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Am J Physiol Renal Physiol 294: F236-F244, 2008. First published October 31, 2007; doi:10.1152/ajprenal.00331.2007
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Homocysteine stimulates monocyte chemoattractant protein-1 expression in the kidney via nuclear factor-{kappa}B activation

Sun-Young Hwang,1,3,* Connie W. H. Woo,2,3,* Kathy K. W. Au-Yeung,3 Yaw L. Siow,2,3,4 Tong Y. Zhu,3 and Karmin O1,2,3

1Department of Animal Science and 2Department of Physiology, University of Manitoba, Winnipeg; 3Canadian Center for Agri-Food Research in Health and Medicine, St. Boniface Hospital Research Center, Winnipeg; and 4Center for Research and Treatment of Atherosclerosis, University of Manitoba, Winnipeg, Manitoba, Canada

Submitted 16 July 2007 ; accepted in final form 22 October 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Hyperhomocysteinemia, or an elevation of blood homocysteine (Hcy) levels, is associated with cardiovascular disorders. Although kidney dysfunction is an important risk factor causing hyperhomocysteinemia, the direct effect of Hcy on the kidney is not well documented. There is a positive association between an elevation of blood Hcy levels and the development of chronic kidney disease. Inflammatory response such as increased chemokine expression has been implicated as one of the mechanisms for renal disease. Monocyte chemoattractant protein-1 (MCP-1) is a potent chemokine that is involved in the inflammatory response in renal disease. Nuclear factor-{kappa}B (NF-{kappa}B) plays an important role in upregulation of MCP-1 expression. We investigated the effect of hyperhomocysteinemia on MCP-1 expression and the molecular mechanism underling such an effect in rat kidneys as well as in proximal tubular cells. Hyperhomocysteinemia was induced in rats fed a high-methionine diet for 12 wk. The MCP-1 mRNA expression and MCP-1 protein levels were significantly increased in kidneys isolated from hyperhomocysteinemic rats. The NF-{kappa}B activity was significantly increased in the same kidneys. Pretreatment of hyperhomocysteinemic rats with a NF-{kappa}B inhibitor abolished hyperhomocysteinemia-induced MCP-1 expression in the kidney. To confirm the causative role of NF-{kappa}B activation in MCP-1 expression, human kidney proximal tubular cells were transfected with decoy NF-{kappa}B oligodeoxynucleotide to inhibit NF-{kappa}B activation. Such a treatment prevented Hcy-induced MCP-1 mRNA expression in tubular cells. Our results suggest that hyperhomocysteinemia stimulates MCP-1 expression in the kidney via NF-{kappa}B activation. Such an inflammatory response may contribute to renal injury associated with hyperhomocysteinemia.

hyperhomocysteinemia

HYPERHOMOCYSTEINEMIA, A CONDITION of elevated homocysteine (Hcy) level in the blood, is an independent risk factor for cardiovascular disorders (7, 17, 21). Impaired kidney function is one of the common factors causing hyperhomocysteinemia (20, 37). In patients with chronic renal failure, there is a marked elevation of plasma Hcy levels and a striking increase in the risk for vascular disease (20). Although renal dysfunction or failure is an important factor causing hyperhomocysteinemia, the effect of Hcy, at abnormally high concentrations, on the kidney is unknown. Recent findings from an epidemiological study in the general population have revealed a positive association between an elevation of serum Hcy levels and the development of chronic kidney disease (20). Results from animal studies also have suggested that hyperhomocysteinemia may cause renal injury (10, 12, 40). In contrast to Hcy-induced atherosclerosis, little information is available regarding the mechanisms by which Hcy may exert adverse effect on the kidney. We have demonstrated that hyperhomocysteinemia causes an increased nitric oxide production leading to the formation of peroxynitrite, a potent oxygen free radical, in rat kidneys (40). Further analysis reveals that activation of a transcription factor, namely, nuclear factor-{kappa}B (NF-{kappa}B) is involved in Hcy-induced inducible nitric oxide synthase expression in the kidney (40). Other investigators have reported that hyperhomocysteinemia causes vascular remodeling and tubulointerstitial injury in rat kidneys and glomerular damage in hypertensive rats with hyperhomocysteinemia (10, 12). Our recent study suggested that elevation of Hcy levels in the kidney during ischemia-reperfusion also contributes to oxidative stress and renal injury (23).

