Can Podocytes Be Regenerated in Adults?

Home , Juxtaglomerular cell, Nephron, Podocyte

REVIEW

CURRENT OPINION Can podocytes be regenerated in adults?

Stuart J. Shankland, Benjamin S. Freedman, and Jeffrey W. Pippin

Purpose of review Podocytes are critical components of the nephron filtration barrier and are depleted in many kidney injuries and disease states. Terminally differentiated adult podocytes are highly specialized, postmitotic cells, raising the question of whether the body has any ability to regenerate lost podocytes. This timely question has recently been illuminated by a series of innovative studies. Here, we review recent progress on this topic of significant interest and debate. Recent findings The innovation of genetic labeling techniques enables fate tracing of individual podocytes, providing the strongest evidence yet that podocytes can be replaced by nearby progenitor cells. In particular, two progenitor pools have recently been identified in multiple studies: parietal epithelial cells and cells of renin lineage. These studies furthermore suggest that podocyte regeneration can be enhanced using ex-vivo or pharmacological interventions. Summary Recent studies indicate that the podocyte compartment is more dynamic than previously believed. Bidirectional exchange with neighboring cellular compartments provides a mechanism for podocyte replacement. Based on these findings, we propose a set of criteria for evaluating podocyte regeneration and suggest that restoration of podocyte number to a subsclerotic threshold be targeted as a potentially achievable clinical goal. Keywords glomerulosclerosis, glomerulus, parietal epithelial cell, progenitor, renin cells, stem cell

INTRODUCTION: PODOCYTE ABSOLUTE AND RELATIVE PODOCYTE REGENERATION IN THE ADULT KIDNEY NUMBER The current review is intended to provide a bal- This principle segues to the notion of absolute anced view on the broad topic of adult podocyte podocyte number versus relative podocyte number regeneration. Podocyte differentiation and regen- [5,6&,7]. The latter, also known as podocyte density, eration during kidney development is well dis- is simply a measure of absolute podocyte number cussed by others [1,2]. For context, nephron divided by the glomerular volume it occupies. The progenitor cells (NPCs), which give rise to the prox- denominator, glomerular volume, is influenced by imal nephron including podocytes during kidney age, nephron number, obesity and other factors [8]. embryogenesis, cease to exist in mammalian kid- A decrease in podocyte number or density below a neys shortly after birth [3,4]. NPCs, also known as certain threshold has clearly been shown to underlie metanephric mesenchyme cells, express SIX2 and the onset of proteinuria and the development of CITED1 and self-renew only during kidney develop- glomerulosclerosis [9]. Moreover, the lower the ment [3,4]. podocyte number/density, the more severe the glo- We will use the dictionary definition of regen- merular scarring [9,10]. eration (biology), which is ‘the ability to recreate lost or damaged tissues, organs and limbs’. We Division of Nephrology, Department of Medicine, University of would argue therefore, in states in which the kidney Washington School of Medicine, Seattle, Washington, USA and/or adult podocytes are damaged, that a simple Correspondence to Stuart J. Shankland, Division of Nephrology, Depart- requirement for podocyte regeneration is an actual ment of Medicine, University of Washington School of Medicine, Box decrease in the number of podocytes. Stated differ- 358058, 750 Republican Street, Seattle, WA 98109, USA. ently, we do not expect podocyte regeneration in Tel: +1 206 543 2346; fax: +1 206 685 8661; e-mail: [email protected] diseases in which podocyte number has not been Curr Opin Nephrol Hypertens 2017, 26:154–164 reduced. DOI:10.1097/MNH.0000000000000311 www.co-nephrolhypertens.com Volume 26 Number 3 May 2017 Copyright © 2017 Wolters Kluwer Health, Inc. All rights reserved. Podocyte replacement Shankland et al.

