Evidence is emerging that intraglomerular growth factors and cytokines .... Glucose Induces PDGF in Human Mesangial Cells 2097. AJP December 1996, Vol.
American Journal of Pathology, Vol. 149, No. 6, December 1996 Copyright ©) American Societyfor Investigative Patbology
High Glucose Concentration Induces the Overexpression of Transforming Growth Factor-f3 through the Activation of a Platelet-Derived Growth Factor Loop in Human Mesangial Cells
Salvatore Di Paolo, Loreto Gesualdo, Elena Ranieri, Giuseppe Grandaliano, and Francesco P. Schena From the Institute ofNephrology, University of Ban, Polyclinic, Ban, Italy
High glucose concentration has been shown to induce the overexpression of transforming growth factor (TGF)-j31 mRNA and protein in different ceUl types, including murine mesangial ceUs, thus possibly accountingfor the expansion of mesangial extracelular matrix observed in diabetic glomerulopathy. In the present study, we evaluated platelet-derived growth factor (PDGF) B-chain and PDGF-18 receptor gene expression in human mesangial ceUs (HMCs) exposed to different concentrations ofglucose and then sought a possible relationship between a PDGF loop and the modulation of TGF- 31 expression. HMC [3H]thymidine incorporation was upregulated by 30 mmol/L glucose (HG) up to 24 hours, whereas it was significantly inhibited at later time points. Neutralizing antibodies to PDGF BB abolished the biphasic response to HG, whereas anti-TGF-,3 antibodies reversed only the late inhibitory effect of hyperglycemic medium. HG induced an early and persistent increase of PDGF B-chain gene expression, as evaluated by reverse transcriptase polymerase chain reaction, whereas PDGF-j3 receptor mRNA increased by twofold after 6 hours, thereafter declining at levels 70% lower than in controls after 24 hours. 125I-Labeled PDGF BB binding studies in HMCs exposed to HG for 24 hours confirmed the decrease of PDGF-g8 receptor expression. TGF-f31specific transcripts showed 43 and 78% increases after 24 and 48 hours of incubation in HG, respectively, which was markedly diminished by anti-PDGF BB neutralizing antibodies or
suramin. We conclude that HG induces an early activation of a PDGF loop that, in turn, causes an increase of TGF-f81 gene expression, thus modulating both HMC proliferation and mesangial matrix production. (Am J Pathol 1996 149:2095-2106)
The expansion of extracellular matrix in the mesangial areas of the glomeruli, without evidence of mesangial cell proliferation, is the dominant histological feature of diabetic nephropathy and leads to glomerulosclerosis and obliteration of the capillary lumen over a period of years.1`3 Although hyperglycemia appears to correlate with the histological and clinical manifestations of diabetic glomerulopathy,1'4- 5the mechanisms whereby it exerts its damaging effect are not fully elucidated. Evidence is emerging that intraglomerular growth factors and cytokines provide the link between the initial glomerular injury and the dysregulation of mesangial cell mitogenesis and matrix expansion that accompanies most progressive glomerular diseases. 9-12 An impressive series of investigations, both in vitro and in vivo, has suggested that two particular growth factors, transforming growth factor (TGF)-f1 and platelet-derived growth factor (PDGF), contribute to the pathophysiological process leading to the develPortions of this work were presented at the 1993 meeting of the American Society of Nephrology and are published in abstract form (J Am Soc Nephrol 1993, 4:792). Supported in part by the Consiglio Nazionale delle Ricerche (CNR) Target Project on Biotechnology and Bioinstrumentation (92.1272 and 93.1102.PF70), by the CNR Joint Program USA-Italy (94.2336.04 and 95.9424.04), and by the Ministero della Ricerca Scientifica e Tecnologica (MURST) grants 93.5382, 94.1937, and 95.3957. Accepted for publication July 30, 1996. Address reprint requests to Dr. F. P. Schena, Institute of Nephrology, University of Bari-Policlinico, Piazza G. Cesare, 11 70124 Bari, Italy.
