GSK3beta mediates renal response to vasopressin by modulating adenylate cyclase activity

Abstract

Glycogen synthase kinase 3beta (GSK3beta), a serine/threonine protein kinase, is a key target of drug discovery in several diseases, including diabetes and Alzheimer disease. Because lithium, a potent inhibitor of GSK3beta, causes nephrogenic diabetes insipidus, GSK3beta may play a crucial role in regulating water homeostasis. We developed renal collecting duct-specific GSK3beta knockout mice to determine whether deletion of GSK3beta affects arginine vasopressin-dependent renal water reabsorption. Although only mildly polyuric under normal conditions, knockout mice exhibited an impaired urinary concentrating ability in response to water deprivation or treatment with a vasopressin analogue. The knockout mice had reduced levels of mRNA, protein, and membrane localization of the vasopressin-responsive water channel aquaporin 2 compared with wild-type mice. The knockout mice also expressed lower levels of pS256-AQP2, a phosphorylated form crucial for membrane trafficking. Levels of cAMP, a major regulator of aquaporin 2 expression and trafficking, were also lower in the knockout mice. Both GSK3beta gene deletion and pharmacologic inhibition of GSK3beta reduced adenylate cyclase activity. In summary, GSK3beta inactivation or deletion reduces aquaporin 2 expression by modulating adenylate cyclase activity and cAMP generation, thereby impairing responses to vasopressin in the renal collecting duct.

Figure 1.

These images show the generation of the renal CD-specific GSK3β KO mice. (A) Immunostaining for GSK3β in renal papillae of WT and KO mice. Original magnification, ×20. (B) Western blot analysis shows reduced GSK3β and not GSK3α levels in acutely isolated CDs from the renal inner medulla and papilla. ^#P < 0.0001. (C) Agarose gel showing PCR products of genomic DNA obtained from renal CDs. KO mice show smaller bands compared with the WT mice (floxed), owing to recombination resulting in deletion of GSK3β.

Figure 2.

GSK3β gene deletion reduced urine osmolality after water deprivation. Urine osmolality values of urine collected during 18 hours of water deprivation. *P < 0.001, n = 8.

Figure 3.

GSK3β gene deletion reduced renal AQP2 levels. (A) Quantitative real-time PCR of renal mRNA levels under basal conditions or after 18-hour water deprivation. ^#P < 0.0001. (B) Western blot analysis for AQP2 expression in the renal papillae at baseline or in water-deprived mice. 55 μg of protein was loaded for basal and 40 μg for water-deprived samples. Densitometry of AQP2 band density. *P < 0.001, ^#P < 0.0001.

Figure 4.

Water-deprived KO and WT mouse kidneys were immunostained for AQP2 and pS256-AQP2. AQP2 labeling in the CDs of inner medulla (A) or cortex (B) of WT mice shows intense labeling and apical localization compared with the inner medulla (C) or cortex (D) of KO mice. pS256-AQP2 labeling of inner medulla (E) or cortex (F) of WT mice shows apical labeling, whereas in the inner medulla (G) or cortex (H) of KO mice, labeling is less intense and more cytoplasmic. Original magnification, ×40.

Figure 5.

GSK3β gene deletion reduced dDAVP-induced urine-concentrating capacity in mice. (a) WT and KO mice were injected with dDAVP (1 μM/kg body wt; intraperitoneally) and spot urine collected. *P < 0.001; n = 8. (b) Δ change in urine osmolality of dDAVP-treated mice compared with basal level. (c) pS256-AQP2 levels in the inner medulla and papilla of WT and KO mice 1 hour after dDAVP treatment. ^@P < 0.01.

Figure 6.

Renal cAMP levels are reduced in KO mice. (A) cAMP levels were measured in the urine of WT and KO mice that were water deprived for 18 hours. ^$P < 0.05. (B) cAMP levels in renal papillae. Renal papillae were isolated, finely minced, and treated for 20 minutes with IBMX followed by dDAVP (1 μM) or forskolin (1 μM) for an additional 10 minutes. ^@P < 0.01; *P < 0.001; WT versus KO mice; n = 8.

Figure 7.

GSK3 inhibition reduced cAMP generation in WT C57BL/6J mice and cultured IMCD3 cells. cAMP levels in response to dDAVP (1 μM) or forskolin (1 μM) were measured in the renal papillae of normal C57BL/6J mice (A) or IMCD cells (B) pretreated with vehicle or SB216763 (5 μM). Mice n = 7; IMCD cells n = 6. ^@P < 0.01; *P < 0.001 vehicle versus SB216763.

Figure 8.

GSK3β gene deletion or inhibition reduced adenylate cyclase activity. (A) Adenylate cyclase activity in response to dDAVP was determined in the intact membrane preparations obtained from renal inner medulla and papillae of WT or KO mice (*P < 0.001 WT versus KO mice treated with dDAVP; n = 5) or (B) forskolin (P < 0.0001; WT versus KO mice, determined by ANOVA; n = 5). (C) Adenylate cyclase activity in the membrane preparation of the renal papillae of WT mice treated with GSK3β inhibitor SB216763. (D) Adenylate cyclase 3 or 5/6 expression in renal medullary lysates of water-deprived WT and KO mice.