Physiological aspects of the glomerular filtration barrier

Abstract: Proteinuria is associated with progression of kidney disease and is an independent risk factor for cardiovascular morbidity and mortality. The pathophysiological alterations resulting in proteinuria are obscure, although proteinuria is often associated with pathological changes in the glomerular filtration barrier (GFB). It is therefore of great interest to study the physiology of the GFB, in order to be able to prevent or reduce proteinuria. The studies in this thesis were performed using an in vivo model in anesthetized rats. The rats were cannulated for blood access and the left ureter cannulated for urine collection through a small abdominal incision. The glomerular permeability was measured using sieving coefficients (θ) for fluorescently labeled Ficoll (FITC-Ficoll) with a molecular radius ranging from 10Å to 80Å. The rats were infused with FITC-Ficoll, and blood- and urine samples were collected and analyzed with high performance size exclusion chromatography (HPSEC). In study I, the GFB charge-selectivity was explored using a negatively charged, conformationally intact anionic Ficoll (CMI-Ficoll), comparing it to neutral Ficoll, of same Stokes-Einstein (SE) radius. The sieving of CMI-Ficoll was reduced across the GFB compared to neutral Ficoll for molecules of radius 20-35Å. The GFB was thus found to be negatively charged. However, the charge was of much less magnitude than previously estimated. In study II, the proposed effects of systemic infusion of protamine sulfate (PS) and hyaluronidase (Hyase) on the GFB charge-selectivity were studied. PS and to some extent Hyase, increased the permeability for Ficoll molecules > 50Å, but had no effect on the charge-selectivity of the GFB. In study III, the permeability effects of extracellular adult (HbA) and fetal hemoglobin (HbF) were studied, showing increases in θ for macromolecules (50-80Å in radius) after infusion of HbF, but not after HbA or cyano-inactivated HbF (CN-HbF) infusions. Tempol (a superoxide radical scavenger) and α-1-microglobulin (A1M; a physiological hemebinding protein and radical scavenger), both prevented the increase in the permeability of the GFB. In study IV, the effects of the pro-inflammatory cytokines, TNF-α, Il-1β and IL-6, on the GFB permeability were studied. TNF-α and Il-1β caused a rapid, reversible, increase in glomerular permeability, while IL-6 generated a more gradual response. These effects could be inhibited with tempol, showing a reactive oxygen species (ROS) dependency of the cytokine effects on the GFB permeability.