Abstract:

Objective

This study aimed to investigate the effect and mechanism of Klotho gene hypermethylation in vascular SMC-osteoblast conversion induced by indoxyl sulfate (IS).

Methods

Human aortic vascular smooth muscle cells (HASMC) were cultured. IS concentrations of 0, 200, 500,1 000 μmol/L were used for 6 days. Alizarin red staining was used for observation of calcium salt deposits.Real-time polymerase chain reaction (PCR) and Western blotting were applied to detect mRNA and protein expressions of α-smooth muscle actin (α-SMA), smoothin, osteopontin (OPN), alkaline phosphatase(ALP), nuclear binding factor alpha 1 (Cbf α1), Klotho, and different DNA methylation transferases(DNMT). Klotho gene methylation level in HASMC was assessed by pyrosequencing assay. In IS 1 000 μmol/L group, different concentrations of 5-Aza-2-deoxycytidine (5Aza-2dc) were added into the culture medium for interference experiment. Mean comparison between groups was done with independent sample t-test, while rate comparison between groups was done with chi-square test.

Results

In IS 1 000 μmol/L group, extracellular matrix of HASMC was stained red, which was concentrated in cell nodules, showing positive calcium salt deposition. IS decreased the expression of α-SMA mRNA and protein as well as smoothin, and increased the mRNA and protein expression of OPN and ALP, making the HASMC to gradually lose the SMC phenotype and transfer to osteoblast. Stimulation of HASMC by IS at the concentration of 200,500, and 1 000 μmol/L for 6 days significantly induced Klotho gene hypermethylation ratio compareal with the control group (9 ±1% vs 4 ±0.7%, t=5.38, P ＜0.01;18 ±1.3 vs 4 ±0.7%,t=6.92, P ＜0.01; 41 ±2% vs 4 ±0.7%, t =9.26, P ＜0.001). IS at the concentration of 500 and 1 000 μmol /L also significantly decreased Klotho mRNA and protein levels in HASMC. At the same time,IS obviously increased the expression of DNMT1 mRNA and protein level. 5Aza-2dc 10 μmol/L reduced the calcium deposition induced by IS, upregulated α-SMA protein and smoothin expression, down-regulated Cbf αl protein level, simultaneously decreased the Klotho gene methylation ratio (20 ±1% vs 41 ±2%,t=6.98, P ＜0.01), and increased the protein level of Klotho in HASMC.

Conclusion

IS induced the vascular SMC-osteoblast conversion. Through upregulating of DNMT1 activity, IS induced Klotho gene hypermethylation, and down-regulated Klotho protein expression. Block of Klotho gene hypermethylation reduced the vascular SMC-osteoblast conversion induced by IS.

Key words: Klotho gene, DNA methylation, Indoxyl sulfate