Sung Chih-Chien, Chen Min-Hsiu, Lin Yi-Chang, Lin Yu-Chun, Lin Yi-Jia, Yang Sung-Sen, Lin Shih-Hua
Division of Nephrology, Department of Medicine, National Defense Medical Center, Tri-Service General Hospital, Taipei, Taiwan.
Division of Cardiovascular Surgery, Department of Surgery, National Defense Medical Center, Tri-Service General Hospital, Taipei, Taiwan.
Front Med (Lausanne). 2021 Jun 9;8:679171. doi: 10.3389/fmed.2021.679171. eCollection 2021.
The utility of urinary extracellular vesicles (uEVs) to faithfully represent the changes of renal tubular protein expression remains unclear. We aimed to evaluate renal tubular sodium (Na) or potassium (K) associated transporters expression from uEVs and kidney tissues in patients with Gitelman syndrome (GS) caused by inactivating mutations in . uEVs were isolated by ultracentrifugation from 10 genetically-confirmed GS patients. Membrane transporters including Na-hydrogen exchanger 3 (NHE3), Na/K/2Cl cotransporter (NKCC2), NaCl cotransporter (NCC), phosphorylated NCC (p-NCC), epithelial Na channel β (ENaCβ), pendrin, renal outer medullary K1 channel (ROMK), and large-conductance, voltage-activated and Ca-sensitive K channel (Maxi-K) were examined by immunoblotting of uEVs and immunofluorescence of biopsied kidney tissues. Healthy and disease (bulimic patients) controls were also enrolled. Characterization of uEVs was confirmed by nanoparticle tracking analysis, transmission electron microscopy, and immunoblotting. Compared with healthy controls, uEVs from GS patients showed NCC and p-NCC abundance were markedly attenuated but NHE3, ENaCβ, and pendrin abundance significantly increased. ROMK and Maxi-K abundance were also significantly accentuated. Immunofluorescence of the representative kidney tissues from GS patients also demonstrated the similar findings to uEVs. uEVs from bulimic patients showed an increased abundance of NCC and p-NCC as well as NHE3, NKCC2, ENaCβ, pendrin, ROMK and Maxi-K, akin to that in immunofluorescence of their kidney tissues. uEVs could be a non-invasive tool to diagnose and evaluate renal tubular transporter adaptation in patients with GS and may be applied to other renal tubular diseases.
尿细胞外囊泡(uEVs)能否如实地反映肾小管蛋白表达的变化仍不清楚。我们旨在评估吉特曼综合征(GS)患者的uEVs和肾组织中与肾小管钠(Na)或钾(K)相关的转运蛋白表达情况,GS由特定基因的失活突变引起。通过超速离心从10例基因确诊的GS患者中分离出uEVs。通过对uEVs进行免疫印迹以及对活检肾组织进行免疫荧光检测,来检测包括钠氢交换体3(NHE3)、钠钾氯共转运体(NKCC2)、氯化钠共转运体(NCC)、磷酸化NCC(p-NCC)、上皮钠通道β(ENaCβ)、pendrin、肾外髓钾通道1(ROMK)以及大电导、电压激活和钙敏感钾通道(Maxi-K)在内的膜转运蛋白。还纳入了健康对照和疾病(贪食症患者)对照。通过纳米颗粒跟踪分析、透射电子显微镜和免疫印迹对uEVs进行了表征。与健康对照相比,GS患者的uEVs显示NCC和p-NCC丰度明显降低,但NHE3、ENaCβ和pendrin丰度显著增加。ROMK和Maxi-K丰度也显著增强。GS患者代表性肾组织的免疫荧光检测结果与uEVs相似。贪食症患者的uEVs显示NCC、p-NCC以及NHE3、NKCC2、ENaCβ、pendrin、ROMK和Maxi-K丰度增加,类似于其肾组织免疫荧光检测结果。uEVs可能是一种用于诊断和评估GS患者肾小管转运蛋白适应性的非侵入性工具,并且可能适用于其他肾小管疾病。