Aprotinin uptake in the proximal tubules in the rat kidney. II. Uptake site relative to glomerulus

Glomerular filtration rates in whole kidney and in outer, middle and inner cortical zones have previously been estimated by measuring the amount of iodinated Aprotinin, filtered and taken up in the first two thirds of the proximal convoluted tubules, in part positioned more superficial than the parent glomerulus. Thus, an appreciable amount of the absorbed Aprotinin may be located superficial to its filtration site and lead to an underestimate of glomerular filtration in deep cortical layers. Therefore, in this study we have measured the distance from the glomerulus to the center of proximal convoluted tubular ball and the site of Aprotinin uptake. Measurements were made on photos of Microfil-injected tubules and on camera lucida drawings of tubular transections from autoradiographs of nephrons containing both Microfil and iodinated Aprotinin. Both techniques showed that the center of the tubular ball was localized more superficial in all cortical layers. The average distance, in percent of cortical thickness, from all proximal convoluted tubular transections to the parent glomerulus was 9% in deep and 13% in middle and superficial cortex. Corresponding distances for tubular transections containing Aprotinin were 7 and 12%. Grain density in five reconstructed proximal convoluted tubules showed a continuous and exponential fall of Aprotinin along the uptake segment. The results may be used to estimate single nephron filtration rate from Aprotinin uptake and glomerular density in outer, middle, and inner cortex.     

Quantitative intravital microscopy using a Generalized Polarity concept for kidney studies

In this article, we describe a ratiometric intravital two-photon microscopy technique for studying glomerular permeability and differences in proximal tubule cell reabsorption. This quantitative approach is based on the Generalized Polarity (GP) concept, in which the intensity difference between two fluorescent molecules is normalized to the total intensity produced by the two dyes. After an initial intravenous injection of a mixture of 3-, 40-, and 70-kDa fluorescently labeled dextrans into live Munich-Wistar-Frömter (MWF) rats, we were able to monitor changes in the GP values between any two dyes within local regions of the kidney, including the glomerulus, Bowman's capsule, proximal tubule lumens and proximal tubule cells, and individual capillary vessels. We were able to quantify accumulations of different dextrans in the Bowman's space and in tubular lumens as well as reabsorption by proximal tubular cells at different time points in the same rat. We found that for 6- to 8-wk-old MWF rats that developed spontaneous albuminuria, the 40- and 70-kDa dextrans, with hydrodynamic radii larger than albumin, were differentially filtered, but both were able to pass the glomerular filtration barrier and enter into the urinary space of the Bowman's capsule within a few seconds after intravenous infusion. Using GP image analysis, we found that negatively charged dextrans of both 40 and 70 kDa were better reabsorbed by the proximal tubule cells than the neutrally charged 40-kDa dextran. These results demonstrate the potential power of the GP imaging technique for quantitative studies of glomerular filtration and tubular reabsorption. glomerular permeability; tubular reabsorption; charge selectivity; two-photon excitation; multiphoton     

Renal filtration and reabsorption of GFP in frog Rana temporaria: effect of arginine-vasotocin

The renal tubular uptake of green fluorescent protein (GFP) in frog Rana temporaria was studied by laser confocal microscopy. The specific green fluorescence was revealed in the proximal tubule cells 30 min after intravenous GFP injection. The GFP fluorescence was distributed predominantly in the apical part of the cytoplasm in the form of the intensively fluorescing vesicles. The GFP injections increased dose-dependently the GFP tubular uptake. This was confirmed by the quantitative assessment of intensity of the specific fluorescence, its relative vesicular density, and by correlation analysis. Preliminary administration of arginine vasotocin into the dorsal lymphatic sac decreased significantly the GFP absorption. The effect of arginine vasotocin was inhibited by pretreatment a vasopressin V1-receptor antagonist. These results suggest that a decrease in the GFP absorption is due to a fall of the AVT-dependent glomerular filtration rate and consequently a decrease in the filtered GFP amount. The effect of arginine vasotocin on the GFP absorption seems to be mediated via the V1-like receptors of preglomerular blood vessels.     

