Rat Kidney Homogenates Research Articles

Вплив кверцетину на показники оксидативнонітрозативного стресу в тканинах нирок щурів за умов гострого десинхронозу та ліпополісахаридіндукованої системної запальної відповіді

The relevance of this study stems from the growing interest in the impact of acute desynchronosis (AD) on oxidative disorders and the insufficiently studied effect of flavonoids on these processes in kidney tissue. This study investigates the effect of quercetin on oxidative and nitrosative stress in rat kidney homogenate under AD and 3 lipopolysaccharide (LPS)-induced systemic inflammatory response (SIR). The animals were randomly divided into five groups: group 1 served as the control, group 2 underwent LPS-induced SIR modeling for 8 weeks, group 3 experienced AD reproduction by shifting the light-dark cycle 6 hours earlier for 7 days, group 4 underwent AD modeling during the last week of LPS-induced SIR, and rats in group 5 were administered a water-soluble form of quercetin (corvitin) intragastrically was administered at a dose of 200 mg/kg (20 mg/kg in terms of quercetin) daily during the AD modeling against LPS-induced SIR. It was found that the modeling of LPS-induced oxidative stress and AD significantly increased the rate of superoxide anion radical production in kidney homogenate by microsomal monooxygenases and NO synthase (NOS), mitochondrial respiratory chain, leukocyte NADPH oxidase, inducible NOS activity, and decreased the activity of the constitutive NOS isoform and its coupling index. Under AD modeling, the rate of superoxide anion radical production by leukocyte NADPH oxidase, total and inducible NOS activity was significantly lower compared with LPS-induced SIR. When modeling AD against the background of LPS-induced SIR, the production of superoxide anion radical by microsomes, mitochondria and leukocyte NADPH oxidase exceeded the respective results of group 2 by 15.9%, 16.0% and 38.5%, and group 3 by 24.4%, 17.5% and 74.6%. The inducible NOS activity was 22.9% higher compared to the value of group 2 and 93.4% higher in group 3, the concentration of peroxynitrites of alkali and alkaline earth metals was 35.5% higher than the respective parameters of group 2 and 57.9% higher than in group 3. Administration of quercetin during AD modeling and LPS-induced oxidative stress significantly reduced the rate of superoxide anion radical production by microsomal monooxygenases by 17.1%, mitochondrial respiratory chain by 31.2%, leukocyte NADPH oxidase by 19.4%, total and inducible NOS activity by 50.4% and 55.5%, respectively; the concentration of peroxynitrites of alkali and alkaline earth metals diminished by 41.4% compared to the value of group 4. It has been concluded that the administration of quercetin significantly reduces the signs of oxidative-nitrosative stress in the kidney homogenate of rats.

Effects of Fenofibrate and Gemfibrozil on Kynurenic Acid Production in Rat Kidneys In Vitro: Old Drugs, New Properties.

Kidney dysfunction significantly increases the cardiovascular risk, even in cases of minor functional declines. Hypertriglyceridemia is the most common lipid abnormality reported in patients with kidney disorders. PPAR-α (peroxisome proliferator-activated receptor-α) agonists called fibrates are the main agents used to lower triglyceride levels. Kynurenic acid (KYNA) is a tryptophan (Trp) derivative directly formed from L-kynurenine (L-KYN) by kynurenine aminotransferases (KATs). KYNA is classified as a uremic toxin, the level of which is correlated with kidney function impairments and lipid abnormalities. The aim of this study was to analyze the effect of the most commonly used triglyceride-lowering drugs, fenofibrate and gemfibrozil, on KYNA production and KAT activity in rat kidneys in vitro. The influence of fenofibrate and gemfibrozil on KYNA formation and KAT activity was tested in rat kidney homogenates in vitro. Fenofibrate and gemfibrozil at 100 µM-1 mM significantly inhibited KYNA synthesis in rat kidney homogenates. Both fibrates directly affected the KAT I and KAT II isoenzyme activities in a dose-dependent manner at similar concentrations. The presented results reveal the novel mechanism of action of fibrates in the kidneys and suggest their potential role in kidney function protection beyond the well-known anti-hyperlipidemic effect.

Antioxidant potential of ethanol extract of Annona muricata leaves and its inhibitory effect on lipid peroxidation in 1,2-dimethylhydrazine induced colon carcinogenesis

