Kidney Side Population Reveals Multilineage Potential and Renal Functional Capacity but Also Cellular Heterogeneity

Kidney Side Population Reveals Multilineage Potential and Renal Functional Capacity but also Cellular Heterogeneity

Grant A. Challen,*§ Ivan Bertoncello,† James A. Deane,‡ Sharon D. Ricardo,‡ and Melissa H. Little* *Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, †The Peter MacCallum Cancer Centre and Department of Pathology, University of Melbourne, Melbourne, and the ‡Monash Immunology and Stem Cell Laboratories, Monash University, Melbourne, Australia; §Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, Texas

Side population (SP) cells in the adult kidney are proposed to represent a progenitor population. However, the size, origin, phenotype, and potential of the kidney SP has been controversial. In this study, the SP fraction of embryonic and adult kidneys represented 0.1 to 0.2% of the total viable cell population. The immunophenotype and the expression profile of kidney SP cells was distinct from that of bone marrow SP cells, suggesting that they are a resident nonhematopoietic cell population. Affymetrix expression profiling implicated a role for Notch signaling in kidney SP cells and was used to identify markers of kidney SP. Localization by in situ hybridization confirmed a primarily proximal tubule location, supporting the existence of a tubular “niche,” but also revealed considerable heterogeneity, including the presence of renal macrophages. Adult kidney SP cells demonstrated multilineage differentiation in vitro, whereas microinjection into mouse metanephroi showed that SP cells had a 3.5- to 13-fold greater potential to contribute to developing kidney than non-SP main population cells. However, although reintroduction of SP cells into an Adriamycin-nephropathy model reduced albuminuria:creatinine ratios, this was without significant tubular integration, suggesting a humoral role for SP cells in renal repair. The heterogeneity of the renal SP highlights the need for further fractionation to distinguish the cellular subpopulations that are responsible for the observed multilineage capacity and transdifferentiative and humoral activities. J Am Soc Nephrol 17: 1896–1912, 2006. doi: 10.1681/ASN.2005111228

he existence of resident stem cells in the mature mam- cells from solid tissues also may possess this SP phenotype. SP malian kidney has not been proved conclusively. The cells now have been isolated from a wide variety of mammalian T multipotentiality of the mesenchyme within the embry- tissues, and stem/progenitor cells can be enriched from bone onic kidney (1,2), the identification of “nephroblast” cells from marrow, skeletal muscle, liver, brain, mammary gland, skin, human fetal kidneys (3), and the transdifferentiation of renal testis, and retina using this dye efflux phenomenon (13). cell types during recovery from transient kidney damage (4,5) The presence of SP cells in kidneys was demonstrated pre- all suggest the existence of progenitor populations. More re- viously in mouse (14,15) and rat (16). Asakura et al. (14) isolated cently, several groups have identified bromodeoxyuridine SP cells from a number of solid organs of the mouse, including (BrdU) label–retaining cells within either the proximal tubules the kidney. They concluded that the kidney SP represented (6) or the papilla (7) of the kidney. approximately 5% of all cells and that these largely were ϩ A defining property of murine hematopoietic stem cells CD45 and likely to represent bone marrow–derived HSC. ϩ (HSC) is low fluorescence after staining with Hoechst 33342 Iwatani et al. (16) isolated GFP SP cells from adult rat kidney (8–10). This dye has proved to be remarkably powerful in the (0.03 to 0.1% of total cells) and transplanted these into recipient purification and characterization of HSC when used alone or in animals to examine both their hematopoietic and nonhemato- combination with antibodies that are directed against stem cell poietic potential. These cells showed little renal potential even epitopes. Hoechst “low” cells are described as side population in response to Thy1 glomerulonephritis, although this study (SP) cells and are identified by their ability to efflux the dye also demonstrated that the kidney SP of rats that had under- rapidly via membrane transport pumps (11,12). Recently, ex- ϩ gone a bone marrow transplant from a GFP donor contained citement has been generated by the findings that putative stem ϩ approximately 10% GFP cells, indicating the presence of hematopoietic cells in this renal cell population. In contrast, Hish- ikawa et al. (15) reported the isolation of an apparently much Received December 4, 2005. Accepted April 9, 2006. larger kidney SP (approximately 5.1%) from mice and sug- Published online ahead of print. Publication date available at www.jasn.org. gested that these cells show multilineage potential and renal Address correspondence to: Prof. Melissa H. Little, Institute for Molecular Bio- regenerative capacity in vivo. Hence, the size, origin, and po- science, Queensland Bioscience Precinct, 306 Carmody Road, The University of Queensland, St. Lucia, Brisbane, QLD, 4072, Australia. Phone: ϩ61-7-3346-2054; tential of the kidney SP remain unclear. Fax: ϩ61-7-3346-2101; E-mail: [email protected] In this study, we sought to clarify these issues via phenotypic

Copyright © 2006 by the American Society of Nephrology ISSN: 1046-6673/1707-1896 J Am Soc Nephrol 17: 1896–1912, 2006 Characterization of Kidney Side Population Cells 1897 and functional analysis of embryonic and adult kidney SP. Our Affymetrix Microarrays results indicate that kidney SP is a resident renal cell popula- Five micrograms of fragmented cRNA was hybridized to Test3 ar- tion with some progenitor cell characteristics but that this pop- rays to confirm amplification labeling, and bias was comparable for all ulation is still heterogeneous after FACS. Hence, to truly assess samples. Ten micrograms of fragmented cRNA for each sample was the potential of this population, more specific cell surface mark- hybridized (16 h, 45°C) to Mouse Genome 430 2.0 arrays in duplicate. Arrays were scanned with a GeneChip scanner 3000 (Affymetrix) using ers will be required to further fractionate the kidney SP so as to GeneChip Operating Software v1.1 (Affymetrix). interrogate the exact phenotype of the cells that are responsible for the progenitor activity. Microarray Data Analysis and Bioinformatics Image data were imported into Genespring v6.1 (Silicon Genetics), Materials and Methods normalized per chip and to the median expression levels of each probe Preparation of Cell Suspension across all arrays, and subjected to one-way ANOVA. Membrane orga- Six- to 8-wk-old outbred CD1 mice were killed by cervical disloca- nization classes for representative sequences of Affymetrix probes were tion, and kidneys were harvested into cold HBSS (Sigma, St. Louis, MO) bioinformatically predicted as described previously (17). All primary and 2% FCS (Invitrogen, San Diego, CA). Femurs and tibias were microarray data and supplementary tables are available at http:// flushed with PBS and 2% FCS to collect bone marrow cells. Embryonic kidney.scgap.org/files (username: reviewer, password: 4321). kidneys were dissected from embryonic day 15.5 (E15.5) embryos in cold Leibovitz’s L15 media (Life Technologies). Adult kidneys were Riboprobe Synthesis, In Situ Hybridization, and minced with scissors and digested enzymatically in HBSS, 7.5 mg/ml Immunohistochemistry collagenase B (Roche), 1.2 U/ml Dispase II (Roche), and 0.01% DNAse Constructs that contained the gene of interest were obtained from the type I (Sigma) for 20 min at 37°C. Collagenase concentration was 1 SRC Microarray facility (Institute for Molecular Bioscience) and se- mg/ml for dissociation of embryonic tissue. Tissue was dissociated quenced verified before use. Digoxigenin-labeled riboprobes were syn- further by gentle trituration with a 23-G needle, red blood cells were thesized as described previously (18). For section in situ hybridization lysed, and the suspension was passed through a 40-␮M cell strainer (ISH), kidneys were fixed overnight in 4% paraformaldehyde (PFA)/ (BD Falcon). PBS at 4°C and embedded in paraffin, and sections were cut at 7 ␮m. Hybridizations were performed using the DIG wash and block buffer Hoechst Staining and Antibody Labeling set (Roche). Immunohistochemistry was performed subsequent to ISH Cells were resuspended in DMEM (Life Technologies), 10 mM color development with the following antibodies: Anti–aquaporin-1 HEPES (BDH Laboratory Supplies), 2% FCS, and 5 ␮g/ml Hoechst (anti-AQP1; Chemicon) and anti–calbindin-D28K (Sigma). For poly- 33342 (Sigma), with or without 50 ␮M verapamil (Sigma), at 1 ϫ 106/ml clonal antibodies, sections were blocked using 2% sheep serum in PBS and incubated at 37°C for 90 min with gentle agitation. After staining, for 1 h then incubated for 1 h with primary antibodies diluted in block. cells were washed with cold PBS and 2% FCS and stained with anti- For mAb, the MOM kit (Vector Laboratories, Burlingame, CA) was bodies for 20 min on ice. The following antibodies were used: CD45- used according to the manufacturer’s instructions. Secondary antibody FITC (clone 30-F11), CD31-PE (clone MEC13.3), sca-1-PE (clone E13– and tertiary streptavidin incubations were performed using an ABC kit 161.7), c-kit-FITC (clone 2B8), CD24a-PE (clone M1/69; all (Vector Laboratories), then visualized with the DAB-Plus Substrate kit Pharmingen), CD34 (clone BI-3C5; Zymed), and isotype controls (Zymed Laboratories). Photographs were taken using an Olympus (Pharmingen). 7-Aminoactinomycin D (7-AAD; 2 ␮g/ml; Sigma) was AX70 compound microscope with Kodak Elite Ektachrome 160T color added to each sample before FACS. reversal film (Rochester, NY).

