Stem Cells and Generation of New Cells in the Adult Kidney

C H A P T E R

29 Stem Cells and Generation of New Cells in the Adult Kidney Juan A. Oliver1 and Qais Al-Awqati2 1

Department of Medicine, Columbia University, New York, NY, USA Departments of Medicine and Physiology, Columbia University, New York, NY, USA

2

EMBRYONIC ORIGIN OF RENAL CELLS The idea that the kidney is an organ needs to be tempered by the realization that it is the nephron that is the organ. Each nephron, as far as we know, is independent from other nephrons, and the kidney is to a first approximation a collection of these mini-organs put together in a complex three-dimensional assembly. The nephrons of all mammals are remarkably similar in size, function, and origin (as far as we know), and the differences among species are largely if not entirely due to the number of these units in the assembly, with mice having 10,000 and whales having 250 million in each kidney. But while the epithelial character of the nephron garners most of the attention, one needs to be reminded that the kidney has a very extensive vascular network with multiple distinct morphological and functional domains, as well as an abundant interstitial cell population that unfortunately is still poorly understood (see Kaissiling and Le Hir1 for a review). While the taxonomy of the renal epithelial cells is nearly complete, that of the cells of the vascular and interstitial compartments awaits detailed characterization. Hence, analysis of renal regeneration after injury, as well as search for putative renal stem cells in the adult kidney, has essentially been restricted to the epithelial compartment. However, as epithelial cells are likely instructed by mesenchymal signals and epithelia-mesenchymal cross-talk is critical for renal epithelial differentiation and function,2 there is a need for deeper understanding of the cell types that comprise the renal vascular and interstitial compartments, and their roles in kidney homeostasis and repair from injury. For example, in many organs including the kidney, mesenchymal cells with characteristics of Seldin and Giebisch’s The Kidney, Fifth Edition. DOI: http://dx.doi.org/10.1016/B978-0-12-381462-3.00029-X

precursor/stem cells have been found to reside near or in the vascular wall,3 5 but the exact origin and normal function of these cells is unknown. The different cellular compartments of the adult kidney have been traditionally recognized by their morphological characteristics or by their embryonic origin, since it was long ago recognized that the adult (metanephric) kidney derives from two distinct elements of the intermediate mesoderm: the metanephric mesenchyme and the ureteric bud. Within the kidney, the ureteric bud gives rise to the collecting duct cells, while some metanephric mesenchyme cells give rise to the rest of the nephron.6 However, over the last few decades, the discovery of several genes that are expressed in the restricted group of cells of the renal anlage has allowed a different taxonomic approach that has greatly illuminated our understanding of the distinct cell populations in the adult kidney. Moreover, it has allowed development of research tools with which it is possible to probe in the adult kidney the function of specific cells, of specific genes in specific cells, and importantly for the present discussion, to identify the daughter cells of different cell types by in vivo genetic cell lineage methods. Thus, we briefly review the embryonic origin of the distinct cells in the adult kidney, emphasizing those aspects that might clarify the origin of new cells in the adult organ.

Epithelial Cells All epithelial cells of the adult kidney are believed to derive from the intermediate mesoderm, from which both the ureteric bud (a branch of the Wolffian duct) and the metanephric mesenchyme originate. Renal

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morphogenesis starts when the ureteric bud invades the metanephric mesenchyme and starts branching. The cells in the tip of each ureteric bud branch give rise to the collecting duct cells and the metanephric mesenchyme cells in contact with each ureteric bud tip give rise, after a series of morphogenic steps, to the cells of the remaining nephron segments spanning from the connecting tubule to the glomerulus. The ureteric bud, like the Wolffian duct, expresses the homeobox gene HoxB7,7 and transgenic mice expressing HoxB7-GFP or HoxB7-Cre recombinase have been used to label most, if not all, of the cells in the ureteric bud branches of the embryonic collecting duct,8 and their progeny in the adult kidney. The metanephric mesenchymal cells undergo simultaneous differentiation (to generate a nephron for each ureteric bud tip) and growth, so that the appropriate number of nephrons will be generated for the branches of the ureteric bud. It was recently found that the metanephric mesenchymal cells that are in contact with the tips of the ureteric bud, referred to as the cap mesenchyme, are the progenitor cells of all nephron epithelia (except the collecting duct). These cells were found to express the transcription factors Cited19 and Six2,10 thereby allowing generation of transgenic mice that label all nephron epithelial cells except those of the collecting ducts. More relevant to the present discussion is that these mice can be used to permanently identify the progeny of adult nephron epithelial cells,9,10 thus providing an invaluable tool for analysis of epithelial cell regeneration after kidney injury and/or disease,11 as discussed below.

defined. Finally, an area of the intermediate mesoderm located ventro lateral to the dorsal aorta generates renal interstitial cells that express the stem cell factor receptor (c-kit).17 During embryogenesis, these cells appear to be involved in the maintenance of the metanephric mesenchyme-derived cells, but identification of their progeny in the adult kidney remains to be established.

Endothelial Cells The renal circulation is both anatomically and functionally complex, and likely contains many types of endothelial cells. Their exact origin is still poorlyunderstood. It is clear that once kidney development starts, the renal anlage contains angioblats18,19 that give rise to endothelial cells. It is currently unknown whether these endothelial precursors migrate into the developing kidney or differentiated from cells that reside in the metanephric mesenchyme. The latter appears more likely, as we found that many cells in the renal anlage express tyrosine kinases that are characteristic of adult endothelial cells.20 Interestingly, in addition to being endothelial precursors, angioblasts in the renal anlage appear to provide signals important for development and differentiation of the metanephric mesenchyme.21 It is currently unknown if there exists interaction in the adult kidney between endothelial cells and either interstitial or epithelial cells that might be involved in maintaining kidney homeostasis or repair from injury. Recent work by Lin et al.22 suggests that endothelial-to-pericyte cross-talk is involved in the generation of kidney myofibroblats, as detailed below.

Mesenchymal/Stromal Cells Like renal epithelial cells, the vast majority of the stroma cells in the adult kidney derive from the intermediate mesoderm12 that in the kidney gives rise to a cell population that expresses the forkhead transcription factor Foxd1.2,13,14 These cells generate many renal interstitial cells, as well as mesangial cells, vascular smooth muscle, pericytes, and renal capsule, and likely mesenchymal stem cells.5 Foxd1-expressing cells are absolutely required for normal kidney development,2,13,14 and their adult progeny is of extreme interest because it likely contains pluripotent MSC5 and pericytes,4 although a clear-cut distinction between these two cell types is not yet possible. In addition, identification of Foxd1 as marker of these cells has made it possible to develop transgenic mice that can be used to label the stroma cell progeny in the adult kidney.15 Another population of renal stromal cells derives from the paraxial mesoderm,16 but the precise contribution of these cells to the interstitial and mesenchymal cell populations of the adult kidney remains to be

NEW CELLS IN THE ADULT KIDNEY Normal Conditions As assayed by a variety of methods, the normal adult kidney has a low rate of cellular proliferation.23 26 Using antibodies to Ki67, a nuclear protein expressed in cycling cells during G0 and G1, between 0.4 1% of all cells were cycling in the adult rat kidney.24,25 Interestingly, age has a profound effect in the abundance of proliferating cells found in the kidney. Vogesterder et al.25 found that while only B0.4% of all renal cells were positive for Ki67 in the kidneys of 16to 20-week-old rats, in animals that were only 4 weeks old the number of cycling cells was B5%. This suggests that in the kidney there is an age-dependent progressive decline in the number of cycling cells, and that workers examining renal cell proliferation should take into account the age of the animal as an important variable.

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Proliferating cells in the renal cortex of 4-week-old rats were found preferentially located to the S3 segment of the proximal tubule, when compared to the S1 and S2 segments.25 This interesting result might have important implications in designing strategies to identify renal stem cells, a subject to which we will return below. In contrast, in 1-year-old rats, we found that most of the kidney parenchyma had a homogeneous fraction of B1% of Ki67 positive cells Figure 29.1. There were two exceptions to this, however, the body of the papilla where there were extremely low numbers of cycling cells (, 0.1% of the cells were positive for Ki67), and the upper part of the papilla (at the papilla medullary junction), where we found the highest frequency of cycling cells (B2.5%),24 indicating that it is an area of privileged cellular proliferation in the adult rat. Detailed morphological observations indicate that terminally differentiated tubular epithelia cells can generate new cells,27,28 but these observations don’t exclude the possibility that there are epithelial stem cells.

