Effect of oleamide on water and sodium ion transport in osmoregulatory organs of vertebrates

In the frog Rana temporaria L., oleamide solution (10 μmole/L) applied to the isolated basal surface of the skin augmented the short-circuit current (SCC) from 59.8 ± 2.5 to 78.2 ± 1.4 μA/cm². When applied to the serous membrane of the urinary bladder, oleamide (1 μmole/L) induced more than a 30-fold increase in osmotic water permeability. The addition of argininevasotocin against the background of oleamide further increased SCC across the skin and osmotic water permeability in the bladder. In Wistar rats, intraperitoneal injection of oleamide (0.1 μmole/L per 100 g of body weight) to non-anesthetized animals after water load reduced diuresis by 22% and increased solute-free water reabsorption and urinary sodium excretion by 31% and 55%, respectively, but did not affect urinary potassium excretion. These findings provide evidence of the similarity between the effects of oleamide and nonapeptide neurohypophyseal hormones on water and ion transport in epithelial cells of osmoregulatory organs in vertebrates.     

Effects of exendin-4 and selenium on the expression of GLP-1R,IRS-1, and preproinsulin in the pancreas of diabetic rats

The mechanisms by which exendin-4 and selenium exert their antidiabetic actions are still unclear. Here, we investigated the effects of exendin-4 or selenium administration on the expression of glucagon-like peptide-1 receptor (GLP-1R), insulin receptor substrate-1 (IRS-1), and preproinsulin in the pancreas of diabetic rats. Diabetes was induced by streptozotocin administration. Diabetic rats were injected intraperitoneally with 0.03 μg exendin-4/kg body weight/daily or treated with 5 ppm selenium in drinking water for a period of 4 weeks. GLP-1R and IRS-1 levels were decreased while the level of preproinsulin messenger RNA (mRNA) was increased in the pancreas of diabetic untreated rats, as compared to that in control rats. Treatment of diabetic rats with exendin-4 increased protein and mRNA levels of GLP-1R, and IRS-1, and the mRNA level of preproinsulin in the pancreas, as compared to their levels in diabetic untreated rats. Selenium treatment of diabetic rats increased the pancreatic mRNA levels of GLP-1R, IRS-1, and preproinsulin. Exendin-4 or selenium treatment of diabetic rats also increased the numbers of pancreatic islets and GLP-1R molecules in the pancreas. Therefore, exendin-4 and selenium may exert their antidiabetic effects by increasing GLP-1R, IRS-1, and preproinsulin expression in the pancreas and by increasing the number of pancreatic islets.     

The mechanism of glucagon-like peptide-1 participation in the osmotic homeostasis

We have found the physiological mechanism of intensification of the excessive fluid removal from the body under the action of glucagon-like peptide-1 and its analog exenatide. Under the water load in rats, exenatide significantly increased the clearance of lithium, reduced fluid reabsorption in the proximal tubule of the nephron and intensified reabsorption of sodium ions in the distal parts, which contributed to the formation of sodium-free water and faster recovery of osmotic homeostasis. Blocking this pathway with a selective antagonist of glucagon-like peptide-1 receptors slowed down the elimination of excessive water from the body.     

Effect of crude extract of Stevia rebaudiana on renal water and electrolytes excretion.

To evaluate the effect of crude extract of Stevia rebaudiana on renal water, Na+ and K+ excretion, male Wistar rats (250-350 g each) under antidiuresis or water diuresis conditions, were evaluated. During intravenous infusion of the extract (0.05 mg/min/100 g) no significant differences were detected in mean arterial pressure or renal hemodynamics parameters. In contrast, fractional water and sodium excretion and solute clearance increased significantly, in both groups of animals. In antidiuresis rats the extract significantly increased reabsorption of water by the collecting duct and in water diuresis animals the extract significantly increased free water clearance. The data suggest preferential action of the extract in the proximal tubular cells involved with salt transport mechanism.     

The effect of intravenous hypertonic saline infusion on renal function and vasopressin excretion in sheep.

