loss of inversin contributes to renal cystic disease through altered cellular processes and ...

259 Pages·2017·3.79 MB·English

by  Messersmith, Ashlee J

LOSS OF INVERSIN CONTRIBUTES TO RENAL CYSTIC
DISEASE THROUGH ALTERED CELLULAR PROCESSES AND
DECREASED SODIUM TRANSPORT IN RENAL EPITHELIAL CELLS
by
Nalini H. Kulkarni

A Dissertation
Submitted to the Faculty of Purdue University
In Partial Fulfillment of the Requirements for the degree of

Doctor of Philosophy

Department of Biological Sciences
Indianapolis, Indiana
May 2017
ii

THE PURDUE UNIVERSITY GRADUATE SCHOOL
STATEMENT OF DISSERTATION APPROVAL
Dr. Bonnie L. Blazer-Yost, Chair
Department of Biological Sciences
Dr. Cynthia V. Stauffacher
Department of Biological Sciences
Dr. Rosamund C. Smith
Department of Biological Sciences
Dr. Simon J Atkinson
Department of Biological Sciences
Dr. Nicolas F. Berbari
Department of Biological Sciences

Approved by:
Dr. Stephen K. Randall
Head of the Departmental Graduate Program
iii

Dedication

This dissertation is dedicated to all the people in my life who have been a positive
force for guidance and support during this entire process. I would like to thank my
parents Hanumantha Rao and Sunanda Bai for all that they have done for me over my
lifetime. You have been a constant source unconditional love and support. Thanks to my
Uncle Late Narayana Kulkarni for his constant encouragement and faith in me. Most
importantly, thanks to my husband, Prahalad for his love and endless support. I am very
thankful for your patience and care. I want to thank my two beautiful children, Neha and
Keshav for making my everyday an absolute joy.
iv

ACKNOWLEDGMENTS
At the outset, I extend my heartfelt thanks to my mentor and committee chair, Dr.
Bonnie Blazer-Yost for giving me the opportunity to work in her laboratory, providing
guidance and constructive feedback during the course of this investigation. I express my
sincere gratitude to my committee member Dr. Rosamund Smith for her constant
encouragement, guidance and support over the years. Thanks to my committee member
Dr. Cynthia Stauffacher for her valuable time and patience. I also thank my other
committee members Dr. Simon Atkinson and Dr. Nicolas Berbari for their time and
suggestions. I would like to thank Dr. Tao Wei for providing bioinformatics assistance
and helpful discussions. My thanks to Dr. Amy Usborne for providing histology support.
Finally, I would like to thank the Lilly Graduate Research Advanced Degrees program
for funding, time and support.
v

TABLE OF CONTENTS
LIST OF TABLES ............................................................................................................ vii
LIST OF FIGURES ........................................................................................................... ix
ABSTRACT ....................................................................................................................... xi
CHAPTER1: INTRODUCTION ........................................................................................ 1
An overview of renal cystic diseases ...................................................................... 1
Dominant forms of inherited renal cystic diseases ................................................. 3
Recessive forms of inherited polycystic kidney disease ......................................... 5
Type II nephronophthisis (NPHP2) ...................................................................... 11
Animal models of NPHP2 .................................................................................... 12
Mouse inversin ...................................................................................................... 13
Inversin in development ........................................................................................ 16
Inversin role in cilia of mature renal epithelial cells ............................................. 18
Mechanism of cyst growth and expansion ............................................................ 19
Research presentation and summary ..................................................................... 21
CHAPTER 2: TRANSCRIPTOME ANALYSIS OF inv/inv MICE KIDNEYS.............. 25
Introduction ........................................................................................................... 25
Materials and methods .......................................................................................... 27
Results ................................................................................................................... 32
Discussion ............................................................................................................. 69
CHAPTER 3: LOSS OF INVERSIN DECREASES TRANSEPITHELIAL SODIUM
TRANSPORT IN MURINE RENAL CELLS OF THE CORTICAL COLLECTING
DUCT ................................................................................................................................ 78
Introduction ........................................................................................................... 78
Materials and methods .......................................................................................... 81
Results ................................................................................................................... 87
Discussion ........................................................................................................... 111
vi

