Original Report: Laboratory Investigation American
Journal of
Nephrology
Am J Nephrol 2014;39:466–475 DOI: 10.1159/000362623
Received: November 11, 2013 Accepted: March 31, 2014 Published online: May 17, 2014
Atorvastatin Improves Survival of Implanted Stem Cells in a Rat Model of Renal Ischemia-Reperfusion Injury Jieru Cai Xiaofang Yu Bingying Zhang Hui Zhang Yi Fang Shaopeng Liu Tongqiang Liu Xiaoqiang Ding Division of Nephrology, Zhongshan Hospital, Shanghai Medical College, Fudan University, Shanghai, China
Abstract Aims: To investigate the impacts of combinatorial atorvastatin (Ator) perioperative administration and mesenchymal stem cell (MSC) implantation on therapeutic effects in the rat experimental acute kidney injury. Methods: The model of renal ischemia-reperfusion (I/R) injury was induced by the release of bilateral renal pedicle clamps following 45 min of occlusion. Immediately after reperfusion, CM-Dil-labeled MSCs (1 × 106 cells) or vehicles only were administered through the carotid artery of the animals pretreated with or without Ator. Results: The combined treatment with Ator and MSCs (Ator+MSCs) markedly reduced the elevated levels of serum creatinine and blood urea nitrogen, as well as the severity of renal damage 24 h after I/R injury. In addition, we also observed inhibition of renal tubular cell apoptosis and promotion of proliferation in the Ator+MSCs group compared with the other groups. Consistent with the improvement in renal function and morphology, Ator pretreatment significantly ameliorated oxidative stress, inhibited inflammation response, and increased the viability of implanted MSCs. With regard to the further mechanism, we found
© 2014 S. Karger AG, Basel 0250–8095/14/0396–0466$39.50/0 E-Mail [email protected] www.karger.com/ajn
that the expression of Toll-like receptor 4 (TLR4) and highmobility group box 1, potential mediators of innate immunity, was significantly decreased in the Ator-treated groups. Conclusion: Ator treatment may protect the kidney undergoing I/R injury through suppression of TLR4 signaling, creating a better environment for the survival of grafted MSCs. The extra benefit of the Ator+MSCs combined therapy may result from the Ator-mediated inhibition of oxidative stress and inflammation in the ischemic kidney. © 2014 S. Karger AG, Basel
Introduction
Acute kidney injury (AKI), predominantly caused by renal ischemia-reperfusion (I/R) injury, is a complex clinical syndrome with high morbidity rate, serious consequences and unsatisfactory therapeutic options [1–3]. Therefore, investigation of alternative therapies is needed. Recently, mesenchymal stem cells (MSCs) have been demonstrated to repair damaged renal structures and improve kidney functions, indicating their therapeutic potential in AKI [4–8]. However, the benefits of MSCs are
J. Cai and X. Yu contributed equally to this work.
Dr. Xiaoqiang Ding Division of Nephrology, Zhongshan Hospital Shanghai Medical College, Fudan University Shanghai 200032 (China) E-Mail ding.xiaoqiang @ zs-hospital.sh.cn
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Key Words Atorvastatin · Mesenchymal stem cells · Renal ischemia-reperfusion injury · Inflammation
Subjects and Methods Animals Male Sprague-Dawley rats, weighing 250–300 g, were obtained from the Animal Center, Shanghai Medical College, Fudan University (Shanghai, China) and housed in a pathogen-free environment with a standard diet. All procedures were approved by the Institutional Animal Care Use Committee of Fudan University. 48 rats were randomly allocated to treatment with or without Ator. I/R surgery was then performed, followed by infusion with MSCs or vehicle only. Each of the following groups contained 12 rats: noAtor+no-MSCs (control), Ator+no-MSCs (Ator), no-Ator+MSCs (MSCs) and Ator+MSCs. Six rats per group were sacrificed per time point, at 24 and 72 h post-I/R, respectively. Isolation and Culture of Bone-Marrow-Derived MSCs MSCs were isolated from Sprague-Dawley rats, weighing 50– 100 g, as previously reported [21]. Cells of passages 6–8 were used for these experiments. At 80% confluence, cells were detached, and labeled with CM-Dil (Invitrogen, USA) according to the manufacturer’s instructions. The labeling efficiency was >98%, as determined by fluorescent microscopy. MSCs were kept on ice in PBS until infusion. I/R Injury Rat Model, Cell Transplantation, and Atorvastatin Administration Rats were anesthetized with pentobarbital sodium (40 mg/kg, i.p.). I/R injury was induced by clamping both renal pedicles for 45 min. Thereafter, the clamps were removed and kidney reperfusion
Ator Treatment Improves MSCs
was visually confirmed. Immediately after reperfusion, CM-Dillabeled MSCs were injected via the left carotid artery (1 × 106 cells in 500 μl PBS). The animals in the control group received an equal volume of cell-free PBS (vehicle). As previously described [22], treatment with Ator (Lipitor, 5 mg/kg/day by gavage) was started 3 days prior to MSCs transplantation and continued until time of sacrifice. Measurement of Renal Function Blood samples for measurement of blood urea nitrogen (BUN) and serum creatinine (Scr) were collected at time of sacrifice. Scr levels were determined using the Quantichrom Creatinine Assay Kit (BioAssay Systems, Hayward, Calif., USA), following the manufacturer’s protocol. BUN was measured by direct quantification of serum urea with a colorimetric assay kit according to the instruction protocol (BioAssay Systems). Staining and Histological Analysis After fixation in 10% buffered formalin, kidneys were dehydrated with ethanol and embedded in paraffin. For analysis of tubular injury, 3-μm sections were stained with hematoxylin and eosin. Post-ischemic tubular injury, scored by assessing the percentage of tubules in the corticomedullary junction that displayed tubular cell flattening, cell necrosis, loss of brush border, and luminal cast formation, was determined as described previously [23]. Twenty non-overlapping fields of each section were evaluated (400×) as follows: 0 = normal, 1 = 10–25%, 2 = 26–50%, 3 = 51– 75%, and 4 = >75%. TUNEL staining (Merck Calbiochem, Germany) was performed to assess apoptosis at the single-cell level according to the manufacturer’s instructions. Tissue sections, counterstained by Harris’ hematoxylin, were examined microscopically (400×) and at least 400 cells were counted in each of a minimum of eight random HPFs in the corticomedullary junction. The percentage of apoptotic cells was termed the apoptotic index. Immunohistochemical staining for Ki-67 (proliferating cells) was performed as described previously [21], with monoclonal anti-Ki-67 (1:100; Abcam). Scoring for Ki-67-positive cells was carried out in 10 randomly chosen areas in the cortex or outer medulla for each section under 400× magnifications. To determine the differentiation potential of implanted MSCs in vivo, frozen sections at 5 μm thickness were randomly chosen for immunofluorescent staining with anti-cytokeratin-18 (CK-18, 1:100; Abcam) or E-cadherin (E-cad, 1:100; Abcam). In order to quantify the degree of engraftment, MSCs, revealed by their red fluorescence from the CM-Dil, were counted from 10 randomized fields (200×) that contained MSCs under a fluorescent microscope. Those CM-Dil-labeled cells (nuclei stained blue with DAPI) positive for CK-18 or E-cad were considered to be differentiated into renal tubular-like cells. Oxidative Stress-Related Product and Enzymes Kidney samples collected at sacrifice were homogenized and analyzed for oxidative stress levels by test kits according to the manufacturer’s instructions (Jiancheng Institute, Nanjing, China). The enzymatic activities of superoxide dismutase (SOD) were measured via xanthine-oxidation method and myeloperoxidase (MPO) by testing the H2O2-dependent oxidation of o-dianisidine solution. Glutathione (GSH) levels were measured by the 5,5-dithiobis-2-nitrobenzoic acid-glutathione disulfide reductase recy-
Am J Nephrol 2014;39:466–475 DOI: 10.1159/000362623
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limited by the low viability of transplanted cells in I/Rinjured tissues [9–11]. The pathological environment produced by I/R, characterized by the elevation of reactive oxygen species (ROS) and inflammatory cytokines, is thought to be at least partially responsible for the poor survival and engraftment of injected cells [12]. To address this problem, it is pivotal to protect transplanted cells from adverse effects of a harmful microenvironment. Statin has been shown to exert several biological activities independent of cholesterol-lowering action, including modulation of anti-apoptosis, antioxidant effects, and anti-inflammatory responses [13, 14]. In experimental models of AKI, perioperative therapy with statin has been shown to significantly lower postoperative pro-inflammatory serum cytokine levels, improve renal microcirculation and preserve renal function [15–18], which may protect grafted MSCs. Moreover, it has been reported that statin may enhance the differentiation potential of MSCs [13, 19, 20]. Based on these findings, we hypothesized that pretreatment of the animals with atorvastatin (Ator) improves the renal microenvironments created by I/R injury, thus facilitating the survival of implanted MSCs in vivo.
