Induced Pluripotent Stem Cell-Derived Conditioned Medium Attenuates Acute Kidney Injury by Downregulating the Oxidative Stress-Related Pathway in Ischemia–Reperfusion Rats

Abstract

Teratoma-like formation addresses a critical safety concern for the potential utility of induced pluripotent stem cells (iPSCs). Therefore, therapy utilizing iPSC-derived conditioned medium (iPSC-CM) for acute kidney injury (AKI) has attracted substantial interest. A recent study showed that iPSC-CM effectively alleviated ventilator-induced lung injury in rats. It prompts us to assess the therapeutic effects of iPSC-CM on ischemic AKI. First, we assessed the changes in renal function and tubular cell apoptosis by intraperitoneal administration of iPSC-CM to ischemia–reperfusion (I/R) rats. Second, we explored the oxidative stress-related pathway in the apoptosis of renal tubular cells subjected to hypoxia–reoxygenation (H/R). Administration of iPSC-CM significantly improved renal function and protected tubular cells against apoptosis in rats with I/R-AKI, and the optimal effect was observed at the 50-fold concentrated iPSC-CM. iPSC-CM also mitigated the H/R-induced apoptosis of NRK-52E cells in vitro. Reactive oxygen species (ROS) production was augmented in kidneys following I/R and in NRK-52E cells subjected to H/R. Meanwhile, expressions of phosphorylated p38 MAPK, TNF-α, and cleaved caspase 3 and NF-κB activity were consistently increased in vivo and in vitro. Following administration of iPSC-CM, ROS production was abolished, and inflammatory cytokine expression was significantly suppressed. Annexin V–propidium iodide flow cytometry and in situ TUNEL assay further showed that iPSC-CM markedly attenuated H/R- or I/R-induced tubular cell apoptosis. Intriguingly, treatment with iPSC-CM significantly improved the survival of rats with I/R-induced AKI. iPSC-CM represents a favorable source of stem cell-based therapy and may serve as a potential therapeutic strategy for kidney repair in ischemic AKI.

Introduction

Acute kidney injury (AKI), characterized by a rapid decline of renal function, is common in critically ill patients with sepsis, major operations, or any form of shock (17). Emerging evidence suggests that AKI is not merely a localized damage but also a trigger for a systemic inflammatory response which eventually leads to adverse effects in other organs such as the heart, lung, and liver (28). This explains why AKI associates with a remarkably high morbidity and mortality, and even a small increase in serum creatinine could predispose patients toward progression of chronic kidney disease and death (14). Although current therapeutic approaches for AKI remain largely supportive (15), to modulate the local AKI-induced systemic inflammatory response is a potentially promising strategy (28).

Renal ischemia is the major etiology and accounts for 50% of clinical AKI cases (32). Emerging evidence suggests that oxidative stress underlies the pathogenesis of ischemia–reperfusion-induced AKI (I/R-AKI) (1,27). Ischemia results in profound tissue hypoxia of the kidney, and subsequent reperfusion causes cellular reoxygenation, which in turn generates reactive oxygen species (ROS), activates the inflammatory response, and promotes tubular cell apoptosis (5,9). Apoptosis of renal tubular cells is the major contributor for the development of I/R-AKI (5,9). The inflammatory response in I/R kidney injury is characterized by increased monocyte infiltration, activation of mitogen-activated protein kinase (MAPK), tumor necrosis factor (TNF)-α, and nuclear factor (NF)-κB (8,25,29). Through the signaling pathway, excess ROS could be involved in tubular epithelial cell apoptosis by triggering the caspases during reoxygenation (21).

Stem cell therapy has recently been demonstrated to markedly facilitate tubular cell proliferation and kidney repair and improve recovery of kidney function in I/R-AKI (2,12,19,26,33). The prospect of cell therapy utilizing induced pluripotent stem cells (iPSCs) for AKI has attracted substantial interest. Our recent study has shown that iPSCs via intrarenal arterial administration to I/R kidneys in rats posed antioxidant, anti-inflammatory, and antiapoptotic properties to attenuate tubular cell injury and kidney failure (20). Our findings highlight the potential of iPSC therapy for I/R-AKI in the future. However, the adverse effects such as pulmonary embolism and teratoma formation warrant concern in iPSC therapy. Intriguingly, the proliferative and regenerative tubular cells actually outnumbered the engrafted iPSCs, indicating the possibility of a paracrine reparative effect (20). Recently it was shown that administration of iPSC-derived conditioned medium (iPSC-CM) alone could effectively improve endotoxin-and ventilator-induced acute lung injury (23,38), which attests the paracrine protective effect of iPSC-CM. Until now, the renoprotective effect of iPSC-CM on I/R-AKI has not been assessed. Therefore, we aimed to examine whether iPSC-CM via intraperitoneal administration could exhibit the systemic protective effect against I/R-AKI in rats. We further assessed the mechanisms of iPSC-CM in protecting renal tubular cells against apoptosis in hypoxia–reoxygenation (H/R) in vitro or I/R in vivo.

