Regrow or Repair: Potential Regenerative Therapies for the Kidney
Melissa H. Little
Institute for Molecular Bioscience, University of Queensland, St. Lucia, Queensland, Australia
Address correspondence to: Prof. Melissa H. Little, Institute for Molecular Bioscience, Queensland Bioscience Precinct, University of Queensland, St. Lucia, Brisbane, Qld, 4072, Australia. Phone: +61-7-3346-2054; Fax: +61-7-3346-2101; m.little{at}imb.uq.edu.au; Web: www.imb.uq.edu.au
Regenerative medicine is being heralded in a similar way asgene therapy was some 15 yr ago. It is an area of intense excitementand potential, as well as myth and disinformation. However,with the increasing rate of end-stage renal failure and limitedalternatives for its treatment, we must begin to investigateseriously potential regenerative approaches for the kidney.This review defines which regenerative options there might befor renal disease, summarizes the progress that has been madeto date, and investigates some of the unique obstacles to suchtreatments that the kidney presents. The options discussed includein situ organ repair via bone marrow recruitment or dedifferentiation;ex vivo stem cell therapies, including both autologous and nonautologousoptions; and bioengineering approaches for the creation of areplacement organ.
The term regenerative medicine straddles cell biology, matrixbiology, and bioengineering with the objective to regrow orrepair a damaged organ or tissue type. It can be defined asthe use of cells for the treatment of disease and encompassesboth organ repair and the de novo regeneration of an entireorgan (Figure 1). Organ repair can be delivered in situ or exvivo. The simplest and most pharmacologically attractive strategyfor organ repair in situ is the delivery of a soluble reparativefactor that improves the ability of the kidney to repair itself.Although such an approach may involve the understanding of thefactors that are produced by stem cells, this is not a cellulartherapy and is not dealt with in this review. Other in situpossibilities include the recruitment of stem cells to the kidneyto elicit repair and the induction of dedifferentiation of residentrenal cells. Whereas some regard in situ approaches as morelikely to be successful for an architecturally and anatomicallyconstrained organ such as the kidney, the other approach isthe ex vivo culture of stem cells for redelivery to the damagedkidney. This might involve autologous or nonautologous stemcells from a variety of sources. Finally, a bioengineering approachthat relies on cells, factors, and matrix may be achievable.Although seemingly the most difficult, it may be the more feasibleapproach for genetic conditions such as polycystic kidney disease.The matrix of options illustrated in Figure 1 could be drawnup for almost any organ. This review investigates each optionand relates it to the function and the structure of the kidneyso as to examine its feasibility and identify the key obstaclesto delivery.
Figure 1. Potential therapeutic options for the treatment of renal disease. The options are presented as predominantly pharmaceutical to predominantly biotechnological from left to right. Illustration by Josh Gramling—Gramling Medical Illustration.
Setting the Stage: Normal Kidney Development and Regeneration in Vertebrates
Regenerative biology draws on an understanding of normal developmentalprocesses. Understanding the molecular basis of kidney developmentwill be the key to the development of regenerative therapiesfor chronic renal disease. During mammalian development, threeseparate excretory organs develop: The pronephros, the mesonephros,and the metanephros. In mammals, it is the paired metanephroithat persist postnatally and constitute the permanent kidney.The permanent kidney arises via reciprocal interactions betweentwo tissues, the ureteric bud (UB) and the metanephric mesenchyme(MM), the latter arising from the intermediate mesoderm (IM)(1). The UB gives rise to the collecting ducts and the ureter.The MM, which shows much broader potential and gives rise toall other elements of the nephrons, the interstitium, and thevasculature, is regarded as the renal progenitor population(2). As the UB reaches the MM, signals from the tips of thebranching UB induce areas of adjacent MM to aggregate and undergoa mesenchyme-to-epithelial (MET) transition. Each MET eventrepresents the birth of a new nephron with the first nephrons"born" in the center of the MM. The peripheral MM, which hasnot yet undergone induction, is referred to as the nephrogeniczone. Nephrogenesis in humans is complete by week 36 of gestation(3), whereas it continues for 1 to 2 wk after birth in the mouseand the rat. At that time, it is assumed that the peripheralnephrogenic zone is exhausted.
Can the kidney regenerate? In simple vertebrates, includingfish and amphibians, metanephroi do not form and the permanentexcretory unit is the mesonephros. Elasmobranchs (sharks, rays,and skates) constitute a unique example of "kidney" regeneration;their mesonephroi can undergo accelerated nephrogenesis afterpartial ablation to replace the missing parts (4). In the mammal,partial nephrectomy stimulates hypertrophy of remaining tissue,even in the contralateral kidney, but not the generation ofnew nephrons (5). However, whereas the resection of an adultkidney does not lead to the regeneration achieved in the liver,the mammalian kidney shares with the majority of organs theability to repopulate and repair structures that have sustainedsome degree of injury. This process, termed cellular repair,can be achieved by reentry into mitosis and proliferation ofneighboring cells. As a result, the kidney can undergo significantremodeling in response to acute damage. For example, obstructionof the ureter can result in the near destruction of the kidneymedulla, but once the obstruction is removed, there is a rapidprocess of reconstruction and repair that will regenerate thetubules of the medulla without forming new nephrons (6). Ithas been proposed that the cells that elicit such repair comefrom interstitial cell transdifferentiation (7), tubular celldedifferentiation and migration into the areas of damage beforeredifferentiation (8,9), the recruitment of stem cells fromthe bone marrow (10–14), or the generation of new tubularcells from an endogenous renal stem cell population (reviewedin reference [15]). Which of these is primarily responsiblefor the cellular repair that is observed after acute damagehas not been proved definitively using lineage tracing. However,the mammalian kidney seems to have a very limited potentialfor structural repair or true regeneration. While nephrogenesisis occurring in the fetus, there is evidence that a systemichumoral response to nephrectomy allows the enhanced nephrogenesisof the remaining organ (16). However, nephrogenesis in mammalsceases just before or shortly after birth (3), and the birthof new nephrons has never been reported after this point intime. Chronic injury of the kidney, which is responsible forthe majority of cases of end-stage renal failure, results inirreversible glomerular and tubular damage and resultant lossof renal function. Hence, mammalian kidneys respond to chronicdamage by fibrosis, scarring, and irreversible functional loss.
Can we improve the capacity of the kidney for cellular repair?The ability of cells that originate from bone marrow to moveinto distant sites within the body, including the kidney, isnow well recognized. Reports have suggested that these cellscan transdifferentiate into tubular epithelial cells (12), mesangialcells (11,13,14), glomerular endothelial cells (17,18), andeven podocytes (12). As in most organs, bone marrow–derivedcells (BMDC) appear in the kidney in response to damage. Thelineage of these cells is unclear, and their ability to elicittransdifferentiation is controversial because the possibilityof cell fusion has not always been eliminated (19) (Figure 2).The use of lineage tracing has been critical to differentiatingthese two possibilities. In the case of the muscle, there isevidence from studies in which bone marrow was derived fromLysM-Cre mice that it is the monocytic lineage that is recruitedand fuses with cells in the target organ (20). This lineagegives rise to the macrophages, which express proteins that areinvolved in fusion processes. This does not answer the questionof the relative value of this fusion process. In the brain,BMDC can fuse with Purkinje cells (21), a cell type that ispresumed to be unable to divide, possibly leading to a "rejuvenation"of such terminally differentiated cell types. Certainly, thefunctional outcome of BMDC recruitment must always be assessed.
