Chronic kidney disease (CKD) remains a highly prevalent condition that affects all ages and has a high mortality rate (Lv and Zhang, 2019), constituting a significant portion of the global disease burden (Romagnani
Mesenchymal stem/stromal cells (MSCs) are attractive candidates for developing cell-based therapies for CKD, as they can contribute to tissue repair or regeneration by releasing appropriate factors related to cell proliferation, survival, and differentiation (Yun and Lee, 2019). Several reports have suggested that the angiogenic and regenerative properties of MSCs can improve various pathological conditions
Melatonin is a peptide hormone released by the pineal gland to modulate several physiological functions, including sleep, but there has been growing interest in its broader therapeutic potential. Accumulating evidence suggests that melatonin not only ameliorates myocardial infarction (Fu
Hexokinase-2 (HK2) catalyzes the ATP-mediated phosphorylation of glucose to glucose-6-phosphate (G6P), which is the rate-limiting step of glucose metabolism. Overexpression of HK2 has been linked to diabetic nephropathy (Rabbani and Thornalley, 2019), or diabetic kidney disease, which is another form of CKD induced by hyperglycemia (Kato and Natarajan, 2019). Elevation of HK2 has also been speculated to be related to ischemic CKD (Rabbani and Thornalley, 2019). Although HK2 isoforms have distinct roles in non-pathologic cellular processes, HK2 overexpression can occur in some pathogenic contexts such as in mediating tumorigenic activity (Liu
In the current study, we investigated whether HK2 overexpression is related to non-diabetic CKD and MSCs from the CKD condition. Our data showed that HK2 expression was markedly elevated in the serum of CKD patients, as well as in MSCs derived from a CKD mouse model (CKD-mMSCs). Thus, we hypothesized that melatonin may influence the accumulation of abnormal glycolytic intermediates, or glycolytic overload, to improve mitochondrial function and protect MSCs against CKD. Melatonin-treated CKD-mMSCs demonstrated lower HK2 expression, a reduced MG concentration, improved mitochondrial function, and normal glycolysis. Collectively, our data show that melatonin downregulates HK2 and MG to improve the therapeutic potential of MSCs for CKD.
The local ethics committee approved this study, and informed consent was obtained from all individuals participating in the study. Explanted sera (n=30) were obtained from patients with CKD at Gyeongsang National University, Jinju, Korea (IRB: SCHUH 2018-04-035-002). Upon fulfilling the transplantation criteria, the control samples were obtained from healthy controls (n=30) at the Korea Institute of Radiological & Medical Sciences (IRB: SCHUH 2018-04-035-002). CKD diagnoses were made based on abnormal kidney function with an estimated glomerular filtration rate<25 mL•min–1•1.73 m–2 for over 3 months (stages 3b-5).
We prepared a CKD mouse model following a previously established protocol (Yoon
The concentrations of MG in the human healthy control group or CKD patient group, or in whole cell lysates of healthy mMSCs, CKD-mMSCs, and CKD-mMSCs treated with melatonin were determined using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (Lifespan Biosciences, Seattle, WA, USA). A total of 100 μL of the sample from each group was used for the experiments. The levels of MG were quantified by measuring the absorbance at 450 nm using a microplate reader (BMG Labtech, Ortenberg, Germany).
Healthy- and CKD-mMSCs were washed twice with phosphate-buffered saline (PBS), and then cultured in fresh alpha-minimal essential medium (HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum (GIBCO, Grand Island, NY, USA). To examine the protective effect of melatonin (Sigma-Aldrich, St. Louis, MO, USA), mMSCs were incubated with melatonin (1 μM) at 37°C for 24 h and then subjected to various experimental assays for respective purposes. For luzindole (Sigma-Aldrich) treatment, the mMSCs were pretreated with luzindole (1 μM for 48 h) before melatonin treatment.
