Epac ameliorates tubulointerstitial inflammation in diabetic nephropathy via C/EBPβ/SOCS3/STAT3 pathway

Abstract

Background: Tubulointerstitial inflammation plays a pivotal role in the progression of diabetic nephrology (DN), and tubular cells act as the driving force in the inflammation cascade. The aim of the study is to investigate whether activation of Epac alleviates tubulointerstitial inflammation through SOCS3/JAK/STAT3 pathway under DN. Methods: The kidney morphological changes and the level of oxidative stress were observed in db/db mice treated with Epac agonist (8-pCPT-2’-O-Me-cAMP). The expression of inflammatory cytokines, fibrosis markers, C/EBPβ, SOCS3 and p-STAT3 in renal tissues were assessed. In in vitro study, the changes of above genes and proteins were also detected in human proximal tubular cell line (HK-2) incubated with high glucose (HG) with and without Epac agonist. Overexpression of SOCS3 plasmid, SOCS3 siRNA and C/EBP-β siRNA were employed and the translocation of C/EBP-β and p-STAT3 were observed by immunofluorescence. Macrophage migration assay were detected in Transwell system. Results: In db/db mice, we observed that the expression of MCP-1 was up-regulated in renal tubular cells, which concurrent with increased influx of macrophages, cytokines production (MCP-1, TNF-α, and IL-6), and tubulointerstitial fibrosis. Moreover, we also found that these changes were accompanied by reduced C/EBP-β expression and increased SOCS3 and phosphorylated STAT3 (p-STAT3) expression. However, after treated db/db mice with 8-O-cAMP, an Epac activator, the expression of SOCS3 further up-regulated while other protein expression profile was partially reversed. In vitro , high glucose (30mM, HG) treatment down-regulated C/EBP-β expression while increased SOCS3, p-STAT3 and MCP-1 expression in HK-2 cells; Activation of Epac by 8-O-cAMP further increased the gene and protein expression of SOCS3 while reversed the changes of the other proteins. Mechanistically, under HG ambience, knockdown of C/EBP-β or SOCS3 in HK-2 cells partially blocked the inhibitory effect of Epac activation on MCP-1 expression. Furthermore, 8-O-cAMP treatment enhanced the nuclear-translocation of C/EBP-β and further increased the transcription of SOCS3 gene, which decreased MCP-1 expression through inhibiting the phosphorylation of STAT3 in HK-2 cells. Conclusions: These data indicate that Epac ameliorates tubulointerstitial inflammation in DN at least partially through C/EBPβ/SOCS3/STAT3 pathway. Therefore, Activation of Epac by 8-O-cAMP may be a novel therapeutic strategy for DN.