adult stem cells in the repair of the injured renal tubule

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Adult stem cells in the repair of the injured renal tubuleLloyd G Cantley

INTRODUCTIONThe requirement for our kidneys to both contin-uously filter the blood to remove accumulated toxins and to concentrate the urine to prevent dehydration generates high demand for cellular oxygen in a region of relatively low blood flow. This makes the renal tubular cell uniquely susceptible to injury. In hospitalized patients this susceptibility is heightened, and manifests as a propensity for acute renal failure to develop following release of endogenous cytokines and toxins (such as in sepsis and rhabdomyolysis), exposure to exogenous toxins (such as amino-glycosides and radiocontrast agents), or episodes of renal hypoperfusion. When patients are younger or when the injury is less severe, renal tubules can regenerate and regain normal or near-normal function within days; however, in more severe cases of injury and in older patients, the repair process can be prolonged or even fail completely. This can result in a requirement for long-term dialysis and a marked increase in patient mortality.

For these reasons, nephrologists have long sought to better understand the process of tubule repair, and to use this understanding to develop strategies for improving the rate and degree of recovery following episodes of acute renal failure. Examination of kidney specimens from humans that have died following such episodes, as well as of kidneys from animal models of acute renal failure induced by ischemia and reperfusion, have led to the conclusion that repair of the tubule after injury is mediated by the surviving tubular cells that border the region of injury (Figure 1).1 After the insult occurs, these cells rapidly lose their brush border and dediffer-entiate into a more mesenchymal phenotype. This process seems to be followed by migration of the de differentiated cells into the regions where cell necrosis, apoptosis or detachment have resulted in denudation of the tubular basement membrane. There they proliferate and eventually redifferentiate into an epi thelial phenotype, completing the repair process. In

The capacity of the kidney to regenerate functional tubules following episodes of acute injury is an important determinant of patient morbidity and mortality in the hospital setting. After severe injury or repeated episodes of injury, kidney recovery can be significantly impaired or even fail completely. Although significant advances have been made in the clinical management of such cases, there is no specific therapy that can improve the rate or effectiveness of the repair process. Recent studies have indicated that adult stem cells, either in the kidney itself or derived from the bone marrow, could participate in this repair process and might therefore be utilized clinically to treat acute renal failure. This review will focus on our current understanding of these stem cells, the controversies surrounding their in vivo capacity to repopulate the renal tubule, and further investigations that will be required before stem cell therapy can be considered for use in the clinical setting.

KEYWORDS acute renal failure, ischemia, kidney, stem cells

LG Cantley is an Associate Professor in the Section of Nephrology at Yale University School of Medicine, New Haven, CT, USA.

CorrespondenceSection of Nephrology, Department of Medicine, Yale University School of Medicine, New Haven, CT 06520, [email protected]

Received 17 May 2005 Accepted 1 September 2005

www.nature.com/clinicalpracticedoi:10.1038/ncpneph0021

REVIEW CRITERIAMaterial for this review was identified by searching PubMed for all articles published prior to August 2005 using the search terms “stem cell”, “kidney”, “bone marrow”, “injury”, “repair” and “ischemia”.

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general, it is thought that the local release of growth factors such as hepatocyte growth factor (HGF), epidermal growth factor, and insulin-like growth factor-1 (IGF-1) coordinates this process of dedifferentiation, migration, proliferation and eventual redifferentiation.2

TISSUE-SPECIFIC RENAL PROGENITOR CELLSDespite several observations supporting the asser-tion that tubular cells are the source of renal tubule repair, it has been thought for many years that the ADULT RENAL STEM CELL might also have a role in the repair process. This concept emerged as our understanding of kidney develop ment became more sophisticated. We now know that during nephron formation, the cells that will eventually make up the glomerular epithelium, proximal tubule, loop of Henlé and distal convoluted tubule all originate as mesenchymal cells, which condense around the tip of the ureteric bud and undergo transformation to epithelial cells.3 If these

mesenchymal cells persisted in the adult renal interstitium, they would provide a reservoir of tubule cell progenitors that could be activated to migrate into the tubule and differentiate into epithelial cells in response to renal injury (Figure 1). A similar process has been observed in elasmo branchs and fish. Mesenchymal cells in the adult kidney of these species can respond to renal injury by increasing their rate of division and augmenting formation of new nephrons.4,5

Although it has been difficult to isolate or even confirm the existence of such cells in the mammalian kidney, several studies support the possibility that renal progenitor cells persist in the adult renal interstitium in rodents and humans. Labeling with the DNA marker BrdU is one of the methods that has been used in attempts to confirm the existence of endo genous renal adult stem cells.6,7 This approach takes advantage of the observation that organ-specific stem cells, unlike most specialized cells, can exist for long periods without dividing.8 When labeled with

