[EFFECT OF DIFFERENT PROTEINS ON THE REABSORPTION OF YELLOW FLUORESCENT PROTEIN IN THE KIDNEY OF BROWN FROG RANA TEMPORARIA].

Protein reabsorption in the proximal tubules (PT) of the frog kidney was studied by the methods of immunohistochemistry, fluorescent and confocal microscopy. Yellow fluorescent protein (YFP) was introduced in combination with other proteins. Reabsorption of YFP introduced simultaneously with ly- sozyme or green fluorescent protein (GFP) did not differ from the result of YFP injection only. Previous lysozyme injection did not change YFP absorption in contrast to YFP uptake reduced after GFP pretreat- ment. Lysozyme loading for 4 days resulted in a significant reduction in YFP absorption. The results show that receptor-mediated endocytosis in the frog kidney depends on the molecular nature of absorbable ligands, conditions of their competitive absorption and lysosomal accumulation in epithelial PT cells.

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 (endosomes), 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 intracellular 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.

[Reabsorption of yellow fluorescent protein in the Rana temporaria kidney by receptor-mediated endocytosis].

The absorption of yellow fluorescent protein (YFP) and the expression of the endocytic receptors, megalin and cubilin, were investigated in the renal proximal tubules (PT) in frogs Rana temporaria after parenteral YFP injections. The methods of confocal microscopy and immunohistochemistry were used. The dynamics of YFP absorption was analyzed 2 h after injection. The logarithmic time dependence of the accumulation of YFP-containing endocytic vesicles in PT cells and the completion of absorption process 90-120 min after injection were shown. Unlike substantial megalin and cubilin expression 15-30 min after YFP introduction, immunolabeled endocytic receptors were not detected in PT cells after 2 h. The re-injection of YFP led to the appearance of apical endocytic vesicles containing megalin or cubilin colocalized with YFP. At the same time, the decrease of YFP uptake associated with reduction in the number of receptor-containing vesicles was demonstrated, suggesting a failure of megalin and cubilin expression. The decrease of absorption capacity of PT cells after YFP re-injection was similar to that found previously under conditions of the competitive absorption of green fluorescent protein (GFP) and YFP injected in different sequences. The data are the further demonstration of the proposed mechanism limiting the tubular protein absorption in the frog kidney and suggest the involvement of megalin and cubilin in uptake and vesicular transport of YFP.

Absorption capacity of renal proximal tubular cells studied by combined injections of YFP and GFP in Rana temporaria L.

The capacity for protein reabsorption in the renal proximal tubule (PT) was studied in Rana temporaria frogs by separate, simultaneous and sequential introduction of yellow fluorescent protein (YFP) and green fluorescent protein (GFP). The uptake patterns of YFP and GFP in PT epithelial cells were investigated 15-120min after their bolus intravenous and intraperitoneal injection. As shown by confocal microscopy, the tubular uptake of YFP and GFP was time- and dose-dependent. These proteins are absorbed in similar way and can be accumulated in the same endocytic vesicles after their combined injections. When GFP was injected 30 and 90min before YFP, and vice versa, the number of vesicles with pre-injected protein increased and the percentage of vesicles with colocalized GFP and YFP reduced. At the same time, the uptake rate of a protein injected later progressively and significantly decreased. Subcellular localization of endocytic receptors, megalin and cubilin, in renal PT cells after intravenous YFP introduction were revealed by immunofluorescent microscopy. Colocalization of internalized YFP with megalin or cubilin in the endocytic vesicles was demonstrated. The data suggest the possibility of protein uptake by receptor-mediated endocytosis and the existence of a mechanism limiting the protein absorption rate in wintering frogs.

[Morpho-functional changes in small intestine epithelium of frog Rana temporaria during hibernation].

Structure and function of small intestinal epithelium were studied in overwintering frogs Rana temporaria at various stages of hibernation. In the process of testing of absorption of arginine vasotocin (AVT) in experiments in vitro it is established that at the period of hibernation there is preserved the capability of the epithelium for absorption of this nonapeptide without hydrolysis. However, as compared with October-December, in January-February and later, a decrease of the AVT absorption takes place, which is the most pronounced in March-April. Changes in epithelial structures appear by the middle of winter and are progressing by spring. In April-May, as compared with the beginning of hibernation, the height of enterocytes, the length of microvilli, and the number of microvilli decrease by 33 %, 40 %, and 57 %, respectively. The absence of features of destruction indicates an adaptive character of the observed changes. Dynamics of the studied parameters indicates morphological plasticity of the small intestine epithelium of R. temporaria at the period of hibernation.

[Absorption of arginine-vasopressin and arginine-vasotocin in small intestine of the frog Rana temporaria].