Annona muricata, as it is scientifically called but known in most parts of Nigeria as soursop. A fruit tree with various therapeutic uses, the plant belongs to the family of Annonaceae and has several medicinal properties. The medicinal properties of Annona muricata are well known, and they can be used to treat a variety of illnesses and conditions. Effects of ethanol extract of Annona muricata leaves (EEAML) on the enzymatic antioxidants of the kidney and colon in Wistar rats induced with 1, 2 Dimethylhydrazine (DMH) at 25 mg/kg bodyweight s.c. were investigated in this study. Thirty-six male Wistar rats weighing 110–170g were acclimated for two weeks and randomized into six groups (six per group). Group A (control), Group B (extract; 120 mg/kg body weight of ethanolic extract of A. muricata), Group C (DMH only), Group D (DMH + EXTRACT), Group E (pretreatment), and Group F (posttreatment). The extract was administered daily via oral gavage, while the DMH was given subcutaneously at a dosage of 25 mg/kg bodyweight. This study reveals the carcinogenic effect of DMH induced oxidative stress were ameliorated by the administration of this leaf extract of EEAML at a dose of 120mg/kg b.w. The colon and kidney homogenates of rats administered EEAML (co-treated, pretreatment and post- treatment) showed increased enzymatic antioxidant; glutathione peroxidase, catalase activities and SOD activities. But a reduction in malondialdehyde levels was observed. These findings validate the use of Annona muricata in traditional medicine by showing that the ethanolic extract of the leaves can reduce DMH-induced oxidative stress and alter the enzymatic antioxidant profile in colon carcinogenesis.

Barleria prionitis L. extracts ameliorate doxorubicin-induced acute kidney injury via modulation of oxidative stress, inflammation, and apoptosis.

Doxorubicin (DOX) is a chemotherapeutic drug with potential nephrotoxic effects on patients who are on cancer chemotherapy. An interest has been observed in using natural products to ameliorate the potential side effects of DOX. The present study is to investigate the cellular mechanisms underlying the protective effects of Barleria prionitis L. (BP) (Acanthaceae) extracts, DOX-induced acute kidney injury (AKI). Hexane (25mg/kg/day), ethyl acetate (80mg/kg/day), n-butanol (70mg/kg/day), and water (120mg/kg/day) extracts of BP, were administered to DOX-induced (5mg/kg (2500μL/kg), ip) Wistar rats for four consecutive weeks. At the end of the study, investigations were carried out for the assessment of biomarkers of nephrotoxicity, oxidative stress, inflammation, and apoptosis. Treatments with BP extracts significantly reversed DOX-induced elevations in serum and urine biochemical markers of nephrotoxicity (serum creatinine; 21-33%, blood urea nitrogen; 26-58%, β2-microglobulin; 19-22% and urine total protein; 47-67%). There was a reduction in the levels of tumor necrosis factor-α, interleukin-1β, and malondialdehyde in kidney homogenates of rats treated with the n-butanol extract (by 43, 62, and 24%) and water extract (by 57%, 85%, and 26%) (p<0.05). Immunohistochemical expression of the pro-apoptotic B-cell associated X protein was reduced while the anti-apoptotic B-cell lymphoma gene product 2 protein was increased in kidney tissues after the treatments with BP extracts. The selected BP extracts significantly ameliorated DOX-induced AKI. The findings would open new vistas for the development of a drug using the BP extracts to minimize DOX-induced AKI in cancer patients.

#3508 FUROSEMIDE DECREASES KYNURENIC ACID SYNTHESIS IN RAT KIDNEY IN VITRO

Abstract Background and Aims Loop diuretics are highly potent agents, particularly beneficial in patients with impaired kidney function or heart failure exacerbation. Furosemide, due to fast onset and short time of action, remains the most commonly used drug from this class of diuretics. Inhibition of sodium-potassium-chloride cotransporter (NKCC2) in the thick ascending limb of Henle's loop is the main mechanism of furosemide's action. Other effects of furosemide, like modulation of potassium channels activity or activation of prostaglandin synthesis, were previously described. It was postulated that furosemide may exacerbate kidney injury in a dose dependent manner. Kynurenic acid (KYNA) is a tryptophan derivative synthesized from L-kynurenine (L-KYN) by kynurenine aminotransferases (KATs). KAT I and KAT II are the main KAT isoenzymes. Non-selective antagonism towards ionotropic glutamatergic receptors, as well as activation of aryl hydrocarbon receptors and the α7 nicotinic acetylcholine receptors are known mechanisms of KYNA’s action. KYNA was shown to regulate water and electrolyte balance through natriuretic effect. By some researchers KYNA, together with other tryptophan metabolites, is classified as uremic toxin. We aimed to analyze the influence of furosemide, widely used loop diuretic, on KYNA’s synthesis and the activity of both KATs in rat kidney in vitro. Method The influence of furosemide on KYNA’s production with KAT I and KAT II activity was tested in rat kidney homogenates in vitro after 2 hours incubation in the presence of KYNA precursor L-KYN and furosemide. Diuretic was examined at increasing concentrations: 1 μM, 10 μM, 50 μM, 100 μM, 500 μM and 1 mM. Enzymatic KYNA’s production was measured by high performance liquid chromatography (HPLC) with fluorometric detector. All applicable international, national and institutional guidelines for the care and use of animals were followed. Results Furosemide at 500 μM and 1 mM lowered KYNA formation in kidney homogenates in vitro to 80% (p &amp;lt; 0.05) and 70% (p &amp;lt; 0.05) of control value, respectively. At 1 mM concentration furosemide inhibited kidney KAT I activity in vitro to 38% (p &amp;lt; 0.01) of control value. Similarly, furosemide at 500 μM and 1 mM concentration decreased kidney KAT II activity in vitro to 53% (p &amp;lt; 0.01) and 18% (p &amp;lt; 0.001) of control value, respectively. Conclusion Our study presents a novel mechanism of action of furosemide. Inhibition of KYNA synthesis in the kidney by furosemide may have influence on kidney function.

