Transcript CFTR Activity Modulation by AMP

Regulation of Epithelial
Transport and the Role of the
Metabolic Sensor AMPK in AKI
Ken Hallows, M.D., Ph.D.
Associate Professor of Medicine
and of Cell Biology and Physiology
Pittsburgh Center for Kidney Research Pittsburgh Center for Kidney Research
5/ 1/ 11 4:57 PM
Renal-Electrolyte Division, University of Pittsburgh
October 24, 2013
5’-Adenosine MonophosphateActivated Protein Kinase (AMPK)
Ubiquitous Metabolic Sensor of Cells
a, b, g heterotrimer: a - catalytic; b and g - regulatory
• Activated by high cellular
[AMP]/[ATP] (metabolic
depletion) and by upstream
AMPKKs
--AICAR (ZMP)
--metformin
Hardie DG, 2007
--Compound C
AMPK regulates a wide variety of cellular processes:
Diseases
Obesity (leptin receptor)
Diabetes (metformin)
Cancer (Peutz-Jeghers
syndrome)
Cardiac (arrhythmias,
ischemic preconditioning)
Hallows, Curr. Opin. Nephrol. Hypertens., 2005
Carling, Trends Biochem. Sci. 29:18, 2004
Potential Role of AMPK in Ischemic
Injury in Various Tissues
AMPK is activated rapidly during myocardial ischemia. In
most studies the net effect of this activation appears to be
beneficial (e.g., Russell et al., J Clin Invest 114:495, 2004).
Activation of AMPK may be deleterious for outcomes in
acute stroke (Li et al., Stroke 38:2992, 2007; McCullough et
al., J Biol Chem 280:20493, 2005 ).
 The role and consequences of AMPK activation in
ischemic injury could depend on the nature of the ischemic
insult, both the timing and severity of AMPK activation, and
the organ or tissue involved.
There is a relative paucity of studies looking at the potential
role of AMPK in the kidney.
Metformin Pretreatment Limits Na+ Pump
Internalization Following Ischemia in MDCK Cells
2DG/AA
Metformin
Seo-Mayer et al.,
AJP-Renal, 2011
Metformin Pretreatment Limits Renal IRI in Vivo
Seo-Mayer et al.,
AJP-Renal, 2011
Combination therapy with AICAR and N-acetylcysteine
attenuates ischemia-reperfusion injury (IRI) in a
canine model of autologous renal transplantation
 AMPK pre-activation might play a protective role
Study confounded by
simultaneous use of
antioxidant N-acetylcysteine.
Lin et al., Transplantation 78:654, 2004
Transport-Metabolism Coupling
• Transport processes account for a large % of cellular
energy expenditure in epithelia (Na+,K+-ATPase utilizes up
to 50% in renal tubular cells under resting conditions).
• Energy sensing by AMPK may be a physiologically
relevant mechanism by which renal tubular cells maintain
tight coupling between energy metabolism and tubular
transport.
• Under pathological conditions of ischemia, the
expression and activity of many transport proteins are
acutely inhibited. AMPK inhibits transport proteins and is
activated by ischemia in the kidney.
• Hypothesis: Inhibition of ion transport by ischemia is an
adaptive response mediated by AMPK to help conserve
cellular energy during the acute injury period.
Model for AMPK-dependent
Regulation of Membrane Transport
Hallows, Curr. Opin. Nephrol. Hypertens., 2005
AMPK Activation Induces b-ENaC
Redistribution Away from Apical Membrane
AMPK Inhibition Blunts ENaC Inhibition
Following Chemical Ischemia
100
Ussing chamber recordings of ENaC-dependent
Isc in mpkCCDc14 cells
% Amiloride-Sensitive Isc
90
p < 0.05 (± Comp C)
80
70
2 DOG 10mM
CompC + 2DOG 10mM
CCCP 5uM
60
CompC + CCCP 5uM
AntiA 0.1uM
CompC + AntiA 0.1uM
50
40
30
0
0.5
1
1.5
2
Time After Ischemia (min)
2.5
Similar results were
obtained with AMPK
knockdown by RNAi.