Materials and Methods

Animals

C57/B6 mice purchased from the Bio-LASCO Taiwan Co., Ltd. (Taipei, Taiwan) and Sprague–Dawley rats obtained from the Laboratory Animal Center of the National Yang-Ming University (Taipei, Taiwan) were raised in a sound-attenuated room with a 12-h light–dark cycle. All experimental procedures and protocols were approved by the Institutional Animal Care and Use Committee of the National Yang-Ming University and complied with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health.

iPSC Culture and Conditioned Medium

For preparation of mouse embryonic fibroblasts (MEFs), C57/B6 mice were mated naturally, and embryos were harvested from the uteri of 13.5-day pregnant females. After decapitation and evisceration, the carcasses were minced with scissors, washed in phosphate-buffered saline (PBS; Gibco, Grand Island, NY, USA), and digested by 0.25% trypsin–EDTA (Life Technologies, Grand Island, NY, USA) solution at 37°C for 20 min. After trypsinization, Dulbecco's modified Eagle medium (DMEM; Sigma-Aldrich, St. Louis, MO, USA) containing 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA) was added with gentle pipetting to help dissociation. After incubating the tissue/medium mixture for 10 min at room temperature, the supernatant was retrieved, and cells were collected by centrifugation (200 × g, for 5 min at 4°C) and resuspended in MEF medium [high-glucose DMEM (Gibco) supplemented with 10% FBS (Invitrogen), 1% penicillin/streptomycin (Invitrogen), 1 mM sodium pyruvate (Life Technologies), and 2 mM L-glutamine (Life Technologies)] and cultured at 37°C with 5% CO₂. At confluence, MEFs were treated with 10 mg/ml mitomycin C (Thermo Fisher Scientific, Rockford, IL, USA) for 3 h at 37°C. The mitotically inactivated MEFs were then dispersed by 0.05% trypsin–EDTA (Life Technologies) and replated at 5 × 10⁴ cells/cm² as feeder cells.

Murine iPSCs were generated from MEFs derived from 13.5-day-old embryos of C57/B6 mice by retroviral transduction of three transcriptional factors, octamerbinding transcription factor-4 (Oct-4), sex-determining region Y-box 2 (Sox2), and Kruppel-like factor 4 (Klf4) as described previously with slight modifications (13,22,31). Briefly, in order to get the viral particles, Plat-E cells (Cell Biolabs Inc., San Diego, CA, USA) were seeded at 3.6 × 10⁶ cells per 100-mm dish (Becton Dickinson, Franklin Lakes, NJ, USA) 1 day before transduction. On the next day, pMX-based retroviral vectors (Academia Sinica, Taipei, Taiwan) encoding mouse complementary deoxyribonucleic acid (DNA) were introduced into Plat-E cells using FuGENE 6 Transfection Reagent (Roche Applied Science, Indianapolis, IN, USA) according to the manufacturer's protocol. Forty-eight hours after transfection, virus-containing supernatants were collected for target cell infection. The virus-containing supernatants were filtered through a 0.45-μm filter (BD) and supplemented with 4 mg/ml polybrene (Millipore, Billerica, MA, USA). On the day before viral infection, MEFs were seeded (8 × 10⁵ cells/well) into six-well dishes (BD). Equal amounts of supernatants containing viruses encoding each of the three genes were mixed and transferred to the MEF dish and incubated overnight. The culture medium was changed every other day. After 6 days, transduced MEFs were cultured on fresh feeder cells. Embryonic stem cell-like colonies appeared from large background colonies 7 days after viral transduction and were manually picked on days 24 to 30. Colonies that maintained their embryonic stem cell-like morphology were further passaged and analyzed for pluripotent potential (7).

Undifferentiated iPSCs (50,000 cells/cm²) were routinely cultured and expanded on mitotically inactivated feeder cells in six-well culture plates (BD) in the presence of 0.3% leukemia inhibitory factor (LIF; StemCell Technologies, Vancouver, Canada) in an iPSC medium consisting of DMEM (Sigma-Aldrich) supplemented with 15% FBS (Invitrogen), 100 mM minimal essential medium nonessential amino acids (Sigma-Aldrich), 0.55 mM 2-mercaptoethanol (Gibco), and penicillin/streptomycin (Invitrogen). Every 3 to 4 days, colonies were detached with 0.2% collagenase IV (Invitrogen), dissociated into single cells with 0.025% trypsin (Sigma-Aldrich) and 0.1% chicken serum (Invitrogen) in PBS, and cultured onto fresh feeder cells.

For preparation of iPSC-CM, iPSCs were placed at 10,000 cells per cm² and incubated in growth medium for 1 day as previously described (38). The attached cells were washed thrice with PBS (Gibco), and the medium was replaced with serum-free basal medium [DMEM–high glucose (Gibco), 10 mM nonessential amino acid (Gibco), 0.3% LIF (StemCell Technologies), and 1% penicillin/streptomycin (Invitrogen)] and incubated for 48 h. After detachment by trypsin (Sigma-Aldrich), the iPSCs with whole culture medium were collected and centrifuged for 10 min at 230 × g with a Beckman centrifuge (C1015 rotor; Beckman Coulter, Inc., Fullerton, CA, USA) to obtain the supernatant as the iPSC-CM. The iPSC-CM was further concentrated 25-fold and 50-fold by ultrafiltration using centrifugal filter units with a molecular weight cutoff of 5 kDa (Millipore) following manufacturer's instructions. Medium was added to culture dishes without iPSCs and processed identically as the normal control medium (NM). Our previous work has indicated that iPSC-CM contained increased levels of various trophic and proangiogenic factors such fibroblast growth factor-2 (FGF-2), vascular endothelial growth factor (VEGF), and angiopoietin-1 (ANGPT1) (23).