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opment of glomerulosclerosis (reviewed in Refs. 1012). TGF-,B induces matrix protein synthesis while inhibiting the production of matrix-degrading proteases and increasing the synthesis of protease inhibitors.12 Such peculiar biological activities justify TGF-3's critical involvement in extracellular matrix accumulation and scarring observed with tissue injury repair. Members of the TGF-,B gene family exert variable effects on cell growth, depending on multiple factors including specific cell type, degree of differentiation, interference of other growth factors in culture, and plating density.10'13 Specifically, the effect of TGF-,B on mesangial cell proliferation is still controversial, although the majority of studies report a general growth-inhibitory effect of TGF-f3.14-18 PDGF displays a strong mitogenic effect on mesangial cells (MCs) in vitro as well as in ViVo19'20 and may likely represent the final common pathway through which a number of mitogens exert their effect on mesangial cells.10 Moreover, it can influence the synthesis and degradation of matrix constituents, either directly or through the induction of other growth factors. 10,21-24 Recently, several reports focused on the expression of TGF-P by resident glomerular cells exposed to a diabetic milieu and supported a role of the growth factor also in the pathogenesis of diabetic nephropathy. In vitro studies demonstrated that a high ambient glucose concentration is able to induce an increased expression of TGF-f1 in different cell types, including murine mesangial cells.25,26 In streptozotocin-treated Sprague-Dawley rats, glomerular TGF-,B1 mRNA and protein levels progressively increase after the onset of hyperglycaemia.27'28 Moreover, diabetic rat glomeruli show increased levels of matrix components known to be induced by TGF-,B, indicating that the growth factor is biologically active.28 Similarly, glomeruli from humans with established diabetic nephropathy also display a striking increase of TGF-,31 protein and deposition of fibronectin.28 Finally, renal cortices isolated from spontaneously diabetic, nonobese mice reveal an increased content of TGF-,B mRNA and protein, TGF-,2 being the predominant isoform, which may reflect a species-selective process.29 Conversely, the possible influence of a diabetic milieu on the expression of PDGF is largely circumstantial. The PDGF system has been implicated in the development of atherosclerosis and microvascular complications associated with diabetes mellitus.30'31 Glomeruli of streptozotocin-induced diabetic rats have been reported to display an increase of PDGF B-chain mRNA levels.27 Indirect in vitro evidence suggests that PDGF mediates the increase of
collagen IV mRNA and peptide synthesis induced by advanced glycosylation end products that accumulate in diabetes.23 Moreover, autocrine and paracrine interactions between TGF-,B1 and PDGF have been documented in several cellular models. 16'1821'32 We therefore investigated whether elevated ambient glucose concentration would influence the gene expression of PDGF B-chain and PDGF-,B receptor (PDGF-f3R) by cultured human MCs (HMCs) and sought a possible relationship between a PDGF loop and TGF-13 gene expression in a hyperglycemic environment.
Materials and Methods Cell Isolation and Culture HMCs were established and characterized as reported previously.33 Cells were allowed to grow until confluent in RPMI 1640 medium (Gibco Laboratories, Grand Island, NY) supplemented with 17% heat-inactivated fetal bovine serum (Hyclone Laboratories, Logan, UT), 100 U/ml penicillin, 100 ,ug/ml streptomycin, 2 mmol/L L-glutamine, 2 mmol/L sodium pyruvate, 1 % (v/v) nonessential amino acids, 5 tLg/ml insulin, 5 ,tg/ml transferrin,and 5 ng/ml selenium. For passage, confluent cells were washed with phosphate-buffered saline (PBS), removed with 0.025% trypsin/0.5 mmol/L EDTA in PBS, and plated in RPMI. Experiments included in this study were performed on cells between the 5th and 10th passages from at least four different cell lines.
Culture Conditions To initiate experiments, HMCs were plated into 10cm2 Petri dishes or 24-well plastic plates, grown to confluence in RPMI 1640 containing 17% fetal bovine serum and 5 ,tg/ml insulin, rested for 48 hours in serum- and insulin-free medium, and then grown in fresh medium containing 10 mmol/L or 30 mmol/L glucose without serum and insulin.
Cell Growth DNA synthesis in response to different glucose concentrations was measured as the amount of [methyl3H]thymidine incorporated into trichloroacetic-acidprecipitable material. Cells were plated in 24-well dishes at a density of 2 x 104 to 4 x 104/well, grown to confluence, and made quiescent by placing them in serum-free medium for 48 hours. Then, cells were incubated with 10 mmol/L or 30 mmol/L glucose, without serum and insulin, for 12 to 72 hours at 37°C.
