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Endocrinology Vol. 143, No. 12 4627-4635
Copyright © 2002 by The Endocrine Society

ARTICLE

Insulin Inhibits Dexamethasone Effect on Angiotensinogen Gene Expression and Induction of Hypertrophy in Rat Kidney Proximal Tubular Cells in High Glucose

Shao-Ling Zhang, Xing Chen, Chih-Chang Wei, Janos G. Filep, Shiow-Shih Tang, Julie R. Ingelfinger and John S. D. Chan

Université de Montréal, Centre Hospitalier de l’Université de Montréal (S.-L.Z., X.C., C.-C.W., J.S.D.C.), Hôtel-Dieu Hospital, Research Center, Montréal, Québec H2W 1T8 Canada; Université de Montréal (J.G.F.), Maisonneuve-Rosemont Hospital, Research Center, Montréal, Québec, Canada, H1T 2M4; and Harvard Medical School (S.-S.T., J.R.I.), Massachusetts General Hospital, Pediatric Nephrology Unit, Boston, Massachusetts 02114-3117

Address all correspondence and requests for reprints to: John S. D. Chan, Université de Montréal, Centre Hospitalier de l’Université de Montréal, Hôtel-Dieu Hospital, Research Center, Pavillon Masson, 3850 Saint Urbain Street, Montréal, Québec H2W 1T8 Canada. E-mail: john.chan{at}umontreal.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The present studies investigated whether insulin inhibits the stimulatory effect of dexamethasone (DEX) on angiotensinogen (ANG) gene expression and induction of hypertrophy in rat immortalized renal proximal tubular cells (IRPTCs) in a high-glucose milieu. Rat IRPTCs were cultured in monolayer. ANG and ANG mRNA expression in IRPTCs were quantified by a specific RIA for rat ANG and by RT-PCR assay, respectively. A fusion gene containing the full length of the 5'-flanking region of the rat ANG gene linked to a chloramphenicol acetyl transferase reporter gene was introduced into IRPTCs. The level of fusion gene expression was determined by cellular chloramphenicol acetyl transferase enzymatic activity. Cellular hypertrophy was assessed by flow cytometry, cellular p27Kip1 protein expression, and protein assay. Our results showed that high glucose (i.e. 25 mM) and DEX (10-7 M) additively stimulated ANG gene expression and induced IRPTC hypertrophy. Insulin inhibited the effect of high glucose and DEX on these parameters. The inhibitory effect of insulin was reversed by PD 98059 (a MAPK inhibitor) but not by wortmannin (a phosphatidylinositol-3-kinase inhibitor). These results demonstrate that insulin is effective in blocking the stimulatory action of high glucose and DEX on ANG gene expression and induction of IRPTC hypertrophy, suggesting its important role in preventing local intrarenal renin-angiotensin system activation and renal proximal tubular cell hypertrophy induced by hyperglycemia and glucocorticoids in vivo.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN ADDITION TO THE circulating renin-angiotensin system (RAS), the existence of a local intrarenal RAS is generally accepted (1, 2). For example, the mRNA components of the RAS, including angiotensinogen (ANG), renin, angiotensin-converting enzyme (ACE) and angiotensin II receptors are expressed in rodent (mouse and rat) immortalized renal proximal tubular cell lines (IRPTCs; Refs. 3, 4, 5, 6, 7, 8). These findings suggest that local intrarenal angiotensin II (Ang II) may play a paracrine and/or an autocrine role in the kidney.

Clinical and animal studies have shown that intensive insulin therapy and chronic treatment with ACE inhibitors or angiotensin II receptor1 antagonists delay the progression of nephropathy in diabetes (9, 10, 11, 12). In vitro experiments have also shown that the incubation of murine proximal tubular cells in high glucose (i.e. 25 mM) medium or in the presence of high levels of Ang II (i.e. >10-8 M) induced cellular hypertrophy and extracellular matrix protein synthesis (13, 14, 15, 16). Such observations suggest that high glucose levels and intrarenal RAS activation may play an important role in the pathogenesis of diabetic nephropathy.

We have previously reported that RU 486 (a glucocorticoid receptor antagonist) blocked the stimulatory effect of dexamethasone (DEX) on immunoreactive (IR)-ANG secretion from rat IRPTCs (17). More recently we reported that high glucose levels stimulated ANG gene expression and induced hypertrophy of IRPTCs (18, 19, 20, 21). The stimulatory effect of high glucose was prevented by inhibitors of aldose reductase and protein kinase C (PKC), blockers of p38 MAPK and the RAS, as well as by stable transfection of antisense rat ANG (rANG) cDNA (18, 19, 20, 21). Taken together, these studies suggest that glucocorticoid and high glucose may act additively to stimulate ANG gene expression and induce RPTC hypertrophy in diabetes.

