Loss and reappearance of gap junctions in regenerating liver

Changes in intercellular junctional morphology associated with rat liver regeneration were examined in a freeze-fracture study. After a two-thirds partial hepatectomy, both gap junctions and zonulae occludentes were drastically altered. Between 0 and 20 h after partial hepatectomy, the junctions appeared virtually unchanged. 28 h after partial hepatectomy, however, the large gap junctions usually located close to the bile canaliculi and the small gap junctions enmeshed within the strands of the zonulae occudentes completely disappeared. Although the zonulae occludentes bordering the bile canaliculi apparently remained intact, numerous strands could now be found oriented perpendicular to the canaliculi. In some instances, the membrane outside the canaliculi was extensively filled with isolated junctional strands, often forming very complex configurations. About 40 h after partial hepatectomy, very many small gap junctions reappeared in close association with the zonulae occludentes. Subsequently, gap junctions increased in size and decreased in number until about 48 h after partial hepatectomy when gap junctions were indistinguishable in size and number from those of control animals. The zonulae occludentes were again predominantly located around the canalicular margins. These studies provide further evidence for the growth of gap junctions by the accretion of particles and of small gap junctions to form large maculae.     

Intercellular junctions during development of the definitive kidney in lampreys: freeze fracture and morphometric analyses

The changing morphology of intercellular junctions in renal morphogenesis during lamprey metamorphosis was followed using freeze-fracture replicas and morphometry. Gap junctions and particle aggregates among strands of occluding junctions are conspicuous in the differentiating podocytes of the renal corpuscle and in the early ciliated neck and proximal segments but not in the distal segment. The cells of the segmented nephron arise from alpha nephrogenic cells which have a focal aggregate (macula occludens) of 4.8 junctional strands. Upon the initiation of metamorphosis the number of strands increases to 8 in undifferentiated cells with either maculae or zonulae occludentes. Differentiation of both neck and proximal segments is accompanied by gradual transformation of a 7-strand zonula occludens to a 4-strand junction but it becomes more shallow in the latter segment. Two types of undifferentiated cell are recognized through five of the seven metamorphic stages. Cells of two types of distal segments begin differentiation at the midpoint of transformation and immediately show zonula occludens of different morphology. Distal segment I (pars recta) has 5 strands and a 0.250 mum depth whereas segment II has 8-9 strands with twice the apical-basal depth. The larval archinephric duct undergoes a moderate transformation in junctional morphology with the addition of 2 strands and no increase in apical-basal depth in zonulae occludentes during metamorphosis. Changes and development of types of intercellular junctions along the nephron in lampreys are discussed with reference to known regional functional specialization in this organism and with the morphology of renal tubular intercellular junctions in other vertebrates.     

Fine structure of the liver in the larval lamprey, Petromyzon marinus L.; hepatocytes and sinusoids

The ultrastructure of hepatocytes, bile canaliculi, and hepatic sinusoids of the larval lamprey, Petromyzon marinus, was examined using thin-sectioned and freeze-fractured tissues. The liver is a "tubular gland" with hepatocytes arranged in a tubular fashion around large bile canaliculi. Hepatocytes are roughly conical in shape, with their tapered apices facing a bile canalicular lumen. They possess extensive rough and smooth endoplasmic reticulum, a well-developed Golgi complex, abundant mitochondria, and varying numbers of large secondary lysosomes. Both secondary lysosomes and the Golgi complex are concentrated in the apical or peribiliary cytoplasm, indicating a possible role in bile secretion. The apical surfaces of the hepatocytes bear numerous elongate microvilli and occasional cilia, which project into the bile canaliculi. The hepatocytes are joined, apically, by junctional complexes composed of zonulae occludentes and adhaerentes. In freeze-fracture, the zonulae occludentes are of variable apicobasal depth and consist of honeycomb-like meshworks of fibrils. Spaces of variable width frequently appear in the P-face grooves, indicating that the zonulae occludentes are "leaky." Numerous communicating (gap) junctions join the hepatocytes laterally. Varying numbers of lateral microvilli project into the intercellular spaces and, basally, the plasma membrane is deeply infolded, resulting in the formation of apparently interdigitating basal processes resting upon a thin basal lamina. Sinusoids are composed of both a heavily-fenestrated, continuous endothelium, and phagocytic reticulo-endothelial (Kupffer) cells. Depsite the difference in arrangement of their hepatocytes, the mammalian and lamprey livers show similar ultrastructural features.     

