High Glucose Stimulates Proliferation and Collagen Type I Synthesis in Renal Cortical Fibroblasts

Mediation by Autocrine Activation of TGF-β

Abstract

Renal tubular epithelial cells and interstitial fibroblasts are active participants in tubulointerstitial fibrosis, the best correlate of decreased glomerular filtration in diabetic nephropathy. It was reported previously that high ambient glucose stimulates transforming growth factor-β (TGF-β) mRNA and bioactivity, promotes cellular hypertrophy, and increases collagen synthesis in proximal tubular cells. This study evaluates the effects of high glucose and TGF-β on the behavior of murine renal cortical fibroblasts (TFB) in culture. High glucose (450 mg/dl) significantly increased [³H]-thymidine incorporation (by 60 to 80% after 24 to 72 h) and cell number, without significantly increasing cell death when compared with normal glucose (100 mg/dl). There also was a transient increase in the mRNA of the c-myc and egr-1 early-response genes. Exogenous TGF-β1 was promitogenic rather than antiproliferative in contrast to other renal cell types. Northern blot analysis demonstrated constitutive expression of TGF-β1, -β2, and -β3 transcripts. Exposure to high glucose increased all three TGF-β isoforms in a time-dependent manner. High glucose as well as exogenous TGF-β1 also increased [³H]-proline incorporation, α2(I) collagen mRNA, and type I collagen protein (measured by immunoassay). Treatment with a neutralizing pan-selective monoclonal anti-TGF-β antibody markedly attenuated the stimulation by high ambient glucose of thymidine incorporation, TGF-β1 mRNA, and type I collagen mRNA and protein levels. It is concluded that high ambient glucose and exogenous TGF-β1 share similar actions on renal fibroblasts. Moreover, the stimulation of cell proliferation and collagen type I synthesis in these cells by high ambient glucose are mediated by activation of an autocrine TGF-β system.

Tubulointerstitial fibrosis is an important component of renal injury in diabetic nephropathy (reviewed in references ¹ and ²). The degree of tubulointerstitial fibrosis in this disease closely correlates with the magnitude of mesangial matrix expansion (³), the decrease in GFR, and the increase in urinary albumin excretion (^4,5). Among diabetic patients with variable degrees of glomerulosclerosis, those revealing a fairly preserved tubulointerstitium have a higher kidney survival rate (⁶). Thus, the elucidation of the behavior of renal tubulointerstitial cells in the diabetic milieu, particularly the changes related to cell growth and extracellular matrix biosynthesis, is an important endeavor in the search for the basis of fibrogenic mechanisms and the nature of progressive kidney failure.

The cellular compartment of the renal tubulointerstitium consists of tubular epithelial cells, interstitial and perivascular cells including fibroblasts, and infiltrating cells. Various extracellular matrix components are elaborated into the tubular basement membranes and the interstitial space of this compartment. The tubulointerstitial fibroblast is one of the predominant cell types in the cortical and outer medullary interstitium (⁷) and is similar in shape and ultrastructure to fibroblasts of other parenchymal organs (⁸). However, it has been demonstrated that renal fibroblasts in culture have their own autocrine/paracrine systems of vasoactive agents and growth factors (^9,10,11,12). For instance, angiotensin II causes hyperplasia and extracellular matrix synthesis via the angiotensin type I receptor in cultured NRK-49F fibroblasts (¹¹). Other studies have demonstrated that thymidine incorporation, cellular protein content, and sodium-hydrogen exchange activity in human proximal tubular cells are all increased by the addition of conditioned media from human renal cortical fibroblasts in culture, and these effects are blocked by the specific neutralization of the insulin-like growth factor-1 receptor (¹²).

We reported previously that the effects of high ambient glucose on proximal tubular epithelial cells and glomerular mesangial cells in culture closely resemble those of transforming growth factor-β (TGF-β) (^13,14,15). In fact, an increase in TGF-β mRNA/bioactivity is demonstrable when these cells are cultured in media containing increasing concentrations of D-glucose. Moreover, treatment with neutralizing anti-TGF-β antibody prevents the cellular hypertrophy and the increase in collagen biosynthesis induced by high glucose (^14,15). We recently expanded on these observations by demonstrating invivo that repeated administration of a neutralizing anti-TGF-β antibody ameliorates some of the early changes seen in the kidneys of streptozotocin-diabetic mice, such as renal and glomerular hypertrophy and the increased mRNA levels of type IV collagen and fibronection (¹⁶).