Enzyme-Linked Immunosorbent Assay (ELISA) The following cytokines and growth factors, associated with the paracrine and/or endocrine effects of MSCs in renal protection, were quantified by ELISA: vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF-1) from kidney homogenates according to the manufacturer’s instructions (R&D Systems), as well as the pro-inflammatory (TNF-α, IL-1β) and anti-inflammatory cytokines (IL-10). Photometric measurements were conducted at 450 nm using microplate reader (Bio-Rad). Western Blot Analysis Homogenized kidney samples were loaded at 30 μg/sample onto 12.5% SDS-PAGE gels for electrophoresis and transferred to PVDF membranes. Membranes were blocked with 5% milk and probed with anti-Bax (1:1,000; Cell Signaling), anti-Bcl2 (1:1,000; Cell Signaling), anti-HMGB1 (1:1,000; Cell Signaling), anti-Tolllike receptor 4 (TLR4) (1:1,000; Abcam), and anti-β-actin (1:2,000; Abcam), followed by incubation with horseradish peroxidase-conjugated secondary antibodies. Specific bands of target proteins were visualized by chemiluminescence. Protein bands were analyzed by ImageJ software and normalized to the β-actin band densities. Statistics Data were collected and analyzed using the SPSS Software (version 13.0), then presented as mean ± SD. Statistical differences between two groups were evaluated with Student’s unpaired t test. One-way ANOVA with the LSD-t test were used for multiple comparisons among four groups. Statistical significance level was defined as p < 0.05.
Results
Therapeutic Potential of Ator and/or MSCs in Renal Ischemia-Reperfusion Injury Post-I/R 24 h, there was a marked reduction in the levels of Scr in the Ator or the MSCs group, compared to the control group (p < 0.05). Similarly, the BUN levels were reduced in the Ator or the MSCs group compared to the control group (p < 0.05). Furthermore, animals in the Ator+MSCs group had sharply decreased Scr and BUN levels when compared to those in the MSCs group (p < 0.05). Post-I/R 72 h, there was a further decline in Scr and BUN levels. Although there was a still notable reduction in the treated groups (the Ator group, the MSCs group, and the Ator+MSCs group) compared with the control group, no significant differences were detected among the treated groups (fig. 1a, b). Histological examination revealed that marked tubular damage occurred in the injured kidney at hour 24 after 468
Am J Nephrol 2014;39:466–475 DOI: 10.1159/000362623
surgery, and resulted in a high injury score (3.30 ± 0.65). Compared with the control group, animals in the Ator or the MSCs group had a significantly reduced injury score (p < 0.05). Animals in the Ator+MSCs group had less tubular damage than those in the MSCs group (1.70 ± 0.92 vs. 2.30 ± 0.80, p = 0.019) (fig. 1c, d). These results indicated a synergistic effect of concomitant therapy with Ator and MSCs in restoring the renal function and ameliorating the I/R-induced tubular damage. We next examined whether double therapy can change the renal proliferative response 72 h after I/R injury. The frequency of Ki-67 was higher in the treated groups than that in the control group, especially in the Ator+MSCs group (p < 0.05) (fig. 1e, f). Then, TUNEL analysis was conducted 24 h post-ischemia to determine whether Ator+MSCs dual treatment increased organ protection by enhancing anti-apoptotic action. Apoptosis of renal tubular cells in the Ator or the MSCs group was obviously lower than that in the control group (p < 0.05). Further analysis indicated that animals in the Ator+MSCs group had a significantly decreased apoptotic index compared with that in the MSCs group (p < 0.05) (fig. 1g, h). The quantitative analysis of apoptosis-regulating proteins revealed that expression of the pro-apoptotic protein Bax was significantly downregulated and anti-apoptotic protein Bcl-2 was upregulated in the Ator or the MSCs group compared with the control group (p < 0.05). Bcl-2 expression reached the peak value in the Ator+MSCs group, but the presence of Ator did not alter expression of Bax in the Ator+MSCs group compared with the MSCs group (fig. 1i). In summary, combined treatment with Ator and MSCs markedly attenuated renal dysfunction and the severity of renal damage, inhibited apoptosis, and ameliorated proliferation in injured kidney compared with MSCs-alone treatment. Improvement of the Survival and Immune-Modulatory Effects of MSCs in vivo in the Presence of Ator Pretreatment Immunofluorescent analysis of renal sections revealed that 24 h post-ischemia, the numbers of CM-Dil-labeled cells in the Ator+MSCs group was significantly higher than that in the MSCs group (32.33 ± 3.51 vs. 22.67 ± 3.79, p = 0.015). At 72 h post-ischemia, few cells were detected in the section and no significant differences were found in the number of labeled cells between the two groups (fig. 2a, b). Further analysis revealed that none of the CMDil-labeled cells were positive for CK-18 or E-cad in either of these groups during the treatment period, which Cai/Yu/Zhang/Zhang/Fang/Liu/Liu/Ding
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cling method. The malondialdehyde (MDA), a secondary product of lipid peroxidation, was determined as thiobarbituric acid (TBA)-reactive substance.
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Fig. 1. Effect of Ator and/or MSCs on I/R-induced renal dysfunction and histological changes. a, b At 24 h post-I/R, all treated animals showed a marked reduction in Scr and BUN levels compared to the control group, with the greatest effect observed in the Ator+MSCs group. c, d Compared with other groups, injured score was significantly decreased in the Ator+MSCs group 24 h post-I/R. HE. ×200. e, f The number of Ki-67-positive cells was higher in the treated group than the control group, especially in the Ator+MSCs group. ×200. g, h The apoptotic index in the Ator
group or the MSCs group was significantly decreased compared with that in the control group, and the Ator+MSCs group had a further decrease in apoptotic cells compared with the MSCs groups. i Compared with the control group, the expression of the pro-apoptotic protein Bax was significantly downregulated and anti-apoptotic protein Bcl-2 was upregulated in treated animals, especially in the Ator+MSCs group. * p < 0.05 vs. control group; # p < 0.05 vs. MSCs group.