Antitoxic Principles from Moringa oleifera (Mo11) and Musa sapientum (Ms06) Ameliorated Cadmium Chloride-Induced Renal Hyperplasia and Apoptosis through Ki67/P53-Mediated Pathway in Rats

Background: Cadmium (Cd) is an established carcinogen. Cd-induced renotoxicity resulted in oxidative stress, loss of excretory kidney functions, and apoptosis of murine kidney cells. Objectives: This study evaluated renoprotective potentials of MO11 (isolated from Moringa oleifera leaves) and MS06 (isolated from Musa sapientum suckers) against Cd chloride (CdCl2)-induced renotoxicity, renal hyperplasia, and apoptosis in rats. Materials and Methods: Twenty-four adult male Wistar rats (average weight of 155 g) were randomly divided into seven groups (n = 4). Group 1 received physiological saline. Groups 2–4 and 6 received single intraperitoneal (i.p) administration of 1.5 mg/kg body weight of CdCl2 (i.p) (Day 1). Groups 3–4 and 6 were posttreated with 15 mg/kg body weight of MO11, 15 mg/kg body weight of MO11 +7 mg/kg body weight of MS06, and 3.35 mg/kg body weight of doxorubicin, respectively (days: 1–17). Group 5 received only olive oil dose (vehicle), respectively (days: 1–17). Kidney histopathology (hematoxylin and eosin technique) and enzyme-linked immunosorbent assay concentrations of biomarkers of proliferation (Ki67) and apoptosis (p53) in kidney homogenates of rats of Groups 1–6 were evaluated. Results: Histopathological analyses showed normal kidney histology in the rats of Groups 1–6. Posttreatments of CdCl2-induced renotoxicity with MO11, MO11+MS06, and doxorubicin resulted in downregulations of Ki67 and p53 in Groups 3, 4, and 6 as compared with Group 2. Conclusion: MO11 and MS06 possess renoprotective, anti-proliferation, and anti-apoptosis potentials.

Tri-Reagent Homogenate Is a Suitable Starting Material for UHPLC-MS Lipidomic Analysis

Background: Transcriptomic and lipidomic dual analyses usually initiate with independent extractive procedures. That entails a difficulty in aligning results from both omics platforms, especially in the case of highly heterogeneous tissues, such as the kidney. Methods: Bligh and Dyer lipid extraction was performed using rat kidney homogenates prepared in PBS or commercially available Tri-reagent used for RNA extraction. Samples were analyzed by ultrahigh performance liquid chromatography-mass spectrometry (UHPLC-MS) lipidomic analysis. Results: Comparison of the lipidome obtained from phosphate-buffered saline (PBS) and Tri-reagent homogenates showed qualitative and quantitative validity of the Tri-reagent homogenate with the exception of ether lipids; the acidic nature of the mix seems to promote the hydrolysis of the ether bond, especially in plasmalogens. We tested several conditions in the sample processing, which allowed to optimize the procedure. Conclusions: Aiming to implement a method that allows the extraction of RNA and lipids from the same tissue homogenate not using external tracers, we here report the use of Tri-reagent homogenates as a suitable starting material for UHPLC-MS lipidomic analysis.

Phytochemical Profiling, Anti-inflammatory, Antioxidant and Antimicrobial Effects of Seven IndianMorinda Species

