TARGETING KIDNEY INFLAMMATION IN A MURINE MODEL OF ALPORT SYNDROME

by

Kyoungheon Ahn

A thesis submitted in conformity with the requirements for the degree of Master of Science

Department of Physiology University of Toronto

© Copyright by Kyoungheon Ahn 2018

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Targeting kidney inflammation in a murine model of Alport Syndrome

Kyoungheon Ahn

Master of Science

Department of Physiology University of Toronto

2018

Abstract

Alport Syndrome is a rare inherited kidney disease that is due to genetic mutations of

type IV collagen. The mutations interfere with normal assembly of the glomerular basement

membrane and this structural abnormality leads to a progressive rise in urinary protein excretion

and eventually to end stage renal failure. Preliminary studies in our laboratory suggest that

inflammation may play an early role in the pathogenesis of glomerular injury.

Thus, the relationship between inflammatory pathways and AS disease progression was

examined in this study by inhibiting NF-κB activation in glomerular cells using MS417. I

hypothesized that the early activation of NF-κB plays a key role in the initiation and progression

of kidney injury in AS and the blockade of NF-κB-mediated gene expression can attenuate

kidney injury in experimental AS. The findings from the analysis of gene expression in a murine

model of AS identified NF-κB as an important target for treatment.

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Acknowledgements

I would like to thank my supervisor, Dr. James Scholey, for providing me with this

opportunity. I am truly grateful for the time and effort you have invested in me and the

experiences I have gained in your lab has helped me grow not only as a scholar but as a human

being. Thank you!

I would also like to thank my committee members, Dr. York Pei and Dr. Scott Heximer,

for their invaluable input and guidance. Their support throughout my degree have challenged and

pushed me to become a more critical and attentive scientist.

A special thanks to my examination committee members, Dr. Zhong-Ping Feng and Dr.

Tianru Jin, for providing me with indispensable input to my thesis work. I am also extremely

grateful for Dr. Feng’s unflagging patience, understanding, and support as the departmental

graduate coordinator.

I must also extend my gratitude to the members of the Pei lab, in particular, Dr. Xuewen

Song, who has lent her expertise to my research work, as well as members of the Scholey lab.

A very special thank you to Veronica Talunay for her unrelenting kindness and patience.

Thank you for all the laughter and chats, I will forever be grateful to you!

I also would like to thank Dr. John He for providing me with the NF-κB inhibiting

compound, MS417, which provided the foundation for my research.

This thesis could not have been completed without the funding provided by the

Department of Physiology, the Alport Syndrome Foundation, and the Kidney Foundation of

Canada.

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Table of Contents

Acknowledgements ...................................................................................................................... iii

Table of Contents ......................................................................................................................... iv

List of Figures ............................................................................................................................... vi

List of Tables .............................................................................................................................. viii

List of Abbreviations ................................................................................................................... ix

Chapter 1 Introduction ................................................................................................................ 1

1.1 Clinical features of Alport Syndrome ....................................................................... 1

1.2 Genotype-phenotype correlation ............................................................................... 4

1.3 Glomerular basement membrane .............................................................................. 6

1.3.1 Laminin ............................................................................................................ 6

1.3.2 Type IV collagen .............................................................................................. 8

1.3.3 Nidogen ............................................................................................................ 9

1.3.4 Heparan sulfate proteoglycans ....................................................................... 10

1.3.5 Glomerular basement memebrane in Alport Syndrome ................................ 11

1.4 Current clinical management options ..................................................................... 12

1.5 Renal inflammation blockade via BRD4 inhibition ................................................ 18

1.6 Experimental design and hypothesis ....................................................................... 23

Chapter 2 Materials and Methods ............................................................................................. 24

2.1 Animals ................................................................................................................... 24

2.2 MS417 treatment ..................................................................................................... 24

2.3 Blood biochemistry ................................................................................................. 25

2.4 Urinary albumin excretion ...................................................................................... 25

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2.5 Isolation of mice glomeruli ..................................................................................... 26

2.6 Microarray data and bioinformatics analysis .......................................................... 30

2.7 Cell culture and NF-κB activity assay .................................................................... 31

2.8 Real-time RT-PCR .................................................................................................. 32

2.9 Statistical analysis ................................................................................................... 32

Chapter 3 Results ........................................................................................................................ 33

3.1 Characterization and breeding of murine model of Alport Syndrome ................... 33

3.2 Early microarray studies in glomeruli ..................................................................... 39

3.3 Targeting NF-κB in vitro ........................................................................................ 47

3.4 Analysis of NF-κB inhibition in vivo using whole kidney samples ....................... 49

3.4.1 Whole animal kidney examination ................................................................ 49

3.4.2 Proinflammatory cytokine expression ........................................................... 54

3.5 Analysis of NF-κB inhibition in vivo via microarray studies ................................. 56

Chapter 4 Discussion .................................................................................................................. 65

4.1 Major changes in gene expression are observed in the glomeruli prior to a decline in GFR in experimental AS ........................................................................................................... 65 4.2 Pathway analysis of the differential gene expressions established that pathways converged on NF-κB ..................................................................................................................... 72 4.3 A novel approach to inhibiting NF-κB-mediated gene expression: BRD4 antagonism .................................................................................................................................... 76 4.4 BRD4 inhibitor treatment led to a decrease in cytokine gene expression and a trend towards improved creatinine levels and albuminuria ................................................................... 78 4.5 Conclusions ............................................................................................................. 80

4.6 Pitfalls and future directions ................................................................................... 81

References .................................................................................................................................... 82

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List of Figures

Figure 1. Molecular mechanism of BRD4-regulated inflammation and the inhibition of inflammatory gene transcription with MS417 .............................................................................. 22 Figure 2. Flow chart of glomeruli isolation from Col4a3-/- mice .................................................. 28

Figure 3. RT-PCR of podocyte and distal tubular markers for microarray sample selections ..... 29

Figure 4. Col4a3 KO mice breeding scheme and PCR genotyping ............................................. 35

Figure 5. Heat map of collagen IV α1-α5 expression levels in Col4a3-/- kidneys ....................... 36

Figure 6. Histopathologic injury in Col4a3-/- and WT kidneys .................................................... 37

Figure 7. Functional manifestations of renal pathology of Col4a3-/- mice ................................... 38

Figure 8. Levels of differential gene expression in kidney tissues in WT and KO mice ............. 41

Figure 9. Schematic summary of aberrant activation of signnaling pathways in AS mice .......... 43

Figure 10. Levels of differentially expressed IL-1β target genes ................................................. 44

Figure 11. Levels of differentially expressed TLR4 and MYD88 target genes ............................ 45

Figure 12. Levels of differentially expressed NF-κB target genes in kidney tissues of WT and KO mice ........................................................................................................................................ 46 Figure 13. MS417-inhibited Ang II-induced NF-κB activation HK-2 cells ................................. 48

Figure 14. Body weight change measurements in WT and KO mice ........................................... 50

Figure 15. Blood urea nitrogen (BUN) levels in 7-week-old WT and KO mice following vehicle or MS417 treatment ...................................................................................................................... 51 Figure 16. Serum creatinine levels assessed in 7-week-old animals following vehicle or MS417 treatment ....................................................................................................................................... 52 Figure 17. Urinary albumin excretion (UAE) assessed in 7-week-old animals ........................... 53

Figure 18. Proinflammatory cytokine levels in kidney tissue in WT and Col4a3 KO mice after sham or MS417 treatment ............................................................................................................. 55 Figure 19. Levels of differential gene expression in glomeruli in WT and KO mice after 10-day treatment with sham or MS417 ..................................................................................................... 61

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Figure 20. Marked differential gene expression levels of IL-1β target genes in glomeruli of WT and KO mice ................................................................................................................................. 62 Figure 21. Levels of marked differential gene expression levels of TLR4 and MYD88 target genes in glomeruli of WT and KO mice ....................................................................................... 63 Figure 22. Marked differential gene expression levels of NF-κB target genes in glomeruli of WT and KO mice ................................................................................................................................. 64

viii

List of Tables

Table 1. 55 Ingenuity Pathway Analysis predicted activated upstream regulators in 4-week-old KO glomeruli ................................................................................................................................ 42 Table 2. Summary of glomerular isolation perfusion performed and RNA yield qualities for analyses ......................................................................................................................................... 58 Table 3. RNA quality of glomeruli samples selected for microarray study .................................. 59 Table 4. Reassignment of experimental groups for microarray study .......................................... 60

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List of Abbreviations

αSMA Alpha-smooth muscle actin

ACE Angiotensin-converting enzyme

ADAS Autosomal dominant Alport Syndrome

ADM Adrenomedullin

AKI Acute kidney injury

Ang II Angiotensin II

ARAS Autosomal recessive Alport Syndrome

ARB Angiotensin receptor blocker

AS Alport Syndrome

AT1R Angiotensin II type 1 receptor

AT2R Angiotensin II type 2 receptor

BET Bromodomain and extra-terminal domain

BM Bone marrow

BMP-7 Bone morphogenic protein-7

BRD4 Bromodomain-containing protein 4

BUN Blood urea nitrogen

CCP-1 Chemokine receptor 1

CD45 Cluster of differentiation 45

CDK9 Cyclin-dependent kinase 9

CKD Chronic kidney disease

COL Collagen

CXCL10 C-X-C motif chemokine 10

CXCR3 C-X-C motif chemokine receptor 3

CYP1B1 Cytochrome P450 family 1 subfamily B member 1

DAMP Damage-associated molecular pattern

DOX Doxycycline

DMSO Dimethyl sulfoxide

EAST Epilepsy, ataxia, sensorineural deafness, and tubulopathy

ECM Extracellular matrix

x

ESCAPE Evaluation Study of Congestive Heart Failure and Pulmonary Artery Catheterization Effectiveness

ESRD End-stage renal disease

ESRF End-stage renal failure

GAPDH Glyceraldehyde 3-phosphate dehydrogenase

GBM Glomerular basement membrane

GFR Glomerular filtration rate

HBSS Hank’s balanced salt solution

HIV Human immunodeficiency virus

HIVAN HIV-associated nephropathy

HK-2 Human kidney-2

HMG-CoA 3-hydroxy-3-methylglutaryl-CoA

HSPG Heparan sulfate proteoglycan

IFN-γ Interferon gamma

IgA Immunoglobulin A

IκB Inhibitor kappa B

IKK IκB kinase

IL Interleukin

IPA Ingenuity Pathway Analysis

KCNJ1 Potassium voltage-gated channel subfamily J member 1; renal outer medullary potassium channel

KCNJ16 Potassium voltage-gated channel subfamily J member 16; inward rectifier potassium channel 16

KLK1 Kallikrein 1

KO Knockout

LCC Laminin coiled-coil

LG Laminin globular

LM-521 Laminin-521

LincRNA Long intergenic non-coding ribonucleic acid

LPS Lipopolysaccharide

MCP-1 Monocyte chemoattractant protein-1

MMP Matrix metalloproteinase

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MSC Mesenchymal stem cell

MT Masson’s trichrome

MYD88 Myeloid differentiation primary response 88

NADPH Nicotinamide adenine dinucleotide phosphate

NC Negative control

NF-κB Nuclear factor-kappa-light-chain-enhancer of activated B cells

NLR Nucleotide-binding oligomerization domain-like receptor

PAMP Pattern-associated molecular pattern

PAS Periodic acid-Schiff

PBS Phosphate-buffered saline

PDGF Platelet-derived growth factor

PI3K Phosphoinositide 3-kinase

PRR Pattern recognition receptor

P-TEFb Positive transcription elongation factor b

RANTES Regulated on activation, normal T cell expressed and secreted

RAS Renin-angiotensin system

RC Renal cortex

RCC Renal cell carcinoma

RHD Rel homology domain

RIN Ribonucleic acid integrity number

ROS Reactive oxygen species

RT-PCR Reverse transcription polymerase chain reaction

SFRP1 Secreted frizzled related protein 1

SM22α Smooth muscle protein 22-alpha

TAGLN Transgelin

TGF-β Transforming growth factor beta

TLR Toll-like receptor

TNF Tumour necrosis factor

TNF-α Tumour necrosis factor alpha

TNFRSF12A Tumour necrosis factor receptor superfamily member 12A

TNFSF12 Tumour necrosis factor superfamily member 12

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TRIF Toll-interleukin-1 receptor domain-containing adapter-inducing interferon-beta

TWEAK TNF-related weak inducer of apoptosis

UAE Urinary albumin excretion

UUO Unilateral ureteral obstruction

Wnt Wingless-related mouse mammary tumour virus

WT Wildtype

XLAS X-linked Alport Syndrome

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Chapter 1

Introduction

1.1 Clinical features of Alport Syndrome

Alport Syndrome (AS) is a rare inherited form of chronic kidney disease (CKD)

characterized by progressive nephropathy, sensorineural loss, ocular defects, and the

development of end stage renal disease (ESRD)1–3. It affects at least one in 50,000 individuals

and it is caused by mutations in genes that encode α3-α4-α5(IV) chains of type IV collagen

(Col4α3, Col4α4, and Col4α5, respectively), which interfere with the normal assembly of the

glomerular basement membrane (GBM)2,4–6. Mutations of the COL4A5 gene on chromosome

Xq22 cause X-linked AS (XLAS), which accounts for approximately 85% of cases, whereas

recessive mutations of COL4A3 or COL4A4 genes on chromosome 2q36 constitute the remaining

cases (autosomal recessive AS; ARAS)7–9. In rare instances (<1%), COL4A3 or COL4A4

mutations may follow an autosomal dominant pattern (autosomal dominant AS; ADAS)10.

Clinically, XLAS and ARAS have similar disease characteristics and prognosis, and are

indistinguishable on clinical grounds or standard assessments of renal biopsy tissue, despite the

very distinct patterns of inheritance6.

