Histochemical studies on the mechanism of macromolecule leakage across the glomerular capillary wall

Summary For the purpose of revealing the barrier effect of the anionic groups of glomerular capillary wall against the serum protein leakage, morphologic and histochemical observations were made on the rat kidney perfused in situ with three kinds of cationic macromolecules different in chemical characteristics followed by blood flow restoration. The polyethyleneimine perfusion resulted in the complete disappearance of ionized anionic groups of glomerular capillary and the massive protein leakage through glomeruli by blood flow restoration. Cationic ferric colloid perfusion induced moderate protein leakage, and avidin perfusion was less in neutralization effect of anionic groups and the protein leakage was of least. The protein leakage from glomeruli, however, was stopped or markedly suppressed soon after the blood flow restoration by the newly formed functioning anionc barrier probably by some particular serum protein deposition. The findings indicate that the deionization of the glomerular capillary wall will not be responsible for the persistent albuminuria.     

Influence of molecular configuration on the passage of macromolecules across the glomerular capillary wall

The influence of molecular configuration on the filtration of macromolecules across glomerular capillary walls was examined by comparing fractional clearances of two uncharged polysaccharides of distinctly different molecular configuration in the Munich-Wistar rat. The macromolecules employed were dextran, a slightly branched polymer of glucopyranose, and ficoll, a highly cross-linked copolymer of sucrose and epichlorohydrin. Differences in effective shape between these two polymers were determined from measurements of several physical properties of aqueous solutions containing either dextran or ficoll. It was found that dextran is best represented as a prolate ellipsoid with axial ratios of 4, 9, and 16 for molecules with Stokes-Einstein radii of 22, 32, and 40 A, respectively. On the other hand, ficoll is more closely approximated as spherical since the axial ratio was found to be between 1 and 2 for all molecular sizes. Fractional clearances of dextran and ficoll ranging in effective radius from 18 to 44 A were determined in each of seven Munich-Wistar rats. Fractional clearances of dextran were found to be greater than those of ficoll, the difference being significant for molecular radii ranging from 24 to 44 A. In addition, as shown previously for dextran, ficoll was found to be neither secreted nor reabsorbed by the renal tubules. These results, therefore, suggest that in addition to molecular size and charge, molecular configuration is also a determinant of the filtration of macromolecules across the glomerular capillary wall.     

Modeling combined transport of water and charged graded-size molecules across the glomerular capillary wall

Clearance studies using various probe molecules established that the passage of molecules/proteins across the glomerular capillary wall of mammalian kidneys is increasingly restricted as their size and net negative charge increase. An extended mathematical model, based on the Fiber Matrix theory, was developed to describe the dynamics of the size- and charge-selective functions of the glomerular capillary barrier using mainly its hemodynamic, morphometric, and electrostatic variables. The glomerular basement membrane was represented as a homogeneous three-dimensional network of fibers of uniform length (L_f), radius (R_f), and packing density (N_fv) and characteristic Darcy permeability. The model was appropriate for simulating fractional clearance data of neutral and charged solutes from an experimental modeling exercise. We believe that the L_f and R_f best-fit numerical values may signify new insights for the diagnosis of some human nephropathies.     

Permselectivity of the glomerular capillary wall to macromolecules. II. Experimental studies in rats using neutral dextran.

