Macrophages are involved in the protective role of human umbilical cord-derived stromal cells in renal ischemia–reperfusion injury

Stem Cell Research (2013) 10, 405–416

Available online at www.sciencedirect.com

www.elsevier.com/locate/scr

Macrophages are involved in the protective role of human umbilical cord-derived stromal cells in renal ischemia–reperfusion injury Wei Li a, c , Qiang Zhang a , Mei Wang a , Huiyi Wu c , Fei Mao a , Bin Zhang a , Runbi Ji a , Shuo Gao a , Zixuan Sun a , Wei Zhu a , Hui Qian a,⁎, Yongchang Chen a , Wenrong Xu a, b,⁎ a

School of Medical Science and Laboratory Medicine, Jiangsu University, 301 Xuefu Road, Zhenjiang, Jiangsu 212013, China The Affiliated Hospital of Jiangsu University, 438 Jiefang Road, Zhenjiang, Jiangsu 212001, China c Department of Central Laboratory, the First People's Hospital of Lianyungang, 182 Tongguan Road, Lianyungang, Jiangsu 222002, China b

Received 5 July 2012; received in revised form 16 January 2013; accepted 19 January 2013 Available online 29 January 2013 Abstract Administration of fibroblastic cells derived from a number of tissues (collectively called “mesenchymal stem cells”) has been suggested to be beneficial for renal repair and mortality reduction in renal ischemia–reperfusion injury (IRI), but the underlying mechanism is not fully understood. In the present study, our objective was to investigate the involvement of macrophages in the therapeutic effect of human umbilical cord-derived stromal cells (hUCSCs) on renal IRI. Twenty-four hours after reperfusion, hUCSCs were injected intravenously and resulted in significant improvements in renal function, with a lower tubular injury score together with more proliferative and fewer apoptotic tubular cells in kidney tissue. Moreover, hUCSCs reduced the infiltration of macrophages into renal interstitium especially at 5 days post-reperfusion, while the proportion of anti-inflammatory M2 macrophages was markedly increased. HUCSCs also alleviated the local inflammatory response in kidneys. The absence of macrophages during the early phase of reperfusion enhanced the therapeutic effect of hUCSCs, whereas macrophage depletion during the late repair phase eliminated the renoprotective role of hUCSCs. In vitro, macrophages cocultured with hUCSCs were switched to the alternatively activated M2 phenotype. Our data indicate that hUCSCs are capable of promoting the M2 polarization of macrophages at injury sites, suggesting a new mechanism for hUCSC-mediated protection in renal IRI. © 2013 Elsevier B.V. All rights reserved.

Introduction ⁎ Corresponding authors at: School of Medical Science and Laboratory Medicine, Jiangsu University, 301 Xuefu Road, Zhenjiang, Jiangsu 212013, China. Fax: + 86 511 85038483. E-mail addresses: [email protected] (H. Qian), [email protected] (W. Xu). 1873-5061/$ - see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.scr.2013.01.005

Acute kidney injury (AKI), which is characterized by impaired renal function, results in a major clinical problem especially in critically ill patients admitted to the intensive care unit. Considering the poor outcomes and high incidence of AKI, early diagnosis and the development of effective therapeutic

406 strategies are urgently needed. Renal ischemia–reperfusion injury (IRI) is a major cause of AKI in both native and transplanted kidneys, and is associated with a high level of mortality and morbidity (Bonventre and Yang, 2011). However, therapeutic regimens which could completely ameliorate injury after ischemia and reperfusion are still unavailable. Administration of a variety of fibroblastic cells (often called “mesenchymal stem cells”) has been suggested to be an effective treatment to promote renal repair after IRI (Tögel et al., 2005), but the mechanism involved has not been clearly defined. There is increasing evidence to suggest that a variety of fibroblastic cells display capabilities of homing to injury sites and exerting their profound immunomodulatory role by regulating both innate and adaptive immune cells (Choi et al., 2012; Nauta and Fibbe, 2007). Among these cells, macrophages have recently been demonstrated to play a potent role in the pathogenesis and progression of renal IRI (Jo et al., 2006; Vinuesa et al., 2008). Recently, increasing numbers of studies have favored the immunomodulatory effects of fibroblastic cells, which were derived from different tissue sources, on macrophages as a critical mechanism in the amelioration of inflammation-related diseases, such as sepsis, wound healing and acute myocardial infarction (Németh et al., 2009; Zhang et al., 2010; Dayan et al., 2011). However, the interactions between human umbilical cord-derived stromal cells (hUCSCs) and macrophages in the repair progress of renal IRI, especially whether hUCSCs can induce the polarization of macrophages in kidney tissues, are poorly understood. Macrophages may play diverse roles in both the injury and repair phase of diseases (Ricardo et al., 2008). Generally, they are categorized into two populations defined as classically and alternatively activated macrophages (M1 and M2, respectively) (Sica and Mantovani, 2012). M1 macrophages play an important role in pathogen clearance and produce high levels of proinflammatory cytokines during injury, whereas M2 macrophages contribute to tissue homeostasis and secrete mainly anti-inflammatory cytokines in the repair phase. In this current study, we tested the hypothesis that macrophages located in the damaged kidney tissue after ischemia–reperfusion can be induced towards the M2 phenotype by the administration of human umbilical cord-derived stromal cells, which may provide a novel explanation for the mechanism underlying the renoprotective role of hUCSCs.

