RESUMEN

In the renal tubules, ATP released from epithelial cells stimulates purinergic receptors, regulating salt and water reabsorption. However, the mechanisms by which ATP is released into the tubular lumen are multifaceted. Pannexin1 (Panx1) is a newly identified. ubiquitously expressed protein that forms connexin-like channels in the plasma membrane, which have been demonstrated to function as a mechanosensitive ATP conduit. Here, we report on the localization of Panx1 in the mouse kidney. Using immunofluorescence, strong Panx1 expression was observed in renal tubules, including proximal tubules, thin descending limbs, and collecting ducts, along their apical cell membranes. In the renal vasculature, Panx1 expression was localized to vascular smooth muscle cells in renal arteries, including the afferent and efferent arterioles. Additionally, we tested whether Panx1 channels expressed in renal epithelial cells facilitate luminal ATP release by measuring the ATP content of urine samples freshly collected from wild-type and Panx1(-/-) mice. Urinary ATP levels were reduced by 30% in Panx1(-/-) compared with wild-type mice. These results suggest that Panx1 channels in the kidney may regulate ATP release and via purinergic signaling may participate in the control of renal epithelial fluid and electrolyte transport and vascular functions.

Asunto(s)

Adenosina Trifosfato/orina , Membrana Celular/metabolismo , Conexinas/metabolismo , Riñón/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Animales , Conexinas/genética , Células Epiteliales/metabolismo , Canales Iónicos/metabolismo , Masculino , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/genética , Transducción de Señal/fisiología

RESUMEN

Connexins (Cxs) are widely-expressed proteins that form gap junctions in most organs, including the kidney. In the renal vasculature, Cx37, Cx40, Cx43, and Cx45 are expressed, with predominant expression of Cx40 in the endothelial cells and Cx45 in the vascular smooth muscle cells. In the tubules, there is morphological evidence for the presence of gap junction plaques only in the proximal tubules. In the distal nephron, Cx30, Cx30.3, and Cx37 are expressed, but it is not known whether they form gap junctions connecting neighboring cells or whether they primarily act as hemichannels. As in other systems, the major function of Cxs in the kidney appears to be intercellular communication, although they may also form hemichannels that allow cellular secretion of large signaling molecules. Renal Cxs facilitate vascular conduction, juxtaglomerular apparatus calcium signaling, and tubular purinergic signaling. Accordingly, current evidence points to roles for these Cxs in several important regulatory mechanisms in the kidney, including the renin angiotensin system, tubuloglomerular feedback, and salt and water reabsorption. At the systemic level, renal Cxs may help regulate blood pressure and may be involved in hypertension and diabetes.

Asunto(s)

Conexinas/fisiología , Uniones Comunicantes/fisiología , Riñón/fisiología , Sistema Renina-Angiotensina/fisiología , Equilibrio Hidroelectrolítico/fisiología , Animales , Humanos

RESUMEN

In the renal tubule, ATP is an important regulator of salt and water reabsorption, but the mechanism of ATP release is unknown. Several connexin (Cx) isoforms form mechanosensitive, ATP-permeable hemichannels. We localized Cx30 to the nonjunctional apical membrane of cells in the distal nephron and tested whether Cx30 participates in physiologically important release of ATP. We dissected, partially split open, and microperfused cortical collecting ducts from wild-type and Cx30-deficient mice in vitro. We used PC12 cells as ATP biosensors by loading them with Fluo-4/Fura Red to measure cytosolic calcium and positioning them in direct contact with the apical surface of either intercalated or principal cells. ATP biosensor responses, triggered by increased tubular flow or by bath hypotonicity, were approximately three-fold greater when positioned next to intercalated cells than next to principal cells. In addition, these responses did not occur in preparations from Cx30-deficient mice or with purinergic receptor blockade. After inducing step increases in mean arterial pressure by ligating the distal aorta followed by the mesenteric and celiac arteries, urine output increased 4.2-fold in wild-type mice compared with 2.6-fold in Cx30-deficient mice, and urinary Na(+) excretion increased 5.2-fold in wild-type mice compared with 2.8-fold in Cx30-deficient mice. Furthermore, Cx30-deficient mice developed endothelial sodium channel-dependent, salt-sensitive elevations in mean arterial pressure. Taken together, we suggest that mechanosensitive Cx30 hemichannels have an integral role in pressure natriuresis by releasing ATP into the tubular fluid, which inhibits salt and water reabsorption.