A theoretical note on exponential flow in the proximal part of the mammalian nephron

On the basis of the experiments of A. M. Walkeret al. (Am. J. Physiol.,134, 580–595, 1941), it is postulated that the fraction of glomerular filtrate reabsorbed up to a given point in the proximal tubule is independent of the rate of filtration. This, combined with the assumption that the proximal tubule is uniform from glomerular to distal end, implies that the volume of flow per unit of time past a given point in the proximal tubule decreases exponentially as a function of distance from the glomerulus. From this it is deduced that the rate of reabsorption of Na⁺ is proportional to the rate of formation of glomerular filtrate—a result established in clearance experiments. The analogy between a nephron and a catalytic flow reactor is indicated, and it is noted that, in both systems, reaction velocity can depend on the rate of flow.     

Megalin-mediated endocytosis of cystatin C in proximal tubule cells

Serum levels of cystatin C, an endogenous cysteine proteinase inhibitor, are often used as an indicator of glomerular filtration rate. Although it is known that cystatin C is filtered by glomeruli and metabolized in proximal tubule cells (PTC), the precise molecular mechanism underlying this process is undetermined. Using quartz-crystal microbalance analyses, we demonstrate that cystatin C binds directly to megalin, an endocytic receptor in PTC, in a Ca(+)-dependent manner. We also find that cystatin C is endocytosed specifically via megalin in rat yolk sac epithelium-derived L2 cells which share a variety of characteristics with PTC. Finally, in vivo studies using kidney-specific megalin knockout mice provide evidence that megalin mediates proximal tubular uptake of cystatin C. We conclude that megalin is an endocytic receptor of cystatin C in PTC.     

Quantitative measurement of glucose-6-phosphate dehydrogenase in cortical fractions of the rabbit nephron

A quantitative fluorimetric method is described for estimating the activity of glucose-6-phosphate dehydrogenase in isolated fractions of rabbit nephron from the superficial part of the renal cortex: macula densa, proximal convoluted tubule, distal convoluted tubule and glomerulus. The mean activity in the macula densa region was 2.5 X 10(-18) mol/micrometers 3/min, which was about twice the mean activity of the proximal and distal tubular cells and four times that of the glomeruli. As glucose-6-phosphate dehydrogenase is located in the cytoplasm, the average cytoplasmic enzyme activity of the different tubular cells was calculated: macula densa activity was 4.0 X 10(-18) mol/micrometers 3/min whilst proximal tubular cells showed about a third, and distal tubular cells about a quarter of this activity.     

Receptor-Mediated Endocytosis of Lysozyme in Renal Proximal Tubules of the Frog Rana Temporaria

The mechanism of protein reabsorption in the kidney of lower vertebrates remains insufficiently investigated in spite of raising interest to the amphibian and fish kidneys as a useful model for physiological and pathophysiological examinations. In the present study, we examined the renal tubular uptake and the internalization rote of lysozyme after its intravenous injection in the wintering frog Rana temporaria using immunohisto- and immunocytochemistry and specific markers for some endocytic compartments. The distinct expression of megalin and cubilin in the proximal tubule cells of lysozyme-injected frogs was revealed whereas kidney tissue of control animals showed no positive immunoreactivity. Lysozyme was detected in the apical endocytic compartment of the tubular cells and colocalized with clathrin 10 min after injection. After 20 min, lysozyme was located in the subapical compartment negative to clathrin (endo-somes), and intracellular trafficking of lysozyme was coincided with the distribution of megalin and cubilin. However, internalized protein was retained in the endosomes and did not reach lysosomes within 30 min after treatment that may indicate the inhibition of intra-cellular trafficking in hibernating frogs. For the first time, we provided the evidence that lysozyme is filtered through the glomeruli and absorbed by receptor-mediated clathrin-dependent endocytosis in the frog proximal tubule cells. Thus, the protein uptake in the amphibian mesonephros is mediated by megalin and cubilin that confirms a critical role of endocytic receptors in the renal reabsorption of proteins in amphibians as in mammals.Key words: Endocytic receptor, frog, kidney, lysozyme, protein uptake, proximal tubule     