Materials and methods Mice and renal ischemia–reperfusion model All experiments were performed in accordance with Chinese legislation regarding experimental animals. Male C57BL/6 mice aged 8–10 weeks were purchased from the Laboratory Animal Center (Yangzhou University, China). Mice were housed in individual microisolator cages under specific pathogen-free conditions, with free access to water and chow. After they were anesthetized with an intraperitoneal injection of 2.0% (w/v) pentobarbital sodium at 4.0 ml/kg, mice were subjected to bilateral flank incisions and both renal pedicles were clamped for 30 min with microaneurysm clamps. During ischemia, mice were hydrated with warm saline and body temperature was maintained at 37 °C with a heating pad. After the clamps were removed, the kidneys were inspected to

W. Li et al. confirm restoration of blood flow and the flank incisions were sutured in two layers. Sham operation was performed in a similar manner, without clamping the renal pedicles.

Isolation and infusion of hUCSCs HUCSCs were isolated as previously described (Qiao et al., 2008), with a minor improvement. Briefly, human umbilical cords freshly obtained with donor consent were washed off the cord blood, and the umbilical artery and vein were removed prior to mincing. After being rinsed in antibiotics for disinfection, the remaining cord tissue was cut into 1 mm 3 pieces and placed directly into culture dishes for 30 min to improve tissue attachment. After that, the growing medium of Dulbecco's modified Eagle's medium with 10% fetal bovine serum (10% FBS-DMEM; Gibco, Carlsbad, CA) containing penicillin (100 U/ml) and streptomycin (100 μg/ml) was added to the tissue explants. Once cells reached subconfluence, tissue pieces were removed and cells adhering to the dishes were digested and passaged into flasks for further expansion. At 24 h following reperfusion, 2× 10 6 hUCSCs (passage 4) suspended in 0.2 ml sterile phosphate buffer solution (PBS) were injected into mice via the caudal vein. Alternatively, human lung tissue-derived fibroblast cells (HFLs, 2×10 6) suspended in 0.2 ml sterile PBS, or 0.2 ml sterile PBS alone were injected i.v. into mice as the controls. HFL cells were purchased from the Institute of Biochemistry and Cell Biology at the Chinese Academy of Sciences (Shanghai, China) and maintained in 15% FBS-DMEM (Gibco).

Homing studies HUCSCs were prelabeled by incubation with the carbocyanine fluorescent dye CM-Dil (Molecular Probes, Carlsbad, CA) for 30 min and 2 × 10 6 cells were infused into animal models at 24 h after reperfusion. Mice were euthanized at 5 days post-ischemia and the whole kidneys were analyzed by the Maestro in-vivo imaging system (CRI Inc., Santa Maria, CA). On the other hand, the tissues of kidney and lung were cut into 8-μm snap-frozen sections, counterstained with DAPI staining solution (Beyotime Institute of Biotechnology, Nantong, China) and visualized using a confocal microscope (Leica TCS SP5, Heidelberg, Germany).

Assessment of renal function The concentrations of creatinine (Cr) and Blood urea nitrogen (BUN) in serum were measured as the marker of renal function by an automated biochemical analyzer (Olympus, Tokyo, Japan).

Histology Kidney tissues were fixed in 4.0% paraformaldehyde, embedded in paraffin and cut into 4-μm sections, which were stained with H&E and viewed by light microscopy (Nikon, Kanagawa, Japan). The degree of tubular damage was scored by calculating the percentage of tubules in the corticomedullary junction and outer medulla which exhibited tubular dilation, cell necrosis and cast deposition as follows: 0, none; 1, ≤10%; 2, 10–25%; 3,

Macrophages are involved in the protective role of human umbilical cord-derived stromal cells 25–45%; 4, 45–75%; and 5, N 75%. Ten randomly chosen, nonoverlapping high-power fields (HPFs, ×200) per section were examined.

407

MO), whereas the supernatants were harvested at 3, 7 and 11 h for further detection of released IL-10 by ELISA. For phenotype identification by flow cytometry, cells in coculture were harvested with a cell scraper after 3 days of incubation.

Immunohistochemistry Reverse transcription and RT-PCR analysis Kidney sections were incubated with 3.0% hydrogen peroxide for 30 min to remove endogenous peroxidase. After antigen retrieval, the sections were blocked in 5.0% bovine serum albumin (BSA) for 20 min. They were then incubated with the diluted primary antibodies at 4 °C overnight: rat anti-mouse F4/80 (1:100) (clone: BM8; eBioscience, San Diego, CA), monocyte chemoattractant protein-1 (MCP-1, 1:200) (sc-1784; Santa Cruz Biotechnology, Santa Cruz, CA) and rabbit anti-mouse PCNA (1:80) (BS1289; Bioworld, Minneapolis, MN). After washing, this was followed by a further incubation with biotin-conjugated secondary antibody for 20 min. The complex was visualized with 3,3′-diaminobenzidine (DAB) and counterstained with hematoxylin for microscopic examination. The numbers of F4/80-, MCP-1- or PCNA-positive cells in the corticomedullary junction and outer medulla of kidneys were counted in 10 randomly selected non-overlapping HPFs (×400).

Detection of apoptosis by TUNEL staining Apoptotic cells in the kidney tissues were visualized with TUNEL staining. The procedure was performed using an in situ cell death detection kit (Boster Bioengineering Co. Ltd., Wuhan, China) according to the manufacturer's protocol. The number of apoptotic cells in the corticomedullary junction and outer medulla of kidneys was counted in 10 randomly selected non-overlapping HPFs (×400).

