Exosomal Fetuin-A identified by proteomics: A novel urinary biomarker for detecting acute kidney injury

Abstract

Urinary exosomes containing apical membrane and intracellular fluid are normally secreted into the urine from all nephron segments, and may carry protein markers of renal dysfunction and structural injury. We aimed to discover biomarkers in urinary exosomes to detect acute kidney injury (AKI), which has a high mortality and morbidity. Animals were injected with cisplatin. Urinary exosomes were isolated by differential centrifugation. Protein changes were evaluated by two-dimensional difference in gel electrophoresis and changed proteins were identified by mass spectrometry. The identified candidate biomarkers were validated by Western blotting in individual urine samples from rats subjected to cisplatin injection; bilateral ischemia and reperfusion (I/R); volume depletion; and intensive care unit (ICU) patients with and without AKI. We identified 18 proteins that were increased and nine proteins that were decreased 8 h after cisplatin injection. Most of the candidates could not be validated by Western blotting. However, exosomal Fetuin-A increased 52.5-fold at day 2 (1 day before serum creatinine increase and tubule damage) and remained elevated 51.5-fold at day 5 (peak renal injury) after cisplatin injection. By immunoelectron microscopy and elution studies, Fetuin-A was located inside urinary exosomes. Urinary Fetuin-A was increased 31.6-fold in the early phase (2-8 h) of I/R, but not in prerenal azotemia. Urinary exosomal Fetuin-A also increased in three ICU patients with AKI compared to the patients without AKI. We conclude that (1) proteomic analysis of urinary exosomes can provide biomarker candidates for the diagnosis of AKI and (2) urinary Fetuin-A might be a predictive biomarker of structural renal injury.

Extracellular Vesicles in Renal Diseases

“…Only exosomal Fetuin-A but not free Fetuin-A was detectable in the urine before morphologic injury developed. 58 Thus, urinary exosomal biomarkers might be more enriched in the urine than its free form and therefore, have a potential to be a more sensitive biomarker in AKI. This notion is supported by another study showing that, although there were no changes in urinary-free neutrophil gelatinaseassociated lipocalin neutrophil gelatinaseassociated lipocalin levels, neutrophil gelatinase-associated lipocalin levels in urinary exosomes (uEXs) were increased in patients with delayed graft function after kidney transplantation.…”

Applicability of 2D gel electrophoresis and liquid chromatography in proteomic analysis of urine using mass spectrometry MALDI-TOF

“…Then, the inhibitors mixture is added to the obtained supernatant to prevent the proteins decomposition. Their concentration is determined by the Bradford method and the whole content is stored at -80 o C for further analyses (Zhou et al 2006). To avoid the risk of sample contamination with outer environmental factor, in some cases cystocentisis, the direct puncture of the urine bladder, is carried out with monitoring by USG.…”

Application of Flow Cytometry in the Studies of Microparticles

“…Other bodily fluids may also serve as a source of information on elevated MP numbers. For instance, recent discovery of elevated MP numbers present in urine in patients developing acute kidney injury may also point to the usefulness of the MP level monitoring [Zhou et al, 2006]. Also, malignant effusions such as ascites fluids were also shown to have elevated MP numbers [Andre et al, 2002].…”