Novel PS-OME miRNA130b-3p Reduces Inflammation and Injury and Improves Survival after Renal Ischemia-Reperfusion Injury

Abstract

Introduction:

Acute kidney injury (AKI) is a prevalent medical disorder characterized by a sudden decline in kidney function, often because of ischemia-reperfusion events. It is associated with significant chronic complications, and currently available therapies are limited to supportive measures. Extracellular cold-inducible RNA-binding protein (eCIRP) has been identified as a mediator that potentiates inflammation following ischemia-reperfusion injury. However, it has been discovered that miRNA 130b-3p acts as an endogenous inhibitor of eCIRP. To address the inherent instability of miRNA in vivo, a chemically modified miRNA mimic called PS-OME miR130 was developed. We hypothesize that administration of PS-OME miR130 following renal ischemia/reperfusion (I/R) can lead to reduced inflammation and injury in a murine model of AKI.

Methods:

C57BL/6 male mice underwent renal I/R by clamping of bilateral renal hilum for 30 min or sham operation. Immediately following closure, mice were intravenously administered vehicle (PBS) or PS-OME miR130 at a dose of 12.5 nmol/mouse. Blood and kidneys were collected after 24 h for further analysis. Separately, mice underwent renal I/R and administered vehicle or treatment and, survival was monitored for 10 days.

Results:

Following renal I/R, mice receiving vehicle showed a significant increase in serum markers of kidney injury and inflammation including BUN, NGAL, KIM-1 and IL-6. Following treatment with PS-OME miR130, these markers were significantly decreased. Kidney tissue mRNA expression for injury and inflammation markers including NGAL, KIM-1, KC, and MIP-2 were increased after renal I/R, however, these markers showed a significant reduction with PS-OME miR130 treatment. Histologically, treatment with PS-OME miR130 showed a significant decrease in neutrophil infiltration and injury severity score, and decreased apoptosis. In the 10-day survival study, mice in the treatment group showed a significant reduction in mortality as compared to vehicle group.

Conclusion:

In a murine renal I/R model, the administration of PS-OME miR130, a direct eCIRP antagonistic miRNA mimic, resulted in the reduction of kidney inflammation and injury, and improved survival. PS-OME miR130 holds promise to be developed as novel therapeutic for AKI as an adjunct to the standard of care.

INTRODUCTION

Acute kidney injury (AKI) is a critical medical condition characterized by a rapid decline in the kidney function, often due to hypovolemic ischemic events, nephrotoxicity, or sepsis [1, 2]. Clinically, AKI is associated with a sudden increase in serum creatinine levels and/or reduced urine output as defined by the RIFLE/AKIN criteria [1, 2]. The RIFLE/AKIN criteria are an acronym for the progressively increasing grades of severity of AKI conditions which correspond to Risk, Injury and Failure. The Loss and End-Stage Renal Disease (ESRD) criteria, represent the outcomes and correspond to duration of kidney dysfunction. AKI affects 10-15% of hospitalized patients and up to 65% of critically ill patients in the intensive care units [1-3]. AKI is associated with high mortality rates (20-50%) and even following the resolution of AKI, 30% of patients may suffer from long-term complications, including increased risk of chronic kidney disease (CKD) and ESRD and ultimately, require long-term dialysis or kidney transplantation [1, 2, 4, 5]. Current AKI therapies focus on supportive measures like fluid management, electrolyte correction, and renal replacement therapy (RRT) in severe cases [1, 2]. These interventions, however, do not target the underlying molecular and cellular processes driving kidney injury and inflammation. The development of novel therapies addressing these pathways could reduce AKI severity, prevent CKD or ESRD progression and improve patient outcomes.

