miR-17-92 ameliorates renal ischemia reperfusion injury

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Kaohsiung Journal of Medical Sciences (2017) xx, 1e11

Available online at www.sciencedirect.com

ScienceDirect journal homepage: http://www.kjms-online.com

Original Article

miR-17-92 ameliorates renal ischemia reperfusion injury Turun Song, Mianzhi Chen, Zhengsheng Rao, Yang Qiu, Jinpeng Liu, Yamei Jiang, Zhongli Huang, Xianding Wang, Tao Lin* Department of Urology, Urology Research Institute and Organ Transplantation Center, West China Hospital, Sichuan University, Chengdu, Sichuan, PR China Received 20 April 2017; accepted 11 September 2017

KEYWORDS Acute kidney injury; Ischemia reperfusion injury; miR-17-92

Abstract There is limited information on the role of miR-17-92 in renal tubular pathophysiology. Therefore, the present study was performed to determine whether miR-17-92 plays a role in ischemia-reperfusion injury (IRI)-induced acute kidney injury. We originally demonstrated that miR-17-92 is up-regulated following IRI in vivo. To explore the roles of miR17-92 in the IRI process, we first generated a renal proximal tubule-specific miR-17-92 deletion (PT-miR-17-92
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) knockout mouse model with Cre driven by the Kap promoter. We found that PT-deficient miR-17-92 mice had more severe renal dysfunction and renal structures than their littermates. Compared with sham-operated mice, both wide-type (WT) mice and PT-miR-17-92
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mice showed increased serum levels of creatinine and urea. However, the levels of serum urea and creatinine in PT-miR-17-92
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mice after the IRI operation were significantly higher than the levels in WT mice. In addition, PT-miR-17-92
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mice showed higher levels of serum potassium and phosphonium after the IRI operation. Histological analysis revealed that PT-miR-17-92
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mice had substantial histopathologic changes, such as tubular dilation and tubular necrosis. Overexpression of miR-17-92 could partially reverse the side-effects of IRI on the proximal tubules in vivo. Furthermore, we employed a quantitative proteomic strategy and identified 16 proteins as potential targets of miR-17-92. Taken together, our findings suggested that miR-17-92 may ameliorates IRI-induced acute kidney injury. Our results indicate that pharmacologic modulation of these miRNAs may have therapeutic potential for acute kidney injury. Copyright ª 2017, Kaohsiung Medical University. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/ by-nc-nd/4.0/).

Conflicts of interest: All authors declare no conflicts of interests. * Corresponding author. Department of Urology, West China Hospital, Sichuan University, Guoxue Xiang #37, Chengdu 610041, Sichuan, PR China. E-mail address: [email protected] (T. Lin). https://doi.org/10.1016/j.kjms.2017.09.003 1607-551X/Copyright ª 2017, Kaohsiung Medical University. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Please cite this article in press as: Song T, et al., miR-17-92 ameliorates renal ischemia reperfusion injury, Kaohsiung Journal of Medical Sciences (2017), https://doi.org/10.1016/j.kjms.2017.09.003

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Introduction Ischemia-reperfusion injury (IRI) of the kidney is one of the primary causes of AKI. It has been shown to be associated with high mortality, morbidity, and cost in patients undergoing major vascular, cardiac, or transplantation surgery [1,2]. In IRI, a transient drop in blood flow to the kidney is followed by a reperfusion period. Ischemia compromises the continuous supply of oxygen. Rapid return of oxygenated blood, which is referred to as reperfusion, is essential for restoring kidney function. However, reperfusion itself contributes to cellular injury and deathda phenomenon referred to as reperfusion injury [3,4]. The proximal tubular (PT) cells within the kidney are highly dependent on energy for the efficient transport of ions, water, and macromolecules across cell layers. In the IRI process, blood flow to the outer medulla is disproportionately reduced with respect to the reduction in total blood flow. Thus, epithelial cell injury is most pronounced in the proximal tubule located in the outer medulla, and this region is particularly susceptible to hypoxia [5], which would lead to dysfunction of the Naþ/Kþ ATPase pump. This allows intracellular accumulation of Naþ ions followed by an influx of water, leading to cell swelling, intracellular disruption, and eventual cell death [6,7]. MicroRNAs (miRNAs) are small non-coding RNA molecules that modulate gene expression either by regulating a specific set of genes or by reinforcing gene-expression programs through suppression of transcriptional noise [8]. The associations between the abnormal expression of miRNAs and many disease processes have been reported [9], and therefore, miRNAs are potentially promising small-molecule drugs [10]. Numerous hallmarks of acute kidney injury, such as apoptosis and TLR signaling, are regulated by miRNAs. A recent study reported that transgenic overexpression of the miR-17-92 cluster in mice resulted in signs of autoimmune injury in the glomeruli [11]. In our preliminary study, miR17-92 was highly expressed in the kidney, especially in the renal cortex, which mainly includes proximal tubules. To our knowledge, there is limited information on the role of miR17-92 in renal tubular pathophysiology. Therefore, the present study was performed to determine whether miR-1792 plays a role in IRI-induced acute kidney injury.

