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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online August 15, 2003 as doi:10.1096/fj.03-0181fje. |
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activation enhances cell surface ENaC
via up-regulation of SGK1 in human collecting duct cells1
Translational Medicine and Technology, GlaxoSmithKline, ACCI, Addenbrooke’s Hospital, Cambridge, CB2 2GG, UK;
* Translational Medicine and Technology, GlaxoSmithKline, Research Triangle Park, North Carolina, USA;
Clinical Pharmacology Unit, Addenbrooke’s Hospital, Cambridge, UK; and
Unite INSERM 489 and Universite Paris 6, Hopital Tenon, Paris, France
2Correspondence: Translational Medicine and Technology, GlaxoSmithKline, ACCI, Addenbrooke’s Hospital, Hills Road, Cambridge, CB2 2GG, UK. E-mail: Guizhu_2_Hong{at}gsk.com
SPECIFIC AIMS
It has been reported that peroxisome proliferator-activated receptor gamma (PPAR
) mRNA is present in human medullary collecting ducts of the kidney, where its role remains unclear. To understand the biological function of PPAR in the kidney, we investigated the effects of PPAR on the expression and activity of serum and glucocorticoid-regulated kinase (SGK1) and epithelial sodium channel (ENaC).
PRINCIPAL FINDINGS
1. PPAR is expressed in human cortical collecting ducts
Immunohistochemical staining of normal human kidney sections revealed that PPAR was expressed in the distal convoluted tubule, cortical collecting ducts (CCDs), and medullary CDs. Consistent with these findings, we detected PPAR expression in a previously characterized human CCD cell line (HCCD). These findings complement and extend previous reports that have detected PPAR mRNA in human medullary CDs.
2. PPAR activation enhances the activity of SGK1 but not protein kinase B
We initially examined the effects of PPAR agonist treatment on PI3-K-mediated signaling in HCCD cells by measuring the activities of two downstream signal transducers: protein kinase B (PKB) and SGK1. Saturating doses of thiazolidinediones, pioglitazone (20 µM), or rosiglitazone (2 µM) were used in all assays; the higher concentration of pioglitazone reflects its 
10-fold lower potency for activating PPAR in a cell-based transactivation assay. TZD treatment of HCCD cells for 4 h (in the presence of 0.5% serum) resulted in a significant increase in the activity of SGK1 but no change in the activity of PKB (Fig. 1
a, b). These findings suggest that the activity of the proximal PI3-K-dependent cascade, and specifically of 3-phosphoinositide-dependent kinase 1 and 2 (PDK1 and PDK2, the upstream activators of both PKB and SGK1), were not significantly regulated by PPAR activation over the time course of our experiments. Therefore, the acute enhancement of SGK1 activity we observed is probably independent of PDK1/2 activation.
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3. SGK1 mRNA expression is induced by PPAR agonists in HCCD cells
To determine whether the enhancement of SGK1 activity by PPAR agonists resulted from an increase in SGK1 expression, we measured levels of SGK1 mRNA in cells treated with rosiglitazone in the presence of 0.5% serum. Consistent with a previous report, SGK1 mRNA was rapidly induced by serum (2-fold within 30 min), reaching maximal levels after 2 h treatment and gradually declining thereafter. Rosiglitazone treatment augmented serum-induced SGK1 expression (Fig. 1c
). Treatment with pioglitazone also resulted in a significant increase in SGK1 transcripts (Fig. 1d
). These findings suggest that the increased activity of SGK1 observed after TZD treatment is mediated via the direct up-regulation of SGK1 mRNA expression.
4. PPAR complexes bind to a predicted PPRE in the promoter of the SGK1 gene
Bioinformatic analysis of the human SGK1 gene predicted six potential PPREs within 1825 bp upstream of the translational start site (27590 bp – 29415 bp of GenBank accession number AL135839). Electromobility shift assays were used to determine whether these putative PPREs bound PPAR; these assays were performed in the presence of retinoic X receptor (RXR), with which PPAR forms a heterodimer to constitute the DNA binding moiety. Only one of the six putative PPREs (corresponding to 1801 bp 1778 bp upstream of the SGK1 translation start) formed a complex with PPAR/RXR.
