Extracellular matrix deposition and cell proliferation in a model of chronic glomerulonephritis in the rat

Sigrid Harendza, André Schneider, Udo Helmchen1 and Rolf A. K. Stahl

Department of Medicine, Division of Nephrology and Osteology and 1 Department of Pathology, University Hospital Eppendorf, Hamburg, Germany

Correspondence and offprint requests to: Sigrid Harendza, Universitätskrankenhaus Eppendorf, Medizinische Klinik, Abt. Nephrologie/Osteologie, Pavillon 61, Martinistr. 52, D-20246 Hamburg, Germany.



   Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Resident glomerular cell proliferation, matrix deposition and secretion of matrix metalloproteinases play a major role in the progression of chronic glomerular disease. These features were studied in a novel approach in a rat model of chronic glomerulonephritis induced by four injections of an anti-Thy 1.1 antiserum at weekly intervals.

Methods. Chronic immune mediated mesangial injury was induced in male Sprague–Dawley rats by repeated intravenous injection of an anti-Thy 1.1 antiserum. One week after the first and fourth injection of the antiserum proteinuria was evaluated and the kidneys were removed. Immunohistology was performed for proliferating cells, monocytes and collagen type IV. Furthermore, mRNA expression of collagen type IV, TGF-ß and the matrix degrading enzyme MMP-2 as well as MMP-2 protein expression were studied.

Results. Urinary protein excretion was dramatically increased after one antiserum injection and stayed elevated at a lower level after the fourth antiserum injection. After the initial induction of nephritis, 7 days following antiserum, resident glomerular cell proliferation was increased whereas with repeated injections of the antiserum cell numbers were not different from controls, as measured 1 week after the fourth injection. In contrast, extracellular matrix accumulation (collagen type IV) increased after the first antiserum injection and further increased after the fourth antiserum injection. The mRNA expression for collagen type IV increased after the first antiserum injection and showed further increase after the fourth antiserum injection. Induction of nephritis also stimulated glomerular mRNA expression of MMP-2 and TGF-ß, both of which remained at a high level after the fourth antiserum injection. Glomerular protein levels of MMP-2 also increased after the first antiserum injection and showed a further slight increase after the fourth injection.

Conclusion. Increased cellular proliferation is involved in an early stage of this disease, while enhanced expression of glomerular matrix and augmented mRNA and protein expression of the matrix degrading enzyme MMP-2 continue into the chronic phase, and contribute to the extensive structural remodeling process that accompanies this form of glomerular injury.

Keywords: chronic glomerulonephritis; collagen type IV; matrix remodelling; metalloproteinases



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Intraglomerular accumulation of extracellular matrix (ECM) proteins and mesangial cell proliferation are important components of pathophysiological changes in various glomerular diseases, often resulting in end-stage renal disease with development of severe glomerulosclerosis [1,2]. The mechanisms leading to ECM synthesis and turnover are still poorly understood. Several studies demonstrate that a matrix degrading enzyme, the matrix metalloproteinase 2 (MMP-2), is involved in the regulation of the mesangial cell inflammatory phenotype and plays a major role in cell growth and matrix deposition in a model of acute mesangial proliferative glomerulonephritis [3,4]. These studies suggest that enhanced MMP-2 expression is an important factor in the remodelling of the excess matrix which accumulates in this model. The in vitro data provide evidence for a conversion of an activated, inflammatory mesangial phenotype to the resting state after inhibition of MMP-2 in these cells [3].

Experiments from other laboratories in different cell systems confirm a close interaction between ECM turnover and modulation of cell growth in vitro [5,6]. In the rat anti-Thy 1.1 glomerulonephritis, induced by a single injection of an antiserum that reacts with a Thy 1-like antigen on the surface of glomerular mesangial cells [7,8], an increase of ECM synthesis during proliferation of mesangial cells could also be demonstrated [9]. The rat anti-Thy 1.1 nephritis is considered to be a self-limited disease with resolution of glomerular hypercellularity with apoptosis acknowledged as a major cell clearing mechanism [10] and increased MMP-2 activity leading to ECM degradation [4]. To date, cell proliferation and ECM turnover, two important pathophysiological features of renal disease, have not been extensively studied in models of chronic glomerulonephritis although a few studies report prolonged glomerular changes after two injections of anti-Thy 1.1 antibody [1115].

