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* Department of Medicine, Division of Nephrology, University of Freiburg, Freiburg, Germany;
Department of Laboratory Medicine and
Institute for Clinical Pathology, University of Vienna, Vienna, Austria; and
Academic Renal Unit, Medical School Building, Southmead Hospital, Bristol, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Chemokines are a group of small peptides that are subdivided into four families, comprising >50 ligands with at least 17 different receptors. These chemokine families are defined by the presence of either a C, a CC, a CXC, or a C'C residue at the amino terminus of the protein. The largest of these subfamilies is the CXC chemokines, in which two amino-terminal cysteines are separated by a nonconserved amino acid, and the CC chemokines, in which two amino-terminal cysteines are juxtaposed (7). Within the glomerulum, chemokines and their receptors are expressed in infiltrating cells as well as in resident glomerular cells. Glomerular-produced chemokines seem not only to induce recruitment of inflammatory cells, but can also alter functions of resident glomerular cells, such as the formation of extracellular matrix (8). Therefore, chemokines may play an important role in the events leading to podocyte injury and proteinuria. In this study, we investigated the expression and function of chemokine receptors in cultured podocytes and in human kidney sections.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human podocytes were isolated from normal human kidney sections obtained from renal carcinoma patients undergoing nephrectomy. Primary cultures of podocytes were established as follows: Intact tissue was passed through steel sieves with decreasing pore sizes of 450 and 180 µm. Glomerula were collected using a third sieve with a pore size of 120 µm and counted under a light microscope (x400). Encapsulated glomerula were absent upon visual inspection. Glomerula were suspended in DMEM containing 10% heat-inactivated FCS, 2.5 mM glutamine , 0.1 mM sodium pyruvate, 5 mM HEPES buffer, 1 mg/ml streptomycin, 100 U/ml penicillin, 0.1x nonessential amino acids (100x; all Seromed, Berlin, Germany), insulin, transferrin, and a 5 mM sodium selenite supplement and then plated at a concentration of 100 glomerula/cm2 onto collagen-coated petri dishes (Greiner, Nürtingen, Germany). Glomerula were incubated at 37°C and 5% CO2 in air. After 4 days, cell colonies began to sprout around the glomerula. Cell colonies were excised and incubated in a tube containing 5 ml 0.2% collagenase IV (Sigma-Aldrich, Deisenhofen, Germany) at 37°C for 30 min and were then washed and plated in 25-cm2 culture flasks.
Cells showed an epithelial morphology with a polyhedral shape when confluency was reached and were immunohistologically characterized as podocytes. They stained positive for Wilm’s tumor Ag (WT1) and expressed nephrin, markers which are only found in podocytes in the adult kidney. Moreover, cells were negative for factor 8 related Ag, a marker for endothelial cells. Cells between passages 15 and 23 were seeded at 37°C onto collagen A (Biochrom, Berlin, Germany)-coated plates and cultured in standard RPMI 1640 medium containing 10% FCS, 100 U/ml penicillin, and 1 mg/ml streptomycin. In another set of experiments, early passages (passage 1 and passage 4) of cultured podocytes were used to confirm the functional expression of the chemokine receptors in short-term cultured cells. In addition, a conditionally immortalized human podocyte cell line demonstrating nephrin and podocin expression was used (9)
Cultures of primary human podocytes were infected with retrovirus-containing supernatants from the packaging cell line (PA317). The retroviral construct consisted of a SV40 large T-Ag gene containing both the tsA58 and the U19 mutations (10). Infection, selection, and continuous culture were conducted at 33°C. A single-cell clone was used for all of the experiments described.
Induction of differentiation
Subsequently, cells were grown on type I collagen-coated flasks layered with glass coverslips for the purpose of immunostaining. Cells were then plated onto the flasks and grown either at the "permissive" temperature of 33°C (in 5% CO2) to promote cell propagation as a cobblestoned phenotype or at the "nonpermissive" temperature of 37°C (in 5% CO2) to inactivate the SV40 T-Ag and allow the cells to differentiate.
