Name: hydrodynamic renal pelvis injection for non-viral expression of proteins in the kidney
Uploaded: 2018-01-08T12:30:00.000+00:00
Duration: 8 min 26 s
Description: Watch this Scientific Journal Video about Hydrodynamic Renal Pelvis Injection for Non-viral Expression of Proteins in the Kidney at JoVE.com

A Career Development Award from the Department of Veterans Affairs [BX002797] supported L.E.W. and the National Institutes of Health [R01-DK095867] and American Heart Association [15GRNT25700209] supported J.C. The National Institutes of Health [DK093660], Department of Veterans Affairs [BX002190], and the Vanderbilt Center for Kidney Disease supported M.H.W. This material is the result of work supported with resources and use of facilities at the VA Tennessee Valley Healthcare System.

<ol>
	<li>Liu, F., Song, Y., Liu, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrodynamics-based+transfection+in+animals+by+systemic+administration+of+plasmid+DNA.">Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA.</a> Gene Ther. 6 (7), 1258-1266 (1999).</li><li>Zhang, G., Budker, V., Wolff, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+levels+of+foreign+gene+expression+in+hepatocytes+after+tail+vein+injections+of+naked+plasmid+DNA.">High levels of foreign gene expression in hepatocytes after tail vein injections of naked plasmid DNA.</a> Human Gene Therapy. 10, 1735-1737 (1999).</li><li>Fumoto, S., Nishimura, K., Nishida, K., Kawakami, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-Dimensional+Imaging+of+the+Intracellular+Fate+of+Plasmid+DNA+and+Transgene+Expression:+ZsGreen1+and+Tissue+Clearing+Method+CUBIC+Are+an+Optimal+Combination+for+Multicolor+Deep+Imaging+in+Murine+Tissues.">Three-Dimensional Imaging of the Intracellular Fate of Plasmid DNA and Transgene Expression: ZsGreen1 and Tissue Clearing Method CUBIC Are an Optimal Combination for Multicolor Deep Imaging in Murine Tissues.</a> PLoS One. 11 (1), e0148233 (2016).</li><li>Yoshino, H., Hashizume, K., Kobayashi, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Naked+plasmid+DNA+transfer+to+the+porcine+liver+using+rapid+injection+with+large+volume.">Naked plasmid DNA transfer to the porcine liver using rapid injection with large volume.</a> Gene Ther. 13 (24), 1696-1702 (2006).</li><li>Kamimura, K., Suda, T., Xu, W., Zhang, G., Liu, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image-guided,+lobe-specific+hydrodynamic+gene+delivery+to+swine+liver.">Image-guided, lobe-specific hydrodynamic gene delivery to swine liver.</a> Mol Ther. 17 (3), 491-499 (2009).</li><li>Kamimura, K., Zhang, G., Liu, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image-guided,intravascular+hydrodynamic+gene+delivery+to+skeletal+muscle+in+pigs.">Image-guided,intravascular hydrodynamic gene delivery to skeletal muscle in pigs.</a> Mol Ther. 18 (1), 93-100 (2010).</li><li>Suda, T., Liu, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrodynamic+gene+delivery:+its+principles+and+applications.">Hydrodynamic gene delivery: its principles and applications.</a> Mol.Ther. 15 (12), 2063-2069 (2007).</li><li>Alino, S. F., 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=Naked+DNA+delivery+to+whole+pig+cardiac+tissue+by+coronary+sinus+retrograde+injection+employing+non-invasive+catheterization.">Naked DNA delivery to whole pig cardiac tissue by coronary sinus retrograde injection employing non-invasive catheterization.</a> J Gene Med. 12 (11), 920-926 (2010).</li><li>Suda, T., Gao, X., Stolz, D. B., Liu, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+impact+of+hydrodynamic+injection+on+mouse+liver.">Structural impact of hydrodynamic injection on mouse liver.</a> Gene Ther. 14 (2), 129-137 (2007).</li><li>Skrypnyk, N. I., Harris, R. C., de Caestecker, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ischemia-reperfusion+model+of+acute+kidney+injury+and+post+injury+fibrosis+in+mice.">Ischemia-reperfusion model of acute kidney injury and post injury fibrosis in mice.</a> J Vis Exp. (78), (2013).