Regeneration of Glomerular Podocytes by Human Renal Progenitors
Elisa Ronconi*,
Costanza Sagrinati*,
Maria Lucia Angelotti*,
Elena Lazzeri*,
Benedetta Mazzinghi*,
Lara Ballerini*,
Eliana Parente*,
Francesca Becherucci*,
Mauro Gacci,
Marco Carini,
Enrico Maggi*,
Mario Serio*,
Gabriella Barbara Vannelli,
Laura Lasagni*,
Sergio Romagnani* and
Paola Romagnani*
* Excellence Center for Research, Transfer and High Education Denothe, Department of Medical and Surgical Critical Care, and Department of Anatomy, University of Florence, Florence, Italy
Correspondence: Dr. Paola Romagnani, Department of Clinical Pathophysiology, Nephrology Section, University of Florence, Viale Pieraccini 6, 50139, Firenze, Italy. Phone: ++39554271356; Fax: ++39554271357; E-mail: p.romagnani{at}dfc.unifi.it
Received for publication July 11, 2008.
Accepted for publication September 21, 2008.
Depletion of podocytes, common to glomerular diseases in general,plays a role in the pathogenesis of glomerulosclerosis. Whetherpodocyte injury in adulthood can be repaired has not been established.Here, we demonstrate that in the adult human kidney, CD133+CD24+cells consist of a hierarchical population of progenitors thatare arranged in a precise sequence within Bowman's capsule andexhibit heterogeneous potential for differentiation and regeneration.Cells localized to the urinary pole that expressed CD133 andCD24, but not podocyte markers (CD133+CD24+PDX– cells),could regenerate both tubular cells and podocytes. In contrast,cells localized between the urinary pole and vascular pole thatexpressed both progenitor and podocytes markers (CD133+CD24+PDX+)could regenerate only podocytes. Finally, cells localized tothe vascular pole did not exhibit progenitor markers, but displayedphenotypic features of differentiated podocytes (CD133–CD24–PDX+cells). Injection of CD133+CD24+PDX– cells, but not CD133+CD24+PDX+or CD133-CD24– cells, into mice with adriamycin-inducednephropathy reduced proteinuria and improved chronic glomerulardamage, suggesting that CD133+CD24+PDX– cells could potentiallytreat glomerular disorders characterized by podocyte injury,proteinuria, and progressive glomerulosclerosis.
Glomerular diseases account for 90% of ESRD at a cost of $20billion/yr in the United States.1 The traditional glomerulardisease classification encompasses a bewildering array of descriptivepathologic entities and their clinical counterparts. However,converging evidence suggests that podocyte depletion, secondaryto toxic, genetic, immune, infectious, oxidant, metabolic, hemodynamic,and other mechanisms, is a common determining factor that canresult in a broad spectrum of clinical syndromes. As long asthe podocyte loss is limited, restitution or repair is possible.By contrast, 20 to 40% podocyte loss results in a scarring response,until at greater than 60% podocyte loss glomeruli become globallysclerotic and nonfiltering. These stages of podocyte depletionare accompanied by corresponding degrees of proteinuria and,as an increasing proportion of glomeruli become involved, bymeasurable reduction in the clearance function of the kidney.1–3To what extent podocytes can be replaced at all during adultlife, and if so, how and at what rate, is still unclear. Indeed,mature podocytes have limited capacity to divide in situ anddisplay all phenotypic and functional features of highly specialized,terminally differentiated cells.4,5 A potential mechanism forpodocyte replacement might be stem cell migration from the bonemarrow, as described in some models.6–8 However, it hasbeen established that the developmental source of podocytesare resident renal progenitors.4,5,9,10
Recently, on the basis of CD24 expression, a surface moleculethat has been used to identify different types of human stemcells,11–13 CD133 (a marker of hematopoietic and othertypes of adult tissue stem cells13,14), and the polycomb groupprotein Bmi-1 (a transcription factor that is critical for maintenanceof stem cell self renewal15,16), we have identified a subsetof cells in adult human kidneys that exhibit stem cell phenotypicand functional features and can regenerate tubular cells ofdifferent portions of the nephron, in vitro and in vivo.17–19These CD133+CD24+ renal progenitors are physically located withinBowman's capsule, the only place in the kidney that appearsto be contiguous with both tubular cells as well as glomerularpodocytes. In this study, we investigated whether CD133+CD24+renal progenitors can also regenerate podocytes through theirdivision and migration along Bowman's capsule toward the glomerulartuft. We demonstrate here that CD133+CD24+ renal progenitorsare a heterogeneous and hierarchical population of progenitorcells arranged in a precise sequence within the Bowman's capsuleof adult human kidneys, which exhibit a heterogeneous differentiativeand regenerative potential for either tubular renal cells orpodocytes.
Heterogeneous Expression of Renal Progenitors and Podocyte Markers among Cells of Bowman's Capsule
To evaluate the possible existence of a hierarchical relationshipbetween CD133+CD24+ renal progenitors and podocytes, we firstanalyzed the expression of the renal progenitor markers CD133and CD24; markers that specifically stain podocytes in the contextof adult healthy human glomeruli, such as nestin;20 and complementreceptor-1 (CR1)21 or podocalyxin (PDX), which stains podocytesand endothelial cells,22 in glomerular structures of adult humankidneys by using laser confocal microscopy. On the basis ofthe expression of CD133, CD24, PDX, nestin, and CR1, we demonstratedby confocal microscopy that CD133+CD24+ renal progenitors area heterogeneous and hierarchical population of undifferentiatedand more differentiated cells that are arranged in a precisesequence within Bowman's capsule (Figure 1). Three distinctpopulations of cells were identified. A subset of more undifferentiatedcells expressing CD133 and CD24 in the absence of CR1, nestin,and PDX localized at the urinary pole of Bowman's capsule (Figure 1).A transition population expressing CD133 and CD24, as well asnestin, CR1, and PDX, usually localized between the urinaryand the vascular pole (Figure 1). Finally, more differentiatedcells expressing neither CD133 nor CD24, but exhibiting thepodocyte markers, localized at the vascular pole of Bowman'scapsule and were contiguous with fully differentiated podocytes(Figure 1). A list of the different markers of renal cells usedin the study is given in Table 1.
