Glomerular Epithelial Stem Cells: the Good, the Bad, and the Ugly

BRIEF REVIEW www.jasn.org

Glomerular Epithelial Stem Cells: The Good, The Bad, and The Ugly

Laura Lasagni* and Paola Romagnani*†

*Excellence Centre for Research, Transfer and High Education for the development of De Novo Therapies (DENOTHE), University of Florence, Florence, Italy; and †Pediatric Nephrology Unit, Meyer University Hospital, Florence, Italy

ABSTRACT Global glomerulosclerosis with loss of podocytes in humans is typical of end- ing podocyte number.23–25 Because resi- stage renal pathology. Although mature podocytes are highly differentiated dent podocytes do not divide, this sug- and nondividing, converging evidence from experimental and clinical data gests that new podocytes derive by suggests adult stem cells within Bowman’s capsule can rescue some of this loss. regeneration. Regression of renal disease Glomerular epithelial stem cells generate podocytes during kidney growth and with remodeling of glomerular architec- regenerate podocytes after injury, thus explaining why various glomerular ture is observed in pancreatic transplant disorders undergo remission occasionally. This regenerative process, however, patients with type 1 diabetes after 10 is often inadequate because of inefficient proliferative responses by glomerular years of normoglycemia26 and in patients epithelial stem cells with aging or in the setting of focal segmental glomerulo- treated chronically with angiotensin- sclerosis. Alternatively, an excessive proliferative response by glomerular epi- converting enzyme inhibitors.23–27 thelial stem cells after podocyte injury can generate new lesions such as Taken together, these results imply there extracapillary crescentic glomerulonephritis, collapsing glomerulopathy and tip are stem cells in adult glomeruli with the lesions. Better understanding of the mechanisms that regulate growth and potential to regenerate podocytes. differentiation of glomerular epithelial stem cells may provide new clues for prevention and treatment of glomerulosclerosis.

J Am Soc Nephrol 21: 1612–1619, 2010. doi: 10.1681/ASN.2010010048 THE GOOD: GLOMERULAR EPITHELIAL STEM CELLS REGENERATE PODOCYTES Global glomerulosclerosis in humans ac- glomerulosclerosis (FSGS).1,8–13 Fi- companies most progressive renal pa- nally, mutations that produce a glomer- Stem cells are functionally defined by thology. Although primary injury to each ulosclerosis occur exclusively among their ability to self-renew and differenti- of the somatic cell types in the glomeru- genes expressed by the podocyte.14–16 ate into cell lineages reflecting their tissue 21 lar tuft associates with some form of glo- Interestingly, depletion of highly spe- of origin. The ability to self-renew stem merular disease, injury to endothelial cialized cells with limited capacity to di- cells is maintained by a process called and mesangial cells repair by prolifera- vide is a common pathway driving many symmetric division, where new daughter tion of adjacent cells.1,2 By contrast, types of organ failure.17–20 In other adult cells maintain all the functional and phe- 21 podocytes are highly differentiated, neu- organs, loss of highly specialized cells notypic properties of stem cells. How- ron-like cells that cannot divide,1,2 which during injury can be replaced by resident ever, once activated, stem cells can also explains why podocyte injury is a key stem cells.21,22 For example, neuronal cell regenerate by asymmetric division, pro- driver of focal or global glomerulosclero- depletion after ischemic injury generates ducing a daughter stem cell and a com- sis.3 Indeed, a large body of evidence brain dysfunction, but neuronal stem from experimental models suggests loss cells in the adult brain also drive replace- Published online ahead of print. Publication date of podocytes over a certain threshold in- ment of lost neurons with some func- available at www.jasn.org. duces glomerulosclerosis.2–7 Podocyte tional recovery.22 Accordingly, severe Correspondence: Dr. Paola Romagnani, Depart- number is also reduced in proportion to podocyte loss and glomerulosclerosis ment of Clinical Pathophysiology, Nephrology Section, University of Florence, Viale Pieraccini 6, the severity of injury and degree of pro- can be rescued occasionally by replace- 50139 Firenze, Italy. Phone: ϩ39554271356; Fax: teinuria, and predicts progression in ment.23–27 Data from experimental mod- ϩ39554271357; E-mail: [email protected] patients with diabetic nephropathy, els also demonstrate that regression of Copyright © 2010 by the American Society of IgA nephropathy, and focal segmental glomerulosclerosis can occur by increas- Nephrology

