BASIC RESEARCH www.jasn.org MicroRNA-489 Induction by Hypoxia–Inducible Factor–1 Protects against Ischemic Kidney Injury † † ‡ † ‡ Qingqing Wei,* Yong Liu, Pengyuan Liu, Jielu Hao,* Mingyu Liang, Qing-sheng Mi, § | Jian-Kang Chen,* and Zheng Dong* *Department of Cellular Biology and Anatomy, Medical College of Georgia at Augusta University and Charlie Norwood Veterans Affairs Medical Center, Augusta, Georgia; †Department of Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin; ‡Kidney Institute, Changzheng Hospital of Second Military Medical University, Shanghai, China; §Departments of Dermatology and Internal Medicine, Henry Ford Health System, Detroit, Michigan; and |The Second Xiangya Hospital, Central South University, Changsha, China ABSTRACT MicroRNAs have been implicated in ischemic AKI. However, the specific microRNA species that regulates ischemic kidney injury remains unidentified. Our previous microarray analysis revealed microRNA-489 induction in kidneys of mice subjected to renal ischemia-reperfusion. In this study, we verified the induction of microRNA-489 during ischemic AKI in mice and further examined the underlying mechanisms. Hypoxia– inducible factor–1a deficiency associated with diminished microRNA-489 induction in cultured rat proximal tubular cells subjected to hypoxia and kidney tissues of mice after renal ischemia-reperfusion injury. Moreover, genomic analysis revealed that microRNA-489 is intronic in the calcitonin receptor gene, and chromatin immunoprecipitation assays showed increased binding of hypoxia–inducible factor–1 to a specificsitein the calcitonin receptor gene promoter after hypoxia. Inhibition of microRNA-489 increased apoptosis in renal tubular cells after ATP depletion injury in vitro, whereas microRNA-489 mimics mediated protection. In mice, inhibition of microRNA-489 enhanced tubular cell death and ischemic AKI without significantly affecting tubular cell proliferation. Deep sequencing identified 417 mRNAs that were recruited to the RNA–induced silencing complex by microRNA-489. Of the identified mRNAs, 127 contain microRNA-489 targeting sites, and of those, 18 are involved in the cellular stress response, including the poly(ADP-ribose) polymerase 1 gene implicated in ischemic kidney injury. Sequence analysis and in vitro studies validated poly(ADP-ribose) poly- merase 1 as a microRNA-489 target. Together, these results suggest that microRNA-489 is induced via hypoxia–inducible factor–1 during ischemic AKI to protect kidneys by targeting relevant genes. J Am Soc Nephrol 27: 2784–2796, 2016. doi: 10.1681/ASN.2015080870 Renal ischemia-reperfusion is a main cause of AKI, important role played by miRs in various cellular whichisassociatedwithhighmortality.1 Recent processes ranging from cell growth and prolifera- work further indicates that AKI may be an impor- tion to cell death. Notably, dysregulation of miRs tant contributing factor in the development and progression of CKD.2,3 Decades of research have Received August 7, 2015. Accepted January 25, 2016. gained significant insights into the pathogenesis of ischemic AKI, including tubular cell injury and Published online ahead of print. Publication date available at death, vascular damage, and inflammatory re- www.jasn.org. sponse.4–7 However, effective approaches for the Correspondence: Dr. Zheng Dong, The Second Xiangya Hospital, diagnosis and treatment of AKI are still lacking.8 Central South University, 139 Middle Renmin Road, Changsha, Hunan 410011, China; or Dr. Qingqing Wei, Department of Cel- MicroRNAs (miRs) are a group of noncoding lular Biology and Anatomy, Medical College of Georgia at Augusta small RNAs of 21–25 nucleotides that regulates tar- University, 1459 Laney Walker Boulevard, Augusta, GA 30912. get gene expression mainly by blocking their Email: [email protected] or [email protected] mRNA translation.9,10 Recent work has shown an Copyright © 2016 by the American Society of Nephrology 2784 ISSN : 1046-6673/2709-2784 JAmSocNephrol27: 2784–2796, 2016 www.jasn.org BASIC RESEARCH has emerged as a critical pathogenic factor in a variety of hu- this induction, we measured miR-489 by quantitative real– man diseases.9,10 In kidneys, miRs have been shown to play time PCR (Figure 1). Compared with sham-operated con- indispensable roles in kidney development.11 For example, trol, miR-489 increased 1.4460.17-fold (n=3; P,0.05) and deletion of Dicer (a key enzyme for the production of func- 1.6960.19-fold (n=6; P,0.01) in kidney cortex and outer tional miRs) from podocytes and juxtaglomerular cells led to medulla after 30 minutes of ischemia and 12 or 48 hours of the loss of homeostasis and function of these cells, resulting in reperfusion, respectively (Figure 1A). Interestingly, kidney defects.12–16 Furthermore, specificmiRs,suchas miR-489 induction was also detected during hypoxic incu- miR-192 and miR-29, were shown to be important regulators bation of cultured rat proximal tubular