During ischemia/reperfusion (I/R), the ischemic environment becomes increasingly acidic, leading to cellular dysfunction, disturbed ionic concentration homeostasis, osmotic dysregulation, cell death, and tissue swelling [5-7]. Reperfusion exacerbates the initial injury by triggering an inflammatory response and via generation of reactive oxygen species (ROS), causing further damage in a compromised environment [5-8]. Regarding renal I/R injury, tubular epithelial tissue in the kidneys is severely affected, releasing cytokines and damage-associated molecular patterns (DAMP), which engage the innate immune system and cause additional injury [5, 8-12]. Understanding the molecular pathways in AKI pathogenesis and inflammation helps identify potential therapeutic targets. We reasoned that one such target is extracellular cold-inducible RNA binding protein (eCIRP). CIRP was initially believed to function exclusively by modifying intracellular mRNA stability [12, 13]. However, we previously discovered that when CIRP was released extracellularly and thus termed eCIRP, it functions as a critical DAMP in various inflammatory conditions, including hypovolemic shock, septic shock, and ischemia/reperfusion injuries [9-11, 14]. Further investigation revealed that eCIRP's signaling pathway involves two target receptors, TLR4/myeloid differentiation factor-2 (MD2) and triggering receptor expressed on myeloid cells-1 (TREM-1) [15, 16]. Given their role in causing significant inflammatory responses, studies were targeted on interrupting those signaling receptors using antibodies and peptide compounds [17, 18].

Another promising strategy involved developing an antagonistic agent that could directly inhibit eCIRP activity. Recently, miRNA 130b-3p was reported as an endogenous inhibitor of eCIRP, and concomitantly found to be elevated in serum samples of septic shock patients [11]. A miRNA 130b-3p mimic was then synthesized, and its effectiveness in reducing inflammation was experimentally confirmed [11]. To improve the in vivo miRNA stability, we have chemically engineered the miRNA 130b-3p mimic and designated as PS-OME miR130 [19, 20]. The selection of modifications was focused on using well studied miRNA modification strategies and the presence of significant nuclease protective attributes. The primary techniques used for PS-OME miR130 were PS and ‘OME modification. Specifically, the PS modification added several phosphorothioate bonds to the terminal ends while the ‘OME modification involved the addition of multiple methyl groups throughout the miRNA mimic. Together, these changes help prevent degradation by reducing nuclease binding sites [21, 22]. Originally, miRNA’s were believed to primarily function intracellularly as gene expression regulators however, studies have shown additional functions related to intercellular signaling, pathologic mechanisms and proinflammatory responses [23-33]. Most miRNA-based therapeutics follow this intracellular paradigm and therefore require extensive modifications to both protect it from degradation and provide intracellular transport. Given that our compound acts specifically on an extracellular target, the focus is on preventing degradation without impeding its eCIRP specific binding affinity [34]. In fact, this modified miRNA was shown to successfully reduce tissue injury and inflammation in rodent models of sepsis and hepatic I/R injury [19, 20]. Previously we have shown that renal I/R results in elevated serum and tissue levels of eCIRP and additionally that in CIRP knockout mice there is an attenuated inflammation and injury response [35]. Therefore, we hypothesize that administering PS-OME miR130 following renal I/R reduces inflammation, injury severity, and improves survival in a murine AKI model.

MATERIALS AND METHODS

PS-OME miRNA 130b-3p Synthesis

A single stranded PS-OME miRNA130 5’mC*mA*mG*mUmGmCmAmAmUmGmAmUmGmAmAmAmGmGmG*mC*mA*mU-3’ was synthesized by Integrated DNA Technologies (Coralville, Iowa). Sample provided as a lyophilized powder was resuspended using nuclease free PBS and stored in −20°C. The symbols in the miRNA sequence represent * for PS bonds and “m” for 2’OME modifications.

Experimental Animals

C57BL/6 male mice (20-28g) (Charles River Laboratories Wilmington, MA) were housed in temperature and light controlled rooms on a 12-h dark/light cycle and fed a standard rodent chow diet. Upon arrival, mice were allowed 7 days of acclimatization to the environment. Experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the Feinstein Institutes for Medical Research and were performed in accordance with the National Institutes of Health and the Guide for the Care and Use of Laboratory Animals.

