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Chronic kidney disease (CKD) is a leading cause of death, and its progression is driven by glomerular podocyte injury and loss, manifesting as proteinuria. Proteinuria includes urinary loss of coagulation zymogens, cofactors, and inhibitors. Importantly, both CKD and proteinuria significantly increase the risk of thromboembolic disease. Prior studies demonstrated that anticoagulants reduced proteinuria in rats and that thrombin injured cultured podocytes. Herein we aimed to directly determine the influence of circulating prothrombin on glomerular pathobiology. We hypothesized that (pro)thrombin drives podocytopathy, podocytopenia, and proteinuria. Glomerular proteinuria was induced with puromycin aminonucleoside (PAN) in Wistar rats. Circulating prothrombin was either knocked down using a rat-specific antisense oligonucleotide or elevated by serial intravenous infusions of prothrombin protein, which are previously established methods to model hypo- (LoPT) and hyper-prothrombinemia (HiPT), respectively. After 10 days (peak proteinuria in this model) plasma prothrombin levels were determined, kidneys were examined for (pro)thrombin co-localization to podocytes, histology, and electron microscopy. Podocytopathy and podocytopenia were determined and proteinuria, and plasma albumin were measured. LoPT significantly reduced prothrombin colocalization to podocytes, podocytopathy, and proteinuria with improved plasma albumin. In contrast, HiPT significantly increased podocytopathy and proteinuria. Podocytopenia was significantly reduced in LoPT vs. HiPT rats. In summary, prothrombin knockdown ameliorated PAN-induced glomerular disease whereas hyper-prothrombinemia exacerbated disease. Thus, (pro)thrombin antagonism may be a viable strategy to simultaneously provide thromboprophylaxis and prevent podocytopathy-mediated CKD progression.
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Idiopathic nephrotic syndrome (NS) is a common glomerular disease. Although glucocorticoids (GC) are the primary treatment, the PPARγ agonist pioglitazone (Pio) also reduces proteinuria in patients with NS and directly protects podocytes from injury. Because both drugs reduce proteinuria, we hypothesized these effects result from overlapping transcriptional patterns. Systems biology approaches compared glomerular transcriptomes from rats with PAN-induced NS treated with GC vs. Pio and identified 29 commonly regulated genes-of-interest, primarily involved in extracellular matrix (ECM) remodeling. Correlation with clinical idiopathic NS patient datasets confirmed glomerular ECM dysregulation as a potential mechanism of injury. Cellular deconvolution in silico revealed GC- and Pio-induced amelioration of altered genes primarily within podocytes and mesangial cells. While validation studies are indicated, these analyses identified molecular pathways involved in the early stages of NS (prior to scarring), suggesting that targeting glomerular ECM dysregulation may enable a future non-immunosuppressive approach for proteinuria reduction in idiopathic NS.
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BACKGROUND: Nephrotic syndrome is associated with an acquired hypercoagulopathy that is thought to drive its predisposition for venous thromboembolism. Previous studies have suggested that urinary antithrombin (AT) loss leading to acquired AT deficiency is the primary mechanism underlying this hypercoagulopathy, but this hypothesis has not been directly tested. The objectives of this study were to test the influence of AT levels on hypercoagulopathy in nephrotic syndrome patient samples and perform meta-analyses to evaluate the likelihood of AT deficiency in patients with nephrotic syndrome. METHODS: Samples from three independent nephrotic syndrome cohorts were analyzed. AT antigen and activity assays were performed using ELISA and amidolytic assays, respectively. Plasma thrombin generation, albumin, and urine protein-to-creatinine ratios were determined using established methods. Meta-analyses were performed by combining these new data with previously published data. RESULTS: AT levels were not consistently related to either plasma albumin or proteinuria. AT was quantitatively related to hypercoagulopathy in adult nephrotic syndrome, whereas AT activity was inconsistently associated with hypercoagulopathy in childhood nephrotic syndrome. Notably, hypercoagulopathy did not differ between patients with normal AT levels and those with levels below the threshold used to define clinical AT deficiency (<70%). Moreover, ex vivo AT supplementation did not significantly alter hypercoagulopathy in AT-deficient plasma samples. The meta-analyses demonstrated that AT deficiency was not a uniform feature of nephrotic syndrome and was more common in children than adults. CONCLUSIONS: These data suggest that AT deficiency plays only a limited role in the mechanisms underlying the acquired hypercoagulopathy of nephrotic syndrome. Moreover, AT deficiency was not present in all patients with nephrotic syndrome and was more likely in children than adults despite the higher risk for venous thromboembolism in adults than children.