Tubular basement membrane

1

23

Influx of residenttubular stem cells Dedifferentiation of surviving tubular cells

Cell migration, proliferation, tubule reconstitution

Protection or repair bycells from a distant site(e.g. bone marrow)

Renal progenitor cellsTubular epithelium

Denudation of tubularbasement membrane

Ischemic injury or acute tubular necrosis

Figure 1 Proposed mechanisms of tubule repair after acute injury. After ischemic or toxic renal injury, cells are lost from the tubular basement membrane. The repair process is effected by new cells migrating into the region and reconstituting a functional tubular epithelium. The predominant pathway of repair seems to be growth factor-dependent dedifferentiation of surviving tubular cells, followed by their migration, proliferation and redifferentiation into normal tubules (2). It has also been suggested that the interstitium of the kidney contains adult renal stem cells capable of migrating into the region of injury and differentiating to form tubular epithelial cells that contribute to repair (1). Another possibility is that cells from distant sites such as the bone marrow could either secrete factors that protect the endogenous cells, or could themselves enter the kidney and migrate to the injured tubule before differentiating into tubular epithelial cells (3).

GLOSSARYBRUSH BORDERCollection of microvilli on the plasma membrane of an epithelial cell that increases the surface area available for absorption

MESENCHYMAL Originating from the mesenchyme; an embryonic tissue that arises from the mesodermal layer

ADULT RENAL STEM CELLA hypothetical undifferentiated cell found in the adult kidney that has the potential to differentiate into the specialized cells of the kidney

ELASMOBRANCHSFish of the class Chondrichthyes that have cartilaginous, non-bony skeletons; e.g. rays, skates and sharks



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BrdU during late organogenesis, only these slow-cycling stem cells retain the label. Oliver et al., who injected 3-day-old rodents with BrdU and analyzed their kidneys several months later, found numerous BrdU-labeled cells in the inter-stitium of the renal papilla.7 They also observed that following transient renal ischemia, the BrdU-positive cells were rapidly lost from the papilla, indicating that they had either migrated out of this region or started dividing. Staining for Ki67, a marker of proliferation, showed that papillary BrdU-positive cells were indeed induced to divide by renal ischemia, as would be expected for a stem cell involved in tubule repair. In addition, these investigators demonstrated that the renal papilla contained non-tubular cells that could be isolated in culture and induced to express neuronal or epithelial markers.

One of the major difficulties of identifying endogenous renal stem cells has been finding a cell-surface marker that facilitates cell isolation and purification. A recent approach to identi-fying organ-specific stem cells is isolation on the basis of the cells’ ability to extrude Hoechst 33342 dye.9 Cells with this property, termed side population (SP) cells, have been found in several organs, including the kidney. SP cell populations seem to be enriched in both hematopoietic and non-hematopoietic stem cells.10,11 Hishikawa and co-workers have shown that the renal inter-stitium contains SP cells, and that intravenous infusion of these cells can counteract the rise in blood urea nitrogen (BUN) and creatinine in rodents with acute tubular injury induced by cisplatin injection.12 SP cells isolated from the acutely injured kidney expressed high levels of messenger RNA for several growth factors implicated in renal development and/or repair, including HGF, vascular endothelial growth factor (VEGF) and leukemia inhibitory factor.

Bussolati and colleagues explored the possibility that endogenous renal stem cells express CD133, a surface marker of endo thelial pro genitor cells, hematopoietic progenitor cells, neural stem cells and embryonic intestinal epithelial cells.13–15 The investigators were able to detect small numbers of CD133+ cells in the interstitium of adult human kidneys—approximately 1% of the total cell population—and isolate these cells in culture.16 Interestingly, the purified cells expressed the early nephron developmental marker PAX2, as well as several markers typical of bone marrow stromal cells (also known as mesenchymal stem cells [MSCs]),17 but were

negative for hematopoietic cell markers such as CD34 or CD45.

When Bussolati and co-workers cultured the CD133+ cells in the presence of HGF and fibro-blast growth factor-4, the cells stopped expressing CD133 and began to express epithelial markers such as cytokeratin, E-cadherin and zona occludens-1. They also continued to express the mesenchymal marker vimentin, indicating that the cells were not fully differentiated. By contrast, culture in the presence of VEGF resulted in expres-sion of endothelial markers including VE-cadherin and von Willebrand factor.16 These in vitro results indicate that CD133+ renal cells might be pluri-potent, having the capacity to differentiate into either tubular cells or vascular cells if presented with the appropriate cues. To explore this poten-tial in vivo, the investigators intra venously injected fluorescently labeled CD133+ cells into mice that had been given an intramuscular injec-tion of glycerol to induce rhabdomyolysis and sub sequent myoglobin-mediated acute renal failure. Examination of the kidneys of these mice 3 days after CD133+ cell injection revealed that the transplanted cells were proliferating and had been incorporated into cortical proximal and distal tubules. So, CD133+ cells derived from the renal inter stitium might also have the capacity to differ-entiate toward an epithelial lineage in vivo. As the human kidney specimens used to derive these cells only included the renal cortex, it is unclear whether the CD133+ cells described in this study16 are the same as the slow-cycling cells that were found primarily in the papilla of the rodent.7

