Sarah Hosgood
University of Cambridge
14th November 2015
Normothermic Kidney Perfusion
Refrigeration
Cooling: to Cell Metabolism
• Anaerobic metabolism
• ATP depleted
•
Physiological
Maintains cellular metabolism
• Oxygenation
• Aerobic metabolism
• ATP regenerated
• Pre-transplant assessment
• Pre-transplant therapies
Preservation: Warm or Cold
Static Cold Storage
Hypothermic Machine
Perfusion
Ex-vivo Normothermic
Perfusion (EVNP)
Flow probe
Centrifugal Pump
Pressure line
Oxygenator/heater
Infusion Pumps
Arterial
Venous
Urine collection
Venous
Reservoir
Ex-Vivo Normothermic Perfusion Circuit
Infusion fluids
Urine collection bag
Organ chamber
Venous reservoir
Oxygenator
Centrifugal pump
Isolated Organ Perfusion System
EVNP in Renal Transplantation
Resuscitation to improve early graft function
Pilot series of EVNP (n=36)
Pre-transplant quality and viability assessment
EVNP discarded human kidneys series (n=92)
Pre-transplant therapies and allograft modulation
Noble gases/ nanoparticles
Aim: To determine the feasibility, safety and efficacy of
normothermic perfusion in marginal donor kidney transplantation
vs
n=36 n=47
ECD 23 all ECD
DCD 8
SCD 5
Pilot Clinical Study
Back-table prep
- in ice
Recipient Anaesthetised Dissection Transplant bed
Kidney
cold flushed
EVNP 60 min
EVNP Human Pilot Study Protocol
• 1 unit cross-matched packed red cells + 250 ml crystalloid
• Mean arterial pressure 75 mmHg
• 34-37oC
• TPN (no lipid); Dexamethasone; PGI2
• Co-amoxiclav
• No leucocytes/ little complement
A period of ‘Ideal Perfusion”
EVNP Perfusate
Ex-vivo Normothermic Perfusion
EVNP CS P value
N=36 n=47
First CIT (hr) 10.6 ± 3.9 11.8 ± 3.8 0.240
EVNP (min) 63 ±16 -
Second CIT (min) 26 ± 20 -
Anastomosis (min) 26 ± 6 31 ± 4 0.060
Total ischaemia (hr) 12.9 ± 4.5 12.3 ± 3.7 0.614
Ischaemic Times
• Basiliximab (20mg days 0 & 4)
• Tacrolimus (7-10 ng/ml)
• MMF 0.5g bd
• Prednisolone 20mg 5mg 6/52
Immunosuppression
0
5
10
15
20
25
30
35
40
45
50
EVNP CS
IF
DGF
Results: Early Graft Function
Number of
patients
DGF: 4/36 vs 17/47
(11%) (36%)
P=0.011
Requirement
for dialysis in
first 7 days
0
5
10
15
20
25
30
35
40
45
50
EVNP CS
No AR
AR
Number of
patients
AR: 6/36 vs 9/47
(17%) (19%)
P=0.755
Results: Acute rejection
EVNP CS P value Graft survival 95. 7% 94.3% 0.510 Patient survival 100% 94.3% 1.000
Renal Function
n=36
Arterial thrombosis 0
Venous thrombosis 0
Renal artery stenosis 0
Ureteric stenosis 1
Infection 0
EVNP: Complications
EVNP (n=36 kidneys)
• Feasible and straightforward
• Appears to be safe
• May improve early graft function
in marginal donor kidneys
Summary
0
2
4
6
8
10
12
14
18h CS NP
AT
P:A
DP
ra
tio
ATP : ADP Ratio
**
** P <0.05
Pre CS 18h CS NP
0
5
10
15
20
25
30
35
40
Ex
pre