Activation of NF-{kappa}B has been shown to be involved in the onset of glomerulosclerosis (14, 27). In the resting cell, NF-{kappa}B resides in the cytoplasm in an inactive form that is associated with an inhibitory protein (I{kappa}B). Although several isoforms of NF-{kappa}B such as p50/p65, (p65)2, and c-rel/p65 protein complexes have been detected in various types of cells, the predominant NF-{kappa}B isoform in kidney cells is thought to be a p50/p65 heterodimer (14, 31). The inhibitory protein I{kappa}B{alpha} is one of the best-characterized forms of I{kappa}B (3, 9, 18, 29). Upon stimulation, there is a rapid phosphorylation of I{kappa}B{alpha} and subsequent degradation of I{kappa}B{alpha} by the proteasome, leading to the release of NF-{kappa}B. After dissociation from I{kappa}B, the free NF-{kappa}B can enter the nucleus, a process termed translocation. Once inside the nucleus, NF-{kappa}B binds to the {kappa}B binding motifs in the promoters or enhancers of its target genes (9, 18, 29). Activation of NF-{kappa}B has been shown in the kidney after ischemia-reperfusion injury (31) as well as in the atherosclerotic lesions (4, 16). Once activated, NF-{kappa}B is able to upregulate gene expression of many inflammatory molecules, including monocyte chemoattractant protein-1 (MCP-1). MCP-1 is a potent chemokine that stimulates the migration of leukocytes, including monocytes, into the intima of arterial wall and other tissues, including kidney. Little MCP-1 is detectable in normal kidneys (8, 24, 25). However, MCP-1 gene expression is greatly increased in kidneys of patients and animal models with kidney diseases (11, 26, 28, 32, 36). Although hyperhomocysteinemia is a common clinical finding in patients with chronic kidney disease, the effect of Hcy on the kidney is not clear. In the present study, we investigated the effect of Hcy on MCP-1 expression in the kidney and the underlying mechanism of such an effect in hyperhomocysteinemic rats and in human proximal tubular cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal model. Male Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA) aged 8 wk were divided into three groups and maintained for 12 wk on the following types of diet: 1) control diet (regular diet), consisting of Lab Diet rat diet 5001 (PMI Nutrition International, St. Louis, MO); 2) high-methionine diet, consisting of regular diet plus 1.7% (wt/wt) methionine; and 3) high-cysteine diet, consisting of regular diet plus 1.2% (wt/wt) cysteine. Each group consisted of 12 rats. Results from our previous studies demonstrated that hyperhomocysteinemia could be induced in rats by feeding a high-methionine diet (2, 35, 39, 40). The serum total Hcy (tHcy) concentration was measured with the IMx Hcy assay, which was based on fluorescence polarization immunoassay technology (Abbott Diagnostics, Abbott Park, IL) (19). All procedures were performed in accordance with the Guide for the Care and Use of Experimental Animals published by Canadian Council on Animal Care and approved by University of Manitoba Protocol Management and Review Committee.

Cell culture. Human kidney cortex proximal tubular cells (HK-2; CRL-2190) were purchased from the American Type Culture Collection (ATCC). Cells were cultured in keratinocyte-serum free medium (GIBCO-BRL 17005-042) with 5 ng/ml recombinant epidermal growth factor and 0.05 mg/ml bovine pituitary extract according to the ATCC instruction. THP-1, a human monocytic cells line, were purchased from ATCC and cultured in RPMI 1640 medium (Hyclone). L-Hcy was prepared from L-Hcy thiolactone (Sigma-Aldrich, St. Louis, MO) as described previously (38). In brief, L-Hcy thiolactone was hydrolyzed in NaOH (5 M) to remove the thiolactone group and then neutralized with HCl. Freshly prepared L-Hcy was used in the experiments.

Measurement of MCP-1 mRNA and protein. Total RNAs were isolated from kidneys or HK-2 cells with TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA). The MCP-1 mRNA was measured by a real-time polymerase chain reaction (PCR) analysis using the iQ5 real-time PCR detection system (Bio-Rad, Hercules, CA). In brief, 2 µg of total RNA were converted to cDNA by reverse transcriptase. The reaction mixture of real-time PCR contained 0.4 µM of 5' and 3' primer and 2 µl of cDNA product in iQ-SYBR green supermix reagent (Bio-Rad). The primers used for rat MCP-1 were (forward) 5'-CAGAAACCAGCCAACTCTCA-3' and (reverse) 5'-AGACAGCACGTGGATGCTAC-3' (GenBank accession no. AF058786), and those used for rat GAPDH were (forward) 5'-TCAAGAAGGTGGTGAAGCAG-3' and (reverse) 5'-AGGTGGAAGAATGGGAGTTG-3' (GenBank accession no. NM_017008). The primers for human MCP-1 were (forward) 5'-CCGAGAGGCTGAGACTAACC-3' and (reverse) 5'-GGAATGAAGGTGGCTGCTAT-3' (GenBank accession no. NM_002982), and those for human GAPDH were (forward) 5'-ATCATCCCTGCCTCTACTGG-3' and (reverse) 5'-GTCAGGTCCACCACTGACAC-3' (GenBank accession no. NM_002046). All primers were synthesized by Invitrogen. The relative change in MCP-1 mRNA expression was determined by the fold change analysis in which the degree of change = Formula, where Ct = (CtMCP-1 – CtGAPDH)treatment – (CtMCP-1 – CtGAPDH)control (13). Ct was the cycle number at which the fluorescence signal crossed the threshold, which was determined by iQ5 Optical System software (version 2; Bio-Rad). The MCP-1 protein in the kidney tissue was quantified using a commercial ELISA kit (Biosource International, Camarillo, CA).

Immunohistochemical staining. For detection of MCP-1 protein, kidneys were excised from rats fed different types of diet. A portion of the kidney was immersion-fixed in 10% neutral-buffered formalin overnight and then embedded in paraffin. Sequential 5-µm paraffin-embedded sections were immunostained using rabbit polyclonal antibodies (1:100) as primary antibodies against rat MCP-1 (PeproTech EC, Rocky Hill, NJ). Endogenous peroxidase was blocked with 0.3% H2O2 for 20 min. The secondary antibodies for immunostaining were biotin-conjugated anti-rabbit immunoglobulins (1:200; Dako Canada, Mississauga, ON, Canada). Sections were then treated with 3,3-diaminobenzidine (DAB)-H2O2 colorimetric substrate solution. The attached peroxidase catalyzed the H2O2-mediated oxidation of DAB to yield brown color. The area displayed brownish color indicating the MCP-1 protein adducts. For a negative control, nonspecific rabbit IgG was used as primary antibodies.

