rapamycin complex 1 (mTORC1). RagD expression along the mammalian nephron included the thick ascending limb and the distal convoluted tubule. The identified RRAGD variants were shown to induce a constitutive activation of mTOR signaling in vitro. CONCLUSIONS: Our findings establish a novel disease, which we call autosomal dominant kidney hypomagnesemia (ADKH-RRAGD), that combines an electrolyte-losing tubulopathy and dilated cardiomyopathy. The condition is caused by variants in the RRAGD gene, which encodes Rag GTPase D; these variants lead to an activation of mTOR signaling, suggesting a critical role of Rag GTPase D for renal electrolyte handling and cardiac function.
Asunto(s)
Cardiomiopatía Dilatada/genética , Hipercalciuria/genética , Enfermedades Renales/genética , Proteínas de Unión al GTP Monoméricas/genética , Mutación Missense , Nefrocalcinosis/genética , Defectos Congénitos del Transporte Tubular Renal/genética , Serina-Treonina Quinasas TOR/metabolismo , Cardiomiopatía Dilatada/metabolismo , Femenino , Células HEK293 , Humanos , Hipercalciuria/metabolismo , Enfermedades Renales/metabolismo , Túbulos Renales Distales/metabolismo , Masculino , Modelos Moleculares , Natriuresis/genética , Nefrocalcinosis/metabolismo , Linaje , Conformación Proteica , Defectos Congénitos del Transporte Tubular Renal/metabolismo , Convulsiones/genética , Convulsiones/metabolismo , Transducción de Señal , Secuenciación del Exoma , Secuenciación Completa del GenomaRESUMEN
Background Dopamine D5 receptor (D5R) plays an important role in the maintenance of blood pressure by regulating renal sodium transport. Our previous study found that human D5R mutant F173L transgenic ( hD 5 R F173L-TG) mice are hypertensive. In the present study, we aimed to investigate the mechanisms causing this renal D5R dysfunction in hD 5 R F173L-TG mice. Methods and Results Compared with wild-type D5R-TG ( hD 5 R WT-TG) mice, hD 5 R F173L-TG mice have higher blood pressure, lower basal urine flow and sodium excretion, and impaired agonist-mediated natriuresis and diuresis. Enhanced reactive oxygen species production in hD 5 R F173L-TG mice is caused, in part, by decreased expression of antioxidant enzymes, including thioredoxin 1 (Trx1). Na+-K+-ATPase activity is increased in mouse renal proximal tubule cells transfected with hD 5 R F173L, but is normalized by treatment with exogenous recombinant human Trx1 protein. Regulation of Trx1 by D5R occurs by the phospholipase C/ protein kinase C (PKC) pathway because upregulation of Trx1 expression by D5R does not occur in renal proximal tubule cells from D1R knockout mice in the presence of a phospholipase C or PKC inhibitor. Fenoldopam, a D1R and D5R agonist, stimulates PKC activity in primary renal proximal tubule cells of hD5R WT -TG mice, but not in those of hD 5 R F173L-TG mice. Hyperphosphorylation of hD5RF173L and its dissociation from Gαs and Gαq are associated with impairment of D5R-mediated inhibition of Na+-K+-ATPase activity in hD 5 R F173L-TG mice. Conclusions These suggest that hD 5 R F173L increases blood pressure, in part, by decreasing renal Trx1 expression and increasing reactive oxygen species production. Hyperphosphorylation of hD5RF173L, with its dissociation from Gαs and Gαq, is the key factor in impaired D5R function of hD 5 R F173L-TG mice.
Asunto(s)
Presión Sanguínea/genética , Hipertensión/genética , Natriuresis/genética , Especies Reactivas de Oxígeno/metabolismo , Receptores de Dopamina D5/genética , Tiorredoxinas/genética , Animales , Presión Sanguínea/efectos de los fármacos , Cromograninas/metabolismo , Diuresis/efectos de los fármacos , Diuresis/genética , Agonistas de Dopamina/farmacología , Fenoldopam/farmacología , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Regulación de la Expresión Génica , Humanos , Hipertensión/metabolismo , Túbulos Renales Proximales/efectos de los fármacos , Túbulos Renales Proximales/metabolismo , Ratones , Ratones Noqueados , Ratones Transgénicos , Natriuresis/efectos de los fármacos , Proteína Quinasa C/metabolismo , Receptores de Dopamina D1/genética , Receptores de Dopamina D5/metabolismo , Transducción de Señal , Tiorredoxinas/metabolismo , Tiorredoxinas/farmacología , Fosfolipasas de Tipo C/metabolismoRESUMEN
BACKGROUND: Recent evidence emphasizes the critical role of inflammation in the development of diabetic nephropathy. Angiotensin-converting enzyme (ACE) plays an active role in regulating the renal inflammatory response associated with diabetes. Studies have also shown that ACE has roles in inflammation and the immune response that are independent of angiotensin II. ACE's two catalytically independent domains, the N- and C-domains, can process a variety of substrates other than angiotensin I. METHODS: To examine the relative contributions of each ACE domain to the sodium retentive state, renal inflammation, and renal injury associated with diabetic kidney disease, we used streptozotocin to induce diabetes in wild-type mice and in genetic mouse models lacking either a functional ACE N-domain (NKO mice) or C-domain (CKO mice). RESULTS: In response to a saline challenge, diabetic NKO mice excreted 32% more urinary sodium compared with diabetic wild-type or CKO mice. Diabetic NKO mice also exhibited 55% less renal epithelial sodium channel cleavage (a marker of channel activity), 55% less renal IL-1ß, 53% less renal TNF-α, and 53% less albuminuria than diabetic wild-type mice. This protective phenotype was not associated with changes in renal angiotensin II levels. Further, we present evidence that the anti-inflammatory tetrapeptide N-acetyl-seryl-asparyl-lysyl-proline (AcSDKP), an ACE N-domain-specific substrate that accumulates in the urine of NKO mice, mediates the beneficial effects observed in the NKO. CONCLUSIONS: These data indicate that increasing AcSDKP by blocking the ACE N-domain facilitates sodium excretion and ameliorates diabetic kidney disease independent of intrarenal angiotensin II regulation.