Given these inter-relationships between high ambient glucose, TGF-β expression, and phenotypic changes in glomerular and tubular cells, we therefore became interested in studying the effects of high glucose concentration on the behavior of renal fibroblasts in tissue culture. We studied a cell line derived from mouse renal cortex, designated TFB (⁸), to examine the effects of media glucose concentration on cell growth, early-response gene expression, proline incorporation, and type I collagen levels. We also assessed TGF-β isoform expression and applied a neutralizing anti-TGF-β antibody to determine whether the effects of high glucose are mediated by activation of an autocrine TGF-β system.

Materials and Methods

Cell Culture

TFB were isolated by the differential sieving technique using kidney cortex from naive SJL/J(H-2^s) mice (⁸). These cells demonstrate many phenotypic features typical of differentiated fibroblasts (⁸), including positive staining for α-smooth muscle actin (¹⁷) and fibroblast specific protein-1 (¹⁸). Furthermore, thymidine incorporation and collagen synthesis are increased in response to TGF-β1 at a dose of 0.04 to 1 ng/ml (⁸). Cells were maintained at 37°C in a humidified incubator with 5% CO₂/95% air and propagated in Dulbecco's modified Eagle's medium (DMEM; Life Technologies, Gaithersburg, MD) containing 10% fetal calf serum (FCS), 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM glutamine. Cells were passaged every 4 to 5 d by light trypsinization.

Cell Proliferation Assays

TFB (1 × 10⁴) were plated in 96-well microtiter plastic plates (Nunclon, Denmark) in DMEM containing 100 mg/dl (5.6 mM) D-glucose and 10% FCS. Cells were rested for 24 h in fresh DMEM containing 1% FCS and 100 mg/dl D-glucose concentration. Cells were then studied for an additional 24- to 72-h period in fresh DMEM containing 1 to 3% FCS and a D-glucose concentration ranging between 100 and 600 mg/dl (5.6 to 33 mM). For some experiments, the osmolarity of the medium was adjusted with D-mannitol. When the cells were examined for 48- and 72-h periods, the FCS in the media was increased to 3% to sustain adequate growth. Some wells were treated with 30 μg/ml monoclonal pan-selective anti-TGF-β neutralizing antibody (gift of Dr. Brian M. Fendly of Genentech) (¹⁹) or normal mouse IgG as control (Sigma, St. Louis, MO) for the same duration as that of high ambient glucose. ³[H]-Thymidine (5 Ci/mmol; Amersham, Arlington Heights, IL) at 1 μCi per well was added for the final 4 h of culture. Cells were lysed and harvested on glass-fiber paper and counted for scintillation as described previously (^20,21). To obtain direct cell counts, TFB were cultured as described above except they were plated onto 24-well plastic plates (3 × 10⁵ cells/well) and then counted in an automated counter (Coulter Electronics, Hialeah, FL).

Cell Survival Analysis

Cells were studied in 96-well plates and maintained in the same cell culture conditions as for the thymidine incorporation studies. Cell survival was measured by a commercial kit based on the colorimetric conversion of a novel tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] to formazan, which detects the proportion of the number of living cells in culture by measuring the absorbance of each well at 490 nm (Promega, Madison, WI). Absorbance at 490 nm was directly proportional to the number of living cells, and the correlation coefficient between cell number and absorbance was 0.99, indicating a linear response between cell number and absorbance. The percent cell survival (or death) of TFB in high ambient glucose was compared relative to the number of cells in 100 mg/dl glucose.

Total ³[H]-Proline Incorporation

To gain insight into the kinetics of high glucose-mediated collagen synthesis in TFB, we examined the time course of incorporation of ³[H]-proline into TCA-precipitable proteins (²²). For these experiments, 1 × 10⁵ cells/well were plated onto 24-well plates in DMEM containing 100 mg/dl D-glucose with 10% FCS, and at confluence were made quiescent by incubation for 24 h in serum-free DMEM. The media were then changed to fresh DMEM with either 100 or 450 mg/dl D-glucose with 2% FCS (cells in 24-well plates required at least 2% FCS for sustained growth). Cells were propagated for an additional 24- to 72-h period, and 1 μCi ³[H]-proline (130 Ci/mmol, Amersham) was added to each well for the last 12 h of culture. Cells were washed twice in ice-cold phosphate-buffered saline (PBS), precipitated twice in ice-cold 10% TCA, redissolved in 500 μl of 0.5 M NaOH + 0.1% Triton X-100, and counted for β-emissions. Additional cells plated in parallel were scraped off the plate after trypsinization and counted in a Coulter counter. Proline incorporation was expressed as counts per minute (cpm)/10⁶ cells.