As a positive control for CXCR2 expression, we used human lung microvascular endothelial cells in one series of experiments (11). Cells were cultured as previously described (11).
Expression of CCR and CXCR mRNA in cultured human podocytes and human glomerula
The RNA preparation, the reverse transcription and the PCR amplification were performed according to the method described recently (12). In brief, the total RNA from cultured human podocytes or human glomerula was isolated with guanidinium/acid phenol/chloroform extraction and the amount of RNA was measured with spectrophotometry. For first-strand synthesis, 2 µg of total RNA was mixed in 5x reverse transcription buffer and incubated with 5 U of DNase I for 15 min. After termination, the reaction was completed with 0.5 mM dNTP, 0.5 µM sequence-specific primers, 10 mM DTT, and 200 U of superscript II transcriptase (reverse transcriptase was omitted in some experiments to control for the amplification of contaminating DNA). The reverse transcription was performed at 42°C for 1 h followed by a denaturation at 95°C for 5 min. cDNA was purified from amplification reaction and solved in 30 µl of 10 mM Tris-buffer (pH 8). PCR was performed in duplicates in a total volume of 20 µl, each containing 2 µl of reverse transcription reaction and 18 µl of PCR master mixture containing 10 pmol each of sense and antisense primer and 2.5 U of Taq DNA polymerase. The cycle profile included denaturation for 30 s at 94°C, annealing for 30 s at 60°C, and extension for 30 s at 72°C. In the experiments for the analysis of the mRNA expression of CXCR1, 36 cycles of PCR were performed. In all other experiments, 30 cycles of PCR were performed.
The amplification products of 10 µl from each PCR were separated on a
1.5% agarose gel, stained with ethidium bromide, and visualized by UV
irradiation. PCR amplification of reverse transcription
reactions without reverse transcriptase revealed no PCR product,
thereby excluding amplification of genomic DNA. The identity of
amplification products was determined by dideoxy sequencing. The
primers used are shown in Table I
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The amplification products of 10 µl of PCR product were separated on a 1.5% agarose gel, stained with ethidium bromide, visualized with UV irradiation, and photographed with Polaroid film 667. The photographs were taken to evaluate the band densities by volume integration using a Hewlett Packard IIcx flatbad scanner and computer based imaging software (Image Quant; Molecular Dynamics, Krefeld, Germany). The data were normalized on the GAPDH mRNA expression.
Measurements of the free cytosolic intracellular Ca2+ concentration ([Ca2+]i)
Measurements of cytosolic calcium with the Ca2+-sensitive dye fura 2 (Sigma-Aldrich) were performed in podocytes on an inverted fluorescence microscope setup (13). The system allows fluorescence measurements at the single-cell level at three excitation wavelengths. The field of measurement can be set between a diameter of 2 and 300 µm with an adjustable pinhole. A time resolution of up to 200 Hz was achieved by using a high-speed filter wheel and a single photon counting tube (Hamamatsu H63460-04; Hamamatsu, Herrsching, Germany). The autofluorescence signal of cells that had not been loaded with fura 2 was measured and subtracted from the results obtained in fura-2-loaded cells. This had no effect on the bandwidth of the measurements. A calibration of the fura-2 fluorescence signal was attempted at the end of each experiment by using Ca2+ ionophore ionomycin (1 µM) and low and high Ca2+ buffers. [Ca2+]i was calculated from the fluorescence ratio 340:380 nm according to the equation described by Grynkiewicz et al. (14).
Measurement of IL-8 release
The podocytes were cultured in six-well plates. They were kept at 37°C for 24 h at different FCS concentrations (0, 1, 10%) before supernatants were taken. IL-8 concentrations were measured with an ELISA (R&D Systems, Wiesbaden, Germany) following the manufacturer’s instructions. The protein content of each well was measured with the Lowry method (15) and used to normalize the IL-8 release on the protein content per well.
