RESUMO
Exposure to chronic hyperglycemia because of diabetes mellitus can lead to development and progression of diabetic kidney disease (DKD). We recently reported that reduced superoxide production is associated with mitochondrial dysfunction in the kidneys of mouse models of type 1 DKD. We also demonstrated that humans with DKD have significantly reduced levels of mitochondrion-derived metabolites in their urine. Here we examined renal superoxide production in a type 2 diabetes animal model, the db/db mouse, and the role of a mitochondrial protectant, MTP-131 (also called elamipretide, SS-31, or Bendavia) in restoring renal superoxide production and ameliorating DKD. We found that 18-week-old db/db mice have reduced renal and cardiac superoxide levels, as measured by dihydroethidium oxidation, and increased levels of albuminuria, mesangial matrix accumulation, and urinary H2O2 Administration of MTP-131 significantly inhibited increases in albuminuria, urinary H2O2, and mesangial matrix accumulation in db/db mice and fully preserved levels of renal superoxide production in these mice. MTP-131 also reduced total renal lysocardiolipin and major lysocardiolipin subspecies and preserved lysocardiolipin acyltransferase 1 expression in db/db mice. These results indicate that, in type 2 diabetes, DKD is associated with reduced renal and cardiac superoxide levels and that MTP-131 protects against DKD and preserves physiological superoxide levels, possibly by regulating cardiolipin remodeling.
Assuntos
Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2 , Nefropatias Diabéticas , Mitocôndrias , Oligopeptídeos/farmacologia , Superóxidos/metabolismo , Animais , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Nefropatias Diabéticas/tratamento farmacológico , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Humanos , Masculino , Camundongos , Camundongos Knockout , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias/patologiaRESUMO
Renal-specific oxido-reductase/myoinositol oxygenase (RSOR/MIOX) is expressed in renal tubules. It catabolizes myo-inositol and its expression is increased in diabetic mice and in LLC-PK(1) cells under high-glucose ambience. Aldose reductase (AR) is another aldo-keto reductase that is expressed in renal tubules. It regulates the polyol pathway and plays an important role in glucose metabolism, osmolyte regulation, and ECM pathobiology via the generation of advanced glycation end products, reactive oxygen species, and activation of transforming growth factor (TGF)-beta. In view of the similarities between AR and RSOR/MIOX, the pathobiology of RSOR/MIOX and some of the cellular pathways affected by its overexpression were investigated. An increased expression of fibronectin was noted by transfection of LLC-PK(1) cells with pcDNA3.1-RSOR/MIOX. Similar changes were observed in LLC-PK(1) cells under high-glucose ambience, and they were notably lessened by RSOR/MIOX-small interfering (si) RNA treatment. The changes in tubulointerstitial fibronectin expression were also observed in the kidneys of db/db mice having high levels of RSOR. The pcDNA3.1-RSOR/MIOX transfectants had an increased NADH/NAD(+) ratio, PKC and TGF-beta activity, Raf1:Ras association, and p-ERK phosphorylation. These changes were significantly reduced by the inhibitors of PKC, aldose reductase, Ras farnesylation, and MEK1. Similar increases in various the above-noted parameters were observed under high-glucose ambience. Such changes were partially reversed with RSOR-siRNA treatment. Expression of E-cadherin and vimentin paralleled in cells overexpressing RSOR/MIOX or subjected to high-glucose ambience. These studies suggest that RSOR/MIOX modulates various downstream pathways affected by high-glucose ambience, and conceivably it plays a role in the pathobiology of tubulointerstitium in diabetic nephropathy.
Assuntos
Aldeído Redutase/metabolismo , Nefropatias Diabéticas/enzimologia , Inositol Oxigenase/metabolismo , Túbulos Renais/enzimologia , Aldeído Redutase/antagonistas & inibidores , Aldeído Redutase/genética , Animais , Caderinas/metabolismo , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/patologia , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fibronectinas/metabolismo , Fibrose , Glucose/metabolismo , Inositol Oxigenase/antagonistas & inibidores , Inositol Oxigenase/genética , Túbulos Renais/patologia , Células LLC-PK1 , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NAD/metabolismo , Fosforilação , Proteína Quinase C-alfa/metabolismo , Prenilação de Proteína , Transporte Proteico , Interferência de RNA , Transdução de Sinais , Suínos , Transfecção , Fator de Crescimento Transformador beta1/metabolismo , Vimentina/metabolismo , Quinases raf/metabolismo , Proteínas ras/metabolismoRESUMO
The aim of this study was to investigate whether high glucose induces aldose reductase (AKR1B1) expression through NFkappaB, which may contribute to the pathogenesis of diabetic nephropathy. 34 Caucasoid patients with type 1 diabetes were recruited; 20 nephropaths and 14 long-term uncomplicated subjects. Peripheral blood mononuclear cells (PBMCs) were cultured under normal or high glucose (25 mmol/l of d-glucose) with or without an aldose reductase inhibitor (ARI). High glucose increased NFkappaB binding activities in the PBMCs from nephropaths compared to the uncomplicated subjects (1.77+/-0.22 vs. 1.16+/-0.04, p=0.02). ARI induced a substantially greater decrease of NFkappaB binding activities in the nephropaths compared to the uncomplicated subjects (0.58+/-0.06 vs. 0.79+/-0.06, p=0.032). AKR1B1 protein levels in the nephropaths were increased under high glucose conditions and decreased in the presence of an ARI, whilst the silencing of the NFkappaB p65 gene in vitro reduced the transcriptional activities of AKR1B1 in luciferase assays. These results show that NFkappaB induces AKR1B1expression under high glucose conditions, and the pattern of expression differs between nephropaths and the uncomplicated subjects.
