Chronic kidney disease (CKD) is a major global public health issue, causing several complications such as hypertension, atherosclerosis, aging, and diabetes [1
]. In particular, kidney dysfunction leads to the excretion of uremic toxins, which accumulate in the blood and have toxic effects on several types of tissue [2
]. Studies using murine CKD models have shown that CKD-mediated oxidative stress is induced in certain regions of the brain, including the cerebral cortex and hippocampus, resulting in cognitive dysfunction [3
-cresol, one of the major uremic toxins, is a protein-bound solute that induces cell apoptosis and senescence [5
]. Recent studies have revealed that P
-cresol induces cellular senescence, inhibition of proliferation, and dysfunction of mitochondria in mesenchymal stem cells (MSCs) [6
]. Therefore, uremic toxins such as P
-cresol are a major hurdle for stem cell-based therapies, and the development of uremic toxin-resistant stem cells to treat CKD patients is urgently needed.
MSCs are a major source of stem cells for regenerative medicine due to their capacity for self-renewal and multipotency [9
]. Although several studies have revealed that the main mechanism of MSC-mediated tissue repair is via their potential for self-renewal and multi-differentiation, their paracrine effect has also been proposed to be an important mechanism of regeneration [10
]. In response to injury, MSCs secrete several types of cytokine and chemokine (immunomodulatory, cytoprotective, angiogenic, arteriogenic, antiapoptotic, antioxidant, cell migratory, and homing factors), as well as microvesicles and exosomes [10
]. Despite their unique characteristics, the severe pathophysiological conditions of several diseases restrict the beneficial effects of MSCs in regenerative medicine.
Cellular prion protein (PrPC
) is a glycosylphosphatidylinositol-anchored glycoprotein whose expression is related to PrPSC
, the protease resistant misfolded prion isoform of PrPC
]. Previous reports suggest that PrPC
is involved in cell survival, proliferation, and signal transduction [7
]. In addition, PrPC
increases antioxidant activity under conditions of oxidative stress, leading to inhibition of reactive oxygen species (ROS) generation [7
]. Therefore, PrPC
is an interesting molecular target for treating CKD-related cognitive dysfunction induced by oxidative stress. Our previous studies have shown that tauroursodeoxycholic acid (TUDCA), a bile acid, protects against oxidative stress-induced apoptosis in MSCs through the upregulation of PrPC
]. In addition, TUDCA-treated stem cells and progenitor cells have a therapeutic effect in ischemic diseases under conditions of oxidative stress [7
]. These findings suggest that TUDCA-treated stem/progenitor cell-based therapeutics may be one candidate for regenerative medicine for several diseases associated with oxidative stress. This study and other recent studies have found that CKD induces ROS generation and endoplasmic reticulum (ER) stress in the hippocampus [19
]. To reveal the effect of TUDCA-treated CKD-MSCs against P
-cresol-induced ROS stress in neural cell line SH-SY5Y cells, we co-cultured SH-SY5Y cells with TUDCA-treated CKD-MSCs. We showed that TUDCA-treated MSCs prevent neural cell death induced by CKD-associated ER stress through the upregulation of PrPC
, highlighting the potential of this pathway for CKD cell-based therapy.
In this study, we provide evidence that the generation of ROS is induced in the hippocampus of an adenine-induced murine model, resulting in ROS-mediated ER stress and cell death in neural cell line SH-SY5Y cells. In addition, we have shown that co-culture of SH-SY5Y cells with TUDCA-treated CKD-hMSCs inhibits uremic toxin-induced ER stress and cell death in SH-SY5Y cells through the upregulation of PrPC
. In particular, in a murine CKD model, injection with TUDCA-treated CKD-hMSCs suppressed ROS production and ER stress, suggesting that TUDCA-treated hMSCs could prevent oxidative stress in the hippocampus of those suffering with CKD. The paracrine effect is one possible mechanism by which TUDCA-treated CKD-hMSCs protect SH-SY5Y cells against cell death due to P
-cresol-induced oxidative stress. In P
-cresol-treated MSCs, TUDCA increases the secretion of IL-10 [7
], which inhibits free radical generation [22
has been detected in the ER and Golgi, suggesting that it could be packaged into secretory vesicles [23
]. Our results have shown that in a co-culture assay, TUDCA-treated CKD-hMSCs increased the level of PrPC
in SH-SY5Y cells, suggesting that PrPC
might be delivered from TUDCA-treated CKD-hMSCs to SH-SY5Y cells by secretory vesicles. However, further studies are needed to determine the precise mechanisms by which cytokines/growth factors are secreted in MSCs in response to TUDCA treatment and how PrPC
is delivered from TUDCA-treated MSCs to SH-SY5Y cells.
