1 Department of Nephrology, Royal Melbourne Hospital, Parkville and 2 Microvascular Biology and Wound Healing Group, RMIT University, Bundoora, Victoria, Australia
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Abstract |
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Methods. Renal fibroblasts were grown from the cortical tissue of surgically removed human kidneys. The ability of human renal fibroblasts to reorganize interstitial collagen I was examined in vitro using solidified collagen I lattices. Integrin function was blocked by incubating fibroblasts with isotype-specific antibodies prior to addition to collagen I lattices.
Results. Human renal fibroblasts embedded in collagen I lattices progressively decreased lattice diameter to 60.6±11.4% of initial diameter at 48 h post-release (P<0.01). Fibroblasts incubated in the presence of antibody to ß1 integrin failed to contract collagen I lattices, whilst fibroblasts incubated with non-specific antibody reduced lattice diameter to 60.1±12.4% of initial diameter at 48 h post-release (P<0.01). Further characterization of integrin subunits showed that blocking
2ß1 integrin prevented lattice contraction (P<0.05,
2ß1 integrin antibody vs non-specific antibody), whilst blocking of
5ß1,
3ß1 and
1ß1 integrins did not influence this process.
Conclusions.We postulate that collagen I fibril rearrangement by human renal fibroblasts in vitro appears to be an integrin-mediated process involving the 2ß1 integrin.
Keywords: collagen lattices; contraction; fibroblast; integrin; renal; scar formation
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Introduction |
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Increasingly it is being recognized that fibrosis is a consequence of several overlapping processes. These include not only increased matrix synthesis (fibrogenesis), but also decreased degradation (fibrolysis) and contraction of surrounding extracellular matrix [24].
The demonstration by us of a temporal relationship between renal myofibroblasts and reducing scar size suggests that fibroblast-mediated contraction of interstitial collagens contributes to renal scar formation by increasing the relative density of extracellular matrix [4]. However, the mechanism by which renal fibroblasts bind to collagenous proteins is unknown.
In skin wounds, cell-mediated adhesion and reorganization of granulation tissue is an integrin-dependent process [5]. The integrins are an ß heterodimeric group of cell-surface receptors that are responsible for both movement through and communication with fibrillar and extracellular matrix (ECM) proteins. They have been implicated in many biological processes including platelet aggregation, immune functions, tumour invasion, and tissue remodelling [6].
The ß1 integrins in turn are a family of cell-surface receptors, which mediate cell-matrix interaction [6,7]. Fibroblasts and other adult mesenchymal cells express the ß1 integrins, specific isotypes of which mediate binding to fibronectin (5ß1,
3ß1), and the collagens (
1ß1,
2ß1 and
3ß1) [6,7]. All three collagen receptors have been shown to interact with collagen I in vitro [8]. Upregulation of integrins has been demonstrated in a number of human renal diseases [911], where they have been shown to correlate with histological damage [12].
We therefore investigated the contractile properties of human renal fibroblasts in vitro and the role of integrins in cellmatrix interactions with collagen I matrix.
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Subjects and methods |
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Specific antibodies were used to block the function of ß1 integrins to assess the role of integrins in the process of fibroblast-mediated ECM contraction and to define the receptor responsible for renal fibroblastcollagen I binding. In addition, leukocyte common antigen (LCA) was used as a non-specific antiserum to control for the effect of antisera addition to the collagen lattice system. Fibroblast populations do not express this protein. To control for intra-assay variation, each set of integrin antisera lattices was poured along with a set of control and non-specific antibody lattices.
The rate of contraction of collagen I lattices by human renal fibroblasts was compared to a homogeneous population of alpha smooth-muscle actin (SMA)-positive rat fibroblasts in an attempt to examine the involvement of
SMA expression in this process.
Patients
Fresh renal cortical tissue was obtained from five patients who underwent surgical nephrectomy at the Royal Melbourne Hospital. The patient study group comprised four males and one female between the ages of 38 and 64 years (mean age 54 years) at the time of operation. Patient details are provided in Table 1. The study protocol was approved by the Royal Melbourne Hospital Clinical Research Ethics Committee.
