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     

Ultrastructural observations on the pars descendens of the proximal tubule in the kidney of the male rat

Summary The pars descendens (pars recta) of the proximal tubule in the male rat kidney, consisting of the terminal part of the second proximal segment (P₂) and of the third proximal segment (P₃), was studied with the electron microscope. A technique of tissue orientation and trimming was used which permitted precise topographic definition of the tubules studied in the electron microscope. The terminal descending part of the P₂ showed some minor differences from the convoluted part of this segment, and ultrastructure also changed along the course of the P₃. In the beginning of the latter segment numerous, shallow interdigitations were observed between adjacent cells; along the course of the segment they decreased in number or disappeared. In the initial part of the P₃ mitochondria were more abundant than in the terminal portion of the segment and at least as numerous as in the straight part of the P₂. Also, the dense, acid phosphatase-positive cytoplasmic bodies decreased somewhat in size along the course of the P₃. The smooth surfaced endoplasmic reticulum reached a higher development in the P₃ than anywhere else in the proximal tubules.Investigation supported by grants from: Fonden til Lægevidenskabens Fremme and the Danish Medical Research Council. — The authors are indebted to Mrs. J. Barslund and Mrs. M. Jacobsen for excellent technical assistance.     

Heterogeneity of tight junctions along the collecting duct in the renal medulla

Summary The tight junctions along the medullary collecting duct in the kidneys of the rat and the rabbit were studied with freeze-fracture electron microscopy and quantitated according to the number of strands and the apico-basal depth (nm) of the junctions.The most elaborate tight junctions were found in the inner stripe of the outer medulla; rat: 10.6±0.8 strands and 205±24nm; rabbit: 11.6±2.4 strands and 291±55 nm.The elaboration of the tight junctions decreased continuously towards the papillary tip. Inner zone I; rat: 9.3±2.6 strands and 186±38nm, rabbit: 9.5±2.3 strands and 247±59nm. Inner zone II; rat: 7.1±2.2 strands and 129±32nm, rabbit: 8.5±1.4 strands and 199±26nm. Inner zone III; rat: 6.0±1.6 strands and 111 + 19 nm, rabbit: 7.0±1.5 strands and 183±43 nm. In the inner zone III comprising the papillary tip tight junctions with only 1–3 strands were not infrequently seen. Preliminary findings in the kidney of the golden hamster indicate a similar decline of junctional tightness along the collecting duct.These morphological observations suggest that the permeability of the paracellular pathway of the medullary collecting duct increases towards the tip of the papilla, especially in the rat. The functional implications for the medullary recycling of urea and electrolytes, and for the urinary concentrating mechanism are discussed.In addition, the tight junctions of the papillary epithelium are described.     

Characteristics of D-alanine transport by luminal membrane vesicles from pars convoluta and pars recta of rabbit proximal tubule

Uptake of D-alanine against a concentration gradient has been shown to occur with isolated luminal-membrane vesicles from pars convoluta or pars recta of rabbit proximal tubule. Renal D-alanine transport systems, displaying the following characteristics, were shown: (1) In vesicles from pars convoluta, the uptake of D-alanine was mediated by both Na+-dependent and Na+-independent transport processes. It was found that an inwardly directed H+-gradient could drive the transport of D-alanine into the vesicles both in the presence and absence of Na+. Thus, in addition to Na+, the transport of D-alanine is influenced by the H+-gradient. (2) In vesicles from pars recta, the transient accumulation of D-alanine was strictly dependent on Na+, since no 'overshoot' was ever observed in the absence of Na+. Although the Na+-dependent uptake of D-alanine was stimulated at acid pH, H+ did not substitute for Na+, as it apparently does in pars convoluta, but instead potentiated the Na+ effect. (3) Addition of L-alanine to vesicle preparations, both from pars convoluta and from pars recta, specifically inhibited renal uptake of D-alanine. A comparison between the transport characteristics of D- and L-alanine indicated that these two isomers of alanine probably share common transport systems located along the proximal tubule of rabbit kidney.     

GTP-binding proteins in luminal and basolateral membranes from pars convoluta and pars recta of rabbit kidney proximal tubule.

