Permselectivity of the glomerular capillary wall to macromolecules. I. Theoretical considerations.

The transport of macromolecules across the renal glomerular capillary wall has been described theoretically using flux equations based on (a) restricted transport through small pores, and (b) the Kedem-Katchalsky formulation. The various assumptions and limitations inherent in these two approaches are discussed. To examine the coupling between macromolecular solute transport and the determinants of glomerular filtration rate, these flux equations were combined with mass balance relations which allow for variations in the transmembrane driving forces along a glomerular capillary. It was predicted, using both pore theory and the Kedem-Katchalsky equations, that fractional solute clearance should be strongly dependent on the determinants of glomerular filtration rate when convection and diffusion both contribute to solute transport. When convection becomes the sole mechanism for transcapillary solute transport, however, fractional solute clearance is essentially independent of changes in the determinants of glomerular filtration rate. Consequently, unless diffusion is absent, fractional solute clearances alone are insufficient to characterize the permselective properties of the glomerular capillary wall, since these values may be altered by changes in glomerular pressures and flows as well as changes in the properties of the capillary wall per se.     

Glomerular endothelium: a porous sieve and formidable barrier

The glomerular capillary endothelium is highly specialized to support the selective filtration of massive volumes of plasma. Filtration is driven by Starling forces acting across the glomerular capillary wall, and depends on its large surface area and extremely high water permeability. Glomerular endothelial cells are extremely flat and perforated by dense arrays of trans-cellular pores, the fenestrae. This phenotype is critical for the high glomerular water permeability and depends on podocyte-derived VEGF, as well as TGF-beta. Endothelial cell-derived PDGFB, in turn, is necessary for the establishment of mesangial cells, which sculpt the glomerular loop structure that underlies the large filtration surface area. In pre-eclampsia, inhibition of the VEGF- and TGF-beta signaling pathways leads to endothelial swelling and loss of fenestrae, reducing the glomerular filtration rate. Similarly, in the thrombotic microangiopathies, glomerular endothelial cell injury coupled with inappropriate VWF activation leads to intracapillary platelet aggregation and loss of the flat, fenestrated phenotype, thus reducing the glomerular filtration rate. Normally, a remarkably small fraction of albumin and other large plasma proteins passes across the glomerular capillary wall despite the massive filtration of water and small solutes. An elaborate glycocalyx, which covers glomerular endothelial cells and their fenestrae forms an impressive barrier that, together with other components of the glomerular capillary wall, prevents loss of plasma proteins into the urine. Indeed, microalbuminuria is a marker for endothelial glycocalyx disruption, and most forms of glomerular endothelial cell injury including pre-eclampsia and thrombotic microangiopaties can cause proteinuria.     

An in vitro model of the glomerular capillary wall using electrospun collagen nanofibres in a bioartificial composite basement membrane

The filtering unit of the kidney, the glomerulus, contains capillaries whose walls function as a biological sieve, the glomerular filtration barrier. This comprises layers of two specialised cells, glomerular endothelial cells (GEnC) and podocytes, separated by a basement membrane. Glomerular filtration barrier function, and dysfunction in disease, remains incompletely understood, partly due to difficulties in studying the relevant cell types in vitro. We have addressed this by generation of unique conditionally immortalised human GEnC and podocytes. However, because the glomerular filtration barrier functions as a whole, it is necessary to develop three dimensional co-culture models to maximise the benefit of the availability of these cells. Here we have developed the first two tri-layer models of the glomerular capillary wall. The first is based on tissue culture inserts and provides evidence of cell-cell interaction via soluble mediators. In the second model the synthetic support of the tissue culture insert is replaced with a novel composite bioartificial membrane. This consists of a nanofibre membrane containing collagen I, electrospun directly onto a micro-photoelectroformed fine nickel supporting mesh. GEnC and podocytes grew in monolayers on either side of the insert support or the novel membrane to form a tri-layer model recapitulating the human glomerular capillary in vitro. These models will advance the study of both the physiology of normal glomerular filtration and of its disruption in glomerular disease.     

