Department of Nephrology, Leiden University Medical Center, Leiden, The Netherlands
Correspondence and offprint requests to: Dr Cees van Kooten, Department of Nephrology, Leiden University Medical Center, C3P, Albinusdreef 2, 2333 ZA Leiden, The Netherlands. Email: kooten{at}lumc.nl
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Abstract |
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Methods. Human renal fibroblasts (TK173) and PTECs (HK2) were used to examine the effects of high glucose (25 mM D-glucose) and TGF-ß1 (1 ng/ml) on collagen type I, III and IV secretion compared with control medium (5.5 mM glucose). Matrix components were measured by enzyme-linked immunosorbent assay (ELISA) and reverse transcriptionpolymerase chain reaction (RTPCR).
Results. Renal fibroblasts are the main producers of the interstitial components collagen type I and type III, while collagen type IV was secreted predominantly by PTECs. However, renal fibroblasts were also able to secrete collagen type IV. Secretion of collagen type IV by fibroblasts was increased upon stimulation with TGF-ß1, reaching levels comparable with those secreted by TGF-ß1-induced PTECs. Moreover, high glucose stimulated increased collagen type IV secretion. Importantly, this could not be attenuated by neutralizing pan-specific anti-TGF-ß antibodies.
Conclusions. These data show that renal fibroblasts secrete collagen type IV, which can be increased by high glucose independent of endogenous TGF-ß. This suggests that as well as the increased expression of interstitial components, renal fibroblasts can contribute to the increased expression of the basement membrane component collagen type IV in tubulointerstitial fibrosis observed during diabetic nephropathy.
Keywords: collagen type IV; diabetic nephropathy; fibroblasts; proximal tubular epithelial cells; TGF-ß; tubulointerstitial fibrosis
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Introduction |
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The cellular compartment of the tubulointerstitium is composed of tubular epithelial cells (TECs) and interstitial fibroblasts. TECs are the predominant cell type of the interstitium, and serve as a major source of pro-inflammatory cytokines, chemokines and profibrotic growth factors during tubulointerstitial fibrosis. Furthermore, TECs contribute to the excessive ECM deposition in the kidney [3]. The resident interstitial fibroblasts are major producers of ECM components. Although normally present in low numbers, fibroblasts are increased during fibrosis [4], which may play a role in the progression of fibrosis by excessive ECM deposition [5,6]. Furthermore, resident cells also undergo morphological and functional changes [7].
In normal kidneys, the production of different collagen molecules is compartimentalized, with TECs as the principal cells of collagen type IV production [8], and fibroblasts producing collagens type I and III [9,10]. In tubulointerstitial fibrosis, matrix components are abundantly upregulated [11,12]. In addition to quantitative changes, there is aberrant expression of novel matrix components. For instance, collagen type IV, normally expressed in glomerular and tubular basement membranes [13], is no longer restricted to the basement membranes, but is also expressed in the interstitium [8,11,12,14,15]. Production of this collagen type IV has been attributed predominantly to TECs [8]. Most studies with renal fibroblasts examined total collagen secretion [16]. There are only a few studies which have examined the expression of collagen type IV in renal fibroblasts [17]. There is still little known about its regulation and the relative contribution of renal fibroblasts and TECs to the aberrant expression of collagen type IV during fibrosis.
Growth factors play an important role in the regulation of ECM production. In particular transforming growth factor-ß (TGF-ß) has been demonstrated to be an important mediator of the fibrotic process [18]. TGF-ß expression is increased in the diabetic kidney, and treatment of streptozotocin-induced diabetic mice with neutralizing anti-TGF-ß antibodies attenuated kidney hypertrophy and ECM gene expression [19]. Although TGF-ß plays a central role in diabetic nephropathy, TGF-ß-independent processes are also thought to play a role. For instance, we have shown recently that the glucose-induced expression of interstitial components fibronectin and collagen type III was TGF-ß independent in human renal fibroblasts [10].
In this study, we examined the effect of high ambient glucose and TGF-ß1 on the secretion of the interstitial collagens type I and III, and the basement membrane component collagen type IV by human renal fibroblasts and proximal tubular epithelial cells (PTECs). We found that renal fibroblasts secrete collagen type IV, which was increased when fibroblasts were stimulated with TGF-ß1 or high glucose, reaching levels comparable with those secreted by TGF-ß1-induced PTECs. Further-more, glucose-induced collagen type IV was independent of endogenous TGF-ß. These results suggest that renal fibroblasts can contribute to the observed aberrant expression of collagen type IV in the tubulointerstitium during fibrosis induced by the hyperglycaemic environment via a TGF-ß-independent pathway.
