The fundamental pathogenesis of some glomerular diseases may result from abnormalities of bone marrow stem cells (BMC), as we and others have demonstrated (1,2). If so, bone marrow transplantation (BMT) could attenuate glomerular injury in affected human patients and experimental animals (3,4). Theoretically, the mechanism underlying such attenuation involves the replacement of destructive immune cells of the recipient with donor BMC.
BMC clearly have the potential to differentiate into not only hematopoietic lineages but also mesenchymal lines (5). Both in vivo and in vitro, BMC have served as precursor cells for bone, cartilage, lung parenchyma, muscle, and hepatic cells in irradiated or immunodeficient animals (6,7,8,9,10,11). In addition, stem cells have been identified in adult tissues undergoing extensive cell replacement because of physiologic turnover or injury (12).
During embryonic glomerulogenesis, cells of the metanephric mesenchyme apparently have the capacity to convert into glomerular epithelial cells, whereas endothelial cells are derived from precursor endothelial cells, rather than the metanephric mesenchyme (13,14,15). In the anti-Thy-1 model of mesangial proliferative glomerulonephritis, repopulation of the mesangium after injury results from the migration of mesangial cell-like cells that reside in the juxtaglomerular apparatus (16,17). However, whether the mesangial cells routinely originate from the metanephric mesenchyme or from the endothelial cell lineage is still debatable.
We hypothesized that glomerular cells in nephritic mice that have been cleansed of their destructive cells and then treated with BMT might be repopulated by donor cells, with prevention of the otherwise obligatory glomerular lesions. To clarify this issue, we transplanted BMC from mice transgenic for green fluorescence protein (GFP) into syngeneic C57BL/6j (B6) mice. Because all tissues from GFP-transgenic mice (green mice), with the exception of erythrocytes and hair, are green under excitation light (18), GFP-positive donor cells in the recipients are readily identifiable. Our findings demonstrated that BMC might have the potential to differentiate into glomerular cells.
Materials and Methods
Mice
Female B6 mice were purchased from Japan Sankyo Labo Service Co. (Tokyo, Japan). Mice transgenic for GFP (GFP mice) were established in the Research Institute for Microbial Diseases, Osaka University, using the previously reported method (18). The enhanced GFP cDNA in these mice is under the control of a chicken β-actin promoter and a cytomegalovirus enhancer. For this study, 8-wk-old hemizygous female GFP mice were selected as donors. All protocols were approved by the ethics committee of our institution, and all surgical procedures conformed to the guidelines established by the United States Department of Health and Human Services, as published by the National Institutes of Health (National Institutes of Health publication 85-23, revised 1985).
BMT
In preliminary experiments, a single 8.0-Gy dose of total-body irradiation from a 60Co source killed all 8-wk-old female B6 mice by 3 wk after the irradiation if subsequent BMT was not performed. In spleens of these irradiated mice, no hematopoietic colonies were macroscopically visible 14 d after irradiation, indicating that no pluripotential stem cells were present (19). On the basis of these results, we irradiated recipient female B6 mice, at 8 wk of age, with 8.0 Gy. BMC obtained from pelvic, femoral, and peroneal bones of donors were incubated with anti-Thy-1.2 antibody (Ab) (F7D5; Serotec Ltd., Oxford, England), at an appropriate concentration, on ice for 30 min and were then reacted with complement (Cedar Lane, Ontario, Canada) at 37°C for 30 min, as described previously (3). Five to 6 h after irradiation, the recipient B6 mice received injections, through the tail vein, of 1 × 107 T cell-depleted BMC from 8-wk-old GFP mice ([GFP→B6]) or from 8-wk-old B6 mice ([B6→B6]).
Cytofluorometric Analyses
Spleen cells were gently homogenized and depleted of red blood cells in Tris-buffered ammonium chloride (pH 7.2). Pelvic, femoral, and peroneal bone BMC were washed three times with phosphate-buffered saline (PBS), after which 1 × 106 splenic cells and BMC were resuspended in 1% paraformaldehyde (PFA)-PBS and analyzed with a flow cytometer (EPICS CS; Coulter Electronics, Hialeah, FL). Cells expressing GFP were detectable at the same wavelength as used for FITC detection (488 nm).
