MicroRNA-187 Reduces Acute Ischemic Renal Podocyte Injury via Targeting Acetylcholinesterase

MicroRNA-187 Reduces Acute Ischemic Renal Podocyte Injury via Targeting Acetylcholinesterase

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MicroRNA-187 Reduces Acute Ischemic Renal Podocyte Injury via Targeting Acetylcholinesterase Jianing Yue, MD, PhD,1 Yi Si, MD,1 Ting Zhu, MD, Jue Yang, MD, Xin Xu, MD, Yuan Fang, MD, PhD, and Weiguo Fu, MD* Department of Vascular Surgery, Zhongshan Hospital, Fudan University, Shanghai, China

article info

abstract

Article history:

Background: Podocyte injury was reported to be involved in the major pathogenesis of

Received 14 December 2018

ischemia/reperfusion (I/R)-induced ischemic acute renal failure. Our purpose was to study

Received in revised form

the mechanism of miR-187 improving I/R-induced podocytes injury.

22 April 2019

Materials and methods: The miR-187 mimics and inhibitor were transfected into the immor-

Accepted 4 June 2019

talized mouse podocyte (MPC-5) cells, and then transfected cells were subjected to hypoxia/

Available online xxx

reoxygenation (H/R, 3/3 h) to establish an H/R cell model. To investigate the effects of miR-187 on H/R-induced cell injury, cell viability and apoptosis were measured by Cell Counting Kit-8

Keywords:

(CCK-8) assay and flow cytometry. Dual-luciferase report system was used to verify whether

Podocyte

miR-187 could directly target acetylcholinesterase (ACHE). The animal ischemia/reperfusion

Hypoxia/reoxygenation injury

model was established and injected with miR-187 agomir. Kidney tissue sections were sub-

Hematoxylin and eosin staining

jected to histological examination by hematoxylin and eosin staining to assess the renal injury.

Creatinine

Real-time quantitative PCR and western blot were performed to determine gene expressions. Results: The transfection of miR-187 mimics contributed to MPC-cells resistance to H/Rinduced cell injury, which was reflected by enhanced cell viability and reduced apoptosis (from 20.05% to 9.43%) in H/R þ negative control group. ACHE was confirmed as a target of miR-187, and ACHE siRNA had a similar efficiency to miR-187 mimic. The injection of miR187 agomir not only effectively protected the kidney from I/R-induced injury, but also reduced the concentrations of serum creatinine. Moreover, nephrin was noticeably increased and desmin was decreased under the effects of agomir. Conclusions: Our findings indicated that miR-187 improved I/R-induced ischemic acute renal failure through protecting glomerular filtration barrier by blocking the expression of ACHE. ª 2019 Elsevier Inc. All rights reserved.

Introduction Renal transplantation is a preferred treatment method for patients with end-stage renal disease.1 However, renal grafts inevitably experience ischemia when they are separated from

blood supply of a donor, and then the reperfusion will induce the generation of reactive oxygen species and activate a series of deleterious cellular responses, ultimately resulting in renal reperfusion injury.2,3 Previous study has proved that renal ischemia-reperfusion injury (IRI) was involved in the

* Corresponding author. Department of Vascular Surgery, Zhongshan Hospital, Fudan University, No.180 Fenglin Road, Xuhui District, Shanghai, 200032, China. Tel.: þ21 64041990; fax: þ21 64038308. E-mail address: [email protected] (W. Fu). 1 These authors contributed equally to this work. 0022-4804/$ e see front matter ª 2019 Elsevier Inc. All rights reserved. https://doi.org/10.1016/j.jss.2019.06.005

