Like the Sgk1
gene transcript and the Sgk1 protein (40
promoter activity proved to be very responsive to increases in extracellular tonicity in IMCD cells. As shown in Figure A, IMCD cells transfected with the Sgk
promoter–driven luciferase reporter demonstrated a 5- to 6-fold increase in promoter activity following exposure to an approximately 150-mOsm/kg increase (i.e., above the tonicity of DMEM medium, which is 296 mOsm/kg H2
O) in extracellular tonicity. The background vector (pGL3-Luc) showed no sensitivity to extracellular tonicity.
Osmotic induction of rat Sgk1 promoter in rat IMCD cells.
The osmotic activation of this promoter was dependent on the p38 MAPK signaling system (Figure B). Treatment of the IMCD cells with the p38 MAPK inhibitor SB203580 led to a dose-dependent suppression of the osmotic induction of Sgk1
-Luc reporter activity. Similarly, cotransfection with a dominant negative mutant of MKK6, an activating kinase immediately upstream from p38 MAPK (41
), resulted in a nearly 50% decrease in the tonicity-dependent induction.
Earlier studies (34
) indicated that osmotic induction of the Sgk1
promoter in NMuMg cells is dependent on a GC-rich region, shown in vitro to associate with the transcription factor Sp1, in close proximity to the transcription start site of the Sgk1
gene. Since Sp1-binding sites have not been linked to induction of osmotically sensitive genes in renal cells, we examined the promoter sequence for the presence of alternative sites that might contribute to the osmotic induction. We identified a consensus tonicity-responsive enhancer (TonE) (44
) approximately 312 bp upstream from the transcription start site. The rat Sgk1
TonE (TGGAAAATCACC) (Figure A) is completely homologous with the murine and human aldose reductase osmoregulatory elements (13
). We introduced a series of point mutations into the putative TonE in the Sgk1
promoter–driven luciferase reporter and transfected these into IMCD cells. As shown in Figure B, one of the mutations (M3) led to complete reversal of the osmotic induction, while 2 of the others, M1 and M2, led to intermediate levels of inhibition; a fourth, M4, with base modifications outside the core TonE, was without effect.
Mutation of TonE blocks osmotic induction of the Sgk1 gene promoter in IMCD cells.
Given the previous association of the putative Sp1-binding element (34
) with the osmotic induction of this promoter’s activity, we created a mutation in this site, alone and in combination with the TonE mutation (Figure A), and introduced the reporters into IMCD cells. As shown in Figure B, virtually the entire osmotic induction appears to flow through TonE in these cells. Mutation of the Sp1-binding element in the GC-rich region did not affect the osmotic induction when tested alone or in the presence of the TonE mutation. Thus, while this Sp1-binding element appears to play a role in controlling the Sgk1
promoter response to tonicity in NMuMg cells (34
), in IMCD cells the TonE site dominates in orchestrating this response.
Proximal Sp1-binding elements do not participate in the osmotic induction of Sgk1 promoter activity in rat IMCD cells.
In the case of the aldose reductase (13
) and betaine transporter (20
) genes, a specific nuclear transcription factor termed NFAT5 has been shown to associate with TonE and stimulate transcriptional activity of the contiguous promoter. To explore the involvement of NFAT5 as a mediator of the osmotic induction of the Sgk1
promoter, we carried out EMSA of nuclear extracts from IMCD cells cultured under isotonic versus hypertonic (NaCl) conditions using radiolabeled Sgk1
TonE sequence as a probe. As shown in Figure A, exposure to the hypertonic environment resulted in an increase in protein association with the oligonucleotide harboring the TonE, here designated as the NFAT5 complex. Incubation with anti-NFAT5 antibody, but not with antibody directed against the p50 subunit of NF-κB, resulted in a supershift of the associated protein. The NFAT5 complex was competed by unlabeled oligonucleotide harboring the wild-type sequence and by the sequence harboring the M4 mutation, but not by oligonucleotides harboring mutations M1, M2, or M3 (Figure B). Thus, the relative affinity of NFAT5 for these mutated osmotic response element (ORE) sequences mirrors the ability of these sequences to signal the osmotic induction of the Sgk1
promoter (see Figure ).
NFAT5 binding to TonE in the Sgk1 promoter in vitro.
Western blot analysis for NFAT5 protein showed that there was a shift of protein from the cytoplasm into the nuclear compartment after 4 hours of exposure to the hypertonic environment (75 mM NaCl), and this nuclear sequestration persisted for 24 hours (Figure , C and D). In addition, there was a net increase in levels of total NFAT5 protein at 24 hours that was not present at the 4-hour time point (Figure E).
