RIP 1 kinase inhibition for acute ischemic kidney injury

Kevin Gallagher MRC/KRUK/GSK PhD fellow University of Edinburgh Supervisors: Ewen Harrison, Jeremy Hughes, Jim Ross, Lorna Marson, Stephen Wigmore, Allison Beal

DCD transplantation

40% get delayed graft

function

Increased rate of graft loss

Healthy Tubular necrosis

Fibrosis leading to

graft loss

92% of DCD grafts show evidence of ATN on day 10 protocol biopsies

Bank et al Nephrology Dialysis Transplantation, December 2017

How do cells die and can we stop them?

NECROSIS APOPTOSIS

PROGRAMMED NECROSIS?

Can we stop tubular cells from necrosing with a drug?

NECROPTOSIS

Programmed cell suicide:

Definition? Caspase independent cell death dependent on RIPK1

IRI

RIPK1 MLKL

Stimulus: Many

MLKL

Necrostatin-1

IDO

T-cell

receptors

Immune

pathways

Overall aim:

To prove that tubular necrosis can be

prevented by inhibiting RIPK1/NECROPTOSIS

in ischemia reperfusion injury

And determine if this is beneficial even if

drug is given AFTER the injury

Left kidney Left renal

blood

vessels

Micro-

clamp

The drug: World first, highly specific and potent RIPK1 inhibitor (human ready)

Human: proximal tubular cell

In-vitro ischemic models

Mouse: IRI

RIPK1 Necrostatin-1

IDO

T-cell

receptors

Immune

pathways MLKL GSK963a

Does specific RIPK1 inhibition improve kidney function and reduce

cell death in IRI?

N=8 Mean and SD shown

Sh

am

Veh

icle

Co

mm

erc

ial R

IPK

1 in

hib

ito

r

Hig

hly

sp

ecif

ic R

IPK

1 in

hib

ito

r

0

2 5

5 0

7 5

1 0 0

1 2 5

1 5 0

1 7 5

2 0 0

Cre

ati

nin

e (

um

ol/

L)

p = 0 .0 0 6

Moderate IRI Severe IRI

Sh

am

Veh

icle

Co

mm

erc

ial R

IPK

1 in

hib

ito

r

Hig

hly

sp

ecif

ic R

IPK

1 in

hib

ito

r

0

2 5

5 0

7 5

1 0 0

1 2 5

1 5 0

1 7 5

2 0 0

Cre

ati

nin

e (

um

ol/

L)

p = 0 .0 1

VEHICLE RIPK1 inhibitor

x200

Moderate

injury

50-60%

CMJ

tubular

necrosis

Severe

injury 80-

90% CMJ

tubular

necrosis

Tubular necrosis scores:

Veh

icle

RIP

K1 in

hib

ito

r

0

1

2

3

4

Sc

ore

1 = 0 - 2 4 %

2 = 2 5 - 4 9 %

3 = 5 0 - 7 4 %

4 = > 7 5 %

p = 0 .0 1

Veh

icle

RIP

K1 in

hib

ito

r

0

1

2

3

4

Sc

ore

p = 0 .0 0 8

Median (25th

, 75th

) shown

Moderate IRI Severe IRI

RIPK1

MLKL

Stimulus:

Ischemia

reperfusion

?

Improves

renal

function

Reduces

necrosis

Green: Activated MLKL

(phosphorylated)

Red: Dead cells (TUNEL)

Healthy kidney (sham): No dead cells (red), no phosphorylated MLKL (green)

x100

Moderate

injury

Severe

injury

Vehicle RIPK1 inhibition

x100

Green: Activated MLKL

(phosphorylated)

Red: Dead cells (TUNEL)

Renal ischemic

injury is associated

with MLKL

phosphorylation

Activated MLKL is found on the apical boarder of IRI injured tubules:

Green: Activated

MLKL

(phosphorylated)

Red: Brightfield

pseudocolour

Blue: DAPI (nuclei)

