The role of AMP-protein kinase in
the genesis of sepsis-induced
acute kidney injury (AKI)
Hernando Gomez
Kui Jin
Jacob Volpe
Daniel Escobar
Brian S. Zuckerbraun
John A. Kellum
Collaborators
Ken Hallows
Nuria Pastor-Soler
Center for Critical Care Nephrology
Department of Critical Care Medicine and Surgery
University of Pittsburgh School of Medicine
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Background
• The pathophysiology of sepsis-induced acute kidney
injury (AKI) remains incompletely understood.
• The paradigm of renal hypoperfusion and renal
blood flow (RBF) has been challenged
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Background
• AKI can occur in the
setting of increased RBF
Langenberg, Bellomo et al. KI 2006
• Transient warm ischemia is
not enough
ALL
31.4%
RIFLE I or F
PRCS* No PRCS
51.7%
6.4%
*PRCS = Post-resuscitation cardiogenic shock
Chua, et al. Resuscitation 2012
• AKI can occur in the
absence of clinical signs of
shock
Murugan, et al. KI 2010
CAP
AKI
Non severe CAP
20.3%
Non severe sepsis
23.8%
Not requiring ICU
25%
• The AKI phenotype is
reproducible in vitro
Mariano, et al. Crit Care 2008
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Is there anything else out
there?
iPhone 10
The tallest iphone yet
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Background
1. Microvascular dysfunction
Microvascular
Dysfunction
Wu et al. JASN 2007
2. Apical tubular epithelial
cell vacuolization and loss of
brush border
Inflammation
DAMPs
PAMPs
Sepsis-induced
AKI is NOT
ATN
Tiwari et al. 2005
3. Inflammation and
oxidative stress
Metabolic
response
AMPK
4. Paucity of
apoptosis/necrosis
Wu et al. JASN 2007
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Background
K12 Grant
Microvascular
Dysfunction
Inflammation
DAMPs
PAMPs
Metabolic
response
AMPK
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Metabolic response
= Energetic balance
• Metabolic dysfunction
– Decrease in ATP?
• Humans (striated muscle): Brealey D, Singer M: Curr Infect Dis Rep 2003.
• After CLP (cardiac myocites): Watts et al. J Molec Cell Card 2004
• Kidney – no change after E.coli infusion: May et al. ICM 2012
• Yet, there is no apoptosis – ???
• Processes that sustain Energy balance may be
important.
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Energy regulating processes
• I/R
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AMP Activated Protein Kinase
H:
OverActivation
(AICAR)
Cytokines Sepsis
Anabolism
Catabolism
Inflammation
Autophagy
Organ
Protection
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Methods
In vivo: C57BL/6 CLP model (n=8-10/group)
24h
CLP
AMPK stimulation
AICAR 100mg/kg
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8h
Sacrifice and
sample collection
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Can external over-stimulation of AMPK
protect against sepsis-induced AKI?
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AMPK exogenous activation prevented the
sepsis-induced AKI phenotype
CLP
CLP+AICAR
*
*
*
…and limited inflammation
*
*
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*<0.05 vs. CLP
What is the effect of AMPK external overstimulation on survival?
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AICAR did not impact mortality
at 7 days
AICAR
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In summary so far
AICAR 24 hours before CLP:
• Decreased clinical markers of AKI: Cr, BUN, CysC at 8
hours after CLP
• Limited the inflammatory response at 8 hours after
CLP
• Had no effect on mortality at 7 days.