Figure 2. Stem cells, whether recruited to the kidney from distant organs or delivered to the kidney after ex vivo expansion of an isolated stem cell population, may contribute to repair via the production of specific cyto/chemokines or growth factors (humoral response), transdifferentiation into specific renal cell types, or cell fusion. It is not always clear which of these events occurred or which event was of the greatest functional significance. Although it has been shown to occur, the regulation of stem cell recruitment to the kidney has not been elucidated. There is increasing evidence for a humoral reparative role being provided by introduced stem cells, but the nature of this response also remains to be investigated. Illustration by Josh Gramling—Gramling Medical Illustration.
In the context of the kidney, several studies have examinedthe recruitment of BMDC to kidney in response to damage signalsand their transdifferentiative and reparative capacity. Theinjury models used include ischemia-reperfusion injury (22),folic acid–induced acute tubular injury (23,24), unilateralureteric obstruction (25), and anti-Thy1 antibody–mediatedglomerulonephritis (13). Bone marrow transplantation into HIgAmice, which have glomerulonephritis, improved renal functionin these mice (26). In the studies in which careful quantificationof recruitment to the tubular epithelium has been performed,donor-derived bone marrow has contributed between 0.06 and 11%of the epithelial cells (22–24). This level does declinewith time. An initial recruitment level of 11% dropped to 0.67%at 28 d after ischemia with a concomitant increase in recruitmentto the interstitium (22). Two seminal papers in this area (22,23)disagreed on whether there was evidence for transdifferentiation,but both concluded that while BMDC recruitment occurs, repairis predominantly elicited via proliferation of endogenous renalcells. Duffield et al. (23) maintain that BDMC contribute aregenerative cytokine environment that may be important in theresulting functional repair (Figure 2). If this process couldbe recapitulated pharmacologically, then repair may occur withoutthe need for recruitment. Pretreatment of animals with stemcell factor and granulocyte colony-stimulating factor (granulocyteCSF) has been shown to improve recovery from ischemic injuryin the absence of transdifferentiation of BMDC (27), and thecombined pretreatment with granulocyte CSF and macrophage CSFprovides renoprotection from cisplatin-induced renal failure(28). It also may prove valuable to improve recruitment. Heldet al. (29) used a genetically induced model of chronic tubulardamage that involved hereditary tyrosinemia (mutations in fumarylacetoacetatehydrolase) and mutations in homogentisic acid dioxygenase andreported significant integration (50%) of introduced BMDC. Hence,a drive for the selection of wild-type cells considerably increasesthe regeneration process (29). More recently, recruitment andapparent podocytic transdifferentiation of male BMDC to theglomeruli of mice that lacked collagen43 has been reported (30).This is a model of Alport syndrome in which there is considerableshedding of protein through the damaged glomerular basementmembrane. Whereas podocytes have not been a reported site ofbone marrow recruitment in other experimental models, this studyclaimed a bone marrow origin for 10% of the podocytes in thesemice with a reduction in protein shedding and evidence of collagenreplacement within the basement membrane. In this case, accessmay have been increased as a result of the altered permeabilityof the basement membrane, but BMDC from mutant mice were notrecruited to the glomeruli of mutant recipients, suggestingan active selection for collagen-producing cells. In all ofthese reports of bone marrow recruitment to damaged kidneys,the lineage of the BMDC that were recruited has not been established.However, adoptive transfer of macrophages into a model of unilateralureteric obstruction significantly reduced fibrosis in the latestages of this damage state (25). This may have involved transdifferentiationor an altered immunologic response. What also has not been investigatedis whether the recruitment of BMDC is good or bad in cases ofchronic renal damage.
Controlled Dedifferentiation as a Treatment of Renal Disease
Can we repair a kidney by recapitulating development? Amongvertebrates, certain amphibians show a unique ability to regeneratecompletely complex organs or body parts (31). Salamanders, newts,and axolotls can reconstitute various anatomic structures suchas limbs, spinal cord, heart, tail, retina, lens, and upperand lower jaws. In the case of the limb, this process involvesdedifferentiation (i.e., loss of a specialized phenotype toreturn to a progenitor phenotype), proliferation of the resultingprimitive blastema, and then redifferentiation of cells in thevicinity of the injury (32) as opposed to the mobilization ofa stem cell population per se. Muscle fibers, Schwann cells,periosteal cells, and cells from the connective tissue undergodedifferentiation and then organize a blastema from which thenew limb arises (Figure 3A). Can this be applied in higher vertebrates?Regeneration within the skate mesonephros is a process thattakes place in an identified nephrogenic zone using a persistentfield of progenitors that can be recruited for regeneration(Figure 3B). Whether these progenitors represent stem cells,as defined as a long-term, self-renewing cell population, hasnot been established. In mammals, there is no persistent blastemain the adult (Figure 3C). In the absence of such a persistentpopulation of renal progenitors, could such a blastemal fieldbe generated via dedifferentiation in the mammalian kidney?In a recent review of the obstacles to limb regeneration inthe mammal (33), it was observed that mammalian limb cells lackthe response of reentry into S-phase in response to thrombin(even though this response still would be present if a mousecell were fused with that of a salamander), and their more compleximmune systems respond to damage via the production of fibrosisand the recruitment of inflammatory cells. Possibly as a resultof these differences, the production of the blastema that isrequired for regeneration does not occur, yet there are examplesof cell types even in humans that show enormous regenerativecapacities, together with more salamander-like properties suchas an ability to recommence cell division and dedifferentiateto regenerate. Oligodendrocyte precursor cells have been revertedto multipotential neural stem cells that are able to proliferateand to give rise to neurons, astrocytes, and oligodendrocytes(34). More striking, highly specialized multinucleated musclecells have been induced to dedifferentiate into mononucleatedmultipotent progenitor cells that are able to adopt the osteogenic,chondrogenic, adipogenic, and myogenic fates (35). In this case,the dedifferentiation was induced by ectopic expression of thetranscriptional repressor Msx1 in combination with growth factorstimulation. Finally, the mouse MRL strain has been shown tohave both a marked capacity not to scar and to restore normalmyocardial tissue without scarring through a process the authorsdescribe as similar to regeneration in amphibians (36). Howfeasible is dedifferentiation as a therapy? Postnatal cell turnoverin the kidney has never been examined thoroughly, but the cellularcomplexity of this organ suggests that a dedifferentiation intoblastema followed by redifferentiation for the purposes of regenerationwould need to be as complex as that seen in the salamander limb.Hence, we need to understand the blastemal progenitors thatgive rise to the kidney and to understand the process that longhas been observed in the kidney in response to short-term localdamage: The epithelial-to-mesenchymal transition of tubularcells. If able to be induced, then dedifferentiation might beevoked in situ or ex vivo (Figure 4). In situ dedifferentiationwould require controllable gene therapy to ensure a cessationof dedifferentiation and subsequent induction of regeneration,or it runs the risk of generating blastemal expansions as fora Wilms’ tumor.
Figure 3. Different approaches to regeneration and repair within vertebrates. (A) Regeneration in the salamander limb in response to resection involves the dedifferentiation of muscle, bone, and connective tissue elements to form an undifferentiated mitotic blastema. This blastema re-patterns and re-differentiates into a limb equivalent only to the region that has been resected. (B) The development of nephrons within the skate mesonephros involves an incorporation into the end of the mesonephric tubules. The maintenance of a persistent blastemal population allows for structural repair via continued nephrogenesis. (C) Development of nephrons in mammalian metanephroi utilizes a mesenchyme-to-epithelial transition from an exhaustible nephrogenic zone, preventing "structural" regeneration after the cessation of nephrogenesis. Illustration by Josh Gramling—Gramling Medical Illustration.
Figure 4. Cellular therapeutic options for the treatment of renal disease include in vivo and ex vivo options and may utilize autologous or nonautologous stem cells or the dedifferentiation of mature adult renal cells. These options currently are hypothetical. Illustration by Josh Gramling—Gramling Medical Illustration.
