A atividade do NHE3 em túbulo proximal é inibida pela sinalização enviesada do receptor de angiotensina II tipo 1/beta-arrestina / Proximal tubule NHE3 activity is inhibited by beta-arrestin-biased angiotensin II type 1 receptor signaling

Os receptores medeiam a maioria das respostas fisiológicas em resposta a diversidade de estímulos. A ativação da sinalização mediada pelo receptor de angiotensina II tipo 1 é o principal responsável pelos efeitos do hormônio angiotensina II (Ang II) nos tecidos alvo. No rim concentrações fisiológicas de Ang II aumentam a atividade no túbulo proximal da isoforma 3 do trocador de Na+/H+ (NHE3). Este efeito é crucial para a manutenção do volume extracelular e pressão arterial. Evidências recentes mostraram que a ativação seletiva da sinalização enviesada da beta-arrestina/ receptor AT1 induz diurese e natriurese independentemente da sinalização via proteína G. Neste estudo testamos a hipótese de que a sinalização enviesada do receptor AT1/ beta-arrestina inibe a atividade do NHE3 no túbulo proximal, bem como investigar os possíveis mecanismos moleculares que medeio este efeito. Para tal, nós determinamos os efeitos do composto TRV120023, que se liga ao receptor AT1, bloqueando o acoplamento da proteína G e estimulando a sinalização da beta-arrestina, na função do NHE3 in vivo e in vitro. A atividade do NHE3 foi medida quer em túbulo proximal nativo, por meio de microperfusão estacionária, bem como em uma linha celular de túbulo proximal de gamba (OKP), por meio de recuperação de pH intracelular dependente de Na+. Os nossos resultados mostram que o TRV120023 na concentração de 10-7 M inibe marcadamente a atividade do NHE3 em túbulo proximal quer in vivo quer in vitro, sendo que este efeito é completamente abolido nas células silenciadas para a beta-arrestina 1 e 2 através de RNA de interferência. Adicionalmente, a estimulação do NHE3 pela Ang II é completamente suprimida pelo TRV120023 quer in vivo quer in vitro. A inibição do NHE3 pelo TRV120023 foi associada com a diminuição do NHE3 expresso na superfície da membrana plasmática em células OKP e com a redistribuição entre o corpo e a base das microvilosidades em túbulo proximal de rato. A...

Cell surface receptors mediate most of our physiological responses to an array of stimulus. The triggering of the angiotensin II type I (AT1) receptor signaling is the major control point in the regulation of the ultimate effects of the peptide hormone angiotensin II (Ang II) on its target tissue. In the kidney physiological concentrations of Ang II upregulate the activity of proximal tubule Na+/H+ exchanger isoform 3 (NHE3). This effect is crucial for maintenance of extracellular fluid volume homeostasis and blood pressure. Recent findings have shown that selective activation of the betaarrestin-biased AT1 receptor signalingpathway induces diuresis and natriuresis independent of G-protein mediated signaling. This study tested the hypothesis that activation of this AT1 receptor/beta-arrestin signaling inhibits NHE3 activity in proximal tubule as well as investigate the underlying molecular mechanisms mediating this effect. To this end, we determined the effects of the compound TRV120023, which binds to the AT1R, blocks G protein coupling, and stimulates beta-arrestin signaling, on NHE3 function in vivo and in vitro. NHE3 activity was measured in both native proximal tubules, by stationary microperfusion, and in opossum proximal tubule (OKP) cells, by Na+-dependent intracellular pH recovery. Our results showed that 10-7 MTRV120023 remarkably inhibited proximal tubule NHE3 activity both in vivo and in vitro, and the effect was completely abolished in OKP cells silenced for beta-arrestin 1 and 2 by small interference RNA. Additionally, stimulation of NHE3 by Ang II was completely suppressed by TRV120023 both in vivo as well as in vitro. Inhibition of NHE3 activity by TRV120023 was associated with a decrease in NHE3 surface expression in OKP cells and with a redistribution from the body to the base of the microvilli in the rat proximal tubule. The decreased surface NHE3 in OKP cells was associated with an increase in NHE3 internalization via...

