Expression of Functional CCR and CXCR Chemokine Receptors in Podocytes

Cell culture

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 (×400). 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, 0.1× nonessential amino acids (100×; 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 5× 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⇓.

Semiquantitative analysis

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 × 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-γ (MIG), stromal cell-derived factor 1α (SDF-1α), 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.

Human cultured podocytes and human glomerula express mRNA for CCR and CXCR subtypes

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. 1⇓A shows ethidium-bromide-stained agarose gel electrophoreses of PCR products for CCR4, CCR8, CCR9, and CCR10 in cultured human podocytes. Fig. 1⇓B 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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FIGURE 1.

Expression of mRNA for CCRs (A) and CXCRs (B) in human cultured podocytes and human glomerula. Fragments of the different chemokine receptors were amplified using specific primers (see Table I⇑) and subjected to agarose gel electrophoresis. Lanes on the left side indicate base pair length. Note that podocyte bands with the expected sizes are present in the CCR4, 8, 9, and 10 lane and in the CXCR1, CXCR3, CXCR4, and CXCR5 lane, whereas in human glomerula (lower panels), bands of CCR1–10 and CXCR1–5 are positive. In both human cultured podocytes and human glomerula, the bands for WT1 and nephrin, which are only expressed in podocytes, were present. Sequence analysis of the resulting amplification products revealed the sequence identity of the fragments.

Sequence analysis of the resulting amplification products revealed the sequence identity of the fragments. mRNA could not be detected for CCR1–3 and CCR5–7 and CXCR2 in human podocytes using 30 or 36 cycles of PCR.

It is known that proinflammatory cytokines such as IL-1β can stimulate the expression of chemokine receptors in other cell types (18). IL-1 β has been shown to activate the transcription factor NF-κB in podocytes (19). In addition, IL-1β expression is up-regulated in podocytes in Heymann nephritis. To test whether IL-1β might induce mRNA expression for those CCRs and CXCRs which could not be found by RT-PCR, podocytes were treated with Il-1β (10 ng/ml) for 6 and 18 h, and mRNA expression of CCRs and CXCRs was studied. IL-1β 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. 1⇑A, lower panel) and CXCR1–CXCR5 (Fig. 1⇑B, 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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FIGURE 2.

Fluorescence recordings showing the effect of CC chemokines on [Ca²⁺]_i in podocytes. Left panel, The original recordings of the CC chemokines induced an increase of [Ca²⁺]_i in human podocytes: CCR4 agonist MDC (A), CCR8 agonist I-309 (B), CCR9 agonists HCC-1 and TECK (C), and CCR10 agonist CCL28 (D). Right panel, Concentration response curves or summary of the effects of the CCR ligands. Numbers of observations are shown above the curve. ∗, Statistical significance.

CXCR ligands increase the free cytosolic calcium concentration [Ca²⁺]_i in human cultured podocytes

The CXCR3 agonist MIG (n = 32), the CXCR4 agonist SDF-1α (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-1α as a positive control induced an increase of [Ca²⁺]_i in podocytes (Fig. 3⇓A, left panel). The [Ca²⁺]_i response to MIG, SDF-1α, and BCA-1 was concentration dependent with an EC₅₀ value of 85 nM for MIG, 0.6 nM for SDF-1α, 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-1α (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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FIGURE 3.

Fluorescence recordings showing the effect of CXC chemokines on [Ca²⁺]_i in podocytes. A, IL-8, an agonist of CXCR1 and CXCR2, respectively, had no effect on [Ca²⁺]_i of human podocytes grown in FCS. After addition of IL-8, SDF-1α as a positive control induced an increase of [Ca²⁺]_i. B–D, left panel, MIG, SDF-1α, and BCA-1, ligands for CXCR3, CXCR4, and CXCR5, respectively, induced an increase of [Ca²⁺]_i in human podocytes. Right panel, Concentration response curves of the effects of the CXCR ligands. Numbers of observations are shown above the curve. ∗, Statistical significance.

To test whether early passages of human podocytes express the same set of functional chemokine receptors, the measurement of [Ca²⁺]_i was performed on these cells and revealed the same pattern of calcium responses to CXCR ligands (passages 1 and 4: IL-8 (3.7 μM, n = 10; increase of [Ca²⁺]_i: 0 ± nM)^; MIG (0, 5 μM, n = 5; increase of [Ca²⁺]_i: 136 ± 30 nM), SDF-1α (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. 4⇓A).

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FIGURE 4.

