Decreased bone density and increased phosphaturia in gene-targeted mice lacking functional serum- and glucocorticoid-inducible kinase 3

Abstract

Insulin and growth factors activate the phosphatidylinositide-3-kinase pathway, leading to stimulation of several kinases including serum- and glucocorticoid-inducible kinase isoform SGK3, a transport regulating kinase. Here, we explored the contribution of SGK3 to the regulation of renal tubular phosphate transport. Coexpression of SGK3 and sodium-phosphate cotransporter IIa significantly enhanced the phosphate-induced current in Xenopus oocytes. In sgk3 knockout and wild-type mice on a standard diet, fluid intake, glomerular filtration and urine flow rates, and urinary calcium ion excretion were similar. However, fractional urinary phosphate excretion was slightly but significantly larger in the knockout than in wild-type mice. Plasma calcium ion, phosphate concentration, and plasma parathyroid hormone levels were not significantly different between the two genotypes, but plasma calcitriol and fibroblast growth factor 23 concentrations were significantly lower in the knockout than in wild-type mice. Moreover, bone density was significantly lower in the knockouts than in wild-type mice. Histological analysis of the femur did not show any differences in cortical bone but there was slightly less prominent trabecular bone in sgk3 knockout mice. Thus, SGK3 has a subtle but significant role in the regulation of renal tubular phosphate transport and bone density.

Figure 1. Coexpression of SGK3 stimulates electrogenic phosphate transport in NaPiIIa-expressing Xenopus oocytes

(a) Arithmetic means±s.e.m. (n = 11–18) of phosphate (3 mmol/l)-induced currents (I_Pi) in Xenopus oocytes injected with water (H₂O) or complementary RNA (cRNA) encoding SGK3 or NaPiIIa, or cRNA encoding both SGK3 and NaPiIIa. *Indicates significant difference from absence of cRNA encoding NaPiIIa (p<0.05). ^###Indicates significant difference from absence of cRNA encoding SGK3 (P<0.001). (b) Arithmetic means±s.e.m. (n = 42–60) of the normalized chemiluminescence intensity of NaPiIIa expression in Xenopus oocytes injected with H₂O (left bar), with cRNA encoding (middle bar), or with cRNA encoding both, NaPiIIa and SGK3 (right bar). *Indicates statistically significant difference from absence of cRNA (P<0.05). ^###Indicates difference from absence of SGK3 cRNA (P<0.001). RLU, relative luminescence.

Figure 2. Protein abundance of renal sodium-dependent phosphate cotransporters and sodium-dependent phosphate transport activity in brush border membrane vesicles (BBMVs) of SGK3 knockout mice (sgk3^KO) and wild-type mice (sgk3^WT) mice

(a) Original western blots for NaPiIIa, NaPiIIc, and Pit2. All membranes were stripped and reprobed for β-actin to control for loading. (b) Arithmetic means±s.e.m. (n = 5–6 each group) of the sodium-dependent transport rates into isolated BBMVs after 1 min in the absence (upper bars) and presence (lower bars) of 6 mmol/l phosphonoformic acid (PFA) to block phosphate transport mediated by SLC34 family members. (c) Arithmetic means±s.e.m. (n = 6 each group) of relative band density of NaPilla (left panel), NaPillc (middle panel), and Pit2 (right panel).

Figure 3. Fractional excretion of calcium and phosphate in SGK3 knockout mice (sgk3^KO) and wild-type mice (sgk3^WT)

(a) Arithmetic means±s.e.m. (n = 10–19 each group) of plasma calcium (left panel) and phosphate (right panel) concentration in sgk3^KO (closed bars) and sgk3^WT (open bars) mice. (b) Arithmetic means±s.e.m. (n = 10–19 each group) of fractional urinary calcium (left panel) and phosphate (right panel) excretion in sgk3^KO (closed bars) and sgk3^WT (open bars) mice. *P<0.05 versus respective value of sgk3^WT mice. FE, fractional excretion.

Figure 4. Plasma parathyroid hormone (PTH), FGF23, and calcitriol (1,25(OH)₂D₃) concentrations in SGK3 knockout mice (sgk3^KO) and wild-type mice (sgk3^WT)

Arithmetic means±s.e.m. of plasma PTH (a; n = 10–12 each group), 10 each group), 1,25(OH)₂D₃ (b; n = and FGF23 (c; n = 8 in each group) concentration in sgk3^KO (closed bars) and sgk3^WT (open bars) mice. *P<0.05 versus respective value of sgk3^WT mice.

Figure 5. Bone density and histological analysis of femur bones from SGK3 knockout mice (sgk3^KO) and wild-type mice (sgk3^WT)

(a) Arithmetic means±s.e.m. (n = 6 each group) of bone density in sgk3^KO (closed bar) and sgk3^WT (open bar) mice. *P<0.05 versus respective value of sgk3^WT mice. (b) Histology of bone in the sgk3^KO and sgk3^WT mice representative for n = 5–6 mice each group. The cortical bone is similar but the trabecular bone is less prominent in sgk3^KO than in sgk3^WT mice.