Measurement of NADPH-oxidase activity
Podocytes were rinsed twice with ice-cold PBS, scraped from the wells, and resuspended in 2 ml of Krebs buffer. After centrifugation at 750 x g for 5 min at 4°C, the pellet was resuspended in 1.5 ml of fresh Krebs buffer (pH 7.35; containing 99 mM NaCl, 4.7 mM KCl, 1.8 mM CaCl2, 1.2 mM MgCl2, 25 mM NaHCO3, 1.03 mM K2HPO4, 20 mM sodium-HEPES, and 11.1 mM glucose). Cells were centrifuged as above and then resuspended in 0.6 ml of Krebs buffer. The luminescence buffer contained 5 µM lucigenin and 0.1 mM NADPH as the substrate.
To calculate the amount of superoxide produced, total counts were generated by integrating the area under the signal curve. These values were compared with a standard curve which was generated by using xanthine/xanthine oxidase as described elsewhere (12).
Superoxide generation was expressed as nanomoles of O2- generated per milligram of cellular protein per minute as described earlier (16). Protein content of the cell suspension was measured with the Lowry method (15).
Immunhistochemistry of cultured human podocytes
The immunolabeling was done as previously described (17). Briefly, coverslips were fixed with 2% paraformaldehyde and 4% sucrose in PBS, for 10 min, and were then permeabilized with 0.3% Triton X-100 (Sigma-Aldrich) in PBS for 10 min. Nonspecific binding sites were blocked with 4% FCS plus 0.1% Tween 20 (Sigma-Aldrich) in PBS for 30 min. Primary and secondary Abs were applied in the appropriate dilutions according to standard techniques, and the coverslips were mounted on glass slides with 15% Mowiol (Calbiochem, La Jolla, CA) and 50% glycerol in PBS. Further washes and mounting were conducted as above. Images were obtained using a Leica photomicroscope attached to a Spot 2 slider digital camera (Diagnostic Instruments, Solms, Germany) and processed with Adobe PhotoShop 5.0 software (Adobe Systems, Unterschleissheim, Germany).
Immunohistochemistry of kidney cortex
Normal kidney cortex and cortex from two patients with membranous nephropathy were obtained from nephrectomy specimens and snap frozen in liquid N2. Cryostat sections (4 µm) were prepared and incubated with Abs to CXCR 1–5 (10 µg/ml) (R&D Systems) for 2 h at room temperature. After being washed with PBS, bound Ab was detected using FITC-conjugated rabbit anti-mouse IgG (Accurate Chemical and Scientific, Westbury, NY). As controls, primary Abs were omitted or replaced by irrelevant mouse Abs. Micrographs were taken on a microscope equipped for epiluminescence (Axiophot; Carl Zeiss, Oberkochen, Germany).
Chemicals
The following agents were used: macrophage-derived chemokine
(MDC), I-309, thymus-expressed chemokine (TECK), hemofiltrate CC
chemokine (HCC-1), human CC chemokine 28 (CCL28), monokine induced by
IFN-
(MIG), stromal cell-derived factor 1
(SDF-1), B
cell-attracting chemokine 1 (BCA-1), and IL-8 (all R&D Systems). The
following Abs were used: anti-human CXCR1, anti-human CXCR2,
anti-human CXCR3, anti-human CXCR4, anti-human CXCR5 (all
R&D Systems), and FITC-conjugated goat anti-mouse (Jackson
ImmunoResearch Laboratories, West Grove, PA); the controls used were
mouse IgG1 for monoclonal anti-CXCR Abs (Santa Cruz Biotechnology,
Santa Cruz, CA) and monoclonal anti-human IL-8 Ab (R&D
Systems).