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Síndrome Nefrótico , Tromboembolia Venosa , Adulto , Niño , Humanos , Síndrome Nefrótico/complicaciones , Antitrombinas , Tromboembolia Venosa/complicaciones , Estudios de Cohortes , Antitrombina III/orinaRESUMEN
The present study determined the independent contributions of temperature, strong ion difference ([SID]), total weak acid concentration ([Atot]) and PCO2 to changes in arterial and mixed venous [H+] and total carbon dioxide concentration ([TCO2]) during 37 min of moderate intensity exercise (~50% of heart rate max) and the first 60 min of recovery. Six horses were fitted with indwelling carotid and pulmonary artery (PA) catheters, had PA temperature measured, and had blood samples withdrawn for immediate analysis of plasma ion and gas concentrations. The increase in core temperature during exercise (+4.5 °C; p < 0.001) significantly (p < 0.05) increased PO2, PCO2, and [H+], but without a significant effect on [TCO2] (p > 0.01). The physicochemical acid-base approach was used to determine contributions of independent variables (except temperature) to the changes in [H+] and [TCO2]. In both arterial and venous blood, there was no acidosis during exercise and recovery despite significant (p < 0.05) increases in [lactate] and in venous PCO2. In arterial blood plasma, a mild alkalosis with exercise was due to primarily to a decrease in PCO2 (p < 0.05) and an increase in [SID] (p < 0.1). In venous blood plasma, a near absence of change in [H+] was due to the acidifying effects of increased PCO2 (p < 0.01) being offset by the alkalizing effects of increased [SID] (p < 0.05). The effect of temperature on PO2 (p < 0.001) resulted in an increased arterio-venous PO2 difference (p < 0.001) that would facilitate O2 transfer to contracting muscle. The simultaneous changes in the PCO2 and the concentrations of the other independent acid-base variables (contributions from individual strong and weak ions as manifest in [SID] and [Atot]) show complex, multilevel control of acid-base states in horses performing even moderate intensity exercise. Correction of acid-base variables to core body temperature presents a markedly different physiological response to exercise than that provided by variables measured and presented at an instrument temperature of 37 °C.
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Glomerular disease manifests as nephrotic syndrome (NS) with high proteinuria and comorbidities, and is frequently refractory to standard treatments. We hypothesized that a selective modulator of PPARγ, GQ-16, will provide therapeutic advantage over traditional PPARγ agonists for NS treatment. We demonstrate in a pre-clinical NS model that proteinuria is reduced with pioglitazone to 64%, and robustly with GQ-16 to 81% of nephrosis, comparable to controls. Although both GQ-16 and pioglitazone restore glomerular-Nphs1, hepatic-Pcsk9 and serum-cholesterol, only GQ-16 restores glomerular-Nrf2, and reduces hypoalbuminemia and hypercoagulopathy. GQ-16 and pioglitazone restore common and distinct glomerular gene expression analyzed by RNA-seq and induce insulin sensitizing adipokines to various degrees. Pioglitazone but not GQ-16 induces more lipid accumulation and aP2 in adipocytes and white adipose tissue. We conclude that selective modulation of PPARγ by a partial agonist, GQ-16, is more advantageous than pioglitazone in reducing proteinuria, NS associated comorbidities, and adipogenic side effects of full PPARγ agonists.
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Oral electrolyte supplementation may influence acid-base state during exercise due to the intestinal absorption of administered water and electrolytes used to mitigating sweat losses. This study examined the effect of pre-exercise electrolyte supplementation (3 and 8 L) on plasma acid-base variables at rest, during moderate intensity exercise and during recovery. It was hypothesized that electrolyte supplementation will result in improved acid-base state compared to the alkalosis typical of prolonged exercise. In randomized crossover fashion, four horses were administered 3 L or 8 L of a hypotonic electrolyte solution (PNW) intended to replace sweat losses, or water alone (CON), 1 h before treadmill exercise to fatigue (at 35% of peak VO2) or for 45 min at 50% peak VO2. Blood was sampled at 10-min intervals before, during and after exercise, and analyzed for dependent and independent acid-base variables. Effects of 3 L of supplementation at low exercise intensities were minimal. In the 8 L trials, plasma [H+] decreased (p < 0.05) during exercise and early recovery in CON but not PNW. Plasma TCO2 decreased (p < 0.05) by 30 min after PNW reaching a nadir of 28.0 ± 1.5 mmol/L during the early exercise period (p = 0.018). Plasma pCO2 and strong ion difference [SID] were the primary contributors to changes in [H+] and [TCO2], respectively. Pre-exercise PNW of 8 L intended to fully replenish sweat loses maintained [H+], decreased [TCO2] and mitigated the mild alkalosis during moderate intensity exercise.
