1. Prevention of Renal Injury by Nitric Oxide in Prolonged Cardiopulmonary Bypass A Double Blind Randomized Controlled Trial. Chong Lei & Lorenzo Berra Emanuele Rezoagli, Binglan Yu, Sabrina Strelow, Francesco Nordio, Joseph V. Bonventre, Lize Xiong, Warren M. Zapol. AHA, Orlando, Florida, 11/11/2015

2. Background Prolonged cardiopulmonary bypass (CPB) for cardiac surgery is associated with a high morbidity and mortality 1,2 Acute kidney injury (AKI) is the most common complication after cardiac surgery with prolonged CPB 3 Prolonged CPB duration is associated with hemolysis and high levels of free hemoglobin (Hb) 4 CPB-induced hemolysis causes vascular Nitric Oxide (NO) depletion, which is associated with AKI 4 1.Une D. et al. Circulation. 2015 2.Wrobel K. et al. Circulation. 2015 3.Karkouti K. et al. Circulation. 2009 4.Vermeulen Windsant IC. et al. Front Physiol. 2014

3. Hypotheses Prolonged CPB produces high levels of plasma ferrous (Fe 2+ ) Oxy-Hb, that depletes vascular NO, thereby causing vasoconstriction Treatment of a patient with NO will oxidize plasma Fe 2+ Oxy-Hb to Fe 3+ Met-Hb, which does not bind NO The oxidation of plasma Hb by the administration of exogenous NO will decrease the incidence of AKI by reducing plasma consumption of NO

4. Study Design Subjects: patients undergoing surgical replacement of multiple heart valves requiring prolonged CPB (>90 min) at a single center (Xijing Hospital, Xi’an, China) Double-blind, randomized trial of NO vs. standard gas mixture (O 2 + N 2 ) Nitric Oxide at a dose of 80 part per million was delivered via the oxygenator during CPB and via the ventilator for 24 h after surgery

5. Endpoint and Power Primary endpoint: Reducing AKI defined as either: An increase in serum creatinine by 50% within 7 days after surgery, or An increase in serum creatinine by 0.3 mg/dL (26.5 μM) within 2 days after surgery Sample size calculation: Observation: 65% incidence of AKI α-error = 0.05, β-error = 0.8, 30% AKI reduction Sample size: 106 patients per group

6. Baseline Characteristics Control group (n=112) NO group (n=105) Age, years48 ± 948 ± 10 Female, n (%)70 (63)59 (56) BMI, kg/m 2 22 ± 3 CPB duration, min132 (111 - 154)137 (122 - 158) NYHA, n (%) II50 (45)43 (41) III62 (55)61 (58) IV0 (0)1 (1) EuroSCORE I2.1 (2.1 - 3.2)2.3 (2.1 - 4.4) EuroSCORE II, (%)1.1 (0.9 - 1.5)1.1 (0.9 - 1.6)

7. Plasma Hemoglobin

8. Measuring Plasma NO Consumption Plasma NO consumption was measured, using a NO chemiluminescence analyzer, to assess the ability of plasma Fe 2+ Oxy-Hb to scavenge NO The level of NO consumption gave us an estimate of the levels of Fe 2+ Oxy-Hb and Fe 3+ Met-Hb in plasma Wang X, et al. Proc Natl Acad Sci USA. 2004

9. Plasma NO Consumption

10. Renal complications Control group (n=112) NO group (n=105) RR (95% CI)p AKI incidence, n (%)71 (63)52 (50)0.78 (0.62 - 0.99)0.04 Stage 160 (54)46 (44)0.82 (0.62 - 1.08)0.15 Stage 28 (7)4 (4)0.53 (0.17 - 1.72)0.29 Stage 33 (3)2 (2)0.71 (0.12 - 4.17)0.71 RRT, n (%)*6 (5)3 (3)0.53 (0.14 - 2.08)0.37 NO Reduces AKI Incidence *RRT: Renal replacement therapy: number of patients requiring continuous venous-venous hemofiltration of hemodialysis after surgery.

11. Mortality and Safety Control group (n=112) NO group (n=105) Mortality, n (%)6 (5)2 (2) Serious adverse events00 Met-Hb >10%00

12. Conclusions NO administration (80 ppm via oxygenator and vent for up to 24 hours) to patients undergoing multiple cardiac valve replacements requiring prolonged CPB decreased the incidence of AKI from 63% to 50% (p=0.04) Administration of 80ppm NO for 24 hours was safe. Blood Met-Hb remained below 10%

13. Conclusions NO administration during CPB reduced the NO consumption of plasma to low levels while decreasing AKI incidence. These findings suggest that interventions that prevent vascular depletion of NO might be a possible target to prevent renal injury associated with hemolysis

14. Acknowledgments MENTORS: Lize Xiong, MD, PhD President of Xijing Hospital Xi’an, China Joseph Bonventre, MD, PhD Chief, Renal Division Brigham Women Hospital Boston, USA Warren M. Zapol, MD Director, Anesthesia Center for Critical Care Research Massachusetts General Hospital Boston, USA GRANT SUPPORT: 1.FAER, Foundation for Anesthesia Education and Research, Mentored Research Training Grant, USA 1.Anesthesia Center Funds, Massachusetts General Hospital, Boston, USA 2.Xijing Hospital and Fourth Military Medical University, China 3.National Natural Science Foundation of China NSFC# 81370011, China

16. Plasma Hemoglobin

17. Secondary Outcomes and Safety Outcomes N 2 group (n=112) NO group (n=105) RR (95% CI)pValue Cardiac Complications Perioperative MI, n (%)4 (4)9 (9)2.40 (0.76 - 7.56)0.13 Non Perioperative MI, n (%)1 (1)0 (0)0.36 (0.01 - 8.63)0.52 Cardiac Arrest, n (%)1 (1) 1.07 (0.07 - 16.84)0.96 Arrhythmia, n (%)13 (12)6 (6)0.49 (0.19 - 1.25)0.14 Heart Failure, n (%)4 (4)1 (1)0.27 (0.03 - 2.35)0.23 Neurological Complications Stroke, n (%)1 (1)2 (2)2.13 (0.20 - 23.18)0.53 Delirium, n (%)4 (4)5 (5)1.33 (0.37 - 4.83)0.66 Respiratory Complications Infective Pneumonia, n (%)50 (45)42 (40)0.90 (0.66 - 1.22)0.49 ALI/ARDS, n (%)28 (25)27 (26)1.03 (0.65 - 1.62)0.90 Non Infective ALI/ARDS, n (%)22 (20)10 (10)0.48 (0.24 – 0.97)0.04 Pleural Effusion, n(%)3 (3)2 (2)0.71 (0.12 - 4.17)0.71 Wound Infection, n (%)3 (3)2 (2)0.71 (0.12 - 4.17)0.71 Bleeding, n (%)3 (3)0 (0)0.15 (0.01 - 2.91)0.21 Reintubation, n (%)6 (5)2 (2)0.36 (0.07 - 3.58)0.2 Reoperation, n (%)2 (2)0 (0)0.21 (0.01 - 4.39)0.32 Readmission, n (%)3 (3)2 (2)0.71 (0.12 - 4.17)0.71 Mortality, n (%)6 (5)2 (2)0.36 (0.07 - 1.72)0.20 Methemoglobinemia >10%, n (%)0 (0) -- Adverse events due to NO, n (%)n.a.0 (0)--