Statistics
The data are given as mean values ± SEM, where
n refers to the number of experiments. The average of the
effect of the agonist before and after the experiment was taken as
control. A paired t test was used to compare mean values
within each experimental series. A p 
0.05 was
accepted to indicate statistical significance.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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By using RT-PCR, we studied the expression of CCR and CXCR
subtypes in human cultured podocytes and human glomerula with
sequence-specific primers for each different receptor subtype (Table I
). Fig. 1A shows
ethidium-bromide-stained agarose gel
electrophoreses of PCR products for CCR4,
CCR8, CCR9, and CCR10 in cultured human podocytes. Fig. 1B
shows ethidium bromide-stained agarose gel electrophoreses of PCR
products for CXCR1, CXCR3, CXCR4, and CXCR5 in cultured human
podocytes. In contrast to all other CCRs and CXCRs, which were
detectable with 30 cycles of PCR, CXCR1 could only be constantly
detected with 36 cycles of PCR.
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It is known that proinflammatory cytokines such as IL-1
can
stimulate the expression of chemokine receptors in other cell types
(18). IL-1 has been shown to activate the
transcription factor NF-
B in podocytes (19). In
addition, IL-1 expression is up-regulated in podocytes in Heymann
nephritis. To test whether IL-1 might induce mRNA expression for
those CCRs and CXCRs which could not be found by RT-PCR,
podocytes were treated with Il-1 (10 ng/ml) for 6 and 18 h, and
mRNA expression of CCRs and CXCRs was studied. IL-1 did not induce
the expression of either CCR1–3 or CCR5–7 nor did it induce the
expression of CXCR2 (n = 3, data not shown).
In human glomerula, the PCR products of CCR1–CCR10 (Fig. 1A, lower panel) and CXCR1–CXCR5 (Fig. 1B,
lower panel) could be detected. In addition, we studied the mRNA
expression of CCRs and CXCRs in conditionally immortalized,
differentiated human podocytes, which may better mimic podocytes in
vivo. These podocytes showed the same expression pattern of CCRs and
CXCRs, namely, the CCR4, CCR8–10, and CXCR1, CXCR3, CXCR4, and CXCR5
(data not shown).
CCR ligands increase the free cytosolic calcium concentration [Ca2+]i [infi] in human cultured podocytes
Addition of the CCR4 agonist macrophage-derived chemokine (MDC,
n = 33), the CCR8 agonist I-309 (n =
33), the CCR9 agonists TECK (n = 6) and HCC-1
(n = 2), and the specific CCR10 agonist human CCL28
(n = 4) (20, 21) to single podocytes
loaded with fura-2 resulted in a reversible increase of
[Ca2+]i. The left
panel of Fig. 2 shows the typical
original fluorescence measurements obtained from single podocytes
exposed to MDC (A, 10 nM), I-309 (B, 100 nM),
HCC-1 and TECK (C, 2.5 µg/ml and 100 nM,
respectively), and CCL28 (D, 2 µg/ml). The right
panel of Fig. 2 gives the concentration response curves
(A, B, and D) or the summary of the
experiments (C and D) for the different CCR
agonists.
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The CXCR3 agonist MIG (n = 32), the CXCR4 agonist
SDF-1 (n = 32), and the CXCR5 agonist
BCA-1(n = 46) induced a reversible increase of
[Ca2+]i in podocytes. In
contrast, different concentrations of IL-8 had no effect on
[Ca2+]i (370 µM,
n = 5; 37 µM, n = 3; 3.7 µM,
n = 2) in podocytes grown in the presence of FCS. After
the addition of IL-8, SDF-1 as a positive control induced an
increase of [Ca2+]i in
podocytes (Fig. 3A, left
panel). The [Ca2+]i
response to MIG, SDF-1, and BCA-1 was concentration dependent with
an EC50 value of 85 nM for MIG, 0.6 nM for
SDF-1, and 10 nM for BCA-1, respectively. The left panel
of Fig. 3 shows typical original fluorescence measurements obtained
from single podocytes exposed to IL-8 (A, 370 µM), MIG
(B, 100 nM), SDF-1 (C, 10 nM), or BCA-1
(D, 100 nM). The right panel of Fig. 3 gives the
concentration response curves for the different CXCR agonists.
