The plasma membrane Ca2+ pump from proximal kidney tubules is exclusively localized and active in caveolae

Plasma membrane Ca2+-ATPase is involved in the fine-tuned regulation of intracellular Ca2+. In this study, the presence of Ca2+-ATPase in caveolae from kidney basolateral membranes was investigated. With the use of a discontinuous sucrose gradient, we show that Ca2+-ATPase is exclusively located and fully active in caveolin-containing microdomains. Treatment with methyl-beta-cyclodextrin--a cholesterol chelator--leads to a spreading of both caveolin and completely inactive Ca2+-ATPase toward high-density fractions. These data support the view that Ca2+ fluxes mediated by Ca2+-ATPase in kidney epithelial cells occur only in caveolae, being strictly dependent on the integrity of these microdomains.     

On the connection between the transverse tubules and the plasma membrane in frog semitendinosus skeletal muscle

The transverse tubular system (TTS) of skeletal muscle fibers represents the morphological basis for the inward spread of conduction of the electrical signal that triggers muscle contraction. A historical account of the main steps contributing to the elucidation of the structure and function of the TSS has been presented by Huxley (1971). While the localization of the TSS and its association with the sarcoplasmic reticulum (SR) is well documented; there is still a need further to develop our knowledge of the morphology of the connection between the TSS and the plasma membrane. It is generally believed that the TSS opens directly to the extracellular space and that there is continuity between its membrane and the sarcolemma. However, direct observation of such a connection has been clearly shown only for the myotome of fish (Franzini-Armstrong and Porter, 1964). In other muscle fibers, only indirect evidence of the connection has been provided by experiments showing penetration of extracellular tracers into the TSS. These extracellular markers were also observed inside another membrane-bounded compartment consisting of round profiles named "caveolae" (Yamada, 1955) or "pinocytotic vesicles" (Ashurst, 1969). The present study deals with the communication between the TTS, caveolae, and plasma membrane (Peachey, 1965); Ezerman and Ishikawa, 1967; Schiaffino and Margreth, 1968; and Rayns et al., 1968). A detailed study of the caveolae compartment was undertaken with ruthenium red as an electron-dense tracer. As a result of this study, we propose that in certain species the caveolae compartment represents the transitional region in the connection between the TSS and the sarcolemma.     

Cells and intercellular contacts in glomeruli and tubules of the frog kidney

Summary By the use of thin sections and freeze-fracture replicas the glomerular and tubular structures of the kidney of the frog (Rana esculenta) were studied with special reference to intercellular junctions.In the glomerulus the filtration barrier is of very variable thickness, and frequent tight and gap junctional contacts occur between podocyte processes.Although structurally less elaborate, the proximal tubule resembles its mammalian counterpart. In the initial part the tight junctions are relatively shallow but become very broad in the mid and distal portions of the proximal tubule. The proximal tubular cells are extensively linked by gap junctions. In some animals the shapes of the cells in the proximal and distal portions of the proximal tubule were markedly different.The distal tubule consists of two segments which differ mainly in the pattern of interdigitations and the structure of the zonulae occludentes. Similarities with the tight junctional morphology of the mammalian distal tubule are striking. In the first part of the distal tubule (diluting segment) a narrow band of parallel tight junctions is found closely resembling that found in the mammalian straight distal tubule; in the more distal part of the distal tubule, however, a broad band of anastomosing tight junctional strands exists, like the zonula occludens of the mammalian convoluted distal tubule.The connecting tubule displays cellular dimorphism: its wall contains a mixture of light and dark (flask) cells. The luminal and basolateral membranes of the flask cells are covered with numerous rod-shaped particles. The tight junctions of the connecting tubule are broad and increase in depth and number of strands along its length; they are typical of a very tight epithelium.In spite of several dissimilarities with phylogenetically younger kidneys our findings suggest that many structural principles of the mammalian kidney are also represented in the kidneys of amphibians. The structural-functional relationships are discussed.     

myo-Inositol transport in plasma membrane of rat kidney

The myo-inositol transport system in kidney plasma mambrane preparation was investigated. myo-Inisitol uptake was more rapid than that due to non-specific uptake. Specific myo-inisitol uptake was temperature dependent and pH sensitive; the optimum was at pH 7.4. Specific myo-insitol uptake was inhibited by scyllitol and inosose-2 but not(+)-inositol, d-glucose, d-galactose or mannitol. Inhibition of myo-inositol uptake by scyllitol was of the competitive type. It showed that the transport system is stereospecific and that myo-inositol shares the transport system with scyllitol. Moreover, the specific myo-inositol uptake was inhibited by phlorizin. Counter transport of myo-inositol was demonstrated. The results indicate that myo-inositol uptake by the membrane preparation represents the entry into the intravesicular spaces rather than binding to the membrane.It was concluded that the plasma membrane of rat kidney has a cyclitol carrier system specific to myo-inositol and scyllitol.     

