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The signal pathway for the repressive effect of dipyridamole on myofibroblast transdifferentiation

Renal fibrosis is the common final pathway of various chronic kidney diseases (CKDs), irrespective of the initial causes of nephropathy. Decline of renal function, glomerular filtration rate, has been exhibited to be highly associated with the progression of tubulointerstitial fibrosis.1 Fibrosis is considered to occur via a variety of events, which increase the level of transforming growth factor‐β1 (TGF‐β1), a profibrotic cytokine, in renal tissue. TGF‐β1 induces the emergence of myofibroblasts, myofibroblast transdifferentiation, followed by the accumulation of extracellular matrices such as type I collagen, ie, fibrosis.2 The myofibroblast is characterized by the expression of α‐smooth muscle actin (α‐SMA) in the cytoplasm. Thus, α‐SMA is generally accepted as a marker of fibrosis.3, 4, 5 Dipyridamole is in clinical use as an anti‐platelet agent. The reno‐protective effects exerted by dipyridamole have been demonstrated in animal models6 and humans with the early stage CKD.7 In almost all of these experiments and studies, the amelioration effects of dipyridamole on proteinuria were indicated to be mediated by the repression of platelet aggregation which was generally recognized in CKD. 
 
Dipyridamole increases cAMP levels by inhibiting phosphodiesterase in a variety of cells including platelets. The cAMP acts through three pathways of its downstream: protein kinase A (PKA), exchange factor directly activated by cAMP (Epac),8 and cyclic nucleotide‐gated (CNG) ion channels.9 In the Epac pathway, the increased intracellular cAMP binds to Epac, a guanine nucleotide exchange factor, followed by activation of the low molecular weight G‐protein Rap. Epac contributes to numerous pathophysiological processes, including proliferation, differentiation, cell adhesion, cell junction formation, and exocytosis. In the present study, the effects of dipyridamole against renal fibrosis, the late stage CKD, were investigated in a system without platelet aggregation. In addition, the mechanism by which dipyridamole exerted its effects was examined.

humans with the early stage CKD. 7 In almost all of these experiments and studies, the amelioration effects of dipyridamole on proteinuria were indicated to be mediated by the repression of platelet aggregation which was generally recognized in CKD.
Dipyridamole increases cAMP levels by inhibiting phosphodiesterase in a variety of cells including platelets. The cAMP acts through three pathways of its downstream: protein kinase A (PKA), exchange factor directly activated by cAMP (Epac), 8 and cyclic nucleotide-gated (CNG) ion channels. 9 In the Epac pathway, the increased intracellular cAMP binds to Epac, a guanine nucleotide exchange factor, followed by activation of the low molecular weight G-protein Rap. Epac contributes to numerous pathophysiological processes, including proliferation, differentiation, cell adhesion, cell junction formation, and exocytosis. In the present study, the effects of dipyridamole against renal fibrosis, the late stage CKD, were investigated in a system without platelet aggregation. In addition, the mechanism by which dipyridamole exerted its effects was examined.

| Immunofluorescence microscopy
In order to identify the cell type, a monolayer culture of the cells was grown on a two-well Labtek chamber slide (AS ONE, Osaka, Japan). Cell cultures were divided into three groups: the control, the 2 ng/mL TGF-β1, and the 2 ng/mL TGF-β1 + 3 μmol/L dipyridamole group. After treatments, the cells of each group were fixed in 4% paraformaldehyde and subjected to indirect immunofluorescence labelling for α-SMA. In brief, the cells were permeabilized with 0.5% Triton X-100 in PBS for 20 minutes, washed with PBS, and incubated with the primary antibody against α-SMA (Sigma-Aldrich, St Louis, MO, USA; 1:800 dilution) for 2 hours at room temperature (RT). After washing, they were incubated with FITC-conjugated goat anti-mouse IgG secondary antibody (Caltag Laboratories, Burlingame, CA, USA; 1:200 dilution) for 1 hour at RT, followed by DAPI nuclear staining. After staining, the cells were rinsed, observed, and photographed using an Olympus BX 60 microscope equipped with epifluorescence optics. As a negative control, the primary antibody was replaced with mouse nonimmune serum.

| Western blots
The cells were exposed to inhibitors of cAMP pathways, 5 μmol/L H89 or 3 μmol/L ESI-05, and activators of cAMP pathways, 30 μmol/ L Phe-cAMP or 10 μmol/L O-Me-cAMP for 1 hour before the addition of 2 ng/ml TGF-β1. They were exposed to 3 μmol/L dipyridamole for 30 minutes before a treatment of TGF-β1. They were cultured for 48 hours after TGF-β1 was added. Then cell lysates were prepared by adding ice cold protein extraction reagent (modified RIPA) with proteinase inhibitors. They (10 μg protein) were applied by 10% SDS-PAGE electrophoresis and blotted to a polyvinylidene difluoride filter membrane. The membranes were incubated with anti-α-SMA, or anti-GAPDH antibody. The membranes were then incubated with appropriate horseradish peroxidase-conjugated secondary antibodies. Immunoreactive bands were visualized by using the ECL system, and the intensity of the bands was measured by Image Quant TL (GE Healthcare, Buckinghamshire, UK).

