Rapid, dynamic changes in glomerular permeability to macromolecules during systemic angiotensin II (ANG II) infusion in rats

The actions of systemic angiotensin II (ANG II) infusions on glomerular permeability were investigated in vivo. In anesthetized Wistar rats (250-280 g), the left ureter was cannulated for urine collection, while simultaneously blood access was achieved. Rats were continuously infused intravenously with either of four doses of ANG II ranging from 16 ng·kg(-1)·min(-1) (Lo-ANG II) to 1.82 μg·kg(-1)·min(-1) (Hi-ANG II), and in separate experiments with aldosterone (Aldo; 0.22 mg·kg(-1)·min(-1)), or with the calcium channel blocker nimodipine, or with the Aldo antagonist spironolactone together with a high ANG II dose (910 ng·kg(-1)·min(-1); Hi-Int-ANG II), respectively, and with polydisperse FITC-Ficoll-70/400 (molecular radius 10-80 ?) and (51)Cr-EDTA. Plasma and urine samples were taken at 5, 15, 30, 60, and 120 min and analyzed by high performance size-exclusion chromatography for determination of glomerular sieving coefficients (θ) to Ficoll. Mean arterial pressure (MAP) and glomerular filtration rate (GFR) were also assessed. For ANG II, there was a rapid, marked, partly reversible increase in glomerular permeability (θ) for Ficoll molecules >34 ? in radius, peaking at 5-15 min, which was completely abrogated by the ANG II blocker candesartan but not affected by spironolactone at 15 and 30 min. For Aldo, the response was similar to that found for the lowest dose of ANG II infused. For the two highest ANG II doses given (Hi-Int-ANG II and Hi-ANG II), GFR decreased transiently, concomitant with marked, sustained increases in MAP. Nimodipine completely blocked all hemodynamic ANG II actions, whereas the glomerular permeability response remained unchanged. Thus ANG II directly increased glomerular permeability independently of its hemodynamic actions and largely independently of the concomitant Aldo response. The ANG II-induced increases in glomerular permeability were, according to a two-pore and a log-normal distributed pore model, compatible with an increased number of "large pores" in the glomerular filter, and, to some extent, an increase in the dispersity of the small-pore radius.     

Regulation of glomerular filtration rate and sodium excretion by angiotensin II

In addition to its extrarenal functions, including the control of arterial pressure and aldosterone secretion, the renin-angiotensin system (RAS) also has multiple intrarenal actions in controlling glomerular filtration rate (GFR) and sodium excretion. Angiotensin II (AngII) helps to prevent excessive decreases in GFR in different physiological and pathophysiological conditions by preferentially constricting the efferent arterioles, an action that can be mediated by either intrarenally formed or circulating AngII. Circulating AngII and intrarenally formed AngII do not appear to directly constrict preglomerular vessels, including the afferent arterioles, when the RAS is activated physiologically. The sodium-retaining action of AngII may be due, in part, to constriction of efferent arterioles and to subsequent changes in peritubular capillary physical forces. However, AngII may also directly stimulate sodium reabsorption in proximal and distal tubules, although the exact site at which AngII increases distal tubular transport is still uncertain. Considerable evidence indicates that the direct intrarenal effects of AngII on tubular reabsorption, including those caused by changes in peritubular capillary physical forces or a direct action on tubular transport, are quantitatively more important than those mediated by changes in aldosterone secretion. Thus, the intrarenal effects of AngII provide a mechanism for stabilizing the GFR and excretion of metabolic waste products while causing sodium and water retention, thereby helping to regulate body fluid volumes and arterial pressure.     

Molecular make-up of the glomerular filtration barrier

The glomerular filtration barrier is composed of glomerular endothelial cells, the glomerulus basement membrane and the podocyte cell layer. The filtration barrier is a target of injury in several systemic and renal diseases, and this often leads to progressive renal disease and kidney failure. Therefore, it is essential to understand the molecular biology of the glomerulus. During the last two decades, a lot of new information about molecular components of the glomerulus filtration barrier has been generated. Many of the key discoveries have been obtained through studies on the genetic background of inherited glomerular diseases. These studies have emphasized the role of podocytes in the filtration barrier function. During the last decade, the use of knockout mouse technology has become more available and given important new insights into the functional significance of glomerular components. Large-scale approaches, such as microarray profiling, have also given data about molecules involved in the biology and pathology of the glomerulus. In the coming decade, the use of global expression profiling platforms, transgenic mouse lines, and other in vivo gene delivery methods will rapidly expand our understanding of biology and pathology of the glomerular filtration barrier, and hopefully expose novel target molecules for therapy in progressive renal diseases.     

