NDT Advance Access originally published online on February 22, 2006
Nephrology Dialysis Transplantation 2006 21(6):1504-1513; doi:10.1093/ndt/gfl017
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© The Author [2006]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org
Original Articles: Experimental Nephrology
Elevated glucose induction of thrombospondin-1 up-regulates fibronectin synthesis in proximal renal tubular epithelial cells through TGF-ß1 dependent and TGF-ß1 independent pathways
Department of Medicine, University of Hong Kong, Hong Kong, China
Correspondence and offprint requests to: Professor Tak Mao Chan and Dr Susan Yung, Department of Medicine, Queen Mary Hospital, University of Hong Kong, Pokfulam Road, Hong Kong SAR, China. Email: dtmchan{at}hkucc.hku.hk
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Abstract |
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Background. TGF-ß1 bioactivation, consequent to the interaction of latent TGF-ß1 with thrombospondin-1 (TSP-1), correlates with matrix accumulation in mesangial cells. Tubulointerstitial damage predicts poor renal survival. There is little data on TGF-ß1 bioactivation and matrix synthesis in human proximal renal tubular epithelial cells under the influence of high glucose concentrations. This study thus investigates the role of TSP-1 in mediating elevated glucose-induction of TGF-ß1 bioactivation and fibronectin (FN) synthesis in human proximal tubular epithelial cells.
Methods. Human proximal renal tubular epithelial cells (HK-2 cells) were incubated with 5, 10, 20 or 30 mM D-glucose for up to 3 weeks either in the presence or absence of TSP-1 blocking peptide. In separate studies HK-2 cells were incubated with exogenous TSP-1 (0–10 ng/ml) or TGF-ß1 (0–10 ng/ml) for 24 h. Cell proliferation was assessed by [3H]-thymidine incorporation. TGF-ß1 transcript, secretion and bioactivity were investigated by quantitative real-time PCR, ELISA and the MLEC bioassay respectively. TSP-1 and FN synthesis were assessed by quantitative real-time PCR, ELISAs and Western blot analysis.
Results. Elevated glucose concentrations increased TSP-1 synthesis, which was associated with reduced cell proliferation, increased TGF-ß1 bioactivity, and stimulation of FN synthesis. The inclusion of TSP-1 blocking peptide to cells stimulated with elevated glucose concentration abrogated activation of TGF-ß1 and induction of FN secretion. Exogenous TSP-1 increased bioactive TGF-ß1 in HK-2 cells to initiate FN accumulation. Of interest is our observation that TSP-1 also increased matrix synthesis through a mechanism independent of TGF-ß1. TGF-ß1 in turn modulated TSP-1 synthesis, indicative of an autocrine loop between TSP-1 and TGF-ß1.
Conclusions. TSP-1 plays an important role in the induction of matrix synthesis by high glucose concentrations in human proximal renal tubular epithelial cells, through TGF-ß1 dependent and TGF-ß1 independent pathways. Pharmacological intervention targeting increased TSP-1 expression may interrupt the pathogenesis of diabetic nephropathy.
Keywords: fibronectin; glucose; proximal renal tubular epithelial cells; TGF-ß1; thrombospondin-1
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Introduction |
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Diabetic nephropathy (DN) is the most common cause of end-stage renal disease worldwide, accounting for more than 40% of patients who require renal replacement therapy. DN is initiated by hyperglycaemia and is characterized by mesangial expansion and thickening of the tubular and glomerular basement membranes due to excessive matrix accumulation, associated with progressive renal deterioration [1]. Much of the research on DN to date has focused on glomerular abnormalities. The rate of progressive renal failure, however, correlates better with the extent of tubulointerstitial than with glomerular damage [2]. During DN, the tubulointerstitium is subjected to both direct and indirect pathogenetic influences such as vasoactive hormones, growth factors and matrix proteins as a consequence of its location in the kidney and its functional properties. These molecules act in concert to amplify the fibrotic process. Proximal renal tubular epithelial cells (PTEC) constitute the predominant cell type within the tubulointerstitium. In addition to the transport of fluid and electrolytes, there is increasing evidence that PTEC are involved in inflammatory and fibrotic processes, thereby playing a pivotal role in the pathogenesis of tubulointerstitial disease in various renal parenchymal diseases [3].
