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Division of Nephrology, Fundación Jiménez Díaz, Universidad Autonoma de Madrid, Spain.
Correspondence to Dr. Alberto Ortiz, Unidad de Diálisis, Fundación Jiménez Díz, Avda Reyes Católicos 2, 28040 Madrid, Spain. Fax : +34 915 494 764 ; E-mail : aortiz{at}fjd.es
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, interferon-
, and lipopolysaccharide)
increased cell surface Fas expression and were sensitized to apoptosis induced
by FasL (FasL 55 ± 5% versus control 8.3 ± 4.1%
apoptotic cells at 24 h, P < 0.05). Cytokine-primed primary
cultures of tubular epithelial cells also acquired sensitivity to FasL-induced
apoptosis. These results suggest that FasL expression by intrinsic renal cells
may play a role in cell homeostasis in the normal kidney and during renal
injury. |
Introduction |
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Most studies on the biologic activity of FasL have relied on the usage of activating anti-Fas antibodies. It has recently become evident that the complex regulation of FasL-Fas apoptosis involves, among other factors, different biologic activities for soluble and membrane-bound FasL (17). Both soluble FasL and agonistic anti-Fas antibodies are less effective than membrane-bound FasL in inducing apoptosis (17). Thus, cells such as freshly isolated human peripheral blood T lymphocytes, previously thought to be resistant to FasL-induced cell death, have now been found to be sensitive to membrane-bound FasL (17).
We have now studied the site of FasL expression in normal and injured kidneys in vivo, the ability of renal tubular cells to express an active form of FasL, and the sensitivity of these cells to recombinant FasL-induced apoptosis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Fas-sensitive A20 and Fas-resistant A20R murine B lymphoma cells were a kind gift from Jürg Tschopp (Lausanne University, Switzerland) (9). L1210 (Fas-negative) and L1210Fas (Fas-transfected) murine leukemia cells were a kind gift from Alberto Anel (Universidad de Zaragoza, Spain) (20).
Polyclonal rabbit anti-FasL antibodies raised against a peptide corresponding to amino acids 2 to 19 mapping at the amino terminus of FasL (13, and the control peptide were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). PE62 polyclonal rabbit anti-FasL antibody was a gift from J. Tschopp (9). The rat IgM monoclonal A11 that binds to the FasL extracellular peptide 196-220 was from Alexis (Läufelfingen, Switzerland) (9), control rat IgM and biotin-anti-rat IgM was from ImmunoKontact (Frankfurt, Germany), and streptavidin phycoerythrin was from Calbiochem (San Diego, CA). Neutralizing anti-FasL antibodies (clone MFL3 ; Pharmingen, San Diego, CA) were used at a concentration of 10 µg/ml (21). All of these antibodies bind to murine FasL (9, 13), and neutralizing MFL3 antibodies inhibit the cytotoxicity of FasL from all strains of mice tested, including SJL mice (21).
Recombinant human FasL (Alexis) was used in the presence of a 10-fold excess of a cross-linking antibody, which by itself was devoid of lethal activity. Cross-linking of FasL restores the biologic activity of soluble FasL and simulates its presence on the cell membrane (22). Human FasL activates the murine Fas receptor (22).
Models of Renal Injury
Inbred female Wistar rats and albino Swiss mice were obtained from the
Fundación
Jiménez Díaz
animal facilities. Studies were conducted in accord with the National
Institutes of Health Guide for the Care and Use of Laboratory Animals. Immune
complex-mediated proliferative glomerulonephritis was induced in rats that
were previously immunized with ovalbumin (Sigma, St. Louis, MO), by daily
intraperitoneal administration of 10 mg of ovalbumin
(23). Samples from rats with
nephritis (n = 3) were obtained when glomerular injury was fully
developed (proteinuria >100 mg/d).
C57BL/6 (B6) and Balb/c mice were obtained from Iffa-CREDO (Barcelona, Spain). Four-week-old MRL-lpr/lpr mice without nephritis (n = 3) and 4-mo-old MRL-lpr/lpr mice with nephritis (n, = 3) were a gift from Carlos Martinez-A (Centro Nacional de Biotecnología, Madrid, Spain). Kidney samples were snap-frozen for RNA and protein studies or fixed in 10% buffered formaldehyde and embedded in paraffin for histologic studies.
