Increased expression of monocyte chemoattractant protein-1 (MCP-1) by renal epithelial cells in culture on exposure to calcium oxalate, phosphate and uric acid crystals

Tohru Umekawa, Nasser Chegini1 and Saeed R. Khan

Department of Pathology and Laboratory Medicine and 1 Institute for Wound Research, Department of Obstetrics and Gynecology, University of Florida College of Medicine, Gainesville, FL, USA



   Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. During the development of non-infectious kidney stones, crystals form and deposit in the kidneys and become surrounded by monocytes/macrophages (M/M). We have proposed that in response to crystal exposure renal epithelial cells produce chemokines, which attract the M/M to the sites of crystal deposition. We investigated the expression of monocyte chemoattractant protein-1 (MCP-1) mRNA and protein by NRK52E rat renal tubular epithelial cells exposed to calcium oxalate (CaOx), brushite (Br, a calcium phosphate) and uric acid (UA) crystals.

Methods. Confluent cultures of NRK52E cells were exposed to CaOx, Br or UA at a concentration of 250 µg/ml (66.7 µg/cm2). They were exposed for 1, 3, 6, 12, 24 and 48 h for isolation of mRNA and 24 h for ELISA to determine the secretion of protein into the culture medium. Since cells are known to produce free radicals on exposure to CaOx crystals we also investigated the effect of free radical scavenger catalase on the crystal induced expression of MCP-1 mRNA and protein.

Results. Exposure of NRK52E cells to the crystals resulted in increased expression of MCP-1 mRNA and production of the chemoattractant. CaOx crystals were most provocative while UA the least. Treatment with catalase had a negative effect on the increased expression of both MCP-1 mRNA and protein, which indicates the involvement of free radicals in up-regulation of MCP-1 production.

Conclusion. Exposure to both CaOx and calcium phosphate crystals stimulates increased production of MCP-1. Free radicals appear to be involved in this up-regulation. Results indicate that MCP-1, which is often associated with localized inflammation, may be one of the chemokine mediators associated with the deposition of various urinary crystals in the kidneys during kidney stone formation. Because of the small number of experiments performed here, results must be confirmed by more extensive studies with larger sample size.

Keywords: calcium oxalate; calcium phosphate; free radicals; macrophages; MCP-1; monocyte chemoattractant protein-1



   Introduction
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Mammalian urine is normally metastable with respect to calcium phosphate (CaP) and calcium oxalate (CaOx) and under suitable conditions, promotes their nucleation in the renal tubules [1]. Animal model studies have shown that most crystals formed in the renal tubular lumens are excreted as crystalluria particles [2]. However, during certain hyperoxaluric conditions, at first crystals remain in the renal tubules and then move into the interstitium. Once in the interstitium, macrophages and multinucleated giant cells surround the crystals. We postulated that this was a protective response to eliminate the crystals and stop the development of kidney stones. Recent studies have confirmed our hypothesis by demonstrating that macrophages in vitro can internalize CaOx crystals and eventually dissolve them [3]. How are the macrophages recruited to the sites where crystals have moved into the interstitium? We as well as other investigators have shown that exposure to moderately high levels of oxalate and CaOx crystals is injurious to renal epithelial cells [46]. We hypothesized that inflammation following crystal-induced injury plays a significant role in the pathogenesis of CaOx nephrolithiasis [2,7] and that oxalate and CaOx crystals stimulate renal epithelial cells to produce chemokines, which attract the macrophages. Support for this hypothesis comes from experiments in which we exposed a rat renal epithelial cell line, the NRK52E to oxalate and CaOx crystals and showed a significant increase in the expression of monocyte chemoattractant protein-1 (MCP-1) mRNA and protein [8]. MCP-1 mRNA expression and protein production increased more significantly after exposure to CaOx crystals than to oxalate alone and reduced significantly after catalase treatment. Since both oxalate and CaOx crystals appear to exert their influence upon the epithelial cells through the production of free radicals [4,5,9], they may also be involved in stimulation of MCP-1 production.