Ator Treatment Improves MSCs
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Fig. 2. Effect of Ator on the survival and immune-modulatory function of MSCs. a, b MSCs were labeled with CM-Dil and appeared red under fluorescent confocal microscopy (nuclei stained blue with DAPI) 24 and 72 h post-ischemia (×20 in the upper panel; ×63 in the lower panel). c At 24 h post-ischemia, the expression levels of IGF-1 in the Ator+MSCs group were significantly higher
than that in the MSCs group. Moreover, there were trends towards upregulation of VEGF and HGF in the Ator+MSCs group compared with the MSCs group. At 72 h post-ischemia, double therapy with Ator and MSCs improved the expressions of these cytokines compared with the control group. * p < 0.05 vs. control group; # p < 0.05 vs. MSCs group.
indicated that MSCs did not differentiate into renal tubular cells after transplantation (data not shown). The immune-modulatory effects of MSCs were examined by ELISA analysis of some cytokines and growth factors involved in kidney repair (fig. 2c). At 24 h post-ischemia, the expression levels of IGF-1 in the Ator+MSCs
group were significantly higher than those in the MSCs group. Moreover, there were trends towards upregulation of VEGF and HGF in the Ator+MSCs group compared with the MSCs group. At 72 h post-ischemia, we noted that the expressions of b-FGF and HGF in the Ator+MSCs group significantly improved (p < 0.05) compared with
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Fig. 3. Assessment of oxidative stress and inflammatory response in the injured kidney at 24 h post-I/R. a Ator treatment inhibited the expression of the inflammatory cytokines (IL-1β and TNF-α) and promoted expression of the anti-inflammatory cytokine (IL10) compared with the control group (p < 0.05). In the Ator+MSCs group, the expression of IL-1β further decreased compared with the MSCs group (p < 0.05). b The MPO activity and the MDA concentration in the Ator group were decreased compared with the control group. Lower MDA concentration and higher GSH level
were observed in the Ator+MSCs group compared with the MSCs group, and higher SOD activity compared with the Ator group. c The expressions of HMGB1 and TLR4 were downregulated in monotherapy groups compared with the control group and further decreased HMGB1 and TLR4 expression were detected in the Ator+MSCs group compared with monotherapy groups, although some differences between groups did not prove to be statistically significant. * p < 0.05 vs. control group; # p < 0.05 vs. MSCs group; † p < 0.05 vs. Ator group.
those in the MSCs group, while no differences in VEGF and IGF-1 were found between these MSCs-implanted groups.
greater effect on inhibiting inflammation than the MSCs group, although significant difference was only noted in IL-1β expression (p < 0.05). Moreover, the benefit of the dual therapy was no better than that of Ator-alone treatment (fig. 3a). With regard to the oxidative stress-related products and enzymes, which included MPO, MDA, SOD and GSH, we found that the MPO activity and the MDA concentration were lower in the Ator group compared with the control group (p < 0.05), while no significant differences were detected in these four parameters between the MSCs group and the control group. In combined therapy group, lower MPO activity and MDA concentration, and higher SOD activity and GSH level were observed compared with monotherapy groups, although most of the differences were not significant (fig. 3b). Thus, we inferred that the protective effects of Ator may
Reduced Oxidative Stress and Inhibition of Inflammatory Cytokine Expression in the Kidney with Ator Treatment via the TLR4 Pathway We assessed the renal microenvironment by analyzing the expression of inflammatory-associated cytokines, and several oxidative stress-related products and enzymes in the kidney at day 1 post-I/R. Compared with the control group, the inflammatory cytokines (TNF-α and IL-1β) were markedly downregulated and the anti-inflammatory cytokine (IL-10) upregulated in the Ator group (p < 0.05), but these cytokines did not significantly improve in the MSCs group. The Ator+MSCs group produced a Ator Treatment Improves MSCs
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Discussion
In the present study, we have demonstrated that Ator+MSCs promotes greater recovery of kidneys undergoing I/R injury than MSCs alone. A low-dose and shortterm administration of Ator to the animals around the time of MSCs transplantation enhances the survival and paracrine/endocrine effects of MSCs in the ischemic kidney. With regard to the potential mechanism, extra benefits from combined treatment may be associated with Ator pretreatment, by improving the renal microenvironment via the TLR4 pathway. The kidney undergoing I/R results in extensive and complex inflammatory/oxidative stress responses [12]. Although MSCs have been shown to demonstrate great therapeutic promise in renal I/R injury [5–8], the harsh post-ischemic renal microenvironment reduces the survival of implanted cells within target sites, thus limiting their benefits [9–11]. To address this problem, a number of studies aimed at promoting the retention and survival of MSCs to increase the number of cells successfully engrafting in the injured kidney have been published. The main strategy utilized has involved the alteration of ‘seeds’ meaning donor cells by preconditioning or genetic modification of MSCs [24–26]. However, these techniques do not always provide sufficient effects for the improvement of the cell survival, and further exploration is required for clinical application due to the complexity of these procedures. In this study, we established an ap472
Am J Nephrol 2014;39:466–475 DOI: 10.1159/000362623
Ator perioperative administration TLR4-HMGB1 pathway ଯ
Soil: kidneys
Oxidative stress ଯ Inflammation ଯ Improve renal microenvironment
Seed: MSCs
MSCs survival ଭ Immune modulatory function ଭ Apoptosis ଯ Proliferation ଭ I/R injury ଯ
Fig. 4. Schema of the mechanisms underlying renal protection conferred by Ator and MSCs treatment. Ator pretreatment downregulates the activation of TLR4-HMGB1 pathway in the kidney, consequently resulting in the reduced oxidative stress and decreased inflammation. The improved renal microenvironment strengthens the renal protective functions conferred by MSCs, through promotion of MSCs survival and generation of renoprotective factors. The improved renal microenvironment and the corresponding increased survival of MSCs further promote the recovery of injured kidney.
proach by improving the ‘soil’, the microenvironment of target organ, with Ator to facilitate survival and biological behavior of implanted cells. Statin, a drug known to inhibit cholesterol biosynthesis, has been shown to possess pleiotropic effects that include enhancement of endothelial nitric oxide production, anti-inflammatory and anti-oxidative responses [27–30]. Therefore, statin may be effective in improving the renal microenvironment generated by I/R injury. Our findings indicated that Ator administration downregulated the expression of oxidative stress and inflammation markers in the injured kidney, specifically MDA, MPO, IL-1β and TNF-α, while upregulating the expression of IL-10. These findings were consistent with previous reports [17, 31, 32]. In order to further investigate the mechanisms by which Ator protects infused MSCs, we concentrated on the TLR signaling pathways, especially TLR4 and its enCai/Yu/Zhang/Zhang/Fang/Liu/Liu/Ding
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be related to modulation of renal inflammation and oxidative stress caused by I/R injury and there was a partial synergism between Ator and MSCs. To further confirm whether the benefit of Ator treatment is associated with the activation of innate immunity via the TLR signaling pathways, we examined the regulation of TLR4 and its endogenous ligand, HMGB1, within the kidney. The expressions of TLR4 and HMGB1 were significantly downregulated in the Ator group compared to the control group (p < 0.05) and the same trend of TLR4 and HMGB1 expression existed in the MSCs group. In the Ator+MSCs group, the levels of TLR4 and HMGB1 were further decreased compared with the monotherapy groups, although there was no significant difference in expression of HMGB1 between the Ator+MSCs group and the Ator group (fig. 3c). Taken together, we inferred that the heightened effects of treatment with Ator may be associated with the downregulation of TLR and HMGB1 expression.
