Stage-dependent localization of F-actin and Na+ /K+ -ATPase in zebrafish embryos detected using optimized cryosectioning immunostaining protocol.

The increasing use of the zebrafish model in biomedical and (eco)toxicological studies aimed at understanding the function of various proteins highlight the importance of optimizing existing methods to study gene and protein expression and localization in this model. In this context, zebrafish cryosections are still underutilized compared with whole-mount preparations. In this study, we used zebrafish embryos (24-120 hpf) to determine key factors for the preparation of high-quality zebrafish cryosections and to determine the optimal protocol for (immuno)fluorescence analyses of Na+ /K+ -ATPase and F-actin, across developmental stages from 1 to 5 dpf. The results showed that the highest quality zebrafish cryosections were obtained after the samples were fixed in 4% paraformaldehyde (PFA) for 1 h, incubated in 2.5% bovine gelatin/25% sucrose mixture, embedded in OCT, and then sectioned to 8 µm thickness at -20°C. Fluorescence microscopy analysis of phalloidin-labeled zebrafish skeletal muscle revealed that 1-h-4% PFA-fixed samples allowed optimal binding of phalloidin to F-actin. Further immunofluorescence analyses revealed detailed localization of F-actin and Na+ /K+ -ATPase in various tissues of the zebrafish and a stage-dependent increase in their respective expression in the somitic muscles and pronephros. Finally, staining of zebrafish cryosections and whole-mount samples revealed organ-specific and zone-dependent localizations of the Na+ /K+ -ATPase α1-subunit. RESEARCH HIGHLIGHTS: This study brings optimization of existing protocols for preparation and use of zebrafish embryos cryosections in (immuno)histological analyses. It reveals stage-dependent localization/expression of F-actin and Na+ /K+ -ATPase in zebrafish embryos.

Long-term effects of melatonin and resveratrol on aging rats: A multi-biomarker approach.

Aging-related impaired body structure and functions may be, at least partially, caused by elevated oxidative stress. Melatonin (MEL) and resveratrol (RSV) may act as antioxidant and anti-aging compounds, but these actions in experimental animals and humans are controversial. Herein, a rat model of aging was used to study the long-term sex-related effects of MEL and RSV treatment on body mass and blood/plasma parameters of DNA damage, oxidative status (glutathione and malondialdehyde levels), and concentrations of sex hormones. Starting from the age of 3mo, for the next 9mo or 21mo male and female Wistar rats (n = 4-7 per group) were given water to drink (controls) or 0.1 % ethanol in water (vehicle), or MEL or RSV (each 10 mg/L vehicle). DNA damage in whole blood cells was tested by comet assay, whereas in plasma, glutathione, malondialdehyde, and sex hormones were determined by established methods. Using statistical analysis of data by ANOVA/Scheffe post hoc, we observed a similar sex- and aging-dependent rise of body mass in both sexes and drop of plasma testosterone in control and vehicle-treated male rats, whose pattern remained unaffected by MEL and RSV treatment. Compared with controls, all other parameters remained largely unchanged in aging and differently treated male and female rats. We concluded that the sex- and aging-related pattern of growth and various blood parameters in rats were not affected by the long-term treatment with MEL and RSV at the estimated daily doses (300-400 µg/kg b.m.) that exceed usual moderate consumption in humans.

Subchronic exposure of individual and combined ochratoxin A and citrinin selectively affects the expression of rat renal organic cation transporters.

Ochratoxin A (OTA) and citrinin (CIT) are nephrotoxins found co-occurring in various human/animal food/feed and recognized as a health threat. However, most studies investigate individual effects and neglect their combined nephrotoxic effects in mammals. Previous studies have indicated that organic anion/cation transporters (OATs/OCTs) localized in renal proximal tubules mediate the transport of OTA and CIT. Still, little is known about the in vivo effects of individual/combined OTA and CIT on protein localization/expression of OCTs, physiologically/pharmacologically important renal transporters. Here, we used Western blot and immunofluorescence microscopy to study the effects of subchronic (21-day) exposure to individual/combined OTA (0.125 and 0.250 mg kg-1 b.w.) and CIT (20 mg kg-1 b.w.) on protein localization/expression of organic cation transporters (rOct1/Slc22a1 and rOct2/Slc22a2) in kidneys of Wistar rats. Since the antioxidant resveratrol (RSV) has shown measurable protective effects against OTA- and CIT-related oxidative stress toxicity in vitro, we investigated the effects of an OTA + CIT + RSV combination on rOct1/2 localization/expression in the same model. Individual OTA induced a dose-dependent decrease of rOct1 but not rOct2 protein expression, whereas their localization pattern remained unchanged. Individual CIT did not affect the renal rOct1/2 protein localization/expression. Combined OTA + CIT exposure induced a significant decrease of rOct1 protein expression by an OTA250 dose, whereas oral co-administration of OTA + CIT + RSV resulted in a significant decrease of rOct1/2 protein expression. Thus, we revealed an OTA-related selective effect on the rOct1/2 protein expression and a non-specific adverse effect of RSV in the OTA + CIT + RSV combination on the renal organic cation transport system in rat.

