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Endocrinology, doi:10.1210/en.2008-0143
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Endocrinology Vol. 149, No. 7 3294-3305
Copyright © 2008 by The Endocrine Society

Epithelial Sodium Channel Is a Key Mediator of Growth Hormone-Induced Sodium Retention in Acromegaly

Peter Kamenicky, Say Viengchareun, Anne Blanchard, Geri Meduri, Philippe Zizzari, Martine Imbert-Teboul, Alain Doucet, Philippe Chanson and Marc Lombès

Institut National de la Santé et de la Recherche Médicale (INSERM) (P.K., S.V,. G.M., P.C., M.L.), Unité (U) 693, Le Kremlin Bicêtre F-94276, France; Université Paris-Sud 11 (P.K., S.V., G.M., P.C., M.L.), Faculté de Médecine Paris-Sud, Unité Mixte de Recherche (UMR)-S693, Le Kremlin Bicêtre F-94276, France; Assistance Publique-Hôpitaux de Paris (A.B.), Hôpital Européen Georges Pompidou, Centre d’Investigation Clinique, Paris F-75908, France; Assistance Publique-Hôpitaux de Paris (G.M.), Hôpital de Bicêtre, Service de Pharmacogénétique, Biochimie Moléculaire et Hormonologie, Le Kremlin Bicêtre F-94275, France; INSERM (P.Z.), U549, Centre Broca, Paris F-75014, France; Université Pierre et Marie Curie (M.I.-T., A.D.), Université Paris 06, UMR 7134 Paris F-75006, France; Centre National de la Recherche Scientifique (M.I.-T., A.D.), UMR 7134, Paris F-75006, France; and Assistance Publique-Hôpitaux de Paris (P.C., M.L.), Hôpital de Bicêtre, Service d’Endocrinologie et Maladies de la Reproduction, Le Kremlin Bicêtre F-94275, France

Address all correspondence and requests for reprints to: Marc Lombès, M.D., Ph.D., Institut National de la Santé et de la Recherche Médicale Unité 693, Faculté de Médecine Paris-Sud, 63, rue Gabriel Péri, 94276 Le Kremlin Bicêtre Cedex France. E-mail: marc.lombes{at}u-psud.fr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Acromegalic patients present with volume expansion and arterial hypertension, but the renal sites and molecular mechanisms of direct antinatriuretic action of GH remain unclear. Here, we show that acromegalic GC rats, which are chronically exposed to very high levels of GH, exhibited a decrease of furosemide-induced natriuresis and an increase of amiloride-stimulated natriuresis compared with controls. Enhanced Na+,K+-ATPase activity and altered proteolytic maturation of epithelial sodium channel (ENaC) subunits in the cortical collecting ducts (CCDs) of GC rats provided additional evidence for an increased sodium reabsorption in the late distal nephron under chronic GH excess. In vitro experiments on KC3AC1 cells, a murine CCD cell model, revealed the expression of functional GH receptors and IGF-I receptors coupled to activation of Janus kinase 2/signal transducer and activator of transcription 5, ERK, and AKT signaling pathways. That GH directly controls sodium reabsorption in CCD cells is supported by: 1) stimulation of transepithelial sodium transport inhibited by GH receptor antagonist pegvisomant; 2) induction of {alpha}-ENaC mRNA expression; and 3) identification of signal transducer and activator of transcription 5 binding to a response element located in the {alpha}-ENaC promoter, indicative of the transcriptional regulation of {alpha}-ENaC by GH. Our findings provide the first evidence that GH, in concert with IGF-I, stimulates ENaC-mediated sodium transport in the late distal nephron, accounting for the pathogenesis of sodium retention in acromegaly.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CHRONIC HYPERSECRETION of GH and subsequent hypersecretion of IGF-I in acromegaly lead to major cardiovascular complications that constitute the main cause (60%) of mortality. Arterial hypertension that occurs in 30–40% of acromegalic patients is one of the most important prognostic factors of increased mortality (1). Plasma volume expansion and increased cardiac output have been well documented and seem to play major roles in the pathophysiology of high blood pressure (2). In addition, increased extracellular volume is constantly present, and accounts for soft tissue swelling and organomegaly consistent with the antinatriuretic effects of GH (3). In contrast, adult GH-deficient patients have a reduction in plasma and extracellular volume that can be normalized by GH substitution (4). However, the molecular mechanisms by which GH controls sodium and water homeostasis remain poorly understood.

The initial concept of indirect effects of GH via activation of the renin angiotensin aldosterone system (5, 6, 7, 8) or decrease of the plasma atrial natriuretic peptide concentration (9, 10, 11) is controversial. Rather, several studies concluded to direct stimulatory effects of GH/IGF-I on renal sodium and water reabsorption (7, 8, 10). Central questions remain whether GH controls tubular sodium transport directly or through its major target IGF-I, and in which segments of the nephron this potential antinatriuretic effect may occur.

The presence of functional GH receptor (GHR) in target cells is a prerequisite for direct renal effects of GH. Indeed, in situ hybridization studies in the rat kidney showed that GHR mRNA expression was confined to the proximal tubule and the thick ascending limb (TAL) of Henle’s loop (12). However, the expression of GHR in the distal nephron remains controversial (12, 13, 14). Recent observations have extended GHR expression to glomerular mesangial cells (15) and podocytes (16). In vitro microperfusion of rabbit proximal tubules exposed to GH and IGF-I (17), as well as lithium clearance measurements, an important index of proximal tubular sodium reabsorption, in GH-treated patients (10) and rats (18), have excluded a prominent role of the proximal tubule in GH-induced sodium transport. Likewise, although a recent study reported that acute GH administration in rats results in increased phosphorylation of Na+,K+,2Cl – cotransporter in the TAL of the Henle’s loop, the lack of a concomitant GH-induced change in sodium transport questions the physiological relevance of this observation (18). Based on human metabolic studies, it has been alternatively suggested that GH may exert its effects in the distal nephron (8, 10), which plays a pivotal role in sodium homeostasis and constitutes the major segment mediating sodium-retaining effects of the mineralocorticoid hormone aldosterone (19). The classical view of aldosterone action is that it binds to the mineralocorticoid receptor (MR), a ligand-dependent transcription factor, to modulate gene expression, resulting in induction of proteins implicated into the transepithelial ionic transport (20). Aldosterone-regulated transepithelial sodium reabsorption in the distal nephron occurs via the amiloride-sensitive epithelial sodium channel (ENaC) located at the apical membrane and the basolateral Na+,K+-ATPase of cortical collecting duct (CDD) cells. ENaC is composed of three subunits ({alpha}, β, and {gamma}) (21), constituting the rate-limiting step of apical Na+ entry. Although the presence of GHR in the distal nephron has been demonstrated in some but not all studies (12, 13, 14), it has thus far never been functionally characterized.

