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.     

Reabsorption of fluorescein-isothiocyanate-labelled-ovalbumin in the kidney of normal and castrated male and female rats

The reabsorption of ovalbumin double labelled with fluorescein isothiocyanate (FITC) in the kidneys of normal and castrated male and female rats was investigated using fluorometry and fluorescence microscopy. The animals received an intravenous injection of either 2 or 7 mg fluorescein-thiocarbamyl (FTC)-ovalbumin per kilogram bodyweight (bw) and were killed 4 or 8 min post-injection. Animals injected with unlabelled ovalbumin (7.0 mg/kg bw) served as controls. Fluorescence microscopy revealed that FTC-ovalbumin was reabsorbed exclusively in the renal proximal tubule, the highest level of reabsorption being observed in its first part. Four and eight minutes after the injection, FTC-ovalbumin was only observed in apical reabsorption vacuoles, with lysosomes exhibiting no specific fluorescence. Fluorometric determinations for the renal homogenate supernatant showed that the renal reabsorption of FTC-ovalbumin was up to 24% higher in normal females than in normal males. Castration resulted in a significant increase in renal reabsorption in male rats (up to 38%; significant), whereas a minor decrease was observed in castrated females. The renal uptake differences in normal and castrated animals are discussed in the light of the sex-hormone-dependent catabolism of lysosomal proteins in the renal proximal tubule of rats.     

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.     

Reabsorption of fluorescein-isothiocyanate-labelled-ovalbumin in the kidney of normal and castrated male and female rats

Summary The reabsorption of ovalbumin double labelled with fluorescein isothiocyanate (FITC) in the kidneys of normal and castrated male and female rats was investigated using fluorometry and fluorescence microscopy. The animals received an intravenous injection of either 2 or 7 mg fluorescein-thiocarbamyl (FTC)-ovalbumin per kilogram bodyweight (bw) and were killed 4 or 8 min post-injection. Animals injected with unlabelled ovalbumin (7.0 mg/kg bw) served as controls. Fluorescence microscopy revealed that FTC-ovalbumin was reabsorbed exclusively in the renal proximal tubule, the highest level of reabsorption being observed in its first part. Four and eight minutes after the mjection, FTC-ovalbumin was only observed in apical reabsorption vacuoles, with lysosomes exhibiting no specific fluoreseence. Fluorometric determinations for the renal homogenate supernatant showed that the renal reabsorption of FTC-ovalbumin was up to 24% higher in normal females than in normal males. Castration resulted in a significant increase in renal reabsorption in male rats (up to 38%; significant), whereas a minor decrease was observed in castrated females. The renal uptake differences in normal and castrated animals are discussed in the light of the sex-hormone-dependent catabolism of lysosomal proteins in the renal proximal tubule of rats.Supported by the Deutsche Forschungsgemeinschaft (SFB 105)     

Sex dependence of clearance rates of aldosterone and its metabolites from plasma of intact rats.

Following I.V. injection of 3H-aldosterone, the rates of clearance of plasma 3H-radioactivity was demonstrated to be sex-dependent in intact rats. Even though the percentages of CH2Cl2-extractable plasma radioactivity are greater in female than in male rats, the quantities of CH2Cl2-extractable label are similar until 60 min post-injection. However, the quantities of non-extractable, polar metabolites of aldosterone (NEPD) are markedly greater in the plasma of males and rapidly reach peak levels 10 min post-injection of aldosterone. In females, these polar metabolites (NEPD) are rapidly cleared from the blood. After bile-duct cannulation, the rate of excretion of aldosterone radiometabolites was demonstrated to be rapid and sex-dependent. Within 1 hr., female rats excreted via the bile 82% of the injected dose of 3H-aldosterone, compared to 49% in male rats. In both sexes, greater than 95% of the total radioactivity excreted in the bile are non-extractable polar metabolites of aldosterone (NEPD). The sex hormones appear to influence not only the nature of metabolism of aldosterone in the liver, but also the rates of clearance of aldosterone and its metabolites from the plasma into the bile.     

