NOVEL DUAL-TARGET APPROACH TO COMBAT PERITONEAL DIALYSIS-ASSOCIATED FIBROSIS: SYNERGISTIC INHIBITION OF PDE5 AND 5-HT2B RECEPTORS

Peritoneal fibrosis (PF) is a leading cause of ultrafiltration failure in patients undergoing peritoneal dialysis (PD). Among the mediators implicated in PF, serotonin (5-hydroxytryptamine, 5-HT) has been shown to promote extracellular matrix (ECM) synthesis in fibroblasts through a transforming growth factor-beta 1 (TGF-β1)-dependent mechanism. TGF-β1 plays a pivotal role in activating resident fibroblasts and driving their transdifferentiation into myofibroblasts—cells distinguished by increased expression of alpha-smooth muscle actin (α-SMA) and excessive ECM deposition. These activated myofibroblasts are central contributors to the development and progression of PF. Sildenafil, a selective phosphodiesterase-5 (PDE5) inhibitor, has been reported to exhibit anti-fibrotic effects in conditions such as systemic sclerosis. In the present study, we explore the synergistic anti-fibrotic potential of Sildenafil and SB204741, a selective 5-HT2B receptor antagonist, using human peritoneal fibroblasts (HPFBs) derived from parietal peritoneal biopsies of PD patients.

Parietal peritoneal biopsies (PB) were obtained from control subjects (n=15) and PD patients (n=15) during laparotomy and incubated overnight with dispase (2.4 U/mL) at 37°C to isolate human peritoneal fibroblasts (HPFBs). Two treatment strategies were employed, Post-treatment strategy – HPFBs were first stimulated with TGF-β1 (10 ng/mL) for 1 hour, followed by treatment with TGF-β1 (10 ng/mL) alone or in combination with Sildenafil (10 µM), SB204741 (1 µM), or both, for 24 hours. Pre-treatment strategy – HPFBs were pre-treated with Sildenafil, SB204741, or their combination for 1 hour, followed by exposure to TGF-β1 (10 ng/mL) for 24 hours. Gene expression analysis of pro-fibrotic markers (COL1A1, COL1A2, ACTA2, CTGF, FN1, TGFβ1, HSPG2, KCNMA1, KRT19) and anti-fibrotic markers (MMP2, MMP7, MMP9, MMP10, TIMP1) was performed using real-time PCR. Cytokine levels in the culture supernatants—including pro-inflammatory (IFN-γ, IL-4, IL-17, IL-1β, IL-6, TNF-α) and anti-inflammatory (IL-10) cytokines—were quantified by ELISA. Protein expression of type I collagen and α-SMA was assessed by immunoblotting. Further we differentiated the naïve CD4 T cells in the presence of serum from the patients and the control.

In this study, the combination of Sildenafil and SB204741 demonstrated a superior anti-fibrotic effect compared to either agent alone. TGF-β1 stimulation of HPFBs significantly upregulated the expression of pro-fibrotic genes. Treatment with the drug combination markedly reduced this expression, with near-complete suppression of ACTA2 (p<0.01). The MMP/TIMP ratio, indicative of anti-fibrotic activity, was restored to near-normal levels (p<0.01). Protein expression of α-SMA and type I collagen was significantly reduced (Table 1). Additionally, the combination therapy led to a decrease in pro-inflammatory cytokine production (p<0.05) and a concomitant increase in the anti-inflammatory cytokine IL-10 (p<0.01) (Figure 1). The Th2 phenotype was observed significantly higher in patients serum as compared to control serum (p<0.001).

The combination of Sildenafil and SB204741 may offer therapeutic potential for treating peritoneal fibrosis during its active phase by effectively preventing the transdifferentiation of resident fibroblasts into myofibroblasts.