Delivery of Exogenous Artificially Synthesized miRNA Mimic to the Kidney Using Polyethylenimine Nanoparticles in Several Kidney Disease Mouse Models

Summary

Here, we deliver exogenous artificially synthesized miRNA mimics to the kidney via tail vein injection of a nonviral vector and polyethylenimine nanoparticles in several kidney disease mouse models. This led to significant overexpression of target miRNA in the kidney, resulting in inhibited progression of kidney disease in several mouse models.

Abstract

microRNAs (miRNAs), small noncoding RNAs (21-25 bases) that are not translated into proteins, inhibit lots of target messenger RNAs (mRNAs) by destabilizing and inhibiting their translation in various kidney diseases. Therefore, alternation of miRNA expression by exogenous artificially synthesized miRNA mimics is a potentially useful treatment option for inhibiting the development of many kidney diseases. However, because serum RNAase immediately degrades systematically administered exogenous miRNA mimics in vivo, delivery of miRNA to the kidney remains a challenge. Therefore, vectors that can protect exogenous miRNA mimics from degradation by RNAase and significantly deliver them to the kidney are necessary. Many studies have used viral vectors to deliver exogenous miRNA mimics or inhibitors to the kidney. However, viral vectors may cause an interferon response and/or genetic instability. Therefore, the development of viral vectors is also a hurdle for the clinical use of exogenous miRNA mimics or inhibitors. To overcome these concerns regarding viral vectors, we developed a nonviral vector method to deliver miRNA mimics to the kidney using tail vein injection of polyethylenimine nanoparticles (PEI-NPs), which led to significant overexpression of target miRNAs in several mouse models of kidney disease.

Introduction

miRNAs, small noncoding RNAs (21-25 bases) that are not translated into proteins, inhibit lots of target messenger RNAs (mRNAs) by destabilizing them and inhibiting their translation in various kidney diseases^1,2. Therefore, gene therapy employing exogenous artificially synthesized miRNA mimics or inhibitors is a potential new option for inhibiting the development of many kidney diseases^3,4,5.

Despite the promise of miRNA mimics or inhibitors for gene therapy, delivery to target organs remains a big hurdle for in vivo experiments to develop their clinical potential. Because artificially synthesized miRNA mimics or inhibitors are subject to immediate degradation by serum RNase, their half-life is shortened upon systemic administration in vivo⁶. Additionally, the efficiency of miRNA mimics or inhibitors to cross the plasma membrane and transfect cytoplasm is generally much lower without appropriate vectors^7,8. These lines of evidence suggest that the development of the miRNA mimics or inhibitors delivery system for the kidney is required, to enable their use in clinical settings and make them a new treatment option for patients with various kidney diseases.

Viral vectors have been used as carriers to deliver exogenous miRNA mimics or inhibitors to the kidney^9,10. Although they have been developed for biosafety and transfection efficacy, viral vectors may still cause an interferon response and/or genetic instability^11,12. To overcome these concerns, we developed an miRNA mimics delivery system for the kidney using polyethylenimine nanoparticles (PEI-NPs), a nonviral vector, in several mouse models of kidney disease^13,14,15.

PEI-NPs are linear polymer-based NPs that can effectively deliver oligonucleotides, including miRNA mimics, to the kidney, and are considered preferable for preparing nonviral vectors because of their long-term safety and biocompatibility^13,16,17.

This study demonstrates the effects of systematic exogenous miRNA mimics delivery with PEI-NPs via tail vein injection in renal fibrosis model mice produced by unilateral ureter obstruction (UUO). Additionally, we demonstrate the effects of systematic exogenous miRNA mimic delivery with PEI-NPs via tail vein injection in diabetic kidney disease model mice (db/db mice: C57BLKS/J Iar -+Lepr^db/+Lepr^db) and acute kidney injury model mice produced by renal ischemia-reperfusion injury (IRI).

Protocol

All animal experimental protocols were approved by the animal ethics committee of Jichi Medical University and performed in accordance with Use and Care of Experimental Animals guidelines from the Jichi Medical University Guide for Laboratory Animals. Here, we demonstrated miRNA mimic delivery to the kidney resulting in its overexpression using UUO mice. This study was approved by the Ethics Committee of Jichi Medical University [Approval Nos. 19-12 for renal fibrosis, 17-024 for acute kidney infection (AKI), and 19-11 for diabetic nephropathy].