Immunofluorescence studies on the expression of CXCR1–5 in immortalized, differentiated human podocytes. Positive staining for CXCR3–5 (D, F, and G). In podocytes cultured in FCS, no signal for the CXCR1 and CXCR2 (B and C) was detectable. Mouse IgG as negative control for these experiments (A).

Effect of FCS on IL-8 release and expression of CXCR1 in podocytes

To prove whether podocytes themselves may release chemokines, the expression of mRNA for IL-8, MIG, IP-10, SDF-1α, and BCA-1 was investigated using RT-PCR. Fig. 5⇓A shows that podocytes grown in the presence of 10% FCS express mRNA for IL-8 but not for MIG, IP-10, SDF-1α, or BCA-1. Fig. 5⇓B 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. 5⇓C) or in human podocytes (data not shown). For the positive control of CXCR2 staining, we used human lung microvascular endothelial cells (Fig. 5⇓C). In FCS-starved podocytes, IL-8 caused a concentration-dependent increase of [Ca²⁺]_i (Fig. 5⇓D).

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FIGURE 5.

Effect of FCS on IL-8 release and expression of CXCR1 in podocytes. A, Expression of mRNA for IL-8 but not MIG, IP-10, SDF-1α, or BCA-1 was detected in human podocytes. B, Detection of the release of IL-8 protein from podocytes after incubation with different concentrations of FCS (1% and 10%). C, Immunofluorescence staining showed expression of CXCR1 but not CXCR2 in human podocytes. Mouse IgG was used as a negative control for CXCR1 staining and human microvascular endothelial cells as positive control for CXCR2 staining. D, Concentration response curve of the effect of IL-8 on [Ca²⁺]_i in podocytes that were grown without FCS for 24 h. Numbers of observations are shown above the curve. ∗, Statistical significance. E, IL-8 induced an increase of [Ca²⁺]_i in the presence of serum-free medium (0% FCS) and in the presence of FCS-containing medium (1% FCS) with the IL-8-neutralizing Ab (n-IL-8 mab). F, Fragments of CXCR1 were amplified using specific primers (see Table I⇑) and subjected to agarose gel electrophoresis under certain culture conditions. Band densities were evaluated by volume integration. The data were normalized on the GAPDH mRNA expression.

To clarify the role of IL-8 in suppressing CXCR1 expression in FCS-containing medium, we investigated the effect of IL-8-neutralizing Ab on the [Ca²⁺]_i response to IL-8. After adding IL-8-neutralizing Ab (10 μg/ml; n = 8) for 24 h to podocytes grown in FCS-containing medium, IL-8 did induce an increase of [Ca²⁺]_i. Fig. 5⇑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. 5⇑F). 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-1α (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-1α (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. 6⇓C). Before stimulation with IL-8, cells were serum starved for 24 h to up-regulate CXCR1.

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FIGURE 6.

Effects of CCR and CXCR ligands on NADPH-mediated superoxide anion production in human podocytes. A, A representative original recording of the time course and magnitude of NADPH-oxidase activation after stimulation with chemokine ligands, in this case SDF-1α (10 nM). B, Control cells and cells that had been treated with the respective CXC chemokine for 4 h. C, Control cells and cells that had been treated with the respective CCR chemokine for 4 h. Superoxide generation by NADPH-oxidase activity was calculated by integrating the total counts during the first 15 min and superoxide generation was expressed as nanomoles of O²⁻ generated per milligram of cellular protein per minute.

Like the CXCR ligands, the CCR ligands MDC (100 nM), I-309 (10 nM), TECK (100 nM), and CCL28 (2 μg/ml) caused a significant but smaller increase of superoxide production in comparison to control conditions by 136 ± 9%, 162 ± 7%, 129 ± 8%, and 129 ± 9%, respectively (Fig. 6⇑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. 6⇑A shows a representative original recording of the time course and magnitude of NADPH-oxidase activation after stimulation with CXCR ligands, in this case, SDF-1α (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. 7⇓B), CXCR3 (Fig. 7⇓F), and CXCR5 (Fig. 7⇓J) 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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FIGURE 7.

Immunofluorescence studies on the expression of CXCR1–5 in control kidney (A, C, E, G, and I) and in a kidney with membranous nephropathy (B, D, F, H, and J). Note that there is no or slight staining of CXCR1 (A), CXCR3 (E), and CXCR5 (I) in the control kidney, whereas in membranous nephropathy a positive fluorescence staining of these CXCRs (B, F, and J) could be detected in podocytes (arrows indicate podocytes).