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NDT Advance Access originally published online on December 27, 2006
Nephrology Dialysis Transplantation 2007 22(3):722-731; doi:10.1093/ndt/gfl668
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© The Author [2006]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Enhancement of epithelial sodium channel expression in renal cortical collecting ducts cells by advanced glycation end products

Chiz-Tzung Chang1,2, Mai-Szu Wu2, Ya-Chung Tian2, Kuan-Hsing Chen2, Chun-Chen Yu2, Chang-Hui Liao1, Cheng-Chieh Hung2 and Chih-Wei Yang2

1Graduate Institute of Clinical Medical Sciences, Chang Gung University and 2Kidney Research Institute, Chang Gung Memorial Hospital and Chang Gung University, Taoyuan, Taiwan

Correspondence and offprint requests to: Cheng-Chieh Hung, MD, PhD, Kidney Research Institute and Department of Nephrology, Chang Gung Memorial Hospital, 5, Fu-Shing St., Kueishan, Taoyuan 333, Taiwan. Email: cchung9651{at}yahoo.com



   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Background. The epithelial sodium channel (ENaC) is a complex, and the {alpha}ENaC subunit has a crucial role in sodium uptake induced by aldosterone in the distal nephron. Although experimental animal models of diabetes have demonstrated up-regulation of {alpha}ENaC expression in renal cortical collecting duct (CCD) cells, the molecular mechanism remains unclear. Advanced glycation end products (AGEs) are by-products of long-term hyperglycaemia and comprise a significant pathogenic factor in diabetic nephropathy. We hypothesize that AGEs play a role in regulating {alpha}ENaC gene expression.

Methods. Mouse CCD cells (mpkCCDcl4) were cultured with AGE to determine the effects of AGE on {alpha}ENaC expression and sodium uptake. Gene expressions of ENaC were measured by real-time PCR and sodium uptake was measured with fluorescent dye as a sodium indicator (SBFI-AM). This study analysed mitogen-activated protein kinases signalling pathways by western blotting. Cells co-transfected with plasmids of the {alpha}ENaC promoter carrying a luciferase reporter and plasmids expressing wild-type or mutant serum- and glucocorticoid-induced kinase 1 (Sgk1) mRNA were stimulated with AGE to identify the signalling pathway.

Results. The AGEs, stimulated in a time- and dose-dependent manner, enhanced {alpha}ENaC mRNA expression and sodium uptake in mpkCCDcl4 cells. The AGEs also significantly stimulated Sgk1 mRNA and Sgk1 activity in a time- and dose-dependent manner. Co-transfected with plasmid expressing mutant Sgk1 significantly limited stimulated {alpha}ENaC promoter-driven luciferase activity by AGEs in mpkCCDcl4 cells.

Conclusion. Experimental results indicate that AGEs induced {alpha}ENaC expression and increased sodium uptake in renal CCD cells. The mechanism through which AGEs activate {alpha}ENaC expression may be via activation of Sgk1 in mpkCCDcl4 cells.

Keywords: advanced glycation end products; cortical collecting duct cells; diabetic nephropathy; epithelial sodium channel; serum- and glucocorticoid-induced kinase



   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Patients with diabetes mellitus (DM) frequently develop diabetic nephropathy manifested by oedema, low urine sodium excretion and hypertension caused by sodium retention [1]. Progression of diabetic nephropathy commonly results in renal failure. The pathophysiological mechanisms of diabetic nephropathy act through multiple dysregulated pathways. Activation of the renin–angiotensin–aldosterone system, stimulated production of cytokines and growth factors, accumulation of sorbitol, enhanced production of advanced glycation end products (AGEs) and induction of carbonyl or oxidative stress are all potential pathways of diabetic nephropathy [2]. These mechanisms are directly or indirectly activated by high serum glucose levels. AGEs form via non-enzymatic glycation reactions between sugars and polypeptides following long-term incubation. Advanced glycation is associated with normal aging and is increased in diabetes as a result of time and sugar concentration. Increasing evidence indicates that AGEs are an important factor involved in development of diabetic nephropathy. Typically, AGEs act directly via formation of protein cross-links that alter the structure and function of cells or via binding to different cell surface receptors [3].

In the kidneys, hormones in the distal nephron regulate the fine control of sodium absorption. In principal connecting and collecting duct cells, the epithelial sodium channel (ENaC) mediates apical entry of sodium which is subsequently extruded from cells through the basolateral Na+, K+-ATPase [4]. Enhanced sodium absorption in renal cortical collecting duct (CCD) cells has been implicated as the pathogenic cause of sodium sensitive hypertension and sodium retention in glomerulonephritis [5]. As a principal component of transepithelial sodium transport in CCD cells, the ENaC is a heteromultimeric complex consisting of {alpha}, ß and {gamma} ENaC subunits. The function of ENaC is inhibited by amiloride. Among these three ENaC subunits, {alpha}ENaC accounts for most ENaC function. Investigations of the xenopus oocyte system showed that transcellular sodium current in oocytes expressing ß and {gamma} ENaC subunits was OR <10% of oocytes expressing {alpha}, ß and {gamma} ENaC. Furthermore, {alpha}ENaC knockout mice died soon after birth [6]. Aldosterone is the principal regulator of {alpha}ENaC, and binding of aldosterone to mineralcorticoid receptors activates serum- and glucocorticoid-induced kinase 1 (Sgk1). Activation of Sgk1 then increases {alpha}ENaC synthesis and transcellular sodium transport [7].