Asunto(s)
Nefropatías Diabéticas/metabolismo , Nefropatías Diabéticas/patología , Peptidil-Dipeptidasa A/química , Peptidil-Dipeptidasa A/deficiencia , Sustitución de Aminoácidos , Angiotensina II/metabolismo , Animales , Dominio Catalítico/genética , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patología , Nefropatías Diabéticas/genética , Canales Epiteliales de Sodio/metabolismo , Inflamación/genética , Inflamación/metabolismo , Inflamación/patología , Interleucina-1beta/metabolismo , Riñón/metabolismo , Riñón/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutagénesis Sitio-Dirigida , Natriuresis/genética , Natriuresis/fisiología , Oligopéptidos/antagonistas & inhibidores , Oligopéptidos/metabolismo , Peptidil-Dipeptidasa A/genética , Dominios Proteicos , Sistema Renina-Angiotensina/fisiologíaRESUMEN
BACKGROUND: Pathogenic variations in HSD11B2 gene triggers the apparent mineralocorticoid excess syndrome (AME). There is scarce information regarding the phenotypes of subjects carrying heterozygous pathogenic variants in HSD11B2 gene. We investigated if serum cortisol/cortisone (F/E) ratio and cortisone are useful for identifying partial 11ßHSD2 deficiency in those heterozygous subjects. METHODS: We studied two patients diagnosed with AME and their families carrying either D223N or R213C mutation. We also evaluated 32 healthy control subjects (13 children and 19 adults) to obtain normal references ranges for all measured variables. Case 1: A boy carrying D223N mutation in HSD11B2 gene and Case 2: A girl carrying R213C mutation. We assessed serum F/E ratio and cortisone by HPLC-MS/MS, aldosterone, plasma-renin-activity(PRA), electrolytes, and HSD11B2 genetic analyses. RESULTS: The normal values (median [interquartile range]) in children for serum F/E and cortisone (µg/dl) were 2.56 [2.21-3.69] and 2.54 [2.35-2.88], and in adults were 4.42 [3.70-4.90] and 2.23 [1.92-2.57], respectively. Case 1 showed a very high serum F/E 28.8 and low cortisone 0.46 µg/dl. His mother and sister were normotensives and heterozygous for D223N mutation with high F/E (13.2 and 6.0, respectively) and low cortisone (2.0 and 2.2, respectively). Case 2 showed a very high serum F/E 175 and suppressed cortisone 0.11 µg/dl. Her parents and sister were heterozygous for the R213C mutation with normal phenotype, but high F/E and low cortisone. Heterozygous subjects showed normal aldosterone, PRA, but lower fractional excretion of sodium and urinary Na/K ratio than controls. CONCLUSION: Serum F/E ratio and cortisone allow to identify partial 11ßHSD2 deficiencies, as occurs in heterozygous subjects, who would be susceptible to develop arterial hypertension.
Asunto(s)
11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 2/genética , Cortisona/sangre , Hidrocortisona/sangre , Síndrome de Exceso Aparente de Mineralocorticoides/sangre , Adolescente , Adulto , Biomarcadores/sangre , Estudios de Casos y Controles , Niño , Preescolar , Estudios Transversales , Femenino , Predisposición Genética a la Enfermedad , Herencia , Heterocigoto , Humanos , Masculino , Persona de Mediana Edad , Síndrome de Exceso Aparente de Mineralocorticoides/diagnóstico , Síndrome de Exceso Aparente de Mineralocorticoides/enzimología , Síndrome de Exceso Aparente de Mineralocorticoides/genética , Mutación , Natriuresis/genética , Linaje , Fenotipo , Valor Predictivo de las PruebasRESUMEN
Many physiological functions have a circadian rhythm, including blood pressure (BP). BP is highest during the active phase, whereas during the rest period, BP dips 10-20%. Patients that do not experience this dip at night are termed "nondippers." Nondipping hypertension is associated with increased risk of cardiovascular disease. The mechanisms underlying nondipping hypertension are not understood. Without the circadian clock gene Per1, C57BL/6J mice develop nondipping hypertension on a high-salt diet plus mineralocorticoid treatment (HS/DOCP). Our laboratory has shown that PER1 regulates expression of several genes related to sodium (Na) transport in the kidney, including epithelial Na channel (ENaC) and Na chloride cotransporter (NCC). Urinary Na excretion also demonstrates a circadian pattern with a peak during active periods. We hypothesized that PER1 contributes to circadian regulation of BP via a renal Na-handling-dependent mechanism. Na-handling genes from the distal nephron were inappropriately regulated in KO mice on HS/DOCP. Additionally, the night/day ratio of Na urinary excretion by Per1 KO mice is decreased compared with WT (4 × vs. 7×, P < 0.001, n = 6 per group). Distal nephron-specific Per1 KO mice also show an inappropriate increase in expression of Na transporter genes αENaC and NCC. These results support the hypothesis that PER1 mediates control of circadian BP rhythms via the regulation of distal nephron Na transport genes. These findings have implications for the understanding of the etiology of nondipping hypertension and the subsequent development of novel therapies for this dangerous pathophysiological condition.
