PDF(Pubmed)
Abstract：
In this study, we assessed the impact of commercially available polymyxin B against VRP-034 (novel formulation of polymyxin B) using a validated in vitro human renal model, aProximateTM. Freshly isolated primary proximal tubule cells (PTCs) were cultured in Transwell plates and treated with various concentrations of the formulations for up to 48 h. The functional expression of megalin-cubilin receptors in PTC monolayers was validated using FITC-conjugated albumin uptake assays. Polymyxin B and VRP-034 were evaluated at six concentrations (0.3, 1, 3, 10, 30, and 60 µM), and nephrotoxicity was assessed through measurements of transepithelial electrical resistance (TEER), intracellular adenosine triphosphate (ATP) levels, lactate dehydrogenase (LDH) release, and novel injury biomarkers [kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), and clusterin]. Additionally, histological analysis using annexin V apoptosis staining was performed. Our results indicated a significant decrease in TEER with polymyxin B at concentrations ≥10 μM compared to VRP-034. Toxic effects were observed from ATP and LDH release only at concentrations ≥30 μM for both formulations. Furthermore, injury biomarker release was higher with polymyxin B compared to VRP-034, particularly at concentrations ≥10 µM. Histologically, polymyxin B-treated PTCs showed increased apoptosis compared to VRP-034-treated cells. Overall, VRP-034 demonstrated improved tolerance in the aProximateTM model compared to polymyxin B, suggesting its potential as a safer alternative for renal protection.
摘要：
在这项研究中,我们使用经过验证的体外人肾模型评估了市售多粘菌素B对VRP-034（多粘菌素B的新型制剂）的影响，ProximateTM。将新鲜分离的原代近端小管细胞（PTC）在Transwell板中培养，并用各种浓度的制剂处理长达48小时。使用FITC缀合的白蛋白摄取测定验证了PTC单层中megalin-cubilin受体的功能表达。多粘菌素B和VRP-034在六个浓度（0.3、1、3、10、30和60µM）下进行评估，通过测量跨上皮电阻（TEER）评估肾毒性，细胞内三磷酸腺苷（ATP）水平，乳酸脱氢酶（LDH）释放，和新型损伤生物标志物[肾损伤分子-1（KIM-1），中性粒细胞明胶酶相关脂质运载蛋白（NGAL），和clusterin]。此外,使用膜联蛋白V凋亡染色进行组织学分析。我们的结果表明，与VRP-034相比，浓度≥10μM的多粘菌素B的TEER显着降低。对于两种制剂,仅在浓度≥30μM时从ATP和LDH释放观察到毒性作用。此外，与VRP-034相比,多粘菌素B的损伤生物标志物释放量更高,特别是在浓度≥10µM时.组织学上,与VRP-034处理的细胞相比,多粘菌素B处理的PTC显示出增加的凋亡。总的来说,与多粘菌素B相比，VRP-034在aproximateTM模型中表现出改善的耐受性，表明其作为肾脏保护更安全的替代方案的潜力。