density will decrease [25]. This occurs following KEY POINTS nephrectomy, in the transplanted kidney, in aging, Adult podocytes are unable to self-renew as they are obesity and certain glomerular diseases following unable to proliferate. the scarring of injured glomeruli [8]. The increased glomerular volume leads to a mismatch in coverage Replacement of adult podocytes that have been lost in by podocytes of underlying glomerular capillaries disease needs to derive from stem/progenitor cells. [8]. This is initially compensated for by hypertrophy Glomerular parietal epithelial cells and cells of renin of podocytes (i.e. an increase in their size without an lineage are two local sources of adult podocyte stem/ increase in their number), that over time becomes progenitor cells. paradoxically maladaptive [8]. Under these circum- To prevent/limit and even reverse glomerular scarring stances, podocyte density is reduced, but podocyte due to podocyte loss, the goal for replacement does number does not change. Whether this is sufficient not need to be 100%. to trigger podocyte regeneration is not yet clear. EVIDENCE THAT ADULT PODOCYTES CAN Although numbers vary somewhat due to meth- BE PARTIALLY OR FULLY REPLACED IN odology, the mean number of podocytes per human DISEASE STATES glomerulus is 558 (range 263–983), and in mice 72 Several lines of evidence support the notion that (range 70–80) [11&&]. Recent human studies have adult podocytes can be replaced following their shown that absolute podocyte number increases depletion. These include the following: during the adolescent period by up to 20% [12]. In contrast, normal kidney aging is accompanied (1) An increase in podocyte number in disease using cell- by reduced podocyte number in man [11&&] and specific markers: In studies using nongenetic cell rodents [13]. In contrast to absolute podocyte num- fate mapping, the identification of podocytes ber, however, podocyte density is markedly relies on several markers such as WT-1 [26], p57 decreased (more than four-fold) in large glomeruli [23] and others [27]. Several studies have shown in adult humans, compared with children [12]. in experimental models of diabetic and non- diabetic kidney disease that podocyte number ABSOLUTE PODOCYTE NUMBER could be raised/increased when animals were DEPLETION given ACE-I, ARBs, steroids, retinoids or normal- Several mechanisms can lead to podocyte depletion ization of their diabetic state [28–31,32&&,33]. In including apoptosis, detachment, necrosis and DNA all these studies, the increase in podocyte num- damage [14–16]. Indeed, viable podocytes can be ber occurred in the absence of podocyte pro- detected in the urine and can be cultured ex vivo liferation. Changes in podocyte-specific [17,18]. It is estimated that patients with kidney proteins can result secondary to injury, and thus disease slough podocytes into their urine at more the specificity of these changes also needs to than 10 cells/mg creatinine [17]. Clinical pro- be considered. gression is in many cases much slower, and it has (2) An increase in podocyte number in disease using been observed that this suggests a regenerative genetic labeling techniques: Inducible conditional mechanism [19]. reporters enable labeling of podocytes for line- The adult terminally differentiated podocyte is age tracing. For example, the mT/mG mouse unable to proliferate, thereby preventing self- crossed with a podocin-rtTA mouse perma- renewal in the face of ongoing losses [20]. Mechan- nently labels all cells in the body with a red isms underlying the inability of adult podocytes to color. When Cre is turned on by activating the proliferate include an increase in expression of cell podocin promoter with doxycycline, all podo- cycle inhibitors (such as the cyclin kinase inhibitors cytes become green in color, whereas other cells p27 [21,22] and p57 [22,23]), DNA damage resulting remain red [34,35]. Wanner et al. [36&&] used in cell cycle arrest [24], mitotic catastrophe and mT/mG mice crossed with inducible diphtheria deficiencies in M-type cyclins [20]. Thus, to replace toxin receptor (DTR) mice to partially deplete adult podocytes, other cell types need to fulfill this podocytes with the diphtheria toxin. Podocytes biological role. were labeled green and expressed DTR following doxycycline administration; when diphtheria RELATIVE PODOCYTE NUMBER toxin was subsequently administered, there DEPLETION was a decrease in green cells (podocytes). When If the glomerular volume increases without any followed over 4 weeks, red cells appeared on change in absolute podocyte number, podocyte the glomerular tuft and expressed podocyte

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proteins. The authors of the manuscript con- (1) migration from their original location to a new cluded that overall their results suggest a podo- location along the glomerular basement mem- cyte renewal of 38% of ablated cells. brane (GBM) in the glomerular tuft (2) de novo expression of several podocyte Using the same mT/mG mice, Lasagni et al. proteins, coincident with loss of their original [37&&] depleted podocytes with the podocyte toxin cell-specific proteins Adriamycin. When followed over time, the percent- (3) expression of epithelial cell markers age of newly generated podocytes was 6% when all (4) acquisition of classic podocyte ultrastructural podocytes were taken in to account or 30.4% in mice features (such as foot process and slit dia- that had remission of their proteinuria. In cell fate phragms) mapping studies with a conditional paired box gene (5) displaying functionality (such as the production 2(PAX2) – confetti mouse, marking parietal epi- of vascular endothelial growth factor and thelial cells (PECs), indicated that PECs adopted specific extracellular matrix proteins, angioten- podocyte characteristics and accounted for 25% sin II responsiveness and limiting albumin per- replacement of depleted podocytes in mice with meability). regression of proteinuria compared with those that did not. Overall, podocyte regeneration was further We also recommend a context for the need for increased to 32.6% when the glycogen synthase podocytes to be replaced and for their progenitors to inhibitor 3 (GSK3)-inhibitor BIO (6-Bromoindirubin- therefore be called upon, that is a decrease in 3’-oxome) was administered [37&&]. absolute podocyte number. Our group used an inducible podocin rtTA Having argued that adult podocytes can be reporter, which permanently labels podocytes with replaced either partially or fully by stem/progenitor ZsGreen only when mice are given doxycycline cells and that this number can be significantly [38&]. The total complement of podocytes, measured increased under certain conditions such as renin by the podocyte markers p57, podocin and synap- angiotensin aldosterone system (RAAS) blockade topodin along with the podocyte reporter ZsGreen, [32&&] and GSK3 inhibition [37&&], important ques- was abruptly depleted by 30–40% with a cytopathic tions arise including what are the sources for podo- antipodocyte antibody. This coincided with similar cyte replacement, what is the evidence in support of decreases in podocyte labeling and DAPI (4’,6- this, and is replacement the cumulative response Diamidino-2-Phenylindole) staining. Importantly, from more than one source in a podocyte depleted following this nadir in podocytes, there was a sub- glomerulus? Although it is tempting to speculate on sequent increase in the number of cells staining for opportunities for podocyte replacement from orga- p57, podocin and synaptopodin as well as DAPI stain- noids [40,41] and sources such as the bone marrow ing, but not ZsGreen. Repeated BrdU (5-Bromo- and amniotic fluid [42], we will address what is 2’-deoxyuridine), administered to measure cumulat- reported regarding two potential endogenous sour- ive proliferation, was not detected in podocytes. ces normally resident in the kidney. Finally, using live animal imaging, Hackl et al. [39] also showed that podocytes can be replaced in experimental models of glomerular disease. Serial Glomerular parietal epithelial cells as adult multiphoton microscopy on the same injured glo- podocyte progenitors merulus revealed the appearance of a new podocyte Several lines of evidence support the rationale that (labeled with a fluorescent reporter) within 24 h of PECs possess biological programs necessary to differ- the previous image, providing direct evidence for entiate into adult podocytes. First, PECs and podo- podocyte regeneration. cytes arise from a common restricted pool of In summary, the studies above strongly support mesenchymal cells during glomerulogenesis [43]. the notion that following a decrease in absolute Second, a PEC subpopulation defined as cells at podocyte number in experimental mouse models, the tubular pole coexpressing the stemness markers podocytes can be at least partially replaced from a CD133 and CD24 has been identified [44]. Third, a nonpodocyte source. subpopulation of PECs lining Bowman’s capsule typically at the glomerular vascular pole coexpress both PEC and podocyte proteins and even show ADULT PODOCYTE PROGENITORS ultrastructural features of podocytes [45]. These cells Based on the preceding literature, we recommend have been named ‘glomerular epithelial transitional that the following criteria be considered as evidence cells’. When such cells along Bowman’s capsule only that a stem/progenitor cell can transdifferentiate to express podocyte proteins under normal conditions, an adult podocyte fate: they have been referred to as ‘ectopic podocytes’