Other potential surface markers of renal progenitor cells have been identified using gene expression profiling of mesenchymal cells from embryonic kidney. Challen and colleagues found that 21 genes are selectively upregulated in cells destined to differentiate into renal structures. Two of these, CD24 and cadherin-11, are surface proteins that might be useful for isolation of viable progenitor cells from the adult kidney.18 Cells expressing CD24 were incorporated into newly forming tubules, whereas cadherin-11 was expressed primarily on cells that formed the interstitium.

BONE MARROW CELLS IN KIDNEY REPAIRIn addition to the repair capabilities of the tubular cell itself, and the possibility that endo genous renal stem cells participate in repair, studies in other organ systems have raised the possibility that adult stem cells from the bone marrow might



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participate in repair following tubular injury. The bone marrow contains two major categories of cells, the hematopoietic cell lineages and the STROMAL CELLS. Hematopoietic cells include the pluripotent hematopoietic stem cells (HSCs) and their progeny from which all the cellular blood elements are derived. These elements are collec-tively termed ‘lineage-positive cells’, and include the precursors of polymorphonuclear leukocytes, T cells, B cells, macrophages, megakaryocytes and erythrocytes. MSCs are less well characterized and their functional role in the bone marrow is not as well understood as that of HSCs. They comprise a heterogeneous group of cells thought to be crucial to maintenance of an environment conducive to survival and maturation of HSCs. Individual cells from this stromal population can also differentiate into other cell types such as adipose, muscle and bone (Figure 2).19,20 Several groups have isolated MSCs by exploiting their capacity to adhere to plastic tissue culture dishes and proliferate in response to serum.19,21 Unfortunately, there is currently no clear consensus on how many individual cell types constitute MSCs, how MSCs should be isolated and purified, or even which MSCs are actually stem cells capable of asymmetric division.

Numerous groups have shown that cells residing in the bone marrow have an un expected degree of plasticity. By transplanting a single male bone-marrow stem cell (BMSC) into a lethally irradiated female mouse, Krause and colleagues showed that all the blood elements were reconstituted, and that small numbers of Y-chromosome-positive epithelial cells were present in the lungs, liver, intestine and skin of the surviving animals.22 These donor-derived cells appeared to have been functionally incorporated into at least some of the recipient organs; for example, those in the lung expressed surfactant B. Similar studies have indicated that cells derived from the bone marrow can differentiate into, or fuse with, other cells including hepatocytes, pancreatic islet cells, endothelial cells and cardiac muscle cells.23–26 Nevertheless, the true level of plasticity of bone marrow-derived cells remains controversial, as other groups have been unable to reproduce some of these findings.27,28

Mobilization of bone marrow-derived cells to the kidney after injuryThe studies described above have attracted substantial attention because they indicate that bone marrow might harbor significant

numbers of cells that, if mobilized appropriately into the circulation, could augment the organ repair process. Renal injury itself can cause a modest increase in the number of circulating bone marrow-derived cells29–31 (although at least one group failed to detect an appreciable increase).32 The mechanism of this mobilization of bone marrow-derived cells is not fully under-stood. Zhang and colleagues have shown that the HSC-mobilizing cytokine granulocyte colony-stimulating factor (G-CSF) is upregulated in the circulation and renal tubule following ischemia–reperfusion injury.33 In addition, Togel and colleagues demonstrated that another stem cell-mobilizing factor, stromal cell-derived factor-1 (SDF-1), is upregulated in the kidney after ischemic injury. SDF-1 can induce stem cell mobilization and homing via activation of its receptor CXCR4.31 It should be noted, however, that several cytokines expressed following ischemic renal injury in rodents (including

HSC lin–, c-kit+, Sca-1+, CD34+

Lineage-positive cells lin+, CD34–, CD45+

MSC lin–, c-kit– (Sca-1+/–, Stro-1+)

