Membrane traffic after inhibition of endocytosis in renal proximal tubules

This study was performed to examine quantitatively the cellular organelles involved in membrane recycling after inhibition of luminal endocytosis in renal proximal tubules. Paraffin oil was microinfused into rat renal proximal convoluted tubules to prevent luminal endocytosis. After 1-2 hr the kidneys were fixed by perfusion and prepared for electron microscopy. Segment 1 proximal tubules infused with paraffin oil and control tubules from the same kidney were studied. In addition we examined proximal tubules from kidneys fixed by immersion 30 sec after removal of the kidney. In the oil-infused tubules the large endocytic vacuoles (greater than 0.5 micron) disappeared, the amount of small endocytic vacuoles (less than 0.5 micron) was reduced to about 10%, and the amount of dense apical tubules was significantly increased. The dense apical tubules were very seldom seen connected to the apical plasma membrane in controls but this was occasionally observed in tubules fixed by immersion and relatively often in oil-infused tubules. An ultrastructural morphometric analysis substantiated and extended the qualitative observations and provided quantitative estimates of volumes and surface areas for large endocytic vacuoles, lysosomes, mitochondria, small endocytic vacuoles, and dense apical tubules in control and experimental tubules. The results strongly support the suggestion that the dense apical tubules located in the apical cytoplasm represent the vehicle for the recycling of membrane from endocytic vacuoles back to the plasma membrane, and show that in renal proximal tubule cells small and large endocytic vacuoles are transformed into dense apical tubules when endocytosis is stopped.     

Ultrastructure of the tubular nephron of the lizard Podarcis (= Lacerta) Taurica

The proximal, intermediate, and distal convoluted tubules of the neprhon of Podarcis (= Lacerta) taurica were examined by electron microscopy. Proximal tubule cells have large, apical cytoplasmic protrusions and microvilli interpreted to function in urate secretion. Adjacent cells are bound apically by tight junctions and desmosomes but interdigitate in their basal region. This situation is repeated in the other tubules with significant differences in intercellular space width. The basal surfaces bear numerous cytoplasmic processes. The intermediate tubule has proximal and distal segments each with dark, ciliated, and light cells, the cuboidal dark cells with dense cytoplasm constituting the main bulk of the wall. As the cells of the proximal and distal segments resemble those of the proximal and distal convoluted tubules, respectively, the intermediate tubule is considered as a transition region. The ciliated cell body has two broad processes extending from the lumen, one to the basement membrane and one to a foot process of a light cell. The light cell is surrounded by dark and ciliated cells. It does not reach the lumen, but contacts the basement membrane through a process running below a ciliated cell to form a mushroom-shaped structure in tubule cross-section, the light cell process forming the stalk and a ciliated cell the cap. The cilia probably propel the glomerular filtrate towards the distal convoluted tubule. This latter tubule has initial, middle, and terminal zones, all nonciliated but with different lumen widths and cell shapes.     

Electron microscopic immunohistochemical localization of components of Na+-cotransporters along the rat nephron

The localization of Na+-cotransport proteins in cortex and outer medulla of rat kidney was investigated with five monoclonal antibodies. Recently, it was found that these antibodies altered Na+-D-glucose cotransport and/or Na+-dependent high affinity phlorizin binding in pig kidney cortex and that three of these antibodies interacted also with Na+-cotransporters for lactate, L-alanine and/or L-glutamate (Koepsell, H., K. Korn, A. Raszeja-Specht, S. Bernotat-Danielowski, D. Ollig, J. Biol. Chem. 263, 18,419-18,429 (1988]. In pig and rat the monoclonal antibodies bind to two brush-border membrane polypeptides with identical molecular weights and isoelectric points of 75,000 and pI 5.5, and 47,000 and pI 5.4. These polypeptides have been previously identified as components of the porcine renal Na+-D-glucose cotransporter (Neeb, M., U. Kunz, H. Koepsell, J. Biol. Chem. 262, 10,718-10,727 (1987] and may also be part of other Na+-cotransporters. The electron microscopic localization of antibody binding was demonstrated by protein A-gold labeling on ultrathin plastic sections. Three antibodies bound to brush-border membranes of proximal convoluted and straight tubules. In the proximal convoluted tubules all antibodies reacted with apical endocytic vacuoles, apical dense tubules and lysosomes. Since dense tubules are supposed to originate from endocytic vacuoles and to fuse with brush-border membranes the data suggest recycling of Na+-cotransporters in the proximal convoluted tubule. In the outer medulla two antibodies bound to apical membranes of descending thin limbs (DTL) of short loops of Henle and to apical and basal membranes of DTL of long loops of Henle. Three antibodies bound to apical membranes of collecting ducts. These data indicate that Na+-cotransporters or homologous proteins exist beyond the proximal tubule.     

