Reactive oxygen species impair Na+ transport and renal components of the renin-angiotensin-aldosterone system after paraquat poisoning.

Paraquat (1,1'-dimethyl-4,4'-bipyridyl dichloride) is an herbicide widely used worldwide and officially banned in Brazil in 2020. Kidney lesions frequently occur, leading to acute kidney injury (AKI) due to exacerbated reactive O2 species (ROS) production. However, the consequences of ROS exposure on ionic transport and the regulator local renin-angiotensin-aldosterone system (RAAS) still need to be elucidated at a molecular level. This study evaluated how ROS acutely influences Na+-transporting ATPases and the renal RAAS. Adult male Wistar rats received paraquat (20 mg/kg; ip). After 24 h, we observed body weight loss and elevation of urinary flow and serum creatinine. In the renal cortex, paraquat increased ROS levels, NADPH oxidase and (Na++K+)ATPase activities, angiotensin II-type 1 receptors, tumor necrosis factor-α (TNF-α), and interleukin-6. In the medulla, paraquat increased ROS levels and NADPH oxidase activity but inhibited (Na++K+)ATPase. Paraquat induced opposite effects on the ouabain-resistant Na+-ATPase in the cortex (decrease) and medulla (increase). These alterations, except for increased serum creatinine and renal levels of TNF-α and interleukin-6, were prevented by 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (tempol; 1 mmol/L in drinking water), a stable antioxidant. In summary, after paraquat poisoning, ROS production culminated with impaired medullary function, urinary fluid loss, and disruption of Na+-transporting ATPases and angiotensin II signaling.

Undernutrition - thirty years of the Regional Basic Diet: the legacy of Naíde Teodósio in different fields of knowledge.

Undernutrition is characterized by an imbalance of essential nutrients with an insufficient nutritional intake, a disorder in which the clinical manifestations in most cases are the result of the economic and social context in which the individual lives. In 1990, the study by the medical and humanitarian Naíde Teodósio (1915-2005) and coworkers, which formulated the Regional Basic Diet (RBD) model for inducing undernutrition, was published. This diet model took its origin from the observation of the dietary habits of families that inhabited impoverished areas from the Pernambuco State. RBD mimics an undernutrition framework that extends not only to the Brazilian population, but to populations in different regions worldwide. The studies based on RBD-induced deficiencies provide a better understanding of the impact of undernutrition on the pathophysiological mechanisms underlying the most diverse prevalent diseases. Indexed papers that are analyzed in this review focus on the importance of using RBD in different areas of knowledge. These papers reflect a new paradigm in translational medicine: they show how the study of pathology using the RBD model in animals over the past 30 years has and still can help scientists today, shedding light on the mechanisms of prevalent diseases that affect impoverished populations.

Histone Deacetylase Activity and the Renin-Angiotensin-Aldosterone System: Key Elements in Cardiorenal Alterations Provoked by Chronic Malnutrition in Male Adult Rats.

