P2X7 receptor and Sepsis- Induced Acute Kidney Injury

276 Pages·2016·18.17 MB·English

P2X receptor and Sepsis- Induced
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Acute Kidney Injury

Nishkantha Arulkumaran
A thesis submitted to University College London in candidature for the degree of
Doctor of Philosophy

Bloomsbury Institute of Intensive Care Medicine
Division of Medicine
University College London
Gower Street, London WC1E 6BT

Affiliations:
Centre for Nephrology (& London Epithelial Group)
Rowland Hill Street, Royal Free Campus, University College London, NW3 2PF

Imperial College Kidney and Transplant Institute
5th Floor Commonwealth Building, Hammersmith Hospital, Du Cane Road,
London W12 0HS
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Declaration of originality

I, Nishkantha Arulkumaran confirm that the work presented in this thesis is my own.
Where information has been derived from other sources, I confirm that this has been
indicated in the thesis.

Signed:

Date:
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Abstract

Acute kidney injury (AKI) is a common clinical problem within the intensive care unit.
Sepsis is implicated in half the cases of AKI; in those patients requiring acute renal
replacement therapy there is an associated mortality of 50%. However, other than
maintenance of an adequate circulation, no specific therapy exists for septic AKI. This is
in large part related to a poor understanding of the underlying pathophysiology..

I used a 72 hr clinically relevant, fluid-resuscitated rat model of sepsis and recovery to
undertake a detailed temporal characterization of the pathophysiology of septic AKI and
relevant biomarkers of kidney injury and dysfunction, and to assess the impact of
targeted treatments. As with human studies, renal histology demonstrated minimal tissue
injury or early inflammatory cell infiltration, however renal recovery was associated with a
marked increase in renal macrophage infiltration. A panel of 8 renal biomarkers revealed
that urine NGAL was the most sensitive marker, having risen by 3 hours’ post-insult and
elevated for 24hrs.

Renal blood flow was maintained over the first 24 hrs, however a fall in renal cortical
oxygenation occurred despite similar renal oxygen delivery and utilization at 24 hrs.
Though electron microscopy showed normal mitochondrial structure, I found an increased
expression of mitochondrial uncoupling protein-2 (UCP-2); this may further uncouple
mitochondrial respiration. Multiphoton imaging of live healthy kidney slices incubated in
either sham or septic serum showed a rise in tubular reactive oxygen species (ROS) and
falls in NADH and mitochondrial membrane potential in the septic serum group, findings
that are consistent with uncoupling. Pre-incubation with the ROS scavenger, 4-OH-
TEMPO, prevented these effects.

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The NLRP3 inflammasome plays an important role in pro-inflammatory cytokine
production. A NLRP3 inflammasome inhibitor, P2X antagonist, prevented LPS-induced
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IL-1β production by peripheral blood monocytes in vitro, however this was related in part
to its diluent vehicle, dimethyl sulfoxide (DMSO). In the kidney, proximal tubular P2X and
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caspase-1 expression was seen both in vivo and ex vivo during sepsis. DMSO/P2X
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antagonist treatment after the onset of sepsis was associated with reduced renal IL-1β
expression and improvements in tachycardia, stroke volume, albumin and lactate
however effects on renal function were inconclusive. Further studies targeting the NLRP3
inflammasome in sepsis are warranted.

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Acknowledgements

I would like to express particular appreciation to my PhD supervisors, Prof Mervyn Singer
(primary supervisor), Prof Robert Unwin and Dr Frederick Tam (co-supervisors) for
providing me with excellent mentorship and opportunities. It has been an honor to work
with three inspirational individuals who have demonstrated the importance of being open
to ideas, hard work and humility. In addition to teaching me the fundamentals of scientific
research and providing timely career guidance, my research mentors have been
exemplary academic clinicians.

Working in Prof Singer’s lab has been a milestone in my career and I have enjoyed his
charisma, enthusiasm and sense of humor. Dr Frederick Tam has been instrumental in
my academic career having guided me throughout my academic clinical fellowship since
2008. Prof Unwin has been able to provide novel insight into the project and has been
instrumental in fostering a number of important collaborations.

Dr Paul Bass (University College London) has assisted me in analysing renal
histopathology. Prof Michael Duchen (University College London) has provided me with
the facilities for confocal microscopy and has given useful insight into the methodology.
Prof Alan Salama (University College London) has been generous with his time in
reading my upgrade report and conducting my upgrade viva.