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Cell culture
Renal fibroblasts were grown by explantation from the cortical tissue of human kidneys (n=5). The specimens were collected separately in Hank's buffered salt solution supplemented with gentamicin (0.025%). Small pieces of cortical tissue (1 mm3 per plate) were minced into the base of 6-cm2 Petri dishes and flooded with Dulbecco's modified Eagle's medium (DMEM) and 20% fetal calf serum (FCS) (Commonwealth Serum Laboratories, Parkville, Australia) containing antibiotics (standard growth media) and L-valine (46 mg/ml) (Sigma Chemical Company, St Louis, MO, USA) and incubated at 37°C with 95% air/5% CO2.
Growth media (DMEM and 20% FCS) was changed every alternate day after cultured cells could be seen in dishes (approximately 10 days). Cells reached confluence in Petri dishes 2028 days after setup. After this time, subcultures were established by transferring cells into sterile flasks (Nunc, Roskilde, Denmark). Cell monolayers were rinsed twice with sterile 0.01 mol/l phosphate-buffered saline (warmed to 37°C). They were lifted from the flask surface using 1xtrypsin/ethylenediamine tetra-acetic acid (1 : 250, 0.25% w/v) (ICN Biochemicals, Costa Mesa, CA, USA) for 5 min at 37°C. Only human cells between passages 3 and 8 (inclusive) were used for experimentation.
All cells were characterized on the basis of immunohistochemistry and in vitro growth patterns. Fibroblasts were defined by positive immunohistochemical staining for D7-FIB (fibroblast marker) (Serotec, Oxford, UK), vimentin (mesenchymal cell marker), and collagens I and III, and negative staining for endothelial (von Willebrand factor (vWF)), mesangial (desmin) and epithelial (cytokeratin) cell markers. In addition, monolayer growth characteristics at confluence were used to delineate cell phenotype, as described previously [16].
A homogeneous population of SMA-positive rat myofibroblasts were grown in DMEM and 20% FCS with antibiotics, and characterized according to methods described previously [13]. HeLa cell cultures (Commonwealth Serum Laboratories) were maintained in Roswell Park Memorial Institute 1640 and 10% FCS supplemented with antibiotics at 37°C with 95% air/5% CO2.
Immunohistochemical characterization of cells
Cells grown on sterile glass coverslips were fixed for 5 min in ice-cold methanol and stored at 4°C before staining [13]. Immunoperoxidase procedures were carried out as described previously [13] using the following primary antibodies: mouse anti-human vimentin, SMA, cytokeratin, desmin, rabbit anti-human vWF (Dako, Glostrup, Denmark), mouse anti-human D7-FIB (fibroblast marker) [17], and goat anti-human collagen I and III (Southern Biotechnology, Birmingham, AL, USA).
Briefly, cells were incubated with primary antibody after endogenous peroxidase activity was quenched using 3% hydrogen peroxide in methanol at room temperature. The cells were then incubated with a biotinylated secondary antibody and detected using an avidinbiotin complex (Vectastain ABC Elite kits, Vector Laboratories, Burlingame, CA, USA). The chromogen substrate used was diaminobenzidine (DAB) (Dako) followed by DAB-enhancing solution (Dako), both prepared according to manufacturer's instructions. Harris's haematoxylin was used to aid definition of cells.
Immunolocalization of integrins on cultured cells
Immunofluorescence procedures were carried out using an anti-mouse Ig-fluorescein isothiocyanate secondary antibody and the following primary antibodies: mouse anti-human 1ß1 integrin (Chemicon International, Temecula, CA, USA), mouse anti-human
2ß1 integrin,
3ß1 integrin,
5ß1 integrin, and ß1 integrin (all Becton Dickinson, Franklin Lakes, NJ, USA). Cellular localization of integrins was viewed using confocal microscopy (Biorad, Hercules, CA, USA).
