Synthesis, molecular modeling Insights, and anticancer assessment of novel polyfunctionalized Pyridine congeners.

The present study describes synthesizing a novel series of polyfunctionalized pyridine congeners 1-18 and assessed for cytotoxic efficacies versus HCT-116, MCF-7, and HepG-2 among one non-cancerous BJ-1 human normal cell. Most compounds were precisely potent anticancer candidate drugs. The molecular impact of the most active compounds 9, 10, 11, 13, 15, and 17 was evaluated after MCF-7 treatment. The gene expression of pro- and ant-apoptosis markers P53, Bax, Caspase-3 and Bcl-2 as well as VEGFR-2 and HER2 were determined. Compounds 13 and 15 induced upregulation of pro-apoptosis of P53, Bax, Caspase-3 and downregulation of anti-apoptosis Bcl-2 gene. However, compound 15 showed higher effect compared to 13 and respective control. Moreover, a slight reduction in HER2 gene expression was detected due to compound 15 treatment, while VEGFR-2 gene was upregulated. In agreement, the immunoblotting analysis showed higher accumulation of P53, Bax, Caspase-3 proteins and of decrease the Bcl-2 protein levels. Furthermore, docking studies united with molecular dynamic simulation exposed compounds 13 and 15 fitting in the middle of the active site at the interface linking the ATP binding site and the allosteric hydrophobic binding pocket. Finally, we performed Petra/Osiris/ Molinspiration (POM) analysis for the newly synthesized compounds. The evaluation of primary in silico parameters revealed significant differences among individual polyfunctionalized pyridine compounds, highlighting the most promising candidates. These preliminary results may help in coordinating and initiating other research projects focused on polyfunctionalized pyridine compounds, especially those with predicted bioactivity, low toxicity, optimal ADME parameters, and promising perspectives.

Development of pyrolo[2,3-c]pyrazole, pyrolo[2,3-d]pyrimidine and their bioisosteres as novel CDK2 inhibitors with potent in vitro apoptotic anti-proliferative activity: Synthesis, biological evaluation and molecular dynamics investigations.

Inhibiting the CDK2/cyclin A2 enzyme has been validated in multiple clinical manifestations related to multiple types of cancer. Herein, novel series of pyrolo[2,3-c]pyrazole, pyrolo[2,3-c]isoaxazole and pyrolo[2,3-d]pyrimidine, pyrolo[3,2-c]pyridine & indole based analogs were designed, synthesized and biologically evaluated for their in vitro antiproliferative activity where the obtained results revealed that most of the newly synthesized compounds showed significant cytotoxic activity towards MCF-7 (breast cancer cell lines) and HepG-2 (hepatocellular carcinoma) with IC50 ranging from 3.20 µM to 10.05 µM & from 2.18 µM to 13.49 µM, respectively, compared to that of Sorafenib (IC50 9.76 & 13.19 µM, respectively). The in vitro inhibitory profile of the most promising compounds (9, 11, 14, 15, 16, 17 and 20) towards CDK2/CyclinA2 was evaluated. Compounds 14 & 15 exhibited potent inhibitory profile against CDK2 with (IC50 0.11 and 0.262 µM, respectively comparable to Sorafenib IC50 0.184 µM. Western blotting of 14 & 15 at MCF-7 cell line confirmed the diminishing activity on CDK2. Furthermore, both compounds exserted a significant cell cycle arrest and apoptosis. Moreover, the normal cell line cytotoxicity for both compounds revealed low cytotoxic results in normal cells rather than cancer cells. Molecular docking and dynamic simulation validated the potentiality of the newly synthesized compounds to have high binding affinity within CDK2 binding pocket. 3DQSAR pharmacophore, in-silico ADME/TOPKAT studies and drug-likeness showed proper pharmacokinetic properties and helped in structure requirements prediction. The obtained model and pattern of substitution could be used for further development of CDK2 inhibitors.