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(10 nM, n = 6;
increase of [Ca2+]i:
155 ± 41 nM), and BCA-1 (100 nM, n = 8; increase
of [Ca2+]i: 173 ±
36 nM); data not shown). Immunolabeling of CXCR1–5 in human cultured podocytes
Immunolabeling was performed with human podocytes and conditionally immortalized human podocytes cultured in 5% FCS. Both cultured human podocytes and immortalized, differentiated podocytes revealed an identical pattern of CXCR staining:
Fig. 4 shows the CXCR staining of the
human immortalized podocyte cell line. We detected a positive staining
for CXCR3–5 (D, F, and G,
n = 4), whereas no staining was seen for CXCR1
(B, n = 4) and CXCR2 (C,
n = 4). As a negative control, we used mouse IgG as the
primary Ab in these experiments (Fig. 4A).
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To prove whether podocytes themselves may release chemokines, the
expression of mRNA for IL-8, MIG, IP-10, SDF-1, and BCA-1 was
investigated using RT-PCR. Fig. 5A shows that podocytes grown
in the presence of 10% FCS express mRNA for IL-8 but not for MIG,
IP-10, SDF-1, or BCA-1. Fig. 5B shows that IL-8 protein
is released from podocytes after incubation with different
concentrations of FCS (1 and 10%) and that only small amounts are
released in 0% FCS (n = 6). FCS alone did not contain
any detectable IL-8 (data not shown). To further investigate whether
CXCR1 or CXCR2 may have been down-regulated by the release of IL-8 in
podocytes that have been grown in the presence of FCS, the protein
expression of CXCR1 and CXCR2 was investigated in FCS-starved
podocytes. Indeed, immunofluorescence staining of starved podocytes
showed an expression of CXCR1 but not CXCR2 in immortalized,
differentiated podocytes (Fig. 5C) or in human podocytes
(data not shown). For the positive control of CXCR2 staining, we used
human lung microvascular endothelial cells (Fig. 5C). In
FCS-starved podocytes, IL-8 caused a concentration-dependent increase
of [Ca2+]i (Fig. 5D).
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E summarizes the data of the effect of neutralizing IL-8 Ab
on IL-8-induced increase of
[Ca2+]i.
To investigate whether an up-regulation of CXCR1 takes place at the
transcriptional level, we performed a series of semiquantitative
RT-PCRs under different cell culture conditions: FCS starvation caused
a slight increase of mRNA expression of CXCR1 but the addition of IL-8
in FCS-free medium or the addition of IL-8-neutralizing Ab in
FCS-containing medium did not result in a change of mRNA expression of
CXCR1 (Fig. 5F). These results indicate that functional
CXCR1 down-regulation in the presence of FCS is not regulated on the
transcriptional level but due to ligand-induced receptor
internalization.
FCS starvation might have also influenced the
[Ca2+]i response to the
other CXCR agonists. To clarify this point, we assessed the
[Ca2+]i response to MIG
(0.5 µM, n = 5), SDF-1 (10 nM,
n = 5), and BCA-1 (100 nM, n = 5) in
FCS-starved cells. When compared with the podocytes grown in FCS, the
[Ca2+]i response to the
agonists was not significantly different from that of FCS-starved
podocytes (data not shown).
CCR and CXCR ligands increase O2- production in podocytes
Activation of the NADPH-oxidoreductase enzyme complex leading to
generation of reactive oxygen species has been demonstrated in
podocytes in vivo and in vitro. It has been suggested that this
generation of reactive oxygen species plays a major role in the
pathogenesis of proteinuria (22). The addition of NADPH
(0.1 mM) to control podocytes led to a small increase of superoxide
anion production (data not shown). After the pretreatment of podocytes
with IL-8 (50 nM, n = 5), MIG (0.5 µM,
n = 8), SDF-1 (10 nM, n = 6), and
BCA-1 (100 nM, n = 4), the addition of NADPH caused a
significant increase of superoxide production in comparison to control
conditions by 262 ± 42%, 225 ± 56%, 228 ± 40%, and
216 ± 61%, respectively (Fig. 6C). Before stimulation with
IL-8, cells were serum starved for 24 h to up-regulate CXCR1.
