Name: delivery of exogenous artificially synthesized mirna mimic to the kidney using polyethylenimine nanoparticles in several kidney disease mouse models
Uploaded: 2022-05-10T14:00:00
Description: 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

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 f...

<ol>
	<li>Mohr, A. M., Mott, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+microRNA+biology.">Overview of microRNA biology.</a> Seminars in Liver Disease. 35 (1), 3-11 (2015).</li><li>Bushati, N., Cohen, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=microRNA+functions.">microRNA functions.</a> Annual Review of Cell and Developmental Biology. 23, 175-205 (2007).</li><li>Simpson, K., Wonnacott, A., Fraser, D. J., Bowen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=microRNAs+in+diabetic+nephropathy:+From+biomarkers+to+therapy.">microRNAs in diabetic nephropathy: From biomarkers to therapy.</a> Current Diabetes Reports. 16 (3), 35 (2016).</li><li>Yheskel, M., Patel, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+microRNAs+in+polycystic+kidney+disease.">Therapeutic microRNAs in polycystic kidney disease.</a> Current Opinion in Nephrology and Hypertension. 26 (4), 282-289 (2017).</li><li>Lv, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+potential+of+microRNAs+for+the+treatment+of+renal+fibrosis+and+CKD.">Therapeutic potential of microRNAs for the treatment of renal fibrosis and CKD.</a> Physiological Genomics. 50 (1), 20-34 (2018).</li><li>Dykxhoorn, D. 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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+risks+related+to+the+use+of+adeno-associated+virus-based+vectors.">Evaluation of risks related to the use of adeno-associated virus-based vectors.</a> Current Gene Therapy. 3 (6), 545-565 (2003).</li><li>Lukashev, A. N., Zamyatnin, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+vectors+for+gene+therapy:+Current+state+and+clinical+perspectives.">Viral vectors for gene therapy: Current state and clinical perspectives.</a> Biochemistry. Biokhimiia. 81 (7), 700-708 (2016).</li><li>Morishita, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delivery+of+microRNA-146a+with+polyethylenimine+nanoparticles+inhibits+renal+fibrosis+in+vivo.">Delivery of microRNA-146a with polyethylenimine nanoparticles inhibits renal fibrosis in vivo.</a> International Journal of Nanomedicine. 10, 3475-3488 (2015).</li><li>Ishii, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+expression+profiling+in+diabetic+kidney+disease.">MicroRNA expression profiling in diabetic kidney disease.</a> Translational Research: The Journal of Laboratory and Clinical. 237, 31-52 (2021).</li><li>Aomatsu, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+expression+profiling+in+acute+kidney+injury.">MicroRNA expression profiling in acute kidney injury.</a> Translational Research: The Journal of Laboratory and Clinical. (21), 00283-00288 (2021).</li><li>Lungwitz, U., Breunig, M., Blunk, T., Gopferich, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyethylenimine-based+non-viral+gene+delivery+systems.">Polyethylenimine-based non-viral gene delivery systems.</a> European Journal of Pharmceutics and Biopharmaceutics. 60 (2), 247-266 (2005).</li><li>Swami, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+unique+and+highly+efficient+nonviral+DNA/siRNA+delivery+system+based+on+PEI-bisepoxide+nanoparticles.">A unique and highly efficient nonviral DNA/siRNA delivery system based on PEI-bisepoxide nanoparticles.</a> Biochemical and Biophysical Research Communications. 362 (4), 835-841 (2007).