Stimulation of TGF-beta type II receptor by high glucose in mouse mesangial cells and in diabetic kidney

Motohide Isono*, András Mogyorósi*, Dong Cheol Han, Brenda B. Hoffman, and Fuad N. Ziyadeh

Renal-Electrolyte and Hypertension Division, Department of Medicine, and Penn Center for the Molecular Studies of Kidney Diseases, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6144


    ABSTRACT
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ABSTRACT
INTRODUCTION
RESEARCH DESIGN AND METHODS
RESULTS
DISCUSSION
REFERENCES

Transforming growth factor-beta (TGF-beta ) is important in the pathogenesis of diabetic nephropathy, but little is known about the regulation of the ligand-binding TGF-beta type II signaling receptor (Tbeta IIR). There were significant increases in Tbeta IIR protein and mRNA levels in kidney cortex after 1-6 wk of streptozotocin-induced diabetes. Mouse mesangial cells cultured in high glucose demonstrated significantly increased Tbeta IIR protein and mRNA levels compared with normal glucose. This effect was independent of stimulation of TGF-beta bioactivity by high glucose. Consistent with transcriptional activation by high glucose, the half-life (~4 h) of Tbeta IIR mRNA was not affected by glucose concentration. Moreover, mouse mesangial cells transiently transfected with reporter constructs containing the first 47- or 274-bp promoter fragments of Tbeta IIR demonstrated significantly increased reporter activity in high glucose. Cells grown in high glucose demonstrated increased responsiveness to a relatively small dose of exogenous TGF-beta 1 (0.5 ng/ml): [3H]proline incorporation and alpha 1(IV) collagen mRNA were significantly greater in cells cultured in high than in normal glucose. Hence, the expression of Tbeta IIR is increased in the diabetic kidney and in mesangial cells cultured in high glucose, primarily because of stimulation of gene transcription. Tbeta IIR upregulation by high ambient glucose may contribute to the increased sensitivity of mesangial cells to the profibrogenic action of TGF-beta 1.

diabetic nephropathy; glomerulosclerosis; type IV collagen; glomerulus; transforming growth factor-beta


    INTRODUCTION
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ABSTRACT
INTRODUCTION
RESEARCH DESIGN AND METHODS
RESULTS
DISCUSSION
REFERENCES

DIABETIC NEPHROPATHY is the single most frequent cause of end-stage renal disease, and hyperglycemia is a crucial factor in the development of diabetic kidney disease in susceptible individuals. Many in vitro studies using kidney-derived cells have demonstrated that the cellular abnormalities seen in diabetic kidney disease, such as cellular hypertrophy, enhanced extracellular matrix production, and altered production of growth factors, can be reproduced by high ambient glucose in the culture media (40). High glucose exerts its deleterious effects by numerous pathways; in particular, the multifunctional cytokine transforming growth factor-beta (TGF-beta ) has been implicated as a principal mediator of diabetic nephropathy (2, 13, 28). We have reported that high ambient glucose in proximal tubular cells and mesangial cells stimulates expression and bioactivity of TGF-beta 1, the most ubiquitous isoform in the mammalian kidney (7, 22, 41). Elevated renal TGF-beta 1 mRNA and protein levels have also been found in various animal models and the human form of diabetic nephropathy (9, 25, 28, 36, 39). There is compelling evidence that the prosclerotic and hypertrophic effects of high ambient glucose in glomerular mesangial cells in culture are mediated by increased production and activation of TGF-beta 1 (5, 33, 41), since they can be prevented by neutralizing anti-TGF-beta antibodies (41).

Virtually all cell types produce one or more isoforms of TGF-beta and also express TGF-beta receptors. Three major types of TGF-beta receptors have been identified: type I (Tbeta IR), type II (Tbeta IIR), and type III (Tbeta IIIR). The latter lacks an identifiable cytoplasmic signaling domain and is thought to act as a reservoir or capacitor of TGF-beta on the cell surface. Tbeta IR and Tbeta IIR are the signaling receptors that belong to the transmembrane serine/threonine kinase receptor family. Tbeta IIR is the primary or ligand-binding receptor, because it forms a high-affinity complex with TGF-beta and then binds the type I receptor, resulting in phosphorylation of Tbeta IR protein by the type II receptor kinase and subsequent transduction of the signal (14, 35).