[46], and when they express WT-1, typical of adult subpopulation of PECs expressing CD133/CD24 podocytes [26], they have been called ‘parietal podo- require retinoids for normal survival and function cytes’ [46]. Moreover, the number and density of [57]. Moreover, when albumin was taken up by PECs glomerular epithelial transitional cells increases [58], endogenous retinoid synthesis was impaired under certain experimental and human glomerular [57]. This might explain in part why podocyte regen- diseases, oftentimes accompanying podocyte eration is limited in albuminuric states. Benigni et al. depletion, such as diabetic kidney disease [29], aging [59] recently showed that a subpopulation of PECs nephropathy [47], focal segmental glomerulosclero- coexpressing the differentiation marker neural cell sis (FSGS) [48] and membranous nephropathy [31]. adhesion molecule also express the angiotensin 1 In addition, the number of transition cells can be receptor and that proliferation of these cells could increased by administration of retinoids [31], corti- be reduced by giving rats the angiotensin-convert- costeroids [49] and angiotensin-converting enzyme ing enzyme inhibitor (ACE inhibitor) Lisinopril inhibitor (ACE inhibition) [30], as well as an [59,60]. Anders’ laboratory showed that a-interfer- improvement in the diabetic milieu [29]. ons and b-interferons reduced the capacity of PEC However, we need to be cautious in these inter- progenitors to induce nephrin mRNA expression pretations, because we [38&] and others [39,50] [61]. have reported that podocytes can migrate from We have reported that abrupt podocyte the glomerular tuft to Bowman’s capsule, where depletion induced by the administration of a cyto- they might begin to coexpress PEC proteins. Thus, toxic antipodocyte antibody is followed by partial we will discuss data supporting and not supporting podocyte replacement, in the absence of podocyte PECs as adult podocyte progenitors. proliferation, and that RAAS inhibitors can further augment podocyte replacement [30,32&&,38&]. To determine if neighboring PECs might serve this Studies supporting a role for parietal role, podocytes were depleted in PEC-reverse tetra- epithelial cells as adult podocyte progenitors cycline-transactivator/LC1/Rosa26 reporter ( pPEC- During normal adolescence, Moeller’s group rtTA/LC1/R26R) mice [62,63]. A subset of labeled reported that in PEC reporter mice, a subset of PECs migrated from Bowman’s capsule onto the podocytes derive from PECs [51&,52]. They suggest glomerular tuft, where they de novo began to express that these are parietal podocytes, distinct from adult several proteins considered podocyte-specific PECs, that are committed to a podocyte fate and (Fig. 1) [64&]. constitute a ‘reserve’ of podocytes for development. Finally, Lasagni et al. [37&&] used two different This was validated recently by the laboratories of conditional PEC reporter mice in which to lower Wanner et al. [37&&] and Lasagni et al. [37&&]. Both podocyte number with Adriamycin. Permanently groups predict that this podocyte reservoir is respon- labeled PECs moved onto the glomerular tuft, where sible for 10% of podocytes. This fits well with recent they began to coexpress three different podocyte studies in humans by Puelles and Bertram [12]. proteins and acquired ultrastructural features that Using normal human kidneys, Romagnani’s typify podocytes. These events can be further laboratory isolated cells from Bowman’s capsule increased by administering a GSK3 inhibitor coexpressing PEC proteins and the surface markers [37&&]. Many of the studies described above fulfill CD133/CD24 and then propagated them ex vivo in a the criteria we proposed above supporting that PECs cell culture system [44], which required a decrease in serve as adult podocyte stem/progenitor cells. Notch signaling [53]. These ‘renal progenitor cells’ could be expanded under certain cell culture conditions and began to coexpress markers of podocytes Studies lacking a supporting a role for and tubular cells [54,55]. Similarly, blockade of the parietal epithelial cells as adult podocyte chemokine stromal-derived factor (SFD/CXCL12) progenitors enhanced the differentiation of renal progenitors An important recent article showed that in humans, toward a podocyte phenotype in culture [56]. podocyte number increased during adolescence by Furthermore, when administered intravenously to up to 20% [12]. This suggested a pool of nonpodo- SCID mice with Adriamycin-induced nephropathy, cyte cells that served a progenitor role, as podocytes renal progenitors were found in the glomerulus, cannot adequately proliferate beyond glomerulo- expressing podocyte proteins, acquiring ultrastruc- genesis. Using a transgenic mouse model to specifi- tural features of podocytes and were accompanied cally label PECs permanently in vivo, Appel et al. [62] by improved disease outcomes [44]. showed that a subset of PECs migrate to the glomer- Several studies have highlighted factors that ular tuft during mouse adolescence, where they inhibit PEC progenitors. Peirid showed that the begin to express podocyte proteins and also take