Studies of the permeation properties of glomerular basement membrane: cross-linking renders glomerular basement membrane permeable to protein

Cross-linking glomerular basement membrane (GBM) has been shown to render it more permeable to protein. Isolated pig GBM was cross-linked with dimethylmalonimidate which reacts selectively with lysine ?-NH₂ groups or with glutaraldehyde, a less selective cross-linking agent. Studies of the ultrafiltration properties of these materials in vitro using cytochrome c, myoglobin, bovine serum albumin and immunoglobulin showed that cross-linking had markedly increased solvent and protein fluxes as compared with native membranes particularly at higher pressures. Filtration studies with serum demonstrated that the cross-linked membranes were more permeable to serum proteins. Thickness measurements under pressure indicated that cross-linked membrane was less compressed than native membrane as pressure was increased. Pore theory did not provide a suitable model for analysis of the results, but analysis of the results using the fibre-matrix hypothesis indicated that cross-linking had the effect of bundling together the fibres (type IV collagen) in the GBM matrix. The effect of cross-linking on filtration could be explained by a combination of contraction of the membrane, fibre bundling and increased rigidity compared with native membrane. Cross-linking of GBM might lead to long-term damage of the glomerular capillary wall in nephritis, so promoting proteinuria.     

Stretch, tension and adhesion - adaptive mechanisms of the actin cytoskeleton in podocytes

Podocytes form an epithelial layer on the outer aspect of the basement membrane of glomerular capillaries. The interdigitating pattern of podocyte foot processes (PFPs) generates a unique and extremely long cell-cell contact area - the filtration slit. Thus, the interdigitating PFPs are the morphological basis for the high hydraulic conductivity of the glomerular capillaries. Any disturbance in this interdigitating pattern results in a drop of glomerular filtration rate impairing renal function. PFPs are based on the actin cytoskeleton, consisting of a subplasmalemmal network and a central core of filament bundles. Besides giving PFPs their morphology, the actin cytoskeleton anchors cell-cell contact and cell-matrix proteins in podocytes. Several human genetic diseases as well as transgenic mouse models provide evidence for the crucial role of the actin cytoskeleton in podocytes. Varying flow rates of the filtrate, increased glomerular capillary pressure in glomerular hypertension, and varying activation states of contractile proteins in PFPs impose a mechanical load on the actin cytoskeleton, challenging the intricate arrangement of PFPs and podocyte adhesion. Here we review data about the actin cytoskeleton of podocytes and the response of podocytes to mechanical load. From these data possible mechanisms are emerging how the actin cytoskeleton may allow podocytes to adapt to states of increased mechanical load.     

Localization and distribution of anionic charges in the glomerular mesangium of normal and nephrotic rats

Sulfated glycosaminoglycans and sialoglycoproteins are thought to play a pivotal role in the glomerular capillary wall barrier to filtration since these anionic charged elements are important in the maintenance of capillary wall integrity and constitute a charge-selective filter. The development of proteinuria in puromycin aminonucleoside (PAN) nephrosis is associated with polyanion loss from the glomerular capillary wall structures. Since in PAN nephrosis the permeability of the mesangial area to plasma proteins and tracer substances has also been shown to be increased, the purpose of this study was to analyse the localization and distribution of anionic charges in the glomerular mesangium in this experimental model. Glycosaminoglycans were labeled by perfusion of the kidneys with ruthenium red solution (RR). Electron microscopic examination revealed the presence of distinct small RR granules ("anionic sites") in the mesangial intercellular matrix substance and in the laminae rarae of the glomerular basement membrane (GBM). The center-to-center spacing of the granules was measured and a frequency distribution of intervals in different interval classes was constructed. In normal glomeruli the anionic sites in the mesangial matrix showed a distribution pattern identical to the GBM with a maximal interval incidence at the 31-40 nm class. In nephrotic rats anionic site distributions in matrix and GBM did not change significantly. Sialoglycoproteins were labeled with colloidal iron (CI). In PAN nephrosis a decrease of CI binding was observed at the epithelial-basement membrane junction of the glomerular capillary wall. However, CI labeling of the mesangial matrix and mesangial cell membranes did not differ from that of normal glomeruli.(ABSTRACT TRUNCATED AT 250 WORDS)     

Optimization of affinity and ion-exchange chromatographic processes for the purification of proteins

