Pioglitazone Protects Mesenchymal Stem Cells against P-Cresol-Induced Mitochondrial Dysfunction via Up-Regulation of PINK-1

Abstract

Mesenchymal stem cells (MSC) could be a candidate for cell-based therapy in chronic kidney disease (CKD); however, the uremic toxin in patients with CKD restricts the therapeutic efficacy of MSCs. To address this problem, we explored the effect of pioglitazone as a measure against exposure to the uremic toxin P-cresol (PC) in MSCs. Under PC exposure conditions, apoptosis of MSCs was induced, as well as PC-induced dysfunction of mitochondria by augmentation of mitofusion, reduction of mitophagy, and inactivation of mitochondrial complexes I and IV. Treatment of MSCs with pioglitazone significantly inhibited PC-induced apoptosis. Pioglitazone also prevented PC-induced mitofusion and increased mitophagy against PC exposure through up-regulation of phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK-1). Furthermore, pioglitazone protected against PC-induced mitochondrial dysfunction by increasing the cytochrome c oxidase subunit 4 (COX4) level and activating complexes I and IV, resulting in enhancement of proliferation. In particular, activation of nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) regulated the pioglitazone-mediated up-regulation of PINK-1. These results indicate that pioglitazone protects MSCs against PC-induced accumulated mitochondrial dysfunction via the NF-κB⁻PINK-1 axis under P-cresol exposure conditions. Our study suggests that pioglitazone-treated MSCs could be a candidate for MSC-based therapy in patients with CKD.

Keywords: PTEN-induced putative kinase 1; cell proliferation; chronic kidney disease; mesenchymal stem cell; pioglitazone.

Figure 1

Pioglitazone-treated mesenchymal stem cells (MSCs) and induced apoptosis on exposure to P-cresol (PC). (A–D) Western blot was used to analyze the expression of apoptosis-associated proteins BCL2, BAX, cleaved caspase-3, and cleaved PARP-1 in MSCs exposed to PC (500 μM) for 0, 24, 48, and 72 h. The expression levels were determined by densitometry relative to β-actin. Values represent the mean ± standard error of the mean (SEM). * p < 0.05, and ** p < 0.01 vs untreated MSCs. (E–H) Western blot was used to analyze MSCs pretreated with pioglitazone (5 μM; 24 h) for the expression of apoptosis-associated proteins BCL2, BAX, cleaved caspase-3, and cleaved PARP-1 on exposure to PC. The expression levels were determined by densitometry relative to β-actin. Values represent the mean ± SEM. ** p < 0.01 vs untreated MSCs, ## p < 0.01 vs MSCs exposed to PC. (I) Annexin V and PI positive cells were detected using fluorescence-activated cell sorting (FACS) analysis. Values represent the mean ± SEM. ** p < 0.01 vs untreated MSCs, ## p < 0.01 vs MSCs exposed to PC.

Figure 2

Pioglitazone-treated MSCs increased the expression of PINK-1 through activation of NF-κB. (A,B) Western blot was used to analyze the activation of p-NF-κB and the expression of PINK-1 in MSCs on exposure to PC (500 μM) for 0, 24, 48, and 72 h. The expression levels were determined by densitometry relative to β-actin. Values represent the mean ± SEM. * p < 0.05 and ** p < 0.01 vs untreated MSCs. (C,D) Western blot was used to analyze the activation of p-NF-κB and the expression of PINK-1 in MSCs treated with pioglitazone (5 μM) for 0, 24, 48, and 72 h. The expression levels were determined by densitometry relative to β-actin. Values represent the mean ± SEM. * p < 0.05 and ** p < 0.01 vs untreated MSCs. (E) Pretreated MSCs with or without NF-κB SN50 cell permeable inhibitory peptide trifluoroacetate salt (SN-50) for 24 h were analyzed via Western blot for the expression of PINK-1 in the treatment with pioglitazone (5 μM; 48 h). The expression levels were determined by densitometry relative to β-actin. Values represent the mean ± SEM. * p < 0.05 and ** p < 0.01 vs untreated MSCs, ## p < 0.01 vs MSCs treated with pioglitazone. (F) MSCs pretreated with or without pioglitazone (5 μM; 48 h) were analyzed via Western blot for the expression of PINK-1 on exposure to PC (500 μM; 48 h). The expression levels were determined by densitometry relative to β-actin. Values represent the mean ± SEM. ** p < 0.01 vs untreated MSCs, ## p < 0.01 vs MSCs exposed to PC.

