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NDT Advance Access published online on November 8, 2008
Nephrology Dialysis Transplantation, doi:10.1093/ndt/gfn614
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© The Author [2008]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org


The role of Sgk-1 in the upregulation of transport proteins by PPAR-{gamma} agonists in human proximal tubule cells

Sonia Saad, David J. Agapiou, Xin-Ming Chen, Veronica Stevens and Carol A. Pollock

Kolling Institute of Medical Research, University of Sydney, Sydney, NSW, Australia

Correspondence and offprint requests to: Carol Pollock, Renal Research Laboratories, Kolling Institute of Medical Research, University of Sydney, Sydney, Australia. Tel: +61-2-9926-7126; Fax: +61-2-9436-3719; E-mail: carpol{at}med.usyd.edu.au



   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Background. Cellular sodium and water transport are dysregulated in diabetes mellitus. Synthetic peroxisome proliferator-activated receptor gamma (PPAR-{gamma}) agonists are currently used in the treatment of type 2 diabetes, but their use is limited by fluid retention. Recent data suggest that PPAR-{gamma} agonists stimulate distal tubular epithelial Na transport, potentially through the serine glucocorticoid kinase-1 (Sgk-1)-dependent regulation of the epithelial Na channel. We have recently demonstrated that Sgk-1 additionally regulates sodium reabsorption through the proximal tubular sodium hydrogen exchanger-3 (NHE3). However, the effects of PPAR-{gamma} agonists on Sgk-1, the water channel proteins aquaporins and on sodium transport in human proximal tubule cells (PTCs) have not previously been studied.

Methods. PTCs were exposed to the PPAR-{gamma} agonists, pioglitazone and the more selective PPAR-{gamma} agonist L-805645 with and without the Sgk inhibitor (GSK650394A). PPAR-{gamma}, Sgk-1, NHE3, AQP 1 and 7 mRNA and protein expression were determined by semi-quantitative PCR and western blot. The Sgk-1-specific effect was determined using Sgk-1 siRNA.

Results. Exposure of PTCs to 10 µM pioglitazone and 8 µM L-805645 increased the mRNA and protein expression of PPAR-{gamma} (P < 0.005), NHE3 and Sgk-1 (both P < 0.05). The expression of AQPs 1 and 7 was increased by pioglitazone and L-805645 (both P < 0.05). The increases in NHE3 and AQPs 1 and 7 were significantly reduced by pharmacological inhibition of Sgk and when cultures were exposed to Sgk-1-specific siRNA.

Conclusions. PPAR-{gamma} agonists enhanced the expression of NHE3, AQP 1 and 7 channels in human proximal tubule cells through Sgk-1-dependent pathways.

Keywords: diabetes mellitus; sodium transport; water channel



   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Serum and glucocorticoid regulated kinase (Sgk) is a novel member of the serine/threonine kinase gene family. To date three isoforms have been reported that share 80% amino acid identity, with tissue expression being highly organ specific. In the kidney, Sgk-1, -2 and -3 have been demonstrated [1,2]. The majority of research to date on Sgk-1 in the kidney has focused on the distal tubules, where Sgk-1 is thought to be a key mediator of aldosterone-induced sodium reabsorption through the epithelial sodium channel (ENaC) [3]. This involves alterations in the ubiquitination status of the ENaC subunits via a mechanism that is likely to involve Sgk-1 phosphorylation of Nedd-4 [4,5]. More recently, Sgk-2 has also been shown to be a potent stimulator of the {alpha}, β and {gamma} subunits of ENaC [2]. Recent data confirm that Sgk-1 mRNA and protein are induced by factors known to be integral to the diabetic state [6]. Results from our laboratory uniquely support the hypothesis that Sgk-1 mediates PTC proliferation and protects against apoptosis under high glucose conditions [7]. We and others have additionally shown that Sgk-1 promotes Na reabsorption via increased NHE3 in the PTC of the human kidney [8] in a process potentially involving the NHE regulatory factor (NHERF) [9] and mediated through the epidermal growth factor receptor (EGF-R) [7].

Peroxisome proliferator-activated receptor gamma (PPAR-{gamma}) is a member of the nuclear hormone receptor superfamily. Activation of the PPAR-{gamma} pathway requires heterodimerization of ligand-bound PPAR-{gamma} with the retinoid X receptor (RXR) to form a transcription factor that binds specific peroxisome proliferator response elements (PPRE) in the promoters of target genes [10]. Despite the extensive literature on the beneficial effects of synthetic PPAR-{gamma} agonists in limiting insulin resistance, protecting pancreatic β cell function and our own data suggesting a protective effect on nephropathy [11–15], salt and water retention pose a key limitation to the uptake of these agents in clinical practice.

