RESUMO
Excessive activation of the mineralocorticoid receptor (MR) is implicated in cardiovascular and renal disease. Decreasing MR activation with MR antagonists (MRA) is effective to slow chronic kidney disease (CKD) progression and its cardiovascular comorbidities in animal models and patients. The present study evaluates the effects of the MR modulator balcinrenone and the MRA eplerenone on kidney damage in a metabolic CKD mouse model combining nephron reduction and a 60% high-fat diet. Balcinrenone and eplerenone prevented the progression of renal damages, extracellular matrix remodeling and inflammation to a similar extent. We identified a novel mechanism linking MR activation to the renal proteoglycan deposition and inflammation via the TLR4 pathway activation. Balcinrenone and eplerenone similarly blunted this pathway activation.
Assuntos
Eplerenona , Matriz Extracelular , Camundongos Endogâmicos C57BL , Antagonistas de Receptores de Mineralocorticoides , Proteoglicanas , Receptores de Mineralocorticoides , Transdução de Sinais , Receptor 4 Toll-Like , Animais , Antagonistas de Receptores de Mineralocorticoides/farmacologia , Antagonistas de Receptores de Mineralocorticoides/uso terapêutico , Receptor 4 Toll-Like/metabolismo , Eplerenona/farmacologia , Eplerenona/uso terapêutico , Receptores de Mineralocorticoides/metabolismo , Matriz Extracelular/metabolismo , Matriz Extracelular/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Masculino , Proteoglicanas/metabolismo , Espironolactona/farmacologia , Espironolactona/análogos & derivados , Insuficiência Renal Crônica/tratamento farmacológico , Insuficiência Renal Crônica/metabolismo , Insuficiência Renal Crônica/patologia , Modelos Animais de Doenças , Rim/efeitos dos fármacos , Rim/metabolismo , Rim/patologia , Camundongos , Inflamação/metabolismo , Inflamação/tratamento farmacológicoRESUMO
Urinary [Formula: see text] excretion is decreased in chronic kidney disease (CKD), but very little is known about fecal [Formula: see text] excretion. Sodium zirconium cyclosilicate (SZC) is a cation exchanger that selectively captures K+ in the gastrointestinal tract. We investigated if SZC can sequester [Formula: see text] in vivo and evaluated the effect of SZC on fecal [Formula: see text] in a mouse model of CKD. Mice with CKD induced by 5/6 kidney ablation were fed either a regular diet or a diet containing SZC (4 g/kg) and followed for 7 days. Fecal [Formula: see text] was measured before and after the addition of 50 meq KCl/L to release [Formula: see text] from SZC. [Formula: see text] sequestered in SZC in the gastrointestinal (GI) tract was estimated from the change in fecal [Formula: see text] observed when KCl was added to liberate the sequestered [Formula: see text]. In mice with CKD, fecal [Formula: see text] excretion was higher than in normal mice and also higher than urine [Formula: see text] excretion measured concurrently. Using data pooled from the SZC diet, the change in [Formula: see text] was 6.5 ± 0.6 compared with 0.6 ± 0.6 µmol/g on the normal diet (P < 0.0001). In conclusion, fecal [Formula: see text] excretion in CKD is increased and about sixfold higher than urine [Formula: see text] excretion, revealing an important route of elimination of [Formula: see text] present in the GI tract. SZC administration sequesters a substantial portion of [Formula: see text] in the GI tract, suggesting that the binding of [Formula: see text] offers therapeutic potential beyond its known primary action as a specific K+ binder.NEW & NOTEWORTHY Fecal [Formula: see text] excretion in chronic kidney disease is increased and about sixfold higher than urine [Formula: see text] excretion, revealing an important route of elimination of [Formula: see text] that is present in the gastrointestinal tract. Sodium zirconium cyclosilicate (SZC) administration sequesters a substantial portion of [Formula: see text], suggesting that binding of [Formula: see text] by SZC in the gastrointestinal tract offers therapeutic potential in chronic kidney disease and other clinical conditions beyond its known primary action of SZC as a specific K+ binder.