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B).
Superoxide generation by NADPH-oxidase activity was calculated by
integrating the total counts during the first 15 min. Superoxide
generation was expressed as nanomoles of O2-
generated per milligram of cellular protein per minute. Fig. 6A shows a representative original recording of the time
course and magnitude of NADPH-oxidase activation after stimulation with
CXCR ligands, in this case, SDF-1 (10 nM).
Immunofluorescence studies show the expression of CXCR1, CXCR3, and CXCR5 in kidneys with membranous nephropathy but not in control kidneys
We examined cryostat sections of normal kidney cortex and cortex from two patients with MGN and nephrectomy. The diagnosis of membranous nephropathy was made on kidney biopsies with the typical findings of light, electron, and immunofluorescence microscopy (data not shown).
In control kidneys, there was no or only slight staining of
CXCR1–CXCR5 (Fig. 7, A,
C, E, G, and I), whereas in
MGN a positive fluorescence staining for CXCR1 (Fig. 7B),
CXCR3 (Fig. 7F), and CXCR5 (Fig. 7J) could be
detected in podocytes (arrows indicate podocytes, Fig. 7). Glomerular
CXCR2 (Fig. 7, C and D) and CXCR4 (Fig. 7, G and
H) were not detectable in these biopsies (Fig. 7).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In podocytes grown in FCS, mRNA of the CXCR subtypes 1, 3, 4, and 5 were detected. Under these conditions, CXCR3–5 were functionally active as was demonstrated by the chemokine-induced increases in [Ca2+]i. The half maximal concentrations fit well with those described in other cell types such as lymphocytes (26, 27, 28). It was only by using highly sensitive and stringent RT-PCR conditions that mRNA for CXCR1 could be detected in podocytes. Immunhistochemically, CXCR1 could not be detected in podocytes grown in a medium with FCS. Also, IL-8, a ligand of CXCR1 and CXCR2, did not induce a [Ca2+]i response in podocytes, suggesting that podocytes do not express functional CXCR1 under these conditions. However, we showed that FCS stimulated an IL-8 release in podocytes. In the absence of FCS, IL-8 release was inhibited and up-regulation of functional CXCR1 but not CXCR2 was induced; i.e., CXCR1 could be detected in immunnolabeling studies and IL-8 induced a [Ca2+]i response. The data suggest that FCS-mediated release of IL-8 suppressed the expression of functional CXCR1 in podocytes. This hypothesis was supported in experiments, in which IL-8 increased [Ca2+]i after a 24-h incubation of podocytes with FCS and IL-8 Abs to neutralize the released IL-8.
The different culture conditions used did not significantly influence
mRNA expression of CXCR1, indicating that CXCR1 is down-regulated by
ligand-induced receptor internalization. IL-8 is known to be a
neutrophil chemotactic factor which promotes leukocyte adhesion and
leads to the recruitment of polymorphonuclear leukocytes to sites of
tissue inflammation (29). IL-8 protein expression along
the capillary walls and in the mesangium has been reported in several
forms of glomerulonephritis. It has been suggested that IL-8 may be
partially responsible for the infiltration of leukocytes during
glomerulonephritis (30, 31). In addition, IL-8 might
induce albuminuria by altering the metabolism of the sulfated compounds
of the glomerular basement membrane (32). In a model of
immune complex nephritis, IL-8 was expressed in affected glomerula and
injection of an anti-IL-8 Ab reduced glomerular neutrophil
infiltration, prevented fusion of foot processes of podocytes, and
inhibited proteinuria (33). IL-8 release of podocytes
during proteinuric diseases may therefore, in an autocrine fashion,
participate in the development of proteinuria in glomerular diseases.
mRNA could be detected in podocytes for IL-8 but not for IP-10,
SDF-1, and BCA-1. However, these and other ligands can be released
from leukocytes and lymphocytes, infiltrating glomerula in the case of
inflammation. In addition, some chemokines are also produced by
resident glomerular cells. Synthesis of MCP-1, IL-8, and IP-10 has been
demonstrated in mesangial cells (34).