Such genetic mutations in the type IV collagen give rise to microhematuria as the first

renal manifestation of AS in childhood, which then gradually progress to proteinuria,

hypertension, and eventually to kidney failure in all males with XLAS and in both sexes with

ARAS4,11. Males with X-linked inheritance may be affected with an early onset of the disease,

generally accompanied by extrarenal features, and develop ESRD before the age of 30, or

experience a late onset with renal failure after 30 years of age, commonly accompanied solely by

hearing loss12,13. High-tone sensorineural hearing loss occurs in 70% of affected males and

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lenticonus in approximately 30% of males with XLAS by age 40, when renal failure, hearing

loss, and retinopathy are already present14. In contrast, the disease characteristics tend to be

predominantly milder in females with XLAS compared to males as they are heterozygous

carriers of the disease. Generally, XLAS female patients have minimal clinical manifestations

such as asymptomatic hematuria by age of 30-40 years, proteinuria (75%), ESRD (15% by age

of 60), hearing loss (40%), or peripheral retinopathy (40%). Lenticonus may not occur and

central retinopathy is rare15. As well, X chromosome inactivation, the process by which the

unmutated copy of one X chromosome counters the effect of the mutation, contributes to

variability among female carriers of XLAS, lending to mosaic disease symptoms16.

Autosomal recessive AS is suspected where the disease is found in only a single

generation or a consanguineous family, where males and females in a family are affected with

equal frequency and severity1,17. In autosomal recessive AS, patients exhibit an early onset of

renal failure, including persistent microhematuria that progresses to significant proteinuria and

ultimately to ESRD, before 30 years of age in both sexes. Individuals with ARAS typically also

exhibit juvenile onset of hearing loss with no gender differences in the incidence of hearing

deficit. Ocular lesions stemming from ARAS are present in 30-40% of individuals with more

frequent occurrences in affected males than females18. Contrastingly, in individuals with

autosomal dominant AS, ESRD is frequently delayed until later adulthood than in those with

XLAS or ARAS, with late onset of cochlear manifestations and rare occurrence of ocular

complicity19,20.

In addition to nephropathy, cochlear and ocular abnormalities observed in AS result from

an absence of the collagen IV networks in the ear and eye14. Hearing loss in AS is never

congenital with defective adhesion of the organ of Corti to the basilar membrane possibly

3

underlying the hearing deficit observed in AS patients21,22. Pathognomonic ocular feature of AS

is bilateral anterior lenticonus in which the anterior surface of the lens assumes abnormal conical

shapes and protrude into the anterior chambers of the eyes, bringing about progressive distortion

of the lens accompanied by increasing myopia and astigmatism23. Other typical ocular

abnormalities include dot-and-fleck retinopathy, consisting of whitish or yellowish flecks or

granulations in the retina, corneal disorders, such as recurrent corneal erosion and posterior

polymorphous corneal dystrophy, which can cause severe ocular pain24–27.

Atypical clinical features of AS include gastroesophageal leiomyomatosis, a condition

characterized by visceral smooth muscle overgrowth leading to benign tumours within the

respiratory, gastrointestinal, and female reproductive tracts28,29. Leiomyomas are rarely

associated with AS; however, when present, it is pathognomonic for AS and affected patients

carry a large deletion in the 5’ ends of the COL4A5 and COL4A6 genes, which is normally

expressed in smooth muscle cells of the gastroesophageal tract30. The pathogenetic correlation

between the deletions of the COL4A5 and COL4A6 genes and the incidence of leiomyomas has

not been elucidated. One study by Dahan et al. (1995) suggests that cell proliferation in

leiomyomatosis could be due to the misalignment that results from the occurrence of repeated

sequences in the large COL4A5 and COL4A6 introns31. Arterial abnormalities, such as aneurysms

in adolescence, have also been reported in patients with AS, pronouncing the importance of

Col4α5 and Col4α6 chains in the maintenance of vascular integrity32–34.

The pathologic lesions for renal biopsies from AS patients are variable when examined

by electron microscopy; however, typically renal biopsies exhibit podocyte hypertrophy and

stiffness of the capillary wall, periodically associated with the presence of tubular red blood cell

casts20. Thereafter, gradual glomerular basement membrane thickening and splitting with

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progressive enlargement of mesangial stalks can be observed20,35. This development of

longitudinal splitting of the lamina densa of GBM bestowing a lamellated appearance is

diagnostic of AS36. The GBM lesion can be intermittent, alternating between segments of thick

or abnormally thin GBM, which is particularly prevalent in children with AS or XLAS females37.

GBM lamellation is also accompanied by fragmentary podocyte foot process effacement,

exhibiting progressive podocyte depletion, a common mechanism in glomerular diseases that

further drives progression38–41. Furthermore, there is segmental or diffuse glomerulosclerosis,

tubular atrophy, and interstitial fibrosis present, along with interstitial infiltration with

lymphocytes and macrophages, correlated with progressive renal failure42,43.

1.2 Genotype-phenotype correlation

The correlation between clinical presentations and the underlying mutations that bring

about AS has been extensively studied and reviewed in order to not only better understand the

mechanisms of disease, but also to potentiate the prognostic value of efficient and noninvasive

screening methods, such as genetic testing44. Extensive studies have been performed globally to

characterize the natural history of AS and it has been established that there is a strong association

between the genetic variants and the renal disease severity45–47. Specifically, patients with large

deletions and nonsense mutations demonstrate more severe manifestations of the disease with

earlier progression to ESRD compared to those with missense mutations, while those with splice

site mutations present with an intermediate disease progression48. Thus, the risk of ESRD by 30

years of age for male XLAS patients with missense mutations, splice site mutations, and large

deletions and nonsense mutations are 50%, 70%, and 90%, respectively, with the average age of

ESRD being 37, 28, and 25 years, respectively12. Correspondingly, large deletions, splice site, or

5

truncating mutations confer a significantly higher risk of developing hearing loss and ocular

lesions in patients than among those with missense mutations12,44,48. Furthermore, Bekheirnia et

al. (2010) demonstrated that there was an association between the mutation position and the

disease outcome, with an earlier onset of ESRD related to pathogenic mutations at the 5’ end of

the gene; the development of hearing impairment and ocular changes showed a similar

association with the mutation location. Though the genotype-phenotype correlation has only

been established in XLAS males, clinical courses of XLAS and ARAS are similar47. In contrast,

no correlation between the degree of the disease progression and the type of underlying mutation

could be substantiated in female carriers of XLAS15,49.

Currently several animal models of Alport Syndrome have been developed and published

in the literature50,51. Such experimental models have yielded significant insight into the

molecular basis of AS as well as the structure and biosynthesis of type IV collagen, thus making

animal models invaluable in the study of pathogenesis and treatment of disease52. So far, these

models are limited to spontaneous AS in dogs and transgenic models of AS generated in mice51.

XLAS models include COL4A5-mutant Samoyed dogs and Navasota dogs, with English cocker

spaniel modelling ARAS, and Bull terrier for ADAS53–57. Murine models are comprised of

COL4A3 knockouts and tg/tg mice58–60. These animal models offer opportunities for

investigation of the molecular pathophysiology of the disease, the roles of type IV collagen

chains in the pathobiology, as well as the applicability of the model in the treatment of AS51.

Understanding the source and extent of the genetic variation underlying AS is crucial in

the management of the disease as clinical care and treatments rely on prognosticating phenotypes

from genetic polymorphisms. Knowledge about the type and location of the mutations adds

significant information about the progress of the renal and extrarenal manifestations of the

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disease, which could potentially aid in clinical counselling and evaluation of therapeutic

approaches.

1.3 Glomerular basement membrane

The glomerular basement membrane (GBM) is a non-cellular meshwork of extracelluar

matrix (ECM) proteins that plays a central role in the glomerular filtration barrier, separating the

vasculature from the urinary space61. It is situated between the interdigitated podocytes (visceral

epithelial cells) and fenestrated endothelial cells, formed during glomerulogenesis by the fusion

of these two cellular membranes62. It provides not only selective permeability across the

glomerular filtration barrier, but also contributes integral structural support for the glomerular

capillaries62. Moreover, it imparts morphogenic cues that determine the polarization of

subcellular constituents and harbours ligands for cell receptors and transporters on the surface of

adjacent endothelial cells, podocytes, and mesangial cells62. As with all basement membranes,

the four major components of the GBM are type IV collagen, laminin, nidogens, and heparan

sulfate proteoglycans (HSPG), which conjunctively assemble an interwoven meshwork that

imparts size and charge-selective properties63. However, the GBM is unusually thicker than most

other basement membranes and consists of ECM macromolecule isoforms that vary from other

basement membranes (laminin-521, collagen α3α4α5(IV), and agrin), presumably due to its

unique functional properties in establishing and maintaining the glomerular filtration barrier64.

1.3.1 Laminin

Laminin is a ubiquitous basement membrane component that is secreted as heterotrimers

of alpha, beta, and gamma chains65. There are five α chains, four β chains, and three γ chains that

7

assemble to form 15 different heterotrimeric macromolecules. Each distinct laminin trimers are

named based on the specific αβγ chain composition; for instance, laminin α5β2γ1 is referred to

as laminin-521 (LM-521)66. The laminin heterotrimeric glycoprotein is structurally cross-shaped

with the lower long arm of the cruciform assembled by the association of all three α, β, and γ

chains intertwining with one another via coiled-coil interactions and disulfide bonding, forming

laminin coiled-coil (LCC) domains67. The laminin globular (LG) domain is situated at the distal

end of the long arm, consisting exclusively of carboxyl-termini of α chains. The LG domains

mediate interactions between laminin trimers and neighbouring cells by serving as ligands to cell

surface receptors, such as integrins and dystroglycan68. The three other short arms are formed by

alternating globular and rod-like domains, including the amino-terminal globular domain, called

LN domain, which mediates the trimer-trimer interactions in the extracellular space that bring

about laminin polymerization and the initiation of basement membrane assembly64,65,69.

The major laminin trimer found in the mature GBM is LM-521, which is secreted by both

podocytes and endothelial cells69. In the early stages of glomerulogenesis, LM-111 and LM-511

are the major laminin components in the nascent GBM; however, as the GBM matures, major

transitions in the laminin trimer deposition occurs in which LM-111 is initially replaced by LM-

511 and then eventually by LM-521 in the mature glomeruli, the composition of which persists

in the GBM throughout life70. The molecular mechanisms responsible for these developmental

laminin isoform substitutions have yet to be elucidated, but this composition transition is crucial

for glomerulogenesis71. The importance of these laminin transitions is particularly highlighted by

the effects of mutations that prevent them from occurring. For example, a null mutation of

laminin α5 in mice prevents the developmental switch in laminin α chain deposition in which α5

replaces α1 in the GBM during glomerular morphogenesis70. Thus, this gives rise to an aberrant

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GBM and the subsequent failure of glomerular vascularization. The GBM breakdown most likely

occurs due to the lack of a sufficient concentration of polymerized laminin trimers, as the

laminin network is required to maintain basement membrane integrity. Furthermore, Lama5-/-

mice were shown to exhibit either renal agenesis in one or both kidneys or if both present, the

kidneys were much diminished in size in comparison to those of the littermate controls70,72. The

absence of laminin α5 leads to progressively atypical metanephrogenesis and eventually result in

embryonic lethality.

1.3.2 Type IV collagen

Type IV collagen is the most abundant protein found in the GBM, composed of six

distinct alpha chains 1 through 6, designated Col4α1-Col4α67. The encoded proteins, α1(IV)-

α6(IV) assemble to form three different heterotrimers referred to as protomers: α1α1α2;

α3α4α5; and α5α5α673. Collagen α1α1α2(IV) is synthesized by endothelial cells, mesangial

cells, as well as podocytes of immature glomeruli, while collagen α3α4α5(IV) originates solely

from podocytes74. Like all collagen chains, type IV collagen chains are characterized by a long

collagenous domain of Gly-X-Y amino acid triplet repeats, with glycine being the only residue

small enough to fit inside the collagen helix75. However, unlike other collagen types, type IV

chains consist of multiple noncollagenous interruptions, which impart flexibility to the protomers

and the overall interwoven network they form in the GBM65. The small noncollagenous amino-

terminal domain of all collagen IV α chains is termed 7S and the larger carboxyl-terminal

noncollagenous domain is called NC1, with both domains involved in promoter interactions and

promoting collagen IV network assembly64. The compositions of the protomers are

9

predetermined by the sequence and structure embedded in the NC1, ensuring that only the three

types of heterotrimers form76.

During embryonic development, the α1α1α2(IV) network is the most abundant collagen

IV network in the GBM, and it is gradually replaced by the α3α4α5(IV) heterotrimer in the

glomerulus, cochlea, and eye77. This transition in the composition of collagen IV chains occurs

concurrently with the transition of trimeric laminin chains, which switches from laminin-511 to

laminin-521, as well as the formation of the capillary loops within the maturing glomeruli76. The

molecular mechanisms for the laminin and collagen IV isoform substitutions in the GBM are

unknown. However, the mature α3α4α5(IV) network is more heavily crosslinked with disulfide

linkages and resistant to proteolytic degradation than the immature α1α1α2(IV) network, which

consists of more proteolytic cleavage sites than the α3α4α5(IV) network24. This isoform switch

may be critical for the establishment and maintenance of the permselective barrier properties in

the glomerulus, especially when accommodating the increased blood pressure in the adult74. In

AS, mutations in type IV collagen genes COL4A3, COL4A4, and COL4A5, which encode the

α3α4α5(IV) chains, interfere with the assembly of the α3α4α5(IV) collagen network in the

GBM and hinder the developmental switch from α1α1α2(IV) to α3α4α5(IV) network77. This

persistence of the nascent GBM results in abnormal membranes and clinical features

characteristic of AS, illustrating the importance of the α3α4α5 type IV collagen network in the

GBM.

1.3.3 Nidogen

Nidogens are two homologous basement membrane glycoproteins, designated nidogen-1

and nidogen-2, that are able to bind to the short arms of laminin γ1 chain as well as to type IV

10

collagen62. Thus, they are thought to act as a molecular bridge between the laminin and collagen

IV networks in the basement membrane78. However, the loss of either or both isoforms of

nidogen have no effect on the basement membrane formation, suggesting that nidogens may

provide extra stability to basement membranes under increased stress, but are not required for

their initial development79. Both nidogen-1 and -2 are present in the GBM, but not much is

known about their developmental expression patterns78.

1.3.4 Heparan sulfate proteoglycans

Heparan sulfate proteoglycans (HSPGs) are glycoproteins with a protein core to which

heparan sulfate side chains are covalently linked62. These sulfated glycosaminoglycan side

chains can impart a highly negative charge, which corresponds to the anionic sites within the

GBM that are detectable by cationic probes78. Whilst perlecan is the most prominent HSPG in

most basement membranes and in the mesangial matrix, agrin is the major HSPG in the mature

GBM. During glomerulogenesis, perlecan and agrin are uniformly distributed; thereafter,

perlecan is substituted by agrin and confined to the subendothelial side of the GBM78.

