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Ischaemia-reperfusion (IR) injury makes a major contribution to graft damage during kidney transplantation and increases the risks of primary non-function, delayed graft function and rejection. Oxidative damage to mitochondria is a key early event in IR injury. The aim of this project was to examine the safety and efficacy of the mitochondria-targeted antioxidant MitoQ in reducing pig and human kidney IR injury using an ex vivo normothermic perfusion (EVNP) system. Over a range of 500 nM to 250 µM using a 150 pig kidneys and 80 declined deceased human kidneys, MitoQ was successfully taken up by pig and human kidneys in a concentration-dependent manner, resulting in stable tissue concentrations over 24 hours of cold storage followed by 6 hours of EVNP. The uptake of MitoQ was increased approximately 2-fold when MitoQ was administered to warm (rather than cold) kidneys and when kidneys were preserved using hypothermic machine perfusion (rather than cold static storage). 50 µM MitoQ, administered to pig kidneys at the end of warm ischaemia, significantly increased renal blood and urine output flow at the end of 6 h EVNP compared to the control group. Creatinine clearance was numerically higher in the 50 µM MitoQ group compared to the control group but the difference did not reach statistical significance. To test the safety and efficacy of MitoQ in human kidney IRI, pairs of declined deceased human kidneys were used, with one kidney in each pair used as control. The total urine output, creatinine clearance and percentage fall of serum creatinine were numerically higher in the 50 µM MitoQ group compared to the control group, although the differences did not reach statistical significance during 3 h of EVNP. There was a significant difference in the renal blood flow between the 50 µM MitoQ group and the control group at the end of the first hour of EVNP. The renal blood flow remained relatively stable during the first hour of EVNP in the 50 µM MitoQ group compared to a significant decrease in renal blood flow in the control group. There was no effect on fractional excretion of sodium or oxidative injury markers (protein carbonyl formation, lipid peroxidation) in pig or human kidneys, which is consistent with previous studies that demonstrated the requirement of >24 hour after reperfusion for manifestation of changes in these parameters. In this thesis, I was able to successfully demonstrate the safety and potential efficacy of MitoQ in ameliorating renal IRI using pig kidneys. While more declined deceased human kidneys need to be analysed to fully explore the potential efficacy of MitoQ in ameliorating renal IRI, this study provides important data that will help inform future studies and ultimately a clinical trial for assessing the efficacy of the mitochondria-targeted antioxidant MitoQ in human kidney transplantation. My findings suggest that MitoQ has the potential to increase the use of marginal kidneys and to improve graft and patient outcomes.

Thesis ( M.Sc.). / Written for the Dept. of Epidemiology and Biostatistics. Title from title page of PDF (viewed 2008/05/29). Includes bibliographical references.