New indole derivatives as multitarget anti-Alzheimer's agents: synthesis, biological evaluation and molecular dynamics.

Background: Alzheimer's disease is a neurological disorder that causes brain cells to shrink and die. Aim: Thirteen novel 'oxathiolanyl', 'pyrazolyl' and 'pyrimidinyl' indole derivatives were designed and synthesized as anti-Alzheimer's disease treatment. Method: In vitro enzyme assay was performed against both AChE and BChE enzymes. In addition, antioxidant assay and cytotoxicity on a normal cell line were determined. Molecular docking and dynamic simulations were conducted to confirm the binding mode in both esterases' active sites. In silico absorption, distribution, metabolism, excretion and toxicity studies were also carried out. Results & conclusion: Compounds 5, 7 and 11 exhibited superior inhibitory activity against acetylcholinesterase and butyrylcholinesterase, with IC50 values of 0.042 and 3.003 µM, 2.54 and 0.207 µM and 0.052 and 2.529 µM, respectively, compared with donepezil.

Synthesis, biological evaluation, and in silico studies of new CDK2 inhibitors based on pyrazolo[3,4-d]pyrimidine and pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine scaffold with apoptotic activity.

Cyclin-dependent kinase inhibition is considered a promising target for cancer treatment for its crucial role in cell cycle regulation. Pyrazolo pyrimidine derivatives were well established for their antitumor activity via CDK2 inhibition. In this research, new series of pyrazolopyrimidine derivatives (4-15) was designed and synthesised as novel CDK2 inhibitors. The anti-proliferative activities against MCF-7, HCT-116, and HepG-2 were used to evaluate their anticancer activity as novel CDK2 inhibitors. Most of the compounds showed superior cytotoxic activity against MCF-7 and HCT-116 compared to Sorafenib. Only compounds 8, 14, and 15 showed potent activity against HepG-2. The CDK2/cyclin A2 enzyme inhibitory activity was tested for all synthesised compounds. Compound 15 showed the most significant inhibitory activity with IC50 0.061 ± 0.003 µM. It exerted remarkable alteration in Pre G1 and S phase cell cycle progression and caused apoptosis in HCT cells. In addition, the normal cell line cytotoxicity for compound 15 was assigned revealing low cytotoxic results in normal cells rather than cancer cells. Molecular docking was achieved on the designed compounds and confirmed the two essential hydrogen binding with Leu83 in CDK2 active site. In silico ADMET studies and drug-likeness showed proper pharmacokinetic properties which helped in structure requirements prediction for the observed antitumor activity.

Discovery of pyrazolo[3,4-d]pyrimidine and pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine derivatives as novel CDK2 inhibitors: synthesis, biological and molecular modeling investigations.

CDK2 inhibition is an appealing target for cancer treatment that targets tumor cells in a selective manner. A new set of small molecules featuring the privileged pyrazolo[3,4-d]pyrimidine and pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine scaffolds (4-13) as well as the thioglycoside derivatives (14, 15) were designed, and synthesized as novel CDK2 targeting compounds. The growth of the three examined cell lines was significantly inhibited by most of the prepared compounds. Results revealed that most of the compounds showed superior cytotoxic activities against MCF-7 and HCT-116 with IC50 range (45-97 nM) and (6-99 nM), respectively, and moderate activity against HepG-2 with IC50 range of (48-90 nM) compared to sorafenib (IC50: 144, 176 and 19 nM, respectively). Of these compounds, 14 & 15 showed the best cytotoxic activities against the three cell lines with IC50 values of 45, 6, and 48 nM and 46, 7, and 48 nM against MCF-7, HCT-116 and HepG-2, respectively. Enzymatic inhibitory activity against CDK2/cyclin A2 was achieved for the most potent anti-proliferative compounds. Compounds 14, 13 and 15 revealed the most significant inhibitory activity with IC50 values of 0.057 ± 0.003, 0.081 ± 0.004 and 0.119 ± 0.007 µM, respectively compared to sorafenib (0.184 ± 0.01 µM). Compound 14 displayed potent dual activity against the examined cell lines and CDK2, and was thus selected for further investigations. It exerted a significance alteration in cell cycle progression, in addition to apoptosis induction within HCT cells. Molecular docking simulation of the designed compounds confirmed the good fit into the CDK2 active site through the essential hydrogen bonding with Leu83. In silico ADMET studies and drug-likeness studies using a Boiled Egg chart showed suitable pharmacokinetic properties which helped in structure requirement prediction for the observed antitumor activity.