Animal Experiments

Adult Sprague–Dawley rats (male, 8 weeks old, approximately 300 g) were anesthetized with intraperitoneal injection of sodium pentobarbital (50 mg/kg; Gane's Chemical Works, New York, NY, USA). For induction of total ischemia in the kidney, the left renal artery was clamped with a nontraumatic vascular clamp (Karl Klappenecker, Tuttlingen, Germany) for 45 min. Afterward, reperfusion was initiated by releasing the clamp, and the right kidney was removed simultaneously. To investigate the effect of iPSC-CM on I/R injury, 1 ml of PBS, NM, and onefold, 25-fold, or 50-fold concentrated iPSC-CM was injected intraperitoneally twice per day after I/R injury. Sham-operated animals underwent similar operative procedures without receiving clamping of the left renal artery and right nephrectomy. Rats were sacrificed 48 h later to collect the blood and kidney tissue samples. The kidney was divided into two parts. One part was stored for histological analysis, and the other part was quickly frozen and stored in liquid nitrogen for protein analysis. The blood samples were allowed to clot and then centrifuged at 1,000xg for 30 min. Serum levels of blood urea nitrogen (BUN) and creatinine were determined by an Olympus AU-2700 autoanalyzer (Olympus Ltd, Tokyo, Japan).

Histology and Tubular Injury Score

Kidney tissues were fixed with 4% phosphate-buffered formalin solution (Macron Chemicals, Center Valley, PA, USA), embedded in paraffin, and cut into 3-μm sections. Sections were stained with hematoxylin (Merck, Darmstadt, Germany) and eosin (Sigma-Aldrich) (H&E) and periodic acid-Schiff reagent (Sigma-Aldrich). Tubular injury (tubular cell swelling, loss of brush border, or nuclear condensation) was scored from 0 to 4 (0, no changes; 1, changes affecting 25%; 2, changes affecting 25% to 50%; 3, changes affecting 50% to 75%; 4, changes affecting 75% to 100% of the section) as previously described (20).

Immunohistochemistry and In Situ Apoptosis Detection

Immunohistochemical staining was performed as previously described (20). Briefly, formalin-fixed paraffin-embedded sections of kidneys were deparaffinized with xylene and rehydrated in graded alcohols. Sections were subjected to heat-mediated antigen retrieval in 0.01 M citrate buffer (pH 6.0; BioShop, Burlington, Ontario, Canada), and endogenous peroxidase was quenched by 3% H₂O₂ (Sigma-Aldrich). Sections were then incubated with anti-active p65 subunit of NF-κB antibody (1:200; Millipore) overnight at 4°C. After washing, the sections were incubated with Envision horseradish peroxidase (HRP)-labeled polymer (Dako, Glostrup, Denmark) for 1 h at room temperature. The sections were visualized with 3,3′-diaminobenzidine (DAB) (Dako) and counterstained with Gill's hematoxylin (Merck). The number of positive cells was quantified in 15 randomly selected high-power fields per section.

To detect apoptosis in the kidney, the terminal deoxynucleotidyl transferase (TdT)-mediated digoxigenin-deoxyuridine triphosphate nick-end labeling (TUNEL) assay was conducted using the Apoptag® Peroxidase In Situ Apoptosis Detection kit (Millipore) according to the manufacturer's instruction. Apoptotic cells were visualized by DAB (Dako). The presence of nuclear brown staining (TUNEL positive) indicated apoptotic cells. Negative controls were stained without TdT enzyme.

Hypoxia–Reoxygenation of Tubular Epithelial Cells

The rat kidney tubular epithelial cell line (NRK-52E) was obtained from the Food Industry Research and Development Institute (Hsinchu, Taiwan). Cells were maintained in DMEM containing 4.5 g/L glucose (Invitrogen) with 10% FBS at 37°C in a 5% CO₂ atmosphere and passaged twice a week. Cells were plated in 35-mm dishes (Corning Inc., Corning, NY, USA) at a density of 1 × 10⁶ cells/dish, reached ~90% confluence by the next day, and were used for experiments. For hypoxia experiments, cells were treated in a hypoxia C-chamber (BioSpherix, Lacona, NY, USA) inside a standard CO₂ incubator (Revco Ultima II; Thermo Fisher Scientific) with a compact gas oxygen controller (ProOx P110; BioSpherix) to maintain oxygen concentration at 1% by introducing a gas mixture of 95% N₂ and 5% CO₂ for 24 h. After hypoxic treatment, the cells were washed with PBS (Gibco) and cultured with NM or iPSC-CM in 95% O₂/5% CO₂ condition for 6 h (reoxygenation). Control cells were incubated in a regular cell culture incubator with 21% O₂. At the end of the study, cells were monitored morphologically or harvested with indicated buffers to collect cell lysates for biochemical analyses.

Cell Viability Assay

NRK-52E cells were seeded into 24-well plates (Costar, Cambridge, MA, USA) with culture medium, and cell viability was measured by the colorimetric 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay (Sigma-Aldrich). After H/R, 10 μl of MTT solution (5 mg/ml) was added to each well and incubated for 1 h at 37°C. Wells were emptied and blue formazan crystals were dissolved with 100 μl of MTT solubilization solution (10% Triton X-100, 0.1 N HCl in 2-propanol). Then the plate was shaken vigorously for 10 min. Optical density was read at 570 nm using a TiterTek Multiskan microplate reader (Flow Laboratories, McLean, VA, USA).

