Impaired renal Na(+) retention in the sgk1-knockout mouse

Abstract

The serum- and glucocorticoid-regulated kinase (sgk1) is induced by mineralocorticoids and, in turn, upregulates heterologously expressed renal epithelial Na(+) channel (ENaC) activity in Xenopus oocytes. Accordingly, Sgk1 is considered to mediate the mineralocorticoid stimulation of renal ENaC activity and antinatriuresis. Here we show that at standard NaCl intake, renal water and electrolyte excretion is indistinguishable in sgk1-knockout (sgk1(-/-)) mice and wild-type (sgk1(+/+)) mice. In contrast, dietary NaCl restriction reveals an impaired ability of sgk1(-/-) mice to adequately decrease Na(+) excretion despite increases in plasma aldosterone levels and proximal-tubular Na(+) and fluid reabsorption, as well as decreases in blood pressure and glomerular filtration rate.

Figure 1

Generation of sgk1^–/– mice. (a) Targeting strategy. The neomycin resistance cassette (gray box) was flanked by two loxP sites (ovals) and inserted into intron 11. Exons 4–11, which code for the Sgk1 kinase domain (open boxes), were “floxed” by inserting a third loxP site into intron 3. N indicates NheI restriction sites, and the small black bar indicates the external 5′ probe used for Southern blot analysis. Expected fragment sizes of the wild-type and targeted sgk1 locus are also indicated. One homologously recombined ES cell clone was transiently transfected with Cre recombinase, and a clone that had undergone recombination between the first and the third loxP site (type I recombination) was chosen for injection. Arrows below the gene indicate PCR primers used for genotyping. Numbers between the arrows indicate the size of the amplified fragments. Crossed bars below a indicate homologous recombination. (b) Southern blot of NheI-digested genomic DNA from ES cell clones after gene targeting hybridized with a 5′ external probe (black bar in a). Lane 5 shows a targeted ES cell line. (c) Genotyping by PCR of genomic tail DNA of homozygous (–/–) and heterozygous (–/+) sgk1-deficient mice and wild-type mice (+/+) using a mix of three specific primers (arrows in a). (d) Autoradiograph of Northern blot analysis of Sgk1-specific transcripts in +/+ and –/– mice. The deletion of the kinase domain from the genome results in a size reduction of 0.9 kb at the mRNA level in sgk1^–/– mice.

Figure 2

Deficient adaptive response to low Na⁺ intake in sgk1^–/– mice. (a) Urinary excretion of Na⁺ and water, and change in body weight in response to a low-NaCl diet (0 g Na⁺/kg) assessed in awake mice in metabolic cages (n = 6 each group). (b) Shown are mean arterial blood pressure and GFR in anesthetized mice on the control diet and at day 3 after initiating the low-NaCl diet (0.15 g Na⁺/kg) (n = 5–6 each group); and plasma aldosterone concentration in mice on control diet and at day 3 after initiating low-NaCl diet (0 g Na⁺/kg) (n = 8–9 each group). Filled circles and bars indicate sgk1^–/– mice, open circles and bars indicate sgk1^+/+ mice. *P < 0.05 versus sgk1^+/+ mice.

Figure 3

Single-nephron GFR and proximal-tubular Na⁺ reabsorption are altered in sgk1^–/– mice on a low-NaCl diet. (a) Schematic diagram of free-flow collection of tubular fluid from the last loop of the proximal tubule (PT) and the first loop of the distal tubule (DT) on the kidney surface in micropuncture experiments in anesthetized mice at day 3 after initiating a low-NaCl diet (0.15 g Na⁺/kg). (b) Fractional delivery of fluid and Na⁺ to proximal tubule and distal tubule as well as urine (n = 13–23 nephrons in 5–6 mice). *P < 0.05 versus sgk1^+/+ mice. (c) Correlation of Na⁺ reabsorption in proximal tubule with single-nephron filtration rate. Note that nephron filtration rate is reduced in sgk1^–/– mice and that for any given nephron filtration rate the Na⁺ reabsorption is higher in sgk1^–/– mice than in sgk1^+/+ mice, indicating a primary increase in proximal reabsorption in sgk1^–/– mice. Symbols are as given in Figure 2 legend.

Figure 4

Amiloride-sensitive transepithelial potential difference is reduced and αENaC is present in the luminal cell membrane of the renal collecting system of sgk1^–/– mice on a low-NaCl diet. (a) Transepithelial potential difference (PD) in isolated perfused collecting ducts at day 3 after initiating a low-NaCl diet (0 g Na⁺/kg) in the absence (n = 12–16 ducts in 3–5 mice) (white bar) and presence (n = 4–5 ducts in 3–5 mice) (black bar) of 10 μM amiloride. *P < 0.05 versus sgk1^+/+ mice. (b) Immunohistochemical localization of αENaC in renal connecting tubule profiles at day 3 after initiating a low-NaCl diet (0 g Na⁺/kg). Arrowheads indicate apical localization of αENaC.