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Published ahead of print on September 7, 2006
J Am Soc Nephrol 17: 2799-2811, 2006
© 2006 American Society of Nephrology
doi: 10.1681/ASN.2006020130
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Pathophysiology of Renal Disease and Progression

Blockade of p38 Mitogen-Activated Protein Kinase and TGF-beta1/Smad Signaling Pathways Rescues Bone Marrow–Derived Peritubular Capillary Endothelial Cells in Adriamycin-Induced Nephrosis

Jinhua Li*, James A. Deane*,{dagger}, Naomi V. Campanale*, John F. Bertram{dagger} and Sharon D. Ricardo*,{dagger}

* Monash Immunology and Stem Cell Laboratories and {dagger} Department of Anatomy and Cell Biology, Monash University, Melbourne, Victoria, Australia

Address correspondence to: Dr. Sharon D. Ricardo, Monash Immunology and Stem Cell Laboratories, Monash University, Clayton, Victoria 3800, Australia. Phone: +61-3-9905-0671; Fax: +61-3-9905-0680; E-mail: sharon.ricardo{at}med.monash.edu.au

Received for publication February 8, 2006. Accepted for publication July 26, 2006.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 References
 
The peritubular capillary (PTC) network is a component of the tubulointerstitium of the kidney with important roles in renal function and hemodynamics. Bone marrow (BM)-derived cells can contribute to repair of the renal PTC network after ischemic injury. However, the cell fate and the regulation of renal BM-derived cell engraftment in comparison with somatic cells during disease progression are unclear. This study characterized the time course and regulation of PTC endothelial cell injury in adriamycin (ADR)-induced nephropathy in mice, a model of chronic, irreversible, progressive renal disease. Enhanced green fluorescence protein–positive BM cells that coexpressed two endothelial cell markers, von Willebrand factor and CD31, were found to engraft into the PTC of chimeric ADR-injected mice in a time-dependent manner. The number of BM-derived PTC endothelial cells peaked 2 wk after ADR injection, then declined dramatically thereafter. In these mice, apoptosis was evident in BM-derived PTC endothelial cells, and the p38 mitogen-activated protein kinase (MAPK) and TGF-beta1/Smad signaling pathways were activated. Blocking both the p38 MAPK and TGF-beta1/Smad signaling pathways by administration of a p38 MAPK inhibitor (SB203580) and a TGF-beta receptor 1 inhibitor (ALK5I) to ADR-injected mice rescued BM-derived PTC endothelial cells from apoptosis, reduced the loss of PTC, and restored kidney function. Investigation into the signaling pathways that regulate the differentiation and survival of BM-derived cells that engraft into the kidney in the proinflammatory setting of progressive renal disease is vital for the successful development of cell-based therapies to promote renal regeneration and repair.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Conclusion
 References
 
The peritubular capillary (PTC) network is a component of the tubulointerstitium of the kidney and plays important roles in maintaining renal function and hemodynamics. The importance of interstitial injury in the progression of renal disease has been investigated extensively (1). Studies of various renal diseases using human biopsy specimens have demonstrated a significant negative correlation between the relative area of the PTC and serum creatinine concentration (2–4). In cortical areas that exhibit tubulointerstitial injury, damage to PTC ranges from partial loss to complete loss of network relative to the progression of disease (5). The loss of PTC in the tubulointerstitium results in renal ischemia, and chronic hypoxia is the final common pathway to end-stage renal failure largely independent of the initial insult (6).

There is increasing evidence that bone marrow (BM)-derived cells can transdifferentiate into various kidney somatic cells, including mesangial cells (7) and endothelial cells (8), under certain pathologic circumstances, although the existence of BM-derived tubular epithelial cells is controversial (9). PTC endothelium that is damaged by vascular rejection after renal transplantation is partially repaired by endothelial cells of the recipient in human and animal models (10,11). PTC endothelial cells also can be repaired by BM-derived cells after ischemic injury (12).

Recent studies have shown that the function of the grafted kidney in a renal transplant recipient determines the number of endothelial progenitor cells (13), and endothelial progenitor cells are deficient in patients with uremia (14). This suggests that kidney function is a major determinant of the fate of endothelial progenitor cells.

The p38 mitogen-activated protein kinase (p38 MAPK) signaling pathway and the TGF-beta1/Smad signaling pathway are activated and play pivotal roles during the progression of renal diseases (15–18). p38 MAPK and TGF-beta1/Smad are involved in endothelial cell apoptosis under pathologic circumstances (19–23). Whether such BM-derived intrarenal endothelial cells ultimately are lost via apoptosis in chronic disease is not known. Equally, the role of p38 MAPK and the TGF-beta/Smad signaling in the fate of such BM-derived cells also is not known.

Our study determined the fate of PTC endothelial cells in a mouse model of adriamycin (ADR)-induced nephropathy that is characterized by severe glomerulosclerosis and interstitial fibrosis that lead to reduced renal function. Enhanced green fluorescence (EGFP)-positive BM-derived cells that expressed von Willebrand factor (vWF) and CD31 were demonstrated to engraft into the PTC of chimeric mice with ADR nephropathy in a time-dependent manner after induction of chronic, irreversible, progressive renal disease. However, apoptosis and activation of p38 MAPK and TGF-beta1/Smad signaling pathways were observed in BM-derived PTC endothelial cells in co-occurrence with somatic renal cell injury. Inhibition of p38 MAPK and TGF-beta/Smad signaling pathways in ADR-injected mice reduced the rate of apoptosis of BM-derived PTC endothelial cells and restored kidney function.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Conclusion
 References
 
Experimental Animals
To investigate whether BM-derived cells can transdifferentiate into PTC endothelial cells, we generated chimeric mice using BM that was transplanted from donor mice that expressed EGFP constitutively (EGFP transgenic mice). The EGFP gene, driven by the chicken beta-actin promoter, is expressed in all tissues and organs except hair and red blood cells (24). Six-week-old BALB/c male mice (20 to 25 g body wt) were irradiated with two split doses of 5 Gy of {gamma}-irradiation separated by 4 h. Whole BM was isolated from the femur and tibia of male BALB/c mice that were of the same age and expressed EGFP by flushing with ISCOVE’s minimal essential medium. Irradiated mice received an injection of 0.1 ml of ISCOVE’s minimal essential medium with or without 1 x 106 BM cells via tail-vein injection. Twelve weeks after BM transplantation (BMT), mice received a single intravenous injection of ADR (10.5 mg/kg; Sigma, St. Louis, MO). Control mice were treated with an equivalent intravenous volume of normal saline vehicle (NS). Mice were killed at 72 h, 2 wk, and 4 wk after ADR or NS injection.

