Hemofiltration induces generation of leukocyte-derived CD31+/CD41- microvesicles in sepsis.

BACKGROUND: Microvesicles (MV) are extracellular vesicles known to be associated with cellular activation and inflammation. Hemofiltration is an effective blood purification technique for patients with renal failure and possibly also eliminates inflammatory mediators in the setting of sepsis. On the other hand, proinflammatory stimuli are induced by blood contacting the artificial membrane during extracorporeal blood purification. In chronic dialysis patients a systemic increase in MV has been described. The aim of the study was to investigate whether hemofilter passage of blood in continuous veno-venous hemofiltration (CVVH) alters MV composition and levels in critically ill patients with sepsis. METHODS: Pre- and postfilter bloods as well as ultrafiltrate samples from intensive care unit patients with severe sepsis were obtained during CVVH with regional citrate anticoagulation. MV subtypes in blood were analyzed by high-sensitivity flow cytometry. Additionally, tissue factor (TF) levels and MV-associated TF activities as well as MV activities were quantified. All parameters were corrected for hemoconcentration applied during CVVH. RESULTS: Twelve patients were analyzed. A significant increase in presumably mostly leukocyte-derived CD31+/CD41- MV (1.32 (1.09-1.93)-fold [median (25th-75th quartiles)], p = 0.021) was observed post- to prefilter, whereas platelet-derived MV as well as AnnexinV-binding MV were unaltered. Increments of AnnexinV+, CD42b+ and CD31+/CD41- MV post- to prefilter correlated with filtration fraction (FF) (all p < 0.05). Significant reductions in MV activity [0.72 (0.62-0.84)-fold, p = 0.002] and TF level [0.95 (0.87-0.99)-fold, p = 0.0093] were detected postfilter compared to prefilter. No MV activity was measurable in ultrafiltrate samples. CONCLUSIONS: Despite clearing a fraction of small PS-exposing MV CVVH does not eliminate larger MV. Concurrently, CVVH induces the release of CD31+/CD4- MV that indicate leukocyte activation during hemofilter passage in septic patients. Increments of several MV subtypes within the hemofilter correlate with FF, which supports common recommendations to keep FF low. A fraction of TF is being cleared by CVVH via ultrafiltration.

Characterization of Microvesicles in Septic Shock Using High-Sensitivity Flow Cytometry.

PURPOSE: Endothelial pathology is considered to play a key role in septic shock. Since endothelial-derived microvesicles (MV) are elevated in various diseases associated with endothelial pathology, they are considered surrogate markers of the endothelial state. By analyzing the signature of circulating MV with high-sensitivity flow cytometry (hsFC), we wanted to test the hypothesis whether endothelial-derived MV are increased in septic shock. METHODS: MV in blood from healthy volunteers and patients with septic shock treated in a medical intensive care unit were quantified by hsFC, which has an improved detection limit of approximately 0.3 µm. RESULTS: Patients with septic shock (n = 30) showed 3-fold higher levels of CD31+/CD41- MV (58.5 (26.4-101.2) [median (25th-75th percentile)] vs. 19.5 (12.8-25.4) MV/µL; P <0.001) compared with healthy volunteers (n = 18). Absolute counts of CD144+, CD62E+, and CD106+ MV, specific for endothelial-derived MV, were low in all groups. The number of CD31+/CD41- MV correlated significantly with leukocyte count (rs = 0.64; P <0.001). Platelet-derived CD41+ MV were significantly elevated in the group dying within 48 h after inclusion (639.1 (321.3-969.7) vs. 221.5 (119.5-456.9) MV/µL; P = 0.037). Patients dying within 48 h had also significantly higher levels of CD31+/CD41-/AnnexinV- MV (51.9 (24.9-259.8) vs. 18.9 (9.7-31) MV/µL; P = 0.028). CONCLUSIONS: Despite an improved detection limit for MV by using hsFC, counts of endothelial-specific MV are unexpectedly low in patients with septic shock. Increased amounts of CD41+ and CD31+/CD41-/AnnexinV- MV indicate release by activated platelets and possibly leukocytes correlating with unfavorable outcome.

Oncostatin M inhibits TGF-ß1-induced CTGF expression via STAT3 in human proximal tubular cells.

Matricellular proteins play a critical role in the development of tubulointerstitial fibrosis and renal disease progression. Connective tissue growth factor (CTGF/CCN2), a CCN family member of matricellular proteins, represents an important mediator during development of glomerular and tubulointerstitial fibrosis in progressive kidney disease. We have recently reported that oncostatin M (OSM) is a potent inhibitor of TGF-ß1-induced CTGF expression in human proximal tubular cells (PTC). In the present study we examined the role of TGF-ß1- and OSM-induced signaling mechanisms in the regulation of CTGF mRNA expression in human proximal tubular HK-2 cells. Utilizing siRNA-mediated gene silencing we found that TGF-ß1-induced expression of CTGF mRNA after 2h of stimulation at least partially depends on SMAD3 but not on SMAD2. In contrast to TGF-ß1, OSM seems to exert a time-dependent dual effect on CTGF mRNA expression in these cells. While OSM led to a rapid and transient induction of CTGF mRNA expression between 15 min and 1h of stimulation it markedly suppressed basal and TGF-ß1-induced CTGF mRNA levels thereafter. Silencing of STAT1 or STAT3 attenuated basal CTGF mRNA levels indicating that both STAT isoforms may be involved in the regulation of basal CTGF mRNA expression. However, knockdown of STAT3 but not STAT1 prevented OSM-mediated suppression of basal and TGF-ß1-induced upregulation of CTGF mRNA expression. Together these results suggest that the inhibitory effect of OSM on TGF-ß1-induced CTGF mRNA expression is mainly driven by STAT3, thereby providing a signaling mechanism whereby OSM may contribute to tubulointerstitial protection.

Oncostatin M is a novel inhibitor of TGF-ß1-induced matricellular protein expression.

Matricellular proteins in the kidney have been associated with the development of tubulointerstitial fibrogenesis and the progression of renal disease. This study investigated potential antifibrotic effects of the cytokine oncostatin M (OSM) in human proximal tubule cells (PTC), particularly with regard to inhibition of profibrotic events initiated by TGF-ß1. In human PTC, OSM diminished transforming growth factor (TGF)-ß1-induced expression of the transcriptional epithelial-mesenchymal transition mediator FoxC2. Furthermore, exposure to OSM attenuated basal and TGF-ß1-induced expression of the matricellular proteins SPARC, TSP-1, TNC, and CTGF regardless of the sequence of ligand administration. OSM was shown to result in rapid and sustained phosphorylation of both Stat1 and Stat3 and also in transient phosphorylation of Smad2/3 in contrast to TGF-ß1, which demonstrated a gradually building phosphorylation of Smad2/3 and a brief phosphorylation of Smad1/5/8. Utilizing receptor-blocking molecules, we found the inhibitory effect of OSM on TGF-ß1-induced CTGF mRNA expression occurs independently of Smad2/3 signaling and present evidence that this effect may be partially driven by OSM receptor-mediated Stat1 and/or Stat3 signaling pathways, thereby providing a mechanism whereby OSM can contribute to tubulointerstitial protection.