Antidiabetic Effects of a Tripeptide That Decreases Abundance of Na+-d-glucose Cotransporter SGLT1 in the Brush-Border Membrane of the Small Intestine.

In enterocytes, protein RS1 (RSC1A1) mediates an increase of glucose absorption after ingestion of glucose-rich food via upregulation of Na+-d-glucose cotransporter SGLT1 in the brush-border membrane (BBM). Whereas RS1 decelerates the exocytotic pathway of vesicles containing SGLT1 at low glucose levels between meals, RS1-mediated deceleration is relieved after ingestion of glucose-rich food. Regulation of SGLT1 is mediated by RS1 domain RS1-Reg, in which Gln-Ser-Pro (QSP) is effective. In contrast to QSP and RS1-Reg, Gln-Glu-Pro (QEP) and RS1-Reg with a serine to glutamate exchange in the QSP motif downregulate the abundance of SGLT1 in the BBM at high intracellular glucose concentrations by about 50%. We investigated whether oral application of QEP improves diabetes in db/db mice and affects the induction of diabetes in New Zealand obese (NZO) mice under glucolipotoxic conditions. After 6-day administration of drinking water containing 5 mM QEP to db/db mice, fasting glucose was decreased, increase of blood glucose in the oral glucose tolerance test was blunted, and insulin sensitivity was increased. When QEP was added for several days to a high fat/high carbohydrate diet that induced diabetes in NZO mice, the increase of random plasma glucose was prevented, accompanied by lower plasma insulin levels. QEP is considered a lead compound for development of new antidiabetic drugs with more rapid cellular uptake. In contrast to SGLT1 inhibitors, QEP-based drugs may be applied in combination with insulin for the treatment of type 1 and type 2 diabetes, decreasing the required insulin amount, and thereby may reduce the risk of hypoglycemia.

Subchronic exposure to individual and combined ochratoxin A and citrinin affects the expression of rat renal organic anion transporters.

Ochratoxin A (OTA) and citrinin (CIT) are mycotoxins known to co-contaminate human/animal food/feed. Their prominent nephrotoxic effects pose a threat to human and animal health. Studies have shown synergistic or additive effects of these two mycotoxins, but a clear consensus on this phenomenon does not exist. In vitro/vivo studies on OTA and CIT effects showed they elevate oxidative stress parameters. Some in vitro studies tested resveratrol (RSV) as a potential antioxidant to counteract these OTA and CIT effects. However, data on the combined effects of OTA + CIT mycotoxins and RSV on their in vivo toxicity is lacking. We used immunofluorescence microscopy and Western blotting to study the subchronic effects of individual/combined OTA (0.125 and 0.250 mg kg-1 b.w.) and CIT (20 mg kg-1 b.w.) on the localization/expression of rat renal organic anion transporters (rOats) (rOat1/Slc22a6, rOat2/Slc22a7, rOat3/Slc22a8, rOat5/Slc22a19) that mediate the secretion/reabsorption of organic anions in kidney proximal tubules. We investigated if RSV (20 mg kg-1 b.w.) can counteract the effects of both mycotoxins on the localization/expression of studied transporters. Results revealed Oat- and dose-dependent changes in protein expression of rOats. When combined with both mycotoxins, RSV decreased the protein expression of all of the studied rOats. Its effect was additive on Oat1/2/5. Thus, RSV failed to ameliorate OTA- and/or CIT-related nephrotoxic effects on the expression of studied rOats in rat kidneys.

Sex-dependent expression of metallothioneins MT1 and MT2 and concentrations of trace elements in rat liver and kidney tissues: Effect of gonadectomy.

