1 Medizinische Poliklinik, Klinikum Innenstadt der Ludwigs-Maximilians-Universität, Munich, Germany and 2 Department of Pathology, University Hospital Nijmegen, Nijmegen, The Netherlands
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Abstract |
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Methods. Kidneys were harvested at day 0, 2 and 7. Induction of nephritis was confirmed by assessment of albuminuria by ELISA and by histological evaluation. RNA was prepared from cortex and isolated glomeruli. RNase protection assays were performed to study the expression of chemokines, RNase protection assays as well as quantitative RT-PCR assays to study the expression of chemokine receptors.
Results. In the cortex of nephritic kidneys mRNA for MCP-1 was increased 5-fold on day 2 and increased 4-fold on day 7 as compared to controls. mRNA for RANTES was increased 5-fold on day 7 and mRNA for IP-10 6-fold on day 7. The increase of mRNA for the chemokine receptors CCR1 and 5 was between 2-fold and 3-fold determined by RNase protection assay and for CCR1, 2 and 5 between 2- and 4-fold as determined by RT-PCR. In isolated glomeruli we found by RT-PCR an increase of CCR1, CCR2 and CCR5 of between 3 and 12-fold.
Conclusion. These results show that chemokines and their specific chemokine receptors are increased in parallel in this model of glomerulonephritis, consistent with the potential role of the chemokine system in leukocyte recruitment to the immune injured kidney.
Keywords: CC-chemokines; chemokine receptors; glomerulonephritis; mouse; nephrotoxic glomerulonephritis
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Introduction |
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The specificity of chemokine action is mediated through selective expression of chemokine receptors by different leukocyte subpopulations. All chemokine receptors are structurally related and belong to the family of seven-transmembrane G-protein coupled receptors. Many chemokine receptors are considered shared receptors, because in vitro they can bind more than one chemokine [3].
Chemokines have been proposed to attract leukocytes in inflammatory kidney diseases. For example MCP-1 mRNA is increased in timely concordance with macrophage infiltration in rat models of anti-Thy1.1 nephritis [4,5] and of nephrotoxic nephritis [610]. Subsequent studies demonstrated persisting elevated levels of MCP-1, MCP-3, MIP-1, MIP1-ß, TCA-3 and RANTES mRNA at 24 h, 3 and 7 days after induction of glomerular injury with anti-GBM antibodies again correlating with persistently elevated numbers of macrophages within the glomeruli [11]. An increase of MCP-1 could also be detected at the protein level in glomerular lysates by ELISA [6]. Pretreatment of rats with a neutralizing anti-MCP-1 antibody before induction of a nephrotoxic nephritis resulted in less morphological damage, a decrease in glomerular macrophage infiltration and a reduction of proteinuria [6,12,13]. The protective effect of anti-MCP-1 and of the RANTES antagonist MetRANTES was confirmed in a murine model of nephrotoxic serum nephritis [14].
Further evidence for a role of chemokines and their receptors in the pathogenesis of experimental nephritis was obtained with the receptor blocker vMIPII. vMIP-II is a chemokine analogue encoded by human herpesvirus 8 with the capacity to bind and block the MCP1-receptor CCR2, the RANTES receptors CCR1, CCR3 and CCR5 and the fractalkine receptor (CX3 CR1). In NTS nephritis in the rat pretreatment with vMIP-II reduced glomerular leukocyte infiltration, morphological damage and proteinuria [15].
A role for chemokines in human kidney diseases is supported by the findings of MCP-1 and RANTES expression in human kidney biopsies correlating with the local infiltration of macrophages [1619]. Furthermore we recently demonstrated the presence of CCR5 positive leukocytes in human biopsies from patients with a variety of renal diseases.
To date, a simultaneous analysis of the expression of chemokines and their receptors in mouse NTS nephritis has not been performed. Such an analysis is the prerequisite to future therapeutic intervention studies targeting the identified chemokines and chemokine receptors. Our results confirm the concerted expression of CC-chemokines and their receptors in murine NTS-nephritis and identify CCR1, CCR2 and CCR5 as potential targets for therapeutic interventions.
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Methods |
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Assessment of albuminuria
Spot urine specimens were obtained from mice at the time of sacrifice for the determination of the urine protein/creatinine ratio (Up/Ucr) as an index of urinary albumin excretion. Urine creatinine was measured using a Hitachi Autoanalyzer, and urine albumin concentration was measured using an ELISA for mouse albumin (EXOCELL, Philadelphia, PE).
Histological analysis
Kidney sections in the coronary plane were fixed in Bouin's solution. Fixed kidneys were dehydrated and embedded in paraplast (Amstelstad B.V., Amsterdam, The Netherlands). Sections (4 µm) were stained with haematoxylin and eosin and periodic acid-Schiff. Sections were examined for glomerular cellularity and necrosis, crescent formation and interstitial infiltration as described. The percentage of glomerular crescents was assessed by counting 40 glomeruli per section and mouse.
