Die Arbeit von Autoren FCO, DF, JN, DJH-und GDS erwähnt durch den Chief Scientist Office, Grantnummer finanziert wird: ETM37 und unterstützt von der Cancer Research UK Experimental Cancer Medicine Centre. Die Arbeit von AL wird von der Royal College of Surgeons of Edinburgh Robertson Trust, die Melville Vertrauen für die Pflege und Heilung von Krebs und dem Medical Research Council finanziert. IO wird durch ein Royal Society of Edinburgh Scottish Government Fellowship by Marie-Curie-Maßnahmen und der UK Medical Research Council kofinanziert unterstützt. Die Autoren möchten SCOTRRCC Mitantragsteller und Mitarbeitern für ihre nützlichen Diskussionen über einige der Themen in diesem Papier diskutiert danken.

Ein. Identifizierung von morphologischen und molekularen Tumorheterogenität <ol><li> Tumore von -80 ° C Gefrierschrank entfernt und auf Trockeneis aufbewahrt. </li><li> Dividieren Tumoren in Abschnitte von etwa 1 cm 3. Karte die ursprüngliche Position von jedem Tumor Abschnitts relativ zu einander und das Etikett mit einem eindeutigen Namen. Lagern Sie die Proben in den einzelnen Kryoröhrchen bei -80 ° C bis zur Verwendung. </li><li> Coat Proben in Oktober und schneiden in einem Kryostaten bei -22 ° C. </li><li> Proben gefärbt mit Hämatoxylin und Eosin Gegenfärbung Methode. </li><li> Mikroskopie Analyse von Gefrierschnitten, um sicherzustellen, sie sind von klarzelligen Natur, lebensfähige Tumorzellen und Planieren (low grade, high grade oder gemischte low / high grade, schwierig zu unterscheiden, Fuhrman Klassen 1 bis 4 auf gefrorenen Abschnitt). Abbildung 1 a &amp; b (H &amp; E-Färbung der hoch niedrig und gemischte Klasse). </li><li> Wählen Sie bis zu 4 Proben von jeder morphologisch unterschiedliche Regionen innerhalb der einzelnen Tumor-Protein-Extraktion. </li></ol> 2. Protein Extraction aus Tumorproben <ol><li> Cut Tumorprobe vom Oktober </li><li> Ort 50-75 mg Gewebe in 2 ml-Röhrchen mit 990 ul Lysepuffer [50 mM Tris-HCl (pH 7,5), 5 mM EGTA (pH 8,5), 150 mM NaCl] mit Aprotinin (Sigma A6279) (10 mg ergänzt / ml), 2-Phosphatase-Inhibitor-Cocktail (Sigma, P5716), Phosphatase-Inhibitor-Cocktail 3 (Sigma, P0044) und einem Protease-Inhibitor-Cocktail (Roche, 11836153001). </li><li> Hinzufügen eines einzelnen 5 mm Stahlkugel in jedes Röhrchen und homogenisieren bei 50Hz für 5 min zweimal mit einem TissueLyser, die Überprüfung der Höhe der Homogenisierung nach jeder 5 min Zeit. </li><li> Übertragen homogenisierte Probe auf neue 2-ml-Tube mit Pipette Verlassen der Stahlkugel hinter sich. </li><li> Fügen Sie 10 ul Triton X-100 zu jeder Probe vor dem Zentrifugieren bei 13.000 × g für 30 min bei 4 ° C. </li><li> Übertragen Überstände an die frische Mikrozentrifugenröhrchen. </li><li> Bestimmen Proteinkonzentration mit BCA-Assay (Abbildung 2). </li><li>Normalisieren Proteinkonzentrationen von 1 mg / ml. </li></ol> 3. Antikörper Validation <ol><li> Planen Proteinproben (extrahiert aus geeigneten Zelllinien oder Gewebe) für Western Blot und auf einem 10% SDS-PAGE-Gel. </li><li> Übertragen Proben auf Nitrozellulosemembran Nacht bei 4 ° C. </li><li> Sperrung der Membran in Li-Cor Odyssey Blocking Buffer (50:50 verdünnt in PBS) für 1 Stunde bei Raumtemperatur. </li><li> Verdünnen Sie die primären Antikörper in Li-Cor Odyssey Blocking Buffer (verwässert 50:50 in PBS) bei den Herstellern empfohlenen Verdünnung typischerweise 1 in 1.000. </li><li> Inkubieren Membran in primären Antikörper über Nacht bei 4 ° C. </li><li> Make-up 0,1% PBS-Tween20 (PBS-T, 1 ml Tween20 / 1L PBS). </li><li> Waschen Membran in PBS-T bei Raumtemperatur für 5 min (x3). </li><li> Verdünnten Sekundärantikörper in Odyssey Blocking Buffer (verwässert 50:50 in PBS) 0,01% SDS bei 1:10,000 Verdünnung (1,5 μl/15 ml) fluoreszenzmarkierten. </li><li> Inkubieren Membran in sekundärenAntikörper bei Raumtemperatur für 45 min unter leichtem Schütteln - es ist wichtig, um die Membran von dem Licht, bis zu dem Zeitpunkt, zu schützen, wie er letztlich gescannt wurden. </li><li> Waschen Membran in PBS-T bei Raumtemperatur für 5 min (x3), Halten der Membran in der Dunkelheit. </li><li> Waschen Membran in PBS bei Raumtemperatur für 5 min (x3), um restliche Tween20 zu entfernen, wieder Halten der Membran in der Dunkelheit. </li><li> Lie Membran flach auf einem Stück Filterpapier in der Dunkelheit und an der Luft trocknen - so die Membran zu trocknen kann das Signal zu verbessern und reduzieren Hintergrund, aber unbrauchbar machen zum Abisolieren und wieder Sondieren. </li><li> Scannen Sie die Membran auf der Licor Odyssey Scanner. Halten der Membran in der Dunkelheit, solange es abgetastet wurde. </li><li> Nur auswählen Antikörpern, die eine einzelne Bande bei der überwiegenden korrekten Molekulargewicht zu erzeugen. 3 zeigt Beispiele für akzeptable und inakzeptable Antikörper zur Verwendung mit RPPA. </li></ol> 4. RPPA Printing <ol><li> Protein-Lysate wurden auf Nitrocellulose-beschichtete Glasobjektträger (Fastslides-Whatman) mit einem MicroGrid II Roboter spotter gesichtet. Die Folien verwendet wurden, enthielten 2 Pads, auf die die Proben wurden gesichtet. Jedes Pad wurde mit identischen Proben entdeckt, in diesem Fall 100 Proben. Andere verfügbare Formate sind 1, 8 und 16 Pad gleitet. Je höher die Anzahl von Pads je kleiner die Anzahl von Proben, die auf die jeweils gesichtet werden kann. </li><li> Eine Reihe von fünf 2-fache Verdünnungen wurden von jeder Probe hergestellt und jedes wurde dann in dreifacher gesichtet (was zu insgesamt 15 Flecken pro Probe). </li></ol> 5. RPPA Protein-Erkennung <ol><li> Feuchten Präparate im Überschuß Licor Blocking Buffer (50:50 verdünnt in PBS). Inkubation bei RT für 1 Stunde auf einem schaukelnden