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(a) (b) (c) (d)

FIGURE 1. Reporter labeled PECs migrate onto the glomerular tuft in focal segmental glomerulosclerosis and express synaptopodin. (a) Synaptopodin staining (green) is restricted to podocytes. (b) b-gal staining (red) is detected in cells lining Bowman’s capsule (dashed arrows) and inside the tuft (solid arrows). (c) DAPI staining is restricted to nuclei (blue). (d) Merge of all three stains, showing that a subset of reporter labeled PECs on the tuft costain with synaptopodin (solid arrows). (Reproduced from [64&], Fig. 4). on their ultrastructural shape. However, these experimental diseases studied. Although a PEC authors have concluded in subsequent studies might well differentiate into an adult podocyte, [51&] that PECs do not serve as podocyte progenitors the magnitude of ‘regeneration’ that results from in adult mice in disease, a notion recently supported this alone may be inadequate to fully replace the by Huber’s laboratory [36&&]. Although these studies number of podocytes depleted, and the precise were robustly performed, the three models studied, cues and mechanisms underlying such events are namely uninephrectomy, 5/6 nephrectomy and not yet well delineated. There may well be bidirec- early middle-aged mice 12 months of age, are typ- tional repair/regenerative process ongoing, in which ically not accompanied by a decrease in absolute PECs differentiate into podocytes and podocytes podocyte number but rather podocyte density, due differentiate into PECs. Also, we must be cautious to an increase in glomerular volume. Additional in that the animal models of PEC fate mapping studies in models of absolute podocyte number have not been fully reproduced in independent depletion are therefore required. studies and that use of temporally expressed In several states of podocyte depletion, the num- markers in tissue sections is not a surrogate for ber of glomerular epithelial transition cells (defined fate mapping. as cells coexpressing PEC and podocyte proteins) is increased both along Bowman’s capsule and in the glomerular tuft [47,65]. Guhr et al. [66] showed that Cells of renin lineage as adult podocyte the de novo expression of podocyte proteins in PECs progenitors was due to reduced ubiquitin-mediated degra- Cells of renin lineage (CoRL) are vascular smooth dation. Studies using reporter mice have suggested muscle cells that produce, or once produced, renin a very different paradigm. Following injury, a subset [69]. Under normal conditions, they are restricted to of labeled podocytes could be detected having the juxtaglomerular compartment (JGC) in adults moved from the glomerular tuft and now lining and comprise approximately 0.01% of the total Bowman’s capsule [38&,39,50,52,67]. In some kidney cell number. They derive from FoxD1- instances, the labeled podocytes coexpressed PEC positive stromal cells [70]. Total CoRL number proteins in addition to podocyte proteins. This decreases in aged mice [71]. CoRL can transdiffer- podocyte-to-PEC migration may occur in response entiate into smooth muscle cells, mesangial cells to injury or loss of PECs [68]. Thus, cells coexpress- and possibly pericytes [72,73]. When VHL is selec- ing PEC and podocyte proteins along Bowman’s tively deleted in CoRL, they can differentiate into capsule could derive from migrating podocytes. erythropoietin-producing cells [74]. CoRL can trans- Taken together, there are compelling data that differentiate into adult mesangial cells in glomeru- support a biological role for PECs as adolescent and lar disease [75] and contribute to the interstitial adult podocyte progenitors. Yet, there is no consist- pericyte pool following ureteral obstruction [76]. ency across all the models and marker systems Adult CoRL themselves can be regenerated by adult (Table 1). We need to consider several variables that renal mesenchymal-like cells that express CD44, might explain these differences such as species and c-Kit and/or CD105 [77]. Adult CoRL are therefore