For immunohistology sections were snap frozen in liquid nitrogen and 24 µm cryostat sections were obtained. Sections were stained with monospecific FITC-labelled goat anti-mouse Ig (heavy and light chains) and goat anti-mouse C3 serum (both from Cappel Laboratories, West Chester, US), goat anti-rabbit Ig (Nordic, Tilburg, The Netherlands), and rabbit anti-human fibrinogen cross-reacting with mouse fibrinogen (Dako, Copenhagen, Denmark). The sections were examined in a fluorescence microscope equipped with epi-illumination (Leica DBMR, Wetzlar, Germany) and the staining intensity was determined as previously described [10].
The number of glomerular and interstitial macrophages was determined in 5 µm frozen kidney sections with a rat anti-mouse macrophage monoclonal FA/11 antibody [20] (kindly donated by Dr G.L.E. Koch, MRC Laboratory of Molecular Biology, Cambridge, UK) as first antibody, a three step avidin-biotin technique with biotinylated goat anti-rat antibodies [21] as second antibodies and ABC complexes with alkaline phosphatase (Vector Laboratories, Burlingame, US) for histochemical detection. The number of glomerular macrophages was assessed as the mean number per glomerular cross-section (GCS) counted in at least 40 glomeruli per section and mouse. The number of interstitial macrophages was determined as mean per high power field (HPF) from 10 HPS chosen at random per section and mouse (magnification: lens objective 40x, ocular 10x).
Isolation of glomeruli
Glomeruli from four animals per group were isolated by sequential sieving as previously reported [22] with minor modifications. These consisted of addition of collagenase (1 mg/ml, type IV Worthington, Freehold, NJ, US) to the preparation buffer and 2% BSA (Fraction V, Boehringer, Mannheim, Germany) as well as 0.02% TWEEN 20 (Fluka, Buchs, Switzerland) to the washing buffer. The purity of the glomerular fraction was determined by light microscopy and was approximately 90±5%.
mRNA isolation, RNase protection assays and quantitative reverse transcriptionPCR
Total RNA was extracted from kidney cortex (half a kidney from two animals per time point) and purified glomeruli fractions (starting material for sieving one and a half kidneys from four animals per time point). Tissues were homogenized in a commercial phenol-guanidine isothiocyanate (GITC) reagent (TRI-Reagent, Molecular Research Center, Cincinatti, OH, US) and total RNA isolated as described by the manufacturer.
Multiprobe template sets for mouse chemokines MCP-1, RANTES, MIP-1, MIP-1ß, MIP-2, Eotaxin, IP-10 and TCA-3 and mouse CCR 15 for use in RNase protection assays were obtained from Pharmingen (San Diego, US). Protection assays were performed according to manufacturers instructions using 5 µg of total RNA from kidney cortex to detect chemokine RNA on day 2 and 20 µg on day 7 and using 25 µg of RNA from kidney cortex to detect chemokine receptor-RNA. Protected fragments were separated by denaturing polyacrylamide gel electrophoresis. After drying gels were exposed at -80°C on X-ray film using intensifying screens or exposed on phosphor screens for analysis with a Storm 840 Phosphorlmager (Molecular Dynamics, Sunnyvale, USA). Relative expression was calculated as x-fold change compared to controls and corrected for expression of housekeeping genes. Relative expression was determined for two separate series of experiments which resulted in comparable results.
Reverse transcription (RT) and quantitative RTPCR were performed as described [23]. Contaminating genomic DNA was removed using 5 U of RNase-free DNase (Promega, Ingelheim, Germany) for 15 min at 37°C per RNA sample containing about 2 µg RNA in the presence of 20 U of RNasin (Promega Biotech, Madison, WI, US). DNase was removed by phenol chloroform extraction and ethanol precipitation. The purified RNA was redissolved in 11 µl diethyl-pyrocarbonate-treated water containing 20 U of RNAsin and denatured for 5 min at 65°C in the presence of 0.5 µl (=0.5 µg) oligo (dT)12 oligonucleotides. Aliquots of 100 U of a modified Moloney murine leukomia virus reverse transcriptase (SuperScriptRT, Life Technologies, Eggenstein, Germany) were added and RT performed for 1 h at 42°C. For reverse transcriptase negative controls (RT minus) no enzyme was added.