FACS Cell Culture and In Vitro Differentiation Assays Cells were plated out in kidney cell culture medium (DMEM, 10% Cells were analyzed with a FACS Vantage SE (Becton Dickinson) and FCS, 5 ␮g/ml transferrin, 5 ␮g/ml insulin, 5 pM thyronine, 50 nM sorted using a MoFlo (DakoCytomation). FITC, PE, and 7-AAD were prostaglandin, 50 nM selenium, ad 50 nM hydrocortisone) and grown excited with the 488-nm laser, and emission signals were detected using in 5% CO at 37°C. For differentiation assays, cells were cultured in 530/30, 575/25, and 670/40 band-pass filters, respectively. Hoechst 2 kidney cell medium for 1 wk before being changed to adipogenic was excited with the 365-nm laser, and emission was detected using (DMEM/F12, 3% FCS, 33 ␮M biotin, 17 ␮M pantothenate, 100 nM 424/44 (blue) and 660/20 (red) filters. Dead cells were discriminated by insulin, 1 ␮M dexamethasone, 0.25 ␮g/ml amphotericin B, and 0.25 7-AAD staining, compensation was adjusted using samples that were mM IBMX) or osteogenic (DMEM, 10% FCS, 0.2 mM ascorbic acid, 25 stained with one fluorochrome only, and the SP gate was set using mM ␤-glycerophosphate, 500 nM dexamethasone, and 20 mM glu- verapamil control samples. FACS data were analyzed with winMDI 2.8. tamine) differentiation medium. Cells were cultured for an additional 3 wk with medium changed every second day, then fixed for 10 min in RNA Isolation and Target Preparation 4% PFA/PBS. Adipocytes were identified by staining with Nile Red Total RNA was prepared from SP cells using Trizol (Gibco BRL) (Molecular Probes; 1:2000 in PBS) for 15 min. Osteocytes were identi- extraction and RNeasy mini kits (Qiagen), including on-column DNAse fied by alkaline phosphatase assay using 0.1 mg/ml Fast Blue BB salt digestion. RNA concentration and quality were determined using bio- (Sigma), 0.1 mg/ml Naphthol AS-MX phosphate (Sigma), and 2 mM analyzer RNA microfluidic chips (Agilent). Twenty nanograms of total MgCl2 in 0.1 M Tris-HCl (pH 8.5). Images were captured with a Nikon RNA was amplified linearly using the message AMP aRNA kit (Am- sight DS-L1 digital camera connected to a Nikon TS 100 ECLIPSE bion). The ENZO BioArray High Yield RNA transcript Labeling Kit inverted microscope. 10T1/2 cells were used as a positive control in (ENZO Life Sciences) was used in the second round of amplification to differentiation assays. Purified macrophages were cultured in RPMI produce biotinylated cRNA. Labeled cRNA was fragmented before (Life Technologies), 10% FCS, and 12.5 ng/ml colony-stimulating factor hybridization. 1 (Chiron). 1898 Journal of the American Society of Nephrology J Am Soc Nephrol 17: 1896–1912, 2006

Microinjection of Cells into Metanephroi Kidney SP and main population (MP) cells were labeled with 1 ␮M CM-DiI (Molecular Probes) for 5 min at 37°C and an additional 15 min on ice. Kidneys were dissected from E12.5 embryos, and labeled cells were microinjected into isolated metanephroi through drawn glass pipettes using a Femtojet microinjector (Eppendorf). Approximately 100 cells were injected into each kidney. Kidneys that received an injection then were cultured as explants for3dat5%CO2 at 37°C on 3.0-␮m polycarbonate transwell filters (Costar) with MEM, 10% FCS, and 20 mM glutamine; fixed in 4% PFA/PBS for 30 min; and processed for paraffin embedding. Sections were cut at 4 mm and analyzed by immunofluorescence (IF) with anti–calbindin-D28K (Sigma), anti-WT1 (Santa Cruz), and anti-Pax2 (Zymed) primary antibodies and Alexa Fluor-488 secondary antibodies (Molecular Probes). Digital images were captured using a Zeiss LSM-510 META confocal microscope and analyzed with Adobe Photoshop 7.0.

Adriamycin Model of Kidney Damage Figure 1. Hoechst staining profiles of bone marrow (A), embry- Recipient BALB/c mice were administered 10 mg/kg body wt Adria- onic day 15.5 (E15.5) kidney (B), and adult kidney (C) cell mycin (doxorubicin; Sigma) via a single tail-vein injection. Just before suspensions. The side population (SP) is enclosed by the re- injection, recipient mice received donor SP and MP cells that were gions indicated. The SP fraction was ablated in the presence of Ͻ isolated from Ccrslc (BALB/c-GFP) mice. Approximately 2 ϫ 105 cells the verapamil being reduced to 0.01% for all tissues (D were injected directly into the left kidney, and 1 ϫ 105 were injected through F). into circulation via the renal vein. Mice were killed 3, 7, and 14 d after Adriamycin or vehicle injection; 24-h urine samples were collected in metabolic cages before the mice were killed. Urine albumin content was determined with a Bio-Rad Protein Assay with BSA standards (Bio- Immunophenotypic analyses showed that although SP cells Rad), and creatinine was determined with a Creatinine Assay kit (Cay- from embryonic and adult kidneys displayed similar cell sur- man Chemical). A one-way ANOVA was used for statistical analysis of face profiles, both were distinct from bone marrow (Figure 2A). urine data. Recipient mice were perfusion-fixed with 4% PFA/PBS, and Although the stem cell marker Sca-1 and renal progenitor cell the kidney that received an injection was processed and embedded in marker CD24a antigen (CD24a) were expressed at high levels in optimal cutting temperature medium (Tissue-Tek). Cryosections were all three populations, the kidney SP contained very low levels cut at 10 ␮m and analyzed by IF with anti-AQP1 (Chemicon), anti- of cells that expressed the markers CD45, CD34, and c-kit, AQP2 (Chemicon), anti–calbindin-D28K (Sigma), anti-CD31 (Pharmin- which were very predominant in the bone marrow SP (Figure gen), and anti-desmin (Sigma) primary antibodies and Alexa Fluor-594 2B). Sca-1/c-kit double labeling, which identifies primitive secondary antibodies (Molecular Probes). ϩ HSC, showed that 88% of bone marrow SP cells were Sca-1 c- ϩ kit , consistent with previous findings (9). Kidney SP cells ϩ ϩ Results rarely fell into the Sca-1 c-kit fraction. Sca-1 and CD24a were Abundance and Phenotype of SP Cells in Embryonic and expressed strongly by all three populations, and double label- Adult Mouse Kidneys ing for these markers showed that 82, 83, and 88% of bone Cell suspensions from embryonic and adult mouse kidneys marrow, E15.5 kidney, and adult kidney SP cells, respectively, that were stained with Hoechst 33342 displayed similar SP ϩ ϩ fell into the Sca-1 CD24a fraction (Figure 2C). All antibodies profiles to bone marrow (Figure 1). The bone marrow SP frac- were assayed individually and are representative of at least tion represented on average 0.10% of the total viable cell pop- three separate experiments, and histograms display a mini- ulation (0.08 to 0.13%), whereas that of E15.5 and adult kidneys mum of 1000 SP cells. were 0.10% (0.06 to 0.12%) and 0.14% (0.08 to 0.23%), respectively. This was much lower than the approximately 5% kidney SP fraction reported in previous mouse studies (14,15) but in SP Gene Expression Profile Analysis agreement with the frequency reported in rat (16). The ability to We sought to examine comprehensively the expression pro- detect SP cells depends on their capacity to efflux Hoechst dye, files of mouse bone marrow and E15.5 and adult kidney SP a process that is inhibited by calcium channel blockers such as using Affymetrix MOE430_2.0 arrays, which represent approx- verapamil. When 50 ␮M verapamil was included in the imately 39,000 transcripts. Two rounds of in vitro transcription Hoechst preparations, the SP fraction was reduced to Ͻ0.01% in were required to obtain sufficient cRNA for hybridization. all three tissues (Figure 1), indicating that the cells being de- Test3 arrays confirmed that amplification bias and labeling tected indeed were true SP cells identified by Hoechst efflux. were comparable among all samples. Image data were normal- Because of the variability in the SP purification strategy, all ized per chip and to the median expression levels of each probe FACS data presented in this study are representative of at least across all arrays to account for variables such as cRNA frag- three individual experiments to account for biologic and sam- mentation efficiency, hybridization conditions, and back- ple preparation variation; 50 to 80% of all FACS events repre- ground signal. Replicate samples were subject to one-way sented a viable cell. ANOVA (P Ͻ 0.02) to display only the most statistically reliable J Am Soc Nephrol 17: 1896–1912, 2006 Characterization of Kidney Side Population Cells 1899