Asunto(s)
Presión Sanguínea , Ritmo Circadiano , Hipertensión/metabolismo , Túbulos Renales Distales/metabolismo , Natriuresis , Proteínas Circadianas Period/metabolismo , Eliminación Renal , Animales , Presión Sanguínea/genética , Ritmo Circadiano/genética , Acetato de Desoxicorticosterona , Modelos Animales de Enfermedad , Canales Epiteliales de Sodio/genética , Canales Epiteliales de Sodio/metabolismo , Predisposición Genética a la Enfermedad , Hipertensión/genética , Hipertensión/fisiopatología , Túbulos Renales Distales/fisiopatología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Natriuresis/genética , Proteínas Circadianas Period/deficiencia , Proteínas Circadianas Period/genética , Fenotipo , Eliminación Renal/genética , Cloruro de Sodio Dietético , Miembro 3 de la Familia de Transportadores de Soluto 12/genética , Miembro 3 de la Familia de Transportadores de Soluto 12/metabolismo , Factores de Tiempo , Regulación hacia ArribaRESUMEN
BACKGROUND: Salt sensitivity of blood pressure (SSBP) increases the risk of cardiovascular complications, and the heritability of SSBP is about 50% in Chinese population. However, studies identifying genes involved in BP responses to acute sodium loading and diuresis shrinkage are still limited. METHOD: A total of 342 essential hypertensives from Beijing were recruited in our study. A modified Sullivan's acute oral saline load and diuresis shrinkage test was conducted to each individual. Medical history and lifestyle risk factors were obtained by questionnaire. Generalized linear model was used to examine the associations of 29 single-nucleotide polymorphisms (SNPs) with SSBP and false discovery rate (FDR) was used to correct P values for multiple testing. RESULTS: In the process of acute sodium loading, after adjusting for age and 24-hour urinary sodium concentration, SNPs in CYP11B2, PRKG1, SLC8A1 genes were significantly associated with systolic BP (SBP) rising in the additive and recessive model; SNPs in CYP4A11, PRKG1, SLC8A1, and ADRB2 genes were significantly associated with diastolic BP (DBP) rising. In the process of diuresis shrinkage, SNPs of CLCNKA, eNOS, PRKG1 gene were associated with SBP and DBP decreasing. After FDR correction, rs434082 in SLC8A1 gene was still significantly associated with blood pressure rising during salt load. In the additive model, A allele increased DBP of 2.8 mm Hg (FDR_q = 0.029) and MAP of 3.1 mm Hg (FDR_q = 0.029) after adjusting for age and 24-hour urinary sodium concentration. CONCLUSION: SLC8A1 gene may contribute to BP change in the process of acute sodium loading in a Han Chinese population.
Asunto(s)
Presión Sanguínea/genética , Hipertensión Esencial/genética , Polimorfismo de Nucleótido Simple , Cloruro de Sodio Dietético/efectos adversos , Intercambiador de Sodio-Calcio/genética , Anciano , Pueblo Asiatico/genética , Beijing/epidemiología , Hipertensión Esencial/diagnóstico , Hipertensión Esencial/etnología , Hipertensión Esencial/fisiopatología , Femenino , Frecuencia de los Genes , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Humanos , Masculino , Persona de Mediana Edad , Natriuresis/genética , Fenotipo , Factores de Riesgo , Cloruro de Sodio Dietético/administración & dosificaciónRESUMEN
Recent studies suggested a direct link between circadian rhythms and regulation of sodium excretion. Endothelin-1 (ET-1) regulates sodium balance by promoting natriuresis through the endothelin B receptor (ETB) in response to increased salt in the diet, but the effect that the time of day has on this natriuretic response is not known. Therefore, this study was designed to test the hypothesis that ETB receptor activation contributes to the diurnal control of sodium excretion and that sex differences contribute to this control as well. Twelve-hour urine collections were used to measure sodium excretion. On day 3 of the experiment, a NaCl load (900 µeq) was given by oral gavage either at Zeitgeber time [ZT] 0 (inactive period) or ZT12 (active period) to examine the natriuretic response to the acute salt load. Male and female ETB-deficient (ETB def) rats showed an impaired natriuretic response to a salt load at ZT0 compared with their respective transgenic controls (Tg cont). Male ETB def rats showed a delayed natriuretic response to a salt load given at ZT12 compared with male Tg cont, a contrast to the prompt response shown by female ETB def rats. Treatment with ABT-627, an ETA receptor antagonist, improved the natriuretic response seen within the first 12 h of a ZT0 salt load in both sexes. These findings demonstrate that diurnal excretion of an acute salt load 1) requires ET-1 and the ETB receptor, 2) is more evident in male vs. female rats, and 3) is opposed by the ETA receptor.
