Functional Purinergic Signalling in Epithelial
Cells of the Proximal Nephron
by
Gareth Price
0917263
Mini-Project 2 Thesis
Supervisors: Dr Paul Squires and Dr Claire Hills
MOAC Doctoral Training Centre
July 2013
Contents
1 Introduction
1
1.1
Diabetic Nephropathy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1
1.2
EMT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1
1.3
TGFβ1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3
1.4
Connexins and Hemichannels . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3
1.5
ATP, adenosine and purinoreceptors . . . . . . . . . . . . . . . . . . . . . . . . .
4
1.6
Aims of the project . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5
2 Materials and Methods
6
2.1
Cell Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6
2.2
Western Blotting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6
2.3
Data Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6
2.4
Calcium Microfluorimetry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6
2.5
MTT Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7
2.6
Crystal Violet Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7
2.7
Lactate Dehydrogenase Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8
3 Results
9
3.1
Protein characterisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9
3.2
Purinergic Receptor Agonist-dependent changes in [Ca2+ ] . . . . . . . . . . . . .
9
3.3
The effect of TGF-β1 on cell viability and cytotoxicity. . . . . . . . . . . . . . . .
12
3.4
The effect of TGF-β1 on protein expression . . . . . . . . . . . . . . . . . . . . .
14
4 Discussion
17
5 Conclusions
19
6 Acknowledgements
20
A Abbreviations
20
i
Abstract
Diabetic Nephropathy is the single most common cause of entry into renal replacement therapy and the leading cause of end-stage renal disease. DN, a chronic
kidney disease, is a micro-vascular complication characterised by the formation of
tubulointerstitial fibres as a result of TGF-β1-induced epithelial-to-mesenchymal
transition. In this study the presence of connexins Cx26, Cx40, Cx43 and purinoreceptors P2 Y1 , P2 Y2 and P2 Y3 was confirmed. Subsequently, elevated TGF-β1 levels
(2-10 ng/ml) were found to result in a significant decrease in Cx26, Cx43, P2 Y1 and
P2 Y6 as determined through densitometry of western blots. Calcium Microfluorimetry was used to produce dose response curves and efficacy profiles, resulting in a
concentration-dependent efficacy profile ATP ≥ UTP > ADP AMP = adenosine.
Cytotoxic assays were performed to ensure that the studied concentrations of TGFβ1 were not harmful to the cells. Except for a potential concern that the number of
viable cells is affected, all other assays resulted in studied TGF-β1 concentrations
proving to be non-toxic.
ii
Section 1. Introduction
1
1.1
Introduction
Diabetic Nephropathy
It has been reported that in 2010, there were 285 million adult sufferers from diabetes
mellitus [1]. By 2030, it is predicted that this number will increase by 54%, to 439 million.
It is not surprising therefore, considering such high numbers, that diabetic nephropathy
(DN), a common complication found in both types of diabetes, is the single most common
cause of entry into renal replacement therapy and the leading cause of end-stage renal
disease (ESRD) [2].
DN refers to the micro-vascular complication resulting from extended gycaemic assault.
There are structural and functional changes in various kidney compartments such as the
glomerulus, tubulointerstitium, proximal tubule and vasculature. Even though DN affects
both type 1 and type 2 diabetics, these changes are virtually indistinguishable, yet their
pathogenesis is distinct [3, 4]. Structural changes include mesangial expansion, glomerular
basement membrane thickening, tubular atrophy and tubolointerstitial fibrosis [1]. These
result in an increased glomerular filtration rate, proteinura, systemic hypertension and
eventually renal failure [2].
Extracellular matrix (ECM) accumulation in the glomerular mesangium and tubular interstitium is fostered by the presence of increased inflammatory and pro-fibrogenic
cytokines. This, combined with a decline in proximal tubule number (due to prolonged
exposure to metabolic and hemodynamic pertubations) leads to tubulointerstitial fibrosis
and excessive renal scarring [1, 5, 6]. The most important of the pro-fibrogenic cytokines
is thought to be TGF-β1, which induces epithelial-to-mesenchymal transition (EMT).
1.2
EMT
EMT stems from a fundamental concept of biology: “cells come from cells” [7, 8] – and
to form the various cell types found in the body, differentiation occurs. Even though cells
are highly specialised and a state of terminal differentiation may appear to be necessary
(at least in adult tissue), this is not found in practice. Epithelial cells possess a plasticity
that allows them to change their phenotype following morphogenic pressure (from tissue
damage and subsequent repair, for example) [9].
Within EMT there are four stages of morphological and phenotypical changes (illustrated in Figure 1): 1) a loss of cell adhesion molecules such as E-cadherin and ZO-1 and
an increase in the E-cadherin repressor SNAIL [10]; 2) an increase in mesenchymal markers α-SMA (smooth muscle actin) and vimentin, an intermediate filament protein; 3) the
loss of cell adhesion due to these expression changes in important adhesion components
and cytoskeletal remodelling, leading to disruption of the attachment of the cells to the
tubular basement membrane (TBM); 4) the acquirement of the ability to migrate from
1
Section 1. Introduction
E-Cadherin
ZO-1
Cytokeratin
TBM
transitional phase
loss of adhesion
alpha-SMA
vimentin
FSP
SNAIL
N-Cadherin
migration
+ invasion
Figure 1: EMT. Epithelial-to-mesenchymal transition is the transdiffiferation between a static, secure cell to a mobile, invasive cell of a mesenchymal fibroblast.
