Reduced ENaC activity and blood pressure in mice with genetic knockout of the insulin receptor in the renal collecting duct.

To elucidate the role of the insulin receptor (IR) in collecting duct (CD), we bred mice with IR selectively deleted from CD principal cells using an aquaporin-2 promoter to drive Cre-recombinase expression. Young, adult male knockout (KO) mice had altered plasma and electrolyte homeostasis under high- (HS) and low-sodium (LS) diets, relative to wild-type (WT) littermates. One week of LS feeding led to a significant reduction in urine potassium (K(+)) and sodium (Na(+)) excretion in KO, and a reduction in the ratio of Na(+) to chloride (Cl(-)) in plasma, relative to WT. HS diet (1 wk) increased plasma K(+) and reduced urine Na(+) to Cl(-) ratio in the KO. Furthermore, KO mice had a significantly (P = 0.025) blunted natriuretic response to benzamil, an epithelial sodium channel (ENaC) antagonist. Western blotting of cortex homogenates revealed modestly, but significantly (∼15%), lower band density for the ß-subunit of ENaC in the KO vs. WT mice, with no differences for the α- or γ-subunits. Moreover, blood pressure (BP), measured by radiotelemetry, was significantly lower in KO vs. WT mice under basal conditions (mmHg): 112 ± 5 (WT), 104 ± 2 (KO), P = 0.023. Chronic insulin infusion reduced heart rate in the WT, but not in the KO, and modestly reduced BP in the WT only. Overall, these results support a fundamental role for insulin through its classic receptor in the modulation of electrolyte homeostasis and BP.

Salt sensitivity of nitric oxide generation and blood pressure in mice with targeted knockout of the insulin receptor from the renal tubule.

To elucidate the role of the insulin receptor (IR) on kidney nitric oxide generation and blood pressure (BP) control, we generated mice with targeted deletion of renal tubule IR using loxP recombination driven by a Ksp-cadherin promoter. Male knockout (KO) and wild-type (WT) littermates (~4 mo old) were transitioned through three 1-wk treatments: 1) low-NaCl diet (0.085%); 2) high-NaCl diet (HS; 5%); and 3) HS diet plus 3 mM tempol, a superoxide dismutase mimetic, in the drinking water. Mice were then switched to medium-NaCl (0.5%) diet for 5 days and kidneys harvested under pentobarbital anesthesia. Twenty-four-hour urinary nitrates plus nitrites were significantly higher in the WT mice under HS (2,067 ± 280 vs. 1,550 ± 230 nmol/day in WT and KO, respectively, P < 0.05). Tempol attenuated genotype differences in urinary nitrates plus nitrites. A rise in BP with HS was observed only in KO mice and not affected by tempol (mean arterial pressure, dark period, HS, 106 ± 5 vs. 119 ± 4 mmHg, for WT and KO, respectively, P < 0.05). Renal outer medullary protein levels of nitric oxide synthase (NOS) isoforms by Western blot (NOS1-3 and phosphorylated-S1177-NOS3) revealed significantly lower band density for NOS1 (130-kDa isoform) in the KO mice. A second study, when mice were euthanized under HS conditions, confirmed significantly lower NOS1 (130 kDa) in the KO, with an even more substantial (>50%) reduction of the 160-kDa NOS1 isoform. These studies suggest that the loss of renal IR signaling impairs renal nitric oxide production. This may be important in BP control, especially in insulin-resistant states, such as the metabolic syndrome.