Studies of Cell Death
Tubular epithelial cells were cultured in 12-well plates. Cells were
cultured in serum-free media in the presence or absence of 30 ng/ml murine
tumor necrosis factor- (TNF-) (Immugenex, Los Angeles, CA), 300
U/ml human interferon- (IFN-) (Immugenex), and 10 µg/ml
bacterial lipopolysaccharide (LPS) (Sigma) for 48 h, with FasL added for the
last 24 h. When specified, some cells were also cultured in the presence of
10% FCS. Hypodiploid apoptotic cells were quantified by flow cytometry of
permeabilized, propidium iodide-stained cells, as described previously
(14). For morphologic
assessment of apoptosis, cells were cultured in chamber slides, fixed with
methanol : acetone (1:1), and stained with propidium iodide in the presence of
RNase A (24).
Reverse Transcription-PCR and Northern Hybridization
Kidney total RNA was isolated using the acid guanidinium-phenol chloroform
method (25), and 40 µg was
separated in 1% agarose gels containing 2.3% formaldehyde. MCT poly(A) RNA was
isolated using the FASTRACK mRNA kit (Invitrogen, San Diego, CA) and 5 µg
was loaded. RNA was transferred to nylon membranes (Genescreen, New England
Nuclear, Boston, MA) and prehybridized and hybridized as described previously
(16).
Reverse transcription (RT)-PCR for mouse FasL detection was carried out as described previously (16), using the following primers : 5'-CACAAATCTGTGGCTACCG, 3'-GCCCATATCTGTCCAGTAG. The resulting PCR product was cloned and sequenced to confirm its identity. It expands several exons, thus excluding the possibility of amplification of genomic DNA. The following primers were used for amplification of rat FasL : 5'-CATAGAGCTGTGGCTACC, 3'-ATGGTCAGCAACGGTAAC. The PCR product had 534 bp. Semiquantitative PCR was performed in rat and mouse kidney samples according to published techniques (26). FasL and G3PDH were amplified in the presence of 32P-dCTP (Amersham). The PCR products were separated in 4% polyacrylamide/urea gels, and bands were measured by densitometry and corrected for G3PDH. The optimal number of amplification cycles was chosen on the basis of experiments that established the exponential range of the reaction.
The murine FasL probe has been described previously (16, 27). Sense and antisense RNA probes for in situ hybridization were labeled with digoxigenin using a commercially available kit (DIG RNA labeling kit ; Boehringer Mannheim, Mannheim, Germany), following the manufacturer's instructions.
Flow Cytometry Analysis of FasL and Fas Expression
To study cell surface, FasL cells were cultured for 3 h in the presence of
5 mM ethylenediaminetetra-acetic acid (EDTA), which inhibits metalloproteases
and decreases the release of FasL from the cell membrane, thus increasing the
availability of this cytokine at the cell surface
(28). Cells were washed with
phosphate-buffered saline (PBS) and resuspended in 0.2% bovine serum
albumin/PBS. A total of 5 x 105 cells was incubated with 20
µg/ml monoclonal All anti-FasL antibody or control rat IgM in 5% FCS, 0.2%
bovine serum albumin, in PBS for 30 min at 4°C, followed by incubation
with 1 : 100 biotin-anti-rat IgM for 30 min at 4°C and by incubation with
1 : 50 streptavidin phycoerythrin. Cells were analyzed on a cytofluorograph,
and debris was excluded from analysis by selective gating based on anterior
and right angle scatter. At least 10,000 events were collected for each
sample, and data were displayed on a logarithmic scale of increasing
fluorescence intensity (14).
Mean cell fluorescence was calculated using LYSIS II software.
To study cell surface Fas expression, cells were cultured in the presence of control medium or cytokines for 48 h and stained with Jo-2 anti-murine Fas antibody or control hamster IgG (Pharmingen), as described (14).
Immunohistochemistry
Immunohistochemistry was carried out as described previously
(29) in paraffin-embedded
tissue sections 5 µm thick. Primary antibodies were 20 µg/ml All rat
monoclonal anti-FasL (Alexis) or 1 µg/ml rabbit polyclonal anti-FasL (Santa
Cruz) (13). Sections were
counterstained with Carazzi's hematoxylin. Some sections were subsequently
incubated with the proximal tubule marker, fluorescein-conjugated,
Tetranogolobus lotus (diluted 1:33) (Sigma) or the collecting tubule
marker, fluorescein-conjugated, Dolichos biflorum (diluted 1:100)
(Sigma) (30). These sections
were mounted in 90% glycerol/PBS and photographed immediately. Negative
controls included incubation with a nonspecific Ig of the same isotype as the
primary antibody in both cases, and competition of the primary antibody with a
10-fold excess of the immunogenic peptide in the case of the rabbit
polyclonal. Positive controls included testis
(7) and eye
(13).