dogenous ligand HMGB1. TLR4 signaling pathways are potential mediators of innate immune responses occurred by I/R injury [33]. Some experiments have shown that this pathway plays a key role in the induction of inflammation response, and oxidative stress injury in a variety of ischemic tissues [34, 35]. Our results confirmed that the expression of TLR4 and HMGB1 was significantly decreased in the Ator group when compared with the control group. With the presence of Ator, MSCs could further decrease the expression of TLR4 and HMGB1. Our preliminary conclusion is that HMGB1-TLR4 signaling pathways may be involved in the improvement of the renal microenvironment. However, in this study the exploration of signaling pathways is limited to protein level expression. Although these results could not fully elucidate the role of Ator in the TLR4 signaling pathway, previous experiments have demonstrated that Ator can attenuate the activation of TLR4 expression and its downstream pathways, including mitogen-activated protein kinase, NF-κB and AMP-activated protein kinase [33, 36–39]. Therefore, it can be speculated that the effect of Ator on the suppression of the TLR-dependent inflammatory pathway may play a key role in promoting the recovery of the kidney. Accumulating evidence has demonstrated that the infusion of MSCs results in anti-inflammatory and antiapoptotic effects in animal models of I/R injury [8, 10, 21, 24, 40]. Our research indicates that MSCs administration at the time of reperfusion could provide a degree of protection against AKI. A major obstacle in using cell therapy is the low survival of transplanted cells in the adverse renal microenvironment and the death of the majority of transplanted cells within the first day after transplantation. Our results confirmed that the number of engrafted MSCs in the Ator+MSCs group was significantly improved compared with the MSCs group 24 h post-ischemia. Moreover, MSCs transplantation, in conjunction with Ator pretreatment of the animals, may dramatically attenuate renal dysfunction and morphological alterations, inhibit apoptosis and enhance proliferation in injured kidney when compared with MSCs transplantation alone after I/R. The extra benefit of combined therapy may stem from improvement in the renal microenvironment created by perioperative treatment the animals with Ator, and may improve the survival of injected MSCs. However, the mechanisms by which MSCs ameliorate I/R injury remain to be elucidated. Several researches have indicated that MSCs possess the capacity to differentiate into renal tubular cells [6, 41]. Although Ator has been shown to increase the potential of differentiation of
implanted cells in post-infarct myocardium [13], our results were consistent with other studies that showed no transdifferentiation of MSCs into renal tubular-like cells [10, 42]. Ator administration improved the survival of MSCs; however, it did not increase the potential of differentiation of implanted cells in post-ischemic kidney. Other studies have suggested that the reparative effects of MSCs can be primarily ascribed to their endocrine/paracrine functions and this result was also confirmed in our study [10, 42]. Compared with MSCs delivery alone, treating the animals with Ator prior to MSCs delivery further increased the secretion of growth factors and cytokines (VEGF, HGF, and IGF-1). These proteins are known to improve renal function in AKI, and exert more powerful multifunctional immune-regulatory activities, such as inhibition of apoptosis and suppression of inflammation. The improved renal microenvironment and the corresponding increased survival of MSCs further promote the recovery of injured kidney. In conclusion, for the first time, this study has documented experimental evidence that the Ator+MSCs combined therapy may have a synergistic effect on the regeneration and repair of renal function and morphology post-ischemia. The benefit may result from the statinmediated inhibition of oxidative stress, and inflammation in the injured kidney via suppression of TLR4 signaling pathway, resulting in a better environment for the survival of implanted MSCs (fig. 4). The data from the present study also offer a potentially new clinical strategy for the application of stem cell transplantation in the treatment of AKI.
Ator Treatment Improves MSCs
Am J Nephrol 2014;39:466–475 DOI: 10.1159/000362623
Acknowledgements We thank Pfizer for donation of atorvastatin. We also thank Dr. Rende Xu, Karen M. Peterson, and Dr. Jia Guo for critically reading the manuscript. This work was supported by National Natural Science Foundation of China (81000307), the Major Project of Basic Research of Technology Committee in Shanghai of China (12DJ1400200).
Disclosure Statement
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The authors have no conflicts of interest to disclose.
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23 Allam R, Scherbaum CR, Darisipudi MN, Mulay SR, Hagele H, Lichtnekert J, Hagemann JH, Rupanagudi KV, Ryu M, Schwarzenberger C, Hohenstein B, Hugo C, Uhl B, Reichel CA, Krombach F, Monestier M, Liapis H, Moreth K, Schaefer L, Anders HJ: Histones from dying renal cells aggravate kidney injury via TLR2 and TLR4. J Am Soc Nephrol 2012;23:1375–1388. 24 Tian HB, Lu Y, Shah SP, Wang QS, Hong S: 14S,21R-dihydroxy-docosahexaenoic acid treatment enhances mesenchymal stem cell amelioration of renal ischemia/reperfusion injury. Stem Cells Dev 2012;21:1187–1199. 25 Yuan L, Wu MJ, Sun HY, Xiong J, Zhang Y, Liu CY, Fu LL, Liu DM, Liu HQ, Mei CL: VEGF-modified human embryonic mesenchymal stem cell implantation enhances protection against cisplatin-induced acute kidney injury. Am J Physiol Renal Physiol 2011; 300:F207–F218. 26 Eliopoulos N, Zhao J, Forner K, Birman E, Young YK, Bouchentouf M: Erythropoietin gene-enhanced marrow mesenchymal stromal cells decrease cisplatin-induced kidney injury and improve survival of allogeneic mice. Mol Ther 2011;19:2072–2083. 27 Cantoni S, Cavallini C, Bianchi F, Bonavita F, Vaccari V, Olivi E, Frascari I, Tassinari R, Valente S, Lionetti V, Ventura C: Rosuvastatin elicits kdr-dependent vasculogenic response of human placental stem cells through PI3K/ AKT pathway. Pharmacol Res 2012; 65: 275– 284. 28 Jahromi GP, Seidi S, Sadr SS, Shabanzadeh AP, Keshavarz M, Kaka GR, Hosseini SK, Sohanaki H, Charish J: Therapeutic effects of a combinatorial treatment of simvastatin and bone marrow stromal cells on experimental embolic stroke. Basic Clin Pharmacol Toxicol 2012;110:487–493. 29 Mottaghi S, Larijani B, Sharifi AM: Atorvastatin: an efficient step forward in mesenchymal stem cell therapy of diabetic retinopathy. Cytotherapy 2013;15:263–266. 30 Song L, Yang YJ, Dong QT, Qian HY, Xu H, Meng XM, Tang Y: Atorvastatin protects swine bone marrow mesenchymal stem cells from apoptosis through AMPK but not PI3K/ AKT pathway. Zhonghua Xin Xue Guan Bing Za Zhi 2011;39:1033–1038. 31 Todorovic Z, Nesic Z, Stojanovic R, Basta-Jovanovic G, Radojevic-Skodric S, Velickovic R, Chatterjee PK, Thiemermann C, Prostran M: Acute protective effects of simvastatin in the rat model of renal ischemia-reperfusion injury: it is never too late for the pretreatment. J Pharmacol Sci 2008;107:465–470. 32 Nesic Z, Todorovic Z, Stojanovic R, Basta-Jovanovic G, Radojevic-Skodric S, Velickovic R, Chatterjee PK, Thiemermann C, Prostran M: Single-dose intravenous simvastatin treatment attenuates renal injury in an experimental model of ischemia-reperfusion in the rat. J Pharmacol Sci 2006;102:413–417.