Northern Hybridization

Quiescent TFB were cultured in 75-cm² flasks for 24 to 72 h in DMEM containing either 100 or 450 mg/dl D-glucose with 3% FCS (cells in flasks required at least 3% FCS for sustained growth). To some flasks was added the neutralizing anti-TGF-β antibody (30 μg/ml), control mouse IgG (30 μg/ml), or recombinant human TGF-β1 (1 ng/ml; R&D Systems, Minneapolis, MN). At the end of the incubation period, cells were harvested, total RNA was isolated, and Northern blotting was performed as described previously (^14,23). The cDNA probes used were: a 275-bp EcoRI fragment from mouse TGF-β1 (²³), a 442-bp EcoRI-XhoI fragment from mouse TGF-β2 clone pm TGFβ2-9A11C (gift from Dr. H. Moses, Vanderbilt University, Nashville, TN) (²⁴), a 609-bp BamHI-XhoI fragment from mouse TGF-β3 clone pmTGFβ3-11C (gift from Dr. Moses) (²⁵), a PstI fragment from the c-myc clone pmyc41 (²⁶), a PstI insert of the mouse egr-1 gene (²⁷), and a 515-bp fragment of rat GAPDH (²⁰). The 450-bp cDNA probe encoding murine α2(I) collagen was synthesized by the PCR using murine cDNA as template and specific oligonucleotide primers based on known sequences. The PCR product was cloned into the pCRII TA cloning system (Invitrogen, La Jolla, CA). Nucleotide sequencing of the PCR product confirmed the identity of the probe. Hybridization and washing were performed as reported previously (¹⁵). The membranes were then autoradiographed with intensifying screens (Kodak, Wilmington, DE) at -70°C for 1 to 4 d. Exposed films were scanned with a laser-densitometer (Hoefer Scientific Instruments, San Francisco, CA), and all mRNA levels were calculated relative to those of GAPDH. Measurements of ratios in normal glucose media (100 mg/dl) were assigned a relative value of 100%.

Enzyme-Linked Immunosorbent Assay

TFB were cultured in 60-mm plastic dishes for 72 h in DMEM containing either 100 or 450 mg/dl D-glucose with 3% FCS. The media contained 50 μg/ml L-ascorbic acid and 50 μg/ml β-aminopropionitrile, to promote collagen synthesis and prevent cross-links, respectively. Some dishes also received 30 μg/ml neutralizing anti-TGF-β antibody or control mouse IgG, or 1 ng/ml recombinant human TGF-β1. The conditioned media were collected, centrifuged, and stored in -20°C. The protein concentration in the media was determined in triplicate (Bio-Rad, Richmond, CA). For enzyme-linked immunosorbent assay, a 96-well immunoplate (Nunc, Roskilde, Denmark) was coated with 150 μl of conditioned media overnight at 4°C. The wells were washed three times with PBS/1% Tween 20 and blocked with PBS/1% BSA for 30 min. Rabbit anti-mouse type I collagen antibody (1:200; Biodesign, Kennebunk, ME) in PBS/1% BSA was used as primary antibody for 2 h at room temperature. After three washes, the wells were incubated with 1:4000 peroxidase-conjugated anti-rabbit antibody (Chemicon, Temecula, CA) in PBS/1% BSA for 1 h at room temperature. Final detection was performed by peroxidase substrate (Bio-Rad) for 20 min and assessed at 405 nm optical density. Mouse type I collagen (Chondrex, Redmond, WA) was used as standard, and a linear correlation was found between optical density and type I collagen at a dose ranging from 3 ng/ml to 200 ng/ml. All results were normalized to 1 mg of protein of conditioned media.

Statistical Analyses

Data are presented as the mean ± SEM with n as the number of different experiments. Groups were compared by ANOVA, and the Mann-Whitney test was used to compare unpaired individual groups. P < 0.05 was considered significant.