In Site Hybridization
Paraffin-embedded tissue sections, 5 µm thick, were fixed in 1.5%
paraformaldehyde-1.5% glutaraldehyde for 10 min, treated with 5 mM levamisole
for 30 min, and deproteinized with 0.2N HCl for 20 min at room temperature
followed by digestion with 25 µg/ml proteinase K in 0.1 M Tris, 0.005 M
EDTA, 0.5% sodium dodecyl sulfate for 30 min at 37°C. They were
subsequently hybridized with 1 ng/ml denatured digoxigenin-11-UTP-labeled FasL
riboprobe in hybridization solution (2x SSC, 1x Denhardt's, 0.1 M
sodium phosphate, pH 6.5, 10% dextran sulfate, 40% deionized formamide, 24 mM
vanadyl ribonuclease complex, and 0.5 µg/ml yeast tRNA) at 42°C
overnight, under sealed coverslips, in a humidified chamber. Slides were
washed in 2x SSC for 5 min and 0.2x SSC for 3 min, and incubated
with 1:750 alkaline phosphatase-conjugated anti-digoxigenin antibody
(Boehringer Mannheim) for 60 min at 37°C. Colorimetric detection of mRNA
was performed with nitroblue tetrazolium and
5-bromo-4-chloro-3-indolylphosphate (X-phosphate) in the dark for 10 to 30
min. Negative controls consisted of matched serial sections hybridized without
RNA probe or with sense probe or pretreated with 25 µg/ml RNase A for 1 h
(Sigma) before hybridization with the corresponding antisense probe. Testis
samples were included as positive controls.
Western Blot
Tissue and cell samples were homogenized in lysis buffer (50 mM Tris-HCl,
150 mM NaCl, 2 mM EDTA, 2 mM ethyleneglycol-bis(ß-aminoethyl
ether)-N,N' -tetra-acetic acid, 0.2% Triton X-100, 0,3%
Nonidet-P40, 0.1 mM phenylmethylsulfonyl fluoride, and 1 µg/ml pepstain A)
and then separated by 12% sodium dodecyl sulfate-polyacrylamide gel
electrophoresis under reducing conditions. After electrophoresis, samples were
transferred to polyvinylidene difluoride membranes. The membranes were blocked
with 5% skim milk in PBS/0.5% vol/vol Tween 20 for 1 h, washed with PBS/Tween,
and incubated with 40 ng/ml PE 62, a polyclonal anti-FasL antibody or
polyclonal anti-murine Fas (1:500) (Santa Cruz), in 5% milk PBS/Tween for 18 h
at 4°C. The blots were washed with PBS/Tween and subsequently incubated
with horseradish peroxidase-conjugated antirabbit IgG (1:2500 ; Amersham).
After washing with PBS/Tween, the blots were developed with the
chemiluminescence method according to the manufacturer's instructions
(Amersham). FasL (a gift from J. Tschopp)-transfected 293 cell lysates were
diluted 10-fold and used as positive controls. As a negative control, the
antibodies were preincubated with a 10-fold excess of the immunogenic peptide
before Western blotting. Similar results were obtained with a polyclonal
anti-FasL antibody from Santa Cruz. Tubulin was used as loading control.
Cytosolic and membrane proteins were obtained by lysing the cells in 2% Triton X-114 precondensed in PBS. After phase separation at 37°C, the lower phase enriched in detergent contains the membrane proteins, and the upper aqueous phase contains the cytosolic proteins. Both phases were separated, precipitated with methanol/chloroform, and resuspended in sample buffer (22).
