Aldosterone requires vasopressin V1a receptors on intercalated cells to mediate acid-base homeostasis

Abstract

Both aldosterone and luminal vasopressin may contribute to the maintenance of acid-base homeostasis, but the functional relationship between these hormones is not well understood. The effects of luminal vasopressin likely result from its interaction with V1a receptors on the luminal membranes of intercalated cells in the collecting duct. Here, we found that mice lacking the V1a receptor exhibit type 4 renal tubular acidosis. The administration of the mineralocorticoid agonist fludrocortisone ameliorated the acidosis by restoring excretion of urinary ammonium via increased expression of Rhcg and H-K-ATPase and decreased expression of H-ATPase. In a cell line of intercalated cells established from transgenic rats expressing the mineralocorticoid and V1a receptors, but not V2 receptors, knockdown of the V1a receptor gene abrogated the effects of aldosterone on H-K-ATPase, Rhcg, and H-ATPase expression. These data suggest that defects in the vasopressin V1a receptor in intercalated cells can cause type 4 renal tubular acidosis and that the tubular effects of aldosterone depend on a functional V1a receptor in the intercalated cells.

Figure 1.

The different effects of acid-load (A–D) and fludrocortisone (E–H) on urinary acid excretion in WT and V1aR^−/− mice. The administration of 0.28 M NH₄Cl decreased the urine pH (A) and increased the titratable acid (B), ammonium (C), and net acid excretion (D) in WT (dotted line) and V1aR^−/− mice (solid line). The increase in urinary ammonium and net acid excretion was greater in WT mice than in V1aR^−/− mice. In contrast, fludrocortisones increased the urine pH (E), ammonium (F), and net acid excretion (G) while decreasing the titratable acid excretion (H). The effects of fludrocortisones were more significant in V1aR^−/− (solid line) than in WT mice (dotted line). Mean ± SEM. n = 4 to 6. *P < 0.05 and **P < 0.01 versus WT mice; ††P < 0.05 versus day 1.

Figure 2.

Smaller physiologic effects of fludrocortisone (flud) on acid-base–related transporters in the kidney of V1aR^−/− mice. V1aR^−/− mice showed higher expression levels of H-ATPase and lower expression levels of H-K-ATPase and Rhcg than WT mice. Fludrocortisone decreased H-ATPase expression, whereas it increased H-K-ATPase, Rhcg, and AE1expression in WT mice. Effects of fludrocortisones were smaller in V1aR^−/− mice. (A) H-ATPase α (116 kD), (B) H-K-ATPase β (67 kD), (C) Rhcg (58 kD), (D) AE1 (96 kD), (E) pendrin (100 kD), and (F) β actin (42 kD). Expression of each transporter in sham of WT was considered as 1. *P < 0.05 and **P < 0.01 versus sham of the WT or V1aR^−/−; ††P < 0.05 versus sham of the WT. Means ± SEM. n = 4 to 7.

Figure 3.

IN-IC cells express V1aR and acid-base-related transporters but lack V2R (A) Photograph of IN-IC cells. Homogeneous cells with large nucleus are observed. (B) Expression of vasopressin and aldosterone-related receptors, channels and transporters in the IN-IC cells (top panel), and rat renal medulla (bottom panel). Although all of the examined receptors, transporters,and channels are present in rat renal medulla, V2R, aquaporin 2,and ENaC β and γ were not expressed in the IN-IC cells. Lane 1, size marker; lane 2, GAPDH (308 bp); lane 3, V1aR (425 bp); lane 4, V2R (578 bp); lane 5, aquaporin 2 (553 bp); lane 6, ENaC α (647 bp); lane 7, ENaC β (619 bp); lane 8, ENaCγ (561 bp); lane 9, size marker; lane 10, H-ATPase α1 (Hα1, 510 bp); lane 11, H-ATPase β1 (Hβ1, 503 bp); lane 12, H-ATPase β2 (Hβ2, 540 bp); lane 13, H-K-ATPase α1 (HKα1, 438 bp); lane 14, H-K-ATPase α2 (HKα2, 472 bp); lane 15, Rhcg (307 bp); lane 16, AE1 (178 bp); lane 17, pendrin (488 bp); lane 18, Foxi 1 (516 bp); lane 19, mineralocorticoid receptor (MR, 380 bp); lane 20, 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2, 361 bp). (C) Vasopressin decreased V1aR mRNA expression in IN-IC cells. Expression of mRNA without vasopressin was considered as 1. (D) Aldosterone dose-dependently decreased V1aR mRNA expression in IN-IC cells. Expression of mRNA without aldosterone was considered as 1. *P < 0.05 versus without vasopressin (C) or aldosterone (D). Means ± SEM. n = 4 to 6.

Figure 4.

Stimulative effects of arginine vasopressin and inhibitory effects of V1aR gene knockdown on the expression of acid-base–related transporters in IN-IC cells. Vasopressin increased the expression of H-ATPase, H-K-ATPase, and Rhcg in a dose-dependent manner. The effects of vasopression were almost completely abolished by a V1aR RNA interference-mediated knockdown experiment. Vasopressin did not affect the expression level of AE1 and pendrin. (A) H-ATPase α (116 kD), (B) H-K-ATPase β (67 kD), (C) Rhcg (58 kD), (D) AE1 (96 kD), (E) pendrin (100 kD), and (F) β actin (42 kD). Expression of each transporter without vasopressin in the absence or presence of siRNA was considered as 1. *P < 0.05 versus the expression in the absence of siRNA. Mean ± SEM. n = 5 to 9.

Figure 5.

Stimulative effects of aldosterone and inhibitory effects of V1aR gene knockdown on the expression of acid-base–related transporters in the IN-IC cells. Aldosterone decreased H-ATPase expression in a dose-dependent fashion and increased the expression of H-K-ATPase, and Rhcg. V1aR gene knockdown abolished the effects of aldosterone on H-K-ATPase and Rhcg and largely inhibited its effect on the expression of H-ATPase. Although aldosterone slightly stimulated the expression of AE1 and pendrin, knockdown of the V1aR did not affect the effects of aldosterone administration. (A) H-ATPase α (116 kD), (B) H-K-ATPase β (67 kD), (C) Rhcg (58 kD), (D) AE1 (96 kD), (E) pendrin (100 kD), and (F) β actin (42 kD). Expression of each transporter without aldosterone in the absence or presence of siRNA was considered as 1. *P < 0.05 versus expression in the absence of siRNA. Mean ± SEM. n = 5 to 9.

Figure 6.

Schematic presentation of vasopressin and aldosterone-induced acid excretion in the intercalated cell. Vasopressin-mediated activation of the V1a receptor stimulates proton secretion by H-ATPase and H-K-ATPase and NH₃ excretion by Rhcg. In contrast, aldosterone via mineralocorticoid receptor stimulated NH₃ excretion via Rhcg and proton secretion by H-K-ATPase, but it reduces proton secretion via H-ATPase. The presence of V1aR is essential for aldosterone-induced effects on H-ATPase, H-K-ATPase, and Rhcg. Bicarbonate excretion via AE1 is also stimulated by aldosterone. The presence of the vasopressin V1a receptor is essential for the effects of aldosterone.