To address the direct impact of GH on the control of sodium handling and to localize its target site of action, we used complementary approaches on various experimental models that all provided converging evidence for direct antinatriuretic effects of GH in the late distal nephron. Metabolic cage studies in an animal model of acromegaly, the GC rats bearing somatotropic cell tumors (22), allowed us to examine the influence of chronic GH hypersecretion on sodium balance in vivo and to identify the aldosterone-sensitive distal nephron as a direct target of GH action. To decipher the mechanisms by which GH stimulated transepithelial sodium transport, we used a highly differentiated CCD cell line, the KC3AC1 cells (23). This cell-based system enabled us to demonstrate, for the first time, the presence of functional GHR in a CCD-derived cell line and to characterize the molecular targets involved in the pathophysiology of extracellular volume expansion in acromegaly.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Hormones and drugs
GH and pegvisomant were kindly provided by Serono (Boulogne, France) and Pfizer (Paris, France), respectively. IGF-I, U0126, and Ly294002 were from Euromedex (Mundolsheim, France), AG490 was from VWR (Strasbourg, France), protein A Sepharose CL-4B was from GE Healthcare (Uppsala, Sweden), and all other reagents were from Sigma-Aldrich (St. Louis, MO).

Animal studies
Animal housing and metabolic studies were performed according to the French legislation. GC rats were generated as previously described (22). Briefly, 12 x 106 GC cells were injected sc into the flank of 8-wk-old female Wistar-Furth rats (Charles River, L’Arbresle, France). Animals were maintained on a regular 12-h light, 12-h dark cycle, fed ad libitum, and weighed weekly for 12 wk. Water intake, baseline 24-h urine volume, urinary creatinine, and electrolyte concentrations determined on an automatic analyzer (Konelab 20i; Thermo, Cergy Pontoise, France) were obtained for 2 consecutive days after 3-d animal adaptation to encaging. Furosemide (40 mg/kg) was injected ip, and urine was collected during consecutive 3-h urinary sample collections. Amiloride was given in the drinking water (150 mg/liter) for 24 h with concomitant 24-h urinary sample collection. Amiloride intake was not different in GC and wild-type (WT) rats when normalized to body weight (9.8 vs. 10.2 mg/kg in controls). Plasma GH concentrations were determined by enzyme immunoassay as previously described (24). IGF-I and aldosterone concentrations were measured by RIA. Three days after metabolic challenges, animals were killed after anesthesia, and kidneys were collected for histology and Western blot analysis.

Na+/K+-ATP-ase assay
Na+/K+-ATP-ase activity was determined using pools of four to six permeabilized proximal convoluted tubules (PCTs), cortical TALs of Henle’s loop, or CCDs as described previously (25). Na+/K+-ATPase activity was taken as the difference between mean total and ouabain-resistant ATPase activities, each determined in triplicate. Values are means ± SEM from several animals.

Cell culture
KC3AC1 cells (passages 10–20) were seeded on collagen I-coated Transwell filters (Costar Corp., Brumath, France) or petri dishes, and routinely cultured at 37 C in a humidified incubator gassed with 5% CO2 within an epithelial medium composed of DMEM/HAM’s F12 (1:1), 2 mM glutamine, 50 nM dexamethasone, 50 nM sodium selenite, 5 µg/ml transferrin, 5 µg/ml insulin, 10 ng/ml epidermal growth factor, 2 nM T3, 100 U/ml penicillin, 100 µg/ml streptomycin, 20 mM HEPES (pH 7.4), and 5% dextran charcoal-treated serum. To study GH actions, epithelial medium was replaced by a minimum medium having the same composition as the epithelial medium with omission of dextran charcoal-treated serum, dexamethasone, and epidermal growth factor. To study IGF-I effects, insulin was omitted to the minimal medium.

RT-PCR and quantitative real-time PCR
Total RNA was extracted from cells with Trizol reagent (Invitrogen, Cergy Pontoise, France), and RNA was processed for RT-PCR analysis. Gene expression was quantified by real-time PCR using an ABI 7300 (Applied Biosystems, Foster City, CA). Results represent the relative expression for a given sample calculated as the ratio (amol of specific gene/fmol of 18S). Murine RNAs were extracted from pools of 20–50 microdissected nephronic segments, and quantitative real-time PCR was performed using a cDNA quantity corresponding to 0.1 mm nephron. Results are expressed as molecules per mm tubule length. Supplemental Table 1, which is published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org, indicates primer sequences of genes analyzed.

