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Peritoneal fibrosis results in ultrafiltration failure in peritoneal dialysis patients. 5-hydroxytryptamine (5-HT; Serotonin) strongly induces extracellular matrix synthesis in peritoneal fibroblasts in a Transforming growth factor beta 1 (TGF-β1) dependent manner. We aimed to evaluate anti-fibrotic role of inhibitors of 5-HT2 and 5-HT2B (Terguride and SB204741), respectively in human peritoneal fibroblasts (HPFBs) isolated from peritoneum of PD patients.
In 5-HT/TGF-β1 stimulated HPFBs, upregulated pro-fibrotic gene expression was observed, which significantly reduced on co-culture with 5-HT2/5-HT2B inhibitors, with no effect on anti-fibrotic genes mRNA expression as well as in western blot. In 5-HT stimulated HPFBs, treatment with both 5-HT inhibitors decreased type 1 collagen and α-SMA. All the proinflammatory cytokines were upregulated in the serum sample as well as in the PBMCs of the patients as compared to control. In both pre treatment and post treatment strategy the profibrotic genes were downregulated upon treatment (p<0.001).Biopsy from parietal peritoneum (PB) of control patients (n=8) and PD patients (n=13) excised during laparotomy was incubated overnight in dispase (2.4 U/mL)/37°C. In post-treatment strategy, cells were incubated with 5-HT (1µM) for 1 hr and later with 5-HT (1µM) and terguride or SB204741 (1µM, each) for 24 hr. In pre-treatment strategy, cells were pre-treated with terguride or SB204741 (1µM, each) for 1 hr and later with only 5-HT (1µM) for 24 hr. HPFBs were also incubated with TGF-β1 (10ng/ml) and 5-HT inhibitors similar to the above strategies. Real time quantitative PCR for pro-fibrotic (TGF-Β1, COL1A1, COL1A2, ACTA2, CTGF and FN1) and anti-fibrotic genes (MMP2/TIMP1) expression was performed. Type 1 collagen and alpha smooth muscle actin (α-SMA) was performed by immunoblotting. The proinflammatory markers like IL-6, MCP-1, IL1-b, IL-17 was measured by ELISA and Flowcytometry.
In 5-HT/TGF-β1 stimulated HPFBs, upregulated pro-fibrotic gene expression(p<0.0001) was observed, which significantly reduced on co-culture with 5-HT2/5-HT2B inhibitors(p<0.0001), with no effect on anti-fibrotic genes mRNA expression as well as in western blot. In 5-HT stimulated HPFBs, treatment with both 5-HT inhibitors decreased type 1 collagen(p<0.001) and α-SMA(p<0.001). All the proinflammatory cytokines were upregulated in the serum sample as well as in the PBMCs of the patients as compared to control (p<0.001). In both pretreatment and post treatment strategy the profibrotic genes were downregulated upon treatment (p<0.001).
5-HT receptor antagonists attenuate 5-HT/TGF-β1 mediated pro-fibrotic potential of HPFBs by attenuating the production of ECM proteins and alpha smooth muscle actin in HPFBs.