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Endocrinology Vol. 144, No. 10 4338-4349
Copyright © 2003 by The Endocrine Society

High Glucose Stimulates Angiotensinogen Gene Expression and Cell Hypertrophy via Activation of the Hexosamine Biosynthesis Pathway in Rat Kidney Proximal Tubular Cells

Tusty-Jiuan Hsieh, Pierre Fustier, Shao-Ling Zhang1, Janos G. Filep, Shiow-Shih Tang, Julie R. Ingelfinger, I. George Fantus, Pavel Hamet and John S. D. Chan

Université de Montréal Centre Hospitalier de l’Université de Montréal-Hôtel Dieu Centre de Recherche Pavillon Masson (T.-J.H., P.F., S.-L.Z., P.H., J.S.D.C.), Montréal, Québec, Canada H2W 1T8; Maisonneuve-Rosemont Hospital, Research Center (J.G.F.), Montréal, Québec, Canada H1T 2M4; Harvard Medical School, Massachusetts General Hospital, Pediatric Nephrology Unit (S.-S.T., J.R.I.), Boston, Massachusetts 02114-3117; and Mount Sinai Hospital, Department of Medicine, University of Toronto (G.F.), Toronto, Ontario, Canada M5G 1X5

Address all correspondence and requests for reprints to: Dr. John S. D. Chan, Université de Montréal Centre Hospitalier de l’Université de Montréal-Hôtel Dieu Centre de Recherche Pavillon Masson, Montréal, Québec, Canada H2W 1T8. E-mail: john.chan{at}umontreal.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The present study investigated whether activation of the hexosamine biosynthesis pathway might mediate at least in part the high glucose effect on angiotensinogen (ANG) gene expression and immortalized renal proximal tubular cell (IRPTC) hypertrophy. IRPTC were cultured in monolayer. ANG, renin, and ß-actin mRNA expression were determined by specific RT-PCR assays. Phosphorylation of p38 MAPK, activating transcription factor-2 (ATF-2), and cAMP-responsive element-binding protein (CREB) was determined by Western blot analysis. Cell hypertrophy was assessed by flow cytometry, intracellular p27kip1 protein levels, and [3H]leucine incorporation into proteins. Glucosamine stimulated ANG and renin mRNA expression and enhanced p38 MAPK, ATF-2, and CREB phosphorylation in normal glucose (5 mM) medium. Azaserine and 6-diazo-5-oxo-L-norleucine (inhibitors of glutamine: fructose-6-phosphate amino transferase enzyme) blocked the stimulatory effect of high glucose, but not that of glucosamine, on ANG gene expression in IRPTCs. SB 203580 (a specific p38 MAPK inhibitor) attenuated glucosamine action on ANG gene expression as well as p38 MAPK and ATF-2 phosphorylation, but not that of CREB. GF 109203X and calphostin C (inhibitors of protein kinase C) blocked the effect of glucosamine on ANG gene expression and CREB phosphorylation, but had no impact on p38 MAPK and ATF-2 phosphorylation. Finally, both glucosamine and high glucose induced IRPTC hypertrophy. The hypertrophic effect of glucosamine was blocked in the presence of GF 109203X, but not azaserine and SB 203580. In contrast, the hypertrophic effect of high glucose was blocked in the presence of azaserine and GF 109203X, but not SB203580. Our studies demonstrate that the stimulatory effect of high glucose on ANG gene expression and IRPTC hypertrophy may be mediated at least in part via activation of hexosamine biosynthesis pathway signaling.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
HIGH LEVELS OF glucose and/or angiotensin II (Ang II) may be responsible, directly or indirectly, for renal proximal tubular hypertrophy and interstitial fibrosis in diabetes. In vitro studies have shown that the incubation of murine proximal tubular cells in high glucose medium (>=25 mM) and/or in the presence of high concentrations of Ang II (>=10-8 M) induces cellular hypertrophy and extracellular matrix protein expression (1, 2, 3, 4, 5). Clinical trials have demonstrated beneficial effects of angiotensin-converting enzyme (ACE) inhibitors or Ang II receptor antagonists in reducing proteinuria and slowing the progression of nephropathy in diabetic patients (6, 7, 8, 9, 10). These studies indicate an important role for Ang II in the development of diabetic nephropathy.

In addition to the systemic or circulatory renin-angiotensin system (RAS), it is now well accepted that there is a local intrarenal RAS (11, 12). The mRNA and protein components of the RAS, including angiotensinogen (ANG), renin, ACE, and Ang II receptors (AT1-R and AT2-R), are expressed in murine (mouse and rat) immortalized proximal tubular cell lines (13, 14, 15, 16, 17, 18). We have reported that high concentrations (25 mM) of glucose stimulate ANG gene expression in rat immortalized renal proximal tubular cells (IRPTCs) (19, 20). Inhibitors of aldose reductase (Tolrestat), protein kinase C (PKC; staurosporine or H-7), and p38 MAPK (SB 203580) blocked the stimulatory effect of high glucose on ANG gene expression (19, 20). These studies indicate that high glucose action on ANG expression gene in IRPTCs is mediated at least in part via polyol/PKC and p38 MAPK signal transduction pathways. We have also reported that blockers of RAS and stable transfection of antisense rat ANG cDNA prevented the induction of IRPTC hypertrophy in high glucose (21, 22). Such studies provided evidence that intrarenal RAS activation plays an important role(s) in the pathogenesis of nephropathy in diabetes.