This study documents several alternative approaches for the optimization of the ion-exchange and affinity chromatographic purification of proteins. In these approaches, the chromatographic process has been treated as a four-stage (adsorption, washing, elution, and regeneration) operation. Central to these investigations has been the elaboration of practical iterative procedures based on the use of theoretical models describing each of these stages. Predictions derived from these models have then been evaluated in terms of experimental data obtained using batch adsorption measurements in finite bath configurations and frontal breakthrough measurements with packed beds of different dimensions, containing nonporous and porous adsorbents of different selectivities and capacities for proteins. Commencing with the kinetic and distribution parameters derived from batch equilibrium measurements, the effect of the initial concentration of the target protein, the solid-liquid volume ratio, the superficial velocity and the column dimensions on the pressure drop, production rate, concentration profile, column utilization, and yield have been determined with packed beds. The potential of these iterative approaches to simplify the determination of key mass transfer and interaction parameters required for scale-up and economic optimization of chromatographic purifications of proteins has been examined using ion exchange, immobilized metal ion affinity, and triazine dye pseudo-affinity adsorbents of different selectivity and adsorption capacities. (c) 1996 John Wiley & Sons, Inc.     

Studies of the permeation properties of glomerular basement membrane: cross-linking renders glomerular basement membrane permeable to protein.

Cross-linking glomerular basement membrane (GBM) has been shown to render it more permeable to protein. Isolated pig GBM was cross-linked with dimethylmalonimidate which reacts selectively with lysine epsilon-NH2 groups or with glutaraldehyde, a less selective cross-linking agent. Studies of the ultrafiltration properties of these materials in vitro using cytochrome c, myoglobin, bovine serum albumin and immunoglobulin showed that cross-linking had markedly increased solvent and protein fluxes as compared with native membranes particularly at higher pressures. Filtration studies with serum demonstrated that the cross-linked membranes were more permeable to serum proteins. Thickness measurements under pressure indicated that cross-linked membrane was less compressed than native membrane as pressure was increased. Pore theory did not provide a suitable model for analysis of the results, but analysis of the results using the fibre-matrix hypothesis indicated that cross-linking had the effect of bundling together the fibres (type IV collagen) in the GBM matrix. The effect of cross-linking on filtration could be explained by a combination of contraction of the membrane, fibre bundling and increased rigidity compared with native membrane. Cross-linking of GBM might lead to long-term damage of the glomerular capillary wall in nephritis, so promoting proteinuria.     

Image analyser as a tool for the study ofin vitro glomerular vasoreactivity

Many drugs used in clinics can dramatically reduce renal hemodynamics. For some years there have been developed in our laboratory twoin vitro glomerular models, isolated glomeruli and mesangial cell cultures, to quantitate, by video image analyzer, the direct glomerular effect of vasoreactive agents. The present study shows the vasoconstrictive effects of angiotensin II and cyclosporin in both models and compares their glomerular vasoconstriction with or without vasodilating agents such as verapamil. This drug-induced glomerular vasoreactivity is time- and dose-dependent; moreover, it can be reversible after perfusion in control conditions. The interest of thesein vitro glomerular models is validated by fair correlations betweenin vivo andin vitro data and between the responses of both. These models can be considered as tools for assessing glomerular vasoreactivity of nephrotoxic agentsAbbreviations AngII angiotensin II - CsA cyclosporin A - GFR glomerular filtration rate - PSA planar surface area - RBF renal blood flow     

Biology of glomerular cells in culture

The glomerulus is a complex structure including four cell types, namely mesangial, visceral epithelial, parietal epithelial and endothelial cells. Mesangial cells resemble smooth muscle cells and play a major role in the synthesis of the components of the glomerular basement membrane and in the vasoreactivity of the glomerular tuft. In particular, they express receptors for angiotensin II which mediate mesangial cell contraction, this effect resulting in the decrease of the filtration area. They are also the site of synthesis of a variety of inflammatory agents which are involved in the development of glomerular injury in glomerulonephritis. Visceral epithelial cells, also referred to a podocytes, also participate in the synthesis of the normal constituents of the glomerular basement membrane. They express receptors for atrial natriuretic factor and possess on their surface a number of ectoenzymes. They also, in concert with mesangial cells, release metalloproteases which contribute to the degradation of the extracellular matrix. Parietal epithelial cells have been little studied. They represent the main constituent of the crescents observed in extracapillary proliferative glomerulonephritis. Endothelial cells secrete vasodilatory agents such as nitric oxide and prostacyclin and vasoconstrictor agents such as endothelin which act on the adjacent mesangial cells. New methods of culture of glomerular cells are in progress. Their aim is to keep as long as possible the physiological phenotype of these cells. Another progress is the availability of stable transformed cell lines which represent an abundant source of material for biochemical studies.     