Differentiation/fusion

HepatocytesIslet cells

Endothelialprogenitor

cells

OsteoblastsChondrocytes

Adipocytes

AngioblastsNeuronal cellsHepatocytes

MAPCs

Red blood cells Lymphocytes Macrophages Megakaryocytes

2 3 4

1

5

Neutrophils

Figure 2 Proposed differentiation capacities of bone-marrow stem cells. Bone marrow contains both hematopoietic stem cells (HSCs) and bone marrow stromal cells (also known as mesenchymal stem cells; MSCs). HSCs are typically described as expressing no lineage-specific markers (lin–), but do express c-Kit, Sca-1, and later CD34, on their surface. Their primary function is as a stem cell reservoir for the lineage-positive precursors for each of the blood elements (1). Studies from some groups indicate that HSCs might also be capable of differentiating into non-blood cells, such as endothelial cells, hepatocytes or pancreatic islet cells (2 and 3). This potential is less well understood; it could reflect fusion of the HSC with an existing cell or the true capacity of transdifferentiation. MSCs are a heterogeneous group of adherent cells that can be cultured from bone marrow aspirates. These cells are believed to contribute to the HSC niche in bone marrow, but can also differentiate to express markers typical of osteoblasts, adipocytes and chondrocytes in vitro (4). It has been reported that occasional clones of MSCs, called multipotent adult progenitor cells (MAPCs),25 can acquire the capacity to be passaged indefinitely and to differentiate into other cell types including neuronal cells, hepatocytes and angioblasts (5).

GLOSSARYSTROMAL CELLSConnective tissue cells of an organ found in the loose connective tissue; usually associated with the uterine mucosa, ovary and hematopoietic system



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Cxcl-1, interleukin-1 and interleukin-6) induce mature inflammatory cells to home to the kidney.34–37 In fact, most bone marrow-derived cells detected in the rodent kidney in the first few days after ischemia–reperfusion injury are leuko-cytes and T cells located in the renal interstitium. Most researchers are of the opinion that these cells actually exacerbate acute renal failure.36–39 The roles of individual factors in preferentially mobilizing inflammatory cells that might worsen tubule injury versus that of other bone marrow-derived cells that could augment tubule repair remain to be elucidated.

Bone marrow-derived cells might populate the renal tubuleTwo studies in humans support the idea that some bone marrow-derived cells which home to the kidney are not inflammatory cells, and can incorporate into the renal tubule. In the tubules of renal biopsy specimens from eight male patients transplanted with female kidneys, Poulsom and co-workers found Y-chromosome-positive cells within the tubules that co-expressed epi thelial markers.40 They noted wide variation in the proportion of these Y-chromosome-positive tubular cells in different biopsy specimens, ranging from 1.8% to 20% of the tubular cells examined. Similarly, Gupta et al. found small numbers of Y-chromosome-positive tubular cells in renal biopsies taken from two men trans-planted with female kidneys.41 These cells—less than 1% of the tubular cells examined—were classified as epithelial because they expressed the epithelial marker cytokeratin but did not express CD45, a membrane protein found on nearly all nucleated hematopoietic cells. Furthermore, in this small study, the investigators only detected Y-chromosome-positive tubular cells in kidneys from patients diagnosed with acute tubular necrosis following transplantation, indicating that tubular injury might specifically mobilize or direct bone marrow-derived cells to the kidney during repair.

The idea that bone marrow contains cells that home to the tubular epithelium has been further explored in mice. Poulsom et al. found that transplantation of male bone marrow into lethally irradiated female mice resulted in the appearance of a small proportion (approxi-mately 5%) of Y-chromosome-positive cells in the recipient animal’s tubular epithelium.40 More recently, the same group determined that the number of Y-chromosome-positive

tubular cells increases following folic acid-induced tubular injury in the recipient mouse, and that some of these cells undergo division within the tubule. They note, however, that most cells involved in tubule repair seem to be Y-chromosome-negative endogenous tubular cells.42 The finding that proliferation of endo-genous tubular cells rather than influx of bone marrow-derived cells provides the bulk of cells involved in tubule repair is supported by the work of Lin et al.43 and Duffield and colleagues.32

In a separate study, transplantation of puri-fied MSCs expressing green fluorescent protein (GFP) into mice in the absence of injury resulted in the appearance of GFP-positive cells in several organs, including liver, lung, muscle and kidney.44 In the kidney these cells were sparsely distributed, being present in only 2 of 13 mice examined and at a frequency of 5–10 cells per kidney section. The cells had a tubular epithelial morphology and bound the tubule-specific lectins from Ricinus communis and Lotus tetragonolobus.

Yokoo and colleagues conducted a particularly interesting study of the capacity of bone marrow-derived cells to differentiate into tubular cells.45 They injected cultured adult human MSCs into embryonic rats to determine the capacity of MSCs to be reprogrammed into renal cells in vivo. They found that if the cells were injected in the vicinity of the embryonic kidney, they were incorporated into the developing tubules and glomerulus, and expressed appropriate tubule-specific markers (aquaporin-1 and NBC-) and glomerulus-specific markers (nephrin and podocin). This process was further augmented if the MSCs were induced to express the embryo nic kidney growth factor glial-derived neurotrophic factor before transplantation.

