Endothelial Progenitor Cells Derived from Wharton's Jelly of Human Umbilical Cord Attenuate Ischemic Acute Kidney Injury by Increasing Vascularization and Decreasing Apoptosis,Inflammation,and Fibrosis

Isolation and Culture of EPCs

This study was approved by the Institutional Review Board of the National Taiwan University Hospital, Taipei, Taiwan. With the consent of the parents, fresh human umbilical cords (n = 10, male fetus: six, female fetus: four) were obtained after birth and stored in Hank's balanced salt solution (Life Technologies, Grand Island, NY, USA) for 1–24 h before processing to obtain MSCs, which were differentiated into EPCs. Wharton's jelly from umbilical cords was diced into cubes of approximately 0.5 cm³ in serum-free Dulbecco's modified Eagle medium (SF-DMEM; Gibco, Grand Island, NY, USA) and centrifuged at 500 × g for 5 min at room temperature (RT). After removal of the supernatant, the pellet (mesenchymal cells) was suspended in collagenase type I (0.2 mg/ml; Sigma-Aldrich, St. Louis, MO, USA) in SF-DMEM and the suspension incubated for 24 h at 37°C. Cells were then centrifuged as above, and the cells washed with SF-DMEM, then suspended in 0.2% trypsin (Life Technologies) in SF-DMEM. The suspension incubated for 30 min at 37°C with agitation. After centrifugation at 250 × g for 5 min, the cells were maintained in endothelial basal growth medium (EGM-2; Lonza Allendale, NJ, USA) supplemented with hydrocortisone, human epidermal growth factor, vascular endothelial growth factor, basic human fibroblast growth factor, human recombinant analog of insulin-like growth factor with substitution of Arg for Glu at position 3, ascorbic acid, heparin, and gentamicin/amphotericin B (all from Lonza) and 20% fetal bovine serum (Biological Industries, Kibbutz Beit Haemek, Israel). The medium was changed every 3 days for 15 days until completion of differentiation had been established by morphology; then the cells were used for immunocytochemical staining and transplantation studies. The cells were characterized as endothelial cell-like by immunocytochemistry, flow cytometry, and Western blotting using antibodies directed against the endothelial cell markers CD34 (BD Pharmingen, CA, USA), kinase insert domain receptor (KDR; Abcam, Cambridge, UK), von Willebrand factor (vWF; Neomarkers, Fremont, CA, USA), CD31, and thrombomodulin (TM; both from Santa Cruz, Santa Cruz, CA, USA), and the absence of the lymphocytic marker CD45 (Santa Cruz). To verify the endothelial-like phenotype, uptake of 1,1′-dioctadecyl 3,3,3′,3′-tetramethylindo-carbocyanine (DiI)-labeled acetylated low-density lipoprotein (LDL), a function of endothelial cells, was measured by incubating the cells for 4 h at 37°C with 10 μg/ml of DiI-acetylated LDL (Invitrogen, Grand Island, NY, USA) and examining them by fluorescence microscopy.

Renal Ischemia–Reperfusion (I/R) Model and WJ-EPC Transplantation

This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, and the protocol was approved by the Committee on the Ethics of Animal Experiments of the National Taiwan University. The animals were housed at a constant temperature and humidity, with a 12:12-h light–dark cycle, and had unrestricted access to a standard diet and tap water. All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering. Male 8-week-old C57BL/6 (n = 8/per group; National Taiwan University, Taipei, Taiwan) mice were used, and the renal I/R model was established as described previously (28, 45), with minor modifications. Briefly, anesthesia was induced with sodium pentobarbital (60 mg/kg body weight IP; Sigma-Aldrich); then, following a midline abdominal incision, right nephrectomy was performed and the left renal pedicle clamped for 40 min using a microvascular clamp (Karl Klappenecker, Tuttlingen-Nendingen, Germany). After inspection for signs of ischemia, the mouse was placed on a heating pad to maintain a stable intraperitoneal temperature of 37°C throughout the subsequent stages. After removal of the clamp, restoration of blood flow was confirmed visually. Sham controls underwent the same surgical procedure, but without vascular occlusion and are hereafter referred to as non-I/R controls.