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Materials and methods |
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For experimental purposes, cells were seeded at a density of 50 000 cells/well in 48-well plates (Costar, Corning, NY) and grown in DMEM containing 10% FCS for 24 h to reach confluence. Subsequently, cells were brought to quiescence by placing them in DMEM with 0.5%
FCS. After 24 h, cells were cultured for 4 days in 0.3 ml of medium alone, i.e. DMEM containing 5.5 mM D-glucose supplemented with 0.5%
FCS, or medium containing the following additives: 25 mM D-glucose (Merck, Darmstadt, Germany), 25 mM L-glucose (Sigma) and 1 ng/ml recombinant human TGF-ß1 (R&D systems, Abingdon, UK). Under all experimental conditions, except where indicated, 50 µg/ml ascorbic acid and 100 µg/ml ß-aminopropionitrile were added to promote collagen synthesis and prevent cross-links, respectively (both purchased from Sigma). Blocking anti-TGF-ß studies were performed by the addition of a neutralizing anti-human TGF-ß1, 2, 3 monoclonal antibody (2G7) (20 or 50 µg/ml) [20] or a control mouse IgG at the start of the culture. After 4 days, culture supernatants were harvested, and measured for secreted proteins by enzyme-linked immunosorbent assay (ELISA). Cells were trypsinized and viable cells were counted using Trypan Blue exclusion.
Collagen type I, type III and type IV inhibition ELISA
Collagen type I, type III and type IV accumulation in culture supernatants was measured using a specific inhibition ELISA as previously described for fibronectin [10]. Briefly, 96-well Nunc Maxisorb microtitre plates (Gibco/Invitrogen) were coated with 0.5 µg/ml human collagen type I, type III or type IV (all purchased from Sigma) in phosphate-buffered saline (PBS) (100 µl/well) overnight at room temperature. Collagen type I, type III or type IV standard and samples were pre-incubated overnight at 4°C with the same volume of goat anti-human collagen type I, goat anti-human collagen type III or goat anti-human collagen type IV, respectively (all purchased from Immunologicals Direct, Oxfordshire, UK) in PBS/0.05% Tween/2% casein (PTC). Plates were washed and, after a blocking step with PTC for 1 h at 37°C, 100 µl of the pre-incubated standard or sample was added to the plates, and incubated at 37°C for 1 h. After washing the plates, a peroxidase-conjugated secondary rabbit anti-goat IgG (Nordic Immunology, Tilburg, The Netherlands) was added and incubated for 1 h at 37°C. The ELISA was developed with ABTS (2,2'-amino-bis-3-ethylbenzthiazoline-6-sulfonic acid)/H2O2 and read at an optical density (OD) of 415 nm.
RNA isolation and semi-quantitative reverse transcriptionpolymerase chain reaction (RTPCR)
Total cellular RNA was extracted using RNAzolB (Campro Scientific, Veenendaal, The Netherlands) according to the manufacturer's description. The quantity and purity of the isolated RNA were measured at OD260 and OD280, and analysed on a 0.5x TBE (0.045 M Tris borate, 0.001 M EDTA) 1% agarose gel.