BACKGROUND/AIMS: Chronic malnutrition (M) affects >1 billion people worldwide. Epidemiological data point to long-term renal and cardiovascular outcomes (e.g. arterial hypertension, cardiorenal syndromes). The renin-angiotensin-aldosterone system (RAAS) has been implicated in the physiopathology of these disturbances, but M-induced alterations in RAAS-modulated renal Na+ handling and their cardiovascular repercussions are not known. Moreover, altered tissue-specific histone deacetylases (HDAC) results in arterial hypertension and the use of sodium Valproate (Val; a HDAC inhibitor) reduces blood pressure. However, there are no reports regarding the renal and cardiovascular effects of HDAC inhibition in M, or on the signaling pathways involved. The central aim of our study has been to investigate whether alterations in the HDAC/RAAS axis underpin alterations in active Na+ transport in the kidney and heart, and affects blood pressure. METHODS: Male rats aged 28 days were given either a control (C) or a multideficient diet (Regional Basic Diet, RBD), which mimics alimentary habits from developing countries. Subgroups received Losartan (Los), a blocker of type 1 Angiotensin II receptors. When the rats reached 70 days, new subgroups received Val until they were 90 days of age. Homogenates and enriched plasma membrane fractions from renal cortex corticis and cardiomyocytes were obtained by differential centrifugation of the tissues. The activity of renal and cardiac deacetylases was assayed by measuring - after incubation with the membranes - the amount of deacetylated lysines in a substrate containing an acetylated lysine side chain. Protein kinases activities were measured following the incorporation of the γ-phosphoryl group of [γ-32P]ATP into Ser/Thr residues of histone type III-S. The activity of Na+-transporting ATPases (kidney and heart) was quantified by measuring the release of Pi from ATP that was sensitive to ouabain ((Na++K+)ATPase), or sensitive to furosemide (Na+-ATPase). Tail-cuff plethysmography was used to measure systolic blood pressure and heart rate. RESULTS: M provoked HDAC downregulation, which was reversed by Los and Val, either alone or in combination, with selective upregulation of protein kinases C and A (PKC, PKA) in renal cortex corticis, but not in left ventricle cardiomyocytes. The 2 kinases were strongly inhibited by Los and Val in both organs. Malnourished rats developed elevated systolic arterial pressure (SAP) and heart rate (HR) at 70 days of age; Los and Val restored the control SAP, but not HR. Functional and the above biochemical alterations were associated with the deregulation of renal and cardiac Na+-transporting ATPases. (Na++K+)ATPase activities were downregulated in M rats in both organs, and were further inhibited by the pharmacological treatments in the renal cortex corticis (C and M groups) and the left ventricle (only in C rats). No additional effect was found in cardiac (Na++K+)ATPase from M rats. Ouabain-resistant Na+-ATPase was upregulated in renal cortex corticis and downregulated in cardiomyocytes, returning to C values after administration of Los and Val. CONCLUSION: The HDAC/RAAS axis appears to be a key regulator of Na+-transporting ATPases in renal cortex corticis and cardiomyocytes via an appropriate balance of PKC and PKA activities. Modifications within the HDAC/RAAS axis provoked by chronic M - with repercussions in renal and cardiac Na+ transport - underpin alterations in bodily Na+ homeostasis that culminate with the onset of arterial hypertension and potential cardiorenal syndrome.

Cardiac Inflammation after Ischemia-Reperfusion of the Kidney: Role of the Sympathetic Nervous System and the Renin-Angiotensin System.

BACKGROUND/AIMS: To investigate the role of the sympathetic nervous system (SNS) and renin-angiotensin system (RAS) in renal ischemia/reperfusion-induced (I/R) cardiac inflammatoryprofile. METHODS: Left kidney ischemia was induced in male C57BL/6 mice for 60 min, followed by reperfusion for 12 days, and treatment with or without atenolol, losartan, or enalapril. The expression of vimentin in kidney and atrial natriuretic factor (ANF) in the heart has been investigated by RT-PCR. In cardiac tissue, levels of ß1-adrenoreceptors, adenylyl cyclase, cyclic AMP-dependent protein kinase (PKA), noradrenaline, adrenaline (components of SNS), type 1 angiotensin II receptors (AT1R), angiotensinogen/Ang II and renin (components of RAS) have been measured by Western blotting and HPLC analysis. A panel of cytokines - tumour necrosis factor (TNF-α), interleukin IL-6, and interferon gamma (IFN-γ) - was selected as cardiac inflammatory markers. RESULTS: Renal vimentin mRNA levels increased by >10 times in I/R mice, indicative of kidney injury. ANF, a marker of cardiac lesion, increased after renal I/R, the values being restored to the level of Sham group after atenolol or enalapril treatment. The cardiac inflammatory profile was confirmed by the marked increase in the levels of mRNAs of TNF-α, IL-6, and IFN-γ. Atenolol and losartan reversed the upregulation of TNF-α expression, whereas enalapril restored IL-6 levels to Sham levels; both atenolol and enalapril normalized IFN-γ levels. I/R mice showed upregulation of ß1-adrenoreceptors, adenylyl cyclase, PKA and noradrenaline. Renal I/R increased cardiac levels of AT1R, which decreased after losartan or enalapril treatment. Renin expression also increased, with the upregulation returning to Sham levels after treatment with SNS and RAS blockers. Angiotensinogen/Ang II levels in heart were unaffected by renal I/R, but they were significantly decreased after treatment with losartan and enalapril, whereas increase in renin levels decreased. CONCLUSION: Renal I/R-induced cardiac inflammatory events provoked by the simultaneous upregulation of SNS and RAS in the heart, possibly underpin the mechanism involved in the development of cardiorenal syndrome.

Alpha-Tocopherol during lactation and after weaning alters the programming effect of prenatal high salt intake on cardiac and renal functions of adult male offspring.

