Activation of Epac by 8-O-cAMP improves renal function and pathologic changes in the kidneys of db/db mice.
To test the role of Epac in the tubular damage in the kidney of db/db mouse, we treated db/db mice with Epac specific activator 8-pCPT-2’-O-Me-cAMP (8-O-cAMP) by intraperitoneal injection for 8 weeks. Db/db mice exhibited higher body weights, increased blood glucose, serum creatinine and urine albumin levels compared to db/m mice (Fig. 1a-d). Slightly but non-significantly increase in body weight was seen in db/db mice treated with 8-O-cAMP. By contrast, notable decreases in blood glucose (Fig. 1b), serum creatinine (Fig. 1c) and urine albumin levels (Fig. 1d) were observed in db/db mice after 8-O-cAMP treatment.
HE and PAS staining revealed mesangial expansion and tubular atrophy in the kidneys of db/db mice compared to db/m mice (Fig. 1e); Masson’s trichrome staining showed increased interstitial fibrosis in the kidney of db/db mice (Fig. 1e). However, Activation of Epac with 8-O-cAMP dramatically ameliorated these abnormal changes (Fig. 1e). Quantitative analysis of tubular and glomerular damage score confirmed this tendency (Fig. 1f, g). These data indicated that Epac activator 8-O-cAMP may exert a therapeutic role in the diabetic kidney injury.
Activation of Epac alleviates the inflammation response, oxidative stress, and interstitial fibrosis in the kidney of db/db mice.
Inflammation and oxidative stress contribute critically to the initiation and extension of DN. We therefore evaluated renal inflammatory cytokines and reactive oxidative stress (ROS) production the kidneys of db/db mice receiving 8-O-cAMP treatment. By IHC and Western blot assay, TNF-α, IL-6 expression was found to be up-regulated in the tubules of db/db mice compared to those of controls (Fig. 2a (e, f vs b, a)); DHE staining demonstrated increased ROS production in the tubular cells of the kidneys of db/db mice (Fig. 2b (b vs a)). Finally, the expression of FN and Col-1 were also detected, as one can see, a notable increase in FN and Col-1 expression in the tubulointerstitium of db/db mice (Fig. 2a (g, h vs c, d)) and similar result was demonstrated by Western blot assays (Fig. 2c). Of note, these above abnormal changes were reversed after treated with 8-O-cAMP (Fig. 2a (i, j, k, l) and Fig. 2c). These results provided compelling evidence supporting a renoprotective role of activation of Epac in DN.
Activation of Epac attenuates renal macrophage infiltration in db/db mice.
To further understand the role of Epac agonist 8-O-cAMP in the inflammation response in the kidney of db/db mice, we detect the expression of macrophage chemotactic factor MCP-1 and macrophage accumulation in the kidney. IHC assay showed that the expression of MCP-1 significantly increased in the tubules of db/db mice compared to the db/m mice (Fig. 3a (b vs a)). Similar results of MCP-1 protein and gene expression were demonstrated by western blot assays (Fig. 3b) and qPCR (Fig. 3c), respectively. Moreover, immunofluorescence staining results showed that the expression of F4/80 was significantly up‐regulated in in renal interstitium of db/db mice, indicating the increased macrophage infiltration, which were significantly inhibited by 8-O-cAMP treatment (Fig. 3a and a2). Besides, M1 macrophages promote the inflammation in kidney of DN. Next, we detected the effect of 8-O-cAMP on the changes of macrophages polarity in kidney of DN by iNOS staining. The result showed that 8-O-cAMP significantly reduced the expression of iNOS (Marker of M1 macrophage) in macrophages (Fig.3a and a3) in db/db mice. These above results indicated that activation of Epac may exert anti‐inflammatory effects by reducing macrophage accumulation and activation.
Activation of Epac increases C/EBP-β, SOCS3 expression while blunts STAT3 activation.