The GTP-binding proteins on luminal and basolateral membrane vesicles from outer cortex (pars convoluta) and outer medulla (pars recta) of rabbit proximal tubule have been examined. The membrane vesicles were highly purified, as ascertained by electron microscopy, by measurements of marker enzymes, and by investigating segmental-specific transport systems. The [35S]GTP gamma S binding to vesicles, and to sodium cholate-extracted proteins from vesicles, indicated that the total content of GTP-binding proteins were equally distributed on pars convoluta, pars recta luminal and basolateral membranes. The membranes were ADP-ribosylated with [32P]NAD+ in the presence of pertussis toxin and cholera toxin. Gel electrophoresis revealed, for all preparations, the presence of cholera toxin [32P]ADP-ribosylated 42 and 45 kDa G alpha s proteins, and pertussis toxin [32P]ADP-ribosylated 41 kDa G alpha i1, 40 kDa G alpha i2 and 41 kDa G alpha i3 proteins. The 2D electrophoresis indicated that Go's were not present in luminal nor in basolateral membranes of pars convoluta or pars recta of rabbit proximal tubule.     

Structural heterogeneity of the basement membrane in the rat proximal tubule

Summary The ultrastructure of the basement membrane of the rat proximal tubule was observed by transmission electron microscopy after the use of a cold dehydration technique. The basement membrane of the P1 segment is thick and possesses several structural specializations that are rare in other basement membranes; these include intraepithelial ridges, dense bars, and basement membrane vesicles. The intraepithelial ridges are found in the intercellular spaces between interdigitating processes of the proximal tubule cells. The ridges and the interdigitating processes run circumferentially around the tubule. The dense bars are frequently found in the intraepithelial ridges. They are especially prominent on the concave side of the tubular bends and to a lesser extent near sites where intracellular actin filaments anchor onto the basal cell membranes. The basement membrane vesicles are bounded by unit membranes; they are variable in both their electron density and their size. They are usually found in association with dense bars, and the grade of their accumulation is positively correlated with the development of the dense bars. These three specializations have no topographical relationship with the interstitial structures, such as fibrobalasts and collagen fibrils. The specializations are best developed on the concave side of tubular bends where the circumferential stresses caused by the intraluminal hydraulic pressure are presumably the largest; we therefore propose that they are an adaptation to, or a manifestation of, the increased wall stress in the proximal tubule.     

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)     

Invaginated apical vacuoles in the cells of the proximal convoluted tubule in the rat kidney

Summary Following perfusion fixation of the rat kidney with glutaraldehyde the proximal tubule cells display small apical vacuoles, large apical vacuoles, and apical vacuoles in which a part of the limiting membrane is invaginated into the vacuole. These invaginated apical vacuoles occur more frequently in proximal convoluted tubules than in proximal straight tubules. One tubular cell may contain apical vacuoles of different sizes and stages of invagination, ranging from larger vacuoles with a wide lumen and a small area of invaginated membrane to smaller elements with no apparent lumen and a large area of invaginated membrane. Invaginated apical vacuoles lie either singly in the cytoplasm or close to the membranes of other apical vacuoles, but never in contact with the cell membrane or the membranes of lysosomes, endoplasmic reticulum, Golgi apparatus, mitochondria and peroxisomes.These findings suggest that the invaginated apical vacuoles are not fixation artifacts, but rather develop in living state in cells of the proximal tubule from spherical endocytotic elements.Supported by the Deutsche Forschungsgemeinschaft (SFB 105)     

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.     

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     

The transepithelial shunt pathway in the renal proximal tubule of newt kidney

The transepithelial shunt pathway of newt proximal tubule was examined with glass micro-electrode and electron microscopic methods. The input resistance of the peritubular (basal) membrane and tubular wall were found to be $\text{4.2 ± 0.1 · 10}{}^6$ ($\text{mean ± S.E.}\text{, n = 16}$) and $\text{11.4 ± 0.2 · 10}{}^4\text{(n = 11)}$, respectively. The input resistance of the peritubular membrane was approximately 40-times larger than that of the tubular wall. When the kidneys were perfused in a lanthanum solution, the lanthanum ions were then observed in the junctional complexes and in the intercellular spaces on both the basal and apical sides. The results indicate that the electrical shunt pathway corresponds to the apical junctional complexes and the intercellular spaces, and that the tight junctions are not truly ‘tight’ for the transepithelial movement of small ions in the proximal tubule of the newt kidney.     