In the present studies, we investigated whether insulin could inhibit the stimulatory effect induced by high glucose and DEX on ANG gene expression and induction of IRPTC hypertrophy. Our findings demonstrated that insulin indeed inhibits the additive action of high glucose and DEX on ANG gene expression and induction of IRPTC hypertrophy. The inhibitory action of insulin was reversed by PD 98059, an inhibitor of MAPK, MAPK kinase (MEK; Ref. 22) but not by wortmannin, an inhibitor of phosphatidylinositol-3-kinase (PI3K; Ref. 23).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
D(+)-Glucose, L-glucose, DEX, and insulin were purchased from Sigma-Aldrich Canada Ltd. (Oakville, Ontario, Canada). RU 486 was a gift from Dr. Paul Robel (Unité 488 Institut National de la Santé et de la Recherche Medicale, Bicêtre, France). Normal glucose (1000 mg/liter or 5 mM) DMEM (pH 7.45) (catalog no. 12320-032) was obtained from Life Technologies, Inc. (Burlington, Ontario, Canada). PD 98059 and wortmannin were purchased from Calbiochem Inc. (La Jolla, CA). {gamma}-[32P-ATP] (3000 Ci/mol), {alpha}-[35S] dATP (>1000 Ci/mmol), Na125I and D-threo-[1,2 14C]choramphenicol were purchased from Amersham-Pharmacia Biotech (Baie d’Urfe, Quebec, Canada).

The expression vectors, plasmid containing the coding sequence for chloramphenicol acetyltransferase (CAT) without the promoter (pOCAT) or with Rous sarcoma virus enhancer/promoter sequence (pRSV/CAT) fused to the 5'-end of the CAT coding sequence, respectively, were a gift from Dr. Joel F. Habener (Molecular Endocrinology, Massachusetts General Hospital, Boston, MA). Thin-layer chromatography plates were purchased from Fisher Scientific (Montréal, Québec, Canada). Restriction and modified enzymes were acquired from Life Technologies, Inc., La Roche Biochemicals (Dorval, Québec, Canada), or Amersham-Pharmacia Biotech.

RIA for rat ANG
The RIA for rANG was developed in our laboratory, and the procedure has been described previously (17). Purified plasma rANG (i.e. >90% pure, as analyzed by SDS-PAGE) and iodinated rANG were used as hormone and tracer, respectively. This RIA is specific for intact rANG (i.e. 62- to 65-kDa rANG) and has no cross-reactivity with pituitary hormone preparations or other rat plasma proteins (17). The lower limit of detection for this RIA is approximately 2 ng rANG. The intra- and interassay coefficients of variation were 9% (n = 10) and 14% (n = 10), respectively.

Cell culture
IRPTCs at passages 12–18 were used in the present studies. The characteristics of the IRPTCs have been described previously (7, 24). These cells are highly differentiated and express proximal tubular characteristics as defined by the presence of antigens including carbonic anhydrase, ecto-ATPase and GLUT-2. The cells also express alkaline phosphatase, aquaporin-1 (CHIP-28), and the megalin (gp330), but they do not express factor VIII. Ninety to 95% of cells are stained positively for these antigens as well as for RAS components, i.e. ANG, renin, ACE, and ANG II receptors. Thus, IRPTCs resemble rat renal proximal tubular cells (RPTCs) in vivo. In contrast to primary culture of nondiabetic rat RPTCs that will not grow after two to three passages, IRPTCs grow continuously, with a population doubling time of 16–18 h. Most importantly, both IRPTCs and primary culture of nondiabetic rat RPTCs respond similarly to both high glucose and insulin on ANG gene expression (18, 19, 20, 21, 25, 26, 27) Hence, the data obtained with IRPTCs support the extrapolation to the normal biology of RPTCs in vivo.

Briefly, IRPTCs were grown in 100 x 20-mm plastic Petri dishes (Life Technologies, Inc.) in 5 mM glucose DMEM, supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 µg/ml streptomycin. The cells were kept in a humidified atmosphere in 95% air, 5% CO2 at 37 C. For subculturing, cells were trypsinized (0.05% trypsin and 0.01% EDTA) and plated at 2.5 x 104 cells/cm2 in 100 x 20-mm Petri dishes.

DNA transfection and CAT assay
The method of construction of the rANG-CAT fusion gene, pOCAT (rANG N-1498/+18) has been described previously (28, 29). Plasmid or ANG-CAT fusion gene were transfected into IRPTCs using Lipofectamine reagent according to the instruction manual provided by the supplier (Life Technologies, Inc.) as described previously (26). We have optimized the DNA concentration for gene transfection at 2–3 µg per 0.5–1 x 106 cells. Thus, in the present studies, a total of 2 µg supercoiled DNA (i.e. 2 µg pOCAT (rANG N-1498/+18) or 1 µg pRSV/CAT and 1 µg pOCAT) was used routinely in cell transfection. Twenty-four hours after the transfection, the media were replaced with fresh media containing 5 mM or 25 mM D-glucose and 1% depleted fetal bovine serum (dFBS, see below) in the absence or presence of various hormones or drugs. The cells were harvested 24–48 h later and assayed for CAT activity (29, 30).

