Identification of cellular origin of type I collagen in glomeruli of rats with crescentic glomerulonephritis induced by anti-glomerular basement membrane antibody
Jin-Song He,
Kayo Hayashi,
Satoshi Horikoshi,
Kazuhiko Funabiki,
Isao Shirato and
Yasuhiko Tomino
Division of Nephrology, Department of Medicine, Juntendo University School of Medicine, Tokyo, Japan
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Abstract
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Background. Type I collagen is an interstitial collagen, which is not present in normal glomeruli. As type I collagen was observed in advanced glomerular lesions, it appears to be associated with deterioration of renal function. However, the origins of cells expressing type I collagen mRNA in glomeruli of diseased kidneys remains controversial.
Methods. We examined the expression of type I collagen in glomeruli at protein and mRNA levels in rat crescentic glomerulonephritis induced by anti-glomerular basement membrane (GBM) antibody. In addition, in situ hybridization and immunohistochemical staining of serial sections were performed to identify the cellular origin of type I collagen in glomeruli.
Results. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) in isolated glomeruli showed that mRNA expression of type I collagen was remarkably increased on days 7, 14, and 28 after anti-GBM antibody injection (12.2±1.4, 20.2±2.1 and 14.6±1.0-fold over day 0, respectively). Immunofluorescence for type I collagen demonstrated marked staining in the fibrocellular and fibrous crescents, and weak staining within glomerular mesangial areas. In close association with mRNA levels analysed by RT-PCR, in situ hybridization revealed predominant presence of
1(I) collagen mRNA in cells within crescentic areas and Bowman's capsules. Serial section analysis for immunostaining and in situ hybridization showed that some
1(I) collagen mRNA-positive cells were also positive for cytokeratin. In contrast, no
1(I) collagen mRNA-positive cells were stained by ED-1 and podocalyxin.
Conclusions. It appears that increased expression of type I collagen at the protein and mRNA levels in glomeruli is involved in the progression of glomerulonephritis. At least in this crescentic model, parietal epithelial cells (PECs) may partially contribute to the dysregulated production of type I collagen, which leads to glomerulosclerosis.
Keywords: crescentic glomerulonephritis; cytokeratin; in situ hybridization; parietal epithelial cell; type I collagen
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Introduction
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Quantitative and qualitative changes in collagen content play an important role in the destruction of organ architecture and function. Type I collagen is the major collagenous component which is normally present in the renal interstitium and blood vessels [1]. While not in normal glomeruli, type I collagen has been identified in diseased glomeruli, especially in advanced sclerotic lesions in human and experimental glomerulonephritis [27]. It appears that abnormal production of type I collagen in glomeruli may contribute to progression of glomerulonephritis. However, relatively little is known about the cellular origin of type I collagen in glomeruli. Previous studies found that type I collagen is deposited in the crescents and mesangium in human renal diseases [2,8]. It has also been demonstrated that type I collagen messenger RNA (mRNA) was localized in glomerular epithelial and mesangial cells [4,5,7]. But these studies did not provide direct evidence that type I collagen originated from intrinsic glomerular cells. Therefore, the contribution of glomerular resident cells and/or migrating cells from outside the glomeruli to production of type I collagen in glomeruli remains unclear. Although Wiggins et al. [3] and Merritt et al. [6] reported that expression of type I collagen mRNA was increased in crescentic glomerulonephritis induced by anti-GBM antibody, none of the previous studies identified the source of type I collagen in this disease. In the present study, in order to investigate the expression of type I collagen, we examined the glomerular expression of type I collagen at the protein and mRNA levels. Furthermore, serial section analysis for in situ hybridization and immunohistochemistry was also performed to identify cells producing type I collagen in rat glomeruli of crescentic nephritis induced by anti-GBM antibody.
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Materials and methods
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Experimental animals
Glomerulonephritis was induced in male Wister-Kyoto (WKY) rats (Japan Charles River Laboratories, Kanagawa, Japan) weighing 140170 g by intravenous (i.v.) injection of 5 µl/100 g body weight of rabbit anti-rat GBM antibody. The method for preparing anti-GBM antibody has been previously described [9]. As a control, the same volume of saline (physiological salt solution, PSS) was injected i.v. at 0, 3, 7, 14 and 28 days after injection, rats were housed in metabolic cages to allow collection of 24 h urinary samples (n=5 at each point). Urine was subjected to protein analysis by the pyrogallol red method.
