Glomerular endothelial derived vesicles mediate podocyte dysfunction: A potential role for miRNA.

MicroRNAs (miRNA) are shown to be involved in the progression of several types of kidney diseases. Podocytes maintain the integrity of the glomerular basement membrane. Extracellular vesicles (EV) are important in cell-to-cell communication as they can transfer cellular content between cells, including miRNA. However, little is known about how extracellular signals from the glomerular microenvironment regulate podocyte activity. Using a non-contact transwell system, communication between glomerular endothelial cells (GEnC) and podocytes was characterised in-vitro. Identification of transferred EV-miRNAs from GEnC to podocytes was performed using fluorescence cell tracking and miRNA mimetics. To represent kidney disease, podocyte molecular profiling and functions were analysed after EV treatments derived from steady state or activated GEnC. Our data shows activation of GEnC alters EV-miRNA loading, but activation was not found to alter EV secretion. EV delivery of miRNA to recipient podocytes altered cellular miRNA abundance and effector functions in podocytes, including decreased secretion of VEGF and increased mitochondrial stress which lead to altered cellular metabolism and cytoskeletal rearrangement. Finally, results support our hypothesis that miRNA-200c-3p is transfered by EVs from GEnC to podocytes in response to activation, ultimately leading to podocyte dysfunction.

Arginase II inhibition prevents nitrate tolerance.

BACKGROUND AND PURPOSE: Nitrate tolerance, the loss of vascular responsiveness with continued use of nitrates, remains incompletely understood and is a limitation of these therapeutic agents. Vascular superoxide, generated by uncoupled endothelial NOS (eNOS), may play a role. As arginase competes with eNOS for L-arginine and may exacerbate the production of reactive oxygen species (ROS), we hypothesized that arginase inhibition might reduce nitrate tolerance. EXPERIMENTAL APPROACH: Vasodilator responses were measured in aorta from C57Bl/6 and arginase II knockout (argII -/-) mice using myography. Uncoupling of eNOS, determined as eNOS monomer : dimer ratio, was assessed using low-temperature SDS-PAGE and ROS levels were measured using L-012 and lucigenin-enhanced chemiluminescence. KEY RESULTS: Repeated application of glyceryl trinitrate (GTN) on aorta isolated from C57Bl/6 mice produced a 32-fold rightward shift of the concentration-response curve. However this rightward shift (or resultant tolerance) was not observed in the presence of the arginase inhibitor (s)-(2-boronethyl)-L-cysteine HCl (BEC; 100 µM) nor in aorta isolated from argII -/- mice. Similar findings were obtained after inducing nitrate tolerance in vivo. Repeated administration of GTN in human umbilical vein endothelial cells induced uncoupling of eNOS from its dimeric state and increased ROS levels, which were reduced with arginase inhibition and exogenous L-arginine. Aortae from GTN tolerant C57Bl/6 mice exhibited increased arginase activity and ROS production, whereas vessels from argII -/- mice did not. CONCLUSION AND IMPLICATIONS: Arginase II removal prevents nitrate tolerance. This may be due to decreased uncoupling of eNOS and consequent ROS production.