EPCs to be used for transplantation were labeled with the red fluorescent nanoparticle dye Q-tracker (Invitrogen) according to the manufacturer's instructions (3). Q-tracker-labeled WJ-EPCs (5 × 10⁵) or saline were injected into the renal subcapsular space using a Hamilton syringe. The incision was then sutured with 5-0 silk (UNIK, Taipei, Taiwan), and the animals were allowed to recover.

Specimen Collection, Morphometric Analysis, and Immunohistochemical Examination of Vascular Density and Inflammation

Mice were killed by intraperitoneal injection of an overdose of sodium pentobarbital at 12 h or 1, 2, or 7 days after cell transplantation, and the left kidney was carefully excised, postfixed overnight at 4°C in 4% paraformaldehyde (Bionovas, Ontario, Canada), and paraffin-embedded.

Morphometric changes were examined on sections of renal tissues stained with hematoxylin and eosin (Sigma-Aldrich). The slides (n = 3 for each kidney) were coded, and renal histopathological changes were evaluated using previously described criteria (10). The three morphological indices used were tubular necrosis, absence of nuclei in tubular cells, and tubular dilation; changes were graded on a 0–2 scale in a double-blind fashion.

In the immunohistochemical studies, all antibodies were diluted in 10% normal goat serum (Gibco) in phosphate-buffered saline (PBS-NGS). For histological analysis of vascularization, paraffin sections (5 μm) were incubated for 16 h at 4°C with a 1:50 dilution of anti-CD31 antibody (Abcam), then for 90 min at RT with a 1:200 dilution of biotinylated goat anti-rabbit IgG antibodies (Vector Labs, Cambridgeshire, UK), followed by development with DAB (Sigma-Aldrich). Five fields from each tissue section were randomly selected and the number of microvessels counted. To detect an inflammatory response, the sections were incubated at 4°C overnight with a 1:100 dilution of polyclonal goat anti-mouse macrophage inflammatory protein (MIP-2) antibody (Santa Cruz) or a 1:50 dilution of polyclonal rabbit anti-mouse keratinocyte-derived chemokine (KC) antibody (BioVision, Milpitas, CA, USA). Sections were then incubated for 90 min at RT with a 1:200 dilution of biotinylated-conjugated goat anti-rabbit IgG antibody. To identify transplanted WJ-EPCs in the kidney, samples were incubated overnight at 4°C with a 1:100 dilution of polyclonal sheep anti-human b2-microglobulin antibody (Biogenesis, Berkshire, UK), then for 90 min at RT with a 1:200 dilution of FITC-conjugated goat anti-sheep IgG antibody (Jackson ImmunoResearch, West Grove, PA, USA). To estimate the amount of fibrosis, sections were incubated overnight at 4°C with a 1:100 dilution of polyclonal rabbit antibody against human S100A4 (Dako Cytomation, Carpinteria, CA, USA), a fibroblast marker (6), or were subjected to Jones silver staining (5% silver nitrate in 3% aqueous methenamine, Sigma), a method detecting the amount of extracellular matrix production (6). The fibrotic score was assigned as 0, no fibrosis; 1, mild fibrosis; 2, moderate fibrosis; and 3, severe fibrosis.

Evaluation of Renal Function

To provide evidence of EPC-induced changes in renal function, serum creatinine and blood urea nitrogen (BUN) levels were measured using, respectively, a colorimetric or a modified urease-Berthelot method using commercial kits (Randox Laboratories, Antrim, UK). Blood samples were collected by eye bleeding at 12 h, 1 day, 2 days, and 7 days after cell transplantation.

Identification of Apoptotic Cells by TUNEL Staining

Apoptotic cells were identified by terminal dUTP nick-end labeling (TUNEL) using a commercial kit (In Situ Cell Death Detection Kit; Roche, Indianapolis, IN, USA) according to the manufacturer's protocol. The sections were counterstained with hematoxylin to visualize all nuclei. The percentage of TUNEL-positive cells was calculated in six randomly nonoverlapping regions of the renal cross section in a high power field.

