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Histone lysine crotonylation during acute kidney injury in mice

View Article: PubMed Central - PubMed

ABSTRACT

Acute kidney injury (AKI) is a potentially lethal condition for which no therapy is available beyond replacement of renal function. Post-translational histone modifications modulate gene expression and kidney injury. Histone crotonylation is a recently described post-translational modification. We hypothesized that histone crotonylation might modulate kidney injury. Histone crotonylation was studied in cultured murine proximal tubular cells and in kidneys from mice with AKI induced by folic acid or cisplatin. Histone lysine crotonylation was observed in tubular cells from healthy murine and human kidney tissue. Kidney tissue histone crotonylation increased during AKI. This was reproduced by exposure to the protein TWEAK in cultured tubular cells. Specifically, ChIP-seq revealed enrichment of histone crotonylation at the genes encoding the mitochondrial biogenesis regulator PGC-1α and the sirtuin-3 decrotonylase in both TWEAK-stimulated tubular cells and in AKI kidney tissue. To assess the role of crotonylation in kidney injury, crotonate was used to increase histone crotonylation in cultured tubular cells or in the kidneys in vivo. Crotonate increased the expression of PGC-1α and sirtuin-3, and decreased CCL2 expression in cultured tubular cells and healthy kidneys. Systemic crotonate administration protected from experimental AKI, preventing the decrease in renal function and in kidney PGC-1α and sirtuin-3 levels as well as the increase in CCL2 expression. For the first time, we have identified factors such as cell stress and crotonate availability that increase histone crotonylation in vivo. Overall, increasing histone crotonylation might have a beneficial effect on AKI. This is the first observation of the in vivo potential of the therapeutic manipulation of histone crotonylation in a disease state.

No MeSH data available.


Crotonate increases histone crotonylation and modifies gene expression in cultured proximal tubular epithelial cells. Cells were stimulated with 0, 50 or 100 mM crotonate for 24 h. (A) Quantification of histone crotonylation (anti-Kcr) and representative western blot. Mean±s.e.m. of three independent experiments; *P<0.05 vs 0 mM crotonate (non-parametric Mann–Whitney U-test). (B,D) PGC-1α and CCL2 mRNA levels. Data from eight independent experiments expressed as mean±s.e.m. *P<0.05 vs control (Student's t-test). (C) PGC-1α western blot of whole-cell extracts. Data from four independent experiments is expressed as mean±s.e.m. *P<0.05 vs control (non-parametric Mann–Whitney U-test). (E) SIRT3 mRNA levels in tubular cells exposed to 50 mM crotonate. Data from four independent experiments expressed as mean±s.e.m. *P<0.05 vs control (non-parametric Mann–Whitney U-test). (F,G) ChIP-seq analysis was performed using a pan anti-Kcr antibody in (F) tubular cells incubated with 100 ng/ml TWEAK for 6 h (data from three independent experiments) and (G) kidney tissue from mouse AKI (six animals per group). Results show the percentage of Kcr enrichment.
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DMM024455F3: Crotonate increases histone crotonylation and modifies gene expression in cultured proximal tubular epithelial cells. Cells were stimulated with 0, 50 or 100 mM crotonate for 24 h. (A) Quantification of histone crotonylation (anti-Kcr) and representative western blot. Mean±s.e.m. of three independent experiments; *P<0.05 vs 0 mM crotonate (non-parametric Mann–Whitney U-test). (B,D) PGC-1α and CCL2 mRNA levels. Data from eight independent experiments expressed as mean±s.e.m. *P<0.05 vs control (Student's t-test). (C) PGC-1α western blot of whole-cell extracts. Data from four independent experiments is expressed as mean±s.e.m. *P<0.05 vs control (non-parametric Mann–Whitney U-test). (E) SIRT3 mRNA levels in tubular cells exposed to 50 mM crotonate. Data from four independent experiments expressed as mean±s.e.m. *P<0.05 vs control (non-parametric Mann–Whitney U-test). (F,G) ChIP-seq analysis was performed using a pan anti-Kcr antibody in (F) tubular cells incubated with 100 ng/ml TWEAK for 6 h (data from three independent experiments) and (G) kidney tissue from mouse AKI (six animals per group). Results show the percentage of Kcr enrichment.

