This work was partially supported by JSPS KAKENHI (Grant No. 21K08233). We thank Edanz (https://jp.edanz.com/ac) for editing drafts of this manuscript.

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-mimic13,14,15 and PEI-NPs-control-miRNA (as a negative control) are described for one mouse.
<ol>
	<li>Prepare the following items:
	<ol>
		<li>Prepare 10 &#181;L of linear PEI-NPs.</li>
		<li>Prepare 50 &#181;L of artificially synthesized miRNAs dissolved in nuclease-free water at a concentration of 100 &#181;M (5 nmol miRNA dissolved in 50 &#181;L of nuclease-free water).</li>
		<li>Prepare 50 &#181;L of artificially synthesized control (non-target) miRNAs dissolved in nuclease-free water at a concentration of 100 &#181;M (5 nmol miRNA dissolved in 50 &#181;L of nuclease-free water).</li>
		<li>Prepare 5% and 10% glucose solutions. Ensure the availability of 1.5 mL microcentrifuge tubes and a vortex mixer.</li>
	</ol>
	</li>
	<li>Dissolve 10 &#181;L of PEI-NPs in 90 &#181;L of 5% glucose solution in a 1.5 mL microcentrifuge tube. Then, vortex the tube gently and spin down.</li>
	<li>Mix 50 &#181;L of artificially synthesized miRNAs (5 nmol) dissolved in nuclease-free water (100 &#181;M concentration) with 50 &#181;M 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.</li>
	<li>Mix 100 &#181;L of PEI-NPs in 5% glucose solution and 100 &#181;L of miRNA mimic (5 nmol) in 5% glucose solution. Then, vortex the tube gently and spin down.</li>
	<li>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.</li>
</ol>
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 &#181;L 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.
<ol>
	<li>Prepare the following items:
	<ol>
		<li>Prepare 2 &#181;L of PEI-NPs and 100 &#181;L of Cy3-labeled double-strand oligonucleotides [Cy3-labeled miRNA mimic (1 nmol; 10 &#181;M)].</li>
		<li>Prepare 5% and 10% glucose solutions. Ensure the availability of 1.5 mL microcentrifuge tubes and a vortex mixer.</li>
		<li>Use a 50 mL plastic centrifuge tube with a small hole in the cap to isolate the tail veins of UUO mice.</li>
		<li>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&#39;,6-diamidino-2-phenylindole (DAPI) may be prepared for optional staining of proximal tubules and cell nuclei.</li>
	</ol>
	</li>
	<li>Dissolve 2 &#181;L of PEI-NPs in 98 &#181;L of 10% glucose solution in a 1.5 mL microcentrifuge tube. Then, vortex the tube gently and spin down.</li>
	<li>Add 100 &#181;L of Cy3-labeled miRNA mimic to 100 &#181;L of PEI-NPs in 10% glucose solution (prepared in step 2.2). Then, vortex the tube gently and spin down.</li>
	<li>Incubate the mixture for 15 min at RT to prepare a stable complex of PEI-NPs-Cy3-labeled-miRNA-mimic.</li>
	<li>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.</li>
	<li>Fill a 1.0 mL syringe with a 27 G needle with 200 &#181;L of PEI-NPs-Cy3-miRNA-mimic complex.</li>
	<li>Inject PEI-NPs-Cy3-miRNA-mimic (200 &#181;L) 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.</li>
	<li>One hour after 200 &#181;L 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.</li>
	<li>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 .</li>
	<li>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.</li>
	<li>Embed the kidney tissue samples in OCT compound and freeze in liquid nitrogen.</li>
	<li>Use a cryostat to prepare sections (5 &#181;m 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.</li>
	<li>Gently wash the slides twice with PBS.</li>
	<li>Visualize the fluorescence staining by fluorescence microscopy at 100x and 400x magnification and appropriate imaging software.</li>
</ol>
3. Confirmation of target miRNA alterations following delivery of miRNA mimic to kidney by PEI-NP via tail vein injection
<ol>
	<li>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-PCR18,19,20. 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&#39;s protocol.</li>
</ol>

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The authors declare that they have no conflicts of interest.