CORTEX

MEDULLA

Upper Middle Tip PAPILLA

Ki67+(%)

3 2 1 0

Cortex Medulla Upper Middle Tip (lateral) Papilla

FIGURE 29.1

Cellular proliferation in adult kidney. Top: Kidney regions were examined for cellular proliferation. Bottom: Fraction of Ki67-positive cells in different kidney regions. Cortex and medulla had a similar number of Ki67-positive cells, but the lateral side of the papilla had significantly more Ki67-positive cells than the cortex and medulla, as well as the other regions of the papilla. The middle and tip of the papilla had significantly fewer Ki67 positive cells than other regions of the kidney. (From ref. [27].)

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Organ Repair from Injury In contrast to normal conditions, the kidney displays a remarkable proliferation capacity shortly after transient injury. For example, injury induced by 30 45 minutes of complete renal artery occlusion in rodents causes functional failure, and widespread cellular apoptosis and necrosis that are followed by diffuse cellular proliferation and functional recovery. What is the origin of these new cells? While the kidney has multiple cell types, studies on the generation of new cells after injury have fundamentally focused on the epithelial cells, likely because of their better-understood functional importance and easier identification. It is now established that the new epithelial cells after kidney injury develop from within the parenchyma, rather than being derived from extrarenal sources such as the bone marrow,29,30 and thus three possibilities appear likely: (1) any surviving terminally differentiated epithelial cell can generate identical cells; (2) there exist kidney epithelial stem cells capable of generating any epithelial cell type, similar to what was found in the interfollicular epidermis;31,32 and (3) pluripotent renal stem cells generate epithelial as well as other cell types, as in the case of the stem cells in the bulge of the skin.33 36 Morphological observations35,37 and functional studies with nucleotide analogs27,38 have provided strong evidence that terminally-differentiated epithelial cells generate new epithelial cells after injury. More recent elegant genetic cell fate-mapping studies have confirmed this suspicion; Humpreys et al.11 used reporter mice in which the renal epithelial cell compartment was labeled by a Cre recombinase driven by the promoter of Six2, a gene that is specifically expressed in embryonic epithelial precursors (see above), and examined their response to acute kidney injury. They found that injury induced massive cellular proliferation, and that all new epithelial cells expressed the reporter gene (Figure 29.2). Since RT-PCR of adult kidney tissue could neither detect Six2 nor Cre, it is apparent that the labeled cells originated from epithelial cells labeled previously during kidney development, thus excluding the interstitial/stroma cell compartment as the origin of new epithelial cells. Needless to say, this experiment does not address whether there exists a group of restricted epithelial cells that are responsible for all the new epithelia generated after injury; these cells could function as adult renal epithelial stem cells. In a recent study, Humphreys et al.39 examined this possibility by labeling cycling cells after transient kidney injury with two different thymidine analogs administered sequentially. Since the number of epithelial cells that were positive for both nucleotides was very low, these workers concluded that surviving epithelial cells repopulate the nephron epithelium in a stochastic manner,

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Repair Labeled tubule

Injury

1

or

2

FIGURE 29.2

Surviving epithelial cells after injury generate new epithelial cells. (Left) New cells after acute kidney injury in mice with genetically labeled epithelial tubules will not carry the label if they derive from cells outside the epithelial cell compartment (#1), but will carry the label if they derive from terminally differentiated epithelial cells (#2). (Right) Fifteen days after transient ischemic injury and repair, there was no dilution on the number of labeled cells (dark) despite marked cellular proliferation, indicating that they derived from epithelial cells. (From ref. [14].)

suggesting that the nephron epithelia has no stem cells.39 However, while control experiments clearly showed appropriate specificity of both antibodies, detection of closely related thymidine analogs during conditions that, unlike in the control experiments, probably result in incorporation of very different amounts of the two analogs in a given cell are fraught with potential problems. More importantly, their conclusion is based on the implicit assumption that a putative population of epithelial stem cells would be a small fraction of the total number of cells. Under these conditions, to repopulate the damaged epithelia the stem cell progeny would need to divide rapidly, and would thus incorporate both nucleotide analogs. However, as detailed below, stem cells in Drosophila Malpighian tubules are a very large fraction of the total cells, and there is no reason why this may not also be the case in mammalian kidneys. Similarly, in organs other than the kidney such as the adult airway epithelia, stem cells account for about one-third of the total number of cells.40,41 Identification of the site where cellular proliferation first starts after transient injury could potentially facilitate identification of precursor/stem cells. Unfortunately, for most insults that cause acute kidney injury with functional failure, cellular proliferation has most often been examined one or a few days afterwards, at which time cell proliferation, while very prominent in the S3 segment of the proximal tubule, is also widespread in other parts of the kidney parenchyma, particularly the medulla.38,42 44 Following acute kidney injury by renal artery occlusion we could not detect proliferating cells until B24 hours later when we examined kidney sections of B5 μm thickness, as is done routinely. However, with

100 μm vibrotome sections, one hour after injury we found that the upper part of the papilla had more proliferative cells than other parts of the kidney24 (see Figure 29.3), suggesting that this is the site of initial cellular proliferation after kidney injury, which we previously found to be the site of enhanced cell cycling under normal conditions (see above). Interestingly, Vinsonneau et al.45 reported that the first cells that they found cycling after ischemia reperfusion injury to the kidney were uro-epithelial cells in the upper part of the urinary (intrarenal) space and neighboring interstitial cells; detailed analysis with Ki67 and BrdU incorporation showed that these cells were proliferating B16 hours after ischemic injury and B4 hours before proliferation could be detected elsewhere in the kidney. The site identified by Vinsonneau et al.45 is where the base of the papilla attaches to the medulla, it is in close proximity to the cortex, and appears to be the same proliferating site we identified in the upper papilla. Needless to say, identification of these “early” proliferating cells and of their progeny would be of extreme interest. An additional observation merits mention. In the few studies that identified the cells that first started proliferating after injury, either after transient ischemic injury44 or aminoglycoside toxicity,46,47 it is remarkable that in all instances they were interstitial cells, perhaps suggesting that some interstitial cell plays a critical role in initiating epithelial regeneration. Indeed, the likelihood of renal functional recovery after injury was found to correlate with increases in the number of interstitial cells, many of which were likely myofibroblasts.43,48,49 Although these results raise the possibility that myofibroblasts might be involved in epithelial regeneration, other interstitial cells such as macrophages are known

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Upper papilla

US Cortex

Medulla

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zinc-α2-glycoprotein (Zag), an adipokine associated with cachexia.58 To examine whether the poor regenerative capacity of the aging kidney is due to a decrease in the numbers or in the functionality of putative renal stem/progenitor cells awaits definitive identification of these cells. In sum, both during normal conditions and particularly after transient kidney injury, the S3 segment of the proximal tubule is a site of intense cell proliferation, suggesting the presence of progenitor cells in this part of the nephron. In addition, the upper part of the papilla and close to the urinary space is also a site of robust cell cycling, both under normal conditions24 and after transient kidney injury.24,45

BrdU

FIGURE 29.3 Cellular proliferation was first detected in the upper papilla following kidney injury. There was a selective increase of cells in S-phase in the upper papilla during the first hour after transient ischemic injury. Note abundant BrdU-labeled cells in the upper papilla, whereas the cortex and medulla revealed very rare BrdU-labeled cells (US: urinary space). Photomicrographs were done in 100 μm tissue sections obtained with a vibratome.(From ref. [27].)

to be involved in kidney repair,50 and a detailed characterization of the interstitial cell compartment following transient kidney injury is lacking.

Effect of Age For many organs, including the kidney,51 the capacity to recover from injury decreases with age, an observation familiar to most practicing nephrologists. In most organs, it remains to be determined whether this is due to a decrease in the number of organ-specific stem cells or to the inability of the stem cells to be activated, but recent work suggest that the latter is more likely (reviewed by Liu and Rando52). For example, it was recently found that aging muscle had a normal number of satellite cells, but the cells failed to activate in response to exercise.53 Interestingly, progenitor/ stem cells in advanced age can display a “young” response by exposure to a young systemic environment,54 indicating that the changes responsible for the functional decrease of precursor/stem cells are potentially reversible. While the effect of age on the renal proliferative capacity after injury has not been studied in detail, it appears that cell proliferation after acute kidney injury decreases with age,55 in agreement with the poor prognosis of recovery from acute kidney injury in elderly patients.51 Mechanisms that could account for these observations include telomere shortening56 and increased expression by renal epithelial cells of

IDENTIFICATION OF POTENTIAL PRECURSOR/STEM CELLS IN THE ADULT KIDNEY Studies from several laboratories have presented evidence characterizing renal precursor cells in the adult kidney, but none of these cells meet strict criteria for traditional adult, organ-specific stem cells; i.e., asymmetric cell division and multipotency. Nonetheless, since our ultimate aim is to understand the origin of new cells in the adult kidney so that the responsible mechanisms might be manipulated, these studies are worth reviewing. In addition, the results of these studies are likely to be useful for future work to identify adult renal stem cells. Several strategies have been used to identify and characterize adult kidney precursor/stem cells, and the robustness of the obtained results varies widely. In our view, the methodological approach used for cell identification and/ or isolation of the cells best separates these studies.