FasL Bioactivity
FasL killing activity was assessed by incubating 1 x 105
to 1 x 102 effector MCT cells, 6 h after seeding, with 1
x 105 bromodeoxyuridine-labeled target A20R and A20 cells
(9) or L1210 and L1210Fas cells
(20) in 48-well plates. Higher
effector : target (E : T) ratios were used in preliminary experiments, but
were abandoned because tubular cells became overconfluent at E : T ratios
>1. The release of bromodeoxyuridine was then determined after a 14-h
coincubation using a cellular DNA fragmentation enzyme-linked immunosorbent
assay (Boehringer Mannheim). As a positive control, MCT cells were replaced by
10 ng/ml recombinant FasL. For the calculation of the percentage of dead
cells, cell death induced in Fas-sensitive cells by recombinant FasL was
considered to be 100%. Flow cytometry confirmed more than 90% apoptosis among
A20-Fas-sensitive cells treated with FasL. Results are expressed as mean
± SEM of independent experiments each consisting of quadruplicate
wells.
Statistical Analyses
Results are expressed as mean ± SEM. Significance at the 95% level
was established using ANOVA and t test. Bonferroni correction for
multiple comparisons was used.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Increased Renal FasL and de Novo Glomerular FasL Expression during
Glomerular Inflammation
We then addressed the question of whether FasL expression changed in the
course of renal inflammation in rodent models. FasL mRNA, as assessed by
semiquantitative RT-PCR, was increased in whole kidneys from rats with
immune-complex proliferative glomerulonephritis and mice with lupus nephritis
(Figure 4). In the course of
murine lupus nephritis, both full-length FasL and an approximately 26-kD
molecule corresponding to the size of soluble FasL were increased
(Figure 4E).
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The pattern of FasL expression also changed during glomerular and tubulointerstitial inflammation. De novo FasL expression was noted in the glomerular mesangium, and FasL immunoreactivity was also prominent in tubular cells and among infiltrating interstitial cells in MRL lpr/lpr mice with lupus proliferative glomerulonephritis and tubulointerstitial nephritis (Figure 5).
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FasL mRNA and protein are also limited to tubular epithelial cells in normal rat kidney (Figure 6). As was the case during mouse renal inflammation, in rats with immune complex proliferative glomerulonephritis, FasL mRNA and protein were expressed de novo by glomerular cells (Figure 6).
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Murine Cultured Tubular Epithelial Cells Express FasL
Proximal tubular epithelial cells were the main source of renal FasL in
vivo. We then studied FasL expression in cultured tubular epithelial
cells. FasL mRNA was detected in cultured murine proximal tubular epithelial
MCT cells by RT-PCR, Northern blot, and in situ hybridization
(Figures 7 and
8). RT-PCR also identified a
FasL transcript in primary cultures of tubular epithelial cells
(Figure 7B). In situ
hybridization showed that FasL mRNA expression was higher in MCT cells grown
at low cell density than when cells were confluent
(Figure 8). Western blot
confirmed the presence of a 36- to 40-kD FasL protein in these cells, which,
after Triton X-114 extraction, appeared in the membrane fraction
(Figure 9A), as corresponds to
the full-length transmembrane form of FasL. As was the case for mRNA,
nonconfluent cells expressed more FasL protein than confluent cells
(Figure 9A), indicating a
regulatory process that deserves further study. Flow cytometry showed that low
levels of FasL protein are present in the cell surface, where they can be
biologically active (mean cell fluorescence 30% higher than control isotype
antibody-stained cells) (Figure
9B). The low amount of FasL in the cell surface is consistent with
data from other cell types
(14,
31), where most FasL is bound
to intracellular membranes
(31).
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A 36- to 40-kD FasL protein was also present in the membrane fraction of primary cultures of mouse tubular epithelial cells (Figure 9C).
Tubular Epithelial Cell FasL Is Biologically Active
Not all FasL-expressing cells are able to promote apoptosis
(11). Thus, we tested the
ability of tubular epithelial MCT cell FasL to promote apoptosis. In
vitro assays using two different sets of Fas-sensitive and Fas-resistant
cells (A20 and A20R, and L1210Fas and L1210) demonstrated that tubular
epithelial cell-derived FasL is able to induce cell death in lymphoid cells.
A20R cells were generated by continuous culture in the presence of FasL, and
have downregulated Fas receptor expression
(22). By contrast, L1210Fas
were made sensitive to FasL apoptosis by transfecting them with a Fas
expression vector (20).