Glomerular RNA isolation and RT-PCR
WKY rats were anaesthetized with intraperitoneal (i.p.) injections of sodium pentobarbital (35 mg/kg body weight) and sacrificed on day 0, 1, 3, 7, 14, and 28 after injection of anti-GBM antibody followed National Research Council guidelines. Both kidneys were removed and partially preserved for histologic analysis. Glomeruli were isolated by differential sieving as described previously [10]. The preparation of glomeruli was more than 90% pure with rare tubular segments. Total RNA was extracted from isolated glomeruli by the acid guanidinium thiocyanate-phenol-chloroform method. One microgram of total RNA was reverse-transcribed using oligo (dT) primers and reverse transcriptase (Superscript II, Life Technologies, Rockville, MD, USA). The single strand complementary DNA (cDNA) product was denatured and amplified by PCR with two primers under the following conditions: 1 min at 94°C, 1 min at 60°C, and 2 min at 72°C for 28 cycles (for type I collagen) and for 24 cycles (for GAPDH). The region amplified by each set of primers are as follows: human
1(I) collagen (Gen bank Z74615) nucleotides 312607, 5'-AAC GGC AAG GTG TTG TGC GAT G, 3'-AGC TGG GGA GCA AAG TTT CCT C and rat GAPDH nucleotide 331 to 1038, 5'-ACC ACC ATG GAG AAG GCT GG, 3'-GGT TTC TTA CTC CTT GGA GG. The PCR product was confirmed to have 88.2% homology to human
1(I) collagen gene. The PCR products were separated by 2.0% agarose gel electrophoresis and visualized by ethidium bromide staining. Gels were scanned using Master Scan (Scanalytics, Billerica, MA, USA) and the signal intensity of bands was counted. Each experiment was accompanied by amplification of GAPDH as an internal control and the intensities of the cDNA bands for type I collagen were normalized to the GAPDH band intensities.
In situ hybridization
The murine
1(I) procollagen cDNA, which is 1.6 kb and codes for residues within the triple helical regions of
1 chain estimated to encompass amino acid number 400950 [11], was inserted into the PstI sites of plasmid vector pBR. A 1.3-kb cDNA fragment of murine
1(I) procollagen cDNA was cloned into the PstI site and BamHI sites of the transcription vector pSPT 18 at a site between the SP6 and T7 promotors. Antisense and sense RNA probes were prepared with an in vitro transcription fragment of murine
1(I) procollagen cDNA using T7 and SP6 RNA polymerase, respectively, in the presence of 350 µM/l digoxigenin(DIG)-link UTP in 20 µl of reaction mixture, according to the manufacture's instructions (Boehringer-Mannheim, Mannheim, Germany). The specificity of probe was confirmed by northern blot and in situ hybridization was performed as described previously [5]. Briefly, 4-µM-fixed frozen sections were treated with 1 µg/ml proteinase K (Sigma, St Louis, MO, USA) in TE (10 mM TrisHCl, 1 mM ethylenediaminetetra-acetic acid) and 0.25% acetic anhydride in 0.1 M triethanolamine (pH 8.0), then dehydrated and air-dried. Hybridization was performed with a digoxigenin-labelled complentary RNA probe (1 µg/ml) in 25 µl of the hybridization solution (50% formamide, 2xSSC, 1xDenhardt's solution, 100 µg/ml salmon sperm DNA, 100 µg/ml heparin, 200 µg/ml yeast transfer RNA (tRNA), 10% dextran sulfate: Iatron, Tokyo, Japan) covered with parafilm at 42°C for 16 h in a humidified chamber. After hybridization, the sections were washed in 50% formamide, 2xSSC at 47°C for 30 min, and then treated with 6 µg/ml RNase A (Sigma) in TNE (10 mM TrisHCl, pH 7.5, 500 mM NaCl, 1 mM ethylenediamine-tetra-acetic acid) at 37°C for 30 min, and washed in two changes of 2xSSC at 47°C for 20 min, followed by 0.2xSSC at room temperature for 20 min. Sections were reacted with anti-digoxigenin antibody conjugated with alkaline phosphatase (1:500, Boehringer-Mannheim, Mannheim, Germany) at 37°C for 1 h. The hybridized signals were developed with nitroblue tetrazolium salt and 5-bromo-4-chloro-3-indolyl phosphate (Boehringer-Mannheim, Mannheim, Germany). In every experiment, both sense and antisense probes with the same specific activity of the labelling and the same concentration were provided. Each tissue was studied in at least three separate experiments.