For RTPCR, 1 µg of total RNA was reverse-transcribed into cDNA by oligo(dT) priming using Moloney murine leukaemia virus reverse transcriptase (all purchased from Invitrogen, Paisley, UK). cDNA was amplified by PCR using primers for human GAPDH as housekeeping gene (forward primer 5'-ACC ACA GTC CAT GCC ATC AC-3', reverse primer 5'-TCC ACC ACC CTG TTG CTG TA-3', yielding a product of 433 bp) and the 1 subunit of collagen type IV (forward primer 5'-ATG TCA ATG GCA CCC ATC AC-3', reverse primer 5'-CTT CAA GGT GGA CGG CGT AG-3', yielding a product of 382 bp). Each PCR was performed in a total volume of 40 µl, containing 10 mM TrisHCl pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.06 mg/ml bovine serum albumin (BSA), 0.25 mM dNTPs, 25 pmol forward primer, 25 pmol reverse primer (all primers were purchased from Invitrogen) and 0.8 U of AmpliTaq (Perkin Elmer, Foster City, CA). For GAPDH, amplification started with 5 min at 95°C. This was followed by 35 cycles consisting of the following steps: 94°C for 1 min, 55°C for 1 min and 72°C for 1 min. The final extension was performed at 72°C for 5 min. For COL4A1, amplification started with 5 min of denaturation at 95°C, followed by 35 PCR cycles. Each cycle consisted of the following steps: 95°C for 1 min, 65°C for 0.5 min and 72°C for 1.5 min. The final extension was performed at 72°C for 5 min. All PCRs were performed in a PTC-200 DNA engine thermal cycler (MJ Research, Waltham, MA). PCR products were analysed on a 0.5x TBE 1% agarose gel. To quantitate mRNA in different conditions, cDNA titrations were performed to ensure that the PCR is in the exponential phase. The intensity of bands was determined by densitometry, using EagleSight software (Stratagene, La Jolla, CA). Analysis of data was performed by calculating collagen
1 (IV) mRNA levels relative to those of GAPDH. Measurements of ratios in 5.5 mM glucose medium without supplements were assigned a relative value of 1, representing control values.
Statistical analysis
The data are presented as means±SD of triplicate cultures and are representative of at least three independent experiments. Differences between various culture conditions were evaluated by analysis of variance (ANOVA) with a Bonferroni correction for multiple comparison. In some experiments, relative differences were tested with a one-sample t-test. A value of P<0.05 was considered to represent a significant difference.
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Results |
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Collagen type IV accumulation in fibroblast cultures from different sources
Since ascorbic acid and ß-aminopropionitrile can influence the processing of collagens, we have also investigated collagen type IV accumulation by renal fibroblasts in the absence of these chemicals. Collagen type IV secretion by renal fibroblasts was substantially lower in the absence of ascorbic acid and ß-aminopropionitrile (Figure 2A). Nevertheless, stimulation of fibroblasts under these culture conditions with either TGF-ß1 or 25 mM D-glucose resulted in increased secretion of collagen type IV.
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To exclude the suggestion that collagen type IV secretion by renal fibroblasts was caused by SV40 transformation of the cells, primary fibroblasts derived from skin were tested. As seen in renal fibroblasts, collagen type IV was secreted by primary fibroblasts, and increased after stimulation with 25 mM D-glucose or TGF-ß1 (Figure 2C).
Collagen 1 (IV) chain mRNA expression in fibroblasts
To investigate further the mechanism of collagen type IV expression in renal fibroblasts, collagen 1 (IV) mRNA expression was measured. To quantitate mRNA in different conditions, a semi-quantitative RTPCR was performed by cDNA titration using GAPDH as housekeeping gene (Figure 3A). Stimulation with TGF-ß1 or 25 mM D-glucose for 3 days, but not with 25 mM L-glucose, resulted in the upregulation of collagen
1 (IV) mRNA expression in renal fibroblasts compared with 5.5 mM glucose conditions (Figure 3B). At day 4, TGF-ß1-induced collagen
1 (IV) mRNA expression returned to basal levels. Glucose-induced collagen type IV mRNA expression was also lowered compared with 3 day incubations, although it was still significantly increased compared with the normoglycaemic cultures.
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Discussion |
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The cellular compartment of the tubulointerstitium is composed of TECs and interstitial fibroblasts. The predominant cell types in the tubulointerstitial area are TECs, while interstitial fibroblasts are normally present in low numbers. However, fibroblasts are present in increased numbers during fibrosis, presumably via two different mechanisms: (i) cellular proliferation by resident interstitial fibroblasts [4]; and (ii) epithelial mesenchymal transformation, whereby TECs can undergo phenotypical changes and transform into fibroblast-like cells [5,6]. The precise role and function of the latter cells, which have been observed in vitro, but also in progressive tubulointerstitial fibrosis [5,6], are as yet unknown. Nevertheless, since fibroblasts are major producers of ECM components, the increased numbers of fibroblasts and their activation into myofibroblasts can enhance the fibrotic process by excessive ECM deposition [5,6].
