Our studies focused on the importance of BMSCs in a model of severe ischemic injury of the kidney, which is characterized by organ failure with subsequent recovery. In this model, many tubular cells of the outer medulla die by necrosis and apoptosis (26
), and in surviving cells, there is widespread cytoskeletal disruption. Injury is followed by cytoskeletal reorganization and intense tubular cell proliferation during the recovery phase. We used 3 different reporters to identify bone marrow–derived cells to evaluate whether these cells contribute directly to the repair of the epithelium after injury. The use of 1 reporter, EGFP, provided no evidence for direct BMSC involvement in tubular cell regeneration. Bone marrow cells did, however, make a small contribution to repopulation of the injured vascular endothelium and could indirectly affect tubule regeneration. These results were supported by examination of sex-mismatched chimeras. There was a low level of false positive results when in situ hybridization was used to detect the Y chromosome. This false positivity was eliminated when deconvolution microscopy was used to define better the 3D tissue relationship of tubule cells to leukocytes and other interstitial cells (27
). The use of another reporter, β-gal, suggested a small contribution from bone marrow–derived cells to tubular cell replacement. Closer examination of this reporter model indicated that endogenous β-gal in the tubular cells of the inflamed postischemic kidney could account for all of the β-gal–positive tubular cells.
Our I/R model resulted in disruption of the actin cytoskeleton in most of the intact tubular cells in the outer stripe of the outer medulla 48 hours after ischemia as reflected by phalloidin staining (18
). Three weeks after ischemia, the disrupted tubules had almost completely returned to normal. We and others have demonstrated that spreading, dedifferentiation, and then proliferation of viable cells occur during recovery from renal I/R injury (3
). In our previous study (3
) and our current study, tubular cell proliferation was demonstrated to be prominent. In the absence of BMSCs differentiating into tubular cells, it is probable that regenerating tubular cells derive from dedifferentiated cells of tubular origin that have survived the initial ischemic insult, divide, and are able to redifferentiate into the mature tubular cell phenotype.
Recent reports suggest that there is significant plasticity in the developmental potential of many different adult cell types, although adult stem cells appear to be more limited than their embryonic counterparts in proliferative capacity and pluripotentiality (8
). Recently, some studies have provided evidence that adult bone marrow cells can give rise to or fuse with cells outside their tissue of origin, including heart, brain, and liver (29
). Several reports also indicate that cells of bone marrow origin can become functioning endothelial cells and that this population is important in revascularization following injury (32
). Our findings are consistent with such observations on the endothelium and focus attention on the renal vasculature as an important site for repair. Although frequently overlooked, evidence for the importance of vascular repair in the injured kidney was provided in recent studies in which direct administration of endothelial cells into postischemic kidney circulation was reported to hasten recovery (37
) and longer-term studies showing that the postischemic kidney is characterized by loss of capillaries in the outer medulla that are not fully regenerated (39
). Bone marrow–derived endothelial cells may possess a greater capacity to proliferate and as such are likely important contributors to vascular recovery from injury (40
). The fact that many fewer endothelial cells in contralateral kidneys are replaced by cells originating from the bone marrow suggests that bone marrow cell differentiation in the mouse kidney contributes more to replenishing the endothelial cells lost due to injury than to the replenishing of endothelial cells during normal cell turnover. Compared with the contribution of bone marrow–derived endothelial cells to vascularization of other organs including hind limb and tumorigenesis (34
), the contribution of bone marrow cells to revascularization in our model of kidney repair appears only to be minor, and it is unlikely, therefore, that bone marrow cells contribute significantly to regeneration. Whether this finding reflects impaired revascularization in the regenerating kidney, as has been suggested in other studies (39
), remains to be explored.
Previous investigation of the role of BMSCs in the injured kidney has focused on tubular cell regeneration. Bone marrow cells have been reported to contribute to both normal turnover of renal epithelium in mice (5% of tubular cells after 13 weeks of chimerism) and regeneration in human renal transplantation (up to 6.8% after 3 months) (14
). These provocative observations have been pursued by others who suggest in 1 study that, 7 days following ischemic injury, 20% of all tubules in the outer medulla are replaced by bone marrow–derived cells (15
). In another study of mild ischemic injury, which was not completely characterized, 80% of all tubules were reported to contain differentiated tubular cells of bone marrow origin only 24 hours following ischemia (16
). Both these studies, which were performed in the same mouse strain we used, relied heavily on staining for β-gal activity with X-gal. Furthermore, in 1 report, replacement with bone marrow cells appeared to occur at a time when cell death and cell sloughing predominate (16
). It is our contention that the tubular cell β-gal staining reported previously may reflect endogenous β-gal. Our study indicates that native mouse kidney tubules have high levels of endogenous β-gal, which, following mild fixation, stains strongly with X-gal if the pH is lower than 7.5. Even at high pH, after mild fixation of thick (0.2 mm) sections, endogenous β-gal activity can be detected weakly in the outer medulla of normal kidneys and diffusely in the cortex in postischemic kidneys. It is possible, therefore, that endogenous β-gal is more easily stained in the postischemic kidney, and this staining can easily be interpreted as bacterial β-gal activity derived from donor cells in this model. When we minimized detection of endogenous but not bacterial β-gal activity, tubular epithelial cells in 5-μm sections did not exhibit β-gal activity. Furthermore, bacterial β-gal–specific antibody did not locate antigen in tubular cells. In our opinion, the LacZ
chimeric mouse expressing bacterial β-gal in bone marrow cells is an unreliable model for tracking bone marrow–derived cells in the adult kidney. When we used sex-mismatched chimeras to identify cells of bone marrow origin, we found very small numbers of tubular cell nuclei that stained for the Y chromosome. The same few apparently positive cells were analyzed by deconvolution microscopy. When this technique was used, it became apparent that aggregates of fluorescent probe were located outside of the nucleus, which indicates that these cells did not have true nuclear Y chromosome positivity. Recently, in a detailed study of epithelial cells in enteric human biopsies from human sex-mismatched bone marrow transplants, it was pointed out that conventional microscopy markedly overestimated the number of bone marrow–derived epithelial cells (27
). Therefore, detailed 3D microscopy of tissue is required for definitive scoring of Y chromosome–positive nuclei. Because tissues from EGFP chimeric mice require no fixation and mice do not endogenously express GFP, we have found the EGFP chimeric model to give superior results, with greater sensitivity and specificity over other approaches. In addition, colocalization studies can be performed using 1-step antibody procedures on these tissues, which further enhances the specificity of findings.
