Background

Renal interstitial fibrosis (RIF) is a pathological change common to a variety of chronic renal diseases, ultimately progressing to end-stage renal failure. It is believed that epithelial cell phenotype inversion plays an important role in RIF, which is characterized by expression of the mesenchymal maker α-SMA, loss of the epithelial maker E-cadherin, and enhanced secretion of extracellular matrix. IL-17, a newly discovered pro-inflammatory cytokine, has recently been reported to play an important role in tissue fibrosis, involving pulmonary, liver, intestine and skin tissues. This study aimed to investigate whether IL-17A, a member of the IL-17 family, can induce epithelial cell phenotype inversion, and to explore the molecular mechanism of this phenotype inversion, in vitro.

Methods

HK-2 cells were cultured and incubated with IL-17A. Cell proliferation was measured by CCK-8 assay, and the secretion of types I and III collagen was detected by ELISA in dose-dependent and time-dependent experiments. To find out whether IL-17A can induce epithelial cell phenotype inversion, HK-2 cells were stimulated with 80 ng/mL of IL-17A and 10 ng/mL of TGF-β1 as a positive control, for 72 h. To explore the potential signaling pathway, anti-TGF-β1 antibody was added before IL-17A treatment. At the same time, anti-TGF-β1 antibody alone was added to the medium as the negative control group. The expression of types I and III collagen, α-SMA and E-cadherin proteins, and mRNA was measured by real-time PCR, western blotting and immuno-histochemistry.

Results

IL-17A promoted the proliferation of HK-2 cells and secretion of types I and III collagen in a dose-dependent and time-dependent manner. Compared with the normal control, IL-17A could stimulate the expression of α-SMA, types I and III collagen, and suppressed the expression of E-cadherin in HK-2 cells. Incubation of IL-17A with TGF-β1 antibody decreased significantly the expression of α-SMA, but increased the expression of E-cadherin in HK-2 cells.

Conclusion

Our results suggest that IL-17A might promote the proliferation of HK-2 cells and secretion of extracellular matrix, and induce epithelial cell phenotype inversion via a TGF-β1-dependent pathway. Blocking the pro-inflammatory cytokine IL-17A might be a potential target for the treatment of fibrotic kidney disease.

中文翻译：

---

促炎性白介素17A对HK-2细胞体外上皮细胞表型转化的影响。

背景

肾间质纤维化（RIF）是多种慢性肾脏疾病常见的病理变化，最终发展为终末期肾衰竭。相信上皮细胞表型倒置在RIF中起重要作用，其特征在于间充质制造者α- SMA的表达，上皮制造者E-钙黏着蛋白的损失以及细胞外基质的分泌增加。IL-17是一种新发现的促炎性细胞因子，最近据报道在涉及肺，肝，肠和皮肤组织的组织纤维化中起着重要作用。本研究的目的是调查IL-17A，该IL-17家族的一个成员是否可以诱导上皮细胞表型反转，并探讨该表型反转的分子机制，在体外。

方法

培养HK-2细胞，并与IL-17A一起孵育。通过CCK-8测定法测量细胞增殖，并在剂量依赖性和时间依赖性实验中通过ELISA检测I型和III型胶原的分泌。为了发现IL-17A是否可以诱导上皮细胞表型转化，以80 ng / mL的IL-17A和10 ng / mL的TGF-β1作为阳性对照刺激HK-2细胞72 h。为了探索潜在的信号通路，在IL-17A治疗之前加入了抗TGF-β1抗体。同时，将单独的抗TGF-β1抗体添加到培养基中作为阴性对照组。通过实时荧光定量PCR，蛋白质印迹和免疫组化检测I和III型胶原蛋白，α -SMA和E-cadherin蛋白以及mRNA的表达。

结果

IL-17A以剂量依赖和时间依赖的方式促进HK-2细胞的增殖以及I型和III型胶原的分泌。与正常对照组相比，IL-17A可以刺激HK-2细胞中α- SMA，I型和III型胶原蛋白的表达，并抑制E-钙黏着蛋白的表达。IL-17A与TGF-β1抗体一起孵育可显着降低HK-2细胞中α- SMA的表达，但可上调E-钙黏着蛋白的表达。

结论

我们的结果表明，IL-17A可能通过TGF-β1依赖性途径促进HK-2细胞的增殖和细胞外基质的分泌，并诱导上皮细胞表型转化。阻断促炎性细胞因子IL-17A可能是治疗纤维化肾病的潜在靶标。