Assuntos
DNA/metabolismo , Nefropatias Diabéticas/metabolismo , Glucose/farmacologia , NF-kappa B/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Aldeído Redutase/antagonistas & inibidores , Aldeído Redutase/genética , Motivos de Aminoácidos , Sequência de Bases , Western Blotting , Nefropatias Diabéticas/enzimologia , Inibidores Enzimáticos/farmacologia , Feminino , Inativação Gênica , Humanos , Proteínas I-kappa B/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Inibidor de NF-kappaB alfa , NF-kappa B/química , NF-kappa B/genética , Fosforilação/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacosRESUMO
A promising area of novel anti-diabetic therapy involves identification of small molecule activators of the glucokinase enzyme to reduce blood glucose and normalize glucose stimulated insulin secretion. Herein, we report the identification and optimization of a series of 4-sulfonyl-2-pyridone activators. The activators were evaluated for in vitro biochemical activation and pharmacokinetic properties. As part of these efforts, a unique metabolic liability of the 4-sulfonyl-2-pyridone ring system was identified wherein this heterocycle readily undergoes conjugation with glutathione under non-enzymatic conditions.
Assuntos
Glucoquinase/efeitos dos fármacos , Hipoglicemiantes/farmacocinética , Piridonas/farmacocinética , Animais , Glicemia , Ativação Enzimática/efeitos dos fármacos , Glutationa/química , Humanos , Hipoglicemiantes/química , Hipoglicemiantes/metabolismo , Microssomos Hepáticos/metabolismo , Piridonas/química , Piridonas/metabolismo , Ratos , Ratos Sprague-Dawley , Relação Estrutura-AtividadeRESUMO
We investigated the role of polyol pathway enzymes aldose reductase (AR) and sorbitol dehydrogenase (SDH) in mediating injury due to ischemia-reperfusion (IR) in Type 2 diabetic BBZ rat hearts. Specifically, we investigated, (a) changes in glucose flux via cardiac AR and SDH as a function of diabetes duration, (b) ischemic injury and function after IR, (c) the effect of inhibition of AR or SDH on ischemic injury and function. Hearts isolated from BBZ rats, after 12 weeks or 48 weeks diabetes duration, and their non-diabetic littermates, were subjected to IR protocol. Myocardial function, substrate flux via AR and SDH, and tissue lactate:pyruvate (L/P) ratio (a measure of cytosolic NADH/NAD+), and lactate dehydrogenase (LDH) release (a marker of IR injury) were measured. Zopolrestat, and CP-470,711 were used to inhibit AR and SDH, respectively. Myocardial sorbitol and fructose content, and associated changes in L/P ratios were significantly higher in BBZ rats compared to non-diabetics, and increased with disease duration. Induction of IR resulted in increased ischemic injury, reduced ATP levels, increases in L/P ratio, and poor cardiac function in BBZ rat hearts, while inhibition of AR or SDH attenuated these changes and protected hearts from IR injury. These data indicate that AR and SDH are key modulators of myocardial IR injury in BBZ rat hearts and that inhibition of polyol pathway could in principle be used as a therapeutic adjunct for protection of ischemic myocardium in Type 2 diabetic patients.
Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Miocárdio/metabolismo , Polímeros/metabolismo , Traumatismo por Reperfusão/metabolismo , Transdução de Sinais/fisiologia , Trifosfato de Adenosina/metabolismo , Aldeído Redutase/metabolismo , Animais , Modelos Animais de Doenças , L-Iditol 2-Desidrogenase/metabolismo , Lactatos/metabolismo , Piruvatos/metabolismo , Ratos , Ratos Endogâmicos BBRESUMO
Previously studied inhibitors of aldose reductase were largely from two chemical classes, spirosuccinamide/hydantoins and carboxylic acids. Each class has its own drawbacks regarding selectivity, in vivo potency, and human safety; as a result, the pathogenic role of aldose reductase in diabetic retinopathy remains controversial. ARI-809 is a recently discovered aldose reductase inhibitor (ARI) of a new structural class, pyridazinones, and has high selectivity for aldose versus aldehyde reductase. To further test the possible pathogenic role of aldose reductase in the development of diabetic retinopathy, we examined the retinal effects of this structurally novel and highly selective ARI in insulinized streptozotocin-induced diabetic rats. ARI-809 treatment was initiated 1 month after diabetes induction and continued for 3 months at a dose that inhibited the polyol pathway in the retina of diabetic rats to a similar extent as sorbinil, a poorly selective hydantoin ARI previously shown to prevent retinopathy in this model. ARI-809 improved survival, inhibited cataract development, normalized retinal sorbitol and fructose, and protected the retina from abnormalities that also occur in human diabetes: neuronal apoptosis, glial reactivity, and complement deposition. Because ARI-809 is a novel chemotype highly selective for aldose reductase, these results support the notion that aldose reductase is the key relay that converts hyperglycemia into glucose toxicity in neural and glial cell types in the retina.
Assuntos
Aldeído Redutase/antagonistas & inibidores , Retinopatia Diabética/prevenção & controle , Inibidores Enzimáticos/uso terapêutico , Piridazinas/uso terapêutico , Retina/metabolismo , Animais , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/patologia , Retinopatia Diabética/tratamento farmacológico , Molécula 1 de Adesão Intercelular/biossíntese , Masculino , Polímeros/metabolismo , Ratos , Ratos Sprague-Dawley , Retina/efeitos dos fármacos , Retina/patologia , Regulação para CimaRESUMO
The expression of aldose reductase is tightly regulated by the transcription factor tonicity response element binding protein (TonEBP/NFAT5) binding to three osmotic response elements (OREs; OREA, OREB, and OREC) in the gene. The aim was to investigate the contribution of NFAT5 to the pathogenesis of diabetic nephropathy. Peripheral blood mononuclear cells (PBMCs) were isolated from the following subjects: 44 Caucasoid patients with type 1 diabetes, of whom 26 had nephropathy and 18 had no nephropathy after a diabetes duration of 20 years, and 13 normal healthy control subjects. In addition, human mesangial cells (HMCs) were isolated from the normal lobe of 10 kidneys following radical nephrectomy for renal cell carcinoma. Nuclear and cytoplasmic proteins were extracted from PBMCs and HMCs and cultured in either normal or high-glucose (31 mmol/l D-glucose) conditions for 5 days. NFAT5 binding activity was quantitated using electrophoretic mobility shift assays for each of the OREs. Western blotting was used to measure aldose reductase and sorbitol dehydrogenase protein levels. There were significant fold increases in DNA binding activities of NFAT5 to OREB (2.06 +/- 0.03 vs. 1.33 +/- 0.18, P = 0.033) and OREC (1.94 +/- 0.21 vs. 1.39 +/- 0.11, P = 0.024) in PBMCs from patients with diabetic nephropathy compared with diabetic control subjects cultured under high glucose. Aldose reductase and sorbitol dehydrogenase protein levels in the patients with diabetic nephropathy were significantly increased in PBMCs cultured in high-glucose conditions. In HMCs cultured under high glucose, there were significant increases in NFAT5 binding activities to OREA, OREB, and OREC by 1.38 +/- 0.22-, 1.84 +/- 0.44-, and 2.38 +/- 1.15-fold, respectively. Similar results were found in HMCs exposed to high glucose (aldose reductase 1.30 +/- 0.06-fold and sorbitol dehydrogenease 1.54 +/- 0.24-fold increases). Finally, the silencing of the NFAT5 gene in vitro reduced the expression of the aldose reductase gene. In conclusion, these results show that aldose reductase is upregulated by the transcriptional factor NFAT5 under high-glucose conditions in both PBMCs and HMCs.
