جائزة تطوير المهنة من إدارة شؤون قدامى المحاربين [BX002797] دعم L.E.W. والمعاهد الوطنية للصحة [R01-DK095867] و "جمعية القلب الأمريكية" [15GRNT25700209] دعم كيركراده المعاهد الوطنية للصحة [DK093660]، وإدارة شؤون قدامى المحاربين في [BX002190]، ومركز فاندربيلت "أمراض الكلي" الدعم M.H.W. هذه المواد هو نتيجة للعمل مدعومة بالموارد واستخدام المرافق في "نظام الرعاية الصحية وادي تينيسي خامسا".

<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>

المؤلفين لا علاقة تكشف وتعلن عدم تضارب المصالح.

ويرد في هذا البروتوكول وسيلة قوية لتحقيق التعبير الجيني استنساخه على وجه التحديد في الكلي. في أيدي جراح معتدلة من ذوي الخبرة وجدنا النسبة المئوية للفئران ترانسفيكتيد بهذا الأسلوب في نطاق 50-100 ٪، تبعاً للعمر الماوس وحساسية قراءات للتحوير. مستوى التعبير الجيني لوسيفراس فوق الخلفية لعدة أشه...

تقنية حقن الوريد ذيل هيدرودينامية أصبحت وسيلة شائعة لتحقيق مستويات عالية من التعبير الجيني في الماوس الكبد1،2. هي أيضا transfected الكلي بهذا الأسلوب على مستوى أقل بكثير، حوالي 100-fold أقل3. حقن الحوض الكلوي هيدرودينامية الموصوفة هنا يوفر طريقة بسيطة للتحكم في خصوصية التعبير الجهاز من خلال الوسائل المادية باستخدام نفس المبادئ هيدرودينامية التي وضعت سابقا في الكبد4،5 ، العضلات6و7،أجهزة أخرى8. هذا الأسلوب ترانسفيكتس الخلايا في الحيوانات الحية في فيفو باستخدام الضغط والسرعة لإجبار السوائل المحتوية على الحمض الخلوي الصبغي في الخلايا، في نفس الوقت الذي يحفز الضرر للجهاز الذي يتم بسرعة حل9. باستخدام التقنيات الجراحية الراسخة لتصور الكلي عن طريق شق الجناح10 جنبا إلى جنب مع حقنه واحدة بحقنه الأنسولين، وقد وجدنا تعداء الناجح لأنواع مختلفة من خلايا الكلي، أساسا من خلالي الليفية، الأنابيب، وجمع القناة11. تشريح لهذه الفئران أظهرت أن الأجهزة الأخرى هي عدم transfected على مستويات عالية بما يكفي لتصور طريق لوسيفراس11من تقنيات التصوير. نظراً للتقنية غير الفيروسية، يسمح استخدام بلازميد الحمض النووي تعداء إعداد سريع وسهل من الكواشف اللازمة للحقن.
وقد استخدمنا حقن هيدرودينامية المترجمة للتعبير عن الجلوتاثيون المضادة للأكسدة A4 S-ترانسفيراز ومستقبلات عامل النمو-1 مثل الأنسولين، وهرمون ارثروبويتين في الكلي، مع كل من الآثار البيولوجية المتوقعة11، 12 , 13-تقييم مفصل لتوجيه الإدارة وحجم الحقن، وجرعة الحمض النووي، واختيار المروج قد أجرى11. بالإضافة إلى ذلك، أظهرت كلا بيجيباك ينقول النظام و/أو السيكلوفوسفاميد علاج لقمع رد الفعل المناعي للتحوير لتحسين نتائج التعبير الجيني طويلة الأجل11. واستخدمت محققون آخرون نهجاً الوريد كلوي في الفئران بالنجاح، وتحقيق كفاءة عالية تعداء لفترات من الوقت أكثر من شهر واحد من14. بيد التصحيح الوراثي لتعمل محاكاة الأمراض البشرية عادة ما تتم في الفئران أولاً كإثبات لمفهوم منذ النماذج الوراثية معظم الثدييات نماذج الماوس. مقارنة حقن الوريد الكلوي إلى حقن الحوض الكلوي، ووجد أن حقن في الحوض الكلوي متفوقة السياق الكلي للتعبير الجيني (حوالي عشرة إضعاف أعلى) وبقاء11. الحوض الكلوي هو طريقا مثاليا للدخول إلى الكلي لأنها مرنة بما يكفي لتحمل التقلبات في إنتاج البول، وقادرة على الحفاظ على سلامتها الهيكلية حتى عندما المتوسعة خلال هيدرونيفروسيس في كثير من الأحيان. بالإضافة إلى ذلك، يسمح حقن في الحوض الكلوي الوصول إلى الكلي دون ثقب كبسولة الكلي، يسمح للسائل المحقون واضح الاحتفاظ بالكلى أفضل من حقن intraparenchymal. أجهزة الماوس الأخرى لم يكن طريقا لدخول خلاف المفرج، ولكن المساحة البولي الكلي موقع حقن مثالية. بالإضافة إلى ذلك، أدى الحقن الوريد الكلوي تسرب الدم في تجويف البطن. وقدرت حجم الكلي الإجمالي الكلي البرية من نوع الماوس بالتصوير بالرنين المغناطيسي تكون حوالي 0.2 سم3من حيث حجم الكلي واحد مساو تقريبا لكمية السوائل التي تحقن بالحوض الكلوي هيدرودينامية حقن (100 ميليلتر)15. وهنا، قد وفرنا جميع الفروق الدقيقة المفصلة البروتوكول حقن الحوض الكلوي الهيدروديناميكي لتحقيق تعداء قابل لإعادة الإنتاج الكلي.