yue et al  mir-187 mitigated podocyte death in iri

induction of a series of severe clinical problems including ischemic acute renal failure (iARF).4,5 Despite advances in immunosuppressive and supportive therapy, iARF is still significantly correlated with high morbidity and mortality, noticeably, in-hospital mortality rate could be higher than 50% among patients who were admitted to the intensive care unit.6,7 Unfortunately, although those patients survived from acute illness, they still are at a high risk of exacerbation of chronic kidney disease that may accelerate the progress of end-stage renal disease.8,9 Currently, most researches were devoted to studying the effects of ischemia/reperfusion (I/R) on the renal tubular epithelial cell injury. Metformin preconditioning could protect renal function from I/R-induced injury by suppressing the production of inflammatory cytokines and inhibiting the apoptosis of renal tubular epithelial cells.10 In recent years, some studies indicated that the major pathogenesis of renal IRI was attributed to the decreased glomerular filtration.11,12 Podocyte is one of the key components of the glomerular filtration barrier, and the dysfunction of podocytes was proved to contribute to more than one proteinuric kidney disease.13 In 2016, Yamashita et al. showed that podocytes occupied a vital place in the pathogenesis of renal IRI. Therefore, podocytes could be a possible target for the improvement of kidney IRI. MicroRNAs (miRNAs) are a type of 20-24 nucleotideelength, small, noncoding RNAs.14 Evidence demonstrated that miRNAs participated in the development of various diseases, for example, Alzheimer’s disease,15 coronary artery disease,16 and various cancers,17-19 by regulating target genes. Recently, it was reported that miR-223 had the ability to improve kidney injury by enhancing the tissueprotective activity of mesenchymal stem cells.20 Meanwhile, several differentially expressed miRNAs were identified by contrasting the expression profiles of miRNAs including miR214, miR-192, miR-187, miR-20a, miR-21, miR-194, miR-146a, miR-199a-3p, and miR-805 before and after renal IRI.21 Among them, the overexpressed miR-21 could prevent renal IRI through protecting the epithelial cell and inhibiting the production of inflammatory cytokines.22 MiR-187 has been reported as an important factor in tumorgenesis, for example, miR-187 is differentially expressed in lung tumors.23 MiR-187 is an independent prognostic factor in breast cancer and leads to an increase in invasive potential in vitro.24 A systematic review and metaanalysis found that miR-187 might be a prognostic molecular signature in renal cell carcinomas.25 MiRNA-187 was found to be downregulated in clear cell renal cell carcinoma and was associated with a lower survival rate and could inhibit cell growth and migration.26 Godwin et al. have shown that miR187 was differentially expressed in IRI mice.21 Hence, we hypothesized that miR-187 functioned in the renal IRI and studied the underlying mechanism.

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(Beijing, China). MPC-5 cells were preliminarily cultured in Roswell Park Memorial Institute-1640 culture medium (RPMI1640; SigmaeAldrich, Merck KGaA, Darmstadt, Germany), which was supplemented with 1% penicillin/streptomycin (100 U/ml), 1% pyruvate and interferon-g (IFN-g, 10 U/mL) and 10% fetal bovine serum in 5% CO2 at 33 C to propagate. Then, the cells differentiated under the medium without IFN-g at 37 C for 10e14 d. The medium was replaced every 5 d. MPC-5 cells were harvested for experiments in this study.

Hypoxia/reoxygenation (H/R) model of MPC-5 cells The MPC-5 cells were seeded into 6-well plates (1  105 cells/ well) containing phosphate-buffered saline (PBS). For hypoxia construction,27 the plates were incubated in a small airtight chamber with 5% CO2 and 95% N2 at 37 C. After 1, 2, 3, and 4 h of incubation, PBS medium was discarded and MPC-5 cells were resuspended by complete culture medium and maintained in an incubator with 5% CO2 and 95% air at 37 C for 3 h to complete the process of H/R-induced cell injury.

Cell transfection The miR-187 mimics, miR-187 inhibitor, negative control (NC), and acetylcholinesterase siRNA (siACHE, sense: 50 -AAAAGGT GGTAGCATCCAATACCTGTCTC-30 and antisense: 50 -AATAT TGGATGCTACCACCTTCCTGTCTC-30 ) were synthesized by Shanghai GenePharma Co, Ltd (Shanghai, China). All the vectors were transfected into MPC-5 cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA).27 24 h after transfection, the transfected cells were transferred to complete medium for 24 h of incubation. The transfection efficiencies of all vectors were determined by real-time quantitative PCR (RT-qPCR).

Cell viability To study how miR-187 affected the H/R-induced injury in MPC-5 cells, all transfected MPC-5 cells were subjected to 3-h hypoxia and then to 3-h reoxygenation. Then, the cells were harvested for cell viability detection, according to the Cell Counting Kit-8 (CCK-8) method (Dojindo, Kumamoto, Japan).27 The cells were plated onto 96-well plates (5  103 cells/well) in 5% CO2 at 37 C. Then, the CCK-8 reaction solution was added into each well. After 30 min of incubation, the optical density was quantified using a microplate reader at 450 nm (Molecular Devices, Sunnyvale, CA).