Myeloperoxidase activity assay. The myeloperoxidase (MPO) activity was measured in the kidney to assess the leukocyte infiltration into the tissue as described previously (5, 31). A portion of the kidney was homogenized in 3 ml of 50 mM potassium phosphate buffer (pH 6.0) containing 0.5% hexadecyltrimethylammoniun bromide. The homogenate was centrifuged at 20,000 g for 20 min, and the supernatant was collected. The reaction mixture containing 0.6 mM 3,3',5,5'-tetramethylbenzidine, 0.03 M hydrogen peroxide, 80 mM sodium phosphate buffer (pH 5.5), and an aliquot of supernatant was incubated at 25°C for 15 min. The reaction was stopped by the addition of 1 ml of 0.5 M sulfuric acid. The absorbance was measured at 450 nm. Peroxidase (Sigma-Aldrich) was used as an external standard. MPO activity was expressed as milliunits of enzyme activity per milligram of protein.

Western immunoblot analysis. Kidney I{kappa}B{alpha} and cyclooxygenase-2 (COX-2) protein levels were determined by Western immunoblot analysis. For the measurement of total I{kappa}B{alpha} and phosphorylated I{kappa}B{alpha} proteins, kidney proteins (100 µg/ml) were separated by electrophoresis on a 12.5% SDS polyacrylamide gel, whereas for COX-2, the electrophoresis was carried out on a 8% SDS polyacrylamide gel. Proteins on the gel were then transferred to a nitrocellulose membrane. The membrane was probed with rabbit anti-I{kappa}B{alpha} polyclonal antibodies or anti-phospho-I{kappa}B{alpha} (Ser32) polyclonal antibodies (New England Biolabs, Beverly, MA) and with rabbit anti-COX-2 polyclonal antibodies (Lab Vision, Fremont, CA). Horseradish peroxidase-conjugated secondary antibodies (Zymed, South San Francisco, CA) were used to develop the membranes. The I{kappa}B{alpha}, phospho-I{kappa}B{alpha}, or COX-2 protein bands were visualized using enhanced chemiluminescence reagents and analyzed with a gel documentation system (Bio-Rad Gel Doc1000 and Multi-Analyst version 1.1).

Electrophoretic mobility shift assay. Nuclear proteins were isolated, and electrophoretic mobility shift assay (EMSA) was performed to determine the NF-{kappa}B/DNA binding activity (2, 40). In brief, nuclear proteins (10 µg) were incubated with the reaction buffer for 15 min at room temperature, followed by incubation with 32P-end-labeled oligonucleotide containing the consensus sequence for the {kappa}B binding site (5'-GGGGACTTTCC-3') (Promega, Madison, WI). The reaction mixture was separated in a nondenaturing polyacrylamide gel (6%) that was later exposed to X-ray film at –80°C. The binding of labeled oligonucleotide to nuclear proteins was blocked by adding 100-fold excess of unlabeled specific competitor oligonucleotide to the reaction mixture. This was to confirm that the binding of 32P end-labeled oligonucleotide to NF-{kappa}B was sequence specific.

Decoy oligodeoxynucleotide transfection. Double-stranded oligodeoxynucleotide (ODNs; Sigma-Genosys) were prepared from complementary single-stranded phosphorothioate-bonded ODNs by melting at 95°C for 5 min, followed by a cool-down phase of 3 h to room temperature (22). The sequence of the single-stranded ODNs was 5'-AGTTGAGGGGACTTTCCCAGGC-3' for wild-type NF-{kappa}B and 5'-CATGTCGTCACTGCGCTCAT-3' for scrambled ODN (underlined letters denote phosphorothioate-bonded bases, whereas bold letters denote the consensus binding site) (6). Proximal tubular cells were treated with 100 nM ODN and 4 µl of Oligofectamine in supplement-free medium for 4 h. After the delivery of ODN into the tubular cells, the medium containing the decoy ODN was replaced with fresh medium containing supplement. Cells were subsequently incubated in the absence or presence of Hcy for a defined time period.

Chemotaxis assay. Monocyte migration was determined by monocyte chemotaxis assay using a 48-well Micro Chemotaxis Chamber (Neuro Probe, Gaithersburg, MD) as described in previous studies (14, 30). In brief, human proximal tubular cells were incubated in the presence or absence of Hcy (100 µM). After incubation, an aliquot of the medium (defined as conditioned medium) was transferred to the lower chamber of the Micro Chemotaxis Chamber. The lower and upper chambers were separated by a 5-µm pore size polycarbonate membrane (Neuro Probe). An aliquot of THP-1 monocyte suspension (2 x 106 cells/ml) was added to the upper chamber, and the monocytes were allowed to transmigrate from the upper chamber to the lower chamber for 2 h. After transmigration, the surface of the membrane facing the THP-1 cell suspension was scraped and washed three times according to the manufacturer's instructions. Cells that had migrated toward the conditioned medium were fixed and then stained with hematoxylin. The number of migrated monocytes was determined by counting the cells in five high-power fields under light microscopy. The results are expressed as a percentage of control.