Functional morphology of the glomerular filtration barrier of Gallus gallus

The anionic charge barrier and the endothelial and epithelial pore sizes on the glomerular filtration barrier (GFB) were examined in white leghorn chickens (Gallus gallus). Ruthenium red was used to stain anionic charge sites on the GFB. The tissue was treated by normal dehydration and freeze substitution dehydration for transmission electron microscopy (TEM). In addition, the basal lamina was isolated for study. The results of our study indicate that G. gallus possess a thick, negatively charged glycocalyx surrounding the podocytes and slit diaphragm and on the endothelium. However, in all cases, little anionic charge is present in the basal lamina. The pores on the endothelium are elliptical and have mean dimensions of 148 × 110 nm. This is in contrast to mammals, which have smaller, round pores. The epithelial pores in G. gallus measure approximately 35 nm in length, approximately 4 times larger than those found in mammals. These results indicate that the avian glomerulus may allow the filtration of larger molecules from the plasma than occurs in mammals and that the charge on the molecule may not be as restrictive a filtration characteristic as in mammals. © 1996 Wiley-Liss, Inc.     

Transmembrane collagen XVII is a novel component of the glomerular filtration barrier

The kidney filtration barrier consists of the capillary endothelium, the glomerular basement membrane and the slit diaphragm localized between foot processes of neighbouring podocytes. We report that collagen XVII, a transmembrane molecule known to be required for epithelial adhesion, is expressed in podocytes of normal human and mouse kidneys and in endothelial cells of the glomerular filtration barrier. Immunoelectron microscopy has revealed that collagen XVII is localized in foot processes of podocytes and in the glomerular basement membrane. Its role in kidney has been analysed in knockout mice, which survive to birth but have high neonatal mortality and skin blistering and structural abnormalities in their glomeruli. Morphometric analysis has shown increases in glomerular volume fraction and surface densities of knockout kidneys, indicating an increased glomerular amount in the cortex. Collagen XVII deficiency causes effacement of podocyte foot processes; however, major slit diaphragm disruptions have not been detected. The glomerular basement membrane is split in areas in which glomerular and endothelial basement membranes meet. Differences in the expression of collagen IV, integrins α3 or β1, laminin α5 and nephrin have not been observed in mutant mice compared with controls. We propose that collagen XVII has a function in the attachment of podocyte foot processes to the glomerular basement membrane. It probably contributes to podocyte maturation and might have a role in glomerular filtration.     

Holey barrier: claudins and the regulation of brain endothelial permeability

Endothelial tight junctions (TJs)* are an important functional part of the blood-brain barrier (BBB). In this issue, Nitta et al. (2003) demonstrate that claudin-5, a transmembrane protein of TJs, is a critical determinant of BBB permeability in mice. Unexpectedly, knockout of claudin-5 did not result in a general breakdown of TJs but in a selective increase in paracellular permeability of small molecules. This suggests that the BBB can be manipulated to allow selective diffusion of small molecules and makes claudin-5 a possible target for the development of drugs for this purpose.     

