Bone Marrow Is a Reservoir of Repopulating Mesangial Cells during Glomerular Remodeling

Reagents and Chemicals

Mouse anti-Thy1 monoclonal antibody (ox-7) was kindly provided by Dr. Seiichi Matsuo (Nagoya University, Japan). All other antibodies were obtained from commercial sources: mouse anti-vimentin antibody (Immunon, Pittsburgh, PA), mouse anti-smooth muscle actin monoclonal antibody (Immunotech S.A., Marseille, France), mouse anti-rat monocyte/macrophage monoclonal antibody (ED1) (Serotec Ltd., Oxford, England), mouse anti-rat CD45 (Common Leukocyte Antigen) monoclonal antibody (Chemicon, Temecula, CA), mouse anti-rat RECA1 monoclonal antibody (Cosmobio, Tokyo, Japan), rabbit antilaminin polyclonal antibody (Sanbio BV, Uden, The Netherlands), rabbit anti-Factor VIII related antigen/von Willebrand Factor polyclonal antibody (NeoMarkers, Fremont, CA), rabbit anti-desmin polyclonal antibody (Bio-Science, Emmenbruecke, Switzerland), and rabbit anti-GFP polyclonal antibody (Molecular Probe, Inc., Eugene, OR). All secondary antibodies come from Vector Laboratories (Burlingame, CA).

Animals

Sprague-Dawley (SD) rats were purchased from Japan SLC, Inc. (Hamamatsu, Japan) and were maintained in a specific pathogen-free environment at the animal facility of Osaka University School of Medicine. They were allowed free access to standard laboratory diet and tap water. All rats weighing 150 to 180 g (6 wk old), anesthetized by intraperitoneal administration of pentobarbital, were subjected to the experiments. All the procedures described here were approved by the animal committee of Osaka University School of Medicine. Transgenic SD rats carrying the EGFP transgene (EGFP rat) were established by use of the same construct and technique described above to produce EGFP transgenic mice (19). The expression of EGFP was under the control of the cytomegalovirus enhancer and the chicken β-actin promoter that are derived from an expression vector, pCAGGS (20).

Whole Body Irradiation and Bone Marrow Transplantation

Bone marrow transplantation (BMT) was performed by use of female normal SD rats as recipients and male EGFP rats as donors. EGFP-expressing bone marrow cells were collected by flushing bone shafts of femurs and tibias of EGFP rats with BM Media (Medium-199 [Life Technologies] supplemented with 2% fetal calf serum and 2 μg/ml gentamicin). After sieved through 50-μm meshes and washed with BM Media twice, the cells were resuspended at a concentration of 1 × 10⁸ cells/ml and kept on ice until use. Then normal SD rats were lethally irradiated at a dose of 10 Gy per animal by use of an x-ray generator. Within 4 h after the irradiation, the bone marrow cells prepared from EGFP rats were administered to each irradiated rat via tail vein (referred to hereafter as chimeric rat). The day of BMT was designated as BMT day 0. In our preliminary experiments, some recipients received tacrolimus (FK506, Fujisawa Pharmaceutical Co., Ltd., Osaka, Japan) intramuscularly at a daily dose of 0.5 or 1 mg/kg body wt from BMT day −1 to day 13. Forty chimeric rats were made and were subjected to the analysis.

Experimental Glomerulonephritis

To induce anti-Thy1 antibody-mediated glomerulonephritis (Thy1 nephritis), anti-Thy1 monoclonal antibody (ox-7) was administered to the chimeric rats via tail vein at a single dose of 0.5 mg/kg body wt on BMT day 21. The day of the administration was designated as BMT day 21/Thy1 day 0.