Determination of In Vitro ROS Production

Lucigenin-enhanced chemiluminescence (CL) was used to detect ROS production in NRK-52E cells following H/R (6). ROS was measured in NRK-52E cells treated with normoxia + NM, normoxia + iPSC-CM, H/R + NM, and H/R + iPSC-CM, respectively. Tubular cells were then washed by PBS for measurement of CL in a completely dark chamber of the Chemiluminescence Analyzing System (CLD-110; Tohoku Electronic Industrial, Sendai, Japan). After background level of CL was determined for 60 s, 1 ml of 0.1 mM lucigenin (Sigma-Aldrich) in PBS was added into each sample. The CL was recorded continuously for an additional 300 s. The total amount of CL was calculated by integrating the area under the curve following subtraction of the background level. The assay was performed in duplicate for each sample and was expressed as CL counts/10 s.

Measurement of Ex Vivo ROS Production

For measurement of ROS production in rat kidney, 50 mg of kidney tissue was homogenized with 1 ml of PBS, and 0.2 ml of tissue solution was used. Tissue ROS was measured using the lucigenin-enhanced CL method as described previously (6). The CL was then measured in the dark chamber of the Chemiluminescence Analyzing System (CLD-110; Tohoku Electronic Industrial). After background level of CL was determined for 60 s, 0.5 ml of 0.1 mM lucigenin (Sigma-Aldrich) in PBS (pH 7.4) was added to the sample, and CL was continuously monitored for an additional 540 s. The total amount of CL was calculated by integrating the area under the curve after subtracting the background CL. The results were expressed as CL counts/10 s.

In Situ ROS Detection

Dihydroethidium (DHE) staining was utilized to analyze the location of ROS production as previously described with some modifications (10). Renal tissue was cut into 3-μm frozen sections and permeabilized by ice-cold acetone for 5 min. Afterward, the section was incubated with mouse anti-rat CD68 antibody (1:100; AbD Serotec, Oxford, UK) at 4°C overnight. Then the section was incubated with goat anti-mouse fluorescein isocyanate (FITC)-conjugated secondary antibody (1:50; Jackson ImmunoResearch Lab oratories, West Grove, PA, USA) at room temperature for 2 h. The section was stained with DHE (100 μM; Sigma-Aldrich) at room temperature for 30 min and counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich). The ethidium–DNA fluorescence was examined at 520-nm excitation and 580-nm emission using an epifluorescence microscope (AX80; Olympus).

Western Blotting

Cytoplasmic and nuclear protein extracts were prepared by a commercial Nuclear Extraction kit (Cayman Chemical, Ann Arbor, MI, USA). Kidney tissues or whole-cell lysates were processed by Western blot using primary rabbit antibodies for p38 MAPK (1:1,000; Cell Signaling, Beverly, MA, USA), phospho-p38 MAPK (1:1,000; Cell Signaling), extracellular signal-regulated kinase (Erk) 1/2 (1:2,000; Cell Signaling), phospho-Erk 1/2 (1:1,000; Cell Signaling), c-Jun N-terminal kinase (JNK, 1:1,000; Cell Signaling), phospho-JNK (1:2,000; Cell Signaling), TNF-α (1:1,000; Abcam, Cambridge, UK), phospho-NF-κB p65 (1:1,000; Cell Signaling), phospho-NF-κB p50 (1:200; Cell Signaling), interleukin-6 (IL-6, 1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA), interferon-γ (1:1,000; Abcam), monocyte chemotactic protein-1 (MCP-1, 1:200; Santa Cruz), β-actin (1:1,000; Cell Signaling), and poly (ADP-ribose) polymerase (PARP, 1:1,000; Cell Signaling) as well as primary mouse antibodies for active cleaved caspase 3 (CPP32, 1:500; Sigma-Aldrich), NF-κB p65 (1:500; Santa Cruz Biotechnology), NF-κB p50 (1:200; Santa Cruz Biotechnology), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, 1:1,000; Abcam) were used. Afterward, species-directed secondary antibodies, goat anti-rabbit (Abcam), or horse anti-mouse (Sigma-Aldrich) HRP-conjugated IgG were used. Data were normalized to GADPH or β-actin expression.

ELISA for NF-κB p65 Activity

Active NF-κB DNA-binding activity in the nucleus was measured with Trans-AM NF-κB p65 transcription factor assay kits (Active Motif Europe, Rixensart, Belgium) according to manufacturer's instructions. Briefly, 2 μg of nuclear extract was added to 96-well plates coated with an oligonucleotide containing the NF-κB consensus binding site. This oligonucleotide then specifically bound to the active form of NF-κB p65 subunit contained in the nuclear extract. Thereafter, primary antibody that recognized the active form of NF-κB p65 was added. Afterward, a secondary HRP-conjugated antibody was used with tetramethylbenzidine substrate. The reaction was stopped with 0.5 M H₂SO₄ and measured at 450 nm.

