RESUMEN
To better understand the role of the inward-rectifying K channel Kir4.1 (KCNJ10) in the distal nephron, we initially studied a global Kir4.1 knockout mouse (gKO), which demonstrated the hypokalemia and hypomagnesemia seen in SeSAME/EAST syndrome and was associated with reduced Na/Cl cotransporter (NCC) expression. Lethality by ~3 wk, however, limits the usefulness of this model, so we developed a kidney-specific Kir4.1 "knockdown" mouse (ksKD) using a cadherin 16 promoter and Cre-loxP methodology. These mice appeared normal and survived to adulthood. Kir4.1 protein expression was decreased ~50% vs. wild-type (WT) mice by immunoblotting, and immunofluorescence showed moderately reduced Kir4.1 staining in distal convoluted tubule that was minimal or absent in connecting tubule and cortical collecting duct. Under control conditions, the ksKD mice showed metabolic alkalosis and relative hypercalcemia but were normokalemic and mildly hypermagnesemic despite decreased NCC expression. In addition, the mice had a severe urinary concentrating defect associated with hypernatremia, enlarged kidneys with tubulocystic dilations, and reduced aquaporin-3 expression. On a K/Mg-free diet for 1 wk, however, ksKD mice showed marked hypokalemia (serum K: 1.5 ± 0.1 vs. 3.0 ± 0.1 mEq/l for WT), which was associated with renal K wasting (transtubular K gradient: 11.4 ± 0.8 vs. 1.6 ± 0.4 in WT). Phosphorylated-NCC expression increased in WT but not ksKD mice on the K/Mg-free diet, suggesting that loss of NCC adaptation underlies the hypokalemia. In conclusion, even modest reduction in Kir4.1 expression results in impaired K conservation, which appears to be mediated by reduced expression of activated NCC.
Asunto(s)
Nefronas/metabolismo , Canales de Potasio de Rectificación Interna/deficiencia , Potasio en la Dieta/sangre , Reabsorción Renal , Alcalosis/sangre , Alcalosis/genética , Alcalosis/fisiopatología , Animales , Acuaporina 3/metabolismo , Técnicas de Silenciamiento del Gen , Genotipo , Hipercalcemia/sangre , Hipercalcemia/genética , Hipercalcemia/fisiopatología , Hiperpotasemia/sangre , Hiperpotasemia/genética , Hiperpotasemia/fisiopatología , Hipernatremia/sangre , Hipernatremia/genética , Hipernatremia/fisiopatología , Capacidad de Concentración Renal , Ratones Endogámicos C57BL , Ratones Noqueados , Nefronas/fisiopatología , Fenotipo , Fosforilación , Canales de Potasio de Rectificación Interna/genética , Miembro 3 de la Familia de Transportadores de Soluto 12/metabolismoRESUMEN
It is recognized that dopamine promotes natriuresis by inhibiting multiple transporting systems in the proximal tubule. In contrast, less is known about the molecular targets of dopamine actions on water-electrolyte transport in the cortical collecting duct (CCD). Epithelial cells in the CCD are exposed to dopamine, which is synthesized locally or secreted from sympathetic nerve endings. Basolateral K(+) channels in the distal renal tubule are critical for K(+) recycling and controlling basolateral membrane potential to establish the driving force for Na(+) reabsorption. Here, we demonstrate that Kir4.1 and Kir5.1 are highly expressed in the mouse kidney cortex and are localized to the basolateral membrane of the CCD. Using patch-clamp electrophysiology in freshly isolated CCDs, we detected highly abundant 40-pS and scarce 20-pS single channel conductances, most likely representing Kir4.1/5.1 and Kir4.1 channels, respectively. Dopamine reversibly decreased the open probability of both channels, with a relatively greater action on the Kir4.1/5.1 heterodimer. This effect was mediated by D2-like but not D1-like dopamine receptors. PKC blockade abolished the inhibition of basolateral K(+) channels by dopamine. Importantly, dopamine significantly decreased the amplitude of Kir4.1/5.1 and Kir4.1 unitary currents. Consistently, dopamine induced an acute depolarization of basolateral membrane potential, as directly monitored using current-clamp mode in isolated CCDs. Therefore, we demonstrate that dopamine inhibits basolateral Kir4.1/5.1 and Kir4.1 channels in CCD cells via stimulation of D2-like receptors and subsequently PKC. This leads to depolarization of the basolateral membrane and a decreased driving force for Na(+) reabsorption in the distal renal tubule.