Tissue Processing and Immunohistochemistry

On the day indicated, the chimeric rats were thoroughly perfused with phosphate-buffered saline (PBS) followed by 10% buffered formalin via the abdominal aorta. Then the tissues were removed and further fixed for 3 h in 10% buffered formalin at 4°C. After being briefly rinsed in PBS, the fixed tissues were processed in one of two ways. For frozen cryostat sections, the tissues were placed in 30% sucrose in PBS overnight, embedded in Tissue-Tek OCT compound (Sakura FineTek, Tokyo, Japan), and frozen in liquid nitrogen. As long as they were kept at −70°C, the green fluorescence of EGFP was well preserved at least for 6 months. For paraffin sections, the tissues were dehydrated and embedded in paraffin wax. Sections cut at 4-μm thickness were incubated in various primary antibodies at room temperature for 1 h. After three washes for 5 min each in PBS, the sections were incubated in the appropriate secondary antibodies. For frozen sections, secondary antibodies were labeled with Texas Red. In paraffin sections, the localization of EGFP was visualized by use of anti-GFP polyclonal antibody in combination with an avidin-biotin detection system (Vector Laboratories). To prevent putative nonspecific binding to rat immunoglobulins, 2% normal rat serum was included in diluents for all secondary antibodies. To visualize nuclei under a fluorescence microscope, the sections were stained with 4′,6-diamidino-2-phenylindole, dihydrochloride (Molecular Probe, Inc., Eugene, OR). These processed samples were observed under a fluorescence microscope (Nikon Eclipse E600, Tokyo, Japan) or a confocal laser-scanning microscope with appropriate filters (Carl Zeiss LSM410, Oberkochen, Germany). All images were captured by a digital imaging system connected to a Macintosh computer.

Culture of Mesangial Cells

A primary culture of mesangial cells was established from isolated glomeruli of the chimeric rats and maintained as described elsewhere (21). The cells cultured on chamber slides (Nunc, Inc., Naperville, IL) were fixed with ice-cold methanol for 5 min and were subjected to immunohistochemistry.

Lodging of Bone Marrow-Derived Cells in the Kidney

EGFP transgenic rats expressed EGFP as nonsecreted cytoplasmic protein throughout the body except peripheral erythrocytes and hair. Bright green fluorescence was observed in all components of the kidney under a fluorescence microscope (Figure 1A). In the chimeric rats, green fluorescence derived from EGFP that was only expressed in the transplanted marrow cells, and their progeny provided a sharp contrast with fluorescence-negative recipient tissues. Less than 5% of animals that were subjected to BMT died within 5 to 10 d, presumably because of graft failure or irradiation toxicity. In the bone marrow and spleen of the established chimeric rats, an average of 80% cells expressed EGFP at BMT day 21 (Figure 1B). FK506 that we used to obtain preliminary data were withdrawn from our protocol because we could not experience any sign of graft-versus-host disease in the absence of FK506.

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Figure 1. Enhanced green fluorescence protein (EGFP)⁺ cells fluoresce brightly in EGFP transgenic rats and the chimeric rats. (A) Frozen kidney sections from the EGFP transgenic rats. Kidney sections taken from 6-wk-old EGFP rats were observed by use of fluorescence microscopy without any staining. Cells with bright green fluorescence were clearly identified throughout the kidney. (B) Bone marrow and spleen taken from the chimeric rats. The chimeric rats on bone marrow transplant (BMT) day 21 were thoroughly perfused and fixed as described in Materials and Methods. Paraffin sections were stained with anti-GFP polyclonal antibody followed by an avidin-biotin-peroxidase detection system. Then the sections were counterstained with hematoxylin. Representative pictures of the bone marrow (a) and the spleen (b) are shown. Cells taken from bone marrow of the chimeric rats were spread onto a glass slide and were fixed with ice-cold methanol. The cells were subjected to Giemsa staining and were observed by use of light (c) and fluorescence microscopy (d). Representative pictures are shown. Magnifications: ×400 in A and B (a and b), ×800 in B (c and d).

In Figure 2, kidney sections taken from the chimeric rats are shown. AT BMT day 5, EGFP⁺ cells were observed in gaps between renal tubules and were evenly scattered from the cortex through the papilla without any foci or cluster (Figure 2). EGFP⁺ cells were also observed in the interstitium of all tissues we looked in, such as heart, liver, skeletal muscle, peritoneal wall, and intestine (data not shown). These interstitial EGFP⁺ cells were not vascular plugs mechanically entrapped during circulation, because they were localized exclusively outside vascular lumens when endothelial cells were stained with anti-Factor VIII antibody or anti-RECA1 antibody (data not shown). They did not belong to leukocytes, hematopoietic lineages, or antigen-presenting cells because a large part of the interstitial EGFP⁺ cells did not react with anti-CD45 antibody or anti-dendritic cell antibody as long as we tested. One distinct property is that about half of the interstitial EGFP⁺ cells expressed vimentin, which implies that they belong to mesenchymal cells (data not shown). Although the number of the interstitial EGFP⁺ cells did not show further increase during our observation up to 12 mo after BMT, they did not lodge in tissues unless irradiated animals were rescued by the successful engraftment of the bone marrow. Therefore, the interstitial EGFP⁺ cells observed in the chimeric rats were likely to be the progeny of the reconstituted marrow.