Glucose Induces PDGF in Human Mesangial Cells 2097 AJP December 1996, Vol. 149, No. 6
Some cells were cultured for 24 to 48 hours in the presence of either 50 ,ug/ml neutralizing polyclonal rabbit anti-human PDGF BB antibody (Genzyme, Cambridge, MA) or 30 ,ug/ml monoclonal mouse anti-TGF-,B antibody, recognizing human TGF-41 and TGF-,f2 (Genzyme). Control experiments were performed in which HMCs were treated with rabbit or mouse nonimmune IgG. At the end of the incubation period, cells were pulsed for 4 hours with 1.0 j.tCi/ml [methyl-3H]thymidine (Amersham, Little Chalfont, UK). The medium was then removed, and the cells were washed twice in ice-cold 5% trichloroacetic acid and incubated in 5% trichloroacetic acid for 5 minutes. The cells were solubilized by adding 0.75 ml of 0.25 N NaOH in 0.1% sodium dodecyl sulfate (SDS). Aliquots of 0.5 ml were then neutralized and counted in scintillation fluid using a beta counter. In parallel experiments, cell proliferation was determined also by direct cell counting, as described previously.33 Then, we wondered whether DNA synthesis in response to different glucose concentrations might be differentially affected at different stages of cell confluency. Therefore, in a separate set of experiments, cells were seeded at a density of 5 x 103 or 20 x 103 cells/well, cultured in RPMI 1640 plus 10% fetal bovine serum for 5 days, made quiescent by serum deprivation for 48 hours, and finally exposed to 10 mmol/L or 30 mmol/L glucose, without serum and insulin, for 12 to 48 hours. Then, the cells were pulsed with [methyl-3H]thymidine for 4 hours and subsequently treated as described above.
RNA Isolation and Northern Blot Analysis For each experiment, 2 x 106 cells were plated and cultured as detailed above. After reaching confluency, HMCs were rested for 48 hours in serum-free medium and then incubated for 0 to 48 hours in RPMI 1640 containing 10 mmol/L or 30 mmol/L glucose without serum and insulin. In some experiments, cells were exposed for 24 hours to 30 mmol/L glucose with or without the addition of 50 ,ug/ml rabbit anti-human PDGF BB neutralizing antibody (Genzyme) or 100 jumol/L suramin. Control experiments were performed in which HMCs were treated with rabbit nonimmune IgG. At the end of incubation, cells were lysed with 4 mol/L guanidinium isothiocyanate containing 25 mmol/L sodium citrate, pH 7.0, 0.5% Sarcosyl, and 0.1 mmol/L 2-13-mercaptoethanol. Total RNA was isolated by the single-step method, using phenol and chloroform/isoamyl alcohol.34 Electrophoresis of 20 ,tg of total RNA was carried out in 1% agarose gel with 2.2 mol/L formal-
dehyde. The RNA was then transferred overnight to a nylon membrane (Schleicher & Schuell, Dassel, Germany). The cDNA probes used were a 2.14-kb fragment encoding the human TGF-,B1, isolated from pBR 327 plasmid with EcoRI, and a 751-bp fragment of the human PDGF-pR cDNA, isolated from pGEM-1 plasmid with Pstl. The cDNAs were labeled with [32P]dCTP (3000 Ci/mmol, Amersham) using random primer extension and added at 1 x 106 cpm/ml. Prehybridization and hybridization were performed for 18 hours at 420C in a buffer containing 50% formamide, 5X standard saline citrate (SSC), 5X Denhardt's solution, 0.1% SDS, and 100 ,ug/ml denatured salmon sperm DNA. The blots were then washed once in 2X SSC, 0.1% SDS at 220C for 5 minutes and once in the same buffer at 550C for 30 minutes. Finally, the membranes were washed in 1X SSC, 0. 1% SDS at 550C for an additional 30 minutes. After drying, membranes were exposed to a Kodak X-Omat film with intensifying screens at -700C. Membranes were subsequently stripped and rehybridized with a 32P-labeled glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probe, added at 2 x 105 cpm/ml, to account for small differences in RNA loading and transfer.
PDGF Receptor-Binding Studies HMCs were plated in 24-well dishes at a density of 5 x 104, grown to confluency, and made quiescent by incubation in serum-free RPMI 1640 for 24 hours. Then, cells were exposed to 10 mmol/L or 30 mmol/L glucose for 24 hours at 370C. After rinsing with 1 ml of binding buffer (RPMI 1640 plus 25 mmol/L Hepes, pH 7.4, and 2 mg/ml bovine serum albumin), cells were incubated with 5 ng/ml 1251-labeled PDGF BB (sp. act., 1000 Ci/mmol; Amity, Milan, Italy) for 2 hours at 40C, with constant gentle rotatory agitation. At the end of the incubation period, cells were washed three times with ice-cold PBS containing 1 mmol/L CaCI2 and 2 mg/ml bovine serum albumin and then solubilized by adding 1.0 ml of 20 mmol/L Hepes, pH 7.4, 1% Triton X-100, 10% (v/v) glycerol, and 0.1 mg/ml bovine serum albumin. Cell-bound radioactivity was counted in a gamma counter. Nonspecific binding was determined in the presence of a 100-fold excess of purified PDGF BB, and it never exceeded 20% of total radioactivity bound. Specific binding was calculated by subtracting nonspecific binding from the total counts bound per well.