Table 1. Summary of published studies on parietal epithelial cells as adult podocyte progenitors

Model system Data supporting PEC as adult podocyte progenitors Reference

Studies strongly supporting PECs PEC reporter mice A subset of labeled PECs is detected in the glomerular tuft in [62] as adolescent podocyte newborn and adolescent mice and coexpress podocyte progenitors markers Labeled PECs migrate to the glomerular tuft following [36&&] podocyte depletion, augmented by administering a GSK3 inhibitor Human kidney A subpopulation of PECs at tubular pole costains for the [53] stem/progenitor markers CD133/CD24 When these cells are isolated, and grown ex vivo, they have [44] self-renewal potential and high cloning efficiency Under certain culture conditions, they acquire podocyte [55] phenotypic features Cells harvested from normal kidneys that double-labeled for PEC and stem/progenitor markers improved disease outcomes when injected into mice with experimental FSGS Studies suggesting, but not Rat model ACE-inhibition affects a subset of cells lining Bowman’s [28] definitively proving, that a capsule that coexpress PEC proteins and NCAM (used as a subset of PECs are adult differentiation marker) podocyte progenitors Diabetic mouse Blockade of the chemokine stromal-derived factor (SFD/ [56] CXCL12) enhanced the differentiation of renal progenitors toward a podocyte phenotype

ACE, angiotensin-converting enzyme; NCAM, neural cell adhesion molecule; PEC, parietal epithelial cell.

multipotent with marked stemness, serving as pro- mice that were not exposed to tamoxifen, no cells genitors for several kidney cell types and can them- were detected either in the JGC or intraglomerular selves be replaced by progenitors as well. compartments [78]. The degree of scarring within individual glomeruli was also significantly lower in glomeruli that contained labeled CoRL. Studies supporting a role for cells of renin Second, a subpopulation of intraglomerular lineage as adult podocyte progenitors CoRL coexpressed one of the following podocyte To determine whether CoRL also can serve as pro- proteins: WT-1, nephrin, podocin or synaptopodin genitors for podocytes, we utilized several constitu- [32&&,78]. CoRL did not express nephrin, podocin or tive and inducible reporter mice that permanently synaptopodin when in a JGC. Some mapped CoRL tag renin-producing cells using Cre/loxP somatic cells that had moved into diseased glomeruli did not DNA recombination [78]. Administering tamoxi- coexpress markers of podocytes. fen-labeled CoRL only in Ren1c-CreER, tdTomatoR Third, when CoRL reporter mice were given animals and labeled cells were confined to the JGC. either the ACE-inhibitor Enalapril or the AT1 Over 95% of labeled cells coexpressed renin, and blocker Losartan in the absence of disease, the pool none was detected in glomeruli of normal mice, as of CoRL increased in the JGC, due to both recruit- expected [32&&,78]. We depleted podocyte number ment and proliferation of existing CoRL; a subset of by 30–40% in reporter mice using a model of classic labeled CoRL migrated to the glomerulus prior to FSGS induced by cytotoxic antipodocyte antibodies podocyte depletion; and the number of CoRL that [32&&,78]. Several lines of evidence support a para- migrated to the glomerulus from the JGC following digm of CoRL serving as adult podocyte stem/pro- podocyte depletion was significantly augmented genitors. [32&&]. The latter was accompanied by overall kidney First, labeled CoRL were detected in a subpopu- function improvement and a higher podocyte lation of injured glomeruli (Fig. 2). In the majority number. of glomeruli containing labeled CoRL, cells in the Fourth, we next asked if these events also occur glomerulus were distributed either within the following progressive podocyte depletion (rather glomerular tuft adherent to the GBM or along than abrupt loss) in two additional models (5/6 Bowman’s capsule. Neither renin protein nor renin nephrectomy and aged kidney). First, a 5/6 neph- mRNA was detected in glomeruli of FSGS mice. In rectomy remnant kidney model was induced

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(a) (b) (c)

(a’) (b’) (c’)

(d) (e) (e’)

FIGURE 2. Reporter labeled cells of renin lineage migrate onto the glomerular tuft in focal segmental glomerulosclerosis and remnant kidney, and express synaptopodin and podocin. (a) Synaptopodin staining (green) is restricted to podocytes. (b) red fluorescent protein staining (red) is detected within glomerular tuft (white arrow). (c) Synaptopodin and RFP staining colocalize (white arrow) in the glomerular tuft. (a0 –c0) Higher magnification of insets shown above. (d) At baseline, podocin staining (green) is restricted to podocytes and RFP staining (red) is restricted to cells of renin lineage in the juxtaglomerular compartment. (e) In the remnant kidney model, reporter labeled cells of renin lineage on the glomerular tuft costain with podocin. (e0) Higher magnification of inset shown in (e) (white arrows indicate costaining). (Reproduced from [32&&], Fig. 5 and [79], Fig. 8).