</li><li>Woodard, L. E., 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=Kidney-specific+transposon-mediated+gene+transfer+in+vivo.">Kidney-specific transposon-mediated gene transfer in vivo.</a> Sci Rep. 7, 44904 (2017).</li><li>Liang, 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=Loss+of+glutathione+S-transferase+A4+accelerates+obstruction-induced+tubule+damage+and+renal+fibrosis.">Loss of glutathione S-transferase A4 accelerates obstruction-induced tubule damage and renal fibrosis.</a> Journal of Pathology. 228 (4), 448-458 (2012).</li><li>Liang, M., 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=Protective+role+of+insulin-like+growth+factor-1+receptor+in+endothelial+cells+against+unilateral+ureteral+obstruction-induced+renal+fibrosis.">Protective role of insulin-like growth factor-1 receptor in endothelial cells against unilateral ureteral obstruction-induced renal fibrosis.</a> Am J Pathol. 185 (5), 1234-1250 (2015).</li><li>Corridon, P. R., 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+method+to+facilitate+and+monitor+expression+of+exogenous+genes+in+the+rat+kidney+using+plasmid+and+viral+vectors.">A method to facilitate and monitor expression of exogenous genes in the rat kidney using plasmid and viral vectors.</a> Am J Physiol Renal Physiol. 304 (9), F1217-F1229 (2013).</li><li>Wallace, D. 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=Tracking+kidney+volume+in+mice+with+polycystic+kidney+disease+by+magnetic+resonance+imaging.">Tracking kidney volume in mice with polycystic kidney disease by magnetic resonance imaging.</a> Kidney Int. 73 (6), 778-781 (2008).</li><li>Woodard, L. E., Wilson, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=piggyBac-ing+models+and+new+therapeutic+strategies.">piggyBac-ing models and new therapeutic strategies.</a> Trends Biotechnol. 33 (9), 525-533 (2015).</li><li>Yeikilis, R., 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=Hydrodynamics+based+transfection+in+normal+and+fibrotic+rats.">Hydrodynamics based transfection in normal and fibrotic rats.</a> World J Gastroenterol. 12 (38), 6149-6155 (2006).</li><li>Thalhofer, C. J., 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+imaging+of+transgenic+Leishmania+parasites+in+a+live+host.">In vivo imaging of transgenic Leishmania parasites in a live host.</a> J Vis Exp. (41), (2010).</li><li>Wooddell, C. I., 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=Muscle+damage+after+delivery+of+naked+plasmid+DNA+into+skeletal+muscles+is+batch+dependent.">Muscle damage after delivery of naked plasmid DNA into skeletal muscles is batch dependent.</a> Hum Gene Ther. 22 (2), 225-235 (2011).</li><li>Crespo, 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=Hydrodynamic+liver+gene+transfer+mechanism+involves+transient+sinusoidal+blood+stasis+and+massive+hepatocyte+endocytic+vesicles.">Hydrodynamic liver gene transfer mechanism involves transient sinusoidal blood stasis and massive hepatocyte endocytic vesicles.</a> Gene Ther. 12 (11), 927-935 (2005).</li><li>Rocca, C. J., Ur, S. N., Harrison, F., Cherqui, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=rAAV9+combined+with+renal+vein+injection+is+optimal+for+kidney-targeted+gene+delivery:+conclusion+of+a+comparative+study.">rAAV9 combined with renal vein injection is optimal for kidney-targeted gene delivery: conclusion of a comparative study.</a> Gene Ther. 21 (6), 618-628 (2014).</li></ol>

The authors have nothing to disclose and declare no conflict of interest.

In this protocol a robust method for achieving reproducible gene expression specifically in the kidney is described. In the hands of a moderately experienced surgeon we have found the percentage of mice transfected by this technique to be in the range of 50-100%, depending on mouse age and the sensitivity of the readout of the transgene. The level of luciferase gene expression was above background for several months in mice receiving piggyBac transposons and completely maintained for several weeks in immunocompr...

