Abstract
Amphiregulin (AREG) is a transmembrane glycoprotein recently implicated in kidney fibrosis. Previously, we reported that the AREG-targeting Self-Assembled-Micelle inhibitory RNA (SAMiRNA-AREG) alleviated fibrosis by stably silencing the AREG gene, and reduced the side effects of conventional siRNA treatment of pulmonary fibrosis. However, the therapeutic effect of SAMiRNA-AREG in renal fibrosis has not been studied until now. We used two animal models of renal fibrosis generated by a unilateral ureteral obstruction (UUO) and an adenine diet (AD) to investigate whether SAMiRNA-AREG inhibited renal fibrosis. To investigate the delivery of SAMiRNA-AREG to the kidney, Cy5-labeled SAMiRNA-AREG was injected into UUO- and AD-induced renal fibrosis models. In both kidney disease models, SAMiRNA-AREG was delivered primarily to the damaged kidney. We also confirmed the protective effect of SAMiRNA-AREG in renal fibrosis models. SAMiRNA-AREG markedly decreased the UUO- and AD-induced AREG mRNA expression. Furthermore, the mRNA expression of fibrosis markers, including α-smooth muscle actin, fibronectin, α1(I) collagen, and α1(III) collagen in the UUO and AD-induced kidneys, was diminished in the SAMiRNA-AREG-treated mice. The transcription of inflammatory markers (tumor necrosis factor-α and monocyte chemoattractant protein-1) and adhesion markers (vascular cell adhesion molecule 1 and intercellular adhesion molecule 1) was attenuated. The hematoxylin and eosin, Masson’s trichrome, and immunohistochemical staining results showed that SAMiRNA-AREG decreased renal fibrosis, AREG expression, and epidermal growth factor receptor (EGFR) phosphorylation in the UUO- and AD-induced models. Moreover, we studied the effects of SAMiRNA-AREG in response to TGF-β1 in mouse and human proximal tubule cells, and mouse fibroblasts. TGF-β1-induced extracellular matrix production and myofibroblast differentiation were attenuated by SAMiRNA-AREG. Finally, we confirmed that upregulated AREG in the UUO or AD models was mainly localized in the distal tubules. In conclusion, SAMiRNA-AREG represents a novel siRNA therapeutic for renal fibrosis by suppressing EGFR signals.
Highlights
The prevalence of chronic kidney disease (CKD) is increasing worldwide
We investigated the mRNA expression of adhesion molecules, vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1), which was dramatically increased on day 4 in the ureteral obstruction (UUO) kidneys compared to the sham kidneys (Fig. 1e)
We found that α-smooth muscle actin (α-SMA) was upregulated in myofibroblasts from the peritubular and the interstitial regions of UUO kidneys compared to those from mice treated with Self-Assembled-Micelle inhibitory RNA (SAMiRNA)-AREG (Fig. 3c)
Summary
The prevalence of chronic kidney disease (CKD) is increasing worldwide. It is increasing in Korea. Remarkable histological changes including interstitial fibrosis and crystal deposition occur in the renal proximal tubules In this model, the mRNA expression of fibrosis markers was increased by tubular fibrosis. The effectiveness of in vivo delivery beyond the liver and nonspecific innate immune stimulation by siRNA are factors limiting the therapeutic development of siRNAs19,21,22. To overcome these limitations, we developed Self-Assembled-Micelle inhibitory RNA (SAMiRNA). SAMiRNA-AREG was developed and tested in pulmonary disease models, such as bleomycin-induced pulmonary fibrosis and transforming growth factor-beta (TGF-β) transgenic mouse models[23]. In damaged vasculature or fibrosis, SAMiRNA-AREG can be selectively delivered to inflamed or fibrotic tissues by the enhanced permeation and retention (EPR) effect[24,25]
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