Can we elicit cellular repair in the kidney via the introductionof stem cells? The development of stem cell therapies for kidneyis in its infancy primarily because of the complexity of theorgan involved, the degree of damage present at the time ofdiagnosis, and the belief that kidney development ceases atbirth. Three sources of stem cells can be envisioned in thedevelopment of such treatments: (1) Renal adult stem cells,(2) nonrenal adult stem cells, and (3) embryonic stem (ES) cells.These options are depicted in Figure 4.
Renal Adult Stem Cells
The existence of multipotent adult stem cells that are criticalfor the ongoing turnover of the skin, bone marrow, stomach,intestine, and cornea have been known for a long time. Therenow is strong evidence for the existence of adult stem cellswith a much greater degree of plasticity in many organs. Thederivation of cells that display apparent pluripotency has nowbeen reported from many adult organs, including brain, bonemarrow, skin, and fat (37–41). Such observations suggestthat "stem cells" exist in all adult tissues. What does a renaladult stem cell look like, and where is it? Although many attemptshave been made to identify such a population, no definitivedata to date establish the existence of a long-term, self-renewingcell population with the capacity to generate distinct daughtercells with renal potential in the adult kidney. However, manyapproaches have been taken to look for such a population. Burrowand Wilson (42) reported the culture of nephrogenic zone cellsfrom the developing human kidney in media from a Wilms’tumor cell line. They termed these cells nephroblasts. The criticalcomponents of the conditioned medium were never identified,and a similar cell type has never been cultured successfullyfrom postnatal kidney. Kitamura et al. (43) screened for stemcell potential in various regions of the postnatal murine kidneyvia dissection of various nephron segments and culture afterdissociation to single cell. In this way, they defined a cellline that was derived from the S3 segment of the proximal tubules.This could be maintained long term without transformation andexpressed Pax2, Wnt4, and WT1. These cells seemed to contributeto renal tubules in a model of ischemia/reperfusion injury,but improvement in renal function was not assessed. Evidenceof clonogenic self-renewal was not presented. With the increasingamount of literature on the expression profile of the developingkidney across time and subcompartment, it may become possibleto dissect better the compartments, such as the nephrogeniczone and the cap mesenchyme, and identify cell surface markercombinations with which to search for a persistent fetal renalprogenitor. In an attempt to define the profile of a renal progenitorpopulation, expression profiling of the 10.5 d postcoitus mouseMM versus adjacent IM was performed (44). This identified thespecific expression of transmembrane proteins such as CD24aand cadherin11 that differentially marked the MM at that timepoint. It remains to be shown whether a CD24a+cadherin11+ populationpersists in the adult kidney, whether such cells self-renew,and whether they show any renal capacity when isolated fromadult tissue. On the basis of reports that CD133 marked hematopoieticstem cells/endothelial progenitors (45), Bussolati et al. (46)isolated CD133+ cells from the adult kidney to examine theirpotential as renal stem cells. These cells did not seem to bederived from the blood (CD34–CD45–), did expresssome mesenchymal stem cell (MSC) markers (CD29+, CD44+, andCD73+), but showed only limited differentiation capacity. However,they did express Pax2, homed to kidneys that were damaged viaintramuscular glycerol injection, and gave rise to endothelialand tubular epithelial cells within these kidneys. The clonogenicityof these cells was not established, but the authors hypothesizedthat these interstitial cells could act as a supply of replacementtubular cells or assist in revascularization after damage.
One way of looking for stem cells in solid organs is a shortadministration (pulse) of bromodeoxyuridine (BrdU) followedby a long chase period. This approach is based on the premisethat stem cells cycle slowly and, having incorporated BrdU intotheir DNA, will retain this label for a long time. Several groupshave used this approach to identify slow-cycling cells in thekidney, which may represent renal stem cells. The timing ofthe pulse, duration of the pulse, and length of the chase areimportant to the interpretation of the results. Maeshima etal. (47) identified BrdU-labeled cells in the renal tubules,which they termed renal progenitor-like tubular cells. Thesecells reenter mitosis in response to renal damage and turn intofibroblasts (48). However, they also show the potential to becomeproximal tubule and collecting duct cells and can form tubularstructures in vitro when cultured in collagen gel (49). Thetiming of the pulse (postnatal) and the length of the chase(2 wk) suggest that these cells are either differentiated tubularcells or tubular progenitors of limited potential. Indeed, carefulimmunohistologic analysis of cortical BrdU-labeled tubular cellssuggests that these cells are unlikely to represent stem cellsbecause they are identical to the surrounding terminally differentiatedrenal tubular cells (50). Oliver et al. (51) identified a populationof BrdU label–retaining cells within the papilla of thekidney. BrdU was pulsed during the first postnatal week, duringwhich nephrogenesis is continuing in the rodent, and the chaseendured for 2 mo, indicating long-term label retention. Thesecells can be cultured under hypoxic conditions to form aggregatesof nestin-positive cells, a marker in other stem cells types.In response to an ischemic insult, these cells seemed to reentermitosis rapidly, particularly within the outer medulla. Althoughmore likely to represent stem cells, clonogenicity was not establishedand these cells also may represent a transiently amplifyingcell population that is recruited during injury rather thantrue stem cells. Definitive markers for the isolation of thesecell types still are required. The above research justifiablyhas generated a great deal of excitement but no rigorous proofof the existence of a pluripotential adult renal stem cell withlong-term self-renewal capacity and clonogenicity.
The ability of hematopoietic stem cells to efflux dyes suchas Hoechst 33342 and Rhodamine 123 has been used as the basisof a single-step hematopoietic stem cell (HSC) isolation protocol(52). The term side population (SP) is used to describe HSCthat are isolated in this way. The observation of cells withthe same efflux profile in solid organs has raised the possibilityof organ-based SP, which also may represent stem cells. Severalgroups have reported the existence of an SP in the adult rodentkidney (53–57). These data remain contradictory in termsof the relative size, origin, and lineage capacity of the renalSP. Iwatani et al. (55) showed no evidence for a capacity totransdifferentiate into renal cells in vivo. Hishikawa et al.(54,56) reported that these cells possess renal and multilineagecapacity in culture. When assessing the effect of renal SP cellintroduction into a model of renal damage, they observed littleevidence for transdifferentiation, with SP cells being locatedin the interstitium of the recipient kidney. Challen et al.(57), using more stringent isolation procedures, demonstratedthat the renal SP represented 0.1% of the kidney. These cellsshowed an immunophenotype that was distinct from that of bonemarrow SP, and these cells demonstrated evidence for multilineagepotential in vitro and in vivo. However, despite stringent selection,this population remains heterogeneous with evidence of a monocyticfraction, which may result in the apparent phenotypic plasticity.In both studies (56,57), the introduction of SP cells into amodel of acute experimental renal damage was reparative, suggestinga paracrine role for this cell type that, if characterized,may obviate the need for a cell at all. While SP cells fromthe bone marrow do represent HSC, it is not correct to assumethat dye efflux activity alone indicates self-renewal capacity(58). No studies of the renal SP have proved self-renewal. Theirreparative activity nevertheless warrants further investigation,and the definition of a marker phenotype that allows isolationwithout the assessment of dye efflux is needed. Musculin/MyoRhas been reported as a marker of renal SP cells (54), but ourown data do not support this (57), raising the question of SPhomogeneity and the reproducibility of current isolation techniques.
