Thompson Austin D, Janda Jaroslav, Schnellmann Rick G
Department of Pharmacology and Toxicology, College of Pharmacy, Bio5 Institute, The University of Arizona, Tucson, AZ, United States.
Southwest Environmental Health Sciences Center, Tucson, AZ, United States.
Front Cardiovasc Med. 2023 Feb 9;10:1114726. doi: 10.3389/fcvm.2023.1114726. eCollection 2023.
During an episode of acute kidney injury (AKI), a sudden and rapid decline in renal function is often accompanied by a persistent reduction in mitochondrial function, microvasculature dysfunction/rarefaction, and tubular epithelial injury/necrosis. Additionally, patients who have experienced an AKI are at an elevated risk of developing other progressive renal, cardiovascular, and cardiorenal related diseases. While restoration of the microvasculature is imperative for oxygen and nutrient delivery/transport during proper renal repair processes, the mechanism(s) by which neovascularization and/or inhibition of microvascular dysfunction improves renal recovery remain understudied. Interestingly, pharmacological stimulation of mitochondrial biogenesis (MB) post-AKI has been shown to restore mitochondrial and renal function in mice. Thus, targeting MB pathways in microvasculature endothelial cell (MV-EC) may provide a novel strategy to improve renal vascular function and repair processes post-AKI. However, limitations to studying such mechanisms include a lack of commercially available primary renal peritubular MV-ECs, the variability in both purity and outgrowth of primary renal MV-EC in monoculture, the tendency of primary renal MV-ECs to undergo phenotypic loss in primary monoculture, and a limited quantity of published protocols to obtain primary renal peritubular MV-ECs. Thus, we focused on refining the isolation and phenotypic retention of mouse renal peritubular endothelial cells (MRPEC) for future physiological and pharmacological based studies. Here, we present a refined isolation method that augments the purity, outgrowth, and phenotypic retention of primary MRPEC monocultures by utilizing a collagenase type I enzymatic digestion, CD326+ (EPCAM) magnetic microbead epithelial cell depletion, and two CD146+ (MCAM) magnetic microbead purification cycles to achieve a monoculture MRPEC purity of ≅ 91-99% by all markers evaluated.
在急性肾损伤（AKI）发作期间，肾功能的突然快速下降通常伴随着线粒体功能的持续降低、微血管功能障碍/稀疏以及肾小管上皮损伤/坏死。此外，经历过AKI的患者患其他进行性肾脏、心血管和心肾相关疾病的风险会升高。虽然在肾脏正常修复过程中，微血管的恢复对于氧气和营养物质的输送/运输至关重要，但新生血管形成和/或抑制微血管功能障碍改善肾脏恢复的机制仍未得到充分研究。有趣的是，在AKI后对线粒体生物发生（MB）进行药理刺激已被证明可恢复小鼠的线粒体和肾功能。因此，靶向微血管内皮细胞（MV-EC）中的MB途径可能为改善AKI后的肾血管功能和修复过程提供一种新策略。然而，研究此类机制的局限性包括缺乏可商购的原代肾周MV-EC、原代肾MV-EC在单培养中的纯度和生长的变异性、原代肾MV-EC在原代单培养中发生表型丧失的趋势以及获取原代肾周MV-EC的已发表方案数量有限。因此，我们专注于优化小鼠肾周内皮细胞（MRPEC）的分离和表型保留，以用于未来基于生理学和药理学的研究。在此，我们提出一种优化的分离方法，该方法通过利用I型胶原酶消化、CD326+（EPCAM）磁性微珠上皮细胞去除以及两个CD146+（MCAM）磁性微珠纯化循环来提高原代MRPEC单培养物的纯度、生长和表型保留，通过所有评估的标志物实现单培养MRPEC的纯度约为91-99%。
