Utilizing siRNA to Minimize Nephrotoxic Injury

描述（由申请人提供）：肾毒素仍然是急性肾损伤（AKI）的主要原因。RNA干扰作为一种治疗多种疾病的新型特异性基因沉默技术具有很大的前景。寡核苷酸，像许多其他小大分子一样，存在于肾小体形成的超滤液中，因此以相对高浓度呈递给衬在肾元的小管上皮细胞。这些细胞，尤其是近端小管段的细胞，会大量吸收寡核苷酸，因此肾脏是迄今为止系统递送寡核苷酸最多的地方。这使得siRNA成为治疗肾脏疾病的一种很有前景的技术，特别是考虑到近端小管细胞对毒性或缺血性损伤的特殊敏感性。鉴于这些观察结果的临床和生物学重要性，肾细胞类型对siRNA的处理需要更好地表征。我们认为siRNA优先积聚在近端小管上皮细胞中，因为这些细胞具有快速内吞传递到滤液中的大分子的特化能力。荧光显微镜的新技术，特别是多光子成像在活体显微镜中的应用，允许在活体动物中实时分析标记的寡核苷酸的细胞和亚细胞分布。我们建议在四个特定目的中使用这种方法，以更好地了解近端小管细胞摄取siRNA的机制，并确定使用siRNA预防中毒性急性肾损伤（AKI）的可行性。我们建议：1)确定siRNA在正常肾脏中细胞和细胞内积累、分解代谢和生物学效应的动力学；2)确定慢性肾脏疾病对肾脏处理siRNA的影响；3)确定内吞阻断对siRNA摄取的影响；4)测试siRNA阻止近端小管细胞摄取氨基糖苷的能力，从而限制肾毒性。

DESCRIPTION (provided by applicant): Nephrotoxins remain a major cause of acute kidney injury (AKI). RNA interference holds great promise as a novel and specific therapeutic gene-silencing technology for a wide range of diseases. Oligonucleotides, like many other small macromolecules, are present in the ultrafiltrate formed at the renal corpuscle, and therefore are presented to the tubular epithelial cells lining the nephron at relatively high concentrations. These cells, particularly those in the proximal tubule segments, avidly take up oligonucleotides, so that the kidney has by far the greatest accumulation of systemically delivered oligonucleotides. This makes siRNA a promising technology for treatment of kidney disease, especially given the particular sensitivity of proximal tubule cells to toxic or ischemic injury. Given the clinical and biological importance of these observations, the handling of siRNA by renal cell types needs to be better characterized. We propose that siRNA is preferentially accumulated in proximal tubule epithelial cells because of the specialization of these cells for rapid endocytosis of macromolecules delivered to the filtrate. New technologies in fluorescence microscopy, particularly the application of multi-photon imaging to intravital microscopy, allow the cellular and subcellular distribution of labeled oligonucleotides to be analyzed in real time in live animals. We propose to use this methodology in four specific aims to better understand the mechanism of uptake of siRNA by proximal tubule cells and to establish the feasibility of using siRNA to prevent toxic acute kidney injury (AKI). We propose: 1) to determine the kinetics of cellular and intracellular accumulation, catabolism and biological effects of siRNA in the normal kidney; 2) to determine the effect of chronic kidney disease on siRNA handling by the kidney; 3) to determine the effect of endocytic blockade on siRNA uptake; and 4) to test the ability of siRNA to prevent aminoglycoside uptake by proximal tubule cells and hence limit renal toxicity.