Expression and activation of STAT3 in chronic proliferative immune complex glomerulonephritis and the effect of fosinopril
Wuxing Zhang,
Xiangmei Chen,
Suozhu Shi,
Ribao Wei,
Jianzhong Wang,
Nobuaki Yamanaka and
Quan Hong
Department of Nephrology, Kidney Center and Key Laboratory of PLA, General Hospital of PLA, Beijing 100853, People's Republic of China
Correspondence and offprint requests to: Prof. Xiangmei Chen, Department of Nephrology, General Hospital of PLA, Beijing 28 Fuxing Road, 100853, P.R. China. Email: xmchen{at}public.bta.net.cn
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Abstract
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Background. Signal transducers and activators of transcription (STATs) are cytoplasmic proteins that are activated in response to stimulation from various cytokines. Among these, STAT3 is an important member that has been implicated in the inflammatory proliferation of cells. We hypothesized that STAT3 may be activated in kidneys of rats having modified chronic immune complex glomerulonephritis, and that angiotensin-converting enzyme (ACE) inhibition with fosinopril may prevent the activation of STAT3 and subsequent upregulation of tissue inhibitor of metalloproteinase-1 (TIMP-1), which are effects that may explain the therapeutic effects of fosinopril on nephritis.
Methods. Fifty-one Wistar rats were randomly divided into three groups that included a control group, a model group and a fosinopril group. Bovine serum albumin (BSA) nephritis was induced by subcutaneous immunization and daily intraperitoneal (i.p.) administration of BSA. To accentuate the nephritis, we performed uni-nephrectomy and gave 100 µg of lipopolysaccharide (LPS) as an i.p. injection. Macrophage infiltration (ED-1) was assessed with immunohistochemistry. The expression and activation of STAT3 and the expression of TIMP-1, one of the STAT3 downstream genes, were observed in renal tissues of rats by means of immunohistochemistry, electrophoretic mobility shift assay (EMSA), western blot and northern blot. The relationships between STAT3 phosphorylation, 24 h urinary protein excretion and TIMP-1 expression were also analysed.
Results. Northern blot showed that the mRNA expression of both STAT3 and TIMP-1 was significantly increased in kidneys from the model group, but significantly decreased in the fosinopril group (P<0.05). Western blot analysis revealed similar increases in the expression of STAT3, phospho-STAT3 (p-STAT3) and TIMP-1 in the model group. Analysis of immunohistochemistry showed that STAT3 and p-STAT3 were expressed in very few cells of normal rats, that expression was strong in model rats and that this increased expression was attenuated in the fosinopril group (P<0.05). The expression of p-STAT3 in glomeruli was positively correlated with 24 h proteinuria as well as with glomerular TIMP-1 expression. Double staining showed that some ED-1-positive cells also contained p-STAT3-positive staining.
Conclusions. The present study showed that STAT3 is expressed and activated in kidneys of rats with modified immune complex glomerulonephritis. These rats also had increased ED-1-positive cells, with some cells showing simultaneous expression of p-STAT3 and ED-1, which may contribute to glomerular inflammatory proliferation and extracellular matrix accumulation. Finally, fosinopril downregulated STAT3 activation and ED-1 influx, which are effects that may attenuate renal damage in this model.
Keywords: ACE inhibitors; immune complex glomerulonephritis; signal transducers and activators of transcription
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Introduction
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Cytokine-driven glomerular mesangial cell proliferation and overproduction of extracellular matrix (ECM) play important roles in the response of the kidney to injury and in the development of glomerulosclerosis [1]. Cytokines exert their effects through specific receptors. Various signal transduction pathways are activated through distinct regions of each receptor's cytoplasmic domains. Among these signalling molecules, signal transducers and activators of transcription (STATs) are proteins that play a central role in transmitting cytokine signals [2]. The inactive cytoplasmic STATs are activated through tyrosine phosphorylation and dimerize before translocating into the nucleus to activate the target gene's transcription [2].