Renal Ischemia-Reperfusion Model

Renal I/R procedure was performed as previously described [14, 35]. Before the surgery, the mice were randomly selected as the sham (n=10), vehicle (n=14), and treatment (n=14) groups. The mice were anesthetized with 2-4% inhaled isoflurane and sedation depth was confirmed throughout the surgical procedure. Dissection was performed to visualize the renal pedicles and vascular clips were placed bilaterally across the renal pedicles. The ischemia time was standardized to the experimental branch; a 24-h experiment undergoing 30 min and a 10-day survival experiment with 34 min [14]. Following ischemia, clamps were removed, and kidney reperfusion was confirmed bilaterally. The surgical incision was closed in two layers using nylon 4-0 sutures. Immediately following the closure, mice were randomly allocated to be administered intravenously, via retroorbital injection, of PBS (vehicle) or 12.5 nmoles of PS-OME miR130 (treatment) in both the 24-h experiment and the 10-d survival experiment. The dosing was based on previous studies showing an effective reduction in inflammatory markers in both unmodified and modified miRNA 130b-3p mimics in other injury models [11, 19, 20]. Analgesia was provided by delayed subcutaneous injection of buprenorphine (50 μg/kg), and resuscitation was provided by 500 μL bolus of normal saline. Mice were placed in separate recovery cages and were provided nutritionally fortified DietGel 76A (Clear H₂O, Westbrook, ME).

For the mice in the 24-h experiment, blood and renal tissues were collected 24 h after reperfusion. Renal samples were divided along the coronal plane, and bilateral sections were harvested. One set of sections were preserved in 10% formalin for histopathologic analysis, while bilateral opposing sections were flash frozen in liquid nitrogen and stored at −80°C, for quantitative polymerase chain reaction (PCR) analysis. Mice in the 10-d survival experiment underwent evaluation for euthanasia by humane criteria twice daily for the first three days and daily for seven additional days (n=20/group). Days survived were measured per group and defined by either early euthanasia due to fulfillment of the humane endpoint criteria or reaching the 10-d endpoint. The humane endpoints were the following: [1] weight loss >20%, [2] minimal or no response to stimuli, [3] Grimace score of 2, [4] body condition score ≤2, [5] labored/agonal breathing. In the presence of 2 or more of the above criteria, the mice were euthanized.

Measurement of Organ Injury Markers

The collected serum was prepared by centrifugation of whole blood at 3000g for 10 min and stored at −80°C. Levels of BUN and creatinine were measured using colorimetric assays per the manufacturer’s instructions (Pointe Scientific, Canton, MI). Serum levels for IL-6, NGAL, and KIM-1 were analyzed using enzyme-linked immunosorbent assay kits per the manufacturer’s instructions (Biosciences, San Jose, CA).

Reverse Transcription-Quantitative PCR (RT-qPCR)

Total RNA was isolated from renal samples from the 24-h experiment using TRIzol reagent (Invitrogen, Thermo Fisher Scientific Inc.) according to the manufacturer's instructions. The extracted RNA was then reverse transcribed into cDNA using reverse transcriptase (Applied Biosystems, Thermo Fisher Scientific Inc.). PCR reactions were set up in a final volume of 24 μL containing 0.08 μM of each forward and reverse primer, appropriate amounts of cDNA, nuclease-free water, and 2 X SYBR Green Master Mix (Applied Biosystems, Thermo Fisher Scientific Inc.). Amplification and analysis were performed using a Step One Plus real-time PCR machine (Applied Biosystems, Thermo Fisher Scientific Inc.). Mouse β-actin mRNA served as an internal control for normalization, and the relative gene expression levels were determined using the 2^-ΔΔCt method. The mRNA expression was calculated as the fold change relative to sham tissue samples. The primer sequences used are as follows:

KIM-1, 5’-TGCTGCTACTGCTCCTTGTG-3’ (forward), and 5’-GGGCCACTGGTACTCATTCT-3’ (reverse)

NGAL, 5’-CTCAGAACTTGATCCCTGCC-3’ (forward), and 5’-TCCTTGAGGCCCAGAGACTT-3’ (reverse)

KC, 5’-GCTGGGATTCACCTCAAGAA-3’ (forward), and 5’-ACAGGTGCCATCAGAGCAGT-3’ (reverse)

MIP-2, 5’-CCCTGGTTCAGAAAATCATCCA-3’ (forward), and 5’-GCTCCTCCTTTCCAGGTCAGT-3’ (reverse)

β-actin, 5’-CGTGAAAAGATGACCCAGATCA-3’ (forward), and 5’-TGGTACGACCAGAGGCATACAG-3’ (reverse)

Renal Histopathology

Renal tissue collected from the 24-h experiment was fixed in 10% formalin and paraffin embedded. The tissue was sectioned to 5 μm and stained using hematoxylin and eosin (H&E). Renal tissue injury was assessed by light microscopy in a blinded fashion. Each prepared sample was evaluated at five randomly selected fields for the following categories: tubular cell injury, tubular cell detachment, loss of brush border, tubular simplification, and cast formation [14, 35]. Each category per tissue section was scored as 1 (<10%), 2 (10% - 25%), 3 (>25% - 50%), 4 (>50% - 75%), and 5 (>75%), for a maximal score of 25.

