Differential expression of collagen type IV alpha-chains in the tubulointerstitial compartment in experimental chronic serum sickness nephritis.

The expression of collagen type IV chains in the renal tubulointerstitium was investigated during the development of chronic serum sickness (CSS) in rats, a model for immune complex-mediated renal disease. Immunohistochemical studies showed increased expression of alpha4(IV) collagen early during disease development, followed by an increase in alpha1(IV) through alpha3(IV) collagen subchain expression, especially in the tubular basement membrane. Dot-blot and in situ hybridization analysis showed a transient increase in steady-state mRNA levels for all collagen IV subchains during the development of CSS, which was most abundant for alpha1(IV), alpha2(IV), and alpha4(IV). Statistical correlations were found between the mRNA levels of alpha1(IV) and alpha2(IV) collagen and between alpha3(IV) and alpha4(IV), in line with the results of others which showed that these chains are co-distributed as heterotrimer collagen type IV molecules. However, additional correlations were found between the mRNA levels coding for alpha1(IV) and alpha3(IV) collagen, and between alpha1(IV) and alpha4(IV) mRNAs in the course of CSS. These abnormal correlations support the hypothesis that changes occur in the co-expression of the collagen IV subchains during the development of CSS. In addition, a strong correlation was found between the presence in the tubulointerstitium of alpha1(IV) and alpha2(IV) collagen chains, on the one hand, and the tubulointerstitial influx of R73+ and ED1+ cells, on the other, suggesting the involvement of inflammatory cells in the observed alterations in matrix production. Changes in the relative abundance of collagen IV chains in disease states may perturb the collagen IV network in the tubulointerstitial compartment and thereby play a role in the development of renal failure.

Differential expression of collagen IV isoforms in experimental glomerulosclerosis.

Expansion of the glomerular mesangial matrix (MM), thickening of the glomerular basement membrane (GBM), and eventually the development of glomerulosclerosis are often seen in immunologically mediated kidney diseases. In addition to quantitative changes in the extracellular matrix (ECM), qualitative changes in ECM molecules may contribute to alterations in the composition of the glomerular matrix. The expression of collagen IV, alpha 1-5(IV) mRNA, and polypeptides was therefore investigated during the development of chronic graft-versus-host disease (GvHD) in mice, a model for lupus nephritis, and in chronic serum sickness (CSS) in rats, a model for membranous nephropathy. Immunohistochemical studies showed increased mesangial expression of alpha 1 and alpha 2 early in the disease, but only late in the GBM. In contrast, alpha 3 and alpha 4 increased in the GBM during disease, but not in the MM. The mRNA levels for all collagen IV chains were increased in isolated glomeruli before morphological alterations were detectable. The mRNA increase was earlier and more profound for alpha 3, alpha 4 and alpha 5 than for alpha 1 and alpha 2. Expression of alpha 3(IV) was greatest in GvHD, whereas expression of alpha 4 was greatest in CSS. As determined by in situ hybridization (ISH), alpha 1 mRNA was observed dispersed in the glomerulus, but alpha 3, alpha 4, and alpha 5 mRNAs were mainly located in cells at the periphery of the glomerular tuft. The changes in the relative abundance of collagen IV mRNA in disease states may perturb the collagen IV network, altering glomerular structure and function, and may thereby play a central role in the development of glomerulonephritis and glomerulosclerosis.

Augmented expression of glomerular basement membrane specific type IV collagen isoforms (alpha3-alpha5) in experimental membranous nephropathy.

In human and experimental membranous nephropathy, new extracellular matrix accumulates between, and eventually surrounds, immune deposits on the subepithelial aspect of the glomerular basement membrane (GBM). To define the nature and source of this newly deposited matrix, we studied by in situ hybridization and immunohistology the production and tissue deposition of the recently defined basement membrane type IV collagen chain isoforms alpha3, alpha4, and alpha5, the mesangium-specific alpha1 and alpha2 isoforms of type IV collagen, and the fibrillar interstitial type I collagen during the development of immunological injury in passive Heymann nephritis (PHN), a rodent model of membranous nephropathy. Our results show that steady-state mRNA levels of alpha3-alpha5 (IV) but not alpha1 (IV) are significantly increased in the glomeruli of rats with PHN at the peak of immunological injury after 14 days. Increased signal for alpha4 (IV) and the new appearance of alpha1 (I) could be clearly localized to glomerular podocytes, the target of injury in this model. In addition, increased levels of immunoreactive alpha3-alpha5 were visible in the peripheral and paramesangial GBM together with de novo deposits of type I collagen. A modest increase in mesangial staining for alpha1/alpha2 (IV) was present in PHN glomeruli. In rats depleted of complement for 5 days after PHN induction, the peak of alpha4 (IV) mRNA expression on day 14 was blunted. In conclusion, we have shown increased production of the intrinsic GBM type IV collagen isoforms alpha3-alpha5 and ectopic production of type I collagen by injured podocytes in PHN. These changes may contribute to the formation of an expanded and disorganized GBM, as seen in experimental and human membranous nephropathy.