Metallothioneins (MTs) exhibit binding affinity for several essential and toxic trace elements. Previous studies in rodents indicated sex differences in the hepatic and renal expression of MTs and concentrations of various elements. The mechanism responsible for these differences has not been resolved. Here, in the liver and kidney tissues of sham-operated and gonadectomized male and female rats we determined the expression of MT1 and MT2 (MT1&2) mRNA by RT-PCR, abundance of MT1&2 proteins by Western blotting and immunocytochemistry, concentrations of essential (Fe, Zn, Cu, Co) and toxic (Cd, Hg, Pb) elements by ICP-MS, and oxidative status parameters (SOD, GPx, MDA, GSH) by biochemical methods. In both organs, the expression of MT1&2 mRNA and MT1&2 proteins was female-dominant, upregulated by castration, and downregulated by ovariectomy. Concentrations of Fe in the liver and Co in the kidneys followed the same pattern. Most other elements (Zn, Cu, Cd, Hg) exhibited female- or male-dominant sex differences, affected by gonadectomy in one or both organs. Pb was sex- and gonadectomy-unaffected. GPx and MDA were elevated and associated with the highest concentrations of Fe only in the female liver. We conclude that the sex-dependent expression of MT1&2 mRNA and proteins in the rat liver and kidneys may include different mechanisms. In the liver, the female-dominant tissue concentrations of Fe may generate oxidative stress which is a potent enhancer of MTs production, whereas in kidneys, the female-dominant expression of MTs may be unrelated to Fe-mediated oxidative stress.

Sodium-glucose cotransporters: new targets of cancer therapy?

Glucose, the key source of metabolic energy, is imported into cells by two categories of transporters: 1) facilitative glucose transporters (GLUTs) and 2) secondary active sodium-glucose cotransporters (SGLTs). Cancer cells have an increased demand for glucose uptake and utilisation compared to normal cells. Previous studies have demonstrated the overexpression of GLUTs, mainly GLUT1, in many cancer types. As the current standard positron emission tomography (PET) tracer 2-deoxy-2-(18F)fluoro-D-glucose (2-FDG) for imaging tumour cells via GLUT1 lacks in sensitivity and specificity, it may soon be replaced by the newly designed, highly sensitive and specific SGLT tracer α-methyl-4-(F-18)fluoro-4-deoxy-Dglucopyranoside (Me-4FDG) in clinical detection and tumour staging. This tracer has recently demonstrated the functional activity of SGLT in pancreatic, prostate, and brain cancers. The mRNA and protein expression of SGLTs have also been reported in colon/colorectal, lung, ovarian, head, neck, and oral squamous carcinomas. So far, SGLTs have been poorly investigated in cancer, and their protein expression and localisation are often controversial due to a lack of specific SGLT antibodies. In this review, we describe current knowledge concerning SGLT1 and SGLT2 (over)expression in various cancer types. The findings of SGLTs in malignant cells may help in developing novel cancer therapies with SGLT2 or SGLT1/SGLT2 inhibitors already used in diabetes mellitus treatment.

Sex-independent expression of chloride/formate exchanger Cfex (Slc26a6) in rat pancreas, small intestine, and liver, and male-dominant expression in kidneys.

Chloride/formate exchanger (CFEX; SLC26A6) mediates oxalate transport in various mammalian organs. Studies in Cfex knockout mice indicated its possible role in development of male-dominant hyperoxaluria and oxalate urolithiasis. Rats provide an important model for studying this pathophysiological condition, but data on Cfex (rCfex) localisation and regulation in their organs are limited. Here we applied the RT-PCR and immunochemical methods to investigate rCfex mRNA and protein expression and regulation by sex hormones in the pancreas, small intestine, liver, and kidneys from intact prepubertal and adult as well as gonadectomised adult rats treated with sex hormones. rCfex cDNA-transfected HEK293 cells were used to confirm the specificity of the commercial anti-CFEX antibody. Various biochemical parameters were measured in 24-h urine collected in metabolic cages. rCfex mRNA and related protein expression varied in all tested organs. Sex-independent expression of the rCfex protein was detected in pancreatic intercalated ducts (apical domain), small intestinal enterocytes (brush-border membrane; duodenum > jejunum > ileum), and hepatocytes (canalicular membrane). In kidneys, the rCfex protein was immunolocalised to the proximal tubule brush-border with segment-specific pattern (S1=S2

Expression profiling and immunolocalization of Na+-D-glucose-cotransporter 1 in mice employing knockout mice as specificity control indicate novel locations and differences between mice and rats.