Zusammenfassung der Arbeit /Dissertation zur Erlangung des akademischen Grades Dr. med. Hyperspectral Imaging (HSI) of Human Kidney Allografts Die HSI Bildgebung ist ein nichtinvasives Instrument zur quantitativen Bewertung der Sauerstoffanreicherung parenchymatöser Organe, der Mikroperfusion von Transplantaten sowie zur Messung der Hämoglobin- und Wasserkonzentration von Organen. Aktuell wird die HSI Bildgebung bereits in der neuronalen Funktionsdiagnostik zur detaillierten Gewebedifferenzierung genutzt18 und dient zur Überprüfung der Perfusion komplexer Anastomosen im Bereich der Ösophagus- und der Leberchirurgie31,32. Die ersten Ergebnisse zur Tumordetektion konnten ebenfalls erfolgversprechend publiziert werden20. Ziel unserer Studie war es das Potenzial dieser neuartigen Bildgebungstechnik im Rahmen der Transplantationsmedizin vorzustellen und zu beschreiben. Es sollte gezeigt werden, ob die HSI Analyse für die intraoperative Qualitäts- und Lebensfähigkeitsbewertung bei Nierentransplantationen verwendet werden kann. Generell hat sich die Komplikationsrate nach erfolgter Nierentransplantation in den letzten Jahrzehnten von bis zu 30% auf weniger als 6% verringert6. Die Einschätzung der Organqualität während der Organentnahme oder Implantation, die potentiellen Funktionalität einer Transplantatniere im Rahmen einer postmortal oder auch lebend gespendeten Niere ist jedoch fortwährend schwer33. Im Rahmen der aktuellen Studie, wurden zur Erläuterung der beschriebenen Zielsetzung 17 postmortale Nierentransplantationen mit der hyperspektralen Kamera 15 und 45 Minuten nach Reperfusion fotografiert. Hierbei wurden mithilfe der HSI- Kamera die Gewebeoxygenierung (StO2), die Perfusion (NIR-Perfusionsindex), der OHI (Organ Hämoglobin Index) und der TWI (Tissue-Water-Index, Gehalt des Wassers des Gewebes) erfasst19,31. Revisionseingriffe, wie die Ureterneuimplantation und eine Thrombektomie wurden ebenfalls begleitet und analysiert. Zwei Patienten (11,8%) entwickelten eine Delayed Graft Function (DGF). Als Ergebnis zeigte sich, dass die optisch ermittelte Oxygenierung und Mikroperfusion bei Patienten mit einer DGF in unserer Studie signifikant verringert war. Gleiches galt für den NIR Perfusionsindex. Bei einer postoperativen Ureternekrose wurde ein deutliches NIR-Perfusionsdefizite des nekrotischen Teils dargestellt. Die Bestätigung erfolgte durch die Histopathologie. Wir konnten zeigen, dass eine genaue, quantifizierbare Aussage über die Mikroperfusion im arteriellen sowie venösen Stromgebiet möglich ist. Eine sichere und nicht invasive Untersuchungsmethode ist somit während einer Nierentransplantation integrierbar ohne den Ablauf der Operation zu beeinflussen. Die hyperspektrale Bildgebung ermöglicht somit intraoperativ die Organfunktion während der Transplantation zu bewerten und eine verzögerte Transplantatfunktion vorherzusagen. Als Kritikpunkt an der neuen Methode könnte angeführt werden, dass die Technik der HSI Visualisierung eine direkte Sicht auf die Niere erfordert. Grund dafür ist, dass die derzeitige maximale Eindringtiefe für Mikrozirkulationsmessungen 4 bis 6 Millimeter beträgt. Die intraoperative Sonographie ist aktuell das Einzige vergleichbare, bildgebende, zugelassene und nicht invasive Verfahren zur Bewertung der Organe. Mit der Ultraschalldiagnostik ist die Makroperfusion darstellbar und Vasospasmen sowie Perfusionsdefizite können frühzeitige detektiert werden34. Auf die Mikrozirkulation und die Oxygenierung und mögliche noch chirurgisch behandelbare Perfusionsdefizite, kann jedoch nur bedingt eingegangen werden35. Grundsätzlich hat die Bedeutung der Ultraschalldiagnostik im prä- und postoperativen Verlauf bei Patienten nach Nierentransplantation einen hohen Stellwert. Nichtsdestotrotz könnte die Kombination von Sonographie und HSI die genaue Beschreibung von Mikro- und Makroperfusion optimieren und das Erfassen und Beschreiben der Organqualität verbessern. Grundsätzlich stünde als weitere Alternative zu Bewertung des Organs die Indocyaningrün-Angiographie zur Verfügung. Hier besticht die hyperspektrale Bildgebung jedoch, da keine intravenöse Gabe von Floreszenzen wie Indocyaningrün (ICG) benötigt wird. So sind Wechselwirkungen und allergische Reaktionen ausgeschlossen28. Zusammenfassend wäre die mögliche Weiterentwicklung der Bildgebung in ein video- gestütztes operatives Tool zur Überwachung der Reperfusion und Fertigung diverser Anastomosen ein Meilenstein in der operativen Bildgebung und könnte wegweisenden Erkenntnisse der vaskulären Versorgung generieren. Nach unseren erfolgversprechenden Ergebnissen könnte es bald möglich sein, die Maschinenperfusion, die eine Verbesserung der Graft-Funktion und auch Verbesserung des Gesamtüberlebens bei Nieren- und Lebertransplantation zeigt5,36, mit hyperspektraler Bildgebung zu observieren und noch feiner zu justieren. Die Erkenntnis der HSI Bildgebung ist in Zeiten der Organknappheit7,11 essentiell und unseren Erachtens nur vorteilhaft zu beschreiben.