Essential regulatory elements for NHE3 gene transcription in renal proximal tubule cells

The objectives of the present study were to identify the cis-elements of the promoter absolutely required for the efficient rat NHE3 gene transcription and to locate positive and negative regulatory elements in the 5’-flanking sequence (5’FS), which might modulate the gene expression in proximal tubules, and to compare this result to those reported for intestinal cell lines. We analyzed the promoter activity of different 5’FS segments of the rat NHE3 gene, in the OKP renal proximal tubule cell line by measuring the activity of the reporter gene luciferase. Because the segment spanning the first 157 bp of 5’FS was the most active it was studied in more detail by sequential deletions, point mutations, and gel shift assays. The essential elements for gene transcription are in the region -85 to -33, where we can identify consensual binding sites for Sp1 and EGR-1, which are relevant to NHE3 gene basal transcription. Although a low level of transcription is still possible when the first 25 bp of the 5’FS are used as promoter, efficient transcription only occurs with 44 bp of 5’FS. There are negative regulatory elements in the segments spanning -1196 to -889 and -467 to -152, and positive enhancers between -889 and -479 bp of 5’FS. Transcription factors in the OKP cell nuclear extract efficiently bound to DNA elements of rat NHE3 promoter as demonstrated by gel shift assays, suggesting a high level of similarity between transcription factors of both species, including Sp1 and EGR-1.

Modulación inflamatoria en el shock traumático: [revisión] / Inflammatory modulation in traumatic shock: [review]

Hemorrhagic hypovolemic shock secondary to trauma is an important cause of morbidity and mortality worldwide. During the last few years, new concepts have emerged and the guidelines of fluid resuscitation in these patients have been redefined. The concept of hypotensive resuscitation has been established and new colloid solutions based on starch have been manufactured, been hydroxyethyl starch in a balanced electrolytic solution, the most studied and successful one. It has been reported, as well, the positive effects of the pharmacologic modulation of the inflammatory pathways in experimental model subjects submitted to hypovolemic shock. Products such as, ethyl pyruvate and the Na+/H+ type 1 inhibitor, BIIB513, have been Studies only experimentally in rodent models using colloids as the primary resuscitation fluid. The significant improvement in the hemodinamyc, pattern and the cardiac and inflammatory indexes and mediators, has created the basis for their use in clinical trials in the near future. The systemic inflammatory response is an important cause of multiple organ failure that increases the late mortality of patients surviving the initial early phases of hypovolemic traumatic shock and its experimental modulation in rodent models with products such as ethyl pyruvate and BIIB513 has produced excellent in vivo and in vitro results.

Universalmente se considera el Shock hipovolémico de origen hemorrágico como una importante causa de morbi-mortalidad. Durante los últimos años se ha redefinido los conceptos de la reanimación con líquidos intravenosos en los pacientes con choque hipovolémico y establecido los conceptos de reanimación hipotensa con el uso de nuevos coloides derivados del almidón, tales como el hidroxietil-almidón en solución electrolítica balanceada (Hextend®). Así mismo, se ha reportado el beneficio que conlleva el uso de modificadores de la cascada inflamatoria en modelos experimentales de sujetos sometidos a choque hipovolémico hemorrágico. Productos como el etil piruvato y la BIIB513, un inhibidor selectivo del intercambiador Na+/H+ tipo 1, han sido estudiados sólo experimentalmente en modelos roedores, empleando coloides como principal elemento de reanimación. Al mejorar el perfil hemodinámico, parámetros cardíacos y niveles de mediadores inflamatorios, estos compuestos constituyen una base cierta para ser incluidos en estudios clínicos en un futuro próximo. La respuesta inflamatoria sistémica está íntimamente implicada en la patogénesis de la Falla Orgánica Múltiple, aumentando la mortalidad tardía de pacientes que sobreviven las etapas tempranas del shock hipovolémico hemorrágico traumático. Su modulación experimental con el etil piruvato o bien la BIIB513 ha dado excelente resultado tanto en modelos experimentales in vivo como in vitro.