Data from animal and in vitro studies have shown that elevated glucose concentration stimulates TGF-ß1 secretion and matrix synthesis in PTEC [4,5]. TGF-ß1 is a multifunctional regulator of cell growth, gene expression, and differentiation. It is secreted by various cell types as an inactive precursor, comprising an active peptide non-covalently associated with a precursor peptide, termed the latency-associated peptide (LAP) [6]. Conversion of inactive TGF-ß1 into its active form is required before it can bind to cell surface receptors and elicit cellular responses. Previous studies have shown that latent TGF-ß1 can be activated by a number of mechanisms, including conformational changes induced by extreme pH, high temperature, limited proteolysis or deglycosylation of LAP, or by binding to various membrane or extracellular matrix components [7,8]. In addition, thrombospondin-1 (TSP-1) derived from platelets or endothelial cells can activate TGF-ß1 [9]. In this context, data from animal models of glomerulonephritis have shown that TSP-1 expression precedes the development of tubulo-interstitial fibrosis. Increased TSP-1 levels have been observed in plasma and platelets of diabetic patients [10]. Furthermore, increased TSP-1 mRNA and protein expression have been demonstrated in mesangial cells cultured under elevated glucose concentrations, and this was associated with TGF-ß1 activation [11]. Previous studies have reported on the observed increase in TGF-ß1 secretion in PTEC upon exposure to high ambient glucose, but the mechanisms of TGF-ß1 bioactivation have not been investigated in human PTEC [4]. In this study, we examined the role of TSP-1 in mediating TGF-ß1 bioactivation and fibronectin (FN) synthesis in elevated glucose-stimulated human PTEC. We demonstrated that TSP-1 could enhance FN synthesis in human PTEC predominantly through a TGF-ß1 independent pathway, and only 25% of matrix synthesized by human PTEC was mediated through TSP-1 induction of TGF-ß1 bioactivation.
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Materials and methods |
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Cell culture
All experiments were performed on HK-2 cells (ATCC, Manassas, USA), which are normal PTEC immortalized by transduction with the human papilloma virus 16 E6/E7 genes [12]. HK-2 cells were cultured in 5 mM DMEM/F12 medium supplemented with 10% FCS. Cells of the 20–25th passage were pre-conditioned for periods of up to 3 weeks with physiological (5 mM) or elevated (10, 20 and 30 mM) concentrations of D-glucose, the latter concentrations mimicking those in diabetic patients. Mannitol at identical concentrations was used as the hexose control in parallel experiments. Cells were growth arrested for the last 72 h of the stimulation period, and stimulated in fresh medium for a further 24 h prior to extraction of total protein or mRNA for Western blot analysis and quantitative real-time PCR, respectively. To determine the role of TSP-1 in mediating elevated glucose-induction of TGF-ß1, cells were pre-incubated with either 5 or 30 mM D-glucose or mannitol for periods of up to 3 weeks in the presence of either a TSP-1 blocking peptide (GGWSHW) or the negative control peptide (GGYSHW) [11]. In separate studies, unstimulated HK-2 cells were incubated with exogenous TSP-1 (0–10 ng/ml) or TGF-ß1 (0–10 ng/ml) for 24 h. To assess whether TSP-1 was able to induce FN synthesis independent of TGF-ß1 induction, HK-2 cells were incubated with TSP-1 in the presence or absence of a neutralizing antibody to TGF-ß1 (R&D Systems, TWC Biosearch International, Hong Kong; 10 µg/ml). This dose was chosen since preliminary studies demonstrated that TGF-ß neutralizing antibody at 1 µg/ml reduced TGF-ß1 mediated FN secretion to levels similar to that of control without demonstrating any cytotoxicity.