Carbon tetrachloride induced proliferation of tight junctions in the rat liver as revealed by freeze-fracturing

Application of carbon tetrachloride produced a progressive proliferation of tight junctions in the rat liver. This system proved to be rapid and highly reproducable and affords the opportunity for tracing the fate of tight junctions in freeze-fracture replicas, facilitating investigations on their formation and function. Beginning on day one carbon tetrachloride treatments resulted in the progressive loosening and fragmentation of the junctional meshwork. After three to four days the membrane outside the zonulae occludentes was extensively filled with proliferated discrete junctional elements often forming complex configurations. From the fifth day on the zonulae occludentes were restricted again predominantly around the bile canaliculus margins. But the junctional meshwork of the zonulae occludentes remained loosened in comparison to those in the control rats. It could be shown that tight junction proliferation on the lateral surface of the plasmalemma occurred both through de novo formation from discrete centers of growth by addition of intramembranous particles and through reorganization of preexistent junctional strands of the fragmented zonulae occludentes bodies. Whereas the large gap junctions close associated with the zonulae occludentes remained more or less unaffected during the experiments, small gap junctions increased in number after five days and were located at the margin or in the tight junction domain. It is assumed that the degeneration of the tight junctions served as a pool for intramembranous particles which form the gap junctions. The results of these observations are discussed in relation to those obtained in other systems.     

Ultrastructural features of degeneration of the gallbladder during lamprey biliary atresia

Thin sections and freeze-fracture replicas were used to examine the fine structural features of degeneration of the gallbladder during lamprey biliary atresia. The cells of the epithelium undergo a progressive accumulation of dense bodies and vacuoles, loss of glycogen, condensation of the filamentous ectoplasm, fragmentation of microvilli, and dilation of cisternae of rough endoplasmic reticulum but eventually disappear by stage 4 of metamorphosis. Zonulae occludentes in the epithelium show a progressive increase in apical-basal depth as the junctional strands fragment. The possibility of an influence of transformed, subepithelial cells on degeneration of epithelial cells is suggested by close contact of the former with the thickened, highly pleated, epithelial basal lamina. The smooth muscle cells of the larval gallbladder are believed to transform during lamprey metamorphosis into these subepithelial cells which shed their external lamina, become intimately associated with collagen and other microfibrils, and which may be capable of phagocytosis. The events of gallbladder degeneration during lamprey metamorphosis show features of apoptosis.     

Liver ultrastructure and a new cell type in the Japanese newt, Cynops pyrrhogaster.

The liver of the Japanese newt, Cynops pyrrhogaster, has been investigated using light, scanning, and transmission electron microscopy. Hepatic parenchyma was composed of clusters and cords or tubules of polyhedral cells separated by a sinusoidal net. Hepatocytes had spherical, euchromatic nuclei with one or more nucleoli and stacked mitochondria with sparse cristae and dense bodies. Rough endoplasmic reticula formed peribiliary stacks and diffusely scattered vesicles and tubules. Smooth endoplasmic reticula were more pronounced in glycogen-rich hepatocytes. Most hepatocytes contained peroxisomes, Golgi complexes and large numbers of fat droplets within the cytoplasm along with glycogen. Some cells were mainly glycogen-storing and contained few or no fat droplets. A special feature of the newt liver was biliary atresia. Bile canaliculi had short, stout microvilli which were entirely atretic in some canaliculi. Canaliculi were sealed off by junctional complexes including zonulae occludentes and maculae adherentes. The latter showed extraordinary wider desmosomal gaps in the vicinity of the atretic bile canaliculi. The sinusoid wall was non-distinctive and contained fenestrated endothelial cells connected to Kupffer cells by zonulae occludentes. A distinctive new cell type (OG cell) was observed in the newt liver. These cells were found individually or in small clusters in proximity with the sinusoidal surfaces. They had small nuclei, a paucity of cytoplasmic organelles, but numerous, unique, osmiophilic granules of two distinct types. Less numerous Type I granules contained homogeneous electron-dense material, and a predominant Type II granule contained circumferentially arranged subparticulation. Granules of both types were detected within the cytoplasm of endothelial cells and within sinusoids together with blood elements. The function of this secretory type cell remains obscure, though it may represent a stage of melanophore.     