The proximal tubule in the pathophysiology of the diabetic kidney

Diabetic nephropathy is a leading cause of end-stage renal disease. A better understanding of the molecular mechanism involved in the early changes of the diabetic kidney may permit the development of new strategies to prevent diabetic nephropathy. This review focuses on the proximal tubule in the early diabetic kidney, particularly on its exposure and response to high glucose levels, albuminuria, and other factors in the diabetic glomerular filtrate, the hyperreabsorption of glucose, the unique molecular signature of the tubular growth phenotype, including aspects of senescence, and the resulting cellular and functional consequences. The latter includes the local release of proinflammatory chemokines and changes in proximal tubular salt and fluid reabsorption, which form the basis for the strong tubular control of glomerular filtration in the early diabetic kidney, including glomerular hyperfiltration and odd responses like the salt paradox. Importantly, these early proximal tubular changes can set the stage for oxidative stress, inflammation, hypoxia, and tubulointerstitial fibrosis, and thereby for the progression of diabetic renal disease.     

Renal handling of inorganic mercury in mice. The early excretion phase following a single intravenous injection of mercuric chloride studied by the Silver Amplification method

The early renal excretion of mercuric mercury was studied in male BALB/c mice between 15 seconds and 30 min following a single intravenous injection of 3 mg HgCl2/kg body weight. The cytochemical Silver Amplification method applied at the light and electron microscopical levels showed mercury to be excreted by glomerular filtration and reabsorbed by proximal tubular epithelial cells by means of adsorptive endocytosis. Mercury was rapidly demonstrated in the lysosomal vacuome of proximal tubular epithelial cells. No uptake was observed from the peritubular side, and there was no evidence of tubular secretion of mercury. It is proposed that mercury is excreted in the form of mercury-protein complexes, assisted by the physiological proteinuria in mice, which is enhanced by mercury-induced damage to the glomerular structures.     

Innervation of the renal proximal convoluted tubule of the rat

Experimental data suggest the proximal tubule as a major site of neurogenic influence on tubular function. The functional and anatomical axial heterogeneity of the proximal tubule prompted this study of the distribution of innervation sites along the early, mid, and late proximal convoluted tubule (PCT) of the rat. Serial section autoradiograms, with tritiated norepinephrine serving as a marker for monoaminergic nerves, were used in this study. Freehand clay models and graphic reconstructions of proximal tubules permitted a rough estimation of the location of the innervation sites along the PCT. In the subcapsular nephrons, the early PCT (first third) was devoid of innervation sites with most of the innervation occurring in the mid (middle third) and in the late (last third) PCT. Innervation sites were found in the early PCT in nephrons located deeper in the cortex. In juxtamedullary nephrons, innervation sites could be observed on the PCT as it left the glomerulus. This gradient of PCT innervation can be explained by the different tubulovascular relationships of nephrons at different levels of the cortex. The absence of innervation sites in the early PCT of subcapsular nephrons suggests that any influence of the renal nerves on the early PCT might be due to an effect of neurotransmitter released from renal nerves reaching the early PCT via the interstitium and/or capillaries.     

Contraluminal uptake of serine in the proximal nephron

Rabbit proximal nephron segments were microperfused in vitro to determine whether active contraluminal uptake of serine occurs in the renal proximal tubule during bath-to-lumen transport (influx) of the L- and D-isomers in the convoluted (pars convoluta) and straight (pars recta) segments. It is known that several amino acids are actively reabsorbed in the proximal nephron by a mechanism involving co-transport with sodium at the luminal membrane. There is some evidence that certain amino acids may also be accumulated across the contraluminal membrane by an energy-dependent mechanism, indicating that net reabsorption is the result of two oppositely directed active transport processes. During in vitro microperfusion of rabbit proximal nephron segments in this study, inward movement of L- and D-serine occurred in a bath-to-cell direction against a concentration gradient in the range 305-2735:1, indicating active uptake at the contraluminal membrane. The concentration gradients were maintained during influx of both isomers of serine in the proximal tubule. L-Serine accumulation by tubular cells was similar in the pars convoluta and recta, and significantly greater than that of D-serine, which was the same in both regions of the proximal tubule. The data support the conclusion that renal handling of serine involves active contraluminal uptake of the L- and D-isomers in both regions of the proximal tubule, and suggest that contraluminal events play an important role in renal handling of amino acids.     