Perlecan, produced primarily by glomerular endothelial cells, is anchored in the basement

membranes by interactions with other constituents, binding to nidogen via its core protein, and to

laminin and collagen IV via its heparan sulfate side chains80. On the other hand, agrin is derived

mainly from podocytes and its N-terminal domain binds to the laminin γ1 subunit, while the C-

terminal LG domain binds to cell surface receptors, such as dystroglycans and integrins63. Thus,

agrin may play a role in cell-matrix adhesion through the affinity of its domain for laminin,

dystroglycan, and integrin.

11

HSPGs in GBM were originally thought to confer charge selectivity in the glomerular

filtration barrier; however, this concept has been recently challenged81. Mice with podocyte-

derived agrin knockout did not lead to any structural or functional defects in the glomerulus,

despite the loss of GBM negativity76. Furthermore, deletions of both agrin and heparan sulfate

side chains on perlecan had little, if any, effects on permselectivity with no signs of proteinuria82.

Hence, no critical role for HSPGs, agrin and perlecan, in the GBM has been proven thus far.

1.3.5 Glomerular basement membrane in Alport Syndrome

Mutations of COL4A3, COL4A4, or COL4A5 interfere with the assembly of the

α3α4α5(IV) network in the GBM, resulting in the impediment of the collagen switch from

α1α1α2(IV) to α3α4α5(IV) network during development4. This persistent distribution of the

primordial α1α1α2(IV) network in the Alport GBM produces abnormal membranes that are less

structurally sound due to fewer intra- and interheterotrimer crosslinks and more proteolytic

cleavage sites than the α3α4α5(IV) network24,83. Thus, the α1α1α2(IV) isoform is more

susceptible to biomechanical strain caused by intraglomerular hypertension and ultrafiltration,

endogenous proteolysis, and eventually results in the characteristic degenerative GBM

splitting84,85. This disposition to proteolytic degradation at the site of glomerular filtration is

augmented by ectopic deposition of laminin chains, α1 and α5, resulting in an overall disruption

of the GBM architecture86. LM-511 increases in regions of focally thickened GBM, which are

more permeable to protein71. Furthermore, the lack of collagen IV and laminin isoform

substitutions in the GBM bring about altered podocyte orientation, podocyte foot process

effacement, and disruption of slit diaphragms. Affected podocytes can transmit this pathology

and injury to adjacent healthy podocytes, giving rise to glomerulosclersosis with an

12

accumulation of ECM, as well as kidney fibrosis due to fibroblast formation by epithelial-

mesenchymal transition, a physiological repair response to injury87,88. Dysregulation of matrix

metalloproteinases (MMPs) in glomerular mesangial cells has also been evidenced in AS, with

their induction shown to contribute to the mechanism of pathobiology, while the blockade of

specific MMP activities has been shown to ameliorate the disease progression89,90. In particular,

MMP-2, MMP-3, MMP-9, MMP-12, and MMP-14 have been most frequently associated with

inflammatory diseases and fibrosis in the literature89–93. Furthermore, a study by Zeisberg et al.

(2006) identified a dichotomous role for MMPs in the progression of AS in Col4α3-/- mice, in

which an MMP upregulation was significantly correlated to deterioration of glomerular function;

however, after the induction of proteinuria and the emergence of fibrosis, MMPs were found to

be renoprotective against disease progression91. Thus, changes in the integrity of the GBM,

brought about by the mutations in the genes encoding collagen type IV α chains, affect cell

signalling mechanisms, and further the pathogenesis of Alport glomerulopathy.

1.4 Current clinical management options

Currently the mechanisms responsible for kidney injury in AS have not been fully

elucidated and the standard of care is limited to nonspecific therapeutic approaches that reduce

renal fibrosis and delay the onset of ESRD94. To date, several treatment options have been

demonstrated to improve outcomes in animal models of AS, including renin-angiotensin system

(RAS) blockade, such as angiotensin-converting enzyme inhibitors (ACE inhibitors) and

angiotensin receptor blockers (ARBs), cyclosporine A, matrix metalloproteinase (MMP)

inhibitors, transforming growth factor beta 1 (TGF-β1) inhibitors, HMG-CoA reductase

inhibition, chemokine receptor 1 (CCP-1) blockade, bone morphogenic protein-7 (BMP-7), stem

13

cell transplantation, and bone marrow irradiation90,91,95–104. Such studies have not only shed light

on an array of potentially effective therapies for AS, but have also contributed significantly in the

understanding of the mechanistic pathways mediating the progression of AS towards ESRD.

Despite the lack of definite treatment for AS, ACE inhibitors are the recommended first

line of treatment for non-immunological therapy of proteinuric glomerular disease105. ACE

inhibitors work by preventing the conversion of angiotensin I to angiotensin II, a growth factor

involved in activating fibroblasts, leading to increased synthesis of matrix proteins, as well as

functioning as a profibrotic cytokine, activating mononuclear cells and increasing

proinflammatory mediators98. The downstream proinflammatory effects of angiotensin II are

mediated via the angiotensin type 2 receptor (AT2R) and the TGF-β pathway, which has been

shown to be important in the renal fibrosis in AS98. Thus, ACE inhibition has been shown to have

antiproteinuric and antifibrotic nephroprotective effects in AS in a time-dependent manner, with

earlier introduction of therapy yielding delayed onset of ESRD and prolonged life

expectancy97,106. Extensive clinical data on the efficacy and safety of ACE inhibitors in patients

with progressive nephropathies are available; for instance, the Evaluation Study of Congestive

Heart Failure and Pulmonary Artery Catheterization Effectiveness (ESCAPE) trial showed that

ACE inhibition with ramipril resulted in low incidence of adverse effects in children with

chronic kidney disease107. However, in the case of patients experiencing adverse reactions to

ACE inhibitors, such as coughs, angioedema, hyperkalemia, as well as decreased glomerular

filtration rate (GFR), ARBs may be used108,109. ARBs inhibit angiotensin II activity specifically

by antagonizing its action on the angiotensin II type 1 receptor (AT1R)110. Long-term treatment

with losartan was found to confer sustained antihypertensive and renoprotective effects with high

tolerance in children with nephropathies111,112. Thus, angiotensin antagonism has been shown to

14

be effective in preserving renal function via its capacity to reduce glomerular hypertrophy and

sclerosis, as well as tubulointerstitial inflammation and fibrosis. This recommended approach to

treatment is well-tolerated and is widely used in patients with AS; however, it does not prevent

ESRD105. Its efficacy is limited and most pronounced when administered at early stages of the

disease, before the onset of microalbuminuria, thus making it a partially effective treatment

option with limitations.

Calcineurin inhibitor, cyclosporine A, has also been experimentally and clinically studied

as a potential treatment option of AS94. Originally introduced into clinical practice as an

immunosuppressant medication, it has been found to have beneficial effects on proteinuria and

renal function when applied in the therapeutic management of AS. A study in a canine model of

XLAS by Chen et al. (2003) showed that cyclosporine induced a significant delay in the onset

and rate of progression of glomerulosclerosis as well as in the deterioration of renal function,

although treatment did not affect the amount of interstitial fibrosis nor yield any beneficial

effects on proteinuria99. In contrast, the examination of the therapeutic efficacy of cyclosporine

in small clinical trials have demonstrated a substantial reduction in proteinuria in Alport patients.

However, cyclosporine usage was also associated with decreased GFR and significant renal

lesions related to cyclosporine nephrotoxicity, thus precluding long-term administration113–115.

Though the mechanisms of actions of cyclosporine in AS have yet to be elucidated, its

antiproteinuric effect seems to stem from its ability to stabilize the podocyte actin cytoskeleton,

independent of its immunosuppressive action116. This discovery sheds further light on the role of

calcineurin signalling in the biology of proteinuric kidney diseases and provides novel

calcineurin substrates, such as synaptopodin, as auspicious starting points for the identification of

antiproteinuric drugs that evade the chronic adverse effects of long-term cyclosporine treatment.

15

Other experimental pharmacological therapies that have been studied with limited clinical

experience include broad spectrum MMP inhibition, TGF-β1 blockade, inhibition of HMG-CoA

reductase, CCP-1 antagonist, and BMP-7. These therapeutic interventions start to give insight

into the pathophysiology of AS and highlight the importance of understanding the underlying

molecular mechanisms of disease before initiating treatment. For instance, the pharmacologic

ablation of MMP-2, MMP-3, MMP-9, and MMP-12, which are responsible for the proteolytic

degradation of GBM, before the onset of proteinuria in Col4α3-/- mice have led to a significant

attenuation in disease progression, marked by delayed proteinuria and prolonged survival91,92.

Likewise, a significant elevation in MMP expression levels were observed in a canine model of

XLAS, implicating their induction with ECM dysregulation and interstitial fibrosis in Alport

kidneys117,118. Thus, MMPs have a contributory role in the pathobiology of Alport renal disease

and their inhibition ameliorate GBM damage, restoring its ultrastructure and function119. TGF-β1

has also been shown to have a potential role in Alport renal disease pathogenesis. A study by

Sayers et al. (1999) observed an induction of TGF-β1 mRNA and proteins in both human and

murine podocytes following the establishment of proteinuria120. Similarly, HMG-CoA reductase

inhibitors, also referred to as statins, which are potent inhibitors of cholesterol synthesis, have

been recognized to have nephroprotective effects in Col4α3-/- mice1. Beyond its role on lipid

reduction, HMG-CoA reductase inhibitor’s pleiotropic effects have been recognized, in which,

for instance in the kidney, it is able to inhibit mesangial cell proliferation, mesangial matrix

deposition, as well as moderate monocyte infiltration and production of TGF-β1101. Thus, the

administration of HMG-CoA reductase inhibitor brought about an attenuation in the expression

of TGF-β, as well as concomitant decreases in proteinuria, renal fibrosis, and inflammatory cell

infiltration, in addition to a prolongation of the lifespan of the KO mice. Other Col4a3-/- mice

16

studies have further illustrated CCP-1 antagonist and the upregulation of BMP-7 as potential new

therapies for use in AS. The usage of BX471, a small molecule CCR-1 inhibitor, was associated

with improved survival of KO mice, as well as decreased interstitial macrophages, tubular

atrophy, and interstitial fibrosis100. Treatment of KO mice with exogenous recombinant human

BMP-7 led to significant decrease in the expression of profibrotic molecules, improved renal

function, histology, and survival in mice, illuminating an overall renal protective effect of BMP-

7102.

Biological interventions, including cell-based therapies and bone marrow irradiation, are

emerging techniques that have been extensively evaluated as a prospective therapeutic options in

AS patients. Cell-based regenerative therapy aims to repair the underlying defect; in the case of

AS, the defective assembly of the α3-α4-α5(IV) chains of collagen in the GBM121. Thus far,

replacement of the mutated genes via gene therapy have not been successful in humans122. In

Alport mice, however, it has been reported that stem cell therapy has yielded therapeutic

benefits, promoting its application for clinical trials. For instance, Ninichuk et al. (2006)

presented that injections of primary bone marrow (BM)-derived multipotent mesenchymal stem

cells (MSCs) into Col4a3 KO mice reduced interstitial fibrosis compared to saline-injected mice,

though marks of renal function, including blood urea nitrogen (BUN), creatinine levels,

proteinuria, and survival were not affected by MSC administration96. Interestingly, MSC was

found to localize to kidneys of the KO mice after injection, but their differentiation into renal

cells was not detected. Similarly, studies by Prodromidi et al. (2006) and Sugimoto et al. (2006)

have independently illustrated that whole BM transplantation is able to ameliorate the chronic

renal injury caused by Col4α3 deficiency in KO mice via differentiation of stem cells into

podocytes that then secrete the missing collagen α3-α4-α5(IV) chains103,104. This is paralleled by

17

decreased serum urea and creatinine levels, reduced interstitial fibrosis, and improved renal

histology. Nonetheless, the extent of podocyte cell engraftment by BM cells was low, suggesting

that some other effect of BM transplantation may be contributing to the functional disease

recovery122. Furthermore, despite reported improvements in renal function as well as glomerular

architecture defects in treated Alport mice compared to control groups, the age at ESRF, a

meaningful endpoint of the disease, was not tested in either studies94,122–124. Contrastingly,

Katayama et al. (2006) reported that BM transplant after lethal irradiation with either WT or KO

BM prolonged the lifespan with similar efficiencies, denoting that the amelioration of disease

observed is conferred most significantly by the irradiation. A positive correlation between the

irradiation dosage and survival time was noted. The work by Katayama et al. challenges the

observations published by Prodromidi and Sugimoto groups and these differences may be

derived from the dissimilar age at the time of BM transplant as well as the varying genetic

backgrounds of the experimental Alport mice, in which Katayama’s study was performed in AS

mice on the 129 SvJ background, while Prodromidi and Sugimoto’s studies were generated in

C57BL/6J strain of Col4a3 KO mice95,103,104. Thus, the different backgrounds and age at which

BM transplantations were completed could have imparted different outcomes, attributable to the

varied severity of GBM defects. Our current knowledge about gene- and cell-based therapies and

their possible efficacies in the treatment of AS is yet insufficient and many experimental

questions need to be addressed before clinical studies are considered in Alport patients125.

Nonetheless, biological interventions, such as regenerative medicine, provide new hope for a

means to restore structural and functional integrity of the diseased kidney in the treatment of AS

and other inherited renal glomerular diseases.

18

Currently there is a lack of therapies that can effectually change the course of disease

progression in patients with AS94. Promising experimental treatments have been developed and

studied that promote the elucidation of the renal pathology of AS. However, despite a variety of

treatment options available, none of these are causal therapeutic strategies that have been as

effective in prolonging survival as ACE inhibition; they merely attenuate and do not prevent the

progression to ESRD1. Hence, for patients with AS who have progressed to renal failure, dialysis

or renal transplantation are the only therapeutic options104. There remains a gap in our

understanding of the early mechanisms that lead to progressive kidney injury in AS which limits

efficacious treatment. Accordingly, the Alport Foundation has issued a call for new studies in the

development of novel treatment approaches to AS.