Modulation of the expression of Tbeta IR and Tbeta IIR is associated with altered cellular responses in disease states. For instance, reduced expression of these receptors contributes to the loss of sensitivity to TGF-beta and the increased proliferation of some cancer cells (10, 31), whereas upregulation of Tbeta IIR is associated with increased sensitivity to TGF-beta -mediated growth inhibition (20). Receptor upregulation has been reported in animal models of glomerulosclerosis: Tbeta IR and Tbeta IIR are increased in experimental membranous nephropathy, and Tbeta IIR and Tbeta IIIR are increased in adriamycin-induced nephropathy (24, 30). In different forms of human glomerulonephritis, there is increased Tbeta IIR expression in the sclerotic lesions (37). We previously reported that the early development of diabetic renal hypertrophy in streptozotocin (STZ)-induced diabetes in mice is likely linked to the coordinate upregulation of TGF-beta 1 and Tbeta IIR in the kidney (27). These observations suggest the possibility that increased Tbeta IIR expression in the kidney may enhance the responsiveness to TGF-beta and contribute to extracellular matrix accumulation.

The aim of our study was to examine the expression of Tbeta IIR protein and mRNA levels in the diabetic mouse kidney and to define the role of high ambient glucose in the regulation of Tbeta IIR in cultured mesangial cells. To further clarify the biological significance of Tbeta IIR upregulation, we examined collagen type IV expression and the relative responsiveness of the cells to exogenous TGF-beta 1 in normal- vs. high-glucose media. We report that the expression of Tbeta IIR is increased in the diabetic kidney and in mesangial cells cultured in high-glucose media, primarily because of stimulation of gene transcription, and that Tbeta IIR upregulation by high glucose may contribute to the increased sensitivity of mesangial cells to the profibrogenic action of TGF-beta 1. The data are consistent with the notion that the upregulation of TGF-beta 1 and Tbeta IIR by high ambient glucose in kidney cells is correlated with the increased bioactivity of the TGF-beta system in diabetic nephropathy.


    RESEARCH DESIGN AND METHODS
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ABSTRACT
INTRODUCTION
RESEARCH DESIGN AND METHODS
RESULTS
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Experimental animals. C57Bl female mice were fed a standard pellet laboratory chow and were provided with water ad libitum. Diabetes was induced in weight-matched 8-wk-old mice by two consecutive daily injections of STZ (200 mg/kg ip; Sigma Chemical, St. Louis, MO) dissolved in 10 mmol/l sodium citrate, pH 5.5; controls were injected with buffer alone. This high dose of STZ is required to induce diabetes in mice (27), as opposed to rats. Once glucosuria was detected, 0.3-0.5 U NPH insulin (Eli Lilly, Indianapolis, IN) was administered daily to prevent ketonuria and maintain the blood glucose concentration at near the moderately hyperglycemic level of 25 mmol/l. In an additional group of diabetic mice, the insulin dose was increased up to 1.0 U daily to maintain the blood glucose concentration at around the normal level of ~6 mmol/l. Groups of diabetic and control mice were killed 1-6 wk after the detection of glucosuria. The kidney cortex was excised, immediately frozen in liquid nitrogen, and stored at -70°C for subsequent RNA and protein extraction.

Cell culture. Murine mesangial cells (MMCs) were isolated and transformed with non-capsid-forming SV-40 virus to establish a permanent cell line with a stable, differentiated phenotype, including the typical spindle-like appearance, positive staining for vimentin and desmin, and contraction in response to ANG II and expression of AT1 receptor (33, 41). The cells were maintained in DMEM (GIBCO BRL, Gaithersburg, MD) containing a normal D-glucose concentration of 5.5 mmol/l, 10% FCS, 100 µg/ml streptomycin, 100 U/ml penicillin, and 2 mmol/l glutamine (42). The cells were incubated in a humidified atmosphere of 5% CO2 at 37°C and passaged every 3-4 days by trypsinization. Subconfluent cells were made quiescent by reducing the concentration of FCS to 0.5% for 24 h. The glucose concentration of experimental conditions was increased to 25 mmol/l by the addition of a small volume of 1.0 mol/l D-glucose. Cells were cultured for different time periods in DMEM containing 5.5 or 25 mmol/l glucose. In some experiments, cells were incubated with a rabbit neutralizing panselective anti-TGF-beta antibody (R & D Systems, Minneapolis, MN) or purified rabbit IgG (Sigma Chemical). Recombinant human TGF-beta 1 protein (R & D Systems) was used to examine the effect of exogenous TGF-beta 1.