Mechanistic study of Hg(II) interaction with three different α-aminophosphonate adsorbents: Insights from batch experiments and theoretical calculations.

Herein, efficient and potential chelating α-aminophosphonate based sorbents (AP-) derived from three different amine origins (aniline/anthranilic acid/O-phenylenediamine) to form AP-H, carboxylated and aminated enhanced aminophosphonate as AP-H, AP-COOH, and AP-NH2 were synthesized via a facile method. The structure of the synthesized sorbents was elucidated using different techniques; elemental analysis (CHNP/O), FT-IR, NMR (1H-, 13C and 31P NMR), TGA and BET. The fabricated sorbents were exploited for Hg(II) removal from aqueous solution via sorption properties. Isotherm fitted by Langmuir equation: the maximum sorption capacities at optimum pH 5.5, and T:25 ± 1 °C, were found to be 1.33, 1.23, and 1.15 mmol Hg g-1 for AP-COOH, AP-NH2, AP-H, respectively, which is roughly correlated with the active sites density and the hard/soft characteristics of adsorbents' reactive groups. Metal-ligand binding affinities are qualitatively rationalized in terms of hard and soft acids and bases (HSAB) theory. The interaction of Hg(II) (soft) has a stronger affinity to AP-COOH can be considered a softer base compared with reference material (AP-H) over than AP-NH2 (hard). This sequence result showed opposite trends consistent with their reciprocal properties according to the steric effect modulates and the specific surface area. Thermodynamics analysis for absolute values of ΔH°, ΔS° and ΔG° afford the selectivity towards Hg(II) sorption with the following order: AP-COOH > AP-NH2 >AP-H. Elution and regeneration was carried out by HCl solution and recycled for a minimum of five cycles, the sorption and desorption efficiencies are greater than 91%. Such sorbents exhibit good durability, stability and promising potential for Hg(II) removal. Finally, a new modelling technique for quantitative non-linear description and comparison of equivalent geographical positions in 3D space of extended relationships. Exothermic and spontaneous behavior were observed using a proposed Floatotherm that included the Van't Hoff parameters model.

Discovery of New Pyrazolopyridine, Furopyridine, and Pyridine Derivatives as CDK2 Inhibitors: Design, Synthesis, Docking Studies, and Anti-Proliferative Activity.