Endocytosis mediated by the mannose receptor in liver endothelial cells. An immunocytochemical study

Immunocytochemical labeling of ultrathin cryosections from rat liver showed that mannose-terminated glycoproteins are removed rapidly from the blood stream mainly by the sinusoidal endothelial cells. The mannose-terminated glycoprotein ovalbumin was injected intravenously into rats 1 min, 6 min, and 24 min before perfusion fixation of the liver. Several minor and at least three major subcellular compartments were shown to be involved in the endocytic process. One minute after injection, ovalbumin was found at the cell surface, in coated pits, in coated vesicles, in tubular structures, and bound to the membrane of large early endosomes of which some showed a cisternal structure. After 6 min, ovalbumin was found in the lumen of large electron-lucent late endosomes and after 24 min in electron-dense structures, presumably lysosomes. The early endosomes have an ultrastructure which, together with the labeling pattern, indicates that this compartment has the same function as the CURL identified in parenchymal liver cells. The results are in accordance with recent biochemical findings indicating that ovalbumin endocytosed by endothelial cells is found sequentially in three different subcellular fractions depending on the time between injection and cooling for fractionation (G. M. Kindberg, T. Berg: Intracellular transport of endocytosed mannose terminated glycoproteins in rat liver endothelial cells. In: E. Wisse, D. L. Knook, K. Decker (eds.): Cells of the Hepatic Sinusoid. Vol. 2. pp. 120-124. Kupffer Cell Foundation. Rijswijk The Netherlands 1989).     

Role of apical tubules in endocytosis in nonciliated cells of the ductuli efferentes of the rat: a kinetic analysis

The apical region of nonciliated cells of the ductuli efferentes of the rat contains tubular coated pits (TCP) connected to the apical plasma membrane, apical tubules (AT) which occasionally show a partial coat, and endosomes which are often continuous with one or more apical tubules. To investigate the formation and fate of TCP and AT, a quantitative analysis was performed on the labeling indices of these structures at various time intervals (0.5-120 min) after a single injection of a tracer, cationic ferritin (CF), into the lumen of the rete testis. The labeling indices of both TCP and AT exhibited similar cyclical patterns, first reaching a peak at 25 min, then dropping to a minimum at 35 min, then rising to a second peak at 60 min. Since TCP were well labeled at 30 sec while AT were not, the tracer must rapidly enter TCP and thence AT. However, since tracer was virtually absent from the lumen by 30 min, it was not possible to reconcile the second peak of labeling index of TCP and AT by this mechanism. In another experiment, rats were injected once as before, injected again at 30 min, and then sacrificed at 30 min following the second injection. The results from this experiment showed that the labeling index of TCP and AT did not drop but was similar to that of the 60-min peak after a single injection. The interpretation is that there was recycling of tracer, which had already migrated from TCP to AT to endosomes, back to the apical plasma membrane via apical tubules. Moreover, when rats were injected once, injected again at 30 min, and sacrificed 3 min following the second injection, the labeling index for TCP and AT was significantly higher (P less than .05) than at the 30-min time interval after a single injection, indicating that recycled apical tubules were functionally capable of binding further CF. Morphological observations on images of transition between TCP and AT and the fact that AT were often found connected to endosomes suggest that TCP detach from the cell surface to give rise to AT, which in turn fuse to form endosomes. The kinetic analysis demonstrates in quantitative terms that a portion of the AT, which fuses to form endosomes, recycles back to the apical plasma membrane and contributes to the formation of new TCP.     