</li><li>Kaneko, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+microRNA+expression+in+the+kidneys+of+immunoglobulin+a+nephropathic+mice.">Detection of microRNA expression in the kidneys of immunoglobulin a nephropathic mice.</a> Journal of Visualized Experiments: JoVE. (161), e61535 (2020).</li><li>Yanai, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+real-time+PCR+evaluation+of+microRNA+expressions+in+mouse+kidney+with+unilateral+ureteral+obstruction.">Quantitative real-time PCR evaluation of microRNA expressions in mouse kidney with unilateral ureteral obstruction.</a> Journal of Visualized Experiments: JoVE. (162), e61383 (2020).</li><li>Aomatsu, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+quantitative+detection+method+for+microRNAs+in+the+kidney+of+an+ischemic+kidney+injury+mouse+model.">A quantitative detection method for microRNAs in the kidney of an ischemic kidney injury mouse model.</a> Journal of Visualized Experiments: JoVE. (163), e61378 (2020).</li><li>Kushibiki, T., Nagata-Nakajima, N., Sugai, M., Shimizu, A., Tabata, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+anti-fibrotic+activity+of+plasmid+DNA+expressing+small+interference+RNA+for+TGF-beta+type+II+receptor+for+a+mouse+model+of+obstructive+nephropathy+by+cationized+gelatin+prepared+from+different+amine+compounds.">Enhanced anti-fibrotic activity of plasmid DNA expressing small interference RNA for TGF-beta type II receptor for a mouse model of obstructive nephropathy by cationized gelatin prepared from different amine compounds.</a> Journal of Controlled Release: Official Journal of the Controlled Release Society. 110 (3), 610-617 (2006).</li><li>Xia, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppression+of+renal+tubulointerstitial+fibrosis+by+small+interfering+RNA+targeting+heat+shock+protein+47.">Suppression of renal tubulointerstitial fibrosis by small interfering RNA targeting heat shock protein 47.</a> American Journal of Nephrology. 28 (1), 34-46 (2008).</li><li>Tanaka, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+gene+transfer+of+hepatocyte+growth+factor+to+skeletal+muscle+prevents+changes+in+rat+kidneys+after+5/6+nephrectomy.">In vivo gene transfer of hepatocyte growth factor to skeletal muscle prevents changes in rat kidneys after 5/6 nephrectomy.</a> American Journal of Transplantation: Official Journal of the American Society of Transplantation and the American Society of Transplant Surgeons. 2 (9), 828-836 (2002).</li><li>Hamar, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+interfering+RNA+targeting+Fas+protects+mice+against+renal+ischemia-reperfusion+injury.">Small interfering RNA targeting Fas protects mice against renal ischemia-reperfusion injury.</a> Proceedings of the National Academy of Sciences of the United States of America. 101 (41), 14883-14888 (2004).</li><li>Ma, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenon+preconditioning+protects+against+renal+ischemic-reperfusion+injury+via+HIF-1alpha+activation.">Xenon preconditioning protects against renal ischemic-reperfusion injury via HIF-1alpha activation.</a> Journal of the American Society of Nephrology: JASN. 20 (4), 713-720 (2009).</li><li>Wei, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short+hairpin+RNA+knockdown+of+connective+tissue+growth+factor+by+ultrasound-targeted+microbubble+destruction+improves+renal+fibrosis.">Short hairpin RNA knockdown of connective tissue growth factor by ultrasound-targeted microbubble destruction improves renal fibrosis.</a> Ultrasound in Medicine and Biology. 42 (12), 2926-2937 (2016).</li></ol>