Northern hybridization
Total cellular RNA was isolated from confluent cell monolayers using Trizol® reagent (Gibco Life Sciences Inc., Grand Island, NY, USA) in accordance with the manufacturer's instructions. Isolated RNA was denatured and electrophoresed through a 1% agarose gel containing formaldehyde (2.2 mol/l). RNA for experimentation was then transferred and UV-fixed onto Hybond® nylon membranes (Amersham International, Buckinghamshire, UK). Northern blot analysis was used to determine expression of ß1 integrin (cDNA probe kindly donated by Dr Alan Wildeman, University of Guelph, Ontario, Canada) [13]. Any differences in RNA loading were corrected by probing blots with an 18s ribosomal RNA cDNA probe. Autoradiograph signals were quantified using densitometry and data is represented as ß1integrin : 18s mRNA ratio for each sample.
Cell-populated collagen I lattices
Collagen lattice contraction assays were performed by modifying the methods of Gotwals et al. [18]. Cells were removed from flasks by trypsinization and counted using a haemocytometer. After being resuspended in standard growth media, 1x106 cells were incorporated into control lattices. As integrins are cell-surface receptors, they can be blocked using function-blocking antibodies before they come into contact with their substrate. In antisera blocking experiments, 1x106 cells were resuspended in media in sterile tubes containing relevant monoclonal antibodies (see Antibody preparation), and incubated at 37°C with 95% air for 15 min prior to collagen lattice preparation [18].
Collagen lattices were prepared by mixing the resuspended cells with 1.8 ml acid-solubilized collagen I (final collagen concentration 2.1 mg/ml) (ICN Biochemicals) in 24-mm diameter cluster dishes (ICN Biochemicals). Lattices were solidified at 37°C with 95% air/5% CO2 before being dislodged from the sides of the dish with a sterile scalpel blade. Five lattices were poured for each of the antibodies tested. To control for interassay variation, five control and five LCA lattices were also poured at this time.
Cell-mediated contraction of collagen I substrate was measured hourly for 6 h beginning immediately after lattice dislodgment (T0T6) and then at 24 and 48 h post-release (T24 and T48 respectively). Contraction, determined by a decrease in lattice diameter, was measured using a metric ruler as described previously [13,14]. In order to validate diameter as a legitimate estimation of contraction, the volume of 22 lattices was determined by water displacement and compared to diameter measurements.
Antibody preparation
Monoclonal monomeric antibodies to human sequences of 1 (clone FB12) (Chemicon International),
2 (clone P1E6),
3 (clone P1B5),
5 (clone mAb16) and ß1 (clone mAb13) integrins (all Becton Dickinson) were dialysed overnight against sterile water to remove sodium azide. These clones had previously been shown to block integrin function in concentration dependency studies using human skin fibroblasts [19,20]. Antibodies were used at the same protein concentration (0.02 mg/ml diluted in standard growth media) and sterilized through a 0.2 µm filter before use.
A monoclonal antibody to human LCA (Dako) was also dialysed and used as a non-specific antiserum. This antibody was also used at 0.02 mg/ml.
Statistics
Data was analysed using one-way ANOVA with correction for multiple comparisons or Wilcoxon rank sum test as appropriate. As data was normally distributed, it is represented as mean±SD. The mean of five lattices was used to determine P values.
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Results |
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The human renal cell cultures grown from these samples were defined as fibroblasts on the basis of immunohistochemistry and in vitro growth patterns. These features were similar in cell cultures derived from all five patients. No qualitative relationships between cell viability and age were noticed. In culture, they appeared to mimic their in vivo counterparts. At confluence, cultured cells grew in fingerprint configurations but did not cluster upon one another, as is a characteristic of mesangial cell growth [13]. Furthermore, at no point did they exhibit the cobblestone appearance associated with epithelial and endothelial cell cultures. When characterized at passage 3, more than 95% of these cells were positive for vimentin and D7-FIB (fibroblast marker) (Figure 1), but were negative for cytokeratin (epithelial cell marker) and vWF (endothelial cell marker). Further, cells were consistently negative for desmin, a cytoskeletal marker used to define cultured mesangial cells [16]. In addition, they stained positively for collagens I and III.
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Integrin expression |
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Lattice contracture |
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Contractile mediators |
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Diameter values for the five sets of control and LCA lattices were found to be within 3 mm of each other.