To confirm that the osmotically dependent NFAT5 association with the Sgk1 promoter takes place within the context of the intact cell, we carried out ChIP analysis of the endogenous rat Sgk1 promoter using an antibody directed against NFAT5. As shown in Figure A, exposure of IMCD cells to hypertonic medium (addition of 75 mM NaCl or 150 mM sucrose to culture medium) led to increased association of both NFAT5 and RNA polymerase II with the native Sgk1 gene relative to the isotonic control. Neither NFAT5 nor RNA polymerase II interacted to any significant degree with sequence lacking the TonE positioned 3.2 kb upstream from the Sgk1 gene. To prove that this interaction between NFAT5 and the Sgk1 gene promoter requires the TonE-binding sequence described above, we introduced reporter plasmids harboring 1,474 bp of rat Sgk1 5′ flanking sequence (–1,430 to +44) into IMCD cells by transient transfection. One of these reporters contained entirely wild-type sequence, while the other harbored a selective mutation (M3) in the TonE. As shown in Figure B, the wild-type sequence showed evidence of substantial osmosensitive association with NFAT5 in the ChIP assay; mutation of the TonE-binding site resulted in near complete loss of this association.
ChIP analysis of the Sgk1 promoter.
To link the NFAT5 protein functionally to the osmotic induction of Sgk1
promoter activity, we used 2 different approaches to inhibit NFAT5 activity. Introduction of U6-N5 ex8, a small inhibitory RNA vector that specifically targets the NFAT5 endogenous transcript (46
), completely reversed the osmotic induction of the Sgk1
promoter, while the empty U6 vector was devoid of activity (Figure ). Similarly, introduction of a dominant negative NFAT5 expression vector (NFAT5-DN) along with the Sgk1
-Luc reporter led to significant inhibition of the osmotic response. Collectively, these findings, along with the results presented above, support the hypothesis that NFAT5 is the relevant transcription factor that binds to the Sgk1
TonE and mediates the osmotic induction of that gene’s promoter in IMCD cells.
Selective inhibition of NFAT5 leads to blockade of the hypertonic induction of the Sgk1 gene promoter.
A previous study by Rozansky et al. (47
) demonstrated that sgk1
gene expression can also be activated by reductions in extracellular tonicity (i.e. hypoosmolality) in A6 cells. To evaluate this phenomenon in cultured IMCD cells and its dependence on the TonE site described above, we introduced the wild-type Sgk1
-Luc or the TonE-mutated Sgk1
-Luc into primary cultures of rat IMCD cells and exposed them to either a hyper- or a hypotonic environment. As shown in Figure , relative to the cells cultured in isotonic medium, cells exposed to either the hypo- or hypertonic medium showed a significant increase in expression of the wild-type Sgk1
promoter. However, while mutation of the TonE, as expected, led to a near complete inhibition of the hypertonic stimulation of promoter activity, it had virtually no effect on the hypotonic stimulation. This implies that the hypotonic stimulation of Sgk1
gene transcription operates over signaling pathways that are largely independent of those used for the hypertonic induction, including NFAT5 and the TonE.
Hypotonic induction of the Sgk1 promoter does not require TonE.
As mentioned above, expression of both Sgk1 and NPR-A has been shown to increase in IMCD cells exposed to increased extracellular tonicity. To establish the link between Sgk1 and the induction of NPR-A gene expression, we employed an siRNA approach. Cultured rat IMCD cells were transfected with one of 3 siRNA sequences, each of which was designed to target the rat Sgk1 gene coding sequence, prior to exposure of the cells to increased extracellular tonicity (75 mM NaCl). As shown in Figure A, exposure to hypertonic culture media resulted in a 4- to 5-fold increase in Sgk1 mRNA expression in these cells. This increment was partially reduced by siSgk1A and siSgk1B and nearly completely eliminated by cotransfection with siSgk1C. Similarly, as shown in the Western blot in Figure B, siSgk1C had little effect on basal levels of Sgk1 protein, but it nearly completely reversed the NaCl-dependent increment in Sgk1. The same level of extracellular tonicity (75 mM NaCl) led to a 3-fold increase in NPR-A mRNA levels (Figure C) and a 4-fold increment in NPR-A protein levels (Figure D) in IMCD cells. Transfection of these cells with siSGK1C inhibited the increase in NPR-A protein and mRNA by approximately 60%. Collectively, these data indicate that the osmotic induction of Sgk1 is a major contributor to the osmotic stimulation of NPR-A gene expression in IMCD cells.
Sgk1 siRNA blocks NaCl-induced Sgk1 and NPR-A gene transcription and translation.
To extend this observation to an in vivo model, we subjected male Sprague-Dawley rats to 24 hours of water deprivation, a procedure that is known to increase urine osmolality (4
) and renal medullary sodium concentration (4
). As shown in Table , water deprivation resulted in a significant increase in urine osmolality (~40% increase over the baseline urine osmolality). There was a modest, but not statistically significant, increase in plasma osmolality, and no significant change in systolic or diastolic blood pressure in these 2 groups. The water-restricted animals lost approximately 10% of their body weight over the 24-hour period, while weights in the control animals were stable.