Assessment of cell proliferation
HK-2 cells were seeded onto 96-well culture plates at a density of 10 000 cells/cm2 and cultured in DMEM/Hams F12 medium containing 5, 10, 20 or 30 mM D-glucose, or mannitol, supplemented with 10% FCS for 24 h. Cells were washed twice with PBS and further incubated in DMEM/Hams F12 containing physiological or elevated glucose or mannitol concentrations for up to 3 weeks. During the last 24 h of the time period, cells were incubated in fresh medium containing methyl-[3H]-thymidine (Amersham Biosciences China Limited, 1 µCi/ml). At the end of the time period, the supernatant was decanted, cells trypsinized with 0.05% trypsin/0.02% EDTA, the incorporated radioactivity was precipitated with 10% trichloroacetic acid (TCA, 10 µ1), washed twice with TCA and dissolved in 0.1 M NaOH (20 µl). The [3H]-thymidine incorporation was detected by beta-scintillation counting.
TGF-ß1, TSP-1 and FN mRNA expression by quantitative real-time polymerase chain reaction
RNA from HK-2 cells was extracted with TriReagent (Onwon Trading Limited, Hong Kong) according to the manufacturer's instructions. One microgram of total RNA was reverse-transcribed to cDNA with M-MLV reverse transcriptase (Invitrogen Life Technologies, Hong Kong) using the random hexamers method. Taqman quantitative real-time PCR reactions were performed in triplicate according to the manufacturer's instructions (Applied Biosystems, Hong Kong) in an ABI Prism 7700 Sequence Detection System using TGF-ß1, TSP-1 and FN primer sets (Assays-on-Demand, Applied Biosystems). The calculation of relative change in mRNA was performed using the delta–delta method [13] standardized to the housekeeping gene GAPDH.
Determination of TGF-ß1 concentration in culture supernatant
One millilitre samples of culture supernatant were lyophilized to 200 µl in PBS containing 1% fatty acid free BSA, and assayed using a commercial TGF-ß1 ELISA kit according to the manufacturer's instructions (R&D Systems, TWC Biosearch International, Hong Kong). Cross-reactivity with other TGF-ß isoforms was less than 2%.
Determination of TGF-ß1 bioactivity
The mink lung epithelial cell (MLEC) bioassay for TGF-ß1 was performed as described previously [14]. Briefly, MLECs (passage 50–55) were cultured in 96-well culture plates at 10 000 cells/cm2 in DMEM supplemented with 10% FCS for 2–4 h. MLECs were exposed to serially diluted recombinant human TGF-ß1 (0–1000 pg/ml) or spent culture media from HK-2 cells obtained under control or experimental conditions for 72 h at 37°C. Thereafter, [3H]-thymidine (1 µCi/ml) was added to each well and the incubation continued for a further 24 h. The cells were then washed thrice with ice-cold PBS, trypsinized with 0.05% trypsin/0.02% EDTA. The incorporated material was precipitated with 10% TCA, washed twice with TCA, and the pellet dissolved with 0.1 M NaOH. The [3H]-thymidine incorporation was assessed by beta scintillation. A standard curve was constructed from the varying degrees of inhibition on MLEC growth observed with the corresponding concentrations of recombinant TGF-ß1 [14].
Measurement of TSP-1 and FN secretion by ELISA
Cells were cultured under control or experimental conditions for periods up to 3 weeks. The supernatants were decanted, centrifuged for 10 min at 1000 g to remove cell debris and then assayed for TSP-1 and FN, using in-house ELISAs [11]. Briefly, 96-well microtitre plates (Immunlon 2; Thermo Labsystems, Hong Kong) were coated in duplicate with TSP-1 or FN (0–500 ng/ml), or HK-2 culture media (starting dilution 1:10) in serial dilution of 15 mM Na2CO3 and 35 mM NaHCO3, pH 9.6, overnight at 4°C. Unless otherwise stated, all incubations were for 1 h at 37°C, and were then washed with PBS/0.1% Tween-20 in between all steps. The plates were blocked with 3% BSA, and incubated with primary antibodies (1:1500 for both). The relevant horseradish peroxidase-conjugated secondary antibody was then added and bound, and immunoglobulin detected by measuring the absorbance at 450 nm, after the addition of Sigma Fast o-phenylenediamine dihydrochloride (Sigma Chemical Co, Tin Hang Technology Ltd). Prior experiments demonstrated <5% cross-reactivity of primary antibodies with other matrix proteins. Intra-assay and inter-assay coefficients of variance were 5.01±1.41 and 5.81±1.95%, respectively, for TSP-1, and 6.08±1.21 and 5.89±3.47%, respectively, for FN.
