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Comparative Proteomic Analysis of Rapamycin Versus Cyclosporine Combination Treatment in Mouse Podocytes
1Division of Nephrology, Department of Internal Medicine, Severance Hospital, Yonsei University Health System, Seoul, Republic of Korea; The Research Institute for Transplantation, Yonsei University College of Medicine, Seoul, Republic of Korea.
Background:
The mechanism of podocyte injury observed with the use of rapamycin (RPM) remains unclear. The conversion from calcineurin inhibitors (CNIs) to RPM in kidney transplant recipients has been associated with a higher incidence of proteinuria and renal injury. In this study, we performed proteomic analyses to investigate the alteration of protein expression in mouse podocytes treated with RPM in comparison with CNI/RPM combination.
Methods:
Immortalized mouse podocytes were treated with 20 nmol/L RPM or 20 nmol/L RPM + 1 μg/mL cyclosporine. Podocyte proteins were separated by 2-dimensional polyacrylamide gel electrophoresis (2DE) and identified by matrix-assisted laser desorption time-of-flight (MALDI-TOF) mass spectrometry and peptide fingerprinting. Selected proteins were analyzed by means of Western blot assay.
Results:
We identified 36 differently expressed proteins after isolated RPM or CNI/RPM combination treatment in cultured mouse podocytes. There are 3 distinct patterns of protein expression: (1) potentiated down- or upregulation of proteins by CNI/RPM treatment compared with isolated RPM treatment (n = 4); (2) partial offset of down-regulation by CNI/RPM in comparison with RPM treatment (n = 25); (3) no difference in down-regulation between RPM and CNI/RPM treatment (n = 5). We found a significant interplay between RPM and CNI on the expression of the selected proteins in mouse podocytes. This might explain the higher incidence of proteinuria by CNI/RPM combination in clinical settings.
Conclusions:
Further study is required to elucidate the target protein associated with RPM-induced podocyte injury.
Insights
Rapamycin (RPM) and calcineurin inhibitors (CNIs) affect kidney podocyte protein expression differently. Combining RPM and CNIs alters protein expression, potentially explaining increased proteinuria in transplant patients.
Area of Science:
- Nephrology
- Proteomics
- Immunology
Background:
- The mechanism of rapamycin-induced podocyte injury is not fully understood.
- Conversion to rapamycin (RPM) from calcineurin inhibitors (CNIs) in kidney transplant recipients correlates with increased proteinuria and renal injury.
Purpose of the Study:
- To investigate alterations in mouse podocyte protein expression following treatment with RPM alone versus a combination of CNI and RPM.
Main Methods:
- Cultured mouse podocytes were treated with RPM or RPM plus cyclosporine (a CNI).
- Proteins were analyzed using 2D-polyacrylamide gel electrophoresis, MALDI-TOF mass spectrometry, and Western blotting.
Main Results:
- 36 differentially expressed proteins were identified in response to RPM or CNI/RPM treatment.
- Distinct patterns of protein expression changes were observed, with significant interplay between RPM and CNI.
- These findings suggest a molecular basis for the increased proteinuria seen with combined CNI/RPM therapy.
Conclusions:
- The study highlights a significant interaction between RPM and CNIs affecting podocyte protein expression.
- Further research is needed to identify specific proteins involved in RPM-induced podocyte injury.
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