Statistical analysis. The results were analyzed using two-tailed independent Student's t-test. The level of statistical significance was set at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Expression of MCP-1 in the kidney. Rats that were fed a high-methionine diet for 12 wk developed hyperhomocysteinemia (Fig. 1A). The high-methionine diet resulted in a significant increase in the serum Hcy levels. There was no significant change in serum tHcy levels in rats fed a high-cysteine diet compared with the control group (Fig. 1A). There was a significant increase in MCP-1 mRNA levels in kidneys isolated from rats fed a high-methionine diet (Fig. 1B). In accordance with these results, the levels of MCP-1 protein were significantly elevated in those kidneys (Fig. 1C). Cysteine is another sulfhydryl (SH)-containing amino acid. There was no change in MCP-1 mRNA and protein levels in rats fed a high-cysteine diet compared with the control group (Fig. 1, B and C). These results suggested that hyperhomocysteinemia-induced MCP-1 expression in the kidney was not due to a general effect produced by SH-containing amino acids. When pyrrolidine dithiocarbamate (PDTC), a known inhibitor for NF-{kappa}B activation (31, 40), was injected into to the high-methionine fed rats, the renal level of MCP-1 protein was reduced to that of the control group (Fig. 1C). These results suggested that increased MCP-1 expression in the kidney during hyperhomocysteinemia might be mediated via NF-{kappa}B activation. In addition, another inflammatory factor, namely, COX-2 protein, was also measured in kidneys by Western immunoblot analysis. There was no significant difference in the levels of COX-2 proteins in kidneys isolated from control, high-methionine-fed, and high-cysteine-fed rats (Fig. 1D).


Figure 1
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  		Fig. 1. Expression of monocyte chemoattractant protein-1 (MCP-1) mRNA in rat kidneys. Rats were fed a control diet (control), a high-methionine diet (Met), or a high-cysteine diet (Cys) for 12 wk. A: serum total homocysteine (tHcy) levels were measured. B: total RNAs were prepared from kidneys, and the MCP-1 mRNA levels were measured using a real-time PCR detection system. C: MCP-1 protein levels in kidneys were determined by ELISA. In 1 set of experiments, rats fed with a high-methionine diet for 12 wk were treated with pyrrolidine dithiocarbamate (PDTC; 100 mg/kg ip daily) for 3 days before euthanasia (Met + PDTC). D: COX-2 protein levels in the kidney tissue were measured by Western immunoblot analysis. β-Actin was used to confirm equal amount of protein loading for each sample. Results are means ± SE (n = 12). *P < 0.05 compared with control.

 
Distribution of MCP-1 protein in the kidney. The distribution of MCP-1 protein in the kidney was examined by immunohistochemical analysis. Compared with the control group, kidneys isolated from rats fed a high-methionine diet displayed an increased staining for MCP-1 protein in the cortex and medulla (Fig. 2). As a negative control, the immunohistochemical staining was performed with nonspecific antibodies (IgG), and no positive staining was observed (Fig. 2). Leukocyte infiltration was assessed by measuring the MPO activity in the kidney. The MPO activity was significantly increased in kidneys isolated from hyperhomocysteinemic rats (Fig. 3), indicating an elevation of leukocyte infiltration into the kidney during hyperhomocysteinemia.


Figure 2
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  		Fig. 2. Immunohistochemical staining of MCP-1 protein in rat kidneys. Rats were fed a control, Met, or Cys diet for 12 wk. Immunohistochemical staining for MCP-1 protein was performed with anti-MCP-1 antibodies. After counterstaining with Mayer's hematoxylin, MCP-1 protein was identified under light microscope at a magnification of x400. Arrowheads point to the areas positively stained with MCP-1 protein. As a negative control, immunohistochemical staining was performed using nonspecific rabbit IgG as primary antibodies. All staining analyses were performed in kidneys isolated from 12 rats of each group. Representative photos are shown.

 

Figure 3
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  		Fig. 3. Determination of myeloperoxidase (MPO) activity in rat kidneys. Kidneys were isolated from rats fed a control, Met, or Cys diet for 12 wk. MPO activity was determined to evaluate leukocyte infiltration. Results are means ± SE (n = 12). *P < 0.05 compared with control.

 
Role of NF-{kappa}B activation in MCP-1 expression in the kidney. The NF-{kappa}B activation in the kidney was examined by EMSA. The NF-{kappa}B/DNA binding activity was significantly elevated in kidneys isolated from rats fed a high-methionine diet compared with the control (Fig. 4A). Western immunoblot analysis was performed to determine the levels of phosphorylated I{kappa}B{alpha} protein and total I{kappa}B{alpha} protein in the kidney. There was a significant increase in the level of phosphorylated I{kappa}B{alpha} protein (Fig. 4B) and a significant reduction in the level of I{kappa}B{alpha} protein (Fig. 4C) in kidneys isolated from rats fed a high-methionine diet. To further investigate the link between NF-{kappa}B activation and hyperhomocysteinemia-induced MCP-1 expression, we treated rats fed a high-methionine diet with PDTC. Such treatment completely blocked hyperhomocysteinemia-induced MCP-1 mRNA expression (Fig. 5B) and elevation of MCP-1 protein levels in kidneys isolated from rats fed a high-methionine diet (Fig. 1C). These results suggested that hyperhomocysteinemia-induced MCP-1 expression in rat kidneys was mediated via NF-{kappa}B activation.