It has recently been reported that Sgk-1 is a target gene for PPAR-{gamma} activation [16,17]. This suggests that excessive Na reabsorption in response to PPAR-{gamma} activation may occur through a regulated downstream activation of SgK-1 in the proximal tubule. However, prior studies have focused on PPAR-{gamma}—induced Na reabsorption in the collecting ducts, with the cellular pathways being almost exclusively studied in collecting tubule cell lines [18,19]. Hence, the functional consequences of PPAR-{gamma} agonists on Sgk-1-mediated sodium retention in the proximal tubule are not known.

Aquaporins (AQPs) are membrane-inserted water channel proteins that mediate water reabsorption from the renal tubular fluid. AQPs 1 and 7 are the key isoforms expressed in the proximal tubule. It is considered that AQP 1 plays the dominant role in proximal tubular water transport, whereas AQP 7 mediates water and glycerol transport [20].

The PPAR-{gamma} agonist, rosiglitazone, has been shown to induce AQPs 2 and 3 in whole kidney homogenates in rats [21], but whether this expression is secondarily regulated by changes in vasopressin is unknown. The effect of PPAR-{gamma} on AQP expression in human proximal tubule cells has been largely unexplored, and the molecular mechanisms that increase the expression and insertion of these channels into the membrane and channel activity are still poorly understood. Although Sgk-1 is considered to be a key modifier of cellular volume under conditions of osmotic stress [22], the parallel changes that occur in water transport as a consequence of altered sodium transport are considerably understudied. The mechanisms by which Sgk-1 interacts with PPAR-{gamma} and activates downstream alterations in salt and water transport in the human proximal tubule is unresolved and explored in the present studies.



   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Human PTC culture
The methods for isolation of primary culture of PTC are previously described [23]. In brief, the kidney cortex was dissected from the medulla, finely minced and then digested by collagenase (383 U/mg, Worthington) for 30 min at 37°C and passed through 100-µm mesh. The filtrate was resuspended in 50 ml of 45% Percoll (Pharmacia, Uppsala, Sweden) and centrifuged at 45 700 g, using a Beckman J2-MC centrifuge, at 4°C for 30 min. The bottommost tissue band containing the highly purified PTC was carefully removed and washed. The PTC fragment pellet was resuspended in serum-free hormonally defined media consisting of 1:1 (vol/vol) DMEM and Hams F-12 (DMEM/F-12; Trace, Noble Park, Australia) supplemented with 10 ng/ml (1.64 nM) epidermal growth factor, 5 mg/ml human transferrin, 5 mg/ml (0.87 mM) bovine insulin, 0.05 mM hydrocortisone, 50 mM prostaglandin E1, 50 nM selenium and 5 pM triiodothyronine (all from Sigma-Aldrich, MO, USA). Passage 2 cells were used for all experiments. The ultrastructure, growth and immunohistochemistry of PTC have been well characterized in our laboratory and shown to reproducibly reflect the biology and physiology of their in vivo counterparts [23,24].

Experimental protocol
Human proximal tubule cells were grown in 10-cm tissue culture dishes (Becton, Dickinson, NJ, USA). The clinically available thiazolidinedione pioglitazone (Cayman Chemical, MI, USA) and the more selective PPAR-{gamma} agonist L-805645 (Merck Biosciences, Darmstadt, Germany) were used to determine the specific effects of PPAR-{gamma} activation. Pioglitazone has a binding activity (IC50) to the recombinant human PPAR-{gamma} isoform of 3000 nM and L-805645 of 50 nM. The binding affinity of pioglitazone to PPAR-{gamma} is 20–40 µM and that of L-805645 is 2000 nM. Hence, L-805645 is more potent and more selective for PPAR-{gamma} [25]. Initial ‘dose-response’ experiments were undertaken to determine the concentration at which pioglitazone and L-805645 maximally stimulated PPAR-{gamma} protein expression. Based on these studies, 10 µM of pioglitazone and 8 µM of L-805645 were used in the experimental protocols as previously described [11,14].