Maternal salt overload programs cardiovascular and renal alterations in the offspring. However, beneficial and harmful effects of high dose vitamin E supplementation have been described in humans and animals. We investigated the hypothesis as to whether cardiac and renal alterations can be programmed by gestational salt overload, and can become further modified during lactation and after weaning. Male Wistar rats were used, being the offspring of mothers that drank either tap water or 0.3 mol/L NaCl for 20 days before and during pregnancy. α-Tocopherol (0.35 g/kg) was administered to mothers daily during lactation or to their offspring for 3 weeks post-weaning. Systolic blood pressure (tcSBP) was measured in juvenile rats aged 210 days. The response of mean arterial pressure (MAP) and heart rate (HR) to intravenous infusion of angiotensin II (Ang II) was also examined. Left ventricle plasma membrane (PMCA) and sarcoplasmic reticulum Ca2+ -ATPase (SERCA) activities, and certain parameters of renal function, were measured. Maternal saline programmed for increased body mass and kidney mass/body mass ratio, increased tcSBP, increased mean arterial pressure and heart rate with anomalous response to infused Ang II. In the heart, saline increased PMCA and α-Tocopherol per se increased PMCA/SERCA. In the kidney, the most remarkable result was the silent saline programming of CrCl , which was sensitized for a sharp decrease after α-Tocopherol. In conclusion, the combination of maternal saline overload and high α-Tocopherol immediately after birth leads to simultaneous cardiovascular and renal alterations in the young offspring, like those encountered in type V cardiorenal syndrome.

Tartrate-resistant phosphatase type 5 in Trypanosoma cruzi is important for resistance to oxidative stress promoted by hydrogen peroxide.

Trypanosoma cruzi (the causative agent of Chagas disease) presents a complex life cycle that involves adaptations in vertebrate and invertebrate hosts. As a protozoan parasite of hematophagous insects and mammalian hosts, T. cruzi is exposed to reactive oxygen species (ROS). To investigate the functionality of T. cruzi tartrate-resistant acid phosphatase type 5 (TcACP5), we cloned, superexpressed and purified the enzyme. Purified TcACP5 exhibited a Vmax and apparent Km for pNPP hydrolysis of 7.7 ±â€¯0.2 nmol pNP × µg-1 × h-1 and 169.3 ±â€¯22.6 µM, respectively. The pH dependence was characterized by sharp maximal activity at pH 5.0, and inhibition assays demonstrated its sensitivity to acid phosphatase inhibitors. Similar activities were obtained with saturating concentrations of P-Ser and P-Thr as substrates. The enzyme metabolizes hydrogen peroxide (H2O2) in vitro, and parasites superexpressing this enzyme were more resistant to oxidative stress promoted by H2O2. Taken together, these results suggest that TcACP5 plays a central role in phosphoryl transfer and redox reactions.

Perinatal α-tocopherol overload programs alterations in kidney development and renal angiotensin II signaling pathways at birth and at juvenile age: Mechanisms underlying the development of elevated blood pressure.

α-Tocopherol (α-Toc) overload increases the risk of dying in humans (E.R. Miller III et al. Meta-analysis: high-dosage vitamin E supplementation may increase all-cause mortality Ann Int Med. 142 (2005) 37-46), and overload during early development leads to elevation of blood pressure at adult life, but the mechanism(s) remains unknown. We hypothesized that α-Toc overload during organogenesis affects the renal renin angiotensin system (RAS) components and renal Na+ handling, culminating with late elevated blood pressure. Pregnant Wistar rats received α-Toc or the superoxide dismutase mimetic tempol throughout pregnancy. We evaluated components of the intrarenal renin angiotensin system in neonate and juvenile offspring: Ang II-positive cells, Ang II receptors (AT1 and AT2), linked protein kinases, O2- production, NADPH oxidase abundance, lipid peroxidation and activity of Na+-transporting ATPases. In juvenile offspring we followed the evolution of arterial blood pressure. Neonates from α-Toc and tempol mothers presented with accentuated retardment in tubular development, pronounced decrease in glomerular Ang II-positive cells and AT1/AT2 ratio, intense production of O2- and upregulation of the α, ε and λ PKC isoforms. α-Toc decreased or augmented the abundance of renal (Na++K+)ATPase depending on the age and α-Toc dose. In juvenile rats the number of Ang II-positive cells returned to control values as well as PKCα, but co-existing with marked upregulation in the activity of (Na++K+) and Na+-ATPase and elevated arterial pressure at 30 days. We conclude that the mechanisms of these alterations rely on selective targeting of renal RAS components through genic and pro-oxidant effects of the vitamin.