Analysis of Protein Expression in Renal Tissues

Renal tissues were homogenized in lysis buffer [20 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 1 mM PMSF, pH 7.4, containing protease inhibitors (Cell Signaling, Danvers, MA, USA)], then the homogenate was centrifuged at 11,800 × g for 30 min at 4°C and the supernatant retained. The protein content of the supernatant was measured using a Bio-Rad protein assay. An aliquot of the supernatant (20 μg/ml of protein) was subjected to 10% SDS-PAGE [30% acrylamide, 1.5 M Tris-HCl (pH 8.8), 10% sodium dodecyl sulfate, 10% ammonium persulfate, 0.04% tetramethylethylene-diamine; Sigma-Aldrich] and the proteins transferred onto PVDF membranes (Millipore, Danvers, MA, USA). The membranes were incubated overnight at 4°C with a 1:1,000 dilution in Tris-buffered saline (0.2% Tween 20, 1.36 M NaCl, 200 mM Tris-base) of polyclonal mouse antibodies against human CD31 (Santa Cruz), CD34 (BD Pharmingen), KDR (Abcam), vWF (Neomarkers), p53 upregulated modulator of apoptosis (PUMA) (Gene Tex, Irvine, CA, USA), Bcl-2 (Gene Tex), or polyclonal rabbit antibodies against human TM or CD45 (both from Santa Cruz). Membranes were then incubated for 1 h at RT with a 1:2,000 dilution in TBST of horseradish peroxidase (HRP)-conjugated goat anti-rabbit/mouse IgG antibodies (Sigma-Aldrich), and bound antibody detected using Chemiluminescence Reagent Plus (Millipore, Danvers, MA, USA). The intensity of each band was quantified using a densitometer. GAPDH, used as the internal control, was detected using a rabbit anti-GAPDH antibody (1:3,000; Sigma-Aldrich) and an HRP-conjugated goat anti-rabbit IgG antibody (1:1,000 dilution).

Measurement of Reactive Oxygen Species (ROS)

The kidney was rapidly isolated, washed twice with PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄.2H₂O, 2 mM KH₂PO₄; Sigma-Aldrich) and placed in a sucrose (Sigma-Aldrich) concentration gradient before cryosectioning into 8-μm frozen sections. Sections were incubated at 37°C for 30 min with the fluorescent dye dihydroethidium (DHE) (1 μM; Sigma-Aldrich), washed with PBS, and observed under a fluorescence microscope.

Mouse Cytokine Array

To measure the expression of cytokines induced by I/R injury and the effects of WJ-EPCs, a commercially available antibody array for the detection of 32 cytokines was used (RayBiotech, Norcross, GA, USA) following the manufacturer's instructions. Briefly, decapsulated kidney tissues were minced at 4°C in RayBio cell lysis buffer (RayBiotech), then equal amounts of total protein (250 μg) were incubated overnight at 4°C with the mouse cytokine antibody array membrane. The membrane was washed, incubated sequentially with the biotinylated anti cytokine antibody mixture and HRP-conjugated streptavidin, and bound antibody was detected with Chemiluminescence Reagent Plus (NEN) and a UVP Biospectrum AC system.

Measurement of KC Levels in Renal Tissues

KC levels in renal tissue homogenates were measured using an ELISA kit specific for human KC (BD Biosciences, Franklin Lakes, NJ, USA). The absorbance at 450 nm was read on an ELISA reader.

Statistical Analysis

Graph Pad Prism 5 (Graph Pad, La Jolla, CA, USA) was used to analyze quantitative data. Where applicable, the results are presented as the mean ± SEM. Statistical analysis was performed by ANOVA, followed by Duncan's multiple comparison posttest. A value of p < 0.05 was considered statistically significant.

EPC Transplantation Restores Renal Function in Mice with I/R Injury

To investigate the potential of umbilical cord MSCs to differentiate into endothelial cells, the pelleted cells after centrifugation of freshly prepared tissue pieces were cultured in EGM-2 medium. Under these culture conditions, many EPC colonies were present, and the cells showed the cobblestone-like morphology characteristic of an endothelial cell monolayer. These cells were capable of taking up DiI-acetylated LDL particles and were assembled into vascular tubelike structures. In contrast, MSCs did not show the phenomena. When EPC characterization was further performed by immunostaining, the majority of the cells expressed the EPC markers CD34 and KDR and the mature endothelial marker CD31. Flow cytometric analysis confirmed that the cells expressed high levels of all three markers. Both EPCs and MSCs did not express CD45, the leukocyte common antigen. The percentage of EPCs within total MSCs was 20 ± 3%. The detailed results are provided as an online supplement (https://drive.google.com/?tab=wo&authuser=0#folders/0B5EVZau1-kredkdBdVMyTGxuRTQ).