AMPK Blocker Blunts Ischemic Current Inhibition
Following Basolateral Nystatin Permeabilization
100
p < 0.05 (± Comp C)
Relative Current (%)
90
80
70
60
Comp C + Anti A
Antimycin A
50
40
30
0
1
2
3
4
5
6
Time after Ischemia (min)
7
8
AMPK Contributes to Acute Inhibition of Apical
ENaC Expression Following Chemical Ischemia
Whole Cell lysate
KDa
Biotinylation
150
β-ENaC
102
76
102
γ-ENaC
76
52
β-Actin
Con
2 DOG
DMSO
pThr172
AMPK-α
AntiA Con
2 DOG
Comp C
AntiA
Con
2 DOG
DMSO
AntiA
Con 2 DOG AntiA
Comp C
ENaC is More Apically Distributed under
Normal Conditions in AMPK b1 -/- Mice
Fraser et al., AJP-Renal, 2013
Rodent Models of Kidney Injury
• Metabolic cage (urine Na+, Cr, volume, etc.)
• Models of acute kidney injury (AKI); renal pedicle
ligation
– Histology and biochemistry
• Ischemia-reperfusion injury (15-30 min ligation;
24-48 h recovery)
– Histology and biochemistry
– Lytes, renal function, etc.
• Assessment of renal oxygen consumption?
• Sepsis models (e.g., cecal ligation and puncture)
Planned Studies: Examine the Role of AMPK
in the Down-regulation of Na+ Transport in
Vivo in Response to Ischemic AKI
 Examine the role of AMPK in mediating the inhibition of transport
proteins (e.g., ENaC activity) in response to chemical ischemia in
collecting ducts isolated from AMPK-b1 KO vs. WT mice  patchclamp measurements.
 Examine the role of AMPK in mediating the acute ischemiaassociated down-regulation of ENaC expression at the apical
membrane using salt-restricted AMPK-b1 KO vs. WT mice  tissue
immunolocalization.
 Repeat above studies with acute (6 h) or chronic (3 d) AICAR or
metformin pre-treatment (to mimic ischemic preconditioning).

IRI protocols with these animal models
Additional Goals
• Characterize the role of AMPK in both the
longer-term Na+ transport response and the
general ability of cells and epithelial tissues
to recover from IRI and septic injury.
• Possible clinical trials with FDA-approved
AMPK modulators (e.g., metformin) in a
clinical setting where IRI to the kidney can be
expected to occur (e.g., abdominal aortic
aneurysm repair, ?IV contrast dye).
Acknowledgments
Pittsburgh
Hui Li
Liz Rush
Eric Morrell
Rodrigo Alzamora
Darwin King
Jeff Lee
Outside Collaborators
Bruce Kemp (Melbourne)
David Power (Melbourne)
Michael Caplan (Yale)
Alexander Staruschenko (MCW)
Vivek Bhalla (Stanford)
Prabhleen Singh (UCSD)
Núria Pastor-Soler
Allison Marciszyn
Fan Gong
Mohammad Albataineh
John Kellum
Support
O’Brien Pittsburgh Center for
Kidney Research
b-ENaC has a More Apical Pole
Distribution in Kidney Principal Cells
of AMPK-b1 KO Mice
AMPK activator AICAR ameliorates ischaemia reperfusion injury in the rat kidney
British Journal of Pharmacology
Volume 166, Issue 6, pages 1905-1915, 27 JUN 2012 DOI: 10.1111/j.1476-5381.2012.01895.x
http://onlinelibrary.wiley.com/doi/10.1111/j.1476-5381.2012.01895.x/full#f1
AMPK activator AICAR ameliorates ischaemia reperfusion injury in the rat kidney
British Journal of Pharmacology
Volume 166, Issue 6, pages 1905-1915, 27 JUN 2012 DOI: 10.1111/j.1476-5381.2012.01895.x
http://onlinelibrary.wiley.com/doi/10.1111/j.1476-5381.2012.01895.x/full#f2
AMPK activator AICAR ameliorates ischaemia reperfusion injury in the rat kidney
British Journal of Pharmacology
Volume 166, Issue 6, pages 1905-1915, 27 JUN 2012 DOI: 10.1111/j.1476-5381.2012.01895.x
http://onlinelibrary.wiley.com/doi/10.1111/j.1476-5381.2012.01895.x/full#f3