Search

NaDC-3 (SLC13A3) generates an in-to-out gradient of dicarboxylates, which is a driving force for several renal exchangers that mediate secretion of endogenous and xenobiotic organic anions (OA). NaDC-3 has been cloned from different species, characterized biochemically and functionally, and localized to the proximal tubule (PT) basolateral membrane (BLM). However, its detailed localization along the nephron, and regulation of its expression in vivo, has been poorly studied. We have raised a novel polyclonal antibody against the synthetic peptide specific for human NaDC-3 (Ab), and characterized the expression and distribution of this transporter in male (M) and female (F) human kidneys (HK) by immunocytochemistry (IC) in tissue cryosections and by immunoblotting of isolated total cell membranes (TCM). The Ab also reacted with pig (PK) and rat (RK) kidneys. Sex differences (SD) and the role of sex hormones were studied in more details in intact, gonadectomized, and testosterone (T)-, estradiol (E)-, or progesterone (P)-treated castrated rats. In the HK, the Ab stained exclusively the BLM of PT, whereas in TCM from various tissue zones, it labelled 3 peptide-blockable protein bands (43, 60, & 80 kDa), possibly representing the degradation product, nonglycosylated, and glycosylated proteins, respectively. The band density and IC staining showed no SD. Similar pattern of protein bands, without SD, was also observed in the PK. However, in the RK, the Ab stained strongly the BLM and intracellular organelles in PT and collecting ducts, and weakly the apical domain of macula densa and distal tubules. In the RK cortex, the density of protein bands (43, 60, & 78 kDa), and the intensity of IC staining, were: a) stronger in M, b) unaffected by castration, c) upregulated by ovariectomy, and d) inhibited weakly by T- and strongly by E- and P-treatment of castrates. The data indicate the presence of species differences in the abundance, distribution, and sex hormone-driven expression of renal NaDC-3.

The organic anion transporter 5 (Oat5, Slc22a19) was previously localized to the brush-border of proximal tubule (PT) S3 segment in rat and mouse kidneys. Here we report on sex hormone-regulated expression of Oat5 in rat kidneys, after reinvestigating: a) expression of its mRNA by end-point and real time RT-PCR in the tissue, b) abundance of its protein by Western blotting (WB) in isolated membranes, and c) immunolocalization in tissue cryosections. In untreated male (M) and female (F) adult rats, the expression of Oat5 mRNA was predominant in the outer stripe (OS), exhibiting sex differences (M

In the mammalian kidneys, handling of various organic compounds is mediated by multispecific organic anion and cation transporters localized in the luminal and contraluminal cell membrane domains of specific nephron segments, largely in proximal tubules. These transporters are responsible for cellular uptake and/or elimination of endogenous and xenobiotic organic compounds, including various anionic and cationic drugs, thus contributing to their reabsorption and/or secretion along the nephron. Recent studies have indicated a pivotal role of these transporters in drug resistance, drug-drug interactions, and drug-induced nephrotoxicity, whereas the presence of disfunctional transporters due to truncated isoforms or point mutations can cause genetic diseases. In rat, mouse and rabbit nephrons, a number of these transporters exhibit sex differences in their protein and/or mRNA expression. In comparison with the expression in rodents and rabbits, in the human nephrons some transporters are absent, some exhibit different localization in the cell membrane domains, and none exhibit the sex-dependent expression. Species differences in some transporters have been further demonstrated concerning substrate selectivity, distribution in cells along the nephron, levels of mRNA and/or protein expression, sensitivity to inhibitors, and regulation. Overall these differences in the mammalian kidneys indicate that: a) data on the membrane transporters-related functions in one species can not simply be regarded as relevant for other species, and b) many physiological, pharmacological, and toxicological findings related to organic anion and cation transport and transporters in rodents and rabbits do not reflect the situation in humans.

Glucose is the main source of metabolic energy for various cellular functions, and thus plays a central role in supporting intermediary metabolism and cellular homeostasis. Since plasma membrane is impermeable to glucose, its cellular uptake is mediated by two distinct processes via specific glucose transporter proteins that belong to the family of solute carriers (SLC) ; the SLC2 family members, GLUTs (glucose transporters), are sodium-independent facilitators of the glucose transport, whereas the SLC5 family members, SGLTs (sodium and glucose transporters) mediate the secondary-active sodium- glucose cotransport. Until now, 14 GLUTs and 12 SGLTs isoforms have been identified in humans of which 5 GLUTs and none SGLTs were detected in the mammalian blood cells. Detailed physiological function, precise mechanism of transport, substrates affinity, exact three-dimensional structures, and a precise tissue distribution of most GLUTs in various mammalian organs, including blood, have been poorly explored. In this review we will focus on GLUTs in the mammalian blood cells, where the data on their expression and functional roles are contradictory or largely missing. Since many GLUTs are associated with diabetes, and are up-regulated in cancers, it is undoubtedly important to further investigate GLUTs expression in different organs/tissues, including the blood cells. Understanding the complexity of glucose homeostasis that includes knowledge about tissue distribution and function of GLUTs, as well as the signaling pathways that regulate glucose metabolism, may help to develop new therapeutic strategies to target specific diseases, such as diabetes mellitus, some autoimmunity diseases, and cancer.