Methods Mouse strains In this study, miR-17-92(flox/flox) mice were crossed with KAP-CRE mice to generate miR-17-92(flox/flox)XCREY mice according to the breeding protocol in Fig. 2A. The miR-1792(flox/flox)XCREY mice showed miR-17-92 deletion in the renal proximal tubules. These mice and their wide-type (WT) littermates (male, 9-weeks-old) were used for IRI experiments. The mice were housed in an animal facility with a 14-h light/10-h dark cycle, and food and water were available ad libitum.

T. Song et al. mirc1-floxed allele was detected with PCR using miR-17-92F (50 -TCGAGTATCTGACAATGTGG-30 ) and miR-17-92-R (50 TAGCCAGAAGTTCCAAATTGG-30 ) primers to amplify a 289bp product for homozygous mice and a 255-bp product for WT mice. The presence of the CRE gene was confirmed by PCR using the primer pair 50 -GCCTGCATTACCGGTCGATGC-30 and 50 -CAGGGTGTTATAAGCAATCCC-30 to amplify a 481-bp product.

Real-time PCR analysis of miRNAs Total RNA from the kidney cortex was extracted using Trizol (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instruction. Then, 2 mg of total RNA was converted to cDNA using the miRNA Reverse Transcription Kit (Fementas, Waltham, MA, USA). Real-time PCR was performed using the RIBOBIO miRNA assay kit (Guangzhou, China), which included sequence-specific primers for cDNA synthesis and probes for real-time PCR. Quantification was performed using DCt values.

Murine model of renal IR injury After Institutional Animal Care and Use Committee approval, adult (20 g) male mice were subjected to 20 min of renal IR. I/R injury was induced by unilateral clamping (micro aneurysm clamps) of one renal pedicle for 30 min under general inhalation anesthesia (3% isoflurane and oxygen) and remove contralateral one. After removal of clamps, kidneys were inspected for restoration of blood flow. For analgesic purposes, mice received a subcutaneous injection of 0.1 mg/kg buprenorphine (Temgesic; Schering-Plough) 30 min before surgery. Sham-operated animals underwent the same procedure excepting clamping of the renal pedicles. We collected the kidneys and plasma samples on days 1, 3, 5, 7, 9, 11, and 13 after renal IRI or sham operation to examine the severity of renal dysfunction.

Histological evaluation and TUNEL assay Mice were killed and perfused with PBS followed by 4% paraformaldehyde. The kidneys were fixed in 10% formalin and then embedded in paraffin. Sections were cut and stained with hematoxylin and eosin. Histologic changes, including tubular necrosis, tubular dilation, cast formations, and loss of brush border, were examined in a blinded manner. Micrographs of representative fields were recorded. Apoptosis in renal tissues was identified using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay with an in situ Cell Death Detection kit (Promega, Madison, WI, USA), according to the manufacturer’s instructions. Five fields per section and three sections per kidney were examined in each experimental group (n Z 3 mice in each group).