Figure 2. (A) Immunophenotypic FACS analysis of SP. Hollow peaks indicate isotype controls and solid fills are marker expression. Kidney SP samples contained very low levels of cell-expressing hematopoietic markers such as CD45, CD34, CD31, and c-kit. (B) Summary of cell surface profiles of bone marrow, embryonic kidney, and adult kidney SP cells. (C) FACS double plots analyzing ϩ ϩ ϩ ϩ coexpression of markers on SP cells. Bone marrow SP cells were highly homogeneous in terms of CD45 CD24a and c-kit Sca-1 Ϫ ϩ Ϫ ϩ expression. Embryonic and adult kidney cells were predominantly CD45 CD24a and c-kit Sca-1 . All three SP samples were ϩ ϩ predominantly Sca-1 CD24a .

genes. A more than two-fold change threshold was used to all genes that were differentially upregulated in E15.5 kidney define differential gene expression between samples. SP and adult kidney SP, respectively (Figure 3A). There were The bone marrow SP gene expression profile that was gen- relatively few differences in direct comparison of embryonic erated in this study correlates extremely well with another (206 nonredundant genes; Supplementary Table 3) and adult recent microarray analysis of Hoechst-effluxing bone marrow kidney SP (325 nonredundant genes; Supplementary Table 4), cells that was performed on a similar Affymetrix platform (19), indicating that this phenotype is relatively conserved through- providing confidence that our method was sound. A total of out renal development. 1122 nonredundant genes showed differentially increased ex- The list of genes that show differentially increased expression pression in the bone marrow SP cell profile compared with that in embryonic and adult kidney SP reveals many genes that may of embryonic and adult kidney (Supplementary Table 1), in- be indicative of renal stem/progenitor cell phenotype, includ- cluding many HSC/hematopoietic genes such as CD34, CD45, ing endoglin/CD105, which is expressed on both hematopoi- CD47, CD48, CD53, c-kit, lymphocyte cytosolic protein 2 etic (20) and mesenchymal stem cells (21), the mesenchymal (Lcp2), lymphocyte antigen 64 (Ly64), and lymphocyte antigen stem cell maker CD44 (21), and Pax8, a critical transcription 86 (Ly86). To identify markers of kidney SP cells, genes that factor that is required for the specification of renal progenitor showed differentially increased expression in both E15.5 and cells (22). The proliferation marker Ki67 showed a 43- and adult kidney SP samples compared with total adult kidney 11-fold increase in expression in E15.5 and adult kidney SP, were identified (734 nonredundant genes; Supplementary Ta- respectively, compared with total kidney, which suggests that ble 2). This gene list encompassed approximately 78 and 70% of these cells are cycling more rapidly than the majority of the 1900 Journal of the American Society of Nephrology J Am Soc Nephrol 17: 1896–1912, 2006

Figure 3. (A) Microarray data analysis strategy for identifying genes enriched in kidney SP cells. Of the genes expressed, more than two-fold in E15.5 and adult kidney SP samples over total kidney, 734 nonredundant overlapping genes were identified to define kidney SP cells. (B) Affymetrix microarray gene expression profiles. CD45, CD34, and c-kit provide support between gene and protein expression data by displaying stronger expression in bone marrow SP compared with kidney SP as seen in immunophenotyping. (C) Localization of cell surface markers enriched in kidney SP cells. RNA in situ hybridization (ISH) of adult kidneys showed expression in proximal tubules for endoglin (Eng), solute carrier family 2, member 1 (Slc2a1), PDZK1 interacting protein 1 (Map17), lymphocyte antigen 6 complex, locus E (Sca-2/Ly6e; also in distal tubules), lymphocyte antigen 6 complex, locus A (Sca-1/Ly6a; also in cortical collecting ducts), hypothetical protein LOC231503 (LOC231503), and RIKEN cDNA 0610009B10 gene (0610009B10Rik). CD24a antigen (CD24a) was expressed in thick ascending loops of Henle, distal tubules, and collecting ducts. Expression of ATP-binding cassette, subfamily G (WHITE), member 2 (ABCG2), one of the major membrane pumps responsible for the SP phenotype in other organs, was observed in collecting ducts but not proximal tubules. The proliferation marker Ki67 was expressed in distinct cells of certain tubules. ϩ Immunohistochemistry after ISH confirmed the localization of Ki67 cells in proximal tubules by aquaporin-1 (AQP1) staining but never in distal tubules or collecting ducts as identified by calbindin-D28K (Calb1) staining. Bar ϭ 50 ␮m. kidney. CD24a and Sca-1 (Ly6e), which were strongly ex- expression data. Moreover, markers that were strongly expressed in kidney SP samples by immunophenotyping, also pressed by bone marrow SP cells but detected only at low levels were in this list, providing good evidence for correlation be- in kidney SP, such as CD45 and c-kit, also showed similar tween gene (microarray) and protein (immunophenotyping) expression profiles at the transcriptional level by Affymetrix J Am Soc Nephrol 17: 1896–1912, 2006 Characterization of Kidney Side Population Cells 1901 profiling (Figure 3B). This provided reassurance that the cell Notch Signaling Pathway surface profile of the samples remained intact during tissue The Notch signaling pathway is an evolutionarily conserved processing for FACS analysis. pathway that is critical for tissue morphogenesis during devel- The ability to purify potential stem/progenitor cells relies opment but is also involved in tissue maintenance and repair in largely on the expression of appropriate cell surface anti- the adult. This pathway has been shown to be critical for gens. Stem cell biology in general but particularly in solid normal self-renewal and cell fate commitment of stem cells tissues suffers from the lack of specific cell surface markers from the blood (23,24), brain (25), muscle (26), mammary gland (27), and epidermis (28,29). A survey of the list of genes that are that unambiguously label all stem cells and/or only stem strongly expressed in kidney SP highlights many members of cells. Bioinformatics was applied to the gene lists of interest the Notch signaling pathway (Figure 4A), such as the receptors to identify genes that encode transmembrane proteins, as Notch1, Notch2, and Notch3; the ligands Jagged1 and Jagged2; described previously (17). The subset of cell surface markers the secreted protein radical fringe gene homologue (Rfng); the that were enriched in kidney SP cells compared with total intracellular signaling molecules Deltex2 (Dtx2) and Deltex3 kidney is shown in Table 1. These molecules represent can- (Dtx3); and the transcriptional regulators transducin-like en- didate cell surface markers of renal SP cells for purification hancer of split (Tle1) and 1700023B02Rik (CBF1 interacting by antibody-based FACS in the absence of or in combination corepressor) as well as noted Notch target genes Gsk3b (30), with Hoechst staining. cyclin D1 (31), and cyclin E1 (32). ISH was performed to determine the localization of these Notch pathway members in the adult kidney (Figure 4B). Most genes showed overlapping ex- Markers of Kidney SP Are Expressed in Proximal Tubules pression in proximal tubules, including Notch1, Notch2, The spatial location (niche) of kidney SP cells was examined Notch3, Jagged2 (also seen in cortical collecting ducts), Rfng, via RNA in situ hybridization in adult kidney sections for cell Dtx2, and Dtx3. Jagged1 was only weakly expressed in the surface markers that were enriched in the kidney SP compared adult kidney and was observed in glomeruli and vascular with total kidney (Table 1). Analysis showed that there was bundles. Expression of 1700023B02Rik was fairly nonspecific expression throughout several tubular components (Figure 3C). but was enriched in distal tubules and cortical collecting ducts. However, most genes were expressed in proximal tubules, in particular the segments at the corticomedullary junction. One Does the Kidney SP Include Resident Renal Macrophages? exception to this was CD24a, which was expressed in thick The list of genes that were strongly expressed in kidney SP ascending loops of Henle, distal tubule, and collecting ducts. cells also seemed to be enriched for many genes that typically Other genes showed an interstitial location that will be dis- are associated with monocytes/macrophages. Macrophages cussed further. ABCG2, one of the major membrane transport- can be identified in embryonic mouse kidneys as early as E12.5 ers responsible for conveying the dye-efflux phenotype in SP (Figure 5, A and B), and 52 of the 734 genes (Table 2) that were cells from other organs, was expressed predominantly in col- expressed more strongly in kidney SP compared with total lecting ducts of the kidney. This gene did not show a significant kidney also were highly expressed by resident renal macro- increase in expression in kidney SP cells compared with total phages (our unpublished data). To investigate this, we pre- kidney, which may suggest that kidney SP cells efflux Hoechst pared cell suspensions from the kidneys of c-fms-GFP trans- using other membrane transporter pumps. genic mice (33) and stained them with Hoechst for SP analysis Although the profiling did not assume that such markers (Figure 5D). The c-fms gene encodes the receptor for macrophage colony-stimulating factor (CSF1). The proportion of c- would be expressed exclusively by the SP compartment of the ϩ kidney, the lack of a restricted expression pattern that may be fms cells in the kidney SP fraction (9.3%) was approximately double the incidence of their normal abundance in the whole indicative of a stem cell population suggests heterogeneity in ϩ organ (4.1%). However, although the c-fms cells were over- the kidney SP. Although these cell surface molecules where represented in the kidney SP fraction, this cell type still was not expressed by proximal tubule cells in general, the approxi- the major constituent of this population. ISH for some of the mately 0.1% detection frequency of kidney SP cells by FACS molecules that were coexpressed by renal macrophages and dictates that not every proximal tubule cell will be an SP cell. kidney SP cells (Figure 5, E through J) showed that although ISH of adult kidneys for the proliferation marker Ki67, which some of these genes were expressed exclusively by macro- was strongly enriched in E15.5 and adult kidney SP cells, again phages (c-fms, Gp49b, Bmp2, and Tde2), others also were more showed expression mainly in proximal tubules but with only widely distributed in various renal tubular components (Lgals3 one or two cells of a particular tubule staining positive. Immu- and Csf2rb1). This again suggests that although kidney SP are nohistochemistry for markers of different kidney segments was low in abundance, this fraction still contains a heterogeneous ϩ performed after ISH to clarify that the Ki67 cells indeed were mix of renal cell types. ϩ in proximal tubules. The vast majority of Ki67 cells were Further FACS analysis was performed to verify that the ϩ observed in proximal tubules that were stained with AQP1, c-fms cells that were being found in kidney SP samples were ϩ with a minor subpopulation being seen in certain interstitial of the monocytic lineage (Figure 5, K through O). Most CD45 ϩ ϩ cells. Ki67 cells were never seen in distal tubules of collecting cells that were present in kidney MP also were Mac-1 (ap- ducts that were identified by calbindin-D28K staining. proximately 70%), confirming that the majority of hematopoi- 1902 Journal of the American Society of Nephrology J Am Soc Nephrol 17: 1896–1912, 2006

