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Kidney International, Vol. 54 (1998), pp. 1872–1878

HORMONES - CYTOKINES - SIGNALING

Sequential effects of high glucose on mesangial cell transforming growth factor-␤1 and fibronectin synthesis

JONG HOON OH,HUNJOO HA,MI RA YU, and HI BAHL LEE

Hyonam Kidney Laboratory, Soon Chun Hyang University and Department of Pharmacology, Yonsei University College of Medicine, Seoul, Korea

Sequential effects of high glucose on mesangial cell transforming to their increased synthesis, (ii) suppressing the production ␤ growth factor- 1 and fibronectin synthesis. of proteases that normally degrade the matrix and increas- Background. Transforming growth factor (TGF)-␤ is recognized as the final common mediator of the principal lesions of diabetic ing synthesis of the inhibitors of the same proteases, (iii) nephropathy such as renal hypertrophy and mesangial expansion. modulating the expression of integrins on the cell’s surface To gain better understanding of the temporal relationships be- in a manner that facilitates attachment to the newly syn- tween high glucose (HG) and mesangial cell (MC) TGF-␤1 thesized matrix, and (iv) autoinducing its own production, ␤ synthesis and between TGF- 1 and extracellular matrix (ECM) which greatly amplifies its action [3–6]. Increased expres- synthesis, the present study examined early and sequential effects ␤ of HG on TGF-␤1 and fibronectin (FN) mRNA expression and sion of TGF- and ECM mRNAs and proteins in the protein synthesis. kidneys of diabetic animals [7–15] and diabetic patients [9, Methods. Confluent primary rat MC was stimulated with 5.6 16, 17] implicates TGF-␤ in the pathogenesis of diabetic (control) or 30 (high) mM glucose after synchronizing the growth nephropathy. by incubation with serum-free media for 48 hours. Results. Mesangial cell TGF-␤1 mRNA expression increased Mesangial cells cultured under high glucose exhibit in- significantly in six hours and continued to increase until 48 hours creased expression of ECM mRNAs and proteins [18, 19] in response to HG. The level of TGF-␤1 mRNA was 1.5-fold and is an accepted in vitro model of diabetic nephropathy. higher than that of control glucose at six hours and 1.8-fold at 48 Expression of TGF-␤ mRNA and bioactivity are also ␤ hours. TGF- activity in heat-activated conditioned media under increased in this in vitro model [20–22]. HG increased 1.5- and 1.6-fold at 24 and 48 hours, respectively, compared to control glucose. FN mRNA increased significantly at Recent studies have demonstrated that neutralizing an- 24 and 48 hours and 1.4-fold that of control glucose at both time tibody against TGF-␤ significantly reduced high glucose- points. FN protein also increased 1.5-fold that of control glucose induced collagen production in mesangial cells [21] and ␤ at 48 hours. Anti-TGF- antibody completely abolished HG- fibronectin production in glomerular epithelial cells [23]. induced FN synthesis. ␤ Conclusions. The present findings demonstrate that HG stimu- TGF- 1 antisense oligonucleotides [24] attenuated porcine lates TGF-␤1 very early and prior to FN production and that mesangial fibronectin and heparan sulfate proteoglycan HG-induced FN production is mediated by TGF-␤. This finding is production stimulated by high glucose. Anti-TGF-␤ anti- consistent with the view that TGF-␤ mediates increased ECM body also attenuated renal hypertrophy and ECM gene accumulation by MC under high glucose conditions. expression in diabetic mice [25]. All these studies suggest that TGF-␤ is the final common mediator of the principal lesions of diabetic nephropathy [6]. Excessive deposition of extracellular matrix (ECM) and Consistent with this hypothesis, increased TGF-␤ expres- subsequent mesangial expansion is the principal structural sion was observed in renal cortex very early after the onset lesion of diabetic kidney [1, 2]. Transforming growth factor of diabetes and before manifestation of ECM expansion. (TGF)-␤ stimulates the deposition of ECM through (i) Streptozotocin-induced diabetic rats showed an increased directly up-regulating the genes of matrix proteins leading TGF-␤ expression at 24 hours after the induction of diabetes as measured by polymerase chain reaction (PCR) analysis [11]. A recent study utilizing in situ hybridization Key words: diabetic nephropathy, TGF-␤1, anti-TGF-␤ antibody, renal demonstrated increased glomerular TGF-␤1 mRNA ex- hypertrophy, extracellular matrix, protein synthesis. pression at three days after injection of streptozotocin [15]. Spontaneous diabetic BB rat and NOD mouse [12] and Received for publication January 2, 1998 and in revised form June 23, 1998 streptozotocin-induced diabetic mice [25] also exhibited Accepted for publication June 25, 1998 increased TGF-␤1 mRNA and protein levels in the renal © 1998 by the International Society of Nephrology cortex a few days after the appearance of glycosuria.