In this study we have developed a model of chronic mesangial proliferative glomerulonephritis in Sprague–Dawley rats by multiple injections of an anti-Thy 1.1 antiserum. The model is characterized by increased proliferation of mesangial cells in the early phase, persistent proteinuria, and persistent and expanded deposition of ECM in the later phase, while the expression of collagen type IV mRNA, of the matrix degrading enzyme MMP-2 mRNA and of TGF-ß mRNA remains elevated at both time points studied.



   Subjects and methods
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Induction of nephritis
Chronic immune mediated mesangial injury was induced in male Sprague–Dawley rats (100 g body weight) by repeated intravenous injection of 0.5 ml of a rabbit anti-rat thymocyte serum (ATS). ATS was generated in New Zealand rabbits by repeated immunization with 2x108 thymocytes from Lewis rats combined with Hunter's Titer Max as described earlier [16].

Experimental protocol
To assess mesangial proliferation and matrix synthesis in the chronic model of mesangio-proliferative nephritis, rats were divided in three groups of 10 animals each (Figure 1Go). Control animals received 0.5 ml of non-antibody rabbit serum intravenously. Nephritic animals received either a single intravenous injection of 0.5 ml ATS or one injection per week for 4 weeks. Animals were sacrificed 1 week after the last antiserum injection. Kidneys were removed and tissue from one kidney from each animal was collected for histologic evaluation. The second kidney was used for isolation of glomeruli using a fractional sieving technique [17] resulting in a 96% purity of glomeruli.



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Fig. 1. Structural diagram of the experimental design of induction of chronic glomerulonephritis. A detailed description is given in Materials and methods. R, injection of non-antibody rabbit serum; A, ATS injection, N, nephrectomy.

 
Urinalysis
Twelve-hour urine specimens were collected from each animal prior to sacrifice and urinary protein was measured according to Lowry [18].

RNA isolation and northern blot hybridization
Total glomerular RNA was isolated by the guanidinium isothiocyanate technique [19]. RNA was electrophoresed through a 1.2% agarose gel containing 2.2 M formaldehyde. Equal loading of the lanes was evaluated by ethidium bromide staining of the 18S and 28S rRNA. RNA was transferred to nylon membranes (Zetabind; Cuno, Meriden, CT, USA) by vacuum blotting and UV cross-linked. Membranes were hybridized with three probes: a 0.8 kb cDNA fragment of rat MMP-2, a 1.3 kb cDNA fragment of {alpha}1 (IV) collagen (American Type Culture Collection) and a 0.52 kb TGF-ß1 cDNA fragment [20]. The cDNA fragments were radiolabelled using hexamer primers and 50 µCi [32P]dCTP (Dupont, NEN Research Products, Boston, MA, USA). Membranes were hybridized with 1x106 c.p.m. per milliliter of a standard hybridization buffer [19] for 18 h at 42°C. The membranes were washed twice in 2xSSC/0.5% SDS for 30 min at room temperature and subsequently in 0.4xSSC/0.5% SDS at 65°C for 30 min. Autoradiography was performed for 24–72 h at -80°C with intensifying screens. Nylon membranes were then stripped for 60 min in a standard stripping buffer [21] at 65°C and rehybridized with a 0.58 kb cDNA fragment of human 18S rRNA to account for small loading and transfer variabilities. Exposed films were scanned with a laser densitometer (Hoefer Scientific Instruments, San Francisco, CA, USA), and relative RNA levels were calculated. All blots were performed in triplicate.