In normal kidneys, collagen types I and III are expressed mainly in the interstitium [8,14], while the expression of collagen type IV is restricted to the glomerular and tubular basement membranes [13]. In tubulointerstitial fibrosis, the composition of the interstitium is altered, including increased presence of collagen type I, III and IV [12,14]. Here, we demonstrate that, while high glucose was able to increase both collagen type I and type III in renal fibroblasts, TGF-ß1 only increased collagen type I secretion. Under no condition was there increased secretion of interstitial collagens by PTECs. In addition to quantitative changes in collagens, expression of novel matrix components in the tubulointerstitium during fibrosis has also been observed: the expression of collagen type IV is no longer restricted to the basement membranes, but is also expressed in the interstitium [8,11,12,14,15]. The aberrant expression of collagen type IV in the interstitium during fibrosis has been attributed predominantly to TECs [8]. Only a few studies have examined the expression of collagen type IV in renal fibroblasts [17], and there is still little known about its regulation and the relative contribution of renal fibroblasts and TECs to the aberrant expression of collagen type IV during fibrosis. We now confirm that human renal fibroblasts secrete collagen type IV. Although basal collagen type IV secretion by fibroblasts was low compared with PTECs, the secretion of collagen type IV by fibroblasts was increased after stimulation with TGF-ß1 and high glucose, reaching levels comparable with those secreted by PTECs. Therefore, we suggest that as well as PTECs, fibroblasts can also contribute to the excessive and aberrant collagen type IV deposition in the renal interstitium.
In our hands, using HK2 cells, exposure to high glucose did not result in an increased collagen type IV production. This is in clear contrast to previous studies with human and murine PTECs [21,22] and might be a specific feature of this cell line. Since our standard protocol to generate primary human PTECs uses DMEM/HAM F-12 which already contains high glucose (17.5 mM), we have not been able to address this question specifically.
The increase in collagen type IV protein secretion was accompanied by an increase in collagen 1 (IV) gene transcription in renal fibroblasts. However, after an initial increase in collagen
1 (IV) mRNA expression by high glucose, the expression levels were already falling after 4 days of incubation. These data seem to conflict with our kinetics study which showed a concomitant continued rise in collagen type IV protein secretion. However, ECM turnover is regulated by synthesis and degradation of matrix components. It is thought that disruption of the balance between synthesis and degradation leads to uncontrolled ECM turnover [24]. Therefore, it is feasible that glucose-induced deposition of ECM is accomplished by an initial increase in ECM synthesis accompanied by increased ECM transcription. Subsequently, ECM accumulation is maintained by a decrease in ECM proteolysis.
One of the most potent mediators of fibrosis is undoubtedly TGF-ß. It has been shown that TGF-ß plays a central role in the development of diabetic nephropathy, and numerous studies have demonstrated the close resemblance of the effect of TGF-ß1 and high ambient glucose in the synthesis of ECM components [19]. Previous studies by our group and others, especially in mesangial cells, have demonstrated that glucose-induced effects were mediated by TGF-ß1 [23]. However, several studies have suggested the existence of TGF-ß-independent pathways. For instance, it has been demonstrated that the expression of the 1 and
5 subunits of collagen type IV induced by glucose is independent of TGF-ß1 in mouse podocytes [25]. We now show that glucose-induced collagen type IV secretion in renal fibroblasts is also independent of endogenous TGF-ß, comparable with our previous findings with fibronectin [10]. Thus, in addition to the interstitial matrix components, basement membrane proteins are also regulated by glucose via a TGF-ß-independent pathway in human renal fibroblasts. These data suggest that the regulation of matrix metabolism under hyperglycaemic conditions is different for renal fibroblasts compared with other renal cell types.
An important question is which alternative factors might be responsible for the observed increased ECM expression in renal fibroblasts. Although direct effects of glucose cannot be excluded, the pronounced delay in kinetics does suggest production of autocrine mediators. Attractive candidates to mediate glucose-induced regulation of ECM in our culture system are angiotensin II, insulin-like growth factor-I and connective tissue growth factor, since these have been implicated as TGF-ß1-independent inducers of ECM [2628].
In conclusion, we have shown that renal fibroblasts, in addition to the interstitial components collagen type I and III, also secrete collagen type IV. Stimulation with glucose resulted in increased secretion of collagen type IV, which was independent of endogenous TGF-ß. These data suggest that renal fibroblasts can contribute to the increased expression of collagen type IV in tubulointerstitial fibrosis observed during diabetic nephropathy.
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Acknowledgments |
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Conflict of interest statement. None declared.
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References |
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