STAT3, a member of the STAT family, is activated by diverse stimuli and is involved in many functions, including cell growth regulation, inflammation and early embryonic development [3]. STAT3 is activated by many of the cytokines that use signalling receptor subunits similar to gp130. However, many factors other than cytokines lead to STAT3 activation, such as angiotensin II (Ang II) [4,5], thrombin [6] and growth factors that are coupled with tyrosine kinase receptors. Many STAT3 target genes have been identified, including those encoding the proliferation-associated proteins cyclin D1 and Myc, the antiapoptotic proteins Bcl-xl, Mcl-l and Bcl-2, the proangiogenic factor vascular endothelial growth factor (VEGF) and tissue inhibitor of metalloproteinase-1 (TIMP-1), which is important in ECM accumulation [6,7]. Activation of STAT3 occurs in many solid and haematological tumours [7], inflammatory arthritis [8], neuronal hypoxia [9], liver regeneration [10], intestinal inflammation [11], acute sepsis and in other areas. Inhibition of constitutively active STAT3 signalling pathways has been repeatedly shown to inhibit tumour cell growth in vitro and in vivo [12], and to attenuate experimental arthritis [8], which may provide a novel means for therapeutic intervention for human cancer and arthritis.
Because STAT3 plays important roles in cell growth, differentiation and survival, several studies have been performed using a number of renal cell types to show that STAT3 can be activated by various stimuli [46]. STAT3 activation in glomeruli has been reported in the Thy1.1 model [13] and the nephrotoxic serum model [14]. However, there is little information about the upregulation of STAT3 signalling in immune complex glomerulonephritis. In the present study, we used a modified immune complex glomerulonephritis rat model to examine the expression and activation of STAT3 in renal tissues.
Ang II is considered to be a growth factor involved in both cell proliferation and ECM accumulation, which are two features observed in renal injury [15]. Emerging data have indicated that Ang II is a proinflammatory mediator that participates in inflammatory responses in several pathological processes. In renal injury models and even in immune complex nephritis, blockade of Ang II actions by angiotensin-converting enzyme (ACE) inhibitors may decrease proteinuria, inflammatory cell infiltration and gene expression of matrix proteins [15]. Moreover, Ang II also activates the JAK/STAT pathway in vascular smooth muscle cells (VSMCs) to then trigger VSMC proliferation, indicating that STAT3 is also a signal protein for Ang II [5,16]. For the current study, we hypothesized that STAT3 may be activated in the kidneys from rats having modified chronic immune complex glomerulonephritis, and that ACE inhibition with fosinopril may prevent STAT3 activation, inflammatory infiltration and subsequent TIMP-1 upregulation, which are actions that may explain the therapeutic effects of these drugs on nephritis.
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Materials and methods
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Materials
Bovine serum albumin (BSA) (product no. A8022), incomplete Freund's adjuvant (IFA) (product no. F5506) and lipopolysaccharide (LPS) (product no. L2880) were purchased from Sigma-Aldrich Inc. (St. Louis, MO). The ACE inhibitor fosinopril (lot no. 020108) was purchased from Sino-American Shanghai Squibb Pharmaceuticals, Ltd. Rabbit polyclonal anti-STAT3 antibody (catalogue no. sc-482), mouse monoclonal anti-p-STAT3 antibody (catalogue no. sc-8059) and rabbit polyclonal anti-TIMP-1 antibody (catalogue no. sc-5538) were purchased from Santa Cruz Biotechnology Inc., CA. Mouse monoclonal anti-macrophage antibody ED-1 (catalogue no. MAB1435) was purchased from Chemicon International Inc., CA. Mouse monoclonal anti-human TIMP-1 antibody (catalogue no. ZM-0430) and mouse monoclonal anti-human
-smooth muscle actin (
-SMA) antibody (catalogue no. ZM-0003) were purchased from Beijing Zhongshan Biotechnology Co., Ltd.
Animals
At least 1 week before the experiment, 51 female Wistar rats weighing 130150 g were obtained from and housed in the animal facility (temperature, 2022°C; humidity, 5556%; 12 h light/12 h dark cycle; unlimited access to food and water) of the Laboratory Animal Center of PLA General Hospital. Rats were divided randomly into three groups: a control group (n = 17), a model group (n = 17) and a fosinopril group (n = 17). Experimental procedures were performed in accordance with the recommendations and policies of our hospital for the protection and care of animals used for experimental and other scientific purposes, and were granted permission by the local authorities.
