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CXCL10 expression induced by Mxi1 inactivation induces mesangial cell apoptosis in mouse Habu nephritis
Cellular Signalling 27 (2015) 943–950
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CXCL10 expression induced by Mxi1 inactivation induces mesangial cell apoptosis in mouse Habu nephritis Lingling Wu a,b, Xiaoniao Chen a,b, Yan Mei a, Quan Hong a, Zhe Feng a, Yang Lv a, Jun Wen a, Xiaoluan Liu a, Guangyan Cai a,⁎, Xiangmei Chen a,⁎ a b
Department of Nephrology, Chinese PLA General Hospital, Chinese PLA Institute of Nephrology, State Key Laboratory of Kidney Diseases, National Clinical Research Center for Kidney Diseases, China Medical College, NanKai University, Tianjin, China
a r t i c l e
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Article history: Received 12 November 2014 Received in revised form 13 January 2015 Accepted 27 January 2015 Available online 12 February 2015 Keywords: Mxi1 CXCL10 Apoptosis Mesangial cells Caspase 3
a b s t r a c t MAX interactor 1 (Mxi1) proteins are c-myc antagonists that primarily exert their biological functions by inhibiting Myc-dependent gene transcription. In this study, Mxi1−/− mice were used to generate a model of mesangial proliferative glomerulonephritis for the first time. In the present study, we demonstrated that Mxi1−/− mice exhibited a more typical and severe pathological phenotype, which was displayed primarily as a noticeable dissolution phenotype with a higher proportion of apoptotic cells and higher chemokine CXCL10 expression during the early days of modeling, compared with wild-type mice. Additionally, we determined that IRF3-mediated TLR4 signaling was likely involved in regulating CXCL10 expression, which might participate in the mesangial dissolution process. We also found increases in CXCL10 expression, caspase 3 activation, and the proportion of apoptotic cells when Mxi1 expression was inhibited in mouse mesangial cells. Furthermore, the proportion of apoptotic cells decreased after inhibiting CXCL10 expression. Therefore, we concluded that the mesangial cell apoptosis observed in this mesangial proliferative glomerulonephritis model was related to CXCL10 expression induced by Mxi1 inactivation. This finding provides a new theoretical basis for the mechanism of mesangial proliferative glomerulonephritis progression and reveals potential intervention targets for the early treatment of this disease. © 2015 Elsevier Inc. All rights reserved.
1. Introduction MAX interactor 1 (Mxi1) is a member of the MAD family of transcriptional repressors that play roles in cell proliferation and differentiation [1–3]. Mxi1 primarily exerts its biological functions by inhibiting Myc-dependent gene transcription [4–6]. Myc is involved in many biological processes, including cell proliferation, growth, metabolism, differentiation, and malignancy. Mxi1 is abnormally expressed in various human tumors and is thought to play a positive regulatory role in cell proliferation [7–9]. Mxi1 expression decreases significantly during the proliferative period in an anti-Thy1 nephritic rat model and gradually increases as proliferation declines, thereby suggesting that Mxi1 plays a role in mesangial proliferation [10]. However, the specific mechanisms underlying the effects of Mxi1 on mesangial proliferative glomerulonephritis (MesPGN) require further investigation. MesPGN, which includes IgA nephropathy (IgAN) and non-IgA MesPGN, is a common chronic kidney disease [11,12]. Many factors
⁎ Corresponding authors at: Department of Nephrology, Chinese PLA Institute of Nephrology & Key Lab, Chinese PLA Hospital, 28 Fuxing Road, Beijing 100853, China. Tel.: +86 10 68211187; fax: +86 10 68130297. E-mail addresses: [email protected] (G. Cai), [email protected] (X. Chen).
http://dx.doi.org/10.1016/j.cellsig.2015.01.019 0898-6568/© 2015 Elsevier Inc. All rights reserved.
are involved in the occurrence and development of MesPGN. Although the specific details of the causes and pathogenesis of MesPGN remain unclear, MesPGN is known to be an autoimmune inflammatory disease that involves various pathways; therefore, the causes of this disease are likely related to the immune-mediated inflammatory response [13–15]. Based on the microarray results from anti-Thy-1 nephritis models, the expression levels of chemokines, such as CCL2, CCL5 and CCL6, are significantly higher during the early stage of modeling, suggesting that inflammatory-reaction-related cytokines play an important role in the pathological progression of MesPGN [16,17]. In the present study, Mxi1 knockout mice (Mxi1−/− mice) were used to generate a mouse model of MesPGN. Both Mxi1−/− and wildtype (Mxi1+/+) mice were compared using periodic acid-Schiff (PAS) staining, which revealed that the knockout of Mxi1 aggravated the pathological phenotype. The Mxi1−/− mice also exhibited the typical dissolution phenotype, while the Mxi1+/+ mice exhibited an extremely light and atypical dissolution phenotype. Using real-time PCR and western blot analyses, we detected changes in chemokine expression and found that the CXCL10 level increased significantly in the Mxi1−/− mice during modeling. Furthermore, the specific mechanisms by which CXCL10 affected mesangial cells were investigated in vitro using cultured mouse mesangial cells (MMCs).
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2. Materials and methods
2.6. Apoptosis studies
2.1. Experimental animals and induction of a Habu nephritis mouse model
Cultured MMC apoptosis was assessed using Hoechst 33342 (SigmaAldrich) staining. The cells were stained with Hoechst 33342 at room temperature for 5 min after being fixed in 4% paraformaldehyde for 10 min at room temperature and then washed 3 times with ice-cold PBS. The MMCs were observed by fluorescence microscopy, and all of the microscopic images were captured using a 12.8-megapixel camera (DP72, Olympus, Japan). Cultured MMC apoptosis was also assessed using TUNEL staining. MMCs were cultured to 70% confluency in special glass-bottom microwell dishes, and then TUNEL staining was performed using an In Situ Cell Death Detection Kit, POD (Roche, Switzerland).