Assuntos
Hiperpotassemia , Insuficiência Renal Crônica , Animais , Camundongos , Potássio , Trato GastrointestinalRESUMO
KEY POINTS: Dietary Na restriction, through the mineralocorticoid aldosterone, acts on epithelial Na channels via both fast (24 h) and slow (5-7 days) mechanisms in the kidney. The fast effect entails increased proteolytic processing and trafficking of channel protein to the apical membrane. It is rapidly reversible by the mineralocorticoid receptor antagonist eplerenone and is largely lost when tubules are studied ex vivo. The slow effect does not require increased processing or surface expression, is refractory to acute eplerenone treatment, and is preserved ex vivo. Both slow and fast effects contribute to Na retention in vivo. Increased Na+ reabsorption in the proximal tubule also promotes Na conservation under conditions of chronic dietary Na restriction, reducing Na+ delivery to the distal nephron. ABSTRACT: Changes in the activity of the epithelial Na channel (ENaC) help to conserve extracellular fluid volume. In rats fed a low-salt diet, proteolytic processing of ENaC increased within 1 day, and was almost maximal after 3 days. The rapid increase in the abundance of cleaved αENaC and γENaC correlated with decreased urinary Na+ excretion and with increased ENaC surface expression. By contrast, ENaC activity, measured ex vivo in isolated cortical collecting ducts, increased modestly after 3 days and required 5 days to reach maximal levels. The mineralocorticoid receptor antagonist eplerenone reversed the increase in cleaved γENaC and induced natriuresis after 1 or 3 days but failed to alter either ENaC currents or Na+ excretion after 7 days of Na restriction. We conclude that Na depletion, through aldosterone, stimulates ENaC via independent fast and slow mechanisms. In vivo, amiloride-induced natriuresis increased after 1 day of Na depletion. By contrast, hydrochlorothiazide (HCTZ)-induced natriuresis decreased gradually over 7 days, consistent with increased ability of ENaC activity to compensate for decreased Na+ reabsorption in the distal convoluted tubule. Administration of amiloride and HCTZ together increased Na+ excretion less in Na-depleted compared to control animals, indicating decreased delivery of Na+ to the distal nephron when dietary Na is restricted. Measurements of creatinine and Li+ clearances indicated that increased Na reabsorption by the proximal tubules is responsible for the decreased delivery. Thus, Na conservation during chronic dietary salt restriction entails enhanced transport by both proximal and distal nephron segments.
Assuntos
Restrição Calórica , Diuréticos/farmacologia , Canais Epiteliais de Sódio/metabolismo , Túbulos Renais Distais/metabolismo , Natriurese , Trocador 3 de Sódio-Hidrogênio/metabolismo , Sódio/deficiência , Aldosterona/farmacologia , Amilorida/farmacologia , Animais , Canais Epiteliais de Sódio/química , Hidroclorotiazida/farmacologia , Túbulos Renais Distais/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Trocador 3 de Sódio-Hidrogênio/antagonistas & inibidoresRESUMO
AIMS: AZD9977 is the first mineralocorticoid receptor modulator in clinical development exerting similar organ protection as eplerenone with minimal urinary electrolyte effects in preclinical studies. The aim was to perform the initial clinical assessment of AZD9977. METHODS: A first-in-human trial explored doses from 5 to 1200 mg. To study effects on urinary electrolyte excretion an additional randomized placebo controlled cross-over four-period clinical trial was performed. Twenty-three healthy volunteers were administered fludrocortisone alone or in combination with AZD9977, eplerenone or both. AZD9977/eplerenone combination was given to assess if AZD9977 can attenuate eplerenone induced natriuresis. RESULTS: AZD9977 at doses from 5 to 1200 mg was safe and well tolerated and pharmacokinetics were compatible with further development. AZD9977 exhibited similar effects on urinary ln [Na+ ]/[K+ ] as eplerenone when using fludrocortisone as mineralocorticoid receptor agonist, and the combination had an additive effect on ln [Na+ K+ ]. CONCLUSIONS: The results in man contradict the results in rodent models driven by aldosterone, in which AZD9977 has minimal electrolyte effects. Future clinical studies with AZD9977 should be performed in presence of endogenous or exogenous aldosterone to assess potential benefit of AZD9977 in patients.
Assuntos
Benzoatos/administração & dosagem , Antagonistas de Receptores de Mineralocorticoides/administração & dosagem , Natriurese/efeitos dos fármacos , Oxazinas/administração & dosagem , Potássio/urina , Sódio/urina , Adulto , Benzoatos/efeitos adversos , Benzoatos/farmacocinética , Estudos Cross-Over , Quimioterapia Combinada/efeitos adversos , Quimioterapia Combinada/métodos , Eplerenona/administração & dosagem , Eplerenona/efeitos adversos , Eplerenona/farmacocinética , Fludrocortisona/administração & dosagem , Fludrocortisona/efeitos adversos , Fludrocortisona/farmacocinética , Voluntários Saudáveis , Humanos , Masculino , Antagonistas de Receptores de Mineralocorticoides/efeitos adversos , Antagonistas de Receptores de Mineralocorticoides/farmacocinética , Oxazinas/efeitos adversos , Oxazinas/farmacocinética , Potássio/metabolismo , Receptores de Mineralocorticoides/metabolismo , Eliminação Renal/efeitos dos fármacos , Método Simples-Cego , Sódio/metabolismoRESUMO
Peroxisome proliferator-activated receptors (PPARs) are ligand-dependent transcription factors regulating lipid and glucose metabolism. Ongoing drug discovery programs aim to develop dual PPARα/γ agonists devoid of the side effects of the marketed antidiabetic agents thiazolidinediones and the dual agonists glitazars. Recently, we described a new dual PPARα/γ ligand, LT175, with a partial agonist profile against PPARγ and interacting with a newly identified region of the PPARγ-ligand binding domain (1). Here we show that LT175 differentially activated PPARγ target genes involved in fatty acid esterification and storage in 3T3-L1-derived adipocytes. This resulted in a less severe lipid accumulation compared with that triggered by rosiglitazone, suggesting that LT175 may have a lower adipogenic activity. Consistent with this hypothesis, in vivo administration of LT175 to mice fed a high-fat diet decreased body weight, adipocyte size, and white adipose tissue mass, as assessed by magnetic resonance imaging. Furthermore, LT175 significantly reduced plasma glucose, insulin, non-esterified fatty acids, triglycerides, and cholesterol and increased circulating adiponectin and fibroblast growth factor 21 levels. Oral glucose and insulin tolerance tests showed that the compound improves glucose homeostasis and insulin sensitivity. Moreover, we demonstrate that the peculiar interaction of LT175 with PPARγ affected the recruitment of the coregulators cyclic-AMP response element-binding protein-binding protein and nuclear corepressor 1 (NCoR1), fundamentals for the PPARγ-mediated adipogenic program. In conclusion, our results describe a new PPAR ligand, modulating lipid and glucose metabolism with reduced adipogenic activity, that may be used as a model for a series of novel molecules with an improved pharmacological profile for the treatment of dyslipidemia and type 2 diabetes.
