American Journal of Pathology, Vol. 152, No. 5, May 1998 Copynight American Society for Investigative Pathology
Tenascin-C Promotes Healing of Habu-Snake Venom-Induced Glomerulonephritis Studies in Knockout Congenic Mice and in Culture
Naoyuki Nakao,*t Noriko Hiraiwa,t Astushi Yoshiki,t Fumio Ike,t and Moriaki Kusakabet From the Renal Division,* Internal Medicine, GenGen do-Kimitu Hospital, Chiba, and the Division of Experimental Animal Research,t Tsukuba Life Science Center, The Institute of Physical and Chemical Research (RIKEN), Ibaraki, Japan
Mice without the gene for tenascin-C, a multifunctional extracellular matrix protein expressed in many important biological events, including wound healing, did not show any phenotype. However, it is now obvious that the phenotype of deletion of one gene frequently depends on the genetic background. Therefore, we have newly generated tenascin-C knockout mice (KO) by backcrossing original KO into three congenic lines: C57BL/6N, BALB/cA, and GRS/A (GR). And we investigated the disease course of reversible kidney injury, Habusnake venom-induced proliferative glomerulonephritis. In all strains, the disease was more severe in KO, but the severity varied with the strain. The KO-GR showed irreversibility; all treated KO-GR died by the 4th month due to renal failure. The diseased KO-GR showed abnormal regenerative reactions, including reduced proliferation of mesangial cells, key players in glomerulonephritis, and reduced production of some kinds of cytokines and matrices, leading to poor formation of granulation tissue. In culture, the mesangial cells from the KO-GR had the same potential for proliferation and response to cytokines as controls, but interestingly, to achieve this potential, they required contact with tenascin-C. These reactions were blocked by an anti-tenascin monoclonal antibody. The results of the present study, the first report showing the most dramatic phenotype so far discovered, have strongly suggested the importance of tenascin-C in the resolution of the renal inflammation and that of the genetic background on which the KO was developed. (AmJPathol 1998, 152:1237-1245)
Tenascin-C (TN), a hexameric extracellular matrix (ECM) glycoprotein,1 has been speculated to play a locally important role in development, oncogenesis, and wound healing, because of its spatiotemporally restricted expression.2 Surprisingly, the originally produced TN knockout mice were reported to show no phenotypic difference in gross organization or histology, which casted doubt on its roles in development.5 However, recent investigations using newly generated congenic or inbred knockout mouse strains have demonstrated some phenotypic differences. A needle wound in the cerebral cortex showed a high number of astrocytes in young knockout mice from the seventh backcrossing into a congenic GRS/A strain.6 In full-thickness skin excision lesions of the 129/Sv inbred knockout mice, the amount of fibronectin appeared to be reduced in granulation tissue, although the lesion itself healed normally.7 In a separate study, the knockout mice that were inbred nine times by sibling mating showed abnormal behavior, such as hyperlocomotion and poor swimming ability.8 These results have indicated that TN may have functions in several organs, which can be revealed in a particular congenic line and with specific experimental probes. Glomerulonephritis (GN) is an inflammation of the kidney. In most types of GN, an inflammation occurs in the glomerular mesangial area, an intercapillary interstitium of glomerulus. Glomerular mesangial cells, which are constituent cells in the mesenchyme, play a central role in its pathogenesis. The cell probably represents a specialized pericyte and possesses many of the functional properties of smooth muscle cells.9 In GN, they alter their phenotype by expressing smooth muscle a-actin and secreting inflammatory molecules, such as cytokines and several ECM molecules, including TN.10 GN merits investigation in the TN knockouts as TN is re-expressed in
Supported in part by Special Coordination Funds of the Science and Technology Agency of the Japanese Government, partly by a Grant-inAid for Scientific Research on Priority Areas and a Grant-in-Aid for Scientific Research from The Ministry of Education, Science, Sports, and Culture. Accepted for publication February 26, 1998. Address reprint requests to Dr. Moriaki Kusakabe, Division of Experimental Animal Research, Tsukuba Life Science Center, The Institute of Physical and Chemical Research (RIKEN), 3-1-1, Koyadai, Tsukuba, Ibaraki 305-0074, Japan. E-mail:
[email protected]
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diverse kinds of GN.11 A model of regenerative GN is easily induced by injection of Habu snake venom.12 The venom destroys the mesangial structure. However, within 1 month, the damage will usually heal spontaneously, where the mesangial cells actively participate in regeneration. Here, we describe the induction of Habu-snakevenom-induced GN into three TN knockout congenic mouse stains, a quantitative analysis of the regenerative reactions, and a study in cell culture of the mesangial cells cell lines.