Glomerulonephritis (GN) induced by Habu snake venom is a selflimiting nephritis model that primarily causes glomerular mesangial injury. This method successfully generates a mesangial proliferative glomerulonephritis mouse model. In this study, Mxi1−/− mice (kindly provided by Professor HW Lee at Yonsei University; the results of Mxi1−/− mouse identification were shown in Supplemental Fig. 1) and Mxi1+/+ mice were given a single intravenous injection of Habu snake venom (HSV) at a dose of 2.5 mg/kg bodyweight to generate a HSV-induced mesangial proliferative glomerulonephritis model. Control mice were given saline injections. The mice were maintained under the following specific pathogen-free conditions: 22 ± 1 °C, 40% humidity, 12/12 h light/dark cycle, five males per cage, and free access to food and water. The mice were sacrificed at 1, 3 and 7 days after injection for experiments (eight mice per group), and the renal cortex was isolated. 2.2. Reagents HSV was purchased from Wako. Hoechst 33342 was purchased from Sigma-Aldrich. The following primary antibodies were used: rabbit anti-active caspase 3 (Cell Signaling), rabbit anti-CXCL10 (Cell Signaling), mouse anti-CXCL10 (R&D), rabbit anti-p-IRF3 (Cell Signaling), anti-toll-like receptor 4 (TLR4, Cell Signaling), mouse anti-β-actin (Sigma-Aldrich) and rabbit anti-Mxi1 (Abcam). The following secondary antibodies were used: horseradish peroxidase-conjugated IgG and mouse IgG (Beyotime). 2.3. Cell culture and transfection MMCs were purchased from ATCC (No. CRL-1927) and were maintained in RPMI 1640 medium (Sigma-Aldrich) supplemented with 10% fetal calf serum (FCS; Life Technologies, Rockville, MD, USA), 100 U/ml penicillin G, and 100 μg/ml streptomycin in a humidified 5% CO2 atmosphere. RNAiMAX (Invitrogen Life Technology) transfection reagent was used with MMCs for siRNA transfection. For the CXCL10 inhibition experiment, the cells were cultured in the presence of CXCL10 antibody at a concentration of 700 ng/ml, and the control group was given IgG as the same dose. All of the experiments were repeated three times (N = 3), and the data were presented as the means ± SD. 2.4. Western blot analysis The glomeruli and cells were lysed in RIPA buffer composed of 50 mM Tris–Cl (pH 7.6), 5 mM EDTA, 150 mM NaCl, 0.5% NP-40, and 0.5% Triton X-100 and contained 1 μg/ml leupeptin, aprotinin, and antipain; 1 mM sodium orthovanadate; and 0.5 mM phenylmethylsulfonyl fluoride. Protein concentrations were determined using the Bradford assay. In total, 50 μg of total protein per sample was separated by 6–15% SDSPAGE and then transferred to membranes, which were blocked with 5% skim milk. The membranes were probed with primary antibody overnight at 4 °C and then incubated with horseradish peroxidaseconjugated secondary antibody. 2.5. Terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end-labeling (TUNEL) analysis Four-micron thick kidney tissue sections were prepared from paraffin-embedded tissue. TUNEL staining was performed using an In Situ Cell Death Detection Kit, POD (Roche, Switzerland).
2.7. Real-time RT-PCR Total RNA was isolated using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). A TaqMan Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) and a Gene Amp® PCR System 9700 (Applied Biosystems) were used to generate cDNA. The gene expression levels were analyzed by quantitative real-time PCR using SYBR Green Master Mix and a 7500 Real-time PCR System (Applied Biosystems). The results were analyzed using the 2−ΔΔCT method with normalization against the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression level (N = 5 for each group). The following primers were used: CXCL10: (Fw) 5′-CTGAGTGGGACTCAAGGGAT-3′ and (Rev) 5′-AGGCTCGCAGGGATGA TTTC-3′; Mxi1: (Fw) 5′-GCAACACCAGCACTGCCAAC-3′and (Rev) 5′AGGAGACTGCATCATGAACC-3′; and GADPH: (Fw) 5′-TGCACCACCAAC TGCTTAGC-3′ and (Rev) 5′-GGCATGGACTGTGGTCATGAG-3′. 2.8. Histological analysis and immunohistochemical (IHC) staining Kidney samples were fixed in 10% formalin and embedded in paraffin, and then 4-μm thick sections were prepared. The sections were deparaffinized and stained with PAS. The total number of nucleated cells in each glomerulus was counted. This count included endothelial cells, mesangial cells, visceral epithelial cells, and any infiltrating cells. For each mouse kidney section, at least 20 glomeruli were used for the cell counting. The total number of cells counted was divided by the total number of glomeruli counted to determine the average number of cells/glomerulus [18]. For the IHC studies, renal sections were incubated with antibody against CXCL10 (1:50), and standard staining techniques were used. 2.9. Statistical analysis All data analyses were performed using SPSS 11.0 software (SPSS Inc., Chicago, IL, USA). The data were expressed as the mean ± SD. Comparisons among the groups were conducted using ANOVA. P b 0.05 was considered significant. 3. Results 3.1. Mxi1−/− mice exhibit a severe pathological phenotype and an increased proportion of apoptotic cells Using histopathological examination techniques, we found that Mxi1−/− mice exhibited a more typical and severe dissolution phenotype on modeling day 1 and fewer cells per glomerulus (32.60 ± 5.29) compared with the Mxi1−/− control mice (43.87 ± 4.05), *P b 0.05, this difference was statistically significant. However, the numbers of glomerular nucleated cells were 37.50 ± 3.56 and 40.17 ± 5.51 per glomerulus on modeling day 1 and day 0 (control), respectively, in Mxi1+/+ mice (Fig. 1A and B). Interestingly, although no difference in the number of cells per glomerulus was observed between the Mxi1+/+ and Mxi1−/− mice on modeling day 0, the
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Fig. 1. Mxi1−/− mice demonstrated a more typical, severe dissolution phenotype on day 1 after HSV injection. Representative images of (A) PAS staining. (B) Glomerular nucleated cells in all types of mice were counted. At least 20 glomeruli were counted for each mouse kidney section. The apoptosis levels were determined using western blot analysis (C and D) and TUNEL staining (E). The data were presented as the means ± SD. *P b 0.05 versus the Mxi1−/− mouse control group.