To trace the distribution of transplanted EPCs in the kidney, Q-tracker fluorescent nanoparticles were used to label the cells before transplantation. The Q-tracker particles were homogenously distributed in the WJ-EPC cytoplasm, as shown by fluorescence microscopy (Fig. 1A). To determine the effects of EPC transplantation on renal function in I/R injury in mice, WJ-EPCs (5 × 10⁵) or saline were injected into the subcapsular space of the left kidney; then the location of the transplanted cells was visualized by Q-tracker and immunostaining for the endothelial cell marker CD31. At 12 h after transplantation, the transplanted cells were only found in the capsular space and the superficial part of the cortex (Fig. 1B, C), whereas they were also detected in the renal cortex at days 1 and 2 and in the cortex and medulla at day 7 (Fig. 1C). EPC transplantation resulted in a significant decrease in serum levels of creatinine (0.8 ± 0.1 mg/dl) and BUN (105.2 ± 16.6 mg/dl) compared saline-treated mice (2.2 ± 0.2 mg/dl for creatinine and 139.5 ± 24.2 mg/dl for BUN; *p < 0.05 vs. I/R + saline group) (Fig. 1D). These results showed that EPCs could move from the renal capsule to the cortex, then to the medulla, and attenuate the renal dysfunctions seen with I/R injury.

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Figure 1.

Transplanted WJ-EPCs move from the renal capsule to the medulla and restore renal function following renal ischemia-reperfusion (I/R) injury. (A) Q-tracker particles are homogenously distributed in the cytoplasm of cultured WJ-EPCs. Scale bar: 25 μm. (B) At 12 h after transplantation, WJ-EPCs are seen in the renal capsule and the superficial part of the renal cortex. The Q-tracker-labeled WJ-EPCs express β2-microglobulin and CD31. Scale bar: 50 μm. (C) Q-tracker-labeled WJ-EPCs move from the renal capsule to the renal medulla in a time-dependent manner. The arrows indicate transplanted WJ-EPCs in the renal capsule (left), cortex (center), and medulla (right). Scale bar: 50 μm. (D) Serum creatinine and BUN levels in the three groups; the data are expressed as the mean+SEM (n = 8). *p<0.05 versus I/R + saline group.

EPC Transplantation Improves Renal Morphology in Mice with I/R Injury

To determine what factors could account for the increased renal function seen after EPC transplantation, we examined kidney morphology using hematoxylin and eosin staining. Compared to the sham-operated group, the I/R + saline group showed dramatic damage to the tissue morphology at 12 h or 1, 2, or 7 days after I/R injury (Fig. 2A), which included tubular necrosis, absence of nuclei, and tubular dilation (Fig. 2B). I/R-induced injury was significantly decreased by EPC treatment (Fig. 2A). To quantify I/R-induced kidney injury, we determined the tissue injury score using three morphological indices (tubular necrosis, absence of nuclei in tubular cells, and tubular dilation) of acute kidney failure in a double-blind manner. As shown in Figure 2C, at 12 h after induction of I/R injury, saline-treated mice showed a significant increase in the renal injury score, and this was significantly reduced by EPC transplantation (*p < 0.05 vs. I/R + saline group). These results show that EPC transplantation reduces I/R-induced renal injury in mice.

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Figure 2.

WJ-EPC transplantation ameliorates tissue injury after I/R injury. (A) Representative H&E-stained images of renal tissues from the sham-operated, I/R + saline, I/R + EPC groups at 12 h, 1, 2, or 7 days after I/R injury. Scale bar: 50 μm. (B) High magnification images showing tubular necrosis, absence of nuclei, and tubular dilation in the I/R + saline group at 12 h. Scale bar: 25 μm. (C) Semiquantitative analysis of tissue damage at 12 h after I/R injury [total injury score based on tubular necrosis (0–2), absence of nuclei in tubular cells (0–2), and tubular dilation (0–2)]. The data are presented as the mean ± SEM (n = 5). *p < 0.05 versus I/R + saline group.