Introduction: Gabapentin (Gabalept_, Katena_, Neurontin_) and vigabatrin (Sabril_) are chemical compounds that represent new-generation antiepileptic drugs. Gabapentin (GBP) has been designed as a structural analogue of GABA, and has been demonstrated to increase GABA concentration. The mechanism of action is related to events modulated through its interaction with a receptor thought to be associated with L-system amino acid carrier protein. Vigabatrin (VGB) is a synthetic GABA derivate. It is an enzyme-activated, irreversible inhibitor of GABA transaminase that resulted from a systematic search into possible ways of increasing GABAergic inhibition through interference with GABA metabolism. GBP and VGA are eliminated renally completely unchanged and the pharmacokinetic varability is more predictable. Material and Method: A simple method was developed for the routine clinical monitoring of the antiepileptic drugs GBP and VGB in the serum of patients with epilepsy using high- performance liqid chromatography (HPLC) with fluorescence detector set at λexc=235 nm and λem=435 nm. After protein precipitation with acetonitril compounds are pre-column derivatized by o-phthaldehyde (OPA). Separations were carried out at 28° C on Nucleodur C18 100-5 column (dp=5_m) from Macherey Nagel. The mobile phase was isocratic, consisting of 0.02 M phosphoric acid and acetonitril (45:55, v/v) with flow-rate 0.6 ml/min. Results: The method was fully validated and linear calibration curves were obtained in the concentration ranges from 21.9 to 146.0 _mol/L for GBP and 57.9 to 386.5 _g/ml for VGB. The limit of detection (LOD) was 0.047 _mol/L for GBP, and 1.724 _mol/L for VGB. The limit of quatitation (LOQ) was 0.123 _mol/L for GBP, and 3.718 _mol/L for VGB. The within-day precision expressed as relative standard deviations (RSD) for two different concentration levels (n = 10) were 10% for both compounds, and day-to-day RSD at two different concentration levels during 3 separate days were 5% for GBA, and 10 % for VGB. Conclusion: The described method was simple, fast, highly sensitive and reproducible, while endogenous compounds did not interfere with the assay. It was used for simultaneous analysis of both antepileptic drugs in therapeutic drug monitoring.

Ochratoxin A (OTA) is a ubiquitous fungal metabolite with nephrotoxic and carcinogenic potential. Various animal models have demonstrated that OTA mainly impairs proximal tubule (PT) function and causes glucosuria, enzymuria, and diminished secretion of organic anion p-aminohippurate (PAH). The last indicates that OTA toxicity may target renal organic anion transporters (Oats ; subfamily of Slc22a drug transporters). Oat1 (Slc22a6) and Oat3 (Slc22a8) proteins reside in the basolateral membrane (BLM) of PT epithelial cells and mediate both OTA and PAH transport in a noncompetitive mode. Previous studies have shown that subchronic intoxication of rats with OTA leads to a dramatic reduction of PAH clearance/excretion, indicating that Oat1 and Oat3 may be involved in OTA nephrotoxicity, possibly via inhibiting their transport activity and/or expression in the cell membrane. However, these possibilities have not been clarified. In order to study effects of OTA on cell/tissue morphology and on cellular expression of renal Oat1 and Oat3, especially in view of male-dominant expression of these transporters in renal proximal tubules in rats and mice, we used an established subchronic in vivo model of OTA intoxication in adult male (M) and female (F) Wistar rats. The animals of both sexes were treated with various doses of OTA (0, 50, 125, 250 & 500 μ g/kg b.w., p.o., every 2nd day for 10 days). Using peptide-specific polyclonal antibodies, Oats were studied by Western blotting (WB) in total cell membranes (TCM) isolated from the cortex (C) and outer stripe (OS) tissues, and by immunofluorescence cytochemistry (IC) in tissue cryosections, whereas the expression of their mRNA was analyzed by RT-PCR using RNA from the renal C and OS. The study of tissue morphology revealed that OTA treatment resulted in damaged PT S3 segments in medullary rays, manifested by the loss of regular tubular structure and cell membranes, cell desquamation and other necrotic events. These phenomena were more severe at higher OTA doses, and were much more pronounced in M. As found in WB experiments, in spite of different levels of the Oat1 and Oat3 protein expression in isolated membranes (M>F), their expression in OTA-treated rats in both sexes followed a similar and dual, dose-dependent pattern. At low OTA doses (50-125 μ g/kg b.w.) the expression of Oat1 and Oat3 protein increased ~50% and ~300%, respectively, whereas high OTA doses (250-500 μ g/kg b.w.) decreased the expression of Oat1 (~70%) but not of Oat3 in respect to controls (vehicle-treated rats). By IC, the staining intensity of both transporters in proximal tubules completely matched the WB data. However, the RT-PCR data showed that the Oat1 mRNA expression was unaffected by low OTA doses, whereas high OTA doses downregulated it, and the expression of Oat3 mRNA was unaffected by any OTA dose. Also the expression of housekeeping gene GAPDH mRNA remained unchanged in all OTA-treated animals. Our data indicate that in rats: 1) the OTA treatment causes dose-related and M-dominant damage to cell morphology in proximal tubule S3 segments, 2) Oat1 and Oat3 have a similar pattern of protein expression for both sexes, with upregulation of both transporters at low but downregulation of only Oat1 at high OTA doses, 3) the upregulation of Oat1 and Oat3 proteins seems to be mRNA-unrelated and post-transcriptional, 4) previously observed inhibition of the renal PAH transport/secretion in rodents treated with high OTA doses may be explained by the loss of cell plasma membrane (transporting surface) and Oat1 protein in this membrane, and 5) upregulated Oat1 and Oat3 proteins at low OTA doses may possibly enhance OTA uptake and accelerate the development of nephrotoxicity.

Introduction: At the cell membrane, phosphoinositide 3-kinase(PI3K)/Akt signaling pathway plays a crucial role in mediating growth factor-induced cell survival and proliferation. However, previous studies have demonstrated a progressive increase in the level and activity of PI3K in nuclei of HL-60 cells differentiated in the presence of all-trans-retinoic acid (ATRA). The aim of the present study is to investigate the changes in subcellular distribution and activities of Akt during agonist-induced differentiation and particular phase of the cell cycle and to determine the role of the nuclear Akt in differentiation and proliferation of HL-60 cells. Materials and methods: HL-60 cells (ECCACC, UK) are maintained in exponential growth and differentiated in the presence of 1 M ATRA, 1, 25% DMSO or 500 nM phorbol miristate acetate (PMA). The expression of CD11b is determined by FACS analysis. Cell lysates and nuclei are analyzed for the presence of phosphorylated and total Akt by Western blot analysis or subjected to Akt kinase assay using Crosstide as the substrate (Matkovic et al, Leukemia, 20:941-951, 2006). Akt protein is down-modulated by commercially available siRNA (Upstate, USA). Cells are synchronized by incubation in the presence of aphidicolin. Results: The Akt-activity is found to be increased in the nuclei and lysates of HL-60 cells incubated in the presence of 1μ M ATRA for 4 days. The kinase assay show no increase in the activity of Akt immunoprecipitated from either postnuclear membranes or cytosol. Time-course study of nuclear Akt-activity in HL-60 cells show progressive increase in the levels of phosphorylated Akt that correlates with an increase in the expression of differentiation marker (CD11b). Down-modulation of Akt protein by siRNA inhibits the expression of Akt protein and decreases the level of CD11b. No increase in the level of the nuclear Akt is observed in HL-60 cells treated with other granulocytic inducers (DMSO) or strong antiproliferative agonists (phorbol miristate acetate, PMA). Preliminary data show a progressive increase in the level of phosphorylated Akt in lysates of aphidicolin-synchronized HL-60 cells. Conclusion: Our results reveal that ATRA increases activity of nuclear Akt in HL-60 cells, and that the activity of Akt is necessary for ATRA-mediated differentiation.