1872 Oh et al: High glucose, TGF-␤1, and fibronectin 1873

However, the temporal relations between high glucose micrograms of total RNA were electrophoresed through a and mesangial cell TGF-␤1 mRNA expression and protein 1.2% agarose gel with 2.2 M formaldehyde. The RNA from synthesis and between TGF-␤1 and fibronectin mRNA the gel was transferred onto nylon membranes using a expression and protein synthesis have not been established. capillary transfer and covalently cross-linked to the mem- The present study examined early and sequential effects of brane with UV light using a gene-linker (Bio-Rad, Rich- high glucose concentration on mesangial cell TGF-␤1 and mond, CA, USA). fibronectin mRNA expression and protein synthesis. In Prehybridization was performed for five hours at 42°C addition, the effect of neutralizing antibody to TGF-␤ on using a commercial prehybridization buffer (GIBCO BRL, fibronectin synthesis was determined to examine the role of Gaitherburg, MD, USA). Hybridization was conducted for TGF-␤ in high glucose-induced fibronectin synthesis by 20 hours at 42°C using excised cDNA inserts as probes after mesangial cells. [32P]dCTP-labeling by a random primer extension method (Pharmacia Biotech, Uppsala, Sweden). Rat cDNA probes METHODS for TGF-␤1 and fibronectin were purchased from the Mesangial cell culture American Type Culture Collection (ATCC, Rockville, MD, USA). Human glyceraldehyde-3-phosphate dehydrogenase Mesangial cells were obtained by culturing glomeruli (GAPDH) cDNA was constructed based on the previously isolated from kidneys of 100 to 150 g male Sprague-Dawley published sequence using the PCR [30]. The membranes rats by conventional sieving methods as previously de- were washed three times for 30 minutes: first in 2ϫ sodium scribed [26, 27]. Mesangial cells were identified by phase chloride and sodium citrate (SSC; 1 ϫ SSC is 0.15 M NaCl contrast microscopy according to the morphological crite- and 0.015 M sodium citrate, pH 7.0) with 0.1% sodium ria and by immunofluorescence technique. Cells were cul- dodecyl sulfate (SDS) at room temperature; the second in tured in Dulbecco’s modified Eagle’s medium (DMEM) 0.2 ϫ SSC with 0.1% SDS at room temperature; and the containing 20% fetal bovine serum (FBS), 100 U/ml peni- third in 0.2 ϫ SSC with 0.1% SDS at 55°C. Autoradiogra- cillin, 100 ␮g/ml streptomycin, 0.5 ␮g/ml fungizone, 44 mM phy was performed by exposing the blots to Kodak X-Omat NaHCO , and 14 mM N-2-hydroxy-ethylpiperazine-NЈ-2- 3 K XK-1 X-ray film with intensifying screens at Ϫ70°C for ethane sulfonic acid (HEPES). Near confluent mesangial one to five days. The blots were then rehybridized with a cells grown in 100 mm culture dish were incubated with 32P-labeled human GAPDH cDNA probe as an internal serum-free media for 48 hours to arrest