<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

حقن هيدرودينامية يخلق بيئة محلية، والضغط ترانسفيكت الأنسجة المختلفة مع بلازميد الحمض النووي وغيرها من المواد. حقن الوريد هيدرودينامية الذيل، على سبيل المثال، هو أسلوب راسخة التي يمكن ترانسفيكتيد في الكبد. ويصف هذه المخطوطة تطبيق المبادئ هيدرودينامية بالحقن الكلي الماوس مباشرة مع بلازميد الحمض النووي للتعبير الجيني الخاصة بالكلى. تم تخديره من الفئران والكلى يتم كشفها بواسطة شق الجناح متبوعاً حقن سريعة بلازميد الحل التي تحتوي على الحمض النووي مباشرة في الحوض الكلوي. ويحتفظ الإبرة في مكان لمدة عشر ثوان وهو خياطة شق الموقع. يعيش في اليوم التالي، تصوير الحيوان أو لطخة غربية، أو immunohistochemistry قد تستعمل للاعتداء التعبير الجيني أو فحوصات أخرى مناسبة للتحوير لخيار تستخدم للكشف عن البروتين للفائدة. أساليب المنشورة لإطالة أمد التعبير الجيني تشمل المعاملة التكامل والسيكلوفوسفاميد التحوير بوساطة ينقول تكبح الاستجابة المناعية للتحوير.

تقنية الجراحة وحقن بسيطة لأداء يتقن مرة واحدة، تتطلب لا معدات رئيسية أو مواد مكلفة. يوم واحد من التدريب على الفئران عدة المقرر للقتل الرحيم إذا جديدة إلى جراحة الكلي شق الجناح، ينبغي أن يسمح فيها الفئران ليست الجراحة التالية تم استردادها لأن المحاولة الأولى في هذه العمل?...

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

حقن الحوض الكلوي الهيدروديناميكي للتعبير غير الفيروسية من البروتينات في الكلي

ويصف هذا البروتوكول طريقة لحقن بلازميد الحمض النووي الكلي الماوس عبر الحوض الكلوي لإنتاج التعبير التحوير على وجه التحديد في الكلي.

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. ويصف هذا البروتوكول طريقة لحقن بلازميد الحمض النووي الكلي الماوس عبر الحوض الكلوي لإنتاج التعبير التحوير على وجه التحديد في الكلي.

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.

تقنية لاستهداف حقن مكروي إلى البلدان النامية<em&gt; القيطم</em&gt; الكلى

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.