Flow Cytometeric Analysis for Apoptotic Tubular Cells

Apoptotic tubular cells were detected by a commercial Annexin V–FITC Apoptosis Detection Kit (eBioscience, San Diego, CA, USA). NRK-52E cells were cultured for 24 h on a 6-cm Petri dish (Becton Dickinson, Oxnard, CA, USA) and incubated in hypoxic or normoxic conditions for 24 h. Then cells were cocultured with NM or iPSC-CM for 6 h in the normoxic condition. Cells were detached by trypsin (Sigma-Aldrich), centrifuged, and washed twice with ice-cold PBS (Gibco). Afterward, cells were resuspended using 1× binding buffer, and the cell number was adjusted to 1 × 10⁶ per 100 μl. One hundred microliters of cell suspension was taken and added into a 5-ml centrifuge tube, followed by adding 5 μl of annexin V–FITC and 10 μl of propidium iodide. Cells were incubated in the dark at room temperature for 30 min and resuspended in 400 μl of 1× binding buffer. Finally, cells were analyzed by a FACSCalibur flow cytometer (Beckman Instrument) with an excitation wavelength at 488 nm. All experiments were performed in triplicate.

Statistical Analysis

Data were shown as means ± standard errors of the mean. Between-group comparisons were analyzed by unpaired t-test or one-way ANOVA followed by the Tukey's post hoc analysis for multiple comparisons where appropriate. Survival curves were generated by Kaplan–Meier's method and compared by log-rank test. A two-tailed value of p < 0.05 was considered statistically significant. All statistical analysis was performed by Statistical Package of Social Science (SPSS 15.0, 2006; IBM, Armonk, NY, USA).

Results

iPSC-CM Ameliorated Renal Tubular Cell Apoptosis in I/R Kidney Injury

To determine the effects of iPSC-CM on I/R-AKI in rats, PBS, NM, as well as 1-fold, 25-fold, and 50-fold concentrated iPSC-CM were administered intraperitoneally twice daily after I/R injury for 2 days, respectively. Forty-eight hours after I/R injury, BUN and serum creatinine levels in the 50-fold iPSC-CM group were significantly reduced compared with PBS- and NM-treated groups (Fig. 1A). Histological examination showed that 50-fold iPSC-CM markedly attenuated the tubular injury in I/R-kidney (Fig. 1B). No significant differences in renal function (Fig. 1A) and tubular injury score (Fig. 1B) were observed among PBS, NM, onefold and 25-fold iPSC-CM-treated groups. Number of TUNEL-positive tubular cells significantly increased in PBS, NM, and onefold CM-treated group compared to the sham-operated group. Among iPSC-CM-treated groups, the number of TUNEL-positive tubular cells was significantly reduced only in the 50-fold iPSC-CM group (Fig. 1C).

Figure 1.

iPSC-CM improved renal function and attenuated tubular injury in rats with I/R-AKI. (A) Blood urea nitrogen (BUN) and serum creatinine levels were determined 48 h after reperfusion in the sham-operated group, I/R + PBS, I/R + NM, and I/R + iPSC-CM (onefold, 25-fold, and 50-fold concentrated) groups via intraperitoneal route. (B) Representative histology and tubular injury score of kidney samples taken from sham-operated, I/R + PBS, I/R + NM, and I/R + iPSC-CM rats 48 h after I/R injury, respectively. (C) Apoptosis of renal tubular cells measured by terminal deoxynucleotidyl transferase-mediated digoxigenin-deoxyuridine triphosphate nick-end labeling (TUNEL) assay. n = 5 in each group. Data were expressed as mean ± SEM. *p < 0.05 versus sham-operated group. #p < 0.05 versus I/R + PBS group.

iPSC-CM Improved Cell Viability in Hypoxia–Reoxygenation

To establish an in vitro model to simulate I/R-related AKI of rats, NRK-52E cells were cultured under hypoxic conditions. Using MTT assay, the viability of NRK-52E cells was significantly suppressed by 24 h of hypoxia followed by 6 h of reoxygenation (Fig. 2A). Impressively, the inhibitory effect of H/R in cell viability was significantly mitigated by coculture with iPSC-CM (Fig. 2B).

Figure 2.

iPSC-CM reduced apoptosis of NRK52E cells after hypoxia–reoxygenation (H/R) via inhibition of ROS production and oxidative stress-related pathway. (A) NRK-52E cell viability, determined by MTT assay, reduced prominently when exposed to hypoxia for 24 h then followed by reoxygenation for 6 h. (B) iPSC-CM (50-fold) significantly improved NRK-52E cell viability subjected to 24 h of hypoxia and 6 h of reoxygenation (H/R) compared with NM. (C) Recording of lucigenin-enhanced chemiluminescence (CL) from NRK-52E cells subjected to H/R. The CL levels of cells cultured in normoxia condition and treated with NM or iPSC-CM were equally low (3,500–4,000 counts per 10 s). An abrupt increase in CL was noted in cells exposed to H/R and treated with NM (H/R + NM group). The H/R-induced surge of CL was abolished by 50-fold iPSC-CM (H/R + iPSC-CM group) and returned to the same level at the normoxia condition. (D) Quantitative amount of ROS in NRK-52E cells exposed to normoxia or H/R and treated with iPSC-CM or NM. (E–G) Expression of MAPKs (p38, JNK, and ERK) from NRK-52E cells by Western blot analysis. (H) Activation of active NF-κB p65 in NRK-52E cells by Trans-AM NF-κB p65 transcription factor assay. (I, J) Expressions of TNF-α and cleaved caspase 3 (CPP32) from NRK-52E cells by Western blot (n = 4). (K) Representative graphs of apoptotic cells analyzed with annexin V–propidium iodide (PI) staining by flow cytometry in the four groups. (L) Quantitative data of apoptotic cells in each group (n = 4). Data were expressed as mean ± SEM. *p < 0.05 versus NRK-52E cells without H/R. #p < 0.05 versus NRK-52E cells exposed to H/R, treated with NM.