Conscious Merino ewes were given an intravenous hypertonic sodium chloride load of 4 mmol.min-1 for 100 min. This resulted in increases in urine flow, sodium and potassium excretion and plasma sodium concentration and osmolality. Urinary vasopressin output and solute-free water reabsorption increased and plasma renin activity declined. Renal plasma flow and glomerular filtration rate (GFR) rose, as did the solute clearance. The change in urinary osmolality was related to the initial urine osmolality such that when the initial urine osmolality was high the urine became more dilute, and vice versa. Tubular sodium reabsorption increased but the fractional reabsorption rate fell. It is suggested that the increase in GFR was at least partly due to the increase in AVP and that the electrolyte loss can be accounted for by the increase in GFR without necessarily involving AVP or other hormonal effects at the tubular level.     

Exenatide can reduce glucose independent of islet hormones or gastric emptying

Exenatide is a long-acting glucagon-like peptide-1 (GLP-1) mimetic used in the treatment of type 2 diabetes. There is increasing evidence that GLP-1 can influence glycemia not only via pancreatic (insulinotropic and glucagon suppression) and gastric-emptying effects, but also via an independent mechanism mediated by portal vein receptors. The aim of our study was to investigate whether exenatide has an islet- and gastric-independent glycemia-reducing effect, similar to GLP-1. First, we administered mixed meals, with or without exenatide (20 microg sc) to dogs. Second, to determine whether exenatide-induced reduction in glycemia is independent of slower gastric emptying, in the same animals we infused glucose intraportally (to simulate meal test glucose appearance) with exenatide, exenatide + the intraportal GLP-1 receptor antagonist exendin-(9-39), or saline. Exenatide markedly decreased postprandial glucose: net 0- to 135-min area under the curve = +526 +/- 315 and -536 +/- 197 mg.dl(-1).min(-1) with saline and exenatide, respectively (P < 0.05). Importantly, the decrease in plasma glucose occurred without a corresponding increase in postprandial insulin but was accompanied by delayed gastric emptying and lower glucagon. Significantly lower glycemia was induced by intraportal glucose infusion with exenatide than with saline (92 +/- 1 vs. 97 +/- 1 mg/dl, P < 0.001) in the absence of hyperinsulinemia or glucagon suppression. The exenatide-induced lower glycemia was partly reversed by intraportal exendin-(9-39): 95 +/- 3 and 92 +/- 3 mg/dl with exenatide + antagonist and exenatide, respectively (P < 0.01). Our results suggest that, similar to GLP-1, exenatide lowers glycemia via a novel mechanism independent of islet hormones and slowing of gastric emptying. We hypothesize that receptors in the portal vein, via a neural mechanism, increase glucose clearance independent of islet hormones.     

Effect of pentoxifylline on CdCl2-induced nephrotoxicity in the rat

We developed a rat model of cadmium (Cd)-induced nephrotoxicity and tried to prevent renal damage by treating the animals with pentoxifylline (PTX). Sprague-Dawley (SD) rats given CdCl₂ 3.0 mg/kg sc, daily for 2 wk showed evidences of renal proximal tubular damage, including significant increases in urine volume, urinary excretion ofN-acetyl-β-D-glucosaminidase (NAG), alanine aminopeptidase (AAP), and fractional excretion of sodium (FENa), and a decrease in the percentage of tubular reabsorption of phosphate (%TRP). PTX significantly improved the urinary excretion of NAG and %TRP. Urine volume was increased threefold in the CdCl₂-treated rats and fivefold in the Cd+PTX-treated rats, respectively, as compared with saline-treated control. Total protein, AAP, and creatinine clearance, showed no change after PTX administration. Concentration of Cd in the renal cortex was three times higher than that in the renal medulla, but there were no differences in concentration between the Cd-treated rats and the Cd+PTX-treated rats. Our animal model was useful in studying the renal tubular damage produced by cadmium. PTX appears useful for improving the nephrotoxicity of Cd.     

Altered reabsorption of protein by the renal cortex in rats treated with hypertonic saline or mannitol.

