Molecular hydrogen: prospective treatment strategy of kidney damage after cardiac surgery.

Cardiac surgery-associated acute kidney injury is a common post-operative complication, mostly due to increasing oxidative stress. Recently, molecular hydrogen (H2 gas) has also been applied to cardiac surgery due to its ability to reduce oxidative stress. We evaluated the potential effect of H2 application on the kidney in an in vivo model of simulated heart transplantation. Pigs underwent cardiac surgery within 3 h while connected to extracorporeal circulation (ECC) and subsequent 60 min of spontaneous reperfusion of the heart. We used two experimental groups: T-pigs after transplantation and TH-pigs after transplantation treated with 4% H2 mixed with air during inhalation of anesthesia and throughout oxygenation of blood in ECC. The levels of creatinine, urea and phosphorus were measured in plasma. Renal tissue samples were analyzed by Western blot method for protein levels of nuclear factor erythroid 2-related factor 2 (Nrf2), Kelch-like ECH-associated protein 1 (Keap-1), and superoxide dismutase (SOD1). After cardiac surgery, selected plasma biomarkers were elevated. However, H2 therapy was followed by the normalization of all these parameters. Our results suggest activation of Nrf2/Keap1 pathway as well as increased SOD1 protein expression in the group treated with H2. The administration of H2 had a protective effect on the kidneys of pigs after cardiac surgery, especially in terms of normalization of plasma biomarkers to control levels.

Effect of handling on ATP utilization of cerebral Na,K-ATPase in rats with trimethyltin-induced neurodegeneration.

Previously it was shown that for reduction of anxiety and stress of experimental animals, preventive handling seems to be one of the most effective methods. The present study was oriented on Na,K-ATPase, a key enzyme for maintaining proper concentrations of intracellular sodium and potassium ions. Malfunction of this enzyme has an essential role in the development of neurodegenerative diseases. It is known that this enzyme requires approximately 50% of the energy available to the brain. Therefore in the present study utilization of the energy source ATP by Na,K-ATPase in the frontal cerebral cortex, using the method of enzyme kinetics was investigated. As a model of neurodegeneration treatment with trimethyltin (TMT) was applied. Daily handling (10 min/day) of healthy rats and rats suffering neurodegeneration induced by administration of TMT in a dose of (7.5 mg/kg), at postnatal days 60-102 altered the expression of catalytic subunits of Na,K-ATPase as well as kinetic properties of this enzyme in the frontal cerebral cortex of adult male Wistar rats. In addition to the previously published beneficial effect on spatial memory, daily treatment of rats was accompanied by improved maintenance of sodium homeostasis in the frontal cortex. The key system responsible for this process, Na,K-ATPase, was able to utilize better the energy substrate ATP. In rats, manipulation of TMT-induced neurodegeneration promoted the expression of the α2 isoform of the enzyme, which is typical for glial cells. In healthy rats, manipulation was followed by increased expression of the α3 subunit, which is typical of neurons.

Alterations in Oxidative Stress Markers and Na,K-ATPase Enzyme Properties in Kidney after Fructose Intake and Quercetin Intervention in Rats.

The study aimed to characterize the consequences of a 15-week intake of 10% fructose on the kidney, with the focus on oxidative stress markers and properties of the Na,K-ATPase enzyme. Various antioxidants naturally occurring in common food were demonstrated to be protective against fructose-induced deterioration of kidneys. Therefore, we also aimed to observe the effect of 6-week quercetin administration (20 mg/kg/day) that was initiated following the 9-week period of higher fructose intake, by determining the concentration of sodium, potassium, creatinine, urea, and glucose in blood plasma and oxidative status directly in the renal tissue. Kinetic studies of renal Na,K-ATPase were utilized for a deeper insight into the molecular principles of expected changes in this enzyme activity under conditions of presumed fructose-induced renal injury. Fructose intake led to increase in body weight gain, plasma glucose and sodium levels, and deterioration of kidney properties, although some compensatory mechanisms were observable. Quercetin administration improved glycemic control in rats exposed to fructose overload. However, an increase in plasma creatinine, a decrease in GSH/GSSG ratio in renal tissue homogenate, and a controversial effect on renal Na,K-ATPase enzyme suggest that quercetin treatment may not be beneficial in the condition of pre-existing renal pathology.