Mentions: Next, we searched for potential target genes of histone crotonylation whose expression is differentially regulated in AKI. PGC-1α is a regulator of mitochondrial biogenesis that is decreased in AKI, whereas SIRT3 is a decrotonylase, and both regulate the expression of each other. Therefore, as a representative downregulated gene we chose PGC-1α, because it regulates SIRT3 expression (Giralt et al., 2011; Kong et al., 2010; Bell and Guarente, 2011). Moreover, TWEAK decreases PGC-1α expression by epigenetic mechanisms involving histone acetylation (Ruiz-Andres et al., 2015). ChIP-seq analysis using the pan anti-crotonyl-lysine antibody showed that PGC-1α and SIRT3 were more enriched in crotonylated histones in tubular cells treated with TWEAK and in kidneys with AKI (Fig. 3F,G). To study the effect of crotonylation on PGC-1α and SIRT3 expression, cells were pretreated with crotonate because exogenous crotonate increased histone crotonylation in cultured tubular cells (Fig. 3A). This is consistent with findings in non-renal cells (Tan et al., 2011; Sabari et al., 2015). Crotonate increased tubular cell PGC-1α mRNA and protein levels (Fig. 3B,C). As representative upregulated gene, we chose CCL2 because it encodes the MCP-1 chemokine, a promoter of kidney injury (Sanz et al., 2010a). Crotonate decreased tubular cell CCL2 mRNA in cultured cells (Fig. 3D). Taken together, these results suggest that histone crotonylation could play an overall protective role in kidney injury by promoting upregulation of some protective genes and downregulation of genes involved in tissue injury. Crotonate also increased SIRT3 mRNA levels in cultured tubular cells in a time-dependent manner (Fig. 3E), suggesting the activation of a negative-feedback loop.Fig. 3.


