Lack of Integrin α1β1 Leads to Severe Glomerulosclerosis after Glomerular Injury
2004; Elsevier BV; Volume: 165; Issue: 2 Linguagem: Inglês
10.1016/s0002-9440(10)63326-3
ISSN1525-2191
AutoresXiwu Chen, Gilbert Moeckel, Jason D. Morrow, Dominic Cosgrove, Raymond C. Harris, Agnes B. Fogo, Roy Zent, Ambra Pozzi,
Tópico(s)Wound Healing and Treatments
ResumoSeverity of fibrosis after injury is determined by the nature of the injury and host genetic susceptibility. Metabolism of collagen, the major component of fibrotic lesions, is, in part, regulated by integrins. Using a model of glomerular injury by adriamycin, which induces reactive oxygen species (ROS) production, we demonstrated that integrin α1-null mice develop more severe glomerulosclerosis than wild-type mice. Moreover, primary α1-null mesangial cells produce more ROS both at baseline and after adriamycin treatment. Increased ROS synthesis leads to decreased cell proliferation and increased glomerular collagen IV accumulation that is reversed by antioxidants both in vivo and in vitro. Thus, we have identified integrin α1β1 as a modulator of glomerulosclerosis. In addition, we showed a novel pathway where integrin α1β1 modulates ROS production, which in turn controls collagen turnover and ultimately fibrosis. Because integrin α1β1 is expressed in many cell types this may represent a generalized mechanism of controlling matrix accumulation, which has implications for numerous diseases characterized by fibrosis. Severity of fibrosis after injury is determined by the nature of the injury and host genetic susceptibility. Metabolism of collagen, the major component of fibrotic lesions, is, in part, regulated by integrins. Using a model of glomerular injury by adriamycin, which induces reactive oxygen species (ROS) production, we demonstrated that integrin α1-null mice develop more severe glomerulosclerosis than wild-type mice. Moreover, primary α1-null mesangial cells produce more ROS both at baseline and after adriamycin treatment. Increased ROS synthesis leads to decreased cell proliferation and increased glomerular collagen IV accumulation that is reversed by antioxidants both in vivo and in vitro. Thus, we have identified integrin α1β1 as a modulator of glomerulosclerosis. In addition, we showed a novel pathway where integrin α1β1 modulates ROS production, which in turn controls collagen turnover and ultimately fibrosis. Because integrin α1β1 is expressed in many cell types this may represent a generalized mechanism of controlling matrix accumulation, which has implications for numerous diseases characterized by fibrosis. Glomerulosclerosis, the process by which glomerular tissue is replaced by extracellular matrix (ECM), is the final common pathway for loss of functioning glomeruli. All three major cell types that constitute the glomerulus contribute to this process. Podocytes and endothelial cells are likely critical for initiation of sclerosis; however, mesangial cells are the major contributor to progression.1Fogo AB Mesangial matrix modulation and glomerulosclerosis.Exp Nephrol. 1999; 7: 147-159Crossref PubMed Scopus (95) Google Scholar The normal glomerular response to injury is rapid synthesis of ECM components (mainly collagens) that are remodeled as the injured glomerulus undergoes repair. In glomerulosclerosis, ECM accumulation, particularly collagens, is uncontrolled. The synthesis of ECM is, in part, regulated by integrins.2Hynes R Integrins: bidirectional, allosteric signaling machines.Cell. 2002; 110: 673-687Abstract Full Text Full Text PDF PubMed Scopus (7054) Google Scholar Integrin α1β1, a major collagen receptor, is expressed in all cell types in the glomerulus.3Voigt S Gossrau R Baum O Loster K Hofmann W Reutter W Distribution and quantification of alpha 1-integrin subunit in rat organs.Histochem J. 1995; 27: 123-132Crossref PubMed Scopus (26) Google Scholar, 4Korhonen M Ylanne J Laitinen L Virtanen I Distribution of beta 1 and beta 3 integrins in human fetal and adult kidney.Lab Invest. 1990; 62: 616-625PubMed Google Scholar This integrin has been associated with renal disease and is overexpressed in proliferating mesangium in glomerulonephritis.5Shikata K Makino H Morioka S Kashitani T Hirata K Ota Z Wada J Kanwar YS Distribution of extracellular matrix receptors in various forms of glomerulonephritis.Am J Kidney Dis. 1995; 25: 680-688Abstract Full Text PDF PubMed Scopus (59) Google Scholar, 6Kuhara T Kagami S Kuroda Y Expression of beta 1-integrins on activated mesangial cells in human glomerulonephritis.J Am Soc Nephrol. 