Excreted urinary mediators in an animal model of experimental immune nephritis with potential pathogenic significance.код для вставкиСкачать
ARTHRITIS & RHEUMATISM Vol. 56, No. 3, March 2007, pp 949–959 DOI 10.1002/art.22556 © 2007, American College of Rheumatology Excreted Urinary Mediators in an Animal Model of Experimental Immune Nephritis With Potential Pathogenic Significance Tianfu Wu,1 Chun Xie,1 Madhavi Bhaskarabhatla,1 Mei Yan,1 Amanda Leone,1 Su Sin Chen,1 Xin J. Zhou,1 Chaim Putterman,2 and Chandra Mohan1 Objective. Currently, proteinuria is viewed as the earliest indicator of renal disease in immune-mediated nephritis. The objective of this study was to determine whether additional mediators may be excreted in the urine during immune-mediated nephritis, using an experimental model with a well-defined disease course. Methods. Urine samples from mice with anti– glomerular basement membrane (anti-GBM) antibody– induced experimental nephritis were screened using a focused immunoproteome array bearing 62 cytokines/ chemokines/soluble receptors. Molecules identified through this screening assay were validated using an enzyme-linked immunosorbent assay. One of these molecules was further evaluated for its pathogenic role in disease, using antibody-blocking studies. Results. Compared with B6 and BALB/c mice, in which moderately severe immune-mediated nephritis develops, the highly nephritis-susceptible 129/Sv and DBA/1 mice exhibited significantly increased urinary levels of vascular cell adhesion molecule 1 (VCAM-1), P-selectin, tumor necrosis factor receptor I (TNFRI), and CXCL16, particularly at the peak of disease. Whereas some of the mediators appeared to be serum derived early in the disease course, local production in the kidneys appeared to be an important source of these mediators later in the course of disease. Both intrinsic renal cells and infiltrating leukocytes appeared to be capable of producing these mediators. Finally, antibodymediated blocking of CXCL16 ameliorated experimental immune nephritis. Conclusion. These studies identified VCAM-1, P-selectin, TNFRI, and CXCL16 as a quartet of molecules that have potential pathogenic significance; the levels of these molecules are significantly elevated during experimental immune nephritis. The relevance of these molecules in spontaneous immune nephritis warrants investigation. The experimental transfer of antibodies to the glomerular basement membrane (anti-GBM) into young healthy mice and rats precipitates glomerular and interstitial disease, with a rapid and reproducible time course (1–3). The immune-mediated nephritis in this experimental model shares several features with autoantibodymediated nephritis in spontaneous lupus, particularly the downstream cellular and molecular players that mediate renal disease. This appears to be important, because antiglomerular antibodies have also been suggested to be of pathogenic relevance in human and murine lupus (4,5). Hence, some of the lessons learned from the experimental immune nephritis model may be applicable to the pathogenic events that mediate spontaneous lupus nephritis. Recently, we observed an interesting straindependent difference in susceptibility to anti-GBM antibody–induced nephritis. Whereas some mouse strains exhibit mild to moderately severe anti-GBM– triggered nephritis, other strains exhibit florid renal disease following an anti-GBM antibody challenge (2,3). 1 Tianfu Wu, PhD, Chun Xie, MD, Madhavi Bhaskarabhatla, MS, Mei Yan, BS, Amanda Leone, BS, Su Sin Chen, MD, Xin J. Zhou, MD, Chandra Mohan, MD, PhD: University of Texas Southwestern Medical School at Dallas; 2Chaim Putterman, MD, PhD: Albert Einstein College of Medicine, Bronx, New York. Drs. Wu and Xie contributed equally to this work. Address correspondence and reprint requests to Chandra Mohan, MD, PhD, Department of Internal Medicine/Rheumatology, University of Texas Southwestern Medical Center, Mail Code 8884, Y8.204, 5323 Harry Hines Boulevard, Dallas, TX 75390-8884. E-mail: Chandra.firstname.lastname@example.org. Submitted for publication May 1, 2006; accepted in revised form November 20, 2006. 