1. Preparation of PEI-NPs-miRNA-mimic complex

NOTE: Here, preparation of PEI-NPs-miRNA-mimic^13,14,15 and PEI-NPs-control-miRNA (as a negative control) are described for one mouse.

1. Prepare the following items:
   1. Prepare 10 µL of linear PEI-NPs.
   2. Prepare 50 µL of artificially synthesized miRNAs dissolved in nuclease-free water at a concentration of 100 µM (5 nmol miRNA dissolved in 50 µL of nuclease-free water).
   3. Prepare 50 µL of artificially synthesized control (non-target) miRNAs dissolved in nuclease-free water at a concentration of 100 µM (5 nmol miRNA dissolved in 50 µL of nuclease-free water).
   4. Prepare 5% and 10% glucose solutions. Ensure the availability of 1.5 mL microcentrifuge tubes and a vortex mixer.
2. Dissolve 10 µL of PEI-NPs in 90 µL of 5% glucose solution in a 1.5 mL microcentrifuge tube. Then, vortex the tube gently and spin down.
3. Mix 50 µL of artificially synthesized miRNAs (5 nmol) dissolved in nuclease-free water (100 µM concentration) with 50 µM of 10% glucose solution in 1.5 mL microcentrifuge tubes. Then, vortex the tube gently and spin down. This process yields miRNA dissolved in 5% glucose solution.
4. Mix 100 µL of PEI-NPs in 5% glucose solution and 100 µL of miRNA mimic (5 nmol) in 5% glucose solution. Then, vortex the tube gently and spin down.
5. Incubate the mixture for 15 min at room temperature (RT) to prepare a stable complex of PEI-NPs-miRNA-mimic. This solution contains PEI-NPs and miRNA mimic (5 nmol) at a ratio of nitrogens (N) in the polymer to phosphate (P) in nucleic acids (N/P ratio) = 6 (Figure 1).
   NOTE: PEI-NPs-control-miRNA complex can be prepared using the same process described in steps 1.1-1.5 for all kidney disease mice models described in this manuscript (UUO, diabetic nephropathy, and AKI). One injection volume of PEI-NP-miRNAs-mimic is the same as 5 nmol of miRNAs-mimic conjugated with PEI-NPs at N/P ratio = 6. The injection duration and frequency of each PEI-NP-miRNAs-mimic depends on the disease model in which it is applied.

2. Confirmation of significant delivery of miRNA mimic to the kidney in UUO mice by PEI-NP via tail vein injection using fluorescent microscopy

NOTE: Here, the delivery method of artificially purified miRNA mimic to the kidney is elaborated, indicating the establishment of therapeutic methods for various renal diseases. In brief, UUO mice were administered cyanine3 carboxylic acid (Cy3)-labeled miRNA mimic added to 100 µL of PEI-NPs via the tail vein. The delivery to the kidneys was confirmed with fluorescence microscopy. PEI-NPs-cyanine3 carboxylic acid (Cy3)-labeled miRNA mimic (oligonucleotides) for one mouse is described. This method can significantly deliver miRNA mimic to the kidney in several mouse models, such as UUO mice, diabetic kidney disease mice, and AKI mice produced by IRI. Here, we used a UUO mouse model for the video demonstration. The induction method of UUO was described previously elsewhere¹³. Follow these protocols within six days after UUO surgery.