Enhanced ENaC expression in CCD cells has been demonstrated in animal models with type 1 and type 2 DM. In streptozotocin-induced type 1 diabetic rats, all three ENaC subunits are increased at mRNA and protein levels [8,9]. In obese Zucker rats with type 2 DM, ENaC expression was also elevated [9]. The molecular mechanism by which ENaC expression is increased in diabetes remains unclear. Patch clamp studies of CCD cells dissected from salamanders demonstrated that insulin increased ENaC current activity [10]. However, type 1 DM rats with an insulin deficiency also had enhanced ENaC expression in CCD cells [8]. Factors other than insulin are likely responsible for altered ENaC expression in a DM setting.

Since AGEs are an important pathogenic factor in diabetic nephropathy and can be filtrated through glomeruli, the question arises as to whether AGE regulates expression of ENaC in CCD cells. To answer this question, experiments were performed on immortalized mpkCCDcl4 cells that have retained the principal characteristics of CCD cells.



   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Acknowledgements
 References
 
Reagents
Fetal calf serum (FCS), Dulbecco's modified Eagle's medium (DMEM) and F-12 nutrient mixture (Ham) were obtained from the Invitrogen Corporation (Carlsbad, CA, USA). The MAPKs pathway inhibitors (PD98095, SP600125 and SB203580) were obtained from Calbiochem Corporation (La Jolla, CA, USA) and stored in dimethylsulphoxide (DMSO) as 50 mM (PD98095), 40 mM (SP600125) and 10 mM (SB203580) stock solutions. The mitogen-activated protein kinases (MAPKs) pathway inhibitors—phospho-specific extracellular signal-regulated kinases (ERK1/2), c-Jun N-terminal kinases (JNKs), p38 kinase, total JNKs and p38 kinase antibodies—were purchased from Cell Signaling Technology (Beverly, MA, USA). Total ERKs antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-alpha tubulin antibody was obtained from Lab Vision Co. (Fermont, CA, USA). Rabbit anti-rat {alpha}ENaC antibody (Q3560 {alpha}ENaC N-terminus) was kindly provided by Prof. Mark Knepper (NIH, Bethesda, USA). This antibody had been documented to cross-reaction with mouse {alpha}ENaC in western blot analysis [9]. The plasmid pGL-3 (pGL3-basic) and plasmid pGL-3 containing the luciferase gene subcloned downstream of the full-length murine {alpha}ENaC promoter (pGL3-basic/{alpha}mENaC) were kindly donated by Dr André Dagenais (Université de Montreal, Montreal, Canada). The plasmids expressing either full-length Sgk1 (Sgk) or a kinase-dead dominant negative (K127M)-Sgk1 (mSgk) were kindly provided by Prof. David Pearce (University of California, San Francisco, California, USA). All other chemicals were obtained from Sigma Chemical Co. (St Louis, MO, USA).

In vitro preparation of AGEs
The AGEs were prepared by incubating bovine serum albumin (BSA) 10 mg/ml at 37°C for 6 weeks with D-glucose (90 g/l) in a 0.4 M phosphate buffer containing azide [3]. The control preparation was treated identically with the exception that glucose was omitted. Preparations were dialysed against phosphate buffer to remove free glucose after incubation for 6 weeks. The degree of glycation was measured using spectrophotometry (excitation 340 and emission 370 nm). Advanced glycation was associated with an approximate 10-fold increase in fluorescence compared with that of controls.

Cultured mouse cortical collecting duct cells and experimental treatments
The mpkCCDcl4 cells were derived from isolated CCD cells microdissected from kidneys of SV-PK/Tag transgenic mice as described previously [11]. The mpkCCDcl4 cells were cultured in porous Costar filters (pore size 0.4 µm) (Corning, NY, USA) in a modified DM medium (DMEM: Ham's F12, 1:1 vol/vol; 60 nM sodium selenate; 5 µg/ml transferrin; 2 mM glutamine; 50 nM dexamethasone; 1 nM triiodothyronine; 10 ng/ml epidermal growth factor; 5 µg/ml insulin; 20 mM D-glucose; 2% FCS and 20 mM HEPES, pH 7.4) at 37°C in 5% CO2–95% air atmosphere for 5 days. Cells were serum-deprived for 18 h and then stimulated with various AGEs or BSA (vehicle) concentrations (0–1000 µg/ml) by directly adding AGEs or BSA to the medium in both apical and basal sides of the filter for a particular time—the medium was not changed in an effort to prevent serum re-feeding on signalling pathways. Other chemicals without specific mention were also added to both sides of the filters. Alternatively, mpkCCDcl4 cells were pre-incubated with specific inhibitors and added 1 h prior to AGE treatment. Total RNA was extracted for reverse transcription polymerase chain reaction (RT-PCR) or real-time PCR. Total cell lysates were extracted for western blot analysis. All measurements were performed at minimum in triplicate.

Cell proliferation and toxicity assay
To analyse potential non-specific cytotoxicity or cell proliferation effect of AGEs on mpkCCDcl4 cells, cell proliferation was determined using the MTT [(3-4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] dye assay. Cells were seeded and grown for 5 days in 96-well trays and then incubated for 24 h in 100 µl defined medium with or without increasing concentrations of AGEs. Cell viability for each AGE concentration was determined by comparison with that of untreated cells; results are expressed as percentages of viable cells. All measurements were performed in duplicate.