Genotyping

Two-dimensional gel electrophoresis and image analysis

Genomic DNA was extracted through mouse tail biopsy for polymerase chain reaction (PCR)-based genotyping. The

Mice were killed and the kidney cortex was harvested. The renal cortex was smashed in liquid nitrogen, lysed in a

Please cite this article in press as: Song T, et al., miR-17-92 ameliorates renal ischemia reperfusion injury, Kaohsiung Journal of Medical Sciences (2017), https://doi.org/10.1016/j.kjms.2017.09.003

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miR-17-92 and IRI

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Figure 1. Identification of the signature for miR-17-92 following IRI. The expression of miRNAs within the miR-17-92 cluster was measured in wide type mice by real-time. Naı¨ve kidney cortex serves as day 0. The data are expressed as mean  SE of three independent experiments. Relative quantitation was calculated by the DCt method normalizing miRNA expression to the endogenous control U6.

lysis buffer (8 mol urea, 2 mol thiourea, 2% CHAPS, 1% DTT, 2% IPG buffer), and then mixed at 4  C for 30 min. After centrifugation at 16,000 g for 60 min at 4  C, the supernatant was collected, aliquoted, and stored at
80  C. Protein levels were determined using the G250 method. The strips were rehydrated for 16 h at room temperature with 340 ml of rehydration buffer (8 mol urea, 2% CHAPS, 0.4% DTT, 0.5% IPG buffer) containing 1.5 mg of protein samples. We used 18-cm IPG strips (pH 3e10, non-linear, Bio-Rad) above 20  C. Isoelectric focusing was manipulated in the Ettan IPGphor IEF System, according to the manufacturer’s instructions. The rehydrated strips underwent a stepwise voltage increment program as follows: 100, 200, and 500 V for 1 h each, 1000 V for 2 h, and 8000 V afterward until 100 kVh. After isoelectric focusing, the strips were incubated in equilibration buffer I (6 M urea, 2% SDS, 30% glycerol, 1% DTT, 50 mM Tris-Cl, pH 6.8) and equilibration buffer II (6 M urea, 2% SDS, 30% glycerol, 4% iodoacetamide, 50 mM TrisCl, pH 6.8) for 20 min separately with gentle agitation.

Subsequently, the strips were loaded onto 12% SDSpolyacrylamide gels. The gels were run at 5 W/gel for 40 min and then 17 W/gel until the dye fronts reached the bottom. The gels were stained using the R250 staining method compatible with MS analysis. All gels were run thrice independently to ensure reproducibility.

Tryptic in-gel digestion The proteins in the gel were digested using trypsin (V5280; Promega), according to the manufacturer’s instructions. In short, the SDS-PAGE gels were excised, spots in the gel were picked out, and the gel was cut into pieces using a razor blade. The 2D gel was destained with 100 mM NH4HCO3/50% acetonitrile (ACN) at room temperature for at least 15 min. This step was repeated twice. After dehydration with 100% ACN, the gels were immersed in 25 ml of trypsin solution (10 ng/ml) for 1 h at 4  C. Digestion buffer (40 mM H4HCO3/10% ACN) was then added until the

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T. Song et al. VAC concentrator (Thermal) at 4  C. The samples were then subjected to mass spectrometry.

MALDI-TOF/TOF MS and MS/MS spectrometry analysis and database search Mass spectra were obtained using a MALDI-TOF/TOF MS fitted with an MS source. MS/MS analysis was performed as described previously. The MS/MS data were acquired, and MASCOT was used for database search. The parameters for the search were as follows: database, Swiss-Port; taxonomy, Mus Muscle; enzyme, trypsin; mass tolerance, 0.1 Da; MS/MS tolerance, 0.05 Da; and an allowance of one missed cleavage. The options of “carbamidomethylation” and “oxidation of methionine” were selected as modifications. Proteins with probability-based MOWSE scores exceeding their threshold (p < 0.05) were considered to have been positively identified.

Statistical analysis Data are expressed as mean  SEM. Biochemical data were analyzed using one-way ANOVA with Bonferroni correction for comparisons. The analyses were performed using GraphPad Software (GraphPad, San Diego, CA, USA). Statistical analyses involved the t-test. A p-value <0.05 was considered statistically significant.

