Glycated albumin stimulates TGF-βbgr;1 production and protein kinase C activity in glomerular endothelial cells1

Kidney International, Vol. 59 (2001), pp. 673–681

Glycated albumin stimulates TGF-␤1 production and protein kinase C activity in glomerular endothelial cells1 SHELDON CHEN, MARGO P. COHEN, GREGORY T. LAUTENSLAGER, CLYDE W. SHEARMAN, and FUAD N. ZIYADEH Renal-Electrolyte and Hypertension Division and Penn Center for Molecular Studies of Kidney Diseases, University of Pennsylvania, and Institute of Metabolic Research and Exocell, University City Science Center, Philadelphia, Pennsylvania, USA

the proliferative capacity, which is likely mediated by PKC and partly by TGF-␤, glycated albumin impedes the ability of the glomerular capillary endothelium to act as a first line of defense against deleterious circulating factors in the diabetic state.

Glycated albumin stimulates TGF-␤1 production and protein kinase C activity in glomerular endothelial cells. Background. The activation of protein kinase C (PKC) and transforming growth factor-␤ (TGF-␤) in glomerular mesangial cells has been linked to mesangial matrix expansion in diabetic nephropathy. The role of these mediators in affecting the changes associated with diabetes in the biology of glomerular endothelial cells (GEnCs), which synthesize components of the glomerular basement membrane, is not known. We postulated that the PKC and TGF-␤ systems promote the increased endothelial cell synthesis of glomerular basement membrane that is evoked by Amadori-modified glycated albumin, which is present in elevated concentrations in diabetes. Methods. We examined the effects of PKC inhibition on collagen IV and TGF-␤1 production by mouse GEnCs incubated with glycated albumin and the influence of glycated albumin on PKC activity, TGF-␤1 production, and proliferation by these cells. Results. In physiologic (5.5 mmol/L) glucose concentrations, glycated albumin caused an increase in type IV collagen production that was totally prevented by a general PKC inhibitor GF 109203X (GFX), but only partly prevented by a neutralizing anti–TGF-␤ antibody. Glycated albumin increased the steadystate level of TGF-␤1 mRNA and stimulated the production of TGF-␤1 protein, which was also prevented by the PKC inhibitor GFX. Of note, glycated albumin significantly stimulated PKC activity, as measured by the phosphorylation of a PKC-specific substrate. Cell proliferation, measured by [3H]-thymidine incorporation and cell counting, was decreased in the presence of glycated albumin. This effect was completely prevented by GFX and partially reversed by anti–TGF-␤ antibody. Exogenous TGF-␤1 inhibited cell proliferation to a degree similar to that of glycated albumin. Conclusions. PKC signaling and consequent TGF-␤1 activation participate in the glycated albumin-induced stimulation of basement membrane collagen production by GEnC. By reducing

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The glomerular lesions in diabetic nephropathy are characterized by mesangial extracellular matrix (ECM) accumulation in association with loss of filtration area and thickening of the peripheral glomerular basement membrane [1–3]. Expansion of the mesangial matrix is a central early feature [4], and because mesangial cells can be readily established in culture, many studies have focused on mesangial cell proliferative and biosynthetic responses to media manipulations to investigate potential diabetes-related nephropathic factors [5–11]. For example, high glucose media concentrations have been shown to cause inhibition of growth, bioactivation of transforming growth factor-␤1 (TGF-␤1), and increased expression of ECM proteins in mesangial cells [5, 7, 9–11]. Albumin modified by Amadori glucose adducts, the principal circulating glycated protein, has analogous influences on mesangial cell biology and has been shown to decrease proliferative capacity and induce coordinated increases in the expression of TGF-␤1, the TGF-␤ type II receptor, type IV collagen, and fibronectin [12–16]. Less well studied, perhaps because they have been difficult to isolate and propagate in culture, are glomerular endothelial cells (GEnCs) [17–19]. Glucose and glycated albumin have been shown to adversely affect the biology of endothelial cells cultured from large blood vessels and umbilical veins [20–24]. In endothelial cells from large vessels, for example, glycated albumin stimulates nitric oxide synthase activity and gene expression [25]. However, endothelial cells from capillary beds such as the glomerular tuft may behave differently in response to pathobiologic factors [18, 26–28]. Relatively little is known about factors that modulate the biology of GEnCs, which produce a portion of the peripheral glomerular basement membrane underlying them [29], or

See Editorial by Striker, p. 799.