number of glomerular nucleated cells decreased more noticeably in the Mxi1−/− mice compared with that in the Mxi1+/+ mice on modeling day 1. We hypothesized that the survival status of the MMCs may not change in the physiological environment after Mxi1 knockout but that the mesangial cells in Mxi1−/− mice may be more easily injured under pathological conditions. Human MesPGN is a disease characterized by glomerular mesangial cell (GMC) apoptosis and proliferation. Liu et al. [19] found that interferon regulatory factor-1 (IRF-1) mediates mesangial cell apoptosis in an anti-Thy-1 nephritic rat model. Caspases are crucial mediators of programmed cell death (apoptosis). Caspase 3 is a frequently activated death protease that catalyzes the specific cleavage of many key cellular proteins [20]. Therefore, cleaved caspase 3 (the activated form of caspase 3) is a good indicator of apoptosis. In this study, western blot analysis and TUNEL staining were used to detect apoptotic-related indicators in both wild-type and knockout mice. A significant change in the activated caspase 3 (acti-caspase 3) expression level was found in Mxi1−/− mice on modeling day 1, which persisted until day 3 (*P b 0.05), while only a slightly elevated activated caspase 3 expression level was observed in Mxi1+/+ mice on modeling day 1 (Fig. 1C and D). The TUNEL staining results demonstrated similar trends in the expression level changes (Fig. 1E). Together, these results suggest that the Mxi1−/− mice exhibit a more severe pathological phenotype. 3.2. Mxi1−/− mice exhibit significantly high CXCL10 expression In a previous study regarding the gene expression profiles in an antiThy-1 nephritic model, the results revealed significantly increased
expression levels of inflammation-related factors, including the chemokines CCL2, CXCL10 and CXCL13 and the cell adhesion molecules ICAM1 and VCAM1, suggesting that the upregulation of inflammationrelated factors is associated with anti-Thy-1 nephritic pathological processes [16,17]. Because the role of CCL2 has been well documented in human and rat MesPGN, we selected CXCL10 to explore the roles of chemokines in the MesPGN pathological process in mice. CXCL10 was initially detected in both Habu nephritis mouse models. CXCL10 expression increased at both the mRNA (Fig. 2A) and protein (Fig. 2B and C) levels in the Mxi1−/− kidney, and the most noticeable change was observed on modeling day 1, while CXCL10 expression decreased slightly in the Mxi1+/+ mice. In biopsies from the two types of mice, positive staining for CXCL10 was only observed in the Mxi1−/− model mice, while the control Mxi1−/− mice and all of the Mxi1+/+ mice showed negative staining (Fig. 2D). The differing trends in CXCL10 expression in the two Habu nephritis models suggest that aggravation of the MesPGN pathological phenotype may be induced by CXCL10 regulation upon Mxi1 inactivation. TLR4 is a pattern recognition receptor that recognizes not only bacterial lipopolysaccharide but also the endogenous danger-associated molecular patterns released from dying or injured cells [21]. TLR4 can induce CXCL10 expression through the MyD88-independent signaling mediated by IRF3, which is downstream of TLR4 activation [22,23]. Therefore, we investigated whether the TLR4–IRF3 pathway is activated in our mouse model. As expected, TLR4 expression increased in the Mxi1−/− mice, and the most noticeable change was observed on modeling day 1, on which phosphorylated (activated) IRF3 demonstrated a
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Fig. 2. Increased CXCL10 expression and activation of the TLR4–IRF3 signaling pathway in the Mxi1−/− mouse MesPGN model. RT-PCR (A) and western blot analyses (B) were used to analyze CXCL10 expression, and the signal intensity was quantified (C). The data were presented as the means ± SD. *P b 0.05 versus the control group. The CXCL10 spatial expression pattern in the Habu Mxi1−/− mouse glomeruli was determined by (D) IHC staining. (E and F) Western blot analysis showed that the TLR4 and phosphorylated IFR3 expression levels increased in model mice on days 1 and 3 after HSV injection compared with the control Mxi1−/− mice. Thus, the TLR4–IRF3 signaling pathway was activated. The data were presented as the means ± SD. *P b 0.05 versus the Mxi1+/+ mice control group, # P b 0.05 versus the Mxi1−/− mice control group.
change that was similar to that in TLR4 (Fig. 2E–F). These results suggest that the TLR4–IRF3 signaling pathway may be involved in regulating CXCL10 expression and the mesangial dissolution process. 3.3. Inactivation of Mxi1 induces apoptosis and elevates CXCL10 expression Because the Mxi1−/− mice demonstrated higher levels of apoptosis during the early stages of the Habu nephritis model, we downregulated Mxi1 expression in MMCs using RNA interference (RNAi) technology with three Mxi1 small interfering RNAs (siRNAs). Based on the Hoechst 33342 and TUNEL staining results, the numbers of apoptotic cells in the Mxi1 RNAi group (si-Mxi1-1, si-Mxi1-2, and si-Mxi1-3; the siRNA transfection efficiencies are shown in Supplemental Fig. 2) were significantly higher (Fig. 3A–C) than those in the control and unrelated RNA transfection groups (negative control, NC). We also detected an elevated
activated caspase 3 level in the Mxi1 RNAi group by western blot analysis (Fig. 3D–E). Finally, to investigate whether Mxi1 inactivation induces CXCL10 expression, we used RT-PCR to detect CXCL10 expression. As expected, Mxi1 knockdown increased CXCL10 expression (Fig. 3F). 3.4. CXCL10 induces Mxi1-inactivated MMC apoptosis upon caspase 3 activation CXCL10, which is also called γ-interferon-inducible protein 10 (IP-10), belongs to the CXC chemokine family, which plays important roles in the occurrence and development of several human diseases [24–26]. Recently, Schulthess et al. reported that CXCL10 could induce beta cell apoptosis and abolish beta cell function [27]. The Mxi1 RNAi group was treated with either a CXCL10 antibody or IgG to determine
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Fig. 3. Effect of Mxi1 inactivation in vitro. Representative pictures of (A) Hoechst 33342- and TUNEL-stained MMCs treated with unrelated RNA (negative control, NC) and three Mxi1 siRNAs: si-Mxi1-1, si-Mxi1-2, and si-Mxi1-3. The apoptosis rate was quantified. The data were presented as the means ± SD. *P b 0.05 versus the control group (B and C). An elevated activated caspase 3 level was detected by (D and E) western blot analysis in the three Mxi1 RNAi groups. The RT-PCR (F) results suggest that the inactivation of Mxi1 induced CXCL10 expression. All of the experiments were repeated three times (N = 3); the data were presented as the means ± SD. *P b 0.05 versus the control group.
whether CXCL10 is responsible for the apoptosis observed after Mxi1 inactivation. Compared with the IgG group (Si-Mxi1 + IgG), the antibody-treated group (Si-Mxi1 + Anti-CXCL10) had a lower proportion of apoptotic cells (Fig. 4A–C) and a decreased activated caspase 3 level (Fig. 4D–E), suggesting that apoptosis decreased upon CXCL10 inhibition. Thus, we concluded that Mxi1-inactivated apoptosis is associated with CXCL10 expression and that CXCL10 induces apoptosis by activating caspase 3.
