Hormones, Growth Factors, Cell Biology and Structure

Essential Role(s) of the Intrarenal Renin-Angiotensin System in Transforming Growth Factor–β1 Gene Expression and Induction of Hypertrophy of Rat Kidney Proximal Tubular Cells in High Glucose

Shao-Ling Zhang, Catherine To, Xing Chen, Janos G. Filep, Shiow-Shih Tang, Julie R. Ingelfinger and John S. D. Chan
JASN February 2002, 13 (2) 302-312;

Abstract

ABSTRACT. These studies investigated the question of whether the intrarenal renin-angiotensin system (RAS) is essential for transforming growth factor–β1 (TGF-β1) gene expression and induction of hypertrophy of renal proximal tubular cells in high glucose in vitro. Antisense and sense angiotensinogen (ANG) cDNAs were stably transfected into rat immortalized renal proximal tubular cells (IRPTC). ANG secretion from rat IRPTC was quantified by a specific RIA for rat ANG. Cellular ANG, TGF-β1, and collagen α1 (type IV) mRNA levels were determined by Northern blot analysis or by reverse transcriptase–PCR assay. Hypertrophy of IRPTC was analyzed by Western blotting of cellular p27Kip1 protein, flow cytometry, and cellular protein assay. The results showed that stable transfer of antisense ANG cDNA into IRPTC suppressed the basal TGF-β1 and collagen α1 (type IV) mRNA expression and blocked the stimulatory effect of high glucose (i.e., 25 mM) on TGF-β1 and collagen α1 (type IV) mRNA expression and induction of IRPTC hypertrophy. In contrast, stable transfer of sense ANG cDNA into IRPTC had no significant effect on these parameters. These data demonstrate that local intrarenal RAS activation is essential for TGF-β1 gene expression and induction of hypertrophy of renal proximal tubular cells in high glucose.

High levels of glucose and/or angiotensin II (AngII) may directly or indirectly be responsible for renal proximal tubular hypertrophy and tubulofibrosis in diabetes. For example, in vitro studies have shown that culture of murine proximal tubular cells in a high-glucose medium (i.e., ≥25 mM) or in the presence of high AngII concentrations (i.e., ≥10−8 M) stimulates transforming growth factor–β1 (TGF-β1) expression and induces cellular hypertrophy with extracellular matrix protein expression (1–9). Clinical investigations have revealed that the administration of angiotensin-converting enzyme (ACE) inhibitors or AngII receptor antagonists reduces proteinuria and slows the progression of nephropathy in diabetes (10–18). All these studies have indicated an important role for AngII in the development of nephropathy in diabetes.

In the past 10 yr, it has been demonstrated that mRNA components of the renin-angiotensin system (RAS), including angiotensinogen (ANG), renin, ACE, and AngII receptors, are expressed in murine (mouse and rat) immortalized renal proximal tubular cell (IRPTC) lines (19–24). We have reported that ANG protein is secreted from rat IRPTC (25), providing evidence that intrarenal AngII is derived from the ANG synthesized within RPT in vivo. Enhanced local formation of AngII may therefore contribute to the pathogenesis of diabetic nephropathy.

We have recently shown that ANG gene expression in IRPTC is stimulated by high glucose levels (i.e., 25 mM) (26,27). Inhibitors of aldose reductase (i.e., tolrestat), protein kinase C (i.e., staurosporine, H-7, or GF 109203 X), and p38 mitogen-activated protein kinase (i.e., SB 208392) blocked the stimulatory action of high glucose (26,27). Such studies have demonstrated that the stimulatory effect of high glucose levels on ANG gene expression in IRPTC is mediated, at least in part, via the activation of both polyol/protein kinase C and p38 mitogen-activated protein kinase signal transduction pathways. Furthermore, we have reported that ACE inhibitors (i.e., perindopril and captopril) and an AngII-receptor antagonist (i.e., losartan) blocked ANG gene expression and induction of hypertrophy in IRPTC stimulated by high glucose levels (28). These data further support the notion that intrarenal RAS activation may play an important role in the development of renal hypertrophy in early diabetes.

In the present studies, we examined the influence of antisense and sense ANG cDNA transfer on renal TGF-β1 and collagen α1 (type IV) gene expression as well as on the induction of hypertrophy in IRPTC stimulated by high glucose levels. Herein we report that stable transfer of antisense ANG cDNA in IRPTC blocked the basal and stimulatory effect of high glucose levels on TGF-β1 and collagen α1 (type IV) gene expression and induction of IRPTC hypertrophy. In contrast, the stable transfer of sense ANG cDNA in IRPTC did not affect the stimulatory action of high glucose levels on these parameters.