Results

Effect of High Glucose on Cell Proliferation

As shown in Figure 1, raising the ambient glucose concentration for 24 h significantly increased thymidine incorporation in near-confluent TFB. This increase in thymidine incorporation was dose-dependent, with an optimal response achieved at a glucose concentration of 450 mg/dl (average increase of 51, 56, and 66% at 300, 450, and 600 mg/dl glucose, respectively, compared with 100 mg/dl, P < 0.05). Therefore, a glucose concentration of 450 mg/dl was tested in all subsequent experiments. The stimulatory effect was not mediated by an increase in osmolarity, since raising the osmolarity by the addition of D-mannitol did not significantly stimulate thymidine incorporation (Figure 1). This effect of high glucose concentration at 24 h was not transient (Table 1), as thymidine incorporation was increased 78% at 48 h and 84% at 72 h. We found consistent stimulation of cellular proliferation by high glucose at various cell confluence states (initial plating density ranging from 1 × 10² to 1 × 10⁴ cell/well). Longer incubations beyond 72 h in the low serum state (1 to 3% FCS) could not be performed because of the need for culture passage and to avoid a tendency for cell detachment. Changes in final cell number were of smaller magnitude but they paralleled the changes seen in thymidine incorporation. Raising the glucose concentration from 100 to 450 mg/dl significantly increased total cell number by 10% at 48 h (13.2 ± 0.1 × 10⁵versus 14.5 ± 0.1 × 10⁵ cells/well; n = 6, P < 0.05) and by 6% at 72 h (14.2 ± 1.2 × 10⁵versus 15.0 ± 1.9 × 10⁵ cells/well; n = 6, P < 0.05).

Figure 1: Raising the ambient glucose concentration causes a dosedependent increase in thymidine incorporation in murine renal cortical fibroblasts (TFB) in culture. Confluent cells were made quiescent for 24 h and then studied for an additional 24 h in fresh Dulbecco's modified Eagle's medium containing 1% fetal calf serum and a D-glucose concentration ranging from 100 to 600 mg/dl (5.6 to 33 mM). The stimulatory effect of high glucose is not mediated by an increase in the osmolarity of the medium since the addition of an equimolar amount of D-mannitol does not significantly stimulate thymidine incorporation. Data are for mean ± SEM, n = 3, 12 replicates each. ^* P < 0.05 versus 100 mg/dl glucose.

Table 1: Effects of ambient glucose and neutralizing anti-TGF-β on thymidine incorporation in cultured murine renal cortical fibroblasts (TFB)^a

Effect of High Glucose on Cell Survival

Because we found a modest increase in cell number after growing TFB in high glucose media, we wondered whether high glucose also increased apoptotic injury and the proportion of cell death, as we have recently reported for proximal tubular epithelial cells (²⁸). Cell survival analysis was measured by a sensitive colorimetric assay from Promega, which detects the proportion of the number of living cells in culture. We found no significant increase in apoptosis under the same experimental conditions that resulted in stimulation of thymidine incorporation by high glucose. Table 2 shows that the proportion of dead cells was not significantly altered when the glucose concentration was increased to 450 mg/dl and when the incubation period was for 24, 48, or 72 h in DMEM containing 3% FCS. Adding 350 mg/dl D-mannitol to 100 mg/dl D-glucose for up to 72 h did not increase the proportion of dead cells. However, a significant increase in apoptosis was observed only when the glucose concentration was increased further, to 600 mg/dl, and for a prolonged incubation period of 72 h (Table 2). Therefore, the simplest explanation for why the steady-state cell count was increased only modestly by high glucose in the face of a relatively high rate of thymidine incorporation is not due to increased cell death but relates to the inherent differences in the sensitivity and kinetics of cell counting versus thymidine incorporation.

Table 2: Effects of D-glucose and D-mannitol on percent cell death in cultured murine renal fibroblasts (TFB)^a

Effect of TGF-β1 on Cell Proliferation

Addition of exogenous TGF-β1 (1 ng/ml) increased thymidine incorporation time-dependently in TFB. For example, the incorporation in control cells cultured in 100 mg/dl glucose for 24 h was 16.7 ± 0.7 × 10³ cpm/well; the incorporation in cells treated with 1 ng/ml TGF-β1 was 20.0 ± 0.9 × 10³ cpm/well (n = 12; P < 0.05). The stimulatory effect of TGF-β1 was persistent beyond 24 h; on average, TGF-β1 increased thymidine incorporation by 42% after 48 h and 257% after 72 h of culture in 100 mg/dl glucose. There was consistent stimulation of cellular proliferation by 1 ng/ml TGF-β1 at different cell confluence states (initial plating density ranging between 1 × 10² to 1 × 10⁴ cell/well). In addition, treatment with TGF-β1 significantly stimulated thymidine incorporation above baseline when the cells were grown in high glucose media, and the effect was persistent after 48 and 72 h. For example, there was an average increase of 51% in thymidine incorporation after 24 h of culture in 450 mg/dl glucose versus 20% in 100 mg/dl glucose.