Asunto(s)
Natriuresis/genética , Receptor de Endotelina B/metabolismo , Sodio/metabolismo , Animales , Atrasentán , Antagonistas de los Receptores de Endotelina/farmacología , Endotelina-1/metabolismo , Femenino , Masculino , Natriuresis/efectos de los fármacos , Pirrolidinas/farmacología , Ratas , Ratas Endogámicas WKY , Ratas Transgénicas , Receptor de Endotelina B/genética , Factores Sexuales , Sodio/farmacología , Sodio/orina , Factores de TiempoRESUMEN
The gene SLC4A5 encodes the Na(+)-HCO3 (-) cotransporter electrogenic 2, which is located in the distal nephron. Genetically deleting Na(+)-HCO3 (-) cotransporter electrogenic 2 (knockout) causes Na(+)-retention and hypertension, a phenotype that is diminished with alkali loading. We performed experiments with acid-loaded mice and determined whether overactive epithelial Na(+) channels (ENaC) or the Na(+)-Cl(-) cotransporter causes the Na(+) retention and hypertension in knockout. In untreated mice, the mean arterial pressure was higher in knockout, compared with wild-type (WT); however, treatment with amiloride, a blocker of ENaC, abolished this difference. In contrast, hydrochlorothiazide, an inhibitor of Na(+)-Cl(-) cotransporter, decreased mean arterial pressure in WT, but not knockout. Western blots showed that quantity of plasmalemmal full-length ENaC-α was significantly higher in knockout than in WT. Amiloride treatment caused a 2-fold greater increase in Na(+) excretion in knockout, compared with WT. In knockout, but not WT, amiloride treatment decreased plasma [Na(+)] and urinary K(+) excretion, but increased hematocrit and plasma [K(+)] significantly. Micropuncture with microelectrodes showed that the [K(+)] was significantly higher and the transepithelial potential (Vte) was significantly lower in the late distal tubule of the knockout compared with WT. The reduced Vte in knockout was amiloride sensitive and therefore revealed an upregulation of electrogenic ENaC-mediated Na(+) reabsorption in this segment. These results show that, in the absence of Na(+)-HCO3 (-) cotransporter electrogenic 2 in the late distal tubule, acid-loaded mice exhibit disinhibition of ENaC-mediated Na(+) reabsorption, which results in Na(+) retention, K(+) wasting, and hypertension.
Asunto(s)
Canales Epiteliales de Sodio/fisiología , Hipertensión Renal/metabolismo , Simportadores de Sodio-Bicarbonato/deficiencia , Amilorida/farmacología , Amilorida/uso terapéutico , Animales , Antihipertensivos/uso terapéutico , Modelos Animales de Enfermedad , Diuréticos/uso terapéutico , Canales Epiteliales de Sodio/efectos de los fármacos , Hematócrito , Hidroclorotiazida/uso terapéutico , Concentración de Iones de Hidrógeno , Hipertensión Renal/tratamiento farmacológico , Hipertensión Renal/genética , Hipopotasemia/etiología , Túbulos Renales Distales/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Ratones , Ratones Congénicos , Ratones Endogámicos C57BL , Ratones Noqueados , Natriuresis/efectos de los fármacos , Natriuresis/genética , Polimorfismo de Nucleótido Simple , Potasio/metabolismo , Sodio/metabolismo , Bloqueadores de los Canales de Sodio/farmacología , Bloqueadores de los Canales de Sodio/uso terapéutico , Simportadores de Sodio-Bicarbonato/genética , Simportadores de Sodio-Bicarbonato/fisiologíaRESUMEN
Caffeine is one of the most widely consumed behavioral substances. We have previously shown that caffeine- and theophylline-induced inhibition of renal reabsorption causes diuresis and natriuresis, an effect that requires functional adenosine A1 receptors. In this study, we tested the hypothesis that blocking the Gi protein-coupled adenosine A1 receptor via the nonselective adenosine receptor antagonist caffeine changes Na(+)/H(+) exchanger isoform 3 (NHE3) localization and phosphorylation, resulting in diuresis and natriuresis. We generated tubulus-specific NHE3 knockout mice (Pax8-Cre), where NHE3 abundance in the S1, S2, and S3 segments of the proximal tubule was completely absent or severely reduced (>85%) in the thick ascending limb. Consumption of fluid and food, as well as glomerular filtration rate, were comparable in control or tubulus-specific NHE3 knockout mice under basal conditions, while urinary pH was significantly more alkaline without evidence for metabolic acidosis. Caffeine self-administration increased total fluid and food intake comparably between genotypes, without significant differences in consumption of caffeinated solution. Acute caffeine application via oral gavage elicited a diuresis and natriuresis that was comparable between control and tubulus-specific NHE3 knockout mice. The diuretic and natriuretic response was independent of changes in total NHE3 expression, phosphorylation of serine-552 and serine-605, or apical plasma membrane NHE3 localization. Although caffeine had no clear effect on localization of the basolateral Na(+)/bicarbonate cotransporter NBCe1, pretreatment with DIDS inhibited caffeine-induced diuresis and natriuresis. In summary, NHE3 is not required for caffeine-induced diuresis and natriuresis.