Figure 4
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  		Fig. 4. NF-{kappa}B activation in rat kidneys. Kidneys were isolated from rats fed a control, Met, or Cys diet for 12 wk. A: NF-{kappa}B/DNA binding activity was determined by EMSA. Levels of phospho-I{kappa}B{alpha} protein (B) and total I{kappa}B{alpha} protein (C) were measured using Western immunoblot analysis. Results are expressed as a percentage of control and are means ± SE (n = 12). *P < 0.05 compared with control.

 

Figure 5
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  		Fig. 5. NF-{kappa}B activation and MCP-1 expression in rat kidneys. Rats were fed a control or Met diet for 12 wk. One group of rats fed with a Met diet for 12 wk was treated with PDTC (100 mg/kg daily) for 3 days before euthanasia. A: NF-{kappa}B/DNA binding activity was determined by EMSA. B: MCP-1 mRNA expression was measured using a real-time PCR detection system. Results are means ± SE (n = 12). *P < 0.05 compared with control. #P < 0.05 compared with Met.

 
Effect of Hcy on MCP-1 expression in tubular cells. The effect of Hcy on MCP-1 expression was also examined in human kidney proximal tubular cells. Treatment of tubular cells with Hcy resulted in a significant increase in cellular MCP-1 mRNA expression (Fig. 6A) and MCP-1 protein secreted into the culture medium (Fig. 6B). To determine whether Hcy-induced MCP-1 production could lead to monocyte chemotaxis, we collected the culture medium from the Hcy-treated tubular cells (defined as conditioned medium) and used it for measuring monocyte migration in the chemotaxis assay (14, 30). The conditioned medium significantly enhanced monocyte migration in the chemotaxis assay (Fig. 6C). Anti-MCP-1 antibodies abolished such a stimulatory effect (Fig. 6C). These results suggested that Hcy-induced MCP-1 production in tubular cells could lead to monocyte chemotaxis. To further investigate the causative relationship between Hcy-induced NF-{kappa}B activation and MCP-1 expression, we transfected tubular cells with decoy NF-{kappa}B ODN to inhibit the activation of NF-{kappa}B. The interaction of decoy ODN with NF-{kappa}B prevents the binding of this transcription factor with the target genes, including MCP-1. EMSA showed that transfection of tubular cells with NF-{kappa}B ODN completely prevented NF-{kappa}B/DNA binding activity (Fig. 7A). Inhibition of NF-{kappa}B activation also abolished Hcy-induced MCP-1 mRNA expression in tubular cells (Fig. 7B). As a control, cells were transfected with scrambled ODN, which had no effect on Hcy-induced NF-{kappa}B activation and MCP-1 mRNA expression in those cells (Fig. 7, A and B). In addition, tubular cells were also incubated with other amino acids. Cysteine treatment did not stimulate MCP-1 expression in tubular cells (Fig. 7C). In another set of experiments, cells were incubated with methionine that was used in diet to induce hyperhomocysteinemia in rats. Methionine treatment did not affect MCP-1 expression in tubular cells (Fig. 7C). Furthermore, addition of S-adenosyl-methionine (Sigma-Aldrich), a metabolite formed during metabolism of methionine to Hcy, to the culture medium did not affect the expression of MCP-1 mRNA in tubular cells (Fig. 8).


Figure 6
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  		Fig. 6. Effect of Hcy on MCP-1 expression in proximal tubular cells and monocyte chemotaxis. Human proximal tubular cells were incubated in the absence or presence of Hcy (100 µM) for various time periods. A: levels of MCP-1 mRNA were measured using a real-time PCR detection system. B: the amount of MCP-1 protein secreted into the culture medium was measured by ELISA. C: for monocyte chemotaxis assay, the culture medium was collected from cells incubated in the absence (control) or presence of Hcy. In 1 set of the experiment, anti-MCP-1 antibodies (0.5 µg/ml) were added to the conditioned medium 10 min before the monocyte chemotaxis assay. Results are expressed as a ratio of MCP-1 mRNA vs. GAPDH mRNA and are means ± SE (n = 5). *P < 0.05 compared with control. #P < 0.05 compared with Hcy.

 

Figure 7
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  		Fig. 7. Effect of Hcy on NF-{kappa}B activation and MCP-1 expression in proximal tubular cells. Human proximal tubular cells were transfected with decoy NF-{kappa}B oligodeoxynucleotides (ODN) or scrambled decoy ODN. Transfected cells were incubated in the absence or presence of Hcy (100 µM). A: after 15 min of incubation, NF-{kappa}B/DNA binding activity was determined by EMSA. B: after 4 h of incubation, MCP-1 mRNA expression was determined using a real-time PCR detection system. C: nontransfected tubular cells were incubated in the absence or presence of Hcy (100 µM), cysteine (100 µM), or methionine (Met; 100 µM) for 4 h. Results are means ± SE (n = 5). *P < 0.05 compared with control.

 

Figure 8
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  		Fig. 8. Effect of S-adenosyl-methionine (SAM) on MCP-1 expression in tubular cells. Human proximal tubular cells were incubated in the absence or presence of SAM (10 or 100 µM) for 4 h. MCP-1 mRNA expression was determined using a real-time PCR detection system. Results are means ± SE (n = 3).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Results from the present study have clearly demonstrated that in the absence of other known risk factors, hyperhomocysteinemia can induce MCP-1 expression in rat kidneys. Activation of NF-{kappa}B due to increased phosphorylation of I{kappa}B{alpha} plays an important role in hyperhomocysteinemia-induced MCP-1 expression in the kidney tissue. Hcy-induced MCP-1 production in tubular cells leads to increased monocyte chemotaxis.