To normalize the efficiency of transfection, 1 µg pTK/hGH [a vector with the thymidine kinase enhancer/promoter fused to the 5'-human GH gene] was cotransfected with 2 µg pOCAT (rANG N-1498/+18) or 1 µg pRSV/CAT and 1 µg pOCAT as described previously (30). The plasmid pRSV/CAT served as a positive control to monitor the efficiency of transfection of rANG-CAT fusion gene in the absence of drugs added. The level of transfection efficiency for pRSV/CAT in IRPTCs ranged from 60–90%, i.e. the percentage of conversion of 14-C chloramphenicol to mono- and diacetyl chloramphenicol. The transfection efficiency of pOCAT (rANG N-1498/+18) in IRPTCs ranged from 25–35%, compared with pRSV/CAT. The inter- and intraassay coefficient variations of transfection for pOCAT (rANG N-1498/+18) in IRPTCs are 25% and 12% (n = 10), respectively. The method for CAT assay has been described previously (29, 30).

Effect of high glucose, DEX, and insulin on IR-rANG secretion from IRPTCs
IRPTCs were plated at a density of 1–2 x 105 cells/well in 6-well plates and incubated overnight in normal glucose DMEM containing 10% FBS. Cell growth was arrested by incubating the cells in serum-free DMEM for 24 h. Various DEX concentrations (10-13 to 10-7 M) in the presence or absence of RU 486 (a glucocorticoid receptor antagonist) were then added to normal (5 mM) or high (25 mM) glucose culture medium containing 1% dFBS, and the cells were incubated for an additional 24 h. To study the effect of insulin on IR-rANG secretion from IRPTCs, the cells were incubated in 5 mM or 25 mM medium plus DEX (10-7 M) in the presence or absence of insulin (i.e. 10-13 to 10-7 M) for 24 h. At the end of the incubation period, the media were collected and stored at -20 C until assayed for IR-rANG. To maintain constant isotonicity or osmolality, the 5 mM glucose media were supplemented with L-glucose to 20 mM final concentration in all experiments. The dFBS (i.e. depleted of endogenous steroid and thyroid hormones) was prepared by incubation with 1% activated charcoal and 1% AG 1 x 8 ion-exchange resin (Bio-Rad Laboratories, Inc., Richmond, CA) for 16–24 h at room temperature, as described by Samuels et al. (31).

Effect of high glucose, DEX, and insulin on ANG mRNA expression in IRPTCs
To study the effect of DEX and insulin on ANG mRNA expression, IRPTCs were incubated in 5 mM glucose or 25 mM glucose medium in the absence or presence of DEX (10-7 M) with or without insulin (10-7 M) for 24 h. At the end of the incubation period, the cells were collected, and total RNA was extracted with Trizol reagent (Life Technologies, Inc.) according to the protocol of the supplier. The total RNA was used in RT-PCR assays for ANG and ß-actin mRNA, as we have described previously (18, 19, 20, 21, 25, 26, 27). The forward primer 5' CCT CGC TCT CTG GAC TTA TC 3' and the reverse primer 5' CAG ACA CTG AGG TGC TGT TG 3', corresponding to the nucleotide sequences N + 676 to N + 695 and N + 882 to N + 901 of rANG cDNA (32), were employed for PCR. Furthermore, primers specific for rat ß-actin (33) (forward and reverse primers 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 nucleotide N + 155 to N + 179 of exon 3 and nucleotide N + 115 to N + 139 of exon 5 of rat ß-actin) served as internal controls in another PCR. The RT-PCR mixtures were then separated on 1.5% agarose gel and transferred onto Hybond XL nylon membranes (Amersham-Pharmacia Biotech). Subsequently, the 32P-labeled oligonucleotides 5' GAG GGG GTC AGC ACG GAC AGC ACC 3' and 5' TCC TGT GGC ATC CAT GAA ACT ACA TTC 3', corresponding to the nucleotides N + 775 to N + 798 of rANG cDNA (28) and nucleotides N + 9 to N + 35 of exon 4 of rat ß-actin (29), respectively, were used to hybridize products on the membrane. Finally, the membrane was washed and exposed to autoradiography. The relative densities of the PCR bands were determined with a computerized laser densitometer.

Effect of high glucose, DEX, and insulin on IRPTC hypertrophy
The effect of high glucose, DEX, and insulin on IRPTC hypertrophy was evaluated by flow cytometry, cellular p27Kip1 expression and protein content. Briefly, IRPTCs were plated at 5 x 104 cells/well in 6-well plates in 5 mM glucose DMEM containing 10% FBS. Then the cells were synchronized in 5 mM glucose for 24 h. Subsequently, the cells were incubated in 5 mM glucose medium, 25 mM glucose medium, or 25 mM glucose medium and DEX (10-7 M) in the absence or presence of RU 486 (10-5 M) or insulin (10-7 M) with or without PD 98059 (10-5 M) at 37 C for 24–72 h. The media were changed every 24 h. The cells were harvested at the end of various incubation periods and titrated to a single cell suspension at a density of 106 cells/ml in PBS. The cells were then subjected to flow cytometry analysis (FACScan, Becton Dickinson and Co., Mountain View, CA) as described previously (20, 21). Briefly, a 14-mW argon laser (emission: 488 nm) was used to obtain 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 10,000 cells per sample was analyzed by the CellQuest Pro software (Becton Dickinson Immunocytometry Systems, San Jose, CA) and gating on physical parameters to exclude cell debris. Cell viability was determined by staining with propidium iodine (0.5 µg/ml).