Introduction: All-trans retinoic acid (ATRA)-based treatment of acute promyelocytic leukemia (APL) patients is the first example of therapy by differentiation. The classical view of the action of ATRA in APL holds that ATRA releases corepressors from the chimeric PML-RARa protein and thus allows the growth inhibition, differentiation and apoptosis in t(15 ; 17) APL cells. The pharmacological inhibitors of phosphoinositide 3-kinase(PI3K)/Akt pathway have been proposed in the treatment of leukemia based on their antiproliferative effects. However, recent studies (Matkovic et al, Leukemia, 20:941-951, 2006) demonstrated Akt-activation in nuclei of ATRA-treated HL-60 cells raising the possibility that PI3K/Akt– inhibitors may block antitumor properties of retinoids. The aim of this study is to compare the sensitivity of two different leukemia cell lines to commercially available PI3K/Akt-inhibitors. NB4 cell line, established from an APL patient, is a typical AML-M3 carrying t(15 ; 17). HL-60 cells, isolated from AML-M2 patient, do not display specific t(15, 17) translocation but share the responsiveness to ATRA and are widely used to demonstrate the differentiative activity of ATRA and to predict its efficiency in vivo. Materials and Methods: HL-60 cells and NB4 cells were maintained in exponential growth and differentiated in the presence of ATRA (1microM). The Akt activity in different cell fractions was determined by Western blot analysis and kinase assays using Crosstide. The following inhibitors were tested: Akt inhibitor I (1L-6-hydroxymethyl-chiro-inositol 2(R)-2-O-methyl-3-O-octadecylcarbonate, Calbiochem), Akt inhibitor II (SH-5, Calbiochem), LY 294002 (Calbiochem) and MEK-inhibitor PD 98059 (Calbiochem). The number of viable cells was quantified using a hemocytometer and trypan blue exclusion. The expression of CD11b and the cell cycle distribution of propidium iodide-labeled cells were determined by FACS analyses. Results: Both HL-60 and NB4 cells show an increase in the activity of Akt in nuclei and lysates after ATRA-mediated differentiation. PI3K inhibitor LY 294002 and MEK-inhibitor PD98059 reduced the number of viable cells and the expression of differentiation marker in both cell lines. The presence of Akt inhibitors inhibited the growth of both control and ATRA-treated NB4 and HL-60 cells and reduced the expression of CD11b in ATRA-treated NB4 cells. In contrast, Akt-inhibitors had no inhibitory effects on the expression of CD11b in ATRA-treated HL-60 cells, but increased the percentage of control cells expressing CD11b. The inhibition of Akt altered the life span of differentiated HL-60 cells as the number of events in sub-G1 measured at 7 days after the addition of ATRA was higher in samples treated with the combination of Akt inhibitors and ATRA that in the cells treated with ATRA alone. Conclusion: Two retinoid-responsive leukemia cell lines respond differently to the presence of PI3K/Akt inhibitors ; the expression of differentiation marker is reduced in ATRA-treated NB4 cells, there are no inhibitory effects on ATRA-induced differentiation of HL-60 cells and the percentage of control HL-60 cells expressing CD11b is increased. These results suggest that the combination of retinoids with Akt-inhibitors could be exploited to improve retinoid therapy in selected cases. In addition, data further suggest that ATRA may mediate a number of nontranscriptional changes in cell signaling by mechanisms apart from the direct effects of the nuclear receptor on transcription.

Major efforts are under way to define reliable biomarkers of Alzheimer’ s disease (AD). With the advent of new drugs such as gamma-secretase inhibitors, early detection of elderly subjects with mild cognitive impairment (MCI) who are destined to develop AD is becoming increasingly important. Highly significant increases of hyperphosporylated tau proteins in cerebrospinal fluid (CSF) have been recently reported in AD patients compared to controls by several independent groups, including ours. These findings support the notion that CSF phosphorylated tau proteins may be useful biomarkers in the early identification of AD in MCI subjects.

Statins, inhibitors of hydroxy-methylglutaryl coenzyme A reductase, are considered first-line therapy for treatment of hypercholesterolemia. However, beneficial effects of statin treatment on cardiovascular morbidity and mortality has been not entirely explained by the reduction in low density lipoproteins (LDL) level but also in antioxidant activity of statins that contribute to their protective cardiovascular effects. There is an extensive evidence that links hypercholesterolemia with increased lipid peroxidation and increased oxidative stress. Several studies correlates levels of LDL oxidation with circulating malondialdehyde (MDA) which is secondary oxidation product . Mainly level of MDA in animal plasma and tissue was determined by the thiobarbituric method which is based on condensation of MDA with two molecules of 2-thiobarbituric acid (TBA) and method was widely used to measure the extent of oxidative deterioration of lipids in various biological systems. The absorbance of the complex is usually measured by spectrophotometry or by spectrofluorometry directly or by specific techniques, also based on TBA adduct, where HPLC separation with spectrophotometric or spectrofluorometric or MS detection was employed. The aim of the present study was to examine effect of simvastatin (SIMV) treatment on LDL oxidation based on the measuring of the rat MDA plasma level as parameter of oxidative stage by two different analytical techniques. Comparison of results between this analytical approaches would contribute in decision which technique is more reliable and accurate for further investigation on statin antioxidant action. Material and Methods. Forty six male Wistar rats (weight 170-200 g), were divided in two control groups (n=7 each) and four experimental groups (n=8 each). Experimental groups were on SIMV treatment which was given orally in the doses 10 and 50 mg/kg/day and for the period of 3 weeks. At the end of drug treatment animals from first two groups were sacrificed and blood samples were taken and other two groups were left additional 9 days and than sacrificed and samples were taken. The level of MDA was measured using the spectrophotometrically or by reverse phase HPLC-MS method. MDA levels are expressed as μ mol concentrations using calibrating curves and expressed as percentage of decrease of MDA level comparing to negative control. Results. SIMV in both doses decreases MDA level in plasma. Spectroscopic analysis of MDA level after SIMV application have shown decrease in plasma concentration by 9, 7% and 16, 1% after administration of 10 mg/kg/day and 50 mg/kg/day dose respectively after 21 day of application. When the same doses and same application period was applied but blood was taken after 30 days, 32, 2% and 64, 5% decrease of MDA level were recorded. However, HPLC-MS analysis has shown 12, 2% and 17, 3% decrease of MDA level after 10 mg/kg/day and 50 mg/kg/day of SIMV dose after examining the same plasma samples. Conclusion. Our results have shown that SIMV shows antioxidant action which was indicated by lowering one of the oxidative markers - MDA. We suggest that both methods, spectroscopic or HPLC-MS gives results that have similar direction, but with difference in magnitude or sensitivity.

