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

Ein Career Development Award vom Department of Veterans Affairs [BX002797] L.E.W. und die National Institutes of Health [R01-DK095867] American Heart Association [15GRNT25700209] unterstützt und j.c. Die National Institutes of Health [DK093660], Department of Veterans Affairs [BX002190] und der Vanderbilt-Zentrum für Nierenerkrankungen unterstützt M.H.W. Dieses Material ist das Ergebnis der Arbeit mit Ressourcen und Nutzung der Einrichtungen im VA Tennessee Valley Healthcare System unterstützt.

<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. 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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>

Die Autoren haben nichts offen zu legen und kein Interessenkonflikt zu erklären.

In diesem Protokoll wird eine robuste Methode zur Erreichung reproduzierbare Genexpression speziell in der Niere beschrieben. In den Händen eines mäßig erfahrener Chirurgen fanden wir den Prozentsatz der Mäuse, die durch diese Technik im Bereich von 50-100 %, je nach Alter der Maus und die Empfindlichkeit der Anzeige des Transgens transfiziert. Das Niveau der Luciferase-gen-Expression war über Hintergrund für mehrere Monate in den Mäusen erhalten PiggyBac Transposons und für mehrere Wochen bei immungesch...

Die hydrodynamischen Schweif Vene Injektionstechnik ist eine häufig verwendete Möglichkeit, hohe Genexpression in Maus Leber1,2geworden. Die Nieren sind auch durch diese Technik auf einem viel niedrigeren Niveau, etwa 100-fold weniger3transfiziert. Die hydrodynamischen Nierenbecken-Injektion, die hier beschriebenen bietet eine einfache Möglichkeit zu steuern, die Besonderheit der Orgel Ausdruck durch physische Mittel nach den gleichen hydrodynamischen Grundsätzen, die zuvor in Leber,4,5 festgelegt wurden , Muskel-6und andere Organe7,8. Diese Methode transfects Zellen in lebenden Tieren in Vivo durch Druck und Geschwindigkeit zwingen Flüssigkeit enthaltende DNA in die Zellen gleichzeitig induzieren Schäden an das Organ, das schnell gelöst9. Mit etablierten Operationstechniken, um die Niere über eine Flanke Schnitt10 zusammen mit einer einzigen Injektion von Insulin Spritze zu visualisieren, haben wir erfolgreiche Transfektion von verschiedenen Arten von Nierenzellen, vor allem interstitielle Fibroblasten gefunden, Tubuli und sammeln Kanal11. Sezieren von diesen Mäusen haben gezeigt, dass andere Organe nicht auf Ebenen hoch genug, um durch Luciferase bildgebende Techniken11visualisieren transfiziert sind. Da die Technik nicht-viralen ist, gestattet die Verwendung von Plasmid DNA zur Transfektion schnelle und einfache Zubereitung der Reagenzien zur Injektion erforderlich.
Wir haben lokalisierte hydrodynamische Injektionen verwendet, um auszudrücken das Antioxidans Glutathion S-Transferase A4, der Insulin-ähnliche Wachstumsfaktor-1 Rezeptor und das Hormon Erythropoietin in der Niere, alle mit der erwarteten biologischen Wirkungen11, 12 , 13. detaillierte Auswertung der Route der Verwaltung, Einspritzmenge, DNA-Dosierung und Projektträger Wahl wurde durchgeführt11. Darüber hinaus sowohl die PiggyBac Transposon System- und/oder Cyclophosphamid Behandlung unterdrücken die Immunreaktion auf das Transgen haben gezeigt, dass langfristige gen Ausdruck Ergebnisse11zu verbessern. Andere Forscher haben einen renale Vene Ansatz in der Ratte mit Erfolg, eine hohe Transfektion Effizienz für Zeiträume von mehr als einem Monat14verwendet. Genetische Korrektur der Phänotypen imitiert menschlichen Krankheiten werden jedoch in der Regel durchgeführt bei Mäusen zuerst als ein Proof-of-Concept da am meisten Säugetieren genetische Modelle Mausmodellen. Wir renal Vene Injektion zu Nierenbecken Injektion verglichen und festgestellt, dass die Injektion in das Nierenbecken, die renal Vene für die Genexpression (etwa um das zehnfache höher) und überleben11überlegen war. Das Nierenbecken ist eine ideale Route für die Einreise in die Niere, weil es flexibel genug, um Schwankungen in der Urinproduktion zu tolerieren ist und ist oft in der Lage, seine strukturelle Integrität auch wenn während Hydronephrose geweitet. Injektion in das Nierenbecken erlaubt darüber hinaus Zugriff auf die Niere ohne piercing die Niere Kapsel, so dass die eingespritzte Flüssigkeit sichtbar durch die Nieren besser als Intraparenchymal Injektion erhalten bleiben soll. Andere Maus Organe haben keine Route Eintrag nicht das Gefäßsystem, aber der Harn Raum der Niere ist eine ideale Injektionsstelle. Darüber hinaus führte in der renalen Vene Austritt von Blut in die Bauchhöhle. Das gesamte Niere Volumen der Wildtyp Maus Nieren ist geschätzt worden, durch Magnetresonanz-Bildgebung zu ca. 0,2 cm3, so ist das Volumen einer einzelnen Niere annähernd gleich die Menge der Flüssigkeit injiziert durch hydrodynamische Nierenbecken Injektion (100 µL)15. Hier haben wir all die detaillierten Nuancen des hydrodynamischen Nierenbecken Injektion Protokolls, reproduzierbare Transfektion der Niere zu erreichen zur Verfügung.