The harvested healthy-mMSCs or CKD-mMSCs were lysed in mitochondria lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA) and were subsequently incubated for 10 min on ice. The mixtures were centrifuged at 5,000
Whole cell lysates, cytosol fraction lysates, or mitochondrial lysates from healthy-mMSCs or CKD-mMSCs (30 μg protein) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on an 8%-12% gel, and the proteins were transferred to a nitrocellulose membrane. After the blots were washed with TBST (10 mM Tris-HCl [pH 7.6], 150 mM NaCl, 0.05% Tween 20), the membranes were blocked with 5% skim milk for 1 h at room temperature and incubated with the appropriate primary antibodies against HK1 (#2024S, Cell Signaling Technology, Beverly, MA, USA), HK2 (#2867S, Cell Signaling Technology), VDAC1 (NB100-695, Novus), CDK2 (sc-6248, Santa Cruz Biotechnology, Santa Cruz, CA, USA), CDK4 (SC-56277, Santa Cruz Biotechnology), cyclin D1 (SC-20044, Santa Cruz Biotechnology), cyclin E (SC-377100, Santa Cruz Biotechnology), and β-actin (sc-47778, Santa Cruz Biotechnology). The membranes were then washed, and the primary antibodies were detected using goat anti-rabbit IgG or goat anti-mouse IgG secondary antibodies (Santa Cruz Biotechnology). The bands were detected by enhanced chemiluminescence (Amersham Pharmacia Biotech, Little Chalfont, UK). Densitometric quantification was performed using ImageJ software 1.48v (National Institutes of Health, Bethesda, MD, USA).
The production of mitochondrial superoxide in mMSCs was measured using MitoSOX (Thermo Fisher Scientific). To measure mitochondrial membrane potential, tetramethylrhodamine, methyl ester, and perchlorate (TMRE; Thermo Fisher Scientific) dye was used. The mMSCs in each group were trypsinized and centrifuged at 1,200 rpm for 3 min. After washing with PBS twice, the cells were treated with either 10 μM MitoSOX in PBS or 200 nM TMRE in PBS at 37°C for 15 min. MitoSOX-positive or TMRE-positive cells were analyzed using flow cytometry (Sysmex, Kobe, Japan). Flow cytometry data were analyzed using FCS Express 5 Flow research software (DeNovo Software, Los Angeles, CA, USA).
OCR and ECAR were measured in mMSCs using an XF96 extracellular flux analyzer (Seahorse Bioscience, MA, USA). In brief, mMSCs were seeded at 45,000 cells per well in XF96 cell culture multi-well plates in Dulbecco’s modified Eagle medium, and incubated for 24 h at 37°C and 5% CO2. The XF96 cartridges were then incubated overnight in an XF calibrant at 37°C in a non-CO2 incubator. After the growth medium of mMSCs was changed with XF medium, the plates were incubated at 37°C in a non-CO2 incubator for 1 h. Inhibitors were diluted to appropriate concentrations in XF medium and loaded into corresponding microwells in the XF96 cartridge plate. Following the equilibration of the sensor cartridges, the XF96 cell culture plate was loaded into the XF96 Extracellular Flux analyzer at 37°C. OCR and ECAR were measured after cycles of mixing and data acquisition (basal), or inhibitor injection, mixing, and data acquisition using Seahorse Wave Desktop software (Agilent Technologies, Santa Clara, CA, USA).
All animal care procedures and experiments were approved by the Institutional Animal Care and Use Committee of Soonchunhyang University Seoul Hospital (Seoul, Korea) (IACUC2013-5) and performed in accordance with the National Research Council (NRC) Guidelines for the Care and Use of Laboratory Animals. All animal experiments were performed using 8-week-old male BALB/c mice (Biogenomics, Seoul, Korea). The animals were maintained on a 12 h light/dark cycle at 25°C, in accordance with the regulations of Soonchunhyang University Seoul Hospital.
