5-HT2 and 5-HT2B inhibitors mitigate fibrotic phenotype of peritoneal fibroblasts via non-canonical signaling in patients on continuous ambulatory peritoneal dialysis

Abstract

Background

Peritoneal fibrosis is a major cause of ultrafiltration failure in continuous ambulatory peritoneal dialysis (CAPD) patients · 5-hydroxytryptamine (5-HT) induces extracellular matrix synthesis in peritoneal fibroblasts in Transforming growth factor beta 1 (TGFβ1) dependent manner· We aimed to evaluate antifibrotic role of 5-HT2 and 5-HT2B inhibitors Terguride and SB 204741 respectively in human peritoneal fibroblasts (HPFB) isolated from omentum and parietal peritoneum of CAPD patients·

Methods

Omentum biopsy (OB) of control patients (n=6) undergone elective abdominal surgery and CAPD patients (n=4) was washed with sterile PBS and incubated in 0.125% trypsin/0.01% EDTA twice for 20 minutes followed by two times for 40 minutes at 37ºC · Biopsy from parietal peritoneum PB excised during laparotomy incubated overnight in dispase (2.4 U/mL)/37ºC· After centrifugation pellet obtained from OB and PB incubated in complete DMEM gave HPFB within 2 weeks · In first strategy HPFB from OB and PB were incubated with (5-HT-1µM) for 1 hr and later with (5-HT-1µM) and (Terguride ¸ SB 204741-1µM each) for 24 hr· In second strategy HPFB from OB and PB were pre-treated with (Terguride¸ SB 204741-1µM each) for 1 hr and later with only (5-HT-1µM) for 24 hr · Real time qPCR for pro-fibrotic (TGFβ1 ¸ Col1a1¸ Col1a2¸ ACTA2¸ CTGF and FN1) and anti-fibrotic genes (MMP2/TIMP1) expression was performed · Type I collagen and αSMA phosphorylation status of Smad3 and ERK1/2 was examined by immunoblotting·

Results

In 5-HT stimulated HPFB upregulated expression of profibrotic genes (p<0.05) mRNA at 24 hr was observed · Co-culture of HPFB with 5-HT2 and 5-HT2B receptor antagonists Terguride and SB 204741 significantly reduced pro-fibrotic genes expression (p<0.05) in both the strategies· Effect on anti-fibrotic genes mRNA in both the strategies was not affected· Pre-treatment with Terguride and SB 204741 decreased the production of type 1 collagen and αSMA significantly (p<0.05)· 5-HT dose dependently increased the mRNA levels of TGFβ1· Terguride and SB 204741did not influence Smad3 phosphorylation (canonical pathway) rather they significantly reduced ERK1/2 phosphorylation (noncanonical pathway) p<0.05 ·

Conclusion

　5-HT2 and 5-HT2B receptor antagonists reduce profibrotic mRNA expression in 5-HT stimulated HPFB by suppressing TGFβ1 mediated noncanonical pathway·