Antioxidant SMe1EC2 may attenuate the disbalance of sodium homeostasis in the organism induced by higher intake of cholesterol.

The study was focused to the influence of higher intake of cholesterol on properties of the renal Na,K-ATPase, a key system in maintaining the homeostasis of sodium in the organism. Feeding for 4 weeks with cholesterol-enriched food for rats afflicted with hereditary hypertriglyceridemia by itself enhanced the activity of Na,K-ATPase, probably as a consequence of higher number of active enzyme molecules as suggested by 32 % increase of V (max) value. This may be hypothesized as a reason for the increased retention of sodium. Three-week-lasting treatment of animals kept on high cholesterol diet with antioxidant SMe1EC2 in a dose of 10 mg kg(-1) day(-1) normalized the function of renal Na,K-ATPase to the level comparable in hypertriglyceridemic rats fed with the standard diet. Therefore, our results suggest that the antioxidant SMe1EC2 in the applied dose seems to be effective in the attenuation of cholesterol-induced retention of sodium. Treatment for 3 weeks with Fenofibrate in a dose of 100 mg kg(-1) day(-1) reversed the function of renal Na,K-ATPase only slightly.

Chronic cardiotoxicity of doxorubicin involves activation of myocardial and circulating matrix metalloproteinases in rats.

AIM: To investigate the role of matrix metalloproteinases (MMPs) in the responses of rats to a prolonged doxorubicin (DOX) treatment. METHODS: Male Wistar rats were used. DOX was administered by intraperitoneal injections of seven doses (cumulative dose was 15 mg/kg). Control animals were treated with saline. Tissue or plasma samples were collected at four and eight weeks after the application of the last dose. Protein levels were determined by immunoblot assay, and MMP activities were measured by gelatin zymography. Superoxide content was analyzed using a lucigenin chemiluminescence assay and superoxide dismutase (SOD) activities with a SOD assay kit. Qualitative structural alterations of the heart were characterized by transmission electron microscopy. RESULTS: Systolic blood pressure was higher in DOX-treated rats as compared with the control rats at 8 weeks after treatment. In contrast, there were no differences in the heart rate between the control and DOX-treated rats. DOX treatment caused marked heterogeneous subcellular alterations of cardiomyocytes and structural disorganizations of the cardiac extracellular space. The effects of DOX were linked to a stimulation of plasma MMP-2 and MMP-9 activities that had already increased by 4 weeks after the end of the treatment. In the left ventricle, however, DOX only led to increased MMP-2 activation at 8 weeks after the end of treatment. These changes in tissue MMP-2 were connected with stimulation of Akt kinase activation, inhibition of SOD, an increase in superoxide levels, induction of iNOS protein expression and caspase-3 activation. CONCLUSION: Our results show that MMPs are involved in the chronic cardiotoxicity of DOX in rats. The data also suggest that reactive oxygen species (superoxide), NO production (iNOS) and the Akt kinase pathway can modulate MMP-2 activities in rat hearts influenced by DOX.

Pharmacology of Catechins in Ischemia-Reperfusion Injury of the Heart.

