Abstract
The activity of the Epithelial Na+ Channel (ENaC) in renal principal cells (PC) fine-tunes sodium excretion and consequently, affects blood pressure. The Gs-adenylyl cyclase-cAMP signal transduction pathway is believed to play a central role in the normal control of ENaC activity in PCs. The current study quantifies the importance of this signaling pathway to the regulation of ENaC activity in vivo using a knock-in mouse that has conditional expression of Gs-DREADD (designer receptors exclusively activated by designer drugs; GsD) in renal PCs. The GsD mouse also contains a cAMP response element-luciferase reporter transgene for non-invasive bioluminescence monitoring of cAMP signaling. Clozapine N-oxide (CNO) was used to selectively and temporally stimulate GsD. Treatment with CNO significantly increased luciferase bioluminescence in the kidneys of PC-specific GsD but not control mice. CNO also significantly increased the activity of ENaC in principal cells in PC-specific GsD mice compared to untreated knock-in mice and CNO treated littermate controls. The cell permeable cAMP analog, 8-(4-chlorophenylthio)adenosine 3′,5′-cyclic monophosphate, significantly increased the activity and expression in the plasma membrane of recombinant ENaC expressed in CHO and COS-7 cells, respectively. Treatment of PC-specific GsD mice with CNO rapidly and significantly decreased urinary Na+ excretion compared to untreated PC-specific GsD mice and treated littermate controls. This decrease in Na+ excretion in response to CNO in PC-specific GsD mice was similar in magnitude and timing as that induced by the selective vasopressin receptor 2 agonist, desmopressin, in wild type mice. These findings demonstrate for the first time that targeted activation of Gs signaling exclusively in PCs is sufficient to increase ENaC activity and decrease dependent urinary Na+ excretion in live animals.
Highlights
The epithelial Na+ channel (ENaC) is expressed in sodiumabsorbing epithelial tissue to include that lining the aldosterone-sensitive distal nephron, which encompasses the late distal convoluted tubule, the connecting tubule and the collecting duct (Garty and Palmer, 1997; Rossier et al, 2002; Verouti et al, 2015; Hanukoglu and Hanukoglu, 2016)
The current findings demonstrate that exclusive activation of Gs signaling in renal principal cells (PC) is sufficient to rapidly increase ENaC activity to decrease Na+ excretion
Targeted Stimulation of GsD Increases cAMP-Response Element Reporter Activity Selectively in the Kidneys of PC-Specific GsD Knockin Mice. We began these studies by testing whether targeted expression of GsD in PCs resulted in selective and temporal cAMP signaling in the kidney
Summary
The epithelial Na+ channel (ENaC) is expressed in sodium (re)absorbing epithelial tissue to include that lining the aldosterone-sensitive distal nephron, which encompasses the late distal convoluted tubule, the connecting tubule and the collecting duct (Garty and Palmer, 1997; Rossier et al, 2002; Verouti et al, 2015; Hanukoglu and Hanukoglu, 2016). Na+ reabsorbed via ENaC finetunes serum Na+ levels, and contributes to the renal axial corticomedullary hypertonic gradient that concentrates urine (Reif et al, 1986; Perucca et al, 2008; Stockand, 2010) This makes the proper activity of ENaC critical to the normal control of blood pressure and for maximizing urinary water concentration (Bugaj et al, 2009). Because of this function, gain- and loss-of-function mutations in ENaC and its upstream regulatory pathways cause disordered blood pressure associated with abnormal renal salt excretion (Garty and Palmer, 1997; Rossier et al, 2002; Verouti et al, 2015; Hanukoglu and Hanukoglu, 2016). This key renal ion channel is a druggable target for diuretics and antihypertensive agents to include triamterene and amiloride
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