Differential regulation of the bumetanide-sensitive cotransporter (NKCC2) by ovarian hormones

Abstract

The Na-K-2Cl cotransporter (NKCC2) regulates sodium transport along the thick ascending limb of Henle's loop and is important in control of sodium balance, renal concentrating ability and renin release. To determine if there are sex differences in NKCC2 abundance and/or distribution, and to evaluate the contribution of ovarian hormones to any such differences, we performed semiquantitative immunoblotting and immunoperoxidase immunohistochemistry for NKCC2 in the kidney of Sprague Dawley male, female and ovariectomized (OVX) rats with and without 17-beta estradiol or progesterone supplementation. Intact females demonstrated greater NKCC2 protein in homogenates of whole kidney (334+/-29%), cortex (219+/-20%) and outer medulla (133+/-9%) compared to males. Ovarian hormone supplementation to OVX rats regulated NKCC2 in the outer medulla only, with NKCC2 protein abundance decreasing slightly in response to progesterone but increasing in response to 17-beta estradiol. Immunohistochemistry demonstrated prominent NKCC2 labeling in the apical membrane of thick ascending limb cells. Kidney section NKCC2 labeling confirmed regionalized regulation of NKCC2 by ovarian hormones. Localized regulation of NKCC2 by ovarian hormones may have importance in controlling sodium and water balance over the lifetime of women as the milieu of sex hormones varies.

Figure 1

Representative control loading gel stained with GelCode Blue. Prior to Western blotting control loading gels were run to confirm equality of loading in each lane. Shown are homogenates from the inner stripe of the outer medulla from the progesterone study. Each lane was loaded with homogenate from a different rat. To ensure equality of loading, three representative bands were quantified by densitometry.

Figure 2

Semiquantitative immunoblots showing sex differences of NKCC2 abundance. For each blot, each lane was loaded with homogenate from a different rat. A preliminary Coomasssie-stained gel demonstrated equality of loading among the lanes. (A) Whole kidney (WK) homogenates compared 6 male and 6 female rats. B) Homogenates from cortex (CTX) and inner stripe of the outer medulla (OM) compared 4 male and 4 female rats. C) Bar graphs summarizes quantification of band densities. Band densities were normalized to control males with males set at 100%. An unpaired T-test was performed. Mean±standard error (SE), * p<0.05

Figure 3

Semiquantitative immunoblots showing effect of 17-β estradiol on NKCC2 abundance. A) Homogenates from whole kidney (WK), cortex (CTX) and inner stripe of the outer medulla (OM) compared 8 intact female, 6 ovariectomized (OVX)+placebo pellet (SC, 21 day pellet – for 7 days) female and 7 OVX +17 β estradiol (SC 0.05 mg, 21 day pellet, for 7 days) rats. B) Band densities are quantified and summarized as bar graphs. Band densities were normalized to intact females with females set at 100%. A one-way ANOVA with a bonferroni post hoc was used to compare groups. Mean±standard error (SE), * versus intact female, # versus OVX; p<0.05

Figure 4

Semiquantitative immunoblots showing effect of progesterone on NKCC2 regulation. Whole kidney (WK), cortex (CTX) and inner stripe of the outer medulla (OM) homogenates from different rats were loaded in each lane. (A) Immunoblots compared intact females, ovariectomized (OVX)+vehicle (SC 200 μl PEG for 7 days) and OVX+progesterone (SC 2.5 mg/200 μl PEG for 7 days). B) Quantification of band densities were normalized to intact females with intact females set at 100%. Band densities are summarized as bar graphs. A one-way ANOVA with a bonferroni post hoc was used to compare groups. Mean±standard error (SE), * versus intact female, # versus OVX; p<0.05

Figure 5

Immunoperoxidase NKCC2 labeling of cortex (CTX). NKCC2 labeling from rats using Vectastain Elite ABC Kit, (rabbit-specific stain). Kidney sections from male, female and ovariectomized (OVX) rats.

Figure 6

Immunoperoxidase NKCC2 labeling of inner stripe of outer medulla (OM). NKCC2 labeling from rats using Vectastain Elite ABC Kit, (rabbit-specific stain). Kidney sections from male, female and ovariectomized (OVX) rats treated with either placebo pellets (SC, 21 day pellet, for 7 days) or with 17-β estradiol (SC 0.05 mg, 21 day pellet, for 7 days) were used.