1997; 8: 1679-1687PubMed Google Scholar In addition, anti-integrin α1 antibodies reduced scarring in rat models of glomerular injury by inhibiting integrin α1β1-dependent (VLA-1) leukocyte function with consequent immune response dampening.7Cook HT Khan SB Allen A Bhangal G Smith J Lobb RR Pusey CD Treatment with an antibody to VLA-1 integrin reduces glomerular and tubulointerstitial scarring in a rat model of crescentic glomerulonephritis.Am J Pathol. 2002; 161: 1265-1272Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar Despite these results, one would expect that lack of integrin α1β1 might predispose to increased glomerulosclerosis because integrin α1β1 is critical for the support of cell proliferation and survival on collagen substrata,8Pozzi A Wary KK Giancotti FG Gardner HA Integrin alpha1beta1 mediates a unique collagen-dependent proliferation pathway in vivo.J Cell Biol. 1998; 142: 587-594Crossref PubMed Scopus (252) Google Scholar as well as for sensing extracellular collagen levels and down-regulating endogenous collagen synthesis.9Gardner H Broberg A Pozzi A Laato M Heino J Absence of integrin alpha1beta1 in the mouse causes loss of feedback regulation of collagen synthesis in normal and wounded dermis.J Cell Sci. 1999; 112: 263-272Crossref PubMed Google Scholar Consequently, loss of α1β1 function results in increased collagen expression9Gardner H Broberg A Pozzi A Laato M Heino J Absence of integrin alpha1beta1 in the mouse causes loss of feedback regulation of collagen synthesis in normal and wounded dermis.J Cell Sci. 1999; 112: 263-272Crossref PubMed Google Scholar and an inability of α1-null cells to proliferate on collagenous substrata.8Pozzi A Wary KK Giancotti FG Gardner HA Integrin alpha1beta1 mediates a unique collagen-dependent proliferation pathway in vivo.J Cell Biol. 1998; 142: 587-594Crossref PubMed Scopus (252) Google Scholar To determine the role of integrin α1β1 in mediating the host response to renal injury, we compared the response of wild-type and integrin α1-null mice to adriamycin (ADR)-induced nephropathy. This is a nonimmunologically mediated injury induced by generation of reactive oxygen species (ROS) that is characterized by mild mesangial sclerosis in BALB/c mice.10Chen A Sheu LF Ho YS Lin YF Chou WY Chou TC Lee WH Experimental focal segmental glomerulosclerosis in mice.Nephron. 1998; 78: 440-452Crossref PubMed Scopus (84) Google Scholar, 11Deman A Ceyssens B Pauwels M Zhang J Houte KV Verbeelen D Van den Branden C Altered antioxidant defence in a mouse adriamycin model of glomerulosclerosis.Nephrol Dial Transplant. 2001; 16: 147-150Crossref PubMed Scopus (56) Google Scholar, 12Wang Y Wang YP Tay YC Harris DC Progressive adriamycin nephropathy in mice: sequence of histologic and immunohistochemical events.Kidney Int. 2000; 58: 1797-1804Crossref PubMed Scopus (265) Google Scholar We demonstrate that α1-null mice on the BALB/c background developed severe glomerular injury, characterized by mesangiolysis within 24 hours of ADR administration and consequent relentless mesangial sclerosis. Excessive ROS production was the major stimulus for the unwarranted excessive synthesis of collagen by both α1-null mice and primary mesangial cells, at steady state levels and after treatment with ADR. Excessive ROS-mediated collagen deposition could be ameliorated, both in vivo and in vitro, by treatment with antioxidants. Together these results demonstrate that integrin α1β1 is a critical modulator of glomerular injury and its functional loss leads to increased sclerosis primarily by stimulating ROS production. All experiments were performed according to institutional animal care guidelines. Wild-type and integrin α1-null BALB/c male mice (5 weeks old, ∼20 g body weight) received a single intravenous injection of ADR (10 mg/kg; Sigma, St. Louis, MO) as described.10Chen A Sheu LF Ho YS Lin YF Chou WY Chou TC Lee WH Experimental focal segmental glomerulosclerosis in mice.Nephron. 