<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

Hydrodynamische Injektion schafft eine lokale, Hochdruck-Umfeld, um verschiedenen Geweben mit Plasmid-DNA und anderen Substanzen zu transfizieren. Hydrodynamische Schweif Vene Injektion, ist zum Beispiel eine etablierte Methode, die durch die Leber transfiziert werden kann. Dieses Manuskript beschreibt eine Anwendung der hydrodynamischen Prinzipien durch Injektion von der Mausniere direkt mit Plasmid DNA für Nieren-spezifische Genexpression. Mäuse werden betäubt und die Niere durch eine Flanke Inzision, gefolgt von einer schnellen Injektion ein Plasmid DNA-haltigen Lösung direkt in das Nierenbecken ausgesetzt ist. Die Nadel ist für zehn Sekunden gehalten und Ortsbild Einschnitt vernäht. Am nächsten Tag live Animal Imaging, Western-Blot oder Immunohistochemistry, Genexpression assay verwendet werden oder anderen Assays geeignet, um das Transgen Wahl dienen zur Erkennung des Proteins des Interesses. Veröffentlichten Methoden der Genexpression zu verlängern zählen Transposon-vermittelten Transgen Integration und Cyclophosphamid behandelt, um die Immunantwort auf das Transgen hemmen.

Die Chirurgie und Injektion Technik sind einfach durchzuführen, einmal gemeistert, erfordert keine große Ausrüstung oder teuren Materialien. Wenn neue zur Flanke-Schnitt Niere Chirurgie, einen Trainingstag auf mehrere Mäuse geplant für Euthanasie dürfen in denen die Mäuse sind nicht wiederhergestellten nach der Operation, weil der erste Versuch dieser Operation wesentlich länger als normal dauern kann. Alternativ können Forscher mit ähnlichen Techniken vertraut ganz einfach find...

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

Hydrodynamische Nierenbecken Injektion für nicht-virale Expression von Proteinen in der Niere

Dieses Protokoll beschreibt eine Methode zum Injizieren von Plasmid DNA in der Mausniere über das Nierenbecken, Transgene Ausdruck speziell in der Niere zu produzieren.

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

Research

JoVE Journal

Biology

18.4K Aufrufe.  Tennessee Valley Healthcare System. Dieses Protokoll beschreibt eine Methode zum Injizieren von Plasmid DNA in der Mausniere über das Nierenbecken, Transgene Ausdruck speziell in der Niere zu produzieren.

Serie: 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.

Technik auf Zielmikroinjektions an die Entwicklungs<em&gt; Xenopus</em&gt; 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 ...

Forschung

Entwicklungsbiologie

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.

Intravenöse Mikroinjektionen mit Zebrabärblingembryonen zu Acute Kidney Injury Study

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

Biologie

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.

Mikrodissektion primäre renale Gewebe Segmente und Einbindung mit neuartigen Gerüst-freie Konstrukt-Technologie

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

Biotechnik

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.

Die intravaskuläre Lieferung von Biologics zur Rattenniere

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

Medizin

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.

Intrarenale Injektion von<em&gt; Escherichia coli</em&gt; In einem Rattenmodell der Pyelonephritis

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.

Direkte Medikamentenabgabe an die Niere über die Nierenarterie

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.

Abgabe von exogenen künstlich synthetisierten miRNA-Nachahmungen an die Niere unter Verwendung von Polyethylenimin-Nanopartikeln in verschiedenen Mausmodellen für Nierenerkrankungen

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.

Nephrotoxin Mikroinjektion in Zebrabärbling akuten Nierenschädigung zu Modell

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 ...

Untersuchung der Proteinexpression während der Entwicklung von Hühnerlinsen

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.

Autor im Rampenlicht: Untersuchung der Linsenentwicklung und -funktion durch Mikroinjektion des RCAS(A)-Retrovirus in embryonaler Hühnerlinse 

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.

Mikroinjektion von rekombinantem RCAS(A)-Retrovirus in embryonale Hühnerlinse

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

Hydrodynamische Nierenbecken Injektion für nicht-virale Expression von Proteinen in der Niere

Zusammenfassung

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