Background: The fruits, leaves and roots of Morinda species are used in the treatment of inflammations, cancers, diabetes, psychiatric disorders, bacterial and viral infections. However, no study has been conducted on chemical profiling, anti-inflammatory, antioxidant and antimicrobial potentials of leaves of seven Indian Morinda species. Aim: The study aimed to investigate the anti-inflammatory, antioxidant and antimicrobial effects of methanol extract of seven Indian Morinda species. Material and Methods: The total contents of iridoids, flavonoids, anthraquinone glycosides, triterpenoids, lignans and coumarins from methanol extract of each species were determined by using different established protocols. The anti-inflammatory activity of methanol extracts of each species was evaluated using carrageenan and CFA-induced arthritis in male Wistar albino rats. In vivo, antioxidant activity was determined by estimating the levels of superoxide dismutase, catalase, glutathione and malondialdehyde in liver and kidney homogenates of male Wistar rats. The antimicrobial activity of methanol extracts of all seven species was determined by using the microdilution method against selected microbes. Results: Different values of total contents of iridoids, flavonoids, anthraquinone glycosides, triterpenoids, lignans, and coumarins were achieved from methanol extract of leaf of M. umbellata, M. jasminoides, M. reticulata, M. parvifolia and M. persicaefolia. The potent anti-inflammatory effect was demonstrated (carrageenan-induced paw oedema model) by methanol extract of leaves of M. umbellata at 50 mg/kg dose. Similarly, M. umbellata methanol extract showed maximum antiarthritic effect against CFA-induced arthritis on the 17th day (p.o.). Maximum SOD levels in liver and kidney homogenates were increased by the methanol extract of M. persicaefolia. The catalase concentration was enhanced by the methanol extract of M. jasminoides. GSH level was raised by the methanol extract of leaves of M. umbellata, but M. royoc reduced the levels of MDA in treated animals. The methanol extract of M. parvifolia leaves displayed maximum antibacterial activity against K. pneumoniae. M. persicaefolia methanol extract showed the strongest antifungal activity against P. chrysogenum. Conclusion: The methanol extract of leaves of M. jasminoides, M. reticulata, M. parvifolia, M. umbellata and M. persicaefoli showed promising anti-inflammatory, antioxidant and antimicrobial effects on the studied experimental models.

11βHSD2 Efficacy in Preventing Transcriptional Activation of the Mineralocorticoid Receptor by Corticosterone.

Affinity of the mineralocorticoid receptor (MR) is similar for aldosterone and the glucocorticoids (GC) cortisol and corticosterone, which circulate at concentrations far exceeding those of aldosterone. 11β-hydroxysteroid dehydrogenase type 2 (11βHSD2) inactivation of GC within the immediate vicinity of the MR is credited with prereceptor specificity for aldosterone in cells coexpressing MR and 11βHSD2. 11βHSD2 efficacy is also critical to other recently described 11βHSD2 substrates. The aim of this work was to address doubts that low levels of expression of 11βHSD2 in aldosterone target tissues suffice to prevent the initiation of gene transcription by the MR activated by physiological concentrations of corticosterone. Cell models stably expressing an MR/Gaussia luciferase reporter and various levels of constitutive or induced 11βHSD2 at concentrations lower than those in rat kidney homogenates and microsomes were produced. Aldosterone and corticosterone were equipotent transactivators of the MR reporter gene in cells without 11βHSD2. Rate of conversion of tritiated corticosterone to 11-dehydrocorticosterone increased and corticosterone-induced nuclear translocation of MR decreased, as 11βHSD2 expression increased. The 50% maximal MR activation for the reporter gene stimulation by corticosterone rose with increasing 11βHSD2 expression, shifting the steroid dose-response curve for corticosterone-induced MR transactivation to the right. Several stable cell lines expressing an easily and reproducibly measured MR reporter system and consistent incremental amounts of 11βHSD2 protein were produced and used to document that 11βHSD2 within low physiological levels inactivates relevant concentrations of GC and decreases MR transactivation by GC in a dose-dependent fashion, laying to rest doubts of the efficacy of this enzyme.

Chenopodium ambrosioides var. ambrosioides leaf extracts possess regenerative and ameliorative effects against mercury-induced hepatotoxicity and nephrotoxicity

Empirical literature reveals the use of Chenopodium ambrosioides var. ambrosioides leaf extract as antipyretic, antimicrobial, antioxidant, cancer chemoprotective, and antidiabetic agent in folklore medicine – with these pharmacological properties linked to its secondary metabolites. There are, however, no adequate information on the possible protective, regenerative, and ameliorative effects on toxicity induced by heavy metals. Hence, the investigations on mercury-induced nephrotoxicity and hepatotoxicity in this study. The leaf extract was profiled using gas chromatographic instrument coupled with mass spectrometer (GC–MS) and revealed 100 metabolites ranging from polyphenols, terpenes, terpenoids, and cardiac glycosides. The antioxidant assays revealed that C. ambrosioides leaf extracts possess antioxidant properties with hexane fraction exhibiting the highest scavenging activities for DPPH (IC50 = 0.02 mg/mL), FRAP (730.92 mg/AAE g), and metal ion chelating ability (IC50 = 3.18 mg/mL). Nephrotoxicity and hepatotoxicity biomarkers using total protein, albumin, total bilirubin, direct bilirubin, catalase, glutathione peroxidase (GPx), reduced glutathione (GSH), creatinine, uric acid, urea, ALT, and AST activities both in the kidney and liver homogenates of Wistar rats revealed significant regenerative improvement which differs significantly at p < 0.05 when compared with experimental rats with toxicant only. In the same vein, the histomorphological analysis showed remarkable hepatocellular regeneration presenting features of normal architectural arrangements of hepatic tissues while acute toxicological studies revealed that the LD50 of C. ambrosioides leaf material is well above 5000 mg/kg. The outcomes of this study showed that the leaf extract exhibited high hepatoprotective and nephroprotective activities as well as being implicated in the discovery of safe, cheaper, and effective therapeutic agents thereby highlighting its importance as a promising industrial crop.

