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. 2013 Jul;27(7):2723-32.
doi: 10.1096/fj.12-223792. Epub 2013 Apr 4.

Regulation of ENaC in mice lacking renal insulin receptors in the collecting duct

Tengis S Pavlov  1 , Daria V Ilatovskaya, Vladislav Levchenko, Lijun Li, Carolyn M Ecelbarger, Alexander Staruschenko
Affiliations

Regulation of ENaC in mice lacking renal insulin receptors in the collecting duct

Tengis S Pavlov et al. FASEB J. 2013 Jul.

Abstract

The epithelial sodium channel (ENaC) is one of the central effectors involved in regulation of salt and water homeostasis in the kidney. To study mechanisms of ENaC regulation, we generated knockout mice lacking the insulin receptor (InsR KO) specifically in the collecting duct principal cells. Single-channel analysis in freshly isolated split-open tubules demonstrated that the InsR-KO mice have significantly lower ENaC activity compared to their wild-type (C57BL/6J) littermates when animals were fed either normal or sodium-deficient diets. Immunohistochemical and Western blot assays demonstrated no significant changes in expression of ENaC subunits in InsR-KO mice compared to wild-type littermates. Insulin treatment caused greater ENaC activity in split-open tubules isolated from wild-type mice but did not have this effect in the InsR-KO mice. Thus, these results suggest that insulin increases ENaC activity via its own receptor affecting the channel open probability. To further determine the mechanism of the action of insulin on ENaC, we used mouse mpkCCDc14 principal cells. Insulin significantly augmented amiloride-sensitive transepithelial flux in these cells. Pretreatment of the mpkCCDc14 cells with phosphatidylinositol 3-kinase (LY294002; 10 μM) or mTOR (PP242; 100 nM) inhibitors precluded this effect. This study provides new information about the importance of insulin receptors expressed in collecting duct principal cells for ENaC activity.

Keywords: aldosterone-sensitive distal nephron; kidney; mTOR.