Table 1. Cell surface markers that were strongly expressed in E15.5 and adult kidney compared with total kidneya

Affymetrix Probe E15.5 SP 1b Adult SP 1c Gene Symbol Gene Name UniGene

1436970_a_at 7.3 39.3 Pdgfrb Platelet derived growth factor receptor, ␤ Mm0.4146 1420125_at 9.5 30.7 Tcta T cell leukemia translocation altered gene Mm0.24670 1449396_at 59.3 30.2 Aoc3 Amine oxidase, copper containing 3 Mm0.67281 1434502_x_at 7.0 26.4 Slc4a1 Solute carrier family 4, member 1 Mm0.7248 1421965_s_at 3.4 23.1 Notch3 Notch gene homolog 3 (Drosophila) Mm0.4945 1420330_at 35.4 20.9 Clecsf9 C-type lectin, superfamily member 9 Mm0.248327 1420394_s_at 5.8 20.2 Gp49b Glycoprotein 49 B Mm0.34408 1458996_at 12.9 19.9 Itga5 Integrin ␣5 (fibronectin receptor ␣) Mm0.16234 1418350_at 23.5 18.7 Dtr Diphtheria toxin receptor Mm0.289681 1416295_a_at 21.7 15.5 Il2rg Interleukin 2 receptor, ␥ chain Mm0.2923 1460253_at 6.5 14.8 Cklfsf7 Chemokine-like factor super family 7 Mm0.35600 1425335_at 5.5 13.2 Cd8a CD8 antigen, ␣ chain Mm0.1858 1424067_at 19.1 12.7 Icam1 Intercellular adhesion molecule Mm0.90364 1454933_at 8.8 11.8 2610027C15Rik RIKEN cDNA 2610027C15 gene Mm0.41569 1455556_at 7.6 11.0 Notch2 Notch gene homolog 2 (Drosophila) Mm0.254017 1424568_at 8.0 10.5 Tspan2 Tetraspan 2 Mm0.27469 1432176_a_at 5.2 10.3 Eng Endoglin Mm0.225297 1416379_at 17.7 9.4 Panx1 Pannexin 1 Mm0.142253 1436212_at 2.8 9.4 Tmem71 Transmembrane protein 71 Mm0.132299 1434773_a_at 10.1 9.3 Slc2a1 Solute carrier family 2, member 1 Mm0.21002 1425268_a_at 4.9 9.3 3110045G13Rik RIKEN cDNA 3110045G13 gene Mm0.158962 1455477_s_at 4.6 9.2 Map17 PDZK1 interacting protein 1 Mm0.30181 1437279_x_at 5.0 8.8 Sdc1 Syndecan 1 Mm0.2580 1420682_at 13.3 8.7 Chrnb1 Cholinergic receptor, ␤ polypeptide 1 Mm0.86425 1456085_x_at 8.7 8.6 Cd151 CD151 antigen Mm0.30246 1428074_at 6.9 8.6 2310037P21Rik RIKEN cDNA 2310037P21 gene Mm0.8569 1446466_at 4.4 8.4 Mrc2 Mannose receptor, C type 2 Mm0.235616 1455660_at 8.2 8.2 Csf2rb1 Colony-stimulating factor 2 receptor, ␤1 Mm0.235324 1436297_a_at 4.4 8.1 Grina Glutamate receptor Mm0.41665 1443037_at 5.3 8.0 Sdfr1 Stromal cell–derived factor receptor 1 Mm0.15125 1448906_at 5.1 7.5 Cdh16 Cadherin 16 Mm0.19423 1436644_x_at 8.0 6.7 Tmem25 Transmembrane protein 25 Mm0.41409 1448793_a_at 4.8 6.1 Sdc4 Syndecan 4 Mm0.3815 1439773_at 3.1 5.9 Ly6e (Sca-2) Lymphocyte antigen 6 complex, locus E Mm0.788 1435064_a_at 3.0 5.8 Tmem27 Transmembrane protein 27 Mm0.143766 1417185_at 7.2 5.7 Ly6a (Sca-1) Lymphocyte antigen 6 complex, locus A Mm0.263124 1436656_at 3.4 5.5 LOC231503 Hypothetical protein LOC231503 Mm0.99790 1453031_at 3.2 5.1 0610009B10Rik RIKEN cDNA 0610009B10 gene Mm0.28766 1418633_at 3.8 5.0 Notch1 Notch gene homolog 1 (Drosophila) Mm0.290610 1426431_at 3.4 4.8 Jag2 Jagged 2 Mm0.186146 1426206_at 2.4 4.8 Robo4 Roundabout homolog 4 (Drosophila) Mm0.293315 1417999_at 2.3 4.7 Itm2b Integral membrane protein 2B Mm0.4266 1424208_at 4.1 4.7 Ptger4 Prostaglandin E receptor 4 (subtype EP4) Mm0.18509 1437502_x_at 4.8 4.1 Cd24a CD24a antigen Mm0.29742 a_at 2.7 4.1 Atp1b1 ATPase, Na؉/K؉ transporting, ␤1 Mm0.4550_1451152 1418394_a_at 3.6 4.0 Cd97 CD97 antigen Mm0.334648 1423341_at 3.5 3.6 Cspg4 Chondroitin sulfate proteoglycan 4 Mm0.41329 1448931_at 4.9 3.5 F2rl1 Coagulation factor II (thrombin) receptor-like 1 Mm0.1614 1418674_at 3.6 3.5 Osmr Oncostatin M receptor Mm0.10760 1427164_at 4.7 3.4 Il13ra1 Interleukin 13 receptor, ␣1 Mm0.24208 1421408_at 19.6 3.4 Igsf6 Immunoglobulin superfamily, member 6 Mm0.160384 1456601_x_at 2.7 3.3 Fxyd2 FXYD domain-containing regulator 2 Mm0.22742 1416236_a_at 5.0 3.0 Eva Epithelial V-like antigen Mm0.33240 1447878_s_at 5.0 2.9 Fgfrl1 Fibroblast growth factor receptor-like 1 Mm0.35691 1419872_at 3.3 2.8 Csf1r Colony-stimulating factor 1 receptor Mm0.22574 1424456_at 3.0 2.7 Pvrl2 Poliovirus receptor-related 2 Mm0.4341 1442821_at 5.6 2.7 Smbp SM-11044 binding protein Mm0.246440 1423506_a_at 3.3 2.7 Nnat Neuronatin Mm0.233903 1449130_at 10.8 2.6 Cd1d1 CD1d1 antigen Mm0.1894 1423760_at 6.0 2.4 Cd44 CD44 antigen Mm0.330428 1438928_x_at 2.2 2.4 Ninj1 Ninjurin 1 Mm0.18503 1437513_a_at 2.7 2.3 Tde2 Tumor differentially expressed 2 Mm0.29344 1423513_at 3.1 2.2 Neo1 Neogenin Mm0.42249