Neonatal maternal separation alters adult eyeblink conditioning and glucocorticoid receptor expression in the interpositus nucleus of the cerebellum

Neonatal maternal separation alters learning and memory. Glucocorticoids also modulate adult learning and memory, and neonatal maternal separation alters forebrain glucocorticoid receptor (GR) concentrations. We used eyeblink classical conditioning to assess the effect of neonatal maternal separation on associative learning. We assessed delay eyeblink conditioning, GR expression, and total neuron number in the interpositus nucleus, a critical site of plasticity in eyeblink conditioning, in adult rats that had undergone either standard animal facilities rearing, handling for 15 min, or maternal separation for either 15 or 60 min per day on postnatal days 2-14. At 2-3 months of age, delay eyeblink classical conditioning was assessed. Brains were processed for GR immunohistochemistry, and GR expression in the interpositus nucleus was assessed using a computer-based densitometry system. Neuron counts and nuclear volumes were obtained from an alternate series of thionin-stained sections. Maternal separation significantly impaired eyeblink conditioning in male but not female rats. Handling and maternal separation did not significantly affect interpositus neuron number and volume. However, prolonged maternal separation significantly increased GR expression in the posterior interpositus in males, and increases were correlated with eyeblink conditioning. In female rats, maternal separation and handling did not significantly alter interpositus neuron number, volume, or GR protein expression, and GR expression did not correlate with eyeblink conditioning. Thus, neonatal maternal separation produces adult deficits in eyeblink conditioning and alterations in GR expression in its neural substrate in a sex-dependent manner.     

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.     

T cell responses to streptococcal antigens in rats: relation to susceptibility to streptococcal cell wall-induced arthritis

In order to investigate the immunological mechanism of the chronic phase of streptococcal cell wall (SCW)-induced arthritis in Lewis rats, we compared the SCW-specific T cell response in arthritis-susceptible (female Lewis) and resistant (F344) rats. We present evidence that this T cell response is absent in F344 rats, while it is clearly present in Lewis rats. The T cell response was analyzed both in the spleen and in lymph nodes. In addition, we show, that injection of SCW in the F344 rat induces a general unresponsiveness in this strain: the response to mitogen was severely suppressed in SCW-injected F344 rats and, furthermore, when SCW was coinjected with ovalbumin, the response to ovalbumin was depressed. The fact that priming with ovalbumin alone induces a normal response in the F344 rat to both mitogen and ovalbumin implies that the observed abnormality after SCW priming is not a general immunological defect in this strain. Additionally, we demonstrate that adherent cells of both Lewis and F344 exert negative effects on an in vitro T cell response after injection with SCW, and that F344-adherent cells are more potent in this effect. Removal of OX8-positive cells leads to a restoration of the SCW-specific T cell response in SCW-injected F344 rats, indicating that the expression of this response is controlled by (SCW-specific?) suppressor T cells. Our results provide suggestive evidence for the obligatory role of SCW-specific T cells in the expression of chronic joint inflammation after systemic injection of SCW.     

Renal filtration and reabsorption of GFP in <Emphasis Type="Italic">Rana temporaria</Emphasis>: 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 cells of proximal tubules 30 min after intravenous GFP injection. The GFP fluorescence was located predominantly in the apical part of the cytoplasm in the form of 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 sack decreased significantly the GFP absorption. The effect of arginine-vasotocin was inhibited by pretreatment with an antagonist of vasopressin V_i-receptors. The results of this work together with literature data allow believing that a decrease in the GFP absorption in the frog kidney under effect of arginine-vasotocin is due to a fall of the AVT-dependent glomerular filtration rate and to a decrease in the input of protein into the lumen of tubules. The action of arginine-vasotocin seems to be mediated via the V_i-like receptors of preglomerular blood vessels.     

Sequential passage of alpha2mu-globulin through the hepatic endoplasmic reticulum and Golgi apparatus during secretion.

Studies on the synthesis and secretion of the sex-dependent urinary protein, alpha2mu-globulin, have been extended by establishing its sequential passage from the rough to the smooth endoplasmic reticulum and Golgi-rich fractions of the liver of adult male rats. After injection of 14C-labeled amino acids, the maximum radioactivity of alpha2mu occurred at 20 min in the rough, 25 min in the smooth microsomes and 30 or 35 min in the Golgi-rich fractions. Radioactive alpha2mu-globulin appeared in the bloodstream and kidneys after a lag of 20--25 min. Results indicate that alpha2mu-globulin follows a secretory pathway similar to that of serum albumin.     