Plattform. </li><li> Planen 800 ul Primärantikörper in Licor Blocking Buffer (50:50 verdünnt in PBS) in den gewünschten Konzentrationen und auf Eis. </li><li> Montieren Sie die Folien in entweder (i) der einzelnen Frame Chip Clipoder (ii) die &quot;FastFrame &#39;vier Bucht Objektträgerhalter so dass eine Abdichtung zwischen dem Schieber und der Inkubationskammer gebildet. </li><li> Reste von Waschpuffer aus Brunnen und fügen Sie 600 ul primären Antikörper in die jeweiligen Vertiefungen. </li><li> Legen Sie die Folie und Kammer in einem verschlossenen nassen Box und inkubieren on rocking-Plattform über Nacht bei 4 ° C. </li><li> Make up 0,1% PBS-Tween20 (PBS-T; 100 ul Tween 20/100 ml PBS). </li><li> Objektträger aus kalten Raum und entfernen Sie vorsichtig die primären Antikörper aus jeder Vertiefung. </li><li> Fügen Sie 600 ul PBS-T und waschen Dias on rocking-Plattform bei RT für 5 min (X3). </li><li> Planen fluoreszenzmarkierten Sekundärantikörper durch Verdünnung in Blockierungspuffer Odyssey (verdünnt in PBS 50:50) 0,01% SDS bei 1:2000 Verdünnung (1 ul / 2 ml) in der ersten Instanz. </li><li> Entfernen Puffer aus Brunnen und fügen Sie 600 ul fluoreszenzmarkierten Sekundärantikörper die jeweiligen Vertiefungen. Sekundärantikörper Inkubieren bei Raumtemperatur für 45 min unter leichtem Schütteln - esWichtig ist, die Membran von dem Licht, solange es endgültig abgetastet schützen. </li><li> Entfernen Sekundärantikörper aus gut waschen und kurz (x3) in 600 ul PBS-T bei Raumtemperatur. Objektträger vom Träger, in einen geeigneten Behälter übertragen und waschen über PBS-T für 15 min, halten die Membran in der Dunkelheit. </li><li> Entfernen PBS-T und ferner Waschen Membran mit PBS bei Raumtemperatur für 15 min, um restliche Tween-20 zu entfernen, wieder Halten der Membran in der Dunkelheit. </li><li> Trocknen Sie die Fastslide in 50 ° C Ofen für 10 min und dann scannen auf dem Li-Cor Odyssey Scanner. Halten Sie die Folie in der Dunkelheit, bis es gescannt wurde. </li><li> Scannen die Folie bei 680 nm und / oder 800 nm in Abhängigkeit von der sekundären Antikörper / Antikörper verwendet. Für zweifarbige Detektion immer hochvernetzten adsorbierten Sekundärantikörper an Kreuzreaktivität minimieren. Eine sorgfältige Auswahl der primären und sekundären Antikörper ist notwendig für Zweifarb-Detektion. Von vorrangiger Bedeutung ist die Auswahl derHost verschiedenen Spezies (z. B. Kaninchen-und Maus) für die beiden primären Antikörper. Dies ermöglicht Diskriminierung durch Anti-Kaninchen und Anti-Maus-Sekundärantikörper, der mit Farbstoff mit leicht unterscheidbare Emissionsspektren markiert sind. </li><li> Bilddateien werden als. Tiff-Dateien gespeichert. Abbildung 4 (Bild der gescannten Dateien). </li></ol> 6. Data Analysis <ol><li> Starten Sie die MicroVigene Software (VigeneTech, Carlisle, MA, USA). </li><li> Zu öffnen. Tiff Bilddatei mit dem Scan des RPPA Folie. </li><li> Auswählen eines vordefinierten Vorlagendatei, die ein Gitter auf dem Bild des Schiebers RPPA überlagern erhalten wird. </li><li> Klicken Sie auf das Definieren Regions of Interest (ROI)-Taste, um das Grid. </li><li> Positionieren Sie den Grid über die RPPA Flecken. 6a (Bild Raster über Bild). </li><li> Klicken Sie auf die Schaltfläche Alle auswählen, um alle ROI markieren. </li><li> Klicken Sie auf Alle. MicroVigene wird autommatisch finden ROI, finden Sie die Flecken, subtrahieren Sie den Hintergrund, entfernen Sie Staub und quantifizieren Flecken. </li><li> Klicken Sie auf die Ansicht Verdünnungskurve Taste, um die Ergebnisse für alle Proben auf der RPPA Folie. </li><li> Klicken Sie auf Speichern Dilution Data. </li><li> Da jede Probe über 5 Verdünnung Punkten gedruckt wird jeweils dreimal gibt es 15 Punkten zu analysieren, wodurch die Gefahr von Fehlern vermindert und verbessert die Qualität der Kurvenanpassung. MicroVigene erzeugt eine 4-Parameter logistic-log-Modell &quot;Supercurve&quot;-Algorithmus (6b), die alle Spots eine Sigmoid-Kurve von Antigen-Antikörper-Bindung Kinetik produzieren enthält. Die Annahme ist, dass die gleichen Antikörper-Antigen-Bindungskinetik findet in jeder Probe vor Ort, auch in den verschiedenen Proben, also, indem alle Punkte auf einem Array eine gemeinsame Antwort-Kurve passen, das Vertrauen der Kurvenanpassung 10,11 zu erhöhen Y = a + ((ba) / (1 + e (c * d-ln (x))) wobei x der dilution Faktor und Y ist die Signalintensität. Proben können unter Verwendung der vergleichsweise y0-Wert, der in der Analyse zu der y-Wert entsprach am Mittelpunkt der x-Werte nach dem Mappen denjenigen auf den supercurve analysiert werden. </li><li> Exportieren Sie die Daten in Microsoft Excel und Grundstück y0 wie in Abbildung 7. </li><li> Intratumorale Protein Varianz wurde getrennt für unbehandelte und behandelte behandelten Patienten in einer ANOVA Rahmen berechnet. Varianz Verteilungen Kombinieren von Daten aus allen analysierten Proteine ​​wurden durch einen Mann-Whitney-Test (MWT) verglichen. False discovery rate (FDR) Korrektur wurde 12; Intratumorale Abweichungen für einzelne Proteine ​​wurden durch einen F-Test, bei denen Annahmen von Normalität und Homoskedastizität statt, jeweils mit den Lillefours und Fligner Tests beurteilt verglichen. Differential Proteinexpression zwischen unbehandelten und behandelten Patienten Proben wurde für jedes Protein unter Verwendung des Student-t-Test, bei dem Normalität und Homoskedastizität Annahmes erfüllt sind, sonst MWT durchgeführt wurde; FDR wurde über kombinierten t-Test und MWT Werte 12 angelegt. Bedeutung der Proteinexpression und Varianz Pearson-Korrelation für die Proteine ​​wurden mit Hilfe eines Standard-Ansatz [R Referenz] und FDR angewendet 12. </li></ol>