Preliminary experiments have determined a maximal protein expression of AQP 1 and AQP 7 after 24 h treatment. Hence, the 24-h time point was used for the determination of cellular protein. Unless otherwise stated, PTCs were exposed to the following experimental conditions for 24 h: (1) 5 mM D-glucose (control media); (2) 10 µM pioglitazone in 5 mM D-glucose and (3) 8 µM L-805645 in 5 mM D-glucose. As L-805645 and pioglitazone were dissolved in 0.016 and 0.13% DMSO, respectively, additional controls were undertaken to evaluate independent effects of the DMSO at 0.13%. As transcriptional regulation precedes protein synthesis, mRNA expression was studied at earlier time points. Preliminary time course studies demonstrated maximal expression at 3 h. Hence mRNA expression is reported at 3 h.

To determine the role of Sgk in mediating observed changes, a recently synthesized Sgk inhibitor (GSK650394A) was used [26]. GSK650394A was provided by GlaxoSmithKline Pharmaceutical, PA, USA, for the purpose of this study. GSK650394A inhibits Sgk-1 and Sgk-2, with IC50 for Sgk-1 being 62 nM and IC50 for Sgk-2 being 103 nM. An initial dose-response experiment for GSK650394A was originally undertaken that defined 2 µM GSK650394A as the optimal dose to inhibit Sgk-1 and Sgk-2. Hence, this dose was used in all subsequent experiments. No change in cell viability could be detected at this concentration (as determined by MTT assay 90.5 ± 7.6% of control; P = 0.3). To ensure that the specificity of any observed effect was due to Sgk-1 inhibition, rather than being due to Sgk-2 or non-specific ‘off-target’ effects, studies were repeated using a specific Sgk-1 siRNA.

SGK-1 gene silencing
siRNA was designed to specifically target SGK-1 mRNA sites (Table 1) (Ambion, Austin, TX, USA). PTCs were transfected with SgK-1 siRNAs (4 nM final concentration) using Ribojuice (Novagen, Darmstadt, Germany), as per manufacturer's instructions. mRNA and protein levels were determined using semi-quantitative PCR and western blot. All siRNA experiments included non-specific control transfection of cells (non-specific control siRNAs, Ambion).


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  		Table 1 SGK-1 siRNA sequences

 
Once Sgk-1 knockdown was confirmed, subsequent experiments were conducted to determine the effect of exposure to pioglitazone and L-805645 on Sgk-1 and AQPs 1 and 7 in PTCs in which Sgk-1 was effectively silenced. Twenty-four hours after SgK-1 knockdown, cells were exposed to control conditions (5 mM glucose), pioglitazone (10 µM) or L-805645 (8 µM) for either 3 or 24 h and total RNA or cell lysates were collected. Semi-quantitative PCR and western blotting experiments were performed as described below. Sgk-1 siRNA had no effect on Sgk-2 mRNA.

SGK-1 RT-PCR
Total RNA was extracted using a RNeasy kit (QIAGEN), according to the manufacturer's instructions. RNA was reverse transcribed using the Superscript II Reverse Transcriptase kit (GibcoBRL, MD, USA). The sequence of the primers is shown in Table 2. PCR conditions were an initial denaturation at 94°C for 1 min, followed by 35 cycles at 94°C for 60 s, 60°C for 1 min and 30 s, 72°C for 2 min, with a final extension at 72°C for 10 min using the Expand High Fidelity PCR System (Roche, Mannheim, Germany). Amplification products were electrophoresed through 2% agarose gels to determine RT-PCR product sizes. All experiments were performed in triplicate, using negative (no template) and positive actin controls. Gels were stained with ethidium bromide and photographed. The photograph was then scanned into a computer and the relative intensities of the individual bands were determined using the Image J analysis software (National Institutes of Health, USA).


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  		Table 2 RT-PCR oligonucleotide primer quantified using NIH Image software v1.60 sequences

 
Western blotting
Western blots were performed on Triton X-100 soluble fractions. Protein concentrations were determined using the Bio-Rad protein assay (Hercules, CA, USA). Samples were run on SDS–PAGE under reducing conditions and transferred to the Hybond ECL nitrocellulose membrane (Amersham Pharmacia, Germany). The membranes were blocked in 5% skim milk, and incubated with the pan-Sgk antibody (Cell Signaling, Beverly, MA, USA), Sgk-1-specific antibody (Abcam, Cambridge, UK), NHE3 antibody (BD Biosciences, Franklin Lakes, NJ, USA) or AQP 1 and 7 antibody (Chemicon International, CA, USA) overnight at 4°C followed by incubation with an anti-rabbit or anti-mouse antibody (Amersham Pharmaceuticals, USA) for 1 h at room temperature. Protein was detected using an ECL Kit (Amersham Pharmaceuticals). The bands corresponding to Sgk-1 (49 kDa), AQP 1 (28 kDa), AQP 7 (30 kDa), PPAR-{gamma} (67 kDa) and NHE3 (85 kDa) were quantified using NIH Image software v1.60. A non-specific band of ~120 kDa was detected using the NHE3 antibody. Doublets were detected when using the Sgk-1-specific antibody as noted in Figure 5B. The identity of NHE3 and Sgk-1 was confirmed by gene expression (data not shown). Results of protein expression are normalized to actin in all figures.