The increase in SGK1 message in response to PPAR activation and the identification of a PPRE in the SGK1 promoter together provide strong evidence that the SGK1 gene is directly regulated by PPAR.
5. PPAR enhances translocation of ENaC
to the plasma membrane of HCCD
Having demonstrated that PPAR activation directly increases SGK1 expression in HCCD cells, we sought to determine the functional consequences of enhanced SGK1 activity. SGK1 activation by aldosterone and insulin results in an increased translocation of preexisting ENaC to the apical membrane of epithelial cells. We assessed the effect of TZDs on levels of cell surface ENaC in HCCD cells by performing cell surface biotinylation, followed by isolation of labeled proteins with streptavidin agarose and detection of ENaC by immunoblotting. TZD treatment for 4 h resulted in significant increases in the cell surface expression of ENaC (Fig. 2
a). This suggests that one functional consequence of PPAR-induced SGK1 activation in HCCD cells is enhanced translocation of ENaC to the cell surface. As shown for regulation by aldosterone, we would expect this to result in enhanced activity of the ENaC channel.
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6. PPAR agonists also stimulate ENaC expression in HCCD cells after 24 h treatment
A significant increase in ENaC mRNA expression induced by PPAR activation was detected after 24 h treatment, but not after 4 h (Fig. 2b
). Consistent with the mRNA data, total ENaC protein levels were elevated by PPAR activation only after 24 h (Fig. 2c
). These findings are consistent with a recent report showing that PPAR agonists increase ENaC protein levels (to 164% of control levels) in rat kidney. It is likely that this regulation of ENaC expression by PPAR is indirect, as no PPREs were predicted in the promoter region of the ENaC gene (data not shown). These findings have two implications: 1) enhanced cell surface ENaC observed after 4 h of PPAR agonist treatment is not due to an increase in total ENaC protein levels and 2) cell surface ENaC may be regulated in two phases by PPAR activation, with a later enhancement caused by increased ENaC protein synthesis after the initial, SGK1-dependent translocation of existing ENaC.
CONCLUSIONS AND SIGNIFICANCE
Previous studies have shown that early activation of SGK1 by aldosterone is sufficient to increase ENaC-mediated current primarily via enhancement of ENaC translocation to the cell membrane and that subsequently synthesis of ENaC is induced by aldosterone (whereas that of ENaCß or ENaC is not). The preliminary data show that aldosterone increases SGK1 and ENaC mRNA expression in an order similar to that seen with TZD treatment in HCCD cells (data not shown). Our results therefore indicate that PPAR activation may enhance sodium reabsorption in the CCD in a manner similar to aldosterone via regulation of cell surface ENaC levels through coordinate effects on SGK1 and ENaC expression (Fig. 3
). Clearly, other possible consequences of PPAR activation in the kidney remain to be determined.
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The use of TZDs for the treatment of type 2 diabetes has been reported to be associated with an occasional syndrome of sodium retention, the mechanism of which has been obscure. A significant implication of our findings is that sodium retention could be caused by TZDs through the aldosterone-like stimulation of ENaC insertion into the plasmalemma that we have described. Our results therefore point to the possibility that the sodium retention sometimes seen with TZD treatment could be most effectively managed by coadministration of diuretics that act by inhibiting the action of aldosterone or by inhibiting ENaC such as spironolactone and amiloride.
In summary, our work demonstrates that 1) the SGK1 gene is a novel target for direct transcriptional regulation through binding of PPAR/RXR to a predicted PPRE in its promoter sequence, 2) activation of SGK1 by PPAR results in an increase in the cell surface localization of ENaC, and 3) prolonged treatment with PPAR agonists leads to increased ENaC expression. The significance of these findings lies not only in their demonstration that PPAR activation is likely to lead to increased sodium reabsorption in the cortical collecting duct; they also suggest a possible mechanism of TZD-associated sodium retention and thus point to potential ways to treat this effect.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0181fje; doi: 10.1096/fj.03-0181fje 
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