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Effects of stromal interaction molecule 1 or Orai1 overexpression on the associated proteins and permeability of
Li Miao1,2, Dongyue Wei2, Yuanyuan Zhang1
1Nanjing Children's Hospital Affiliated to Nanjing Medical University, Nanjing, China.
Aim:
The present study was conducted to determine whether two important signalling molecules of store-operated channel (SOC), stromal interaction molecule 1 (STIM1) and Orai1, were involved in glomerular podocyte injury. We explored the effects of STIM1/Orai1 overexpression on podocyte associated proteins and cell permeability.
Methods:
The expression of STIM1 and Orai1 were examined in the renal cortex of adriamycin-induced nephropathy mice by real-time RT-PCR. The recombinant plasmid of STIM1/Orai1, identified by restriction enzyme digestion and PCR, was transfected into MPC5 cells via lipofectamine 2000. The transfecting efficiency was observed by a fluorescence microscope. RT-PCR and Western blotting were used to evaluate the expression levels of STIM1, Orai 1 and some podocyte-associated molecules in the transfected MPC5 cells. In addition, we examined the diffusion of FITC-dextran across the podocyte monolayer to investigate whether STIM1/Orai1 overexpression could affect cell permeability.
Results:
We found that the mRNA levels of STIM1 and Orai1 were increased in adriamycin-induced nephropathy mice. STIM1/Orai1 overexpression significantly decreased the expression of podocin and CD2-associated protein (CD2AP), whereas it increased the expression of α-actinin-4. The permeability was significantly increased in the STIM1/Orai1 overexpression group.
Conclusion:
Our findings suggested that STIM1/Orai1 overexpression could affect the cell permeability and the expression of partial podocyte-associated proteins, which may ultimately result in podocyte injury.
Insights
Stromal interaction molecule 1 (STIM1) and Orai1 overexpression increased kidney cell permeability and altered proteins, suggesting a role in podocyte injury.
Area of Science:
- Nephrology
- Cell Biology
- Molecular Biology
Background:
- Glomerular podocyte injury is a key factor in kidney disease progression.
- Store-operated channel (SOC) proteins, including stromal interaction molecule 1 (STIM1) and Orai1, are crucial for calcium signaling.
- The specific role of STIM1 and Orai1 in podocyte injury remains to be fully elucidated.
Purpose of the Study:
- To investigate the involvement of STIM1 and Orai1 in glomerular podocyte injury.
- To explore the effects of STIM1/Orai1 overexpression on podocyte-associated proteins and cell permeability.
Main Methods:
- Examined STIM1 and Orai1 mRNA levels in adriamycin-induced nephropathy mice using real-time RT-PCR.
- Transfected MPC5 cells with STIM1/Orai1 recombinant plasmid and assessed expression via RT-PCR and Western blotting.
- Evaluated FITC-dextran diffusion across podocyte monolayers to determine cell permeability changes.
Main Results:
- STIM1 and Orai1 mRNA levels were elevated in adriamycin-induced nephropathy mice.
- Overexpression of STIM1/Orai1 decreased podocin and CD2-associated protein (CD2AP) expression.
- STIM1/Orai1 overexpression led to increased α-actinin-4 expression and significantly elevated cell permeability.
Conclusions:
- STIM1/Orai1 overexpression impacts cell permeability and alters key podocyte-associated proteins.
- These changes suggest that STIM1 and Orai1 play a significant role in the pathogenesis of podocyte injury.