Cell apoptosis

Materials and methods

The MPC-5 cell apoptosis was determined by flow cytometry assay (Beyotime Institute of Biotechnology).27 In brief, all transfected cells subjected to H/R were stained with 25 mg/mL Annexin V-fluorescein isothiocyanate and 25 mg/mL propidium iodide. The apoptosis rate were analyzed under a flow cytometer (BD FACSCalibur; BD Biosciences)

Cell culture

Dual-luciferase report system

The immortalized mouse podocyte (MPC-5) cell line was obtained from Cell Resource Center/Peking Union Medical College

The sequence of ACHE-30 -untranslated regions (UTR) was predicted to contain a possible binding-site of miR-187 by

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Targetscan7.2 (http://www.targetscan.org/vert_72/). Dualluciferase report system was performed to verify whether miR-187 specifically targeted ACHE.27 Briefly, the wild-type and mutant ACHE-30 -UTR sequences (mut) obtained from GenePharma (Shanghai GenePharma Co, Ltd) were inserted into the luciferase vector pGL3-Basic vector (Promega, Madison, WI). MiR-187 mimics were then cotransfected with 50 nM of ACHE-30 -UTR or mut into HEK293T cells (American Type Culture Collection, ATCC, Manassas, VA) using Lipofectamine 2000. 48 h after the transfection, luciferase activity of every type of transfected cells was quantified by a dual-luciferase reporter assay system (Promega) and renilla-luciferase was used for normalization.

Animal I/R model A total of 60 5-wk-old male SpragueeDawley rats (weighing 180-220 g) were purchased from the Slac Laboratory Animal Center (Shanghai, China). All rats were kept in temperatureand humidity-controlled cages, with free access to water and rodent food. All protocols were approved by the Institutional Animal Care and Use Committee of Zhongshan Hospital, Fudan University, and the experiments were conducted strictly following the NIH Guide for the Care and Use of Laboratory Animals. To investigate the effects of miR-187 overexpression on renal IRI in vivo, the rats were randomly divided into sham control, I/R group, agemir-NC þ I/R group and miR-187 agomir þ I/R group.21 The miR-187 agomir and NC agomir were obtained from GenePharma. The 50 nmoles of antagomir or agomir-NC were dissolved in 375 mL saline and injected via tail vein (Guangzhou RiboBio., Co). The rats in sham and I/R groups were injected with saline, whereas the Sprague Dawley rats were intraperitoneally anesthetized with 50 mg/kg 5% barbitone and subjected to an abdominal incision. A thermistor was connected to a servo-controlled heating pad (model D1-L; Haake, Tokyo, Japan) to maintain the rectal temperatures of rats at 37 C. After the right renal arteriovenous ligation, right nephrectomy was performed, whereas the left renal ischemia was then induced by clamping the left renal artery occlusion using nontraumatic clamps for 45 min, and the change of the color of the left kidney was synchronously observed. Subsequently, the clamps were removed and the blood supply of the left kidney was recovered. After 96 h of reperfusion, the intraperitoneally anesthetized rats were euthanized and the blood was collected by heart puncture and the renal tissues were carefully dissected for subsequent experiments. Sham-operated control mice were also subjected to the same operation, without clipping left renal artery. Blood samples were collected for the examination of serum creatinine (CREA) concentrations by an automatic biochemistry analyzer (Hitachi 7060; Hitachi Koki Co, Ltd, Hitachinaka City, Japan).

serially sliced into 4-mm-thick sections for HE staining. The paraffin-embedded sections were stained with hematoxylin (Solarbio, Beijing, China) and eosin (Solarbio) for pathological analysis.