Accompanying the processes is the acquisition of mesenchymal markers such as αSMA and vimentin, and the loss of important epithelial adhesion components such
as E-cadherin. This process in entirely reversible.
the TBM into the interstitium, accompanied by increased invasiveness and resistance to
apoptosis [2].
Normally, the tubular epithelial cells form a strongly coupled epithelial sheet held
together by E-cadherin. Once EMT starts to occur and expression of E-cadherin declines, cellular adhesion decreases and the sheet begins to dissociate . The increase in
α-SMA, accompanied by an increase in Ca2+ -binding fibroblast-specific protein 1 (FSP)
and vimentin intermediate fibres (in exchange for normal cytokeratin), leads to the morphological change from a cobblestone appearance to a thin fibrous appearance. This
change in appearance can be seen in Figure 2 (images provided by PES).
Figure 2: The change in appearance of HK2 cells under differing concentrations of TGF-β1. HK2 cells undergo vast morphological changes when
grown in different concentrations of TGF-β1. Normally described as possessing a
cobblestone appearance, their shape changes to being more thin and fibrous.
2
Section 1. Introduction
1.3
TGFβ1
TGF-β1 and its downstream SMAD signalling cascade is thought to be the most important inducer of EMT and plays a key role in DN [11]. TGF-β1 is a broad-spectrum
cytokine that is involved in several important biological processes such as cell growth,
differentiation, adhesion, proliferation, tissue repair and apoptosis [12, 13]. In diabetic
models such as rats and mice, it is found that TGF-β1 levels (both gene expression and
secretion) are elevated [14, 15, 16]. This is also confirmed through histological analysis
of patient biopsies where EMT has been observed. TGF-β1 affects the expression of important cadherins, catenins and other epithelial recognition and adhesion proteins [17],
inducing the aforementioned EMT morphological changes.
TGF-β1 binds to its receptor (TGF-β1 receptor II, TβRII) via SMAD dependent
pathways, which activate the TGF-β1 receptor type 1 kinase. Normally these SMAD
pathways are stringently controlled so that unwanted TGF-β1 responses are prevented.
Control is performed by both inhibitory SMADs and transcriptional co-repressors [18]
such as SnoN(Ski-related novel gene, non Alu-containing), Ski (Sloan-Kettering Institute
proto-oncogene), and TGIF (TG-interacting factor). In models of diabetes and DN,
levels of these three co-repressors are diminished, further providing evidence for SMAD
dependent TGF-β1 involvement in EMT [2].
1.4
Connexins and Hemichannels
While the fibrotic response and EMT is modulated and regulated by a number of genes,
signals from neighbouring cells also drive these processes. There is a direct flow of information between physically coupled cells through gap junctions (GJ). GJs are made up
of two connexons (as shown in Figure 3), one contributed by each cell, which are aligned
so that a 2nm channel appears between them. Each connexon is made of six individual
transmembrane connexins and is functionally active in the plasma membrane of the cell
[19]. However, if there are no neighbouring cells or the connexons are not aligned perfectly (as may be the case in DN), each connexon makes a hemichannel, through which
secondary messengers such as cAMP and inositol 1,4,5-trisphospate (IP3) and inorganic
ions including Ca2+ and Na+ can be transported [20]. It is these small molecules that act
as autocrine or paracrine signals. Transport through the hemichannel is limited to 1 kDa
[21].
There are 20 isoforms of connexins in humans and rodents, all of which are referred
to as CxMW, where MW is the molecular weight. Each connexin has four membrane
spanning domains connected by two extracellular loops and one intracellular loop [20].
Although there is some homology between the different isoforms, each connexin has different specificity and function, depending on the shape/size of the pore and the residues that
line it [22]. Post translational modifications such as phosphorylation and S-nitrosylation
3
Section 1. Introduction
can also affect these properties [23].
A
ATP
Connexon
(”Hemichannel”)
Cell 1 Cytoplasm
Extracellular Space
Cell 2 Cytoplasm
HC
B
GJ
Figure 3: Connexins are the basis of gap junctions and hemichannels.
A. 6 connexins assemble to form a hexameric pore found in the plasma membrane
of cells. These “connexons” can align with another of a different cell to produce
gap junctions, or exist by itself as a hemichannel. B. Gap junctions are seen as
2nm plaques in EM images, and are found between two physically coupled cells.
Hemichannels exist on their own. Reproduced from [21].