Figure 1. Heterogeneous expression of renal progenitors and podocytes markers by cells of Bowman's capsule in adult human kidney. (A) Triple-label immunofluorescence for CD133 (red), CD24 (blue), and PDX (green) showing co-expression of CD133 and CD24 on one subset (red arrow) and co-expression of CD133, CD24, and PDX on another subset of parietal epithelial cells in Bowman's capsule of a human kidney (white arrows). CD133–CD24–PDX+ cells are also visible (yellow arrow). Sectioning of the glomerulus does not allow its polarity to be established. Objective 20x, zoom 1.7. (B) Triple-label immunofluorescence for CD133, PDX, and CR1 showing a subset of CD133+ cells that co-express PDX and CR1 (white arrow), and more differentiated cells that co-express PDX and CR1 but lack CD133 (yellow arrow) and localize at the vascular pole (VP). Objective 20x, zoom 2.4. (C) Triple-label immunofluorescence for CD133 (green), nestin (blue), and CR1 (red) demonstrating the existence of cells expressing CD133 in absence of the podocyte markers nestin and CR1, which localize to the urinary pole (UP) of Bowman's capsule, and cells expressing CD133, nestin, and CR1 (white arrow), which localize between the urinary and the vascular pole (VP) of Bowman's capsule. Glomerular podocytes also appear as CD133–nestin+CR1+. Objective 20x, zoom 2.8.
Table 1. Different markers of renal cells used in the study and their localization in adult healthy human kidneys
Identification of CD133+CD24+PDX–, CD133+CD24+PDX+, and CD133–CD24–PDX+ Cells in Adult Human Kidneys
To recover each of the three populations, we took advantageof the surface expression by these cells of CD133 and/or PDX.PDX is considered a podocyte marker but, at least in human kidney,its expression has also been described in some parietal cellsof Bowman's capsule,23 and on a subset of endothelial cells.22Therefore, total renal cells were first depleted of CD45+ cells(leukocytes)17,18 and then analyzed for the contemporaneousexpression of CD133, CD24, PDX, and CD31 (an endothelial cellmarker) by triple-label immunofluorescence. FACS analysis demonstratedthat CD133+CD24+ cells were 1 to 4% of total renal cells, aspreviously reported,17–19 whereas CD133+CD24+PDX+ cellswere 0.2 to 0.7% of total renal cells. In addition, none ofthese two populations expressed the endothelial cell markerCD31.17,18 These results suggest that CD133+CD24+ cells containa transition population co-expressing markers of both renalprogenitors and podocytes (CD133+CD24+PDX+) and a more undifferentiatedpopulation that does not express PDX, in agreement with thefindings obtained by confocal microscopy (Figure 2A). To providefurther support to this possibility, we separated PDX+ and PDX–cells by immunomagnetic cell sorting, which allowed recoveryof the two populations with a purity of more than 98% (Figure 2B).The assessment of renal progenitor markers on PDX+ cells revealedthat a relevant percentage (4 to 8%) of these cells co-expressedCD133 and CD24. Moreover, CD133+CD24+PDX+ cells did not expressCD31, whereas they consistently co-expressed the podocyte markerWT-1 (Figure 2B, left). These findings provide additional evidencefor the existence of a rare, distinct population characterizedby an intermediate phenotype between renal progenitors and differentiatedpodocytes. Of note, CD133+CD24+ cells also represented 1 to5% of PDX- cells (Figure 2B, right). In previous studies, wedemonstrated that the combined surface expression of CD133 andCD24 in healthy adult human kidneys is a selective propertyof renal progenitor cells of Bowman's capsule.17–19 Thus,CD133+CD24+PDX+, CD133+CD24+PDX–, and CD133–CD24–PDX+populations were directly purified from total renal cell suspensionsby means of immunomagnetic techniques and analyzed by real-timequantitative reverse transcription PCR (RT-PCR). CD133+CD24+PDX–cells expressed the renal progenitor markers CD133 and Bmi-1,17–19but not the podocyte-specific markers nephrin, podocin, andWilms’ tumor antigen-1 (WT-1). CD133+CD24+PDX+ cells expressedboth renal progenitor and podocyte markers. CD133–CD24–PDX+cells did not express renal progenitor markers but high levelsof podocyte-specific transcripts (Figure 3, A through C). Takentogether, these results suggest that CD133+CD24+ renal progenitorsrepresent a heterogeneous population, including a subset ofcells (CD133+CD24+PDX+) that displays initial signs of podocytecommitment.
Figure 2. Identification of CD133+CD24+PDX–, CD133+CD24+PDX+, and CD133–CD24–PDX+ cells in adult human kidney cell suspensions. (A) After depletion of CD45+ cells, total renal cells were analyzed for the contemporaneous expression of CD133, CD24, PDX, and CD31 by triple-label immunofluorescence using FACS analysis, demonstrating that CD133+CD24+ cells represent 3.7%, whereas CD133+CD24+PDX+ cells represent 0.6 to 0.7% of total renal cells. Neither CD133+CD24+ cells nor CD133+CD24+PDX+ cells express the endothelial cell marker CD31. (B) Expression of CD133, CD24, CD31, and WT-1, as assessed by flow cytometry on PDX+ cells (left) and on PDX– cells (right). Purification of PDX+ cells confirms the existence of a population of PDX+ cells that co-express CD133 and CD24 but do not express CD31 and represent a subset of WT-1-expressing podocytes.
