Clin Exp Nephrol DOI 10.1007/s10157-015-1123-1
ORIGINAL ARTICLE
Glomerular cytokine expression in murine lupus nephritis Kadiombo Anastasie Tshilela1 • Hidekazu Ikeuchi1 • Takayuki Matsumoto2 Takashi Kuroiwa3 • Noriyuki Sakurai2 • Toru Sakairi1 • Yoriaki Kaneko1 • Akito Maeshima1 • Keiju Hiromura1 • Yoshihisa Nojima1
•
Received: 30 January 2015 / Accepted: 7 May 2015 Ó Japanese Society of Nephrology 2015
Abstract Background Aberrant expression of T helper cell (Th) cytokines is believed to play a central role in the pathogenesis of systemic lupus erythematosus (SLE). While the glomerulus is one of the major targets of lupus inflammation, little is known about the cytokine expression in glomeruli. The current study aimed to explore the profiles of Th cytokine gene expressions in isolated glomeruli of lupus-prone mice. Methods Glomeruli were purified from lupus-prone MRL/lpr mice using the magnetic microbead method. Expressions of cytokine genes representing the Th subset and FoxP3 were examined using real-time polymerase chain reaction. Serum levels of these cytokines were also measured by enzyme-linked immunosorbent assay. MRL/n mice were used as controls. Histologic glomerular damages were scored semiquantitatively. To examine the role of TNF-a in glomerular damage, we administered etanercept, a TNF-a antagonist, into the subjects. Results Glomerular gene expressions of TNF-a in lpr mice increased with week postpartum and reached statistically significant levels at 16 weeks compared with those of the glomeruli from control mice. Expressions of IFN-c, IL-4 and FoxP3 also increased, but the difference was not & Hidekazu Ikeuchi [email protected] 1
Department of Medicine and Clinical Science, Gunma University Graduate School of Medicine, 3-39-22 Showa, Maebashi, Gunma 371-8511, Japan
2
Dialysis and Rheumatology Center, Toho Hospital, 1155 Kasagake, Midori, Gunma 379-2311, Japan
3
Gunma Rheumatism Clinic, 1040 Ino, Takasaki, Gunma 370-0004, Japan
significant. There was a significant increase in serum levels of TNF-a, IFN-c, and IL-17 and decrease in those of IL-4. Among the genes examined, TNF-a significantly correlated with glomerular damage score. Administration of etanercept did not affect glomerular cytokine expressions or proteinuria and failed to ameliorate histologic glomerular damages. Conclusion Our data suggest that Th1 cytokines, especially TNF-a, are dominantly expressed in the glomeruli of lupus-prone mice, but its pathophysiological role remains unclear. Keywords Lupus nephritis Kidney glomerulus Th1–Th2 balance Tumor necrosis factor-alpha
Introduction Systemic lupus erythematosus (SLE) is a chronic autoimmune disease affecting multiple organ systems, including the skin, joint, lung, nervous system, and kidney. Lupus nephritis (LN) is one of the most prevalent and serious complications of SLE and predicts a poor outcome [1]. LN is a representative of immune complex-mediated disease, which is characterized by depositions of immunoglobulins and complements, various degrees of glomerular hypercellularity, and thickening of glomerular basement membranes. Disruption of immunologic tolerance and acquired autoimmunity causes SLE and LN, in which aberrant expressions of T helper cell (Th) cytokines play a pivotal role [2]. Previous studies showed that Th1 cytokines (e.g., TNFa and IFN-c) were dominantly expressed in proliferative LN and that Th2 cytokines (e.g., IL-4) were dominant in other forms of SLE [2, 3]. Recent studies suggested a pathogenic role of Th17 [4, 5] and FoxP3 ? regulatory T
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cell (Treg) in LN [2, 6]. However, these data were obtained from studies on blood or whole kidney samples, and few studies have been conducted using isolated glomeruli. In the present study, we examined glomerular gene expressions of Th cytokines from murine LN by isolating glomeruli using magnetic microbeads technique. We found that TNF-a was a dominant cytokine expressed by glomeruli of lupus-prone mice, but its pathophysiologic role is unclear because etanercept, an inhibitor of TNF-a, failed to ameliorate glomerular damages.
Materials and methods Animals Female MRL/lpr and MRL/n mice were obtained from Japan SLC, Inc. (Hamamatsu, Japan) and were acclimatized in the laboratory 1 week before use. MRL/lpr mice were killed at 4, 8, 12, 16, and 20 weeks (n = 3–5 every week). We used MRL/n mice as normal controls and killed them at 10 weeks. Each group had 5 mice. Individual mice were placed in metabolic cages for 24 h urine collections every 4 weeks. All experiments were conducted under animal care and used committee-approved protocols.