Effect of Anti-TGF-β Antibodies on Cell Proliferation

To test the hypothesis that the effect of high glucose on cell growth is mediated by endogenous TGF-β activation, we added a monoclonal pan-selective anti-TGF-β neutralizing antibody to cultured TFB. Table 1 shows that the stimulatory effect of high glucose on basal thymidine incorporation after 48 h was virtually prevented by the addition of 30 μg/ml anti-TGF-β antibody (P < 0.05). The addition of an irrelevant mouse IgG as control did not inhibit thymidine incorporation in either normal or high glucose media (P = NS). Prevention of the proliferative effect of high glucose by the anti-TGF-β antibody persisted after 72 h (Table 1). The presence of anti-TGF-β antibody in 100 mg/dl glucose media did not suppress thymidine incorporation at 24 h, but significantly decreased it after 48 and 72 h (Table 1) (n = 6, P < 0.05), suggesting that the basal rate of cell proliferation, even in low glucose concentration, is due partly to constitutive production and bioactivation of TGF-β.

Effect of Glucose on Early-Response Gene Expression

To further evaluate the proliferative response of TFB to elevated glucose concentration, we studied the expression of the proto-oncogene c-myc and egr-1 by Northern analysis. As shown in Figure 2, exposure of quiescent TFB for 24 h to 450 mg/dl glucose resulted in increased c-myc and egr-1 mRNA levels when compared with cells grown in 100 mg/dl glucose (41 and 180% increase, respectively, P < 0.05, n = 3). However, and as is the case often for early-response genes, the stimulatory response was transient, reverting to baseline after 48 h.

Figure 2: Raising the ambient glucose concentration causes an increase in c-myc and egr-1 expression in murine renal cortical fibroblasts (TFB) in culture. The autoradiogram shows results of a representative Northern analysis (n = 3) of RNA isolated from fibroblasts grown for 24 h in media containing either 100 mg/dl (N) or 450 mg/dl (H) D-glucose concentration and hybridized sequentially with cDNA probes for c-myc, egr-1, and GAPDH.

Effects of High Glucose on Proline Incorporation

Collagen protein is rich in proline, and incorporation of radiolabeled proline into cells can be taken as a rough estimate of collagen synthesis rate. Raising the ambient glucose concentration from 100 to 450 mg/dl induced a significant increase in ³[H]-proline incorporation into TCA-precipitable proteins (average increase of 37% after 48 h; from 33.4 ± 2.3 × 10³versus 45.8 ± 8.1 × 10³ cpm/10⁶ cells; n = 6, P < 0.05). This stimulatory effect persisted after 72 h (not shown).

Effects of High Glucose on TGF-β Isoform Expression

TFB expressed all isoforms of TGF-β (-β1, -β2, and -β3) including four TGF-β2 transcripts measuring 5.0, 3.5, 3.0, and 2.2 kb, as expected for this isoform (²⁹). Culture in high ambient glucose concentration, after a lag period of 48 h, increased all TGF-β isoforms (Figure 3). There was an 83% increase in TGF-β1 mRNA expression compared with normal glucose concentration (Figure 3, top panel). All TGF-β2 transcripts were increased in high glucose, by an average of 105% (Figure 3, second panel). The TGF-β3 mRNA was increased by 137% (Figure 3, third panel). After 72 h in culture, all isoforms of TGF-β were also increased in high glucose (TGF-β1: 65% increase; TGF-β2: 45% average increase; TGF-β3: 127% increase; P < 0.05 compared with normal glucose; n = 3).

Figure 3: Raising the ambient glucose concentration causes an increase in transforming growth factor-β1 (TGF-β1), -β2, and -β3 in murine renal cortical fibroblasts (TFB) in culture. The autoradiogram shows results of a Northern blot representing three independent experiments. TFB were grown for 48 h in the presence of either 100 mg/dl (N) or 450 mg/dl (H) D-glucose concentration, and the isolated RNA was probed sequentially with cDNA encoding TGF-β1, -β2, -β3, and GAPDH. High glucose (H), compared with normal glucose (N), increased steady-state mRNA levels for TGF-β1, -β2, and -β3 relative to the housekeeping gene GAPDH. Note that the exogenous addition of TGF-β1 (+T1) at a dose of 1 ng/ml also caused a modest increase in TGF-β1, -β2, and -β3 mRNA levels.

