Background

Urinary extracellular vesicles (EVs) have gained increasing interest in recent years as a potential source of noninvasive biomarkers of diseases related to urinary organs, but knowledge of the mechanism is still limited. The current study sought to clarify the mechanism of urinary EVs behind di-(2-ethylhexyl) phthalate (DEHP)-induced hypospadias via PFN2 delivery.

Method

PFN2 expression in hypospadias was predicted by bioinformatics analysis. Following the induction of a hypospadias rat model using DEHP, rats were injected with EVs and/or underwent alteration of PFN2 and TGF-β1 to assess their effects in vivo. The extracted rat urothelial cells (UECs) were co-cultured with EVs extracted from urine for in vitro experiments.

Result

Microarray analysis predicted poor PFN2 expression in hypospadias. Upregulated PFN2 was found in urinary EVs, and restrained epithelial-mesenchymal transition (EMT) was observed in DEHP-exposed rats. Urinary EVs or PFN2 overexpression increased SMAD2, SMAD3, and TGF-β1 protein expression and SMAD2 and SMAD3 phosphorylation in UECs and DEHP-exposed rats. UEC migration, invasion, and EMT were augmented by EV co-culture or upregulation of PFN2. Of note, the silencing of TGF-β1 counterweighed the effect of PFN2. Besides, EV co-culture or overexpression of PFN2 or TGF-β1 elevated the body weight, anal–genital distance (AGD), anal–genital index (AGI), and EMT of DEHP-exposed rats.

Conclusion

In summary, urinary EVs activated the SMAD/TGF-β1 pathway to induce EMT via PFN2 delivery, thus protecting against DEHP-induced hypospadias.

Graphical Abstract

(1) EMT in epithelial cells inhibits DEHP-induced hypospadias.

(2) Urine-derived EVs deliver PFN2 to promote EMT in epithelial cells.

(3) PFN2 can activate the SMAD/TGF-β1 signaling axis.

(4) Urine-derived EVs can transmit PFN2 to activate the SMAD/TGF-β1 signaling axis, thus promoting EMT and inhibiting the occurrence of hypospadias.

中文翻译：

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尿细胞外囊泡通过 PFN2 递送促进上皮-间质转化，预防邻苯二甲酸二（2-乙基己基）诱发的尿道下裂

背景

近年来，泌尿细胞外囊泡（EV）作为泌尿器官相关疾病的非侵入性生物标志物的潜在来源引起了越来越多的关注，但对其机制的了解仍然有限。目前的研究试图阐明邻苯二甲酸二(2-乙基己基)酯 (DEHP) 通过 PFN2 传递诱发尿道下裂的尿 EV 背后的机制。

方法

通过生物信息学分析预测尿道下裂中 PFN2 的表达。使用 DEHP 诱导尿道下裂大鼠模型后，给大鼠注射 EV 和/或进行 PFN2 和 TGF-β1 的改变，以评估它们的体内效果。提取的大鼠尿路上皮细胞（UEC）与从尿液中提取的EV共培养用于体外实验。

结果

微阵列分析预测尿道下裂中 PFN2 表达较差。在尿液 EV 中发现 PFN2 上调，并且在暴露于 DEHP 的大鼠中观察到上皮间质转化 (EMT) 受到抑制。在UEC和暴露于DEHP的大鼠中，尿液EV或PFN2过度表达增加了SMAD2、SMAD3和TGF-β1蛋白表达以及SMAD2和SMAD3磷酸化。EV 共培养或 PFN2 上调增强了 UEC 迁移、侵袭和 EMT。值得注意的是，TGF-β1 的沉默抵消了 PFN2 的作用。此外，EV共培养或PFN2或TGF-β1的过度表达升高了DEHP暴露大鼠的体重、肛门-生殖器距离（AGD）、肛门-生殖器指数（AGI）和EMT。

结论

总之，尿液 EV 激活 SMAD/TGF-β1 通路，通过 PFN2 递送诱导 EMT，从而预防 DEHP 诱导的尿道下裂。

图形概要

(1)上皮细胞EMT抑制DEHP诱导的尿道下裂。

(2) 尿源性 EV 递送 PFN2 以促进上皮细胞的 EMT。

(3)PFN2可以激活SMAD/TGF-β1信号轴。

(4)尿源性EVs可传递PFN2激活SMAD/TGF-β1信号轴，从而促进EMT，抑制尿道下裂的发生。