For blocking of the p38 MAPK and TGF-beta1/Smad signaling pathways, 2 wk after ADR injection, mice were treated with a p38 MAPK inhibitor (SB203580; Calbiochem, La Jolla, CA) and a TGF-beta receptor I (ALK5) inhibitor (ALK5I; Calbiochem), via implantation of ALZET osmotic pumps (DURECT Corp., Cupertino, CA) until the experimental end point. A preliminary experiment was carried out to determine the effective dose range of SB203580 and ALK5I in ADR-induced nephropathy. Doses of SB203580 and ALK5I from 0.25 to 2 mg/kg were administered respectively to eight groups of three mice. On the basis of the results, a larger experiment was performed to confirm that ADR-injected mice that received SB203580 1 mg/kg per d + ALK5I 1 mg/kg per d can achieve maximal renoprotective effects without obvious adverse effects compared with vehicle alone, SB203580 1 mg/kg per d alone, or ALK5I 1 mg/kg per d alone. Mice received either the vehicle (DMSO) or combination of SB203580 (SB) 1 mg/kg per d + ALK5I 1 mg/kg per d. The solvents of SB203580 and ALK5I were water and DMSO, respectively. SB203580 and ALK5I were stored separately in two minipumps. In treated groups, two minipumps were implanted in each mouse. Mice were killed 2 wk after the initiation of vehicle or SB203580+ALK5I treatment (n = 6/group per time point). All experiments were performed with the approval of a Monash University Animal Ethics Committee, which adheres to the "Australian Code of Practice for the Care and Use of Animals for Scientific Purposes."

FACS Analysis
FACS analysis for EGFP expression in peripheral blood leukocytes was performed on a Becton Dickinson FACS Scan and analyzed with cell Quest (Becton Dickinson, Franklin Lakes, NJ).

Measurement of Proteinuria and Creatinine
Mice were housed in metabolic cages, with free access to food and water on the days of urine collection. Protein from 24-h urine samples and serum creatinine levels were measured by Detergent Compatible protein assay kit (Bio-Rad, Hercules, CA) and Creatinine assay kit (Cayman Chemical, Ann Abor, MI), respectively, according to instructions supplied.

Antibodies
The following antibodies were used for immunofluorescence: Rabbit anti-phosphorylated p38 (p-p38) raised against the dual phosphorylated tyrosine and threonine residues of the p38 peptide (1:100; Sigma-Aldrich, St. Louis, MO), rat anti-CD31 (1:100; BD Biosciences-Pharmingen, San Diego, CA), rabbit anti-vWF (1:400; DakoCytomation, Glostrup, Denmark), rabbit anti–phosphorylated Smad2 (p-Smad2; 1:100), and rabbit anti-GFP (1:800; Chemicon, Temecula, CA).

Histology and Immunofluorescence Microscopy
Renal histology was assessed in 10% buffered formalin–fixed, paraffin-embedded tissue sections (4 µm) stained with periodic acid-Schiff (PAS). For immunofluorescence, tissues were fixed in 4% paraformaldehyde (Sigma-Aldrich) for 8 h, transferred to PBS that contained 30% sucrose for overnight incubation at 4°C, embedded in O.C.T. (TissueTek, Tokyo, Japan) and stored at –80°C. Frozen sections were cut (5 µm) using a cryostat (Leica, Bensheim, Germany) and blocked with 2% BSA in PBS and incubated with rabbit anti-GFP (1:800) and either rabbit anti–p-p38 MAPK (1:100) or rabbit anti–p-Smad2 (1:100) antibodies, respectively, for 60 min at room temperature. Sections were probed with goat anti-rabbit and Alexa Fluor 647 conjugate (1:2000; Molecular Probes, Eugene, OR) and mounted with Fluorescence Mounting Medium (DakoCytomation). Kidney tubular epithelial cells or endothelial cells in GFP chimeric mice were identified by the expression of E-cadherin and CD31 or vWF, respectively. Sections were incubated with rat anti–E-cadherin, rat anti-CD31, or rabbit anti-vWF for 60 min followed by goat anti-rat or goat anti-rabbit, Alexa Fluor 647 conjugate (1:2000; Molecular Probes), or Alexa Fluor 594 conjugate (1:2000; Invitrogen, Melbourne, Australia). Sections were analyzed with an Olympus Fluoview 1000 confocal microscope (Olympus, Tokyo, Japan), FV10-ASW software (version 1.3c; Olympus), oil UPLFL x60 objective (NA 1.25; Olympus) at 2x or 3x digital zoom, and step size at 0.5 µm when serial confocal microscopy analysis was applied. Contrast and brightness of the images were adjusted further in Adobe Photoshop 7.0 (Adobe Systems, Mountain View, CA).

Identification of Apoptosis
Apoptotic cells were identified using the terminal deoxynucleotidyl transferase–mediated digoxigenin-dNTP nick end-labeling (TUNEL) method (ApopTag Red In Situ Apoptosis Detection Kit; Chemicon International). Cryosections (5 µm) were incubated with working-strength terminal deoxynucleotidyl transferase enzyme at 37°C for 1 h followed by anti-digoxigenin and rhodamine conjugate at room temperature for 30 min and counterstained with DAPI (Molecular Probes). Apoptotic endothelial cells were identified by double labeling using the TUNEL method and anti-CD31 or anti-vWF antibody.

Evaluation of PTC Endothelial Cell Injury
In each sample group, 40 randomly selected microscopic fields were examined under x400 magnification for assessment of PTC changes and the number of apoptotic cells (25). PTC changes and apoptotic cell numbers were expressed per mm2 (25). The percentage of apoptosis cells in BM-derived and endogenous PTC endothelial cells was expressed as CD31+/TUNEL+/EGFP+ in total CD31+/EGFP+ or CD31+/TUNEL+/EGFP– in total CD31+/EGFP– cells, respectively.

Identification of Apoptosis with Activation of the p38 MAPK or TGF-beta1/Smad Signaling in PTC Endothelium
Six-week-old BALB/c male mice (20 to 25 g body wt) received a single intravenous injection of ADR (10.5 mg/kg). Control mice received an equivalent intravenous volume of NS. Mice were killed at 2 wk after ADR or NS injection. Kidney tissues were fixed in 4% paraformaldehyde and embedded in O.C.T. as described above. Apoptosis with activation of p38 MAPK or TGFbeta1/Smad2 signaling in PTC endothelial cells was identified by quadruple labeling using the TUNEL method (In Situ Cell Death Detection Kit, Fluorescein; Roche Applied Science, Castle Hill, Australia), anti-CD31, anti–p-p38 MAPK, or anti–p-Smad2 antibody and counterstained with DAPI. Goat anti-rat Alexa Fluor 647 conjugate (1:2000) and goat anti-rabbit Alexa Fluor 594 conjugate (1:2000) were used. Four-channel, serial confocal microscopy analysis was applied as described above.

