Sex-related differences in the handling of fluorescent ovalbumin by the proximal tubule of the rat kidney

Sex-dependent protein handling in the rat renal tubular system was studied both qualitatively and quantitatively using the method of direct fluorescent protein tracing. The protein tracer, fluorescent ovalbumin, was synthesized by conjugating hen ovalbumin with fluorescein isothiocyanate (FITC), and the fluorescence characteristics of fluoresceinthiocarbamyl (FTC)-ovalbumin conjugates with different degrees of labelling were studied. Heavily labelled tracer was intravenously injected into male and female rats, and both kidneys were perfused; the right kidney was then homogenized and used for quantitative fluorometric measurements, while the left kidney was perfusion fixed and prepared for fluorescence microscopy. The tubular reabsorption of fluorescent ovalbumin was studied 4 min and 10 min after the injection of different doses (1.4, 7.0 and 14.0 mg/kg body weight) of the tracer, and the tubular catabolism was investigated in animals killed 60 and 120 min after the injection. Fluorescence microscopy demonstrated that, in both sexes and regardless of the dose administered and the time after injection, specifically fluorescent protein or its degradation products was only present in the epithelial cells of the proximal tubule. With regard to sex-dependent differences in protein handling, fluorometry indicated that at 4 min (7.0 mg) and at 10 min (all doses) after injection, female animals had reabsorbed more fluorescent protein than males. With regard to the catabolic phase, both the fluorescence microscopy and the fluorometric results showed that the female rats had degraded the fluorescent tracer at a significantly higher rate than males. The results are discussed in connection with sex-dependent proteinuria in rats.     

Sex-related differences in the handling of fluorescent ovalbumin by the proximal tubule of the rat kidney

Summary Sex-dependent protein handling in the rat renal tubular system was studied both qualitatively and quantitatively using the method of direct fluorescent protein tracing. The protein tracer, fluorescent ovalbumin, was synthesized by conjugating hen ovalbumin with fluorescein isothiocyanate (FITC), and the fluorescence characteristics of fluores-ceinthiocarbamyl (FTC)-ovalbumin conjugates with different degrees of labelling were studied. Heavily labelled tracer was intravenously injected into male and female rats, and both kidneys were perfused; the right kidney was then homogenized and used for quantitative fluorometric measurements, while the left kidney was perfusion fixed and prepared for fluorescence mciroscopy. The tubular reabsorption of fluorescent ovalbumin was studied 4 min and 10 min after the injection of different doses (1.4, 7.0 and 14.0 mg/kg body weight) of the tracer, and the tubular catabolism was investigated in animals killed 60 and 120 min after the injection. Fluorescence microscopy demonstrated that, in both sexes and regardless of the dose administered and the time after injection, specifically fluorescent protein or its degradation products was only present in the epithelial cells of the proximal tubule. With regard to sex-dependent differences in protein handling, fluorometry indicated that at 4 min (7.0 mg) and at 10 min (all doses) after injection, female animals had reabsorbed more fluorescent protein than males. With regard to the catabolic phase, both the fluorescence microscopy and the fluorometric results showed that the female rats had degraded the fluorescent tracer at a significantly higher rate than males. The results are discussed in connection with sex-dependent proteinuria in rats.In honour of Prof. P. van DuijnSupported by the Deutsche Forschungsgemeinschaft (SFB 105)     

Lactate oxidation by three segments of the rabbit proximal tubule

Oxidation of [U14C]lactate to 14CO2 was measured in vitro, in nonperfused anatomically defined segments of rabbit proximal tubule (S1, proximal convoluted, and S2 and S3, proximal straight tubules). The rate of lactate oxidation was similar in S2 and S3 segments, and within the range of lactate oxidation rates measured in vivo. In contrast, the oxidation rate of S1 segments was significantly lower than that of S2 or S3. In proximal straight tubules, lactate oxidation was inhibited by incubation at 0 degrees C, or by application of 1 mM ouabain. To determine if the rate of transepithelial transport affected the rate of lactate oxidation, lactate oxidation was measured in proximal straight tubules after the lumen had been opened by perfusion with Ringer's containing 10 mM polyethylene glycol. No difference in lactate oxidation rate was observed between tubules with patent lumina and nonperfused tubules. These results suggest that the various segments of the renal proximal tubule have different metabolic characteristics, and that the rate of substrate oxidation is related to the activity of the Na+, K+-ATPase.     