Altered signaling pathways linked to angiotensin II underpin the upregulation of renal Na(+)-ATPase in chronically undernourished rats.

This study has investigated the participation of altered signaling linked to angiotensin II (Ang II) that could be associated with increased Na(+) reabsorption in renal proximal tubules during chronic undernutrition. A multideficient chow for rats (basic regional diet, BRD) was used, which mimics several human diets widely taken in developing countries. The Vmax of the ouabain-resistant Na(+)-ATPase resident in the basolateral membranes increased >3-fold (P<0.001) accompanied by an increase in Na(+) affinity from 4.0 to 0.2mM (P<0.001). BRD rats had a >3-fold acceleration of the formation of phosphorylated intermediates in the early stage of the catalytic cycle (in the E1 conformation) (P<0.001). Immunostaining showed a huge increase in Ang II-positive cells in the cortical tubulointerstitium neighboring the basolateral membranes (>6-fold, P<0.001). PKC isoforms (α, ε, λ, ζ), Ang II type 1 receptors and PP2A were upregulated in BRD rats (in %): 55 (P<0.001); 35 (P<0.01); 125, 55, 11 and 30 (P<0.001). PKA was downregulated by 55% (P<0.001). With NetPhosK 1.0 and NetPhos 2.0, we detected 4 high-score (>0.70) regulatory phosphorylation sites for PKC and 1 for PKA in the primary sequence of the Na(+)-ATPase α-subunit, which are located in domains that are key for Na(+) binding and catalysis. Therefore, chronic undernutrition stimulates tubulointerstitial activity of Ang II and impairs PKC- and PKA-mediated regulatory phosphorylation, which culminates in an exaggerated Na(+) reabsorption across the proximal tubular epithelium.

Renal molecular mechanisms underlying altered Na+ handling and genesis of hypertension during adulthood in prenatally undernourished rats.

In the present study, we investigated the development of hypertension in prenatally undernourished adult rats, including the mechanisms that culminate in dysfunctions of molecular signalling in the kidney. Dams were fed a low-protein multideficient diet throughout gestation with or without α-tocopherol during lactation. The time course of hypertension development followed in male offspring was correlated with alterations in proximal tubule Na+-ATPase activity, expression of angiotensin II (Ang II) receptors, and activity of protein kinases C and A. After the establishment of hypertension, Ang II levels, cyclo-oxygenase 2 (COX-2) and NADPH oxidase subunit expression, lipid peroxidation and macrophage infiltration were examined in renal tissue. Lipid peroxidation in undernourished rats, which was very intense at 60 d, decreased at 90 d and returned to control values by 150 d. During the prehypertensive phase, prenatally undernourished rats exhibited elevated renal Na+-ATPase activity, type 2 Ang II receptor down-regulation and altered protein kinase A:protein kinase C ratio. Stable late hypertension coexisted with highly elevated levels of Ang II-positive cells in the cortical tubulointerstitium, enhanced increase in the expression of p47phox (NADPH oxidase regulatory subunit), marked down-regulation of COX-2 expression, expanded plasma volume and decreased creatinine clearance. These alterations were reduced when the dams were given α-tocopherol during lactation. The offspring of well-nourished dams treated with α-tocopherol exhibited most of the alterations encountered in the offspring of undernourished dams not treated with α-tocopherol. Thus, alterations in proximal tubule Na+ transport, subcellular signalling pathways and reactive oxygen species handling in renal tissue underpin the development of hypertension.

Different antihypertensive and metabolic responses to rostafuroxin in undernourished and normonourished male rats: Outcomes on bodily Na+ handling.