Assuntos
Adipogenia/efeitos dos fármacos , Compostos de Bifenilo/administração & dosagem , Hipoglicemiantes/farmacologia , Resistência à Insulina , Insulina/farmacologia , PPAR alfa/agonistas , PPAR gama/agonistas , Fenilpropionatos/administração & dosagem , Células 3T3-L1 , Animais , Compostos de Bifenilo/metabolismo , Glicemia/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Diabetes Mellitus Tipo 2/tratamento farmacológico , Dislipidemias/tratamento farmacológico , Glucose/metabolismo , Teste de Tolerância a Glucose , Hipoglicemiantes/metabolismo , Insulina/sangue , Ligantes , Metabolismo dos Lipídeos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Correpressor 1 de Receptor Nuclear/metabolismo , PPAR alfa/metabolismo , PPAR gama/metabolismo , Fenilpropionatos/metabolismoRESUMO
OBJECTIVE: To evaluate if previously found associations between low serum bilirubin concentration and kidney function decline is independent of hemoglobin and other key confounders. RESEARCH DESIGN AND METHODS: Clinical trial data from the SAVOR-TIMI 53 trial as well as the UK primary care electronic healthcare records, Clinical Practice Research Datalink (CPRD), were used to construct three cohorts of patients at risk of chronic kidney disease (CKD). The randomized clinical trial (RCT) cohort from the subset of SAVOR-TIMI 53 trial consisted of 10,555 type-2 diabetic patients with increased risk of cardiovascular disease. The two observational data cohorts from CPRD consisted of 71,104 newly diagnosed type-2 diabetes (CPRD-DM2) and 82,065 newly diagnosed hypertensive (CPRD-HT) patients without diabetes. Cohorts were stratified according to baseline circulating total bilirubin levels to determine association on the primary end point of a 30% reduction from baseline in estimated glomerular filtration rate (eGFR) and the secondary end point of albuminuria. RESULTS: The confounder adjusted hazard ratios of the subpopulation with lower than median bilirubin levels compared to above median bilirubin levels for the primary end point were 1.18 (1.02-1.37), 1.12 (1.05-1.19) and 1.09 (1.01-1.17), for the secondary end point were 1.26 (1.06-1.52), 1.11 (1.01-1.21) and 1.18 (1.01-1.39) for SAVOR-TIMI 53, CPRD-DM2, CPRD-HT, respectively. CONCLUSION: Our findings are consistent across all cohorts and endpoints: lower serum bilirubin levels are associated with a greater kidney function decline independent of hemoglobin and other key confounders. This suggests that increased monitoring of kidney health in patients with lower bilirubin levels may be considered, especially for diabetic patients.
Assuntos
Diabetes Mellitus Tipo 2 , Diabetes Mellitus , Insuficiência Renal Crônica , Albuminúria/complicações , Bilirrubina , Progressão da Doença , Taxa de Filtração Glomerular , Humanos , Rim , Fatores de RiscoRESUMO
Obesity and/or metabolic diseases are frequently associated with chronic kidney disease and several factors associated with obesity may contribute to proteinuria and extracellular matrix production. Mineralocorticoid receptor antagonists have proven their clinical efficacy in diabetic kidney disease with preclinical data suggesting that they may also be efficient in non-diabetic chronic kidney disease associated to metabolic diseases. In the present study we developed a novel mouse model combining severe nephron reduction and High Fat Diet challenge that led to chronic kidney disease with metabolic alterations. We showed that the Mineralocorticoid Receptor antagonist canrenoate improved metabolic function, reduced albuminuria and prevented the synergistic effect of high fat diet on renal fibrosis and inflammation in chronic kidney disease mice.
