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

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

Hernando Gomez, MD

Mentors: Michael R. Pinsky, MDJohn A. Kellum, MDBrian Zuckerbraun, MD


A Unifying Theory of Sepsis-Induced Acute Kidney Injury

Work in progress…


The classic conceptual model

AKI

Hypovolemia Heart failure Major surgery Sepsis

“Classic conception”

Hypoperfusion Ischemia/hypoxia

Shock


•Exposure to warm ischemia (cardiac arrest) doesn’t always result in AKI

AKI and warm ischemia

Chua, et al. Resuscitation 2012

No PRCSPRCS*

RIFLE I or F 31.4% 51.7%

ALL

*PRCS = Post-resuscitation cardiogenic shock

6.4%


•Sepsis-induced AKI can occur in the absence of shock

AKI in the absence of overt shock

CAP AKINon severe CAP 20.3%Non severe sepsis 23.8%Not requiring ICU 25%

1Murugan Kidney Int 2010

AKI (n=302)

No AKI (n=962)

AKI (n=386)

No AKI (n=1158)


•AKI is independent of renal blood flow2

1Langenberg, Bellomo Crit Care 2005, 2Langenberg et al. Kidney Int 2006

Systematic review of studies measuring Renal Blood Flow (RBF) in sepsis1

Species Studies (n,%) RBF n(%) ~ or RBF n(%)Human 3 - 3Animals 159 99 (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 RBF2

Baseline

Sepsis

Baseline

Sepsis

Sepsis-induced AKI


•Exposure to septic plasma causes AKI-like changes in tubular epithelial cells in vitro1

Podocytes and Proximal tubular epithelial cells

Burned septic patient

Sepsis-induced AKI

a. Alteration in cell polarity

Vehi

cle

Hea

lthy

Bur

n+se

ptic

AK

I

b. Decreased cell-cell interactions(tight junction ZO-1 protein)

No stimuli Healthy plasma

Burned+ Septic AKI plasma

c. Increased apoptosis (TUNEL)

Seps

is(S

)

S+A

KI

Bur

n+S

Bur

n+S+

AK

I

1Mariano Crit Care 2008


Sepsis-induced AKIConsistent histology findings

Tiwari et al

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

(S-AKI) is NOT

ATN


Sepsis-induced AKI… is there anything else out there?


Sepsis-induced AKI

Microvascular dysfunction

InflammationMetabolic response

Lack of functionLack of cell death


Sepsis-induced AKIUnifying 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”

1

2

1. El-Achkar 20082. Wu 2007

Reference


Sepsis-induced AKIUnifying theory modelSluggish microvascular flow amplifies the “danger alarm signal” on the capillary side, and TLR-4-mediates recognition of DAMPs/PAMPs on the tubular side

= Amplification H1

1 2

4

H1

1. Tiwari 2005, Wu 20072. Goddard 1995, Holthoff 20123. Singbartl 20114. Kalakeche 2011, El-Achkar 2008

Reference

HypothesisH1

3

1

1 TNFα 4


Sepsis-induced AKITEC response: 1. Prioritization of energy consumption, 2. quality control, and 3. cell cycle arrest

Tubular Lumen

NHE1

Cl-Endocytosis

Mitochondria

Protein synthesis

Regulation of energy metabolism• Prioritization of

utilization• Mitophagy• Cell cycle arrest

G1

G2

S

M

G0

Tubular epithelial cellS2 segment and beyond

Cl-

Inflammatory mediators from blood

• Altered energy balance: AMP:ATP

• Uncoupled respiration

• ROS/RNS• ѱ

Apoptosis

Nucleus

1

1

2

H1

H1

1

S1 Tubular epithelial cell

Signal from S1 cells Filtered mediators


Sepsis-induced AKI

DAMPs

NaCl

Afferent arteriole Efferent arteriole

Peritubular capillary

TAL

Loop of Henle

PT

Macula Densa

DT

CD

GFR

Inju

red

tubu

lar c

ells

The Tubulo-glomerular feedback (TGF) may be the link between tubular injury and decline in GFR

* *


Sepsis-induced AKIPreliminary data

Sepsis = Alteration in energy metabolism

How is energy metabolism regulated in the cell?



AMP activated protein kinase (AMPK)

AutophagyInflammation

AnabolismCatabolism

Over-Activation(AICAR)

Organ Protection

Cytokines Sepsis



• 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



0

0.1

0.2

0.3

0.4

0.5

0.6

mg/

dL

Groups

Creatininep<0.05

0500

1000150020002500300035004000

pg/m

L

Groups

Cystatin C

0102030405060708090

mg/

dL

Groups

BUNp<0.05

p<0.05p<0.05 p<0.05

Activation of AMPK by AICAR protects against cecal ligation and puncture-induced kidney injury



Activation of AMPK by AICAR reverses cecal ligation and puncture-induced increases in serum cytokine levels


Sepsis-induced AKI: K12 developmentAims

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.

Approach: Animal model (rat CLP) and TEC culture.

1. Determine the differences in microvascular dysfunction, AMPK activation and mitophagy in animals with and without AKI. (CLP rat model)

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)

H11: Microvascular dysfunction precedes AMPK and mitophagy activationH12: 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



Metabolic responseLack of function

Lack of cell death



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

1. Determine the role of inflammation on AMPK and mitophagy activation, and on energy balance in the TEC (CLP rat model: TLR+/+, TLR4 -/-, Leukocyte depletion model; TEC culture with exposure to septic serum)

H11: AMPK and mitophagy will not be activated in TLR4-/- TEC

H12: TLR4-/- mice will not develop microvascular dysfunction after CLPH13: 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)

H11: AMPK over-activation is associated with higher AMP:ATP ratio and less oxidative stress

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)

H12: Activation of AMPK and mitophagy will increase with increasing CLP severity sera.


InflammationMetabolic response



Inflammation

Metabolic response




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


Thank you


Thoughts??

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

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