1.5
ATP, adenosine and purinoreceptors
One of the signalling molecules that enter cells via hemichannels is the ubiquitous adenosine triphosphate (ATP) molecule and its metabolite adenosine nucleoside. These are
released as paracrine signals from one cell and activate the purinergic receptors on another, and are considered to be major modulators of renal function [24]. There are two
broad classes of purinoreceptors, first proposed in 1978 by Burnstock et al : those that
are selective for adenosine, P1 , and those that are selective for ATP, P2 [25, 26]. Further classification of the P2 receptors led to the establishment of two subclasses, P2 X –
ionotropic ligand gated ion channels and P2 Y – metabotropic g-protein coupled receptors
(GPCRs). Full descriptions and further information can be found in a review by G. Burnstock in 2006 [27]. P2 purinoreceptors are unsurprisingly found in many components of
the kidney, including glomerular cells, renal tubular cells, renal vascular (endothelial and
smooth muscle) cells, and interstitial cells [24].
4
Section 1. Introduction
It has recently been shown that in cardiac fibroblasts, ATP is transported through
connexin hemichannels (especially through the most abundant rat Cx43 and Cx45 proteins) and that pro-fibrosis, determined through correlated α-SMA, a major component
in EMT, is induced through the activation of P2 receptors (especially P2 Y2 , the most
abundant purinoreceptor) [28]. This data implies that the release of ATP and its metabolites is an important mechanism for fibroblast homeostasis in both the basal and activated
state.
1.6
Aims of the project
This study aims to determine whether the increased levels of TGF-β1, brought about by
high glucose levels in diabetics, have an effect on the expression of three of the major
connexin isoforms, Cx26, Cx40 and Cx43. We will use an established HK2 cell line that
models the cells of the proximal tubule of the kidney. It is already known that TGF-β1
decreases Cx43 expression [29]. We postulate that if whole-cell expression of connexins is
decreased, paracrine signalling between neighbouring cells would also decrease due to the
decrease in hemichannels.
Further to studying connexin expression, this study also investigates the role of P2 Yx
purinergic receptors and the effects of ATP and its metabolites (ADP, AMP, adenosine,
and also UTP) on the concentration of Ca2+ within the cell. Combined with determining if
there are changes in expression of P2 Y1 , P2 Y2 and P2 Y6 , we can start to characterise how
communication in the proximal tubule changes in diabetic nephropathy. If the mechanisms
of cell adhesion and communication are fully known, potential therapeutic candidates are
more likely to be unearthed.
Finally, to establish whether any expression changes are due to cytotoxic effects from
elevated TGF-β1 levels, we will run LDH, MTT and Crystal Violet assays. These determine cell plasma membrane damage, cell viability and cell number respectively.
5
Section 2. Materials and Methods
2
Materials and Methods
HK2 cells were obtained from ATCC Bio-Resource Centre (LGC Standards. Middlesex,
UK). Tissue culture media (DMEM/F12) and supplies were from Invitrogen (Paisley,
UK). All antibodies were purchased from Santa Cruz (CA, USA). Immobilon P membrane
was from Millipore (Watford, UK) and Enhanced Chemiluminescence reagents were from
Amersham Biosciences (Buckinghamshire, UK). TGFβ was obtained from Sigma (Poole,
UK).
2.1
Cell Maintenance
Cells were maintained in DMEM/Ham’s F12 medium, containing 10% FCS, EGF (5 ng/ml),
glucose (17.5 mM) and calcium (0.5 mM). They were incubated at 37 ◦C with 5% CO2 .
Cells were split when 80% confluent, every 3-4 days using trypsin. Prior to treatment,
cells were placed in fresh DMEM/F12 media containing 5 mM glucose, and then quiesced
by changing the media to serum free DMEM/F12 (5 mM glucose).
2.2
Western Blotting
All Western Blots were run following usual instructions. The gels were made up of 10%
resolving gel and 4% stacking gel, consisting of 30% Bis/Acrylamide mix; 1.5 M Tris-HCl
(pH8.8); 1 M Tris-HCl (pH6.8); 10% Sodium Dodecyl Sulphate (SDS); 10% Ammonium
Persulphate (APS) and TEMED (N,N,N,N-tetramethylethylenediamine). The gels were
run for approximately 1 hour at 125 V, and transfer to the Immobilon P membrane was
performed at 100 V (for 1 hour). Primary antibodies were used at a 1:10000 dilution, and
secondary antibodies at 1:40000.
2.3
Data Analysis
Densitometry of the Western Blot autoradiographs was performed with TotalLab Quant
2003. Statistical tests were performed using Prism GraphPad software version 6.0 (San
Diego, CA, USA). Statistical importance was determined using a one-way ANOVA test
with Tukey’s multiple comparison post-test. Data are expressed as mean + standard error
of the mean, and n denotes the number of separate experiments. Statistical significance
was determined when P < 0.05 and are further explained in the figure captions.
2.4
Calcium Microfluorimetry
HK2 cells were transferred onto coverslips in 6-well plates and incubated overnight in
17.5 mM Glucose DMEM/F12 to allow them to adhere. Following this, the media was replaced with 5 mM Glucose DMEM/F12 so that high glucose levels did not affect results.