1998; 78: 440-452Crossref PubMed Scopus (84) Google Scholar Mice were sacrificed 0, 24, and 72 hours, or 1, 2, 4, 6, and 8 weeks after ADR injection. Seven mice/genotype/treatment were used for this set of experiments. Three independent experiments were performed. When antioxidants were used, mice were treated daily with α-tocopherol acetate (100 mg/kg daily i.p., Sigma) and ascorbic acid (10 mg/ml in drinking water daily, Sigma) beginning 7 days before ADR (ADR + AOX group) until sacrifice (1 week after ADR injection). Some mice received ADR only (ADR group) and others only the antioxidants (AOX group). One week after ADR injection was chosen as an end point because matrix deposition and hyalinosis were primarily observed in α1-null, but not wild-type mice, at this time (Figure 1). Ascorbic acid and α-tocopherol acetate were used at the indicated doses because they have been shown to ameliorate ROS-mediated injury in rodents.13Ricardo SD Bertram JF Ryan GB Antioxidants protect podocyte foot processes in puromycin aminonucleoside-treated rats.J Am Soc Nephrol. 1994; 4: 1974-1986PubMed Google Scholar, 14Naziroglu M Cay M Ustundag B Aksakal M Yekeler H Protective effects of vitamin E on carbon tetrachloride-induced liver damage in rats.Cell Biochem Funct. 1999; 17: 253-259Crossref PubMed Google Scholar Four mice/genotype/treatment were used for this set of experiments. Two independent experiments were performed. Urine was collected 24 hours before sacrifice and urinary albumin was measured by the ELISA Albuwell M test kit (Exocel Inc., Philadelphia, PA) and expressed as mg/L. F2-isoprostanes were measured in urine using stable isotope dilution methods, gas chromatography negative ion chemical ionization-mass spectrometry,15Morrow JD Zackert WE Yang JP Kurhts EH Callewaert D Dworski R Kanai K Taber D Moore K Oates JA Roberts LJ Quantification of the major urinary metabolite of 15-F2t-isoprostane (8-iso-PGF2alpha) by a stable isotope dilution mass spectrometric assay.Anal Biochem. 1999; 269: 326-331Crossref PubMed Scopus (144) Google Scholar and expressed as ng/mg urine creatinine. Blood was collected immediately at sacrifice and serum creatinine was measured using a commercially available colorimetric kit (Sigma) and expressed as mg/dL/g body weight. Kidneys, removed immediately at sacrifice, were either fixed in 4% formaldehyde and embedded in paraffin for both morphological and immunohistochemistry analysis, or depleted of medulla and frozen for mRNA analysis. Paraffin tissue sections were stained with periodic acid-Schiff or Jones' for the evaluation of glomerular injury. Mesangial matrix expansion index (MMEI) was evaluated in 100 glomeruli/kidney by a single blinded observer (G.M.) and expressed by percentage as normal, mild, and severe (0%, 1 to 30%, and >30% of glomerular tuft with visible expanded mesangial matrix, respectively). Kidneys (n = four or seven/genotype) were evaluated and the MMEI expressed as mean ± SD for each group. Portions of renal cortex were fixed in 2.5% glutaraldehyde in phosphate buffer. Samples were postfixed in OsO4, dehydrated in ethanol, and embedded in resin. Thin sections were assessed for mesangiolysis, mesangial matrix deposition, and podocyte injury. Foot process effacement (FPE) was semiquantitatively assessed (percent of effacement in 10 peripheral capillary loops/glomerulus with a total of seven kidneys/genotype evaluated) and expressed as mean ± SD for each study group. Immunohistochemistry on paraffin kidneys sections was done using rabbit anti-mouse collagen IV antibodies (1:100; Biodesign, Saco, ME), or rabbit anti-mouse proliferating cell nuclear antigen (PCNA) (1:100; Santa Cruz, Santa Cruz, CA), followed by horseradish peroxidase-conjugated goat secondary antibody to rabbit IgG (1:200; Jackson Immunoresearch, West Grove, PA) and Sigma Fast DAB chromogenic tablets (Sigma). The glomerular proliferation index was expressed as (number of PCNA-positive glomerular cells/total number of glomerular cells) × 100. Glomerular apoptosis was evaluated by staining paraffin section with the Dead End colorimetric terminal dUTP nick-end labeling (TUNEL) system (Promega, Madison, WI) using diaminobenzidine as the chromogenic substrate. The glomerular apoptotic index was expressed as (number of TUNEL-positive glomerular cells/total number of glomerular cells) × 100. Ten glomeruli/kidney were evaluated with a total of seven kidneys/genotype/treatment. To