RESUMO
Thiazolidinedione PPARγ agonists such as rosiglitazone and pioglitazone are effective antidiabetic drugs, but side effects have limited their use. It has been posited that their positive antidiabetic effects are mainly mediated by the inhibition of the CDK5-mediated Ser273 phosphorylation of PPARγ, whereas the side effects are linked to classical PPARγ agonism. Thus compounds that inhibit PPARγ Ser273 phosphorylation but lack classical PPARγ agonism have been sought as safer antidiabetic therapies. Herein we report the discovery by virtual screening of 10, which is a potent PPARγ binder and in vitro inhibitor of the CDK5-mediated phosphorylation of PPARγ Ser273 and displays negligible PPARγ agonism in a reporter gene assay. The pharmacokinetic properties of 10 are compatible with oral dosing, enabling preclinical in vivo testing, and a 7 day treatment demonstrated an improvement in insulin sensitivity in the ob/ob diabetic mouse model.
RESUMO
This phase Ib study compared the effects of AZD9977, a selective mineralocorticoid receptor modulator with predicted low hyperkalemia risk, with spironolactone on serum potassium (sK+ ) in patients with heart failure (HF) with preserved or mildly reduced ejection fraction (EF; ≥40%), and renal impairment. Patients with HF with EF greater than or equal to 40% and estimated glomerular filtration rate of 40-70 ml/min/1.73 m2 were randomized to once-daily AZD9977 100 mg or spironolactone 25 mg for 14 days, up-titrated to AZD9977 200 mg or spironolactone 50 mg for another 14 days. The primary end point was relative change (%) in sK+ for AZD9977 versus spironolactone (baseline to day 28). Serum/urinary electrolytes, fractional excretion (FE) of Na+ /K+ , plasma aldosterone, cortisol, and renin, and safety were also assessed. Sixty-eight patients were randomized (AZD9977, n = 33; spironolactone, n = 35). Mean (SD) age was 73.0 (8.5) years, 51.5% men. Mean sK+ change from baseline to day 28 was 5.7% (AZD9977) and 4.2% (spironolactone), and 1.5% and 4.2% at day 14. Relative change (95% confidence interval) in sK+ with AZD9977 versus spironolactone was -0.3% (-5.3% to 4.4%; day 28), and 3.4% (-0.8% to 7.5%; day 14). Median increase from baseline in plasma aldosterone at day 28 was 89.8 pmol/L for AZD9977 and 67.4 pmol/L for spironolactone. Median FE of K+ was 12.9% (AZD9977) and 10.1% (spironolactone). AZD9977 was well-tolerated. No discontinuations due to hyperkalemia occurred with either treatment. Evidence of target engagement for AZD9977 with a favorable safety profile, supports further evaluation of AZD9977 in patients with HF and renal impairment.
Assuntos
Insuficiência Cardíaca , Antagonistas de Receptores de Mineralocorticoides , Espironolactona , Idoso , Feminino , Humanos , Masculino , Aldosterona , Eletrólitos , Insuficiência Cardíaca/diagnóstico , Insuficiência Cardíaca/tratamento farmacológico , Hidrocortisona , Hiperpotassemia/induzido quimicamente , Hiperpotassemia/diagnóstico , Hiperpotassemia/tratamento farmacológico , Antagonistas de Receptores de Mineralocorticoides/efeitos adversos , Potássio , Receptores de Mineralocorticoides , Renina , Espironolactona/efeitos adversos , Volume Sistólico , Resultado do TratamentoRESUMO
BACKGROUND: The most profound effect of vasopressin on the kidney is to increase water reabsorption through V2-receptor (V2R) stimulation, but there are also data suggesting effects on calcium transport. To address this issue, we have established an isolated perfused kidney model with accurate pressure control, to directly study the effects of V2R stimulation on kidney function, isolated from systemic effects. METHODS: The role of V2R in renal calcium handling was studied in isolated rat kidneys using a new pressure control system that uses a calibration curve to compensate for the internal pressure drop up to the tip of the perfusion cannula. RESULTS: Kidneys subjected to V2R stimulation using desmopressin (DDAVP) displayed stable osmolality and calcium reabsorption throughout the experiment, whereas kidneys not administered DDAVP exhibited a simultaneous fall in urine osmolality and calcium reabsorption. Epithelial sodium channel (ENaC) inhibition using amiloride resulted in a marked increase in potassium reabsorption along with decreased sodium reabsorption. CONCLUSIONS: A stable isolated perfused kidney model with computer-controlled pressure regulation was developed, which retained key physiological functions. The preparation responds to pharmacological inhibition of ENaC channels and activation of V2R. Using the model, the dynamic effects of V2R stimulation on calcium handling and urine osmolality could be visualised. The study thereby provides evidence for a stimulatory role of V2R in renal calcium reabsorption.
