Abstract
Objective: Podocytes play an important role in maintaining the structural integrity and function of the glomerular filtration barrier. Adriamycin (ADR) -induced podocyte injury has been extensively studied, however, its mechanism remains unclear. In the present study, we aimed to explore novel genes associated with podocyte injury induced by ADR.
Methods: A microarray assay was carried out on isolated podocytes with and without ADR treatment and analyzed using Genespring GX software. Two genes, Fos-like antigen 1 (FOSL1) and Regulator of G-protein signaling 2 (RGS2) were selected as candidate genes because their levels showed the most significant changes after ADR-induced podocyte injury. To confirm the changes in mRNA levels and their protein expression, ADR was added to cultured mouse podocytes and analyzed at 1, 2, 6, and 24 hours using real-time PCR and immunofluorescence. Finally, we also confirmed the expression of both proteins in vivo, by staining mouse kidney tissue at 0, 3, and 15 days after ADR injection.
Results: Microarray data showed that after ADR treatment, FOSL1 was up-regulated, while RGS2 was down-regulated. Real-time PCR analysis confirmed that ADR induced a significant increase of FOSL1 at 24 hours (p < 0.001, 24 h vs. control), and a marked down-regulation of RGS2 (p < 0.001, at each time point vs. control). Immunofluorescence analysis of podocytes treated with ADR for 24 hours showed an up-regulation tendency for FOSL1 protein and a down-regulation tendency for RGS2. On ADR-induced injury mice specimens, the expression of FOSL1 was increased in a time dependent manner after the injection; on the other hand, RGS2 was down-regulated, especially 15 days after injection.
Conclusion: FOSL1 and RGS2 were both expressed in podocytes, and were significantly regulated after ADR treatment, revealing a possible role in podocyte injury induced by ADR. These results could help guide research further to finally elucidate the underlying mechanisms in podocyte injury.