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,<su...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>4&#8217;,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&#8217;-UUCUCCGAACGUGUCACGUTT- 3&#8217; (sense) 
			5&#8217;-ACGUGACACGUUCGGAGAATT-3&#8242; (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&#8217;-UGAGAACUGAAUUCCAUGGGU 
			UT-3&#8242; (sense) 5&#8217;-CCCAUGGAAUUCAGUUCUCAUU -3&#8242; (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&#8217;-AACAUUCAUUGCUGUCGGUGG 
			GUU-3&#8217;</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&#8217;-UCGAAUCCCAGCGGUGCCUCU -3&#8242;</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 
Fluo...

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 ...

This method can help answer key questions in therapy of microRNA to the kidney, which remains a challenge. The main advantage of this technique is that it's enabled the therapy of microRNA mimics to the kidney without causing an interferon response or genetic instability. Begin by dissolving 10 microliters of PEI-NPs in 90 microliters of 5%glucose solution in a 1.5-milliliter microcentrifuge tube.Then vortex the tube gently and spin down. Mix 50 microliters of artificially synthesized miRNAs dissolved in 100 micromolar of nuclease-free water with 50 micromolar of 10%glucose solution in 1.5-milliliter microcentrifuge tubes. Vortex the tube gently and spin down.This process yields miRNA dissolved in 5%glucose solution. Then mix 100 microliters of PEI-NPs in 5%glucose solution and 100 microliters of miRNA mimic in 5%glucose solution. Vortex the tube gently and spin down.Incubate the mixture for 15 minutes at room temperature to prepare a stable complex of PEI-NPs miRNA mimic. Dissolve two microliters of PEI-NPs in 98 microliters of 10%glucose solution in a 1.5-milliliter microcentrifuge tube. Vortex the tube gently and spin down.Then add 100 microliters of Cy3-labeled miRNA mimic to 100 microliters of PEI-NPs in 10%glucose solution and vortex the tube gently. Incubate the mixture for 15 minutes at room temperature to prepare a stable complex of PEI-NPs-Cy3-labeled-miRNA-mimic. Place the UUO mouse headfirst in a 50-milliliter centrifuge tube without anesthesia and then place the tail through a prepared hole in the cap.Fill a one-milliliter syringe with a 30-gauge needle with 200 microliters of PEI-NPs-Cy3-labeled-miRNA-mimic complex and inject the complex via the tail vein of the mouse. 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 until the kidney color changes to pale yellow.This indicates that the whole mouse body is perfused with PBS. After collecting the kidneys, remove the renal capsules, and wash them twice with PBS. Embed the kidney tissue samples in OCT compound and freeze them in liquid nitrogen.Use a cryostat to prepare sections and mount the sections onto silane-coated glass slides. Fix them with 4%paraformaldehyde and gently wash the slides twice with PBS. Finally, visualize the fluorescence staining by fluorescence microscopy at 100x and 400x magnification and appropriate imaging software.The representative image shows the structure of the PEI-NPs-miRNA-mimic complex. The delivery and effects of miRNA-146a-5p-mimic using PEI-NPs in renal fibrosis are depicted in these images. 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 and the glomerulus. qRT-PCR analysis showed that injection of PEI-NPs-miRNA-146a-5p-mimic induced significant overexpression of miRNA-146a-5p in the kidney. The histological analysis with Sirius red staining is shown here.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 mice in vivo. 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. Histological analysis with Periodic acid-Schiff staining showed that injection of PEI-NPs-miRNA-181b-5p-mimic once per week from 10 to 20 weeks of age inhibited the progression of diabetic kidney disease in mice.Delivery and effects of PEI-NPs-miRNA-5100-mimic in acute kidney injury model mice produced by ischemia reperfusion injury are shown here. The data of the fluorescent microscopy analysis qRT-PCR analysis to evaluate the overexpression effects of miRNA-5100 following injection of PEI-NPs-miRNA-5100-mimic and the histological analysis of hematoxylin-eosin stained kidney are depicted in these images. PEI-NPs injected via the tail vein two days before the ischemia reperfusion injury, or IRI, procedure prevent acute kidney injury progression induced by IRI.When performing this technique, prepare five-micrometer thick sections which include as much kidney tissue as possible. You can also prepare seven-micrometer thick sections.

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Research

JoVE Journal

Medicine

Spectral Karyotyping to Study Chromosome Abnormalities in Humans and Mice with Polycystic Kidney Disease

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being analyzed 1, 2. This classical banding technique, however, is not reliable in identifying complex chromosomal aberrations such as those associated with cancer. To overcome the limitations of the banding technique, Spectral Karyotyping (SKY) is introduced to provide much reliable information on chromosome abnormalities.
SKY is a multicolor fluorescence in-situ hybridization (FISH) technique to detect metaphase chromosomes with spectral microscope 3, 4. SKY has been proven to be a valuable tool for the cytogenetic analysis of a broad range of chromosome abnormalities associated with a large number of genetic diseases and malignancies 5, 6. SKY involves the use of multicolor fluorescently-labelled DNA probes prepared from the degenerate oligonucleotide primers by PCR. Thus, every chromosome has a unique spectral color after in-situ hybridization with probes, which are differentially labelled with a mixture of fluorescent dyes (Rhodamine, Texas Red, Cy5, FITC and Cy5.5). The probes used for SKY consist of up to 55 chromosome specific probes 7-10.
The procedure for SKY involves several steps (Figure 1). SKY requires the availability of cells with high mitotic index from normal or diseased tissue or blood. The chromosomes of a single cell from either a freshly isolated primary cell or a cell line are spread on a glass slide. This chromosome spread is labeled with a different combination of fluorescent dyes specific for each chromosome. For probe detection and image acquisition,the spectral imaging system consists of sagnac interferometer and a CCD camera. This allows measurement of the visible light spectrum emitted from the sample and to acquire a spectral image from individual chromosomes. HiSKY, the software used to analyze the results of the captured images, provides an easy identification of chromosome anomalies. The end result is a metaphase and a karyotype classification image, in which each pair of chromosomes has a distinct color (Figure 2). This allows easy identification of chromosome identities and translocations. For more details, please visit Applied Spectral Imaging website (<a href="http://www.spectral-imaging.com/" target="_blank">http://www.spectral-imaging.com/</a>).
SKY was recently used for an identification of chromosome segregation defects and chromosome abnormalities in humans and mice with Autosomal Dominant Polycystic Kidney Disease (ADPKD), a genetic disease characterized by dysfunction in primary cilia 11-13. Using this technique, we demonstrated the presence of abnormal chromosome segregation and chromosomal defects in ADPKD patients and mouse models 14. Further analyses using SKY not only allowed us to identify chromosomal number and identity, but also to accurately detect very complex chromosomal aberrations such as chromosome deletions and translocations (Figure 2).