Assuntos
Fatores de Transcrição/metabolismo , Adulto , Idoso , Aldeído Redutase/genética , Diabetes Mellitus , Nefropatias Diabéticas , Feminino , Regulação Enzimológica da Expressão Gênica , Mesângio Glomerular/fisiologia , Mesângio Glomerular/fisiopatologia , Humanos , Leucócitos Mononucleares/fisiologia , Masculino , Pessoa de Meia-Idade , Valores de Referência , Fatores de Transcrição/genética , Regulação para Cima , População BrancaRESUMO
The exaggerated flux through polyol pathway during diabetes is thought to be a major cause of lesions in the peripheral nerves. Here, we used aldose reductase (AR)-deficient (AR(-/-)) and AR inhibitor (ARI)-treated mice to further understand the in vivo role of polyol pathway in the pathogenesis of diabetic neuropathy. Under normal conditions, there were no obvious differences in the innervation patterns between wild-type AR (AR(+/+)) and AR(-/-) mice. Under short-term diabetic conditions, AR(-/-) mice were protected from the reduction of motor and sensory nerve conduction velocities observed in diabetic AR(+/+) mice. Sorbitol levels in the sciatic nerves of diabetic AR(+/+) mice were increased significantly, whereas sorbitol levels in the diabetic AR(-/-) mice were significantly lower than those in diabetic AR(+/+) mice. In addition, signs of oxidative stress, such as increased activation of c-Jun NH(2)-terminal kinase (JNK), depletion of reduced glutathione, increase of superoxide formation, and DNA damage, observed in the sciatic nerves of diabetic AR(+/+) mice were not observed in the diabetic AR(-/-) mice, indicating that the diabetic AR(-/-) mice were protected from oxidative stress in the sciatic nerve. The diabetic AR(-/-) mice also excreted less 8-hydroxy-2'-deoxyguanosine in urine than diabetic AR(+/+) mice. The structural abnormalities observed in the sural nerve of diabetic AR(+/+) mice were less severe in the diabetic AR(-/-) mice, although it was only mildly protected by AR deficiency under short-term diabetic conditions. Signs of oxidative stress and functional and structural abnormalities were also inhibited by the ARI fidarestat in diabetic AR(+/+) nerves, similar to those in diabetic AR(-/-) mice. Taken together, increased polyol pathway flux through AR is a major contributing factor in the early signs of diabetic neuropathy, possibly through depletion of glutathione, increased superoxide accumulation, increased JNK activation, and DNA damage.
Assuntos
Aldeído Redutase/deficiência , Dano ao DNA , Diabetes Mellitus Experimental/fisiopatologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Neurônios Motores/fisiologia , Condução Nervosa/fisiologia , Aldeído Redutase/genética , Animais , Diabetes Mellitus Experimental/genética , Ativação Enzimática , Frutose/metabolismo , Genes Reporter , Glucose/metabolismo , Glutationa/metabolismo , Inositol/metabolismo , Camundongos , Camundongos Knockout , Poli Adenosina Difosfato Ribose/metabolismo , Valores de Referência , Sorbitol/metabolismo , Superóxidos/metabolismo , Nervo Sural/fisiopatologiaRESUMO
OBJECTIVE: This 7-year longitudinal study examines the potential impact of aldose reductase gene (AKR1B1) polymorphisms on the decline of nerve function in an adolescent diabetic cohort. RESEARCH DESIGN AND METHODS: Patients with type 1 diabetes (n = 262) were assessed with three cardiovascular autonomic tests (heart rate variation during deep breathing, Valsalva maneuver, and during standing from a lying position) and pupillometry (resting pupil diameter, constriction velocity, and reflex amplitude), thermal, and vibration thresholds on the foot. Genotyping was performed for promoters (C-106T and C-12G), (CA)(n) dinucleotide repeats, and intragenic BamH1 polymorphism. RESULTS: Median time between first and last assessment was 7.0 years (interquartile range 5.1-11.1), with a median of five assessments (four to seven) per individual. At first assessment, median age was 12.7 years (11.7-13.9), median duration was 5.3 years (3.4-8.0), and median HbA(1c) was 8.5% (7.8-9.3). All tests declined over time except for two cardiovascular autonomic tests and vibration discrimination. Faster decline in maximum constriction velocity was found to associate with the Z-2 allele (P = 0.045), Z-2/Z-2 (P = 0.026). Slower decline in hot thermal threshold discrimination associated with Z+2 (P = 0.044), Z+2/Z+2 (P < 0.0005), Z+2/T (P = 0.038), and bb (P = 0.0001). CONCLUSIONS: Most autonomic and quantitative sensory nerve testings declined over time. AKR1B1 polymorphisms were strongly associated with the rate of decline of these complications.
