Subfractionation, characterization, and in-depth proteomic analysis of glomerular membrane vesicles in human urine.

Urinary exosome-like vesicles (ELVs) are a heterogenous mixture (diameter 40-200 nm) containing vesicles shed from all segments of the nephron including glomerular podocytes. Contamination with Tamm-Horsfall protein (THP) oligomers has hampered their isolation and proteomic analysis. Here we improved ELV isolation protocols employing density centrifugation to remove THP and albumin, and isolated a glomerular membranous vesicle (GMV)-enriched subfraction from 7 individuals identifying 1830 proteins and in 3 patients with glomerular disease identifying 5657 unique proteins. The GMV fraction was composed of podocin/podocalyxin-positive irregularly shaped membranous vesicles and podocin/podocalyxin-negative classical exosomes. Ingenuity pathway analysis identified integrin, actin cytoskeleton, and Rho GDI signaling in the top three canonical represented signaling pathways and 19 other proteins associated with inherited glomerular diseases. The GMVs are of podocyte origin and the density gradient technique allowed isolation in a reproducible manner. We show many nephrotic syndrome proteins, proteases, and complement proteins involved in glomerular disease are in GMVs and some were only shed in the disease state (nephrin, TRPC6, INF2 and phospholipase A2 receptor). We calculated sample sizes required to identify new glomerular disease biomarkers, expand the ELV proteome, and provide a reference proteome in a database that may prove useful in the search for biomarkers of glomerular disease.

Impact of left ventricular function on immediate and long-term outcomes after pericardiectomy in constrictive pericarditis.

OBJECTIVE: Most patients with constrictive pericarditis have normal measures of left ventricular function when assessed by the ejection phase index of ejection fraction, yet there is a wide spectrum of outcome after pericardiectomy. We hypothesized that parameters of non-ejection indexes of cardiac function (+dP/dt and tau) may predict postoperative prognosis. METHODS: The immediate and long-term outcomes of pericardiectomy were analyzed in 40 patients (30 male, mean age 62 years) with surgically confirmed constrictive pericarditis who underwent preoperative cardiac catheterization using high-fidelity micromanometer pressures. Left ventricular pressures were digitized at 5-msec intervals during end expiration, from which peak positive dp/dt and tau measurements were obtained. Patients were classified into 3 groups: Group 1 (n = 13) included those with abnormal +dP/dt and tau (defined as +dP/dt < 1200 mm Hg/s, tau > 50 msec); group 2 (n = 11) included those with either abnormal +dP/dt or tau; and group 3 (n = 16) included those with normal +dP/dt and tau. RESULTS: There were no significant differences of gender, New York Heart Association class, duration of symptoms, and underlying cause among the 3 groups. Group 1 patients had lower preoperative ejection fraction and higher left and right ventricular end-diastolic pressures. Postoperative inotropic support was more frequently needed in group 1, and postoperative mortality was higher in group 1 than in groups 2 and 3. All postoperative deaths but 1 were in group 1. The median postoperative follow-up was 2.4 years. The postoperative long-term survival of group 1 was significantly lower compared with that of groups 2 and 3. CONCLUSION: In patients with constrictive pericarditis undergoing pericardiectomy, those with abnormal left ventricular contractility and relaxation properties assessed by cardiac catheterization before surgery incur higher operative mortality and poor long-term outcome after surgery.