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 mixed13,14,15,16,17 (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 instability11,15,16.
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 miRNAs9,10. However, viral vectors have potential problems such as causing an interferon response and/or genetic instability11,12. Therefore, several alternative delivery methods using nonviral vectors, such as Gelatin-NPs21,22, electroporation23, hydrodynamic injection24,25, 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.

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 diseases1,2. Therefore, gene therapy employing exogenous artificially synthesized miRNA mimics or inhibitors is a potential new option for inhibiting the development of many kidney diseases3,4,5.
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 vectors7,8. 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 kidney9,10. Although they have been developed for biosafety and transfection efficacy, viral vectors may still cause an interferon response and/or genetic instability11,12. 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 disease13,14,15.
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 biocompatibility13,16,17.
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 -+Leprdb/+Leprdb) and acute kidney injury model mice produced by renal ischemia-reperfusion injury (IRI).

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

delivery of exogenous artificially synthesized mirna mimic to the kidney using polyethylenimine nanoparticles in several kidney disease mouse models

microRNAs&#160;(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.

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 publications13,14,15.
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).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63302/63302fig01.jpg" /> 
Figure 1: Structure of the PEI-NPs-miRNA-mimic complex. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63302/63302fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63302/63302fig02.jpg" /> 
Figure 2: Delivery and effects of PEI-NPs-miRNA-146a-5p-mimic in renal fibrosis mice. (A) Fluorescent microscopy analysis. Scale bar = 200 &#181;m. (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 &#181;m).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,&#160;unilateral ureter obstruction; OAS1, 5&#8242;-oligoadenylate synthase; STAT1, signal transduction and activator of transcription 1. Values represent mean &#177; standard error (error bars). *P &#60; 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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63302/63302fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63302/63302fig03.jpg" /> 
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 &#181;m. (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).&#160;(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 &#181;m. We used an analysis of variance (ANOVA) for multiple comparison analysis testing among&#160;groups. If we detected statistical significance by the ANOVA, we carried out Turkey&#39;s test to compare the means of two groups as a post hoc analysis.&#160;Abbreviations:&#160;PEI-NPs, polyethylenimine nanoparticles; qRT-PCR, quantitative real-time polymerase chain reaction; miRNA, microRNA. *P &#60; 0.05. This figure has been modified from Ishii&#160;et al.14. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63302/63302fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63302/63302fig04.jpg" /> 
Figure 4: Delivery and effects of PEI-NPs-miRNA-5100-mimic in acute kidney injury model mice produced by IRI. (A) Fluorescent microscopy analysis.&#160;Scale bar = 100 &#181;m. (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 &#956;m. 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 &#60; 0.01,*P &#60; 0.05. This figure has been modified from Aomatsu&#160;et al.15. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63302/63302fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Delivery of Exogenous Artificially Synthesized miRNA Mimic to the Kidney Using Polyethylenimine Nanoparticles in Several Kidney Disease Mouse Models at JoVE.c

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

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.

microRNAs&#160;(miRNAs), small noncoding RNAs (21-25 bases) that are not translated into proteins, inhibit lots of target messenger RNAs (mRNAs) by ...

delivery-exogenous-artificially-synthesized-mirna-mimic-to-kidney

Nanyang Technological University

Research

JoVE Journal

Medicine

1.5K Views.  Jichi Medical University. Deze methode kan helpen bij het beantwoorden van belangrijke vragen in de therapie van microRNA naar de nier, wat een uitdaging blijft. Het belangrijkste voordeel van deze techniek is dat het de therapie van microRNA-nabootsingen naar de nier mogelijk maakt zonder een interferonrespons of genetische instabiliteit te veroorzaken. Begin met het oplossen van 10 microliter PEI-NP's in 90 microliter 5% glucose-oplossing in een microcentrifugebuis van 1,5 milliliter.Draai vervolgens de buis ...

