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

Liang Wu,1,2 Martijn H van Heugten,3 Thierry PP van den Bosch,4 Hans Duimel,5 Carmen López-Iglesias,5 Dennis A Hesselink,2 Carla C Baan,2 Karin Boer2 1Department of Nephrology, the First Affiliated Hospital of Shaoyang University, Shaoyang, Hunan, People’s Republic of China; 2Erasmus MC Transplant Institute, University Medical Center Rotterdam, Department of Internal Medicine, Division of Nephrology and Transplantation, Rotterdam, the Netherlands; 3University Medical Center Rotterdam, Department of Internal Medicine, Division of Nephrology and Transplantation, Rotterdam, the Netherlands; 4Department of Pathology, University Medical Center Rotterdam, Rotterdam, the Netherlands; 5The Microscopy CORE Laboratory at the Faculty of Health, Medicine and Life Sciences, Maastricht University, Maastricht, the NetherlandsCorrespondence: Liang Wu, Erasmus MC Transplant Institute, Department of Internal Medicine, University Medical Center Rotterdam Erasmus MC, Room No. Na-514, Doctor Molewaterplein 40, 3015 GD, Rotterdam, the Netherlands, Tel +31 0622145029, Email [email protected]: 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 tubul

The lipid droplet coat protein perilipin 5 also localizes to muscle mitochondria

Perilipin 5 (PLIN5/OXPAT) is a lipid droplet (LD) coat protein mainly present in tissues with a high fat-oxidative capacity, suggesting a role for PLIN5 in facilitating fatty acid oxidation. Here, we investigated the role of PLIN5 in fat oxidation in skeletal muscle. In human skeletal muscle, we observed that PLIN5 (but not PLIN2) protein content correlated tightly with OXPHOS content and in rat muscle PLIN5 content correlated with mitochondrial respiration rates on a lipid-derived substrate. This prompted us to examine PLIN5 protein expression in skeletal muscle mitochondria by means of immunogold electron microscopy and Western blots in isolated mitochondria. These data show that PLIN5, in contrast to PLIN2, not only localizes to LD but also to mitochondria, possibly facilitating fatty acid oxidation. Unilateral overexpression of PLIN5 in rat anterior tibialis muscle augmented myocellular fat storage without increasing mitochondrial density as indicated by the lack of change in protein content of five components of the OXPHOS system. Mitochondria isolated from PLIN5 overexpressing muscles did not possess increased fatty acid respiration. Interestingly though, 14C-palmitate oxidation assays in muscle homogenates from PLIN5 overexpressing muscles revealed a 44.8% (P = 0.05) increase in complete fatty acid oxidation. Thus, in mitochondrial isolations devoid of LD, PLIN5 does not augment fat oxidation, while in homogenates containing PLIN5-coated LD, fat oxidation is higher upon PLIN5 overexpression. The presence of PLIN5 in mitochondria helps to understand why PLIN5, in contrast to PLIN2, is of specific importance in fat oxidative tissues. Our data suggests involvement of PLIN5 in directing fatty acids from the LD to mitochondrial fatty acid oxidation

Reduced mitochondrial density in the vastus lateralis muscle of patients with COPD

Skeletal muscle dysfunction is a well-recognised hallmark of COPD, leading to exercise intolerance. The vastus lateralis of these patients is characterised by reduced mitochondrial enzyme activity, whereas this is not the case in the tibialis anterior. It is however unclear if the comprised oxidative capacity in the vastus is due to reduced mitochondrial volume density.Therefore, muscle biopsies were obtained from the vastus lateralis of 6 COPD patients and 4 healthy age-matched controls and from the tibialis anterior of another 6 patients and 6 controls. Mitochondrial number, fractional area and morphometry, as well as Z-line width (as a surrogate marker of fibre type), were analysed using transmission electron microscopy.Mitochondrial number (0.34 vs. 0.63 microm(-2)) and fractional area (1.95% vs. 4.25%) were reduced in the vastus of COPD patients compared to controls, respectively. Despite a reduced mitochondrial number (0.65 vs. 0.88 microm(-2)), mitochondrial fractional area was maintained in the tibialis.It can be concluded that reduced mitochondrial fractional area is likely contribute to the decreased oxidative capacity in the vastus of COPD patients, whereas the maintained mitochondrial fractional area in the tibialis may explain the normal oxidative capacity