There are three major types of non-infectious kidney stones, CaOx, CaP and uric acid (UA) stones [1]. We have already shown that CaOx crystals provoke the renal epithelial cells. To further assess the implication of MCP-1 expression in abnormalities associated with non-infectious kidney stones the current study examined whether CaP and UA crystals also stimulate renal epithelial cells to produce MCP-1. Since brushite (Br) is considered most likely to form in the renal tubules we used the Br form of CaP in our studies. CaOx crystals were used for comparison purposes.



   Subjects and methods
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Cell culture
A normal rat kidney epithelial-derived cell line, NRK52E, was obtained from American Type Culture Collection (CRL-1571; Manasses, VA). They were maintained as continuously growing monolayers in 75 cm2 Falcon T-flask (Fisher, Atlanta, GA) in culture in 1:1 ratio Dulbecco's modified essential medium nutrient mixture and F-12 (DMEM/F-12, Gibco BRL, Grand Island, NY) containing 4% fetal calf serum, 15 mmol/l HEPES, 20 mmol/l sodium bicarbonate, 0.5 mmol/l sodium pyruvate, 17.5 mmol/l glucose, streptomycin and penicillin at 37°C in a 5% CO2 air atmosphere incubator. Under these conditions, cells achieved confluence, they were washed with serum- and sodium pyruvate-free DMEM/F-12 media and then the cells were exposed to calcium oxalate monohydrate (COM), calcium phosphate (Br) and UA crystal (Sigma, St Louis, MO) in the same medium (20 ml). Based on our earlier experience [911] the cells were incubated for 1, 3, 6, 12, 24 and 48 h for isolation of mRNA and culture medium for ELISA were collected after an exposure to COM, Br or UA for 24 h. COM, Br or UA were added to the culture medium at a concentration of 250 µg/ml (66.7 µg/cm2). Untreated cells served as the control.

Reverse transcriptase for real-time PCR
The transcription of MCP-1 mRNA in NRK52E was determined using real-time PCR. Total RNA was isolated from treated and untreated cells (1x106 cells) using TRIZOL Reagent (Gibco BRL) according to the manufacturer's protocol. Two micrograms of total RNA was reverse transcribed to complementary DNA (cDNA). In brief, 50 µl reactions contained 3 µl of 100 mM MgCl, 1.25 µl of RNase inhibitor, 5 µl of 10x PCR buffer, 10 µl of 10 mM dNTP mix, 1.3 µl of Oligo d(T), 1.5 µl of reverse transcriptase (Gibco BRL). This mixture was incubated for 60 min at 37°C. Reaction was stopped by heating to 94°C for 5 min.

Primers for real-time PCR
Primers were designed using Primer Express software (PE Applied Biosystems, Foster City, CA). According to the cDNA sequences of rat MCP-1 (accession no. M57441) and rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (accession no. NM_17008) as follows: 5'-CAGATCTCTCTTCCTCCACCACTAT-3' (forward primer, bases 17–41) and 5'-CAGGCAGCAACTGTGAACAAC-3' (reverse primer, bases 69–89) for MCP-1; 5'-TGCCAAGTATGATGACATCAAGAA-3' (forward primer, bases 780–803) and 5'-AGCCCAGGATGCCCTTTAGT-3' (reverse primer, bases 831–850) for GAPDH.

Real-time quantitative PCR
PCR product was directly monitored by measuring the increase in fluorescence of dye (SYBR GREEN, PE Applied Biosystems) bound to the amplified double-stranded DNA. The parameter of threshold cycle (CT) was defined as the fractional cycle number at which fluorescence exceeds a threshold level. The comparative CT method quantifies the amount of mRNA relative to that of a reference sample, termed the calibrator, for comparison of the expression level of every unknown sample. The calibrator sample was analysed on every assay plate with. To normalize the relative amount of MCP-1 mRNA, GAPDH was chosen as an internal reference. The changes in expression are given by unknown samples of interest.