Experimental protocol and treatment
To induce immune complex glomerulonephritis, we modified the method of Arisz et al. [17]. In brief, 34 rats in the model group and fosinopril group were given left nephrectomy followed 1 week later by intracutaneous immunization at 2 week intervals with 1.5 mg of BSA diluted in 0.15 ml of phosphate-buffered saline (PBS) (pH 7.4) emulsified with 0.15 ml of IFA, until precipitating antibodies to BSA with titres (log2)
16 were produced. Daily intraperitoneal (i.p.) injections of 3.0 mg of BSA in 1 ml of PBS were given over a period of 2 weeks (n = 6 for each group) or 4 weeks (n = 11 for each group). The rats were then killed. At the 21st day of BSA, 100 µg of LPS in 1 ml of PBS was given i.p. The 17 rats in the control group were given left nephrectomy, pre-immunization with adjuvant alone, and daily i.p. injections with 1 ml of PBS with BSA or LPS. At the beginning of the i.p. BSA injection, the 17 rats in the fosinopril group were given daily gavage of 10 mg/kg of fosinopril dispersed in 1 ml 0.9% of saline, while the rats in the control and model groups received saline gavage.
Blood pressure measurement
Systolic blood pressure (SBP) was measured in conscious, restrained rats using a tail-cuff sphygmomanometer (Natume KN-210, made in Japan). The SBP for each rat was calculated from the average of three separate measurements at each session.
Analytical and histological studies
Before the rats were sacrificed, urine was collected from animals kept individually in metabolism cages for 24 h with free access to water but deprived of food. Urinary protein was assessed by the Coomassie brilliant blue method. Serum samples were collected by orbital plexus bleeding, and serum levels of total protein (TP), albumin (ALB), total cholesterol (Tch), triglycerides (TG), creatinine (Cr) and blood urea nitrogen (BUN) were determined by routine laboratory tests on the day of sacrifice with a Hitachi 7150 automatic biochemical analytical instrument.
At the time of sacrifice, animals were anaesthetized with ether. The kidneys were perfused in vivo through the abdominal aorta with 50 ml of normal saline at 4°C. They were then immediately removed and processed further for histological studies and for protein RNA extraction. A portion of each kidney was snap-frozen in liquid nitrogen and stored at 70°C for protein and RNA extraction. Tissues for histological study were fixed in 10% buffered formalin, routinely processed, embedded in paraffin, sectioned in 1 µm samples and stained with periodic acidSchiff. Histological examination was performed under light microscopy without the examiners having knowledge of the experimental protocol. The distributions of IgG and C3 were analysed in frozen sections by immunofluorescence tests with fluorescein isothiocyanate (FITC)-conjugated goat anti-rat IgG and FITC-conjugated goat anti-rat C3 antibody.
Electrophoretic mobility shift assay (EMSA)
For the EMSA, we used the STAT-binding double-stranded consensus oligonucleotides probe m67 (5'-CATTTCCCGTAAATC-3'), which binds STAT3. This probe was end-labelled with [
-32P]ATP by T4 polynucleotide kinase (Gibco-BRL) according to the manufacturer's protocol. For the binding reaction, 50 000 c.p.m. of the end-labelled STAT1- or STAT3-binding probe was co-incubated with a cocktail of 20 µg of nuclear proteins in gel shift binding buffer [10 mM TrisHCl pH 7.5, 50 mM NaCl, 4% glycerol, 1.0 mM MgCl2, 5.0 mM EDTA, 0.5 mM dithiothreitol (DTT)] and 2 µg of poly(dIdC) at room temperature for 30 min. EMSAs were performed on a non-denaturing 4% polyacrylamide gel in 0.5x TBE buffer at 100 V. Dried gels were subjected to autoradiography at 70°C.
