Nephrol Dial Transplant (2009) 24: 1753–1758
doi: 10.1093/ndt/gfn714
Advance Access publication 7 January 2009
Rat mesangial cells exhibit sex-specific profibrotic and
proinflammatory phenotypes
Izabella Z. A. Pawluczyk1,2 , Eddie K. C. Tan1,2 and Kevin P. G. Harris1,2
1
Department of Infection, Immunity and Inflammation, University of Leicester and 2 John Walls Renal Unit, Leicester General
Hospital, Leicester, UK
Correspondence and offprint requests to: Izabella Z. A. Pawluczyk; E-mail: [email protected]
Abstract
Background. Chronic renal disease progresses more
rapidly in males compared to females. This study investigated whether there were any inherent differences between
male and female mesangial cells that could contribute to
this phenomenon and whether these differences could be
modulated by sex hormones.
Methods. Experiments were carried out on cultured mesangial cells derived from adult male and female Wistar rat kidneys. Fibronectin, TNFα and IL-1β levels were measured
in control and macrophage-conditioned medium (MCM)injured cells in the presence and absence of 17β estradiol
or testosterone.
Results. Male mesangial cells expressed higher baseline
fibronectin levels compared to female cells. Similarly,
basal levels of the proinflammatory cytokines TNFα and
IL-1β were higher in male cells. Fibronectin and IL-1β
levels were enhanced proportionately between the sexes
in response to MCM stimulation, whilst the increase in
TNFα levels was greater in MCM-stimulated female cells.
Treatment with 10−8 M estradiol down-regulated baseline fibronectin levels in female mesangial cells but had
no effect on basal levels in male cells. Estradiol had
no effect on MCM-stimulated fibronectin levels in female mesangial cells but further increased stimulated levels in male cells. Testosterone had no effect on basal
fibronectin levels of either sex but further enhanced
MCM-stimulated fibronectin levels in mesangial cells of
both sexes. Sex hormone treatment had no effect on cytokine levels in male mesangial cells. However, in female
cells estradiol decreased TNFα levels and increased IL1β levels, while testosterone increased the levels of both
cytokines.
Conclusion. These data would suggest that male mesangial cells inherently exhibit greater profibrotic and proinflammatory characteristics than female cells. The inherent
gender phenotypes are further modulated by sex hormones.
This sexual dimorphism in mesangial cells may play a contributory role in the faster rate of progression to end-stage
renal disease in males.
Keywords: cytokines; 17β estradiol; fibronectin; mesangial cells;
testosterone
Introduction
Male patients with diverse types of primary chronic kidney
disease exhibit a more rapid rate of decline in renal function
than do female patients [1]. This has been shown to be the
case in primary inflammatory glomerular disease [2], diabetic nephropathy [3] and non-glomerular disease [4]. In
prepubescent patients with hereditary renal disease, there is
no sex-linked difference in age at time of death from renal
disease [4] suggesting that sex hormones are the underlying determining factor in the development of renal failure
rather than chromosomal gender per se. A meta-analysis using 68 studies and evaluating over 11 000 patients supported
the hypothesis that men with non-diabetic chronic renal disease, irrespective of aetiology, show a more rapid decline in
renal function than women [5]. However, the meta-analysis
was unable to assess whether the presence of testosterone
or the absence of oestrogen was the determining factor.
Many studies in experimental animals have examined
the direct effects of sex hormone treatment or effects of sex
hormone withdrawal via ‘gonadectomy’ [6–8]. The majority of these studies support the hypothesis that oestrogens
are generally protective and androgens are detrimental to
the course of renal disease. However, in some models of
renal disease, oestrogens have also been associated with an
acceleration of the renal injury [9,10].
The development of glomerulosclerosis is one of the
classic histological hallmarks of end-stage renal disease.
The glomerulus has been shown to be an oestrogen-target
tissue. Oestrogens acting via ERα receptors are thought to
modulate the turnover of extracellular matrix protein in a
manner that would be expected to protect against progressive scarring and the development of glomerulosclerosis
[11,12]. Mesangial cells are thought to be primarily responsible for modulating the levels of extracellular matrix in the
glomerulus. A number of in vitro studies have been conducted to examine the effects of sex hormones on mesangial cell pathophysiology [13–17]. Of note, the reduced
expression of collagens 1 and IV in response to estradiol or
selective oestrogen receptor modulators (SERM) has been
demonstrated.
However, to our knowledge, no studies have been performed specifically to evaluate the role of chromosomal
C The Author [2009]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.