Western blot analysis and immunoprecipitation assay
Total protein extracts were prepared from WT and GHR knockout mouse kidney and liver or from KC3AC1 cells. When GH or IGF-I treatment was tested, cells were deprived for 3 or 24 h in the appropriate minimal media as indicated previously. Tissues or cells were washed twice with ice-cold PBS and lysed at 4 C with lysis buffer. Immunoblots were incubated overnight with the following dilutions of primary antibodies: anti-GHR AL-47, 1:1000 (kindly provided by Professor S. J. Frank, Birmingham, AL); anti-IGF-IRa N20:sc-712, 1:1000 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA); anti-phospho-p44/42 MAPK E10 (1:2000), anti-p44/42 MAPK E10 (1:1000), anti-phospho-AKT (1:1000), and anti-AKT (1:1000) (all from Cell Signaling Technology, Inc., Beverly, MA); and anti-{alpha}, β, and {gamma}-ENaC subunit antibodies, 1:1000 (kindly provided by Dr. M. van Bemmelen and Professor L. Schild, Lausanne, Switzerland). This was followed by incubation with peroxidase-conjugated goat antirabbit antibody, dilution 1:15,000 or antimouse antibody, dilution 1:20,000, respectively (Vector Laboratories, Burlingame, CA), and visualized by the ECL+ detection kit (GE Healthcare). When indicated, immunoprecipitation was performed by incubating 1 mg total proteins with anti-signal transducer and activator of transcription (STAT5) C17 antibody (Santa Cruz Biotechnology) overnight at 4 C, and the immunoprecipitates were collected using protein A Sepharose CL-4B beads, reduced in Laemmli buffer, and subjected to 7.5% acrylamide SDS-PAGE and Western blot as described previously. Electrotransferred proteins were incubated with monoclonal anti-phospho-tyrosine antibody (1:5000, clone 4G10; Upstate Biotechnology Inc., Lake Placid, NY), followed by incubation with the secondary antibody. For loading normalization, membranes were incubated with anti-STAT5 or anti-{alpha}-tubulin (1:10,000, DM1A; Sigma-Aldrich). Quantitative analysis of specific signals was performed using Quantity One software (Bio-Rad Laboratories, Inc., Hercules, CA).

Immunocytochemistry
Cells cultured on Lab-Tek (Nunc, Roskilde, Denmark) were washed in PBS, then fixed with 10% buffered formol in PBS (pH 7.3) for 10 min, washed three times in PBS before processing for immunocytochemistry. Cells cultured on filters were fixed in 10% buffered formol in PBS (pH 7.3) for 60 min, dehydrated, and embedded in paraffin blocks. Serial sections were deparaffinized, and antigen retrieval was performed in a microwave oven in pH 6 citrate buffer for 15 min. After blocking with 2.5% normal horse serum, cells were incubated overnight with dilution of primary antibodies as follows: anti-GHR AL-47, 1:500; anti-IGF-IR{alpha}, 1:400; anti-phospho-STAT5 sc-11761, 1:200 (Santa Cruz Biotechnology). After endogenous peroxidase quenching, immunodetection was performed using the ImmPress reagent kit (Vector Laboratories). Irrelevant rabbit immunoglobulins were used as negative controls.

Ionic transport measurements
Cells seeded on collagen I-coated Transwell filters were cultured for 6 d in the epithelial medium that was replaced by the appropriate minimal medium 24 h before hormonal stimulation. The next day, 500 µl supernatant was recovered from the medium bathing the apical and basolateral surface of KC3AC1 cells. Ionic concentrations were measured.

EMSA
Differentiated KC3AC1 cells were starved for 3 h in insulin-containing minimal medium and exposed to GH stimulation. Nuclear protein extraction and gel mobility shift assays were performed as described (26). Purified oligonucleotides were annealed and labeled with [32P]deoxycytidine triphosphate (GE Healthcare) using the Klenow fragment of DNA polymerase (Life Technologies, Inc., Paisley, UK) to a specific activity of approximately 108 cpm/µg DNA. Oligonucleotide sequences were as follows: {alpha}-ENaC-forward, 5'-GCTTCTCTTCTCGGAACCTCA-3'; and {alpha}-ENaC-reverse, 5'-GAAGAGAAGAGCCTTGGAGTG-3'. Protein-DNA complexes were separated from free DNA by electrophoresis on nondenaturing 4.5% polyacrylamide gel in 0.25 x Tris-borate-EDTA buffer at 200 V for 1 h. Gels were dried and exposed to x-ray film at –80 C. Underlined nucleotides correspond to the consensus sequences of the STAT response element.

Statistical analysis
Data were analyzed for statistical significance using the software Prism 4 (GraphPad Software Inc., San Diego, CA). We performed either nonparametric Mann-Whitney U test, Kruskal-Wallis multivariance analysis followed by a posttest analysis of Dunn’s comparison test or nonparametric Spearman correlation. Levels of statistical significance were fixed for P values less than or equal to 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
GH enhances sodium transport in the late distal nephron in GC rats
To address the impact of GH hypersecretion on renal electrolyte balance and to clarify the site of altered sodium handling along the nephron, metabolic studies coupled to pharmacological tests were performed in GC rats, an animal model of acromegaly. After 12-wk sc GC cell implantation leading to chronic GH hypersecretion, GC rats presented with a significant increase in body weight (434.7 ± 16.4 g vs. 207.0 ± 3.7 g for WT animals; P < 0.001), consistent with the very high GH concentrations in GC rats as compared with the control group (WT 10.0 ± 1.4 ng/ml and GC 8217.0 ± 1088.2 ng/ml; P < 0.01). As expected, this was also accompanied by a significant increase in IGF-I levels (WT 395 ± 38 ng/ml and GC 1707 ± 146 ng/ml; P < 0.001). Interestingly, we constantly observed a dramatic 3.5-fold renal hypertrophy leading to a 1.6-fold increase in kidney weight to body weight ratio in acromegalic animals, suggesting a specific impact of chronic GH hypersecretion on kidney remodeling (Fig. 1Go, A and B). In addition, histological examination disclosed a major interstitial edema associated with a 1.7-fold increase in glomerular size (WT 89.9 ± 3.2 vs. GC 155.9 ± 3.5 µm; P < 0.01). Despite this glomerular hypertrophy that paralleled the renal hypertrophy, we did not observe any modification in the diameter of the microdissected CCDs of the acromegalic animals (Fig. 1DGo).


Figure 1
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  		FIG. 1. Renal hypertrophy in GC rats is not accompanied by modification of tubular diameter. A, Morphological features of WT and GC rat kidney; the scale is in mm. B, Comparison of kidney weight (left panel) and kidney weight to body weight ratio (right panel) in WT and GC rats; body weight of WT rats was 207 ± 3.7 g and of GC rats was 434.7 ± 16.4 g (mean ± SEM, n = 10; **, P < 0.01). C, Histological features of WT and GC rat kidneys (magnification, x10; bar, 100 µm). D, Representative microdissected CCD of WT and GC rats (magnification, x2.5; bar, 100 µm).