The dark side of the mycorrhiza

Plant association with arbuscular mycorrhizal (AM) fungi is usually regarded as mutualistic. However, this positive effect could disappear if the benefit of the fungal-plant association changes with colonization density. In order to test the conditionality of this interaction, we evaluated plant performance and tolerance to defoliation across five levels of commercial AM fungal inoculum concentrations. Additionally, we evaluated if plant performance and tolerance were similarly affected by a whole soil community collected under a native congener. Along the gradient of inoculation, plant performance exhibited a peak at intermediate inoculum concentration, indicating the presence of an optimum level of AM fungal concentration that maximized AM fungal benefit. Root colonization by fungal hyphae increased linearly across the experimental inoculation gradient. Paralleling root colonization, plant tolerance to defoliation decreased linearly along the inoculum gradient. Plant performance was similar under the whole soil and commercial treatments. Our results show a negative correlation between tolerance to defoliation and AM fungal inoculum concentration, indicating that AM fungi colonization could constrain the evolution of plant tolerance to herbivory.Key words: compensation, defences, ecological interactions, herbivory, multitrophic interactions, mycorrhizal fungi, toleranceArbuscular mycorrhizal (AM) fungi occur in all ecosystems of the world and associate with the roots of about 70% of all vascular plants.¹ This association is typically regarded as mutualistic, because there is a bidirectional transfer of nutrients between the host plant and its fungal partners. Carbon compounds are passed from the plant to the fungus and, in return, there is a transfer of mineral nutrients, principally nitrate and phosphate.² However, this association also entails costs. The amount of carbon allocated to AM fungi is estimated to range from 4% to 20% of a plant''s total carbon budget.² Throughout the literature, there are examples of the conditionality of this relationship exemplified by a continuum of the effects of AM fungal colonization on hosts from positive, through null to negative.^3–5 Moreover, it has been suggested that the benefit of a plant associating with fungal symbionts depends not only on the identity of AM fungi⁴ and plant genotypes⁶ but also on hyphal colonization density in roots.⁷ In a recent greenhouse study, we examined components of the conditionality of plant interactions with soil biota.⁸ We were interested in knowing how the performance and tolerance to defoliation of the annual plant Datura stramonium varied along a concentration gradient of commercial AM fungal inoculum containing four Glomus species (Mycorrhizal Applications, Grants Pass, OR USA).We found a curvilinear relationship between AM fungal inoculum concentration and plant performance, as predicted by previous models.⁷ The quadratic decelerating function between inoculum concentration and plant performance indicates an optimum level of AM fungal concentration (1/24^th total pot volume) that maximizes AM fungal benefit (Fig. 1A). This result suggests that, in D. stramonium, positive associations between AM fungi and plant fitness may not be proportional and, that at high colonization densities, mycorrhizae may have detrimental effects, perhaps by competing with plants for nutrients, or by interfering with other essential interactions.^4,5 We also found, from root examination, that hyphal colonization of roots increased linearly with AM fungi inoculum concentration. Moreover, we found that tolerance to herbivory decreased linearly with increasing AM fungal inoculum concentration (r² = −0.40; F_1,27 = 5.89; p = 0.0222; Fig. 1B), suggesting that, in our system, at high densities, mycorrhizae may become parasitic and may compete for resources (e.g., carbon) with the induced host plant response to leaf damage.Open in a separate windowFigure 1Effect of a gradient in AM fungi inoculum concentration on D. stramonium performance. (A) Non-linear relation between seed production and inoculum concentration. In general, plants achieved their maximal performance at an inoculum concentration of 1/24^th total pot volume. (B) Tolerance to defoliation decreased linearly with inoculum concentration. Tolerance was calculated as the difference in standardized seed production between related damaged and undamaged genetically related plants corresponding to six genetic full-sib families.In order to know whether the effects we found in the greenhouse using commercial inoculum could be expected in the field, we addressed whether or not D. stramonium performance and tolerance were similarly influenced by whole soil field communities; including AM fungi, pathogens, root herbivores, etc. Unfortunately, D. stramonium is not native to the area where this research was undertaken, so we collected soil immediately below plants of a native congener Datura wrightii, a perennial herb that grows at the Putah Creek Reserve (UC, Davis). Pots were inoculated at a 1/12^th total pot volume with this live soil and plants were grown concurrently with those in the previous experiment. We compared plant performance and tolerance under the live soil treatment and the last level of the commercial AMF inoculum gradient (both inoculated at a 1/12^th total pot volume). Results indicated no differences in foliar area (F_1,94 = 1.18; p = 0.2782), root mass (F_1,94 = 0.99; p = 0.3222), flowering day (χ² = 0.31; p = 0.5804) and fitness (χ² = 0.03; p = 0.8691). Moreover, root colonization levels were (F_1,94 = 0.75; p = 0.3877) in both 1/12^th volume vs. live soil, as well as in the 0 AMF and sterilized soil (F_1,94 = 2.56; p = 0.1130). Despite these similarities, plant tolerance did differ significantly between AMF and live soil treatments (F_1,94 = 5.49; p = 0.0411), tolerance being greater under the live soil treatment (0.3755 ± 0.0311 tolerance) relative to the 1/12^th AM fungal treatment (−0.5744 ± 0.2714 tolerance). This result suggests that the expression of plant tolerance may also depend on the identity of AMF colonizing roots or the number and identities of soil bacteria. We did not know which microbial species were in the soils we collected.We show that, when inoculated over a gradient of abundance, Glomus AM fungal colonization consistently decreased tolerance to herbivory. The presence of mycorrhizae could therefore decrease the adaptive value of traits increasing tolerance. We also show here that though live soil inoculum had similar effects in magnitude and direction to those of commercial AMF incoculum on growth and fitness, live soil biota collected under a congener of D. stramonium increased tolerance to herbivory at the same levels of root colonization. Overall, the results of this study indicate that the interaction between soil biotic components and the response of D. stramonium to leaf damage is highly conditional; and can depend on amounts of root colonization, as well as perhaps identities of AM fungi and bacteria. In both cases, soil biota affected the impact of damage to leaves aboveground. AM fungi may mediate the efficacy of tolerance as a defense, and this effect may be especially important in light of herbivore adaptation, when tolerance may be favored over resistance as a plant defense strategy.¹⁰     

