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Renalase is a recently identified FAD/NADH-dependent amine oxidase mainly expressed in kidney that is secreted into blood and urine where it was suggested to metabolize catecholamines. The present study evaluated central and peripheral dopaminergic activities in the renalase knockout (KO) mouse model and examined the changes induced by recombinant renalase (RR) administration on plasma and urine catecholamine levels. Compared with wild-type (WT) mice, KO mice presented increased plasma levels of epinephrine (Epi), norepinephrine (NE), and dopamine (DA) that were accompanied by increases in the urinary excretion of Epi, NE, DA. In addition, the KO mice presented an increase in urinary DA-to-L-3,4-dihydroxyphenylalanine (L-DOPA) ratios without changes in renal tubular aromatic-L-amino acid decarboxylase (AADC) activity. By contrast, the in vivo administration of RR (1.5 mg/kg sc) to KO mice was accompanied by significant decreases in plasma levels of Epi, DA, and L-DOPA as well as in urinary excretion of Epi, DA, and DA-to-L-DOPA ratios notwithstanding the accompanied increase in renal AADC activity. In addition, the increase in renal DA output observed in renalase KO mice was accompanied by an increase in the expression of the L-type amino acid transporter like (LAT) 1 that is reversed by the administration of RR in these animals. These results suggest that the overexpression of LAT1 in the renal cortex of the renalase KO mice might contribute to the enhanced L-DOPA availability/uptake and consequently to the activation of the renal dopaminergic system in the presence of renalase deficiency. [ABSTRACT FROM AUTHOR]

Natural killer (NK)-cells are a lymphocyte population playing a critical role in the immune surveillance against tumors and virally infected cells. The development of human hematopoietic stem cells (hHSC) into fully differentiated NK-cells pass through discrete stages of differentiation involving a variety of factors such as cytokines, membrane factors, and transcription factors (TFs). Because there is lack of studies in this field, we decided to perform an extended analysis of TFs during in vitro differentiation of NK-cells. At several points of differentiation, cells were characterized by their mRNA expression either for NK-cell cell markers, for a number of mature NK-cell receptors or a large panel of TFs. Our data suggests that some TFs (ID2, EGR-2 and T-BET) play a role in NK-cell commitment, differentiation and maturation. Although delayed on its expression, BLIMP1 also seems to be involved in differentiation and maturation of NK cells, but not in NK-cell commitment. E4BP4 and TOX are more related with initial stages of NK-cell commitment. PU.1, MEF, Ikaros, EGR-1, BCL11B and IRF-2 revealed less involvement in maturation and were more associated with NK-cell commitment and pNK cell production. GATA-3 showed a differential role during the ontogeny of NK-cells. We show that assessment of the transcripts present in the differentiating NK-cells demonstrated, a pattern of preserved and differential gene expression remarkably similar to that seen in mice except for E4BP4 that showed constant downregulation throughout the culture period. A thorough understanding of NK-cell developmental mechanisms is important as it may enable future therapeutic manipulation. [ABSTRACT FROM AUTHOR]

Natural Killer (NK)-cells are peripheral blood lymphocytes that represent an important arm of the innate immune system. NK-cells play a critical role in the immune surveillance against tumors and virally infected cells in a major histocompatibiliy complex (MHC)-unrestricted fashion. We have explored such capacities of NK-cells after differentiation from hematopoietic stem and progenitor cells derived from human umbilical cord blood. Several culture conditions have been established supporting proliferation and subsequent differentiation of these cells in terms of receptor expression and specific lysis depending on the growth conditions in the presence and absence of supportive stromal feeders. We show that acquisition of Killer Immunoglobulin Receptor (KIR) as well as NK Cytotoxicity Receptor expressions is independent of culture condition whereas absence of stromal feeders did not support acquisition of CD94/NKG2A expression. Such KIR-positive/NKG2A-negative cells generated under different culture conditions showed strong and specific cytolytic activity which could have impact on further immunotherapeutic strategies. [ABSTRACT FROM AUTHOR]