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.     

Ultrastructure of the Protonephridium in Acteonemertes bathamae Pantin (Rhynchocoela: Enopla: Hoplonemertini)

The protonephridial system consists of terminal cell, protonephridial capillary, protonephridial tubule and efferent duct. The terminal cell is an elongated, thin-walled, fenestrated basket containing a ciliary flame circumscribed by a palisade of straight microvilli. The filtration area is confined to the terminal cell and consists of slits bridged by a filtration membrane. The cilia, as well as the microvilli, projects into the proximal bell-shaped part of the thin-walled protonephridial capillary. The terminal cells are often found in pairs connected to the same capillary, which has a very narrow lumen. The proximal part of the thick-walled, convoluted protonephridial tubule is ciliated and shows characteristic foldings of the luminal plasma membrane and numerous small vesicles in the cytoplasm. The cells of the following, non-ciliated part of the tubule have interdigitating lateral surfaces and the bases deeply invaginated to form compartments with numerous mitochondria; in the cytoplasm are many large vesicles, possibly containing lipid droplets, and small amounts of glycogen. The distal protonephridial tubule resembles various epithelia with an osmoregulatory function, including the vertebrate nephron.     

Study on the origin of apical tubules in ileal absorptive cells of suckling rats using concanavalin-A as a membrane-bound tracer

Summary The ileal absorptive cells of suckling rats exhibit high levels of endocytic activity being engaged in nonselective uptake of macromolecules from the intestinal lumen. The apical cytoplasm usually contains an extensive network of small, membrane-limited tubules (apical tubules: AT), in addition to newly formed endocytic vesicles and large endocytic vacuoles. To determine whether the AT are directly involved in the endocytic process by carrying the tracer into the cell, we have analysed movements of the apical cell membrane of the ileal absorptive cells by using a membrane-bound tracer (horseradish peroxidase-labelled cancanavalin-A: Con-A HRP). The ileal absorptive cells were exposed in vitro to Con-A HRP for 10 min at 4° C, incubated for different times in Con-A free medium at 37° C, and prepared for electron microscopy. After 1 min incubation at 37° C, invaginations of the apical cell membrane, including coated pits, and endocytic vesicles were labelled with HRP-reaction product, whereas the AT and large endocytic vacuoles were negative. After 2.5 min, almost all the large endocytic vacuoles were labelled with reaction product, which was seen in their vacuolar lumen and along the luminal surface of their limiting membrane. A few AT with reaction product were seen in the apical cytoplasm; they were in frequent connection with the reaction-positive large endocytic vacuoles. With increasing incubation time, the number of the labelled AT increased. Thus, after 15 min at 37° C, the apical cytoplasm was fully occupied by the reaction-positive AT. The ends of these AT were often continuous with small spherical coated vesicles. No reaction product was detected in the Golgi complex at any time after incubation. These observations indicate that the AT located in the apical cytoplasm probably originate by budding off from the large endocytic vacuoles, rather than being involved in the process of endocytosis.     