To detect cDNA for CCR 15, sequence-specific 1822 bp oligonucleotide primers were designed for each receptor based on the published sequences (Table 1). A 1 µl sample of the cDNA was then used to perform PCR reactions in 25 µl containing 2.5 µl 10x PCR buffer with 15 mM MgCl2, 4 µl 1.25 mM dNTP, 0.25 µl (=2.5 pmol) forward primer, 0.25 µl (=2.5 pmol) reverse primer, 15.525 µl H2O and 0.125 µl AmpliTaq polymerase (Perkin Elmer, Foster City, CA). Amplifications were performed in a Perkin Elmer Cetus using the following conditions. After denaturation for 3 min at 94°C, samples were cycled 30 times for 1 min at 94°C, 1 min at 56°C, and for 2 min at 72°C; final extension was performed for 7 min at 72°C. To control for variation in tissue mass and RT reaction efficiency, cDNA concentration was adjusted assuming constant adenine-nucleotide-carrier (ANC) concentrations in equal amounts of RNA extracted from tissue (GAPDH and ß-actin-expressions corresponded to ANC expression in multiple experiments in this modeldata not shown). For this purpose PCR was performed using oligonucleotide primers selected from published sequences (Table 1
) on a serial 5-fold dilution series of each cDNA, with assays performed over a 23 log range. PCR was then repeated using the standardized cDNA templates to verify equivalent cDNA concentrations at each time point.
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To assess product abundance the amplified cDNA was analysed on a non-denaturing 5% polyacrylamide gel, stained with VistraGreen (Amersham, Braunschweig, Germany) and analyzed with ImageQuant Software on a Storm 840 Phosphorlmager. Relative product abundance was evaluated by densitrometric analysis and expressed in relation to controls and corrected for ANC-housekeeper expression. Relative expression was determined for each of two independent series of experiments, which resulted in comparable results.
Statistical analysis
Data were expressed as means±SEM and analysed by Student's t-test for unpaired data. Statistical significance was defined as P<0.05.
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Results |
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Morphological analysis
Light microscopy revealed focal segmental deposition of homogenous or finely granular periodic acid-Schiff-positive material in the capillary loops. Capillary dilatation and endothelial cell damage were also apparent in nephritic animals at day 2. An increase of this damage and focal segmental capillary loop necrosis was observed on day 7 (Figure 1). No hypercellularity and only few cellular crescents (5%) were noted.
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Immunohistochemistry for FA/11 positive cells showed occasional macrophages within glomeruli (0.4±0.2/GCS) and some macrophages in the interstitium (3.8±0.3/HPF) of control mouse kidneys. Interstitial macrophages were increased especially around Bowmans capsule in NTS injected animals on day 2 (5.2±0.2/HPF) and day 7 (5.3±0.3/HPF) as compared to controls (3.5±0.2/HPF in controls; P<0.05) (Figure 1). No significant increase of macrophages within glomeruli of NTS injected animals was found either on day 2 (0.3±0.1/GCS) or on day 7 (0.5±0.3/GCS vs 0.5±0.2/GCS in controls).
Chemokine expression
Chemokine transcript levels from cortical extracts were determined by RNase protection assay on day 2 and 7. Levels of mRNA for MCP-1 were increased 5-fold on day 2 as compared to controls, whereas RANTES and IP-10 were detectable, but not increased as compared to controls (Figure 2). On day 7 an increase of MCP-1 (4-fold), RANTES (5-fold), and IP-10 (6-fold) occurred as compared to controls (Figure 2
). MIP-1
and MIP-2 were not detectable in mouse kidney extracts. MIP-1ß and TCA-3 were detectable on day 2 in experimental animals but expression levels were low. We also noted a slight basal expression of lymphotactin and eotaxin in both NTS and control groups (Figure 2
). These results were confirmed in two series of independent experiments. The MCP-1 signal shows several bands. This does not reflect different splice variants but is caused by a principle problem of the multi-probe protection assay system. As eleven different probes are used in parallel in one reaction, the RNase digestion conditions are not optimal for each probe. This can result in the appearance of several bands for a probe. In fact in control experiments the MCP-1 signal could be reduced to one single band by reducing the concentration of RNase. However, under these conditions hybrids with other probes are not digested to completion. Clearly in the case of MCP-1 the multiple bands can be assigned to MCP-1.
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Chemokine receptor expression
Chemokine receptor transcripts were determined by RT-PCR using total RNA both from renal cortex and isolated glomeruli. In renal cortex cDNA for CCR1, CCR2 and CCR5 could be detected. Nephritic kidneys showed higher cDNA levels at day 2 and day 7, respectively, for CCR1 (2-fold/2-fold), CCR2 (4-fold/4-fold), and CCR5 (2-fold/5-fold) (Figure 3). We could not detect cDNA for CCR4 using standard PCR-conditions. Results were confirmed in two series of independent experiments which were analysed twice. Analysing isolated glomeruli by RT-PCR, cDNA for CCR1, 2 and 5 could be detected. According to densitrometric analysis glomeruli from nephritic kidneys showed elevated cDNA levels on day 2 and day 7, respectively, for CCR1 (6-fold/4-fold), CCR2 (5-fold/5-fold), and CCR5 (8-fold/12-fold) (Figure 3
).
