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Ischemic reperfusion injuries in transplantation: mechanisms and prevention

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  1. Ischemic reperfusion injuries in transplantation: mechanisms and prevention Pr L Badet MD , PhD / Dr Ricardo Codas MD Department of Kidney and pancreas Transplantation. Lyon France Pr Benoit BARROU, Pr T Hauet On behalf of the FLIRT Group Fédération pour l’étude des lésions d’ischémie reperfusion en transplantation

  2. No DGF no AR AR DGF DGF + AR Ischemia reperfusion injuries • Group of lesions inflicted to the graft during the time between donor and recipient surgery • Short period of time in the case of a living donor • Much longer in the case of Brain dead donor • Average CIT in Kx Tx : 20 hours • Impact both short and long term graft outcome

  3. Key role of the mitochondria Na+ Na+ • Each day an adult products and uses a quantity of ATP equivalent to 75 % of his body weight • In basal situation (without physical activity) , 30 % of the ATP production is used for Na+ and K+ transmembranal pumps • ATP is mainly product by the oxadative phosphorylation K+ K+

  4. Efficiency of ATP production dramatically decrease 1 mole de glucose Aerobic Metabolism Anaerobic Metabolism 38 moles d’ATP 2 moles d’ATP 2 mol. d’a lactique

  5. Acidosis lactate pyruvate glucose acétyl CoA cycle de Krebs Ischemia = Stop the oxydative phosphorylation by ATP NADH, H+ glycolyse - O2 +O2 NADH, H+ FADH2 B Barrou, T Hauet EFPMO Juin 2009

  6. ISCHEMIA-REPERFUSION PHYSIOPATHOLOGY HYPOXY A.T.P DEPLETION MITOCHONDRIAL IMPAIRMENT INTRA CELLULAR ACCUMULATION of Na+, Ca++, and R.O.S (reactive oxygen species)

  7. ENZYMEACTIVATION Protease, N.O Synthase, Phospholipase, Endonucléase Lesions : Cytosqueleton, ADN, Cellular membrane Cellular Death : Apoptosis, Necrosis

  8. Biochemichal Consequences Lack of O2 and substrates Waste accumulation Ischemia oxidative metabolism Inhibition anaerobic Glycolysis ATP depletion hypoxanthine Inhibition pompes Na/K lysosome Instability Lactic Acide Electrolytes losses intraCel Œdema lytic Enzymes release Ph ↓ • proteases and phospholipases activity Ca++ chanel opening  Ca++ cytosqueleton proteins alterations membrane lipid Peroxydation Mitochondrial swelling ROS

  9. Paradoxal effect of reperfusion Ischemia Reperfusion : things are getting worse

  10. Reperfusion Syndrome X 100 Perico N, Lancet 2004, 364: 1814-1827

  11. Immunological consequencesInsights of „Innate Immunity“

  12. Toll receptors ARN sb TIR domain

  13. TLR and DAMPs • Toll Like Receptors, including TLR2 and TLR4, could function as: • detectors of sterile (not pathogen-associated) injury upon binding to endogenous ligands released by damaged cells • DAMPs = „danger - associated molecular patterns“ [ Matzinger ] or „damage - associated molecular patterns“ [Land ].. • Examples of putative endogenous ligands • heat-shock proteins, • high-mobility group box 1 (HMGB1), • heparan sulfate, • hyaluronan fragments, • fibronectin . • ….

  14. TLR4 Expression Human kidney biopsy before reperfusion Cold isch: 0.5 h (n=15) Cold isch: 20 h (n=9) • TLR4 is expressed in normal human kidneys • TLR4 is significantly upregulated by ischemic injury Krüger et al. Blood 2009

  15. Witness of „Oxidative Stress“ 1. In the brain dead donor 2. During cold storage preservation 3. During postischemic reperfusion(free oxygen radical generation) in the recipient „Stress“

  16. Initiation of “Adaptive Alloimmunity” by Reperfusion Injury to Allografts via Initiation of Innate Immunity : the Hypothesis modified(Land W:Eur Surg Res March2002; 34:145 - 153) ROS-mediated Injury Damaged donor cell = non-native proteins Expression of HSPs T- and B- Cell proliferation in alloimmunity =„Danger“ Signal ! „SOS“-Signal Toll-like Receptors resting DC Signal 2: costimulation activated T-Cells B7 CD 28 Signal 3: IL2R CD 40 CD 40L Il-2 TCR MHC Signal 1: MHC/peptid Recognition by TCR activated ,mature DC Innate Immunity: MHC – independent; Adaptive Alloimmunity : MHC – dependent; purely injury – driven and MHC-differences - driven WL,2002