Experiments in vitro demonstrated a partial absorption of arginine-vasopressin (AVP) in the frog small intestine. Dynamics and efficiency of the nonapeptide absorption are studied with use of hydroosmotic method of recording of the osmotic permeability of the frog urinary bladder epithelium and immunoenzyme analysis. In the process of absorption there were preserved intactness of the hormone cyclic structure and its physiological activity, like in the case of the arginine-vasotocin (AVT) absorption. The AVP absorption increased at its administration into the gut with inhibitor of proteases. By methods of immunoelectron and immunofluorescent microscopy with use of polyclonal antibody to AVP, location of the label to the hormone was shown in the enterocyte cytoplasm. Thus, there was obtained a morphological evidence for the AVP absorption and transepithelial transfer in the frog small intestine. These data enlarge the concept of the poorly studied properties of the absorbing epithelium of the vertebrate intestine with respect to absorption of intact molecules of polypeptides.

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.

[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.

[Green fluorescent protein absorption and accumulation in the renal proximal tubule cells after its increased entry into blood].

The uptake of green fluorescent protein (GFP) by the proximal renal tubules was studied in the anaesthetized rats using laser confocal microscopy after GFP intravenous injection or administration into the small intestine lumen. The specific green fluorescence revealed in the proximal tubule cells after intravenous injection correlated with the logarithm of GFP dose injected intravenously (r = 0.96, p < 0.05). GFP fluorescence after its intravenous injection was higher than that one after GFP infusion into the small intestine (p < 0.05). Following the increase of injected GFP dose, the epitheliocyte cytoplasm, in addition to diffuse fluorescence, demonstrated large intensely fluorescent vesicles, that was confirmed by a graphical analysis. The reported changes in the intensity and pattern of specific fluorescence indicate the enhancement of GFP absorption by the cells of proximal tubules and GFP accumulation in the intracellular compartments during its increased entry into circulation.

[The effect of glucose on antidiuretic hormone absorption in the rat and frog small intestine].

Administration of 5 ml/100 g body weight of 1% glucose solution to stomach produced the same diuretic kidney response in fasted Wistar rats as administration of the same amount of water. Intragastric administration of arginine vasopressin along with the water load evoked an antidiuretic response. Arginine vasopressin in the same volume of glucose induced no kidney response difference as compared with the hormone action in experiments with water load. 0.1 nmol of arginine vasotocin, having been itroduced into the rat isolated ileum, prevented the effect of glucose on the hormone absoption. 0.1 nmol of arginine vasotocin, having been introduced into the frog isolated ileum along with isotonic glucose solution, increased the hormone absorption; fructose did not affect this process whereas mannitol decreased absorption ofarginine vasotocin. This absorption was also reduced by intraileal introduction of arginine vasotocin with the hypotonic Ringer solution. The findings suggest that glucose in the rat gastrointestinal tract does not affect arginine vasopressin absorption in vivo, whereas in the frog ileum glucose increases arginine vasotocin absorption in vitro.

[Immunocytochemical localization of vasopressin at its absorption by cells of rat small intestine]. / Immunotsitokhimicheskaia lokalizatsiia vazopressina pri ego vsasyvanii kletkami épiteliia tonkoi kishki krysy.

Morpho-physiological characteristics of the transport of cyclic nonapeptide arginine vasopressin (AVP) across the rat intestinal epithelium was studied in experiments in vitro. A partial absorption of physiologically active AVP was followed when filling the isolated intestinal lumen by hormone solution. By methods of immunoelectron and immunofluorescence confocal microscopy, using polyclonal anti-AVP antibodies, cytoplasmic localization of AVP label was shown in enterocytes. The AVP label was also observed in the intercellular space in the basal area of epithelium. No label was revealed in the intercellular junctions, and no predominant label accumulation was found in any cytoplasmic structures of the epithelial cells. The obtained results are considered as evidence for the transcellular pathway of partial AVP absorption in rat small intestine.

[Antidiuretic reaction of the human and rat kidney to peroral administration of arginine-vasopressin and desmopressin]. / Antidiureticheskaia reaktsiia pochek cheloveka i krysy pri peroral'nom vvedenii arginin-vazopressina i desmopressina.

Water in amount of 5 ml/100 g body weight was administered through a gastric probe into the stomach in alert rats; subjects-volunteers drank 20 ml of water per 1 kg of body weight. This resulted in diuresis at the peak of which the excreted water fraction reached 23% in rats and 12.4% in human subjects, whereas excretion of the osmotically free water amounted to 0.103 +/- 0.018 ml/min/100 g body weight and 10.0 +/- 1.8 ml/min/1.73 m2 of the body surface, respectively. These data indicate a practically complete inhibition of the arginine vasopressin secretion. On intragastric administration of 10 micrograms of arginine vasopressin or 0.2 microgram of desmopressin, with water in rats, a prolonged and quite obvious antidiuretic response occurred, with a marked increase of reabsorption of the osmotically free water in kidneys. A direct correlation has been found between the dose of the intragastrically administered vasopressin in the dose range from 0.1 to 10 micrograms/100 g body weight and a decrease of clearance of the osmotically free water. In subjects volunteers, an antidiuretic reaction to administration of 0.2 mg of desmopressin with water, was found. The data obtained provide a direct proof of intestinal absorption of nanopeptides without loss of their physiological activity. Significance of the data obtained for physiology of digestion and for clinical medicine, is discussed.