Cadmium stimulation of Na-independent organic acid transport in the kidney tubules

The influence of Cd++ (as well as of Hg++ and Cu++) on the uptake of an organic acid (fluorescein) in superficial proximal tubules of the surviving rat kidney was studied at 20 degrees C, when the active transport of fluorescein does not depend on the external Na. In contrast to mercury and copper, cadmium stimulated the uptake of fluorescein from the beginning of incubation. The minimal effective concentration of Cd++ was 5 X 10(-6)M, the relative effect of Cd++ on the uptake being the same within the concentration range from 5 X 10(-6) to 10(-3) M. A 60 minutes pre-incubation with Cd++ at 20 degrees C resulted in a significant increase in the stimulatory effect of acetate on the fluorescein transport. The stimulation of the fluorescein transport by cadmium was prevented by ouabain or by omissing Na from the incubating medium, although neither ouabain nor the absence of Na affected the transport of fluorescein under these conditions. It is supposed that the stimulation by Cd++ of the fluorescein transport may result from the activated oxidation of NAD-linked substrates due to acceleration of the active transepithelial transport of Na ions.     

Comparison between calcium transport and adenosine triphosphatase activity in membrane vesicles derived from rabbit kidney proximal tubules

Characteristics of Ca2+ uptake were studied in a vesicular preparation of proximal tubule plasma membranes from rabbit kidney and compared with the properties of both membrane-bound and solubilized Ca2+-ATPase activities. Calcium uptake required both ATP and MgCl2 and revealed two kinetic components with respect to Ca2+ concentration requirements, one with a high affinity for Ca2+ (1.8 microM), operative in the range of cytosolic Ca2+ activity, and one with a low affinity for Ca2+ (250 microM) which may become active only at abnormally high cytosolic Ca2+ concentrations. The high- and low-affinity components were stimulated to similar extents by phosphate, and required similar concentrations of ATP (0.6 mM) for half-maximal activity. The amount of membrane-bound phosphoenzyme formed from ATP in the presence of Ca2+ was the same regardless of whether only one or both sites were saturated, suggesting that occupancy of the second Ca2+ binding site accelerates the enzyme turnover. Inhibition of Ca2+ transport by Na+ was reversed by the addition of ouabain or an ATP-regenerating system, indicating that this inhibitory effect of Na+ on Ca2+ uptake may be due to the accumulation of ADP in the medium as a result of Na+ pump activity. Low concentrations of carbonyl cyanide p-trifluoromethoxyphenylhydrazone and valinomycin (2.5 and 1 microM, respectively) were without effect on Ca2+ uptake in the presence of phosphate, whereas higher concentrations of the ionophores (200 and 100 microM, respectively) reduced uptake by 60% or more. The calmodulin antagonist 48/80 also reduced Ca2+ uptake with half-maximal effectiveness at 100 micrograms/ml. None of these drugs affected either ATPase activity or the EGTA-induced Ca2+ efflux from preloaded vesicles. The Ca2+ dependence of ATP hydrolysis by the membrane-bound enzyme preparation was similar to that observed for Ca2+ uptake by the vesicles. However, with solubilized enzyme, concentrations of Ca2+ similar to that found in the plasma reduced Ca2+-stimulated ATP hydrolysis to one-half of its maximal rate. This indicates that peritubular Ca2+ may play a role in the regulation of Ca2+ transport across the tubular epithelium. ATP could not be replaced by ITP as a substrate for Ca2+ uptake, and the (Ca2+ + Mg2+)ITPase activity of soluble enzyme was 25-fold lower than in the presence of ATP. This is an indication that the active Ca2+ pumping mechanism in proximal tubules is critically dependent on the nucleoside moiety of the substrate.     

Osmotically reactive plasma membrane vesicles prepared from rabbit kidney tubules by mild hypotonic lysis

