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).MethodsBiobanked 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.ResultsUrine 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.ConclusionsUrinary 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.
Highlights
Urinary stone disease (USD) is common, painful, and costly to manage, affecting approximately 1 in 11 people in the United States and 5–15 % of the population worldwide[1,2,3]
Urinary excretion of extracellular vesicles (EVs) derived from specific types of activated immune cells and EVs with proteins related to calcium/phosphorus regulation differed between calcium oxalate stone formers (CSFs) and non-stone formers (NSFs)
Urinary EVs expressing biomarkers of plasma membrane vesicle generation and endocytosis mediated exosome generation (Huntington interacting protein 1;HIP1) Urinary excretion of Anoctamin 4 (ANO4) expressing EVs was significantly lower in CSFs than NSFs (P < 0.05), while EVs expressing HIP1 trended lower in CSFs compared to NSFs (P = 0.07, Table 3 and Supplemental Figure 2)
Summary
Urinary stone disease (USD) is common, painful, and costly to manage, affecting approximately 1 in 11 people in the United States and 5–15 % of the population worldwide[1,2,3]. RP appear to serve as a nidus for deposition of protein and crystal layers from the urine in the renal pelvis, leading to anchored CaP and/or CaOx urinary stones[2, 8, 9].much is still not clear about the initiation and progression of RP and calcium stone formation. This lack of an in-depth mechanistic understanding has hindered the development of potential therapies [5]
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