Establishment of rat model of anti-glomerular basement membrane nephritis induced by pCol (24-38) / 解放军医学杂志

ABSTRACT

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 120-150g, 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 4th week in anti-GBM model group than in control group [(52.27±10.50)mg/24h vs. (4.87±0.64)mg/24h, P<0.001], and then rose gradually until the 7th week [(255.80±9.79)mg/24h vs. (5.78±0.39)mg/24h, 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 anti-GBM 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.