TUDCA is one of the bile acids which has been used for the treatment of several liver diseases, such as primary sclerosing cholangiolitis and biliary cirrhosis [25
]. In neurodegenerative diseases, TUDCA also protects neural cell apoptosis and neuronal loss by upregulating cell survival pathways [26
]. Furthermore, recent studies indicate that TUDCA-stimulated stem/progenitor cells enhance their functionalities and therapeutic effects in ischemic diseases [7
]. TUDCA-stimulated endothelial progenitor cells (EPCs) increase angiogenesis in a murine hind limb ischemia model by enhancing recruitment of EPCs to ischemic injured tissues [18
]. In addition, TUDCA rescues cellular senescence in aged EPCs through reduction of cyclin-dependent kinase inhibitor 1A (p21) and ROS levels, with the result that TUDCA-treated senescent EPCs enhance blood vessel regeneration in a hind limb model [18
]. Under ischemia-mediated ER stress conditions, TUDCA inhibits ER stress-induced apoptosis in MSCs through the RAC (Rho family of GTPases)—alpha serine/threonine-protein kinase (Akt)-PrPC
signaling pathway [17
]. In particular, TUDCA-treated MSCs increase functional recovery in a murine hind limb ischemia model by enhancing the activity of the anti-oxidative enzyme, manganese-dependent superoxide dismutase (MnSOD) [17
]. Our recent study has shown that TUDCA exerts a protective effect against oxidative stress in MSCs under the presence of uremic toxin via the upregulation of PrPC
]. This study indicates that treatment with TUDCA significantly increases the expression of PrPC
in CKD-hMSCs, compared with that in non-treated CKD-hMSCs. PrPC
plays a role in several intracellular signal transduction pathways and is also released to other cells during cell–cell interaction by exosomes [27
]. Our results have shown that co-culture of SH-SY5Y cells with TUDCA-treated CKD-hMSCs increases the PrPC
level in SH-SY5Y cells, with the result that P
-cresol-induced ER stress and apoptosis in SH-SY5Y cells are inhibited by increased anti-oxidant enzyme activities. These findings suggest that TUDCA-stimulated CKD-MSCs could protect against neural cell death induced by uremic toxin-induced oxidative stress through the secretion of PrPC
. However, further investigation is needed to elucidate the precise mechanism of TUDCA-mediated secretion of PrPC
is a glycoprotein which attaches to the cell membrane [28
]. Abnormal conversion of PrPC
generates prions, leading to neurodegenerative diseases [29
]. Although the abnormal form, PrPSc
, causes dysfunction of the nervous system, PrPC
is associated with various cellular and physiological processes, such as neural differentiation, protection of oxidative stress, inhibition of neurodegeneration, and self-renewal of stem cells [30
]. Previous studies have revealed that P
-cresol increases the production of ROS in MSCs [17
]. In particular, oxidative stress is induced by an imbalance in ROS and increases as CKD progresses [33
], indicating that ROS generation is closely related to the stage of CKD. In MSCs, the upregulation of PrPC
protects against apoptosis caused by ischemia-induced oxidative stress [12
also regulates the expression of MnSOD, resulting in protection of MSCs against ROS-mediated ER stress in an Akt-dependent manner [17
]. In addition, PrPC
prevents apoptosis of MSCs under conditions of uremic toxin-mediated ER stress [7
]. Furthermore, PrPC
contributes to long-term neuroprotection in the ischemic brain and PrPC
deficiency causes sensitivity to oxidative stress in brain ischemia [34
]. Our results indicate that co-culture of SH-SY5Y cells with TUDCA-treated CKD-hMSCs increases PrPC
expression in SH-SY5Y cells, resulting in induction of catalase and SOD activity. Augmentation of anti-oxidant enzyme activities results in a reduction of ROS and inhibition of ER stress, leading to protection against apoptosis in SH-SY5Y cells under the presence of uremic toxins. These results indicate that the augmentation of PrPC
levels by co-culture with TUDCA-treated CKD-hMSCs protects SH-SY5Y cells against uremic toxin-induced ER stress through the activation of anti-oxidant enzymes, suggesting that PrPC
plays a key role in protection against ER stress in neural cells under conditions of CKD-mediated oxidative stress. Moreover, these findings suggest that TUDCA might be a powerful priming agent for enhancing the antioxidant effect of MSCs in patients with CKD.