Cellular Markers Several workers have isolated cells from the adult kidney using a candidate gene approach. For example, Busolatti et al.59 used the transmembrane glycoprotein Cd133, which is expressed in hematopoietic stem cells, endothelial progenitor cells, neuronal, glial, and glioblastoma stem cells, as well as some other cell types (see Mizrak et al.60 for a review), to select cells from the adult human kidney. The isolated cells showed strong clonogenic capacity, and when subcutaneously transplanted to SCID mice formed tubules which expressed some renal epithelial markers. Finally, when the cells were injected intravenously into mice with glycerol-mediated acute kidney injury, they integrated into tubules. However, the functional significance of the integration was not explored.

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Using a somewhat similar approach, Dekel et al.61 used Sca-1, a protein identified in several progenitor cells, to isolate a cell population from mouse kidneys. However, because Sca-1 is also expressed in renal epithelia, these workers used a collagenase digestion approach that appeared to yield interstitial cells. The in vitro differentiation of these cells suggested that they were mesenchymal-like stem cells, since they differentiated into myogenic, osteogenic, adipogenic, and neural lineages. When injected into kidneys with acute kidney injury, they also incorporated into tubules.

In Vitro Growth Behavior Several laboratories have isolated cells from adult kidneys based on their characteristics when cultured in vitro. For example, cells were selected because of their high clonogenic potential, ability to outlast all other cultured cells or their ability to change their phenotype. For example, Gupta et al.,62 cultured cells from collagenase digested rat kidneys, and after B5 weeks in culture they obtained a cell population with substantial proliferative potential (which in fact was the characteristic that lead to isolation of the cells) and expressing transcription factors such as Pax-2 and Oct4 that are either involved in renal development or in determining cell fate. When the cells were injected into the subcapsular space of normal kidneys, some of them incorporated into tubules. Kitamura et al.63 used a slightly different strategy by isolating single nephrons from adult rat kidneys and culturing them in vitro. Cells growing out from these nephrons were harvested and individually cultured; and the clone with the fastest proliferation capacity was characterized. These clonal cells expressed several proteins important in kidney development and proteins characteristic of mature nephrons. When the cells were injected in the subcapsular region of a kidney with acute kidney injury, the cells engrafted into renal tubules. More recently, Lee et al.64 used a mouse with a targeted mutation in which GFP is expressed under the control of Myh9, a gene expressed in interstitial cells (among others). After isolation of the GFP1 cells from the kidney and subsequent culture for eight weeks, they isolated a cell line that expressed renal embryonic or stem cell markers such as Oct-4, Pax-2, Wnt-4, and WT-1. Immunolocation of the endogenous Oct-41 and GFP1 cells in the kidney found them in the interstitial space of the medulla and papilla. Finally, when the cells were injected directly into kidneys after acute kidney injury, they incorporated into tubules and partially decreased the functional consequences of the injury.

In Vivo Growth Behavior The hypothesis that adult, organ-specific stem cells divide infrequently has been a powerful tool in locating and identifying several stem/precursor cells. According to this hypothesis, due to their low cycling behavior, stem cells retain S-phase labels (traditionally 3H-Thymidine or a thymidine analog such as BrdU) when given as a short pulse followed by a long time of chase. Recent work has, however, demonstrated that there is more complexity than originally thought65 67 (see Fuchs68 for an informed discussion), as not all stem cells are low cycling and “label retention” experiments identify a heterogeneous group of cells. Nonetheless, populations of “label-retaining cells” from several organs are enriched for stem cells,66,69 72 and identification of a “label-retaining” group of cells is of great interest. To detect low cycling cells, an S-phase label is only administered during a short period of intense growth (e.g., embryonic life or shortly after birth), so that all cells are cycling and incorporate the label; during the subsequent period of chase the label is diluted and lost by the progeny of dividing cells and selectively retained by low- or non-cycling cells. Cells cycling infrequently were thus named “label-retaining cells” (LRC). Several investigators have used this approach in the kidney. Maeshima et al.38 administered BrdU for one week to adult rats that were then “chased” for two weeks, and found that there were many BrdU-retaining cells the kidney parenchyma, particularly in the proximal tubules. Following transient ischemic injury, most of the tubular cells that were proliferating (expressing proliferating nuclear antigen; PCNA) were also BrdUlabeled cells. These results led Maeshima et al.38 to conclude that a “label-retaining” cell population was the precursor of new cells during kidney repair from injury. However, the long pulse and short period of chase complicate the interpretation of these results. In addition, the increased number of BrdU-labeled cells after injury was likely due to detection of the cells loaded with BrdU during the “pulse” plus their immediate progeny after injury. Nonetheless, because a relatively small population of cells were labeled during the one-week “pulse” and many of these cells were also PCNA positive after injury, this experiment suggests that both during normal conditions and during repair from injury a group of renal epithelial cells cycles at a much higher frequency that most other epithelial cells. This raises the possibility that these cells could be adult, renal epithelial precursors. We exploited the low cycling frequency of adult stem cells by giving BrdU to newborn rats or mice during a short pulse, and “chased” the animals to

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adulthood.73 In addition, to circumvent the use of BrdU, we24 also followed the suggestion of Tumbar et al.74 and used transgenic mice that expressed a fusion protein of histone 2B and GFP (referred as H2BGFP mice) under the control of a tetracycline-responsive element. Pregnant females given doxycycline give birth to pups where all cells are GFP1 cells, but when these pups are chased to adulthood, only low-cycling cells remain GFP1. As shown in the Figure 29.4, after 2 3 months of chase, label-retaining cells were only found in the kidney papilla. When isolated, the cells showed pluripotency and, like other stem cells, formed spheres.24,73 Under normal conditions, most of these papillary label-retaining cells (LRC) in the adult kidney are, unsurprisingly, in growth arrest, but we found in the upper papilla a small population of them that were cycling24 (see Figure 29.5), indicating that they generate new cells and explaining that the number of papillary LRC slowly decreased with age. In marked contrast to the normally low-cycling frequency of the papillary LRC, many of these cells proliferated shortly after acute kidney injury, likely generating many daughter cells, since after a few weeks the S-phase label disappeared from the papilla (see Figure 29.6). Hence, the cells we identified in the kidney papilla showed two cycling characteristics: first, during normal homeostasis, the cells are low-cycling (and thus they are LRC) except for a small population of them that are cycling in the upper papilla; second, and in marked contrast to normal conditions, may of the papillary LRC start cycling shortly after injury. These results strongly suggest that the population of LRC of the kidney papilla is involved in kidney repair, and that within the LRC cells are renal stem/precursor cells. Because the renal capsule contains a population of nestin-expressing cells,75 and this intermediate filament protein has been found in a variety of precursor cells,76 Park and co-workers77 searched for low-cycling cells in this part of the kidney. They administered a short pulse of BrdU to 4-week-old mice, and followed the pulse by a chase period of two months. They found that the kidney capsule had a sparse population of BrdU-retaining cells which further characterization showed them to be CD291, Sca-11, vimentin1, and nestin1, but that did not express hematopoietic or endothelial markers. These cells showed marked proliferative and clonogenic capacities, and could differentiate towards adipogenic, chondrogenic, and osteogenic lineages, suggesting that they were MSC or MSC-like. The function of these cells in vivo remains unknown, and it would be of great interests to determine whether kidney injury induces their proliferation.

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Cortex

Papilla

Perlecan

GFP

FIGURE 29.4 Papillary LRCs in mice expressing histone 2BGFP. The kidney of an 11-week-old mouse pulsed with doxycycline during embryonic life showed low cycling cells (i.e., GFP- retaining) in the papilla but not the cortex or medulla. (From ref. [27].)