Morphometric Analyses
At 2 (n = 3), 4 (n = 3), 8 (n = 3), and 24 (n = 5) wk after BMT, we performed histologic analyses of the kidneys from [GFP→B6] mice, which had been sufficiently perfused with 25 ml of PBS and 25 ml of 3% formalin-PBS for removal of circulating cells from the glomeruli. For light microscopy, tissue samples were then fixed with 3% formalin-PBS and embedded in paraffin. After sectioning, the tissues were stained with periodic acid-Schiff reagent. The numbers of cells in the glomeruli were counted individually for >20 glomeruli/cross-section, and a mean value was determined for each section. GFP-positive cells in the glomeruli were observed with a laser-scanning confocal microscope (LSM) (LSM410; Zeiss, Oberkochen, Germany) at 2, 4, 8, and 24 wk after BMT. Formalin-fixed sections (0.5 mm) were mounted in 10% gelatin-PBS. The numbers of GFP-positive cells in the glomeruli were counted for >20 glomeruli, and a mean value was determined for each section.
For clear identification of the locations of GFP-positive cells within glomeruli, mice at the 24th week after BMT (n = 3) were perfused with 25 ml of PBS and 25 ml of 2% PFA-PBS. Tissue samples were fixed with 2% PFA-PBS for 2 h at 4°C and then washed with 6.8% sucrose-PBS overnight at 4°C. Tissues were then embedded in Tecknovit 8100 (cold polymerizing resin; Heraeus Kulzer, Wehrheim, Germany), according to the instructions provided by the manufacturer. Finally, the sections (2 μm) were observed with the LSM, and digitized images were obtained by using LSM software version 3.5 (based on Windows version 3.1).
Immunohistochemical Analyses
Tissue samples from the chimeric mice at the 24th week after BMT (n = 3) were fixed with 2% PFA-PBS, washed with 6.8% sucrose-PBS (same samples used in the Tecknovit 8100 analysis described above), embedded in OCT compound (Miles Scientific, Naperville, IL), and quickly frozen in dry ice-acetone. Cryostat sections (4 μm) were fixed in cold acetone for 10 min. After being washed with PBS, the sections were incubated with polyclonal rabbit anti-desmin Ab (ICN Pharmaceuticals, Cleveland, OH) at 4°C overnight, followed by rinsing in PBS and incubation at 37°C for 60 min with rhodamine-conjugated sheep anti-rabbit IgG Ab (ICN Pharmaceuticals). The sections were rinsed again in PBS, mounted with SlowFade antifade kits (Molecular Probes, Eugene, OR), and observed with the LSM. The detection of infiltrating F4/80-positive macrophages or Thy-1-positive T cells relied on immunohistochemical analyses based on the avidin-biotin-peroxidase method. First, the endogenous biotins in the sections were blocked, as directed, with an avidin-biotin blocking kit (Vector Laboratories, Burlingame, CA). Next, the sections were incubated with primary Ab overnight at 4°C. Primary Ab were used in this study as follows: rat anti-mouse F4/80 monoclonal Ab (mAb) (BMA Biomedicals AG, Augst, Switzerland) for macrophage staining and rat anti-mouse Thy-1.2 mAb (30-H12; Pharmingen, San Diego, CA) for T cell staining. The sections were then incubated with biotinylated mouse anti-rat IgG (κ chain) mAb (MARK-1; Zymed Laboratories, San Francisco, CA) for 60 min at room temperature and were further incubated with avidin-biotin-peroxidase complex (Vector ABC Elite staining kit; Vector Laboratories). The peroxidase was developed with a diaminobenzidine substrate solution (peroxidase substrate kit; Vector Laboratories). Both kits were used according to the instructions provided by the manufacturer. Finally, the sections were counterstained with methyl green. For the staining of macrophage scavenger receptors and MHC class II molecules, we used rat anti-mouse macrophage scavenger receptor mAb (clone 2f8; Serotec) and rat anti-mouse MHC class II mAb (clone ER-TR3; BMA Biomedicals), respectively, as primary Ab. Texas red-labeled mouse anti-rat IgG Ab was used as the second Ab (Jackson ImmunoResearch, West Grove, PA). Four-micrometer sections were fixed in acetone for 10 min at 4°C and rinsed in PBS. Primary Ab were allowed to bind during a 1-h incubation at 37°C. Sections were then washed in PBS and incubated with the second Ab for 1 h at 37°C. Negative control experiments were performed by replacing the firststep Ab with incubation buffer only or with isotype-matched Ab. We counted the number of GFP-positive/scavenger receptor-positive cells and GFP-positive/MHC class II-positive cells in the glomeruli of the chimeric mice at the 24th week after BMT.