1612 ISSN : 1046-6673/2110-1612 J Am Soc Nephrol 21: 1612–1619, 2010 www.jasn.org BRIEF REVIEW mitted progenitor.21 In their normal environment, committed progenitors retain the capacity to divide and differentiate toward a particular lineage. Urinary pole Recently, we provided the first evidence C Detaching that adult human glomeruli contain a hier- podocytes archical population of stem and commit- A ted progenitor cells.28–33 These resident stem and progenitor cells localize within B the Bowman’s capsule and are identified by the presence of both CD24 and CD133, two surface molecules that are shared by different types of human adult ϩ ϩ stem cells.34,35 CD24 , CD133 cells localize at the urinary pole of Bowman’s capsule and exhibit self-renewal proper- Vascular stalk ties and also the potential to differentiate into podocytes or proximal tubular cells (Figure 1).33 Clonal analyses demonstrate this subset of parietal epithelial cells represent multipotent epithelial stem cells and not simply a mixture of Figure 1. The good: Glomerular epithelial stem cells regenerate podocytes. Glomer- ular epithelial stem cells (red) are localized at the urinary pole. A transitional cell unipotent progenitors.33 This feature population (podocyte progenitors, red/light blue) displays features of either glomer- was demonstrated by first culturing ular epithelial stem cells or podocytes (light blue) and localize between the urinary pole progeny derived from single CD24ϩ, ϩ and the vascular stalk. Cells that express only podocyte markers and the phenotypic CD133 cells and then transplanting features of differentiated podocytes localize at the vascular stalk of the glomerulus or them into SCID mice with focal segmen- within the glomerular tuft (light blue). Proposed mechanisms of podocyte regeneration tal glomerulosclerosis (FSGS).33 are depicted in more detail in (A), (B), and (C). (A) Glomerular epithelial stem cells can ϩ ϩ CD24 , CD133 stem cells follow a self-renew and also generate novel podocytes by progressively proliferating and phenotypical and functional hierarchy to differentiating toward the vascular stalk. This occurs as the kidney grows, during generate a population of podocyte-com- childhood and adolescence, and might also occur after an injury, which allows for a mitted progenitors between the urinary slow generation of novel podocytes, such as after uninephrectomy. (B and C) In and the vascular pole of Bowman’s capsule, glomerular disorders characterized by severe podocyte death or detachment, glomerular epithelial stem cells generate cell bridges between the Bowman’s capsule and the expressing both stem cells and podocyte glomerular tuft, which may allow a quick replacement of lost podocytes. (B) Cell markers (Figure 1). These progenitors dif- bridges may provide a slide for the migration, proliferation, and differentiation of an ferentiate only toward the podocyte lineage adjacent progenitor and a quick replacement of lost podocytes. (C) Bridging parietal 33 and lack the properties of self-renewal. epithelial cells might also acquire podocyte markers after injury and directly replace the Previous studies show the existence of lost podocytes. The directions of migration, proliferation, and differentiation of glo- transitional cells exhibiting a mixed phe- merular epithelial stem cells to regenerate lost podocytes are indicated by the arrows. notype between parietal epithelial cells and neo-podocytes in proximity of the vascular (Figure 1).38 Genetic labeling also supports podocytes. Indeed, the possibility that pa- stalk of the glomerulus.36,37 Podocyte- the notion that this parietal epithelial cell rietal epithelial cells also migrate from committed progenitors proliferate and dif- population regenerates itself.38 Thus, pari- Bowman’s capsule to the capillary tuft in ferentiate into cells that loose stem cell etal epithelial cells have the ability not only regions different than the vascular pole is markers and acquire high levels of podo- to generate differentiated podocytes but suggested by adhesions and also bridges cyte-specific markers as they progressively also to self-renew, which further demon- representing new migratory tracks be- migrate toward the vascular stalk of the strates they represent stem cells. A contin- tween Bowman’s capsule and the tuft (Fig- Bowman’s capsule (Figure 1).33 These uous generation of novel podocytes occurs ure 1).25,39 Interestingly, a recent study us- findings in humans were also confirmed in as the kidney grows,38 and might also occur ing genetic tagging of parietal epithelial parallel studies performed in rodents. In- during enlargement of a contralateral kid- cells demonstrates that bridges between deed, using genetic tagging of parietal epi- ney after uninephrectomy. Bowman’s capsule and the glomerular tuft thelial cells, Appel et al.38 demonstrated However, in glomerular disorders char- in experimental models of glomerular dis- that such cells proliferate and differentiate acterized by acute or severe podocyte loss, orders are exclusively generated by parietal along the urinary space and move to the regeneration may require other pathways epithelial cells.40 These bridges provide a vascular stalk generating neo-podocytes that allow faster replacement of injured pathway for the migration and differentia-

