Clin Exp Immunol 1999; 117:355–360
Low-density lipoproteins enhance transforming growth factor-beta 1
(TGF-b1) and monocyte chemotactic protein-1 (MCP-1) expression
induced by cyclosporin in human mesangial cells
S. DI PAOLO, G. GRANDALIANO, L. GESUALDO, E. RANIERI & F. P. SCHENA Department of Emergency and
Organ Transplant, Division of Nephrology, University of Bari, Policlinico, Bari, Italy
(Accepted for publication 31 March 1999)
SUMMARY
Cyclosporin (CsA) is widely used in the treatment of renal disease and transplantation, which are often
complicated by alterations of lipid metabolism. Both chronic administration of CsA and hyperlipidaemia have been shown to evoke an early macrophage influx and have progressively led to glomerular and
interstitial sclerosis. MCP-1 is the major monocyte chemoattractant secreted by stimulated mesangial
cells and TGF-b1 is a key mediator of fibrogenesis in chronic progressive renal fibrosis. Thus, the
combined effect of CsA and low-density lipoprotein (LDL) on the gene and protein expression of
MCP-1 and TGF-b1 in cultured human mesangial cells (HMC) was explored. Both agents induced an
early and persistent increase of MCP-1 and TGF-b1 mRNA levels and protein release. The
simultaneous addition of CsA and LDL did not display any additive effect on target gene expression,
but it caused a synergistic effect on MCP-1 and TGF-b1 protein secretion into culture medium. On the
other hand, CsA and LDL had different effects on cell proliferation: the latter increased DNA
synthesis, whereas CsA inhibited both spontaneous and mitogen-stimulated mesangial cell growth.
The study concludes that CsA and LDL display an additive effect on TGF-b1 and MCP-1 synthesis
and release by HMC, thus possibly co-operating to induce an early macrophage influx and the
subsequent mesangial expansion and increased extracellular matrix deposition. However, in contrast
they seem to modulate HMC proliferation differently, which is a further critical event intimately
involved in the development of glomerulosclerosis.
Keywords cyclosporin low-density lipoprotein
factor-beta 1 monocyte chemotactic protein-1
INTRODUCTION
Cyclosporin (CsA) is a cornerstone in the management of organ
transplantation and a number of primary renal and non-renal
disease states as well [1]. A major complication of long-term
therapy is chronic CsA nephropathy which is characterized by
prominent striped interstitial fibrosis, focal and segmental glomerulosclerosis, tubular atrophy and vascular injury [1,2]. The pathogenesis of chronic focal fibrosis remains incompletely elucidated.
Chronic renal vasoconstriction with impaired oxygen delivery has
been suggested to contribute to the genesis of sclerotic lesions
[1,3]. CsA has been shown to directly stimulate collagen transcription and synthesis [4–6] and inhibit proliferation of cultured renal
cells [4,7]. Moreover, in the salt-depleted rat model of chronic CsA
Correspondence to: Professor F. P. Schena MD, Department of
Emergency and Organ Transplant, Division of Nephrology, University of
Bari, Policlinico, Piazza Giulio Cesare, 11-70124 Bari, Italy.
E-mail: fp.schena@nephro.uniba.it
q 1999 Blackwell Science
human mesangial cells
transforming growth
nephropathy, whose features resemble the human lesions, it has
been documented that macrophage infiltration precedes interstitial
fibrosis and may be central to the later development of progressive
fibrotic disease [8].
On the other hand, patients with renal transplant [9] or chronic
renal disease [10] often display abnormalities in plasma lipoproteins and hyperlipidaemia. It has long been accepted that the
described changes in lipid metabolism in renal disease may be a
predisposing factor for atherosclerotic lesions in these patients
[11]. Moreover, there is strong evidence that lipid alterations
may influence the rate of progression of renal disease in humans
[12]. In animal models of renal disease several studies have
demonstrated an association between circulating cholesterol
levels and indices of glomerular injury, including the degree of
glomerulosclerosis, mesangial expansion and hyalinosis [13].
More direct evidence that hyperlipidaemia can contribute to
glomerular injury has been furnished by studies of diet-induced
hypercholesterolaemia in a variety of normal animals. Rats fed
355
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S. Di Paolo et al.
with a cholesterol-supplemented diet for 3 months show glomerular enlargement, mesangial expansion and hypercellularity,
albuminuria, and a modest degree of focal glomerulosclerosis [14].