The cellular levels of p27Kip1 protein in IRPTCs were measured by Western blotting, using monoclonal antibodies against p27Kip1 (Transduction Laboratories, Inc., Mississauga, Ontario, Canada). Cellular p27Kip1 level is an indicator of cellular hypertrophy in murine proximal tubular cells (34, 35, 36). Furthermore, p27Kip1 regulates growth arrest in response to TGFß, rapamycin, cAMP, and contact inhibition. Thus, studies of p27Kip1 expression combined with assessment of relative cell size by flow cytometry (FACScan, Becton Dickinson and Co.) and cellular protein quantification will give a better assessment of high-glucose effect on IRPTC hypertrophy. Briefly, the cells were incubated for 4 h. Then the cells were lysed in 300 µl lysis buffer [62.5 mM Tris-HCl, pH 6.8; containing 2% SDS (wt/vol); 10% glycerol; 50 mM dithiothreitol; and 0.1% bromophenol blue (wt/vol)] before transfer into Eppendorf (Ultident Scientific, St. Laurent, Québec, Canada) tubes. The cell lysates were sonicated for 30 sec, heated at 95 C for 5 min, and centrifuged at 12,000 x g for 10 min at 4 C. Small aliquots (20–30 µl) of the supernatants were subjected to 10% PAGE containing SDS and then transferred onto a polyvinyl difluoride membrane (Hybond-P, Amersham-Pharmacia Biotech). The membrane was first blotted with p27Kip1 monoclonal antibodies and then reblotted with ß-actin monoclonal antibodies and chemiluminescent developing reagent (La Roche Biochemicals). The relative densities of p27kip1 and ß-actin bands were determined with a computerized laser densitometer.

To assess the cellular protein content, the cells were rendered quiescent for 24 h and then harvested with 0.05% EDTA. The number of cells per well were counted, lysed in 100 µl 2 M NaOH, and cellular protein content determined by a modified method of Markwell et al. (37). BSA was used as a standard. Cellular protein content is an indicator of cellular hypertrophy, as shown by Mackovic-Basic et al. (38).

Statistical analysis
Three to four separate experiments were performed for each protocol, and each treatment group was assayed in triplicate. The data were subjected to t test or ANOVA followed by Bonferroni analysis to compare the control and treatment groups in the same experiment. A probability level of P <= 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of high glucose, DEX, and insulin on IR-rANG secretion in IRPTCs
A concentration-dependent relationship between DEX and the stimulation of IR-rANG secretion was observed between 10-13 and 10-7 M DEX in both normal-glucose (5 mM) and high-glucose (25 mM) medium (Fig. 1Go). Maximal stimulation of IR-rANG secretion was apparent at 10-7 M DEX. The stimulatory effect of DEX (10-7 M) in combination with high-glucose (25 mM) medium was significantly greater than that of high-glucose (25 mM) medium alone.



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  		Figure 1. Effect of DEX on IR-rANG secretion from rat IRPTCs. Cells were incubated for 24 h in the presence of 5 mM glucose medium or 25 mM glucose medium and various DEX concentrations (10-13 to 10-7 M). Media were harvested after 24 h of incubation and assayed for IR-rANG. The concentration of IR-rANG in 5 mM glucose medium and the absence of DEX represents the control level (i.e. 100%). Each point represents the mean ± SD of three independent experiments (*, P <= 0.05; and **, P <= 0.01).

 
Figure 2AGo shows that Dex (10-7 M) stimulated ANG expression in IRPTCs in 5 mM glucose. Both insulin (10-7 M) and RU486 (10-5 M) blocked the stimulatory effect of DEX on ANG expression in 5 mM glucose, whereas insulin alone had no effect on ANG expression in 5 mM glucose. These studies support the notion that insulin and RU 486 could directly block the DEX effect on ANG expression in IRPTCs. Insulin was effective in inhibiting the stimulatory effect of DEX (10-7 M) and high glucose on IR-rANG secretion from IRPTCs in a dose-dependent manner (Fig. 2BGo) with a maximal effect observed at 10-7 M. This effective dose of insulin, i.e. 10-7 M, was therefore routinely used in all subsequent experiments. The inhibitory effect of insulin on IR-rANG secretion from IRPTCs in high glucose and DEX was blocked by PD 98059 but not by wortmannin. These studies demonstrate that the insulin action in attenuating the stimulatory effect of high glucose and DEX on IR-rANG secretion from IRPTCs is probably mediated via activation of the p44/42 MAPK signal transduction pathway but not via the PI3K signaling pathway.