Assuntos
Cálcio/metabolismo , Canais Epiteliais de Sódio/metabolismo , Rim/metabolismo , Receptores de Vasopressinas/metabolismo , Animais , Transporte Biológico , Calibragem , Desamino Arginina Vasopressina/metabolismo , Eletrólitos , Taxa de Filtração Glomerular , Masculino , Concentração Osmolar , Perfusão , Ratos , Ratos Sprague-Dawley , Receptores de Vasopressinas/efeitos dos fármacosRESUMO
INTRODUCTION:: AZD9977 is a novel mineralocorticoid receptor (MR) modulator, which in preclinical studies demonstrated organ protection without affecting aldosterone-regulated urinary electrolyte excretion. However, when tested in humans, using fludrocortisone as an MR agonist, AZD9977 exhibited similar effects on urinary Na+/K+ ratio as eplerenone. The aim of this study is to understand whether the contradictory results seen in rats and humans are due to the mineralocorticoid used. MATERIALS AND METHODS:: Rats were treated with single doses of AZD9977 or eplerenone in combination with either aldosterone or fludrocortisone. Urine was collected for five to six hours and total amounts excreted Na+ and K+ were assessed. RESULTS:: AZD9977 dose-dependently increased urinary Na+/K+ ratio in rats when tested against fludrocortisone, but not when tested against aldosterone. Eplerenone dose-dependently increased urinary Na+/K+ ratio when tested against fludrocortisone as well as aldosterone. CONCLUSIONS:: The data suggest that the contrasting effects of AZD9977 on urinary electrolyte excretion observed in rats and humans are due to the use of the synthetic mineralocorticoid fludrocortisone. Future clinical studies are required to confirm the reduced electrolyte effects of AZD9977 and the subsequent lower predicted hyperkalemia risk.
Assuntos
Aldosterona/farmacologia , Benzoatos/farmacologia , Fludrocortisona/farmacologia , Mineralocorticoides/farmacologia , Oxazinas/farmacologia , Receptores de Mineralocorticoides/metabolismo , Aldosterona/administração & dosagem , Animais , Benzoatos/administração & dosagem , Eplerenona/farmacologia , Fludrocortisona/administração & dosagem , Humanos , Oxazinas/administração & dosagem , Potássio/urina , Ratos , Sódio/urinaRESUMO
The bile acid-sensing transcription factor farnesoid X receptor (FXR) regulates multiple metabolic processes. Modulation of FXR is desired to overcome several metabolic pathologies but pharmacological administration of full FXR agonists has been plagued by mechanism-based side effects. We have developed a modulator that partially activates FXR in vitro and in mice. Here we report the elucidation of the molecular mechanism that drives partial FXR activation by crystallography- and NMR-based structural biology. Natural and synthetic FXR agonists stabilize formation of an extended helix α11 and the α11-α12 loop upon binding. This strengthens a network of hydrogen bonds, repositions helix α12 and enables co-activator recruitment. Partial agonism in contrast is conferred by a kink in helix α11 that destabilizes the α11-α12 loop, a critical determinant for helix α12 orientation. Thereby, the synthetic partial agonist induces conformational states, capable of recruiting both co-repressors and co-activators leading to an equilibrium of co-activator and co-repressor binding.
Assuntos
Receptores Citoplasmáticos e Nucleares/agonistas , Receptores Citoplasmáticos e Nucleares/química , Animais , Linhagem Celular , Proteínas Correpressoras/genética , Proteínas Correpressoras/metabolismo , Humanos , Ligação de Hidrogênio , Ligantes , Fígado/metabolismo , Espectroscopia de Ressonância Magnética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ligação Proteica , Conformação Proteica em alfa-Hélice , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismoRESUMO
The mechanism-based risk for hyperkalemia has limited the use of mineralocorticoid receptor antagonists (MRAs) like eplerenone in cardio-renal diseases. Here, we describe the structure and property-driven lead generation and optimization, which resulted in identification of MR modulators ( S)-1 and ( S)-33. Both compounds were partial MRAs but still demonstrated equally efficacious organ protection as eplerenone after 4 weeks of treatment in uni-nephrectomized rats on high-salt diet and aldosterone infusion. Importantly, and in sharp contrast to eplerenone, this was achieved without substantial changes to the urine Na+/K+ ratio after acute treatment in rat, which predicts a reduced risk for hyperkalemia. This work led to selection of ( S)-1 (AZD9977) as the clinical candidate for treating MR-mediated cardio-renal diseases, including chronic kidney disease and heart failure. On the basis of our findings, we propose an empirical model for prediction of compounds with low risk of affecting the urinary Na+/K+ ratio in vivo.