The reabsorption of horseradish peroxidase (HRP) by the proximal tubule cells of rat kidneys was investigated by measuring the concentration of HRP in total particulate fractions of the cortex 1/4 and 1 hr after intravenous injection, and by correlated cytochemical observations. When compared to the corresponding values of the control animals, the concentration of HRP 1 hr after injection was decreased approximately 10-fold in the renal cortex of rats which had received an intravenous injection of hypertonic saline or two subcutaneous injections of mannitol. The plasma clearance and the urinary excretion of HRP were not altered significantly after injection of hypertonic saline, but the plasma clearance was decreased and the urinary excretion increased after injection of mannitol. When the dose of injected HRP was varied, the reabsorption of HRP by the renal cortex was proportional to the dose in the experimental and the control animals. Cytochemical staining for peroxidase activity also showed that the phagosomes and phagolysosomes of the proximal tubule cells contained much less peroxidase in the experimental rats than in the control rats. After injection of mannitol, large vacuoles appeared in the proximal tubule cells. The vacuoles often contained peroxidase-positive granules (phagosomes) which varied in diameter from the limit of microscopic visibility up to several microns. Most of the vacuoles did not react for acid phosphatase activity, but lysosomes were often aggregated around the vacuoles and seemed to release acid phosphatase into the cytoplasm. Certain analogies between the reabsorption of protein and that of water by the proximal tubule cells are discussed.     

The role of the kidneys in the regulation of osmolality and concentrations of cations in the blood serum in hyperglycemia

The role of the kidneys in the restoration of osmotic and ionic homeostasis during persistent hyperosmia caused by hyperglycemia was analyzed. The study was performed in children with diabetes with a disease duration of five months to 17 years. The physical and chemical parameters of their blood serum (such as the osmolality and the concentrations of Na, K, Ca, and Mg) were within standard ranges. Hyperglycemia was shown to be accompanied by decreased concentrations of sodium (r = ?0.791, p < 0.01) and magnesium (p < 0.001) in the blood serum. The calculated clearance of sodium-free water indicates that the compensatory reaction is based on greater water reabsorption with increased diuresis. The regulation of water-salt balance keeps the serum potassium and calcium at the levels of the control group. The results indicate that the stability of the cell volume is the predominant aim of the human regulatory system and that increased renal reabsorption of sodium-free water is a physiological compensatory mechanism in hyperglycemia.     

Diet and water-salt balance in rats

We compared parameters of water-salt balance in Wistar female rats fed normal chows during more than 2 weeks. Potassium content was 1.4-fold higher in diet I than in diet II, and sodium end water content was 3.3- and 7.5-fold higher in diet II than in diet I. Blood osmolality and concentration of Na+, K+, Mg2+ were equal in rats fed different chow. In water-loaded rats (5 ml of water/100 bw per os) fed different chow, urine flow rate did not differ, but solute-free water excretion was higher by 40.2% in the rats fed diet II vs. diet I. The sort of diet did not affect the renal sodium excretion during oral administration of 5 ml 0.9% NaCl per 100 g bw to rats. After vasopressin injection solute-free water reabsorption was 1.5-fold higher in rats fed diet II. Natriuretic and hydruretic effect of exenatide, glucagon-like peptide 1 mimetic, was weaker in rats fed diet I. The data obtained indicate that organism can effectively maintain blood parameters. The modulation of hormone regulatory effects on water and sodium balance was found to depend on the state of organism under diet consumed continuously.     

rs11567842 SNP in <Emphasis Type="Italic">SLC13A2</Emphasis> gene associates with hypocitraturia in Thai patients with nephrolithiasis

Hypocitraturia is a profound risk for kidney stone formation and recurrence. Sodium-dicarboxylate cotransporter-1 (NaDC-1) is a main transporter responsible for citrate reabsorption in renal proximal tubules. To investigate an association of sodium-dicarboxylate cotransporter-1 (NaDC-1) polymorphism with hypocitraturia in Thai patients with nephrolithiasis (NL). Exonic SNPs in NaDC-1 were screened in peripheral blood DNA of 13 NL patients. The rs11567842 (A/G) variant was found and further genotyped in 145 NL patients and 115 non-stone forming controls. NL patients had significantly lower level of urinary citrate than the controls. Based on logistic regression, hypocitraturia was significantly associated with urinary stone formation (adjusted OR 8.34, 95% CI 4.63–15.04). Significant association of urinary citrate level with rs11567842 genotype was found only in the NL group. NL patients with GG genotype had significantly higher urinary citrate than those with AA and AG genotypes. GG carrying patients had significantly reduced risk for hypocitraturia (adjusted OR 0.15; 95% CI 0.05–0.48, AA as reference). In selected 15 calcium oxalate stone patients, AA carriers had significantly higher intrarenal NaDC-1 mRNA than GG and AG carriers. Homozygous GG of rs11567842 SNP in NaDC-1 gene was a protective genotype for hypocitraturia in kidney stone patients. The findings suggested that patients with AA genotypes were more susceptible to hypocitraturia than those with GG, hence carrying a higher risk for kidney stone recurrence.     