Northern blot analysis
Total tissue RNA was extracted with Trizol reagent (Gibco-BRL) according to the manufacturer's instructions. Briefly,
80 mg of tissue was homogenized with 0.8 ml of Trizol solution, placed on ice for 15 min, transferred into sterile centrifugation tubes, mixed with chloroform and centrifuged at 12 000 r.p.m. for 15 min at 4°C. The upper transparent layer was transferred to another centrifuge tube and mixed with an equal volume of isopropanol, followed by centrifugation again at 4°C. The RNA pellets were dissolved in diethylpyrocarbonate (DEPC)-treated water and quantified with a spectrophotometer. A 20 µg aliquot of total RNA was electrophoresed on a 1% agarose gel containing 2.2% formaldehyde and transferred onto a nylon membrane (Hybond, Amersham, UK) by capillary blotting, cross-linked in an ultraviolet cross-linker, pre-hybridized and then hybridized with 32P-labelled rat STAT3 and rat TIMP-1 probe. Probes of rat STAT3 anti-TIMP-1 for northern blotting were prepared by reverse transcriptionpolymerase chain reaction (RTPCR). The primers used for RTPCR were as follows: rat TIMP-1 sense sequence, 5'-GCCCCAACCCACCCACAGA-3'; antisense, 5'-TTTGCAAGGGATGGCTGAACAG-3'; rat STAT3 sense sequence, 5'-TGGAAGAGGCGGCAGCAGATAGC-3'; and antisense, 5'-CACGGCCCCCATTCCCACAT-3'. The PCR products were purified with glassmilk (Bio-Rad) and identified by DNA sequencing.
Western blot analysis
Tissues were lysed in RIPA buffer (50 mM TrisHCl at pH 7.4, 1% NP-40, 150 mM NaCl, 1 mM EDTA and 0.25% deoxycholic acid) containing 1 mM phenylmethylsulfonyl fluoride (Sigma Aldrich, St. Louis, MO), 1 mg/ml leupeptin and 1 mg/ml aproptinin, then homogenized with a glass homogenizer and centrifuged at 15 000 g for 15 min. The protein concentrations of the supernatants were quantified using the Micro BCATM Protein Assay Reagent Kit (Pierce, Rockford, IL). The protein extracted was mixed with 2x SDS sample buffer (125 mM TrisHCl, pH 6.8, 4% SDS, 20% glycerol, 10% ß-mercaptoethanol) at a 1:1 ratio and was boiled for 5 min. Proteins were separated by SDSPAGE (8 or 12%) and transferred onto a nitrocellulose membrane (Amersham, Hybond-C). Membranes were subsequently blocked with TBST [10 mM TrisHCl, pH 8.0, 150 mM NaCl, 0.05% (v/v) Tween-20] and 5% (w/v) non-fat dry milk at room temperature for 1 h, and then probed using primary antibodies for STAT3, p-STAT3 and TIMP-1 overnight at 4°C. After washing, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody and visualized by an enhanced chemiluminescence (ECL) detection system (Santa Cruz Biotechnology). The levels of STAT3, p-STAT3 and TIMP-1 were quantitated by densitometric scanning of the X-ray films with the UVP Gel Documentation System GDS8000 and 1D Gel Analysis Software.