<ol>
	<li>Stewart, G. D., O'Mahony, F. C., Powles, T., Riddick, A. C., Harrison, D. 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=What+can+molecular+pathology+contribute+to+the+management+of+renal+cell+carcinoma.">What can molecular pathology contribute to the management of renal cell carcinoma.</a> Nat. Rev. Urol. 8, 255-265 (2011).</li><li>Rini, B. I., Michaelson, M. D., Rosenberg, J. E., Bukowski, R. M., Sosman, J. 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=Antitumor+activity+and+biomarker+analysis+of+sunitinib+in+patients+with+bevacizumab-refractory+metastatic+renal+cell+carcinoma.">Antitumor activity and biomarker analysis of sunitinib in patients with bevacizumab-refractory metastatic renal cell carcinoma.</a> J. Clin. Oncol. 26, 3743-3748 (2008).</li><li>Swanton, C., Larkin, J. M., Gerlinger, M., Eklund, A. C., Howell, 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=Predictive+biomarker+discovery+through+the+parallel+integration+of+clinical+trial+and+functional+genomics+datasets.">Predictive biomarker discovery through the parallel integration of clinical trial and functional genomics datasets.</a> Genome Med. 2, 52 (2010).</li><li>Cortes, J., Jabbour, E., Kantarjian, H., Yin, C. C., Shan, 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=Dynamics+of+BCR-ABL+kinase+domain+mutations+in+chronic+myeloid+leukemia+after+sequential+treatment+with+multiple+tyrosine+kinase+inhibitors.">Dynamics of BCR-ABL kinase domain mutations in chronic myeloid leukemia after sequential treatment with multiple tyrosine kinase inhibitors.</a> Blood. 110, 4005-4011 (2007).</li><li>Gerlinger, M., Rowan, A. J., Horswell, S., Larkin, J., Endesfelder, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intratumor+heterogeneity+and+branched+evolution+revealed+by+multiregion+sequencing.">Intratumor heterogeneity and branched evolution revealed by multiregion sequencing.</a> N. Engl. J. Med. 366, 883-892 (2012).</li><li>Fisher, R., Larkin, J., Swanton, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inter+and+intratumour+heterogeneity:+a+barrier+to+individualized+medical+therapy+in+renal+cell+carcinoma.">Inter and intratumour heterogeneity: a barrier to individualized medical therapy in renal cell carcinoma.</a> Front. Oncol. 2, 49 (2012).</li><li>Yachida, S., Jones, S., Bozic, I., Antal, T., Leary, 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=Distant+metastasis+occurs+late+during+the+genetic+evolution+of+pancreatic+cancer.">Distant metastasis occurs late during the genetic evolution of pancreatic cancer.</a> Nature. 467, 1114-1117 (2010).</li><li>O'Mahony, F. C., Faratian, D., Varley, J., Nanda, J., Theodoulou, 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=The+use+of+automated+quantitative+analysis+to+evaluate+epithelial-to-mesenchymal+transition+associated+proteins+in+clear+cell+renal+cell+carcinoma.">The use of automated quantitative analysis to evaluate epithelial-to-mesenchymal transition associated proteins in clear cell renal cell carcinoma.</a> PLoS One. 7, e31557 (2012).</li><li>Spurrier, B., Ramalingam, S., Nishizuka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reverse-phase+protein+lysate+microarrays+for+cell+signaling+analysis.">Reverse-phase protein lysate microarrays for cell signaling analysis.</a> Nat. Protoc. 3, 1796-1808 (2008).</li><li>Hu, J., He, X., Baggerly, K. A., Coombes, K. R., Hennessy, B. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-parametric+quantification+of+protein+lysate+arrays.">Non-parametric quantification of protein lysate arrays.</a> Bioinformatics. 23, 1986-1994 (2007).</li><li>Wang, X., Dong, Y., Jiwani, A. J., Zou, Y., Pastor, 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=Improved+protein+arrays+for+quantitative+systems+analysis+of+the+dynamics+of+signaling+pathway+interactions.">Improved protein arrays for quantitative systems analysis of the dynamics of signaling pathway interactions.</a> Proteome Sci. 9, 53 (2011).</li><li>Benjamini, Y., Hochberg, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlling+the+False+Discovery+Rate:+A+Practical+and+Powerful+Approach+to+Multiple+Testing.">Controlling the False Discovery Rate: A Practical and Powerful Approach to Multiple Testing.</a> Journal of the Royal Statistical Society Series B (Methodological). 57, 289-300 (1995).</li></ol>