Table 2. Summary of published studies on cells of renin lineage as adult podocyte progenitors

Data supporting CoRL as adult Model system podocyte progenitors Reference

Studies supporting CoRL as adult CoRL reporter mice following abrupt A subset of labeled PECs is detected [32&&]; [79]; [78] podocyte progenitors podocyte depletion in the glomerular tuft in newborn and adolescent mice and coexpress podocyte markers CoRL reporter mice following chronic Remnant model of FSGS contain [79]; [71] podocyte depletion labeled CoRL in glomeruli coexpressing podocyte proteins Aging kidney Human kidney No data to date

CoRL, cells of renin lineage; FSGS, focal segmental glomerulosclerosis; PEC, parietal epithelial cell.

FIGURE 3. Podocyte loss and regeneration and their relationship to structural glomerular changes. The Y axis shows the normal podocyte number and range per glomerulus in man and mouse. Three scenarios represented by different colors are shown for podocyte depletion, based on studies by Wiggins [8]. A decrease in podocyte number by 20% (represented by the green area) corresponds to a decrease in podocyte number per glomerulus by 112 in man and 14 in mice. A decrease in podocyte number by 21–40% (represented by the yellow area) corresponds to a decrease in podocyte number per glomerulus by 117–224 in man and 15–29 in mice. A decrease in podocyte number by greater than 40% (represented by the red area) corresponds to a decrease in podocyte number per glomerulus by more than 228 in man and more than 30 in mice. A decrease in podocyte number by 20% (represented by the red dashed arrow A) is not accompanied by any serious decline in kidney function, at least over the short term, unless there is subsequent podocyte depletion. In a steady state, we argue therefore that there is no major biological need to replace these podocytes. There is however a need to replace podocytes when their number is depleted between 21 and 40% to limit further synchial formation and focal segmental glomerulosclerosis and to even reverse these processes. However, podocyte replacement does NOT need to return to the normal number for this to happen. Rather, we propose that the goal to replace podocyte number is above the threshold for scarring. As shown in the graph, this is typically less than 20% of normal (represented by the dashed arrow B). For example, to reach 20% of normal from a nadir of 40% of normal to prevent/reverse scarring in an individual glomerulus requires only a 20% increase in podocyte number for that glomerulus (red arrow B). The mean number of glomeruli in a mouse kidney is 12 083 Æ 1009 per kidney or 24 166 glomeruli total per mouse [80]. In focal segmental glomerulosclerosis, for example, if 20% of glomeruli are affected, the number of podocytes that need to be replaced in one kidney to return to the original baseline is 67 665 (20% of glomeruli Â 24 166 glomeruli Â 14 podocytes lost per glomerulus). If podocyte number drops by 40% (decrease of 29 podocytes in a single glomerulus), global glomerulosclerosis will ensue. Thus, still using the example of 20% of glomeruli affected implies that 20% of glomeruli Â 24 166 glomeruli Â 29 podocytes lost per glomerulus ¼ 140 162 total podocytes needed. Thus, for repair from a 40% drop in podocytes to a 20% drop in podocytes (above which scarring does not occur), 72 497 podocytes are needed, whereas complete restoration would require 140 162 podocytes. If podocyte number drops below the 40% threshold to 50% for example, then an immediate goal for replacement would be to increase podocyte number by 10% (white arrow C) to limit the progression from focal to global sclerosis and to reverse global to focal sclerosis. The second goal is to increase podocyte number by another 20% to limit focal sclerosis (red arrow B). Finally, several therapies (shown on the right side of the graph) increase podocyte number and in doing so, limit and/or reverse the progression of scarring by regenerative repair (black line) (example is red arrow D), whereas no therapy leads to nonregenerative repair (blue line). surgically in CoRL reporter mice [79]. As expected, Fifth, we studied the aged mouse kidney, as it is mice developed mild FSGS [79]. However, in a characterized by a progressive segmental and global subpopulation of glomeruli that did not have glomerulosclerosis, accompanied by a slow decline obvious segmental scarring, labeled CoRL could be in podocyte number [80]. In CoRL reporter mice detected, and a subset of these cells coexpressed aged 1 year, a decrease in podocyte number was several podocyte proteins (Fig. 2) [79]. Glomerulo- accompanied by the presence of CoRL in the intra- sclerosis was substantially reduced in glomeruli with glomerular compartment, and some of these cells labeled CoRL. coexpressed podocyte proteins [71]. CoRL in the