Matrix and adhesion molecules in kidney pathology: recent observations.

The purpose of this article is to review a set of recently obtained data concerning matrix and matrix adhesion molecules in renal disease. Our goal is not to cover the entire topic, but rather to focus on findings obtained with an experimental model for chronic lupus nephritis, evoked in mice by inducing graft-versus-host disease (GVHD). The overall aim of these studies was to investigate the role of adhesion molecules as targets for autoantibodies, in the recruitment of inflammatory cells, and in the accumulation of matrix in kidney disorders. In addition, we set out to discover how matrix proteins in renal diseases differ from normal matrix molecules both quantitatively, in their increased frequency, and qualitatively, in their intramolecular structure. The advances in understanding and methodology described in this review imply a substantial capability for greater insight into the pathogenesis of kidney disease; for making better use of renal biopsies, such as in applying competitive reverse-transcriptase-polymerase chain reaction (RT-PCR) in RNA analysis for matrix; and in developing more effective treatment strategies for patients with kidney disease.

Fatal attraction: adhesion molecules and disease.

Knowledge of molecular mechanisms in cell-cell and cell-matrix adhesion has increased rapidly in the past decade. Adhesion mechanisms are of prime importance in both physiology and pathology. With respect to the kidney, expression of adhesion molecules has been studied in a variety of diseases, including various forms of glomerulonephritis. Hitherto, these descriptive studies have merely launched extensive speculation regarding the role of adhesion mechanisms in renal pathology. A logical next step is to correlate adhesion molecule expression to alterations in structures which may possibly be affected by altered adhesion, for example gap junctions. Current studies linking structural to functional adhesion expand our understanding of cell biology in health and disease.

Differential effects of sex hormones on autoantibody production and proteinuria in chronic graft-versus-host disease-induced experimental lupus nephritis.

In patients with systemic lupus erythematosus, the female-to-male ratio is as high as 10:1. Sex hormones are thought to play a role in this difference in susceptibility. In a previous study, we demonstrated a high susceptibility of female mice to the development of glomerulonephritis after induction of chronic graft-versus-host disease (GVHD), compared with male mice. In order to unravel further this gender-related difference (C57B1/10*DBA/2)F1 hybrid mice were either castrated or ovariectomized and treated with 17beta-ethinyloestradiol or testosterone-decanoate preceding the induction of chronic GVHD. Testosterone-decanoate reduced significantly the development of albuminuria in females. In contrast, proteinuria of 17beta-ethinyloestradiol-treated female mice was in the same range as that of sham-operated mice. Autoantibody levels against glomerular basement membrane, renal tubular epithelium, dsDNA and ssDNA, as determined by ELISA, were higher in 17beta-ethinyloestradiol-treated female mice than in all other groups. Immunofluorescence studies showed the presence of immunoglobulin and complement deposits in glomeruli of all animals, without significant differences between the experimental groups. Our findings confirm earlier observations, in that testosterone-decanoate is shown to be an inhibitory compound, whereas 17beta-ethinyloestradiol has stimulating properties in autoimmunity. Moreover, our results show for the first time differential hormonal effects on autoantibody levels and proteinuria in experimental lupus nephritis.

Cloning of the mouse fibronectin V-region and variation of its splicing pattern in experimental immune complex glomerulonephritis.