Nonrenal Adult Stem Cells
With an increasing number of adult cells with seeming pluripotency,can these cells be encouraged to turn into renal cell typesand assist in the treatment of renal damage? This review concentrateson the MSC. The term MSC refers to an adult stem cell that ispresent in the bone marrow in low numbers and has a capacityto differentiate into a wide range of mesenchymal tissue types,including cartilage, bone, muscle, stroma, fat, tendon, andother connective tissues. This term more recently has been appliedto plastic adherent fibroblastic cells that are isolated fromthe bone marrow and other tissues that show mesenchymal multipotency.Few definitive markers identify these plastic-adherent mesenchymalcells, and in most studies, there is little definitive proofthat these cells are true "stem cells." Hence, it has been proposedthat these should be referred to more properly as multipotentmesenchymal stromal cells (59), although the acronym MSC canapply to both. Unlike HSC, once isolated, these mesenchymalstromal cells can be grown in culture for many population doublingsand now have been shown also to have a much broader potential,including neural differentiation. In some studies, the surfacephenotype of an MSC has been investigated. They are negativefor markers that include CD34, CD45, and CD14 and positive forCD166, CD105, CD29, and CD44 (60). Several groups have now investigatedthe effect of delivery of MSC in models of acute renal damage.Herrera et al. (61) induced damage using an intramuscular injectionof glycerol in C57/BL6 mice and monitored the fate of greenfluorescent protein–MSC that were introduced intravenously.These cells homed selectively to damaged kidneys and seemedto differentiate into tubular epithelial cells. There also seemedto be evidence for a trophic role as shown by an increase inproliferating cell nuclear antigen–positive tubular cellsthroughout the kidney. A reduction in creatinine levels in thegreen fluorescent protein–MSC–treated mice suggestedthat a functional improvement was elicited by MSC. Morigi etal. (62) investigated the renoprotective capacity of MSC usinga cisplatin model for acute renal damage and tracing the introducedcells using Y-chromosome fluorescence in situ hybridization.They also reported MSC integration into tubular epithelium andevidence for increased tubular proliferation that was not elicitedby the introduction of HSC. More recently, another group usingthe ischemia/reperfusion model of acute damage in the rat alsoshowed evidence for improved renal function after infusion ofMSC. They attributed this renoprotective effect to a paracrinemechanism, because there was no evidence of transdifferentiation(63,64). MSC were iron dextran–labeled and then trackedusing magnetic resonance imaging, suggesting that they werelocated primarily in the glomerular capillaries, as might beexpected after an intravenous infusion. In light of their abilityto be cultured; their ready accessibility from blood; and theirapparent homing, transdifferentiation, and paracrine protectiveactivities, this seems to be a very strong candidate for useas an adult stem cell in the treatment of renal disease. A greaterunderstanding of the factors that regulate their homing andrenoprotective activity may prove equally fruitful in deliveringa cell-free approach to renal disease. To this end, Luttichauxet al. (65) have begun to define the chemokine receptors thatare expressed by MSC in vivo and in culture. Togel et al. (64)highlighted a reduction in the production of proinflammatorycytokines and a concomitant increase in anti-inflammatory cytokinessuch as IL-10, TGF-, Bcl2, and basic fibroblast growth factorafter infusion of MSC into an ischemia/reperfusion model ofacute damage.
Human ES Cells
The concept of stem cell–based therapy has grown rapidlysince the derivation of human ES cells (hESC). ES cells arepluripotent cells that are derived from the inner cell massof a developing embryo and have the capacity to divide indefinitelywhile retaining a pluripotent phenotype. Excitement about thepotential to use ES cells to repair or regrow organs has increasedsince the derivation of ES cells or embryonic gonadal stem cellsfrom human tissue (66–68). What potential is there forES cells to develop into renal progenitors? Although more difficultto maintain and propagate than their murine counterparts (69),hESC have the ability to develop along ectodermal, mesodermal,and endodermal lineages. Cells of mesodermal origin are foundin spontaneously differentiating cultures of hESC, and hESCnow can be induced readily to undergo sequential hematopoietic(70) and cardiomyocyte differentiation under the control ofmembers of the TGF- superfamily (reviewed in reference [71]).This potential to derive mesodermal tissue bodes well for renaldifferentiation. The introduction of undifferentiated ES cellsinto a tissue usually results in teratoma formation. Yamamotoet al. (72) used this approach to provide evidence that murineES cells had the potential to give rise to mesonephric ductsand UB in teratomas. In contrast, Steenhard et al. (73) reported50% integration of undifferentiated ES cells into the tubulesof embryonic kidneys without evidence for teratomas. Kobayashiet al. (74) created Wnt4-transformed murine ES cells and showedin vitro that these had the capacity to form aquaporin 2–positiverenal tubules. Kim and Dressler (75) sought to direct renaldifferentiation of murine ES cells by relying on previous researchon commitment to early IM in Xenopus (76). They demonstratedthat murine embryoid bodies (EB) that were cultured in a combinationof activin A and retinoic acid expressed a number of markersof IM (Eya1 and Lim1), early kidney development (Pax2, WT1,Wnt4, Six2, and GDNF), and renal tubule–specific markers(cadherin-6) in vitro. Using lacZ to trace their progress, EBthat were primed with retinoic acid, activin A, and bone morphogenicprotein 7 when injected into embryonic kidney cultures showed100% incorporation into developing renal tubules. There wasno evidence that this process was occurring via cell fusion,because not a single fusion event was observed when an ES cellline that contained a loxP-flanked EYFP construct in the Rosa26locus was injected into kidney explants that were isolated froma mouse line that expressed Cre recombinase in the developingrenal tubules (Ksp-Cre). While impressive, the developing kidneymay better provide the full signals required to direct onwardrenal tubular development than an adult kidney. Yamamoto etal. (72) did not direct their murine ES cells in any way. ConventionalEB culture also induced the expression of most of the markersthat were seen by Kim and Dressler (75). Indeed, these cellswent on to form renal structures within the peritoneum of nudemice, suggesting a level of spontaneous renal induction in murineES cells.
A number of obstacles would remain even once the directed differentiationof hESC toward a renal progenitor fate were achieved. The developmentof cell isolation techniques will be required to ensure progenitorpurity, thereby overcoming the possibility of teratoma formation.Delivery remains an issue, as for other stem cells. In addition,legal barriers and ethical debate about the derivation of hESCremain. Without somatic cell nuclear transfer to generate autologoushESC that are tailored for individual patients, an hESC-basedtherapy is likely to require immunosuppression, although thedata discussed later in this report showing the immunologicprotection of embryonic material may mean that immune rejectionis less of an issue than expected. Conversely, there is considerablescientific debate over the ability to derive hESC safely usingsomatic cell nuclear transfer because of our lack of understandingand inability to reprogram genomic imprinting (77). These areobstacles to the adoption of hESC technology in all tissues.
Our kidneys filter our entire blood volume 30 times a day, reabsorbing>95% of what is filtered to produce only 1 to 2 L of urine.The kidneys also regulate pH and fluid balance and maintainred blood cell count, BP, and bone density via the productionof key hormones. The architecture of the kidney is such thatthe nephrons are aligned with the corresponding tubular sectionsthat are adjacent to each other. This arrangement establishesthe countercurrent mechanism that is essential for urinary concentrationand, in turn, fluid maintenance and ion balance. These uniquespatial constraints and the cellular complexity of this organmake bioengineering a major challenge. A replacement "kidney"can be envisaged as either the complete reengineering of theexisting organ or the creation of an alternative structure(s)that is designed to carry out one or more kidney functions.