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1 Thadhani R, Pascual M, Bonventre JV: Acute renal failure. N Engl J Med 1996; 334: 1448– 1460. 2 Fang Y, Ding X, Zhong Y, Zou J, Teng J, Tang Y, Lin J, Lin P: Acute kidney injury in a Chinese hospitalized population. Blood Purif 2010;30:120–126. 3 Devarajan P: Update on mechanisms of ischemic acute kidney injury. J Am Soc Nephrol 2006;17:1503–1520. 4 Lin F: Stem cells in kidney regeneration following acute renal injury. Pediatr Res 2006; 59:74R–78R. 5 Herrera MB, Bussolati B, Bruno S, Fonsato V, Romanazzi GM, Camussi G: Mesenchymal stem cells contribute to the renal repair of acute tubular epithelial injury. Int J Mol Med 2004;14:1035–1041. 6 Morigi M, Imberti B, Zoja C, Corna D, Tomasoni S, Abbate M, Rottoli D, Angioletti S, Benigni A, Perico N, Alison M, Remuzzi G: Mesenchymal stem cells are renotropic, helping to repair the kidney and improve function in acute renal failure. J Am Soc Nephrol 2004;15: 1794–1804. 7 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 2005;115:1743–1755. 8 Lange C, Togel F, Ittrich H, Clayton F, NolteErnsting C, Zander AR, Westenfelder C: Administered mesenchymal stem cells enhance recovery from ischemia/reperfusion-induced acute renal failure in rats. Kidney Int 2005;68: 1613–1617. 9 Burst VR, Gillis M, Putsch F, Herzog R, Fischer JH, Heid P, Muller-Ehmsen J, Schenk K, Fries JWU, Baldamus CA, Benzing T: Poor cell survival limits the beneficial impact of mesenchymal stem cell transplantation on acute kidney injury. Nephron Exp Nephrol 2010;114:E107–E116. 10 Togel F, Hu ZM, Weiss K, Isaac J, Lange C, Westenfelder C: Administered mesenchymal stem cells protect against ischemic acute renal failure through differentiation-independent mechanisms. Am J Physiol Renal Physiol 2005;289:F31–F42. 11 Togel F, Yang Y, Zhang P, Hu ZM, Westenfelder C: Bioluminescence imaging to monitor the in vivo distribution of administered mesenchymal stem cells in acute kidney injury. Am J Physiol Renal Physiol 2008; 295:F315–F321. 12 Chen YT, Sun CK, Lin YC, Chang LT, Chen YL, Tsai TH, Chung SY, Chua S, Kao YH, Yen CH, Shao PL, Chang KC, Leu S, Yip HK: Adipose-derived mesenchymal stem cell protects kidneys against ischemia-reperfusion injury through suppressing oxidative stress and inflammatory reaction. J Transl Med 2011;9:51.
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37 Ajamieh H, Farrell G, Wong HJ, Yu J, Chu E, Chen J, Teoh N: Atorvastatin protects obese mice against hepatic ischemia-reperfusion injury by Toll-like receptor-4 suppression and endothelial nitric oxide synthase activation. J Gastroenterol Hepatol 2012;27:1353–1361. 38 Calisto KL, Carvalho Bde M, Ropelle ER, Mittestainer FC, Camacho AC, Guadagnini D, Carvalheira JB, Saad MJ: Atorvastatin improves survival in septic rats: effect on tissue inflammatory pathway and on insulin signaling. PLoS One 2010;5:e14232. 39 Zhang Q, Yang YJ, Wang H, Dong QT, Wang TJ, Qian HY, Xu H: Autophagy activation: a novel mechanism of atorvastatin to protect mesenchymal stem cells from hypoxia and serum deprivation via AMP-activated protein kinase/mammalian target of rapamycin pathway. Stem Cells Dev 2012;21:1321–1332.
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33 Eltzschig HK, Eckle T: Ischemia and reperfusion–from mechanism to translation. Nat Med 2011;17:1391–1401. 34 Mkaddem SB, Bens M, Vandewalle A: Differential activation of Toll-like receptor-mediated apoptosis induced by hypoxia. Oncotarget 2010;1:741–750. 35 Klune JR, Tsung A: Molecular biology of liver ischemia/reperfusion injury: established mechanisms and recent advancements. Surg Clin North Am 2010;90:665–677. 36 Wang L, Zhang X, Liu L, Yang R, Cui L, Li M: Atorvastatin protects rat brains against permanent focal ischemia and downregulates HMGB1, HMGB1 receptors (RAGE and TLR4), NF-κB expression. Neurosci Lett 2010;471:152–156.
Journal of
Nephrology
Am J Nephrol 2014;39:466–475 DOI: 10.1159/000362623
Received: November 11, 2013 Accepted: March 31, 2014 Published online: May 17, 2014
Atorvastatin Improves Survival of Implanted Stem Cells in a Rat Model of Renal Ischemia-Reperfusion Injury Jieru Cai Xiaofang Yu Bingying Zhang Hui Zhang Yi Fang Shaopeng Liu Tongqiang Liu Xiaoqiang Ding Division of Nephrology, Zhongshan Hospital, Shanghai Medical College, Fudan University, Shanghai, China
Abstract Aims: To investigate the impacts of combinatorial atorvastatin (Ator) perioperative administration and mesenchymal stem cell (MSC) implantation on therapeutic effects in the rat experimental acute kidney injury. Methods: The model of renal ischemia-reperfusion (I/R) injury was induced by the release of bilateral renal pedicle clamps following 45 min of occlusion. Immediately after reperfusion, CM-Dil-labeled MSCs (1 × 106 cells) or vehicles only were administered through the carotid artery of the animals pretreated with or without Ator. Results: The combined treatment with Ator and MSCs (Ator+MSCs) markedly reduced the elevated levels of serum creatinine and blood urea nitrogen, as well as the severity of renal damage 24 h after I/R injury. In addition, we also observed inhibition of renal tubular cell apoptosis and promotion of proliferation in the Ator+MSCs group compared with the other groups. Consistent with the improvement in renal function and morphology, Ator pretreatment significantly ameliorated oxidative stress, inhibited inflammation response, and increased the viability of implanted MSCs. With regard to the further mechanism, we found
© 2014 S. Karger AG, Basel 0250–8095/14/0396–0466$39.50/0 E-Mail [email protected] www.karger.com/ajn
that the expression of Toll-like receptor 4 (TLR4) and highmobility group box 1, potential mediators of innate immunity, was significantly decreased in the Ator-treated groups. Conclusion: Ator treatment may protect the kidney undergoing I/R injury through suppression of TLR4 signaling, creating a better environment for the survival of grafted MSCs. The extra benefit of the Ator+MSCs combined therapy may result from the Ator-mediated inhibition of oxidative stress and inflammation in the ischemic kidney. © 2014 S. Karger AG, Basel
Introduction
Acute kidney injury (AKI), predominantly caused by renal ischemia-reperfusion (I/R) injury, is a complex clinical syndrome with high morbidity rate, serious consequences and unsatisfactory therapeutic options [1–3]. Therefore, investigation of alternative therapies is needed. Recently, mesenchymal stem cells (MSCs) have been demonstrated to repair damaged renal structures and improve kidney functions, indicating their therapeutic potential in AKI [4–8]. However, the benefits of MSCs are