Liposome preparation and in vivo depletion of macrophages Clodronate [dichloromethylene bisphosphonate (Cl2MBP)] liposome (lipo-Cl2MBP) was prepared according to a previously described method (Rooijen and Sanders, 1994). Mice were injected with a bolus of 100 μl/10 g body weight of lipo-Cl2MBP or liposomal vehicle (lipo-PBS) via the caudal vein. Two days after injection, immunochemistry was performed to validate the effect of macrophage depletion in mouse kidney and spleen tissues.

Coculture of macrophages and hUCSCs RAW 264.7 cells were first inoculated into 6-well flat-bottom plates at 8 × 10 5 cells/well in high-glucose DMEM (Gibco) supplemented with 10% heat-inactivated FBS (Gibco). After 6 h of incubation, the supernatant was removed and hUCSCs were added to the same wells at 2 × 10 5 cells/well for an additional incubation of 3 days. For indirect-contact coculture, 0.4-μm pore size transwell inserts (Corning, Lowell, MA) were used to partition macrophages and hUCSCs. RAW 264.7 cells or hUCSCs cultured alone were provided as controls. Cells were collected for gene analysis of inflammatory cytokines at either 5 h (for IL-6 and arginase-1) or 24 h (for IL-1β and IL-10) after stimulation with lipopolysaccharide (LPS, Sigma, St. Louis,

After purification, 1 μg total RNA was reverse transcribed in a 20 μl reaction volume, which was then processed using a commercial kit for RT-PCR (Thermo Scientific, Rockford, IL) according to the manufacturer's instructions. For cocultured cells, expressions of IL-1β, IL-6, arginase-1 and IL-10 were detected by PCR, with GAPDH as the housekeeping gene. For mouse kidney tissues, IL-1β, IL-6 and IL-10 mRNA normalized to GAPDH were measured by real-time PCR, which was performed in a reaction mixture containing 10 μl 2 × SYBG Mix, 0.4 μl 10 μM of each primer, 0.1 μl Taq DNA polymerase (Invitrogen, Carlsbad, CA) and 1 μl cDNA. Primers for mouse IL-1β, IL-6, arginase-1, IL-10 and GAPDH were designed using the Primer Software and produced by Invitrogen company (Table 1). Real-time PCR was performed in a reaction mixture, containing 10 μl 2 × SYBG Mix (Invitrogen), 0.4 μl 10 μM of each primer, 0.1 μl Taq DNA polymerase (Invitrogen) and 1 μl cDNA. All reactions were performed in triplicate on a CFX-96 (Bio-Rad, Hercules, CA) according to the manufacturer's instructions.

Measurement of IL-10 secretion by ELISA The concentration of the cytokine IL-10 was measured in the supernatant of macrophages cocultured with hUCSCs or cultured alone, using a mouse-specific ELISA kit (ExCell Biology, Shanghai, China) following the manufacturer's protocol. The detection limit was 7.0 pg/ml.

Flow cytometry Kidney tissue was minced, incubated with 0.3% collagenase IV (Sigma), ground gently, and filtered through a 40-μm cell strainer prewetted with PBS. After washing, the pellet was incubated with ACK (160 mM NH4Cl, 10 mM KHCO3 and 0.1 mM EDTA, pH 7.4) to remove erythrocytes and resuspended in PBS for flow cytometric detection on a FACSCalibur (BD Biosciences, Sparks, MD). In brief, kidney cells were first incubated with PE-conjugated anti-CD11b (1:100) (clone: M1/70; eBioscience) and FITC-conjugated anti-CD206 (1:100) (MR5D3; Santa Cruz Biotechnology) for 30 min at 4 °C in the dark. The labeled cells were then washed and resuspended in PBS for analysis. As negative controls, isotype-matched antibodies with the corresponding fluorescent labeling were used in our study. Cells in the coculture experiment were also analyzed according to the same protocol.

Statistical analysis Data are expressed as mean ± SEM. Statistics analysis was performed by the non-parametric Mann–Whitney U test using SPSS 16.0 software. Statistical significant level was defined as P b 0.05.

408 Table 1

W. Li et al. Primer sequences for the amplification of target genes.

Genes

Accession number

Primer sequence (5′–3′)

Product size (bp)

Annealing temperature (°C)

IL-1β

NC_000068

212

58

IL-6

NC_000071

279

52

Arginase-1

NC_000076

191

56

IL-10

NC_000067

106

64

GAPDH

NC_000072

For: 5′-CTTCCTTGTGCAAGTGTCTG-3′ Rev: 5′-GCCTGAAGCTCTTGTTGATG-3′ For: 5′-GGAGACTTCACAGAGGATAC-3′ Rev: 5′-CTCCAGGTAGCTATGGTACT-3′ For: 5′-CCAGATGTACCAGGATTCTC-3′ Rev: 5′-AGCAGGTAGCTGAAGGTCTC-3′ For: 5′-CTGAGGCGCTGTCATCGATT-3′ Rev: 5′-TGGCCTTGTAGACACCTTGG-3′ For: 5′-AAGGTCGGTGTGAACGGATT-3′ Rev: 5′-CATTCTCGGCCTTGACTGTG-3′

162

62

Results HUCSCs preserve renal function and morphology in mice with renal IRI Twenty-four hours after reperfusion, 2 × 10 6 hUCSCs were injected into mice with renal IRI. Confocal microscopy showed

that hUCSCs prelabeled with the red fluorescent dye CM-Dil were localized in the lung and kidney tissues at 5 days after reperfusion (Fig. 1A). An in vivo imaging study also showed the homing of large numbers of hUCSCs into the impaired kidney (Fig. 1B). The level of serum Cr and BUN was markedly increased at 1 day post-ischemia and gradually reduced from 3 days after reperfusion in IRI mice given PBS (Figs. 1C, D).