More recently, we reported that high glucose evoked the generation of reactive oxygen species in IRPTCs (23). Antioxidants and superoxide scavengers, inhibitors of mitochondrial oxidation, a manganese superoxide dismutase mimetic, and catalase suppressed ANG gene expression and p38 MAPK phosphorylation evoked by high glucose (23). Hydrogen peroxide also stimulates p38 MAPK phosphorylation and ANG gene expression in IRPTCs, and its effect is inhibited by catalase and SB 203580 (23). These studies demonstrated that the high glucose action on ANG gene expression is mediated at least in part via reactive oxygen species (ROS) generation and activation of p38 MAPK signaling. The molecular mechanism(s) of ROS action on ANG gene expression, however, remains largely undefined.

A major function of the hexosamine biosynthesis pathway (HBP) is diverting hexose phosphate and glutamine to the glycosylation pathway and synthesis of glycoproteins, mainly in the liver. Normally, this pathway accounts for only a small percentage (1–3%) of the glucose metabolized and is an ideal candidate as a cellular nutrient sensor responding to energy availability (see reviews in Refs. 24 and 25). Recent studies have reported that glucosamine also stimulates ANG gene expression in rat liver and adipose tissue (26). Thus, the aim of present experiments was to investigate whether the HBP could also mediate the high glucose effect on ANG gene expression and hypertrophy in IRPTCs. Our results provide evidence that high glucose-induced ANG gene expression and IRPTC hypertrophy are indeed mediated at least in part via activation of the HBP signal transduction pathway.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Glucosamine, D-mannitol, azaserine [O-diazoacetyl-L-serine, an inhibitor of glutamine:fructose-6-phosphate amidotransferase (GFAT) enzyme], 6-diazo-5-oxo-L-norleucine (DON; an inhibitor of GFAT), GF 109203X (an inhibitor of PKC), calphostin C (an inhibitor of PKC), and monoclonal antibodies against ß-actin were purchased from Sigma-Aldrich Canada Ltd. (Oakville, Canada). SB 203580 (a specific inhibitor of p38 MAPK) was obtained from Calbiochem (La Jolla, CA). Normal glucose (5 mM), DMEM (catalog no. 12320), 100x penicillin/streptomycin, and fetal bovine serum (FBS) were bought from Invitrogen (Burlington, Canada). [3H]Leucine was bought from Amersham Pharmacia Biotech (Baie d’Urfé, Canada). Monoclonal antibodies against p27Kip1 and rat ANG were obtained from Transduction Laboratories, Inc. (Mississauga, Canada) and Research Diagnostics, Inc. (Flanders, NJ), respectively.

The PhosphoPlus p38 MAPK, PhosphoPlus activating transcription factor-2 (ATF-2), and PhosphoPlus cAMP-responsive element binding protein (CREB) antibody kits were purchased from New England Biolabs, Inc. (Mississauga, Canada). The PhosphoPlus p38 MAPK kit was used for the rapid analysis of p38 MAPK (Thr180)/Tyr182) phosphorylation status, which functions in the stress-activated protein kinase cascade. The PhosphoPlus CREB and PhosphoPlus ATF-2 were deployed for the rapid analysis of CREB (Ser133) and ATF-2 (Thr71) phosphorylation status, respectively.

Oligonucleotides were synthesized by ALPHA DNA (Montréal, Canada). Restriction and modifying enzymes were purchased from Invitrogen-La Roche Biochemicals, Inc. (Laval, Canada) or Amersham Pharmacia Biotech.

Cell culture
IRPTCs (cell line 93-p-2-1) at passages 11–18 were used in the present studies. The characteristics of this cell line have been described previously (27).

IRPTCs were grown in 100 x 20-mm plastic petri dishes (Life Technologies, Inc.) in normal glucose (i.e. 5 mM) DMEM (pH 7.45) supplemented with 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin,, in a humidified atmosphere in 95% air and 5% CO2 at 37 C. For subculturing, the cells were trypsinized (0.05% trypsin and EDTA) and plated at 2.5 x 104 cells/cm2 in 100 x 20-mm petri dishes.

Effect of glucosamine (GlcN), azaserine (AZA), DON, SB 203580, GF 109203X, and calphostin C on ANG mRNA expression in IRPTCs
To study the effect of GlcN on the expression of ANG mRNA in IRPTCs, these cells were incubated in 5 mM glucose medium containing 1% depleted FBS in the absence or presence of various concentrations of GlcN (10-10–10-4 M) or in 25 mM glucose medium in the absence or presence of various concentration of azaserine (AZA) or DON (10-9–10-7 M) for 24 h. At the end of the incubation period, the cells were collected, and total RNA was extracted using TRIzol reagent according to the protocol of the supplier (Invitrogen). Total RNA was used in RT-PCR to quantify the amount of ANG, renin, and ß-actin mRNA as described previously (23). Briefly, 2 µg total RNA were used to synthesize first-strand cDNAs by employing the SuperScript preamplification system according to the supplier’s protocol (Invitrogen). Then, first strand cDNA was diluted with water to a ratio of 1:4, and aliquots were processed to amplify the rat ANG, renin, and ß-actin cDNA fragments with the PCR core kit according to the supplier’s instructions (Invitrogen). First strand cDNA (5 µl) and primers of rat ANG (400 nM) and rat ß-actin (100 nM) were added in a final volume of 50 µl PCR mixture (final concentration, 1x PCR buffer, 0.2 mM deoxy-NTP, 1.5 mM MgCl2, and 2.0 U Taq DNA polymerase; Life Technologies, Inc.). The PCR mixture was amplified in a PerkinElmer/Cetus 2400 thermocycler (Norwalk, CT). After denaturation at 94 C for 3 min, ANG or renin and ß-actin cDNA were coamplified in the same tube under the following conditions: 94 C for 45 sec, 60 C for 45 sec, and 72 C for 1 min and 30 sec. PCRs were further extended at 72 C for 7 min after 30 cycles.