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

Quantitative Real-Time PCR Evaluation of microRNA Expressions in Mouse Kidney with Unilateral Ureteral Obstruction

MicroRNAs (miRNAs) are single stranded, non-coding RNA molecules that typically regulate gene expression at the post-transcriptional level by binding to partially complementary target sites in the 3&#39; untranslated region (UTR) of messenger RNA (mRNA), which reduces the mRNA&#39;s translation and stability. The miRNA expression profiles in various organs and tissues of mice have been investigated, but standard methods for the purification and quantification of miRNA in mouse kidney have not been available. We have established an effective and reliable method for extracting and evaluating miRNA expression in mouse kidney with renal interstitial fibrosis by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The protocol required five steps: (1) creation of sham and unilateral ureteral obstruction (UUO) mice; (2) extraction of kidney samples from the UUO mice; (3) extraction of total RNA, which includes miRNA, from the kidney samples; (4) complementary DNA (cDNA) synthesis with reverse transcription from miRNA; and (5) qRT-PCR using the cDNA. Using this protocol, we successfully confirmed that compared to the controls, the expression of miRNA-3070-3p was significantly increased and those of miRNA-7218-5p and miRNA-7219-5p were significantly decreased in the kidneys of a mouse model of renal interstitial fibrosis. This protocol can be used to determine the miRNA expression in the kidneys of mice with UUO.

We describe a method for evaluating the microRNA expression in the kidneys of mice with unilateral ureteral obstruction (UUO) by quantitative reverse-transcription polymerase chain reaction. This protocol is suitable for studying kidney microRNA expression profiles in mice with UUO and in the context of other pathological conditions.

MicroRNAs (miRNAs) are single stranded, non-coding RNA molecules that typically regulate gene expression at the post-transcriptional level by binding to ...

Genetics

Quantitative Real-Time Polymerase Chain Reaction Evaluation of MicroRNA Expression in Kidney and Serum of Mice with Age-Dependent Renal Impairment

MicroRNAs (miRNAs) are small, noncoding RNAs consisting of 21-25 bases. They are not translated into proteins but rather work to impede the functioning of their target messenger RNAs (mRNAs) by destabilizing them and disrupting their translation. Although the miRNA expression profiles in various mouse organs and tissues have been investigated, there have been no standard methods for purifying and quantifying mouse kidney and serum miRNAs. We have established an effective and reliable method for extracting and evaluating the miRNA expression in the serum and kidney of mice with age-dependent renal impairment. 
The method uses quantitative reverse-transcription-polymerase chain reaction (qRT-PCR), and the protocol requires six steps: (1) preparing senescence-accelerated mouse resistance 1 (SAMR1) mice and senescence-accelerated mouse prone (SAMP1) mice; (2) extracting serum samples from these mice; (3) extracting a kidney sample from each mouse; (4) extracting total RNA (including miRNA) from kidney and serum samples from each mouse; (5) the synthesis of complementary DNA (cDNA) with reverse transcription from the miRNA; (6) conducting a qRT-PCR using the cDNA obtained. 
This protocol was used to confirm that, compared to the controls, the expression of miRNA-7218-5p and miRNA-7219-5p was significantly changed in the kidney and serum of a mouse model of age-dependent renal impairment. This protocol also clarified the relationship between the kidney and serum of the mouse model of age-dependent renal impairment. This protocol can be used to determine miRNA expression in the kidney and serum of mice with age-dependent renal impairment.

We present a method for evaluating microRNA expression in the kidney and serum of mice with age-dependent renal impairment by quantitative reverse-transcription polymerase chain reaction.

MicroRNAs (miRNAs) are small, noncoding RNAs consisting of 21-25 bases. They are not translated into proteins but rather work to impede the functioning of ...

