The role of microcirculatory and mitochondrial dysfunction in sepsisinduced acute kidney injury (AKI): a. model of sepsis-induced organ dysfunction

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1 The role of microcirculatory and mitochondrial dysfunction in sepsisinduced acute kidney injury (AKI): a model of sepsis-induced organ dysfunction Hernando Gomez, MD Mentors: Michael R. Pinsky, MD John A. Kellum, MD Brian Zuckerbraun, MD Exemplary Care Cutting-edge Research World-class Education

2 A Unifying Theory of Sepsis-Induced Acute Kidney Injury Work in progress

3 The classic conceptual model AKI Hypovolemia Heart failure Major surgery Sepsis Shock Hypoperfusion Ischemia/hypoxia Classic conception

4 AKI and warm ischemia Exposure to warm ischemia (cardiac arrest) doesn t always result in AKI ALL PRCS* No PRCS RIFLE I or F 31.4% 51.7% 6.4% *PRCS = Post-resuscitation cardiogenic shock Chua, et al. Resuscitation 2012

5 AKI in the absence of overt shock 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% AKI (n=302) AKI (n=386) No AKI (n=962) No AKI (n=1158) 1 Murugan Kidney Int 2010

6 AKI is independent of renal blood flow 2 Systematic review of studies measuring Renal Blood Flow (RBF) in sepsis 1 Species Studies (n,%) RBF n(%) ~ or RBF n(%) Human 3-3 Animals (62%) 60 (38%) Small 65 (41%) 19 (29%) 46 (71%) Large 94 (59%) 53 (56%) 41 (44%) AKI can occur in the setting of increased RBF 2 Sepsis Sepsis Baseline Baseline 1 Langenberg, Bellomo Crit Care 2005, 2 Langenberg et al. Kidney Int 2006

7 Exposure to septic plasma causes AKI-like changes in tubular epithelial cells in vitro 1 Burned septic patient a. Alteration in cell polarity b. Decreased cell-cell interactions (tight junction ZO-1 protein) Vehicle Healthy Burn+ septic AKI No stimuli c. Increased apoptosis (TUNEL) Healthy plasma Burned+ Septic AKI plasma Podocytes and Proximal tubular epithelial cells Sepsis(S) S+AKI Burn+S Burn+ S+AKI 1 Mariano Crit Care 2008

8 Consistent histology findings 1. Microvascular dysfunction Wu et al. JASN Apical tubular epithelial cell vacuolization and loss of brush border Sepsisinduced AKI (S-AKI) is NOT ATN Tiwari et al 3. Inflammation and oxidative stress Wu et al. JASN Paucity of apoptosis/necrosis

9 is there anything else out there?

10 Microvascular dysfunction Inflammation Metabolic response Lack of function Lack of cell death

11 Unifying theory model DAMPs/PAMPs and other inflammatory mediators gain access to the tubular epithelial cell through filtration and peritubular capillary-tec interactions The danger alarm signal Reference 1. El-Achkar Wu

12 Unifying theory model Sluggish microvascular flow amplifies the danger alarm signal on the capillary side, and TLR-4- mediates recognition of DAMPs/PAMPs on the tubular side Reference 1. Tiwari 2005, Wu Goddard 1995, Holthoff Singbartl Kalakeche 2011, El- Achkar 2008 TNFα H 1 = Amplification H 1 Hypothesis H 1

13 TEC response: 1. Prioritization of energy consumption, 2. quality control, and 3. cell cycle arrest Tubular Lumen Signal from S1 cells Filtered mediators Cl - Tubular epithelial cell S2 segment and beyond Endocytosis 1 H 1 H 1 Regulation of energy metabolism Prioritization of utilization Mitophagy Cell cycle arrest Mitochondria Altered energy balance: AMP:ATP Uncoupled respiration ROS/RNS ѱ Apoptosis S1 Tubular epithelial cell 1 Protein synthesis Inflammatory mediators from blood 1 NHE1 Cl - 2 G2 M S G1 G0 Nucleus

