METHODS: We established a mouse model by surgically ligating the unilateral ureter to induce renal injury in vivo. And we utilized renal in situ electroporation of a plasmid with low LncRNA A33 expression to establish the unilateral ureteral obstruction(UUO)mouse model. In vitro, we stimulated primary tubular epithelial cells(pTEC) injury using TGF-β1, siRNA-A33, and pcDNA3.1-A33 plasmids were transfected into pTECs to respectively knockdown and overexpress LncRNA A33, and both in vitro and in vivo models were intervened with A&P.
RESULTS: The results demonstrated that A&P effectively alleviated renal fibrosis in mice. Subsequent findings indicated high expression of LncRNA A33 in the kidneys of UUO mice and TGF-β1-induced renal tubular cells. In situ, renal electroporation of a plasmid with reduced LncRNA A33 expression revealed that inhibiting LncRNA A33 significantly improved renal fibrosis in UUO mice. Moreover, A&P effectively suppressed LncRNA A33 expression both in vitro and in vivo. Subsequent downregulation of LncRNA A33 in renal tubular epithelial cells resulted in the downregulation of numerous fibrotic markers, a significant inhibition of LncRNA A33, and a notable reduction in downstream ferroptosis signaling. Cell experiments demonstrated that A&P improved renal fibrosis in UUO mice by inhibiting LncRNA A33 and downregulating ferroptosis signaling.
CONCLUSIONS: Through the inhibition of LncRNA A33 and subsequent downregulation of ferroptosis signaling, A&P showed potential as a therapeutic approach for improving renal fibrosis in UUO mice, providing a potential treatment avenue for CKD.
方法:我们通过手术结扎单侧输尿管以体内诱导肾损伤建立了小鼠模型。我们利用低LncRNAA33表达质粒的肾脏原位电穿孔建立单侧输尿管梗阻(UUO)小鼠模型。体外,我们使用TGF-β1,siRNA-A33和pcDNA3.1-A33质粒刺激原代肾小管上皮细胞(pTEC)损伤,将质粒转染到pTEC中,分别敲低和过表达LncRNAA33,并在体外和体内模型中进行干预。
结果:结果表明A&P能有效缓解小鼠肾纤维化。随后的发现表明LncRNAA33在UUO小鼠的肾脏和TGF-β1诱导的肾小管细胞中高表达。在原地,LncRNAA33表达降低的质粒的肾电穿孔显示,抑制LncRNAA33显著改善UUO小鼠的肾纤维化。此外,A&P在体外和体内均有效抑制LncRNAA33表达。随后LncRNAA33在肾小管上皮细胞中的下调导致许多纤维化标志物的下调,LncRNAA33的显着抑制,以及下游铁凋亡信号的显着减少。细胞实验表明,A&P通过抑制LncRNAA33和下调铁凋亡信号传导来改善UUO小鼠的肾纤维化。
结论:通过抑制LncRNAA33和随后下调铁凋亡信号,A&P显示出作为改善UUO小鼠肾纤维化的治疗方法的潜力,为CKD提供潜在的治疗途径。