The sense and antisense rat ANG primers used were 5'-CCT CGC TCT CTG GAC TTA TC-3' and 5'-CAG ACA CTG AGG TGC TGT TG-3', corresponding to the nucleotide sequences +676 to +695 and +882 to +901 of rat ANG cDNA (28), respectively. The sense and antisense rat renin primers used were 5'-CTG CCA CCT TGT TGT GTG AG-3' and 5'-CCA GTA TGC ACA GGT CAT CG-3', corresponding to the nucleotide sequences +1033 to +1052 and +1277 to +1296 of rat ANG cDNA (29), respectively. The sense and antisense rat ß-actin primers were 5'-ATG CCA TCC TGC GTC TGG ACC TGG C-3' and 5'-AGC ATT TGC GGT GCA CGA TGG AGG G-3', corresponding to the nucleotide sequences +155 to +179 of exon 3, and nucleotide sequences of +115 to +139 of exon 5 of the rat ß-actin gene (30), respectively.

To identify rat ANG, renin, and ß-actin cDNA fragments, 10 µl of the PCR product were electrophoresed on 1.2% agarose gel and transferred onto a Hybond-N+ nylon membrane (Amersham Pharmacia Biotech). Digoxigenin (DIG)-labeled oligonucleotide 5'-GAG GGG GTC AGC ACG GAC AGC ACC-3' or 5'-TCC CAG GGC TTG CAT GAT CA-3' corresponding to nucleotides +775 to +798 of rat ANG cDNA (28) and nucleotides +1119 to +1139 of rat renin cDNA (29), respectively, prepared with a DIG oligonucleotide 3' end labeling kit (La Roche Biochemicals, Inc.) was used to hybridize the PCR products on the membrane. After stringent washing, the membrane was detected with a DIG luminescent detection kit (La Roche Biochemicals, Inc.) and exposed to Kodak BMR film (Eastman Kodak Co., Rochester, NY). After ANG or renin mRNA analysis, the same membrane was stripped and rehybridized with a ß-actin oligonucleotide probe (sequence, 5'-TCC TGT GGC ATC CAT GAA ACT ACA TTC-3'; corresponding to nucleotides +9 to +35 of exon 4 of the rat ß-actin gene) (30). ANG or renin mRNA levels were normalized by corresponding ß-actin mRNA levels.

To determine the putative signaling pathway(s) downstream of GlcN action on ANG mRNA expression in IRPTCs, the cells were incubated in 5 mM glucose medium plus GlcN (10-6 M) in the absence or presence of AZA, DON, SB 203580, GF 109203X, and calphostin C for 24 h. At the end of the incubation period, they were collected, and total RNA was extracted for RT-PCR analysis to quantify the amounts of ANG and ß-actin mRNA expressed in IRPTCs.

The depleted FBS was prepared by incubation with 1% activated charcoal and 1% AG 1 x 8 ion exchange resin (Bio-Rad Laboratories, Richmond, CA) for 16–24 h at room temperature as described by Samuel et al. (31). This procedure removes endogenous steroid and thyroid hormones from the FBS.

Effect of GlcN on ANG expression, p38 MAPK, ATF-2, and CREB phosphorylation in IRPTCs
The effects of GlcN on ANG protein expression, p38 MAPK, ATF-2, and CREB phosphorylation were determined by Western blots with monoclonal antibodies against rat ANG (1:500 dilutions), PhosphoPlus p38 MAPK, PhosphoPlus ATF-2, and PhosphoPlus CREB antibody kits, respectively, according to the instruction of the supplier. Briefly, 1 x 107 cells were plated in 100-mm petri dishes in 5 mM glucose DMEM containing 10% FBS, cultured until reaching 80–90% confluence, and then synchronized in 5 mM glucose medium for 24 h. Subsequently, the cells were incubated in 5 mM glucose in the presence or absence of SB 203580 (10-6 M) or GF 109203X (10-6 M) for 10 min and then further incubated for various periods with GlcN (10-6 M). Then, cells were lysed in 300 µl lysis buffer [50 mM Tris-HCl (pH 8.0) containing 1% Nonidet P-40, 250 mM NaCl, 2 mM EDTA, 1 mM phenylmethylsulfonylfluoride, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM sodium orthovanadate, and 50 mM NaF] and transferred into Eppendorf tubes. The cell lysates were sonicated for 15 sec, heated at 95 C for 5 min, and centrifuged at 12,000 x g for 5 min. Thirty-five microliters of the supernatants were subjected to SDS-10% PAGE, then transferred onto a polyvinylidene difluoride membrane (Hybond-P, Amersham Pharmacia Biotech). The membrane was initially blotted for anti-ANG antibodies, phosphorylated p38 MAPK, phosphorylated ATF-2, or phosphorylated CREB and then reblotted for ß-actin, total p38 MAPK, total ATF-2, or total CREB, respectively, according to the instructions of the suppliers.