EPC Transplantation Increases Microvascular Density in Mice with I/R Injury

Since a previous study demonstrated that renal microvascular injury is a critical factor in the development of kidney injury (5), we examined the effects of EPCs on the microvascular density using CD31 immunostaining. As shown in Figure 3A and B, I/R injury caused a dramatic decrease in microvessel density in the I/R + saline group compared to the sham group over the 7 days, and this was completely prevented by WJ-EPC transplantation (*p < 0.05 vs. I/R + saline group). In addition, some of the transplanted WJ-EPCs appeared to surround, and be incorporated into, microvascular structures at day 7 (Fig. 3C).

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Figure 3.

WJ-EPC transplantation inhibits the decrease in microvascular density in mice with I/R injury. (A) Kidney sections from the sham-operated, I/R + saline, and I/R + EPC groups stained with anti-CD31 antibody at 1 day after transplantation; the arrows indicate CD31-positive cells. Scale bar: 50 μm. (B) Graph showing microvascular density per high power field in the three groups at 12 h or 1, 2, or 7 days (anti-CD31 antibody immunostaining); the values are presented as the mean ± SEM (n = 5). *p < 0.05 versus I/R + saline group. (C) On day 7, transplanted WJ-EPCs (arrows) surround, and are incorporated into, the microvascular structure (arrows). Serial sections of kidney from the I/R + EPC group stained with H&E (left) and showing Q-tracker-positive cells (right, arrows). Scale bar: 25 μm.

EPC Transplantation Decreases Cell Apoptosis and ROS Production

Since we found that EPC transplantation reduced I/R-induced injury, we investigated whether this was due to decreased cell apoptosis. As shown in Figure 4A, little TUNEL staining was seen in specimens from the sham-operated group, but numerous apoptotic cells were observed in the I/R + saline group and, as shown on serial sections in Figure 4B, the apoptotic cells colocalized with renal tubular cells, endothelial cells, and glomerular cells. EPC transplantation dramatically decreased the number of apoptotic cells in the ischemic kidney (Fig. 4A). The quantitative analysis in Figure 4C showed that, at 12 h, the percentage of apoptotic cells was increased from 0.3± 0.1% in the sham-operated animals to 11.7 ± 3.2% in the I/R + saline group, but to only 2.8 ± 1.2% with WJ-EPC transplantation. The percentage of apoptotic cells was highest at 12 h after I/R injury. Moreover, as shown in Figure 4D, Western blotting demonstrated that, at 12 h, expression of PUMA, a proapoptotic protein, in the I/R kidney was increased, while Bcl-2, an antiapoptotic protein, was decreased. Both effects were significantly reduced in the EPC-treated I/R group (*p < 0.05 vs. I/R + saline group). These results show that EPC implantation prevented I/R-induced cell apoptosis.

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Figure 4.

WJ-EPC transplantation reduces cell apoptosis following I/R injury. (A) Detection of apoptosis (arrows) by TUNEL staining (upper) or H&E staining (lower) in the sham-operated, I/R + saline, and I/R + EPC groups at the indicated times. Scale bar: 100 μm. (B) Overlap of TUNEL-positive cells (arrows) and renal tubular cells, endothelial cells, or glomerular cells (H&E staining) in serial sections at 12 h after transplantation. Scale bar: 25 μm. (C) Quantitative analysis of apoptotic cells at 12 h or 1, 2, or 7 days after transplantation by TUNEL staining expressed as a percentage of the total cell number. (D) Expression of PUMA and Bcl2 at 12 h after I/R injury in renal tissues from the sham-operated, I/R + saline, and I/R + EPC groups determined by Western blotting. In C and D, n = 5 per group. *p < 0.05 for I/R + EPC versus I/R + saline.

To determine whether ROS contributed to the cell apoptosis in response to I/R injury (33), we measured ROS production by DHE staining. As shown in Figure 5A, at 12 h after transplantation, ROS was significantly higher in the I/R + saline group than in the sham-operated group, and this effect was decreased by EPC transplantation. Quantitative analysis showed that the number of DHE-positive cells was increased from 131 ± 9 per high-power field in the sham-operated animals to 452 ± 42 in the I/R + saline group, but to only 160 ± 43 in the animals with WJ-EPC transplantation (*p < 0.05 vs. I/R + saline group) (Fig. 5B).

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Figure 5.