iPSC-CM Inhibited the ROS Production in Tubular Cells After H/R

Using the lucigenin-enhanced CL method to measure ROS production, baseline CL count was around 2,000 per 10 s in all groups (Fig. 2C). In normoxic condition, the CL count increased modestly after adding lucigenin and reached approximately 3,800 per 10 s both in the NM- and iPSC-CM-treated groups. By contrast, in H/R condition, the CL count abruptly rose to the highest level at about 10,000 per 10 s in NM-treated group. Remarkably, coculture with iPSC-CM significantly restored the CL count back to 4,000 per 10 s compared with NM treatment group (Fig. 2C). Similarly, while integrating the area under the curve from 60 s to 160 s, total CL count in H/R had an approximately twofold increase compared with that in normoxia in the NM-treated groups. The augmented ROS generation in H/R was abolished by coculture of iPSC-CM and total CL count restored to the same level in the normoxia group (Fig. 2D).

iPSC-CM Inhibited H/R-Elicited Tubular Cell Apoptosis

H/R significantly increased the protein expression of phospho-p38 MAPK in NRK-52E cells. Coculture of iPSC-CM in reoxygenation phase suppressed the H/R-mediated expression of phospho-p38 MAPK (Fig. 2E). However, there was no significant difference in the expression of ERK and JNK between NM- and iPSC-CM-treated groups (Fig. 2F, G). In addition, intranuclear active NF-κB p65 activity was significantly higher in the H/R condition than that in the normoxia condition. Treatment of iPSC-CM could significantly suppress the activation of NF-κB activity induced by H/R (Fig. 2H).

The signaling pathway of H/R-induced apoptosis was also investigated in NRK-52E cells. Protein expression of TNF-α and cleaved caspase 3 were significantly increased in the H/R groups. Treatment with iPSC-CM significantly suppressed the H/R-induced expression of TNF-α (Fig. 2I) and cleaved caspase 3 (Fig. 2J). Annexin V/propidium iodide assay further showed that H/R caused prominent apoptosis in NRK-52E cells (Fig. 2K) and the number of apoptotic cells was significantly lowered by iPSC-CM treatment (Fig. 2L).

iPSC-CM Reduced ROS Production in Tubular Cells with I/R Kidney Injury

ROS production in kidney tissues was measured 48 h after I/R in rats. By adding lucigenin, the CL count slightly increased to 1,500 per 10 s in the sham group treated with NM. In I/R groups treated with PBS or NM, the CL count surged significantly to the highest level around at 7,000 per 10 s. In I/R rats treated with iPSC-CM, the CL count dropped to 3,000 per 10 s (Fig. 3A). Furthermore, I/R groups treated with PBS or NM resulted in a twofold increase in total CL count compared with the sham-operated group. Intraperitoneal administration of iPSC-CM to rats with I/R injury eliminated the increased total CL amount (Fig. 3B). Using DHE staining, we further localized the origin of ROS production in rat kidney tissues after I/R-AKI. DHE fluorescence indicating ROS production predominantly existed in renal tubular cells, and only a few DHE-positive cells colocalized with CD68⁺ macrophages in PBS- or NM-treated groups (Fig. 3C). DHE fluorescence in renal tubular cells was significantly reduced in the iPSC-CM-treated group.

Figure 3.

iPSC-CM reduced ROS production in renal tubule cells of rats with ischemia–reperfusion (I/R)-AKI. (A) ROS production measured by lucigenin-enhanced chemiluminescence (CL) assay and (B) quantitative ROS amount in the sham-operated rats, rats with I/R injury receiving either PBS, NM, or iPSC-CM. *p < 0.05 versus sham group, #p < 0.05 versus I/R + PBS group. (C) Immunofluorescence staining for in situ ROS by dihydroethidium (DHE) staining and macrophage by anti-CD68 staining in renal tissues of the sham, I/R + PBS, I/R + NM, and I/R + iPSC-CM groups. DHE-positive cells were predominantly renal tubular cells and in some macrophages (arrowhead); *renal tubule. 4′,6-Diamidino-2-phenylindole (DAPI) represented nuclear staining. Scale bars: 50 μm.