1. Prepare the following items:
   1. Prepare 2 µL of PEI-NPs and 100 µL of Cy3-labeled double-strand oligonucleotides [Cy3-labeled miRNA mimic (1 nmol; 10 µM)].
   2. Prepare 5% and 10% glucose solutions. Ensure the availability of 1.5 mL microcentrifuge tubes and a vortex mixer.
   3. Use a 50 mL plastic centrifuge tube with a small hole in the cap to isolate the tail veins of UUO mice.
   4. For fluorescence microscopy, prepare optimal cutting temperature (OCT) compound, a cryostat, liquid nitrogen, phosphate-buffered saline (PBS), 1.0 mL syringes with a 27 G needle, and silane-coated glass.
      NOTE: Fluorescein-labeled Lotus tetragonolobus lectin (a proximal tubule marker) and 4',6-diamidino-2-phenylindole (DAPI) may be prepared for optional staining of proximal tubules and cell nuclei.
2. Dissolve 2 µL of PEI-NPs in 98 µL of 10% glucose solution in a 1.5 mL microcentrifuge tube. Then, vortex the tube gently and spin down.
3. Add 100 µL of Cy3-labeled miRNA mimic to 100 µL of PEI-NPs in 10% glucose solution (prepared in step 2.2). Then, vortex the tube gently and spin down.
4. Incubate the mixture for 15 min at RT to prepare a stable complex of PEI-NPs-Cy3-labeled-miRNA-mimic.
5. Place the UUO mouse headfirst in a 50 mL centrifuge tube without anesthesia, and then place the tail through the prepared hole in the cap.
6. Fill a 1.0 mL syringe with a 27 G needle with 200 µL of PEI-NPs-Cy3-miRNA-mimic complex.
7. Inject PEI-NPs-Cy3-miRNA-mimic (200 µL) via the tail vein of the mouse using the 1.0 mL syringe with 27 G needle.
   NOTE: Wipe the tail vein with cotton wool saturated in ethanol (70%-80%) to dilate the tail vein and disinfect the injection area.
8. One hour after 200 µL of PEI-NPs-Cy3-miRNA-mimic complex injection, anesthetize the mouse with isoflurane (4%-5%) using an appropriate anesthetic device for small animals. Confirm the depth of anesthesia with an absence of toe pinch reflex. Maintain the anesthesia with isoflurane (3%) throughout the surgery.
9. Next, make an incision into the skin, muscles, and ribs with surgical scissors and forceps to expose the heart. After making an incision into the right atrium, inject PBS into the left ventricle to draw out blood throughout the body until the kidney color changes to pale yellow (indicating that the whole mouse body is perfused with PBS).
   NOTE: Cardiac perfusion is performed to efficiently remove the blood throughout the body .
10. Stop isoflurane and remove the kidneys from the mice and wash them with PBS. After that, remove renal capsules from the kidneys and wash the kidneys twice with PBS.
11. Embed the kidney tissue samples in OCT compound and freeze in liquid nitrogen.
12. Use a cryostat to prepare sections (5 µm thick). Mount the sections onto silane-coated glass slides and fix them with 4% paraformaldehyde.
    NOTE: Kidney tissues can optionally be stained with fluorescein-labeled Lotus tetragonolobus lectin (2 mg/mL) at RT for 2 h for proximal tubules, followed by DAPI (30 nM in PBS) at RT for 15 min for cell nuclei.
13. Gently wash the slides twice with PBS.
14. Visualize the fluorescence staining by fluorescence microscopy at 100x and 400x magnification and appropriate imaging software.

3. Confirmation of target miRNA alterations following delivery of miRNA mimic to kidney by PEI-NP via tail vein injection

1. Perform quantitative real-time polymerase chain reaction (qRT-PCR) to confirm the target miRNA alternations.
   NOTE: Refer to the previously published article for the details of qRT-PCR^18,19,20. The qRT-PCR system used to generate the representative results provided below was changed by the manufacturer. qRT-PCR should be conducted in accordance with the latest manufacturer's protocol.

Representative Results

The target miRNAs for renal fibrosis, diabetic nephropathy, and AKI described below were selected based on the microarray, qRT-PCR, and/or database research for gene therapy applications. For further details, refer to the previous publications^13,14,15.