Of note, the increased mesangial cellularity was in part a result of
an increased influx of macrophages, preceding mesangial expansion and albuminuria.
Consequently, both chronic CsA administration and hyperlipidaemia lead to early macrophage infiltration, followed by
diffuse glomerular and interstitial fibrosis. Since MCP-1 is the
major monocyte chemoattractant secreted by stimulated mesangial
cells [15] and TGF-b1 is a well known fibrogenic factor stimulating the synthesis of different collagens in a wide variety of cell
types, including mesangial cells [16–18], this study has explored
the combined effects of CsA and low-density lipoprotein (LDL) on
the gene and protein expression of MCP-1 and TGF-b1 in cultured
human mesangial cells (HMC), in the attempt to verify whether
CsA and lipids can co-operate to induce glomerular damage.
MATERIALS AND METHODS
Cell culture and characterization
Normal appearing portions of human kidneys surgically removed
for renal carcinoma were used to culture mesangial cells from
outgrowths of collagenase-treated glomeruli. Glomerular mesangial cells were extensively characterized as previously described
[19]. The cells were grown in RPMI 1640 containing 17% heatinactivated fetal bovine serum (FBS), 100 U/ml penicillin,
100 mg/ml streptomycin, 2 mM L-glutamine, 2 mM sodium
pyruvate, 1% (v/v) non-essential amino acids, 5 mg/ml insulin,
5 mg/ml transferrin and 5 ng/ml selenium. All the experiments
included in this study were performed on mesangial cells
between the fifth and the tenth passage from at least four
different cell lines.
Measurement of DNA synthesis
DNA synthesis was measured as the incorporation of methyl-3Hthymidine into trichloroacetic acid (TCA)-insoluble material.
Mesangial cells, plated in 24-well dishes at a density of
4 × 104 cells/well, were grown to confluence and then starved in
serum-free medium for 48 h. LDL (Sigma, Milan, Italy) and/or CsA
(Sandoz, Basel, Switzerland) at the indicated concentrations were
added to quadruplicate wells for 28 h. CsA was first re-dissolved as a
stock solution of 10 mg/ml in absolute ethanol and further diluted in
medium. Control cells received the appropriate amount of solvent
(absolute ethanol) only. During the last 4 h of incubation the cells
were pulsed with 3H-thymidine (1 mCi/ml, SA 70 Ci/mmol; Amersham, Aylesbury, UK). The medium was then removed, the cells
were washed twice in 5% TCA and then incubated in 5% TCA for
5 min. The cells were solubilized by adding 0·75 ml of 0·25 N NaOH
in 0·1% SDS. Aliquots (0·5 ml) were then neutralized and counted
in scintillation fluid using a b-counter.
RNA isolation
Human mesangial cells, plated in 10-cm2 Petri dishes at a density of
2 × 106 cells/dish, were grown to confluence and rested in serum-free
medium for 48 h. LDL (75 mg/ml) and/or CsA (2·5 mg/ml) were then
added for the indicated time periods. At the end of incubation,
mesangial cells were lysed with 4 M guanidinium isothiocyanate
containing 25 mM sodium citrate pH 7·0, 0·5% sarcosyl and 0·1 mM
2-b-mercaptoethanol and total RNA was isolated by the single-step
method, using phenol and chloroform/isoamyl alcohol [20].
Northern blot
TGF-b1 and MCP-1 gene expression were studied by Northern
blotting, as previously described [21]. Briefly, 20 mg of total RNA
from each experimental condition were electrophoresed through a
1% agarose-formaldehyde gel. The gel was transferred to a
nitrocellulose membrane (Schleicher & Schuell, Dassel,
Germany). The membrane was first stained with ethidium bromide
to evaluate the 28S and 18S ribosomal bands and then prehybridized at 428C for 2 h in 50% formamide, 0·5% SDS, 2×
Pipes–NaCl–EDTA buffer and 0·1 mg/ml salmon sperm DNA.