Since previous studies in other cell systems have described that TGF-β1 can autoinduce its own production through a transcriptional mechanism (³⁰), we assessed the effects of adding exogenous TGF-β1 on the expression of TGF-β1 mRNA and the other TGF-β isoforms. Upon addition of 1 ng/ml TGF-β1 for 48 h, there was a small degree of stimulation of the mRNA levels of TGF-β1, TGF-β2, and TGF-β3 (ranging between 10 and 70%) in the presence of either normal or high glucose (Figure 3). Furthermore, and consistent with the autoinduction of TGF-β1, Figure 4 shows that the addition of the pan-selective neutralizing anti-TGF-β antibody partially attenuated the high glucose-stimulated increase in TGF-β1 mRNA level: 39% increase, versus 83% increase in the high glucose media containing control IgG (Figure 4, lane 4 versus lane 6).

Figure 4: A representative Northern blot demonstrating the effects of high glucose, neutralizing anti-TGF-β antibody (+αT), and irrelevant Ig (+Ig) on TGF-β1 mRNA expression in murine renal cortical fibroblasts (TFB) in culture. TFB were grown for 48 h in the presence of either 100 mg/dl (N) or 450 mg/dl (H) D-glucose concentration, and the isolated RNA was probed sequentially with cDNA encoding TGF-β1 and GAPDH. Note that the stimulation of TGF-β1 mRNA by high glucose was partially prevented by the anti-TGF-β antibody, but not by the control IgG (39% versus 83% increase, P < 0.05, n = 3).

Effects of High Glucose, Exogenous TGF-β1, and Anti-TGF-β Antibody on α2(I) Collagen Gene Expression and Type I Collagen Protein Production

After a lag period of 48 h, TFB cultured in 450 mg/dl glucose demonstrated 81% stimulation of α2(I) collagen mRNA level compared with cells grown in 100 mg/dl glucose, and this effect persisted (114% increase) after 72 h (Figure 5). As expected, exogenous TGF-β1 (1 ng/ml) also stimulated α2(I) collagen mRNA level, and this effect was additive to that of high ambient glucose (Figure 6, lane 4, and Figure 7). Treatment with the neutralizing anti-TGF-β antibody markedly attenuated the high glucose-stimulated increase in α2(I) mRNA level compared with no supplement or IgG treatment (Figure 6, lane 6, and Figure 7). In normal glucose-containing media, the anti-TGF-β antibody had no inhibitory effect on α2(I) collagen mRNA (Figure 6, lane 5, and Figure 7). As control, the addition of an irrelevant mouse IgG showed no inhibitory effect on the high glucose induced-increase in α2(I) collagen mRNA (Figure 6, lanes 7 and 8, and Figure 7).

Figure 5: Raising the ambient glucose concentration causes an increase in α2(I) collagen in murine renal cortical fibroblasts (TFB) in culture. The autoradiogram shows results of a representative Northern blot probed sequentially with cDNA encoding α2(I) collagen and GAPDH. High glucose increased α2(I) collagen mRNA levels by 81 and 114% at 48 and 72 h, respectively (P < 0.05, n = 3).

Figure 6: A representative Northern blot demonstrating the effects of high glucose, recombinant human TGF-β1 (+T1), neutralizing anti-TGF-β antibody (αT), and irrelevant Ig (+Ig) on α2(I) collagen mRNA expression in murine renal cortical fibroblasts (TFB) in culture. The autoradiogram shows results of a representative Northern blot probed sequentially with cDNA encoding α2(I) collagen and GAPDH. TFB were grown for 48 h in the presence of normal (N; 100 mg/dl) or high (H; 450 mg/dl) glucose. Note that the stimulation in α2(I) mRNA level by high glucose was virtually abolished by 30 μg/ml anti-TGF-β antibody but not by the same dose of control IgG. Exogenous addition of TGF-β1 at a dose of 1 ng/ml to media containing the high glucose concentration resulted in a marked increase in α2(I) collagen mRNA expression.

