ELABELA and an ELABELA Fragment Protect against AKI

Abstract

Renal ischemia-reperfusion (I/R) injury is the most common cause of AKI, which associates with high mortality and has no effective therapy. ELABELA (ELA) is a newly identified 32-residue hormone peptide highly expressed in adult kidney. To investigate whether ELA has protective effects on renal I/R injury, we administered the mature peptide (ELA32) or the 11-residue furin-cleaved fragment (ELA11) to hypoxia-reperfusion (H/R)-injured or adriamycin-treated renal tubular cells in vitro ELA32 and ELA11 significantly inhibited the elevation of the DNA damage response, apoptosis, and inflammation in H/R-injured renal tubular cells and suppressed adriamycin-induced DNA damage response. Similarly, overexpression of ELA32 or ELA11 significantly inhibited H/R-induced cell death, DNA damage response, and inflammation. Notably, treatment of mice with ELA32 or ELA11 but not an ELA11 mutant with a cysteine to alanine substitution at the N terminus (AE11C) inhibited I/R injury-induced renal fibrosis, inflammation, apoptosis, and the DNA damage response and markedly reduced the renal tubular lesions and renal dysfunction. Together, our results suggest that ELA32 and ELA11 may be therapeutic candidates for treating AKI.

Keywords: DNA damage response; ELABELA; acute kidney injury; inflammation.

Copyright © 2017 by the American Society of Nephrology.

Figures

Figure 1.

ELA level is downregulated after renal I/R injury in vivo and in vitro. (A) Sequences of ELA peptides used in this study. (B) RT-PCR results of Apela, Apln, Aplnr, and Rn18s in different tissues of fetal and adult mice. (C) qPCR results of Apela in the kidneys of different experimental groups (n=3–4 per group). CT, noninjured mice. (D) Representative images of ELA (red), TGN38 (green), and 4,6-diamidino-2-phenylindole (DAPI; blue) staining in NRK-52E cells. NC, noninjured cells. Scale bar, 10 μm. *P<0.05.

Figure 2.

ELA32 and ELA11 treatments suppress H/R injury–induced inflammation, DNA damage, and apoptosis in cultured renal tubular cells. (A) Experimental design chart of H/R. S, serum; G, Glucose. (B) qPCR results of Kim1, Il6, Icam1, Tnfa, and Tgfb1 in different experimental groups. (C) Representative Western blots (left panel) with densitometric quantitative results (right panel) of p-ATR, p-Chk1, p-H2A.X, and β-actin in different experimental groups. (D) Representative images of p-H2A.X in different groups. Scale bar, 10 μm. (E) Representative images of TUNEL assay and (F) quantitative results of TUNEL-positive cells in different experimental groups. Scale bar, 100 μm. (G) Representative Western blots (left panel) with densitometric quantitative results (right panel) of caspase3, c-Cas3, and β-actin in different experimental groups. Each experiment was performed in duplicate or triplicate and repeated at least three times. A representative result is shown. DAPI, 4,6-diamidino-2-phenylindole; E11, 300 pM ELA11-treated H/R-injured cells; E32, 300 pM ELA32-treated H/R-injured cells; NC, noninjured cells; PC, positive cells. *P<0.05; **P<0.01.

Figure 3.

Overexpression of E32-GFP and E11-GFP inhibits H/R injury–induced DNA damage, apoptosis, and inflammation in cultured renal tubular cells. (A) qPCR results of Tnfa, Icam1, Vcam1, and Il6 in different experimental groups. (B) Relative cell viability measured by MTT assay. (C) Representative Western blots (left panel) with densitometric quantitative results (right panel) of p-ATR, p-Chk1, p-H2A.X, and β-actin in different experimental groups. (D) Representative Western blots (left panel) with densitometric quantitative results (right panel) of caspase3, c-Cas3, PARP-1, c-PARP-1, and β-actin in different experimental groups. Each experiment was performed in duplicate or triplicate and repeated at least three times. A representative result is shown. E11-GFP, cells transfected with the E11-GFP plasmid and cultured under H/R condition; E32-GFP, cells transfected with the E32-GFP plasmid and cultured under H/R condition; H/R+GFP, cells transfected with the pRK-GFP plasmid and cultured under H/R condition; NC, cells transfected with the pRK-GFP plasmid and cultured under normal condition. *P<0.05; **P<0.01.

Figure 4.