The cDNA probes used were a 2·14-kb fragment encoding the
human TGF-b1, isolated from pBR 327 plasmid with EcoRI, and a
0·7-kb fragment of the baboon MCP-1 cDNA, isolated from
SKþbluescript plasmid with EcoRI and SacI. The two probes
were labelled by random priming using a commercial kit
(Amersham) and 32P-dCTP (S.A.3000 Ci/mmol; Amersham). The
probe (2 × 106 ct/min) was added to 10 ml of prehybridization
solution, and the blots were hybridized for 16 h at 428C in a
buffer containing 50% formamide, 5× SSC, 5× Denhart’s solution,
0·1% SDS, and 100 mg/ml denatured salmon sperm DNA. The
blots were then washed once in 2× SSC, 0·1% SDS at 228C for
5 min, and placed once again in the same buffer at 558C for 30 min.
Finally, the membranes were washed in 1× SSC, 0·1% SDS at 558C
for a further 30 min. After drying, membranes were exposed to a
Kodak X-OMAT film with intensifying screens at ¹708C.
ELISA
Confluent mesangial cells in six-well dishes were starved in serumfree medium for 24 h and then LDL (75 mg/ml) and/or CsA (2·5 mg/
ml) were added in triplicate wells for the indicated time periods. At
the end of incubation the supernatants were harvested, centrifuged
for 10 min at 1000 g to remove the cell debris and stored at ¹808C
until use. MCP-1 measurement in the supernatant was performed
using a commercial human MCP-1 ELISA kit (Quantikine; R&D,
Abingdon, UK). This is a multiple sandwich solid-phase enzyme
immunoassay, which uses MoAbs raised against human MCP-1.
The sensitivity of the ELISA is 5 pg/ml. TGF-b1 protein concentration was measured using human TGF-b1 ELISA system
(Biotrak, Amersham, UK), a multiple sandwich solid-phase
enzyme immunoassay, which uses monoclonal and polyclonal
antibodies raised against human TGF-b1. Latent TGF-b1 was
activated before the assay by heating the samples at 808C for
10 min, as previously described [22]. The lower limit of detection
of the assay is 4 pg/ml. The enzymatic reaction was detected in an
automatic microplate photometer (Titertek; Flow Labs, USA). The
MCP-1 and TGF-b1 concentration of the unknown samples was
determined by interpolation into a standard curve developed with
known amounts of recombinant human MCP-1 and TGF-b1
protein. Protein levels were normalized to cell counts.
Statistical analysis
Data are presented as mean 6 s.d. Data were compared using
ANOVA and Tukey HSD test. P < 0·05 was considered significant.
RESULTS
Effect of CsA and LDL on HMC proliferation
CsA inhibited, in a dose-dependent manner, the proliferation of
HMC grown in serum-free RPMI 1640. DNA synthesis was
reduced to 50% of control cells with CsA concentrations between
1 and 5 mg/ml (Fig. 1a). Trypan blue dye exclusion test, lactic
q 1999 Blackwell Science Ltd, Clinical and Experimental Immunology, 117:355–360
Cyclosporin, LDL and human mesangial cells
Fig. 1. (a) Effect of cyclosporin (CsA) on 3H-thymidine incorporation into
DNA in human mesangial cells. Confluent cells were made quiescent by
incubation in serum-free medium for 48 h, followed by the addition of
increasing concentrations of CsA for a total of 28 h. Cells were pulsed with
3
H-thymidine during the last 4 h of incubation. Data represent the mean
6 s.d. from four experiments performed in quadruplicate. *P < 0·02;
**P < 0·001; ***P < 0·0001. (b) Effect of native low-density lipoprotein
(LDL) on the proliferation of human mesangial cells. *P < 0·05; **P < 0·01;
***P < 0·001; ****P < 0·0001 (n ¼ 4).
dehydrogenase (LDH) assay of cell supernatants as well as the
rapid increase of cell proliferation following the challenge with fresh
10% FBS-supplemented medium demonstrated that the drug was
devoid of any cytotoxic effect on HMC in culture (data not shown).
The addition of increasing concentrations of LDL to quiescent
HMC resulted in a biphasic change in 3H-thymidine incorporation
(Fig. 1b). With concentrations of LDL up to 75 mg/ml DNA
synthesis was increased above baseline by 80%, whereas higher
concentrations of LDL were less stimulatory with 3H-thymidine
incorporation lower than the peak value (Fig. 1b).