X400 magnification

Moderate IRI

pMLKL quantification TUNEL positive cells

Moderate IRI Severe IRI N=6

Sh

am

Veh

icle

RIP

K1 in

hib

ito

r

0

2

4

6

8

1 0

1 2

1 4

1 6

1 8

2 0

To

tal

pM

LK

L s

tain

ing

pe

r l

ow

po

we

r f

ield

(ra

win

tde

n i

n m

illi

on

s)

p = 0 .0 0 4

Sh

am

Veh

icle

RIP

K1 in

hib

itio

r

Veh

icle

RIP

K1 in

hib

ito

r

0

5 0

1 0 0

1 5 0

2 0 0

2 5 0

3 0 0

3 5 0

4 0 0

4 5 0

Nu

mb

er o

f T

UN

EL

po

sit

ive

nu

cle

i p

er l

ow

po

we

r f

ield

p = 0 .0 3

p = 0 .0 3

T=0 T=24

pMLKL

RIP inhibitor

N=8

P=0.03 P=0.01

Creatinine 48 hours after injury Urea 48 hours after injury

Drug first given after 4 hours of reperfusion

IRI+

Veh

icle

IRI+

RIP

K1 in

hib

itio

n

0

5 0

1 0 0

1 5 0

2 0 0

2 5 0

Se

ru

m c

re

ati

nin

e (

um

ol/

L)

IRI+

Veh

icle

IRI+

RIP

K1 in

hib

itio

n

0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

8 0

9 0

1 0 0

Se

ru

m u

re

a (

mm

ol/

L)

IRI with 48 hours reperfusion: Drug given 4 hours after injury

Whole kidney flow cytometry macrophage panel (8 antibodies)

N=3

Sh

am

IRI+

veh

icle

IRI+

RIP

K1 in

hib

itio

r

0

2 0 0

4 0 0

6 0 0

8 0 0

1 0 0 0

Ab

so

lute

nu

mb

er f

ro

m

10

00

00

liv

e c

ell

s

Inflammatory macrophages

p-=0.001

CD45+ve/F480+ve/Ly6c+ve/CD206-ve

Sh

am

IRI+

veh

icle

IRI+

RIP

K1 in

hib

ito

r0

1

2

3

4

5

6

%o

f a

ll l

ive

ce

lls

All leucocytes

CD45+ve

SHAM IRI+Vehicle IRI+RIPK1 inhibitor

Ly6C positive: highly inflammatory

CD

206 positive: A

nti-

inflam

matory

Each dot = a macrophage. Split by: Inflammatory activation.

Findings support significantly reduced acute injury but importantly –

Drug given 4 hours after

Negative control Vehicle RIPK1 inhibition

Injury = Inhibit ATP

production (antimycin A)

and glucose deprivation.

N = 6

.

Blue = live cell Red/Pink=dead cell

Veh

icle

RIP

K1 in

hib

itio

n

ML

KL

in

hib

itio

n

Casp

ase in

hib

itio

n

0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

8 0

H u m a n tu b u la r c e ll d e a th a t 1 2 h o u rs o f in ju ry

% D

ea

d c

ell

s * * * * * *

*

6 810

12

0

2 0

4 0

6 0

8 0

H u m a n tu b u la r c e ll d e a th

(L o s s o f m e m b ra n e in te g r ity : D N A re le a s e )

T im e p o in t ( h o u r s )

% D

ea

d c

ell

s

p = 0 .0 1

V e h ic le R IP K 1 in h ib it io n

6 810

0

2 0

4 0

6 0

8 0

H u m a n tu b u la r c e ll s u rv iv a l

(C e ll m e ta b o lic a c tiv ity )

T im e p o in t ( h o u r s )

% L

ivin

g c

ell

s

V e h ic le R IP K 1 In h ib it io n

p = 0 .0 1

1. Is beneficial in

mouse IRI even when

given AFTER injury

2. Reduces mouse

tubular cell death by

necroptosis

3. Reduces HUMAN ischemic tubular cell

death by necroptosis in-vitro

?

The first evidence

that specific RIPK1

inhibition is

beneficial in IRI

The first evidence

that necroptosis

occurs in IRI and that

RIPK1 inhibition

reduces this

Early evidence

That this process may

be relevant in human

kidney cells

• MRC, KRUK and GSK

• My supervisors

• Jim Black, Kathryn Sangster, Jyoti

Nanda, Anwar Palakka , Gary

Borthwick, Laura Denby, Carolynn

Cairns