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.

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.

Iron Uptake Controls Trypanosoma cruzi Metabolic Shift and Cell Proliferation.

(1) Background: Ionic transport in Trypanosoma cruzi is the object of intense studies. T. cruzi expresses a Fe-reductase (TcFR) and a Fe transporter (TcIT). We investigated the effect of Fe depletion and Fe supplementation on different structures and functions of T. cruzi epimastigotes in culture. (2) Methods: We investigated growth and metacyclogenesis, variations of intracellular Fe, endocytosis of transferrin, hemoglobin, and albumin by cell cytometry, structural changes of organelles by transmission electron microscopy, O2 consumption by oximetry, mitochondrial membrane potential measuring JC-1 fluorescence at different wavelengths, intracellular ATP by bioluminescence, succinate-cytochrome c oxidoreductase following reduction of ferricytochrome c, production of H2O2 following oxidation of the Amplex® red probe, superoxide dismutase (SOD) activity following the reduction of nitroblue tetrazolium, expression of SOD, elements of the protein kinase A (PKA) signaling, TcFR and TcIT by quantitative PCR, PKA activity by luminescence, glyceraldehyde-3-phosphate dehydrogenase abundance and activity by Western blotting and NAD+ reduction, and glucokinase activity recording NADP+ reduction. (3) Results: Fe depletion increased oxidative stress, inhibited mitochondrial function and ATP formation, increased lipid accumulation in the reservosomes, and inhibited differentiation toward trypomastigotes, with the simultaneous metabolic shift from respiration to glycolysis. (4) Conclusion: The processes modulated for ionic Fe provide energy for the T. cruzi life cycle and the propagation of Chagas disease.

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.

Bone Marrow Mononuclear Cells Restore Normal Mitochondrial Ca2+ Handling and Ca2+-Induced Depolarization of the Internal Mitochondrial Membrane by Inhibiting the Permeability Transition Pore After Ischemia/Reperfusion.

Acute kidney injury due to ischemia followed by reperfusion (IR) is a severe clinical condition with high death rates. IR affects the proximal tubule segments due to their predominantly oxidative metabolism and profoundly altered mitochondrial functions. We previously described the impact of IR on oxygen consumption, the generation of membrane potential (ΔΨ), and formation of reactive oxygen species, together with inflammatory and structural alterations. We also demonstrated the benefits of bone marrow mononuclear cells (BMMC) administration in these alterations. The objective of the present study has been to investigate the effect of IR and the influence of BMMC on the mechanisms of Ca2+ handling in mitochondria of the proximal tubule cells. IR inhibited the rapid accumulation of Ca2+ (Ca2+ green fluorescence assays) and induced the opening of the cyclosporine A-sensitive permeability transition pore (PTP), alterations prevented by BMMC. IR accelerated Ca2+-induced decrease of ΔΨ (Safranin O fluorescence assays), as evidenced by decreased requirement for Ca2+ load and t1/2 for complete depolarization. Addition of BMMC and ADP recovered the normal depolarization profile, suggesting that stabilization of the adenine nucleotide translocase (ANT) in a conformation that inhibits PTP opening offers a partial defense mechanism against IR injury. Moreover, as ANT forms a complex with the voltage-dependent anion channel (VDAC) in the outer mitochondrial membrane, it is possible that this complex is also a target for IR injury-thus favoring Ca2+ release, as well as the supramolecular structure that BMMC protects. These beneficial effects are accompanied by a stimulus of the citric acid cycle-which feed the mitochondrial complexes with the electrons removed from different substrates-as the result of accentuated stimulus of citrate synthase activity by BMMC.

Diminazene Aceturate, an angiotensin converting enzyme 2 (ACE2) activator, promotes cardioprotection in ischemia/reperfusion-induced cardiac injury.