Transition Path Times Measured by Single-Molecule Spectroscopy

The transition path is a tiny fraction of a molecular trajectory during which the free-energy barrier is crossed. It is a single-molecule property and contains all mechanistic information of folding processes of biomolecules such as proteins and nucleic acids. However, the transition path has been difficult to probe because it is short and rarely visited when transitions actually occur. Recent technical advances in single-molecule spectroscopy have made it possible to directly probe transition paths, which has opened up new theoretical and experimental approaches to investigating folding mechanisms. This article reviews recent single-molecule fluorescence and force spectroscopic measurements of transition path times and their connection to both theory and simulations.     

Long-Term Responses to Loss of Renal Mass: Glomerular Changes: Adaptation of Glomerular Forces and Flows to Renal Injury

The mechanism of glomerular ultrafiltration in normal kidneys or after renal injury is reviewed. The role of increased glomerular plasma flow in mediating increases of nephron filtration rate is evidenced under experimental conditions resulting in filtration pressure disequilibrium along glomerular capillaries. The increase of nephron filtration in hypertrophied kidneys appears to be due mainly to a rise of glomerular plasma flow and, to a smaller extent, to an increase of glomerular capillary hydrostatic pressure, the ultrafiltration coefficient remaining unchanged. In contrast, in the early phases of experimentally induced nephrotoxic serum nephritis, a decrease of the ultrafiltration coefficient was observed; nephron filtration rate, however, remained within the normal range, as a consequence of a higher hydrostatic pressure in the glomerular capillaries of the nephritic kidneys.     

The endoendothelial fibrin lining as the crucial barrier and the role of fibrin(ogenin) gels in controlling transcapillary transport

The interface between the two portions of the 'vessel-blood organ', viz., the vessel wall and the circulating blood, is considered by the author to be the endoendothelial fibrin lining (EEFL). The view that the endothelium, consisting of the endothelial cells and the interendothelial cement substance, is the primary filtration barrier in capillary permeability (CP) is no longer tenable. There is considerable evidence that the primary barrier is an endocapillary protein layer, originally postulated by Danielli in 1940. Copley considered this layer to be identical with the EEFL formed in the more or less immobile portion of the plasmatic zone in close proximity to the vessel wall. Processes of fibrin formation and fibrinolysis can occur there homeostatically, undisturbed by the flow of blood. The fibrinopeptides and plasminopeptides, freed at this site by the conversion of fibrinogen to fibrin and of plasminogen to plasmin, respectively, were reported by Copley et al in 1966 to augment CP. These peptides thus take part in the steadily occurring normal physiological CP. This is facilitated by the porosity of the EEFL due to the network or gel structure of fibrin strands. The author's concept that the EEFL acts as the primary barrier, controlling transendothelial transport and transport across the basement membrane (BM), is discussed on the basis of older and recent findings by several investigators. In particular, the BM is dealt with in some detail as a barrier. Emphasis is placed on the existence of fibrin as a main constituent of the BM, hitherto not generally known. This was demonstrated by direct evidence in the production of (non-thrombocytopenic) vascular purpura with fibrin antiserum. Numerous tiny foci of fibrin(ogenin) gels are expected to stud the BM. Augmented capillary fragility (CF) due to increased fibrinolysis of many of these focal fibrin gels result in petechial hemorrhages. CF and CP are physical properties of the blood capillary wall which behave antagonistically and are controlled by fibrin formation and fibrinolysis, steadily occurring in the vascular layers including the BM. This barrier secures the integrity of the capillary wall by preventing extravasation of blood or hemorrhages. New experimental approaches to verify the detection of fibrin in the microstructure of the capillary wall are proposed. Moreover, hemorheological experimentation, models and treatments are needed to establish whether or not the EEFL is the crucial, critical barrier in CP, as proposed.     