The present study examined the expression of type 1 L-amino acid transporter (LAT1) and its associated glycoprotein 4F2hc in freshly isolated renal proximal tubules and immortalized renal proximal tubular epithelial (PTE) cells from spontaneously hypertensive (SHR) and normotensive (WKY) rats. The study also examined the inward and outward transport of [14C]-L-leucine, the preferred substrate of LAT1. The abundance of LAT1 and 4F2hc was greater in SHR than in WKY, both in freshly isolated renal proximal tubules and immortalized renal proximal tubular cells. In the absence of extracellular Na+ the BCH (2-aminobicyclo(2,2,1)-heptane-2-carboxylic acid)-sensitive [14C]-L-leucine uptake in SHR PTE cells was ñ50% that observed in WKY PTE cells (77±4 vs 164±7 pmol/mg protein). In the absence of extracellular Na+ the affinity of the transporter for the substrate in WKY PTE cells was 7.7-fold that in SHR cells, as evidenced by lower K0.5 values. Gene silencing with a LAT1 siRNA and a 4F2hc siRNA signficantly reduced LAT1 and 4F2hc expression, which was accompanied by a marked reduction in Na+-independent [14C]-L-leucine uptake in both SHR and WKY PTE cells. The spontaneous and L-leucine-stimulated outward transfer of [14C]-L-leucine was Na+-independent in both SHR and WKY PTE cells. The spontaneous [14C]-L-leucine efflux was higher in WKY than in SHR PTE cells and the potency of L-leucine to stimulate [14C]-L-leucine efflux in WKY (EC50 = 9 μM) was greater than in SHR PTE cells (EC50 = 41 μM). It is concluded that the SHR kidney overexpress LAT1/4F2hc units which display low affinity for L-leucine transport. Copyright © 2007 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

This study examined the inward transport of L-[14C]alanine, an ASCT2 preferential substrate, in monolayers of immortalized renal proximal tubular epithelial (PTE) cells from Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats. The expression of ASCT2 in WKY and SHR PTE cells and kidney cortices from WKY and SHR was also evaluated. L-[14C]alanine uptake was highly dependent on extracellular Nat Replacement of NaCl by LiCI or choline chloride abolished transport activity in SHR and WKY PTE cells. In the presence of the system L inhibitor BCH, Na+-dependent L-alanine uptake in WKY and SHR PTE cells was inhibited by alanine, serine, and cysteine, which is consistent with amino acid transport through ASCT2. The saturable component of Nat-dependent L-alanine transport under V+ax conditions in SHR PTE cells was one-half of that in WKY PTE cells, with similar K,,+ values. Differences in magnitude of Nat- dependent L-alanine uptake through ASCT2 between WKY and SHR PTE cells correlated positively with differences in ASCT2 protein expression, this being more abundant in WKY PTE cells. Abundance of ASCT2 transcript and protein in kidney cortices of SHR rats was also lower than that in normotensive WKY rats. In conclusion, immortalized SHR and WKY PTE cells take up L-alanine mainly through a high-affinity Na+-dependent amino acid transporter, with functional features of ASCT2 transport. The activity and expression of the ASCT2 transporter were considerably lower in the SHR cells. [ABSTRACT FROM AUTHOR]

This study evaluated in spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) the response to salt loading of the renal dopaminergic system and transcript abundance of Nat-independent (LAT1 and LAT2) and Na+-dependent (ASCT2 and B°AT1) amino acid transporters potentially involved in renal tubular uptake of L-DOPA. Rats were fed normal (NS)- or high (HS; 1% saline as drinking water)-salt intake for 24 h. Transcript abundance of amino acid transporters was age dependent, differently regulated in WKY and SHR and responded differently to salt intake. HS intake similarly increased urinary dopamine in 4-wk-old SHR and WKY. At 12 wk of age, HS intake increased urinary dopamine in SHR, but not in WKY. Changes in urinary dopamine paralleled changes in the uptake of L-DOPA in isolated renal tubules from 4- and 1 2-wk-old WKY and SHR on NS and HS intake. At 12 wk of age, HS intake was accompanied by decreases in LAT1 and LAT2 transcript abundance in WKY and SHR. ASCT2 and B°AT1 expression was significantly decreased in both 4- and 12-wk-old WKY and in 4-wk-old SHR on HS intake. By contrast, HS intake increased ASCT2 and B°AT1 expression in 12-wk-old SHR. It is concluded that salt-sensitive mechanisms influence LAT1, LAT2, ASCT2, and B°AT1 gene transcription. Differences in urinary dopamine and tubular uptake of L-DOPA between WKY and SHR during HS intake, namely in 12-wk-old animals, may result from increases in the ASCT2 and B°AT1 mRNA levels and less pronounced decreases in LAT2 expression. [ABSTRACT FROM AUTHOR]