RNA extraction and RT-PCR
Total RNA was extracted from confluent mpkCCDcl4 cells using the guanidium thiocyanate–phenol–chloroform method (Cinna/Biotecx Laboratories International, Inc., Friendwood, TX, USA) and treated with RNase-free DNase I (Boehringer Mannheim, Germany) at 37°C for 30 min. The RNA (1 µg) was reverse-transcribed using the avian myeloblastosis virus reverse transcriptase (RT AMV, Boehringer Mannheim) at 42°C for 60 min. Complementary DNA was amplified for 30–42 cycles in 100 µl total volume containing 50 mM KCl, 20 mM Tris–HCl (pH 8.4), 10 mM dNTP, 1.5–3.0 mM MgCl2, 1 unit Taq polymerase and 10 pmol of specific PCR primers. The thermal cycling protocol was as follows: 94°C for 1 min 60°C for 1 min, and 72°C for 3 min. Amplification products were separated on a 4% agarose gel with ethidium bromide and then photographed. Primers for mouse {alpha}ENaC, Sgk1 and ß-actin were as follows: {alpha}ENaC, sense 5-CTA ATA TGC TGG ACA CAC C-3 and anti-sense 5-AAA GCG TCT GGA TCC-3 (564 bps); Sgk1, sense 5-GCA CTT CGA TCC CGA GTT TA-3 and anti-sense 5-TTG AGA GGA GGG TGT GCT CT-3 (348 bps); ß-actin, sense 5-GCC AGG ATA GAG CCA CCA ATC-3 and ß-actin, anti-sense 5-ACT GCC CTG GCT CCT AGC A-3 (460 bps).

Real-time PCR and primers
Total RNA was isolated from mpkCCDcl4 cells and reverse-transcribed to DNA according to protocols described in the previous subsection. Real-time PCR was performed in triplicate on an ABI-Prism 7700 using SYBR Green I as a double-stranded DNA-specific dye according to the manufacturer's instructions (PE-Applied Biosystems, Cheshire, Great Britain) using SYBR® Green PCR Master Mix (1:1, PE-Applied Biosystems), forward and reverse primers (200 nM each) and sample RNA (90 ng). To standardize quantification of selected genes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH)—as the standard housekeeping gene—from each sample was quantified by real-time PCR. The TaqMan rodent GAPDH control reagents (Cat. No. 4308313) containing the GAPDH primers and VIC-probe were purchased from Applied Biosystems. Primers were constructed to be compatible with a single RT-PCR thermal profile (95°C for 10 min, 40 cycles of 95°C for 30 s and 60°C for 1 min) to allow for simultaneous analysis of multiple transcripts. Accumulation of the PCR product was monitored in real time (PE-Applied Biosystems), and the crossing threshold (Ct) was determined using PE-Applied Biosystems software. For each primer set, a no-template control and a no-reverse amplification control were utilized. Post-amplification dissociation curves were assessed to verify the presence of a single amplification product in the absence of DNA contamination. The ratio of the target gene to GAPDH expression levels (relative gene expression numbers) was determined by subtracting the threshold cycle number (Ct) of the target gene from the Ct of GAPDH expression and increasing the difference to the power of 2. The Ct values are defined as the number of PCR cycles at which the fluorescent signal during the PCR achieves a fixed threshold. Thus, target gene mRNA expression is expressed relative to GAPDH expression. Fold changes in gene expression were determined in comparison with controls. Primers for {alpha}ENaC are sense 5-ACC GCA TGA AGA CGG CC-3 and anti-sense 5-CCA GTA CAT CAT GCC GAA GGT-3; for ßENaC, sense 5-GCC AGT GAA GAA GTA CCT CC-3 and anti-sense 5-CCT GGG TGG CAC TGG TGA A-3 and, for {gamma}ENaC, sense 5-CAC TGG TCG AAG CGG AAA-3 and anti-sense 5-GCA CAG TCA GAG GTG TCA TT-3 [12].

Sodium uptake study
Sodium-binding benzofuran isophthalate acetoxymethyl ester (SBFI-AM, Molecular Probes, OR, USA) was employed as a sodium indicator to measure [Na+]i; the protocol was a modified version of that described by Schlatter et al. [13]. Briefly, SBFI 10 µM mixed with Pluronic F127 (Molecular Probes, OR, USA) (1:1) were placed at the apical side of AGEs- or BSA-stimulated mpkCCDcl4 for 1 h. Cells were then washed with PBS to remove residual SBFI. After aspirating out the PBS, cells were disrupted by repeat freezing–thawing for 15 min and dissolved in 400 µl (10%) Triton-100. Cell lysate 100 µl was then activated with wavelengths alternating at 340/380 nm. Fluorescence was detected at 515 nm using a Victor 3 multiple channel fluorescence reader (PerkinElmer, Boston, MA). All measurements were performed in duplicate for 12 separate filters. The ratio of fluorescence (340/380 nm) was used as specific signal. The other set of experiments were performed simultaneously by incubating amiloride 10–5 M at the apical side of AGEs- or BSA-stimulated cells 10 min prior to adding SBFI. The differences in [Na+]i with and without amiloride were applied to estimate the rate of sodium uptake. Nystatin (360 µg/ml) was used to calibrate [Na+]i. Data are expressed as fold change compared with that of control cells without treatment with AGEs or other chemicals.