14 DAMPs * * The Tubulo-glomerular feedback (TGF) may be the link between tubular injury and decline in GFR Afferent arteriole GFR Efferent arteriole DT Macula Densa CD TAL Injured tubular cells Loop of Henle NaCl PT Peritubular capillary

15 Preliminary data Sepsis = Alteration in energy metabolism How is energy metabolism regulated in the cell?

16 Preliminary data Over- Activation (AICAR) AMP activated protein kinase (AMPK) Cytokines Sepsis Anabolism Catabolism Inflammation Autophagy Organ Protection

17 Preliminary data Model: Rodent (mice) Cecal Ligation and Puncture (CLP). 22 gauge needle / 2 perforations 4 Groups and interventions (n=5-8/group): CLP Sham CLP+AICAR (100mg/kg) Sham + AICAR

18 Preliminary data Activation of AMPK by AICAR protects against cecal ligation and puncture-induced kidney injury mg/dl Creatinine p<0.05 p<0.05 mg/dl BUN p<0.05 pg/ml Cystatin C p<0.05 p<0.05 Groups Groups Groups

19 Preliminary data Activation of AMPK by AICAR reverses cecal ligation and punctureinduced increases in serum cytokine levels

20 : K12 development Aims Aim 1: To determine the role of microvascular dysfunction on activation of energy regulating pathways in the tubular endothelial cell (TEC), namely, AMPK activation and induction of mitophagy. Microvascular dysfunction Approach: Animal model (rat CLP) and TEC culture. Metabolic response Lack of function Lack of cell death 1. Determine the differences Microvascular in microvascular dysfunction, AMPK activation and mitophagy in animals with and without AKI. (CLP rat model) dysfunction H1: Microvascular dysfunction, and activation of AMPK and mitophagy in AKI > Non AKI 2. Determine the temporal and spatial relationship between microvascular dysfunction and activation of AMPK and mitophagy. (CLP rat model) H1 1 : Microvascular dysfunction precedes AMPK and mitophagy activation H1 2 : Microvascular dysfunction is associated with AMPK and mitophagy activation 3. Determine if microcirculatory dysfunction is associated with energy failure in the TEC. (CLP rat model) H1: Microvascular dysfunction is associated with an increase in AMP/ATP ratio Metabolic response Lack of function Lack of cell death

21 Inflammation Microvascular dysfunction Lack of function Lack of cell death Sepsis-induced AKI: K12 development Aims Aim 2: To determine the signaling mechanisms regulating recognition and response of the TEC to inflammatory mediators Approach: Animal model (rat CLP) and TEC culture. Focus: AMPK signaling, mitophagy activation, energy failure Metabolic response 1. Determine the role of inflammation on AMPK and mitophagy activation, and on energy balance in the TEC (CLP rat model: Microvascular TLR+/+, TLR4 -/-, Leukocyte depletion model; TEC culture with exposure to septic serum) H1 1 : AMPK and mitophagy will not be activated in TLR4-/- TEC dysfunction H1 2 : TLR4-/- mice will not develop microvascular dysfunction after CLP H1 3 : CLP after leukocyte depletion will be associated with a lack of AKI 2. Determine the role of AMPK over-activation on TEC response to inflammation (CLP rat model and TEC culture) Inflammation H1 1 : AMPK over-activation is associated with higher AMP:ATP ratio and less oxidative stress Lack of function Lack of cell death 3. Determine the effect of sera from animals subjected to different severities of CLP (low, moderate, severe) on TEC AMPK and mitophagy activation, and on AMP:ATP ratio in vitro (TEC culture exposed to septic sera from different severity models of AKI) H1 2 : Activation of AMPK and mitophagy will increase with increasing CLP severity sera. Metabolic response

22 : K12 development Aims Aim 3: To define the different clinical/biochemical phenotypes of presentation of sepsis, and their relation to sepsis-induced AKI. Approach: Database exploration HiDenIC and Astute. 1. Determine S-AKI subpopulations based on clinical variables refinement of prior epidemiologic descriptions 2. Determine S-AKI subpopulations using biochemical variables such as urine NGAL, TIMP-2 and IGFBP-7

23 Thank you

24 Thoughts??