Bioartificial Glomeruli and Renal Tubules
The kidney was the first organ whose partial function was replacedby an artificial device (78). However, extracorporeal (outsideof the body) hemodialysis replaces only the filtration activityof the kidney and not very efficiently. There has been considerableresearch into the adaptation of this approach via the bioengineeringof devices to replace filtration or reabsorption (78–83).In the case of filtration, microporous synthetic biocompatiblehollow fibers that were coated with MDCK cell extracellularmatrix and then seeded with autologous endothelial cells thatwere harvested from the patient’s circulating blood wereshown to decrease albumin loss (78,79). For mimicking tubularfunction, notably resorptive capacity, a renal assist device(RAD) in which renal parenchymal cells are harvested and seededonto the internal surface of hemodialysis hollow fibers wasdeveloped. Blood from the patient is passed along the outsideof such fibers. The viability of the seeded cells is maintainedvia oxygen and substrates that are provided by the passing bloodand ultrafiltrate (79,84). When tested in animals, these bioartificialtubules provided 40% of normal resorptive capacity. Initially,these two units were used in concert. More recently, the productionof a RAD with human cells has been completed successfully andused on humans in an extracorporeal setting (84,85). Here, conventionaldialysis was combined with a RAD that contained 109 human renalproximal tubular cells that were harvested from donated humankidneys (85). The lack of direct contact between the blood ofthe patient and these cells allowed a nonautologous cell sourceto be used. After passing through the hemofilter, patient bloodpassed through the RAD before being returned to the patient.Ultrafiltrate from the hemofilter also was shunted partiallythrough the RAD to allow reabsorption. This now has been usedin phase I/II clinical trials on intensive care unit patientswith multiple organ failure, including acute renal failure (85).Such patients normally show a >70% mortality rate even whenprovided with dialysis. While proving to be safe and apparentlyimproving patient survival, cells within the RAD also demonstratedmetabolic and endocrine functions that were appropriate forrenal cells and presumably produced chemokines that possiblywere critical to patient response. This bioartificial approachusing human renal epithelial cells now is referred to by thedeveloping company, RenaMed Biologics, as Renal BioreplacementTherapy (http://www.nephrostherapeutics.com/) and is being movedinto a phase III clinical trial. Could such a unit be implantedfor use in chronic renal disease? The challenges to this includethe maintenance of patency, reaching a size that is small enoughfor implantation, and providing the other functions that normallyare provided by the kidney. The last may be overcome via genetherapy of the cells that are used to seed this apparatus. Seededcells may even be manipulated to produce their own anticoagulantto assist in maintaining patency. Even if this challenge isnot reached, the use of renal bioreplacement therapy in an extracorporealsetting well may revolutionize the treatment of intensive careunit patients with multiple organ failure.
Recapitulating Development to Create a Kidney De Novo
More than a decade of research already has been devoted to thedevelopment of xenotransplantation as an alternative to organdonation (86,87). If all immunologic obstacles could be overcome,then this approach will have a significant impact on the treatmentof humans with chronic renal disease. More recently, the useof fetal renal tissue for xenotransplantation has revealed asurprising lack of rejection (88–90). Xenotransplantedembryonic kidney tissue seems to be immunologically protectedfrom the recipient. When transplanted into the abdominal cavity,the embryonic kidney becomes vascularized from the omentum (91,92)and development of functional nephrons proceeds. Such materialalso can be transplanted successfully into the renal subcapsularspace (88,92). The vascularization of such renal primordia islikely to be driven by the expression of vascular endothelialgrowth factor, which normally acts to draw in developing vasculaturefrom the adjacent aorta during normal kidney formation (93).The immune protection that is afforded such embryonic kidneysseems to exist across concordant (between rodents) or nonconcordant(pig to rodent) barriers. Indeed, anephric rats that were supportedby the renal function of a single transplanted metanephros drainedby virtue of an ureteroureterostomy have been shown to survive(94). Observations of immune protection of fetal tissue alsohave been made with liver and pancreas anlage (95). In earlierstudies, other groups experienced rejection when transplantingembryonic metanephric slices from one animal to another (96,97).This may be explained by the observation that lack of rejection,subsequent vascularization, and lack of teratoma formation oforgan primordia is governed by the collection of that primordiawithin a defined window of development (98,99). Rogers et al.(100) reported that immune protection relates to the absenceof donor dendritic cells in early rat anlagen, although immunosuppressionis required for rejection to be prevented when crossing moredisparate immunologic barriers, such as pig to human (101).Dekel et al. (92) investigated the basis of this immune protectionvia expression profiling of adult kidney versus fetal grafts.This suggested a reduced expression of a variety of chemokinesand proinflammatory factors, suggesting a reduction or immaturityof the innate immune response (90,92).
The knowledge that is gained from metanephric transplantationwill be critical for the bioengineering of a de novo replacementorgan. The advantage of using such fetal material as opposedto stem cells is the inherent organ-specific identity of thistissue, which obviates the need for directed differentiation.Availability also is a significant advantage. However, the possibilityof retroviral transmission remains as a question mark over theadoption of such an approach. With the appropriate cells andenvironment, could nephrogenesis be recapitulated so as to createone or more de novo replacement organs in the peritoneal cavityof the patient? Three components would be required: Extracellularmatrix, secreted factors, and cells. The potential sources ofcells would be the same as for ex vivo organ repair with thesecells needed to mimic very early MM or even IM. The abilityof MSC and undergo nephrogenesis during development was demonstratedby Yokoo et al. (60) in whole rodent embryo cultures. HumanMSC, engineered to produce GDNF and lacZ using a replication-defectiveadenoviral construct, were injected into embryonic day 9.5 and11.5 embryos in the region of the IM that gives rise to thedeveloping kidney. After onward development ex vivo for up to48 h, these cells showed complete contribution to the developingkidney. Understanding how such MSC were directed along a renaldevelopmental program would be needed for their successful usein a de novo organ. Yoo et al. (102) reported the harvest ofadult renal cells that were expanded in culture and seeded ontocollagen-coated cylindrical polycarbonate membranes to createan artificial renal tubule. This then was implanted subcutaneouslyin recipient animals with vascularization occurring from thehost. Silicone catheters were connected to these constructs,and after 1 wk, a urine-like filtrate was seen collected inthe terminal reservoir. This approach, as anticipated, resultedin an acute rejection. Whatever cell is used, an ability tomanipulate these cells genetically also may allow for the regulationof red cell count and bone density. The onward development andneovascularization of transplanted metanephroi can be enhancedvia the addition of growth hormone, IGF1, hepatocyte growthfactor, and fibroblast growth factor 2 (90,103–105). Suchresearch starts to define growth factors that may assist inde novo organ generation. The most optimal location for suchan organ would be the peritoneal cavity of the patient/recipientwith vascularization from the host as for metanephric xenotransplants.Metanephric transplantation vascularized from the ventral bodywall mesothelium has been established in the mouse (106). Thiswill enable the use of transgenic mice to examine cell lineageand the relative contribution of recipient to a mini-kidney.
The remaining obstacle to the viability and functionality ofa de novo peritoneal mini-kidney is the requirement for a ureterplumbed into a bladder. The construction of ureters and bladdersis a considerably simpler bioengineering task. There alreadyis a high demand for urinary organ replacements for patientswho undergo resections for bladder or ureteric cancer; childrenwho require surgery to repair congenital dysplasia; and patientswith urinary incontinence, vesicoureteral reflux, congenitaldysplasia, and erectile dysfunction (107). These patients traditionallyhave had sections of gut used for reconstructions. The use ofsuch mucous-secreting, permeable tissues in an organ such asthe bladder creates problems. More recently, silicone or polyglycolicacid–based scaffolds have been used to create replacementparts (107). Replacement bladders have been generated usingpremolded biodegradable polyglycolic acid fiber matrices ontowhich urothelial cells and smooth muscle cells have been culturedon the luminal surface (108,109). Replacement bladders thatare generated in this way from autologous biopsies now havebeen implanted successfully in seven patients who required cystectomyfor treatment of myelomeningocele (110) (http://www.tengion.com).These patients have been monitored for up to 60 mo and showedoptimal bladder function in those whose implants had the mentumwrapped around them. These patients also displayed normal renalfunction, no evidence of mucus production, and normal adjacentbowel function (110). A similar approach has been applied tourethras in animal models (111).