The transcription factor, C/EBP-β, has been reported to bind directly to the SOCS3 promoter region(33). Here, we found that the expression of C/EBP-β in db/db mice was significantly reduced compared with db/m mice (Fig. 4a (b vs a)). Interestingly, the expression C/EBP-β was significantly increased by the treatment of 8-O-cAMP (Fig. 4a (c) ). Considering that the MCP-1 is a classical STAT-responsive inflammatory gene and SOCS3 is known as a negative regulator of STAT3 signaling pathway. Next, we examined the effect of 8-O-cAMP on the expression of SOCS3 and p-STAT (Tyr705). Immunohistochemistry revealed a lower level of SOCS3 and p-STAT3 expression in db/m mice (Fig. 4a (d, g)), and broad distribution of them in db/db mice (Fig. 4a (e, h)). Moreover, activation of Epac with 8-O-cAMP further up-regulated SOCS3 expression (Fig. 4a (f)), while inhibited p-STAT3 expression in db/db mice (Fig. 4a (i)). Similar results were observed with respect to expression of C/EBP-β, SOCS3 and p-STAT3, as detected by western blot assays (Fig. 4c). These data suggested that activation of Epac might increase SOCS3 expression and inhibit STAT3/MCP-1 pathway by recovering C/EBP-β expression.
Sequential changes in expression of SOCS3/STAT3/MCP-1 in HK-2 cells exposed to high glucose.
Western blot assays and real-time PCR demonstrated that HG (30 mM, D-glucose)-induced the mRNA and protein expression levels of SOCS3 was highest at 8h and then gradually decreased to normal level (Fig. 5a and b). However, the protein expression of p-STAT3 was increased in a time-dependent manner, peaking at 24-48h following treatment with HG (Fig. 5a1).In line with this, a time-dependent increase in the mRNA and protein expression of MCP-1 was observed and was maximal at 48h (Fig. 5a and c).
Activation of Epac reduces MCP-1 expression and decreases macrophage recruitment via SOCS3.
In order to confirm the effect of Epac on p-STAT expression in HK-2 cells expose to HG. HK-2 cells were pretreated with 8-O-cAMP (100μM) for 5 hours before treated with HG. Immunofluorescence assay revealed that the p-STAT3 expression was quite low in HK-2 cells under basal conditions (5.6 mM D-glucose) and HG treatment resulted in the activation of STAT3 (Fig. 6a (a, b) and b), while it was largely reduced by the treatment with 8-O-cAMP (Fig. 6a (c) and b).
To further determine whether the inhibitory effect of Epac on the STAT3 activation was relied on SOCS3, we transfected HK-2 cells with SOCS3 siRNA contaminant with 8-O-cAMP treatment. As shown in Figure 6B, western blot results showed the inhibitory effect of Epac activation on p-STAT3 and MCP-1 expression was abolished by knockdown SOCS3 expression in HK-2 cells exposed to HG ambience. Likewise, we also observed that HG-induced increased p-STAT3 and MCP-1 expression in HK-2 cells were significantly reduced by SOCS3 overexpression, suggesting modulation of SOCS3 expression is sufficient to mimic the activation of Epac. Real-time PCR demonstrated the mRNA expression of SOCS3 and MCP-1 was in accordance with the protein expression. (Fig. 6c and d). These results suggested that Epac-induced SOCS3 expression reduced MCP-1 expression by preventing the STAT phosphorylation in HK-2 cells under HG condition.
Furthermore, macrophage migration assay was used to demonstrate activation of Epac-reduced MCP-1 expression can effectively decrease the recruitment of macrophages. The number of THP-1 cells migrated from the upper side to the bottom side of the membrane were increased by adding the hyperglycemia-stimulated HK-2 cells supernatant (Fig. 6f (b)).While supernatant from HK-2 cells treated with 8-O-cAMP under HG condition, the number of migrated-macrophages were significantly decreased (Fig. 6f (c)).
Activation of Epac increases SOCS3 expression via C/EBP-β.
By IF staining, C/EBP-β was found to be highly expressed in the nuclear in HK-2 cells under basal conditions (Fig. 7a (a)). The HG exposure reduced nuclear fluorescence of C/EBP-β (Fig. 7a (b)), which was restored by 8-O-cAMP treatment (Fig. 7a (c)). The transcription factor, C/EBP-β, has been reported to bind directly to the SOCS3 promoter region(33), we next determined whether Epac modulates the expression of SOCS3 by C/EBP-β. HK-2 cells were transfected with C/EBP-β siRNA before HG treatment. Western blot analysis showed that the expression of C/EBP-β significantly decreased in HK-2 cells exposure to HG, and activation of Epac by 8-O-cAMP restored C/EBP-β and SOCS3 expression, while this effect was neutralized by C/EBP-β siRNA transfection (Fig. 7b). Similar results of SOCS3 mRNA expression were demonstrated using real-time PCR (Fig. 7c). These data confirmed the role of C/EBP-β in controlling the Epac-dependent induction of the SOCS3 gene in HK-2 cells.