Junctional complexes of the tubular cells in the human kidney as revealed with freeze-fracture

Intercellular junctions between human kidney tubular cells were studied by the freeze-fracture technique. The number of strands of the zonulae occludentes increased gradually from the proximal segment to the collecting tubule. Only one strand was visible in the proximal segment (in contrast to 2-4 strands of the neighbouring Bowman's capsule). In the thin segment 2-4 strands were revealed. In the distal segment 1-5 strands were present in the pars recta, 4-6 in the pars convoluta. The most extensive and complex zonulae occludentes were found in the collecting tubule. Gap junctions were seen only between proximal tubular cells. The extent of the zonulae occludentes along the tubules in human kidneys is very similar to that observed in the kidney tubules of other mammals. The findings accord well with electrophysiological measurements and with the results of tracer studies on experimental animals.     

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)     

Vitamin-A-induced mucous metaplasia. An in vitro system for modulating tight and gap junction differentiation

Stratified squamous epithelia from 14-day chick embryo shank skin contain rare tight-junctional strands and only small gap junctions. Exposure of this tissue to retinoic acid (vitamin-A) (20 U/ml) in organ culture, however, induces mucous metaplasia, accompanied by tight-junction formation and gap-junction growth; untreated specimens continue to keratinize. To investigate sequential stages of junctional assembly and growth, we examined thin sections and freeze-fracture replicas at daily intervals for 3 days. During the metaplastic process, tight junctions assemble in midepidermal and upper regions, beginning on day 1 and becoming maximal on day 3. Two tight-junctional patterns could be tentatively identified as contributing to the emergence of fully formed zonulae occludentes: (a) the formation of individual ridges along the margins of gap junctions; (b) de novo generation of continuous ramifying strands by fusion of short strand segments and linear particulate aggregates near cellular apices. Gap junction enlargement, already maximal at day 1, occurs primarily three to four cell layers deep. Growth appears to occur by annexation of islands of 20-40 8.5-nm particles into larger lattices of islands separated by particle-free aisles. Eventually, a single gap junction may occupy much of the exposed membrane face in freeze-fractured tissue, but during apical migration of the cells such junctions disappear. The vitamin- A chick-skin system is presented as a responsive model for the controlled study of junction assembly.     

Morphological changes in the junctional complex of cells in the arachnoid layer of the rat after cold injury

Summary The junctional complexes of cells in the outer arachnoid layer overlying the cerebral cortex of 2-week-old rats were examined with freeze-fracture electron microscopy up to 60 min after transcranial cold injury to the dorsal surface of the brain. Within 30 min after injury, areas of gap and tight junctions with morphological features characteristic of junction formation and/or junction disruption were found scattered among normal junctional complexes in some arachnoid cells. Within 60 min after injury, tight junctions with features typical of less leaky zonulae occludentes were present in all arachnoid cells examined. These morphological features include increases in the number of tight junctional strands and the number of strand-to-strand anatomoses. Gap junctions were interspersed among the tight junctional strands, and many were completely encircled by the strands. The increase in the number and complexity of the tight junctional strands in response to brain injury may be the morphological basis for the maintenance of the cerebrospinal fluid-blood dural barrier.This study was supported by the National Institute of Neurological and Communicative Disorders and Stroke Grant NS20590. The opinions or assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the DoD or the USUHS. The experiments reported herein were conducted according to the principles set forth in the Guide for Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, National Research Council, DHEW Pub. No. (NIH) 78-23     

Freeze-fracture study of the junctional complexes of human and rabbit thyroid follicles

Summary Zonulae occludentes, gap junctions and desmosomes have been demonstrated in replicas of freeze-fractured follicular cells of normal human and rabbit thyroid glands. The zonulae occludentes between the human follicular cells are composed of two to eight strands, which completely separate the intercellular space from the follicular lumen. Four to twelve or more strands are visible between the follicular cells of the rabbit thyroid gland.In the meshes of the zonulae occludentes as well as below them, gap junctions are present. They are numerous on the fracture faces of the human follicular cell membranes, but infrequent in those of the rabbit.Aggregates of particles related to desmosomes are found in the deeper meshes of the zonulae occludentes or close to them.     