Figure 3. Phenotypic characterization and distinct differentiative properties of clonal progenies of CD133+CD24+PDX– and CD133+CD24+PDX+ cells. Assessment of mRNA levels for CD133, Bmi-1, nephrin, WT-1, and podocin by real-time quantitative RT-PCR on (A) freshly isolated CD133+CD24+PDX– cells, (B) freshly isolated CD133+CD24+PDX+ cells, and (C) freshly isolated CD133–CD24–PDX+ cells. Data are mean ± SEM of triplicate assessment of one representative from independent experiments performed on sorted cells obtained from four different donors; fgs = femtograms. (D) Representative micrograph of clonally expanded CD133+CD24+PDX– cells, objective 10x. Left: Expression of tubular markers before (day 0) and after (day 21) culture in tubular differentiation medium was assessed by confocal microscopy analysis for LTA and by real-time quantitative RT-PCR comparison of multiple tubular specific markers before and after differentiation. Data are mean ± SEM of values obtained in 20 different clones. TO-PRO-3 counterstains nuclei (blue). Objective 20x, zoom 1.8. Right: Expression of podocyte markers before (day 0) and after (day 7) culture of CD133+CD24+PDX– clones in VRAD medium was assessed by confocal microscopy for CD133 (green), synaptopodin (red), nephrin (green), GLEPP (red), and by real-time quantitative RT-PCR comparison of nephrin, WT-1, podocin, and PDX mRNA levels before and after differentiation. TO-PRO-3 counterstains nuclei (blue). Objective 20x, zoom 0.9. (E) Representative micrograph of CD133+CD24+PDX+ cells (objective 20x) and staining for CD133 (green), synaptopodin (red), WT-1 (green), nephrin (green), and GLEPP-1 (red) as assessed by confocal microscopy. One representative of 20 distinct clones analyzed is shown. TO-PRO-3 counterstains nuclei (blue). Objective 20x, zoom 0.9.
Clonally Expanded CD133+CD24+PDX– Cells Generate In Vitro Tubular Cells and Podocytes, whereas CD133+CD24+PDX+ Cells Can Generate Only Podocytes
The functional characterization of the three populations provideddefinitive evidence that CD133+CD24+PDX– cells representa multipotent population, whereas CD133+CD24+PDX+ cells representtransition cells showing features of podocyte progenitors andthe CD133–CD24–PDX+ cells are fully differentiatedpodocytes. Indeed, CD133+CD24+PDX– cells could be cloned,maintained in culture, and expanded in a medium that allowsthe growth of undifferentiated renal progenitors [microvascularendothelial growth medium (EGM-MV)].17–19 More importantly,clonally expanded CD133+CD24+PDX– cells could not onlybe differentiated toward the tubular lineage if cultured inthe tubular differentiating medium [renal epithelial cell growthmedium (REGM+HGF)],17,18 but also toward the podocyte lineageif cultured in the podocyte-maintaining medium, VRAD (VitaminD3, retinoic-acid-supplemented DMEM)24 (Figure 3D). Differentiationtoward tubular cells resulted in the acquisition of bindingproperties of Lotus tetragonolobus agglutinin (LTA), a specificproperty of proximal tubular epithelia (Figure 3D). Real-timeRT-PCR demonstrated strong upregulation of other markers ofdifferent portions of the nephron, such as aminopeptidase Aand the Na/Gluc1 cotransporter, -glutamyltransferase, the Na/Hexchanger, aquaporin-1, aquaporin-3, or the thiazide-sensitiveNa/Cl transporter (Figure 3D). By contrast, when the same clonesof CD133+CD24+PDX– cells were cultured in VRAD, they beganto express the podocyte markers nephrin, WT-1, synaptopodin,podocin, PDX, and anti-glomerular epithelial protein 1 (GLEPP-1)at both mRNA and protein levels, as demonstrated by real-timeRT-PCR and confocal microscopy, respectively. Differentiationof CD133+CD24+PDX– cells associated with a progressivedownregulation of CD133 expression, as already reported.17 Ofnote, when single cell suspensions obtained from ten cloneswere recloned by limiting dilution, the resulting second-roundsubclones displayed the same capacity for multidifferentiationas the original clone, providing additional evidence that CD133+CD24+PDX–cells are indeed multipotent and exhibit self renewal in culture.On the other hand, CD133+CD24+PDX+ cells could not be expandedor cloned in EGM-MV, whereas if plated in VRAD medium they generatedclones comprised of a maximum of 800 to 1000 cells that couldnever be subcloned, suggesting that these cells did not displayself-renewal capacities. Confocal microscopy and real-time RT-PCRdemonstrated that CD133+CD24+PDX+ clones showed the phenotypeof transition cells co-expressing CD133, CD24, WT-1, synaptopodin,GLEPP-1, and nephrin (Figure 3E). Accordingly, clones couldnever be obtained from CD133+CD24+PDX+ cells in the presenceof REGM+HGF medium, suggesting that these cells cannot generatetubular cells. Finally, CD133–CD24–PDX+ were unableto generate clones even in VRAD medium and survived in culturefor only a few days, further confirming their nature as terminallydifferentiated cells. These findings suggest that CD133+CD24+PDX–cells in Bowman's capsule represent an uncommitted populationof cells with extensive self-renewal potential that can generateboth tubular cells and podocytes. By contrast, CD133+CD24+PDX+cells do not display the potential to differentiate in tubularcells, suggesting that they represent a progenitor already committedtoward the podocyte lineage.