Results Identification of a signature for miR-17-92 following IRI

Figure 2. Mouse model of target depletion of miR-17-92 from renal proximal tubules. (A) A schematic description for the production of proximal tubule-specific miR-17-92 knockout (PTmiR-17-92
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) mice. This study uses only male littermate mice with confirmed genotypes for experiment. (B) PCR-based genotyping to confirm miR-17-92 depletion from proximal tubules. Genomic DNA is isolated from kidney cortex of PT-miR-1792
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, PT-miR-17-92þ/
and PT-miR-17-92þ/þ mice. GAPDH is also amplified as control. (C) quantitative real-time PCR of miR17-92 expression in kidney cortex. Kidney cortex is dissected from PT-miR-17-92
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, PT-miR-17-92þ/
and PT-miR-17-92þ/þ mice for collect total RNA for qRT-PCR of miR-17-92 and U6.

gels were covered. The gels were incubated overnight at 37  C. Digestion products were collected through double extraction with 50% ACN/5% trifluoroacetic acid for 1 h each time. The combined extracts were dried in a speed-

Total RNA from IRI and sham-control kidney cortex samples were obtained separately, and real-time PCR was performed. Cohorts of mice in each group (n Z 4 per time point) were killed at days 1, 3, 5, 7, 9, 11, and 13. Data suggested that the miRNAs were differentially regulated following IRI. The miRNAs were also differentially expressed in sham controls, apparently because of inflammation induced by surgery. The seven miRNAs within the cluster could be placed into four groups based on their expression patterns (Fig. 1). miR-17 and miR-175p were rapidly up-regulated following IRI when compared with control samples and the levels observed in control (day 0) kidneys, and they showed a dramatic decrease at day 3 and began to return to normal levels (Group 1). miR-18a and miR-19b were rapidly downregulated after IRI during the first few days and began to return to normal levels from day 7 (Group 2). miR-19a and miR-20a showed a sharp increase following IRI, and then, they reduced from day 5 (Group 3). Among these miRNAs, the expression pattern of miR-92a was unique and was not influenced by IRI.

Generation of a mouse model for proximal tubulespecific depletion of miR-17-92 A mouse model with targeted miR-17-92 depletion in renal proximal tubules was established to demonstrate the role

Please cite this article in press as: Song T, et al., miR-17-92 ameliorates renal ischemia reperfusion injury, Kaohsiung Journal of Medical Sciences (2017), https://doi.org/10.1016/j.kjms.2017.09.003

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miR-17-92 and IRI of miR-17-92 in the pathogenesis of IRI-induced renal injury. miR-17-92flox mice were crossed with Kap-Cre mice. The Kap promoter drives Cre expression predominantly in renal PT cells. As shown in Fig. 2A, after two rounds of breeding, we obtained miR-17-92flox/floxCreKap mice that showed knockout of miR-17-92 in the renal proximal tubules (PT-miR-17-92
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), as expected. Because the expression level of Cre is regulated by the expression level of androgen, we used only male mice in this study to ensure the depletion of miR-17-92. To verify the reduction of miR-17-92 in the proximal tubule, we extracted genomic DNA from the kidney cortex, which consists mainly of proximal tubules. As exhibited in Fig. 2B, PCR clearly amplified a 250-bp fragment from the WT mice (PT-miR-17-92þ/þ) cortex. In contrast, all PTmiR-17-92
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mice exhibited a clear 287-bp fragment, indicating Cre-mediated deletion of miR-17-92 from the proximal tubules. Quantitative real-time PCR was also performed to examine miR-17-92 expression in the renal cortex with regard to the RNA level. As shown in Fig. 2C, PT-miR-17-92
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tissues showed a remarkable decrease in miR-17-92 expression in the renal cortex. Taken together, these results validated the PT-miR-17-92
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conditional knockout model.

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Preservation of renal function and structure by miR-17-92 after IRI To assess the role of miR-17-92 in IRI-induced acute kidney injury, WT and PT-miR-17-92
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mice underwent IRI operation, and renal function was compared between these mice. Compared with sham-operated mice, both WT and PT-miR-17-92
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mice showed increased serum levels of creatinine and urea (Fig. 3A and B); however, the levels of serum urea and creatinine in PT-miR-17-92
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mice after IRI was significantly higher than the levels in WT mice, suggesting a remarkable prevention of renal dysfunction associated with renal IRI. Furthermore, the magnitudes of renal dysfunction, as evaluated by the increase in the serum creatinine and urea levels, were aggravated by over 57% and 240%, respectively, in PT-miR-17-92
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mice (Fig. 3A and B). Because IRI mainly involves the renal tubules and affects potassium retention (3) and solute reabsorption (14), serum potassium and phosphonium levels were also measured. Both WT and PT-miR-17-92
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mice showed up-regulation of serum potassium and phosphonium levels, in accordance with the results mentioned above, and PT-miR-17-92
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mice displayed higher levels of serum