Culture of Glomerular Cells
Kidneys were obtained from [GFP→B6] mice 24 wk after BMT (n = 3). The glomeruli were isolated by differential sieving, as reported previously (20), and were then plated on chamber slides coated with collagen I (BIOCOAT culture slides; Falcon, Becton Dickinson Labware, Mountain View, CA). In our experiments, the purity of the glomerulus preparation was >90% and few tubular cells were contaminating the cultures, as determined by microscopy. The culture medium was Dulbecco's modified Eagle's medium (DMEM)/F12 (1:1) (Life Technologies, Grand Island, NY) supplemented with 20% fetal bovine serum (FBS) (REHATUIN; Intergen, Purchase, NY). The cells were maintained in a humidified atmosphere with 5% CO2 at 37°C. Within 2 wk, most primary cultures exhibited an irregular stellate shape. The identity of the cultured cells was confirmed by immunofluorescence detection with specific Ab and assessment of responses against angiotensin II (AngII) (see below).
Cell Culture Immunofluorescence Assays
Cells grown on chamber slides were fixed with 2% PFA-PBS for 10 min at room temperature. After being washed with PBS, cells were permeabilized with 0.1% Triton X-100 (Sigma Chemical Co., St. Louis, MO) in PBS for 1 min at room temperature and were washed again. Nonspecific binding of avidin and biotin was prevented by using an ABC blocking kit, as recommended by the manufacturer (Vector Laboratories). After being washed with PBS, the cells were incubated for 2 h at 37°C with one of the following Ab: rabbit antiserum against desmin (ICN Pharmaceuticals), rabbit antiserum against factor VIII-related antigen (Zymed), or mouse IgG mAb against cytokeratin (Zymed). The cells were rinsed in PBS and incubated with biotin-labeled sheep anti-rabbit IgG Ab (American Qualex, La Mirada, CA) or biotin-labeled rabbit anti-mouse IgG Ab (American Qualex) for 1 h at 37°C, followed by incubation with rhodaminelabeled avidin (Molecular Probes, Eugene, OR) for 1 h at 37°C. The cells were again rinsed in PBS, mounted using SlowFade antifade kits, and observed with a LSM. Negative control experiments were performed by replacing the first-step Ab with incubation buffer only or with isotype-matched Ab.
AngII Stimulation of Cells in Culture
Cells grown on chamber slides were washed with serum-free DMEM and left on slides for 10 min at room temperature. The cells were then exposed to 10-6 M AngII (Sigma Chemical Co., St. Louis, MO) at room temperature, as reported previously (20). Before and after stimulation with AngII for 10 min, photographs of the same field were taken under high magnification (×2400). In control experiments, cells were exposed to the vehicle without AngII; photographs were taken before and after exposure to the vehicle for 10 min.
Results
BMC from Green Mice Reconstituted Recipient BMC and Peripheral Lymphocytes
To verify whether BMT from GFP donors successfully reconstituted recipients that had been irradiated to remove pluripotential stem cells, the expression of GFP in splenic lymphocytes and BMC was analyzed by flow cytometry at 2, 4, 8, and 24 wk after BMT. As shown in Table 1, although splenic lymphocytes and BMC from non-BMT-treated B6 mice or syngeneic [B6→B6] chimeric mice were negative for GFP, the cells from GFP-transgenic mice were GFP-positive (86.9 ± 6.2% in splenic lymphocytes and 64.3 ± 6.0% in BMC). Two weeks after BMT, 85.2 ± 5.0% of splenic lymphocytes and 64.3 ± 3.6% of BMC from [GFP→B6] chimeric mice were positive for GFP. The percentages of GFP-positive spleen cells and BMC of [GFP→B6] mice were comparable to those of GFP mice and remained consistently so until 24 wk after BMT, indicating that the recipient B6 mice had been completely reconstituted with BMC of GFP mouse origin.