3.5. TLR4–IRF3 signaling induces CXCL10 expression in Mxi1-inactivated MMCs The TLR4–IRF3 signaling pathway, which is activated in vivo, is also thought to be involved in CXCL10 expression and in mesangial dissolution. The IRF3 and Mxi1 expression levels were simultaneously downregulated in MMCs using RNAi to verify this proposal. The group transfected with the two siRNAs (Si-Mxi1 + Si-IRF3) demonstrated
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Fig. 4. Effect of the CXCL10 antibody on Mxi1-inactivated MMCs. Representative pictures of (A) Hoechst 33342- and TUNEL-stained control cells, NC cells, NC cells treated with IgG (NC + IgG), Si-Mxi1-treated cells (Si-Mxi1), antibody-treated cells (Si-Mxi1 + Anti-CXCL10), and Si-Mxi1 plus IgG treated cells (Si-Mxi1 + IgG). The apoptosis rate was quantified. The data were presented at the means ± SD. *P b 0.05 versus the control group, #P b 0.05 versus the Si-Mxi1 or Si-Mxi1 + IgG group (B and C). The activated caspase 3 level was detected by (D and E) western blot analysis. All experiments were repeated three times (N = 3); the data were presented as the means ± SD. *P b 0.05 versus the control group, #P b 0.05 versus the Si-Mxi1 or Si-Mxi1 + IgG group.
a lower proportion of apoptotic cells (Fig. 5A–C) and a decreased activated caspase 3 level (Fig. 5D–E) compared with the Mxi1 single knockdown group (Si-Mxi1). In contrast, the IRF3 single knockdown group (Si-IRF3, the siRNA transfection efficiency is shown in Supplemental Fig. 3) demonstrated an apoptotic cell number that was similar to that of the control group. Moreover, the CXCL10 expression level was lower in the Si-Mxi1 + Si-IRF3 group (Fig. 5F). All of these results suggest that interfering with the TLR4–IRF3 signaling pathway reduces apoptosis and CXCL10 expression induced by Mxi1 inactivation; specifically, the TLR4–IRF3 signaling pathway influences CXCL10 expression and is involved in Mxi1-inactivated MMC apoptosis. 4. Discussion MesPGN is an important cause of end-stage renal disease. Currently, the pathogenesis of MesPGN remains unclear but may be related
primarily to genetic immune abnormalities and to other factors. Our previous study found that Mxi1 plays an important role in a rat MesPGN model [10]. In the present study, Mxi1−/− mice were used to generate a murine MesPGN model. We found that the inactivation of Mxi1 aggravated the pathological phenotype, suggesting that Mxi1 is highly important in mice. Glomerulonephritis is an autoimmune disease that is primarily associated with the immune-mediated inflammatory response. MesPGN is the most common type of glomerulonephritis, and inflammationrelated cytokines play an important role in its pathology [14]. Traditional anti-Thy-1 nephritis is a classic, reversible model of MesPGN. Certain studies have shown that 2–3 days after antibody injection, rat kidneys demonstrate acute mesangial injury that primarily results in mesangial dissolution and mesangial cell apoptosis [16,17]. The MesPGN model induced by HSV injection (Habu nephritis) is a self-limiting nephritic model that results in acute focal damage and mesangial cell
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Fig. 5. The TLR4–IRF3 signaling pathway plays a role in CXCL10 expression in Mxi1-inactivated MMCs. Representative pictures of (A) Hoechst 33342- and TUNEL-stained control cells, NC cells, Si-IRF3-treated cells (Si-IRF3), Si-Mxi1-treated cells (Si-Mxi1), and Si-Mxi1- plus Si-IRF3-treated cells (Si-Mxi1 + Si-IRF3). The apoptosis rate was quantified. The data were presented as the means ± SD. *P b 0.05 versus the control group, #P b 0.05 versus the Si-Mxi1 group (B and C). Activated caspase 3 levels were detected by (D and E) western blot analysis. The RT-PCR (F) results suggest that inhibiting IRF3 expression decreases Mxi1-inactivation-induced CXCL10 expression. All of the experiments were repeated three times (N = 3); the data were presented as the means ± SD. *P b 0.05 versus the control group, #P b 0.05 versus the Si-Mxi1 group.
proliferation in rats, rabbits and mice [28–30]. Therefore, HSV was used to establish a mouse model of MesPGN. Similar to the rat MesPGN model, the MesPGN mice also demonstrated a dissolution phenotype during the early stages of modeling, and the Mxi1−/− mice exhibited a more severe pathological phenotype. Considering that inflammationrelated cytokines play an important role in the pathological process of MesPGN, we investigated the CCL2 expression levels (data not shown), which have been frequently reported, and CXCL10 expression levels, which were found to be elevated during the early stage of anti-Thy 1 nephritis. We found a significant increase in the CXCL10 level in the Mxi1−/− MesPGN mice but not in the Mxi1+/+ mice. CXCL10, which is also called IP-10, belongs to the CXC chemokine family. CXCL10 is thought to participate in chemotaxis, to induce apoptosis, to regulate cell growth, and to inhibit angiogenesis. CXCL10 is also associated with several human diseases, including infectious diseases and chronic inflammation [31–33]. Elevated levels of apoptosis were found in Mxi1−/− mice on modeling day 1 and continued until modeling day 3, while only slightly elevated levels were observed on modeling day 1 in the Mxi1+/+ mice. The higher level of apoptosis in the Mxi1−/− mice may be because CXCL10 can induce apoptosis. siRNA-mediated knockdown of Mxi1 was performed in MMCs to
investigate this hypothesis. As expected, Mxi1 knockdown increased CXCL10 expression, the proportion of apoptotic cells, and the active caspase 3 level. After treating the cells with CXCL10 antibody, the proportion of apoptotic cells and the activated caspase 3 level decreased, suggesting that the apoptosis resulting from Mxi1 inactivation in MMCs is indeed related to CXCL10 expression. TLRs play a central role in both the innate and adaptive immune responses to microbial ligands. Some previous evidence has suggested that these receptors are stimulated by endogenous ligands. In addition to leucocytes, TLRs are also expressed in parenchymal cells. Therefore, renal disease could be affected by the stimulation of TLRs in leucocytes or renal cells. Zhai et al. reported that TLR4–IRF3 activation triggers CXCL10 expression in ischemia reperfusion injury (IRI) mouse liver [22], and Furuichi et al. found that CXCL10 was expressed in the kidney after IRI [34]. Thus, we investigated the expression of TLR4 and phosphorylated (activated) IRF3 in both Mxi1−/− and Mxi1+/+ mice. TLR4 expression increased in both the Mxi1−/− and Mxi1+/+ mice on modeling day 1, while an elevated expression level of phosphorylated IRF3 was only detected in the Mxi1−/− mice (data not shown for Mxi1+/+ mice). These findings suggest that the TLR4–IRF3 pathway may be involved in regulating CXCL10 expression in Mxi1−/− mice. To
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References
Fig. 6. The graphic overview of cell injury induced by mxi 1.
determine whether this pathway is involved in regulating CXCL10 expression, IRF3 expression was knocked down using an RNAi technique, which successfully decreased CXCL10 expression. Because TLR4 is involved in pathological processes via different pathways, such as MyD88, this receptor may be involved in the pathological process in the Mxi1+/+ mouse MesPGN model via a pathway other than the TLR4–IRF3 pathway. 5. Conclusions In this study, Mxi1−/− mice were used to generate a MesPGN model for the first time. Through comparisons with Mxi1+/+ mice, we found that CXCL10 expression positively correlates with Mxi1−/− MesPGN mesangial cell apoptosis and that the TLR4–IRF3 pathway is involved in regulating CXCL10 expression. Furthermore, we verified that the CXCL10 expression induced by Mxi1 inactivation causes MMC apoptosis by activating caspase 3 in vitro and that this finding reveals potential intervention targets for the treatment of mesangial cell injury disease (Fig. 6). Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.cellsig.2015.01.019. Disclosure of potential conflicts of interest All the authors declared no competing interests. Acknowledgments We thank professor HW Lee for the generous gifts of Mxi1 −/− mice. This work was supported by grants from Programs of the National Natural Science Foundation of China (81330019, 81270794, 81470949), 973 Program (2011CBA01003) and 863 program (2012AA02A512).