Assuntos
Homeostase/efeitos dos fármacos , Antagonistas de Receptores de Mineralocorticoides/farmacologia , Oxazinas/farmacologia , Potássio/metabolismo , Substâncias Protetoras/farmacologia , Sódio/metabolismo , Animais , Coração/efeitos dos fármacos , Humanos , Rim/efeitos dos fármacos , Masculino , Antagonistas de Receptores de Mineralocorticoides/síntese química , Antagonistas de Receptores de Mineralocorticoides/metabolismo , Estrutura Molecular , Oxazinas/síntese química , Oxazinas/metabolismo , Potássio/urina , Substâncias Protetoras/síntese química , Substâncias Protetoras/metabolismo , Ratos Sprague-Dawley , Ratos Wistar , Receptores de Mineralocorticoides/metabolismo , Insuficiência Renal Crônica/tratamento farmacológico , Sódio/urina , Relação Estrutura-AtividadeRESUMO
Excess mineralocorticoid receptor (MR) activation promotes target organ dysfunction, vascular injury and fibrosis. MR antagonists like eplerenone are used for treating heart failure, but their use is limited due to the compound class-inherent hyperkalemia risk. Here we present evidence that AZD9977, a first-in-class MR modulator shows cardio-renal protection despite a mechanism-based reduced liability to cause hyperkalemia. AZD9977 in vitro potency and binding mode to MR were characterized using reporter gene, binding, cofactor recruitment assays and X-ray crystallopgraphy. Organ protection was studied in uni-nephrectomised db/db mice and uni-nephrectomised rats administered aldosterone and high salt. Acute effects of single compound doses on urinary electrolyte excretion were tested in rats on a low salt diet. AZD9977 and eplerenone showed similar human MR in vitro potencies. Unlike eplerenone, AZD9977 is a partial MR antagonist due to its unique interaction pattern with MR, which results in a distinct recruitment of co-factor peptides when compared to eplerenone. AZD9977 dose dependently reduced albuminuria and improved kidney histopathology similar to eplerenone in db/db uni-nephrectomised mice and uni-nephrectomised rats. In acute testing, AZD9977 did not affect urinary Na+/K+ ratio, while eplerenone increased the Na+/K+ ratio dose dependently. AZD9977 is a selective MR modulator, retaining organ protection without acute effect on urinary electrolyte excretion. This predicts a reduced hyperkalemia risk and AZD9977 therefore has the potential to deliver a safe, efficacious treatment to patients prone to hyperkalemia.
Assuntos
Benzoatos/farmacologia , Antagonistas de Receptores de Mineralocorticoides/farmacologia , Oxazinas/farmacologia , Administração Oral , Aldosterona , Animais , Benzoatos/química , Benzoatos/farmacocinética , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Eplerenona , Humanos , Rim/efeitos dos fármacos , Rim/metabolismo , Rim/patologia , Masculino , Camundongos Mutantes , Antagonistas de Receptores de Mineralocorticoides/química , Antagonistas de Receptores de Mineralocorticoides/farmacocinética , Estrutura Molecular , Oxazinas/química , Oxazinas/farmacocinética , Potássio/urina , Ratos Sprague-Dawley , Receptores de Mineralocorticoides/genética , Receptores de Mineralocorticoides/metabolismo , Insuficiência Renal Crônica/tratamento farmacológico , Insuficiência Renal Crônica/metabolismo , Insuficiência Renal Crônica/patologia , Sódio/urina , Sódio na Dieta , Espironolactona/análogos & derivados , Espironolactona/química , Espironolactona/farmacocinética , Espironolactona/farmacologiaRESUMO
The development of the dual peroxisome proliferator-activated receptor (PPAR) alpha/gamma agonist tesaglitazar as an oral antidiabetic was recently discontinued. Here we present tumor data from a 2-year carcinogenicity study in rats given 0.3, 1, 3, and 10 micromol/kg tesaglitazar is presented with focus on the findings of subcutaneous fibrosarcomas. To investigate the mechanism for induction of fibrosarcomas, replicative DNA synthesis (immunohistochemical detection of BrdU-labeled cells) and expression of PPARgamma (immunohistochemistry and reverse transcription-polymerase chain reaction) in subcutaneous adipose tissues was assessed in rats administered 1 or 10 micromol/kg for 2 weeks or 3 months. Poorly differentiated subcutaneous mesenchymal sarcomas with a predominant spindle cell appearance occurred at the highest dose level of 10 micromol/kg in both sexes, and these tumors were diagnosed as fibrosarcomas. The 10-micromol/kg dose was at or above the maximum tolerated dose and caused considerable cardiovascular mortality. Tesaglitazar stimulated DNA synthesis mainly in subcutaneous interstitial mesenchymal cells. The percentage of BrdU-labeled interstitial cells was increased at 1 and 10 micromol/kg after 2 weeks. The increase in DNA synthesis was still significant at the end of the 12-week treatment at 10 mumol/kg, the dose producing fibrosarcoma. However, at 1 micromol/kg, a dose below the no-observed-effect level for fibrosarcoma, the level of DNA synthesis was similar to control levels at 12 weeks. Immunohistochemical analyses showed no detectable PPARgamma protein in the majority of BrdU-labeled interstitial mesenchymal cells in white and brown fat. This indicates that stimulation of DNA synthesis is not mediated via direct activation of PPARgamma in these cells. The results suggest that the induction of rat fibrosarcoma by tesaglitazar, at exposures 100-fold above the human therapeutic exposure, may involve proliferation of undifferentiated mesenchymal cells in subcutaneous tissues.