Histology and immunohistochemistry
For light microscopy, the kidneys were fixed with 10% neutral-buffered formaldehyde for 2 days, dehydrated and embedded in paraffin. Paraffin sections were made at 2 µm and stained with periodic acid-Schiff (PAS). Four-micrometre fresh frozen sections were provided for staining with goat anti-type I collagen antibody. After blocking for 30 min, the sections were incubated with goat anti-type I collagen antibody (1:40, Southern Biotechnology Associates, Inc., USA) at 37°C for 1 h, and then stained with FITC-conjugated anti-goat IgG antibody at room temperature for 30 min. Negative controls were induced by omission of the primary antibody in the staining procedure. For identification of the cellular origin of type I collagen in the glomerular cells, specimens from day 14 after anti-GBM antibody injection, were sectioned serially. The sections adjacent to those studied by in situ hybridization were stained with cell makers for PEC (anti-pan cytokeratin antibody, 1:100, Sigma, USA), podocyte (anti-podocalyxin, 1:1000, kindly provided by Dr Hidetake Kurihara, Department of Anatomy, Juntendo University School of Medicine, Tokyo, Japan) [12], monocyte/macrophage (anti-rat ED-1 antibody, 1:400, Serotec Ltd, UK) and glomerular mesangial cell (anti-Thy-1 antibody, 1:400, Serotec Ltd, UK). For detection of PEC by anti-cytokeratin antibody, immunostaining was performed after microwave pre-treatment in 0.01 M citrate buffer (pH 6.0) for 8 min and digestion of the sections in 0.0125% trypsin in 0.05 M TrisHCl (pH 7.6) for 30 s. Immunohistochemical staining was performed as follows: briefly, endogenous peroxidase was blocked with 0.3% (v/v) H2O2 in methanol at room temperature for 15 min. After washing with phosphate buffered saline (PBS), non-specific binding sites were blocked with an avidin-biotin blocking kit (Vector Laboratories, Burlingame, CA, USA) and the blocking solution (2% fetal calf serum, 2% fetal bovine serum, 0.2% fish-gelatin in PBS). Incubation with anti-pan cytokeratin antibody was performed at 4°C overnight and with the other primary antibodies at room temperature for 1 h. After washing with PBS, the sections were incubated with biotin-conjugated horse anti-mouse IgG antibody (Vector Laboratories) as a secondary antibody for 30 min. Then sections were incubated with avidin and biotinylated horseradish peroxidase solution (Vector Laboratories) for 30 min. Immunoglobulin complexes were visualized by incubation with 3,3'-diaminobenzidine in PBS (0.5 mg/ml) with 0.03% H2O2. The sections were counterstained with haematoxylin.
Statistical analysis
Results are presented as the mean±SD. The unpaired t-test was used to compare the data. Differences were considered a significant at P<0.05.
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Results
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Urinary protein excretion and histologic changes
Urinary protein levels in the normal rats were <20 mg/24 h. In rats injected with anti-GBM antibody, urinary protein excretion increased significantly from day 3 (102.2±36.9 mg/24 h) and persisted during the experimental period. Pathological findings revealed that infiltrating of leukocytes was observed in glomeruli on day 3. From day 3 to 7, the accumulation of monocytes was marked in the glomerular capillary lumens and mesangial areas for replacing neutrophil infiltration. On day 7, both glomerular mesangial hypercellularity and extracellular matrix accumulation began with crescent formation. Thereafter, the cellular crescents progressed to fibrocellular or fibrous crescents. Increased cellularity was resolved by day 28 although hyalinized substances emerged in glomerular mesangial areas or Bowman's space. It appeared that the glomerular lesions were in the chronic phase (data not shown).
Gene expression of type I collagen in isolated glomeruli
Glomerular gene expression of type I collagen mRNA was analysed by RT-PCR as shown in Figure 1
. mRNA expression was hardly detected in control rats (Figure 1a
). Semi-quantitative analysis demonstrated that type I collagen mRNA increased from day 7 (12.2±1.4-fold over day 0), reached maximum levels on day 14 (20.2±2.1-fold over day 0) and remained elevated on day 28 (14.6±1.0-fold over day 0) (n=4 at each point) (Figure 1b
).