This study aimed to investigate whether the Diminazene Aceturate (DIZE), an angiotensin-converting enzyme 2 (ACE2) activator, can revert cardiac dysfunction in ischemia reperfusion-induced (I/R) injury in animals and examine the mechanism underlying this effect. Wistar rats systemically received DIZE (1 mg/kg) for thirty days. Cardiac function in isolated rat hearts was evaluated using the Langendorff technique. After I/R, ventricular non-I/R and I/R samples were used to evaluate ATP levels. Mitochondrial function was assessed using cardiac permeabilized fibers and isolated cardiac mitochondria. Cardiac cellular electrophysiology was evaluated using the patch clamp technique. DIZE protected the heart after I/R from arrhythmia and cardiac dysfunction by preserving ATP levels, independently of any change in coronary flow and heart rate. DIZE improved mitochondrial function, increasing the capacity for generating ATP and reducing proton leak without changing the specific citrate synthase activity. The activation of the ACE2 remodeled cardiac electrical profiles, shortening the cardiac action potential duration at 90 % repolarization. Additionally, cardiomyocytes from DIZE-treated animals exhibited reduced sensibility to diazoxide (KATP agonist) and a higher KATP current compared to the controls. DIZE was able to improve mitochondrial function and modulate cardiac electrical variables with a cardio-protective profile, resulting in direct myocardial cell protection from I/R injury.

An Iron Transporter Is Involved in Iron Homeostasis, Energy Metabolism, Oxidative Stress, and Metacyclogenesis in Trypanosoma cruzi.

The parasite Trypanosoma cruzi causes Chagas' disease; both heme and ionic Fe are required for its optimal growth, differentiation, and invasion. Fe is an essential cofactor in many metabolic pathways. Fe is also harmful due to catalyzing the formation of reactive O2 species; for this reason, all living systems develop mechanisms to control the uptake, metabolism, and storage of Fe. However, there is limited information available on Fe uptake by T. cruzi. Here, we identified a putative 39-kDa Fe transporter in T. cruzi genome, TcIT, homologous to the Fe transporter in Leishmania amazonensis and Arabidopsis thaliana. Epimastigotes grown in Fe-depleted medium have increased TcIT transcription compared with controls grown in regular medium. Intracellular Fe concentration in cells maintained in Fe-depleted medium is lower than in controls, and there is a lower O2 consumption. Epimastigotes overexpressing TcIT, which was encountered in the parasite plasma membrane, have high intracellular Fe content, high O2 consumption-especially in phosphorylating conditions, high intracellular ATP, very high H2O2 production, and stimulated transition to trypomastigotes. The investigation of the mechanisms of Fe transport at the cellular and molecular levels will assist in elucidating Fe metabolism in T. cruzi and the involvement of its transport in the differentiation from epimastigotes to trypomastigotes, virulence, and maintenance/progression of the infection.

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.

The Functioning of Na+-ATPases from Protozoan Parasites: Are These Pumps Targets for Antiparasitic Drugs?

The ENA ATPases (from exitus natru: the exit of sodium) belonging to the P-type ATPases are structurally very similar to the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA); they exchange Na+ for H+ and, therefore, are also known as Na+-ATPases. ENA ATPases are required in alkaline milieu, as in the case for Aspergillus, where other transporters cannot mediate an uphill Na+ efflux. They are also important for salt tolerance, as described for Arabidopsis. During their life cycles, protozoan parasites might encounter a high pH environment, thus allowing consideration of ENA ATPases as possible targets for controlling certain severe parasitic diseases, such as Chagas' Disease. Phylogenetic analysis has now shown that, besides the types IIA, IIB, IIC, and IID P-type ATPases, there exists a 5th subgroup of ATPases classified as ATP4-type ATPases, found in Plasmodium falciparum and Toxoplasma gondii. In malaria, for example, some drugs targeting PfATP4 destroy Na+ homeostasis; these drugs, which include spiroindolones, are now in clinical trials. The ENA P-type (IID P-type ATPase) and ATP4-type ATPases have no structural homologue in mammalian cells, appearing only in fungi, plants, and protozoan parasites, e.g., Trypanosoma cruzi, Leishmania sp., Toxoplasma gondii, and Plasmodium falciparum. This exclusivity makes Na+-ATPase a potential candidate for the biologically-based design of new therapeutic interventions; for this reason, Na+-ATPases deserves more attention.

A ferric reductase of Trypanosoma cruzi (TcFR) is involved in iron metabolism in the parasite.