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Die RPPA hier vorgestellte Methode stellt einen hohen Durchsatz Alternative zu den weit verbreiteten, aber vergleichsweise geringen Durchsatz Western-Blot-Technik von Protein-Analyse. Das Verfahren erlaubt Hunderte von Proben werden semiquantitativ ausgewertet und gleichzeitig zur direkten Vergleich der wichtigsten Proteine ​​in einem breiten Auswahl von Zelllinien und Gewebeproben. Multiplexen mit unterschiedlichen Antikörperspezies weiter erhöht die Leistung der Technik so dass mehrere Antikörper gleichzeitig e...

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td></tr><tr><td> Aprotinin </td><td> Sigma </td><td> A6279 </td></tr><tr><td> Phosphatase-Inhibitor-Cocktail 2 </td><td> Sigma </td><td> P5726 </td></tr><tr><td> Phosphatase-Inhibitor-Cocktail 3 </td><td> Sigma </td><td> P0044 </td></tr><tr><td> Proteaseinhibitorcocktail </td><td> Roche </td><td> 11836153001 </td></tr><tr><td> Triton X-100 </td><td> Triton-X </td><td> T8787 </td></tr><tr><td> Li-Cor Odyssey Blocking Buffer </td><td> Li-Cor </td><td> 927-40000 </td></tr><tr><td> TissueLyser </td><td> Qiagen </td><td> 85600 </td></tr><tr><td> MicroGrid II Roboter spotter </td><td> Biorobotics </td><td></td></tr><tr><td> FastFrame &quot;vier bay Diahalter </td><td> Whatman </td><td> 10486001 </td></tr><tr><td> FAST Slide - 2-Pad </td><td> Whatman </td><td> 10485317 </td></tr><tr><td> IRDye 680LT Goat anti-Mouse IgG </td><td> Licor </td><td> 926-68020 </td></tr><tr><td> IRDye 800CW Ziege anti-Kaninchen IgG </td><td> Licor </td><td> 926-32211 </td></tr></tbody></table>

the use of reverse phase protein arrays (rppa) to explore protein expression variation within individual renal cell cancers