949 950 WU ET AL Examples of the former include the C57BL/6 (B6) and BALB/c mouse strains, whereas the 129/Sv, BUB, NZW, and DBA/1 mouse strains develop severe proliferative renal disease following an anti-GBM antibody challenge (2,3). The genetic and molecular players that account for this strain difference currently remain unknown. In this study, we compared the proteins excreted in the urine of mice with severe nephritis with those in the urine of mice with mild nephritis, using immunoproteomic analysis. Interestingly, compared with the B6 and BALB/c strains, DBA/1 and 129/Sv mice exhibited high urinary levels of vascular cell adhesion molecule 1 (VCAM-1), P-selectin, soluble tumor necrosis factor receptor I (TNFRI), and CXCL16, correlating well with the level of disease severity. Importantly, these molecules were noted to be expressed within the kidneys during disease, being elaborated by intrinsic renal cells as well as infiltrating leukocytes. Finally, we demonstrate that at least 1 of these molecules, CXCL16, is essential for renal disease in this experimental model. MATERIALS AND METHODS Mice. B6, 129/Sv, BALB/c, and DBA/1 mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Urine samples from the mice subjected to accelerated experimental nephritis were collected on day 1, day 7, and day 14/15 or day 21 after disease induction, as described previously (3). Female mice were used for all experiments, and 24-hour urine collection using metabolic cages was carried out for all mice. All collected urine samples were aliquoted and rapidly frozen away without any protease inhibitors. This was done deliberately, to enrich for potential urinary biomarkers that were not labile and that may be robustly detected in any laboratory setting. All animal experiments were carried out in accordance with the institutional guidelines. Screening of urine using proteomic arrays. Membranes bearing antibodies to 62 mouse cytokines/chemokines/ soluble receptors were purchased from Ray Biotech (Norcross, GA [online at http://www.raybiotech.com]) and were used to screen urine obtained from B6, BALB/c, 129/Sv, and DBA/1 mice (5 mice per strain) 14 days after induction of experimental nephritis, as detailed previously (3). The specific molecules that were assayed on the arrays were Axl, Blc, CD30 ligand (CD30L), CD30T, CD40, Crg2, cutaneous T cell–attracting chemokine (CTACK), CXCL16, eotaxin, eotaxin 2, Fas ligand, fractalkine, granulocyte colony-stimulating factor (G-CSF), granulocyte–macrophage CSF (GM-CSF), interferon- ␥ (IFN␥), insulin-like growth factor binding protein 3 (IGFBP3), IGFBP-5, IGFBP-6, interleukin-1␣ (IL-1␣), IL-1␤, IL-2, IL-3, IL-3Rb, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12 p40/p70, IL-12 p70, IL-13, IL-17, KC, leptin receptor (leptin R), leptin (OB), LiX, L-selectin, lymphotactin, monocyte chemotactic protein 1 (MCP-1) MCP-5, macrophage CSF (M-CSF), MIG, macrophage inflammatory protein 1␣ (MIP-1␣), MIP-1␤, MIP-2, MIP-3␣, MIP-3␤, platelet factor 4 (PF-4), P-selectin, RANTES, stem cell factor (SCF), stromal cell–derived factor 1␣ (SDF-1␣), thymus and activation–regulated chemokine (TARC), T cell–attracting chemokine 3 (TCA-3), thymusexpressed chemokine (TECK), tissue inhibitor of metalloproteinases 1 (TIMP-1), TNF␣, TNFRI, TNFRII, thrombopoietin (TPO), VCAM-1, and vascular endothelial growth factor (VEGF). Screening was carried out as recommended by the manufacturer. Briefly, after blocking the membranes, they were incubated with urine samples diluted at a ratio of 1:5 in dilution buffer. Next, biotin-conjugated primary antibodies to the different mediators on the membrane and horseradish peroxidase (HRP)–conjugated streptavidin were applied to develop the membranes. The intensity of spots on the membrane were imaged using ImageJ software (http:// rsb.info.nih.gov/ij). Selected reactivities were confirmed using enzyme-linked immunosorbent assays (ELISAs), as described below. ELISA detection of TNFRI, CXCL16, P-selectin, and VCAM-1. The following ELISA kits were purchased from R&D Systems (Minneapolis, MN) and were used according to the recommendations of the manufacturer: mouse TNFRI/ TNFRSF1A DuoSet (catalog no. DY425), mouse CXCL16 DuoSet (catalog no. DY503), mouse P-Selectin/CD62P DuoSet (catalog no. DY737), and mouse VCAM-1/CD106 DuoSet (catalog no. DY643). The detection limits of these 4 ELISAs were 4 pg/ml, 16 pg/ml, 62 pg/ml, and 125 pg/ml, respectively. All urine samples were diluted at a ratio of 1:5 or higher for the ELISAs, and concentrations of the respective molecules were ascertained using manufacturer-supplied standards. Likewise, all culture supernatants were diluted at a ratio of 1:2 or 1:10 