WJ-EPC transplantation attenuates the increase in oxidative stress at 12 h after I/R injury. (A) Intracellular ROS production visualized by DHE fluorescent staining in frozen renal sections from the sham-operated, I/R + saline, and I/R + EPC groups. Scale bar: 50 μm. (B) Quantitative analysis of the number of DHE-positive cells per high power field. The data are expressed as the mean ± SEM (n = 5). *p < 0.05 versus I/R + saline group.

EPC Transplantation Decreases Renal Inflammation and Fibrosis in Mice with I/R Injury

Since inflammation plays a significant role in the pathogenesis of AKI (1), we examined the effects of I/R injury and WJ-EPC transplantation on the expression of inflammatory cytokines using an antibody array. At 12 h after transplantation, there were no obvious changes in other cytokines, but MIP-2 and KC expression was higher in the I/R + saline group than in the I/R + EPC group (insets in Fig. 6A), and these results were confirmed by immunohistochemistry (main panels in Fig, 6A). Figure 6B shows that, in the I/R + saline group, KC expression was seen in tubular cells (left panel), vascular cells (center panel), and glomerular cells (right panel). We then measured KC levels in the kidney homogenate by ELISA and found that they were increased from 11.3 ± 3.7 pg/ml in the sham-operated animals to 123.4 ± 10.3 pg/ml in the I/R + saline group, but to only 77.7 ± 10.7 pg/ml in the I/R + EPC group (*p < 0.05 vs. I/R + saline group) (Fig. 6C).

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Figure 6.

WJ-EPC transplantation inhibits the increase in inflammatory cytokine expression at 12 h after I/R injury. (A) MIP-2 and KC expression in the three groups was screened using a commercial mouse cytokine antibody array (upper right insert). Representative photomicrographs of immunohistochemical staining for MIP-2 (scale bar: 25 μm) and KC (scale bar: 50 μm) in renal tissues from the sham-operated, I/R + saline, and I/R + EPC groups. (B) High magnification images showing KC expression (arrows) in the I/R + saline group in tubular cells, vascular cells, and glomerular cells. Scale bar: 20 μm. (C) KC levels in renal tissues from the sham-operated, I/R + saline, I/R +EPC groups measured by ELISA. The data are expressed as the mean ± SEM (n = 5). *p < 0.05 versus I/R + saline group.

Since long-term loss of peritubular capillaries is associated with the development of kidney fibrosis (5, 6), we next evaluated the effects of I/R injury and WJ-EPC transplantation on fibrosis by immunostaining for the fibroblast marker, S100A4. As shown in Figure 7A, the number of S100A4-positive cells increased in a time-dependent manner following I/R injury, and this effect was decreased by WJ-EPC transplantation. At day 7 after transplantation, S100A4-positive cells were associated with glomerular, interstitial, renal tubular, and endothelial cells (Fig. 7B). As shown in Figure 7C, the fibrotic score on day 7 after I/R injury was 0.17 ± 0.08 in the sham-operated animals, 2.12 ± 0.03 in the I/R + saline group, and 1.23 ± 0.17 in the WJ-EPC transplanted animals (*p < 0.05 vs. I/R + saline group), and fibrosis was progressive. To further demonstrate that renal fibrosis was decreased by EPC transplantation, the Jones silver staining method was used to detect the amount of extracellular matrix (6). As shown in Figure 7D, interstitial silver staining was seen at all time points in the I/R + saline group, but was strongest at day 7 in terms of both area and intensity of staining, and these effects were attenuated by EPC transplantation.

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Figure 7.

WJ-EPC transplantation inhibits the increase in renal fibrosis following I/R injury. (A) Paraffin sections of kidneys from the sham-operated, I/R + saline, and I/R + EPC groups at different time points stained with antibodies against S100A4, a fibroblast marker. The arrows indicated S 100A4-positive cells. Scale bar: 50 μm. (B) Higher magnification of images in the I/R + saline group at day 7 after transplantation. The arrows indicate S100A4-positive cells associated with glomerular cells, interstitial cells, renal tubular cells, and endothelial cells. Scale bar: 50 μm. (C) Quantitative analysis of the fibrotic score for the three groups at different time points. The data are presented as the mean + SEM (n = 5). *p < 0.05 versus I/R + saline group. (D) Paraffin sections of kidney from the sham-operated, I/R + saline, and I/R + EPC groups at different time points stained using the Jones silver stain method to detect the amount of extracellular matrix (arrows). Scale bar: 50 μm.