Characterization of <Emphasis Type="Italic">SWEET</Emphasis> family members from loquat and their responses to exogenous induction

The plant SWEET family is a sugar transporter family that plays a significant role in plant development. Here, seven loquat SWEET family members were identified by RNA-seq. These were designated as EjSWEET1, EjSWEET2a, EjSWEET2b, EjSWEET2c, EjSWEET4, EjSWEET15, and EjSWEET17. Phylogenetic and predictive functional annotation analyses suggest that the loquat SWEETs are classified as having sucrose, glucose and fructose transportation features. The in vivo responses of loquat SWEETs to exogenous sugar or NaCl was investigated by applying high concentrations of sugar or salt to 7-month-old loquat seedlings cultured in a nutrient medium. The results showed that most loquat SWEET genes can respond to exogenous applications of sucrose, glucose, fructose and salt. The response of EjSWEET1 to exogenous fructose was faster than the others, indicating that EjSWEET1 is more sensitive to exogenous fructose compared with other loquat SWEETs. EjSWEET15 can be induced by sucrose, but is suppressed by glucose. This indicates its possible role in sucrose transporting. The response of loquat SWEETs to NaCl showed broadly similar patterns compared to sugars. However, after a longer time of NaCl treatment, most loquat SWEETs are upregulated, especially EjSWEET15. This indicates its long-term response to high salinity.     

Larval skipper frogs recognise kairomones of certain predators innately

Recognising potential predators is critical for the survival and reproduction of prey animals. However, prey animals may possess an innate ability to recognise the signature odours (kairomones) of only certain native, sympatric predators, while requiring learning to recognise others. Our observations have shown that larval skipper frogs (Euphlyctis cyanophlyctis) fail to recognise kairomones of dragonfly nymph, a common predator of amphibian tadpoles with a cosmopolitan distribution. Hence, we wanted to determine if larval skipper frogs totally lack an innate mechanism to recognise kairomones of all aquatic predators, or have an innate ability to recognise kairomones of only certain predators. In a series of experiments, we tested the antipredator response of larval skipper frogs to kairomones of dragonfly nymph (Bradinopyga geminata); walking catfish (Clarias batrachus); Mozambique tilapia (Oreochromis mossambicus); two species of predatory tadpoles, Indian bullfrog (Hoplobatrachus tigerinus) and Jerdon’s bullfrog (Hoplobatrachus crassus); and the checkered keel back snake (Xenochrophis piscator). The results clearly indicate that larval skipper frogs have the innate ability to recognise kairomones of the walking catfish, both species of larval bullfrog and checkered keel back snake. However, they lack the innate ability to recognise kairomones of dragonfly nymph and Mozambique tilapia. Prey choice of the Mozambique tilapia and gape-limitation of dragonfly nymphs could be responsible for the lack of innate responses of larval skipper frogs to them. The study provides empirical evidence for the notion that prey can innately recognise certain predators.     

Renal effects of angiotensin II in the newborn period: role of type 1 and type 2 receptors

Background

Evidence suggests a critical role for the renin-angiotensin system in regulating renal function during postnatal development. However, the physiological relevance of a highly elevated renin-angiotensin system early in life is not well understood, nor which angiotensin receptors might be involved. This study was designed to investigate the roles of angiotensin receptors type 1 (AT1R) and type 2 (AT2R) in regulating glomerular and tubular function during postnatal development.