New pyridine, pyrazoloyridine, and furopyridine derivatives substituted with naphthyl and thienyl moieties were designed and synthesized starting from 6-(naphthalen-2-yl)-2-oxo-4-(thiophen-2-yl)-1,2-dihydropyridine-3-carbonitrile (1). The chloro, methoxy, cholroacetoxy, imidazolyl, azide, and arylamino derivatives were prepared to obtain the pyridine--C2 functionalized derivatives. The derived pyrazolpyridine-N-glycosides were synthesized via heterocyclization of the C2-thioxopyridine derivative followed by glycosylation using glucose and galactose. The furopyridine derivative 14 and the tricyclic pyrido[3',2':4,5]furo[3,2-d]pyrimidine 15 were prepared via heterocyclization of the ester derivative followed by a reaction with formamide. The newly synthesized compounds were evaluated for their ability to in vitro inhibit the CDK2 enzyme. In addition, the cytotoxicity of the compounds was tested against four different human cancer cell lines (HCT-116, MCF-7, HepG2, and A549). The CDK2/cyclin A2 enzyme inhibitory results revealed that pyridone 1, 2-chloro-6-(naphthalen-2-yl)-4-(thiophen-2-yl)nicotinonitrile (4), 6-(naphthalen-2-yl)-4-(thiophen-2-yl)-1H-pyrazolo[3,4-b]pyridin-3-amine (8), S-(3-cyano-6-(naphthaen-2-yl)-4-(thiophen-2-yl)pyridin-2-yl) 2-chloroethanethioate (11), and ethyl 3-amino-6-(naphthalen-2-yl)-4-(thiophen-2-yl)furo[2,3-b]pyridine-2-carboxylate (14) are among the most active inhibitors with IC50 values of 0.57, 0.24, 0.65, 0.50, and 0.93 µM, respectively, compared to roscovitine (IC50 0.394 µM). Most compounds showed significant inhibition on different human cancer cell lines (HCT-116, MCF-7, HepG2, and A549) with IC50 ranges of 31.3-49.0, 19.3-55.5, 22.7-44.8, and 36.8-70.7 µM, respectively compared to doxorubicin (IC50 40.0, 64.8, 24.7 and 58.1 µM, respectively). Furthermore, a molecular docking study suggests that most of the target compounds have a similar binding mode as a reference compound in the active site of the CDK2 enzyme. The structural requirements controlling the CDK2 inhibitory activity were determined through the generation of a statistically significant 2D-QSAR model.

Critical evaluation of functional aspects of evaporation barriers through environmental and economics lens for evaporation suppression - A review on milestones from improved technologies.

Climate change models predict an increase in rainfall variability, leading to floods and drought events, hence intensifying the need for reservoirs. However, up to 50% of reservoirs' capacity is lost by evaporation, affecting their function of ensuring water availability and stability. Over decades biological, chemical and physical barriers "covers" were developed for inhibiting evaporation. Such barrier's efficiency and applicability are still a matter of discussion, given their economic efficiency, environmental consequences, and operational difficulties are accounted for. In this review, we discussed the efficiency, applicability, and environmental suitability of these covers. Compared to the physical covers, the chemical and biological solutions tend to be less efficient. However, the use of physical covers is multidisciplinary, involving climate, material, and hydrological sciences, and are more efficient. Among the physical covers, the use of suspended covers and free-floating elements decreases evaporation to the tune of 85 and 80.0%, respectively. However, the economic efficiency of free-floating elements remains an open question since all studies overlooked their water footprint (water used in the manufacturing process of these covers), which was found to be very high. The use of these covers decreases heat storage, gas exchange rate, and light availability that could adversely influence dissolved oxygen, water quality, aquatic organisms, and the water ecosystem's function. These ecological consequences have not yet been investigated. The exception is the suspended covers, which have had determinate effects on dissolved oxygen and algae growth. Due to light weight, floating elements' operation is unstable and vulnerable to move due to wind effects. Therefore, such covers must be engineered to increase their stability. Free-floating elements could provide a visible and scalable solution to evaporation suppression when considering their economic visibility, environmental effects, and stability against wind and wave effects under the field conditions. However, these covers can be viable only when water availability is the limiting factor in crop production. We found that studies at reservoir scale are highly limited, therefore, investigations at reservoirs' scale emphasizing ecological aspects, cover stability and cost efficiency, are urgently needed.

Synthesis of new uracil derivatives and their sugar hydrazones with potent antimicrobial, antioxidant and anticancer activities.

