Polarized HLA Class I Expression on Renal Tubules Hinders the Detection of Donor-Specific Urinary Extracellular Vesicles

Abstract

Purpose 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). Patients and Methods 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. Results 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. Conclusion 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.

Urinary extracellular vesicles in healthy individuals: positive correlation between podocyte and tubular vesicles independent of kidney function

“…Spot urine samples were collected from 30 healthy kidney transplant donors at the Erasmus MC, Rotterdam, The Netherlands, between August 2018 and August 2021 (clinical parameters are summarized in Supplementary Table S1). Urine processing protocols, including centrifugation and addition of a proteinase inhibitor to the supernatant, were described previously [5].…”

“…The acquisition of Imaging Flow Cytometry (IFCM) data was conducted using an ImageStreamX Mark II instrument (ISx; Cytek Biosciences, Fremont, CA, USA) installed with INSPIRE® software (version 200.1.0.765; Cytek Biosciences), following established protocols for analyzing unprocessed urine and plasma [5,6]. The INSPIRE® settings were configured as follows: a flow speed velocity of 40 mm/s, a flow core diameter of 6 μm, 60× magnification, and laser wavelengths of 405 nm (channel 01), 488 nm (channel 02), 642 nm (channel 05), and 785 nm for side scatter (SSC) excitation (channel 06).…”

“…Fluorescence signals from eFluor450, Alexa488, and APC (or Alexa647) were detected in channels 01, 02, and 05, respectively. Quantification of samples was performed using Amnis IDEAS software (version 6.2; Cytek Biosciences) [5,6].…”

“…However, research on the discrimination of single kd-uEVs from different nephron segments and their correlation with kidney biology remains limited. Imaging flow cytometry (IFCM) can identify single kd-uEVs by labeling them with CD63 (uEV marker), combined with PODXL or AQP2 [4,5].…”