Asunto(s)
Cafeína/farmacología , Diuresis/efectos de los fármacos , Diuréticos/farmacología , Túbulos Renales/efectos de los fármacos , Natriuresis/efectos de los fármacos , Intercambiadores de Sodio-Hidrógeno/efectos de los fármacos , Animales , Diuresis/genética , Femenino , Tasa de Filtración Glomerular/efectos de los fármacos , Túbulos Renales/metabolismo , Masculino , Ratones , Natriuresis/genética , Sodio/metabolismo , Intercambiador 3 de Sodio-Hidrógeno , Intercambiadores de Sodio-Hidrógeno/genética , Intercambiadores de Sodio-Hidrógeno/metabolismoRESUMEN
Thiazide diuretics are used to treat hypertension; however, compensatory processes in the kidney can limit antihypertensive responses to this class of drugs. Here, we evaluated compensatory pathways in SPAK kinase-deficient mice, which are unable to activate the thiazide-sensitive sodium chloride cotransporter NCC (encoded by Slc12a3). Global transcriptional profiling, combined with biochemical, cell biological, and physiological phenotyping, identified the gene expression signature of the response and revealed how it establishes an adaptive physiology. Salt reabsorption pathways were created by the coordinate induction of a multigene transport system, involving solute carriers (encoded by Slc26a4, Slc4a8, and Slc4a9), carbonic anhydrase isoforms, and V-type Hâº-ATPase subunits in pendrin-positive intercalated cells (PP-ICs) and ENaC subunits in principal cells (PCs). A distal nephron remodeling process and induction of jagged 1/NOTCH signaling, which expands the cortical connecting tubule with PCs and replaces acid-secreting α-ICs with PP-ICs, were partly responsible for the compensation. Salt reabsorption was also activated by induction of an α-ketoglutarate (α-KG) paracrine signaling system. Coordinate regulation of a multigene α-KG synthesis and transport pathway resulted in α-KG secretion into pro-urine, as the α-KG-activated GPCR (Oxgr1) increased on the PP-IC apical surface, allowing paracrine delivery of α-KG to stimulate salt transport. Identification of the integrated compensatory NaCl reabsorption mechanisms provides insight into thiazide diuretic efficacy.
Asunto(s)
Presión Sanguínea/fisiología , Cloruros/orina , Síndrome de Gitelman/fisiopatología , Natriuresis/fisiología , Nefronas/metabolismo , Reabsorción Renal/fisiología , Amilorida/análogos & derivados , Amilorida/farmacología , Amoníaco/metabolismo , Animales , Transporte Biológico , Anhidrasas Carbónicas/genética , Anhidrasas Carbónicas/fisiología , Modelos Animales de Enfermedad , Activación Enzimática , Canales Epiteliales de Sodio/fisiología , Perfilación de la Expresión Génica , Redes Reguladoras de Genes , Síndrome de Gitelman/genética , Ácidos Cetoglutáricos/metabolismo , Glomérulos Renales/metabolismo , Masculino , Ratones , Ratones Noqueados , Natriuresis/genética , Comunicación Paracrina , Fosforilación , Procesamiento Proteico-Postraduccional , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores Notch/fisiología , Receptores Purinérgicos P2/fisiología , Transducción de Señal , Cloruro de Sodio/farmacocinética , Simportadores de Cloruro de Sodio-Potasio/genética , Simportadores de Cloruro de Sodio-Potasio/fisiología , Miembro 3 de la Familia de Transportadores de Soluto 12/metabolismoRESUMEN
The Na(+)/Ca(2+) exchanger (NCX) is a bidirectional transporter that is controlled by membrane potential and transmembrane gradients of Na(+) and Ca(2+). Although two isoforms of NCX1 and NCX2 are coexpressed on the basolateral membrane of the distal nephron, the functional significance of these isoforms is not entirely clear. Therefore, we used NCX1- and NCX2-heterozygote knockout mice (KO) and their double KO, as well as isoform-selective NCX inhibitors, to determine the roles of NCX isoforms in urine formation and electrolyte excretion in mice. NCX inhibitors, particularly NCX2-sensitive inhibitors, caused a dose-dependent natriuresis and in a higher dose, moreover, hypercalciuria. Consistently, NCX1-KO possessed normal renal function similar to wild-type mice (WT), whereas NCX2-KO and double KO exhibited moderate natriuresis and hypercalciuria. Notably, renal responses to YM-244769 were equivalently observed in NCX1-KO and WT, but disappeared in NCX2-KO and double KO. Thus, functional inhibition of NCX2 initially causes natriuresis, and further inhibition of NCX2 produces hypercalciuria, suggesting that the functional significance of NCX2 lies in Na(+) and Ca(2+) reabsorption of the kidney.
Asunto(s)
Hipercalciuria/fisiopatología , Natriuresis/fisiología , Intercambiador de Sodio-Calcio/metabolismo , Compuestos de Anilina/farmacología , Animales , Técnicas de Inactivación de Genes , Hipercalciuria/genética , Riñón/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Natriuresis/efectos de los fármacos , Natriuresis/genética , Niacinamida/análogos & derivados , Niacinamida/farmacología , Éteres Fenílicos/farmacología , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Intercambiador de Sodio-Calcio/antagonistas & inhibidores , Intercambiador de Sodio-Calcio/genética , Tiourea/análogos & derivados , Tiourea/farmacologíaRESUMEN
Defective pressure-natriuresis related to abnormalities in the natriuretic response has been associated with hypertension development. A major signaling pathway mediating pressure natriuresis involves the cGMP-dependent protein kinase 1 (PRKG1) that, once activated by Src kinase, inhibits renal Na(+) reabsorption via a direct action on basolateral Na-K ATPase and luminal Na-H exchanger type 3, as shown in renal tubuli of animals. Because a clear implication of PRKG1 in humans is still lacking, here we addressed whether PRKG1 polymorphisms affect pressure-natriuresis in patients. Naive hypertensive patients (n = 574), genotyped for PRKG1 rs1904694, rs7897633, and rs7905063 single nucleotide polymorphisms (SNPs), underwent an acute Na(+) loading, and the slope of the pressure-natriuresis relationship between blood pressure and Na(+) excretion was calculated. The underlying molecular mechanism was investigated by immunoblotting protein quantifications in human kidneys. The results demonstrate that the PRKG1 risk haplotype GAT (rs1904694, rs7897633, rs7905063, respectively) associates with a rightward shift of the pressure-natriuresis curve (0.017 ± 0.004 µEq/mm Hg per minute) compared with the ACC (0.0013 ± 0.003 µEq/mm Hg per minute; P = 0.001). In human kidneys, a positive correlation of protein expression levels between PRKG1 and Src (r = 0.83; P<0.001) or α1 Na-K ATPase (r = 0.557; P<0.01) and between α1 Na-K ATPase and Na-H exchanger type 3 (r = 0.584; P<0.01) or Src (r = 0.691; P<0.001) was observed in patients carrying PRKG1 risk GAT (n = 23) but not ACC (n = 14) variants. A functional signaling complex among PRKG1, α1 Na-K ATPase, and Src was shown by immunoprecipitation from human renal caveolae. These findings indicate that PRKG1 risk alleles associate with salt-sensitivity related to a loss of the inhibitory control of renal Na(+) reabsorption, suggestive of a blunt pressure-natriuresis response.