The hydrodynamic tail vein injection technique has become a commonly used way to achieve high levels of gene expression in mouse liver1,2. The kidneys are also transfected by this technique at a much lower level, approximately 100-fold less3. The hydrodynamic renal pelvis injection described here provides a simple way to control the specificity of organ expression through physical means using the same hydrodynamic principles that have been established previously in liver4,5, muscle6, and other organs7,8. This method transfects cells in live animals in vivo by using pressure and speed to force fluid containing DNA into the cells, simultaneously inducing damage to the organ that is quickly resolved9. Using well-established surgical techniques to visualize the kidney via a flank incision10 along with a single injection by insulin syringe, we have found successful transfection of various types of kidney cells, mainly interstitial fibroblasts, tubules, and collecting duct11. Dissection of these mice has shown that other organs are not transfected at levels high enough to visualize by luciferase imaging techniques11. Since the technique is non-viral, use of plasmid DNA for transfection permits fast and easy preparation of the reagents required for injection.
We have used localized hydrodynamic injections to express the antioxidant glutathione S-transferase A4, the insulin-like growth factor-1 receptor, and the hormone erythropoietin in the kidney, all with the expected biological effects11,12,13. Detailed evaluation of route of administration, injection volume, DNA dosage, and promoter choice has been performed11. Additionally, both the piggyBac transposon system and/or cyclophosphamide treatment to suppress the immune reaction to the transgene have been shown to improve long-term gene expression outcomes11. Other investigators have used a renal vein approach in rat with success, achieving high transfection efficiency for time periods of greater than one month14. However, genetic correction of phenotypes mimicking human disease are usually performed in mice first as a proof-of-concept since most mammalian genetic models are mouse models. We compared renal vein injection to renal pelvis injection and found that injection into the renal pelvis was superior to the renal vein for gene expression (approximately ten-fold higher) and survival11. The renal pelvis is an ideal route of entry into the kidney because it is flexible enough to tolerate fluctuations in urine production and is often able to maintain its structural integrity even when dilated during hydronephrosis. Additionally, injection into the renal pelvis allowed access to the kidney without piercing the kidney capsule, allowing the injected fluid to be visibly retained by the kidney better than intraparenchymal injection. Other mouse organs do not have a route of entry other than the vasculature, but the urinary space of the kidney is an ideal injection site. Additionally, injection into the renal vein resulted in leakage of blood into the abdominal cavity. The total kidney volume of wild-type mouse kidneys has been estimated by magnetic resonance imaging to be approximately 0.2 cm3, so the volume of a single kidney is approximately equal to the amount of fluid injected by renal pelvis hydrodynamic injection (100 &#181;L)15. Herein, we have made available all of the detailed nuances of the hydrodynamic renal pelvis injection protocol to achieve reproducible transfection of the kidney.

