The tight junctions of renal tubules in the cortex and outer medulla

Summary Quantitative aspects of tight junction morphology were systematically studied in the cortical and outer medullary segments of the distal urinary tubules of rat, hamster, rabbit, cat, dog and the primitve primate Tupaia belangeri.Only minor differences in junctional architecture were found between straight and convoluted portions of the distal tubule. In contrast, the collecting duct in cortex and outer medulla, in all species, exhibits the most elaborate tight junctions observed along the uriniferous tubule.The present and previous findings from this laboratory indicate that increasing tightness of the junctional complexes is apparent along the course of the nephron in all species studied.The proposed relationship between quantitative aspects of the zonula occludens and presently available values for transepithelial electrical resistance was re-examined for the renal tubules. It was found that for the mammalian kidney a satisfactory correlation exists between the tight junction morphology and presently known functional parameters. This relationship is the more evident the more additional dimensional characteristics of the intercellular clefts are taken into consideration.It may therefore be concluded that, at least for the mammalian kidney, the assumption of differences in the molecular organization of the tight junctions is not needed to explain so far unresolved discrepancies between tubular morphology and function.Parts of these findings were presented at the 72nd Meeting of the Anatomical Society, Aachen; April 1977 (see Verh. Anat. Ges. 72:229–234 [1978])Supported by the Deutsche Forschungsgemeinschaft     

A freeze-fracture study of tight junctions in the pars convoluta and pars recta of the renal proximal tubule

Summary The morphology of tight junctions of the renal proximal tubule was studied comparing the pars convoluta and pars recta of rat, golden hamster, rabbit, cat, dog and tupaia. Though some interspecies variations were observed, the convoluted portions of the proximal tubules revealed quite uniformly very leaky tight junctions with mainly 1–2 strands.Along the whole proximal tubule of the rabbit kidney including the pars recta only minor differences of the zonulae occludentes were found. By contrast, the tight junctions of the pars recta in other species were much more elaborate, especially in cat and tupaia, having up to 6 strands and an overall depth of more than 150 nm. The implications of these findings are discussed with special regard to the functional differences between the pars convoluta and pars recta of the proximal tubule.This work was supported by the Deutsche Forschungsgemeinschaft     

Morphology of tight junctions in the ciliary epithelium of rabbits during arachidonic acid-induced breakdown of the blood-aqueous barrier

Summary A reversible breakdown of the blood-aqueous barrier in the iridial processes of rabbits has been induced by arachidonic acid as demonstrated by the passage of horseradish peroxidase at places through the tight junctions. Freeze-fracture images reveal very discontinuous Pface ridges. However, the analysis of complementary replicas demonstrates that discontinuities of P-face ridges are always complemented by particles or short bars found in the E-face furrows. Though the problem exists of correlating freeze-fracture images of the junctional structure to the focal passage of horseradish peroxidase, the data suggest that the discontinuities of P-face ridges cannot be the structural counterpart of the passage of horseradish peroxidase. Alternative pathways of horseradish peroxidase are discussed in context with the offset bifibrillary model of the junction.Supported by a research fellowship from the Deutsche Forschungsgemeinschaft Present address: Universitäts Augen- und Poliklinik der Freien Universität, Klinikum Steglitz, Hindenburgdamm 30, 1000 Berlin 45This paper was presented in part at the International Symposium on Membrane Transport Mechanisms in the Eye, September 1984, Berlin     

Gap junctions between the follicle cells and the oocyte during oogenesis in an insect,Tribolium destructor/Coleoptera

Summary Oocyte-follicle cell gap junctions inTribolium occur in all oogenetic stages studied. During early previtellogenesis the junctions are found exclusively between lateral membranes of oocyte microvilli and the membrane of prefollicle cells. In late previtellogenesis and vitellogenesis the junctions are located between the tips of oocyte microvilli and the flat membranes of the follicle cells. During previtellogenesis gap junctions are infrequent, whereas in the phase of yolk accumulation their number increases considerably, exceeding 17 junctions/m² of the follicle cell membrane. It could be shown by microinjection of a fluorescent dye that gap junctions are in a functional state during vitellogenesis. Possible roles of heterologous gap junctions in oogenesis are discussed.     

Interendothelial junctions in kidney vessels

Summary The interendothelial junctions of all segments of the renal vasculature have been studied in eight species using the freeze-fracture technique. Three types of junctions have been found. Combinations of tight and gap junction elements are characteristic for interlobular arteries and proximal afferent arterioles. Continuous tight junction strands not subdivided into individual particles are typical for the glomerular arterioles close to the glomerulus and the vasa recta. The interendothelial junctions of glomerular and peritubular capillaries and cortical veins are characterized by slight elevations decorated with sparse arrays of particles on the P-face of the endothelial cell plasma membrane.These studies were supported by the German Research Foundation within the SFB 90 Cardiovascular System.     