Cellular Function Since first used to enrich for hematopoietic stem cells,78 80 the Side Population discrimination assay has been used to identify stem cells and progenitor cells in a variety of organs and tumors.81 The assay can be useful when stem cell-specific markers are not available, and it is based on the differential ability of cells to efflux a lipophilic fluorescent dye via the ATP-binding cassette transporter protein ABCG2. Many stem/precursor cells rapidly efflux the Hoechst 33342 dye, a process that can be blocked with ABC transporter inhibitors. By flow cytometry, such cells are recognized as being “Hoechst low” (versus “Hoechst high”) and as they are a very small fraction of the total cells, they were termed side population (SP) cells. Because the assay is dependent on normal cellular metabolic function in freshly isolated cells, it is extremely challenging (for a detailed discussion see Golebiewska et al.81), and uncertainty exists about the significance of some of the results. Nonetheless several workers have isolated SP cells from the adult kidney.82 85 The frequency of these cells has been reported to vary from B5%84 to B0.1%.83,85 In one study, exogenous administration of the isolated cells to mice with cisplatin-induced acute kidney injury improved the kidney’s functional response.84 These provocative results need further analysis, particularly because the SP of the kidney

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GFP

GFP

Ki67

US

Ki67

US

US

Merge

FIGURE 29.5 LRC in the upper papilla from chains of proliferating cells. LRCs (GFP1: top) and proliferating cells (Ki67-positive: middle) in the upper papilla. The merged image shows that several cells are positive for both Ki67 and GFP (arrows). Broken white line depicts papillary edge (US: urinary space). (From ref. [27].)

FIGURE 29.6 Proliferation of papillary LRC after transient ischemic injury. Cellular proliferation of papillary LRC after transient renal ischemia. Thirty-six hours after ischemic injury, many LRC of the papilla (FITC fluorescin) were cycling and labeled by a Ki67 antibody (rhodamine) (Scale bars: 50 μm). (From ref. [76].)

appears to show considerable heterogeneity.83 However, the kidney is a transporting organ with a large number of ABC-type transporters expressed in several of its segments including ABCG2, the putative transporter of the Hoechst33342 dye. Hence, the presence of cells that pump the dye out may not be the best marker for progenitor cells. Another cellular function that has been exploited to isolate putative renal stem/precursor cells is the aldehyde dehydrogenase activity (ALDH). This enzyme oxidizes intracellular aldehydes (for a review see Sophos and Vasiliou86), and it is believed to have a role in cellular differentiation by its ability to oxidize retinol to retinoic acid.87 Hematopoietic, neural, and some cancer stem/precuror cells were found to have high ALDH enzymatic activity.88 Similarly, Lindgren et al.89 recently isolated from the human kidneys cells with progenitor characteristics. Using an ALDH enzymatic

assay in suspensions of renal cortical cells deprived of glomeruli, they separated the cells into ALDHlow and ALDHhigh populations. Interestingly, transcription analysis of the populations showed that one of the most upregulated genes in the ALDHhigh cells was Cd133, a membrane protein found in some progenitor/ stem cells and used to isolate other renal progenitors (see above and the section on podocyte precursors next). In vitro, the cells showed some stem/precursor cell characteristics, but of particular interest is their finding by immunohistochemistry that there is a population of Cd133-positive cells scattered through the proximal tubule, clearly showing that there is some heterogeneity in the proximal tubular cells. Analyses of kidney biopsies of patients with acute tubular necrosis showed that in areas of regeneration tubular cells positive for Cd133 in their luminal membrane were frequently found to be also positive for vimentin in their

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basolateral membrane. Since tubular epithelial cells expressing vimentin have been observed during the recovery phase of acute kidney injury,90,91 it is possible that these Cd133- and vimentin-expressing cells might be specialized epithelial precursors.

Podocyte Precursors It had long been assumed that in adult kidneys podocytes, the specialized epithelial cells that surround the glomerular capillary loops, have extremely low or no cycling activity. Yet, these cells are clearly renewed because they can be captured in the urine of normal subjects.92 Interest in the origin of podocytes and their life cycle has been spurred by observations that their numbers can decrease during diseases caused by several mutations that disrupt their proteins, leading to glomerular sclerosis (reviewed by D’Agati93). In addition, it was recently found that a variable fraction of the cells in the glomerular “crescents” (layers of abnormal cells attached to the Bowman’s capsule) often present in glomerulonephritis were found to express nestin.94 Since normal podocytes express nestin, these results raised the possibility that cells in crescents might derive from podocytes or that podocytes and cells in crescents share a common lineage. Regardless, these interesting observations suggested that both podocyte loss and/or their uncontrolled proliferation might be involved in renal diseases. Analyzing human kidney sections, Sagrinati et al.95 found that a subpopulation of the parietal epithelial cells of Bowman’s capsule expressed Cd24 and Cd133, two cell surface proteins found in a variety of adult stem cells. Isolation of these cells from glomerular cultures showed them to possess high cloning efficiency and some differentiation potential. In addition, when the cells were intravenously administered to mice with acute kidney injury, there was improvement in the morphological and functional renal response.95,96 The observation that some epithelial cells in the parietal side of the Bowman’s capsule had progenitor characteristics led to the hypothesis that these cells could be podocyte precursors.97,98 With this in mind, Ronconi et al.98 analyzed human kidneys sections, and confirmed that a group of cells in the parietal epithelia of the Bowman’s capsule expressed Cd133 and Cd24 and that, in contrast, fully differentiated podocytes only expressed proteins used to identify them such as nestin, complement receptor-1, and podocalyxin. Interestingly, they also identified a third cell population that expressed Cd133, Cd24, and podocyte markers, suggesting that it could be a podocyte precursor. Isolation of these three different cell populations by FACS using Cd133, Cd24, and podocalyxin and their

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culture in vitro showed that a cell clone derived from a cell only positive for Cd133 and Cd24 contained cells that expressed both tubular epithelial markers and podocyte markers. In contrast, clones of cells derived from cells positive for Cd133, Cd24 plus podocalyxin only expressed podocyte markers. When these two different types of cells were intravenously administered to mice that had received the podocyte toxin adriamycin, they found that cells only positive for Cd133 and Cd24, but not cells positive for Cd133, Cd24, and podocalyxin, were capable of reducing urinary protein excretion and morphologic damage. Finally, labeling the cells with vital dyes showed that the former, but not the latter, localized to glomeruli. These results were interpreted as indicating that in adult kidney, podocytes derive from a precursor cell population that resides in the parietal epithelium of Bowman’s capsule. One might conclude that the study shows heterogeneity in the parietal epithelial cells of Bowman’s capsule, and that in vitro these cell types express different genes. The mechanism responsible for the functional recovery after cell injection and its significance is harder to define, since the large majority of epithelial cells administered intravenously are likely to lodge into the lung vascular bed. The hypothesis that parietal epithelial cells might migrate into the glomerular capillaries and differentiate into podocytes was also examined by Appel et al.97 in a detailed and elegant study. These authors reported that in adult rats, cells that had morphological characteristics of both glomerular parietal epithelial cells and podocytes could be detected by electron microscopy in the glomerular vascular stacks. That these cells could be transitional cells between parietal epithelial cells and podocytes was examined in 10-day-old mice with a variety of approaches. By immune-detection they found that some cells had markers of both parietal epithelial cells and podocytes. Administration of BrdU to young rats for two weeks showed that, while a substantial number of parietal epithelial cells were labeled with BrdU, only B0.5% of podocytes were labeled. However, as the animals aged, the number of BrdU1 parietal epithelial cells decreased, while that of BrdU1 podocytes increased, suggesting that the latter derived from the former. In a final genetic fate-mapping experiment, these workers generated triple-transgenic mice in which labeling of the parietal epithelial cells and their progeny could be temporarily controlled by doxycycline administration. In 5-day-old and 10-day-old mice they found that the number of podocytes that derived from parietal epithelial cells increased as the animals aged. This elegant study provides strong evidence that podocytes derive from parietal epithelial cells, at least in the young. This is a very exciting result, because even if this process only occurs at very young