A reverse genetic screen in the zebrafish identifies crb2b as a regulator of the glomerular filtration barrier

The glomerular filtration barrier is necessary for the selective passage of low molecular weight waste products and the retention of blood plasma proteins. Damage to the filter results in proteinuria. The filtration barrier is the major pathogenic site in almost all glomerular diseases and its study is therefore of clinical significance. We have taken advantage of the zebrafish pronephros as a system for studying glomerular filtration. In order to identify new regulators of filtration barrier assembly, we have performed a reverse genetic screen in the zebrafish testing a group of genes which are enriched in their expression within the mammalian glomerulus. In this novel screen, we have coupled gene knockdown using morpholinos with a physiological glomerular dye filtration assay to test for selective glomerular permeability in living zebrafish larvae. Screening 20 genes resulted in the identification of ralgps1, rapgef2, rabgef1, and crb2b. The crumbs (crb) genes encode a family of evolutionarily conserved proteins important for apical-basal polarity within epithelia. The crb2b gene is expressed in zebrafish podocytes. Electron microscopic analysis of crb2b morphants reveals a gross disorganization of podocyte foot process architecture and loss of slit diaphragms while overall polarity is maintained. Nephrin, a major component of the slit diaphragm, is apically mis-localized in podocytes from crb2b morphants suggesting that crb2b is required for the proper protein trafficking of Nephrin. This report is the first to show a role for crb function in podocyte differentiation. Furthermore, these results suggest a novel link between epithelial polarization and the maintenance of a functional filtration barrier.     

Random-coil model for glomerular sieving of dextran

Dextran has been the most commonly employed test molecule for probing the selectivity of glomerular filtration to macromolecules of varying size. The usual theories for hindered transport of solid spheres through pores have limited utility in interpreting clearance data for dextran or other linear polymers because such polymers in solution more closely resemble random, solvent-filled coils than solid spheres. To provide a model for glomerular filtration of random-coil macromolecules, the equilibrium partitioning of random coils between cylindrical pores and bulk solution was simulated using Monte Carlo calculations, and those results were combined with a hydrodynamic theory for restricted motion of solvent-filled polymer coils in pores. The rates of transport predicted for either neutral random coils or for solid spheres of the same Stokes-Einstein radius were significantly lower than observed transport rates of dextran through the glomerular capillary wall or across synthetic porous membranes. This facilitation of dextran transport was modeled by postulating weak, attractive interactions between dextran monomers and the pore wall. The random-coil model with attractive interactions, modeled using a short-range, square-well potential, was found to adequately represent dextran sieving data in normal rats. Various limitations of this approach are discussed.     

Endothelial cell and podocyte autophagy synergistically protect from diabetes-induced glomerulosclerosis

The glomerulus is a highly specialized capillary tuft, which under pressure filters large amounts of water and small solutes into the urinary space, while retaining albumin and large proteins. The glomerular filtration barrier (GFB) is a highly specialized filtration interface between blood and urine that is highly permeable to small and midsized solutes in plasma but relatively impermeable to macromolecules such as albumin. The integrity of the GFB is maintained by molecular interplay between its 3 layers: the glomerular endothelium, the glomerular basement membrane and podocytes, which are highly specialized postmitotic pericytes forming the outer part of the GFB. Abnormalities of glomerular ultrafiltration lead to the loss of proteins in urine and progressive renal insufficiency, underlining the importance of the GFB. Indeed, albuminuria is strongly predictive of the course of chronic nephropathies especially that of diabetic nephropathy (DN), a leading cause of renal insufficiency. We found that high glucose concentrations promote autophagy flux in podocyte cultures and that the abundance of LC3B II in podocytes is high in diabetic mice. Deletion of Atg5 specifically in podocytes resulted in accelerated diabetes-induced podocytopathy with a leaky GFB and glomerulosclerosis. Strikingly, genetic alteration of autophagy on the other side of the GFB involving the endothelial-specific deletion of Atg5 also resulted in capillary rarefaction and accelerated DN. Thus autophagy is a key protective mechanism on both cellular layers of the GFB suggesting autophagy as a promising new therapeutic strategy for DN.     