and synchronize control to assess RNA quantity and integrity. Quantitation the cell growth. After this time period, the media were of mRNA signals was performed by densitometry using changed to fresh serum-free DMEM containing 5.6 or 30 MCID (Imaging Research Inc., St. Catharines, Ontario, mM glucose for 3 to 48 hours. Cells were also incubated Canada) and normalized with GAPDH mRNA signals. with 5.6 and 30 mM glucose media in the presence of 25 ␮g/ml neutralizing mouse anti-bovine TGF-␤ antibody (Ig ␤ ␤ ␤ G fraction, neutralizes TGF- 1, - 2, and - 3; Genzyme Bioassay for TGF-␤ activity Corporation, Cambridge, MA, USA). After additional in- cubation, the media were collected, centrifuged to remove The activity of TGF-␤ present in the conditioned media ␤ cell debris, and used for bioassay of TGF- 1 and immuno- was measured using the mink lung epithelial cell (MLEC) blot analysis of fibronectin. After removing the media, the growth inhibition assay, where human recombinant mesangial cells were washed with phosphate buffered sa- TGF-␤1 (Genzyme; 0.64 to 10,000 pg/ml) was used to line (PBS), then lysed using 1 ml of a mixture consisting of generate a standard curve [31]. Preliminary study revealed 4 M guanidium thiocyanate, 25 mM sodium citrate pH 7.0, undetectable TGF-␤ activity in the conditioned media 0.5% sarcosyl, and 0.1 M 2-mercaptoethanol for RNA under our experimental condition. The activation of latent isolation. All experiments were performed using cells be- TGF-␤ was performed by heating conditioned media at th th tween the 6 and 8 passages. 80°C for 15 minutes. In brief, CCL-64 cells (ATCC) were grown in 96-well culture plates with RPMI containing 5% Northern blot analysis FBS and conditioned media for 96 hours. MTT (3-[4,5- Standard Northern blot was performed as previously dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; Thia- described [28] after isolation of total RNA using the zolyl blue) was then added and cells were incubated for method of Chomczynski and Sacchi [29]. In brief, cells were another eight hours. Elution buffer was added and cells were homogenized with 4 M guanidium thiocyanate. Total pro- incubated for additional 48 hours and optical density was tein and DNA were extracted with acid phenol, and RNA measured at 562 nm. Under our experimental condition, precipitated with isopropanol. After washing with ethanol, half-maximally effective concentrations of TGF-␤1 inhibiting the amount of RNA was quantitated by measuring the MLEC growth (EC50) was around 200 ng/liter. Uncondi- absorbance at 260 nm using a spectrophotometer (Ultra- tioned media containing 30 mM glucose did not exhibit spec 3000; Pharmacia Biothech, England, UK). Twenty discernible effect on MLEC growth inhibition assay. 1874 Oh et al: High glucose, TGF-␤1, and fibronectin