determine collagen deposition by mesangical cells, 3 × 104 cells were plated for 48 hours on uncoated four-well chamber slides in the presence of Dulbecco's modified Eagle's medium (DMEM) containing 2% fetal calf serum (FCS) and 0, 0.1, 0.5, and 1 μmol/L ADR in the presence of the antioxidants TEMPOL (10 μmol/L) and DPI (1 μmol/L) (both from Sigma). The doses of TEMPOL, a superoxide mimetic, and DPI, a specific NADPH oxidase inhibitor, were chosen because they represent the maximum dose able to inhibit ROS generation in wild-type and α1-null mesangial cells without cytotoxic effects (data not shown). Immunofluorescence on acetone-fixed cells was done using the rabbit anti-mouse collagen IV antibodies described above (1:100) followed by fluorescein isothiocyanate-conjugated goat secondary antibody to rabbit IgG (1:100; Jackson) and 2 μg/ml 4,6-diamidino-2-phenylindole to visualize nuclei. To determine collagen deposition by mesangial cells, 3 × 105 cells were plated on uncoated six-well plates in the presence of DMEM containing 2% FCS and in the absence or presence of TEMPOL (10 μmol/L) and DPI (1 μmol/L). After 48 hours, cells were scraped, suspended in 50 mmol/L Hepes, pH 7.5, 150 mmol/L NaCl, 1% Triton X-100, and centrifuged 10 minutes at 14,000 rpm. Cell lysates were resolved by 5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Gels were either stained with Coomassie Blue to evaluate equal loading, or transferred to Immobilon-P membranes (Millipore, Billerica, MA). Membranes were incubated with a rabbit anti-collagen IV antibody (Biodesign) and immunoreactive proteins visualized using a peroxidase-conjugated goat anti-rabbit and an ECL kit (Pierce, Rockford, IL). Total RNA from renal cortices (n = seven kidneys/genotype) was isolated using a single-step isolation kit (TRIzol; Invitrogen, Carlsbad, CA). Ten μg of total RNA was separated in formaldehyde-containing 1% agarose gels, transferred to nylon membranes (Nytran Supercharge; Schleicher & Schuell, Keene, NH), and hybridized with 32P-labeled cDNAs for mouse α1(IV) collagen. Mouse β2-tubulin was used for normalization. Collagen IV and tubulin bands were quantified by densitometry analysis using an Alpha Imager 2000 (Alpha Innotech, San Leandro, CA) and collagen IV signal expressed as collagen IV/tubulin. Primary mesangial cells were isolated and characterized as previously described.16Striker GE Killen PD Farin FM Human glomerular cells in vitro: isolation and characterization.Transplant Proc. 1980; 12: 88-99PubMed Google Scholar For proliferation assays, 5 × 103 primary mesangial cells were plated in DMEM containing 2% FCS on to 96-well plates coated with 10 μg/ml fibronectin or collagen IV (both from Sigma). In some experiments, mesangial cells were cultured in DMEM containing 2% FCS in the presence of different concentrations of ADR (0, 0.1, 0.5, 1, and 5 μmol/L) alone or in combination with TEMPOL (10 μmol/L) and DPI (1 μmol/L). After 2 days the medium was replaced, the cells were then pulsed for an additional 48 hours with 3H-thymidine (1 μCi/well) and processed as described.17Pozzi A Moberg PE Miles LA Wagner S Soloway P Gardner HA Elevated matrix metalloprotease and angiostatin levels in integrin alpha 1 knockout mice cause reduced tumor vascularization.Proc Natl Acad Sci USA. 2000; 97: 2202-2207Crossref PubMed Scopus (351) Google Scholar Manual cell counts paralleled the results of 3H-thymidine incorporation (data not shown). A suspension of wild-type or α1-null mesangial cells was incubated with monoclonal antibodies of the appropriate integrin (ie, anti-α1, -α2, -α5 and -β1 integrins all from Pharmingen, San Diego, CA) (1:100 dilution), followed by incubation with the appropriate secondary antibodies (fluorescein isothiocyanate-coupled rabbit anti-rat or hamster immunoglobulin) (1:100 dilution). Flow cytometry was performed with a FACScan instrument (Becton Dickinson, Franklin Lakes, NJ). Cell suspensions incubated with secondary antibody only were used as a negative control for integrin expression. Two independent experiments were performed in duplicate. Microtiter plates (96-well) were coated with fibronectin or collagen IV at the indicated concentrations in phosphate-buffered saline (PBS) for 1 hour at 37°C. Plates were then washed with PBS and incubated with PBS containing 