Granulocyte Receptor-1 (GR-1) Immunohistochemistry

Gr-1 immunohistochemistry was performed by dewaxing paraffin-embedded sections in xylene and graded series of ethanol washes. Slides were then heated in 0.92% citric acid buffer (Vector Laboratories; Burlingame, CA) at 95°C for 15 min. Slides were allowed to cool to room temperature, incubated in 2% H₂O₂/60% methanol and blocked using 5% normal rat serum/Tris buffered saline (TBS). Sections were then incubated overnight with anti-Gr-1 antibody (1:50 dilution) (Abcam; Cambridge, MA) in 2% normal horse serum/TBS with 0.02% Triton X-100 at 4°C. Immunohistochemical reaction was detected using Vectastain ABC reagent and DAB kit (Vector laboratories). Slides were then counterstained with H&E before imaging. Each prepared sample was evaluated at five randomly selected fields and Gr-1 positive staining cells were quantified using ImageJ software.

Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL) Assay

To assess apoptotic cell death, TUNEL staining was performed and quantified as TUNEL positive staining cell counts. Paraffin-embedded sections of renal tissue were dewaxed, rehydrated, and followed by permeabilization using proteinase K solution. The fluorescent staining was performed using a commercially available In Situ Cell Death Detection Kit (Roche Diagnostics, Indianapolis, IN) per the manufacturer’s instructions. Sections were stained using 4’, 6-diamidino-2-phenylindole (DAPI) to provide nuclear counterstain. Each prepared sample was evaluated at five randomly selected fields, and TUNEL positive staining cells were quantified using ImageJ software.

Statistical Analysis

Data are expressed as mean ± standard error of the mean (SEM), compared via one-way analysis of variance (ANOVA) and the difference between groups were analyzed by the Student Newman Keul’s test. Analysis of the survival study was carried out using the Kaplan-Meier estimator and Log-Rank test for survival. All statistical calculations were performed using the GraphPad Software. The significance was considered as p<0.05.

RESULTS

PS-OME miR130 Attenuated Systemic and Renal Specific Injury Markers

To provide evidence that PS-OME miR130 can specifically improve inflammation in a renal I/R model, serum was tested for kidney injury and inflammation markers. Serum levels of BUN and creatinine are important biomarkers that provide valuable information about kidney function and help in the evaluation and monitoring of AKI. The mice in the vehicle group showed a significant 4.0-fold increase in BUN levels from sham (22.1 ± 2.6 vs. 90.9 ± 9.3 mg/dL), while the levels in the PS-OME miR130 group reduced to 33.9 ± 7.0 mg/dL which was a significant 63% reduction from the vehicle (Fig. 1A). Although there was a significant increase in creatinine in the vehicle group, from 0.52 ± 0.03 mg/dL in sham to 1.14 ± 0.2 mg/dL in vehicle, PS-OME miR130 treatment reduced these levels to 0.78 ± 0.07 mg/dL, but the decrease was not statistically significant (Fig. 1B). Interestingly, serum NGAL and KIM-1, two major kidney injury markers, had significant increases from 0.10 ± 0.01 mg/dL and 0.07 ± 0.05 mg/dL in sham to 27.4 ± 6.6 mg/dL and 22.2 ± 4.5 mg/dL in the vehicle group, while PS-OME miR130 treatment significantly decreased these markers to 13.3 ± 1.6 mg/dL and 12.0 ± 1.7 mg/dL respectively (Fig. 1C-D). Cytokines such as IL-6 are key indicators of systemic inflammation. Serum IL-6 levels were also increased from 4.5 ± 1.9 mg/dL in sham to 280.2 ± 30.1 mg/dL in the vehicle group whereas PS-OME miR130 treatment significantly decreased these levels to 199.0 ± 21.1 mg/dL which was a 29% reduction from the vehicle (Fig. 1E). These results suggest PS-OME miR130 attenuates renal I/R-induced systemic inflammation and injury markers in AKI.