Fig. 1. Representative Northern blot analysis of mesangial cell expression of TGF-␤1 and fibronectin mRNAs. After incubation of quiescent mesangial cells with control (C; 5.6 mM) or high (H; 30 mM) glucose for the given period, total RNA was isolated, electrophoresed and transferred onto nylon membranes. Northern blots were hybridized with [32P]- labeled cDNA probe for TGF-␤1, fibronectin, and GAPDH. Autoradiographs were developed by exposing blots to X-ray films. Mesangial cell TGF-␤1 mRNA expression increased significantly at six hours and continued to increase until 48 hours in response to high glucose. Compared to TGF-␤1, the increase in fibronectin mRNA in response to high glucose was delayed, significant only at 24 and 48 hours.

␤ Immunoblot analysis of fibronectin protein Table 1. Effects of high glucose on mesangial cell TGF- 1 and fibronectin mRNAs Immunoblot analysis was performed to determine the production and secretion of fibronectin into the mesangial Time after stimulaiton hours cell conditioned media [32]. After measuring the concen- 036 122448 trations of protein by the Bradford method [33] using the TGF-␤1 1.0 1.0 Ϯ 0.1 1.5 Ϯ 0.3a 1.5 Ϯ 0.1a 1.7 Ϯ 0.2a 1.8 Ϯ 0.4a Bio-Rad assay, aliquots of conditioned media were mixed Fibronectin 1.0 1.0 Ϯ 0.1 1.2 Ϯ 0.1 1.2 Ϯ 0.2 1.4 Ϯ 0.2a 1.4 Ϯ 0.1a with sample buffer containing SDS and ␤-mercaptoethanol Values are the intensity of densitometric readings of TGF-␤1 and and heated at 95°C for 15 minutes. Samples were applied to fibronectin mRNAs corrected for GAPDH and relative to that of control glucose at the given time point. Values are expressed as mean Ϯ SE of four 5% polyacrylamide gel and electrophoresed. A prestained experiments. SDS-PAGE standard (broad range, Bio-Rad) was also a P Ͻ 0.05 compared to control glucose electrophoresed as a molecular weight marker. After elec- trophoresis, the proteins were transferred onto a nitrocel- lulose membrane using a transblot chamber with Tris (Fig. 1). The steady state levels of TGF-␤1 and GAPDH buffer. Western blots were incubated with rabbit anti- mRNAs remained constant for an additional 48 hours mouse fibronectin (GIBCO BRL) for two hours at room incubation in serum-free control glucose media. Fibronec- temperature. The membranes were washed with PBS- tin mRNA expression tended to decrease, although the Tween-20 for one hour and incubated with peroxidase- difference between 0 and 48 hours did not reach statistical conjugated secondary antibody, rabbit anti-IgG (Sigma significance (P Ͼ 0.05). The mRNA level in response to Chemical Company, St. Louis, MO, USA) for two hours at high glucose was compared with that in response to control room temperature. After washing, the membranes were glucose at a given time. As summarized in Table 1, the time Ј incubated with a color reagent (50 mg 3,3 -diaminobenzi- sequence of high glucose-induced TGF-␤1 mRNA was dine, 0.1 ml H2O2 in 100 ml PBS) for 15 minutes or ECL different from that of fibronectin. Mesangial cell TGF-␤1 detection reagents (Amersham Life Science, Little Chal- mRNA expression increased significantly at six hours (P Ͻ font, England, UK) for precisely one minute followed by 0.05) and continued to increase until up to 48 hours in autoradiography. Positive immunoreactive bands were response to high glucose. The level of TGF-␤1 mRNA was quantitated densitometrically and compared to controls. 1.5ϫ that of control glucose at six hours and 1.8ϫ at 48 Analysis of data hours. Compared to TGF-␤1, the increase in fibronectin All results are expressed as mean Ϯ standard error (SE). mRNA expression in response to high glucose was some- ϫ Analysis of variance was used to assess the differences what delayed, significant only at 24 and 48 hours and 1.4 between multiple groups. If the F statistic was significant, that of control glucose at both time points. the mean values obtained from each group were then Effects of high glucose on mesangial cell TGF-␤ compared by Fisher’s least significant difference method. A bioactivity P value Ͻ 0.05 was used as the criterion for a statistically significant difference. MLEC growth inhibition assay of mesangial cell condi- tioned media was performed to determine the translation RESULTS of TGF-␤1 mRNA into protein synthesis and secretion. Time sequence of high glucose-induced TGF-␤1 and Heat-activated conditioned media showed a time-depen- fibronectin mRNAs in mesangial cells dent increase in TGF-␤1 activity in both control and high Quiescent mesangial cells grown in serum-free media for glucose (P Ͻ 0.05; Fig. 2) in the face of little change in total two days expressed both TGF-␤1 and fibronectin mRNAs protein concentration (Table 2). High glucose significantly Oh et al: High glucose, TGF-␤1, and fibronectin 1875

tured under high glucose conditions. Figure 4 demonstrates that anti-TGF-␤ antibody at 25 ␮g/ml completely abolished high glucose-induced fibronectin synthesis without a signif- icant effect in basal production of fibronectin in control glucose.