Analisis ecocardiografico del efecto de diferentes inhibidores del intercambiador Na+/H+ sobre la estructura y funcion sistolica del ventriculo izquierdo en ratas espontaneamente hipertensas / Echocardiographic analysis of the effect of different Na+/H+ exchanger inhibitors on left

El presente estudio fue proyectado para analizar mediante ecocardiograma los efectos del HOE 642 (cariporide) (HOE) y del BIIB 723 (BIIB) sobre la estructura y función sistólica del ventrículo izquierdo en ratas espontáneamente hipertensas (SHR)- 8 con 30 mg/kg/día de HOE, 8 con 30 mg/kg/día de BIIB durante 30 días y 4 sin tratamiento (grupo control) durante esos 30 días. Los distintos parámetros analizados no mostraron cambios durante ese período en las ratas controles. Si bien el HOE determinó un leve descenso de la presión arterial (C: 184 ± 1.75 mm Hg; 30d:176.20 ± 2.60 mm Hg, p <0.01), no detectada con BIIB,ambas drogas provocaron un aumento del estrés sistólico pico (HOE C:166 ± 29 kdinas/cm2; 30d: 204 ± 34kdinas/cm2, p <0.04. BIIB C: 164 ± 25.90 kdinas/cm2; 30d: 234 ± 29.30 kdinas/cm2, p<0.02).Tanto HOE comoBIIB redujeron significativamente la masa ventricular izquierda (MVI) (HOE C: 612.50±50 mg; 30d:452 ± 37 mg,p <0.01; BIIB C: 544 ± 16mg; 30 d: 374 ± 25 mg, p<0.01). El porcentaje de acortamiento endocárdico no semodificó luego del tratamiento con HOE (C: 62.30 ± 2.75; 30d 65.50 ± 2.40%, ns) y BIIB (C: 63.20 ± 2.39%;30d 67.20 ± 1.62%, ns). Los resultados analizados permiten concluir que ambos inhibidores determinaron similarreducción de la MVI. Esa reducción se acompañó de mejoría en los índices de evaluación de la función sistólica ventricular izquierda, pese al incremento del estrés sistólico pico. Estas evidencias sugieren que independientemente del inhibidor utilizado se encuentra un aumento del inotropismo, previamente comprometidodurante el desarrollo de la hipertrofia

The aim of this study was to analyze by echocardiogram, the action of two Na+/H+ exchange, inhibitors, HOE 642 (HOE) and BIIB 723 (BIIB) on left ventricular (LV) mass and LV systolic function. We studied 16 spontaneously hypertensive rats (SHR), 8 treated with HOE 30 mg/kg/day, 8 with 30 mg/kg/day of BIIB during 30 days and 4 SHR as controls during those 30 days. Results are expressed as mean values ± SEM. The systolic blood pressure and theechocardiograpic parameters examined did not evidence changes during that period in the controls rats. Eventhough HOE determined a slight decrease in blood pressure (HOE C: 184 ± 1.75 mm Hg; HOE 30d: 176.20 ±2.60 mm Hg - p <0.01) which was not detected with BIIB, both drugs provoked an increase of peak systolic stress (HOE C: 166 ± 29 kdynes/cm2; HOE 30d: 204 ± 34 kdynes/cm2, p <0.04; BIIB C: 164 ± 25.90 kdynes/cm2; BIIB 30d: 234 ± 29.30 kdynes/cm2, p <0.02). HOE and BIIB reduced LV mass after 30 days of administration (HOE C: 612.50 ± 50 mg; 30d: 452 ± 37 mg, p <0.01. BIIB C: 544 ± 16mg; 30d: 374 ± 25 mg, p <0.01). LV endocardial shortening was similar independently of the NHE inhibitors used (HOE C: 62.30 ± 2.75%; 30d: 65.50 ± 2.40%, ns. BIIB C: 63.20 ± 2,39%; 30d 67,20 ± 1.62%, ns). These data demonstrate that long-treatment with HOE or BIIB produced similar LV mass regression without changes in endocardial fractional shortening in spite of the increase of peak systolic stress. This finding could represent an increased inotropism previously depressed by the development of hypertrophy