Only CD133+CD24+PDX– Cells Reduce Proteinuria and Improve Glomerular and Tubular Injury in Severe Combined Immunodeficiency Mice Suffering from Nephrotic Syndrome
The ability of CD133+CD24+PDX– and CD133+CD24+PDX+ cellsto regenerate injured renal cells was then assessed in a modelof adriamycin-induced renal injury, which resembles focal segmentalglomerulosclerosis, a human disorder characterized by podocytedepletion and tubular damage.25 To this end, CD133+CD24+PDX–,CD133+CD24+PDX+ cells, or saline were injected into adriamycin-treatedSevere Combined Immunodeficiency (SCID) mice. As an additionalcontrol, adriamycin-treated SCID mice were injected with a mixtureof CD133–CD24– renal cells. Because persistent podocytedepletion induces proteinuria, urinary albumin/creatinine ratiolevels were measured in all mice 7 d after adriamycin injection.Mice with adriamycin-induced nephropathy showed high urinaryalbumin/creatinine ratio levels that were unaffected by injectionof saline, CD133–CD24– cells, or CD133+CD24+PDX+cells (Figure 4A). By contrast, injection of CD133+CD24+PDX–cells strongly reduced proteinuria, as reflected by significantlylower urinary albumin/creatinine ratios (Figure 4A). Similarresults were obtained when single clones of CD133+CD24+PDX–cells were used (Figure 4A). Because adriamycin-induced nephropathyis a chronic disorder characterized by persistent proteinuriaand progressive glomerulosclerosis with tubulointerstitial injury,we also tested the effect of CD133+CD24+PDX– cell treatmentover long periods of time. As shown in Figure 4B, treatmentwith repeated injections of CD133+CD24+PDX– cells significantlyreduced urinary albumin/creatinine ratios at all time pointsanalyzed. In chronically injured kidneys, the improvement ofproteinuria induced by injection of CD133+CD24+PDX– cellswas also associated with reduced glomerular and tubulointerstitialinjury, as demonstrated in periodic acid-Schiff (PAS)-stainedsections at day 28 (Figure 4, C through E). In saline-treatedmice, glomerulosclerosis was significantly increased in associationwith reduction of glomerular surface area, whereas relativeinterstitial volume was expanded. Moreover, the degree of tubularatrophy, as characterized by a decrease in the height of tubularepithelial cells, loss of brush border, and vacuolization, wasaggravated in the saline-treated group as compared with CD133+CD24+PDX–cells at day 28 after adriamycin injection (Figure 4, C throughE).
Figure 4. CD133+CD24+PDX– cells, but not CD133+CD24+PDX+ or CD133-CD24– cells, reduce proteinuria and improve glomerular and tubulointerstitial injury in SCID mice affected by adriamycin-induced nephropathy. (A) Albumin/creatinine ratio as measured at day 7 in adriamycin-treated mice that received saline (n = 12), CD133–CD24– (n = 6), CD133+CD24+PDX+ (n = 6), and CD133+CD24+PDX– (n = 12) bulk of cells or two representative clones of CD133+CD24+PDX– cells (n = 3 for each clone). Data are expressed as mean ± SEM. a versus b, a versus c, b versus c, d versus e, d versus f, e versus f, NS; a versus d, b versus d, c versus d, P < 0.001; a versus e, a versus f, P < 0.01; b versus e, b versus f, c versus e, c versus f, P < 0.05. (B) Time course experiments of albumin/creatinine ratio as measured in healthy (black square) or adriamycin-treated mice that received saline (red circle) or CD133+CD24+PDX– cells (blue triangle). Red arrow points to the day of adriamycin injection. Black arrows point to the days of CD133+CD24+PDX– cell injection. Data are expressed as mean ± SEM. (n = 12 mice at each time point for each group of treatment). P < 0.0001 between the two groups of treatment (ANOVA for multiple comparisons). (C) Left: PAS staining of renal cortical sections of mice with adriamycin-induced nephropathy treated with CD133+CD24+PDX– cells; objective 20x. Right: High-power magnification of a glomerulus; objective 40x. (D) Left: PAS staining of renal cortical sections of mice with adriamycin-induced nephropathy treated with saline.; objective 20x. Right: High-power magnification of a glomerulus; objective 40x. (E) Quantitation of glomerular and tubulointerstitial injuries in renal cortical sections of saline-treated and CD133+CD24+PDX– treated mice after the induction of adriamycin nephropathy. Data are expressed as mean ± SEM. g versus i and h versus j, P < 0.001.
CD133+CD24+PDX– and CD133+CD24+PDX+ Cells Exhibit Different Regenerative and Differentiative Potential in SCID Mice Suffering from Nephrotic Syndrome
To further investigate their ability to regenerate injured podocytesand tubular cells, CD133+CD24+PDX–, CD133+CD24+PDX+, andCD133–CD24– renal cells were labeled with the redfluorescent dye PKH26 before their injection into adriamycin-treatedSCID mice. Labeled CD133+CD24+PDX– cells localized toglomerular structures, where they acquired the podocyte-specificmarkers synaptopodin, WT-1, nephrin, and podocin (Figure 5,A through F). In addition, double-label immunohistochemistryfor human HLA-I antigen and podocin confirmed the engraftmentof CD133+CD24+PDX– cells into the glomerular structures(Figure 5, G through I). Furthermore, relevant numbers of labeledCD133+CD24+PDX– cells were also observed in tubular structureslabeled with LTA (Figure 5K). Identical results were obtainedwhen single clones of CD133+CD24+PDX– cells were used.By contrast, when labeled CD133+CD24+PDX+ cells were injected,only rare red-labeled podocytes were observed (Figure 5L). Finally,red labeling was never observed in mice injected with CD133–CD24–renal cells (Figure 5M), with saline solution (Figure 5J), orin healthy mice injected with CD133+CD24+PDX– cells (SupplementaryFigure 1). Quantitation of the number of PKH26-positive cellsexpressing markers of differentiated podocytes or tubular cellswas performed on sections stained with podocin or LTA, respectively.On day 7 after injury, the number of cells that showed PKH26labeling was equal to 11.08 ± 3.3% of all podocytes,and to 7.5 ± 1.9% of all proximal tubular cells in micetreated with CD133+CD24+PDX– cells, whereas no tubularcells and only 0.8 ± 0.4% of all podocytes were replacedby CD133+CD24+PDX+ cells. After 45 d the number of CD133+CD24+PDX–cells engrafted in the kidney of mice with adriamycin-inducednephropathy remained similar (Supplementary Figure 2), thusconfirming the different regenerative and differentiative potentialof CD133+CD24+PDX– and CD133+CD24+PDX+ cells.
