pCol(24-38)诱导抗肾小球基底膜肾病大鼠模型的建立
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摘要
目的以含大鼠Ⅳ型胶原α3链的非胶原区[α3(Ⅳ)NC1]的多肽pCol(24-38)作为抗原建立抗肾小球基底膜(GBM)病大鼠模型,研究抗GBM病的发病机制。方法选择雄性WKY大鼠20只(8～10周龄),体重120～150g,随机分为对照组和抗GBM病模型组,每组10只。抗GBM病模型组使用多肽pCol(24-38)作为抗原,对照组使用多肽pCol(38-52)作为抗原,两组均用完全弗氏佐剂乳化后,于大鼠背部皮下三点注射免疫。免疫后每周定时收集24h尿,测尿蛋白含量,所有动物在免疫后49d处死,取血清,采用肌氨酸氧化酶法检测血清肌酐,脲酶法检测尿素氮浓度,并用间接ELISA法检测血清抗pCol(24-38)抗体相对滴度。取肾脏组织,一部分石蜡切片后行PAS染色及免疫组化染色,另一部分经OCT包埋冷冻切片后用直接免疫荧光法检测IgG在肾小球的沉积情况。结果与对照组比较,抗GBM病模型组的24h尿蛋白含量自第4周开始明显升高[(52.27±10.50)mg/24h vs.(4.87±0.64)mg/24h,P<0.001],之后逐渐上升,至第7周,抗GBM病模型组的24h尿蛋白含量仍明显高于对照组[(255.80±9.79)mg/24h vs.(5.78±0.39)mg/24h,P<0.001];抗GBM病模型组血清肌酐水平[(145.3±22.60)μmol/L vs.(36.81±2.21)μmol/L,P<0.001]、尿素氮水平[(26.59±5.01)mmol/L vs.(6.92±0.27)mmol/L,P<0.001]均明显升高;抗GBM病模型组的循环抗pCol(24-38)IgG抗体水平明显高于对照组[(1.59±0.18) vs.(0.09±0.01),P<0.05]。抗GBM病模型组PAS染色可见肾小球硬化,弥漫性新月体形成,免疫组化染色可见肾小球有明显的巨噬细胞浸润,荧光染色可见IgG在GBM呈明显线性沉积;而对照组PAS染色未见异常肾小球,免疫组化染色未见巨噬细胞浸润,荧光染色在GBM上未发现IgG沉积。结论使用多肽pCol(24-38)作为抗原免疫WKY大鼠能成功构建抗GBM病动物模型。该抗原制备方法简单,对于设计新的抗GBM病的治疗策略有重要价值。
Objective Polypeptide pCol(24-38) containing rat non-collagen region of collagen type Ⅳ alpha 3 chain[α3(Ⅳ)NC1] was used as antigen to establish the rat model of anti-glomerular basement membrane(GBM) disease, and study the possible pathogenesis of anti-GBM disease. Methods Twenty male WKY rats, 8-10 weeks old and weight 120-150 g, were randomly divided into control group and anti-GBM model group(10 each). Polypeptide pCol(24-38) was used as antigen in anti-GBM model group, while pCol(38-52) was used as antigen in control group. After emulsification with complete Freund's adjuvant, rats in the both groups were immunized by subcutaneous injection at three points on the back. 24-hour urine was collected regularly every week after immunization to measure the urinary protein content. All animals were sacrificed 49 days after immunization, and serum was taken. The serum creatinine level was detected by creatine oxidase method, urea nitrogen concentration was determined by urease method, and the relative titer of serum anti-pCol(24-38) antibodies was detected by indirect ELISA. Part of kidney tissue was stained with PAS and immunohistochemically treatment in paraffin section, and the other part was frozen section after OCT embedding to detect IgG deposition in glomeruli by direct immunofluorescence method. Results The 24-hour urinary protein levels increased significantly from the 4 th week in anti-GBM model group than in control group [(52.27±10.50)mg/24 h vs.(4.87±0.64)mg/24 h,P<0.001], and then rose gradually until the 7 th week [(255.80±9.79)mg/24 h vs.(5.78±0.39)mg/24 h, P<0.001]. Both the serum creatinine level and urea nitrogen level increased obviously in anti-GBM model group than in control group [(145.3±22.60)μmol/L vs.(36.81±2.21)μmol/L;(26.59±5.01)mmol/L vs.(6.92±0.27)mmol/L] with statistically significant difference(P<0.001).Compared with the control group, rats in anti-GBM model group produced high levels of circulating anti-pCol(24-38) IgG antibodies [(1.59±0.18) vs.(0.09±0.01)] with statistically significant difference(P<0.05). PAS staining showed glomerular sclerosis and diffuse crescent formation in anti-GBM model group; immunohistochemistry showed macrophage infiltration in glomerular; fluorescence staining showed obvious linear deposition of IgG in glomerular basement membrane. However, in the control group, PAS staining showed no abnormal glomerular, immunohistochemistry showed no macrophage infiltration, and fluorescence staining showed no IgG deposits on the glomerular basement membrane. Conclusions The animal model of antiGBM disease may be successfully constructed by using polypeptide pCol(24-38) as antigen to immunize WKY rats. The preparation method of the antigen is simple, and so is valuable for designing new therapeutic strategies against anti-GBM disease.