J. Cai and X. Yu contributed equally to this work.
Dr. Xiaoqiang Ding Division of Nephrology, Zhongshan Hospital Shanghai Medical College, Fudan University Shanghai 200032 (China) E-Mail ding.xiaoqiang @ zs-hospital.sh.cn
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Key Words Atorvastatin · Mesenchymal stem cells · Renal ischemia-reperfusion injury · Inflammation
Subjects and Methods Animals Male Sprague-Dawley rats, weighing 250–300 g, were obtained from the Animal Center, Shanghai Medical College, Fudan University (Shanghai, China) and housed in a pathogen-free environment with a standard diet. All procedures were approved by the Institutional Animal Care Use Committee of Fudan University. 48 rats were randomly allocated to treatment with or without Ator. I/R surgery was then performed, followed by infusion with MSCs or vehicle only. Each of the following groups contained 12 rats: noAtor+no-MSCs (control), Ator+no-MSCs (Ator), no-Ator+MSCs (MSCs) and Ator+MSCs. Six rats per group were sacrificed per time point, at 24 and 72 h post-I/R, respectively. Isolation and Culture of Bone-Marrow-Derived MSCs MSCs were isolated from Sprague-Dawley rats, weighing 50– 100 g, as previously reported [21]. Cells of passages 6–8 were used for these experiments. At 80% confluence, cells were detached, and labeled with CM-Dil (Invitrogen, USA) according to the manufacturer’s instructions. The labeling efficiency was >98%, as determined by fluorescent microscopy. MSCs were kept on ice in PBS until infusion. I/R Injury Rat Model, Cell Transplantation, and Atorvastatin Administration Rats were anesthetized with pentobarbital sodium (40 mg/kg, i.p.). I/R injury was induced by clamping both renal pedicles for 45 min. Thereafter, the clamps were removed and kidney reperfusion
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was visually confirmed. Immediately after reperfusion, CM-Dillabeled MSCs were injected via the left carotid artery (1 × 106 cells in 500 μl PBS). The animals in the control group received an equal volume of cell-free PBS (vehicle). As previously described [22], treatment with Ator (Lipitor, 5 mg/kg/day by gavage) was started 3 days prior to MSCs transplantation and continued until time of sacrifice. Measurement of Renal Function Blood samples for measurement of blood urea nitrogen (BUN) and serum creatinine (Scr) were collected at time of sacrifice. Scr levels were determined using the Quantichrom Creatinine Assay Kit (BioAssay Systems, Hayward, Calif., USA), following the manufacturer’s protocol. BUN was measured by direct quantification of serum urea with a colorimetric assay kit according to the instruction protocol (BioAssay Systems). Staining and Histological Analysis After fixation in 10% buffered formalin, kidneys were dehydrated with ethanol and embedded in paraffin. For analysis of tubular injury, 3-μm sections were stained with hematoxylin and eosin. Post-ischemic tubular injury, scored by assessing the percentage of tubules in the corticomedullary junction that displayed tubular cell flattening, cell necrosis, loss of brush border, and luminal cast formation, was determined as described previously [23]. Twenty non-overlapping fields of each section were evaluated (400×) as follows: 0 = normal, 1 = 10–25%, 2 = 26–50%, 3 = 51– 75%, and 4 = >75%. TUNEL staining (Merck Calbiochem, Germany) was performed to assess apoptosis at the single-cell level according to the manufacturer’s instructions. Tissue sections, counterstained by Harris’ hematoxylin, were examined microscopically (400×) and at least 400 cells were counted in each of a minimum of eight random HPFs in the corticomedullary junction. The percentage of apoptotic cells was termed the apoptotic index. Immunohistochemical staining for Ki-67 (proliferating cells) was performed as described previously [21], with monoclonal anti-Ki-67 (1:100; Abcam). Scoring for Ki-67-positive cells was carried out in 10 randomly chosen areas in the cortex or outer medulla for each section under 400× magnifications. To determine the differentiation potential of implanted MSCs in vivo, frozen sections at 5 μm thickness were randomly chosen for immunofluorescent staining with anti-cytokeratin-18 (CK-18, 1:100; Abcam) or E-cadherin (E-cad, 1:100; Abcam). In order to quantify the degree of engraftment, MSCs, revealed by their red fluorescence from the CM-Dil, were counted from 10 randomized fields (200×) that contained MSCs under a fluorescent microscope. Those CM-Dil-labeled cells (nuclei stained blue with DAPI) positive for CK-18 or E-cad were considered to be differentiated into renal tubular-like cells. Oxidative Stress-Related Product and Enzymes Kidney samples collected at sacrifice were homogenized and analyzed for oxidative stress levels by test kits according to the manufacturer’s instructions (Jiancheng Institute, Nanjing, China). The enzymatic activities of superoxide dismutase (SOD) were measured via xanthine-oxidation method and myeloperoxidase (MPO) by testing the H2O2-dependent oxidation of o-dianisidine solution. Glutathione (GSH) levels were measured by the 5,5-dithiobis-2-nitrobenzoic acid-glutathione disulfide reductase recy-
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limited by the low viability of transplanted cells in I/Rinjured tissues [9–11]. The pathological environment produced by I/R, characterized by the elevation of reactive oxygen species (ROS) and inflammatory cytokines, is thought to be at least partially responsible for the poor survival and engraftment of injected cells [12]. To address this problem, it is pivotal to protect transplanted cells from adverse effects of a harmful microenvironment. Statin has been shown to exert several biological activities independent of cholesterol-lowering action, including modulation of anti-apoptosis, antioxidant effects, and anti-inflammatory responses [13, 14]. In experimental models of AKI, perioperative therapy with statin has been shown to significantly lower postoperative pro-inflammatory serum cytokine levels, improve renal microcirculation and preserve renal function [15–18], which may protect grafted MSCs. Moreover, it has been reported that statin may enhance the differentiation potential of MSCs [13, 19, 20]. Based on these findings, we hypothesized that pretreatment of the animals with atorvastatin (Ator) improves the renal microenvironments created by I/R injury, thus facilitating the survival of implanted MSCs in vivo.
Enzyme-Linked Immunosorbent Assay (ELISA) The following cytokines and growth factors, associated with the paracrine and/or endocrine effects of MSCs in renal protection, were quantified by ELISA: vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF-1) from kidney homogenates according to the manufacturer’s instructions (R&D Systems), as well as the pro-inflammatory (TNF-α, IL-1β) and anti-inflammatory cytokines (IL-10). Photometric measurements were conducted at 450 nm using microplate reader (Bio-Rad). Western Blot Analysis Homogenized kidney samples were loaded at 30 μg/sample onto 12.5% SDS-PAGE gels for electrophoresis and transferred to PVDF membranes. Membranes were blocked with 5% milk and probed with anti-Bax (1:1,000; Cell Signaling), anti-Bcl2 (1:1,000; Cell Signaling), anti-HMGB1 (1:1,000; Cell Signaling), anti-Tolllike receptor 4 (TLR4) (1:1,000; Abcam), and anti-β-actin (1:2,000; Abcam), followed by incubation with horseradish peroxidase-conjugated secondary antibodies. Specific bands of target proteins were visualized by chemiluminescence. Protein bands were analyzed by ImageJ software and normalized to the β-actin band densities. Statistics Data were collected and analyzed using the SPSS Software (version 13.0), then presented as mean ± SD. Statistical differences between two groups were evaluated with Student’s unpaired t test. One-way ANOVA with the LSD-t test were used for multiple comparisons among four groups. Statistical significance level was defined as p < 0.05.