Materials and Methods

D(+)-glucose, l-glucose, and human AngII were purchased from Sigma-Aldrich Canada Ltd. (Oakville, Ontario, Canada). Normal-glucose (5 mM) Dulbecco’s modified Eagle medium (DMEM, catalog 12320) was obtained from Life Technologies, Inc. (Burlington, Ontario, Canada). Gamma-[32P-ATP](3000 Ci/mol) and Na125I were purchased from Amersham-Pharmacia Biotech (Baie d’Urfé, Quebec, Canada). Oligonucleotides were synthesized by Life Technologies, Inc. Restriction and modifying enzymes were purchased from either Life Technologies, Inc., Boehringer-Mannheim (Dorval, Quebec, Canada), or Amersham-Pharmacia Biotech.

The plasmid pGEM-3 (rANG cDNA) containing full-length rat ANG cDNA was a gift from Dr. Joel F. Habener (Massachusetts General Hospital, Boston, MA). Rat ANG cDNA (nucleotides N+1 to N+1434) was subcloned into the mammalian expression vector pRC/RSV in sense and antisense orientations by standard methods. The vector pRC/RSV was purchased from Invitrogen Inc. (La Jolla, CA). Rat TGF-β1 cDNA was cloned from IRPTC in our laboratory (by J.S.D.C.) by conventional reverse transcriptase–PCR (RT-PCR). Sense and antisense primers corresponding to nucleotides N+401 to N+424 (5′ GCC GCC TCC CCC ATG CCG CCC TCG 3′) and N+1585 to N+1565 (5′ TCA GCT GCA CTT GCA GGA GCG 3′) of rat TGF-β1 cDNA (29) were used in PCR.

RIA for Rat ANG

The RIA for rANG developed in our laboratory (by J.S.D.C.) has been described elsewhere in detail (25). Purified rat plasma ANG (>90% pure, as analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis), and iodinated rANG were used as hormone standard and tracer, respectively. This RIA is specific for intact rANG (i.e., 62 to 65 kD rANG) and has no cross-reactivity with pituitary hormone preparations or other rat plasma proteins (25). The lower limit of detection of the RIA is ∼2 ng of rANG. The intra-assay and interassay coefficients of variation were 9% (n = 10) and 14% (n = 10), respectively.

Cell Culture

IRPTC (cell line 93-p-2–1) from passage 11 were used in stable gene-transfection studies. The characteristics of this cell line have been described elsewhere (24). These cells express the mRNA and protein of ANG, renin, ACE, and AngII receptors (24).

IRPTC were grown in 100 × 20 mm plastic Petri dishes (Life Technologies, Inc.) in normal glucose (i.e., 5 mM) DMEM (pH 7.45) supplemented with 10% fetal bovine serum (FBS), 100 U/ml of penicillin, and 100 μg/ml of streptomycin. The cells were grown in a humidified atmosphere in 95% air–5% CO2 at 37°C. For subculturing, the cells were trypsinized (0.05% trypsin and ethylenediaminetetraacetic acid) and plated at 2.5 × 104 cells/cm2 in 100 × 20 mm Petri dishes.

Stable Gene Transfection in IRPTC

IRPTC were plated at a density of 1 to 2 × 105 cells/well in six-well plates and incubated overnight in normal glucose (i.e., 5 mM) DMEM that contained 10% FBS. The pasmids pRSV/rANG (+) and pRSV/rANG (−) that contained the ANG cDNA in sense and antisense orientations, respectively, were stably transfected into IRPTC by use of Lipofectamine reagents (Life Technologies, Inc.). The plasmid pRC/RSV was also stably transfected into IRPTC as a negative control. The stable transformants were selected in the presence of geneticin (G418) (500 μg/ml) (Life Technologies. Inc.) according to the method we described elsewhere (30).

Basal Immunoreactive rANG Secretion in Stable Transformants

Stable transformants were synchronized by incubating the cells in serum-free medium with normal glucose DMEM for 24 h. The stable transformants were then incubated in normal (5 mM) glucose culture medium that contained 1% depleted FBS (dFBS), and the cells were incubated for an additional 24 h. At the end of the incubation period, the media were collected and stored at −20°C until assayed for basal immunoreactive rANG (IR-rANG).

Basal Rat ANG, TGF-β1, and Collagen α1 (Type IV) mRNA Expression in Stable Transformants

To assess the basal rANG, TGF-β1, and collagen α1 (type IV) mRNA expression, stable transformants were incubated in a normal (5 mM) glucose culture medium that contained 1% dFBS for 24 h. The cells were then harvested and extracted for total RNA with Trizol reagent (Life Technologies, Inc.). Total RNAs were used for the quantification of ANG, TGF-β1, and β-actin mRNAs by Northern blot analysis (31). Cellular collagen α1 (type IV) mRNA was quantified by RT-PCR as described below.