Objective Polypeptide pCol(24-38) containing rat non-collagen region of collagen type Ⅳ alpha 3 chain[α3(Ⅳ)NC1] was used as antigen to establish the rat model of anti-glomerular basement membrane(GBM) disease, and study the possible pathogenesis of anti-GBM disease. Methods Twenty male WKY rats, 8-10 weeks old and weight 120-150 g, were randomly divided into control group and anti-GBM model group(10 each). Polypeptide pCol(24-38) was used as antigen in anti-GBM model group, while pCol(38-52) was used as antigen in control group. After emulsification with complete Freund's adjuvant, rats in the both groups were immunized by subcutaneous injection at three points on the back. 24-hour urine was collected regularly every week after immunization to measure the urinary protein content. All animals were sacrificed 49 days after immunization, and serum was taken. The serum creatinine level was detected by creatine oxidase method, urea nitrogen concentration was determined by urease method, and the relative titer of serum anti-pCol(24-38) antibodies was detected by indirect ELISA. Part of kidney tissue was stained with PAS and immunohistochemically treatment in paraffin section, and the other part was frozen section after OCT embedding to detect IgG deposition in glomeruli by direct immunofluorescence method. Results The 24-hour urinary protein levels increased significantly from the 4 th week in anti-GBM model group than in control group [(52.27±10.50)mg/24 h vs.(4.87±0.64)mg/24 h,P<0.001], and then rose gradually until the 7 th week [(255.80±9.79)mg/24 h vs.(5.78±0.39)mg/24 h, P<0.001]. Both the serum creatinine level and urea nitrogen level increased obviously in anti-GBM model group than in control group [(145.3±22.60)μmol/L vs.(36.81±2.21)μmol/L;(26.59±5.01)mmol/L vs.(6.92±0.27)mmol/L] with statistically significant difference(P<0.001).Compared with the control group, rats in anti-GBM model group produced high levels of circulating anti-pCol(24-38) IgG antibodies [(1.59±0.18) vs.(0.09±0.01)] with statistically significant difference(P<0.05). PAS staining showed glomerular sclerosis and diffuse crescent formation in anti-GBM model group; immunohistochemistry showed macrophage infiltration in glomerular; fluorescence staining showed obvious linear deposition of IgG in glomerular basement membrane. However, in the control group, PAS staining showed no abnormal glomerular, immunohistochemistry showed no macrophage infiltration, and fluorescence staining showed no IgG deposits on the glomerular basement membrane. Conclusions The animal model of antiGBM disease may be successfully constructed by using polypeptide pCol(24-38) as antigen to immunize WKY rats. The preparation method of the antigen is simple, and so is valuable for designing new therapeutic strategies against anti-GBM disease.
引文
[1] McAdoo SP, Pusey CD. Antiglomerular basement membrane disease[J]. Semin Respir Crit Care Med, 2018, 39(4):494-503.
[2] Gulati K, McAdoo SP. Anti-glomerular basement membrane disease[J]. Rheum Dis Clin North Am, 2018,44(4):651-673.
[3] Ooi JD, Chang J, O'Sulivan KM, et al. The HLA-DRB1*15:01restricted Goodpasture's T cell epitope induces GN[J].J Am Soc Nephrol, 2013, 24(3):419-431.
[4] Goodpasture EW. The significance of certain pulmonary lesions in relation to the etiology of influenza[J]. Am J Med Sci, 2009,338(2):148-151.
[5] Wilson CB, Dixon FJ. Anti-glomerular basement membrane antibody-induced glomerulonephritis[J]. Kidney Int, 1973,3(2):74-89.
[6] Reynolds J, Preston GA, Pressler BM, et al. Autoimmunity to the alpha 3 chain of type IV collagen in glomerulonephritis is triggered by'autoantigen complementarity'[J]. J Autoimmun,2015, 59:8-18.
[7] Reynolds J, Cook PR, Behmoaras J, et al. Genetic susceptibility to experimental autoimmune glomerulonephritis in the Wistar Kyoto rat[J]. Am J Pathol, 2012, 180(5):1843-1851.
[8] Yu XH, Petersen F. A methodological review of induced animal models of autoimmune diseases[J]. Autoimmun Rev, 2018,17(5):473-479.
[9] Reynolds J, Albouainain A, Duda MA, et al. Strain susceptibility to active induction and passive transfer of experimental autoimmune glomerulonephritis in the rat[J]. Nephrol Dial Transplant, 2006, 21(12):3398-3408.
[10] Jia XY, Cui Z, Yang R, et al. Antibodies against linear epitopes on the Goodpasture autoantigen and kidney injury[J]. Clin J Am Soc Nephrol, 2012, 7(6):926-933.