J Am Soc Nephrol 21: 1612–1619, 2010 New Insights into Glomerular Epithelial Stem Cells 1613 BRIEF REVIEW www.jasn.org tion of an adjacent progenitor and a quick of glomerular epithelial stem cells has sible explanation for the progressive in- replacement of lost podocytes (Figure 1B), or limits.29,38,44,45 Wiggins and co-work- crease in prevalence of global glomerulo- alternatively, bridging parietal epithelial cells ers1,3 find that repair of podocytes occurs sclerosis with aging, which may be may acquire podocyte markers after injury41 when Ͻ20% of podocytes are lost; 20 to related to an exhaustion of the self-re- and directly replace lost podocytes (Figure 40% podocyte loss results in a scarring newal potential of glomerular epithelial 1C). Accordingly, generation of bridges by response and Ͼ60% podocyte loss pro- stem cells. Indeed, reduced potential for parietal epithelial cells to replace lost podo- duces globally sclerotic and nonfiltering self-renewal with aging is described in cytes has also been recently reported using in glomeruli. The amount of podocyte in- other adult stem cells.46–50 Accumulated vivo multiphoton microscopy in rat models jury seems greatly influenced by the re- DNA damage and loss of DNA repair of PAN nephritis.42 In summary, a large body generative capacity of glomerular epithe- may be one of the mechanisms underly- of evidence indicates the Bowman’s capsule lial stem cells, and glomerulosclerosis ing age-dependent stem cell decline.47 of adult kidneys contains a population of glo- leading to ESRD may occur in those glo- However, the most important modula- merular epithelial stem cells, which replace merular disorders where the amount of tor of the regenerative potential of stem lost podocytes through multiple mecha- podocyte injury exceeds the possibility of cells is likely to be the surrounding envi- nisms of glomerular regeneration. regeneration (Figure 2).24,26 In addition, ronment.48–50 After birth, adult stem glomerular epithelial stem cells display cells reside in a specialized microenvi- a different regenerative potential at ronment called a “niche,” which regu- THE BAD: LIMITS AND DEFAULTS distinct stages of life,38 exhibiting the lates the delicate balance between self-re- IN THE REGENERATIVE highest regenerative potential through newal and differentiation.21,48–52 POTENTIAL OF GLOMERULAR adolescence,38 which might explain The localization of glomerular epithe- EPITHELIAL STEM CELLS why glomerular disorders have a better lial stem cells at the urinary pole of the prognosis during childhood whereas Bowman’s capsule suggests that the adult Several studies indicate, as already re- FSGS is more frequent at an older age glomerulus contains such a stem cell ported for other types of adult stem (Figure 2).1 niche. This hypothesis is supported by cells,20,21,43 that the regenerative capacity This observation also provides a pos- the observation that embryonic stem cells, after commitment toward renal lineages, migrate to the urinary pole of Bowman’s capsule after injection into developing kidneys53—a selective prop- Urinary pole erty of stem cell niches.21,48–52 Previous