P0016EFFECT OF GEMFIBROZIL ON KYNURENINE AMINOTRANSFERASE ACTIVITY AND KYNURENIC ACID PRODUCTION IN RAT KIDNEY

Abstract Background and Aims Hypertriglyceridemia is the most common lipid disorder in chronic kidney disease. Lifestyle changes and fibrates administration are main methods of lowering triglycerides’ level. PPARα (peroxisome proliferator-activated receptor-α) activation is the primary mechanism of action of fibrates. A tryptophan metabolite, kynurenic acid (KYNA), is produced from L-kynurenine (L-KYN) by kynurenine aminotransferases (KATs). KAT I and KAT II isoenzymes are the best analyzed KATs. KYNA acts predominantly as a nonselective antagonist of ionotropic glutamatergic receptors, particularly N-methyl-D-aspartate (NMDA) type, which are highly expressed in the kidney. Natriuretic and hypotensive effects of KYNA are well described. Diet rich in fatty acids was reported to elevate central and peripheral KYNA level. The goal of presented study was to analyze the influence of gemfibrozil, one of the fibrates given to treat hypertriglyceridemia, on KYNA production and the activity of KAT isoenzymes: KAT I and KAT II, in rat kidney in vitro. Additionally, the molecular docking of gemfibrozil to KAT I and KAT II structures was performed to study drug-enzyme interaction. On the final step the microarray datamining was carried out to investigate whether gemfibrozil affects the expression of KAT-coding genes. Method The effect of gemfibrozil on KYNA synthesis together with KAT I and KAT II activity was tested in rat kidney homogenates in vitro after 2 hours incubation in the presence of L-KYN and gemfibrozil. The drug was examined at the concentration of 1 µM, 10 µM, 50 µM, 100 µM, 500 µM and 1 mM. Production of KYNA was analyzed using the high performance liquid chromatography (HPLC) with fluorometric detector. Results Gemfibrozil at 100 µM, 500 µM and 1 mM decreased KYNA production in kidney homogenates in vitro to 66% (P &amp;lt; 0.05), 58% (P &amp;lt; 0.01) and 41% (P &amp;lt; 0.01) of control value, respectively. At 100 µM, 500 µM and 1 mM concentration gemfibrozil lowered renal KAT I activity in vitro to 68% (P &amp;lt; 0.05), 56% (P &amp;lt; 0.01) and 52% (P &amp;lt; 0.01) of control value, respectively. Moreover, gemfibrozil at 500 µM and 1 mM concentration decreased kidney KAT II activity in vitro to 47% (P &amp;lt; 0.001) and 26% (P &amp;lt; 0.001) of control value, respectively. Results of the molecular docking suggested that gemfibrozil may affect the active site of both KAT I and KAT II. Publicly available microarray datasets suggested that the expression of KAT-coding genes does not change after gemfibrozil administration. Conclusion Gemfibrozil decreases KYNA production in rat kidney in vitro through inhibition of KAT I and KAT II isoenzymes. Presented results indicate a novel mechanism of gemfibrozil’s action in the kidney. Its potential role in nephrotoxicity needs verification in upcoming studies.

Curcumin Improves Biomolecules Associated with Renal Function and Attenuates Oxidative Injury and Histopathological Changes in Potassium-Induced Toxicity in Rats' Kidney.

The protective effect of curcumin on potassium bromate (KBrO3)-induced renal damage was investigated in vivo. Treatment with KBrO3 (20mg/kgbw) caused a significant increase in arginase and adenosine deaminase (ADA) activities in rats' kidney. However, oral administration of curcumin (20mg/kg bw) caused a significant reduction in ADA and arginase activities in KBrO3 + CUR group. Furthermore, nitric oxide level was significantly low in KBrO3 group compared with the control. After treatment with curcumin in KBrO3 + CUR group, nitric oxide levels increased significantly (P < 0.05). Determination of some kidney biomarkers revealed elevated levels of creatinine, serum urea, and electrolytes (Na+ and Cl-) in KBrO3-treated rats. Curcumin effectively reduced the levels of these renal function parameters in KBrO3 + CUR groups and were not significantly different from the control. Antioxidant enzyme activities such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX) activities as well as glutathione (GSH) levels were significantly low with concomitant higher levels of malondialdehyde (MDA) after treatment with KBrO3. Curcumin caused a significant increase in SOD, CAT, and GPX activities including GSH levels with lower production of MDA in kidney homogenates of rats in KBrO3 + CUR. Curcumin ameliorated corpuscular degeneration in the kidney tissue and exhibited protection against tubular necrosis. These results revealed the protective effect of curcumin against KBrO3-induced renal toxicity by preventing degradation of ADA and arginine, improving antioxidant status and histopathological changes in rats' kidney.

