Department of Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
Physiol Genomics. 2012 Sep 1;44(17):829-42. doi: 10.1152/physiolgenomics.00060.2012. Epub 2012 Jul 17.
The renal medullary thick ascending limb (mTAL) of the Dahl salt-sensitive (SS) rat is the site of enhanced NaCl reabsorption and excess superoxide production. In the present studies we isolated mitochondria from mTAL of SS and salt-resistant control strain SS.13(BN) rats on 0.4 and 8% salt diet for 7 days and performed a proteomic analysis. Purity of mTAL and mitochondria isolations exceeded 93.6 and 55%, respectively. Using LC/MS spectral analysis techniques we identified 96 mitochondrial proteins in four biological mTAL mitochondria samples, run in duplicate, as defined by proteins with a false discovery rate <5% and scan count ≥2. Seven of these 96 proteins, including IDH2, ACADM, SCOT, Hsp60, ATPA, EFTu, and VDAC2 were differentially expressed between the two rat strains. Oxygen consumption and high-resolution respirometry analyses showed that mTAL cells and the mitochondria in the outer medulla of SS rats fed high-salt diet exhibited lower rates of oxygen utilization compared with those from SS.13(BN) rats. These studies advance the conventional proteomic paradigm of focusing exclusively upon whole tissue homogenates to a focus upon a single cell type and specific subcellular organelle. The results reveal the importance of a largely unexplored role for deficiencies of mTAL mitochondrial metabolism and oxygen utilization in salt-induced hypertension and renal medullary oxidative stress.
Dahl 盐敏感（SS）大鼠的肾髓质升支粗段（mTAL）是增强 NaCl 重吸收和过量超氧化物产生的部位。在本研究中，我们从 SS 和盐抵抗对照品系 SS.13（BN）大鼠的 mTAL 中分离出线粒体，这些大鼠分别在 0.4%和 8%盐饮食下饲养 7 天。mTAL 和线粒体的分离纯度分别超过 93.6%和 55%。使用 LC/MS 光谱分析技术，我们在四个生物学 mTAL 线粒体样本中鉴定出 96 种线粒体蛋白，这些样本重复运行两次，定义为假发现率<5%且扫描计数≥2 的蛋白质。这 96 种蛋白质中的 7 种，包括 IDH2、ACADM、SCOT、Hsp60、ATPA、EFTu 和 VDAC2，在两种大鼠品系之间存在差异表达。氧消耗和高分辨率呼吸测定分析表明，高盐饮食喂养的 SS 大鼠的 mTAL 细胞和外髓质的线粒体的耗氧量低于 SS.13（BN）大鼠的耗氧量。这些研究将传统的蛋白质组学范式从仅关注整个组织匀浆扩展到关注单一细胞类型和特定的亚细胞细胞器。研究结果揭示了 mTAL 线粒体代谢和氧利用缺陷在盐诱导高血压和肾髓质氧化应激中的重要作用。