Studies of the regulation of renal gluconeogenesis in normal and Pi depleted proximal tubule cells

Pi depletion of proximal tubule cells isolated from mouse kidney results in a decrease in the cell content of fructose-2,6-bisphosphate and an increase in the rate of gluconeogenesis from pyruvate, malate and succinate. Gluconeogenesis from glycerol is unaffected by Pi depletion. Introduction of fructose-2,6-bisphosphate into the cytosol of ATP-permeabilized cells is accompanied by a fall in gluconeogenesis. The presence of external Ca2+ stimulates gluconeogenesis. When cytosolic Ca2+ is raised to 1.8 microM by permeabilization, the resealed cells still require 2.5 mM Ca2+ in the bathing medium in order to perform gluconeogenesis at the maximum rate. Cells permeabilized in the presence of cAMP show a decreased rate of glucose production. Phorbol ester stimulates gluconeogenesis provided that the phorbol treatment is performed in the absence of Ca2+ ions. It is suggested that Pi depletion may stimulate pyruvate carboxylase activity and facilitate the entry of certain gluconeogenic substrates into mitochondria. It is also proposed that important aspects of the control of renal gluconeogenesis by parathyroid hormone are mediated by protein kinase C.     

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     

Freeze-fracture and tracer experiments on the permeability of the zonulae occludentes in the olfactory mucosa of vertebrates

Summary The junctional belt around the sensory cells in the nasal olfactory mucosa of the frog and in the vomeronasal organ of the mouse appears as a network of interconnected ridges in freeze-fracture replicas. Numerous open-ended ridges were observed and, consequently, open routes from the region below the junctional belt to that above it. Lanthanum nitrate permeates the junctional belt when administered from the surface of the epithelium as well as from the vascular system. When applied at a concentration of 1–3%, the tracer is deposited within the junctional belt forming facets which are visible in tangential sections. These facets correspond to the areas defined by the network or ridges seen in freeze-fracture replicas. Various aspects of these observations are discussed, such as the replacement of cells in the sensory epithelium, the stimulation of extrinsic fibers and the generation of a transepithelial potential.     

A model for de novo synthesis and assembly of tight intercellular junctions. Ultrastructural correlates and experimental verification of the model revealed by freeze-fracture

The structure and function of intercellular tight (occluding) junctions, which constitute the anatomical basis for highly regulated interfaces between tissue compartments such as the blood-testis and blood-brain barriers, are well known. Details of the synthesis and assembly of tight junctions, however, have been difficult to determine primarily because no model for study of these processes has been recognized. Primary cultures of brain capillary endothelial cells are proposed as a model in which events of the synthesis and assembly of tight junctions can be examined by monitoring morphological features of each step in freeze-fracture replicas of the endothelial cell plasma membrane. Examination of replicas of non-confluent monolayers of endothelial cells reveals the following intramembrane structures proposed as 'markers' for the sequential events of synthesis and assembly of zonulae occludentes: development of surface contours consisting of elongate terraces and furrows (valleys) orientated parallel to the axis of cytoplasmic extensions of spreading endothelial cells, appearance of small circular PF face depressions (or volcano-like protrusions on the EF face) that represent cytoplasmic vesicle-plasma membrane fusion sites, which are positioned in linear arrays along the contour furrows, appearance of 13-15 nm intramembrane particles at the perimeter of the vesicle fusion sites, and alignment of these intramembrane particles into the long, parallel, anastomosed strands characteristic of mature tight junctions. These structural features of brain endothelial cells in monolayer culture constitute the morphological expression of: reshaping the cell surface to align future junction-containing regions with those of adjacent cells, delivery and insertion of newly synthesized junctional intramembrane particles into regions of the plasma membrane where tight junctions will form, and aggregation and alignment of tight junction intramembrane particles into the complex interconnected strands of mature zonulae occludentes. The distribution of filipin-sterol complex-free regions on the PF intramembrane fracture face of junction-forming endothelial plasmalemmae corresponds precisely to the furrows, aligned vesicle fusion sites and anastomosed strands of tight junctional elements.(ABSTRACT TRUNCATED AT 400 WORDS)     