Semiquantitative scoring Glomerular histological damage was evaluated by a semiquantitative scoring system as previously described [8]. Paraffin sections. (4 lm) were stained with periodic acidSchiff stain. Each glomerulus was scored on a semiquantitative scale of glomerular cross-sections (gcs): 0 = normal (35–40 cells/gcs, Fig. 1a, b); 1 = mild, glomeruli with few lesions showing slight proliferative changes and mild hypercellularity (41–50 cells/gcs, Fig. 1c); 2 = moderate, glomeruli with moderate hypercellularity (51–60 cells/gcs, Fig. 1d), including segmental and/or diffuse proliferative changes and hyalinosis; and 3 = severe, glomeruli with segmental or global sclerosis and/or severe hypercellularity ([60 cells/gcs, Fig. 1e), necrosis, and crescent formation. An average score of 20 glomeruli was calculated in each mouse.
Preparation of cDNA from collected glomeruli Glomeruli were isolated using microbeads as described previously [7]. Briefly, 4.5 lm magnetic beads (Dynal A.S., Oslo, Norway) were perfused by intracardiac injection. Kidneys were subsequently removed, and renal cortexes were minced. After 30 min of digestion with 1 mg/mL of collagenase A and 100 U/mL of deoxyribonuclease I in Hank’s balanced salt solution (HBSS; Invitrogen AB, Lidingo, Sweden) at 37 °C, glomeruli were collected using a magnetic particle concentrator (Dynal). Total RNA was isolated from purified glomeruli using RNeasy Mini Kit (Qiagen, Chatsworth, CA). Reverse transcriptase for converting RNA to cDNA was obtained from QuantiTect Reverse Transcription Kit (Qiagen). Quantitative real-time polymerase chain reaction Quantitative real-time Polymerase Chain Reaction (PCR) was performed using commercially available TaqMan Gene Expression Assays (Applied Biosystems, Foster City, CA, USA) of mice TNF-a (Mm00443258_m1), IFN-c (Mm00801778_m1), IL-4 (Mm00445259_m1), IL-17A (Mm00439619_m1), FoxP3 (Mm00475162_m1), and mice endogenous control according to the manufacturer’s instructions. Threshold cycle was calculated as the maximum number of PCR cycle (45) in case the gene was undetectable.
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Fig. 1 Histological pictures. a MRL/n mouse at 10 weeks, scored as 0 (normal), b MRL/lpr mouse at 4 weeks, scored as 0. c MRL/lpr mouse at 8 weeks, scored as 1 (mild). d MRL/lpr mouse at 12 weeks, scored as 2 (moderate). e MRL/lpr mouse at 16 weeks, scored as 3 for segmental sclerosis (severe). Brown magnetic beads can be seen in c and d
Clin Exp Nephrol
Urine and serum analyses
samples were collected for 24 h every 4 weeks. All mice were killed at 18 weeks; serum and kidney were analyzed as above.
Urinary protein, urinary creatinine, and serum creatinine were assessed by Hitachi 7180 autoanalyser (Hitachi HighTechnologies Corp., Tokyo, Japan). Serum concentration of cytokines was measured by multiplex bead array assays using the LUMINEX xMAP technology by Luminex 200 Analyzer (luminex corporation, Austin, TX, USA), as previously described [9]. When values were below detection limit, a value of 0.01 pg/ml was assigned. AntidsDNA antibody was measured using a commercially available ELISA kit (Shibayagi, Gunma, Japan).
Statistical analysis Statistical analysis was performed using Mann–Whitney U test or Kruskal–Wallis analysis followed by Dunn’s post test for comparisons between groups. Correlations between glomerular damage and gene expressions or serum cytokines were analyzed by Spearman’s rank correlation coefficient. SPSS Statistics 22.0 (Chicago, IL, USA) was used for statistical analysis. P values of \0.05 were considered statistically significant.
The effect of TNF-a antagonist on lupus-prone mice Etanercept (Takeda pharmacy, Japan), a fusion protein composed of a TNF receptor and the constant end of an IgG1 antibody, can block mouse TNF-a activities [10]. It was subcutaneously administered weekly to MRL/lpr mice from 8 to 16 weeks. Human IgG1 was similarly administered to lpr mice, which were analyzed as the control group. Urine
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Weeks box TNF-a, white box IFN-c. c The fold expression of IL-4, IL-17A, and FoxP3 in the glomeruli of normal control (NC) and MRL/lpr mice, relative to NC, is shown after correction for internal control. Black box IL-4, white box FoxP3, bar box IL-17A (IL-17) d Level of serum cytokines in normal control (NC) and MRL/lpr mice. Black box TNF-a, white box IFN-c, bar box IL-4, dot box IL-17A *P \ 0.05 versus normal control **P \ 0.001 versus normal control
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control (Figs. 2b, c). Serum levels of cytokines, except IL4, in lpr mice were significantly higher than those in control mice (Fig. 2d). Serum levels of IL-4 in lpr mice were significantly low at 16 weeks (Fig. 2d).