El intercambiador Na+/Ca2+ como responsable del atontamiento miocardio / The Na+/Ca2+ exchanger as responsible of myocardial stunning

Nuestro objetivo fue examinar la participación de los intercambiadores Na+/H+ (NHE) y Na+/Ca+2 (NCX) sobre las alteraciones sistólicas propias del atontamiento miocárdio. Se utilizó el modelo de corazón aislado isovolúmico de rata el cual fue sometido a 20 minutos de isquemia global (Is) y 30 minutos de perfusión (R). Este protocolo se repitió en prasencia de 1microM de HOE 642, bloqueante específico del NHE-1 y de KB-R7943, bloqueante del modo inverso del NCX (entrando Ca+2 y sacando Na de la célula) administrados antes de la Is, y/o en la R. En los controles isquémicos la contractilidad, evaluada a través de la +dP/dtmax se recuperó aproximadamente un 60 porciento. La recuperación postisquémica fue total cuando el bloqueo de NHE fue efectuado antes de la Is o al comienzo de la R y mejoró significativamente cuando en Is y R bloqueó el NCX ( 95 + o - 7 porciento). La contractura isquémica disminuyó com ambos tratamientos cuando fueron realizados previos a la Is. El aumento de la PDF observado en la R (24 + o - 6 y 12 + o - 2 mmHg) y por el bloqueo del NCX realizado durante Is y R (12 + o - 6 mmHg). Estos resultados indican que la activación del modo inverso del NCX secundaria a una activación del NHE (que incrementa el Na+ intracelular) durante la isquemia y reperfusión es el mecanismo culpable de la sobrecarga de Ca+2 involucrado en la disminuición de la contractilidad que caracteriza al miocardio atontado.

Cell Biology of H+ Transport in Epithelia

Cell homeostasis of H+ ions has been an object of wide interest in the last two decades, which has led to extended knowledge about a considerable number of membrane transport mechanisms responsible for keeping cell pH within physiological limits. Among these mechanisms the most important are Na+/H+ exchange, the vacuolar H+-ATPase, the H+-K+-ATPase, Cl-/HCO3 - exchange and Na+/HCO3 - cotransport. The present review covers both cellular function and molecular aspects of these transporters, starting from a discussion of the methods used for the determination of cell pH and epithelial H+ transport, and analysing their molecular constitution, cloning and known isoforms, as well as their functional role in the maintenance of cell pH and epithelial transport.

The role of the distal nephron in the regulation of acid-base equilibrium by the kidney

The present paper reviews mechanisms by which the kidney controls systemic acid-base balance, with emphasis on the role of the distal nephron, and particularly of the cortical distal tubule. These mechanisms are essentially based on H-ion transport along the whole nephron. In proximal tubule cells, approximately 80 of H-ion secretion is mediated by Na+/H+ exchange, and 20 by H(+)-ATPase. In the distal nephron, acid-base transport mechanisms are located mainly in intercalated cells. H-ion secretion is effected by vacuolar H(+)-ATPase in alpha-intercalated cells and, in K-depleted animals, also by the gastric type H/K ATPase. In animals in alkalosis, beta-intercalated cells secrete bicarbonate by an apical Cl-/HCO3- exchanger, while a basolateral H-ATPase transfers H-ions into the interstitium. In cortical distal tubule, these mechanisms have been shown to be present in the intercalated cells of the connecting segment and of the initial collecting duct (the late distal tubule of micropuncture experiments). In the convoluted distal tubule (early distal tubule), most H-ion secretion occurs by means of the Na+/H+ exchanger. These data show that the distal nephron, including the cortical distal tubule, is a nephron segment responsible for a sizeable portion of bicarbonate reabsorption and titratable acid generation, as well as for bicarbonate secretion under appropriate metabolic conditions, being therefore the site of fine regulation of renal mechanisms that maintain acid-base homeostasis.