Arginase-II negatively regulates renal aquaporin-2 and water reabsorption

Abstract

Type-II l-arginine:ureahydrolase, arginase-II (Arg-II), is abundantly expressed in the kidney. The physiologic role played by Arg-II in the kidney remains unknown. Herein, we report that in mice that are deficient in Arg-II (Arg-II^-/-), total and membrane-associated aquaporin-2 (AQP2) protein levels were significantly higher compared with wild-type (WT) controls. Water deprivation enhanced Arg-II expression, AQP2 levels, and membrane association in collecting ducts. Effects of water deprivation on AQP2 were stronger in Arg-II^-/- mice than in WT mice. Accordingly, a decrease in urine volume and an increase in urine osmolality under water deprivation were more pronounced in Arg-II^-/- mice than in WT mice, which correlated with a weaker increase in plasma osmolality in Arg-II^-/- mice. There was no difference in vasopressin release under water deprivation conditions between either genotype of mice. Although total AQP2 and phosphorylated AQP2-S256 levels (mediated by PKA) in kidneys under water deprivation conditions were significantly higher in Arg-II^-/- mice compared with WT animals, there is no difference in the ratio of AQP2-S256:AQP2. In cultured mouse collecting duct principal mCCD_cl1 cells, expression of both Arg-II and AQP2 were enhanced by the vasopressin type 2 receptor agonist, desamino- d-arginine vasopressin (dDAVP). Silencing Arg-II enhanced the expression and membrane association of AQP2 by dDAVP without influencing cAMP levels. In conclusion, in vivo and in vitro experiments demonstrate that Arg-II negatively regulates AQP2 and the urine-concentrating capability in kidneys via a mechanism that is not associated with the modulation of the cAMP pathway.-Huang, J., Montani, J.-P., Verrey, F., Feraille, E., Ming, X.-F., Yang, Z. Arginase-II negatively regulates renal aquaporin-2 and water reabsorption.

Keywords: collecting duct; copeptin; osmolality; urine; vasopressin.

Figure 1

Ablation of Arg-II enhances total and membrane-associated AQP2 levels in the kidney under WD conditions in mice. Total kidney lysates and crude membrane fractions were prepared from WT and Arg-II^−/− mice under either basal or WD conditions for 24 h. Total kidney lysates (40 µg; A), and crude membrane fractions (15 μg; B) were loaded and subjected to immunoblotting analysis of AQP2. Tubulin and Na⁺/K⁺-ATPase were used as loading control for total kidney proteins and crude membrane proteins, respectively. Quantifications of immunoblotting signals (n = 6 animals in each group) are presented as dot plots in the right panels. Basal, basal condition; KO, Arg-II^−/−. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2

WD enhances Arg-II and AQP2 expression in the collecting ducts of inner medulla. Renal paraffin sections were prepared from WT and Arg-II^−/− mice under either basal or WD conditions for 24 h and subjected to immunofluorescence staining of AQP2 (red) and Arg-II (green) followed by counterstaining of the nuclei with DAPI (blue). Representative images are from 4 independent series of experiments. WT-Basal, WT-basal condition; KO-Basal, Arg-II^−/−-basal condition; KO-WD, Arg-II^−/−-WD.

Figure 3

WD does not change Arg-II expression in proximal straight tubules. A) Kidney outer medulla paraffin sections were prepared from WT and Arg-II^−/− mice under either basal or WD conditions for 24 h and subjected to immunofluorescence staining of AQP2 (red) and Arg-II (green). Shown are representative images obtained from 4 independent series of mice. B, C) Immunoblotting analysis of Arg-II in inner medulla (50 µg/lane, n = 4 animals in each group; B) and kidney tissue without inner medulla (40 µg/lane, n = 4 animals in each group; C). Tubulin was used as loading control. Basal, basal condition; KO, Arg-II^−/−. *P < 0.05.

Figure 4

Water balance in mice under basal and WD conditions. Metabolic cage experiments were performed as described in Materials and Methods. Water intake (A), urine volume (B), urine osmolality (C), plasma copeptin (D), plasma osmolality (E), and plasma Na⁺ concentration (F) were measured in WT and KO mice under basal or WD conditions. Data are presented from 8 animals in each group. Basal, basal condition; KO, Arg-II^−/−. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5

Silencing Arg-II enhances dDAVP-induced AQP2 expression. mCCD_cl1 cells were transduced with rAd/U6-LacZ^shRNA as control or rAd/U6-Arg-II^shRNA to silence the Arg-II gene. Forty-eight hours post-transduction, cells were serum starved overnight, then incubated in the absence or presence of 10⁻⁸ M dDAVP for 24 h. A) Immunoblotting analysis of Arg-II and AQP2 was performed with total cell lysates (40 µg of total cell lysate/lane). Lysates of total kidney and inner medulla were used as positive control (pos) for Arg-II and AQP2, respectively. Tubulin served as loading control. Shown are representative blots of Arg-II and AQP2 expression. B, C) Quantifications for Arg-II (B) and AQP2 (C) immunoblotting signals are shown in dot plots. Data are presented from 5 independent experiments. N.d., not detectable. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6

WD-induced AQP2 Ser256 phosphorylation is enhanced in Arg-II^−/− mice. Total kidney lysates were prepared from WT and Arg-II^−/− mice under WD conditions for 24 h. Total kidney lysates (40 μg) were loaded and subjected to immunoblotting analysis of Arg-II, pSer256-AQP2, and AQP2. Tubulin was used as loading control. Quantifications of immunoblotting signals (n = 3 animals in each group) and the ratio of pSer256-AQP2 to AQP2 are presented as dot plots in the right panels. KO-WD, Arg-II^−/−-WD. *P < 0.05, **P < 0.01.

Figure 7

No effect of Arg-II silencing on cAMP stimulated by dDAVP was noted. There was no significant difference in the elevation of cAMP concentration in response to dDAVP after knockdown of Arg-II in principal cells. mCCD_cl1 cells were plated onto 96-well plates and grown to confluence, then transduced with rAd/U6-LacZ^shRNA as control or rAd/U6-Arg-II^shRNA to silence Arg-II gene. Forty-eight hours post-transduction, cells were serum starved overnight, then incubated with or without 10⁻⁸ M dDAVP for the last 0.5, 4, or 24 h. During the last 30 min, 0.5 mM of the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX), was added. Cells were then lysed and intracellular cAMP was measured. N.s., not significant. *P < 0.001 compared with LacZ^shRNA without dDAVP, ^#P < 0.001 compared with Arg-II^shRNA without dDAVP.

Figure 8

Inhibition of PKA prevents the augmentation of dDAVP-induced AQP2 by Arg-II silencing. mCCD_cl1 cells were transduced with rAd/U6-LacZ^shRNA as control or rAd/U6-Arg-II^shRNA to silence Arg-II gene. Forty-eight hours post-transduction, cells were serum starved overnight, then incubated in the absence or presence of 10⁻⁸ M dDAVP for 24 h. For inhibition of PKA, cells were pretreated with 20 µM PKA inhibitor (PKi), a selective PKA inhibitor, for 1 h, then challenged continuously with PKi for 24 h. Immunoblotting analyses of AQP2 in total cell lysates (40 µg/lane; A) and AQP2 in crude membrane fractions (15 µg/lane; B) were performed. Tubulin and Na⁺/K⁺-ATPase served as loading control for total kidney lysates and crude membrane fractions, respectively. Shown are representative blots from 4 independent experiments. Data are expressed as fold change to LacZ^shRNA plus dDAVP group. *P < 0.05, **P < 0.01, ***P < 0.001.