Assuntos
Aldeído Redutase/genética , Diabetes Mellitus Tipo 1/genética , Polimorfismo Genético/genética , Adolescente , Alelos , Sistema Nervoso Autônomo/fisiopatologia , Diabetes Mellitus Tipo 1/fisiopatologia , Repetições de Dinucleotídeos/genética , Feminino , Frequência do Gene , Predisposição Genética para Doença/genética , Genótipo , Frequência Cardíaca/fisiologia , Humanos , Estudos Longitudinais , Masculino , Reflexo Pupilar/fisiologia , Manobra de Valsalva/fisiologiaRESUMO
Sorbitol dehydrogenase (hSDH) and aldose reductase form the polyol pathway that interconverts glucose and fructose. Redox changes from overproduction of the coenzyme NADH by SDH may play a role in diabetes-induced dysfunction in sensitive tissues, making SDH a therapeutic target for diabetic complications. We have purified and determined the crystal structures of human SDH alone, SDH with NAD(+), and SDH with NADH and an inhibitor that is competitive with fructose. hSDH is a tetramer of identical, catalytically active subunits. In the apo and NAD(+) complex, the catalytic zinc is coordinated by His69, Cys44, Glu70, and a water molecule. The inhibitor coordinates the zinc through an oxygen and a nitrogen atom with the concomitant dissociation of Glu70. The inhibitor forms hydrophobic interactions to NADH and likely sterically occludes substrate binding. The structure of the inhibitor complex provides a framework for developing more potent inhibitors of hSDH.
Assuntos
Cristalografia por Raios X , L-Iditol 2-Desidrogenase/química , Sítios de Ligação , Humanos , Cinética , L-Iditol 2-Desidrogenase/metabolismo , Funções Verossimilhança , Ligação Proteica , Conformação ProteicaRESUMO
Diabetes mellitus (DM) is a major health problem with devastating effects on ocular health in both industrialized and developing countries. The control of hyperglycemia is critical to minimizing the impact of DM on ocular tissues because inadequate glycemic control leads to ocular tissue changes that range from a temporary blurring of vision to permanent vision loss. The biochemical mechanisms that promote the development of diabetic complications have been extensively studied. As a result, a number of prominent biochemical pathways have been identified. Among these, the two-step sorbitol pathway has been the most extensively investigated; nevertheless, it remains controversial. To date, long-term pharmacological studies in animal models of diabetes have demonstrated that the onset and development of ocular complications that include keratopathy, retinopathy and cataract can be ameliorated by the control of excess metabolic flux through aldose reductase (AR). Clinically the alleles of AR have been linked to the rapidity of onset and severity of diabetic ocular complications in diabetic patient populations around the globe. In spite of these promising preclinical and human genetic rationales, several clinical trials of varying durations with structurally diverse aldose reductase inhibitors (ARIs) have shown limited success or failure in preventing or arresting diabetic retinopathy. Despite these clinical setbacks, topical ARI Kinostat(®) promises to find a home in clinical veterinary ophthalmology where its anticipated approval by the FDA will present an alternative treatment paradigm to cataract surgery in diabetic dogs. Here, we critically review the role of AR in diabetes mellitus-linked ocular disease and highlight the development of Kinostat(®) for cataract prevention in diabetic dogs. In addition to the veterinary market, we speculate that with further safety and efficacy studies in humans, Kinostat(®) or a closely related product could have a future role in treating diabetic keratopathy.
Assuntos
Aldeído Redutase/antagonistas & inibidores , Catarata/tratamento farmacológico , Doenças da Córnea/tratamento farmacológico , Complicações do Diabetes , Retinopatia Diabética/tratamento farmacológico , Inibidores Enzimáticos/uso terapêutico , Imidazolidinas/uso terapêutico , Administração Tópica , Animais , Diabetes Mellitus/metabolismo , Inibidores Enzimáticos/administração & dosagem , Humanos , Imidazolidinas/administração & dosagem , Soluções OftálmicasRESUMO
Increased flux of glucose through the polyol pathway may cause generation of excess reactive oxygen species (ROS), leading to tissue damage. Abnormalities in expression of enzymes that protect against oxidant damage may accentuate the oxidative injury. The expression of catalase (CAT), CuZn superoxide-dismutase (CuZnSOD), glutathione peroxidase (GPX), and Mn superoxide-dismutase (MnSOD) mRNA was quantified in peripheral blood mononuclear cells-obtained from 26 patients with type 1 diabetes and nephropathy, 15 with no microvascular complications after 20 years' duration of diabetes, and 10 normal healthy control subjects-that were exposed in vitro to hyperglycemia (HG) (31 mmol/l D-glucose). Under HG, there was a twofold increase in the expression of CAT, CuZnSOD, and GPX mRNA in the patients without complications and the control subjects versus patients with nephropathy (P < 0.0001), and MnSOD did not change in any of the groups. The aldose reductase inhibitor zopolrestat partially restored the levels of CAT, CuZnSOD, and GPX mRNA in the patients with nephropathy (P < 0.05). There was a highly significant correlation between increased aldose reductase (ALR2) expression, CAT, CuZnSOD, and GPX mRNA levels under HG conditions and polymorphisms of ALR2 in the patients with nephropathy (P < 0.00001). In conclusion, these results suggest that high glucose flux through aldose reductase inhibits the expression of antioxidant enzymes.