Asunto(s)

Adenosina Trifosfato/metabolismo , Conexinas/metabolismo , Túbulos Renales Colectores/metabolismo , Natriuresis , Animales , Conexina 30 , Canales Epiteliales de Sodio/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Presión , Receptores Purinérgicos/metabolismo

RESUMEN

Several isoforms of connexin (Cx) proteins have been identified in a variety of tissues where they play a role in intercellular communication, either as the components of gap junctions or as large, nonselective pores known as hemichannels. This investigation seeks to identify the localization and regulation of Cx30.3 in mouse, rat, and rabbit kidney using a Cx30.3(+/lacZ) transgenic approach and immunofluorescence. Cx30.3 was detected in all three species and predominantly in the renal medulla. Both the nuclear lacZ staining indicative of Cx30.3 expression and indirect immunohistochemistry provided the same results. Cx30.3 immunolabeling was mainly punctate in the mouse, typical for gap junctions. In contrast, it showed continuous apical plasma membrane localization in certain tubule segments in the rat and rabbit kidney, suggesting that it may also function as hemichannels. In the cortex, Cx30.3 was localized in the intercalated cells of the cortical collecting duct, because the immunoreactive cells did not label for AQP2, a marker for principal cells. In the medulla, dense Cx30.3 staining was confined to the ascending thin limbs of the loop of Henle, because the immunoreactive cells did not label for AQP1, a marker of the descending thin limbs. Immunoblotting studies indicated that Cx30.3 expression was unchanged in response to either high or low salt intake or in spontaneously hypertensive rats. Cx30.3 appears to be constitutively expressed in certain renal tubular segments and cells and its role in overall kidney function remains to be investigated.

Asunto(s)

Conexinas/genética , Hipertensión/metabolismo , Riñón/metabolismo , Cloruro de Sodio Dietético/metabolismo , Animales , Línea Celular , Conexinas/biosíntesis , Túbulos Renales/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , ARN Mensajero/metabolismo , Conejos , Ratas , Ratas Sprague-Dawley

RESUMEN

Connexin (Cx) proteins are known to play a role in cell-to-cell communication via intercellular gap junction channels or transiently open hemichannels. Previous studies have identified several connexin isoforms in the juxtaglomerular apparatus (JGA), but the vascular connexin isoform Cx45 has not yet been studied in this region. The present work aimed to identify in detail the localization of Cx45 in the JGA and to suggest a functional role for Cx45 in the kidney using conditions where Cx45 expression or function was altered. Using mice that express lacZ coding DNA under the control of the Cx45 promoter, we observed beta-galactosidase staining in cortical vasculature and glomeruli, with specific localization to the JGA region. Renal vascular localization of Cx45 was further confirmed with the use of conditional Cx45-deficient (Cx45fl/fl:Nestin-Cre) mice, which express enhanced green fluorescence protein (EGFP) instead of Cx45 only in cells that, during development, expressed the intermediate filament nestin. EGFP fluorescence was found in the afferent and efferent arteriole smooth muscle cells, in the renin-producing juxtaglomerular cells, and in the extra- and intraglomerular mesangium. Cx45fl/fl:Nestin-Cre mice exhibited increased renin expression and activity, as well as higher systemic blood pressure. The propagation of mechanically induced calcium waves was slower in cultured vascular smooth muscle cells (VSMCs) from Cx45fl/fl:Nestin-Cre mice and in control VSMC treated with a Cx45 gap mimetic peptide that inhibits Cx45 gap junctional communication. VSMCs allowed the cell-to-cell passage of the gap junction permeable dye Lucifer yellow, and calcium wave propagation was not altered by addition of the ATP receptor blocker suramin, suggesting that Cx45 regulates calcium wave propagation via direct gap junction coupling. In conclusion, the localization of Cx45 to the JGA and functional data from Cx45fl/fl:Nestin-Cre mice suggest that Cx45 is involved in the propagation of JGA vascular signals and in the regulation of renin release and blood pressure.