Plasma membrane vesicles were obtained by hypotonic lysis in an ice-cold medium containing EDTA and MgCl₂. The vesicles were isolated by differential centrifugation. Compared to a total kidney homogenate, a 10–12-fold enrichment of trehalase and alkaline phosphatase (marker enzymes for renal brush border), and a 6-fold enrichment of (Na⁺---:K⁺)-stimulated ATPase, (a marker enzyme for the basal and lateral plasma membrane of the tubule cell), was achieved. Contamination by other cell organelles was very low. The plasma membrane vesicles enclosed small amounts of the cytoplasmic enzymes lactate dehydrogenase and malate dehydrogenase, which exhibited full activity only after their release into the medium by sonication.Electron micrographs of this preparation showed microvilli with drumstick-like expansions, but also spherical vesicles. By measuring the distribution of radio-actively labelled compounds of different molecular weight in a packed sediment of the plasma membranes under isotonic conditions, an intravesicular volume of 82% or 9 μl/mg of membrane protein was estimated. The intravesicular volume decreased when the osmolality of the medium was augmented by raffinose. The scattering of light by the vesicular suspension could be used to monitor rapid volume changes. By this method, the following sequence of flux rates was established: glycerol>erythritol> adonitol>mannitol. The fluxes of LiCl, NaCl, and KCl were almost identical, but faster than those of adonitol and mannitol. The data indicate, that a large fraction of the plasma membrane isolated in this preparation have formed vesicles, and also that they have retained, as far as investigated, the permeability characteristics of the plasma membrane.     

Characterization of ATP-dependent Ca2+ transport in the basolateral membrane vesicles from proximal and distal tubules of the rabbit kidney

Basolateral membrane vesicles were prepared from purified proximal and distal tubules of the rabbit kidney. The properties of the ATP-dependent Ca2+ transport were investigated. In both membranes, there was a high affinity, ATP-dependent Ca2+ transport system (Km = 0.1 microM). The optimal concentration of Mg2+ was 0.5 mM and the optimal concentration of ATP was 1 mM. The nucleotide specificity and pH dependence of the Ca2+ transport in both membranes were similar. In basolateral membrane vesicles, calmodulin had no effect on Ca2+ transport. However, in basolateral membrane vesicles depleted of calmodulin, exogenous calmodulin increased the Ca2+ transport by increasing maximal velocity. There were no major differences in the properties of the ATP-dependent Ca2+ transport system in these two membranes. These findings are discussed in relation to why parathyroid hormone differentially modulates Ca2+ transport in these two segments of the nephron.     

Urate transport across the apical membrane of renal proximal tubules

Since the molecular cloning of the renal apical urate/anion exchanger URAT1 (SLC22A12), several membrane proteins relevant to urate transport have been identified. In addition, the identification of PDZ (PSD-95, DglA, and ZO-1) domain protein PDZK1 as a binding partner of URAT1, and the emerging role of PDZ scaffold for renal apical transporters have led to a new concept of renal urate transport: urate-transporting multimolecular complex, or "urate transportsome," that may form an ultimate functional unit at the apical membrane of renal proximal tubules. Elucidation of urate transportsome will lead to the new drug development for hyperuricemia.     

Double dependence of organic acid active transport in proximal tubules of surviving frog kidney on sodium ions II. Relationship between counter-flows of fluorescein and sodium ion across cell layer

With the aid of a direct microfluorimetric method a dependence of organic onion (fluorescein) transport into proximal tubules of surviving frog kidney on Na⁺-flow in the opposite direction was studied. It was shown that the complete removal of Na⁺ from the tubules lumen resulted in inhibition of fluorescein transport of about 30%. After a specific inhibitor of sodium channels, amiloride (10^-3M) having been introduced into lumen of the tubules, the fluorescein transport was inhibited to the same extent. Amiloride affects only when Na⁺ is present in the tubular lumen. S present in the tubular lumen. Strophantin K (5 · 10^?5 M), a specific inhibitor of (Na⁺, K⁺)-ATPase, reduced fluorescein transport about twice. Substances increasing the 3′,5′-AMP level in cells (theophylline, NaF) and exogenous 3′,5′-AMP inhibited fluorescein transport while substance that decreased the 3′,5′-AMP level intracellularly (carbachol) stimulated it. For realization of these effects Na⁺ should be present in proximal tubules lumen.Thus, the various effects on the Na⁺ flow from lumen of the tubules to medium at the level of both the basal and apical membranes alter the rate of organic acid active transport from medium to lumen as a result of changes in the maximum rate of transport ($\text{V}$) with unchanged $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$. It is suggested that the system of Na⁺ extrusion from proximal tubules produces peritubular membrane-side (near the membrane) gradient of Na⁺ concentration which may be higher than the summary Na⁺ gradient between the medium and the cytoplasm. The magnitude of this gradient affects the maximal rate value of Na⁺-dependent organic acid transport. So, there is a double dependence of the active transport system on Na⁺, and the stages where Na⁺ is needed are: (1) the formation of a carrier-substrate-Na⁺ complex and (2) the production of substantial membrane-side Na⁺ gradient at the expense of Na⁺ extrusion from the tubules.     