Increased chemokine expression in the tissue is one of the key steps in the inflammatory response. Chemokines including MCP-1 play an important role in leukocyte infiltration and activation during the inflammatory process. Similar to its role in the pathogenesis of atherosclerosis, elevated MCP-1 production contributes significantly to the recruitment of leukocytes into the kidney during the development of glomerulosclerosis (1, 11, 14, 15, 26, 28, 32, 36). An increase in chemokine expression is also responsible for leukocyte recruitment into allografts, which contributes to allograft rejection in kidney transplantation (8, 24, 25). We previously reported that Hcy stimulated MCP-1 production in vascular smooth muscle cells (34), endothelial cells (30), and macrophages (33). We have postulated that Hcy-induced MCP-1 expression in vascular cells may play an important role in the development of atherosclerosis in patients with hyperhomocysteinemia (6, 22). However, little information is available regarding the effect of hyperhomocysteinemia on chemokine expression in the kidney. The present study clearly demonstrated that Hcy, at elevated concentrations, also stimulated the expression of MCP-1 mRNA and protein in the kidney. The MPO activity, used as an index of leukocyte infiltration, was increased in kidneys of hyperhomocysteinemic rats. A majority of patients with chronic renal disease have displayed hyperhomocysteinemia (20, 37). Epidemiological studies have revealed an inverse correlation between serum levels of Hcy and renal function (20). However, the impact of Hcy, at pathologically high concentrations, on the kidney itself is unknown. To the best of our knowledge, this is the first study demonstrating that hyperhomocysteinemia can induce MCP-1 expression in the kidney. It is plausible that such a stimulatory effect may play an important role in inflammatory response, which in turn, aggravates renal dysfunction in patients with hyperhomocysteinemia.

The activation of NF-{kappa}B is intimately involved in inflammatory reaction. NF-{kappa}B activation has been implicated to play an important role in chemokine expression (30, 33, 34). This transcription factor can be activated by diverse pathogenic signals. In general, a rapid phosphorylation and degradation of its inhibitor protein (I{kappa}B{alpha}) lead to nuclear translocation of NF-{kappa}B. Once inside the nucleus, the NF-{kappa}B binds to the promoter region of the target genes including MCP-1 and regulates gene expression. Several lines of evidence obtained from the present study suggested that the activation of NF-{kappa}B plays an important role in MCP-1 expression in the kidney during hyperhomocysteinemia. First, the levels of total I{kappa}B{alpha} were markedly reduced in the kidneys of hyperhomocysteinemic rats as a result of increased phosphorylation of this inhibitor protein. Second, results from the EMSA revealed that the binding activity of NF-{kappa}B/DNA was significantly increased, suggesting an activation of this transcription factor during hyperhomocysteinemia. Pretreatment of hyperhomocysteinemic rats with a known NF-{kappa}B inhibitor reduced MCP-1 expression in the kidneys to the level similar to those found in the control group. Third, the involvement of NF-{kappa}B was further examined in proximal tubular cells transfected with NF-{kappa}B decoy oligodeoxynucleotide to inhibit the activation of this transcription factor. Indeed, inhibition of NF-{kappa}B activation completely abolished Hcy-induced MCP-1 expression in tubular cells. Together, these results indicated that NF-{kappa}B activation was necessary for Hcy-induced chemokine expression in the kidney. We previously demonstrated that Hcy could induce MCP-1 expression via NF-{kappa}B activation in vascular cells (30, 33–35). Our results together with others suggest that Hcy-induced MCP-1 expression in vascular cells serves as one of the important mechanisms contributing to Hcy-induced atherosclerosis. The present study provides evidence that hyperhomocysteinemia activates NF-{kappa}B, leading to increased MCP-1 expression in the kidney. Increased chemokine expression, in turn, facilitates leukocyte recruitment into the kidney. Those infiltrated cells are capable of producing inflammatory factors that exacerbate kidney injury. Together, these results suggest that Hcy-induced MCP-1 expression via NF-{kappa}B activation may serve as one of the mechanisms contributing to kidney injury.

In summary, the present study clearly demonstrates a direct link between hyperhomocysteinemia and inflammatory response in the kidney. Our results suggest that activation of NF-{kappa}B pathway is essential for Hcy-induced MCP-1 expression in the kidney. Increased chemokine expression may represent one of the important mechanisms that contribute to renal injury in patients with hyperhomocysteinemia.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported, in part, by the Heart & Stroke Foundation of Canada, Natural Sciences and Engineering Research Council of Canada, Canadian Institutes for Health Research, and the St. Boniface Hospital & Research Foundation.