1% bovine serum albumin for 60 minutes to block nonspecific adhesion. One hundred μl of single-cell suspensions (5 × 105 mesangial cells/ml) in serum-free DMEM containing 0.1% bovine serum albumin were added in quadruplicate to 96-well plates and incubated for 60 minutes at 37°C. In some experiments, cell suspensions were preincubated with anti-integrin antibodies (anti-α1, -α2, and -β1 integrins) (10 μg/ml final concentration) on ice for 30 minutes before the assay. Nonadherent cells were removed by washing the wells with PBS. Cells were then fixed with 4% formaldehyde, stained with 1% crystal violet, solubilized in 20% acetic acid, and then read at 570 nm. Background cell adhesion to 1% bovine serum albumin-coated wells was subtracted from the values obtained on ECM proteins. Two independent experiments were performed in quadruplicate. ROS in mesangial cells were measured using the substrate dihydro-rhodamine (Molecular Probes, Eugene, OR).18Zent R Ailenberg M Waddell TK Downey GP Silverman M Puromycin aminonucleoside inhibits mesangial cell-induced contraction of collagen gels by stimulating production of reactive oxygen species.Kidney Int. 1995; 47: 811-817Crossref PubMed Scopus (18) Google Scholar Briefly, 15 × 104 mesangial cells were plated in six-well plates coated with 10 μg/ml of fibronectin or collagen IV in DMEM containing 10% FCS. After 24 hours, cells were incubated for a total of 6 hours with DMEM containing 2% FCS, 30 U/ml horseradish peroxidase, 2 μmol/L dihydro-rhodamine, and different concentrations of ADR (0, 0.1, 1, and 5 μmol/L) alone or together with TEMPOL (10 μmol/L) and/or DPI (1 μmol/L). Cells were then trypsinized, washed twice in PBS and the generation of fluorescent rhodamine 123 was analyzed with a FACScan (λex = 488 nm, λem = 525 nm) as described.18Zent R Ailenberg M Waddell TK Downey GP Silverman M Puromycin aminonucleoside inhibits mesangial cell-induced contraction of collagen gels by stimulating production of reactive oxygen species.Kidney Int. 1995; 47: 811-817Crossref PubMed Scopus (18) Google Scholar Three independent experiments were performed in duplicate. We used the t-test for comparisons between two groups, and analysis of variance using Sigma-Stat software for statistical differences between multiple groups. P ≤ 0.05 was considered statistically significant. A single injection of ADR (10 mg/kg), previously shown to induce renal injury in BALB/c mice,11Deman A Ceyssens B Pauwels M Zhang J Houte KV Verbeelen D Van den Branden C Altered antioxidant defence in a mouse adriamycin model of glomerulosclerosis.Nephrol Dial Transplant. 2001; 16: 147-150Crossref PubMed Scopus (56) Google Scholar, 12Wang Y Wang YP Tay YC Harris DC Progressive adriamycin nephropathy in mice: sequence of histologic and immunohistochemical events.Kidney Int. 2000; 58: 1797-1804Crossref PubMed Scopus (265) Google Scholar was given to both wild-type and integrin α1-null mice on the BALB/c background. The wild-type mice developed an increase in serum creatinine 2 weeks after ADR injection (Figure 1A), which persisted until week 6 and started to recover by week 8. These mice also developed significant proteinuria that peaked 4 weeks after ADR but was still present at week 8. In the α1-null mice, the levels of serum creatinine and albuminuria were significantly worse when compared to wild-type mice at all time points (Figure 1A). Although integrin α1-null mice develop normally and have no overt renal phenotype,19Gardner H Kreidberg J Koteliansky V Jaenisch R Deletion of integrin alpha 1 by homologous recombination permits normal murine development but gives rise to a specific deficit in cell adhesion.Dev Biol. 1996; 175: 301-313Crossref PubMed Scopus (230) Google Scholar their glomeruli are smaller with reduced glomerular tuft area and evidence of mild mesangial matrix accumulation (in 20% of glomeruli), when compared to wild-type controls (Figure 1, B and C). Within 24 hours of ADR injection, kidneys from α1-null mice, but not wild-type mice, showed evidence of mesangial expansion, focal mesangiolysis with multiple small electron-lucent mesangial lesions, and increased FPE (31 ± 6.7% in the α1-null versus 10 ± 6.5% in wild-type; P < 0.007; n = 3) (Figure 1, B and D). By 72 hours the mesangial expansion and FPE had further increased in α1-null versus wild-type mice (67 ± 12% in the α1-null versus 30 ± 10% in wild-type; P < 0.01; n = 3) (Figure 1, B and D). At 1 week glomerular matrix deposition and hyalinosis became evident in α1-null mice, but not in their wild-type counterparts. Although wild-type kidneys showed mesangial expansion because of both mesangial cell proliferation and increased mesangial matrix deposition (measured by MMEI) by week 4 as previously reported,11Deman A Ceyssens B Pauwels M Zhang J Houte KV Verbeelen D Van den Branden C Altered antioxidant defence in a mouse adriamycin model of glomerulosclerosis.Nephrol Dial Transplant. 