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Stem Cells 2005;23:44-54 www.StemCells.com
© 2005 AlphaMed Press

Rare Incorporation of Bone Marrow-Derived Cells Into Kidney After Folic Acid-Induced Injury

Mark S. Szczypkaa, Angela J. Westovera, Shawn G. Clouthierc, James L.M. Ferrarac, H. David Humesa,b,c

a Research Division, Nephros Therapeutics Inc., Ann Arbor, Michigan;
b University of Michigan Comprehensive Cancer Center and
c Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan, USA

Key Words. Bone marrow transplant • Transdifferentiation • Kidney • Bone marrow-derived Folic acid • Adult stem cells • Acute renal failure

Correspondence: Mark S. Szczypka, 1995 Highland Drive, Suite F, Ann Arbor, MI 48108, USA. Telephone: 734–975–0080; Fax: 734–975–0090; e–mail: mszczypka{at}nephrostherapeutics.com


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Results obtained in recent experiments suggest that bone marrow-derived cells (BMDCs) engraft into tissues and differentiate into various somatic cell types. However, it is unclear whether injury is required for the phenomenon to occur at appreciable frequencies. In this study we tested whether BMDCs engraft into kidneys and differentiate into renal cells in the absence or presence of toxic injury. Renal damage was induced by delivery of folic acid (FA) to bone marrow (BM)-recipient mice 1 or 9 months after bone marrow transplant, and kidneys were examined for donor-derived cells 2,4, or 8 weeks after injury. Donor-derived cells were abundant in the renal interstitium of injured kidneys and were detected in glomeruli of vehicle- and FA-treated mice. Most of these cells expressed the common leukocyte antigen CD45 and display morphological characteristics of white blood cells. No donor-derived renal tubule cells (RTCs) were detected in kidney sections of BM-recipient mice. However, in cell culture, a cluster of seven donor-derived cells of 4 x 106 RTCs examined (~ 0.0002%) displayed morphological characteristics of RTCs. CD45+ cells of donor origin were also detected in glomeruli and glomerular outgrowths. Nested polymerase chain reaction analysis for the male-specific Sry gene in cultured RTCs and glomerular outgrowths confirmed the presence of donor-derived cells. These results suggest that BMDCs may incorporate into glomeruli as specialized glomerular mesangial cells; however, BMDCs rarely contribute to the repair of renal tubules in uninjured or FA-treated mouse kidneys.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The concept of bone marrow-derived cells (BMDCs) engrafting and differentiating into functional somatic cells of various organs is appealing because it provides potential novel therapy for many disease states. Evidence that BMDCs differentiate into somatic cells in vitro and in vivo exists, but frequency is variable and functionality of the cells has not been fully addressed [1–4]. Studies investigating whether BMDCs engraft and differentiate into kidney cells have yielded mixed results [5–14]. Extrarenal cells seem to differentiate into renal tubule cells (RTCs) at low frequency in humans [5]. In rodents, remarkable levels of donor-derived renal tubules (RTs) have been reported in kidneys after renal ischemia-reperfusion injury [7, 8]. BMDCs also seem to repopulate injured glomeruli of rodents at low frequency [10, 11], and unfractionated bone marrow (BM) from oligosyndactyly mutant mice (ROPOs/+) reportedly transmits glomerulosclerosis to wild-type (WT) mice after bone marrow transplant (BMT), suggesting that BMDCs transmit disease from mutant to healthy animals [9].

Multiple studies have failed to detect BM-derived renal cells in uninjured animals. Wagers et al. [12] did not detect donor-derived renal cells in mice after hematopoietic reconstitution with green fluorescent protein-positive (GFP+) hematopoietic stem cells (HSCs) or in parabiotic GFP+:GFP– mice. Purified BMDCs exhibiting morphological characteristics of liver, lung, gastrointestinal tract, and skin cells were detected after hematopoeitic reconstitution of mice with purified HSCs, but no renal cells were found [13, 14]. However, multipotent adult progenitor cells purified from BM and neural stem cells incorporate into kidneys of chimeric mice after injection into 3.5-day-old blastocysts. This observation suggests that these adult stem cells (ASCs) can incorporate into kidney when placed in the appropriate environment [14, 15].