Hypertension is a pandemic nowadays. We aimed to investigate whether chronic undernutrition modifies the response to the antihypertensive drug rostafuroxin in juvenile hypertensive rats. Chronic undernutrition was induced in male rats using a multideficient diet known as the Regional Basic Diet (RBD), mimicking alimentary habits in impoverished regions worldwide. Animals were given RBD-or a control/CTRL normal diet for rodents-from weaning to 90 days, and rostafuroxin (1 mg/kg body mass) was orally administered from day 60 onwards. For the last 2 days, the rats were hosted in metabolic cages to measure food/energy, water, Na+ ingestion, and urinary volume. Rostafuroxin increased food/energy/Na+ intake in CTRL and RBD rats but had opposite effects on Na+ balance (intake minus urinary excretion). The drug normalized the decreased plasma Na+ concentration in RBD rats, increased urinary volume in RBD but not in CTRL, and decreased and increased urinary Na+ concentration in the RBD and CTRL groups, respectively. Rostafuroxin decreased the ouabain-sensitive (Na+ +K+ )ATPase and increased the ouabain-resistant Na+ -ATPase from proximal tubule cells in both groups and normalized the systolic blood pressure in RBD without effect in CTRL rats. We conclude that chronic undernutrition modifies the response of blood pressure and metabolic responses to rostafuroxin.

Liver steatosis, cardiac and renal fibrosis, and hypertension in overweight rats: Angiotensin-(3-4)-sensitive hepatocardiorenal syndrome.

Overweight/obesity is a growing pandemic that affects many organs and tissues. We have investigated whether a high-lipid diet provokes an imbalance between type 1 and type 2 angiotensin II (Ang II) receptors signaling, leading to liver alterations associated with cardiovascular and kidney disturbances. Chronic administration of a high-lipid diet can provoke hepatocardiorenal syndrome resulting from activation of the Ang II→type 1 receptor axis, which is entirely counteracted by Ang-(3-4), the allosteric enhancer of the Ang II→type 2 receptor pathway.

Angiotensin-(3-4) normalizes the elevated arterial blood pressure and abnormal Na+/energy handling associated with chronic undernutrition by counteracting the effects mediated by type 1 angiotensin II receptors.

We investigated the mechanisms by which chronic administration of a multideficient diet after weaning alters bodily Na+ handling, and culminates in high systolic blood pressure (SBP) at a juvenile age. From 28 to 92 days of age, weaned male Wistar rats were given a diet with low content and poor-quality protein, and low lipid, without vitamin supplementation, which mimics the diets consumed in impoverished regions worldwide. We measured food, energy and Na+ ingestion, together with urinary Na+ excretion, Na+ density (Na+ intake/energy intake), plasma Na+ concentration, SBP, and renal proximal tubule Na+-transporting ATPases. Undernourished rats aged 92 days had only one-third of the control body mass, lower plasma albumin, higher SBP, higher energy intake, and higher positive Na+ balance accompanied by decreased plasma Na+ concentration. Losartan or Ang-(3-4) normalized SBP, and the combination of the 2 substances induced an accentuated negative Na+ balance as a result of strong inhibition of Na+ ingestion. Na+ density in undernourished rats was higher than in control, irrespective of the treatment, and they had downregulated (Na++K+)ATPase and upregulated Na+-ATPase in proximal tubule cells, which returned to control levels after Losartan or Ang-(3-4). We conclude that Na+ density, not only Na+ ingestion, plays a central role in the pathophysiology of elevated SBP in chronically undernourished rats. The observations that Losartan and Ang-(3-4) normalized SBP together with negative Na+ balance give support to the proposal that Ang II⇒AT1R and Ang II⇒AT2R axes have opposite roles within the renin-angiotensin-aldosterone system of undernourished juvenile rats.

Renal ischemia-reperfusion leads to hypertension and changes in proximal tubule Na+ transport and renin-angiotensin-aldosterone system: Role of NADPH oxidase.

Acute renal injury (AKI) is a risk factor for the development of hypertension, which involves oxidative stress, changes in Na+ handling, and the intrarenal renin-angiotensin-aldosterone system (RAAS) as underlying mechanisms. We investigated in rats whether renal ischemia-reperfusion (IR) leads to changes in the proximal tubule ATP-dependent Na+ transport and the intrarenal content of RAAS components, as well as the role of NADPH oxidase. Rats weighing 300-350 g were submitted to AKI by bilateral IR (n = 25). After IR injury, the animals were followed up for 4 weeks. One part (n = 7) received daily treatment with the NADPH oxidase inhibitor apocynin (100 mg/kg, drinking water), while another part (n = 9) received apocynin 24 h before and after IR. One group was submitted to sham surgery (n = 8). Four weeks after IR, the rats presented elevated systolic blood pressure, as well as increased lipid peroxidation, NADPH oxidase activity, (Na++K+)ATPase activity, and upregulation of type 1 angiotensin II receptor in the renal cortex. On the other hand, there was a decrease in Na+-ATPase activity and downregulation of the isoforms 1 and 2 of the angiotensin-converting enzyme, type 2 angiotensin II receptor, and of the α and ε isoforms of protein kinase C. Most of these alterations was prevented by both apocynin treatment protocols. Thus, we conclude that AKI-induced by IR may induce changes in proximal tubule ATPases and RAAS components compatible with renal Na+ retention and hypertension. These data also indicate that the NADPH oxidase represents a key factor in the origin of these alterations.