Two groups, including our own, have used purified bone marrow cells transgenically expressing bacterial β-galactosidase to inves-tigate the role of renal injury in regulating reprogramming of BMSCs.29,46 The cells were transplanted into mice that had been subjected to transient renal ischemia and reperfusion. Both groups found that, after injury and transplantation, mice that had undergone renal ischemia and reperfusion had a signifi-cant proportion of tubules containing at least one β-galactosidase-positive cell, unlike the sham-operated control mice. As these cells co-expressed proximal tubule markers such as

GLOSSARYNBC-1An electrogenic sodium-bicarbonate co-transporter



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megalin and the type II Na/Pi co-transporter, but did not display blood cell markers such as CD45, both groups concluded that they were bone marrow cells that had differentiated into, or fused with, tubular epithelial cells. It was proposed that these cells participate in the repair process after ischemic renal injury.

Careful examination of our β-galactosidase-stained sections revealed the absence of β-galactosidase-positive interstitial inflamma-tory cells.29 By contrast, other studies have shown that lymphocytes, leukocytes and macrophages infiltrate the interstitium of the kidney following ischemia–reperfusion injury.38,47 To pursue this surprising observation, we repeated our ischemia–reperfusion experiments in female mice transplanted with male bone marrow-derived cells. In sections of kidney from these animals, Y-chromosome-positive cells were common in the renal interstitium, mostly in the outer medulla where the majority of tubular injury occurs in this model. By contrast, only rarely were tubular cells found to contain the Y chromosome (Figure 3). These cells were observed in the outer medulla of injured kidneys, and accounted for less than 0.01% of the tubular epithelial cell population that we examined in that region.

Similarly, Lin and co-workers noted that transplantation of male bone marrow into female recipients resulted in a substantially lower proportion of bone marrow-derived Y-chromosome-positive cells in the tubules after ischemic injury than they had detected with β-galactosidase staining (only 8.3% of tubules contained cells that were Y-chromosome-positive, compared with nearly 80% that contained at least one β-galactosidase-positive cell).46 These results indicate a substantial difference between the proportion of tubular cells detected with β-galactosidase staining following transplantation of β-galactosidase-positive bone marrow cells, and the proportion detected with Y-chromosome staining after transplantation of male bone marrow. It has been suggested that this differ-ence might be due to pH-dependent detection of endogenous β-galactosidase activity or uptake of the β-galactosidase enzyme by the injured endogenous tubular cells.32,48

The rarity of the Y-chromosome-positive tubular epithelial cells that we observed raises the question of whether bone marrow cells truly differentiate into tubular epithelial cells in vivo, or whether the rare Y-chromosome-positive cells could be artifacts of imaging. In a model of

folic acid-induced tubular injury in female mice transplanted with male bone marrow, Szczypka et al. failed to detect marrow-derived tubular cells in kidney sections. The authors were able to isolate and culture a few cells from these kidneys that were Y-chromosome-positive and expressed epithelial markers.49 Similarly, Duffield and co-workers failed to find examples of bone marrow-derived renal tubular cells in two mouse models of bone marrow transplantation: ischemic renal injury of male-to-female transplant recipient, and GFP-transgenic bone marrow donation to wild-type recipient.32

Conflicting results reported by different groups, as well as conflicting results obtained when the same group compared two different methods of tracking bone marrow-derived cells, have generated uncertainty as to whether cells from the bone marrow actually become tubular cells in vivo. Purification and charac-terization of bone marrow-derived kidney cells at both the phenotypic and genetic levels will be needed to convincingly demonstrate that they

A B C

Figure 3 Bone-marrow stem cells may rarely contribute to renal tubule repair. Female Bl/6 mice underwent 25 min of unilateral renal ischemia, followed by reperfusion and transplantation of 5 × 105 male lineage-negative bone marrow cells. Animals were sacrificed on day 7 and kidney sections analyzed for nuclei (stained blue with DAPI) expressing the Y chromosome (red). Multiple sections from the ischemically injured kidneys and from the contralateral control kidneys of five mice were examined. The majority of Y-chromosome-positive cells were located within the renal interstitium in the region of maximal tubular injury (outer medulla). Rare cells that were Y-chromosome-positive and also appeared to express tubule-specific markers were found in the outer medullary tubules in two out of the five mice examined. No Y-chromosome-positive tubular cells were found in the contralateral kidneys. The two best examples of Y-chromosome-positive cells that seem to express a tubule-specific marker are shown. (A) Y-chromosome-positive cell co-expressing the proximal tubule marker megalin (green) at its apical surface (arrow). (B) Y-chromosome-positive cell expressing the thick ascending limb marker Tamm-Horsfall protein (arrow). Note that most Y-chromosome-positive cells are surrounding the injured tubule, and are not within the tubule (arrowheads). (C) Sections of injured kidney stained with a cocktail of antibodies directed against all lineage-positive blood cells (green) reveal that although most Y-chromosome-positive cells in the renal interstitium are inflammatory cells (arrows), occasional non-inflammatory bone marrow-derived cells are seen close to the injured tubules (arrowheads).