Sex different cytochrome-c uptake in the proximal tubule of the rat kidney

The present study deals with the dose- and time-dependent uptake of cytochrome c (CYT c) in the proximal tubule of the rat kidney, and shows that there are segment and sex differences in the reabsorption of CYT c. Rats of both sexes were intravenously injected with different doses of CYT c (0.75-9.0 mg per 100 g body weight), and the kidneys were investigated by light and electron microscopy at different times (3 min, 10 min, and 2 h) after the injection. After 3 and 10 min, CYT c was demonstrated in apical vacuoles of different sizes and in some lysosomes of the S1 and S2 segments, whereas after 2 h, CYT c was found only in lysosomes of all three segments of the proximal tubule. At these times, the S1 segment contained more CYT c than the S2 and S3 segments. However, 2 h after the injection of 6 or 9 mg CYT c, the differences between the S1 and S2 segments disappeared almost completely, due to a strong lysosomal accumulation of CYT c in the S2 segment. At all studied times and CYT-c doses, the S3 segment contained less CYT c than the S1 and S2 segments. On the whole, different levels of CYT-c reabsorption were found in the different segments of the proximal tubule, which was saturable with increasing CYT-c doses, i.e. firstly in the proximal and then in the distal parts of the proximal tubule. Two hours after the injection of CYT c, a difference between males and females was observed, with the lysosomes of the S1 and S2 segments of females containing more CYT c than those of males. Thus, more CYT c was reabsorbed in the proximal tubule of females than in that of males.     

Cofilin interacts with ClC-5 and regulates albumin uptake in proximal tubule cell lines

Receptor-mediated endocytosis is a constitutive high capacity pathway for the reabsorption of proteins from the glomerular filtrate by the renal proximal tubule. ClC-5 is a voltage-gated chloride channel found in the proximal tubule where it has been shown to be essential for protein uptake, based on evidence from patients with Dent's disease and studies in ClC-5 knockout mice. To further delineate the role of ClC-5 in albumin uptake, we performed a yeast two-hybrid screen with the C-terminal tail of ClC-5 to identify any interactions of the channel with proteins involved in endocytosis. We found that the C-terminal tail of ClC-5 bound the actin depolymerizing protein, cofilin, a result that was confirmed by GST-fusion pulldown assays. In cultured proximal tubule cells, cofilin was distributed in nuclear, cytoplasmic, and microsomal fractions and co-localized with ClC-5. Phosphorylation of cofilin by overexpressing LIM kinase 1 resulted in a stabilization of the actin cytoskeleton. Phosphorylation of cofilin in two proximal tubule cell models (porcine renal proximal tubule and opossum kidney) was also accompanied by a pronounced inhibition of albumin uptake. This study identifies a novel interaction between the C-terminal tail of ClC-5 and cofilin, an actin-associated protein that is crucial in the regulation of albumin uptake by the proximal tubule.     

Terrestrial adaptation and diversity of the kidney functions in the evolution of vertebrates, Amphibia]

The Amphibia bridge the phyletic gap between the aquatic fishes and the terrestrial vertebrates. This transition has involved many interesting changes of metabolisms. In this short review, we have attempted to summarize the kidney structure and functions on the osmoregulations in the Amphibia. Amphibians excrete the water absorbed through their skin as a dilute urine. Pronephros of tadpoles may start to work in the hatching stages and metanephros is well developed and functions. Glomerular filtration rate is relatively large and glomerular intermittency is important to regulate urine production. The proximal tubule reabsorbs approximately 20-45% of filtered water and sodium. Absorption is driven by the basolateral Na+, K(+)-ATPase common to all tubular cells. The diluting segment, early parts of distal nephron, highly develops basolateral interdigitation and reabsorbs approximately 40% of filtered Na+, K+, and Cl-, but is impermeable to water, thus this part results in the formation of hypo-osmotic tubular fluid. In the late distal tubule, the primary mechanism of reabsorption may be via a luminal NaCl synporter, driven by the ubiquitous Na+, K(+)-ATPase on basolateral membrane. In collecting tubule, there are two types of cells, the principal cells and the intercalated cells. Many hormonal and nervous regulations are involved in the glomerular filtration rate and reabsorptions in the amphibian nephrons.     