Key words: Amadori, nonenzymatic glycation, basement membrane, type IV collagen, cell proliferation, diabetic nephropathy. Received for publication January 20, 2000 and in revised form August 2, 2000 Accepted for publication September 5, 2000

 2001 by the International Society of Nephrology

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about influences in these cells that are relevant to the pathogenesis of diabetic nephropathy. Compared with mesangial cells, GEnCs express more ␣3(IV) collagen mRNA [30], and they respond to a high glucose media concentration with increased synthesis of type IV collagen [19]. We have shown that glycated albumin, in concentrations found in clinical specimens, stimulates the production of type IV collagen and fibronectin in a glomerular endothelial cell line, even in the absence of high ambient glucose [31]. However, the molecular mediators underlying the increased matrix production by glycated albumin in these cells have not been elucidated. Since glycated albumin in increased concentrations bathes the glomerular vascular bed in diabetes [16], the mechanisms by which the glycated protein modulates glomerular endothelial biology warrant investigation. Because the effects of glycated albumin on ECM production in GEnC resemble those observed in mesangial cells, we postulated that the cytokines and signaling factors mediating increased matrix synthesis might be common to both cell types. In mesangial cells, stimulation of type IV collagen, laminin, and fibronectin expression are associated with increased expression of the fibrogenic cytokine TGF-␤1 [11, 14] and activation of the signal transduction factor protein kinase C (PKC) [32–36]. Other evidence linking the TGF-␤ system and PKC activation to altered glomerular cell biology in diabetes includes studies in experimental animal models showing that renal TGF-␤1 expression and PKC activity are increased, that renal overexpression of matrix proteins can be lessened by treatment with anti–TGF-␤ antibody, and that glomerular hyperfiltration and albuminuria can be ameliorated by inhibiting the PKC-␤ isoform [34, 37–45]. These considerations prompted the present experiments, which attempt to determine whether glycated albumin modulates PKC activity in GenC, and whether activation of PKC and increases in TGF-␤1 are involved in the stimulated ECM expression that occurs when GEnCs are incubated with glycated albumin [31]. The compound GF 109203X (GFX) was used to inhibit the various PKC isoforms, and a pan-selective monoclonal antibody was used to neutralize TGF-␤. Since high ambient glucose concentrations have been reported to activate PKC and to augment the expression of TGF-␤1 and ECM proteins [11, 32, 35, 36, 46–48], all incubations were conducted in 5.5 to 10 mmol/L glucose media (except where specifically indicated) to ensure that observed responses did not accrue from an independent effect of elevated glucose. Parallel incubations with nonglycated albumin, in the same concentration as glycated albumin, served as controls. METHODS Cell culture All studies were performed on a murine glomerular endothelial cell line that is spontaneously immortalized