Ontogenesis of glomerular basement membrane: structural and functional properties

Protein A-gold immunocytochemistry was applied in combination with morphometrical approaches to reveal the alpha 1(IV), alpha 2(IV), and alpha 3(IV) chains of type IV collagen as well as entactin on renal basement membranes, particularly on the glomerular one, during maturation. The results have indicated that a heterogeneity between renal basement membranes appears during the maturation process. In the glomerulus at the capillary loop stage, both the epithelial and endothelial cell basement membranes were labeled for the alpha 1(IV) and alpha 2(IV) chains of type IV collagen and entactin. After fusion, both proteins were present on the entire thickness of the typical glomerular basement membrane. At later stages, the labeling for alpha 1(IV) and alpha 2(IV) chains of type IV collagen decreased and drifted towards the endothelial side, whereas the labeling for the alpha 3(IV) chain increased and remained centrally located. Entactin remained on the entire thickness of the basement membrane during maturation and in adult stage. The distribution of endogenous serum albumin in the glomerular wall was studied during maturation, as a reference for the functional properties of the glomerular basement membrane. This distribution, dispersed through the entire thickness of the basement membrane at early stages, shifted towards the endothelial side of the lamina densa with maturation, demonstrating a progressive acquisition of the permselectivity. These results demonstrate that modifications in the content and organization of the different constituents of basement membranes occur with maturation and are required for the establishment of the filtration properties of the glomerular basement membrane.     

Potential use of isolated glomeruli and cultured mesangial cells as in vitro models to assess nephrotoxicity

The purpose of this short review is to present the potential of using isolated glomeruli and cultured mesangial cells as two differentin vitro models to assess the glomerular effect of molecules with nephrotoxic properties. The advantage of using isolated renal glomeruli is that they conserve the architecture of this anatomical region of the kidney; moreover, they are free of any vascular, nervous or humoral influences derived from other regions of the kidney. Mesangial cells are perivascular pericytes located within the central portion of the glomerular tuft between capillary loops. Mesangial cells have a variety of functions including synthesis and assembly of the mesangial matrix, endocytosis and processing of plasma macromolecules, and control of glomerular hemodynamics, mainly the ultrafiltration coefficient K _f, via mesangial cell contraction or release of vasoactive hormones. Most authors agree that mesangial cells play a major role in glomerular contraction, filtration surface area, and K _f regulation. One of the major effects of toxicants on glomerular structures is contraction. We can assess quantitatively the degree of toxicant-induced mesangial cell contraction or glomerular contraction by measuring the changes in planar cell surface area or apparent glomerular cross-sectional area after exposition to the toxicant. Thesein vitro models can also reveal glomerular effects of xenobiotics that are difficult or impossible to observe in vivo. In addition, these studies permit a fundamental examination of the mechanism of action of xenobiotics on glomerular cells, including the possibility that at least a part of their effects are mediated by local mediators released by glomerular cells. We review the effects and the mechanisms of action of several toxicants such as gentamicin, cyclosporin, cisplatin, and cadmium on isolated glomeruli or cultured mesangial cells. As suchin vitro results confirmin vivo renal hemodynamic changes caused by toxicants, we conclude that these models are fruitful tools for the study of renal toxicity. Thesein vitro systems might also serve as a predictive tool in the evaluation of drugs inducing changes in glomerular filtration rate and as a way to propose protective agents against these dramatic hemodynamic effects. This revised version was published online in July 2006 with corrections to the Cover Date.     

Characterization of protein behavior in high-performance capillary electrophoresis using a novel capillary system

Original calculations of over a million theoretical plate efficiency for macromolecular solutes in the open tubular high-performance capillary electrophoresis experiment considered axial diffusion to be the efficiency limiting factor. In practice, interactions of biopolymers, such as proteins, with the capillary wall has had a significant impact on readily achieving high efficiencies for a wide variety of proteins. This paper reports a capillary system in which protein-surface interactions have been minimized, resulting in high efficiencies (greater than or equal to 300,000 theoretical plates). This system allows the analysis of a set of protein standards over a wide pI range at neutral pH and moderate ionic strength. The characterization of the behavior of those protein standards in this capillary system is described.