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

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.

Figure 1

Results of Masson's trichrome staining and the serum levels of indoxyl sulfate (IS) and p-cresyl sulfate (PCS) in experimental mice. (a) Masson's trichrome staining showed that both IS- and PCS-injected mice had significantly increased kidney fibrosis than control mice. The total IS concentrations of control, IS, and PCS mice were 2.39±0.15, 8.55±0.37, and 5.61±0.60 mg/l, respectively. The total PCS concentrations of control, IS, and PCS study groups were <0.225, 0.66±0.04, and 1.82±0.03 mg/l, respectively. (b) The total IS and PCS concentrations of IS- and PCS-injected mice were significantly higher than those of control mice. The free IS levels of control, IS, and PCS study groups were 0.87±0.14, <0.50, and <0.50 mg/l, respectively. The free IS levels of IS-injected mice were significantly higher than control mice (P<0.001). The free PCS levels of control, IS, and PCS study groups were <0.15, <0.15, and 0.21±0.01 mg/l, respectively. (b) The free PCS levels of PCS-injected mice were significantly higher than control mice (P<0.001). ^*P<0.05 vs. control; ^#P<0.05 vs. IS.

Figure 2

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.

Figure 3

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.

Figure 4

DNA methyltransferase 1 (DNMT 1) inhibitor demethylated the Klotho gene and increased Klotho expression in vivo. (a) Methylation-specific PCR (MSP) with mouse Klotho set 1 primers showed that simultaneous treatment with 5-aza-2′-deoxycytidine (5Aza-2dc) in indoxyl sulfate (IS)- and p-cresyl sulfate (PCS)-injected mice significantly demethylated the Klotho gene. (b) Western blotting showed that 5Aza-2dc significantly increased the Klotho expression in IS- and PCS-injected mice.

Figure 5

Indoxyl sulfate (IS) and p-cresyl sulfate (PCS) increased DNA methylation of the Klotho gene in vitro. Chromosome DNA samples from cultured HK2 cells treated with IS or PCS for 72 h were analyzed. Methylation-specific PCR (MSP) with human Klotho primer sets (a) 1 and (b) 2 revealed that the methylation indexes of HK2 cells treated with IS or PCS were significantly increased. ^*P<0.05 vs. lane 1.

Figure 6

Indoxyl sulfate (IS) and p-cresyl sulfate (PCS) reduced Klotho expression in vitro. Western blotting showed that the Klotho protein expression was significantly decreased in HK2 cells treated with IS or PCS. ^*P<0.05 vs. lane 1.

Figure 7

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.

Figure 8

DNA methyltransferase 1 (DNMT 1) inhibitor demethylated the Klotho gene. Methylation-specific PCR (MSP) with human Klotho set 1 primers showed that 5-Aza-2′-deoxycytidine (5Aza-2dc) inhibited Klotho gene hypermethylation in cultured HK2 cells treated with 50 mg/l indoxyl sulfate (IS) or p-cresyl sulfate (PCS). ^*P<0.05 vs. lane 1.

Figure 9

DNA methyltransferase 1 (DNMT 1) inhibitor increased Klotho expression in vitro. Western blotting showed that 5-aza-2′-deoxycytidine (5Aza-2dc) at concentrations of 1 and 10 μmol/l increased the Klotho protein expression significantly in HK2 cells treated with 50 mg/l indoxyl sulfate (IS) or p-cresyl sulfate (PCS). ^*P<0.05 vs. lane 1.

Figure 10

The flow diagram for animal study. 5Aza-2dc, 5-aza-2′-deoxycytidine; IS, indoxyl sulfate; PBS, phosphate-buffered saline; PCS, p-cresyl sulfate.

Figure 11

Plots of CpG islands of the Klotho gene in mice and humans. The genomic region near the transcription start site (+1) of the Klotho gene (mouse: −300 to +900 bp; human: −600 to +200 bp) was inputted for CpG island analysis. The predictive CpG island of the mouse Klotho gene was 931 bp (−123 to +808 bp). The predictive CpG island of the human Klotho gene was 629 bp (−482 to +147 bp). The locations of the methylation-specific PCR (MSP) primers relative to transcription starting site are presented.