The cells were loaded for 30 minutes in 5 mM glucose at 37 ◦C with 5 µM Fura-2/AM
(Sigma, UK). Fure-2 is used because it is a ratiometric dye. This means that photo6
Section 2. Materials and Methods
bleaching will not affect the results and the basal lines always go back to the same value
each time. It is not a single emmission dye, however, so two different exposures have to
be taken. All experiments were carried out at 37 ◦C. Coverslips, held in a separate stage,
were then placed in the centre of a stainless steel bath placed onto a heating platform on
the microscope stage (Axiovert 200 Research Inverted microscope, Carl Zeiss Ltd., Welwyn Garden City, UK). The standard extracellular medium used for washing was a Na+
rich balanced salt solution (137 mM NaCl, 5.4 mM KCl, 1.3 mM CaCl2 , 0.8 mM MgSO4 ,
0.3 mM Na2 HPO4 , 0.4 mM KH2 PO4 , 4.2 mM NaHCO3 , 10 mM HEPES and 5 mM glucose,
pH 7.4).
A low-pressure flow system (flow rate 1-2ml/min) was used to change the solutions in
the bath to allow for the addition of ATP, UTP, ADP, AMP and adenosine. Cells were
illuminated alternatively at 340nm and 380nm using a Metaflour imaging workbench
(Universal Imaging Corp Ltd., Marlow, Bucks, UK). Emitted light was filtered using a
510nm long-pass barrier filter and detected using a Cool Snap HQ CCD camera (Roper
Scientific). Cells were selected as regions of interest and data was collected at 3 second
intervals.
2.5
MTT Assay
The 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Figure 4) assay
is a common way of determining the cytotoxic effects of treatments on cell viability. It is
a colourmetric assay that utilises the reduction of MTT to purple, insoluble formazan by
cellular enzymes in the mitochondria of viable cells. HK2 cells were cultured in a 96-well
plate in DMEM/F12 media with 5 mM glucose and left for 48 hours, then serum starved
for a further 24 hours. The cells were then treated with 0-10 ng/ml TGFβ1 overnight.
After being treated with a solubilisation solution, the 96-well plate was read by a plate
reader, and each cell’s viability determined following the manufacturer’s instructions.
Results are expressed as a percentage of the control cells.
Br -
HN
N
N+
S
N
N
Cellular Enzymes
N
N
N
N
N
S
Figure 4: MTT
2.6
Crystal Violet Assay
Crystal Violet (Figure 5) provides quantitative information about the density of cells
adhering to 12-well plates. Crystal Violet stains the DNA in cells and can provide quan7
Section 2. Materials and Methods
titative data due to the colourmetric nature of the assay. The media was removed and
the cells were washed with PBS, then fixed with paraformaldehyde for 10 minutes. After
several washes with PBS to remove the PFA, the cells were incubated with a 1% Crystal
Violet solution for 10 minutes at room temperature. After further washes, the stain was
solubilised with 1% SDS, and the density of cells for each treatment was determined using
a plate reader. All data are presented as a percentage of the control stain absorbence.
N
C+
ClN
N
Figure 5: Crystal Violet
2.7
Lactate Dehydrogenase Assay
Plasma membrane damage is a common method of assessing cell death or cytotoxicity.
Lactate Degydrogenase (LDH) is released into media when the plasma membrane is damaged. HK2 cells were grown in 12 well plates in 5 mM glucose containing media for 48
hours prior to a 24 hour period of serum starvation. The cells were then treated with
0-10 ng/ml TGFβ overnight and the LDH levels assayed using the LDH-cytoxicity assay
kit II (Abcam) following manufacturer’s instructions. The values were presented as a
percentage of the LDH release that was observed as compared to control cells.
8
Section 3. Results
3
Results
P2Y1
P2Y2
P2Y6
50kDa
CX26
52kDa
CX40
43kDa
CX43
26kDa
49kDa
40kDa
Figure 6: Characterisation blots. Western Blots confirmed the presence of
Cx26,40,43 and P2 Y1 , P2 Y2 and P2 Y6 . All bands are marked with their position
on the membrane as determined relative to a marker column. Most are higher than
the actual molecular weights, but are consistent with documentation.
3.1
Protein characterisation
HK2 cells express connexin and P2 Y purinergic receptor protein.
Western blots confirmed that HK2 cells express a) P2 Y1 , b) P2 Y2 , c) P2 Y6 , d) Cx26, e)
Cx40 and f) Cx43 protein. Bands can be seen at 50 kDa, 52 kDa, 43 kDa, 26 kDa, 49 kDa
and 45 kDa respectively (Figure 6). Although most are higher than their actual molecular
weights, the bands are consistent with those in the documentation for the antibodies.
3.2
Purinergic Receptor Agonist-dependent changes in [Ca2+ ]
In HK2 cells, ATP, UTP and ADP all significantly increase [Ca2+ ] in a concentration dependent manner.
We have examined the effects of purinergic receptor agonists ATP, ADP, AMP, adenosine and UTP on [Ca2+ ]. To determine the optimum concentration of agonist to use,
a concentration dependent dose response was performed between 0.1-100 µM (Figure 7
Panel A). The optimal concentration was found to be 10 µM.