Trypanosoma cruzi is a parasitic protozoan that infects various species of domestic and wild animals, triatomine bugs and humans. It is the etiological agent of American trypanosomiasis, also known as Chagas Disease, which affects about 17 million people in Latin America and is emerging elsewhere in the world. Iron (Fe) is a crucial micronutrient for almost all cells, acting as a cofactor for several metabolic enzymes. T. cruzi has a high requirement for Fe, using heminic and non-heminic Fe for growth and differentiation. Fe occurs in the oxidized (Fe3+) form in aerobic environments and needs to be reduced to Fe2+ before it enters cells. Fe-reductase, located in the plasma membranes of some organisms, catalyzes the Fe3+⇒ Fe2+ conversion. In the present study we found an amino acid sequence in silico that allowed us to identify a novel 35 kDa protein in T. cruzi with two transmembrane domains in the C-terminal region containing His residues that are conserved in the Ferric Reductase Domain Superfamily and are required for catalyzing Fe3+ reduction. Accordingly, we named this protein TcFR. Intact epimastigotes from the T. cruzi DM28c strain reduced the artificial Fe3+-containing substrate potassium ferricyanide in a cell density-dependent manner, following Michaelis-Menten kinetics. The TcFR activity was more than eightfold higher in a plasma membrane-enriched fraction than in whole homogenates, and this increase was consistent with the intensity of the 35 kDa band on Western blotting images obtained using anti-NOX5 raised against the human antigen. Immunofluorescence experiments demonstrated TcFR on the parasite surface. That TcFR is part of a catalytic complex allowing T. cruzi to take up Fe from the medium was confirmed by experiments in which DM28c was assayed after culturing in Fe-depleted medium: (i) proliferation during the stationary growth phase was five times slower; (ii) the relative expression of TcFR (qPCR) was 50% greater; (iii) intact cells had 120% higher Fe-reductase activity. This ensemble of results indicates that TcFR is a conserved enzyme in T. cruzi, and its catalytic properties are modulated in order to respond to external Fe fluctuations.

Acute Myocardial Infarction Reduces Respiration in Rat Cardiac Fibers, despite Adipose Tissue Mesenchymal Stromal Cell Transplant.

Adipose-derived mesenchymal stromal cell (AD-MSC) administration improves cardiac function after acute myocardial infarction (AMI). Although the mechanisms underlying this effect remain to be elucidated, the reversal of the mitochondrial dysfunction may be associated with AMI recovery. Here, we analyzed the alterations in the respiratory capacity of cardiomyocytes in the infarcted zone (IZ) and the border zone (BZ) and evaluated if mitochondrial function improved in cardiomyocytes after AD-MSC transplantation. Female rats were subjected to AMI by permanent left anterior descending coronary (LAD) ligation and were then treated with AD-MSCs or PBS in the border zone (BZ). Cardiac fibers were analyzed 24 hours (necrotic phase) and 8 days (fibrotic phase) after AMI for mitochondrial respiration, citrate synthase (CS) activity, F0F1-ATPase activity, and transmission electron microscopy (TEM). High-resolution respirometry of permeabilized cardiac fibers showed that AMI reduced numerous mitochondrial respiration parameters in cardiac tissue, including phosphorylating and nonphosphorylating conditions, respiration coupled to ATP synthesis, and maximal respiratory capacity. CS decreased in IZ and BZ at the necrotic phase, whereas it recovered in BZ and continued to drop in IZ over time when compared to Sham. Exogenous cytochrome c doubled respiration at the necrotic phase in IZ. F0F1-ATPase activity decreased in the BZ and, to more extent, in IZ in both phases. Transmission electron microscopy showed disorganized mitochondrial cristae structure, which was more accentuated in IZ but also important in BZ. All these alterations in mitochondrial respiration were still present in the group treated with AD-MSC. In conclusion, AMI led to mitochondrial dysfunction with oxidative phosphorylation disorders, and AD-MSC improved CS temporarily but was not able to avoid alterations in mitochondria function over time.

Extracellular Vesicles Derived from Induced Pluripotent Stem Cells Promote Renoprotection in Acute Kidney Injury Model.

Induced pluripotent stem cells (iPSC) have been the focus of several studies due to their wide range of application, including in cellular therapy. The use of iPSC in regenerative medicine is limited by their tumorigenic potential. Extracellular vesicles (EV) derived from stem cells have been shown to support renal recovery after injury. However, no investigation has explored the potential of iPSC-EV in the treatment of kidney diseases. To evaluate this potential, we submitted renal tubule cells to hypoxia-reoxygenation injury, and we analyzed cell death rate and changes in functional mitochondria mass. An in vivo model of ischemia-reperfusion injury was used to evaluate morphological and functional alterations. Gene array profile was applied to investigate the mechanism involved in iPSC-EV effects. In addition, EV derived from adipose mesenchymal cells (ASC-EV) were also used to compare the potential of iPSC-EV in support of tissue recovery. The results showed that iPSC-EV were capable of reducing cell death and inflammatory response with similar efficacy than ASC-EV. Moreover, iPSC-EV protected functional mitochondria and regulated several genes associated with oxidative stress. Taken together, these results show that iPSC can be an alternative source of EV in the treatment of different aspects of kidney disease.

