Patients with chronic kidney disease (CKD) experience a progressive loss of kidney function over a period of months or years. Consequently, the diseased kidneys fail to metabolize and excrete toxic metabolites and organic waste solutes, which are normally removed by healthy kidneys. It is known that such retained toxic products, commonly referred to as “uremic toxins,” are transported by the blood to other organs, causing adverse effects throughout the body (D’Hooge
When cells age or become damaged by external stimuli, they are replaced by new cells that differentiate from adult stem cells into corresponding tissue-specific cell types to maintain tissue stability (Krause
Individuals with CKD experience disturbances of sleep, which may be linked to impaired endogenous melatonin secretion during nighttime (Maung
Human adipose tissue-derived MSCs were obtained from the American Type Culture Collection (Manassas, VA, USA). MSCs were confirmed to be pathogen- and mycoplasma-free. MSCs expressed specific cell surface markers [cluster of differentiation (CD) 73 and CD105, but not CD31]. MSCs were cultured in α-minimum essential medium (Gibco, Gaithersburg, MD, USA) supplemented with 10% (v/v) fetal bovine serum (Gibco), 100 U/mL penicillin, and 100 μg/mL streptomycin. MSC cultures were grown in a humidified atmosphere of 95% air and 5% CO2 at 37°C.
MSCs were exposed to PC (0, 50, 100, 500 μM; Sigma-Aldrich, St. Louis, MO, USA) for 72 h or PC (500 μM; for 72 h) with preincubation melatonin (0, 1, 10, 100 μM; for 30 min; Sigma-Aldrich) or phosphate-buffered saline (PBS; vehicle). Cell proliferation parameters were assessed using a cell proliferation 5-bromo-2′-deoxyuridine (BrdU) ELISA colorimetric kit (Roche, Mannheim, Germany). To perform ELISA, 100 μg/mL BrdU was added to cultured MSCs and incubated at 37°C for 6 h. Then, 1 M H2SO4 was added to stop the reaction. Light absorbance of the samples was measured by an ELISA reader (BMG labtech, Ortenberg, Germany) at 450 nm.
Senescence was assessed by measuring the percentage of cultured cells that stained positively for senescence-associated beta-galactosidase (SA-β-gal) activity as described previously (Cong
MSCs were harvested from the culture dish by scraping with a rubber policeman on ice. To measure catalase activity, cell lysate protein extracts (40 μg) were incubated with 20 mM H2O2 (Sigma-Aldrich) in 0.1 M Tris-HCl (Sigma-Aldrich) for 30 min. Then, 50 mM Amplex Red reagent (Thermo Scientific, Waltham, MA, USA) and 0.2 U/mL of horseradish peroxidase (Sigma-Aldrich) was added and incub ated for 30 min at 37°C. Changes in absorbance values associated with H2O2 degradation were measured by an ELISA reader (BMG labtech) at 563 nm.
MSCs homogenates were extracted by using RIPA lysis buffer (Thermo Scientific). Cell lysates (20 μg of total protein) were separated by using 6–15% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the proteins were transferred to nitrocellulose membrane (Thermo Scientific). After the blots had been washed with a solution comprising 10 mM Tris-HCl (pH 7.6; Sigma-Aldrich), 150 mM NaCl (Sigma-Aldrich), and 0.05% Tween-20 (Sigma-Aldrich), the membranes were incubated with 5% bovine serum albumin for 1 h at room temperature and then, incubated with appropriate primary antibodies against phosphoinositide 3-kinase (PI3K), phosphorylated PI3K (p-PI3K), Akt, phosphorylated Akt (p-Akt), phosphorylated 5′-AMP-activated protein kinase (AMPK), mechanistic target of rapamycin (mTOR), phosphorylated mTOR (p-mTOR), LC3, beclin 1, ATG7, p62/Sequestosome 1 (p62/SQSTM1), senescence marker protein 30 (SMP30), and β-actin (all from Santa Cruz Biotechnology, Dallas, TX, USA). The membranes were then washed more than twice, and the primary antibodies were detected using a goat anti-rabbit IgG or a goat anti-mouse IgG conjugated to horseradish peroxidase (Santa Cruz Biotechnology). The bands were visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech, Little Chalfont, UK).
To measure superoxide anion levels in cultured MSCs, the cells were incubated with 10 μM dihydroethidium (DHE; Sigma-Aldrich) for 30 min at 37°C. After washing with PBS three times, samples were visualized by fluorescence microscopy (Zeiss, Oberkochen, Germany) or analyzed by flow cytometry (Cyflow Cube 8 FACS; Sysmex Partec, Görlitz, Germany). Data were analyzed using standard FSC Express software (De Novo Software, Los Angeles, USA).
All data are expressed as the mean ± standard error of the mean (SEM). Results of all experiments were analyzed by one-way analysis of variance (ANOVA). Comparisons of more than three groups were made by using the Bonferroni-Dunn test. Differences were considered to be statistically significant if
Exposure of MSCs to a range of PC concentrations (0–500 μM) for 72 h induced cell senescence as was indicated by decreased BrdU incorporation levels (Fig. 1A). At 500 μM PC, the lowest BrdU incorporation in MSCs was observed (Fig. 1A). It has been shown previously that treatment with 500 μM PC increased reactive oxygen species (ROS) production and inhibited cell viability, proliferation, and migration (Chang
The cytoprotective effect of melatonin is thought to be mediated via its binding to melatonin receptors and subsequent activation of various signaling pathways that promote cell survival (Lee
The increase in ROS is closely related to the activity of AMP-activated protein kinase (AMPK) (Li
Excessive autophagy is associated with the inhibition of cell proliferation and increase in the number of senescent cells (Yang
Expression of senescence marker protein 30 (SMP30) decreases with age (Maruyama
In our study, we for the first time demonstrated that the uremic toxin PC, found in individuals with CKD, caused MSC senescence. Furthermore, we demonstrated that pretreatment with melatonin prevented PC-induced senescence of MSCs by activating the PI3K and Akt pathways and by inhibiting ROS-dependent, AMPK pathway-mediated autophagy.
PC, a metabolite of environmental toxins, is a uremic toxic solute normally excreted by the kidneys. In conditions such as CKD, PC accumulates in the body (D’Hooge
Previous studies suggested that PC induced oxidative stress in CKD patients by activating catalase and NADPH oxidase (Watanabe
Next, we attempted to uncover consequences of mTOR dysregulation for other cellular responses that are important in cell senescence. PC has been shown to induce autophagy in human renal proximal tubular cells (Lin
Overall, our experimental results highlight a considerable therapeutic potential of melatonin anti-autophagic effects that reverse PC-induced MSC senescence. By showing that the protective action of melatonin against PC-induced MSC senescence is sensitive to the incubation with an Akt inhibitor, we demonstrated key significance of the Akt signaling pathway activation in melatonin effects. Therefore, our findings reveal a novel role of melatonin and Akt pathway in autophagy and in MSC senescence associated with CKD. Although our study was performed
The authors declare that they have no conflicts of interest.
This work was supported by National Research Foundation grant funded by the Korean government (NRF-2016R1D-1A3B01007727, NRF-2017M3A9B4032528). The funders had no role in the study design, data collection or analysis, the decision to publish, or preparation of the manuscript.