Derzeit gibt es keine kurative Behandlung für metastasierten klarzelligen Nierenzellkarzinoms, der häufigsten Variante der Krankheit. Ein wichtiger Faktor in diesem Therapieresistenz wird gedacht, um die molekulare Komplexität der Erkrankung 1 sein. Gezielte Therapie wie die Tyrosinkinase-Inhibitor (TKI)-Sunitinib genutzt wurden, aber nur 40% der Patienten reagieren, mit der überwältigenden Mehrheit dieser Patienten schubförmig innerhalb von 1 Jahr 2. Als solche ist die Frage der intrinsischen und erworbenen Resistenz in Nierenzellkrebskranken ist höchst relevant 3. Um den Widerstand gegen TKI zu studieren, mit dem Ziel der Entwicklung wirksamer, individuell abgestimmte Behandlungen wird sequentielle Gewebe nach einer bestimmten Zeit der gezielten Therapie erforderlich, ein Konzept, das sich bewährt bei chronischer myeloischer Leukämie 4 hatte. Doch die Anwendung einer solchen Strategie in Nierenzellkarzinom ist durch das hohe Niveau der b kompliziertoth inter-und intratumorale Heterogenität, die ein Merkmal des Nierenzellkarzinoms 5,6 sowie anderen soliden Tumoren 7 ist. Intertumoral Heterogenität aufgrund der Transkriptom-und genetische Unterschiede ist gut, auch bei Patienten mit ähnlichen Präsentation, Stadium und Grad des Tumors festgestellt. Darüber hinaus ist es klar, dass es große morphologische (intratumorale) Heterogenität in RCC, die wahrscheinlich noch größere molekulare Heterogenität repräsentieren. Detaillierte Kartierungen und Kategorisierung von RCC Tumoren durch kombinierte morphologische Analyse und Fuhrman Einstufung ermöglicht die Auswahl von repräsentativen Flächen für Proteomanalyse. Protein-basierte Analyse der RCC 8 ist attraktiv wegen seiner breiten Verfügbarkeit in Pathologie-Laboratorien, aber ihre Anwendung problematisch sein kann aufgrund der begrenzten Verfügbarkeit von spezifischen Antikörpern 9. Aufgrund der Dot-Blot Art der Reverse Phase Protein Arrays (RPPA), Antikörper-Spezifität muss be vorvalidierten, als solche strengen Qualitätskontrolle von Antikörpern verwendet wird, ist von größter Bedeutung. Trotz dieser Einschränkung der Dot-Blot-Format nicht zulässt Assay Miniaturisierung, so dass für den Druck von Hunderten von Proben auf einen einzelnen Nitrocellulose Folie. Bedruckte Folien können dann in einer ähnlichen Weise wie Western-Analyse werden unter Verwendung von Ziel-spezifischen primären Antikörper und fluoreszenzmarkierten Sekundärantikörper analysiert, so dass für Multiplexing. Differential Proteinexpression in allen Proben auf einer Folie kann dann gleichzeitig durch Vergleichen der relativen Pegel der Fluoreszenz in einer kostengünstigeren und Weise mit hohem Durchsatz untersucht werden.

Ein Beispiel eines gescannten RPPA Schieber kann in 4 (i) sowohl mit 680 und 800 nm dargestellt Kanäle erkennen. Trennen der Bilder durch Wellenlänge, Abbildung 4 (ii) kann jedes Pad auf der RPPA Schieber zu analysierenden und individuelle Proteinexpression bestimmt Abbildung 4 (iii). Wie in 4 gesehen werden kann, (iii) die Expression von Proteinen in den Proben ist einzigartig mit Gelsolin mit einem hohen Grad der Expression auf den ...

Watch this Scientific Journal Video about The Use of Reverse Phase Protein Arrays RPPA to Explore Protein Expression Variation within Individual Renal Cell Cancers at JoVE.com

The Use of Reverse Phase Protein Arrays RPPA to Explore Protein Expression Variation within Individual Renal Cell Cancers

RPPA ermöglicht die Proteinexpression von Hunderten von Proben auf Nitrozellulose Folien gedruckt, um gleichzeitig abgefragt werden, mit fluoreszenzmarkierten Antikörpern. Diese Technik angewendet worden ist, um die Wirkung der medikamentösen Behandlung Heterogenität innerhalb klarzelligen Nierenkarzinom studieren.

Der Einsatz von Reverse Phase Protein Arrays (RPPA) an Protein Expression Variation innerhalb der einzelnen Renal Cell Cancers erkunden

Currently there is no curative treatment for metastatic clear cell renal cell cancer, the commonest variant of the disease. A key factor in this treatment ...

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The Use of Reverse Phase Protein Arrays (RPPA) to Explore Protein Expression Variation within Individual Renal Cell Cancers

33.5K Aufrufe.  University of Edinburgh. RPPA ermöglicht die Proteinexpression von Hunderten von Proben auf Nitrozellulose Folien gedruckt, um gleichzeitig abgefragt werden, mit fluoreszenzmarkierten Antikörpern. Diese Technik angewendet worden ist, um die Wirkung der medikamentösen Behandlung Heterogenität innerhalb klarzelligen Nierenkarzinom studieren.

Serie: The Use of Reverse Phase Protein Arrays RPPA to Explore Protein Expression Variation within Individual Renal Cell Cancers

Use of a Hanging-weight System for Isolated Renal Artery Occlusion

In hospitalized patients, over 50% of cases of acute kidney injury (AKI) are caused by renal ischemia 1-3. A recent study of hospitalized patients revealed that only a mild increase in serum creatinine levels (0.3 to 0.4 mg/dl) is associated with a 70% greater risk of death than in persons without any increase 1. Along these lines, surgical procedures requiring cross-clamping of the aorta and renal vessels are associated with a renal failure rates of up to 30% 4. Similarly, AKI after cardiac surgery occurs in over 10% of patients under normal circumstances and is associated with dramatic increases in mortality. AKI are also common complications after liver transplantation. At least 8-17% of patients end up requiring renal replacement therapy 5. Moreover, delayed graft function due to tubule cell injury during kidney transplantation is frequently related to ischemia-associated AKI 6. Moreover, AKI occurs in approximately 20% of patients suffering from sepsis 6.The occurrence of AKI is associated with dramatic increases of morbidity and mortality 1. Therapeutic approaches are very limited and the majority of interventional trials in AKI have failed in humans. Therefore, additional therapeutic modalities to prevent renal injury from ischemia are urgently needed 3, 7-9. 
To elucidate mechanisms of renal injury due to ischemia and possible therapeutic strategies murine models are intensively required 7-13. Mouse models provide the possibility of utilizing different genetic models including gene-targeted mice and tissue specific gene-targeted mice (cre-flox system). However, murine renal ischemia is technically challenging and experimental details significantly influence results. We performed a systematic evaluation of a novel model for isolated renal artery occlusion in mice, which specifically avoids the use of clamping or suturing the renal pedicle 14. This model requires a nephrectomy of the right kidney since ischemia can be only performed in one kidney due to the experimental setting. In fact, by using a hanging-weight system, the renal artery is only instrumented once throughout the surgical procedure. In addition, no venous or urethral obstruction occurs with this technique. We could demonstrate time-dose-dependent and highly reproducible renal injury with ischemia by measuring serum creatinine. Moreover, when comparing this new model with conventional clamping of the whole pedicle, renal protection by ischemic preconditioning is more profound and more reliable. Therefore his new technique might be useful for other researchers who are working in the field of acute kidney injury.