Quantification of total cellular protein
Cells cultured under control or experimental conditions were lysed with 20 mM sodium acetate, pH 6.0 containing 4 M urea, 1% Triton X-100 (100 µl) [14]. Cell lysates were desalted and buffer-exchanged into PBS using Ultrafree centrifugal concentrators (Millipore China Ltd, Hong Kong). Total protein was measured using the Bio-Rad DC protein assay (Bio-Rad Laboratories, Hong Kong).
Western blot analysis for cell-associated TSP-1 and FN
Whole cell lysate (10 µg total protein content) was denatured in sample buffer at 95°C for 5 min and subjected to SDS-PAGE on 10% polyacrylamide gels. Proteins were transferred onto nitrocellulose membrane (Schleicher & Schuell, Gene Company, Hong Kong) using a mini-gel transfer system (Bio-Rad, Hong Kong) at 100 V for 1 h at 4°C. Equal loading of proteins was confirmed by staining of membranes with 1% Ponceau S solution [14]. Membranes were immunoblotted as previously described using respective primary (1:1000 dilution for both TSP-1 and FN antibodies) and secondary antibodies. Bands were visualized by enhanced chemiluminescence, semi-quantitated by densitometry using ChemiGenius analysis software (Syngene Ltd, Cambridge, United Kingdom) and unless otherwise stated, expressed as arbitrary densitometric unit (DU). Expression of TSP-1 and FN was normalized to ß-actin.
Statistical analysis
Unless otherwise stated, all experiments were repeated three times. The results are expressed as mean±SD. Statistical analysis was performed using GraphPad Prism version 3.00 for Windows, (GraphPad Software, San Diego, CA, USA). Between-group comparisons were made by ANOVA. P<0.05 was considered statistically significant.
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Effect of elevated glucose concentrations on HK-2 cell proliferation
HK-2 cell proliferation, assessed by [3H]-thymidine incorporation was similar upon exposure to D-glucose at 5 or 10 mM. Higher D-glucose concentrations reduced cell proliferation in a time-dependent manner, which became significant after 96 h for 20 and 30 mM D-glucose (Figure 1A). Mannitol at identical concentrations did not alter cell proliferation compared with 5 mM D-glucose (Figure 1B).
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), 10 mM (
), 20 mM 
and 30 mM 
D-glucose (A) or mannitol (B) for up to 3 weeks. PTEC proliferation was determined by [3H]-thymidine incorporation assay. The values are expressed as mean±SD from three separate experiments. *P<0.05 compared to 5 mM D-glucose.Effect of elevated glucose concentrations on TSP-1 and TGF-ß1 synthesis
HK-2 cells constitutively secreted TSP-1 (Figure 2A). Elevated glucose concentrations increased TSP-1 secretion in a dose-dependent manner (Figure 2A). Cells exposed to 30 mM D-glucose showed a significant increase in TSP-1 secretion after 24 h, while 48 h of incubation was required before such induction by 10 or 20 mM D-glucose became significant. Maximum induction of TSP-1 secretion occurred after 1 week of stimulation for all three concentrations of D-glucose (514.2±24.5, 1048.9±38.6, 1240.9±31.6, and 1508.6±64.8 ng/µg cellular protein for 5, 10, 20 and 30 mM D-glucose respectively, P<0.05 comparing 5 mM with the other D-glucose concentrations). Mannitol did not affect TSP-1 secretion (Figure 2B).
