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.

Figure 1. Serum creatinine (SCr) and blood urea nitrogen (BUN) in cisplatin-induced AKI in rats (n=56)

SCr (A) and BUN (B) increased significantly on day 3, peaked at day 5, and returned to baseline at day 14 after cisplatin injection. Data are means ± SEM. *P<0.01, #p<0.05 versus day 0.

Figure 2. Histology of the kidney sections stained with periodic acid-Schiff reagent in the outer stripe of the outer medulla (OSOM) after renal injury

Temporal morphologic change of OSOM before (A), and at day 1 (B), day 2 (C), day 3 (D), day 5 (E) after cisplatin injection. Kidney 8 hr (F) and 24 hr (G) reperfusion after 37 min of bilateral ischemia.

Figure 3

Two dimensional differential in-gel electrophoresis (2D-DIGE) and the fold change of urinary exosome-associated proteins of rats before and after cisplatin injection. Urinary exosome-associated proteins were isolated from pooled 8 hr urine samples from 12 rats before and after cisplatin injection. (A) 2D-DIGE: shows increase (red), decrease (green) or no change (yellow) in abundance of exosome-associated proteins after cisplatin injection. Black numbers were spots identified by MALDI-TOF-TOF, white numbers were spots identified by LC-MS/MS. (B) Fold change of urinary exosome-associated proteins that were increased or decreased after cisplatin. The grey bars are membrane-associated proteins. Numbers (1) to (15) match the black numbers in 2D-DIGE (A).

Figure 4. Identification of Fetuin-A protein by LC-MS/MS

MS spectra of a Fetuin-A peptide (sequence: LGGEEVSVACK). The red peaks indicate matched b-ion series, the blue peaks indicate matched y-ion series, and the black peaks indicated unmatched ions. The majority of peaks are matched in agreement with the high cross-correlation score (Xcorr = 4).

Figure 5. Temporal excretion of urinary exosomal Fetuin-A in AKI animal models and human subjects by western blotting analysis

(A) Cisplatin-induced AKI rats (typical result from 3 replicates), (B) I/R-induced AKI rats (in 15, 30, and 40 mins of bilateral ischemia and reperfusion), (C) one blot of above three types of AKI rats (one rat pre-, day 0, day 1, and day 2 after cisplatin; one rat from pre- and 8 hr after I/R; one rat from pre- and 24–30 hr after VD), and (D) ICU patients with and without AKI compared to healthy volunteers.

Figure 6. The distribution of Fetuin-A in rat urine 2 days after cisplatin injection

(A) The secretion of Fetuin-A in different urinary fractions from the same urine sample (lane 1: 200,000 × g pellets, lane 2: 17,000 × g pellets, lane 3: whole urine, lane 4: 17,000 × g supernatant, and lane 5: 200,000 × g supernatant) by western blot analysis. The lower label indicates the original volume of urine used for each lane. (B) Comparison of urinary exosomal Fetuin-A in the 200,000 × g pellets without and with washing by isolation solution (lane 1: unwashed pellet; lane 2: washed pellets). (C) Fetuin-A located inside of urinary vesicles in 200,000 × g pellets by immunoelectron microscopy, Bar=100 nm. Inset shows a magnified image of a urinary vesicle labeled with gold-conjugated anti-Fetuin-A.

Figure 7. Location of Fetuin-A in outer stripe of the outer medulla (OSOM) of rat kidney

Left panel: Temporal immunohistochemistry staining for Fetuin-A in OSOM of kidneys before (A) and at day 2 (B), day 3 (C), and day 5 (D) after cisplatin injection in rats. Right panel: Negative control (injured kidney section at day 3 after cisplatin) (E), Sham surgery for Ischemia and reperfusion (I/R) (F), I/R rats at 8 hr (G), and 24 hr (H) after I/R.