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are in fact functional tubular epithelial cells. On the basis of most current data, it seems that even if adult bone marrow-derived cells retain the capacity to differentiate into, or fuse with, renal tubular cells, it is an uncommon event and does not have a predominant role in the acute tubule regenerative process.

Functional effects of bone marrow-derived cells in renal repair In parallel with these investigations into the phenotypic characteristics of bone marrow-derived cells in the kidney, several groups have begun to explore the capacity of bone marrow cells to promote tubule repair after injury. Two approaches have been employed by these inves-tigators: infusion of large numbers of bone marrow cells from a donor animal, and mobiliza-tion of endogenous bone marrow cells into the circulation.

Infusion of bone marrow-derived cellsTo determine if cells from the bone marrow have a functional role in the renal recovery process, we examined BUN levels and renal histology in control mice and mice whose bone marrow had been ablated by lethal irradiation, followed in each case by bilateral ischemia–reperfusion. We found that bone marrow ablation signifi-cantly increased the degree and duration of the rise in BUN levels after ischemia–reperfusion, and resulted in more severe histological injury 7 days later.29 When ablated mice received an infusion of 5 × 105 lineage-negative bone marrow cells (containing small numbers of both HSCs and MSCs, but no mature inflamma-tory cells) after injury, the rise in BUN and the degree of patho logic injury were indistinguish-able from that in the injured control animals. Although infusion of the bone marrow cells did not improve renal function to greater than control levels, it did limit the initial rise in BUN, indicating a possible protective role of lineage-negative bone marrow cells that was lost from bone marrow-ablated animals.

Morigi and co-workers separated bone marrow cells into HSCs and MSCs, and compared the effects of these two cell popula-tions in a cisplatin-induced murine model of tubular injury.50 As MSCs are a rare compo-nent of adult bone marrow (making up less than 0.01% of the total bone marrow cell population), the cells were first amplified in culture for several weeks. These experiments,

performed in the absence of bone marrow ablation, showed that infusion of 2 × 105 purified MSCs from a male mouse into a female mouse markedly diminished the initial rise in BUN, minimized tubular damage and accel-erated pro liferation of tubule cells following cisplatin injection. Purified HSCs had little beneficial effect. Morigi et al. reported the presence of Y-chromosome-positive cells in the injured tubules of the MSC-transplanted mice. These were classified as epithelial on the basis of morphology and expression of the tubule lectin from Lens culinaris. The finding that MSCs can protect renal tubules from acute injury and might be directly incor-porated into the regenerating tubule has been supported by a study from Herrera et al. in a glycerol-induced murine rhabdomyolysis model of acute renal failure.51

The capacity of ex vivo-amplified MSCs to protect the rodent kidney from ischemia–reperfusion injury has been confirmed by two other groups, but with an important difference from the earlier studies.32,52 Neither Togel and colleagues nor Duffield et al. were able to detect direct engraftment of renal tubules by MSCs. The study by Togel et al. showed that infused MSCs partially reverse the increase in creatinine in rats subjected to ischemia–reperfusion injury. These MSCs were, however, only transiently present in the renal vasculature, and were not detected within the renal parenchyma for up to 3 days after infusion.52 Examination of gene expression in the MSC-treated kidneys revealed a decrease in proinflammatory cytokines and an increase in several growth factors, including basic fibroblast growth factor and transforming growth factor-α. So, these authors concluded that MSCs protect the kidney via paracrine and/or endocrine effects rather than through direct engraftment in the renal tubules. Similarly, Duffield et al. failed to detect infused MSCs in the mouse kidney, even though MSCs that had been cultured on a basement-membrane prep-aration substantially counteracted the rise in creatinine after ischemia–reperfusion.32 These authors found that proliferation of endogenous tubular cells, rather than engraftment of trans-planted MSCs, accounted for the recovery of renal function.

Mobilization of bone marrow-derived cellsSeveral groups have explored the mobilization of endogenous BMSCs as a therapeutic option



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for acute renal failure. Historically, bone marrow cell-mobilizing regimens were devel-oped to promote an increase in the number of circulating HSCs and allow peripheral stem cell harvest for bone marrow transplanta tion. Several of these regimens have been used in mice subjected to acute tubular injury, with varying results. Togel and colleagues found that attempts to mobilize HSCs with cyclo-phosphamide and G-CSF resulted in an increased inflammatory response in the ischem-ically injured kidney and actually worsen ed the progression of acute renal failure in mice subjected to ischemia–reperfusion.30 This response was thought to be due to the large increase in circulating leukocyte levels that occurs after G-CSF administration.