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ages, understanding of its controlling mechanisms might allow its modulation. An additional conclusion appears also important; it is clear that as our understanding of the homeostasis of the renal cell populations advances, the distinction between embryonic and adult lives is becoming less clear-cut than previously believed (i.e., “development” appears to be a life-long process), a subject to which we return below. Because a variety of glomerulonephritis types are associated with hyperplasia of Bowman’s capsule (a histological finding long ago termed “crescents”), the possibility that unregulated proliferation of a precursor cell could be responsible for their generation was examined. In a study of human kidney biopsies, the hypercellular lesions of crescentic glomerulonephritis were analyzed with antibodies and RT-PCR after laser capture of the lesions.99 The authors found that these lesions contained three kinds of cells: Cd1331, Cd241, podocalyxin2 and nestin2 (presumed podocyte precursors), Cd1331, Cd241, podocalyxin1 and nestin1 (named intermediate cells), and finally, Cd1332, Cd242 but podocalyxin1 and nestin1 (same profile as that of differentiated podocytes). When the lesions were examined for the presence of proliferating cells with a Ki67 antibody, most of the cells that were cycling were only positive for Cd1331, Cd241. Further, in vitro, Cd1331, Cd241, podocalyxin2 and nestin2 cells responded robustly to TGFβ induction of extracellular proteins. These results were taken as evidence that generation of glomerular crescents is likely due to proliferation of “podocyte progenitors.” In a related experimental study, Smeets et al.100 induced crescentic glomerulonephritis in triple-transgenic mice in which parietal epithelial cells and their progeny could be labeled by the administration of doxycycline. In these mice, “crescents” could be detected to be positive by enzymatic X-gal staining, strongly suggesting that these cells derived from epithelial cells of the Bowman’s capsule. In contrast, podocyte genetic labeling experiments did not show that the cells in the crescents derived from podocytes. These interesting results strongly suggest that some cells in the parietal epithelium of the Bowman’s capsule, and not the podocytes, proliferate in response to injury. However, in a fascinating study, Ding et al.101 found that deletion of the product of the von Hippel-Lindau gene (Vhlh, encoding VHL) specifically in podocytes caused glomerulonephritis with crescent formation. Loss of VHL stabilizes the hypoxia-inducible factor (HIF) α-subunit and activates hypoxia-response genes. Of great interest is their finding that the cells in the crescents were derived from podocytes; this was elegantly shown by laser-capture of the crescents and PCR demonstrating the rearrangement of the genomic DNA induced in the podocytes (a tour de force control

experiment which unfortunately is rarely done). In addition, because the gene expression profile revealed induction of Cxcr4 expression in glomeruli, Ding and co-workers overexpressed this receptor in podocytes; this resulted in podocyte proliferation and glomerulonephritis. This extremely interesting study therefore indicates that under some conditions, podocytes can proliferate, likely by the effect of Cxcl12 (SDF-1) the ligand of Cxcr4, and that their proliferation can lead to “crescent” formation.

Renal Mesenchymal Stem Cells and/or Pericytes Mesenchymal stem cells (MSC) from the bone marrow were originally characterized by their ability to adhere to culture surfaces and when cultured with appropriate differentiation media, by their differentiation potential into adipocytes, osteoblasts, chondrocytes, and, sometimes, myoblasts. In other organs including the kidneys, MSC identification and characterization remains to be formalized, since they might be a heterogeneous population that expresses a variety of cell surface markers.5 Nonetheless, there is widespread interest in MSC because, in addition to their differentiation potential, they appear to be present in most if not all organs4,5 including the kidney,5,102 and their precise function remains to be defined. They preferentially locate in the perivascular space4,5 of even large blood vessels such as the aorta and vena cava,5 and have pericycte-like characteristics.3 In the kidney, mesangial cells also have MSC-like characteristics,5 and might be the glomerular cells that express Cd1461 and Cd1332 found by Bruno et al.103 in glomeruli. Because of their perivascular location and mesenchymal origin, pericytes are difficult to distinguish from MSCs, and how these two cells differ and relate is still poorlyunderstood. Although true renal pericytes can only be unambiguously identified by their anatomical location surrounding capillary endothelial cells, these cells have been implicated in myofibrobalst generation during renal fibrogenesis,15 and are discussed in detail below. In organs other than the kidney, pericytes have been found to have MSC-like characteristics,104 and in the most detailed study to date, perivascular cells from skeletal muscle, bone marrow, and adipose tissue were found to express several pericyte markers, as well as MSC markers and MSC differentiation potential,4 suggesting that MSC and pericytes might be the same cells in different locations. It has been shown that during tooth growth and in response to injury, pericytes can give rise to MSC, although in the same organ MSC have an additional, pericyte-independent origin,105 so that lineage analysis between MSC and pericytes is likely to be complex. In the kidney, mesangial cells,

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POTENTIAL SITES HARBORING RENAL STEM CELLS

vascular smooth muscle, pericytes, and likely, MSC derive from the Foxd1 embryonic cellular compartment described above but as stated, the exact relationship among these different cells remains to be defined. It is currently unknown whether renal pericytes/ MSCs generate daughter cells important for normal kidney homeostasis or for repair from injury. However, recent studies showing that pericytes characterized as Cd731, platelet-derived growth factor receptor1 and smooth muscle actin negative, give rise to myofibroblasts during renal fibrosis15 have generated a great deal of interest, a finding that we discuss below. However, morphological observations indicating the transient presence of myofibroblasts after transient kidney injury43,106 suggest that these cells, long studied from a pathogenic perspective, might also be involved in organ repair, a possibility that we believe deserves attention. Regardless, since like other organs, the kidney contains perivascular cells that are pericytes, MSC or their immediate progenitors,15 their full characterization and role in normal renal homeostasis and if any, in organ repair from injury, would be of great interest.

POTENTIAL SITES HARBORING RENAL STEM CELLS Given the kidney’s anatomical complexity, identification of stem cells within it might be facilitated by focusing on sites that might harbor them. Several strategies are possible to locate candidate sites, but we believe that current evidence already suggests the presence of sites likely to contain stem cells.

Highly Proliferative Regions Because many adult stem cells are low-cycling (see and 107 for recent reviews), to generate needed new cells they frequently give rise to an immediate progeny that experience several “transit amplifying” (TA) divisions prior to their differentiation. In this way, many new cells can be regenerated from infrequent stem cell cycling. Hence, adult stem cells can be located close to sites of increased cell cycling. Indeed a seminal discovery in this field showed that there was postnatal neurogenesis in the granular zone of the dentate gyrus in the hippocampus,57 and led to the discovery that the subgranular zone harbored neural stem cells (reviewed in Zhao et al.108). Hence, proliferative regions during normal homeostasis or after kidney injury are good candidates to harbor renal stem cells. After nephrogenesis is completed in young rats and during normal homeostasis, it was found that the S3

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segment of the proximal tubule displayed high cycling activity25,28: over a two-week period in 4-week-old rats, 90% of the cells of the S3 segment had cycled, a remarkable rate of proliferation. While this may reflect morphogenetic requirements of a growing kidney, the high proliferative capacity of these cells raises the possibility that they could be “transit amplifying” daughter cells of a proximal tubule stem cell. Other findings support that the S3 segment of the proximal tubule might contain stem cells. Shortly after several types of kidney injury, high proliferation activity was particularly prominent in cells from the S3 segment.25,42,43 Moreover, Gupta et al.62 found cells in the cortico medullary junction which expressed Oct-4 and had progenitor characteristics, and Kitamura et al.63 isolated a highly proliferative cell type from the S3 segment of rat kidneys that, at least in vitro, also expressed precursor cell markers. Whether these putative stem cells relate to the recently described podocyte precursor (see above) or renal proximal tubule progenitors95 remains to be determined. The S3 segment of the proximal tubule is of additional interest as a potential site to harbor renal stem cells, because indirect determinations of oxygen tension have shown that this part of the nephron surprisingly exists in a hypoxic environment quite different from other parts of the kidney cortex.109 A low oxygen tension is characteristic of sites where stem cells in other organs reside, a subject that we discuss below. To determine whether the adult kidney contained regions of increased cell proliferation, we used kidneys of one-year-old rats, because we reasoned that fully grown older animals would not have proliferating cells needed for organ growth. We found cycling cells by staining for Ki67, a marker of cellular proliferation.24 As shown in Figure 29.1, Ki67-positive cells were rare in the cortex and medulla, and were solitary and of a similar frequency (B1 % of the total cells). In contrast, the papilla showed marked heterogeneity in its abundance of Ki67-positive cells; they were extremely rare in its tip and middle part (B0.1 %), but were readily apparent in the upper part of the peripheral papilla, adjacent to the urinary space (US) where the kidney parenchyma forms a narrow angle which provides an easily identifiable landmark and the papilla meets the medulla; it is also very close to the kidney cortex. In this region, and unlike other areas of the kidney, Ki67positive cells frequently formed chain-like structures and accounted for B2.5 % of the total cells, a significant difference from other parts of the kidney. When cellular proliferation was assayed 24 hours after labeling cells in S-phase by administration of a single dose of BrdU, proliferating cells were very rarely detected in the cortex and medulla but again, the lateral part of the upper papilla consistently contained cycling cells; i.e.,

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BrdU-labeled (not shown). We also obtained similar results in kidney of six-month-old rats. Interestingly, after acute kidney injury, the lateral part of the upper papilla is also the site in which we first detected the increased cell cycling that follows injury; rats were subjected to 45 minutes of unilateral kidney ischemia, following which were given a single dose of BrdU and sacrificed one hour later. Thus, only cells that were in S-phase during this hour could be labeled, and using 5 μm kidney sections, we saw only equally rare labeled cells in normal kidneys or in the kidneys subjected to ischemia. However, examination of 100 μm vibratome sections showed abundant cells in S-phase in the upper papilla (Figure 29.3), next to the urinary space (US) only in the kidneys subjected to ischemia. In the aggregate, these results indicate that the lateral upper part of the kidney papilla contains more proliferating cells than other parts of the kidney under normal conditions, and that it contains the first cells that start cycling in response to transient injury. As reviewed above, Vinsonneau et al.45 found that the first intrarenal area that contains Ki67 positive cells after ischemia reperfusion injury was the uro-epithelium and adjacent interstitial space in the upper part of the urinary space at the papilla medullary junction. In sum, because of their proliferative behavior, both under homeostatic conditions and during repair from several types of kidney injury, the S3 segment of the proximal tubule and the lateral part of the upper papilla are domains of the kidney where stem cells appear likely to reside.