Figure 5. Distinct regenerative potential of CD133+CD24+PDX–, CD133+CD24+PDX+, or CD133–CD24– cells for podocytes and tubular cells in SCID mice affected by adriamycin-induced nephropathy. (A) Representative micrograph of kidney sections of mice with adriamycin-induced nephropathy treated with PKH26-labeled CD133+CD24+PDX– cells (red) and stained with synaptopodin (SYN, green), which demonstrates engraftment of CD133+CD24+PDX– cells in both glomerular and tubular structures and differentiation toward podocytes and tubular cells at day 7. TO-PRO-3 counterstains nuclei (blue). Objective 40x, zoom 1.4. (B) High magnification of the split image showed in panel a detailing PKH26 labeling in glomerular cells. TO-PRO-3 counterstains nuclei (blue). Objective 40x, zoom 1.7. (C) High magnification of the split image showed in panel a, detailing synaptopodyn (SYN) labeling in glomerular cells. TO-PRO-3 counterstains nuclei (blue). Objective 40x, zoom 1.7. (D) Representative micrograph of kidney sections of mice with adriamycin-induced nephropathy treated with PKH26-labeled CD133+CD24+PDX– cells (red) and stained with WT-1 (green), which demonstrates engraftment of CD133+CD24+PDX– cells and tubular structures and differentiation toward podocytes (arrow) and tubular cells at day 28. TO-PRO-3 counterstains nuclei (blue). Objective 40x, zoom 1.2. (E) Representative micrograph of kidney sections of mice with adriamycin-induced nephropathy treated with PKH26-labeled CD133+CD24+PDX– cells (red) and stained with nephrin (green), which demonstrates engraftment of CD133+CD24+PDX– cells in glomerular structures and differentiation toward podocytes (arrow) at day 28. TO-PRO-3 counterstains nuclei (blue). Objective 40x, zoom 1.7. (F) Representative micrograph of kidney sections of mice with adriamycin-induced nephropathy treated with PKH26-labeled CD133+CD24+PDX– cells (red) and stained with podocin (green), which demonstrates engraftment of CD133+CD24+PDX– cells in glomerular structures and differentiation toward podocytes (arrow) at day 28. TO-PRO-3 counterstains nuclei (blue). Objective 40x, zoom 1.8. (G) Double-label immunohistochemistry for podocin (AEC, red) and HLA-I human antigen (vector SG, dark blue) in a section adjacent to panel f from kidneys of SCID mice with adriamycin-induced nephropathy treated with CD133+CD24+PDX– cells, which confirms that the PKH26-labeled cells shown in panel f also exhibit HLA-I immunostaining (arrow) at day 28. Objective 40x. (H) Double-label immunohistochemistry for podocin (AEC, red) and HLA-I human antigen (vector SG, dark blue) in kidneys of SCID mice with adriamycin-induced nephropathy treated with CD133+CD24+PDX– cells, which demonstrates engraftment of a CD133+CD24+PDX– cell in a glomerulus and its differentiation toward a podocyte (arrow) at day 28. Objective 40x. (I) Double-label immunohistochemistry for podocin (AEC, red) and HLA-I human antigen (vector SG, dark blue) in kidneys of SCID mice with adriamycin-induced nephropathy treated with CD133+CD24+PDX– cells, which demonstrates engraftment of several CD133+CD24+PDX– cells and their differentiation toward podocytes (arrows) at day 28. Objective 40x. (J) Double-label immunohistochemistry for podocin (AEC, red) and HLA-I human antigen (vector SG, dark blue) in kidneys of SCID mice with adriamycin-induced nephropathy treated with saline, which shows absence of HLA-I immunostaining at day 28. Objective 40x. (K) Representative micrograph of kidney sections of mice with adriamycin-induced nephropathy treated with PKH26-labeled CD133+CD24+PDX– cells (red) and stained with LTA (green), which demonstrates engraftment of CD133+CD24+PDX– cells in proximal tubular structures and differentiation toward tubular cells at day 28. TO-PRO-3 counterstains nuclei (blue). Objective 40x. (L) Representative micrograph of kidney sections of mice with adriamycin-induced nephropathy treated with PKH26-labeled CD133+CD24+PDX+ cells (red) and stained with WT-1 (green), which demonstrates rare CD133+CD24+PDX+ cells in glomerular structures at day 28. TO-PRO-3 counterstains nuclei (blue). Objective 40x, zoom 1.7. (M) Representative micrograph of kidney sections of mice with adriamycin-induced nephropathy treated with PKH26-labeled CD133–CD24– cells (red) and stained with WT-1 (green), which demonstrates the absence of CD133–CD24– cells in glomerular or tubular structures at day 28. TO-PRO-3 counterstains nuclei (blue). Objective 20x.
Recent insights have defined a unified concept of glomerulardiseases in which podocyte injury or loss is a common determiningfactor, which suggests the need for rational clinical effortsto allow podocyte preservation.1,3 Mature podocytes are postmitoticcells that can undergo DNA synthesis to a limited degree butdo not proliferate because they arrest in the G2/M phase ofthe cell cycle.1,3 However, in most adult epithelia, replacementof damaged or dead cells is maintained through the presenceof stem/progenitor cells.26 Unless the epithelial stem/progenitorcells are permanently damaged, most epithelia are able to repairtheir tissues after injuries.26 Although glomerular disordersrepresent the most prominent cause of ESRD, remission of thedisease and regression of renal lesions have been observed inexperimental animals and even in humans.27 This shows that remodelingof glomerular architecture is possible, which would imply regenerationof the injured podocytes and reconstitution of the glomerulartuft. However, the inability of the podocyte to proliferateand replace injured cells suggests the existence of potentialstem/progenitor cells within the adult glomerulus.
