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Suppression of Klotho expression by protein-bound uremic toxins is associated with increased DNA methyltransferase expression and DNA hypermethylation.

Sun CY, Chang SC, Wu MS - Kidney Int. (2012)

Bottom Line: The expression of DNA methyltransferases 1, 3a, and 3b isoforms in HK2 cells treated with indoxyl sulfate or p-cresyl sulfate was significantly increased.Specific inhibition of DNA methyltransferase isoform 1 by 5-aza-2'-deoxycytidine caused demethylation of the Klotho gene and increased Klotho expression in vitro.Thus, inhibition of Klotho gene expression by uremic toxins correlates with gene hypermethylation, suggesting that epigenetic modification of specific genes by uremic toxins may be an important pathological mechanism of disease.

View Article: PubMed Central - PubMed

Affiliation: School of Medicine, Chang Gung University, Taoyuan, Taiwan.

ABSTRACT
The expression of the renoprotective antiaging gene Klotho is decreased in uremia. Recent studies suggest that Klotho may be a tumor suppressor, and its expression may be repressed by DNA hypermethylation in cancer cells. Here we investigated the effects and possible mechanisms by which Klotho expression is regulated during uremia in uninephrectomized B-6 mice given the uremic toxins indoxyl sulfate or p-cresyl sulfate. Cultured human renal tubular HK2 cells treated with these toxins were used as an in vitro model. Injections of indoxyl sulfate or p-cresyl sulfate increased their serum concentrations, kidney fibrosis, CpG hypermethylation of the Klotho gene, and decreased Klotho expression in renal tubules of these mice. The expression of DNA methyltransferases 1, 3a, and 3b isoforms in HK2 cells treated with indoxyl sulfate or p-cresyl sulfate was significantly increased. Specific inhibition of DNA methyltransferase isoform 1 by 5-aza-2'-deoxycytidine caused demethylation of the Klotho gene and increased Klotho expression in vitro. Thus, inhibition of Klotho gene expression by uremic toxins correlates with gene hypermethylation, suggesting that epigenetic modification of specific genes by uremic toxins may be an important pathological mechanism of disease.

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Related in: MedlinePlus

Indoxyl sulfate (IS)- and p-cresyl sulfate (PCS)-injected mice had increased DNA methyltransferase 1 (DNMT 1) expression and DNA hypermethylation of the Klotho gene. (a) The results of western blot analysis and immunostaining showed that IS and PCS significantly increased the DNMT 1 expression. (b) Methylation-specific PCR (MSP) analysis with primer sets 1 and 2 showed that both IS- and PCS-injected mice had significantly higher methylation indexes of Klotho genes than control mice. *P<0.05 vs. control.
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fig2: Indoxyl sulfate (IS)- and p-cresyl sulfate (PCS)-injected mice had increased DNA methyltransferase 1 (DNMT 1) expression and DNA hypermethylation of the Klotho gene. (a) The results of western blot analysis and immunostaining showed that IS and PCS significantly increased the DNMT 1 expression. (b) Methylation-specific PCR (MSP) analysis with primer sets 1 and 2 showed that both IS- and PCS-injected mice had significantly higher methylation indexes of Klotho genes than control mice. *P<0.05 vs. control.

Mentions: The western blot results revealed that the expression of DNMT 1 in IS- and PCS-injected mice significantly increased compared with the control mice. It was also noted by immunohistochemistry staining that IS and PCS significantly increased the DNMT 1 expression in the nuclei of the glomerular and tubular cells (Figure 2a). Methylation-specific PCR (MSP) analysis with primer sets 1 and 2 showed that both IS- and PCS-injected mice had significantly higher methylation indexes of the Klotho gene than control mice (Figure 2b). The relative methylation indexes of IS- and PCS-injected mice vs. control mice were 5.3:1 and 4.4:1, respectively, in the position of primer set 1. The relative methylation indexes of IS- and PCS-injected mice vs. control mice were 3.8:1 and 2.7:1, respectively, in the position of primer set 2.