Western blot analysis
Cells were lysed in a solution containing 70 mM ß-glycerophosphate (pH 7.2), 0.1 mM sodium orthovanadate, 2 mM MgCl2, 1 mM EDTA, 1 mM DTT, 0.5% Triton X-100, 10% glycerol and protease inhibitors (2 µg/ml aprotinin, 2 µg/ml leupeptin and 100 µg/ml PMSF) for 30 min. Lysates were centrifuged at 14 000 rpm for 10 min at 4°C, and protein concentrations in supernatants were measured using the Bradford method (Bio-Rad Laboratories, Hercules, CA, USA). Samples of 50 µg protein were mixed with sample buffer [500 mM Tris–HCl (pH 6.8), 10% sodium dodecyle sulphate (SDS), 30% glycerol, 0.012% bromophenol blue and 0.6M DTT] and boiled at 95°C for 5 min. Samples were loaded onto 10% SDS–polyacrylamine gels and separated using a Tris buffer saline (TBS). Following eletrophoresis, gels were immersed in transfer buffer containing 25 mM Tris–HCl, 0.2 M glycine and 20% methanol, electrotransferred to Immunobilon-P membranes (Millipore Bedford, MA, USA) and blotted with appropriate antibodies at 4°C for 16 h in TBS containing 0.1% Tween-20 and 5% non-fat milk. Following washing, membranes were incubated with horseradish peroxidase-conjugated antibodies at room temperature for 45 min. Signals were detected using electrochemiluminescence immunoassay (ECL) (Amersham, Arlington Heights, IL, USA) according to the manufacturer's instructions, and exposed to X-OMAT AR film (Eastman Kodak, Rochester, NY, USA). Stripping the initial antibody probe was accomplished by submerging membranes in 100 mM 2-ß-mercaptoethanol, 20% SDS and 62.5 mM Tris–HCl (pH 6.8) at 55°C for 50 min, washed twice in TBS/0.1% Tween-20 for 10 min each, and re-blotted with appropriate antibodies to determine equal sample loading [14].

Sgk1 kinase activity assay
The Sgk1 kinase activity was assayed using Sgk activity assay kits according to the manufacturer's instructions (Stressgen, Canada). Briefly, mpkCCDcl4 cells cultured with AGEs or BSA were scraped off, collected in Tris-buffered saline (TBS), centrifuged at 150g for 5 min and homogenized in 50 µl lysis buffer (10 mM KCl, 1.5 mM MgCl2, 10 mM Tris–HCl, pH 7.4, 0.1 mM PMSF and 100 µg/ml leupeptin). Protein lysate (20 µl) was added to ready-to-use Sgk1 pre-coated 96-well plates and incubated with 5 µl (100 µM) ATP for 60 min at 4°C. The reaction was stopped by removing the medium and adding 5 µl (1 µM) phosphospecific substrate antibody. Anti-rabbit IgG HRP was added, washed and then incubated with prepared 2,2'-Azino-bis-(3-ethyl-benzthiazoline-6-sulphonic acid) (ABTS). Activity of Sgk1 was assayed by measuring colour intensity using a micropippet reader at 450 nm.

Transient transfection and {alpha}ENaC promoter-driven luciferase activity assay
The mpkCCDcl4 cells grown in a porous filter for 72 h in DMEM:Ham's F12 medium were transfected with either pGL3-basic, pGL3-basic/{alpha}mENaC, full-length Sgk1 (mSgk1/pcDNA3) or a kinase-dead dominant negative Sgk1 (mSgk1/K127M/pcDNA3) plasmid alone or in combination using lipofectamine according to LipofectamineTM2000 transfection protocol (Life Technologies, Inc., Carlsbad, CA, USA). Transfected cells were cultured for 48 h and then serum-deprived for 12 h prior to incubation with AGEs or BSA for an additional 24 h. The luciferase activity was measured with a dual luciferase assay kit (Promega, Madison, WI, USA) using a luminometer (Lumat LB 9506, Berthold Technology) and was calibrated with the amount of extracted protein.

Statistical analysis
Results are expressed as means ± SD from (n) experiments performed in duplicate or triplicate. Differences between groups were analysed by the Student's t-test or Tukey's analysis of variance. Statistical analysis was performed using StatviewTM (Macintosh version). A value of P < 0.05 was considered significant.



   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Acknowledgements
 References
 
Cell proliferation or toxicity by advanced glycation end products in mpkCCDcl4 cells
Confluent mpkCCDcl4 cells were grown in serum-free medium in the presence of increasing concentrations of BSA or AGEs (0–1000 µg/ml for 24 h). The percentage of viable cells did not differ significantly between BSA- or AGE-treated and untreated control cells (97 ± 4% and 98 ± 8% vs untreated cells, respectively, n = 6). These findings indicate that cultured mpkCCDcl4 cells remain viable following 24 h incubation with AGEs or BSA.

Advanced glycation end products enhanced {alpha}ENaC expression in mpkCCDcl4 cells
Confluent mpkCCDcl4 cells were incubated with AGEs or BSA to determine their affect on {alpha}ENaC expression. The AGEs enhanced mRNA expression of {alpha}ENaC at 8 h as compared with that of BSA-treated cells (Figure 1A). The levels of {alpha}ENaC mRNA expressions correlated positively with the AGE concentrations added for 24 h. Incubation with AGEs for 24 h induced an expression of {alpha}ENaC mRNA higher than that of control values at a concentration of 250 µg/ml and peaked at a concentration of 500 µg/ml (Figure 1B). To verify if the increase in {alpha}ENaC mRNA also occurred at protein expression, confluent mpkCCDcl4 cells were incubated with increasing concentrations of AGEs for 24 h. Total cell lysates were extracted for {alpha}ENaC protein expression. Consistent with RT-PCR findings, western blot analysis result demonstrated that AGEs promoted {alpha}ENaC protein expression at 100 µg/ml AGE concentration and peaked at 500 µg/ml in a dose-dependent manner (Figure 1C).