Dr Holly Courteneidge (University College London), Ms Gurgeet Bhangal (Imperial
College, London), Dr Simona Deplano (Imperial College, London), Dr Alex Dyson
(University College London), and Dr Clare Turner (Imperial College, London) have
worked in the laboratory with me at various points over the past few years and have
provided me with invaluable assistance in different laboratory techniques. Mr Jahm
Persaud (Royal Free Hospital) has kindly run samples in the bioechemistry lab.
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I have also been fortunate to work with a number of fellow medical trainees from different
European countries who have come to work in Prof Singer’s lab. This includes Dr Marijie
Sixma (Holland), Dr Elisabetta Greco (Italy), Dr Elias Carevello (Italy), and Ms Elisa
Jentho (Germany).

I am most grateful to the Welcome Trust for providing me with a 3- year PhD fellowship
funding, and to the Intensive Care Society (UK) for providing me with a project grant. I am
also grateful to Abbvie Pharmaceuticals (Chicago, USA) for providing me with the P2X
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receptor antagonist.

Finally I would like to acknowledge my parents for their inspiration, my in-laws for always
taking an interest, and my wife, Sophie, for her endless support and putting up with my
work obsession.

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Funding and Awards

2014: Intensive Care Society (UK)- Research Gold Medal Award

2013: Intensive Care Society (UK)- New Investigator Award (£13,800)

2011: Wellcome Trust Fellowship (£242,000)
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Presented Work

ORAL PRESENTATIONS

1. Novel insights into sepsis induced acute kidney injury
Gold Medal Award, Intensive Care Society
Intensive Care Society State of the Art Meeting, London, Dec 2014

2. Mitochondrial dysfunction contributes to sepsis-induced acute kidney
injury
E Greco, N Arulkumaran, M Sixma, H Courtneidge, M Duchen, F Tam, R J
Unwin, M Singer
27th Annual Congress of the European Society of Intensive Care Medicine,
Barcelona, Spain, October 2014

3. Mitochondrial uncoupling contributes to fever in sepsis
E Greco, N Arulkumaran, A Dyson, M Singer
27th Annual Congress of the European Society of Intensive Care Medicine,
Barcelona, Spain, October 2014

4. Temporal changes in renal haemodynamics and oxygenation in a rat model
of sepsis
N Arulkumaran, M Sixma, P Bass, F Tam, RJ Unwin, M Singer
26th Annual Congress of the European Society of Intensive Care Medicine,
Paris, France, October 2013

5. Renal macrophage infiltration in a rat model of sepsis and recovery
N. Arulkumaran, M. Sixma, S Saeed, G Bhangal, P Bass, F Tam, M. Singer
26th Annual Congress of the European Society of Intensive Care Medicine,
Paris, France, October 2013
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POSTER PRESENTATIONS

1. Temporal changes in systemic and renal inflammation and histology in a
72-hour rat model of faecal peritonitis
N Arulkumaran, M Sixma, E Ceravola, E Jentho, P Bass, RJ Unwin, F Tam, M
Singer.
27th Annual Congress of the European Society of Intensive Care Medicine,
Barcelona, Spain, October 2014

2. P2X Receptor and Haemorrhage-reperfusion- Induced Acute Tubular Injury
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M Sixma, A Dyson, N Arulkumaran, P Bass, RJ Unwin, F Tam, M Singer
26th Annual Congress of the European Society of Intensive Care Medicine,
Paris, France, October 2013

3. P2X receptor and sepsis- induced acute tubular injury
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N Arulkumaran, A Dyson, C Turner, R Unwin, FW Tam, M Singer
25th Annual Congress of the European Society of Intensive Care Medicine,
Lisbon, Portugal, 13-17 October 2012

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Published Work

1. C Turner, N Arulkumaran, M Singer, RJ Unwin, FWK Tam. Is the inflammasome
a potential therapeutic target in renal disease? BMC Nephrology. Jan 2014.

2. N Arulkumaran, C Turner, M Sixma, M Singer, RJ Unwin and FWK Tam.
Purinergic signaling in inflammatory renal disease. Frontiers in physiology. Aug
2013

3. Arulkumaran N, Unwin RJ, Tam FW. A potential therapeutic role for P2X
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receptor (P2X R) antagonists in the treatment of inflammatory diseases. Expert
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Opin Investig Drugs 2011; 20, 897-915

Content from these manuscripts (text, tables, and figures), written by myself but edited by
colleagues and supervisors, has been included in the thesis.
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