(Fig. 1). Glomerular damage score increased with week postpartum in lpr mice (Fig. 2a). Analysis of glomerular expressions of genes, and level of serum cytokines
Correlation between glomerular histological scores and glomerular gene expressions or serum cytokine levels
Relative expressions of glomerular TNF-a genes in lpr mice increased by 8.6- and 23.0-folds with week and reached statistically significant levels at 12 and 16 weeks, respectively, compared with those from the glomeruli of control mice (Fig. 2b). The expressions of IFN-c, IL-4, IL17 and FoxP3 were high at 16 weeks, but the difference was not statistically significant when compared with Fig. 3 Correlation between glomerular damage scores and glomerular gene expressions TNF-a (a) IFN-c (b) IL-4 (c) IL-17A (d) and FoxP3 (e). Data were accumulated from all time points and were analyzed together
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Next, we examined the relationship of glomerular histologic damage with glomerular gene expression or serum level of cytokines in lpr mice. Histologic and biochemical data at each time point were collected and analyzed
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altogether. Glomerular expressions of TNF-a, serum TNFa, and serum IFN-c were positively correlated with the extent of glomerular damages (Figs. 3a, 4a, b). The effect of etanercept on lupus-prone mice We hypothesized that TNF-a may have a central role in the pathogenesis of LN because of the significant relationship between glomerular and serum TNF-a with glomerular damage. Thus, we examined the effect of etanercept on lpr nephritis; however, its administration did not change levels of proteinuria (Fig. 5a), serum anti-ds DNA (Fig. 5b), and serum creatinine (data not shown). In addition, etanercept administration did not affect glomerular damage (Fig. 5c) or glomerular gene expressions, except IFN-c (Fig. 5d). In contrast, serum TNF-a and IL-17A were significantly upregulated (Fig. 5e).
Discussion In this study, we examined gene expressions of Th cytokines in isolated glomeruli of lupus-prone mice and found increased expressions of TNF-a and IFN- c, suggesting a Th1 dominancy in lupus glomeruli. We also found a significant correlation between glomerular damage
2
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scores with relative expression of glomerular TNF-a gene as well as with serum levels of TNF-a. Previous studies demonstrated that Th1 cytokines, mainly IFN-c, were dominant in serum and in proliferative LN kidneys [2, 3, 11]. Direct analysis of the glomerulus by laser microdissection disclosed upregulation of IFN-c, but the expression of TNF-a were not examined by these studies [3, 12, 13]. In this regard, this is the first report to show, by direct examination, the dominant expression of TNF-a in isolated lupus glomeruli. Regardless of the significant correlation between TNF-a and glomerular damage, systemic inhibition of TNF-a failed to ameliorate nephritis. Thus, the role of TNF-a in LN remains unknown. Recent studies suggested a pathogenic role of Th17 in human and murine LN [2, 4, 5]. However, the number of cells expressing IL-17A was low in MRL/lpr kidney, and genetic disruption of IL-17A gene did not affect morphologic or functional parameters in MRL/lpr mice with LN [14]. In accordance with this, we confirmed a very low expression of IL-17A gene in MRL/lpr kidney and found no association between serum and glomerular expression of IL-17A with histologic damage scores. These results stand against the hypothesis of a critical role of IL-17A in the pathophysiology of LN, at least in MRL/lpr model mice. Upregulation of Th2 cytokines have been reported in proliferative LN [2, 3, 11]. In the present study, we noted
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Fig. 5 Effects of etanercept on MRL/lpr nephritis. a Comparison of proteinuria between IgG and etanercept in MRL/lpr mice. Each group had 10 mice. Etanercept or control IgG1 was subcutaneously administrated weekly on MRL/ lpr mice from 8 to 16 weeks; all mice were killed at 18 weeks. b Comparison of dsDNA antibodies between IgG and etanercept in MRL/lpr mice at 18 weeks. c Comparison of glomerular damage between IgG and etanercept in MRL/lpr mice at 18 weeks. d, e The effect of IgG or etanercept on glomerular gene expression (d) or serum cytokine expression (e). Black box IgG; white box Etanercept; IgG: Human IgG1, TNF: TNF-a IFN: IFN-c, IL-17: IL-17A
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higher expression of IL4 in lpr glomeruli, although at insignificant levels. Serum levels of IL-4 were lower in lpr mice. No correlation was observed between glomerular IL-4 expression and glomerular damage. While upregulation of FoxP3-positive cells had been demonstrated in peripheral blood of human and murine LN [15, 16], immunohistochemical analysis failed to show a significant
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increase in lupus glomeruli compared with normal control [17]. The expression of FoxP3 was clearly detected in our assay on lpr glomeruli, but correlation with glomerular damage was not found. Taken together, it is hard to consider that IL-4 and FoxP3 primarily contribute to the development and progression of glomerular damages in MRL/lpr mice.