Delivery and effects of miRNA-146a-5p-mimic using PEI-NPs in renal fibrosis mice¹³
Fluorescent microscopy analysis showed that tail vein injection of PEI-NPs could deliver the miRNA mimic to the tubulointerstitial space. In renal fibrosis mice produced by UUO, this included renal tubular cells, which do not have a regular form in the kidney (Figure 2A arrow), and the glomerulus (Figure 2A). qRT-PCR analysis showed that injection of PEI-NPs-miRNA-146a-5p-mimic induced significant overexpression of miRNA-146a-5p in the kidney (Figure 2B). Moreover, injection inhibited the progression of renal fibrosis in the model mice produced by UUO, as estimated with Sirius red staining (red indicates fibrotic areas) in vivo. Injection of PEI-NPs-miRNA-146a-5p-mimic was conducted 1 day before, 1 day after, and 3 days after UUO treatment (estimated 6 days after UUO treatment) (Figure 2C). Injection of PEI-NPs-control miRNA did not show preventative effects on renal fibrosis (Figure 2B,C). Administration of PEI-NPs-miRNA-146a-5p-mimic and PEI-NPs-miRNA-control-miRNAs did not cause an IFN response, such as increased expression of 5-oligoadenylate synthase and signal transduction and activator of transcription 1 in mice (Figure 2D).

Delivery and effects of miRNA-181b-5p-mimic using PEI-NPs in diabetic kidney disease model mice¹⁴
To assess the therapeutic potential of miRNA-181b-5p for diabetic kidney disease, we injected miRNA-181b-5p-PIE-NPs or control miRNA-PIE-NPs into 10-week-old db/db mice weekly for 10 weeks. Fluorescent microscopy analysis showed that tail vein injection of PEI-NPs could deliver miRNA mimic to the glomerulus and tubulointerstitial space in kidneys of diabetic kidney model (db/db) mice in vivo (Figure 3A). In addition, the qRT-PCR analysis showed that injection of PEI-NPs-miRNA-181b-5p-mimic induced significant overexpression of miRNA-181b-5p in the kidney (Figure 3B). Histological analysis with Periodic acid-Schiff staining showed that injection of PEI-NPs-miRNA-181b-5p-mimic once per week from 10-20 weeks of age inhibited the progression of diabetic kidney disease in db/db mice in vivo (Figure 3C). In contrast, injection of PEI-NPs-control miRNA did not yield preventative effects on renal fibrosis (Figure 3B,C).

Delivery and effects of PEI-NPs-miRNA-5100-mimic in acute kidney injury model mice produced by IRI¹⁵
Fluorescent microscopy analysis showed that the tail vein injection of PEI-NPs could deliver miRNA mimic to the glomerulus and tubulointerstitial space of AKI model mice kidneys in vivo (Figure 4A). qRT-PCR analysis showed that the injection of PEI-NPs-miRNA-5100-5p-mimic induced significant overexpression of miRNA-5100 in kidneys of AKI mice produced by IRI (Figure 3B). Histological analysis with hematoxylin-eosin staining showed that a single injection of PEI-NPs-miRNA-5100 reduced tubular cell apoptosis (black arrow), which prevented AKI development in the AKI mice model produced by IRI. PEI-NPs-miRNA-5100-mimic was injected via the tail vein 2 days before the IRI procedure (Figure 4B). Injection of PEI-NPs-control miRNA-did not show preventative effects on renal fibrosis (Figure 4B,C).

Figure 1: Structure of the PEI-NPs-miRNA-mimic complex. Please click here to view a larger version of this figure.

Figure 2: Delivery and effects of PEI-NPs-miRNA-146a-5p-mimic in renal fibrosis mice. (A) Fluorescent microscopy analysis. Scale bar = 200 µm. (B) qRT-PCR analysis of miRNA-146a-5p expression levels in each group (n = 6 per group). (C) Histological analysis with Sirius red staining (red indicates the fibrotic area, scale bar = 100 µm).Injection of PEI-NPs-miRNA-146a-5p-mimic 1 day before, 1 day after, and 3 days after UUO treatment inhibited the development of renal fibrosis in mice in vivo. (D) qRT-PCR analysis of kidney to investigate potential IFN response effects to PEI-NPs-miRNA-146a-5p in UUO mice (n = 6 per group). Abbreviations: DAPI, 4,6-diamidino-2-phenylindole; FITC, fluorescein isothiocyanate; qRT-PCR, quantitative real-time polymerase chain reaction; NS, not significant; PEI-NPs, polyethylenimine nanoparticles; miRNA, microRNA; UUO, unilateral ureter obstruction; OAS1, 5′-oligoadenylate synthase; STAT1, signal transduction and activator of transcription 1. Values represent mean ± standard error (error bars). *P < 0.05. Morishita et al. (International Journal of Nanomedicine 2015 10 3475-3488. Originally published by and used with permission from Dove Medical Press Ltd).¹³. Please click here to view a larger version of this figure.