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  		Figure 2. Inhibitory effect of insulin and RU 486 on IR-rANG secretion from rat IRPTCs. A, Cells were incubated for 24 h in the presence of 5 mM glucose plus RU 486 (10-5 M) or insulin in the presence or absence of PD 98059 or wortmannin. Media were harvested and assayed for IR-rANG. The levels of IR-rANG in medium containing 5 mM glucose are expressed as control (i.e. 100%). The inhibitory effect of insulin is compared with cells that were incubated in 5 mM glucose (without insulin). The results are expressed as the percentage of controls (each point represents the mean ± SD of four independent experiments). B, Cells were incubated for 24 h in the presence of 5 mM glucose, 25 mM glucose, or 25 mM glucose plus various concentrations of insulin with or without PD 98059 or wortmannin. Media were harvested and assayed for IR-rANG. The levels of IR-rANG in medium containing 5 mM glucose are expressed as control (i.e. 100%). The inhibitory effect of insulin is compared with cells that were incubated in 25 mM glucose (without insulin). The results are expressed as the percentage of controls. Each point represents the mean ± SD of three independent experiments (*, P <= 0.05; **, P <= 0.01; and ***, P <= 0.005).

 
Effect of high glucose, DEX, and insulin on cellular ANG mRNA levels in IRPTCs
Figure 3Go shows that DEX (10-7 M) alone stimulated ANG mRNA expression in 5 mM glucose medium. Insulin (10-7 M) or RU 486 (10-5 M) inhibited the stimulatory effect of DEX on ANG mRNA expression in IRPTCs. Insulin alone had no effect on ANG mRNA expression in IRPTCs in 5 mM glucose medium.



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  		Figure 3. Effect of DEX, RU 486, and insulin on ANG mRNA expression in rat IRPTCs. After a 24-h incubation in media with 5 mM glucose, 5 mM glucose, and DEX (10-7 M) in the absence or presence of RU 486 (10-5 M) or insulin (10-7 M), cells were harvested and assayed for ANG mRNA levels by RT-PCR. DNA fragments of the RT-PCR mixture were separated on 1.5% agarose gel and then transferred onto a nylon membrane. Subsequently, the membrane was blotted with a 32P-labeled oligonucleotide corresponding to the nucleotide N + 775 to N + 798 of rat ANG and N + 9 to N + 35 of exon 4 of rat ß-actin, respectively. The relative densities of the PCR band of ANG were compared with the ß-actin control (A). The rANG mRNA level in cells normalized in 5 mM glucose (i.e. ratio of ANG/ß-actin) was considered as the control (100%) (B). Each point represents the mean ± SD of three independent experiments (*, P <= 0.05; **, P <= 0.01; and ***, P <= 0.005).

 
High glucose (25 mM) stimulated the accumulation of ANG mRNA to levels that were 1.5-fold higher than those in control cells cultured in 5 mM glucose medium (P < 0.05; Fig. 4Go). A combination of high glucose (25 mM) and DEX (10-7 M) further enhanced ANG mRNA accumulation, resulting in 2.0-fold higher ANG mRNA levels than in control cells (5 mM). The addition of RU 486 (10-5 M) or insulin (10-7 M) inhibited the stimulatory effect of high glucose and DEX on ANG mRNA accumulation in IRPTCs. The inhibitory effect of insulin was reversed in the presence of PD 98059 (10-5 M).



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  		Figure 4. Effect of high glucose, DEX, RU 486, insulin, and PD 98059 on ANG mRNA expression in rat IRPTCs. After a 24-h incubation in media with 5 mM glucose, 25 mM glucose, 25 mM glucose, and DEX in the absence or presence of RU 486 or insulin with or without PD 98059, cells were harvested and assayed for ANG mRNA levels by RT-PCR as described above in Fig. 3Go. The relative densities of the PCR band of ANG were compared with the ß-actin control (A). The rANG mRNA level in cells normalized in 5 mM glucose (i.e. ANG/ß-actin) was considered as the control (100%) (B). Each point represents the mean ± SD of three independent experiments. (*, P <= 0.05; and **, P <= 0.01).

 
Effect of high glucose, DEX, and insulin on rANG gene promoter activity
Figure 5Go shows that high glucose significantly stimulated rANG gene promoter, compared with that observed in 5 mM glucose (P <= 0.05). A combination of high glucose and DEX further enhanced rANG gene promoter activity. RU 486 or insulin inhibited the stimulatory effect of high glucose and DEX on ANG/CAT fusion gene expression in IRPTCs. The inhibitory effect of insulin was reversed in the presence of PD 98059 but not wortmannin. These studies support the notion that high glucose, DEX, and insulin modulate ANG gene expression at the transcriptional level.



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  		Figure 5. Effect of high glucose, DEX, RU 486 and insulin on the expression of pOCAT (rANG N-1498/+18) in IRPTCs. After a 24-h incubation in media with 5 mM glucose, 25 mM glucose, 25 mM glucose, and DEX in the presence of RU 486 or insulin with or without PD 98059, cells were harvested and assayed for CAT activity. The relative activity in cells normalized in 5 mM glucose was given relative value of 100% (control). A, A typical transient expression (autoradiogram) of the fusion gene pOCAT (rANG N-1498/+18) in IRPTCs. B, Results of transient gene expression as the percentage of conversion of the [14C] chloramphenicol per 20 µg cellular proteins. Each point represents the mean ± SD of three independent experiments (*, P <= 0.05; *, P <= 0.05; and **, P <= 0.01.

