Rapid analyses of proteomes and interactomes using an integrated solid-phase extraction-liquid chromatography-MS/MS system.

Here, we explore applications of a LC system using disposable solid-phase extraction (SPE) cartridges and very short LC-MS/MS gradients that allows for rapid analyses in less than 10 min analysis time. The setup consists of an autosampler harboring two sets of 96 STAGE tips that function as precolumns and a short RP analytical column running 6.5 min gradients. This system combines efficiently with several proteomics workflows such as offline prefractionation methods, including 1D gel electrophoresis and strong-cation exchange chromatography. It also enables the analysis of interactomes obtained by affinity purification with an analysis time of approximately 1 h. In a typical shotgun proteomics experiment involving 36 SCX fractions of an AspN digested cell lysate, we detected over 3600 protein groups with an analysis time of less than 5.5 h. This innovative fast LC system reduces proteome analysis time while maintaining sufficient proteomic detail. This has particular relevance for the use of proteomics within a clinical environment, where large sample numbers and fast turnover times are essential.

No effect of remote ischaemic conditioning on inflammation in a porcine kidney transplantation model.

MAIN PROBLEM: Delayed graft function after kidney transplantation is associated with decreased graft survival and increased patient mortality but the pathogenesis is poorly understood. Remote ischaemic conditioning (rIC) may prevent delayed graft function by an anti-inflammatory effect. In a porcine model of transplantation from adults to children, we investigated the inflammatory response in the transplanted kidney and the effect of rIC. METHODS: Kidneys were recovered from brain dead donor pigs(63kg) and transplanted into two groups of recipient pigs(15kg) after 22h of cold ischaemia. Recipients were randomised to either: rIC (n=8) performed before the 10-h reperfusion period or no-rIC (n=8). Non-transplanted kidneys from eight brain dead pigs served as controls. RESULTS: Compared to controls, transplantation increased the number of apoptotic cells, macrophages and neutrophils in the kidney. After transplantation, IL-10 levels increased and IL-6 levels decreased in the kidney, whereas levels of TNF-α and IL-8 were not affected. A significant rise in plasma IL-1ß and IL-6 was observed in the recipients after transplantation. Plasma IL-10 was not affected by transplantation and TNF-α and IL-8 were below detection limit. No effect of rIC was found with regards to cell infiltration or cytokine production. CONCLUSION: Renal transplantation elicits an inflammatory response in the kidney manifested as apoptotic cell death, macrophage and neutrophil infiltration, and an anti-inflammatory cytokine response 10h after transplantation. This response was not modified by rIC.

Improved GFR and renal plasma perfusion following remote ischaemic conditioning in a porcine kidney transplantation model.

Delayed graft function (DGF) complicates approximately 25% of kidney allografts donated after brain death (DBD). Remote ischaemic conditioning (rIC) involves brief, repetitive, ischaemia in a distant tissue in connection with ischaemia/reperfusion in the target organ. rIC has been shown to induce systemic protection against ischaemic injuries. Using a porcine kidney transplantation model with donor (63 kg) recipient (15 kg) size mismatch, we investigated the effects of recipient rIC on early renal plasma perfusion and GFR. Brain death was induced in donor pigs (n = 8) and kidneys were removed and kept in cold storage until transplantation. Nephrectomized recipient pigs were randomized to rIC (n = 8) or non-rIC (n = 8) with one kidney from the same donor in each group. rIC consisted of 4 × 5 min clamping of the abdominal aorta. GFR was significantly higher in the rIC group compared with non-rIC (7.2 ml/min vs. 3.4 ml/min; ΔGFR = 3.7 ml/min, 95%-CI: 0.3-7.2 ml/min, P = 0.038). Renal plasma perfusion in both cortex and medulla measured by dynamic contrast-enhanced magnetic resonance imaging (MRI) was significantly higher over time in the rIC group compared with non-rIC. This experimental study demonstrated a positive effect of rIC on early graft perfusion and function in a large animal transplantation model.