<table><tbody><tr><td>AnaSed Xylazine</td><td>Patterson Veterinary</td><td>07-808-1947</td><td>Anesthetic - Not controlled substance</td></tr><tr><td>BD Insulin Syringe 0.5 mL 29G 1/2 Inch</td><td>Cardinal Health</td><td>309306</td><td>Required syringes</td></tr><tr><td>Buprenex</td><td>Pharmacist/Veterinarian</td><td></td><td>Analgesia - Controlled Substance</td></tr><tr><td>Dynarex Disposable Towel Drape</td><td>Thermo Fisher Scientific</td><td>19-310-671</td><td>Place over heat pad</td></tr><tr><td>EndoFree Plasmid Maxi Kit</td><td>Qiagen</td><td>12362</td><td>Use only endotoxin-free plasmid DNA</td></tr><tr><td>Endosafe Gel-Clot LAL Rapid Positive Control</td><td>Charles River</td><td>PC200</td><td>Positive control for endotoxin test</td></tr><tr><td>Endosafe Gel-Clot LAL Rapid Single Test Vial</td><td>Charles River</td><td>R13500</td><td>Endotoxin test</td></tr><tr><td>Extra Fine Micro Dissecting Scissors</td><td>Roboz Surgical Instrument</td><td>RS-5882</td><td>Surgical tool</td></tr><tr><td>Fisherbrand Instant Sealing Sterilization Pouch - 9&quot;</td><td>Thermo Fisher Scientific</td><td>01-812-51</td><td>For autoclaving surgical tools</td></tr><tr><td>Gaymar Heat Pump</td><td>Paragon Medical</td><td>TP-700</td><td>Water-circulating heat pump</td></tr><tr><td>Germinator 500</td><td>Roboz Surgical Instrument</td><td>DS-401</td><td>To reuse surgical tools during surgery</td></tr><tr><td>Graefe Forceps</td><td>Roboz Surgical Instrument</td><td>RS-5136</td><td>Surgical tool</td></tr><tr><td>Graefe Tissue Forceps</td><td>Roboz Surgical Instrument</td><td>RS-5153</td><td>Surgical tool</td></tr><tr><td>Halsey Needle Holder, 5&quot; Length</td><td>Roboz Surgical Instrument</td><td>RS-7841</td><td>Surgical tool</td></tr><tr><td>Heat pads - 15&quot; x 21&quot; - need at least 3</td><td>Paragon Medical</td><td>TP22G</td><td>For use with Gaymar Heat Pump</td></tr><tr><td>IsoFlo (Isoflurane, USP)</td><td>Abbott Animal Health</td><td>5260-04-05</td><td>For imaging and euthanasia</td></tr><tr><td>Isotec Isoflurane Delivery System Vaporizor</td><td>Smiths Medical</td><td>VCT3K2</td><td>For imaging and euthanasia</td></tr><tr><td>Ketamine</td><td>Pharmacist/Veterinarian</td><td></td><td>Anesthetic - Controlled Substance</td></tr><tr><td>Kimwipes</td><td>Kimberly-Clark Professional</td><td>34120</td><td>Laboratory tissues</td></tr><tr><td>Living Image software</td><td>Caliper Life Sciences</td><td></td><td>For live animal imaging</td></tr><tr><td>Luciferin</td><td>Perkin Elmer</td><td>122796</td><td>For live animal imaging</td></tr><tr><td>Nanodrop 2000</td><td>Thermo Scientific</td><td>ND-2000-US-CAN</td><td>Spectrophotometer for DNA measurement</td></tr><tr><td>Prevantics Swabs</td><td>Thermo Fisher Scientific</td><td>23-100-110</td><td>For skin surgery prep</td></tr><tr><td>Prolene 5-0 sutures Taper 30&quot;</td><td>Thermo Fisher Scientific</td><td>NC0256891</td><td>Non-absorbable sutures for skin</td></tr><tr><td>Puralube Brand Opthalmic Ointment</td><td>Patterson Veterinary</td><td>07-888-2572</td><td>To keep eyes moist during surgery</td></tr><tr><td>Trans IT - QR Hydrodynamic Delivery Solution</td><td>Mirus Bio</td><td>MIR-5240</td><td>Hydrodynamic delivery buffer for diluting DNA</td></tr><tr><td>Vicryl 5-0 Sutures J303H</td><td>Thermo Fisher Scientific</td><td>NC9816710</td><td>Absorbable sutures for muscle layer</td></tr><tr><td>Wahl Mini Arco Clipper</td><td>Med-Vet International</td><td>8787-1550</td><td>Shaver for skin prep</td></tr><tr><td>Xenogen IVIS 200</td><td>Caliper Life Sciences</td><td></td><td>For live animal imaging</td></tr></tbody></table>

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.

The surgery and injection technique are simple to perform once mastered, requiring no major equipment or expensive materials. If new to flank-incision kidney surgery, one training day on several mice scheduled for euthanasia should be allowed in which the mice are not recovered following surgery because the first attempt at this surgery may take much longer than normal. Alternatively, investigators familiar with similar techniques may find it quite simple. Carefully follow the illustratio...

Watch this Scientific Journal Video about Hydrodynamic Renal Pelvis Injection for Non-viral Expression of Proteins in the Kidney at JoVE.com

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

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 ...

hydrodynamic-renal-pelvis-injection-for-non-viral-expression-proteins

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

18.4K Views.  Tennessee Valley Healthcare System. 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.