Assuntos
Catalase/genética , Diabetes Mellitus Tipo 1/enzimologia , Nefropatias Diabéticas/enzimologia , Glutationa Peroxidase/genética , Hiperglicemia/enzimologia , Superóxido Dismutase/genética , Adulto , Idoso , Aldeído Redutase/antagonistas & inibidores , Aldeído Redutase/genética , Antioxidantes/metabolismo , Benzotiazóis , Glicemia/metabolismo , Células Cultivadas , Diabetes Mellitus Tipo 1/genética , Nefropatias Diabéticas/genética , Inibidores Enzimáticos/farmacologia , Feminino , Expressão Gênica , Predisposição Genética para Doença , Humanos , Leucócitos Mononucleares/enzimologia , Masculino , Pessoa de Meia-Idade , Ftalazinas/farmacologia , Polimorfismo Genético , RNA/sangue , RNA Mensageiro/sangue , Tiazóis/farmacologiaRESUMO
Discovery of a highly selective, potent, and safe non-carboxylic acid, non-hydantoin inhibitor of aldose reductase (AR) capable of potently blocking the excess glucose flux through the polyol pathway that prevails under diabetic conditions has been a long-standing challenge. In response, we did high-throughput screening of our internal libraries of compounds and identified 6-phenylsulfonylpyridazin-2H-3-one, 8, which showed modest inhibition of AR, both in vitro and in vivo. Initial structure-activity relationships concentrated on phenyl substituents and led to 6-(2,4-dichlorophenylsulfonyl)-2H-pyridazin-3-one, 8l, which was more potent than 8, both in vitro and in vivo. Incorporation of extant literature findings with other aldose reductase inhibitors, including zopolrestat, resulted in the title inhibitor, 19m, which is one of the most potent and highly selective non-carboxylic acid, non-hydantoin inhibitors of AR yet described (IC50, 1 nM; ED90 vs sciatic nerve sorbitol and fructose, respectively, 0.8 and 4.0 mg/kg). In rats, its oral bioavailability is 98% and it has a favorable plasma t(1/2) (26 +/- 3 h).
Assuntos
Aldeído Redutase/antagonistas & inibidores , Aldeído Redutase/química , Diabetes Mellitus Experimental/tratamento farmacológico , Hipoglicemiantes/síntese química , Piridazinas/síntese química , Sulfonas/síntese química , Administração Oral , Animais , Disponibilidade Biológica , Células CACO-2 , Frutose/metabolismo , Humanos , Hipoglicemiantes/química , Hipoglicemiantes/farmacologia , Cristalino/metabolismo , Masculino , Piridazinas/química , Piridazinas/farmacologia , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/antagonistas & inibidores , Nervo Isquiático/efeitos dos fármacos , Nervo Isquiático/metabolismo , Sorbitol/metabolismo , Relação Estrutura-Atividade , Sulfonas/química , Sulfonas/farmacologiaRESUMO
Sorbitol dehydrogenase (SDH) is a polyol pathway enzyme that catalyzes conversion of sorbitol to fructose. Recent studies have demonstrated that activation of aldose reductase, the first enzyme of the polyol pathway, is a key response to ischemia and that inhibition of aldose reductase reduces myocardial ischemic injury. In our efforts to understand the role of pathway in affecting metabolism under normoxic and ischemic conditions, as well as in ischemic injury in myocardium, we investigated the importance of SDH by use of a specific inhibitor (SDI), CP-470,711. SDH inhibition increased glucose oxidation, whereas palmitate oxidation remained unaffected. Global ischemia increased myocardial SDH activity by approximately 1.5 fold. The tissue lactate/pyruvate ratio, a measure of cytosolic NADH/NAD+, was reduced by SDH inhibition under both normoxic and ischemic conditions. ATP was higher in SDI hearts during ischemia and reperfusion. Creatine kinase release during reperfusion, a marker of myocardial ischemic injury, was markedly attenuated in SDH-inhibited hearts. These data indicate that myocardial SDH activation is a component of ischemic response and that interventions that inhibit SDH protect ischemic myocardium. Furthermore, these data identify SDH as a novel target for adjunctive cardioprotective interventions.