Asunto(s)
Proteína Quinasa Dependiente de GMP Cíclico Tipo I/genética , Hipertensión/genética , Riñón/metabolismo , Natriuresis/genética , Polimorfismo Genético , Sodio/metabolismo , Adulto , Anciano , Alelos , Presión Sanguínea/genética , Proteína Quinasa Dependiente de GMP Cíclico Tipo I/metabolismo , Femenino , Predisposición Genética a la Enfermedad , Genotipo , Humanos , Hipertensión/metabolismo , Hipertensión/fisiopatología , Riñón/fisiopatología , Masculino , Persona de Mediana Edad , Sodio en la Dieta/metabolismoRESUMEN
Gamma(2)-melanocyte-stimulating hormone (γ2MSH) is a peptide hormone released by the pituitary gland which is thought to act directly on the renal inner medulla to promote increased sodium excretion into urine (natriuresis). The aim of this study was to determine if a stable analog, [Nle(3), D-Phe(6)]-γ2MSH (NDP-γ2MSH), of the native peptide regulated the activity, expression and cellular localization of epithelial sodium channel (ENaC) in a murine inner medullary collecting duct (mIMCD-3) cell line. Our results indicate that expression of the γ2MSH receptor, melanocortin receptor 3 receptor (MC3R), is up-regulated by culturing the cells in media with an increased osmolality (â¼400mOsm/kg). Furthermore, stimulation of cAMP signaling and sodium transport by 1nM NDP-γ2MSH occurs only in cells cultured in the high osmolality media. Finally, treatment of mIMCD-3 cells cultured in high osmolality medium for 1h with 1nM NDP-γ2MSH causes a reduction in expression of serum- and glucocorticoid-induced kinase (sgk1) and a reduction in expression and cell surface abundance of the alpha subunit of ENaC. Collectively, this data suggest that γ2MSH directly regulates both ENaC expression and cellular localization in the inner medulla to exert its natriuretic effect.
Asunto(s)
Células Epiteliales/metabolismo , Canales Epiteliales de Sodio/genética , Túbulos Renales Colectores/metabolismo , gamma-MSH/genética , Animales , Línea Celular , Medios de Cultivo , AMP Cíclico/metabolismo , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Canales Epiteliales de Sodio/metabolismo , Regulación de la Expresión Génica , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/metabolismo , Transporte Iónico , Túbulos Renales Colectores/citología , Túbulos Renales Colectores/efectos de los fármacos , Ratones , Natriuresis/genética , Concentración Osmolar , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Receptor de Melanocortina Tipo 3/genética , Receptor de Melanocortina Tipo 3/metabolismo , Transducción de Señal , Sodio/metabolismo , alfa-MSH/análogos & derivados , alfa-MSH/farmacología , gamma-MSH/metabolismoRESUMEN
Renal medullary hypoxia-inducible factor (HIF)-1α and its target genes, such as haem oxygenase and nitric oxide synthase, have been indicated to play an important role in the regulation of sodium excretion and blood pressure. HIF prolyl hydroxylase domain-containing proteins (PHDs) are major enzymes to promote the degradation of HIF-1α. We recently reported that high salt intake suppressed the renal medullary PHD2 expression and thereby activated HIF-1α-mediated gene regulation in the renal medulla in response to high salt. To further define the functional role of renal medullary PHD2 in the regulation of renal adaptation to high salt intake and the longer term control of blood pressure, we transfected PHD2 expression plasmids into the renal medulla in uninephrectomized rats and determined its effects on pressure natriuresis, sodium excretion after salt overloading and the long-term control of arterial pressure after high salt challenge. It was shown that overexpression of PHD2 transgene increased PHD2 levels and decreased HIF-1α levels in the renal medulla, which blunted pressure natriuresis, attenuated sodium excretion, promoted sodium retention and produced salt sensitive hypertension after high salt challenge compared with rats treated with control plasmids. There was no blood pressure change in PHD2-treated rats that were maintained in low salt diet. These results suggested that renal medullary PHD2 is an important regulator in renal adaptation to high salt intake and a deficiency in PHD2-mediated molecular adaptation in response to high salt intake in the renal medulla may represent a pathogenic mechanism producing salt sensitive hypertension.