Northern blot analysis. Total RNA (20 µg) was electrophoresed through a 1.2% agarose gel with 0.67 mol/l formaldehyde, blotted onto Gene-Screen Plus nylon membranes (NEN Research Products, Boston, MA) by the capillary method, and subjected to ultraviolet cross-linking (17). Membranes were prehybridized for 1 h at 65°C in a buffer containing 10% dextran sulfate, 1% SDS, and 1 mol/l NaCl. Murine Tbeta IIR and alpha 1(IV) collagen cDNA probes were synthesized by PCR, with mouse kidney cDNA as template, and then cloned into the pCRII TA cloning system (Invitrogen, La Jolla, CA). Nucleotide sequencing of the probes confirmed their identity. The cDNA inserts were separated from the plasmid in low-melt agarose and labeled with 32P-labeled dCTP (3,000 Ci/mmol; Amersham Pharmacia, Piscataway, NJ) with use of a DNA labeling kit (Amersham Pharmacia). The membranes were hybridized with 1 × 106 cpm/ml probe in hybridization buffer (same as prehybridization buffer) for 16 h at 65°C. The membranes were washed for 10 min twice in 2× saline-sodium citrate (SSC; 20× SSC = 3 mol/l NaCl, 0.3 mol/l sodium citrate, pH 7.0) at room temperature and in 2× SSC with 0.1% SDS for 15 min at 65°C; then the membranes were subjected to two 15-min high-stringency washes in 0.1% SSC-0.1% SDS at 65°C if necessary. The membranes were autoradiographed with intensifying screens at -70°C (Kodak, Wilmington, DE). Blots were stripped and rehybridized with a probe encoding mouse ribosomal protein L32 (mrpL32) (16) to account for small loading and transfer variations. Exposed films were scanned with a densitometer (Hoefer Scientific Instruments, San Francisco, CA), and RNA levels relative to those of mrpL32 were calculated.

Immunoblot analysis. Kidney tissue and cells were homogenized in a lysis buffer containing 50 mmol/l Tris · HCl (pH 8.0), 150 mmol/l NaCl, 1% NP-40, 0.1% SDS, 1 mmol/l EDTA, 0.5 mmol/l dithiothreitol, 1 mmol/l phenylmethylsulfonyl fluoride, and 5 µg/ml each of aprotinin and leupeptin. Protein concentrations of samples were quantitated by protein assay kit (Bio-Rad, Hercules, CA). For immunoblots, 30 µg of samples were subjected to SDS-PAGE (12% acrylamide gel). After electrophoretic transfer to nitrocellulose membrane and blocking, the membranes were incubated with rabbit antibody directed against Tbeta IIR protein (Santa Cruz Biotechnology, Santa Cruz, CA) at room temperature for 3 h. A horseradish peroxidase-conjugated anti-rabbit IgG was used to allow the detection of immunoreactive bands with the enhanced chemiluminescence detection system (Amersham Pharmacia). Equal loading and transfer of protein samples were assessed by staining with Ponceau S. Blocking experiments to determine the specificity of the Tbeta IIR immunoreactive bands were performed using blocking peptide (Santa Cruz Biotechnology). Densitometric analysis was performed as described above.

Message stability assay. Quiescent MMCs were cultured for 48 h in DMEM containing 5.5 or 25 mmol/l glucose. The cells were then treated with 5 µg/ml actinomycin D (Sigma Chemical) to inhibit transcription, and total RNA was extracted after specified time periods (between 0 and 10 h). Northern analysis was performed using Tbeta IIR and mrpL32 as probes. The ratio of Tbeta IIR to mrpL32 in normal glucose media at time 0 (i.e., before actinomycin D treatment) was assigned a relative value of 100%.

Chloramphenicol acetyltransferase constructs, transfection, and chloramphenicol acetyltransferase assay. Human Tbeta IIR promoter-reporter chimeric constructs were kindly provided by Dr. S.-J. Kim (National Cancer Institute, Bethesda, MD). The chloramphenicol acetyltransferase (CAT) constructs employed were pTbeta IIR47 and pTbeta IIR274 containing -47 and -274 bp from the transcription start site of the human Tbeta IIR gene (1). The beta -galactosidase-containing plasmid pCH110 was used to control for transfection efficiency. The transient transfection method was essentially as previously described (42). Briefly, 10 µg of test plasmid and 5 µg of pCH110 were used to transfect MMCs by use of the calcium phosphate DNA precipitation technique. After 16 h of incubation in 5% CO2 at 37°C, the medium was removed, cells were washed twice with PBS, and fresh serum-free DMEM containing 5.5 or 25 mmol/l glucose was added for an additional 48 h. Cells were harvested in TEN buffer (40 mmol/l Tris · HCl, pH 7.5, 1 mmol/l EDTA, 150 mmol/l NaCl), lysed with three cycles of freeze-thaw, and spun down, and the supernatant was assayed for beta -galactosidase activity (26). On the basis of equivalent amounts of beta -galactosidase activity, the cell lysates were added to initiate the CAT enzyme assay with use of [14C]chloramphenicol (Amersham Pharmacia), as previously described (7, 42). CAT enzyme activity was expressed as percentage of acetylated chloramphenicol compared with total chloramphenicol.