Results obtained in A20/A20R cells are shown in
Figure 10. An increased rate
of cell death was noted among Fas-sensitive A20 cells cocultured with tubular
epithelial cells, which was not present among Fas-resistant A20R cells under
the same culture conditions. The apoptotic nature of death was confirmed by
morphologic evaluation of the cells. Similar results were obtained with
Fas-sensitive L1210Fas and Fas-resistant L1210 cells (not shown). Apoptosis
induced by tubular epithelial cells in A20 cells was reduced by 73% when
neutralizing anti-FasL antibodies were added to the coculture
(Figure 10).
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FasL Induces Apoptosis in Activated Tubular Epithelial Cells
Tubular epithelial cells have been reported to be relatively resistant to
Fas-induced death (15,
16). However, Fas deficiency
protects tubular epithelial cells from apoptosis in the course of renal
ischemia-reperfusion (6). This
suggests that sensitivity to FasL in tubular epithelial cells is a regulated
process. It has been observed that the survival factors present in serum
protect tubular epithelial cells from the lethal effect of TNF-
(32). Indeed, tubular
epithelial MCT cells were completely resistant to FasL-induced apoptosis when
grown in the presence of serum (Figure
11A). In addition, nonstimulated MCT cells grown in serum-free
media were still relatively resistant to FasL-induced apoptosis. Only the
highest concentration of FasL induced a significant, albeit low, amount of
apoptosis (Figure 11A) (100
ng/ml FasL 12.8 ± 2.4% versus control 3.2 ± 1.0%
apoptotic cells at 24 h, P < 0.05). Under these conditions, MCT
cells were at least 100-fold more resistant to recombinant FasL than the
Fas-sensitive lymphoid cell lines used in our studies, as 1 ng/ml FasL already
induced significant apoptosis in these lymphoid cell lines. As expected from
these data, neutralizing anti-FasL antibodies did not modify spontaneous
apoptosis in tubular epithelial cells after 24 or 48 h in culture in
serum-free media (data not shown). However, during renal injury tubular
epithelial cells are exposed to inflammatory mediators. Thus, we studied their
sensitivity to FasL in the presence of a cocktail of inflammatory mediators
(30 ng/ml TNF-, 300 U/ml IFN-, and 10 µg/ml bacterial LPS).
Upon activation by inflammatory mediators, the sensitivity of MCT cells to
apoptosis induced by FasL was increased : 10 ng/ml FasL already increased the
rate of apoptosis, and the death rate of cells exposed to 100 ng/ml FasL was
6.6-fold that of control cells (Figure
11A). The occurrence of apoptosis was confirmed by morphologic
criteria (fragmented, condensed, and pyknotic nuclei) of fixed, propidium
iodidestained cells (not shown). MCT cells activated by these inflammatory
mediators have an upregulated expression of Fas receptor
(Figure 11, B and C), which
can contribute to their increased susceptibility to FasL-mediated apoptosis.
Serum deprivation also increased Fas expression
(Figure 11B), as it does in
other renal cells (14).
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We then checked the sensitivity of primary cultures of tubular epithelial
cells to FasL-induced apoptosis. Tubular cells were resistant to FasL under
basal conditions ; however, they were sensitized by prestimulation with 30
ng/ml TNF-, 300 U/ml IFN-, and 10 µg/ml bacterial LPS
(Figure 11D). Similar results
were obtained with freshly isolated mouse tubules (not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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FasL-induced apoptosis is a complex phenomenon. Not all FasL-expressing cells can kill FasL-sensitive cells (11,33). In leukocytes, FasL is stored in the membranes of intracellular granules and only becomes exposed in the cell surface, where it has killing activity, upon cell activation (31,33,34). Cultured tubular epithelial cells contain full-length FasL in cell membranes, and the amount of FasL is subject to regulation by cell density. Although additional studies are required to characterize the regulation of FasL expression in tubular epithelium, flow cytometry showed that tubular cell surface FasL was low. The low cell surface expression of FasL is also observed in other FasL-expressing cells (14,31), and is consistent with the potentially dangerous lethal activity of high concentrations of cell surface FasL. However, cell surface FasL from cultured tubular epithelial cells was able to kill lymphoid target cells. The biologic activity was similar to that of melanoma cells expressing FasL (9). The lower killing activity at higher MCT cell effector cell density could be attributed to decreased FasL expression under these culture conditions. Expression of FasL by tumor cells is thought to limit the immune response against the tumor (9, 10). We might hypothesize that tubular cell FasL keeps in check the renal immune response. Indeed, local FasL expression by epithelial cells has been implicated in immune privilege in the eye and testis (12, 13). Nevertheless, this latter view has been called into question (35), and transgenic expression of FasL by islet ß cells did not protect them against rejection (36).