To investigate the effect of antisense ANG cDNA on endogenous ANG mRNA expression in stable transformants, RT-PCR specific for the 3′ noncoding sequence of ANG mRNA was performed. Briefly, cells were harvested after incubation in 5 mM glucose medium that contained 1% dFBS for 24 h and then extracted for total RNA with Trizol reagent and used in RT-PCR for ANG and β-actin mRNA. The forward primer 5′ CCT GTG TAG CCA TGG AGA CAA GGC CAG CGT 3′ and the reverse primer 5′ GTC CTC CTA TAC AGA GTG CCA GCT CCT GGC 3′, corresponding to the 3′ noncoding nucleotide sequences N+1440 to N+1467 and N+1640 to N+1659 of rat ANG mRNA (32), respectively, were used in the RT-PCR assay. The radioactively labeled oligonucleotide that contained the nucleotide sequence N+1494 to N+1523 was used as an internal probe. The methods for RT-PCR for β-actin mRNA were described below.

Effect of High Glucose on TGF-β1 and Collagen α1 (Type IV) mRNA Expression in Stable Transformants

To study the effect of high glucose on TGF-β1 and collagen α1 (type IV) mRNA expression in stable transformants, cells were incubated for 24 h in media with 5 or 25 mM glucose that contained 1% dFBS. At the end of each incubation period, cells were harvested and extracted for total RNA with Trizol reagent and used in RT-PCR for TGF-β1, collagen α1 (type IV), and β-actin mRNA. The forward primer 5′ CTT CAG CTC CAC AGA GAA GAA CTG C 3′ and the reverse primer 5′ TCA GCT GCA CTT GCA GGA GCG 3′, corresponding to the nucleotide sequences N+1267 to N+1297 and N+1585 to N+1565 of rat TGF-β1 cDNA (29), respectively, were used in the RT-PCR assay. Similarly, the forward primer 5′ TAG GTG TCA GCA ATT AGG CAG 3′ and the reverse primer 5′ TCA CTT CAA GCA TAG TGG TCC G 3′, corresponding to the nucleotide sequences N+5808 to N+5828 and N+6291 to N+6270 of mouse collagen α1 (type IV) gene (33), were used in the RT-PCR assay. Finally, primers specific for rat β-actin (34) (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+139 of exon 3 and nucleotide N+115 to N+139 of exon 5 of rat β-actin) were applied in another PCR as internal controls. Briefly, 1 μg of total RNA was used to synthesize first-strand cDNA by the Super-Script preamplification system, following the protocol of the supplier (Life Technologies, Inc.). Then, 2 μl of the cDNA reaction mixture was used to amplify the rat TGF-β1, collagen α1 (type IV), or β-actin cDNA fragment with the PCR-core kit, according to the protocol of the supplier (Life Technologies, Inc.). The RT-PCR mixture was separated on 1.5% agarose gel and transferred onto a nitrocellulose membrane. Subsequently, 32P-labeled oligonucleotides corresponding to the nucleotide N+1451 to N+1474 (5′ AAC CCG GGT GCT TCC GCA TCA CCG 3′) of rat TGF-β1 cDNA (29), the nucleotide N+6172 to N+6193 (5′ GCA TTT CAC ACC TGA GCA CAC A 3′) of the mouse collagen α1 (type IV) gene (33), and the nucleotide N+9 to N+35 (5′ TCC TGT GGC ATC CAT GAA ACT ACA TTC 3′) of rat β-actin (34) were applied to hybridize 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.

To maintain constant isotonicity or osmolality, the 5-mM glucose media was supplemented with l-glucose (20 mM) (final concentration). The dFBS was prepared by incubation with 1% activated charcoal and 1% AG 1 × 8 ion-exchange resin (Bio-Rad Laboratories, Richmond, CA) for 16 to 24 h at room temperature (35). This procedure removes endogenous steroid and thyroid hormones from the FBS (35).

Effect of High Glucose on Cellular Hypertrophy of Stable Transformants

The effect of high glucose (25 mM) on cellular hypertrophy of the stable transformants was evaluated by cellular p27Kip1 expression, flow cytometry, and cellular protein content. The cellular levels of p27Kip1 protein in stable transformants were determined by Western blotting that used monoclonal antibodies against p27Kip1 (Transduction Laboratories, Inc., Mississauga, Ontario, Canada). The cellular p27Kip1 level is an indicator of cellular hypertrophy in murine proximal tubular cells (36–38). Briefly, the cells were incubated in medium that contained 5 or 25 mM glucose for 4 h. Then cells were lysed in 300 μl of lysis buffer (62.5 mM Tris-HCl [pH 6.8] that contained 2% sodium dodecyl sulfate [wt/vol], 10% glycerol, 50 mM dithiothreitol, and 0.1% bromophenol blue [wt/vol]) and transferred into Eppendorf tubes. The cell lysates were sonicated for 20 s, heated at 95°C for 5 min, and centrifuged at 12,000 × g for 10 min at 4°C. Small aliquots (20 to 50 μl) of the supernatants were subjected to 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then transferred onto a PVDF membrane (Hybond-P, Amersham Pharmacia Biotech). The membrane was then blotted by p27Kip1 monoclonal antibodies and ECL-chemiluminescent developing reagent (Amersham-Pharmacia Biotech). To normalize the amount of proteins applied, the same membrane was reblotted by monoclonal antibodies against β-actin (Sigma-Aldrich Corp. Canada Ltd., Ontario, Canada).