Real-time quantitative PCR Total RNA from the transfected MPC-5 cells and collected kidney tissues was extracted using RNAiso Plus (Takara, Dalian, China).27 Total RNAs were reversely transcribed using M-MLV MicroRNA Reverse Transcription Kit (Promega). The relative miRNA levels were detected by SYBR Premix Ex TaqTM (Takara) and normalized to U6. For the analysis of interested gene expressions, cDNA was reversely transcribed from 2.0 mg total RNA using Reverse Transcription Kit (Takara) and reacted at 65 C for 5 min, followed by 30 C for 6 min and 50 C for 55 min. RT-qPCR was performed with Power SYBR Green PCR Master mix (Applied Biosystems, Foster City, CA). A 10 mL final reaction consists of 0.25 mL of the RT reaction, 3-5 pmol of each primer and 1  Power SYBR Green PCR Master Mix and reacted initially at 94 C for 4 min, followed by 40 cycles at 94 C for 30 s, 55 C for 30 s, and 72 C for 1 min. Relative expressions were normalized by the Ct value of b-actin and expression levels were calculated by the 2DDCt formula.28 The primers used in this study were shown in Table.

Western blot The collected kidneys or transfected MPC-5 cells were resuspended by Radioimmunoprecipitation assay buffer (Beyotime, China). The lysate concentration of each sample was quantified using the bicinchoninic acid method (Beyotime). The protein samples were subjected to electrophoresis on sodium dodecyl sulfate-polyacrylamide gel and then transferred to polyvinylidene fluoride membranes. After blocking with 5% nonfat milk in Tris-HCl buffer solution with Tween buffer for 1 h at room temperature, the membranes were cultured with various primary antibodies overnight at 4 C and subsequently incubated with the secondary antibody (1:2000, #ab205718; Abcam, Cambridge, UK). Protein was normalized with GAPDH

Table e Primers for qRT-PCR. Gene Name miR-187

Forward: 50 -TCGTGTCTTGTGTTGCAGC-30 Reverse: 50 -GTGCAGGGTCCGAGGT-30

ACHE

Forward: 50 -CGGGTCTATGCCTACATC-30 Reverse: 50 -GCTCGGTCGTATTATATCCCA-30

Nephrin

Forward: 50 - CCCAGGTACACAGAGCACAA-30 Reverse: 50 - CTCACGCTCACAACCTTCAG-30

Desmin

Forward: 50 - GCGTGACAACCTGATAGACG-30 Reverse: 50 - GTTGGATTTCCTCCTGTAGTTTG-30

Hematoxylin and eosin (HE) staining assay U6

One part of collected rat kidney tissues was fixed with 4% paraformaldehyde (SigmaeAldrich) for longer than 48 h. After being washed with PBS, the kidney tissues were dehydrated with a series of graded ethanol, embedded in paraffin and

Primer sequences

Forward: 50 -CTCGCTTCGGCAGCACA-30 Reverse: 50 - AACGCTTCACGAATTTGCGT-30

b-Actin

Forward: 50 -ACTCGTCATACTCCTGCT-30 Reverse: 50 -GAAACTACCTTCAACTCC-30

yue et al  mir-187 mitigated podocyte death in iri

and visualized using the ECL Plus detection system (GE Healthcare, Waukesha, WI). The primary antibodies (ACHE [1:1000, 68 kDa, #ab183591], nephrin [1:1000, 200 kDa, #ab216341], desmin [1:100000, 53 kDa, #ab32362], and b-actin [1:1000, 42 kDa, #ab8227]) used in this research were purchased from Abcam.

Statistical analysis Quantitative data were expressed as mean  S.E.M and analyzed by Student’s t-test and one-way ANOVA, followed by the Tukey’s multiple comparison test. P < 0.05 was considered as significant.

Results The increasing hypoxia time induced the sustained decline of miR-187 and cell viability in MPC-5 cells The MPC-5 cells were cultured and differentiated for the subsequent experiments (Fig. 1A). The undifferentiated MPC5 cells were triangular and fusiform. After the induction of differentiation, cell volume became larger and cytoplasm grew to cardinal process and secondary podocyte process, and the differentiated MPC-5 cells could be used in experimental studies. Then, the cells were subjected to different hours of hypoxia and to 3 h of reoxygenation. We observed that the

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level of miR-187 reduced gradually as the hypoxia time prolonged, and that 2 h of hypoxia induced a significant decreased at miR-187 level (P < 0.01, Fig. 1B). Meanwhile, 3 h of hypoxia could induce an obvious inhibition in cell viability (P < 0.05, Fig. 1C). Together, the H/R model of MPC-5 cells was constructed successfully, and the hypoxia time was found to be negatively associated with the miR-187 level and MPC-5 cell viability.