Histologic Assessment
At week 4, kidneys from NS, ADR+vehicle, and ADR+SB+ALK5I groups mice were removed. A coronal slice was fixed in 4% paraformaldehyde and embedded in paraffin. Tissue was cut at 5 µm and stained with hematoxylin, PAS, and Masson’s trichrome. The degrees of glomerulosclerosis and interstitial fibrosis were measured (26) using Image J software (http://rsb.info.nih.gov/ij/). The percentage of glomerulosclerosis was calculated by dividing the total area of PAS-positive staining in the glomerulus by the total area of the glomerulus. Interstitial fibrosis was quantified by dividing the area of trichrome-stained interstitium by the total cortical area. The mean value of 20 randomly selected glomeruli or five cortical fields was determined for each section. Five sections were selected from each kidney.

Statistical Analyses
Data are mean ± SD with statistical analyses performed using one-way ANOVA from GraphPad Prism 3.0 (GraphPad Software, San Diego, CA) and post test Tukey analysis when appropriate. P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Conclusion
 References
 
Characteristics of PTC Injury in ADR-Induced Nephropathy
In the NS-treated group, the architecture of renal glomeruli, tubules, and interstitium was intact (Figure 1A). In comparison with the NS-treated group at week 4, the ADR-treated group showed severe glomerular and tubulointerstitial injury, glomerulosclerosis, and interstitial fibrosis (Figure 1B). Proteinuria was significantly increased by 7 d after ADR administration, peaking at 14 d and remaining at a similar level throughout the study period (Figure 1C). Elevated serum creatinine levels also were observed in ADR-injected mice and peaked at 28 d (Figure 1D).


Figure 1
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  		Figure 1. Characteristics of peritubular capillary (PTC) injury in adriamycin (ADR)-induced nephropathy. Morphologic alterations in ADR-induced nephropathy. Normal saline (NS)-treated kidney (A) and ADR-induced nephropathy kidney 28 d after ADR injection (B) were stained with periodic acid-Schiff (PAS) and counterstained with hematoxylin. (B) shows significant glomerular, tubulointerstitial pathology. ADR injection resulted in significant proteinuria (C) and increased serum creatinine (D). Morphologic alteration of PTC in ADR-induced nephropathy after bone marrow transplant (BMT). (E) CD31+ (red) PTC endothelial cells in NS-treated group. (F) Four weeks after ADR injection, there is a marked decrease in CD31+ PTC staining. The PTC that remain appear compressed and misshapen. (G) Time course of the number of CD31+ PTC lumina in ADR and NS-injected mice. Values in histograms with different letters are significantly different. Magnification, x400 in A and B, x600 in E and F.

 
CD31 immunostaining demonstrated a decreased number of CD31-positive PTC lumina associated with areas of tubulointerstitial expansion with progressive ADR nephropathy. Compared with the NS-treated group (Figure 1E), PTC in ADR-injected mice (Figure 1F) were compressed and disintegrated, particularly in areas of tubulointerstitial fibrosis. In ADR-treated mice, the number of CD31-positive PTC lumina at days 14 (706 ± 95 lumina/mm2) and 28 (399 ± 58 lumina/mm2) was significantly decreased compared with days 14 (888 ± 61 lumina/ mm2, P < 0.05) and 28 (874 ± 55 lumina/mm2, P < 0.001) in the NS-treated group, respectively (Figure 1G).

Time Course of BM-Derived PTC Endothelial Cells in ADR-Induced Nephropathy
Twelve weeks after BMT, mice received a single ADR injection. Chronic progressive glomerulosclerosis and interstitial fibrosis were evident 4 wk after ADR injection. FACS analysis showed that the proportion of EGFP-positive total blood leukocytes was 54 ± 5% (n = 8; Figure 2, A and B) in recipient mice after BMT, compared with 68 ± 5% (n = 3) in donor EGFP transgenic mice. Anti-GFP antibody immunofluorescence staining confirmed that EGFP-positive cells expressed EGFP that was detectable by immunohistochemistry (Figure 2, C through E).


Figure 2
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  		Figure 2. Confirmation of chimerism in mice that received BMT from enhanced green fluorescence (EGFP) transgenic mice. Representative FACS analysis for EGFP fluorescence of peripheral blood leukocytes from BALB/c (A) and chimeric mice 12 wk after BMT (B). (C, D, and E) An anti-GFP antibody was used to confirm the detection of EGFP+ cells in a kidney 14 d after ADR injection in EGFP chimeric mice. Magnification, x400 in C, D, and E.

 
To investigate whether BM cells can transdifferentiate into PTC endothelial cells, we examined sections from recipient kidneys at days 3, 14, and 28 after ADR injection. Co-localization of EGFP expression with the endothelial cell markers CD31 and vWF was evident from day 7. The percentage of CD31+ cells that also were EGFP+ peaked at 14 d (4.3 ± 0.8%) but had decreased to 1.5 ± 0.4% 28 d after ADR injection (Figure 3, A through E, and Supplemental Video 1). In comparison, in the NS-treated group, the percentage of CD31+/EGFP+ cells was only 0.2 to 0.4% throughout the time course of analysis (Figure 3E). The percentage of vWF+ cells that were EGFP+ also peaked at 14 d and then decreased (Figure 3, F through J, and Supplemental Video 2).


Figure 3
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  		Figure 3. Time course of BM-derived PTC endothelial cells in ADR-induced nephropathy in kidneys from 14-d ADR-injected EGFP chimeric mice. Confocal images immunolabeled with an anti-CD31 antibody (A), EGFP (B), and DAPI (C) and merged image (D). Arrows demonstrate coexpression of CD31 in an EGFP+ cell. The triple-labeled cell is shown at higher magnification in inset. (E) Quantification of BM-derived cells that expressed CD31 up to 28 d after ADR injection. Values are means ± SD. (F through I) Confocal images from kidney 14 d after ADR injection in EGFP chimeric mice labeled with anti–von Willebrand factor (anti-vWF) antibody (F), EGFP (G), and DAPI (H) and merged image (I). The triple-labeled cell in I is enlarged in inset. (J) Quantification of BM-derived cells that expressed vWF. Values are means ± SD. (K) Confocal image from kidney 14 d after ADR injection in EGFP chimeric mouse labeled with anti-CD31 antibody. A CD31+ cell that expressed EGFP is seen in a severely damaged glomerulus (arrow). This triple-labeled cell is enlarged in inset. Such glomerular cells were observed infrequently compared with those in the tubulointerstitium. Values in histograms with different letters are significantly different. Magnifications: x1200 in A through D; x3600 in D inset, I inset, and K inset; x1800 in F through I; and x600 in K.