6-(4-Chloro-3-nitrophenyl)-4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile (4) was prepared and was reacted with ethyl chloroacetate, hydrazine hydrate, 4-chloroaniline, formaldehyde, acetic anhydride, formic acid, carbon disulfide, 4-cyanobenzaldehyde, triethyl orthoformate, D-sugars, 4-aminoacetophenone, benzoyl choride and cyclohexanone to afford a series of new uracil derivatives (5-18). Examination of some of the prepared compounds for their antimicrobial, antioxidant and anticancer activities was conducted. Among the tested samples, compound 17 was the most active substance against the gram-positive bacteria and was more potent than the reference drug Cefoperazone. Moreover, the antibacterial activity of 17 was higher against gram-negative bacteria. Compounds 6 and 13 reached a higher scavenging ability toward DPPH radicals and are better candidates for antioxidant activity. Also, compounds 6 and 13 had no significant anticancer activity toward liver cancer (Hep G2) and breast cancer (MCF-7) cell lines.

Synthesis and Anticancer Activity of New ((Furan-2-yl)-1,3,4-thiadiazolyl)-1,3,4-oxadiazole Acyclic Sugar Derivatives.

New sugar hydrazones incorporating furan and/or 1,3,4-thiadiazole ring systems were synthesized by reaction of the corresponding hydrazide with different aldose sugars. Heterocyclization of the formed hydrazones afforded the derived acyclic nucleoside analogues possessing the 1,3,4-oxadiazoline as modified nucleobase via acetylation followed by the heterocyclization process. The anticancer activity of the synthesized compounds was studied against human liver carcinoma cell (HepG-2) and at human normal retina pigmented epithelium cells (RPE-1). High activities were revealed by compounds 3, 12 and 14 with IC50 values near to that of the reference drug doxorubicin.

Synthesis, Docking Studies into CDK-2 and Anticancer Activity of New Derivatives Based Pyrimidine Scaffold and Their Derived Glycosides.

BACKGROUND & OBJECTIVE: New diaryl-substituted pyrimidinedione compounds, their thioxo derivatives as well as their bicyclic thiazole compounds were synthesized and characterized. METHODS: The glycosylamino derivatives of the synthesized disubstituted derivatives of the pyrimidine scaffold were also prepared via reaction of the N3-amino derivatives with a number of monosaccharides followed by acetylation. RESULTS: The anticancer activity of the synthesized compounds was studied against human liver cancer (HepG2) and RPE-1cell lines. Compounds 2a, 2b, 3a and 12 showed potent activities with IC50 results comparable to that of doxorubicin. CONCLUSION: Docking investigations into Cyclin-dependent kinase 2 (CDK-2) enzyme, a potential target for cancer medication, were also reported showing the possible binding interaction into the enzyme active site to support their activity behavior.

Design, Synthesis of New Pyridine and Pyrimidine Sugar Compounds as Antagonists Targeting the ERα via Structure-Based Virtual Screening.

BACKGROUND: New aryl substituted cyclohepta[b]pyridine and cyclohepta[d]pyrimidine derivatives were synthesized. The sugar hydrazones of the synthesized pyridine and pyrimidine compounds were also prepared. METHOD: In addition, the 1,3,4-oxadiazolyl acyclic C-nucleoside analogs of the pyridine system were prepared. The hemolytic, prebiotic, anticancer and antimicrobial activities of some of the synthesized compounds were also studied. Compounds 10 and 12 showed high activity against MCF-7, HEPG-2 and HCT-116 cell lines with IC50 at range 3.56-8.55 µg/mL. In addition, the synthesized condensed thiopyrimidine derivative 10 exhibited more potent bactericidal activity while compound 7 demonstrated potent antifungal activity against Aspergillus niger. Furthermore, the synthetic compounds of the pyrimidine base promoted the growth of lactic acid bacteria. RESULTS: The predicted binding patterns of three of the prepared derivatives as possible antagonists against ERα were investigated which showed good binding patterns.

Histopathological patterns of renal diseases in egyptian children: A single-center experience.