In this study, we provide the first evidence that podocytescan be regenerated from a resident population of renal progenitorslocalized within the parietal epithelium of Bowman's capsuleof the human renal glomerulus. On the basis of expression ofthe renal progenitor markers CD133 and CD24, as well as of thepodocyte markers PDX, nestin, synaptopodin, and CR1, we demonstratedby confocal microscopy and FACS analysis that CD133+CD24+ renalprogenitors are a heterogeneous and hierarchical populationof undifferentiated and more differentiated cells that are arrangedin a precise sequence within Bowman's capsule. A subset of moreundifferentiated cells expressing renal progenitor markers inthe absence of podocyte markers localized at the urinary poleof Bowman's capsule. As demonstrated by clonal analysis, thesecells could act as bipotent progenitors for both tubular cellsand podocytes in vitro and in vivo and exhibited self-renewalpotential. A transition population expressing both renal progenitorsand podocyte markers was localized between the urinary and thevascular pole of Bowman's capsule and exhibited differentiativeproperties only toward the podocyte lineage and a limited potentialof clonogenicity and amplification (Figure 6). Finally, moredifferentiated cells, that did not express renal progenitormarkers but exhibited high levels of the podocyte specific markerslocalized at the vascular pole of Bowman's capsule, contiguousto podocytes. These cells shared with podocytes all of the propertiesof postmitotic cells and could not be cloned or amplified inculture, consistent with their nature as terminally differentiatedpodocytes, thus showing agreement with previous studies thatthere are capsular parietal cells localized at the vascularpole of the glomerulus analogous in size, shape, and phenotypeto visceral podocytes, but whose function and role were unknown.23,28,29The discovery that CD133+CD24+ renal progenitors represent apotential source for podocyte replacement provides the basisfor a novel concept that podocyte injuries can be repaired inprinciple by a resident stem cell compartment.4 In addition,the results of this study provide an intriguing explanationfor the genesis of crescents and pseudocrescents, which areknown to reflect uncontrolled proliferation of parietal epithelialcells in response to injury.30,31 Indeed, it is tempting tospeculate that CD133+CD24+ renal progenitors proliferate inan attempt to replace injured podocytes, but if regenerationoccurs in a dysregulated manner it can generate hyperplasticlesions that can lead to renal progenitor depletion, scar formation,and nephron loss.
Figure 6. Schematic representation of the hierarchical distribution of CD133+CD24+PDX– and CD133+CD24+PDX+ cells within human glomeruli. CD133+CD24+PDX– renal progenitors (red) are localized at the urinary pole in close contiguity with tubular renal cells (yellow). A transitional cell population (CD133+CD24+PDX+, red/green) displays features of either renal progenitors (red) or podocytes (green) and localizes between the urinary pole and the vascular pole. At the vascular pole of the glomerulus, the transitional cells are localized in close continuity with cells that lack CD133 and CD24, but exhibit the podocyte markers and the phenotypic features of differentiated podocytes (green).
Finally, the demonstration that CD133+CD24+ renal progenitorscan regenerate injured podocytes suggests that these cells mightprovide a novel tool for cell therapy of proteinuric renal disorders.However, only CD133+CD24+PDX– cells displayed the potentialto regenerate podocytes and tubular cells and functionally improveglomerular injury, which suggests that these cells can behaveas bipotent progenitors. By contrast, CD133+CD24+PDX+ cellsdid not induce improvement of glomerular function and rarelygenerated podocytes, suggesting that these cells display a verylimited engraftment capacity in agreement with their lack ofself-renewal potential. Finally, CD133–CD24– cellsdid not engraft within injured human kidneys at all and didnot induce functional improvement of glomerular injury, whichis consistent with their nature of terminally differentiatedcells. Taken together, these results suggest that CD133+CD24+PDX–renal progenitors may be ideal for stem-cell-based kidney regenerationbecause of their broad differentiation potential, which allowsreplacement of both podocytes and tubular cells because of theirinherent organ-specific identity. Although we cannot excludethe possibility that the engraftment observed might at leastin part be related to cell fusion and exchange of PKH26 dyebetween cells, the observation that CD133+CD24+PDX– renalprogenitors reduce proteinuria is of potential clinical utility.Indeed, several studies have examined the possibility that bone-marrow-derivedstem cells might be used for renal repair.32–36 However,their beneficial role can be offset by their abnormal localdifferentiation into adipocytes accompanied by glomerular sclerosis.33In conclusion, the results of this study provide the first demonstrationthat glomerular injury can be repaired by using resident renalprogenitor cells and suggest that the kidney might contain a"renopoietic system" (Figure 6) with a bipotent progenitor localizedat the urinary pole of Bowman's capsule, where it can initiatethe replacement and regeneration of glomerular and tubular epithelialcells.
Antibodies
The following antibodies were used: anti-CD24 mAb (SN3), anti-WT-1mAb (F6), and anti-nephrin mAb (C17) (Santa Cruz Biotechnology,Santa Cruz, California); anti-human HLA-I mAb (W6/32) (Sigma-Aldrich,Saint Louis, Missouri); anti-CD133/2 mAb (293C3) and PE-conjugatedanti CD133/2 mAb (293C3) (Miltenyi Biotec GmbH, Bergisch Gladbach,Germany); anti-synaptopodin mAb (G1D4) (Progen, Heidelberg,Germany); anti-podocin pAb (Alpha-Diagnostic, San Antonio, Texas);anti-nestin pAb (Chemicon, Temecula, California); anti-CD31mAb (WM-59), anti-CR1 mAb (E11), PE-conjugated mouse anti-IgG2b(MPC-11), and anti-IgG1 (E11) (BD Biosciences, San Diego, California);anti-PDX mAb (222328) and PE-conjugated anti-PDX mAb (222328)(R&D Systems, Minneapolis, Minnesota); anti-GLEPP-1 mAb(5C11) (BioGenex, San Ramon, California); and anti IgG2a (HOPC-1)and PE-conjugated goat anti rat IgG (H+L) (Southern Biotech,Birmingham, Alabama). Alexa Fluor 633-labeled goat anti-mouseIgG1, Alexa Fluor 488-labeled goat anti-mouse IgG2a, Alexa Fluor488-labeled goat anti-mouse IgG1, Alexa Fluor 488-labeled goatanti-rabbit IgG, Alexa Fluor 546-labeled goat anti-mouse IgG2b,and Alexa Fluor 546-labeled goat anti-mouse IgG2a were fromMolecular Probes (Eugene, Oregon).
