Maria João Pinho

DNA methylation imprinting marks and DNA methyltransferase expression in human spermatogenic cell stages

Paternal imprinting marks were shown to be erased in the mouse primordial germ cells and progressively re-established throughout the male germ line development, starting in fetal prospermatogonia and continuing post-natally through the onset of meiosis. We here evaluated imprinting marks in human adult spermatogenic cells and analyzed mRNA and protein expression of DNA Methyltransferases (DNMTs). Spermatogonia A, primary and secondary spermatocytes, round spermatids and elongated spermatids/spermatozoa were isolated by micromanipulation from testicular biopsies of men with normal spermatogenesis. DNA methylation at two imprinted genes, H19 and MEST/PEG1, was analyzed using bisulphite genomic sequencing and DNMTs expression was determined by qRT-PCR and immunofluorescence. H19 was completely methylated at the spermatogonia stage in the analyzed individuals and MEST/PEG1 was completely demethylated, with the exception of few CpGs. The analysis of DNMT1, DNMT3A and 3B expression showed peaks of mRNA transcripts in primary spermatocytes and in mature ejaculated spermatozoa, with DNMT1 transcript level being the most abundant in all cell stages. Immunolocalization showed that DNMT proteins are present throughout the spermatogenic cycle, with stage-specific shuttling between the nucleus and cytoplasm. We conclude that, in humans, methylation imprints are established in spermatogonia A and are maintained in subsequent stages up to elongated spermatid/spermatozoa. Additionally, DNA methyltransferases are expressed throughout human spermatogenesis, possibly maintaining the methylation patterns in order to avoid the transmission of imprinting errors by the male gamete.

Expression of LAT1 and LAT2 amino acid transporters in human and rat intestinal epithelial cells

Summary.The present study evaluated the presence of LAT1 and LAT2 amino acid transporters in human Caco-2 cells and rat IEC-6 cells along the mucosa of the rat digestive tract. The LAT1 cDNA was amplified by PCR using two sets of primers (one specific for rat LAT1 and another simultaneously specific for human, rat and mice). The LAT2 cDNA was amplified by PCR using one set of primers simultaneously specific for human, rat and mice LAT2. The presence of LAT1 and LAT2 protein was examined by means of immunoblotting using an antibody raised against the rat LAT1 and mouse LAT2. Caco-2 and IEC-6 cells, as well as the rat intestinal mucosa, are endowed with both LAT1 and LAT2 transporter transcripts and protein. LAT1 protein is most abundant in IEC-6 cells, which is in agreement with functional data previously reported. The findings in the rat intestinal mucosa indicate that LAT1 protein is most abundant in the colon and its abundance markedly decreases at the level of jejunum and ileum, which contrast with relative homogeneous presence of LAT2 across the digestive tract. In conclusion, Caco-2 and IEC-6 cells, as well as the rat intestinal mucosa, are endowed with both LAT1 and LAT2 amino acid transporter transcripts and protein.

Oxidative stress and the genomic regulation of aldosterone-stimulated NHE1 activity in SHR renal proximal tubular cells

This study evaluated the effects of aldosterone upon Na+/H+ exchange (NHE) activity in immortalized proximal tubular epithelial (PTE) cells from the spontaneously hypertensive rat (SHR) and the normotensive controls (Wistar Kyoto rat; WKY). Increases in NHE activity after exposure to aldosterone occurred in time- and concentration-dependent manner in SHR PTE cells, but not in WKY PTE cells. The aldosterone-induced increases in NHE activity were prevented by spironolactone, but not by the glucocorticoid receptor antagonist Ru 38486. The presence of the mineralocorticoid receptor transcript was confirmed by PCR and NHE1, NHE2, and NHE3 proteins were detected by immunoblot analysis. Cariporide and EIPA, but not S3226, inhibited the aldosterone-induced increase in NHE activity, indicating that NHE1 is the most likely involved NHE isoform. Pretreatment of SHR PTE cells with actinomycin D attenuated the aldosterone-induced increases in NHE activity. The SHR PTE cells had an increased rate of H2O2 production when compared with WKY PTE cells. Treatment of cells with apocynin, a NADPH oxidase inhibitor, markedly reduced the rate of H2O2 production. The aldosterone-induced increase in NHE activity SHR PTE cells was completely prevented by apocynin. In conclusion, the aldosterone-induced stimulation of NHE1 activity is a genomic event unique in SHR PTE cells, which involves the activation of the mineralocorticoid receptor, but ultimately requires the availability of H2O2 in excess.