Recent data suggest that injury enhances incorporation of BMDCs into certain tissues [16]. In humans tissue damage seems to be required for rare incorporation of BMDCs into renal tubules. In contrast, BM-derived RTCs have been detected at appreciable frequencies in both uninjured and injured rodent kidneys [5–8]. Acute renal failure (ARF) results from ischemia, drug, or toxicant exposure [17, 18]. Each of these renal insults may cause specific types of renal injury; however, folic acid (FA)-induced renal injury is considered an accepted model of ARF, because FA administration induces insults that are observed in other model systems of ARF [19–21]. After FA treatment, acute and chronic injury occurs in the kidney, including tubular obstruction, apoptosis, necrosis, tubular denudation, and synthesis of molecules that may promote repair processes [22–25]. Resident macrophages are activated, and additional white blood cells (WBCs) are recruited to the injured regions to contribute to regeneration. Cellular proliferation is abundant after FA-induced injury, and biomarkers of ARF such as serum creatinine and blood urea nitrogen (BUN) are elevated [26].

We developed a mouse model using FA-induced ARF to determine if BMDCs incorporate into uninjured or injured kidneys. The FA model was chosen because acute and chronic injury accompany this insult. We sought to determine the mechanism and frequency of incorporation of BMDCs into kidneys and to test functionality of differentiated cells. Although we used multiple techniques to rigorously examine kidneys for donor-derived renal cells, our data demonstrate that extrarenal cells rarely contribute to repopulation of RTs or glomeruli in uninjured or FA-damaged kidneys. The rare frequency of incorporation of BMDCs into kidney in this study precluded further analysis.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animal Maintenance and Bone Marrow Transplant
Mice were maintained and used in accordance with guidelines for animal care and experimentation established by the National Institutes of Health and the University of Michigan Unit for Laboratory Animal Medicine. BMT procedures used are a modification of those previously published [27] and are described herein. Female C57BL/6 (B6) mice (National Cancer Institute, NCI, Frederick, MD, or Jackson Laboratories, Bar Harbor, ME) were transplanted at a minimum age of 10 weeks (25 g body weight [BW]) with BM from 8-week-old male B6 or B6; 129S-Gt(ROSA)26Sor(ROSA26) mice. BM (5.0 x 106) was harvested from femurs and tibias, resuspended in Leibovitz’s L-15 serum-free medium (Gibco Life Technologies, Grand Island, NY), and injected into the recipient’s tail vein (0.25 ml/injection). Before transplant, host mice received a single 900-cGy TBI dose using a 137Cs source Gammacell 40 model C-161 type 8 irradiator (Nordion International, Inc.; Ontario, Canada) (delivered at 92–94 cGy/min). Mice were housed in sterile microisolator cages and fed normal chow and autoclaved, acidified water for the first 3 weeks after BMT and filtered water thereafter.

Folic Acid-Induced Acute Renal Failure
One or 9 months after BMT, FA (F7876, Sigma, St. Louis) (90 – 270 mg/kg BW) in 300 mM bicarbonate buffer was administered via tail vein. Percent survival was monitored, and quantitative determination of BUN concentration was performed on serum samples obtained from tail bleeds 1 week before FA injection, after injection on experimental days 1, 3, 5, and 7, then weekly (1020–101, Stanbio Laboratories, Boerne, TX).

Experimental Design
Twenty-one female B6 BM-recipient mice were divided by FA dose into four groups: 270 mg/kg BW (n = 3), 240 mg/kg BW (n = 5), 90 mg/kg BW (n = 6), and vehicle (n = 7). All members of this cohort were injected 1 month after transplant. No significant differences in BUN and percent survival were observed between the 240 and 270 mg/kg BW cohorts, so these animals were pooled and treated as one group (≥ 240 mg/kg BW)

Twenty-two female ROSA26 BM-recipient mice were divided into groups by FA dose and time of injection after transplant. Mice treated 1 month (young) after transplant were divided into the following three groups: 240 (n = 8) or 90 mg/kg BW FA (n = 4) and vehicle (n = 1). Mice treated 9 months (old) after transplant were divided into the following three groups: 180 (n = 3) or 240 (n = 3) mg/kg BW FA and vehicle (n = 3). The 180 and 240 mg/kg BW groups did not exhibit significant differences in BUN or percent survival and were therefore pooled and treated as one group (≥ 180 mg/kg). Naive, vehicle, and FA-injected ROSA26 and B6 mice were used as controls.