Production and functional roles of nitric oxide in the proximal tubule

A significant role for nitric oxide (NO) in proximal tubule physiology and pathophysiology has been revealed by a series of in vivo and in vitro studies. Whether the proximal tubule produces NO under basal conditions is still controversial; however, evidence suggests that the proximal tubule is constantly exposed to NO that might include NO from nonproximal tubule sources. When challenged with a variety of stimuli, including hypoxia, the proximal tubule is able to produce large quantities of NO. In vivo studies generally indicate that NO inhibits fluid and sodium reabsorption by the proximal tubule. However, the final effect of NO on proximal tubular reabsorption appears to depend on the concentration of NO and involve interaction with other regulatory mechanisms. NO regulates Na(+)-K(+)-ATPase, Na(+)/H(+) exchangers, and paracellular permeability of proximal tubular cells, which may contribute to its effect on proximal tubular transport. Enhanced production of NO, perhaps depending on macrophage type inducible NO synthase, participates in hypoxic/ischemic proximal tubular injury. In conclusion, NO plays a fundamental role in both physiology and pathophysiology of the proximal tubule.     

Evidence for the presence of functional beta-adrenoceptor along the proximal tubule of the rat kidney

Evidence for the presence of beta adrenoceptors on proximal tubules from the rat kidney has been obtained using enriched tubule suspensions prepared by Percoll centrifugation. Intact tubules demonstrated simultaneous enrichment of parathyroid hormone and isoproterenol sensitive cAMP production with no enrichment of antidiuretic hormone sensitive cAMP production. Both norepinephrine and epinephrine were less potent than isoproterenol and the stimulatory effect of catecholamines could be blocked with propranolol but not phentolamine. The stimulatory effect of norepinephrine on cellular phenylalanine uptake is blunted by co-addition of isoproterenol suggesting that the beta receptor may modulatory catecholamine stimulated transport.     

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.     

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.     

Fine structure of the elasmobranch renal tubule: neck and proximal segments of the little skate

This is the first in a series of studies that examines the renal tubular ultrastructure of elasmobranch fish. Each subdivision of the neck segment and proximal segment of the renal tubule of the little skate (Raja erinacea) has been investigated using electron microscopy of thin sections and freeze-fracture replicas. Flagellar cells, characterized by long, wavy, flagellar ribbons, were observed in both nephron segments. They were found predominantly in the first subdivision of the neck segment, which suggests that propulsion of the glomerular filtrate is a primary function of this part of the renal tubule. In the non-flagellar cells of the neck segment (subdivisions I and II), there were bundles of microfilaments, a few apical cell projections, and, in subdivision II, numerous autophagosomes. In the proximal segment, the non-flagellar cells varied in size, being low in subdivision I, cuboidal in II, tall columnar in III, and again low in IV. Apical cell projections were low and scattered in subdivisions I and IV and were highest in III where the basolateral plasma membrane was extremely amplified by cytoplasmic projections. Furthermore, in these cells the mitochondria were numerous with an extensive matrix and short cristae. A network of tubules of the endoplasmic reticulum characterized the apical region of the non-flagellar cells in subdivisions I, II, and IV. In the late part of subdivision II and the early part of III, the cells were characterized by numerous coated pits and vesicles, large subluminal vacuoles, and basally located dense bodies, all of which are structures involved in receptor-mediated endocytosis. Freeze-fracture replicas revealed gap junctions restricted to the cells of the first three subdivisions of the proximal segment. The zonulae occludentes were not different in the neck and proximal segments, being composed of several strands, suggesting a moderately leaky paracellular pathway.     

Cytoskeletal proteins of the rat kidney proximal tubule brush border

Cytoskeletal components backing the brush border of the rat kidney proximal tubule cell were identified and compared with those of the well characterized intestinal brush border by immuneoverlay and immunocytochemistry. Antibodies reactive against the intestinal microvillus core components, villin and fimbrin, as well as against the terminal web components, spectrin (fodrin) and myosin, were used. Proteins of similar molecular weight to these intestinal brush border cytoskeletal components were identified in isolated kidney brush borders by immuneoverlay. Spectrin, a major component of the terminal web region of both cell types, was more concentrated in the kidney brush border relative to both actin and myosin. By immunofluorescence, villin and fimbrin were localized in the microvilli, and spectrin and myosin were localized to the terminal web region of the brush border. In addition, spectrin was found along the basolateral membranes of the proximal tubule cell, and myosin was detected in a punctate staining pattern throughout its cytoplasm. By immunoelectron microscopy using immunogold labeling procedures, fimbrin and villin were localized in the terminal web as well as in microvilli, and spectrin and myosin were localized to fibrils in the terminal web. A key difference between the epithelia of the two organs is the extensive network of clathrin coated pits found in the terminal web region of the kidney but not the intestinal brush border. The clathrin-rich terminal web region of the kidney, like the intestinal brush border, proved to be quite stable and resistant to disruption by non-ionic detergents and harsh mechanical treatment.     

