Physiological and morphological aspects of the glomerular barrier in health and disease. Results based on experimental studies in the rat

Abstract: Tremendous amounts of fluid are filtered every day through the kidneys. The anastomosing capillary loops surrounded by Bowman s capsule, forming the glomerulus, function in accordance with the interplay between the hydrostatic and the colloid osmotic pressure. The result of the action of these mechanisms is the production of primary urine. The glomerular barrier functions as an entity, each of its components contributing to the overall glomerular permselectivity. However, the specific role of each of these components is not entirely clear. This thesis focuses on the description and functional estimation of a hitherto neglected structure of the glomerular barrier: the endothelial surface layer (ESL). Another aim was to evaluate the size-selectivity of the glomerular barrier in vivo compared to that in the cooled isolated perfused kidney. Finally, we wanted to explore the functional and morphological alterations in the nephrotic syndrome induced by puromycin aminonucleoside (PAN) and to evaluate the protective role of orosomucoid (alfa1- acid glycoprotein).Our observations suggest that the ESL has a highly labile molecular structure, making it difficult to preserve for electron microscopy (EM). However, using an oxygen-carrying fixative and special contrast-enhancing techniques it is possible to visualize and characterize the layer. Our results indicate the ESL to have a width similar to that of the basement membrane (~200 nm) and to uniformly cover the endothelial cells, including the endothelial fenestrae. The ESL was found to behave as a size- and charge-selective gel, reducing the concentration of solutes prior to the functional pore entrance. Moreover, studies in isolated perfusion-fixed kidneys perfused with different ionic strength (IS) solutions (low IS = 34 mM and normal IS = 151 mM) showed that the low IS perfusions reduced the charge density, suggesting a significant volume expansion of the charge selective barrier. Thus, we propose the ESL (one of the few components able to expand in the rigid glutaraldehyde-fixed kidney) to represent the location for most of the charge density of the glomerular capillaries. Glomerular size-selectivity was similar in vivo and in cooled isolated perfused kidneys (cIPK) both in control rats and in rats with PAN nephrosis. PAN induced extensive size- and charge-selectivity alterations, combined with important modifications of podocyte morphology. The intraperitoneal administration of orosomucoid partially protected the glomerular function and the podocyte morphology from the dramatic changes induced by PAN, but did not restore normal kidney function.These results strongly support the concept of a glomerular barrier with pronounced size-and charge-discriminative properties, in qualitative agreement with the classical theory. Novel morphological evidence reveals the details of the ESL and functional data indicate it to be highly responsible for glomerular charge selectivity. Taken together, these findings represent a step forward in our understanding of the glomerular barrier that may help to improve the treatment of patients with nephrotic syndrome.