Assuntos
Alcanossulfonatos/farmacologia , DNA/biossíntese , Fibrossarcoma/induzido quimicamente , Hipoglicemiantes/farmacologia , Mesoderma/metabolismo , PPAR alfa/agonistas , PPAR gama/agonistas , Fenilpropionatos/farmacologia , Neoplasias Cutâneas/induzido quimicamente , Animais , Antimetabólitos , Bromodesoxiuridina , Colesterol/sangue , Replicação do DNA/efeitos dos fármacos , Feminino , Fibrossarcoma/patologia , Imuno-Histoquímica , Masculino , Mesoderma/efeitos dos fármacos , Microdissecção , Tamanho do Órgão/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , RNA/biossíntese , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Cutâneas/patologia , Triglicerídeos/sangueRESUMO
Olf-1/early B-cell factor (O/E-1) is a transcription factor important for B-lymphocyte and neuronal gene regulation. Here we report that all three known O/E genes (O/E-1, -2, and -3) are expressed in mouse adipose tissue and are upregulated during adipocyte differentiation. Forced expression of O/E-1 in either the preadipocyte cell line 3T3-L1 or mouse embryonic fibroblasts augmented adipogenesis, and constitutive expression of O/E-1 in uncommitted NIH 3T3 fibroblasts led to initiation of adipocyte differentiation. Furthermore, a dominant negative form of O/E-1 partially suppressed 3T3-L1 adipogenesis, indicating that expression from endogenous O/E target genes is required for 3T3-L1 terminal differentiation. Thus, our data point to the importance of O/E target genes for adipocyte differentiation and suggest a novel role for O/E-1 as an initiator and stimulator of adipogenesis.
Assuntos
Adipócitos/fisiologia , Tecido Adiposo/metabolismo , Diferenciação Celular/fisiologia , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica no Desenvolvimento , Transativadores/genética , Tecido Adiposo/citologia , Animais , Biomarcadores , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Embrião de Mamíferos/anatomia & histologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Genes Reporter , Camundongos , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Retroviridae/genética , Retroviridae/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Peroxisome proliferator-activated receptor γ (PPARγ) is a ligand-dependent transcription factor involved in many aspects of metabolism, immune response, and development. Total-body deletion of the two Pparg alleles provoked generalized lipoatrophy along with severe type 2 diabetes. Herein, we explore the appearance and development of structural and functional alterations of the kidney, comparing Pparg null-mice to their littermate controls (carrying Pparg floxed alleles). We show that renal hypertrophy and functional alterations with increased glucosuria and albuminuria are already present in 3 weeks-old Pparg null-mice. Renal insufficiency with decreased creatinine clearance progress at 7 weeks of age, with the advance of the type 2 diabetes. At 52 weeks of age, these alterations are accompanied by signs of fibrosis and mesangial expansion. More intriguingly, aged Pparg null-mice concomitantly present an anti-phospholipid syndrome (APS), characterized by the late appearance of microthrombi and a mesangioproliferative pattern of glomerular injury, associated with significant plasmatic levels of anti-ß2- glycoprotein1 antibodies and renal deposition of IgG, IgM, and C3. Thus, in line with the role of PPARγ in metabolic homeostasis, Pparg null-mice first represent a potent model for studying the initiation and the development of diabetic nephropathy. Second, and in relation with the important PPARγ activity in inflammation and in immune system, these mice also highlight a new role for PPARγ signaling in the promotion of APS, a syndrome whose pathogenesis is poorly known and whose current treatment is limited to prevention of thrombosis events.