Figure 5
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  		Fig. 5 (A). Decreased expression of Sgk-1 in gene-silenced cells. PTCs were transfected with either non-specific siRNA control (NSC) or Sgk-1 siRNA. RT-PCR was performed after 24 h. Upper panel: representative image for Sgk-1 and actin bands. Lower panel: normalized results expressed as means ± SE; n = 4. {ddagger}P < 0.0005 versus control. (B). Sgk-1 protein expression in sgk-1-silenced cells. Cells were transfected with either NSC or Sgk-1 siRNA. Sgk-1 protein was detected using the Sgk-1-specific antibody as described in the Materials and methods section. Upper panel: representative images for Sgk-1 and actin bands. Lower panel: normalized results expressed as means ± SE; n = 3. {ddagger}P ≤ 0.0005, #P < 0.005 and *P < 0.05 versus NSC.

 
Confocal immunofluorescence
Confocal microscopy was performed on PTCs grown on Cell-Tak (BD Biosciences, Bedford, MA, USA)-coated cover slips as follows. PTCs were fixed with 2% paraformaldehyde in PBS for 20 min. The Cells were washed with PBS and then blocked with 1% (w/v) bovine serum albumin (BSA) in PBS at room temperature. The cells were then incubated either with anti-NHE3 antiserum (1:1000), anti-AQP 1 antibody (1:50) or anti-AQP 7 antibody (1:50) for 2 h at room temperature, washed and incubated with the relevant FITC antibody (1:200) for 1 h. Slides were sealed with a mounting medium (Dako, Carpenteria, CA, USA) and visualized under a Leica TCS NT laser confocal microscope (Leica, Solms, Germany) with excitation at 488 nm and emission at 570 nm.

Statistical analysis
In vitro experiments were performed at least in triplicate on three to nine different patients’ cell culture preparations. Unless otherwise stated, results are expressed as a percentage of control values (cells exposed to 5 mM glucose). Statistical comparisons between groups were made by analysis of variance (ANOVA) or paired t-tests where appropriate. Analyses were performed using the software package, Statview version 4.5 (Abacus Concepts Inc., Berkley, CA, USA). All data are reported as mean ± SEM. P-values <0.05 were considered significant.



   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
PPAR-{gamma} agonists induced increased PPAR-{gamma} expression
Twenty-four-hour exposure to 10 µM pioglitazone and 8 µM of L-805645 induced a significant upregulation of the PPAR-{gamma} protein expression to 142.3 ± 6.2% (n = 3; P = 0.002) and 401 ± 30.2% (n = 3; P = 0.0006) of control values, respectively (Figure 1).


Figure 1
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  		Fig. 1 Pioglitazone and L-805645 induce PPAR-{gamma} protein expression. PTCs were incubated for 24 h with control media, 10 µM pioglitazone or 8µM L-805645 in control media. Western blotting is as described in the Materials and methods section: representative image for PPAR-{gamma} and actin bands and its graphical representation. Normalized results are expressed as mean ± SEM; n = 3. #P < 0.005 versus control.

 
PPAR-{gamma} agonists induced increased Sgk-1 mRNA and protein expression
Sgk-1 mRNA expression was significantly increased following short-term exposure to pioglitazone and L-805645 [147.5 ± 12.0% (n = 4; P = 0.007) and 215 ± 38.8% (n = 4; P = 0.02; Figure 2A]. This increase in the mRNA level was reflected in the significant increase in the protein level following 24-h exposure to pioglitazone and L-805645 [150 ± 15.2% (n = 4) and 203 ± 32.6% (n = 4); both P = 0.01; Figure 2B]. Pioglitazone and L-805645 had no effect on Sgk-2 protein and mRNA expression (data not shown).


Figure 2
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  		Fig. 2 (A) Pioglitazone induces Sgk-1 expression. PTCs were incubated for 3 h with control media or 10 µM pioglitazone or 8 µM L-805645 in control media. RT-PCR is as described in the Materials and methods section. Upper panel: representative image for Sgk-1 and actin bands. Lower panel: normalized results expressed as means ± SE; n = 4. *P < 0.05 versus control. (B) Pioglitazone and L-805645 induce Sgk-1 protein expression. PTCs were incubated for 24 h with control media, 10 µM pioglitazone or 8µM L-805645 in control media. Western blotting using a pan-Sgk antibody is as described in the Materials and methods section. Upper panel: representative image for Sgk-1 and actin bands. Lower panel: normalized results expressed as means ± SE; n = 4. *P < 0.05 versus control.