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1754
gender per se on renal disease progression. Moreover, no
studies have directly compared the profibrogenic potential
of male and female kidney cells in vitro either basally or
in response to injury. We have therefore examined the effects of gender on renal scarring using mesangial cells in
culture. Specifically, we have evaluated whether there is an
inherent sex-specific difference in their profibrotic potential. In addition, we have assessed how sex hormones might
modulate any profibrotic responses both under basal conditions and under conditions in which mesangial cells have
been stimulated by the macrophage-conditioned medium
(MCM). We have previously demonstrated that exposure
of mesangial cells to macrophage products (as might be
expected to occur in vivo following renal injury) stimulates
a profound profibrotic response [18] allowing the in vitro
study of factors regulating the development of mesangial
scarring in an environment devoid of confounding circulating and haemodynamic factors.
Materials and methods
Unless otherwise stated, all reagents were purchased from Sigma-Aldrich
Chemical Co. (Dorset, UK).
Cell culture
Glomerular mesangial cells were cultured from either the glomerular explants of age-matched adult male or female Wistar rat kidneys (University of Leicester colony) using standard techniques. The cells were
cultured in RPMI 1640 (Invitrogen, Paisley, UK) supplemented with
20% heat inactivated foetal calf serum (FCS), 100 U/ml penicillin,
100 µg/ml streptomycin, 5 µg/ml insulin and 2mM glutamine. Cultured
cells were characterized as mesangial by their typical stellate, fusiform
morphology, their positive staining for the Thy-l antigen and their resistance to the toxic effects of D-valine and PAN.
In all experiments, mesangial cells of passages 2 through 10 were cultured in 24-well plates (Costar-Corning, Buckinghamshire, UK) or 25 cm2
flasks (Costar-Corning), allowed to grow to confluence, then rendered quiescent in the RPMI medium containing 0.5% FCS for 48 h prior to stimulation. In preliminary experiments, we found that there was no significant
difference in fibronectin production between cells grown in phenol red
free and phenol red containing RPMI.
Unless otherwise stated, experiments were conducted on mesangial
cells derived from five different pairs of age- and sex-matched male and
female rats.
Preparation of the MCM
The MCM was prepared from LPS-stimulated, peritoneal macrophages
from either male or female rats as previously described [18].
Culture of mesangial cells in the presence of MCM
In order to examine the effects of mesangial cell injury confluent, quiescent male and female mesangial cells were exposed to a 50% solution
of MCM in the presence or absence of 10−8 M17-β estradiol or 10−8 M
testosterone. The cultures were maintained in this medium for 1 or 3 days.
The tissue culture supernatants were harvested and stored at −20◦ C for
subsequent analysis. In these experiments, female mesangial cells were
stimulated with MCM derived from female rats and male mesangial cells
were stimulated with MCM derived from male rats. Cross-over experiments were also carried out where mesangial cells were stimulated with
MCM generated from macrophages derived from animals of the opposite
sex.
Preparation of cell lysates
After removal of tissue culture supernatants, cell monolayers were washed
with PBS, scraped into 1.0% IGEPAL (BDH, Merck, UK) in a wash buffer
(PBS containing 0.3 M NaCl, 0.1% Tween 20) and then incubated at room
I. Z. A. Pawluczyk et al.
temperature for 30 min. The cell scrapings were then transferred into 2 ml
tubes and sonicated for 5 s using a Jencons 50 W sonicator. The cell lysates
were then centrifuged for 30 s at 11 600 × g before determination of the
protein concentration.
Measurement of fibronectin production
Culture supernatants or cell lysates were assayed for fibronectin using an
inhibition ELISA as previously described [18].
Measurement of IL-1β and TNFα
Rat IL-1β and TNFα were measured using Quantikine ELISA kits (R&D
Systems, Oxford, UK) according to manufacturer’s instructions.
Protein assay
The detergent-lysed cell monolayer protein was assayed using a commercial BioRad DC protein assay kit using BSA standards (BioRad, UK).
Northern blotting
Northern analysis was carried out using a method previously described
[18]. Briefly, total RNA was extracted using TRIzol reagent (Invitrogen)
according to the manufacturer’s instructions. Thirty microgram aliquots of
RNA were electrophoresed on a 1% agarose gel. The resolved RNA was
transferred on to Hybond-N nylon by the capillary action. The membranes
were then hybridized with a 32 P-CTP cDNA probe that had been labelled
using a random primer labelling system (Prime-a-Gene, Promega). Following hybridization, the membranes were washed and exposed to X-ray
film with intensifier screens at −70◦ C. Densitometric analysis of the autoradiographs was carried out on a BioRad GS 700 imaging laser scanner.