and fulfills criteria for endothelial cells as previously described [49]. The cells stain positive for factor VIII and CD31, express angiotensin-converting enzyme, and release endothelin-1 in response to angiotensin II [49]. We have also demonstrated that GEnCs express the novel ␣3 and ␣5 chains of collagen IV, which are components of the glomerular basement membrane (data not shown). Cells were maintained at 37⬚C in a humidified incubator with 5% CO2/95% air and were propagated in Dulbecco’s modified Eagle’s medium (DMEM; GIBCO BRL, Grand Island, NY, USA) containing 10% fetal calf serum (FCS), 100 U/mL penicillin, 100 ␮g/mL streptomycin, and 2 mmol/L glutamine. Cells were passaged every four to five days by light trypsinization. To initiate experiments under varying culture conditions, cells were seeded into 96-well (thymidine incorporation and cell counting) or 24-well (collagen IV, TGF-␤1, and PKC assays) plastic plates or into 100 mm Petri dishes (RNA analysis), grown for 24 to 48 hours in media containing 10 mmol/L glucose and 2% FCS, and then incubated for 24 to 72 hours in fresh media under the experimental conditions described later in this article. Experimental conditions The experimental culture conditions were introduced upon the addition of fresh media containing 5.5, 10 (thymidine incorporation and cell counting), or 25 (TGF-␤1 assay) mmol/L glucose and 2 or 10% (RNA analysis) FCS, without or with the described supplements in the indicated concentrations. Media supplements consisted of purified glycated or nonglycated albumin (500 ␮g/mL), prepared as described later in this article, the PKC inhibitor GFX (500 nmol/L; Calbiochem, La Jolla, CA, USA), TGF-␤1 (1 ng/mL; R&D Systems, Minneapolis, MN, USA), and a pan-selective neutralizing monoclonal anti– TGF-␤ antibody (30 ␮g/mL; gift of Dr. Brian M. Fendly of Genentech, South San Francisco, CA, USA) [37] or an irrelevant murine IgG (30 ␮g/mL). Albumin preparation Glycated albumin was prepared by separation of human serum albumin with chromatography on Affi-gel blue and DEAE-Sepharose, followed by incubation for five days at 25⬚C in buffered saline containing 28 mmol/L glucose. After dialysis to remove free glucose, the glycated species were separated from nonglycated albumin by affinity chromatography on phenylboronate agarose (PBA), which binds the Amadori glucose adducts. This procedure has been shown to yield glycated albumin containing 1 to 2 mol glucose/mol albumin, and glycated moieties are represented as deoxyfructosyllysine [13, 50]. The PBA pass-through, which was used as source material for nonglycated albumin, contained ⬍0.05 mol glucose/mol albumin. The purified glycated and nonglycated albumin preparations migrated on sodium dodecyl sul-

Chen et al: Glycated albumin and GEnC biology

fate-polyacrylamide gel electrophoresis (SDS-PAGE) as homogeneous bands of approximately 66 kD and had distinct mobilities on agarose gel electrophoresis, wherein the glycated protein exhibited greater electronegativity consequent to glycation of lysine amino groups [50]. The concentration of glycated albumin used in these experiments has been shown to modulate mesangial cell biology and is in the range of concentrations found in diabetic specimens [13, 14]. Cell proliferation The [3H]-thymidine incorporation assay was performed in 96-well microtiter plates in 200 ␮L total volume. At the end of a 48-hour incubation period, the cells were pulsed for four hours with [3H]-thymidine (1 ␮Ci/ well, 5 to 7 Ci/mmol; Amersham, Arlington Heights, IL, USA). After trypsinization, cells were collected onto glass microfiber filters (Whatman, Maidstone, UK) using a cell harvester, and radioactivity was assayed by placing the filters in a liquid scintillation fluid (ICN) and counting the [3H] decay in a liquid scintillation counter [10, 51]. Proliferation of cells grown in 96-well microtiter plates was also determined by direct cell counting. After 72 hours of growth, GEnCs were subjected to three minutes of trypsinization followed by resuspension in fresh media supplemented with 10% FCS to neutralize the trypsin. An inverted microscope was used to count the cells in a Bright Line Counting Chamber (Hausser Scientific, Horsham, PA, USA). PKC activity Protein kinase C activity was measured with the Pep Tag assay system (Promega, Madison, WI, USA), in which the change in charge of a fluorescent-tagged PKC-specific substrate PLSRTLSVAAK that occurs with phosphorylation is detected upon separation with agarose gel electrophoresis at neutral pH. At the end of the incubation, the cells were washed with Hank’s balanced salt solution containing 1 mmol/L phenylmethylsulfonyl fluoride (PMSF) and 1 ␮mol/L leupeptin, placed in 100 ␮L of the same buffer, and then freeze thawed for lysis. Aliquots were taken for the assay, which was performed according to the manufacturer’s instructions. Photographs of the gels were scanned into a densitometer program (Scion Image; National Institutes of Health, Bethesda, MD, USA) for quantitation. Each assay included a positive control for PKC activity wherein PKC supplied by the manufacturer was simultaneously subjected to the assay and electrophoretic procedure. A negative control was also run with each assay. Type IV collagen measurement Type IV collagen was measured by competitive enzyme-linked immunosorbent assay (ELISA) [12, 13] using type IV collagen from an Engelbreth-Holm-Swarm