Plasma membrane electron transport in frog blood vessels

In an attempt to see if frog blood vessels possess a plasma membrane electron transport system, the postcaval vein and aorta isolated from Rana tigrina were tested for their ability to reduce ferricyanide, methylene blue, and 2,6-dichloroindophenol. While the dyes remained unchanged, ferricyanide was reduced to ferrocyanide. This reduction was resistant to inhibition by cyanide and azide. Heptane extraction or formalin fixation of the tissues markedly reduced the capability to reduce ferricyanide. Denuded aortas retained only 30% of the activity of intact tissue. Our results indicate that the amphibian postcaval vein and aorta exhibit plasma membrane electron transport.     

rab4 regulates transport to the apical plasma membrane in Madin-Darby canine kidney cells.

The small GTPase rab4 is associated with early endosomes and regulates membrane recycling in fibroblasts. rab4 is present in epithelial cells; however, neither its localization nor function has been established in this cell type. We transfected Madin-Darby canine kidney cells with rab4, the GTPase-deficient mutant rab4Q67L, and the dominant negative mutant rab4S22N that poorly binds guanine nucleotides. Confocal immunofluorescence microscopy showed that rab4 was concentrated on internal structures at the lateral side of the cell around the nucleus. Quantitative immunoelectron microscopy revealed that the majority of rab4 was localized in the upper third of the cytoplasm. In cell surface binding experiments with (125)I-transferrin, we found a redistribution of transferrin receptor from the basolateral to the apical plasma membrane in cells expressing rab4 and rab4Q67L. After accumulation of transferrin at 16 degrees C in basolateral early endosomes, rab4 and rab4Q67L increased the amount of apically targeted transferrin receptor. A qualitatively similar effect was obtained in control cells treated with brefeldin A. The effects of brefeldin A and rab4 on apical targeting of transferrin receptor were not additive, suggesting that brefeldin A and rab4 may act in the same transport pathway from common endosomes.     

Proximo-distal specialization of epithelial transport processes within the Xenopus pronephric kidney tubules

The embryonic kidneys of larval aquatic vertebrates such as fish and frogs serve as excellent model systems for exploring the early development of nephric organs. These experimental systems can easily be manipulated by microsurgery, microinjection, genetics, or combinations of these approaches. However, little is known about how physiologically similar these simple kidneys are to the more complex mammalian adult kidneys. In addition, almost nothing is known about proximo-distal patterning of nephrons in any organism. In order begin to explore the physiological specialization of the pronephric tubules along the proximo-distal axis, a combination of uptake assays using fluorescently tagged proteins, LDL particles and dextrans, and an informatics-targeted in situ screen for transport proteins have been performed on embryos of the frog, Xenopus laevis. Genes identified to be expressed within unique subdomains of the pronephric tubules include an ABC transporter, two amino acid cotransporters, two sodium bicarbonate cotransporters, a novel sodium glucose cotransporter, a sodium potassium chloride cotransporter (NKCC2), a sodium chloride organic solute cotransporter (ROSIT), and a zinc transporter. A novel combination of colorimetric and fluorescent whole-mount in situ hybridization (FCIS) was used to precisely map the expression domain of each gene within the pronephros. These data indicate specialized physiological function and define multiple novel segments of the pronephric tubules, which contain at least six distinct transport domains. Uptake studies identified functional transport domains and also demonstrated that early glomeral leakage can allow visualization of protein movement into the pronephric tubules and thus establish a system for investigating experimentally induced proteinuria and glomerulonephritis.     

Structure of the kidney in the crab-eating frog, Rana cancrivora

The structure of the nephron in the ranid frog, Rana cancrivora, was studied by light and electron microscopy. This frog is the only amphibian species to live in mangrove swamps of very high salinity. The nephron consists of the following parts: renal corpuscle, ciliated neck segment, proximal tubule, ciliated intermediate segment, distal tubule, connecting tubule, and collecting duct. The distal tubule is located in the ventromedial region of the kidney, and the other tubules are situated in the dorsolateral region. Renal corpuscles are found between the two regions. Some renal corpuscles have a wide Bowman's space because of the small glomerulus within them. The proximal tubules are composed of columnar cells with a dense luminal brush border of long microvilli and numerous apical vesicles and vacuoles. The initial part of the distal tubule consists of heavily interdigitated cells, characterized by a very regular palisade arrangement of mitochondria. In the terminal part of the distal tubule, shorter mitochondria of the infolding cells are situated irregularly around the nucleus. The connecting tubule consists of principal cells and canaliculus cells. The collecting duct consists of columnar or cuboidal cells; cytoplasmic organelles are relatively sparse. The canaliculus cells are intercalated between principal cells from the terminal distal tubule to the proximal part of the collecting duct. Our findings indicate that the kidney of R. cancrivora is structurally similar to kidneys of other amphibians. These findings are discussed with regard to probable correlations between ultrastructure and function in R. cancrivora.