Assuntos
L-Iditol 2-Desidrogenase/antagonistas & inibidores , Miocárdio/enzimologia , Animais , Cardiotônicos/uso terapêutico , Quimioterapia Adjuvante , Sistemas de Liberação de Medicamentos , Inibidores Enzimáticos/uso terapêutico , Glucose/metabolismo , Coração/efeitos dos fármacos , Modelos Cardiovasculares , Isquemia Miocárdica/enzimologia , Isquemia Miocárdica/prevenção & controle , Miocárdio/metabolismo , Técnicas de Cultura de Órgãos , Pirimidinas/uso terapêutico , RatosRESUMO
Aldose reductase, a member of the aldo-keto reductase family, has been implicated in the development of vascular and neurological complications in diabetes. Despite recent studies from our laboratory demonstrating protection of ischemic hearts by an aldose reductase inhibitor, the presence and influence of aldose reductase in cardiac tissue remain unknown. Our goal in this study was to isolate and characterize the kinetic properties of cardiac aldose reductase, as well as to study the impact of flux via this enzyme on glucose metabolism and contractile function in hearts subjected to ischemia-reperfusion. Results demonstrate that ischemia increases myocardial aldose reductase activity and that these increases are, in part, due to activation by nitric oxide. The kinetic parameter of cardiac aldose reductase (Kcat) was significantly higher in ischemic tissues. Aldose reductase inhibition increased glycolysis and glucose oxidation. Aldose reductase inhibited hearts, when subjected to ischemia/reperfusion, exhibited less ischemic injury and was associated with lower lactate/pyruvate ratios (a measure of cytosolic NADH/NAD+), greater tissue content of adenosine triphosphate, and improved cardiac function. These findings indicate that aldose reductase is a component of ischemic injury and that pharmacological inhibitors of aldose reductase present a novel adjunctive approach for protecting ischemic hearts.
Assuntos
Aldeído Redutase/metabolismo , Isquemia Miocárdica/fisiopatologia , Miocárdio/enzimologia , Penicilamina/análogos & derivados , Aldeído Redutase/antagonistas & inibidores , Aldeído Redutase/efeitos dos fármacos , Animais , Benzotiazóis , Inibidores Enzimáticos/farmacologia , Glucose/metabolismo , Isquemia Miocárdica/prevenção & controle , Miocárdio/metabolismo , Miocárdio/patologia , Óxido Nítrico/metabolismo , Doadores de Óxido Nítrico/farmacologia , Penicilamina/farmacologia , Ftalazinas/farmacologia , Ratos , Tiazóis/farmacologiaRESUMO
Aldose reductase (AR), a member of the aldo-keto reductase family, has been implicated in the development of vascular and neurological complications of diabetes. Recently, we demonstrated that aldose reductase is a component of myocardial ischemic injury and that inhibitors of this enzyme protect rat hearts from ischemia-reperfusion injury. To rigorously test the effect of aldose reductase on myocardial ischemia-reperfusion injury, we used transgenic mice broadly overexpressing human aldose reductase (ARTg) driven by the major histocompatibility complex I promoter. Hearts from these ARTg or littermate mice (WT) (n=6 in each group) were isolated, perfused under normoxic conditions, then subjected to 50 min of severe low flow ischemia followed by 60 min of reperfusion. Creatine kinase (CK) release (a marker of ischemic injury) was measured during reperfusion; left ventricular developed pressure (LVDP), end diastolic pressure (EDP), and ATP were measured throughout the protocol. CK release was significantly greater in ARTg mice compared with the WT mice. LVDP recovery was significantly reduced in ARTg mice compared with the WT mice. Furthermore, ATP content was higher in WT mice compared with ARTg mice during ischemia and reperfusion. Infarct size measured by staining techniques and myocardial damage evaluated histologically were also significantly worse in ARTg mice hearts than in controls. Pharmacological inhibition of aldose reductase significantly reduced ischemic injury and improved functional recovery in ARTg mice. These data strongly support key roles for AR in ischemic injury and impairment of functional and metabolic recovery after ischemia. We propose that interventions targeting AR may provide a novel adjunctive approach to protect ischemic myocardium.
Assuntos
Aldeído Redutase/fisiologia , Isquemia Miocárdica/enzimologia , Traumatismo por Reperfusão Miocárdica/enzimologia , Trifosfato de Adenosina/metabolismo , Aldeído Redutase/antagonistas & inibidores , Aldeído Redutase/biossíntese , Aldeído Redutase/genética , Animais , Vasos Coronários , Citosol/metabolismo , Inibidores Enzimáticos/farmacologia , Glucose/metabolismo , Glicólise , L-Iditol 2-Desidrogenase/farmacologia , Ligadura , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Isquemia Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/patologia , Miocárdio/metabolismo , NAD/metabolismo , Niacina/farmacologia , Técnicas de Cultura de Órgãos , Oxirredução , Ácido Palmítico/metabolismo , Proteínas Recombinantes de Fusão/fisiologia , Função Ventricular EsquerdaRESUMO
Optimization of a previously disclosed sorbitol dehydrogenase inhibitor (SDI, II) for potency and duration of action was achieved by replacing the metabolically labile N,N-dimethylsulfamoyl group with a variety of heterocycles. Specifically, this effort led to a series of novel, in vitro potent SDIs with longer serum half-lives and acceptable in vivo activity in acutely diabetic rats (e.g., 62, 67, and 69). However, the desired in vivo potency in chronically diabetic rats, ED(90) < or = 5 mg/kg/day, was achieved only through further modification of the piperazine linker. Several members of this family, including 86, showed better than the targeted potency with ED(90) values of 1-2 mg/kg/day. Compound 86 was further profiled and found to be a selective inhibitor of sorbitol dehydrogenase, with excellent pharmacodynamic/pharmacokinetic properties, demonstrating normalization of sciatic nerve fructose in a chronically diabetic rat model for approximately 17 h, when administered orally at a single dose of 2 mg/kg/day.
