Blocking the histone lysine 79 methyltransferase DOT1L alleviates renal fibrosis through inhibition of renal fibroblast activation and epithelial-mesenchymal transition

Abstract

Disruptor of telomeric silencing-1 like (DOT1L) protein specifically catalyzes the methylation of histone H3 on Lys79 (H3K79) and is implicated in tumors. But its role in tissue fibrosis remains unclear. Here we demonstrated that injury to the kidney increased DOT1L expression and H3K79 dimethylation in renal tubular epithelial cells and myofibroblasts in a murine model of unilateral ureteral obstruction. Administration of EPZ5676, a highly selective inhibitor of DOT1L, attenuated renal fibrosis. Treatment with EPZ5676 or DOT1L small interfering RNA also inhibited TGF-β1 and serum-induced activation of renal interstitial fibroblasts and epithelial-mesenchymal transition (EMT) in vitro. Moreover, blocking DOT1L abrogated injury-induced epithelial G₂/M arrest; reduced expression of Snail, Twist, and Notch1; and inactivated several profibrotic signaling molecules in the injured kidney, including Smad3, epidermal growth factor receptor, platelet-derived growth factor receptor, signal transducer and activator of transcription 3, protein kinase B, and NF-κB. Conversely, DOT1L inhibition increased expression of phosphatase and tensin homolog, a protein associated with dephosphorylation of tyrosine kinase receptors, and prevented decline in levels of Klotho and Smad7, 2 renoprotective factors. Thus, our data indicate that targeting DOT1L attenuates renal fibrosis through inhibition of renal fibroblasts and EMT by suppressing activation of multiple profibrotic signaling pathways while retaining expression of renoprotective factors.-Liu, L., Zou, J., Guan, Y., Zhang, Y., Zhang, W., Zhou, X., Xiong, C., Tolbert, E., Zhao, T. C., Bayliss, G., Zhuang, S. Blocking the histone lysine 79 methyltransferase DOT1L alleviates renal fibrosis through inhibition of renal fibroblast activation and epithelial-mesenchymal transition.

Keywords: ERSD; Notch1; Smad3; Snail; transforming growth factor.

Figure 1

Expression of DOT1L and H3K79me2 and the effect of EPZ5676 in obstructed kidneys. A) Photomicrographs illustrate costaining of α‐SMA and DOT1L or H3K79me2 in the tissue section of the obstructed kidney (original magnification, ×600). B) The prepared tissue lysates from sham‐operated or obstructed kidneys of mice administered with or without EPZ5676 were subjected to immunoblot analysis with antibodies against H3K79me2, DOT1L, or GAPDH. C, D) The levels of H3K79me2, DOT1L, and GAPDH were quantified by densitometry, and DOT1L (C) and H3K79me2 (D) levels were normalized to GAPDH. Values are means ± sd (n = 6). Bars with different letters (a‐b) are significantly different from one another (P < 0.05).

Figure 2

Administration of EPZ5676 attenuates development of renal fibrosis and deposition of ECM in obstructed kidneys. A) Photomicrographs illustrating Masson trichrome staining (blue) of kidney tissue (original magnification, ×200). B) The Masson trichrome‐positive tubulointerstitial area was analyzed relative to the whole area from 10 random cortical fields. Data are represented as means ± sd (n = 6). C) Kidney tissue lysates were subjected to immunoblot analysis with antibodies against α‐SMA, collagen I, fibronectin, or GAPDH. D‐F) Expression levels of fibronectin, collagen I, α‐SMA, or GAPDH were quantified by densitometry, and the levels of fibronectin (D), collagen I (E), and α‐SMA (F) were normalized with GAPDH. Values are means ± sd (n =6). Bars with different letters (a‐c) are significantly different from one another (P < 0.05).

Figure 3

Treatment with EPZ5676 inhibits TGF‐β1‐induced renal fibroblast activation. Serum‐starved NRK‐49F cells were pretreated with 50 μM EPZ5676 for 1 h and then exposed to TGF‐β1 (2 ng/ml) for an additional 24 h. Cell lysates were then prepared and subjected to immunoblot analysis with antibodies against fibronectin, collagen I, α‐SMA, or α‐tubulin (A) or DOT1L, H3K79me2, or α‐tubulin (E). The levels of fibronectin (B), type 1 collagen (C), α‐SMA (D), DOT1L (F), or H3K79me2 (G) were quantified by densitometry and normalized with α‐tubulin. Values are the mean ± sd of at least 3 independent experiments. Bars with different letters (a‐d) for each molecule are significantly different from one another (P < 0.05).

Figure 4

Knockdown of DOT1L with siRNA inhibits renal fibroblast activation. Serum‐starved NRK‐49F cells were transfected with siRNA targeting DOT1L or scrambled siRNA [control (Con)] and then incubated in TGF‐b1 (2 ng/ml) for an additional 24 h. Cell lysates were prepared for immunoblot analysis with antibodies against fibronectin, collagen I, α‐SMA, or α‐tubulin (A) or DOT1L, H3K79me2, or α‐tubulin (E). Expression levels of fibronectin (B), collagen I (C), α‐SMA (D), DOT1L (F), or H3K79me2 (G) were quantified by densitometry and normalized with α‐tubulin. Values are the mean ± sd of at least 3 independent experiments. Bars with different letters (a‐d) for each molecule are significantly different from one another (P < 0.05).