Catechins represent a group of polyphenols that possesses various beneficial effects in the cardiovascular system, including protective effects in cardiac ischemia-reperfusion (I/R) injury, a major pathophysiology associated with ischemic heart disease, myocardial infarction, as well as with cardioplegic arrest during heart surgery. In particular, catechin, (-)-epicatechin, and epigallocatechin gallate (EGCG) have been reported to prevent cardiac myocytes from I/R-induced cell damage and I/R-associated molecular changes, finally, resulting in improved cell viability, reduced infarct size, and improved recovery of cardiac function after ischemic insult, which has been widely documented in experimental animal studies and cardiac-derived cell lines. Cardioprotective effects of catechins in I/R injury were mediated via multiple molecular mechanisms, including inhibition of apoptosis; activation of cardioprotective pathways, such as PI3K/Akt (RISK) pathway; and inhibition of stress-associated pathways, including JNK/p38-MAPK; preserving mitochondrial function; and/or modulating autophagy. Moreover, regulatory roles of several microRNAs, including miR-145, miR-384-5p, miR-30a, miR-92a, as well as lncRNA MIAT, were documented in effects of catechins in cardiac I/R. On the other hand, the majority of results come from cell-based experiments and healthy small animals, while studies in large animals and studies including comorbidities or co-medications are rare. Human studies are lacking completely. The dosages of compounds also vary in a broad scale, thus, pharmacological aspects of catechins usage in cardiac I/R are inconclusive so far. Therefore, the aim of this focused review is to summarize the most recent knowledge on the effects of catechins in cardiac I/R injury and bring deep insight into the molecular mechanisms involved and dosage-dependency of these effects, as well as to outline potential gaps for translation of catechin-based treatments into clinical practice.

Influence of sub-chronic diabetes mellitus on functional properties of renal Na(+),K(+)-ATPase in both genders of rats.

For characterization of Na(+),K(+)-ATPase, a key enzyme involved in maintenance of intracellular sodium homeostasis, expression of alpha1 subunit and the ATP- and Na(+)-binding properties were investigated by Western blot analysis and by enzyme kinetics, respectively. Previous studies documented time-dependent alteration of properties of renal Na(+),K(+)-ATPase from its mobilization after 8 days to serious deteriorations after 16 weeks of diabetes in rats. Characterizing the critical period during development of the disease, when mobilization of Na(+),K(+)-ATPase observed in the acute phase turns to its damage, we examined the enzyme properties after 8 weeks lasting diabetes which was induced by a single intraperitoneal administration of streptozotocin in a dose of 65 mg.kg(-1). The unchanged expression of Na(+),K(+)-ATPase alpha1-subunit in both genders indicates that 8 weeks represent the time when the mobilization of enzyme synthesis observed previously in acute diabetes is lost. In this time the renal Na(+),K(+)-ATPase undergoes structural changes in the vicinity of Na(+)-binding site resulting in worsened affinity to sodium in both genders as indicated by 13% and 18% increase of K(Na) value in female and male rats, respectively. However, gender specific was the diabetes-induced decrease in affinity to ATP by 18% which occurred in female rats only.

Mitochondrial function in heart and kidney of spontaneously hypertensive rats: influence of captopril treatment.

Effect of captopril treatment on capability of heart and kidney mitochondria to produce ATP was investigated in spontaneously hypertensive rats (SHR). Heart mitochondria from SHR responded to hypertension with tendency to compensate the elevated energy demands of cardiac cells by moderate increase in mitochondrial Mg2+-ATPase activity, membrane fluidity (MF) and in majority of functional parameters of the mitochondria (p>0.05). Significant increase exhibited only the oxygen consumption (QO2; p<0.01-0.001) and oxidative phosphorylation rate (OPR; p<0.003) with glutamate+malate (GLUT+MAL) as substrates. Lowering the blood pressure (p<0.02) captopril also eliminated the above compensatory response and impaired the oxidative ATP production by decreasing OPR (p<0.001). Kidney mitochondria of SHR experienced serious disarrangement in parameters of oxidative ATP production: increase in Mg2+-ATPase activity (p<0.05) but, also scattered QO2 values (p<0.03-0.01) leading to decrease in OPR and the ADP:O (p<0.05-0.01) values with both GLUT+MAL and succinate as substrates. Captopril treatment does not alleviated but even worsened the above alterations. Mg2+-ATPase became also decreased and the depression of ADP:O became aggravated (p<0.0001).