How might activation of CCRs and CXCRs influence podocyte function? In Heymann nephritis, proteinuria is dependent on Ab-induced formation of the complement C5b-9 membrane attack complex. It has been demonstrated that sublytic C5b-9 attack on podocytes causes up-regulation of expression of the NADPH-oxidoreductase enzyme complex by podocytes, which is then translocated to their cell surfaces. Subsequently, reactive oxygen species are produced locally which reach the glomerular basement membrane matrix. Reactive oxygen species initiate lipid peroxidation and subsequent degradation of glomerular basement membrane collagen IV, leading to proteinuria (35, 36). In the present study, we show that CCR and CXCR ligands induce the activation of NADPH-oxidase, a major source for the production of superoxide anions in podocytes. Activation of chemokine receptors in podocytes may therefore contribute to the pathogenesis of proteinuria via activation of NADPH-oxidases, leading to a release of superoxide anions.
In contrast to the ample information about the release of chemokines during renal injury, little is known about the expression of chemokine receptors on glomerular cells in vivo. To our knowledge, only CXCR3 have been detected in mesangial cells of patients with IgA nephropathy, membranoproliferative glomerulonephritis, and rapidly progressive glomerulonephritis, indicating that CXCR3 might contribute to mesangial cell proliferation in these diseases (37). It is well known that cultured cells frequently reflect pathological conditions rather than a physiological situation. With this in mind, chemokine receptor expression may be the result of cell culture conditions. For example, CXCR3 were shown to be functionally active in cultured human mesangial cells. However, slight or no signal of immunological staining for CXCR3 could be detected in normal glomerula, whereas a positive signal was seen in injured glomerula, suggesting that CXCR3 expression is up-regulated during glomerular inflammation (37). In agreement with these findings, only a slight immunohistological staining for CXCRs could be detected in normal human glomerula. This slight and diffuse signal was seen for CXCR3 and CXCR5 in healthy human glomerula. However in patients with MGN, a positive staining for CXCR1, CXCR3, and CXCR5 was detected in podocytes, but not for CXCR2 or CXCR4. With regard to the release of superoxide anions after activation of these receptors in podocytes in culture, these receptors may be involved in the pathogenesis of podocyte injury and proteinuria in MGN. The difference in expression of CXCR4 in cultured podocytes and in podocytes during MGN (podocytes in culture possess CXCR4, whereas during MGN podocytes do not express CXCR4) might be due to the following: 1) CXCR4 might not be present in MGN but instead in other glomerular diseases associated with injury of the podocyte. 2) CXCR4 might only be found under cell culture conditions.
The proinflammatory cytokine IL-1, which has been shown to induce
chemokine receptor expression in astroglioma cells (18),
did not stimulate mRNA expression for those CCRs and CXCRs which have
not been detected in unstimulated podocytes. This suggests that no
additional chemokine receptor expression can be induced by Il-1 in
podocytes.
In comparison to the strong staining of the CXCR1 and CXCR5 in podocytes in MGN, CXCR3 staining was weaker. In a recently performed semiquantitative evaluation of immunohistochemical staining for the CXCR3, Romagnani et al. (37) found only a slight positive staining for CXCR3 in two of four kidneys from patients with MGN, a finding that was not different from normal kidneys. The difference in our present study, where a small increase of CXCR3 staining could be observed in podocytes from patients with MGN, might be explained by methodical differences; i.e., in the study by Romagnani et al. (37), paraformaldehyde was used to fix kidney tissue and the avidin biotin-peroxidase method was used to detect Ab reaction. In addition, it might be difficult to quantify positive staining in podocytes. Podocytes represent only 5% of all glomerular cells and are sometimes difficult to localize.