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Table 1: GFP expression in splenic lymphocytes and bone marrow cells of chimeric micea
The Number of GFP-Positive Cells in the Glomeruli from [GFP→B6] Mice Increased in a Time-Dependent Manner after BMT
After sufficient perfusion of chimeric mice with PBS to remove circulating GFP-positive cells from their glomeruli, the histologic appearance of the kidneys was assessed at 2, 4, 8, and 24 wk after BMT. As assessed by light microscopy, there was neither mesangial hypercellularity of the glomeruli nor tubulointerstitial injury in [GFP→B6] mice (Figure 1, A and B). Similarly, in the quantitative analysis, the number of glomerular cells in these mice did not change throughout the observation period (Figure 2). As assessed with a LSM, few GFP-positive cells were observed in [GFP→B6] mice 2 wk after BMT (Figure 1C). However, GFP-positive cells were clearly visible in the glomeruli, the periglomerular space, and the interstitium of [GFP→B6] mice 4 wk after BMT, and these green cells were consistently observed in the kidneys of the recipients until 24 wk after BMT (Figure 1D). Moreover, the number of glomerular GFP-positive cells in [GFP→B6] mice increased in a time-dependent manner (2 wk, 0.67 ± 0.67/glomerular cross-section; 4 wk, 4.18 ± 0.22/glomerular cross-section; 8 wk, 8.20 ± 2.15/glomerular cross-section; 24 wk, 12.78 ± 1.68/glomerular cross-section; means ± SEM) (Figure 2). However, [B6→B6] mice (n = 3 at each observation point) exhibited no GFP-positive cells in glomeruli at any observation time.
Figure 1: Light microscopic findings with periodic acid-Schiff staining (A and B) and fluorescence photographs (C and D) of glomeruli from lethally irradiated C57BL/6j (B6) mice given transplants of bone marrow stem cells from syngeneic mice transgenic for green fluorescence protein (GFP) ([GFP→B6] mice), 2 wk (A and C) and 24 wk (B and D) after bone marrow transplantation (BMT). Microscopically, there was neither mesangial hypercellularity of the glomeruli nor tubulointerstitial injury in [GFP→B6] mice 2 wk (A) or 24 wk (B) after BMT. Two weeks after BMT (C), no GFP-positive cells were observed in glomeruli (G). However, GFP-positive cells were clearly visible in the glomeruli (red arrowheads), the periglomerular space, and the interstitium in [GFP→B6] mice 24 wk after BMT (D). Magnification, ×400.
Figure 2: Changes in the numbers of glomerular cells and GFP-positive cells in the glomeruli of [GFP→B6] mice. The numbers of glomerular cells and GFP-positive cells in the glomeruli were assessed 2 wk (n = 3), 4 wk (n = 3), 8 wk (n = 3), and 24 wk (n = 5) after BMT, as described in the Materials and Methods section. The numbers of cells/glomerular cross-section exhibited no significant changes throughout the observation period, whereas the numbers of GFP-positive cells in the glomeruli increased in a time-dependent manner. [UNK], number of GFP-positive cells per glomerulus; ○, number of nuclei per glomerular cross section.
GFP-Positive Cells in the Glomeruli of [GFP→B6] Mice Were Neither Infiltrating Macrophages nor T Cells
To investigate whether the GFP-positive cells in glomeruli were adherent macrophages or T cells, we performed F4/80 staining for macrophages (monocytes) and Thy-1 staining for pan-T cells. F4/80-positive cells and Thy-1-positive cells were barely observed in glomeruli of [GFP→B6] mice at 24 wk after BMT (means ± SEM of 0.19 ± 0.09/glomerular cross-section and 0.49 ± 0.05/glomerular cross-section, respectively) (Figure 3, A and B). Furthermore, the expression of macrophage scavenger receptors and MHC class II molecules was examined. Only 5% of glomerular GFP-positive cells were positive for macrophage scavenger receptors (Figure 3, C1 and C2), and only 6% of glomerular GFP-positive cells were positive for MHC class II molecules (data not shown). These data suggest that most GFP-positive cells in the glomeruli might be neither macrophages nor T cells.
