Adenylate Cyclase 6 Determines cAMP Formation and Aquaporin-2 Phosphorylation and Trafficking in Inner Medulla

Abstract

Arginine vasopressin (AVP) enhances water reabsorption in the renal collecting duct by vasopressin V 2 receptor (V 2 R)-mediated activation of adenylyl cyclase (AC), cAMP-promoted phosphorylation of aquaporin-2 (AQP2), and increased abundance of AQP2 on the apical membrane. Multiple isoforms of adenylate cyclase exist, and the roles of individual AC isoforms in water homeostasis are not well understood. Here, we found that levels of AC6 mRNA, the most highly expressed AC isoform in the inner medulla, inversely correlate with fluid intake. Moreover, mice lacking AC6 had lower levels of inner medullary cAMP, reduced abundance of phosphorylated AQP2 (at both serine-256 and serine-269), and lower urine osmolality than wild-type mice. Water deprivation or administration of the V 2 R agonist dDAVP did not increase urine osmolality of AC6-deficient mice to the levels of wild-type mice. Furthermore, AC6-deficient mice lacked dDAVP-promoted inner medullary cAMP formation and phosphorylation of serine-269 and had attenuated increases in both phosphorylation of serine-256 and apical membrane AQP2 trafficking. In summary, AC6 expression determines inner medullary cAMP formation and AQP2 phosphorylation and trafficking, the absence of which causes nephrogenic diabetes insipidus. The anti-diuretic hormone arginine-vasopressin (AVP) is the primary regulator of water reabsorption in the renal collecting duct (CD) and is critically involved in the regulation of water balance and maintenance of plasma osmolality. 1 AVP acts on the CD through the G s protein-coupled vasopressin V 2 receptor (V 2 R) to stimulate adenylyl cyclase (AC) and thus the synthesis of cAMP. 2 cAMP activates protein kinase A (PKA), which phosphorylates the water channel aquaporin-2 (AQP2) in its COOH-terminal tail on serine residue 256 (S256), thereby resulting in apical plasma membrane accumulation of AQP2. [3][4][5][6] In addition, cAMP-independent activation of AQP2 has been reported, which may involve V 2 R action of phosphoinositide-specific phospholipase C. 7 Other non-PKA-targeted AQP2 phosphorylation sites include S261, S264, and S269. 8 Phosphorylation of AQP2 at S264 and S269 requires prior phosphorylation of S256. 9 Immunohistochemistry showed that pS269-AQP2 localizes exclusively in the apical plasma

AKAP220 manages apical actin networks that coordinate aquaporin-2 location and renal water reabsorption

“…Because the actin barrier forms in the absence of vasopressin stimulation (13,33), we overhydrated the animals to block vasopressin release. Animals were water-loaded by oral gavage (3% total body weight) as described previously (38). Kidneys were harvested 90 min after water loading and fixed in formalin.…”

Natriuretic effect by exendin-4, but not the DPP-4 inhibitor alogliptin, is mediated via the GLP-1 receptor and preserved in obese type 2 diabetic mice

“…We found that the EX4-induced and GLP-1R-mediated natriuresis and NHE3 phosphorylation in the mouse are not associated with an increase in urinary cAMP excretion and persisted in AC6-deficient mice. AC6 is the most abundant AC isoform in rat whole kidney (36) and mouse medulla (33) on the basis of mRNA expression and the dominant AC isoform with regard to forskolin-induced cAMP formation in mouse kidney (4) and inner medulla (33). It could be that the impact of proximal tubule release on total urinary cAMP excretion is lower in mice than rats, although application of parathyroid hormone, which is thought to activate proximal tubular AC, increased urinary cAMP-to-creatinine ratios threefold in mice (43), a finding confirmed in the present studies.…”

“…Mice were randomized to application of EX4 (10 g/kg ip; Sigma-Aldrich, St. Louis, MO), ALG (10 mg/kg ip; Takeda Pharmaceuticals, Oak Grove, IL), or vehicle (0.85% NaCl, 2 l/g body wt ip). After their bladders were emptied, the mice were NaCl-loaded by oral gavage (0.85% NaCl, 30 l/g body wt, ϳ30% of daily NaCl intake) and placed in metabolic cages for quantitative urine collection over 3 h without access to food or water (32,33); then blood glucose was measured by tail snip.…”

“…Plasma and urine were analyzed for Na ϩ and K ϩ concentrations by flame photometry (ColeParmer Instrument, Vernon Hills, IL). cAMP concentrations were assessed by radioimmunoassay (33). In a separate set of two-period clearance studies in WT mice, parathyroid hormone (10 g/kg iv bolus) was administered, and 5 min later the second period was started to measure urinary cAMP excretion.…”

“…Kidneys were harvested 1 h after application of EX4 (10 g/kg ip), ALG (10 mg/kg ip), or vehicle (0.85% NaCl, 2 l/g body wt). Renal membranes were prepared in the presence of protease and phosphatase inhibitors, as previously described (33,39). Immunoblotting was performed at 4°C overnight with the primary NHE3 Ab (Millipore, Billerica, MA), phosphorylated (S552) NHE3 Ab (Novus Biologicals, Littleton, CO), and phosphorylated (S605) NHE3 Ab (Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:1,000, 1:1,000, and 1:200, respectively.…”