Figure 1
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  		Fig. 1. AGEs enhanced {alpha}ENaC mRNA expression in renal CCD cells. Confluent renal CCD (mpkCCDcl4) cells grown in porous Costar filters with serum-free medium for 18 h were then incubated with increasing concentrations of AGEs or BSA (0–1000 µg/ml) for various periods. Expression of {alpha}ENaC and ß-actin mRNAs were analysed by RT-PCR in mpkCCDcl4 cells incubated with 500 µg/ml AGEs or BSA as a function of time (A, left panel for AGEs; right panel for BSA) or increasing concentrations of AGEs or BSA for 24 h (B, left panel for AGEs; right panel for BSA). As a control, ß-actin was employed as the internal standard. The AGEs enhanced expression of {alpha}ENaC mRNA in a time- and dose-dependent manner. Alternatively, confluent mpkCCDcl4 cells were incubated with increasing concentrations of AGEs for 24 h. Total cell lysates (50 µg) were extracted for {alpha}ENaC protein expression by western blot analysis. To specify the antibody for mouse {alpha}ENaC, 10 µg of tissue homogenates from rat kidney cortex (rat) was used as positive control. Alpha-tubulin is shown as a loading control. The blot shown is representative of two separate experiments (n = 2) in which similar results were observed (C).

 
Murine CCD cells expressed ENaC subunits {alpha}, ß and {gamma}. Real-time PCR was performed to confirm RT-PCR results and to determine AGE effects on other ENaC subunits in murine mpkCCDcl4 cells. Real-time PCR results indicated that enhanced expression of {alpha}ENaC mRNA by AGE 500 µg/ml was statistically significant at 4 h of incubation and peaked at 8 h. The mpkCCDcl4 cells incubated with various AGEs concentrations for 24 h also increase {alpha}ENaC mRNA expression in a dose-dependent manner. Consistent with RT-PCR findings, real-time PCR results indicated that AGEs specifically promoted expression of {alpha}ENaC mRNA in a time- and dose-dependent manner. In both cases, the levels of ßENaC and {gamma}ENaC mRNAs remained unchanged (Figure 2A and B).


Figure 2
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  		Fig. 2. The AGEs specifically enhanced expression of {alpha}ENaC mRNA in renal CCD cells. The expressions of {alpha}, ß and {gamma}ENaC mRNAs stimulated by AGEs were analysed in mpkCCDcl4 cells by real-time PCR. The relative {alpha} (black circle), ß (white circle) and {gamma} (black triangle) ENaC mRNAs expression were analysed by real-time PCR in mpkCCDcl4 cells incubated with 500 µg/ml AGEs for various times (A) or with increasing concentrations of AGEs for 24 h (B). The plotted points indicate the fold increase in ENaC mRNA expression compared with that of untreated cells (Time, 0; AGEs or BSA, 0). Values, expressed as relative mRNA fold increase over that of untreated cells, are mean ± SE of duplicate measurements from six independent experiments. *P < 0.05 or **P < 0.01 vs time 0 or AGE 0 values.

 
The advanced glycation end products increased sodium uptake in mpkCCDcl4 cells
Experiments were performed to determine whether AGEs stimulate sodium uptake by mpkCCDcl4 cells. The SBFI-AM was utilized as an indicator of sodium uptake. The AGEs or BSA 500 µg/ml were employed for time-course experiments. Compared with that of BSA, sodium uptake significantly increased at 4 h after AGE incubation; this enhancement increased as incubation time increased (Figure 3A). Concentrations of BSA up to 1000 µg/ml had no affect on sodium uptake; however, AGEs had a dose-dependent effect on sodium uptake. No sodium uptake change existed for cells treated with AGEs or BSA at concentrations of 0, 50 and 100 µg/ml. Sodium uptake increased at 250 µg/ml AGE concentration and peaked at 500 µg/ml (Figure 3B). These analytical results indicate that AGEs elevated sodium uptake in mpkCCDcl4 cells.


Figure 3
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  		Fig. 3. The AGEs increased sodium uptake in renal CCD cells. Confluent mpkCCDcl4 cells incubated with serum-free medium for 18 h were then incubated with increasing concentrations of AGEs or BSA (50–1000 µg/ml) for various periods. Using SBFi-AM as a sodium indicator, sodium uptake was measured in mpkCCDcl4 cells as a function of time (A) or increasing concentrations of AGE (B) with a Victor 3 multiple channel fluorescence reader. Values, expressed as relative fluorescence intensity fold increase over that of untreated cells, are mean ± SE of duplicate measurements from 12 independent experiments. *P < 0.05 or **P < 0.01 vs Time 0 or AGEs 0 values.