 
Effect of high glucose, DEX, and insulin on IRPTC hypertrophy
Figure 6Go shows that a high glucose concentration (25 mM) alone (Fig. 6AGo) induced a 5% (range 5–12%) increase in cell size, compared with the controls (i.e. 5 mM; Fig. 6AGo). DEX at 10-7 M further enhanced the stimulatory effect of high-glucose medium (Fig. 6AGo). RU 486 (10-5 M) prevented the induction of hypertrophy in IRPTCs by DEX (10-7 M) in high glucose (25 mM; Fig. 6BGo). Insulin (10-7 M) attenuated the increase in size of IRPTCs incubated in the presence of DEX and high glucose (Fig. 6CGo). This inhibitory effect of insulin was blocked in the presence of PD 98059 (10-5 M; Fig. 6CGo).



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  		Figure 6. Effect of high glucose, DEX, RU 486, and insulin on cellular size in rat IRPTCs. After a 48-h incubation in media containing 5 mM, 25 mM, or 25 mM plus 10-7 M DEX (A), media containing 25 mM glucose plus DEX with or without RU 486 (B), or media containing 25 mM glucose plus DEX in the presence or absence of insulin with or without PD 98059 (C), the cells were analyzed by flow cytometry (FACScan). Cell viability (assessed by propidium iodide staining) was greater than 90% in each experiment. FSC was expressed as arbitrary units using the CellQuest Pro software. A right shift of the histogram on the x-axis indicates an increase in cellular size.

 
Figure 7Go shows that cellular p27Kip1 protein content was significantly increased in IRPTCs incubated in 25 mM glucose and 25 mM glucose plus DEX (10-7 M), compared with control cells (incubated in 5 mM glucose). RU 486 (10-5 M) or insulin (10-7 M) inhibited the stimulatory effect of a combination of high glucose and DEX on p27Kip1 expression. PD 98059 (10-5 M) reversed the inhibitory effect of insulin on p27Kip1 protein content in IRPTCs.



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  		Figure 7. Effect of high glucose, DEX, RU 486, and insulin on cellular p27Kip1 levels in IRPTCs. After a 24-h incubation in media containing 5 mM or 25 mM glucose, 25 mM glucose, and DEX in the absence or presence of RU 486 or insulin with or without PD 98059, the cells were harvested and assayed for p27Kip1 and ß-actin levels by Western blot analysis (A). The ratio of relative densities of p27kip1 to ß-actin in IRPTCs incubated in 5 mM glucose medium represent the control (100%) (B). Each point represents the mean ± SD of three independent experiments (*, P <= 0.05 and **, P <= 001).

 
Protein content was also significantly increased in IRPTCs incubated in high glucose (25 mM) medium, compared with control (5 mM) cells as shown in Fig. 8Go. DEX at 10-7 M further augmented protein content in IRPTCs incubated in 25 glucose medium. The addition of RU 486 (10-5 M) or insulin (10-7 M) blocked the stimulatory effect of a combination of DEX and high glucose on total cellular protein. PD 98059 again blocked the inhibitory effect of insulin.



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  		Figure 8. Effect of high glucose, DEX, RU 486, and insulin on cellular protein content in rat IRPTCs. Total protein content in IRPTCs was determined by a modified Lowry method and expressed per 106 cells. Cells were incubated for 48 h in the presence of 5 mM glucose, 25 mM glucose, 25 mM glucose, and DEX in the absence or presence of RU 486 or insulin with or without PD 98059 or wortmannin and then harvested and analyzed for total cellular content. Cellular protein content in IRPTCs incubated in 5 mM glucose in the absence of drugs represents the control level. Each point represents the mean ± SD of three independent experiments (*, P <= 0.05 and **, P <= 0.01).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present studies demonstrate that insulin is effective in preventing the stimulatory effect of high glucose and glucocorticoid on ANG gene expression and induction of IRPTC hypertrophy. The insulin action is mediated, at least in part, via the p44/42 MAPK signal transduction pathway and not via the PI3K signaling pathway.

DEX stimulated IR-rANG secretion from IRPTCs in a dose-dependent manner (10-9 to 10-7 M) in normal glucose (i.e. 5 mM) and high glucose (i.e. 25 mM) medium (Fig. 1Go). These DEX concentrations are analogous to the physiological concentrations of plasma cortisol in humans (i.e. 3.75 x 10-7 M) in morning plasma samples (39). Our studies confirm the studies of Loghman-Adham et al. (8), who reported that DEX (10-6 M) stimulated the expression of mouse ANG in immortalized mouse proximal tubular cells. Our present results also confirm our previous studies in which DEX (10-10 to 10-6 M) stimulated IR-rANG secretion and rANG gene promoter activity in a dose-dependent manner in rat IRPTCs and opossum kidney proximal tubular cells, respectively (17, 40, 41, 42). The secretion of IR-rANG from IRPTCs was increased by 1.5-fold in high level of glucose (25 mM) medium, compared with a low-level glucose (5 mM) medium (Fig. 1Go). This level of stimulation by high glucose (1.5-fold) is similar to our previous observations that high glucose induced a 1.5-fold increase in IR-rANG secretion from IRPTCs (18, 19, 20, 21) and a 1.5-fold increase in rANG gene promoter activity in opossum kidney cells (38). The addition of DEX (10-7 M) further enhanced the stimulatory effect of high glucose (25 mM) resulting in 2.0-fold higher levels than in the control (5 mM glucose). These studies demonstrate that high glucose and DEX act additively to stimulate IR-rANG secretion from IRPTCs.