A precise murine model for acute kidney injury (AKI) due to ischemia is an important tool to investigate acute kidney injury and possibly find therapeutic tools to treat renal injury. The hanging weight system offers a tool for immediate and reliable renal artery occlusion and reperfusion without causing renal congestion.

Die Verwendung eines Hanging-weight-System für Isolated Nierenarterie Occlusion

In hospitalized patients, over 50% of cases of acute kidney injury (AKI) are caused by renal ischemia 1-3. A recent study of hospitalized patients ...

Forschung

Medizin

Heterogeneity Mapping of Protein Expression in Tumors using Quantitative Immunofluorescence

Morphologic heterogeneity within an individual tumor is well-recognized by histopathologists in surgical practice. While this often takes the form of areas of distinct differentiation into recognized histological subtypes, or different pathological grade, often there are more subtle differences in phenotype which defy accurate classification (Figure 1). Ultimately, since morphology is dictated by the underlying molecular phenotype, areas with visible differences are likely to be accompanied by differences in the expression of proteins which orchestrate cellular function and behavior, and therefore, appearance. The significance of visible and invisible (molecular) heterogeneity for prognosis is unknown, but recent evidence suggests that, at least at the genetic level, heterogeneity exists in the primary tumor1,2, and some of these sub-clones give rise to metastatic (and therefore lethal) disease. 
Moreover, some proteins are measured as biomarkers because they are the targets of therapy (for instance ER and HER2 for tamoxifen and trastuzumab (Herceptin), respectively). If these proteins show variable expression within a tumor then therapeutic responses may also be variable. The widely used histopathologic scoring schemes for immunohistochemistry either ignore, or numerically homogenize the quantification of protein expression. Similarly, in destructive techniques, where the tumor samples are homogenized (such as gene expression profiling), quantitative information can be elucidated, but spatial information is lost. Genetic heterogeneity mapping approaches in pancreatic cancer have relied either on generation of a single cell suspension3, or on macrodissection4. A recent study has used quantum dots in order to map morphologic and molecular heterogeneity in prostate cancer tissue5, providing proof of principle that morphology and molecular mapping is feasible, but falling short of quantifying the heterogeneity. Since immunohistochemistry is, at best, only semi-quantitative and subject to intra- and inter-observer bias, more sensitive and quantitative methodologies are required in order to accurately map and quantify tissue heterogeneity in situ.
We have developed and applied an experimental and statistical methodology in order to systematically quantify the heterogeneity of protein expression in whole tissue sections of tumors, based on the Automated QUantitative Analysis (AQUA) system6. Tissue sections are labeled with specific antibodies directed against cytokeratins and targets of interest, coupled to fluorophore-labeled secondary antibodies. Slides are imaged using a whole-slide fluorescence scanner. Images are subdivided into hundreds to thousands of tiles, and each tile is then assigned an AQUA score which is a measure of protein concentration within the epithelial (tumor) component of the tissue. Heatmaps are generated to represent tissue expression of the proteins and a heterogeneity score assigned, using a statistical measure of heterogeneity originally used in ecology, based on the Simpson's biodiversity index7.
To date there have been no attempts to systematically map and quantify this variability in tandem with protein expression, in histological preparations. Here, we illustrate the first use of the method applied to ER and HER2 biomarker expression in ovarian cancer. Using this method paves the way for analyzing heterogeneity as an independent variable in studies of biomarker expression in translational studies, in order to establish the significance of heterogeneity in prognosis and prediction of responses to therapy.

Here we describe a method to quantify molecular heterogeneity in histological sections of tumor material using quantitative immunofluorescence, image analysis, and a statistical measure of heterogeneity. The method is intended for use in clinical biomarker development and analysis.

Heterogenität Mapping von Protein Expression in Tumoren mittels quantitativer Immunfluoreszenz

Morphologic heterogeneity within an individual tumor is well-recognized by histopathologists in surgical practice. While this often takes the form of ...

Production and Detection of Reactive Oxygen Species (ROS) in Cancers

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive molecules contain an oxygen and include H2O2 (hydrogen peroxide), NO (nitric oxide), O2- (oxide anion), peroxynitrite (ONOO-), hydrochlorous acid (HOCl), and hydroxyl radical (OH-).
Oxidative species are produced not only under pathological situations (cancers, ischemic/reperfusion, neurologic and cardiovascular pathologies, infectious diseases, inflammatory diseases 1, autoimmune diseases 2, etc&hellip;) but also during physiological (non-pathological) situations such as cellular metabolism 3, 4. Indeed, ROS play important roles in many cellular signaling pathways (proliferation, cell activation 5, 6, migration 7 etc..). ROS can be detrimental (it is then referred to as "oxidative and nitrosative stress") when produced in high amounts in the intracellular compartments and cells generally respond to ROS by upregulating antioxidants such as superoxide dismutase (SOD) and catalase (CAT), glutathione peroxidase (GPx) and glutathione (GSH) that protects them by converting dangerous free radicals to harmless molecules (i.e. water). Vitamins C and E have also been described as ROS scavengers (antioxidants).
Free radicals are beneficial in low amounts 3. Macrophage and neutrophils-mediated immune responses involve the production and release of NO, which inhibits viruses, pathogens and tumor proliferation 8. NO also reacts with other ROS and thus, also has a role as a detoxifier (ROS scavenger). Finally NO acts on vessels to regulate blood flow which is important for the adaptation of muscle to prolonged exercise 9, 10. Several publications have also demonstrated that ROS are involved in insulin sensitivity 11, 12.
Numerous methods to evaluate ROS production are available. In this article we propose several simple, fast, and affordable assays; these assays have been validated by many publications and are routinely used to detect ROS or its effects in mammalian cells. While some of these assays detect multiple ROS, others detect only a single ROS.