Fig. 1. PS-OME miR130 reduces serum markers of kidney injury and inflammation.

Blood was collected from sham, Veh (PBS) or PS-OME miR130 (miR) treated mice at 24 h after renal I/R. BUN (A) and Creatinine (B) were analyzed from serum samples using the respective colorimetric assays (Sham, n = 6; Vehicle and miR treatment, n = 10-12/group). NGAL (C), KIM-1 (D), and IL-6 (E) serum levels were analyzed using the respective ELISAs (Sham, n = 10; Vehicle and miR treatment, n = 8-13/group). Data are expressed as mean ± SE and compared by one-way ANOVA and SNK method (*P<0.05 vs. Sham; ^#P<0.05 vs. Veh).

PS-OME miR130 Attenuated Renal Injury and Inflammation

To determine that the changes seen in the serum levels of these injury and inflammation markers are caused by local kidney injury, the kidney mRNA expression of the injury markers were quantified using RT-qPCR. The mRNA levels for KIM-1 and NGAL were increased by 170.8 ± 19.8-, and 53.4 ± 7.4-fold in the vehicle compared to sham, while the PS-OME miR130 treatment decreased these levels to 82.8 ± 26.2- and 20.9 ± 6.3-fold, a 52% and a 61% decrease from the vehicle mice, respectively (Fig. 2A-B). In addition, KC, and MIP-2 mRNA expressions were significantly increased by 10.9 ± 2.1- and 91.8 ± 14.0-fold in the vehicle group compared to sham mice while the expression of these mRNAs was 5.2 ± 1.0- and 54.7 ± 2.1-fold, respectively, in the mice that received PS-OME miR130 which were 52% and 40% reduced from the vehicle, respectively (Fig. 2C-D).

Fig. 2. PS-OME miR130 reduces renal tissue injury and inflammation.

Renal tissue was harvested 24 h after renal I/R from different groups. Following a total RNA isolation and a reverse transcription, mRNA levels of KIM-1 (A), NGAL (B), KC (C), and MIP-2 (D) were analyzed using a RT-qPCR (n = 4-6/group). The mRNA results were standardized to β-actin levels. Data are expressed as mean ± SE and compared by one-way ANOVA and SNK method (*P<0.05 vs. Sham; ^#P<0.05 vs. Veh).

PS-OME miR130 Reduced Neutrophil Infiltration and Renal Injury Severity

Gr-1 immunohistochemistry was used to assess the degree of neutrophil infiltration, a key indicator of localized inflammation, in the renal histology sections. There was an abundance of Gr-1 staining cells in the vehicle group, with minimal staining in the sham group. Positive staining cell count results were 0.60 ± 0.9, 66.6 ± 27.1 and 25.1 ± 20 for sham, vehicle, and treatment groups respectively. When compared to the vehicle group, the PS-OME miR130 treatment group had a significant reduction in Gr-1 staining cells, with a decrease of 62% in the average cell count indicating treatment decreased neutrophil infiltration (Fig. 3A-B). Furthermore, injury in the kidneys was evaluated using H&E-stained sections measuring severity scoring for tubular cell injury, tubular cell detachment, loss of brush border, tubular simplification, and cast formation. The results showed a grossly increased degree of tissue injury in the vehicle group with an average score of 16.9 ± 3.2 compared to the sham sections with a score of 5.38 ± 2.6. When scoring was compared to the treatment group, 10.3 ± 2.6, there was a significant 39% reduction over the vehicle group (Fig. 3C-D).

Fig. 3. PS-OME miR130 reduces renal neutrophil infiltration and tissue injury severity.

Renal tissue harvested from different groups was sectioned and stained immunohistochemically with a Gr-1 antibody for neutrophil infiltration quantification (A) and stained with H&E for injury severity scoring (C). Representative images at 100x magnification are shown. Neutrophil infiltration was quantified from Gr-1 positively staining cells using Image J from randomized fields (B) (n = 5–7/group). The severity of injury was scored by a blinded investigator (D) (n = 9–10/group). Data are expressed as mean ± SE and compared by one-way ANOVA and SNK method (*P<0.05 vs. Sham; ^#P<0.05 vs. Veh).

