Cytochromes in the rat kidney brush border membrane.

Spectrophotometric studies of the brush border membrane fraction of the rat kidney as compared with those of its mitochondria and microsomes were carried out. Occurrence of cytochromes has been demonstrated in the brush border membranes. Either in the brush border membranes and in the mitochondria evidence for the presence of cytochromes of the types $\text{a}\text{,}\text{b}$ and $\text{c}$ was found, whereas in the microsomes only cytochrome $\text{b}$ was demonstrated.     

Luxol Fast Blue Combined With the Periodic Acid-Schiff Procedure for Cytological Staining of Kidney

Rat kidneys fixed in Regaud's fluid were stained by luxol fast blue (LFB), by the periodic acid-Schiff (PAS) method, and by LFB combined with PAS. When used separately the PAS stains the brush border, hyaline droplets and basement membranes reddish, the LFB stains the mitochondria, hyaline bodies and basement membranes greenish-blue. The combined LFB-PAS method stains the brush border reddish and the mitochondria dark blue, while the hyaline bodies and basement membranes are purplish colored. The LFB-PAS method provides color contrasts which show cytological features that are particularly significant in the kidney.     

The fine structure of the nephronic tubule of the mudskipper Periophthalmus koelreuteri (Pallas)

Ultrastructural examination of the head kidney of Periophthalmus koelreuteri (Pallas) (Teleostei, Gobiidae) revealed that the nephronic tubule cells are bound by tight junctions and desmosomes with little intercellular space. The first proximal segment (PI) consists of low columnar cells with well developed brush borders, indented nuclei, and numerous apical endocytic vesicles and lysosomes. A second cell type possessing clusters of apical cilia and lacking brush border and lysosomes is occasionally found between PI cells. The second proximal segment (PII) is formed of high columnar cells with brush border, regular spherical nuclei and numerous mitochondria located between well developed infoldings of the basal membrane. Single ciliary structures protrude into the lumen from PI and PII cells. The distal segment is lined by low columnar epithelium with few microvilli, regular spherical nuclei, numerous scattered mitochondria, and microbodies. The collecting tubule cells are cuboidal with few euchromatic nuclei, some mitochondria, and secondary lysosomes.     

Analysis of the Heymann nephritogenic glycoprotein in rat, mouse, and human kidney

Passive Heymann nephritis is induced in rats by intravenous administration of antiserum raised against antigens of the renal proximal tubule. Evidence by Kerjaschki and Farquhar indicates that the critical nephritogenic is a high molecular weight glycoprotein (HMWgp) of rat renal brush border membrane. Their immunocytochemical studies also localize the nephritogenic antigen to the glomerular epithelial cell surface and may explain in situ formation of immune complexes at this locus in Heymann nephritis. We have confirmed the observations of Kerjaschki and Farquhar by demonstrating the HMWgp in extracts of rat brush border membrane and isolated glomeruli on sodium dodecyl sulfate-polyacrylamide (SDS-PA) (5%) gels. An antiserum raised to purified rat HMWgp identifies the antigen from rat or mouse kidney on Western blots. However, unlike rodent kidney, we were unable to detect a comparable HMWgp in extracts of human kidney on SDS-PA gels and found no cross-reactive material on Western blots of human brush border membrane proteins. Our observations suggest that human kidney lacks the nephritogenic antigen critical to initiation of Heymann nephritis in rodents.     

Deficiency of a mouse kidney metalloendopeptidase activity: immunological demonstration of an altered gene product

Meprin, an 85,000 molecular weight metalloendopeptidase is a major component of the kidney brush border membrane in mice. Some inbred mouse strains exhibit low levels of meprin activity. These strains were characterized by little, if any, protein in brush border preparations corresponding to the native enzyme. However, material exhibiting partial identity to meprin was identified by Ouchterlony immunodiffusion. Immunoblots of brush border proteins confirmed that this immunoreactive material was present but of higher molecular weight than the native enzyme. The implication of these data is that the structural gene for meprin is expressed, albeit incorrectly, in the low-meprin strains.     