Stem Cell “Niches” Adult stem cells receive critical signaling from their immediate microenvironment, a domain that is distinct from the rest of the organ (see Morrison and Spradling110 for a review). In these locations (referred to as “niches”), stem cells interact with other cells and the extracellular matrix, a process that is best understood in the Drosophila germline stem cells (see Fuller and Spradling for a review111). The stem cell “niche” provides positional information that regulates both proliferation activity and cell fate identity, and in fact it can even redirect cell fate similar to what often happens during embryogenesis.112 While characterization of mammalian stem cell niches is still very limited, some common motifs are emerging that might be useful to identify the location of renal stem cells. Among those, a hypoxic environment is a prominent characteristic. Several organ-specific stem cells such as hematopoietic stem cells113 115 and neural stem cells116 reside in hypoxic regions, and a large body of in vitro work has shown the critical role of oxygen on stem cell

regulation (see Mohyeldin et al.117 for a review). Moreover, in both hematopoietic stem cells and neural stem cells, it was recently found that regulation of the oxygen-sensitive gene HIF-1α is critical for the cells’ maintenance and fate decisions.114 116,118 The kidney parenchyma possesses steep oxygen gradients,119 and variations in renal oxygenation are undoubtedly sensed as they regulate erythropoietin synthesis. While direct determinations of oxygen tension in the kidney papilla have not been reported, it is likely that papillary pO2 ranges are B4 10 mmHg.119 The presence of low-cycling cells in this very hypoxic environment24,39,73,120 and that many of these cells express nestin,24 an intermediate filament protein identified in a variety of progenitor/stem cells,76 suggests that the papilla harbors renal stem cells. Interestingly, using a nestin-GFP-expressing mouse,75 nestin1 cells were detected in large clusters within the papilla, along the vasa rectae and, at least in vitro, these papillary nesting-expressing cells were found to migrate upwards in the papilla towards the cortex, an observation compatible with our in vivo experiments with vital dyes in which we found upward migration of papillary cells, some of which were “label-retaining cells”.24 Several other findings also suggest that the kidney papilla is a niche for renal stem/precursor cells. Dekel and co-workers61 isolated a population of purportedly renal stem cells from the renal interstitium that expressed the stem cell antigen-1 (Sca-1) and were enriched for β1-integrin; when the cells were located in vivo, they were found to be particularly abundant in the renal papilla. Using a different approach, Lee et al.64 identified a renal interstitial cell that expressed several potential renal progenitor cell markers, such as Oct-4, Pax-2, Wnt-4, and WT-1. In vivo, Oct-41 cells were located in the medulla as well as in the papilla, leading these authors to suggest that the interstitium in these regions of the kidney is a “niche” for renal stem cells. Finally, and with a very different approach, Curtis et al.121 examined whether cells from different regions of the kidneys were equally capable of contributing to cell regeneration after acute kidney injury. These workers isolated from mice constitutively expressing β-galactosidase in certain cells from the kidney cortex, medulla or papilla, and implanted them under the capsule of kidneys subjected to ischemiareperfusion injury. Seven days later, implanted cells deriving from the three different regions of the kidney could be detected in the tubules of the renal medulla, but cells obtained from the papilla exhibited the most robust engraftment. These results suggest that cells deriving from all regions of the kidney might contribute to the reparative response of the organ after injury, and that some cells in the papilla are particularly capable of contributing to repair and might be stem cells.

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RENAL STEM CELLS IN NON-MAMMALS

RENAL STEM CELLS IN NON-MAMMALS Renal Progenitors in Fish Unlike mammals, fish appear to be capable of generating new nephrons during adult life, and their kidneys possess a clearly identifiable “nephrogenic zone”.122 This zone decreases in volume as the animal ages, likely because the nephron number increases with age.123 In addition and of great interest, the nephrogenic zone can be stimulated in adult fish. For example, in the little skate the cellular proliferation and morphological complexity of the zone increased after partial nephrectomy,122 a process that resembled embryonic nephrogenesis in mammals. While this morphological response strongly suggests de novo generation of nephrons, confirmation that new and functional nephrons developed remains to be established. Following acute kidney injury due to gentamicin, zebrafish can regenerate their nephrons, a process that starts shortly after injury with expression of wt1b (the zebrafish homolog of Wilm’s Tumor 1) in individually dispersed cells of the mesonephric interstitium.123 These studies suggest that the fish could be a useful model to explore mechanisms of kidney repair where one can derive information that might facilitate identification of mammalian adult renal stem cells. Indeed, in a recent elegant study, Diep et al.124 used zebrafish to search for potential nephron precursor cells. They reasoned that if adult kidney contains nephron progenitors these cells could be transplanted. They induced acute kidney injury by gentamicin administration, and performed a series of transplant experiments using cells from transgenic fish expressing reporters such as EGFP or Cherry with cdh17, thereby locating the fluorescent reporters in the distal nephron. When they injected unpurified whole kidney marrow cells (5 3 102 cells), mostly comprised of non-tubular interstitial cells, they found that the donor cells generated new nephrons in the host in 100% of the cases (Figure 29.7). Nephron number increased with time after injection and at later times, donor-derived nephrons were found at sites distal from the injection site, indicating that the cells migrated. Importantly, they found that these donor-generated nephrons connected to the host renal tubules and to the host’s circulation, as shown by the fact that they could filter fluorescent dextran. Because in cell transplantation experiments it is frequently impossible to be certain that fusion of donor and recipient cells did not occur, to exclude this possibility Diep et al.124 injected Cherry labeled cells into EGFP recipients and found that all engrafted nephrons were Cherry1 and EGFP negative, indicating that cell-to-cell fusion did not take place. To identify the cells responsible for the genesis of new

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nephrons, the authors examined the zebrafish homologs of transcription factors know to be expressed in the mammalian renal vesicles (the precursors of the nephron). They found that kidneys from adult zebrafish transgenics expressing lhx1:EFGP (the homolog of Lh1/Lim 1125,126) contained B100 aggregates of B30 EGFP1 cells per kidney. Examination of the evolution of these aggregates showed that they emerged from the assembly of B3-4lhx1:EFGP1 cells that expanded to form a renal vesicle (expressing wt1b, the homolog of mammalian WT1), which in turn elongated into a nephron. Interestingly, laser ablation of a single aggregate of lhx1:EFGP1 cells prevented nephron formation without affecting neighboring nephrons, indicating that lhx1:EFGP1 cells are required for nephrogenesis. However, transplantation of single lhx1:EGFP1 cells to conditioned hosts failed to engraft, but transplantation of aggregates of lhx1:EFGP1 cells generated nephrons. Thus, lhx1:EFGP1 aggregates contain nephron progenitors and several cells are needed to generate a nephron. Whether the cell aggregates are clonal or contain several nephron progenitors with more restrictive differentiation potential is unclear. Nonetheless, demonstration in adult zebrafish of nephron progenitors capable of kidney regeneration and identification of their “genetic signature” is likely to facilitate design of new studies searching for renal stem cells in adult mammalian kidney.