Effect of GlcN and high glucose on IRPTC hypertrophy
The effect of GlcN and high glucose on IRPTC hypertrophy was evaluated by flow cytometry, cellular p27Kip1 expression, and [3H]leucine incorporation. To determine cell size, the cells were plated at 5 x 104 cells/well in six-well plates in 5 mM glucose/DMEM containing 10% FBS. The cells were then synchronized in 5 mM glucose medium for 24 h and subsequently incubated in 5 mM glucose medium, 5 mM glucose plus GlcN (10-6 M), or 25 mM glucose medium in the presence of AZA (10-7 M), GF 109203X (10-6 M), or SB 203580 (10-6 M). The cells were harvested at the end of the incubation period, dispersed to a single cell suspension, resuspended at a density of 106 cells/ml in PBS, and subjected to flow cytometric analysis (FACScan, BD Biosciences, Mountain View, CA) with CellQuest Pro software as described previously (21, 22). Briefly, a 14-milliwatt argon laser (emission, 488 nm) delivered forward angle light scatter (FSC)-side angle light scatter histograms (voltage, E-1; amplification gain, 4; threshold, 100 channel units based on FSC). FSC, which is proportional to relative cell size for 104 cells/sample, was analyzed with CellQuest Pro software while gating on physical parameters to exclude cell debris. Changes in FSC served to assess relative cell size for 104 cells/sample. Cell viability was determined by staining with propidium iodide (0.5 µg/ml).

The intracellular levels of p27Kip1 protein, an indicator of renal proximal tubular cell hypertrophy (32, 33), were measured by Western blotting using monoclonal antibodies against p27Kip1. Briefly, the cells were incubated in medium containing 5 mM glucose, 5 mM glucose plus GlcN (10-6 M), or 25 mM glucose in the absence or presence of AZA (10-7 M), GF 109203X (10-6 M), or SB 203580 (10-6 M) for 4 h. Then cells were lysed in 300 µl lysis buffer. Fifty micrograms of cellular proteins were subjected to 10% SDS-PAGE and transferred onto a Hybond-P membrane. The membrane was blotted by p27Kip1 monoclonal antibody and enhanced chemiluminescent developing reagent (ECL, Amersham Pharmacia Biotech). To normalize the amount of proteins applied, the same membrane was reblotted by monoclonal antibody against ß-actin.

[3H]Leucine incorporation was used to assess cellular protein synthesis. Briefly, 1 x 104 cells were plated in 24-well plates and kept quiescent for 24 h. Then the cells were incubated with 0.5 µCi/ml [3H]leucine in 5 mM glucose, 5 mM glucose plus GlcN (10-6 M), or 25 mM glucose medium in the absence or presence of AZA (10-7 M), GF 109203X (10-7 M), or SB 203580 (10-6 M) at 37 C for 24 h. At the end of the incubation period, the cells were washed with PBS, dissolved in 100 µl 2 M NaOH, and counted for radioactivity ([3H]leucine incorporation).

Statistical analysis
Four to six separate experiments per protocol were performed, and each treatment group was assayed in duplicate or triplicate. The data were analyzed by one-way ANOVA and Bonferroni test. P <= 0.05 was regarded as statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of GlcN, AZA, and DON on ANG mRNA and ANG protein expression in IRPTCs
Figure 1Go, A and B, shows that GlcN at 10-8–10-6 M in medium containing 5 mM D-glucose significantly stimulated the accumulation of ANG mRNA and renin mRNA to a level more than 1.5- to 2.5-fold higher than that found in control cells (without glucosamine), respectively. High glucose enhanced ANG mRNA expression, and this effect was inhibited by AZA and DON in a dose-dependent manner, with the maximal effect observed at 10-7 M (Fig. 2Go, A and B). D-Mannitol (20 mM) was added in 5 mM D-glucose medium to normalize the osmolarity in comparison with 25 mM D-glucose medium.



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  		FIG. 1. Effect of glucosamine (GLcN) on rat ANG and renin mRNA expression in IRPTCs. Cells were incubated for 24 h in normal glucose (5 mM D-glucose) DMEM in the absence or presence of various concentrations of GlcN. Cells were collected and assayed for rat ANG mRNA (A) and renin mRNA (B) by RT-PCR as described in Materials and Methods. The relative densities of the ANG were normalized with the ß-actin control. ANG and renin mRNA levels in IRPTCs incubated in 5 mM D-glucose DMEM (A and B) were considered the control (100%). Each point represents the mean ± SD of four independent experiments performed in duplicate in A and B, respectively. *, P <= 0.05; **, P <= 0.01; ***, P <= 0.005.