PS-OME miR130 Decreased Apoptosis

As a measurement of apoptotic cell death, renal samples that underwent TUNEL staining and fluorescent microscopy showed an increase in TUNEL positive staining cells in the vehicle group compared to the sham on gross inspection. Following quantification of cell counts, results showed 14.8 ± 6.2, 269.6 ± 99.0, and 119.6 ± 62.6 for sham, vehicle, and treatment groups respectively. This represents a significant 56% decrease in TUNEL positive staining nuclei in the PS-OME miR130 group from vehicle mice (Fig. 4A-B).

Fig. 4. PS-OME miR130 reduces renal apoptosis following renal I/R.

Renal tissue harvested from different groups was sectioned and stained with TUNEL (A). Representative images for samples imaged at 100x magnification are shown. Fields were randomly selected and quantified for TUNEL positive staining cells using Image J (B) (n=5/group). Data are expressed as mean ± SE and compared by one-way ANOVA and SNK method (*P<0.05 vs. Sham; ^#P<0.05 vs. Veh).

PS-OME miR130 Improved Survival

In the final branch of the experiment, a 10-day survival study was conducted after treatment with either the vehicle (PBS) or PS-OME miR130. The results showed that the mice in the vehicle group had a survival rate of 25%. The mice in the treatment group had a survival advantage with a survival rate of 60%. These results showed a significant 35% increase in the survival of mice that received treatment over the vehicle (Fig. 5).

Fig. 5. PS-OME miR130 reduces mortality following renal I/R.

Following renal I/R, mice received either the vehicle or PS-OME miR130 (n =20/group). The survival rate was measured over the course of 10 days and, statistical analysis was performed using the Kaplan-Meier estimator using a log-rank test (*P<0.05 vs. Vehicle).

DISCUSSION

Ischemia-reperfusion injury is a serious medical problem with potentially life-threatening and debilitating clinical manifestations. Given its acute onset and complex pathophysiology involving multiple cellular pathways and processes, it has posed a significant challenge for physicians [1-4, 36-38]. However, due to recent advances in elucidating the pathways involving inflammation, innate immunity and DAMPs, promising progress has been made in attenuating the initial injury [8, 39, 40] . One such DAMP is eCIRP which was initially discovered to be elevated in the circulation of critical care patients during hemorrhagic shock or septic shock [16]. Therefore, for this study, we focused on attempting to antagonize the activity of eCIRP directly using a novel engineered miRNA mimic which was developed based on the miRNA 130b-3p [19]. Originally miRNA 130b-3p, an endogenous 22 base-pair miRNA, was found to be specifically elevated during sepsis and following screening studies was also shown to have a high binding affinity to eCIRP [11]. Initially, administration of a miRNA 130b-3p mimic resulted in the reduction of eCIRP mediated inflammation in sepsis [11]. However, given that any miRNA is unstable in vivo due to its susceptibility to degradation by nucleases, miRNA 130b-3p was chemically engineered in an in effort to improve its stability and thereby increase its potential efficacy [41]. Recently, this modified miRNA which we termed PS-OME miR130, has shown to reduce inflammation and tissue injury in mice which underwent either hepatic I/R or cecal ligation puncture (CLP) sepsis [19, 20]. It has been shown that using surface plasmon resonance and three-dimensional computational modeling, PS-OME miR130 retained its binding affinity to eCIRP and that the binding was comparable to that which was observed with the unmodified miRNA 130b-3p [19]. Importantly, studies on the half-life of the unmodified mimic were unsuccessful as it was undetectable in serum while the modified miRNA was found to have a half-life of 277.2 minutes (18,19).