ULTRASTRUCTURAL STUDIES OF VASOPRESSIN EFFECT ON ISOLATED PERFUSED RENAL COLLECTING TUBULES OF THE RABBIT

Isolated cortical collecting tubules from rabbit kidney were studied during perfusion with solutions made either isotonic or hypotonic to the external bathing medium. Examination of living tubules revealed a reversible increase in thickness of the cellular layer, prominence of lateral cell membranes, and formation of intracellular vacuoles during periods of vasopressin-induced osmotic water transport. Examination in the electron microscope revealed that vasopressin induced no changes in cell structure in collecting tubules in the absence of an osmotic difference and significant bulk water flow across the tubule wall. In contrast, tubules fixed during vasopressin-induced periods of high osmotic water transport showed prominent dilatation of lateral intercellular spaces, bulging of apical cell membranes into the tubular lumen, and formation of intracellular vacuoles. It is concluded that the ultrastructural changes are secondary to transepithelial bulk water flow and not to a direct effect of vasopressin on the cells, and that vasopressin induces osmotic flow by increasing water permeability of the luminal cell membrane. The lateral intercellular spaces may be part of the pathway for osmotically induced transepithelial bulk water flow.     

Evidence for a decreased membrane recycling in the cells of renal proximal tubules exposed to high concentrations of ferritin

Summary The present study was performed to investigate whether membrane recycling via the dense apical tubules in cells of renal proximal tubules could be modified after exposure to large amounts of cationized ferritin. Proximal tubules in the rat kidney were microinfused in vivo with cationized ferritin for 10 or 30 min and then fixed with glutaraldehyde by microinfusion, or proximal tubules were microinfused with ferritin for 30 min and then fixed 2 h thereafter. The tubules were processed for electron microscopy, and the surface density and the volume density of the different cell organelles involved in endocytosis were determined by morphometry. The morphometric analyses showed that after loading of the endocytic vesicles with ferritin the surface density of dense apical tubules decreased to about 50% of the original value. However, 2 h later when ferritin had accumulated in the lysosomes the surface density of dense apical tubules had returned to control values. Furthermore, cationized ferritin was virtually absent from the Golgi region, indicating that the Golgi apparatus in these cells does not participate in membrane recycling. In conclusion, the present study shows that membrane recycling in renal proximal tubule cells can in part be inhibited by loading the endocytic vacuoles with ferritin.     

Ion exchangers mediating NaCl transport in the renal proximal tubule

We have studied the mechanisms of NaCl transport in the mammalian proximal tubule. Studies of isolated brush-border membrane vesicles confirmed the presence of Na+-H+ exchange and identified Cl(-)-formate and Cl(-)-oxalate exchangers as possible mechanisms of uphill Cl- entry. We found that formate and oxalate each stimulate NaCl absorption in microperfused proximal tubules. Stimulation of NaCl absorption by formate was blocked by the Na+-H+-exchange inhibitor EIPA, whereas stimulation by oxalate was blocked by omission of sulfate from the perfusion solutions. These observations were consistent with recycling of formate from lumen to cell by H+-coupled formate transport in parallel with Na+-H+ exchange and recycling of oxalate by oxalate-sulfate exchange in parallel with Na+-sulfate cotransport. Using isoform-specific antibodies, we found that NHE1 is present on the basolateral membrane of all nephron segments, whereas NHE3 is present on the apical membrane of cells in the proximal tubule and the loop of Henle. The inhibitor sensitivity of Na+-H+ exchange in renal brush-border vesicles and of HCO3- absorption in microperfused tubules suggested that NHE3 is responsible for most, if not all, apical membrane Na+-H+ exchange in the proximal tubule. The role of NHE3 in mediating proximal tubule HCO3- absorption and formate-dependent Cl- absorption was confirmed by studies in NHE3 null mice. Finally, we cloned and functionally expressed CFEX, an anion transporter expressed on the apical surface of proximal tubule cells and capable of mediating Cl(-)-formate exchange.     