Stem cells studies also demonstrate that factors Extracellular present in young niche environments re- matrix store proliferative and regenerative capacity of aged stem cells in the niches of other adult tissues.50 Accordingly, very recent data finds Detaching that the regenerative potential of glomer- podocytes ular epithelial stem cells is enhanced or inhibited by different culture conditions.54 More importantly, injection of Detaching Podocyte podocytes glomerular epithelial stem cells under progenitors the contralateral kidney capsule of uni- laterally nephrectomized mice generate Vascular stalk novel renal tissue, including neo-glomerular and tubular structures, a finding that is not observed after injection under the capsule of normal kidneys.54 This latter finding suggests the regenerative potential of glomerular epithe- Figure 2. The bad: Limits and defaults of the regenerative potential of glomerular lial stem cells is strictly dependent on the epithelial stem cells. Age, genetic alterations, and environmental factors limit the regenerative response of glomerular epithelial stem cells (red), thus impairing podo- surrounding environment and the un- cyte (light blue) replacement when the amount of injured cells is extensive. If regen- derlying process of kidney growth gener- eration is impaired, podocyte loss typically results in the deposition of extracellular ates favorable conditions for regenera- matrix (pink), which can be produced by glomerular epithelial stem cells in response to tion.38,54 Although a recent study describes TGF-␤ that is secreted by injured podocytes. the phenotype of glomerular epithelial

1614 Journal of the American Society of Nephrology J Am Soc Nephrol 21: 1612–1619, 2010 www.jasn.org BRIEF REVIEW stem cells,55 we still have little information of these stem cells. This would easily experiments performed in transgenic mice about which other cells support their plain the presence in these lesions of with genetically labeled parietal epithe- growth and differentiation and what cells with an intermediate phenotype lial cells in the nephrotoxic nephritis paracrine factors maintain their function between parietal epithelial cells and model of crescentic glomerulonephritis and number. Thus, further experiments podocytes.59–69 Accordingly, the ma- and also the Thy-1.1 transgenic mouse are necessary to pinpoint this relation- jority of cells present in the hyperplastic model of collapsing glomerulopathy.40 ship and how it changes during progres- lesions of patients with collapsing glo- In both models, genetically labeled pari- sive glomerulosclerosis or aging. merulopathy or crescentic glomerulone- etal epithelia constitute the majority of phritis exhibit the glomerular epithelial cells that compose early extracapillary stem cell markers, CD133 and CD24, proliferative lesions and almost all of the THE UGLY: DYSREGULATED with or without co-expression of podo- proliferating cells.40 Interestingly, Le Hir GLOMERULAR EPITHELIAL STEM cyte markers.70 Therefore, we suggest et al. suggest the development of the cres- CELLS CREATE THEIR OWN that glomerular hyperplastic lesions are cent is initiated by cell bridges that are LESIONS generated by stem/renal progenitor cells formed between the tuft and Bowman’s from Bowman’s capsule at different capsule.71 Because