Urinary extracellular vesicle-associated MCP-1 and NGAL derived from specific nephron segments differ between calcium oxalate stone formers and controls

Abstract

Randall’s plaque (RP; subepithelial calcification) appears to be an important precursor of kidney stone disease. However, RP cannot be noninvasively detected. The present study investigated candidate biomarkers associated with extracellular vesicles (EVs) in the urine of calcium stone formers (CSFs) with low (<5% papillary surface area) and high (≥5% papillary surface area) percentages of RP and a group of nonstone formers. RPs were quantitated via videotaping and image processing in consecutive CSFs undergoing percutaneous surgery for stone removal. Urinary EVs derived from cells of different nephron segments of CSFs ( n = 64) and nonstone formers ( n = 40) were quantified in biobanked cell-free urine by standardized and validated digital flow cytometer using fluorophore-conjugated antibodies. Overall, the number of EVs carrying surface monocyte chemoattractant protein (MCP)-1 and neutrophil gelatinase-associated lipocalin (NGAL) were significantly lower in CSFs compared with nonstone former controls ( P < 0.05) but did not differ statistically between CSFs with low and high RPs. The number of EVs associated with osteopontin did not differ between any groups. Thus, EVs carrying MCP-1 and NGAL may directly or indirectly contribute to stone pathogenesis as evidenced by the lower of these populations of EVs in stone formers compared with nonstone formers. Validation of EV-associated MCP-1 and NGAL as noninvasive biomarkers of kidney stone pathogenesis in larger populations is warranted.

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

“…Differences in the urinary biochemical pro le of the CSF versus NSF group in the current study were not surprising, including lower urine pH and greater calcium and phosphorous excretion and uric acid SS (Table 1) in CSF [4,16,27,44]. Urine calcium and phosphorus excretion in high RP participants were markedly higher compared with NSFs, however those of low RP were not signi cantly different.…”

“…Interestingly, in the current study the number of urinary EVs bearing immune cell markers CD68, CD45, and CD15 were reduced in CSFs compared with NSFs. Previously we had demonstrated that urinary EVs carrying the in ammatory mediator, monocyte chemoattractant protein-1 was also lower in CSFs compared to NSFs [16]. Although matching kidney tissue was not available from CSFs in the current study in order to quantitate immune/in ammatory cell populations and correlate with urinary EV populations, previously published studies do suggest that the number of immune cells within the kidney bearing CD68 may differ in CSFs compared to NSFs [27].…”

“…Thus, in the present study we characterized populations of urinary EVs derived from activated immune cells in CSFs compared to NSFs (controls). Our previous studies demonstrated that urinary excretion of speci c EV populations derived from different nephron segments varied between cohorts of CSFs and NSFs [5,19]. Speci cally we quanti ed urinary EVs bearing markers of immune/in ammatory cells activation, calcium/phosphorus physiology and EV generation from the plasma membrane and endocytic vesicles.…”

“…A standardized and validated ow cytometry (BD FACSCanto™) method was used to de ne EVs by size (≥ 200 nm to ≤ 1000 nm) and annexin-V-uorescence for quanti cation of selected surface biomarker carrying urinary EVs as previously described in detail [5,15,16]. The absolute number of urinary EVs positive for selected speci c biomarkers was calculated as the number of EVs per µL of urine and then…”

“…EVs appear to transmit signals between cells under both physiological and pathological conditions [14]. The concentration and content of bioactive molecules in urinary EVs, including microRNA, DNA, mRNA, lipids and proteins all depend on their cell of origin or stimulus [14][15][16]. Previous studies have reported that EVs participate in signal communication during renal regenerative and pathological processes [14].…”