Increased mRNA and protein expression of extracellular matrix (ECM) components, including fibronectin, occurs during the development of glomerulonephritis and glomerulosclerosis in immunologically mediated kidney diseases. However, in addition to these quantitative changes in ECM expression, qualitative changes in these molecules may contribute to malformations in the composition of the glomerular matrix. These qualitative changes may include alterations in the splicing pattern of the V-region of fibronectin, since this region plays a role in its accumulation. The splicing patterns of this region have been studied in chronic graft-versus-host disease (GvHD) in mice, a model of lupus nephritis, and in chronic serum sickness (CSS) in rats, a model of immune complex nephritis. Cloning of the mouse fibronectin V-region from kidney tissue revealed 96.1 per cent homology with the corresponding domain in rat fibronectin. PCR (polymerase chain reaction) analysis of RNA from isolated glomeruli revealed three isoforms of this region in both mouse and rat fibronectin, namely inclusion or exclusion of the whole region, or exclusion of only the CS1 domain. In both models, increased exclusion of the V-region was observed early in the disease. However, in GvHD the splicing pattern returned to normal, whereas in CSS the shift persisted during the course of the experiment. Differentiated expression of fibronectin isoforms may exert an important effect on the structure and biological function of the glomerulus and may thus play a role in the development of glomerulonephritis and glomerulosclerosis.

Role of the extracellular matrix in the development of glomerulosclerosis in experimental chronic serum sickness.

Glomerulosclerosis is a severe complication of many immunologically mediated kidney diseases and is associated with a poor prognosis with respect to renal function. The aim of this study was to elucidate the role of the extracellular matrix (ECM) in the development of glomerulosclerosis in experimental immune complex glomerulonephritis. Induction of chronic serum sickness by repetitive injections of human IgG into preimmunized Wistar rats leads to the development of immune complex nephritis and glomerulosclerosis. At an early stage of the disease fibrinogen accumulation was observed along the endothelial cells, presumably related to damage of the endothelial lining. mRNA levels for several collagen types, laminin B1 and B2, and fibronectin were increased in both whole-kidney tissue and in isolated glomeruli, but morphological changes were not observed. In situ hybridization experiments demonstrated increased ECM mRNA levels in glomerular and tubular cells. Starting at week 15, glomerular mesangial matrix expansion and thickening of the glomerular basement membrane (GBM) was observed. ECM components were abundantly present. Coagulation factors were not observed at this point. ECM mRNA levels were decreased as compared to week 0, but were still above normal. Focal and segmental end-stage sclerotic lesions developed at weeks 25-30, in which fibronectin and fibrinogen were the major constituents. Other ECM components were found peripherally from these lesions in the remnants of the mesangial matrix and GBM. Sclerotic matrices did not demonstrate an increase of cellular-fibronectin, and other constituents from the circulation were not present in the lesions. Glomerular ECM mRNA was decreased to normal levels. However, a dramatic increase of ECM mRNA expression was observed at sites of inflammatory infiltrate in the perivascular, interstitial, and periglomerular regions. In conclusion, the development of glomerulosclerosis in chronic serum sickness rats is preceded by mesangial matrix expansion in which several ECM components are increasingly expressed. Steady state mRNA levels for these components are increased before morphological changes are detectable. In the final stage there is a specific accumulation of exogenous fibronectin in the glomerular end-stage sclerotic lesions. Simultaneously, an interstitial inflammatory reaction takes place leading to increased ECM production in the tissue surrounding the damaged glomeruli.

Qualitative alterations in laminin expression in experimental lupus nephritis.

Previous studies have revealed quantitative alterations in laminin-1 expression at the mRNA and protein levels during the development of glomerulonephritis and glomerulosclerosis in chronic graft-versus-host disease in mice, a model for lupus nephritis. We have now studied the qualitative alterations in laminin expression with two monoclonal antibodies that recognize epitopes on either the E8 or the P1 fragment of laminin-1. Both of these fragments are involved in cell-matrix and matrix-matrix interactions. In normal glomeruli these laminin epitopes are present only in the mesangial matrix; during embryogenesis, however, they are also present in the glomerular basement membrane. The distribution of laminin epitopes was first studied by using immunofluorescence in kidneys of mice with graft-versus-host disease at different points in time after disease induction. Reflection contrast and immunoelectron microscopy were performed after in vivo injection of the horseradish peroxidase-coupled monoclonal antibodies. In glomeruli of mice 8 weeks after disease induction, both injected antibodies bound specifically in electron-dense immune deposits in the mesangium and subepithelially along the glomerular basement membrane as well as in the expanded mesangial matrix. At 11 and 12 weeks after disease induction, when focal and segmental glomerulosclerosis had developed, the antibodies additionally bound in the matrix subendothelially along the glomerular basement membrane and at the periphery of end-stage sclerotic lesions. To study changes in the distribution of laminin epitopes over time, mice were injected with either monoclonal antibody before induction of graft-versus-host disease. The antibodies were detected 8 and 12 weeks later in the mesangial matrix of mice with lupus nephritis. Once segmental glomerulosclerosis had developed, the antibodies were additionally detected within the thickened glomerular capillary wall. The specific binding of anti-laminin monoclonal antibodies in electron-dense immune deposits further substantiates the hypothesis that anti-laminin autoantibodies participate in glomerular immune complex formation in this model, as suggested by earlier studies. Furthermore, our results show that the distribution of glomerular laminin epitopes in the matrix is altered during the development of glomerular disease. These changes in the structure of the glomerular basement membrane may contribute to the abnormal cell-matrix and matrix-matrix interactions during the development of glomerular disease in this model for lupus nephritis.