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Fig. 1. Gene expression of type I collagen and GAPDH analysed by semiquantitative RT-PCR. (a) Gel shown is representative of one experiment. Lane 1, day 0; lanes 25, PSS-injected controls, day 1, 3, 7, 14 and 28, respectively; lanes 611, anti-GBM antibody-induced glomerulonephritis, day 1, 3, 7, 14 and 28, respectively; lane 12, molecular weight marker (X174/HaeIII digest). Ordinate molecular size in base pairs. (b) The bands were quantified by densitometric scanning and corrected by GAPDH expression. Values are expressed as n-fold of increase vs day 0. , PSS injected controls; , anti-GBM antibody induced glomerulonephritis. Data represent the mean±SD of experiment. *P<0.05 vs PSS-injected controls.
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Immunofluorescence of type I collagen
In the normal rat kidney sections, type I collagen was localized in the renal interstitium and blood vessels, but was not observed in the glomeruli (Figure 2a
). In sections from rats on day 3 after anti-GBM antibody injection, no specific staining was observed in glomeruli (Figure 2b
). On day 7, the staining of type I collagen was marked in the cellular crescents and weak in the glomerular mesangial areas (Figure 2c
). On day 14 and 28, marked and diffuse staining of type I collagen was observed in the fibrocellular and fibrous crescents (Figures 2d
and e
).

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Fig. 2. Immunofluorescene micrographs showing the glomerular localization of type I collagen in the rat kidney sections from control (a), day 3 (b), day 7 (c), day 14 (d) and day 28 (e) nephritic rats. Note that type I collagen is detected only in the interstitial area and vessels (a and b). Magnification (ae) x400.
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In situ hybridization of type I collagen and immunostaining of cytokeratin, podocalyxin, ED-1 and Thy-1: identification of cells expressing type I collagen
In order to identify the cells expressing type I collagen in glomeruli, in situ hybridization was performed using cRNA probe. The antisense probe only demonstrated signals of type I collagen in the vessels of control rats (Figure 3a
) and no signal was detected in glomeruli of rats 3 days after injection of anti-GBM antibody (Figure 3b
). On days 7, 14 and 28,
1(I) mRNA-positive cells were observed in glomeruli, Bowman's capsules and interstitial areas (Figure 3c
e
), and the transcripts in the glomeruli were mainly present in the areas of crescents and Bowman's capsules on day 14 and 28 (Figure 3d
and e
). Their location suggested that they were parietal epithelial cells. As a negative control, sense probe showed that no signal of type I collagen mRNA was observed in renal tissues (Figure 3f
). Immunohistochemical staining of serial sections was performed for further analysis of cells expressing type I collagen using anti-pan cytokeratin, anti-podocalyxin, anti-ED-1, and anti-Thy-1 antibodies. As shown in Figures 4
and 5
, some
1(I) mRNA-positive cells in the crescent areas were stained by cytokeratin (Figures 4
and 5a
, b
). However, only a few
1(I) mRNA-positive cells were stained by Thy-1 (Figures 4
and 5c
); and no
1(I) mRNA-positive cells were stained by ED-1 and podocalyxin (Figure 4d
and e
). These results indicated that type I collagen mRNA in glomeruli was at least partly expressed by PECs.

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Fig. 3. In situ hybridization of type I collagen using digoxigenin-labelled complementary RNA probe. (ae) Antisense probe. (a) Day 0, (b) day 3, (c) day 7, (d) day 14, and (e) day 28 in anti-GBM antibody-induced glomerulonephritis. Intense signals of type I collagen mRNA were observed in the crescents at day 14 and 28, a weak signal was found in the mesangial area at day 7, 14 and 28. (f) Day 14 no signal was observed with the sense probe. Magnification x400.
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Discussion
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In the present study, immunofluorescence showed that type I collagen was detected in the crescents and weakly in the mesangial areas after day 7. In keeping with the immunofluorescence results, semi-quantitative RT-PCR revealed that type I collagen mRNA in isolated glomeruli was significantly increased from day 7 and peaked at day 14. The observations were in accordance with previous reports [3,6]. In situ hybridization showed that mRNA expression of type I collagen was increased and mainly present in cells within crescents and Bowman's capsules at day 7, 14 and 28, which was closely associated with mRNA levels analysed by RT-PCR and immunofluorescence results. These data demonstrated that expression of type I collagen in glomeruli was increased at protein and mRNA levels.