Methods

The renal effects of the selective antagonist to AT1R, ZD 7155 and to AT2R, PD 1233319 were evaluated in two groups of conscious chronically instrumented lambs aged?~?one week (N?=?8) and?~?six weeks (N?=?10). Two experiments were carried out in each animal and consisted of the assessment of renal variables including glomerular and tubular function, for 30 min before (Control) and 60 min after infusion of ZD 7155 and PD 123319, respectively. Statistical significance was determined using parametric testing (Student t-test, analysis of variance ANOVA) as appropriate.

Results

ZD 7155 infusion was associated with a significant decrease in glomerular filtration rate and filtration fraction at one but not six weeks; urinary flow rate decreased significantly in older animals, whereas sodium excretion and free water clearance were not altered. There was an age-dependent effect on potassium handling along the nephron, potassium excretion decreasing after ZD 7155 infusion in younger but not in older lambs. PD 123319 had no significant effects on glomerular filtration rate and tubular function in either age group.

Conclusions

These results provide evidence to support an important role for AT1Rs in mediating the renal effects of angiotensin II during postnatal maturation in conscious developing animals. In contrast to a role for AT2Rs later in life, there appears to be no role for AT2Rs in influencing the renal effects of Angiotensin II in the postnatal period.

     

Glucagon-like peptide 1 receptor expression in primary porcine proximal tubular cells

BACKGROUND: GLP-1 is secreted into the circulation after food intake. The main biological effects of GLP-1 include stimulation of glucose dependent insulin secretion and induction of satiety feelings. Recently, it was demonstrated in rats and humans that GLP-1 can stimulate renal excretion of sodium. Based on these data, the existence of a renal GLP-1 receptor (GLP-1R) was postulated. However, the exact localization of the GLP-1R and the mechanism of this GLP-1 action have not yet been investigated. METHODS: Primary porcine proximal tubular cells were isolated from porcine kidneys. Expression of GLP-1R was measured at the mRNA level by quantitative RT-PCR. Protein expression of GLP-1R was verified with immunocytochemistry, immunohistochemistry and Western blot analysis. Functional studies included transport assessments of sodium and glucose using three different GLP-1 concentrations (200 pM, 2 nM and 20 nM), 200 pM exendin-4 (GLP-1 analogue) and an inhibitor of the dipeptidylpeptidase IV (DPPIV) enzyme (P32/98 at 10 microM). Finally, the expression of NHE3, the predominant Na(+)/H(+) exchanger in proximal tubular cells, was also investigated. RESULTS: GLP-1R, NHE3 and DPPIV were expressed at the mRNA level in porcine proximal tubular kidney cells. GLP-1R expression was confirmed at the protein level. Staining of human and pig kidney cortex revealed that GLP-1R was predominantly expressed in proximal tubular cells. Functional assays demonstrated an inhibition of sodium re-absorption with GLP-1 after 3 h of incubation. Exendin-4 and GLP-1 in combination with P32/98 co-administration had no clear influence on glucose and sodium uptake and transport. CONCLUSION: GLP-1R is functionally expressed in porcine proximal tubular kidney cells. Addition of GLP-1 to these cells resulted in a reduced sodium re-absorption. GLP-1 had no effect on glucose re-absorption. We conclude that GLP-1 modulates sodium homeostasis in the kidney most likely through a direct action via its GLP-1R in proximal tubular cells.     

Tubular protein uptake pattern in the frog model (<Emphasis Type="Italic">Rana temporaria</Emphasis>): The effect of previous protein loading

The effect of increasing protein load on subsequent receptor-mediated protein uptake was studied in the kidney of the common frog Rana temporaria L. Results of in vivo experiments were analyzed in fixed kidney sections using fluorescent or confocal microscopy and immunohistochemistry. Lysozyme was used for daily tubular loading in short-term experiments. Reabsorption of yellow fluorescent protein (YFP) in the proximal tubule (PT) was tested 60 min after introduction into the dorsal lymphatic sac. YFP uptake decreased progressively with increasing duration of lysozyme preload from 2 to 4 days. Lysozyme loading and single protein injections did not change the morphological characteristics of frog glomeruli and PTs, as shown by light and electron microscopy and morphometric analysis. Cessation of loading led to a decrease in the amount of lysozyme accumulated in PT cells. Reduced YFP uptake gradually recovered after cessation of the 4-day load. Restoration of YFP reabsorption was accompanied by increasing expression of endocytic receptors, megalin and cubilin. Based on the data obtained, the frog model can be successfully used for studying both morphological and functional changes in the nephron caused by tubular or glomerular proteinuria and molecular mechanisms involved in the process of renal protein reabsorption.     