Immunohistochemistry
Macrophage infiltration (marked by the surface marker for monocytes/macrophages, ED-1) and expression of STAT3, p-STAT3, TIMP-1 and
-SMA proteins were assessed by immunohistochemistry using the labelled avidinbiotin method with corresponding HistostainTM-SP kits from Zymed Laboratories, Inc. For this purpose, 4 µm thick paraffin sections of formalin-fixed tissues were used, and were deparaffinized and rehydrated. Endogenous peroxidase was blocked by incubating in 3% H2O2/methanol (1:1) at 25°C for 15 min, followed by pre-treatment with 0.05% trypsin in 0.1 M TrisHCl (pH 7.6) with 0.1% CaCl2 for 15 min at 37°C for antigen retrieval. The sections were then incubated in serum blocking solution provided with the kits to block non-specific binding, and then incubated overnight at 4°C with a 1:100 dilution of the primary antibodies, incubated for 20 min at 37°C with biotinylated second antibody, incubated for 20 min at 37°C with streptavidinenzyme conjugate, and the immunoreactive product was visualized by the diaminobenzidine (DAB) reaction. In order to examine the specificity of the immunoreactivity, the primary antibody was omitted to provide a non-specific control. Macrophage infiltration and expression of STAT3 and p-STAT3 were assessed by counting the number of ED-1-, STAT3- or p-STAT3-positive cells, respectively, in 20 glomerular profiles and 20 0.25 x 0.25 mm areas of tubulointerstitium randomly chosen for each kidney. We calculated the mean number in one glomerulus and per 0.0625 mm2 areas of tubulointerstitium, and the expression of TIMP-1 and
-SMA was assessed by measuring the positive areas in 20 glomerular profiles and 20 0.25 x 0.25 mm areas of tubulointerstitium randomly chosen using a real colour image analysis system (TIPAS/88 type, which was designed by the PLA computer research centre) and calculated the mean ratio of positive area in one glomerulus and per 0.0625 mm2 areas of tubulointerstitium. For measurement of TIMP-1 and
-SMA stainings, the negative background staining was calibrated to zero, and the area of positive staining above the background level in each glomerulus or area chosen was measured. Each measurement was derived from computer analysis of the integrated logarithm of the inverse grey value, which is proportional to the total amount of absorbing material in the light path. This system enables the percentage area of positive staining in each glomerulus and per area to be quantified accurately. The p-STAT3/STAT3 ratio for each rat was calculated for evaluation of the STAT3 activation level.
Statistical analysis
All data are presented as means±SD. Differences were evaluated by one-way ANOVA. The relationship between the number of positive cells per glomerulus and 24 h urinary protein excretion and the ratio of TIMP-1-positive areas per glomerulus were assessed by linear regression analysis. Statistical significance was defined as P<0.05.
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Results
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Time course of blood pressure, proteinuria, serum biochemical and renal function parameters
The time course changes in mean levels of systolic blood pressure, urinary protein excretion, serum TP, ALB, Tch, TG, BUN and Cr are shown in Table 1. There were no significant differences in SBP among the three groups. In the model group, serum levels of Tch, TG, BUN and Cr at 2 weeks as well as at 4 weeks were significantly higher than those of the control group, whereas the levels of serum TP and ALB were lower than in controls. In the fosinopril group, serum levels of Tch, TG, BUN, Cr and urinary protein excretion at 2 and 4 weeks were significantly reduced compared with those of the model group (P<0.05).
Histological findings
Light microscopic examination of renal tissue revealed the degrees of hypercellularity, glomerular infiltration of polymorphonuclear leukocytes, fibrinoid necrosis, focal and segmental proliferation and interstitial infiltration in each group (Figure 1). Animals treated with fosinopril showed significantly improved glomerular changes compared with the model group. Immunofluorescence microscopy showed fine to coarse granular deposits of rat IgG and C3, which were extensively localized along the glomerular capillary walls in the model group, whereas IgG and C3 immunofluorescence was weaker in the fosinopril group.

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Fig. 1. Light microscopic examination of renal tissues revealed apparent hypercellularity, glomerular infiltration of polymorphonuclear leukocytes, fibrinoid necrosis, focal segmental proliferation and interstitial infiltration in the model group, which was abated in the fosinopril group. (A) Control group; (B) model group; (C) fosinopril group (PAS, x200).
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DNASTAT3 binding activity
The EMSA showed that the DNASTAT binding activity of STAT3 was apparently increased in the model group, but was markedly decreased after fosinopril treatment (Figure 2).

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Fig. 2. DNASTAT binding activity of STAT3 was significantly increased in the model group, but decreased after fosinopril treatment. Lanes 1 and 2, model group; lanes 3 and 4, fosinopril group; lanes 5 and 6, control group. SIF = sis-inducing factor.
