CRISMA 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 Center for Critical Care Nephrology Department of Critical Care Medicine and Surgery University of Pittsburgh School of Medicine Center for Critical Care Nephrology Collaborators Ken Hallows Nuria Pastor-Soler

CRISMA 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 Center for Critical Care Nephrology

CRISMA Background Center for Critical Care Nephrology 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 CAPAKI Non severe CAP20.3% Non severe sepsis23.8% Not requiring ICU25% 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

CRISMA Is there anything else out there? Center for Critical Care Nephrology iPhone 10 The tallest iphone yet

CRISMA Background Center for Critical Care Nephrology Tiwari et al Apical tubular epithelial cell vacuolization and loss of brush border 4. Paucity of apoptosis/necrosis 1. Microvascular dysfunction Wu et al. JASN Inflammation and oxidative stress Wu et al. JASN 2007 Sepsis-induced AKI is NOT ATN Inflammation DAMPs PAMPs Metabolic response AMPK Microvascular Dysfunction

CRISMA Background Center for Critical Care Nephrology Inflammation DAMPs PAMPs Metabolic response AMPK Microvascular Dysfunction K12 Grant

CRISMA Metabolic response Center for Critical Care Nephrology = Energetic balance Metabolic dysfunction – Decrease in ATP? Humans (striated muscle): Brealey D, Singer M: Curr Infect Dis Rep 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.

CRISMA Energy regulating processes Center for Critical Care Nephrology I/R

CRISMA AMP Activated Protein Kinase Center for Critical Care Nephrology Autophagy Inflammation Anabolism Catabolism Over- Activation (AICAR) Over- Activation (AICAR) Organ Protection Cytokines Sepsis H:

CRISMA Methods Center for Critical Care Nephrology CLP 24h AMPK stimulation AICAR 100mg/kg 8h Sacrifice and sample collection In vivo: C57BL/6 CLP model (n=8-10/group)

CRISMA Center for Critical Care Nephrology Can external over-stimulation of AMPK protect against sepsis-induced AKI?

CRISMA AMPK exogenous activation prevented the sepsis-induced AKI phenotype Center for Critical Care Nephrology *<0.05 vs. CLP * * CLP CLP+AICAR * …and limited inflammation * * *

CRISMA Center for Critical Care Nephrology What is the effect of AMPK external over- stimulation on survival?

CRISMA AICAR did not impact mortality at 7 days Center for Critical Care Nephrology AICAR

CRISMA In summary so far Center for Critical Care Nephrology 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.

CRISMA Center for Critical Care Nephrology 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

CRISMA Time-course: Creatinine Center for Critical Care Nephrology

CRISMA Time-course: NGAL Center for Critical Care Nephrology

CRISMA Time-course: IL-6 Center for Critical Care Nephrology

CRISMA Time-course: AMPK Center for Critical Care Nephrology Total AMPK CLP Sham CLP Sham 6 hours 24 hours

CRISMA Time-course: Quality control processes Center for Critical Care Nephrology Mitophagy and Biogenesis PGC-1a Biogenesis Sibylle et al. PNAS, 2007;104(29):12017 Choi et al. NEJM 2013

CRISMA Time-course: Quality control processes Center for Critical Care Nephrology Mitophagy: LC3 Mitophagy and Biogenesis LC3 A/B ~14-16 kD CLP Sham CLP Sham 6 hours 24 hours Biogenesis: PGC-1alpha CLP Sham CLP Sham 6 hours 24 hours

CRISMA Mitochondrial function: Respiratory control Ratio Center for Critical Care Nephrology State 3 State 4 ADP ADP consumed O 2 consumed O2O2 Time RCR = State 3:state 4 = Mitochondrial coupling

CRISMA Time-course: Mitochondrial respiratory control ratio Center for Critical Care Nephrology

CRISMA In summary… Center for Critical Care Nephrology 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.

CRISMA Center for Critical Care Nephrology What are the differences in terms of AKI, inflammation and mitochondrial respiration between WT and AMPK β1 β1 KO?

CRISMA CLP-induced AKI WT vs. AMPK KO Center for Critical Care Nephrology

CRISMA CLP-induced inflammation WT vs. AMPK KO Center for Critical Care Nephrology

CRISMA Mitochondrial function: Respiratory control Ratio Center for Critical Care Nephrology

CRISMA In summary… Center for Critical Care Nephrology 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.

CRISMA 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. Center for Critical Care Nephrology

CRISMA 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. Center for Critical Care Nephrology

CRISMA Thank you Center for Critical Care Nephrology Kellum lab John Kellum Kui Jin Jacob Volpe Don Maberry Xiaoyan Wen David Emlet Zuckerbraun lab Brian Zuckerbraun Daniel Escobar Ana Maria Botero Ken Hallows Nuria Pastor-Soler Michael Pinsky Shiva lab Sruti Shiva Catherine Corey NHLBI K12 Emergency Medicine Don Yealy Clif Callaway

CRISMA Thank you Center for Critical Care Nephrology

CRISMA Center for Critical Care Nephrology