Assuntos
Síndrome Antifosfolipídica/genética , Nefropatias Diabéticas/metabolismo , PPAR gama/genética , Animais , Anticorpos/imunologia , Síndrome Antifosfolipídica/imunologia , Síndrome Antifosfolipídica/patologia , Complemento C3/imunologia , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/patologia , Homeostase , Imunoglobulina G/metabolismo , Imunoglobulina M/metabolismo , Rim/imunologia , Rim/metabolismo , Rim/patologia , Camundongos , PPAR gama/metabolismo , Transdução de Sinais , beta 2-Glicoproteína I/imunologiaRESUMO
The mineralocorticoid receptor (MR) is a nuclear hormone receptor involved in the regulation of body fluid and electrolyte homeostasis. In this study we explore selectivity triggers for a series of nonsteroidal MR antagonists to improve selectivity over other members of the oxosteroid receptor family. A biaryl sulfonamide compound was identified in a high-throughput screening (HTS) campaign. The compound bound to MR with pKi =6.6, but displayed poor selectivity over the glucocorticoid receptor (GR) and the progesterone receptor (PR). Following X-ray crystallography of MR in complex with the HTS hit, a compound library was designed that explored an induced-fit hypothesis that required movement of the Met852 side chain. An improvement in MR selectivity of 11- to 79-fold over PR and 23- to 234-fold over GR was obtained. Given the U-shaped binding conformation, macrocyclizations were explored, yielding a macrocycle that bound to MR with pKi =7.3. Two protein-ligand X-ray structures were determined, confirming the hypothesized binding mode for the designed compounds.
Assuntos
Desenho de Fármacos , Antagonistas de Receptores de Mineralocorticoides/química , Antagonistas de Receptores de Mineralocorticoides/farmacologia , Receptores de Glucocorticoides/química , Receptores de Glucocorticoides/metabolismo , Receptores de Mineralocorticoides/metabolismo , Relação Dose-Resposta a Droga , Ensaios de Triagem em Larga Escala , Humanos , Antagonistas de Receptores de Mineralocorticoides/síntese química , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Alanine aminotransferase (ALT) in serum is the standard biomarker for liver injury. We have previously described a clinical trial with a novel selective peroxisome proliferator-activated receptor α (PPARα) agonist (AZD4619), which unexpectedly caused increased serum levels of ALT in treated individuals without any other evidence of liver injury. We pinpointed a plausible mechanism through which AZD4619 could increase serum ALT levels; namely through the PPARα-specific activation of the human ALT1 gene at the transcriptional level. In the present study, we present data from the preceding rat toxicity study, demonstrating that AZD4619 had no effect on rat serum ALT activity levels, and further experiments were performed to elucidate the mechanisms responsible for this species-related difference. Our results revealed that AZD4619 increased ALT1 protein expression in a dose-dependent manner in human, but not in rat primary hepatocytes. Cloning of the human and rat ALT1 promoters into luciferase vectors confirmed that AZD4619 induced only the human, but not the rat ALT1 gene promoter in a dose-dependent manner. In PPARα-GAL4 reporter gene assays, AZD4619 was >100-fold more potent on the human vs. rat PPARα levels, explaining the differences in induction of the ALT1 gene between the species at the concentration range tested. These data demonstrate the usefulness of the human and rat ALT1 reporter gene assays for testing future drug candidates at the preclinical stage. In drug discovery projects, these assays elucidate whether elevations in ALT levels observed in vivo or in the clinic are due to metabolic effects rather than a toxic event in the liver.
Assuntos
Alanina Transaminase/metabolismo , Hepatócitos/efeitos dos fármacos , PPAR alfa/agonistas , Propionatos/farmacologia , Sulfonas/farmacologia , Xenobióticos/farmacologia , Alanina Transaminase/sangue , Alanina Transaminase/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Linhagem Celular Tumoral , Células Cultivadas , Feminino , Expressão Gênica/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Luciferases/genética , Luciferases/metabolismo , Masculino , Regiões Promotoras Genéticas/genética , Ratos Wistar , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Especificidade da EspécieRESUMO
Although blockade of the renin-angiotensin-system (RAS) has become standard therapy for diabetic nephropathy (DN), decline in kidney function towards end-stage renal disease is seen in many patients. Elevated plasma aldosterone often accompanies RAS blockade by a phenomenon known as "aldosterone escape" and activates the mineralocorticoid receptor (MR). We therefore examined whether addition of the MR antagonist eplerenone to an ACEI would enhance the efficacy in slowing the progression of DN. Untreated uninephrectomized diabetic db/db mice developed progressive albuminuria and glomerulosclerosis between weeks 18 and 22, associated with decreased number of podocytes and increased renal expression of fibrotic markers. The therapeutic effect of eplerenone at 100 mg/kg BW/d on albuminuria, podocyte injury and renal fibrosis was similar to that of enalapril given alone at maximally effective doses. Adding eplerenone to enalapril resulted in further reduction in these measurements. Renal expressions of TNF-α, MCP-1, Nox2 and p47phox and renal TBARS levels, markers of inflammation and oxidative stress, were increased during disease progression in diabetic mice, which were reduced by eplerenone or enalapril given alone and further reduced by the two drugs given in combination. However, there were no treatment related effects on plasma K+. Our results suggest that eplerenone is effective in slowing the progression of DN in db/db mice and that the effect is additive to an ACEI. The addition of an MR antagonist void of effects on plasma K+ to an ACEI may offer additional renoprotection in progressive DN via blocking the effects of aldosterone due to escape or diabetes-induction.