crisma the role of amp-protein kinase in the genesis of sepsis-induced acute kidney injury (aki)...

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The role of AMP-protein kinase in the genesis of sepsis-induced acute kidney injury (AKI)

Hernando GomezKui JinJacob VolpeDaniel EscobarBrian S. ZuckerbraunJohn A. Kellum

Center for Critical Care Nephrology

Department of Critical Care Medicine and Surgery

University of Pittsburgh School of Medicine


CollaboratorsKen Hallows

Nuria Pastor-Soler

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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

No PRCSPRCS*

RIFLE I or F

31.4% 51.7%ALL

*PRCS = Post-resuscitation cardiogenic shock

6.4%

Chua, et al. Resuscitation 2012

CAP AKI

Non severe CAP 20.3%

Non severe sepsis

23.8%

Not requiring ICU 25%

• AKI can occur in the absence of clinical signs of shock

Murugan, et al. KI 2010

• The AKI phenotype is reproducible in vitro

Mariano, et al. Crit Care 2008

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Is there anything else out there?


iPhone 10The tallest iphone yet

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Background


Tiwari et al. 2005

2. Apical tubular epithelial cell vacuolization and loss

of brush border

4. Paucity of apoptosis/necrosis

1. Microvascular dysfunction

Wu et al. JASN 2007

3. Inflammation and oxidative stress

Wu et al. JASN 2007

Sepsis-induced AKI is NOT

ATN

InflammationDAMPsPAMPs

Metabolic response

AMPK

Microvascular Dysfunction

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Background


InflammationDAMPsPAMPs

Metabolic response

AMPK

Microvascular Dysfunction

K12 Grant

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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


AutophagyInflammation

AnabolismCatabolism

Over-Activation(AICAR)

Organ Protection

Cytokines Sepsis

H:

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Methods


CLP

24h

AMPK stimulationAICAR 100mg/kg

8h

Sacrifice and sample

collection

In vivo: C57BL/6 CLP model (n=8-10/group)

C R I S M ACenter for Critical Care Nephrology

Can external over-stimulation of AMPK protect against sepsis-induced AKI?

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AMPK exogenous activation prevented the sepsis-induced AKI phenotype


*<0.05 vs. CLP

*

*

CLPCLP+AICAR

*

…and limited inflammation

*

*

*


What is the effect of AMPK external over-stimulation 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.


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


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Time-course: NGAL


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Time-course: IL-6


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Time-course: AMPK


Total AMPK

CLP Sham CLPSham

6 hours 24 hours

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Time-course: Quality control processes


Mitophagy and Biogenesis

PGC-1aBiogenesisSibylle et al. PNAS, 2007;104(29):12017

Choi et al. NEJM 2013

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Time-course: Quality control processes


Mitophagy: LC3

Mitophagy and Biogenesis

LC3 A/B ~14-16 kD

CLP Sham CLPSham

6 hours 24 hours

Biogenesis: PGC-1alpha

CLP Sham CLPSham

6 hours 24 hours

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Mitochondrial function: Respiratory control Ratio


State 3

State 4

ADP

ADP consumed

O2 consumed

O2

Time

RCR = State 3:state 4

= Mitochondrial coupling

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Time-course: Mitochondrial respiratory control ratio


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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.


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


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CLP-induced inflammationWT vs. AMPK KO


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Mitochondrial function: Respiratory control Ratio


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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

John KellumKui JinJacob VolpeDon MaberryXiaoyan WenDavid Emlet

Zuckerbraun lab

Brian ZuckerbraunDaniel EscobarAna Maria Botero

Ken Hallows Nuria Pastor-Soler

Michael Pinsky

Shiva lab

Sruti ShivaCatherine Corey

NHLBI K12 Emergency Medicine

Don YealyClif Callaway

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Thank you


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www.ccm.pitt.edu


crisma the role of amp-protein kinase in the genesis of sepsis-induced acute kidney injury (aki)...

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