Collecting duct (CD) adenylyl cyclase VI (AC6) has been implicated in arginine vasopressin (AVP)-stimulated renal water reabsorption. To evaluate the role of CD-derived AC6 in regulating water homeostasis, mice were generated with CD-specific knockout (KO) of AC6 using the Cre/loxP system. CD AC6 KO and controls were studied under normal water intake, chronically water loaded, or water deprived; all of these conditions were repeated in the presence of continuous administration of 1-desamino-8-d-arginine vasopressin (DDAVP). During normal water intake or after water deprivation, urine osmolality (U(osm)) was reduced in CD AC6 KO animals vs. controls. Similarly, U(osm) was decreased in CD AC6 KO mice vs. controls after water deprivation+DDAVP administration. Pair-fed (with controls) CD AC6 KO mice also had lower urine osmolality vs. controls. There were no detectable differences between KO and control animals in fluid intake or urine volume under any conditions. CD AC6 KO mice did not have altered plasma AVP levels vs. controls. AVP-stimulated cAMP accumulation was reduced in acutely isolated inner medullary CD (IMCD) from CD A6 KO vs. controls. Medullary aquaporin-2 (AQP2) protein expression was lower in CD AC6 KO mice vs. controls. There were no differences in urinary urea excretion or IMCD UT-A1 expression; however, IMCD UT-A3 expression was reduced in CD AC6 KO mice vs. controls. In summary, AC6 in the CD regulates renal water excretion, most likely through control of AVP-stimulated cAMP accumulation and AQP2.

The epithelial Na + channel (ENaC) in the aldosterone-sensitive distal nephron (ASDN) is under negative-feedback regulation by the renin-angiotensin-aldosterone system in protection of sodium balance and blood pressure. We test here whether aldosterone is necessary and sufficient for ENaC expression and activity in the ASDN. Surprisingly, ENaC expression and activity are robust in adrenalectomized (Adx) mice. Exogenous mineralocorticoid increases ENaC activity equally well in control and Adx mice. Plasma [AVP] is significantly elevated in Adx vs. control mice. Vasopressin (AVP) stimulates ENaC. Inhibition of the V 2 AVP receptor represses ENaC activity in Adx mice. The absence of aldosterone combined with elevated AVP release compromises normal feedback regulation of ENaC in Adx mice in response to changes in sodium intake. These results demonstrate that aldosterone is sufficient but not necessary for ENaC activity in the ASDN. Aldosterone-independent stimulation by AVP shifts the role of ENaC in the ASDN from protecting Na + balance to promoting water reabsorption. This stimulation of ENaC likely contributes to the hyponatremia of adrenal insufficiency.epithelial transport | hypertension | sodium excretion | diabetes insipidus | sodium wasting R enal sodium excretion is fine-tuned in the aldosterone-sensitive distal nephron (ASDN). Here, the activity of the epithelial Na + channel (ENaC) is limiting for sodium reabsorption (reviewed in refs. 1 and 2). ENaC serves as the apical entry pathway for electrogenic Na + reabsorption through principal cells. Normal ENaC function is required for proper sodium balance and, thus, normal blood pressure. Gain-of-function mutations in ENaC cause inappropriate renal sodium retention and consequent increases in mean arterial pressure (2, 3). Inhibition of ENaC corrects the renal and blood pressure phenotypes resulting from such mutations. Loss-of-function mutations in ENaC, in contrast, cause renal sodium wasting and corresponding decreases in blood pressure (2, 4).The activity of ENaC is under negative-feedback regulation by the renin-angiotensin-aldosterone system (RAAS; ref. 1). The mineralocorticoid, aldosterone, is the final hormone in this cascade. This antinatriuretic factor is essential for proper Na + balance (5, 6). Decreases in blood pressure evoke via reninAngII signaling secretion of aldosterone from the adrenal gland. Aldosterone through the mineralocorticoid receptor (MR) stimulates ENaC in the ASDN to minimize renal sodium excretion in protection of Na + balance and vascular volume (2, 4). Pathological increases in aldosterone elevate blood pressure by promoting inappropriate renal sodium retention (7,8). Inhibition of ENaC ameliorates inappropriate renal sodium retention. In contrast, pathological decreases in aldosterone result in sodium wasting arising from inappropriate increases in renal sodium excretion (4,8,9). MR agonism and antagonism increase and decrease ENaC activity, respectively (10-12). There is strong support for a tight positive relation between...

ABSTRACTcAMP is an important mediator of cystogenesis in polycystic kidney disease (PKD). Several adenylyl cyclase (AC) isoforms could mediate cAMP accumulation in PKD, and identification of a specific pathogenic AC isoform is of therapeutic interest. We investigated the role of AC6 in a mouse model of PKD that is homozygous for the loxP-flanked PKD1 gene and heterozygous for an aquaporin-2-Cre recombinase transgene to achieve collecting duct-specific gene targeting. Collecting duct-specific knockout of polycystin-1 caused massive renal cyst formation, kidney enlargement, and severe kidney failure, with a mean survival time of 2 months. In contrast, coincident collecting duct-specific knockout of polycystin-1 and AC6 (also homozygous for the floxed ADCY6 gene) markedly decreased kidney size and cystogenesis, improved renal function, reduced activation of the B-Raf/ERK/MEK pathway, and greatly increased survival. Absence of collecting duct AC6 did not alter urinary cAMP excretion or kidney cAMP concentration. In conclusion, AC6 is a key mediator of cyst formation and renal injury in a model of PKD.