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Figure 2. Bone marrow-derived EGFP⁺ cells lodged in the recipient kidney. (A) EGFP⁺ cells fluoresced green in the recipient kidney. (B) The basement membrane was stained with antilaminin polyclonal antibody followed by Texas-Red-labeled secondary antibody to visualize the basic structure of the kidney (red). (C) Green EGFP and red laminin were merged. (D) To identify nucleated cells, the image shown in panel C was further merged with 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI)-stained nuclei. Nucleated EGFP⁺ cells are sky-blue, whereas EGFP⁻ cells are dark purple. Slight background due to auto-fluorescence is observed in tubules. Representative pictures are shown at a higher magnification Magnifications, ×400.

Number and Subpopulation of Intraglomerular EGFP⁺ Cells Changed in Response to Mesangiolysis

In contrast to the interstitium, glomeruli of the chimeric rats rarely carried EGFP⁺ cells (Figure 2). It is a relevant and intriguing question whether bone marrow-derived cells are involved in the remodeling process of glomeruli. To elucidate the relationship between bone marrow-derived cells and the remodeling of the glomerulus, we adopted anti-Thy1 antibody-mediated glomerulonephritis (Thy1 nephritis), a well-established self-limiting disease. In Thy1 nephritis, normal mesangium is disrupted (mesangiolysis), which is followed by glomerular remodeling to restore the original structure. Histologic changes of Thy1 nephritis observed in the chimeric rats were consistent with those in normal rats and in studies published elsewhere (Figure 3 A through C). Seven days after the administration of anti-Thy1 antibody (BMT day 28/Thy1 day 7), the number of both EGFP⁺ cells and nucleated cells in glomeruli significantly increased (Figures 3, A through C and 4). It is obvious that the engrafted bone marrow contributed to glomerular hypercellularity as well as EGFP-negative cells that were derived from the recipient kidney itself and/or the recipient marrow surviving the irradiation. When compared with BMT day 21/Thy1 day 0, five times more EGFP⁺ cells were observed in glomeruli on BMT day 28/Thy1 day 7 (Figure 4). Interestingly, only half of the EGFP⁺ cells expressed CD45, which is expressed on the cell surface of various hematopoietic cells, including lymphocytic lineages, monocytic lineages, granulocytic lineages, and erythroid lineages at different levels (22). Normal glomerular structure was restored by 8 wk after onset of the disease (BMT day 77/Thy1 day 56) (Figure 3, C and F). Consistently, the number of intraglomerular CD45⁺EGFP⁺ cells and nucleated cells on BMT day 77/Thy1 day 56 returned to the baseline value (Figure 4). However, the total number of intraglomerular EGFP⁺ cells still remained four times higher than that estimated on BMT day 21/Thy1 day 0, and 60% to 70% of intraglomerular EGFP⁺ cells were CD45-negative (Figure 4). These results indicate that glomeruli on BMT day 77/Thy1 day 56 comprise a greater number of bone marrow-derived cells without CD45-positive hematopoietic properties than glomeruli on BMT day 21/Thy1 day 0. Between BMT day 77/Thy1 day 56 and BMT day 344/Thy1 day 326, there were no further histologic changes, and the number of EGFP⁺ cells did not change (data not shown).

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Figure 3. Recruitment of EGFP⁺ cells to glomeruli was promoted by the induction of Thy1 nephritis. Kidneys obtained from the chimeric rats on Thy1 days 0 (A, D, G, and J), 7 (B, E, H, and K), and 56 (C, F, I, L) were subjected to the histologic analysis. Representative images are shown at a higher magnification. (A through C) Light microscopic images of the kidneys are shown. Paraffin-embedded sections were stained with periodic acid-Schiff reaction. (D through F) Paraffin-embedded sections were stained with anti-GFP polyclonal antibody followed by an avidin-biotin-peroxidase detection system. Cells expressing EGFP display brown color. The sections were counterstained with hematoxylin-eosin. (G through I) Cryostat sections were stained with anti-rat CD45 antibody followed by Texas-Red-labeled secondary antibody and were observed under a fluorescence microscope. EGFP and CD45 display green and red, respectively. As a result, CD45⁺EGFP⁺ cells are identified as orange cells. (J through L) Cryostat sections were stained with anti-Thy1 antibody followed by Texas-Red-labeled secondary antibody. EGFP and Thy1 antigen display green and red, respectively. Thy1⁺EGFP⁺ cells are identified as orange cells. Magnifications, ×400