Tissue Processing
Mice were euthanized by CO2 asphyxiation and subsequently perfused with ice-cold phosphate buffered saline (PBS) (pH = 7.4) containing 1 U/ml heparin. Kidneys were excised, and one half kidney and representative samples from other organs including spleen were cut into 1-mm-thick sections. Tissues were fixed with 2% paraformaldehyde (15700, Electron Microscopy Sciences, Fort Washington, PA) in PBS for one hour, rinsed with PBS, and divided equally, and one half was incubated at 37°C for 24 hours with x-gal solution (X4281C, Gold BioTechnologies, St. Louis). X-gal-stained and unstained tissues were paraffin embedded (butanol substituted for xylene), and 5-µm-thick sections were prepared for immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH).

Renal Tubule and Glomerular Cell Isolations
Kidneys (1.5 kidneys from each animal) were pooled into ice-cold Dulbecco’s modified Eagle’s medium (12-917F, Biowhittaker, Walkersville, MD) containing 100 U/ml penicillin G and 100 mg/ml streptomycin sulfate (9366, Irvine Scientific, Santa Ana, CA) for isolation of RT and glomeruli by the following conditions: naive ROSA26 male (n = 2), naive ROSA26 female (n = 1), vehicle-injected B6 BM–recipient (n = 2), FA-injected B6 BM–recipient (n = 4), and FA-injected ROSA26 BM-recipient (n = 4) mice.

Proximal Tubule and Glomerular Explant Cell Culture
Capsule was removed, and the cortex was excised from the medulla and minced. For proximal tubule (PT) isolation, the resulting slurry underwent collagenase type IV (LS004189, Worthington Biochemical, Lakewood, NJ) digestion followed by density separation on a Percoll gradient [28]. Isolated PTs were dispensed onto plates coated with collagen IV (354233, BD Biosciences, San Jose, CA) (0.5 mg/cm2) and adsorbed fetal calf serum proteins [29]. Media consisted of Ham’s F12/Dulbecco’s modified Eagle’s medium (9438, Irvine, Santa Ana, CA) supplemented with arginine-vasopressin (0.25 ng/ml) (V9879, Sigma), cholesterol (3.9 ng/ml) (C3045, Sigma), epidermal growth factor (60 ng/ml) (100–15, Peprotech, Rocky Hill, NJ), ethanolamine (31 ng/ml) (E0135 Sigma), hydrocortisone (37 ng/ml) (H0888 Sigma), insulin (5 µg/ml) (I8882 Sigma), prostaglandin E1 (10 ng/ml) (P7527, Sigma), selenium (5 ng/ml) (S5261, Sigma), transferrin (5 ng/ml) (T4382, Sigma), triiodothyronine (0.7 ng/ml) (T6397, Sigma), 100 U/ml penicillin G, and 100 mg/ml streptomycin sulfate. Isolation of decapsulated glomeruli was accomplished by differential sieving. Minced cortex was gently pushed through a 100-µm cell strainer, and decapsulated glomeruli were captured on top of a 70-µm cell strainer (352360 and 352350, Falcon/BD Biosciences). Glomeruli were dispensed to fibronectin-coated (F-2006, Sigma) (0.5 µg/cm2) [30] plates in minimum essential Eagle’s medium (M0268, Sigma)/Ham’s F12 nutrient mix (H6760, Sigma) 3:1 supplemented with 10% Nuserum (355100, BD Biosciences) [31], 100 U/ml penicillin G, and 100 mg/ml streptomycin sulfate, with subsets receiving 500 mg/ml heparin (245041, Elkins Sinn, Cherry Hill, NJ) or 10 ng/ml platelet-derived growth factor blocking buffer (BB) (P3201, Sigma) [32, 33]. Glomeruli were incubated undisturbed at 37°C, with partial media changes performed weekly. Outgrowths from PT and glomeruli were plated onto eight-well permanox slides (177445, Nunc) for IHC and onto 100-mm culture plates for x-gal staining as indicated above or harvested for DNA analysis.