Figure 3: Immunohistochemical staining for macrophages and T cells in the glomeruli of [GFP→B6] mice at 24 wk after BMT. F4/80-positive cells (macrophages) (A) and Thy-1-positive cells (T cells) (B) were barely observed within the glomeruli of [GFP→B6] mice 24 wk after BMT. In addition, most GFP-positive cells (C1) in the glomeruli (G) were negative for macrophage scavenger receptors (C2). C1 and C2 depict the same field. Magnifications: ×200 in A and B; ×400 in C1 and C2.
Reconstitution of the Mesangium with BMC of Donor Origin Occurred in [GFP→B6] Mice
To clarify the localization of GFP-positive cells in the glomeruli, we next examined the differential interference contrast images of glomeruli with fluorescence imaging. As shown in Figure 4, some GFP-positive cells were located within the mesangium 24 wk after BMT. To identify the character of these cells, we examined the expression of desmin, which is a marker for mesangial cells, using indirect immunofluorescence with a specific Ab (anti-desmin). As shown in Figure 5, some of the desmin-positive cells were also positive for GFP in the nuclei, suggesting that the mesangial cells of the recipients consisted of donor BMC.
Figure 4: Glomeruli from [GFP→B6] mice observed by differential interference imaging, without (A) or with (B) a digitized fluorescence image, 24 wk after BMT (obtained using LSM software version 3.5, based on Windows version 3.1). GFP-positive cells were observed within the mesangial area (red arrowheads). Magnification, ×1600.
Figure 5: Desmin staining of the glomeruli from [GFP→B6] mice 24 wk after BMT. Glomerular desmin staining revealed a typical mesangial pattern (A). Some desmin-positive mesangial cells were positive for GFP in the nuclei (yellow arrowheads) (B). Magnification, ×800.
Cultured GFP-Positive Cells Isolated from the Glomeruli of [GFP→B6] Mice Were Positive for Desmin Staining and Exhibited Contractile Properties in Response to AngII Stimulation
To confirm that GFP-positive cells within the mesangium in [GFP→B6] mice exhibited the characteristics of mesangial cells, we isolated glomeruli from these chimeric mice at 24 wk after BMT and cultured them in DMEM/F12 with 20% FBS. Two weeks later, GFP-positive cells, which exhibited stellate morphologic features, were clearly observed to be migrating from the glomeruli (Figure 6A). In immunofluorescence assays, the majority of cultured cells (approximately 84%) stained for desmin and approximately 60% of desmin-positive cells expressed GFP in their nuclei (Figure 6, B through D). These cultured cells did not exhibit staining when anti-desmin Ab was replaced by incubation buffer or by isotype-matched Ab. In addition, most of the cultured cells were negative for cytokeratin and factor VII-related antigen staining (data not shown). Therefore, these GFP-positive cells cultured from glomeruli of [GFP→B6] mice exhibited several characteristic features of mesangial cells (20,21).
Figure 6: Characteristics of glomerular cells from [GFP→B6] mice 24 wk after BMT. Two wk after culture inception, GFP-positive cells with stellate morphologic features had grown out from the glomeruli (G) (A). Most of the cultured cells were positive for desmin (red) (B), and some cultured cells expressed GFP in the nuclei (C). Approximately 60% of these desmin-positive cells exhibited GFP (green) expression in the nuclei (D). Magnifications: ×1200 in A; ×2400 in B, C, and D.
To further investigate whether the cultured GFP-positive cells exhibited functional properties similar to those of contractile mesangial cells, we examined the morphologic changes of these cells after AngII exposure (20). The GFP-positive cells from isolated glomeruli of [GFP→B6] mice did not exhibit any morphologic changes when exposed to the vehicle without AngII for 10 min (Figure 7, A and B). In contrast, compared with findings before AngII exposure (Figure 7C), the GFP-positive cells exhibited structural alterations, i.e., sharpening or disappearance of the cytoplasmic processes or withdrawal of the forefront of the cytoplasm (Figure 7D). Therefore, the GFP-positive cells exhibited properties functionally similar to those of mesangial cells in response to AngII stimulation.