By contrast, two groups have found that HSC mobilization with either stem cell factor (SCF) plus G-CSF or macrophage colony-stimulating factor (M-CSF) plus G-CSF protects mice from acute renal failure.53,54 Iwasaki et al. reported that G-CSF plus M-CSF accelerates the drop in BUN and creatinine 4 days after cisplatin injection. Increased numbers of bone marrow-derived cells were found in the renal tubules of these mice.54 Interestingly, Stokman and colleagues failed to detect incorporation of mobilized bone marrow-derived cells into the ischemically injured tubular epithelium after G-CSF plus SCF injection. These authors attributed the protective effect to a decrease in inflammatory cell infiltration.53 This assertion is in concert with the gene expression studies by Togel and colleagues of ischemically injured kidneys infused with MSCs.52

It is important to note that it is not clear whether these regimens mobilize MSCs in addition to HSCs, or whether MSCs are physio-logically mobilized to exit the bone marrow in significant numbers under any conditions. Furthermore, each of these regimens included G-CSF, a factor that directly affects cell types other than BMSCs. For example, cardio myocytes express the G-CSF receptor and respond to G-CSF by inducing anti-apoptotic pathways. This process might account for at least some of the cardioprotective effect of G-CSF detected in rodent models of myocardial infarction.55 The role of G-CSF in direct regulation of renal tubular cell responses to injury is unknown. Further exploration is required before we fully understand the protective effects of these stem cell-mobilizing regimens.

Current understanding of the role of bone marrow cells in kidney tubule repairMany of the studies described above were undertaken in the belief that the most likely function of bone marrow cells in the injured kidney would be to repopulate the denuded tubule, and that the HSC was the cell type most likely to be responsible for repopula-tion. Data collected to date, however, support several strong arguments against this hypoth-esis. First, although the quali tative question of whether bone marrow cells can ever become functional tubular cells in vivo remains in doubt, current data consistently show that the numbers of such cells, if they exist, are far too low to explain the overall improvement in renal function following infusion of BMSCs. It is therefore highly unlikely that the functional effects of bone marrow-derived cells in the kidney can be explained by direct repopulation of the tubule. It is, however, worth noting that if bone marrow-derived cells are functionally incorporated into the tubule in low numbers, they could theoretically serve as a reservoir for more significant tubule repopulation following subsequent episodes of injury.

A second argument against the idea that direct repopulation of the tubule accounts for the effects of bone marrow-derived cells is the pattern of change in renal function observed in animals that receive these cells. If bone marrow cells acted primarily by being func-tionally incorporated into denuded tubules, one would expect the initial drop in glomerular filtration rate (GFR) following acute injury to be un affected by infusion or mobilization of such cells, and the rate of recovery to be greatly increased. By contrast, virtually every func-tional study has shown that the improvement observed following infusion of bone marrow cells is primarily due to prevention of the initial decline in GFR.29,32,52 This is most consistent with a protective effect of the bone marrow cells. It does not, however, rule out an addi-tional effect of enhancing the proliferation of endogenous cells that survive the initial insult, or stimulating the proliferation or differentia-tion (or both) of endogenous renal progenitor cells (Figure 4).

The mechanism of the protective effect of bone marrow cells is not yet well understood. Duffield and co-workers observed that small numbers of medullary vascular endothelial cells were replaced by bone marrow-derived cells



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following ischemia–reperfusion.32 Although this raises the possibility that some bone marrow-derived cells protect or maintain the vascular endothelium, thereby improving medullary blood flow and decreasing overall ischemia time, failure to detect infused MSCs in the injured kidney indicates that the primary protective effect of these cells is more likely to be endo-crine or paracrine in nature. The demonstration by Togel and co-workers that MSCs can secrete several growth factors, including VEGF, HGF and IGF-1, strongly supports this possibility.52 In addition, MSCs are known to inhibit inflam-matory responses,56 and thus might ameliorate acute injury by preventing inflammatory cell infiltration, thereby inhibiting apoptotic tubular cell death.39,52

Unequivocal identification of the bone marrow cell subtype or subtypes that exerts the protective effect on the kidney is yet to be achieved. Current data indicate that MSCs, rather than HSCs, have the most important role in tubule protection, but MSCs are believed to be quite rare in the bone marrow and are

not yet a well-defined cell population. Even the manner and duration of ex vivo amplifi-cation of MSCs might be essential to their protective efficacy.32,52

Therapeutic potential of bone-marrow stem cells in acute renal failureThe great interest in bone marrow-derived cells as therapeutic tools has centered around their availability relative to organ-specific stem cells. Theoretically, bone marrow aspirates or pharmacologic regimens that mobilize bone marrow-derived cells into the circulation could make available large numbers of cells with the capacity to aid organ repair. Unfortunately, recent advances in our understanding of the role of adult stem cells in tubule repair indicate that this approach could be too simplistic.