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. O, Integrative Biology Laboratory, St. Boniface Hospital Research Center, 351 Tache Ave., Winnipeg, Manitoba, Canada R2H 2A6 (e-mail: karmino{at}sbrc.ca)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* S.-Y. Hwang and C. W. Woo contributed equally to this work. Back


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

  1. Anders HJ, Vielhauer V, Kretzler M, Cohen CD, Segerer S, Luckow B, Weller L, Grone HJ, Schlondorff D. Chemokine and chemokine receptor expression during initiation and resolution of immune complex glomerulonephritis. J Am Soc Nephrol 12: 919–931, 2001.[Abstract/Free Full Text]
  2. Au-Yeung KK, Woo CW, Sung FL, Yip JC, Siow YL, O K. Hyperhomocysteinemia activates nuclear factor-{kappa}B in endothelial cells via oxidative stress. Circ Res 94: 28–36, 2004.[Abstract/Free Full Text]
  3. Baldwin AS Jr. The NF-{kappa}B and I{kappa}B proteins: new discoveries and insights. Annu Rev Immunol 14: 649–683, 1996.[CrossRef][Web of Science][Medline]
  4. Brand K, Page S, Rogler G, Bartsch A, Brandl R, Knuechel R, Page M, Kaltschmidt C, Baeuerle PA, Neumeier D. Activated transcription factor nuclear factor-kappa B is present in the atherosclerotic lesion. J Clin Invest 97: 1715–1722, 1996.[Web of Science][Medline]
  5. Chatterjee PK, Cuzzocrea S, Brown PA, Zacharowski K, Stewart KN, Mota-Filipe H, Thiemermann C. Tempol, a membrane-permeable radical scavenger, reduces oxidant stress-mediated renal dysfunction and injury in the rat. Kidney Int 58: 658–673, 2000.[CrossRef][Web of Science][Medline]
  6. Choy PC, Siow YL, Mymin D, O K. Lipids and atherosclerosis. Biochem Cell Biol 82: 212–224, 2004.[CrossRef][Web of Science][Medline]
  7. Clarke R, Daly L, Robinson K, Naughten E, Cahalane S, Fowler B, Graham I. Hyperhomocysteinemia: an independent risk factor for vascular disease. N Engl J Med 324: 1149–1155, 1991.[Abstract]
  8. Grandaliano G, Gesualdo L, Ranieri E, Monno R, Stallone G, Schena FP. Monocyte chemotactic peptide-1 expression and monocyte infiltration in acute renal transplant rejection. Transplantation 63: 414–420, 1997.[CrossRef][Web of Science][Medline]
  9. Karin M. The beginning of the end: I{kappa}B kinase (IKK) and NF-{kappa}B activation. J Biol Chem 274: 27339–27342, 1999.[Free Full Text]
  10. Kumagai H, Katoh S, Hirosawa K, Kimura M, Hishida A, Ikegaya N. Renal tubulointerstitial injury in weanling rats with hyperhomocysteinemia. Kidney Int 62: 1219–1228, 2002.[CrossRef][Web of Science][Medline]
  11. Kuusniemi AM, Lapatto R, Holmberg C, Karikoski R, Rapola J, Jalanko H. Kidneys with heavy proteinuria show fibrosis, inflammation, and oxidative stress, but no tubular phenotypic change. Kidney Int 68: 121–132, 2005.[CrossRef][Web of Science][Medline]
  12. Li N, Chen YF, Zou AP. Implications of hyperhomocysteinemia in glomerular sclerosis in hypertension. Hypertension 39: 443–448, 2002.[Abstract/Free Full Text]
  13. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the Formula method. Methods 25: 402–408, 2001.[CrossRef][Web of Science][Medline]
  14. Lynn EG, Siow YL, Frohlich J, Cheung GT, O K. Lipoprotein-X stimulates monocyte chemoattractant protein-1 expression in mesangial cells via nuclear factor-{kappa}B. Kidney Int 60: 520–532, 2001.[CrossRef][Web of Science][Medline]
  15. Lynn EG, Siow YL, OK. Very low-density lipoprotein stimulates the expression of monocyte chemoattractant protein-1 in mesangial cells. Kidney Int 57: 1472–1483, 2000.[CrossRef][Web of Science][Medline]
  16. Marumo T, Schini-Kerth VB, Fisslthaler B, Busse R. Platelet-derived growth factor-stimulated superoxide anion production modulates activation of transcription factor NF-{kappa}B and expression of monocyte chemoattractant protein 1 in human aortic smooth muscle cells. Circulation 96: 2361–2367, 1997.[Abstract/Free Full Text]
  17. McCully KS. Homocysteine and vascular disease. Nat Med 2: 386–389, 1996.[CrossRef][Web of Science][Medline]
  18. Mercurio F, Zhu H, Murray BW, Shevchenko A, Bennett BL, Li J, Young DB, Barbosa M, Mann M, Manning A, Rao A. IKK-1 and IKK-2: cytokine-activated I{kappa}B kinases essential for NF-{kappa}B activation. Science 278: 860–866, 1997.[Abstract/Free Full Text]
  19. Mudd SH, Finkelstein JD, Refsum H, Ueland PM, Malinow MR, Lentz SR, Jacobsen DW, Brattstrom L, Wilcken B, Wilcken DE, Blom HJ, Stabler SP, Allen RH, Selhub J, Rosenberg IH. Homocysteine and its disulfide derivatives: a suggested consensus terminology. Arterioscler Thromb Vasc Biol 20: 1704–1706, 2000.[Free Full Text]
  20. Ninomiya T, Kiyohara Y, Kubo M, Tanizaki Y, Tanaka K, Okubo K, Nakamura H, Hata J, Oishi Y, Kato I, Hirakata H, Iida M. Hyperhomocysteinemia and the development of chronic kidney disease in a general population: the Hisayama study. Am J Kidney Dis 44: 437–445, 2004.[Web of Science][Medline]
