Maternal dexamethasone exposure does not affect glucose tolerance but alters renal haemodynamics in F1 rats in a sex-dependent manner.

INTRODUCTION: Prenatal programming with dexamethasone increases the risk of the development of hyperglycaemia and insulin resistance, leading to diabetes in adulthood. Dexamethasone also causes a decline in renal glomerular filtration in the adult offspring. Sodium-glucose cotransporter-2 (SGLT2) plays a significant role in regulating blood glucose and renal haemodynamics in diabetic patients. However, the role of SGLT2 in dexamethasone-induced programming and the putative sex-dependent effects on the changes named earlier is unknown. Therefore, this study aimed to investigate the impact of maternal dexamethasone treatment on glucose tolerance, insulin sensitivity, renal perfusion and renal function in adult male and female offspring and the possible contribution of SGLT2 to these changes. METHODS AND RESULTS: Pregnant Sprague Dawley rats (F0 ) were treated with either vehicle or dexamethasone (0.2 mg/kg ip) from gestation Day 15 to 20. F1 males and F1 females were randomly selected from each mother at 4 months of age. There was no change in serum Na+ , Na+ excretion rate, glucose tolerance or insulin sensitivity in F1 male or female rats. However, dexamethasone caused significant glomerular hypertrophy and decreases in CSinistrin and CPAH indicating decreased glomerular filtration rate and renal plasma flow, respectively, in dexamethasone-treated F1 male but not female rats. Dexamethasone did not affect SGLT2 mRNA or protein expression in F1 males or females. CONCLUSION: We conclude that dexamethasone-mediated prenatal programming of glomerular volume, renal function and haemodynamics is sex-dependent, occurring only in adult male offspring.

Viability and Contractility of Rat Brain Pericytes in Conditions That Mimic Stroke; an in vitro Study.

Reopening of the cerebral artery after occlusion often results in "no-reflow" that has been attributed to the death and contraction (rigor mortis) of pericytes. Since this hypothesis still needs to be confirmed, we explored the effects of oxygen glucose deprivation (OGD) on viability and cell death of primary rat pericytes, in the presence or absence of neurovascular unit-derived cytokines. Two morphodynamic parameters, single cell membrane mobility (SCMM) and fractal dimension (Df), were used to analyze the cell contractions and membrane complexity before and after OGD. We found a marginal reduction in cell viability after 2-6 h OGD; 24 h OGD caused a large reduction in viability and a large increase in the number of apoptotic and dead cells. Application of erythropoietin (EPO), or a combination of EPO and endothelial growth factor (VEGFA1-165) during OGD significantly reduced cell viability; application of Angiopoietin 1 (Ang1) during OGD caused a marginal, insignificant increase in cell viability. Simultaneous application of EPO, VEGFA1-165, and Ang1 significantly increased cell viability during 24 h OGD. Twenty minutes and one hour OGD both significantly reduced SCMM compared to pre-OGD values, while no significant difference was seen in SCMM before and after 3 h OGD. There was a significant decrease in membrane complexity (Df) at 20 min during the OGD that disappeared thereafter. In conclusion, OGD transiently affected cell mobility and shape, which was followed by apoptosis in cultured pericytes. Ang1 may have a potentiality for preventing from the OGD-induced apoptosis. Further studies could clarify the relationship between cell contraction and apoptosis during OGD.

Effects of hypoxia, glucose deprivation and recovery on the expression of nucleoside transporters and adenosine uptake in primary culture of rat cortical astrocytes.

The aim of this study was to explore effects of hypoxia, glucose deprivation (HGD) and recovery on expression and activities of equilibrative nucleoside transporters (rENT) and concentrative nucleoside transporters (rCNT) in rat astrocytes in primary culture. Amounts of cellular ATP in the control group (CG, 5% CO(2) in air, medium containing 7 mM D-glucose, 1 mM Na(+)-pyruvate, 1 h), HGD group (2% O(2)/5% CO(2) in N(2), pyruvate-free medium containing 1.5 mM D-glucose and 10 mM 2-deoxy-D-glucose, 1 h) and recovery group (RG, HGD for 1 h, followed by 1 h exposure to the same conditions as the CG) were (nmol/mg protein, n = 4) 18 +/- 1.6, 4.9 +/- 0.6 and 10.1 +/- 0.8, respectively. Extracellular adenosine concentrations increased from (nM, n = 3) 42 +/- 4 in the CG, to 99 +/- 8 in the HGD group and 86 +/- 3 in the RG. Real-time PCR and immunoblotting revealed that in the HGD group and RG, the amounts of rENT1 mRNA and protein were reduced to 40 and 50%, when compared to the CG, respectively. Astrocyte cultures took up [(3)H]adenosine by concentrative and equilibrative transport processes; however, rENT1-mediated uptake was absent in the RG and cultures from the RG took up significantly less [(3)H]adenosine by equilibrative mechanisms than cultures from the CG.