aGenes in boldface were detected by more than one probe set on the Affymetrix microarray. E15.5, embryonic day 15.5; SP, side population. bFold change increase in expression of gene in E15.5 kidney SP compared with total kidney. cFold change increase in expression of gene in adult kidney SP compared with total kidney. J Am Soc Nephrol 17: 1896–1912, 2006 Characterization of Kidney Side Population Cells 1903

Figure 4. (A) Schematic representation of the Notch signaling pathway. Genes in blue were strongly expressed in kidney SP cells by microarray. (B) ISH of adult kidneys for members of the Notch signaling pathway upregulated in kidney SP cells. Most members of this pathway showed expression in proximal tubules, including Notch1, Notch2, Notch3, Jagged2 (also cortical collecting ducts), radical fringe gene homologue (Rfng), Deltex2 (Dtx2), and Deltex3 (Dtx3). Jagged1 was only weakly expressed in adult kidneys but was observed in glomeruli and vascular bundles. Expression of RIKEN cDNA 1700023B02 gene (1700023B02Rik) was fairly nonspecific, but upregulation was observed in distal tubules and cortical collecting ducts. Bar ϭ 50 ␮m.

etic cells that were present in this kidney fraction were macro- mately 6% of total SP cells and approximately 3% of total MP ϩ Ϫ Ϫ ϩ phages, although there were some CD45 Mac-1 cells present cells) but also contained a population of CD45 Mac-1 cells, that were not of the monocyte/macrophage lineage (approxi- suggesting that different monocyte/macrophage populations mately 30%). The kidney SP fraction contained a higher fre- were present. This also accounts for the apparent discrepancy ϩ ϩ ϩ quency of CD45 Mac-1 cells compared with MP (approxi- between CD45 cells (4 to 6%) and macrophages (9 to 10%) in 1904 Journal of the American Society of Nephrology J Am Soc Nephrol 17: 1896–1912, 2006

the kidney SP because it seems that not all the macrophages in this cell population expressed CD45. There were no ϩ Ϫ CD45 Mac-1 cells in the kidney SP, suggesting that no other ϩ bone marrow–derived cells were present. All c-fms cells in ϩ kidney SP also were Mac-1 , but only approximately 80% of ϩ ϩ these c-fms cells were F4/80 . This again suggests that multiple macrophage/monocyte populations were present or that ϩ some of these c-fms cells were premonocytic or nonmacroph- ϩ age. Culture of purified kidney SP c-fms cells confirmed that these cells indeed were macrophages as determined by morphology and marker staining (Figure 5P).

Kidney SP Cells Display Multipotentiality In Vitro Primary culture of SP and MP cells that were isolated from adult mouse kidneys showed that both cell populations clearly were heterogeneous but revealed some contrasts in growth morphology. After 3 wk of culture, MP cell cultures consisted mostly of large, flattened epithelial sheets of cells (Figure 6A). Although some cells with epithelial-like morphology also were seen in SP cell cultures, many gave rise to free-floating balls or clusters of small, round cells (Figure 6, B and C). To determine whether SP cells that were isolated from adult mouse kidneys possessed extrarenal differentiation potential in vitro, we cultured SP and MP cells for 1 wk in kidney primary cell culture medium before exposing them to either osteogenic or adipogenic medium for an additional 3 wk. After 3 wk, cultures were assayed for alkaline phosphatase activity to identify osteocytes, and positive cells were seen clearly in SP cultures (Figure 6E, inset). Cultured SP cells that stained blue often were seen to have adopted morphology similar to that of 10T1/2 cell–derived osteocytes (Figure 6F). After 3 wk of induction, SP cell cultures also showed conversion to adipocytes as identified by Nile Red staining to identify lipid droplets (Figure 6, H and K), similar to those seen in 10T1/2-derived adipocytes (Figure 6, I and L). No osteocytes (Figure 6O) or adipocytes (Figure 6, G and J) were seen from MP cell differentiation.

Figure 5. Interrogation of kidney SP macrophages. Macro- Incorporation of SP Cells into Developing Kidneys phages can be detected in the developing kidney as early as The metanephric organ culture system was exploited re- E12.5. (A and B) Brightfield (A) and epifluorescent (B) views of cently to examine the renal potential of mouse embryonic stem ϭ ␮ E12.5 metanephros from c-fms-GFP mouse. Bar 300 m. (C) cells (34). We used a similar method to determine whether ISH of E15.5 kidney section for c-fms shows localization of macrophages in the developing kidney. Bar ϭ 50 ␮m. (D) Analysis of kidney SP from c-fms-GFP mice. Macrophages represented approximately 4% of the total kidney but up to sion of macrophage genes (E through H). Further FACS anal- approximately 10% of all cells in the kidney SP. (E through J) ysis demonstrated the presence of phenotypically distinct ISH of adult kidneys for overlapping markers of resident renal monocyte/macrophage populations in the kidney SP. (K) macrophages and kidney SP. Bar ϭ 50 ␮m. Whereas expression Hoechst profile of c-fms-GFP kidneys shows selection of SP and ϩ of some genes, such as colony stimulating factor 1 receptor main population (MP) regions. Most of the CD45 cells in the ϩ (CSF1r/c-fms), glycoprotein 49B (Gp49b), bone morphogenetic MP (L) were Mac-1 . This population was doubled in preva- protein 2 (Bmp2), and tumor differentially expressed 2 (Tde2), lence in the kidney SP (M), and the SP fraction also contained a Ϫ ϩ was restricted to macrophages, others, such as lectin, galactose CD45 Mac-1 population, suggesting the presence of more ϩ binding, soluble 3 (Lgals3; also in collecting ducts), and colony- than one macrophage subtype. Further analysis of the c-fms ϩ ϩ stimulating factor 2 receptor, ␤1 (Csf2rb1; also in proximal cells from the kidney SP showed that all were c-fms Mac-1 ϩ tubules and cortical collecting ducts), were expressed in other (N), but only approximately 80% of c-fms cells in this fraction ϩ ϩ compartments of the kidney as well as macrophages. Arrows were F4/80 (O). (P) Collection and in vitro culture of c-fms indicate positions of macrophages. AQP1 immunohistochemis- cells from kidney SP demonstrated that these cells were mac- try was performed after ISH to highlight the interstitial expres- rophages by morphology and F4/80 staining. Bar ϭ 20 ␮m. J Am Soc Nephrol 17: 1896–1912, 2006 Characterization of Kidney Side Population Cells 1905

Table 2. Genes that were strongly expressed in E15.5 and adult kidney SP compared with total kidney and also were highly expressed in resident renal macrophages