U radu su istraženi hematološki i biokemijski krvni pokazatelji zdravlja creskih ovaca uzgajanih po ekološkim principima poljoprivredne proizvodnje. Dobiveni rezultati pokazali su da vrijednosti istraživanih pokazatelja mogu biti dobra osnova za utvrđivanje fizioloških profila koji služe za praćenje zdravlja stada u ovaca uzgajanih po ekološkim standardima. Posebice je to važno za izvorne pasmine, kao što je creska ovca, koje čine osnov ekološke ovčarske roizvodnje.

Introduction: Many authors have reported that organic lesions and metabolic disorders of many organs, especially the liver, are followed by changes of some enzyme activities in blood plasma of domestic animals and poultry. It is also known that the liver in birds is equally affected by radiation as the intestine, bone-marrow and sex glands, which is different in mammals. In our previous investigation we showed that the activities of some enzymes in blood plasma of chickens are significantly changed after a single parenteral injection of 32P. In this paper an attempt was made to investigate the influence of gamma ray irradiation of whole body of chickens upon activity of several enzyme (enzymatic profile) in blood plasma. We also wanted to evaluate whether investigation of the enzymatic profile in blood plasma can help in the diagnosis of organic or functional liver damages caused by gamma ray in the chickens.Materials and Methods: The experiments were performed on hybrid chickens of heavy Jata breeds of booth sexes. Chickens were irradiated by gamma ray in the dose of 7, 23± 0.95 Gy. Blood samples were taken from the wing vein on days 1, 3, 5, 7, 9 and 15 after irradiation.The activity of aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyl transpeptidase (GGT), leucine aminopeptidase (LAP), lactate dehydrogenase (LDH) and alpha-hydroxy-butyrate dehydrogenase (HBDH) in blood plasma of irradiated chicken was determined spectrophotometrically by using Boehringer Mannheim GmbH optimized kits. At the end of the experiment all birds were sacrificed and the organs were pathomorphologically and histologically investigated. Results: Throughout the experimental period, only ALT and GGT activity did not statistically significantly change. All other enzymes activity showed a decreased tendency during the experiment ; statistically significant decreases were recorded as follows: on the 1st day of the experiment – LAP and LDH activity ; on the 3rd day - LAP activity ; on the 5th day - LDH activity ; on the 7th day – AST, LDH and HBDH activity, on the 9th day – LAP and HBDH activity, and on the 15th day – LAP activity.Conclusion: The determination of a so-called enzyme profile in blood plasma which include measurement AST, LAP, LDH and HBDH activities may serve as an additional test for functional liver damages in chickens caused by ionizing radiation before the appearance of clinical simptoms.

It has been suggested that the G-protein-coupled receptors GPR91 (receptor for succinate) and GPR99 (receptor for  -ketoglutarate), that are localized in the mammalian nephron, may sense local levels of succinate and  -ketoglutarate, respectively, and then function as controlers of blood pressure via affecting the activity of renin-angiotensin system. In the mouse kidney, mRNA for these receptors was highly expressed and localized to the proximal (GPR91) and distal (GPR99) tubules, but the exact localization of receptor proteins along the mammalian nephron is not known. In order to screen localization of these receptors in the human kidney, in this study we used several commercial polyclonal antibodies against the peptide sequences specific for the human GPR91 and GPR99 and tested their efficiency in detecting the respective proteins by Western blotting (WB) of total cell membranes isolated from the tissue homogenates, and by immunofluorescence cytochemistry (IC) in tissue cryosections. In WB studies, the optimal antibodies from both groups labeled a single protein band of ~90 kDa (reducing conditions at 37 oC) that dropped to ~50 kDa at higher heating temperatures (reducing conditions at 65-95 oC). In IC studies, the optimal GPR91-antibody stained: a) the basolateral domain of thick ascending limbs of Henle, which were distinguished from the high affinity Na+-glucose cotransporter (SGLT1)-positive proximal tubule S3 segments, and b) intercalated cells in the inner medullary collecting duct, where it colocalized with the vacuolar H+-ATPase. The GPR99-antibodies stained only the brush-border of cortical proximal tubules. In conclusion, GPR91 and GPR99 proteins exhibit specific localization in the human nephron which is different from that in the mouse nephron, and thus indicate possible different, species-specific function(s) of these receptors.

Although radiation is a known producer of reactive oxygene species in living systems it has been shown that acute low dose ionizing irradiation induced various antioxidant mechanisms, enzymatic and non-enzymatic, that defend cells from oxidative stress. It is also shown in increased level of antioxsidant substances such as superoxide dismutase, catalase, glutathione peroxidase and glutathione in different organs after low dose irradiation. However, newly studies of low dose radiation in chickens were performed mostly on hatchability, body weight and egg fertility in commercial meat chicken line. Whereas, there are lack of results on antioxsidant status in chicken embryo and chicks hatched from irradiated eggs the aim of this study was to investigate low dose radiation effects upon antioxidant status in chicken liver after low dose gamma irradiation. The eggs of a heavy breeding chickens COBB 500 were irradiated with the dose of 0, 3 Gy gamma radiation (source 60Co) on the 19th day of incubation. Liver samples were taken on 3, 6, 24, 48, 72 and 96 h after irradiation. The thiobarbituric acid reactive substances (TBARS), glutathione levels (GSH) and activity of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and catalase (CAT) were measured spectrophotometrically. The GSH levels increased in the liver of chickens hatched from irradiated eggs at 6 hours after irradiation and decreased 48 h after irradiation. Antioxsidant enzyme activities for GSH-Px and SOD significantly decreased in the liver of chickens hatched from irradiated eggs 48 h after exposure to the dose of 0.3 Gy. TBARS levels and CAT activity during the experiment were not statistically different when they compared to controls. Obtained results indicated that acute irradiation of chicken eggs on the 19th day of incubation with the dose of 0.3 Gy gamma radiation could have a different effects on antioxidant parameters in embryo liver of chicken, i.e. the enzyme activity of SOD and GSH-Px were decreased, however, catalase and TBARS were not affected by the irradiation. The level of glutathione was firstly increased and after that it decreased. This findings suggest that glutathione is an important antioxidant in the protection of liver cells against ionizing radiation and that this dose does not stimulate antioxidants enzymes in the same tissue.