Isolation, purification and characteristics of brush border and basolateral membranes from cattle intestinal epithelium cells

The surface membrane of cattle intestine epithelium cells is separated into vesicated membrane fractions of the brush border and of basolateral membranes. The brush border membrane fraction is deposited with centrifugation (15,000 g) and is localized in the layers of 45, 56.5 and 48% in the density gradient of sucrose (105,000 g). Basolateral membranes, obtained at 70,000 g, in the density gradient of sucrose are in the layers of 30 and 31.5%. The brush border membranes are 8.5 times purified, basolateral membranes--9.1 times with their insignificant contamination with subcellular elements. The both fractions are deprived of mitochondria impurities.     

Cytoskeletal proteins of the rat kidney proximal tubule brush border

Cytoskeletal components backing the brush border of the rat kidney proximal tubule cell were identified and compared with those of the well characterized intestinal brush border by immuneoverlay and immunocytochemistry. Antibodies reactive against the intestinal microvillus core components, villin and fimbrin, as well as against the terminal web components, spectrin (fodrin) and myosin, were used. Proteins of similar molecular weight to these intestinal brush border cytoskeletal components were identified in isolated kidney brush borders by immuneoverlay. Spectrin, a major component of the terminal web region of both cell types, was more concentrated in the kidney brush border relative to both actin and myosin. By immunofluorescence, villin and fimbrin were localized in the microvilli, and spectrin and myosin were localized to the terminal web region of the brush border. In addition, spectrin was found along the basolateral membranes of the proximal tubule cell, and myosin was detected in a punctate staining pattern throughout its cytoplasm. By immunoelectron microscopy using immunogold labeling procedures, fimbrin and villin were localized in the terminal web as well as in microvilli, and spectrin and myosin were localized to fibrils in the terminal web. A key difference between the epithelia of the two organs is the extensive network of clathrin coated pits found in the terminal web region of the kidney but not the intestinal brush border. The clathrin-rich terminal web region of the kidney, like the intestinal brush border, proved to be quite stable and resistant to disruption by non-ionic detergents and harsh mechanical treatment.     

Maltase-glucoamylase and trehalase in the rabbit small intestine and kidney brush border membranes during postnatal development, the effects of hydrocortisone

Kidney and intestinal brush border membranes were isolated from 14-day-old rabbits and papa?n solubilized maltase-glucoamylase was purified to almost homogeneity from both membranes. Maltase-glucoamylase from kidney and intestine have the same molecular weight (669,000 daltons by AcA 22 gel filtration) and the same Km (4 mM, for maltose). Tris (Ki = 12.5 mM, for maltose) is a non-competitive inhibitor for both enzymes. In intestine, maltase and glucoamylase have low activity during the first two postnatal weeks and then undergo a sharp increase during the next 2 weeks. In contrast, for trehalase, adult levels are reached about 6 days after birth. Hydrocortisone injection to 10 days rabbits causes precocious increases in the specific activities of trehalase (3.6 x), maltase (5.2 x) and glucoamylase (7.4 x). Conversely, kidney maltase, glucoamylase and trehalase activities rise gradually from birth, reaching adult levels by the end of the third week. Administration of hydrocortisone to suckling rabbit does not affect either trehalase or maltase and glucoamylase in kidney brush border membrane.     

Transport of p-aminohippuric acid, uric acid and glucose in highly purified rabbit renal brush border membranes.

A procedure for preparing highly purified brush border membranes from rabbit kidney cortex using differential and density gradient centrifugation is described. Brush border membranes prepared by this procedure were substantially free of basal-lateral membranes, mitochondria, endoplasmic reticulum and nuclear material as evidenced by an enrichment factor of less than 0.3 for (Na+ + K+)-ATPase, succinate dehydrogenase, NADPH-cytochrome c reductase and DNA. Alkaline phosphatase was enriched ten fold indicating that the membranes were enriched at least 30 fold with respect to other cellular organelles. The yield of brush border membranes was 20%. Transport of D-glucose by the membranes was identical to that previously reported except that the Arrhenius plot for temperature dependence of transport was curvilinear (EA = 11.3--37.6 kcal/mol) rather than biphasic. Transport of p-aminohippuric acid and uric acid were increased by the presence of NaCl, either gradient or preequilibrated. However, no overshoot was obtained in the presence of a NaCl gradient, and KCl and LiCl also produced equivalent stimulation of transport suggesting a nonspecific ionic strength effect. Uptakes of p-aminohippuric acid and uric acid were not saturable, and were increased markedly by reducing the pH from 7.5 to 5.6. Probenecid (1 mM) reduced p-aminohippuric acid and uric acid (50 muM) uptake by 49% and 21%, respectively. We conclude that the uptake of uric acid and p-aminohippuric acid by renal brush border membranes of the rabbit occurs primarily by a simple solubility-diffusion mechanism.     