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Cellular Signalling journal homepage: www.elsevier.com/locate/cellsig
CXCL10 expression induced by Mxi1 inactivation induces mesangial cell apoptosis in mouse Habu nephritis Lingling Wu a,b, Xiaoniao Chen a,b, Yan Mei a, Quan Hong a, Zhe Feng a, Yang Lv a, Jun Wen a, Xiaoluan Liu a, Guangyan Cai a,⁎, Xiangmei Chen a,⁎ a b
Department of Nephrology, Chinese PLA General Hospital, Chinese PLA Institute of Nephrology, State Key Laboratory of Kidney Diseases, National Clinical Research Center for Kidney Diseases, China Medical College, NanKai University, Tianjin, China
a r t i c l e
i n f o
Article history: Received 12 November 2014 Received in revised form 13 January 2015 Accepted 27 January 2015 Available online 12 February 2015 Keywords: Mxi1 CXCL10 Apoptosis Mesangial cells Caspase 3
a b s t r a c t MAX interactor 1 (Mxi1) proteins are c-myc antagonists that primarily exert their biological functions by inhibiting Myc-dependent gene transcription. In this study, Mxi1−/− mice were used to generate a model of mesangial proliferative glomerulonephritis for the first time. In the present study, we demonstrated that Mxi1−/− mice exhibited a more typical and severe pathological phenotype, which was displayed primarily as a noticeable dissolution phenotype with a higher proportion of apoptotic cells and higher chemokine CXCL10 expression during the early days of modeling, compared with wild-type mice. Additionally, we determined that IRF3-mediated TLR4 signaling was likely involved in regulating CXCL10 expression, which might participate in the mesangial dissolution process. We also found increases in CXCL10 expression, caspase 3 activation, and the proportion of apoptotic cells when Mxi1 expression was inhibited in mouse mesangial cells. Furthermore, the proportion of apoptotic cells decreased after inhibiting CXCL10 expression. Therefore, we concluded that the mesangial cell apoptosis observed in this mesangial proliferative glomerulonephritis model was related to CXCL10 expression induced by Mxi1 inactivation. This finding provides a new theoretical basis for the mechanism of mesangial proliferative glomerulonephritis progression and reveals potential intervention targets for the early treatment of this disease. © 2015 Elsevier Inc. All rights reserved.
1. Introduction MAX interactor 1 (Mxi1) is a member of the MAD family of transcriptional repressors that play roles in cell proliferation and differentiation [1–3]. Mxi1 primarily exerts its biological functions by inhibiting Myc-dependent gene transcription [4–6]. Myc is involved in many biological processes, including cell proliferation, growth, metabolism, differentiation, and malignancy. Mxi1 is abnormally expressed in various human tumors and is thought to play a positive regulatory role in cell proliferation [7–9]. Mxi1 expression decreases significantly during the proliferative period in an anti-Thy1 nephritic rat model and gradually increases as proliferation declines, thereby suggesting that Mxi1 plays a role in mesangial proliferation [10]. However, the specific mechanisms underlying the effects of Mxi1 on mesangial proliferative glomerulonephritis (MesPGN) require further investigation. MesPGN, which includes IgA nephropathy (IgAN) and non-IgA MesPGN, is a common chronic kidney disease [11,12]. Many factors
⁎ Corresponding authors at: Department of Nephrology, Chinese PLA Institute of Nephrology & Key Lab, Chinese PLA Hospital, 28 Fuxing Road, Beijing 100853, China. Tel.: +86 10 68211187; fax: +86 10 68130297. E-mail addresses: [email protected] (G. Cai), [email protected] (X. Chen).
http://dx.doi.org/10.1016/j.cellsig.2015.01.019 0898-6568/© 2015 Elsevier Inc. All rights reserved.
are involved in the occurrence and development of MesPGN. Although the specific details of the causes and pathogenesis of MesPGN remain unclear, MesPGN is known to be an autoimmune inflammatory disease that involves various pathways; therefore, the causes of this disease are likely related to the immune-mediated inflammatory response [13–15]. Based on the microarray results from anti-Thy-1 nephritis models, the expression levels of chemokines, such as CCL2, CCL5 and CCL6, are significantly higher during the early stage of modeling, suggesting that inflammatory-reaction-related cytokines play an important role in the pathological progression of MesPGN [16,17]. In the present study, Mxi1 knockout mice (Mxi1−/− mice) were used to generate a mouse model of MesPGN. Both Mxi1−/− and wildtype (Mxi1+/+) mice were compared using periodic acid-Schiff (PAS) staining, which revealed that the knockout of Mxi1 aggravated the pathological phenotype. The Mxi1−/− mice also exhibited the typical dissolution phenotype, while the Mxi1+/+ mice exhibited an extremely light and atypical dissolution phenotype. Using real-time PCR and western blot analyses, we detected changes in chemokine expression and found that the CXCL10 level increased significantly in the Mxi1−/− mice during modeling. Furthermore, the specific mechanisms by which CXCL10 affected mesangial cells were investigated in vitro using cultured mouse mesangial cells (MMCs).
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2. Materials and methods
2.6. Apoptosis studies
2.1. Experimental animals and induction of a Habu nephritis mouse model
Cultured MMC apoptosis was assessed using Hoechst 33342 (SigmaAldrich) staining. The cells were stained with Hoechst 33342 at room temperature for 5 min after being fixed in 4% paraformaldehyde for 10 min at room temperature and then washed 3 times with ice-cold PBS. The MMCs were observed by fluorescence microscopy, and all of the microscopic images were captured using a 12.8-megapixel camera (DP72, Olympus, Japan). Cultured MMC apoptosis was also assessed using TUNEL staining. MMCs were cultured to 70% confluency in special glass-bottom microwell dishes, and then TUNEL staining was performed using an In Situ Cell Death Detection Kit, POD (Roche, Switzerland).