The expression and localization of sodium-D-glucose cotransporter SGLT1 (SLC5A1), which is involved in small intestinal glucose absorption and renal glucose reabsorption, is of high biomedical relevance because SGLT1 inhibitors are currently tested for antidiabetic therapy. In human and rat organs, detailed expression profiling of SGLT1/Sglt1 mRNA and immunolocalization of the transporter protein has been performed. Using polyspecific antibodies and preabsorption with antigenic peptide as specificity control, in several organs, different immunolocalizations of SGLT1/Sglt1 between human and rat were obtained. Because the preabsorption control does not exclude cross-reactivity with similar epitopes, some localizations remained ambiguous. In the present study, we performed an immunocytochemical localization of Sglt1 in various organs of mice. Specificities of the immunoreactions were evaluated using antibody preabsorption with the Sglt1 peptide and the respective organs of Sglt1 knockout mice. Because staining in some locations was abolished after antibody preabsorption but remained in the knockout mice, missing staining in knockout mice was used as specificity criterion. The immunolocalization in mouse was identical or similar to rat in many organs, including small intestine, liver, and kidney. However, the male-dominant renal Sglt1 protein expression in mice differed from the female-dominant expression in rats, and localization in lung, heart, and brain observed in rats was not detected in mice. In mice, several novel locations of Sglt1, e.g., in eyes, tongue epithelial cells, pancreatic ducts, prostate, and periurethral glands were detected. Using end-point and quantitative RT-PCR in various organs, different Sglt1 expression in mice and rats was confirmed.

Macro- and microelements in the rat liver, kidneys, and brain tissues; sex differences and effect of blood removal by perfusion in vivo.

Concentrations of macro- and microelements in animal organs indicate the animal health status and represent reference data for animal experiments. Their levels in blood and tissues could be different between sexes, and could be different with and without blood in tissues. To test these hypotheses, in adult female and male rats the concentrations of various elements were measured in whole blood, blood plasma, and tissues from blood-containing (nonperfused) and blood-free liver, kidneys, and brain (perfused in vivo with an elements-free buffer). In these samples, 6 macroelements (Na, Mg, P, S, K, Ca) and 14 microelements (Fe, Mn, Co, Cu, Zn, Se, I, As, Cd, Hg, Pb, Li, B, Sr) were determined by inductively coupled plasma mass spectrometry following nitric acid digestion. In blood and plasma, female- or male-dominant sex differences were observed for 6 and 5 elements, respectively. In nonperfused organs, sex differences were observed for 3 (liver, brain) or 9 (kidneys) elements, whereas in perfused organs, similar differences were detected for 9 elements in the liver, 5 in the kidneys, and none in the brain. In females, perfused organs had significantly lower concentrations of 4, 5, and 2, and higher concentrations of 10, 4, and 7 elements, respectively, in the liver, kidneys, and brain. In males, perfusion caused lower concentrations of 4, 7, and 2, and higher concentrations of 1, 1, and 7 elements, respectively, in the liver, kidneys, and brain. Therefore, the residual blood in organs can significantly influence tissue concentrations of various elements and their sex-dependency.

Distribution of organic anion transporters NaDC3 and OAT1-3 along the human nephron.

The initial step in renal secretion of organic anions (OAs) is mediated by transporters in the basolateral membrane (BLM). Contributors to this process are primary active Na(+)-K(+)-ATPase (EC 3.6.3.9), secondary active Na(+)-dicarboxylate cotransporter 3 (NaDC3/SLC13A3), and tertiary active OA transporters (OATs) OAT1/SLC22A6, OAT2/SLC22A7, and OAT3/SLC22A8. In human kidneys, we analyzed the localization of these transporters by immunochemical methods in tissue cryosections and isolated membranes. The specificity of antibodies was validated with human embryonic kidney-293 cells stably transfected with functional OATs. Na(+)-K(+)-ATPase was immunolocalized to the BLM along the entire human nephron. NaDC3-related immunostaining was detected in the BLM of proximal tubules and in the BLM and/or luminal membrane of principal cells in connecting segments and collecting ducts. The thin and thick ascending limbs, macula densa, and distal tubules exhibited no reactivity with the anti-NaDC3 antibody. OAT1-OAT3-related immunostaining in human kidneys was detected only in the BLM of cortical proximal tubules; all three OATs were stained more intensely in S1/S2 segments compared with S3 segment in medullary rays, whereas the S3 segment in the outer stripe remained unstained. Expression of NaDC3, OAT1, OAT2, and OAT3 proteins exhibited considerable interindividual variability in both male and female kidneys, and sex differences in their expression could not be detected. Our experiments provide a side-by-side comparison of basolateral transporters cooperating in renal OA secretion in the human kidney.