cells (RPTCs). As 17–20 21 in diabetic nephropathy. In 2010, we and Goldwin shown in Figure 1B, hypoxia (1% O2) induced miR-489 et al.22 revealed significant changes of miR expression profiles time dependently, reaching 1.8460.65-fold (n=5; P,0.05) during ischemic AKI in mice. Furthermore, we showed that at 6 hours and then decreasing toward the basal level (Figure ischemic AKI was markedly attenuated by the deletion of 1B). The in vitro datasuggestthatmiR-489islikelytobe Dicer and consequent depletion of miRs in proximal tubular induced in renal tubules by hypoxia in ischemic AKI. To cells.21 Although these studies showed a role of miRs in the localize miR-489 induction in kidneys after ischemia- pathogenesis of ischemic AKI, the specificpathogenicmiR reperfusion injury, we conducted in situ hybridization species remained unknown. There are .1000 miR species in (Figure 1C). Low levels of miR-489 were detected in most humans. In the last couple of years, several miRs have been renal tubules in control kidneys. After 30 minutes of ische- implicated in the regulation of ischemic AKI.23–26 For exam- mia and 48 hours of reperfusion, there was an overall in- ple, miR-24 was shown to promote renal tubular cell injury crease of miR-489 in renal tubules, and the increase was by targeting H2A histone family, member X, and heme more in injured tubules mainly in the outer stripe of outer oxygenase 1,23 whereas miR-21 was upregulated by ischemic medulla (arrows in Figure 1C). Interestingly, we also de- preconditioning to protect kidneys against subsequent injury tected some strong interstitial signals that appeared in by inhibiting programmed cell death protein 4.25 blue, but the interstitial staining did not change significantly miR-489 is an miR that has been reported to act as a tumor after ischemic injury. It was suggested that miR-489 was suppressor and play roles in muscle stem cell quiescence and mainly induced in renal tubular cells. cardiac hypertrophy. It is also upregulated in clear cell papillary renal cell carci- noma.27–30 In ischemic AKI, our miR profiling suggested miR-489 induction in kidney tissues after renal ischemia- reperfusion.21 However, the mechanism of miR-489 induction in these disease con- ditions was unclear, and the role of miR-489 in ischemic AKI was unknown. In this study, we show that miR-489 is in- duced via hypoxia–inducible factor–1 (HIF-1) during hypoxia of tubular cells in vitro and ischemic AKI in mice. We further show a renoprotective role of miR-489 under these conditions. Deep RNA sequencing (RNA-seq) has further identi- fied multiple target genes of miR-489 that may be responsible for its protective effect. RESULTS Figure 1. miR-489 is induced in ischemic AKI and hypoxic renal tubular cells. (A) miR-489 Is Induced in Ischemic AKI C57BL/6 mice were subjected to 30 minutes of bilateral renal ischemia with 12 or 48 hours of reperfusion or sham operation. miR-489 in the kidney cortex and outer and Hypoxic Renal Tubular Cells fi medulla was detected by real-time PCR. (B) RPTCs were treated with 1% oxygen, and Our microarray analysis identi ed miR-489 miR-489 level was examined by quantitative real–time PCR (n=3–6). *P,0.05 compared fi as one of the miRs that was signi cantly with sham operation or no hypoxia controls; **P,0.01 compared with sham. (C) C57BL/6 induced in kidney tissues after 30 minutes mice were subjected to 30 minutes of bilateral ischemia with 48 hours of reperfusion of renal ischemia and 12–48 hours of or sham operation. miR-489 in the kidney was detected by in situ hybridization. The reperfusion in mice.21 To fur ther confirm arrows show the significant induction of miR-489 in injured tubules. Scale bar, 0.2 mm. J Am Soc Nephrol 27: 2784–2796, 2016 MicroRNA-489 in Ischemic Kidney Injury 2785 BASIC RESEARCH www.jasn.org HIF-1 Contributes to miR-489 Induction in Ischemic AKI with previous work,32–34 HIF-1a was also induced in kidney tis- and Hypoxic Incubation sues during renal ischemia-reperfusion in our study (Figure 2B). Because we observed miR-489 induction by hypoxia in RPTCs, To determine the role of HIF-1 in miR-489 induction we hypothesized that miR-489 induction during renal cell during renal hypoxia and ischemia-reperfusion, we initially hypoxia and ischemic AKI may involve HIF-1, the master established stable human embryonic kidney (HEK293) cell regulator of gene expression in hypoxia.31 HIF-1 consists of lines with HIF-1a knockdown by two different shRNAs a-andb-subunits. Although HIF-1b is constitutively (shRNA-A and shRNA-B). Compared with scrambled expressed, HIF-1a is induced by hypoxia and forms a sequence–transfected cells, HIF-1 inhibitory shRNA– heterodimer with HIF-1b to translocate to the nucleus to in- transfected cells showed a lower HIF-1a induction during duce hypoxic gene expression. Accordingly, HIF-1a induction hypoxic incubation (Figure 2C). Importantly, 6 hours of hyp- is a good indication of HIF-1 activation.