Sodium-sensitive, probenecid-insensitive p-aminohippuric acid uptake in cultured renal proximal tubule cells of the rabbit.

In the intact kidney, renal proximal tubule cells accumulate p-aminohippurate (PAH) via a basolateral, probenecid- and sodium-sensitive transport system. Primary cultures of rabbit proximal tubule cells retain sodium-glucose co-transport in culture, but little is known about PAH transport in this system. Purified proximal tubule cells from a rabbit were grown in culture and assessed for PAH and alpha-methyl-D-glucoside uptake capacities as well as proximal tubule marker enzyme activities. Control PAH uptake on collagen-coated filters (20 +/- 3 pmol/mg protein.min; n = 8) was not significantly different from uptake in the presence of 1 mM probenecid (19 +/- 4 pmol/mg protein.min; n = 8). Uptake from the basal side of the cell was 3.9 +/- 0.7 times greater than that from the apical side. In multi-well plate studies, the uptake was significantly reduced by removing sodium from the medium and stimulated by coating the wells with collagen. Glutarate (10 mM) had no effect on the uptake of PAH. Other differentiated proximal tubule characteristics were retained in culture, including the ability to form domes and to transport glucose by a phlorizin-sensitive system. Phlorizin-sensitive 1 mM alpha-methyl-D-glucoside uptake was 134 +/- 42 pmol/mg protein.min (n = 7; P less than 0.02). The proximal tubule marker enzymes alkaline phosphatase and gamma-glutamyltranspeptidase, increased in activity in the cultures after confluence. It was concluded that whereas some differentiated properties were retained during primary culture of rabbit proximal tubule cells, the PAH transport system was selectively lost or modified from that present in the intact kidney.     

Megalin mediates selenoprotein P uptake by kidney proximal tubule epithelial cells

Selenoprotein P (Sepp1) contains most of the selenium in blood plasma, and it is utilized by the kidney, brain, and testis as a selenium source for selenoprotein synthesis. We recently demonstrated that apolipoprotein E receptor-2 (ApoER2) is required for Sepp1 uptake by the testis and that deletion of ApoER2 reduces testis and brain, but not kidney, selenium levels. This study examined the kidney Sepp1 uptake pathway. Immunolocalization experiments demonstrated that Sepp1 passed into the glomerular filtrate and was specifically taken up by proximal tubule epithelial cells. Neither the C terminus selenocysteine-rich domain of Sepp1 nor ApoER2 was required for Sepp1 uptake by proximal tubules. Tissue ligand binding assays using cryosections of Sepp1-/- kidneys revealed that the proximal tubule epithelium contained Sepp1-binding sites that were blocked by the receptor-associated protein, RAP, an inhibitor of lipoprotein receptor-ligand interactions. Ligand blotting assays of kidney membrane preparations fractionated by SDS-PAGE revealed that Sepp1 binds megalin, a lipoprotein receptor localized to the proximal tubule epithelium. Immunolocalization analyses confirmed the in vivo co-localization of Sepp1 and megalin in wild type kidneys and demonstrated the absence of proximal tubule Sepp1 uptake in megalin null mice. These results demonstrate that kidney selenium homeostasis is mediated by a megalin-dependent Sepp1 uptake pathway in the proximal tubule.     

Quantitative measurement of glucose-6-phosphate dehydrogenase in cortical fractions of the rabbit nephron

Summary A quantitative fluorimetric method is described for estimating the activity of glucose-6-phosphate dehydrogenase in isolated fractions of rabbit nephron from the superficial part of the renal cortex: macula densa, proximal convoluted tubule, distal convoluted tubule and glomerulus. The mean activity in the macula densa region was 2.5×10^–18 mol/m³/min, which was about twice the mean activity of the proximal and distal tubular cells and four times that of the glomeruli. As glucose-6-phosphate dehydrogenase is located in the cytoplasm, the average cytoplasmic enzyme activity of the different tubular cells was calculated: macula densa activity was 4.0×10^–18 mol/m³/min whilst proximal tubular cells showed about a third, and distal tubular cells about a quarter of this activity.     