To determine cell size, cells were plated at 5 × 104 cells/well in 6-well plates in 5 mM glucose DMEM that contained 10% FBS. The cells then were synchronized in 5 mM glucose medium for 24 h and subsequently incubated in 5 or 25 mM glucose medium. The media were changed every 48 h. The cells were harvested at the end of the incubation period, titrated to a single cell suspension at a density of 106 cells/ml in phosphate-buffered saline, and subjected to flow cytometry analysis (FACScan) (Becton-Dickinson, Mountain View, CA) with Lysis II software, as described elsewhere (28). Changes in forward light scatter served to assess relative cell size for 104 cells per sample. Cell viability was determined by staining with propidium iodide (0.5 μg/ml).

To assess cellular protein content, the cells were rendered quiescent for 24 h in serum-free media and then incubated in 5 or 25 mM glucose medium. At the end of the incubation period, the cells were harvested with 0.05% ethylenediaminetetraacetic acid. The number of cells per well was counted; after lysis in 100 μl of 2 M NaOH, cellular protein content was determined according to the method of Markwell et al. (39), with the use of bovine serum albumin as a standard. Cellular protein content is an indicator of cellular hypertrophy (40).

Statistical Analyses

Three to four separate experiments per protocol were performed, 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 as statistically significant.

Results

IR-rANG Secretion from Stable Transformants

IR-rANG secretion (Figure 1) from IRPTC stably transfected with pRSV/rANG (−) was significantly lower (P < 0.05) than those released from IRPTC transfected with pRC/RSV or with pRSV/rANG (+).

Figure1

Figure 1. Secretion of basal immunoreactive rat angiotensinogen (IR-rANG) from immortalized renal proximal tubular cells (IRPTC). IRPTC stably transfected with pRSV/rANG (+) or pRSV/rANG (−) or pRC/RSV were incubated for 24 h in medium that contained 5 mM glucose and 1% depleted fetal bovine serum (dFBS). Media were collected and assayed for IR-rANG. The IR-rANG concentration from pRC/RSV-transfected IRPTC in medium that contained low glucose (5 mM) (i.e., 3.1 ± 1.8 ng/ml per 106 cells) was considered the control level (100%). Each bar represents the mean ± SD of four clones (*P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.005). Similar results were obtained in two independent experiments.

Basal Expression of ANG, TGF-β1, and Collagen α1 (Type IV) mRNAs in Stable Transformants

The mRNA levels of cellular ANG and TGF-β1 were quantified by Northern blot analysis. It is apparent that the basal ANG mRNA levels in IRPTC stably transfected with pRSV/rANG (+) were significantly higher (P < 0.05) than those in IRPTC stably transfected with pRSV/rANG (−) or IRPTC stably transfected with pRC/RSV plasmid (Figure 2). ANG mRNA levels in IRPTC stably transfected with pRSV/rANG (−) did not differ from those IRPTC stably transfected with pRC/RSV when analyzed by Northern blot. On the other hand, ANG mRNA levels in IRPTC stably transfected with pRSV/rANG (−) were significantly lower from those IRPTC stably transfected with pRC/RSV or pRSV/rANG (−) when analyzed by RT-PCR specific for the 3′-noncoding region of ANG mRNA (Figure 3). These results demonstrate that the transfected antisense ANG cDNA effectively suppressed the expression of endogenous ANG mRNA in IRPTC.

Figure2

Figure 2. Northern blot analysis of rANG mRNA and β-actin mRNA expression in IRPTC stable transformants. Cells incubated in 5 mM glucose medium and 1% dFBS for 24 h were harvested and assayed by conventional Northern blotting. Total RNAs from IRPTC transformants (30 μg) were separated by agarose gel electrophoresis and transferred to a Hybond-N nylon membrane. The membrane was hybridized with 32P-labeled rat ANG cDNA probe for the rANG gene. The same blot was also hybridized with the 32P-labeled hamster β-actin cDNA probe, to standardize the amount of total RNA used. The relative densities of the ANG band were compared with the β-actin control (A). The level of ANG mRNA in pRC/RSV–transfected IRPTC was considered to be the control (100%) (B). Each point represents the mean ± SD of four different clones (*P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.005). Similar results were obtained in two other experiments.