Expression and functional activity of nucleoside transporters in human choroid plexus.

BACKGROUND: Human equilibrative nucleoside transporters (hENTs) 1-3 and human concentrative nucleoside transporters (hCNTs) 1-3 in the human choroid plexus (hCP) play a role in the homeostasis of adenosine and other naturally occurring nucleosides in the brain; in addition, hENT1, hENT2 and hCNT3 mediate membrane transport of nucleoside reverse transcriptase inhibitors that could be used to treat HIV infection, 3'-azido-3'-deoxythymidine, 2'3'-dideoxycytidine and 2'3'-dideoxyinosine. This study aimed to explore the expression levels and functional activities of hENTs 1-3 and hCNTs 1-3 in human choroid plexus. METHODS: Freshly-isolated pieces of lateral ventricle hCP, removed for various clinical reasons during neurosurgery, were obtained under Local Ethics Committee approval. Quantification of mRNAs that encoded hENTs and hCNTs was performed by the hydrolysis probes-based reverse transcription real time-polymerase chain reaction (RT-qPCR); for each gene of interest and for 18 S ribosomal RNA, which was an endogenous control, the efficiency of PCR reaction (E) and the quantification cycle (Cq) were calculated. The uptake of [(3)H]inosine by the choroid plexus pieces was investigated to explore the functional activity of hENTs and hCNTs in the hCP. RESULTS: RT-qPCR revealed that the mRNA encoding the intracellularly located transporter hENT3 was the most abundant, with E(-Cq )value being only about 40 fold less that the E(-Cq )value for 18 S ribosomal RNA; mRNAs encoding hENT1, hENT2 and hCNT3 were much less abundant than mRNA for the hENT3, while mRNAs encoding hCNT1 and hCNT2 were of very low abundance and not detectable. Uptake of [(3)H]inosine by the CP samples was linear and consisted of an Na(+)-dependent component, which was probably mediated by hCNT3, and Na(+)-independent component, mediated by hENTs. The latter component was not sensitive to inhibition by S-(4-nitrobenzyl)-6-thioinosine (NBMPR), when used at a concentration of 0.5 muM, a finding that excluded the involvement of hENT1, but it was very substantially inhibited by 10 muM NBMPR, a finding that suggested the involvement of hENT2 in uptake. CONCLUSION: Transcripts for hENT1-3 and hCNT3 were detected in human CP; mRNA for hENT3, an intracellularly located nucleoside transporter, was the most abundant. Human CP took up radiolabelled inosine by both concentrative and equilibrative processes. Concentrative uptake was probably mediated by hCNT3; the equilibrative uptake was mediated only by hENT2. The hENT1 transport activity was absent, which could suggest either that this protein was absent in the CP cells or that it was confined to the basolateral side of the CP epithelium.

Blood-brain barrier efflux transport of pyrimidine nucleosides and nucleobases in the rat.

The brain efflux index (BEI), a measurement of blood-brain barrier (BBB) efflux transport, was estimated at 15 s, 30 s, 1 min, 3 min and 10 min after intracerebral injection of [14C]pyrimidines. An initial steep increase of the BEI values over time was observed for [14]uracil and [14C]thymine, followed by a more moderate increase after 1 min. For the corresponding nucleosides, [14C]uridine and [14C]thymidine, the increase of BEI values over time was less steep and linear between 30 s and 3 min. The apparent BBB efflux clearances for [14C]uridine, [14C]thymidine, [14C]uracil and [14C]thymine were (microl/min/g): 95.2 +/- 12.1, 125.3 +/- 18.4, 290.4 +/- 28 and 358.5 +/- 32.5, respectively, which is at least several folds higher than the predicted BBB influx clearances of uridine, uracil and thymidine. Quick depletion of brain parenchyma from brain microvasculature has revealed that [14C] radioactivity accumulated in brain microvessels after injection of nucleosides [14C]thymidine and [14C]uridine, but that was not observed when nucleobases, [14C]thymine and [14C]uracil, were injected. Reverse transcriptase-PCR revealed that the rat brain and liver (positive control) express dihydropyrimidine dehydrogenase, a key enzyme in pyrimidine nucleobase catabolism. Two bands representing spliced variants have been detected with the relative density of the bands (expressed relative to the density of glyceraldehyde3-phosphate dehydrogenase bands, mean +/- SEM from 3 separate samples) 0.16 +/- 0.06 and 0.04 +/- 0.01 (brain) and 0.49 +/- 0.1 and 0.07 +/- 0.01 (liver). Overall, these results indicate that the net direction of pyrimidine BBB transport is the efflux transport; rapid BBB efflux transport and metabolic breakdown of pyrimidine nucleobases appear to be important for brain homeostasis.