Assuntos
Inibidores Enzimáticos/síntese química , L-Iditol 2-Desidrogenase/antagonistas & inibidores , Piperazinas/síntese química , Pirimidinas/síntese química , Animais , Doença Crônica , Diabetes Mellitus Experimental/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Frutose/metabolismo , Masculino , Piperazinas/química , Piperazinas/farmacocinética , Piperazinas/farmacologia , Pirimidinas/química , Pirimidinas/farmacocinética , Pirimidinas/farmacologia , Ratos , Ratos Sprague-Dawley , Nervo Isquiático/metabolismo , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
We report here a novel sorbitol dehydrogenase inhibitor, 16, that shows very high oral potency (50 microg/kg) in normalizing elevated fructose levels in the sciatic nerve of chronically diabetic rats and sustained duration of action (>24 h). Furthermore, 16 shows attractive pharmaceutical properties, including good solubility in simulated human gastric fluid, excellent Caco-2 Papp, moderate lipophilicity, and metabolic stability for achieving good oral absorption and long duration of action.
Assuntos
Inibidores Enzimáticos/síntese química , L-Iditol 2-Desidrogenase/antagonistas & inibidores , Pirimidinas/síntese química , Triazinas/síntese química , Administração Oral , Animais , Células CACO-2 , Diabetes Mellitus/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Frutose/metabolismo , Humanos , Pirimidinas/química , Pirimidinas/farmacologia , Ratos , Nervo Isquiático/efeitos dos fármacos , Nervo Isquiático/metabolismo , Solubilidade , Estereoisomerismo , Relação Estrutura-Atividade , Triazinas/química , Triazinas/farmacologiaRESUMO
We report here on the discovery path that led to a structurally unprecedented non-hydantoin, non-carboxylic acid aldose reductase inhibitor, 24, which shows remarkably potent oral activity in normalizing elevated sorbitol levels and, more significantly, fructose levels in the sciatic nerve of chronically diabetic rats, with ED(90) values of 0.8 and 3 mpk, respectively. It is well absorbed in rats (oral bioavailability, 98%) and has a long plasma t(1/2) (26 +/- 3 h).
Assuntos
Aldeído Redutase/antagonistas & inibidores , Diabetes Mellitus Experimental/metabolismo , Inibidores Enzimáticos/síntese química , Hipoglicemiantes/síntese química , Piridazinas/síntese química , Sulfonas/síntese química , Administração Oral , Aldeído Redutase/química , Animais , Disponibilidade Biológica , Células CACO-2 , Técnicas de Química Combinatória , Cristalografia por Raios X , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Frutose/sangue , Humanos , Hipoglicemiantes/química , Hipoglicemiantes/farmacologia , Cristalino/efeitos dos fármacos , Cristalino/enzimologia , Permeabilidade , Piridazinas/química , Piridazinas/farmacologia , Ratos , Nervo Isquiático/efeitos dos fármacos , Nervo Isquiático/enzimologia , Sorbitol/sangue , Relação Estrutura-Atividade , Sulfonas/química , Sulfonas/farmacologiaRESUMO
Diabetic kidney disease, or diabetic nephropathy, is the leading cause of kidney failure in developed countries and is projected to place an increasingly heavy burden on medical, social and economic systems worldwide. Existing therapies can slow, but do not stop, disease progression. Recent data from preclinical models and patients with diabetes emphasize the need for reducing excess metabolic flux through aldose reductase, an enzyme that plays a critical role in transducing the metabolic abnormalities that cause fibrosis in the diabetic kidney. The background and developmental history of aldose reductase inhibitors are reviewed briefly, as are metabolic, hemodynamic and genetic data linking aldose reductase to diabetic kidney disease. A new paradigm defining the pathogenic role of aldose reductase, the 'metabolic flux hypothesis', is presented, along with updated pharmacological goals for achieving success with this class of inhibitors in diabetic kidney disease.