Measurement of [3H]proline incorporation. Cells (105/well) were plated into 24-well plates (Nunclon), and the media were changed the next day so that cells could be exposed for another 72 h to 5.5 or 25 mmol/l glucose. [3H]proline incorporation was then determined to test the responsiveness of the cells to exogenous TGF-beta 1. For this purpose, all media were changed to fresh DMEM containing 5.5 mmol/l glucose with or without the addition of TGF-beta 1 (0.5 ng/ml) for 24 h. For the last 16 h, cells were pulsed with 1 µCi of [3H]proline {L-(2,3,4,5)-[3H]proline; Amersham Pharmacia}. Radiolabeled MMCs were washed twice in ice-cold PBS and precipitated twice in ice-cold 10% TCA redissolved in 0.5 ml of 0.5 N NaOH with 0.1% Triton X-100. After neutralization with 0.5 N HCl, the incorporated radioactivity was counted in a liquid scintillation counter. Proline incorporation was corrected for the protein content of cells and expressed as counts per million per microgram of cellular protein.

Statistical analysis. Values are means ± SE. ANOVA followed by Scheffé's test was used for multiple comparisons. Two groups were compared by Student's unpaired t-test. P < 0.05 was considered significant.


    RESULTS
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ABSTRACT
INTRODUCTION
RESEARCH DESIGN AND METHODS
RESULTS
DISCUSSION
REFERENCES

Renal Tbeta IIR expression in diabetic mouse. We first examined Tbeta IIR protein and mRNA expression in the kidney of STZ-induced diabetic mice. The characteristics of the experimental mice are presented in Table 1. The plasma glucose levels of diabetic mice were maintained at ~25 mmol/l. The body weights were significantly smaller in diabetic than in control mice. The kidney-to-body weight ratios were also significantly greater in diabetic than in control mice. All these changes were normalized when sufficient insulin was given daily for 3 wk to keep the blood glucose near the normal level (~6 mmol/l).

                              
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Table 1.   Parameters of the experimental mice

Immunoblot analysis of kidney cortex (Fig. 1) clearly shows increased Tbeta IIR protein level after 1 and 3 wk of diabetes in mice compared with control mice. This increase in Tbeta IIR protein in diabetic mice was prevented when sufficient insulin was given daily for 3 wk to maintain the blood glucose level within the normal range (Fig. 1).


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Fig. 1.   Immunoblot analysis of transforming growth factor-beta (TGF-beta ) type II signaling receptor (Tbeta IIR) protein from kidney of diabetic mice. Protein samples (30 µg/lane) from mouse kidney cortex were resolved on 12% SDS-PAGE, transferred to nitrocellulose membranes, and probed with anti-Tbeta IIR antibody. Samples were derived from nondiabetic control mice (N), STZ-induced diabetic mice after 1 and 3 wk of diabetes (D1 and D3, respectively), or after 3 wk of diabetes but treated with a high dose of insulin (Ins) to normalize blood glucose concentration.

Northern blot analysis also demonstrated significant upregulation of Tbeta IIR mRNA in the mouse kidney at 1, 2, 3, and 6 wk of diabetes compared with nondiabetic controls (Fig. 2). The stimulation was ~2-fold after 1 wk of diabetes, peaked at 2.6-fold after 2 wk, and was persistent after 6 wk. These results extend our previous observation showing upregulation of renal Tbeta IIR mRNA after 9 days of diabetes (27). The effects of diabetes on Tbeta IIR mRNA may not be generalized, since we did not observe any change in cardiac Tbeta IIR expression after 1 or 3 wk of diabetes (data not shown).



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Fig. 2.   Northern blot analysis of Tbeta IIR mRNA from kidney of diabetic mice. Total RNA was isolated from kidney cortex of nondiabetic control mice (N) and STZ-induced diabetic mice after 1, 2, 3, and 6 wk of diabetes (D1, D2, D3, and D6, respectively). A: representative Northern blot of Tbeta IIR mRNA. Mouse ribosomal protein L32 (mrpL32) was used for standardization. B: quantitative results of hybridizations demonstrating persistently increased Tbeta IIR mRNA-to-mrpL32 ratio in kidneys of diabetic mice. Values are means ± SE (n = 5). * P < 0.05 vs. control.

Tbeta IIR expression in MMCs cultured in high glucose. To explore the role of high ambient glucose in Tbeta IIR expression, we opted to utilize cultured mesangial cells as a study system given the central role of these cells in the development of diabetic glomerulosclerosis (11, 40). For immunoblot analysis, protein was isolated from MMCs cultured under normal (5.5 mmol/l)- or high (25 mmol/l)-glucose conditions for 72 h, and the blot was probed with a polyclonal rabbit anti-Tbeta IIR antibody. Figure 3 demonstrates that Tbeta IIR protein level was significantly increased in cells cultured in high ambient glucose compared with normal glucose concentration. In parallel studies, Northern blot analysis also showed significant increases in Tbeta IIR mRNA levels in MMCs cultured for 48 or 72 h in high-glucose media (Fig. 4).