Glomerulonephritis (GN) induced by Habu snake venom is a selflimiting nephritis model that primarily causes glomerular mesangial injury. This method successfully generates a mesangial proliferative glomerulonephritis mouse model. In this study, Mxi1−/− mice (kindly provided by Professor HW Lee at Yonsei University; the results of Mxi1−/− mouse identification were shown in Supplemental Fig. 1) and Mxi1+/+ mice were given a single intravenous injection of Habu snake venom (HSV) at a dose of 2.5 mg/kg bodyweight to generate a HSV-induced mesangial proliferative glomerulonephritis model. Control mice were given saline injections. The mice were maintained under the following specific pathogen-free conditions: 22 ± 1 °C, 40% humidity, 12/12 h light/dark cycle, five males per cage, and free access to food and water. The mice were sacrificed at 1, 3 and 7 days after injection for experiments (eight mice per group), and the renal cortex was isolated. 2.2. Reagents HSV was purchased from Wako. Hoechst 33342 was purchased from Sigma-Aldrich. The following primary antibodies were used: rabbit anti-active caspase 3 (Cell Signaling), rabbit anti-CXCL10 (Cell Signaling), mouse anti-CXCL10 (R&D), rabbit anti-p-IRF3 (Cell Signaling), anti-toll-like receptor 4 (TLR4, Cell Signaling), mouse anti-β-actin (Sigma-Aldrich) and rabbit anti-Mxi1 (Abcam). The following secondary antibodies were used: horseradish peroxidase-conjugated IgG and mouse IgG (Beyotime). 2.3. Cell culture and transfection MMCs were purchased from ATCC (No. CRL-1927) and were maintained in RPMI 1640 medium (Sigma-Aldrich) supplemented with 10% fetal calf serum (FCS; Life Technologies, Rockville, MD, USA), 100 U/ml penicillin G, and 100 μg/ml streptomycin in a humidified 5% CO2 atmosphere. RNAiMAX (Invitrogen Life Technology) transfection reagent was used with MMCs for siRNA transfection. For the CXCL10 inhibition experiment, the cells were cultured in the presence of CXCL10 antibody at a concentration of 700 ng/ml, and the control group was given IgG as the same dose. All of the experiments were repeated three times (N = 3), and the data were presented as the means ± SD. 2.4. Western blot analysis The glomeruli and cells were lysed in RIPA buffer composed of 50 mM Tris–Cl (pH 7.6), 5 mM EDTA, 150 mM NaCl, 0.5% NP-40, and 0.5% Triton X-100 and contained 1 μg/ml leupeptin, aprotinin, and antipain; 1 mM sodium orthovanadate; and 0.5 mM phenylmethylsulfonyl fluoride. Protein concentrations were determined using the Bradford assay. In total, 50 μg of total protein per sample was separated by 6–15% SDSPAGE and then transferred to membranes, which were blocked with 5% skim milk. The membranes were probed with primary antibody overnight at 4 °C and then incubated with horseradish peroxidaseconjugated secondary antibody. 2.5. Terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end-labeling (TUNEL) analysis Four-micron thick kidney tissue sections were prepared from paraffin-embedded tissue. TUNEL staining was performed using an In Situ Cell Death Detection Kit, POD (Roche, Switzerland).
2.7. Real-time RT-PCR Total RNA was isolated using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). A TaqMan Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) and a Gene Amp® PCR System 9700 (Applied Biosystems) were used to generate cDNA. The gene expression levels were analyzed by quantitative real-time PCR using SYBR Green Master Mix and a 7500 Real-time PCR System (Applied Biosystems). The results were analyzed using the 2−ΔΔCT method with normalization against the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression level (N = 5 for each group). The following primers were used: CXCL10: (Fw) 5′-CTGAGTGGGACTCAAGGGAT-3′ and (Rev) 5′-AGGCTCGCAGGGATGA TTTC-3′; Mxi1: (Fw) 5′-GCAACACCAGCACTGCCAAC-3′and (Rev) 5′AGGAGACTGCATCATGAACC-3′; and GADPH: (Fw) 5′-TGCACCACCAAC TGCTTAGC-3′ and (Rev) 5′-GGCATGGACTGTGGTCATGAG-3′. 2.8. Histological analysis and immunohistochemical (IHC) staining Kidney samples were fixed in 10% formalin and embedded in paraffin, and then 4-μm thick sections were prepared. The sections were deparaffinized and stained with PAS. The total number of nucleated cells in each glomerulus was counted. This count included endothelial cells, mesangial cells, visceral epithelial cells, and any infiltrating cells. For each mouse kidney section, at least 20 glomeruli were used for the cell counting. The total number of cells counted was divided by the total number of glomeruli counted to determine the average number of cells/glomerulus [18]. For the IHC studies, renal sections were incubated with antibody against CXCL10 (1:50), and standard staining techniques were used. 2.9. Statistical analysis All data analyses were performed using SPSS 11.0 software (SPSS Inc., Chicago, IL, USA). The data were expressed as the mean ± SD. Comparisons among the groups were conducted using ANOVA. P b 0.05 was considered significant. 3. Results 3.1. Mxi1−/− mice exhibit a severe pathological phenotype and an increased proportion of apoptotic cells Using histopathological examination techniques, we found that Mxi1−/− mice exhibited a more typical and severe dissolution phenotype on modeling day 1 and fewer cells per glomerulus (32.60 ± 5.29) compared with the Mxi1−/− control mice (43.87 ± 4.05), *P b 0.05, this difference was statistically significant. However, the numbers of glomerular nucleated cells were 37.50 ± 3.56 and 40.17 ± 5.51 per glomerulus on modeling day 1 and day 0 (control), respectively, in Mxi1+/+ mice (Fig. 1A and B). Interestingly, although no difference in the number of cells per glomerulus was observed between the Mxi1+/+ and Mxi1−/− mice on modeling day 0, the
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Fig. 1. Mxi1−/− mice demonstrated a more typical, severe dissolution phenotype on day 1 after HSV injection. Representative images of (A) PAS staining. (B) Glomerular nucleated cells in all types of mice were counted. At least 20 glomeruli were counted for each mouse kidney section. The apoptosis levels were determined using western blot analysis (C and D) and TUNEL staining (E). The data were presented as the means ± SD. *P b 0.05 versus the Mxi1−/− mouse control group.