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What is the time-course of events for
sepsis-induced AKI in wild type animals in
terms of:
1. AKI signal (Creatinine, CysC, NGAL)
2. Inflammation (IL-6)
3. Energy regulation:
–
AMPK activation
–
Quality control processes (Mitophagy and Biogenesis)
–
Mitochondrial respiration
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Time-course: Creatinine
Creatinine
Creatinine
Creatinine (nmol/ul)
(nmol/ul)
Creatinine
44
33
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8h r
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am
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_ 7 ou
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m LP
m LP
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a
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m
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a
L
a CL
a CL
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am C
Sh
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Sh
Sh
Sh
Sh
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SShha
m
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CC _22hho
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P__ u r
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_
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P__ yyss
77d
daay
yss
NGAL
NGAL (pg/ul)
(pg/ul)
Time-course: NGAL
NGAL
30000
20000
10000
0
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am
_
C
CL 2h
LPP o
__ u
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am ouur
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aay
yss
Sh
IL-6 (pg/ml)
Time-course: IL-6
IL-6
800
600
400
200
0
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Time-course: AMPK
Total AMPK
Sham
CLP
Sham
6 hours
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CLP
24 hours
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Time-course: Quality control
processes Mitophagy and Biogenesis
PGC-1a
Biogenesis
Sibylle et al. PNAS,
2007;104(29):12017
Choi et al. NEJM 2013
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Time-course: Quality control
processes Mitophagy and Biogenesis
Mitophagy: LC3
Biogenesis: PGC-1alpha
LC3 A/B ~14-16 kD
Sham
CLP
Sham
6 hours
Sham
CLP
24 hours
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CLP
6 hours
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CLP
24 hours
A
Mitochondrial function:
Respiratory control Ratio
RCR = State 3:state 4
ADP
O2
= Mitochondrial
coupling
ADP
consumed
O2
consumed
State 3
State 4
Time
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Time-course: Mitochondrial
respiratory control ratio
4
Kidney RCR at 24 and 72 hrs
after CLP in WT
Sham
CLP
RCR
3
2
1
0
24H
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In summary…
• There was a clear difference between CLP and
sham in terms of clinical AKI (Cr, NGAL) and
inflammation (IL-6) markers.
• Although NGAL detected injury as early as 6 hours,
and Cr peaked at 24h.
• The timeline of AMPK activation, as well as
mitophagy and biogenesis remains inconclusive.
• RCR was higher in CLP animals at 24 hours and 72
hours.
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What are the differences in terms of AKI,
inflammation and mitochondrial respiration
between WT and AMPK β1 KO?
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CLP-induced AKI
WT vs. AMPK KO
AMPK Knock out and wild type
NGAL(pg/ul)
4000
AMPK KO
Wild Type
3000
2000
2517 ± 327.0, n=5
1000
2027 ± 183.9, n=4
A
M
W
ild
PK
K
O
Ty
pe
0
150
AMPK KO
Wild Type
100
91.74 ± 7.231, n=5
50
74.27 ± 9.971, n=4
Ty
pe
0
A
M
W
ild
PK
K
O
Cystatin C (nmol/ul)
CLP_25%
hourtype
AMPK Knock
out21G
and24wild
CLP_25% 21G 24 hour
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CLP-induced inflammation
WT vs. AMPK KO
AMPK Knock out and wild type
800
AMPK KO
359.6±204.6
IL-6 (pg/ul)
Wild Type
600
p=0.06
400
166.1±44.18
200
0
A
M
PK
K
O
ild
W
pe
y
T
CLP_25% 21G 24 hour
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Mitochondrial function:
Respiratory control Ratio
Kidney RCR 24 hours after CLP
4
WT
AMPK-/-
RCR
3
2
1
0
WT
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In summary…
• AMPK KO animals displayed trends of higher
creatinine, NGAL, Cystatin C and IL-6 than WT after
CLP.
• There were no differences in the RCR.
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Conclusions
• AICAR 24 hours before CLP prevented AKI and limited
inflammation, but had no effect over 7 day mortality.
• It is unclear what the mechanisms is, as AMPK activation was
not confirmed.
• Although markers of AKI were present after CLP, the timeline of
AMPK activation, as well as of mitophagy and biogenesis
remains inconclusive.
• AMPK KO displayed a trend towards higher markers of AKI and
inflammation, but no differences in mitochondrial coupling.
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Next steps
• Understand the effect of AICAR over AMPK activation,
mitophagy and biogenesis in the setting of sepsis.
• Evaluate the effects of AICAR and AMPK activation on energy
status in the tubular epithelial cell (ATP levels/ATP turnover?).
• Understand the effects of CLP on proximal tubular epithelial
cell function, and the effect that AMPK stimulation may have
upon this both in WT and AMPK KO.
• Evaluate the effect of administration of AICAR to AMPK KO
animals in terms of AKI markers, immune response and cellular
ATP.
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Thank you
Kellum lab
Ken Hallows
Nuria Pastor-Soler
John Kellum
Kui Jin
Jacob Volpe
Don Maberry
Xiaoyan Wen
David Emlet
Michael Pinsky
Shiva lab
Sruti Shiva
Catherine Corey
Zuckerbraun lab
NHLBI K12 Emergency Medicine
Brian Zuckerbraun
Daniel Escobar
Ana Maria Botero
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Don Yealy
Clif Callaway
Center for Critical Care Nephrology
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Thank you
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Center for Critical Care Nephrology
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www.ccm.pitt.edu
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