 
Actinomycin D and cycloheximide suppressed the effect of AGEs on sodium uptake
As AGEs increased sodium uptake in mpkCCDcl4 cells, actinomycin D (a transcription inhibitor) and cycloheximide (a translation inhibitor) were applied to determine whether the AGE effect can be suppressed. Using SBFI-AM as sodium indicator, incubation with AGEs (500 µg/ml for 24 h) increased sodium uptake compared with that in untreated cells. Incubation with actinomycin D (10–6 M) alone did not reduce the sodium uptake. Conversely, pre-incubation with actinomycin D for 1 h prior to adding AGEs suppressed the stimulatory effect of AGEs on sodium uptake (Figure 4). Similar results were noted for cultured cells pre-incubated with cycloheximide (0.3 µg/ml) (Figure 4). These experimental results suggested that AGEs can stimulate sodium uptake at the transcriptional and translational levels.


Figure 4
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  		Fig. 4. Increased sodium uptake induced by AGEs is suppressed by actinomycin D and cycloheximide. Confluent mpkCCDcl4 cells incubated with serum-free medium for 18 h were pre-incubated with or without actinomycin D (10–6 M) or cyclohemimide (0.3 µg/ml) for 1 h before adding AGEs (500 µg/ml) for incubation for a further 24 h. Using SBFI-AM as a sodium indicator, sodium uptake in mpkCCDcl4 cells was analysed using a Victor 3 multiple channel fluorescence reader. Values, expressed as fold increase relative fluorescence intensity over that of untreated cells, are mean ± SE of duplicate measurements from 12 independent experiments. *P < 0.05 vs AGEs alone.

 
AGE effects on {alpha}ENaC were independent of MAPKs signalling pathways
MAPKs kinases are known to affect ENaC function [15]. Further experiments were performed to elucidate the effects of AGEs on phosphorylation of ERK1/2, JNK1/2 and p38 in mpkCCDcl4 cells. Notably, phosphorylated ERK1/2 significantly increased after mpkCCDcl4 cells were incubated with AGEs for 30 min (Figure 5A, upper panel). Similarly, Western blot analysis indicated that AGEs stimulated JNKs; this finding was determined based on the increase in phosphorylated bands detected at 60 min (Figure 5A, middle panel). Adding AGEs for 30 min also significantly enhanced phosphorylation of p38 (Figure 5A, lower panel). In all cases, AGEs, which did not affect total ERKs, JNKs and p38 proteins, had a maximal stimulatory effect on phosphorylated kinases following 30–60 min incubation. To determine which MAPKs pathway(s) is/are specifically involved in expression of {alpha}ENaC mRNA generated by AGEs, mpkCCDcl4 cells were pre-incubated with different specific inhibitors of MAPKs, stimulated with AGEs for 24 h, and {alpha}ENaC mRNA expression was measured by real-time PCR. Neither PD98059 (40 µM), SP600125 (40 µM) nor SB203580 (20 µM) alone significantly affected {alpha}ENaC mRNA expression in mpkCCDcl4 cells. Pre-incubation of mpkCCDcl4 cells with PD98095, SP600125 or SB203580 for 30 min did not alter the stimulatory effect of AGEs (500 µg/ml for 24 h) on {alpha}ENaC mRNA expression (Figure 5B). These experimental findings suggest that the effects of AGEs on {alpha}ENaC mRNA expression were independent of MAPK signalling pathways.


Figure 5
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  		Fig. 5. The effects of AGEs on {alpha}ENaC mRNA expression were independent of MAPKs pathways. Confluent mpkCCDcl4 cells grown in serum-free medium for 18 h were incubated with 500 µg/ml AGEs for various times. Total (T) and phosphorylated (p) ERK1/2, JNK1/2 and p38 MAPKs were examined in cell lysates using specific antibodies directed against total or phosphorylated kinases (A). Alternatively, confluent mpkCCDcl4 cells grown in serum-free medium for 18 h were then pre-incubated with 40 µM PD98095, 40 µM SP600125 or 40 µM SB203580 for 1 h prior to adding AGEs (500 µg/ml for 24 h). The relative {alpha}ENaC mRNA expression in mpkCCDcl4 cells was analysed by real-time PCR. The plotted points denote the fold increase in ENaC mRNA expression compared with that of untreated cells (B). The blot shown is representative of three separate experiments (n = 3) in which similar results were observed. Values, expressed as relative mRNA fold increase over that of untreated cells, are mean ± SE of duplicate measurements from six independent experiments.

 
AGEs increased Sgk1 mRNA expression and kinase activity
As Sgk1 has been shown to regulate ENaC expression, and is an early-induced aldosterone–protein [7], this study determined whether AGEs stimulate Sgk1 expression and activity in cultured mpkCCDcl4 cells. Compared with BSA-treated cells, RT-PCR results suggested that AGEs rapidly induced Sgk1 mRNA expression at 2 h and remained stable at enhanced levels during the following 24 h (Figure 6A). Consistent with these findings, the Sgk kinase activity assay results indicated that AGEs stimulated total cellular Sgk activity in a time-dependent manner (Figure 6B). In both cases, the degrees of Sgk1 mRNA expression and activity in BSA-treated mpkCCDcl4 cells remained unchanged throughout the entire time course.


Figure 6
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  		Fig. 6. The AGEs increased Sgk1 mRNA expression and kinase activity. Confluent mpkCCDcl4 cells grown in serum-free medium for 18 h were incubated with 500 µg/ml AGEs or BSA for various times. Expression of Sgk1 and ß-actin mRNAs were analysed by RT-PCR in mpkCCDcl4 cells as a function of time (A, left panel for AGEs; right panel for BSA). As a control, ß-actin was employed as the internal standard. Alternatively, Sgk kinase activity was measured in cell lysates using Sgk kinase activity assay kits according to the manufacturers’ instructions. Values, expressed as relative colour intensity fold increase over that of untreated cells, are mean ± SE of duplicate measurements from five independent experiments. *P < 0.05 or **P < 0.01 vs time 0 values.