Affymetrix E15.5 Adult Gene Probe SP 1b SP 1c Symbol Gene Name UniGene 1449773_s_at 76.6 189.0 Gadd45b Growth arrest DNA-damage-inducible 45 ␤ Mm0.1360 1424638_at 297.7 87.9 Cdkn1a Cyclin-dependent kinase inhibitor 1A (P21) Mm0.195663 1416039_x_at 19.3 38.9 Cyr61 Cysteine-rich protein 61 Mm0.1231 1447830_s_at 6.1 37.8 Rgs2 Regulator of G-protein signaling 2 Mm0.28262 1455269_a_at 7.3 26.7 Coro1a Coronin, actin binding protein 1A Mm0.290482 1426808_at 42.1 26.4 Lgals3 Lectin, galactose binding, soluble 3 Mm0.248615 1419691_at 16.1 24.0 Camp Cathelicidin antimicrobial peptide Mm0.3834 1417395_at 16.9 24.0 Klf4 Kruppel-like factor 4 (gut) Mm0.4325 1427126_at 5.4 22.2 Hspa1a Heat-shock protein 1A Mm0.6388 1448830_at 5.9 21.1 Dusp1 Dual specificity phosphatase 1 Mm0.239041 1420330_at 35.4 20.9 Clecsf9 C-type lectin, superfamily member 9 Mm0.248327 1420394_s_at 5.8 20.2 Gp49b Glycoprotein 49 B Mm0.34408 1448881_at 3.2 16.1 Hp Haptoglobin Mm0.26730 1437811_x_at 19.7 15.7 Cotl1 Coactosin-like 1 (dictyostelium) Mm0.141741 1419209_at 13.5 15.3 Cxcl1 Chemokine (C-X-C motif) ligand 1 Mm0.21013 1444987_at 3.3 15.2 Ctsb Cathepsin B Mm0.236553 1424067_at 19.1 12.7 Icam1 Intercellular adhesion molecule Mm0.90364 1429012_at 4.5 12.7 Arhgef6 Rac/Cdc42 nucleotide exchange factor 6 Mm0.261443 1423622_a_at 13.9 12.0 Ccnl1 Cyclin L1 Mm0.175612 1438143_s_at 12.2 11.1 Sca2 Spinocerebellar ataxia 2 homolog (human) Mm0.260900 1437527_x_at 12.2 10.1 Mcl1 Myeloid cell leukemia sequence 1 Mm0.1639 1429433_at 24.2 9.5 Bat2d BAT2 domain containing 1 Mm0.245446 1456094_at 8.3 9.2 2700002L06Rik RIKEN cDNA 2700002L06 gene Mm0.232293 1435652_a_at 8.5 8.9 Gnai2 Guanine nucleotide binding protein, a inhibiting 2 Mm0.196464 1443037_at 5.3 8.0 Sdfr1 Stromal cell derived factor receptor 1 Mm0.15125 1416326_at 5.5 7.7 Crip1 Cysteine-rich protein 1 (intestinal) Mm0.272368 1452117_a_at 2.1 7.5 Fyb FYN binding protein Mm0.170905 1418641_at 2.9 7.3 Lcp2 Lymphocyte cytosolic protein 2 Mm0.265350 1423635_at 11.9 7.2 Bmp2 Bone morphogenetic protein 2 Mm0.103205 1439426_x_at 12.3 6.0 Lyzs Lysozyme Mm0.45436 1450857_a_at 4.8 5.9 Col1a2 Procollagen, type I, ␣2 Mm0.277792 1448361_at 12.3 5.9 Ttc3 Tetratricopeptide repeat domain 3 Mm0.213408 1436202_at 2.9 5.5 2210401K01Rik RIKEN cDNA 2210401K01 gene Mm0.298256 1439407_x_at 5.2 4.8 Tagln2 Transgelin 2 Mm0.271711 1442014_at 8.1 4.5 Ifrd1 Interferon-related developmental regulator 1 Mm0.168 1428520_at 2.2 4.1 1110032A13Rik RIKEN cDNA 1110032A13 gene Mm0.186936 1436980_x_at 3.1 3.9 Cnot2 CCR4-NOT transcription complex, subunit 2 Mm0.351553 1458690_at 5.0 3.5 Axot Axotrophin Mm0.260635 1421408_at 19.6 3.4 Igsf6 Immunoglobulin superfamily, member 6 Mm0.160384 1419194_s_at 3.7 3.2 Gmfg Glia maturation factor, ␥ Mm0.194536 1438169_a_at 3.6 3.1 Frmd4b FERM domain containing 4B Mm0.27789 1430295_at 2.7 3.0 Gna13 Guanine nucleotide binding protein, ␣13 Mm0.193925 1419872_at 3.3 2.8 Csf1r Colony-stimulating factor 1 receptor Mm0.22574 1421802_at 17.4 2.8 Ear1 Eosinophil-associated ribonuclease 1 Mm0.86948 1449222_at 3.0 2.5 Ebi3 Epstein-Barr virus–induced gene 3 Mm0.256798 1450350_a_at 2.0 2.5 Jundm2 Jun dimerization protein 2 Mm0.103560 1438928_x_at 2.2 2.4 Ninj1 Ninjurin 1 Mm0.18503 1437513_a_at 2.7 2.3 Tde2 Tumor differentially expressed 2 Mm0.29344 1436736_x_at 8.7 2.3 D0H4S114 DNA segment, human D4S114 Mm0.128733 1421525_a_at 2.0 2.3 Birc1e Baculoviral IAP repeat-containing 1e Mm0.290476 1416256_a_at 5.5 2.1 Tubb5 Tubulin, ␤5 Mm0.273538 1437708_x_at 6.3 2.0 Vamp3 Vesicle-associated membrane protein 3 Mm0.273930

aGenes in boldface were detected by more than one probe set on the Affymetrix microarray. bFold change increase in expression of gene in E15.5 kidney SP compared with total kidney. cFold change increase in expression of gene in adult kidney SP compared with total kidney. kidney SP cells had greater renal differentiation potential than still were viable after FACS. Cells were labeled with the li- MP cells in the early metanephric environment. Equal numbers pophilic tracker CM-DiI and microinjected into metanephroi of SP and MP cells were isolated from adult kidneys by FACS. that were dissected from E12.5 mouse embryos through glass Trypan blue staining of sorted cells indicated that Ͼ90% of cells capillaries. Injected metanephroi were grown as explants in 1906 Journal of the American Society of Nephrology J Am Soc Nephrol 17: 1896–1912, 2006

Figure 6. Culture and in vitro differentiation of adult kidney SP cells. Cultures of MP (A) and SP (B and C) cells clearly contained heterogeneous populations. In differentiating cultures, MP cells showed no evidence of conversion to osteocytes (D). Although some SP cells with an epithelial morphology stained positive blue for alkaline phosphatase activity (E), also present were some examples that seemed to take on the osteocyte morphology (E, inset) witnessed in control 10T1/2 cultures (F). MP cells showed no evidence of conversion to adipocytes (H [bright field] and K [red fluorescence]) as identified by Nile red staining. Whereas SP cells showed adipogenic differentiation (I and L), the lipid droplets were either small or few in number compared with 10T1/2 cells (J and M). Bar ϭ 40 ␮m. vitro for 3 d; sectioned; and analyzed by IF for Wilms’ tumor tures, whereas only 1% of donor cells showed engraftment into homologue (WT1) to identify metanephric mesenchyme (MM), ureteric epithelium. Kidneys that received an injection of SP calbindin-D28K (Calb1) to identify ureteric bud (UB), or Pax2 cells showed much higher engraftment into embryonic renal (expressed in both UB and MM) to determine into which struc- structures, with approximately 13 and 28% of donor cells in- tures of the developing kidney the injected cells had incorpo- corporating into UB and MM structures, respectively. This ϩ rated. CM-DiI cells were scored as positive for either UB represents a 13-fold enrichment of UB-forming activity in SP ϩ ϩ ϩ ϩ (Calbindin /Pax2 ) or MM (WT1 /Pax2 ) incorporation or cells compared with MP cells and a 3.5-fold enrichment for negative when they did not express any of these markers. Three MM-forming activity (Table 3). separate injection experiments were conducted (n Ͼ 12 total kidneys for each cell type), and care was taken to discriminate In Vivo Assessment of SP Renal Potential ϩ the same CM-DiI cell in serial confocal sections such that it The ability of kidney SP cells to act as functional renal stem was not counted more than once. cells in vivo was assessed using an Adriamycin model of mouse Section IF analysis clearly showed that donor cells had in- kidney damage. This is an established model of progressive corporated into MM- and UB-derived structures (Figure 7). In renal damage (35). After a single tail-vein injection of Adria- kidneys that received an injection of MP cells, approximately mycin (10 mg/kg), mice develop an acute glomerular injury 9% of the donor cells had become incorporated into MM struc- that manifests as severe proteinuria and podocyte loss into the J Am Soc Nephrol 17: 1896–1912, 2006 Characterization of Kidney Side Population Cells 1907

Control animals received an equivalent volume of PBS vehicle injection. Mice from each treatment group (n ϭ 3) were killed at three time points after surgery: 3, 7, and 14 d. Twenty-four-hour urine samples were collected from mice the day before they were killed to evaluate renal function. Urine function was evaluated by determining the ratio of urine albumin (␮g):urine creatinine (␮g). There was significant proteinuria in the ADR group (21.3) compared with control mice (6.9) 3 d after treatment (Figure 8A). Urine protein levels were decreased in mice that received SP (8.8) and MP (11.8) injections, returning to levels that were not significantly different from controls. One week after treatment, ADR mice again were significantly higher (26.5) compared with controls (8.0), and both mice that received MP and SP injections showed a minor improvement. Although the MP group (20.6) seemed to have higher urine albumin:urine creatinine levels than SP mice (13.6), this difference was not statistically significant. IF was performed on the kidneys that directly received an injection of donor cells to determine whether cells had en- grafted into the damaged organ (Figure 8B). The majority of donor cells remained in the renal interstitium, primarily con- centrated around the site of injection. Both MP and SP cells showed no evidence of migration to sites of injury throughout the kidneys. SP cells were shown to engraft at low frequency into proximal tubules, distal tubules, and collecting ducts and very rarely into glomeruli. No evidence for engraftment into blood vessels or capillaries was seen in any case. It is interesting that there also were very rare instances of MP cell engraftment into distal tubules and collecting ducts but never in proximal tubules, blood vessels, or glomeruli (data not shown).