Figure3

Figure 3. Reverse transcriptase–PCR (RT-PCR) analysis of endogenous rat ANG mRNA expression in IRPTC stable transformants. After a 24-h incubation period in media with 5 mM glucose plus 1% dFBS, cells were harvested and assayed for rat ANG mRNA levels by RT-PCR. DNA fragments of the RT-PCR reaction mixture were separated on 1.5% agarose gel and then transferred onto a Hybond-XL nylon membrane. Subsequently, the membrane was blotted with a 32P-labeled oligonucleotide corresponding to the nucleotide N+1494 to N+1523 (5′ CAG TGC CTT CAC CCC TGG CTT CCC GTC ACT 3′) of rat ANG mRNA and the nucleotide N+9 to N+35 of exon 4 (5′ TCC TGT GGC ATC CAT GAA ACT ACA TTC 3′) of rat β-actin, respectively. The relative densities of the PCR band of ANG mRNA were compared with the β-actin control (A). The level of ANG mRNA in pRC/RSV–transfected IRPTC was considered to be the control (100%) (B). Each bar represents the mean ± SD of four different clones (*P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.005). Similar results were obtained in another experiment.

Basal TGF-β1 mRNA levels were also significantly lower (P < 0.05) in IRPTC stably transfected with pRSV/rANG (−) compared with IRPTC stably transfected with pRSV/rANG (+) or stably transfected with pRC/RSV (Figure 4). There was no significant difference in TGF-β1 mRNA levels between IRPTC stably transfected with pRSV/rANG (+) or stably transfected with pRC/RSV incubated in 5 mM glucose medium.

Figure4

Figure 4. Northern blot analysis of transforming growth factor–beta 1 (TGF-β1) mRNA and β-actin mRNA in IRPTC stable transformants. Cells were incubated in 5 mM glucose medium and 1% dFBS for 24 h. Total RNAs (30 μg) from IRPTC stable transformants were separated by agarose gel electrophoresis and transferred to a Hybond-N nylon membrane. The membrane was hybridized with 32P-labeled TGF-β1 cDNA probes for the TGF-β1 gene. The same membrane was also hybridized with 32P-labeled hamster β-actin cDNA probes, to standardize the amount of total RNA used. The relative densities of the TGF-β1 band were compared with β-actin controls (A). The level of TGF-β1 mRNA in pRC/RSV–transfected IRPTC was considered to be the control (100%) (B). Each bar represents the mean ± SD of four different clones (*P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.005). Similar results were obtained in two other experiments.

Collagen α1 (type IV) mRNA levels in IRPTC were quantified by specific RT-PCR. Basal collagen α1 (type IV) mRNA levels in IRPTC stably transfected with pRSV/rANG (−) were also significantly lower than those in IRPTC stably transfected with pRSV/rANG (+) (P < 0.05) or stably transfected with pRC/RSV (P < 0.05) (Figure 5). There was no significant difference in collagen α1 (type IV) mRNA levels, however, between IRPTC stably transfected with pRSV/rANG (+) or stably transfected with pRC/RSV.

Figure5

Figure 5. RT-PCR analysis of rat collagen α1 (type IV) mRNA expression in IRPTC stable transformants. After a 24-h incubation period in media with 5 mM glucose plus 1% dFBS, cells were harvested and assayed for rat collagen α1 (type IV) mRNA levels by RT-PCR. DNA fragments of the RT-PCR reaction mixture were separated on 1.5% agarose gel and then transferred onto a Hybond-XL nylon membrane. Subsequently, the membrane was blotted with a 32P-labeled oligonucleotide corresponding to the nucleotide N+6172 to N+6193 (5′ GCA TTT CAC ACC TGA GCA CAC A 3′) of the mouse collagen α1 (type IV) gene and the nucleotide N+9 to N+35 of exon 4 (5′ TCC TGT GGC ATC CAT GAA ACT ACA TTC 3′) of rat β-actin, respectively. The relative densities of the PCR band of collagen α1 (type IV) were compared with the β-actin control (A). The level of collagen α1 (type IV) mRNA in pRC/RSV–transfected IRPTC was considered to be the control (100%) (B). Each bar represents the mean ± SD of four clones (*P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.005). Similar results were obtained in two other experiments.

Effect of High Glucose on Cellular TGF-β1 and Collagen α1 (Type IV) mRNA Expression in IRPTC Stable Transformants

Figure 6 shows that a high glucose concentration (25 mM) stimulated TGF-β1 mRNA expression in nontransfected IRPTC (Figure 6A) as well as in IRPTC stably transfected with pRC/RSV (Figure 6B) or pRSV/rANG (+) (Figure 6C) to levels that were 150% to 200% higher than those detected in control cells incubated in 5 mM glucose medium (P ≤ 0.05, 0.05, and 0.01, respectively). In contrast, high glucose (25 mM) did not stimulate TGF-β1 mRNA expression in IRPTC stably transfected with pRSV/rANG (−) (Figure 6D).

