Adenylyl cyclase VI mediates vasopressin-stimulated ENaC activity

Abstract

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 α and γ 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 vasopressin-stimulated 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.

Figure 1.

Effect of CD AC6 KO on daily urinary Na excretion (UNaV). Control and CD AC6 KO mice are given low (0.01%), normal (0.3%), or high (3.15%) Na diets. Data are shown as UNaV on days 2 (A), 3 (B), and 7 (C) after starting each of the different diets (n=8–16 both groups). Not all days are from the same groups of rats, so data are not shown as a line graph comparing values between days.

Figure 2.

Effect of CD AC6 KO on arterial pressure and pulse. Control and CD AC6 KO mice are given low (0.01%), normal (0.3%), or high (3.15%) Na diets. Systolic BP (A), diastolic BP (B), and pulse rate (C) are determined by radiotelemetry on each diet (n=12 both groups). Hemodynamic values are determined daily for 7 days on each diet and do not vary within the 7-day period; the values shown are the composite of the 7 days on each diet.

Figure 3.

Effect of CD AC6 KO on plasma renin concentration and urinary aldosterone excretion. Control and CD AC6 KO mice are given low (0.15%), normal (0.3%), or high (3.15%) Na diets. PRC (A) or urinary aldosterone excretion (B) is determined after 7 days on each diet (n=12 both groups). *P<0.05 versus control animals on the same diet.

Figure 4.

Effect of valsartan (5 mg/kg per day) on mean arterial pressure in CD AC6 KO and control mice. Arterial pressure is determined by radiotelemetry for 5 days on a normal Na diet, followed by subcutaneous insertion of an osmotic minipump containing valsartan. Two days are allowed for recovery, followed by arterial pressure determination for the next 5 days (n=5 each group). ^†P<0.05 versus control values on same day.

Figure 5.

Effect of CD AC6 KO on urinary AVP excretion. Control and CD AC6 KO mice are given low (0.15%), normal (0.3%), or high (3.15%) Na diets. Urine AVP excretion (24 h) is determined after 7 days on each diet (n=12 both groups).

Figure 6.

Effect of CD AC6 KO on renal cortical ENaC isoform mRNA content. ENaC isoform mRNA is determined in control and CD AC6 KO mice eating a normal (0.3%) Na diet (n=6 both groups). ENaC isoform mRNA is determined by real-time PCR; data are expressed as CD AC6 KO as a percentage of control values. All PCR data are normalized to glyceraldehyde-3-phosphate dehydrogenase to control for total RNA analyzed. ^‡P<0.05 versus control.

Figure 7.

AC6 knockout in the CD reduces ENaC mRNA and protein expression. (A) Effect of CD AC6 KO on renal cortical ENaC isoform protein content. ENaC isoform protein content is determined in control and CD AC6 KO mice eating a normal (0.3%) Na diet (n=6 both groups). (B) ENaC protein expression is determined in total renal cortex and (C) in renal cortical plasma membrane-enriched fractions; data are expressed as CD AC KO as a percentage of control values and are normalized to β-actin to control for loading. Blots are analyzed by densitometry. The larger molecular mass bands for ENaC-α and ENaC-γ are 88 and 85 kD, respectively; the smaller molecular mass bands for ENaC-α and ENaC-γ are 30 and 70–80 kD, respectively. ^‡P<0.05 versus control.

Figure 8.

AC6 is necessary for AVP stimulation of ENaC. Representative gap-free current traces from cell-attached patches made on the apical membrane of principal cells in split-open CDs from control (A and B) and CD AC6 KO (C and D) mice on a normal (0.3%) Na diet before (A and C) and after (B and D) treatment with 1 μM AVP for 30 minutes. The closed state (c) is denoted with a dashed line. Inward current is downward. The holding potential for these patches is −V_p = −60 mV. Summary graphs of NP_o (E), N (F), and P_o (G) for ENaC show control (gray) and CD AC6 KO (black) mice in the absence (hatched bars) and presence (filled bars) of AVP. ^§Significantly greater compared with the absence of AVP within the same group (P<0.05). **Significantly less than control under identical conditions (P<0.05). Sample sizes are shown in Table 1.