Abstract:

【ABSTRACT】 Objecive: To prepare a polyclonal antibody against zebrafish podocin and evaluate podocyte injury with immunofluorescence using an in vivo zebrafish model of inducible podocyte injury. Methodology: (1) Zebrafish podocin peptides as antigen were designed and synthesized to immunize New Zealand white rabbits after coupling with keyhole limpet hemocyanin (KLH). The polyclonal antibody in rabbit sera was purified by protein A column. (2) Titer and specificity of the polyclonal antibody was determined by ELISA and immunohistochemistry. (3) Transgenic zebrafish Tg (pod;gal4;UAS:NTR-mCherry) was generated as inducible podocyte injury model in vivo by nitroreductase (NTR)/metronidazole (MTZ) system. Morpholino antisense oligonucleotides (MO) was designed against translation of zebrafish embryo podocin mRNA. The synthesized podocin antibody was used to evaluate podocyte injury in the two podocyte injury models with immunofluorescence. Result: (1)The purify of synthesized podocin peptides was 92.6%, which met the immunogen standard. (2)The ELISA and immunohistochemistry showed that the titers of anti-podocin antibody reached up to 1：5.12×105 and 1:106. (3) The staining of podocin was revealed as fine contiguous linear-like pattern along the glomerular capillary loop both in normal pronephros and mesonephros of zebrafish. (4)After 24hrs of MTZ treatment, the distribution of podocin became coarse granular pattern. The expression of podocin was significantly reduced at 48h after MTZ treatment. (5)Podocin morpholino-injected embryos exhibited pericardial edema and body-axis curvature. The expression of podocin was remarkably decreased and only barely visible 3dpf. However, the expression and distribution of podocin was recovered gradually and completely restored to normal at 5dpf. Conclusion: The anti-zebrafish podocin antibody we prepared possesses high titer and specificity, and is correlated with the severity of podocyte injury, suggesting a great potential of using podocin antibody for podocyte researches in zebrafish model.

Key words: zebrafish, podocin, podocyte