 
PPAR-{gamma} agonists induced NHE3 mRNA and protein expression
The NHE3 mRNA level was significantly increased following exposure to pioglitazone and L-805645 [153 ± 17.3% (n = 3; P = 0.03) and 198 ± 21.8% (n = 3; P = 0.01; Figure 3A]. Similarly, treating the cells with pioglitazone and L-805645 induces the NHE3 protein expression to 184 ± 24.3% (n = 9; P = 0.005) and 200 ± 40.9% (n = 9; P = 0.02; Figure 3B).


Figure 3
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  		Fig. 3 (A) Pioglitazone induces NHE3 expression. PTCs were incubated for 3 h with control media or 10 µM pioglitazone. RT-PCR is as described in the Materials and methods section. Upper panel: representative image for NHE3 and actin bands. Lower panel: normalized results expressed as means ± SE; n = 3. *P < 0.05 versus control. (B) Pioglitazone and L-805645 induce NHE3 protein expression. PTCs were incubated for 24 h with control media, 10 µM pioglitazone or 8 µM L-805645 in control media. Western blotting is as described in the Materials and methods section. Top: representative image for NHE3 and actin bands. Bottom: normalized results expressed as means ± SE; n = 9. *P < 0.05 and #P ≤ 0.005 versus control.

 
Induction of NHE3 after exposure to PPAR-{gamma} agonists is dependent on Sgk in PTCs
We have previously demonstrated an increase in NHE3 expression following Sgk-1 overexpression in human PTCs [7]. In order to determine the role of Sgk in PPAR-{gamma} agonists’ induction of NHE3, the Sgk-1/-2 inhibitor GSK650394A was used. GSK650394A significantly reduced the NHE3 protein expression to 85 ± 5.9% (n = 4; P = 0.04). The combination of GSK650394A with pioglitazone or L-805645 abrogated the observed increase in NHE3 expression with the PPAR-{gamma} agonists to 101 ± 11.1% (n = 4; P = 0.03) and 88 ± 9.0% (n = 4; P = 0.001) of their control values, respectively (Figure 4). These results suggested that the upregulation of NHE3 following exposure to PPAR-{gamma} agonists is Sgk mediated and subsequent experiments using siRNA confirmed the dependence on the Sgk-1 isoform.


Figure 4
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  		Fig. 4 GSK650394A inhibits PPAR-induced NHE3 protein expression. PTCs were incubated for 24 h with control media, 10 µM pioglitazone or 8 µM L-805645 in control media with or without 2 µM GSK650394A. Western blotting is as described in the Materials and methods section. Upper panel: representative image for NHE3 and actin bands. Lower panel: normalized results expressed as means ± SE; n = 4. *P < 0.05 and #P < 0.005 versus relevant control.

 
Induction of NHE3 after exposure to PPAR-{gamma} agonists is dependent on Sgk-1 in PTCs
GSK650394A inhibits both Sgk-1 and Sgk-2 isoforms and is known to have additional off-target effects, e.g. it may inhibit the JNK pathway in high concentrations. Hence, in order to determine the dependence of the PPAR-{gamma}—induced increase in Na reabsorption on Sgk-1, experiments were repeated using Sgk-1-specific siRNA. The silencing of Sgk-1 was confirmed by mRNA expression. A significant reduction of Sgk-1 mRNA was shown to be 43.5 ± 5.0% of control (n = 4; P < 0.0001; Figure 5A). Sgk-2 mRNA levels were not affected by Sgk-1 siRNA (data not shown). Sgk-1 protein expression was also significantly reduced in cells with Sgk-1 siRNA compared to cells transfected with non-specific control (NSC) siRNA to 35 ± 6.1% of NSC siRNA (n = 3; P = 0.0005). The previously demonstrated increase of the Sgk-1 protein following pioglitazone and L-805645 treatment was also significantly reduced with Sgk-1siRNA to 40 ± 8.5% of NSC siRNA (P = 0.002) and 31 ± 10.8% of NSC siRNA (P = 0.003, respectively; n = 3) (Figure 5B).