Figure 3. miR-17-92 partially preserve of renal function following IRI. (A & B) Renal function as indicated by serum creatinie level (A) and serum urea nitrogen level (B) in WT and PT-miR-17-92
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mice. Serum creatinine levels were measured 48 h after IRI or sham operation. (C & D) Proximal tubule function as indicated by serum Kþ and P in WT and PT-miR-17-92
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mice. Data are mean  SEM (n Z 7 mice in each group). *P<0.01, WT versus PT-miR-17-92
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. Please cite this article in press as: Song T, et al., miR-17-92 ameliorates renal ischemia reperfusion injury, Kaohsiung Journal of Medical Sciences (2017), https://doi.org/10.1016/j.kjms.2017.09.003

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Figure 4. miR-17-92 protects against IRI-induced renal tubular dilation and tubular disruption. PT-miR-17-92
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and WT mice underwent a sham operation (A & B) or IRI operation (C & D). Renal sections were stained with hematoxylin and eosin for histological examination. No apparent differences were observed in renal histology between sham controls of PT-miR-17-92
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mice (A) and WT mice (B). PT-miR-17-92
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kidneys showed marked degree of renal injury (C), including extensive tubular dilation (black arrow) and tubular necrosis (red arrow), whereas WT kidney exhibited a marked reduction in the severity of these features (D). (E & F) Score of tubular dilatation (E) and total ATN (F). *P<0.01, PT-miR-17-92
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versus WT. Figures are representative of three experiments (n Z 3 mice in each group).

potassium and phosphonium, which increased by 80.4% and 56%, respectively. Sham-operated WT and PT-miR-17-92
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mice showed healthy and histologically comparable tubular systems (Fig. 4A and B); however, PT-miR-17-92
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mice showed substantial histopathologic changes, such as tubular dilation and tubular necrosis after IRI (Fig. 4C). In contrast, in WT mice after IRI operation, most of the tubular structure and integrity were maintained and histologic damage was milder when compared to that in PTmiR-17-92
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mice (Fig. 4D). The acute tubular necrosis

score was used to evaluate injury in PT-miR-17-92
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and WT mice (Fig. 4E and F).

Attenuation of tubular epithelial cell apoptosis following IRI in PT-miR-17-92L/L mice TUNEL assay was performed to evaluate the role of Mirc1 in tubular epithelial cell apoptosis after IRI because apoptosis has been frequently noted in the pathogenesis of acute

Please cite this article in press as: Song T, et al., miR-17-92 ameliorates renal ischemia reperfusion injury, Kaohsiung Journal of Medical Sciences (2017), https://doi.org/10.1016/j.kjms.2017.09.003

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Figure 5. miR-17-92 attenuates IRI-reduced renal tubular epithelial cell apoptosis. PT-miR-17-92
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(A & C) and WT (B & D) mice underwent a sham operation (A & B) or IRI operation (C & D). TUNEL staining of representative kidney sections from each experimental group is shown. (EeH) DAPI staining of reprehensive kidney sections from each experimental group is exhibited. (IeL) Merge images for representative kidney sections from each experimental group. (M) Quantitative analysis of TUNELpositive renal epithelial nuclei per total nuclei in WT and PT-miR-17-92
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mice subjected to sham operation or IRI (n Z 3 mice per group).

Figure 6. Over-expression of miR-17-92 partly inverses IRI induced renal dysfunction. Groups of WT and PT-miR-17-92
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mice were subjected to tail vein injection of vehicle or miR-17-92 plasmids 4 days before IRI. Samples were harvested every other day. Over-expression miR-17-92 in WT mice and in PT-miR-17-92
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mice ameliorates the renal dysfunction in both serum creatinie level (A & C) and serum urea nitrogen level (B & D). *P<0.01, PT-miR-17-92
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versus WT. Figures are representative of three experiments (n Z 5 mice in each group).