lineage-tracing exper- It is widely recognized that disruption in stages of differentiation toward mature iments demonstrate that bridging be- the regulated balance between self-re- podocytes (Figure 3A).70 tween the Bowman’s capsule and the tuft newal and differentiation of stem cells Additional confirmation of this hy- are generated by parietal epithelial not only impairs regenerative mecha- pothesis comes from lineage-tracing ex- cells,40 we hypothesize that after massive nisms but also can even create new prob- lems.48,52 For example, myeloproliferative A B diseases arise as a result of aberrant prolif- 56,57 eration of hematopoietic stem cells, Urinary pole whereas a number of hematopoietic stem Stem cells cells are reduced in aplastic anemia, re- Bridges sulting in fatty replacement of bone mar- Detaching row with pancytopenia.58 These stem podocytes cells–related disorders are generated by Macrophages intrinsic genetic alterations or by alterations of the surrounding environment.56–58 In the glomerulus, the response to podocyte injury may cause aberrant epi- Podocyte thelial cell proliferation, the formation of progenitors hypercellular lesions, and the oblitera- tion of Bowman’s space, as seen in col- Vascular stalk lapsing glomerulopathy or crescentic glomerulonephritis.59–63 Until now, the- ories explaining the origin of aberrant epithelial cells in collapsing glomerulopathy and crescentic glomerulonephri- Figure 3. The ugly: Dysregulated glomerular epithelial stem cells create their own 59–69 tis have been controversial. One lesions. Aberrant proliferation of glomerular epithelial stem cells can generate hyper- possibility is these cells are exclusively of plastic lesions. (A) After massive podocyte injury, glomerular epithelial stem cells (red) parietal epithelial origin,59–64 whereas generate cell bridges with the glomerular tuft in several areas of the glomerulus to others suggest some dedifferentiated quickly replace lost podocytes (light blue). However, numerous areas of podocyte podocytes acquire markers of parietal injury distort glomerular structural integrity, thus altering the polarity of glomerular epithelial cells.65–69 epithelial stem cell division and initiating their abnormal proliferation and the devel- After the identification of a popula- opment of extracapillary hyperplastic lesions as well as crescents. Macrophages (green) tion of glomerular epithelial stem cells can also be included within the lesions. Similar processes might occur in crescentic glomerulonephritis and collapsing glomerulopathy. (B) Replacement of podocytes along Bowman’s capsule that generate under physiologic conditions follows a gradient, with neo-podocytes progressively new podocytes, we have explored the added at the vascular stalk. Thus, the tip podocytes represent the “oldest” podocytes possibility that hyperplastic epithelial of the glomerular tuft, which suggests they might be more susceptible to injury related cells in crescentic glomerulonephritis or to heavy proteinuria. Glomerular epithelial stem cells may also proliferate and migrate collapsing glomerulopathy might result from the urinary pole of the Bowman’s capsule toward the tuft in an attempt to replace from an aberrant proliferative response the podocytes lost in response to heavy proteinuria, and generate the tip lesion.