Figure 1 Tracking of hUCSCs in vivo and effect of hUCSC administration on renal function after reperfusion. Twenty-four hours after reperfusion, 2 × 10 6 hUCSCs were administered intravenously into mice with renal IRI by caudal vein and renal function was daily observed. (A): Confocal images of hUCSCs prelabeled with CM-Dil (red) in the lung and impaired kidney at 5 days after reperfusion. The nuclei were visualized by DAPI staining (blue) (magnification, × 200). (B): In-vivo imaging of CM-Dil-hUCSC location (red) in the injured kidney at 5 days after reperfusion. Three animals were analyzed. (C): Quantification of serum Cr at different time points after reperfusion in mice with sham-, PBS-, hUCSC- or HFL-treatment (n = 5). (D): Quantification of serum BUN at different time points after reperfusion in mice with sham-, PBS-, hUCSC- or HFL-treatment (n = 5). ( #P b 0.01: compared with sham group; ⁎P b 0.01: compared with PBS group; ns: no significance).

Macrophages are involved in the protective role of human umbilical cord-derived stromal cells However, infusion of hUCSCs resulted in significantly lower serum Cr and BUN levels than PBS controls, and there was no statistical significance in the levels of Cr and BUN between the groups treated with PBS and HFL (Figs. 1C, D). Renal histology of IRI mice given PBS showed severe impairments, including necrosis and loss of tubular epithelial cells, dilation of tubular lumens, formation of casts, and infiltration of inflammatory cells (Fig. 2A). The tubular injury score peaked at 1 day after reperfusion and remained high during the following course (Fig. 2B). By contrast, hUCSC treatment significantly attenuated tubular impairments and inflammation in kidney tissues (Fig. 2A) and tubular injury score in hUCSC-treated mice was markedly reduced at 3, 5, 7 and 10 days post-injury (Fig. 2B). However, treatment with

409

HFL did not result in any improved amelioration of the renal injury (Fig. 2A) and showed no statistical significance in the level of tubular injury score compared with PBS group (Fig. 2B).

HUCSCs accelerate proliferation of tubular cells and protect tubular cells from apoptosis Kidney tissues from PBS-treated IRI mice showed a slight increase in the number of PCNA-positive cells, which peaked at 3 days after reperfusion (Figs. 3A, B). After infusion of hUCSCs, the number of PCNA-positive cells increased significantly at 3 days after reperfusion and remained high during

Figure 2 Effect of hUCSC administration on renal histology after ischemia–reperfusion. Twenty-four hours after reperfusion, 2 × 10 6 hUCSCs were administered intravenously into mice with renal IRI and renal histology was daily observed. (A): Representative images of H&E-stained kidney sections from sham-, PBS-, hUCSC- or HFL-treated mice at different time points after reperfusion (magnification, × 200). (B): Tubular injury score of the injured kidneys at different time points after reperfusion with PBS, hUCSC- or HFL-treatment (n = 5). ( # P b 0.01: compared with sham group; *P b 0.05, **P b 0.01: compared with PBS group; ns: no significance).

410 the following course. Treatment with HFL did not lead to the promotion of proliferation in tubular epithelial cells, and there was no statistical significance in the number of PCNA-positive cells between PBS and HFL treated groups (Figs. 3A, B). On the other hand, a marked increase in the number of TUNEL-positive cells was detected in IRI mice given PBS which peaked at 1 day after reperfusion, and hUCSC treatment significantly reduced the number of apoptotic tubular cells at 3, 5 and 10 days post-reperfusion (Figs. 3A, C). Moreover, animals treated with HFL showed no statistical significance in the number of TUNEL-positive tubular cells compared with PBS group (Figs. 3A, C).

HUCSC therapy reduces the infiltration of F4/80positive macrophages in the injured kidney Immunochemistry showed that F4/80-positive macrophages were mainly located in the interstitial area of kidney sections (Fig. 4A). The number of F4/80-positive macrophages in IRI mice given PBS significantly increased throughout the observation period and peaked at 5 days after reperfusion (Fig. 4E). However when infused with hUCSCs, the number of F4/80positive macrophages was markedly reduced at 5, 7 and 10 days after reperfusion (Figs. 4B, E). The role of MCP-1, a major

W. Li et al. macrophage chemokine, in the infiltration of macrophages was taken into account and investigated in the kidney sections treated with or without hUCSCs. Immunostaining of MCP-1 in the tubular epithelial cells (Fig. 4C) showed that the number of MCP-1-positive cells significantly increased in PBS-treated IRI mice, peaking at 1 day after reperfusion and lasting through the following course (Fig. 4F). After hUCSC infusion, the number of MCP-1-positive cells was markedly reduced at 3, 5 and 7 days (Figs. 4D, F).

Depletion of macrophages during injury accelerates the therapeutic effect of hUCSCs Immunostaining showed that macrophages were detected in the spleen and kidney at 2 days after lipo-PBS infusion, but that infiltrated macrophages were significantly depleted at 2 days after lipo-Cl2MBP injection (Fig. 5A). Five days after reperfusion, severe histopathology was observed in control mice given PBS, and this impairment was largely reversed by hUCSC infusion (Fig. 5B). In mice treated with lipo-Cl2MBP 24 h before surgery, hUCSC administration provided a further protective effect on renal damage with the best preserved tubular integrity (Figs. 5B, C).