Distribution of calcitonin gene-related peptide-containing fibers in the urinary bladder of the rat and their origin

Summary Using horseradish peroxidase (HRP) as a tracer, we have investigated if the so-called apical tubules (AT) in the kidney proximal tubule cells are directly involved in the endocytic process by carrying the tracer into the cells, or if they are derived from the intracellular membrane compartments. Rat kidney was fixed by vascular perfusion at different time intervals after intravenous injection of HRP and prepared for electron microscopy. An analysis revealed that 0.5 min after injection, invaginations of the plasma membrane and small apical endocytic vesicles, including coated vesicles, were labelled with reaction product, whereas almost all large apical endocytic vacuoles and the AT were negative. The endocytic vacuoles and about 18% of the AT were labelled 1 min after injection. The reaction product in the large endocytic vacuoles was usually seen along the luminal surface of the vacuoles. The AT with reaction product appeared as a branched network, and were frequently connected with the labelled endocytic vacuoles. Three min after injection, reaction product was detected in about 38% of the AT, and thereafter, the percentage increased to about 74% after 7 min. No reaction product was detected in the Golgi complex at any time after HRP-injection. These findings indicate that the AT are probably formed by budding off from the large endocytic vacuoles, rather than being directly involved in the endocytic process.     

SERUM ALBUMIN UPTAKE IN ISOLATED PERFUSED RENAL TUBULES : Quantitative and Electron Microscope Radioautographic Studies in Three Anatomical Segments of the Rabbit Nephron

Proximal convoluted, proximal straight, and cortical collecting tubular segments isolated from rabbit kidney were perfused with I 125-labeled rabbit serum albumin (RSA-I 125) in ultrafiltrate of serum for up to 3 hr After perfusion, the segments were fixed with glutaraldehyde, embedded in Epon, and either counted with a gamma spectrometer to quantitate protein accumulation or analyzed by electron microscope radioautography to sequentially localize radioactivity Proximal convoluted and proximal straight segments accumulate RSA-I 125 nearly linearly as a function of time whereas cortical collecting segments do not accumulate measurable amounts of protein. The rate of accumulation of RSA-I 125 in the proximal convoluted tubule is 2 6 times as great as that in the proximal straight tubule. Electron microscope radioautography of the isolated proximal tubule demonstrated that RSA-I 125 is taken up via small apical vesicles and tubular invaginations, released into large cytoplasmic vacuoles, and finally concentrated in membrane-bounded structures, some of which are acid phosphatase positive These results show that albumin is absorbed by proximal tubules and may be degraded intracellularly within lysosomes. In addition, less radioactivity was located at all times over the lateral intercellular and basilar labyrinthine spaces, suggesting that labeled albumin and/or its breakdown products may be transported across the peritubular cell membrane.     

Helical structure in the apical tubules of several absorbing epithelia

Summary Unique and highly ordered structures were discovered in the so-called apical tubules of several absorbing epithelia (kidney proximal tubule, visceral yolk sac and ductuli efferentes) fixed in situ with a mixture of formaldehyde, glutaraldehyde and osmium tetroxide. The apical tubules were especially numerous in the apical cytoplasm, in addition to the invaginations of the apical plasma membrane, newly formed endocytic vesicles and large endocytic vacuoles. They showed a cylindrical structure (80 nm in diameter) limited by a smooth membrane. Helically wound parallel rows of particles (11 nm in diameter) were found in the apical tubules in close proximity to their limiting membrane. The structure of the helix was determined by following the rows through serial sections and semithin sections, and was found to be a left-handed quadruple helix. These particles surround an electron-lucent cylinder (35 nm in diameter), containing at its center a single row of particles (9 nm in diameter). The apical tubules with the luminal specializations were not seen in continuity with the apical plasma membrane, but were frequently connected with the large endocytic vacuoles, which were present in the deeper levels of the apical cytoplasm. From these observations, it is suggested that the apical tubules are not derivatives of the apical plasma membrane; rather, they represent an intracellular compartment, which is morphologically related to the large endocytic vacuoles.     

