BACKGROUND Heart disease and renal dysfunction are often mutually reinforcing conditions, although the factors underlying this relationship are not fully understood. Cardiac remodelling resulting from disease is partly caused by the reactivation of developmental programmes. By unravelling the mechanisms that drive kidney development and function, it may be possible to gain novel insight into remodelled kidney states that are linked to disease. In this study, we have investigated the interplay between renal and cardiovascular systems during nephrogenesis at the level of the blood filter. METHODOLOGY Owing to the optical transparency and rapid external development of the embryo, the zebrafish provides a research model for advanced imaging technologies, allowing us to visualise structures located deep within living specimens. Here, we combine deep-tissue live imaging and novel functional assays to study development and function of the pronephric kidney - the first and most basic kidney to form in the embryo. RESULTS Using two-photon excitation microscopy, we have successfully established methodology for performing, deep-tissue, time-lapse imaging in living embryos of the two primary cell types forming the kidney – endothelial and epithelial cells. Fluorescence angiograms were performed using supra-vital dye agents to visualise circulatory flow in relation to the pronephric vasculature and the process of blood-filtration. Observations from live imaging studies, supported by immunostaining, were used to create a comprehensive model of the developing glomerular morphology, and interactions at the endothelial-epithelial cell interface, where glomerular epithelial primordia merge around the vascular component of the pronephric kidney. To investigate renal function, we devised a novel assay of pronephric filtration, by tracking the accumulation of injected fluorescent tracers within the excreted filtrate of embryos. This allowed us to relate our time-lapse observations to maturation of the glomerulus, and the evolution of perm-selective function. Finally, we explored methods of mechanically obstructing blood-flow in order to investigate whether altered hemodynamic forces would influence pronephric development. We found that in those embryos with severely disrupted circulatory flow, the glomerular morphology was affected. CONCLUSIONS In summary, the combination of these techniques has allowed us to visualise the multi-cellular organisation of the pronephric kidney over time, which has previously been limited to primarily fixed-tissue approaches. A detailed model of pronephric development has been developed, which could ultimately be used to dissect the molecular mechanisms underlying embryonic kidney development.

The distribution of neurokinin receptors in rat kidney, renal artery, renal vein, and proximal ureter was evaluated by autoradiography after in vitro labeling of NK1 sites with [125I]Bolton-Hunter substance P (BHSP) or NK3 sites with [125I][MePhe7]neurokinin B ([MePhe7NKB). Film autoradiography using [125I][MePhe7]NKB revealed specific binding sites associated with the renal vein and its large branches, the renal pelvis, the inner strip of outer renal medulla, and the proximal ureter. High-resolution autoradiograms demonstrated that these sites were localized to the smooth muscle layer in the veins, pelvis, and ureter. Neither the renal arterial system nor the renal cortex contained specific [125I][MePhe7]NKB binding sites although a high level of nonspecific binding was associated with the renal artery. Specific binding of [125I]BHSP was associated with the renal artery and renal pelvis but not the renal veins. Arterial NK1 receptors appeared to be localized to the adventitia. The results indicate that at least two types of tachykinin receptor are present in the rat kidney. The distinct localization observed for most of the NK1 and NK3 receptors suggests that they have different functions.