Triptolide Attenuates Podocytes Injury And Mechanism Analysis In Transgenic Zebrafish

Part 1. Development and preliminary Application of Anti-zebrafish Podocin AntibodyObjecive:To prepare a polyclonal antibody against zebrafish podocin and evaluate podocytes injury with immunofluorescence using an in vivo zebrafish model of inducible podocyte injury.Methodolgy:(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 rabbits 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-m Cherry) 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 podocytes injury in the two podocyte injury models with immunofluorescence.Result: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 24 hours of MTZ treatment, the distribution of podocin became coarse granular pattern. The expression of podocin significantly reduced was 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.Part 2. Triptolide attenuates podocytes injury and mechanism analysis in transgenic zebrafish:role of gadd45Objecive:To explore the protective effects of triptolide on podocytes and analysis the underlying mechanism of antiproteinuric effects in transgenic zebrafish model.Methodolgy:(1) Transgenic zebrafish Tg(pod;gal4;UAS:NTR-mCherry) was generated as an inducible podocyte injury model in vivo by nitroreductase(NTR)/metronidazole (MTZ) system. The embryos were treated with triptolide. (2) Observed and quantified the percentage of zebrafish larvae periorbital and sac yolk edema after 48h of MTZ treatment.(3) Performed proteinuria assay in Tg((pod:Gal4;UAS:NTR-mCherry;lfabp:VDBP-GFP) embryos using ELISA against GFP after MTZ treatment. (4) Examined the expression and distribution of silt diaphragm molecule podocin using whole mount immunofluorescence staining.(5) Examined the foot process alteration by transmission electron microscopy.(6)Performed Caspase-3 staining to investigate the effects of triptolide on podocyte apoptosis. (7) Carried out a genome-wide transcriptome microarray on RNA samples extracted from triptolide-treated and none-treated human podocytes. (8) Validated the results from the genome-wide transcriptome microarray by qRT-PCR in vitro and in vivo.Result:(1) Edema ratio:triptolide attenuates periorbital and sac yolk edema significantly comparing to MTZ model group. (2) Proteinuria:Triptolide decreased proteinuria level remarkly induced by MTZ in Tg((pod:Gal4;UAS:NTR-mCherry;lfabp:VDBP-GFP) embryos. (3) Silt diaphragm molecule:The distribution of podocin changed to coarse granular pattern manner in MTZ model group after 24 hours and a seriously declined expression of podocin later, but triptolide can restore the expression and distribution of podocin. (4) Foot process: The podocyte foot process effacement was effectively ameliorated in triptolide treatment group. (5) The caspase-3 staining showed that triptolide inhibited apoptosis of podocyte induced by MTZ. (6) Triptolide could downregulate GADD45 members by genome-wide transcriptome analysis in triptolide-treated human podocytes. (7) qRT-PCR validation that gadd45 mRNA level was indeed downregulated by triptolide in vitro and in vivo.Conclusion:Triptolide effectively reduced the proteinuria induced by NTR/MTZ system in transgenic zebrafish, stabilized the expression of podocin, and ameliorated podocyte foot process effacement. The mechanism may associate with inhibiting podocyte apoptosis and downregulating GADD45 gene expression. Otherwise, our study indicated that zebrafish embryos could be a robust experiment vertebrate model to identify new podocyte protective drugs and perform fast and reproducible pharmacological assays with excellent results.