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Hipertensión/genética , Médula Renal/fisiopatología , Procolágeno-Prolina Dioxigenasa/genética , Cloruro de Sodio Dietético/farmacología , Adaptación Fisiológica/efectos de los fármacos , Adaptación Fisiológica/genética , Animales , Presión Sanguínea/efectos de los fármacos , Presión Sanguínea/genética , Regulación de la Expresión Génica , Hipertensión/etiología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Prolina Dioxigenasas del Factor Inducible por Hipoxia , Médula Renal/metabolismo , Masculino , Natriuresis/genética , Presión , Procolágeno-Prolina Dioxigenasa/metabolismo , Ratas , Ratas Sprague-Dawley , Ratas Transgénicas , Sodio/orina , TransgenesRESUMEN
Urotensin II (UII), a peptide hormone which influences glomerular filtration rate and urine concentration, and its receptor, UT, are expressed in the adult rat kidney. The ability of the kidney to reabsorb sodium and water starts to develop in utero and matures during early postnatal life in the rat, yet little is known about the ontogeny of the renal UII system. This study mapped renal expression of the urotensin system during the fetal and postnatal periods and determined renal activity of UII in the immature rat. Urotensin II peptide and mRNA were present in Sprague-Dawley (SD) rat metanephroi from the earliest stage examined, embyonic day 19 (E19; rat gestation 22 days); levels increased to peak at 4 weeks of age. In contrast, UT protein and mRNA expression declined rapidly between E19 and birth and remained at a similar level postnatally. Infusion of rat UII [6-60 pmol min(-1) (100 g body weight)(-1)] or rat urotensin-related peptide [6 pmol min(-1) (100 g body weight)(-1)] in anaesthetized 4-week-old SD rats had no influence on measured renal parameters; however, infusion of UT antagonist, SB-706375 (0.01 mg kg(-1) min(-1)), provoked a pronounced diuresis [vehicle 23.5 ± 1.9 versus antagonist 75.3 ± 12.5 µl min(-1) (100 g body weight)(-1); P < 0.001] and natriuresis, accompanied by modest increases in effective renal blood flow and glomerular filtration rate [vehicle 0.4 ± 0.1 versus antagonist 1.1 ± 0.2 ml min(-1) (100 g body weight)(-1); P < 0.0001] and a significant increase in fractional sodium excretion. These results indicate that the endogenous rat UII system may influence renal sodium and water excretion before the onset of full urine concentrating capacity in the SD rat.
Asunto(s)
Tasa de Filtración Glomerular/fisiología , Riñón/irrigación sanguínea , Riñón/fisiología , Urotensinas/genética , Urotensinas/metabolismo , Animales , Femenino , Feto/metabolismo , Tasa de Filtración Glomerular/genética , Riñón/metabolismo , Masculino , Natriuresis/genética , Natriuresis/fisiología , Hormonas Peptídicas/genética , Hormonas Peptídicas/metabolismo , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Flujo Sanguíneo Regional/genética , Flujo Sanguíneo Regional/fisiología , Sodio/metabolismo , Urotensinas/antagonistas & inhibidores , Agua/metabolismoRESUMEN
OBJECTIVES: The aim of this study was to compare the single-dose effects of thiazide-type diuretics cicletanine and hydrochlorothiazide (HCTZ), on natriuresis and kaliuresis in prehypertensive and treatment-naïve, stage 1 hypertensive patients and to explore the impact of GRK4 gene polymorphisms on thiazide-induced urinary electrolyte excretion. METHODS: The study was a randomized, double-blind, placebo-controlled, three-period, four-treatment, balanced incomplete block, cross-over study in male patients assigned to treatment sequences consisting of placebo, cicletanine 50 mg, cicletanine 150 mg, and HCTZ 25 mg, doses used to treat hypertension. Cumulative urine samples were collected predosing and over 24 h after dosing in each period to compare urine electrolyte excretion profiles of potassium (UKV), sodium (UNaV), magnesium, calcium, phosphate, chloride, and pH among groups. Each treatment was administered to 18 different patients in each period, and an equal number of patients had less than and at least three GRK4 allele variants. RESULTS: Compared with placebo, mean UKV was significantly increased with HCTZ 25 mg (12.7 mmol/day; Pâ≤â0.001), cicletanine 50 mg (4.6 mmol/day; Pâ=â0.026), and cicletanine 150 mg (5.5 mmol/day; Pâ=â0.011), and mean UNaV was significantly increased with HCTZ 25 mg (102.2 mmol/day; Pâ≤â0.001), cicletanine 50 mg (21.7 mmol/day; Pâ=â0.005), and cicletanine 150 mg (57.9 mmol/day; Pâ≤â0.001). CONCLUSION: All treatments had more natriuresis, diuresis, and kaliuresis than placebo, and both doses of cicletanine had less kaliuresis than HCTZ. These findings suggest that cicletanine is a favorable and well tolerated option for the treatment of hypertension with an improved safety profile compared with HCTZ.