Is angiotensin-(3-4) (Val-Tyr), the shortest angiotensin II-derived peptide, opening new vistas on the renin-angiotensin system?

Angiotensin-(3-4) (Ang-(3-4) or Val-Tyr) is the shorter angiotensin (Ang) II-derived peptide, formed through successive hydrolysis that culminates with the release of Val-Tyr as a dipeptide. It is formed both in plasma and in kidney from Ang II and Ang III, and can be considered a component of the systemic and organ-based renin-angiotensin system. It is potently antihypertensive in humans and rats, and its concerted actions on proximal tubule cells culminate in the inhibition of fluid reabsorption, hyperosmotic urinary excretion of Na+. At the renal cell signaling level, Ang-(3-4) counteracts Ang II-type 1 receptor-mediated responses by acting as an allosteric enhancer in Ang II-type 2 receptor populations that target adenosine triphosphate-dependent Ca2+ and Na+ transporters through a cyclic adenosine monophosphate-activated protein kinase pathway.

Knockout of Toll-Like Receptors 2 and 4 Prevents Renal Ischemia-Reperfusion-Induced Cardiac Hypertrophy in Mice.

We investigated whether the pathways linked to Toll-like receptors 2 and 4 (TLRs) are involved in renal ischemia-reperfusion (I/R)-induced cardiac hypertrophy. Wild type (WT) C57BL/6J, TLR2-/- and TLR4-/- mice were subjected to left kidney ischemia for 60 min followed by reperfusion for 5, 8, 12 and 15 days. Proton density magnetic resonance showed alterations in the injured kidney from WT mice, together with signs of parenchymal edema and higher levels of vimentin mRNA, accompanied by: (i) small, but significant, increase in serum urea after 24 h, (ii) 100% increase in serum creatinine at 24 h. A serum peak of inflammatory cytokines occurred after 5 days of reperfusion. Heart weight/body weight and heart weight/tibia length ratios increased after 12 and 15 days of reperfusion, respectively. Cardiac hypertrophy markers, B-type natriuretic peptide (BNP) and α-actin, left ventricle mass, cardiac wall thickness and myocyte width increased after 15 days of reperfusion, together with longer QTc and action potential duration. Cardiac TLRs, MyD88, HSP60 and HSP70 mRNA levels also increased. After 15 days of reperfusion, absence of TLRs prevented cardiac hypertrophy, as reflected by similar values of left ventricular cardiac mass and heart weight/body weight ratio compared to the transgenic Sham. Renal tissular injury also ameliorated in both knockout mice, as revealed by the comparison of their vimentin mRNA levels with those found in the WT on the same day after I/R. The I/R TLR2-/- group had TNF-α, IFN-γ and IL-1ß levels similar to the non-I/R group, whereas the TLR4-/- group conserved the p-NF-κB/NF- κB ratio contrasting with that found in TLR2-/-. We conclude: (i) TLRs are involved in renal I/R-induced cardiac hypertrophy; (ii) absence of TLRs prevents I/R-induced cardiac hypertrophy, despite renal lesions seeming to evolve towards those of chronic disease; (iii) TLR2 and TLR4 selectively regulate the systemic inflammatory profile and NF- κB activation.

Rats undernourished in utero have altered Ca2+ signaling and reduced fertility in adulthood.

