Dogan Yunus E, Bala Niharika, Chacko Kevin M, Tuna Kubra M, Alli Abdel A
Department of Physiology and Aging, College of Medicine, University of Florida Gainesville Florida 32610, USA.
Department of Pediatrics, Faculty of Medicine, Erciyes University Kayseri, Turkey.
Am J Transl Res. 2023 Dec 15;15(12):6690-6700. eCollection 2023.
Hypertension exacerbates the progression and severity of diabetic kidney disease. In this study, we addressed the hypothesis that tempol acts at multiple segments of the nephron to normalize the abundance of sodium coupled epithelial transport proteins in the luminal plasma membrane to mitigate high blood pressure in salt-loaded hypertensive diabetic db/db mice.
Soluble and membrane fractions from freshly homogenized kidney cortex tissue samples were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and probed for specific proteins by Western blotting. Western blotting for specific urinary extracellular vesicle (uEV) markers and nanoparticle tracking analysis was performed to characterize each uEV preparation from each group. A one-way ANOVA was performed to determine statistical significance between three groups (hypertensive diabetic db/db mice treated with vehicle, hypertensive diabetic db/db mice treated with tempol, and wild-type mice).
Tempol treatment reduced systolic blood pressure in hypertensive diabetic db/db mice compared to db/db mice that received vehicle. We observed attenuated membrane protein expression of the sodium hydrogen exchanger 3 (NHE3), sodium potassium chloride co-transporter (NKCC2), sodium chloride cotransporter (NCC), and epithelial sodium channel (ENaC) in the kidney of salt-loaded hypertensive diabetic db/db mice infused with tempol by osmotic minipump for 5 days compared to hypertensive diabetic db/db mice infused with vehicle. Also, the infusion of tempol in hypertensive diabetic db/db mice reduced the augmented protein expression of protein kinase c (PKC) epsilon observed in the vehicle treated hypertensive diabetic db/db kidney when compared to the healthy wild-type kidney. The amount of uEV and their size profiles were comparable between the three groups.
This study demonstrates that tempol down-regulates epithelial transport mechanisms in each segment of the nephron and normalizes salt-induced high blood pressure in diabetic animals presumably in a PKC dependent manner.
高血压会加剧糖尿病肾病的进展和严重程度。在本研究中，我们探讨了这样一个假设，即Tempol作用于肾单位的多个节段，使管腔质膜中钠偶联上皮转运蛋白的丰度正常化，以减轻盐负荷高血压糖尿病db/db小鼠的高血压。
将新鲜匀浆的肾皮质组织样本的可溶性和膜部分通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）进行分离，并用蛋白质免疫印迹法检测特定蛋白质。对每组的每个尿细胞外囊泡（uEV）制剂进行特定uEV标志物的蛋白质免疫印迹和纳米颗粒跟踪分析，以进行表征。进行单因素方差分析以确定三组（接受赋形剂治疗的高血压糖尿病db/db小鼠、接受Tempol治疗的高血压糖尿病db/db小鼠和野生型小鼠）之间的统计学显著性。
与接受赋形剂的db/db小鼠相比，Tempol治疗降低了高血压糖尿病db/db小鼠的收缩压。我们观察到，与接受赋形剂的高血压糖尿病db/db小鼠相比，通过渗透微型泵输注Tempol 5天的盐负荷高血压糖尿病db/db小鼠肾脏中，钠氢交换体3（NHE3）、钠钾氯共转运体（NKCC2）、氯化钠共转运体（NCC）和上皮钠通道（ENaC）的膜蛋白表达减弱。此外，与健康野生型肾脏相比，在高血压糖尿病db/db小鼠中输注Tempol可降低在接受赋形剂治疗的高血压糖尿病db/db肾脏中观察到的蛋白激酶C（PKC）ε增强的蛋白表达。三组之间uEV的数量及其大小分布相当。
本研究表明，Tempol下调肾单位各节段的上皮转运机制，并可能以PKC依赖的方式使糖尿病动物盐诱导的高血压正常化。