Made available in DSpace on 2015-07-22T20:50:27Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-09-29 / Objetivo: investigar a participação da imunidade inata, através da análise de expressão dos genes TLR4 e MYD88, na disfunção precoce do enxerto renal em humanos. Métodos: A quantificação do mRNA foi realizada por PCR em tempo real, em biópsias pré-implantação do enxerto, na urina e no sangue (leucócitos) do primeiro dia póstransplante, em 75 transplantes (TX) com doador falecido (DF) e 18 transplantes com doador vivo (DV). Os desfechos considerados foram DGF (delayed graft function) e retardo na recuperação da função renal, independentemente de necessidade de diálise. Resultados: Em biópsias, os níveis de expressão de TLR4 e MyD88 foram maiores em rins de DF do que de DV; mas não se correlacionaram com disfunção precoce do enxerto; na urina, não diferiram entre DF e DV, e tenderam a ser mais elevados em casos de TX-DF com disfunção precoce do enxerto; no sangue, não diferiram entre DF e DV e foram mais baixos em casos com disfunção precoce. Conclusão: os resultados sugerem uma participação de TLR4 e MYD88 na patogenia das alterações que ocorrem em rins de DF e que baixos níveis de expressão gênica de TLR4 e MYD88, mensurados em amostra de leucócitos do sangue periférico colhida nas primeiras 24 horas pós-transplante, podem predizer retardo na recuperação da função do enxerto e, portanto, poderão vir a ter utilidade clínica no tratamento de receptores de transplante renal de doador falecido. / TEDE / BV UNIFESP: Teses e dissertações

Improving the short-term results of kidney transplantation did not result in improving the long-term function and survival of kidney allograft. Organ shortage and increasing number of marginal donors remains the key problem in transplant today. The quality of donor organ is critical for graft function development and survival. The aim is to improve understanding to ischemia/reperfusion injury and its consequences, predict delayed graft function and rejection, improve organ allocation strategy and identify patients suitable for safe drug minimization or complete withdrawal of immunosuppressive therapy. Analysis of donor kidneys identified poor tubular cell quality and low survival factor, Netrin-1 expression levels, to be associated with delayed graft function. We confirmed that reperfusion phase of ischemia/reperfusion injury leads to minimal morphological but significant molecular abnormalities. Dissociation observed in histology and molecular pathology finding calls for an integrated approach in donor quality organ evaluation and allocation for transplantation. Significant heterogeneity within donors with expanded criteria was shown and subgroup of organs at low risk of delayed graft function was identified. We suggested donor biopsies to be performed as a routine praxis in all kidneys...

Improving the short-term results of kidney transplantation did not result in improving the long-term function and survival of kidney allograft. Organ shortage and increasing number of marginal donors remains the key problem in transplant today. The quality of donor organ is critical for graft function development and survival. The aim is to improve understanding to ischemia/reperfusion injury and its consequences, predict delayed graft function and rejection, improve organ allocation strategy and identify patients suitable for safe drug minimization or complete withdrawal of immunosuppressive therapy. Analysis of donor kidneys identified poor tubular cell quality and low survival factor, Netrin-1 expression levels, to be associated with delayed graft function. We confirmed that reperfusion phase of ischemia/reperfusion injury leads to minimal morphological but significant molecular abnormalities. Dissociation observed in histology and molecular pathology finding calls for an integrated approach in donor quality organ evaluation and allocation for transplantation. Significant heterogeneity within donors with expanded criteria was shown and subgroup of organs at low risk of delayed graft function was identified. We suggested donor biopsies to be performed as a routine praxis in all kidneys...

Aim: Complex arterial reconstruction in kidney transplantation (KT) using kidneys from deceased donors (DD) warrants additional study since little is known about the effects on the mid- and long-term outcome and graft survival. Methods: A total of 451 patients receiving deceased donor KT in our department between 1993 and 2017 were included in our study. Patients were divided into three groups according to the number of arteries and anastomosis: (A) 1 renal artery, 1 arterial anastomosis (N = 369); (B) >1 renal artery, 1 arterial anastomosis (N = 47); and (C) >1 renal artery, >1 arterial anastomosis (N = 35). Furthermore, the influence of localization of the arterial anastomosis (common iliac artery (CIA), versus non-CIA) was analyzed. Clinicopathological characteristics, outcome, and graft and patient survival of all groups were compared retrospectively. Results: With growing vascular complexity, the time of warm ischemia increased significantly (groups A, B, and C: 40 ± 19 min, 45 ± 19 min, and 50 ± 17 min, respectively; p = 0.006). Furthermore, the duration of operation was prolonged, although this did not reach significance (groups A, B, and C: 175 ± 98 min, 180 ± 35 min, and 210 ± 43 min, respectively; p = 0.352). There were no significant differences regarding surgical complications, post-transplant kidney function (delayed graft function, initial non-function, episodes of acute rejection), or long-term graft survival. Regarding the localization of the arterial anastomosis, non-CIA was an independent prognostic factor for deep vein thrombosis in multivariate analysis (CIA versus non-CIA: OR 11.551; 95% CI, 1.218–109.554; p = 0.033). Conclusion: Multiple-donor renal arteries should not be considered a contraindication to deceased KT, as morbidity rates and long-term outcomes seem to be comparable with grafts with single arteries and less complex anastomoses.