Epidemiological and animal studies have shown that placental undernutrition impairs reproduction in adult offspring, but the underlying molecular mechanisms within the male genital tract remain unknown. Due to its special physiological characteristics in transport and the modulation of the environment to which its luminal content is exposed, we hypothesized that the vas deferens would be a highly sensitive target. The goals were to investigate whether intrauterine malnutrition affects molecular mechanisms related to Ca(2+)- and oxidative stress-modulated processes and causes structural alterations in the adult rat vas deferens that could attenuate fecundity and fertility. Male adult rats malnourished in utero had increased vas deferens weight associated with thickening of the muscular coat, a decrease in the total and haploid germ cells, a marked increase in the immature cells, and a decline in the numbers of pregnant females and total offspring per male rat. The ex vivo response of vas deferens from malnourished rats demonstrated an accentuated decrease in the contractile response to phenylephrine. The vas deferens had a marked decrease in Ca(2+) transport due to the uncoupling of Ca(2+)-stimulated ATP hydrolysis and ATP-driven Ca(2+) flux, and the downregulation of both sarco-endoplasmic reticulum Ca(2+)-ATPase 2 and the coupling factor 12-kDa FK506-binding protein. An increase in protein carbonylation (a marker of oxidative damage) and an imbalance between protein kinases C and A were observed as a legacy of undernutrition in early life. These results provide the structural and molecular basis to explain at least in part how maternal undernutrition affects fecundity and fertility in adult male rats.

Mechanisms associated to impaired activity of cardiac P-type ATPases in endothelial nitric oxide synthase knockout mice.

The effect of long-lasting in vivo restriction of nitric oxide (NO) bioavailability on cardiac and renal P-type ATPases critical for intracellular ion homeostasis is controversial. Previous work has shown in eNOS knockout (eNOS(-/-)) mice hearts that Na(+)/K(+)- and Ca(2+)-ATPase activities were depressed but the underlying mechanisms are still unclear. The goal of this study was to characterize potential alterations responsible for impaired enzyme activity in eNOS(-/-) mice. Na(+)/K(+)-ATPase activity from crude preparations of adult male eNOS(-/-) mice hearts and kidneys was reduced compared with wild-type animals (32 %, p < 0.05 and 16 %, p < 0.0001, respectively). Immunoblot analysis showed that although the expression of the predominant (or exclusive, for the kidney) Na(+)/K(+)-ATPase α1 isoform was not significantly changed, there was an important downregulation of the less abundant α2 isoform in the heart (57 %, p < 0.0001). In addition, although cardiac Ca(2+)-ATPase activity was unaltered, the expression of sarco/endoplasmic reticulum Ca(2+)-ATPase 2 protein in eNOS(-/-) mice was very high (290 % compared with wild-type animals, p < 0.0001) without any significant change in phospholamban expression. Consistent with these findings, the content of cardiac and renal free sulfhydryl groups, essential for the catalytic function of such ATPases, was decreased (23 %, p < 0.01 and 35 %, p < 0.05, respectively). Altogether, the present results suggest that the absence of eNOS promotes a compartmentalized altered redox balance that affects the activity and expression of ion transport ATPases.

Undernutrition affects cell survival, oxidative stress, Ca2+ handling and signaling pathways in vas deferens, crippling reproductive capacity.

BACKGROUND: The aim of this work was to investigate the mechanisms by which chronic malnutrition (CM) affects vas deferens function, leading to compromised reproductive capacity. Previous studies have shown that maternal malnutrition affects the reproductive tracts of adult male offspring. However, little is known about the effects of CM, a widespread life-long condition that persists from conception throughout growth to adult life. METHODOLOGY/PRINCIPAL FINDINGS: Young adult male rats, which were chronically malnourished from weaning, presented decreased total and haploid cells in the vas deferens, hypertrophy of the muscle layer in the epididymal portion of the vas deferens and intense atrophy of the muscular coat in its prostatic portion. At a molecular level, the vas deferens tissue of CM rats exhibited a huge rise in lipid peroxidation and protein carbonylation, evidence of an accentuated increase in local reactive oxygen species levels. The kinetics of plasma membrane Ca(2+)-ATPase activity and its kinase-mediated phosphorylation by PKA and PKC in the vas deferens revealed malnutrition-induced modifications in velocity, Ca(2+) affinity and regulation of Ca(2+) handling proteins. The severely crippled content of the 12-kDa FK506 binding protein, which controls passive Ca(2+) release from the sarco(endo) plasmic reticulum, revealed another target of malnutrition related to intracellular Ca(2+) handling, with a potential effect on forward propulsion of sperm cells. As a possible compensatory response, malnutrition led to enhanced sarco(endo) plasmic reticulum Ca(2+)-ATPase activity, possibly caused by stimulatory PKA-mediated phosphorylation. CONCLUSIONS/SIGNIFICANCE: The functional correlates of these cellular and molecular hallmarks of chronic malnutrition on the vas deferens were an accentuated reduction in fertility and fecundity.

FKBP12 depletion leads to loss of sarcoplasmic reticulum Ca(2+) stores in rat vas deferens.