Renal Progenitors in Invertebrates The nephron is the basic structure of the vertebrate kidney, but many insects have tubular excretory systems with “nephron-like” features, suggesting that at least some components of the vertebrate kidneys derive from invertebrate ancestors. In addition, to regulate the composition of hemolymph, Drosophila has filtration cells that possess fly orthologs of the major components of the slit diaphragm, indicating that these cells are podocyte-like.127 The similarity of the fly’s excretory system to that of the nephron in vertebrates suggests that analysis of renal stem cells in Drosophila might be of considerable interest. Indeed, Singh et al.128 used lineage tracing, molecular marking analysis, and BrdU incorporation to follow the fate of the different cells in the Malpigian tubules of adult flies. These tubules contain four types of cells, and Singh et al.128 found that one type of cell was multipotent and could generate all the cell types of the Malpigian tubules of adult flies, indicating that they are renal stem cells. Singh et al. additionally found that autocrine JAK-STAT signaling in the renal stem cells regulated their renewal, and absence of signaling promoted their differentiation with loss of the stem cell

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FIGURE 29.7 The kidney of the adult zebrafish contains progenitor cells that generate new nephrons. (a) Transplantation assay. Whole kidney marrow cells mostly comprising interstitial cells were isolated from genetically labeled donors. (b) Injection of the cells into recipients with damaged nephrons due to genatmicin injection resulted in donor-derived nephrons. (From ref. [127].)

population. Of great interest was their additional finding that these stem cells are present in the lower tubules and ureters, very much resembling the initial location of the nephron precursors recently identified in zebrafish,124 and raising the possibility that the sharp distinction currently being made in mammalian adult kidneys between collecting ducts and the rest of the nephron might require re-evaluation. Also remarkable is that these stem cells are a large fraction of the total number of cells of the Malpighian tubules, rather than the anticipated small minority, another characteristic worth noting for workers on the renal stem field.

NEW CELLS IN DISEASED KIDNEYS Renal Fibroblast and Myofibroblasts In addition to dendritic cells, the normal adult kidney contains a network of interstitial fibroblasts and other cells that remain poorly-characterized, as they are technically difficult to detect and there is no general agreement on markers that identify them.1 However, during many kidney diseases the renal interstitial compartment changes dramatically, with a marked increase in the number of renal fibroblasts and the invasion of the interstitium by a new type of cell called a myofibroblast. Myofibroblasts have fibroblast-like characteristics, but also express α smooth muscle actin (αSMA), and are believed to have great migratory capacity. It is currently thought that the marked proliferation of fibroblasts and myofibroblasts plus the expansion of their secreted extracellular matrix, i.e., renal fibrogenesis, is itself pathogenic as it disrupts the normal renal architecture and is the final common pathway of many kidney diseases. Indeed, expansion of the fibroblast and myofibroblast populations and the extracellular matrix are tightly correlated with the slow and

inexorable progressive loss-of-function that characterizes many kidney diseases. There is thus great interest in the mechanisms responsible for appearance of these cells during diseases. Several possibilities have attracted attention. First, the cells might derive from resident renal interstitial “fibroblasts.” In this view, the disease process induces fibroblast proliferation and in some of them, a change in their phenotype that results in expression of αSMA. Detailed morphological studies suggest that at least in some forms of renal fibrosis, such as that due to unilateral ureteral obstruction (UUO), this is likely the case.129 More recent genetic cell fate tracing studies by Humphreys et al.15 have suggested that myofibroblasts after unilateral ureteral obstruction (UUO) or after acute ischemic kidney injury derive from interstitial cells that are likely pericytes, since they were positive for Cd73 and platelet-derived growth factor receptor β (PGDFR β). Interestingly, pericyte differentiation and proliferation appears to be dependent on endothelial cell action, since blockade of their VEGF receptor 2 attenuates the fibrogenic process.22 However, while Cd73 and PGDFR β appear to be present in most pericytes, there is no definitive marker profile for these cells, as they can only be unambiguously identified by their location around capillary endothelium. Moreover, new studies have shown that most, if not all, organs possess a population of perivascular mesenchymal stem cells,4,5 and the distinction between these cells and pericytes is poorly-understood (see Corselli et al.130 for a review). Hence, whether myofibroblasts solely derive from resident interstitial fibroblasts, pericytes, mesenchymal stem cells or from a combination of these cells remains to be clarified, and must await a detailed characterization of these three interstitial cell types. Another possibility is that myofibroblasts could derive from bone marrow mesenchymal stem cells

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KIDNEY REPAIR BY EXOGENOUS STEM/PRECURSOR CELLS

(MSC); i.e., cells characterized by their ability to differentiate into adipocytes, osteoblasts, chondrocytes, and, sometimes, myoblasts. While Iwano et al.131 found a small number of bone marrow-derived fibroblasts in the renal interstitium, little additional evidence has been obtained to support that bone marrow precursor cells could be the origin of the myofibroblasts. Another possible origin of the renal fibroblasts and myofibroblasts found in kidney diseases is that they derive from renal epithelial cells by epithelial-mesenchymal transition (EMT), a process in which epithelial cells lose the ability to maintain close contacts with their neighbors as well as apico basal polarity, and instead develop a mesenchymal phenotype that includes migratory capacity. The process of EMT is of course widely used during embryogenesis and in some disease processes, particularly cancer (for a recent review see Thiery et al.132). It is of great interest that in nonrenal cells, it has been found that EMT might generate cells with the properties of stem cells,133 but this possibility appears not to have been examined in the kidney. There is little doubt, at least in vitro, that epithelial cells from both embryonic134 and adult kidneys15,135 can undergo EMT. However, there is currently a debate whether this process might generate fibroblasts and myofibroblasts in vivo (see Kapus and Quaggin136 and Kriz et al.137 for informed discussions). Morphological and cell marker studies during renal fibrosis have provided some evidence supporting EMT in vivo, both in experimental models90,138 142 and in human diseases,90,91,143,144 but other studies have failed to support this.129,145 The most robust method currently to determine whether myofibroblasts might derive from renal epithelial cells is based on studies using genetic cell fate analysis. In these studies epithelial cells are genetically labeled so that their progeny (e.g., fibroblasts or myofibroblasts) can be easily identified. Using the UUO model of renal fibrogenesis, Iwano et al.131 found that a substantial number of renal fibroblasts derived from renal epithelial cells. However, three subsequent studies with UUO plus other models of renal fibrogenesis and using different epithelial genes to drive Cre recombinase and label epithelial cells in reporter mice found no evidence that renal fibroblasts and myofibroblasts derived from epithelial cells.15,146,147 There is currently no clear explanation for these conflicting results, and it is apparent that additional studies are needed. Finally, another potential origin of renal fibroblasts and myofibroblasts are endothelial cells. Genetic cell fate experiments have shown that Cre recombinase driven by Tie2 (a tyrosine kinase receptor believed to be endothelial-specific in the adult) identified a reporter gene in renal fibroblasts in UUO and

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streptozotocin-induced renal fibrogenesis.148,149 This unexpected result is of particular interest, because adult endothelial cells have been shown to have the capacity to transform into mesenchymal stem-like cells by activation of activin-like kinase-2,153 and two ligands well-known to be involved in renal fibrogenesis, TGFβ and BMP4, phosphorylated activin-like kinase-2 and induced a mesenchymal phenotype in cultured endothelial cells.150 This brief survey shows that the precise origin of the renal fibroblasts and myofibroblasts during renal diseases remains undefined, and given the medical importance of this issue additional studies are needed. Needless to say, it is also likely that these cells might have more than one origin. While studies based on morphology and/or cell identification by cell-specific markers offer at best indirect evidence, genetic-based cell lineage tracing experiments have their own potential pitfalls (see the review by Matthaei151). In particular, two potential problems might complicate their interpretation. The first is that it is assumed that the reporter gene is expressed in all cells and for the duration of the animal’s life, as well as during the disease process. Definitive proof of this is lacking and, for example, in the adult kidney, the robustness of the reporter expression in the Rosa26 locus (frequently used to introduce the reporter gene) was found to be less than anticipated (152 and our unpublished results). In addition, cell fate-mapping studies can give markedly different results depending on the genetic construct of the reporter.153 The second, and potentially more confounding, problem in the interpretation of genetic cell-fate analyses is that the gene used to activate the reporter gene is chosen because of its restricted expression to a specific cell type during embryogenesis, and lack of expression during adult life and after injury, when the analysis is done. However, the possibility that the gene is activated later in life or by the disease process in a small number of cells might be very difficult to detect.

KIDNEY REPAIR BY EXOGENOUS STEM/PRECURSOR CELLS While analysis of the kidney’s ability to repair from injury has naturally focused on the intrinsic renal cell population, reports that cells derived from the bone marrow could generate new renal cells and perhaps contribute to organ repair generated a great deal of interest.154 157 However, subsequent studies have firmly established that most if not all the new cells generated after kidney injury derived from other renal cells.29,30 Nonetheless, and stimulated from work in other organs, the possibility that exogenous cells might

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either facilitate kidney repair or diminish the consequences of injury has been examined in multiple studies. Indeed, clinical trials with bone marrow-derived MSC are already underway or in planned stages in a variety of diseases, particularly for myocardial infarction, but also including the kidney (http://www3. niddk.nih.gov/fund/other/akiworkshop/). In experimental animals, the potential therapeutic effect of several cell types in acute kidney injury has been examined under a variety of protocols.