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Reverse Transcription (RT) and Polymerase Chain Reaction (PCR) HMCs express extremely low levels of PDGF B-chain-specific transcript, which does not allow conventional analysis by Northern blot. Therefore, in preliminary experiments, we tried to analyze the target gene expression using a highly sensitive and specific RNAse protection assay performed exactly as described.35 Unfortunately, also this technical approach failed to identify measurable amounts of PDGF B-chain mRNA in more than 100 ,tg of total RNA extracted from unstimulated (ie, without serum or other mitogens) HMCs. Thus, we resolved to address this issue by adopting semiquantitative RTPCR, which definitely allows the comparison of the relative amounts of target gene transcripts in the different experimental conditions (ie, 10 and 30 mmol/L glucose) selected. A 1-,ug amount of total RNA from cultured HMCs was used in a RT reaction, and 20 ,il of the reaction mixture containing 1 ,tg of total RNA, PCR buffer (10 mmol/L Tris/HCI, pH 8.3, 50 mmol/L KCI), 5 mmol/L MgCO2, 1 mmol/L dNTPs, 20 U of RNAsin, 2.5 mmol/L of oligo (dT), and 100 U of Moloney murine leukemia virus reverse transcriptase was incubated at 42°C for 30 minutes and then heated to 950C for 5 minutes to inactivate the enzyme activity and to denature RNAcDNA hybrids. All samples were reverse transcribed in the same set of experiments, and the efficiency of the reaction was checked by GAPDH amplification. PCR was performed with two separate sets of oligonucleotide primers, specific for human PDGF B-chain and GAPDH, respectively: PDGF B-chain, 5'-GAA GGA GCC TGG GTT CCC TG-3' upstream and 5'-TTT CTC ACC TGG ACA GGT CG-3' downstream; and GAPDH, 5'-TGG TAT CGT GGA AGG ACT CAT GAC-3' upstream and 5'-ATG CCA GTG AGC TTC CCG TTC AGC-3' downstream. PDGF B-chain and GAPDH cDNA amplification were run simultaneously in the same set of experiments. The reaction was performed at a final concentration of 1X PCR buffer, 2 mmol/L MgCl2, 200 ,umol/L dNTPs, 0.15 ,umol/L PDGF primers or 0.25 ,tmol/L GAPDH primers, and 1.25 U of AmpliTaq DNA polymerase (Perkin Elmer Cetus, Norwalk, CT) in a total volume of 50 .lI. The amplification profile involved denaturation at 950C for 30 seconds, primer annealing at 550C for 1 minute, and extension at 720C for 1 minute. In preliminary experiments, aliquots (10 ,ul) were taken at five-cycle intervals and then electrophoresed in 1.5% agarose gels in Tris borate/EDTA buffer to establish the linear range of the reaction, thus allowing us to choose the optimal
number of amplification cycles for each of the mRNA species studied (35 cycles for PDGF cDNA and 25 cycles for GAPDH cDNA). The expected size of the amplified fragments was 226 and 450 bp for PDGF B-chain and GAPDH, respectively.
Southern Blot Analysis To confirm the specificity of PCR products, 1 ,ul of the amplified cDNA was electrophoresed on 1.5% agarose gel, blotted onto a nylon membrane (Schleicher & Schuell), and cross-linked by exposure to ultraviolet light. After prehybridization, the filter was hybridized with fluorescein-labeled cDNA probes specific for human PDGF B-chain or GAPDH (enhanced chemiluminescence random prime labeling system, Amersham). Hybridization was performed at 600C in 5X SSC, 0.1% SDS, 5% dextran sulfate, and 100 jtg/ml denatured salmon sperm DNA. Thereafter, the filter was washed once in 1X SSC, 0.1% SDS and once in 0.5X SSC, 0.1% SDS at 600C for 15 minutes each. After the stringency washes, the filter was blocked and incubated with horseradish-peroxidase-conjugated anti-fluorescein antibody. The blot was then covered with the detection buffer, containing luminol, and exposed for 3 minutes to Kodak X-Omat x-ray film. The bands obtained were quantified by densitometric analysis. Results were expressed as PDGF B-chain to GAPDH ratios, normalized to the first time point of each experiment.
Statistical Analysis Data are presented as mean ± SD. Data were compared using a two-tailed unpaired t-test. A P value