Characterization of an in vitro system of human renal papillary collecting duct cells

Summary We have developed an in vitro model of human papillary collecting duct cells isolated from cadaver kidneys using methods similar to those we previously reported for the isolation of human proximal tubule cells. To date we have isolated papillary collecting duct cells from 100 normal human kidneys. Papillae were dissected and digested in Cellgro containing 400 U/ml collagenase. Cells were plated on fibronectin-coated culture flasks at a density of 10⁴ live cells/ml in Cellgro supplemented with insulin and 10% fetal bovine serum. Confluent monolayers, which were able to withstand 600 mOsm for 8 h, were obtained within 10 to 15 d. Cells of primary isolates and first passages exhibited epithelial cell ultrastructure including cell junctions, microvilli, and cilia. A dark-brown reaction product was observed in these cells when stained by the immunoperoxidase method with peroxidase-labeled peanut lectin (Arachis hypogaea), which binds specifically to human distal tubule and collecting duct cells. These cells were negative for Factor-VIII (a marker for endothelial cells) and γ-glutamyltransferase (a marker for proximal tubule cells). High activities of the glycolytic enzyme pyruvate kinase and arginine vasopressin-stimulated cAMP production in these cells are consistent with a distal nephron origin. The results indicate that human collecting duct cells can be isolated and cultured to provide an in vitro system to probe pathogenetic mechanisms of potential nephrotoxins. Part of this work was presented at a Symposium of the Center for Alternatives to Animal Testing, April 4–5, 1989, Johns Hopkins Medical Institutions, Baltimore, MD 21205. This work was supported in part by grants R01-AI24179, PO1-A804393 for the Public Health Service, U.S. Department of Health and Human Services, and by a grant from the National Kidney Foundation, Baltimore, MD affiliate.     

Reformation of gap and tight junctions in regenerating liver after cholestasis

Summary Morphometric analysis of the alterations in interhepatocyte junctions induced by bile duct ligation revealed that after 48 h, during which time the serum bilirubin increased 6 to 8 fold, the membrane area occupied by gap junctions on the apico-lateral and medio-lateral sides decreased from 3.6% in controls to 0.02% in the ligated group. The strands of the zonulae occludentes were reduced in number and showed increased discontinuities.Within 45 min of recanalization of the common bile duct, clusters of particles appeared within and adjacent to the tight junctional areas or in the lateral hepatocyte membrane. Subsequently, the particle aggregations localized in the apico-lateral membrane areas increased in number and size becoming finally indistinguishable from those of controls within 96 h after the onset of recanalization. The zonulae occludentes also rearranged and reestablished their original structure during this period. The serum bilirubin fell to normal within 24 h of recanalization. It is concluded that metabolic and ultrastructural restitution associated with the recanalization of the ligated bile duct have no strict temporal correlation to one another.These studies provide further evidence that alterations in gap and tight junctions induced by pathological processes, e.g. during bile duct ligation, are completely reversible when regeneration occurs.Summer student from Harvard Medical School, Boston (USA)     

Regulation by nectin of the velocity of the formation of adherens junctions and tight junctions

Cadherins are key Ca(2+)-dependent cell-cell adhesion molecules at adherens junctions (AJs) in fibroblasts and epithelial cells, whereas claudins are key Ca(2+)-independent cell-cell adhesion molecules at tight junctions (TJs) in epithelial cells. The formation and maintenance of TJs are dependent on the formation and maintenance of AJs. Nectins are Ca(2+)-independent immunoglobulin-like cell-cell adhesion molecules which comprise a family of four members, nectin-1, -2, -3, and -4, and are involved in the formation of AJs in cooperation with cadherins, and the subsequent formation of TJs. We show here that the velocity of the formation of the E-cadherin-based AJs is increased by overexpression of nectin-1 and is reduced by addition of the nectin-1 inhibitors to the medium in L cells stably expressing E-cadherin and Madin-Darby canine kidney cells. Moreover, the velocity of the formation of the claudin-based TJs is increased by overexpression of nectin-1 and is reduced by addition of the nectin-1 inhibitors to the medium in Madin-Darby canine kidney cells. These results indicate that nectins regulate the velocity of the formation of the E-cadherin-based AJs and the subsequent formation of the claudin-based TJs.     