To determine the permselectivity characteristics of the glomerular capillary wall, known molecular size fractions of [3H]dextran, prepared by gel chromatography, were infused into normally hydrated Wistar rats, thus permitting simultaneous measurement of Bowman's space/plasma water (BS/P) and urine/plasma water (U/P) concentration ratios, along with glomerular pressures and flows. Since (BS/P)inulin = 1.01 +/- 0.01 SE(n = 34, radius = approximately 14 A) and since (BS/P)dextran/(BS/P)inulin equaled (U/P)dextran/(U/P)inulin for dextrans ranging in molecular radius from 21 to 35 A, these findings validate that dextrans are neither secreted nor reabsorbed. For dextran radii of 20, 24, 28, 32, 36, 40, and 44 A, (U/P)dextran/(U/P)inulin averaged 0.99, 0.92, 0.69, 0.42, 0.19, 0.06, and 0.01, respectively. In accord with theoretical predictions that these fractional dextran clearances should vary appreciably with changes in glomerular transcapillary pressures and flows, an increase in glomerular plasma flow rate, induced in these same rats by plasma volume expansion, resulted in a highly significant lowering of fractional clearance of all but the smallest and largest dextrans studied. These findings emphasize that fractional solute clearances alone are inadequate to describe the permselective properties of the glomerular capillary wall unless glomerular pressures and flows are also known. This sensitivity of fractional dextran clearance to changes in plasma flow indicates that dextrans are transported across the capillary not only by bulk flow but also to an important extent by diffusion.     

The role of capillary wall in the histogenesis of glomerular diseases.

The glomerular capillary is the most solicited renal component of the local and general systems of integration both under normal and pathological conditions. The alterative and reactive modifications of the glomerular capillary may be the expression of a systematic capillary disease of the initial involvement of ths structure of glomerular corpuscles.The sum of certain kind of lesions of the complex glomerular structure realizes diverse morphofunctional entities of proliferative, membranous, and sclerocicatricial glomerular diseases. The glomerular diseases, irrespective of their nature, induce changes in the balance of the intra-and internephronal relationships, realizing new vicious circles which amplify and maintain the tissular destructions.     

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.     

Interactions of metal cations with anionic groups on the cell wall of the macroalga Vaucheria sp.

The aim of this article was to investigate the interactions of metal cations in aqueous solutions with the biomass of the freshwater macroalga Vaucheria sp. This problem is important when elaborating new applications of biosorption, e.g. the production of mineral feed additives for livestock from the biomass of algae enriched with microelement ions. Potentiometric titration was applied as a quick and cheap screening test to search for new efficient biosorbents. It revealed a variety of functional groups capable of cation exchange on the macroalgal surface, including carboxyl, phosphate, hydroxyl or amino groups. Fourier transform infrared spectroscopy on natural and chromium‐loaded Vaucheria sp. confirmed that carboxyl groups played a dominant role in the biosorption. The study also showed that Ca(II), Na(I), K(I), and Mg(II) ions were released from the biomass after biosorption of Cu(II), Mn(II), Zn(II), and Co(II) ions, indicating that ion exchange was a key mechanism in the biosorption of metal ions by Vaucheria sp. biomass. It was noticed that the mass of the microelement cations bound by the macroalga was proportional to the total mass of light metal ions [Na(I), K(I), Ca(II), and Mg(II)] released from the biomass.     

Morphofunctional studies of the glomerular wall in mice lacking entactin-1.

The architecture of the basement membranes is essential for proper function. This architecture is based on interactions among its components, which assemble in a complex network. Entactin-1 appears to be the mastermind of this assembling. In entactin-1-null transgenic mice, immunocytochemistry established the absence of entactin-1 in the glomerular basement membrane, and morphological thickening of this membrane was demonstrated. This prompted us to investigate the organization of other components of the glomerular basement membrane in the transgenic animals. The distribution of type IV collagen and laminin remained unchanged, whereas that of anionic charges was significantly altered. We also evaluated the impact of the absence of entactin-1 on cell relays by studying the alpha(3)- and the alpha(v)-integrins along the endothelial and epithelial glomerular cell plasma membranes. Only the density of alpha(v) was found to be increased. Finally, the filtration properties of the glomerular wall were evaluated by revealing endogenous albumin distribution across the basement membrane. This was altered in transgenic animals, suggesting changes in permselectivity properties. Entactin-1 appears to be an essential component in basement membranes because its absence appears to modify the molecular organization leading to alterations in functional properties.     

