Renal Unit, Department of Medicine, Verona University Hospital, Verona, Italy.
Nephrology and Dialysis Division, Columbus-Gemelli Hospital, Catholic University School of Medicine, Rome, Italy.
Nephrol Dial Transplant. 2017 Jul 1;32(7):1145-1154. doi: 10.1093/ndt/gfw403.
Epithelial-to-mesenchymal transition (EMT) of peritoneal mesothelial cells induced by high glucose (HG) levels is a major biological mechanism leading to myofibroblast accumulation in the omentum of patients on peritoneal dialysis (PD). Heparanase (HPSE), an endoglycosidase that cleaves heparan sulfate chains, is involved in the EMT of several cell lines, and may have a major role in this pro-fibrotic process potentially responsible for the failure of dialysis. Its specific inhibition may therefore plausibly minimize this pathological condition.
An in vitro study employing several biomolecular strategies was conducted to assess the role of HPSE in the HG-induced mesothelial EMT process, and to measure the effects of its specific inhibition by SST0001, a N-acetylated glycol-split heparin with a strong anti-HPSE activity. Rat mesothelial cells were grown for 6 days in HG (200 mM) culture medium with or without SST0001. Then EMT markers (VIM, α-SMA, TGF-β) and vascular endothelial growth factor (VEGF) (a factor involved in neoangiogenesis) were measured by real-time PCR and immunofluorescence/western blotting. As a functional analysis, trans-epithelial resistance (TER) and permeability to albumin were also measured in our in vitro model using a Millicell-ERS ohmmeter and a spectrophotometer, respectively.
Our results showed that 200 mM of glucose induced a significant gene and protein up-regulation of VEGF and all EMT markers after 6 days of culture. Intriguingly, adding SST0001 on day 3 reversed these biological and cellular effects. HPSE inhibition also restored the normal TER and permeability lost during the HG treatment.
Taken together, our data confirm that HG can induce EMT of mesothelial cells, and that HPSE plays a central part in this process. Our findings also suggest that pharmacological HPSE inhibition could prove a valuable therapeutic tool for minimizing fibrosis and avoiding a rapid decline in the efficacy of dialysis in patients on PD, though clinical studies and/or trials would be needed to confirm the clinical utility of this treatment.
高糖（HG）水平诱导的腹膜间皮细胞上皮-间充质转化（EMT）是导致腹膜透析（PD）患者网膜中肌成纤维细胞积累的主要生物学机制。肝素酶（HPSE）是一种内切糖苷酶，可裂解肝素硫酸链，参与几种细胞系的 EMT，并且可能在这种潜在导致透析失败的促纤维化过程中起主要作用。因此，其特异性抑制可能合理地最小化这种病理状况。
采用多种生物分子策略进行体外研究，以评估 HPSE 在 HG 诱导的间皮 EMT 过程中的作用，并测量其特异性抑制剂 SST0001 的作用，SST0001 是一种具有强抗 HPSE 活性的 N-乙酰化糖分裂肝素。将大鼠间皮细胞在 HG（200mM）培养基中培养 6 天，有或没有 SST0001。然后通过实时 PCR 和免疫荧光/蛋白质印迹测量 EMT 标志物（VIM、α-SMA、TGF-β）和血管内皮生长因子（VEGF）（参与新生血管形成的因子）。作为功能分析，还使用 Millicell-ERS 欧姆计和分光光度计分别在我们的体外模型中测量跨上皮电阻（TER）和白蛋白通透性。
我们的结果表明，200mM 的葡萄糖在培养 6 天后诱导 VEGF 和所有 EMT 标志物的基因和蛋白表达显著上调。有趣的是，在第 3 天添加 SST0001 逆转了这些生物学和细胞效应。HPSE 抑制也恢复了 HG 处理期间丢失的正常 TER 和通透性。
综上所述，我们的数据证实 HG 可以诱导间皮细胞 EMT，HPSE 在这个过程中起着核心作用。我们的发现还表明，药理学 HPSE 抑制可能是一种有价值的治疗工具，可以最小化纤维化并避免 PD 患者透析效果的迅速下降，尽管需要临床研究和/或试验来确认这种治疗的临床实用性。