Renal FasL was increased in two models of renal inflammation : murine lupus
nephritis and rat immune complex-mediated proliferative glomerulonephritis. In
addition to tubular epithelial cells, local sources of FasL during
tubulointerstitial renal injury include infiltrating leukocytes and
interstitial fibroblasts (16).
Indeed, we noted that interstitial cells expressed FasL in lupus mice with
combined glomerular and tubulointerstitial nephritis. Moreover, de
novo glomerular FasL expression was observed in immune complex-mediated
proliferative glomerulonephritis in mice and rats. The cellular origin of
glomerular FasL is not clear from our studies. Infiltrating T cells or
macrophages may be sources of FasL in this model
(23). However, cultured
glomerular mesangial cells also express FasL transcripts, and inflammatory
cytokines released during glomerular injury, such as TNF-, increase
mesangial cell FasL mRNA levels
(16).
During renal damage, FasL may promote apoptosis of renal parenchymal cells. In this sense, glomerular mesangial cells undergo apoptosis when challenged with agonistic anti-Fas antibodies both in vitro and in vivo (4, 5). By contrast, nonstimulated tubular epithelial cells of human or murine origin have been reported to be resistant to cell death induced by agonistic anti-Fas antibodies (15, 16). This feature is shared by murine tubular epithelial cells in vivo, as systemic injection of agonistic anti-Fas antibodies induced apoptosis in glomerular cells but not in tubular epithelium (4). Recombinant FasL is a more potent inducer of apoptosis than agonistic anti-Fas antibodies (17), and cells that have been previously reported to be resistant to anti-Fas-induced apoptosis were later found to be sensitive to FasL-induced apoptosis (17). Our present studies confirm that nonstimulated murine tubular epithelial cells are quite resistant not only to anti-Fas antibodies (16), but also to recombinant, cross-linked FasL when grown in the presence of survival factors. Even when the survival factors present in serum were removed, only high concentrations of FasL increased significantly, although mildly, the rate of apoptosis in MCT tubular epithelial cells. This fact, together with the low spontaneous apoptotic rate of cultured tubular epithelial cells, suggests that under basal conditions FasL does not act as an autocrine death factor. Indeed, neutralizing anti-FasL antibodies did not modify the rate of spontaneous apoptosis in these cells. This is also the case for other cell types with constitutive expression of FasL (2).
Several factors may contribute to the relative resistance of tubular
epithelium to FasL-induced death. The amount of cell surface Fas receptor is
one of them. Indeed, the basal expression of Fas is low in tubular epithelial
cells, below the limit of detection of the flow cytometry technique we used.
Inflammatory stimuli that increase Fas expression prime mesangial cells to
undergo apoptosis induced by anti-Fas antibodies
(16). In this regard,
increased Fas expression has been noted in tubular epithelial cells in the
ischemia-reperfusion model of renal injury and during chronic tubular atrophy
(6,
37). Under these
circumstances, tubular epithelial cells may undergo apoptosis upon Fas
stimulation, in a manner analogous to other FasL-expressing epithelia, such as
thyrocytes, during thyroid inflammation
(2,
37). Indeed, Fas deficiency
protected tubular epithelial cells from apoptosis induced by renal
ischemia-reperfusion (6).
In vitro, a combination of inflammatory mediators that may be present
in the kidney during renal injury increased tubular epithelium Fas expression
and sensitivity to FasL-induced apoptosis both in MCT cells and primary
cultures of tubular epithelial cells. By contrast, the survival factors
present in serum decreased tubular cell Fas and protected against FasL-induced
apoptosis. In addition to the amount of Fas receptors, endogenous
intracellular proteins may protect from Fas-induced death, as inhibition of
protein synthesis promoted anti-Fas-induced apoptosis in nonstimulated
cultured human tubular epithelial cells
(15). Candidate proteins
include members of the Bcl2 family of anti-apoptotic proteins, such as BclxL.
Constitutive expression of BclxL by tubular epithelial cells and its absence
from glomerular cells (38,
39) may be one of the factors
that contributes to their different susceptibility to FasL apoptosis in
vivo. Indeed, both TNF- and serum deprivation decreased BclxL
levels in tubular epithelial cells
(32).