Although there is excitement about the application of many ofthese novel regenerative approaches, many hurdles remain, someunique to this organ. These include research obstacles, suchas a paucity of assays for clonogenicity and renal potential,which hamper our ability to assess adequately potential renalstem cell populations. The unique architecture of the kidneycreates substantial obstacles to the functional integrationof a stem cell–derived nephron. Indeed, the functionalcapacity of a bioengineered organ to provide anything like thefiltering and resorptive capacity of the endogenous kidney isdoubtful.
The final major obstacle is the degree of damage that is presentin a patient with chronic renal disease. It is unlikely thanany organ-based repair process will overcome the extent of damagethat is seen in a patient who has reached end-stage renal failure.This has major implications for the adoption of any autologoustherapy. Even if an adult stem cell population does exist inthe adult kidney, would it remain in an end-stage kidney? Indeed,the adoption of any organ-based cellular therapy is likely tosucceed only if chronic renal disease can be diagnosed earlyand if such therapies are implemented well before end-stagerenal failure is reached. As we move closer to that point intime, the ethical debate about whether trials can proceed beforeESRD will become critical. A lack of surrogate end points withwhich to assess the success of a cellular therapy in renal diseasewill make clinical trails long and expensive, eroding the willof the developers to continue to support the trials. However,the imperative to continue to forge such novel approaches isclear from the rate at which the incidence of chronic renalfailure is rising in both the developed and the developing world(112–114). In the end, it is unlikely that any such therapieswill produce a physiologic outcome that is equivalent to thatof a healthy kidney, but as patient numbers inevitably increasethe use of dialysis for treatment, a novel therapy that createsan improvement over dialysis will become not only a major achievementbut also a necessity.
Acknowledgments
M.H.L. is a National Health and Medical Research Council PrincipalResearch Fellow and is supported by the National Institute ofDiabetes and Digestive and Kidney Diseases.
I thank Miranda Free for editorial support and my family formoral support.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
Saxen L: Organogenesis of the Kidney, Cambridge, Cambridge University Press, 1987
Herzlinger D, Koseki C, Mikawa T, al-Awqati Q: Metanephric mesenchyme contains multipotent stem cells whose fate is restricted after induction. Development 114
: 565–572, 1992[Abstract]
Potter SJ, Thierstein ST: Glomerular development in the kidney as an index of fetal maturity. J Pediatr 22
: 695–706, 1943[CrossRef]
Elger M, Hentschel H, Litteral J, Wellner M, Kirsch T, Luft FC, Haller H: Nephrogenesis is induced by partial nephrectomy in the elasmobranch Leucoraja erinacea. J Am Soc Nephrol 14
: 1506–1518, 2003[Abstract/Free Full Text]
Nagaike M, Hirao S, Tajima H, Noji S, Taniguchi S, Matsumoto K, Nakamura T: Renotropic functions of hepatocyte growth factor in renal regeneration after unilateral nephrectomy. J Biol Chem 266
: 22781–22784, 1991[Abstract/Free Full Text]
Cochrane AL, Kett MM, Samuel CS, Campanale NV, Anderson WP, Hume DA, Little MH, Bertram JF, Ricardo SD: Renal structural and functional repair in a mouse model of reversal of ureteral obstruction. J Am Soc Nephrol 16
: 3623–3630, 2005[Abstract/Free Full Text]
Strutz F, Okada H, Lo CW, Danoff T, Carone RL, Tomas-zewski JE, Neilson EG: Identification and characterization of a fibroblast marker: FSP1. J Cell Biol 130
: 393–405, 1995[Abstract/Free Full Text]
Witzgall R, Brown D, Schwarz C, Bonventre JV: Localization of proliferating cell nuclear antigen, vimentin, c-Fos, and clusterin in the postischemic kidney. Evidence for a heterogenous genetic response among nephron segments, and a large pool of mitotically active and dedifferentiated cells. J Clin Invest 93
: 2175–2188, 1994[Medline]
Bonventre JV: Dedifferentiation and proliferation of surviving epithelial cells in acute renal failure. J Am Soc Nephrol 14[Suppl 1]
: S55–S61, 2003
Cornacchia F, Fornoni A, Plati AR, Thomas A, Wang Y, Inverardi L, Striker LJ, Striker GE: Glomerulosclerosis is transmitted by bone marrow-derived mesangial cell progenitors. J Clin Invest 108
: 1649–1656, 2001[CrossRef][Medline]
Imasawa T, Utsunomiya Y, Kawamura T, Zhong Y, Nagasawa R, Okabe M, Maruyama N, Hosoya T, Ohno T: The potential of bone marrow-derived cells to differentiate to glomerular mesangial cells. J Am Soc Nephrol 12
: 1401–1409, 2001[Abstract/Free Full Text]
Poulsom R, Forbes SJ, Hodivala-Dilke K, Ryan E, Wyles S, Navaratnarasah S, Jeffery R, Hunt T, Alison M, Cook T, Pusey C, Wright NA: Bone marrow contributes to renal parenchymal turnover and regeneration. J Pathol 195
: 229–235, 2001[CrossRef][Medline]
Ito T, Suzuki A, Imai E, Okabe M, Hori M: Bone marrow is a reservoir of repopulating mesangial cells during glomerular remodeling. J Am Soc Nephrol 12
: 2625–2635, 2001[Abstract/Free Full Text]
Ito T, Suzuki A, Okabe M, Imai E, Hori M: Application of bone marrow-derived stem cells in experimental nephrology. Exp Nephrol 9
: 444–450, 2001[CrossRef][Medline]
Ricardo SD, Deane, JA: Adult stem cells in renal injury and repair. Nephrology (Carlton) 10
: 276–282, 2005[Medline]
Douglas-Denton R, Moritz KM, Bertram JF, Wintour EM: Compensatory renal growth after unilateral nephrectomy in the ovine fetus. J Am Soc Nephrol 13
: 406–410, 2002[Abstract/Free Full Text]
Hayakawa M, Ishizaki M, Hayakawa J, Migita M, Murakami M, Shimada T, Fukunaga Y: Role of bone marrow cells in the healing process of mouse experimental glomerulonephritis. Pediatr Res 58
: 323–328, 2005[CrossRef][Medline]
Ikarashi K, Li B, Suwa M, Kawamura K, Morioka T, Yao J, Khan F, Uchiyama M, Oite T: Bone marrow cells contribute to regeneration of damaged glomerular endothelial cells. Kidney Int 67
: 1925–1933, 2005[CrossRef][Medline]
Camargo FD, Chambers SM, Goodell MA: Stem cell plasticity: From transdifferentiation to macrophage fusion. Cell Prolif 37
: 55–65, 2004[CrossRef][Medline]
Camargo FD, Finegold M, Goodell MA: Hematopoietic myelomonocytic cells are the major source of hepatocyte fusion partners. J Clin Invest 113
: 1266–1270, 2004[CrossRef][Medline]
Weimann JM, Johansson CB, Trejo A, Blau HM: Stable reprogrammed heterokaryons form spontaneously in Purkinje neurons after bone marrow transplant. Nat Cell Biol 5
: 959–966, 2003[CrossRef][Medline]
Lin F, Moran A, Igarashi P: Intrarenal cells, not bone marrow-derived cells, are the major source for regeneration in postischemic kidney. J Clin Invest 115
: 1756–1764, 2005[CrossRef][Medline]