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Figure 4. Intraglomerular Thy1⁺ EGFP⁺ cells kept increasing in number during the time course of Thy1 nephritis. The number of nucleated cells in glomeruli was counted by staining 4-μm-thick sections with DAPI. The results were obtained from 30 glomerular sections at each time point. (a) BMT day 21/Thy1 day 0 (Control); (b) BMT day 84 (Disease Control); (c) BMT day 28/Thy1 day 7; and (d) BMT day 77/Thy1 day 56. Each value is expressed as mean ± SD per glomerular section. (A) The average number of intraglomerular nucleated cells in a 4-μm-thick sections. (B) The number of EGFP⁺ cells, CD45⁺EGFP⁺ cells, and Thy1⁺EGFP⁺ cells in glomerular sections were counted by use of fluorescence microscopy equipped with a two-channel filter. The white bar is the number of EGFP⁺ cells, the solid bar is the number of CD45⁺EGFP⁺ cells, and the hatched bar is the number of Thy1⁺EGFP⁺ cells. The results were analyzed by nonpaired t test (*P < 0.01).

As expected, macrophages participated in the pathologic change that was observed in Thy1 nephritis. When stained with anti-monocyte/macrophage antibody (ED1), ∼30% of EGFP⁺ cells in glomeruli on BMT day 28/Thy1 day 7 reacted with ED1 (Figure 5), and ED1⁺EGFP⁺ cells were responsible for half of the ED1⁺ cells in glomeruli at this time point. EGFP⁺ cells on BMT day 77/Thy1 day 56 were almost ED1-negative (Figure 5). We could not stain EGFP⁺ cells with anti-CD45 antibody and anti-monocyte/macrophage antibody simultaneously, because both belong to mouse IgG. Nevertheless, our data indicate that CD45⁺EGFP⁺ cells and ED1⁺EGFP⁺ cells overlapped.

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Figure 5. Infiltration of ED1⁺EGFP⁺ cells into glomeruli. Kidney specimens taken from Thy1 day 7 (A, C, and E) and Thy1 day 56 (B, D, and F) were subjected to the analysis. (A and B) Macrophages were stained with anti-monocyte/macrophage monoclonal antibody (ED1) (red). (C and D) ED1⁺EGFP⁺ cells were identified especially in glomeruli on Thy1 day 7 (orange). (E and F) Nuclei were visualized with DAPI (dark purple) and merged with the image shown in (C) and (D). Nucleated EGFP⁺ cells show sky-blue.

Additionally, no rat IgG was deposited in the kidney of the chimeric rats as far as we tested using anti-rat Ig G antibodies. It indicates that EGFP that could be released from dying EGFP⁺ cells was not immunogenic in the chimeric rats (data not shown).

EGFP⁺ Cells Expressing Thy1 Were Identified in Glomeruli of the Chimeric Rats

Thy1 antigen is a glycerophosphatidylinositol-anchored protein expressed on the cell surface. In rats, Thy1 antigen is unique to mesangial cells within the kidney in vivo and in vitro (23,24). Thy1 antigen has been extensively used as a reliable marker for the rat mesangial cell. In this study, we identified mesangial cells in vivo by the expression of Thy1 antigen.