Differences in RNA loading were normalized with cyclophillin cDNA.
Statistics
Mesangial cell fibronectin, TNFα and IL-1β levels were corrected for cell
protein. Representative northern blots are shown. Results are expressed as
means ± SEM. For comparison of means between two groups, an unpaired
t-test was employed. To compare values between multiple groups, an
analysis of variance (ANOVA) with Bonferroni correction was applied.
Statistical significance was defined as P < 0.05.
Results
Fibronectin expression in mesangial cells
Mesangial cells derived from male rats constitutively secreted more fibronectin into culture supernatants than those
derived from female rats [164.6 ± 21.8 versus 84.3 ±
9.2 µg/mg cell protein (P = 0.001) (Figure 1A)]. Cellassociated fibronectin levels demonstrated a similar pattern
of expression (155 ± 20.8 versus 70.4 ± 18.4 µg/mg cell
protein, P < 0.02) (Figure 1A). Consistent with this observation, northern analysis demonstrated that male rat mesangial cells expressed higher baseline fibronectin mRNA levels than female rat mesangial cells (Figure 1B).
When mesangial cells were stimulated with the MCM,
fibronectin levels increased to 303 ± 31.6 µg/mg cell protein in males and 170 ± 31 µg/mg cell protein in females
(P = 0.006) (Figure 2A). However, there was no significant difference in the observed fold increase between the
sexes (3.89 ± 1.2 and 3.13 ± 0.9 in male and female cells,
respectively).
In order to ascertain whether there was a sex-specific
difference in the ability of MCM to induce a response,
mesangial cells were treated with solutions of MCM derived
from macrophages of the opposite sex. The data showed that
there was no sex-specific difference in the potency of the
Rat mesangial cells exhibit sex-specific profibrotic and proinflammatory phenotypes
Fig. 1. (A) Basal fibronectin levels in male versus female mesangial
cells. Basal fibronectin levels were measured in cultured male and female
mesangial cell supernatants and cell lysates. Results are means ± SEM
expressed as µg fibronectin per mg of cell protein. Male cells expressed
higher baseline levels of fibronectin than female cells in both supernatants
(P = 0.001, n = 5) and cell lysates (P < 0.02, n = 5). (B) Autoradiograph of a northern blot showing expression of fibronectin mRNA in
male and female mesangial cells (representative autoradiograph of three
is presented).
male and female MCM to induce fibronectin, implying that
the response was inherent in the mesangial cells themselves
(Figure 2B).
TNFα and IL-1β expression
TNFα and IL-1β are pleiotropic cytokines involved in inflammation and scarring. Basal TNFα (Figure 3A) and
IL-1β (Figure 3B) levels were higher in male than in
female mesangial cells (165 ± 53 versus 82.5 ± 7.3 and
14.3 ± 5.3 versus 10 ± 1.4 pg/mg cell protein, respectively).
MCM stimulation further elevated TNFα (Figure 3A) and
IL-1β (Figure 3B) levels in male and female mesangial cells
(412 ± 96 versus 281 ± 8.5 and 25.9 ± 5.6 versus 17.6 ±
0.8 pg/mg cell protein, for TNFα and IL-1β, respectively).
However, the fold increase in TNFα levels was greater in
female cells than in males (3.4 versus 2.5). There was no
difference in the fold increase between the sexes in IL-1β
levels (1.8 versus 1.7).
Effect of sex hormones on fibronectin levels
Control and MCM-stimulated male and female mesangial cells were treated with 10−8 M 17-β estradiol or
1755
Fig. 2. MCM-stimulated fibronectin levels in male versus female mesangial cells. Mesangial cells were stimulated with MCM derived from
macrophages of the same sex (A) or the opposite sex (B). Fibronectin
levels were measured. Results are means ± SEM expressed as µg fibronectin per mg cell protein. MCM-stimulated fibronectin levels in male
mesangial cells were higher than in female cells (P = 0.006, n = 5).
There was no sex-specific difference in the potency of the male- and
female-derived MCM to induce fibronectin in mesangial cells.
testosterone. 10−8 M 17-β estradiol down-regulated basal
fibronectin levels in female mesangial cells by 16 ±
2.4% (P < 0.001) but had no effect on MCM-stimulated
female cells (Figure 4A). In male cells, estradiol had no
effect on basal fibronectin levels but unexpectedly induced
a small increase in MCM-stimulated cells (17.7 ± 2%,
P < 0.02) (Figure 4A). Testosterone had no effect on basal
fibronectin levels in cells of either sex (Figure 4B). However, fibronectin levels were further increased by 10−8 M
testosterone in MCM-stimulated cells of both sexes (23 ±
6.9%, P < 0.03 and 32 ± 8.55%, P = 0.02 in male and
female cells, respectively) (Figure 4B).