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(EHS) tumor as the standard, rabbit anti-mouse type IV collagen as the primary antibody (both from Collaborative Research, Medford, MA, USA), and horseradish peroxidase-conjugated goat anti-rabbit IgG (Bio-Rad, Hercules, CA, USA) as secondary antibody. The assay was performed on media collected at the end of the experimental incubation period and on cell contents after lysis according to the previously mentioned procedure. In GEnCs, the cell-associated collagen IV fraction was substantial, and therefore, media and cell lysate measurements for each incubation were summed and normalized to total protein measured with the Bio-Rad protein assay. Pilot studies have shown that both fractions are stimulated proportionately by glycated albumin. TGF-␤1 measurement Transforming growth factor-␤1 levels in spent media collected at the end of the experimental period were measured by immunoassay (Genzyme, Cambridge, MA, USA). Samples were acidified to separate the latencyassociated peptide from the TGF-␤1 dimer, allowing measurement of total TGF-␤1 (active plus latent components) and then neutralized with NaOH [38, 52]. According to the vendor, the primary antibody issued in this assay has negligible cross-reactivity with TGF-␤2 and TGF-␤3. RNA hybridization analysis RNA was extracted from endothelial cells after culture under the described conditions and hybridized with [32P]-labeled cDNA probe as previously described [12, 14]. Briefly, cells were denatured in TRIzol reagent (GIBCO BRL), and RNA was purified according to the manufacturer’s instructions. Twenty micrograms of total RNA were electrophoresed on a 1.2% agarose gel, transferred onto nylon membranes, and ultraviolet wave cross-linked. Integrity and equal loading of samples were assessed by methylene blue staining of the transferred RNA. After hybridization with the TGF-␤1 cDNA probe, blots were successively washed in 2 ⫻ standard saline citrate (SSC) at room temperature, 2 ⫻ SSC/1% SDS at 62⬚C, and 0.1 ⫻ SSC/0.1% SDS at 62⬚C. The membranes were autoradiographed with intensifying screens at ⫺70⬚C for up to three days. Blots were then stripped for two hours at 62⬚C with 5 mmol/L Tris, 0.2 mmol/L ethylenediaminetetraacetic acid (EDTA; pH 8.0), and 5% sodium pyrophosphate and subsequently rehybridized with the mrpL32 cDNA probe to account for small variations in RNA loading and transfer. Exposed films were scanned into a densitometer program (Scion Image; National Institutes of Health) for quantitation of TGF-␤1 mRNA, which was later corrected for the relative amount of mrpL32 mRNA. Preparation of the cDNA probe encoding TGF-␤1 was as previously described [38].

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Fig. 1. Effect of glycated albumin and protein kinase C (PKC) inhibition on collagen IV production. Glomerular endothelial cells (GEnCs) were cultured for 48 hours in DMEM containing 5.5 mmol/L glucose/2% FCS and no supplement (media), nonglycated albumin (NA), or glycated albumin (GA) at 500 ␮g/mL, without (䊐) or with (䊏) 500 nmol/L of the PKC inhibitor GF 109203X (GFX). From each condition, media and cell lysates were collected for immunoassay of collagen IV as described in the text. Results represent the mean ⫾ SEM of four independent cultures and are expressed as micrograms of collagen IV normalized to milligrams of protein. *P ⬍ 0.05 compared with nonglycated albumin; #P ⬍ 0.05 compared with glycated albumin, no GFX.