The total number of intraglomerular Thy1⁺EGFP⁺ cells and the proportion of Thy1⁺EGFP⁺ cells to all EGFP⁺ cells continuously increased over the disease process and reached the maximum value on BMT day 77/Thy1 day 56 (Figures 3, J and K, and 4). This pattern was quite distinct from that of the intraglomerular CD45⁺EGFP⁺ cells. Because the number of nucleated cells already returned to the basal value on BMT day 77/Thy1 day 56, the net increase of Thy1⁺EGFP⁺ cells between BMT day 28/Thy1 day 7 and BMT day 77/Thy1 day 56 were unlikely to result from persistent migration or proliferation of EGFP⁺ cells. Simple calculation based on Figure 4 suggests that Thy1⁺EGFP⁺ cells belong to the intraglomerular CD45-negative EGFP⁺ cells. There was another EGFP⁺ population with a characteristic of CD45⁻Thy1⁻. On BMT day 77/Thy1 day 56, at least 20% of intraglomerular EGFP⁺ cells seemed to be negative for both CD45 and Thy1 (Figure 4). CD45⁻Thy1⁻EGFP⁺ cells might include endothelial lineages, but no EGFP⁺ cells in glomeruli expressed endothelial markers such as Factor VIII and RECA1 as long as we tested (data not shown).

Thy1⁺ EGFP⁺ Cells Provided Structural Support for Glomerular Capillaries

Even though green fluorescence that overlapped red fluorescence of stained Thy1 antigen is a conclusive evidence to prove the existence of marrow-derived mesangial cells, superposition of cell bodies or of intricate processes might be present at different levels in specimens. To obtain higher resolution, we scanned tissue sections using confocal laser-scanning microscopy. Pictures shown in Figure 6 depict the representative image of Thy1⁺EGFP⁺ cells. A sky-blue cell located at the peripheral area of the mesangial stalk is nucleated marrow-derived cell because the dark purple nucleus stained with 4′, 6-diamidino-2-phenylindole, dihydrochloride displays sky-blue in combination with EGFP fulfilling the cytoplasm (Figure 6, arrow). It is obvious that the sky-blue cell expresses Thy1 antigen on its cell surface, and that the cell does not overlap with other Thy1⁺ cells (Figure 6, arrow). This Thy1⁺EGFP⁺ cell extends terminal processes that border capillary lumen. At the same time, dark purple nuclei of endothelial cells within the capillary lumen are easily detected (Figure 6, arrowhead). As a whole, the Thy1⁺EGFP⁺ cell seems to provide structural support to a pair of capillary lumens. We have concluded that this cell is a bone marrow-derived mesangial cell because all these findings meet the geometry of the mesangial cell (25). In vitro culture of glomeruli isolated from the chimeric rats yielded desmin⁺EGFP⁺ cells, which also meet the mesangial property (26) (data not shown).

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Figure 6. Analysis of glomeruli by a confocal laser-scanning microscope. Four-micron-thick cryostat sections from the kidneys on BMT day 77/Thy1 day 56 were stained with anti-Thy1 antibody and DAPI, as described above. Then the sections were observed by use of confocal laser-scanning microscopy. EGFP⁺ cells and nuclei present as green and dark purple, respectively. Nucleated EGFP⁺ cells are identified as sky-blue cells. Thy1 antigen fluoresces red. The white arrow indicates nucleated Thy1⁺EGFP⁺ cells, and the white arrowhead indicates a pair of capillaries containing dark purple nuclei of endothelial cells. A representative picture is shown in a three-color channel mode with a scale bar.

EGFP⁺ Cells Formed a Cluster at the Vascular Pole

We are intrigued with routes that bone-marrow-derived cells take before reaching glomeruli. At least two possibilities can be hypothesized. One possibility is that cells in blood circulation pass through the glomerular endothelial barrier and get in the mesangium. Alternatively, cells may enter glomeruli through the interstitial tissue and the juxtaglomerular zone. Although it is technically difficult to prove which routes they take, the latter possibility was reported by Hugo et al. (13). Consistent with their report, we occasionally met a cluster of EGFP⁺ cells at the glomerular vascular pole after the induction of Thy1 nephritis (Figure 7). Cells forming the clusters expressed vimentin (Figure 7) but not Thy1. This population might be derived from vimentin⁺EGFP⁺ cells that lodged in the interstitial area soon after BMT. So far, we do not have any evidence to prove that vimentin⁺EGFP⁺ cells at the vascular pole enter glomeruli and subsequently change into Thy1⁺EGFP⁺ cells.

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Figure 7. EGFP⁺ cells ormed clusters at vascular poles of glomeruli. Cryostat sections from the kidneys on BMT day 24/Thy1 day 3 were stained and were observed by use of fluorescence microscopy. (A) EGFP, (B) vimentin, (C) the merging of EGFP and vimentin, and (D) the merging of EGFP, vimentin, and DAPI. A representative image is shown. Magnifications, ×400.