The tissue and cell damage induced by traumatic brain injury (TBI) results from primary damage and from a complex secondary cascade of events including oxidative stress, inflammation, increased vascular permeability, mitochondrial dysfunction and excitotoxic damage. There is no standard pharmacological treatment that blocks the progression of this secondary injury and the current management of TBI is mainly supportive. The peroxisome proliferator-activated receptor-γ (PPAR-γ ) is a member of nuclear receptor superfamily involved in different cellular processes such as regulation of oxidative stress and inflammatory response. Agonists of PPAR-γ have already shown the beneficial effects in the stroke and the spinal cord injury models. This work was performed in order to determine the effects of pioglitazone, a PPAR-γ agonist, on the level of antioxidant enzyme activities and the lipid oxidative damage in the parietal cortex 24 hrs after the induction of TBI in the rat. Experiments were performed on adult male Wistar rats. TBI of moderate severity was induced using the lateral fluid percussion (LFP) brain injury model. Briefly, rats were placed in a stereotactic frame and surgically prepared for LFP brain injury or sham operation as previously described (Mcintosh et ai, 1989). A 5-mm craniotomy was performed over the left parietal cortex, between lambda and bregma sutures, leaving the dura mater intact. A hollow female Luer Lock fitting was positioned over the craniotomy and held in place with dental cement. Animals were attached to the LFP device and brain injury was induced by a rapid injection of a pressure pulse of saline. Animals were i.p. injected with either pioglitazone (1 mg/kg) or vehicle 10 min after the TBI. Sham-operated, vehicle-treated animals were used as the control group. Rats were sacrificed 24 hrs after the TBI induction. Activities of antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPX) were determined by standard spectrophotometric measurements. Levels of the brain lipid peroxidation were determined by thiobarbituric acid reactive substances (TBARS) assay. TBI caused the significant increases of the GPX activity and the TBARS level, and a slight decrease of the SOD activity in the injured parietal cortex. Pioglitazone treatment resulted in the additional increase of GPX activity and in the decrease of the TBARS level in the examined cortical region of injured rats. Our findings demonstrate the activation of the antioxidant enzymatic system and the intense oxidative damage of lipids in ipsilateral parietal cortex following TBI. These results also suggest a protective role of pioglitazone in the brain oxidative damage in our experimental conditions.

Introduction: The activity of phosphatidylinositol-specific phospholipase C (PI-PLC) increases in nuclei of cells at temporally distinct phases of the cell cycle. In HL-60 cell line, previous studies demonstrated a PI-PLC inhibitor-sensitive increase in the level of the nuclear DAG at 8 h after aphidicolin block (G2-phase) and an early (30 min) increase in the PI-PLC activity in serum-starved HL-60 cells stimulated with IGF. Our recent study demonstrated two peaks of an increase in the nuclear PI-PLC activities in nocodazole-synchronized HL-60 cells that occur at 1 h and 8.5 h after G2/M-block (Lukinovic-Skudar et al., Biochim Biophys Acta 1733:148-156, 2005). To further prove that the late increase in the PI-PLC activity observed in nocodazole-synchronized cells is a late G1-phase-related event, the PI-PLC activity was measured in serum-starved cells after the re-addition of serum. Materials and Methods: HL-60 cells (ECCACC, UK) are maintained in exponential growth. For synchronization, exponentially growing cells were washed and maintained in medium containing no serum for 24h and then stimulated with FBS (10%, v/v). Cells were labelled by propidium-iodide or BrdU FITC set (556028, BD, USA) and the cell cycle analysis or BrdU incorporation is determined by using FACSCalibur system. Cell fractions were isolated as described (Lukinovic-Skudar et al., Biochim Biophys Acta 1771:514-521, 2007) and PLC activity was determined using [3H] (PtdIns(4, 5)P2) as a substrate. PLCβ 1b was immunoprecipitated from nuclear fractions and immunoprecipitates were subjected to Western blot analysis using anti-P-serine antibodies or anti-PLCβ 1b antibodies. Results: In HL-60 serum-stimulated cells, two distinct peaks of the nuclear PI-PLC activation were detected at 30 minutes and 11 h with no parallel increase in PLC activity in total cell lysates, cytosol and postnuclear membranes. All the peaks of the nuclear PI-PLC activities were completely abolished in the presence of PI-PLC inhibitor ET-18-OCH3 and MEK inhibitor PD98059. The increase in the activity correlates with a PD98059-sensitive increase in the serine phosphorylation of b splicing variant of PI-PLCbeta1 and no change in the amount of PI-PLCbeta1b was detected in nuclei isolated at any time points. The number of cells entering the S-phase was reduced after the addition of inhibitors either immediately or 6 h after the re-addition of serum. Conclusion: Two distinct peaks of nuclear PI-PLCbeta1b activity occur in serum-stimulated HL-60 cells and both early and late G1-phase increases in the nuclear PI-PLC are important for the progression of cells into the S phase