Serial section analysis showed that some
1(I) collagen mRNA positive cells in glomeruli were also positive for cytokeratin, but not positive for podocalyxin. Osada et al. [5] and Minto et al. [7] reported that glomerular epithelial cells expressed type I collagen mRNA and in vitro studies also demonstrated that cultured glomerular epithelial cells expressed mRNA for type I collagen [13]. These results support the hypothesis that type I collagen in glomeruli is partially derived from glomerular epithelial cells. But these studies did not reveal if the type I collagen was from visceral or parietal epithelial cells. Our results showed that cells producing type I collagen were mostly of PEC origin. Although mesangial cells have been found to produce type I collagen in vivo and in vitro [4,14], the present results showed that only a few
1(I) collagen mRNA expressing cells in glomeruli were positive for anti-Thy-1. This is different from the report that mesangial cells were the main source of type I collagen in glomeruli of anti-Thy-1 antibody-induced mesangial proliferative nephritis [4]. The discrepancy may be attributable to the difference of experimental models. As cellular crescents are largely composed of macrophages and PECs [15], ED-1, a cell marker of monocytes and macrophages, was also stained on serial sections. Macrophages have been reported to synthesize type I collagen in vitro [16], and infiltrating macrophages were observed in crescentic nephritis, our results did not show that
1(I) collagen mRNA positive cells coincide with ED-1 positive cells. The results were consistent with another report stating that infiltraing macrophages may not be the source of type I collagen in glomeruli [5]. However, some
1(I) collagen mRNA positive cells were not positive for the cell markers that we used. These cells may be interstitial fibroblasts, which infiltrated through ruptures of Bowman's capsules, although this has not been proven yet. Taking these results together, our observations suggest that type I collagen in glomeruli originate, at least in part, from PECs in rat crescentic nephritis and enhanced production of type I collagen in glomeruli may play an important role in the progression of this disease.
Glomerular epithelial cells undergoing phenotypic changes in vivo have been proposed to explain the synthesis of type I collagen in glomeruli [5,7]. Tubular and glomerular cells have been reported to transdifferentiate into myofibroblasts under certain pathological conditions [17,18]. Myofibroblasts have been shown to synthesize type I collagen [19]. PECs are derived from metanephric mesenchyme that has the capacity to transdifferentiate. The factors involved in the phenotypic changes in these cells remain unknown although growth factors such as platelet-derived growth factor and transforming growth factor-ß may play a role. These factors are known to be involved in the activation of cells into myofibroblasts [20]. Similar effects can be anticipated within the glomeruli because these growth factors are known to be up-regulated in the scarred glomeruli of experimental animals and humans [21]. Maybe these factors activate proliferative and fibrogenic responses of PECs, inducing them to transdifferentiate into myofibroblasts and synthesize type I collagen. It is assumed that phenotypic changes of PECs such as myofibroblast transdifferentiation contribute to production of type I collagen in glomeruli of rats with crescentic glomerulonephritis.
In summary, the present study demonstrated that expression of type I collagen in glomeruli was increased at the protein and mRNA levels. It also provided further evidence for contribution of glomerular resident cells to the production of interstitial type collagen, although further studies are needed to elucidate the mechanisms responsible for inducing PECs synthesis of type I collagen. It is postulated that PECs may change their phenotypes under pathological conditions to synthesize abnormal extracellular matrices including type I collagen which participate in the progression of glomerulosclerosis.
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Acknowledgments
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We are grateful to Dr Hidetake Kurihara (Department of Anatomy, Juntendo University School of Medicine, Tokyo, Japan) for kindly providing anti-podocalyxin antibody and Dr Michio Nagata (Department of Pathology, Institute of Clinical Medical Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan) for his help in immunostaining of cytokeratin.
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Notes
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Correspondence and offprint requests to: Prof. Yasuhiko Tomino, Division of Nephrology, Department of Medicine, Juntendo University School of Medicine, 211 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan. 
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Received for publication: 7.10.99
Revision received 13.11.00.