Although MSCs are one of the powerful sources for autologous stem cell-based therapeutics, pathophysiological conditions in several chronic diseases decrease their therapeutic efficacy. Our findings indicate that TUDCA-treated CKD-hMSCs protect against CKD-mediated ROS generation and ER stress in the hippocampus of a murine CKD model in a PrPC
-dependent manner. These results suggest that treatment with TUDCA might be an effective strategy for the development of autologous MSC-based therapeutics in patients with CKD. However, since there are several murine CKD models which are induced by various etiologies (genetic, autoimmune, infectious, environmental, dietary, and medication), each with differing characteristics, they cannot exactly simulate and predict responses in patients with CKD [37
]. Therefore, further studies using other CKD models are needed to reveal the full protective effects of TUDCA-treated CKD-hMSCs against ROS generation in the hippocampus. Taken together, our study indicates that TUDCA-treated CKD-MSCs protect against neural cell death induced by ROS-associated ER stress in CKD through the PrPC
-catalase/SOD axis (Figure 5
). These findings suggest that TUDCA-treated MSCs could be a strong candidate for autologous stem cell-based therapeutics for CKD, and that PrPC
might be a key molecule for protection against ROS-associated ER stress in the hippocampus of CKD patients.
4. Materials and Methods
4.1. Cell Culture of Human MSCs
Human adipose tissue-derived normal MSCs (hMSCs) and MSCs isolated from CKD patients (CKD-hMSCs) were obtained from Soonchunhyang University Seoul Hospital (IRB: SCHUH 2015-11-017, 2 November 2015). CKD-hMSCs were derived from the adipose tissue of CKD patients, according to the estimated glomerular filteration rate [eGFR] < 35 mL/(min⋅1.73 m2) for more than 3 months (stage 3b). The supplier certified that CD44 and Sca-1 were expressed on the surface of hMSCs. Normal hMSCs and CKD-hMSCs were cultured in α-Minimum Essential Medium (Gibco BRL, Gaithersburg, MD, USA) supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco BRL) and 100 U/mL penicillin/streptomycin (Gibco BRL). Both cell types were grown in a humidified 5% CO2 incubator at 37 °C.
4.2. Culture of SH-SY5Y Cells
The SH-SY5Y cell line was purchased from Seoul University Hospital (Seoul, Korea) and cultured in DMEM (Gibco BRL) supplemented with 10% (v/v) FBS (Gibco BRL) in a humidified 5% CO2 incubator at 37 °C. To establish CKD conditions, SH-SY5Y cells were washed twice with PBS and placed in fresh DMEM with P-cresol (500 μM) for 48 h.
4.3. Co-Culture of SH-SY5Y Cells with hMSCs
SH-SY5Y cells and hMSCs were co-cultured in Millicell Cell Culture Plates (Millipore, Billerica, MA, USA) in a humidified 5% CO2 incubator at 37 °C. SH-SY5Y cells were seeded in the lower compartments, followed by exposure to P-cresol for 24 h. MSCs were then seeded onto the transwell membrane inserts and the plates were incubated for a further 24 h, following which the SH-SY5Y cells were assessed by several assays.
4.4. Western Blot Assay
Protein extracts were separated via 8% to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by transfer to nitrocellulose membrane. After blocking membranes with 5% skim milk for 1 h, glucose-regulated protein 78 (GRP78) primary antibodies against PrPC, protein kinase-like endoplasmic reticulum kinase (PERK), phospho-PERK (p-PERK), inositol-requiring enzyme 1α (IRE1α), p-IRE1α, activating transcription factor 4 (ATF4), and β-actin (Santa Cruz Biotechnology, Dallas, TX, USA) were incubated overnight. After washing with PBS, the membranes were incubated with goat anti-rabbit immunoglobulin g (IgG) or goat anti-mouse IgG conjugated to horseradish peroxidase (Santa Cruz Biotechnology). The bands were detected by enhanced chemiluminescence (Amersham Pharmacia Biotech, England, UK).