1.5 Renal inflammation blockade via BRD4 inhibition

The role of inflammation as a major driver in the pathophysiology of CKD has been

widely and compellingly recognized in recent years126. Inflammation contributes to progressive

kidney damage by inducing the release of proinflammatory cytokines and adhesion molecules,

which eventually brings about a loss of glomeruli, tubular atrophy and fibrosis, accompanied by

a decrease in glomerular filtration rate127. Therefore, an understanding of the molecular pathways

driving renal inflammation is indispensable in the elucidation of the pathogenesis of chronic

renal diseases as well as for the development of novel, targeted therapeutics in hopes of

preventing ESRD in renal pathology128.

During the initial stages of renal disease, inflammatory changes are detected with the

concomitant involvements of several inflammatory cytokines, such as tumour necrosis factor-

alpha (TNF-α) and various interleukins (IL-1, IL-6, IL-18), which then activate a number of

19

signalling pathways129,130. The expression of such inflammatory mediators is regulated by the

transcription factor, nuclear factor-kappaB (NF-κB), which has been shown to activate a number

of proinflammatory genes in both human and experimental models of progressive

nephropathies126. The mammalian NF-κB family consists of five members, RelA (p65), RelB, c-

Rel, NF-κB1 (p50; precursor p105), and NF-κB2 (p52; precursor p100), which all share a highly

conserved DNA-binding domain in the N-terminal region called the Rel homology domain

(RHD)131. Through the association of the RHD, the NF-κB proteins are able to form various

homo- and heterodimers with each other, with the most common and best characterized dimer

being the p65/p50 heterodimer132. In addition, the RHD also binds with the inhibitory κB

proteins (IκB), through which the NF-κB heterodimer is sequestered as an inactive complex in

the cytoplasm. In response to a stimulus, such as cytokines, oxidative stress, or bacterial

endotoxin (LPS), the IκB subunit is rapidly phosphorylated by IκB kinase (IKK) and ultimately

degraded133. The resulting release of the NF-κB dimers and their subsequent translocation to the

nucleus promotes the transcription of target proinflammatory genes, such as RANTES, IL-1, IL-

2, IL-6, MCP-1, TNF-α, as well as adhesion molecules, thereby promoting downstream renal

inflammation126.

Recently, it has come to light that the acetylation of the RelA subunit of NF-κB at lysine-

310 is crucial for the transcriptional activation of NF-κB and the succeeding expression of

inflammatory genes (Figure 1)134. Stimulus-coupled acetylation of RelA by p300/CBP at lysine-

310 promotes a direct interaction with bromodomain-containing protein 4 (BRD4) via its two

bromodomains, which then recruits the acetylated NF-κB to the promoter regions of its target

genes134,135. The binding of the BRD4 to acetylated lysine-310 also promotes the kinases within

the positive transcription elongation factor b (P-TEFb) complex, such as cyclin-dependent kinase

20

9 (CDK9), to phosphorylate RNA polymerase II for the transcriptional activation of NF-κB

target genes, thus driving transcription of proinflammatory genes136,137. This masterly epigenetic

regulator, BRD4, belongs to the bromodomain and extra-terminal domain (BET) family,

consisting of BRD2, BRD3, BRD4, and BRDT. A family of transcriptional coactivators and

elongation factors, BET proteins are characterized by the presence of two conserved tandem

bromodomains and an extra-terminal domain138. The N-terminal bromodomains of BET proteins

are able to recognize and bind to acetylated lysine residues on histones and other nuclear proteins

in the regulation of gene transcriptional activity of cellular proliferation and differentiation

processes, while the extra-terminal domain interacts with histone modifiers to bring about

changes in the chromatin structure, thus effecting epigenetic modulations via chromatic dynamic

and nucleosome assembly modifications139.

Of all the mammalian BET proteins, BRD4 is the most extensively studied isoform and is

broadly implicated in the regulation of renal inflammation140. Given its role in the regulation of

the innate and adaptive immune responses, however, a selective and temporal inhibition of NF-

κB’s proinflammatory activity has been explored as of late, as a complete suppression of NF-κB

may lead to adverse effects141. Recent studies have demonstrated that targeted inhibition of NF-

κB transcriptional activity brings about an attenuation of inflammation in experimental models

of kidney injury. For instance, Zhang et al. (2012) have shown that BET-specific bromodomain

inhibitor, MS417, designed to block BRD4 from binding to the acetylated NF-κB, significantly

downregulates the expression of proinflammatory cytokine and chemokine genes in human renal

tubular epithelial cells, as well as reducing proteinuria, improving renal function, and effectively

ameliorating NF-κB acetylation and in parallel, the expression of its target genes in Tg26 mice,

an established animal model for HIV-associated nephropathy (HIVAN)140. Likewise,

21

administration of the same NF-κB inhibitor, MS417, in db/db mice was shown to attenuate

proteinuria and podocyte injury in diabetic kidney disease142. Hence, these findings strongly

suggest that the selective inhibition of NF-κB activity via BET bromodomain blockade,

represents a new therapeutic approach for treating NF-κB-mediated inflammation and kidney

injury.

Currently the specific effects of NF-κB signalling in experimental AS have not been

extensively studied in the literature. However, recent findings involving the usage of this

aforementioned novel compound, MS417, in the blockade of NF-κB activation in other renal

disease models seem to be promising140,142. The inhibition of BRD4 not only illustrates the

pathophysiologic functions of NF-κB, but it also highlights the significant role of inflammation

in mediating renal disease progression. Thus, these data motivate a pivotal need to investigate the

collaborative roles of NF-kB and BRD4 in the regulation of proinflammatory activation in the

pathogenesis of AS in hopes that targeting epigenetic regulators such as BET proteins may

contribute in the elucidation of the etiology and progression of AS.

22

Figure 1. Molecular mechanism of BRD4-regulated inflammation and the inhibition of inflammatory gene transcription with MS417. Inflammatory stimulus consisting of the binding of ligand to a cell surface receptor, such as Toll-like receptor 4 (TLR4), leads to the recruitment of I kappa B kinase (IKK) complex, which in turn leads to the phosphorylation and degradation of the IκB inhibitor. This frees and activates the NF-κB dimers, p65 and p50. Active NF-κB is translocated to the nucleus, where it binds to consensus sequences in the regulatory elements of gene promoters. The transcription factor recruits BRD4 and together NF-κB and BRD4 control the transcription of inflammatory response genes. BRD4 also helps recruit RNA polymerase II to initiate gene transcription. However, MS417, a BRD4 inhibitor, prevents BRD4 from binding to NF-κB, limiting the recruitment of RNA polymerase II to the transcriptional complex140,142. Thus, the blockade of BRD4 binding to the acetylated NF-κB using MS417 can effectively attenuate NF-κB transcriptional activation of proinflammatory genes.

23

1.6 Experimental design and hypothesis

Sterile inflammation is an innate immune response that is vital for tissue and wound

repair in response to trauma or ischemia-reperfusion injury in the absence of any

microorganisms143. Similar to pathogen-induced inflammation, sterile inflammation is marked by

the recruitment of neutrophils and macrophages, bringing about the production of

proinflammatory cytokines and chemokines, such as TNF and IL-1144. Although inflammation is

important in the resolution of the initial insult, chronic inflammation may lead to emergence of

disease, contributing to the pathophysiology127. Therefore, this study sought to examine the

pathways that result in sterile inflammation in experimental AS, in particular the impact of NF-

κB inhibition via the administration of a BRD4 antagonist.

In order to discern the changes in the patterns of gene expression in the Alport kidney, a

microarray was performed using 4-week-old KO and WT glomeruli as described in Chapter 2.

IPA approach was subsequently used to analyze the differential expression data in hopes of

better understanding the biological changes that occur with the onset of the disease, as well as to

predict the upstream transcriptional regulators to uncover potential therapeutic targets. Once the

molecular alterations of the disease were determined, pharmacologic inhibitor of BRD4 was

administered in HK-2 cells and in KO mice to better define the role of NF-κB in the

pathogenesis of AS.

In summary, I hypothesize that NF-κB will attenuate kidney injury in mice with AS.

24

Chapter 2

Materials and Methods

2.1 Animals

Col4a3+/+ (wildtype; WT) and Col4a3-/- (KO) mice of 129/SvJ background were

purchased from The Jackson Laboratory (Bar Harbor, ME, USA)58. All animals were housed at

the Division of Comparative Medicine at the University of Toronto, and fed standard chow diet

with free access to food and water. Only male mice were used for experiments to avoid the

potential confounding effects of female sex hormones. All experiments were conducted under the

guidelines of the University of Toronto Animal Care Committee.

Male mice were followed from 4 weeks of age until sacrifice and tissue harvest at 7

weeks of age. 24-hour urine samples were obtained at 7 weeks of age by placing animals

individually in metabolic cages in order to perform urinary albumin measurements. At sacrifice,

mice were anesthetized with inhaled isoflurane and their body weights and kidney weights were

recorded. Blood samples were collected from the carotid artery and both kidneys were dissected

out. The kidneys were cross-sectioned into 3 parts, with two polar sections snap-frozen in liquid

nitrogen and stored at -80°C until use. Middle sections of kidneys were fixed in buffered

formalin and embedded in paraffin for histological analysis. The genotypes of study mice were

verified by tail clip genotyping PCR via the usage of the following primers: common, 5’-

CCAGGCTTAAAGGGAAATCC-3’; WT reverse, 5’-TGCTCTCTCAAATGCACCAG-3’; and

mutant reverse, 5’-GCTATCAGGACATAGCGTTGG-3’.

2.2 MS417 treatment

Male WT and KO mice were administered with either a control vehicle (0.1% DMSO in

25

PBS) or MS417 by daily oral gavage at a dosage of 0.0016 mg/kg. MS417 was provided by Dr.

John Cijiang He (Mount Sinai School of Medicine, New York, NY, USA)140,142. The mice were

fed this compound daily from the age of 4 weeks to sacrifice at 7 weeks of age. The mice were

euthanized at 7 weeks of age for blood, urine, and kidney collection. Body weight was recorded

daily from the start of the experiment till sacrifice for all experimental groups.

For the glomerular microarray study, male WT and KO mice were administered with

either a control vehicle (0.1% DMSO in PBS) or MS417 by daily oral gavage at a dosage of

0.0016 mg/kg. The mice were fed this compound daily from the age of 4 weeks till they were

sacrificed 10 days later.

2.3 Blood biochemistry

Blood samples were collected from carotid artery with Microvette (Sarstedt Inc.,

Montreal, QC, Canada) according to the manufacturers’ protocol at the time of sacrifice. Serum

was isolated by centrifuging blood samples at 2000 x g for 5 minutes at room temperature and

was then stored at -80°C until use. Levels of blood biochemical parameters, including blood urea

nitrogen, creatinine, sodium, potassium, chloride, phosphorus, calcium, and total proteins, were

measured at the Toronto Centre for Phenogenomics (Toronto, ON, Canada).

2.4 Urinary albumin excretion

24-hour urine samples were obtained by placing experimental animals individually in

metabolic cages (Nalgene, Nalge Nunc International, Rochester, NY, USA). Urine samples were

centrifuged at 8000 x g for 5 min at room temperature and the supernatants were collected and

stored at -80°C until use. Albumin levels were determined with Albuwell M Kit (Exocell,

26

Philadelphia, PA, USA) following the manufacturers’ guide. Urinary albumin levels were used to

calculate the 24h urinary albumin excretion (UAE) rate.

2.5 Isolation of mice glomerli

The glomerular isolation procedure was adapted from a magnetic bead-based isolation

method described by Takemoto et al. (2002) and Schwarz et al. (2013). An illustrated flow chart

of the protocol is shown in Figure 2145,146.

M-450 tosylactivated, 4.5 µm diameter Dynabeads (Invitrogen, Carlsbad, CA, USA)

were inactivated according to manufacturer’s instructions. Congenic male WT and KO mice at 4

weeks of age were used. At the time of kidney glomeruli harvest, mice were anesthetized and

perfused with 10mL of Hank’s balanced salt solution (HBSS; Invitrogen, Carlsbad, CA, USA)

and then 10mL of 8 x 107 magnetic Dynabeads diluted in HBSS through the left ventricle of the

heart at a constant perfusion speed of 2.3ml/min using Syringe Infusion Pump (Harvard

Apparatus, Holliston, MA, USA). Once the perfusion was completed, kidneys were removed,

minced into 1mm3 pieces, and digested with 1mg/ml collagenase A (Roche Diagnostics,

Indianapolis, IN, USA) in 3ml of HBSS at 37°C for 30 minutes with gentile agitation. The

digested tissue was passed through a 100 µm cell strainer (Thermo Fisher Scientific, Waltham,

MA, USA) using a flattened syringe pestle and the cell strainer was then washed 3 times with 1

ml of PBS. The cell suspension containing intact glomeruli with trapped paramagnetic

Dynabeads was placed into a magnetic particle concentrator (Invitrogen, Carlsbad, CA, USA) for

7 minutes. The glomeruli containing Dynabeads were gathered on the wall of tubes; the

supernatant (containing smaller tubular fragments, single tubular cells, a variety of polymorphic

interstitial cells, and blood cells) and pellets (containing larger tubular fragments) were then

27

carefully pipetted into separate tubes and stored on ice. The glomeruli and larger tubular

fragments were washed for at least three times with PBS. All procedures were performed on ice

except the collagenase digestion. The purity of the glomerular and tubular isolates was verified

under light microscopy. To prove the glomerular and tubulointerstitial origin of separated tissue

fractions, the mRNA expression levels of glomerular (Nphs1 for podocyte) and tubular (Fxyd2

for distal tubules) markers were also examined (Figure 3). Isolated glomeruli samples were

resuspended in QIAzol Lysis Reagent and stored in -80°C.

28

Figure 2. Flow chart of glomeruli isolation from Col4a3-/- mice. Anesthetized mice are perfused with Dynabeads (depicted by blue circles) diluted in HBSS through the left ventricle of the heart. The kidneys are removed, minced into 1mm3 pieces, and then chemically digested in collagenase for 15min at 37°C. The collagenase-digested tissue is then gently filtered through a 100µm cell strainer. The supernatant is discarded and the cell pellet resuspended in HBSS. The glomeruli containing Dynabeads are then collected by a magnetic particle concentrator145,146.