RESULTS Collagen IV production Incubation with glycated albumin in 5.5 mmol/L glucose significantly stimulated collagen IV production, whereas incubation with nonglycated albumin had no effect relative to control (Fig. 1). This stimulation by glycated albumin was prevented in the presence of GFX, a general inhibitor of PKC isoforms (Fig. 1). A neutralizing anti-TGF-␤ antibody was used to determine whether activation of endogenous TGF-␤ mediated the stimulation of collagen IV production. In the presence of glycated albumin (500 ␮g/mL for 48 h), immunoreactive collagen IV levels were increased by 88 ⫾ 10% above control (N ⫽ 4, P ⬍ 0.05). Concurrent anti–TGF-␤ antibody treatment (30 ␮g/mL) of cells incubated in glycated albumin reduced the collagen IV protein level by 47 ⫾ 4% (N ⫽ 4, P ⬍ 0.05), suggesting that TGF-␤ was partly responsible for the glycated albumin-induced collagen IV production. It should be noted that supplementation of cultures with 1 ng/mL TGF-␤1 in the absence of glycated albumin provided only a modest stimulation (37 ⫾ 7%) of collagen IV synthesis by GEnC (N ⫽ 4, P ⬍ 0.05). Protein kinase C activity The ability of glycated albumin to increase PKC activity was assessed in the substrate-specific phosphorylation assay. Visual inspection of gels from experiments in

Fig. 2. Protein kinase C (PKC) activity in glomerular endothelial cells (GEnCs). GEnCs were cultured for 48 hours in DMEM containing 5.5 mmol/L glucose/2% FCS and no supplement (media), nonglycated albumin (NA), or glycated albumin (GA) at 500 ␮g/mL, without or with 500 nmol/L of the PKC inhibitor GF 109203X (GFX). In each lane of the gel, nonphosphorylated substrate is shown on the left and phosphorylated substrate on the right. Negative control (lane A) shows no phosphorylated substrate. Positive control (lane B), obtained with PKC supplied by Promega, shows abundant phosphorylated substrate. Lane C ⫽ media; lane D ⫽ media ⫹ GFX; lane E ⫽ nonglycated albumin; lane F ⫽ nonglycated albumin ⫹ GFX; lane G ⫽ glycated albumin; lane H ⫽ glycated albumin ⫹ GFX.

which cells were incubated for 48 hours in 5.5 mmol/L glucose indicated that PKC activity was increased in cells cultured in media containing glycated albumin compared with nonglycated albumin in the same concentration (Fig. 2). Densitometric scanning of the gel photographs confirmed that exposure to glycated albumin significantly stimulated PKC activity in 5.5 mmol/L glucose media (Fig. 3). This stimulation was prevented by the PKC inhibitor GFX (Figs. 2 and 3). TGF-␤1 production To determine whether changes in TGF-␤1 production accompanied the increases in collagen IV synthesis and

Chen et al: Glycated albumin and GEnC biology

Fig. 3. Effect of glycated albumin and PKC inhibition on PKC activity. GEnCs were cultured and analyzed for PKC activity as described in the legend to Figure 2 and in the text. Data were obtained by densitometric scanning of photographs of gels. Results represent the mean ⫾ SEM of four independent cultures and are expressed as the relative ratio of the densitometry of phosphorylated substrate in control cells (media, no GFX), arbitrarily assigned a value of 1. *P ⬍ 0.05 compared with nonglycated albumin; #P ⬍ 0.05 compared with glycated albumin, no GFX.