Tissues
Normal kidney fragments were obtained from 15 patients who underwentnephrectomy because of renal tumors, in accordance with therecommendations of the Ethical Committee of the Azienda Ospedaliero-UniversitariaCareggi in Florence, Italy.
Confocal Microscopy
Confocal microscopy was performed on 5-µm sections offrozen renal tissues, or on cells cultured on chamber slidesby using a LSM510 META laser confocal microscope (Carl Zeiss,Jena, Germany), as described.37 Staining with FITC-labeled LTA(Vector Laboratories, Burlingame, California) was performedas described.17
Immunomagnetic Cell Sorting and Flow Cytometry
To obtain PDX+ and PDX– cells, cortex from normal kidneyfragments was minced and digested with collagenase IV (750 U/ml;Sigma) for 45 min at 37°C, then depleted of leukocytes usinganti-CD45 MicroBeads (Miltenyi), as described previously.17,18The CD45– fraction was collected and analyzed by flowcytometry. PDX+ and PDX– cells were isolated by incubatingthe CD45– fraction with anti-PDX mAb (R&D Systems)and using rat anti-mouse IgG2a+b MicroBeads (Miltenyi) as thesecondary antibody. The separation was performed by magneticcell sorting using LS columns (Miltenyi), and the efficiencyof separation was evaluated by cell labeling with goat anti-ratIgG (H+L) PE (Southern Biotech). The recovered fractions wereused for flow cytometric analysis. Flow cytometric analysisof surface molecules was performed as described.17,18 To assessthe expression of cytoplasmic WT-1, after incubation with anti-CD133/2mAb, cells were fixed for 15 min with formaldehyde (2% in PBS),permeabilized with PBS containing 0.5% BSA and 0.5% saponin,and then incubated with the specific mAbs. Each area of positivitywas determined by gating on the same cells stained with isotype-matchedmAbs. A total of 104 events for each sample was acquired. Becausethe combined surface expression of CD133 and CD24 in healthyadult human kidneys is a selective property of renal progenitorscells of Bowman's capsule,17–19 CD133+CD24+PDX+, CD133+CD24+PDX–,and CD133–CD24–PDX+ populations were directly recoveredfrom total renal cell suspensions. To recover CD133+CD24+PDX+,CD133+CD24+PDX–, and CD133–CD24–PDX+ populations,total renal cells were first depleted of CD45 and CD31 and thenlabeled with PE-conjugated anti-PDX mAb, followed by magneticlabeling with anti-PE Multisort microbeads using the anti-PEMultisort Kit (Miltenyi), according to the manufacturer's instructions.To obtain CD133+CD24+PDX+ and CD133–CD24–PDX+ cells,magnetic beads were removed by using Multisort release reagent,and cells were treated with a second magnetic separation forCD133 (CD133 Cell Isolation Kit, containing the anti-CD133/1mAb, clone AC133, also used for hematopoietic stem cell sorting).By contrast, to obtain CD133+CD24+PDX– cells, PDX–cells were directly treated with a second magnetic separationfor CD133. The purified cell fractions consisted of more than98% of CD133+CD24+PDX+, CD133+CD24+PDX–, or CD133–CD24–PDX+cells. To recover CD133–CD24– cells, total renalcells depleted of CD45 and CD31 were also sequentially depletedof CD133 and CD24 by magnetic cell sorting. The purified cellfractions consisted of more than 99% of CD133–CD24–cells.
Cell Cultures and In Vitro Differentiation
Cells were plated in EGM-MV (Lonza Ltd., Basel, Switzerland)with 20% FBS (Hyclone, Logan, Utah) or in VRAD medium24 containingDMEM-F12 (Sigma) supplemented with 10% FBS, vitamin D3 100 nM(Sigma), and all-trans retinoic acid (100 µM; Sigma).Generation of clones was achieved by limiting dilution in 96-wellplates and in four-chamber glass slides (VWR International,West Chester, Pennsylvania). Tubulogenic differentiation wasinduced as described elsewhere with REGM (Lonza Ltd.).17,18For podocyte differentiation, cells were treated for 3–7d with VRAD medium.24
Real-Time Quantitative RT-PCR
Taq-Man RT-PCR was performed as described.37,38 CD133, Bmi-1,WT-1, nephrin, podocin, PDX, Na/Cl transporter, Na/Gluc1 cotransporter,aminopeptidase A, -glutamyltransferase, Na/H exchanger, aquaporin-1,and aquaporin-3 quantification was performed using Assay onDemand kits (Applied Biosystems, Warrington, United Kingdom).
Immunohistochemistry
Double immunohistochemistry for HLA-I and podocin was performedas detailed elsewhere.17,18,39 Briefly, after a 30-min preincubationwith normal horse serum (Vectastain ABC kit), sections werelayered for 30 min with anti-human-podocin pAb, followed bybiotinylated anti-rabbit IgG horse antibody, and the avidin-biotin-peroxidasecomplex (Vectastain ABC kit), and 3-amino-9-ethylcarbazole (AEC)(red color) as peroxidase substrate. Sections were subsequentlyexposed to anti-HLA-I mAb (Sigma), followed by biotinylatedanti-mouse IgG horse antibody and the avidin-biotin-peroxidasecomplex (Vectastain ABC kit). Vector SG (bluish-gray color,Vector Laboratories) was used as a chromogen. Colocalizationof AEC and vector SG in the same cell resulted in a final purple/darkblue color.
Xenograft in SCID Mice Model of Adriamycin Nephropathy
Adriamycin nephropathy was induced in female SCID mice (Harlan,Udine, Italy) at the age of 6 wk by a single intravenous injectionof adriamycin (doxorubicin hydrochloride, 6 mg/kg in PBS, Sigma)on day 0 in the tail vein. Animal experiments were performedin accordance with institutional, regional, and state guidelinesand in adherence to the National Institutes of Health Guidefor the Care and Use of Laboratory Animals. On day 1, and againon days 4, 9, 18, and 25 after adriamycin injection, two groupsof mice received intravenous administration as follows: group1, saline (n = 60 mice); and group 2, PKH26-labeled CD133+CD24+PDX–cells (n = 60 mice; 0.75 x 106 cells/d). Twelve mice were killedat each time point after adriamycin injection (day 7, 14, 21,28, and 45) for each group.