 
As shown in Fig. 2Go, A and B, basal Na+ and K+ excretion did not differ in WT and GC rats. Administration of furosemide, an inhibitor of the Na+/K+/2Cl– cotransporter, led to lower natriuretic responses and lower urinary Na+/K+ ratio in GC compared with WT rats (Fig. 2AGo). Given that furosemide increased luminal Na+ delivery downstream of the Henle’s loop, this difference might be accounted for by enhanced sodium reabsorption in the distal parts of the nephron. This hypothesis was validated by showing that the natriuretic response to amiloride, a potent inhibitor of ENaC, was enhanced 1.5-fold in GC rats, and was associated with decreased kaliuresis (urinary Na+/K+ ratio increased 3.8-fold vs. WT) (Fig. 2BGo). We clearly demonstrated an enhanced capacity of the late distal nephron of GC rats to reabsorb Na+ and secrete K+, despite a significant reduction in plasma aldosterone concentrations in GC rat compared with controls (Fig 2CGo). We next measured the Na+/K+-ATP-ase activity, the motor of sodium reabsorption along the nephron, in discrete nephron segments and showed that the Na+/K+-ATP-ase activity was specifically increased in the CCD of GC rats, whereas it was not altered in the proximal tubule (PCT) and the Henle’s loop [cortical TAL of the Henle’s loop (cTAL)] (Fig. 2DGo). Together, these in vivo studies provide direct evidence that chronic GH/IGF-I excess enhances sodium transport in the late distal nephron in acromegaly, independently of the renin-angiotensin-aldosterone system.


Figure 2
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  		FIG. 2. The amiloride-sensitive distal nephron is the site of enhanced sodium transport in GC rats independently of aldosterone action. A and B, Metabolic studies of renal Na+ and K+ handling in basal conditions, and 3 h after furosemide injection (40 mg/kg) (A) and 24 h after oral amiloride administration (150 mg/liter water) (B). Left panels represent basal and furosemide- (A) or amiloride- (B) induced natriuresis normalized by urinary creatinine (creat) in WT ({square}) and GC ({blacksquare}) rats; right panels show urinary Na+/K+ ratio in the same experiments. Data are expressed as mean ± SEM; n = 10 (**, P < 0.01; ***, P < 0.001). C, Plasma aldosterone concentrations in WT and GC rats. Values are means ± SEM, from eight control and six GC rats (**, P < 0.01). D, Na+/K+-ATP-ase activity in PCT, cTAL, and CCD from WT and GC rats. Values (in pmol/mm·h) are means ± SEM from five controls and six GC rats (***, P < 0.001).

 
Chronic GH excess enhances ENaC maturation by proteolytic cleavage of {alpha} and {gamma}-subunits
Because sodium reabsorption in the aldosterone-sensitive distal nephron is mainly mediated by ENaC, we examined the renal expression of its three subunits by Western blot analysis in the GC and WT rats. Figure 3AGo shows unchanged expression of the 95- and 55-kDa forms of {alpha}-subunit but a distinct electrophoretic pattern of lower molecular mass bands observed in GC rats. Indeed, the expression of the 36-kDa fragment was decreased in GC rats, associated with a shift of the 38- to a 40-kDa band (Fig. 3AGo, arrowhead). The expression of the 100- to 110-kDa forms of the β-subunit of ENaC remained unchanged (Fig. 3BGo). Furthermore, the abundance of the 85- and 75-kDa forms of the {gamma}-subunit was not altered. Strikingly, an intermediate approximately 80-kDa fragment clearly appeared in GC rats compared with controls (Fig. 3CGo, arrowhead). Altogether, these results demonstrate that enhanced sodium transport prevailing in the distal nephron of GC rats correlates with a significant modification of ENaC maturation involving distinct proteolytic processing of the {alpha}- and {gamma}-subunits.


Figure 3
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  		FIG. 3. ENaC subunit expression and processing are altered in GC rats. A, {alpha}-Subunit. B, β-Subunit. C, {gamma}-Subunit. Representative immunoblots of WT (the first three lanes) and GC rat (the last four lanes) kidneys. Twenty micrograms of protein from whole kidney homogenates were loaded onto each lane. Immunoblots were incubated with 1:1000 dilutions of anti-{alpha}, -β, and -{gamma} ENaC antibodies. Protein loadings were normalized by reblotting with anti-{alpha}-tubulin antibody. Arrowheads correspond to a 38- to 40-kDa band shift of the {alpha}-subunit of ENaC (A) and to the intermediate 80-kDa fragment of the {gamma}-subunit of ENaC (C) observed in GC rats.