Clin Exp Nephrol
Except for a limited number of reports, majority of trials on TNF-a antagonists in human and murine have failed LN [18–21]. Although there was a significant correlation between glomerular damage and TNF-a expression, we failed to show a beneficial effect of etanercept on LN. There are several explanations for this discrepancy. First, TNF-a may be just a marker of damaged glomeruli, and not a causative mediator. It has been reported that glomerular cells, such as mesangial cells, release TNF-a in response to immunoglobulin deposition [22]. In fact, previous immunohistochemical analysis shows that the site of TNF-a expression in glomerulus from MRL/lpr mice is in mesangial area [23]. Second, TNF-a inhibition resulted to feedback upregulation of glomerular and serum TNF-a as shown in Fig. 5b, c); this may have overcome the effect of etanercept. Third, other cytokines were either upregulated or downregulated in response to administration of TNF-a antagonist, perturbed cytokine networks, and masked the efficacy of etanercept for LN. We did not evaluate cytokine expression by tubular or interstitial cells in the current study; future investigations on this would be necessary to understand more about the pathogenetic role of cytokine network and to seek therapeutic targets in lupus nephritis. In conclusion, we demonstrated dominant expression of TNF-a gene in MRL/lpr nephritis by directly examining isolated glomeruli. Although a significant correlation was observed between glomerular damage and TNF-a expression in glomeruli and serum, systemic inhibition of TNF-a by etanercept failed to show beneficial effects, suggesting a complex role of TNF-a in the development and progression of LN. Acknowledgments We thank Ms Rumiko Koitabashi for her excellent technical assistance. Conflict of interest interest exists.
The authors have declared that no conflict of
References 1. Waldman M, Appel GB. Update on the treatment of lupus nephritis. Kidney Int. 2006;70:1403–12. 2. Miyake K, Akahoshi M, Nakashima H. Th subset balance in lupus nephritis. J Biomed Biotechnol. 2011;2011:980286. 3. Uhm WS, Na K, Song GW, et al. Cytokine balance in kidney tissue from lupus nephritis patients. Rheumatology (Oxford). 2003;42:935–8. 4. Crispin JC, Oukka M, Bayliss G, et al. Expanded double negative T cells in patients with systemic lupus erythematosus produce IL17 and infiltrate the kidneys. J Immunol. 2008;181(12):8761–6.
5. Pisitkun P, Ha HL, Wang H, et al. Interleukin-17 cytokines are critical in development of fatal lupus glomerulonephritis. Immunity. 2012;37:1104–15. 6. Xing Q, Su H, Cui J, Wang B. Role of treg cells and TGF-beta1 in patients with systemic lupus erythematosus: a possible relation with lupus nephritis. Immunol Invest. 2012;41:15–27. 7. Takemoto M, Asker N, Gerhardt H, et al. A new method for large scale isolation of kidney glomeruli from mice. Am J Pathol. 2002;161:799–805. 8. Kikawada E, Lenda DM, Kelley VR. IL-12 deficiency in MRLFas(lpr) mice delays nephritis and intrarenal IFN-gamma expression, and diminishes systemic pathology. J Immunol. 2003;170:3915–25. 9. Elshal MF, McCoy JP. Multiplex bead array assays: performance evaluation and comparison of sensitivity to ELISA. Methods. 2006;38:317–23. 10. Liu R, Bal HS, Desta T, Behl Y, Graves DT. Tumor necrosis factor-a mediates diabetes-enhanced apoptosis of matrix-producing cells and impairs diabetic healing. Am J Pathol. 2006;168:757–64. 11. Masutani K, Akahoshi M, Tsuruya K, et al. Predominance of Th1 immune response in diffuse proliferative lupus nephritis. Arthritis Rheum. 2001;44:2097–106. 12. Wang Y, Ito S, Chino Y, et al. Use of laser microdissection in the analysis of renal-infiltrating T cells in MRL/lpr mice. Mod Rheumatol. 2008;18:385–93. 13. Wang Y, Ito S, Chino Y, et al. Laser microdissection-based analysis of cytokine balance in the kidneys of patients with lupus nephritis. Clin Exp Immunol. 2010;159:1–10. 14. Schmidt T, Paust HJ, Krebs CF, et al. Function of the T 17/IL17A immune response in murine lupus nephritis. Arthritis Rheumatol. 2015;67:475–87. 15. Hondowicz BD, Fields ML, Nish SA, et al. Autoantibody production in lpr/lpr gld/gld mice reflects accumulation of CD4 ? effector cells that are resistant to regulatory T cell activity. J Autoimmun. 2008;31:98–109. 16. Bonelli M, Goschl L, Bluml S, et al. CD4(?)CD25(-)Foxp3(?) T cells: a marker for lupus nephritis? Arthritis Res Ther. 2014;16:R104. 17. Yazici MU, Orhan D, Kale G, et al. Studying IFN-gamma, IL-17 and FOXP3 in pediatric lupus nephritis. Pediatr Nephrol. 2014;29:853–62. 18. Zhu LJ, Yang X, Yu XQ. Anti-TNF-alpha therapies in systemic lupus erythematosus. J Biomed Biotechnol. 2010;2010:465898. 19. Williams VL, Cohen PR. TNF alpha antagonist-induced lupuslike syndrome: report and review of the literature with implications for treatment with alternative TNF alpha antagonists. Int J Dermatol. 2011;50:619–25. 20. Almoallim H, Al-Ghamdi Y, Almaghrabi H, Alyasi O. Anti-tumor necrosis factor-alpha induced systemic lupus erythematosus. Open Rheumatol J. 2012;6:315–9. 21. Yahya TM, Dhanyamraju S, Harrington TM, et al. Spontaneous resolution of lupus nephritis following withdrawal of etanercept. Ann Clin Lab Sci. 2013;43:447–9. 22. Kelley VR, Wuthrich RP. Cytokines in the pathogenesis of systemic lupus erythematosus. Semin Nephrol. 1999;19:57–66. 23. Yokoyama H, Kreft B, Kelley VR. Biphasic increase in circulating and renal TNF-alpha in MRL-lpr mice with differing regulatory mechanisms. Kidney Int. 1995;47:122–30.