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Northern blot analysis of STAT3 and TIMP-1
Northern blot analysis showed that total mRNA of STAT3 and TIMP-1 in kidney tissues was significantly increased in the model group compared with controls (P<0.01), and that these were decreased in the fosinopril group (P<0.01) (Figures 3 and 4).

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Fig. 3. Northern blot analysis showed that gene expression of both STAT3 and TIMP-1 was increased in renal tissues of the model group, but decreased in the fosinopril group. Lanes 1 and 2, model group; lanes 3 and 4, fosinopril group; lanes 5 and 6, control group.
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Fig. 4. Gene expression of STAT3 and TIMP-1 quantified by phosphoroimager analysis relative to GAPDH levels. *P<0.05 vs control, P<0.01 vs the model group.
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Western blot analysis of STAT3, p-STAT3 and TIMP-1
Kidney tissues from the control group contained a small amount of STAT3, p-STAT3 and TIMP-1 proteins, while kidney tissues from the model group had significantly higher levels of STAT3, p-STAT3 and TIMP-1 proteins (P<0.05). In the fosinopril group, kidney tissues had significantly lower levels of STAT3, p-STAT3 and TIMP-1 proteins (P<0.05) (Figures 5 and 6).

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Fig. 5. Western blot analysis showed that the protein expression of STAT3, p-STAT3 and TIMP-1 was increased in renal tissues of the model group, but decreased in the fosinopril group. Lanes 1 and 2, model group; lanes 3 and 4, fosinopril group; lanes 5 and 6, normal control.
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Fig. 6. Protein expression of STAT3, p-STAT3 and TIMP-1 quantified by phosphoroimager analysis relative to ß-actin levels. *P<0.01 vs control, P<0.01 vs the model group.
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Immunohistochemistry findings
Representative glomerular stainings for ED-1, STAT3, p-STAT3, TIMP-1 and
-SMA are shown in Figure 7. The time course analysis results of ED-1, STAT3, p-STAT3, TIMP-1 and
-SMA expression at 2 and 4 weeks are shown in Table 2.
The effect of fosinopril therapy on macrophage infiltration in glomeruli was examined with ED-1 immunohistochemistry. There were more glomerular ED-1-positive cells in the model group than in the control group (P<0.05). This glomerular macrophage infiltration was largely prevented in the fosinopril group. Macrophage infiltration in periglomerular and tubulointerstitial regions was also observed (data not shown). Finally, macrophage infiltration in the tubulointerstitial region was also decreased in kidneys from the fosinopril group.
Immunohistology confirmed the results of western blot analysis showing that expression and activation of STAT3 were enhanced in kidney tissues from the model group. STAT3 and p-STAT3 were detected in both the cytoplasm and nucleus from kidneys in each group. STAT3 and p-STAT3 expression was strongly increased in the glomeruli, tubules and interstitium from kidneys in the model group compared with those in the control group (P<0.05). However, in the fosinopril group, the expression of STAT3 and p-STAT3 was markedly reduced in the glomeruli and interstitium (P<0.05). Furthermore, the p-STAT3/STAT3 ratio, which was significantly increased in the model group (P<0.05), was reduced significantly by fosinopril treatment (P<0.05).
Staining for TIMP-1 was present in mesangial cells and in tubular epithelial cells in kidneys from the model group. The expression of TIMP-1 in glomeruli or in tubulointerstitia was significantly lower in the fosinopril group than in the model group (P<0.05).
In the control group, a small amount of
-SMA was detected in glomeruli and tubulointerstitia, which contrasted with strong staining in VSMCs. In kidneys from the model group, there was strong staining of
-SMA in both the expanded mesangial areas and the tubulointerstitia. The expression of
-SMA in the glomeruli and tubulointerstitia of the fosinopril group was significantly reduced (P<0.05).
Double stainings of p-STAT3, ED-1 and TIMP-1
Double immunostaining in kidney tissues revealed the co-existence of ED-1 (blue colour) and p-STAT3 (red colour), as well as the co-existence of p-STAT3 (red colour) and TIMP-1 (blue colour). Some of the ED-1-positive cells also had p-STAT3-positive stainings, and some cells positive for p-STAT3 also had TIMP-1-positive stainings (Figure 8).