Values are expressed as the mean ± SEM and were evaluated by Student’s t-test, or one- or two-way analysis of variance to compute the significance of differences between groups. Dunnett’s test was used for comparisons of three or more groups. Data were considered significantly different at
To analyze the relationship between HK2 expression and CKD, we measured HK2 expression levels in the serum of patients with CKD. ELISA revealed a high level of HK2 in the serum of CKD patients compared with that of the healthy control group (Fig. 1A). To confirm that HK2 expression leads to metabolic dysregulation, we examined the serum concentration of MG, a cytotoxic glycolytic intermediate. MG levels were also significantly elevated in CKD patients compared with those in the healthy control group (Fig. 1B). These results are consistent with previous reports suggesting that HK2 overexpression may play a significant role in kidney injury (Smith
To examine the relationship between HK2 and MSCs in the context of CKD, MSCs were isolated from both healthy and CKD mice. Immunoblotting revealed that CKD mice-derived MSCs exhibited elevated HK2 levels compared with those of their healthy counterparts, whereas HK1 expression remained unaffected (Fig. 1C). In addition, CKD-mMSCs had increased levels of mitochondrial reactive oxygen species (mtROS) and significantly decreased mitochondrial membrane potential, as assessed using the MitoSOX assay and TMRE staining, respectively (Fig. 1D, 1E). Taken together, these results indicate that in patients with CKD, increased HK2 expression promotes toxic glycolytic intermediates such as MG, and the effect of these intermediates might carry over to MSCs, undermining the functionality of mitochondria in harvested MSCs and their therapeutic potential.
Our data verifying the high HK2 levels in patients with CKD as well as in those with CKD-mMSCs suggested that CKD-mMSCs may suffer from impaired mitochondrial function related to the role of HK2 in metabolism. Hence, we hypothesized that decreasing the expression of HK2 may increase the functionality of MSCs by positively influencing mitochondrial activity. On a different note, we also questioned whether melatonin can modulate glycolytic overload via HK2. We previously reported that melatonin treatment provides protective effects against CKD, and improves mitochondrial functions in renal proximal tubular cells (Han
After confirming the melatonin-mediated reduction of HK2 expression in CKD-mMSCs, we questioned whether the functional improvements observed with mitochondria in melatonin-treated CKD-mMSCs were related to the role of HK2 in glycolytic processes. Consistent with the differences in HK2 expression levels, the MG concentrations were elevated in CKD-mMSCs compared with those of mMSCs harvested from healthy mice (Nor-mMSCs), and this abnormal elevation of MG concentrations was inhibited by melatonin treatment (Fig. 3A).
To determine whether this change in MG concentration is reflective of the larger shift in the bioenergetic profile of MSCs in response to melatonin treatment, we employed a seahorse assay to measure the OCR and ECAR. Plotting the OCR vs. ECAR for CKD-mMSCs, melatonin-treated CKD-mMSCs, and Nor-mMSCs (Fig. 3B) showed a consistently positive correlation with similar slopes, which suggests a similar metabolic profile across different conditions, and that the increase in OCR and ECAR values corresponded to an increase in the metabolic activity of mMSCs. Melatonin-treated CKD-mMSCs showed improved metabolic activity compared with that of the Nor-mMSC group, whereas CKD-mMSCs, which have no efficacy, and melatonin inhibitors were grouped together given their low metabolic activity. Specific measurements of ECAR (Fig. 3C) and OCR (Fig. 3D) demonstrated improved glycolysis and mitochondrial respiration, respectively. Overall, experimental mMSC groups with high MG levels were metabolically less active with worse performance in glycolysis and mitochondrial respiration, and melatonin treatment was able to enhance the metabolic performance of CKD-mMSCs closer to that of Nor-mMSCs through regulation of MG by influencing HK2 expression.