Nanodrug Targeting Oncogenic miRNA-10b for Treating Metastatic Breast Cancer

Monitoring of Nanodrug Accumulation in Murine Breast Cancer Metastases

Metastatic breast cancer is a devastating disease with very limited therapeutic options, calling for new therapeutic strategies. Oncogenic miRNAs have been shown to be associated with the metastatic potential of breast cancer and are implicated in tumor cell migration, invasion, and viability. However, it can be difficult to deliver an inhibitory RNA molecule to the tissue of interest. To overcome this challenge and deliver active antisense oligonucleotides to tumors, we utilized magnetic iron oxide nanoparticles as a delivery platform. These nanoparticles target tissues with increased vascular permeability, such as sites of inflammation or cancer. Delivery of these nanoparticles can be monitored in vivo by magnetic resonance imaging (MRI) due to their magnetic properties. Translation of this therapeutic approach into the clinic will be more accessible because of its compatibility with this relevant imaging modality. They can also be labeled with other imaging reporters such as a Cy5.5 near-infrared optical dye for correlative optical imaging and fluorescence microscopy. Here, we demonstrate that nanoparticles labeled with Cy5.5 and conjugated to therapeutic oligomers targeting oncogenic miRNA-10b (termed MN-anti-miR10b, or &#34;nanodrug&#34;) administered intravenously accumulate in metastatic sites, opening a possibility for therapeutic intervention of metastatic breast cancer.

Here, we describe the protocol for in vivo delivery of magnetic iron oxide nanoparticles carrying RNA oligomers to metastatic breast cancer in animal models, providing a clinically viable approach for the therapeutic silencing of oncogenic nucleic acids.

Metastatic breast cancer is a devastating disease with very limited therapeutic options, calling for new therapeutic strategies. Oncogenic miRNAs have ...

Cancer Research

Detection of MicroRNA Expression in the Kidneys of Immunoglobulin A Nephropathic Mice

Immunoglobulin A (IgA) nephropathy is a type of primary glomerulonephritis characterized by the abnormal deposition of IgA, leading to the end-stage renal failure. In recent years, the involvement of microRNAs (miRNAs) has been reported in the pathogenesis of IgA nephropathy. However, there is no established method for profiling miRNAs in IgA nephropathy using small animal models. Therefore, we developed a reliable method for analyzing miRNA in the kidney of an IgA mouse model (HIGA mouse). The goal of this protocol is to detect the altered expression levels of miRNAs in the kidneys of HIGA mice when compared with the levels in kidneys of control mice. In brief, this method consists of four steps: 1) obtaining kidney samples from HIGA mice; 2) purifying total RNA from kidney samples; 3) synthesizing complementary DNA from total RNA; and 4) quantitative reverse transcription polymerase chain reaction (qRT-PCR) of miRNAs. Using this method, we successfully detected the expression levels of several miRNAs (miR-155-5p, miR-146a-5p, and miR-21-5p) in the kidneys of HIGA mice. This new method can be applied to other studies profiling miRNAs in IgA nephropathy.

microRNAs are involved in the pathogenesis of IgA nephropathy. We have developed a reliable method for detecting microRNA expression levels in the kidneys of an IgA nephropathy mouse model (HIGA mice). This new method will facilitate to check for miRNAs involvement in IgA nephropathy.

Immunoglobulin A (IgA) nephropathy is a type of primary glomerulonephritis characterized by the abnormal deposition of IgA, leading to the end-stage renal ...

Immunology and Infection

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA probes. This protocol, originally developed by Kloosterman and colleagues for broad use with Exiqon miRNA probes1, has been modified to overcome challenges inherent in miRNA analysis in kidney tissues. These include issues such as structure identification and hard to remove residual probe and antibody. Use of relatively thin, 5 mm thick, tissue sections allowed for clear visualization of kidney structures, while a strong probe signal was retained in cells. Additionally, probe concentration and incubation conditions were optimized to facilitate visualization of microRNA expression with low background and nonspecific signal. Here, the optimized protocol is described, covering the initial tissue collection and preparation through the mounting of slides at the end of the procedure. The basic components of this protocol can be altered for application to other tissues and cell culture models.