Previous studies have shown that treating rats or mice with some nephrotoxic metals (NTM) inhibited renal transport/secretion of organic anion (OA) p-aminohippurate (PAH) and its accumulation in incubated kidney cortex slices. Moreover, nephrotoxicity of these metals was prevented by probenecid, a compound that inhibits the transport of various OA, including PAH, thus indicating a possible interaction of NTM and OA at the level of membrane transporters. Major PAH transporters in the mammalian kidney have recently been cloned and characterized, and include Oat1 and Oat3 proteins localized to the basolateral membrane (BLM) of proximal tubule (PT) cells. The interaction between NTM and Oat1 and Oat3, that inhibits PAH secretion, has not been clarified. The aim of this study was to establish if cadmium (Cd), mercury (Hg), and cisplatin (cisPt) affected the expression of PAH transporters Oat1 and Oat3 at the protein and mRNA levels. We compared the expression of these transporters in vehicle-treated male rats (control) with that in rats treated with CdCl2 (2 mg Cd/kg b.m., s.c., daily for 2 weeks), HgCl2 (a single dose of 1 mg Hg/kg b.m., s.c., daily for 2 days) or cisPt (a single dose of 5 mg cisPt/kg b.m., i.p., 5 days before sacrifice). The methods applied were immunobloting (IB) in isolated total cell membranes (TCM) from the kidney cortex, immunocytochemistry (IC) in frozen kidney tissue sections, and RT-PCR with renal cortical tissue mRNA. IB and IC studies were performed using specific polyclonal antibodies, whereas RT-PCR was performed using commercial mRNA probes for specific transporters. In IB studies, the abundance of Oat1 and Oat3 proteins in TCM from the cortex of NTM-treated rats significantly decreased (Cd>Hg>cisPt). In IC studies, both transporters were localized to the PT BLM in control animals, exhibiting different intensities in specific PT segments (Oat1: S2>S3, negative S1 ; Oat3: S1=S2, negative S3). In the NTM-treated rats, the staining intensity for both transporters clearly diminished in the sequence Cd>Hg>cisPt. In addition, heavily damaged cells and loss of BLM invaginations were observed in S1 and S2 of Cd-treated rats, and in S3 of Hg- and cisPt-treated rats. In RT-PCR studies, the expression of mRNA for both transporters was downregulated strongly in Cd- and Hg-treated, and weakly in cisPt-treated rats. To conclude, previously observed inhibition of the renal PAH secretion/transport in NTM-treated animals may result from: a) loss of the PT BLM (transporting membrane surface), and b) loss of Oat1 and Oat3 protein (transporting proteins for PAH) in the PT BLM.

Introduction: Oxcarbazepine (Trileptal) is a keto-derivative of carbamazepine. It was developed by introducing minimal changes in the structure of carbamazepine, which altered the metabolism to avoid the production of epoxide metabolite. Oxcarbazepine is rapidly metabolized to a pharmacologically active monohydroxy metabolite (MDH ; 10-hydroxy-carbazepine, licarbazepine). 10-hydroxy-carbazepine has independent anticonvulsant properties and is mainly responsible for the effect of oxcarbazepine. The aim of this study was to develop and validate a HPLC method for measuring oxcarbazepine and 10-hydroxy-carbazepine simultaneously with other antiepileptics (lamotrigine, carbamazepine, phenobarbitone, phenytoin) which are already in routine use in our laboratory. Materials and Methods: Serum sample (250 _L) pretreatment was based on liquid extraction with ethylacetate/hexane, evaporated and desolved in 200 _L mobile phase. The separation was obtained on reverse-phase column (C18, 250X4, 6 mm I.D. 5_m) using a phosphate buffer/acetonitrile/methanol mixture (pH 6, 8) as a mobile phase, at the flow rate of 1.3 ml/min andDADdetector at 237nm ; total time for chromatographic separation was 13 min. Results: The calibration curves were linear for both analytes (r= 0.9975) in the expected therapeutic range. Intermediate precision (inter-day) expressed as relative standard deviations was less than 3% for 10-hydroxy-carbazepine and oxcarbazepine at two concentration levels. Accuracy, expressed as percent error, ranged from 94.5-103.8% for 10-hydroxycarbazepine and from 88-100% for oxcarbazepine. The limit of quantitation was 2.07 _mol/L for 10-hydroxy-carbazepine and 0.08 _mol/L for oxcarbazepine. Conclusion: Based of analytical parameters (linearity, precision, accuracy, limit of quantitation), the presented method is suitable for pharmacokinetic studies and therapeutic drug monitoring. The method shows good specificity with other prescribed drugs. While less pronounced than for carbamazepine, pharmacokinetic variability for oxcarbazepine was still considerable, and it can be affected by age, pregnancy, concurrent disease and drug-drug interaction. These characteristics suggest that therapeutic drug monitoring may be of value in treatments with oxcarbazepine.

The Na+-independent sulfate (S) transporter Sat-1 has been localized to the basolateral membrane (BLM) of proximal tubules in the rat kidney (K). The same transporter may also mediate S transport in the gastrointestinal tract (GIT) and liver (L), but its presence and the exact cellular localization in these organs has not been described. In this work we used a monoclonal antibody to Sat-1 protein (Sat-1-Ab) and performed localization studies by immunofluorescence cytochemistry (IC) and Western blotting (WB) in the L and in various GIT segments in rats. The Sat-1-Ab was verified in the K by achieving a positive staining of BLM in the proximal tubule S1 and S2 segments, and by labeling a single, ~85 kDa protein band in BLM isolated from renal cortical homogenate. In the L, the Sat-1-Ab strongly stained the sinusoidal membrane ; the canalicular membrane and other structures in hepatocytes remained unstained. However, the double staining in various GIT segments with the Sat-1-Ab and a polyclonal antibody to cell adhesion molecule 105 (CAM-105) indicated the localization of Sat-1 in an intracellular vesicular compartment. In the tested GIT segments, the intensity of intracellular Sat-1 staining showed the following pattern: esophagus < duodenum < jejunum < ileum < colon < stomach. The localization of Sat-1 in endocytic vesicles was disproven by an in vivo assay of FITC-dextran uptake in the colon cells, whereas the double-staining for Sat-1 and the mitochondrial marker cytochrome C in the colon and stomach revealed a complete colocalization of these proteins in mitochondria of enterocytes (colon) and oxyntic cells (stomach). However, in WB of total cell membranes, isolated from the L and various GIT segments, the Sat-1-Ab labeled weakly the ~40 kDa protein band, whereas the K-specific 85 kDa protein band was not labeled at all in these membranes. We conclude that in the rat L and GIT cells, the Sat-1 protein is different from that in the K and may exist as a shorter (truncated) isoform. In various GIT cells, the transporter may be localized in mitochondria.

