THE EFFECTS OF GENERAL ANESTHESIA ON URINARY BIOMARKERS OF KIDNEY INJURY ...

69 Pages·2016·1.28 MB·English

The Pennsylvania State University

The Graduate School

Department of Comparative Medicine
THE EFFECTS OF GENERAL ANESTHESIA ON URINARY BIOMARKERS OF
KIDNEY INJURY: HEPATITIS A VIRUS CELLULAR RECEPTOR-1 (HAVCR1) AND

LIPOCALIN-2 (LCN2)
A Thesis in

Laboratory Animal Medicine

by

Krista M. Hernon

 2016 Krista M. Hernon
Submitted in Partial Fulfillment
of the Requirements
for the Degree of
Master of Science

August 2016
The thesis of Krista M. Hernon was reviewed and approved* by the following:

Ronald P. Wilson
Professor, Department of Comparative and Laboratory Animal Medicine
Professor and Chair, Department of Comparative Medicine

Timothy K. Cooper
Associate Professor, Department of Comparative Medicine

Jenelle M. Izer
Assistant Professor, Department of Comparative Medicine

W. Brian Reeves
Professor, Department of Medicine
Thesis Advisor

*Signatures are on file in the Graduate School
iii

ABSTRACT

The continued development and ongoing use of urinary biomarkers for sensitive
prediction of kidney injury in both preclinical and clinical studies is an active area of research.
As new markers are identified and validated, it is important to utilize them in an effort to
reevaluate the safety of medications delivered to animals during research. Given the frequent use
of anesthesia in various animal models of disease, including in the evaluation of kidney injury,
information regarding the effects of anesthesia alone is important in both guiding the appropriate
selection of anesthetic agents, as well as in the interpretation of data. For this purpose, a
prospective study utilizing male C57BL/6J (n=45) mice exposed to a single anesthetic episode of
one of several commonly used anesthetics was developed. Three injectable agents
(ketamine/xylazine, tiletamine-zolazepam, and pentobarbital) and two inhalational agents
(isoflurane and sevoflurane) were evaluated, and urine was collected at various time points (0, 6,
12, 24, 36, 48, and 72 hr) following anesthesia. Expression of hepatitis A virus cellular receptor 1
(HAVCR1, AKA KIM-1) and lipocalin 2 (LCN2, AKA NGAL) were measured in the collected
urine samples by ELISA. HAVCR1 levels were significantly elevated over baseline in the
ketamine/xylazine group at 6 hr following recovery from anesthesia. The remaining groups did
not increase over baseline during the first 24 hr following anesthesia. Conversely, LCN2 levels
were significantly decreased from baseline at 6 hr following ketamine/xylazine anesthesia.
Surprisingly, LCN2 levels steadily increased over the first 24 hrs following both isoflurane and
sevoflurane anesthesia, with significant elevations present at 24 hr after sevoflurane. These
results suggest an immediate effect of injectable anesthesia on HAVCR1 and LCN2, while the
inhalational effect on these biomarkers increases with time.
iv

TABLE OF CONTENTS
List of Figures .......................................................................................................................... vi
List of Tables ........................................................................................................................... vii
List of Abbreviations ............................................................................................................... viii
Acknowledgements .................................................................................................................. x
Chapter 1 Introduction ............................................................................................................. 1
Chapter 2 Materials and Methods ............................................................................................ 5
Animals ............................................................................................................................ 5
Equipment and Housing ................................................................................................... 6
Anesthesia Preparation, Administration, and Monitoring ................................................ 7
Ischemia-Reperfusion Injury Surgery .............................................................................. 9
Urine Collection and Storage ........................................................................................... 11
Euthanasia, Tissue Collection and Processing ................................................................. 11
Biomarker Quantification................................................................................................. 13
Renal Scoring ................................................................................................................... 13
Statistical Analysis ........................................................................................................... 14
Chapter 3 Results ..................................................................................................................... 16
Clinical Condition for Duration of Study ......................................................................... 16
Anesthetic Induction and Duration .................................................................................. 18
Serum Chemistry .............................................................................................................. 19
Urinary Biomarkers .......................................................................................................... 21
HAVCR1 .................................................................................................................. 21
LCN2 ........................................................................................................................ 23
Renal Histology ................................................................................................................ 25
Chapter 4 Discussion ............................................................................................................... 28
Chapter 5 Future Directions ..................................................................................................... 36
References ................................................................................................................................ 37
Appendix A Supplemental Information .................................................................................. 42
Acute Kidney Injury ......................................................................................................... 42
Biomarkers of Kidney Injury ........................................................................................... 44
Serum Creatinine ...................................................................................................... 44
Blood Urea Nitrogen ................................................................................................ 44
HAVCR1 .................................................................................................................. 45
v