ELA32 and ELA11 treatments inhibit ADR-induced DNA damage in cultured renal tubular cells. (A) Representative Western blots with (B) densitometric quantitative results of p-ATR, p-Chk1, p-H2A.X, and β-actin in different experimental groups. (C) Representative images of p-H2A.X staining in different groups. Scale bar, 10 μm. (D) Relative cell viability measured by MTT assay. (E) Representative Western blots with (F) densitometric quantitative results of caspase3, c-Cas3, PARP-1, c-PARP-1, and β-actin in different experimental groups. Each experiment was performed in duplicate or triplicate and repeated at least three times. A representative result is shown. ADR, 0.5 μM ADR-treated NRK-52E cells; DAPI, 4,6-diamidino-2-phenylindole; E11, 300 pM ELA11-treated ADR-injured cells; E32, 300 pM ELA11-treated ADR-injured cells; NC, noninjured cells; PBS, PBS-treated NRK-52E cells. *P<0.05; **P<0.01.

Figure 5.

ELA may not bind to APJ in renal I/R injury. cAMP assay of ELA32 and ELA11 in (A) normoxia and (B) H/R-injured NRK-52E cells. (C) Internalization of APJ by ELA32 and ELA11 in HEK293 cells. Scale bar, 10 μm. (D) Cell viability of different groups. (E) qPCR results of Mcp1 and Il6 in different experimental groups. CT, noninjured mice; NC, noninjured cells. *P<0.05.

Figure 6.

ELA treatments inhibit I/R injury–induced morphologic changes, inflammation, and fibrosis. (A) Experimental design. (B) Representative images of hematoxylin and eosin (HE) and (C) quantitative results of renal damage score in different experimental groups. (D) Quantitative results of F4/80 staining and (E) representative images of F4/80 in different experimental groups. (F) qPCR results of Mcp-1, Il6, and Il8 in different experimental groups. (G) Representative images of Masson, α-smooth muscle actin (α-SMA), and vimentin staining in different experimental groups. (H) qPCR results of Vimentin, Tgfb1, Fibronetin, and Collagen1a in different experimental groups. AE11C, AE11C-treated I/R-injured mice (n=3–7 per group); CT, noninjured mice; E11, ELA11-treated I/R-injured mice (n=3–7 per group); E32, ELA32-treated I/R-injured mice (n=3–7 per group). Scale bar, 100 μm. *P<0.05; **P<0.01.

Figure 7.

ELA treatments suppress I/R injury–induced DDR and apoptosis in the kidneys. (A) Representative Western blots with (B) densitometric quantitative results of p-ATR, p-Chk1, p-H2A.X, and β-actin in different experimental groups. (C) Representative images of p-H2A.X (brown) in different groups. (D) Representative images of TUNEL assay and (E) quantitative results of TUNEL-positive cells in different experimental groups. (F) Representative Western blots (left panel) with densitometric quantitative results (right panel) of caspase3, c-Cas3, and β-actin in different experimental groups. CT, noninjured mice; DAPI, 4,6-diamidino-2-phenylindole; E11, ELA11-treated I/R-injured mice (n=3–7 per group); E32, ELA32-treated I/R-injured mice (n=3–7 per group); PC, positive cells. Scale bar, 50 μm. *P<0.05; **P<0.01.

Figure 8.

ELA11 treatment suppresses I/R injury–induced autophagy in the kidneys. (A) Representative Western blots with (B) densitometric quantitative results of Atg5, Beclin1, Atg7, Atg12, LC3B-II, and β-actin in different experimental groups. CT, noninjured mice; E11, ELA11-treated I/R-injured mice (n=3–7 per group); E32, ELA32-treated I/R-injured mice (n=3–7 per group). (C) Representative Western blots with densitometric quantitative results of Atg5, Beclin 1, LC3B-II, and HSP70 in different experimental groups. E11, 300 pM ELA11-treated H/R-injured cells; NC, noninjured cells. (D) Relative cell viability measured by MTT assay in NRK-52E cells treated with rapamycin (Rapa). (E) Representative images of LC3B-II in different groups. DAPI, 4,6-diamidino-2-phenylindole. Scale bar, 10 μm. (F) Representative Western blots with densitometric quantitative results of LC3B-II and β-actin in different experimental groups. Each experiment was performed in duplicate or triplicate and repeated at least three times. A representative result is shown. DMSO, DMSO-treated NRK-52E cells; Rapa+E11, 300 pM ELA11-treated rapamycin-injured cells. *P<0.05.