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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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Indoxyl sulfate (IS) and p-cresyl sulfate (PCS) increased DNA methyltransferase (DNMT)1, 3a, and 3b expression in vitro. (a) Compared with HK2 cells without IS/PCS treatment, real-time PCR analysis showed that IS and PCS significantly increased DNMT 1, 3a, and 3b expression. (b) Results of western blotting for DNMT 1 showed that IS and PCS at concentrations of 1, 5, and 50 mg/l significantly increased DNMT 1 protein expression in cultured HK2 cells. *P<0.05 vs. lane 1.
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fig7: Indoxyl sulfate (IS) and p-cresyl sulfate (PCS) increased DNA methyltransferase (DNMT)1, 3a, and 3b expression in vitro. (a) Compared with HK2 cells without IS/PCS treatment, real-time PCR analysis showed that IS and PCS significantly increased DNMT 1, 3a, and 3b expression. (b) Results of western blotting for DNMT 1 showed that IS and PCS at concentrations of 1, 5, and 50 mg/l significantly increased DNMT 1 protein expression in cultured HK2 cells. *P<0.05 vs. lane 1.

Mentions: The results of real-time PCR indicated that IS and PCS could significantly increase DNMT 1 mRNA expression in cultured HK2 cells at a concentration of 1, 5, and 50 mg/l. The mRNA expressions of DNMT 3a and 3b were significantly increased in HK2 cells treated with IS or PCS at concentrations of 5 and 50 mg/l (Figure 7a). Western blotting for DNMT 1 revealed that IS and PCS could significantly increase DNMT 1 protein expression in vitro (Figure 7b).


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) increased DNA methyltransferase (DNMT)1, 3a, and 3b expression in vitro. (a) Compared with HK2 cells without IS/PCS treatment, real-time PCR analysis showed that IS and PCS significantly increased DNMT 1, 3a, and 3b expression. (b) Results of western blotting for DNMT 1 showed that IS and PCS at concentrations of 1, 5, and 50 mg/l significantly increased DNMT 1 protein expression in cultured HK2 cells. *P<0.05 vs. lane 1.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig7: Indoxyl sulfate (IS) and p-cresyl sulfate (PCS) increased DNA methyltransferase (DNMT)1, 3a, and 3b expression in vitro. (a) Compared with HK2 cells without IS/PCS treatment, real-time PCR analysis showed that IS and PCS significantly increased DNMT 1, 3a, and 3b expression. (b) Results of western blotting for DNMT 1 showed that IS and PCS at concentrations of 1, 5, and 50 mg/l significantly increased DNMT 1 protein expression in cultured HK2 cells. *P<0.05 vs. lane 1.
Mentions: The results of real-time PCR indicated that IS and PCS could significantly increase DNMT 1 mRNA expression in cultured HK2 cells at a concentration of 1, 5, and 50 mg/l. The mRNA expressions of DNMT 3a and 3b were significantly increased in HK2 cells treated with IS or PCS at concentrations of 5 and 50 mg/l (Figure 7a). Western blotting for DNMT 1 revealed that IS and PCS could significantly increase DNMT 1 protein expression in vitro (Figure 7b).

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