Excretion of urine extracellular vesicles bearing markers of activated immune cells and calcium/phosphorus physiology differ between calcium kidney stone formers and non-stone formers

Abstract

Backgrounds: Previous studies have demonstrated that excretion of urinary extracellular vesicles (EVs) from different nephron segments differs between kidney stone formers and non-stone formers (NSFs), and could reflect pathogenic mechanisms of urinary stone disease. In this study we quantified selected populations of specific urinary EVs carrying protein markers of immune cells and calcium/phosphorus physiology in calcium oxalate stone formers (CSFs) compared to non-stone formers (NSFs). Methods Biobanked urine samples from CSFs (n = 24) undergoing stone removal surgery and age- and sex- matched NSFs (n = 21) were studied. Urinary EVs carrying proteins related to renal calcium/phosphorus physiology (phosphorus transporters (PiT1 and PiT2), Klotho, and fibroblast growth factor 23 (FGF23); markers associated with EV generation (anoctamin-4 (ANO4) and Huntington interacting protein 1 (HIP1)), and markers shed from activated immune cells were quantified by standardized and published method of digital flow cytometry. Results Urine excretion of calcium, oxalate, phosphorus, and calcium oxalate supersaturation (SS) were significantly higher in CSFs compared to NSFs (P < 0.05). Urinary excretion of EVs with markers of total leukocytes (CD45), neutrophils (CD15), macrophages (CD68), Klotho, FGF23, PiT1, PiT2, and ANO4 were each markedly lower in CSFs than NSFs (P < 0.05) whereas excretion of those with markers of monocytes (CD14), T-Lymphocytes (CD3), B-Lymphocytes (CD19), plasma cells (CD138 plus CD319 positive) were not different between the groups. Urinary excretion of EVs expressing PiT1 and PiT2 negatively (P < 0.05) correlated with urinary phosphorus excretion, whereas excretion of EVs expressing FGF23 negatively (P < 0.05) correlated with both urinary calcium and phosphorus excretion. Urinary EVs with markers of HIP1 and ANO4 correlated negatively (P < 0.05) with clinical stone events and basement membrane calcifications on papillary tip biopsies. Conclusions Urinary excretion of EVs derived from specific types of activated immune cells and EVs with proteins related to calcium/phosphorus regulation differed between CSFs and NSFs. Further validation of these and other populations of urinary EVs in larger cohort could identify biomarkers that elucidate novel pathogenic mechanisms of calcium stone formation in specific subsets of patients.

Differences of Uric Acid Transporters Carrying Extracellular Vesicles in the Urine from Uric Acid and Calcium Stone Formers and Non-Stone Formers

“…The absolute EV count either in the absence or presence of single or dual fluorophore-conjugated antibody staining was calculated using a previously validated method [ 20 , 21 , 23 , 24 ]. The number of EVs/µL urine was used to calculate EV excretion per 24 h and also normalized to EVs/mg urine creatinine [ 20 , 21 , 24 , 26 , 27 , 28 ]. All antibodies were fully validated with reagent controls and appropriate isotype control antibodies conjugated with the same fluorophores together with negative controls, as per our previous publications [ 20 , 21 , 23 , 24 ].…”

Hepatocellular carcinoma-derived exosomal miRNA-761 regulates the tumor microenvironment by targeting the SOCS2/JAK2/STAT3 pathway

“…The increase in N-cadherin plays an important role in cancer metastasis [26,27] and may enhance cell migration even without a decrease in E-cadherin. [12] EMT promotion may also contribute to the mechanism by which HCC-derived exosomes induce the metastasis of recipient HCC cells.…”

“…[11] The immune cell markers of exosomes even reflect renal calcium/phosphorus physiology. [12] Cancer cells grow in an environment that supports tumor growth; the tumor microenvironment (TME) contains normal fibroblasts (NFs), blood vessels, endothelial cells, immune cells, surrounding extracellular matrix (ECM), and other components. [13] Cancer cells interact with the microenvironment to promote tumor growth and metastasis.…”

Extracellular Vesicle-Based Therapy for COVID-19: Promises, Challenges and Future Prospects

“…MVBs bind to the inner plasma membrane, releasing their ILVs in the extracellular environment in the form of exosomes. There are several proteins involved in exosome biogenesis such as endosomal sorting complex required for transport (ESCRT), vacuolar ATPase, and Vps4, which segregate and sort ubiquitylated proteins into ILVs (Figure 1) [13][14][15][16][17][18]. Microvesicles are a little larger (40-1000 nm) in size and are released through pinching-off the plasma membrane via a direct budding process.…”