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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) injection reduced the Klotho expression in renal tubules. (a) Real-time PCR and (b) western blotting revealed that Klotho mRNA and protein expression were significantly lower in kidneys treated with IS or PCS. (c) Immunofluorescence staining indicated that the intensity of positive Klotho staining in renal tubules was significantly decreased in mice treated with IS or PCS. *P<0.05 vs. control.
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fig3: Indoxyl sulfate (IS) and p-cresyl sulfate (PCS) injection reduced the Klotho expression in renal tubules. (a) Real-time PCR and (b) western blotting revealed that Klotho mRNA and protein expression were significantly lower in kidneys treated with IS or PCS. (c) Immunofluorescence staining indicated that the intensity of positive Klotho staining in renal tubules was significantly decreased in mice treated with IS or PCS. *P<0.05 vs. control.

Mentions: Real-time PCR and western blotting analysis showed that mRNA and protein expression of the Klotho gene were significantly decreased in the renal cortex of IS- and PCS-injected mice when compared with the ½-nephrectomized mice without IS and PCS injection (Figure 3a and b). Immunofluorescence staining also showed that the intensity of positive Klotho staining in renal tubules was significantly decreased in IS and PCS mice (Figure 3c). The results of western blotting showed that the expression of α-glutathione S-transferases increased significantly in IS- and PCS-injected mice when compared with the control mice (Supplementary Figure S1a online). The results of immunofluorescence double staining with Klotho and α-glutathione S-transferases showed that IS and PCS significantly decreased Klotho expression and increased α-glutathione S-transferase expression in the renal tubule cells in vivo (Supplementary Figure S1b online).


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) injection reduced the Klotho expression in renal tubules. (a) Real-time PCR and (b) western blotting revealed that Klotho mRNA and protein expression were significantly lower in kidneys treated with IS or PCS. (c) Immunofluorescence staining indicated that the intensity of positive Klotho staining in renal tubules was significantly decreased in mice treated with IS or PCS. *P<0.05 vs. control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3: Indoxyl sulfate (IS) and p-cresyl sulfate (PCS) injection reduced the Klotho expression in renal tubules. (a) Real-time PCR and (b) western blotting revealed that Klotho mRNA and protein expression were significantly lower in kidneys treated with IS or PCS. (c) Immunofluorescence staining indicated that the intensity of positive Klotho staining in renal tubules was significantly decreased in mice treated with IS or PCS. *P<0.05 vs. control.
Mentions: Real-time PCR and western blotting analysis showed that mRNA and protein expression of the Klotho gene were significantly decreased in the renal cortex of IS- and PCS-injected mice when compared with the ½-nephrectomized mice without IS and PCS injection (Figure 3a and b). Immunofluorescence staining also showed that the intensity of positive Klotho staining in renal tubules was significantly decreased in IS and PCS mice (Figure 3c). The results of western blotting showed that the expression of α-glutathione S-transferases increased significantly in IS- and PCS-injected mice when compared with the control mice (Supplementary Figure S1a online). The results of immunofluorescence double staining with Klotho and α-glutathione S-transferases showed that IS and PCS significantly decreased Klotho expression and increased α-glutathione S-transferase expression in the renal tubule cells in vivo (Supplementary Figure S1b online).

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