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ORIGINAL ARTICLE
Glomerular cytokine expression in murine lupus nephritis Kadiombo Anastasie Tshilela1 • Hidekazu Ikeuchi1 • Takayuki Matsumoto2 Takashi Kuroiwa3 • Noriyuki Sakurai2 • Toru Sakairi1 • Yoriaki Kaneko1 • Akito Maeshima1 • Keiju Hiromura1 • Yoshihisa Nojima1
•
Received: 30 January 2015 / Accepted: 7 May 2015 Ó Japanese Society of Nephrology 2015
Abstract Background Aberrant expression of T helper cell (Th) cytokines is believed to play a central role in the pathogenesis of systemic lupus erythematosus (SLE). While the glomerulus is one of the major targets of lupus inflammation, little is known about the cytokine expression in glomeruli. The current study aimed to explore the profiles of Th cytokine gene expressions in isolated glomeruli of lupus-prone mice. Methods Glomeruli were purified from lupus-prone MRL/lpr mice using the magnetic microbead method. Expressions of cytokine genes representing the Th subset and FoxP3 were examined using real-time polymerase chain reaction. Serum levels of these cytokines were also measured by enzyme-linked immunosorbent assay. MRL/n mice were used as controls. Histologic glomerular damages were scored semiquantitatively. To examine the role of TNF-a in glomerular damage, we administered etanercept, a TNF-a antagonist, into the subjects. Results Glomerular gene expressions of TNF-a in lpr mice increased with week postpartum and reached statistically significant levels at 16 weeks compared with those of the glomeruli from control mice. Expressions of IFN-c, IL-4 and FoxP3 also increased, but the difference was not & Hidekazu Ikeuchi [email protected] 1
Department of Medicine and Clinical Science, Gunma University Graduate School of Medicine, 3-39-22 Showa, Maebashi, Gunma 371-8511, Japan
2
Dialysis and Rheumatology Center, Toho Hospital, 1155 Kasagake, Midori, Gunma 379-2311, Japan
3
Gunma Rheumatism Clinic, 1040 Ino, Takasaki, Gunma 370-0004, Japan
significant. There was a significant increase in serum levels of TNF-a, IFN-c, and IL-17 and decrease in those of IL-4. Among the genes examined, TNF-a significantly correlated with glomerular damage score. Administration of etanercept did not affect glomerular cytokine expressions or proteinuria and failed to ameliorate histologic glomerular damages. Conclusion Our data suggest that Th1 cytokines, especially TNF-a, are dominantly expressed in the glomeruli of lupus-prone mice, but its pathophysiological role remains unclear. Keywords Lupus nephritis Kidney glomerulus Th1–Th2 balance Tumor necrosis factor-alpha
Introduction Systemic lupus erythematosus (SLE) is a chronic autoimmune disease affecting multiple organ systems, including the skin, joint, lung, nervous system, and kidney. Lupus nephritis (LN) is one of the most prevalent and serious complications of SLE and predicts a poor outcome [1]. LN is a representative of immune complex-mediated disease, which is characterized by depositions of immunoglobulins and complements, various degrees of glomerular hypercellularity, and thickening of glomerular basement membranes. Disruption of immunologic tolerance and acquired autoimmunity causes SLE and LN, in which aberrant expressions of T helper cell (Th) cytokines play a pivotal role [2]. Previous studies showed that Th1 cytokines (e.g., TNFa and IFN-c) were dominantly expressed in proliferative LN and that Th2 cytokines (e.g., IL-4) were dominant in other forms of SLE [2, 3]. Recent studies suggested a pathogenic role of Th17 [4, 5] and FoxP3 ? regulatory T
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cell (Treg) in LN [2, 6]. However, these data were obtained from studies on blood or whole kidney samples, and few studies have been conducted using isolated glomeruli. In the present study, we examined glomerular gene expressions of Th cytokines from murine LN by isolating glomeruli using magnetic microbeads technique. We found that TNF-a was a dominant cytokine expressed by glomeruli of lupus-prone mice, but its pathophysiologic role is unclear because etanercept, an inhibitor of TNF-a, failed to ameliorate glomerular damages.
Materials and methods Animals Female MRL/lpr and MRL/n mice were obtained from Japan SLC, Inc. (Hamamatsu, Japan) and were acclimatized in the laboratory 1 week before use. MRL/lpr mice were killed at 4, 8, 12, 16, and 20 weeks (n = 3–5 every week). We used MRL/n mice as normal controls and killed them at 10 weeks. Each group had 5 mice. Individual mice were placed in metabolic cages for 24 h urine collections every 4 weeks. All experiments were conducted under animal care and used committee-approved protocols.