Then, the effect of CsA on HMC proliferation induced by
mitogens was evaluated. The addition of CsA blunted 10% FCSstimulated DNA synthesis: 10 mg/ml CsA caused a 50% decrease
of FCS effect (Fig. 2a). Two-hour preincubation of HMC with CsA
prior to the addition of FCS markedly increased such an inhibitory
effect (not shown). LDL-stimulated HMC showed a higher sensitivity to CsA inhibition: 2·5 mg/ml CsA completely abolished the
mitogenic effect of 75 mg/ml LDL and reduced 3H-thymidine
incorporation to baseline values (Fig. 2b).
Thus, 75 mg/ml LDL maximally stimulated HMC proliferation
and 2·5 mg/ml CsA caused at least 50% inhibition of HMC growth,
both spontaneous and LDL-stimulated. Therefore, the above concentrations of LDL and CsA were chosen for all the subsequent
experiments. Of note, such concentrations are compatible with
357
Fig. 2. (a) Effect of cyclosporin (CsA) on fetal calf serum (FCS)-stimulated
human mesangial cells. Confluent cells were serum-deprived for 48 h, then
they were exposed to 10% FCS and increasing concentrations of CsA for
24 h, prior to the addition of 3H-thymidine. *P < 0·0001 versus cell grown
with 10% FCS and without CsA (n ¼ 3). (b) Effect of CsA on human
mesangial cell proliferation stimulated by native low-density lipoprotein
(LDL). Quiescent cells were stimulated with 75 mg/ml LDL in the presence
of various concentrations of CsA. After 24 h, 3H-thymidine was added and
DNA synthesis was measured. *P < 0·05; **P < 0·001; ***P < 0·0001
versus cell grown with 75 mg/ml LDL and without CsA (n ¼ 4). B, Control
cells, grown in the absence of mitogens and CsA.
those achieved by LDL and CsA in vivo (plasma LDL-cholesterol:
approx. 1 mg/ml, corresponding to approx. 200 mg LDL protein/ml;
CsA: approx. 100–200 (trough level) or > 1000 (Cmax) ng/ml
whole blood; the kidney largely exceeds the concentration in
blood [23,24]).
Secretion of TGF-b1 and MCP-1 by HMC
We next tested whether CsA and LDL would cause stimulation of
TGF-b1 and MCP-1 protein in HMC. A sandwich ELISA was used
to measure target proteins in cell culture supernatants 48 h after a
single dose of CsA and/or LDL. The amount of protein measured
was corrected for the number of cells in each sample and expressed
as pg per 105 cells. Figure 3 shows that a single dose of 75 mg/ml
LDL or 2·5 mg/ml CsA led to synthesis and release of TGF-b1 and
MCP-1 from HMC. The simultaneous addition of both agents
resulted in a remarkable additive effect. Of note, TGF-b1 protein
was almost entirely in latent form, since it was detected by ELISA
only after heat activation of culture supernatants.
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S. Di Paolo et al.
Fig. 3. (a) Secretion of TGF-b1 in supernatants from human mesangial
cells exposed to low-density lipoprotein (LDL) and/or cyclosporin (CsA).
Cells were made quiescent by serum deprivation for 48 h, then exposed to
either 75 mg/ml LDL or 2·5 mg/ml CsA or both for 48 h. Culture media were
collected, heat-activated for 20 min and assayed for TGF-b1 by ELISA.
*P < 0·0001; **P < 0·005 versus control (n ¼ 4). (b) Measurement of
MCP-1 released by human mesangial cells stimulated with CsA and/or
LDL for 48 h. MCP-1 protein was measured by ELISA. *P < 0·005;
**P < 0·01; ***P < 0·05 versus control (n ¼ 3).
Effect of CsA and LDL on steady-state TGF-b1 and MCP-1 mRNA
To test whether the observed synthesis of TGF-b1 and MCP-1 was
associated with an increase in steady-state mRNA, Northern blot
analysis of cultured HMC was used. Quiescent HMC expressed
low levels of TGF-b1, which slightly increased over time in
culture. By activating HMC with a single dose of CsA, a two-tothree-fold increase of TGF-b1 mRNA level was observed after 6 h
and continued up to 48 h (Figs 4 and 5). LDL induced a brisk increase
of TGF-b1 transcript after 6 h which slowly declined over time
(Figs 4 and 5). It is noteworthy that by 48 h TGF-b1 mRNA levels
in stimulated HMC were still higher than in untreated cells.