Western blot analysis of MMP-2
For western blot analysis, glomeruli from each group were isolated and centrifuged in 1xPBS. The pellets were resuspended in 100 µl of Laemmli buffer 1 (33% 0.5 mM Tris–HCl pH 6.8, 66% SDS 10%). Samples were boiled for 10 min and then centrifuged. Protein concentration was determined with a Protein DC-Assay (Bio-Rad, Munich, Germany). To equal amounts of protein (75 µg), 1/2vol% loading buffer (0.5 mM Tris–HCl pH 6.8, 20% glycerol, 10% SDS, 0.5 M EDTA, 1% bromphenolblue) was added. The samples were electrophoresed on a 10% polyacrylamide SDS gel. Proteins were transferred electrophoretically to a nitrocellulose membrane (Hybond ECL, Amersham, Freiburg, Germany) at 100 V for 1 h using a Bio-Rad Mini Trans-Blot Apparatus according to the manufacturer's recommendations. The membrane was blocked with 5% non-fat dry milk in washing buffer (1xPBS, 0.1% Tween 20) for 1 h at room temperature and then incubated for another hour with a monoclonal anti-human MMP-2 antibody (ICN, Eschwege, Germany) at a concentration of 1:1000 in the same buffer. After rinsing the membrane in washing buffer for 3x5 min, the secondary antibody, a rabbit anti-mouse IgG antibody conjugated to horseradish-peroxidase (Southern Biotechnology, Birmingham, AL, USA) was added at a concentration of 1:2000 in the same buffer for 1 h at room temperature. After washing the membrane again for 3x5 min the luminescence detection of peroxidase was performed with the ECL system according to the manufacturer's recommendations (Amersham, Freiburg, Germany). Films were exposed for 1–10 min at room temperature.

Histology
Kidney tissues were either fixed in 1% buffered formaldehyde or in Methyl Carnoy's solution. Tissue sections (2 µm) were stained with the following antibodies: a goat anti-type IV collagen antibody (Biozol, Eching, Germany), a monoclonal mouse anti-proliferating cell nuclear antigen (PCNA) antibody (Dakopatts, Hamburg, Germany) and a monoclonal mouse anti-ED-1 antibody (Chemicon International, Temecula, CA, USA). Tissue sections were stained with the alkaline phosphatase anti-alkaline phosphatase (APAAP) technique. PCNA and ED-1 stains were done as double stains on the same tissue section. After incubation with the mouse anti-ED-1 antibody, kidney sections were incubated with a biotinilated anti-mouse-IgG and developed with the peroxidase antiperoxidase reaction. Afterwards a second incubation was performed with the PCNA antibody which was developed with the APAAP complex. All quantitative morphological analyses were performed in a blinded fashion. Evaluation of PCNA and/or ED-1 positive cells was performed by counting positive cells in at least 40 glomeruli of one kidney section per animal at each time point.

For the assessment of glomerular collagen type IV deposition a score was applied to the stained tissue as has been described elsewhere [21], which was adopted from Raij et al. [22] and from Olson et al. [23]. Damage ranged from grade 0 (0–5%) to grade 4 (75–100%). The glomerular damage index was calculated by multiplying the number of glomeruli with their score number. These numbers were added and divided by the total number of glomeruli assessed. At least 40 glomeruli per cross section and animal were evaluated.

Statistical analysis
Results are given as means±SD. Statistical analysis was performed with the unpaired Student's t-test. Statistical significance was defined as P<0.05.



   Results
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Kidney histology and proteinuria
A single injection of anti-Thy 1.1 serum in Sprague–Dawley rats resulted in an acute mesangial proliferative glomerulonephritis when kidneys were investigated 1 week after the injection, similar to findings in Wistar rats [8,13,21]. The following parameters assessed are also in accord with the acute model in Wistar rats [21]. Proteinuria was significantly increased in these animals (Figure 2Go). Renal histological findings showed a high number of proliferating cells assessed by immunostaining of tissue for PCNA. Compared with controls, nephritic animals revealed a 2.2-fold increase in PCNA positive cells (Figure 3A and BGo, Table 1Go). Tissues double stained for PCNA and ED-1 showed no cells positive for both markers in glomeruli. This suggests that the PCNA positive cells were glomerular resident cells rather than monocytes/macrophages. Furthermore, an increased accumulation of collagen type IV was found in glomeruli from nephritic animals (Figure 4BGo), while tissue from normal animals was only weakly positive for this collagen in the glomerulus (Figure 4AGo). The quantitative evaluation of the immunohistology demonstrated a 4.0-fold increase in nephritic rats for collagen type IV compared with controls (Table 1Go).