Cell type-specific glycoconjugates of collecting duct cells during maturation of the rat kidney

Summary The ontogeny of lectin-positive epithelial cell types and the maturation of polarized expression of the glycocalyx of the collecting ducts (CD) of the rat kidney were studied from samples of 18th-day fetal and neonatal kidneys of various ages. Lectins from Dolichos biflorus (DBA) and Vicia villosa (VVA), with preferential affinity to principal cells, stained virtually all CD cells of the fetal kidneys. However, within two days postnatally, the number of cells positive for DBA and VVA decreased to amounts found in the adult kidneys. Moreover, a characteristic change occurred rapidly after birth in the intracellular polarization of the reactive glycoconjugates, from a uniform plasmalemmal to a preferentially apical staining. In contrast, lectins from Arachis hypogaea (PNA), Maclura pomifera (MPA) and Lotus tetragonolobus (LTA), reacting indiscriminatively with principal and intercalated cells of adult kidneys, stained most CD cells in the fetal kidneys, and failed to show any postnatal change in the amount of positive cells or in the intracellular polarization.The immunocytochemical tests for (Na + K)-ATPase and carbonic anhydrase (CA II) revealed the characteristic postnatal decrease in the amount of principal cells and simultaneous increase in the amount of CA II rich intercalated cells. DBA and VVA reactive cells also decreased postnatally, paralleling the changes observed in the (Na + K)-ATPase positive principal cells. The present results suggest that the expression of the cell type-specific glycocalyx of principal and intercalated cells is developmentally regulated, undergoes profound changes during maturation, and is most likely associated with electrolyte transport phenomena.     

Marked reduction in intramembranous particle clusters in the terminal portion of inner medullary collecting ducts of antidiuretic rats

Summary Antidiuretic hormone (ADH) induces an aggregation of intramembranous particles (IMP) into discrete clusters in the luminal plasma membrane of rat renal papillary collecting duct cells (Harmanci et al. 1978). The correlation between an elevated dose of ADH, increased urine osmolality, and greater IMP cluster frequency has led to speculation that the water permeability of the luminal plasma membrane is reflected by the IMP cluster density (Harmanci et al. 1980). The present study indirectly evaluated this water permeability by quantitating collecting duct IMP cluster frequency from freeze fracture replicas in two regions of the renal papilla, at its base and at its tip, in antidiuretic and in water diuretic rats. During antidiuresis there was a high frequency of IMP clusters (189/100 m² of luminal plasma membrane) in cells from the papilla base but not at the papilla tip (9.0/100 m²). During water diuresis there were few IMP clusters in either cells from the papilla base (5.9/100 m²) or at the papilla tip (1.4/100 m²). Most significantly these results suggest that the water permeability of the terminal portion of the inner medullary collecting duct of antidiuretic rats is significantly lower than that of the collecting duct epithelium higher in the papilla.Preliminary findings of this study were presented at the Second International Congress of Cell Biology, West Berlin 1980     

Microheterogeneity of the collecting duct system in rabbit kidney as revealed by monoclonal antibodies

Summary This report describes the immunolocalization of three monoclonal antibodies along the collecting duct system in rabbit kidney. The antibodies were raised against antigens derived from a membrane fraction of homogenized papillary tissue. Western Blot analysis demonstrated that each of the antibodies recognized a single band of about 190000 (P_CD1), 210000 (P_CD2) and 50000 (P_CD3) daltons. In renal tissue, the antibodies bound specifically to the epithelia of the connecting tubule (CNT), the collecting duct (CD) and the papillary surface epithelium. Differences in the binding patterns of the antisera were limited to the cortex. p_CD1 labeled only a few scattered cells in the CNT, and exhibited a heterogeneous binding along the cortical collecting duct (CCD). P_CD2 and P_CD3 binding patterns were similar. In the CNT, these antibodies bound to the intercalated cells (IC-cells) but not to the CNT-cells proper. In the CCD, both IC-cells and principal cells were labeled. The binding to the medullary collecting duct by all three antisera was identical. The ureter was labeled only by P_CD2 and P_CD3, and none of the antisera bound to the bladder epithelium.The antibody binding patterns provide information concerning tubular axial heterogeneity and embryogenetic aspects of the CNT and the CCD. These antibodies may be used as differentiation markers in studies of the developing kidney and of renal tissue culture systems.These studies were supported by Deutsche Forschungsgemeinschaft, Forschergruppe Niere, Kr 546/5-1     

Junctions between interstitial cells of the renal medulla: A freeze-fracture study