2001; 16: 147-150Crossref PubMed Scopus (56) Google Scholar mesangial expansion was far more severe in the α1-null mice at this time (Figure 1, B and C). By week 8 glomerulosclerosis was well developed in the wild-type mice and 40% of glomeruli demonstrated severe MMEI scores (Figure 1, B and C). The pathology in the α1-null mice was significantly worse at the same time point and 60% of glomeruli showed severe MMEI scores (Figure 1, B and C). There was also worse interstitial damage of the kidneys in the α1-null mice particularly evident at 4 and 8 weeks (Figure 1B). Ultrastructural examination of kidneys from wild-type mice showed segmental FPE 4 weeks after ADR administration, which became more widespread by week 8 (Figure 1D). In contrast, there was severe diffuse FPE involving all of the capillary loops in the α1-null mice at week 4 and by week 8, the FPE was near complete (Figure 1D). Although there was evidence of increased mesangial ECM deposition in wild-type mice at 4 weeks that progressed by week 8, the increase in ECM accumulation in α1-null mice was significantly greater at both these time points. To determine the etiology of the mesangial expansion in the ADR-treated mice we initially assessed the proliferation and apoptosis index in glomeruli of untreated and ADR-treated mice. We found a significant increase in the number of PCNA-positive cells primarily in the glomeruli of wild-type mice. This increase was evident 1 week after ADR injection (40 ± 7% in wild-type versus 25 ± 8% in α1-null) and persisted throughout the duration of the study period (Figure 2A). Because leukocyte infiltration has been described in the ADR-induced injury model,11Deman A Ceyssens B Pauwels M Zhang J Houte KV Verbeelen D Van den Branden C Altered antioxidant defence in a mouse adriamycin model of glomerulosclerosis.Nephrol Dial Transplant. 2001; 16: 147-150Crossref PubMed Scopus (56) Google Scholar, 12Wang Y Wang YP Tay YC Harris DC Progressive adriamycin nephropathy in mice: sequence of histologic and immunohistochemical events.Kidney Int. 2000; 58: 1797-1804Crossref PubMed Scopus (265) Google Scholar and integrin α1-null mice show a defect in leukocyte infiltration to the site of injury,20de Fougerolles AR Sprague AG Nickerson-Nutter CL Chi-Rosso G Rennert PD Gardner H Gotwals PJ Lobb RR Koteliansky VE Regulation of inflammation by collagen-binding integrins alpha1beta1 and alpha2beta1 in models of hypersensitivity and arthritis.J Clin Invest. 2000; 105: 721-729Crossref PubMed Scopus (190) Google Scholar, 21Meharra EJ Schon M Hassett D Parker C Havran W Gardner H Reduced gut intraepithelial lymphocytes in VLA1 null mice.Cell Immunol. 2000; 201: 1-5Crossref PubMed Scopus (47) Google Scholar we assessed whether the differences in number of PCNA-positive cells could be because of lymphocyte and/or macrophage infiltration. Both macrophage and lymphocyte infiltration (assessed by staining kidney sections with anti-F4/80 and CD3 antibody, respectively) within glomeruli was minimal in both genotypes and no significant differences were observed at any time point examined (data not shown). Because early mesangiolysis was observed within 24 hours of ADR administration in the α1-null background, we assessed apoptosis at this early time point in both genotypes. As shown in Figure 2B, there was significantly more apoptosis in the α1-null mice compared to their wild-type counterparts at early time points. At later stages (weeks 1 to 8) apoptotic cells were difficult to visualize in the glomeruli of both genotypes. Taken together, these results suggest that increased glomerular proliferation of mesangial cells was not the etiology of the excessive mesangial expansion in the α1-null mice. One of the characteristics of ADR-induced glomerular injury is increased expression and accumulation of collagen type IV in the glomerular tuft and Bowman's capsule.10Chen A Sheu LF Ho YS Lin YF Chou WY Chou TC Lee WH Experimental focal segmental glomerulosclerosis in mice.Nephron. 