Please cite this article in press as: Song T, et al., miR-17-92 ameliorates renal ischemia reperfusion injury, Kaohsiung Journal of Medical Sciences (2017), https://doi.org/10.1016/j.kjms.2017.09.003

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Figure 7. Proteomic analysis of total protein of renal cortex between PT-miR-17-92þ/þ mice and PT-miR-17-92
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mice. (A,B) Representative 2-DE gel images of PT-miR-17-92
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mice renal cortex and the control. Total protein was extracted from PT-miR-1792
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mice kidney cortex and PT-miR-17-92þ/þ mice kidney cortex and then separated on pH3-10 nonlinear IPG strips in the first dimension followed by 12% SDS-PAGE as the second dimension and visualized by CBB staining. Approximate 900e1000 protein spots were detected. A total of 51 spots (marked with arrow and number) were identified as differentially expressed. Among these spots, 26 spots were up-regulated meanwhile 25 spots were down-regulated in PT-miR-17-92
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mice. (C) 51 distinct proteins were divided into three different groups according to their cellular locations: membrane (37.2%), cytoplasm (35.3%) and nucleus (27.5%). (D) 51 distinct proteins were classified into 12 groups based on their biological functions: cell adhesion (11%), immune response (7%), ion transport and binding (15%), transcription regulation (9%), signal transduction (9%), fibroblasts (6%) and cytoskeleton (10%) and chemotoxin stimulation (6%). (E) Western blotting for Pten in PT-miR-17-92þ/þ mice and PT-miR-17-92
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mice renal cortex before and after IRI operation. a-Tublin was used as in internal control. (F) RT-PCR for Kcnj13 and Fmn1 from total RNA from PTmiR-17-92þ/þ mice and PT-miR-17-92
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mice renal cortex. GAPDH was employed as an internal control. Lane 1: total protein from PT-miR-17-92þ/þ mice after IRI. Lane 2: total protein from PT-miR-17-92
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mice after IRI. Lane 3: total protein from PT-miR-1792þ/þ mice before IRI. Lane 4: total protein from PT-miR-17-92
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mice before IRI.

Please cite this article in press as: Song T, et al., miR-17-92 ameliorates renal ischemia reperfusion injury, Kaohsiung Journal of Medical Sciences (2017), https://doi.org/10.1016/j.kjms.2017.09.003

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miR-17-92 and IRI kidney injury. There were only a few TUNEL-positive cells in the kidneys of sham-operated WT and PT-miR-17-92
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mice (Fig. 5A & B). After IRI, a significant number of TUNELpositive cells was observed in PT-miR-17-92
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mice (Fig. 5C). Importantly, dramatically fewer TUNEL-positive cells were noted in WT mice following the IRI operation (Fig. 5D). The percentage of TUNEL-positive cells in Mirc1 mice was 57% lower than that in WT mice (p < 0.01, Fig. 5M).

Validation of the antiapoptotic role of miR-17-92 in vivo Gain-of-function by overexpression of miR-17-92 through tail vein injection was performed to further address the antiapoptotic role of miR-17-92. After overexpression of miR-17-92 in vivo, the serum creatinine (Fig. 6A & C) and serum urea levels (Fig. 6B & D) exhibited milder increases following IRI in both WT (Fig. 6A & C) and PT-miR-17-92
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mice (Fig. 6B & D). Taken together, these results validated the antiapoptotic role of miR-17-92 in IRI-induced renal injury.