ssio
n o
f 7
0K
D H
SP
70
vo
lum
e d
ensi
ty (
mm
2)
HSP 70
**
** P <0.05
Reconditioning
0.0
20.0
40.0
60.0
80.0
100.0
120.0
5 10 15 30 60 90 120 150 180
ml/
min
/10
0g
Reperfusion time (minutes)
Mean Renal Blood Flow
Experimental Evidence
RCT of EVNP in DCD kidney Transplantation
CS Tx 1h NP
3 Centres
Cambridge, Newcastle and Guy’s
Primary Outcome Measure
Delayed graft function – Dialysis in first 7 days
Number of patients
Total sample size = 400 patients across the 3 centres
Tx CS
Degree of injury does not necessarily correlate (inversely) with
viability
What is required is a measure of the potential for recovery from injury
- Restoration of function ex-vivo
EVNP: Viability/Quality Assessment of Discarded Human Kidneys
October 2012 – May 2014
92 Kidneys Declined for Transplantation
Reasons for Decline
Poor Flush 20
Malignancy 18
PMH 11
Donor Age 11
Technical/Anatomical 12
HMP Parameters 8
Histology 6
Prolonged Ischaemia 5
No suitable recipient 1
Demographics (n= 74)
DBD 33
DCD 41
Donor Age (y) 60 ± 15 (31 – 78)
CIT (h) 28.7 ± 13.8 (8.2 – 76.5)
WIT (min) 13.3 ± 3.3 (9 – 20)
Declined Human Kidneys
EVNP for pre-transplant quality assessment
Declined Kidneys from DCD aged 42 years
Right Kidney: Arrival
Left Kidney: Arrival
5 min EVNP
5 min EVNP
60 min EVNP
60 min EVNP
Each kidney assessed after 60 minutes of EVNP
1. Visual assessment
1. Measurement of renal blood flow
2. Total urine output
Quality Assessment Score
Score I: Excellent perfusion (Pink and evenly perfused)
Score II: Moderate perfusion (Pink/purple and patchy appearance)
Score III: Poor perfusion (Global mottled purple appearance)
Score I Score II Score III
n = 27 n = 31 n = 16
Grading: Macroscopic Assessment
Area Sensitivity Specificity Threshold
RBF 0.892 88% (CI 62 - 99%)
72% (CI 59 - 83%)
< 63ml/min/100g
U/O 0.876 94% (CI 68 - 100%)
81% (CI (69 - 90%)
< 50ml
EVNP Assessment Score Macroscopic Score 1, 2, 3 = 1 - 3 RBF <63ml/min/100g = 1 U/O <50ml = 1 Total scores range from 1 to 5
Perfusion Parameter Thresholds
Declined kidneys (n=92)
•18 suspected malignancies (excluded)
•35 kidneys scored (1 - 2)
•25 kidneys scored (3 – 4)
•14 kidneys scored (5)
• 262 Unused kidneys (2013 – 2014)
81% suitable for transplantation
Potential extra transplants last year
= 170
Declined Kidney Series
QUALITY ASSESSMENT SCORE
EVNP Clinical Series
Score 1 – 2 n = 26
Score 3 – 4 n = 10
P value
Donor Age (y) 56.3 ± 11.4 57.3 ± 19.6 0.462
M : F 8 : 18 6 : 4 0.140
ECD 18 5 0.440
DCD 4 4 0.179
SCD 4 1 1.000
Score 1 – 2 Score 3 - 4 P value
Recipient Age (y) 55.9 ± 12.5 61.2 ± 10.4 0.261
M : F 18 : 8 6 : 4 0.700
Pre Dialysis 6 1 0.645
HD 14 4 0.711
PD 6 5 0.224
Demographics
Score 1 – 2 n = 26
Score 3 – 4 n = 10
P value
WIT (min) 13.8 ± 2.8 11.3 ± 2.1 0.196