Chang and Perlman (43) and Aubert et al. (44) have shown that insulin attenuates ANG secretion and ANG mRNA expression in rat hepatoma cells and cultured adipose tissue in vitro, respectively. Consistent with these results, we have observed that insulin blocked the stimulatory action of DEX on IR-rANG secretion from IRPTCs in normal- or high- glucose medium (Fig. 2Go, A and B). The maximum and half-maximum effective doses of insulin were 10-7 M and 10-9 M, respectively. We previously demonstrated that IGF-I and IGF-II did not display any significant inhibition of IR-rANG secretion in IRPTCs (25). Furthermore, we found that antibodies against IGF-1 receptor did not affect the insulin action on ANG expression in IRPTCs (our unpublished results). These studies demonstrate that the inhibitory effect of insulin on IR-rANG secretion from IRPTCs is specific for insulin and the insulin receptor. Most interestingly, PD 98059 at 10-5 M completely blocked the inhibitory action of insulin on IR-rANG secretion from IRPTCs, whereas wortmannin had no effect (Fig. 2BGo). These results are in agreement with our previous report that insulin inhibited the stimulatory action of high glucose on ANG secretion from IRPTCs via the MAPK signal transduction pathway (25). Our data strongly support the notion that the MAPK signaling pathway but not PI3K signaling pathway is involved in the inhibition of ANG gene expression in IRPTCs by insulin.

The stimulatory effect of high glucose and DEX appears to occur at the mRNA level. DEX stimulated ANG mRNA expression in IRPTCs in 5 mM glucose medium (Fig. 3Go). The addition of insulin or RU 486 blocked the stimulatory effect of DEX on ANG mRNA expression in 5 mM glucose medium. In contrast, insulin had no effect on ANG mRNA expression in IRPTCs in 5 mM glucose. These studies support the notion that insulin and RU 486 directly block the DEX effect on ANG mRNA expression in IRPTCs. Furthermore, exposure of IRPTCs to high glucose (25 mM) increased the expression of ANG mRNA by 1.5-fold (P < 0.05), compared with levels in control cells (i.e. 5 mM glucose) (Fig. 4Go), that was further enhanced (2.5-fold increase) by DEX. RU 486 is an antagonist of glucocorticoids that competes with glucocorticoid receptors (45). RU 486 (10-5 M) inhibited the effect of DEX (10-7 M) and high glucose (25 mM) on ANG mRNA expression in IRPTCs, suggesting that the effect of DEX is mediated via the activation of glucocorticoid receptor in IRPTCs. Similarly, insulin at 10-7 M blocked the stimulatory effect of high glucose and DEX on ANG mRNA expression in IRPTCs, and PD 98059 reversed the inhibitory effect of insulin. These data further support that the inhibitory effect of insulin on ANG gene expression is MAPK dependent.

To investigate whether high glucose, DEX, and insulin regulate ANG gene expression at the transcriptional level, we transiently transfected the ANG-CAT fusion gene containing the full-length rANG promoter fused with the CAT reporter gene into IRPTCs. It is apparent that high glucose significantly stimulated ANG/CAT fusion gene expression, compared with those detected in 5 mM glucose (Fig. 5Go). The stimulatory effect of high glucose could be further enhanced in the presence of DEX. This stimulatory effect of high glucose and DEX was inhibited in the presence of RU 486 or insulin. Again the inhibitory effect of insulin was reversed in the presence of PD 98059. Thus, these studies support the notion that high glucose, DEX, and insulin regulate ANG gene expression at the transcriptional level. Nevertheless, studies such as nuclear run-on are definitely warranted to further confirm the transcriptional effect of high glucose, DEX, and insulin on ANG gene expression in IRPTCs.

Our present data show that incubation of IRPTCs in 25 mM glucose induced an increase in cell size (Fig. 6AGo), enhanced p27Kip1 expression (Fig. 7Go), and augmented cellular protein content (Fig. 8Go). These results are in agreement with previous studies, including ours, showing that high-glucose media (i.e. 25 mM) induced cellular hypertrophy of murine proximal tubular cells (13, 14, 20, 21). It is apparent that culture of IRPTCs in high glucose with DEX at 10-7 M led further increases in cell size (Fig. 6AGo), p27Kip1 expression (Fig. 7Go) and protein content (Fig. 8Go). The additive stimulatory effect of high glucose and DEX was blocked by RU 486 (10-5 M) and insulin (10-7 M). These data demonstrate that high glucose and DEX act additively to induce IRPTC hypertrophy, and insulin could block their additive effect.