Results
Therapeutic Potential of Ator and/or MSCs in Renal Ischemia-Reperfusion Injury Post-I/R 24 h, there was a marked reduction in the levels of Scr in the Ator or the MSCs group, compared to the control group (p < 0.05). Similarly, the BUN levels were reduced in the Ator or the MSCs group compared to the control group (p < 0.05). Furthermore, animals in the Ator+MSCs group had sharply decreased Scr and BUN levels when compared to those in the MSCs group (p < 0.05). Post-I/R 72 h, there was a further decline in Scr and BUN levels. Although there was a still notable reduction in the treated groups (the Ator group, the MSCs group, and the Ator+MSCs group) compared with the control group, no significant differences were detected among the treated groups (fig. 1a, b). Histological examination revealed that marked tubular damage occurred in the injured kidney at hour 24 after 468
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surgery, and resulted in a high injury score (3.30 ± 0.65). Compared with the control group, animals in the Ator or the MSCs group had a significantly reduced injury score (p < 0.05). Animals in the Ator+MSCs group had less tubular damage than those in the MSCs group (1.70 ± 0.92 vs. 2.30 ± 0.80, p = 0.019) (fig. 1c, d). These results indicated a synergistic effect of concomitant therapy with Ator and MSCs in restoring the renal function and ameliorating the I/R-induced tubular damage. We next examined whether double therapy can change the renal proliferative response 72 h after I/R injury. The frequency of Ki-67 was higher in the treated groups than that in the control group, especially in the Ator+MSCs group (p < 0.05) (fig. 1e, f). Then, TUNEL analysis was conducted 24 h post-ischemia to determine whether Ator+MSCs dual treatment increased organ protection by enhancing anti-apoptotic action. Apoptosis of renal tubular cells in the Ator or the MSCs group was obviously lower than that in the control group (p < 0.05). Further analysis indicated that animals in the Ator+MSCs group had a significantly decreased apoptotic index compared with that in the MSCs group (p < 0.05) (fig. 1g, h). The quantitative analysis of apoptosis-regulating proteins revealed that expression of the pro-apoptotic protein Bax was significantly downregulated and anti-apoptotic protein Bcl-2 was upregulated in the Ator or the MSCs group compared with the control group (p < 0.05). Bcl-2 expression reached the peak value in the Ator+MSCs group, but the presence of Ator did not alter expression of Bax in the Ator+MSCs group compared with the MSCs group (fig. 1i). In summary, combined treatment with Ator and MSCs markedly attenuated renal dysfunction and the severity of renal damage, inhibited apoptosis, and ameliorated proliferation in injured kidney compared with MSCs-alone treatment. Improvement of the Survival and Immune-Modulatory Effects of MSCs in vivo in the Presence of Ator Pretreatment Immunofluorescent analysis of renal sections revealed that 24 h post-ischemia, the numbers of CM-Dil-labeled cells in the Ator+MSCs group was significantly higher than that in the MSCs group (32.33 ± 3.51 vs. 22.67 ± 3.79, p = 0.015). At 72 h post-ischemia, few cells were detected in the section and no significant differences were found in the number of labeled cells between the two groups (fig. 2a, b). Further analysis revealed that none of the CMDil-labeled cells were positive for CK-18 or E-cad in either of these groups during the treatment period, which Cai/Yu/Zhang/Zhang/Fang/Liu/Liu/Ding
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cling method. The malondialdehyde (MDA), a secondary product of lipid peroxidation, was determined as thiobarbituric acid (TBA)-reactive substance.
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group or the MSCs group was significantly decreased compared with that in the control group, and the Ator+MSCs group had a further decrease in apoptotic cells compared with the MSCs groups. i Compared with the control group, the expression of the pro-apoptotic protein Bax was significantly downregulated and anti-apoptotic protein Bcl-2 was upregulated in treated animals, especially in the Ator+MSCs group. * p < 0.05 vs. control group; # p < 0.05 vs. MSCs group.
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than that in the MSCs group. Moreover, there were trends towards upregulation of VEGF and HGF in the Ator+MSCs group compared with the MSCs group. At 72 h post-ischemia, double therapy with Ator and MSCs improved the expressions of these cytokines compared with the control group. * p < 0.05 vs. control group; # p < 0.05 vs. MSCs group.
indicated that MSCs did not differentiate into renal tubular cells after transplantation (data not shown). The immune-modulatory effects of MSCs were examined by ELISA analysis of some cytokines and growth factors involved in kidney repair (fig. 2c). At 24 h post-ischemia, the expression levels of IGF-1 in the Ator+MSCs
group were significantly higher than those in the MSCs group. Moreover, there were trends towards upregulation of VEGF and HGF in the Ator+MSCs group compared with the MSCs group. At 72 h post-ischemia, we noted that the expressions of b-FGF and HGF in the Ator+MSCs group significantly improved (p < 0.05) compared with
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Fig. 3. Assessment of oxidative stress and inflammatory response in the injured kidney at 24 h post-I/R. a Ator treatment inhibited the expression of the inflammatory cytokines (IL-1β and TNF-α) and promoted expression of the anti-inflammatory cytokine (IL10) compared with the control group (p < 0.05). In the Ator+MSCs group, the expression of IL-1β further decreased compared with the MSCs group (p < 0.05). b The MPO activity and the MDA concentration in the Ator group were decreased compared with the control group. Lower MDA concentration and higher GSH level
were observed in the Ator+MSCs group compared with the MSCs group, and higher SOD activity compared with the Ator group. c The expressions of HMGB1 and TLR4 were downregulated in monotherapy groups compared with the control group and further decreased HMGB1 and TLR4 expression were detected in the Ator+MSCs group compared with monotherapy groups, although some differences between groups did not prove to be statistically significant. * p < 0.05 vs. control group; # p < 0.05 vs. MSCs group; † p < 0.05 vs. Ator group.
those in the MSCs group, while no differences in VEGF and IGF-1 were found between these MSCs-implanted groups.
greater effect on inhibiting inflammation than the MSCs group, although significant difference was only noted in IL-1β expression (p < 0.05). Moreover, the benefit of the dual therapy was no better than that of Ator-alone treatment (fig. 3a). With regard to the oxidative stress-related products and enzymes, which included MPO, MDA, SOD and GSH, we found that the MPO activity and the MDA concentration were lower in the Ator group compared with the control group (p < 0.05), while no significant differences were detected in these four parameters between the MSCs group and the control group. In combined therapy group, lower MPO activity and MDA concentration, and higher SOD activity and GSH level were observed compared with monotherapy groups, although most of the differences were not significant (fig. 3b). Thus, we inferred that the protective effects of Ator may
Reduced Oxidative Stress and Inhibition of Inflammatory Cytokine Expression in the Kidney with Ator Treatment via the TLR4 Pathway We assessed the renal microenvironment by analyzing the expression of inflammatory-associated cytokines, and several oxidative stress-related products and enzymes in the kidney at day 1 post-I/R. Compared with the control group, the inflammatory cytokines (TNF-α and IL-1β) were markedly downregulated and the anti-inflammatory cytokine (IL-10) upregulated in the Ator group (p < 0.05), but these cytokines did not significantly improve in the MSCs group. The Ator+MSCs group produced a Ator Treatment Improves MSCs
Am J Nephrol 2014;39:466–475 DOI: 10.1159/000362623
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Discussion
In the present study, we have demonstrated that Ator+MSCs promotes greater recovery of kidneys undergoing I/R injury than MSCs alone. A low-dose and shortterm administration of Ator to the animals around the time of MSCs transplantation enhances the survival and paracrine/endocrine effects of MSCs in the ischemic kidney. With regard to the potential mechanism, extra benefits from combined treatment may be associated with Ator pretreatment, by improving the renal microenvironment via the TLR4 pathway. The kidney undergoing I/R results in extensive and complex inflammatory/oxidative stress responses [12]. Although MSCs have been shown to demonstrate great therapeutic promise in renal I/R injury [5–8], the harsh post-ischemic renal microenvironment reduces the survival of implanted cells within target sites, thus limiting their benefits [9–11]. To address this problem, a number of studies aimed at promoting the retention and survival of MSCs to increase the number of cells successfully engrafting in the injured kidney have been published. The main strategy utilized has involved the alteration of ‘seeds’ meaning donor cells by preconditioning or genetic modification of MSCs [24–26]. However, these techniques do not always provide sufficient effects for the improvement of the cell survival, and further exploration is required for clinical application due to the complexity of these procedures. In this study, we established an ap472
Am J Nephrol 2014;39:466–475 DOI: 10.1159/000362623
Ator perioperative administration TLR4-HMGB1 pathway ଯ
Soil: kidneys
Oxidative stress ଯ Inflammation ଯ Improve renal microenvironment
Seed: MSCs
MSCs survival ଭ Immune modulatory function ଭ Apoptosis ଯ Proliferation ଭ I/R injury ଯ
Fig. 4. Schema of the mechanisms underlying renal protection conferred by Ator and MSCs treatment. Ator pretreatment downregulates the activation of TLR4-HMGB1 pathway in the kidney, consequently resulting in the reduced oxidative stress and decreased inflammation. The improved renal microenvironment strengthens the renal protective functions conferred by MSCs, through promotion of MSCs survival and generation of renoprotective factors. The improved renal microenvironment and the corresponding increased survival of MSCs further promote the recovery of injured kidney.