When Sgk-1 was significantly silenced, the previously demonstrated increase of NHE3 following pioglitazone and L-805645 treatment was significantly reduced to 82 ± 7.3% (n = 3; P = 0.001) and 78 ± 11.7% (n = 3; P = 0.0004), respectively. Sgk-1 silencing also significantly reduced basal NHE3 expression to 65 ± 12.4% of control values (n = 3; P = 0.04; Figure 6).


Figure 6
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  		Fig. 6 PPAR-induced NHE3 is Sgk-1 mediated. PTCs were incubated for 24 h with control media, 10 µM pioglitazone or 8 µM L-805645. NHE3 protein expression was shown in cells with NSC or Sgk-1 siRNA. Upper panel: representative image for NHE3 and actin bands. Lower panel: normalized results expressed as means ± SE; n = 4. {ddagger}P < 0.0005, #P < 0.005 and *P < 0.05 versus relevant control.

 
PPAR-{gamma} agonists induced AQP 1 and 7 expressions
Both AQP 1 and 7 mRNA expression were induced after 3-h treatment to 140.3 ± 9.4 (n = 3; P = 0.01) and 136.4 ± 7.6 (n = 3; P = 0.008), respectively (Figure 7A and B).


Figure 7
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  		Fig. 7 Pioglitazone induces AQP 1 and 7 mRNA levels. PTC were treated with control (5 mM glucose) media or pioglitazone. (A) Upper panel: representative image for AQP 1 and actin bands. Lower panel: normalized results expressed as means ± SE; n = 3; *P < 0.05 versus control. (B) Upper panel: representative image for AQP 7 and actin bands. Lower panel: normalized results expressed as means ± SE; n = 3. #P < 0.01.

 
The increase of AQP 1 following exposure to PPAR-{gamma} agonists is Sgk-1 mediated
Pioglitazone and L-805645 significantly induced protein expression of AQP 1 to 168 ± 21.3% (n = 3; P = 0.003) and 162 ± 5.1% (n = 3; P = 0.005), respectively. This induction was significantly reduced when cells were concomitantly treated with the Sgk inhibitor GSK650394A to 60 ± 11.5% (P < 0.0001) and 67 ± 17.2% (P < 0.0005), respectively (n = 3) (Figure 8A). GSK650394A reduced the basal expression of AQP 1 to 67.6 ± 12.6% of control (n = 3), although this difference was not statistically significant (P = 0.06). The role of Sgk-1 in the effects of PPAR-{gamma} agonist on AQP 1 was confirmed using Sgk-1 siRNA. The upregulation of AQP 1 with pioglitazone and L-805645 was reduced in Sgk-1-silenced cells compared to wild-type cells to 79 ± 6.3% (n = 3; P = 0.01) and 71 ± 3.0% (n = 3; P = 0.005), respectively. Sgk-1 silencing also significantly reduced basal levels of AQP 1 to 78 ± 6.3% of control (n = 3; P = 0.02). These results clearly demonstrate a role of Sgk-1 in the PPAR-{gamma}—induced AQP 1 expression (Figure 8B).


Figure 8
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  		Fig. 8 (A) PPAR-{gamma}-induced AQP 1 expression is Sgk-1 mediated. PTCs were incubated for 24 h with control media, 10 µM pioglitazone or 8 µM L-805645 in control media with or without 2 µM GSK650394A (n = 3) (A) or with NSC or Sgk-1 siRNA (n = 3) (B). Western blotting is as described in the Materials and methods section. Upper panel: representative images for AQP 1 and actin bands. Lower panel: normalized results expressed as means ± SE; {ddagger}P ≤ 0.0005, #P ≤ 0.005 and *P < 0.05 versus relevant control.

 
The increase of AQP 7 following exposure to PPAR-{gamma} agonists is Sgk-1 mediated
Pioglitazone and L-805645 significantly induced the AQP 7 protein expression to 192 ± 40.6% (n = 4; P = 0.01 and 198 ± 29.4% (n = 4; P = 0.01), respectively. This induction was reduced when cells were treated with the Sgk inhibitor GSK650394A to 67 ± 12.0% (n = 4; P = 0.002) and 76 ± 13.5% (n = 4; P = 0.002), respectively (Figure 9A). The upregulation of AQP 7 with pioglitazone and L-805645 in Sgk-1-silenced cells was reduced to 59 ± 13.7% (n = 3; P < 0.0001) and 72 ± 9.7% (n = 3; P = 0.0002), respectively. These results demonstrate a role of Sgk-1 in PPAR-{gamma}—induced AQP 7 expression (Figure 9B).