before assaying for the respective molecules. Treatment of anti-GBM antibody–induced nephritis using anti-CXCL16. A total of 18 129/Sv mice were subjected to the accelerated model of anti-GBM nephritis, as described previously (3), using adjuvant sensitization on day 0 and rabbit anti-mouse GBM sera on day 5. These 18 mice were divided into 3 groups of 6 each. On days 1, 4, 8, 12, 16, and 20, rat anti-mouse CXCL16 antibody (R&D Systems; catalog no. MAB503), 500 g intraperitoneally (n ⫽ 6 mice), or rat IgG2A isotype control (R&D Systems; catalog no. MAB9371) (n ⫽ 6 mice) was injected into mice in the experimental group and the isotype-control group, respectively. In addition, 6 mice were subjected to anti-GBM nephritis but were not treated with any additional antibodies. Urine and blood samples were collected from all groups of mice on days 0, 7, 14, 17, and 21. Mice were killed on day 21, and the kidneys were processed, analyzed, and graded for histopathology, as detailed elsewhere (3). Real-time reverse transcription–polymerase chain reaction (PCR). Anti-GBM antibody–challenged 129/Sv mice were killed on day 0, day 7, or day 15. RNA was extracted from the renal cortex (after stripping it away from the medulla) for real-time PCR assays using the following primers, according to the previously described protocols (6). For Pselectin, AGATCTGGGCTTCCTGGGTGAAAT and AACGCAAGGACAGGTATCCAGTGA; for VCAM-1, TACCTGCCATCGGGATGATCGTTT and ATTTCTGTG- VCAM-1, P-SELECTIN, TNFRI, AND CXCL16 IN NEPHRITIC URINE CCTCCACCAGACTGT; for CXCL16, TGTCTTCTTCCTCACTGCAGCCAT and ACAGGAGTGTACCAGAGCTGCAAA; for TNFRI, TCAGGCAGTGTCTCAGTTGCAAGA and TCCACCTGGGACATTTCTTTCCGA. The fold change was calculated as described previously (6). In vitro cell culture. Mesangial cells from glomeruli and macrophages from the bone marrow of 2-month-old B6 or 129/Sv mice were derived as described elsewhere (6,7) and stimulated with or without lipopolysaccharide (LPS) (10 g/ml) for 24 hours. Released VCAM-1, CXCL16, TNFRI, and P-selectin were analyzed by ELISA, as described above. Immunohistochemical analysis. Kidney sections obtained from 129/Sv mice 14 days after anti-GBM antibody challenge were stained with the following primary antibodies: goat anti-mouse CXCL16 antibody (R&D Systems; catalog no. AF503), goat anti-mouse TNFRI antibody (R&D Systems; catalog no. BAF425), goat anti-mouse VCAM-1 antibody (R&D Systems; catalog no. BAF643), or goat anti-mouse P-selectin antibody (Santa Cruz Biotechnology, Santa Cruz, CA; catalog no. SC-6941) or the isotype control antibodies. The sections were then developed as described previously (6). Briefly, antigen retrieval was performed by using sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) in a pressure cooker, followed by protein blocking for 20 minutes and peroxidase blocking for 20 minutes. For primary incubation, anti-TNFRI antibody (0.5 g/ml, 60 minutes), anti–P-selectin antibody (0.2 g/ml, 60 minutes), anti-CXCL16 antibody (0.25 g/ml, 60 minutes), or anti-VCAM-1 antibody (0.3 g/ml, 30 minutes) was added. This was followed by incubation with biotinylated anti-goat IgG (2 g/ml), avidin– biotin–peroxidase complex staining (Vectastain Elite ABC Kit; Vector, Burlingame, VT), HRP, diaminobenzidine (DAB) chromagen, and hematoxylin. The protein block, DAB chromagen, and the peroxidase block were from the Dako Cytomation Envision kit (Dako, Carpinteria, CA; catalog no. K4011). Statistical analysis. All comparisons were performed using Student’s t-test for normally distributed data and the Mann-Whitney U test otherwise, using SigmaStat (SPSS, Chicago, IL) or GraphPad Prism (GraphPad Software, San Diego, CA) software. RESULTS Higher amounts of urinary VCAM-1, P-selectin, TNFRI, and CXCL16 in strains with severe nephritis. We recently noted that certain strains of mice, such as NZW, DBA/1, BUB, and 129/Sv, develop severe nephritis in response to an anti-GBM antibody insult, compared with 16 other inbred strains tested (2,3). As a quick screen for mediators that may be excreted in the urine of nephritic mice, we assayed urine from 2 strains of mice with mild experimental nephritis (i.e., B6 and BALB/c), and 2 strains of mice with severe experimental nephritis (i.e., DBA/1 and 129/Sv), 14 days following the nephrotoxic insult (the peak of disease) for the presence 951 Figure 1. Screening of excreted molecules in nephritic urine using commercial protein arrays. A, Representative