αvβ6 Integrin Regulates Renal Fibrosis and Inflammation in Alport Mouse
2007; Elsevier BV; Volume: 170; Issue: 1 Linguagem: Inglês
10.2353/ajpath.2007.060158
ISSN1525-2191
AutoresKyungmin Hahm, Matvey Lukashev, Yi Luo, William J. Yang, Brian Dolinski, Paul H. Weinreb, Kenneth J. Simon, Li‐Chun Wang, Diane R. Leone, Roy R. Lobb, Donald J. McCrann, Norm Allaire, Gerald Horan, Agnes B. Fogo, Raghu Kalluri, Charles F. Shield, Dean Sheppard, Humphrey Gardner, Shelia M. Violette,
Tópico(s)Caveolin-1 and cellular processes
ResumoThe transforming growth factor (TGF)-β-inducible integrin αvβ6 is preferentially expressed at sites of epithelial remodeling and has been shown to bind and activate latent precursor TGF-β. Herein, we show that αvβ6 is overexpressed in human kidney epithelium in membranous glomerulonephritis, diabetes mellitus, IgA nephropathy, Goodpasture's syndrome, and Alport syndrome renal epithelium. To assess the potential regulatory role of αvβ6 in renal disease, we studied the effects of function-blocking αvβ6 monoclonal antibodies (mAbs) and genetic ablation of the β6 subunit on kidney fibrosis in Col4A3−/− mice, a mouse model of Alport syndrome. Expression of αvβ6 in Alport mouse kidneys was observed primarily in cortical tubular epithelial cells and in correlation with the progression of fibrosis. Treatment with αvβ6-blocking mAbs inhibited accumulation of activated fibroblasts and deposition of interstitial collagen matrix. Similar inhibition of renal fibrosis was observed in β6-deficient Alport mice. Transcript profiling of kidney tissues showed that αvβ6-blocking mAbs significantly inhibited disease-associated changes in expression of fibrotic and inflammatory mediators. Similar patterns of transcript modulation were produced with recombinant soluble TGF-β RII treatment, suggesting shared regulatory functions of αvβ6 and TGF-β. These findings demonstrate that αvβ6 can contribute to the regulation of renal fibrosis and suggest this integrin as a potential therapeutic target. The transforming growth factor (TGF)-β-inducible integrin αvβ6 is preferentially expressed at sites of epithelial remodeling and has been shown to bind and activate latent precursor TGF-β. Herein, we show that αvβ6 is overexpressed in human kidney epithelium in membranous glomerulonephritis, diabetes mellitus, IgA nephropathy, Goodpasture's syndrome, and Alport syndrome renal epithelium. To assess the potential regulatory role of αvβ6 in renal disease, we studied the effects of function-blocking αvβ6 monoclonal antibodies (mAbs) and genetic ablation of the β6 subunit on kidney fibrosis in Col4A3−/− mice, a mouse model of Alport syndrome. Expression of αvβ6 in Alport mouse kidneys was observed primarily in cortical tubular epithelial cells and in correlation with the progression of fibrosis. Treatment with αvβ6-blocking mAbs inhibited accumulation of activated fibroblasts and deposition of interstitial collagen matrix. Similar inhibition of renal fibrosis was observed in β6-deficient Alport mice. Transcript profiling of kidney tissues showed that αvβ6-blocking mAbs significantly inhibited disease-associated changes in expression of fibrotic and inflammatory mediators. Similar patterns of transcript modulation were produced with recombinant soluble TGF-β RII treatment, suggesting shared regulatory functions of αvβ6 and TGF-β. These findings demonstrate that αvβ6 can contribute to the regulation of renal fibrosis and suggest this integrin as a potential therapeutic target. Progressive fibrosis is a common process leading to the development of end-stage renal disease and promoted by epithelial remodeling, fibroblast activation, inflammation, and reorganization of cellular interactions with the extracellular matrix (ECM). Molecular mechanisms contributing to these events are complex and include misregulation of the transforming growth factor (TGF)-β axis, aberrant ECM remodeling, and altered expression and function of cell adhesion receptors of the integrin superfamily.1Okada H Kalluri R Cellular and molecular pathways that lead to progression and regression of renal fibrogenesis.Curr Mol Med. 2005; 5: 467-474Crossref PubMed Scopus (67) Google Scholar, 2Sheppard D Functions of pulmonary epithelial integrins: from development to disease.Physiol Rev. 2003; 83: 673-686Crossref PubMed Scopus (124) Google Scholar, 3Norman JT Fine LG Progressive renal disease: fibroblasts, extracellular matrix, and integrins.Exp Nephrol. 1999; 7: 167-177Crossref PubMed Scopus (65) Google Scholar, 4Border WA Noble NA Interactions of transforming growth factor-beta and angiotensin II in renal fibrosis.Hypertension. 1998; 31: 181-188Crossref PubMed Google Scholar, 5Wang W Koka V Lan HY Transforming growth factor-beta and Smad signalling in kidney diseases.Nephrology. 2005; 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Known ligands for αvβ6 include fibronectin, tenascin, and the latency-associated peptides 1 and 3 (LAP1 and LAP3), the N-terminal fragments of the latent precursor forms of TGF-β1 and -β3.16Huang XZ Wu J Spong S Sheppard D The integrin αvβ6 is critical for keratinocyte migration on both its known ligand, fibronectin, and on vitronectin.J Cell Sci. 1998; 111: 2189-2195Crossref PubMed Google Scholar, 17Munger JS Huang X Kawakatsu H Griffiths MJD Dalton SL Wu J Pittet JF Kaminski N Garat C Matthay MA Rifkin DB Sheppard D The integrin αvβ6 binds and activates latent TGFβ1: a mechanism for regulating pulmonary inflammation and fibrosis.Cell. 1999; 96: 319-328Abstract Full Text Full Text PDF PubMed Scopus (1649) Google Scholar, 18Yokosaki Y Monis H Chen A Sheppard D Differential effects of the integrins alpha9beta1, alphavbeta3, and alphavbeta6 on cell proliferative responses to tenascin: roles of the beta subunit extracellular and cytoplasmic domains.J Biol Chem. 1996; 271: 24144-24150Crossref PubMed Scopus (132) Google Scholar, 19Annes JP Rifkin DB Munger JS The integrin αvβ6 binds and activates latent TGFβ3.FEBS Lett. 2002; 511: 65-68Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar As a result of binding to these ligands, αvβ6 can mediate cell adhesion, spreading, migration, and activation of latent TGF-β. TGF-β is synthesized as a latent protein that is cleaved and secreted with the N-terminal LAP noncovalently associated with the mature active C-terminal TGF-β cytokine. The latent TGF-β complex cannot bind to its cognate receptor and thus remains biologically inactive until converted to the active form by one of several alternative mechanisms that include cleavage by proteases, exposure to low pH or ionizing radiation, and conformational changes in the latent complex, allowing it to bind to its cognate receptors.20Munger JS Harpel JG Gleizes PE Mazzieri R Nunes I Rifkin DB Latent transforming growth factor-β: structural feature and mechanisms of activation.Kidney Int. 1997; 51: 1376-1382Crossref PubMed Scopus (442) Google Scholar, 21Khalil N TGF-beta: from latent to active.Microbes Infect. 1999; 1: 1255-1263Crossref PubMed Scopus (278) Google Scholar, 22Barcellos-Hoff MH Latency and activation in the control of TGF-β.J Mammary Gland Biol Neoplasia. 1996; 1: 353-363Crossref Scopus (101) Google Scholar An activating conformational change can be induced by αvβ6 involving direct binding of the integrin to an RGD motif contained within LAP1 and LAP3. This binding converts the TGF-β precursor into a receptor binding-competent state.17Munger JS Huang X Kawakatsu H Griffiths MJD Dalton SL Wu J Pittet JF Kaminski N Garat C Matthay MA Rifkin DB Sheppard D The integrin αvβ6 binds and activates latent TGFβ1: a mechanism for regulating pulmonary inflammation and fibrosis.Cell. 1999; 96: 319-328Abstract Full Text Full Text PDF PubMed Scopus (1649) Google Scholar, 19Annes JP Rifkin DB Munger JS The integrin αvβ6 binds and activates latent TGFβ3.FEBS Lett. 2002; 511: 65-68Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar These findings suggest that up-regulation of αvβ6 expression on the surface of epithelial cells can lead to local TGF-β activation followed by paracrine activation of TGF-β-dependent events in bystander cells. This would include the possibility for indirect downstream effects on TGF-β activity that could be mediated by altering inflammation and fibrosis initially at sites of αvβ6 expression. Because TGF-β has been implicated as a central regulator of renal fibrosis, we hypothesized that its local activation by αvβ6 may be an important process in the onset and progression of renal disease and blockade of αvβ6 function could suppress the development of kidney fibrosis. In the studies described herein, we show that αvβ6 is highly up-regulated in a mouse model of kidney fibrosis and in human kidney samples with fibrotic pathology. Using Col4A3−/− mice, a model of progressive kidney disease similar to that observed in the human Alport syndrome, we show that monoclonal antibodies (mAbs) blocking the ligand binding and TGF-β activation functions of αvβ6,23Weinreb PH Simon KJ Rayhorn P Yang WJ Leone DR Dolinski BM Pearse BR Yokota Y Kawakatsu H Atakilit A Sheppard D Violette SM Function-blocking integrin alphavbeta6 monoclonal antibodies.J Biol Chem. 2004; 279: 17875-17887Crossref PubMed Scopus (120) Google Scholar as well as genetic ablation of β6, potently inhibit both glomerular and tubulointerstitial fibrosis and delay destruction of kidney tissue architecture. We show that although the αvβ6 integrin has restricted expression in the kidney to tubular epithelial cells, it can provide protective effects at distal sites in the tissue. These findings raise the possibility that the antifibrotic effects may also be mediated in part via indirect extrarenal effects in addition to direct effects of blocking αvβ6 on tubular epithelial cells. Delayed treatment studies indicate that therapeutic blockade of αvβ6 not only inhibits the progression of kidney fibrosis but has the potential to allow resolution of existing fibrotic lesions. Our analysis of molecular signatures associated with kidney disease progression and affected by αvβ6 inhibition indicates that the therapeutic impact of the αvβ6 blocking antibodies is similar to that of systemic TGF-β blockade and is mechanistically related to decreased TGF-β activity. These data suggest that αvβ6 is involved in the regulation of renal fibrosis and could provide a novel molecular target for its therapeutic modulation. αvβ6 mAbs were generated as previously described.23Weinreb PH Simon KJ Rayhorn P Yang WJ Leone DR Dolinski BM Pearse BR Yokota Y Kawakatsu H Atakilit A Sheppard D Violette SM Function-blocking integrin alphavbeta6 monoclonal antibodies.J Biol Chem. 2004; 279: 17875-17887Crossref PubMed Scopus (120) Google Scholar Human/mouse chimeric 2A1 and 3G9 cDNAs were generated from the respective parent hybridoma total RNAs with constant region primers CDL-739 for the heavy chain and CDL-738 for the light chain using the First Strand cDNA synthesis kit (Amersham/Pharmacia, Piscataway, NJ). The heavy and light chain variable regions were amplified by the polymerase chain reaction using the same 3′ primers used for cDNA synthesis and pools of degenerate primers specific for most murine antibody gene signal sequences (sequences available on request) and Pfu DNA polymerase (Stratagene, La Jolla, CA). Cloned heavy and light chain variable regions were ligated into mammalian expression vectors with human IgG1 constant regions. Recombinant soluble murine TGF-β receptor type II-Ig fusion protein (rsTGF-βRII-Ig) was generated as previously described7Cosgrove D Rodgers K Meehan D Miller C Bovard K Gilroy A Gardner H Kotelianski V Gotwals P Amattucci A Kalluri R Integrin α1β1 and transforming growth factor-β1 play distinct roles in Alport glomerular pathogenesis and serve as dual targets for metabolic therapy.Am J Pathol. 2000; 157: 1649-1659Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar and purchased from R&D Systems (532-R2; Minneapolis, MN). Antibodies were purchased as indicated: fluorescein isothiocyanate-conjugated pan anti-cytokeratin mAb (C-11; F3418) from Sigma-Aldrich (St. Louis, MO); anti-laminin B1 chain mAb (LT3; MAB1928) from Chemicon (Temecula, CA); phycoerythrin (PE)-conjugated anti-αv mAb (RMV7; CBL1346P) from Chemicon; rabbit anti-αv from Chemicon (AB1930); PE-rat IgG1 (553925) from BD Biosciences (San Jose, CA); and anti-smooth muscle actin (SMA)-Cy3 from Sigma-Aldrich (C-6198). We identified rabbit polyclonal anti-TGF-β (sc-146; Santa Cruz Biotechnology, Santa Cruz, CA) as an antibody that preferentially binds xenograft sections of 293 cells expressing a constitutively active form of TGF-β compared with xenograft sections of 293 cells expressing latent TGF-β.24Barcellos-Hoff MH Ehrhart EJ Kalia M Jirtle R Flanders KC Tsang ML-S Immunohistochemical detection of active transforming growth factor-β in situ using engineered tissue.Am J Pathol. 1995; 147: 1228-1237PubMed Google Scholar Murine NIH3T3 cells stably transfected with β6 (NIH3T3b6) were generated as previously described.23Weinreb PH Simon KJ Rayhorn P Yang WJ Leone DR Dolinski BM Pearse BR Yokota Y Kawakatsu H Atakilit A Sheppard D Violette SM Function-blocking integrin alphavbeta6 monoclonal antibodies.J Biol Chem. 2004; 279: 17875-17887Crossref PubMed Scopus (120) Google Scholar Cells were harvested by trypsinization, washed in phosphate-buffered saline (PBS), and resuspended in flow cytometry (FC) buffer (1× PBS, 2% FBS, 0.1% NaN3, 1 mmol/L CaCl2, and 1 mmol/L MgCl2). Cells (0.2 × 105) were incubated on ice for 1 hour in FC buffer containing purified primary antibodies in a total volume of 100 μl. After incubation, cells were washed two times with ice-cold FC buffer and resuspended in 100 μl of FC buffer containing 5 μg/ml PE-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch) and incubated on ice for 30 minutes. For monitoring αv expression, cells were incubated with a PE-conjugated rat anti-mouse αv mAb (RMV-7) and a PE-conjugated rat IgG1 control. Cells were washed two times with ice-cold FC buffer, and binding of the labeled secondary antibody was monitored by flow cytometry. Tissue sections were deparaffinized in xylene and ethanol, rehydrated in distilled water, and then immersed in methanol containing 0.45% H2O. Tissues were incubated with pepsin (00-3009; Zymed, San Francisco, CA) and blocked with avidin and biotin (SP-2001; Vector Laboratories, Burlingame, CA). Primary antibody was diluted in PBS containing 0.1% bovine serum albumin, and tissues were incubated overnight at 4°C. For immunostaining β6 on mouse tissue, sections were incubated with a human/mouse chimeric form of the anti-αvβ6 mAb, 2A1,23Weinreb PH Simon KJ Rayhorn P Yang WJ Leone DR Dolinski BM Pearse BR Yokota Y Kawakatsu H Atakilit A Sheppard D Violette SM Function-blocking integrin alphavbeta6 monoclonal antibodies.J Biol Chem. 2004; 279: 17875-17887Crossref PubMed Scopus (120) Google Scholar and an anti-human biotinylated secondary antibody (PK-6103; Vector Laboratories). For immunostaining β6 on human tissue, sections were incubated with murine 2A1,23Weinreb PH Simon KJ Rayhorn P Yang WJ Leone DR Dolinski BM Pearse BR Yokota Y Kawakatsu H Atakilit A Sheppard D Violette SM Function-blocking integrin alphavbeta6 monoclonal antibodies.J Biol Chem. 2004; 279: 17875-17887Crossref PubMed Scopus (120) Google Scholar and an anti-mouse-biotinylated secondary antibody (PK-6102; Vector Laboratories). Avidin-biotin complex-horseradish peroxidase (Vector kit PK-6102) was applied to sections and incubated for 30 minutes at room temperature, and 3,3′-diaminobenzidine substrate was prepared as directed (SK-4100; Vector Laboratories) and applied to sections for 5 minutes at room temperature. Tissue sections were stained with Mayer's hematoxylin for 1 minute and rinsed in water and PBS. Frozen tissue sections embedded in O.C.T. compound (Sakura, Tokyo, Japan) were fixed in acetone and blocked with 0.5% casein/0.05% thimerosal in PBS. For immunostaining β6 on human tissue, sections were incubated with murine 2A123Weinreb PH Simon KJ Rayhorn P Yang WJ Leone DR Dolinski BM Pearse BR Yokota Y Kawakatsu H Atakilit A Sheppard D Violette SM Function-blocking integrin alphavbeta6 monoclonal antibodies.J Biol Chem. 2004; 279: 17875-17887Crossref PubMed Scopus (120) Google Scholar and an anti-mouse Alexa Fluor 594 secondary antibody (A-11032; Molecular Probes, Eugene, OR). For immunostaining β6 on mouse tissue, sections were incubated with a human/mouse chimeric form of 2A1 and an anti-human Alexa Fluor 594-conjugated secondary antibody (A-11014; Molecular Probes). For laminin and αv immunostaining, an anti-rat Alexa Fluor 488-conjugated secondary antibody (A-11006; Molecular Probes) was used. All other antibodies were directly conjugated as indicated previously. All images were taken at ×20 with the exception of Figure 2A, which was taken at ×40. All human tissue samples were obtained under approval of local institutional review and patient approval. SMA immunostaining area was quantitated using MetaMorph v5.0 (Universal Imaging Corporation, Sunnyvale, CA) and expressed as percent area relative to total image size. For SMA immunostaining, ×20 images from at least five cortical and one to two medullary sections from each animal were analyzed. Statistical analysis of treatment groups was performed using analysis of variance. Tubular damage and interstitial volume were assessed as previously described25Vielhauer V Anders HJ Mack M Cihak J Strutz F Stangassinger M Luckow B Grone H-J Schlondorff D Obstructive nephropathy in the mouse: progressive fibrosis correlates with tubulointerstitial chemokine expression and accumulation of CC chemokine receptor 2- and 5-positive leukocytes.J Am Soc Nephrol. 2001; 12: 1173-1187PubMed Google Scholar, 26Anders HJ Vielhauer V Frink M Linde Y Cohen CD Blattner SM Kretzler M Strutz F Mack M Grone H-J Onuffer J Horuk R Nelson PJ Schlondorff D A chemokine receptor CCR-1 antagonist reduces renal fibrosis after unilateral ureter ligation.J Clin Invest. 2002; 109: 251-259Crossref PubMed Scopus (209) Google Scholar with modifications. In brief, ×20 images were taken from sections stained with Trichrome-Masson. A grid containing 117 points (13 × 9) was set up 35 μm apart in Stereo Investigator 6.5 (MBF Bioscience, Williston, VT). The number of grid points overlying healthy appearing tubules (healthy tubule volume index) and degenerated tubules and interstitial space (interstitial volume index) was counted and expressed as a percentage of all sampling points. For tubular dilation index, the number of dilated tubules per image was counted. For each kidney, 10 randomly selected, nonoverlapping fields were analyzed by a blinded observer. Col4A3+/− mice in a 129Sv/J background were bred to generate Col4A3−/− mice. Mice were injected intraperitoneally with proteins three times a week from 3 weeks of age to 7 or 8.5 weeks of age, as indicated. mAbs were injected intraperitoneally at 4 mg/kg, and rsTGF-βRII-Ig was injected at 2 mg/kg. Mice were euthanized, and kidneys were collected for RNA and immunostaining. All animal studies were approved and performed in accordance with the Institutional Animal Care and Use Committee. Kidneys were homogenized directly into TRIzol (155-96-018; Invitrogen, Carlsbad, CA), and RNA was extracted according to manufacturer's protocol with an additional 1 ml of acidic phenol/chloroform/isoamyl alcohol (25:24:1, pH 6.6) extraction. Purified total RNA was resuspended in diethylpyrocarbonate-treated H2O (Ambion Inc., Austin, TX) and 260 and 280 recorded (Spectra max Plus; Molecular Devices, Sunnyvale, CA). Residual DNA was removed using 5 units of DNase I amplification grade (Invitrogen) at 20°C for 15 minutes. cDNA was generated using a high-capacity cDNA archive kit according to the manufacturer's protocol (Applied Biosystems Inc., Foster City, CA). Oligonucleotide primers and Taqman MGB probes were designed from Affymetrix consensus sequences using Primer Express version 2.0.0 (Applied Biosystems Inc.). Taqman MGB probes were designed with a 5′ covalently linked fluorescent reporter dye (FAM) and a minor groove binder/nonfluorescent quencher covalently linked to the 3′ end. Oligonucleotide standard templates were designed by the addition of 10 bp of gene-specific sequence to the 5′ and 3′ ends of the amplicon. Reverse-phase high-performance liquid chromatography-purified primers and oligonucleotide standard templates were purchased from Biosearch Technologies Inc. (Novato, CA). High-performance liquid chromatography-purified primers and probe for murine glyceraldehyde-3-phosphate dehydrogenase were synthesized at Biogen Idec (5′-CATGGCCTTCCGTGTTCCTA-3′, 5′-GCGGCACGTCAGATCC-3′, and 6FAM-5′-CCCCAATGTGTC-CGTC-3′). Quadruplicate polymerase chain reactions for samples and standards were cycled in a 7900HT thermal cycler (Applied Biosystems Inc.) under the following conditions: 50°C for 2 minutes (uracil N-deglycosylase digest), 95°C for 10 minutes (activation of Taq thermostable polymerase), and 40 cycles of 95°C for 15 seconds and 60°C for 60 seconds. The fluorescence emission was collected every 7 seconds for the length of the run for each reaction well. Relative transcript quantities were determined for each sample by comparison with oligonucleotide standard curve using Sequence Detection Software (Applied Biosystems Inc.). Total RNA was isolated from kidney samples of each experimental group of mice (five animals per group). The quality of the resulting RNA preparations was verified by capillary electrophoresis on Bioanalyzer 2000 (Agilent). Experimental groups of Col4A3−/− mice analyzed included no treatment, or treatment with one of the following proteins: negative control mAb 1E6, nonblocking anti-αvβ6 mAb 8B3, blocking anti-αvβ6 mAb 3G9, blocking anti-αvβ6 mAb 8G6, and rsTGFβRII-Ig. Additional control groups included wild-type mice of the same genetic background treated with 3G9 and naive wild-type mice. Hybridization probes were prepared from each of the RNA samples and profiled on separate U74Av2 Gene Chip oligonucleotide arrays (Affymetrix, Santa Clara, CA). Hybridization probe synthesis, hybridization, and microarray scanning were performed using the manufacturer's protocols. The array scans were converted into Affymetrix.CEL files, and the resulting data set (group of CEL files representing the complete experiment) was normalized using the robust microarray average method. Statistical and clustering analyses were done using the GeneSpring (Agilent) and Spotfire (Spotfire) data mining tools. We used a Two-strp analysis of variance and fold-change filtering to identify probe sets whose signal intensity was altered by experimental treatment compared with the untreated Col4A3−/− group at P < 0.05 and at least by twofold. Likewise, disease-associated transcripts were selected for differential expression between untreated Col4A3−/− and naive wild-type groups using the statistical cutoff of P < 0.01 and the signal fold-change cutoff of 2. The profiles of the resulting group of genes and the grouping of experimental conditions were analyzed and visualized by hierarchical clustering. Virtual pathway analysis was performed using the Ingenuity Pathway Analysis database (Ingenuity Systems). Several different types of human kidney disease, associated with inflammatory/fibrotic pathology, have shown a corresponding increased expression of TGF-β in the kidney tissue.27Yamamoto T Nakamura T Noble NA Ruoslahti E Border WA Expression of transforming growth factor beta is elevated in human and experimental diabetic nephropathy.Proc Natl Acad Sci USA. 1993; 90: 1814-1818Crossref PubMed Scopus (814) Google Scholar, 28Yamamoto T Noble NA Cohen AH Nast CC Hishida A Gold LI Border WA Expression of transforming growth factor-beta isoforms in human glomerular diseases.Kidney Int. 1996; 49: 461-469Crossref PubMed Scopus (421) Google Scholar, 29Shihab FS Yamamoto T Nast CC Cohen AH Noble NA Gold LI Border WA Transforming growth factor-beta and matrix protein expression in acute and chronic rejection of human renal allografts.J Am Soc Nephrol. 1995; 6: 286-294PubMed Google Scholar Using immunohistochemical analysis, we examined the expression of αvβ6 in human kidney biopsy samples associated with chronic inflammation and fibrosis as a potential mechanism leading to increased activation of TGF-β (Figure 1, A and B). Tissue samples from membranous glomerulonephritis, diabetes mellitus, IgA nephropathy, Goodpasture's syndrome, Alport syndrome, and lupus all showed prominent αvβ6 staining in the epithelial lining of dilated and damaged tubules. In contrast, samples of mor
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