 


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  		FIG. 2. Effect of AZA and DON on rat ANG mRNA expression in IRPTCs. Cells were incubated in normal glucose (5 mM) with or without 20 mM D-mannitol or in high glucose (25 mM D-glucose) DMEM in the absence or presence of different concentrations of AZA (A) or DON (B). Cells were collected and assayed for rat ANG and ß-actin mRNAs by RT-PCR as described in Materials and Methods. The relative densities of the ANG were normalized with the ß-actin control. ANG mRNA levels in IRPTCs incubated in 5 mM D-glucose DMEM (A and B) were considered the control (100%). Each point represents the mean ± SD of four independent experiments performed in duplicate in A and B, respectively. *, P <= 0.05; **, P <= 0.01; ***, P <= 0.005.

 
The stimulatory effect of GlcN on ANG mRNA and protein expression was suppressed in the presence of GF 109203X (10-6 M), SB 203580 (10-6 M), or calphostin C (10-7 M), but not in the presence of AZA (10-7 M) and DON (10-7 M; Fig. 3Go, A and B). These studies indicate that the effect of GlcN on ANG gene expression is mediated via PKC and p38 MAPK signaling.



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  		FIG. 3. Effects of SB 203580, GF 109203X, and calphostin C on rat ANG mRNA and ANG expression in IRPTCs cultured in the presence of GlcN. Cells were incubated for 24 h in normal glucose DMEM and GlcN (10-6 M) in the absence or presence of SB 203580 (10-6 M), GF 109203X (10-6 M), or calphostin C (10-7 M). Cells were harvested and assayed for ANG and ß-actin mRNA levels by RT-PCR (A) or for ANG levels by Western blot (B) as described in Materials and Methods. The ANG mRNA and ANG levels in IRPTCs incubated in normal glucose DMEM in the absence of GlcN were considered the control (100%). The inhibitory effects of SB 203580, GF 109203X, and calphostin C were compared with those of cells incubated in normal DMEM in the presence of GlcN. Each point represents the mean ± SD of three independent experiments performed in duplicate in A and B. *, P <= 0.05; **, P <= 0.01; ***, P <= 0.005.

 
Effect of GlcN on p38 MAPK and ATF-2 phosphorylation in IRPTCs
We tested the hypothesis that p38 MAPK signaling mediates the GlcN effect by assaying p38 MAPK and ATF-2 phosphorylation. Figures 4Go and 5Go show that GlcN (10-6–10-5 M) significantly stimulates p38 MAPK and ATF-2 phosphorylation in IRPTCs (Figs. 4AGo and 5AGo), respectively, which can be suppressed by SB 203580 (10-6 M; Figs. 4BGo and 5BGo), whereas GF 109203X had no effect. These studies confirm that the GlcN action on ANG gene expression is mediated at least in part via activation of p38 MAPK signaling and ATF-2 phosphorylation in IRPTCs.



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  		FIG. 4. Effect of GlcN on p38 MAPK phosphorylation in IRPTCs. Cells were incubated for 10 min in normal glucose DMEM in the absence or presence of various concentrations of GlcN (i.e. 10-10–10-5 M), then harvested and assayed for phosphorylated p38 MAPK (A), or, after 10-min incubation in normal glucose DMEM with or without GlcN (10-6 M) in the absence or presence of SB 203580 or GF 109203X, cells were harvested and assayed for p38 MAPK (B). Phosphorylated p38 MAPK levels in normal glucose DMEM are considered the control (100%). The results are given as percentages of the controls [mean ± SD; n = 3 independent experiments performed in duplicate (A) and in triplicate (B)]. **, P <= 0.01; ***, P <= 0.005.

 


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  		FIG. 5. Effect of GlcN on ATF-2 phosphorylation in IRPTCs. A, Cells were incubated for 10 min in normal glucose DMEM in the absence or presence of various concentrations of GlcN (i.e. 10-8–10-4 M), then harvested and assayed for phosphorylated ATF-2. B, After 10-min incubation in normal glucose DMEM with or without GlcN (10-6 M) in the absence or presence of SB 203580 or GF 109203X, cells were harvested and assayed for ATF-2. The phosphorylated ATF-2 level in cells incubated in normal glucose DMEM was considered the control (100%). Each point represents the mean ± SD of four independent experiments performed in duplicate (A) and six independent experiments performed in duplicate (B). **, P <= 0.01; ***, P <= 0.005.

 
Effect of GlcN on CREB phosphorylation in IRPTCs
Figure 6Go shows that GlcN at 10-6–10-5 M also significantly stimulated CREB phosphorylation in IRPTCs 2.0-fold over control levels (without GlcN; Fig. 6AGo). This GlcN action was inhibited by the presence of GF 109203X (10-6 M), but not by SB 203580 (10-6 M; Fig. 6BGo). These studies suggest that the effect of GlcN on ANG gene expression is mediated at least in part via activation of PKC signaling and CREB phosphorylation in IRPTCs.