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KEY POINTS: Exercise results in rapid and large extracellular to intracellular fluid shifts, as well as significant sweating losses of water and ions. It is unknown whether ions within oral electrolyte supplements are taken up by muscle (and other soft tissues) and whether oral supplementation can effectively offset sweating losses. Pre-loading with 8 L of a balanced hypotonic electrolyte supplement attenuated extracellular fluid losses, increased exercise duration and increased sweating fluid and ion losses during submaximal exercise. Supplemented electrolytes appear in skeletal muscle within 1 h after administration. Electrolyte supplementation increased exercise performance, improved maintenance of extracellular fluid volumes, and attenuated body fluid losses while maintaining sweating rates. ABSTRACT: This study used radioactive sodium (24 Na) and potassium (42 K) in a balanced, hypotonic electrolyte supplement to trace their appearance in skeletal muscle, and also quantified extracellular and whole-body fluid and ion changes during electrolyte supplementation, exercise and recovery. In a randomized crossover design, 1 h after administration of 1 to 3 L of water or electrolyte supplement with 24 Na, horses were exercised at 35% VO2max to voluntary fatigue or, after administration of 8 L of water or electrolyte supplement with 42 K were exercised at 50% peak VO2 for 45 min (n = 4 in each trial). Pre-exercise electrolyte supplementation was associated with decreased loss of fluid and electrolytes from the extracellular fluid compartments during exercise and recovery compared with water alone. The improved fluid and ion balance during prolonged exercise was associated with increased exercise duration, despite continuing sweating losses of fluid and ions. Nasogastric administration of radiotracer 24 Na+ and 42 K+ showed rapid absorption into the blood with plasma levels peaking 45 min after administration, followed by distribution into the extracellular space and intracellular fluid of muscle within 1 h. Following exercise, virtually all Na+ remained within the extracellular compartment, while the majority of K+ underwent intracellular uptake by 2 h of recovery. It is concluded that pre-loading with a large volume, balanced electrolyte supplement helps maintain whole-body fluid and ion balance and support muscle function during periods of prolonged sweat ion losses.
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Condicionamiento Físico Animal , Animales , Electrólitos , Caballos , Sodio , Sudoración , Agua , Equilibrio HidroelectrolíticoRESUMEN
NEW FINDINGS: What is the central question of this study? What are the mechanisms by which equine sweat glands transport sodium, potassium and water into sweat? What is the main finding and its importance? The flux of sodium into sweat does not have an active transport component, the flux of potassium into sweat is partially dependent on an active transport mechanism, and there is no evidence for paracellular transport. ABSTRACT: In two series of experiments, this study used radioactive sodium (Na+ ) and potassium (K+ ) to trace the net flux, and calculate the unidirectional fluxes, of these ions from extracellular fluid into sweat of horses during exercise and recovery. The effect of an oral electrolyte supplement (PNW) on the sweating responses and ion fluxes was also examined. Compared to 8 litres of water (controls), provision of 8 litres of PNW resulted in significantly increased sweating duration (P < 0.001). Two hours before exercise, 99 Tc-labelled diethylene-triamine-pentaacetate (DTPA) was administered i.v. to determine if there was paracellular flux of this molecule in sweat glands during the period of sweating. One hour before beginning moderate-intensity exercise, horses were nasogastrically administered either 24 Na (1-3 litres) or 42 K (8 litres) with water (control) or an electrolyte supplement. Both radiotracers appeared in sweat within 10 min of exercise onset, and the sweat specific activity of both ions increased during exercise (P < 0.001), approaching plasma specific activities. There was no appearance of 99 Tc-DTPA in sweat. The activities of 24 Na and 42 K, together with the concentrations Na+ , K+ and Cl- , argued against significant paracellular flux of these ions into the lumen of sweat glands. The flux analysis for 24 Na indicated a small intracellular pool within sweat gland cells, and no evidence for an active transport component. The flux analysis for 42 K indicated a relatively large intracellular equilibration pool within sweat gland cells, with evidence for an active transport component. The results are discussed with respect to the current understanding of sweat gland epithelial cell ion transport mechanisms at both the basal and the apical membranes. It appears likely that the majority of ions appearing in sweat pass through sweat gland epithelial cells by transcellular mechanisms that include ion transporting pathways as well as apical vesicular exocytosis.