1998; 78: 440-452Crossref PubMed Scopus (84) Google Scholar Because integrin α1 is a primary receptor for collagen IV19Gardner H Kreidberg J Koteliansky V Jaenisch R Deletion of integrin alpha 1 by homologous recombination permits normal murine development but gives rise to a specific deficit in cell adhesion.Dev Biol. 1996; 175: 301-313Crossref PubMed Scopus (230) Google Scholar and is critical in down-regulating endogenous collagen synthesis,9Gardner H Broberg A Pozzi A Laato M Heino J Absence of integrin alpha1beta1 in the mouse causes loss of feedback regulation of collagen synthesis in normal and wounded dermis.J Cell Sci. 1999; 112: 263-272Crossref PubMed Google Scholar we analyzed and compared collagen IV deposition in the glomeruli of ADR-treated wild-type and α1-null mice. Collagen IV mRNA and protein accumulation in α1-null mice was increased 1 week after ADR injection and persisted throughout the study (Figure 2, C and D). Significantly less collagen IV synthesis and deposition were observed in wild-type mice at all time points examined (Figure 2, C and D). There was little or no increase in glomerular collagen I deposition in either the wild-type or α1-null mice at any of the time points (data not shown), suggesting that the primary cause of the increased mesangial expansion in the α1-null mice was because of excessive collagen IV expression. Our in vivo observation that ADR leads to early mesangiolysis with increased apoptosis and consequent mesangial expansion and glomerulosclerosis in α1-null mice (Figure 1, B and D; Figure 2B) strongly suggested that mesangial cells are a target for ADR.18Zent R Ailenberg M Waddell TK Downey GP Silverman M Puromycin aminonucleoside inhibits mesangial cell-induced contraction of collagen gels by stimulating production of reactive oxygen species.Kidney Int. 1995; 47: 811-817Crossref PubMed Scopus (18) Google Scholar We therefore isolated primary mesangial cells to investigate the mechanisms for the increased ADR-induced mesangial injury in α1-null mice. We initially determined the role of integrin α1β1 on mesangial cell adhesion to collagen IV. We established that mesangial cells express both integrins α1β1 and α2β1 and integrin α1-null cells do not compensate for lack of α1β1 by overexpressing the other major collagen receptor α2β1 (Figure 3A). Furthermore, the expression of integrin α5 (fibronectin binding receptor) and β1 subunits was comparable in both genotypes (Figure 3A). We then tested the ability of mesangial cells of both genotypes to adhere to collagenous substrata. As demonstrated in Figure 3B, no differences in adhesion were observed between wild-type and integrin α1-null cells plated on fibronectin (an integrin α5β1-dependent ligand) (Figure 3B). In contrast, despite expressing the integrin α2 subunit, α1-null mesangial cells showed significantly reduced adhesion on collagen IV compared to their wild-type counterparts (Figure 3C), suggesting that integrin α1β1 is the major collagen IV-binding receptor in the mesangium. To further verify that mesangial cell adhesion on collagen IV was mediated by integrin α1β1, mesangial cells of both genotypes were plated on collagen IV in the absence or presence of anti-α1, -α2, and -β1 integrin antibodies. As shown in Figure 3D, adhesion of wild-type mesangial cells was significantly impaired after incubation with anti-α1 or -β1 but not anti-α2 antibodies and was similar to that of α1-null mesangial cells. Taken together, these results suggest that mesangial cell adhesion to collagen IV is an integrin α1β1-dependent event. We subsequently tested the ability of mesangial cells to proliferate on both collagenous and noncollagenous substrata, by assaying 3H-thymidine incorporation.17Pozzi A Moberg PE Miles LA Wagner S Soloway P Gardner HA Elevated matrix metalloprotease and angiostatin levels in integrin alpha 1 knockout mice cause reduced tumor vascularization.Proc Natl Acad Sci USA. 2000; 97: 2202-2207Crossref PubMe
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