array profile depicting the reactivity patterns of urine from 4 strains of mice, 14 days after challenge with antiglomerular antibodies, using the accelerated model, as described previously (3). Black dots indicate the presence of particular proteins. The 4 black dots at the top left represent the positive control, and the 4 blank spots immediately below represent the negative control. Colored circles represent the 4 molecules that were reproducibly elevated in urine from DBA/1 and 129/Sv mice, but not in urine from BALB/c and B6 mice, following nephrotoxic insult. Each array is representative of 2 independent experiments, each of which was conducted using 5 urine samples pooled from each strain of mice. B, Relative abundance of the different proteins, as gauged by measuring the image intensity using ImageJ software. The profile shown is representative of 2 independent experiments. These results were further validated using a second approach, enzyme-linked immunosorbent assay, as detailed in the text and in Figure 2. VCAM-1 ⫽ vascular cell adhesion molecule 1; TNFRI ⫽ tumor necrosis factor receptor I; AU ⫽ arbitrary unit. 952 of 62 cytokines/chemokines or soluble mediators, using a commercially available reverse proteomic array from Ray Biotech. Figures 1A and B show representative array profiles for urine obtained from these 4 strains of mice. The 2 highly disease-prone strains of mice, DBA/1 and 129/Sv, exhibited increased levels of several molecules in their urine, including VCAM-1, P-selectin, TNFRI, and CXCL16, that were not prominent in the urine of the “control” strains of mice. These findings were confirmed using an orthogonal method, ELISA, as detailed below. In contrast, urine from these 4 strains of mice exhibited similar levels of Axl, Blc, CD30L, CD30T, CD40, Crg2, CTACK, eotaxin, eotaxin-2, Fas ligand, fractalkine, G-CSF, GM-CSF, IFN ␥ , IGFBP-3, IGFBP-5, IGFBP-6, IL-1␣, IL-1␤, IL-2, IL-3, IL-3Rb, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12 p40/p70, IL-12 p70, IL-13, IL-17, KC, leptin R, leptin (OB), LIX, L-selectin, lymphotactin, MCP-1, MCP-5, M-CSF, MIG, MIP-1␣, MIP-1␤, MIP-2, MIP-3␣, MIP-3␤, PF-4, RANTES, SCF, SDF-1␣, TARC, TCA-3, TECK, TIMP-1, TNF␣, TPO, and VEGF, following anti-GBM antibody– induced immune nephritis (Figure 1). Finally, the levels of TNFRII (positioned next to TNFRI on the arrays shown in Figure 1A) were elevated not only in the urine of DBA/1 and 129/Sv mice but also in the urine of B6 mice, in which milder disease develops; therefore, TNFRII was not studied further. Excretion of urinary VCAM-1, P-selectin, TNFRI, and CXCL16 early in disease but peaking with disease crescendo. Next, the above array findings were confirmed by ELISA. Interestingly, in the 2 severe disease– prone strains, urinary levels of VCAM-1, P-selectin, TNFRI, and CXCL16 were relatively high on day 7, with a peak on day 14, relative to the levels in B6 and BALB/c mice (Figure 2). In contrast, urinary protein or albumin excretion in these 2 strains of mice was not increased on day 7 (Figure 2, top row). Hence, these 4 molecules appear to be particularly attractive as potential early biomarkers of renal disease, because they appear in the urine before proteinuria sets in. As is evident in Figure 2, day 14 urine from the DBA/1 and 129/Sv mice had significantly higher levels of TNFRI (P ⬍ 10⫺8), P-selectin (P ⬍ 10⫺7), VCAM-1 (P ⬍ 10⫺7), and CXCL16 (P ⬍ 0.004) compared with the corresponding day 14 urinary levels in the B6 and BALB/c control mice. Similarly, day 21 urine from the DBA/1 and 129/Sv mice had significantly higher levels of TNFRI (P ⬍ 0.01), P-selectin (P ⬍ 0.03), VCAM-1 (P ⬍ 10⫺5), and CXCL16 (P ⬍ 0.001) compared with the corresponding day 21 urinary levels of these molecules in the B6 and WU ET AL BALB/c control mice. Interestingly, these 4 molecules exhibited quite different excretion profiles over time. Whereas VCAM-1 levels continued to be high past day 14 (similar to the level of protein), levels of the other 3 markers, particularly TNFRI, dropped rapidly after day 14. Because acute glomerular disease in this model reaches its peak at about day 14 (2,3), it is tempting to propose that whereas the urinary levels of P-selectin, CXCL16, and particularly TNFRI may be good markers of acute renal disease, urinary VCAM-1 may be a better marker of past or chronic nephritis. Similar serum levels of the 4 molecules in all 