Abstract: Objectives Transforming growth factor- 1 (TGF- 1) is one of the key mediators of the peritoneal fibrosis. Short hairpin RNAs (shRNA) transcribed by vectors contained U6 or H1 promoter can trigger sequence-selective gene silencing in mammalian cells. This study investigated the effects of shRNA targeting TGF- 1 on the expression of TGF- 1 and ultrastructure in human peritoneal mesohelial cells (HPMCs) induced by peritoneal dialysis solution (PD solution). Methods All tests were performed on the human peritoneal mesothelial cell (HMrSV5). TGF- 1 specific shRNA expression vectors were constructed and introduced to HPMCs stimulated with two commercially available PD solution (1.50% and 4.25% Dianeal) or DMEM to 48h. The dialysis solutions were diluted twofold with DMEM. Expression of TGF- 1 mRNA was detected by semiquantitative reverse transcription polymerase chain reaction (RT-PCR). The TGF- 1 protein level in the culture supernatant was analyzed by enzyme-linked immunosorbent assay. Ultrastructure changes of HPMCs were observed by transmission electron microscopy. Results The expression of TGF- 1 was upregulated significantly in HPMCs stimulated with 4.25% PD solution (P＜0.01). TGF- 1 expression in pcDU6 plasmid vector-mediated shRNA group were obviously downregulated when compared to the 4.25% PD solution group and the pcDU6 void vector group (P＜0.01), with no significant difference among pcDU6 plasmid vector-mediated shRNA groups (P＜0.01). Ultrastructural studies of HPMCs exposed to 4.25% PD solution showed cell flattening, reduced microvilli, and intracellular organelles compatible with dysfunctional mitochondria. In contrast, the ultrastructural morphology of HPMCs was relatively preserved after introduction with TGF- 1 shRNA vectors. Conclusions TGF- 1 specific shRNA can significantly inhibit the expresson of TGF- 1 and ameliorates the ultrastructural abnormalities in HPMCs stimulated with 4.25% PD solution. Our results of this in vitro study suggest that TGF- 1 specific shRNA expression vectors transfer might be of the therapeutic benefit in peritoneal fibrosis. However, further studies in vivo are needed to confirm this hypothesis.

Key words: RNAi, peritoneal dialysate, Human peritoneal mesothelial cells