J Am Soc Nephrol 21: 1612–1619, 2010 New Insights into Glomerular Epithelial Stem Cells 1615 BRIEF REVIEW www.jasn.org podocyte injury, glomerular epithelial ficient proliferation of glomerular epi- teins promotes podocyte dysfunction and stem cells generate cell bridges with the thelial stem cells, which impairs the cor- injury followed by tuft adhesion and scle- glomerular tuft in several areas of the rect replacement of injured podocytes rosis.75,80 Thus, glomerular epithelial glomerulus to quickly replace lost podo- and defaults to alternative replacement stem cells may proliferate and migrate cytes (Figure 3A). with extracellular matrix (Figure 2).70 In- from the urinary pole of the Bowman’s Numerous areas of podocyte injury terestingly, when glomerular epithelial capsule toward the tuft in an attempt to along with glomerular epithelial stem stem cells are exposed to TGF-␤ secreted replace the podocytes lost in response to cells proliferation heavily distort glomer- by podocytes exposed to proteinuria,74 heavy proteinuria, generating the tip le- ular architecture, thus altering the polar- they also produce and deposit higher sion (Figure 3B). Taken together, the re- ity of stem cells division. Polarized pro- amounts of extracellular matrix.70 In ad- sults of these recent studies suggest the liferation is a critical determinant of dition, the regenerative potential of glo- clinicopathologic features of different correct stem cell differentiation,48–52 and merular epithelial stem cells is reduced in glomerular disorders more likely repre- this might explain why disruption in the aging,38 when FSGS is more frequent sent distinct patterns of injury or repair polarity of glomerular epithelial stem (Figure 2).1,61 rather than diseases. cells initiates abnormal proliferation and Other reasons underlying the differ- the development of hyperplastic glomer- ent response of glomerular epithelial ular lesions impairing recovery (Figure stem cells to massive podocyte injury in CONCLUSIONS 3A). Interestingly, both crescentic glo- various glomerulonephridities are cur- merulonephritis and collapsing glo- rently unknown, but might be related to Podocyte loss is a central determinant merulopathy are characterized by death the type of injury or to the different ge- of progression to glomerulosclero- of numerous podocytes over a short time netic backgrounds of patients. Accord- sis.1,3–16 Podocytes cannot divide,1,2 interval and by an aberrant proliferation ingly, a recent study demonstrates that but regression of glomerulosclerosis is of glomerular epithelial stem cells, which podocyte damage leads to glomerular in- possible, as indicated by experimental suggests they might not be pathogeni- jury with a complete histologic pattern of models and also clinical evidence.24–28 cally distinct but rather two faces of the collapsing glomerulopathy related to The discovery that a population of glo- same disorder.72 That is, crescentic glo- high parietal epithelial cell proliferation merular epithelial stem cells represent merulonephritis presents with podocyte in mice with null alleles for the cell cycle a potential source for podocyte regen- damage in an inflammatory environ- inhibitor, p21, compared with segmental eration establishes an entirely novel ment characterized by nephritic fea- lesions and mild intraglomerular prolif- view that changes the way we think of tures, whereas collapsing glomeru- eration in wild-type mice.75 normal renal cell biology or patho- lopathy often presents with nephrotic Finally, glomerular epithelial stem physiology (Figure 1).29–33,38,39 Indeed, features.62,63,72,73 cells are also the main constituents of the the first main outcome of the discovery Although epithelial cell proliferation tip lesion.70 Interestingly, the tip lesion is of glomerular epithelial stem cells is is most characteristic and prominent in described in several proteinuric condi- that regeneration or the promotion of crescentic glomerulonephritis or col- tions, including FSGS, membranous ne- functional repair after glomerular in- lapsing glomerulopathy, some epithelial phropathy,76 postinfectious glomerulo- jury, and even prevention or treatment cell proliferation is also observed in his- nephritis,77 and diabetic nephropathy.78 of glomerulosclerosis, may be possible. topathologic lesions typically found in Because replacement of podocytes under However, this regenerative process is other podocytopathies, such as the tip- physiologic conditions follows a gradi- sometimes inadequate because of an lesion.62 Interestingly, we recently dem- ent, with neo-podocytes progressively inefficient proliferative response by onstrated that in the tip lesion, as well as added from the vascular stalk, the tip glomerular epithelial stem cells, as it in those FSGS which are characterized by podocytes likely represent the “oldest” may occur in aging patients or after mild levels of hyperplasia, glomerular podocytes in the glomerular tuft, as al- FSGS (Figure 2).40,70 epithelial stem cells are the main constit- ready suggested.38 This should make the In addition, in some situations, an ex- uents of the proliferative lesion.70 This tip podocytes more susceptible to injury cessive proliferative response by glomer- raises the question of how distinct patho- and thus suggests they might be the first ular epithelial stem cells will initiate new genic factors initiate abnormal regenera- to die in response to heavy proteinuria lesions, such as crescentic glomerulone- tive processes. (Figure 3B). On this basis, Haas and phritis or collapsing glomerulopathy FSGS is induced after a 40 to 60% Yousefzadeh79 argue that the tip lesion is (Figure 3).40,70 Thus, converging evi- podocyte loss.1,3 However, in the face of a response to prolonged heavy protein- dence indicates the type of pathologic or massive podocyte injury in this disorder, uria. clinic presentation, or even the outcome hyperplastic glomerular lesions gener- Consistently, experimental evi- of glomerular disorders, may depend on ated by glomerular epithelial stem cells dence in in vitro and in vivo models of the balance between injury1–16 and re- are usually mild.61 This suggests that disease documents that exposure of podo- generation provided by glomerular epi- FSGS might be the consequence of insuf- cytes to excessive amounts of plasma pro- thelial stem cells.29–33,38–40,70 Accord-

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