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Fig. 3.   Immunoblot analysis of Tbeta IIR protein from mouse mesangial cells (MMCs) cultured in high glucose. Protein samples (30 µg/lane) from cells were resolved on 12% SDS-PAGE, transferred to nitrocellulose, and probed with polyclonal rabbit anti-Tbeta IIR antibody. A: representative immunoblotting of MMCs cultured for 72 h in media containing normal glucose (N, 5.5 mmol/l) or high glucose (H, 25 mmol/l). B: quantitative results demonstrating significantly increased immunoreactive Tbeta IIR protein in MMCs cultured in high glucose. Values are means ± SE (n = 3). * P < 0.05 vs. normal glucose.




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Fig. 4.   Northern blot analysis of Tbeta IIR mRNA from MMCs cultured in high glucose. Total RNA was isolated from MMCs cultured for 24-72 h in media containing normal glucose (5.5 mmol/l; open bar) or high glucose (25 mmol/l; closed bar) and hybridized with a 32P-labeled cDNA encoding Tbeta IIR. A: representative Northern blot. Standardization was performed with mrpL32. B: quantitative results of hybridizations demonstrating increased Tbeta IIR mRNA-to-mrpL32 ratio in MMCs cultured in high glucose. Values are means ± SE (n = 4). * P < 0.01 vs. normal glucose.

Tbeta IIR message stability and ambient glucose concentration. To determine whether the increased Tbeta IIR mRNA level is transcriptional in origin or is secondary to increased posttranscriptional stability, Northern blot analysis was performed in the presence of an inhibitor of gene transcription. MMCs were first grown in normal- or high-glucose media for 48 h until the steady-state Tbeta IIR mRNA level was increased by the high-glucose media, and then the cells were exposed to actinomycin D (5 µg/ml) for up to 10 h, which results in a progressive decay in message level over time. Figure 5 shows that the rate of decay of the Tbeta IIR message was of equivalent magnitude in cells grown in normal- and high-glucose media, and the message half-life was ~4 h in normal- and high-glucose media. Thus the increase in the steady-state level of Tbeta IIR mRNA induced by high ambient glucose in mesangial cells is unlikely to be due to increased mRNA stability, although a small component of message stabilization cannot be excluded.



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Fig. 5.   Message stability assay of Tbeta IIR mRNA in MMCs. MMCs were made quiescent by reducing concentration of FCS to 0.5%, grown in normal (5.5 mmol/l)- or high (25 mmol/l)-glucose media for 48 h, and then exposed to actinomycin D (5 µg/ml) to inhibit gene transcription. A: representative Northern blot for hybridizations with Tbeta IIR and mrpL32 probes. B: graphic profile of Tbeta IIR mRNA decay (relative to mrpL32) after treatment with actinomycin D for cells grown in normal (open circle ) or high () glucose media. Data points represent mean values from 3 experiments. Measurements at time 0 (before actinomycin D treatment) were assigned a relative value of 100%. Curves represent best-fit lines.

Tbeta IIR promoter activity in mesangial cells. To confirm that the increase in the steady-state level of Tbeta IIR mRNA by high ambient glucose is likely due to increased gene transcription rate, we performed a reporter assay using chimeric Tbeta IIR promoter/CAT reporter constructs. MMCs were transiently transfected with the human promoter constructs pTbeta IIR47 and pTbeta IIR274. To account for transfection efficiency, the cells were also transfected with the beta -galactosidase-containing plasmid pCH110. On the next day the media were changed to 5.5 or 25 mmol/l glucose, and the cells were harvested 48 h later. As shown in Fig. 6, there was a significant stimulation in Tbeta IIR promoter activity as reflected by CAT activity when the cells were transfected with pTbeta IIR47 or pTbeta IIR274 constructs and then grown for 48 h in high-glucose media.



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Fig. 6.   Transfection of MMCs with chloramphenicol acetyltransferase (CAT) reporter constructs. MMCs were transiently transfected with plasmids containing fragments of human Tbeta IIR promoter (pTbeta IIR47 and pTbeta IIR274) linked to CAT gene and a beta -galactosidase-containing plasmid. Cells were then grown in 5.5 mmol/l (open bar) or 25 mmol/l (solid bar) glucose for 48 h. After measurement of beta -galactosidase activity in each extract, identical amounts were used for CAT assay. A: representative thin-layer chromatography plate after separation of acetylated [14C]chloramphenicol (CAT activity) from unacetylated fraction of extracts. NG, normal glucose; HG, high glucose. B: quantitative results for CAT activity of pTbeta IIR47 and pTbeta IIR274 constructs transiently transfected into MMCs grown in normal and high glucose. CAT activity of each construct is expressed relative to total activity. Values are means ± SE (n = 4). * P < 0.05 vs. normal glucose.