The elevated miR-187 expression decreased H/R-induced MPC-5 cell apoptosis To reveal the functional effects of miR-187 on the H/R-induced MPC-5 cell injury, the miR-187 mimics and inhibitor vectors were transfected into MPC-5 cells before undergoing H/R. As shown in Figure 2A, those vectors have stably expressed in MPC-5 cells, and miR-187 was significantly increased in mimics group and downregulated in inhibitor group. We also found a slightly positive association between miR-18 level and MPC-5 cell viability (Fig. 2B). Subsequently, the transfected cells were subjected to H/R (3 h/3 h) treatment, and the cell viability and apoptosis rate were measured to assess the effects of miR-187 on the H/R-induced cell injury. According to Figure 2C, miR-187 mimics could effectively mitigate the inhibitory effect of H/R at miR-187 level; however, the level of miR-187 in H/R þ inhibitor group decreased obviously, compared with the inhibitor group (P < 0.01). Meanwhile, the increased miR-187 induced by mimics vector also contributed

Fig. 1 e The increasing hypoxia time induced the sustained decline of miRNA-187 and cell viability in MPC-5 cells. (A) The cellular morphologies of MPC-5 cells were observed under an inverted microscope (bar: 100 mm). (B) The relative levels of miR-187 were measured after cell receiving different hours of hypoxia and 3 h of reoxygenation. (C) The effects of different hypoxia time on the cell viability were measured by CCK-8 assay. Each value represented mean ± SEM (n [ 3). #P < 0.05, ## P < 0.01 versus control group. (Color version of figure is available online.)

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Fig. 2 e The elevated miRNA-187 expression reduced H/R-induced MPC-5 cell apoptosis. (A) The transfection efficiency of miR-187 mimics and inhibitor were assessed by RT-qPCR. (B) The cell viability of transfected cells was detected by CCK-8 assay. (C) RT-qPCR was used to measure the level of miR-187 in transfected MPC-5 cells subjected to H/R. (D) The cell viability was measured to assess the effects of H/R on the transfected cells. (E and F) The changes of cell apoptosis were analyzed by flow cytometry. (G-H) The mRNA and protein levels of ACHE were determined by RT-qPCR and western blot. Each value represented mean ± SEM (n [ 3). ##P < 0.01 versus control group; ^P < 0.01 versus NC group; *P < 0.05, **P < 0.01. (Color version of figure is available online.)

yue et al  mir-187 mitigated podocyte death in iri

to the increased cell viability and survival rate (Fig. 2D-F). The miR-187 mimics inhibited cell apoptosis rate from 20.05% of H/R þ NC group to 9.43% of H/R þ mimics group (P < 0.01), while the transfection of miR-187 inhibitor further weakened the capacity of MPC-5 cell resistance to H/R-cause cell death and enhanced apoptosis rate to 26.04% (P < 0.01). In addition, we also measured ACHE relative levels, and found that H/R could induce the expression of ACHE, which was negatively related with miR-187 level. Therefore, these findings indicated that the excessive level of miR-187 could partially mitigate the H/R-induced injury in MPC-5 cells.

ACHE was a target of miR-187 We also investigated the relationship between miR-187 and ACHE through a series of functional studies. As shown in Figure 3A, computational analysis of Targetscan7.2 predicted that the 30 -UTR of the ACHE gene contained a conserved binding site for miR-187. The wild-type or mutant ACHE 30 UTR was cloned into luciferase report plasmids to verify that miR-187 could directly target ACHE mRNA. When miR-187 mimics were cotransfected with wild-type ACHE 30 -UTR luciferase reporter vector, luciferase activity largely decreased in comparison with the transfection of luciferase reporter vector. However, miR-187 mimics failed to affect the luciferase activity of mutant ACHE 30 -UTR luciferase reporter vector (Fig. 3B). Therefore, our data indicated that ACHE was an effective target of miR-187.