Specific accumulation of exogenous fibronectin in experimental glomerulosclerosis.

The prognosis of patients showing glomerulosclerosis as a complication of an immunologically mediated kidney disease is poor. To improve the diagnosis and treatment of these patients, it is important to understand the processes involved in the development of glomerulosclerosis. In this study, we investigated the molecular composition of experimental end-stage glomerular sclerotic lesions and their pathogenesis in chronic graft-versus-host disease (GvHD) in the mouse and chronic serum sickness in the rat. Accumulation studies were performed to determine the degree of specific trapping of constituents from the circulation. Two different models were investigated to determine whether differences in disease initiation resulted in different compositions of the glomerulosclerotic lesions. In both models, glomerulosclerosis was preceded by expansion of the mesangial matrix and thickening of the glomerular basement membrane (GBM). The end-stage sclerotic lesions consisted mainly of fibronectin, which appeared to displace the other extracellular matrix (ECM) components peripherally in the mesangial matrix and GBM. The abundance of fibronectin in the lesions was not reflected in the mRNA levels for this component. Indeed, antibodies directed against the cellular form of fibronectin did not stain positive in the end-stage lesions. These findings, together with accumulation studies, suggest that specific accumulation rather than de novo synthesis of fibronectin plays a major role in the development of experimental glomerulosclerosis, which appears to be independent of the pathway of induction.

Prevention of glomerulosclerosis by early cyclosporine treatment of experimental lupus nephritis.

The influence of cyclosporine A (CsA) treatment on the development of glomerulonephritis and glomerulosclerosis was investigated in chronic graft-versus-host disease (GvHD), a murine model for lupus nephritis. The renal disease is characterized by the formation of IgG-containing electron-dense deposits along the glomerular basement membrane (GBM) and in the mesangium, followed by the onset of proteinuria which starts, varying per individual mouse, about six weeks after the induction of the disease. Glomerular mRNA levels for matrix molecules were increased from week 4, preceding mesangial matrix expansion and GBM thickening which occurred from week 6. These initial events finally led to development of glomerulosclerosis, and end-stage renal failure. Groups of mice received three intraperitoneal (i.p.) injections per week with different doses of CsA, and treatment was started 2, 4, or 6 weeks after induction of the disease. Treatment with 10 or 50 mg CsA/kg/week did not influence the development of glomerulonephritis or glomerulosclerosis. Injection of 100 mg CsA/kg/week delayed the onset of proteinuria only when treatment was started in week 2. In week 6 some mice had already developed proteinuria whereas others had not. Treatment with 250 mg CsA/kg/week starting in week 6 abrogated glomerulonephritis and glomerulosclerosis only in those animals which were not yet proteinuric at that time. This, despite comparable increased autoantibody levels against DNA, GBM, and renal tubular epithelium (RTE) in both treated and untreated GvHD mice. Further increase in proteinuria and development of glomerulosclerosis could not be prevented if the mice already had developed proteinuria when CsA treatment was started. Dot blot analysis and in situ hybridization showed significantly decreased mRNA levels for alpha 1(I) and alpha 1(IV) collagen in kidneys of CsA-treated mice as compared to those of untreated mice 12 weeks after induction of the disease, if the highest dose of CsA was administered before the onset of proteinuria. No effect on these whole-kidney mRNA levels was observed in mice which had already developed proteinuria before CsA injections were started. Increased mRNA expression for matrix molecules in this group and in untreated GvHD mice was observed mainly in the interstitium. The kidneys of the treated GvHD mice and those of mice injected with 250 mg CsA/kg/week without induction of GvHD showed no morphological signs of CsA nephrotoxicity. We conclude that treatment with 250 mg CsA/kg/week prevents the development of glomerulonephritis and glomerulosclerosis in this model of lupus nephritis, if started before the onset of proteinuria.(ABSTRACT TRUNCATED AT 400 WORDS)

Optimal method for RNA extraction from mouse glomeruli.