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  		FIG. 6. Effect of GlcN on CREB phosphorylation in IRPTCs. A, Cells were incubated for 30 min in normal glucose DMEM in the absence or presence of various concentrations of glucosamine (i.e. 10-12–10-6 M), then harvested and assayed for phosphorylated CREB. B, After 30 min of incubation in normal glucose DMEM with or without glucosamine (10-6 M) in the absence or presence of SB 203580 or GF 109203X, cells were harvested and assayed for CREB. The relative density of the phosphorylated CREB band was compared with that of nonphosphorylated CREB. The phosphorylated CREB level in cells incubated in 5 mM D-glucose medium was considered the control (100%). Each point represents the mean ± SD of three independent experiments performed in duplicate (A) and four independent experiments performed in triplicate (B). *, P <= 0.05; **, P <= 0.01; ***, P <= 0.005.

 
Effect of GlcN and high glucose on IRPTC hypertrophy
Figures 7AGo and 8AGo show a right shift in FSC of cells cultured in 5 mM glucose medium plus GlcN (10-6 M) or in 25 mM glucose medium alone, respectively, compared with the scatter of control cells (5 mM glucose medium). The cell diameter of the cells treated with GlcN or 25 mM glucose medium was about 8% (range, 5–12%) larger than that of cells cultured in 5 mM glucose medium, indicating a considerable increase in cell size. AZA (10-7 M) could not prevent the hypertrophic effect of GlcN (Fig. 7BGo), but prevented the hypertrophic effect of high glucose (Fig. 8BGo). GF 109203X (10-6 M) blocked the hypertrophic effect of both GlcN (Fig. 7CGo) and high glucose (Fig. 8CGo). In contrast, SB 203580 could not prevent the increase in cell diameter stimulated by GlcN (Fig. 7DGo) or 25 mM glucose medium (Fig. 8DGo).



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  		FIG. 7. Effects of GlcN, AZA, GF 109203X, and SB 203580 on cellular hypertrophy in IRPTCs. After 48 h of incubation in medium containing 5 mM D-glucose with or without GlcN (10-6 M; A) or 5 mM glucose plus GlcN in the absence or presence of AZA (10-7 M; B), GF 109203X (10-6 M; C), or SB 203580 (10-6 M), IRPTCs were analyzed by flow cytometry. Propidium iodide was used to assess cell viability. Forward light scatter was expressed in arbitrary units. A rightward shift of the plot on the x-axis indicates an increase in cell size.

 


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  		FIG. 8. Effect of high glucose, AZA, GF 109203X, and SB 203580 on cellular hypertrophy in IRPTCs. After 48 h of incubation in medium containing 5 mM D-glucose plus 20 mM D-mannitol or 25 mM D-glucose (A), or 25 mM D-glucose in the absence or presence of AZA (10-7 M; B), GF 109203X (10-6 M; C), or SB 203580 (10-6 M; D), IRPTCs were analyzed by flow cytometry. Propidium iodide was used to assess cell viability. Forward light scatter was expressed in arbitrary units. A rightward shift of the plot on the x-axis indicates an increase in cell size.

 
The cellular p27Kip1 protein content was significantly augmented in IRPTCs incubated either in 5 mM glucose plus GlcN (10-6 M) or in 25 mM glucose compared with that of control cells (incubated in 5 mM glucose; Fig. 9Go, A and B). AZA (10-7 M) could not inhibit the stimulatory effect of GlcN (Fig. 9AGo), but inhibited the stimulatory effect of high glucose on p27Kip1 expression (Fig. 9BGo). GF 109203X (10-6 M) inhibited the stimulatory effect of GlcN and high glucose on p27Kip1 expression (Fig. 9Go, A and B), whereas SB 203580 had no significant inhibitory effect (Fig. 9Go, A and B).



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  		FIG. 9. Effects of GlcN, high glucose, AZA, and GF 109203X on cellular p27Kip1 levels in IRPTCs. After 4 h of incubation in medium containing 5 mM D-glucose with or without GlcN and in the absence or presence of AZA, GF 109203X, or SB 203580 (A), the cells were harvested and assayed for p27Kip1 levels by Western blotting. Similarly, cells were incubated in 25 mM D-glucose in the absence or presence of AZA, GF 109203X, or SB 203580 for 4 h (B) and then assayed for p27Kip1 levels. The relative densities of the p27Kip1 band were normalized with ß-actin. The p27Kip1 protein levels in cells cultured in 5 mM D-glucose medium represent the control (100%). Each bar is the mean ± SD of six independent experiments. **, P <= 0.01; ***, P <= 0.005.

 
Protein synthesis was also significantly elevated in IRPTCs incubated in 5 mM glucose medium plus GlcN (10-6 M) and in high glucose medium (Fig. 10Go, A and B). AZA (10-7 M) could not inhibit the stimulatory effect of GlcN (Fig. 10AGo), but prevented the stimulatory effect of high glucose on protein synthesis (Fig. 10BGo). GF109203X (10-6 M) inhibited the stimulatory effect of GlcN and high glucose on cellular protein synthesis (Fig. 10Go, A and B), whereas SB 203580 has no significant effect (Fig. 10Go, A and B).