Figure 7 shows that there is a significance increase in [Ca2+ ] when ADP, ATP and
UTP (10 µM) are added to HK2 cells. There is no signifiant decrease between UTP, ATP
and ADP. The purinergic agonists lead to basal to peak values (corresponding to ∆[Ca2+ ])
of 0.008 ± 0.005, 0.015 ± 0.007, 0.400 ± 0.028, 0.589 ± 0.040 and 0.48 ± 0.032 for adenosine,
AMP, ADP, ATP and UTP respectively. Thus, there is a clear agonist-dependent response
in the manner ATP ≥ UTP > ADP AMP = adenosine.
In most cases the biggest response is the first, so to circumvent this we ran this
experiment each time adding the agonists in different orders. Thus, any bias that the first
response has with respect to its basal to peak amplitude should be removed and a proper
conclusion can be drawn.
9
Section 3. Results
A
200ms
0.1µM
10µM
1µM
100µM
B
200ms
AMP
Adenosine
[Ca2+] expressed
as 340:380nm
C
ATP
ADP
****
****
****
0.8
UTP
D
0.6
0.4
0.2
0.0
A
AMP
ADP
ATP
UTP
Antagonist
Figure 7: Calcium Microfluorimetry with HK2 cells produces a clear
agonist-dependent efficacy profile in the manner: ATP ≥ UTP > ADP AMP = adenosine. To determine the optimum concentration of agonist to use,
a concentration dependent dose response was performed between 0.1-100 µM (Panel
A). It was decided to take 10 µM for all future experiments. Arrows show times
of addition. n=2 In Panel B, a sample profile trace of HK2 cells showing changes
in [Ca2+ ] in response to the addition of ATP, UTP, ADP, AMP and adenosine (all
10 µM) is shown. After each addition a wash of Na+ rich balanced salt solution was
applied. In Panel C, the addition of ADP, ATP and UTP shows significant increase in
[Ca2+ ] changes, as determined through the measurement of base-to-peak amplitude.
Although there is no significant statistical difference between ADP, ATP and UTP,
ATP ≥ UTP ADP AMP = adenosine. Data is expressed as mean ± SEM
with n = 4 (each n consisting of multiple cells). Where shown, **** P < 0.0001.
An example viewport of the cells analysed using Calcium Microfluorimetry can be
seen in Panel D. λ = 380 nm.
10
Section 3. Results
A
B
% Crystal Violet stain
as compared to control
125
100
75
50
25
0
0
2
4
100
75
50
25
0
10
0
TGF 1 (ng/ml)
2
4
10
TGF 1 (ng/ml)
C
****
****
****
100
% MTT uptake as
compared to control
% LDH released as
compared to control
150
75
50
25
0
0
2
4
10
TGF 1 (ng/ml)
Figure 8: Impact of TGFβ1 on HK2 cell viability, assessed by MTT
uptake, Crystal Violet staining and LDH release. HK2 cells were cultured in
5mM glucose-containing DMEM/F12 media for 48 hours, prior to an overnight serum
starvation. Following this, the cells were treated with 0-10ng/ml TGFβ1 in serumfree media. Cell membrane damage was quantified by measuring LDH concentration
in the surrounding media. Panel A shows that there is no significant difference
between any of the concentrations of TGF-β1. Crystal Violet staining was used to
determine cell density. Panel B shows that no concentrations of TGF-β1 produce
any significant difference in LDH release. As assessed by MTT uptake, shown in
panel C, cell viability significantly decreases when treated with any concentration
of TGF-β1 compared to the control. There is no significant difference between
any of the non-control concentrations. All values are expressed as a percentage of
the control (0 ng/ml), and results are representative of three separate experiments.
Significance: ∗ ∗ ∗∗ P < 0.0001. n=3
11
Section 3. Results
3.3
The effect of TGF-β1 on cell viability and cytotoxicity.
TGF-β1 leads to no cell membrane damage or a reduction in numbers, but
may lead to a reduction in the number of viable cells.
To quantify cytotoxicity, we used the Lactate Dehydrogenase assay as a marker of cell
membrane damage. HK2 cells were stimulated for 24 hours with TGF-β1 (0-10 ng/ml)
in serum-free conditions before the assay was performed. No concentration resulted in a
significant change. Shown as a percentage of LDH release compared to the control, incubation with 2, 4 and 10 ng/ml TGF-β1 yielded 114.2%±1.9%, 99.5%±5.7% and 99.9%±8.3%
respectively, as shown in Figure 8, panel A (n=3). This data suggests that no cell membrane damage occurred.
The Crystal Violet assay was used to quantify the number of HK2 cells after a 24 hour
incubation with 0-10 ng/ml TGF-β1. This assay, like the LDH assay, suggests that there
is no change in cell number. The amount of dye uptake, as compared as a percentage to
the control, was 89.7% ± 1.5%, 88.6% ± 2.4% and 91.1% ± 4.6% for 2, 4 and 10 ng/ml of
TGF-β1., as shown in Figure 8, panel B (n=3).