[Increase of water permeability and recovery of water impermeability of the frog urinary bladder]. / Dinamika uvelicheniia osmoticheskoi pronitsaemosti i vosstanovleniia vodonepronitsaemosti mochevogo puzyria liagushki.

The experiments carried out on the urinary bladder of the frog Rana temporaria L. have shown that the period of recovery of water impermeability after an increase of osmotic water permeability induced by arginine-vasotocin, desmopressin, or cAMP depends on the degree of increase of the osmotic permeability but not on the nature of the substance stimulating the increase of osmotic water permeability. The removal of the hormone in the absence of autacoids fails to recover the water impermeability. After cessation of the vasotocin action the water permeability decrease is delayed if phospholipase A2 and cyclooxygenase are inhibited by qiunacrine and voltaren, respectively. An agonist of V1-receptors has no effect on dynamics of the recovery of water impermeability. This recovery has been shown to depend on PGE2 concentration in the serosal solution after the hormone removal. The obtained results indicate that decrease of water permeability depends not only on removal of vasotocin or cAMP but also on involvement of autacoids.

Possible role of prostaglandins in pathogenesis of nocturnal enuresis in children.

OBJECTIVE: To compare excretion of ions and prostaglandins by the kidney in children with noctural enuresis. MATERIAL AND METHODS: Thirty-two children with primary nocturnal enuresis and 23 normal children were examined. Osmolality and sodium and potassium concentrations were measured in their urine and blood serum. Prostaglandins E2, E1, and F2alpha were determined using kits for immunoenzyme analysis. Luminal and contraluminal prostaglandin secretions were studied in frog urinary bladder. RESULTS: Children with nocturnal enuresis have increased nocturnal diuresis and renal sodium excretion, but no increase was found in excretion of prostaglandins E2, E1, and F2alpha. Administration of sodium diclofenac before bed-time eliminated episodes of nocturnal enuresis in 37% of children; intranasal administration of Adiuretin SD had a positive effect in 69% of enuretics. In children with nocturnal enuresis there is a correlation between renal excretion of PGE2 and sodium ions; this correlation is absent in the control group children, and disappears in enuretics treated with desmopressin. To evaluate the representativeness of the data on prostaglandin secretions to urine as compared with their release to extracellular fluid, experiments on frog urinary bladder were performed: a correlation was found between prostaglandin secretion to the urinary bladder lumen and to the extracellular fluid. CONCLUSIONS: The results of the study suggest that changes in renal function are due not to a higher secretion of prostaglandins in nocturnal enuresis but to the relative dominance of their effect as compared with other physiologically active substances that simultaneously act on renal tubular cells.

[Functional role of V1- and V2-receptors of the apical and basolateral membranes of the frog bladder epithelial cells]. / Izuchenie funktsional'noi roli V1- i V2-retseptorov apikal'noi i bazolateral'noi membran kletok épiteliia mochevogo puzyria liagushki.

Arginine vasotocin, 0.02--1 nM, increases osmotic water permeability of frog urinary bladder, arginine vasotocin after a simultaneous addition to the mucosal and serosal Ringer solutions rises the water permeability to a lesser degree than on the hormone addition only to the serosal solution. 1 nM remestyp, an agonist of V1-receptors, from the apical membrane decreases the hydroosmotic effect of arginine vasotocin added to the serosal Ringer solution. When added to the mucosal solution, combination of the same concentrations of arginine vasotocin and SR 49059, an antagonist of V--receptors, or desmopressin, agonist of V2-receptor alone, increases the effect of the same concentration of arginine vasotocin added to the serosal solution. 1 nM arginine vasotocin at the luminal membrane increases secretion into the Ringer solution of prostaglandin E, and prostaglandin E1 but not of prostaglandin F2 alpha. The data obtained indicate the presence of the arginine vasotocin receptors responsible for the hydroosmotic effect only in the basolateral membranes, while arginine prostaglandin E, participation is shown in modulation of the arginine vasotocin effect.

[Mechanism f kidney participation in maintaining osmotic and ion homeostasis in chronic renal failure]. / Mekhanizm uchastiia pochki v obespechenii osmoticheskogo i ionnogo gomeostaza pri khronicheskoi pochechnoi nedostatochnosti.

In examination of patients with chronic renal failure (CRF) at glomerular filtration rate below 30 ml/min and blood serum ion concentration within limits of normal values hyperosmia has been found. Under the natural regimen essential differences have been revealed neither in variation limits of renal excretion of ions nor osmotically active substances in CRF patients as compared with healthy controls. Diuresis correlated with renal excretion of osmotically active substances. It is shown that a decrease in reabsorption of osmotically active substances depends on secretion and excretion of prostaglandin E2. A suggestion is made about the role of prostaglandins in regulation of renal tubular function at terminal CRF stages.