 
AGEs stimulated {alpha}ENaC expression through Sgk1 activation in mpkCCDcl4 cells
Since AGEs stimulated {alpha}ENaC and Sgk1 expressions in mpkCCDcl4 cells, we proposed that AGEs stimulate {alpha}ENaC mRNA expression via a Sgk1-dependent pathway. To test this hypothesis, luciferase assays were performed using the {alpha}ENaC promoter-driven luciferase reporter gene and either wild-type Sgk1 (Sgk) or mutant Sgk1 with kinase-dead dominant negative plasmids (mSgk). First, the mpkCCDcl4 cells either transfected with pGL-3 (pGL, as a negative control) or pGL-3/{alpha}mENaC (ENaC) were incubated with AGEs or BSA for different time periods (500 µg/ml for 0–24 h) or various concentrations (0–1000 µg/ml for 24 h) to assess the specificity of the luciferase activity assay. Similar to results obtained with real-time PCR studies, AGEs stimulated {alpha}ENaC promoter-driven luciferase activities, which increased at 4 h following AGE incubation and remained stable at enhanced levels during the following 24 h (Figure 7A). Luciferase activity results also demonstrated that the {alpha}ENaC gene can be activated by AGEs in a dose-dependent manner (Figure 7B). Luciferase activity remained unchanged in pGL-transfected cells after BSA or AGE incubation or in EnaC-transfected cells with BSA incubation over different time periods and concentrations models (Figure 7A and B). These experimental results confirmed the effects of AGEs on {alpha}ENaC via induction of {alpha}ENaC transcription.


Figure 7
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  		Fig. 7. The AGEs enhanced {alpha}ENaC expression through Sgk1 activation in mpkCCDcl4 cells. The 60–70% confluent mpkCCDcl4 cells either transfected with pGL-3 (pGL, mock vector, as a negative control) or pGL/{alpha}mENaC (ENaC) were incubated with AGEs or BSA for different time periods (500 µg/ml) (A) or various concentrations (0–1000 µg/ml) (B) to examine the specificity of the luciferase activity assay. Alternatively, the 60–70% confluent mpkCCDcl4 cells transfected with ENaC alone (black bar) or co-transfected ENaC with Sgk1 (gray bar) or mSgk1 (kinase-dead dominant negative plasmids) (striped gray bar) were incubated with AGEs (500 µg/ml for 24 h), then luciferase activity was measured (C). Values, expressed as relative luciferase intensity fold increase over that of untreated cells, are mean ± SE of duplicate measurements from five independent experiments. *P < 0.01 vs time 0 (A) or concentration 0 (B) and AGEs alone (C).

 
Next, this study examined the role of Sgk1 activation in AGE-enhanced {alpha}ENaC expression. The mpkCCDcl4 cells co-transfected with ENaC and Sgk1 or ENaC and mSgk1 were incubated with AGEs (500 µg/ml for 24 h), and then luciferase activity was measured. Compared with that of cells transfected with ENaC alone (Figure 7C, black bar), mpkCCDcl4 cells co-transfected with ENaC and Sgk1 had higher luciferase activity following incubation of AGEs (Figure 7C, gray bar); however, these differences were not statistically significant. Conversely, the mpkCCDcl4 cells co-transfected with ENaC and mSgk1 (kinase-dead dominant negative plasmid) had a significantly lower luciferase activity than that of cells transfected with ENaC alone or co-transfected with ENaC and Sgk1 following AGEs activation (Figure 7, striped gray bar). These analytic results suggested that AGEs stimulated {alpha}ENaC gene expression via Sgk1 activation in mpkCCDcl4 cells.



   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Acknowledgements
 References
 
Excessive water and sodium losses are associated with uncontrolled DM. To compensate for these losses, the kidney increases the number of key sodium and water transporters and channels via the renin–angiotensin–aldosterone system. It is believed that such comprehensive changes decrease volume contraction accompanying large-solute (sugar) and water losses associated with DM. However, diabetic patients with euglycaemia on strict sugar control still developed sodium retention, subtle oedema and hypertension. These clinical observations indicate that, except for the compensatory mechanism mentioned previously, other unknown mechanisms may exist that regulate sodium balance in diabetic nephropathy.

AGEs are produced by long-term high glucose levels with polypeptides. AGEs are known as an important pathogenic factor in diabetic nephropathy; however, almost all studies have focused on glomerular and vascular cells [3]. The effect of AGEs on renal tubule cells, particularly on collecting ducts, and renal tubule segments responsible for fine sodium absorption, has rarely been explored. In diabetic patients, a concentration has been reported of circulating AGE corresponding to about 50 µg/ml of AGE [16]. Because AGE may be concentrated in renal tissue in vivo, and the corresponding levels in vitro have not been determined conclusively, we also examined concentrations that appear supraphysiological (up to 1000 µg/ml). This study provides experimental evidence that AGEs promote {alpha}ENaC gene expression, protein level and sodium uptake in mpkCCDcl4 cells. Notably, Sgk1 has a central role in this process. These experimental results are compatible with up-regulation of {alpha}ENaC expression in diabetic animal studies [8]. As AGEs enhanced {alpha}ENaC gene expression—measured by real-time PCR and {alpha}ENaC promoter-driven luciferase activity assay—in a time- and dose-dependent manner, the increase in {alpha}ENaC expression and sodium uptake in mpkCCDcl4 cells may result from enhanced {alpha}ENaC transcription. This hypothesis is strengthened further by the fact that mpkCCDcl4 cells pre-incubated with transcription inhibitor actinomycin D were refractory to AGE activation. As surface ENaC protein accounts for most of transcellular sodium transport in renal CCD cells [4], increased {alpha}ENaC transcription by AGE likely results in increased {alpha}ENaC protein translation and sodium uptake. The finding that translation inhibitor cycloheximide suppressed the effect of AGEs on sodium uptake in mpkCCDcl4 cells implies that AGEs may also increase {alpha}ENaC protein expression, which in turn increases sodium uptake.