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Figures

Figure 1.
Figure 1.
Insulin increases ENaC Po in freshly isolated split-open CCDs of WT (C57BL/6J) mice fed a sodium-deficient diet for 1 wk. A) Representative current traces from a cell-attached patch in the control and after application of 100 nM insulin. B) Summary graph of ENaC Po changes in response to application of 0, 20, 40, and 100 nM insulin. For each individual experiment, the changes were normalized to ENaC Po in the control; fold increase is shown. Dashed lines are the best fit of the Hill equation. N varies from 5 to 14 experiments for each insulin concentration.
Figure 2.
Figure 2.
Characterization of the principal cell InsR KO. A) Western blots of cortex and inner medullary (as indicated) whole-cell homogenates (20 μg protein/lane) from 2 WT and 2 KO mice probed with an antibody against InsR (α subunit). Densitometry of major 125-kDa band was significantly reduced in the inner medulla from KO mice (n=6 mice/genotype). *P < 0.05; unpaired t test. B) Immunofluorescence of InsR (β subunit, green) using AQP2 (red) as a marker for collecting duct principal cells. Bottom panel shows absent or reduced basolateral staining for IR in principal (yellow arrows) but not in intercalated cells (pink arrows) in the KO vs. WT mice. C) Body weight of WT and KO mice at 13 wk of age was not different. D) Blood pressure (measured by radiotelemetry) was modestly lower in KO mice under basal dietary conditions (significant for systolic, n=8 mice/group). *P < 0.05.
Figure 3.
Figure 3.
ENaC activity is down-regulated in CCDs isolated from InsR-KO mice. A) Representative current traces from cell-attached patches containing ENaC and recorded from the apical membrane of split-open CCD cells of WT and InsR-KO mice fed Na+-deficient (<0.01%) diet for 1 wk. Holding potential is −60 mV. B) Summary graphs of ENaC NPo in cell-attached patches. *P < 0.05 vs. WT littermates.
Figure 4.
Figure 4.
KO of InsR does not affect expression profiles of ENaC subunits in the kidney cortex. Western blot analysis of α-ENaC (A), β-ENaC (B), and γ-ENaC (C) subunit expression in mouse kidney cortex. Cell lysates were collected from InsR-KO mice and their WT littermates and analyzed using anti-ENaC antibodies; equal loading was verified by anti-β-actin antibodies. Summary graphs (right panels) represent densitometric analysis of relative ENaC subunit abundance.
Figure 5.
Figure 5.
Expression and localization of ENaC subunits in the kidney from the InsR-KO mice and their WT littermates. Representative immunohistochemical staining for ENaC subunits in the kidney cortical sections of InsR-KO mice and their WT littermates at ×40 view. Top panels show negative control tissue stained with secondary antibodies in the absence of primary antibodies. Scale bar = 50 μm. Note the edges of the kidney and glomeruli, confirming the cortex localization.
Figure 6.
Figure 6.
ENaC activity and expression in CCDs isolated from InsR-KO mice fed a normal-salt (0.49%) diet. A, B) Representative current traces (A) and summary graphs of ENaC NPo (B) from cell-attached patches containing ENaC and recorded from the apical membrane of split-open CCD cells of WT and InsR-KO mice fed normal-Na+ diet. Holding potential is −60 mV. *P < 0.05 vs. WT littermates. C) Representative immunohistochemical staining for ENaC subunits in the kidney cortical sections of InsR-KO and their WT littermate mice at ×40 view. Scale bar = 50 μm. D) Summary graphs of Western blot analysis of α-ENaC, β-ENaC, and γ-ENaC subunit expression in mouse kidney cortex. Cell lysates were collected from InsR-KO mice and their WT littermates and analyzed using anti-ENaC antibodies; equal loading was verified by anti-β-actin antibodies.
Figure 7.
Figure 7.
Insulin acutely increases ENaC activity in CCDs via InsR. A, C) Effect of insulin on ENaC activity in freshly isolated CCDs of WT (A) and InsR-KO mice (C). Continuous current traces from representative cell-attached patches were recorded on the apical membrane of principal cells before and after treatment with insulin (100 nM). Areas before (I) and after (II) treatment are shown at middle and bottom with an expanded time scale. These patches were held at a −60 mV test potential during the course of the experiment. c, closed current level; on, open current level. B, D) Summary graphs of ENaC NPo changes from WT (B) and InsR-KO mice (D) in response to insulin application in patch-clamp experiments similar to those shown in panels A and C. *P < 0.05 vs. before insulin application.
Figure 8.
Figure 8.
Inhibition of PI3-kinase prevents effect of insulin on ENaC activity in mpkCCDc14 cells. Pretreatment with LY29004, a PI3-kinase inhibitor, prevents ability of insulin to increase ENaC activity in mpkCCDc14 cell monolayer. Cells were serum starved overnight, and measurements were performed as described in Materials and Methods. Insulin (20 nM) was added basolaterally at time 0. Cells were pretreated with 10 μM of LY294002 or LY303511 (inactive negative control compound for LY29004) 30 min before the application of insulin or vehicle. Amiloride (10 μM) was added to the apical membrane at the end of experiment.
Figure 9.
Figure 9.
Inhibition of mTOR prevents effect of insulin on ENaC activity in mpkCCDc14 cells. A) Time course of relative Na+ transport across monolayers of mpkCCDc14 cells in the absence and presence of treatment with various concentrations of PP242, an mTOR inhibitor. Values are means ± sem; n =5–12/concentration. PP242 and vehicle (control) were added bilaterally at time 0, and current was normalized to the starting level. Amiloride (10 μM) was added to the apical membrane at the end of experiment. B) Dose-response curve for PP242 effect on relative current after 8 h treatment. Dashed line is the best fit of the Hill equation, yielding IC50 = 198.0 ± 34.1 nM. C) Pretreatment with PP242 prevents ability of insulin to increase ENaC activity in mpkCCDc14 cell monolayer. Cells were serum starved overnight, and measurements were performed as described in Materials and Methods. Insulin (20 nM) was added basolaterally at time 0. PP242 at concentration of 100 nM was added bilaterally either in presence or absence of insulin at time 0.
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References

    1. Staruschenko A. (2012) Regulation of transport in the connecting tubule and cortical collecting duct. Compr. Physiol. 2, 1541–1584 - PMC - PubMed
    1. Pavlov T. S., Imig J. D., Staruschenko A. (2010) Regulation of ENaC-mediated sodium reabsorption by peroxisome proliferator-activated receptors. PPAR Res. 2010, 703735. - PMC - PubMed
    1. Guan Y., Hao C., Cha D. R., Rao R., Lu W., Kohan D. E., Magnuson M. A., Redha R., Zhang Y., Breyer M. D. (2005) Thiazolidinediones expand body fluid volume through PPARgamma stimulation of ENaC-mediated renal salt absorption. Nat. Med. 11, 861–866 - PubMed
    1. Kotchen T. A., Zhang H. Y., Covelli M., Blehschmidt N. (1991) Insulin resistance and blood pressure in Dahl rats and in one-kidney, one-clip hypertensive rats. Am. J. Physiol. 261, E692–E697 - PubMed
    1. Tiwari S., Riazi S., Ecelbarger C. A. (2007) Insulin's impact on renal sodium transport and blood pressure in health, obesity, and diabetes. Am. J. Physiol. Renal Physiol. 293, F974–F984 - PubMed

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