Asunto(s)

Presión Sanguínea , Conexinas/metabolismo , Aparato Yuxtaglomerular/metabolismo , Renina/metabolismo , Animales , Arteriolas/metabolismo , Señalización del Calcio , Células Cultivadas , Conexinas/genética , Uniones Comunicantes/metabolismo , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas de Filamentos Intermediarios/genética , Aparato Yuxtaglomerular/irrigación sanguínea , Aparato Yuxtaglomerular/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Músculo Liso Vascular/metabolismo , Proteínas del Tejido Nervioso/genética , Nestina , Regiones Promotoras Genéticas , Antagonistas del Receptor Purinérgico P2 , Suramina/farmacología , beta-Galactosidasa/genética

RESUMEN

Diabetes mellitus is the most common and rapidly growing cause of end-stage renal disease in developed countries. A classic hallmark of early diabetes mellitus includes activation of the renin-angiotensin system (RAS), which may lead to hypertension and renal tissue injury, but the mechanism of RAS activation is elusive. Here we identified a paracrine signaling pathway in the kidney in which high levels of glucose directly triggered the release of the prohypertensive hormone renin. The signaling cascade involved the local accumulation of succinate and activation of the kidney-specific G protein-coupled metabolic receptor, GPR91, in the glomerular endothelium as observed in rat, mouse, and rabbit kidney sections. Elements of signal transduction included endothelial Ca2+, the production of NO and prostaglandin (PGE2), and their paracrine actions on adjacent renin-producing cells. This GPR91 signaling cascade may serve to modulate kidney function and help remove metabolic waste products through renal hyperfiltration, and it could also link metabolic diseases, such as diabetes, or metabolic syndrome with RAS overactivation, systemic hypertension, and organ injury.

Asunto(s)

Hiperglucemia/metabolismo , Riñón/metabolismo , Receptores Acoplados a Proteínas G/fisiología , Renina/metabolismo , Animales , Señalización del Calcio/efectos de los fármacos , Señalización del Calcio/fisiología , Citratos/farmacología , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/orina , Dinoprostona/antagonistas & inhibidores , Dinoprostona/metabolismo , Células Endoteliales/metabolismo , Femenino , Glucosa/farmacología , Aparato Yuxtaglomerular/efectos de los fármacos , Aparato Yuxtaglomerular/metabolismo , Masculino , Malonatos/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Óxido Nítrico/antagonistas & inhibidores , Óxido Nítrico/metabolismo , Comunicación Paracrina/efectos de los fármacos , Comunicación Paracrina/fisiología , Conejos , Ratas , Receptores Acoplados a Proteínas G/genética , Renina/sangre , Ácido Succínico/metabolismo , Ácido Succínico/farmacología , Ácido Succínico/orina

RESUMEN

Macula densa (MD) cells express the Na(+)/H(+) exchanger (NHE) isoform NHE2 at the apical membrane, which may play an important role in tubular salt sensing through the regulation of cell volume and intracellular pH. These studies aimed to determine whether NHE2 participates in the MD control of renin synthesis. Renal renin content and activity and elements of the MD signaling pathway were analyzed using wild-type (NHE2(+/+)) and NHE2 knockout (NHE2(-/-)) mice. Immunofluorescence studies indicated that NHE2(-/-) mice lack NHE3 at the MD apical membrane, so the other apical NHE isoform has not compensated for the lack of NHE2. Importantly, the number of renin-expressing cells in the afferent arteriole in NHE2(-/-) mice was increased approximately 2.5-fold using renin immunohistochemistry. Western blotting confirmed approximately 20% higher renal cortical renin content in NHE2(-/-) mice compared with wild type. No-salt diet for 1 wk significantly increased renin content and activity in NHE2(+/+) mice, but the response was blunted in NHE2(-/-) mice. Renal tissue renin activity and plasma renin concentration were elevated three- and twofold, respectively, in NHE2(-/-) mice compared with wild type. NHE2(-/-) mice also exhibited a significantly increased renal cortical cyclooxygenase-2 (COX-2) and microsomal prostaglandin E synthase (mPGES) expression, indicating MD-specific mechanisms responsible for the increased renin content. Significant and chronic activation of ERK1/2 was observed in MD cells of NHE2(-/-) kidneys. Removal of salt or addition of NHE inhibitors to cultured mouse MD-derived (MMDD1) cells caused a time-dependent activation of ERK1/2. In conclusion, the NHE2 isoform appears to be important in the MD feedback control of renin secretion, and the signaling pathway likely involves MD cell shrinkage and activation of ERK1/2, COX-2, and mPGES, all well-established elements of the MD-PGE(2)-renin release pathway.

Asunto(s)

Riñón/enzimología , Renina/genética , Intercambiadores de Sodio-Hidrógeno/genética , Animales , Western Blotting , Ciclooxigenasa 2/metabolismo , Cartilla de ADN , Riñón/efectos de los fármacos , Corteza Renal/enzimología , Ratones , Ratones Noqueados , Renina/sangre , Renina/metabolismo , Tiopental/análogos & derivados , Tiopental/farmacología