  21. Nygard O, Nordrehaug JE, Refsum H, Ueland PM, Farstad M, Vollset SE. Plasma homocysteine levels and mortality in patients with coronary artery disease. N Engl J Med 337: 230–236, 1997.[Abstract/Free Full Text]
  22. O K Siow YL. Biochemical mechanisms of hyperhomocysteinemia in atherosclerosis: role of chemokine expression In: Atherosclerosis, Hypertension and Diabetes, edited by Pierce GN, Nagano M, Zahradka P, and Dhalla NS. Boston, MA: Kluwer Academic, 2003.
  23. Prathapasinghe GA, Siow YL, O K. Detrimental role of homocysteine in renal ischemia-reperfusion injury. Am J Physiol Renal Physiol 292: F1354–F1363, 2007.[Abstract/Free Full Text]
  24. Prodjosudjadi W, Daha MR, Gerritsma JS, Florijn KW, Barendregt JN, Bruijn JA, van der Woude FJ, van Es LA. Increased urinary excretion of monocyte chemoattractant protein-1 during acute renal allograft rejection. Nephrol Dial Transplant 11: 1096–1103, 1996.[Abstract/Free Full Text]
  25. Robertson H, Morley AR, Talbot D, Callanan K, Kirby JA. Renal allograft rejection: β-chemokine involvement in the development of tubulitis. Transplantation 69: 684–687, 2000.[CrossRef][Web of Science][Medline]
  26. Rovin BH, Doe N, Tan LC. Monocyte chemoattractant protein-1 levels in patients with glomerular disease. Am J Kidney Dis 27: 640–646, 1996.[Web of Science][Medline]
  27. Schachter AD, Strehlau J, Zurakowski D, Vasconcellos L, Kim YS, Zheng XX, Gunshin Y, Overstreet SL, Benfield MR, Tejani A, Harmon WE, Herrin JT, Strom TB. Increased nuclear factor-{kappa}B and angiotensinogen gene expression in posttransplant recurrent focal segmental glomerulosclerosis. Transplantation 70: 1107–1110, 2000.[CrossRef][Web of Science][Medline]
  28. Sekiguchi M, Natori Y, Iyonaga K, Takeya M, Natori Y. Expression of monocyte chemoattractant protein-1 in experimental crescentic glomerulonephritis in rats. J Lab Clin Med 129: 239–244, 1997.[CrossRef][Web of Science][Medline]
  29. Simeonidis S, Stauber D, Chen G, Hendrickson WA, Thanos D. Mechanisms by which I{kappa}aB proteins control NF-{kappa}B activity. Proc Natl Acad Sci USA 96: 49–54, 1999.[Abstract/Free Full Text]
  30. Sung FL, Slow YL, Wang G, Lynn EG, O K. Homocysteine stimulates the expression of monocyte chemoattractant protein-1 in endothelial cells leading to enhanced monocyte chemotaxis. Mol Cell Biochem 216: 121–128, 2001.[CrossRef][Web of Science][Medline]
  31. Sung FL, Zhu TY, Au-Yeung KK, Siow YL, O K. Enhanced MCP-1 expression during ischemia/reperfusion injury is mediated by oxidative stress and NF-{kappa}B. Kidney Int 62: 1160–1170, 2002.[CrossRef][Web of Science][Medline]
  32. Tang WW, Qi M, Warren JS, Van GY. Chemokine expression in experimental tubulointerstitial nephritis. J Immunol 159: 870–876, 1997.[Abstract]
  33. Wang G, Siow YL, O K. Homocysteine induces monocyte chemoattractant protein-1 expression by activating NF-{kappa}B in THP-1 macrophages. Am J Physiol Heart Circ Physiol 280: H2840–H2847, 2001.[Abstract/Free Full Text]
  34. Wang G, Siow YL, O K. Homocysteine stimulates nuclear factor {kappa}B activity and monocyte chemoattractant protein-1 expression in vascular smooth-muscle cells: a possible role for protein kinase C. Biochem J 352: 817–826, 2000.[CrossRef][Web of Science][Medline]
  35. Wang G, Woo CW, Sung FL, Siow YL, O K. Increased monocyte adhesion to aortic endothelium in rats with hyperhomocysteinemia: role of chemokine and adhesion molecules. Arterioscler Thromb Vasc Biol 22: 1777–1783, 2002.[Abstract/Free Full Text]
  36. Wenzel U, Schneider A, Valente AJ, Abboud HE, Thaiss F, Helmchen UM, Stahl RA. Monocyte chemoattractant protein-1 mediates monocyte/macrophage influx in anti-thymocyte antibody-induced glomerulonephritis. Kidney Int 51: 770–776, 1997.[Web of Science][Medline]
  37. Wheeler DC. Cardiovascular disease in patients with chronic renal failure. Lancet 348: 1673–1674, 1996.[CrossRef][Web of Science][Medline]
  38. Woo CW, Siow YL, O K. Homocysteine activates cAMP-response element binding protein in HepG2 through cAMP/PKA signaling pathway. Arterioscler Thromb Vasc Biol 26: 1043–1050, 2006.[Abstract/Free Full Text]
  39. Woo CW, Siow YL, Pierce GN, Choy PC, Minuk GY, Mymin D, O K. Hyperhomocysteinemia induces hepatic cholesterol biosynthesis and lipid accumulation via activation of transcription factors. Am J Physiol Endocrinol Metab 288: E1002–E1010, 2005.[Abstract/Free Full Text]
  40. Zhang F, Siow YL, O K. Hyperhomocysteinemia activates NF-{kappa}B and inducible nitric oxide synthase in the kidney. Kidney Int 65: 1327–1338, 2004.[CrossRef][Web of Science][Medline]



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