Summary Cell junctions between interstitial cells of the renal medulla were studied in freeze-fracture replicas of kidneys from rat, rabbit, hamster and the tree-shrew Tupaia belangeri. In all species studied a composite type of intercellular junction was found comprising elements of tight junctions and irregular gap junctions of highly variable size and shape. The number of these junctions increased towards the tip of the papilla.Our findings suggest that the composite junctions observed play a role in the maintainance of the ladder-like arrangement of the interstitial cells in the inner zone. The existence of irregular gap junctions raises the possibility that the functions of the interstitial cells are coordinated, especially during alterations of the functional state of the kidney.Supported by the Deutsche Forschungsgemeinschaft     

Maturation of tight junctions in guinea-pig cecal epithelium

Summary By means of the freeze-fracture technique and in tracer studies it is demonstrated that the structure of tight junctions and the permeability to lanthanum of the guinea-pig cecal epithelium change during maturation of cells. Height and strand number of tight junctions in the apical-basal direction increase as crypt cells migrate to the surface of the epithelium. Likewise, the interlacing of continuous strands was greater in surface than in crypt junctions. The numerous free-ends, isolated individual freestrands and maculae occludentes found in crypt cells were absent in surface epithelial cells. Goblet cells, located at the bottom of crypts, displayed tight junctions similar in characteristics to those of cells located in the middle region of crypts. Cells at the surface and in middle regions of crypts possess tight junctions impermeable to lanthanum, whereas junctions between cells located at the bottom of crypts often were permeable to the tracer, indicating that permeability decreases as the epithelial cells mature. Genesis and maturation mechanisms related to structural configuration of tight junctions are discussed.     

Morphogenesis of tight junctions in the peritoneal mesothelium of the mouse embryo

The peritoneal mesothelium of mouse embryos (12 to 18 day of gestation) was studied by freeze-fracture and in sections in order to reveal the initial formation of the tight junctions. Freeze-fracture observations showed three types of tight junctions. Type I consists of belt-like meshworks of elevations on the P face and of shallow grooves on the E face. No tight junctional particle can be seen either on the elevations or in the grooves. Type II shows rows of discontinuous particles on the elevations on the P face. Type III consists of strands forming ridges on the P face. On the E face, the grooves of Type II and III appear to be narrower and sharper than those of Type I. Quantitatively, Type I junctions are most numerous during the early stages (day 12-13) of embryonic development, while Type III junctions become more common in the later stages, and are the only type seen by day 18. Observations on sections, however, fail to distinguish between the three types. The results suggest that an initial sign of tight junction formation is close apposition of the two cell membranes in the junctional domain, without tight junctional particles. Later, the particles appear to be incorporated in the tight junctions and the strands form by fusion of the particles.     

Cell junctions in early embryos of squid (Loligo pealei)

Summary Squid embryos examined by freeze-fracture and thin-section electron microscopy exhibit identifiable gap junctions during mid-cleavage stages (stages 7–8), and junctional complexes composed of adherent appositions, elaborate septate junctions and gap junctions at slightly later stages (stages 12–13). During germinal layer establishment (stages 12–13) cytoplasmic bridges frequently link the embryonic cells. The presence of gap junctions in cleavagestage embryos provides the morphological substrate for a demonstrated pathway of direct cell-cell communication that is modifiable by experimental treatments and may be physiologically regulatable. The existence of septate junctions and adherent contacts at later stages suggests that some functional specialization, perhaps the establishment of a strongly joined framework of cells at the surface of the embryo, accompanies the formation of germinal layers.     

Novel cellular mechanism that mediates the collecting duct formation during postnatal renal development

We have previously demonstrated that kidney embryonic structures are present in rats, and are still developing until postnatal Day 20. Consequently, at postnatal Day 10, the rat renal papilla contains newly formed collecting duct (CD) cells and others in a more mature stage. Performing primary cultures, combined with immunocytochemical and time-lapse analysis, we investigate the cellular mechanisms that mediate the postnatal CD formation. CD cells acquired a greater degree of differentiation, as we observed that they gradually lose the ability to bind BSL-I lectin, and acquire the capacity to bind Dolichos biflorus. Because CD cells retain the same behavior in culture than in vivo, and by using DBA and BSL-I as markers of cellular lineage besides specific markers of epithelial/mesenchymal phenotype, the experimental results strongly suggest the existence of mesenchymal cell insertion into the epithelial CD sheet. We propose such a mechanism as an alternative strategy for CD growing and development.     