Effect of sex hormones on cytokine levels
Estradiol induced a small reduction in MCM-stimulated
TNFα levels in female cells (10.7 ± 2.7%, P < 0.04).
Testosterone, on the other hand, slightly up-regulated TNFα
levels by 7 ± 0.6% (P < 0.005) in female MCM-stimulated
mesangial cells (Figure 5A). IL-1β levels were elevated by
both estradiol and testosterone in MCM-stimulated female
1756
Fig. 3. TNFα (A) and IL-1β (B) levels in male versus female mesangial cells. TNFα and IL-1β levels were measured in control and MCMstimulated male and female mesangial cells. Results are shown as means ±
SEM (n = 3) expressed as pg cytokine per mg cell protein. Male mesangial
cells expressed higher cytokine levels than female cells.
cells (22 ± 8.6%, P < 0.05 and 25 ± 8%, P < 0.04, respectively) (Figure 5B). Cytokine levels in MCM-stimulated
male mesangial cells were not significantly affected by
treatment with either hormone (Figure 5B).
Discussion
Historically, gender differences in renal disease susceptibility have been attributed to the protective effects of
oestrogen, a hormone with known anti-inflammatory
[19,20] anti-oxidant [21] and anti-fibrotic properties [22].
Investigations in mesangial cells have demonstrated that
estradiol and its metabolites have the potential to be renoprotective inhibiting apoptosis and transforming growth
factor (TGFβ) activity and expression [23], increasing the
expression of extracellular matrix degrading metalloproteinases [24], reducing the synthesis of collagen types I and
IV [15] and inhibiting cell proliferation [16]. In parallel experiments, addition of exogenous testosterone to mesangial
cell cultures has shown a little effect on collagen synthesis
or cell proliferation [16]. Few cell culture studies have assessed inherent differences in male and female cells in the
context of their susceptibility to injury, and to our knowledge no one has compared the effects of gender per se
on renal cell phenotypes. Such differences could, in part,
I. Z. A. Pawluczyk et al.
Fig. 4. Effect of sex hormones on mesangial cell fibronectin levels. Fibronectin levels were measured in male and female mesangial cells treated
with estradiol (A) or testosterone (B). Results are means ± SEM (n = 3)
expressed as µg fibronectin per mg cell protein. E = 17β estradiol, T =
testosterone.
account for the more rapid progression to end-stage renal
disease seen in males with kidney disease.
The current study has demonstrated that male mesangial cells in culture exhibit a more fibrotic phenotype
than female mesangial cells, secreting significantly more
fibronectin, TNFα and IL-1β both constitutively and following injury, suggesting that male chromosomal gender
bestows an inherent propensity to develop a fibrotic phenotype. Cultured lymphocytes from males have similarly
been shown to produce more TNFα and IL-1β than those
from female lymphocytes [25].
Intrinsic differences between male and female mesangial cells may be further modified in vivo by the prevailing hormonal environment. TNFα has been implicated in a
number of chronic renal diseases [26] and is known to be
involved in inducing renal fibrosis. Expression and function of this cytokine are also known to be modulated by
sex hormones. For example, premenopausal women have
lower lipopolysaccharide-induced TNFα levels than men
or postmenopausal women [27]. Furthermore, levels have
been shown to vary with the stage of menstrual cycle [28].
The results from the current study suggest that estradiol
generally reduces and testosterone increases the profibrotic
phenotype of cultured female mesangial cells. Sex hormone
Rat mesangial cells exhibit sex-specific profibrotic and proinflammatory phenotypes
Fig. 5. Effect of sex hormones on mesangial cell TNFα and IL-1β levels.
TNFα (A) and IL-1β (B) levels were measured in male and female mesangial cell supernatants. Results are means ± SEM (n = 3) expressed as pg
cytokine per mg cell protein. E = 17β estradiol, T = testosterone.
effects were less obvious in male mesangial cells although
fibronectin levels in MCM-stimulated cells were increased
by testosterone and to a lesser degree (and unexpectedly) by
estradiol. Metcalfe et al. have previously demonstrated that
testosterone up-regulates TNFα and profibrotic signalling
in animals of both sexes in a model of obstructive renal injury when compared to control females or castrated males
[29]. Other studies have shown that androgens up-regulate
atherosclerotic genes in macrophages from males but not
from females [30]. While oestrogen generally appears to
convey protection, this is not universally the case for all experimental models of renal disease. In diabetic nephropathy and hyperlipidaemia, for example, female gender has
been found to be a risk factor for renal disease [10,31].