Figure6

Figure 6. Effect of high glucose on rat TGF-β1 mRNA expression in IRPTC stable transformants. After a 24-h incubation period in media with 5 or 25 mM glucose media plus 1% dFBS, cells were harvested and assayed for TGF-β1 mRNA levels by RT-PCR. DNA fragments of the RT-PCR mixture were separated on 1.5% agarose gel and then transferred onto a Hybond-XL nylon membrane. Subsequently, the membrane was blotted with a 32P-labeled oligonucleotide corresponding to the nucleotide N+1451 to N+1474 (5′ AAC CCG GGT GCT TCC GCA TCA CCG 3′) of the rat TGF-β1 cDNA and the nucleotide N+9 to N+35 of exon 4 (5′ TCC TGT GGC ATC CAT GAA ACT ACA TTC 3′) of rat β-actin, respectively. The relative densities of the PCR band of rat TGF-β1 were compared with the β-actin control. The level of TGF-β1 mRNA in cells normalized in 5 mM glucose was considered to be the control (100%). Each bar represents the mean ± SD of five dishes (*P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.005). Similar results were obtained in three other experiments.

Similarly, high glucose (25 mM) stimulated collagen α1 (type IV) mRNA expression >150% in nontransfected IRPTC (Figure 7A) and in IRPTC stably transfected with pRC/RSV (Figure 7B) or pRSV/rANG (+) (Figure 7C), compared with control cells incubated in 5 mM glucose medium (P ≤ 0.05, 0.01, and 0.01, respectively). High glucose had no effect, however, on collagen α1 (type IV) mRNA expression in IRPTC stably transfected with pRSV/rANG (−) compared with control cells incubated in 5 mM glucose medium (Figure 7D).

Figure7

Figure 7. Effect of high glucose on rat collagen α1 (type IV) mRNA expression in IRPTC stable transformants. After 24 h of incubation in media with 5 or 25 mM glucose plus 1% dFBS, cells were harvested and assayed for collagen α1 (type IV) mRNA levels by RT-PCR, as described in the Figure 5 legend. The relative densities of the PCR band of collagen α1 (type IV) were compared with the β-actin control. The level of collagen α1 (type IV) mRNA in cells normalized in 5 mM glucose was considered to be the control (100%). Each bar represents the mean ± SD of five dishes (*P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.005). Similar results were obtained in three independent experiments.

Effect of High Glucose on Cellular Hypertrophy of IRPTC Stable Transformants

Figure 8 shows that cellular p27Kip1 protein content was significantly increased in nontransfected IRPTC (Figure 8A) and in IRPTC stably transfected with pRC/RSV (Figure 8B) or pRSV/rANG (+) (Figure 8C) and incubated in 25 mM glucose medium, compared with cells incubated in 5 mM glucose medium (P ≤ 0.05, 0.01, and 0.005, respectively). There was no apparent increase of cellular p27Kip1 protein content in IRPTC stably transfected with pRSV/rANG (−) and incubated in 25 mM glucose medium, compared with cells incubated in 5 mM glucose medium (Figure 8D).

Figure8

Figure 8. Stimulatory effect of high glucose on cellular p27Kip1 levels in IRPTC stable transformants. After 4 h of incubation in media that contained 5 or 25 mM glucose, the cells were harvested and assayed for p27Kip1 levels by Western blotting. The relative densities p27Kip1 band were compared with the β-actin. The levels of p27Kip1 protein in cells normalized in 5 mM glucose medium represent the control level (100%) (*P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.005; n = 5). Similar results were obtained in three independent experiments.

Figure 9 depicts a shift to the right in forward-angle light scatter for nontransfected IRPTC grown in high-glucose medium (25 mM) alone (Figure 9A). Cell diameter was ∼8% (range, 5% to 12%) larger than that of cells cultured in 5 mM glucose medium (Figure 9A), which indicates an increase in cell size. Similar observations were made in IRPTC stably transfected with pRC/RSV (Figure 9B) or pRSV/rANG (+) (Figure 9C). By contrast, high glucose (25 mM) failed to induce an increase in cell diameter in IRPTC stably transfected with pRSV/rANG (−), compared with cells cultured in normal glucose (5 mM) (Figure 9D). Cell viability was >95% viability in all experiments.

Figure9

Figure 9. Effect of high glucose levels on cellular hypertrophy in IRPTC stable transformants. After 48 h of incubation in media that contained 5 or 25 mM glucose, IRPTC stable transformants were analyzed by flow cytometry. DNA was stained with propidium iodide to determine the cell viability. Forward light scatter was expressed in arbitrary units. Rightward shift of the plot on the x-axis indicates an increase in cellular size. (A) Nontransfected IRPTC; (B) IRPTC stably transfected with pRC/RSV; (C) IRPTC stably transfected with pRSV/rANG (+); and (D) IRPTC stably transfected with pRSV/rANG (−). The blank and black histograms represent the effect of 5 and 25 mM glucose medium, respectively.