Intra-endosomal pH-sensitive recruitment of the Arf-nucleotide exchange factor ARNO and Arf6 from cytoplasm to proximal tubule endosomes

Kidney proximal tubule epithelial cells have an extensive apical endocytotic apparatus that is critical for the reabsorption and degradation of proteins that traverse the glomerular filtration barrier and that is also involved in the extensive recycling of functionally important apical plasma membrane transporters. We show here that an Arf-nucleotide exchange factor, ARNO (ADP-ribosylation factor nucleotide site opener) as well as Arf6 and Arf1 small GTPases are located in the kidney proximal tubule receptor-mediated endocytosis pathway, and that ARNO and Arf6 recruitment from cytosol to endosomes is pH-dependent. In proximal tubules in situ, ARNO and Arf6 partially co-localized with the V-ATPase in apical endosomes in proximal tubules. Arf1 was localized both at the apical pole of proximal tubule epithelial cells, but also in the Golgi. By Western blot analysis ARNO, Arf6, and Arf1 were detected both in purified endosomes and in proximal tubule cytosol. A translocation assay showed that ATP-driven endosomal acidification triggered the recruitment of ARNO and Arf6 from proximal tubule cytosol to endosomal membranes. The translocation of both ARNO and Arf6 was reversed by V-type ATPase inhibitors and by uncouplers of endosomal intralumenal pH, and was correlated with the magnitude of intra-endosomal acidification. Our data suggest that V-type ATPase-dependent acidification stimulates the selective recruitment of ARNO and Arf6 to proximal tubule early endosomes. This mechanism may play an important role in the pH-dependent regulation of receptor-mediated endocytosis in proximal tubules in situ.     

Evidence for intrarenal kallikrein storage during chromate-induced acute renal failure in rat

Summary During the course of chromate-induced acute renal failure (ARF), urinary kallikrein excretion (UKE), a serine protease of distal tubule origin in the normal animal was decreased but tissue kallikrein concentration (TK) was increased, suggesting intracellular accumulation. Severe morphological lesions were observed in proximal tubular cells which showed brush border damage, numerous vesicles, necrosis and liquefaction of cytoplasmic material. Less marked changes were also present in distal tubules: large apical vacuoles and swollen mitochondria. Compared to normal rats, using the peroxidase-anti-peroxidase (PAP) method for light microscopy, greater kallikrein immunoreactivity was detected along the apical pole in distal tubules, on the membrane and in the cytoplasm as well as in the glomerulus. By immunoelectron microscopy, kallikrein was found in the connecting apical area, along the luminal, basolateral and basement membranes, in some vesicles, in Golgi apparatus and on ribosomes bound to endoplasmic reticulum. In the glomerulus, kallikrein was observed along the luminal surface of endothelial cell. After 14 days a progressive recovery of renal function, tissue morphology and UKE towards control values was observed. The presence of immunoreactive kallikrein in the glomerulus observed only during ARF confirmed the previous demonstration of kallikrein mRNA in the glomerulus. The cellular accumulation results more likely from a dysfunction of a general secretory mechanism due to cell membrane alteration than from a specific inhibition of kallikrein production and secretion.     

Evidence for intrarenal kallikrein storage during chromate-induced acute renal failure in rat.