في الوريد من يرقات اسماك الزرد Microinjections لدراسة إصابة الكلى الحاد

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

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.

تسليم داخل الأوعية الدموية من البيولوجيات إلى الكلى الجرذ

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.

حقن إنترارينال من<em&gt; الإشريكية القولونية</em&gt; في نموذج الجرذ من التهاب الحويضة والكلية

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

Transient Expression of Proteins by Hydrodynamic Gene Delivery in Mice

Efficient expression of transgenes in vivo is of critical importance in studying gene function and developing treatments for diseases. Over the past years, hydrodynamic gene delivery (HGD) has emerged as a simple, fast, safe and effective method for delivering transgenes into rodents. This technique relies on the force generated by the rapid injection of a large volume of physiological solution to increase the permeability of cell membranes of perfused organs and thus deliver DNA into cells. One of the main advantages of HGD is the ability to introduce transgenes into mammalian cells using naked plasmid DNA (pDNA). Introducing an exogenous gene using a plasmid is minimally laborious, highly efficient and, contrary to viral carriers, remarkably safe. HGD was initially used to deliver genes into mice, it is now used to deliver a wide range of substances, including oligonucleotides, artificial chromosomes, RNA, proteins and small molecules into mice, rats and, to a limited degree, other animals. This protocol describes HGD in mice and focuses on three key aspects of the method that are critical to performing the procedure successfully: correct insertion of the needle into the vein, the volume of injection and the speed of delivery. Examples are given to show the application of this method to the transient expression of two genes that encode secreted, primate-specific proteins, apolipoprotein L-I (APOL-I) and haptoglobin-related protein (HPR).

In vivo transfection of naked DNA by hydrodynamic gene delivery introduces genes into the tissue of an animal with minimal inflammatory response. Sufficient amounts of gene product are generated such that gene function and regulation as well as protein structure and function can be analyzed.

التعبير عابرة من البروتينات عن طريق هيدرودينامي الجينات في الفئران التسليم

Efficient expression of transgenes in vivo is of critical importance in studying gene function and developing treatments for diseases. Over the past ...

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.

توصيل الأدوية مباشرة إلى الكلى عبر الشريان الكلوي

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.

توصيل miRNA الخارجي المركب صناعيا إلى الكلى باستخدام جسيمات البولي إيثيلينيمين النانوية في العديد من نماذج الفئران لأمراض الكلى

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.

سم كلوي حقن مكروي في الزرد لنموذج قصور كلوي حاد

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 ...

دراسة شفط البروتين أثناء تطوير عدسة الدجاج

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.

تسليط الضوء على المؤلف: التحقيق في تطوير العدسة ووظيفتها من خلال الحقن المجهري للفيروس القهقري RCAS (A) في عدسة الدجاج الجنينية 

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.

الحقن المجهري للفيروس القهقري RCAS (A) المؤتلف في عدسة الدجاج الجنينية

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

حقن الحوض الكلوي الهيدروديناميكي للتعبير غير الفيروسية من البروتينات في الكلي

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تقنية لاستهداف حقن مكروي إلى البلدان النامية

في الوريد من يرقات اسماك الزرد Microinjections لدراسة إصابة الكلى الحاد

تسليم داخل الأوعية الدموية من البيولوجيات إلى الكلى الجرذ

حقن إنترارينال من

التعبير عابرة من البروتينات عن طريق هيدرودينامي الجينات في الفئران التسليم

توصيل الأدوية مباشرة إلى الكلى عبر الشريان الكلوي

توصيل miRNA الخارجي المركب صناعيا إلى الكلى باستخدام جسيمات البولي إيثيلينيمين النانوية في العديد من نماذج الفئران لأمراض الكلى

سم كلوي حقن مكروي في الزرد لنموذج قصور كلوي حاد

الحقن المجهري للفيروس القهقري RCAS (A) المؤتلف في عدسة الدجاج الجنينية

الحقن المجهري للفيروس القهقري RCAS (A) المؤتلف في عدسة الدجاج الجنينية