iPSC-CM Suppressed p38 MAPK/TNF-α/NF-κB Signaling in I/R Kidney Injury

In I/R rats treated with PBS or NM, protein expressions of phospho-p38 MAPK and TNF-α were markedly increased in kidney tissues. Expression levels of nuclear phosphorylated p65 and p50 subunits of NF-κB, the downstream target of TNF-α signaling, were also elevated significantly compared with sham-operated animals. While treated with iPSC-CM, the expressions of phospho-p38 MAPK, TNF-α, and nuclear phosphorylated NF-κB p65 were significantly suppressed compared with PBS- or NM-treated rats (Fig. 4A–C). Nonetheless, the expression levels of nuclear NF-κB p50 did not differ significantly among PBS-, NM- and iPSC-CM-treated groups (Fig. 4C, D). Immunohistochemical staining further confirmed that active phospho-NF-κB p65 expression was conspicuous in the nuclei of renal tubular cells in PBS- or NM-treated groups and was less obvious in the iPSC-CM group (Fig. 4E). We then examined the expression of NF-κB-induced proinflammatory cytokines (Fig. 4F). The expression levels of IL-6 and MCP-1 were increased in PBS and NM groups and diminished by iPSC-CM treatment. The expression levels of IFN-γ showed no difference among the three groups. Finally, the expression level of proapoptotic CPP32 was increased in the PBS and NM groups and suppressed in the iPSC-CM group (Fig. 4F).

Figure 4.

Expression of p38 mitogen-activated protein kinase (MAPK), tumor necrosis factor (TNF)-α, nuclear factor (NF)-κB signaling, and cleaved caspase 3 (CPP32) in renal tubules of rats with ischemia–reperfusion (I/R)-AKI. (A, B) Western blots of p38 MAPK and TNF-α in renal tissue of the indicated groups. (C, D) Western blots of nuclear and cytosolic expressions of p65 and p50 subunits of NF-κB. PARP and β-actin represented nuclear and cytosolic loading control, respectively. (E) Immunohistochemical staining of active NF-κB p65 in the kidneys of the indicated groups. HPF, high-power field. Scale bars: 100 μm. (F) Western blots of interleukin-6 (IL-6), monocyte chemotactic factor-1 (MCP-1), interferon-γ (IFN-γ) and CPP32 in renal tissue of the indicated groups. Sham denoted sham-operated group, I/R + PBS, I/R + NM, and I/R + iPSC-CM represented the rats with I/R-AKI receiving PBS, NM, and iPSC-CM groups, respectively (n = 5 in each group). Data were expressed as means ± SEM. *p < 0.05 versus sham-operated group. #p < 0.05 versus I/R + PBS group. §p < 0.05 versus I/R + NM group.

iPSC-CM Improved the Survival of Rats with I/R-Induced AKI

Since mortality is the ultimate unambiguous outcome measure for treatment of AKI, we analyzed survival rate in the sham-operated rats, I/R rats receiving PBS, NM, and iPSC-CM, respectively. Around 70% of rats treated with PBS or NM died within 7 days whereas the mortality rate of I/R rats treated with iPSC-CM reduced to 30% (Fig. 5A), leading to a significantly better survival rate compared with PBS- or NM-treated rats (p < 0.05 for trend). No difference in survival was observed between PBS and NM groups. Additionally, BUN (Fig. 5B) and serum creatinine (Fig. 5C) levels of the rats that survived were measured for 1 week in I/R rats treated with NM and iPSC-CM, respectively. The peak of BUN or serum creatinine occurred 2 days after I/R injury, in accordance with the high mortality in NM-treated rats. The peak deterioration of renal function was significantly attenuated in I/R rats treated with iPSC-CM.

Figure 5.

iPSC-CM protected rats from AKI-induced lethality. The rats were subject to renal ischemia–reperfusion (I/R) operation at day 0, and intraperitoneal injection of 1 ml of PBS, NM, or iPSC-CM (50-fold) twice daily, and then sacrificed at day 7 after surgery. (A) Survival curves in the sham-operated, I/R + PBS, I/R + NM, and I/R + iPSC-CM rats were assessed by log-rank test. (B) BUN and (C) serum creatinine levels in I/R + NM and I/R + iPSC-CM rats were measured at days 0, 1, 2, 4, and 7. The numbers in parentheses denote the number of survived rats. n = 9 in each group. Data were expressed as mean ± SEM. *p < 0.05 versus I/R + NM group.

Discussion

I/R kidney injury is one of the leading causes of AKI, and current treatment options remain scarce except for supportive care (15,17,32). Recently, stem cell therapy has attracted considerable interest in the development of potential remedial strategies for AKI (2,12,19,26,33). Previous studies have shown the therapeutic potential of mesenchymal stem cells (MSCs) in experimental AKI models (12,19,26,33). Nonetheless, most MSCs are derived from bone marrow, a source that is not available in large volume. Recently, Yamanaka et al. (30) have demonstrated that iPSCs could be generated from somatic cells of individual patients, and these patient-specific tailor-made stem cells could alleviate immune rejection, obviate ethical concerns, and overcome the shortage of donor organs. Therefore, iPSCs represent a favorable source for stem cell-based transplantation and could be a safe and beneficial stem cell therapy in kidney diseases. We have previously demonstrated that administration of iPSCs could attenuate AKI in rats with I/R injury (20). Nonetheless, the engrafted iPSCs are disproportionally low in contrast to substantially increased proliferating renal tubular epithelial cells (20), which implies the advantageous effects of iPSCs in AKI are mediated primarily by paracrine/endocrine mechanisms. The present study further corroborated the endocrine/paracrine beneficial role of iPS cells and found that iPSC-CM could attenuate I/R injury-related deterioration of renal function and improve survival. To the best of our knowledge, the present study is the first to unveil the therapeutic effect of iPSC-CM in an I/R-AKI experimental model.