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

Technique to Target Microinjection to the Developing Xenopus Kidney

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus embryos are large, develop externally, and can be easily manipulated by microinjection or surgical procedures. In addition, fate maps have been established for early Xenopus embryos. Targeted microinjection into the individual blastomere that will eventually give rise to an organ or tissue of interest can be used to selectively overexpress or knock down gene expression within this restricted region, decreasing secondary effects in the rest of the developing embryo. In this protocol, we describe how to utilize established Xenopus fate maps to target the developing Xenopus kidney (the pronephros), through microinjection into specific blastomere of 4- and 8-cell embryos. Injection of lineage tracers allows verification of the specific targeting of the injection. After embryos have developed to stage 38 - 40, whole-mount immunostaining is used to visualize pronephric development, and the contribution by targeted cells to the pronephros can be assessed. The same technique can be adapted to target other tissue types in addition to the pronephros.

Technique to Target Microinjection to the Developing Xenopus Kidney

Here, we present a protocol to use fate maps and lineage tracers to target injections into individual blastomeres that give rise to the kidney of Xenopus laevis embryos.

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus ...

Developmental Biology

Intravenous Microinjections of Zebrafish Larvae to Study Acute Kidney Injury

In this video article we describe a zebrafish model of AKI using gentamicin as the nephrotoxicant.  The technique consists of intravenous microinjections on 2 dpf zebrafish. This technique represents an efficient and rapid method to deliver soluble substances into the bloodstream of zebrafish larvae, allowing for the injection of 15-20 fish per hour. In addition to AKI studies, this microinjection technique can also be used for other types of experimental studies such as angiography.  We provide a detailed protocol of the technique from equipment required to visual measures of decreased kidney function. In addition, we also demonstrate the process of fixation, whole mount immunohistochemistry with a kidney tubule marker, plastic embedding and sectioning of the larval zebrafish. We demonstrate that zebrafish larvae injected with gentamicin show morphological features consistent with AKI: edema, loss of cell polarity in proximal tubular epithelial cells, and morphological disruption of the tubule.

We describe a technique of microinjecting the aminoglycoside, gentamicin, into 2 days post-fetilization (dpf) zebrafish larvae to induce acute kidney injury (AKI). We also describe a method for whole mount immunohistochemistry, plastic embedding and sectioning of zebrafish larvae to visualize the AKI mediated damage.

In this video article we describe a zebrafish model of AKI using gentamicin as the nephrotoxicant.  The technique consists of intravenous microinjections ...

Microdissection of Primary Renal Tissue Segments and Incorporation with Novel Scaffold-free Construct Technology

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only one-fourth of these patients achieving transplantation, there is a dire need for alternatives for those with failing organs. In order to decrease the harmful consequences of dialysis along with the overall healthcare costs it incurs, active investigation is ongoing in search of alternative solutions to organ transplantation. Implantable tissue-engineered renal cellular constructs are one such feasible approach to replacing lost renal functionality. Here, described for the first time, is the microdissection of murine kidneys for isolation of living corticomedullary renal segments. These segments are capable of rapid incorporation within scaffold-free endothelial-fibroblast constructs which may enable rapid connection with host vasculature once implanted. Adult mouse kidneys were procured from living donors, followed by stereoscope microdissection to obtain renal segments 200 - 300 &#181;m in diameter. Multiple renal constructs were fabricated using primary renal segments harvested from only one kidney. This method demonstrates a procedure which could salvage functional renal tissue from organs that would otherwise be discarded.

Tissue-engineered renal constructs provide a solution for the organ shortage and deleterious effects of dialysis. Here, we describe a protocol to micro dissect murine kidneys for isolation of cortico-medullary segments. These segments are implanted into scaffold-free cellular constructs, forming renal organoids.

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only ...

Bioengineering

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 ...

Medicine

Intrarenal Injection of Escherichia coli in a Rat Model of Pyelonephritis

Pyelonephritis is a bacterial infection of the kidney and is most commonly caused by Escherichia coli. Recurrent infections can cause significant renal inflammation and fibrosis ultimately resulting in declining kidney function. Before improved clinical management and prevention of pyelonephritis can be instituted, a reliable animal model must be established in order to study the mechanisms of progression, recurrence, and therapeutic efficacy. The transurethral infection model closely mimics human pyelonephritis but exhibits considerable variation due to its reliance on urethral reflux to transport the bacteria to the kidney. Herein, a detailed surgical protocol for performing bacterial injections into the rat renal pelvis is provided and confirmed by non-invasive Magnetic Resonance Imaging (MRI). Using this protocol, animals receive direct exposure to a desired concentration of E. coli bacteria and can fully recover from the surgical procedure with adequate post-operative care. This facilitates subsequent longitudinal MRI assessments of the experimental animal models for comparison with saline (sham) controls. Using this direct delivery approach, the severity of infection is controllable and applicable for mechanistic studies of progression as well as development of novel treatment strategies.