Semiquantitative scoring Glomerular histological damage was evaluated by a semiquantitative scoring system as previously described [8]. Paraffin sections. (4 lm) were stained with periodic acidSchiff stain. Each glomerulus was scored on a semiquantitative scale of glomerular cross-sections (gcs): 0 = normal (35–40 cells/gcs, Fig. 1a, b); 1 = mild, glomeruli with few lesions showing slight proliferative changes and mild hypercellularity (41–50 cells/gcs, Fig. 1c); 2 = moderate, glomeruli with moderate hypercellularity (51–60 cells/gcs, Fig. 1d), including segmental and/or diffuse proliferative changes and hyalinosis; and 3 = severe, glomeruli with segmental or global sclerosis and/or severe hypercellularity ([60 cells/gcs, Fig. 1e), necrosis, and crescent formation. An average score of 20 glomeruli was calculated in each mouse.
Preparation of cDNA from collected glomeruli Glomeruli were isolated using microbeads as described previously [7]. Briefly, 4.5 lm magnetic beads (Dynal A.S., Oslo, Norway) were perfused by intracardiac injection. Kidneys were subsequently removed, and renal cortexes were minced. After 30 min of digestion with 1 mg/mL of collagenase A and 100 U/mL of deoxyribonuclease I in Hank’s balanced salt solution (HBSS; Invitrogen AB, Lidingo, Sweden) at 37 °C, glomeruli were collected using a magnetic particle concentrator (Dynal). Total RNA was isolated from purified glomeruli using RNeasy Mini Kit (Qiagen, Chatsworth, CA). Reverse transcriptase for converting RNA to cDNA was obtained from QuantiTect Reverse Transcription Kit (Qiagen). Quantitative real-time polymerase chain reaction Quantitative real-time Polymerase Chain Reaction (PCR) was performed using commercially available TaqMan Gene Expression Assays (Applied Biosystems, Foster City, CA, USA) of mice TNF-a (Mm00443258_m1), IFN-c (Mm00801778_m1), IL-4 (Mm00445259_m1), IL-17A (Mm00439619_m1), FoxP3 (Mm00475162_m1), and mice endogenous control according to the manufacturer’s instructions. Threshold cycle was calculated as the maximum number of PCR cycle (45) in case the gene was undetectable.
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Fig. 1 Histological pictures. a MRL/n mouse at 10 weeks, scored as 0 (normal), b MRL/lpr mouse at 4 weeks, scored as 0. c MRL/lpr mouse at 8 weeks, scored as 1 (mild). d MRL/lpr mouse at 12 weeks, scored as 2 (moderate). e MRL/lpr mouse at 16 weeks, scored as 3 for segmental sclerosis (severe). Brown magnetic beads can be seen in c and d
Clin Exp Nephrol
Urine and serum analyses
samples were collected for 24 h every 4 weeks. All mice were killed at 18 weeks; serum and kidney were analyzed as above.
Urinary protein, urinary creatinine, and serum creatinine were assessed by Hitachi 7180 autoanalyser (Hitachi HighTechnologies Corp., Tokyo, Japan). Serum concentration of cytokines was measured by multiplex bead array assays using the LUMINEX xMAP technology by Luminex 200 Analyzer (luminex corporation, Austin, TX, USA), as previously described [9]. When values were below detection limit, a value of 0.01 pg/ml was assigned. AntidsDNA antibody was measured using a commercially available ELISA kit (Shibayagi, Gunma, Japan).
Statistical analysis Statistical analysis was performed using Mann–Whitney U test or Kruskal–Wallis analysis followed by Dunn’s post test for comparisons between groups. Correlations between glomerular damage and gene expressions or serum cytokines were analyzed by Spearman’s rank correlation coefficient. SPSS Statistics 22.0 (Chicago, IL, USA) was used for statistical analysis. P values of \0.05 were considered statistically significant.