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

This article describes an in situ hybridization protocol optimized for colormetric detection of microRNA expression in formalin fixed kidney sections.

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA ...

Biology

A Quantitative Detection Method for MicroRNAs in the Kidney of an Ischemic Kidney Injury Mouse Model

MicroRNAs (miRNAs) are involved in various disease states and are effective biomarkers for the early diagnosis of diseases and treatment in mice. However, standard protocols for the purification of miRNAs and detection of their expression in the kidneys of acute kidney injury (AKI) mice have not been well established. This study developed an effective and simple protocol to purify and quantify miRNAs in the kidneys of an AKI mouse model induced by renal ischemia-reperfusion using quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR). This protocol comprises five steps: 1) induction of AKI by renal ischemia-reperfusion, 2) harvesting of kidneys, 3) purification of total RNA, including miRNAs, from kidneys, 4) cDNA synthesis by reverse transcription of miRNA, and 5) qRT-PCR to detect miRNA expression. Using this protocol, the renal ischemia-reperfusion injury model can be generated with mild to severe forms of AKI. Additionally, if the procedure is followed properly, a consistent AKI model with minimal individual differences can be obtained. This qRT-PCR assay shows a very wide dynamic range and enables the discrimination of mature miRNAs, which can be accurately quantified with high specificity. This protocol can be used to study the miRNA expression profile in AKI kidneys.

This article presents a protocol for detecting microRNA expression in the kidneys of an acute kidney injury mouse model using quantitative real-time reverse-transcription polymerase chain reaction. This protocol emphasizes an ischemic kidney injury mouse model and the careful extraction of microRNA samples.

MicroRNAs (miRNAs) are involved in various disease states and are effective biomarkers for the early diagnosis of diseases and treatment in mice. However, ...

Direct Drug Delivery to Kidney via the Renal Artery

There is a need for directed injections to enable increased and specific renal exposure for efficient evaluation of drug targets in the renal research field. Accumulation of drugs in certain organs may give rise to adverse and unwanted effects, depending on the nature of injectate. To minimize spillover and/or accumulation in other tissues, the herein described method directs the formulation into the renal artery bloodstream by inserting a catheter in the infra renal aorta, just below where it branches into the renal artery, resulting in the kidney as first reached organ and distributing of formulation throughout the kidney.
This manuscript provides a detailed description of the method, as well as its challenges and difficulties. It guides the experimenter to become skillful with this type of microsurgery that requires accuracy under sterile conditions. Speed is crucial for minimizing the ischemia and practicing the procedure will increase the chance of successful injections without adverse effects. By modulating the time between injection and reperfusion as well as the injected volume, the risk of spillover to other organs is mitigated. 
Note that this technique is suitable for single dosing strategies.

This manuscript describes a method for targeted delivery to a single kidney via a catheter placed in the infrarenal abdominal aorta in the mouse.

There is a need for directed injections to enable increased and specific renal exposure for efficient evaluation of drug targets in the renal research ...

Hydrodynamic Renal Pelvis Injection for Non-viral Expression of Proteins in the Kidney

Hydrodynamic injection creates a local, high-pressure environment to transfect various tissues with plasmid DNA and other substances. Hydrodynamic tail vein injection, for example, is a well-established method by which the liver can be transfected. This manuscript describes an application of hydrodynamic principles by injection of the mouse kidney directly with plasmid DNA for kidney-specific gene expression. Mice are anesthetized and the kidney is exposed by a flank incision followed by a fast injection of a plasmid DNA-containing solution directly into the renal pelvis. The needle is kept in place for ten seconds and the incision site is sutured. The following day, live animal imaging, Western blot, or immunohistochemistry may be used to assay gene expression, or other assays suited to the transgene of choice are used for detection of the protein of interest. Published methods to prolong gene expression include transposon-mediated transgene integration and cyclophosphamide treatment to inhibit the immune response to the transgene.