Diencephalo-telencephalic changes of tyrosine hydroxylase in rats and grass frogs after sleep deprivation

Based on sleep deprivation-produced changes of electrographic parameters of the wakefulness-sleep cycle (WSC) in rats and frogs (Rana temporaria), dynamics of activity of tyrosine hydroxylase, the key enzyme of dopamine synthesis, was studied immunohistochemically in substantia nigra and nigrostriatal pathway in rats and in striatum, paraventricular organ, and extrahypothalamic pathways in frogs. Changes in dynamics of tyrosine hydroxylase in rats and in frogs are revealed after the 6-h sleep deprivation and after 2 h of postdeprivation sleep. This allows determining the degree of participation of corticostriatal neuroregulatory and hypothalamo-pituitary neurosecretory systems and their role in regulation of WSC. Possible evolutionary peculiarities of morphofunctional differences in homoiothermal and poikilothermal animals are discussed.     

Role of Arabidopsis <Emphasis Type="Italic">ABF1</Emphasis>/<Emphasis Type="Italic">3</Emphasis>/<Emphasis Type="Italic">4</Emphasis> during <Emphasis Type="Italic">det1</Emphasis> germination in salt and osmotic stress conditions

Key message

Arabidopsis det1 mutants exhibit salt and osmotic stress resistant germination. This phenotype requires HY5, ABF1, ABF3, and ABF4.

Abstract

While DE-ETIOLATED 1 (DET1) is well known as a negative regulator of light development, here we describe how det1 mutants also exhibit altered responses to salt and osmotic stress, specifically salt and mannitol resistant germination. LONG HYPOCOTYL 5 (HY5) positively regulates both light and abscisic acid (ABA) signalling. We found that hy5 suppressed the det1 salt and mannitol resistant germination phenotype, thus, det1 stress resistant germination requires HY5. We then queried publically available microarray datasets to identify genes downstream of HY5 that were differentially expressed in det1 mutants. Our analysis revealed that ABA regulated genes, including ABA RESPONSIVE ELEMENT BINDING FACTOR 3 (ABF3), are downregulated in det1 seedlings. We found that ABF3 is induced by salt in wildtype seeds, while homologues ABF4 and ABF1 are repressed, and all three genes are underexpressed in det1 seeds. We then investigated the role of ABF3, ABF4, and ABF1 in det1 phenotypes. Double mutant analysis showed that abf3, abf4, and abf1 all suppress the det1 salt/osmotic stress resistant germination phenotype. In addition, abf1 suppressed det1 rapid water loss and open stomata phenotypes. Thus interactions between ABF genes contribute to det1 salt/osmotic stress response phenotypes.

     

<Emphasis Type="Italic">Ex vivo</Emphasis> analysis of renal proximal tubular cells

Background

Experimental models are inevitably a compromise between accurately reproducing a pathological situation and schematically simplifying it, which is intended to provide both relevance and conclusiveness. In-vivo models are very relevant, but multiple cell-types undergoing various changes may hinder the observation of individual molecular events.

Results

Here, we describe a method for analyzing and isolating specific cell types from the kidney and studying the phenotype they have acquired in vivo. Using flow cytometry, immunofluorescence, and RT-PCR, we show that our method is suitable for studying and isolating proximal tubular cells with an anti Prominin-1 antibody. Kidneys are subjected to mechanical dissociation followed by flow-cytometry analysis. Hundreds of thousands of proximal tubular cells are then isolated by magnetic separation followed by direct analysis or primary cell culture. Using our method, we detect phenotypic changes in the proximal tubular cells after renal ischemia reperfusion, and we isolate the proximal tubular cells, with a purity over 80%.

Conclusions

This method is efficient, quick, simple, and cheap, and should be useful for studying cell-type specific parameters after in vivo experimental studies. It is also a simple method to obtain a specific primary cell culture from any animal strain.