Fluorescein di-ß-D-Galactoside and Fluorescence-Activated Cell Sorting
Blood (300 µl) drawn via cardiac puncture or tail bleed was used for fluorescein di-ß-D-galactoside (FDG) (F-1179, Molecular Probes Inc., Eugene, OR) analysis of peripheral blood mononuclear cells (PBMCs). PBMCs were isolated [34] from ROSA26 BM-recipient mice (n = 5), and 5 x 105 cells were analyzed for ß-galactosidase (ß-gal) activity by hypotonic loading of FDG. Naive ROSA26 and B6 BM-recipient mice were used as controls, and nonviable cells were excluded from analysis by the addition of 1 µg/ml propidium iodide. Cell labeling was analyzed on a FACSCalibur Cytometer (BD Biosciences).

Immunohistochemistry and Fluorescent In Situ Hybridization
Dewaxed and rehydrated sections were permeablized for 10 min in PBS/0.1% triton x-100. Antigen retrieval was accomplished by incubation at 37°C in trypsin-versene (17–161, Biowhittaker) for 20 min. Slides were blocked in 5% normal goat serum/1% bovine serum albumin in PBS for one hour followed by a 1-hour incubation with antibodies against the common leukocyte antigen, CD45 (1:50) (550539, BD Pharmingen). Antibodies were detected with conjugated Alexa Fluors (A-11006 and S-11227, Molecular Probes) (1:100) and counterstained with 4',6-Diamidino-2-phe-nylindole dihydrochloride (DAPI) (1 µg/ml). Incubations were performed at room temperature unless otherwise indicated. BB was used as diluent, with exhaustive PBS rinsing between steps.

Glomerular outgrowths were similarly stained for CD45 and {alpha}-smooth muscle actin (MS-113-B0, NeoMarkers Inc., Fremont, CA) (1:100). Isolated RTCs were analyzed with antibodies against {alpha}-acetylated tubulin-1 (AT1) (1:500) and zonna occludens-1 (ZO1) (32-2700 and 61-7300, Zymed, South San Francisco, CA) and visualized with Alexa Fluors (A11008 [GenBank] and A11005, Molecular Probes). Antigen retrieval was not required for PT cells (PTCs) or glomerular outgrowths.

FISH for Y-chromosome was performed per the manufacture’s direction (1187-YMB-01, Cambio LTD, Cambridge, UK). Identification of PT in sections was accomplished by incubation with biotinylated lotus tetragonolobus lectin (LTA) (Sigma) (1:100), followed by detection with strepavidin, Alexa Fluor 488 conjugate (S-11223, Molecular Probes) (1:100), and DAPI counterstain.

Slides were visualized through x 20 and x 40 objectives of a fluorescent microscope (IX71 Olympus, Melville, NY) in conjunction with a Pixlefly (Cooke, Auburn Hills, MI) camera system. Entire sections were mapped and selected, and identical locations were photographed before FISH and after LTA staining. Photographs were precisely aligned using DAPI staining patterns and merged using Adobe Photoshop Elements software, and Y-chromosome+ cells were identified and scored by three independent observers blinded to the sample treatment.

Detection of Sry by Polymerase Chain Reaction
Genomic DNA was extracted from PTCs and glomerular cells using DNeasy tissue kits (69504, Qiagen, Valencia, CA) according to the manufacturer’s instructions. Polymerase chain reaction (PCR) amplified an 801-bp sequence of the Sry gene [7]. Nested PCR was subsequently performed, resulting in a 262-bp product. Glyceraldehyde-3-phosphate dehydrogenase (GADPH) PCR was performed as a control [35].