 
We also examined glomerular nephropathy in kidneys from BMT mice at days 3, 14, and 28 after ADR injection. The number of endothelial cells was reduced in damaged glomeruli. CD31+/GFP+ cells within glomeruli were observed infrequently at these time points (Figure 3K).

Apoptosis Occurs in BM-Derived PTC Endothelial Cells in ADR-Induced Nephropathy
As described in the previous section, the percentage of CD31+/EGFP+ cells declined markedly by day 28. To study endothelial cell apoptosis, we performed TUNEL staining and anti-CD31 labeling. Apoptosis of endogenous PTC endothelial cells was observed from day 3 onward (Figure 4H). In addition, apoptosis of PTC endothelial cells that were derived from BM was observed (Figure 4, A through G, and Supplemental Videos 3 and 4). The percentage of CD31+/EGFP– and CD31+/EGFP+ PTC endothelial cells that underwent apoptosis 28 d after ADR injection was similar (Figure 4I). This indicates a similar level of apoptosis in endogenous PTC endothelial cells and BM-derived PTC endothelial cells in ADR-induced nephropathy.


Figure 4
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  		Figure 4. Apoptosis in PTC endothelial cells in ADR-induced nephropathy. (A through D) BM-derived apoptotic PTC endothelial cells, triple labeled with terminal deoxynucleotidyl transferase-mediated digoxigenin-dNTP nick end-labeling (TUNEL; A), EGFP (B), and anti-CD31 (C), were detected within PTC lumina at week 2 after ADR injection in chimeric mice kidney. Positive apoptotic cells (E) were counterstained with DAPI (F and G) nuclear staining. (H) Quantification of CD31+ and TUNEL+ cells in PTC endothelial cells. (I) Quantification of CD31+/TUNEL+ cells with or without EGFP expression within PTC lumina at week 2 after ADR injection in chimeric mice kidney, showing no significant difference. Values in histograms with different letters are significantly different. Magnification, x1200.

 
p38 MAPK and TGF-beta/Smad Signaling Pathways Are Activated in BM-Derived PTC Endothelial Cells in ADR-Induced Nephropathy
To detect whether the p38 MAPK signaling pathway is activated in BM-derived EGFP+/CD31+ cells in ADR-injected mice, we used four-channel confocal microscopic analysis to detect p-p38 in these cells. Nuclear expression, confirmed by DAPI staining, of p-p38 expression was detected in EGFP+/CD31+ cells (Figure 5, A through G). Of the total number of CD31+ cells at day 14 after ADR administration, there was no significant difference between the percentage of EGFP+/p-p38+ and EGFP–/p-p38+ endothelial cells (Figure 5H).


Figure 5
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  		Figure 5. p38 mitogen-activated protein kinase (MAPK) and TGF-beta1/Smad signaling pathway activation in BM-derived PTC endothelial cells in ADR-induced nephropathy. (A through G) BM-derived positive nuclear staining of phosphorylated p38 (p-p38) MAPK (A) and CD31+ (B) endothelial cells were detected within PTC at week 2 after ADR injection in chimeric mice kidney (arrows). The p-p38+/CD31+/EGFP+ triple-labeled cell in D is enlarged in inset. (H) Quantification of p-p38 expression in EGFP+ or EGFP–/CD31+ PTC endothelial cells. (I through O) BM-derived positive nuclear staining of phosphorylated Smad2 (p-Smad2; I) and CD31+ (J) endothelial cells were detected within PTC at week 2 after ADR injection in the kidneys of chimeric mice (arrows). (L) p-Smad2+/EGFP+/CD31+ triple-labeled cell. (O) DAPI+/p-Smad2+ double- labeled cell. (P) Quantification of p-Smad2 expression in EGFP+ or EGFP–/CD31+ PTC endothelial cells. Values with different letters are significantly different. Magnifications: 1200 in A through G and I through O; x1800 in D inset, G inset, and O inset; and x3600 in L inset.

 
This trend also was observed in the activation of the TGF-beta1/Smad pathway in CD31+ cells. p-Smad2 expression was detected in nuclei of CD31+ cells (Figure 5, I through O). There also was no significant difference in the percentage of EGFP+/p-Smad2+ and EGFP–/p-Smad2+ cells in ADR-injected mice (Figure 5P). This indicates that the p38 MAPK and TGF-beta1/Smad signaling pathways are activated in BM-derived PTC endothelial cells in ADR-induced nephropathy. Importantly, there is no difference in the fate of BM-derived PTC endothelial cells and endogenous PTC endothelial cells that express p-p38 MAPK and p-Smad2.

Activation of p38 MAPK and TGF-beta1/Smad Signaling Pathways Leads to Apoptosis in PTC Endothelial Cells
To investigate whether activation of the p38 MAPK and TGF-beta1/Smad pathways leads to apoptosis of PTC endothelial cells in ADR-induced nephropathy, we used four-channel confocal microscopy to detect TUNEL+/CD31+/p-p38 MAPK and TUNEL+/CD31+/p-Smad2+ cells in sections of 2-wk ADR-injected kidneys. Nuclear staining was confirmed by DAPI staining. No completely apoptotic PTC endothelial cells stained with p-p38 MAPK or p-Smad2. However, partially apoptotic endothelial cells that stained with p-p38 MAPK (Figure 6, A through H, and Supplemental Videos 5 and 6) or p-Smad2 (Figure 6, I through P, and Supplemental Videos 7 and 8) were observed. These cells accounted for between 0.01 and 0.03% of all PTC endothelial cells. These results suggest that activation of the p38 MAPK and TGF-beta1/Smad signaling pathways may lead to apoptosis in PTC endothelial cells in ADR-induced nephropathy.


Figure 6
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  		Figure 6. Activation of p38 MAPK and TGF-beta1/Smad signaling pathways leads to apoptosis in PTC endothelial cells. (A through H) Partial TUNEL-positive staining (A and E) together with positive nuclear staining of p-p38 MAPK (B and F) and CD31+ (G) endothelial cells were detected within PTC at week 2 after ADR injection in BALB/c mice kidney (arrows). The nuclear staining for TUNEL and p-p38 MAPK was confirmed by DAPI staining (C and D). (I through P) Partial TUNEL-positive (I and M) staining together with positive nuclear staining of p-Smad2 (J) and CD31+ (O) endothelial cells was detected within PTC at week 2 after ADR injection in BALB/c mice kidney (arrows). The nuclear staining of TUNEL and p-Smad2 was confirmed by DAPI staining (K, L, N, and P). (D and H) TUNEL+/p-p38 MAPK+/DAPI+ and TUNEL+/p-p38 MAPK+/CD31+ triple-labeled cells, respectively. (L and P) TUNEL+/p-Smad2+/DAPI+ and TUNEL+/DAPI+/ CD31+ triple-labeled cells. Magnifications: x1800 in A through O; and x4800 in D, H, L, and P inset.