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Fig. 8. Double immunostainings of ED-1, p-STAT3 and TIMP-1 were performed. In kidney tissues, the co-existence of ED-1 (blue colour) and p-STAT3 (red colour) was detected, and so was the co-existence of p-STAT3 (red colour) and TIMP-1 (blue color). It was found that some ED-1-positive cells also had p-STAT3-positive staining, and that some cells positive for p-STAT3 also had TIMP-1-positive staining (inlets are cells amplified showing the apparent co-existence of p-STAT3 and TIMP-1, in which red staining of p-STAT3 was located in the nucleus while blue staining of TIMP-1 was located in the cytoplasm of the same cell).
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Time course of p-STAT3 expression
The time course of p-STAT3 expression was examined in the three experimental groups. The expression of p-STAT3 was observed at both 2 weeks (n = 6 in each group) and 4 weeks (n = 11 in each group). At 2 weeks, p-STAT3 expression increased compared with expression at 0 weeks, and at 4 weeks, p-STAT3 expression was significantly higher than that at 2 weeks (Figure 9).

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Fig. 9. Time course of p-STAT3 expression in different groups. Before = before the experiment; 2 weeks = daily i.p. injection of BSA for 2 weeks; 4 weeks = daily i.p. injection of BSA for 4 weeks. *P<0.05 vs results of the same group before the experiment. P<0.05 vs results of the same group at 2 weeks.
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Correlation of p-STAT3 protein expression with 24 h urinary protein, and TIMP-1 protein expression
We performed association analysis to determine whether activation of STAT3 was associated with severity of glomerular injury. We found that p-STAT3 protein expression was positively correlated with both 24 h proteinuria and TIMP-1 protein expression. A robust and significant association was found between glomerular p-STAT3 protein expression and urinary protein excretion (r = 0.842, P<0.01; Figure 7). A strong correlation was also found between glomerular p-STAT3 expression and glomerular TIMP-1 expression (r = 0.791, P<0.01; Figures 10 and 11).

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Fig. 10. Relationship analysis displayed a positive correlation between p-STAT3 expression and 24 h urinary protein excretion in the model group as well as the fosinopril group (y = 4.321, x = 19.268, r = 0.842, P<0.01).
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Fig. 11. Relationship analysis showed a positive correlation between p-STAT3 and TIMP-1 expression in both the model group and the fosinopril group (y = 0.256x + 11.652, r = 0.791, P<0.01).
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Discussion
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In the present study, we evaluated whether STAT3 is expressed and activated in kidney tissues from a glomerulonephritis model and whether these effects are modified by fosinopril. To do this, we used a modified immune complex glomerulonephritis model in rats. This model shows glomerulonephritis with immune deposits and glomerular macrophage infiltration [18]. Our results showed that kidney tissues from the model group had increased expression and constitutive activation of STAT3, and that the expression of TIMP-1 was increased and positively correlated with STAT3 phosphorylation. In addition, fosinopril treatment inhibited the activation and expression of STAT3 as well as the expression of TIMP-1. These findings may provide some new insights into the mechanisms that determine the development and progression of glomerulonephritis.
We modified the traditional rat chronic serum sickness glomerulonephritis by adding unilateral nephrectomy and i.p. LPS injection. These measures were introduced to obtain an accelerated nephritis. One distinguishing immunopathological feature of this model was the progressive increase in glomerular macrophages, which was marked by the accumulation of ED-1-positive cells in glomeruli. Double staining showed that some ED-1-positive cells also had p-STAT3-positive staining, indicating that macrophages may be important in the activation of STAT3. During the process of glomerular injury, a variety of cytokines may be expressed in glomeruli, and these are synthesized either by resident mesangial cells or by infiltrating inflammatory cells [18,19]. Since JAK/STAT is the main intracellular signalling pathway of cytokines and growth factors, and since STAT3 acts as an important member, it is likely that STAT3 is activated during this process.