Duffield JS, Park KM, Hsiao LL, Kelley VR, Scadden DT, Ichimura T, Bonventre JV: Restoration of tubular epithelial cells during repair of the postischemic kidney occurs independently of bone marrow-derived stem cells. J Clin Invest 115
: 1743–1755, 2005[CrossRef][Medline]
Fang TC, Alison MR, Cook HT, Jeffery R, Wright NA, Poulsom R: Proliferation of bone marrow-derived cells contributes to regeneration after folic acid-induced acute tubular injury. J Am Soc Nephrol 16
: 1723–1732, 2005[Abstract/Free Full Text]
Nishida M, Okumura Y, Fujimoto S, Shiraishi I, Itoi T, Hamaoka K: Adoptive transfer of macrophages ameliorates renal fibrosis in mice. Biochem Biophys Res Commun 332
: 11–16, 2005[CrossRef][Medline]
Imasawa T, Nagasawa R, Utsunomiya Y, Kawamura T, Zhong Y, Makita N, Muso E, Miyawaki S, Maruyama N, Hosoya T, Sakai O, Ohno T: Bone marrow transplantation attenuates murine IgA nephropathy: Role of a stem cell disorder. Kidney Int 56
: 1809–1817, 1999[CrossRef][Medline]
Stokman G, Leemans JC, Claessen N, Weening JJ, Florquin S: Hematopoietic stem cell mobilization therapy accelerates recovery of renal function independent of stem cell contribution. J Am Soc Nephrol 16
: 1684–1692, 2005[Abstract/Free Full Text]
Iwasaki M, Adachi Y, Minamino K, Suzuki Y, Zhang Y, Okigaki M, Nakano K, Koike Y, Wang J, Mukaide H, Taketani S, Mori Y, Takahashi H, Iwasaka T, Ikehara S: Mobilization of bone marrow cells by G-CSF rescues mice from cisplatin-induced renal failure, and M-CSF enhances the effects of G-CSF. J Am Soc Nephrol 16
: 658–666, 2005[Abstract/Free Full Text]
Held PK, Al-Dhalimy M, Willenbring H, Akkari Y, Jiang S, Torimaru Y, Olson S, Fleming WH, Finegold M, Grompe, M: In vivo genetic selection of renal proximal tubules. Mol Ther 13
: 49–58, 2006[CrossRef][Medline]
Sugimoto H, Mundel TM, Sund M, Xie L, Cosgrove D, Kalluri R: Bone-marrow-derived stem cells repair basement membrane collagen defects and reverse genetic kidney disease. Proc Natl Acad Sci U S A 103
: 7321–7326, 2006[Abstract/Free Full Text]
Brockes JP, Kumar A: Plasticity and reprogramming of differentiated cells in amphibian regeneration. Nat Rev Mol Cell Biol 3
: 566–574, 2002[CrossRef][Medline]
Brockes JP: Amphibian limb regeneration: Rebuilding a complex structure. Science 276
: 81–87, 1997[Abstract/Free Full Text]
Brockes JP, Kumar A: Appendage regeneration in adult vertebrates and implications for regenerative medicine. Science 310
: 1919–1923, 2005[Abstract/Free Full Text]
Kondo T, Raff M: Oligodendrocyte precursor cells reprogrammed to become multipotential CNS stem cells. Science 289
: 1754–1757, 2000[Abstract/Free Full Text]
Odelberg SJ, Kollhoff A, Keating MT: Dedifferentiation of mammalian myotubes induced by msx1. Cell 103
: 1099–1109, 2000[CrossRef][Medline]
Leferovich JM, Bedelbaeva K, Samulewicz S, Zhang XM, Zwas D, Lankford EB, Heber-Katz E: Heart regeneration in adult MRL mice. Proc Natl Acad Sci U S A 98
: 9830–9835, 2001[Abstract/Free Full Text]
Azizi SA: Exploiting nonneural cells to rebuild the nervous system: From bone marrow to brain. Neuroscientist 5
: 353–361, 2000
Mezey E, Chandross KJ, Harta G, Maki RA, McKercher SR: Turning blood into brain: Cells bearing neuronal antigens generated in vivo from bone marrow. Science 290
: 1779–1782, 2000[Abstract/Free Full Text]
Rietze RL, Valcanis H, Brooker GF, Thomas T, Voss AK, Bartlett PF: Purification of a pluripotent neural stem cell from the adult mouse brain. Nature 412
: 736–739, 2001[CrossRef][Medline]
Toma JG, Akhavan M, Fernandes KJ, Barnabe-Heider F, Sadikot A, Kaplan DR, Miller FD: Isolation of multipotent adult stem cells from the dermis of mammalian skin. Nat Cell Biol 3
: 778–784, 2001[CrossRef][Medline]
Zuk PA, Zhu M, Mizuno H, Huang J, Futrell JW, Katz AJ, Benhaim P, Lorenz HP, Hedrick MH: Multilineage cells from human adipose tissue: Implications for cell-based therapies. Tissue Eng 7
: 211–228, 2001[CrossRef][Medline]
Burrow CR, Wilson PD: A putative Wilms tumor-secreted growth factor activity required for primary culture of human nephroblasts. Proc Natl Acad Sci U S A 90
: 6066–6070, 1993[Abstract/Free Full Text]
Kitamura S, Yamasaki Y, Kinomura M, Sugaya T, Sugiyama H, Maeshima Y, Makino H: Establishment and characterization of renal progenitor like cells from S3 segment of nephron in rat adult kidney. FASEB J 19
: 1789–1797, 2005[Abstract/Free Full Text]
Challen GA, Martinez G, Davis MJ, Taylor DF, Crowe M, Teasdale RD, Grimmond SM, Little MH: Identifying the molecular phenotype of renal progenitor cells. J Am Soc Nephrol 15
: 2344–2357, 2004[Abstract/Free Full Text]
Bussolati B, Bruno S, Grange C, Buttiglieri S, Deregibus MC, Cantino D, Camussi G: Isolation of renal progenitor cells from adult human kidney. Am J Pathol 166
: 545–555, 2005[Abstract/Free Full Text]
Maeshima A, Yamashita S, Nojima Y: Identification of renal progenitor-like tubular cells that participate in the regeneration processes of the kidney. J Am Soc Nephrol 14
: 3138–3146, 2003[Abstract/Free Full Text]
Yamashita S, Maeshima A, Nojima Y: Involvement of renal progenitor tubular cells in epithelial-to-mesenchymal transition in fibrotic rat kidneys. J Am Soc Nephrol 16
: 2044–2051, 2005[Abstract/Free Full Text]
Maeshima A, Sakurai H, Nigam SK: Adult kidney tubular cell population showing phenotypic plasticity, tubulogenic capacity, and integration capability into developing kidney. J Am Soc Nephrol 17
: 188–198, 2006[Abstract/Free Full Text]
Vogetseder A, Karadeniz A, Kaissling B, Le Hir M: Tubular cell proliferation in the healthy rat kidney. Histochem Cell Biol 124
: 97–104, 2005[CrossRef][Medline]
Oliver JA, Maarouf O, Cheema FH, Martens TP, Al-Awqati Q: The renal papilla is a niche for adult kidney stem cells. J Clin Invest 114
: 795–804, 2004[CrossRef][Medline]
Goodell MA, Brose K, Paradis G, Conner AS, Mulligan RC: Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo. J Exp Med 183
: 1797–1806, 1996[Abstract/Free Full Text]
Asakura A, Rudnicki MA: Side population cells from diverse adult tissues are capable of in vitro hematopoietic differentiation. Exp Hematol 30
: 1339–1345, 2002[CrossRef][Medline]
Hishikawa K, Marumo T, Miura S, Nakanishi A, Matsuzaki Y, Shibata K, Ichiyanagi T, Kohike H, Komori T, Takahashi I, Takase O, Imai N, Yoshikawa M, Inowa T, Hayashi M, Nakaki T, Nakauchi H, Okano H, Fujita T: Musculin/MyoR is expressed in kidney side population cells and can regulate their function. J Cell Biol 169
: 921–928, 2005[Abstract/Free Full Text]
Iwatani H, Ito T, Imai E, Matsuzaki Y, Suzuki A, Yamato M, Okabe M, Hori M: Hematopoietic and nonhematopoietic potentials of Hoechst(low)/side population cells isolated from adult rat kidney. Kidney Int 65
: 1604–1614, 2004[CrossRef][Medline]
Hishikawa K: Do we really need "stem" cell for renal regeneration? Kidney Int 68
: 1966–1967, 2005
Challen GA, Bertoncello I, Deane JA, Ricardo SD, Little MH: Kidney side population reveals multilineage potential and renal functional capacity but also cellular heterogeneity. J Am Soc Nephrol 17