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Condicionamiento Físico Animal , Sudor , Animales , Cloruros/metabolismo , Caballos , Condicionamiento Físico Animal/fisiología , Potasio/metabolismo , Sodio/metabolismo , Sudor/metabolismo , Sudoración , AguaRESUMEN
INTRODUCTION: Nephrotic syndrome (NS) is associated with an acquired hypercoagulopathy that drives its strong predilection for life-threatening thrombosis. We previously demonstrated that hypercoagulopathy is proportional to NS disease severity in animal models. Therefore, hypercoagulopathy and disease severity may inform thrombosis risk and better guide therapeutic decision making. The objective of this study was thus to establish the relationship between disease severity and hypercoagulopathy in human NS. MATERIALS AND METHODS: Thrombin generation assays (TGA) were performed on biorepository plasma samples from a prospective longitudinal NS cohort study. TGA was also determined on a separate cohort of incident NS patients. Multivariable regression was used to build NS-hypercoagulopathy relationship models. RESULTS: Endogenous thrombin potential (ETP) was the TGA parameter most strongly correlated with NS severity and was proportional to conventional measures of NS disease activity including proteinuria, hypercholesterolemia, and hypoalbuminemia. The overall disease activity model was well correlated with ETP (R2 = 0.38). The relationship with disease activity was confirmed in the second cohort. These models further revealed that ETP is related to disease activity in a manner dependent on remission status. CONCLUSION: Consistent with our previously reported animal model observations, we found that the combination of proteinuria, hypercholesterolemia, and hypoalbuminemia correlated with ETP-defined hypercoagulopathy. Hypercoagulopathy improved significantly with partial or complete NS remission. These data are expected to inform studies designed to stratify thrombotic risk for patients with NS.
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Síndrome Nefrótico , Animales , Pruebas de Coagulación Sanguínea , Estudios de Cohortes , Humanos , Síndrome Nefrótico/complicaciones , Estudios Prospectivos , ProteinuriaRESUMEN
Venous thromboembolism (VTE) is the third most common cardiovascular disease and optimizing treatment is essential. In this single-center pilot study, we sought to investigate the effects of statins in addition to anticoagulation in patients with acute VTE. We enrolled patients over 18 with an acute proximal lower extremity deep vein thrombosis with or without pulmonary embolism. Patients were randomized to anticoagulation alone (with either warfarin or rivaroxaban) or anticoagulation and atorvastatin 40âmg daily and followed for 9 months. The primary objective was to determine if adjunct atorvastatin reduced thrombin generation, measured by endogenous thrombin potential and/or peak thrombin concentration. Secondary endpoints included recurrent VTE, arterial thrombosis, bleeding events, lipidomic profiles, and symptoms of post thrombotic syndrome. A total of 21 patients were enrolled (11 anticoagulation only and 10 anticoagulation and atorvastatin) over 3.5 years. Endogenous thrombin potential or peak thrombin was not significantly recued with the addition of atorvastatin. Atorvastatin did significantly reduce the mean LDLs at 3 months, without reduction of either d-dimer or high-sensitivity-C reactive protein. Given the low recruitment rate, continuation of the study was deemed futile and the study was terminated early. Barriers to enrollment and completion of study included the many ineligible patients by exclusion criteria (e.g., preexisting statin use, active malignancy, etc.) and high rate of lost follow-up. The pilot study was terminated early but could inform obstacles for future studies investigating the effects of statins in the management of patients with VTE.