PKC activity observed with glycated albumin, concentrations of the translated TGF-␤1 protein were measured by ELISA in spent media. TGF-␤1 was significantly greater in the supernatants from cells incubated with glycated albumin compared with nonglycated albumin (Fig. 4). This increase was prevented by the PKC inhibitor GFX (Fig. 4). For comparative purposes and because the effect of elevated media glucose on TGF-␤1 production by GEnCs has not been previously examined, additional experiments were performed with media containing 25 mmol/L glucose and nonglycated versus glycated albumin. In the presence of nonglycated albumin, TGF-␤1 concentrations were significantly higher in spent media from incubations in high glucose than in normal glucose (Fig. 4, B vs. A), and this increase was significantly attenuated by the PKC inhibitor GFX. TGF-␤1 production was further augmented when cells were incubated in the combination of glycated albumin and 25 mmol/L glucose, consistent with an additive effect (Fig. 4B). When GFX was added to incubations containing glycated albumin in 25 mmol/L glucose media, TGF-␤1 concentrations were reduced to those of incubations containing nonglycated albumin in 25 mmol/L glucose media (Fig. 4B). Corroboration that glycated albumin stimulated the TGF-␤ system in GEnC was obtained with hybridization analysis for mRNA encoding TGF-␤1. Northern blot analysis showed that the steady-state level of TGF-␤1 mRNA (factored for the housekeeping gene mrpL32) was increased by an average of 30% (N ⫽ 4, P ⬍ 0.001)

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Fig. 4. TGF-␤1 production in GEnCs. GEnCs were cultured for 48 hours in DMEM containing 5.5 mmol/L glucose (A) or 25 mmol/L glucose (B) and nonglycated albumin (NA) or glycated albumin (GA) at 500 ␮g/mL, without (䊐) or with (䊏) 500 nmol/L of the PKC inhibitor GF 109203X (GFX). From each condition, media were collected for immunoassay of TGF-␤1 as described in the text. Results represent the mean ⫾ SEM of four to six independent cultures and are expressed as ng TGF-␤1 normalized to cell number. *P ⬍ 0.05 compared with the respective nonglycated albumin; #P ⬍ 0.05 compared with the respective glycated albumin, no GFX; † P ⬍ 0.05 compared with the respective nonglycated albumin, no GFX.

in cells incubated with glycated albumin compared with nonglycated albumin (Fig. 5). Cell proliferation Experiments to assess the effect of glycated albumin on the proliferation of GEnC were conducted in 10 mmol/L glucose, using [3H]-thymidine incorporation and cell counts as measures of replication. Compared with incubations in media alone or media supplemented with nonglycated albumin, incubations in media with glycated albumin significantly inhibited [3H]-thymidine incorporation (Fig. 6) and reduced cell number (Fig. 7). Moreover, the decrease in [3H]-thymidine incorporation associated with exposure to glycated albumin was prevented when the PKC inhibitor GFX was added to the incubation (Fig. 6A). The addition of anti–TGF-␤ antibody dampened the inhibition of proliferation caused by glycated albumin (Fig. 6B), an effect that was almost statistically significant (N ⫽ 5, P ⫽ 0.06 vs. glycated albumin alone). Consistent with the known antiproliferative effect of TGF-␤ in GEnCs [53], the addition of exogenous TGF-␤1 (1 ng/mL) caused significant inhibition of [3H]-thymidine incorporation (Fig. 6B) and reduction of cell number (Fig. 7). DISCUSSION In these studies, we adhered to conditions in which the glycated protein was represented by albumin modified by

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Fig. 5. TGF-␤1 gene expression in GEnCs. GEnCs were cultured for 48 hours in DMEM containing 5.5 mmol/L glucose/10% FCS and nonglycated albumin (NA) or glycated albumin (GA) at 500 ␮g/mL. Data were obtained by densitometric scanning of autoradiographs of Northern blots successively probed with TGF-␤1 and mrpL32 cDNA. A representative autoradiograph is shown on top. Results represent the mean ⫾ SEM of four independent cultures and are expressed as the percentage of the densitometric ratio of TGF-␤1 to mrpL32 mRNA in control cells (NA), arbitrarily assigned a value of 100. *P ⬍ 0.05 compared with nonglycated albumin.