 
Blocking of p38 MAPK and TGF-beta/Smad Signaling Pathways Reduces Renal Injury and Rescues BM-Derived PTC Endothelial Cells
To investigate the role of p38 MAPK and TGF-beta1/Smad signaling pathways in BM-derived PTC endothelial cells in ADR-induced nephropathy 2 wk after ADR injection, we co-administered the p38 MAPK inhibitor SB203580 and TGF-betaRI inhibitor ALK5I by osmotic pump to mice for an additional 2 wk. Compared with vehicle-treated controls, co-administration of SB203580 and ALK5I reduced ADR-induced p38 MAPK and TGF-beta1/Smad2 activation (Figure 7), glomerulosclerosis (Figure 8, A through C), and interstitial fibrosis (Figure 8, D through F). In addition, the number of PTC endothelial cells was preserved as demonstrated by vWF immunostaining (Figure 8, G and H). These mice also displayed decreased proteinuria (Figure 8I) and reduced serum creatinine levels (Figure 8J). Importantly, co-administration of SB203580 and ALK5I to ADR-injected mice reduced the percentage of BM-derived PTC endothelial cells that underwent apoptosis and preserved BM-derived PTC endothelial cells as demonstrated by an increased percentage of CD31+/EGFP+ cells in total CD31+ cells and a decreased percentage of CD31+/EGFP+/TUNEL+ cells in a total CD31+/EGFP+ compared with the ADR mice with vehicle injection (Figures 9, A through D, and 10 and Supplemental Video 9). Co-expression of vWF/CD31/EGFP in PTC endothelial cells confirmed that these cells were preserved by co-administration of SB 203580 and ALK5I (Figure 9, E through H, and Supplemental Video 10). Taken together, these data demonstrate that blocking the p38 MAPK and TGF-beta/Smad signaling pathways in mice with ADR nephropathy reduces renal injury and rescues BM-derived PTC endothelial cells from apoptosis, leading to restoration of renal function.


Figure 7
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  		Figure 7. Co-administration of SB203580 and ALK5I inhibited activation of the p38 MAPK and TGF-beta/Smad signaling pathways in ADR-induced nephropathy in chimeric mice. Quantification of p-ATF2 (A) and p-Smad2 expression in PTC endothelial cells (B). Values with different letters are significantly different.

 

Figure 8
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  		Figure 8. Blocking p38 MAPK and TGF-beta/Smad signaling pathways reduced ADR-induced injury and rescued BM-derived PTC endothelial cells in ADR-induced nephropathy. PAS (A and B) and Masson’s trichrome (D and E) sections from kidneys: ADR+vehicle (A and D) and ADR+SB203580+ALK5I mice (B and E). Quantification of glomerulosclerosis (C) and interstitial fibrosis (F). Co-administration of SB203580 and ALK5I to ADR mice preserved endogenous and BM-derived PTC endothelial cells as evidenced by coexpression of anti-vWF antibody staining (red) and EGFP (green) in chimeric mice: ADR+vehicle (G) and ADR+SB203580+ALK5I (H). Effects of SB203580 and ALK5I on proteinuria (I) and serum creatinine (J). Values with different letters are significantly different. Magnifications: x1000 in A, B, D, and E; and x600 in G and H.

 

Figure 9
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  		Figure 9. Co-administration of SB203580 and ALK5I preserved BM-derived PTC endothelial cells in ADR-treated mice. Detection of BM-derived PTC endothelial cells by anti-CD31 antibody (A) and EGFP (B) in mice that had ADR-induced nephropathy and were treated with SB203580+ALK5I. (D) The triple-labeled CD31+/EGFP+/DAPI+ cells are enlarged in inset. BM-derived PTC endothelial cells by anti-vWF antibody (E), anti-CD31 antibody (F), and EGFP (G) in ADR-injected mice that were treated with SB203580+ALK5I. (H) One of the vWF+/EGFP+/CD31+ triple-labeled cells is enlarged in inset. Magnifications: x600 in A through H, x3600 in D inset; x1200 in H inset.

 

Figure 10
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  		Figure 10. Quantification of BM-derived CD31+ PTC endothelial cells (A) and BM-derived CD31+/TUNEL+ PTC endothelial cells (B) in ADR-induced nephropathy with vehicle or combination therapy. Values with different letters are significantly different.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Conclusion
 References
 
Our study investigated PTC injury and repair in an ADR-induced nephropathy model that exhibited chronic progressive glomerulosclerosis and interstitial fibrosis. Using ADR-injected chimeric mice that received BMT and expressed EGFP, BM-derived cells were found to engraft into the PTC endothelium and coexpress vWF and CD31 in a time-dependent manner after initiation of renal injury. These findings demonstrate that the p38 MAPK and TGF-beta1/Smad signaling pathways are activated in BM-derived PTC endothelial cells that undergo apoptosis. These pathways also are activated in somatic renal cells that undergo apoptosis. Blockade of the p38 MAPK and TGF-beta1/Smad signaling pathways by co-administration of SB203580 and ALK5I to ADR-injected mice rescued BM-derived PTC endothelial cells from apoptosis, reduced the loss of PTC, and restored kidney function.

The PTC network is known to play important roles in maintaining function and hemodynamics within the kidney. The mechanism(s) that is involved in PTC endothelial cell repair has been the focus of research in various pathophysiologic settings. In recent years, increasing evidence has indicated that adult BM cells have the capability to transdifferentiate into different kinds of cells, such as cardiomyocytes (27), endothelial cells, hepatocytes (28), and neurons (29,30). Numerous studies have investigated the ability of BM-derived endothelial progenitor cells to repair injured endothelium in cardiovascular systems. In the kidney, BM cells can transdifferentiate into mesangial cells (7), podocytes (31), and endothelial cells (8), although the derivation of tubular epithelial cells from BM cells remains unclear (9,12). However, little is known about the regulation of BM-derived PTC endothelial cells during the development of chronic, progressive kidney disease. Furthermore, the fate of BM-derived PTC endothelial cells and the signaling pathways that are involved in this process have not been determined. For the successful development of cell-based therapies for patients with chronic renal disease, this information is vital for the effective timing and delivery of therapeutic intervention in a disease setting (31) in combination with factors that promote engraftment, differentiation, and cell survival.

Numerous studies in human and animal models have shown that BM-derived cells contribute to vascular repair (32–34). Many studies have confirmed that BM cells can transdifferentiate into endothelial cells (8,12,31). The frequency of this transdifferentiation from BM cells to glomerular endothelial cells can reach up to 10% in anti-Thy1 glomerulonephritis (8). However, in the present ADR nephropathy model of chronic, irreversible, progressive glomerulosclerosis and tubulointerstitial fibrosis, there was a very low frequency of BM cell transdifferentiation into glomerular endothelial cells. Our result is consistent with the study that demonstrated that none of the BM-derived cells in glomeruli express endothelial markers such as Factor VIII and rat endothelial cell antigen-1 in anti-Thy1 glomerulonephritis (7). It has been documented that in progressive glomerulonephritis, glomerular capillary regeneration rarely occurs (35). The diverse animal models that were analyzed and different endothelial cell markers that were used may account for this discrepancy in results.