Production and Detection of Reactive Oxygen Species ROS in Cancers

Here we propose simple methods to test and evaluate the presence of reactive oxygen species in cells.

Produktion und Nachweis von reaktiven Sauerstoffspezies (ROS) in Cancers

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive ...

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

Biomarker in einem Tiermodell für die Aufdeckung Neural, hämatologischen und Behavioral Korrelate der PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

Purification and Aggregation of the Amyloid Precursor Protein Intracellular Domain

Amyloid precursor protein (APP) is a type I transmembrane protein associated with the pathogenesis of Alzheimer's disease (AD). APP is characterized by a large extracellular domain and a short cytosolic domain termed the APP intracellular domain (AICD). During maturation through the secretory pathway, APP can be cleaved by proteases termed &alpha;, &beta;, and &gamma;-secretases1. Sequential proteolytic cleavage of APP with &beta; and &gamma;-secretases leads to the production of a small proteolytic peptide, termed A&beta;, which is amyloidogenic and the core constituent of senile plaques. The AICD is also liberated from the membrane after secretase processing, and through interactions with Fe65 and Tip60, can translocate to the nucleus to participate in transcription regulation of multiple target genes2,3. Protein-protein interactions involving the AICD may affect trafficking, processing, and cellular functions of holo-APP and its C-terminal fragments. We have recently shown that AICD can aggregate in vitro, and this process is inhibited by the AD-implicated molecular chaperone ubiquilin-14. Consistent with these findings, the AICD has exposed hydrophobic domains and is intrinsically disordered in vitro5,6, however it obtains stable secondary structure when bound to Fe657. We have proposed that ubiquilin-1 prevents inappropriate inter- and intramolecular interactions of AICD, preventing aggregation in vitro and in intact cells4. While most studies focus on the role of APP in the pathogenesis of AD, the role of AICD in this process is not clear. Expression of AICD has been shown to induce apoptosis8, to modulate signaling pathways9, and to regulate calcium signaling10. Over-expression of AICD and Fe65 in a transgenic mouse model induces Alzheimer's like pathology11, and recently AICD has been detected in brain lysates by western blotting when using appropriate antigen retrieval techniques12. To facilitate structural, biochemical, and biophysical studies of the AICD, we have developed a procedure to produce recombinantly large amounts of highly pure AICD protein. We further describe a method for inducing the in vitro thermal aggregation of AICD and analysis by atomic force microscopy. The methods described are useful for biochemical, biophysical, and structural characterization of the AICD and the effects of molecular chaperones on AICD aggregation.

A method for large-scale purification of the APP intracellular domain (AICD) is described. We also describe methodology to induce in vitro AICD aggregation and visualization by atomic force microscopy. The methods described are useful for biochemical/structural characterization of the AICD and the effects of molecular chaperones on its aggregation.

Reinigung und Aggregation des Amyloid Precursor Protein intrazelluläre Domäne

Amyloid precursor protein (APP) is a type I transmembrane protein associated with the pathogenesis of Alzheimer's disease (AD). APP is characterized by a ...

Microarray-based Identification of Individual HERV Loci Expression: Application to Biomarker Discovery in Prostate Cancer

The prostate-specific antigen (PSA) is the main diagnostic biomarker for prostate cancer in clinical use, but it lacks specificity and sensitivity, particularly in low dosage values1&#8203;&#8203;. &#8216;How to use PSA&#39; remains a current issue, either for diagnosis as a gray zone corresponding to a concentration in serum of 2.5-10 ng/ml which does not allow a clear differentiation to be made between cancer and noncancer2 or for patient follow-up as analysis of post-operative PSA kinetic parameters can pose considerable challenges for their practical application3,4. Alternatively, noncoding RNAs (ncRNAs) are emerging as key molecules in human cancer, with the potential to serve as novel markers of disease, e.g. PCA3 in prostate cancer5,6 and to reveal uncharacterized aspects of tumor biology. Moreover, data from the ENCODE project published in 2012 showed that different RNA types cover about 62% of the genome. It also appears that the amount of transcriptional regulatory motifs is at least 4.5x higher than the one corresponding to protein-coding exons. Thus, long terminal repeats (LTRs) of human endogenous retroviruses (HERVs) constitute a wide range of putative/candidate transcriptional regulatory sequences, as it is their primary function in infectious retroviruses. HERVs, which are spread throughout the human genome, originate from ancestral and independent infections within the germ line, followed by copy-paste propagation processes and leading to multicopy families occupying 8% of the human genome (note that exons span 2% of our genome). Some HERV loci still express proteins that have been associated with several pathologies including cancer7-10. We have designed a high-density microarray, in Affymetrix format, aiming to optimally characterize individual HERV loci expression, in order to better understand whether they can be active, if they drive ncRNA transcription or modulate coding gene expression. This tool has been applied in the prostate cancer field (Figure 1).

