P - cresol and Indoxyl Sulfate Impair Osteogenic Differentiation by Triggering Mesenchymal Stem Cell Senescence

Abstract

Chronic kidney disease (CKD) patients obtained high levels of uremic toxins progressively develop several complications including bone fractures. Protein-bound uremic toxins especially p-cresol and indoxyl sulfate are hardly eliminated due to their high molecular weight. Thus, the abnormality of bone in CKD patient could be potentially resulted from the accumulation of uremic toxins. To determine whether protein-bound uremic toxins have an impact on osteogenesis, mesenchymal stem cells were treated with either p-cresol or indoxyl sulfate under in vitro osteogenic differentiation. The effects of uremic toxins on MSC-osteoblastic differentiation were investigated by evaluation of bone phenotype. The results demonstrated that p-cresol and indoxyl sulfate down-regulated the transcriptional level of collagen type I, deceased alkaline phosphatase activity, and impaired mineralization of MSC-osteoblastic cells. Furthermore, p-cresol and indoxyl sulfate gradually increased senescence-associated beta-galactosidase positive cells while upregulated the expression of p21 which participate in senescent process. Our findings clearly revealed that the presence of uremic toxins dose-dependently influenced a gradual deterioration of osteogenesis. The effects partially mediate through the activation of senescence-associated gene lead to the impairment of osteogenesis. Therefore, the management of cellular senescence triggered by uremic toxins could be considered as an alternative therapeutic approach to prevent bone abnormality in CKD patients.

Keywords: cellular senescence; chronic kidney disease; indoxyl sulfate; mesenchymal stem cells; osteogenesis; p-cresol.

Figure 1

Mesenchymal stem cell characteristics: Bone marrow-derived mesenchymal stem cells exhibited fibroblast-like morphology (A) and were positive for alizarin red S staining (B) and oil red O staining (C) after cultured in osteogenic induction medium and adipogenic induction medium, respectively (Scale bar: 200 µm). Histograms demonstrated the expression of CD34, CD45, CD73, CD90, and CD105 of bone marrow-derived mesenchymal stem cell population (D).

Figure 2

Cytotoxic effects of p-cresol and indoxyl sulfate: The morphology of MSCs was observed after incubation MSCs with various concentrations of p-cresol (A) and indoxyl sulfate (B) for 24 hours (Scale bar: 200 µm). The cell viability was determined at 24, 48, and 72 hours of incubation with p-cresol (C) and indoxyl sulfate (D) using MTT assay. The data were presented as percent viable cells of control from four individual experiments (N=4). * P-value < 0.05 and ** P-value < 0.001 as analyzed by one-way ANOVA.

Figure 3

The effects of p-cresol on osteogenesis of MSC: Osteogenic differentiation of MSCs were determined which included the expression of osteogenic-associated genes, ALP activity, and mineralization. The expression levels of osteogenic-associated genes; RUNX2, OPN, and COL1A1 were examined after treatment with p-cresol for 7 days and 14 days of osteogenic differentiation (A). The alkaline phosphatase (ALP) activity was assessed after treatment with p-cresol (10, 40, 80, and 160 µg/ml) for 7 day and 10 day of differentiation (B). The data were presented as relative ALP activity (U/ml) from four individual experiment (N=4). * P-value < 0.05 comparison with control. Alizarin red S staining was investigated for calcium deposition at day 14 of differentiation (C) (Scale bar: 200 µm).

Figure 4

Effects of indoxyl sulfate (IS) on osteogenic differentiation potential of MSCs: Bar graph demonstrate the expression levels of osteogenic-associated genes; RUNX2, OPN, and COL1A1 at day 7 and 14 of differentiation after treatment with indoxyl sulfate relative to control (A). Alkaline phosphatase activity assessed after treatment with indoxyl sulfate (25, 50, 100, and 200 µg/ml) for 7 day and 10 day of differentiation (B). The data were presented as relative ALP activity (U/ml) from four individual experiment (N=4). * P value < 0.05 comparison with control. Alizarin red S staining was assessed for calcium deposition at day 14 of differentiation (C) (Scale bar: 200 µm).

Figure 5

The role of p-cresol and indoxyl sulfate on MSC senescence during osteogenic differentiation: Senescence-associated betagalactosidase staining demonstrated the blue color developed in senescent cells after exposure to p-cresol (A) and indoxyl sulfate (B) on day 7 of osteogenic differentiation (Scale bar: 200 µm). The number of β-galactosidase positive cells were counted and presented as β-galactosidase positive cells relative to control (C-D). * P-value < 0.05 and ** P-value < 0.01 as compared with control.

Figure 6

The transcriptional levels of senescence-associated genes after p-cresol and indoxyl sulfate treatment: The expression of senescence-associated genes including p16, p53, and p21 was investigated after exposure to p-cresol (A) and indoxyl sulfate (B) at day 7 and day 14 of osteogenic differentiation. * P-value < 0.05 as compared with control.