Spectral Karyotyping (SKY) is an advanced cytogenetics technique to identify genomic and chromosomal aberrations. This technique takes advantage of chromosome painting probes, which allow classification of all chromosomes. SKY can also identify complex chromosome aberrations and segregation defects in mice and humans with various diseases, including polycystic kidney disease.

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being ...

Assessment of Vascular Function in Patients With Chronic Kidney Disease

Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this is only partially explained by traditional CVD risk factors. Vascular dysfunction is an important non-traditional risk factor, characterized by vascular endothelial dysfunction (most commonly assessed as impaired endothelium-dependent dilation [EDD]) and stiffening of the large elastic arteries. While various techniques exist to assess EDD and large elastic artery stiffness, the most commonly used are brachial artery flow-mediated dilation (FMDBA) and aortic pulse-wave velocity (aPWV), respectively. Both of these noninvasive measures of vascular dysfunction are independent predictors of future cardiovascular events in patients with and without kidney disease. Patients with CKD demonstrate both impaired FMDBA, and increased aPWV. While the exact mechanisms by which vascular dysfunction develops in CKD are incompletely understood, increased oxidative stress and a subsequent reduction in nitric oxide (NO) bioavailability are important contributors. Cellular changes in oxidative stress can be assessed by collecting vascular endothelial cells from the antecubital vein and measuring protein expression of markers of oxidative stress using immunofluorescence. We provide here a discussion of these methods to measure FMDBA, aPWV, and vascular endothelial cell protein expression.

The degree of vascular dysfunction and contributing physiological mechanisms can be assessed in patients with chronic kidney disease by measuring brachial artery flow-mediated dilation, aortic pulse-wave velocity, and vascular endothelial cell protein expression.

Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this ...

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The molecular mechanisms leading to cyst formation in PKD remain somewhat unclear, but many genes are thought to be involved. Wnt5a is a non-canonical glycoprotein that regulates a wide range of developmental processes. Wnt5a works through the planar cell polarity (PCP) pathway that regulates oriented cell division during renal tubular cell elongation. Defects of the PCP pathway have been found to cause kidney cyst formation. Our paper describes a method for developing a zebrafish cystic kidney disease model by knockdown of the wnt5a gene with wnt5a antisense morpholino (MO) oligonucleotides. Tg(wt1b:GFP) transgenic zebrafish were used to visualize kidney structure and kidney cysts following wnt5a knockdown. Two distinct antisense MOs (AUG - and splice-site) were used and both resulted in curly tail down phenotype and cyst formation after wnt5a knockdown. Injection of mouse Wnt5a mRNA, resistant to the MOs due to a difference in primary base pair structure, rescued the abnormal phenotype, demonstrating that the phenotype was not due to &#8220;off-target&#8221; effects of the morpholino. This work supports the validity of using a zebrafish model to study wnt5a function in the kidney.

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

We describe a method of generating a possible zebrafish model of polycystic kidney disease. We used Tg(wt1b:GFP) fish to visualize kidney structure. Knockdown of wnt5a was by morpholino injection. Pronephric cyst formation after wnt5a knockdown was observed in this GFP transgenic zebrafish.

Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The ...

Mouse Kidney Transplantation: Models of Allograft Rejection

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely replicates both the technical and pathological processes that occur in human renal transplantation. Although mouse models of allogeneic rejection in organs other than the kidney exist, and are more technically feasible, there is evidence that different organs elicit disparate rejection modes and dynamics, for instance the time course of rejection in cardiac and renal allograft differs significantly in certain strain combinations. This model is an attractive tool for many reasons despite its technical challenges. As inbred mouse strain haplotypes are well characterized it is possible to choose donor and recipient combinations to model acute allograft rejection by transplanting across MHC class I and II loci. Conversely by transplanting between strains with similar haplotypes a chronic process can be elicited were the allograft kidney develops interstitial fibrosis and tubular atrophy. We have modified the surgical technique to reduce operating time and improve ease of surgery, however a learning curve still needs to be overcome in order to faithfully replicate the model. This study will provide key points in the surgical procedure and aid the process of establishing this technique.

