scholarly journals The tight junction protein ZO-1 is concentrated along slit diaphragms of the glomerular epithelium.

1990 ◽  
Vol 111 (3) ◽  
pp. 1255-1263 ◽  
Author(s):  
E Schnabel ◽  
J M Anderson ◽  
M G Farquhar
Keyword(s):  
Tight Junction ◽  
Rat Kidney ◽  
Freeze Fracture ◽  
Kidney Tissue ◽  
Slit Diaphragm ◽  
Kidney Cortex ◽  
Adult Rats ◽  
Junction Protein ◽  
Collecting Tubules ◽  
Lateral Cell

The foot processes of glomerular epithelial cells of the mammalian kidney are firmly attached to one another by shallow intercellular junctions or slit diaphragms of unknown composition. We have investigated the molecular nature of these junctions using an antibody that recognizes ZO-1, a protein that is specific for the tight junction or zonula occludens. By immunoblotting the affinity purified anti-ZO-1 IgG recognizes a single 225-kD band in kidney cortex and in slit diaphragm-enriched fractions as in other tissues. When ZO-1 was localized by immunofluorescence in kidney tissue of adult rats, the protein was detected in epithelia of all segments of the nephron, but the glomerular epithelium was much more intensely stained than any other epithelium. Among tubule epithelia the signal for ZO-1 correlated with the known fibril content and physiologic tightness of the junctions, i.e., it was highest in distal and collecting tubules and lowest in the proximal tubule. By immunoelectron microscopy ZO-1 was found to be concentrated on the cytoplasmic surface of the tight junctional membrane. Within the glomerulus ZO-1 was localized predominantly in the epithelial foot processes where it was concentrated precisely at the points of insertion of the slit diaphragms into the lateral cell membrane. Its distribution appeared to be continuous along the continuous slit membrane junction. When ZO-1 was localized in differentiating glomeruli in the newborn rat kidney, it was present early in development when the apical junctional complexes between presumptive podocytes are composed of typical tight and adhering junctions. It remained associated with these junctions during the time they migrate down the lateral cell surface, disappear and are replaced by slit diaphragms. The distribution of ZO-1 and the close developmental relationship between the two junctions suggest that the slit diaphragm is a variant of the tight junction that shares with it at least one structural protein and the functional property of defining distinctive plasmalemmal domains. The glomerular epithelium is unique among renal epithelia in that ZO-1 is present, but the intercellular spaces are wide open and no fibrils are seen by freeze fracture. The presence of ZO-1 along slit membranes indicates that expression of ZO-1 alone does not lead to tight junction assembly.

scholarly journals Studies on the orientation of brush-border membrane vesicles

1978 ◽  
Vol 172 (1) ◽  
pp. 57-62 ◽  
Author(s):  
W Haase ◽  
A Schäfer ◽  
H Murer ◽  
R Kinne
Keyword(s):  
Brush Border ◽  
Brush Border Membrane ◽  
Rat Kidney ◽  
Freeze Fracture ◽  
Membrane Vesicles ◽  
Brush Border Membrane Vesicles ◽  
Immunological Methods ◽  
Kidney Cortex ◽  
Asymmetric Distribution ◽  
Rat Kidney Cortex

Orientation of rat renal and intestinal brush-border membrane vesicles was studied with two independent methods: electron-microscopic freeze-fracture technique and immunological methods. With the freeze-fracture technique a distinct asymmetric distribution of particles on the two membrane fracture faces was demonstrated; this was used as a criterion for orientation of the isolated membrane vesicles. For the immunological approach the accessibility or inaccessibility of aminopeptidase M localized on the outer surface of the cell membrane to antibodies was used. With both methods we showed that the brush-border membrane vesicles isolated from rat kidney cortex and from rat small intestine for transport studies are predominantly orientated right-side out.

scholarly journals Transplantation of Fetal Kidney Tissue Reduces Cerebral Infarction Induced by Middle Cerebral Artery Ligation

1999 ◽  
Vol 19 (12) ◽  
pp. 1329-1335 ◽  
Author(s):  
Yung-Hsiao Chiang ◽  
Shinn-Zong Lin ◽  
Cesario V. Borlongan ◽  
Barry J. Hoffer ◽  
Marisela Morales ◽  
...  
Keyword(s):  
Cerebral Cortex ◽  
Kidney Transplantation ◽  
Middle Cerebral Artery ◽  
Mrna Expression ◽  
Kidney Tissue ◽  
Kidney Cortex ◽  
Fetal Kidney ◽  
Adult Rats ◽  
Adult Kidney ◽  
The Right