Phagocytosis of erythrocytes by the proximal tubule of the rat kidney

Summary Morphological examination of kidney biopsies from patients with glomerulonephritis and hematuria has revealed the presence of erythrocytes within epithelial cells of the proximal tubule. This observation suggested that the proximal tubule might be capable of phagocytizing morphologically intact erythrocytes. To examine this possibility small quantities of heparinized autologous blood were injected into surface convolutions of proximal tubules of the rat kidney using standard micropuncture techniques. At time intervals ranging from 10 min to 120 h after injection, the kidneys were preserved for light and transmission electron microscopy by drip-fixation with a half-strength Karnovsky's glutaraldehyde-formaldehyde fixative.During the initial 6 h there was a flattening of the brush border and accumulation of electron-dense material representing hemoglobin in apical vacuoles and in lysosome-like structures. From 6 to 15 h after micropuncture, there was progressive loss of the brush border and the simultaneous formation of pseudopodia-like evaginations that extended from the apical plasma membrane and surrounded the individual erythrocytes. By 18 and 24 h, erythrocytes were observed in the proximal tubule cells. At later time intervals, edema, lymphocytic infiltration, and fibrosis were observed in the interstitium. In addition, crystalline structures were present in the lumen and the cells of both proximal and distal tubules. These findings suggest that in addition to their well-established ability to pinocytize hemoglobin and other proteins, the cells of the proximal tubule are capable of phagocytizing morphologically intact autologous erythrocytes. It is possible that phagocytosis by the proximal tubule cells may play a role in the disposal of erythrocytes from the tubular fluid in hematuric conditions.     

Are there specialized junctions in the pars maculata of the distal tubule?

Summary In the present study the tight junctions at the macula densa were compared to those of the adjacent straight and convoluted segments of the distal tubule using freeze fracturing and thin sectioning techniques. Only insignificant differences were found in the number of strands and the apico-basal depth of the tight junctions in the three distal tubular segments of rat, dog and tree shrew. In experiments with horseradish peroxidase on mice and tree shrews, the tracer did not penetrate the apical junctions in any of the distal tubular segments. Our findings do not support the concept of considerably higher permeability of the tight junctions at the macula densa, as previously reported. Gap junctions were never observed in the distal nephron. The present results suggest that the glomerulo-tubular feedback is more likely to be mediated by transcellular resorption of solutes than by passive diffusion through a leaky paracellular shunt pathway.These studies were supported by the German Research Foundation within the SFB 90 Cardiovasculäres System     

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.     

Structure of occluding junctions in ileal epithelial cells of suckling rats

Summary Two kinds of occluding junctions are found between ileal epithelial cells of suckling rats: apical zonulae occludentes (ZO) and fasciae occludentes (FO) which are associated with the lateral plasma membranes of many epithelial cells. In unfixed preparations, glycerol treatment induces the further proliferation of extensive fasciae occludentes. Both kinds of junction have identical structural elements when visualized in freeze fracture replicas, although the arrangement of these elements differs. Zonulae occludentes consist of networks of branching and anastomosing linear ridges or rows of 10 nm particles with 20–30 nm spaces between the rows which form narrow belt-like structures around the apical region of adjacent cells. Fasciae occludentes, on the other hand, consist of similar linear ridges or rows of particles but the junction strands are often discontinuous, open ended and only occasionally intersect with each other. Several different fracture planes through the plasma membrane in the region of the occluding junctions have been observed and these provide further evidence that two components, one from each membrane, fused at the level of the extracellular space, form the junction sealing element. Furthermore, we present evidence which indicates a staggered rather than an in-register arrangement of these two components.This study was supported in part by National Institutes of Health Program Project No. NS10299 and National Institutes of Health Sciences Advancement Award No. RR06148 (J.D.R.) and by the Cancer Research Campaign (S.K.) and Medical Research Council (A.R.L.)