Glucose transport in a model of the rat proximal tubule epithelium

A mathematical model of the rat proximal tubule epithelium has been extended to include terms for glucose-sodium cotransport, as well as the passive permeability properties of urea. Except for a metabolically driven Na⁺-K⁺ exchanger at the cell basolateral membrane, all membrane transport is represented by the relations of linear nonequilibrium thermodynamics. Use of this formalism permits the explicit calculation of the intracellular depolarization immediately following the luminal application of glucose, and shows the magnitude of this potential deflection proportional to the glucose chemical-potential change. The steady-state glucose transport by this model epithelium, like experimental data, is fitted remarkably well by a three-parameter pump-leak model of transport. In view of the nonsaturability of the cotransporter of the model epithelium, the goodness of fit to the three-parameter model is surprising and underscores the uncertainty in extracting individual membrane properties from whole epithelial data. Experiments are simulated in which hypertonic glucose placed in the bath induces cell swelling and K⁺ uptake; a hypertonic impermeant induces cell shrinkage and K⁺ loss. Although this parallels the observations in vivo, the large K⁺ shifts predicted by the model suggest the absence of important volume-regulatory mechanisms from the model scheme.     

Nonequilibrium thermodynamic model of the rat proximal tubule epithelium.

The rat proximal tubule epithelium is represented as well-stirred, compliant cellular and paracellular compartments bounded by mucosal and serosal bathing solutions. With a uniform pCO2 throughout the epithelium, the model variables include the concentrations of Na, K, Cl, HCO3, H2PO4, HPO4, and H, as well as hydrostatic pressure and electrical potential. Except for a metabolically driven Na-K exchanger at the basolateral cell membrane, all membrane transport within the epithelium is passive and is represented by the linear equations of nonequilibrium thermodynamics. In particular, this includes the cotransport of Na-Cl and Na-H2PO4 and countertransport of Na-H at the apical cell membrane. Experimental constraints on the choice of ionic conductivities are satisfied by allowing K-Cl cotransport at the basolateral membrane. The model equations include those for mass balance of the nonreacting species, as well as chemical equilibrium for the acidification reactions. Time-dependent terms are retained to permit the study of transient phenomena. In the steady state the energy dissipation is computed and verified equal to the sum of input from the Na-K exchanger plus the Gibbs free energy of mass addition to the system. The parameter dependence of coupled water transport is studied and shown to be consistent with the predictions of previous analytical models of the lateral intercellular space. Water transport in the presence of an end-proximal (HCO3-depleted) luminal solution is investigated. Here the lower permeability and higher reflection coefficient of HCO3 enhance net sodium and water transport. Due to enhanced flux across the tight junction, this process may permit proximal tubule Na transport to proceed with diminished energy dissipation.     

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.     

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.     

Establishment of renal cell lines derived from S2 segments of the proximal tubule.

The aim of this study was to establish epithelial cell lines derived from defined nephron segments. Primary cultures were prepared from dissected proximal S2 segments of the rabbit kidney, and grown in monolayers. Immortalization was observed after nuclear microinjection of the cells with simian virus 40 DNA and resulted in the development of cell lines of epithelial morphology. These cell lines were maintained in culture for at least 24 passages, then cells were frozen. One of the cell lines, the RKPC-2, was selected and further characterized. RKPC-2 cells formed domes on impermeable supports, indicating fluid and solute transport. RKPC-2 cells formed continuous monolayers of low transepithelial resistance on collagen-coated filters. They were able to accumulate tetraethylammonium, an organic cation; however, no significant transcellular transport could be measured. We conclude that this cell line which shows characteristics of epithelial cells has maintained certain properties of intact proximal tubules, in particular the capacity to accumulate organic cations.