Materials and Methods Generation of Three TN Knockout Congenic Mice Strains The originally produced TN knockout mouse5 was backcrossed with the following three inbred strains for 10 generations: C57BL/6N, BALB/cA, and GRS/A. Genomic DNA was analyzed with polymerase chain reactions using a mixture of two sets of primers: exon 2 primer pair was sense 5'GAA-AAT-CAT-CAG-GCA-CAA-GCG3'and antisense 5'TCT-TCG-CTA-CGC-CAG-C3'; the other was sense 5'TGG-GGA-CAT-CCA-ACT-GTA-TTC-C3' and antisense 5'TCT-TCG-CTA-TTA-CGC-GAG-C3'. With the former set of primers, normal mice showed polymerase chain reaction products of 208 bp, whereas with the latter sets, the KO showed reaction products of 299 bp (data not shown). The KO displayed no TN expression at the protein or gene level. The animals used in this experiment were maintained in the animal facility in the Tsukuba Life Science Center, The Institute of Physical and Chemical Research (RIKEN).
Habu-Snake-Venom-lnduced GN and Experimental Design GN was induced in three mouse strains, 2 months old, by intravenous injection of venom from Trimeresurus flavovitidis (Wako, Osaka, Japan) at a dose of 4 mg/kg body weight. In the first experiment, to compare the clinical courses between the treated normal and KO mice, 30 mice of each strain (15 normal and 15 KO mice) received the venom. At days 0, 3, 7, 14, 21, 28, and 36, blood and 24-hour urine samples were collected from three mice of each group. Urine protein and serum urea nitrogen concentration was measured using commercial kits (Pierce, Rockford, IL and Wako). After collecting these samples at days 0, 3, 7, 21, and 36, the mice were used for pathological examination. In addition, to determine survival rate, seven treated GRS/A mice of each group were followed up for 4 months. Second, to investigate the underlying mechanism how TN influences the disease, 33 GRS/A mice of each group were used for detail histological and morphometric analysis (see below). The organs, including kidney, liver, gut, lung, and aorta, were obtained from three treated mice of each group on days 2, 3, 5, 7, 14, 21, 28, 35, and 42. Finally, to examine
sequential changes in the amount of cytokines and ECM within the diseased glomeruli, the glomeruli from the treated GRS/A mice of both groups were prepared for Western blotting analysis (see below for more detail).
Histology and Morphometric Analysis The dissected organs were treated as described.13 TN expression was detected using rat monoclonal antibody (MAb) 3-6 (IgG2a) to human TN that was raised in our laboratory. To identify components of reactive glomerular cells, the following antibodies were used: PC10 (Dako, Glostrup, Denmark), anti-proliferating cell nuclear antigen; ASM1 (Boehringer Mannheim Biochemica, Mannheim, Germany), anti-smooth muscle a-actin (SMA), as a marker of proliferating glomerular mesangial cells; RB68C5 (PharMingen, San Diego, CA), a rat polyclonal antibody to infiltrating polymorphnuclear cells; M3/38 (Boehringer Mannheim), a mouse MAb to macrophageassociated antigen (Mac2); and F8/86 (Dako), a mouse MAb to human von Willebrand factor, as a marker of endothelial cells. The antigen-positive cells were detected using the avidin-biotin complex (ABC) kit (Vector Laboratories, Burlingame, CA). In situ hybridization was performed as described.14 Apoptotic cells were visualized using a commercial kit (Oncor, Gaithersburg, MD). The numbers of specific antigen-positive and apoptotic cells were divided by total glomerular cell number and expressed as a percentage.
Assay of Cytokines and Extracellular Matrix Proteins Induced in the Diseased Glomeruli by Western Blotting To examine sequential changes in the amount of plateletderived growth factor (PDGF)-BB, transforming growth factor (TGF)-11, collagen type IV, fibronectin, and tenascin-C within the diseased glomeruli, the glomeruli from the six treated GRS/A mice of both genotypes were isolated on days 0, 1, 3, 5, 7, 14, 21, 35, and 42 by a sieving method (see below) and prepared for Western blotting analysis.14 The following antibodies were used: MAb 3-6 (see above); AB200NA, anti-human PDGF-BB (R&D Systems, Minneapolis, MN); 40091, anti-human TGF-f31 (Becton Dickinson, San Jose, CA); AB1943, anti-mouse fibronectin (Chemicon, Temecula, CA); and AB756, antimouse collagen type IV (Chemicon). The reaction was detected with enhanced chemiluminescence (Amersham, Arlington Heights, IL). The density of each band was expressed as relative density to that of the normal mouse at day 0. At each lane, 0.4 mg wet weight of isolated glomeruli obtained from 12 kidneys was loaded (see below). For this assay, one series of experiment has been performed.