Detection and identification of candidate targets of miR-17-92 To identify and quantify the differentially expressed proteins in PT-miR-17-92
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mice and their littermates, equal amounts of proteins from the renal cortex were used for proteomic analyses. Three independent 2D gels were run for each sample, and highly reproducible gel images were obtained. A total of 49 up-regulated proteins were noted, extracted, digested with trypsin, and successfully identified with LC-MS/MS (Fig. 7A & B). We paid attention to the up-regulated proteins rather than the down-regulated proteins because the up-regulated proteins are potential direct targets of miR-17-92. Among the identified upregulated proteins, we focused on 16 proteins that had miR-17, miR-17-5p, miR-18a, miR-19a, miR-19b, or miR-20a binding sites in the 30 untranslated region. Table 1 shows the up-regulated proteins identified in PT-miR-17-92
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mice, and Table 2 shows the candidate proteins identified by LC-MS/MS and provides further details. Pten, Dpysl2, and 14 other proteins were identified as direct target candidates for the miR-17-92 cluster. The classification of the altered proteins based on their biological functions (Fig. 7C) and subcellular localization is shown in Fig. 7D. Western bolting for Pten was performed to validate the LCMS/MS results (Fig. 7E). Because Kcnj13 and Fmn1 are not targets of miR-17-92, the expression with regard to RNA level is the same as the expression with regard to the protein level. Real-time PCR for Kcnj13 and Fmn1 was performed to further validate the LC-MS/MS results (Fig. 7F).

Discussion In this study, we created a conditional knockout mouse model with target ablation in renal proximal tubules. Interestingly, these mice were vulnerable to IRI-induced

9 PT injury. PT-deficient miR-17-92 mice experienced a more severe condition compared to the condition in their littermates with regard to both renal function and renal structures. Compared with sham-operated mice, both WT mice and PT-miR-17-92
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mice showed increased serum levels of creatinine and urea, which indicated renal

Table 1 Up-regulated proteins identified in PT-miR-1792
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mice. Gene name

Uniport access No.

Dis3l2 mCG128585 Pcdha5 TYK2 Kcnj13 Kcnj2 C78339 Fat2 Efcab5 Tsnaxip1 Ccl21a Rrp8 Dpf1 Klc3 Kctd13 Tnfaip1 Fmn1 Myh1 C2cd2 Pde8b Wdr33 Pde8b Fam205a Tcea1 Dock6 Pten Dock4 Mfap1 Rrs1 GM10797 Slc7a10 Anapc1 Gstm4 Wars Atp2b1 Snrpe MC1R PQBP-1 Atp2b2 Pisa 4 Krt77 Mina Fgfr1or2 Sipa1l2 Tcf 20 Kif9 Kif26b Sipa1l3 Olfr368

Q99816 Q8111W P61106 Q9R117 P86046 Q543W5 Q3URQ4 Q75731 A0JP43 Q99P25 P84444 Q9DB85 Q9QX66 Q91W40 Q8BGV7 O70749 Q05860 B2RWX0 Q5DTR6 Q8BIA0 Q8K4P0 E9Q8J6 A2APU8 P107112 Q6ZPS1 O08586 B2RUG6 Q9CQU1 Q9CYH6 Q3V073 P63115 Q6PG65 Q8R5I6 P32921-2 Q6PG79 P62305 Q01726 Q9VHQ8 Q6DIB8 Q9JM05 Q678L1 Q8CD15 Q9CRA9 B9EK76 Q9EPQ8 Q9WV04 Q7TNC6 Q4VBF3 Q8VF22

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10 Table 2

T. Song et al. Candidate proteins identified by LC-MS/MS.

Gene name

Pcdha5 TYK2 Kcjn2 Fat2 Tnfaip1 Fmn1 C2cd2 Tcea1 Pten Dock4 Dpysl2 Anapc1 Atp2b2 Fqfr1op2 Kif26b Sipa1l3

Uniport access No

P61106 Q9R117 Q543W5 Q75731 O70749 Q05860 Q5DTR6 A2APU8 O08056 B2RUG6 Q16555 Q6PG65 Q6DIB8 Q9CRA9 Q7TNC6 Q4VBF3

MS/MS analysis

Target prediction

MS/MS score

Peptides

Aaa (%)

PicTar

TargetScan

MiRanda

34.2 35.7 112.7 115 39.4 47.4 53 83.1 115 38.5 25.3 43.6 51.7 58.1 62.1 34

5 2 1 5 2 2 4 4 5 3 2 2 2 3 3 1

3 1 2 3 1 1 1 2 16 1 3 2 2 1 2 1

17-5p,19a,19b,20a 18a 19a,19b 17,20a 17,20a 17 17,20a 17,19a,19b 17,19a,19b,20a 17,20a 20 18a 17,19a,20a 17,19a,19b,20a 17,20a 17,20a