Extraction of RNA has been described for rat and rabbit glomeruli but not for mouse glomeruli. Due to their small size, mouse glomeruli cannot be isolated by relatively simple sieving techniques. Based on recently reported methods for the isolation of mouse glomeruli, we developed an RNA isolation technique by performing comparative methodological studies. Two standard RNA extraction methods were compared. In addition in separate experiments the influence was studied of protease inhibitors and freezing and thawing of whole kidney prior to glomeruli isolation, on the yield and degradation of RNA. Therefore kidneys were perfused with 10 ml 0.01 M PBS containing 1.25% Fe3O4 through the aorta. Kidneys were decapsulated and passed through a 75-microns metal screen. After pelletting and washing, tubes were placed against a magnet and pelleted glomeruli were washed three times. In a second experiment protease inhibitors were added to the PBS. As a third method, kidneys were frozen before the isolation of glomeruli. From isolated glomeruli RNA was extracted using either caesium chloride or lithium chloride method. The yields of RNA (OD 260) were highest using the lithium chloride method. Hybridization of Northern blots of extracted RNA with cDNA probes showed the best results when RNA was extracted using the lithium chloride method, while the caesium chloride method led to considerable degradation of RNA. Freezing of kidney tissue prior to RNA extraction led to the virtual absence of any signal. We then applied this method successfully in an in-vivo model of experimental lupus nephritis. This is the first description of an optimal protocol for the extraction of RNA from mouse glomeruli.(ABSTRACT TRUNCATED AT 250 WORDS)

Gender-related influences on the development of chronic graft-versus-host disease-induced experimental lupus nephritis.

Autoimmune diseases are far more common in women than in men. In the incidence of systemic lupus erythematosus (SLE), the female-to-male ratio is as high as 10:1. This suggests that sex hormones may play a fundamental role in determining the susceptibility to these diseases. In order to investigate the sex-related differences in the inducibility of chronic graft-versus-host disease-related experimental lupus nephritis, lymphocytes from female DBA/2 donor mice were administered to either male or female (C57BL10 x DBA/2)F1 recipients. An additional group of male recipients received lymphocytes from male DBA/2 donors. After four cell transfers, female recipients developed a significantly higher albuminuria than both male groups. Serum concentrations of autoantibodies against glomerular basement membrane (GBM), collagen IV, and laminin were significantly higher in females 2-4 weeks after induction. Levels of circulating autoantibodies against renal tubular epithelial antigens (RTE) and nuclear antigens were not different between the sexes. In transfer studies, the necessity of the presence of anti-GBM and anti-RTE autoantibodies for the development of glomerulonephritis was confirmed. These findings indicate that: (i) in this model of lupus nephritis, susceptibility to glomerulonephritis is strongly influenced by sex-related genes; and (ii) among the variety of autoantibodies occurring in this model of SLE, both anti-GBM and anti-RTE autoantibodies play a key role in the pathogenesis of glomerulonephritis.

A molecular biologic study of extracellular matrix components during the development of glomerulosclerosis in murine chronic graft-versus-host disease.