Figure 3 Effect of hUCSC administration on the proliferation and apoptosis of tubular epithelium cells. (A): Representative images of PCNA and TUNEL immunostaining in the injured kidneys at 5 days after reperfusion treated with PBS, hUCSCs or HFL (magnification, × 400). (B): Quantification of PCNA-positive tubular epithelium cells in the kidney sections at different time points after reperfusion with PBS-, hUCSC- or HFL-treatment (n = 5). (C): Quantification of TUNEL-positive tubular epithelium cells in the kidney sections at different time points after reperfusion with PBS-, hUCSC- or HFL-treatment (n = 5). ( #P b 0.01: compared with sham group; ⁎P b 0.01: compared with PBS group; ns: no significance).

Macrophages are involved in the protective role of human umbilical cord-derived stromal cells

411

Figure 4 Effect of hUCSC administration on macrophage recruitment and expression of MCP-1 in the kidney after IRI. (A): Representative images of interstitial macrophages labeled with F4/80 in the kidney of mice treated with PBS at 5 days after reperfusion. (B): Representative images of interstitial macrophages labeled with F4/80 in the kidney of mice treated with hUCSCs at 5 days after reperfusion. (C): Representative images of MCP-1-positive tubular epithelial cells in the kidneys of mice treated with PBS at 5 days after reperfusion. (D): Representative images of MCP-1-positive tubular epithelial cells in the kidneys of mice treated with hUCSCs at 5 days after reperfusion (magnification, × 200 and × 400). (E): Quantification of F4/80-positive macrophages in the kidney sections at different time points after reperfusion with PBS or hUCSC treatment (n = 5). (F): Quantification of MCP-1-positive tubular epithelial cells in the kidney sections at different time points after reperfusion with PBS or hUCSC treatment (n = 5). ( #P b 0.01: compared with sham group; ⁎P b 0.05, ⁎⁎P b 0.01: compared with PBS group).

Depletion of macrophages during repair eliminates the therapeutic effect of hUCSCs As the behavior of macrophages is affected by the inflammatory microenvironment at different stages after reperfusion, the possible role of macrophages in the therapeutic action of hUCSCs during the late repair phase was also investigated. In mice treated with lipo-Cl2MBP 24 h after surgery, depletion of macrophages eliminated the preservation of renal integrity by hUCSCs at 5 days following reperfusion,

which led to a failure in the repair progress following renal injury (Figs. 5B, C).

HUCSCs modulate the gene expression of cytokines in RAW 264.7 cells RT-PCR showed that the expression of IL-1β and IL-6 in RAW 264.7 cells cocultured with hUCSCs was markedly inhibited, whereas the expression of arginase-1 and IL-10 was enhanced

412

W. Li et al.

Figure 5 Renal histology after macrophage depletion in hUCSC-treated mice with IRI. (A): Immunohistochemical images of F4/80-positive macrophages in the spleen and kidney tissues at 2 days after lipo-PBS or lipo-Cl2MBP injection (magnification: spleen, ×200; kidney, ×400). (B): Representative images of H&E-stained kidney sections at 5 days after reperfusion from mice treated with PBS or hUCSCs and hUCSC-treated mice injected with lipo-Cl2MBP 24 h before or after surgery (magnification, ×200 and ×400). (C): Quantification of tubular injury score in the kidney sections at 5 days after reperfusion from mice with distinct treatments (n=5). (⁎Pb 0.05; ⁎⁎P b 0.01). LC, liposome clodronate (lipo-Cl2MBP).

following stimulation with LPS (Fig. 6A). Due to the genus specific primers of mouse-origin, no expression of cytokines was detected in hUCSCs which were cultured alone. In addition, the cytokine profile of RAW 264.7 cells cocultured with hUCSCs in a transwell system was similar to that of RAW 264.7 cells cocultured in direct contact with hUCSCs (Fig. 6A). This data suggest that both the direct contact and paracrine activity of hUCSCs contribute to alterations in the cytokine profile of macrophages in vitro. ELISA analysis demonstrated that the level of IL-10 highly increased in the supernatant of RAW 264.7 cells at 7 h after LPS addition and persisted for at least 11 h (Fig. 6B). However, IL-10 level in the supernatant of RAW 264.7 cells cocultured with hUCSCs was found to be significantly higher than that of RAW 264.7 cells cultured alone at 7 and 11 h after LPS stimulation (Fig. 6B).

HUCSCs alleviate the local inflammatory response in the injured kidneys Compared with PBS controls, hUCSC therapy resulted in a significant reduction in the mRNA expression of IL-1β and IL-6

in the impaired kidneys. By contrast, the gene expression of IL-10 was greatly increased by hUCSC treatment (Fig. 6C). In mice with macrophage absence during injury, the expression of cytokines was comparable with mice treated with hUCSCs alone. On the other hand, mice with macrophage absence during repair were characterized by a high expression of IL-1β and IL-6, and a low expression of IL-10 after hUCSC therapy, with no significant difference from PBS controls (Fig. 6C).

HUCSCs switch macrophages to the M2 phenotype in vitro or in vivo RAW 264.7 cells cocultured with hUCSCs in a direct contact manner or in a transwell system exhibited a significant increase in the percentage of CD206 + cells among CD11b + macrophages, compared with RAW 264.7 cells cultured alone (Fig. 7A). In vivo, we also demonstrated that hUCSCs led to a significant increase in the percentage of CD206 + and CD11b + macrophages among the cells of impaired kidneys at 5 days after reperfusion, compared with PBS controls (Fig. 7B).