Vasopressin modulates sodium reabsorption in the collecting duct through adenylyl cyclase-stimulated cyclic AMP, which exists as multiple isoforms; the specific isoform involved in vasopressin-stimulated sodium transport is unknown. To assess this, we studied mice deficient in adenylyl cyclase type VI specifically in the principal cells of the collecting duct. Knockout mice had increased urine volume and reduced urine sodium concentration, but regardless of the level of sodium intake, they did not exhibit significant alterations in urinary sodium excretion, arterial pressure, or pulse rate. Plasma renin concentration was elevated in knockout mice, however, suggesting a compensatory response. Valsartan significantly reduced arterial pressure in knockout mice but not in controls. Knockout mice had decreased renal cortical mRNA content of all three epithelial sodium channel (ENaC) isoforms, and total cell sodium channel isoforms a and g were reduced in these animals. Patch-clamp analysis of split-open cortical collecting ducts revealed no difference in baseline activity of sodium channels, but knockout mice had abolished vasopressinstimulated ENaC open probability and apical membrane channel number. In summary, these data suggest that adenylyl cyclase VI mediates vasopressin-stimulated ENaC activity in the kidney. Arginine vasopressin (AVP) modulation of water flux across the renal collecting duct (CD) is well known; however, it is becoming increasingly apparent that AVP stimulation of epithelial Na channel (ENaC)-mediated Na transport is of substantial importance in Na and water homeostasis. Recent studies indicate that AVP-induced Na reabsorption is important in the hormone's stimulation of water reabsorption (reviewed in Stockand 1 ). Exogenous AVP enhances renal expression of all ENaC subunits in Brattleboro rats, 2 increases ENaC open probability (Po) in MDCK cells, 3 and stimulates Na flux in isolated perfused CD. 4 A 2-to 3-minute exposure to AVP was shown to increase ENaC Po in the split-open isolated mouse cortical CD (CCD); in addition, 30-minute exposure to AVP increased the ENaC apical membrane channel number (N). 5 Recent studies by our group showed that adrenalectomy surprisingly did not reduce renal ENaC abundance, whereas adrenalectomy and AVP V2 receptor antagonism markedly decreased renal ENaC levels. 6 The stimulatory effects of AVP on ENaC are mediated by cAMP. In the split-open mouse CCD, the AVP effect was dependent upon adenylyl cyclase (AC) activity, whereas AVPenhanced apical membrane Na conductance in perfused rat CCD was mediated by cAMP. 4 cAMP increases ENaC Po and N in pulmonary epithelial cells, transfected oocytes, CCD cell lines, cultured CDs, and MDCK cells. [7][8][9][10][11][12] The effect of cAMP on ENaC trafficking is due, at least in part, to inhibition of Nedd4-2-induced ENaC ubiquitination via phosphorylation of Nedd4-2 on Ser 327 , Ser 221 , and Thr 246 ; notably, these are the same residues that are phosphorylated by serum and glucocorticoid-inducible

Arginine vasopressin (AVP) activates the epithelial Na(+) channel (ENaC). The physiological significance of this activation is unknown. The present study tested if activation of ENaC contributes to AVP-sensitive urinary concentration. Consumption of a 3% NaCl solution induced hypernatremia and plasma hypertonicity in mice. Plasma AVP concentration and urine osmolality increased in hypernatremic mice in an attempt to compensate for increases in plasma tonicity. ENaC activity was elevated in mice that consumed 3% NaCl solution compared with mice that consumed a diet enriched in Na(+) with ad libitum tap water; the latter diet does not cause hypernatremia. To determine whether the increase in ENaC activity in mice that consumed 3% NaCl solution served to compensate for hypernatremia, mice were treated with the ENaC inhibitor benzamil. Coadministration of benzamil with 3% NaCl solution decreased urinary osmolality and increased urine flow so that urinary Na(+) excretion increased with no effect on urinary Na(+) concentration. This decrease in urinary concentration further increased plasma Na(+) concentration, osmolality, and AVP concentration in these already hypernatremic mice. Benzamil similarly compromised urinary concentration in water-deprived mice and in mice treated with desmopressin. These results demonstrate that stimulation of ENaC by AVP plays a critical role in water homeostasis by facilitating urinary concentration, which can compensate for hypernatremia or exacerbate hyponatremia. The present findings are consistent with ENaC in addition to serving as a final effector of the renin-angiotensin-aldosterone system and blood pressure homeostasis, also playing a key role in water homeostasis by regulating urine concentration and dilution of plasma.