Further studies on the distribution of some serum protein and enzyme groups in South India

Summary The distribution of electrophoretic variants of 2 serum proteins and 5 red cell enzyme systems have been examined in 4 population groups from Tamilnadu.In the Haptoglobin system Hp ¹ was found in low frequency for all the 4 groups, Nadar (0.037), Reddiar (0.182), Thevar (0.100) and Miscellaneous (0.104). All samples were found to be of the TfC phenotype for the Transferrin system.The AK ² gene in the Adenylate kinase system is relatively more frequent in Miscellaneous (0.075) and Thevar (0.065), and has a lower frequency among the Nadar (0.051) and Reddiar (0.034). No variants were found at the PGM₂ locus in the Phosphoglucomutase system. For the PGM₁ locus, PGM ₁ ¹ frequencies were 0.721, 0.804, 0.796 and 0.639 for the Nadar, Reddiar, Thevar and Miscellaneous groups respectively.For the 6-Phosphogluconate dehydrogenase system, PGD ^c frequencies were 0.048, 0.007, 0.006 and 0.012 for the Nadar, Reddiar, Thevar and Miscellaneous respectively. A single case of the Hackney was found among the Miscellaneous group. Calcutta-1 variant of the Lactate dehydrogenase was frequent in the Nadar (0.024) and Miscellaneous (0.019) and absent in the Reddiar and Thevar.The PHs ^B frequencies in the Acid Phosphatase system were 0.800, 0.794, 0.639 and 0.764 for the Nadar, Reddiar, Thevar and Miscellaneous groups respectively. 1 AC and 1 BC were found among the Nadar and Thevar respectively.

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Zusammenfassung An 4 Populationsstichproben aus Tamilnadu (Südindien) wurde die Verteilung der elektrophoretischen Varianten von 2 Serumprotein- und 5 erythrocytären Enzymsystemen untersucht. Dabei ergaben sich die folgenden wichtigen Ergebnisse. Das Hp¹-Allel ist in allen 4 Stichproben nur in geringer Häufigkeit vertreten (Nadar 0,037, Reddiar 0,182, Thevar 0,100, Sonstige 0,104). In keiner dieser Stichproben fanden sich Transferrin-Varianten. Das AK²-Allel läßt bei den Thevar (0,065) sowie in der Gruppe der Sonstigen (0,075) eine höhere Frequenz erkennen als bei den Nadar (0,051) und den Reddiar (0,034). Varianten am PGM₂-Locus konnten nicht beobachtet werden. Bezüglich des PGM ₁ ¹ -Allels wurden folgende Frequenzen ermittelt: 0,721 (Nadar), 0,804 (Reddiar), 0,796 (Thevar) und 0,639 (Sonstige). Die PGD^c-Frequenzen betragen: 0,048 (Nadar), 0,007 (Reddiar), 0,006 (Thevar) und 0,012 (Sonstige). Darüber hinaus wurde in der Gruppe der Sonstigen ein einziger Hackney-Fall gefunden. Die Calcutta-1-Variante im LDH-System fehlt in den Stichproben der Reddiar und Thevar, während ihre Häufigkeit bei den Nadar und der Gruppe der Sonstigen 0,024 bzw. 0,019 beträgt. Das Allel ph^B des Systems der sauren Erythrocytenphosphatasen kommt bei den Nadar zu 0,800 vor, bei den Reddiar zu 0,794, bei den Thevar zu 0,639 und bei den Sonstigen zu 0,764. Jeweils ein AC-bzw. BC-Phänotyp wurde bei den Nadar und Thevar beobachtet.

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Formed part of a Ph. D. Thesis. The Australian National University, Canberra, Australia.     

Experimental studies on the mechanism of the nitroglycerin effect.

The regional decrease of the speed and extent of the contraction and the relaxation of the canine myocardium induced by coronary ligation was enhanced by intravenously administered nitroglycerin (GTN), simultaneously with the decrease of the left ventricular pressure, while the increase of myocardial fiber length caused by that procedure was partly abolished. The above changes could be reproduced by GTN applied directly onto the ischaemic myocardial surface even before the decrease of the left ventricular pressure. These changes induced by GTN might play a role in the decrease or abolishment of regional asynergic wall motion.     