Dual effect of polyphenolic compounds on cardiac Na+/K+-ATPase during development and persistence of hypertension in rats.

The enzyme kinetics of cardiac Na(+)/K(+)-ATPase were used for characterizing the ATP- and Na(+)-binding sites after administration of red wine polyphenolic compounds (Provinol) during developing and sustained hypertension. Hypertension was induced in rats (LN group) by the nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME, 40 mg*kg(-1)*day(-1)). Provinol (40 mg*kg(-1)*day(-1)) was applied during developing hypertension (LNPF4 group) and sustained hypertension (LNPF7/3 group). Provinol reduced the number of active Na(+)/K(+)-ATPase molecules in cardiac tissue, as indicated by decreased V(max) values (by 33% in LNPF4 and 26% in LNPF7/3 compared with LN). Concerning qualitative properties of the enzyme, Provinol induced different effects on the ATP- and Na(+)-binding sites of Na(+)/K(+)-ATPase. The ATP-binding site was impaired by Provinol, as indicated by increased K(m) value (by 52% in LNPF4 vs. LN), suggesting worsened utilization of substrate by the enzyme. In sustained hypertension, however, Provinol had no effect on the ATP-binding site, as indicated by unchanged K(m) value (LNPF7/3 vs. LN). On the other hand, the Na(+)-binding site was protected by Provinol, as suggested by decreased K(Na) value (by 72% in LNPF4 and 69% in LNPF7/3 vs. LN), indicating an increased affinity of the enzyme for sodium. Thus, Provinol appeared to stimulate the extrusion of Na(+) from cardiac cells, especially in the physiologically important range of sodium concentrations (2-10 mmol*L(-1)), during both developing and sustained hypertension.

Acute diabetes mellitus and its influence on renal Na,K-ATPase in both genders.

Due to the importance of renal Na,K-ATPase in maintaining the sodium homeostasis in the organism, its activity and abundance is intensively studied in condition of diabetes mellitus. The main subject of this study was the investigation of properties of renal Na,K-ATPase and abundance of its alpha1 subunit in view of possible gender-dependent differences in male and female diabetic rats. Diabetes was induced by a single intraperitoneal dose of streptozotocin in a dose of 65 mg.kg(-1). The acute diabetes lasting 8 days induced a significant increase in Na,K-ATPase activity accompanied by significant gender specific increase in K(m) value indicating a worsened affinity of ATP-binding site in female rats. In addition, our present experiments, revealed a significantly higher abundance of renal Na,K-ATPase alpha1 subunit in diabetic rats of both genders amounting 94% increase in males and 107% in females. But, not all of the newly synthesized enzyme molecules are fully active, as the increase in the number of active molecules is smaller (representing 23% in males and 20% in females) as indicated by lower increase in V(max) values.

Gender difference in functional properties of Na,K-ATPase in the heart of spontaneously hypertensive rats.

The aim of present study was the investigation of functional properties of the cardiac Na,K-ATPase in 16 weeks old male and female spontaneously hypertensive rats (SHR). The Na,K-ATPase activity in the presence of increasing concentrations of ATP, as well as Na(+) was lower in SHR of both genders, as compared to respective normotensive controls. Evaluation of kinetic parameters revealed a significant decrease of the maximum velocity (V(max)) in males (30% for ATP-activation, 40% for Na(+)-activation), as well as in females (24% for ATP, 29% for Na(+)), indicating a hypertension-induced diminution of the number of active enzyme molecules in cardiac sarcolemma. Insignificant changes were observed in the value of Michaelis-Menten constant (K(m)) in both cases. The concentration of sodium that gives half-maximal reaction velocity (K(Na)), increased by 38% in male and by 70% in female SHR. This impairment in the affinity of the Na(+)-binding site together with decreased number of active Na,K-ATPase molecules are probably responsible for the deteriorated enzyme-function in hearts of SHR. Direct comparison of SHR of both genders showed, that the enzyme from female hearts seems to be adapted better to hypertension as documented by its increased activity as a consequence of improved ability to bind and utilize ATP, as suggested by 32% decrease of K(m) value in females. In addition, the enzyme from female hearts is able to increase its activity (by 41%) in the presence of increasing sodium concentration even in the range where the enzyme from male hearts is already saturated.