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Elevated glucose concentrations increased total TGF-ß1 secretion by HK-2 cells in a time and dose-dependent manner, with maximum secretion observed after 3 weeks of incubation (6.4±0.3, 8.7±0.2, 13.5±0.6 and 16.5±2.1 pg/µg cellular protein for 5, 10, 20 and 30 mM respectively, P<0.05 compared to control, Figure 3A). After stimulation with D-glucose at 5, 10, 20 and 30 mM, respectively, 94.8±1.4, 95.2±2.4, 94.5±2.1 and 95.1±1.8% of TGF-ß1 in the culture supernatant was detected and only after acid-activation, indicating that most of the TGF-ß1 was synthesized in its latent form and approximately 5% was secreted in an active form. The bioactivity of TGF-ß1 synthesized by HK-2 cells was investigated using the MLEC bioassay (Figure 3C), which confirmed that bioactive TGF-ß1 was induced by elevated D-glucose concentrations. Compared to cells incubated with 5 mM D-glucose, a significant increase in TGF-ß1 bioactivity was first observed after incubation with 10 mM D-glucose for 1 week, or after 72 h of incubation with 20 or 30 mM D-glucose (Figure 3C). Mannitol at identical concentrations did not affect total or bioactive TGF-ß1 secretion (Figure 3B and D). The increase in TGF-ß1 was accompanied by the induction of FN mRNA and increased FN secretion (Figure 4).
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The role of elevated glucose-induced TSP-1 in mediating TGF-ß1 bioactivation and subsequently increased FN synthesis was further investigated using TSP-1 blocking peptides. Incubation of HK-2 cells with either TSP-1 blocking peptide or control peptide at concentrations up to 5 µM in the presence of either 5 or 30 mM D-glucose for up to 3 weeks had no effect on TSP-1 secretion, and was not cytotoxic to cells as determined by lactate dehydrogenose (LDH) release (data not shown). Whilst TSP-1 blocking peptide had no effect on constitutive TGF-ß1 bioactivation or FN secretion in HK-2 cells stimulated with 5 mM D-glucose, TSP-1 blocking peptide (5 µM) abrogated elevated glucose-mediated activation of TGF-ß1 and induction of FN secretion (Figure 5).
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. The supernatants were collected, centrifuged and aliquots used to assess the role of TSP-1 blocking peptide on TGF-ß1 bioactivation (A) and FN secretion (B). Data represents mean±SD from three separate experiments. *P<0.05 compared to 5 mM DMEM/F12 medium.Effect of TSP-1 on TSP-1 transcript and the synthesis of TGF-ß1 and FN
To investigate the direct effect of TSP-1 on TGF-ß1 activation and FN synthesis, separate studies were undertaken in which cells were exposed to exogenous TSP-1 for 24 h. Addition of exogenous TSP-1 induced TSP-1 mRNA (Figure 6A), with maximum induction observed with a TSP-1 concentration of 0.5 ng/ml (6.32±0.18-fold increase compared to control, P<0.05). Exogenous TSP-1 also increased TGF-ß1 mRNA in a dose-dependent manner with maximum induction occurring with 5 ng/ml TSP-1 (Figure 6B). TSP-1 mediated increase in TGF-1 transcript was accompanied by increased TGF-1 secretion and bioactivity (Figures 6C and D). TSP-1 also induced FN mRNA, FN secretion and FN accumulation in the cell-associated fraction, the latter two in a dose-dependent manner (Figure 7). Separate experiments were conducted to determine whether the effect of TSP-1 on FN was mediated through TGF-ß1, in which HK-2 cells were incubated with TGF-ß1 neutralizing antibody (10 µg/ml) for 1 h prior to stimulation with exogenous TSP-1 (1 ng/ml). This dose of TGF-ß1 neutralizing antibody was chosen since it abrogated TGF-ß1 (1 ng/ml) mediated induction of FN secretion (Figure 8A). Whilst neutralizing antibody to TGF-ß1 alone did not alter constitutive FN secretion in HK-2 cells, co-incubation of cells with both TGF-ß1 neutralizing antibody and TSP-1 resulted in a reduction in FN secretion that was significantly apparent after 12 h incubation (49.4±2.4, 46.0±5.4, 67.1±6.2 and 59.3±2.1 ng/µg cellular protein for medium alone, medium and TGF-ß neutralizing antibody, TSP-1 alone, and TSP-1 and TGF-ß neutralizing antibody respectively; P<0.05, TSP-1 or TSP-1 and TGF-ß neutralizing antibody vs medium alone). After 48 h, 26.5±5.7% reduction in FN secretion was observed compared with cells stimulated with TSP-1. Incubation of HK-2 cells with higher concentrations of TGF-ß1 neutralizing antibody (25 µg/ml) did not further inhibit FN secretion, indicating that induction of FN by TSP-1 was not mediated solely through TGF-ß1 (Figure 8B).