proach by improving the ‘soil’, the microenvironment of target organ, with Ator to facilitate survival and biological behavior of implanted cells. Statin, a drug known to inhibit cholesterol biosynthesis, has been shown to possess pleiotropic effects that include enhancement of endothelial nitric oxide production, anti-inflammatory and anti-oxidative responses [27–30]. Therefore, statin may be effective in improving the renal microenvironment generated by I/R injury. Our findings indicated that Ator administration downregulated the expression of oxidative stress and inflammation markers in the injured kidney, specifically MDA, MPO, IL-1β and TNF-α, while upregulating the expression of IL-10. These findings were consistent with previous reports [17, 31, 32]. In order to further investigate the mechanisms by which Ator protects infused MSCs, we concentrated on the TLR signaling pathways, especially TLR4 and its enCai/Yu/Zhang/Zhang/Fang/Liu/Liu/Ding
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be related to modulation of renal inflammation and oxidative stress caused by I/R injury and there was a partial synergism between Ator and MSCs. To further confirm whether the benefit of Ator treatment is associated with the activation of innate immunity via the TLR signaling pathways, we examined the regulation of TLR4 and its endogenous ligand, HMGB1, within the kidney. The expressions of TLR4 and HMGB1 were significantly downregulated in the Ator group compared to the control group (p < 0.05) and the same trend of TLR4 and HMGB1 expression existed in the MSCs group. In the Ator+MSCs group, the levels of TLR4 and HMGB1 were further decreased compared with the monotherapy groups, although there was no significant difference in expression of HMGB1 between the Ator+MSCs group and the Ator group (fig. 3c). Taken together, we inferred that the heightened effects of treatment with Ator may be associated with the downregulation of TLR and HMGB1 expression.
dogenous ligand HMGB1. TLR4 signaling pathways are potential mediators of innate immune responses occurred by I/R injury [33]. Some experiments have shown that this pathway plays a key role in the induction of inflammation response, and oxidative stress injury in a variety of ischemic tissues [34, 35]. Our results confirmed that the expression of TLR4 and HMGB1 was significantly decreased in the Ator group when compared with the control group. With the presence of Ator, MSCs could further decrease the expression of TLR4 and HMGB1. Our preliminary conclusion is that HMGB1-TLR4 signaling pathways may be involved in the improvement of the renal microenvironment. However, in this study the exploration of signaling pathways is limited to protein level expression. Although these results could not fully elucidate the role of Ator in the TLR4 signaling pathway, previous experiments have demonstrated that Ator can attenuate the activation of TLR4 expression and its downstream pathways, including mitogen-activated protein kinase, NF-κB and AMP-activated protein kinase [33, 36–39]. Therefore, it can be speculated that the effect of Ator on the suppression of the TLR-dependent inflammatory pathway may play a key role in promoting the recovery of the kidney. Accumulating evidence has demonstrated that the infusion of MSCs results in anti-inflammatory and antiapoptotic effects in animal models of I/R injury [8, 10, 21, 24, 40]. Our research indicates that MSCs administration at the time of reperfusion could provide a degree of protection against AKI. A major obstacle in using cell therapy is the low survival of transplanted cells in the adverse renal microenvironment and the death of the majority of transplanted cells within the first day after transplantation. Our results confirmed that the number of engrafted MSCs in the Ator+MSCs group was significantly improved compared with the MSCs group 24 h post-ischemia. Moreover, MSCs transplantation, in conjunction with Ator pretreatment of the animals, may dramatically attenuate renal dysfunction and morphological alterations, inhibit apoptosis and enhance proliferation in injured kidney when compared with MSCs transplantation alone after I/R. The extra benefit of combined therapy may stem from improvement in the renal microenvironment created by perioperative treatment the animals with Ator, and may improve the survival of injected MSCs. However, the mechanisms by which MSCs ameliorate I/R injury remain to be elucidated. Several researches have indicated that MSCs possess the capacity to differentiate into renal tubular cells [6, 41]. Although Ator has been shown to increase the potential of differentiation of
implanted cells in post-infarct myocardium [13], our results were consistent with other studies that showed no transdifferentiation of MSCs into renal tubular-like cells [10, 42]. Ator administration improved the survival of MSCs; however, it did not increase the potential of differentiation of implanted cells in post-ischemic kidney. Other studies have suggested that the reparative effects of MSCs can be primarily ascribed to their endocrine/paracrine functions and this result was also confirmed in our study [10, 42]. Compared with MSCs delivery alone, treating the animals with Ator prior to MSCs delivery further increased the secretion of growth factors and cytokines (VEGF, HGF, and IGF-1). These proteins are known to improve renal function in AKI, and exert more powerful multifunctional immune-regulatory activities, such as inhibition of apoptosis and suppression of inflammation. The improved renal microenvironment and the corresponding increased survival of MSCs further promote the recovery of injured kidney. In conclusion, for the first time, this study has documented experimental evidence that the Ator+MSCs combined therapy may have a synergistic effect on the regeneration and repair of renal function and morphology post-ischemia. The benefit may result from the statinmediated inhibition of oxidative stress, and inflammation in the injured kidney via suppression of TLR4 signaling pathway, resulting in a better environment for the survival of implanted MSCs (fig. 4). The data from the present study also offer a potentially new clinical strategy for the application of stem cell transplantation in the treatment of AKI.
Ator Treatment Improves MSCs
Am J Nephrol 2014;39:466–475 DOI: 10.1159/000362623
Acknowledgements We thank Pfizer for donation of atorvastatin. We also thank Dr. Rende Xu, Karen M. Peterson, and Dr. Jia Guo for critically reading the manuscript. This work was supported by National Natural Science Foundation of China (81000307), the Major Project of Basic Research of Technology Committee in Shanghai of China (12DJ1400200).
Disclosure Statement
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The authors have no conflicts of interest to disclose.
References
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