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.

Adipose-Derived Mesenchymal Stromal Cells Under Hypoxia: Changes in Extracellular Vesicles Secretion and Improvement of Renal Recovery after Ischemic Injury.

BACKGROUND/AIMS: The therapeutic potential of extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) in kidney injury has been largely reported. However, new approaches are necessary to optimize the efficacy in the treatment of renal diseases. MSCs physiologically are under a low O2 partial pressure (pO2), and culturing adipose-derived MSCs (ADMSCs) in hypoxia alters their secretory paracrine properties. The aim of this study was to evaluate whether hypoxia preconditioning of ADMSCs alters the properties of secreted EVs to improve renal recovery after ischemia-reperfusion injury (IRI). METHODS: The supernatants of ADMSCs cultivated under 21% pO2 (control) or 1% pO2 (hypoxia) were ultracentrifuged for EVs isolation that were posteriorly characterized by flow cytometry and electron microscopy. The uptake and effects of these EVs were analyzed by using in vitro and in vivo models. HK-2 renal tubule cell line was submitted do ATP depletion injury model. Proteomic analyses of these cells treated with EVs after injury were performed by nano-UPLC tandem nano-ESI-HDMSE method. For in vivo analyses, male Wistar rats were submitted to 45 min bilateral ischemia, followed by renal intracapsular administration of ADMSC-EVs within a 72 h reperfusion period. Histological, immunohistochemical and qRT-PCR analysis of these kidneys were performed to evaluate cell death, inflammation and oxidative stress. Kidney function was evaluated by measuring the blood levels of creatinine and urea. RESULTS: The results demonstrate that hypoxia increases the ADMSCs capacity to secrete EVs that trigger different energy supply, antiapoptotic, immunomodulatory, angiogenic and anti-oxidative stress responses in renal tissue compared with EVs secreted in normoxia. Proteomic analyses of renal tubule cells treated with EVs from ADMSCs in normoxia and hypoxia give a specific signature of modulated proteins for each type of EVs, indicating regulation of distinct biological processes. CONCLUSION: In summary, hypoxia potentially offers an interesting strategy to enhance the properties of EVs in the treatment of acute kidney disease.

L-Tyr-Induced Phosphorylation of Tyrosine Hydroxylase at Ser40: An Alternative Route for Dopamine Synthesis and Modulation of Na+/K+-ATPase in Kidney Cells.

BACKGROUND/AIMS: Dopamine (DA) is a natriuretic hormone that inhibits renal sodium reabsorption, being Angiotensin II (Ang II) its powerful counterpart. These two systems work together to maintain sodium homeostasis and consequently, the blood pressure (BP) within normal limits. We hypothesized that L-tyrosine (L-tyr) or L-dihydroxyphenylalanine (L-dopa) could inhibit the Na+/K+-ATPase activity. We also evaluated whether L-tyr treatment modulates Tyrosine Hydroxylase (TH). METHODS: Experiments involved cultured LLCPK1 cells treated with L-tyr or L-dopa for 30 minutes a 37°C. In experiments on the effect of Dopa Descarboxylase (DDC) inhibition, cells were pre incubated for 15 minutes with 3-Hydroxybenzylhydrazine dihydrochloride (HBH), and them L-dopa was added for 30 minutes. Na+/K+-ATPase activity was quantified colorimetrically. We used immunoblotting and immunocytochemistry to identify the enzymes TH, DDC and the dopamine receptor D1R in LLCPK1 cells. TH activity was accessed by immunoblotting (increase in the phosphorylation). TH and DDC activities were also evaluated by the modulation of the Na+/K+-ATPase activity, which can be ascribed to the synthesis of dopamine. RESULTS: LLCPK1 cells express the required machinery for DA synthesis: the enzymes TH, and (DDC) as well as its receptor D1R, were detected in control steady state cells. Cells treated with L-tyr or L-dopa showed an inhibition of the basolateral Na+/K+-ATPase activity. We can assume that DA formed in the cytoplasm from L-tyr or L-dopa led to inhibition of the Na+/K+-ATPase activity compared to control. L-tyr treatment increases TH phosphorylation at Ser40 by 100%. HBH, a specific DDC inhibitor; BCH, a LAT2 inhibitor; and Sch 23397, a specific D1R antagonist, totally suppressed the inhibition of Na+/K+-ATPase activity due to L-dopa or L-tyr administration, as indicated in the figures. CONCLUSION: The results indicate that DA formed mainly from luminal L-tyr or L-dopa uptake by LAT2, can inhibit the Na+/K+-ATPase. In addition, our results showed for the very first time that TH activity is also significantly increased when the cells were exposed to L-tyr.