4 mouse strains on day 7. A possible factor contributing to the early rise in urine levels of VCAM-1, P-selectin, TNFRI, and CXCL16 could have been potentially high serum levels of these same molecules. Therefore, serum levels of these 4 molecules were next assayed at serial time points following the glomerular insult. In all 4 strains of mice, serum levels of VCAM-1, P-selectin, and CXCL16 reached a peak on day 7 (not day 14), to a fairly similar degree in all 4 strains. Representative ELISA results are shown in Figure 3. Hence, the content of these molecules in urine on day 7 might have been derived from serum, at least in part. After day 7, however, the serum and urine levels of these molecules pursued discordant kinetics, as is evident by comparing Figures 2 and 3. Thus, the differential expression of these molecules in day 14 and day 21 urine samples from the DBA/1 and 129/Sv mice on the one hand, and from the BALB/c and B6 mice on the other, is unlikely to be attributable to differences in the serum levels of these molecules in these strains of mice; rather, local production of these mediators in diseased (DBA/1 and 129/Sv) kidneys may be a major source of these molecules at these later time points. In contrast to the above 3 molecules, serum levels of TNFRI peaked on day 14 and subsided thereafter, similar to the profiles of TNFRI in urine. Indeed, the absolute day 14 serum levels of TNFRI were also highest in 129/Sv mice, followed by DBA/1 mice, and were substantially lower in B6 and BALB/c mice, again reflecting the corresponding urinary profiles. Based on these patterns, it appears that the urinary TNFRI profiles in these 4 strains of mice may be dictated by their corresponding serum levels, to a significant extent. Hyperexpression of VCAM-1, TNFRI, P-selectin, and CXCL16 within diseased kidneys. To determine whether these molecules were expressed within the kidneys, a couple of complementary approaches were adopted. First, real-time PCR studies indicated that levels of these molecules were indeed up-regulated VCAM-1, P-SELECTIN, TNFRI, AND CXCL16 IN NEPHRITIC URINE Figure 2. Urine levels of VCAM-1, CXCL16, P-selectin, and TNFRI, by enzyme-linked immunosorbent assay (ELISA). DBA/1 and 129/Sv mice develop renal disease that is severe compared with that in B6 and BALB/c mice, when challenged with antiglomerular antibodies. In this model of severe disease, 24-hour urine levels of protein and albumin peak on day 14 and tend to remain high until day 21 (top row). Typically, healthy B6 mice do not excrete ⬎1 mg of protein per 24-hour period. The 24-hour urine levels of VCAM-1, CXCL16, TNFRI, and P-selectin followed a very similar time course, as depicted in the second through fifth rows. Each plot was derived by monitoring 5–6 mice per strain through day 21. Within each row, the data from the 4 strains are plotted using the same y-axis scale, in order to highlight the differences between strains. Day 14 and day 21 levels of all 4 molecules were significantly higher in DBA/1 and 129/Sv mice compared with the 2 control strains, BALB/c and B6. Broken lines connect the mean values. ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍ 0.01; ⴱⴱⴱ ⫽ P ⬍ 0.001 by Student’s t-test (or Mann-Whitney nonparametric test for non-normally distributed data), day 7, day 14, and day 21 versus day 0. NS ⫽ not significant (see Figure 1 for other definitions). 953 954 WU ET AL Figure 3. Serum levels of VCAM-1, CXCL16, P-selectin, and TNFRI during anti–glomerular basement membrane (anti-GBM) antibody–induced nephritis. Serum levels of CXCL16, P-selectin, TNFRI, and VCAM-1 were assayed in the same sets of mice (n ⫽ 5–6 mice per strain) described in Figure 2, on days 0, 7, 14, and 21 after the anti-GBM antibody insult. Broken lines connect the mean values. ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍ 0.01; ⴱⴱⴱ ⫽ P ⬍ 0.001 by Student’s t-test (or Mann-Whitney nonparametric test for non-normally distributed data), day 7, day 14, and day 21 versus day 0. NS ⫽ not significant (see Figure 1 for other definitions). within the renal cortex as disease evolved following the nephrotoxic insult, with the most dramatic differences being noted in the expression of VCAM-1 and CXCL16 (Figure 4). Furthermore, we could demonstrate the increased presence of these molecules within the diseased kidneys using immunohistochemistry (Figure 5). Whereas VCAM-1 and CXCL16 showed increased staining in the glomeruli, tubules, and vascular endothelium, TNFRI staining was stronger in the tubules and interstitium. P-selectin staining was strongest within the glomeruli (Figure 5). Although these studies indicate that these molecules can be hyperexpressed within the diseased kidney, they do not clearly distinguish whether these molecules are expressed within renal-intrinsic cells or within infiltrating leukocytes. In vitro studies, however, demonstrated that intrinsic renal cells (as exemplified by mesangial cells) and systemic leukocytes (as exemplified by macrophages) were both capable of synthesizing these products, particularly upon stimulation with LPS (data not shown). Importantly, the levels of all 4 molecules elaborated by the 129/Sv-derived macrophages and mesangial cells were significantly higher than the levels elaborated by B6 cells, which is in resonance with the contrasting urinary profiles of these molecules in these 2 strains of mice, with one exception. Although LPStriggered mesangial cells from 129/Sv mice were a rich source of VCAM-1, CXCL16, and P-selectin, no signif- VCAM-1, P-SELECTIN, TNFRI, AND CXCL16 IN NEPHRITIC URINE Figure 4. Expression of CXCL16, P-selectin, TNFRI, and VCAM-1 within nephritic kidneys. RNA from the renal cortex of 129/Sv mice (n ⫽ 5) with anti–glomerular basement membrane antibody–induced nephritis was isolated on days 0, 7, and 15, and mRNA levels of the 4 molecules were assayed by real-time polymerase chain reaction. The fold change is with respect to mRNA levels on day 0 and was calculated after normalization against GAPDH message, as detailed elsewhere (27). Values are the mean and SD. ⴱ ⫽ P ⬍ 0.05; ⴱⴱⴱ ⫽ P ⬍ 0.001 by Student’s t-test (or Mann-Whitney nonparametric test for nonnormally distributed data), day 7 and day 15 versus day 0. NS ⫽ not significant (see Figure 1 for other definitions). icant increase in TNFRI production was noted. It remains possible that yet other glomerular cell types (e.g., glomerular endothelial cells and podocytes) may be additional sources of some of these mediators; this possibility needs to be examined further. 955 Assessing the role of CXCL16 in immune nephritis. Finally, we sought to determine the pathophysiologic relevance of these 4 molecules in immune nephritis. Some information is already available concerning the role of VCAM-1, P-selectin, and TNFRI in renal disease (8–19), as discussed below. However, much less is known about the role of CXCL16, which is a chemokine expressed on dendritic cells and macrophages. This chemokine has been shown to be important for the recruitment of T cells into various tissues and for cell–cell interactions via the CXCR6 counterreceptor (20–22). Although CXCL16 has been suggested to be important in arthritis, its role in immune nephritis is currently unknown. Given that it can be expressed within the kidneys (Figures 4 and 5), we wondered whether this chemokine might have an obligatory role in engendering renal disease following an immune trigger. Because CXCL16⫺/⫺ mice are not yet available, we examined whether blockade of CXCL16 function using blocking antibodies might have the potential to ameliorate nephritis. As depicted in Figure 6, anti-CXCL16 antibody treatment resulted in a significant reduction in proteinuria, glomerulonephritis, tubulointerstitial disease, and crescent formation following the experimental induction of anti-GBM disease in susceptible 129/Sv mice, compared with placebo-treated mice. Figure 5. Immunohistochemical localization of CXCL16, P-selectin, tumor necrosis factor receptor I (TNFRI), and vascular cell adhesion molecule 1 (VCAM-1) within diseased kidneys. Formalin-fixed kidney sections from 129/Sv mice, obtained on day 0 or day 15 following the anti–glomerular basement membrane antibody challenge, were stained with antibodies to P-selectin, CXCL16, TNFRI, or VCAM-1, or with the secondary control antibody. Results are representative of 3–5 independent kidney sections. 956 WU ET AL Figure 6. Effect of CXCL16 on amelioration of experimental immune nephritis. 