The authors, and others, have recently reported that intracerebral administration of glial cell line-derived neurotrophic factor (GDNF) or osteogenic protein-1 protects against ischemia-induced injury in the cerebral cortex of adult rats. Because these trophic factors are highly expressed in the fetal, but not adult, kidney cortex, the possibility that transplantation of fetal kidney tissue could serve as a cellular reservoir for such molecules and protect against ischemic injury in cerebral cortex was examined. Fetal kidneys obtained from rat embryos at gestational day 16, and adult kidney cortex, were dissected and cut into small pieces. Adult male Sprague-Dawley rats were anesthetized with chloral hydrate and placed in a stereotactic apparatus. Kidney tissues were transplanted into three cortical areas adjacent to the right middle cerebral artery (MCA). Thirty minutes after grafting, the right MCA was transiently ligated for 90 minutes. Twenty-four hours after the onset of reperfusion, animals were evaluated behaviorally. It was found that the stroke animals that received adult kidney transplantation developed motor imbalance. However, animals that received fetal kidney grafts showed significant behavioral improvement. Animals were later sacrificed and brains were removed for triphenyltetrazolium chloride staining, Pax-2 immunostaining, and GDNF mRNA expression. It was noted that transplantation of fetal kidney but not adult kidney tissue greatly reduced the volume of infarction in the cerebral cortex. Fetal kidney grafts showed Pax-2 immunoreactivity and GDNF mRNA in the host cerebral cortex. In contrast, GDNF mRNA expression was not found in the adult kidney grafts. Taken together, our data indicate that fetal kidney transplantation reduces ischemia/reperfusion-induced cortical infarction and behavioral deficits in adult rats, and that such tissue grafts could serve as an unique cellular reservoir for trophic factor application to the brain.

scholarly journals Connexin-Occludin Chimeras Containing the Zo-Binding Domain of Occludin Localize at Mdck Tight Junctions and Nrk Cell Contacts

1999 ◽  
Vol 146 (3) ◽  
pp. 683-693 ◽  
Author(s):  
Laura L. Mitic ◽  
Eveline E. Schneeberger ◽  
Alan S. Fanning ◽  
James Melvin Anderson
Keyword(s):  
Tight Junction ◽  
Tight Junctions ◽  
Mdck Cells ◽  
Transmembrane Protein ◽  
Rat Kidney ◽  
Freeze Fracture ◽  
Binding Domain ◽  
Permeability Barrier ◽  
Cell Contacts ◽  
Cytoplasmic Proteins

Occludin is a transmembrane protein of the tight junction that functions in creating both an intercellular permeability barrier and an intramembrane diffusion barrier. Creation of the barrier requires the precise localization of occludin, and a distinct family of transmembrane proteins called claudins, into continuous linear fibrils visible by freeze-fracture microscopy. Conflicting evidence exists regarding the relative importance of the transmembrane and extracellular versus the cytoplasmic domains in localizing occludin in fibrils. To specifically address whether occludin's COOH-terminal cytoplasmic domain is sufficient to target it into tight junction fibrils, we created chimeras with the transmembrane portions of connexin 32. Despite the gap junction targeting information present in their transmembrane and extracellular domains, these connexin-occludin chimeras localized within fibrils when expressed in MDCK cells, as assessed by immunofluorescence and immunogold freeze-fracture imaging. Localization of chimeras at tight junctions depends on the COOH-terminal ZO-binding domain and not on the membrane proximal domain of occludin. Furthermore, neither endogenous occludin nor claudin is required for targeting to ZO-1–containing cell–cell contacts, since in normal rat kidney fibroblasts targeting of chimeras again required only the ZO-binding domain. These results suggest an important role for cytoplasmic proteins, presumably ZO-1, ZO-2, and ZO-3, in localizing occludin in tight junction fibrils. Such a scaffolding and cytoskeletal coupling function for ZO MAGUKs is analogous to that of other members of the MAGUK family.

scholarly journals Effects of experimental nephrosis on basement-membrane components and enzymes of collagen biosynthesis in rat kidney

1985 ◽  
Vol 226 (1) ◽  
pp. 243-250 ◽  
Author(s):  
A Jukkola ◽  
J Risteli ◽  
H Autio-Harmainen ◽  
L Risteli
Keyword(s):  
Membrane Proteins ◽  
Basement Membrane ◽  
Indirect Immunofluorescence ◽  
Type Iv Collagen ◽  
Rat Kidney ◽  
Kidney Tissue ◽  
Kidney Cortex ◽  
Collagen Biosynthesis ◽  
Type Iv ◽  
Basement Membrane Proteins