Macrophages are involved in the protective role of human umbilical cord-derived stromal cells

Discussion In recent years, stromal cells derived from bone marrow, adipose tissue, or fetal membrane have been demonstrated to be beneficial for recovery from acute kidney injury (Morigi et al., 2008; Feng et al., 2010; Tsuda et al., 2010). Previously, we found that bone marrow stromal cells (BMSCs) ameliorated acute renal failure in a rat model induced by glycerol injection (Qian et al., 2008). However, human umbilical cord has recently become an attractive source of stromal cells, since a larger number of cells can be easily obtained from it without raising any ethical objections (Chao et al., 2008). A previous study reported that hUCSCs are morphologically and immunophenotypically similar to BMSCs, but are more committed to matrix remodeling and angiogenesis (Panepucci et al., 2004). Thus, hUCSCs may be an ideal source of stromal cells for cell-based therapies and we have

413

established a method to isolate stromal cells from human umbilical cord (Qiao et al., 2008). In this current study, we observed that exogenous hUCSCs could home to and localize at the site of lung and injured kidney and play a beneficial role in renal functional and histological improvements, which is in agreement with our previous findings (Qian et al., 2008; Cao et al., 2010; Chen et al., 2011). Ischemia–reperfusion injury results in a characteristic appearance of necrosis/apoptosis of tubular epithelial cells in the kidneys (Kennedy and Erlich, 2008). In this study, we found that hUCSCs promoted the proliferation of tubular epithelial cells, whereas they inhibited the apoptosis of tubular epithelial cells. This demonstrates that hUCSCs can ameliorate mouse renal IRI by improving tissue regeneration and reducing tissue necrosis in the injured kidneys, which may therefore be responsible for the renoprotective effect of hUCSCs. However, the mechanisms underlying these roles remain incompletely understood.

Figure 6 Cytokine expression profiles of macrophages with hUCSC treatment. (A): RT-PCR analysis of IL-1β, IL-6, arginase-1 and IL-10 expression in RAW 264.7 cells after coculture either in direct or in indirect contact with hUCSCs for 3 days in response to LPS stimulation. (B): ELISA analysis of the anti-inflammatory cytokine IL-10 secreted by RAW 264.7 cells cocultured with hUCSCs. The results were representative of three independent experiments (mean ± SEM). (C): Real-time RT-PCR analysis of cytokine mRNA expression in the kidney tissues collected from mice at 5 days post-reperfusion following different treatments. The results were representative of three independent experiments (mean ± SEM). (⁎P b 0.05). TW, transwell; LC, liposome clodronate (lipo-Cl2MBP).

414

W. Li et al.

Figure 7 Proportion of CD206-positive macrophages with hUCSC treatment. (A): Flow cytometric analysis for the proportion of CD206positive cells among CD11b-labeled RAW 264.7 cells after being cocultured with hUCSCs for 3 days either in direct or in indirect contact compared with RAW 264.7 cells cultured alone. (B): Five days after reperfusion, cells collected from kidney tissues were immunostained with PE-CD11b and FITC-CD206 antibodies and subjected to flow cytometry analysis. TW, transwell.

Previously, we demonstrated that injured kidney tissues can induce bone marrow stromal cells to differentiate into renal tubular epithelial-like cells in vitro and in vivo (Qian et al., 2008). However, another work reported that the beneficial effects of BMSCs are primarily mediated by complex paracrine actions, but not by their differentiation into target cells (Tögel et al., 2005). Recently, there is increasing evidence to indicate that immunomodulation does play a critical role in the therapeutic effect of multipotential stromal cells, since tissue injury is generally inflammation-related (Semedo et al., 2009; Aronin and Tuan, 2010; Jang et al., 2009). In particular, many studies have focused on the modulatory effect of stromal cells on macrophages (Kim and Hematti, 2009; Chen et al., 2008; Maggini et al., 2010). One study demonstrated that BMSCs can beneficially modulate the response of the host immune system to sepsis and interact with both circulating and tissue macrophages (Németh et al., 2009). Another report indicated that human gingiva-derived stromal cells can induce M2 polarization of macrophages, which may contribute to a marked acceleration of cutaneous wound healing (Zhang et al., 2010). In the present study, macrophage recruitment occurred rapidly in the interstitial area of injured kidneys and peaked at 5 days after reperfusion, which was reduced significantly following hUCSC treatment. Furthermore, we sought to find out the role of chemokine MCP-1 in macrophage recruitment

effected by hUCSCs. Our results showed that the number of MCP-1-positive tubular epithelial cells was also markedly reduced by hUCSC administration. Thus, these findings provide the first evidence that hUCSCs are capable of reducing macrophage infiltration in the injured kidneys, at least in part by downregulating the expression of MCP-1 in tubular epithelial cells. In vivo, we performed macrophage depletion by lipo-Cl2MBP and chose to deplete macrophages at different stages of reperfusion, to find out whether they have distinct functions which would result in conflicting conclusions (Duffield et al., 2005). Our results show that macrophage abolishment during the early injury phase promotes the ameliorating effect of hUCSCs on renal IRI. This finding is in agreement with current evidence demonstrating the contribution of macrophages to the initiation of renal damage (Jo et al., 2006; Day et al., 2005). Conversely, macrophage depletion during the late repair phase led to a loss of the therapeutic effect of hUCSCs on kidney damage. One group reported that macrophages are involved in the repair phase and are beneficial for kidney repair after IRI (Vinuesa et al., 2008). Here, we provide the first evidence that macrophage infiltration during recovery may be essential for the protective role of hUCSCs in renal IRI. In a coculture experiment, we found that hUCSCs suppressed the transcription of IL-1β and IL-6 mRNA in