Alterations in intercellular junctions of the uterine epithelium during the preimplantation phase in the rabbit

Summary In the rabbit, the pseudopregnant uterus has been used as a model for studying alterations characteristic of the preimplantation phase. Alterations in intercellular junctions of the uterine epithelium were investigated during early pseudopregnancy (day 0 to day 6) by means of the freeze-fracture technique.In the uterine epithelium of oestrous females the zonula occludens belongs to the tight type of tight junctions. During pseudopregnancy an impressive proliferation of tight junctional belts can be observed. The basal strands proliferate, forming loops perpendicular to the luminal surface, whereas the more or less parallel arrangement of the luminal strands is maintained. At day 4 of pseudopregnancy macular tight junctions begin to develop on the lower portions of the lateral plasmalemma and are extensive by day 6 post hCG.Small gap junctions are infrequent between cells of the uterine epithelium and show no significant changes during the preimplantation phase.The physiological significance of the present morphological observations is discussed in the light of changes occurring during the preimplantation period.Supported by grant Kü 210/9 from the Deutsche Forschungsgemeinschaft     

Apico-basal osmotic gradient induces transcytosis in cultured renal collecting duct epithelium

Summary The present experiments report the existence of an apico-basal plasma membrane shuttle in cultured renal collecting duct principal cell epithelium. Apical and basal perfusion under isotonic conditions, 290 mosm phosphate-buffered saline (PBS), has no effect on the shape of the epithelium. In contrast, gradient perfusion bf the epithelium with 75 mosm PBS on the apical side and 290 mosm PBS on the basal side for 10 min alters the morphology of the epithelium by causing the originally columnar epithelial cells to become lower, the intercellular spaces to dilate, and the intracellular vesicles to enlarge. Perfusion of the epithelium with isotonic PBS in the presence of electron-dense cellular markers such as gold-coupled GP_CDI antibody, recognizing a glycoprotein in the plasma membrane of collecting duct cells (W.W. Minuth, G. Lauer, S. Bachman and W. Kriz,Histochemistry 80:171–182, 1984), cationized ferritin (CF), horseradish peroxidase (HRP) and native ferritin (NF) for 10 min reveals their binding at the apical plasma membrane. Little endocytosis is observable. However, after labeling the luminal side by the cellular markers and following exposure to apical hypotonicity, 75 mosm PBS for 10 min, endocytosis of all markers is enhanced to a high degree. Furthermore, the gold-coupled GP_CDI antibody and cationized ferritin are transported within vesicles unidirectionally through the epithelium and are exocytosed at the basolateral aspect, indicating the retrieval and possible translocation of apical plasma membrane. In contrast, volume markers such as NF and HRP are also endocytosed under osmotic gradient exposure, but are not seen to be transcytosed. Therefore, the function of this membrane pathway seems not to be related to water reabsorption, but may be part of a cellular response as protection against the osmotic gradient.     

Fibrillar and cytoskeletal substructure of tight junctions: Analysis of single-stranded tight junctions linking fibroblasts of the lamina fusca in hamster eyes

Summary The lamina fusca of the hamster eye contains layers of flattened, slightly overlapping fibroblasts. Thin sections of the overlapping margins reveal punctate, tight-junction-like membrane appositions associated with accumulation of cytoplasmic filaments, 5–7 nm in diameter. Intermediate filaments are present in the surrounding cytoplasm. A diffuse dense substance occurs in adjacent intercellular space. Freeze-fracture replicas show that the membrane appositions are mainly single-stranded tight junctions, each composed of two fibrils (micelles), and each continuous or nearly continuous around the fibroblastic perimeter. Fracturing characteristics of these junctions offer a unique opportunity to gain further insight into tight junctional morphology. When exposed, the fibrils adhere to the P-face, measure 9.2±0.3 nm in diameter, and are accompanied by a narrow band of membrane differing in texture from non-junctional membrane. Characteristically, the junctional fibrils themselves mark the deviation line along which fracture planes pass from one membrane of the junction to the other. This pattern exposes, over long distances, the P-face of one membrane on one side of this line and E-face of the adjacent membrane on the other. Analysis of any single junction over such distances reveals that the juxtaposition of the fibrils may gradually twist or undulate over a range of at least 180° within the two involved membranes. The fracture plane appears preferentially to pass between the two junctional fibrils; association of the cytoskeleton with junctional fibrils may govern this route of fracture. Cytoskeletal attachment appears to be to a single fibril and may alternate from one fibroblast to the next depending on which cytoplasmic leaflet is nearest a given fibril.Parts of this work have been presented at meetings of the Association for Research in Vision and Ophthalmology (Kelly and Hageman 1983) and the American Association of Anatomists (Hageman and Kelly 1984)