Cell junctions in the early chick embryo--a freeze etch study

Cell junctions in the early chick embryo have been examined in freeze-etch specimen. Well developed zonulae occludentes are found in the epiblast as early as stage 4. Large gap junctions are also found in the epiblast at this stage. In those cells which have left the surface to form mesenchymal structures (Hensen's node, juxtanodal mesenchyme, primitive streak mesenchyme), one finds not only gap, but also tight, junctions. These junctions do not form continuous belts, but appear as fragments, often reduced to single strands, of typical tight junctions. They probably correspond to the focal tight junctions described earlier in sectioned material. The origin and possible significance of these contacts is discussed, and it is suggested that they represent remnants of junctions between neighboring cells in the epiblast. These junctional remnants slowly disappear by “dilution,” either through cell division and/or cell movement. The appearance of newly formed gap junctions is also described.     

Formation of tight and gap junctions in the inner enamel epithelium and preameloblasts in human fetal tooth germs

Human fetal primary tooth germs in the cap stage were fixed with a glutaraldehyde-formaldehyde mixture, and formative processes of tight and gap junctions of the inner enamel epithelium and preameloblasts were examined by means of freeze-fracture replication. Chains of small clusters of particles on the plasma membrane P-face of the inner enamel epithelium and preameloblasts were the initial sign of tight junction formation. After arranging themselves in discontinuous, linear arrays in association with preexisting or forming gap junctions, these particles later began revealing smooth, continuous tight junctional strands on the plasma membrane P-face and corresponding shallow grooves of a similar pattern on the E-face. Although they exhibited evident meshwork structures of various extents at both the proximal and distal ends of cell bodies, they formed no zonulae occludentes. Small assemblies of particles resembling gap junctions were noted at points of cross linkage of tight junctional strands; but large, mature gap junctions no longer continued into the tight junction meshwork structure. Gap junctions first appeared as very small particle clusters on the plasma membrane P-face of the inner enamel epithelium. Later two types of gap junctions were recognized: one consisted of quite densely aggregated particles with occasional particle-free areas, and the other consisted of relatively loosely aggregated particles with particle-free areas and aisles. Gap junction maturation seemed to consist in an increase of particle numbers. Fusion of gap junctions in the forming stage too was recognized. The results of this investigation suggest that, from an early stage in their development, human fetal ameloblasts possess highly differentiated cell-to-cell interrelations.     

Cell junctions in the epithelium of Bowman's capsule

Summary The intercellular connections between the epithelial cells of Bowman's capsule were investigated. It could be demonstrated that typical zonulae occludentes (tight junctions) are present in the species (rat, hamster, and Tupaia) studied. Freeze-fracturing shows a network of anastomizing strands; some species variations are described. In the rat two strands are common. In the golden hamster mostly two to four and occasionally five strands occur. In Tupaia regularly three tight junction strands are found and also gap junctions associated with the zonulae occludentes. In thin sections the goniometric analysis confirms the freeze-fracturing results and reveals attachment zones of macular shape, which are classified as intermediate junctions and desmosomes. The functional role of these cell junctions observed in the epithelium of Bowman's capsule is discussed.     