Figure 7: Changes in the shapes of cells from the glomeruli of [GFP→B6] mice in response to angiotensin II (AngII) stimulation. Cultured GFP-positive cells exposed to vehicle alone exhibited no remarkable morphologic changes (B), compared with features before exposure (A). In contrast, compared with features before AngII stimulation (C), GFP-positive cells exhibited remarkable structural alterations (D). The cytoplasmic processes were sharpened (arrows) or disappeared (arrowheads). Moreover, the forefront of the cytoplasm was withdrawn after AngII stimulation, leading to an increase in the area between the margin of the cell and the black circle (red points). Magnification, ×2400.
Discussion
Because the origin of glomerular mesangial cells that appear during glomerulogenesis and after injury in adult glomeruli is unknown, we investigated the possibility that cells from bone marrow might differentiate into glomerular cells. For this purpose, we devised a BMT model in which mice transgenic for GFP were the source of donor BMC, because the green color of these cells can be detected in the tissues of recipients by using a fluorescence microscope (18). In this system, we can identify the GFP-positive cells in the recipients as donor bone marrow-derived cells. In addition, to examine the central role of donor BMC in the repopulation of recipient glomeruli, we needed to exclude the involvement of resident stem cells. For this purpose, we performed BMT with lethal irradiation of the recipients, for which we previously confirmed the safety and stability of BMT (3). As a result, from 2 to 24 wk after BMT, the percentages of GFP-positive spleen cells and BMC of [GFP→B6] mice were comparable to those of GFP mice (Table 1), suggesting that the B6 recipients had been almost completely reconstituted with BMC of GFP mouse origin. Light microscopic findings revealed that the glomeruli in [GFP→B6] mice demonstrated no leukocyte infiltration and no mesangial hypercellularity (Figures 1, A and B, and 3). Throughout the observation period, no albuminuria was detected in any animals with the single radial immunodiffusion method (data not shown). These observations indicated that BMT with 8.0-Gy, total-body irradiation did not induce remarkable changes in renal function and structure.
Of note, at 4 wk after BMT, GFP-positive cells were clearly observed in the glomeruli, the periglomerular space, and the interstitium in [GFP→B6] mice. Two weeks after BMT, peripheral lymphocytes were almost completely replaced by donor-derived cells. Therefore, if circulating cells had remained in glomeruli after perfusion with our method, GFP-positive cells should have been observed in glomeruli 2 wk after BMT. In fact, very few GFP-positive cells were present in glomeruli 2 wk after BMT (Figure 1C). Therefore, we speculated that the kidney perfusion should be sufficient to remove circulating and nonadherent cells from glomeruli, and most GFP-positive cells in glomeruli from 4 wk after BMT were other cell types. Furthermore, the number of glomerular GFP-positive cells in these chimeric mice increased with time (Figure 2) but was independent of the GFP-positive cellular content of the splenic lymphocytes and bone marrow (Table 1). Twenty-four weeks after BMT, the mean number of GFP-positive cells/glomerular section was 12.78 ± 1.68. One year after BMT, many more GFP-positive cells were observed in the glomeruli of [GFP→B6] mice (data not shown). Previously, bone marrow-derived, Ia-positive cells were also noted in the mesangium (22). For this study, we sufficiently perfused the chimeric mice with PBS to remove circulating GFP-positive cells from their glomeruli. The number of glomerular cells in these mice did not change throughout the observation period (Figures 1 and 2). Immunohistologic studies revealed that few F4/80-positive or Thy-1-positive cells were observed in the glomeruli of [GFP→B6] mice 24 wk after BMT (Figure 3, A and B). In addition, most GFP-positive cells (Figure 3C1) in the glomeruli of [GFP→B6] mice did not express macrophage scavenger receptors (Figure 3C2) or MHC class II molecules (data not shown). These results suggest that the GFP-positive cells in the glomeruli of these chimeric mice were neither adherent macrophages nor T cells; rather, donor green BMC replenished resident glomerular cells.