Cellular protein content was also increased significantly in nontransfected IRPTC and in IRPTC stably transfected with pRC/RSV or pRSV/rANG (+) cultured in high glucose (25 mM) medium, compared with cells in control (5 mM) glucose medium (Figure 10). High glucose (25 mM) had no effect on cellular protein content in IRPTC stably transfected with pRSV/rANG (−), compared with cells cultured in normal glucose (5 mM) medium.

Figure10

Figure 10. Effect of high glucose on cellular protein levels in IRPTC stable transformants. Total protein content in IRPTC stable transformants was determined by a modified Lowry method and expressed per 106 cells. The cells were incubated for 48 h in 5 or 25 mM glucose medium and were then harvested and analyzed for total cellular content. Cellular protein content of in IRPTC incubated in 5 mM glucose medium represents the control level (100%). Each bar represents the mean ± SD of five determinations (*P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.005). Similar results were obtained in three independent experiments.

Discussion

These studies demonstrate that the stable transfer of antisense ANG cDNA in IRPTC abrogates TGF-β1 and collagen α1 (type IV) mRNA expression and hypertrophy in IRPTC induced by high levels of glucose in vitro. The inhibitory action of antisense ANG cDNA gene transfer appears to be mediated, at least in part, via suppression of the local intrarenal RAS and subsequent prevention of formation of TGF-β1.

We have reported elsewhere that ANG is synthesized and secreted by IRPTC (25). These studies showed that IR-rANG levels in culture media with IRPTC stably transfected with pRSV/rANG (+) were significantly higher than in IRPTC stably transfected with pRC/RSV or pRSV/rANG (−) (Figure 1). These data demonstrate that the plasmid pRSV/rANG (+) expressed the ANG protein and secreted it into the medium. On the other hand, antisense ANG cDNA expressed by the plasmid pRSV/rANG (−) was effective in blocking ANG mRNA translation and, subsequently, ANG protein expression. To confirm that IRPTC stably transfected with pRSV/rANG (+) or pRSV/rANG (−) express high and low ANG mRNA levels, respectively, we used conventional Northern blotting to probe ANG mRNA expression in IRPTC. We observed that the transformants with pRSV/rANG (+) stably transfected into their genomes expressed higher ANG mRNA levels compared with IRPTC stably transfected with pRSV/rANG (−) or pRC/RSV (Figure 2) and nontransfected IRPTC (data not shown). We have selected four clones, each with the highest and lowest ANG mRNA levels for these studies, as shown in Figure 2. In contrast, IRPTC stably transfected with pRSV/rANG (−) into their genomes did not display lower ANG mRNA levels, compared with IRPTC stably transfected with pRC/RSV when analyzed by Northern blot (Figure 2), despite the fact that they secreted lower amount of IR-rANG than nontransfected IRPTC and IRPTC stably transfected with pRC/RSV (Figure 1). These results indicate that conventional Northern blotting that uses the ANG cDNA probe is not sensitive enough to detect differences in ANG mRNA levels in these cells. Therefore, we used a more sensitive RT-PCR to quantify the expression of endogenous ANG mRNA in stable transformants. This RT-PCR is specific for 3′-noncoding region of ANG mRNA, i.e., N+1440 to N+1659 of rat ANG mRNA (32). The nucleotide sequence N+1440 to N+ 1659 is not presented in the plasmid, pRSV/rANG (+), or pRSV/rANG (−). The internal radioactively labeled probe N+1494 to N+1523 did not hybridized with either plasmid (not shown). Indeed, our results revealed that endogenous ANG mRNA levels are significantly lower in IRPTC stably transfected with pRSV/rANG (−), compared with IRPTC stably transfected with pRC/RSV or pRSV/rANG (+) (Figure 3). These studies demonstrate that stable transfer of antisense ANG cDNA is an effective method to suppress the endogenous ANG mRNA and ANG protein levels in IRPTC.

Renal hypertrophy and matrix protein synthesis is associated with upregulation of TGF-β1 gene expression in diabetic rats and mice (41–43). There is compelling evidence that AngII-induced RPTC hypertrophy and the stimulated synthesis of collagen type IV are mediated by increased transcription and production of TGF-β1 (2–9). This was demonstrated by the elegant studies of Wolf et al. (7) and Sharma et al. (42), who showed that neutralizing anti–TGF-β1 antibodies significantly reduced the high AngII-stimulated collagen production and hypertrophy in murine proximal tubular cells in vitro and in vivo, respectively. Our experiments have demonstrated that both basal TGF-β1 and collagen α1 (type IV) mRNA levels in IRPTC stably transfected with pRSV/rANG (−) were lower than those in nontransfected IRPTC and IRPTC stably transfected with pRC/RSV or pRSV/rANG (+) (Figures 4 and 5). These data show that both TGF-β1 and collagen α1 (type IV) mRNA expression in IRPTC depends, at least in part, on the presence of endogenous AngII. Thus, renal AngII acts in an autocrine manner to stimulate TGF-β1 expression and, subsequently, TGF-β1 enhances cellular hypertrophy and collagen α1 (type IV) expression in RPTC. Indeed, this possibility is supported by the observations that high glucose levels stimulated both TGF-β1 and collagen α1 (type IV) mRNA levels in nontransfected IRPTC (Figures 6A and 7A) and in IRPTC stably transfected with pRC/RSV (Figures 6B and 7B) or with pRSV/rANG (+) (Figures 6C and 7C) but had no effect on IRPTC stably transfected with pRSV/rANG (−) (Figures 6D and 7D). These data further indicate that high glucose per se is not sufficient to stimulate TGF-β1 and collagen α1 (type IV) mRNA expression in IRPTC. Indeed, the high glucose effect requires the presence of AngII to act synergistically to enhance TGF-β1 and collagen α1 (type IV) mRNA expression in IRPTC.