Figure 3: Delivery and effects of PEI-NPs-miRNA-181b-5p-mimic in diabetic kidney disease model mice¹⁴. (A) Fluorescent microscopy analysis. Scale bar = 200 µm. (B) qRT-PCR analysis to evaluate overexpression effects of miRNA-181b-5p following injection of PEI-NPs-miRNA-181b-5p-mimic (n = 3 per group). (C) Phenotypical changes were observed by light microscopy. Injection of PEI-NPs-miRNA-181b-5p-mimic once per week from 12-20 weeks of age inhibited the progression of diabetic kidney disease in db/db mice in vivo. Scale bar = 50 µm. We used an analysis of variance (ANOVA) for multiple comparison analysis testing among groups. If we detected statistical significance by the ANOVA, we carried out Turkey's test to compare the means of two groups as a post hoc analysis. Abbreviations: PEI-NPs, polyethylenimine nanoparticles; qRT-PCR, quantitative real-time polymerase chain reaction; miRNA, microRNA. *P < 0.05. This figure has been modified from Ishii et al.¹⁴. Please click here to view a larger version of this figure.

Figure 4: Delivery and effects of PEI-NPs-miRNA-5100-mimic in acute kidney injury model mice produced by IRI. (A) Fluorescent microscopy analysis. Scale bar = 100 µm. (B) qRT-PCR analysis to evaluate the overexpression effects of miRNA-5100 following injection of PEI-NPs-miRNA-5100-mimic. (C) Histological analysis of hematoxylin-eosin stained kidney. Scale bar = 100 μm. PEI-NPs injected via the tail vein 2 days before the IRI procedure prevent AKI progression induced by IRI. Abbreviations: PEI-NPs, polyethylenimine nanoparticles; qRT-PCR, quantitative real-time polymerase chain reaction; miRNA, microRNA; AKI, acute kidney injury; IRI, ischemia-reperfusion injury. **P < 0.01,*P < 0.05. This figure has been modified from Aomatsu et al.¹⁵. Please click here to view a larger version of this figure.

Discussion

Using the protocol presented in this manuscript, PEI-NPs can deliver miRNA mimics to the kidney to induce overexpression of target miRNAs, resulting in treatment effects in in vivo mouse models of several renal diseases, including renal fibrosis, diabetic kidney disease, and AKI.

The method to prepare the complex of PEI-NPs and miRNA mimic is very simple. The positively charged surface of PEI-NPs entraps the miRNA mimic when they are just mixed^{13,14,15,16,17} (Figure 1). Additionally, as a linear polyethylenimine-based nonviral vector, PEI-NPs avoid issues associated with the use of viral vectors, such as IFN response and oncogenesis induced by genetic instability^11,15,16.

Injection of PEI-NPs-mimic via tail vein may require training since the tail veins of mice with kidney disease are sometimes difficult to puncture because of dehydration, especially in AKI and obese db/db mice. Additionally, repeated injection of PEI-NPs-miRNA-mimic may be required for several mice models. Injection intervals should be determined by pre-experience of how overexpression by PEI-NPs-miRNA-mimic occurs in the context of each kidney disease mouse model.

Viral vectors are frequently used for transfection of miRNA mimic in kidneys and can lead to significant transfection of miRNAs^9,10. However, viral vectors have potential problems such as causing an interferon response and/or genetic instability^11,12. Therefore, several alternative delivery methods using nonviral vectors, such as Gelatin-NPs^21,22, electroporation²³, hydrodynamic injection^24,25, and transfection using ultrasound²⁶, have been developed. In this manuscript, we introduced PEI-NPs as a nonviral vector for the kidney. Some nonviral vectors are highly invasive (electroporation and hydrodynamic injection), and therefore may be difficult to apply for clinical use. However, a comparison of PEI-NPs with other nonviral vectors, such as lipid-based nanoparticles and Gelatin-NPs, should be evaluated in further studies.