Macrophages are involved in the protective role of human umbilical cord-derived stromal cells macrophages, whereas aginase-1 and IL-10 were upregulated. These findings suggest that hUCSCs can switch the cytokine expression of macrophages into an anti-inflammatory phenotype, characteristic of M2 macrophages, in vitro. In vivo, hUCSC-treated kidneys also showed a significant suppression of the expression of IL-1β and IL-6, and an upregulation of IL-10. Thus, our observations indicate that hUCSCs are capable of modulating the cytokine expression of macrophages, which may contribute to the resolution of inflammation at injury sites in renal IRI. On the other hand, flow cytometric analysis showed increased CD206 expression on the surface of macrophages cocultured with hUCSCs, and that the proportion of CD206 + macrophages was upregulated during recovery following hUCSC treatment in the kidney tissues. Taken together, our findings suggest that hUCSCs elicit the M2 polarization of macrophages, which may result in an accelerated recovery of renal IRI.

Conclusions In conclusion, we demonstrated in this study that hUCSCs can migrate into injured kidneys, and improve renal function and morphology in an established mouse model of renal IRI. Our findings indicate, for the first time to our knowledge, that hUCSCs can reduce the infiltration of macrophages into injured kidneys, but also increase the proportion of M2-like macrophages during repair, which is beneficial for an accelerated recovery. These findings suggest that hUCSCs are effective in ameliorating mouse renal IRI possibly by transforming the infiltrated macrophages into the M2 phenotype, which may provide new insight into the mechanisms underlying the therapeutic effect of hUCSCs and supply a foundation assistant for therapeutic strategies.

Acknowledgments This work was supported by the Major Research Plan of the National Natural Science Foundation of China (Grant No. 91129718), the National Natural Science Foundation of China (Grant no. 31140063, 81000181, 81272481, and 81071421), the Scientific and Technological Supporting Program of Jiangsu Province (Grant no. BE2010703), the Transfer of Scientific and Technological Achievements Foundation of Jiangsu Province (Grant no. BA2009124), and Jiangsu Province’s Project of Scientific and Technological Innovation and Achievements Transformation (Grant no.BL2012055), Jiangsu Province’s Outstanding Medical Academic Leader and Sci-tech Innovation Team Program (Grant no.LJ201117).

References Aronin, C.E.P., Tuan, R.S., 2010. Therapeutic potential of the immunomodulatory activities of adult mesenchymal stem cells. Birth Defects Res. 90, 67–74. Bonventre, J.V., Yang, L., 2011. Cellular pathophysiology of ischemic acute kidney injury. J. Clin. Invest. 121, 4210–4221. Cao, H., Qian, H., Xu, W., Zhu, W., Zhang, X., Chen, Y., Wang, M., Yan, Y., Xie, Y., 2010. Mesenchymal stem cells derived from human umbilical cord ameliorate ischemia/reperfusion-induced acute renal failure in rats. Biotechnol. Lett. 32, 725–732. Chao, K.C., Chao, K.F., Fu, Y.S., Liu, S.H., 2008. Islet-like clusters derived from mesenchymal stem cells in Wharton's jelly of the human umbilical cord for transplantation to control type 1 diabetes. PLoS One 1, e1451.