Previous work from our laboratory and others have shown that STAT3 is expressed and activated by various stimuli in different renal cells. STAT3 was shown to be involved in thrombin-induced expression of the TIMP-1 gene in cultured human mesangial cells [4]. STAT3 also mediated angiotensin-induced TIMP-1 mRNA expression in human renal proximal tubular epithelial cells, which implicated a role for STAT3 signalling pathways in the pathogenesis of renal tubulointerstitial fibrosis [6]. Amiri et al. [5] reported that activation of the JAK/STAT pathway by high glucose, Ang II or both may be of importance in the increased proliferation of glomerular mesangial cells in vitro and synthesis of collagen IV, which is observed in diabetic nephropathy [5]. These findings demonstrated that STAT3 can be activated in renal cells by different stimuli. Moreover, STAT3 activation in glomeruli was also reported in the Thy1.1 model [13] and in the nephrotoxic serum model [14].
The present study demonstrated, using a series of methods, including northern blot, EMSA, western blot and immunohistochemistry, that STAT3 is expressed in vivo and activated in renal tissues during rat modified immune complex glomerulonephritis. Furthermore, the increased ratio of p-STAT3/STAT3 expression in nephritis tissues, determined by immunohistochemistry, also confirmed the existence of STAT3 activation. Phosphorylation of STAT3 can lead to the formation of STAT3 dimers, followed by translocation of these dimers into the nucleus to regulate transcription of target genes, leading to inflammatory injury of renal tissues [2]. Our results also suggested that the enhanced expression and activation of STAT3 in nephritis tissues may be suppressed by fosinopril treatment. We postulate that this may be due to at least two factors. First, since Ang II can activate STAT3 in renal cells [4,5], ACE inhibition may reduce this activation by blocking Ang II production. Secondly, given that ACE inhibitors can reduce macrophage infiltration and downregulate many pro-inflammatory mediators including IL-6 in various nephritis models [15], in which STAT3 is one of the major mediating signalling proteins, it is possible that ACE inhibitors may inhibit inflammation by suppressing STAT3 activation. Our results demonstrated in kidneys of rats with modified immune complex glomerulonephritis that STAT3 is expressed and activated, that ED-1-positive cells were increased and that some ED-1-positive cells during double staining also contained p-STAT3-positive staining. These results indicate that fosinopril may have ameliorated renal damage by downregulating STAT3 activation and ED-1 influx.
Since many STAT3 target genes are proliferation-associated proteins, anti-apoptotic proteins, ECM modulators and proangiogenic factors [7], the activation of STAT3 in nephritis tissues may be closely related to the pathogenesis of renal injury. In this study, STAT3 phosphorylation was positively correlated with 24 h urinary protein, indicating a contribution of STAT3 phosphorylation to the progression of glomerulonephritis. We also analysed the relationship between STAT3 phosphorylation and the expression of TIMP-1, a potent inhibitor of the key matrix-degrading enzymes, such as matrix metalloproteinase [6]. These studies revealed that glomerular phosphorylation of STAT3 was positively correlated with the glomerular expression of TIMP-1, indicating that STAT3 activation may expedite the accumulation of ECM by upregulation of TIMP-1 expression, and that fosinopril may decrease proteinuria and matrix protein expression through a suppression of STAT3 activation.
The present study has provided in vivo evidence that STAT3 is expressed and activated in both glomerular and tubulointersitial regions in kidneys of rats having a modified immune complex glomerulonephritis. This activation may lead to increases in TIMP-1 expression and subsequent injury to renal tissues, whereas treatment with fosinopril may reduce both STAT3 expression and macrophage influx, leading to attenuation of glomerular proliferation and extracellular matrix accumulation. Interference of abnormal STAT3 signalling in kidneys with nephritis may provide a novel therapeutic molecular target for kidneys with nephritis.
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Acknowledgments
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This study was supported by the Main STAT Basic Research Development Program of P.R. China (G2000057000), and by the Creative Research Group Fund from the National Foundation Committee of Natural Science of P.R. China (30121005).
Conflict of interest statement. None declared.
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References
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Received for publication: 1. 1.04
Accepted in revised form: 15. 9.04