CIT 1 (h) 10.7 ± 4.9 12.3 ± 3.4 0.332
EVNP (min) 65.2 ± 9.9 51.5 ± 13.9 0.002*
CIT 2 (h) 23.4 ± 17.2 33.3 ± 15.0 0.120
Anastomosis (min)
29.5 ± 7.4 26.5 ± 6.7 0.266
Demographics
Score 1 -2 n = 26
Score 3 – 4 n = 10
P value
PNF 0 0
DGF 1 3 0.056
SGF <10% Cr fall 5 3 0.149
Acute Rejection 3 4 0.076
0
100
200
300
400
500
600
700
800
Pre 1 2 3 4 5 6 7
Cr
(µ
mo
l/L
)
Post-transplant (Days)
Serum Creatinine Levels
Score 1-2
Score 3-4
P = 0.032
*
Outcome
Score 1 – 2 n = 26
Score 3 – 4 n = 10
P value
Complications 0 0
Patient Survival 100% 100% 1.000
Graft Survival 96.2% 100% 1.000
0
50
100
150
200
250
300
Score 1-2 Score 3-4
Cr
()µ
mo
l/L
)
Serum Creatinine (12 months)
P = 0.018
0
10
20
30
40
50
60
70
80
Score 1-2 Score 3-4
eG
FR
(m
l)
eGFR (12 months)
P = 0.047
Graph Function
Ischaemia Reperfusion Injury Directly administered to the kidney during EVNP
Transplantation Therapies
Nature Reviews Gastroenterology & Hepatology 10, 79-89 (February 2013)
Therapies
Mechanisms of Tissue Protection • TNFα mediated ICAM/VCAM
expression • Inhibition of NFκB activity • HIF1α dependent (EPO/VEGF)*
• Anti-apoptotic signalling
*Ma D et al JASN 2009;20:713 Zhao H et al Am J Transplant 2013;13:2006
Irani Y et al. Nephron Extra. 2011;1:272.
Therapies: Argon and Xenon
17h CS 1h EVNP
10m WIT
1: Argon 70% (n = 5) 2: Control 95%O2 (n = 5) 3: Nitrogen 70% (n = 6)
Reperfusion 3h
Effects of Argon Post-treatment on Renal Ischaemia-reperfusion Injury
0
10
20
30
40
50
60
70
Argon Control NitrogenRB
F (
ml/
min
/10
0g
)
EVNP Mean Renal Blood Flow
P = 0.020
0,0
10,0
20,0
30,0
40,0
50,0
60,0
0 5 10 15 20 25 30 35 40 45 50 55 60
RB
F (
ml/
min
/10
0g
)
Mean Renal Blood Flow - EVNP Argon
Control
Nitrogen
EVNP Results
0
50
100
150
200
250
300
350
400
Argon Control NitrogenRB
F (
ml/
min
/10
0g
)
EVNP Total Urine Output
P = 0.030
0
2
4
6
8
10
Argon Control NitrogenCr
Cl
(ml/
min
/10
0g
) EVNP Creatinine Clearance
P = 0.040
EVNP Results
0
10
20
30
40
50
60
Argon Control NitrogenRB
F (
ml/
min
/10
0g
)
Mean Renal Blood Flow
P = 0.055
0,00
20,00
40,00
60,00
80,00
100,00
0 5 10 15 30 60 90 120 150 180
RB
F (
ml/
min
/10
0g
) Renal Blood Flow
ArgonControlNitrogen
Reperfusion
0
100
200
300
400
500
600
Argon Control Nitrogen
Ur
ine
Ou
tpu
t (m
l)
Total Urine Output
P = 0.008
0
2
4
6
8
10
Argon Control Nitrogen
Cr
CL
(m
l/m
in/1
00
g).
h
AUC Creatinine Clearance
P = 0.016
Reperfusion
Therapeutics and Allograft Modulation during EVNP
CD31 (green) Nanoparticles (red) Overlay
Nanoparticles
Resuscitation
Viability/
Quality
Therapies
Ex-vivo Normothermic
Perfusion
Optimise early graft function
Increased use of
marginal kidneys
Stem cells
Gene therapy
Small molecules
Potential Roles for EVNP
The ice age is ending