Effect of addition of anti-TGF-beta antibody or exogenous TGF-beta 1 on Tbeta IIR mRNA level. High glucose concentration is known to increase TGF-beta 1 level and bioactivity in mesangial cells (33), and previous studies have been conflicting as to whether TGF-beta 1 can modulate the expression of its receptors; exogenous TGF-beta 1 modestly inhibited the expression of Tbeta IIR mRNA in rat mesangial cells (3), but there was no change in Tbeta IIR in the TGF-beta 1 transgenic mouse (19). To assess the contribution of endogenous TGF-beta 1 production in mesangial cells under high-glucose conditions, we examined the effect of a neutralizing panselective anti-TGF-beta antibody on Tbeta IIR expression. Cells were incubated with control IgG or anti-TGF-beta antibody and exposed for 72 h to normal- or high-glucose media. As shown in Fig. 7A, the increased Tbeta IIR expression in high glucose was largely unaffected by the addition of anti-TGF-beta antibody. This suggests that high glucose is capable of increasing the mRNA level of Tbeta IIR independent of the influence of endogenous stimulation of TGF-beta bioactivity. However, addition of exogenous TGF-beta 1 inhibited the expression of Tbeta IIR, especially at a high dose of 2 ng/ml, in cells grown in basal media containing a normal glucose concentration (Fig. 7B), and in accord with previous results in rat mesangial cells (3).



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Fig. 7.   Effects of anti-TGF-beta antibody and exogenous TGF-beta 1 on Tbeta IIR expression. A: quiescent MMCs were cultured for 72 h in media containing normal (5.5 mmol/l) or high glucose (25 mmol/l) with control rabbit IgG (control; 30 µg/ml) or rabbit neutralizing anti-TGF-beta antibody (alpha T, 30 µg/ml). Blots were hybridized with cDNA encoding Tbeta IIR and mrpL32. Northern blot is representative of 3 different experiments. B: quiescent MMCs in normal glucose were treated with TGF-beta 1 (0.5 and 2.0 ng/ml) for 24 h and then subjected to total RNA isolation. Blots were hybridized with cDNA encoding Tbeta IIR and mrpL32. Northern blot is representative of 3 different experiments.

Sensitivity to exogenous TGF-beta 1 protein. We next examined the possibility that increased expression of Tbeta IIR is related to increased sensitivity of mesangial cells to exogenous TGF-beta 1 protein. To test for enhanced TGF-beta 1 action, [3H]proline incorporation and type IV collagen gene expression were measured in MMCs that were grown in 5.5 or 25 mmol/l glucose and then stimulated with a submaximal dose of TGF-beta 1 (0.5 ng/ml). Proline incorporation (Fig. 8A) was significantly increased in cells grown in high glucose, as would be expected (41). Addition of 0.5 ng/ml TGF-beta 1 stimulated proline incorporation into MMCs grown in normal- or high-glucose media, but the extent of this increase was significantly greater in high-glucose media (average increase of 51% in 25 mmol/l glucose vs. 24% in 5.5 mmol/l glucose; Fig. 8A). Similar results were obtained for type IV collagen expression. Figure 8B shows that alpha 1(IV) collagen mRNA was increased to a greater extent by treatment with TGF-beta 1 if the cells were grown in high rather than normal glucose concentration.



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Fig. 8.   Response of MMCs to TGF-beta 1. A: summary of results of [3H]proline incorporation. MMC were exposed for 72 h to media containing normal (5.5 mmol/l) or high (25 mmol/l) glucose, and then all media were changed to fresh DMEM containing 5.5 mmol/l glucose with or without TGF-beta 1 (T, 0.5 ng/ml) for an additional 24 h. For last 16 h, cells were pulsed with [3H]proline. Values are means ± SE (n = 4, 6 replicates each). * P < 0.05 vs. normal glucose without TGF-beta 1; dagger  P < 0.01 vs. high glucose without TGF-beta 1. B: Northern blot of alpha 1(IV) collagen. MMCs were grown for 72 h in normal or high glucose. Media were then changed to fresh DMEM containing 5.5 mmol/l glucose with or without TGF-beta 1 for an additional 24 h and subjected to total RNA isolation. Blots were hybridized with cDNA encoding murine alpha 1(IV) collagen and mrpL32. Cells grown in high glucose demonstrated marked stimulation of alpha 1(IV) collagen compared with normal glucose.