CHAPTER 4: TRANSCRIPTOME ANALYSIS OF INVERSIN-DEPLETED
RENAL EPITHELIAL CELLS: COMPARSION OF IN VITRO WITH IN VIVO
ARRAY DATA .............................................................................................................. 116
Introduction ......................................................................................................... 116
Materials and Methods ........................................................................................ 118
Results ................................................................................................................. 123
Discussion ........................................................................................................... 156
CONCLUSIONS............................................................................................................. 162
REFERENCES ............................................................................................................... 165
APPENDIX ..................................................................................................................... 204
VITA ............................................................................................................................... 238
PUBLICATIONS ............................................................................................................ 240
vii

LIST OF TABLES
Table 1: Types of inherited renal cystic diseases in humans .............................................. 2
Table 2: Genetic heterogeneity and overlap of NPHP, Senior-Loken, Joubert and
Meckel-Gruber syndrome ................................................................................................... 9
Table 3: Kidneys in NPHP2 have overlapping features with NPH and ADPKD ............. 10
Table 4: List of primer-probe sets for validation of array findings by real-time
quantitative PCR ............................................................................................................... 30
Table 5: GO biological process terms considered significantly enriched for the
genes regulated in inv/inv mice kidneys compared to wild-type ...................................... 37
Table 6: GO cellular component terms considered significantly enriched for the
genes regulated in inv/inv mice kidneys compared to wild-type ...................................... 41
Table 7: GO molecular function terms considered significantly enriched for the
genes regulated in inv/inv mice kidneys compared to wild-type ...................................... 42
Table 8: Immune/inflammatory genes upregulated in inv/inv mice kidneys .................... 47
Table 9: Regulation of genes affecting cell cycle progression and proliferation in
inv/inv mice kidney ........................................................................................................... 52
Table 10: Regulation of genes representing a decrease in apoptosis and increase
in cell survival in inv/inv mice kidneys ............................................................................. 55
Table 11: Genes associated with development altered in inv/inv mice kidneys ............... 57
Table 12: Downregulation of genes that affect migration and trafficking in
inv/inv mice kidneys ......................................................................................................... 59
Table 13: Regulation of genes affecting Na+ reabsorption and Na+-dependent
transporters in inv/inv mice kidneys ................................................................................. 62
Table 14: Regulation of genes affecting organic anion and cation transporters
and acid-base balance in inv/inv mice kidneys ................................................................. 65
Table 15: Genes associated with metabolic pathways altered in inv/inv mice kidneys .... 67
Table 16: Sequences of siRNA targeting mouse Invs ....................................................... 83
Table 17: Sequences of shRNA constructs targeting mouse Invs..................................... 84
viii

Table 18: Percent levels of inversin knockdown with different doses of inversin
shRNA lentiviral particles in mCCD cells compared to NTC ......................................... 93
Table 19: Percent inversin knockdown in stable clones and non-targeting control ......... 95
Table 20: Criteria for gene array analysis and potential outcome in inversin-depleted
renal epithelial cells in vitro ............................................................................................ 120
Table 21: Number of genes altered in control and inversin-depleted renal epithelial
cells in vitro..................................................................................................................... 124
Table 22: GO biological process terms considered significantly enriched for the
genes regulated in inversin-depleted renal epithelial cells ............................................. 126
Table 23: GO cellular component terms considered significantly enriched for the
genes regulated in inversin-depleted renal epithelial cells compared to controls ........... 130
Table 24: GO molecular function terms considered significantly enriched for the
genes regulated in inversin-depleted renal epithelial cells compared to controls ........... 131
Table 25: Transcription regulators and the downstream targets affected in
inversin-depleted renal epithelial cells............................................................................ 135
Table 26: Genes affecting cell cycle progression are altered in inversin-depleted
renal epithelial cells ........................................................................................................ 138
Table 27: Genes in the upregulated canonical signaling pathways in
inversin-depleted renal epithelial cells............................................................................ 143
Table 28: Genes involved in ion transport altered in inversin-depleted renal
epithelial cells ................................................................................................................. 146
Table 29: Genes affecting renal fibrosis altered in inversin-depleted renal
epithelial cells ................................................................................................................. 147
Table 30: Altered genes common to inversin-depleted renal epithelial cells and
inv/inv mice cystic kidneys ............................................................................................. 150
Table 31: Upregulated genes inv/inv mice cystic kidneys .............................................. 204
Table 32: Downregulated genes in inv/inv mice cystic kidneys .................................... 211
Table 33: Upregulated genes in inversin-depleted renal epithelial cells ....................... 220
Table 34: Downregulated genes in inversin-depleted renal epithelial cells .................. 230
ix