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Hematopoietic Reconstitution of Transplant Recipient Mice
We transplanted BM from syngeneic B6 or allogeneic ROSA26 male mice into host female B6 mice, allowed sufficient time for hematopoietic reconstitution (1 or 9 months), and tracked incorporation of BMDCs into kidney. Transgenic ROSA26 male donor mice express bacterial ß-gal in all tissues, and its presence can be detected with chromogenic (x-gal) or fluorescent (FDG) substrates. In addition, donor-derived male cells can be identified with FISH to detect the Y-chromosome. Hematopoietic reconstitution was assessed in a subset of ROSA26 BM-recipient mice with x-gal and FDG staining. FACS analysis indicated that FDG+ PBMCs in BM recipients ranged from 28% – 100% compared with ROSA26 controls. Reconstitution within subject was similar between repeated measurements that occurred at least 1 month apart. To confirm successful reconstitution, spleens collected at the time of euthanasia were stained with x-gal and probed with FISH. Spleens retrieved from ROSA26 BM-recipient mice were ß-gal+and FISH+, confirming successful reconstitution. No ß-gal+ cells were detected in the spleens of controls or B6 BM recipients (data not shown).

Folic Acid-Induced Acute Renal Failure
BM-recipient mice were injected with FA 1 or 9 month after BMT, and the dose of FA that promoted survival yet caused ARF in experimental cohorts was determined empirically. The mice injected 1 month after BMT were designated as young, and those injected 9 months after BMT were designated as old. Young vehicle-treated B6 to B6 BM-recipient mice (n = 7) survived without evidence of renal injury (Fig. 1AGo; B6 to B6, vehicle). Eighty-three percent of female B6 BM recipients that received 90 mg/kg FA (n = 6) and 75% of those injected with ≥ 240 mg/kg FA (n = 8) survived treatment. Plasma BUN concentrations in both groups of FA-treated mice peaked 3 to 5 days after FA injection (Fig. 1BGo; B6 to B6).



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  		Figure 1. Percent survival and BUN of vehicle- and FA-treated BM-recipient mice. (A): Percent survival of BM-recipient mice was monitored after administration of vehicle ({bullet}),90 mg/kg FA ({square}),≥ 240 mg/kg FA({triangleup}),≥ 180mg/kg FA(old)({square}), or 240 mg/kg FA(young) ({blacktriangleup}). Data represent mean ± standard error of the mean. (B): BUN levels were monitored after injection of vehicle and FA to BMT mice. Abbreviations: BM, bone marrow; BUN, blood urea nitrogen; FA, folic acid.

 
All young and old vehicle-treated ROSA26 BM-recipient mice (n = 7) survived without evidence of renal injury and were treated as a single group (Fig. 1AGo; ROSA26 to B6, vehicle). All ROSA26 BM recipients injected 1 month (young, n = 4) after BMT with 90 mg/kg BW FA survived treatment (Fig. 1AGo; ROSA26 to B6, 90 mg/kg). An increase in BUN that peaked 3 days after injection and returned to normal levels 14 days after treatment occurred in these mice (Fig. 1BGo; ROSA26 to B6). A dose of 240 mg/kg BW FA (n = 9) administered to a cohort of young ROSA26 BM-recipient mice 1 month after transplant caused rapid and severe elevation of BUN, and these mice perished (Figs. 1A, 1BGo). Old ROSA26 BMT mice did not respond to the 90-mg/kg dose; therefore, they were divided into two groups and injected with either 180 (n = 3) or 240 (n = 3) mg/kg FA. These animals survived and experienced increases in BUN (Figs. 1A, 1BGo; ROSA26 to B6, ≥ 180 mg/kg [old]).

Histopathologic changes were observed in the kidneys of FA-treated mice (Fig. 2Go). Degenerative lesions containing areas of necrosis, marked mononuclear infiltration, and interstitial nephritis were observed. Areas of acute tubular necrosis contained depolarized and flattened tubular cells, and regions of disorganized tissue indicative of tubular obstruction were evident. Kidneys retrieved from young vehicle-treated mice appeared normal; however, all older ROSA26 BM-recipient mice exhibited thickening of the glomerular basement membrane and contained cells with enlarged nuclei (data not shown).