The occluding junctions of mouse duodenal enterocytes during development

Summary The architecture of occluding junctions during the differentiation of the mouse duodenum was studied in freeze-fractured material. Irregular zonulae occludentes (ZO) (Type I) are numerous during fetal life, and are characterized by their irregular width, and by the presence of basal open-ended extensions fused with the discontinuous basal strand of the ZO. Regular ZOs (Type II), typical of the adult villous epithelium, appear after Type I junctions by day 16 of gestation. Two patterns are distinguishable: in the first, parallel strands of ridges and furrows are found without crossing branches; in the second pattern, the junction zone is organized like a network of short branches forming various types of polygons. In fetal and adult mice fasciae occludentes (FO) (Type III) are present on the lateral cell membranes; in unfixed specimens particles are found in the furrows of the E-face and pits on the ridges of the P-face. In fixed tissues, the particles are aligned on the ridges of the P-face. These results indicate that fixation with glutaraldehyde modifies considerably the affinity of junctional particles toward the P-face during the fracture process. Moreover, the presence of numerous large FOs on the lateral cell membranes of enterocytes during late fetal life and in the adult, is possibly related to cell movement along the intestinal villi.     

The formation and distribution of intercellular junctions in the rhesus monkey optic cup: The early development of the cilio-iridic and sensory retinas

The eyes of prenatal monkeys from 30 to 102 ± 2 days old were examined by light microscopy, conventional electron microscopy, and the freeze-fracturing technique. At 30 days, invagination of the optic vesicle has begun, and the inner and outer walls of the forming optic cup are closely apposed anteriorly; invagination is complete at 45 days. By 58 days, the rudiment of the ciliary body and iris has appeared; at 71 days, primitive ciliary processes are present and retinal photoreceptors begin to differentiate. The distribution of intercellular junctions varies both in different regions of the optic cup and at different stages of development. At 30 days, adjacent ventricular and adjacent pigmented cells are joined throughout the optic cup by zonulae adhaerentes and gap junctions. The anterior region of the cup, however, contains two additional junctional specializations: (1) fasciae occludentes between ventricular cells and (2) intermediate and gap junctions between the apposing luminal surfaces of ventricular and pigmented cells. By 36 days the fasciae occludentes between ventricular cells in the anterior optic cup become zonular, signaling the morphological development of the blood-aqueous barrier. In the posterior optic cup, zonulae occludentes appear between adjacent pigmented cells at 36 days; furthermore, with the continuing obliteration of the optic ventricle, luminal junctions spread toward the optic stalk but do not reach the optic disc until 45 days, when invagination is complete. Between 58 and 102 days there are no further changes in the distribution of the junctions anteriorly between the primitive cilio-iridial epithelial cells, whereas in the posterior optic cup the luminal gap and intermediate junctions between pigmented cells and differentiating photoreceptors decrease in number and finally disappear. Two main conclusions can be drawn from this study. (1) In the optic cup, intermediate junctions are consistently present in regions of the plasma membrane which later contain junctional complexes. The temporal and spatial pattern of junctional development suggests that intermediate junctions are necessary for the establishment of tight and gap junctions. (2) Twenty days before the ciliary body-iris anlage becomes visible in the light microscope, the distribution of junctions in the anterior part of the optic cup is identical to that in the adult cilio-iridic retina. The time-honored view that the cilio-iridic retina appears late in development is, therefore, no longer tenable. In the monkey, the optic cup is divided into a cilio-iridic and a sensory region soon after the onset of invagination.     

Morphological changes in the liver of the sea lamprey, Petromyzon marinus L., during metamorphosis. II. Canalicular degeneration and transformation of the hepatocytes

Degeneration of all bile canaliculi takes place in the liver of the sea lamprey, Petromyzon marinus, during metamorphosis. Disintegration of microvilli is observed during earlier stages, and membranous debris ultimately accumulates within the canalicular lumina. Complete occlusion of the lumina and disorganization of junctional complexes is followed by a complete loss of the exocrine biliary pole of hepatocytes and a reorganization of these cells into solid cords. An increase in the size and number of acid phosphatase-containing cytoplasmic bodies coincides with the events of canalicular degeneration. These secondary lysosomes apparently participate in some manner in the isolation and disposal of iron and other bile constituents which no longer can be excreted in bile canaliculi. The loss of the exocrine biliary pole of hepatocytes is concomitant with vascular disturbances in the form of disordered arrangements of sinusoidal endothelial cells and an increase in the population of activated Kupffer cells involved in erythrophagocytosis. The significance of the shift in functional organization of the liver in adult lampreys is discussed in relation to physiological changes in this organism and to human hepatic cholestasis, for which this organism is a potentially valuable experimental model.     

