In the renal collecting duct, vasopressin increases osmotic water permeability (P f ) by triggering trafficking of aquaporin-2 vesicles to the apical plasma membrane. We investigated the role of vasopressin-induced intracellular Ca 2؉ mobilization in this process. In isolated inner medullary collecting ducts (IMCDs) Arginine vasopressin (AVP) 1 regulates water transport across the epithelium of the renal collecting duct, allowing precise control of water excretion. Water transport across the collecting duct is mediated by molecular water channels, the aquaporins (1, 2). Aquaporin-2 provides the water transport pathway across the apical plasma membrane of the collecting duct principal cells, whereas aquaporins-3 and -4 facilitate water transport across the basolateral plasma membrane. AVP increases the osmotic water permeability (P f ) of the collecting duct cells by triggering translocation of intracellular vesicles containing aquaporin-2 to the apical plasma membrane (3), thus increasing the number of water channels in the ratelimiting barrier for transepithelial water transport. This response depends on the binding of AVP to V 2 vasopressin receptors in the basolateral plasma membrane. These receptors couple to the heterotrimeric G protein, G s , which activates the effector enzyme adenylyl cyclase type VI (4) and increases cyclic AMP levels in the cells. Vasopressin, acting via the V 2 receptor, also causes a transient increase in intracellular Ca 2ϩ (5-8). Little is known about the mechanism of the vasopressininduced increase in intracellular Ca 2ϩ , although previous studies establish that it occurs in the absence of activation of the phosphoinositide signaling pathway (9). Little is known also about the physiological role of the vasopressin-induced increase in intracellular Ca 2ϩ in the regulation of aquaporin-2 trafficking. However, studies of a wide variety of vesicular-trafficking processes have pointed to a key role for localized increases in intracellular Ca 2ϩ in triggering the fusion of vesicles with their target membranes (10), raising the possibility that the same could be true for aquaporin-2 vesicle trafficking. One calciumdependent mediator that has been suggested to play a role in water permeability regulation in the vasopressin-responsive toad bladder epithelium is calmodulin (11). Based on recent studies of homotypic fusion of yeast vacuoles, Peters and Mayer conclude that a critical final step in the process of vesicle fusion is dependent on calmodulin (12), and calmodulin actions can be postulated at other steps involved in vasopressin signaling or aquaporin-2 trafficking. In the present study, we investigate the role of intracellular Ca 2ϩ and calmodulin in the AVPmediated regulation of aquaporin-2 trafficking, assessed through the measurement of osmotic water permeability (P f ) in isolated perfused inner medullary collecting duct (IMCD) segments and through immunofluorescence localization of aquaporin-2 in cultured IMCD cells. The results support the view that stimulation of aquaporin-2...

Retinal degenerative diseases, such as glaucoma and macular degeneration, affect millions of people worldwide and ultimately lead to retinal cell death and blindness. Cell transplantation therapies for photoreceptors demonstrate integration and restoration of function, but transplantation into the ganglion cell layer is more complex, requiring guidance of axons from transplanted cells to the optic nerve head in order to reach targets in the brain. Here we create a biodegradable electrospun (ES) scaffold designed to direct the growth of retinal ganglion cell (RGC) axons radially, mimicking axon orientation in the retina. Using this scaffold we observed an increase in RGC survival and no significant change in their electrophysiological properties. When analyzed for alignment, 81% of RGCs were observed to project axons radially along the scaffold fibers, with no difference in alignment compared to the nerve fiber layer of retinal explants. When transplanted onto retinal explants, RGCs on ES scaffolds followed the radial pattern of the host retinal nerve fibers, whereas RGCs transplanted directly grew axons in a random pattern. Thus, the use of this scaffold as a cell delivery device represents a significant step towards the use of cell transplant therapies for the treatment of glaucoma and other retinal degenerative diseases.

Retinal ganglion cells (RGCs) are responsible for the transfer of signals from the retina to the brain. As part of the central nervous system, RGCs are unable to regenerate following injury, and implanted cells have limited capacity to orient and integrate in vivo. During development, secreted guidance molecules along with signals from extracellular matrix and the vasculature guide cell positioning, for example, around the fovea, and axon outgrowth; however, these changes are temporally regulated and are not the same in the adult. Here, we combine electrospun cell transplantation scaffolds capable of RGC neurite guidance with thermal inkjet 3D cell printing techniques capable of precise positioning of RGCs on the scaffold surface. Optimal printing parameters are developed for viability, electrophysiological function and, neurite pathfinding. Different media, commonly used to promote RGC survival and growth, were tested under varying conditions. When printed in growth media containing both brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF), RGCs maintained survival and normal electrophysiological function, and displayed radial axon outgrowth when printed onto electrospun scaffolds. These results demonstrate that 3D printing technology may be combined with complex electrospun surfaces in the design of future retinal models or therapies.

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