| Quantitative real-time PCR
The cell lysates were subjected to RT-PCR for α-SMA at 24 hour after the addition of TGF-β1. Total RNA was isolated from the cells F I G U R E 1 Effect of dipyridamole on expression of α-SMA. After incubation with the reagents, the cells were subjected to immunofluorescence or Western blots for α-SMA. A, Immunofluorecence for α-SMA in the cells treated with or without TGF-β1 in the presence or absence of dipyridamole. Bar = 30 μm. B, The NRK49F cell lysates from three groups were subjected to immunoblot analysis with antibodies against α-SMA and GAPDH. The experiments were repeated three times, and one representative blot is shown. GAPDH reprobe is shown to demonstrate lane load. The molecular weights in kD are on the left. Bar graph shows relative α-SMA band densities normalized to GAPDH signal.*P < 0.01 vs control;**P < 0.05 vs TGF-β1 group YAN ET AL. | 1609

| EIA
The amount of intracellular cAMP at 1 hour after addition of dipyridamole was measured by using a cAMP enzyme immunoassay kit from Cayman Chemical according to the manufacturer's instructions.

| Statistics
Quantitative data were compared using Wilcoxon's test or Student's t test of the software package JMP (SAS Institute, Cary, NC, USA). P-values less than 0.05 were considered statistically significant.
F I G U R E 2 Contribution of cAMP pathways to suppressive effects of dipyridamole on α-SMA expression. A, Alteration of cAMP levels in the cytoplasm yielded by dipyridamole. After incubation the lysates of the cells were subjected to EIA of cAMP. Bar graph shows relative intracellular cAMP levels. *P < 0.01 vs control. B, Effect of the inhibitor of PKA or Epac pathway on α-SMA expression. After incubation with the reagents for 48 hours, the cell lysates were subjected to Western blots for α-SMA. Representative Western blots and relative quantification are provided. *P < 0.005 vs control;**P < 0.005 vs TGF-β1 group; NS not significant vs TGF-β1 + dipyridamole group; ***P < 0.01 vs TGF-β1 + dipyridamole group. C, Effect of the activator of PKA or Epac pathway on α-SMA expression. After incubation with the reagents the cell lysates were subjected to Western blots for α-SMA. Representative Western blots and relative quantification of α-SMA are shown. *P < 0.001 vs control; **P < 0.05; ***P < 0.001 vs TGF-β1 group. D, Effect of the inhibitor or the activator of Epac pathway on α-SMA mRNA expression. The cell lysates were subjected to RT-PCR. Bar graph shows relative values of α-SMA mRNA levels normalized to GAPDH signal. *P < 0.01 vs control; **P < 0.05 vs TGF-β1 group; ***P < 0.05 vs TGF-β1 + dipyridamole group

| Results and discussion
Immunofluorescence for α-SMA revealed that TGF-β1 induced, to a large extent, expression of α-SMA forming stress fibre ( Figure 1A).
Dipyridamole repressed TGF-β1-induced α-SMA expression by the normal range (almost none) as shown by immunofluorescence (Figure 1A) and Western blots for α-SMA ( Figure 1B). Dipyridamole suppressed TGF-β1-induced myofibroblast transdifferentiation in the in vitro system without platelets (Figure 1). To our knowledge, this is the first report that dipyridamole exerted protective effects against fibrosis corresponding to tubulointerstitial lesions in CKD.
Dipyridamole caused the elevation of intracellular cAMP levels (Figure 2A). To determine which pathway of cAMP was involved in the repressive effects of dipyridamole against fibrosis, the cells were exposed to inhibitors or activators of each pathway. H89, a selective PKA inhibitor, did not influence the suppressive action of dipyridamole against TGF-β1-induced α-SMA expression ( Figure 2B). Also, N 6 -Phenyl-cAMP, a PKA activator, exacerbated TGF-β1-stimu- both the PKA and Epac pathways suppressed fibrosis. 10 However, the PKA activator was shown to attenuate fibrosis yielded by exposure of pulmonary fibroblasts to TGF-β1, while the Epac activator did not affect it. 11 In contrast, it was demonstrated that the PKA activator induced fibrosis by mesangial cells. 12 The different effects between the results of the present study and the findings of these reports might be because of the difference in the cells used. The question of which process of the TGF-β1 signalling pathways did dipyridamole block via Epac pathway activation will be resolved in the next paper.
In conclusion, dipyridamole suppressed TGF-β1-induced α-SMA expression via Epac pathway activation. This protective effect of dipyridamole against fibrosis was not mediated by platelet aggregation and was directly exerted on fibroblasts. The Epac pathway provides a new pharmacological approach of dipyridamole for treatment of fibrosis in CKD.

CONFLI CT OF INTEREST
The authors confirm that there are no conflicts of interest.