415

Chen, L., Tredget, E.E., Wu, P.Y.G., Wu, Y., 2008. Paracrine factors of mesenchymal stem cells recruit macrophages and endothelial lineage cells and enhance wound healing. PLoS One 3, e1886. Chen, Y., Qian, H., Zhu, W., Zhang, X., Yan, Y., Ye, S., Peng, X., Li, W., Xu, W., 2011. Hepatocyte growth factor modification promotes the amelioration effects of human umbilical cord mesenchymal stem cells on rat acute kidney injury. Stem Cells Dev. 20, 103–113. Choi, Y.S., Jeong, J.A., Lim, D.S., 2012. Mesenchymal stem cellmediated immature dendritic cells induce regulatory T cell-based immunosuppressive effect. Immunol. Invest. 41, 214–229. Day, Y.J., Huang, L., Ye, H., Linden, J., Okusa, M.D., 2005. Renal ischemia–reperfusion injury and adenosine 2A receptor-mediated tissue protection: role of macrophages. Am. J. Physiol. Renal Physiol. 288, F722–F731. Dayan, V., Yannarelli, G., Billia, F., Filomeno, P., Wang, X.H., Davies, J.E., Keating, A., 2011. Mesenchymal stromal cells mediate a switch to alternatively activated monocytes/macrophages after acute myocardial infarction. Basic Res. Cardiol. 106, 1299–1310. Duffield, J.S., Forbes, S.J., Constandinou, C.M., Clay, S., Partolina, M., Vuthoori, S., Wu, S., Lang, R., Iredale, J.P., 2005. Selective depletion of macrophages reveals distinct, opposing roles during liver injury and repair. J. Clin. Invest. 115, 56–65. Feng, Z., Ting, J., Alfonso, Z., Strem, B.M., Fraser, J.K., Rutenberg, J., Kuo, H.C., Pinkernell, K., 2010. Fresh and cryopreserved, uncultured adipose tissue-derived stem and regenerative cells ameliorate ischemia–reperfusion-induced acute kidney injury. Nephrol. Dial. Transplant. 25, 3874–3884. Jang, H.R., Ko, G.J., Wasowska, B.A., Rabb, H., 2009. The interaction between ischemia–reperfusion and immune responses in the kidney. J. Mol. Med. 87, 859–864. Jo, S.K., Sung, S.A., Cho, W.Y., Go, K.J., Kim, H.K., 2006. Macrophages contribute to the initiation of ischaemic acute renal failure in rats. Nephrol. Dial. Transplant. 21, 1231–1239. Kennedy, S.E., Erlich, J.H., 2008. Murine renal ischaemia–reperfusion injury. Nephrology 13, 390–396. Kim, J., Hematti, P., 2009. Mesenchymal stem cell-educated macrophages: a novel type of alternatively activated macrophages. Exp. Hematol. 37, 1445–1453. Maggini, J., Mirkin, G., Bognanni, I., Holmberg, J., Piazzón, I.M., Nepomnaschy, I., Costa, H., Cañones, C., Raiden, S., Vermeulen, M., Geffner, J.R., 2010. Mouse bone marrow-derived mesenchymal stromal cells turn activated macrophages into a regulatory-like profile. PLoS One 5, e9252. Morigi, M., Introna, M., Imberti, B., Corna, D., Abbate, M., Rota, C., Rottoli, D., Benigni, A., Perico, N., Zoja, C., Rambaldi, A., Remuzzi, A., Remuzzi, G., 2008. Human bone marrow mesenchymal stem cells accelerate recovery of acute renal injury and prolong survival in mice. Stem Cells 26, 2075–2082. Nauta, A.J., Fibbe, W.E., 2007. Immunomodulatory properties of mesenchymal stromal cells. Blood 110, 3499–3506. Németh, K., Leelahavanichkul, A., Yuen, P.S.T., Mayer, B., Parmelee, A., Doi, K., Robey, P.G., Leelahavanichkul, K., Koller, B.H., Brown, J.M., Hu, X., Jelinek, I., Star, R.A., Mezey, É., 2009. Bone marrow stromal cells attenuate sepsis via prostaglandin E2-dependent reprogramming of host macrophages to increase their interleukin-10 production. Nat. Med. 15, 42–49. Panepucci, R.A., Siufi, J.L.C. Silva, W.A. Jr., Proto-Siquiera, R., Neder, L., Orellana, M., Rocha, V., Covas, D.T., Zago, M.A., 2004. Comparison of gene expression of umbilical cord vein and bone marrow-derived mesenchymal stem cells. Stem Cells 22, 1263–1278. Qian, H., Yang, H., Xu, W., Yan, Y., Chen, Q., Zhu, W., Cao, H., Yin, Q., Zhou, H., Mao, F., Chen, Y., 2008. Bone marrow mesenchymal stem cells ameliorate rat acute renal failure by differentiation into renal tubular epithelial-like cells. Int. J. Mol. Med. 22, 325–332. Qiao, C., Xu, W., Zhu, W., Hu, J., Qian, H., Yin, Q., Jiang, R., Yan, Y., Mao, F., Yang, H., Wang, X., Chen, Y., 2008. Human mesenchymal stem cells isolated from the umbilical cord. Cell Biol. Int. 32, 8–15.

416 Ricardo, S.D., Goor, H.V., Eddy, A.A., 2008. Macrophage diversity in renal injury and repair. J. Clin. Invest. 118, 3522–3530. Rooijen, N.V., Sanders, A., 1994. Liposome mediated depletion of macrophages: mechanism of action, preparation of liposomes and applications. J. Immunol. Methods 174, 83–93. Semedo, P., Palasio, C.G., Oliveira, C.D., Feitoza, C.Q., Gonçalves, G.M., Cenedeze, M.A., Wang, P.M.H., Teixeira, V.P.A., Reis, M.A., Pacheco-Silva, A., Câmara, N.O.S., 2009. Early modulation of inflammation by mesenchymal stem cell after acute kidney injury. Int. Immunopharmacol. 9, 677–682. Sica, A., Mantovani, A., 2012. Macrophage plasticity and polarization: in vivo veritas. J. Clin. Invest. 122, 787–795. Tögel, F., Hu, Z., Weiss, K., Isaac, J., Lange, C., Westenfelder, C., 2005. Administered mesenchymal stem cells protect against ischemic acute renal failure through differentiation-independent mechanisms. Am. J. Physiol. Renal Physiol. 289, F31–F42.

W. Li et al. Tsuda, H., Yamahara, K., Ishikane, S., Otani, K., Nakamura, A., Sawai, K., Ichimaru, N., Sada, M., Taguchi, A., Hosoda, H., Tsuji, M., Kawachi, H., Horio, M., Isaka, Y., Kangawa, K., Takahara, S., Ikeda, T., 2010. Allogenic fetal membranederived mesenchymal stem cells contribute to renal repair in experimental glomerulonephritis. Am. J. Physiol. Renal Physiol. 299, F1004–F1013. Vinuesa, E., Hotter, G., Jung, M., Herrero-Fresneda, I., Torras, J., Sola, A., 2008. Macrophage involvement in the kidney repair phase after ischaemia/reperfusion injury. J. Pathol. 214, 104–113. Zhang, Q.Z., Su, W.R., Shi, S.H., Wilder-Smith, P., Xiang, A.P., Wong, A., Nguyen, A.L., Kwon, C.W., Le, A.D., 2010. Human gingiva-derived mesenchymal stem cells elicit polarization of M2 macrophages and enhance cutaneous wound healing. Stem Cells 28, 1856–1868.