Figure 5

EPZ5676 inhibits renal epithelial cells arrested in the G₂/M phase of the cell cycle in obstructed kidneys. A) Photomicrographs illustrate staining of pH3Ser10 in the tissue section of the kidney after treatments as indicated (original magnification, ×600). B) The tubular cells with positive staining of pH3Ser10 were calculated in 10 high‐power fields and expressed as means ± sd. C) Kidney tissue lysates were subjected to immunoblot analysis with antibodies against pH3Ser10, vimentin, Snail, Twist, or GAPDH. D‐G) Expression levels of pH3Ser10 (D), vimentin (E), Snail (F), and Twist (G) were quantified by densitometry and normalized with GAPDH. Values are means ± sd (n =6). Bars with different letters (a‐c) are significantly different from one another (P < 0.05).

Figure 6

Treatment with EPZ5676 inhibits TGF‐β1‐induced renal EMT. A) Serum‐starved RPTCs were pretreated with 50 μM EPZ5676 for 1 h and then exposed to TGF‐β1 (2 ng/ml) for an additional 24 h. Cell lysates were then prepared and subjected to immunoblot analysis with antibodies against fibronectin, collagen I, α‐SMA, α‐tubulin, DOT1L, H3K79me2, or α‐tubulin. B‐F) The levels of fibronectin (B), collagen I (C), α‐SMA (D), E‐cadherin (E), or H3K79me2 (F) were quantified by densitometry and normalized with α‐tubulin. Values are the mean ± sd of at least 3 independent experiments. Bars with different letters (a‐d) for each molecule are significantly different from one another (P < 0.05).

Figure 7

EPZ5676 treatment inhibits activation of the Smad signaling in obstructed kidneys and cultured renal interstitial fibroblasts. Kidney tissue lysates were prepared and subjected to immunoblot analysis with antibodies to phospho‐Smad3 (p‐Smad3), Smad3, Smad7, or GAPDH (A). Serum‐starved NRK‐49F cells were treated with 50 μM EPZ5676 for 1 h followed by exposure of cells to TGF‐β1 (2 ng/ml) for an additional 24 h. Cell lysates were subjected to immunoblot analysis with antibodies against phospho‐Smad3, Smad3, Smad7, or α‐tubulin (E). Expression levels of all of those proteins were quantified by densitometry. Phospho‐Smad3 was normalized to its total protein level (D, F); Smad3 (C, G) and Smad7 (D, H) levels were normalized to GAPDH. The values are means ± sd (n = 6). Bars with different letters (a‐c) are significantly different from one another (P < 0.05).

Figure 8

Blockade of DOT1L inhibits phosphorylation of EGFR, PDGFR‐β, and AKT but retains expression of PTEN in obstructed kidneys. A) Kidney tissue lysates were prepared and subjected to immunoblot analysis with antibodies against phospho‐EGFR (p‐EGFR) (Tyr1068), phospho‐PDGFR‐β (p‐PDGFRβ) (Tyr751), EGFR, PDGFR‐β, phospho‐AKT (p‐AKT) (Ser473), AKT, or GAPDH. All of those proteins were quantified by densitometry, and phospho‐EGFR (B), phospho‐PDGFR‐β (D), and phospho‐AKT (F) were normalized to their total protein levels; EGFR (C), PDGFR‐β (E), and PTEN (G) were normalized to GAPDH. Values are means ± sd (n = 6). Bars with different letters (a‐c) are significantly different from one another (P < 0.05).

Figure 9

Blockade of DOT1L inhibits Notch1 but increases Klotho protein expression in obstructed kidneys. A) Kidney tissue lysates were prepared and subjected to immunoblot analysis with antibodies against Notch1, Klotho, or GAPDH. B, C) All of those proteins were quantified by densitometry, and the levels of Notch1 (B) and Klotho (C) were normalized to GAPDH. Values are means ± sd (n = 6). Bars with different letters (a‐c) are significantly different from one another (P < 0.05).

Figure 10

Blockade of DOT1L inhibits phosphorylation of STAT3 and NF‐κB as well as in obstructed kidneys. A) Kidney tissue lysates were prepared and subjected to immunoblot analysis with antibodies against phospho‐STAT3 (p‐STAT3) (Tyr705), phospho‐NF‐κB (p‐NF‐κB) (p65), or GAPDH. All of those proteins were quantified by densitometry, and phospho‐STAT3 (B) and phospho‐NF‐κB(D) were normalized to their total protein levels; STAT3 (C) and NF‐κB(E) were normalized to GAPDH. Values are means ± sd (n =6). Bars with different letters (a, b) are significantly different from one another (P < 0.05). F) Photomicrographs (original magnification, ×400) illustrating infiltration of leukocytes in kidney sections with naphthol‐AS‐d‐chloroacetate esterase staining on d 7 after various treatments as indicated (arrows). G) Positively stained cells were counted in 10‐15 fields, and mean numbers per field are shown.

Figure 11

Proposed mechanisms of DOT1L inhibition mediated antifibrotic effects in the kidney. Injury to the kidney results in increased expression of DOT1l and H3K79me2 in renal cells, which leads to activation of TGF‐β‐Smad3, EGFR, PGFR, NF‐κB, and STAT3 signaling and down‐regulation of Klotho and bone morphogenetic protein 7. As a result, EMT, G2/M arrest, fibroblast activation, and inflammation occur and contribute to renal fibrosis. Inhibition of DOT1L with EPZ5676 reverses those profibrotic responses and attenuates renal fibrosis.