MCP-1 mRNA level increased four-fold after 6 h exposure to
LDL (Figs 4 and 5). However, this initial increase progressively
declined toward baseline at 48 h. In CsA-treated cells, steady-state
MCP-1 transcript peaked at 12 h, and then decreased over time. By
48 h, there were no significant differences between treated and
untreated cells (Figs 4 and 5).
The simultaneous addition of CsA and LDL to HMC seemingly
failed to show any additive effect on either TGF-b1 or MCP-1
steady-state transcript levels (Figs 4 and 5).
DISCUSSION
The present study used human glomerular mesangial cells to
Fig. 4. (a) Upper panel: Northern blot analysis on mRNA isolated from
human mesangial cells grown for different times in serum-free medium
containing either 2·5 mg/ml cyclosporin (CsA) or 75 mg/ml low-density
lipoprotein (LDL) or both, hybridized with a cDNA probe for human TGFb1. Represented is one of four separate experiments. Lower panel: the
ethidium bromide illustration of the membrane shows 18S and 28S
ribosomal bands. (b) Upper panel: representative autoradiograph of a
Northern blot showing the effect of cyclosporin or LDL or both on
steady-state mRNA levels of MCP-1 in human mesangial cells. Similar
results were obtained in four separate experiments. Lower panel: ethidium
bromide illustration of the membrane.
investigate whether CsA and lipids have the potential to synergistically induce glomerular damage.
Both LDL and CsA induced the gene and protein expression of
TGF-b1 and MCP-1 in HMC. The simultaneous addition of both
agents failed to display any additive effect on specific mRNA
levels, whereas it markedly increased the protein synthesis of
TGF-b1 and MCP-1 evoked by either agent. This would suggest
that LDL and CsA seemingly activate the gene expression of
TGF-b1 and MCP-1 through a common intracellular pathway, but
differentially interfere with TGF-b1 and MCP-1 protein synthesis
at a post-transcriptional level.
Perico et al. elegantly demonstrated that chronic administration
of CsA to rats undergoing renal isograft induces segmental
glomerulosclerosis, a lesion comparable to the one reported in
human renal or heart transplants [25]. CsA has been shown to
enhance the synthesis of extracellular matrix proteins by renal cells
in culture, including MC [4–6]. Previous studies demonstrated that
CsA stimulates the expression of TGF-b1 in cultured murine
proximal tubular cells and syngeneic tubulointerstitial fibroblasts
[7] as well as in human T lymphocytes [22,26]. Here it is
demonstrated that CsA is able to induce the expression of
TGF-b1 also in cultured HMC. Since TGF-b has well characterized fibrogenic effects on various renal cells by activating
collagen transcription and inhibiting collagenases, one may
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Cyclosporin, LDL and human mesangial cells
Fig. 5. Densitometric evaluation of TGF-b1 (a) and MCP-1 (b) mRNA in
human mesangial cells stimulated with low-density lipoprotein (LDL) and
cyclosporin (CsA). The hybridization signals were quantified by scanning
of autoradiograms, normalized to 18S ribosomal RNA band and expressed
as fold increase over control (¼ unstimulated cells). A, 75 mg/ml LDL; B,
2·5 mg/ml CsA; hatched bars, LDL þ CsA. *P < 0·05; **P < 0·01;
***P < 0·001 (n ¼ 4).
surmise that CsA-associated fibrosis could be mediated, at least in
part, by endogenous TGF-b synthesis. Salt-depleted rats chronically given CsA displayed a dramatic increase of kidney matrix
proteins, together with mRNA expression of TGF-b1 and PAI-1, a
protease inhibitor stimulated by TGF-b1, which further supported
the role of TGF-b1 as a key fibrogenic cytokine involved in the
development of chronic CsA nephropathy, by enhancing extracellular matrix deposition and inhibiting its degradation [27].