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Antihipertensivos/uso terapéutico , Hidroclorotiazida/uso terapéutico , Hipertensión/tratamiento farmacológico , Natriuresis/efectos de los fármacos , Potasio/orina , Prehipertensión/tratamiento farmacológico , Piridinas/uso terapéutico , Adulto , Anciano , Método Doble Ciego , Quinasa 4 del Receptor Acoplado a Proteína-G/genética , Quinasa 4 del Receptor Acoplado a Proteína-G/metabolismo , Humanos , Hipertensión/genética , Hipertensión/orina , Masculino , Persona de Mediana Edad , Natriuresis/genética , Polimorfismo de Nucleótido Simple , Prehipertensión/genética , Prehipertensión/orinaRESUMEN
Collecting duct (CD)-derived endothelin-1 (ET-1) acting via endothelin B (ETB) receptors promotes Na(+) excretion. Compromise of ET-1 signaling or ETB receptors in the CD cause sodium retention and increase blood pressure. Activity of the epithelial Na(+) channel (ENaC) is limiting for Na(+) reabsorption in the CD. To test for ETB receptor regulation of ENaC, we combined patch-clamp electrophysiology with CD-specific knockout (KO) of endothelin receptors. We also tested how ET-1 signaling via specific endothelin receptors influences ENaC activity under differing dietary Na(+) regimens. ET-1 significantly decreased ENaC open probability in CD isolated from wild-type (WT) and CD ETA KO mice but not CD ETB KO and CD ETA/B KO mice. ENaC activity in WT and CD ETA but not CD ETB and CD ETA/B KO mice was inversely related to dietary Na(+) intake. ENaC activity in CD ETB and CD ETA/B KO mice tended to be elevated under all dietary Na(+) regimens compared with WT and CD ETA KO mice, reaching significance with high (2%) Na(+) feeding. These results show that the bulk of ET-1 inhibition of ENaC activity is mediated by the ETB receptor. In addition, they could explain the Na(+) retention and elevated blood pressure observed in CD ET-1 KO, CD ETB KO, and CD ETA/B KO mice consistent with ENaC regulation by ET-1 via ETB receptors contributing to the antihypertensive and natriuretic effects of the local endothelin system in the mammalian CD.
Asunto(s)
Canales Epiteliales de Sodio/metabolismo , Túbulos Renales Colectores/metabolismo , Receptor de Endotelina B/deficiencia , Receptor de Endotelina B/genética , Regulación hacia Arriba/genética , Amilorida/farmacología , Animales , Endotelina-1/metabolismo , Endotelina-1/fisiología , Bloqueadores del Canal de Sodio Epitelial , Femenino , Hipertensión/genética , Hipertensión/metabolismo , Masculino , Ratones , Ratones Noqueados , Natriuresis/genética , Sodio/metabolismoRESUMEN
The importance of excess salt intake in the pathogenesis of hypertension is widely recognized. Blood pressure is controlled primarily by salt and water balance because of the infinite gain property of the kidney to rapidly eliminate excess fluid and salt. Up to fifty percent of patients with essential hypertension are salt-sensitive, as manifested by a rise in blood pressure with salt loading. We conducted a two-stage genetic analysis in hypertensive patients very accurately phenotyped for their salt-sensitivity. All newly discovered never treated before, essential hypertensives underwent an acute salt load to monitor the simultaneous changes in blood pressure and renal sodium excretion. The first stage consisted in an association analysis of genotyping data derived from genome-wide array on 329 subjects. Principal Component Analysis demonstrated that this population was homogenous. Among the strongest results, we detected a cluster of SNPs located in the first introns of PRKG1 gene (rs7897633, pâ=â2.34E-05) associated with variation in diastolic blood pressure after acute salt load. We further focused on two genetic loci, SLC24A3 and SLC8A1 (plasma membrane sodium/calcium exchange proteins, NCKX3 and NCX1, respectively) with a functional relationship with the previous gene and associated to variations in systolic blood pressure (the imputed rs3790261, pâ=â4.55E-06; and rs434082, pâ=â4.7E-03). In stage 2, we characterized 159 more patients for the SNPs in PRKG1, SLC24A3 and SLC8A1. Combined analysis showed an epistatic interaction of SNPs in SLC24A3 and SLC8A1 on the pressure-natriuresis (p interactionâ=â1.55E-04, p modelâ=â3.35E-05), supporting their pathophysiological link in cellular calcium homeostasis. In conclusions, these findings point to a clear association between body sodium-blood pressure relations and molecules modulating the contractile state of vascular cells through an increase in cytoplasmic calcium concentration.
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Hipertensión/genética , Hipertensión/fisiopatología , Cloruro de Sodio Dietético/farmacología , Vasoconstricción/genética , Vasodilatación/genética , Presión Sanguínea/efectos de los fármacos , Presión Sanguínea/genética , Estudios de Cohortes , Femenino , Predisposición Genética a la Enfermedad , Humanos , Riñón/efectos de los fármacos , Riñón/fisiopatología , Masculino , Persona de Mediana Edad , Natriuresis/efectos de los fármacos , Natriuresis/genética , Polimorfismo de Nucleótido Simple/genética , Cloruro de Sodio Dietético/administración & dosificación , Vasoconstricción/efectos de los fármacos , Vasodilatación/efectos de los fármacosRESUMEN
Stanniocalcin-1 (STC-1) is made by kidney collecting duct cells for targeting of nephron mitochondria to promote respiratory uncoupling and calcium uniport activity. However, the purpose of these actions and how the renal gene is regulated are poorly understood. This study has addressed the latter issue by monitoring renal STC-1 gene expression in different models of kidney function. Unilateral nephrectomy and over-hydration had no bearing on renal gene activity in adult Wistar rats. Dehydration, on the other hand, had time-dependent stimulatory effects in male and female kidney cortex, where STC-1 mRNA levels increased 8-fold by 72h. Medullary gene activity was significantly increased as well, but muted in comparison ( approximately 2-fold). Gene induction was accompanied by an increase in mitochondrial sequestration of STC-1 protein. Aldosterone and angiotensin II had no bearing on STC-1 gene induction, although there was evidence of a role for arginine vasopressin. Gene induction was unaltered in integrin alpha1 knockout mice, which have an impaired tonicity enhancer binding protein (TonEBP) response to dehydration. The STC-1 gene response could be cytoprotective in intent, as dehydration entails a fall in renal blood flow and a rise in medullary interstitial osmolality. Alternatively, STC-1 could have a role in salt and water balance as dehydration necessitates water conservation as well as controlled natriuresis and kaliuresis.