number of glomerular nucleated cells decreased more noticeably in the Mxi1−/− mice compared with that in the Mxi1+/+ mice on modeling day 1. We hypothesized that the survival status of the MMCs may not change in the physiological environment after Mxi1 knockout but that the mesangial cells in Mxi1−/− mice may be more easily injured under pathological conditions. Human MesPGN is a disease characterized by glomerular mesangial cell (GMC) apoptosis and proliferation. Liu et al. [19] found that interferon regulatory factor-1 (IRF-1) mediates mesangial cell apoptosis in an anti-Thy-1 nephritic rat model. Caspases are crucial mediators of programmed cell death (apoptosis). Caspase 3 is a frequently activated death protease that catalyzes the specific cleavage of many key cellular proteins [20]. Therefore, cleaved caspase 3 (the activated form of caspase 3) is a good indicator of apoptosis. In this study, western blot analysis and TUNEL staining were used to detect apoptotic-related indicators in both wild-type and knockout mice. A significant change in the activated caspase 3 (acti-caspase 3) expression level was found in Mxi1−/− mice on modeling day 1, which persisted until day 3 (*P b 0.05), while only a slightly elevated activated caspase 3 expression level was observed in Mxi1+/+ mice on modeling day 1 (Fig. 1C and D). The TUNEL staining results demonstrated similar trends in the expression level changes (Fig. 1E). Together, these results suggest that the Mxi1−/− mice exhibit a more severe pathological phenotype. 3.2. Mxi1−/− mice exhibit significantly high CXCL10 expression In a previous study regarding the gene expression profiles in an antiThy-1 nephritic model, the results revealed significantly increased
expression levels of inflammation-related factors, including the chemokines CCL2, CXCL10 and CXCL13 and the cell adhesion molecules ICAM1 and VCAM1, suggesting that the upregulation of inflammationrelated factors is associated with anti-Thy-1 nephritic pathological processes [16,17]. Because the role of CCL2 has been well documented in human and rat MesPGN, we selected CXCL10 to explore the roles of chemokines in the MesPGN pathological process in mice. CXCL10 was initially detected in both Habu nephritis mouse models. CXCL10 expression increased at both the mRNA (Fig. 2A) and protein (Fig. 2B and C) levels in the Mxi1−/− kidney, and the most noticeable change was observed on modeling day 1, while CXCL10 expression decreased slightly in the Mxi1+/+ mice. In biopsies from the two types of mice, positive staining for CXCL10 was only observed in the Mxi1−/− model mice, while the control Mxi1−/− mice and all of the Mxi1+/+ mice showed negative staining (Fig. 2D). The differing trends in CXCL10 expression in the two Habu nephritis models suggest that aggravation of the MesPGN pathological phenotype may be induced by CXCL10 regulation upon Mxi1 inactivation. TLR4 is a pattern recognition receptor that recognizes not only bacterial lipopolysaccharide but also the endogenous danger-associated molecular patterns released from dying or injured cells [21]. TLR4 can induce CXCL10 expression through the MyD88-independent signaling mediated by IRF3, which is downstream of TLR4 activation [22,23]. Therefore, we investigated whether the TLR4–IRF3 pathway is activated in our mouse model. As expected, TLR4 expression increased in the Mxi1−/− mice, and the most noticeable change was observed on modeling day 1, on which phosphorylated (activated) IRF3 demonstrated a
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Fig. 2. Increased CXCL10 expression and activation of the TLR4–IRF3 signaling pathway in the Mxi1−/− mouse MesPGN model. RT-PCR (A) and western blot analyses (B) were used to analyze CXCL10 expression, and the signal intensity was quantified (C). The data were presented as the means ± SD. *P b 0.05 versus the control group. The CXCL10 spatial expression pattern in the Habu Mxi1−/− mouse glomeruli was determined by (D) IHC staining. (E and F) Western blot analysis showed that the TLR4 and phosphorylated IFR3 expression levels increased in model mice on days 1 and 3 after HSV injection compared with the control Mxi1−/− mice. Thus, the TLR4–IRF3 signaling pathway was activated. The data were presented as the means ± SD. *P b 0.05 versus the Mxi1+/+ mice control group, # P b 0.05 versus the Mxi1−/− mice control group.
change that was similar to that in TLR4 (Fig. 2E–F). These results suggest that the TLR4–IRF3 signaling pathway may be involved in regulating CXCL10 expression and the mesangial dissolution process. 3.3. Inactivation of Mxi1 induces apoptosis and elevates CXCL10 expression Because the Mxi1−/− mice demonstrated higher levels of apoptosis during the early stages of the Habu nephritis model, we downregulated Mxi1 expression in MMCs using RNA interference (RNAi) technology with three Mxi1 small interfering RNAs (siRNAs). Based on the Hoechst 33342 and TUNEL staining results, the numbers of apoptotic cells in the Mxi1 RNAi group (si-Mxi1-1, si-Mxi1-2, and si-Mxi1-3; the siRNA transfection efficiencies are shown in Supplemental Fig. 2) were significantly higher (Fig. 3A–C) than those in the control and unrelated RNA transfection groups (negative control, NC). We also detected an elevated
activated caspase 3 level in the Mxi1 RNAi group by western blot analysis (Fig. 3D–E). Finally, to investigate whether Mxi1 inactivation induces CXCL10 expression, we used RT-PCR to detect CXCL10 expression. As expected, Mxi1 knockdown increased CXCL10 expression (Fig. 3F). 3.4. CXCL10 induces Mxi1-inactivated MMC apoptosis upon caspase 3 activation CXCL10, which is also called γ-interferon-inducible protein 10 (IP-10), belongs to the CXC chemokine family, which plays important roles in the occurrence and development of several human diseases [24–26]. Recently, Schulthess et al. reported that CXCL10 could induce beta cell apoptosis and abolish beta cell function [27]. The Mxi1 RNAi group was treated with either a CXCL10 antibody or IgG to determine
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Fig. 3. Effect of Mxi1 inactivation in vitro. Representative pictures of (A) Hoechst 33342- and TUNEL-stained MMCs treated with unrelated RNA (negative control, NC) and three Mxi1 siRNAs: si-Mxi1-1, si-Mxi1-2, and si-Mxi1-3. The apoptosis rate was quantified. The data were presented as the means ± SD. *P b 0.05 versus the control group (B and C). An elevated activated caspase 3 level was detected by (D and E) western blot analysis in the three Mxi1 RNAi groups. The RT-PCR (F) results suggest that the inactivation of Mxi1 induced CXCL10 expression. All of the experiments were repeated three times (N = 3); the data were presented as the means ± SD. *P b 0.05 versus the control group.
whether CXCL10 is responsible for the apoptosis observed after Mxi1 inactivation. Compared with the IgG group (Si-Mxi1 + IgG), the antibody-treated group (Si-Mxi1 + Anti-CXCL10) had a lower proportion of apoptotic cells (Fig. 4A–C) and a decreased activated caspase 3 level (Fig. 4D–E), suggesting that apoptosis decreased upon CXCL10 inhibition. Thus, we concluded that Mxi1-inactivated apoptosis is associated with CXCL10 expression and that CXCL10 induces apoptosis by activating caspase 3.