At present, we do not understand the exact molecular mechanism(s) of additive effect of DEX and high glucose on ANG gene expression in IRPTCs. One possibility might be that high glucose stimulates de novo synthesis of diacylglycerol from metabolized glucose via the polyol pathway and then increases PKC activity (18). Once PKC is activated, it may phosphorylate cAMP-responsive element-binding protein (CREB) or CREB-like nuclear protein(s) because CREB contains the site of phosphorylation by PKC (46), and studies by Kreisberg et al. (47) have shown that phorbol ester and high-glucose levels stimulate CREB phosphorylation. The phosphorylated CREB then binds to the cAMP-responsive element of the rat ANG gene (TGACGTAC, nucleotides N-795 to N-788) (48) and interacts with activated glucocorticoid receptor(s) that are bound to glucocorticoid-responsive element(s) (GREs) (i.e. GRE-I, nucleotides N-591 to N-563, and GRE-II, nucleotides N-485 to N-465) in the 5'-flanking region of rANG gene (40, 41). Subsequently, the bound CREB and the activated GR complex act synergistically to stimulate the expression of the rANG gene. This possibility is supported by the studies of Imai et al. (49), who showed that the activated glucocorticoid receptor interacted with CREB in vitro.

Similarly, we do not understand the precise molecular mechanism(s) of the inhibitory effect of insulin on ANG gene expression in IRPTCs stimulated by high glucose and DEX. One possibility might be that insulin activates the Ras/MAPK signal transduction pathway and stimulates the recruitment of the S6 kinase pp90rsk to the signal-dependent coactivator, CREB-binding protein (CBP) (50) as demonstrated by Nakjima et al. (51). Formation of the pp90rsk-CBP complex then inhibits the binding of phosphorylated CREB and activated glucocorticoid receptors to CBP as well as to their respective DNA cis-element in the 5'-flanking region of the ANG gene. Subsequently, ANG gene expression is attenuated. This possibility is supported by our data showing that PD 98059 reverses the inhibitory effect of insulin on ANG gene expression (Figs. 3Go and 4Go). PD 98059 is an inhibitor of MEK (22), and MEK is required for the phosphorylation of p44/42 MAPK and, subsequently, the phosphorylation of the S6 kinase pp90rsk. The second possibility might be that insulin down-regulates the expression of insulin-responsive element (IRE)-binding protein(s) that bind(s) to the IRE in the 5'-flanking region of rat ANG gene. The down-regulation of IRE-binding protein(s) might then attenuate ANG gene transcription in IRPTCs. Indeed, our most recent studies have shown that insulin inhibits the expression of two nuclear proteins with an approximate molecular mass of 48 and 70 kDa that bind to the IRE of rANG gene (26). Clearly, more studies are warranted to elucidate the molecular mechanism(s) of insulin action on ANG gene expression stimulated by high glucose and DEX in IRPTCs.

Finally, the clinical relevance of our present findings to type 2 diabetes remains to be elucidated. Because hyperinsulinemia is frequently found in type 2 diabetes, it would be of interest to know whether insulin could suppress renal ANG gene expression or hyperinsulinemia might induce insulin resistance on ANG gene expression in kidney proximal tubular cells in type 2 diabetes. Clearly, more experiments alone these lines are warranted.

In summary, our studies show that exposure of IRPTCs to high glucose (25 mM) or DEX (10-7 M) directly stimulates rANG gene expression and induces IRPTC hypertrophy, and the effects of high glucose and DEX are additive. This stimulatory effect of high glucose and DEX is blocked by insulin. Our studies suggest that expression of the renal ANG gene may be stimulated in hyperglycemia and hyperglucocorticoidism in vivo. The increased local formation of renal Ang II might then induce the hypertrophy of proximal tubular cells in high glucose. Such results lead us to speculate that the local renal RAS may contribute to the development of nephropathy in diabetes by contributing to renal hypertrophy and subsequently to inflammation and tissue scarring. Finally, insulin appears to be effective in preventing hypertrophy in proximal tubular cells and attenuating nephropathy in diabetes.


   Acknowledgments
 
The authors thank Mr. Ovid M. Da Silva, Éditeur-Rédacteur, Bureau d’aide à la recherche, Research Centre, Centre Hospitalier de l’Université de Montréal, for editing this manuscript.


   Footnotes
 
This work was supported by grants from the Canadian Diabetes Association (1061); Canadian Institutes of Health Research Grants 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 NIH Grants HL-48455 (to J.R.I.) and DK-50836 (to S.-S.T.). S.-L.Z. is the recipient of a Canadian Institutes for Health Research Doctoral Research Award.

Abbreviations: ACE, Angiotensin-converting enzyme; ANG, angiotensinogen; Ang II, angiotensin II; CAT, chloramphenicol acetyltransferase; CBP, CREB-binding protein; CREB, cAMP-responsive element-binding protein; DEX, dexamethasone; dFBS, depleted fetal bovine serum; FBS, fetal bovine serum; FSC, forward-angle light scatter; IR, immunoreactive; IRE, insulin-responsive element; IRPTC, immortalized renal proximal tubular cell; MEK, MAPK kinase; PI3K, phosphatidylinositol-3-kinase; PKC, protein kinase C; pOCAT, chloramphenicol acetyltransferase without the promoter; pRSV/CAT, chloramphenicol acetyltransferase with Rous sarcoma virus enhancer/promoter sequence; rANG, rat ANG; RAS, renin-angiotensin system; RPTC, renal proximal tubular cell.

Received April 16, 2002.

Accepted for publication August 28, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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