[11] Hu SY, Jia XY, Gu QH, et al. T cell responses to peptides of Goodpasture autoantigen in patients with anti-glomerular basement membrane disease[J]. Nephrology(Carlton), 2018,23(4):345-350.
[12] Luo AM, Fox JW, Chen LL, et al. Synthetic peptides of Goodpasture's antigen in antiglomerular basement membrane nephritis in rats[J]. J Lab Clin Med, 2002, 139(5):303-310.
[13] Robertson J, Wu J, Arends J, et al. Activation of glomerular basement membrane-specific B cells in the renal draining lymph node after T cell-mediated glomerular injury[J]. J Am Soc Nephrol, 2005, 16(11):3256-3263.
[14] Robertson J, Wu J, Arends J, et al. Characterization of the T-cell epitope that causes anti-GBM glomerulonephritis[J]. Kidney Int, 2005, 68(3):1061-1070.
[15] Bolton WK, Chen L, Hellmark T, et al. Epitope spreading and autoimmune glomerulonephritis in rats induced by a T cell epitope of Goodpasture's antigen[J]. J Am Soc Nephrol, 2005,16:2657-2666.
[16] Chen LL, Hellmark T, Pedchenko V, et al. A nephritogenic peptide induces intermolecular epitope spreading on collagen IV in experimental autoimmune glomerulonephritis[J]. J Am Soc Nephrol, 2006, 17(11):3076-3081.
[17] Arends J, Wu J, Borillo J, et al. T cell epitope mimicry in antiglomerular basement membrane disease[J].J Immunol,2006, 176(2):1252-1258.
[2] Gulati K, McAdoo SP. Anti-glomerular basement membrane disease[J]. Rheum Dis Clin North Am, 2018,44(4):651-673.
[3] Ooi JD, Chang J, O'Sulivan KM, et al. The HLA-DRB1*15:01restricted Goodpasture's T cell epitope induces GN[J].J Am Soc Nephrol, 2013, 24(3):419-431.
[4] Goodpasture EW. The significance of certain pulmonary lesions in relation to the etiology of influenza[J]. Am J Med Sci, 2009,338(2):148-151.
[5] Wilson CB, Dixon FJ. Anti-glomerular basement membrane antibody-induced glomerulonephritis[J]. Kidney Int, 1973,3(2):74-89.
[6] Reynolds J, Preston GA, Pressler BM, et al. Autoimmunity to the alpha 3 chain of type IV collagen in glomerulonephritis is triggered by'autoantigen complementarity'[J]. J Autoimmun,2015, 59:8-18.
[7] Reynolds J, Cook PR, Behmoaras J, et al. Genetic susceptibility to experimental autoimmune glomerulonephritis in the Wistar Kyoto rat[J]. Am J Pathol, 2012, 180(5):1843-1851.
[8] Yu XH, Petersen F. A methodological review of induced animal models of autoimmune diseases[J]. Autoimmun Rev, 2018,17(5):473-479.
[9] Reynolds J, Albouainain A, Duda MA, et al. Strain susceptibility to active induction and passive transfer of experimental autoimmune glomerulonephritis in the rat[J]. Nephrol Dial Transplant, 2006, 21(12):3398-3408.
[10] Jia XY, Cui Z, Yang R, et al. Antibodies against linear epitopes on the Goodpasture autoantigen and kidney injury[J]. Clin J Am Soc Nephrol, 2012, 7(6):926-933.
[11] Hu SY, Jia XY, Gu QH, et al. T cell responses to peptides of Goodpasture autoantigen in patients with anti-glomerular basement membrane disease[J]. Nephrology(Carlton), 2018,23(4):345-350.
[12] Luo AM, Fox JW, Chen LL, et al. Synthetic peptides of Goodpasture's antigen in antiglomerular basement membrane nephritis in rats[J]. J Lab Clin Med, 2002, 139(5):303-310.
[13] Robertson J, Wu J, Arends J, et al. Activation of glomerular basement membrane-specific B cells in the renal draining lymph node after T cell-mediated glomerular injury[J]. J Am Soc Nephrol, 2005, 16(11):3256-3263.
[14] Robertson J, Wu J, Arends J, et al. Characterization of the T-cell epitope that causes anti-GBM glomerulonephritis[J]. Kidney Int, 2005, 68(3):1061-1070.
[15] Bolton WK, Chen L, Hellmark T, et al. Epitope spreading and autoimmune glomerulonephritis in rats induced by a T cell epitope of Goodpasture's antigen[J]. J Am Soc Nephrol, 2005,16:2657-2666.
[16] Chen LL, Hellmark T, Pedchenko V, et al. A nephritogenic peptide induces intermolecular epitope spreading on collagen IV in experimental autoimmune glomerulonephritis[J]. J Am Soc Nephrol, 2006, 17(11):3076-3081.
[17] Arends J, Wu J, Borillo J, et al. T cell epitope mimicry in antiglomerular basement membrane disease[J].J Immunol,2006, 176(2):1252-1258.