Based on these observations, we hypothesized that PS-OME miR130 could reduce inflammation and kidney injury in a renal I/R model in mice. Our results were in line with the previous findings with reductions to systemic and localized markers of injury and inflammation. Firstly, PS-OME miR130 treatment resulted in a significant reduction of serum levels of BUN and a trend in reduction in creatinine which are the most common metrics for AKI severity in human patients [1, 2]. Following the growing evidence in favor of using biomarkers related to kidney injury, we measured serum concentrations of KIM-1 and NGAL, in addition to the generalized proinflammatory cytokine IL-6 [42-44]. Interestingly, the results correlated well with BUN and creatinine concentrations and showed that treated mice had significant reductions in KIM-1, NGAL and IL-6 in serum when compared to those markers in mice that received vehicle. Furthermore, this suggests that PS-OME miR130 acted to antagonize eCIRP both in circulation and at the tissue injury site. To provide evidence that the serum concentration change in the kidney injury markers was attributed to a localized kidney injury, we measured kidney mRNA levels of these markers using qPCR. The results were consistent with the serum findings and significant reductions in the mRNA expressions of NGAL, KIM-1, KC and MIP-2 were observed in the PS-OME miR130 treatment group compared to the vehicle group.

We then demonstrated if differences in injury could be observed through direct observation of tissue microscopy. H&E-stained sections of the vehicle group showed histological evidence of kidney injury with a high injury score. The treatment with PS-OME miR130 significantly reduced the injury severity score compared to the vehicle score, which was in line with the observations of the circulating kidney specific biomarkers we had previously observed. To assess the activation of immune signaling, we sought to measure the degree of neutrophil infiltration to the kidneys. Tissue sections observed following Gr-1 antibody immunohistochemical staining displayed a greater Gr-1 positive staining cell count in the vehicle group compared to the PS-OME miR130 treated group. An integral part of the ongoing inflammatory signaling is the initiation of apoptosis which we measured using the TUNEL assay. The results showed a significant increase in the quantity of TUNEL positive staining cells in the vehicle group in relation to sham sections and a significant reduction in treatment sections in relation to the vehicle sections. Of note, H&E-stained kidney sections for mice that underwent renal I/R showed significant evidence of acute tubular necrosis which is consistent with previous studies seeking to observe the histological changes resulting from renal I/R. The presence of necrotic tissue represents an important contributory factor through the release of cytokines and DAMPs to the overall kidney tissue injury [42, 45, 46]. The attenuation of these pathways is evidence of the substantial benefit of interrupting the role of DAMPs in the inflammation cascade. Finally, the survival study showed a significant survival benefit in treated mice compared to vehicle mice. Of note, the decision to use 34 minutes of ischemic time in the survival experiments was due to the insufficient mortality observed in the renal I/R mice with the 30 min ischemia which was used in the 24-h experiments [14, 35].

We have previously shown that eCIRP functions as a DAMP in various inflammatory conditions, including hypovolemic shock, septic shock, and ischemia/reperfusion injuries [16-18, 35]. Furthermore, in studies involving CIRP knockout mice undergoing renal I/R, a reduction in inflammation and injury severity was observed [35]. A relationship between eCIRP and AKI was demonstrated in a study where patients undergoing cardiac surgery and whose serum had higher levels of circulating eCIRP showed an increased risk of AKI in the post-operative period [47]. Additionally, eCIRP has been shown to increase other DAMP’s such as HMGB1 further augmenting the inflammatory activity [16, 35]. The mechanistic pathway of DAMPs involves initial activation of pattern recognition receptors (PRRs) such as toll-like receptor (TLR); of which the TLR4/MD2 receptor has been extensively studied to be a key component of the inflammatory pathway [44]. Signaling pathways of eCIRP involves two target receptors: TLR4/MD2 and TREM-1 [15, 16]. The TLR4/MD2 receptor has additionally been studied to be activated by eCIRP and results in the activation of the nuclear factor kB (NFkB) pathway [16, 35]. Given its role in causing significant inflammatory responses, studies were targeted on interrupting the TLR4/MD2 signaling using anti-CIRP antibodies and CIRP-peptide compounds [16]. Initial studies on eCIRP showed significant reduction in inflammation markers using eCIRP binding antibodies [16]. In the current study we demonstrated an alternative strategy in which eCIRP’ activity was blocked by using PS-OME miR130, a novel modified miRNA mimic that acts as a direct eCIRP antagonist, to reduce inflammation and injury in AKI.

