MicroRNA-668 represses MTP18 to preserve mitochondrial dynamics in ischemic acute kidney injury
Qingqing Wei, … , Changlin Mei, Zheng Dong
Qingqing Wei, … , Changlin Mei, Zheng Dong
Published October 16, 2018
Citation Information: J Clin Invest. 2018;128(12):5448-5464. https://doi.org/10.1172/JCI121859.
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Research Article Nephrology

MicroRNA-668 represses MTP18 to preserve mitochondrial dynamics in ischemic acute kidney injury

Abstract

The pathogenesis of ischemic diseases remains unclear. Here we demonstrate the induction of microRNA-668 (miR-668) in ischemic acute kidney injury (AKI) in human patients, mice, and renal tubular cells. The induction was HIF-1 dependent, as HIF-1 deficiency in cells and kidney proximal tubules attenuated miR-668 expression. We further identified a functional HIF-1 binding site in the miR-668 gene promoter. Anti–miR-668 increased apoptosis in renal tubular cells and enhanced ischemic AKI in mice, whereas miR-668 mimic was protective. Mechanistically, anti–miR-668 induced mitochondrial fragmentation, whereas miR-668 blocked mitochondrial fragmentation during hypoxia. We analyzed miR-668 target genes through immunoprecipitation of microRNA-induced silencing complexes followed by RNA deep sequencing and identified 124 protein-coding genes as likely targets of miR-668. Among these genes, only mitochondrial protein 18 kDa (MTP18) has been implicated in mitochondrial dynamics. In renal cells and mouse kidneys, miR-668 mimic suppressed MTP18, whereas anti–miR-668 increased MTP18 expression. Luciferase microRNA target reporter assay further verified MTP18 as a direct target of miR-668. In renal tubular cells, knockdown of MTP18 suppressed mitochondrial fragmentation and apoptosis. Together, the results suggest that miR-668 is induced via HIF-1 in ischemic AKI and that, upon induction, miR-668 represses MTP18 to preserve mitochondrial dynamics for renal tubular cell survival and kidney protection.

Authors

Qingqing Wei, Haipeng Sun, Shuwei Song, Yong Liu, Pengyuan Liu, Man Jiang Livingston, Jianwen Wang, Mingyu Liang, Qing-Sheng Mi, Yuqing Huo, Norris Stanley Nahman, Changlin Mei, Zheng Dong

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Figure 4

Inhibition of miR-668 exacerbates ischemic AKI.

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Inhibition of miR-668 exacerbates ischemic AKI. (A) Schematic diagram de...
(A) Schematic diagram depicting the animal treatment procedure. Anti–miR-668 or scrambled-sequence LNAs were delivered to C57BL/6J male mice through tail vein injection 2 days before ischemic surgery. The mice were subjected to 28 minutes of bilateral renal ischemia followed by 48 hours of reperfusion (I28/48h), or sham operation. (B) qPCR analysis of miR-668 in kidneys verifying the inhibitory effect of anti–miR-668 (n = 3 for sham groups and n = 4 for I28/48h groups; **P < 0.0001, ##P < 0.0001, 2-way ANOVA with Fisher’s LSD). (C) BUN measurement showing ischemic AKI exacerbated by anti–miR-668 (n = 3 for sham groups, n = 6 for I28/48h groups; *P = 0.0318, 2-tailed Student’s t test). (D) Serum creatinine measurement showing ischemic AKI exacerbated by anti–miR-668 (n = 3 for sham groups, n = 6 for I28/48h groups; **P = 0.007, 2-tailed Student’s t test). (E) Representative images of renal histology by H&E staining. Bottom panels are enlarged images of the boxed areas in the top panels. Scale bars: 0.2 mm. (F) Representative images of TUNEL staining. The arrows indicate TUNEL-positive nuclei. Scale bar: 0.2 mm. (G) Counting of TUNEL-positive cells to show that anti–miR-668 increased apoptosis in ischemic AKI (n = 4; *P = 0.0386, 2-tailed Student’s t test). (H) Immunoblots showing the induction of active caspase-3 by anti–miR-668 in ischemic AKI. S, scramble; A, anti–miR-668. Cyclophilin B was used as loading control.