29

Figure 3. RT-PCR of podocyte and distal tubular markers for microarray sample selections. Representative gels illustrating whether glomeruli RNA samples isolated from WT and KO mice had tubular contamination was evaluated via RT-PCR using podocyte (Nphs1) and tubular (Fxyd2) markers. The first column shows an isolated glomeruli sample along with cDNA controls from glomeruli, tubules, renal cortex (RC) sample from a 7-week-old mouse, and RC sample from a 4-week-old mouse, along with a negative control (NC).

30

2.6 Microarray data and bioinformatics analysis

By using Affymetrix Mouse Gene 2.0 ST Arrays (Affyetrix, Santa Clara, CA, USA), the

global gene profiling of glomerular samples from male WT and KO mice at 4 weeks of age (n =

8 per group) was performed. Then in order to test whether the early genetic changes observed in

the disease model could be reversed by the anti-inflammatory drug, MS417, the global gene

profiling of glomerular samples from male vehicle-treated WT mice (n = 5), vehicle-treated KO

mice (n = 9), and MS417-treated KO mice (n = 2) was performed. For all microarray studies, the

total RNA containing small RNA was extracted using RNeasy Mini Kit (Qiagen Inc.,

Mississauga, ON, Canada) following the manufacturer’s protocol with an on-column DNA

digestion step to minimize genomic DNA contamination. The sample integrity of the RNA was

assessed using the RNA 6000 Nano Assay on 2100 Bioanalyzer (Agilent Technologies, Inc.,

Santa Clara, CA, USA) to ensure that RNA integrity number (RIN) was greater than 7.

Microarray experiments were performed at the Microarray Analysis and Gene Expression

Facility at The Hospital for Sick Children (Toronto, Ontario, Canada). Following the

manufacturer’s protocol, 125 ng of total RNA was labelled using the GeneChip WT PLUS

Reagent Kit (Affymetrix, Santa Clara, CA, USA). Fragmented and biotin-labelled ss-cDNAs

were then hybridized to GeneChip Mouse Gene 2.0 ST Arrays for 16 hours at 45°C. GeneChip

Mouse Gene 2.0 ST Arrays feature probe sets for 28,137 coding transcripts, 7,103 non-coding

transcripts, 2,000 long intergenic non-coding transcripts (lincRNA), with an average of 21

unique probes across each gene. Hybridized arrays were then stained and washed in the

Affymetrix Fluidics Station 450. Thereafter, the arrays were scanned on an Affymetrix GeneChip

Scanner 3000 and the image (.DAT) files were preprocessed using the Affymetrix GeneChip

Command Console (AGCC) software v.4.3 to generate cell intensity (.CEL) files. After image

31

processing, the array data was uploaded to the Affymetrix Expression Console software v1.4.1

for further processing and quality control. All quality assessment metrics, including spike-in

controls during target preparation and hydrization, were found within the boundaries. The probe

set signal intensities were then extracted and normalized using the robust multi-array average

(RMA) algorithm embedded in the Expression Console software, which consists of convolution

background correction, quantile normalization, and median polish summarization. Downstream

supervised statistical analysis was carried out via Partek Genomics Suite 6.6 (Partek Inc.,

Chesterfield, MO, USA) to determine differentially expressed genes affected by the genotype

and treatment using a one-way ANOVA and a false discovery rate (FDR) equal to or less than

10%. Ingenuity Pathway Analysis (IPA; Qiagen Silicon Valley, Redwood City, CA, USA) was

used to determine enriched biological processes, disease or toxicological functions, signalling

and metabolic canonical pathways, upstream regulators, and gene pathways and networks in the

context of the gene expression changes in experimental Alport Syndrome.

2.7 Cell culture and NF-κB activity assay

Human kidney-2 cells were sub-cultured in 6-well plates for 24 hours and co-transfected

with 0.4 ug/well of pNF-κB-Luc plasmid (Stratagene, Agilent Technologies, Inc., Santa Clara,

CA, USA) and 3 ug/well of pRL-TK reporter vector (Promega Corp., Madison, WI, USA) using

the Effectene Transfection Reagent Kit (Qiagen, GmbH, Hilden, Germany) according to the

manufacturers’ guides. Cells were incubated for 17 hours then subjected to Angiotensin II (10-7

mol/L) treatment with or without MS417 (1 uM) for 24 hours. Luciferase activity was

determined with the Dual-Luciferase Reporter Assay System (Promega Corp., Madison, WI,

USA) and the light emission was read by FB12 Luminometer (Titertek-Berthold, Berthold

32

Detection System GmbH, Pforzheim, Germany). Total protein measured with Bradford Assay

(Bio-Rad, Hercules, CA, USA) was used to normalize the activity results.

2.8 Real-time RT-PCR

Total RNAs from cortical and glomerular samples were isolated using RNeasy Mini Kit

and reverse transcribed into cDNA with the QuantiTect Reverse Transcription Kit (Qiagen Inc.,

Misssissauga, ON, Canada). For the cortical samples, the mRNA expression levels for TNF-α,

TGF-β1, IL-6, and IL-1β were quantified by real-time PCR (TaqMan; Thermo Fisher Scientific,

Waltham, MA, USA) by using a sequence detection system (ABI Prism 7900; Applied

Biosystems, Foster City, CA). GAPDH was used as internal control. For the glomerular samples,

the mRNA expression levels for Fxyd2 and Nphs1 were quantified by semi-quantitative PCR.

2.9 Statistical analysis

All results are shown as mean ± S.E. Statistical analyses were performed using GraphPad

Prism 5 (GraphPad Software, Inc. La Jolla, CA, USA). For multiple group comparisons, one-

way ANOVAs with Tukey’s range test were performed, and for two-group comparisons,

unpaired t-tests were performed. Statistical significance was set at a p-value of less than 0.05.

33

Chapter 3

Results

3.1 Characterization and breeding of murine model of Alport Syndrome

In order to better understand the molecular pathways associated with renal disease

progression and address the call for new therapeutic options, a well-characterized experimental

murine model of AS with a Col4a3 knockout raised on a 129/SvJ genetic background was

employed for the study. To produce a cohort of mice for experiments, two heterozygous mutant

mice were bred together and succeeding heterozygous progenies were used for further breeding.

The genotypes of study mice were verified by PCR and only male Col4a3-/- mice and wildtype

(WT) littermate control mice were used for the study (Figure 4). The developmental collagen IV

switch has been demonstrated in the Col4a3-/- mice between 4 to 7 weeks of age (Figure 5). It is

evident that by 7 weeks of age, the kidneys of Col4a3-/- mice fail to undergo the normal collagen

IV network switch; Figure 5 illustrates the markedly decreased expression levels of collagen IV

α3, α4, and α5 genes in the Col4a3-/- kidneys, while the expressions of both α1 and α2 genes

were increased compared to that of the age-matched WT controls.

The structural and functional manifestations of the renal pathology of Col4a3-/- mice have

been comprehensively reviewed in the literature, establishing the experimental disease model

extensively and solidly58,147–154. Histopathologic kidney injury was pronounced in the 7-week-old

Col4a3-/- mice on Periodic acid-Schiff- and Masson’s trichrome-stained sections, along with

tubular dilatation, tubulointerstitial scarring, and fibrosis compared with the WT mice (Figure 6).

Furthermore, Col4a3-/- mice exhibited albuminuria with a significant increase in urinary albumin

excretion (UAE) as early as 4 weeks of age compared to WT mice (Figure 7A). Serum creatinine

levels were progressing in an increasing trend in the 7-week-old Col4a3-/- mice, however, the

34

difference did not reach statistical significance (Figure 7B, p = 0.18). Thus, the macroscopic and

microscopic renal examinations illustrated how this Col4a3 knockout model mirrors that of the

human AS.

35

Figure 4. Col4a3 KO mice breeding scheme and PCR genotyping. The breeding scheme for generating experimental groups as well as the maintenance of the mice colonies were effectuated via pairing of two heterozygous mutants (A). Only Col4a3+/+ and Col4a3-/- mice were utilized as experimental groups for the study. Panel B then shows the representative PCR blot of the genotypes of mice used for the study as well as in generation of the Col4a3-/- mouse line verified by PCR (B). The first loaded sample is of Col4a3 KO sample with WT sample loaded next. The third column is of the heterozygous sample152.

309 Bp

230 Bp

Col+/- Col-/- Col+/+

A

B

36

Figure 5. Heat map of collagen IV α1-α5 expression levels in Col4a3-/- kidneys. The gene expression levels for collagen IV α1-α5 in kidney tissue derived from the microarray analysis of 4- and 7-week-old WT and Col4a3-/- mice (latter 7-week-old dataset highlighted with a green box). It is evident that there is an impaired collagen IV network switch in the KO mice between 4 to 7 weeks of age. In the 7-week-old KO kidney, there is an absence of Col4a3, Col4a4, and Col4a5, indicated by the blue squares, as well as a persistence of Col4a1 and Col4a2, indicated by the red squares, compared to the age-matched WT kidney. Each column represents an individual mouse sample and each row represents an individual collagen gene. Red colour indicates upregulation and blue represents downregulation152.

37

Figure 6. Histopathologic injury in Col4a3-/- and WT kidneys. The renal phenotypes of Col4a3-/- (KO) and WT mice at 4 and 7 weeks of age presented in Periodic acid-Schiff-stained and Masson’s trichrome-stained sections. The KO mice display minimal glomerular abnormalities and normal tubulointerstitium compared to WT mice at 4 weeks of age. However, by 7 weeks of age, moderately severe glomerular and tubulointerstitial scarring was evident in the KO but not WT mice. Similarly, there was very little fibrosis evident in 4-week-old KO kidneys compared to the age-matched WT kidneys. By 7 weeks of age, the KO kidneys exemplify extensive tubulointerstitial fibrosis compared to WT mice evidenced by the green protein casts in MT-stained samples152. Insets in PAS-stained sections show the glomerulus. Original magnification: 100x, insets 200x.

38

Figure 7. Functional manifestations of renal pathology of Col4a3-/- mice. Urinary albumin excretion (A) and serum creatinine levels (B) in WT and KO mice at 4 and 7 weeks of age. At 4 weeks of age, a slight increase in urinary albumin excretion, but normal serum creatinine levels in the KO mice as compared to control WT mice. However, by 7 weeks of age, there was a marked increase in albuminuria excretion measure in KO mice, as well as an increase trend in plasma creatinine level, although it was not statistically significant (p = 0.18) Data are expressed as means ± SEM. *p < 0.05 versus WT152.

A B

39

3.2 Early microarray studies in glomeruli

To elucidate the molecular pathways associated with renal disease progression, total RNA

was extracted from glomeruli of 4-week-old male wildtype (WT) and Col4a3-/- (KO) mice

(n=8/group) and a microarray performed using Affymetrix GeneChip Mouse Gene 2.0 ST Array.

The purity of glomeruli samples was evaluated via RT-PCR using podocyte (Nphs1) and tubular

(Fxyd2) markers prior to the microarray analysis (Figure 3). The purity of the RNA glomeruli

samples without any tubular contamination was also quality checked via Bioanalyzer and only

those with RNA of greater 7 were utilized for the microarray study.

Figure 8 shows the heat map highlighting the marked differential expressions of genes in

the glomeruli of 4-week-old WT and KO mice. It presents a very distinct pattern of genes that

are upregulated or downregulated even at 4 weeks of age in the glomeruli of WT and KO mice,

comprising of genes which are involved in a wide variety of biological processes. Table 1 shows

the Ingenuity Pathway Analysis (IPA) predicted activated upstream regulators that may be

responsible for the gene expression changes observed. It identified 55 unique transcription

factors and proteins in the Col4a3 KO glomeruli, with the transcription factor, NF-κB, predicted

by IPA to be the most activated of all upstream regulators. Figures 10, 11, and 12 show the heat

maps of differentially expressed target genes of IL-1β, TLR4 and MYD88, and NF-κB in the

glomeruli of 4-week-old WT and KO mice, respectively. They all highlight the distinct patterns

of gene upregulation and downregulation even at 4 weeks of age. Figure 9 maps the activated

signal transduction pathways in experimental AS as predicted by IPA in 4-week-old KO

glomeruli. It was shown that there are changes in the collagen protein levels in the extracellular

matrix, levels of cytokines, as well as a number of membrane receptors, such as TLR4, TNF-α,

40

and IL-1β receptors, which all converge on the transcription factor, NF-κB. NF-κB, thereafter,

influences a number of inflammatory genes.

41

Figure 8. Levels of differential gene expression in kidney tissues in WT and KO mice. Heat map of marked differential gene expression levels in the glomeruli isolated from 4-week-old WT and KO mice (n = 8 for both groups). Columns represent kidney samples, rows present different genes, and each cell indicates the expression value of the gene in that sample. Red colour indicates increased expression relative to mean (white colour), whereas blue colour indicates decreased expression relative to mean. Courtesy of Dr. Xuewen Song and Dr. York Pei.

42

Table 1. 55 Ingenuity Pathway Analysis predicted activated upstream regulators in 4-week-old KO glomeruli. 55 Ingenuity Pathway Analysis (IPA) predicted activated upstream regulators in Col4a3-/- glomeruli. The upstream regulators are arranged by the z-score, which predicts the activation state of specific upstream regulators using expression patterns of downstream target genes. An upstream regulator is considered activated and significant if equal to or greater than 2. A z-score of equal to or less than -2 is considered an inhibited upstream regulator. Bolded upstream regulators suggest activation of Toll-like receptor (TLR) signalling. Regulators indicated with an asterisk (*) signal downstream of FREM1. Courtesy of Dr. Song and Dr. Pei.

43

Figure 9. Schematic summary of aberrant activation of signalling pathways in AS mice. Map of signalling pathways in AS as predicted by IPA. Upregulated genes are labelled in red and downregulated genes are labelled in green. Genes that were not differentially expressed at the mRNA level are labelled in black. Genes that are predicted to be activated by the IPA upstream regulator analysis is indicated by a red asterisk and genes that are predicted to be inhibited is marked by a green asterisk. Genes highlighted in blue (a-c) are genes whose altered levels of differentially expressed target genes were further examined and visualized via heat maps (presented in subsequent figures). Courtesy of Dr. Xuewen Song and Dr. York Pei.

44

Figure 10. Levels of differentially expressed IL-1β target genes. Heat map of differentially expressed target genes of IL-1β in the glomeruli of 4-week-old KO and WT mice (n = 8 for both groups). Columns represent kidney samples, rows present different genes, and each cell indicates the expression value of the gene in that sample. Upregulated genes are labelled in red and downregulated genes are labelled in blue. Courtesy of Dr. Xuewen Song and Dr. York Pei.