Frequency of cocaine administration affects behavioral and endocrine responses in male and female Fischer rats.

Accumulating evidence has shown disparate behavioral responses to cocaine in male and female rats. To date, there is a lack of understanding of how cocaine administration frequency affects sexually dimorphic behavioral responses. In the present study we investigated the behavioral and endocrine responses to single (1 x 15 mg/kg) and "binge" (3 x 15 mg/kg) cocaine administration in male and female Fischer rats. Overall, females showed a more prolonged and robust behavioral response to both acute and "binge" pattern cocaine administration. Furthermore, sex-dependent behavioral topographies emerged during binge-pattern cocaine administration; female rearing activity increased across "binge" injections while ambulatory activity decreased. In contrast, male ambulatory and rearing behaviors remained constant across injections of "binge" cocaine. At the hormonal level, both single and "binge" pattern cocaine administration decreased testosterone levels in male rats. However, cocaine's modulation of testosterone levels was transient since testosterone levels were decreased by cocaine 30 min but not 3 hr following a single injection. In both male and female rats, "binge" cocaine increased plasma progesterone levels. However, acute cocaine administration increased progesterone levels transiently in only female rats. Our results show that pattern of administration affects both cocaine-stimulated behavioral and endocrine responses in male and female rats.     

The glomerular mesangium: a comparative study of mesangial handling of iron dextran complex and endogenous IgG in mice and rats

The function and channel system of the glomerular mesangium in mice and rats was investigated by studying the uptake and transport of intravenously injected iron-dextran particles, and the localization of endogenous IgG. Animals were killed at 30 min, 8 h, 1 and 3 days and 1 and 2 weeks after intravenous injection of iron dextran complex. It was found that the tracer was present maximally in the mesangium of the mouse at one day after injection whereas a maximum was not reached until the third day in the rat. Maximal levels of tracer particles in the extra-glomerular lacis area were found at three days in the mouse and at 2 weeks in the rat. Disappearance of the tracer from the blood as indicated by the measured serum iron levels did not seem to differ significantly in the two species. Using an ultrastructural immunoperoxidase technique, considerable amount of endogenous IgG were localized in the mesangial channel system in the stalk region and in the extraglomerular lacis area of mice, whereas in rats only very scanty endogenous IgG was present in these locations. It is suggested that the difference in mesangial handling of macromolecular material in mice and rats is more likely to be due to a different rate of transport through the mesangial channel system than to primary differences in mesangial phagocytotic activity.     

The fluorometric assay of rat plasma corticosterone: evaluation of nonspecific fluorescence

An analysis of 168 plasma samples from intact rats, one to 35 days of age, was performed using both brief and specific fluorometric procedures. The amount of fluorescence produced by the brief procedure which could be attributed to corticosterone ranged from a maximum of 72% to a minimum of 16% of the total fluorescence value. Corticosterone represented 50% or more of the brief assay value in only five out of 18 groups of animals assayed. Following statistical analysis of the nonspecific fluorescence, a significant variation was found due to the age of the animal. A highly significant increase in nonspecific fluorescence was found in 21-day old animals following histamine injection. It was concluded that the brief fluorometric assays for corticosterone were of little value if specificity was desired.     

Uptake of exogenous protein tracer in cells of the rat renal papilla

In order to study the phagocytic potential of different cell types of the rat renal papilla with special emphasis on interstitial cells, horseradish peroxidase (HRP) (8 mg/100 g body weight) was injected intravenously into adult rats. The distribution of peroxidase was studied in animals perfusion-fixed 60 and 180 min after injection and was found to be similar after both time intervals. The epithelial cells of the collecting ducts took up the largest amounts of the tracer. HRP was mainly located in large lysosome-like bodies in the basal part of the cytoplasm, suggesting peritubular uptake from the interstitial space. However, small amounts of the tracer were also seen in apical vesicles close to the luminal plasma membrane. The interstitial cells of peroxidase-injected animals were ultrastructurally altered and had large irregular invaginations of the cell membrane. The cells had taken up only small amounts of the tracer which were located in small round lysosome-like bodies. Thus, the interstitial cells displays no macrophage characteristics, either in the native state or when challenged with an extracellular protein.     