  17. The Story of the Munich „Injury Hypothesis“: 1994 and 2002 (Land W: Transplant Rev. 2002; 16: 192) ROS allograft reperfusion injury to allograft any injury (e.g. brain death condition, risk factors for chronic allograft dysfunction) activation of innate immunity activation activation APCs ECs, SMCs activation of TLR-bearing DCs activation of TLR-bearing vascular cells alloactivation of T cells (increased) adaptive alloimmune response alloatherosclerosis (transplant vasculopathy) acute rejection chronic rejection

  18. Danger Signal Theory…seen by a surgeon Allorecognition is a dangerous game But in a context of inflammation, it is no longer a game

  19. Clinical consequences • Renal transplantation • Accute tubular necrosis • Reversersible lesions : DGF • Irreversible lesions: PNF • Pancreas transplantation • Reversible lesion: • Swelling of the pancreatic parenchyma • Coagulation cascade activation • pancreatitis • Irreversible lesions • secondary vein thrombosis • Pancreas loss

  20. Marginal organs • Major factor influencing the severity of clinical consequences of IR injuries • For several years Internationally • Fall in organ quality along the time • 40 % of Marginal donors (Unos criteria): • More than 60 • NHBD • 50 to 60 • Stroke • Diabetes • Arterial Hypertention • Creat level >1,5 mg /dl

  21. How to improve preservation and limit ischemic reperfusion injuries ?Relevance in Kidney and Pancreas Transplantation

  22. 4 different ways • by Graft Preconditioning • by improving the solutions of conservation • by using the best method of preservation • by post conditioning the organ at the time of transplantation

  23. 1- By preconditioning the donor or the organ at the time of procurement

  24. Preconditioning Principles • Play a role on the donor before or during the organ procurement • Phamacological preconditioning in intensive care unit • Heme Oxygénase • NO synthase • N acétyl cystéine • Halogéné: Sevoflurane • FK506 • Vit D3 • EPO +++ • … • Ischemic préconditioning during the organs procurement • By exposing the organ to repeated short period of warm ischemia before final interruption of blood supply

  25. Ischemic preconditioning:Prepare the organ to IR Injuries • By Triggering the biological, biochemical enzymatic ways protecting the organs against IR injuries • Protein kinase C • Stimulation de gène protecteur de l’apoptosis • HSP Induction • Superoxyde dismutase induction • NO synthase induction: iNOS • NFKB modulation • … • Remote effect: Humoral Mechanism is transferable • « cell homing » modifications: reparation of endothelium damages • Burne-Taney MJ et al,. J Immunol 2006 ; 176:7015.

  26. Main target: heart • Two windows of protection • Early Window: described in 1986 within the first 4 hours: the most efficient • Late window described in1993: appears 12 hours after IPC, less powerful than the earliest one

  27. Possible ways of efficiency • Adénosine regulation • Prot Kinase C Translocation from the cytosol to the membrane • p38 MAPK and JNK activation • HSP activation link to TLR4 • also • NFKB stimulation • Regulation of K+ and Ca++ entrance in the mitochondria

  28. Humoral mechanism transferable • Discovered in 1993 for the heart • Przyklenk K et al Circulation 87:893. • Uncertain mechanism but potentially linked to • Adenosine • Bradykinine • Opioïdes • HO-1 expression • Protection of the endothelial cells • Renal protection also probably possible by prolonged ureteral obstruction

  29. Clinical Application • Randomized study • Cardiac surgery in children • By femoral occlusion (4 cycles of 5 min) before myocardic ischemia • Protector effect demonstrated Cheung M Mand al Cardiol 2006.47:2277

  30. An experimental Approach: Kidney • In rat models • Improves renal function recovery • Decreased ROS production and tissular damage • Decreased renal fibrosis on kidney biopsies performed lately • In canine or porcine models • Contentious results • Difficulties to find define the good sequence of preconditioning • No experience in clinical setting

  31. 2- By using an adequate solution of conservation

  32. Improvement of the solution along the time: • Collins (Intracellular) then Eurocollins • UW , Celsior , HTK • New generation : extracellular with PEG