There are several limitations to consider in our study. Firstly, due to differences in the inflammatory response between male and female mice, only male mice were used in this study; therefore, this could result in sufficient changes to inflammation potentially affecting the significance of PS-OME miR130’s efficacy [48-50]. Regarding the administration of treatment, neither the timing nor the dosage of PS-OME miR130 was modified, from the initial parameters used in our previous studies, given that the primary aim was to provide evidence of its effectiveness in a renal IR model. The disadvantage to this approach is having a limited view on the potential for delayed treatment benefits and the optimal timing to ensure maximal disruption of the eCIRP mediated inflammatory pathway activation. Additionally, due to the controlled ischemic time used for the biochemical analysis, there could be significant differences in the availability of PS-OME miR130 at the site of injury relating to the physiologic changes that would be proportional to the ischemic time. An example could be changes in delivery secondary to clot burden and the time required for recovery of tissue debris [51]. Likewise, the differences in perfusion, drug delivery and serum and tissue eCIRP concentrations following treatment may have important implications that have not been fully investigated in our study. Although we have not directly measured the specific changes to eCIRP in serum or tissues following treatment with PS-OME miR130, we have previously shown that eCIRP is increased in both serum and tissue following renal IR (20). In conjunction with our findings in the CIRPKO mice (35) and the results of our study showing a significant reduction in inflammation and injury, it can be reasoned that the initial strong binding affinity of the PS-OME miR130 with eCIRP would result in a decrease in eCIRP mediated inflammatory signaling and thus indirectly reduce eCIRP levels in the treated mice. Future studies measuring the concentration of PS-OME miR130 in serum and tissue at various time points would be beneficial in establishing treatment windows and potential clinical applications. A longer bioavailability could permit premedicating in clinical scenarios where injury is expected, such as preoperatively with emphasis on transplant procedures due to the serious implications of inflammation on transplant failure. In addition, further studies on multiple dosing to maintain an anti-inflammatory state in cases of critical care where it would be difficult to assess the optimal timing of administration.

We have observed the mice only for 10 days after the renal IR surgery. Any mice which reached the 10-day end point post-procedure were euthanized in accordance with the study design. The decision to limit survival to 10 days was based on multiple factors. Firstly, since the scope of the study was to assess acute kidney injury, we reasoned that for bilateral renal IR there is acceptable correlation with human AKI and with the proportional resulting of CKD. However, this resulted in a limitation to both the long-term therapeutic effects for PS-OME miR130 as well as observing potential adverse effects [52]. Secondly, following literature review and our labs previous experiences with bilateral I/R we found that the model carries significant mortality, especially so in our severe AKI model of 34 min ischemia, and the mortality is significantly greatest in the first 3 days post-procedure. Previous studies using similar models without intervention show steady mortality past 10 days (52). However, we acknowledge that not following the survival study beyond 10 days as a limitation of the study and the chronic effects of the disruption of the inflammatory injury is an area we intend on pursuing in future studies. Consequently, further studies on toxicity and pharmacokinetics would be required prior to justifying continuous or multiple dosing regimens of PS-OME miR130. Third, elucidating the exact binding sites between eCIRP and PS-OME miR130 would allow a better understanding of the effects of the modifications on its function and if optimization could be made to, for example, modifying any off-target binding or amplification of target site binding. In the experiments to date, there have been no observable toxicity at current dosing with either the unmodified miRNA 130b-3p or PS-OME miR130 including the observations made in this study. Further studies on toxicity, long term outcomes, and identification of undiscovered alternative binding targets are essential before future therapeutic development.

Considering these recent findings showing the efficacy of basic modifications to miRNA mimics and their ability to exert significant reductions in inflammation through an extracellular mechanism, there is justification for further screening of alternative inflammation associated miRNA. Following modification, these compounds used either alone or in conjunction with other miRNAs may result in significant therapeutic reductions in inflammation associated injury.

CONCLUSION

Using a novel chemically engineered modification to miRNA 130b-3p designated as PS-OME miR130, we demonstrated attenuation of inflammation and injury severity in a murine model undergoing renal I/R. This attenuation was observed in the experimental analysis of the inflammatory cytokines and the kidney injury biomarkers both in serum concentrations and in tissue mRNA expression. Additionally, this effect correlated with significant reductions for treated mice in histologic findings in the short-term experiments and mortality observed in a 10-day survival study. These results suggest that PS-OME miR130 can be further developed as a therapeutic for attenuating acute kidney injury.