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  		Figure 2. Renal damage in FA-treated ROSA26 BM-recipient mice. (A): Representative kidney sections from vehicle (left) and FA-treated BM-recipient mice. (B): Higher power image of boxed region in vehicle-treated animal. (C): Higher power image of boxed region in FA-treated animal. Black arrows depict glomerular hypercellularity. Some x-gal-stained cells are evident. Arrowheads demarcate areas of disorganized tissue and flattened tubule cells. (D): Representative x-gal-stained kidney section from ROSA26 BM-recipient mouse. (E): Immunohistochemistry for the CD45 performed on x-gal-stained section shown in (D). CD45 staining is green, and nuclear DAPI staining is blue. (F): Representative photo of x-gal-stained glomerulus. (G): CD45 immunostaining of x-gal-stained section shown in (F). Abbreviations: BM, bone marrow; FA, folic acid.

 
Donor-Derived Renal Tubule Cells Are Not Detected in Kidney Sections of Bone Marrow-Recipient Mice
FA-and vehicle-treated mice were euthanized 2,4, or 8 weeks after injection, and kidneys were examined for donor-derived renal cells using x-gal staining, FISH, and immunohistochemistry. Numerous x-gal+ cells were detected in kidneys of FA-treated ROSA26 BM recipient mice, but none were found in B6 to B6 mice. These cells were concentrated in areas of intense damage (Figs. 2B, 2CGo). Few x-gal+ cells were detected in vehicle-treated ROSA26 BM recipients, and none in controls. Morphological and immunohistochemical analysis indicated that most x-gal+ cells were WBCs because they stained positively for CD45 and were not located within tubules (Figs. 2D, 2EGo). Numerous x-gal+ cells that express CD45 were also found in glomeruli of BM-recipient mice (Figs. 2F, 2GGo). However, no x-gal+ positive tubule cells were detected in the 14 ROSA26 BM-recipient mice examined.

FISH to detect Y chromosome coupled with immunohistochemical analysis with LTA to detect PTs and CD45 to detect WBCs was performed on tissue sections from BM recipients and controls (Fig. 3Go). No convincing evidence of widespread incorporation of BM-derived cells into kidney was observed after examination of 31 BM-recipient mice; therefore, FISH-stained tissue sections from male and female controls and 15 BM-recipient mice were examined and counted by three individuals blinded to treatment. In male controls, 69.3 ± 4.4% of PTCs were Y chromosome+ (Fig. 4Go, Table 1Go). LTA+ renal PTCs accounted for approximately 30% of nuclei counted in BM-recipient mice. A small percentage of Y chromosome+ tubule cells were identified in transplant recipients (0.8 ± 0.4% to 1.2 ± 0.9%), but, surprisingly, observers also identified positive PTCs in female controls (3.7 ± 2.2%). Rigorous examination of these putative positives in both groups revealed that fluorescent signals were often not associated with nuclei or were located in the interstitium adjacent to the tubular basement membrane and therefore represented staining artifacts. Therefore, no definitive Y chromosome-positive renal tubule cells were identified in female controls or BM-recipient mice (Table 1Go). Putative positive renal tubule cells were identified in tissue sections of BM-recipient mice that arose from merging of nuclei of Y chromosome+ interstitial cells with nuclei of tubule cells after FISH (Fig. 5Go).



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  		Figure 3. Immunohistochemical and FISH analysis of vehicle-treated (left) and folic acid-treated (right) ROSA26 bone marrow transplant-recipient mice. (A): Nuclear DAPI staining. (B): Y-chromosomes detected by FISH appear as red punctuate spots. (C): Lotus tetragonolobus lectin staining of proximal tubules. (D): CD45 labeling of white blood cells. (E): Composite overlay of multiple staining techniques displayed in (A)–(D). Abbreviation: FISH, fluorescent in situ hybridization.