Additional groups of mice were treated with saline (n = 12),CD133–CD24– (n = 6), CD133+CD24+PDX+ (n = 6), CD133+CD24+PDX–(n = 12), or with clonally expanded PKH26-labeled CD133+CD24+PDX–cells (n = 3 mice for each clone) (0.75 x 106 cells/d at day1 and 4 after adriamycin injection). A total number of fivedistinct clones obtained from three different donors was used.Mice were killed at day 7 after adriamycin injection. All ofthe organs of the mice were examined for cells trapping or engraftment.After injection, a limited number of cells were entrapped inthe lung, whereas no cells were observed in the other organs.
As an additional control, PKH26-labeled CD133+CD24+PDX–cells were injected in healthy mice (n = 6 mice; 0.75 x 106cells/d on day 1, and again on days 4, 9, 18, and 25). Micewere killed at day 28.
In all mice, urinary albumin and creatinine in 24-h urine weredetermined with Albuwell M kit (Exocell, Philadelphia, Pennsylvania)and Creatinine Assay kit (Cayman Chemical, Ann Arbor, Michigan).Normal range of urinary albumin or creatinine in our experimentswas calculated in eight additional untreated mice per day. Kidneyswere collected from all animals.
Analysis of Renal Morphology
For analysis of mouse renal morphology, kidney sections of 5-µmthickness of animals killed at day 28 were fixed in ethanoland stained with PAS reagent (Carlo Erba, Milan, Italy). Twentyhigh-power fields (400x) of renal cortex were randomly selectedfor assessing tubular (atrophy, casts, and vacuolization) andinterstitial changes (fibrosis and inflammation) and gradedfrom 0 to 5 (tubulointerstitial area in the cortex was gradedas follows: 0, normal; 1, area of interstitial inflammationand fibrosis, tubular atrophy, and vacuolization involving <10%;2, lesion area between 10 and 20%; 3, lesion area between 20and 30%; 4, lesion area between 30 and 40%; and 5, lesions involving>40% of the field). Fifty randomly selected glomeruli wereassessed for glomerular damage (well developed exudative, mesangialproliferation and glomeruli hypertrophy) and graded as follows:0, normal; 1, slight glomerular damage of the mesangial matrixand/or hyalinosis with focal adhesion involving <10% of theglomerulus; 2, sclerosis of 10 to 20%; 3, sclerosis of 20 to30%; 4, sclerosis of 30 to 40%; and 5, sclerosis >40% ofthe glomerulus. All scoring was performed in a blinded manner.
Statistical Analysis
The results were expressed as mean ± SEM. Comparisonbetween groups was performed by the Mann-Whitney test or byANOVA for multiple comparisons (ANOVA for repeated measures),as appropriate. P < 0.05 was considered to be statisticallysignificant.
The research leading to these results has received funding fromthe European Community under the European Community's SeventhFramework Programme (FP7/2007-2013), grant number 223007, andfrom the European Research Council Starting Grant under theEuropean Community's Seventh Framework Programme (FP7/2007-2013),ERC grant number 205027. This study was supported by the TuscanyMinistry of Health, the Ministero dell’Istruzione, dell’Universita'e della Ricerca (MIUR), the Foundation of Ente Cassa di Risparmiodi Firenze, and the Associazione Italiana per la Ricerca sulCancro. E. Parente is a recipient of a Fondazione Italiana perla Ricerca sul Cancro fellowship. E. Ronconi and C. Sagrinaticontributed equally to this work.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
Supplemental information is available for this article onlineat http://www.jasn.org/.
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Top
Abstract
Introduction
-glutamyltransferase, the Na/H exchanger, aquaporin-1, aquaporin-3, or the thiazide-sensitive Na/Cl transporter (Figure 3D). By contrast, when the same clones of CD133+CD24+PDX– cells were cultured in VRAD, they began to express the podocyte markers nephrin, WT-1, synaptopodin, podocin, PDX, and anti-glomerular epithelial protein 1 (GLEPP-1) at both mRNA and protein levels, as demonstrated by real-time RT-PCR and confocal microscopy, respectively. Differentiation of CD133+CD24+PDX– cells associated with a progressive downregulation of CD133 expression, as already reported.17 Of note, when single cell suspensions obtained from ten clones were recloned by limiting dilution, the resulting second-round subclones displayed the same capacity for multidifferentiation as the original clone, providing additional evidence that CD133+CD24+PDX– cells are indeed multipotent and exhibit self renewal in culture. On the other hand, CD133+CD24+PDX+ cells could not be expanded or cloned in EGM-MV, whereas if plated in VRAD medium they generated clones comprised of a maximum of 800 to 1000 cells that could never be subcloned, suggesting that these cells did not display self-renewal capacities. Confocal microscopy and real-time RT-PCR demonstrated that CD133+CD24+PDX+ clones showed the phenotype of transition cells co-expressing CD133, CD24, WT-1, synaptopodin, GLEPP-1, and nephrin (Figure 3E). Accordingly, clones could never be obtained from CD133+CD24+PDX+ cells in the presence of REGM+HGF medium, suggesting that these cells cannot generate tubular cells. Finally, CD133–CD24–PDX+ were unable to generate clones even in VRAD medium and survived in culture for only a few days, further confirming their nature as terminally differentiated cells. These findings suggest that CD133+CD24+PDX– cells in Bowman's capsule represent an uncommitted population of cells with extensive self-renewal potential that can generate both tubular cells and podocytes. By contrast, CD133+CD24+PDX+ cells do not display the potential to differentiate in tubular cells, suggesting that they represent a progenitor already committed toward the podocyte lineage. 