Ultrastructure of the crayfish kidney--coelomosac, labyrinth, nephridial canal

Regions of the crayfish kidney were examined by electron microscopy. Coelsmosac cells are loosely bound together by desmosome-like spot junctions, and connected to the basal lamina via characteristic pedicels. The cytoplasm contains numerous vesicles and vacuoles of various sizes and is often crowded with large, lysosome-like granules or dense bodies. The morphology suggests a filtration mechanism with reabsorption of materials such as protein from the filtrate and secretion of other substances into the lumen. The labyrinth is composed of cuboidal to columnar cells which possess a brush border, long and narrow intercellular spaces, basal plasmalemmal invaginations and typical cytoplasmic components. Two sub-regions are distinguishable. The morphology of labyrinth I suggests that these cells move fluid isotonically across the epithelium. Labyrinth II, in addition to isotonic transport, appears to be more active in the endocytic uptake and intracellular digestion of large molecules such as protein. The nephridial canal consists of cells which lack a brush border, but display extensive basal invaginations associated with elongated mitochondria. A proximal and distal region are cytologically distinguishable. Proximally, the cells are small and filled with mitochondria throughout. Scattered within the cytoplasm are vesicles, vacuoles, diffuse glycogen, free ribosomes, dense bodies and some rough endoplasmic reticulum. Distally, the cells are less compact, larger, and cuboidal to columnar in shape. The cytoplasm is similar to that of the proximal cells, but the basal invaginations are even larger and more extensive. The morphology of cells in both regions of the nephridial canal is highly suggestive of active solute reabsorption, probably occurring against an osmotic gradient.     

Localization of dehydropeptidase-I, an enzyme processing glutathione, in the rat kidney

An antibody prepared against dehydropeptidase-I, one of the glutathione processing enzymes, from the renal membrane fraction of rats was employed to localize at the light microscopic level the enzyme in the kidney using the avidin/biotin-peroxidase complex method. Dehydropeptidase-I was found to be present on both the brush border and the basolateral membranes of proximal tubular cells. Furthermore, the enzyme activity in the isolated brush border and basolateral membrane fractions was also determined. Distribution profiles of the enzyme activity showed good agreement with the results of the immunohistochemical observations.     

Characterization of a processing protease that converts the precursor form of gamma-glutamyltranspeptidase to its subunits

Previously it was found that the proteolytic processing of precursors of gamma-glutamyltranspeptidase takes place on the brush border membrane of the kidney. The activity of the processing protease in purified brush border membranes was examined using endogenous substrates labeled with [3H]fucose and [35S]methionine. On incubation with brush border membranes in vitro, the precursors were converted stoichiometrically to two subunits, and the reaction followed first order kinetics with a rate constant k of -0.048 min-1. The enzyme responsible for this conversion was membrane-bound, had a weakly basic optimum pH and was inhibited by serine protease inhibitors. These results suggest that the precursor of gamma-glutamyltranspeptidase is processed to the mature form by a serine protease bound to the brush border membrane of kidney.     

Analysis of the pH dependence of folate binding and transport by rat kidney brush border membrane vesicles

Folate reabsorption by the mammalian kidney occurs following a tight binding reaction with the renal brush border membrane. Previous studies have shown that transport of folic acid (PteGlu) by rat kidney brush border membrane vesicles occurs maximally at pH 5.6 via a saturable system that is associated with a binding component. The present studies have shown that the pH dependency of transport was due to the development of the transmembrane pH gradient (7.3 in/5.6 out), not to the acidic pH per se. The pH gradient-mediated transport was stimulated by an inwardly directed ionic gradient, either of NaCl or choline chloride. These gradients also stimulated the membrane binding of PteGlu suggesting that NaCl and choline chloride may have increased PteGlu transport by altering binding to the brush border membrane. Renal brush border membrane vesicular transport of PteGlu was not affected by induction of a relatively positive intravesicular space. Transport was inhibited by 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene, an anion exchange inhibitor. The results suggest that rat kidney brush border membrane transport of PteGlu is initiated by association with a specific membrane protein, followed by transfer of folate across the membrane. The overall activity is influenced by a transmembrane pH gradient.     