All PCR reactions were performed using an ABI Prism 7700 Sequence Detection System (PE Applied Biosystems). For each PCR run, a master mix was prepared: 1x SYBR PCR buffer, 3 mM MgCl2, 200 µM dATP, dCTP and dGTP, 400 µM dUTP, 300 µM primer set for MCP-1 and GAPDH, and 1.25 U of AmpliTaq Gold DNA polymerase. Five microlitres of diluted (1:20) cDNA was added to 45 µl of the PCR master mix. After an initial 10 min denaturation at 95°C, the thermal cycling comprised 40 cycles of denaturation at 95°C for 15 s, and annealing and extension at 60°C for 1 min.

MCP-1 measurements by enzyme linked-immunosorbent assay (ELISA)
Cells were maintained as continuously growing monolayers in 25 cm2 Falcon T-flask (Becton Dickinson Labware, Franklin Lakes, NJ) and cultured in the same condition for mRNA isolation, which was mentioned before. Under these conditions, cells achieved confluence, they were washed with serum- and sodium pyruvate-free DMEM/F-12 media and then the cells were exposed to COM, Br or AU in the same medium (10 ml). The content of MCP-1 in the culture supernatants was determined by ELISA using ELISA kit for rat MCP-1 (Endogene, Woburn, MA) according to the manufacturer's instructions, repeated four times. Protein concentration was determined using BCA Protein Assay Kit (Pierce, Rockford, IL). Data are presented by mean±SD in which values were determined in duplicate for two different samples.

Experiment with free radical scavengers
To evaluate the protective effect of free radical scavengers, COM, Br or UA were exposed to NRK-52E with pre- (24 h) and co-treatment (6 h for mRNA and 24 h for ELISA and LDH analysis) with catalase (Sigma; 2000 U/ml) in the incubation medium.

Statistical analysis
All the experiments were performed twice in duplicates and the results are reported as mean±SD. Statistical analysis for comparison between the treatment groups was performed using non-parametric Kruskal–Wallis and Wilcoxian rank tests to address group and pair wise comparisons. A P value of <0.05 denotes the presence of statistically significant difference.



   Results
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Detection of MCP-1 mRNA transcripts in NRK52E
Figure 1Go shows the standard curves and dissociation curves of the real-time PCR for GAPDH and MCP-1, indicating the optimal conditions of the assays.



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Fig. 1.  (A and B) Standard curve and dissociation curve of real-time PCR for GAPDH. (C and D) Standard curve and dissociation curve of real-time PCR for MCP-1.

 
NRK52E was treated with COM, Br or UA (66.7 µg/cm2) for 1–48 h. As presented in Figure 2Go, the MCP-1 mRNA level increased significantly after stimulation with all three types of crystals. The peak levels of MCP-1 mRNA were observed in the first 6 h after the exposure returning to control levels after 24 h. COM exposure resulted in the largest increase in MCP-1 mRNA, >200-fold compared with control. Exposure to Br and UA resulted in a significant increase in MCP-1 mRNA but at much lower levels, ~45-fold compared with control with Br stimulation and ~2-fold compared with control after UA stimulation.



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Fig. 2.  Relative quantity of MCP-1 mRNA stimulated with various crystals for 1–48 h normalized to GAPDH. The MCP-1 mRNA value was determined by dividing the average MCP-1 value by the average GAPDH value. (A) Exposure to COM crystals (mean±SD, *P<0.05 vs control). (B) Exposure to Br crystals (mean±SD, *P<0.05 vs control). (C) Exposure to UA crystals (mean±SD, *P<0.05 vs control).

 
The catalase treatment had a significant inhibitory effect on COM and Br crystal-induced increases in the MCP-1 mRNA and not so significant an effect on the UA induced increase in MCP-1 mRNA (Figure 3Go). The increases in MCP-1 mRNA were lower in catalase-treated cells. However, the levels of MCP-1 mRNA in cells stimulated with crystals were still higher than those in the control cells, which were not exposed to various crystals.



