Complement Fragment C3a Is Involved In Changing The Phenotype Of Mice Podocytes

Objective: To investigate the effect on temperature-sensitive conditionally immortalized mouse podocyte Epithelial-Mesenchymal Transition by C3 a. Methods: using different concentrations(0.001μM, 0.003μM, 0.01μM, 0.03μM, 0.1μM)C3a to stimulate maturation podocytes for 24 hours, RT-PCR was used to analyze the m RNA transcription of synaptopodin, podocin, nephrin, CD2 AP, FSP-1, α-SMA, vimentin and Type I collagen. Western Blot was used to analyze the protein expression of nephrin, podocin, CD2 AP, FSP-1 and α-SMA. Immunohistochemistry and immunofluorescence was used to analyze the protein expression and distribution of podocin, CD2 AP, synaptopodin, FSP-1, vimentin and α-SMA. Results: 1 proliferative podocytes’ s appearance like cobblestone,however at 37 ℃ without IFN-γ cultured 14 days,podoctyte lose proliferative capacity and expressed synaptopodin, podoctyes became larger and had lots of primary and second processes. 2 immunohistochemistry and immunofluorescence: mature podocytes express synaptopodin, podocin and CD2 AP. But they decreased expression by 0.1μM C3 a stimulation after 24 h,or distribution change. mature Podocytes also expressed a little FSP-1, α-SMA and vimentin, they all increased expression after 0.1μM C3 a stimulation. 3 RT-PCR results: RT-PCR results: Compared with the control group, with C3 a concentration increase synaptopodin, podocin, CD2 AP and nephrin m RNA transcription decreased,while FSP-1,α-SMA,vimentin and collagen I m RNA transcription upregulation.with C3 a concentration(0.001-0.1μM) increase synaptopodin m RNA transcription decreased(P<0.05);(compared 0.003-0.1μM C3 a with 0.001μM C3a;0.003-0.1μM C3 a with 0.003-0.01μM C3a)podocytes’ synaptopodin m RNA transcription decreased(P <0.05).(compared 0.03-0.1μM C3 a with 0-0.01;0.1μM C3 a with 0.03μM C3a)podocytes’ podocin m RNA transcription decreased(P <0.05).(compared 0.01-0.1μM C3 a with 0-0.003μM C3a;0.03-0.1μM C3 a with 0.01μMC3a;0.1μM C3 a with 0.03μM C3a)podocytes’ CD2 AP and nephrin m RNA transcription decreased(P<0.05).With C3 a concentration(0.001-0.1μM)podocytes’ FSP-1 m RNA transcription increased(P<0.05);(compared 0.003-0.1μM C3 a with 0.001μM C3a;0.1μM C3 a with 0.003-0.03μM C3a)podocytes’ FSP-1 m RNA transcription increased(P <0.05).(compared 0.001-0.1μM C3 a with control group;0.003-0.1μM C3 a with 0.001μM C3a;0.01-0.1μM C3 a with 0.003μM C3a)podocytes’ α-SMA m RNA transcription increased(P<0.05).(compared 0.003-0.1μM C3 a with control group;0.01-0.1μM C3 a with 0.003μM C3a)podocytes’ vimentin m RNA transcription increased(P <0.05).(compared 0.01-0.1μM C3 a with 0-0.003μM C3a;0.03μM C3 a with 0.01μM C3a;0.1μM C3 a with 0.03μM C3a)podocytes’ collagen I m RNA transcription increased(P <0.05). 4 Wenstern blot: Compared with the control group, with C3 a concentration increases podocin, CD2 AP and nephrin protein expressed reduction, while FSP-1, α-SMA protein expression increased.(compared 0.01-0.1μM C3 a with control group;0.03-0.1μM C3 a with 0.1μM C3 a group)nephrin expressed decrease(P<0.05). With C3 a concentration increased podocin protein expressed decrease(P<0.05).(compared 0.03-0.1μM group with 0-0.01μM C3a;0.1μM C3 a with 0.03μM C3a)CD2AP expressd decrease(P <0.05).(compared 0.01-0.1μM C3 a with control group;0.1μM C3 a with 0.01-0.03μM C3a)FSP-1 expressed increase(P<0.05).(compared 0.01-0.1μM C3 a with control group;0.03-0.1μM C3 a with 0.01μM C3a)α-SMA expressed increase(P <0.05). Conclusions: 1,mature podocytes had transdifferentiation in vitro. 2 with the C3 a concentration increase,the nephrin, podocin, CD2 AP and synaptopodin expressed decreased, while FSP-1, α-SMA, vimentin, collagen I expressed upregulate. Complement fragment C3 a is involved in changing the phenotype of mice podocytes.