Human endogenous retroviruses (HERV), which occupy 8% of the human genome, retain scarce coding capacities but a hundred thousand long terminal repeats (LTRs). A custom Affymetrix microarray was designed to identify individual HERV locus expression and was used on prostate cancer tissues as a proof of concept for future clinical studies.

Microarray-basierte Identifizierung von Einzel HERV Expression Loci: Anwendung auf Biomarker Discovery in Prostatakrebs

The prostate-specific antigen (PSA) is the main diagnostic biomarker for prostate cancer in clinical use, but it lacks specificity and sensitivity, ...

Three-Dimensional Culture Assay to Explore Cancer Cell Invasiveness and Satellite Tumor Formation

Mammalian cell culture in monolayers is widely used to study various physiological and molecular processes. However, this approach to study growing cells often generates unwanted artifacts. Therefore, cell culture in a three-dimensional (3D) environment, often using extracellular matrix components, emerged as an interesting alternative due to its close similarity to the native in vivo tissue or organ. We developed a 3D cell culture system using two compartments, namely (i) a central compartment containing cancer cells embedded in a collagen gel acting as a pseudo-primary macrospherical tumor and (ii) a peripheral cell-free compartment made of a fibrin gel, i.e. an extracellular matrix component different from that used in the center, in which cancer cells can migrate (invasion front) and/or form microspherical tumors representing secondary or satellite tumors. The formation of satellite tumors in the peripheral compartment is remarkably correlated to the known aggressiveness or metastatic origin of the native tumor cells, which makes this 3D culture system unique. This cell culture approach might be considered to assess cancer cell invasiveness and motility, cell-extracellular matrix interactions and as a method to evaluate anti-cancer drug properties.

Cancer cells are embedded in a collagen gel and then sandwiched in an acellular fibrin gel to generate a 3D culture system in which the invasiveness and formation of satellite tumors may be monitored.

Dreidimensionale Kulturtest auf Krebszelle Invasivität und Satelliten Tumorbildung erkunden

Mammalian cell culture in monolayers is widely used to study various physiological and molecular processes. However, this approach to study growing cells ...

An Efficient Sieving Method to Isolate Intact Glomeruli from Adult Rat Kidney

Preservation of glomerular structure and function is pivotal in the prevention of glomerulonephritis, a category of kidney disease characterized by proteinuria which can eventually lead to chronic and end-stage renal disease. The glomerulus is a complex apparatus responsible for the filtration of plasma from the body. In disease, structural integrity is lost and allows for the abnormal leakage of plasma contents into the urine. A method to isolate and examine glomeruli in culture is critical for the study of these diseases. In this protocol, an efficient method of retrieving intact glomeruli from adult rat kidneys while conserving structural and morphological characteristics is described. This process is capable of generating high yields of glomeruli per kidney with minimal contamination from other nephron segments. With these glomeruli, injury conditions can be mimicked by incubating them with a variety of chemical toxins, including protamine sulfate, which causes foot process effacement and proteinuria in animal models. Degree of injury can be assessed using transmission electron microscopy, immunofluorescence staining, and western blotting. Nephrin and Wilms Tumor 1 (WT1) levels can also be assessed from these cultures. Due to the ease and flexibility of this protocol, the isolated glomeruli can be utilized as described or in a way that best suits the needs of the researcher to help better study glomerular health and structure in diseased states.

The main focus of this protocol is to efficiently isolate viable primary glomeruli cultures with minimal contaminants for use in a variety of downstream applications. The isolated glomeruli retain structural relationships between component cell types and can be cultured ex vivo for a short time.

Eine effiziente Methode der Absiebung, intakten Glomeruli von Erwachsenen Ratte Niere zu isolieren

Preservation of glomerular structure and function is pivotal in the prevention of glomerulonephritis, a category of kidney disease characterized by ...

Precision Implementation of Minimal Erythema Dose (MED) Testing to Assess Individual Variation in Human Inflammatory Response

Minimal erythema dose (MED) testing is frequently used in clinical settings for determining the smallest amount of ultraviolet (UV) irradiation necessary to produce erythema (inflammatory reddening) on the surface of the skin. In this context, the MED is regarded as a key factor in determining starting doses for UV phototherapy for common skin conditions such as psoriasis and eczema. In research settings, MED testing also has potential to be a powerful tool for assessing within- and between-persons variation in inflammatory responses. However, MED testing has not been widely adopted for use in research settings, likely owing to a lack of published guidelines, which is a barrier to obtaining reproducible results from this assay. Also, protocols and equipment for establishing MED vary widely, making it difficult to compare results across laboratories. Here, we describe a precise and reproducible method to induce and measure superficial erythema using newly designed protocols and methods that can easily be adapted to other equipment and laboratory environments. The method described here includes detail on procedures that will allow extrapolation of a standardized dosage schedule to other equipment so that this protocol can be adapted to any UV radiation source.

Precision Implementation of Minimal Erythema Dose MED Testing to Assess Individual Variation in Human Inflammatory Response

Minimal erythema dose (MED) testing is used to establish dosage schedules for ultraviolet radiation phototherapy. It can assess individual variation in inflammatory response but lacks methodology for achieving reproducible results. Here, we present a precision implementation of MED and demonstrate its ability to capture individual variation in inflammatory response.

Präzisionsimplementierung von Minimal Erythema Dose (MED)-Tests zur Beurteilung individueller Variationen in der menschlichen Entzündlichen Reaktion

Minimal erythema dose (MED) testing is frequently used in clinical settings for determining the smallest amount of ultraviolet (UV) irradiation necessary ...

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 ...