Pharmacologic regimens aimed at mobi-lizing BMSCs are likely to mobilize many other cell types, enhancing inflammatory cell infil-tration of the kidney and possibly worsen ing the outcome following acute injury. It is not known whether MSCs can be mobilized from the bone marrow, or whether the bone marrow even contains sufficient numbers of MSCs to confer the protective effects observed after ex vivo amplification and infusion. Studies published to date indicate that the number of MSCs infused could be a critical determinant of their capacity to protect the kidney. A recent study by Lin and colleagues failed to detect any improvement in renal function after infusion of 1 × 106 whole bone marrow cells (predicted to contain 5 × 104 lineage-negative cells and only a few hundred MSCs) at the time of ischemia–reperfusion.43 In our experiments, infusion of 5 × 105 lineage-negative cells (predicted to contain several thousand MSCs) resulted in only partial improvement.29 Significant protection has only been demonstrated when between 2 × 105 and 5 × 105 purified MSCs were infused.32,50 Although the recent studies using SCF, G-CSF and M-CSF for stem cell mobiliza-tion are encouraging, reproduction of these results and careful exploration of the exact effects on MSC mobilization versus inflam-matory cell mobilization will be needed before such regimens can be considered for clinical use in acute renal failure. As previously noted, factors such as G-CSF and erythro poietin that are used to mobilize stem cells might also exert protective effects directly on renal epithelial cells, making it difficult to determine if the

Bone-marrow stem cell

Endothelial cells

Differentiation/fusion

Proliferation/differentiationof adult renalstem cells

Protectionviaendocrineeffect

Cell–cell/paracrineinteractions

ProtectionDifferentiationProliferation

Epithelial cells

Regeneration of functional renal tubule

1

2 3 4

Figure 4 Possible roles for bone-marrow stem cells in facilitating renal repair. Current data support several putative roles for bone marrow-derived cells in recovery from acute renal failure. Some studies indicate that bone-marrow stem cells (BMSCs) can differentiate into small numbers of tubular epithelial cells, peritubular vascular endothelial cells, or both (1). A second possibility is that BMSCs secrete factors that can either augment the capacity of resident renal stem cells to proliferate and enter the tubule during the repair process (2), or that act to prevent tubular cell death and/or enhance proliferation by an endocrine effect on the tubular cell itself, or suppression of inflammatory responses (3). Finally, BMSCs that enter the kidney and surround the injured tubules could act in a paracrine or direct fashion to mediate cell protection and proliferation (4).



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improved outcomes observed in animal studies are due to the tubular cell effects or stem cell mobilization.55,57,58

A second approach is to amplify adult stem cells in the laboratory and then reintroduce them into recipients at the time of injury. Although this approach is practical in animal models of renal injury, it is much less attractive as a therapeutic tool in humans. It would require patients to undergo bone marrow harvest or renal biopsy to obtain MSCs or renal stem cells, respectively, weeks before the injury in order to allow sufficient amplification of the cells. Alternatively, if MSCs could be collected from healthy donors, and amplified and maintained in long-term culture, large numbers of allo-geneic stem cells would be available for trans-plantation at the time of injury. This approach has the disadvantage that immunosuppressive therapy might be required to prevent rejection of the transplanted cells.

The third, and potentially most interesting approach, would be to determine exactly how MSCs protect the renal tubule from injury, and then to mimic this protective or reparative effect pharmacologically. If the primary role of MSCs is to secrete a cytokine or growth factor in response to injury, then the cells themselves might not be required. By determining the exact nature of the MSCs’ protective action and defining the culture conditions necessary to support this protective effect, we should be able to identify this factor (or factors) and either administer it directly or develop pharmaco-logic strategies to stimulate its production by endo genous cells.

CONCLUSIONThe field of adult stem cell research continues to both tantalize and frustrate investigators as conflicting results are reported. At present, it seems that the bone marrow probably harbors a population of poorly understood cells, the MSCs, that has a protective effect in animal models of acute renal failure when infused in large numbers. It is also possible that the kidney contains a population of endogenous tubule progenitor cells that participates in tubule regeneration. Further study of both of these cell types is warranted, with the long-term goal of develop ing strategies that will minimize the severity of tubular injury and increase the capacity for tubular repair in patients with acute renal failure.

KEY POINTS■ A population of tubule progenitor cells may persist in the renal interstitium of adults

■ Stem cells from the bone marrow mobilize to the kidney after injury

■ Mobilizing or infusing bone-marrow stem cells (BMSCs) can have a protective effect in animal models of acute renal failure

■ It is not clear whether BMSCs differentiate to form tubule cells, or fuse with existing tubule elements

■ The low frequency of BMSC differentiation and fusion make it unlikely that these processes account for functional recovery of injured tubules

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AcknowledgmentsThe author would like to thank M Egalka for the images in Figure 3, and D Krause for her helpful comments on the manuscript.

Competing interestsThe author declared he has no competing interests.



adult stem cells in the repair of the injured renal tubule

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