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Fig. 2. Urinary protein excretion was measured for 12 h and showed a marked increase 1 week after the first ATS injection. After four ATS injections proteinuria was reduced, but still significantly above baseline level (P<0.05).

 


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Fig. 3. Immunohistologic double staining for PCNA (long arrows) and ED-1 (short arrows) demonstrates several positive cells in the glomerulus of nephritic rats in the acute phase of the disease 1 week after one ATS injection (B), while glomeruli from control animals (A) and from nephritic animals in the chronic phase of the disease after four ATS injections (C) show hardly any staining. Furthermore, the staining revealed no cells which were positive for both markers, suggesting that the proliferating cells were not monocytes/macrophages. Magnification x100.

 

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Table 1. Quantitative analysis of glomerular extracellular collagen type IV depositiona and glomerular cell proliferationb
 


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Fig. 4. Immunostaining of kidney tissue from control animals with antibodies against collagen type IV (A) was weakly positive in the glomerulus. In nephritic rats one week after one ATS injection accumulation of collagen type IV (B) was markedly increased. Animals with the chronic nephritis showed an even higher increase in collagen type IV (C) after four injections of ATS. Magnification x125.

 
In contrast, rats receiving weekly injections of ATS over 4 weeks developed chronic glomerulonephritis with almost no cell proliferation in the PCNA stain when kidneys were removed 1 week after the last injection (Figure 3CGo). A progressive build-up of glomerular matrix (collagen type IV) was noted (Figure 4CGo, Table 1Go) compared with animals from the acute model receiving only one injection of ATS. Proteinuria persisted in animals with the chronic disease although at a lower level than in the acute phase (Figure 2Go).

Expression of collagen type IV mRNA, MMP-2 mRNA and TGF-ß mRNA
In a northern blot analysis, mild expression of collagen type IV mRNA was found in glomeruli from normal animals (Figure 5AGo). A significant increase was observed in glomeruli from animals after one ATS injection (3.2-fold by densitometry), and an additional increase in collagen type IV mRNA expression was manifest in glomeruli after four ATS injections (8.5-fold compared with controls). There was marginal expression of the matrix degrading enzyme MMP-2 in glomeruli from control animals (Figure 5BGo). MMP-2 expression increased markedly in glomeruli after a single injection of ATS (10.8-fold) and remained at a high level after four injections of ATS (9.9-fold compared with controls). Expression of mRNA for TGF-ß is present in glomeruli of control animals (Figure 5CGo). After a single ATS injection increased TGF-ß mRNA expression was observed (3.1-fold) which persisted at this higher level after four ATS injections (2.7-fold compared with controls).



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Fig. 5. (A) The mRNA expression for collagen type IV showed a significant increase after one ATS injection (3.2-fold by densitometry). Collagen type IV expression increased even further when animals received four injections of ATS (8.5-fold). (B) Glomerular mRNA levels of MMP-2 increased markedly after one ATS injection when compared with controls (10.8-fold by densitometry). MMP-2 expression remained at a high level after four ATS injections (9.9-fold). (C) Expression of TGF-ß mRNA was significantly increased after one ATS injection (3.1-fold by densitometry) and high expression persisted after four ATS injections (2.7-fold).

 
Protein expression of MMP-2
MMP-2 protein levels as assessed by western blotting were very low in glomeruli from control animals (Figure 6Go). In glomeruli from animals with a single ATS injection a significant increase in MMP-2 protein expression could be detected (18-fold). MMP-2 protein expression increased even further in glomeruli of rats after four ATS injections (38-fold versus controls, 2.3-fold versus single ATS injection).



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Fig. 6. Expression of MMP-2 protein levels is low in glomeruli of control animals. Increased glomerular MMP-2 protein expression occured after a single ATS injection (18-fold by densitometry) and a further increase was observed after four ATS injections (38-fold).