1062-4821 Copyright ß 2017 Wolters Kluwer Health, Inc. All rights reserved. www.co-nephrolhypertens.com 161 Copyright © 2017 Wolters Kluwer Health, Inc. All rights reserved. Renal immunology and pathology intraglomerular compartment showed character- Financial support and sponsorship istic ultramorphological features of podocytes, The study was supported by 5 R01 DK 056799-10, 5 namely foot processes and slit diaphragms [71]. R01 DK 056799-12, 1 R01 DK097598-01A1, K01 With increasing age, the density of labeled CoRL DK102826 and American Society of Nephrology Career within the JGC decreased significantly. Development Award. Taken together, in three settings of podocyte depletion (one acute and two chronic), these find- Conflicts of interest ings support the notion that CoRL might serve as There are no conflicts of interest. adult podocyte progenitors and for partial regeneration in states of podocytes depletion (Table 2). REFERENCES AND RECOMMENDED READING Evidence not supporting a role for cells of Papers of particular interest, published within the annual period of review, have been highlighted as: renin lineage as adult glomerular epithelial & of special interest cell progenitors && of outstanding interest As stated above, there is increasing evidence that 1. Little MH. The life cycle of the nephron progenitor. Dev Cell 2015; 35:5–6. CoRL have multicellular differentiation potential, 2. Little MH, McMahon AP. Mammalian kidney development: principles, pro- that is likely context dependent. One view might be gress, and projections. Cold Spring Harb Perspect Biol 2012; 4:1–18. 3. Hartman HA, Lai HL, Patterson LT. Cessation of renal morphogenesis in mice. that there are simply too few CoRL to be able to Dev Biol 2007; 310:379–387. provide any meaningful contribution to podocyte 4. Kobayashi A, Valerius MT, Mugford JW, et al. Six2 defines and regulates a multipotent self-renewing nephron progenitor population throughout mam- replacement. We therefore believe that multiple malian kidney development. Cell Stem Cell 2008; 3:169–181. progenitors must function to replace podocytes. 5. Ashraf S, Gee HY, Woerner S, et al. ADCK4 mutations promote steroid- resistant nephrotic syndrome through CoQ10 biosynthesis disruption. J Clin Moreover, the number of CoRL does increase under Invest 2013; 123:5179–5189. certain circumstances such as when mice are given 6. Puelles VG, van der Wolde JW, Schulze KE, et al. validation of a three- & dimensional method for counting and sizing podocytes in whole glomeruli. ACE-inhibitors or AT1 blockers, thus providing an J Am Soc Nephrol 2016; 27:3093–3104. increased reservoir size. Methods for quantifying podocytes are limited. In this study, the authors introduce a new method that utilizes optical clearing and immunofluorescence to enable counting of podocytes in intact, whole glomeruli. The technique is validated in dose-dependent mouse models of podocyte depletion, establishing a new tool for WHAT MIGHT BE THE GOALS FOR ADULT studies of podocyte regeneration. PODOCYTE REGENERATION? 7. Lemley KV, Bertram JF, Nicholas SB, White K. Estimation of glomerular podocyte number: a selection of valid methods. J Am Soc Nephrol 2013; 24:1193–1202. Eloquent experimental studies have shown the close 8. Wiggins RC. The spectrum of podocytopathies: a unifying view of glomerular correlation between the magnitude of podocyte diseases. Kidney Int 2007; 71:1205–1214. 9. Wharram BL, Goyal M, Wiggins JE, et al. Podocyte depletion causes depletion and the ensuing development of glomer- glomerulosclerosis: diphtheria toxin-induced podocyte depletion in rats ex- ular scarring [8,9,81]. How much podocyte replace- pressing human diphtheria toxin receptor transgene. J Am Soc Nephrol 2005; 16:2941–2952. ment is required to recover/repair normal structure 10. Fukuda A, Chowdhury MA, Venkatareddy MP, et al. Growth-dependent and function? We argue that a reasonable goal podocyte failure causes glomerulosclerosis. J Am Soc Nephrol 2012; 23:1351–1363. should be to simply increase podocyte number to 11. Puelles VG, Cullen-McEwen LA, Taylor GE, et al. Human podocyte depletion that above the critical scarring threshold (20% && in association with older age and hypertension. Am J Physiol Renal Physiol 2016; 310:F656–F668. podocyte loss), which prevents segmental sclerosis The authors utilize a combination of microscopy techniques to measure the number progressing to global (Fig. 3). and density of glomerular cell populations in human children and adults. The results reveal that children have significantly lower absolute podocyte numbers but much greater podocyte density, compared with adults. This suggests age-related podocyte replacement from an unknown source that serves to partially offset a CONCLUSION net loss in podocyte density over time. 12. Puelles VG, Douglas-Denton RN, Cullen-McEwen LA, et al. Podocyte number In conclusion, it is clear that podocyte replacement in children and adults: associations with glomerular size and numbers of other glomerular resident cells. J Am Soc Nephrol 2015; 26:2277–2288. cells exist in adults. It is now important to investi- 13. Roeder SS, Stefanska A, Eng DG, et al. Changes in glomerular parietal gate more thoroughly whether their enhancement epithelial cells in mouse kidneys with advanced age. Am J Physiol Renal Physiol 2015; 309:F164–F178. can functionally improve outcomes such as albu- 14. Tharaux PL, Huber TB. How many ways can a podocyte die? Semin Nephrol minuria. Improved understanding of the mechan- 2012; 32:394–404. 