4.5. Silencing of PrPC Expression by RNA Interference
CKD-hMSCs (2 × 105) seeded in 60-mm dishes were transfected with siRNA in serum-free Opti-MEM (Gibco BRL) using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. At 48 h post-transfection, total protein was extracted, and gene expression was determined through western blot analysis. The siRNA used to target PRNP and a scrambled sequence were synthesized by Bioneer (Daejeon, Korea).
4.6. Flow Cytometric Analysis
Cell apoptosis was assessed using a Cyflow Cube 8 (Partec, Münster, Germany) after staining the cells with propidium iodide (PI) and Annexin V-fluorescein isothiocyanate (FITC). Data analysis was performed using standard FSC Express (De Novo Software, Los Angeles, CA, USA).
Mouse brain tissue was fixed with 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MS, USA) and embedded in paraffin. Immunofluorescence staining was performed using primary antibodies against GRP78 (Santa Cruz Biotechnology) and secondary antibodies Alexa-488 (Thermo Fisher Scientific). Nuclei were stained with 4′,6-diaminido-2-phenylindol (DAPI; Sigma-Aldrich), and immunostained samples were observed using confocal microscopy (Olympus, Tokyo, Japan).
4.8. DHE Staining
DHE (Sigma-Aldrich) was used to measure superoxide anions in CKD mouse brain sections. Each group of sections was exposed to DHE (10 μM) for 30 min at 37 °C. After washing with PBS three times, samples were visualized by confocal microscopy (Olympus) at 488 nm excitation and 590 nm emission.
4.9. Catalase activity
Cells were seeded in 100-mm tissue culture plates and grown to 70% to 75% confluence. After washing twice in PBS, cells were resuspended in lysis buffer (1% Triton X-100 in 50 mM Tris-HCl [pH 7.4] containing 150 mM NaCl, 5 mM EDTA, 2 mM Na3VO4, 2.5 mM Na4PO7, 100 mM NaF, and protease inhibitors). Samples were incubated for 30 min on ice and were centrifuged at 14000 rpm for 30 min at 4 °C. After measuring the protein concentration of the supernatant fraction, catalase activity was measured using a Catalase Assay Kit (Sigma-Aldrich) according to the manufacturer’s instructions.
4.10. Superoxide Dismutase Activity
Cells were harvested, and protein was isolated using extraction buffer. Cell lysates (50 µg) were treated with SOD, and the signal was immediately measured each minute for 15 min using an ELISA reader (BMG labtech, Ortenberg, Germany) at 450 nm.
4.11. Ethics Statement
All animal care procedures and experiments were approved by the Institutional Animal Care and Use Committee of Soonchunhyang University Seoul Hospital (IACUC2013-5, 16 February 2014) and were performed in accordance with the National Research Council (NRC) Guidelines for the Care and Use of Laboratory Animals. The experiments were performed on 8-week-old male BALB/c nude mice (Biogenomics, Seoul, Korea) maintained on a 12 h light/dark cycle at 25 °C in accordance with the regulations of Soonchunhyang University Seoul Hospital.
4.12. The CKD Model
Eight-week-old male BALB/c nude mice were fed an adenine-containing diet (0.25% adenine) for 1 to 2 weeks. Mouse body weight was measured every week. The mice were randomly assigned to 1 of 4 groups containing 10 mice each. After euthanasia, blood was stored at −80 °C to measure blood urea nitrogen (control: 15.8939 ± 1.2360 mg/dL, CKD: 71.8677 ± 2.1999 mg/dL) and creatinine (control: 0.3657 ± 0.0264 mg/dL, CKD: 1.9483 ± 0.1573 mg/dL). In addition, enlarged kidneys were observed.
4.13. Statistical Analysis
Results are expressed as the mean ± SEM. All of the experiments were analyzed by one-way analysis of variance (ANOVA). Some comparisons of ≥3 groups were made using the Bonferroni-Dunn test. A p value < 0.05 was considered statistically significant.