LCN2 ........................................................................................................................ 46
Anesthesia ........................................................................................................................ 48
Ketamine/Xylazine ................................................................................................... 48
Pentobarbital ............................................................................................................ 49
Tiletamine-zolazepam .............................................................................................. 49
Volatile Anesthetics: Isoflurane and Sevoflurane .................................................... 50
Appendix B Raw Data ............................................................................................................ 52
Urinary Biomarkers – Individual Mice ............................................................................ 52
HAVCR1 Group Averages .............................................................................................. 57
LCN2 Group Averages .................................................................................................... 58
vi

LIST OF FIGURES
Figure 1. Change in body weight over duration of the study. .................................................. 17
Figure 2. Serum BUN at endpoint ........................................................................................... 20
Figure 3. Serum Creatinine at endpoint. .................................................................................. 20
Figure 4. Urinary HAVCR1 expressed as a fold change from baseline. ................................. 22
Figure 5. Urinary LCN2 expressed as a fold change from baseline. ....................................... 24
Figure 6. Renal histology scores of individual mice at endpoint. ............................................ 26
Figure 7. Representative renal histology of postive control mice at endpoint. ........................ 27
vii

LIST OF TABLES
Table 1. Drug dilution and dosing scheme .............................................................................. 8
Table 2. Criteria for grading the severity of nephropathy ........................................................ 14
Table 3. Average time of induction and duration of injectable anesthesia .............................. 18
Table 4. Serum BUN and creatinine levels at endpoint ........................................................... 19
Table 5. Average urinary HAVCR1 fold change from baseline .............................................. 23
Table 6. Average urinary LCN2 fold change from baseline .................................................... 25
Table 7. Average renal histology scores at endpoint ............................................................... 26
viii

LIST OF ABBREVIATIONS

AAALAC Association for Assessment and Accreditation
of Laboratory Animal Care, International
AKI Acute Kidney Injury
BUN Blood Urea Nitrogen
Cis Cisplatin, control group name
g Grams
GFR Glomerular Filtration Rate
HAVCR1 Hepatitis A Virus Cellular Receptor 1
hr Hour
IACUC Institutional Animal Care and Use Committee
IP Intraperitoneal
Iso Isoflurane, experimental group name
kg Kilogram
KIM-1 Kidney Injury Molecule 1
KX Ketamine/Xylazine, experimental group name
LCN2 Lipocalin 2
LORR Loss of Righting Reflex
MBC Metabolic Cage
min Minutes
ml Milliliter
NC Negative Control Group Name
NGAL Neutrophil Gelatinase Associated Lipocalin
Pent Pentobarbital, experimental group name
ix

RORR Return of Righting Reflex
SEM Standard Error of the Mean
Sevo Sevoflurane, experimental group name
Sx Renal Ischemia-Reperfusion Surgery, positive
control group name
Tel Tiletamine/Zolazepam (Telazol®),
experimental group name
x

ACKNOWLEDGEMENTS

First I would like to thank my committee members Drs. Ronald Wilson, Timothy Cooper,
Jenelle Izer and W. Brian Reeves for their patience, support, guidance, and expertise throughout
the development and completion of this project. Your probing questions have sculpted the way I
think about science, and have empowered me to ask deeper questions about research.
Next, I would like to thank Allen Kunselman (Senior Instructor and Director of
Biostatistical Consulting) and Emily Wasserman (Biostatistics Intern) of the Penn State Hershey
College of Medicine, Public Health Sciences Department for their assistance in helping me to
select the most appropriate statistical tests, and in understanding the interpretation of their results.
I would also like to thank the Department of Comparative Medicine at Penn State
Hershey for the facilities and financial support for this project. Our animal caretakers and
technicians are irreplaceable in the care they provide to the animals within our institution. In
addition, I am indebted to my resident-mates Drs. John Knouse, Rebecca LaFleur, and Regina
Munden not only for your willingness to cover for me when I was performing research, but
mostly for your friendship. I have learned so much from each of you, and am lucky to have you
each in my life.
A special thank you goes out to our laboratory support staff Ellen Mullady, Gretchen
Snavely, Sherry Cooper, Nancy Harner, and Laura Cos. Your assistance and patience regarding
blood sample and histologic processing was priceless. In addition, thank you to Lisa Willing for
your laboratory support, technical expertise, and encouragement during long days. All of you
ladies made every day a little brighter with your humor, kindness, and chocolate breaks.
Finally, I could not have accomplished any of this without the ongoing love and support
of my family. To my husband, Chris, thank you for your unrelenting support in allowing me to
accomplish my goals. To my parents for raising me to believe I can do anything, and for making