During the course of chromate-induced acute renal failure (ARF), urinary kallikrein excretion (UKE), a serine protease of distal tubule origin in the normal animal was decreased but tissue kallikrein concentration (TK) was increased, suggesting intracellular accumulation. Severe morphological lesions were observed in proximal tubular cells which showed brush border damage, numerous vesicles, necrosis and liquefaction of cytoplasmic material. Less marked changes were also present in distal tubules: large apical vacuoles and swollen mitochondria. Compared to normal rats, using the peroxidase-anti-peroxidase (PAP) method for light microscopy, greater kallikrein immunoreactivity was detected along the apical pole in distal tubules, on the membrane and in the cytoplasm as well as in the glomerulus. By immunoelectron microscopy, kallikrein was found in the connecting apical area, along the luminal, basolateral and basement membranes, in some vesicles, in Golgi apparatus and on ribosomes bound to endoplasmic reticulum. In the glomerulus, kallikrein was observed along the luminal surface of endothelial cell. After 14 days a progressive recovery of renal function, tissue morphology and UKE towards control values was observed. The presence of immunoreactive kallikrein in the glomerulus observed only during ARF confirmed the previous demonstration of kallikrein mRNA in the glomerulus. The cellular accumulation results more likely from a dysfunction of a general secretory mechanism due to cell membrane alteration than from a specific inhibition of kallikrein production and secretion.     

Transtubular transport of proteins in rabbit proximal tubules

The purpose of the present experiments was to study possible different pathways of intracellular transport of proteins after luminal and basolateral uptake in isolated rabbit proximal tubules. Tubules were exposed to cationized ferritin (CF) in the perfusion fluid and horseradish peroxidase (HRP) in the bath simultaneously or to HRP in the bath alone for 30 min. The peritubular fluid (bath) and perfusion fluid were then exchanged and the tubules either fixed immediately or allowed to function during chase-periods for 10, 20, 30, or 60 min before fixation to follow the migration of the proteins through the cells. The proteins were to a large extent found separated in different vacuoles and lysosomes at all time periods studied, indicating separate pathways after uptake via the luminal and basolateral membranes respectively. About 0.5% of the CF taken up by the cells was transported through the cells and became located in the intercellular spaces. HRP was transported from the peritubular fluid to the apical cytoplasm of the tubules indicated by a gradual accumulation of small HRP-containing vesicles, first in the basal part of the cells and then in the apical cytoplasm. In tubules perfused with both CF and HRP in the perfusate, the CF and HRP were found together in apical vacuoles and lysosomes. After perfusion with HRP alone, this tracer was found in similar large vacuoles and lysosomes in the apical cytoplasm, in contrast to the small HRP-filled vacuoles seen after uptake from the bath.     

Nephron structure and immunohistochemical localization of ion pumps and aquaporins in the kidney of frogs inhabiting different environments

Amphibians inhabit areas ranging from completely aqueous to terrestrial environments and move between water and land. The kidneys of all anurans are similar at the gross morphological level: the structure of their nephrons is related to habitat. According to the observation by light and electron microscopy, the cells that make up the nephron differ among species. Immunohistochemical studies using antibodies to various ATPases showed a significant species difference depending on habitat. The immunoreactivity for Na+,K(+)-ATPase was low in the proximal tubules but high in the basolateral membranes of early distal tubules to collecting ducts in all species. In the proximal tubule, apical membranes of the cells were slightly immunoreactive to H(+)-ATPase antibody in aquatic species. In the connecting tubule and the collecting duct, the apical membrane of intercalated cells was immunoreactive in all species. In aquatic species, H+,K(+)-ATPase immunoreactivity was observed in cell along the proximal, distal tubule to the collecting duct. However, H+,K(+)-ATPase was present along the intercalated cells of the distal segments from early distal to collecting tubules in terrestrial and semi-aquatic species. In the renal corpuscle, the neck segment and the intermediate segment, immunoreactivities to ion pumps were not observed in any of the species examined. Taking together our observations, we conclude that in the aquatic species, a large volume of plasma must be filtered in a large glomerulus and the ultrafiltrate components are reabsorbed along a large and long proximal segment of the nephron. Control of tubular transport may be poorly developed when a small short distal segment of the nephron is observed. On the contrary, terrestrial species have a long and well-developed distal segment and regulation mechanisms of tubular transport may have evolved in these segments. Thus, the development of the late distal segments of the nephron is one of the important factors for the terrestrial adaptation.     