: 1896–1912, 2006[Abstract/Free Full Text]
Challen GA, Little MH: A side order of stem cells: The SP phenotype. Stem Cells 24
: 3–12, 2006[Abstract/Free Full Text]
Horwitz EM, Le Blanc K, Dominici M, Mueller I, Slaper-Cortenbach I, Marini FC, Deans RJ, Krause DS, Keating A: Clarification of the nomenclature for MSC: The International Society for Cellular Therapy position statement. Cytotherapy 7
: 393–395, 2005[CrossRef][Medline]
Yokoo T, Ohashi T, Shen JS, Sakurai K, Miyazaki Y, Utsunomiya Y, Takahashi M, Terada Y, Eto Y, Kawamura T, Osumi N, Hosoya T: Human mesenchymal stem cells in rodent whole-embryo culture are reprogrammed to contribute to kidney tissues. Proc Natl Acad Sci U S A 102
: 3296–3300, 2005[Abstract/Free Full Text]
Herrera MB, Bussolati B, Bruno S, Fonsato V, Romanazzi GM, Camussi G: Mesenchymal stem cells contribute to the renal repair of acute tubular epithelial injury. Int J Mol Med 14
: 1035–1041, 2004[Medline]
Morigi M, Imberti B, Zoja C, Corna D, Tomasoni S, Abbate M, Rottoli D, Angioletti S, Benigni A, Perico N, Alison M, Remuzzi G: Mesenchymal stem cells are renotropic, helping to repair the kidney and improve function in acute renal failure. J Am Soc Nephrol 15
: 1794–1804, 2004[Abstract/Free Full Text]
Lange C, Togel F, Ittrich H, Clayton F, Nolte-Ernsting C, Zander AR, Westenfelder C: Administered mesenchymal stem cells enhance recovery from ischemia/reperfusion-induced acute renal failure in rats. Kidney Int 68
: 1613–1617, 2005[CrossRef][Medline]
Togel F, Hu Z, Weiss K, Isaac J, Lange C, Westenfelder C: Administered mesenchymal stem cells protect against ischemic acute renal failure through differentiation-independent mechanisms. Am J Physiol Renal Physiol 289
: F31–F42, 2005[Abstract/Free Full Text]
Luttichaux IV, Notohamiprodjo M, Wechselberger A, Peters C, Henger A, Seliger C, Djafarzadeh R, Huss R, Nelson PJ: Human adult CD34-progenitor cells functionally express the chemokine receptors CCR1, CCR4, CCR7, CXCR5, and CCR10 but not CXCR4. Stem Cells Dev 14
: 329–336, 2005[CrossRef][Medline]
Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM: Embryonic stem cell lines derived from human blastocysts. Science 282
: 1145–1147, 1998[Abstract/Free Full Text]
Reubinoff BE, Pera MF, Fong CY, Trounson A, Bongso A: Embryonic stem cell lines from human blastocysts: Somatic differentiation in vitro. Nat Biotechnol 18
: 399–404, 2000[CrossRef][Medline]
Shamblott MJ, Axelman J, Wang S, Bugg EM, Littlefield JW, Donovan PJ, Blumenthal PD, Huggins GR, Gearhart JD: Derivation of pluripotent stem cells from cultured human primordial germ cells. Proc Natl Acad Sci U S A 95
: 13726–13731, 1998[Abstract/Free Full Text]
Pera MF: Human pluripotent stem cells: A progress report. Curr Opin Genet Dev 11
: 595–599, 2001[CrossRef][Medline]
Kouskoff V, Lacaud G, Schwantz S, Fehling HJ, Keller G: Sequential development of hematopoietic and cardiac mesoderm during embryonic stem cell differentiation. Proc Natl Acad Sci U S A 102
: 13170–13175, 2005[Abstract/Free Full Text]
Singla DK, Sobel BE: Enhancement by growth factors of cardiac myocyte differentiation from embryonic stem cells: A promising foundation for cardiac regeneration. Biochem Biophys Res Commun 335
: 637–642, 2005[CrossRef][Medline]
Yamamoto M, Cui L, Johkura K, Asanuma K, Okouchi Y,
Top
Regenerative Approaches to Renal...
Setting the Stage: Normal...
3 has been reported (30). This is a model of Alport syndrome in which there is considerable shedding of protein through the damaged glomerular basement membrane. Whereas podocytes have not been a reported site of bone marrow recruitment in other experimental models, this study claimed a bone marrow origin for 10% of the podocytes in these mice with a reduction in protein shedding and evidence of collagen replacement within the basement membrane. In this case, access may have been increased as a result of the altered permeability of the basement membrane, but BMDC from mutant mice were not recruited to the glomeruli of mutant recipients, suggesting an active selection for collagen-producing cells. In all of these reports of bone marrow recruitment to damaged kidneys, the lineage of the BMDC that were recruited has not been established. However, adoptive transfer of macrophages into a model of unilateral ureteric obstruction significantly reduced fibrosis in the late stages of this damage state (25). This may have involved transdifferentiation or an altered immunologic response. What also has not been investigated is whether the recruitment of BMDC is good or bad in cases of chronic renal damage. 
superfamily (reviewed in reference [71]). This potential to derive mesodermal tissue bodes well for renal differentiation. The introduction of undifferentiated ES cells into a tissue usually results in teratoma formation. Yamamoto et al. (72) used this approach to provide evidence that murine ES cells had the potential to give rise to mesonephric ducts and UB in teratomas. In contrast, Steenhard et al. (73) reported 50% integration of undifferentiated ES cells into the tubules of embryonic kidneys without evidence for teratomas. Kobayashi et al. (74) created Wnt4-transformed murine ES cells and showed in vitro that these had the capacity to form aquaporin 2–positive renal tubules. Kim and Dressler (75) sought to direct renal differentiation of murine ES cells by relying on previous research on commitment to early IM in Xenopus (76). They demonstrated that murine embryoid bodies (EB) that were cultured in a combination of activin A and retinoic acid expressed a number of markers of IM (Eya1 and Lim1), early kidney development (Pax2, WT1, Wnt4, Six2, and GDNF), and renal tubule–specific markers (cadherin-6) in vitro. Using lacZ to trace their progress, EB that were primed with retinoic acid, activin A, and bone morphogenic protein 7 when injected into embryonic kidney cultures showed 100% incorporation into developing renal tubules. There was no evidence that this process was occurring via cell fusion, because not a single fusion event was observed when an ES cell line that contained a loxP-flanked EYFP construct in the Rosa26 locus was injected into kidney explants that were isolated from a mouse line that expressed Cre recombinase in the developing renal tubules (Ksp-Cre). While impressive, the developing kidney may better provide the full signals required to direct onward renal tubular development than an adult kidney. Yamamoto et al. (72) did not direct their murine ES cells in any way. Conventional EB culture also induced the expression of most of the markers that were seen by Kim and Dressler (75). Indeed, these cells went on to form renal structures within the peritoneum of nude mice, suggesting a level of spontaneous renal induction in murine ES cells. 