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Fig. 3.  Relative quantity of MCP-1 mRNA in cells pre- and co-treated with catalase and stimulated with various crystals for 6 h normalized to GAPDH. The MCP-1 mRNA value was determined by dividing the average MCP-1 value by the average GAPDH value. (A) Exposure to COM crystals (mean±SD, *P<0.05 vs control and COM-treated cells only). (B) Exposure to Br crystals (mean±SD, *P<0.05 vs control as well as Br treated-cells only). (C) Exposure to UA crystals (mean±SD, *P<0.05 vs control).

 

Production of MCP-1 protein by NRK52E cells
There was marked up-regulation of MCP-1 protein synthesis by NRK52E cells after 24 h exposure to COM, Br and UA crystals (Figure 4Go). The amount of MCP-1 protein production with COM crystal stimulation was significantly higher compared with exposure to the Br or UA crystals. Catalase treatment significantly reduced the production of MCP-1 protein by crystal-exposed cells. The amount of protein synthesized was however, still higher than that synthesized by control cells not exposed to various crystals.



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Fig. 4.  The effect of COM, Br or UA with or without catalase treatment on MCP-1 released into the culture media of NRK52E after 24 h of incubation (mean±SD, *P<0.05 vs control as well as COM or Br exposed).

 



   Discussion
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Leukocyte infiltration of the renal interstitium is a common occurrence in inflammatory diseases and is mediated by numerous chemotactic cytokines and chemokines [10], such as IL10, IP10, MCP-1 and RANTES. Most chemokines can be produced by a wide variety of cell types after proper stimulation with pro-inflammatory cytokines and exert their effect on one or more target cell populations. A diverse population of renal cells including proximal tubular epithelial cells, cortical epithelial cells, glomerular endothelial cells and mesangial cells expresses these cytokines and chemokines. MCP-1 is a member of the CC chemokine subfamily and attracts monocytes and other leukocytes. Pro-inflammatory stimuli such as TNF{alpha}, IL1-ß, IFN-{gamma} and lipopolysaccharide rapidly induce MCP-1 expression and production. Production of MCP-1 is influenced by the presence of reactive oxygen species [11,12]. MCP-1 mRNA expression is increased in mouse mesangial cells in the presence of superoxide ions. On the other hand, antioxidants inhibit the IL-1 induced expression of MCP-1 in human mesangial cells [13]. MCP-1 levels are elevated in urine of patients with many glomerulonephropathies [10] and correlate with glomerular injury and monocyte infiltration. Patients with active lupus nephritis show higher urinary levels of MCP-1 than controls or patients with inactive disease. MCP-1 expression and production is also increased in animal models of renal diseases such as nephrotoxic serum nephritis in mice and rats, anti-thymocyte-1 nephritis in rats, systemic lupus nephritis in mice and rabbits, glomerulonephritis in rabbits and rats, etc.

Diverse cell types have been shown to express and produce MCP-1 when exposed to a variety of crystals. Silica stimulates alveolar macrophages and alveolar type II cells to produce a host of cytokines and chemokines including MCP-1 [12,14]. The cellular response is mediated in part by oxidant stress and can be attenuated by antioxidant treatment. Both crocidolite and chrysotile asbestos [15] as well as talc [16] stimulate mesothelial cells lining the pleura to produce of MCP-1. Upon interaction with monosodium urate (MSU) synovial lining cells produce MCP-1 [17].

Results of our studies presented here and earlier show that NRK52E cells express MCP-1. Its expression and production is up-regulated by exposure to CaOx, calcium phosphate and to a limited degree to UA crystals. To our knowledge this is the first report of up-regulation of MCP-1 production by renal epithelial cells in response to stimulation by Br and UA crystals. CaOx monohydrate crystals elicited the most response, both in term of mRNA expression and protein production. Because of the small sample size and in vitro nature of our studies our results need to be confirmed using larger sample size and in vivo studies in animal models.