Figure 9
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  		Fig. 9 (A) PPAR-{gamma}-induced AQP 7 expression is Sgk-1 mediated. PTCs were incubated for 24 h with control media, 10 µM pioglitazone or 8 µM L-805645 in control media with or without 2 µM GSK650394A (A) or with NSC or Sgk-1 siRNA (B). Western blotting is as described in the Materials and methods section. Upper panel: representative images for AQP 7 and actin bands. Lower panel: normalized results expressed as means ± SE; n = 4. {ddagger}P < 0.0005, #P < 0.005 and *P < 0.05 versus relevant control.

 
The increase in NHE3, AQP 1 and 7 expressions and their distribution following pioglitazone treatment
The increase in the protein levels of NHE3, AQP 1 and AQP 7 following pioglitazone treatment was confirmed using confocal microscopy (Figure 10). Immunofluorescence staining revealed intracellular and apical distribution for NHE3, AQP 1 and AQP 7 in PTCs. This is consistent with the known distribution of these channels.


Figure 10
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  		Fig. 10 Pioglitazone induces NHE3, AQP 1 and AQP 7 expression. Representative images of confocal immunofluorescence microscopy showing distribution of NHE3 (A), AQP 7 (B) and AQP 1 (C) in PTCs with and without pioglitazone. Images are representative of three separate experiments. Scans through the cells, at the same plane, showing intracellular and apical distribution of NHE3, AQP 7 and AQP 1 that are increased with pioglitazone. (B) Distribution of AQP 7 in PTCs with and without piogliazone.

 
PPAR-{gamma} agonist increase in NHE3, AQP 1 and AQP 7 is persistent over time
Since PPAR-{gamma} agonists develop their full effect on body weight and fluid retention over multiple days, the effect of pioglitazone on the expression of NHE3, AQP 1 and AQP 7 over a period of 7 days was determined. NHE3, AQP 1 and AQP 7 proteins significantly increased when treated with pioglitazone to 193 ± 28.2% (n = 3; P < 0.05), 180 ± 26.2% (n = 4; P < 0.05) and 155 ± 19.2% (n = 3; P < 0.05), respectively (Figure 11A–C).


Figure 11
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  		Fig. 11 Pioglitazone-induced NHE3 (A), AQP 1 (B) and AQP 7 (C) with their graphical representation over a period of 7 days. Western blotting is as described in the Materials and methods section. Normalized results expressed as means ± SE; *P < 0.05 versus relevant control.

 


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
It is recognized from our own studies [29–31] and those of others [32,33] that cellular Na transport is dysregulated in patients with diabetes mellitus. As a result of its wide distribution and regulation by diverse stimuli, it is thought that Sgk is an important focal point for the convergence of intracellular cross-talk pathways that homeostatically regulates both sodium and water transport in the kidney. Sgk-1 is now recognized to be activated at both the transcriptional and post-translational levels by a number of extracellular signals integral to the diabetic state, including high glucose, angiotensin II and elevated transforming growth factor beta. We have recently demonstrated that high glucose regulates the key transporter of proximal tubular Na reabsorption, NHE3, through an epidermal growth factor-receptor (EGF-R)-mediated upregulation of Sgk-1 [7].

In parallel, we have also recently demonstrated that PPAR-{gamma} in human models of proximal tubular cells are activated by exposure to high glucose [11], as well as by the insulin-sensitizing agents, thiazolidinediones (TZDs), which are currently used for the treatment of type 2 diabetes [34]. The propensity for these drugs to cause fluid retention, resulting in pulmonary and peripheral oedema, has emerged recently as the most common, serious adverse drug reaction associated with these compounds [35]. The causes of oedema and fluid retention with the use of TZDs are speculative at this stage [16].

In the present study, we observed that PPAR-{gamma} is expressed on human PTCs and its level is upregulated following treatment with PPAR-{gamma} ligands, the clinically available TZD (pioglitazone) and the more selective PPAR-{gamma} agonist L-805645. This is consistent with our previous study using a primary human proximal tubule cell line HK-2 cells [11]. Because PPAR-{gamma} is well recognized to dysregulate renal water and sodium transport [36], and due to the fact that the proximal tubules mediate 50–75% of total tubular Na and water reabsorption, the role of PPAR-{gamma} in sodium and water transport in human PTCs was investigated.