The precise phenotype of BMSC precursors that differentiate into endothelial cells is currently unclear. Contrasting reports show that bone marrow stromal cells, c-kit+
BMSCs, or monocytes have the potential to become endothelial cells (42
). Nevertheless, bone marrow stromal cells have been reported to differentiate into endothelial cells in vitro and assist in tumor vascularization in vivo (44
). We therefore injected bone marrow stromal cells i.v. and found that they did not home to the injured kidney, even following differentiation into capillary structures in vitro. Interestingly, they could not be found in other organs (lung, heart, spleen, liver) 2 weeks after injection, but small numbers homed to the bone marrow, which was in keeping with previous observations (46
). Although MSCs did not differentiate into renal structures in vivo, they had the capacity to reduce the severity of ARF following I/R injury. This protective capacity was not restricted to MSCs, since fibroblasts grown under identical conditions had the same efficacy. By contrast, MSCs grown on plastic had no such protective effect. Further work will be required to determine the cause of this protective effect. Some possibilities include cytokine release by injected cells, inadvertent injection of cell substrate, the release of antiinflammatory cytokines such as IL-10 following apoptotic cell death of injected cells, or the immunomodulatory effects of clearance of circulating cells by splenic phagocytes (47
Although we were unable to positively identify MSCs as the stem cells responsible for replacement of endothelial cells in the postischemic kidney, it remains possible that HSCs were mobilized from the bone marrow, as reported by others, or that they derive from a subset of circulating monocytes (15
). We searched carefully for an increased population of circulating leukocytes lacking lineage markers CD11b, GR-1, CD4, CD8, B220, Ter-119 at 24 and 48 hours following induction of injury in our model. We have failed repeatedly to see a mobilized, distinctly lin–
population of circulating leukocytes. Although there is no mobilized population of lin–
cells in the circulation, up to 1% of peripheral leukocytes are lin–
in both healthy and diseased mice and therefore represent a large enough pool to account for the cell replacement we have observed.
Recently, controversy over the importance of BMSCs in solid organ repair has been generated by findings that cardiomyocyte replacement following infarction and pancreas β cell regeneration can both proceed without the involvement of stem cells (49
). Our findings in the kidney reinforce our hypothesis that the tubule has an enormous capacity to repair itself as a result of dedifferentiation of proximal tubule epithelial cells followed by proliferation and reestablishment of a differentiated phenotype (52
). Our findings that bone marrow–derived cells can differentiate into kidney endothelial cells or fuse with these cells suggests that the recovering kidney may benefit from circulating endothelial cell precursors. In the injured pancreas, liver, and heart, studies point to progressive replacement with BMSC-derived endothelial cells over a number of weeks (32
). It is possible therefore that further repair of peritubular capillaries by BMSCs in the postischemic kidney occurs in ensuing weeks.
Our studies do not definitively exclude a role for kidney-derived mesenchymal stem cells in repopulation of the ischemically injured renal tubule. The identification of a population of cells within the kidney that has the capacity to differentiate into tubular cells has remained elusive (54
), although there is a recent report of multipotent stem cells in the renal papilla (55
). In our studies, very early after injury, a large number of proliferating cells could be seen at diverse sites in the regenerating tubules. In order for large numbers of proliferating cells to be derived from endogenous kidney stem cells, a large population of stem cells would have to be present and adjacent to or within proximal tubules, and the intrinsic low proliferation rate of such stem cells would have to be enhanced dramatically. Our findings that bone marrow–derived cells differentiate into or fuse with peritubular vascular endothelial cells suggests possible therapeutic strategies focused on enhancing this process.
We conclude that bone marrow–derived cells do not make a significant contribution to the restoration of epithelial integrity after an ischemic insult and that epithelial cells that restore tubular integrity in the postischemic kidney primarily originate in resident surviving epithelial cells that undergo a phenotypic transition resulting in dedifferentiation, proliferation, and subsequent differentiation of the parent and daughter cells into mature polar epithelial cells. It is hoped that understanding the factors regulating this behavior of the kidney epithelial cell will lead to insight into therapeutic approaches to activate and potentiate this response in patients where it seems to be impaired.