LIST OF FIGURES
Figure 1: Domain architecture of mouse inversin............................................................ 14
Figure 2: Histology and inversin mRNA levels in kidneys from one-day old
wild-type and inv/inv mice. ............................................................................................... 33
Figure 3: Heat map of hierarchial clustering analysis of 646 differentially expressed
genes ................................................................................................................................. 36
Figure 4: Overrepresented GO biological processes altered in inv/inv kidneys .............. 39
Figure 5: Canonical pathways altered in inv/inv mice kidneys analyzed using IPA .... 44
Figure 6: Validation of select immune/inflammatory genes from in vivo
array analysis .................................................................................................................... 49
Figure 7: Validation of select cell cycle genes from in vivo array analysis ..................... 53
Figure 8: Validation of select Na+ reabsorption genes from in vivo array analysis ......... 66
Figure 9: Long-term gene silencing in mCCD cells using inversin Accell siRNA ..... 88
Figure 10: Transepithelial ion transport in inversin knockdown mCCD cells
obtained from Accell siRNA technology ..................................................................... 91
Figure 11: Expression levels of inversin in stable inversin knockdown using
shRNA and untransfected mCCD cells............................................................................. 96
Figure 12: Transepithelial ion transport in stably transduced inversin knockdown
in mCCD cells ................................................................................................................. 100
Figure 13: Loss of inversin decreases transepithelial sodium transport ........................ 104
Figure 14: Expression levels of epithelial sodium channel (ENaC) subunits and
genes that regulate ENaC in inversin knockdown mCCD cells ..................................... 107
Figure 15: Regulation of Crtc2, Sgk1 and Nedd4l protein expression in inversin
knockdown mCCD cells ................................................................................................. 109
Figure 16: Proposed model for epithelial sodium channel (ENaC) regulation
upon inversin loss in renal epithelial cells ...................................................................... 115
Figure 17: Overrepresented GO biological processes altered in inversin-depleted
renal epithelial cells pathway annotations ...................................................................... 128
x

Figure 18: Canonical pathways altered in inversin-depleted renal epithelial
cells analyzed using IPA. ............................................................................................. 133
Figure 19: Network of genes represented in the estrogen-mediated S-phase
entry of cell cycle identified by pathway analysis in IPA. ........................................... 136
Figure 20: Network of genes represented in the mitotic roles of polo-like kinase
identified by pathway analysis in IPA .......................................................................... 137
Figure 21: Assessment of cell proliferation in inversin-depleted renal
epithelial (mCCD) cells .................................................................................................. 140
Figure 22: IPA analysis of genes common to inv/inv mice cystic kidneys and
inversin-depleted renal epithelial cells predicted decrease in organismal death. ........... 152
Figure 23: IPA analysis of genes common to inv/inv mice cystic kidneys and
inversin-depleted renal epithelial cells predicted increased body size. .......................... 154
Figure 24: Assessment of caspase 3/7 activity in inversin-depleted renal
epithelial cells ................................................................................................................. 155
Figure 25: Proposed model of cyst growth and expansion in NPHP2 ........................... 164