Here, we present a protocol to study the immunology of rejection. The surgical model presented reports a short operating time and a concise technique. Depending on the donor-recipient strain combination, the transplanted kidney may develop acute cellular rejection or chronic allograft damage, defined by interstitial fibrosis and tubular atrophy.

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely ...

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular mechanisms responsible for the disease, and are very convenient for rapid drug screening and testing of promising therapies. A limiting factor in these studies is often the lack of efficient non-invasive methods for sequentially analyzing the anatomical and functional changes in the kidney. Magnetic resonance imaging (MRI) is the current gold standard imaging technique to follow autosomal dominant polycystic kidney disease (ADPKD) patients, providing excellent soft tissue contrast and anatomic detail and allowing Total Kidney Volume (TKV) measurements.A major advantage of MRI in rodent models of PKD is the possibility for in vivo imaging allowing for longitudinal studies that use the same animal and therefore reducing the total number of animals required. In this manuscript, we will focus on using Ultra-high field (UHF) MRI to non-invasively acquire in vivo images of rodent models for PKD. The main goal of this work is to introduce the use of MRI as a tool for in vivo phenotypical characterization and drug monitoring in rodent models for PKD.

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

The use of ultra-high field MRI as a non-invasive way to obtain phenotypic information of rodent models for polycystic kidney disease and to monitor interventions is described. Compared with the traditional histological approach, MRI images can be acquired in vivo, allowing for longitudinal follow-up.

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular ...

Murine Kidney Transplant Technique

The first mouse kidney transplant technique was published in 19731 by the Russell laboratory. Although it took some years for other labs to become proficient in and utilize this technique, it is now widely used by many laboratories around the world. A significant refinement to the original technique using the donor aorta to form the arterial anastomosis instead of the renal artery was developed and reported in 1993 by Kalina and Mottram 2 with a further advancement coming from the same laboratory in 1999 3. While one can become proficient in this model, a search of the literature reveals that many labs still experience a high proportion of graft loss due to arterial thrombosis. We describe here a technique that was devised in our laboratory that vastly reduces the arterial thrombus reported by others 4,5. This is achieved by forming a heel-and-toe cuff of the donor infra-renal aorta that facilitates a larger anastomosis and straighter blood flow into the kidney.

The goal of this manuscript is to describe the steps required to perform a kidney transplant in a mouse, paying particular attention to the details of the arterial anastomosis.

The first mouse kidney transplant technique was published in 19731 by the Russell laboratory. Although it took some years for other labs to become ...

Normothermic Ex Vivo Kidney Perfusion for the Preservation of Kidney Grafts prior to Transplantation

Kidney transplantation has become a well-established treatment option for patients with end-stage renal failure. The persisting organ shortage remains a serious problem. Therefore, the acceptance criteria for organ donors have been extended leading to the usage of marginal kidney grafts. These marginal organs tolerate cold storage poorly resulting in increased preservation injury and higher rates of delayed graft function. To overcome the limitations of cold storage, extensive research is focused on alternative normothermic preservation methods.
Ex vivo normothermic organ perfusion is an innovative preservation technique. The first experimental and clinical trials for ex vivo lung, liver, and kidney perfusions demonstrated favorable outcomes.
In addition to the reduction of cold ischemic injury, the method of normothermic kidney storage offers the opportunity for organ assessment and repair. This manuscript provides information about kidney retrieval, organ preservation techniques, and isolated ex vivo normothermic kidney perfusion (NEVKP) in a porcine model. Surgical techniques, set up for the perfusion solution and the circuit, potential assessment options, and representative results are demonstrated.

Normothermic Ex Vivo Kidney Perfusion for the Preservation of Kidney Grafts prior to Transplantation

The severe organ shortage has resulted in increased use of marginal kidney grafts for transplantation. This has triggered interest in alternative storage methods, since marginal grafts especially tolerate cold storage poorly. The technique of normothermic ex vivo kidney perfusion (NEVKP) represents a novel preservation method for kidney grafts prior to &#160;transplantation.