17-5p. 19b 18a 19a,19b 17-5p 17-5p,20a 17,20a 17-5p 17,20a 17,19a,19b,20a 17,20a 19a,20 18a 17,19a,19b,20a 17,19a,19b,20a 17-5p,20a 17-5p

17,19a,19b,20a 17,18a 19a,19b 17.20a 20a 17 17,20a 17,19a,19b,20a 17,19a,19b,20a 17,20a 17,20a 18a 17,19a,19b,20a 17,19a,19b,20a 17,20a 17

dysfunction. However, the levels of serum urea and creatinine in PT-miR-17-92
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mice after IRI were significantly higher than the levels in WT mice. In addition, PTmiR-17-92
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mice showed higher levels of serum potassium and phosphonium after IRI. Histological analysis revealed that PT-miR-17-92
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mice had substantial histopathologic changes, such as tubular dilation and tubular necrosis. Overexpression of miR-17-92 could partially reverse the side-effects of IRI on the proximal tubules. Our results provide initial evidence of the role of miR-17-92 in IRI-induced acute kidney injury. In this study, we showed for the first time that the miR17-92 cluster played antiapoptotic roles in IRI-induced acute tubular injury. miR-17-92, also called mironc1, consisting of seven miRNAs, is markedly and frequently involved in various pathological processes and is reported to enhance cell proliferation and growth. It is interesting to note that miR-17-92 also enhanced cell proliferation in the renal proximal tubules in response to IRI-induced acute tubular injury. Recent studies have shown that miRNAs play important roles in kidney development and pathology. Our study clearly showed that the miR-17-92 cluster, except miR-92a, may play an antiapoptotic role in IRI-induced acute tubular injury. We performed quantitative proteomics to assess directly targeted proteins of miR-17-92 and identified 16 proteins as potential targets. Recently, the association between abnormal expression of miRNAs and acute kidney injury has been reported. However, the molecular mechanisms by which miRNAs modulate the process of acute kidney injury remain largely unknown. One of the reasons may be our limited knowledge of target recognition by miRNAs. In most cases, miRNAs function as modulators at the post-transcriptional level [8]. Considering this fact, we demonstrated that a comprehensive proteomics approach is useful for detecting direct targets of miRNAs. Among the 16 candidate targets with miRNA-binding sites of miR-17, miR-17-5p, miR-18a, miR-19a, miR-19b, and miR-20a detected with proteomics analysis in our study, Pten was

reported as one of the target genes of miR-19a. Recently, it was shown that miR-19a repressed the tumor suppressor Pten gene and hence activated Akt-mTOR signaling [12]. Among the identified target proteins, besides Pten, Dyps2 was reported to be directly regulated by miR-20 [13]. These previous findings provided strong support for our results. Among the identified proteins, Kcjn2 is a potassium transporter responsible for potassium homeostasis. Pcdha5, which shares a highly identical sequence in humans, is a calcium-regulated protein [14]. Atp2b2 is a protein responsible for calcium ion transport [15]. A previous study has shown that point mutations of Atp2b2 cause reduction in Ca2þ influx [16]. In addition, Tnfaip1 plays important roles in immune responses in diabetic nephron diseases, indicating that miR-17-92 may also function in other nephron diseases [17]. In conclusion, miR-17-92 may antagonize the sideeffects of IRI in different pathways, and miR-17-92 could serve as a crosstalk factor in the IRI-induced pathological process. Thus, miR-17-92 is a promising potential antagonist for both acute kidney injury.

Acknowledgment This study was supported by the National Natural Science Foundation of China (Grant No. 81470980, 81600584), and 1.3.5 project for disciplines of excellence, West China Hospital, Sichuan University.

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Please cite this article in press as: Song T, et al., miR-17-92 ameliorates renal ischemia reperfusion injury, Kaohsiung Journal of Medical Sciences (2017), https://doi.org/10.1016/j.kjms.2017.09.003

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Please cite this article in press as: Song T, et al., miR-17-92 ameliorates renal ischemia reperfusion injury, Kaohsiung Journal of Medical Sciences (2017), https://doi.org/10.1016/j.kjms.2017.09.003