    DISCUSSION
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ABSTRACT
INTRODUCTION
RESEARCH DESIGN AND METHODS
RESULTS
DISCUSSION
REFERENCES

Our study is the first to demonstrate a significant increase in Tbeta IIR protein and message levels in the kidney of STZ-induced diabetes mellitus in the mouse and in glomerular mesangial cells cultured under high-glucose conditions. The upregulation of Tbeta IIR in mesangial cells is transcriptional and is not due to increased mRNA stability. In addition, the functional counterpart of the upregulated Tbeta IIR is an enhanced sensitivity to the stimulation by TGF-beta 1, since the incorporation of [3H]proline and the expression of alpha 1(IV) collagen mRNA are significantly greater in mesangial cells cultured in high than in normal glucose concentration.

The single most important factor in the development of diabetic kidney disease is hyperglycemia (18). In general, the effects of high glucose have been attributed to metabolic alterations related to several humoral and biochemical processes. Because TGF-beta has been widely recognized to be a major promoter of extracellular matrix production (40), we have postulated that this autacoid may mediate the stimulatory effects of high glucose on the synthesis of extracellular matrix in mesangial cells (28). In MMCs, we previously demonstrated that the addition of neutralizing anti-TGF-beta antibody markedly attenuates the prosclerotic and hypertrophic effects of high ambient glucose (41). In STZ-induced diabetic kidney disease in mice, we also demonstrated that inhibition of TGF-beta activity with neutralizing anti-TGF-beta antibody leads to significant attenuation of kidney hypertrophy and increased mRNA levels encoding alpha 1(IV) collagen and fibronectin (27). Ample evidence has been gathered to support the notion that increased renal expression of TGF-beta 1 plays an important role in experimental models of diabetic kidney disease as well as in patients with established diabetic nephropathy (9, 18, 25, 29, 36, 39). However, increased TGF-beta bioactivity may be a result of the upregulation of the ligand itself and/or of its signaling receptors. Unlike the situation with TGF-beta 1, little is known about the regulation in the diabetic state of the signaling receptors for TGF-beta 1.

In our study we found a sustained increase in the protein and mRNA levels of Tbeta IIR in the kidney after 1-6 wk of diabetes in the STZ mouse model. The cellular localization of this effect in the kidney cannot be inferred, but it may include the tubular compartment. Our mesangial cell culture studies are in accord with a very recent report that demonstrated that mechanical stretch, especially in conjunction with high-glucose media, can amplify the expression of TGF-beta receptors in rat mesangial cells (21). Previous studies in LLC-PK1 tubular epithelial cells (6) and Madin-Darby canine kidney epithelial cells (38) demonstrated increased TGF-beta receptor protein binding in high glucose. In preliminary studies using mouse proximal tubular epithelial cells, we found a ~50% increase in Tbeta IIR mRNA when the cells were cultured in high-glucose media for 48 or 72 h (data not shown). However, whether the diabetic state can further upregulate Tbeta IIR mRNA level in tubular epithelia in vivo may depend on ambient factors in addition to hyperglycemia, such as ANG II (34).

Upregulation of TGF-beta receptors in other experimental animal models of glomerulosclerosis has been reported. In passive Heymann nephritis in rats, a model of membranous nephropathy, levels of Tbeta IR and Tbeta IIR protein and mRNA were increased in glomeruli, and these changes correlated with fibrotic lesions (25). Tamaki et al. (30) also reported an increase in Tbeta IIR and Tbeta IIIR in adriamycin-induced nephropathy with parallel changes in TGF-beta 1 and fibronectin in the renal cortex. In addition to these observations, increased levels of Tbeta IR, Tbeta IIR, and Tbeta IIIR have been reported in the sclerotic lesions of human glomerulonephritis such as IgA nephropathy and lupus nephritis (37). These results and our observations suggest that the increase in receptors for TGF-beta 1 as well as the upregulation of TGF-beta 1 itself might play an important role in various diseases causing renal fibrosis.

We investigated the basis for the stimulation of Tbeta IIR expression by high ambient glucose. By applying actinomycin D, we showed that this stimulation is not due to an increase in message stability. In fact, the half-life (~4 h) of Tbeta IIR mRNA in mesangial cells was not affected by the glucose concentration in culture. In addition, use of CAT/Tbeta IIR promoter constructs in a reporter assay provided further evidence for transcriptional activation of Tbeta IIR by high ambient glucose.