Discussion These results represent a detailed molecular and functional characterization of the embryonic and adult kidney SP. Unlike purification using combinations of cell surface epitopes, isolation of SP cells is based on dye efflux. This is a dynamic process whereby slight variations in tissue dissociation, cell counting, Hoechst concentration, staining time and temperature, and Figure 7. Section immunofluorescence analysis of embryonic kidneys that received a microinjection of CM-DiI–labeled SP stringency in selection of SP cells by FACS gating can dramat- cells. Donor SP cells showed the ability to engraft into both ically affect the viability, homogeneity, and apparent yield of ϩ ϩ ureteric bud (UB)-derived (calbindin /Pax2 ) and metaneph- SP cells (36). In our study, the SP fraction from embryonic and ϩ ϩ ric mesenchyme (MM)-derived (WT1 /Pax2 ) structures. adult mouse kidneys represented on average 0.10 and 0.14% of Bars ϭ 50 ␮m. Arrows indicate CM-DiI–labeled SP cells in the total viable cell populations, respectively, which is a similar overlays. abundance to the SP reported from bone marrow and other nonhematopoietic tissues in general (13). The existence of SP cells in the mammalian kidney is not a novel finding. However, urinary space. There is rapid accumulation of tubular casts the abundance of SP cells in adult mouse kidneys reported in associated with tubular cell loss and progressive renal failure. previous studies (14,15) is up to 40 times greater than reported Animals were divided into four treatment groups: Control (no here and therefore is likely to represent a significantly different Adriamycin, PBS injection), ADR (Adriamycin, PBS injection), cellular content. We believe that our data are a more accurate MP (Adriamycin, MP cells injected), and SP (Adriamycin, SP estimate of the size of this population. cells injected). Donor cells were isolated from the kidneys of Immunophenotypic analysis showed that the kidney SP con- Ccrslc mice (BALB/c-GFP), and approximately 2 ϫ 105 cells tained only low levels of cells that expressed CD45, CD34, were injected directly into the renal parenchyma of the outer CD31, and c-kit. Combined with the observation that the kid- cortex of the left kidney. An additional bolus of 1 ϫ 105 cells ney SP phenotype is relatively conserved throughout renal was introduced into the circulation via the renal vein just before development as a result of comparatively few differences in administration of Adriamycin by a single tail-vein injection. gene expression profile between E15.5 and adult kidney SP, 1908 Journal of the American Society of Nephrology J Am Soc Nephrol 17: 1896–1912, 2006

Table 3. Engraftment of injected kidney MP and SP cells into embryonic renal structuresa

ϩ ϩ ϩ Marker Marker /DiI Total DiI % Engraftment

Injected kidney MP cells calbindin 4 310 approximately 1% UB WT1 34 336 approximately 9% MM Pax2 24 (22 MM, 2 UB) 272 approximately 8% MM, approximately 1% UB Injected kidney SP cells Calbindin 34 240 approximately 14% UB WT1 98 342 approximately 29% MM Pax2 140 (96 MM, 44 UB) 372 approximately 26% MM, approximately 12% UB

aMM, metanephric mesenchyme; MP, main population; UB, ureteric bud.

these data suggest that this cell fraction is a resident renal cell MyoR as a marker of kidney SP cells. This gene was not population rather than a bone marrow–derived constituent. enriched in the kidney SP fraction in our study; however, the Although 3 to 5% of the cells in the kidney SP were found to be discrepancy in SP size between that study and this (approxi- ϩ ϩ ϩ CD45 , it was determined later that these were a Mac-1 c-fms mately 5 versus 0.1%) suggests that the cellular composition of renal macrophage population. However, it is possible that this the SP was likely to have been very different in that study. Even population is seeded continually from the bone marrow. A with our stringent selection criteria, there still was variability in previous study demonstrated that approximately 10% of kid- the expression patterns of markers that were highly expressed ney SP cells were bone marrow derived (16), a frequency very by kidney SP cells, suggesting that this population still is het- ϩ similar to that of the c-fms macrophage fraction of the kidney erogeneous. It therefore is misleading to think of “kidney SP SP reported here and hence possibly represented renal macro- cells” as a single cell type. phages. Although predominantly displaying a distinct immu- Notch signaling is an evolutionarily conserved and widely nophenotype to bone marrow SP, this does not preclude the used intercellular signaling pathway that influences cellular possibility that hematopoietic cells invaded the metanephros differentiation, proliferation, regeneration, and repair. The very early during organogenesis or are recruited from circula- pathway comprises a set of core components, including the tion in the adult, adopt a renal phenotype, and thereby con- receptors Notch1 through 4; the ligands Delta-like 1 (Dll1), Dll3, tribute to the kidney SP. A careful lineage-tracing study would and Dll4; Jagged1 and Jagged2; proteins that are involved in be required to answer this question comprehensively. receptor cleavage; and co-factors for binding to genomic se- Affymetrix expression profiling was performed to identify quences to regulate transcription of target genes (38). Many markers of kidney SP cells, specifically cell surface molecules, members of this signaling pathway were upregulated in kidney which may prove useful for the purification of these cells in the SP cells, and ISH on adult kidneys again revealed that the absence of or in combination with Hoechst staining. By RNA in members of this pathway were expressed principally in prox- situ hybridization, the expression pattern of most of these mol- imal tubules. Notch signaling also has been implicated in spec- ecules was observed in proximal tubule segments, particularly ification of podocytes and proximal tubules in the developing at the corticomedullary junction. The proliferation antigen Ki67 kidney (39,40) as well as organ regeneration in bone marrow was strongly (ϫ40) upregulated in renal SP cells compared (41), muscle (42), skin (43), heart (44), and liver (45). The kidney ϩ with the total kidney, and Ki67 cells were found in proximal has substantial inherent ability to recover from many types of ϩ tubules in a pattern similar to that reported for BrdU renal acute damage, and the involvement of Notch signaling in this ϩ progenitor-like tubular cells (6). These BrdU cells were pro- process warrants investigation with the caveat that this path- posed to be stem/progenitor cells because they had the ability way is highly expressed by the kidney SP, a potential progen- to proliferate and regenerate the kidney in response to damage. itor population. Another recent study reported isolation of renal progenitor-like In our study, adult kidney SP cells showed the ability to form cells from the S3 proximal tubule segment from rat kidneys several nonrenal mesodermal lineages (osteocyte and adipo- (37). Taken together, these data might suggest that the kidney cyte) in vitro when subjected to specific differentiating culture proximal tubule harbors cells with progenitor properties. A conditions. MP cells never showed such conversion to nonrenal previous study identified a population of potential stem/pro- lineages. Although this suggests evidence for multilineage dif- genitor cells from the papilla of the adult kidney (7). Although ferentiation capacity of kidney SP cells, the cellular heteroge- none of the kidney SP markers that were analyzed by ISH in neity in this population makes it impossible to identify whether this analysis were expressed in such a pattern, this does not all or only a subfraction of the kidney SP has this potential. preclude the existence of stem cells in the renal papilla because Indeed, the list of genes that are enriched in kidney SP cells it is possible that there are multiple progenitor populations contained many genes that are characteristic of monocytes/ present in the mature kidney, each with its own unique prop- macrophages. Tissue macrophages have been shown to play a erties and potentials. Hishikawa et al. (15) identified musculin/ critical trophic role in the development of many organs, includ- J Am Soc Nephrol 17: 1896–1912, 2006 Characterization of Kidney Side Population Cells 1909