The use of hepatitis B immunoglobulin in the Netherlands

Hepatitis B immunoglobulin (HBIg) was administered to 241 patients contaminated with HBAg positive material; 116 persons were followed up for 7 months after the HBIg injection. Only 4 (3.4%) cases of hepatitis B with jaundice and demonstrable HBAg occurred and 15 (12.9%) cases of subclinical hepatitis B were observed. HBIg is well tolerated. Passively transferred HBAb were demonstrable for 2-3 months and no chronic carriers of HBAg were seen after administration of HBI

Time course of atrial fibrillation-induced cellular structural remodeling in atria of the goat.

Background: Previously we documented cellular structural changes of a non-degenerative nature in atrial myocytes after atrial fibrillation (AF) in the goat. The time course of these changes was not studied. Methods and Results: Cellular structural changes were studied by light- and electron microscopy and immunohistochemistry in goat atria after 0-16 weeks AF. The first sign of cellular structural remodeling was a more homogeneous chromatin distribution, at 1 week of AF. Sub-structural changes in mitochondria and sarcoplasmic reticulum occurred gradually. Cellular degeneration was absent. The degree of myolysis and glycogen accumulation increased till 8 weeks of AF and did not increase further from thereon. After 16 weeks of AF, 42% of the myocytes in the right atrial free wall were affected by myolysis. The diameter of the atrial myocytes increased. Dedifferentiation of the atrial myocytes was suggested by altered expression patterns of structural proteins, such as the disappearance of cardiotin (1 week), the A-I junctional part of titin (4 weeks), desmin at the intercalated disk (ID) (8 weeks) and a gradual re-expression of alpha-smooth muscle actin. Conclusion: Remodeling of the cellular ultrastructure in atrial myocardium of the goat develops progressively during AF. Re-expression of fetal proteins indicate dedifferentiation of atrial myocytes, analogous to observations in hibernating myocardium of the ventricle

Increased expression of rapsyn in muscles prevents acetylcholine receptor loss in experimental autoimmune myasthenia gravis.

Myasthenia gravis is usually caused by autoantibodies to the acetylcholine receptor (AChR). The AChR is clustered and anchored in the postsynaptic membrane of the neuromuscular junction (NMJ) by a cytoplasmic protein called rapsyn. We previously showed that resistance to experimental autoimmune myasthenia gravis (EAMG) in aged rats correlates with increased rapsyn concentration at the NMJ. It is possible, therefore, that endogenous rapsyn expression may be an important determinant of AChR loss and neuromuscular transmission failure in the human disease, and that upregulation of rapsyn expression could be used therapeutically. To examine first a potential therapeutic application of rapsyn upregulation, we induced acute EAMG in young rats by passive transfer of AChR antibody, mAb 35, and used in vivo electroporation to over-express rapsyn unilaterally in one tibialis anterior. We looked at the compound muscle action potentials (CMAPs) in the tibialis anterior, at rapsyn and AChR expression by quantitative radioimmunoassay and immunofluorescence, and at the morphology of the NMJs, comparing the electroporated and untreated muscles, as well as the control and EAMG rats. In control rats, transfected muscle fibres had extrasynaptic rapsyn aggregates, as well as slightly increased rapsyn and AChR concentrations at the NMJ. In EAMG rats, despite deposits of the membrane attack complex, the rapsyn-overexpressing muscles showed no decrement in the CMAPs, no loss of AChR, and the majority had normal postsynaptic folds, whereas endplates of untreated muscles showed typical AChR loss and morphological damage. These data suggest not only that increasing rapsyn expression could be a potential treatment for selected muscles of myasthenia gravis patients, but also lend support to the hypothesis that individual differences in innate rapsyn expression could be a factor in determining disease severity