The aim of the present study was to find out whether the basement-membrane proteins laminin and type IV collagen are involved in the development of aminonucleoside-induced nephrosis. These proteins were measured by specific radioimmunoassays in serum, urine and kidney-cortex samples, and they were localized in the glomeruli by indirect immunofluorescence. Nephrosis was induced in rats with a single intraperitoneal injection of puromycin aminonucleoside. Serum laminin concentrations, detected by a radioimmunoassay for the P2 domain of the protein, increased to reach a maximum at days 5-7, and they remained elevated until at least day 14. The increase preceded the development of proteinuria, suggesting a role for laminin in glomerular function. Concomitant with proteinuria, increasing amounts of laminin antigenicity were also found in the urine. The size of the laminin antigen in serum was estimated by gel filtration, and the serum forms were found to contain both the P1 and the P2 regions of the intact laminin molecule. On the other hand, there were no changes in the serum or urinary concentrations of type-IV-collagen-derived antigens, as detected by a radioimmunoassay for the 7S collagen domain of this protein. The total content of laminin in kidney cortex, measured after digestion of the tissue with trypsin and collagenase, was, at day 9, still comparable with normal values, and the distribution of both basement-membrane proteins in the glomeruli, studied by indirect immunofluorescence, was similar to that in the controls. The tissue damage induced by aminonucleoside, however, seems to stimulate collagen biosynthesis, as the activities of prolyl 4-hydroxylase, lysyl hydroxylase and galactosylhydroxylysyl glucosyltransferase in kidney tissue increased significantly, with maxima at days 8-10.

HOCl effects on the tight junctions of rabbit tracheal epithelium

1996 ◽  
Vol 270 (2) ◽  
pp. L224-L231 ◽  
Author(s):  
Y. Guo ◽  
M. Krumwiede ◽  
J. G. White ◽  
O. D. Wangensteen
Keyword(s):  
Tight Junction ◽  
Morphological Changes ◽  
Cell Damage ◽  
Freeze Fracture ◽  
Tracheal Epithelium ◽  
Epithelial Tissue ◽  
Laser Confocal Microscopy ◽  
Transmission Electron ◽  
Junction Protein ◽  
Before And After

We previously found that HOCl, produced from neutrophil products in infected airways, decreases electrical resistance (R) of rabbit tracheal epithelium. Interestingly, HOCl at 6 mM, a reasonable concentration in diseased airways, decreased R without apparent cell damage. This study sought to determine whether this noncytotoxic dose of HOCl causes morphological changes that correlate with the decrease in R. Excised rabbit tracheas were treated with 6 mM HOCl for 15 min, before and after which epithelial R was determined. Epithelial tissue was then fixed and prepared for transmission electron microscopy, immunofluorescent labeling of F-actin or the tight junction protein ZO-1, or freeze fracture to examine tight junction strands. HOCl treatment caused a 50% decrease in R. Electron micrographs showed no cell, cell membrane, or tight junction changes. By laser confocal microscopy, 6 mM HOCl did not affect the distribution of F-actin or ZO-1. However, morphometric analysis of freeze-fracture replicas showed that tight junction strand number was significantly decreased from 7.06 +/- 0.09 to 4.79 +/- 0.11 and junctional width was significantly decreased from 0.306 +/- 0.007 to 0.214 +/- 0.006 microns. These latter changes may have contributed to the observed decrease in epithelial R.

Freeze-fracture replica electron microscopy combined with SDS digestion for cytochemical labeling of integral membrane proteins. Application to the immunogold labeling of intercellular junctional complexes

1995 ◽  
Vol 108 (11) ◽  
pp. 3443-3449 ◽  
Author(s):  
K. Fujimoto
Keyword(s):  
Membrane Proteins ◽  
Tight Junction ◽  
Plasma Membranes ◽  
Freeze Fracture ◽  
Immunogold Labeling ◽  
Integral Membrane Proteins ◽  
Tissue Slices ◽  
Electron Microscopic ◽  
Junction Protein ◽  
Junction Proteins

We propose a new electron microscopic method, the sodium dodecylsulphate (SDS)-digested freeze-fracture replica labeling technique, to study the two-dimensional distribution of integral membrane proteins in cellular membranes. Unfixed tissue slices were frozen with liquid helium, freeze-fractured, and replicated in a platinum/carbon evaporator. They were digested with 2.5% SDS to solubilize unfractured membranes and cytoplasm. While the detergent dissolved unfractured membranes and cytoplasm, it did not extract fractured membrane halves. After SDS-digestion, the platinum/carbon replicas, along with attached cytoplasmic and exoplasmic membrane halves, were processed for cytochemical labeling, followed by electron microscopic observation. As an initial screening, we applied this technique to the immunogold labeling of intercellular junction proteins: connexins (gap junction proteins), occludin (tight junction protein), desmoglein (desmosome protein), and E-cadherin (adherens junction protein). The immunogold labeling was seen superimposed on the image of a fracture face visualized by platinum/carbon shadowing. The immunoreaction was specific, and only the structures where the proteins were expected were labeled. For instance, anti-occludin immunogold complexes were observed immediately adjacent to the tight junction strands on the protoplasmic and exoplasmic fracture faces. No significant levels of gold label were associated with non-tight-junctional regions of plasma membranes. The procedures of the SDS-digested freeze-fracture replica labeling and its potential significance are discussed.

scholarly journals Transport ATPase cytochemistry: ultrastructural localization of potassium-dependent and potassium-independent phosphatase activities in rat kidney cortex.