129/Sv mice were subjected to anti–glomerular basement membrane antibody–induced nephritis, as detailed previously (2,3). Whereas mice in the experimental group (n ⫽ 6) received intraperitoneal injections of anti-CXCL16 (500 g per injection) 3 times weekly throughout the 21-day experimental period, while mice in the placebo groups received an equivalent amount of isotype control antibody (n ⫽ 6) or no antibody (n ⫽ 6). All mice were examined for the degree of proteinuria over 24 hours, the degree of glomerular disease, the extent of glomerular crescent formation, and the degree of interstitial disease. Values are the mean ⫾ SD. ⴱⴱ ⫽ P ⬍ 0.01; ⴱⴱⴱ ⫽ P ⬍ 0.001 by Student’s t-test (or Mann-Whitney nonparametric test for non-normally distributed data), experimental group versus placebo groups. GN ⫽ glomerulonephritis. DISCUSSION Experimental immune nephritis has proven to be a reasonable tool with which to model and study clinical diseases marked by spontaneous immune nephritis, such as Goodpasture’s syndrome and lupus nephritis. Use of this experimental tool has been instrumental in demonstrating the potential pathogenic role of several mediators, including a variety of adhesion molecules, chemokines, and cytokines (23–29). Proteinuria is typically the first indicator that surfaces in both experimental and spontaneous immune nephritis (1–5). In this study, we used 2 resources to determine whether other molecules might become evident in urine before the appearance of overt proteinuria. First, we had previously completed a strain distribution study of antiGBM disease susceptibility (2,3), which in effect allowed us to compare urine from mice that experience severe anti-GBM disease with that from control strains of mice. Second, we took advantage of a commercially available proteome array that allows screening for the presence of 62 different molecules in any given sample. In compar- ing urine from DBA/1 and 129/Sv mice subjected to experimental immune nephritis with urine from the BALB/c and B6 control mice, we identified CXCL16, P-selectin, TNFRI, and VCAM-1 as 4 molecules that are hyperexcreted in mice with severe nephritis. Whereas some of these molecules have previously been studied in the context of renal disease, others have not. VCAM-1 is an adhesion molecule that is expressed on a large number of cell types, including macrophages, dendritic cells, and endothelial cells, and plays an important role in cell recruitment into tissues mediated by VCAM-1–very late activation antigen 4 interactions. Increased expression of VCAM-1 has also been noted in arthritis and other immune disorders, in which it has also been identified as a therapeutic target (30,31). Endothelial hyperexpression of VCAM-1 has previously been noted in experimental immune nephritis, although antibody-mediated blockade was shown to have only a minimal impact on disease (32). VCAM-1 has also been observed to be expressed in the kidneys, in both murine and human lupus (8–10). The above find- VCAM-1, P-SELECTIN, TNFRI, AND CXCL16 IN NEPHRITIC URINE ings are reinforced by our present study, the results of which show that mesangial cells may be an additional source of VCAM-1. In addition, it is clear that infiltrating leukocytes may be another good source of this molecule. The present findings indicate the VCAM-1 may be a potential urinary marker of renal disease in mice with experimental immune-mediated nephritis, concordant with earlier studies of lupus nephritis (33). In addition, it appears that elevated urinary VCAM-1 levels may persist even after active renal inflammation subsides (Figure 2). P-selectin is an adhesion molecule that is expressed on platelets and several other cell types, and the level of P-selectin has been reported to be elevated in other autoinflammatory diseases, in which it has also been recognized as a circulating disease marker (11–13). The prominence of P-selectin in experimentally induced immune nephritis has also been quite extensively studied (34–37). Interestingly, a protective role for endothelial P-selectin in immune nephritis has been suggested (37). It has also been observed to be expressed within the kidneys, in both mice and patients with spontaneous lupus nephritis (14,15). However, it has not been specifically measured in the urine of mice or in the urine of patients with lupus or immune nephritis. These data represent the first demonstration of elevated levels of urinary P-selectin in immune nephritis. TNFRI is a cytokine receptor that plays various functions in the immune system. Interestingly, the expression of TNFRII (but not TNFRI) on intrinsic renal cells has recently been shown to be important in experimental immune nephritis (38). Additionally, levels of serum TNFRI have been noted to