 


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  		Figure 4. Immunohistochemical and FISH analysis of naive control female (left) and male (right) ROSA26 mice. (A): Nuclear DAPI staining. (B): FISH analysis to detect the Y chromosome. White arrowheads depict examples of putative positive staining detected in kidneys of female control mouse. (C): Lotus tetragonolobus lectin staining of proximal tubules. (D): Bright field image of selected area. (E): Composite overlay of multiple staining techniques displayed in (A) through (C). Abbreviation: FISH, fluorescent in situ hybridization.

 

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  		Table 1. Fluorescent in situ hybridization analysis of kidneys.
 


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  		Figure 5. Example of false positive proximal tubule cell observed in bone marrow–recipient mouse. (A): Images represent photos taken of an individual field before FISH. BF, DAPI staining to identify nuclei, and CD45 staining to detect leukocytes. (B): This section was processed for FISH and stained with LTA to identify proximal tubules. Post–FISH BF, DAPI, FISH, and LTA images. Note that some nuclei of DAPI pre–FISH image appear to merge after FISH due to spreading of nuclei (arrows in DAPI post–FISH image). (C): Precisely aligned composite of pre–FISH CD45 staining and post–FISH DAPI, FISH, and LTA staining. Arrow in composite image depicts Y chromosome+ merged nuclei within tubule. Abbreviations: BF, bright field; FISH, fluorescent in situ hybridization; LTA, lotus tetragonolobus lectin.

 
Donor-Derived Renal Cells Are Detected in PTC Cultures and Glomerular Outgrowths
Because no definitive positive donor-derived tubule cells were detected in tissue sections of BMT mice, we isolated PTCs and prepared glomerular explant cultures from BM-recipient and control mice in three separate BMT experiments to determine if BM-derived renal cells could be detected in cell culture. Control PTCs exhibited the expected x-gal staining patterns (data not shown), but of approximately 4 x 106 PTCs isolated from vehicle- and FA-treated ROSA26 BM-recipient mice, only a single cluster of seven ß-gal+ cells was found (Fig. 6AGo). This corresponds to approximately 0.0002% of total cells examined. These cells were CD45– and expressed a component of central cilium (AT1) and an epithelial tight junction protein (ZO1) (Fig. 6BGo). In contrast, ß-gal+ cells that costained with CD45 were routinely found in glomerular outgrowths (Figs. 6C, 6DGo). Nested PCR was required to detect the Y chromosome in PTC cultures and glomerular outgrowths isolated from BM recipients (Fig. 6EGo).



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  		Figure 6. Immunohistochemical and cytochemical analysis of cultured RTCs and glomerular outgrowths. (A): Cluster of donor-derived x-gal-positive RTCs. (B): IHC for acetylated tubulin (AT1) (red; arrows) and zona occludens 1 (ZO1) (green). Nuclear DAPI staining is blue. AT1 is a component of the central cilium found on RTCs, and ZO1 is an integral component of epithelial tight junctions. (C): Positive x-gal staining of cells cultured from glomerular explants. Diffuse and punctuate staining pattern was observed in this cell type. (D): IHCs for CD45 performed on x-gal+ cells shown in (C). CD45 staining is green, and nuclear DAPI staining is blue. (E): First-round PCR (Sry) and nested PCR (Sry N) of DNA obtained from RTC cultures. Female ()and male () controls; B6 male to B6 female BM-recipient mice (B6); and ROSA26 male to B6 female BM recipients (ROSA26). Top panel represents product obtained with primers to GAPDH during first-round PCR analysis. Abbreviations: IHC, immunohistochemistry; RTC, renal tubule cell.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The data presented here demonstrate that BMDCs rarely contribute to remodeling of RTs in vehicle- and FA-treated mice. Our findings contrast with reports demonstrating that hematopoietic cells contribute to regeneration of RTs in mice in the absence or presence of renal injury but are consistent with results from human studies in which patients experienced acute tubular necrosis [5–8]. We observed extensive donor-derived leukocyte infiltration in kidneys of mice treated with FA. This observation is consistent with the known mechanism of progression and recovery from renal injury, in which extensive interstitial leukocyte infiltration occurs 5 to 90 days after the insult [36, 37]. We observed a differential sensitivity to FA treatment between young and old mice injected with similar doses of FA. Mice injected with FA 1 month after BMT experienced increased mortality, greater elevation in BUN, and more extensive kidney damage than mice treated 9 months after BMT. This could result from differential sensitivity to irradiation, recovery time after irradiation and BMT, or the age of the animal at the time of FA administration. Although the amount of renal damage varied between groups, all FA-treated mice experienced similar types of FA-induced injuries.

