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.

Keywords:

• kidney transplantation
• donor-specific biomarker
• HLA
• extracellular vesicles
• human urine
• renal tubule

Acknowledgments

We acknowledge Guido Jenster (Department of Urology, EMC) for TR-FIA and the sponsorship from the China Scholarship Council (grant number 202008430154).

Disclosure

D.A. Hesselink has received lecture and consulting fees from Astellas Pharma, Chiesi Pharma, Medincell, Novartis Pharma, and Vifor Pharma. He has received grant support from Astellas Pharma, Bristol-Myers Squibb, and Chiesi Pharma [paid to his institution]. D.A. Hesselink does not have employment or stock ownership at any of these companies, nor does he have patents or patent applications. Other authors declare no conflict of interest in this work.