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Discussion |
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Chemokines are supposed to play a role in attracting leukocytes to sites of injury in NTS nephritis [2]. Therefore it was of interest to find elevated CC-chemokine levels in nephritic kidneys, as noted in previous studies: MCP-1 is increased at 24 h [24] and up to 48 h [25] in rat models of nephrotoxic nephritis. MCP-1 protein was detected immunohistochemically in glomeruli, vascular endothelial cells, and tubular epithelial cells on day 3 and 6 of rat NTS nephritis but not in controls [26]. Studies of murine NTS nephritis found that MCP-1 and RANTES were increased at day 3 [10] and remained elevated until day 14 [14]. In concordance with the latter study we could also detect an increase of RANTES levels. RANTES expression has been shown to be increased in the rat model [11] and generally seems to start late after the induction of disease [11,14]. Again in concordance with Lloyd et al., we could not detect an increase of MIP-1, MIP-1ß or TCA-3 in nephritic kidneys. This is in contrast to results obtained in rat NTS [11,24,27]. We also observed an increase of IP-10 in mouse NTS nephritis as previously reported in rat nephritis [28]. These differences of the chemokine expression profile between rat and mouse may indicate species specificities.
The effect of a chemokine depends on the presence of chemokine receptors on target cells. Whereas chemokines can be produced by multiple cells in inflammatory states, CCR expression is mostly restricted to leukocytes. The low basal level of expression of CCR1, CCR2 and CCR5 which was observed in control kidneys is most likely due to the presence of leukocytes in the renal tissue. It should be noted that kidneys were not perfused free of blood prior to processing so that blood leukocytes would be present in the tissue. In the accelerated model of NTS nephritis we found predominantly an interstitial and periglomerular leukocyte infiltration. It is reasonable to assume that the increase of CCR transcripts in nephritic cortex arises from the interstitial macrophage and T-cell infiltrate, an assumption that will need future immunohistological evaluation. Isolated glomeruli were only studied by RT-PCR because of the small, limited amount of material isolated. Interestingly, the upregulation of CCR transcripts in nephritic glomeruli was about 3-fold higher than the upregulation observed in renal cortex. As the number of glomerular mononuclear cells is not markedly increased in this mouse model [29], the marked increase in glomerular mRNA levels for CCR either results from their upregulation on the mononuclear cells present, or perhaps from adherent periglomerular leukocytes in the isolated glomerular preparations.
CCR1 (receptor for MIP-1, MCP-3 and RANTES) is expressed on monocytes and activated T-cells. Interestingly we could recently show that human mesangial cells express this receptor after stimulation with IFN-
[30]. Potentially the increase of CCR1 receptor expression in our model could occur on mesangial cells. As CCR1 knockout mice have recently been reported to have worse NTS disease than their wild-type controls [31], the CCR1 receptor might even act in a counter-regulatory fashion.
CCR2 is so far the only known receptor for MCP-1. MCP-1 is known to play a functional role in NTS-nephritis as blocking of MCP-1 improved mononuclear cell infiltration, glomerular and interstitial damage and proteinuria [6,12,13,26,32]. Our results show increased CCR2 levels by RT-PCR but not in RNase protection assay in nephritic renal cortex. This may be due to the higher sensitivity of RT-PCR. In any case there is a definite upregulation of CCR-2 transcripts in isolated glomeruli by RTPCR.
CCR5 (receptor for RANTES, MIP-1ß and MIP-1) can be expressed on monocytes, activated T-cells and NK cells. By in situ hybridization Eitner et al. reported CCR5 expression only on infiltrating cells in human allograft rejection but could not detect CCR5 on intrinsic renal cells [33]. By immunohistochemistry we also detected CCR5 only on CD3-positive T cells in human transplant biopsies as well as in a variety of human glomerular and tubulointerstitial diseases [34].
Unfortunately at present the lack of appropriate antibodies for murine chemokine receptors does not allow assignment of the various CCRs to specific cell types in the mouse kidneys. It should be noted, however, that there is a reasonable correlation between the upregulation of mRNA for the chemokine receptors and their respective ligands in the murine NTS nephritis studied (Figure 5). The future assignment of CC-chemokine receptors to specific cell types will be a first step towards identification of potential therapeutic targets for chemokine receptor blockade. Because of binding of CC-chemokines to multiple receptors and the presence of multiple chemokine receptors an ideal receptor antagonist should be able to block the binding of several chemokines to multiple receptors in parallel. Such broad-spectrum antagonists may prove to be of considerable therapeutic value in the future.
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Acknowledgments |
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Notes |
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References |
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