  33. intra or extra cellular solution ? Na+ Na+ K+ K+ ATP level In ischemic condition : Membranal lesion • 02  ATP  of pumps activity B Barrou, EFPMO Juin 2009

  34. Laboratoire de RMN - Physiologie - Cryobiologie- Michel EUGENE 31P Magnetic Resonance Spectroscopy of Isolated perfused rat kidney(37°C + continuous perfusion 95%O2 5% CO2) Effect of depolarizing (high K+) solutions ATP (%) low K+ 120 100 80 Krebs - PEG 30 g/L high K+ 60 40 Eurocollins 20 U W 0 C 1 2 3 4 5 6 7 8 time (h) Bauza G, Hauet T, Eugene M

  35. How to limit the cellular lesions ? • extracellular solution: K+ low • fight against the arterial spasm Effects of elevation in external K+ concentration on contractile force and membrane potential in rabbit MCA B Barrou, M Eugène EFPMO Juin 2009 Gokina et al Am.J Physiol Heart Circ.Physiol 278:H2105-H2114, 2000

  36.  Na-K ATPase activity  Passive entrance of Na+  Cellular oedema Impermeants Colloides Play a role on the vascular compartment Play a role on the interstitium and on the membrane • HES (amidon)  UW • PM > 106 daltons •  viscosity, red cells aggregation • Dextran : • PEG • immunomasquage B Barrou, EFPMO Juin 2009

  37. Study In Vivo Pig autotransplantation b 1500 1000 the solution Na-Uw-PEG improve renal function recovery Créatinine Clearance (µl/min/kg) a 500 0 Control IGL-1 UW

  38. Creatinine decrease was significantly faster in IGL-1 group from 4 to 15 days post-transplant

  39. Histology • less fibrosis • less apoptotic cells • Less level of expression of CMH II

  40. PEG and immunological consequences of IR ischémie lésions tissulaires Ligands endogènes des TLR • Immunité innée Engagement TLR sur DC Immunomasquage Maturation DC • CD80 CD86 sur DC Alloantigène Migration DC vers OL II Activation des LT naifs Ag spécifique  Alloreconnaissance Evènements Ag dépendants, spécifiques Evènements Ag indépendants, non spécifiques

  41. Immunomasquage : Role of polyethylene glycol (PEG)

  42. Negative control RPMI with no PEG RPMI with PEG 20 kD Immunomasking effect of PEGImmunofluorescence study • Islets were cultured overnight in RPMI ± PEG 20 kD • Incubation with anti MHC Class I antibody (mouse anti mouse H2-Kk) Flirt 2007 Epifluorescent microscopy: magnification x400

  43. 10 days prolonged islet allograft survivalin a "superacute" rejection model (InsHA to CL4) Allografts (InsHA to CL4) Allo HBSS Allo PEG MST = 2.4 ± 0.4 days MST = 12.5 ± 0.5 days P< 0.0001 Flirt 2007

  44. 3- By using the best method of preservation:static incubation vs. perfusion machine

  45. Preservation PrinciplesAdvantages of Static (Ice) System and Pulsatile Perfusion System Static Ice System Simple, reproducible technique Inexpensive Few supply needs Little technical expertise needed Allows for easy long distance transportation Acceptable immediate function rates with short ischemic times Pulsatile Perfusion More complicated to set up More expensive Need machine, perfusion cassette and liquid especially design Technical expertise Provides a monitoring capability Allows transportation

  46. Flow effects on the endothelial cell Kamm R, Annu. Rev. Fluid Mech. 2002. 34:211–32 Endothelial cells under no-flow conditions (a) and after exposure to 1.2 Pa of steady, laminar shear stress for 24 h (b). - Michel EUGENE Lab. Physiologie / Centre de recherche en transplantation INRA Le Magneraud & Université de Poitiers

  47. Oxygenation : Maintain ATP reserves t’Hart N, Liver transplant. 2005: 1403-1411 Slice of rat liver preserved in l’UW gluconate with : 0% d’O2 21% d’O2 95% d’O2 *: p<0.05 vs. Groupes B et C

  48. P < 0.05 vs. KPS perf. P < 0.05 vs. KPS perf. Leucocyte InfiltrationFlirt 2008 Pig model of renal autotransplantation with 60 mn WI and 24 H preservation on the machine