Localization of aquaporin-7 in rat and mouse kidney using RT-PCR, immunoblotting, and immunocytochemistry

To establish the segmental, cellular, and subcellular localization of AQP7 in rat and mouse kidney, we used RT-PCR, immunocytochemical, and immunoblotting approaches. RT-PCR of rat and mouse kidney zones revealed AQP7 mRNA in cortex and outer stripe of the outer medulla. RT-PCR on microdissected nephron segments revealed AQP7 mRNA in proximal convoluted and straight tubules. Immunoblotting using peptide-derived rabbit antibodies to either rat or mouse AQP7 revealed a 28-kDa band in kidney and testes from rat and mouse, respectively. Immunocytochemistry revealed strong AQP7 labeling of segment 3 proximal tubules and weaker labeling of proximal convoluted tubules in both rat and mouse kidneys. The labeling was almost exclusively confined to the brush border with no basolateral labeling. No labeling was observed of thin descending limbs or collecting duct. Immunolabeling controls were negative. The presence of AQP7 in the proximal tubule brush border indicates a role of AQP7 in proximal tubule water reabsorption.     

ULTRATHIN FROZEN SECTIONS : II. Demonstration of Enzymic Activity

Endogenous enzyme activity can be readily and routinely demonstrated in ultrathin, frozen sections for electron microscopy. The procedure employed to obtain the best structural preservation as well as enzyme activity in thin sections involved fixation in glutaraldehyde, embedding in thiolated gelatin or pure gelatin, partial dehydration in glycerol, and sectioning in a cryostat at -35°C with a slightly modified Porter-Blum microtome on which the tissue is maintained at -70°C and the knife at -23°C. Kidney cortex was used as test tissue, but a few other organs were occasionally used. Thin sections were floated on the surface of several incubation media routinely employed for enzyme cytochemistry. Positive, specific reactions were obtained for alkaline phosphatase in kidney brush border, for adenosine triphosphatase in brush border and in basal membranes of distal tubules, for acid phosphatase and esterase in lysosomes, and for NADH diaphorase in mitochondria. Mitochondrial ATPase was sporadically evident only in the distal tubule of the kidney. Localizations of enzyme activity reported by other technical approaches were confirmed and in some cases somewhat improved.     

Transport of p-aminohippuric acid,uric acid and glucose in highly purified rabbit renal brush border membranes

A procedure for preparing highly purified brush border membranes from rabbit kidney cortex using differential and density gradient centrifugation is described. Brush border membranes prepared by this procedure were substantially free of basal-lateral membranes, mitochondria, endoplasmic reticulum and nuclear material as evidenced by an enrichment factor of less than 0.3 for (Na⁺ + K⁺)-ATPase, succinate dehydrogenase, NADPH-cytochrome c reductase and DNA. Alkaline phosphatase was enriched ten fold indicating that the membranes were enriched at least 30 fold with respect to other cellular organelles. The yield of brush border membranes was 20%.Transport of d-glucose by the membranes was identical to that previously reported except that the Arrhenius plot for temperature dependence of transport was curvilinear (E_A = 11.3–37.6 kcal/mol) rather than biphasic. Transport of p-aminohippuric acid and uric acid were increased by the presence of NaCl, either gradient or preequilibrated. However, no overshoot was obtained in the presence of a NaCl gradient, and KCl and LiCl also produced equivalent stimulation of transport suggesting a nonspecific ionic strength effect. Uptakes of p-aminohippuric acid and uric acid were not saturable, and were increased markedly by reducing the pH from 7.5 to 5.6. Probenecid (1 mM) reduced p-aminohippuric acid and uric acid (50 μM) uptake by 49% and 21%, respectively. We conclude that the uptake of uric acid and p-aminohippuric acid by renal brush border membranes of the rabbit occurs primarily by a simple solubility-diffusion mechanism.     