A bulk of D-glucose reabsorption in the mammalian kidney (K) is mediated by the low affinity, high capacity Na+-glucose cotransporter SGLT2. In various transport and hybridization studies, this transporter has been localized in the cells of proximal tubule segments S1 and S2. The transporter exhibits the 1:1 Na+:glucose coupling ratio, and unlike SGLT1, a high affinity Na+-glucose cotransporter localized mainly to the proximal tubule S3 segments, it does not transport D-galactose. cDNA for SGLT2 has been identified first in humans and later in rats and mice. The expression of its mRNA has been detected in most human tissues, the highest levels being present in kidney. A convincing immunolocalization of SGLT2 along the mammalian nephron has not been reported. In this work we generated a novel polyclonal antibody (Ab) specific for peptide sequence in the rat SGTL2 protein, and performed detailed Western blotting (WB) and immunocytochemical (IC) studies of the expression of SGLT2 protein along the nephron in variously-treated adult (3 months) and prepubertal (3 weeks) male (M) and female (F) rats. WB experiments were performed with brush-border (BBM) and total cell membranes isolated from tissue homogenates, whereas the IC studies were performed in tissue cryosections. In addition, the expression of SGLT2 mRNA was determined by RT-PCR using RNA isolated from various tissue zones. In WB of isolated membranes, the Ab labeled a single protein band at ~75 kDa. The protein band and the immunostaining was absent in isolated membranes and tissue cryosections, respectively, from the rat small intestine, indicating a specific localization of SGLT2 in the K. In membranes from the K cortex (C) and outer stripe (OS) of intact adult rats, the density of 75 kDa protein band: a) exhibited strong zonal (C>OS) and gender differences (F>M), and b) increased weakly by castration and remained unaffected by ovariectomy. In castrated rats, the protein band was downregulated by testosterone and upregulated by estradiol treatment. By IC, the Ab stained BBM of S1 and S2 (S1>S2) with gender-dependent manner (F>M). The staining intensity in gonadectomized animals, and in sex hormone-treated castrated M matched the pattern of WB data. In prepubertal rats, the respective protein band in isolated K membranes and the staining intensity in PT were weaker than in adult animals, and gender-independent. The expression of SGLT2 mRNA was found to be high in the C and low in the OS, but gender-independent. Our data indicate that in the rat K: 1) SGLT2 is localized to the BBM of proximal tubule S1 and S2 segments (S1>S2) in the C, 2) at the protein level, SGLT2 expression exhibits zonal (C>OS) and gender differences (F>M) that occur after puberty, and 3) at the mRNA level, the expression of SGLT2 exhibits no gender differences, thus indicating the involvement of posttranslational mechanisms in sex hormone-regulated expression of SGLT2 protein.

Mycophenolate mofetil (MMF) is an immunosupressive drug used in prophylaxis of rejection of transplanted solid organs in combination with cyclosporine A (CyA) and steroids. It is metabolised in the liver to the active component mycophenolic acid (MPA), an irreversible noncompetitive inhibitor of cell enzime 5'-monophosphate (IMP)dehydrogenase. The aim of the study was...

The kidneys maintain body fluid and electrolyte homeostasis using polarized localization of various transporters/carriers in the apical (luminal) and basolateral membrane domains of the cells along nephron. These transporters use ATP or transmembrane ion gradients to drive vectorial transport of their substrates in direction of secretion or reabsorption. The ATP-dependent carriers, termed ATP-binding casette transporters (Abc/ABC for animal/human transporters ; primary active transporters) and ATP-independent solute carriers (Slc/SLC for animal/human carriers ; secondary and terciary active transporters) are important for: a) handling of endogenous organic compounds that are produced during normal metabolism, such as organic anions (OA) and cations (OC), peptides, nucleosides, and their products, b) handling of exogenous (xenobiotic) organic compounds, such as food constituents (flavonoids, mycotoxins, pesticides, other alimentary organic substances), and drugs (various antibiotics and chemotherapeutics), c) drug-drug interactions, and d) development of nephrotoxicity and specific transporter-related diseases. Because of their major importance in drug transport and drug-mediated nephrotoxicity relevant to humans, a bulk of current research in the field of renal transporters has been dedicated to the roles of OA and OC transporters (Oats for animal/OATs for human, and Octs for animal/OCTs for human transporters, respectively). However, most of these data were obtained from the experiments in animals, largely rats and mice ; only a limited amount of data is from other animal species and humans. Recent observations in experimental animals and humans have indicated that renal secretory and reabsorptive functions under various physiological, pharmacological, and toxicological conditions may be different in males (M) and females (F), and that these differences may be related to sex hormone-regulated expression and action of specific transporters in one of the membrane domains of (mainly) proximal tubules (PT). Accordingly, a) M-dominant gender differences (GD) in renal excretion/clearance in rats and mice were observed for some OA, such as p-aminohippurate (PAH) and furosemide, due to androgen-stimulated expression of their carriers Oat1 and Oat3 in the PT basolateral membrane (BLM), b) renal accumulation and toxicity of mercury in rodents is M-dominant, because this toxic metal can enter the PT cells by molecular mimicry, e.g., bound to SH groups in some OA and thus transported via basolateral Oat1 and/or Oat3, c) M-dominant GD in renal handling of some OC in rats, such as tetraethylammonium (TEA), amantadine, and cisplatin, are determined by androgen-stimulated expression of Oct2 in the PT BLM, d) F-dominant renal clearance/excretion of various metabolic products and xenobiotics in rats (cyclacillin, carnitine, egualen sodium, perfluorooctanoic acid, estradiol-17 -glucuronide, taurocholate, dibromosulfophtalein, zenarestat) may be related to the lower expression of the respective transporters in the PT brush-border membrane (BBM) (Pept1, Octn2, Oatp1, Oat2), resulting in lower reabsorption of the filtered compounds in F. Where studied, the expression of several transporter (Oat1, Oat2, Oat3, Oat5, Oct2) were low and similar in M and F before the puberty ; GD occurred after the puberty. Contrary to the numerous cases of gender-dependent, carrier-mediated transport of specific organic compounds in rats and mice, a very few reports have been related to other species, including humans. In rabbits the expression of Oat1, Oat2, Oat3, and Oct2, in the PT significantly increased after the puberty, however without exhibiting sex differences in adult animals. Accordingly, rabbits exhibit no GD in urinary excretion of OA PAH and OC TEA. Some comparable data were also found in dogs. Furthermore, studies in humans showed that: a) the plasma level of acetylsalicylate, which is a substrate of several OATs (OAT1-4), was higher in F, b) the renal clearance of urate in F is higher than in M, possibly due to lower reabsorption of urate via weakly expressed URAT1 in the PT brush-border membrane, c) the renal clearances of the OA ciprofloxacin (a substrate of OATs), and of the OC amantadine (a substrate of OCT2), were found to be higher in M, but gender-related expression of the respective carriers in the human nephron has not been reported. Some other therapeutic drugs in humans (verapamil, metronidazole, vecuronium) also exhibit GD in their blood clearance, but a possible contribution of renal excretion/relevant transporters are not known. Humans and animals exhibit a similar set of renal transporters, but the presence, localization along the nephron, and intracellular localization of some carriers seem to be different from those in experimental animals. For example: a) Oat2 in rats and mice was localized to the PT BBM, whereas in humans, OAT2 was localized to the PT BLM, b) Oct1 was localized in the rat kidney largely to the PT BLM, whereas in the human kidney, OCT1 was not detected, c) Oct2 in the rat kidney was localized predominantly to the BLM of PT S3 segment, whereas OCT2 in the human kidney was localized to the BLM along the entire PT, and d) OAT4 in the human kidney was localized to the PT BBM, whereas a similar protein was absent from the rat nephron. These species differences in the expression and/or cellular localization of some transporters may influence the secretory and/or reabsorptive direction of renal transport of their substrates and thus may affect their urinary excretion. This further indicates that the findings regarding GD and effects of sex (and, possibly, other) hormones upon renal transporters in one species can not simply be regarded as relevant for other species.