3.5. TLR4–IRF3 signaling induces CXCL10 expression in Mxi1-inactivated MMCs The TLR4–IRF3 signaling pathway, which is activated in vivo, is also thought to be involved in CXCL10 expression and in mesangial dissolution. The IRF3 and Mxi1 expression levels were simultaneously downregulated in MMCs using RNAi to verify this proposal. The group transfected with the two siRNAs (Si-Mxi1 + Si-IRF3) demonstrated
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Fig. 4. Effect of the CXCL10 antibody on Mxi1-inactivated MMCs. Representative pictures of (A) Hoechst 33342- and TUNEL-stained control cells, NC cells, NC cells treated with IgG (NC + IgG), Si-Mxi1-treated cells (Si-Mxi1), antibody-treated cells (Si-Mxi1 + Anti-CXCL10), and Si-Mxi1 plus IgG treated cells (Si-Mxi1 + IgG). The apoptosis rate was quantified. The data were presented at the means ± SD. *P b 0.05 versus the control group, #P b 0.05 versus the Si-Mxi1 or Si-Mxi1 + IgG group (B and C). The activated caspase 3 level was detected by (D and E) western blot analysis. All experiments were repeated three times (N = 3); the data were presented as the means ± SD. *P b 0.05 versus the control group, #P b 0.05 versus the Si-Mxi1 or Si-Mxi1 + IgG group.
a lower proportion of apoptotic cells (Fig. 5A–C) and a decreased activated caspase 3 level (Fig. 5D–E) compared with the Mxi1 single knockdown group (Si-Mxi1). In contrast, the IRF3 single knockdown group (Si-IRF3, the siRNA transfection efficiency is shown in Supplemental Fig. 3) demonstrated an apoptotic cell number that was similar to that of the control group. Moreover, the CXCL10 expression level was lower in the Si-Mxi1 + Si-IRF3 group (Fig. 5F). All of these results suggest that interfering with the TLR4–IRF3 signaling pathway reduces apoptosis and CXCL10 expression induced by Mxi1 inactivation; specifically, the TLR4–IRF3 signaling pathway influences CXCL10 expression and is involved in Mxi1-inactivated MMC apoptosis. 4. Discussion MesPGN is an important cause of end-stage renal disease. Currently, the pathogenesis of MesPGN remains unclear but may be related
primarily to genetic immune abnormalities and to other factors. Our previous study found that Mxi1 plays an important role in a rat MesPGN model [10]. In the present study, Mxi1−/− mice were used to generate a murine MesPGN model. We found that the inactivation of Mxi1 aggravated the pathological phenotype, suggesting that Mxi1 is highly important in mice. Glomerulonephritis is an autoimmune disease that is primarily associated with the immune-mediated inflammatory response. MesPGN is the most common type of glomerulonephritis, and inflammationrelated cytokines play an important role in its pathology [14]. Traditional anti-Thy-1 nephritis is a classic, reversible model of MesPGN. Certain studies have shown that 2–3 days after antibody injection, rat kidneys demonstrate acute mesangial injury that primarily results in mesangial dissolution and mesangial cell apoptosis [16,17]. The MesPGN model induced by HSV injection (Habu nephritis) is a self-limiting nephritic model that results in acute focal damage and mesangial cell
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Fig. 5. The TLR4–IRF3 signaling pathway plays a role in CXCL10 expression in Mxi1-inactivated MMCs. Representative pictures of (A) Hoechst 33342- and TUNEL-stained control cells, NC cells, Si-IRF3-treated cells (Si-IRF3), Si-Mxi1-treated cells (Si-Mxi1), and Si-Mxi1- plus Si-IRF3-treated cells (Si-Mxi1 + Si-IRF3). The apoptosis rate was quantified. The data were presented as the means ± SD. *P b 0.05 versus the control group, #P b 0.05 versus the Si-Mxi1 group (B and C). Activated caspase 3 levels were detected by (D and E) western blot analysis. The RT-PCR (F) results suggest that inhibiting IRF3 expression decreases Mxi1-inactivation-induced CXCL10 expression. All of the experiments were repeated three times (N = 3); the data were presented as the means ± SD. *P b 0.05 versus the control group, #P b 0.05 versus the Si-Mxi1 group.
proliferation in rats, rabbits and mice [28–30]. Therefore, HSV was used to establish a mouse model of MesPGN. Similar to the rat MesPGN model, the MesPGN mice also demonstrated a dissolution phenotype during the early stages of modeling, and the Mxi1−/− mice exhibited a more severe pathological phenotype. Considering that inflammationrelated cytokines play an important role in the pathological process of MesPGN, we investigated the CCL2 expression levels (data not shown), which have been frequently reported, and CXCL10 expression levels, which were found to be elevated during the early stage of anti-Thy 1 nephritis. We found a significant increase in the CXCL10 level in the Mxi1−/− MesPGN mice but not in the Mxi1+/+ mice. CXCL10, which is also called IP-10, belongs to the CXC chemokine family. CXCL10 is thought to participate in chemotaxis, to induce apoptosis, to regulate cell growth, and to inhibit angiogenesis. CXCL10 is also associated with several human diseases, including infectious diseases and chronic inflammation [31–33]. Elevated levels of apoptosis were found in Mxi1−/− mice on modeling day 1 and continued until modeling day 3, while only slightly elevated levels were observed on modeling day 1 in the Mxi1+/+ mice. The higher level of apoptosis in the Mxi1−/− mice may be because CXCL10 can induce apoptosis. siRNA-mediated knockdown of Mxi1 was performed in MMCs to
investigate this hypothesis. As expected, Mxi1 knockdown increased CXCL10 expression, the proportion of apoptotic cells, and the active caspase 3 level. After treating the cells with CXCL10 antibody, the proportion of apoptotic cells and the activated caspase 3 level decreased, suggesting that the apoptosis resulting from Mxi1 inactivation in MMCs is indeed related to CXCL10 expression. TLRs play a central role in both the innate and adaptive immune responses to microbial ligands. Some previous evidence has suggested that these receptors are stimulated by endogenous ligands. In addition to leucocytes, TLRs are also expressed in parenchymal cells. Therefore, renal disease could be affected by the stimulation of TLRs in leucocytes or renal cells. Zhai et al. reported that TLR4–IRF3 activation triggers CXCL10 expression in ischemia reperfusion injury (IRI) mouse liver [22], and Furuichi et al. found that CXCL10 was expressed in the kidney after IRI [34]. Thus, we investigated the expression of TLR4 and phosphorylated (activated) IRF3 in both Mxi1−/− and Mxi1+/+ mice. TLR4 expression increased in both the Mxi1−/− and Mxi1+/+ mice on modeling day 1, while an elevated expression level of phosphorylated IRF3 was only detected in the Mxi1−/− mice (data not shown for Mxi1+/+ mice). These findings suggest that the TLR4–IRF3 pathway may be involved in regulating CXCL10 expression in Mxi1−/− mice. To
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References
Fig. 6. The graphic overview of cell injury induced by mxi 1.
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