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A Unified Theory of Sepsis-Induced

Acute Kidney Injury:


Inflammation, microcirculatory dysfunction,
bioenergetics and the tubular cell adaptation
to injury
Hernando Gomez, MD
Mentors: John A. Kellum, MD
Michael R. Pinsky, MD
Brian Zuckerbraun, MD
Cliff Callaway, MD
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Conflicts of Interest
1. Research Career Development Programs in Emergency Medicine. University
of Pittsburgh Emergency Medicine (5K12 HL109068-03) K12 Scholar
2. Impact of Blood Storage Duration on Physiologic Measures: RECESS
Ancillary Study (5RO1 HL101382-03) Site PI

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The classic conceptual model

AKI
Hypovolemia

Heart failure

Major surgery

Sepsis

Shock

Hypoperfusion
Ischemia/hypoxia

Classic conception

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The classic conceptual model


Exposure to warm ischemia does not necessarily cause AKI
RIFLE I or F

ALL

PRCS*

No PRCS

31.4%

51.7%

6.4%

*PRCS = Postresuscitation
cardiogenic shock

Chua, et al. Resuscitation 2012

Sepsis-induced AKI can occur in the absence of shock


CAP

AKI

Non severe CAP

20.3%

Non severe sepsis

23.8%

Not requiring ICU

25%

Murugan, et al. Kidney Int 2010

Exposure to septic plasma causes AKI-like changes in tubular


epithelial cells in vitro

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Sepsis-induced AKI is there anything else


out there?

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Sepsis-induced AKI
Consistent histology findings
1. Microvascular dysfunction
Microvascular
Dysfunction

Wu et al. JASN 2007

Inflammation

2. Apical tubular epithelial cell


vacuolization and loss of
brush border

DAMPs
PAMPs

Sepsisinduced AKI
(S-AKI)
is NOT
ATN

Tiwari et al. 2005

3. Inflammation and
oxidative stress
Metabolic
response
Mitophagy
AMPK

Wu et al. JASN 2007

4. Paucity of apoptosis/
necrosis

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Sepsis-induced AKI
Conceptual framework
1. Amplification
Microvascular
dysfunction

Inflammation
DAMPs
PAMPs

Hypoxia
Wu et al. JASN 2007

Amplification

Alarm

signal
Immune system

2. TEC response to the


amplified alarm signal

AKI phenotype

Decreased GFR
Tubular injury
Paucity of necrosis
and/or apoptosis

Metabolic
response

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Mitophagy
AMPK

Sepsis-induced AKI
Conceptual framework: 1. Amplification of the alarm signal

1
3
Reference

4
2

1. Tiwari 2005, Wu
2007
2. Goddard 1995,
Holthoff 2012
3. Singbartl 2011
4. Kalakeche 2011,
El-Achkar 2008
H1

Hypothesis

H1

= Amplification
1

H1

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Gomez et al. Shock. In press.

Sepsis-induced AKI
Conceptual framework: 2. TEC response to the alarm signal

Reference
1. Tiwari 2005, Wu
2007
2. Yang 2009, Finkel
2009, Mitra 2009
H1

Tubular injury

H1

Hypothesis

H1

Paucity of necrosis
and/or apoptosis

AKI phenotype
1

1
2

Decreased GFR

Gomez
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et al. Shock. In press.

Sepsis-induced AKI
Preliminary work

Hypothesis
Exogenous stimulation of autophagy improves renal recovery
during sepsis.
Stimulation of autophagy:
1. AICAR (5Aminoimidazole-4carboxamide 1-b-Dribofuranoside,
Acadesine, N1-(b-DRibofuranosyl)-5aminoimidazole-4carboxamide)

1
3

2. Temsirolimus

Inhibition of autophagy:
3. Compound C
4. VPS34 SiRNA

Kilonski D. Nature Rev 2007;8:931-937

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Sepsis-induced AKI
Model 1A: Cecal ligation and punture (mice)
Primary outcome: Renal Function (Creatinine, BUN, Cystatin C)
Zuckerbraun Lab