Rosmarinic acid administration attenuates diabetes-induced vascular dysfunction of the rat aorta.

OBJECTIVES: Oxidative stress as well as inflammation processes are engaged in diabetic vascular complications. Rosmarinic acid, a natural phenol antioxidant carboxylic acid, was found to have multiple biological activity, including anti-inflammatory and antitumour effects, which are a consequence of its inhibition of the inflammatory processes and of reactive oxygen species scavenging. The aim of this work was to study effects of rosmarinic acid administration on vascular impairment induced by experimental diabetes in rats. METHODS: Diabetes was induced by streptozocin (3 × 30 mg/kg daily, i.p.) in Wistar rats. Rosmarinic acid was administered orally (50 mg/kg daily). Ten weeks after streptozocin administration, the aorta was excised for functional studies, evaluation by electron microscopy and real time PCR analysis. KEY FINDINGS: In the aorta of diabetic rats, decreased endothelium-dependent relaxation was accompanied by overexpression of interleukin-1ß, tumour necrosis factor-α, preproendothelin-1 and endothelin converting enzyme-1. Structural alterations in the endothelium, detected by electron microscopy, indicated aortic dysfunction caused by diabetes. The diabetes-induced aortic disorders were prevented by rosmarinic acid administration. CONCLUSIONS: Rosmarinic acid protected aortic endothelial function and ultrastructure against diabetes-induced damage. Both antioxidant and anti-inflammatory effects of rosmarinic acid seemed to participate in the mechanism of this protection.

Supplementation with n-3 polyunsaturated fatty acids to lipopolysaccharide-induced rats improved inflammation and functional properties of renal Na,K-ATPase.

Measurements of enzyme kinetics of renal Na, K-ATPase were used for characterization of ATP- and Na⁺-binding sites in rats that were subjected to 10 days of moderate inflammation that was induced by a single dose of Escherichia coli lipopolysaccharides (LPSs) at a dose of 1 mg kg⁻¹ body weight. We hypothesized that LPSs might initiate a malfunction of renal Na, K-ATPase, which is a key enzyme involved in regulation of sodium homeostasis in the organism. We also investigated the potential effect that fish oil (FO) has in the prevention of Na, K-ATPase alterations by administering FO daily at a dose of 30 mg kg⁻¹. Alone, LPS elevated the level of C-reactive protein by more than 500% and free radicals by 36% in plasma, as indicated by an increased level of malondialdehyde. The Na, K-ATPase was slightly altered in the vicinity of the ATP-binding site as suggested by the 9% increase of the concentration of ATP necessary for half-maximal activation of the enzyme, thus indicating a deteriorated binding of ATP as a consequence of inflammation. Daily supplementation of FO partly attenuated LPS-induced injury, as observed by a significant decrease in the plasma levels of C-reactive protein and free radicals, hence maintaining the activity of renal Na, K-ATPase to the level of healthy control animals. In conclusion, our findings showed that FO prevented an excessive malondialdehyde production in LPS-treated animals and stabilized renal Na, K-ATPase.

Sex differences in endothelial function of aged hypertriglyceridemic rats - effect of atorvastatin treatment.