be increased in human systemic lupus erythematosus (17,18). There is a single report of hyperexpression of TNFRI in the kidneys of patients with proliferative lupus nephritis (19), but TNFRI has not yet been examined in the urine of patients with lupus or immune nephritis. Thus, this report is the first to describe elevated levels of urinary TNFRI in immune nephritis. Based on our findings, however, it appears that urinary TNFRI in immune nephritis may be originating, at least in part, from serum (Figures 2 and 3). CXCL16 is a chemokine that is reported to be expressed on dendritic cells and macrophages and is important for the recruitment of T cells and natural killer T cells into various tissues and for cell–cell interactions via the CXCR6 counterreceptor (20–22). CXCL16 also appears to be important in arthritis; however, it has not been studied previously in human 957 nephritis, in either the kidneys or urine. This represents the first report implicating a pathogenic role for this molecule in immune nephritis. Our in vitro studies indicate that both macrophages and mesangial cells constitute potential sources of this chemokine in nephritic kidneys, although additional cell types may also be contributors. The results of these studies have several important implications. First, given that all of these molecules are expressed within diseased or injured kidneys (Figures 4 and 5), they may be playing critical roles in disease pathogenesis. Indeed, in this study, we have formally demonstrated a pathogenic role for CXCL16 in experimental immune nephritis. Bone marrow transfer studies using CXCL16-deficient mice are warranted to ascertain the respective contributions of infiltrating versus resident renal cells toward the renal expression of CXCL16 (and the other implicated molecules) during immune nephritis. Although these 4 molecules have been discussed as independent entities, it is certainly possible that they may be coordinately regulated during disease, and there is some evidence to support this possibility (39–41). Second, because these 4 molecules appear early in experimental immune nephritis, prior to the onset of proteinuria, and correlate well with disease, they may also have the potential to serve as harbingers of renal disease in spontaneous immune-mediated nephritis (e.g., in lupus). In parallel studies, we recently demonstrated that levels of these 4 molecules are also elevated in the urine of mice and patients with lupus, correlating well with proteinuria and disease activity (Wu T, et al: unpublished observations). It would also be important to expand these “biased” proteomic studies (focused on only 62 molecules) to unbiased proteomic screens, in order to uncover additional mediators that may be excreted in the urine during the course of the disease. One hopes that such urine-based proteomic studies coupled with serum-based proteomics, as exemplified by the recently reported “glomerular proteome arrays” (42), may collectively pave the way toward a panel of “biomarkers” that may help identify individuals at the incipience of immune-mediated renal damage. Finally, given the observation that at least some of these molecules may play critical roles in the pathogenesis of nephritis, they also represent potential targets for therapeutic intervention. Indeed, VCAM-1 has been suggested as a potential therapeutic target in end-organ disease (30,31), although antibody-mediated blocking in experimental immune nephritis has not been very effective (32). Likewise, it has been suggested that glomerular 958 WU ET AL disease may be amenable to therapy by blocking P-selectin (16), although conflicting results have been noted in experimental immune nephritis (37). Based on the antibody-blocking studies described here, CXCL16 may turn out to be yet another therapeutic target in immune nephritis. Finally, it remains to be seen whether the coordinate blocking of 1 or more of these molecules may offer therapeutic relief that is even more superior in immune nephritis. AUTHOR CONTRIBUTIONS Dr. Mohan had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study design. Wu, Xie, Mohan. Acquisition of data. Wu, Xie, Bhaskarabhatla, Yan, Leone, Chen, Zhou. Analysis and interpretation of data. Wu, Xie, Putterman. Manuscript preparation. Wu, Xie, Putterman, Mohan. Statistical analysis. Wu, Xie. REFERENCES 1. Masugi M. 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