Molecular mechanisms of the nephroprotective action of polyphenols in experimental chronic kidney disease. Association with hydrogen sulfide system

Annotation. The results of a study of the effects of genistein, resveratrol and quercetin on the main markers of oxidative stress, thiol disulfide equilibrium and the state of the nitric oxide system in rat kidneys after modelling chronic kidney disease (CKD) (5/6 of a single kidney nephrectomy) is shown. Blood and single rat kidney homogenate were used for biochemical studies. Statistical processing of the obtained results was performed in the program “STATISTICA 6.1”. The results are presented as M±m. The significance of the difference between the scores was evaluated using the Student's t-test or the Mann-Whitney U-test. A nonparametric Spearman correlation coefficient was used to determine the correlation between two independent indicators. It was shown that the studied substances, especially quercetin, caused antioxidant activity in CKD, which was manifested by a significant decrease of MDA, CGP and NADPH oxidase activity (by 21.0–40.4%), as well as an increase in SOD activity (by 29.7–38.7%) in kidney compared with untreated animals. Reliable strong correlation (r=ǀ0.65–0.74ǀ) between the H2S content and oxidative stress markers was established. The use of polyphenols in CKD reduced the thiol-disulfide imbalance in rat kidneys: the activity of thioredoxin reductase significantly increased, the content of reduced thiol groups of proteins (by 16.0–66.4%) and the level of disulfides decreased (by 21.1–25.3%), in comparison with untreated animals. Stabilization of the thiol disulfide equilibrium is one of the mechanisms of the effect of polyphenols on the H2S system in the kidneys, since significant and strong correlation relationships were recorded between these indicators and the H2S content (r=ǀ0.63–0.75ǀ). The administration of resveratrol, and especially genistein, was accompanied by an increase in the activity of the eNOS (by 36.3–44.7%) and a decrease in the activity of iNOS (by 15.4–20.9%), in comparison with untreated animals. The NO system in the kidneys is an important molecular target for influence of genistein and resveratrol on the production of H2S during CKD is realized, since reliable strong correlation were recorded between the H2S content and the activity of NO synthase isoforms (r=ǀ0.70–0.75ǀ).

Oxidatively modified proteins in kidneys of rats fed with glyphosate-resistant genetically modified soybean and the herbicide Roundup

Genetic technologies have become a tool for achieving the desired properties of plant crops instead of traditional breeding in recent decades. They consist in artificial editing of a plant genome (genetic modification) by inserting the genes encoding desired features from the DNA of one organism in another, often unrelated, species. One of the most popular crops is soybean containing up to 38–42% of proteins in its seeds, and its most common type is GTS 40-3-2 (Monsanto Canada Inc.) line of transgenic soybean. The genetically modified soybean "Roundup Ready" is resistant to the action of herbicide "Roundup" (it continues to grow when it is cultivated with this herbicide). Therefore, the study of individual and combined effects of both factors on the free radical oxidation processes in biomolecules is very relevant. Experimental research was performed on 4-month Wistar rats to study the long-term effects of feeding with genetically modified soybean and herbicide "Roundup", both separately and together, on the rat kidneys. The results of the study showed that after 12 months of feeding with genetically modified soybean treated with herbicide "Roundup" (IV group) and receiving the herbicide with drinkable water (V group), there was an increase in the level of carbonyl derivatives in the rat kidney homogenates in the first (F0) and in subsequent generations (F1, F2) of rats. The research results showed that the highest level of carbonyl derivatives was noted in the kidneys of the third generation of rats. Along with the increase in oxidatively modified proteins in the rat kidney homogenates, there was a decrease in the content of sulfhydryl groups and proteolytic enzymes in the IV and V groups, the lowest level was observed in the third generation. The use of the same transgenic soybean variety not treated with any herbicide did not lead to an increase in the level of carbonyl derivatives and a decrease in the content of sulfhydryl groups compared to control group rats. Thus, the obtained experimental data indicate that both feeding with the genetically modified soybean treated with the herbicide and receiving the herbicide "Roundup" with drinkable water lead to the initiation of free radical processes in the kidneys of rats of all three generations and imbalance of the oxidant–antioxidant system, most notably in the third generation of rats. Such research results indicate the negative effects of the investigated factors and indicate that the herbicide "Roundup" may be accumulated in the seeds of transgenic soybean and also it may increase the oxidative modification of proteins in the rat kidneys. Hence, it is necessary to carry out a detailed study of the effects of these factors on histochemical changes in the kidney and liver structure and an investigation of antioxidant enzyme activity in these organs.