 
GHR and IGF-IR are expressed in CCD cells
To establish whether GH exerts direct effects in the distal parts of the nephron, we first analyzed the expression profile of GHR (Fig. 4AGo, left panel) and IGF-IR (right panel) mRNA on microdissected murine nephronic segments by quantitative real-time PCR. Beside the well-described expression of GHR and IGF-IR mRNAs in the proximal tubule and Henle’s loop, we detected substantial amounts of GHR and IGF-IR in the distal nephron with an expression gradient from the cortex to the medulla. To investigate further the molecular mechanisms involved in the antinatriuretic effects of GH and/or IGF-I in the CCD, we used the KC3AC1 cells that maintain aldosterone responsiveness and functional MR signaling (23). When cultured on collagen-coated petri dishes, KC3AC1 cells develop at confluence numerous domes, a characteristic morphological feature of highly differentiated polarized epithelial cells (Fig. 4BGo). The relative abundance of GHR and IGF-IR mRNA in undifferentiated and fully differentiated dome-forming cells was measured by quantitative real-time PCR and compared with that determined in microdissected murine CCD. We showed that the levels of receptor transcripts increase as a function of the cell differentiation (Fig. 4CGo), and the relative GHR transcript abundance in KC3AC1 cells was comparable to that of endogenous GHR expressed in isolated CCDs, estimated at approximately 0.25 amol/µg RNA or 10,000 molecules/µg cellular proteins. Western blot analysis using anti-GHR AL47 antibody revealed a specific band migrating at 115 kDa in KC3AC1 whole cell lysates, also present in murine kidney and liver protein extracts. In contrast, it was absent in GHR knockout mouse organs in which only the 82-kDa nonspecific bands were detected, thus confirming the specificity of the higher molecular mass band (Fig. 4DGo). These results are consistent with the presence of the GHR protein in KC3AC1 cells. IGF-IR protein expression was also analyzed by Western blot. Figure 4EGo reveals two specific bands migrating at 130 and 200 kDa in KC3AC1 cell lysates and in murine kidney, providing evidence for the presence of the {alpha}-subunit of IGF-IR protein. GHR and IGF-IR protein expression in KC3AC1 cells was further analyzed by immunocytochemistry. Figure 4Go, F and G, illustrates GHR and IGF-IR immunostainings in KC3AC1 grown on petri dishes, as well as in a monolayer of KC3AC1 cells cultured on filters. The GHR and IGF-IR immunoreactivity was predominantly localized to the cell membrane and the adjacent cytoplasm, and was enhanced in the dome areas. Finally, KC3AC1 cells did not express IGF-I either in basal conditions or after GH stimulation (data not shown). Collectively, these results demonstrate conclusively that KC3AC1 cells express GHR and IGF-IR both at mRNA and protein level but not IGF-I, enabling us to distinguish direct GH-induced effects from those mediated via IGF-I.


Figure 4
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  		FIG. 4. GHR and IGF-IR are expressed in KC3AC1 cells. A, Relative GHR and IGF-IR expression along different segments of the mouse nephron was evaluated by quantitative real-time PCR. Results (molecule per mm tubule length) are expressed as means ± SEM from five mice. Note that the scale for GHR expression in PCT and proximal straight tubule (PST) (white bars) is 10 times higher than that for the other tubular segments (black bars). B, Morphological features of subconfluent (left panel) and differentiated dome-forming (right panel) KC3AC1 cells on phase-contrast micrographs (magnification, x20). C, GHR and IGF-IR mRNA expression in undifferentiated (Undiff) and differentiated (Diff) KC3AC1 cells were quantified by quantitative real-time PCR analysis. A 2.5-fold increase in GHR transcript levels was observed in dome-forming cells compared with undifferentiated cells, whereas IGF-IR expression was only increased by 50%. Results are mean ± SEM of at least nine independent determinations and represent fold induction above basal expression, which was 0.091 ± 0.015 and 0.305 ± 0.046 amol /fmol 18S for GHR and IGF-IR, respectively (***, P < 0.001; **, P < 0.01). D and E, Western blot analysis of GHR (D) and IGF-IR{alpha} (E) expression in KC3AC1 cells. Thirty µg of proteins from KC3AC1 cell homogenates, and WT and GHR knockout kidney and liver were processed for immunoblotting with anti-GHR. Note the presence of a specific 115-kDa band for GHR in KC3AC1 cells. Ten or 50 µg proteins extracted from KC3AC1 cells or whole kidney, respectively, was used for IGF-IR detection, which revealed a 200- and 130-kDa specific band for IGF-IR. F and G, Immunocytochemical detection of GHR (F) and IGF-IR (G) in dome-forming cells (upper panels) and cells cultured on filters (lower panels). CNT, Connecting segment; DCT, distal convoluted tubule; MTAL, CTAL, medullary, cortical TAL of the Henle’s loop; OMCD, outer medulla collecting duct; PCT, proximal convoluted tubule; PST, proximal straight tubule; CCD, cortical collecting duct.

 
GHR and IGF-IR signaling in KC3AC1 cells
To assess the functional integrity of GHR in KC3AC1 cells, we examined the activation of the classical GHR-associated signaling pathways. GH treatment led to a rapid STAT5 phosphorylation visible as early as 5 min and sustained until 60 min after hormonal stimulation (Fig. 5AGo). This dose-dependent effect was maximal at a GH concentration of 1000 ng/ml, and was strongly antagonized (70%) by the Janus kinase (JAK) 2 inhibitor AG490 and partially inhibited (40%) by the GHR antagonist pegvisomant, as determined by image density quantification (Fig. 5BGo). Immunocytochemistry with an antiphospho-STAT5 antibody showed a transient nuclear translocation of phosphorylated STAT5 in KC3AC1 cells 30 min after GH stimulation (Fig. 5CGo). GH exposure also induced a rapid and transient ERK1/2 phosphorylation, with the maximal response 2 min after stimulation (Fig. 5DGo). This effect was completely inhibited by the GHR antagonist pegvisomant and the MEK inhibitor U0126 (Fig. 5EGo). The IGF-IR signaling in KC3AC1 cells was also examined by activation of its canonical signaling pathways. As shown by Western blot analysis, IGF-I treatment at a concentration of 10 nM resulted in rapid and transient ERK1/2 phosphorylation, with maximal response 2 min after hormonal stimulation (Fig. 5FGo), and prolonged AKT phosphorylation, lasting 5–30 min (Fig. 5GGo). Full inhibition of these effects by the respective kinase inhibitors U0126 and Ly294002 proved the specificity of the IGF-IR signaling. Together, these studies provide strong evidence for the functional integrity of the GHR and IGF-IR signaling in KC3AC1 cells, thus constituting a suitable cell-based system to study biological effects of GH and IGF-I in the CCD.