1975 ◽  
Vol 66 (3) ◽  
pp. 586-608 ◽  
Author(s):  
S A Ernst
Keyword(s):  
Alkaline Phosphatase ◽  
Atpase Activity ◽  
Rat Kidney ◽  
Proximal Tubules ◽  
Rabbit Kidney ◽  
Kidney Cortex ◽  
Rat Kidney Cortex ◽  
Distal Tubules ◽  
Extracellular Side ◽  
Collecting Tubules

A cytochemical method for the light and electron microscope localization of the K- and Mg-dependent phosphatase component of the Na-K-ATPase complex was applied to rat kidney cortex, utilizing p-nitrophenylphosphate (NPP) as substrate. Localization of K-N-ATPase activity in kidneys fixed by perfusion with 1% paraformaldehyde -0.25% glutaraldehyde demonstrated that distal tubules are the major cortical site for this sodium transport enzyme. Cortical collecting tubules were moderately reactive, whereas activity in proximal tubules was resolved only after short fixation times and long incubations. In all cases, K-NPPase activity was restricted to the cytoplasmic side of the basolateral plasma membranes, which are characterized in these neplron segments by elaborate folding of the cell surface. Although the rat K-NPPase appeared almost completely insensitive to ouabain with this cytochemical medium, parallel studies with the more glycoside-sensitive rabbit kidney indicated that K-NPPase activity in these nephron segments is sensitive to this inhibitor. In addition to K-NPPase, nonspecific alkaline phosphatase also hydrolyzed NPP. The latter could be differentiated cytochemically from the specific phosphatase, since alkaline phosphatase was K-independent, insensitive to ouabain, and specifically inhibited by cysteine. Unlike K-NPPPase, alkaline phosphatase was localized primarily to the extracellular side of the microvillar border of proximal tubules. A small amount of cysteine-sensitive activity was resolved along peritubular surfaces of proximal tubules. Distal tubules were unreactive. In comparative studies, Mg-ATPase activity was localized along the extracellular side of the luminal and basolateral surfaces of proximal and distal tubules and the basolateral membranes of collecting tubules.

Utilization of stereo in the freeze-fracture study of the ultrstructure of rat kidney cortex

Micron (1969) ◽  
1978 ◽  
Vol 9 (3) ◽  
pp. 127-132
Author(s):  
S.E. Coleman ◽  
J. Duggan ◽  
H.C. Aldrich ◽  
R.L. Hackett
Keyword(s):  
Rat Kidney ◽  
Freeze Fracture ◽  
Kidney Cortex ◽  
Rat Kidney Cortex ◽  
Fracture Study

A dominant mutant of occludin disrupts tight junction structure and function

1999 ◽  
Vol 112 (12) ◽  
pp. 1879-1888 ◽  
Author(s):  
S.D. Bamforth ◽  
U. Kniesel ◽  
H. Wolburg ◽  
B. Engelhardt ◽  
W. Risau
Keyword(s):  
Tight Junction ◽  
Freeze Fracture ◽  
Integral Membrane Protein ◽  
Epithelial Cell Line ◽  
Permeability Barrier ◽  
Cell Monolayers ◽  
Junction Protein ◽  
N Terminus ◽  
Freeze Fracture Electron Microscopy ◽  
And Function

The tight junction is the most apical intercellular junction of epithelial cells and forms a diffusion barrier between individual cells. Occludin is an integral membrane protein specifically associated with the tight junction which may contribute to the function or regulation of this intercellular seal. In order to elucidate the role of occludin at the tight junction, a full length and an N-terminally truncated murine occludin construct, both FLAG-tagged at the N terminus, were stably introduced into the murine epithelial cell line CSG 120/7. Both constructs were correctly targeted to the tight junction, as defined by colocalization with another tight junction protein, ZO-1. The construct lacking the N terminus and extracellular domains of occludin was found to exert a dramatic effect on tight junction integrity. Cell monolayers failed to develop an efficient permeability barrier, as demonstrated by low transcellular electrical resistance values and an increased paracellular flux to small molecular mass tracers. Furthermore, gaps were found to have been induced in the P-face associated tight junction strands, as visualized by freeze-fracture electron microscopy. These findings demonstrate an important role for the N-terminal half of occludin in tight junction assembly and maintaining the barrier function of the tight junction.

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