Figure 7: Summary of the studies on α2(I) collagen mRNA expression in TFB. Exposed films were scanned with a laser-densitometer, and mRNA levels were calculated relative to those of GAPDH. Measurements of ratios in normal glucose media without supplements (-) were assigned a relative value of 100% representing control values. The stimulation in α2(I) mRNA level by high glucose was virtually abolished by anti-TGF-β antibody but not by control IgG (9% versus 81% increase, P < 0.05, n = 3). Exogenous addition of TGF-β1 at a dose of 1 ng/ml resulted in a marked increase in α2(I) collagen mRNA expression. Data are for mean ± SEM, n = 3. ^* P < 0.05 versus normal glucose.

To assess type I collagen protein production, enzyme-linked immunosorbent assay was performed on supernatants of TFB grown in the presence of L-ascorbic acid and β-aminopropionitrile. TFB cultured in 450 mg/dl glucose for 72 h secreted 69% more type I collagen than cells grown in 100 mg/dl glucose (Table 3). Treatment with the neutralizing anti-TGF-β antibody markedly attenuated the high glucose-stimulated increase in type I collagen protein (9% increase only, P < 0.05) (Table 3). In normal glucose-containing media, the anti-TGF-β antibody had no significant effect on type I collagen protein (Table 3). As control, the addition of an irrelevant mouse IgG did not modify type I collagen protein production compared with no supplement (Table 3).

Table 3: Effects of ambient glucose, TGF-β1, and neutralizing anti-TGF-β antibody on type I collagen protein production (ELISA) in cultured murine renal cortical fibroblasts (TFB)^a

Discussion

The present study demonstrates that high ambient glucose, independent of media osmolarity, stimulates cell proliferation in a murine renal cortical fibroblast cell line as evidenced by increased thymidine incorporation and cell number without significantly increasing cell death. High glucose also increases type I collagen protein synthesis in these cells as indicated by an increase in tritiated proline incorporation, α2(I) mRNA level, and type I collagen protein production. The rationale for conducting these studies relates to the results of our previous investigations on the effects of ambient glucose on renal proximal tubular cells (^13,31) and glomerular mesangial cells (^14,15). We demonstrated previously that the effects of high glucose on cell growth of these cells, particularly the inhibition of proliferation and the induction of hypertrophy (^13,14), are mediated by an autocrine system involving production and activation of TGF-β. The stimulation of collagen expression by high ambient glucose in mesangial cells is also mediated by TGF-β activation (¹⁵). Our present investigation in renal fibroblasts suggests that high glucose concentration results in stimulation rather than inhibition of cellular proliferation. This is a novel finding that underscores the diversity of cell-specific metabolic responses to environmental factors. Nonetheless, this promitogenic effect of high glucose in renal fibroblasts is similar to that of exogenous TGF-β1, and both high glucose and exogenous TGF-β1 can also promote collagen type I production in these cells. Interestingly, these effects of high glucose and TGF-β1 on renal fibroblasts are similar to those reported for angiotensin II in NRK fibroblasts (¹¹).

The effects of high glucose to stimulate proliferation and collagen synthesis in renal fibroblasts not only mimic the actions of exogenous TGF-β1, but are in fact mediated by the bioactivation of endogenous TGF-β, as evidenced by the ability of a pan-selective anti-TGF-β antibody to reverse the high glucose-induced actions. Of interest is the reversal of the high glucose-induced TGF-β1 mRNA expression by the neutralizing anti-TGF-β antibody, which is consistent with the known autoregulatory stimulation by TGF-β1 itself (³⁰). We also demonstrate in this study that the immediate early-response genes c-myc and egr-1, which are transcription factors known to be expressed in the G₁ phase of the cell cycle, are transiently increased in renal fibroblasts cultured in high glucose media. This stimulation is short-lived perhaps because of a more delayed effect of high glucose to increase endogenous TGF-β1 activity (after 48 and 72 h in culture), which can lead to subsequent c-myc suppression (³²).

The stimulatory effect of TGF-β on fibroblast proliferation (rather than inhibition) is not totally surprising, since the biologic actions of TGF-β are known to vary considerably depending on cell type and culture conditions. Signal transduction as a result of TGF-β binding to the type II (or primary) signaling receptor can result in either proliferation or growth inhibition (³³). In general, TGF-β inhibits epithelial and endothelial cell proliferation but can either stimulate or inhibit mesenchymally derived cell proliferation depending on the concentration of TGF-β, degree of confluence, and the presence or absence of other growth factors (^34,35). The cellular mechanisms explaining the stimulatory effects of TGF-β1 on fibroblast growth in this study remain to be elucidated, but we cannot exclude the possibility of activation of other mitogenic systems such as platelet-derived growth factor (or upregulation of its receptor) (³⁴) and fibroblast growth factor-2. Recently, it has been demonstrated that TGF-β stimulates fibroblast growth factor-2 mRNA and protein levels in human renal fibroblasts derived from fibrotic kidneys (³⁶).