45

Figure 11. Levels of differentially expressed TLR4 and MYD88 target genes. Heat map of differentially expressed target genes of TLR4 and MYD88 in the glomeruli of 4-week-old KO and WT mice (n = 8 for both groups). Columns represent kidney samples, rows present different genes, and each cell indicates the expression value of the gene in that sample. Upregulated genes are labelled in red and downregulated genes are labelled in blue. Courtesy of Dr. Xuewen Song and Dr. York Pei.

46

Figure 12. Levels of differentially expressed NF-κB target genes in kidney tissues of WT and KO mice. Heat map of differentially expressed target genes of NF-κB in the glomeruli of 4-week-old KO and WT mice with red depicting upregulation and blue representing downregulation (n = 8 for both groups). Columns represent kidney samples, rows present different genes. Interestingly, the heat map shows that mRNA levels for several genes regulated by NF-κB actually decreased. The mechanism responsible for these changes were not addressed in this current study. It possibly represents secondary effects downstream of the original stimulus for the activation of NF-κB. Courtesy of Dr. Xuewen Song and Dr. York Pei.

47

3.3 Targeting NF-κB in vitro

In order to determine whether there was any biological effect of MS417 on NF-κB

inactivation, the NF-κB luciferase activity was measured in human kidney-2 (HK-2) cells

cultured in medium containing angiotensin II (Ang II), with or without the NF-κB-blocking

compound, MS417. HK-2 cells were transfected with an NF-κB reporter construct and the NF-

κB activation activity was determined by measuring the luciferase activity. As expected, Ang II

caused a robust increase in NF-κB activity in HK-2 cells transfected with the NF-κB reporter

construct. This effect was attenuated with MS417 treatment, which inhibited activation of Ang

II-induced NF-κB (Figure 13).

48

Figure 14. MS417-inhibited Ang II-induced NF-κB activation in HK-2 cells. Human-kidney 2 cells were transfected with pNF-κB-Luc plasmid as described previously. After 24 hours of incubation with Ang II (10-7 mol/L) with or without MS417 (1 uM), cells were lysed and the lysates were used for luciferase activity assay. Ang II stimulated the luciferase activity and the treatment with MS417 attenuated the Ang II-induced rise in NF-κB activation in the HK-2 cells. n = 3 for all groups. *p < 0.001 vs. transfection control; † p < 0.001 vs. Ang II-treated group.

49

3.4 Analysis of NF-κB inhibition in vivo using whole kidney samples

3.4.1 Whole animal kidney examination

Compared to corresponding sham animals, there was no significant change in body

weights after MS417 treatment in either WT mice or Col4a3 KO mice, indicating no apparent

toxicity observed based on body weight measurements (Figure 14). No mortality in either

untreated and treated groups were observed during the course of the experiment (data not

shown). BUN and creatinine levels were measured to assess kidney function. Plasma BUN was

increased in vehicle-treated WT kidneys (Figure 15). However, compared to control, the plasma

BUN level was not significantly altered with MS417 treatment in the KO kidneys. Plasma

creatinine values were changed to a similar extent in both WT and MS417-treated KO animals

(Figure 16).

To determine if MS417 treatment would help alleviate kidney function impairment, the

urinary albumin excretion rates were measured for all experimental groups (UAE, Figure 17).

The vehicle-treated KO mice had elevated UAE, but despite a trend of a decrease in the change

in albuminuria level, the UAE failed to reach statistical significance when compared to the WT

mice (Figure 17).

50

Figure 14. Body weight change measurements in WT and KO mice. Body weight measurements were recorded from the start of the treatment at 4 weeks of age till sacrifice at 7 weeks of age for all experimental groups. There were no significant differences among the three experimental groups in their body weight measurements at the time of sacrifice. Results are shown as mean ± SE. n = 4 for vehicle-treated WT; n = 3 for vehicle-treated KO; n = 5 for MS417-treated KO.

51

Figure 15. Blood urea nitrogen (BUN) levels in 7-week-old WT and KO mice following vehicle or MS417 treatment. Plasma samples were obtained at sacrifice at 7 weeks of age after 3 weeks of daily treatment. BUN levels were measured with frozen plasma samples. MS417 treatment trended towards a reduction in the BUN levels in the KO kidneys compared to the vehicle-treated KO kidneys, but the difference failed to reach statistical significance. Results are presented as mean ± SE. n = 13 for WT sham; n = 9 in vehicle-treated KO group; n = 9 in MS417-treated KO group. *p < 0.05 compared to WT sham; p = 0.12 for MS417-treated KO group vs vehicle-treated KO group.

52

Figure 16. Serum creatinine levels assessed in 7-week-old animals following vehicle or MS417 treatment. Creatinine levels were measured with frozen plasma samples. There was a clear rise in the serum creatinine level in the vehicle-treated KO kidneys, which was attenuated with the administration of MS417; however, the difference failed to reach statistical significance. Results are presented as mean ± SE. For WT with vehicle treatment, n = 13; n = 9 for KO with vehicle treatment; n = 9 for KO with MS417 treatment. *p < 0.05 compared to WT sham; p = 0.21 for MS417-treated KO kidneys compared to vehicle-treated KO kidneys.

53

Figure 17. Urinary albumin excretion (UAE) assessed in 7-week-old animals. Body weight measurements were recorded from the start of the treatment at 4 weeks of age till sacrifice at 7 weeks of age for all experimental groups. The rise in albuminuria observed in the vehicle-treated KO group is decreased with MS417 treatment, though the difference failed to reach statistical difference. Results are shown as mean ± SE. n = 10 for all groups. *p < 0.05 compared to WT sham; p = 0.07 for MS417-treated KO vs vehicle-treated KO.

*

54

3.4.2 Proinflammatory cytokine expression

Renal inflammation and the accumulation of macrophages play an important role in

kidney injury. Thus, the expression profiles of proinflammatory cytokines, interleukein-6 (IL-6)

and interleukin-1β (IL-1β) were assessed in cortical kidney samples as indicators of

inflammation and disease progression (Figure 18). The measurement of mRNA levels of

proinflammatory cytokines, IL-6 and IL-1β, showed dramatic induction of all genes in vehicle-

treated KO mice compared to the kidneys of WT mice. However, the mean mRNA levels of IL-6

and IL-1β were lowered in the kidneys of the MS417-treated KO mice compared to the kidneys

of WT mice with a statistically significant difference for IL-1β (Figure 18). The differences were

not statistically significance when the proinflammatory mRNA expression levels of MS417-

treated kidneys were compared to vehicle-treated KO kidneys.

55

Figure 18. Proinflammatory cytokine levels in kidney tissue in WT and Col4a3 KO mice after sham or MS417 treatment. mRNA expression levels of IL-6 (A) IL-1β and (B) TNF-α in WT and KO mouse kidney after vehicle or MS417 treatment. GAPDH was used as internal control. Results are normalized to WT sham and presented as mean ± SE. n ≥ 10 in each experimental group for IL-6 and IL-1β. n values are varying per experimental group as during the performance of the experiments, amplifications failed in some of the wells and were omitted for the data analysis. *p < 0.05 compared to WT sham; p = 0.19 for MS417-treated KO vs vehicle-treated KO (A); p = 0.54 for MS417-treated KO kidney vs vehicle-treated KO kidney (B).

A B

56

3.5 Analysis of NF-κB inhibition in vivo via microarray studies of isolated glomeruli

To determine whether there were any changes in the molecular pathways linked to the

disease progression of Alport Syndrome with the administration of MS417, the total RNA was

extracted from the glomeruli of 38-day-old male mice from three experimental groups: WT with

vehicle treatment; KO with vehicle treatment; and KO with MS417 treatment (n = 7 for all

experimental groups). As previously described, the purity of the glomerular RNA samples to

ensure the absence of tubular contamination was evaluated via RT-PCR using podocyte and

tubular markers (Figure 3). Table 2 shows the summary of the glomerular isolation perfusions

performed, the number of glomeruli RNA samples with RIN higher than 6, the number of RNA

samples sent for Bioanalyzer analysis, and the number of RNA samples used for the microarray

analysis. Table 3 lists the RNA glomeruli samples that were selected for the microarray study.

When the microarray analysis was performed, it became evident that the assignment of the

genotypes to the experimental animals was incorrect. Table 4 then lists the newly assigned

genotype and treatment grouping after an inaccuracy in the genotype assignment in the

experimental group was discerned. A total of 21 glomeruli samples were utilized for the study.

A microarray was then performed using Affymetrix GeneChip Mouse Gene 2.0 ST Array

on these samples. Figure 19 shows the heat map of the differential gene expression levels in the

glomeruli of WT and KO mice after 10 days of either sham or MS417 treatment. The initial

changes in gene expression that was previously observed in the glomeruli of 4-week-old mice in

vehicle-treated WT and KO groups were sustained in this experiment. However, there was no

significant marked differential expressions of genes in the MS417-treated KO group when

compared to the vehicle-treated KO group.

Figures 20, 21, and 22 illustrate the heat maps of differentially expressed target genes of

57

IL-1β, TLR4 and MYD88, and NF-κB in the glomeruli of WT and KO mice treated for 10 days

with sham or MS417, respectively. Once again, the distinct patterns of marked differential gene

expression noted in the 4-week-old WT and KO glomeruli are conserved in the glomeruli after

the 10-day treatment with sham or MS417. The 10-day treatment of MS417 in KO mice did not

alter the gene expression patterns of NF-κB, IL-1β, and TLR4 and MYD88 targets compared to

that of the vehicle-treated KO mice.

58

Table 2. Summary of glomerular isolation perfusions performed and RNA yield qualities for analyses. A total of 34 perfusions were completed on three experimental groups of WT vehicle (n = 15), KO vehicle (n = 8), and KO treatment (n = 11) mice, which yielded n = 11, n = 7, and n = 7 of acceptable-quality RNA samples (RIN > 9) for WT vehicle, KO vehicle, and KO treatment groups, respectively. RIN score of greater than or equal to 6 is considered a good quality RNA suitable for analysis155. A total of 25 RNA samples were submitted for Bioanalyzer analysis and a total of 21 RNA samples were utilized for the microarray analysis. Courtesy of Dr. Xuewen Song and Dr. Pei York.

59

Table 3. RNA quality of glomeruli samples selected for microarray study. The RNA concentration and the sample purity levels (260/280 and 260/230) levels are shown for the individual RNA samples that were selected for the microarray study. In total, 21 RNA samples were submitted, which is comprised of n = 7 for each experimental group of WT vehicle, KO vehicle, and KO treatment. A 260/280 ratio of approximately 2.0 or higher is acceptable as pure for RNA and a 260/230 range of 2.0 to 2.2 is considered pure for RNA samples156. Courtesy of Dr. Xuewen Song and Dr. Pei York.

60

Table 4. Reassignment of experimental groups for microarray study. The genotypes of the three experimental groups, WT vehicle, KO vehicle, and KO treatment (n = 7 per group), were reanalyzed. The first column shows the RNA ID of the individual samples. The second column shows the treatment assignment to the WT and KO animals; however, when the microarray analysis was performed, it became evident that the assignment of the genotypes of the animals was incorrect. The corrected genotype and treatment is shown in the third column. The RNA samples were then reassigned experimental grouping as WT vehicle (n = 5), WT treatment (n = 5), KO vehicle (n = 9), and KO treatment (n =2). In total, 21 individual samples of glomeruli were experimented on. Courtesy of Dr. Xuewen Song and Dr. Pei York.

61

Figure 19. Levels of differential gene expression in glomeruli in WT and KO mice after 10-day treatment with sham or MS417. Heat map of marked differential gene expression levels in the glomeruli isolated from 38-day-old WT and KO mice treated with either sham or MS417 for 10 days after which the mice were sacrificed. n = 9 for vehicle-treated KO; n = 2 for MS417-treated KO; n = 5 for vehicle-treated WT; n = 5 for MS417-treated WT. Columns represent kidney samples, rows present different genes, and each cell indicates the expression value of the gene in that sample. Upregulated genes are shown in red and downregulated genes are shown in blue. Courtesy of Dr. Xuewen Song and Dr. York Pei.

62

Figure 20. Marked differential gene expressions of IL-1β target genes in glomeruli of WT and KO mice. Heat map of marked differential gene expression levels of IL-1β targets in the glomeruli isolated from WT and KO mice treated with vehicle or MS417 for 10 days. n = 9 for vehicle-treated KO; n =2 for MS417-treated KO; n = 5 for vehicle-treated WT; n = 5 for MS417-treated WT. Columns represent kidney samples, rows present different genes, and each cell indicates the expression value of the gene in that sample. Upregulated genes are labelled in red and downregulated genes are labelled in blue. Courtesy of Dr. Xuewen Song and Dr. York Pei.

After10-daytreatmentwithMS417

63

Figure 21. Levels of marked differential gene expression of TLR4 and MYD88 targets in glomeruli in WT and KO mice. Heat map of TLR4 and MYD88 target gene expression levels in the glomeruli isolated from WT and KO mice after a 10-day treatment with vehicle or MS417. n = 9 for vehicle-treated KO group; n = 2 for MS417-treated KO; n = 5 for vehicle-treated WT; n = 5 for MS417-treated WT. Upregulated genes are shown in red and downregulated genes are shown in blue. Courtesy of Dr. Xuewen Song and Dr. York Pei.

After10-daytreatmentwithMS417

64

Figure 22. Marked differential gene expression levels of NF-κB target genes in glomeruli of WT and KO mice. Heat map of marked differentially expressed NF-κB target genes in the glomeruli isolated from WT and KO mice treated with vehicle or MS417 for 10 days before sacrificing. n = 9 for vehicle-treated KO; n = 2 for MS417-treated KO; n = 5 for vehicle-treated WT; n = 5 for MS417-treated WT. Columns represent kidney samples, rows present different genes, and each cell indicates the expression value of the gene in that sample. Upregulated genes are labelled in red and downregulated genes are labelled in blue. Courtesy of Dr. Xuewen Song and Dr. York Pei.