PTC endothelium that is damaged by vascular rejection after renal transplantation is partially repaired by endothelial cells of the recipient in patients and in experimental animal models (10,11). BM cells have been reported to transdifferentiate into PTC endothelial cells after ischemic injury (12,16). In our study, the kidneys of ADR-treated mice showed a more than six-fold increase in the number of donor-derived PTC endothelial cells compared with NS-treated controls. This suggests that injury can enhance BM-derived PTC endothelial cell transdifferentiation. BM-derived PTC endothelial cell transdifferentiation peaked at 14 d after ADR injection and declined by 28 d. In anti–glomerular basement membrane and unilateral ureteral obstruction models, PTC regression with endothelial cell apoptosis has been demonstrated during the progression of disease (5,25). This suggests that PTC endothelial cells that are derived from BM also may undergo apoptosis. Our studies using TUNEL and CD31 coexpression confirmed that PTC apoptosis occurs in BM-derived PTC endothelial cells. There was no significant difference in the percentage of apoptotic cells between endogenous intrarenal and BM-derived PTC endothelial cells, indicating that BM-derived cells undergo pathologic changes in a fibrotic setting where ongoing inflammation is evident.

ADR-induced nephropathy is characterized by overt proteinuria, glomerular and interstitial inflammation, and fibrosis (36). Recently, Koshikawa et al. (37) demonstrated that ADR injection led to podocyte injury and p38 MAPK activation in podocytes. Treatment with FR167653, a p38 MAPK inhibitor, dramatically reduced ADR-induced podocyte injury and prevented glomerulosclerosis and renal dysfunction in the chronic phase of ADR nephropathy. ADR also can cause vascular damage directly (38). Our study demonstrated that PTC endothelial cells undergo apoptosis, peaking at day 3, with an elevated incidence continuing until day 28 after ADR injection. This is consistent with persistently overt proteinuria in ADR nephropathy. Our results support the notion that ADR not only causes endothelial injury directly but also induces proteinuria via podocyte injury, thereby damaging PTC endothelium. Blocking p38 MAPK and TGF-beta1/Smad signaling reduced proteinuria and thereby preserved PTC endothelium.

p38 MAPK and TGF-beta/Smad signaling pathways play important roles in the progression of renal disease (15,16,35,39). Activation of both of these signaling pathways can induce apoptosis (19,20,40,41). The findings from our study demonstrate that the p38 MAPK and TGF-beta/Smad signaling pathways are activated in PTC endothelial cells that are derived from BM. Moreover, no difference was observed in the percentage of p-p38 and p-Smad2 cells between endogenous intrinsic and BM-derived cells. More important, partially apoptotic endothelial cells stain with p-p38 MAPK or p-Smad2, suggesting the sequential relationship between the activation of the p38 MAPK or TGF-beta/Smad signaling pathways and apoptosis. Blocking p38 MAPK and TGF-beta/Smad signaling pathways reduced renal injury and prevented apoptosis and thereby preserved the number of PTC endothelial cells that were derived from BM. This suggests that in the ADR model of chronic progressive renal disease, kidney function determines the fate of BM-derived PTC endothelial cells. This is consistent with a recent study that demonstrated that endothelial progenitor cell number in renal transplant recipients is determined by kidney graft function (12) and that uremia causes endothelial progenitor cell deficiency (13). Recently, Seeger et al. (42) demonstrated that p38 MAPK plays a pivotal role in regulating the number of endothelial progenitor cells ex vivo. They observed that SB203580 can reduce the negative effects of TNF-{alpha} and glucose on endothelial progenitor cell survival. In our study, blocking p38 MAPK and TGF-beta1/Smad signaling pathways in ADR-injected mice preserved the number of PTC endothelial cells that were derived from BM. This indicates that blocking p38 MAPK and TGF-beta1/Smad signaling pathways may prevent the negative effects of activation of p38 MAPK and TGF-beta1/Smad signaling pathways in ADR-induced nephropathy and increase the number of BM-derived PTC endothelial cells.

de Groot et al. (13) demonstrated that the function of the grafted kidney determines the number of endothelial progenitor cells, and endothelial progenitor cells are deficient in patients with uremia (14). In our study, it is not clear whether inhibition of p38 MAPK and TGF-betaRI has a direct or an indirect effect leading to increased numbers of BM-derived PTC endothelial cells that are evident with improved renal structure and functional recovery in ADR-induced nephropathy. It cannot be ruled out that SB203580 and ALK5I may have direct effects on the number of BM-derived endothelial progenitor cells.

Some studies have suggested that the process of cell–cell fusion rather than cell transdifferentiation may contribute to BM-derived Purkinje neurons, liver hepatocytes, and cardiac myocytes (43–47). However, cell–cell fusion events are observed to occur at a very low frequency (<1% of cardiomyocytes and <0.1% of hepatocytes or Purkinje cells [43]). In our study, the percentage of BM-derived CD31+ cells was estimated to be 4.3% in 2-wk ADR-injected mice, compared with 0.2 to 0.4% in the NS-injected mice. This suggests that it is unlikely that BM cell–cell fusion is the major contributor to tissue regeneration in the kidney.


   Conclusion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
References
 
This study demonstrates that p38 MAPK and TGF-beta1/Smad signaling pathways are activated in BM-derived PTC endothelial cells in the ADR model of chronic, progressive renal disease. In chimeric mice, BM-derived PTC endothelial cells were found to undergo apoptosis via the same pathways as intrinsic renal somatic cells. Blocking the p38 MAPK and TGF-beta1/Smad signaling pathways in ADR-injected mice rescued intrinsic and BM-derived PTC endothelial cells from apoptosis, increased the number of intrinsic and BM-derived PTC endothelial cells, reduced the loss of PTC, and ameliorated renal injury, leading to restoration of renal function. The study provides insights into the regulation of the fate of BM cells during renal injury that may improve the efficiency of cell-based therapy for the treatment of chronic, progressive kidney disease.


   Acknowledgments
 
These studies were supported by a Kidney Health Australia Bootle Bequest. We acknowledge the members of the Renal Regeneration Consortium for their support. J.L. is the recipient of a Chinese Government Scholarship for Outstanding Oversees Students and a Monash Graduate Scholarship.

Confocal imaging was performed at the Monash MicroImaging facility at Monash University.