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, and TSP-1 with TGF NA Effect of TGF-ß1 on TGF-ß1 transcript, and the synthesis of TSP-1 and FN
Exogenous TGF-ß1 at concentrations of 0.01–0.5 ng/ml significantly induced TGF-ß1 mRNA expression in HK-2 cells (1.88±0.2, 1.70±0.15 and 2.03±0.36-fold increase compared to control, P<0.05 for all) (Figure 9A). Addition of exogenous TGF-ß1 to HK-2 cells also increased TSP-1 mRNA expression, TSP-1 secretion, and cell-associated TSP-1 (Figure 9B–D). Exogenous TGF-ß1 also induced FN mRNA with maximum induction occurring with 0.1 ng/ml TGF-ß1 (4.5±0.45-fold increase compared to control, P<0.05), with a concomitant increase in both secreted and cell-associated FN (Figure 10).
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Discussion |
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The renal tubulointerstitium accounts for more than 90% of the kidney volume and comprises the tubular epithelium, blood vessels and interstitium, with PTEC as the predominant cell type. The importance of tubulointerstitial inflammation, matrix accumulation and fibrosis as major determinants in the progression of renal failure is increasingly recognized in a variety of renal diseases, including DN. In this regard, PTEC can be a direct target for the harmful actions of elevated glucose concentrations in diabetes mellitus. Glucose uptake by PTEC is independent of insulin but directly related to the level of plasma glucose. Excessive glucose in the glomerular filtrate also leads to increased glucose reabsorption by PTEC, which further exacerbates the effects of hyperglycaemia. Data from animal and in vitro studies have implicated elevated glucose concentration in mediating pathological changes in the tubular basement membrane and in altering the composition of matrix components, both being characteristic features in DN [3–5]. Acute or chronic exposure of PTEC to glucose at high concentrations induces TGF-ß1 transcription and bioactivity. Although the role of TGF-ß1 in the pathogenesis of DN is undisputed, the post-transcriptional mechanisms that regulate TGF-ß1 activation in PTEC are less well characterized.
TGF-ß1 is secreted in many cell types as a latent, inactive procytokine complex consisting of a mature active TGF-ß1 protein non-covalently bonded to a disulphide-linked dimer of its N-terminal propeptide known as the LAP. The secreted latent TGF-ß complex is unable to bind to TGF-ß receptors until the biologically active peptide is dissociated from the N-terminal portion of its LAP. Most processes that induce activation of the latent complex either degrade the LAP or alter the interaction of the LAP with the mature domain. The activation of TGF-ß1 is a prerequisite for binding to its cellular receptors and for its biological activities. Since TGF-ß1 is ubiquitously expressed, the rate at which active TGF-ß1 is produced is central to the regulation of TGF-ß1 activity. In vitro, active TGF-ß1 can be released from the latent complex by proteases, glycosidases, extreme pH, heat or chaotropic reagents [7–9]. Physiological mechanisms of activation are not totally understood, although exposure to reactive oxygen species, binding to 
vß6 integrin, and interaction with TSP-1 have been implicated [8,9,11].
TSP-1 is a multidomain protein synthesized and secreted by a variety of cells including mesangial cells, endothelial cells, skin fibroblasts, and platelets. TSP-1 plays an important role in cell adhesion, proliferation and angiogenesis [9]. Previous studies have shown that TSP-1 could activate latent TGF-ß1 in endothelial cells and fibroblasts. Recent studies have highlighted the ability of TSP-1 to activate TGF-ß1 in human and rat mesangial cells exposed to elevated glucose concentrations [11]. In this context, diabetes mellitus is associated with increased TSP-1 in the kidneys, and the development of diabetic organ damage such as nephropathy is associated with increased plasma TSP-1 levels [10]. The effect of TSP-1 on TGF-ß1 in human PTEC under elevated glucose concentrations has not been investigated.