Regional specialization in the malpighian tubules of the new zealand glow-worm Arachnocampa luminosa (diptera: Mycetophilidae). The structure and function of type I and II cells

The Malpighian tubules of the glow-worm Arachnocampa luminosa are divided into four morphologically distinct regions (Parts 1--4) each comprised of a different cell type (Types I--IV). The ultrastructure of Type II cells is indicative of a transport function. The basal cell surface is highly invaginated and at the apical surface the lumen is lined with microvilli about 80% of which contain mitochondria. Spherites contained in these cells are formed from small vesicles produced by the Golgi apparatus. They have a central uric acid core enclosed by laminations of phosphates of calcium and magnesium. Cells of Part 2 of the tubule secrete a fluid high in potassium (173 mM) and low in sodium (18 mM). The cell is 30 mV negative and the lumen 44 mV positive to the bathing solution. This is consistent with the proposal of an apical cation pump. The secretion produced by Part 2 of the tubules is modified by the Type I cells by the reabsorption of potassium (162 mM) and the addition of sodium (24 mM) to the primary excretory fluid. Type I cells are 20 mV negative and the lumen 22 mV positive with respect to the bathing medium. From ultrastructural observations, Type I cells exhibit features characteristic of transporting cells thought to have an absorptive function. The basal and apical cell surfaces are extensively folded, and mitochondria are found in bands above the basal infoldings and below the microvilli. Mitochondria do not penetrate the microvilli. On comparative grounds, the fine structure of Type I cells suggest that they reabsorb ions from the tubule lumen. Energy for these processes may come from the breakdown of lipids by microperoxisomes contained within these cells. Alternatively, the fluid produced by Part 2 of the tubule may be modified passively by diffusional processes across Type I cells.     

Membrane receptors involved in endocytosis in rat renal proximal tubule

In conclusion the present study has demonstrated the localization of 3 receptors for endocytosis in rat renal proximal tubule cells. The three receptors are located in the membranes of the vacuolar compartment involved in endocytosis in these cells and in addition in dense apical tubules responsible for membrane recycling in the proximal tubule.     

The membrane composition of coated pits, microvilli, endosomes, and lysosomes is distinctive in the rat kidney proximal tubule cell

The distribution of a number of membrane proteins on plasmalemmal microdomains (microvilli, coated pits) and in endosomes and lysosomes of the proximal tubule epithelial cell was determined in normal rat kidneys by immunofluorescence and immunoelectron microscopy. Two major brush border proteins, 130 and 94 kD, and gamma-glutamyl transpeptidase were detected on the membranes of the microvilli but were not found on membranes of coated pits. Gp330, the Heymann nephritis antigen, and clathrin were localized in coated pits. The lysosomal membrane glycoprotein, lgp120 (Lewis, V., S. A. Green, M. Marsh, P. Vihko, A. Helenius, and I. Mellman, 1985, J. Cell Biol., 100: 1839-1847) was restricted to lysosomes where it co-localized with beta-glucuronidase. Endosomes, identified by preloading with HRP injected 5-15 min before rats were killed, did not contain detectable amounts of any antigen tested. The distribution of the same proteins was also determined in rats given sodium maleate, which is known to slow or reduce protein absorption by the proximal tubule and to cause vacuolation of the endocytic apparatus. After maleate treatment the distribution of microvillar and lysosomal markers was unchanged, but the coated pit markers were redistributed--gp330 was concentrated in newly formed apical vacuoles, and clathrin was diffusely distributed in the apical cytoplasm or on apical coated vesicles. These findings indicate that the membrane composition of microvilli, coated pits, endosomes, and lysosomes is distinctive in the proximal tubule cell; and that gp330, unlike other known coated pit membrane components, is not transferred to endosomes during endocytosis. After maleate treatment, the coated pits lose their clathrin coats, and the corresponding membrane is internalized.