The MTT assay was used to assess cell viability after 24 hour stimulation with 010 ng/ml TGF-β1 in serum-free conditions. The MTT assays enables quantification as
the amount of MTT uptake correlates directly with the number of viable cells. After 24
hours of incubation with TGF-β1, there is a significantly decreased cell viability (49.5% ±
2.3%, 43.3% ± 5.2%, 41.5 ± 5.5% for 2, 4 and 10 ng/ml respectively, as shown in Figure 8,
panel C, all values percentages of the control). The decreases were all significant with
P< 0.0001, n=3.
12
P2Y1 Expression
(% as compared to control)
A
13
0
2
***
4
*
TGF 1 (ng/ml)
*
**
10
B
P2Y2 Expression
(% as compared to control)
0
50
100
150
0
4
TGF 1 (ng/ml)
2
10
(55kDa)
Tubulin
(42kDa)
P2Y2
C
0
50
100
150
0
4
TGF 1 (ng/ml)
2
*
10
(55kDa)
Tubulin
(36kDa)
P2Y6
Figure 9: TGF-β1 evoked changes in Purinergic Receptors in HK2 cells, assessed by western blots. HK2 cells were
cultured in 5mM glucose-containing DMEM/F12 media for 48 hours prior to an overnight serum starvation. The cells were then treated
with 0-10ng/ml TGF-β1 for 24 hours under serum-free conditions. Expression was quantified using densitometry. Panel A shows that
increases in TGF-β1 produce a significant decrease in P2 Y1 expression. There is a significant decreasing trend in expression with
increasing concentrations of TGF-β1. Each increase in TGF-β1 concentration is more significant than the last. TGF-β1 produces a
similar, but lesser decreasing trend for P2 Y6 expression (Panel C). There is a significant decrease when HK2 cells were incubated with
10ng/ml TGF-β1. Panel B shows that there is no significant change when HK2 cells were incubated with 0-10ng/ml TGF-β1. Each
panel shows the band of interest at the top, with a house keeping band (α-tubulin) that was used to account for differences in loading
underneath it. The graphs in the lower part show the mean + SEM, expressed as percentages as compared to the control. Significance:
∗ P<0.05, ∗∗ P<0.01, ∗ ∗ ∗ P<0.001,∗ ∗ ∗∗ P<0.0001. n=3
0
50
100
150
(55kDa)
Tubulin
(45kDa)
P2Y6 Expression
(% as compared to control)
P2Y1
Section 3. Results
Section 3. Results
3.4
The effect of TGF-β1 on protein expression
TGF-β1 significantly down regulates P2 Y1 and P2 Y6 expression, and causes
no significant change in P2 Y2 expression.
HK2 cells were cultured for 48 hours in 5 mM glucose containing DMEM/F12 media
prior to an overnight serum starvation. The cells were then stimulated for 24 hours with
0-10ng/ml TGF-β1 and the resulting western blots were analysed using densitometry
(Figure 9).
TGF-β1 invoked a concentration-dependent decrease in expression for the P2 Y1 purinergic receptor protein. As a percentage compared to the control, 2, 4 and 10ng/ml resulted
in 75.3% ± 3.9% (P < 0.05, n=3), 58.0% ± 4.1% (P < 0.01, n=3) and 48.2% ± 8.7%
(P<0.001, n=3).
TGF-β1 invokes a similar, but lesser response in P2 Y6 expression. As a percentage compared to the control, 2 and 4ng/ml TGF-β1 produced lower, but insignificant
decreases of 81.8% ± 10.5% and 56.7% ± 13.1% respectively. At 10ng/ml, there is a significant drop to 40.5%±9.9% (P<0.05, n=3). Due to the appearance of a definite decreasing
trend, one might expect the statistical significance to increase if further repeats are carried
out.
Unlike P2 Y1 and P2 Y6 , however, TGF-β1 invokes no significant change in P2 Y2 expression, another metabotropic GPCR protein. The resulting changes in expression was (as a
percentage compared to the control) 132.3% ± 15.0%, 133.4% ± 16.1% and 93.1% ± 9.0%
for 2, 4 and 10ng/ml respectively.
14
Cx26 Expression
(% as compared to control)
15
0
2
****
4
TGF 1 (ng/ml)
***
****
10
B
0
50
100
150
0
4
TGF 1 (ng/ml)
2
10
(55kDa)
Tubulin
(40kDa)
Cx40
C
0
50
100
150
0
2
***
4
TGF 1 (ng/ml)
*
**
10
(55kDa)
Tubulin
(43kDa)
Cx43
Figure 10: TGF-β1 down regulates Cx26 and Cx43 expression, and has no effect on Cx40 expression in HK2 cells.
HK2 cells were cultured in 5mM glucose containing DMEM/F12 media for 48 hours prior to an overnight serum starvation. The cells
were then treated with 0-10ng/ml TGF-β1 for 24 hours under serum-free conditions. Expression was quantified using densitometry.
Panel A shows that when HK2 cells were incubated in 2-10ng/ml TGF-β1 there was a significant decrease in Connexin 26 expression.