DISCUSSION Increased glomerular TGF-␤ expression in experimental diabetic animals [7–15] and diabetic patients [9, 16, 17], attenuation of renal hypertrophy and enhanced ECM gene expression in diabetic animals with anti-TGF-␤ antibody [25], and inhibition of high glucose-induced mesangial cell fibronectin and heparan sulfate proteoglycan synthesis by TGF-␤ antisense oligonucleotides [24] suggest that TGF-␤ is the final common mediator of diabetic nephropathy. High glucose-induced collagen production by mesangial Fig. 2. Effects of high glucose on mesangial cell TGF-␤1 bioactivity. cells [21] and fibronectin production by glomerular epithe- After incubation of quiescent mesangial cells with control (5.6 mM; Ⅺ)or lial cells [23] have been effectively inhibited by anti-TGF-␤ high (30 mM; ) glucose for the given period, aliquots of mesangial cell antibody. However, the role of TGF-␤1 in high glucose- conditioned media were analyzed by a mink lung epithelial cell (MLEC) growth inhibition assay. Values are expressed as mean Ϯ SE from three induced mesangial fibronectin production and the temporal separate experiments. *P Ͻ 0.05 compared to control glucose. Heat- relations among high glucose, TGF-␤1, and fibronectin in ␤ activated conditioned media showed time-dependent increase in TGF- 1 mesangial cells have not been studied. The present study protein in both control and high glucose conditions. High glucose signif- icantly increased TGF-␤1 secretion at 24 and 48 hours when compared to examined effects of high glucose concentration on mesan- control glucose. gial cell TGF-␤1 and fibronectin mRNA expression and protein synthesis from 3 to 48 hours after stimulation. Mesangial cells cultured under high glucose is an accepted increased TGF-␤1 secretion by mesangial cells when com- in vitro model of diabetic nephropathy and has been used to pared to control glucose at 24 and 48 hours, with the investigate pathogenetic mechanisms of diabetic nephrop- respective total TGF-␤1 at 0.77 Ϯ 0.22 and 0.80 Ϯ 0.10 athy at molecular and cellular levels. ng/ml in control glucose and 1.20 Ϯ 0.22 and 1.34 Ϯ 0.06 In agreement with previous studies [34, 35], there was ng/ml in high glucose (P Ͻ 0.05). Conditioned media constitutive expression of TGF-␤1 mRNA in quiescent without heat activation exhibited undetectable inhibition of mesangial cells. In response to high glucose, TGF-␤1 MLEC growth, suggesting that rat mesangial cells secreted mRNA increased 1.5-fold above control at six hours and TGF-␤1 protein mostly in inactive or latent forms. 1.8-fold at 48 hours. The extent of high glucose-induced TGF-␤1 mRNA expression was comparable to the findings ␤ Effects of high glucose and anti-TGF- antibody on of Wolf et al [20], who reported a 50% increase at 48 hours fibronectin protein production by mesangial cells after stimulation, and Mogyorosi et al [22], who reported a To confirm the existence of fibronectin protein in condi- 100% increase at 72 hours after stimulation. In contrast, tioned media, we performed immunoblot analysis. Cumu- when synchronized mesangial cells were stimulated with lative production of fibronectin by MC cultured in both serum or phorbol ester, TGF-␤1 mRNA began to increase control and high glucose increased progressively with time as early as one hour and reached the maximum at nine as summarized in Figure 3. Fibronectin was always detect- hours to several-fold that of pre-stimulation values [34]. able at six hours after the addition of serum-free control Mesangial cells used in the present study also exhibited a glucose media and was assigned a relative value of 1. fivefold increase in TGF-␤1 mRNA in response to phorbol Relative increase in fibronectin at 6, 12, 24 and 48 hours ester (data not shown), comparable to the findings of was 1, 2.3 Ϯ 0.9, 3.1 Ϯ 0.2 and 4.7 Ϯ 0.3 under control Kaname et al [34]. Angiotensin II caused a threefold glucose, and 1.4 Ϯ 0.4, 2.6 Ϯ 0.8, 4.5 Ϯ 0.9 and 7.2 Ϯ 1.4 increase in mesangial TGF-␤1 mRNA expression at four under high glucose, respectively. Thus, fibronectin protein hours, a sixfold at eight hours, and a threefold increase at production in high glucose at 6, 12, 24, and 48 hours was 48 hours compared to control values [35]. 1.40ϫ, 1.13ϫ, 1.45ϫ, and 1.53ϫ that of control glucose, Our study demonstrates that high glucose induces respectively. The difference between control and high TGF-␤1 mRNA very early and significantly at six hours. glucose was significant only at 48 hours (P Ͻ 0.05). Wolf et al reported that high glucose increased TGF-␤1 Studies with neutralizing antibody to TGF-␤ were per- mRNA expression at 48 hours but not at 24 hours [20]. The formed in order to examine the involvement of TGF-␤ on differences in mesangial cells used in the two studies, increased fibronectin production by mesangial cells cul- primary rat mesangial cells in the present study versus 1876 Oh et al: High glucose, TGF-␤1, and fibronectin

Table 2. Concentration of total protein in mesangial cell conditioned media

Time after stimulation hours 0 3 6 12 24 48 Control glucose ␮g/ml 36.0 Ϯ 6.6 31.1 Ϯ 7.9 30.5 Ϯ 7.5 35.1 Ϯ 7.4 33.9 Ϯ 7.9 32.5 Ϯ 8.5 High glucose ␮g/ml 26.0 Ϯ 5.0 27.6 Ϯ 4.2 32.2 Ϯ 4.2 30.9 Ϯ 5.4 36.0 Ϯ 2.1 36.1 Ϯ 3.6

Values are expressed as mean Ϯ SE of three separate experiments.