Similarly, in vitro, the differential response between male
and female cells may depend on the culture conditions
and the type of stimulus applied. It is clear that the response of experimental animals or cells of different gender
to sex hormones is also a multifactoral process that may
vary according to the cell type and function, and disease
aetiology.
As to the mechanism of the observed gender dimorphism,
it is clear that the sex-specific phenotype is inherent in the
mesangial cells themselves as male cells, outside of the
influence of sex hormones, constitutively produce more
fibronectin than females. The ‘sex’ of the derived MCM
does not appear to play a role as in the cross-over MCM
1757
experiments, where male and female mesangial cells were
exposed to MCM derived from animals of different sexes,
there was no sex-specific difference in the ability of the
MCM to induce fibronectin in mesangial cells. In further
support of this, Huang et al. have shown that there is no
significant difference between cytokine expression levels
in peritoneal macrophages from male and female mice.
Moreover, they found that even though oestrogen decreased,
and progesterone increased cytokine expression levels by
macrophages, there was no significant difference in the
magnitude of the response to sex steroids between male
and female peritoneal macrophages [32].
TGFβ is a fibrogenic growth factor known to be affected by estradiol [23], and estradiol has been shown to
reverse the profibrotic effects of TGFβ in experimental
animal models [19]. However, neither estradiol nor testosterone has previously been shown to affect steady state
TGFβ mRNA levels in mesangial cells [16]. Moreover, we
have previously shown that TGFβ only accounts for 20%
of the fibronectin response of rat mesangial cells to MCM
(as determined by neutralizing TGFβ antibodies) [18]. Increased fibronectin production by male mesangial cells
undoubtedly involves a number of mechanisms, one of
which may involve increased production of a number of cytokines and growth factors acting concert. However, what
specific aspect of the male genotype that results in the production of increased cytokines is yet to be determined.
In our in vitro study, the hormonal environment to which
cells were exposed in culture was regulated. However, we
cannot rule out effects of hormonal preconditioning of the
kidneys in vivo prior to mesangial cell preparation and the
role this may play in the observed effects. Unfortunately,
delineation of these effects is beyond the scope of the current study, although the observations provide a platform for
further investigations. It is of note that others have shown
that male kidneys do not seem to be influenced by lack of
ERα suggesting that the ‘kidney knows its sex’ [33].
Traditionally, culture conditions for studying steroid hormone effects have involved the use of phenol red-free media. This is because a lipophilic impurity contained in the
phenol red preparation has been described as a weak oestrogen agonist in oestrogen sensitive cells [34]. However,
in preliminary experiments we found that there was no significant difference in fibronectin production between cells
grown in phenol red free and phenol red containing RPMI.
Therefore, all of our experiments were carried out in RPMI
containing phenol red.
Other investigators in this field have used charcoalstripped FCS in their culture media. However, charcoal
stripping not only removes sex hormones but also other
polypeptide growth factors whose activity may be required
for normal healthy growth. For instance, charcoal-stripped
sera have been shown to alter proliferation rates of MCF7
cells [35]. In preliminary studies, we found that mesangial
cells are very sensitive to the culture conditions in which
they are grown and we therefore used batch-tested FCS to
ensure consistency.
To our knowledge, this is the first study to directly
compare the differences in profibrotic responses between
male and female mesangial cells. These data would suggest that male rat mesangial cells inherently exhibit greater
1758
profibrotic and proinflammatory characteristics than female rat mesangial cells. However, what specific aspect
of the male genotype results in the profibrotic phenotype
is yet to be determined. This sexual dimorphism in mesangial cells may play a contributory role in the faster rate of
progression of glomerulosclerosis leading to end-stage renal disease in males. The inherent gender phenotypes are
further influenced by circulating sex hormones that play an
additional role in modulating the phenotype of the cells.
Acknowledgements. Parts of this manuscript were presented in abstract
form at the International Society of Nephrology Conference in Singapore
2005. Eddie Tan was supported by a Baxter Fellowship.
Conflict of interest statement. None declared.
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Received for publication: 16.6.08; Accepted in revised form: 27.11.08