 


   Discussion
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Chronic glomerular diseases are accompanied by a number of characteristic features. Destruction of the glomerular integrity followed by a decrease of glomerular function is most frequently associated with proliferation of resident glomerular cells and extensive matrix accumulation and remodelling [1,2]. While these pathological observations are well described for human chronic kidney diseases, little is known about these major components in experimental chronic kidney diseases. Since enhanced mesangial cell growth and elevated extracellular matrix formation are typical features of the acute anti-Thy 1.1 model of glomerulonephritis [21], we developed a chronic form of this nephritis by multiple ATS injections in Sprague–Dawley rats, and have used this model to study the characteristic phenotypes of this chronic immune lesion, as well as the regulation of genes for collagen type IV, the matrix degrading enzyme MMP-2 and TGF-ß at the mRNA and protein level in comparison with the acute phase of this model.

A single ATS injection results in acute glomerular injury with increased proliferation of resident glomerular cells, increased collagen accumulation and upregulation in the mRNA expression of collagen type IV and MMP-2 as well as TGF-ß. This acute form of glomerulonephritis has been reported to be self-limiting, and changes in kidney tissue can return to normal [24]. After weekly ATS injections for 4 weeks, a chronic disease developed and the number of proliferating glomerular cells was reduced. Furthermore, collagen type IV mRNA expression remained at high levels while glomerular collagen type IV accumulation increased compared with a single injection of ATS. The changes in histology and mRNA production were accompanied by persistent proteinuria. These alterations suggest a chronic lesion in glomeruli of animals receiving four ATS injections. In addition, the mRNA and protein expression for the matrix degrading enzyme MMP-2 was still upregulated in animals with the chronic disease, a finding consistent with the observation that the activity of MMP-2 is upregulated in glomeruli from mice transgenic for bovine growth hormone in a murine model for glomerulosclerosis [25].

The decrease in mesangial cell proliferation with a simultaneous increase in collagen type IV accumulation is a prominent feature in the chronic model of glomerulonephritis induced by four injections of ATS. Mesangial cell growth could not be restimulated by multiple ATS injections, while mesangial hypercellularity has been described after two consecutive injections of the monoclonal antibody 1-22-3 [13]. The inverse correlation of cell proliferation and matrix accumulation has been described in other animal models and glomerular cells, in which TGF-ß was indicated as being a potent mediator of these processes [26,27]. In the chronic model of glomerulonephritis induced by four ATS injections TGF-ß mRNA expression remained elevated. TGF-ß was found to have an inhibitory effect on mesangial cell proliferation [26], and to induce the production of extracellular matrix proteins in mesangial cells grown in a diabetic milieu [27]. Inhibition of MMP-2 by antisense DNA in mesangial cells in vitro lead to an increase in TGF-ß secretion accompanied by stimulated collagen type IV production in these cells [28]. When TGF-ß is inhibited by an antibody in this setting, collagen type IV secretion is reduced as well [28]. In addition, the proliferation of mesangial cells treated with MMP-2 antisense is significantly reduced [3]. Elevated levels of TGF-ß mRNA and protein expression have been observed in anti-Thy 1.1 glomerulonephritis 5 and 10 days after a single ATS injection [29]. Furthermore, TGF-ß has been shown to increase MMP-2 mRNA expression in mesangial cells in vitro and in the acute phase of the anti-Thy 1.1 nephritis [30]. The net effect of matrix accumulation and degradation in this experimental setting resulted in a matrix increase in the glomerulus. Hence TGF-ß is a strong candidate for inducing increased matrix accumulation, increased MMP-2 mRNA expression and decreased cell proliferation in the chronic model.

In summary, we were able to establish a model of chronic Thy-1.1 nephritis in rats by using a multiple ATS-injection approach leading to persistent proteinuria. Chronic glomerular changes were accompanied by increased matrix accumulation and decreased glomerular cell proliferation. In addition to high expression of collagen type IV mRNA in the chronic phase of the Thy-1.1 nephritis a persistent elevation of mRNA and protein expression of the matrix degrading enzyme MMP-2 was observed as well as a persistent high expression of TGF-ß mRNA.



   Acknowledgments
 
We gratefully thank Ursula Kneißler for excellent technical assistance with the immunohistology. This work was supported by grants Ha 2056/3-1 and Sta 193/6-4 from the Deutsche Forschungsgemeinschaft.



   References
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 22.10.98
Accepted in revised form: 7. 6.99