Is there any further evidence that CXC chemokines may participate in the development of glomerular damage and proteinuria? It was recently demonstrated that IP-10 expression is up-regulated in resident glomerular cells in vitro and in vivo in Adriamycin nephropathy. Levels of glomerular IP-10 mRNA expression and glomerular and tubulointerstitial IP-10 protein coincidenced with maximal proteinuria (38). Therefore, IP-10 may participate in the pathogenesis of podocyte injury and proteinuria during Adriamycin nephrosis. In addition to their possible role in glomerular inflammation, the chemokine receptors CCR5 and CXCR4 are also known to serve as coreceptors for the cellular entry of HIV-1 (39). Moreover, CXCR5 has been demonstrated to act as a specific coreceptor for HIV-2 (40). The podocyte plays a crucial role in the pathogenesis of HIV nephropathy and it has recently been shown that the loss of podocyte maturity markers and podocyte proliferation occur in HIV-associated nephropathy (41). Very recently, it was demonstrated that CCR5 and CXCR4 were not expressed in resident glomerular cells in HIV-associated renal disease (42). The possible role of CXCR5 in HIV-2-induced nephropathy has to be investigated in future studies. Expression of CXCR5 was primarily demonstrated on B lymphocytes (43). Interestingly, we show here that CXCR5 is also expressed on a nonhemopoietic cell type; the podocyte. Several earlier studies have shown that podocytes are able to express other leukocyte-associated markers, such as CD2-associated protein or CD68 under normal and pathological conditions, respectively, indicating that there are some similarities between protein expression of leukocytes and podocytes (44, 45). Recently, it has also been shown that injured podocytes undergo a process of transdifferentiation with the acquisition of epitopes that are characteristic of activated macrophages (46). The presence of distinct chemokine receptors on podocytes further supports the idea that podocytes can turn into inflammatory cells which are actively involved in the inflammatory processes that occur in glomerular diseases.
In conclusion, cultured podocytes express functional CCR and CXCR receptors. Activation of these receptors may contribute to podocyte damage and proteinuria during glomerular diseases. Therefore, it will be interesting to find out whether chemokine receptor antagonists can reduce proteinuria in patients with inflammatory glomerulopathy.
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Acknowledgments |
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Footnotes |
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2 T.B.H. and H.C.R. contributed equally to this manuscript.
3 Address correspondence and reprint requests to Dr. Hermann Pavenstädt, Medizinische Universitätsklinik Freiburg, Abteilung IV, Labor C4, Hugstetter Strasse 55, D-79106 Freiburg, Germany. E-mail address: paven{at}mm41.ukl.uni-freiburg.de
4 Abbreviations used in this paper: MGN, membranous nephropathy; IP-10, IFN-inducible protein; MCP, monocyte chemoattractant protein; WT1, Wilm’s tumor Ag; [Ca2+]i, intracellular calcium; MIG, monokine induced by IFN-; SDF-1, stromal cell-derived factor 1; BCA-1, B cell-attracting chemokine 1; MDC, macrophage-derived chemokine; TECK, thymus-expressed chemokine; CCL28, C chemokine ligand 28; HCC, hemofiltrate CC chemokine.
Received for publication February 2, 2002. Accepted for publication April 19, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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K. Bedard and K.-H. Krause The NOX Family of ROS-Generating NADPH Oxidases: Physiology and Pathophysiology Physiol Rev, January 1, 2007; 87(1): 245 - 313. [Abstract] [Full Text] [PDF]
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E. Galkina and K. Ley Leukocyte Recruitment and Vascular Injury in Diabetic Nephropathy J. Am. Soc. Nephrol., February 1, 2006; 17(2): 368 - 377. [Abstract] [Full Text] [PDF]
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M. P. Winn, N. Daskalakis, R. F. Spurney, and J. P. Middleton Unexpected Role of TRPC6 Channel in Familial Nephrotic Syndrome: Does It Have Clinical Implications? J. Am. Soc. Nephrol., February 1, 2006; 17(2): 378 - 387. [Full Text] [PDF]
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