The LSM revealed that some GFP-positive green cells resided within the mesangium of glomeruli from [GFP→B6] mice (Figure 4). To characterize these cells, we used indirect immunofluorescence assays and an Ab against desmin, a well known marker for mesangial cells (20,21). With this method, a portion of the desmin-positive mesangial cells were also positive for GFP, as shown in Figure 5. These data indicate that mesangial cells in the B6 recipients had been replenished by GFP donor BMC.
To confirm that GFP-positive cells in the glomeruli exhibited the characteristics of mesangial cells, we isolated glomeruli from [GFP→B6] mice at 24 wk after BMT and cultured them in DMEM/F12 with 20% FBS. Subsequently, most of these cultured cells stained for desmin, and approximately 60% of the desmin-positive cells expressed GFP (Figure 6). The presumed source of these desmin-positive/GFP-positive cells was the donor BMC, whereas the desmin-positive/GFP-negative cells may have originated from the B6 recipients. In addition, most of the cultured cells were negative for both cytokeratin, a marker for glomerular epithelial cells, and factor VIII-related antigens, which typify glomerular endothelial cells (20). Finally, after exposure to AngII stimulation, these GFP-positive cells demonstrated the contractile properties of mesangial cells (Figure 7). These characteristic features suggest that the GFP-positive cells from the glomeruli of [GFP→B6] mice are mesangial cells. It was recently reported that the hematopoietic transcription factor PU.1 protein was identified within nuclear extracts of mesangial cells (23). That study supports our observations that glomerular mesangial cells may be derived from BMC.
How we can explain the restoration of mesangial cells in the BMT-treated mice by donor BMC? Several recent studies demonstrated that BMC serve as precursor cells for various mesenchymal tissues (5,6,7,8,9,10,11). Takahashi et al. (24) previously reported that the proportion of endothelial progenitor cells in the circulation was approximately 10% in normal B6 mice. We then considered that a mesangial progenitor cell-enriched population might exist in the circulation, and we hypothesized that these precursors might journey into the mesangial area and differentiate into mesangial cells as a normal event or after injury. The renewal rate of mesangial cells has been estimated to be approximately 1%/d (25). Because in this study recipient mice were irradiated, we could not exclude the effect of radiation on mesangial cells and their turnover rates. Another possibility is that bone marrow-derived mesangial precursor cells may reside in the extraglomerular mesangium. Recently, Hugo et al. (16) indicated that the juxtaglomerular apparatus has a role in maintaining the size of the mesangial cell population after injury in the anti-Thy-1 model of mesangial proliferative glomerulonephritis. Therefore, in this study, bone marrow-derived mesangial progenitor cells might reside in the juxtaglomerular apparatus and migrate into the mesangium. Additional studies are needed to investigate the origin of mesangial progenitor cells.
GFP-positive cells were also found in the heart, liver, and vessels in our [GFP→B6] mice. Recent studies demonstrated that platelet-derived growth factor and vascular endothelial growth factor and their receptors are essential molecules for glomerulogenesis (17,26,27). In addition, mice deficient in laminin α3 exhibit abnormalities in the development of glomerular endothelial and mesangial cells (28). Although we still have no data on the mechanisms underlying the differentiation of BMC into mesangial cells, it is thought that some growth factors and extracellular matrix components locally produced by resident glomerular cells in BMT recipients might play an important role in the supplementation by donor BMC.
Furthermore, the data presented here may support the notion that BMT from normal donors can attenuate glomerular injury by replacing harmful immune cells and replenishing glomerular cells in nephritic recipients (3). Therefore, BMT may offer new insights into the mechanisms that promote glomerular diseases and may also provide a new approach for the treatment of these diseases.
We thank Tomoko Murata, Hisako Arai, and Emiko Kikuchi for technical support in preparing tissue samples and using the LSM and Drs. Minori Kamada and Miyuki Agawa for flow cytometric analyses. We also acknowledge Dr. Tatsuo Shimosawa and Phyllis Minick for critical editing of the manuscript, as well as the radiotherapeutics staff at the Tokyo Metropolitan Institute of Gerontology.
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