In female rats, ethylene glycol treatment elevates protein expression of hepatic and renal oxalate transporter sat-1 (Slc26a1) without inducing hyperoxaluria.

AIM: To investigate whether the sex-dependent expression of hepatic and renal oxalate transporter sat-1 (Slc26a1) changes in a rat model of ethylene glycol (EG)-induced hyperoxaluria. METHODS: Rats were given tap water (12 males and 12 females; controls) or EG (12 males and 12 females; 0.75% v/v in tap water) for one month. Oxaluric state was confirmed by biochemical parameters in blood plasma, urine, and tissues. Expression of sat-1 and rate-limiting enzymes of oxalate synthesis, alcohol dehydrogenase 1 (Adh1) and hydroxy-acid oxidase 1 (Hao1), was determined by immunocytochemistry (protein) and/or real time reverse transcription polymerase chain reaction (mRNA). RESULTS: EG-treated males had significantly higher (in µmol/L; mean±standard deviation) plasma (59.7±27.2 vs 12.9±4.1, P<0.001) and urine (3716±1726 vs 241±204, P<0.001) oxalate levels, and more abundant oxalate crystaluria than controls, while the liver and kidney sat-1 protein and mRNA expression did not differ significantly between these groups. EG-treated females, in comparison with controls had significantly higher (in µmol/L) serum oxalate levels (18.8±2.9 vs 11.6±4.9, P<0.001), unchanged urine oxalate levels, low oxalate crystaluria, and significantly higher expression (in relative fluorescence units) of the liver (1.59±0.61 vs 0.56±0.39, P=0.006) and kidney (1.77±0.42 vs 0.69±0.27, P<0.001) sat-1 protein, but not mRNA. The mRNA expression of Adh1 was female-dominant and that of Hao1 male-dominant, but both were unaffected by EG treatment. CONCLUSIONS: An increased expression of hepatic and renal oxalate transporting protein sat-1 in EG-treated female rats could protect from hyperoxaluria and oxalate urolithiasis.

Sex-dependent expression of water channel AQP1 along the rat nephron.

In the mammalian kidney, nonglycosylated and glycosylated forms of aquaporin protein 1 (AQP1) coexist in the luminal and basolateral plasma membranes of proximal tubule and descending thin limb. Factors that influence AQP1 expression in (patho)physiological conditions are poorly known. Thus far, only angiotensin II and hypertonicity were found to upregulate AQP1 expression in rat proximal tubule in vivo and in vitro (Bouley R, Palomino Z, Tang SS, Nunes P, Kobori H, Lu HA, Shum WW, Sabolic I, Brown D, Ingelfinger JR, Jung FF. Am J Physiol Renal Physiol 297: F1575-F1586, 2009), a phenomenon that may be relevant for higher blood pressure observed in men and male experimental animals. Here we investigated the sex-dependent AQP1 protein and mRNA expression in the rat kidney by immunochemical methods and qRT-PCR in tissue samples from prepubertal and intact gonadectomized animals and sex hormone-treated gonadectomized adult male and female animals. In adult rats, the overall renal AQP1 protein and mRNA expression was ∼80% and ∼40% higher, respectively, in males than in females, downregulated by gonadectomy in both sexes and upregulated strongly by testosterone and moderately by progesterone treatment; estradiol treatment had no effect. In prepubertal rats, the AQP1 protein expression was low compared with adults and slightly higher in females, whereas the AQP1 mRNA expression was low and similar in both sexes. The observed differences in AQP1 protein expression in various experiments mainly reflect changes in the glycosylated form. The male-dominant expression of renal AQP1 in rats, which develops after puberty largely in the glycosylated form of the protein, may contribute to enhanced fluid reabsorption following the androgen- or progesterone-stimulated activities of sodium-reabsorptive mechanisms in proximal tubules.