Figure 3

Pioglitazone-treated MSCs increased mitochondrial fission by increasing expression of PINK-1 on exposure to PC. (A,B) After PC (500 μM) exposure for 72 h, mitochondria were measured using Mitotracker in si-PRNP-transfected MSCs after treatment with pioglitazone (5 μM) for 24 h. Values represent the mean ± SEM. * p < 0.05 and ** p < 0.01 vs untreated MSCs, ## p < 0.01 vs MSCs exposed to PC, $$ p < 0.01 vs MSCs treated with pioglitazone. (C–E) After the 72 h PC (500 μM) exposure, Western blot was used to analyze the activation of phosphorylated dynamin-related protein 1 (p-DRP1) and the expression of mitofusin-1 (MFN1) and dynamin-like 120 kDa protein (OPA1) in MSCs after 24 h of treatment of small interfering-RNA PRioN Protein (si-PRNP)-transfected MSCs with pioglitazone (5 μM; 24 h). The expression levels were determined by densitometry relative to β-actin. Values represent the mean ± SEM. ** p < 0.01 vs untreated MSCs, # p < 0.05 and ## p < 0.01 vs MSCs exposed to PC, $$ p < 0.01 vs MSCs treated with pioglitazone.

Figure 4

Pioglitazone-treated MSCs increased mitophagy by increasing PINK-1 on exposure to PC. (A) Autophagy was measured by lysosomal/autophagic vacuole fluorescent staining. Values represent the mean ± SEM. ** p < 0.01 vs untreated MSCs, ## p < 0.01 vs MSCs exposed to PC, $$ p < 0.01 vs MSCs treated with pioglitazone. (B,C) After the 72 h PC (500 μM) exposure, Western blot was used to analyze the expression of LC3B and P62 in MSCs after 24 h treatment of si-PRNP-transfected MSCs with pioglitazone (5 μM; 24 h). The expression levels were determined by densitometry relative to β-actin. Values represent the mean ± SEM. ** p < 0.01 vs untreated MSCs, ## p < 0.01 vs MSCs exposed to PC, $$ p < 0.01 vs MSCs treated with pioglitazone.

Figure 5

Pioglitazone-treated MSCs restored mitochondrial function by increasing PINK-1. (A) Western blot was used to analyze the expression of cytochrome c oxidase subunit 4 (COX4) in MSCs on exposure to PC (500 μM) for 0, 24, 48, and 72 h. The expression levels were determined by densitometry relative to β-actin. Values represent the mean ± SEM. ** p < 0.01 vs untreated MSCs. (B) After the 72 h PC (500 μM) exposure, Western blot was used to analyze the expression of COX4 in MSCs after the 24 h treatment in si-PRNP-transfected MSCs with pioglitazone (5 μM; 24 h). The expression levels were determined by densitometry relative to β-actin. Values represent the mean ± SEM. * p < 0.05 and ** p < 0.01 vs untreated MSCs, ## p < 0.01 vs MSCs exposed to PC, $$ p < 0.01 vs MSCs treated with pioglitazone. (C,D) After exposure to PC (500 μM; 72 h), complex I and IV activity was measured in the MSCs treated with pioglitazone (5 μM; 24 h) by evaluating the expression of PINK-1. Values represent the mean ± SEM. ** p < 0.01 vs untreated MSCs, ## p < 0.01 vs MSCs exposed to PC, $$ p < 0.01 vs MSCs treated with pioglitazone. (E) Mitochondrial ROS was detected by MitoSOX™. The positive MitoSOX™ of MSCs was measured using a flow cytometer. Values represent the mean ± SEM. ** p < 0.01 vs untreated MSCs, ## p < 0.01 vs MSCs exposed to PC, $$ p < 0.01 vs MSCs treated with pioglitazone.

Figure 6

Pioglitazone-treated MSCs increased cell proliferation by regulation of PINK-1. (A–D) Western blot was used to analyze the expression of cell-cycle-associated proteins cyclin D1, cyclin-dependent kinase 4 (CDK4), cyclin E, and CDK2 in MSCs on exposure to PC (500 μM) for 0, 24, 48, and 72 h. The expression levels were determined by densitometry relative to β-actin. Values represent the mean ± SEM * p < 0.05 and ** p < 0.01 vs untreated MSCs. (E–H) After the 72 h PC (500 μM) exposure, Western blot was used to analyze the expression of COX4 in MSCs after the 24 h treatment of si-PRNP-transfected MSCs with pioglitazone (5 μM; 24 h). The expression levels were determined by densitometry relative to β-actin. Values represent the mean ± SEM. * p < 0.05 and ** p < 0.01 vs untreated MSCs, ## p < 0.01 vs MSCs exposed to PC, $$ p < 0.01 vs MSCs treated with pioglitazone.

Figure 7

Schematic representation of possible mechanisms by which pioglitazone prevents PC-induced dysfunction of mitochondria in MSCs through the NF-κB–PINK-1 signal pathway. Under the PC exposure condition, the activation of NF-κB and the expression of PINK-1 are decreased, resulting in apoptosis induced by dysfunction of mitochondria. Treatment with pioglitazone activates NF-κB and increases the expression of PINK-1 under the PC exposure condition, leading to the reinstatement of function of mitochondria and the augmentation of MSC proliferation. Red thick arrow means up-regulation, and blue thick arrow means down-regulation.