Tubular GFP uptake pattern in the rat and frog kidneys

The renal tubular uptake of green fluorescent protein (GFP) after its bolus intravenous injection was studied in both frogs and rats. GFP fluorescence in the proximal tubule (PT) was revealed by fluorescent and confocal microscopy. Granular GFP fluorescence was observed nearby in the apical membrane of PT cells featuring distribution over the cytoplasm. GFP was internalized into endosomes and lysosomes as determined by immunocytochemistry in frogs. The tubular uptake and accumulation of GFP were dose- and time-dependent in both rats and frogs. Intralymphatic sac injection of arginine vasotocin (AVT) decreased the uptake of GFP in hydrated frogs. A high negative correlation between the AVT dose and the uptake of GFP was revealed. The effect of AVT was inhibited by a V(1)-receptor antagonist. A noted decrease in the average number of fluorescent PT profiles per kidney section and their irregular distribution after AVT injections suggest that not all of the glomeruli or preglomerular vessels are equally responsive to AVT. GFP may serve as a good marker for tubular uptake and intracellular traffic in the amphibian kidney for use in in vivo studies.     

Tubular proteinuria in mice and humans lacking the intrinsic lysosomal protein SCARB2/Limp-2

Deficiency of the intrinsic lysosomal protein human scavenger receptor class B, member 2 (SCARB2; Limp-2 in mice) causes collapsing focal and segmental glomerular sclerosis (FSGS) and myoclonic epilepsy in humans, but patients with no apparent kidney damage have recently been described. We now demonstrate that these patients can develop tubular proteinuria. To determine the mechanism, mice deficient in Limp-2, the murine homolog of SCARB2, were studied. Most low-molecular-weight proteins filtered by the glomerulus are removed in the proximal convoluted tubule (PCT) by megalin/cubilin-dependent receptor-mediated endocytosis. Expression of megalin and cubilin was unchanged in Limp-2(-/-) mice, however, and the initial uptake of injected Alexa Fluor 555-conjugated bovine serum albumin (Alexa-BSA) was similar to wild-type mice, indicating that megalin/cubilin-dependent, receptor-mediated endocytosis was unaffected. There was a defect in proteolysis of reabsorbed proteins in the Limp-2(-/-) mice, demonstrated by the persistence of Alexa-BSA in the PCT compared with controls. This was associated with the failure of the lysosomal protease cathepsin B to colocalize with Alexa-BSA and endogenous retinol-binding protein in kidneys from Limp-2(-/-) mice. The data suggest that tubular proteinuria in Limp-2(-/-) mice is due to failure of endosomes containing reabsorbed proteins to fuse with lysosomes in the proximal tubule of the kidney. Failure of proteolysis is a novel mechanism for tubular proteinuria.     

Tissue pressures and fluid dynamics in the kidney.

The kidney has several characteristics which make renal pressures and fluid dynamics unique when compared to other organs. Renal blood flow is roughly 100 times that of skeletal muscle. The renal circulation consists of two distinct capillary beds in series: a high pressure system in the glomerulus that favors filtration and a low pressure system in the peritubule network that favors reabsorption. The hydrostatic pressure in the glomerular capillary is 4-6 times higher than the hydrostatic pressure in the peritubule capillary so that approximately 25% of the plasma is filtered. The bulk of the filtrate is subsequently reabsorbed by the peritubule capillary network. Micropuncture techniques have been used to obtain quantitative measurements of the pressures and fluid dynamics of the peritubule microcirculation. The net force for uptake of all the fluid reabsorbed by a single proximal tubule up to the point of micropuncture is 21 mm Hg acting over a capillary bed with a permeability surface area product of 2 nl/min per mm Hg. In contrast to subcutaneous tissue and muscle, the renal interstitial fluid pressure is positive. The consequence of a positive interstitial fluid pressure is that normal lymph flow is relatively high and changes in interstitial fluid pressure have relatively little effects on lymph flow.