65

Chapter 4

Discussion

Alport Syndrome is a progressive nephropathy of the glomerular basement membrane, a

major component of the glomerular filtration barrier87. It is caused by mutations in the genes

encoding the α3/α4/α5 chains of type IV collagen, which is one of the principal constituents of

the GBM93,157. Thus, the dysregulation of the collagen IV alpha chains manifests in a clinical

course present with microscopic hematuria, which progresses to proteinuria, and eventually

culminating in renal failure5,87. Renal biopsies from patients also typically show segmental or

diffuse glomerulosclerosis, tubular atrophy, interstitial fibrosis, as well as interstitial infiltration

of lymphocytes and macrophages, correlating with progressive renal failure8,158. Yet, the exact

molecular mechanisms by which the mutations exert their pathogenic effects on the glomerulus

remain elusive. A greater understanding of the mechanisms behind the gradual degeneration of

the GBM in AS would lead to the discovery of renoprotective methods, which could potentially

prevent glomerulopathies in the future. Therefore, using in vitro and in vivo models, this study

examined the possibility of inflammation as a major driving force behind the glomerular injury

in AS. Furthermore, the impact of NF-κB inhibition via competitive blockade of BRD4 on the

renal disease progression in AS was investigated.

4.1 Major changes in gene expression are observed in the glomeruli prior to a decline in

GFR in experimental AS

The molecular effects of COL4A3/A4/A5 mutations were investigated in the glomeruli of

WT or mutant (Col4a3-/-) mice via microarray analysis. Microarray technology is a powerful tool

that allows the analysis and quantification of thousands of gene expression levels

66

simultaneously159,160. Thus, such high-throughput approach has been utilized in recent years to

discern a global viewpoint of the disease at the molecular level, enabling the evaluation of the

cellular and metabolic derangement associated with the diseased state161. The gene expression

profiles in this study give information with regards to both upregulated and downregulated genes

in the early phase of disease progression in this experimental model. Figure 8 shows the heat

map of differentially expressed genes with twofold greater expression in 4-week-old KO

compared to WT kidneys, showcasing a prominent pattern of pathway activation and inactivation

that is consonant with the COL4A3 mutation. The heat map revealed marked overexpression of

genes involved in inflammation mediated by chemokine and cytokine signalling pathways as

well as PDGF signalling pathway in 4-week-old KO glomeruli compared to the age- and sex-

matched control glomeruli. Genes involved in a variety of non-immune pathways, such as

angiogenesis, blood coagulation, gonadotropin-releasing hormone receptor, integrin signalling,

PI3 kinase, as well as Wnt signalling, were downregulated in the early timepoint of COL4A3

deletion.

Inflammation has been well-established as an important driver of pathophysiology across

a broad range of diseases in the literature, including in nephropathies162,163. The expression levels

of proinflammatory mediators, such as cytokines and chemokines, are upregulated in the

diseased state in comparison to healthy controls, conveying their crucial role in the pathogenesis

of CKD. For instance, TNFRSF12A (tumour necrosis factor receptor superfamily member 12A)

is a receptor for tumour necrosis factor ligand superfamily member 12 (TNFSF12; also called

TNF-related weak inducer of apoptosis (TWEAK)), a cytokine that is widely expressed with

overlapping signalling function with tumour necrosis factor (TNF)164,165. Excessive activation of

67

the TNF/TWEAK pathway has been observed in kidney injury to promote NF-κB-dependent

inflammatory response, fibrosis, angiogenesis, as well as apoptosis165,166.

Likewise, C-X-C motif chemokine 10 (CXCL10) is a cytokine belonging to the CXC

chemokine family that has been implicated in inflammatory diseases, which is seen to be

strongly expressed in 4-week-old KO mice167. It was seen in the study by Petrovic-Djergovic et

al. (2015) that stimulation of cultured podocytes with IFN-γ and TNF-α markedly induced the

expression of Cxcl10 mRNA and CXCL10 secretion, illustrating its role in the propagation of

innate and adaptive immunity, as well as inflammation168. Kaffe et al. (2018) also investigated

the pathogenetic role of CXCL10 in disease progression in vivo by blocking the CXCR3-

CXCL10 axis, inhibiting macrophage recruitment in congenital hepatic fibrosis in the presence

of polycystic kidney disease169. It was found that CXCL10 inhibition slowed the progression of

the disease by reducing CD45+ immune cell infiltration, collagen deposition, the total number of

macrophages, as well as proinflammatory T cells.

Adrenomedullin (ADM) is a vasodilatory peptide abundantly expressed in the kidney

with antiproliferative effects on glomerular mesangial cells170. Its production has been seen to be

activated in several pathophysiological states, including cardiac, hepatic, respiratory, renal

failure, tumour growth, as well as during inflammatory states171,172. A body of evidence indicates

that inflammatory cytokines, such as TNF-α and IL-1β, upregulates ADM mRNA expression,

suggesting that inflammatory cytokines may trigger ADM gene expression during

inflammation171. A rise in ADM level was observed to significantly ameliorate proteinuria,

glomerular and tubular injury, as well as renal dysfunction in salt-related renal hypertension173.

Thus the increase in delivery of ADM gene in the kidney suggests a renoprotective role,

68

producing a wide spectrum of beneficial effects in compensation for the impaired renal function

in the diseased state174.

Another gene that was overexpressed in the KO glomeruli is CYP1B1, which is a

member of the cytochrome P450 enzyme family I, subfamily B, polypeptide 1175,176. It is

expressed in cardiovascular and renal tissues, with a major role in the mediation of Ang II-

induced activation of NADPH oxidase, generation of reactive oxygen species (ROS),

proliferation, hypertrophy and/or deactivation of a wide range of xenobiotics176–178. CYP1B1

expression has been implicated in pathophysiology, particularly in tumours as well as

cardiovascular and renal abnormalities179. For instance, Zordoky and group reported that acute

doxorubicin (DOX) toxicity, a chemotherapy drug that causes oxidative damage and induces

inflammatory changes in the heart, kidney, and liver tissues, caused an induction of CYP1B1

expression in both the kidneys and liver in rats in a mechanism which has yet to be fully

elucidated177. Similarly, the deleterious pathophysiological effects of Ang II infusion in the

kidneys were observed to be minimized by the inhibition of CYP1B1 activity by treatment with a

selective CYP1B1 inhibitor, TMS, in rats, and by Cyp1b1 gene disruption in the mice175,180,181.

Transgelin (TAGLN), also designated SM22α, is a cytoskeletal protein whose expression

has been known to be induced in kidney injury182,183. A differentiation marker of smooth muscle

cells during embryogenesis, TAGLN is present transiently in the glomeruli during development

and later absent in normal adult glomeruli184. However, the reappearance of TAGLN within

injured glomeruli is a major finding that links its expression to the initiation and/or progression

of renal diseases characterized by podocyte injury and proteinuria, corresponding to the

overexpression seen in the KO glomeruli in the microarray analysis185. The literature has

documented marked TAGLN upregulation in experimental models of nephropathies, as well as in

69

renal tissue of patients with proteinuric diseases, such as diabetic nephropathy and IgA

nephropathy186. Furthermore, microarray and proteomic analyses in obstructive nephropathy

model suggest TAGLN is upregulated during fibrosis development, an important pathological

feature that eventually leads to renal failure187.

Genes downregulated in diseased animals compared to controls were mainly required for

cell structure and localization, cell cycle, metabolic processes, and developmental processes

(Figure 8). Among the 16 genes belonging to this group were, for example, secreted frizzled

related protein 1 (SFRP1), kallikrein 1 (KLK1), and klotho. SFRP1, a secreted antagonist of Wnt

signalling, is a known tumour suppressor that is involved in cell growth188. Though the role of

SFRP1 in CKD has not yet been elucidated, the loss of its expression and the contribution its

absence has on pathological events have been documented in a variety of cancers, including

renal cell carcinomas (RCC), in which the loss of SFRP1 is often observed in human

cancers189,190. This deregulation of SFRP1 expression level in RCC may allude to its role in

abberant Wnt signalling in the development of solid tumours190,191. In addition, study by

Matsuyama and group found that kidneys from Sfrp1-/- mice showed significant expression of

myofibroblast markers, aSMA, as well as fibroblast genes, vimentin, after unilateral ureteral

obstruction (UUO), giving rise to significant expansion of renal fibrosis192. The inactivation of

SFRP1 also brought about an elevation of phosphorylated c-Jun and JNK levels, which are

mediators of non-canonical Wnt signalling activation, corroborating SFRP1’s role as a modulator

of renal damage through the non-canonical Wnt/PCP pathway. Thus, SFRP1 may be a key player

in regulation and development of Wnt/PCP-mediated renal interstitial fibrosis in CKD192.

KLK1 deficiency has been documented in experimental models of hypertension, diabetic

nephropathy, and kidney injury193. KLK1 is a highly-conserved serine protease expressed in the

70

kidney, which cleaves low molecular weight kininogen into kinin, which is then involved in

kidney function and blood pressure regulation via vasodilatory and natriuretic effects194. In

experimental acute kidney injury (AKI), renal KLK1 activity is markedly diminished, and the

gene transfer of KLK1 can abrogate renal injury by minimizing apoptosis and inflammatory cell

recruitment193,195,196. Recent research has also identified the kidney-secreted hormone, klotho, as

a central player in CKD-related inflammation with anti-aging and nephroprotective properties197.

A decrease in klotho level has been observed not only in chronic or acute animal models of renal

injury characterized by renal inflammation, such as diabetic nephropathy, but also in patients

with G1 category of CKD, in which normal GFR is accompanied by pathological albuminuria or

other evidence of kidney injury198,199. Furthermore, interestingly, studies have found that

inflammatory cytokines, including TWEAK and TNF-α, downregulate renal klotho gene

transcription via NF-κB both in vitro and in vivo200. Likewise, exogenous provision of klotho to

the kidneys of db/db mice negatively regulated NF-κB activation and the suppression of

subsequent production of proinflammatory cytokines, such as RANTES, MCP-1, IL-6, and IL-8,

in response to TNF-α stimulation, showcasing a bidirectional relationship between klotho and

inflammation197,201,202.

Downregulation of inward rectifier potassium channel 16 (KCNJ16; Kir5.1) was also

observed in Figure 8, which mirrors the findings reported in the literature, in which disruption of

KCNJ16 induced severe hypokalemia203. Human mutations in KCNJ16 are associated with renal

pathologies characterized by salt-wasting, renal tubulopathy, and hypokalemia, known as EAST

syndrome, highlighting the critical role of the Kir5.1 potassium channel in the distal

nephron203,204. Similarly, KCNJ1 (Kir1.1) loss brings about type II Bartter syndrome, which

features polyuria, renal salt wasting, and hypotension; such clinical manifestations are largely

71

recapitulated in Kcnj1-null mice205. Schmidt et al. (2007) further uncovered that

proinflammatory cytokines, IL-1β and TNF-α, extensively suppressed the expression of KCNJ1,

postulating the cytokines may mediate LPS-induced downregulation of tubular sodium

transporters206.

To fully delineate the molecular mechanisms behind the progressive deterioration of the

GBM in the early stages of AS, the changes in the gene expression in the glomeruli were

analyzed by microarrays in experimental model of AS immediately following the onset of

albuminuria. Microarray analysis has been extensively applied in the field of experimental

nephrology due to its efficiency in the analysis of changes in the transcriptome207. The results in

this study showed that there was a distinct pattern of differentially expressed genes between the

Col4a3 KO and WT kidneys before the materialization of major structural and functional

changes at 4 weeks of age (Figure 8). Many of the upregulated differentially expressed genes in

the KO glomeruli were implicated in chemokine- and cytokine-induced inflammatory pathways,

whereas those that were downregulated in diseased animals were involved in cell structure, cell

cycling, as well as metabolic and developmental processes. The serial changes in glomerular

gene expression in early experimental AS were in accordance with previous findings in other

nephropathies. Thus, it was found that at the onset of proteinuria, the kidneys of experimental AS

were marked with changes in gene expression prior to the development of structural and

functional manifestations of the disease compared to WT controls.

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4.2 Pathway analysis of the differential gene expression established that pathways

converged on NF-κB

In this study, the gene expression profile of Col4a3-/- glomeruli was further investigated

to better understand the biological mechanisms of AS. The microarray dataset, which established

that the onset of proteinuria is marked with a change in gene expression with the identification of

771 differentially expressed genes in mice with experimental AS compared to controls, was

further analyzed for potent activated upstream regulators. This was done with the application of

the Ingenuity Pathway Analysis (IPA), which calculates the activation z-score that represents the

bias in gene regulation, thus reducing the chance that random data will generate significant

predictions208–210. With the z-scores filtered at 2.0, the IPA is then able to identify the cascade of

upstream transcriptional regulators that are predicted to be activated based on the expression

pattern of the target genes211,212. Table 1 shows the 55 predicted putative upstream regulators,

with NF-κB revealed to be the most activated upstream regulator, along with a multitude of

regulators that are implicated in Toll-like receptor (TLR) signalling213–216. Figure 8 then

schematically summarizes the signalling pathways in experimental AS as predicted by IPA,

emphasizing the central role of NF-κB in the modulation of inflammatory pathways in AS; its

function as a major downstream convergent point for a multitude of proinflammatory

transducers, including TLR4, is clearly recapitulated.

Renal inflammation is a universal response to injury, in which the the innate immune

system is stimulated to bring about an array of inflammatory responses as the first line of

defense215. The innate immune system, composed of germlike-encoded receptors expressed by

macrophages, dendritic cells, monocytes, neutrophils, and natural killer cells, collectively serves

to monitor extracellular and intracellular compartments for signs of infection or tissue injury217.

73

These sensors, appropriately called pattern recognition receptors (PRRs), recognize common

molecular patterns and translate these diverse triggers of renal inflammation into induction of

cell activation and secretion of proinflammatory cytokines and chemokines. PRRs may respond

to various bacterial and viral pathogen-associated molecular patterns (PAMPs), as well as non-

microbial host-derived stimuli, such as damage-associated molecular patterns (DAMPs), which

are endogenous molecules released by injured tissues during inflammation218. The activation of

the PRRs mediates downstream pathways through transcription factors that modulate the

expression of genes with proinflammatory phenotype219.