Histochemical and ultrastructural studies of the innervation of the lymph-vessel and blood-vessel wall. 1. Adrenergic innervation

The adrenergic innervation of the lymph vascular wall was studied by means of the Falck fluorescence histochemical tecnique and electron microscopy with Tranzer and Richards' histochemical tecnique. The lymph vessels wall, compared with that of blood vessels, shows very few adrenergic nerve fibers located in the adventitia outside the smooth muscle cells. The possible role of the nervous system in the motor control of the lymph vessels is discussed.     

Histochemical and ultrastructural studies of the innervation of the lymph-vessel and blood-vessel wall. II. Cholinergic innervation

Using the Acetylcholinesterase (AChE) tecnique applied to light and electron microscopy, was observed that the lymph vascular wall shows very few and inconstant AChE-positive fibers. The cholinergic fibers run prevalently longitudinal in the perivascular connective tissue, only brief segments show a loose network. The results are discussed and compared with blood vessels innervation.     

Population genetic studies of the Philippine Negritos. I. A pilot survey of red cell enzyme and serum protein groups

Electrophoretic surveys of red cell enzyme and serum protein systems representing 21 genetic loci were carried out on 129 blood samples of the Negritos of Pampanga, Central Luzon, the Philippines. Nine (out of 16) red cell enzyme loci and four (out of five) serum protein loci showed polymorphic variation. Low frequencies of ACP 1A, GPTs1, ESD2, and Hp1, and a markedly high frequency of PGM12 were contrasted to those in non-Negrito Filipinos. Variant ESD phenotypes with a slowly migrating isozyme occurred in high frequency. The new allele designated as ESD3Negrito (ESD3N) had a frequency of .10 +/- .019. In AK, a variant phenotype indistinguishable from AK 2-1 was observed in 14% of the sample. In the Gc system, a fast migrating variant was discovered in high frequency which was distinct from Gc Ab and Gc J. The variant allele, denoted GcNegrito (GcN), had a frequency of .21 +/- .025. A relatively high degree of allelic diversity in the Negrito sample was also suggested by the average heterozygosity for 21 loci screened (.165), which is compared to that of the Japanese population (.140).     

Identification of a defence mechanism in vivo against the leakage of enterokinase into the blood.

1. The serum proteinase inhibitors alpha 1-antitrypsin, alpha 2-macroglobulin, inter-alpha-trypsin inhibitor and C1-esterase inhibitor were found not to affect the catalytic activity of human enterokinase, whereas bovine trypsin activity was modified essentially as expected. Enterokinase was also not inhibited by Trasylol (trypsin inhibitor from bovine lung) or bovine pancreatic trypsin inhibitor. No other component in human or mouse serum complexing with enterokinase was identified. 2. Human enterokinase administered intravenously into mice was rapidly cleared from the circulation with a half-life of 2.5 min. This removal was not the result of the difference in species, since partially purified mouse enterokinase was cleared at the same rate as the human enzyme. Clearance was mediated by recognition of the carbohydrate portion of enterokinase and not through specific recognition of its catalytic site. Immunofluorescent staining showed that the enzyme accumulated in the liver. Attempts to block the clearance by the simultaneous infusion of competing glycoproteins suggested that enterokinase was taken up by hepatocytes. Of the glycoproteins tested only two, human lactoferrin (terminal fucosyl alpha 1 leads to 3 N-acetylglucosamine) and bovine asialo-fetuin (terminal galactosyl beta 1 leads to 4 N-acetylglucosamine) were weakly competitive. Two inhibitors of endocytosis, Intralipid and Triton WR1339, failed to delay the removal of enterokinase. It is proposed that enterokinase is cleared from the circulation by an as yet uncharacterized hepatocyte receptor.