   Footnotes
 
Published online ahead of print. Publication date available at www.jasn.org.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 References
 

  1. Bohle A, Mackensen-Haen S, Wehrmann M: Significance of postglomerular capillaries in the pathogenesis of chronic renal failure. Kidney Blood Press Res 19 : 191 –195, 1966[Medline]
  2. Ridson RA, Sloper JC, Wardener HE: Relationship between renal function and histological changes found in renal biopsy specimens from patients with persistent glomerular nephritis. Lancet 2 : 363 –366, 1968[Medline]
  3. Striker GE, Schainuck LI, Cutler RE, Benditt EP: Structural-functional correlations in renal disease. I. A method for analyzing and classifying histopathological changes in renal disease. Hum Pathol 1 : 615 –630, 1970[Medline]
  4. Schainuck LI, Striker GE, Cutler RE, Benditt EP: Structural-functional correlations in renal disease. II. The correlations. Hum Pathol 1 : 631 –641, 1970[Medline]
  5. Ohashi R, Kitamura H, Yamanaka N: Peritubular capillary injury during the progression of experimental glomerulonephritis in rats. J Am Soc Nephrol 11 : 47 –56, 2000[Abstract/Free Full Text]
  6. Nangaku M: Chronic hypoxia and tubulointerstitial injury: A final common pathway to end-stage renal failure. J Am Soc Nephrol 17 : 17 –25, 2006[Abstract/Free Full Text]
  7. Ito T, Suzuki A, Imai E, Okabe M, Hori M: Bone marrow is a reservoir of repopulating mesangial cells during glomerular remodeling. J Am Soc Nephrol 12 : 2625 –2635, 2001[Abstract/Free Full Text]
  8. Rookmaaker MB, Smits AM, Tolboom H, Van ’t Wout K, Martens AC, Goldschmeding R, Joles JA, Van Zonneveld AJ, Grone HJ, Rabelink TJ, Verhaar MC: Bone-marrow-derived cells contribute to glomerular endothelial repair in experimental glomerulonephritis. Am J Pathol 163 : 553 –562, 2003[Abstract/Free Full Text]
  9. Lin F, Moran A, Igarashi P: Intrarenal cells, not bone marrow-derived cells, are the major source for regeneration in postischemic kidney. J Clin Invest 115 : 1756 –1764, 2005[CrossRef][Medline]
  10. Lagaaij EL, Cramer-Knijnenburg GF, van Kemenade FJ, van Es LA, Bruijn JA, van Krieken JH: Endothelial cell chimerism after renal transplantation and vascular rejection. Lancet 357 : 33 –37, 2001[CrossRef][Medline]
  11. Xu W, Baelde HJ, Lagaaij EL, De Heer E, Paul LC, Bruijn JA: Endothelial cell chimerism after renal transplantation in a rat model. Transplantation 74 : 1316 –1320, 2002[CrossRef][Medline]
  12. Duffield JS, Park KM, Hsiao LL, Kelley VR, Scadden DT, Ichimura T, Bonventre JV: Restoration of tubular epithelial cells during repair of the postischemic kidney occurs independently of bone marrow-derived stem cells. J Clin Invest 115 : 1743 –1755, 2005[CrossRef][Medline]
  13. de Groot K, Bahlmann FH, Bahlmann E, Menne J, Haller H, Fliser D: Kidney graft function determines endothelial progenitor cell number in renal transplant recipients. Transplantation 79 : 941 –945, 2005[CrossRef][Medline]
  14. de Groot K, Bahlmann FH, Sowa J, Koenig J, Menne J, Haller H, Fliser D: Uremia causes endothelial progenitor cell deficiency. Kidney Int 66 : 641 –646, 2004[CrossRef][Medline]
  15. Adhikary L, Chow F, Nikolic-Paterson DJ, Stambe C, Dowling J, Atkins RC, Tesch GH: Abnormal p38 mitogen-activated protein kinase signalling in human and experimental diabetic nephropathy. Diabetologia 47 : 1210 –1222, 2004[Medline]
  16. Stambe C, Atkins RC, Tesch GH, Masaki T, Schreiner GF, Nikolic-Paterson DJ: The role of p38alpha mitogen-activated protein kinase activation in renal fibrosis. J Am Soc Nephrol 15 : 370 –379, 2004[Abstract/Free Full Text]
  17. Hong SW, Isono M, Chen S, Iglesias-De La Cruz MC, Han DC, Ziyadeh FN: Increased glomerular and tubular expression of transforming growth factor-beta1, its type II receptor, and activation of the Smad signaling pathway in the db/db mouse. Am J Pathol 158 : 1653 –1663, 2001[Abstract/Free Full Text]
  18. Zeisberg M, Hanai J, Sugimoto H, Mammoto T, Charytan D, Strutz F, Kalluri R: BMP-7 counteracts TGF-beta1-induced epithelial-to-mesenchymal transition and reverses chronic renal injury. Nat Med 9 : 964 –968, 2003[CrossRef][Medline]
  19. Yilmaz A, Kliche S, Mayr-Beyrle U, Fellbrich G, Waltenberger J: p38 MAPK inhibition is critically involved in VEGFR-2-mediated endothelial cell survival. Biochem Biophys Res Commun 306 : 730 –736, 2003[CrossRef][Medline]
  20. Nakagami H, Morishita R, Yamamoto K, Yoshimura SI, Taniyama Y, Aoki M, Matsubara H, Kim S, Kaneda Y, Ogihara T: Phosphorylation of p38 mitogen-activated protein kinase downstream of bax-caspase-3 pathway leads to cell death induced by high D-glucose in human endothelial cells. Diabetes 50 : 1472 –1481, 2001[Abstract/Free Full Text]
  21. Yue TL, Ni J, Romanic AM, Gu JL, Keller P, Wang C, Kumar S, Yu GL, Hart TK, Wang X, Xia Z, DeWolf WE, Feuerstein GZ: TL1, a novel tumor necrosis factor-like cytokine, induces apoptosis in endothelial cells. Involvement of activation of stress protein kinases (stress-activated protein kinase and p38 mitogen-activated protein kinase) caspase-3-like protease. J Biol Chem 274 : 1479 –1486, 1999[Abstract/Free Full Text]
  22. Solovyan VT, Keski-Oja J: Apoptosis of human endothelial cells is accompanied by proteolytic processing of latent TGF-beta binding proteins and activation of TGF-beta. Cell Death Differ 12 : 815 –826, 2005[CrossRef][Medline]