Amadori glucose adducts and not by advanced glycation end-products (AGE), because the former is the principal physiologic construct in which glycated albumin exists in vivo. Previous work has shown that the Amadori-modified protein can influence functional properties of mesangial and endothelial cells in vitro and in vivo [12, 14, 16, 25, 36, 54, 55]. In GEnCs, we found that PKC signaling predominantly mediated the increased synthesis of TGF-␤1 and collagen IV in response to glycated albumin. This interpretation was based on the findings that glycated albumin could activate the PKC system and that the PKC inhibi-

Fig. 6. Effect of glycated albumin and TGF-␤ modulation on [3H]-thymidine incorporation. GEnCs were cultured for 48 hours in DMEM containing 10 mmol/L glucose/2% FCS and no supplement (Media), nonglycated albumin (NA), or glycated albumin (GA) at 500 ␮g/mL. (A) Cells were incubated without (䊐) or with (䊏) 500 nmol/L of the PKC inhibitor GF 109203X (GFX). (B) Cells were incubated without other addition (䊐) or with 1 ng/mL TGF-␤1 (T, ), 30 ␮g/mL irrelevant murine IgG (IgG, 䊏), or 30 ␮g/mL neutralizing anti-TGF-␤ antibody (␣T, ). From each condition, cells were collected for scintillation counting of [3H]-thymidine incorporation as described in the text. Results represent the mean ⫾ SEM of five independent cultures and are expressed as counts per minute (cpm) of [3H] decay. *P ⬍ 0.05 compared with the respective Media or nonglycated albumin; #P ⬍ 0.05 compared with the respective glycated albumin, no GFX.

tor GFX totally prevented the glycated albumin-induced increases in translated TGF-␤1 and type IV collagen proteins. The existence of a PKC-responsive activated protein-1 (AP-1) site in the promoter of the TGF-␤1 gene provides a plausible molecular basis for PKC signaling of TGF-␤1 production [56, 57]. Although glycated albumin-induced PKC activation may also stimulate collagen IV gene transcription through direct AP-1 binding, the involvement of the profibrotic cytokine TGF-␤ as an intermediary between these events should be considered. We found, however, that a neutralizing antibody against TGF-␤ did not fully prevent the increased collagen IV synthesis in response to PKC acti-

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Fig. 7. Effect of glycated albumin and TGF-␤1 on cell counts. GEnCs were cultured for 48 hours in DMEM containing 10 mmol/L glucose/2% FCS and no supplement (Media), nonglycated albumin (NA), or glycated albumin (GA) at 500 ␮g/mL, without (䊐) or with ( ) 1 ng/mL TGF-␤1. From each condition, cells were collected for cell counting as described in the text. Results represent the mean ⫾ SEM of five independent cultures and are expressed as the number of cells per well. *P ⬍ 0.05 compared with Media, no TGF-␤1; #P ⬍ 0.05 compared with nonglycated albumin.

vation, and exogenous TGF-␤1 only modestly stimulated collagen IV production in the absence of glycated albumin. Others have also reported that the addition of TGF-␤1 to cultured GEnCs did not significantly stimulate collagen IV expression [30]. Nevertheless, our data suggest that PKC-signaled endogenous TGF-␤ bioactivity mediated some stimulation of collagen IV synthesis in response to glycated albumin. In this respect, our findings with GEnCs are analogous to those with mesangial cells in which the glycated albumin-induced increase in TGF-␤1 has been linked to PKC activation and increased ECM protein synthesis [14, 36]. This interpretation is consistent with reports that TGF-␤1 up-regulates fibronectin expression in umbilical vein endothelial cells [58] and that glycated albumin stimulates fibronectin production in GEnCs [31]. Additional experiments are necessary to define the role of TGF-␤ isoforms and their receptors in glomerular capillary basement membrane and fibronectin synthesis and to identify other potential mediators of matrix production in GEnCs. In a study of human umbilical vein endothelial cells, Cagliero et al found that high glucose did not augment TGF-␤ production, measured by bioassay as total TGF-␤, but both high glucose and exogenous TGF-␤1 did inhibit cell replication [58]. The growth-inhibitory effect of high glucose was reversed after the cells were incubated in normal glucose media, but the inhibitory effect of TGF-␤1 was not reversed after cells were incubated without the cytokine. The investigators concluded that high glucose does not significantly stimulate TGF-␤1 production in umbilical vein endothelial cells, that the growth-inhibitory effects of high glucose are independent of TGF-␤1,