ing the gut and mammary gland (46–48). Resident renal macrophages are present within the developing kidney from E12.5 and are located in the interstitium closely associated with the tubules (our unpublished data). Analysis of c-fms-GFP mice showed that resident renal macrophages were more than twice as prevalent in the kidney SP fraction as in the total kidney cell population. Although the converse argument is that such cells represent only approximately 10% of the kidney SP, there is clear evidence that the monocytic lineage contributes to apparent transdifferentiation in other organs (49,50). The identification and quantification of this subfraction clearly establishes the heterogeneity of the kidney SP and raises the question of whether this subfraction of the SP population is actively involved in the apparent multilineage or renal regenerative potential. Injection of adult kidney SP cells into embryonic kidneys resulted in a much higher engraftment of donor cells into developing nephron structures than injection of MP cells. How- ever, unlike a recent study that injected embryonic stem cells into metanephroi (34), kidney SP cells were not able to generate entire renal structures de novo, possibly suggesting they have substantial differentiative but not proliferative capacity. Using a mouse model of acute kidney damage, we also investigated the ability of kidney SP cells to integrate into the kidney in vivo. Once again, we saw the recruitment and occasional integration of SP cells into the kidney but did not see evidence for proliferation. This finding is in accordance with that of Hishikawa et al. (15), which suggested that the ability of kidney SP cells to contribute positively to renal repair in vivo may not require transdifferentiation or integration into tubular compartments but may involve a humoral response. This does suggest an enrichment for renal capacity in the SP fraction; however, MP cells also were observed to integrate into distal tubules and collecting ducts. It was not determined whether the observed donor cell integration into renal structures was due to a transdifferentiation event or cell fusion. The importance of using a control cell population in functional models of renal damage is underscored here by the observation that MP cells not only showed an ability to integrate into regenerating tubules but showed evidence of improved renal function as determined by albuminuria. Although there is no evidence to suggest that progenitor cells would be located exclusively in the SP fraction, many studies of this nature investigate only the potential of

animals subjected to Adriamycin nephrotoxicity and killed after 14 d. Antibody co-localization (antibody, red; donor SP cell, Figure 8. In vivo assessment of the renal regenerative capacity of green; DAPI, blue) was analyzed using antibodies to AQP1 kidney SP cells. (A) Urine albumin:urine creatinine ratio levels (proximal tubules [B and C]), Calb1 (distal tubules, collecting were assessed at 3 and 7 d after delivery of Adriamycin in duct [D and E]), AQP2 (collecting duct [F and G), desmin control (sham injection), ADR (no cells), MP (injected with MP (glomerular mesangial cells [H and I]), and CD31 (endothelial cells), and SP (injected with SP cells) mice. There was a statis- cells [J and K]). SP cells were identified in the recipient kidneys tically significant increase in urinary albumin at both 3 and 7 d and were seen to integrate into tubular structures and rarely in the Adriamycin-treated mice in which no cells were admin- into glomeruli (yellow arrows) but never into blood vessels. istered. Although not statistically significant, the introduction More commonly, though, the injected cells were located in the of either SP or MP cells reduced albuminuria (*P ϭ 0.05). (B interstitium between renal tubules (white arrows). Images on ϩ through K) Confocal analysis of the integration of SP cells the right show localization of GFP donor SP cells in a bright- ϩ derived from GFP mice and delivered into the kidney of field image. Bar ϭ 50 ␮m. 1910 Journal of the American Society of Nephrology J Am Soc Nephrol 17: 1896–1912, 2006 their cell of interest without considering cells that lack the Acknowledgments desired phenotype. The mature kidney contains Ͼ26 differen- This work was performed as part of the Renal Regeneration Consor- tiated cell types, and it would be interesting to fractionate the tium (http://www.renalregeneration.com/) and was supported by the MP to determine the nature of the cells that are responsible for National Institute of Diabetes and Digestive and Kidney Diseases, this positive effect. The mechanism by which donor cells ex- National Institutes of Health (DK63400) as part of the Stem Cell Ge- erted their beneficial effects warrants more investigation to nome Anatomy Project (http://www.scgap.org/). G.A.C. was sup- explore the possibility of using this as the basis for a cellular ported by an Australian Postgraduate Award. M.H.L. is an NHMRC therapy in the treatment of renal disease. Principal Research Fellow. The in vitro and in vivo SP injection experiments presented We thank Rohan Teasdale and Melissa Davis for bioinformatic analysis, Sean Grimmond and Darrin Taylor for data management, David here specifically sought to assess the potential of uncultured SP Hume for c-fms-GFP mice (all Institute for Molecular Bioscience), and cells. These approaches were challenging because of the small Grace Chojnowski (Queensland Institute of Medical Research, Bris- size of the kidney SP and hence the number of donor cells that bane, Australia) for FACS. were available. This was compounded by the fact that only approximately 70% of the total cells that were harvested from ϩ constitutional GFP mice were actually GFP . It would have been possible to increase the number of cells that were available References for injection by culturing sorted SP cells for a time period before 1. Oliver JA, Barasch J, Yang J, Herzlinger D, Al-Awqati Q: injection. This strategy was not used to avoid the possibility of Metanephric mesenchyme contains embryonic renal stem artificially extending or altering the phenotype and potential of cells. Am J Physiol Renal Physiol 283: F799–F809, 2002 these cells via in vitro culture. Our observation of heterogeneity 2. Dekel B, Burakove T, Arditti FD, Reich-Zeliger S, Milstein in this population also would compromise the interpretation of O, Aviel-Ronen S, Rechavi G, Friedman N, Kaminski N, Passwell JH, Reisner Y: Human and porcine early kidney results that are gained from cultured cells because it is highly precursors as a new source for transplantation. Nat Med 9: likely that specific subpopulations would be selected during 53–60, 2003 this process. 3. Burrow CR, Wilson PD: 1994: Renal progenitor cells— Is the renal SP a stem/progenitor cell population? Adult Problems of definition, isolation and characterization. Exp kidney SP cells showed some progenitor characteristics such as Nephrol 2: 1–12, 1994 multilineage differentiation in vitro and an enriched capacity 4. Nadasdy T, Laszik Z, Blick KE, Johnson DL, Silva FG: for integration into developing metanephric structures in vivo, Tubular atrophy in the end-stage kidney: A lectin and but clonogenicity remains to be proved. Far from establishing immunohistochemical study. Hum Pathol 25: 22–28, 1994 that the renal SP is a stem cell population, this rigorous molec- 5. Ng YY, Huang TP, Yang YC: Tubular epithelial-myofibro- ular and functional characterization suggests that the renal SP blast transdifferentiation in progressive tubulointerstitial is a heterogeneous population that, although showing some fibrosis in 5/6 nephrectomized rats. Kidney Int 56: 1266– promising functional properties in vitro, shows little evidence 1269, 1998 for in vivo transdifferentiation when compared with MP cells. 6. Maeshima A, Yamashita S, Nojima Y: Identification of One of the most specific markers for the SP was Ki67, a marker renal progenitor-like tubular cells that participate in the regeneration processes of the kidney. J Am Soc Nephrol 14: of proliferation that normally is associated with a transit-am- 3138–3146, 2003 plifying population rather than a progenitor. In vivo, the renal 7. Oliver JA, Maarouf O, Cheema FH, Martens TF, Al-Awqati SP shows an apparent humoral activity that might indicate Q: The renal papilla is a niche for adult kidney stem cells. regenerative properties that do not require any transdifferen- J Clin Invest 114: 795–804, 2004 tiation. It is interesting to note that in recent Phase I/II clinical 8. Goodell MA, Brose K, Paradis G, Conner AS, Mulligan RC: trial of patients with acute renal failure, renal parenchymal cells Isolation and functional properties of murine hematopoi- that were seeded along a hollow fiber renal-assist device etic stem cells that are replicating in vivo. J Exp Med 183: seemed to provide these patients with proproliferative, anti- 1797–1806, 1996 inflammatory cytokines (51). 9. Goodell MA, Rosenzweig M, Kim H, Marks DF, DeMaria M, Paradis G, Rupp SA, Sieff CA, Mulligan RC, Johnson RP: Dye efflux studies suggest that hematopoietic stem Conclusion cells expressing low or undetectable levels of CD34 antigen We have provided a careful genomic, phenotypic, and func- exist in multiple species. Nat Med 3: 1337–1345, 1997 10. Goodell MA: Stem cell identification and sorting using the tional characterization of a stringently selected renal SP fraction Hoechst 33342 side population (SP). In: Current Protocols in from embryonic and adult mouse kidneys. 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