  23. Hyman KM, Seghezzi G, Pintucci G, Stellari G, Kim JH, Grossi EA, Galloway AC, Mignatti P: Transforming growth factor-beta1 induces apoptosis in vascular endothelial cells by activation of mitogen-activated protein kinase. Surgery 132 : 173 –179, 2002[CrossRef][Medline]
  24. Hadjantonakis AK, Gertsenstein M, Ikawa M, Okabe M, Nagy A: Generating green fluorescent mice by germline transmission of green fluorescent ES cells. Mech Dev 76 : 79 –90, 1998[CrossRef][Medline]
  25. Ohashi R, Shimizu A, Masuda Y, Kitamura H, Ishizaki M, Sugisaki Y, Yamanaka N: Peritubular capillary regression during the progression of experimental obstructive nephropathy. J Am Soc Nephrol 13 : 1795 –1805, 2002[Abstract/Free Full Text]
  26. Vleming LJ, Baelde JJ, Westendorp RG, Daha MR, van Es LA, Bruijn JA: The glomerular deposition of PAS positive material correlates with renal function in human kidney diseases. Clin Nephrol 47 : 158 –167, 1997[Medline]
  27. Makino S, Fukuda K, Miyoshi S, Konishi F, Kodama H, Pan J, Sano M, Takahashi T, Hori S, Abe H, Hata J, Umezawa A, Ogawa S: Cardiomyocytes can be generated from marrow stromal cells in vitro. J Clin Invest 103 : 697 –705, 1999[Medline]
  28. Mitchell C, Fausto N: Bone marrow-derived hepatocytes: Rare but promising. Am J Pathol 161 : 349 –350, 2002[Free Full Text]
  29. Mezey E, Chandross KJ, Harta G, Maki RA, McKercher SR: Turning blood into brain: Cells bearing neuronal antigens generated in vivo from bone marrow. Science 290 : 1779 –1782, 2000[Abstract/Free Full Text]
  30. Brazelton TR, Rossi FM, Keshet GI, Blau HM: From marrow to brain: Expression of neuronal phenotypes in adult mice. Science 290 : 1775 –1779, 2000[Abstract/Free Full Text]
  31. Poulsom R, Forbes SJ, Hodivala-Dilke K, Ryan E, Wyles S, Navaratnarasah S, Jeffery R, Hunt T, Alison M, Cook T, Pusey C, Wright NA: Bone marrow contributes to renal parenchymal turnover and regeneration. J Pathol 195 : 229 –235, 2001[CrossRef][Medline]
  32. Takahashi T, Kalka C, Masuda H, Chen D, Silver M, Kearney M, Magner M, Isner JM, Asahara T: Ischemia- and cytokine-induced mobilization of bone marrow-derived endothelial progenitor cells for neovascularization. Nat Med 5 : 434 –438, 1999[CrossRef][Medline]
  33. Kawamoto A, Gwon HC, Iwaguro H, Yamaguchi JI, Uchida S, Masuda H, Silver M, Ma H, Kearney M, Isner JM, Asahara T: Therapeutic potential of ex vivo expanded endothelial progenitor cells for myocardial ischemia. Circulation 103 : 634 –637, 2001[Abstract/Free Full Text]
  34. Kocher AA, Schuster MD, Szabolcs MJ, Takuma S, Burkhoff D, Wang J, Homma S, Edwards NM, Itescu S: Neovascularization of ischemic myocardium by human bone-marrow-derived angioblasts prevents cardiomyocyte apoptosis, reduces remodeling and improves cardiac function. Nat Med 7 : 430 –436, 2001[CrossRef][Medline]
  35. Shimizu A, Kitamura H, Masuda Y, Ishizaki M, Sugisaki Y, Yamanaka N: Rare glomerular capillary regeneration and subsequent capillary regression with endothelial cell apoptosis in progressive glomerulonephritis. Am J Pathol 151 : 1231 –1239, 1997[Abstract]
  36. Wang Y, Wang YP, Tay YC, Harris DC: Progressive Adriamycin nephropathy in mice: Sequence of histologic and immunohistochemical events. Kidney Int 58 : 1797 –1804, 2000[CrossRef][Medline]
  37. Koshikawa M, Mukoyama M, Mori K, Suganami T, Sawai K, Yoshioka T, Nagae T, Yokoi H, Kawachi H, Shimizu F, Sugawara A, Nakao K: Role of p38 mitogen-activated protein kinase activation in podocyte injury and proteinuria in experimental nephrotic syndrome. J Am Soc Nephrol 16 : 2690 –2701, 2005[Abstract/Free Full Text]
  38. Pastorino F, Brignole C, Marimpietri D, Cilli M, Gambini C, Ribatti D, Longhi R, Allen TM, Corti A, Ponzoni M: Vascular damage and anti-angiogenic effects of tumor vessel-targeted liposomal chemotherapy. Cancer Res 63 : 7400 –7409, 2003[Abstract/Free Full Text]
  39. Choi ME: Mechanism of transforming growth factor-beta1 signaling. Kidney Int Suppl 77 : S53 –S58, 2000[CrossRef][Medline]
  40. Bottinger EP, Bitzer M: TGF-beta signaling in renal disease. J Am Soc Nephrol 13 : 2600 –2610, 2002[Abstract/Free Full Text]
  41. Schiffer M, Bitzer M, Roberts IS, Kopp JB, ten Dijke P, Mundel P, Bottinger EP: Apoptosis in podocytes induced by TGF-beta and Smad7. J Clin Invest 108 : 807 –816, 2001[CrossRef][Medline]
  42. Seeger FH, Haendeler J, Walter DH, Rochwalsky U, Reinhold J, Urbich C, Rossig L, Corbaz A, Chvatchko Y, Zeiher AM, Dimmeler S: p38 mitogen-activated protein kinase downregulates endothelial progenitor cells. Circulation 111 : 1184 –1191, 2005[Abstract/Free Full Text]
  43. Alvarez-Dolado M, Pardal R, Garcia-Verdugo JM, Fike JR, Lee HO, Pfeffer K Lois C, Morrison SJ, Alvarez-Buylla A: Fusion of bone-marrow-derived cells with Purkinje neurons, cardiomyocytes and hepatocytes. Nature 425 : 968 –973, 2003[CrossRef][Medline]
  44. Vassilopoulos G, Wang PR, Russell DW: Transplanted bone marrow regenerates liver by cell fusion. Nature 422 : 901 –904, 2003[CrossRef][Medline]
  45. Wang X, Willenbring H, Akkari Y, Torimaru Y, Foster M, Al-Dhalimy M, Lagasse E, Finegold M, Olson S, Grompe M: Cell fusion is the principal source of bone-marrow-derived hepatocytes. Nature 422 : 897 –901, 2003[CrossRef][Medline]
  46. Weimann JM, Johansson CB, Trejo A, Blau HM: Stable reprogrammed heterokaryons form spontaneously in Purkinje neurons after bone marrow transplant. Nat Cell Biol 5 : 959 –966, 2003[CrossRef][Medline]
  47. Wagers AJ, Weissman IL: Plasticity of adult stem cells. Cell 116 : 639 –648, 2004[CrossRef][Medline]



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