There is no significant difference between the three concentrations. Likewise, there is a significant decrease in expression in Connexin
43 (as shown in Panel C), although 2 and 4ng/ml are not as statistically significant. There is no significant difference in Connexin 40
expression, shown in Panel B. Each panel shows the band of interest at the top, with a house keeping band (a-tubulin) that was used
to account for differences in loading underneath it. The graphs in the lower part show the mean + SEM, expressed as percentages as
compared to the control. Significance: ∗ P<0.05, ∗∗ P<0.01, ∗ ∗ ∗ P<0.001,∗ ∗ ∗∗ P<0.0001. n=3
0
50
100
150
(55kDa)
Tubulin
(26kDa)
Cx40 Expression
(% as compared to control)
Cx26
Cx43 Expression
(% as compared to control)
A
Section 3. Results
Section 3. Results
TGF-β1 significantly decreases Cx26 and Cx43 expression, and causes no significant change in Cx40 expression.
HK2 cells were cultured for 48 hours in 5 mM glucose containing DMEM/F12 media
prior to an overnight serum starvation. The cells were then stimulated for 24 hours with
0-10ng/ml TGF-β1 and the resulting western blots were analysed using densitometry
(Figure 10).
TGF-β1, at the studied concentration, invokes a highly significant decrease in Cx26
expression (Panel A). 2ng/ml of TGF-β1 resulted in a significant decrease to 44.0%±7.5%
as compared to the control (P<0.001. n=3). Furthermore, 4 and 10ng/ml invokes a
greater decrease to 38.9% ± 11.0% and 25.8% ± 6.3% respectively (P<0.0001).
TGF-β1 has no significant effect on Cx40 expression (Panel B). After stimulation with
2, 4 and 10ng/ml TGF-β1, expression of Cx40 was 100.4%±7.6%, 103.9%±11.3% and 100.6%±
20.32% respectively.
Like P2 Y1 , TGF-β1 also down regulates Cx43 in a concentration dependent manner
(Panel C). 2 ng/ml TGF-β1 leads to 61.6% ± 7.8% expression as compared to the control
(P<0.05, n=3). Further, 4 and 10ng/ml resulted in 53.4% ± 11.4% and 33.1% ± 11.2%
respectively (P<0.01 and P<0.001, n=3). This correlates with results in the literature
[29].
16
Section 4. Discussion
4
Discussion
In diabetic nephropathy, the pro-fibrotic cytokine TGF-β1 induces epithelial-to-mesenchymal
transition (EMT), resulting in the accumulation of extracellular matrix (ECM). The cells
of the kidney begin to lose important adhesion components responsible for the direct
physical coupling between them, and to the attachment and organisation to the tubular
basement membrane, the basis of the epithelial sheet that plays a large role in water
retention and other functions of the kidney.
We have confirmed in HK2 cells, a cell line that models the cells of the human proximal tubule, the existence of the major connexin family members Cx26, Cx40 and Cx43.
Connexins are the monomer protein units that combine to make a hexameric assembly
with a pore through the centre. Two of these hexameric “connexon” assemblies from
neighbouring, coupled cells can align to form Gap Junctions (GJs), or can exist in isolation as hemichannels. Both GJs and CxHCs are routes by which inorganic ions and small
secondary messengers can enter the cell, either by direct exchange from one neighbour to
another (GJs), or via a concentration gradient from the surrounding area (hemichannels).
We have determined that elevated levels of TGF-β1 lead to a significant down regulation of Cx26 and Cx43. Cx40 is unchanged with the studied TGF-β1 concentrations
(0-10ng/ml). The fact that the data in this study correlates directly with previously
reported data is very encouraging and gives confidence to any data that has not been
published before [29]. Reduction in Cx26 and Cx43 expression possibly limits the types
of connexon forms, thus limiting the types of molecules that are allowed passage through
the pore. It is not established whether there is a specific secondary messenger molecule
that is directly affected by a significant decrease in Cx26 and Cx43 expression. It is also
not established how other isoforms of connexins respond – there are approximately 20
isoforms that join in different combinations to form connexons of different specificities. It
may be that the decrease in some connexin isoforms are compensated for by an increase
in others. A full study involving all isoforms found in the kidney would be, although
tedious, very informative.
We also considered the effects of TGF-β1 on the accepting cell. It has been proven that
ATP and its metabolites activate purinoreceptors, leading to a SMAD cascade ultimately
ending in fibrosis. It has only recently been accepted that ATP, despite its ubiquitousness
as an energy storage molecule, is an important signalling molecule. There are two types
of purinoreceptors: P1 – receptors that are specific for adenosine, and P2 – receptors that
are specific for ATP. P2 receptors are found on the surface of nearly all cell types in the
kidney – indicative of its importance in signalling. We determined that elevated levels
TGF-β1 lead to a significant decrease in P2 Y1 at all concentrations, and a significant
decrease in P2 Y6 at 10ng/ml.