Organ-specific overexpression of type 2 L-amino acid transporter (LAT2) in the kidney of the spontaneous hypertensive rat (SHR), accompanied by an enhanced ability to take up L-DOPA, may constitute the basis for the enhanced renal production of dopamine in the SHR in an attempt overcome the deficient dopamine-mediated natriuresis. To understand the physiological role of LAT2-mediated L-DOPA handling, we used 21-nucleotide small interfering RNA duplexes (siRNA) to specifically suppress LAT2 expression in LLC-PK1 cells, a cell line that retains several properties of proximal tubular epithelial cells and takes up L-DOPA largely through Na+-independent transporters. After cloning the LLC-PK1 LAT2 gene, one target region of LAT2 mRNA (nt 97-117) was selected by scanning the length of the LAT2 gene for AA-dinucleotide sequences and downstream 19 nucleotides. Levels of LAT2 cDNA, determined by real-time quantitative RT-PCR, were markedly (P < 0.05) reduced by LAT2 siRNA but not by the mismatch LAT2 siRNA. The LAT2 siRNA but not the mismatch LAT2 siRNA, reduced by 85% [14C]-L-DOPA accumulation, a time- and concentration-dependent effect. The efflux of intracellular [14C]-L-DOPA was markedly increased (P < 0.05) by L-DOPA and L-leucine. The [14C]-L-DOPA outward transport was decreased 90% by LAT2 siRNA, but not by the mismatch LAT2 siRNA. However, treatment with the siRNA LAT2 did not affect the L-DOPA-induced fractional outflow of [14C]-L-DOPA. The Na+independent and pH-sensitive L-DOPA transporter may include the hetero amino acid exchanger LAT2, whose activation results in trans-stimulation of L-DOPA outward transfer. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are regulators of gene expression and play a key role in the pathophysiology of various disease processes, namely cardiovascular disease. This study aimed at the identification of the miRNA expression pattern of peripheral blood mononuclear cells derived from hypertensive subjects (HT) and non-hypertensive subjects with cardiac disease (CD) using a genome-wide approach. Furthermore, we explored the potential of orthogonal partial least squares-discrimination analysis (OPLS-DA) to analyze miRNA expression data. The homogeneity of the population was assessed with Principal Component Analysis (PCA) and verified by the clear separation of the three groups on the basis of the miRNA expression levels. Moreover, local PCA of HT group indicates the splitting of this group in two distinct subgroups. This distinction correlates with two subject’s characteristics (smoking habits and history of myocardial infarction) that are closest to the CD group. OPLS-DA analysis confirms the separation of three groups and identified the most important miRNA to discrimination between groups. Among the most influential miRNA to the positive differentiation of HT from CD and control, several of them are relevant to endothelial dysfunction. On the other hand, miRNAs associated with angiogenesis positively differentiate CD from HT and control. Mir-186, a regulator of myogenin, is the most important miRNA to discriminate HT from CD. These results provide evidence of altered miRNA profile in hypertensives and cardiac disease and demonstrate that PCA and OPLS-DA are very useful and robust tools to analyze how different miRNAs contribute to the separations of groups and to find interesting patterns within the multidimensional data. [ABSTRACT FROM AUTHOR]