Isolation and Cloning of Mesangial Cells To obtain isolated glomeruli, cortical tissue of kidney was minced and successively filtered through a 149-, 105-,
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and 74-gm steel sieve.14 Glomerular cells grew out several days after culture of the isolated glomeruli. Then, after a short digestion with 0.05% trypsin and 0.53 mmol/L EDTA (GIBCO BRL, Gaithersburg, MD), we carefully picked up single cells and transferred them into 96-well plates in Dulbecco's modified Eagle's medium with 25% fetal bovine serum (Filtron, Brooklyn, Australia). The selected cells were allowed to grow in 96-well plates, subpassaged three times, and stored in liquid nitrogen until use. To identify the cells as glomerular mesangial cells, they were stained with the following antibodies: ASM1; MAK6, anti-cytokeratin (Triton, Alameda, CA); DER1 1, anti-desmin (Dako); and F8/86. The cells displayed smooth-muscle-actin-positive, cytokeratin-negative, desmin-positive, von-Willebrand-factor-negative reactions (data not shown).
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The same number of mesangial cells from normal and KO mice (5 x 102) were plated in plastic or ECM-coated 48-well dishes. For coating, 100 mg/ml murine collagen type IV (Becton Dickinson), 50 mg/ml murine fibronectin (UCB Biogenesis, Poole, UK), and 10 mg/ml human TN purified from A375 melanoma culture medium were used. For antibody perturbation assays, dishes were preincubated with the 3-6 MAb for at least 2 hours at room temperature. For cytokine stimulation, 5 ng/ml PDGF-BB or 25 ng/ml TGF-,31 (R&D Systems) was added to the culture system. For an estimation of growth of the mesangial cells and their response to the cytokines, we counted the number of living cells using a counting kit (Dojindo Laboratories, Kumamoto, Japan). A TGF-B31 ELISA kit (Morinaga, Kanagawa, Japan) was used to measure the TGF-f1 concentration in culture media and in cultured cells.
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NM KO NM KO NM KO NM KO NM KC Day 0 1 mo. 2 mo. 3 mo. 4 mo. Figure 1. Clinical courses of Have-snake venom-induced glomerulonephritis in tenascin-C knockout congenic (KO) and normal inbred (NM) mouse (B6, C57BL/6N; BALB, BALB/cA; GR, GRS/A). A: Daily proteinuria. B: Serum urea concentration. C: Mice survival of GRS/A strain.
Statistics The values are expressed as the mean ± SD. The onetailed Student's t-test or two-way analysis of variance was appropriately used.
Results No Phenotype of TN Knockout (KO) Mice, Regardless of Strain Our three KO congenic lines (C57BL/6N, BALB/cA, and GRS/A) developed normally. There were no obvious differences between the KO and their normal counterparts (NM) in several physical and anatomical studies, regardless of the strain (data not shown).
Experimental Glomerulonephritis (GN) Did Not Heal Normally After injection of venom, each strain showed renal damage indicated by increased proteinuria and elevated se-
rum urea nitrogen concentration (Figure 1, A and B). In each strain, the damage of the normal mouse was transient (open symbols). However, these indices continued to increase in the KO and never returned to a normal level through the experimental period (closed symbols). Thus, for all strains, the GN was much more severe in the KO. In addition, we found a prominent strain difference in the degree of the GN. The kidney damage was mild in C57BL/6N, moderate in BALB/cA, and severe in GRS/A. The kidney pathology reflected these strain differences (data not shown). We therefore followed the course of the disease more thoroughly in the GRS/A strain (Figure 1 C). Remarkably, all KO-GR died due to renal dysfunction by the 4th month, whereas none of the normal mice died.
Defective Granulation Tissue in the Damaged Glomeruli of the KO-GR At day 1, most glomeruli of normal mice displayed glomerular cystic lesions (Figure 2B). From day 3 to day 7, the cysts were rapidly replaced by a prominent prolifer-
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Figure 2. Glomerular pathology of normal (NM-GR) and knockout (KO-GR) GRS/A strain (H&E staining). The same degree of the lesions was induced by intravenous administration of Habu-snake venom (asterisk in G). In the normal strain (NM-GR), regeneration occurred, whereas in the knockout strain (KO-GR), destruction of the kidney is seen. Bars, 0.3 mm (A, B, E, F, and G) and 0.4 mm (C, D, H, 1, and J).
ation and an accumulation of ECM, forming a granulation (Figure 2C). After day 14, the granulation was gradually reconstructed with a resolution of the hypercellularity and remodeling of ECM (Figure 2D) to finally restore its normal structure (Figure 2E). Remarkably, these regenerative reactions were almost completely missing in the KOGR. On day 1, the cysts formed in the KO-GR were the same in incidence and severity as those in normal mice
(Figure 2G). There were no statistical differences detected in the incidence and severity of the cysts (incidence, 78.6 +- 0.9% in NM-GR versus 79.0 + 0.5% in KO-GR; diameter as severity, 0.8 + 0.03 mm in NM-GR versus 0.8 + 0.04 mm in KO-GR). However, both the hypercellularity and the ECM deposition were very small, so that the granulation appeared immature (Figure 2H). Later, hemorrhage occurred in the granulation and the
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