The Sgk1 is a protein kinase widely distributed in numerous tissues. In the kidney, Sgk1 is induced in the aldosterone sensitive distal nephron where it stimulates sodium uptake, in part by suppressing ubiquitin ligase Nedd4-2-mediated retrieval of ENaC from the luminal membrane [17]. In CCD cells, ENaC is induced principally by the aldosterone hormone; however, non-hormonal factors, such as osmotic stress, can also induce Sgk1 expression [18]. In contrast to aldosterone, through which stimulation of ENaC transport can be explained simply by an increase in channel synthesis, Sgk1 effects are complex and involve increases in ENaC open probability, subunit abundance within apical membranes and intracellular compartments, and activation of one or more pools of pre-existing channels within apical membranes and/or intracellular compartments in A6 renal epithelial cell lines [19]. The Sgk1 mRNA expression was selectively enhanced in diabetic and hypertensive kidneys [20,21]. The Sgk1 plays a pivotal role in insulin-mediated sodium retention and salt-sensitizing hypertensive effect of high fructose intake [22]. The Sgk1 was up-regulated in diabetic nephropathy and actively participated in the stimulation of matrix protein deposition in this common complication of diabetic hyperglycaemia [23]. Experimental results in this study demonstrated that Sgk1 mRNA expression and activity were increased by AGEs. Additionally, activation of {alpha}ENaC gene promoter luciferase activity by AGEs was hindered by mutant Sgk1 in mpkCCDcl4 cells. These experimental findings imply that the effect of AGEs on {alpha}ENaC gene expression in mpkCCDcl4 cells was via activation of Sgk1. Although the mechanisms by which AGEs activated Sgk1 in this model are unclear, the association between AGEs and Sgk1 activation has rarely been elucidated.

In this study, the mechanisms by which AGEs regulate Sgk1 and {alpha}ENaC gene expression are still unknown. Many, but not all, of the effects of AGE are mediated by their engagement with the receptor for AGE (RAGE), a member of the immunoglobulin superfamily that is a signal transduction receptor [24]. It is interesting to test if enhanced {alpha}ENaC mRNA expression by AGEs in mpkCCDcl4 cells is through RAGE. Our preliminary data that S100/calgranulins, a specific ligand for RAGE, does not enhance Sgk and {alpha}ENaC mRNA expression in mpkCCDcl4 cells implied that RAGE may not play a central role in mediating {alpha}ENaC mRNA transcription stimulated by AGEs (unpublished data). However, this preliminary data could not conclusively exclude the role of RAGE. Further experiments, including applying soluble RAGE to interrupt AGEs–RAGE signalling or RAGE siRNA to blocking RAGE expression, are necessary to verify this hypothesis. AGEs activate MAPKs pathways in numerous tissues such as renal tubule cells. The ERK1/2 pathways can crosstalk with Sgk1 and transiently affect sodium transport in mpkCCDcl4 cells [15]. Activation of p38 MAPK has been shown to activate Sgk1 in HEK293 cells [25]. Although mpkCCDcl4 cells in this study exhibited significant MAPKs activity by AGEs, experimental results obtained using specific inhibitors did not find a direct relationship between activated MAPK kinases by AGE and {alpha}ENaC mRNA expression. Therefore, enhanced {alpha}ENaC mRNA expression by AGEs in mpkCCDcl4 cells was not through activation of MAPK pathways.

In conclusion, this study indicated that AGEs can up-regulate {alpha}ENaC mRNA expression and protein level, and enhance sodium uptake in murine renal CCD cells. Notably, Sgk1 likely plays a central role in regulating {alpha}ENaC mRNA transcription stimulated by AGEs.



   Acknowledgements
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
References
 
Pro. D. Pearce (California, USA) and Dr A. Dagenais (Montreal, Canada) are appreciated for kindly providing the plasmids used in this study. Prof. M. Knepper (NIH, USA) is appreciated for kindly providing anti-{alpha}ENaC antibody. Prof. A. Vandewalle (INSERM, France) is also appreciated for his valuable discussions. The authors would like to thank the National Science Council of the Republic of China, Taiwan and Chang Gung Memorial Hospital for financially supporting this research under Contract No. NSC 94-2314-B-182A-186 (to C.C.H.), grant from INSERM (France)-NSC (Taiwan) (to A. Vandewalle and C.C.H.) and CMRPG 33104 (to C.T.C.). The authors also like to thank YC Ko, CT Huang and HM Yu for their technical assistance.

Conflict of interest statement. None declared.



   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 

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Received for publication: 11. 7.06
Accepted in revised form: 17.10.06


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