Suppression of Klotho expression by protein-bound uremic toxins is associated with increased DNA methyltransferase expression and DNA hypermethylation.

Sun CY, Chang SC, Wu MS - Kidney Int. (2012)

Indoxyl sulfate (IS)- and p-cresyl sulfate (PCS)-injected mice had increased DNA methyltransferase 1 (DNMT 1) expression and DNA hypermethylation of the Klotho gene. (a) The results of western blot analysis and immunostaining showed that IS and PCS significantly increased the DNMT 1 expression. (b) Methylation-specific PCR (MSP) analysis with primer sets 1 and 2 showed that both IS- and PCS-injected mice had significantly higher methylation indexes of Klotho genes than control mice. *P<0.05 vs. control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3306006&req=5

fig2: Indoxyl sulfate (IS)- and p-cresyl sulfate (PCS)-injected mice had increased DNA methyltransferase 1 (DNMT 1) expression and DNA hypermethylation of the Klotho gene. (a) The results of western blot analysis and immunostaining showed that IS and PCS significantly increased the DNMT 1 expression. (b) Methylation-specific PCR (MSP) analysis with primer sets 1 and 2 showed that both IS- and PCS-injected mice had significantly higher methylation indexes of Klotho genes than control mice. *P<0.05 vs. control.
Mentions: The western blot results revealed that the expression of DNMT 1 in IS- and PCS-injected mice significantly increased compared with the control mice. It was also noted by immunohistochemistry staining that IS and PCS significantly increased the DNMT 1 expression in the nuclei of the glomerular and tubular cells (Figure 2a). Methylation-specific PCR (MSP) analysis with primer sets 1 and 2 showed that both IS- and PCS-injected mice had significantly higher methylation indexes of the Klotho gene than control mice (Figure 2b). The relative methylation indexes of IS- and PCS-injected mice vs. control mice were 5.3:1 and 4.4:1, respectively, in the position of primer set 1. The relative methylation indexes of IS- and PCS-injected mice vs. control mice were 3.8:1 and 2.7:1, respectively, in the position of primer set 2.

Bottom Line: The expression of DNA methyltransferases 1, 3a, and 3b isoforms in HK2 cells treated with indoxyl sulfate or p-cresyl sulfate was significantly increased.Specific inhibition of DNA methyltransferase isoform 1 by 5-aza-2'-deoxycytidine caused demethylation of the Klotho gene and increased Klotho expression in vitro.Thus, inhibition of Klotho gene expression by uremic toxins correlates with gene hypermethylation, suggesting that epigenetic modification of specific genes by uremic toxins may be an important pathological mechanism of disease.

View Article: PubMed Central - PubMed

Affiliation: School of Medicine, Chang Gung University, Taoyuan, Taiwan.

ABSTRACT
The expression of the renoprotective antiaging gene Klotho is decreased in uremia. Recent studies suggest that Klotho may be a tumor suppressor, and its expression may be repressed by DNA hypermethylation in cancer cells. Here we investigated the effects and possible mechanisms by which Klotho expression is regulated during uremia in uninephrectomized B-6 mice given the uremic toxins indoxyl sulfate or p-cresyl sulfate. Cultured human renal tubular HK2 cells treated with these toxins were used as an in vitro model. Injections of indoxyl sulfate or p-cresyl sulfate increased their serum concentrations, kidney fibrosis, CpG hypermethylation of the Klotho gene, and decreased Klotho expression in renal tubules of these mice. The expression of DNA methyltransferases 1, 3a, and 3b isoforms in HK2 cells treated with indoxyl sulfate or p-cresyl sulfate was significantly increased. Specific inhibition of DNA methyltransferase isoform 1 by 5-aza-2'-deoxycytidine caused demethylation of the Klotho gene and increased Klotho expression in vitro. Thus, inhibition of Klotho gene expression by uremic toxins correlates with gene hypermethylation, suggesting that epigenetic modification of specific genes by uremic toxins may be an important pathological mechanism of disease.

Show MeSH
Related in: MedlinePlus