BACKGROUND: We studied the development of glomerulosclerosis in murine chronic graft-versus-host disease, a model for human systemic lupus erythematosus. EXPERIMENTAL DESIGN: The disease was induced in (C57BL10 x DBA/2)F1 hybrids by injection of DBA/2 lymphocytes leading to deposition of auto-antibodies in the glomeruli, and a lupus type of nephritis morphologically. We have determined the levels of mRNA coding for laminin (B1 and B2), a 67 kilodalton laminin binding protein, and types I and IV collagen, in control and graft-versus host disease mice at various times after disease induction. RESULTS: Laminin and collagen mRNAs were increased in whole kidneys 4 weeks after induction of the disease. At week 10, all animals displayed dramatic stimulation of alpha 1(I), alpha 1(IV), laminin B1, and B2 mRNAs. The 67 kilodalton laminin binding protein mRNA was also doubled from week 4 to 16. In isolated glomeruli, the mRNA level coding for laminin B2 was already significantly increased from week 8. This enhancement of laminin synthesis corresponds to the mesangial expansion and to the development of laminin-containing spike formations of the glomerular basement membrane at week 8. CONCLUSIONS: The expansion of the mesangial matrix in murine chronic graft-versus-host disease is caused at least in part, by an increased production of extracellular matrix components by glomerular cells. These results demonstrate that the increase of specific extracellular matrix components mRNAs precedes light microscopic changes. Quantitative evaluation of the mRNA levels coding for extracellular matrix proteins may reveal a useful method for the early detection of the development of glomerular sclerosis at the stage preceding the onset of anatomo-clinical changes.

A histologic study of the extracellular matrix during the development of glomerulosclerosis in murine chronic graft-versus-host disease.

The development of glomerulosclerosis was studied in murine chronic graft-versus-host disease (GvHD), which is a model for human systemic lupus erythematosus. The authors investigated the distribution patterns of six components of the extracellular matrix (ECM), i.e., laminin, fibronectin, collagen types I, III, IV, and VI during the course of the disease. All of these ECM components except collagen type I were found in the glomeruli of normal mice, where all of them were intrinsic constituents of the mesangium. Laminin, fibronectin, and collagen type IV were also found in the glomerular capillary walls. Starting 6 weeks after the induction of GvHD and continuing at week 8, the onset of an expansion of the mesangial matrix was observed. At the same time, the amounts of laminin, fibronectin, and collagen types IV and VI increased. Ten weeks after the onset of the disease, glomerulosclerosis developed. Traces of the interstitial collagen type I were found in sclerotic glomeruli. The levels of four ECM components, i.e., collagens III, IV, VI, and laminin were markedly decreased in the sclerotic glomeruli as compared with week 8. In contrast, the amount of fibronectin in the sclerotic glomeruli increased dramatically. Immunoelectron microscopic examination showed fibronectin in the sclerotic lesions, in contrast to laminin, collagen type I, and collagen type IV. It is concluded that the sclerotic lesions in murine chronic GvHD contain fibronectin. The small amounts of the ECM components laminin, as well as collagens III, IV, and VI in the sclerotic glomeruli in GvHD, might represent remnants of mesangial material and collapsed capillary walls. These components are probably replaced by increased production and/or accumulation of collagen type I and fibronectin.

Isolation and characterization of mouse glomerular basement membrane.

Glomerular basement membrane (GBM) antigens used for immunological studies have until now been isolated from human and rat glomeruli but not from mice. However, given the growing awareness of extracellular matrix species-specificity, the need for a purification method of mouse GBM exists. We now report the purification of GBM from isolated mouse glomeruli. 10 ml 0.01 M phosphate buffered saline (PBS) containing 1.25% Fe3O4 was perfused through the aortae of (C57BL10 x DBA/2)F1 mice over 1 min, kidneys were decapsulated and passed through a 0.075 mm mesh metal screen and collected. After pelletting and washing, the tube was placed against one pole of a magnet and the pelleted glomeruli were washed. This procedure was repeated three times. The glomerular suspension was examined by light microscopy, sonicated, and lyophilized. In suspension no free fragments of tubuli or Bowman's capsule were present as observed by light microscopy. Mouse GBM was prepared from the isolated glomeruli using a modification of earlier described methods for human and rat GBM by enzymatic digestion. Elisa studies showed the presence of laminin, type IV collagen, and fibronectin. Monoclonal antibody directed against tubular epithelial antigen gp160 did not react to either mouse or rat GBM. Sera and glomerular eluates from (C57BL10 x DBA/2)F1 mice suffering from chronic graft-versus-host autoimmune disease gave a higher signal against mouse GBM than against rat GBM. Normal rabbit serum, normal mouse serum and normal mouse eluate did not show significant activity against mouse GBM. Immunoblotting showed the presence of both laminin B1 and B2 chains as well as type IV collagen alpha 1 and alpha 2 subunits. In summary, we describe a method by which it is possible to extract mouse GBM with a high purity.