Treatment by testicular sperm extraction and intracytoplasmic sperm injection of 65 azoospermic patients with non‐mosaic Klinefelter syndrome with birth of 17 healthy children

The aim of this work was to present the clinical and embryological outcomes of 65 azoospermic patients with non‐mosaic Klinefelter syndrome (KS), treated by testicular sperm extraction (TESE), followed by intracytoplasmic sperm injection (ICSI), either with fresh or cryopreserved testicular spermatozoa. In total, spermatozoa were recovered in 25/65 (38.5%) of the cases. Of the 48 patients who choose to perform TESE followed by ICSI using fresh testicular spermatozoa (treatment TESE), spermatozoa was recovered in 19 patients (40%), with birth of 12 newborn. Of the 17 patients who choose to perform TESE followed by testicular sperm cryopreservation, spermatozoa were recovered in six patients (35%), with birth of one child. Of the patients who performed treatment TESE, nine went for a new cycle using cryopreserved spermatozoa. Of these, five patients had a previous failed treatment cycle (two patients, three newborn) and four with a previous success went for a new cycle (one patient, one newborn). Overall, the embryological and clinical rates were as follows: 52% of fertilization, 41% of blastocyst, 27% of implantation, 39% of live birth delivery and 47% of newborn. Of the 16 clinical pregnancies, 14 had a successful delivery (12 girls and 5 boys). The 17 newborns had a mean gestation time of 37.2 weeks (35.3% pre‐term) and a mean newborn weight of 2781.3 g (37.5% low weight). Comparisons between cycles with fresh and frozen‐thaw spermatozoa revealed higher fertilization and clinical pregnancy rates with fresh spermatozoa, with no differences regarding implantation or newborn rates. Of the 17 newborns, no abnormal karyotypes (n = 3) or numerical abnormalities in chromosomes 13, 18, 21, X and Y (n = 14) as evaluated by Multiplex Ligation–dependent Probe Amplification were observed. In conclusion, this study presents further data that reassures that men with KS have no increased risk of transmitting their genetic problem to the offspring.

Age-related changes in renal expression of oxidant and antioxidant enzymes and oxidative stress markers in male SHR and WKY rats

Oxidative stress has been hypothesized to play a role in aging and age-related disorders, such as hypertension. This study compared levels of oxidative stress and renal expression of oxidant and antioxidant enzymes in male normotensive Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR) at different ages (3 and 12 months). In the renal cortex of 3-month old SHR increases in hydrogen peroxide (H(2)O(2)) were accompanied by augmented expression of NADPH oxidase subunit Nox4 and decreased expression of antioxidant enzymes SOD1 and SOD3. A further increase in renal H(2)O(2) production and urinary TBARS was observed in 12-month old WKY and SHR as compared with 3-month old rats. Similarly, expressions of NADPH oxidase subunit p22(phox), SOD2 and SOD3 were markedly elevated with age in both strains. When compared with age-matched WKY, catalase expression was increased in 3-month old SHR, but unchanged in 12-month old SHR. Body weight increased with aging in both rat strains, but this increase was more pronounced in WKY. In conclusion, renal oxidative stress in 12-month old SHR is an exaggeration of the process already observed in the 3-month old SHR, whereas the occurrence of obesity in 12-month old normotensive rats may partially be responsible for the age-related increase in oxidative stress.

Organ-Specific Overexpression of Renal LAT2 and Enhanced Tubular L-DOPA Uptake Precede the Onset of Hypertension

Abstract—Spontaneously hypertensive rats (SHR) might have increased renal production of dopamine. l-3,4-Dihydroxyphenylalanine (l-DOPA) uptake in renal epithelial cells is promoted through the type 2 L-type amino acid transporter (LAT2), and this might rate-limit the synthesis of renal dopamine. The present study evaluated l-DOPA uptake in isolated renal proximal tubules of SHR and normotensive controls (Wistar-Kyoto rats [WKY]). Expression of LAT1 and LAT2 in the renal cortex and intestinal mucosa was also evaluated. Tubular uptake of l-DOPA in WKY and SHR was a saturable process, being greater in the latter than the former at both 4 and 12 weeks of age. cDNA fragments (LAT1, 688 bp; LAT2, 729 bp) labeled with 32P were used as probes for Northern blot analysis. Expression of LAT2 in SHR kidneys was higher than in WKY kidneys. This increase was more marked at 4 than at 12 weeks of age. Intestinal LAT2 expression, however, was identical in SHR and WKY at both 4 and 12 week of age. By Northern blot analysis, the LAT1 transcript was not identified in either the kidney or intestine. Kidney total RNA was then reverse-transcribed and amplified by polymerase chain reaction with specific primers for LAT1. The presence of a fragment of the expected size for LAT1 led to the conclusion that LAT1 mRNA is a rare message in kidney. We conclude that overexpression of LAT2 in the SHR kidney might contribute to the enhanced l-DOPA uptake, which is organ specific and precedes the onset of hypertension.