Modeling combined transport of water and test macromolecules across the glomerular capillary barrier: dynamics of the permselectivity

The structure, function, and composition of the basement membrane of the glomerular capillaries of the mammalian kidney have been extensively studied, in light of the membrane's important physiological role in glomerular filtration of macromolecules and of its frequent involvement in renal diseases. An analytical mathematical model, based on the fiber matrix theory, was developed to describe the dynamics of the permselective function of the glomerular capillary barrier using mainly its hemodynamic and morphometric variables. The glomerular basement membrane was represented as a homogeneous three-dimensional meshwork of fibers of uniform length (L(f)), radius (R(f)), and packing density (N(fv)) and characterized by a local Darcy permeability (a measure of the intrinsic hydraulic conductance of the glomerular basement membrane). The model was appropriate for simulating in vivo fractional clearance data of neutral test macromolecules from an experimental rat model. We believe that the L(f) and R(f) best-fit numerical values, characterizing a glomerular basement membrane geometrical arrangement, may represent diagnostic measures for renal function in health and disease. That is, these parameters may signify new insights for the diagnosis of some human nephropathies and possibly may explain the beneficial effects and/or sites of action of some pharmacological modifiers (e.g., angiotensin converting enzyme inhibitors).     

From Daedalus to Mengele: the dark side of human genetics

Until recently, the role of scientists in society has been considered sacrosanct. Wherever scientists had been involved in crimes, either the crimes or the quality of science was denied (that is, the science was considered merely pseudoscience). As a result it has been claimed that science can only flourish in democracies but is doomed in other states. So far experience contradicts this opinion. In this paper I discuss the origins of the view that scientists and geneticists in particular, are sacrosanct. I trace it back to the earliest mythological scientists, such as Daedalus and Dr. Faustus. I view the well-known Dr. Mengele as a successor in this tradition.     

The dark side of cohesin: the carcinogenic point of view

Genome instability is a hallmark of cancer cells and how it arises is still not completely understood. Correct chromosome segregation is a pre-requisite for preserving genome integrity. Cohesin helps to ensure faithful chromosome segregation during cell cycle, however, much evidence regarding its functions have come to light over the last few years and suggest that cohesin plays multiple roles in the maintenance of genome stability. Here we review our rapidly increasing knowledge on the involvement of cohesin pathway in genome stability and cancer.     

Electron microscopic study of glomerular filtration barrier in the rat kidney embedded in polymerized glutaraldehyde-urea.

Embedding kidney in polymerized glutaraldehyde-urea favors the retention of glycoprotein matrix of the cell coat and the basement membrane of the glomeruli. The basement membrane appears as a single layer with uniform amorphous matrix. Thick glycoprotein coat covers the whole surface of prodocytes and their foot processes. In areas other than the slits and the portion of the foot processes which touch on the basement membrane, the coat is a continuous layer with an average thickness of 490 A. In the slits between the foot processes of podocytes there is an actual fusion of glycoprotein coats; the average width of the slit is 415 A. The glycoprotein 'plugs' in the slit may be a significant portion of the glomerular filtration barrier against macromolecules, together with the basement membrane and the slit diaphragms.     

The Repeat Expansion Diseases: The dark side of DNA repair

DNA repair normally protects the genome against mutations that threaten genome integrity and thus cell viability. However, growing evidence suggests that in the case of the Repeat Expansion Diseases, disorders that result from an increase in the size of a disease-specific microsatellite, the disease-causing mutation is actually the result of aberrant DNA repair. A variety of proteins from different DNA repair pathways have thus far been implicated in this process. This review will summarize recent findings from patients and from mouse models of these diseases that shed light on how these pathways may interact to cause repeat expansion.