Our results also indicated that iPSC-CM could increase viability of renal tubular cells by suppressing I/R-elicited production of ROS. Oxidative stress plays a crucial role in the pathogenesis of I/R-AKI (1), and we did find that ROS was predominantly produced by the renal tubular cells in I/R-AKI, which was in accordance to previous literature (6). Emerging new evidence indicates that local renal damage in AKI may bring about the initiation and perpetuation of systemic inflammatory response and multiple organ failure (4,28). Previously, we have shown that local intrarenal arterial administration of iPSCs could reduce the oxidative damage markers, including malondialdehyde and 4-hydroxynonenal (20). To investigate the systemic therapeutic role of iPSC-CM, the present study confirmed that intraperitoneally administered iPSC-CM could lessen oxidative stress. Moreover, iPSC-CM could suppress the activation of proinflammatory and proapoptotic pathways during I/R-AKI as evidenced by a reduction in NF-κB activity and decreased expressions of p38 MAPK, TNF-α, IL-6, MCP-1 and CPP-32 in the iPSC-CM-treated rats. The in vitro study also revealed that iPSC-CM could inhibit apoptosis of NRK-52E cells subjected to H/R and suppress the H/R-provoked NF-κB activity and expression of p38 MAPK, TNF-α, and CPP-32 (Fig. 6). In line with our findings, MSC-derived conditioned medium and microvesicles also protect against I/R-AKI and cisplatin-induced AKI by inhibiting apoptosis and promoting proliferation of renal tubular cells (3,11). In addition, administration of embryonic MSC-derived conditioned medium could ameliorate progression of renal function and reduce glomerular injury in subtotally nephrectomized rats (36), supporting the notion that stem cell-derived protective factors could be exploited as a therapeutic option for kidney diseases. Taken together, iPSC-CM exhibited a systemic antiapoptotic, antioxidant, and anti-inflammatory effect in rats with I/R-AKI.

Figure 6.

Schematic illustration for the pivotal role of iPSC-CM in protection of renal tubular cells from apoptosis after ischemia–reperfusion (I/R) injury. The beneficial effects of iPSC-CM possibly include reduction in ROS production, suppression in p38-MAPK activation, and inhibition in TNF-α-induced cell death and its downstream NF-κB signaling.

Some debates exist regarding whether the beneficial effect of stem cells is through direct replacement of damaged tissue by transdifferentiated homing stem cells or through the paracrine/endocrine action by trophic factors released by stem cells (2). The evidence that transplanted stem cells migrate to the damaged tissue and subsequently transdifferentiate into target cells remains scarce (35). Therefore, the currently favored hypothesis regarding the reparative effect of stem cells in I/R-AKI is the paracrine/endocrine effect (2,35). Nonetheless, these notions are largely rooted in the research of MSCs (2,35), and little is known about the exact restorative mechanism of iPSCs in I/R-AKI. Yang et al. (38) and Liu et al. (24) have shown that treatment with iPSC-CM could equally effectively ameliorate endotoxin-related and ventilator-induced acute lung injury compared to iPSCs. We previously also demonstrated increased levels of various trophic and proangiogenic factors in the iPSC-CM, including fibroblast growth factor-2, insulin-like growth factor-binding protein, vascular endothelial growth factor, and angiopoietin-1 (23). These molecules have been shown to participate in the regeneration process in ischemic AKI (18,37). Angiopoietin-1 contributes to preservation of peritubular capillaries in I/R-AKI (16), and vascular endothelial growth factor has also been shown to be directly renoprotective in the pathological cascade of AKI (34). Therefore, these trophic factors of iPSC-CM can account for, at least in part, the protection of the kidney against I/R injury. Notably, administration of onefold or 25-fold concentrated iPSC-CM resulted in a progressive, yet insignificant, reduction of BUN, creatinine, and tubular injury. The significant improvement of renal function and tubular injury occurred in the 50-fold concentrated iPSC-CM-treated group, suggesting that a sufficient amount of iPSC-secreted factors was required to alleviate I/R-AKI (Fig. 1). Collectively, our finding substantiated the hypothesis that iPSCs mediated the reparative effects through the paracrine/endocrine manner, and further studies are needed to determine the optimal dosage of the iPSC-CM.

There have been arguments as to the disadvantages of iPSC therapy, including the potential malignant risk, low reprogramming efficiency, and the possible thrombotic effect. In fact, we did observe a deleterious thrombotic effect of intra-arterial administration of high-dose iPSCs in I/R kidney injury (20). Given that the reparative action of iPSCs was primarily via paracrine/endocrine effect, administration of iPSC-CM instead of iPSCs could reasonably obviate the thrombotic risk. Furthermore, collection of conditioned medium from the preexisting successfully reprogrammed iPSCs would be less cumbersome compared to generating and expanding the new iPSCs.

Conclusions

The current study demonstrated a pivotal role of iPSC-CM in renoprotective effects on I/R-AKI as shown by improved renal function and survival, lower ROS production, suppressed proinflammatory signaling, and reduced tubular epithelial cell apoptosis. Our findings indicate that iPSC-CM is a potential resource for stem cell-based therapy against I/R-induced AKI. Administration of iPSC-CM could provide impetus on the advancement in treating I/R-induced AKI. Further research may help translate the bench in vivo findings to clinical practice.