This protocol describes a method to inject plasmid DNA into the mouse kidney via the renal pelvis to produce transgene expression specifically in the kidney.

Hydrodynamic injection creates a local, high-pressure environment to transfect various tissues with plasmid DNA and other substances. Hydrodynamic tail ...

In Vivo Nanovector Delivery of a Heart-specific MicroRNA-sponge

MicroRNA (miRNA) is small non-coding RNA which inhibits post-transcriptional messenger RNA (mRNA) expression. Human diseases, such as cancer and cardiovascular disease, have been shown to activate tissue and/or cell-specific miRNA expression associated with disease progression. The inhibition of miRNA expression offers the potential for a therapeutic intervention. However, traditional approaches to inhibit miRNAs, employing antagomir oligonucleotides, affect specific miRNA functions upon global delivery. Herein, we present a protocol for the in vivo cardio-specific inhibition of the miR-181 family in a rat model. A miRNA-sponge construct is designed to include 10 repeated anti-miR-181 binding sequences. The cardio-specific &#945;-MHC promoter is cloned into the pEGFP backbone to drive the cardio-specific miR-181 miRNA-sponge expression. To create a stable cell line expressing the miR-181-sponge, myoblast H9c2 cells are transfected with the &#945;-MHC-EGFP-miR-181-sponge construct and sorted by fluorescence-activated cell sorting (FACs) into GFP positive H9c2 cells which are cultured with neomycin (G418). Following stable growth in neomycin, monoclonal cell populations are established by additional FACs and single cell cloning. The resulting myoblast H9c2-miR-181-sponge-GFP cells exhibit a loss of function of miR-181 family members as assessed through the increased expression of miR-181 target proteins and compared to H9c2 cells expressing a scramble non-functional sponge. In addition, we develop a nanovector for the systemic delivery of the miR-181-sponge construct by complexing positively charged liposomal nanoparticles and negatively charged miR-181-sponge plasmids. In vivo imaging of GFP reveals that multiple tail vein injections of a nanovector over a three-week period are able to promote a significant expression of the miR-181-sponge in a cardio-specific manner. Importantly, a loss of miR-181 function is observed in the heart tissue but not in the kidney or the liver. The miRNA-sponge is a powerful method to inhibit tissue-specific miRNA expression. Driving the miRNA-sponge expression from a tissue-specific promoter provides specificity for the miRNA inhibition, which can be confined to a targeted organ or tissue. Furthermore, combining nanovector and miRNA-sponge technologies permits an effective delivery and tissue-specific miRNA inhibition in vivo.

In Vivo Nanovector Delivery of a Heart-specific MicroRNA-sponge

Tissue-specific microRNA inhibition is a technology that is underdeveloped in the microRNA field. Herein, we describe a protocol to successfully inhibit the miR-181 microRNA family in myoblast cells from the heart. Nanovector technology is used to deliver a microRNA sponge that demonstrates significant in vivo cardio-specific miR-181 family inhibition.

MicroRNA (miRNA) is small non-coding RNA which inhibits post-transcriptional messenger RNA (mRNA) expression. Human diseases, such as cancer and ...

Modelling Intratumoral Treatment with miRNA Inhibitors: A Method to Analyze the Effect of miRNA Inhibitors on Tumor Growth in Murine Model

Source: Ramirez-Moya, J. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/59322" target="_blank">In Vivo Inhibition of MicroRNA to Decrease Tumor Growth in Mice.</a> J. Vis. Exp. (2019)
This video demonstrates the in vivo delivery of miRNA inhibitors as therapeutic agents against tumor growth in orthotopic mouse models. The intratumoral administration of synthetic miRNA inhibitors or antagomiRs allows them to bind and inhibit endogenous miRNAs, thereby causing mRNA upregulation and suppression of tumor growth.

Source: Ramirez-Moya, J. et al. In Vivo Inhibition of MicroRNA to Decrease Tumor Growth in Mice. J. Vis. Exp. (2019)
This video demonstrates the in vivo ...

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

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