Kidney transplantation has become a well-established treatment option for patients with end-stage renal failure. The persisting organ shortage remains a ...

Intravascular Delivery of Biologics to the Rat Kidney

The renal microvascular compartment plays an important role in the progression of kidney disease and hypertension, leading to the development of End Stage Renal Disease with high risk of death for cardiovascular events. Moreover, recent clinical studies have shown that renovascular structure and function may have a great impact on functional renal recovery after surgery. Here, we describe a protocol for the delivery of drugs into the renal artery of rats. This procedure offers significant advantages over the frequently used systemic administration as it may allow a more localized therapeutic effect. In addition, the use of rodents in pharmacodynamic analysis of preclinical studies may be cost effective, paving the way for the design of translational experiments in larger animal models. Using this technique, infusion of rat recombinant Vascular Endothelial Growth Factor (VEGF) protein in rats has induced activation of VEGF signaling as shown by increased expression of FLK1, pAKT/AKT, pERK/ERK. In summary, we established a protocol for the intrarenal delivery of drugs in rats, which is simple and highly reproducible.

The administration of drugs for recovery of kidney function requires control of the localization and distribution of the therapeutic compound. Here, we describe in detail a simple technique for intrarenal delivery of drugs in rats. This procedure may be easily performed with no mortality and high reproducibility.

The renal microvascular compartment plays an important role in the progression of kidney disease and hypertension, leading to the development of End Stage ...

Assessment of Kidney Function in Mouse Models of Glomerular Disease

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function in diabetic nephropathy and podocyte-specific VEGF-A knock-out mice; therefore, this protocol describes the full kidney work-up used in our lab to assess these mouse models of glomerular disease, enabling a vast amount of information regarding kidney and glomerular function to be obtained from a single mouse. In comparison to alternative methods presented in the literature to assess glomerular function, the use of the method outlined in this paper enables the glomerular phenotype to be fully evaluated from multiple aspects. By using this method, the researcher can determine the kidney phenotype of the model and assess the mechanism as to why the phenotype develops. This vital information on the mechanism of disease is required when examining potential therapeutic avenues in these models. The methods allow for detailed functional assessment of the glomerular filtration barrier through measurement of the urinary albumin creatinine ratio and individual glomerular water permeability, as well as both structural and ultra-structural examination using the Periodic Acid Schiff stain and electron microscopy. Furthermore, analysis of the genes dysregulated at the mRNA and protein level enables mechanistic analysis of glomerular function. This protocol outlines the generic but adaptable methods that can be applied to all mouse models of glomerular disease.

This protocol describes a full kidney work-up that should be carried out in mouse models of glomerular disease. The methods allow for detailed functional, structural, and mechanistic analysis of glomerular function, which can be applied to all mouse models of glomerular disease.

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function ...

Estimation of Nephron Number in Whole Kidney using the Acid Maceration Method

Nephron endowment refers to the total number of nephrons an individual is born with, as nephrogenesis in humans is completed by 36 weeks of gestation and no new nephrons are formed post-birth. Nephron number refers to the total number of nephrons measured at any point in time post-birth. Both genetic and environmental factors influence both nephron endowment and number. Understanding how specific genes or factors influence the process of nephrogenesis and nephron loss or demise is important as individuals with lower nephron endowment or number are thought to be at a higher risk of developing renal or cardiovascular disease. Understanding how environmental exposures over the course of a person's lifetime affects nephron number will also be vital in determining future disease risk. Thus, the ability to assess whole kidney nephron number quickly and reliably is a basic experimental requirement to better understand mechanisms that contribute to or promote nephrogenesis or nephron loss. Here, we describe the acid maceration method for the estimation of whole kidney nephron number based on the procedure described by Damadian, Shawayri, and Bricker, with slight modifications. The acid maceration method provides fast and reliable estimates of nephron number (as assessed by counting glomeruli) that are within 5% of those determined using more advanced, albeit expensive, methods such as magnetic resonance imaging. Moreover, the acid maceration method is an excellent high-throughput method to assess nephron number in large numbers of samples or experimental conditions.

Estimates of whole kidney nephron number are important clinically and experimentally, as there is an inverse association between nephron number and an enhanced risk of renal and cardiovascular disease.Herein, the use of the acid maceration method, which provides fast and reliable estimates of whole kidney nephron number, is demonstrated.

Nephron endowment refers to the total number of nephrons an individual is born with, as nephrogenesis in humans is completed by 36 weeks of gestation and ...