Although little is known about the mechanisms regulating Tbeta IIR, it is likely that AP-1 and/or Sp-1 binding sites in the Tbeta IIR promoter are important for stimulation of gene transcription by high glucose. The CAT constructs that we used contain an Sp-1 binding site in pTbeta IIR47 and an AP-1 (or cAMP-response element) site in addition to Sp-1 sites in pTbeta IIR274 (1, 8). It has been reported that high glucose stimulates AP-1 binding and expression of c-fos and c-jun, components of the AP-1 complex, in cultured mesangial cells (11, 32). Sp-1 has also been shown to mediate expression of the acetyl-CoA carboxylase by high glucose (4). Longer promoter segments as well as additional studies are required to establish a link between increased activity of cis-acting glucose-response elements and transcriptional activation of the Tbeta IIR promoter by high glucose.

Our observations demonstrating that anti-TGF-beta antibody failed to significantly modify Tbeta IIR mRNA levels suggest that the stimulatory effects of high glucose on receptor expression are independent of the associated stimulation of endogenous TGF-beta 1 bioactivity. This finding is also supported by our previous report showing that treatment with anti-TGF-beta antibody in diabetic mice failed to significantly inhibit the renal Tbeta IIR expression (27). Moreover, Mozes et al. (19) recently reported no changes in Tbeta IIR in the TGF-beta 1 transgenic mouse (19). However, these findings do not rule out an inhibitory effect of high concentrations of TGF-beta 1 on Tbeta IIR expression. We showed in the present study that sufficiently high concentrations of TGF-beta 1, up to 2 ng/ml, can dampen the level of Tbeta IIR mRNA in cells grown in normal-glucose media.

The number of Tbeta IIR may be regulated by a variety of hormones and growth factors. In rat prostate epithelial cells, Tbeta IIR number is negatively regulated by androgens (12). In IK-90, a human endometrial cancer cell line, epidermal growth factor increases TGF-beta 1 binding sites (23). Modulation of the receptor is associated with changes in the sensitivity to TGF-beta 1 protein. For instance, reduced expression of Tbeta IR and/or Tbeta IIR in some cancer cell lines contributes to the abnormal cell growth (10, 31). On the other hand, stimulation of collagen synthesis by TGF-beta 1 is enhanced in vascular smooth muscle cells from atherosclerotic lesions that exhibit a high Tbeta IR-to-Tbeta IIR ratio (15). Furthermore, regenerating liver cells, which express more TGF-beta receptors than resting cells, exhibit high sensitivity to growth inhibition by TGF-beta 1 (20). From these observations and several in vivo studies demonstrating increased expression of TGF-beta receptors in experimental renal disease (24, 30, 37), we hypothesize that the coordinate upregulation of TGF-beta receptors, as well as of TGF-beta 1 itself (7, 33), may play an important role in progressive renal fibrosis.

In this study we examined whether high ambient glucose, in addition to its known upregulating effect on TGF-beta 1 protein and message (33, 41), would also increase the sensitivity of mesangial cells to exogenous TGF-beta 1 treatment in conjunction with the increased Tbeta IIR. Indeed, we found that cells grown in high glucose exhibit greater responsiveness than cells grown in normal glucose, as assessed by [3H]proline incorporation and type IV collagen expression. These observations suggest that receptor upregulation by high glucose in mesangial cells leads to increased responsiveness to TGF-beta 1 and, in turn, promotes the fibrotic response. Thus the increased production of TGF-beta 1 in the diabetic state and the greater abundance of Tbeta IIR in renal cells exposed to high ambient glucose provide an explanation for the heightened bioactivity of the TGF-beta system and the resultant tissue injury in diabetic nephropathy.


    ACKNOWLEDGEMENTS

The authors thank Dr. Jia Guo for technical assistance.


    FOOTNOTES

* M. Isono and A. Mogyorósi contributed equally to this work.

This study was supported in part by Grants DK-44513, DK-45191, and DK-54608 and Training Grant DK-07006 from the National Institute of Diabetes and Digestive and Kidney Diseases. M. Isono and A. Mogyorósi are supported by fellowships from the Juvenile Diabetes Foundation International. D. C. Han is a visiting scholar at the University of Pennsylvania and is supported by the Korean Research Foundation and the Hyonam Kidney Laboratory at Soon Chun Hyang University Hospital (Seoul, Korea). B. B. Hoffman is supported by an Individual National Research Service Award from the National Institutes of Health.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: F. N. Ziyadeh, 700 Clinical Research Bldg., Renal-Electrolyte and Hypertension Div., University of Pennsylvania, 415 Curie Blvd., Philadelphia, PA 19104-6144 (E-mail: ziyadeh{at}mail.med.upenn.edu).

Received 17 May 1999; accepted in final form 7 December 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
RESEARCH DESIGN AND METHODS
RESULTS
DISCUSSION
REFERENCES

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