Histone lysine crotonylation during acute kidney injury in mice
Crotonate increases histone crotonylation and modifies gene expression in cultured proximal tubular epithelial cells. Cells were stimulated with 0, 50 or 100 mM crotonate for 24 h. (A) Quantification of histone crotonylation (anti-Kcr) and representative western blot. Mean±s.e.m. of three independent experiments; *P<0.05 vs 0 mM crotonate (non-parametric Mann–Whitney U-test). (B,D) PGC-1α and CCL2 mRNA levels. Data from eight independent experiments expressed as mean±s.e.m. *P<0.05 vs control (Student's t-test). (C) PGC-1α western blot of whole-cell extracts. Data from four independent experiments is expressed as mean±s.e.m. *P<0.05 vs control (non-parametric Mann–Whitney U-test). (E) SIRT3 mRNA levels in tubular cells exposed to 50 mM crotonate. Data from four independent experiments expressed as mean±s.e.m. *P<0.05 vs control (non-parametric Mann–Whitney U-test). (F,G) ChIP-seq analysis was performed using a pan anti-Kcr antibody in (F) tubular cells incubated with 100 ng/ml TWEAK for 6 h (data from three independent experiments) and (G) kidney tissue from mouse AKI (six animals per group). Results show the percentage of Kcr enrichment.
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DMM024455F3: Crotonate increases histone crotonylation and modifies gene expression in cultured proximal tubular epithelial cells. Cells were stimulated with 0, 50 or 100 mM crotonate for 24 h. (A) Quantification of histone crotonylation (anti-Kcr) and representative western blot. Mean±s.e.m. of three independent experiments; *P<0.05 vs 0 mM crotonate (non-parametric Mann–Whitney U-test). (B,D) PGC-1α and CCL2 mRNA levels. Data from eight independent experiments expressed as mean±s.e.m. *P<0.05 vs control (Student's t-test). (C) PGC-1α western blot of whole-cell extracts. Data from four independent experiments is expressed as mean±s.e.m. *P<0.05 vs control (non-parametric Mann–Whitney U-test). (E) SIRT3 mRNA levels in tubular cells exposed to 50 mM crotonate. Data from four independent experiments expressed as mean±s.e.m. *P<0.05 vs control (non-parametric Mann–Whitney U-test). (F,G) ChIP-seq analysis was performed using a pan anti-Kcr antibody in (F) tubular cells incubated with 100 ng/ml TWEAK for 6 h (data from three independent experiments) and (G) kidney tissue from mouse AKI (six animals per group). Results show the percentage of Kcr enrichment.
Mentions: Next, we searched for potential target genes of histone crotonylation whose expression is differentially regulated in AKI. PGC-1α is a regulator of mitochondrial biogenesis that is decreased in AKI, whereas SIRT3 is a decrotonylase, and both regulate the expression of each other. Therefore, as a representative downregulated gene we chose PGC-1α, because it regulates SIRT3 expression (Giralt et al., 2011; Kong et al., 2010; Bell and Guarente, 2011). Moreover, TWEAK decreases PGC-1α expression by epigenetic mechanisms involving histone acetylation (Ruiz-Andres et al., 2015). ChIP-seq analysis using the pan anti-crotonyl-lysine antibody showed that PGC-1α and SIRT3 were more enriched in crotonylated histones in tubular cells treated with TWEAK and in kidneys with AKI (Fig. 3F,G). To study the effect of crotonylation on PGC-1α and SIRT3 expression, cells were pretreated with crotonate because exogenous crotonate increased histone crotonylation in cultured tubular cells (Fig. 3A). This is consistent with findings in non-renal cells (Tan et al., 2011; Sabari et al., 2015). Crotonate increased tubular cell PGC-1α mRNA and protein levels (Fig. 3B,C). As representative upregulated gene, we chose CCL2 because it encodes the MCP-1 chemokine, a promoter of kidney injury (Sanz et al., 2010a). Crotonate decreased tubular cell CCL2 mRNA in cultured cells (Fig. 3D). Taken together, these results suggest that histone crotonylation could play an overall protective role in kidney injury by promoting upregulation of some protective genes and downregulation of genes involved in tissue injury. Crotonate also increased SIRT3 mRNA levels in cultured tubular cells in a time-dependent manner (Fig. 3E), suggesting the activation of a negative-feedback loop.Fig. 3.

View Article: PubMed Central - PubMed

ABSTRACT

Acute kidney injury (AKI) is a potentially lethal condition for which no therapy is available beyond replacement of renal function. Post-translational histone modifications modulate gene expression and kidney injury. Histone crotonylation is a recently described post-translational modification. We hypothesized that histone crotonylation might modulate kidney injury. Histone crotonylation was studied in cultured murine proximal tubular cells and in kidneys from mice with AKI induced by folic acid or cisplatin. Histone lysine crotonylation was observed in tubular cells from healthy murine and human kidney tissue. Kidney tissue histone crotonylation increased during AKI. This was reproduced by exposure to the protein TWEAK in cultured tubular cells. Specifically, ChIP-seq revealed enrichment of histone crotonylation at the genes encoding the mitochondrial biogenesis regulator PGC-1&alpha; and the sirtuin-3 decrotonylase in both TWEAK-stimulated tubular cells and in AKI kidney tissue. To assess the role of crotonylation in kidney injury, crotonate was used to increase histone crotonylation in cultured tubular cells or in the kidneys in vivo. Crotonate increased the expression of PGC-1&alpha; and sirtuin-3, and decreased CCL2 expression in cultured tubular cells and healthy kidneys. Systemic crotonate administration protected from experimental AKI, preventing the decrease in renal function and in kidney PGC-1&alpha; and sirtuin-3 levels as well as the increase in CCL2 expression. For the first time, we have identified factors such as cell stress and crotonate availability that increase histone crotonylation in vivo. Overall, increasing histone crotonylation might have a beneficial effect on AKI. This is the first observation of the in vivo potential of the therapeutic manipulation of histone crotonylation in a disease state.

No MeSH data available.