Among the several distinct classes of PRRs is the membrane-bound TLR family, which

effectuate inflammatory responses by being able to detect a wide array of PAMPs and DAMPs in

the extracellular space163. TLRs are expressed on a variety of immune cells, including

macrophages, neutrophil granulocytes, mast cells, dendritic cells, and T and B lymphocytes, as

well as on other cell types, such as parenchymal and epithelial cells194,220. The binding of a

ligand to a TLR activates numerous signalling pathways, in which TLR activates adaptor

proteins, which in turn initiate NF-κB in the actuation of inflammatory responses. Two main

TLR signalling pathways are myeloid differentiation factor 88 (MYD88)-dependent pathway and

the TIR-domain-containing adaptor protein-inducing IFN-β (TRIF)-dependent pathway221. The

MYD88-dependent pathway is activated by all known TLRs except TLR3, and leads to the

production of proinflammatory cytokines, such as TNF, IL-1β, IL-12, IL-6, IL-8, and CC-motif

chemokine ligand 2 (CCL2), whereas TLR3 and TLR4 can trigger the TRIF-dependent pathway,

which brings about the production of type I interferons (IFNs), IFN-α and IFN-β, against viral

infection222,223. Generally, all TLRs can induce the expression of NF-κB-dependent genes,

including most proinflammatory cytokines and chemokines, in addition to being able to induce

74

the expression of procytokines, IL-1 and IL-18163. Recent studies have suggested that TLRs have

an important role in the pathogenesis of renal diseases, with their exaggerated activation

documented in renal ischemia, acute kidney injury, ESRF, acute tubulointerstitial nephritis, renal

transplant rejection, as well as delayed allograft function224–227. Thus, although inflammation

mediated by PRRs can help to resolve and repair an initial insult, it has been recognized that

overactivation of DAMP signalling may trigger inappropriate sterile inflammation, thereby

contributing to unnecessary organ damage and dysfunction225. The data listed on Table 1 agrees

with previous reports that TLR and NF-κB signalling are important in the regulation of

inflammatory and repair processes in the kidneys of AS, with predicted upstream regulators

implicated in the activation of TLR signalling indicated in bolded letters222.

TLR4 is the most extensively studied amongst the numerous TLRs that have been

described in the literature, with significant implications in renal diseases194. TLR4 has been

shown to have an active role in the development of tubulointerstitial injury, albuminuria, as well

as glomerular damages. For instance, in patients with diabetic nephropathy, TLR4 was found to

be highly expressed in the renal tubules, positively correlating with interstitial macrophage

infiltration214. Furthermore, in animal models of glomerulosclerosis and mesangioproliferative

glomerulonephritis, it was shown that TLR4 activation in podocytes could induce

proinflammatory cytokines and promote podocyte injury228. Similarly, TLR4-deficient mice

exhibited less albuminuria, mesangial expansion, infiltration of macrophages, and decreased

proinflammatory and extracellular matrix-associated gene expression in the glomeruli compared

to wildtype mice229. Subsequently downstream of that, mice that lacked the adaptor protein,

MYD88, which is required for TLR signal transduction, presented a marked reduction in

inflammation as well230,231. This finding then led to an examination of the role of IL-1 receptor 1

75

(IL-1R1), which is a MYD88-dependent transmembrane receptor232. In IL-1R1 mutant mice, a

significant reduction in neutrophil response was observed, emphasizing IL-1R1’s essential

function in neutrophil recruitment in response to inflammation and injury221.

One of the ligands of IL-1R1 that was seen to be upregulated in the microarray data of

the Col4a3-/- mice (Table 1, Figure 10) is IL-1β, a potent multifunctional proinflammatory

cytokine that has been implicated in the sterile inflammatory response233,234. Unlike most

inflammatory cytokines that are mainly transcriptionally regulated, IL-1β is produced in the

cytosol as a biologically inactive pro-IL-1β, which is activated intracellularly via proteolytic

cleavage by caspase-1233. In turn, the activation and conversion of caspase-1 from its zymogen

form, pro-caspase-1, is under the direction of Nod-like receptor (NLR) protein family members,

which collectively forms an inflammasome complex235. This stimulation of inflammasomes

cleaves caspase-1, which then hydrolyzes pro-IL-1β into its mature and functional form, exciting

a proinflammatory response through the cleavage and thus activation of IL-1β, IL-18, as well as

pyroptosis, a programmed lytic cell death. Hence, IL-1β is yet another key mediator that drives

acute neutrophilic inflammatory response, with the microarray datasets mirroring that of the

observations documented in other studies in the literature.

Accordingly, heat maps representing the expression patterns of IL-1β, TLR4/MYD88,

and NF-κB target genes were generated in order to ascertain the cellular manifestations of

inflammation in the experimental model of AS (Figures 10-12). NF-κB target genes that are

differentially expressed in the 4-week-old KO glomeruli compared to their age-matched WT

controls confirmed the upregulation of genes involved in apoptosis signalling pathway, B and T

cell activation, inflammatory pathways mediated by chemokine and cytokine signalling, p53

pathway, as well as Toll receptor signalling pathway to name a few (Figure 12). This pattern of

76

differentially expressed gene profiling of IL-1β target genes (Figure 10) and TLR4 and MYD88

target genes (Figure 11) in the glomeruli of 4-week-old KO mice are also reiterated in the heat

maps generated by the microarray studies, illustrating the enhancement of pathways that are

involved in the recruitment and development of sterile inflammation that initiates renal injury

and dysfunction223.

4.3 A novel approach to inhibiting NF-κB-mediated gene expression: BRD4 antagonism

These findings suggest that the TLR4 pathway is a prominent mediator of sterile

inflammation in the pathophysiology of CKD and the inhibition of its signalling pathway could

block the damage brought about by the acute inflammatory response. However, blocking TLR4,

as a central endotoxin receptor that is the innate immune system’s primary responder to

pathogens, could also have detrimental effects in infectious and immunological settings,

compromising the host’s immunogenicity236. Therefore, targeting the downstream mediator of

TLR4 signalling, NF-κB, which is a point of convergence for a myriad of other signalling

pathways relevant to inflammation, may limit the risks of disrupting the rapid innate immunity

against microbial infections.

Numerous factors have been discovered to contribute to the transcriptional activation of

NF-kB target genes and with it multitudinous methods in which NF-κB has been inhibited have

been described in the literature, including genetic and pharmacologic maneuvers132,237–239.

However, due to its protective role in innate immune response to infection, a complete inhibition

of NF-κB may bring about adverse effects; thus, a selective blockade of its activity would help

maximize the beneficial effects240. Recently, emerging evidence has suggested that

posttranslational modifications of NF-κB may play a critical role in the transcriptional regulation

77

of NF-κB-mediated inflammatory response240,241. In particular, the acetylation of NF-κB’s RelA

subunit has emerged as a significant component of NF-κB signalling linked to the regulation of

DNA binding, IκB binding, as well as transactivation potential241. The acetylation at lysine-310

is recognized by two bromodomains of BRD4, which in turn recruits and activates CDK9 to

phosphorylate RNA polymerase II for the transcription of NF-κB target genes140,142,242. Hence,

lysine-310 acetylation, through its capacity to create docking sites for the recruitment of NF-κB

transcriptional cofactors, is required for the full transcriptional activity of NF-κB242.

Correspondingly, in order to study the biological effects of NF-κB inhibition via

pharmacological BRD4 blockade, an in vitro NF-κB luciferase study was carried out. NF-κB

stimulation was effected using Ang II in HK-2 cells and MS417/BRD4 activity was interfered

with the administration of MS417. The NF-κB luciferase assay revealed that MS417 was able to

efficiently inhibit NF-κB after induction with Ang II, supporting BRD4’s capability as an

effective NF-κB inhibitor (Figure 13). Other studies have demonstrated the efficacy of MS417 in

the potent suppression of NF-κB activity. For instance, Zhang et al. showed that MS417

effectively attenuated the NF-κB transcriptional activation of proinflammatory genes in kidney

cells treated with TNF or infected with HIV140. Moreover, MS417 treatment was able to

ameliorate inflammation and kidney injury in HIV-1 transgenic mice. Similarly, MS417 was

shown to inhibit NF-κB acetylation and its transcriptional activation in a murine model of

diabetic nephropathy142. Consistent with these findings, this study also clearly delineates and

exemplifies the role of acetylation of NF-κB and the effect of its inhibition via BRD4 blockade.

78

4.4 Treatment of BRD4 inhibitor led to a decrease in cytokine gene expression and a trend

towards improved creatinine levels and albuminuria

BET bromodomain-specific inhibitor, MS417, and its role in the modulation of NF-κB

transcriptional activity has been studied in other experimental models of nephropathies, such as

HIVAN and diabetic nephropathy; however, bromodomain inhibition has not been previously

examined in AS140,142. Thus, in this study, the effect of BRD4 antagonism on NF-κB-moderated

inflammatory gene expression was further investigated in vivo using Col4a3 KO and WT mice.

This pilot study was able to confirm that the drug, MS417, was first and foremost, well-tolerated

in the KO mice compared to the respective control groups via body weight measurements—

alterations in body weight is considered an indicator of in vivo toxicity (Figure 14)243,244.

Moreover, MS417 promoted a modest but therapeutic trend towards recovery in BUN, serum

creatinine, as well as UAE levels in the KO mice compared to the vehicle-treated WT mice

(Figures 15-17). Taken together, these results suggest that MS417 does not cause drug toxicity,

and is able to comparatively reduce proteinuria and improve renal function through its capacity

to inhibit NF-κB acetylation and transcriptional activation in the kidneys of the AS mice.

MS417 was discovered to also attenuate the expression of NF-κB target genes. The

analysis of the mRNA levels of proinflammatory cytokines, IL-6 and IL-1β suggested that BET

bromodomain inhibition downregulates NF-κB-mediated transcription of proinflammatory

cytokine and chemokine genes in Col4a3-/- mice (Figure 18). This finding corroborates previous

literature positing that selective inhibition of acetylated NF-kB binding to the transcriptional

cofactor, BRD4, does effectively attenuate NF-κB transcriptional activation of proinflammatory

genes, which are markers of kidney injury severity245,246. Elevated levels of IL-6 and IL-1β in

CKD have been observed in other studies in vivo and in vitro, in which their increased expression

79

levels were positively correlated with the severity of the renal disease progression119,247–249. One

study in 2014 found that upon analyzing the glomerular mRNA from an Alport mouse model,

there was a significant induction of proinflammatory cytokine, IL-6, that correlated with GBM

destruction119. Koga and group have examined the expression of proinflammatory cytokines, IL-

6, IL-1β, TNF-α, and TGF-β in a murine experimental model of AS, in which they were

discovered to be markedly induced in Alport kidneys compared with WT250. The elevation in

cytokine levels seen in the kidney tissues of Col4a3 KO mice were found to be significantly

preventable for IL-1β, while moderately ameliorated for IL-6 with the usage of NF-kB inhibitor,

MS417 (Figure 18).

To further investigate the mechanism of BRD4 inhibition in the pathophysiology of

experimental AS, a microarray study was performed to discern the effects of MS417 treatment on

the KO glomeruli compared to WT kidneys (Figures 19-22). However, perchance due to the

fallacious dosage administered to the mice, conflicting results were yielded in which there was

not a significant alteration in the gene expression levels in the treated mice compared to the KO.

4.5 Conclusions

Using a combination of in vitro and in vivo approaches, this thesis examined the potential

for NF-κB inhibition via pharmacological approach to BRD4 antagonism in experimental

models of AS, and the impact of that molecular obstruction on the pathologic manifestations. A

microarray study of the differentially expressed gene levels in the isolated glomeruli from

Col4a3 KO and WT mice revealed a defining genetic profile as early as 4 weeks of age in the

diseased state in comparison to healthy controls. A bioinformatic analysis via IPA of the

microarray dataset predicted activated upstream regulators, uncovering NF-κB as the most

80

activated transcription factor as well as its role as a key convergence centre for multiple affected

pathways, indicating sterile inflammation as a potential driver of the renal pathogenesis in AS—

an exaggerated reaction of the innate immune system gone awry. A novel BRD4 inhibitor,

MS417, was demonstrated to be an effective inhibitor of NF-κB activation both in vitro and in

vivo without effecting any biotoxicity in the HK-2 cells and Col4a3 mice. MS417 was

demonstrated to trend towards an attenuation of BUN, plasma creatinine, and UAE levels

compared to WT mice. Furthermore, MS417 administration attenuated the augmented

proinflammatory cytokine expression levels of IL-6 and IL-1β, with statistical significance for

the latter. Further microarray studies were accomplished with isolated glomeruli from 4-week-

old KO and WT mice with vehicle or MS417 treatment, which did not cause a similar attenuation

of inflammation previously observed, possibly due to the dosage error. However, overall, these

findings demonstrate the role of NF-κB-mediated sterile inflammation on the disease progression

in experimental AS, guiding the development of therapeutic strategies specifically targeting the

signalling pathway responsible for the deleterious effects of NF-κB.

4.6 Pitfalls and future directions

Several other hypotheses may be derived from the IPA analysis (Figure 9), for instance, it

is possible that early interaction between the podocytes and the abnormal basement membrane

leads to the activation of MMPs, subsequently bringing about a degradation of the ECM and the

generation of DAMPs that activate the innate immunity mediate in part by TLR4. However, this

aspect of the IPA analysis was not examined in this study and future experiments will be

necessary to address this hypothesis.

81

The study was based on Zhang et al. (2012) and Liu et al. (2014), both papers which used

an in vivo dosage of 0.08 mg/kg when treating db/db and HIV-1 transgenic mice with the BRD4-

antagonizing compound, MS417140,142. However, post-treatment renal analyses revealed that the

mice were dosed at a differing dosage of 0.0016 mg/kg due to a calculation error made. In

addition, the genotypes of the mice samples were rechecked during the microarray study using

the Col4a3 expression and it was discovered that there was an issue with the initial assignment of

the mice genotypes. At the time of the glomeruli sample submission for the microarray study, it

was presumed that n = 7 for each vehicle-treated WT, vehicle-treated KO, and MS417-treated

KO experimental groups were originally submitted (Table 2). However, the repeated genotyping

analysis reassigned vehicle-treated WT as n = 5, vehicle-treated KO n = 9, and MS417- treated

KO n = 2 (Table 4). For future studies, the correct dosage of MS417 as well as accurate

genotyping, resulting in a higher n value for each experimental group, would help in yielding

more effectual and precise outcomes. Despite the experimental errors, these analyses and the

yielded negative results still provided data that plays a part in the effort to uncover novel

therapies for AS.

82

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