Renal amino acid transport systems and essential hypertension

Several clinical and animal studies suggest that “blood pressure goes with the kidney,” that is, a normotensive recipient of a kidney genetically programmed for hypertension will develop hypertension. Intrarenal dopamine plays an important role in the pathogenesis of hypertension by regulating epithelial sodium transport. The candidate transport systems for L‐DOPA, the source for dopamine, include the sodium‐dependent systems B0, B0,+, and y+L, and the sodium‐independent systems L (LAT1 and LAT2) and b0+. Renal LAT2 is overexpressed in the prehypertensive spontaneously hypertensive rat (SHR), which might contribute to enhanced L‐DOPA uptake in the proximal tubule and increased dopamine production, as an attempt to overcome the defect in D1 receptor function. On the other hand, it has been recently reported that impaired arginine transport contributes to low renal nitric oxide bioavailability observed in the SHR renal medulla. Here we review the importance of renal amino acid transporters in the kidney and highlight pathophysiological changes in the expression and regulation of these transporters in essential hypertension. The study of the regulation of renal amino acid transporters may help to define the underlying mechanisms predisposing individuals to an increased risk for development of hypertension.—Pinto, V., Pinho, M. J., Soares‐da‐Silva, P., Renal amino acid transport systems and essential hypertension. FASEB J. 27, 2927–2938 (2013). www.fasebj.org

Cloning and gene silencing of LAT2, the L-3,4-dihydroxyphenylalanine (l-DOPA) transporter, in pig renal LLC-PK1 epithelial cells

Organ‐specific overexpression of type 2 l‐amino acid transporter (LAT2) in the kidney of the spontaneous hypertensive rat (SKR), 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 irans‐stimulation of l‐DOPA outward transfer.—Soares‐da‐Silva, P., Serrao, M. P., João Pinho, M., Bonifacio, M.J. Cloning and gene silencing of LAT2, the L‐3,4‐dihydroxyphenylalanine (l‐DOPA) transporter, in pig renal LLC‐PK1 epithelial cells. FASEB J. 18, 1489–1498 (2004)

Characterization of rat heart alkaline phosphatase isoenzymes and modulation of activity

Alkaline phosphatase (ALP) is important in calcification and its expression seems to be associated with the inflammatory process. We investigated the in vitro acute effects of compounds used for the prevention or treatment of cardiovascular diseases on total ALP activity from male Wistar rat heart homogenate. ALP activity was determined by quantifying, at 410 nm, the p-nitrophenol released from p-nitrophenylphosphate (substrate in Tris buffer, pH 10.4). Using specific inhibitors of ALP activity and the reverse transcription-polymerase chain reaction, we showed that the rat heart had high ALP activity (31.73 +/- 3.43 nmol p-nitrophenol.mg protein-1.min-1): mainly tissue-nonspecific ALP but also tissue-specific intestinal ALP type II. Both ALP isoenzymes presented myocardial localization (striated pattern) by immunofluorescence. ALP was inhibited a) strongly by 0.5 mM levamisole, 2 mM theophylline and 2 mM aspirin (91, 77 and 84%, respectively) and b) less strongly by 2 mM L-phenylalanine, 100 mL polyphenol-rich beverages and 0.5 mM progesterone (24, 21 to 29 and 11%, respectively). beta-estradiol and caffeine (0.5 and 2 mM) had no effect; 0.5 mM simvastatin and 2 mM atenolol activated ALP (32 and 36%, respectively). Propranolol (2 mM) tended to activate ALP activity and corticosterone activated (18%) and inhibited (13%) (0.5 and 2 mM, respectively). We report, for the first time, that the rat heart expresses intestinal ALP type II and has high total ALP activity. ALP activity was inhibited by compounds used in the prevention of cardiovascular pathology. ALP manipulation in vivo may constitute an additional target for intervention in cardiovascular diseases.

Increased Arterial Blood Pressure and Vascular Remodeling in Mice Lacking Salt-Inducible Kinase 1 (SIK1)

Rationale: In human genetic studies a single nucleotide polymorphism within the salt-inducible kinase 1 (SIK1) gene was associated with hypertension. Lower SIK1 activity in vascular smooth muscle cells (VSMCs) leads to decreased sodium-potassium ATPase activity, which associates with increased vascular tone. Also, SIK1 participates in a negative feedback mechanism on the transforming growth factor-&bgr;1 signaling and downregulation of SIK1 induces the expression of extracellular matrix remodeling genes. Objective: To evaluate whether reduced expression/activity of SIK1 alone or in combination with elevated salt intake could modify the structure and function of the vasculature, leading to higher blood pressure. Methods and Results: SIK1 knockout (sik1−/−) and wild-type (sik1+/+) mice were challenged to a normal- or chronic high-salt intake (1% NaCl). Under normal-salt conditions, the sik1−/− mice showed increased collagen deposition in the aorta but similar blood pressure compared with the sik1+/+ mice. During high-salt intake, the sik1+/+ mice exhibited an increase in SIK1 expression in the VSMCs layer of the aorta, whereas the sik1−/− mice exhibited upregulated transforming growth factor-&bgr;1 signaling and increased expression of endothelin-1 and genes involved in VSMC contraction, higher systolic blood pressure, and signs of cardiac hypertrophy. In vitro knockdown of SIK1 induced upregulation of collagen in aortic adventitial fibroblasts and enhanced the expression of contractile markers and of endothelin-1 in VSMCs. Conclusions: Vascular SIK1 activation might represent a novel mechanism involved in the prevention of high blood pressure development triggered by high-salt intake through the modulation of the contractile phenotype of VSMCs via transforming growth factor-&bgr;1-signaling inhibition.