The effect of TNF-a antagonist on lupus-prone mice Etanercept (Takeda pharmacy, Japan), a fusion protein composed of a TNF receptor and the constant end of an IgG1 antibody, can block mouse TNF-a activities [10]. It was subcutaneously administered weekly to MRL/lpr mice from 8 to 16 weeks. Human IgG1 was similarly administered to lpr mice, which were analyzed as the control group. Urine
(b) Relave mRNA expression, TNF-α
*
2
1
0
NC
4
8
12
50
*
400 300
30
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20 *
100
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Weeks
Weeks
(c)
500
TNF -α IFN -γ
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(d) 600 IL-4
50
400
IL-17
40
300
FoxP3
30
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10 NC
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Weeks Fig. 2 Glomerular damage scores, glomerular gene expressions, and serum cytokines in MRL/lpr mice. a Glomerular damage scores of control and MRL/lpr mice. Bar indicates median score. Groups for each week consisted of 3–5 mice per week. Normal control (NC) mice were killed and examined at 10 weeks. b The fold expression of Th1 cytokines in glomeruli of normal control (NC) and MRL/lpr mice, relative to NC, is shown after correction for internal control. Black
Serum cytokines (pg/ml)
500
0
50
60
IL-17, FoxP3
Glomerular damage
Representation image of glomeruli from lpr mice was shown at each week with glomerular damage score
**
3
Relave mRNA expression, IL-4
Analysis of glomerular damage
IFN-γ
(a)
Results
TNF -α
40
IFN -γ 30
IL-4
20
IL-17
*
* *
10
*
0
NC
4
8
12
16
Weeks box TNF-a, white box IFN-c. c The fold expression of IL-4, IL-17A, and FoxP3 in the glomeruli of normal control (NC) and MRL/lpr mice, relative to NC, is shown after correction for internal control. Black box IL-4, white box FoxP3, bar box IL-17A (IL-17) d Level of serum cytokines in normal control (NC) and MRL/lpr mice. Black box TNF-a, white box IFN-c, bar box IL-4, dot box IL-17A *P \ 0.05 versus normal control **P \ 0.001 versus normal control
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control (Figs. 2b, c). Serum levels of cytokines, except IL4, in lpr mice were significantly higher than those in control mice (Fig. 2d). Serum levels of IL-4 in lpr mice were significantly low at 16 weeks (Fig. 2d).
(Fig. 1). Glomerular damage score increased with week postpartum in lpr mice (Fig. 2a). Analysis of glomerular expressions of genes, and level of serum cytokines
Correlation between glomerular histological scores and glomerular gene expressions or serum cytokine levels
Relative expressions of glomerular TNF-a genes in lpr mice increased by 8.6- and 23.0-folds with week and reached statistically significant levels at 12 and 16 weeks, respectively, compared with those from the glomeruli of control mice (Fig. 2b). The expressions of IFN-c, IL-4, IL17 and FoxP3 were high at 16 weeks, but the difference was not statistically significant when compared with Fig. 3 Correlation between glomerular damage scores and glomerular gene expressions TNF-a (a) IFN-c (b) IL-4 (c) IL-17A (d) and FoxP3 (e). Data were accumulated from all time points and were analyzed together
(a)
Next, we examined the relationship of glomerular histologic damage with glomerular gene expression or serum level of cytokines in lpr mice. Histologic and biochemical data at each time point were collected and analyzed
(b)
TNF-α
IFN-γ
10000
1000
r = -0.377 P = 0.101
1000
U 3
100
100
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1
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2
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(d)
IL-4
1
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IL-17
100
10000 1000
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r = -0.357 P = 0.122
100 10
r = 0.210 P = 0.374
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0.1
FoxP3
(e) 100
r = -0.404 P = 0.078 10
1 0
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123
0.5
1
1.5
2
2.5
0.5
1
1.5
2
2.5
Clin Exp Nephrol Fig. 4 Correlation between glomerular damage scores and serum cytokines TNF-a (a) IFN-c (b) IL-4 (c) and IL17A (d). Data were accumulated from all time points and were analyzed together
(a)
(b)
Serum TNF-α
Serum IFN-γ
100
100
r = 0.650 P = 0.012
10
10
r = 0.675 P = 0.008
1
1 0
0.5
1
1.5
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0
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0.1
0
0.01
0
(c)
0.5
(d)
Serum IL-4
100
1
1.5
2
2.5
2
2.5
Serum IL-17
100
10
r = 0.035 P = 0.906
10
r = -0.065 P = 0.825
1 0
0.5
1
1.5
1 0
0.5
1
1.5
0.1
altogether. Glomerular expressions of TNF-a, serum TNFa, and serum IFN-c were positively correlated with the extent of glomerular damages (Figs. 3a, 4a, b). The effect of etanercept on lupus-prone mice We hypothesized that TNF-a may have a central role in the pathogenesis of LN because of the significant relationship between glomerular and serum TNF-a with glomerular damage. Thus, we examined the effect of etanercept on lpr nephritis; however, its administration did not change levels of proteinuria (Fig. 5a), serum anti-ds DNA (Fig. 5b), and serum creatinine (data not shown). In addition, etanercept administration did not affect glomerular damage (Fig. 5c) or glomerular gene expressions, except IFN-c (Fig. 5d). In contrast, serum TNF-a and IL-17A were significantly upregulated (Fig. 5e).
