Indoxyl Sulfate Enhance the Hypermethylation of Klotho and Promote the Process of Vascular Calcification in Chronic Kidney Disease

Abstract

Chronic kidney disease (CKD) is a state of Klotho deficiency. The Klotho expression may be suppressed due to DNA hypermethylation in cancer cells so we have investigated the effects and possible mechanisms by which Klotho expression is regulated in human aortic smooth muscle cells (HASMCs). The vascular Klotho hypermethylation in radial arteries of patients with end-stage renal disease was described. Cultured HASMCs and 5/6-nephrectomized Sprague Dawley (SD) rats treated with indoxyl sulfate (IS) were used as in vitro and in vivo models, respectively. IS increased CpG hypermethylation of the Klotho gene and decreased Klotho expression in HASMCs, and potentiated HASMCs calcification. The expression of DNA methyltransferase (DNMT) 1 and 3a in HASMCs treated with IS was significantly increased and specific inhibition of DNA methyltransferase 1 by 5-aza-2'-deoxycytidine(5Aza-2dc) caused demethylation of the Klotho gene and increased Klotho expression. In rats, injection of IS potentiated vascular calcification, increased CpG hypermethylation of the Klotho gene and decreased Klotho expression in the aortic medial layer and all of these changes could be reverted by 5Aza-2dc treatment. Transcriptional suppression of vascular Klotho gene expression by IS and epigenetic modification of Klotho by IS may be an important pathological mechanism of vascular calcification in CKD.

Vascular calcification is associated with Wnt‐signaling pathway and blood pressure variability in chronic kidney disease rats

“…Addition of Wnt pathway inhibitor secreted frizzeled‐related protein 5 could inhibit high phosphate‐induced calcification of VSMCs . Dysregulation of klotho, which is an intrinsic inhibitor of Wnt‐signaling, was also shown to be associated with CKD VC . Wnt signaling may induce enhanced expression of matrix metalloproteinase (MMP)‐2 and ‐9, which then foster the development of arteriosclerosis and calcification .…”

“…23 Dysregulation of klotho, which is an intrinsic inhibitor of Wnt-signaling, was also shown to be associated with CKD VC. 27,28 Wnt signaling may induce enhanced expression of matrix metalloproteinase (MMP)-2 and -9, which then foster the development of arteriosclerosis and calcification. 26 We found Wnt3a and β-catenin expression enhanced in calcified vascular in CKD rats.…”

Amelioration of Uremic Toxin Indoxyl Sulfate-Induced Osteoblastic Calcification by SET Domain Containing Lysine Methyltransferase 7/9 Protein

“…In terms of calcification, Adijiang et al [23] demonstrated that IS promoted aortic calcification and accelerated differentiation of VSMCs into osteoblast-like cells with expression of osteoblast-specific proteins in hypertensive rats. A previous study showed that IS enhanced Klotho gene methylation in VC [8]. Epigenetic changes were implicated in the persisting vascular effects of IS.…”

“…Notably, SET7/9 contributes to the transcriptional activation of fibrotic genes in CKD [17]. Our recent study found that IS could regulate specific DNA methylation in human artery smooth muscle cells (HASMCs) [8]. Moreover, the expression level of SET7/9 is frequently downregulated in the medial layer of radial arteries from CKD patients.…”

“…In addition, conventional hemodialysis cannot efficiently remove IS because of its high binding affinity to albumin [6]. Moreover, IS has been demonstrated to act as a vascular toxin [7,8]. All these findings suggested that IS might be one of the major contributors to VC in patients with CKD.…”