17
Section 4. Discussion
If both connexin and purinoreceptor expression are decreased simultaneously this may
significantly affect vital cellular processes such as proliferation and growth [30, 31]. This
is particularly devastating when groups of cells (i.e. tissues) live in a co-ordinated manner
and signalling between cells is important for flow of information. It is therefore important to fully characterise the effect of elevated TGF-β1, and to determine what different
components of the ATP signalling pathway contribute. Further to this, connexins have
also been considered to be tumour surpressors, and have other non gap-junction mediated
effects [32].
While it is useful to determine whether increased TGF-β1 levels have any direct cytotoxic effect on HK2 cells, it is also imperative in assigning roles for different protein
expression. We confirm that the studied TGF-β1 concentrations have no cytotoxic effects on cell number and no significant damage to plasma membranes occurred. It is
concerning, however, that the number of viable cells significantly decreases. This must
be remembered when considering the effects of TGF-β1 on connexin and purinoreceptor
expression. However, because connexins and purinoreceptors are found on the plasma
membrane, where no damage was found, it is still reasonable to make conclusions based
on the densitometry results in this study.
The calcium microfluorimetry results show that the HK2 cells possess purinoreceptors
that respond to ATP-like signals. We confirmed that HK2 cells respond to ATP in a
concentration dependent manner. 10 µM was decided to be the optimal concentration due
to the lack of significant change in higher concentrations. There is a significant increase
in [Ca2+ ] when ATP, UTP and ADP are added to the cells, indicating the presence of
P2 purinoreceptors. Although there is no significant response to adenosine/AMP, this
may be due to the concentrations studied being too low. With such a marked efficacy
profile, it is imperative that this experiment is repeated but with TGF-β1 treatments
over 24 and 48 hours – if the results follow the expression changes shown by the western
blot densitometry then one would expect a severely muted response to ATP, UTP and
ADP. It would be interesting to determine whether there is an increase in sensitivity to
adenosine/AMP as part of a rescue mechanism to restore cellular signalling.
It must be remembered that all experiments were performed using HK2 cells – a model
cell line that is, purposely, far simpler than proximal tubule cells from primary human
tissue. To that effect, the results found in this study may not provide the full story of what
happens in diabetics (although for all intents and purposes this cell line is thought to be
adequate, at least initially). Therefore for these results to have full translational benefit
the main findings from this study would need to be reproduced using primary human
tissue. Of course, obtaining human tissue is both expensive and ethically challenging, so
one might use a more complex model cell line to determine whether the results from this
study can be confirmed. There are experimental setups which include a three dimensional
18
Section 5. Conclusions
mesh and electrical potentials, where cells can grow in an environment more representative
of tissue.
There is a wealth of potential work that stems from this study. Although we have
found that whole-cell expression of some connexins and purinoreceptors decrease, this
does not take into account the location of these proteins. Compartmentalisation Western
Blotting and immunoblotting would indicate whether the connexins and receptors move
from the plasma surface, where they are functionally active, to elsewhere in the cell.
Using siRNA, it would also be possible to knock down particular connexin or purinoreceptor isoforms, which would allow us to establish whether any particular isoforms
are especially crucial for cellular communication and whether any form of rescue attempts
are made by the cell. Model cell lines such as HK2 are very adaptable and genetically
modifiable – and so no major problems are anticipated. Lastly, there is the possibility
to use biosensors that are able to detect minute concentrations of ATP molecules. This
would be particularly useful to directly link ATP with the maintenance of cellular adhesion, coupling and communication while confirming the location and movement of ATP
molecules. All of these described possibilities have been put forward in a PhD proposal.
5
Conclusions
TGF-β1 is an important cytokine which is elevated in diabetics. In this study, it has
been implicated in an associated decrease in connexin 26 and 43 expression, along with
a decrease in purinoreceptors P2 Y1 and P2 Y6 . Simultaneous down regulation of these
important proteins that contribute to signal release and activation have heavy implications
in diabetic nephropathy. Furthermore, we determine that HK2 cells respond to ATP in
a concentration dependent manner, and they respond with a general efficacy profile of
ATP ≥ UTP > ADP AMP = adenosine. Finally, we confirmed that the studied
concentrations of TGF-β1 do not affect cell number or cause plasma membrane damage,
but the MTT assay suggests that the number of viable cells significantly decreases.
19
Section 6. Acknowledgements
6
Acknowledgements
I would like to extend many thanks to my supervisors Dr. Paul Squires and Dr. Claire
Hills for their expert guidance and patience throughout the project.
I would also like to thank the MOAC DTC and our generous funders, EPSRC.
A Abbreviations
DMEM
Dulbecco’s Modified Eagle Medium
DN
Diabetic Nephropathy
EGF
Epidermal Growth Factor
EMT
Epithelial-to-mesenchymal transition
ESRD
End Stage Renal Disease
FCS
Fetal Calf Serum
FSP
Fibroblast-Specific Protein
GJ
Gap Junctions
HC
Hemichannels
HK2
Human Kidney Cells 2
siRNA
Small Interfering RNA
SMAD
Small mothers against decapentaplegic
TBM
Tubular Basement Membrane
TGFβ1
Transforming Growth Factor beta, isoform 1
20
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