Effect of 24-hour water restriction on plasma aldosterone levels, plasma and urine osmolality, blood pressure, and body weight (before and after dehydration)
Water restriction was associated with a statistically significant increase in urinary sodium excretion (Figure A), confirming the presence of the dehydration natriuresis observed by others in rats, rabbits, sheep, mice, dogs, and humans (1
) following short-term water restriction. This was accompanied by a significant increase in Sgk1
mRNA (Figure B) and Sgk1 protein (Figure C) in the renal IMCD. Because Sgk1
gene expression is known to be regulated by aldosterone, we measured plasma aldosterone levels at the end of the water deprivation period. As reported in Table , there was no change in plasma aldosterone levels after 24-hour water deprivation, a finding that is consistent with previous dehydration studies (3
). Since some studies (48
) have shown that increased Sgk1 expression is linked to increased epithelial sodium channel (ENaC) expression, we asked whether dehydration-induced Sgk1 expression was associated with increased ENaC expression. As shown in Figure D, water restriction modestly increased expression of the α subunit of ENaC (αENaC). However, this modest elevation in αENaC expression was accompanied by increased sodium excretion rather than sodium absorption (see Figure A), implying that Sgk1-mediated activation of natriuresis plays a dominant physiological role in this special circumstance.
Water restriction increases urine sodium excretion, Sgk1 mRNA, and protein levels in rat IMCD.
The increase in Sgk1 expression was accompanied by an increase in NPR-A mRNA (Figure A) and protein (Figure B), which, in turn, were linked to a 3-fold increase in urinary cyclic GMP excretion (Figure C). Cyclic GMP is the biological end product of NPR-A activation (i.e., ligand-dependent guanylyl cyclase activation).
Dehydration activates the natriuretic peptide system in rat IMCD.
To investigate a possible role for the ligand itself (i.e., ANP) in contributing to this increase in urinary cyclic GMP, we measured ANP mRNA transcript levels in the atria of the control versus water-restricted rats, as well as plasma ANP levels in the 2 groups. As shown in Figure , D and E, water restriction reduced, rather than increased, expression of the atrial ANP gene and circulating levels of ANP.
The kidney itself is also thought to be a source of ANP gene expression. Cells of the distal nephron have been shown to harbor proANP gene transcripts and ANP immunoreactivity (50
), and ANP immunoreactivity has been identified in the urine (51
). Urinary ANP, termed urodilatin, is a product of the renal ANP gene with a 4-amino-acid N-terminal extension (supplied from the proANP precursor) linked to the core ANP peptide (52
). As shown in Figure F, expression of proANP mRNA in the inner medulla is increased following 24 hours of water restriction, as is the excretion of urodilatin in urine (Figure G) (urodilatin concentration in urine: control, 4.37 ± 0.64 pg/μl vs. dehydrated, 9.33 ± 1.01 pg/μl; P
< 0.01), suggesting that locally generated ligand as well as increased expression of the NPR-A receptor (see above) contribute to the increased urinary excretion of sodium in the acutely dehydrated state.
To confirm the mechanistic link between dehydration natriuresis and increased NPR-A expression, we examined the effect of 24-hour water restriction in wild-type and Npr1–/–
(i.e., NPR-A gene–deleted) mice. As noted previously (53
mice display significant hypertension compared with control littermates (systolic BP, 112.8 ± 10.6 vs. 103.8 ± 6.9 mmHg, P
< 0.01; diastolic BP, 96.8 ± 12.5 vs. 83.3 ± 8.1 mmHg, P
< 0.05). Twenty-four hours of water deprivation resulted in weight loss (12% in wild-type and 13% in Npr1–/–
mice) and increased urine osmolality in wild-type and Npr1–/–
mice compared with their respective control littermates with free access to water (wild-type, 3,123.4 ± 606.4 vs. 1,817.9 ± 401.5 mOsm/kg H2
< 0.01; Npr1–/–
, 2,415.6 ± 549.1 vs. 1,568.8 ± 578.5 mOsm/kg H2
< 0.01). This was accompanied by a significant increase in Sgk1 protein expression in IMCD (Figure A), suggesting that hyperosmolality activates Sgk1 expression in this nephron segment in both wild-type and Npr1–/–
mice. Urinary sodium excretion was markedly increased during 24 hours of water restriction in wild-type mice (Figure B); however, this natriuresis did not occur in Npr1–/–
mice (Figure B). As expected, dehydration dramatically increased NPR-A expression in wild-type mice; there was no NPR-A protein expression in Npr1–/–
mice (Figure C). NPR-A activity, assessed as urinary cyclic GMP excretion, was significantly increased in dehydrated wild-type but not in dehydrated Npr1–/–
mice (Figure D). These findings support a mechanistic link between dehydration-induced natriuresis and NPR-A signaling activity.
Effect of water restriction on the Sgk1/NPR-A signaling pathway in wild-type and Npr1–/– mice.