Elevated expressions of myeloid-related proteins-8 and -14 are danger biomarkers for lupus nephritis.

Myeloid-related proteins, MRP-8 and -14, which have been identified as molecules that mediate the danger signaling in innate immune response, are also known as the DAMPs (damage associated molecular pattern molecules). The proteins were found in infiltrating macrophages and neutrophils at inflammatory sites. Their expression was correlated with severe forms of glomerulonephritis. Therefore, this study examined whether or not MRP-8 and -14 can be used as biomarkers for identifying severely active lupus nephritis (LN). Total blood leukocyte samples and renal biopsy tissues from a prospective cohort of LN patients were used to determine mRNA and protein expression levels of MRP-8 and -14. The mRNA levels of MRP-8 and -14 in total blood leukocytes were significantly higher in active LN patients than quiescent LN patients and healthy controls. Moreover, the mRNA levels of MRP-8 and -14 in the total blood leukocytes and kidney tissues were significantly correlated with therapeutic response and the mRNA expression levels in the kidney were associated with an early loss of the kidney function. MRP-8 and -14 can be used as non-invasive prognostic biomarkers in patients with LN.

Exosomes in urine: who would have thought...?

Normal urine contains thousands of proteins, largely due to the presence of 'exosomes,' tiny vesicles secreted into the urine by renal epithelial cells. These exosomes, demonstrated by Keller and colleagues to be also retrievable from amniotic fluid, offer great promise for future disease biomarker discovery studies.

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

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.

Collection, storage, preservation, and normalization of human urinary exosomes for biomarker discovery.

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 studied methods for collection, storage, and preservation of urinary exosomal proteins. We collected urine from healthy volunteers, added protease inhibitors, and stored urine samples at 4, -20, and -80 degrees C for 1 week or 7 months. Samples were thawed with and without extensive vortexing, and three fractions were isolated: urinary sediment, supernatant, and exosome fraction. Protein concentration, electrophoresis patterns, and abundance of seven exosome-associated proteins were measured. Exosome-associated proteins were not detected in sediment or supernatant fractions. Protease inhibitors prevented degradation of exosome-associated proteins. Freezing at -20 degrees C caused a major loss in exosomes compared to fresh urine. In contrast, recovery after freezing at -80 degrees C was almost complete. Extensive vortexing after thawing markedly increased exosome recovery in urine frozen at -20 or -80 degrees C, even if frozen for 7 months. The recovery from first and second morning urine was similar. The abundance of cytosolic exosome-associated proteins did not decrease during long-term storage. We concluded: (1) protease inhibitors are essential for preservation; (2) storage at -80 degrees C with extensive vortexing after thawing maximizes the recovery of urinary exosomes; (3) the difference between first and second morning urine exosome-associated protein was small, suggesting minimal protein degradation in the urinary tract/bladder; (4) urinary exosomes remain intact during long-term storage. These urine collection, storage, and processing conditions may be useful for future biomarker discovery efforts.

Convective-controlled double high flux hemodiafiltration: a novel blood purification modality.

Convective-controlled double high flux hemodiafiltration (CC-DHF) was set-up using two high flux dialyzers. The convection occurred in the first while the fluid replacement took place in the second dialyzer. The system of CC-DHF basically resembled that of hemodiafiltration. CC-DHF was performed in 9 chronic hemodialysis Thai patients who had been treated with high flux hemodialysis for at least 6 months. When compared with high flux hemodialysis, CC-DHF could provide higher Kt/Vurea (2.4+/-0.4 vs. 2.0+/-0.4, p<0.05) and beta2-microglobulin clearance (106.2+/-15.4 vs. 48.9+/-6.1 ml/min, p<0.01). Following 6-month therapy of CC-HDF, the predialysis beta2-microglobulin levels were reduced by 12.7% while the values of Kt/Vurea were consistently higher than 2.7. The quality of life consistently improved during the 6 months of CC-DHF treatment. There were no differences in clinical and technical complications between CC-DHF and high flux hemodialysis. In conclusion, CC-DHF could provide performance comparable to hemodiafiltration without the need for expensive hemodiafiltration machines.