15. Lim BJ, Yang JW, Do WS, Fogo AB. Pathogenesis of focal segmental isms involved will guide the development of glomerulosclerosis. J Pathol Transl Med 2016; 50:405–410. interventions, such as small molecules, that increase 16. Fogo AB. Decade in review – glomerular disease: the glomerulus reveals some secrets. Nat Rev Nephrol 2015; 11:633–634. the number of podocyte replacement cells above 17. Vogelmann SU, Nelson WJ, Myers BD, Lemley KV. Urinary excretion of viable the threshold required to prevent the onset of podocytes in health and renal disease. Am J Physiol Renal Physiol 2003; 285:F40–F48. glomerulosclerosis. 18. Petermann AT, Krofft R, Blonski M, et al. Podocytes that detach in experimental membranous nephropathy are viable. Kidney Int 2003; 64:1222– Acknowledgements 1231. 19. Appel D, Kershaw DB, Smeets B, et al. Recruitment of podocytes from None. glomerular parietal epithelial cells. 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J Am Soc Nephrol 2014; 25:693–705. migration of CoRL to the intraglomerular compartment following podocyte The authors showed that in PEC reporter mice, a subset of PECs serve as depletion. Third, this leads to an overall increase in the transdifferentiation of podocyte progenitors during adolescence, but not in adult mice, nor during CoRL to podocytes. glomerular hypertrophy following partial nephrectomy. In humans aged 2 weeks 33. Wang L, Tang Y, Howell DN, et al. A novel mouse model of podocyte to 2 years, some cells on Bowman’s capsule express the podocyte marker depletion. Nephron Exp Nephrol 2012; 121:e10–e22. synaptopodin, which was not detected at 7 years of age. The authors conclude 34. Romagnani P, Rinkevich Y, Dekel B. The use of lineage tracing to study kidney that a small fraction of committed podocytes reside on Bowman’s capsule close to injury and regeneration. Nat Rev Nephrol 2015; 11:420–431. the vascular stalk and is recruited onto the glomerular tuft during infancy to 35. Lombardi D, Lasagni L. Transgenic strategies to study podocyte loss and adolescence in mice and humans. regeneration. Stem Cells Int 2015; 2015:678347. 52. Schulte K, Berger K, Boor P, et al. Origin of parietal podocytes in atubular 36. Wanner N, Hartleben B, Herbach N, et al. Unraveling the role of podocyte glomeruli mapped by lineage tracing. J Am Soc Nephrol 2014; 25:129–141. && turnover in glomerular aging and injury. J Am Soc Nephrol 2014; 25:707– 53. Lasagni L, Ballerini L, Angelotti ML, et al. Notch activation differentially 716. regulates renal progenitors proliferation and differentiation toward the podo- The authors genetically labeled glomerular parietal epithelial cells (PECs) in cyte lineage in glomerular disorders. Stem Cells 2010; 28:1674–1685. inducible hPODXL1.rtTA;tetO.Cre;mT/mG mice exposed to doxycycline from 54. Angelotti ML, Ronconi E, Ballerini L, et al. Characterization of renal progeni- embryonic day 8.5 to postnatal day 28. They confirmed studies by the group of tors committed toward tubular lineage and their regenerative potential in renal Moeller that during adolescence, PECs serve as podocyte progenitors under tubular injury. Stem Cells 2012; 30:1714–1725. physiological conditions. Using a model of acute podocyte depletion by genetically 55. Sagrinati C, Netti GS, Mazzinghi B, et al. Isolation and characterization of inserting the diphtheria toxin receptor into podocytes, the authors noted podocyte multipotent progenitor cells from the Bowman’s capsule of adult human regeneration, but the results suggested a source other than PECs. PECs did not kidneys. J Am Soc Nephrol 2006; 17:2443–2456. serve a role for podocyte regeneration in the unilateral nephrectomy model, which 56. Darisipudi MN, Kulkarni OP, Sayyed SG, et al. Dual blockade of the homeo- is typically not accompanied by a marked decrease in podocyte number. Finally, static chemokine CXCL12 and the proinflammatory chemokine CCL2 has using young middle-aged mice (12 months old), the authors noted no role for PECs additive protective effects on diabetic kidney disease. Am J Pathol 2011; progenitors. 179:116–124. 37. Lasagni L, Angelotti ML, Ronconi E, et al. Podocyte regeneration driven by 57. Peired A, Angelotti ML, Ronconi E, et al. Proteinuria impairs podocyte && renal progenitors determines glomerular disease remission and can be regeneration by sequestering retinoic acid. J Am Soc Nephrol 2013; pharmacologically enhanced. Stem Cell Reports 2015; 5:248–263. 24:1756–1768. The authors evaluated whether the regenerative response to podocyte injury 58. Ohse T, Chang AM, Pippin JW, et al. A new function for parietal epithelial influences chronic kidney disease outcome. In models of focal segmental glomer- cells: a second glomerular barrier. 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Am J Pathol ment with BIO, a GSK3 inhibitor, significantly increased disease remission by 2013; 183:1769–1778. enhancing renal progenitor sensitivity to the differentiation effect of endogenous 61. Migliorini A, Angelotti ML, Mulay SR, et al. The antiviral cytokines IFN-a and retinoic acid. These results establish renal progenitors as critical determinants of IFN-b modulate parietal epithelial cells and promote podocyte loss: implica- glomerular disease outcome and a pharmacological enhancement of their differ- tions for IFN toxicity, viral glomerulonephritis, and glomerular regeneration. Am entiation as a possible therapeutic strategy. J Pathol 2013; 183:431–440.

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