The knockdown of ACHE diminished the H/R-caused MPC5 cell apoptosis To confirm whether miR-187 mitigated H/R-induced MPC5 cell injury was mediated through the inhibition of ACHE expression, siACHE was transfected into MPC-5 cells before undergoing H/R. As shown in Figure 4A, siRNA has effectively blocked the expression of ACHE in MPC-5. Meanwhile, the results of cell viability and apoptosis detections showed that siACHE had a similar efficacy to miR-187 mimics (Fig. 4B-D). The inhibition of ACHE could partially diminish the H/Rinduced reduction of cell viability (P < 0.05); in the meantime, siACHE transfection also decreased H/R-induced

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apoptosis from 19.55% in H/R þ NC group to 9.2% in H/R þ siACHE group (P < 0.01). Collectively, our studies confirmed that the improvement of miR-187 on the H/Rinduced MPC-5 cell injury was attributed to the blocking of the expression of ACHE.

The injection of miR-187 agomir improved renal IRI in vivo The animal I/R model was established to further verify the treatment effects of elevated miR-187 on the renal IRI. We examined the concentration of serum CREA in each experimental group rat, and found that rat I/R model had a significantly higher concentration of serum CREA than that in the sham group; however, rats injected with miR-187 agomir had a lower concentration of serum CREA than those in NC þ I/R group (n ¼ 13, P < 0.01, Fig. 5A). Then, kidney tissue sections were subjected to histological examination by HE staining to assess I/R-induced renal injury. After 96 h of reperfusion, the kidney tissue of rat I/R model showed substantial changes in tubular structure and integrity, and the number of apoptotic cell in I/R group also increased obviously, compared with the sham group. However, agomir injection could partly protect the kidney from I/R-caused tubular necrosis and damage (Fig. 5B). A high level of miR-187 was also found in agomir þ I/R group, indicating that miR-187 agomir was efficiently expressed in kidney tissues (n ¼ 13, Fig. 5C). In addition, we also measured the expressions of nephrin and desmin, which acted as the markers of podocytes, and we found that miR-187 agomir could significantly promote the mRNA and protein levels of nephrin and inhibit desmin and ACHE expressions (n ¼ 13, Fig. 5D-F), indicating that the injection of agomir could also protect podocytes from I/R-induced injury. Taken together, the in vivo experiments indicated that the excessively expressed miR-187 could significantly improve I/Rcaused kidney tubule and podocyte injury via blocking the expression of ACHE.

Discussion The study on metabolic and ultrastructural changes in the kidney suffering I/R injury observed acute kidney injury (AKI)

Fig. 3 e ACHE was a target of miRNA-187. (A) Computational analysis of Targetscan7.2 predicted that the 30 -UTR of the ACHE gene contained a conserved binding site for miR-187. (B) Dual-luciferase report system performed to verify the close relationship between ACHE and miR-187. Each value represented mean ± SEM (n [ 3). **P < 0.01. (Color version of figure is available online.)

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Fig. 4 e The knockdown of ACHE diminished H/R-caused MPC-5 cell apoptosis. (A) The transfection efficiency of siACHE was detected by RT-qPCR. (B) The changes of cell viability were measured by CCK-8 assay. (C and D) Cell apoptosis was analyzed by flow cytometry. (E and F) The mRNA and protein levels of ACHE were determined by RT-qPCR and western blot. Each value represented mean ± SEM (n [ 3). b-actin was set as an internal control. *P < 0.05, **P < 0.01. (Color version of figure is available online.)

protein biomarkers in proximal tubules and glomeruli, the flattening of podocytes and pedicals surrounding glomerular capillaries and the effacement of podocyte pedicals.29 The phenomena pointed to the key role of podocytes in the pathogenesis of I/R-induced AKI. To investigate the functional roles of miR-187 in the renal IRI, we established an H/R podocyte model in vitro, and the miR-187 mimics and inhibitor were transfected into podocytes before undergoing H/R. We found that the excessive expression of miR-187 could enhance the capacity of podocytes resistance to IRI and reduced I/Rinduced cell apoptosis. In addition, ACHE 30 -UTR was confirmed to contain a binding site of miR-187, and the transfection of siACHE had a similar efficacy to miR-187 mimics in podocyte viability and apoptosis; therefore, we rationally speculated that the renal protection of miR-187 was mediated through the regulation of ACHE. Podocytes was an essential component of the glomerular filtration barrier and involved in maintaining the structural integrity of the glomerular basement membrane.30 Podocytes are terminally differentiated epithelial cells and are incapable of