ACTIVITY OF FREE RADICAL OXIDATION AND CYCLOOXYGENASE EXPRESSION IN RATS KIDNEYS UNDER EXPERIMENTAL THERAPY OF OXALATE NEPHROLITHIASIS WITH TETRAPEPTIDE Leu-Ile-Lys-Met

The aim of the study was to investigate the effect of Leu-Ile-Lys-Met tetrapeptide on the activity of free radical oxidation (FRO) and the level of cyclooxygenase-1 (COX-1) and COX-2 expression in rat kidney with experimental oxalate nephrolithiasis. Material and methods . The experiments were carried out on 30 male rats Wistar runoff, divided into 3 groups: the comparison group (10 intact rats), the control group (6 weeks of experimental oxalate nephrolithiasis simulation, n = 10), the experimental group (6 weeks of experimental simulation oxalate nephrolithiasis + oral administration of Leu-Ile-Lys-Met tetrapeptide at a dose of 12 mg/kg from the 3rd to the 6th week, n = 10). The oxalate nephrolithiasis was simulated according to conventional ethylene glycol model. COX-1 and COX-2 concentration in rat urine at baseline and after 6 weeks of experimental nephrolithiasis was determined by enzyme immunoassay using the prostaglandin-endoperoxide synthase 1 and 2 (PTGS 1, 2) kit from «Cloud-Clone Corp.». In rat kidney homogenate, the activity indicators of FRO processes were determined by common methods. Results and discussion . At the background of the Leu-Ile-Lys-Met tetrapeptide, free radical kidney damage was weakened, which was manifested in the complete absence of morphological signs of nephrolithiasis, decrease of thiobarbituric acid-reactive product concentration and overall prooxidant activity as compared to the control group; glutathione peroxidase activity, on the contrary, increased by 1.5 times.

Angiotensin II type 1 receptor blockers decrease kynurenic acid production in rat kidney in vitro

Glutamate (GLU) mainly through N-methyl-d-aspartate (NMDA) receptors plays pivotal role in kidney function regulation. Kynurenic acid (KYNA), a GLU receptors antagonist, is synthesized from kynurenine by kynurenine aminotransferases (KATs). Previously, it was shown that angiotensin II type 1 receptor blockers (ARBs) decrease KYNA production in rat brain in vitro. The aim of this study was to examine the influence of six ARBs: candesartan, irbesartan, losartan, olmesartan, telmisartan, and valsartan on KYNA production on rat kidney in vitro. The effect of ARBs was determined in kidney homogenates and on isolated KAT II enzyme. Among tested ARBs, irbesartan was the most effective KYNA synthesis inhibitor with IC50 of 14.4 μM. Similar effects were observed after losartan (IC50 45.9 μM) and olmesartan administration (IC50 108.1 μM), whereas candesartan (IC50 475.3 μM), valsartan (IC50 513.9 μM), and telmisartan (IC50 669.5 μM) displayed lower activity in KYNA synthesis inhibition in rat kidney homogenates in vitro. On the other hand, valsartan (IC50 27.5 μM) was identified to be the strongest KAT II inhibitor in rat kidney in vitro. Candesartan, losartan, and telmisartan suppressed KAT II activity with IC50 equal to 83.2, 83.3, and 108.3 μM, respectively. Olmesartan and irbesartan were the weakest KAT II inhibitors with IC50 values of 237.4 and 809.9 μM, respectively. Moreover, molecular docking suggested that studied ARBs directly bind to an active site of KAT II. In conclusion, our results indicate that ARBs decrease KYNA synthesis in rat kidney through enzymatic inhibition of KAT II, which may have impact on kidney function.

Omega-3 polyunsaturated fatty acids alleviate adenine-induced chronic renal failure via regulating ROS production and TGF-β/SMAD pathway.

To explore the role of omega-3 polyunsaturated fatty acids (ω-3 PUFAs) in adenine-induced rat chronic renal failure and its underlying mechanism. 30 Sprague Dawley (SD) rats were randomly assigned into three groups, namely sham group, adenine induction group (adenine group) and adenine induction + ω-3 PUFAs treatment group (ω-3 PUFAs group), with 10 rats in each group. Serum and kidney samples were collected after rats were sacrificed. Serum levels of Cr (creatinine) and BUN (urea nitrogen) were detected using commercial kits. HE (hematoxylin and eosin) staining was performed to evaluate the pathological changes of kidneys. Levels of oxidative stress indicators in rat kidney homogenate were detected by relative commercial kits, including SOD (superoxide dismutase), GSH (reduced glutathione), CAT (catalase), and T-AOC (total antioxidant capacity). Reactive oxygen species (ROS) production was also detected by immunofluorescence. Protein expressions of nuclear factor E2 related factor 2 (Nrf2) and transforming growth factor-beta (TGF-β)/SMAD pathway-related genes were detected by Western blot. Serum levels of Cr and BUN in ω-3 PUFAs group were remarkably decreased compared with those of adenine group. Higher contents of SOD, GSH, CAT and T-AOC were observed in ω-3 PUFAs group compared with those of adenine group. Besides, MAD content and ROS production were lower in ω-3 PUFAs group than those of adenine group. Pathological changes of kidneys were alleviated after ω-3 PUFAs treatment. Western blot results demonstrated that ω-3 PUFAs treatment remarkably upregulates Nrf2, HO-1, NQO1, but downregulates relative genes in TGF-β/SMAD pathway. ω-3 PUFAs alleviated adenine-induced chronic renal failure through enhancing antioxidant stress and inhibiting inflammatory response via regulating Nrf2 and TGF-β/SMAD pathway.