Our data showed that exposure of HK-2 cells to elevated glucose concentrations resulted in the up-regulation of TSP-1 synthesis and secretion. This was accompanied by an increase in total and bioactive TGF-ß1, as well as increased FN mRNA and translation, and reduced cell proliferation. The observation that FN mRNA was increased in PTEC stimulated with elevated glucose concentrations is in contrast to published data [15]. Using reverse transcription and polymerase chain reaction (RT–PCR), Phillips and colleagues [15] showed that elevated glucose-induction of FN synthesis was independent of FN mRNA up-regulation in porcine PTEC (LLC-PK1 cells). In our preliminary studies using RT–PCR, no alterations in FN mRNA were observed in HK-2 cells exposed to elevated glucose concentration, whilst quantitative real-time PCR highlighted an increase in FN mRNA in these cells. It is possible that RT–PCR is not sufficiently sensitive to detect transcriptional changes in FN mRNA under these experimental conditions. Elevated glucose concentrations also significantly reduced cell proliferation. This was not a result of cytotoxicity, however, since high ambient glucose did not alter LDH release in these cells.
To ascertain whether TSP-1 could modulate the fibrotic pathway in HK-2 cells, a TSP-1 blocking peptide was used in conjunction with elevated glucose concentrations. The sequence (GGWSHW) used for the blocking peptide is also localized to the type 1 domain of the TSP-1 molecule, and is essential for the correct orientation of the TSP-1 molecule prior to its interaction with the mature portion of the TGF-ß1 molecule. The addition of the GGWSHW peptide to HK-2 cells, whilst able to bind to the latent TGF-ß1 complex was unable to activate the growth factor, and prevented the activation of TGF-ß1 and subsequent induction of matrix synthesis by TSP-1. The direct action of TSP-1 on increasing bioactive TGF-ß1 and FN was also demonstrated by experiments using exogenous TSP-1. Interestingly, TGF-ß neutralizing antibody did not completely abrogate the stimulation of FN synthesis by TSP-1 despite the addition of excess neutralizing antibody. The results indicated that both TGF-ß1 dependent and TGF-ß1 independent pathways operate in mediating the TSP-1 effect on FN synthesis in PTEC. Further studies are ongoing to investigate the relative importance of the different pathways under various circumstances and the factors that could modulate the individual pathways, including the effects of blocking peptides. Other investigators have observed that TSP-1 induced an increase in FN synthesis in human mesangial cells, which was accompanied by increased bioactive TGF-ß1 while total TGF-ß1 remained unaltered [11,16]. However, these effects were completely abrogated by TGF-ß1 neutralizing antibody. Collective data thus highlight the importance of TSP-1 in the mediation of TGF-ß1 actions which vary between cell types, and indicate that distinct mechanisms mediate the effects of elevated glucose in different cell types. In this respect, independent researchers have also demonstrated that the increase in matrix synthesis upon stimulation with high glucose concentrations was independent of TSP-1 and TGF-ß1 in human skin fibroblasts and renal fibroblasts [17,18].
Our finding that TGF-ß1 induced TSP-1 mRNA and protein expression in PTEC implicates an autocrine relationship between TSP-1 and TGF-ß1. This is corroborated by similar data from cultured smooth muscle cells [19]. Recent studies have highlighted the role of ERK1/2 and p38 MAPK in the regulation of TSP-1 by TGF-ß1 in rat proximal tubular cells and mouse fibroblasts [20]. In addition, we have demonstrated in this study that TGF-ß1 can modulate its own synthesis in HK-2 cells.
In conclusion, we have demonstrated that TSP-1 mediates, at least in part, the increased levels of bioactive TGF-ß1 in human PTEC upon stimulation by elevated glucose concentrations, and thus the downstream increase in matrix synthesis. In addition, TSP-1 can induce FN synthesis in PTEC independent of TGF-ß1. These findings underscore the importance of TSP-1 and perturbed renal tubular epithelial cell function in the pathogenesis of DN.
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Acknowledgments |
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We thank Mr Jack KH Leung for his invaluable assistance in the culture of HK-2 cells. This work was supported by a grant from the University of Hong Kong (CRCG 10 206 116) and the Wai Hung Charity Foundation.
Tak Mao Chan and Susan Yung contributed equally to this work.
Conflict of interest statement. The authors of this manuscript have had no involvement that might raise the question of bias in the work reported or in the conclusions, implications, or opinions stated.
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[Abstract/Free Full Text]
Accepted in revised form: 13. 1.06
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