Short-term functional adaptation of aquaporin-1 surface expression in the proximal tubule, a component of glomerulotubular balance

Transepithelial water flow across the renal proximal tubule is mediated predominantly by aquaporin-1 (AQP1). Along this nephron segment, luminal delivery and transepithelial reabsorption are directly coupled, a phenomenon called glomerulotubular balance. We hypothesized that the surface expression of AQP1 is regulated by fluid shear stress, contributing to this effect. Consistent with this finding, we found that the abundance of AQP1 in brush border apical and basolateral membranes was augmented >2-fold by increasing luminal perfusion rates in isolated, microperfused proximal tubules for 15 minutes. Mouse kidneys with diminished endocytosis caused by a conditional deletion of megalin or the chloride channel ClC-5 had constitutively enhanced AQP1 abundance in the proximal tubule brush border membrane. In AQP1-transfected, cultured proximal tubule cells, fluid shear stress or the addition of cyclic nucleotides enhanced AQP1 surface expression and concomitantly diminished its ubiquitination. These effects were also associated with an elevated osmotic water permeability. In sum, we have shown that luminal surface expression of AQP1 in the proximal tubule brush border membrane is regulated in response to flow. Cellular trafficking, endocytosis, an intact endosomal compartment, and controlled protein stability are the likely prerequisites for AQP1 activation by enhanced tubular fluid shear stress, serving to maintain glomerulotubular balance

Inhibition of Notch signaling induces extensive intussusceptive neo-angiogenesis by recruitment of mononuclear cells

Notch is an intercellular signaling pathway related mainly to sprouting neo-angiogenesis. The objective of our study was to evaluate the angiogenic mechanisms involved in the vascular augmentation (sprouting/intussusception) after Notch inhibition within perfused vascular beds using the chick area vasculosa and MxCreNotch1(lox/lox) mice. In vivo monitoring combined with morphological investigations demonstrated that inhibition of Notch signaling within perfused vascular beds remarkably induced intussusceptive angiogenesis (IA) with resultant dense immature capillary plexuses. The latter were characterized by 40% increase in vascular density, pericyte detachment, enhanced vessel permeability, as well as recruitment and extravasation of mononuclear cells into the incipient transluminal pillars (quintessence of IA). Combination of Notch inhibition with injection of bone marrow-derived mononuclear cells dramatically enhanced IA with 80% increase in vascular density and pillar number augmentation by 420%. Additionally, there was down-regulation of ephrinB2 mRNA levels consequent to Notch inhibition. Inhibition of ephrinB2 or EphB4 signaling induced some pericyte detachment and resulted in up-regulation of VEGFRs but with neither an angiogenic response nor recruitment of mononuclear cells. Notably, Tie-2 receptor was down-regulated, and the chemotactic factors SDF-1/CXCR4 were up-regulated only due to the Notch inhibition. Disruption of Notch signaling at the fronts of developing vessels generally results in massive sprouting. On the contrary, in the already existing vascular beds, down-regulation of Notch signaling triggered rapid augmentation of the vasculature predominantly by IA. Notch inhibition disturbed vessel stability and led to pericyte detachment followed by extravasation of mononuclear cells. The mononuclear cells contributed to formation of transluminal pillars with sustained IA resulting in a dense vascular plexus without concomitant vascular remodeling and maturatio

Interaction of hemodynamic and metabolic parameters during angiogenesis and angioadaptation

Die strukturelle Adaptation der Gefäßbetten ist mit funktioneller Anpassung, bedingt durch Interaktionen rheologischer, metabolischer, hämodynamischer und molekularer Faktoren, assoziiert. Infolgedessen wachsen Gefäße oder werden zurückgebildet, was eine sich ständig ändernde Sauerstoffverteilung zur Folge hat. Aufgrund der engen Beziehung zwischen dem Sauerstoffangebot und der Angioadaptation, ist die Bestimmung lokaler Sauerstoffkonzentration von sehr großer Bedeutung. Um die lokale Sauerstoffsättigung (SO2) messen zu können, wurde eine nichtinvasive multispektrale Methode entwickelt. Dieses Verfahren macht sich die Unterschiede in den Absorptionscharakteristika zwischen Oxy- und Desoxyhämoglobin für die Messung von SO2 und Hämatokrit während Intravitalmikroskopie zunutze. Die Sauerstoffsättigung und der Hämatokrit wird zweidimensional kartiert, die Kalkulation der Werte und die Erstellung der Bilder erfolgt mithilfe einer Software, die speziell zu diesem Zweck entwickelt wurde (Saturation of Oxygen Analysis Program=SOAP). Weiterhin wurde ein mathematisches Modell ausgearbeitet, das die Flusseigenschaften der roten Blutzellen beschreibt . Eine Analyse ausgewählter, für die Angioadaptation relevanter, Gene wurde durchgeführt.The structural adaptation of vascular beds is associated with functional alignment caused by the interaction of hemorheology, metabolics, hemodynamics and gene expression. In consequence, vessels grow or degenerate, resulting in an altered oxygen distribution in vascular networks. Because of the close connection between oxygen availability and angioadaptation, the local oxygen saturation in microvessels is of prime importance for intravital studies in terminal vascular beds. In order to obtain vital status of tissues at the local level a non invasive multispectral approach was developed. This method based on differences in absorption spectra between oxygenated and deoxygenated haemoglobin and allows oxygen saturation (SO2) and hematocrit measurement during trans- and epi-ilumination intravital microscopy. The SO2 and hematocrit values as 2D map of area under investigation could be calculated using for this purpose developed analysis software (SOAP). This technique allows generation of intravascular SO2 and hematocrit images for all vessels in a microscopic field of view in vivo in different tissues and under different conditions. Furthermore a two-dimensional computer simulation to predict trajectories of single red blood cells was developed, since rheological behaviour of erythrocytes influence oxygen distribution. Supplementary an analysis angioadaptation related genes VEGFA, TIE2, ANG2 and ADAMTS was carried out

Avian area vasculosa and CAM as rapid in vivo pro-angiogenic and antiangiogenic models.