Occluding junctions in the epithelia of the gut-associated lymphoid tissue (GALT) of the rabbit ileum and caecum

Summary The zonulae occludentes of the dome epithelia and adjacent non-dome epithelia in four locations of the gut-associated lymphoid tissue (GALT) in the rabbit ileum and caecum (Peyer's patches, sacculus rotundus, caecal lymphoid patches, appendix) were studied in freeze-fracture replicas. In all locations the zonulae occludentes of the dome epithelium are composed of more junctional strands than in the corresponding non-dome epithelium. In the dome epithelia of Peyer's and caecal lymphoid patches the zonulae occludentes show considerable structural variation; the number of superimposed strands is 10 (range 5–18). In the dome epithelia of sacculus rotundus and appendix, in addition to zonulae occludentes, extended networks of junctional strands (fasciae occludentes) are present particularly between M-cells and enterocytes. The zonulae occludentes consist of 8 to 9 (range 5–15) superimposed strands; the fasciae occludentes extend up to a depth of 20m on the lateral membranes. The presence of the fasciae occludentes correlates with the appearance of regularly shaped clusters of lymphocytes, which are most developed in the dome epithelia of sacculus rotundus and appendix. These results suggest (1) that in contrast to the dome epithelia of Peyer's and caecal lymphoid patches those of sacculus rotundus and appendix are compartmentalized, and (2) that the mobility of lymphocytes and diffusion of antigens in the dome epithelia of sacculus rotundus and appendix is restricted.     

Nonparenchymal liver cells and granulomas during lamprey biliary atresia

Transmission (thin sections and freeze-fracture replicas) and scanning electron microscopy were used to describe the nonparenchymal liver cells during the seven (1-7) stages of metamorphosis in the sea lamprey, Petromyzon marinus L., when bile ducts and canaliculi degenerate. The biliary atresia is accompanied by an increased diameter of fenestrae in the endothelium, an active phagocytosis by Kupffer cells in the sinusoids, and large lipid inclusions in perisinusoidal lipocytes (fat-storing or Ito cells). Plasma-like cells and foci of nonparenchymal cells (granulomas) are present in the liver interstitium during at least four stages of metamorphosis. The fenestrae in the sinusoidal wall are wider (up to 2.8-micron diameter) than normally reported for vertebrate livers but are likely a reflection of the morphogenetic and physiological events and consequences of the biliary atresia. Kupffer cells are involved in an extensive erythrophagocytosis, the storage of iron, and perhaps the incorporation of cellular components from hepatocytes. Lipocytes are the vitamin A-storing cells of the transforming liver and may be responsible for some perisinusoidal fibrosis. Granulomas are present during stages 3-6 and are focal areas where mononuclear leukocytes (lymphocytes and plasmalike cells), macrophages, and neutrophils have infiltrated the hepatic parenchyma. The function of the granulomas is not known; but their presence may be related to the porous nature of the sinusoidal wall, the tissue degeneration, and/or the physiological change (e.g., bile stasis) during biliary atresia.     

Cell junctions in the renal tubule of a fresh-water teleost,Salmo gairdneri Rich.

Summary Cell junctions in the renal tubule of the fresh-water rainbow trout were studied with thin-section and freeze-fracture techniques. Gap junctions were restricted to the proximal tubule, which is consistent with other vertebrate classes. Segments I and II of the proximal tubule and the collecting tubule/collecting duct system exhibited a well-developed zonula occludens with anastomosing strands. The distal segment showed a narrow zonula occludens composed of few parallel strands. The structure of the occluding junctions along the renal tubule of this teleost displays several similarities with the pattern of the zonulae occludentes in the amphibian and the mammalian nephron. From these observations, in conjunction with available data from other vertebrate classes, it can be concluded that in the proximal tubule the development of a deep and complex zonula occludens is a general feature of cold-blooded vertebrates.