Fig. 3. Immunoblot analysis of fibronectin production by mesangial cell. After incubation of quiescent mesangial cells with control (C; 5.6 mM; Ⅺ) or high (H; 30 mM ) glucose for the given period, aliquots of mesangial cell conditioned media were electrophoresed under reducing conditions and transferred onto nitrocellulose membranes. Western blots were performed as described in the text. Values are expressed as mean Ϯ SE of three experiments. *P Ͻ 0.05 compared to control glucose. The inset Western blot is a representative of three individual experiments. Shown here is cumulative amount of fibronectin produced by mesangial cells that progressively increased with time in both control and high glucose. High glucose induced significantly more fibronectin synthesis than control glucose at 48 hours.

transformed mouse mesangial cell line in the previous the same cell. The rates of TGF-␤1 and fibronectin mRNA study, and the presence versus absence of serum depriva- degradation following actinomycin D (5 ␮g/ml) were simi- tion period to synchronize the cell cycle before stimulation lar between mesangial cells cultured under control glucose may explain the apparent difference. and under high glucose (data not shown), suggesting that Compared to TGF-␤1, the increase in fibronectin mRNA high glucose did not alter the stabilities of TGF-␤1 and expression in response to high glucose was delayed, that is, fibronectin mRNAs in rat mesangial cells. it was significant only at 24 and 48 hours, which is consistent Elevation of TGF-␤1 mRNA at six hours in response to with the view that high glucose-induced fibronectin synthe- high glucose was followed by increased protein synthesis at sis is mediated by TGF-␤1. Ayo et al reported that high 24 and 48 hours. Elevated fibronectin mRNA at 24 hours glucose increased mRNAs of ECM including fibronectin at was followed by a significant increase in fibronectin protein three to seven days but not at one and two days [19]. In at 48 hours. In addition, the amount of TGF-␤ protein their study, cells between the 20th and 40th passages were secreted by mesangial cells grown under control and high used, and this may give a different time course in high glucose conditions during the first 24 hours were compara- glucose-induced fibronectin mRNA expression [36]. The ble to the reported concentrations of TGF-␤1 stimulating significance of the present study is that high glucose synthesis of fibronectin [24] and collagen [21]. Further, induced TGF-␤1 mRNA expression prior to fibronectin in neutralizing antibody to TGF-␤ completely abolished the Oh et al: High glucose, TGF-␤1, and fibronectin 1877