To investigate whether the stimulatory action of high glucose on induction of IRPTC hypertrophy is indeed mediated via the expression of endogenous AngII, we examined p27Kip1 expression, cell size, and protein in stable transformants. Our results showed that high glucose (25 mM) induced p27Kip1 expression in nontransfected IRPTC (Figure 8A) and in IRPTC stably transfected with pRC/RSV (Figure 8B) or pRSV/rANG (+) (Figure 8C) but not in IRPTC stably transfected with pRSV/rANG (−) (Figure 8D). Furthermore, culture of nontransfected IRPTC and IRPTC stably transfected with pRC/RSV or pRSV/rANG (+) in 25 mM glucose medium induced cell hypertrophy, as evidenced by the rightward shift in forward light scatter on flow cytometry (Figure 9, A through C) and increased cellular protein content (Figure 10). Although flow-cytometry analysis does not allow the precise measurement of cell size, an 8% to 12% augmentation in cell diameter observed in our experiments (Figure 9, A through C) would indicate a considerable increase in the average size of IRPTC exposed to high glucose. These results are consistent with our results elsewhere (28) and those of others that have shown that high glucose (i.e., ≥25 mM) induces cellular hypertrophy of murine and porcine proximal tubular cells by 5% to 10% as analyzed by flow cytometry (1–7). Our data also reveal that the stimulatory effect of high glucose was abrogated in IRPTC stably transfected with pRSV/rANG (−) (Figures 9D and 10). These studies indicate that blockade of the local renal RAS is an effective method in attenuating or preventing hypertrophy of RPTC evoked by hyperglycemia. Indeed, this notion is supported by our previous investigations, which demonstrated that the addition of ACE inhibitors (i.e., perindopril and captopril) and of AngII-receptor antagonists (i.e., losartan) inhibited IRPTC hypertrophy induced by 25 mM glucose (28), confirming that renal AngII mediated the effect of high glucose (25 mM) on the induction of increase in cellular size and protein levels. High glucose had no effects, however, on these parameters in IRPTC stably transfected with pRSV/rANG (−). Moreover, we have found that high glucose does not stimulate thymidine incorporation in nontransfected IRPTC or stable transformants, compared with normal glucose (data not shown). These studies confirm that high glucose and AngII are capable of inducing cellular hypertrophy but not cell proliferation.

In summary, our studies showed that exposure of nontransfected IRPTC and IRPTC stably transfected with pRC/RSV or pRSV/rANG (+) to 25 mM glucose stimulated TGF-β1 mRNA expression and enhanced IRPTC hypertrophy. The stimulatory effect of high glucose on these parameters was prevented in IRPTC stably transfected with pRSV/rANG (−). These results suggest that local renal RAS activation may be a critical step for renal TGF-β1 expression and the development of renal hypertrophy in early diabetes. Therefore, inhibition of renal ANG gene expression and AngII formation may be an important step whereby RAS blockers could prevent or attenuate the development of hypertrophy of renal proximal tubular cells in diabetes.

Acknowledgments

This work was supported by grants from Canadian Institutes of Health Research (MT-13420 to J.S.D.C, MT-15070 to J.S.D.C. and J.G.F., and MT-12573 to J.G.F.) and the National Institutes of Health (HL-48455 to J.R.I. and DK-50836 to S.S.T.). The authors thank Dr. Tian-Tian Wang for the studies of thymidine incorporation in IRPTC and Mr. Ovid M. Da Silva, Éditeur-Rédacteur, Bureau d’aide à la recherche, Research Center, CHUM, for editing this manuscript.

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Essential Role(s) of the Intrarenal Renin-Angiotensin System in Transforming Growth Factor–β1 Gene Expression and Induction of Hypertrophy of Rat Kidney Proximal Tubular Cells in High Glucose
Shao-Ling Zhang, Catherine To, Xing Chen, Janos G. Filep, Shiow-Shih Tang, Julie R. Ingelfinger, John S. D. Chan
JASN Feb 2002, 13 (2) 302-312;
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