In the mammalian liver, elimination of endogenous and exogenous organic anions (metabolic products, various drugs, environmental toxins) is mediated by various organic anion transporters (OATs), transmembrane proteins that belong to solute carrier family Slc22. The rat liver organic anion transporter 3 (rOat3, Slc22a8) has been cloned, characterized as a dicarboxylate exchanger, and defined as “ male predominant” due to much higher expression of its mRNA in males (M) than in females (F). However, cellular localization and the levels of rOat3 protein expression in rat hepatocytes are unknown. This study was designed to examine the expression of rOat3 in the rat liver at the level of: a) mRNA in the tissue using RT-PCR, and b) protein in isolated total cell membranes (TCM) using Western blotting (WB) and in tissue cryosections using immunocytochemistry (IC). For this purpose we used the following experimental groups of rats: a) intact adult M and F, b) castrated M treated with oil (O ; control) or with oil solutions of testosterone (T), progesterone (P) and estradiol (E), and c) adult M treated with O, E and P. The RT-PCR data confirmed the male-predominant expression of rOat3 mRNA in the rat liver. Castration markedly decreased the expression of Oat3 mRNA ; the treatment of castrated rats with T restored it to the level of intact M, while the treatment with E and P had no effect. However, the treatment of adult M with E and P strongly downregulated the expression of rOat3 mRNA as compared to that in O-treated M. By WB in TCM prepared in non-reducing conditions, a polyclonal anti-rOat3 antibody labeled a single peptide-blockable protein band of ~50 kDa. In various experimental conditions the density pattern of this protein resembled the pattern of rOat3 mRNA. In IC studies, the anti-rOat3 antibody strongly stained some intracellular organelles in the M but not F liver. The double staining studies with organelle-specific marker antibodies (plasma membranes, endocytic vesicles, mitochondria, peroxisomes, lysosomes) indicated that rOat3 might be localized in lysosomes. We conclude that rOat3 in the rat liver exhibits strong gender differences (M>F) at both mRNA and protein levels due to testosterone stimulation and estradiol and progesterone inhibition, and may be localized in lysosomes.

Introduction. A role of inflammation in the brain, both in human epilepsy and in animal epilepsy models, has been recently recognized. Agonists of the peroxisome proliferator-activated receptors gamma (PPAR-γ ) have been shown to be neuroprotective in models of various neurological diseases such as Parkinson’ s disease, Alzheimer’ s disease, multiple sclerosis, focal cerebral ischemia, mostly through their anti-inflammatory actions. The aim of our study was to investigate the effects of a synthetic PPAR-γ agonist pioglitazone on the 24 hrs-survival rate, as well as on the COX-2 and HSP70 protein expressions in brains of rats with lithium plus pilocarpine-induced status epilepticus (SE). Materials and Methods. SE was induced by administration of pilocarpine hydrochloride (30 mg/kg, i.p.) 20-24 hrs after LiCl (127 mg/kg, i.p) and 30 min after methylscopolamine nitrate (1 mg/kg, s.c.) in 80 to 90 days old male Wistar rats. The onset of SE was defined as uninterrupted generalized motor seizures lasting over 5 min or occurring with intervals lesser than 2 min. Rats were administered i.p. either pioglitazone (1 ; 3 mg/kg) or equivalent volume of the vehicle (DMSO), 10 min after the SE onset. SE was interrupted 2 hrs after its onset by an injection of diazepam (10 mg/kg, i.p.). Control animals received LiCl, saline instead of pilocarpine hydrochloride and methylscopolamine nitrate, DMSO and diazepam. Rats were sacrificed 24 hrs after the SE onset and Western blotting analyses were performed in different rat brain regions. Results. Pioglitazone treatment increased the rate of 24 hrs-survival in rats with lithium-pilocarpine induced SE, in a dose-dependent manner from 27% (vehicle treated rats) to 45.8% (pioglitazone 1 mg/kg) and 55% (pioglitazone 3 mg/kg). Western blotting analyses showed a strong induction of the COX-2 expression in animals with SE, as well as its attenuation in pioglitazone treated rats. Additionally, pioglitazone treatment led to an overexpression of the HSP70. Conclusion. The results of our study suggest a protective role of a single dose of pioglitazone in the lithium-pilocarpine model of SE in the rat. Namely, these data indicate that the inhibition of the post-SE COX-2 overexpression and an enhancement in the HSP70 expression may be involved in the protection mechanisms underlying the prominent increase of the 24 hrs-survival rate of rats in this epilepsy model.