Mesenchymal Stem Cells (MSC) When HSC and bone marrow-derived MSC were administered to mice it was found that the latter, but not the former, protected the animals from the functional consequences of acute kidney injury due to cisplatin.156 Multiple studies have confirmed that extrarenal MSC can either diminish the functional consequences of acute kidney injury or accelerate recovery of kidney function after injury. In addition to MSC derived from the bone marrow,23,158 161 MSC from human cord blood162 or even the kidney163 have been shown to be effective. In these studies, acute kidney injury was induced by several methods and the cells were administered through a variety of routes: intravenously, intra-arterially and by direct intrarenal injection. What are the potential mechanisms by which exogenous MSC might protect the kidney from injury or facilitate its repair? Although initial observations suggested incorporation of the cells into the renal parenchyma,156 more recent studies have shown that their mechanism of action is likely to be that the cells provide endocrine/ paracrine factors, perhaps growth factors23,161,164 or chemokines such as IL10.161 In this view, the mechanism of action of the MSC would be similar to that of directly delivering growth factors in transient kidney injury (reviewed by Hammerman and Miller165). Unfortunately, very little information is available on the identity of these beneficial factors, and whether MSC are more or less effective than isolated growth factors alone. Another possible mechanism is that the transplanted MSC might modulate the host immune response by effects on macrophages,166,167 dendritic cells,168,169 T-cells168 or B-cells.170 Given the complexity of the kidney’s response to the insults used to induce functional acute renal failure, it is apparent that there might be multiple mechanisms whereby exogenous cells could protect this organ from injury or facilitate its repair. An additional problem complicating analysis of these studies is that because MSC are identified a posteriori (based on a variety of characteristics such as high proliferation capacity and ability to differentiate into several mesenchymal lineages) there are no strict identification

criteria for the administered cells, making comparison across studies extremely difficult. Regardless, given the clinical severity and epidemiological importance of acute kidney failure, the finding that MSC can improve its evolution is of extreme interest, and studies that illuminate their mechanisms of action are urgently needed. Work in other organs has begun to provide answers. In an elegant study in myocardial repair from ischemic myocardial infarction, Lee et al.171 found that human MSC (hMSC) administered intravenously acutely embolized into the lungs where they upregulated multiple genes, including the antiinflamatory protein TSG-6 (the product of tumor necrosis factor-stimulated gene 6). Similar to the observations in kidney injury, the injected hMSC reduced the myocardial inflammatory response to ischemia, decreased infarct size, and improved myocardial function. Administration of recombinant TSG-6 had similar effects as hMSC, but hMSC that were transduced with TSG-6 siRNA did not. This interesting study provides a detailed mechanistic explanation of why hMSC facilitate myocardial repair without engraftment into the organ, and opens new therapeutic avenues for research.

Other Bone Marrow-Derived Cells Earlier experiments with bone marrow-derived stem cells (other than MSC) suggested that they could ameliorate the functional consequences of transient acute kidney injury.154 While the possibility that these cells exerted this effect by generating significant numbers of renal epithelia cells has been ruled out,29,30 their mechanism of action remains unexplained and little explored. Lie et al.172 used human Cd341 hematopoietic stem/progenitor cells isolated from peripheral blood after granulocyte colony stimulating factor mobilization, and injected them into mice. They found that, whereas the cells did not localize to normal kidneys, they did so in injured kidneys. Moreover, administration of the cells increased cellular proliferation in the injured kidneys, accelerated kidney functional recovery, and increased animal survival. The exact mechanism(s) whereby these cells exerted these effects remain to be determined, and many possibilities exist. For example, in the heart where bone marrow-derived cells also had significant regenerative effects, their action appears to be mediated by resident cardiac stem cells. In a model of myocardial infarction by coronary ligation, Loffredo et al.173 administered a population of purified c-kit1 bone marrow-derived cells and found that they stimulated endogenous cardiomyocite progenitors. These progenitors are capable of differentiating into several cell types, including cardiac muscle cells, and their stimulation resulted in improved

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cardiac repair and enhanced function. In contrast, MSC were unable to stimulate endogenous myocardial progenitors.

Kidney Cells A variety of studies have examined whether administration of cells derived from the kidney might facilitate this organ’s repair from injury. All the cells used in these studies possess some “precursor/stem” characteristics, but were poorly-characterized and even their exact in vivo location is somewhat uncertain. Administration of cells derived from the S3 segment of the proximal tubule had no effect on the functional response to acute kidney injury due to ischemia-reperfusion.62,63 However, when the kidney was injured by cisplatin, subcapsular transplantation of the cells facilitated kidney functional recovery.174 Several studies have used cells from the kidney that were captured by their expression of Cd24 and Cd133, either from embryonic kidney175 or adult kidneys.95,96 In these studies, intravenous administration of the cells facilitated kidney functional recovery from rhabdomyolysis induced by glycerol administration. Lee et al.64 used a renal interstitial cell population expressing Oct4, Pax2, Wnt 4, and WT1 (discussed above), and found that intrarenal injection of the cells markedly blunted the functional consequences of kidney injury from transient ischemia. What is to be concluded form these experiments with exogenous cells? That the administration of MSC might improve the kidney’s functional response to injury appears well-established; however, that of other bone marrow cells or renal-derived cells appears to be less robust. Significant problems in many of these studies include: (1) poor characterization of the cell population used; (2) less than ideal monitoring of the kidney functional response, notoriously difficult to do in mice; and (3) lack of analysis of where the majority of the injected cells locate. Most cells injected into the renal artery are retained by glomeruli176 and the vast majority of intravenously injected cells remain in the lungs.171 The recent discovery that under specific culture conditions, MSC reduce their size such that when they are injected intravenously they can cross the pulmonary circulation166 is a finding that may allow the systemic delivery of MSC to the kidney. It is of course possible that injected cells that locate at sites other than the kidney might be responsible for the observed beneficial effects on renal function. Regardless of the location and potential mechanism of action of the injected cells, these initial results suggesting that exogenous cell therapy might be beneficial in acute kidney injury need confirmation and further analysis.

CONCLUDING REMARKS Whether the adult mammalian kidney contains stem cells that self-renew and are pluripotent is currently unknown. However, given its multiple cell types and by analogy with other organs, it is very likely that renal stem cells might indeed be present. In contrast to the mammalian kidney, it is established that the Malpighian tubule of Drosophila contains true stem cells, and that the zebrafish kidney can be regenerated from a small number of specialized epithelial precursor cells, strongly suggesting that it also contains stem cells. Most work searching for a renal stem cell has focused on the epithelial cellular compartment, likely because of its understood important functional role and the relative ease with which it can be identified. However, the adult kidney also contains an abundant population of interstitial cells that are just now beginning to be understood; the discovery that the transcription factor Foxd1 (Bcl2) is expressed during embryonic development in many of these cells has provided an important new research tool that should illuminate their role. While bona fide adult stem cells in the mammalian kidney remain to be identified, several observations suggest that some stem/precursor cells exist in several locations. Perhaps the best evidence that a specialized precursor exists is in the glomerular podocytes, although it also appears likely that, at least under pathological conditions, podocytes themselves can generate other cell types. Perhaps podocytes are not solely terminally-differentiated cells, but can give rise to other cell progeny. A surprisingly variable cell identity was recently discovered in some of the progeny of hair follicle stem cells; these daughter cells were found to return to the stem cell niche and serve as future stem cells.177 Confirming classical morphological observations, new genetic cell lineage tracing experiments have provided strong evidence that after acute kidney injury epithelial cells give rise to new epithelia cells, but it remains unclear whether there exist distinct epithelial stem/precursor cells. In fact, multiple observations are compatible with the presence of specialized epithelial progenitors/stem cells in the S3 segment of the proximal tubule. Is it possible that some of the conflicting results could be explained by the presence of two types of stem/precursor cells: one that normally divides to provide new cells during homeostatic conditions and a low-cycling stem cell that is activated during organ repair, as appears to be the case in other organs (see Fuchs68 and Li and Clevers107 for reviews)? Finally, several lines of evidence strongly suggest that the kidney papilla is a “niche” for renal stem cells. This very hypoxic part of the kidney contains many

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low-cycling cells (identified as “label-retaining cells”) that slowly disappear as the animal ages, and that after acute kidney injury quickly start cycling and markedly decrease in number after repair. While in vitro characterization of these cells has shown them to have many properties of stem/precursor cells, no array of specific cell markers can yet identify them, so that their identity and the identity of their progeny remains to be defined. It is our hope that genetic cell lineage analysis will allow us to answer these questions.

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