Donor-derived CD45+ cells were also detected within glomeruli of vehicle- and FA-treated mice and in glomerular outgrowths. A subpopulation of CD45+ glomerular mesangial cells (GMCs) has been characterized [38, 39]. These GMCs represent approximately 1%–2% of total cells found within the glomerulus. Although x-gal+, Y chromosome-containing glomerular cells were detected in this study, our data cannot elucidate whether these cells represent this specialized population of GMCs or are WBCs.

What is the source of discrepancies among experimental observations of incorporation of BMDCs into RTs? Experimental design, type of injury induced or encountered by the model organism, differences in sensitivities among assays, or combination of these are potential sources. Disparate observations between this and other studies are difficult to reconcile. For example, some existing data suggest that donor-derived RTCs are present in uninjured rodent kidneys. We found no definitive evidence of donor-derived RTCs in kidney sections obtained from vehicle- or FA-treated mice even though multiple sections from numerous transplant recipients were examined with x-gal cytochemistry and FISH. When no convincing evidence was uncovered 2 weeks after injury, we extended renal recovery time to 4 and 8 weeks. We only detected extremely rare BMDCs (approximately 0.0002%) that adopted morphological characteristics of RTCs in cell cultures 8 weeks after injury. Finally, we extended the time for hematopoietic reconstitution from 1 to 9 months before inducing renal injury and were still unable to find BM-derived RTCs in kidney sections.

If transdifferentiation occurs in adult kidneys, a requisite first step in this process is that ASCs from another organ migrate and engraft into the kidney. Circulating side population (SP) cells have been isolated from peripheral blood, but these cells differentiate into common lymphoid and dendritic cells when grown in culture, suggesting that they have limited differentiation potential [40]. It is possible that circulating ASCs continually engraft into uninjured kidneys but only proliferate and subsequently differentiate into renal cells if they receive appropriate environmental cues. Our data do not support this contention, because BMDCs were rarely found in uninjured kidneys.

Alternatively, cellular damage may stimulate recruitment of stem cells to the kidney. Increases in circulating Lin-Sca1+ hematopoietic cells have been found after renal injury [8]. These cells may be mobilized by yet unidentified molecules produced by kidneys after ARF. If multipotent ASCs migrate and engraft into the kidney, the engrafted cells must receive signals that direct proliferation and subsequent differentiation into renal cells. Molecules synthesized in embryonic metanephric mesenchyme are expressed in damaged kidneys, and this environment promotes functional repair of injured kidneys. Therefore, proliferation, reprogramming, and differentiation of engrafted ASCs into renal cells may occur under appropriate conditions, but the frequency may depend on type of injury.

Data demonstrating that BMDCs fuse in vitro and in vivo with various cell types have provided an alternative explanation for the suggested expanded differentiation capacity of ASCs. Cell fusion is the principle source of BM-derived hepatocytes found in fumarylacetoacetate hydrolase-deficient (Fah–/–) mice that regain normal liver function after transplant of WT BM [41, 42]. BMDCs spontaneously fuse in vitro with neural stem cells and at a low frequency in vivo with select cell types in liver, brain, and heart [43]. We could not elucidate whether the rare donor-derived renal cells detected in this study arose from transdifferentiation or fusion events. Regardless of the mechanism, further rigorous experimental approaches using sensitive and reproducible methods for tracking donor-derived cells are required to confirm results obtained in previous experiments, to ascertain the mechanism of incorporation of BMDCs into renal cells, and to firmly establish the reliability and robustness of this phenomenon.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We thank Chris Strayhorn, David Splan, Mike Smith, Tomiyo Weymert, and Steve Greenway for technical assistance. This work was supported in part by National Institutes of Health grant CA39542


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received on May 13, 2004; accepted for publication on August 24, 2004.




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