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.

Mesenchymal stem cells and cell-derived extracellular vesicles protect hippocampal neurons from oxidative stress and synapse damage induced by amyloid-ß oligomers.

Alzheimer's disease (AD) is a disabling and highly prevalent neurodegenerative condition, for which there are no effective therapies. Soluble oligomers of the amyloid-ß peptide (AßOs) are thought to be proximal neurotoxins involved in early neuronal oxidative stress and synapse damage, ultimately leading to neurodegeneration and memory impairment in AD. The aim of the current study was to evaluate the neuroprotective potential of mesenchymal stem cells (MSCs) against the deleterious impact of AßOs on hippocampal neurons. To this end, we established transwell cocultures of rat hippocampal neurons and MSCs. We show that MSCs and MSC-derived extracellular vesicles protect neurons against AßO-induced oxidative stress and synapse damage, revealed by loss of pre- and postsynaptic markers. Protection by MSCs entails three complementary mechanisms: 1) internalization and degradation of AßOs; 2) release of extracellular vesicles containing active catalase; and 3) selective secretion of interleukin-6, interleukin-10, and vascular endothelial growth factor to the medium. Results support the notion that MSCs may represent a promising alternative for cell-based therapies in AD.

Bioactive lipids are altered in the kidney of chronic undernourished rats: is there any correlation with the progression of prevalent nephropathies?

BACKGROUND: Undernutrition during childhood leads to chronic diseases in adult life including hypertension, diabetes and chronic kidney disease. Here we explore the hypothesis that physiological alterations in the bioactive lipids pattern within kidney tissue might be involved in the progression of chronic kidney disease. METHODS: Membrane fractions from kidney homogenates of undernourished rats (RBD) were submitted to lipid extraction and analysis by thin layer chromatography and cholesterol determination. RESULTS: Kidneys from RBD rats had 25% lower cholesterol content, which disturb membrane microdomains, affecting Ca2+ homeostasis and the enzymes responsible for important lipid mediators such as phosphatidylinositol-4 kinase, sphingosine kinase, diacylglicerol kinase and phospholipase A2. We observed a decrease in phosphatidylinositol(4)-phosphate (8.8 ± 0.9 vs. 3.6 ± 0.7 pmol.mg-1.mim-1), and an increase in phosphatidic acid (2.2 ± 0.8 vs. 3.8 ± 1.3 pmol.mg-1.mim-1), being these lipid mediators involved in the regulation of key renal functions. Ceramide levels are augmented in kidney tissue from RBD rats (18.7 ± 1.4 vs. 21.7 ± 1.5 fmol.mg-1.min-1) indicating an ongoing renal lesion. CONCLUSION: Results point to an imbalance in the bioactive lipid generation with further consequences to key events related to kidney function, thus contributing to the establishment of chronic kidney disease.

Extracellular vesicles as regulators of tumor fate: crosstalk among cancer stem cells, tumor cells and mesenchymal stem cells.

The tumor microenvironment comprises a heterogeneous population of tumorigenic and non-tumorigenic cells. Cancer stem cells (CSCs) and mesenchymal stem cells (MSCs) are components of this microenvironment and have been described as key regulators of different aspects of tumor physiology. They act differently on the tumor: CSCs are described as tumor initiators and are associated with tumor growth, drug resistance and metastasis; MSCs can integrate the tumor microenvironment after recruitment and interact with cancer cells to promote tumor modifications. Extracellular vesicles (EVs) have emerged as an important mechanism of cell communication under the physiological and pathological conditions. In cancer, secretion of EVs seems to be one of the main mechanisms by which stem cells interact with other tumor and non-tumor cells. The transfer of bioactive molecules (lipids, proteins and RNAs) compartmentalized into EVs triggers different responses in the target cells, regulating several processes in the tumor as angiogenesis, tumor invasiveness and immune escape. This review focuses on the role of CSCs and MSCs in modulating the tumor microenvironment through secretion of EVs, addressing different aspects of the multidirectional interactions among stem cells, tumor and tumor-associated cells.

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.