gamma-Glutamyltranspeptidase activity in newborn rat kidney brush border.

gamma-Glutamyltranspeptidase, known to be localized in the proximal tubule cell brush border in the rat, is a membrane-bound enzyme which transfers the gamma-glutamyl moiety of glutathione or its analogue gamma-glutamyl-p-nitroanilide to an amino acid or dipeptide acceptor. Brush borders were isolated from the kidneys of newborn and adult Sprague-Dawley rats and assayed for gamma-glutamyltranspeptidase activity. There is an increase in specific activity in the brush border with maturation. Newborn and adult brush border preparations exhibit similar pH optima, substrate affinities, apparent Km values, patterns of heat inactivation, inhibition by glutathione, and migration on polyacrylamide gels. Polyacrylamide gel electrophoresis of a deoxycholate extract of brush border proteins and subsequent reaction with substrate within the gel reveal the presence of two bands, suggesting the presence of two forms of gamma-glutamyltranspeptidase in the rat kidney brush border.     

Phlorizin receptors in isolated kidney brush border membranes Differential enzymatic modification of high-affinity receptors and unspecific binding sites

High-affinity phlorizin receptors in isolated kidney brush border membranes are destroyed by the proteolytic enzymes trypsin and papain. The digested membranes show increased unspecific phlorizin binding. It is proposed, that both enzymes expose a deeper, more hydrophobic layer in the brush border membrane to explain the latter finding.     

Monoclonal antibodies against rat kidney antigens.

The proximal tubule of the nephron is subdivided into three structurally and functionally distinct segments, which can be differentiated with the help of special methods. With the aim of producing selective markers for these three portions of the proximal tubule, we raised monoclonal antibodies against the brush border membranes of the rat kidney. Immunohistochemistry was carried out with eleven different monoclonal antibodies to sections of rat kidney and other tissues at the light- and electron-microscopical level. These monoclonal antibodies mainly detect antigens located on the brush border of the proximal tubule, and they allow a distinction between its three segments. However, some antibodies also recognize other portions of the nephron, or even the glomerulus or stromal elements. Sites recognized by the antibodies are not limited to the kidney, but staining is observed on the intestinal brush border, the intralobular ducts of the pancreas, the bile canaliculi of the liver and on the macrophage clusters of the spleen. These antibodies are interesting reagents which can be applied to study biochemical differences between brush border membranes. In addition, they recognize antigenically related sites in other organs with reabsorptive or secretory tasks.     

Characterization of antigenic sialoglycoprotein subunits of the placental brush border membranes: Comparison with liver and kidney membrane subunits by two-dimensional electrophoresis

The sialoglycoprotein subunits of human placental brush border membranes were labeled by sequential treatment with periodate and (³H)-sodium borohydride, which trititates sialic acid, and by lactoperoxidase-catalyzed (¹²⁵I) iodination of tyrosine residues. The labeled subunits were characterized with respect to their affinity for antisera raised against Triton X-100 extracts of placental brush border membranes. The immunochemically reactive components were analyzed by two-dimensional electrophoresis according to a modification of the O'Farrell technique [20] enabling the assignment of estimated M_r? and pI. Of the 33 ³H-labeled brush border subunits present in Triton X-100-solubilized membrane preparations, 18 subunits reacted with antiplacental brush border antisera insolubilized on CNBr-activated Sepharose or in immunoprecipitates. Fourteen of these tritiated subunits were also labeled with ¹²⁵I, confirming that these are glycoproteins. The plasma membranes of normal human liver and microsomes from kidney were examined for the placental brush border glycoprotein subunits by reaction with insolubilized antiplacental brush border antisera and two-dimensional electrophoresis of the reacting tritium-labeled subunits. Comparison of the two-dimensional electrophoretic maps of the immunochemically reacting glycoproteins from liver, kidney, and placenta resulted in the identification of seven placental subunits in common with liver and kidney on the basis of antigenic cross-reactivity, M_r?, and pI. Four placental glycoproteins were not found in the other tissues and are potentially specific to the placenta. Three of the placental subunits were only seen in placenta and kidney. Three of the subunits ran at the dye front and could not be assigned molecular weights. One of the subunits was poorly labeled by tritiation of sialic acid and was not considered.