Intrarenal Injection of Escherichia coli in a Rat Model of Pyelonephritis

This manuscript describes a rat surgical model of pyelonephritis using direct intra-renal infection by Escherichia coli into the renal pelvis. The experimental procedure can be utilized to study the pathogenesis of pyelonephritis as well as the associated inflammation and fibrosis.

Pyelonephritis is a bacterial infection of the kidney and is most commonly caused by Escherichia coli. Recurrent infections can cause significant renal ...

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 ...

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.

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 ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

Studying Protein Expression During Chicken Lens Development

Microinjection of Recombinant RCAS(A) Retrovirus into Embryonic Chicken Lens

Embryonic chicken (Gallus domesticus) is a well-established animal model for the study of lens development and physiology, given its high degree of similarity with the human lens. RCAS(A) is a replication-competent chicken retrovirus that infects dividing cells, which serves as a powerful tool to study the in situ expression and function of wild-type and mutant proteins during lens development by microinjection into the empty lumen of lens vesicle at early developmental stages, restricting its action to surrounding proliferating lens cells. Compared to other approaches, such as transgenic models and ex vivo cultures, the use of an RCAS(A) replication-competent avian retrovirus provides a highly effective, rapid, and customizable system to express exogenous proteins in chick embryos. Specifically, targeted gene transfer can be confined to proliferative lens fiber cells without the need for tissue-specific promoters. In this article, we will briefly overview the steps needed for recombinant retrovirus RCAS(A) preparation, provide a detailed, comprehensive overview of the microinjection procedure, and provide sample results of the technique.

Author Spotlight: Investigating Lens Development and Function Through Microinjection of RCAS(A) Retrovirus in Embryonic Chicken Lens 

This protocol paper describes the methodology of embryonic chicken lens microinjection of an RCAS(A) retrovirus as a tool for studying in situ function and expression of proteins during lens development.

Embryonic chicken (Gallus domesticus) is a well-established animal model for the study of lens development and physiology, given its high degree of ...

Hydrodynamic Renal Pelvis Injection: A Technique to Directly Deliver DNA into Kidney Cells of Murine Model

Source: Woodard, L. E. et al. <a href="https://jove.unicartagenaproxy.elogim.com/video/56324" target="_blank">Hydrodynamic Renal Pelvis Injection for Non-viral Expression of Proteins in the Kidney.</a> J. Vis. Exp. (2018)
In this video, we present the protocol for hydrodynamic injection of plasmids into the renal pelvis of the mouse kidney for nucleic acid transfection. This technique allows organ-specific delivery of plasmids by utilizing the principle of high hydrostatic pressure, which forcefully drives the plasmids into the specific site without causing tissue injury.

Source: Woodard, L. E. et al. Hydrodynamic Renal Pelvis Injection for Non-viral Expression of Proteins in the Kidney. J. Vis. Exp. (2018)
In this video, ...

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

Summary

Explore More Videos

Chapters in this video

Technique to Target Microinjection to the Developing Xenopus Kidney

Intravenous Microinjections of Zebrafish Larvae to Study Acute Kidney Injury

Microdissection of Primary Renal Tissue Segments and Incorporation with Novel Scaffold-free Construct Technology

Intravascular Delivery of Biologics to the Rat Kidney

Intrarenal Injection of Escherichia coli in a Rat Model of Pyelonephritis

Direct Drug Delivery to Kidney via the Renal Artery

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

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

Microinjection of Recombinant RCAS(A) Retrovirus into Embryonic Chicken Lens

Hydrodynamic Renal Pelvis Injection: A Technique to Directly Deliver DNA into Kidney Cells of Murine Model