Fig. 4. Effects of anti-TGF-␤ antibody on high glucose-induced mesangial cell fibronectin production. Quiescent mesangial cells were cultured for 48 hours in serum-free medium containing 5.6 or 30 mM glucose in the presence or absence of 25 ␮g/ml anti-TGF-␤ antibody. After the incubation period, aliquots of mesangial cell conditioned media were electrophoresed under reducing conditions and transferred onto nitrocellulose membranes. Western blots using ECL detection system were performed as described in the text. Values are expressed as means Ϯ SE of three experiments. *P Ͻ 0.05 compared to 5.6 mM glucose in the absence of anti-TGF-␤ antibody. #P Ͻ 0.05 compared to 30 mM glucose in the absence of anti-TGF-␤ antibody. The inset Western blot is a representative of three individual experiments. Anti-TGF-␤ antibody completely abolished high glucose-induced fibronectin production without significant change in basal production of fibronectin in control glucose. high glucose-induced mesangial cell fibronectin production tetraacetic acid; FBS, fetal bovine serum; FN, fibronectin; GAPDH, without a significant change in basal production of fi- glyceraldehyde-3-phosphate dehydrogenase; HEPES, N-2-hydroxy-eth- ylpiperazine-NЈ-2-ethane sulfonic acid; HG, high glucose; MC, mesangial bronectin in control glucose, indicating that high glucose- cell; MLEC, mink lung epithelial cell; MTT, 3-[4, 5-dimethyl-2-yl]-2,5- induced but not basal production of fibronectin by mesan- diphenyl tetrazolium bromide; PBS, phosphate buffered saline; PCR, gial cells is due to an increase in TGF-␤ bioactivity. The polymerase chain reaction; SDS, sodium dodecyl sulfate; SSC, sodium chloride and sodium citrate; TGF, transforming growth factor. present data are thus consistent with the previous studies [21, 23–25] demonstrating that increased ECM synthesis associated with diabetes is mediated by TGF-␤. REFERENCES In conclusion, our data demonstrate that high glucose ␤ 1. MAUER SM, STEFFES MW, ELLIS EM, SURTHERLAND DM, BROWN induces TGF- 1 mRNA and protein synthesis very early DM, GRETH FC: Structural-functional relationships in diabetic ne- and prior to fibronectin production by mesangial cell. phropathy. J Clin Invest 74:1143–1155, 1984 ␤ 2. STEFFES MW, OSTERBY R, CHAVERS B, MAUER SM: Mesangial TGF- 1 mRNA is induced as early as six hours after expansion as a central mechanism for loss of kidney function in stimulation with high glucose, while fibronectin mRNA is diabetic patients. Diabetes 38:1077–1081, 1989 induced at 24 hours. TGF-␤1 protein synthesis was signif- 3. BORDER WA, RUOSLAHTI E: Transforming growth factor-␤ in tissue icantly increased at 24 hours and fibronectin protein at 48 repair. J Clin Invest 90:1–7, 1992 4. BORDER WA, NOBLE NA: Transforming growth factor-␤ in tissue hours. These findings along with complete inhibition by fibrosis. N Engl J Med 331:1286–1292, 1994 ␤ 5. BORDER WA, YAMAMOTO T, NOBLE NA: Transforming growth fac- anti-TGF- antibody of high glucose-induced fibronectin ␤ production support the view that high glucose-induced tor- in diabetic nephropathy. Diabetes/Metabol Rev 12:309–339, 1996 6. ZIYADEH FN, HAN DC: Involvement of transforming growth factor-␤ fibronectin synthesis is mediated by TGF-␤. and its receptors in the pathogenesis of diabetic nephropathy. Kidney Int 52(Suppl 60):S7–S11, 1997 ACKNOWLEDGMENTS 7. BOLLINENI JS, RADDI AS: Transforming growth factor-␤1 enhances glomerular collagen synthesis in diabetic rats. Diabetes 42:1673–1677, This work was supported in part by a grant from Korea Science and 1993 Engineering Foundation (KOSEF 94-0403-07-01-3).We are grateful to 8. NAKAMURA T, FUKUI M, EBIHARA I, OSADA S, NAGAOKA I, TOMINO Dr. Choon Sik Park and Miss Mi Ho Kim of Hyonam Kidney Laboratory Y, KOIDE H: mRNA expression of growth factors in glomeruli from for technical assistance with TGF-␤1 bioassay. diabetic rats. Diabetes 42:450–456, 1993 9. YAMAMOTO T, NAKAMURA T, NOBLE NA, RUOSLAHTI E, BORDER Reprint requests to Hi Bahl Lee, M.D., Hyonam Kidney Laboratory, Soon WA: Expression of transforming growth factor beta is elevated in Chun Hyang University, 657 Hannam Dong, Yongsan ku, Seoul 140-743, human and experimental diabetic nephropathy. Proc Natl Acad Sci Korea. USA 90:1814–1818, 1993 E-mail: [email protected] 10. PANKEWYCZ OG, GUAN J-X, BOLTON WK, GOMEZ A, BENEDICT JF: Renal TGF-␤ regulation in spontaneously diabetic NOD mice with APPENDIX correlation in mesangial cells. Kidney Int 46:748–758, 1994 11. SHANKLAND SJ, SCHOLEY JW: Expression of transforming growth Abbreviations used in this article are: DMEM, Dulbecco’s modified factor-␤1 during diabetic renal hypertrophy. Kidney Int 46:430–442, eagle’s medium; ECM, extracellular matrix; EDTA, ethylenediamine 1994 1878 Oh et al: High glucose, TGF-␤1, and fibronectin

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