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
AKI
Hypovolemia
Heart failure
Major surgery
Sepsis
Shock
Hypoperfusion
Ischemia/hypoxia
Classic conception
ALL
PRCS*
No PRCS
31.4%
51.7%
6.4%
*PRCS = Postresuscitation
cardiogenic shock
AKI
20.3%
23.8%
25%
Sepsis-induced AKI
Consistent histology findings
1. Microvascular dysfunction
Microvascular
Dysfunction
Inflammation
DAMPs
PAMPs
Sepsisinduced AKI
(S-AKI)
is NOT
ATN
3. Inflammation and
oxidative stress
Metabolic
response
Mitophagy
AMPK
4. Paucity of apoptosis/
necrosis
Sepsis-induced AKI
Conceptual framework
1. Amplification
Microvascular
dysfunction
Inflammation
DAMPs
PAMPs
Hypoxia
Wu et al. JASN 2007
Amplification
Alarm
signal
Immune system
AKI phenotype
Decreased GFR
Tubular injury
Paucity of necrosis
and/or apoptosis
Metabolic
response
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
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
Exemplary Care Cutting-edge Research World-class Education
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
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)
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
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
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
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
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
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
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
Sepsis-induced AKI
Model 1B: LPS (mice)
Temsirolimus inhibited mTOR and
induced autophagy
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
Sepsis-induced AKI
Model 1B: LPS (mice)
VPS34 SiRNA inhibited autophagy and decreased renal recovery at 48
hours after LPS
Groups
Non target
VPS34
Cystatin C
29
636
133*
1367*
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
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
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300"
2000"
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1400"
1200"
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600"
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Escobar,Education
Gomez, Zuckerbraun.
Unpublished data
Exemplary Care Cutting-edge Research World-class
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
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
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.
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.
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
Exemplary Care Cutting-edge Research World-class Education
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
Acknowledgements
Thank you