Figure 5
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  		FIG. 5. GHR and IGF-IR signaling in KC3AC1 cells. A and B, KC3AC1 cells were stimulated with 1000 ng/ml GH for 0–60 min (A) or increasing concentrations of GH for 15 min (B) or with 10 µg/ml pegvisomant (Peg) or 10 µM AG490 alone (–), or in the presence of 1000 ng/ml GH (+ GH). Lysates were prepared at the indicated time, and 1 mg proteins was immunoprecipitated (IP) with anti-STAT5 antibody, followed by Western blotting (WB) using anti-phospho-tyrosine antibody. Membranes were reblotted with anti-STAT5 antibody for loading control. C, KC3AC1 cells grown on Lab-Tek were incubated or not with 100 ng/ml GH for 15 min. Immunocytochemistry with an anti-phospho-STAT5 antibody revealed a specific nuclear staining in the GH-treated cells (inset). D and E, For ERK1/2 pathway, cells were treated with 1000 ng/ml GH for 0–30 min, and direct Western blotting on 30-µg protein lysates was performed with anti-phospho-p44/42 antibody (D). Similar experiments were performed in the absence or presence of 1000 ng/ml GH alone or with 10 µg/ml pegvisomant or 10 µM U0126 for 2 min (E). Protein loadings were normalized by reblotting with anti-p44/p42 antibody. F and G, KC3AC1 cells were stimulated with 10 nM IGF-I for 0–30 min alone, or in the presence of 10 µM U0126 or 10 µM Ly294002 (Ly) for 5 min. Thirty micrograms of protein lysates were submitted to direct Western blotting using anti-phospho-p44/42 (F) or anti-phospho-AKT antibodies (G). Protein loadings were normalized with the corresponding antibodies.

 
GH and IGF-I stimulate ionic transports in KC3AC1 cells
To investigate the potential impact of GH and IGF-I on ionic transport in CCD cells, we used KC3AC1 cells cultured on Transwell filters that exhibited a high transepithelial resistance (5.27 ± 0.17 K{Omega}/cm2) and an amiloride-sensitive short-circuit current (Isc) 23.95 ± 5.27 µA consistent with an ENaC-dependent sodium transport (23). Ion concentrations in the apical and basolateral compartments were measured after GH exposure (100 ng/ml). Under basal conditions, KC3AC1 cells generated apical to basolateral Na+ and Cl– gradients (Na+: 4.40 ± 0.61 and Cl–: 3.60 ± 0.34 mmol/ml·24 h), indicating a Na+/Cl– reabsorption, accompanied by a K+ secretion with basolateral to apical gradient of 1.22 ± 0.21 mmol/ml·24 h. GH treatment (100 ng/ml) induced a 60% increase in Na+ reabsorption and comparable increase in Cl– reabsorption (Fig. 6Go, A and C). The effects were completely inhibited by the GHR antagonist pegvisomant, establishing unambiguously the involvement of GH/GHR activation cascade. Of note, GH did not significantly influence potassium transport under these experimental conditions (Fig. 6BGo). On the other hand, IGF-I (10 nM) induced a 100% increase in Na+ with a 50% increase in Cl– transepithelial gradient. Importantly, it was accompanied by a sharp 3.5-fold increase in K+ transepithelial gradient (Fig. 6Go, D–F). These findings demonstrate that GH exerts direct stimulatory effects on Na+ and Cl– reabsorption in CCD-derived cells, whereas IGF-I induces Na+/Cl– reabsorption with a marked K+ secretion.


Figure 6
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  		FIG. 6. GH and IGF-I stimulate ionic transport in KC3AC1 cells. A–C, KC3AC1 cells seeded on collagen I-coated Transwell filters were starved for 24 h in minimum medium and treated or not (Basal) for 24 h with 100 ng/ml GH alone or with 10 µg/ml pegvisomant (Peg). D–F, Similar experiments after 24-h starvation in minimum medium with insulin omission were performed with 10 nM IGF-I treatments. Supernatants were recovered from the apical (A) and basolateral (B) compartments, and Na+, Cl–, and K+ concentrations were measured. Ionic gradients representing the differences between basolateral vs. apical Na+ and Cl– concentrations or the differences between apical vs. basolateral K+ concentrations were calculated. Results are mean ± SEM of independent determinations and represent fold induction above basal ionic gradient (*, P < 0.05; **, P < 0.01; ***, P < 0.001). A–C, Basal Na+, Cl–, and K+ concentration gradients in mmol/liter·24 h were 4.40 ± 0.61, 3.60 ± 0.34, and 1.22 ± 0.21, respectively (n = 10). D–F, Basal Na+, Cl –, and K+ concentration gradients in mmol/liter·24 h were 4.76 ± 0.43, 3.53 ± 0.33, and 1.46 ± 0.17, respectively (n = 13). Ub, Ubiquitin; MRE, mineralocorticoid response element; STAT-RE, STAT response element.

 
{alpha}-ENaC is a direct GH target gene in CCD cells
To decipher the molecular bases of the GH-induced increase in Na+ reabsorption, gene expression analyses were performed by quantitative real-time PCR experiments on GH-treated KC3AC1 cells. We first demonstrated that GH induced a 2- to 3-fold increase in the expression of suppressor of cytokine signaling (SOCS) 2 and cytokine-inducible SH2-containing protein (CIS), two classical GH target genes (Fig. 7AGo, upper and middle panels). GH exposure also led to a significant 30% increase in {alpha}-ENAC but not β-ENAC and {gamma}-ENAC mRNA steady-state levels, suggesting that {alpha}-ENAC is a novel renal target of GH (Fig. 7AGo, lower panel). Because GH treatment of KC3AC1 cells induced STAT5 phosphorylation (Fig. 5Go, A–C), we analyzed the promoter sequence of SCNN1A and identified a perfectly conserved STAT5 response element located –2097 bp upstream of the transcription initiation start site of the gene. As shown in Fig. 7BGo, electromobility shift assays with nuclear proteins extracted from untreated or GH-treated KC3AC1 cells demonstrated the presence of specific STAT5-RE-protein complexes in GH-treated extracts, suggesting that GH regulates {alpha}-ENaC transcription via STAT5 activation. Collectively, our results clearly demonstrate that {alpha}-ENaC expression is directly stimulated by acute exposure to GH, a finding consistent with the enhanced capacity of CCD cells to reabsorb sodium accounting for hydroelectrolytic retention observed in acromegaly. Because IGF-I also regulated transepithelial ionic transport in this cell model, we examined the putative transcriptional control of IGF-I on candidate target gene expression. At variance with GH, {alpha}-ENAC mRNA expression was not affected, whereas the expression of serum- and glucocorticoid-induced kinase (Sgk) 1 transcripts was rapidly and transiently stimulated as early as 30 min after IGF-I exposure (Fig. 7CGo). Sgk1 expression was not modified by GH treatment (data not shown). Together, we demonstrated that GH and IGF-I modulate distinct target genes implicated in the regulation of ionic transport in the CCD cells, a finding suggesting that both hormones could cooperate to control sodium balance.