Prenatal exposure to valproate induces sex-, age-, and tissue-dependent alterations of cholesterol metabolism: Potential implications on autism

Here, we investigated the protein network regulating cholesterol metabolism in the liver and brain of adolescent and adult male and female rats prenatally exposed to valproate (VPA), a well validated experimental model of autism spectrum disorders (ASD). We were aimed at studying whether prenatal VPA exposure affected the proteins involved in cholesterol homeostasis in a sex-dependent manner. To this aim the protein network of cholesterol metabolism, in term of synthesis and plasma membrane trafficking, was analyzed by western blot in the liver and different brain areas (amygdala, cerebellum, cortex, hippocampus, nucleus accumbens, and dorsal striatum) of adolescent and adult male and female rats prenatally exposed to VPA. Our results show that physiological sex-dependent differences are present both in the liver and in brain of rats. Interestingly, VPA affects specifically the brain in an age- and region-specific manner; indeed, cerebellum, cortex, hippocampus and nucleus accumbens are affected in a sex-dependent way, while this does not occur in amygdala and dorsal striatum. Overall, we demonstrate that each brain area responds differently to the same external stimulus and males and females respond in a different way, suggesting that this could be related to the diverse incidences, between the sexes, of some neurodevelopmental pathologies such as autism, which displays a 3:1 male to female ratio.     

A selective fluorescence reaction for peptides and chromatographic analysis

A novel and selective fluorescence reaction is proposed for the quantitative determination of peptides by reversed-phase liquid chromatography (RPLC). A single fluorescent product was formed when a peptide was heated at 120 degrees C for 20 min in a neutral aqueous medium (pH 7.0) containing catechol, sodium periodate, and sodium borate. The fluorescent products of four peptides such as Leu-Gly, Ala-Leu-Gly, Tyr-Gly-Gly-Phe-Leu, and Leu-Leu-Leu were easily separated on a reversed-phase column by gradient elution of methanol in a mobile phase containing sodium borate (pH 7.0), and then quantitatively detected by fluorimetry. The lower limits (S/N=3) of the detection for the tested peptides were 0.5-1.0 pmol per an injection volume (40 microl). In addition, the fluorescent products of phenylalanine amide and Leu-Leu-Leu were identified by electrospray ionization-time of flight-mass spectrometry (ESI-TOF/MS) for the elucidation of their chemical structures.     

Uptake of exogenous protein tracer in cells of the rat renal papilla

Summary In order to study the phagocytic potential of different cell types of the rat renal papilla with special emphasis on interstitial cells, horseradish peroxidase (HRP) (8 mg/100 g body weight) was injected intravenously into adult rats. The distribution of peroxidase was studied in animals perfusion-fixed 60 and 180 min after injection and was found to be similar after both time intervals. The epithelial cells of the collecting ducts took up the largest amounts of the tracer. HRP was mainly located in large lysosome-like bodies in the basal part of the cytoplasm, suggesting peritubular uptake from the interstitial space. However, small amounts of the tracer were also seen in apical vesicles close to the luminal plasma membrane. The interstitial cells of peroxidase-injected animals were ultrastructurally altered and had large irregular invaginations of the cell membrane. The cells had taken up only small amounts of the tracer which were located in small round lysosome-like bodies. Thus, the interstitial cells displays no macrophage characteristics, either in the native state or when challenged with an extracellular protein.Supported by Karolinska Institutet and the Swedish Medical Research Council (proj. no. 05937)