Objective: There is a rising demand for assisted reproductive medicine, including sperm, oocyte and embryo donation. Besides medical and legal considerations, genetic testing, including carrier screening for multiple autosomal and X-linked recessive disorders plays an essential role in evaluating hereditary risk among donors and therefore exclude them from the donation process., Methods: A retrospective study was conducted on oocyte donors from a private clinic of assisted reproduction who underwent genetic testing between June 2014 and September 2023. Pre and post-test procedures were performed at the private clinic while karyotyping and carrier screening for Cystic Fibrosis, Fragile X syndrome and Spinal Muscular Atrophy were performed at the Genetic Unit of Faculty of Medicine, University of Porto., Results: Among 581 donors, 81 women were excluded from the donation process since 5/563 had an alteration in karyotype, 57/581 were carriers of a Cystic Fibrosis Transmembrane conductance Regulator pathogenic variant or had a 5T allele, 11/394 had Survival of Motor Neuron 1 deletion and 8/426 had an intermediate or premutation allele in Fragile X Messenger Ribonucleoprotein gene. While recommendations from fertility societies advocate for comprehensive screening, opinions differ on the mandatory implementation of expanded carrier screening., Conclusions: In conclusion, the genetic tests and the pre and post-test counseling is imperative to optimize reproductive outcomes in the oocyte donation process.

Natural killer (NK)-cells are a lymphocyte population playing a critical role in the immune surveillance against tumors and virally infected cells. The development of human hematopoietic stem cells (hHSC) into fully differentiated NK-cells pass through discrete stages of differentiation involving a variety of factors such as cytokines, membrane factors, and transcription factors (TFs). Because there is lack of studies in this field, we decided to perform an extended analysis of TFs during in vitro differentiation of NK-cells. At several points of differentiation, cells were characterized by their mRNA expression either for NK-cell cell markers, for a number of mature NK-cell receptors or a large panel of TFs. Our data suggests that some TFs (ID2, EGR-2 and T-BET) play a role in NK-cell commitment, differentiation and maturation. Although delayed on its expression, BLIMP1 also seems to be involved in differentiation and maturation of NK cells, but not in NK-cell commitment. E4BP4 and TOX are more related with initial stages of NK-cell commitment. PU.1, MEF, Ikaros, EGR-1, BCL11B and IRF-2 revealed less involvement in maturation and were more associated with NK-cell commitment and pNK cell production. GATA-3 showed a differential role during the ontogeny of NK-cells. We show that assessment of the transcripts present in the differentiating NK-cells demonstrated, a pattern of preserved and differential gene expression remarkably similar to that seen in mice except for E4BP4 that showed constant downregulation throughout the culture period. A thorough understanding of NK-cell developmental mechanisms is important as it may enable future therapeutic manipulation.

Cl(-) /HCO (3)(-) exchanger and Na(+) /H(+) exchanger 3 are the main transporters responsible for NaCl reabsorption in kidney proximal tubules (PT). It is well accepted that membrane exchangers can be regulated by reactive oxygen species (ROS). In the kidney, ROS are known to contribute to decreases in Na(+) excretion and consequently increase blood pressure. The present study investigated mechanisms by which H(2) O(2) -induced stimulation of Cl(-) /HCO (3)(-) exchanger activity is enhanced in proximal tubular epithelial (PTE) cells immortalized from spontaneously hypertensive rats (SHR) as compared to normotensive Wistar Kyoto (WKY). H(2) O(2) decreased K(m) values for Cl(-) /HCO (3)(-) exchanger activity in SHR PTE cells, but had no effect on the kinetic parameters in WKY cells. DTDP stimulated in a concentration-dependent manner Cl(-) /HCO (3)(-) exchanger activity in both cell lines, but SHR PTE cells were 2.4-fold more responsive to this oxidant. In contrast, thimerosal had no effect on exchanger activity in both cell lines. The effects of H(2) O(2) and DTDP upon the exchanger activity were blocked by DTT in WKY and SHR PTE cells. Similar to H(2) O(2), DTDP decreased K(m) values for Cl(-) /HCO (3)(-) exchanger activity in SHR PTE cells. Basal content of free thiol groups was higher in WKY PTE cells than in SHR. Upon H(2) O(2) treatment the free thiol groups decreased in both cell lines; however, this decrease was more pronounced in WKY cells. In conclusion, in SHR PTE cells H(2) O(2) stimulates Cl(-) /HCO (3)(-) exchanger activity via modification of thiol groups of intracellular and/or transmembrane protein. Furthermore, the thiol oxidation-dependent pathway also increases the HCO (3)(-) affinity in SHR PTE cells., (Copyright © 2011 Wiley Periodicals, Inc.)

