Inducible renal‐tubule insulin receptor (IR) knockout affects sodium transporter/channel expression and activity in a sex‐dependent manner

Abstract

Insulin has been demonstrated to be natriferic in thick ascending limb (TAL), distal convoluted tubule (DCT), and collecting duct (CD). Direct activation of sodium (Na+) transporters and channels by the insulin receptor (IR) likely underlies this effect. To elucidate the role of tubular IR on blood pressure (BP) and apical transporter/channel activity at these sites, we knocked out (KO) IR from the renal tubule, in male (M) and female (F) mice (C57Bl6 background) using a tetracycline‐inducible system. Doxycycline (DOX) was fed to all mice ad libitum (625 mg/kg·diet) for two weeks to induce KO in homozygously IR floxed mice harboring both inducible Cre and paired box gene 8 (PAX8) promoter transgenes. WT mice were negative for Cre, PAX8 or both transgenes. BP and sensitivity to select diuretics was assessed in the pre‐DOX and post‐DOX states. Four weeks post‐DOX, mice were euthanized 4 hours after an acute injection of insulin (0.5 U/kg·bw) and blood and kidneys collected for analysis. Two‐way ANOVA (sex X genotype) revealed DOX marginally reduced kidney weight (p = 0.059) and increased blood potassium (K+, p = 0.02). These differences were abrogated in F. Final blood glucose was also lower in F (p = 0.001) and in KO mice (p = 0.016). Pre‐Dox BP (mm Hg) measured by tail‐cuff plethysmography was similar in M (103.1 ± 3.2) and F (100.2 ± 6.0). DOX caused a fall in MAP (~ 5 mm Hg) in F, but not M KO mice. Natriuretic responses to furosemide (FUR), thiazide (TZD), and benzamil (BNZ) were used as indexes of Na‐K‐2Cl cotransporter (NKCC2), Na‐Cl cotransporter (NCC), and epithelial sodium channel (ENaC) activity, respectively, and were all marginally‐to‐significantly higher in F versus M mice in the pre‐Dox state (18, 300, and 44% higher, respectively), suggesting greater basal activity of these Na+ reabsorptive routes in F (n = 12–17/sex). After DOX, there was a significant interaction (p = 0.010, sex X genotype) for the response to FUR, with M KO responding with an increase, and F KO, with a decrease. The response to TZD tended to increase in the M KO, but decrease in the F KO (relative to same sex WT). The response to BNZ remained higher in the F, and was slightly increased in both sexes by KO (~20–50%). In cortex homogenates (CTXH), western blotting revealed band density for the β‐subunit of IR decreased on average 60% with no sex difference. F had significantly higher band density for α‐ENaC (90‐kDa band), as compared to M (p < 0.0001), and KO led to a significant interactive term in that band density was decreased in F, but increased in M. Both sexes showed reduced band densities for β‐ and γ‐ENaC (85 kDa) and outer medullary NKCC2 in KO. NCC band density was not affected by KO, but significantly higher in F (p < 0.0001). Taken together these results demonstrate differential sensitivity between the sexes to renal tubular IR KO in regard to homeostatic control of Na+ and K+ handling and BP. KO led to modest reductions in kidney weight and transporter/channel expression levels, supporting the role for insulin in growth and maintenance of cell size and differentiation. Little to no reduction in measurable activity could result from compensation, which appeared more effective in males.Support or Funding InformationAmerican Diabetes Association (MF)Georgetown University Medical CenterThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.