Mesangial matrix expansion occurs in lipid-mediated glomerular injury, and anti-lipidaemic therapy reduces the degree of
mesangial expansion [12]. In vitro, LDL stimulates collagen IV
production [28] and fibronectin synthesis [29,30] in cultured
mesangial cells. Studer and colleagues reported an increase of
TGF-b bioactivity in the medium of rat MC cultured with LDL for
24–48 h [30]. The experiments reported here demonstrate that
LDL directly stimulates the gene and protein expression of
TGF-b1 by cultured HMC, similar to the effect described by Ding
et al. in human glomerular epithelial cells [31]. Furthermore, the
present study demonstrates for the first time that the simultaneous
exposure of HMC to CsA and LDL markedly increases TGF-b1
protein synthesis induced by either agent. Recently, one study
investigated the effect of dietary cholesterol on CsA nephrotoxicity in the rat. Cholesterol feeding significantly aggravated CsAinduced renal function impairment and enhanced the renal
expression of PAI-1 induced by CsA treatment [32].
Animals fed with a cholesterol-supplemented diet display
expanded mesangial matrix and increased mesangial cellularity,
which seems to result from an increased influx of macrophages as
359
well as from glomerular MC proliferation [12,33]. Rovin & Tan first
described a dose-dependent increase of MCP-1 in MC exposed to
LDL-cholesterol, thus suggesting one mechanism whereby hyperlipidaemia can cause the recruitment of macrophages and subsequent glomerular injury [29]. One novel finding of our study is the
ability of CsA to induce the expression of MCP-1 in cultured HMC.
Recently, Young et al. studied a rat model of chronic CsA nephrotoxicity and found that a significant macrophage influx precedes the
development of cortical interstitial fibrosis and afferent arteriolar
hyalinosis [8]. Our data lend support to the hypothesis that CsA,
similar to LDL, may induce an early influx of macrophages through
the stimulation of MCP-1 gene and protein expression by HMC.
However, LDL and CsA appear to display divergent effects on
HMC proliferation: the former seems to stimulate cell growth,
whereas the latter has been shown to inhibit DNA synthesis by
HMC, both in the absence and in the presence of mitogens (fetal
calf serum (FCS) or LDL). Recently, Wolf et al. suggested that
intrarenal synthesis and release of TGF-b1 may explain CsAinduced growth arrest, since the growth factor is a well known
growth inhibitor for various renal cells [7]. This hypothesis,
although attractive, does not seem fully to explain the actions of
CsA on cell cycle. Indeed, LDL enhances HMC proliferation in
spite of its ability to induce the synthesis and release of TGF-b1.
Golay et al. found that CsA and TGF-b1 independently inhibit B
cell proliferation by differential inhibition of the induction of
proto-oncogenes, which probably code for transcription factors
[34]. Exposure of HMC to native LDL consistently increased
mRNA encoding two transcription factors, c-fos and c-jun, as
well as platelet-derived growth factor (PDGF) A and B chain,
which, in turn, enhanced the proliferative effect of LDL [35]. It
may then be supposed that CsA inhibits LDL-stimulated HMC
proliferation by directly interfering with intracellular activation of
immediate early genes and growth factors. On the other hand, the
possibility that CsA anti-proliferative effect could be simply
ascribed to the increase of LDL-induced release of TGF-b1 protein
cannot be ruled out.
In conclusion, CsA and LDL display an additive effect on
TGF-b1 and MCP-1 synthesis and release by HMC, and may thus
co-operate to induce an early macrophage influx and the subsequent mesangial expansion and increased extracellular matrix
deposition. However, they seem to modulate HMC proliferation
differently, which is a further critical event intimately involved in
the development of glomerulosclerosis.
ACKNOWLEDGMENTS
This study was partly supported by the Associazione Progresso Scientifico
Nefrologia e Trapianto (APSNT), the Baxter Extramural Program Grant (eight
round 1995–98), the Istituto Superiore di Sanità (96.7019) and by grants from
the Ministero dell’ Universita’ e della Ricerca Scientifica e Tecnologica (40%:
96.7404 and 60%: 95.3957, 96.8187). A preliminary report of these data was
presented at the meeting: New Dimensions in Transplantation, held in
Florence (Italy), February 16–19, 1998 (Transplant Proc 1998; 30: 2051).
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