The aim of this study was to investigate the putative influence of some pharmacological agents and drugs of abuse upon the apical uptake of butyrate (BT) into Caco-2 cells. The apical uptake of (14)C-BT by Caco-2 cells was (1) time and concentration dependent, (2) pH dependent, (3) Na(+) independent and Cl(-) dependent, (4) energy dependent, (5) inhibited by several BT structural analogues (acetate, propionate, alpha-ketobutyrate, pyruvate, lactate), (6) insensitive to the anion exchange inhibitors DIDS and SITS and (7) inhibited by the monocarboxylate transport (MCT) inhibitors NPPB and pCMB. These characteristics are compatible with an involvement of MCT1-mediated transport. Acutely, uptake of a low concentration of (14)C-BT (10 microM) was reduced by acetaldehyde, acetylsalicylic acid, indomethacin, caffeine and theophylline and increased by MDMA. Chronically, uptake was increased by caffeine and decreased by tetrahydrocannabinol and MDMA; reverse transcription quantitative real-time PCR analysis showed that these three compounds decreased the mRNA levels of MCT1. Acutely, acetaldehyde, indomethacin and MDMA reduced the uptake of a high concentration of (14)C-BT (20 mM), and acetylsalicylic acid increased it. Chronically, none of the compounds affected uptake. Acetaldehyde, indomethacin and propionate seem to be competitive inhibitors of (14)C-BT uptake. Acetylsalicylic acid simultaneously increased the K (m) and the V (max) of (14)C-BT uptake. In conclusion, MCT1-mediated transport of (14)C-BT in Caco-2 cells is modulated by either acute or chronic exposure to some pharmacological agents and drugs of abuse (acetaldehyde, acetylsalicylic acid, indomethacin, caffeine, theophylline and the drugs of abuse tetrahydrocannabinol and MDMA).

Organ-specific overexpression of type 2 L-amino acid transporter (LAT2) in the kidney of the spontaneous hypertensive rat (SHR), accompanied by an enhanced ability to take up L-DOPA, may constitute the basis for the enhanced renal production of dopamine in the SHR in an attempt overcome the deficient dopamine-mediated natriuresis. To understand the physiological role of LAT2-mediated L-DOPA handling, we used 21-nucleotide small interfering RNA duplexes (siRNA) to specifically suppress LAT2 expression in LLC-PK1 cells, a cell line that retains several properties of proximal tubular epithelial cells and takes up L-DOPA largely through Na+-independent transporters. After cloning the LLC-PK1 LAT2 gene, one target region of LAT2 mRNA (nt 97-117) was selected by scanning the length of the LAT2 gene for AA-dinucleotide sequences and downstream 19 nucleotides. Levels of LAT2 cDNA, determined by real-time quantitative RT-PCR, were markedly (P<0.05) reduced by LAT2 siRNA but not by the mismatch LAT2 siRNA. The LAT2 siRNA but not the mismatch LAT2 siRNA, reduced by 85% [14C]-L-DOPA accumulation, a time- and concentration-dependent effect. The efflux of intracellular [14C]-L-DOPA was markedly increased (P<0.05) by L-DOPA and L-leucine. The [14C]-L-DOPA outward transport was decreased 90% by LAT2 siRNA, but not by the mismatch LAT2 siRNA. However, treatment with the siRNA LAT2 did not affect the L-DOPA-induced fractional outflow of [14C]-L-DOPA. The Na+-independent and pH-sensitive L-DOPA transporter may include the hetero amino acid exchanger LAT2, whose activation results in trans-stimulation of L-DOPA outward transfer.-Soares-da-Silva, P., Serrão, M. P., João Pinho, M., Bonifácio, M. J. Cloning and gene silencing of LAT2, the L-3,4-dihydroxyphenylalanine (L-DOPA) transporter, in pig renal LLC-PK1 epithelial cells.