24h

AICAR 100mg/kg
CoC

8h

CLP

Sacrifice and
sample collection
Primary outcome
Creatinine, BUN, Cystatin C
Secondary outcome
Cytokine expression
Endothelial adhesion molecule expression
Leukocyte expression
Induction of mitophagy (Atg7)

Howell, Gomez, et al. PLoS ONE 8(7): e69520. doi:10.1371


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Sepsis-induced AKI
Model 1A: Cecal ligation and punture (mice)
Primary outcome: Renal Function
Activation of AMPK by AICAR protects against cecal ligation and
puncture-induced kidney injury

BUN

Creatinine

Cystatin C

P=0.02

p<0.05

0.4

mg/dL

mg/dL

0.5
0.3
0.2
0.1
0

Groups

90
80
70
60
50
40
30
20
10
0

pg/mL

0.6

4000
3500
3000
2500
2000
1500
1000
500
0

Groups

Groups

Howell, Gomez, et al. PLoS ONE 8(7): e69520. doi:10.1371


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Sepsis-induced AKI
Model 1A: Cecal ligation and punture (mice)
Secondary outcomes: Cytokine expression
Activation of AMPK by AICAR reverses cecal ligation and punctureinduced increases in serum cytokine levels

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Education

Escobar,
Gomez, Zuckerbraun.
Unpublished data

Sepsis-induced AKI
Model 1: Cecal ligation and punture (mice)
Secondary outcomes: Endothelial activation - ICAM/VCAM
Activation of AMPK by AICAR decreases ICAM expression induced by CLP

ICAM
40x

CONTROL

AICAR

Compound C

Sham

CLP

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Education

Escobar,
Gomez, Zuckerbraun.
Unpublished data

Sepsis-induced AKI
Model 1: Cecal ligation and punture (mice)
Secondary outcomes: Leukocyte infiltration CD45
Activation of AMPK by AICAR reduces leukocyte presence after cecal
ligation and puncture

CD45
40x

CONTROL

AICAR

Compound C

Sham

CLP

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Education

Escobar,
Gomez, Zuckerbraun.
Unpublished data

Sepsis-induced AKI
Model 1: Cecal ligation and punture (mice)
Secondary outcomes: Leukocyte infiltration CD3
Activation of AMPK by AICAR reduces leukocyte presence after cecal
ligation and puncture

CD3
40x

CONTROL

AICAR

Compound C

Sham

CLP

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Education

Escobar,
Gomez, Zuckerbraun.
Unpublished data

Sepsis-induced AKI
Model 1: Cecal ligation and punture (mice)
Secondary outcomes: Induction of mitophagy (Atg7)
Activation of AMPK by AICAR increases induction of mitophagy beyond
the effect of CLP

Actin
Atg7
Control AICAR
Sham

CoC Control

AICAR

CoC

CLP

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Education

Escobar,
Gomez, Zuckerbraun.
Unpublished data

Sepsis-induced AKI
Model 1B: LPS (mice)
Primary outcome: Renal Function (BUN, Cystatin C)
Rosengart Lab

2h

2h

Temsirolimus
(5mg/kg)

LPS
(1.5mg/kg)

Temsirolimus
(5mg/kg)

Sacrifice and
sample collection
Primary outcome
BUN, Cystatin C
Secondary outcome

LC3B (mitophagy induction)

P-Ser2448 mTOR (Inhibition of


mTOR)

Howell, Gomez, et al. PLoS ONE 8(7): e69520. doi:10.1371


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Sepsis-induced AKI
Model 1B: LPS (mice)
Temsirolimus inhibited mTOR and
induced autophagy

Temsirolimuis fascilitated renal


recovery (BUN, Cystatin C)

Howell, Gomez, et al. PLoS ONE 8(7): e69520. doi:10.1371


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Sepsis-induced AKI
Model 1B: LPS (mice)
Primary outcome: Renal Function (BUN, Cystatin C)
Rosengart Lab

48h

72h

VPS34 SiRNA or
Non target SiRNA
Tail vein injection
(6mg/kg)

Allocated to LPS
vs. Control

Sacrifice and
sample collection
Primary outcome
BUN, Cystatin C
Secondary outcome

LC3B (mitophagy induction)