Differences in responses of renal epithelial cells to exposure to the three types of crystals reported here may depend upon their tendencies to induce free radical generation. As we mentioned above, mediation of MCP-1 production by free radicals has been demonstrated in many systems [1619]. Renal epithelial cell response to oxalate ions and CaOx crystals involves the production of free radicals [4,5,9]. Interaction between cultured human umbilical vein endothelial cells and crystals of CaOx, MSU, hydroxyapatite and calcium pyrophosphate dihydrate results in the production of superoxide anions [18]. Here we show that the inflammatory response of renal epithelium to not only CaOx crystals but also Br may be mediated by free radical production. Expression and production of MCP-1 was significantly reduced by catalase treatment but not totally neutralized. Catalase reduces hydrogen peroxide and is only one of the three major enzymes which detoxify reactive oxygen species. Since catalase treatment did not completely neutralize the response, many other reactive oxygen species may be produced during the interactions. Catalase also showed only a limited effect on MCP-1 production by cells on exposure to UA crystals; a different mechanism may be involved. UA exposure had only a limited effect on the production of MCP-1, perhaps because encounter with UA may not involve free radical production. Moreover, UA acid itself is an antioxidant. The limited production of MCP-1 by cells exposed to UA crystals as seen here may be a result of physical interaction, since both obstruction and stretching have been shown to induce MCP-1 production [19,20].

Crystal deposition diseases are generally associated with localized inflammation. Intra-articular deposition of hydroxyapatite, octacalcium phosphate, tri-calcium phosphate or calcium pyrophosphate dihydrate lead to inflammation and are established causes of inflammatory arthritis [21]. Tissue culture as well as rat model studies have shown that both oxalate and CaOx crystals induce renal epithelial cells to increase the production of a variety of macromolecules such as osteopontin, prothrombin, heparan sulfate and various members of the inter-{alpha}-inhibitor family including bikunin [7,22]. Almost all these molecules play some role in inflammatory cascade. CaOx crystal deposition in the kidneys begins with retention in the renal tubules followed by migration to the interstitium. Intratubular crystals are seen in association with membranous vesicles and other cellular degradation products while macrophages, monocytes and polymorphonuclear leukocytes surround the interstitial crystals [2,23]. Crystals are also seen inside the giant cells.

Deposition of urate crystals in the kidneys during chronic gouty arthritis is already known to cause intense inflammatory reaction. Microtophi containing urate crystals surrounded by macrophages, lymphocytes and giant cells develop in the renal interstitium. In a rat model of urate deposition disease induced by feeding UA and oxonic acid, animals quickly developed amorphous as well as crystalline deposits in dilated tubules of the renal papillae [24]. Amorphous material was identified as UA and crystalline material, which was often organized in tophi, as urate. Many tubules were filled with cellular debris. Tubular epithelial cells appeared damaged with flattened morphology, scant microvilli, and intercellular as well as intracellular spaces, which at later time periods frequently contained crystal ghosts. Mitotic figures were also, though rarely, seen. Inflammatory cells including leukocytes and neutrophils were seen in the interstitium near the damaged tubules. In separate studies, response of renal epithelial cells (MDCK line) to an exposure to UA and MSU crystals was characterized by release of lysosomal and cytosolic enzymes into the medium, crystal attachment to the apical cell surfaces, their endocytosis and movement beneath the monolayer [25]. UA crystals were less reactive than MSU crystals. It was concluded those interstitial deposits of UA and urate crystals in the kidneys could be derived from intratubular deposits and provoke inflammatory response. During UA nephrolithiasis, supersaturation of urine with undissociated UA may induce precipitation of UA and/or MSU in the renal tubules, from where they most probably move into the interstitium. Chronic localized inflammation associated with interstitial crystal deposition may cause significant morbidity and damage with eventual sloughing of the dead tissue and ulceration of the crystals to papillary surface to form a stone nidus. This proposed sequence of events is similar to what we have suggested for the development of CaOx nephrolithiasis [2].



   Acknowledgments
 
Supported in part by the National Institutes of Health grants #RO1 DK53962 and DK59765.



   Notes
 
Correspondence and offprint requests to: Saeed R. Khan, Department of Pathology and Laboratory Medicine, University of Florida College of Medicine, Box 100275, Gainesville, FL 32610-0275, USA. Email: khan{at}pathology.ufl.edu Back



   References
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 15. 5.02
Accepted in revised form: 3.10.02