Discussion In this study, we examined gene expressions of Th cytokines in isolated glomeruli of lupus-prone mice and found increased expressions of TNF-a and IFN- c, suggesting a Th1 dominancy in lupus glomeruli. We also found a significant correlation between glomerular damage
2
2.5
0
0
scores with relative expression of glomerular TNF-a gene as well as with serum levels of TNF-a. Previous studies demonstrated that Th1 cytokines, mainly IFN-c, were dominant in serum and in proliferative LN kidneys [2, 3, 11]. Direct analysis of the glomerulus by laser microdissection disclosed upregulation of IFN-c, but the expression of TNF-a were not examined by these studies [3, 12, 13]. In this regard, this is the first report to show, by direct examination, the dominant expression of TNF-a in isolated lupus glomeruli. Regardless of the significant correlation between TNF-a and glomerular damage, systemic inhibition of TNF-a failed to ameliorate nephritis. Thus, the role of TNF-a in LN remains unknown. Recent studies suggested a pathogenic role of Th17 in human and murine LN [2, 4, 5]. However, the number of cells expressing IL-17A was low in MRL/lpr kidney, and genetic disruption of IL-17A gene did not affect morphologic or functional parameters in MRL/lpr mice with LN [14]. In accordance with this, we confirmed a very low expression of IL-17A gene in MRL/lpr kidney and found no association between serum and glomerular expression of IL-17A with histologic damage scores. These results stand against the hypothesis of a critical role of IL-17A in the pathophysiology of LN, at least in MRL/lpr model mice. Upregulation of Th2 cytokines have been reported in proliferative LN [2, 3, 11]. In the present study, we noted
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(a)
(b) 400
,J*
DsDNA Ab (mU/ml)
Proteinuria (mg/dL)
400
300
200
100
0
8
10
12
14
16
350 300 250 200 150 100 50
18
Weeks
(c)
P = 0.3 450
(WDQHUFHSW
0
IgG
(d)
P = 0.8 4
Etanercept
P = 0.3 16
Glomerular mRNA expression
Glomerular damage
Fig. 5 Effects of etanercept on MRL/lpr nephritis. a Comparison of proteinuria between IgG and etanercept in MRL/lpr mice. Each group had 10 mice. Etanercept or control IgG1 was subcutaneously administrated weekly on MRL/ lpr mice from 8 to 16 weeks; all mice were killed at 18 weeks. b Comparison of dsDNA antibodies between IgG and etanercept in MRL/lpr mice at 18 weeks. c Comparison of glomerular damage between IgG and etanercept in MRL/lpr mice at 18 weeks. d, e The effect of IgG or etanercept on glomerular gene expression (d) or serum cytokine expression (e). Black box IgG; white box Etanercept; IgG: Human IgG1, TNF: TNF-a IFN: IFN-c, IL-17: IL-17A
3
2
1
14
IgG
Etanercept
Etanercept
10 8 6 4
P = 0.8 P < 0.01
P = 0.6 P = 0.3
2 0
0
IgG
12
TNF
IFN
IL-4
IL-17 FoxP3
(e) 8000
P = 0.04
6HUXPF\WRNLQHV SJPO
7000 6000
IgG Etanercept
5000 4000
P = 0.01
P = 0.5
3000 2000
P = 0.1
1000 0
71)
,)1
higher expression of IL4 in lpr glomeruli, although at insignificant levels. Serum levels of IL-4 were lower in lpr mice. No correlation was observed between glomerular IL-4 expression and glomerular damage. While upregulation of FoxP3-positive cells had been demonstrated in peripheral blood of human and murine LN [15, 16], immunohistochemical analysis failed to show a significant
123
,/
,/
increase in lupus glomeruli compared with normal control [17]. The expression of FoxP3 was clearly detected in our assay on lpr glomeruli, but correlation with glomerular damage was not found. Taken together, it is hard to consider that IL-4 and FoxP3 primarily contribute to the development and progression of glomerular damages in MRL/lpr mice.
Clin Exp Nephrol
Except for a limited number of reports, majority of trials on TNF-a antagonists in human and murine have failed LN [18–21]. Although there was a significant correlation between glomerular damage and TNF-a expression, we failed to show a beneficial effect of etanercept on LN. There are several explanations for this discrepancy. First, TNF-a may be just a marker of damaged glomeruli, and not a causative mediator. It has been reported that glomerular cells, such as mesangial cells, release TNF-a in response to immunoglobulin deposition [22]. In fact, previous immunohistochemical analysis shows that the site of TNF-a expression in glomerulus from MRL/lpr mice is in mesangial area [23]. Second, TNF-a inhibition resulted to feedback upregulation of glomerular and serum TNF-a as shown in Fig. 5b, c); this may have overcome the effect of etanercept. Third, other cytokines were either upregulated or downregulated in response to administration of TNF-a antagonist, perturbed cytokine networks, and masked the efficacy of etanercept for LN. We did not evaluate cytokine expression by tubular or interstitial cells in the current study; future investigations on this would be necessary to understand more about the pathogenetic role of cytokine network and to seek therapeutic targets in lupus nephritis. In conclusion, we demonstrated dominant expression of TNF-a gene in MRL/lpr nephritis by directly examining isolated glomeruli. Although a significant correlation was observed between glomerular damage and TNF-a expression in glomeruli and serum, systemic inhibition of TNF-a by etanercept failed to show beneficial effects, suggesting a complex role of TNF-a in the development and progression of LN. Acknowledgments We thank Ms Rumiko Koitabashi for her excellent technical assistance. Conflict of interest interest exists.
The authors have declared that no conflict of
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