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  		FIG. 10. Effects of GlcN, high glucose, AZA, and GF 109203X on cellular protein levels in IRPTCs. Protein synthesis in IRPTCs was determined by [3H]leucine incorporation and expressed per 4 x 104 cells. The cells were incubated for 24 h in 5 mM D-glucose with or without GlcN (10-6 M) and in the absence or presence of AZA (10-7 M), GF 109203X (10-6 M), or SB 203580 (10-6 M), then were harvested and analyzed for protein synthesis (A). Similarly, cells were incubated in 25 mM D-glucose in the absence or presence of AZA, GF 109203X, or SB 203580 for 24 h (B) and then assayed for [3H]leucine protein levels. Cellular protein synthesis in IRPTCs incubated in 5 mM D-glucose medium represents the control (100%). Each bar is the mean ± SD of six independent experiments. *, P <= 0.05; **, P <= 0.01.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present studies demonstrate that high glucose action on ANG gene expression and induction of IRPTC hypertrophy is mediated at least in part by activation of the HBP. This stimulatory action of HBP appears to be p38 MAPK and PKC dependent.

ROS have increasingly been implicated in the progression of renal disease (34, 35). Although the high glucose milieu may induce oxidative stress in the diabetic kidney (36, 37), the underlying mechanism(s) of ROS-induced damage remains incompletely understood. Recently, Brownlee’s group (38, 39) proposed a unifying concept to explain the mechanisms of high glucose in tissue injury. This concept suggests that increased glucose metabolism via mitochondrial oxidation results in excess ROS generation. Elevation of ROS partially inhibits glyceraldehyde-3-phosphate dehydrogenase, thereby slowing glucose flux through the glycolytic-tricarboxylic acid cycle pathway. The elevated ROS simultaneously promotes glucose metabolism via alternate pathways, such as the polyol/PKC pathway and the HBP, as well as through nonenzymatic conversion of glucose to advanced glycation end products. All of these effects subsequently influence gene expression. Indeed, studies have shown that activation of the HBP stimulated the expression of several genes, including TGFß1 (40, 41), leptin (42, 43), plasminogen activator inhibitor-1 (44, 45), and fibronectin (46). Recent studies have also revealed that GlcN stimulates ANG gene expression in rat liver and adipose tissue (26), supporting the role of this pathway in ANG gene expression. Thus, we speculated that HBP may mediate some of the high glucose effect on ANG gene expression and may play a role in the induction of hypertrophy in kidney proximal tubular cells.

We have chosen rat IRPTCs for the present studies. Ninety to 95% of cells are stained positively for antigens specific to the proximal tubule as well as for RAS components, including ANG, renin, ACE, and Ang II receptors (27). Thus, IRPTCs resemble rat RPTCs in vivo. In contrast to primary cultures of nondiabetic rat RPTCs that will not grow after two or three passages. IRPTCs grow continuously, with a population doubling time of 16–18 h. Most importantly, both IRPTCs and primary cultures of nondiabetic rat RPTCs respond similarly to high glucose and insulin on ANG gene expression (19, 20, 21, 22, 23, 47, 48, 49, 50). Hence, the data obtained with IRPTCs could be extrapolated to the normal biology of RPTCs in vivo.

Our present studies demonstrate that GlcN (10-8–10-4 M) stimulates ANG and renin mRNA expression in IRPTCs in normal glucose medium (5 mM), with a maximum effect observed at 10-6 M. These stimulatory concentrations of GlcN are lower than those used by Gabriel et al. (26), who reported that GlcN at 2 mM enhanced ANG gene expression in diabetic liver and adipose tissue. In contrast, we observed that GlcN at 10-4 M or higher started to inhibit ANG mRNA and renin mRNA expression in IRPTCs. At present, we do not have an explanation for this discrepancy. One possibility is that high concentrations of GlcN (i.e. >=10-4 M) are toxic to IRPTCs, but not to liver or adipose tissue. Indeed, we have found that GlcN (10-4–10-3 M) increases the percentage of cell death from 5–12% in IRPTCs after 24 h of incubation, as determined by the trypan blue exclusion method (unpublished results). These data suggest that the renal tubular cells might be more sensitive to GlcN toxicity than liver or adipose tissue. More studies are definitely warranted to clarify these discrepancies. It is interesting that GlcN also stimulates renin mRNA expression. To our best knowledge, this is the first report that GlcN could enhance renin gene expression in kidney proximal tubular cells. Thus, our data indicate that GlcN could directly activate intrarenal RAS to yield Ang II and subsequently affects cell hypertrophy. Furthermore, our results reveal that AZA and DON block the stimulatory effect of high glucose (25 mM) on ANG mRNA expression in IRPTCs in a dose-dependent manner. Taken together, these studies suggest that a major portion of the stimulatory effect of high glucose on ANG gene expression is mediated at least in part via activation of the HBP.

Interestingly, the addition of GF 109203X, calphostin C, and SB 203580 inhibited the stimulatory effect of GlcN on ANG mRNA and ANG expression in IRPTCs, whereas AZA and DON had no effect. These studies indicate that the GlcN effect on ANG gene expression in IRPTCs could by-pass the GFAT enzyme and is PKC and p38 MAPK dependent. At present it is unclear whether GlcN could affect the half-life or stability of ANG mRNA, or if it has an effect at the transcriptional level in IRPTCs.

Our results showed that GlcN stimulated p38 MAPK, ATF-2, and CREB phosphorylation in IRPTCs. SB 203580 inhibited p38 MAPK and ATF-2 phosphorylation, whereas it did not affect CREB phosphorylation. In contrast, GF 109203X had no effect on p38 MAPK and ATF-2 phosphorylation, but significantly inhibited CREB phosphorylation stimulated by GlcN. These data further support the idea that GlcN-stimulated ANG gene expression is mediated via both p38 MAPK and PKC signal transduction pathways.

