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[Establishment of a podocyte cell injury model induced by puromycin aminonucleoside]
Shu Fang Liu1, Jie Ding, Qing Feng Fan
1Department of Pediatrics, Peking University First Hospital, Beijing, China.
Objective:
To establish a podocyte cell injury model induced by puromycin aminonucleoside (PAN), an in vitro model for studying the role of podocytes, especial the slit diaphragm molecules in proteinuria at the cellular and molecular levels.
Methods:
MPC5 were treated for 24 and 48 hours by 15, 45 and 75 mg/L PAN, respectively. The podocyte molecular behavior during podocyte injury was evaluated: the apoptotic podocyte cells were revealed with FITC-Annexin V and Propidium Iodide (PI) assay and the proliferative podocyte cells detected with MTT assay after PAN treatment. The distribution of Nephrin and Podocin was revealed with indirect-immunofluorescent staining under confocal microscope. The distribution of F-actin was revealed with direct-immunofluorescent staining under microscope.
Results:
The percentage of apoptotic podocyte cells was increased in a dose- and time-dependent manner after PAN treatment. In PAN-treated group, the apoptosis was obviously increased at hour 48, the PAN-45 treated group was 33.48% +/- 14.55% and PAN-75 treated group 38.01% +/- 12.13% vs the control group 6.38% +/- 0.50% (P<0.01).
Conclusion:
We set up an in vitro podocyte injury model treated with PAN for the first time. This reliable cell model is a good basis for further studies on podocyte injury.
Insights
This study establishes a novel in vitro podocyte cell injury model using puromycin aminonucleoside (PAN). This reliable model aids in studying podocyte injury and proteinuria at the molecular level.
Area of Science:
- Nephrology
- Cell Biology
- Molecular Biology
Context:
- Podocyte injury is a key factor in proteinuria.
- Existing in vitro models have limitations in studying podocyte slit diaphragm molecules.
Purpose:
- To establish a reliable in vitro podocyte cell injury model induced by puromycin aminonucleoside (PAN).
- To investigate the cellular and molecular mechanisms of podocyte injury and proteinuria.
Summary:
- MPC5 cells were treated with varying concentrations of PAN for 24 and 48 hours.
- Apoptosis, proliferation, and the distribution of key podocyte proteins (Nephrin, Podocin, F-actin) were assessed.
- PAN treatment significantly increased podocyte apoptosis in a dose- and time-dependent manner.
Impact:
- This study presents the first successful in vitro podocyte injury model using PAN.
- The established model provides a robust platform for future research into podocyte dysfunction and therapeutic strategies for proteinuria.