The aim of the study was to test the hypothesis that the effect of atorvastatin on endothelium-dependent relaxation of the superior mesenteric artery (SMA) may differ in male vs. female aged hypertriglyceridemic rats (HTGs). Experiments were performed on 11-month-old male and female Prague hereditary HTGs. Atorvastatin (ATO) was administered p.o. in the dose of 0.30 mg/100g/day. Controls received vehiculum. After two months of ATO administration blood pressure, serum triglycerides (TG) and total cholesterol (CHOL) were determined. Endothelial function of SMA was studied in vitro using evaluation of relaxant responses of precontracted SMA to acetylcholine. The serum TG of control male HTGs were found to be statistically higher than those of female controls, while CHOL and blood pressure did not share gender differences. Responses of SMA of female control HTGs were statistically decreased compared to their male counterparts. ATO treatment induced decrease in blood pressure and TG of both males and females, yet CHOL values were reduced only in females. The protective effect of ATO on SMA endothelial function was much more pronounced in females compared to males. We conclude that vascular endothelial dysfunction of aged HTG rats is more severe and more attenuated by ATO in females compared to males. The protective effect of ATO on vascular endothelial function does not seem to depend solely on its lipid lowering action.

Effect of saliva from horse fly Hybomitra bimaculata on kinetic properties of Na,K-ATPase: possible role in regulation of relaxation.

The possible involvement of salivary gland extract (SGE) from horse flies in modifying hyperpolarization and relaxation via alterations in functional properties of sarcolemmal Na,K-ATPase in the host tissue was tested in vitro by application of various amounts of SGE from Hybomitra bimaculata.SGE in the amount of 3 µg proteins representing approximately the equivalent of one salivary gland of Hybomitra bimaculata induced a stimulatory effect on Na,K-ATPase at all ATP concentrations applied. This effect resulted from the improved ATP-binding site affinity in the Na,K-ATPase molecule, as implicated by the reduction in K(M). Increasing the amount of SGE to 6.5 µg resulted in inhibition of the enzyme, which was characterized by reduction in V(max) and also K(M). This suggests that in the presence of relatively high Hybomitra bimaculata SGE concentration some SGE components affect Na,K-ATPase, when ATP is already bound to the enzyme.Our results indicate that SGE from the horse fly Hybomitra bimaculata contain at least two different biologically active compounds modifying the acute recovery and maintenance of excitability during contractile activity in the host tissue by affecting Na,K-ATPase with opposite effects, depending on the ratio of SGE-proteins to proteins of the host tissue.

Effect of maturation on renal Na+/K+-atpase and its susceptibility to nitric oxide-deficient hypertension in rats.

1. The present study deals with the effect of maturation on the kinetic properties of renal Na(+)/K(+)-ATPase and its susceptibility to nitric oxide (NO)-deficient hypertension induced by the NO synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME). 2. Immature (4-week-old) and adult (12-week-old) male Wistar rats were administered L-NAME (40 mg/kg per day) in their drinking water for 4 weeks. 3. The properties of the ATP- and Na(+)-binding sites of Na(+)/K(+)-ATPase were investigated by activation of the enzyme with increasing concentrations of the energy substrate ATP and/or cofactor Na(+). Unchanged values of K(m) suggest that energy utilization by the enzyme in the kidney of control rats remains unaffected during maturation. Conversely, the decrease in K(Na) values (the concentration of Na(+) necessary to achieve half-maximal reaction velocity) indicates improved affinity for Na(+) in the older group of control rats. 4. Application of L-NAME to all young animals had no significant effect on the functional properties of Na(+)/K(+)-ATPase. 5. In adult animals, the V(max) values remained unchanged after treatment with L-NAME, but the affinities of the ATP- and Na(+)-binding sites were decreased, as indicated by significant increase in K(m) and K(Na) values. 6. Maturation of control rats was accompanied by an increase in the Na(+) affinity of renal Na(+)/K(+)-ATPase without affecting ATP utilization. However, maturation increased the susceptibility of renal Na(+)/K(+)-ATPase to the harmful effects of L-NAME.