The immunophilin 12-kDa FK506 binding protein (FKBP12) stabilizes intracellular Ca(2+) release channel (CRC) activity in different tissues. In this work, the presence of FKBP12 in rat vas deferens (RVD) and its possible contribution to RVD function was investigated. Treatment under appropriate pH, temperature, and ionic conditions was used to strip FKBP12 from CRC binding sites; Western blotting revealed FKBP12 in control but not in treated homogenates. Disruption of the FKBP12-CRC complex in RVD decreased the Ca(2+) content of sarcoplasmic reticulum (SR) by increasing Ca(2+) leakage through the ryanodine receptor (RyR3 isoform) but not through 1,4,5-inositol trisphosphate receptors (IP(3)R1 and IP(3)R3 isoforms). The decrease of SR Ca(2+) content was not related to inhibition of SERCA ATPase. It seems that dissociation of FKBP12-RyR leads to conformational changes in RyR that make it difficult for ryanodine to access its binding site. Rapamycin, which is commonly used as a pharmacological tool to disrupt the FKBP12-RyR complex, decreased phenylephrine-induced contractions in RVD epididymal halves. The data suggest that FKBP12 is expressed in RVD in a labile association with RyR3. Disruption of the FKBP12-RyR3 complex may lead to modifications of RVD physiology and in consequence may compromise male fertility.

Ivermectin as an inhibitor of the plasma membrane and the sarcoplasmic/endoplasmic reticulum Ca2+-ATPases in rat vas deferens / Ivermectina como um inibidor das Ca2+-ATPases de membrana plasmática e de retículo sarco/endoplasmático no ducto deferente de rato

Objective: The present work investigated the effect of ivermectin on Ca2+ content and on the Ca2+-ATPase activity (represented by the plasma membrane Ca2+-ATPase and the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase present in rat vas deferens. Methods: The assays were carried out using ultracentrifuged homogenate preparations from rat vas deferens in the presence or absence of the 12-kDa FK506-binding protein-Ca2+ release channel complex. Measures of Ca2+ content and Ca2+ ATPase activity were then carried out in function of different concentrations of ivermectin. Results: The data show that ivermectin (10 µM) reduces the sarcoplasmic reticulum Ca2+ content in FK506-binding protein (+) and FK506-binding protein (-) fractions of ultracentrifuged homogenate from rat vas deferens (inhibition of 50 and 40%, respectively, p<0.05) and inhibits both the activities of sarcoplasmic/endoplasmic reticulum Ca2+-ATPase and plasma membrane Ca2+-ATPases pumps (33 and 16%, respectively, p<0.05). Conclusion: These data suggest that ivermectin effects Ca2+ handling in the rat vas deferens, indicating that this drug could alter the contractility of this smooth muscle. Therefore, ivermectin could be an interesting pharmacological tool to alter the physiological function of vas deferens and to manipulate the fertility status of male rats.

Objetivo: O presente trabalho investigou o efeito da ivermectina no conteúdo de Ca2+ e na atividade Ca2+-ATPásica (representada pela Ca2+-ATPase de membrana plasmática e pela Ca2+-ATPase de retículo sarco/endoplasmático presente no ducto deferente de rato. Métodos: Os ensaios foram realizados por meio de preparações de homogeneizado ultracentrifugado de duto deferente de rato na presença ou ausência do complexo proteína de ligação ao FK506 de 12 kDa-canal liberador de Ca2+. Após esse procedimento, avaliações do conteúdo de Ca2+ e da atividade Ca2+-ATPásica foram realizadas em função de diferentes concentrações de ivermectina. Resultados: Os dados mostram que a ivermectina (10 µM) reduz o conteúdo de cálcio no retículo sarcoplasmático de frações FK506-binding protein (+) e FK506-binding protein (-) de homogeneizado ultracentrifugado de duto deferente de rato (50 e 40% de inibição, respectivamente, p<0,05) e inibe as atividades das enzimas Ca2+-ATPase de retículo sarco/endoplasmático e Ca2+-ATPase de membrana plasmática (33 e 16%, respectivamente, p<0,05). Conclusão: Os dados sugerem que a ivermectina afeta a mobilização de cálcio no duto deferente de rato, o que indica que esse fármaco pode alterar a contratilidade desse músculo liso. Dessa forma, ivermectina pode ser ferramenta farmacológica interessante para alterar a função fisiológica do duto deferente e manipular o estado de fertilidade de ratos machos.