Angiogenesis, the development of new blood vessels from preexisting ones, is driven by coordinated signaling pathways governed by specific molecules, hemodynamic forces, and endothelial and periendothelial cells. The processes involve adhesion, migration, and survival machinery within the target endothelial and periendothelial cells. Factors that interfere with any of these processes may therefore influence angiogenesis either positively (pro-angiogenesis) or negatively (antiangiogenesis). The avian area vasculosa (AV) and the avian chorioallantoic membrane (CAM) are two useful tools for studying both angiogenesis and antiangiogenesis since they are amenable to both intravascular and topical administration of target, agents, are relatively rapid assays, and can be adapted very easily to study angiogenesis-dependent processes, such as tumor growth. Both models provide a physiological setting that permits investigation of pro-angiogenic and antiangiogenic agent interactions in vivo

Logarithmic line graphs showing growth rates of CAM (a) and the various coarse components of the CAM (B).

<p><b>a:</b> The CAM grew rapidly in phase I (E8-E13), moderately at phase II (E13-18) and was undergoing regression in phase III (E18-E20). CAM volume was significantly correlated to body mass (P = 0.01). CAM growth was strongly positively related to body mass increase up to E18, but it had a strong negative correlation until time of hatching. <b>b:</b> Logarithmic line graphs showing growth regression analysis of the coarse CAM components. The mesoderm was the fastest growing component while the large vessels were the slowest. Both the chorion and allantois grew at the same rate. When viewed on individual basis at various growth phases, all components were growing fastest in phase I and except the mesoderm, they were regressing in phase III. All components were significantly correlated to body mass during the entire period (P ≤ 0.05). The chorion, allantois and vessels were regressing in phase III (showed a strong negative correlation with body mass).</p

TEM micrographs showing the changing ultrastructure of the allantoic layer between E8 and E20.

<p>Note that in all cases the microvilli project into the allantoic lumen (asterisks) and this is important in identifying the mesenchyme (Me) in cases where mesenchymal cells are not evident. <b>a and b</b>: The allantois at E8 consists of a single layer of squamous fibroblast like cells (f) with numerous filopodia (white arrowheads) extending into the mesenchyme and short microvilli (black arrowheads) towards the allantoic lumen. The mesodermal layer has loose connective tissue and fibroblasts (white arrows). Some cells in the allantois show mitotic figures (black arrow in B). <b>c and d:</b> Thickening of the allantois is notable by E12 where it starts to recruit cells to form a two cell layer separated by enormous intercellular spaces (arrow in c). The first few granules start to appear in the outer layer of the allantois (arrowhead in c). A mesenchymal cell (Mc) is closely aligned with the inner layer of the epithelium. By E15, a two-cell layer is well accomplished and formation of the third layer begins (see layers 1, 2, 3 in d). Notice the granules in the outer layer of the allantois (arrowheads) are well developed. The basal border looks irregular and the basement membrane is amorphous (black arrows) and there are cells close to the epithelium (white arrow). <b>e and f:</b> The allantois has 3–4 well-formed cell layers at E18 and the basement membrane (black arrows) is neatly formed. The outer cell layer has abundant glycogen granules (arrowheads) and short microvilli. By E20, the layers of the allantois look shrunken and the glycogen granules (arrowheads in f) are depleted.</p

Growth phases and percentage changes in body mass and CAM volume in the developing chick embryo.

<p>Growth phases and percentage changes in body mass and CAM volume in the developing chick embryo.</p

TEM micrographs showing the changing structure of the chorionic layers.

<p><b>a and b</b>: The chorion at E8 is thin with large capillaries (Ca) separated by undifferentiated epithelial cells (arrowhead in a). The mesenchyme (Me) is also shown. By E11 there is recruitment of cells from the subchorionic layer of the mesenchyme (Me). Such cells (white arrow) are loosely attached to primary cells of the chorion (black arrow in b). At these stages the various cell types of the chorionic layer are not differentiated. <b>c and d</b>: The typical chorionic cells such as the villous cavity (VC) cells and basal (BC) cells become recognizable by E12 and by E15 (d) such cells are well differentiated. The chorionic capillaries (Ca) remain on the external aspect of the epithelium. <b>e and f</b>: By E18 the first signs of degeneration are evident in the VC, with loss of microvilli although some cells in the basal layer show some mitotic activity (arrowhead). Clear signs of cell degeneration at E20 include dissolution of basal cells (BC) and crumbling of VC cells.</p

Bar graphs showing the variation in body mass (g), CAM volume (cm³) and body mass standardized CAM volume (cm³g^-1).

<p><b>a</b>: Notice that body mass increases steadily between E8-E13, sharply between E13-E15 and then steadily between E15-E20, marking the three growth phases. <b>b</b>: The CAM volume increases fast between E8-E13, steadily between E13-E15 and then declined between E18-E20, the latter was thus a phase of regression. The phase of rapid CAM growth precedes that of embryonic growth. On Scheffé's test (p ≤0.05) CAM volume at E8 and E11 were significantly lower than at all the other subsequent days (asterisks). <b>c:</b> Body-mass standardized CAM volume decreased gradually through the entire period.</p