Howell, Gomez, et al. PLoS ONE 8(7): e69520. doi:10.1371


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Sepsis-induced AKI
Model 1B: LPS (mice)
VPS34 SiRNA inhibited autophagy and decreased renal recovery at 48
hours after LPS

Groups

Non target
VPS34

48 hours after LPS


BUN

Cystatin C

29

636

133*

1367*

Howell, Gomez, et al. PLoS ONE 8(7): e69520. doi:10.1371


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Sepsis-induced AKI
Model 2: Cell culture
2A. Macrophages cytokine expression
2B. Renal endothelial cells adhesion molecule expression: ICAM/VCAM
Zuckerbraun Lab

1h

AICAR (1mM)
CoC (10uM)

3h

LPS
100ng/mL

Harvest
Primary outcome
Macrophages: IL-6, TNF-a, INF-g
Endothelial cells: ICAM, VCAM

Escobar, Gomez, Zuckerbraun. Unpublished data


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Sepsis-induced AKI
Model 2: Macrophage culture
Cytokine expression
Activation of AMPK by AICAR reverses release of IL-6, INF-gamma and
TNF-alpha from Macrophages in cell culture
3.5"

250"

3"

200"

2.5"

INF9gamma"

150"
100"
50"

2"
1.5"
1"
0.5"

LP
S+
Co
C"

oC
"

Co
nt
ro
l+C

ica
r"
LP
S+
A

Co
nt
ro
l+A
ica
r"

Co
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ro
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co
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l+A

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S"

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nt
ro
l"

0"

LP
S"

IL66"

300"

2000"
1800"

Cells stimulated with LPS

TNF8alpha"

1600"

Control

1400"
1200"
1000"
800"
600"
400"
200"

oC
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Escobar,Education
Gomez, Zuckerbraun.
Unpublished data
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Sepsis-induced AKI
Model 2: Renal endothelial cell culture
Expression of ICAM
Activation of AMPK by AICAR decreases ICAM expression induced by
LPS in renal endothelial cells

ICAM
60x

CONTROL

AICAR

Compound C

Nucleus
ICAM

Sham

LPS

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Escobar,Education
Gomez, Zuckerbraun.
Unpublished data

Sepsis-induced AKI
Model 2: Renal endothelial cell culture
Expression of VCAM
Activation of AMPK by AICAR decreases ICAM expression induced by
LPS in renal endothelial cells

VCAM
60x

CONTROL

AICAR

Compound C

Nucleus
VCAM

Sham

LPS

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Escobar,Education
Gomez, Zuckerbraun.
Unpublished data

Sepsis-induced AKI
Summary of findings
1. Over-stimulation of mitophagy via AMPK stimulation or mTOR
inhibition, reduces the clinical manifestation of AKI and facilitates
recovery
2. Inhibition of mitophagy caused a decrease in recovery of the renal
function after LPS.
3. Stimulation of AMPK decreased the inflammatory response as
measured by cytokine release and leukocyte infiltration.
4. Stimulation of AMPK decreased the expression of endothelial
adhesion molecules.

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Sepsis-induced AKI
Conclusions
1. Mitophagy seems to be an important mechanism through which the
kidney responds and recovers from sepsis-induced injury.
2. There was however an important difference on how AICAR and
Temsirolimus protected the kidney. AICAR decreased the initial
injury, whereas Temsirolimus improved its recovery after injury.

Different models: LPS vs. CLP


Mechanism of action may be different AICAR decreased inflammatory
response.

3. The effect of AMPK may have been exerted through modulation of:

Inflammation:
Reduces systemic inflammatory mediators
Reduces Leukocyte presence in the kidney
Microvascular function:
Reduces expression of adhesion molecules
Mitochondrial quality control processes
Over-induction of mitophagy
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Sepsis-induced AKI
Future directions
1. Leukocytes
2. TL4 -/-

Inflammation

Microvascular
dysfunction

4
Tubular
epithelial cell
response

Effects of:
1. Leukocyte depletion
2. TLR-4-/3. Autophagy stimulation

H1

1. AMPK -/2. Acute vs. Chronic


stimulation

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Gomez et al. Shock. In press.

Acknowledgements

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Thank you

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