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Effect of ![]() |
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Discussion |
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Human renal fibroblasts possess features that enable them to remodel collagen I and we have shown previously, along with others, the importance of cytoskeletal actin reorganization in the contraction of collagen I lattices [13]. By blocking the rearrangement of actin filaments using cytochalasin D, we were able to prevent cell-mediated contraction of collagen I lattices.
The upregulation of cellular integrin expression has been documented in a number of renal fibrocontractive diseases [9,10,12]. In particular, the change in distribution of ß1 integrins by cells within the glomerulus has been associated with human progressive glomerulonephritis, including IgA nephritis and minimal-change nephrotic syndrome [10]. Further, the association of 2ß1 integrin expression on cells in areas of cortical scarring has been described in human renal biopsies [12]. Mesangial cells express low level ß1 integrins in the normal glomerulus in vivo but express
SMA and higher levels of
1ß1 and
5ß1 integrins in response to injury [21]. The upregulation of these markers suggest a phenotypic change that results in the activation of mesangial cells, and is accompanied by production of matrix proteins. Further, upregulation of rat mesangial cell
1ß1 integrin is associated with increased matrix-contracting ability of cells and appears to be modulated at least in part by cytokines [21].
In vitro, human renal fibroblasts express all three integrins of the ß1 family known to bind collagen I matrix: 1ß1,
2ß1 and
3ß1. Within the limits of this study, we have indicated that
2ß1 integrin mediates human real fibroblast-collagen I matrix interactions. Similarly, when human dermal fibroblasts are cultured within collagen I matrix, an increase in
2ß1 integrin is observed [22]. However, more than one collagen receptor may be involved in the process of fibroblast-mediated collagen I lattice contraction.
Cultured cells may express various ß1 integrins simultaneously and alter their expression in response to changes in the local environment [11,23]. During wound healing, increased deposition of fibronectin and its receptor 5ß1 integrin occur simultaneously in skin fibroblast cultures [5,24]. Clark and co-workers observed that the increased expression of fibronectin receptors on skin fibroblasts could be attributed to increased platelet-derived growth factor concentrations in culture [24]. In addition, both fibronectin and
5ß1 integrin appear to be upregulated in response to injury in both tracheal wound healing and during the formation of granulation tissue, where they are associated with periods of local cell proliferation and migration [25]. Further, fibronectin has long been thought of as an intermediary protein which serves to bind and link interstitial collagens to the cell via actin [5]. For this reason, we examined the effect of blocking
5ß1 integrin expression on collagen I lattice contraction. As human renal fibroblasts contracted collagen I lattices in the presence of
5ß1 integrin antiserum, our data suggests that the
5ß1 fibronectin receptor is not involved in this instance. Further, current literature suggests that integrin expression and use is both a species- and cell- specific phenomenon [21,26,27]. In light of this, and given the promiscuity of integrins, other fibronectin receptors not tested in this study may have a role in fibroblast contraction of collagen I.
While previous studies have documented the importance of SMA expression in cells able to contract lattices in vitro [28], our work suggests that the expression of matrix receptors may be of greater importance in the process of matrix manipulation and subsequent fibril contraction in this instance. It appears that the
SMA-positive cells were not responsible for the lattice contraction observed using human fibroblast cultures. Equal numbers of rat myofibroblasts and human renal fibroblasts contracted collagen I lattices to a similar extent. However, less contraction was observed when a reduced number of rat myofibroblasts were treated under the same conditions. This preliminary data suggests that a sufficient number of
SMA-positive cells did not exist within human renal fibroblast cultures to generate contraction of collagen I lattices under the experimental conditions described. However, further experiments are required to test this hypothesis. Cultured human fibroblasts derived from skin biopsies failed to express
SMA, yet were able to rearrange solidified collagen I matrices in response to growth factors present in serum [24]. This preliminary work therefore supports the hypothesis that fibroblast traction through collagen lattices is thought to result in physical rearrangement of collagen fibres into bundles [14]. However, the lack of fibroblast-specific markers has been one of the limitations of working with cultured human renal cortical interstitial cells. Given that renal fibroblasts cannot currently be distinguished from their counterparts in other organs, this may not be a renal-specific phenomenon.
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Conclusions |
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Acknowledgments |
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Notes |
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References |
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