Our studies clearly demonstrate an increase in NHE3 expression following exposure to PPAR-{gamma} agonists in human proximal tubule cells, and this increase is persistent over time. Similarly, Song et al. have demonstrated an increase in the NHE3 protein expression following the use of rosiglitazone in the whole kidney of Sprague Dawley rats through an unknown mechanism [21]. Zanchi et al. have demonstrated a lower level of lithium clearance, as an indirect measurement of renal proximal sodium absorption, during administration of pioglitazone suggesting an increased reabsorption of sodium in the proximal tubule [37]. Recently, a number of reports have sought to address the effects of TZDs on kidney function in rodent models [18,19]. Most of these studies specifically assessed sodium transport in the distal nephron. A highly potent and selective PPAR-{gamma} agonist, Farglitazar, was shown to increase sodium reabsorption in the distal nephron in rats through a mechanism potentially involving stimulation of the ENaC and the Na-K-ATPase pump [17]. However, the in vivo treatment with Farglitazar did not result in major changes in any ENaC subunits, even though fluid retention was clearly observed [17]. In addition, Chen et al. have also reported that amiloride (an inhibitor of collecting duct salt absorption acting through ENaC) did not prevent but rather enhanced the TZD-induced blood volume expansion [17]. Although available studies clearly demonstrate that the distal nephron is involved in the fluid retention following the use of PPAR-{gamma} agonists, the concurrent work has suggested that Sgk-1 is mechanistically involved [16,17], which suggests a possible role for the proximal tubule. Our data support the hypothesis that PPAR-{gamma} agonists increase sodium sensitive transporters in the proximal tubule.

Hong et al. have demonstrated that PPAR-{gamma} can bind to specific response elements in the Sgk-1 promoter. In addition, Sgk-1 has been shown to mediate PPAR-{gamma}—induced increases in cell surface ENaC alpha in the human distal tubule [16]. In contrast, Nofziger et al. did not observe any PPAR-{gamma}—induced changes in the amount of Sgk transcript or protein expression when using mouse principal kidney cortical collecting duct cells (mpkCCDc14) [38]. Their studies concluded that the TZDs are not contributing to the fluid retention through direct regulation of ENaC or insulin-stimulated ENaC activity. These studies supported the possibility that the agonists may be acting on water and Na transporting machinery located prior to the distal convoluted tubule [38]. Similarly, Guan et al. could find no evidence for increased Sgk-1 mRNA expression in cultured mouse inner medullary collecting duct cells [18]. However, in these prior studies, the proximal tubular contribution to sodium and water retention was not specifically studied in in vivo models or indirectly assessed in cell culture experiments. We have uniquely demonstrated in the present study an increase in Sgk mRNA and protein levels following PPAR-{gamma} activation and that the PPAR-{gamma}—mediated NHE3 expression in human PTCs is Sgk-1 mediated.

Exposure to PPAR-{gamma} has been shown to increase the selective water channel aquaporin-2 (AQP 2) in the distal tubule [17]. The effect of PPAR-{gamma} on AQP expression in human PTCs has been largely unexplored, and the molecular mechanisms that increase channel activity are still poorly understood. We have demonstrated that PPAR-{gamma} agonists induce the constitutively active AQP 1 and AQP 7 (key isoforms expressed in the proximal tubule) and that this increase is Sgk-1 mediated. The increase in AQPs 1 and 7 following PPAR-{gamma} agonist is persistent over a period of 7 days. This finding is of clinical relevance especially since PPAR-{gamma} agonists develop their full effect on body weight and fluid retention over multiple days.

Schnermann et al. demonstrated that deficiencies in NHE3 and in the water channel aquaporin 1 (AQP 1) cause reductions in proximal fluid absorption that are accompanied by proportionate decrements in glomerular filtration rate [39,40]. In addition, the water permeability of the proximal straight tubule brush border membrane measured by the stopped flow method was reduced in AQP 7 knockout mice compared to wild-type mice [20]. These studies support our findings that highlight the importance of the proximal tubule in sodium and water transport following the use of TZDs.

In conclusion, prior studies have suggested that PPAR-{gamma} agonists stimulate Sgk-1-mediated sodium transport in the distal tubule. The present in vitro study demonstrates that PPAR-{gamma} agonists enhanced the expression of NHE3, AQP 1 and 7 channels in human proximal tubule cells through Sgk-1-dependent pathways. The in vivo relevance of these findings remains to be established.



   Acknowledgments
 
We acknowledge the support of the National Health and Medical Research Council of Australia, Merck Laboratories for generously providing L-805645 and GlaxoSmithKline pharmaceutical for generously providing GSK65039A. We thank the Electron Microscopy Unit at Sydney University for scanning the confocal images.

Conflict of interest statement. None declared.



   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received for publication: 2. 6.08
Accepted in revised form: 9.10.08


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