Figure 7
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  		FIG. 7. GH and IGF-I increase the expression of specific target genes in KC3AC1 cells. A, Induction of SOCS2, CIS, and {alpha}ENaC mRNA expression in KC3AC1 cells by GH. Cells were incubated in minimal medium 3 h before exposure to GH (100 ng/ml) for 24 h. mRNA expression was measured by quantitative real-time PCR. Results expressed as amol/fmol of 18S are mean ± SEM of six independent determinations (*, P < 0.05; **, P < 0.01; ***, P < 0.001). B, Specific binding to a STAT5 response element located in the {alpha}ENAC promoter region. The sequence of the STAT5-RE oligonucleotide is underlined in bold. Nuclear extracts (5 µg proteins) from GH-treated (lane 1) and untreated KC3AC1 cells (lane 3) were analyzed for binding to a double-stranded STAT5-RE oligonucleotide. After a 15-min incubation of proteins with 105 cpm [32P]-radiolabeled STAT5-RE, protein-DNA complexes were separated on a nondenaturing 4.5% polyacrylamide gel and detected by autoradiography. Specific complexes were identified by competition experiments in which 100-ng unlabeled STAT5-RE (Cp = competitor) was added (lanes 2 and 4). C, KC3AC1 cells were starved for 24 h in minimum medium without insulin and exposed to 10 nM IGF-I for various periods of time. Relative expression levels of Sgk1 and {alpha}ENAC transcripts were determined by quantitative real-time PCR analysis. Results are mean ± SEM of six independent determinations and represent fold induction above basal arbitrary set at one (*, P < 0.05; **, P < 0.01). Basal expression of Sgk1 is 0.386 ± 0.014 amol/fmol 18S, and of {alpha}ENaC 0.143 ± 0.004 amol/fmol 18S.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although the antinatriuretic effects of GH have been proposed a long time ago (3), the mechanisms by which GH and/or IGF-I regulate sodium and water balance remain poorly understood. In the present study, several lines of evidence indicate direct antinatriuretic effects of GH in the late distal nephron. We demonstrate that acromegaly leads to enhanced amiloride-sensitive sodium transport in the CCD associated with GH-induced transcriptional activation and proteolytic maturation of ENaC.

Renal metabolic studies in GC rats permitted to exclude a major contribution of Henle’s loop in sodium-retaining effects of GH excess. Instead, the increase in amiloride-stimulated natriuresis and the decrease in furosemide-induced natriuresis together with the specific increase in Na+/K+-ATP-ase activity in CCD clearly revealed the pivotal role of the distal nephron as the major site of GH/IGF-I induced sodium retention in acromegaly. Of interest, 6 wk after tumor removal in GC rats with normal circulating GH concentrations, the natriuretic responses to furosemide and amiloride challenge returned to the normal ranges (data not shown), indicating that chronic GH and/or IGF-I exposure are indeed responsible for altered Na+ excretion. However, these functional renal investigations do not permit to precise the relative contribution of GH and IGF-I to the increased sodium transport prevailing in the CCD. Considering the distinct half-lives of GH (18 min) and IGF-I (8–9 h) (27, 28), the massive polyuria and dramatic tissue swelling regression generally observed a few hours after pituitary tumor removal in acromegalic patients support a direct and rapid control of renal sodium reabsorption by GH. Because GH concentrations in GC rats are about 2 orders of magnitude higher than those measured in acromegalic patients, one has to be cautious when translating these observations to human pathology.

The molecular mechanisms by which GH exerts its antinatriuretic effects are not entirely clear. The major effect of amiloride on sodium excretion demonstrated in GC rats points to a prominent role played by ENaC as the key mediator of GH action. Here, we show that chronic GH excess leads to major modifications in the proteolytic maturation of both {alpha} and {gamma}-subunits of ENaC in GC rat kidneys. Indeed, proteolytic cleavage of {alpha} and {gamma}-ENaC subunits by furin, a serine protease, is associated with increased channel activity (29, 30). Recent studies suggested that a second cleavage event within the {gamma}-ENaC subunit, by prostasin (31) or by neutrophil elastase (32), releasing an inhibitory peptide domain, was required to activate fully the channel (33). Along this line, the intermediate 80-kDa {gamma}-ENaC band observed in GC rats could represent an increased pool of furin-cleaved {gamma}-subunit. Finally, ENaC processing in the kidney is regulated by distinct physiological or pathological stimuli, including hyperaldosteronism, due to either sodium restriction or aldosterone administration (34, 35). However, increased sodium reabsorption in the CCD accompanied by ENaC proteolytic maturation despite low plasma aldosterone concentrations in acromegalic rats provides additional evidence that GH/IGF-I directly controls ENaC activity, regardless of the mineralocorticoid status.