Cultured fibroblasts derived from the skin of patients with type 1 diabetes mellitus and nephropathy have been demonstrated to exhibit increased cellular proliferation (³⁷) and collagen synthesis (³⁸) compared with fibroblasts from diabetic patients without nephropathy. More pronounced effects were observed when the cells were cultured in high glucose concentration (³⁸). Skin fibroblasts also retained their phenotypic characteristics even after repeated passage in culture, thus underscoring the importance of intrinsic as well as environmental factors. It remains unproven whether the abnormal behavior of skin fibroblasts is reflective of the behavior of renal cells such as interstitial fibroblasts or glomerular mesangial cells derived from diabetic kidney lesions.

Fibroblasts perform many functions such as maintenance of tissue architecture, synthesis of contractile machinery, and participation in intercellular communication (³⁹). Renal fibroblasts are connected to other cellular components of the tubulointerstitium by specific attachments to basement membranes, and they are also in close contact with lymphatics, nerves, and dendritic cells (^7,40). Tubulointerstitial fibrosis is an important component of renal injury in diabetic nephropathy (^1,2,41,42). In patients with diabetic nephropathy, there is a good correlation between the expression of α-smooth muscle actin, a cytoskeletal protein marker of interstitial fibroblasts, and the decrement in renal function (^41,42), tubulointerstitial fibrosis (⁴²), and the intensity of type III collagen staining (⁴¹). The results of the current study suggest that an environmental factor such as high ambient glucose concentration not only increases the average collagen production per individual fibroblast, but also increases the total number of fibroblasts. Increased numbers of fibroblasts in this space can produce excess amounts of fibrotic material. Hyperglycemia may therefore be an important cause of the expansion of the tubulointerstitial space in diabetic nephropathy. The high-glucose stimulation of TGF-β production and the subsequent stimulation in cell proliferation and collagen synthesis represent a potential explanation for the tubulointerstitial fibrosis of diabetic kidney disease.

Significant increases in the immunoreactivity of all TGF-β isoforms have been demonstrated previously in the tubulointerstitium of various renal diseases including diabetic nephropathy in humans (⁴³). It has been reported that TGF-β1 and TGF-β3 mRNA are constitutively expressed in several mesenchymal cells, whereas TGF-β2 has a more restricted pattern of expression (^29,44). We demonstrate in this study the constitutive expression of all three mammalian isoforms of TGF-β in renal fibroblasts. High glucose can increase the level of the mRNA of these different isoforms, including the multiple transcripts encoding TGF-β2. The genes for TGF-β1, TGF-β2, and TGF-β3 are located on different chromosomes and have different promoter sequences, suggesting that the increased expression of each isoform in response to high glucose may occur either through different transcriptional mechanisms or perhaps through some other common, posttranscriptional mechanism. Additional studies will be required to explore these possibilities. We recently reported that high glucose stimulates TGF-β1 production in mouse mesangial cells through predominantly transcriptional activation (⁴⁵).

We conclude that high ambient glucose and exogenous TGF-β1 share similar actions in cultured renal fibroblasts. Both high glucose and TGF-β1 are promitogenic in these cells rather than antiproliferative as is typically the case in epithelial, endothelial, and other renal cell types. Moreover, the stimulation of cell proliferation and the increase in collagen type I synthesis in renal fibroblasts by high ambient glucose are mediated largely by activation of an autocrine TGF-β system. These studies underscore the importance of metabolic factors such as hyperglycemia in altering the behavior of renal fibroblasts, and they further implicate TGF-β in the genesis of tubulointerstitial fibrosis.

Acknowledgments

This work was supported in part by National Institutes of Health (Grants DK-44513 and DK-45191 and Training Grant DK-07006). Dr. Han is a visiting scholar at the University of Pennsylvania, and is supported by the Korean Research Foundation, the Hyonam Kidney Laboratory, and Soon Chun Hyang University Hospital, Seoul, Korea. Dr. Isono is supported by a fellowship from the Juvenile Diabetes Foundation. Dr. Hoffman is supported by an Individual National Research Service Award from National Institutes of Health.