proliferation, and podocyte dysfunction or injury could cause proteinuria and glomerulosclerosis, subsequently contributing to the pathogenesis of multiple nephropathies including AKI.31,32 In our study, H/R caused a sharp decrease in podocyte viability and survival rate, which suggested that the glomerular filtration barrier was damaged and may contribute to the pathogenesis of iARF. The previous study showed that autophagy activated by rapamycin could significantly inhibit the adriamycin-induced podocyte apoptosis and protect podocyte from the effects of pathological conditions of glomerular disease.33 The transfection of miR-187 mimics could also effectively protect podocytes from H/R-induced cell injury and death; however, the underlying mechanism still remained to be investigated. Some studies demonstrated that miR-187, which acts as a tumor suppressor, was involved in the regulation of various cancer progressions; for instance, Liang et al. demonstrated that miR-187 exerted its tumor-suppressive activity through regulating glia-activating factor mRNA in cervical cancer,34 and that it also could inhibit the osteosarcoma growth and metastasis by blocking the expression of mitogen-activated

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Fig. 5 e The injection of miRNA-187 agomir improved I/R-induced kidney injury in vivo. (A) The concentrations of CREA were analyzed using an automatic biochemistry analyzer. (B) Kidney tissue sections were subjected to histological examination by HE staining to assess the renal injury. (bar: 100 mm and 50 mm). (C) The level of miR-187 was detected by RT-qPCR. (D-F) The expressions of podocyte biomarkers (nephrin and desmin) and ACHE were analyzed by RT-qPCR and western blot. Each value represented mean ± SEM (n [ 13). U6 served as the internal control for miR-187 and b-actin was set as an internal control for target genes. **P < 0.01. (Color version of figure is available online.) protein kinase 12.35 This article revealed a new role of miR-187, which protected podocytes against H/R-induced cell injury. To further verify the protective effects of miR-187 on cell survival rate of podocytes in vivo, we established an animal renal IRI model, and miR-187 agomir was injected into rat before the operation. We found that miR-187 could also exert its protective effects on podocytes in vivo, and subsequently diminish the renal injury caused by I/R. Taken together, our findings indicated that miR-187 could be a possible therapeutic target to keep podocyte number under the pathological conditions of IRI. Furthermore, our data identified that ACHE was an effective target of miR-187. ACHE is known as an enzyme that rapidly hydrolyzes neurotransmitter acetylcholine and terminates cholinergic neurotransmission.36 Recently, Xie et al. found that although wild-type and ACHE knock-out heterozygous mice were subjected to the same surgical procedure of I/R treatment, the kidneys of the wild-type mice showed more obvious accumulation of the cleaved poly (ADP-ribose) polymerase (a biomarker for apoptosis) than the kidney tissues from

heterozygous mice, showing that ACHE was also an important contributor in cell apoptosis.37 In our study, a significant upregulation of ACHE observed in H/R-treated podocytes demonstrated that ACHE was involved in the pathogenesis of IRI in the kidney. In addition, the in vivo experiments showed remarkably reduced mRNA and protein levels of ACHE under the effects of miR-187 agomir. Meanwhile, the changes of nephrin and desmin levels indicated that the injection of agemir contributed to the mitigation of I/R-caused podocyte injury and damage of glomerular filtration barrier. Therefore, our data indicated that miR-187 could effectively protect podocytes both in vivo and in vitro, and such a protective effect were mediated through the regulation of ACHE expression.

Conclusions In conclusion, our study reveals, for the first time, the underlying mechanism of miR-187 mitigating I/R-induced renal

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injury was attributed to suppressing the expression of ACHE. The transfection of miR-187 mimics obviously enhanced the ability of podocytes resistance to I/R-induced cell injury and increased cell survival rate, while the injection of agomir can not only protect podocytes, but also further diminish I/Rinduced renal injury. Therefore, miR-187 might be a novel therapeutic target for the improvement of renal IRI.

12.

13. 14.

Acknowledgment This work was supported by the National Natural General Foundation of China [81470581]. Authors’ contributions: J.Yu. contributed to conception of this research and final approval of the version to be published; T.Z. contributed to design of this research and revising on the manuscript; J.Ya. and Y.S. contributed to data acquisition. X.X. and Y.F. contributed to data interpretation. W.F. contributed to data analysis, article drafting and agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy.

Disclosure

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The authors declare no conflicts of interest. 19.

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