Department of Nephrology, the First Affiliated Hospital of Shaoyang University, Shaoyang, Hunan, People's Republic of China.
Erasmus MC Transplant Institute, University Medical Center Rotterdam, Department of Internal Medicine, Division of Nephrology and Transplantation, Rotterdam, the Netherlands.
Int J Nanomedicine. 2024 Apr 12;19:3497-3511. doi: 10.2147/IJN.S446525. eCollection 2024.
Kidney transplantation is the optimal treatment for patients with end-stage kidney disease. Donor-specific urinary extracellular vesicles (uEVs) hold potential as biomarkers for assessing allograft status. We aimed to develop a method for identifying donor-specific uEVs based on human leukocyte antigen (HLA) mismatching with the kidney transplant recipients (KTRs).
Urine and plasma were obtained from HLA-A2+ donors and HLA-A2- KTRs pre-transplant. CD9 (tetraspanin, EV marker) and HLA-A2 double-positive (CD9+ HLA-A2+) EVs were quantified using isolation-free imaging flow cytometry (IFCM). Healthy individuals' urine was used to investigate CD9+ HLA-class-I+ uEV quantification using IFCM, time-resolved fluoroimmunoassay (TR-FIA), and immunogold staining cryo-electron microscopy (cryo-EM). Culture-derived CD9+ HLA-class-I+ EVs were spiked into the urine to investigate urine matrix effects on uEV HLA detection. Deceased donor kidneys and peritumoral kidney tissue were used for HLA class I detection with histochemistry.
The concentrations of CD9+ HLA-A2+ EVs in both donor and recipient urine approached the negative (detergent-treated) control levels for IFCM and were significantly lower than those observed in donor plasma. In parallel, universal HLA class I+ uEVs were similarly undetectable in the urine and uEV isolates compared with plasma, as verified by IFCM, TR-FIA, and cryogenic electron microscopy. Culture supernatant containing HLA class I+ vesicles from B, T, and human proximal tubule cells were spiked into the urine, and these EVs remained stable at 37°C for 8 hours. Immunohistochemistry revealed that HLA class I was predominantly expressed on the basolateral side of renal tubules, with limited expression on their urine/apical side.
The detection of donor-specific uEVs is hindered by the limited release of HLA class I+ EVs from the kidney into the urine, primarily due to the polarized HLA class I expression on renal tubules. Identifying donor-specific uEVs requires further advancements in recognizing transplant-specific uEVs and urine-associated markers.
肾移植是治疗终末期肾病患者的最佳方法。供体特异性尿细胞外囊泡（uEVs）具有作为评估同种异体移植物状态的生物标志物的潜力。我们旨在开发一种基于与肾移植受者（KTR）的人类白细胞抗原（HLA）不匹配来识别供体特异性 uEVs 的方法。
在移植前从 HLA-A2+供体和 HLA-A2- KTR 获得尿液和血浆。使用无分离成像流细胞术（IFCM）定量 CD9（四跨膜蛋白，EV 标志物）和 HLA-A2 双阳性（CD9+HLA-A2+）EVs。使用 IFCM、时间分辨荧光免疫测定（TR-FIA）和免疫金染色冷冻电子显微镜（cryo-EM）对健康个体的尿液进行 CD9+HLA 类-I+uEV 定量研究。将培养衍生的 CD9+HLA 类-I+EV 掺入尿液中，以研究尿液基质对 uEV HLA 检测的影响。使用组织化学检测已故供体肾脏和肿瘤周围肾脏组织中 HLA 类 I 的表达。
供体和受者尿液中 CD9+HLA-A2+EVs 的浓度接近 IFCM 的阴性（去污剂处理）对照水平，并且明显低于供体血浆中观察到的水平。同时，通过 IFCM、TR-FIA 和冷冻电子显微镜验证，普遍的 HLA 类-I+uEV 在尿液和 uEV 分离物中也同样无法检测到，与血浆相比。从 B、T 和人近端肾小管细胞的培养上清液中加入 HLA 类 I+囊泡，这些 EV 在 37°C 下 8 小时内保持稳定。免疫组织化学显示 HLA 类 I 主要在肾小管的基底外侧表达，在其尿液/顶端侧表达有限。
由于 HLA 类 I 在肾小管上的极化表达，HLA 类 I+EV 从肾脏释放到尿液中有限，这阻碍了供体特异性 uEVs 的检测。识别供体特异性 uEVs 需要进一步开发识别移植特异性 uEVs 和尿液相关标志物的方法。