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and that high glucose and TGF-␤1 exert their effects through distinct pathways and at different loci. Our findings and those of others [18, 26–28] suggest that endothelial cells of glomerular origin respond to potential modulating factors in ways that are both similar to and different from the behavior of endothelial cells of larger vessels such as the aorta or umbilical vein. Both cell types, for example, show decreased proliferation with addition of TGF-␤1 or glycated albumin [24, 58]. In contrast to the situation with umbilical vein endothelial cells, however, high glucose and glycated albumin significantly stimulated TGF-␤1 production by GEnC. Antagonism of endogenous TGF-␤ with a neutralizing antibody partly restored the proliferative capacity of the cells in glycated albumin (P ⫽ 0.06), but perhaps more complete inhibition of TGF-␤ could have normalized GEnC proliferation. This possibility is supported by the finding that the PKC inhibitor GFX, which almost normalized TGF-␤1 production in cells incubated with glycated albumin, also successfully reversed the inhibition of [3H]-thymidine incorporation caused by glycated albumin. Our data indicate that glycated albumin exerts at least some of its antiproliferative action through induction of TGF-␤, but this does not exclude the involvement of other PKC-activated factors. The glomerular capillary endothelium is strategically positioned between the circulation and other glomerular elements, and it forms the first line of defense against deleterious metabolic, biochemical, and hemodynamic factors presented to the kidney. In the diabetic state, influences such as hyperglycemia and elevated glycated serum protein levels trigger maladaptive biosynthetic programs that contribute to pathobiologic processes [20–24]. High glucose, for instance, increases the expression of basement membrane components in large vessel endothelial cells [20] and stimulates the production of TGF-␤1 in GEnCs. Glycated albumin, as seen in this study, increases PKC activity, TGF-␤1 levels, and ECM expression in GEnCs, but it does not require high ambient glucose to achieve these effects. Finally, glycated albumin stimulates TGF-␤1 and ECM protein production in mesangial cells incubated with normal glucose media, further documenting that glycated albumin exerts its own potent biologic effects [12, 14, 15, 36]. In general, we have found that mesangial cells respond more vigorously to glycated albumin than GEnCs. In similar experiments with comparable levels of glycated albumin, mesangial cells raised their TGF-␤1 mRNA levels by an average of 160% above control [14] versus 30% for GEnCs in this study. Mesangial cells also increased their collagen IV production by as much as 100% above control [36] versus 50% for GEnCs. The significance of the quantitative differences in the effects of glycated albumin on the two glomerular cell types is uncertain. In addition, the extent to which these in vitro data

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in GEnC can be extrapolated to the in vivo situation would need to be determined. The observation that glycated albumin, independent of glycemic status, inhibits the proliferative capacity of the endothelial cell barrier and promotes the production of basement membrane collagen strengthens the concept that glycated albumin substantively contributes to the pathogenesis of glomerular lesions in diabetes. Future investigations will define further the intracellular mediators and signaling processes underlying the actions of glycated albumin. Coupled with our previous work, the results of the present study suggest that reducing the biologic activity of this Amadori-modified protein, in addition to control of hyperglycemia, may be beneficial in forestalling diabetic nephropathy. ACKNOWLEDGMENTS This study was supported in part by grants no. DK 44513, DK 54608, and EY 11825 from the National Institutes of Health. Dr. S. Chen is supported by a fellowship grant from the Juvenile Diabetes Foundation International. Reprint requests to Fuad N. Ziyadeh, M.D., 700 Clinical Research Building, Renal-Electrolyte and Hypertension Division, University of Pennsylvania, 415 Curie Boulevard, Philadelphia, Pennsylvania 191046144, USA. E-mail: [email protected]

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