The present cross-sectional, retrospective study was aimed to determine the histopathological spectrum of renal diseases in Egyptian children and to evaluate the indications, safety, and efficacy of percutaneous renal biopsy (PRB) in a large tertiary center in Egypt. PRBs performed at the Department of Pediatrics, Tanta University Hospital over a period of nine years (from January 2007 to December 2015) were included. Light microscopic (LM) examination was performed in all cases while immunofluorescence and electron microscopic examination were performed in selected cases. Two hundred and thirty renal biopsies were performed during the study duration. Nine biopsies were excluded from the study due to insufficient sample (<7 glomeruli per specimen) giving a PRB efficacy rate of 96.1%. Results of 221 renal biopsies performed on 210 patients from native kidneys were described. Ninety-seven patients were male (46.19%) and 113 were female (53.81%) with age ranging from three months to 18 years (mean 10.51 ± 3.81 years). The main indications of renal biopsy were nephrotic syndrome (NS) (43.89%), lupus nephritis (23.53%), and recurrent or persistent hematuria (10.41%). The most common finding on LM examination of renal biopsies from children with NS was minimal change disease (22.17%). Secondary nephropathies were mostly due to lupus (23.53%). IgA nephropathy was found in eight patients (3.62%). Local pain at the site of biopsy was the most common minor complication seen postbiopsy (60.58%). Transient gross hematuria was seen in 13 patients (5.88%) without urinary retention. Major complications that required surgical intervention or blood transfusion did not occur. NS was the main indication and minimal change disease was the most common histological finding of renal biopsies from Egyptian children. Complications and efficacy of renal biopsy procedure were comparable to that reported from developed countries.

Design, synthesis and anticancer evaluation of novel pyrazole, pyrazolo[3,4-d]pyrimidine and their glycoside derivatives.

The chalcone derivatives 3a,b were cyclized upon reaction with thiourea to give the pyrazolo[3,4-d]pyrimidine derivatives 5a,b. Condensation of 5a,b and their hydrazide derivatives 8a,b with cyclic and acyclic glucose gave the condensed S- and N-glycosides 7a,b and 9a,b, respectively. Reaction of 3b with ethyl cyanoacetate followed by reaction with cyclic glucose afforded a mixture of the O- and/or N-glycoside isomers 12 and 13, respectively. The pyrazolo[3,4-c]pyrazole derivative 14 was also obtained from the reaction of 3b with hydrazine hydrate. A number of the synthesized compounds were screened for their antitumor activity against three different tumor cell lines HEPG2 (liver), HCT116 (colon) and MCF-7 (breast) with a docking study against CDK2.

A convenient synthesis and molecular modeling study of novel pyrazolo[3,4-d]pyrimidine and pyrazole derivatives as anti-tumor agents.

An efficient method to obtain ethyl 5-amino-1-tosyl-1H-pyrazole-4-carboxylate (3) was outlined using condensation reactions of 4-methylbenzenesulfonylhydrazide with (E)-ethyl 2-cyano-3-ethoxyacrylate. The cyclocondensation reaction of this substrate and its hydrazide derivative with urea, thiourea, formamide, formic acid, d-glucose, o-phenylenediamine, 4-dimethylaminobenzaldehyde, anthracene-9-carbaldehyde, thioglycolic acid and carbon disulphide then with hydrazine hydrate analogues furnished a series of pyrazolo[3,4-d]pyrimidine, pyrazolo[3,4-d]oxazin-4-one, pyrazole-4-glucoside, 4-benzo[d]imidazole, 1,3-thiazolidinone, 1,3,4-oxadiazol-2(3H)-thione and 1,2,4-triazol-5(4H)-thione derivatives respectively. The structure of the compound 3 was supported by X-Ray crystallographic data. Orally administrated, one of each of the series of pyrazoles showed significant effects in mouse tumor model cancer cell lines (EAC) and two human cancer cell lines of Colon cancer (HCT-29) and Breast cancer (MCF-7) with docking studies.

Enhanced hydrogen production from water via a photo-catalyzed reaction using chalcogenide d-element nanoparticles induced by UV light.