To gain more insight into the molecular events underlying GH-induced regulation of sodium reabsorption in the distal nephron, we used the renal KC3AC1 cell line. To the best of our knowledge, our study is the first report demonstrating functional GHR expression in CCD-derived cells, the site of the aldosterone-sensitive sodium transport (36). In addition, because KC3AC1 cells did express functional IGF-IR but not IGF-I, this cell-based system also enabled us to assess directly the impact of IGF-I actions on CCD cells, independently from those induced by GH. As expected, GH treatment of KC3AC1 cells resulted in activation of canonical JAK2/STAT5 and ERK1/2 signaling pathways (37), and in the induction of classical GH target genes such as SOCS2 and CIS (38, 39). One of the main findings is that GH also exerts direct stimulatory effects on transcriptional regulation of {alpha}ENaC expression. This effect appears to be linked to JAK2/STAT5 activation because we have identified a STAT5 response element in the promoter of SCNN1A gene conserved in mice, rats, and humans. Because a cross talk between cytokine receptor signaling pathways and steroid receptors has been already documented (40), a functional link between MR, the key transcriptional regulator in CDD cells, and STAT5 is very likely given the vicinity of their respective response elements on the SCNN1A promoter. Whether MR and STAT5 physically interact and how they functionally cooperate to activate transcription remain to be addressed experimentally.

Our study conclusively demonstrates that GH exposure not only stimulates transcription of {alpha}ENaC but also affects ENaC subunits proteolytic maturation. Considering that ENaC activity depends on other complex regulatory mechanisms, including posttranscriptional regulation by Nedd4–2-mediated ubiquitinylation that controls apical membrane abundance as well as alteration of the open probability (Po) of functional channels (34, 36, 41), it remains to investigate whether the antinatriuretic effects of GH may also involve modification in the residency time and functional properties of the apical channels.

From a physiological perspective, it is interesting to note that lactogens and GH signaling play essential roles in the osmoregulation and in the salinity adaptation of teleost fish (42, 43). Consistent with our finding, a recent study suggests that prolactin induces sodium transport via stimulation of ENaC and Na+/K+-pump activities in the amphibian skin (44). Lactogen/GH-dependent ENaC activation could represent an important phylogenetic axis in the control of the "milieu intérieur" conserved throughout hundreds of millions of years of evolution.

Besides GH, IGF-I also stimulates transepithelial sodium transport, in accordance with previous studies on other mammalian and amphibian cell lines (45, 46, 47, 48). It has been proposed that, like insulin (49), IGF-I controls sodium transport via an increase in Sgk1 protein level and phosphatidylinositol 3-kinase (PI-3K)-mediated Sgk1 phosphorylation (48), leading to subsequent inactivation of the ubiquitin ligase Nedd4–2 (41). Here, we provide the first evidence that IGF-I also regulates Sgk1 transcription that, together with its well-known aldosterone-dependent induction (50), represents an additional cross talk between aldosterone and IGF-I signaling pathway.

Based on our results, we propose a model of cooperative GH and IGF-I action in CCD cells leading to an intricate control of transepithelial sodium reabsorption that is schematized in Fig. 8Go. GH binding to GHR triggers activation of JAK2/STAT5 and MAP kinase pathways, leading to transcriptional activation of the classical and kidney specific GH target genes, including {alpha}ENaC. ENaC activity is also modulated by posttranscriptional and posttranslational events, including proteolytic maturation of {alpha} and {gamma}-subunits. IGF-I, locally synthesized in the kidney or produced in the liver, binds to IGF-IR and regulates apical membrane abundance of ENaC via PI-3K-dependent Sgk1 activation (47, 48).


Figure 8
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  		FIG. 8. Proposed model of cooperative GH and IGF-I action in CCD cells.

 
In summary, our findings reveal the critical role of GHR signaling in the sodium transport of CCD cells. The demonstration of a direct GH action mediated by ENaC activation provides a novel mechanism that, in concert with IGF-I, accounts for the regulation of sodium homeostasis. Given the importance of sodium balance in the pathogenesis of arterial hypertension, our study opens new therapeutic strategies to prevent or cure cardiovascular complications in acromegalic patients.


   Acknowledgments
 
We thank Professor Stuart Frank (University of Alabama, Birmingham, AL) for his gift of anti-GH receptor AL-47 antibody and helpful advice for immunodetection assays, Dr. Miguel van Bemmelen and Professor Laurent Schild (University of Lausanne, Lausanne, Switzerland) for their gifts of anti-epithelial sodium channel antibodies, and Professor John Kopchick (Ohio University, Athens, OH) for his permission to use GH receptor knockout mice. We also thank Dr. Marie-Thérèse Bluet-Pajot [Institut National de la Santé et de la Recherche Médicale (INSERM) Unité (U) 549, Paris, France] for her assistance in the initial investigation of GC rats and Dr. Nadine Binart (INSERM U 845, Paris, France) for critical and helpful discussions. We thank Dr. Anne Davit-Spraul (Department of Biochemistry, Hôpital de Bicêtre, Bicêtre, France) for her assistance with ionic measurements, Annick Ganieux (Institut Fédératif de Recherche 93 Bicêtre, Bicêtre, France) for plasmid preparation, and Roman Bogorad (INSERM U 845, Paris, France) for immunoprecipitation assays.


   Footnotes
 
This work was supported by funding from Institut National de la Santé et de la Recherche Médicale and Université Paris-Sud 11, and a grant from Pfizer, France. Part of this study was supported by a research grant from Pfizer Laboratory (2008–2009, to P.C. and M.L.). P.K. was a recipient of fellowship from the Gouvernement Français (Egide) and from the Ministère de l’Enseignement Supérieur et de la Recherche, France.

Disclosure Statement: P.C. is a consultant of and received lecture fees from Novartis, Pfizer, and Ipsen Laboratories. P.K., S.V., A.B., G.M., P.Z., M.I.-T., and A.D. have nothing to declare.

First Published Online April 3, 2008

Abbreviations: CCD, Cortical collecting duct; CIS, cytokine-inducible SH2-containing protein; cTAL, cortical thick ascending limb of the Henle’s loop; ENaC, epithelial sodium channel; GHR, GH receptor; JAK, Janus kinase; MR, mineralocorticoid receptor; PCT, proximal convoluted tubule; PI-3K, phosphatidylinositol 3-kinase; Sgk, serum- and glucocorticoid-induced kinase; SOCS, suppressor of cytokine signaling; STAT, signal transducer and activator of transcription; TAL, thick ascending limb; WT, wild type.

Received January 30, 2008.

Accepted for publication March 21, 2008.


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Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
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Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals