Increased Goodpasture Antigen-Binding Protein Expression Induces Type IV Collagen Disorganization and Deposit of Immunoglobulin A in Glomerular Basement Membrane
2007; Elsevier BV; Volume: 171; Issue: 5 Linguagem: Inglês
10.2353/ajpath.2007.070205
ISSN1525-2191
AutoresFernando Revert, Ramón Merino, Carlos Monteagudo, Jesús Macías, Amando Peydró, Javier Alcácer, Pedro Muniesa, Regina Marquina, Mario Blanco, Marcos Iglesias, Francisco Revert‐Ros, Jesús Merino, Juan Saus,
Tópico(s)Complement system in diseases
ResumoIncreased expression of Goodpasture antigen-binding protein (GPBP), a protein that binds and phosphorylates basement membrane collagen, has been associated with immune complex-mediated pathogenesis. However, recent reports have questioned this biological function and proposed that GPBP serves as a cytosolic ceramide transporter (CERTL). Thus, the role of GPBP in vivo remains unknown. New Zealand White (NZW) mice are considered healthy animals although they convey a genetic predisposition for immune complex-mediated glomerulonephritis. Here we show that NZW mice developed age-dependent lupus-prone autoimmune response and immune complex-mediated glomerulonephritis characterized by elevated GPBP, glomerular basement membrane (GBM) collagen disorganization and expansion, and deposits of IgA on disrupted GBM. Transgenic overexpression of human GPBP (hGPBP) in non-lupus-prone mice triggered similar glomerular abnormalities including deposits of IgA on a capillary GBM that underwent dissociation, in the absence of an evident autoimmune response. We provide in vivo evidence that GPBP regulates GBM collagen organization and its elevated expression causes dissociation and subsequent accumulation of IgA on the GBM. Finally, we describe a previously unrecognized pathogenic mechanism that may be relevant in human primary immune complex-mediated glomerulonephritis. Increased expression of Goodpasture antigen-binding protein (GPBP), a protein that binds and phosphorylates basement membrane collagen, has been associated with immune complex-mediated pathogenesis. However, recent reports have questioned this biological function and proposed that GPBP serves as a cytosolic ceramide transporter (CERTL). Thus, the role of GPBP in vivo remains unknown. New Zealand White (NZW) mice are considered healthy animals although they convey a genetic predisposition for immune complex-mediated glomerulonephritis. Here we show that NZW mice developed age-dependent lupus-prone autoimmune response and immune complex-mediated glomerulonephritis characterized by elevated GPBP, glomerular basement membrane (GBM) collagen disorganization and expansion, and deposits of IgA on disrupted GBM. Transgenic overexpression of human GPBP (hGPBP) in non-lupus-prone mice triggered similar glomerular abnormalities including deposits of IgA on a capillary GBM that underwent dissociation, in the absence of an evident autoimmune response. We provide in vivo evidence that GPBP regulates GBM collagen organization and its elevated expression causes dissociation and subsequent accumulation of IgA on the GBM. Finally, we describe a previously unrecognized pathogenic mechanism that may be relevant in human primary immune complex-mediated glomerulonephritis. Basement membrane collagen (type IV collagen) is composed of six distinct α chains (α1 to α6) that apparently form only three types of triple-helical molecules: α1.α1.α2(IV), α3.α4.α5(IV), and α5.α5.α6(IV). The structure of the renal glomerulus is maintained by the glomerular basement membrane (GBM), a peripheral wrapping sheet, and the mesangial matrix, a mesh that cements the core of the capillary tuft. A membrane-organized α3.α4.α5(IV) collagen network supports GBM, and a mesh-organized α1.α1.α2(IV) network scaffolds the mesangial matrix. However, when GBM contacts the capillary wall (capillary GBM), its α3.α4.α5(IV) network (epithelial component) fuses with a membrane-organized version of the α1.α1.α2(IV) network (endothelial component), a phenomenon that is critical to assemble the glomerular filtration barrier and does not occur when GBM contacts the mesangial matrix (mesangial GBM).1Bonsib SM Renal anatomy and histology.in: Jennette JC Olson JL Schwartz MM Silva FG Heptinstall's Pathology of the Kidney. Lippincott Williams and Wilkins Publishers, Philadelphia2007: 1-70Google Scholar, 2Hudson BG Tryggvason K Sundaramoorthy M Neilson EG Alport's syndrome. Goodpasture's syndrome, and type IV collagen.N Engl J Med. 2003; 348: 2543-2556Crossref PubMed Scopus (781) Google Scholar Goodpasture antigen-binding protein (GPBP) is a nonconventional Ser/Thr kinase that targets the NC1 domain,3Raya A Revert F Navarro S Saus J Characterization of a novel type of serine/threonine kinase that specifically phosphorylates the human Goodpasture antigen.J Biol Chem. 1999; 274: 12642-12649Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 4Raya A Revert-Ros F Martinez-Martinez P Navarro S Rosello E Vieites B Granero F Forteza J Saus J Goodpasture antigen-binding protein, the kinase that phosphorylates the Goodpasture antigen, is an alternatively spliced variant implicated in autoimmune pathogenesis.J Biol Chem. 2000; 275: 40392-40399Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar a key structure in the molecular and supramolecular organization of type IV collagen.2Hudson BG Tryggvason K Sundaramoorthy M Neilson EG Alport's syndrome. Goodpasture's syndrome, and type IV collagen.N Engl J Med. 2003; 348: 2543-2556Crossref PubMed Scopus (781) Google Scholar In humans, GPBP is associated with GBM, and increased expression levels of this kinase have been linked with induction of the proautoimmune inflammatory cytokine tumor necrosis factor-α and with Goodpasture and systemic lupus erythematosus diseases,4Raya A Revert-Ros F Martinez-Martinez P Navarro S Rosello E Vieites B Granero F Forteza J Saus J Goodpasture antigen-binding protein, the kinase that phosphorylates the Goodpasture antigen, is an alternatively spliced variant implicated in autoimmune pathogenesis.J Biol Chem. 2000; 275: 40392-40399Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 5Granero F Revert F Revert-Ros F Lainez S Martinez-Martinez P Saus J A human-specific TNF-responsive promoter for Goodpasture antigen-binding protein.FEBS J. 2005; 272: 5291-5305Crossref PubMed Scopus (15) Google Scholar suggesting that GPBP plays a role in GBM collagen organization and in associated immune complex-mediated diseases. However, it has been postulated recently that GPBPΔ26, a GPBP isoform generated by mRNA alternative splicing, is a cytosolic transporter of ceramide between endoplasmic reticulum and Golgi apparatus, and thus, this isoform has been renamed as CERT.6Hanada K Kumagai K Yasuda S Miura Y Kawano M Fukasawa M Nishijima M Molecular machinery for non-vesicular trafficking of ceramide.Nature. 2003; 426: 803-809Crossref PubMed Scopus (859) Google Scholar Based on structural homology and in vitro studies using recombinant materials, these authors proposed a similar role for GPBP (CERTL) and questioned the biological significance of GPBP binding and phosphorylating type IV collagen3Raya A Revert F Navarro S Saus J Characterization of a novel type of serine/threonine kinase that specifically phosphorylates the human Goodpasture antigen.J Biol Chem. 1999; 274: 12642-12649Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar and immunohistochemical evidence revealing GPBP association with human GBM.4Raya A Revert-Ros F Martinez-Martinez P Navarro S Rosello E Vieites B Granero F Forteza J Saus J Goodpasture antigen-binding protein, the kinase that phosphorylates the Goodpasture antigen, is an alternatively spliced variant implicated in autoimmune pathogenesis.J Biol Chem. 2000; 275: 40392-40399Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar Thus, the role of GPBP in vivo has remained not only unknown but also controversial. Predominant IgA deposits at the glomerular mesangium are the histopathological hallmark of IgA nephropathy, the most common primary glomerulonephritis in humans.7Donadio JV Grande JP IgA nephropathy.N Engl J Med. 2002; 347: 738-748Crossref PubMed Scopus (710) Google Scholar Systemic lupus erythematosus is a complex disease displaying IgG autoantibody deposits in multiple organs and tissues including the renal glomerulus (lupus nephritis).8Balow JE Boumpas DT Austin III, HA Systemic lupus erythematosus and the kidney.in: Lahita RG Systemic Lupus Erythematosus. Academic Press, San Diego1999: 657-685Google Scholar In both instances, glomerulonephritis is thought to be mediated by GBM-associated immune complexes, although the mechanisms responsible for immune complex deposits have not been defined. In Goodpasture disease, IgG autoantibodies bind to GBM in an antigen-antibody manner; however, the mechanisms underlying autoantibody production and binding also remain unknown because the pathogenic epitope(s) residing in the noncollagenous-1 (NC1) domain of the α3 chain of type IV collagen (the Goodpasture antigen) is cryptic in the quaternary structure.2Hudson BG Tryggvason K Sundaramoorthy M Neilson EG Alport's syndrome. Goodpasture's syndrome, and type IV collagen.N Engl J Med. 2003; 348: 2543-2556Crossref PubMed Scopus (781) Google Scholar Nevertheless, deposits of immune complexes associated with GBM cause glomerulonephritis in all three diseases, suggesting the existence of common pathogenic mechanisms. New Zealand White (NZW) mice are considered healthy animals, although they convey a genetic predisposition for lupus nephritis.9Theofilopoulos AN Dixon FJ Murine models of systemic lupus erythematosus.Adv Immunol. 1985; 37: 269-390Crossref PubMed Scopus (1459) Google Scholar Accordingly, historical reports reveal that aging in NZW mice is associated with autoantibody production and clinically silent immune complex-mediated glomerulonephritis.10Braverman IM Study of autoimmune disease in New Zealand mice. I. Genetic features and natural history of NZB, NZY and NZW strains and NZB-NZW hybrids.J Invest Dermatol. 1968; 50: 483-499Abstract Full Text PDF PubMed Scopus (52) Google Scholar, 11Hahn BH Shulman LE Autoantibodies and nephritis in the white strain (NZW) of New Zealand mice.Arthritis Rheum. 1969; 12: 355-364Crossref PubMed Scopus (21) Google Scholar, 12Kelley VE Winkelstein A Age- and sex-related glomerulonephritis in New Zealand white mice.Clin Immunol Immunopathol. 1980; 16: 142-150Crossref PubMed Scopus (42) Google Scholar More recently, a glomerulonephritis with predominant glomerular deposits of IgA and IgM have been reported in NZW mouse-derived models.13Marquina R Diez MA Lopez-Hoyos M Buelta L Kuroki A Kikuchi S Villegas J Pihlgren M Siegrist CA Arias M Izui S Merino J Merino R Inhibition of B cell death causes the development of an IgA nephropathy in (New Zealand white × C57BL/6)F(1)-bcl-2 transgenic mice.J Immunol. 2004; 172: 7177-7185Crossref PubMed Scopus (42) Google Scholar These data suggest that genetic background in NZW mice predisposes for all three IgG, IgA, and IgM glomerular deposits. This condition may also extend to human patients because glomerular IgG and IgM deposits are common abnormalities in primary IgA nephropathy, and glomerular IgA and IgM deposits are frequently detected in lupus nephritis.7Donadio JV Grande JP IgA nephropathy.N Engl J Med. 2002; 347: 738-748Crossref PubMed Scopus (710) Google Scholar, 8Balow JE Boumpas DT Austin III, HA Systemic lupus erythematosus and the kidney.in: Lahita RG Systemic Lupus Erythematosus. Academic Press, San Diego1999: 657-685Google Scholar Here, we report that aging in NZW mice correlates with increased glomerular GPBP expression, GBM disruption and associated IgA deposits, and matrix expansion in the context of a lupus-prone autoimmune response. Moreover, transgenic expression of hGPBP in non-lupus-prone mice induced similar GBM abnormalities, albeit in the absence of an autoimmune response. Thus, our observations provide evidence that in vivo GPBP regulates type IV collagen organization and elevated expression of this kinase induces immune complex-mediated glomerulonephritis. All of the procedures were performed according to institutional guidelines for the use of animals in experimentation. We used NZW, C57BL/6, and BALB/c inbred mice and transgenic mice expressing hGPBP (Tg-hGPBP) and non-Tg-hGPBP littermate mice. For morphological studies, 60 NZW mice between 2 and 14 months of age were analyzed using standard histochemical and immunofluorescence procedures. Groups of at least three mice representing unaffected (young mice 7 month of age) were analyzed by confocal microscopy, and at least two mice from each group were further analyzed by electron microscopy (EM). Control morphological studies were also performed with C57BL/6 mice of 4, 8, and 12 months of age, and, other than IgA mesangial deposits, we found no significant glomerular abnormalities in these mice by light microscopy (LM), confocal, or EM procedures. To generate Tg-hGPBP mice, we produced pCAGG-FLAG-GPBP by inserting FLAG-GPBP encoding cDNA into EcoRI site of CAGG expression vector provided by Jun-ichi Miyazaki (Osaka University Medical School, Osaka, Japan).14Niwa H Yamamura K Miyazaki J Efficient selection for high-expression transfectants with a novel eukaryotic vector.Gene. 1991; 108: 193-199Crossref PubMed Scopus (4683) Google Scholar A SnaBI-HindIII fragment of pCAGG-FLAG-GPBP was subcloned in the pVAX vector (Invitrogen, Carlsbad, CA) to facilitate digestion with NruI and PmeI. The resulting DNA fragment (4.2 kb) was isolated and used for pronuclear injection of zygotes, as previously described.15Hogan B Constantini F Lacy E Manipulation of the Mouse Embryo: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Plainview1986Google Scholar We screened the resulting offspring for transgene transmission by polymerase chain reaction (PCR) analysis of genomic DNA extracted from mouse tails using specific GPBP primers (see below). Tg-hGPBP mice (F1, 50% C57BL/6 and 50% DBA2) were backcrossed with C57BL/6 mice for six generations to acquire C57BL/6 genetic background (F2 to F7, 75 to 99.25% C57BL/6 and 25 to 0.75% DBA2). Samples from mice representing each individual generation were analyzed by standard immunofluorescence and confocal microscopy. Mice from F3 to F5 generations were further characterized by real-time reverse transcription (RT)-PCR, immunoprecipitation, histochemistry, EM, and enzyme-linked immunosorbent assay and used in the study. In general, mice were sacrificed monthly commencing at 4 months of age until type IV collagen-based GBM lesions were evident by confocal microscopy, and the progression of glomerulonephritis was followed for 1 or 2 additional months in littermate mice. Non-Tg-hGPBP littermates raised in parallel were used as controls in the studies. A total of 36 Tg-hGPBP and 22 non-Tg-hGPBP littermates were characterized using confocal microscopy. Moreover, 16 Tg-hGPBP and 6 non-Tg-hGPBP were analyzed by EM. The presence of clinical glomerulonephritis was assessed by estimating proteinuria by determination of urine albumin as previously described.13Marquina R Diez MA Lopez-Hoyos M Buelta L Kuroki A Kikuchi S Villegas J Pihlgren M Siegrist CA Arias M Izui S Merino J Merino R Inhibition of B cell death causes the development of an IgA nephropathy in (New Zealand white × C57BL/6)F(1)-bcl-2 transgenic mice.J Immunol. 2004; 172: 7177-7185Crossref PubMed Scopus (42) Google Scholar Anti-FLAG M2 monoclonal antibody coupled to agarose beads or horseradish peroxidase (Sigma Chemical Co., St. Louis, MO) were used for immunoprecipitation or Western blot, respectively. To detect α1-α2(IV) we used a goat anti-type IV collagen polyclonal antibody (AB769; Chemicon, Temecula, CA), unlabeled or labeled with Alexa Fluor 647 (Invitrogen). To detect α3(IV) we used biotin-labeled mAb3 provided by Jorgen Wieslander (Wieslab AB, Lund, Sweden). For GPBP-specific detection we used chicken antibodies recognizing the 26-residues (GPBPpep1) present in GPBP and absent in GPBPΔ26 (CERT) previously characterized.4Raya A Revert-Ros F Martinez-Martinez P Navarro S Rosello E Vieites B Granero F Forteza J Saus J Goodpasture antigen-binding protein, the kinase that phosphorylates the Goodpasture antigen, is an alternatively spliced variant implicated in autoimmune pathogenesis.J Biol Chem. 2000; 275: 40392-40399Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar Specific antibodies were further purified by adsorption on a GPBPpep1-Sepharose column and their specificity determined by Western blot and indirect immunofluorescence using GPBPpep1 for competing antibody binding (Supplemental Figure S1, see http://ajp.amjpathol.org). The production and characterization of mAb 14, a mouse monoclonal antibody for GPBP have been reported.3Raya A Revert F Navarro S Saus J Characterization of a novel type of serine/threonine kinase that specifically phosphorylates the human Goodpasture antigen.J Biol Chem. 1999; 274: 12642-12649Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar Fluorescein isothiocyanate (FITC)-labeled antibodies for specific immunoglobulins and secondary antibodies were from Sigma. Other conjugates were tetramethyl-rhodamine isothiocyanate (TRITC)-labeled ExtrAvidin (Sigma) and FITC-labeled avidin (Vector Laboratories, Burlingame, CA). Kidneys were removed from mice, immediately frozen in liquid nitrogen and ground with pestle and mortar. The resulting kidney powder (50 mg) was homogenized in 10 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl, 0.1% sodium dodecyl sulfate, 1% Nonidet P-40, 1% sodium deoxycholate, 2 mmol/L ethylenediaminetetraacetic acid, 1 mmol/L phenylmethyl sulfonyl fluoride, and 10 μg/ml leupeptin with a rotor-stator homogenizer (Omni International, Marietta, GA). Homogenates were cleared by centrifugation (20,000 × g, 15 minutes at 4°C), and supernatants were diluted with 2.5 vol of 10 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl (Tris-buffered saline) supplemented with 1 mmol/L phenylmethyl sulfonyl fluoride and extracted with 30 μl of agarose-anti FLAG M2 beads for 3 hours at 4°C under continuous end-to-end agitation. Beads were washed three times with ice-cold Tris-buffered saline, and samples were eluted twice with 0.1 mg/ml of FLAG peptide in Tris-buffered saline. Finally, eluted material was analyzed by Western blot using anti-FLAG M2-horseradish peroxidase or mAb 14. Serum levels of total IgG and IgA were determined as previously described,13Marquina R Diez MA Lopez-Hoyos M Buelta L Kuroki A Kikuchi S Villegas J Pihlgren M Siegrist CA Arias M Izui S Merino J Merino R Inhibition of B cell death causes the development of an IgA nephropathy in (New Zealand white × C57BL/6)F(1)-bcl-2 transgenic mice.J Immunol. 2004; 172: 7177-7185Crossref PubMed Scopus (42) Google Scholar and results were expressed in mg/ml in reference to a standard curve obtained with a mouse reference serum (MP Biomedicals, Fountain Parkway, OH) (Supplemental Figure S2, see http://ajp.amjpathol.org). Circulating IgG and IgA anti-nucleosome or anti-ssDNA autoantibodies were determined following procedures described elsewhere,13Marquina R Diez MA Lopez-Hoyos M Buelta L Kuroki A Kikuchi S Villegas J Pihlgren M Siegrist CA Arias M Izui S Merino J Merino R Inhibition of B cell death causes the development of an IgA nephropathy in (New Zealand white × C57BL/6)F(1)-bcl-2 transgenic mice.J Immunol. 2004; 172: 7177-7185Crossref PubMed Scopus (42) Google Scholar, 16Cohen PL Caricchio R Abraham V Camenisch TD Jennette JC Roubey RA Earp HS Matsushima G Reap EA Delayed apoptotic cell clearance and lupus-like autoimmunity in mice lacking the c-mer membrane tyrosine kinase.J Exp Med. 2002; 196: 135-140Crossref PubMed Scopus (530) Google Scholar and results were expressed in titration units based on a standard curve obtained from serial dilutions of a pool of serum from 6- to 8-month-old MRL lpr/lpr mice. Circulating IgG and IgA anti-dsDNA autoantibodies were measured by the standard Crithidia luciliae immunofluorescence assay (Zentech, Angleur, Belgium), and results were expressed in an scale ranging from 0 to 3 depending on their reactivity at 1:10 (1), 1:50 (2), and 1:250 (3) serum dilutions (Supplemental Figure S2, see http://ajp.amjpathol.org). Circulating anti-basement membrane autoantibodies were detected using standard indirect enzyme-linked immunosorbent assay procedures by coating the plates with 2 μg/ml of purified bovine testis NC1 domain, gifted by Billy G. Hudson (Vanderbilt University Medical Center, Nashville, TN), and using Amplex UltraRed reagent (Invitrogen), a fluorogenic horseradish peroxidase substrate, for detection. The coating material contains the hexameric form of the NC1 domain of type IV collagen from α1.α1.α2(IV) and α3.α4.α5(IV) networks. When indicated the hexamer was denatured for 30 minutes at 100°C in the presence of 6 mol/L guanidine-HCl and similarly used for coating purposes. In these assays, results were expressed as arbitrary units of intensity fluorescence (Supplemental Figure S2, see http://ajp.amjpathol.org). The ability of IgG and IgA from Tg-hGPBP and non-Tg-hGPBP mice to form glomerular deposits was assessed by intravenous injection of serum pools from either of these mouse strains into 2 month-old BALB/c mice. Individual mice received a total volume of 1.5 ml of phosphate-buffered saline or serum pool injected during 3 consecutive days. One day after the last injection, mice were sacrificed, and the presence of IgA and IgG glomerular deposits was examined by immunofluorescence (Supplemental Figure S2, see http://ajp.amjpathol.org) following procedures previously described.13Marquina R Diez MA Lopez-Hoyos M Buelta L Kuroki A Kikuchi S Villegas J Pihlgren M Siegrist CA Arias M Izui S Merino J Merino R Inhibition of B cell death causes the development of an IgA nephropathy in (New Zealand white × C57BL/6)F(1)-bcl-2 transgenic mice.J Immunol. 2004; 172: 7177-7185Crossref PubMed Scopus (42) Google Scholar Serum pools contained similar IgG or IgA levels. Kidneys were fixed in 10% formalin, embedded in paraffin, and sliced on an electronic rotary microtome (Microm, Walldorf, Germany) to generate sections for standard histochemical procedures. Sections of 6 to 7 μm were obtained by cryostat (Microm) from frozen kidneys embedded in OCT (Sakura, Tokyo, Japan). Cryosections were blocked with an avidin/biotin blocking kit (Vector Laboratories) and also with an irrelevant ascites (1:10). Sections were subsequently incubated with primary antibodies and with anti-chicken IgY-TRITC, streptavidin-FITC, fluorescein-avidin, anti-goat TRITC, or combinations thereof and mounted for observation using standard or confocal microscopy. All steps were for 1 hour at room temperature. Standard fluorescence microscopy was performed with an Axioskop 2 plus microscope (Carl Zeiss, Oberkochen, Germany) combined with a Spot camera and software v2.2 (Diagnostic Instruments, Sterling Heights, MI). All images were acquired using the same settings. Confocal images were acquired using a TCS-SP2 laser-scanning confocal spectral microscope (Leica Microsystems Heidelberg GmbH, Mannheim, Germany) equipped with argon and helium-neon laser beams and attached to a Leica DM1RB inverted microscope. The excitation wavelengths for fluorochromes were 488 nm for FITC, 543 nm for TRITC, and 633 for Alexa Fluor 647. To ensure specificity of fluorochromes, the emission aperture for fluorescence detection were 500 to 543 nm for FITC, 559 to 615 nm for TRITC, and 650 to 750 nm for Alexa Fluor 647. Distribution of fluorescence was analyzed using the Leica Confocal Software version 2.61. Images were acquired using the same settings either for young and aged NZW mice or for Tg-hGPBP and non-Tg-hGPBP mice when comparing intensity of fluorescence. Slices from mouse kidney were fixed overnight at 4°C in 2% glutaraldehyde and 0.1 mol/L cacodylate, pH 7.5, and rinsed with 0.1 mol/L cacodylate and 0.1 mol/L saccharose, pH 7.5, and kept at 4°C. Postfixation was performed with 1% osmium tetroxide in the same buffer for 1 hour. Dehydration through a graded acetone series was followed by embedding in Epon 812 resin. Selection of the samples was made in semithin sections stained with toluidine blue and evaluated in a light microscope. Ultrathin sections, stained with uranyl acetate and lead citrate, were examined with a 1010 Jeol electron microscope (JEOL Ltd., Tokyo, Japan) at 60 kV. Cryosections of NZW mouse kidney were stained with hematoxylin and eosin following the manufacturer's recommendations for RNA preparation. Approximately 250 to 300 glomerular sections from each mouse were dissected and pooled using a PALM microdissector (P.A.L.M. Instruments, Bernried, Germany). Total RNA was extracted from individual mouse sections using RNeasy Protect mini kit (Qiagen, Valencia, CA). cDNA was synthesized using random hexamer (Applied Biosystems, Foster City, CA) and RTG You Prime RXN beads (Amersham Pharmacia Biotech, Piscataway, NJ) according to the manufacturers' recommendations. Real-time PCRs for GPBP and GAPDH were performed in duplicate with SYBR Green PCR Master Mix and a SDS 5700 apparatus (Applied Biosystems). Primers used were 5′-GGGAAGCCCATCACCATCT-3′ (forward) and 5′-CGACATACTCAGCACCGGC-3′ (reverse) for GAPDH and 5′-GCTGTTGAAGCTGCTCTTGACA-3′ (forward) and 5′-CCTGGGAGCTGAATCTGTGAA-3′ (reverse) for GPBP. To determine GPBP expression we used the ΔΔCt method and GAPDH as a normalizer. Relative expression of GPBP was calculated using 5-month-old mice as reference (GPBP expression = 1). In Tg-hGPBP mice, hGPBP and murine GPBP levels were determined using individual melting curves. Data are presented as scatter plots with a bar indicating the mean of each series; a dot or circle represents the mean value measured in individual mouse samples. We used unpaired Student's t-test or Mann-Whitney test to assess differences between series. A P value <0.05 was considered significant. Prism 4.0 software (GraphPad Software, San Diego, CA) was used for all calculations. LM studies revealed that commencing with 7-months of age, NZW mice developed a glomerulonephritis characterized by hyaline deposits and mesangial matrix expansion (Figure 1), which progressively affected an increasing number of glomeruli in the absence of significant proteinuria (not shown). Hyaline deposits and matrix components occupied the mesangium or the peripheral region of the glomerulus displaying two distinct morphological patterns, mesangial or nodular, which remained differentiated until glomerular collapse. In general, the nodular pattern was more focal and the mesangial pattern more diffuse, affecting 20 to 40% and 40 to 90% of the glomeruli, respectively. The pathology was more evident with Masson trichrome staining, which revealed that as hyaline fuchsinophilic material (lipstick color) increased, there was also a matricial anilinophilic material (blue color) that constrained and progressively substituted hyaline fuchsinophilic material. The latter virtually disappeared when glomerular architecture was disrupted, as noted by a significant reduction in cells and the collapse of capillary spaces (glomerulosclerosis) (Figure 1A). The pathological pattern varied among mice, and thus, we noted mice with either a prevalent nodular (20 to 30%) or mesangial (70 to 80%) glomerulonephritis, although mice and glomeruli with mixed patterns were also observed. Mesangial cell proliferation (more than three cells per peripheral mesangial area) was found in affected glomeruli of the mice undergoing mesangial glomerulonephritis. In contrast, no mesangial cell proliferation was detected in mice with prevalent nodular glomerulonephritis (not shown). Other histopathological findings included wire loops, hyaline thrombi, subendothelial deposits, silver-stained spikes, and occasional crescents (not shown), all of which are common findings in immune complex-mediated glomerulonephritis. Finally, no inflammatory cell infiltrates (macrophages, lymphocytes, or neutrophils) were detected in these animals. EM analysis revealed that, whereas in young NZW mice the glomerular structure was virtually preserved (not shown), there existed abundant abnormalities in old NZW mice (Figure 1B). The dominant ultrastructural finding included mesangial deposits intimately associated with the lamina densa of a lengthened (para)mesangial GBM (Figure 1Ba). These deposits were either electron dense and homogenous or less electron dense and vacuolated (Figure 1Bb). As the pathology progressed, these deposits could be found filling paramesangium and mesangium and even occluding capillary walls, although with differing proportions depending on the morphological pattern. In mesangial glomerulonephritis, electron-dense homogeneous deposits were predominant (Figure 1Bc) whereas in nodular glomerulonephritis the predominant deposits were of vacuolated material (Figure 1, Bd and Be). Additional ultrastructural findings included electron-dense deposits associated with lamina rara of (para)mesangial GBM (Figure 1Bf) and with the subepithelial or the subendothelial side or within the lamina densa of capillary GBM (not shown). All of these abnormalities are commonly found in human immune complex-mediated glomerulonephritis including IgA nephropathy and systemic lupus erythematosus.8Balow JE Boumpas DT Austin III, HA Systemic lupus erythematosus and the kidney.in: Lahita RG Systemic Lupus Erythematosus. Academic Press, San Diego1999: 657-685Google Scholar, 17Haas M IgA nephropathy and Henoch-Schönlein purpura nephritis.in: Jennette JC Olson JL Schwartz MM Silva FG Heptinstall's Pathology of the Kidney. Lippincott Williams and Wilkins Publishers, Philadelphia2007: 423-486Google Scholar Finally, fibrillar collagen was not noted at any disease stage. LM and EM studies supported that NZW mice underwent age-dependent glomerulonephritis characterized by GBM structural alterations and mesangial matrix expansion. This was further investigated by indirect immunofluorescence procedures in NZW mice of different ages and types of glomerulonephritis (Figure 2). Mesangial matrix expansion in NZW mouse glomerulonephritis was confirmed using s
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