It is noteworthy that both full-length transmembrane FasL as well as the soluble form of the cytokine were increased in murine lupus nephritis. Local expression in the cell surface is important for the ability of FasL to induce cell death, as the apoptotic-inducing capacity of naturally, enzymatically processed soluble FasL is reduced by >1000-fold compared with cell surface transmembrane FasL (22). Soluble FasL may even function as a death antagonist, by competing with the more active cell membrane FasL (17). The presence of soluble FasL during renal inflammation may limit the lethal activity of transmembrane FasL. Indeed, in contrast to results in acute renal failure (6), in the course of lupus nephritis Fas deficiency did not protect tubular epithelial cells from apoptosis (40).
We have explored the relationship between renal FasL and apoptosis. However, FasL may have additional functions. Recent evidence suggests that soluble FasL can promote neutrophil chemotaxis (41). In addition, FasL itself can transduce intracellular signals (42). It is currently unknown whether this is another pathway for FasL to influence tubular epithelial cell biology.
In summary, FasL is expressed by tubular cells in normal kidney and de novo in the glomeruli during renal injury. Renal cell FasL promotes apoptosis of lymphoid cells. It had been previously demonstrated that Fas activation induced apoptosis of mesangial cells in vitro and in vivo. We now show that while under basal conditions tubular epithelial cells are relatively resistant, recombinant FasL induces apoptosis of tubular epithelial cells activated by inflammatory stimuli. Taken together, these results suggest that FasL and Fas play a role in normal kidney homeostasis and renal injury that deserves further study.
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Acknowledgments |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 P. Justo, A. B. Sanz, J. Egido, and A. Ortiz 3,4-Dideoxyglucosone-3-ene Induces Apoptosis in Renal Tubular Epithelial Cells Diabetes, August 1, 2005; 54(8): 2424 - 2429. [Abstract] [Full Text] [PDF]
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C. Lorz, P. Justo, A. Sanz, D. Subira, J. Egido, and A. Ortiz Paracetamol-Induced Renal Tubular Injury: A Role for ER Stress J. Am. Soc. Nephrol., February 1, 2004; 15(2): 380 - 389. [Abstract] [Full Text] [PDF]
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P. Justo, C. Lorz, A. Sanz, J. Egido, and A. Ortiz Intracellular Mechanisms of Cyclosporin A-Induced Tubular Cell Apoptosis J. Am. Soc. Nephrol., December 1, 2003; 14(12): 3072 - 3080. [Abstract] [Full Text] [PDF]
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 L. M. Blanco-Colio, B. Munoz-Garcia, J. L. Martin-Ventura, C. Lorz, C. Diaz, G. Hernandez, and J. Egido 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Inhibitors Decrease Fas Ligand Expression and Cytotoxicity in Activated Human T Lymphocytes Circulation, September 23, 2003; 108(12): 1506 - 1513. [Abstract] [Full Text] [PDF]
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 C. Dai, J. Yang, and Y. Liu Transforming Growth Factor-beta 1 Potentiates Renal Tubular Epithelial Cell Death by a Mechanism Independent of Smad Signaling J. Biol. Chem., March 28, 2003; 278(14): 12537 - 12545. [Abstract] [Full Text] [PDF]
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M. P. CATALAN, A. REYERO, J. EGIDO, and A. ORTIZ Acceleration of Neutrophil Apoptosis by Glucose-Containing Peritoneal Dialysis Solutions: Role of Caspases J. Am. Soc. Nephrol., November 1, 2001; 12(11): 2442 - 2449. [Abstract] [Full Text] [PDF]
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 H. A. Manley and V. A. Lennon Endoplasmic Reticulum Membrane-sorting Protein of Lymphocytes (BAP31) Is Highly Expressed in Neurons and Discrete Endocrine Cells J. Histochem. Cytochem., October 1, 2001; 49(10): 1235 - 1244. [Abstract] [Full Text] [PDF]
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S. K. Jo, S. Y. Yun, K. H. Chang, D. R. Cha, W. Y. Cho, H. K. Kim, and N. H. Won {alpha}-MSH decreases apoptosis in ischaemic acute renal failure in rats: possible mechanism of this beneficial effect Nephrol. Dial. Transplant., August&nb |