Culture of IRPTCs in 5 mM glucose medium plus GlcN or in 25 mM glucose medium induced cell hypertrophy, as evidenced by the rightward shift in FSC on flow cytometry compared with 5 mM glucose medium, the enhancement of cellular p27Kip1 expression, and protein synthesis. Although flow cytometry does not allow the precise measurement of cell size, the 5–10% increase in cell diameter observed in our studies would indicate a considerable increment in the average size of IRPTCs cultured in GlcN and high levels of glucose. These results are in agreement with previous observations, including ours, that high glucose levels (i.e. >=25 mM) induce hypertrophy of murine and porcine proximal tubular cells by 5–10%, as analyzed by flow cytometry (1, 2, 3, 4, 5). Our present experiments also show that the addition of AZA did not block the GlcN effect, but prevented the effect of high glucose on IRPTC hypertrophy, p27Kip1 expression and cellular protein synthesis. In contrast, GF 109203X was effective in preventing the stimulatory effect of GlcN and high glucose on IRPTC hypertrophy, p27Kip1 expression, and cellular protein synthesis, whereas SB 203580 had no significant effect (i.e. P >= 0.05) on these parameters. We were surprised that SB 203580 inhibited the stimulatory effect of GlcN and high glucose on ANG gene expression, but not on cell hypertrophy. At present, we do not have a good explanation for these observations. One possibility might be that SB 203580 does not inhibit the expression of other, as yet unidentified factors involved in cell hypertrophy, whereas the expression of these factors can be inhibited by PKC inhibitors. On the other hand, our findings are similar to those of Hannken et al. (51), who reported that SB 203580 failed to abolish Ang II-stimulated p27Kip1 expression in mouse renal proximal tubular cells. Indeed, more studies are needed along this line. Nevertheless, our data indicate that the blockade of activation of the local renal RAS by HBP or PKC signaling is an effective method of attenuating or preventing renal proximal tubular cell hypertrophy induced by hyperglycemia.

At present we do not know the exact molecular mechanism(s) underlying the stimulatory action of high glucose (i.e. the downstream pathway after p38 MAPK and PKC activation) on ANG gene expression in IRPTCs. One possibility is that high glucose induces CREB phosphorylation via the PKC signaling pathway, as demonstrated by our present data and reported by Kreisberg et al. (52). The phosphorylated CREB might have dimerized to form a homodimer or interacts with phosphorylated nuclear ATF-2 to form a heterodimer that binds to the cAMP-responsive element in the 5'-flanking region of the rat ANG gene (53) and subsequently enhances gene expression. This possibility is supported by our present finding that high glucose levels augmented the phosphorylation of both CREB and ATF-2 in IRPTCs. Furthermore, our preliminary studies showed that GlcN enhances ANG mRNA expression in IRPTCs that have been cotransfected with sense CREB cDNA and ATF-2 cDNA, but not in IRPTCs that have been cotransfected with antisense CREB cDNA and sense ATF-2 cDNA (unpublished results). Nevertheless, more studies are warranted to confirm this possibility.

In summary, our studies reveal that exposure of IRPTCs to GlcN or 25 mM glucose enhances ANG gene expression and induces IRPTC hypertrophy. This stimulatory effect of high glucose is blocked by inhibitors of HBP and PKC. These results suggest that HBP blockade might offer a novel therapeutic approach to prevent or attenuate glucose-induced ANG gene expression and, consequently, the development of diabetic nephropathy. However, it remains to be seen whether long-term HBP blockade may indeed be beneficial in the treatment of diabetic nephropathy.


   Acknowledgments
 
We thank Mr. Ovid M. Da Silva (Éditeur-Rédacteur, Research Support Office, Research Center, CHUM) for editing this manuscript.


   Footnotes
 
This work was supported by grants from the Canadian Diabetes Association (1061, to J.S.D.C. and P.H.), the Kidney Foundation of Canada, the Canadian Institutes of Health Research [MOP-13420 (to J.S.D.C.), MT-15070 (to J.S.D.C. and J.G.F.), and MOP-12573 (to J.G.F.) and NET-54012 (I.G.F.)], the NIH [HL-48455 (to J.R.I.) and DK-50836 (to S.S.T.)], and Juvenile Diabetes Research Foundation (to I.G.F.).

1 S.L.-Z. is the recipient of a Canadian Institutes of Health Research Doctoral Research Award. Back

Abbreviations: ACE, Angiotensin-converting enzyme; ANG, angiotensinogen; Ang II, angiotensin II; ATF-2, activating transcription factor-2; AZA, azaserine (O-diazoacetyl-L-serine); CREB, cAMP-responsive element-binding protein; DIG, digoxigenin; DON, 6-diazo-5-oxo-L-norleucine; FBS, fetal bovine serum; FCS, forward angle light scatter; GFAT, glutamine:fructose-6-phosphate amidotransferase; HBP, hexosamine biosynthesis pathway; IRPTCs, immortalized renal proximal tubular cells; PKC, protein kinase C; RAS, renin-angiotensin system; ROS, reactive oxygen species.

Received February 18, 2003.

Accepted for publication July 2, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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