Glycine mitigates renal oxidative stress by suppressing Nox4 expression in rats with streptozotocin-induced diabetes

Glycine exerts renoprotective effects, but the mechanism remains unclear. Glycine is increasingly recognized as a factor that attenuates oxidative stress, a key mechanism underlying diabetic nephropathy. In this study, we investigated the effects of glycine on diabetic renal injury and oxidative stress by adding 1% glycine in the drinking water of rats with streptozotocin-induced diabetes for 20 weeks. Glycine levels decreased in the plasma and kidney homogenates of diabetic rats but were restored by oral glycine administration. In these diabetic rats, glycine attenuated renal damage, as evidenced by the decreased mesangial expansion, tubular interstitial fibrosis, and neutrophil gelatinase-associated lipocalin (NGAL) expression. Glycine also ameliorated the raise in urinary malondialdehyde (MDA) levels and partially restored renal glutathione levels in diabetic rats. Renal levels of the Nox4 mRNA and protein, a major source of renal oxidative stress, were suppressed by the treatment with glycine. Immunohistological analysis revealed that glycine had protective effects on the tubular area rather than the glomerular area. Our results strongly suggest that the protective effect of glycine on renal oxidative stress and structural damage may be linked to enhancement of GSH synthesis and suppression of renal Nox4 expression in diabetic rats.

Evaluating prodrug characteristics of a novel anticoagulant fusion protein neorudin, a prodrug targeting release of hirudin variant 2-Lys47 at the thrombosis site, by means of in vitro pharmacokinetics

Recombinant neorudin (EPR-hirudin, EH), a low-bleeding anticoagulant fusion protein, is an inactive prodrug designed to be converted to the active metabolite, hirudin variant 2-Lys47 (HV2), locally at the thrombus site by FXa and/or FXIa, following activation of the coagulation system. Our aim was to evaluate the prodrug characteristics of EH by comparing the biotransformation of EH and HV2 in biological matrices, including rat blood, liver, and kidney homogenates, demonstrating the cleavage of EH to HV2 by FXa and FXIa, and comparing the conversion of EH to HV2 between fresh whole blood and whole-blood clot homogenate, using ultra-performance liquid chromatography-mass spectrometry (UPLC-MS/MS). Both EH and HV2 were stable in blood and unstable in the liver and kidney homogenates. Eight EH metabolites and eight HV2 metabolites identified as N-terminal fragments were found in the liver and kidney. C-terminal proteolysis is therefore the major metabolic pathway, with serine/cysteine carboxypeptidases and metallocarboxypeptidases being responsible for the degradation of EH and HV2 in the liver and kidney, respectively. EH was cleaved to release HV2 by FXIa. Higher levels of HV2 were produced from EH in the whole-blood clot homogenate, in which the coagulation system was activated compared with those in fresh whole blood. In conclusion, the metabolism of EH and HV2 shares the same cleavage pattern, and EH is transformed into HV2 when the coagulation system is activated, where FXIa is a specific enzyme. Our in vitro study revealed the anticipated prodrug characteristics of EH newly designed as an inactive prodrug of hirudin.

Dodecanoyl isothiocyanate and N′-(2-cyanoacetyl) dodecanehydrazide as precursors for the synthesis of different heterocyclic compounds with interesting antioxidant and antitumor activity

ABSTRACTDodecanoyl isothiocyanate 1 was utilized for the construction of thioureido, 2-thioxo-2,3-dihydroquinazolin-4(1H)-one, benzo[d]-1,3-thiazin-4(H)-one and 3-amino quinazolin-4(3H)-one derivatives 2, 3, 4, and 5 respectively. However, N′-(2-cyanoacetyl) dodecanehydrazide 6 was also used as a key starting material for the construction of a variety of new pyrazolone, pyrazole, thiadiazole, pyridazine, chromeno[2,3-c]pyrazole, and dithiolane derivatives 7, 9, 12, 15, 18, and 20, respectively. The newly synthesized compounds were characterized by elemental analyses and spectral data (IR, 1H NMR, and mass spectra). Some of the newly synthesized compounds bearing heterocyclic moieties were tested for antioxidant activity as reflected in their ability to inhibit oxidation in rat brain and kidney homogenates using 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) inhibition. Also, in vitro anticancer activity was examined using the standard (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) (MTT) method against a panel of two human tumor cell lines namely; hepatocellular carcinoma HepG2 and mammary gland breast cancer MCF-7. Compounds 12 and 16b exhibited the highest activities as antioxidant and antitumor agents. Meanwhile, compounds 7 and 20 showed moderate activities and the rest of the tested compounds showed weak activities.