This protocol has the following limitations. First, although PEI-NPs-miRNAs are suitable for mouse models (at least as a first stage), large amounts of PEI-NPs-miRNAs are required for large animal models (such as rats, rabbits, monkeys, and dogs) used for preclinical research. Additionally, it should be noted that PEI-NPs did not deliver miRNA mimic specifically to the kidney because PEI-NPs, like viral vectors, are not a targeted delivery system. Therefore, the phenotype of other organs may need to be investigated.

Materials

Name	Company	Catalog Number	Comments
4’,6-diamidino-2-phenylindole for staining to nucleus	Thermo Fisher Scientific	D-1306
Buffer RPE	Qiagen	79216	Wash buffer 2
Buffer RWT	Qiagen	1067933	Wash buffer 1
Control-miRNA-mimic (artificially synthesized miRNA)	Thermo Fisher Scientific	Not assigned	5’-UUCUCCGAACGUGUCACGUTT- 3’ (sense) 5’-ACGUGACACGUUCGGAGAATT-3′ (antisense)
Cy3-labeled double-strand oligonucleotides	Takara Bio Inc.	MIR7900
Fluorescein-labeled Lotus tetragonolobus lectin	Vector Laboratories Inc	FL-1321
In vivo-jetPEI	Polyplus	101000021
MicroAmp Optical 96-well reaction plate for qRT-PCR	Thermo Fisher Scientific	4316813	96-well reaction plate
MicroAmp Optical Adhesive Film	Thermo Fisher Scientific	4311971	Adhesive film for 96-well reaction plate
miRNA-146a-5p mimic (artificially synthesized miRNA)	Thermo Fisher Scientific	Not assigned	5’-UGAGAACUGAAUUCCAUGGGU UT-3′ (sense) 5’-CCCAUGGAAUUCAGUUCUCAUU -3′ (antisense)
miRNA-146a-5p primer	Qiagen	MS00001638	Not available because Qiagen has changed qRT-PCR kits (from miScript miRNA PCR system to miRCURY LNA miRNA PCR System from May 2021)
miRNA-181b-5p mimic (artificially synthesized miRNA)	Gene design	Not assigned	5’-AACAUUCAUUGCUGUCGGUGG GUU-3’
miRNA-181b-5p primer	Qiagen	MS00006083	Not available because Qiagen has changed qRT-PCR kits (from miScript miRNA PCR system to miRCURY LNA miRNA PCR System from May 2021)
miRNA-5100-mimic (artificially synthesized miRNA)	Gene design	Not assigned	5’-UCGAAUCCCAGCGGUGCCUCU -3′
miRNA-5100-primer	Qiagen	MS00042952	Not available because Qiagen has changed qRT-PCR kits (from miScript miRNA PCR system to miRCURY LNA miRNA PCR System from May 2021)
miRNeasy Mini kit	Qiagen	217004	Membrane anchored spin column in a 2.0-mL collection tube
miScript II RT kit	Qiagen	218161	Not available because Qiagen has changed qRT-PCR kits (from miScript miRNA PCR system to miRCURY LNA miRNA PCR System from May 2021)
miScript SYBR Green PCR kit	Qiagen	218073	Not available because Qiagen has changed qRT-PCR kits (from miScript miRNA PCR system to miRCURY LNA miRNA PCR System from May 2021)
QIA shredder	Qiagen	79654	Biopolymer spin columns in a 2.0-mL collection tube
QIAzol Lysis Reagent	Qiagen	79306	Phenol/guanidine-based lysis reagent
QuantStudio 12K Flex Flex Real-Time PCR system	Thermo Fisher Scientific	4472380	Real-time PCR instrument
QuantStudio 12K Flex Software version 1.2.1.	Thermo Fisher Scientific	4472380	Real-time PCR instrument software
RNase-free water	Qiagen	129112
RNU6-2 primer	Qiagen	MS00033740	Not available because Qiagen has changed qRT-PCR kits (from miScript miRNA PCR system to miRCURY LNA miRNA PCR System from May 2021)
Tissue-Tek OCT (Optimal Cutting Temperature Compound)	Sakura Finetek Japan Co.,Ltd.	Not assigned