Since melatonin was found to be capable of regulating HK2 expression and MG concentration in CKD-mMSCs to restore their mitochondrial function and metabolic activity, we next analyzed whether these improvements translated into an enhanced proliferative capacity of MSCs, which is an important feature for autologous MSC treatment. To assess the improvement in cell proliferation induced by melatonin in CKD-mMSCs, we determined the cell cycle of CKD-mMSCs using flow cytometry. CKD-mMSCs showed a decrease in the cell population in the S phase compared with that of Nor-mMSCs. However, melatonin treatment effectively restored the cell population in the S phase (Fig. 4A). We further determined the expression of cell cycle-associated proteins in CKD-mMSCs. Western blotting data showed marked downregulation in the expression of CDK2, cyclin E, CDK4, and cyclin D1, which are all important for the G1-phase to S-phase transition, whereas melatonin treatment successfully restored the expression of these proteins in CKD-mMSCs (Fig. 4B). Densitometric analysis showed that pre-treatment with luzindole almost completely negated the protective effect of melatonin, indicating that all improvements observed with CKD-mMSCs were attributable to the melatonin treatment. These results suggest that the ability of melatonin to regulate HK2 and its downstream metabolite MG may have implications for restoring cell proliferation in CKD-mMSCs.
After confirming that melatonin treatment improved the mitochondrial function and cell proliferation of CKD-mMSCs, we tested whether melatonin could improve the therapeutic efficacy of CKD-mMSCs in the CKD mouse model. We examined the renal function recovery in CKD mice after the injection of melatonin-treated CKD-mMSCs by measuring serum blood urea nitrogen (BUN) and creatinine levels. When Nor-mMSCs were injected into CKD mice, BUN and creatinine levels decreased, whereas the injection of CKD-mMSCs did not decrease BUN and creatinine levels. However, when melatonin-treated CKD-mMSCs were injected into CKD mice, BUN and creatinine levels significantly increased (Fig. 5A, 5B). Further, treatment with luzindole reversed these effects, confirming that the observed effects were due to melatonin treatment (Fig. 5A, 5B). These results suggest that melatonin enhances the mitochondrial function and cell proliferation of CKD-mMSCs, and improves the therapeutic efficacy of CKD-mMSCs in a CKD mouse model.
Sustained damage to the kidney tissues leads to CKD and progressive loss of kidney function. The compromised glomerular filtration, excretion, and reabsorption causes the increased circulation of uremic toxins. These effects can be detrimental to the viability and normal functions of MSCs and limit their application in regenerative therapy for CKD patients. Although previous studies to improve the efficacy of MSC-based therapy for CKD have demonstrated that melatonin treatment provides a broad range of protective effects (Han
Although recent studies on the mechanisms underlying the therapeutic effect of melatonin against kidney diseases have focused on its effect on mitochondrial function (Yoon
hMSCs are among the most commonly used adult stem cells in experimental cell therapies (Pittenger
Although melatonin is primarily known for its role in sleep regulation, the broad impact of melatonin on regenerative processes in various tissues provides an excellent entry point for exploring different regulatory mechanisms for therapeutic purposes (Tordjman
In this study, we demonstrated that melatonin restores mitochondrial function and promotes cell proliferation by inhibiting the expression of HK2 and the accumulation of MG. There are conflicting reports on the correlation between mitochondrial function and cell proliferation. In cancer cells with active cell division, mitochondrial function is decreased and glycolysis is increased (Galli
Given that the clinical treatment of CKD patients with hMSC-based cell therapy requires substantial improvements in efficacy, we examined the efficacy of the melatonin-mediated inhibition of HK2 expression in CKD-mMSCs derived from a CKD mouse model. Not only were abnormal elevations of HK2 and its downstream metabolite MG inhibited by melatonin treatment, but also mitochondrial function as assessed by the respiratory OCR, and cell proliferation as measured by the expression of pro-proliferative proteins, were also improved. This experimental outcome may reflect the significant role of HK2 in the cellular activity and metabolism of MSCs. It is noteworthy that the broad impact of melatonin on CKD-mMSCs influences other metabolic processes in addition to hexokinase-induced glycolysis. Therefore, identification and characterization of the various intertwined metabolic regulators under the influence of melatonin with respect to MSCs in a CKD condition may build a foundation for developing more effective therapeutic strategies, cell-based or otherwise, for treating CKD and other kidney disorders.
This research was supported by the Bio & Medical Technology Development Program of the National Research Foundation (NRF) funded by the Korean government (MSIT) (2019M3A9H1103495).
The authors have no conflict of interest to declare.