Hydrogen has the potential to meet the requirements as a clean non-fossil fuel in the future. The photocatalytic production of H2 through water splitting has been demonstrated and enormous efforts have been published. The present work is an attempt to enhance the production of H2 during water splitting using synthesized nanoparticles based on chalcogenide d-element semiconductors via a photochemical reaction under UV-light in the presence of methanol as a hole-scavenger. In general, the enhanced activity of a semiconductor is most likely due to the effective charge separation of photo generated electrons and holes in the semiconductors. Hence, the utilization of different semiconductors in combination can consequently provide better hydrogen production. Accordingly in this research work, two different semiconductors, with different concentrations, either used individually or combined together were introduced. They in turn produced a high concentration of H2 as detected and measured using gas chromatography. Herein, data revealed that the nano-structured semiconductors prepared through this work are a promising candidate in the production of an enhanced H2 flux under visible UV radiation.

Ethyl 5-amino-1-[(4-methyl-phen-yl)sulfon-yl]-1H-pyrazole-4-carboxyl-ate.

In the title mol-ecule, C13H15N3O4S, the benzene and pyrazole rings are inclined to each other at 77.48 (3)°. Two amino H atoms are involved in bifurcated hydrogen bonds, viz. intra-molecular N-H⋯O and inter-molecular N-H⋯O(N). The inter-molecular hydrogen bonds link the mol-ecules related by translation in [100] into chains. A short distance of 3.680 (3) Šbetween the centroids of benzene and pyrazole rings from neighbouring mol-ecules shows the presence of π-π inter-actions, which link the hydrogen-bonded chains into layers parallel to the ab plane.

N-aryl pyrazoles: DFT calculations of CH acidity and deprotonative metallation using a combination of lithium and zinc amides.

A series of N-aryl and N-heteroaryl pyrazoles have been deproto-metallated using a 2,2,6,6-tetramethylpiperidino-based mixed lithium-zinc combination. Mono-, di-, and tri-iodides have been obtained after subsequent trapping with iodine, depending on the substrate and on the quantity of base used. The results have been discussed in the light of the CH acidities of the substrates, determined both in the gas phase and in THF solution using the DFT B3LYP method.

Renal ACE2 expression and activity is unaltered during established hypertension in adult SHRSP and TGR(mREN2)27.

Differential renal expression of a homolog of the angiotensin-converting enzyme (ACE), that is, ACE2, has been implicated as a genetic basis of polygenetic hypertension in the stroke-prone spontaneously hypertensive rat model. However, data on the role of ACE2 in hypertension are still inconclusive. Therefore, we analyzed kidney ACE2 mRNA, ACE2 protein and ACE2 enzyme activities in the adult polygenetic stroke-prone spontaneously hypertensive rat (SHRSP) and the monogenetic TGR(mREN2)27 rat models, in comparison with their normotensive reference strains, Wistar-Kyoto (WKY) and Spraque-Dawley (SD) rats, respectively. Kidney ACE2 mRNA was studied using quantitative real-time reverse transcriptase-PCR (RT-PCR) in cortex and medulla, whereas protein expression was scored semiquantitatively in detail in different renal structures using immunohistochemistry. Furthermore, total renal tissue ACE2 activity was measured using a fluorimetric assay that was specified by the ACE2 inhibitor DX600. In SHRSP and homozygous TGR(mREN2)27 rats with established hypertension, kidney ACE2 mRNA, protein and tissue ACE2 activities were not different from their respective WKY and SD reference strain, respectively. In addition, when we looked at renal localization, we found ACE2 protein to be predominantly present in glomeruli and endothelium with weak staining in distal and negative staining in proximal tubuli. Thus, our data challenge previous work that implicates ACE2 as a candidate gene for hypertension in SHRSP by reporting a significant reduction of ACE2 in the kidneys of SHRSP. Taken together, renal ACE2 is not altered in the SHRSP and TGR(mREN2)27 genetic rat models with established hypertension.