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Atorvastatina/uso terapéutico , Inhibidores de Hidroximetilglutaril-CoA Reductasas/uso terapéutico , Tromboembolia Venosa/tratamiento farmacológico , Enfermedad Aguda , Adulto , Anciano , Atorvastatina/farmacología , Femenino , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Masculino , Persona de Mediana Edad , Proyectos Piloto , Adulto JovenRESUMEN
BACKGROUND: Thrombosis is a potentially life-threatening nephrotic syndrome (NS) complication. We have previously demonstrated that hypercoagulopathy is proportional to NS severity in rat models and that pioglitazone (Pio) reduces proteinuria both independently and in combination with methylprednisolone (MP), a glucocorticoid (GC). However, the effect of these treatments on NS-associated hypercoagulopathy remains unknown. We thus sought to determine the ability of Pio and GC to alleviate NS-associated hypercoagulopathy. METHODS: Puromycin aminonucleoside-induced rat NS was treated with sham, Low- or High-dose MP, Pio, or combination (Pio + Low-MP) and plasma was collected at day 11. Plasma samples were collected from children with steroid-sensitive NS (SSNS) and steroid-resistant NS (SRNS) upon presentation and after 7 weeks of GC therapy. Plasma endogenous thrombin potential (ETP), antithrombin (AT) activity, and albumin (Alb) were measured using thrombin generation, amidolytic, and colorimetric assays, respectively. RESULTS: In a rat model of NS, both High-MP and Pio improved proteinuria and corrected hypoalbuminemia, ETP and AT activity (p < .05). Proteinuria (p = .005) and hypoalbuminemia (p < .001) were correlated with ETP. In childhood NS, while ETP was not different at presentation, GC therapy improved proteinuria, hypoalbuminemia, and ETP in children with SSNS (p < .001) but not SRNS (p = .330). CONCLUSIONS: Both Pio and GC diminish proteinuria and significantly alleviate hypercoagulopathy. Both Pio and MP improved hypercoagulopathy in rats, and successful GC therapy (SSNS) also improved hypercoagulopathy in childhood NS. These data suggest that even a partial reduction in proteinuria may reduce NS-associated thrombotic risk.
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Coagulación Sanguínea/efectos de los fármacos , Glucocorticoides/uso terapéutico , Síndrome Nefrótico/tratamiento farmacológico , Pioglitazona/uso terapéutico , Trombosis/tratamiento farmacológico , Animales , Niño , Femenino , Glucocorticoides/administración & dosificación , Glucocorticoides/farmacología , Humanos , Masculino , Síndrome Nefrótico/complicaciones , PPAR gamma/agonistas , Pioglitazona/administración & dosificación , Pioglitazona/farmacología , Ratas , Ratas Wistar , Receptores de Glucocorticoides/agonistas , Trombosis/etiologíaRESUMEN
Diabetes is a global epidemic disease, which leads to multiorgan dysfunction, including heart disease. Diabetes results from the limited absorption of glucose into insulin-sensitive tissues. The heart is one of the main organs to utilize glucose as an energy substrate. Glucose uptake into striated muscle is regulated by a family of membrane proteins called glucose transporters (GLUTs). Although calcium channel blockers, including diltiazem, are widely prescribed drugs for cardiovascular diseases, including in patients with diabetes, their pharmacological effects on glucose metabolism are somewhat controversial. We hypothesized that diltiazem treatment will exhibit detrimental effects on whole body glucose homeostasis and glucose transport in the striated muscle of patients with diabetes. Healthy and streptozotocin-treated rats were randomly assigned to receive diltiazem treatment or a placebo for 8 weeks. Blood glucose was significantly increased in the untreated diabetic groups, which worsened after diltiazem treatment. Diabetes decreased protein content of both GLUT4 (the predominate insulin-sensitive glucose transporter) and AS160 (Akt Substrate at 160 kDa, the downstream protein in the signaling cascade that regulates GLUT4 trafficking) in striated muscle of diabetic rats, with a more pronounced alteration after diltiazem treatment. We additionally reported that diabetic rodents displayed marked systolic dysfunction, which was not rescued by diltiazem treatment. In conclusion, diltiazem treatment worsened the effects of diabetes-induced hyperglycemia and diabetes-induced alterations in the regulation of glucose transport in striated muscle.
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Glucemia/efectos de los fármacos , Bloqueadores de los Canales de Calcio/efectos adversos , Diltiazem/efectos adversos , Animales , Cardiomiopatías Diabéticas , Modelos Animales de Enfermedad , Homeostasis/efectos de los fármacos , Masculino , Músculo Esquelético/efectos de los fármacos , Distribución Aleatoria , Ratas , Ratas WistarRESUMEN
Nephrotic syndrome is characterized by massive proteinuria and injury of specialized glomerular epithelial cells called podocytes. Studies have shown that, whereas low-concentration thrombin may be cytoprotective, higher thrombin concentrations may contribute to podocyte injury. We and others have demonstrated that ex vivo plasma thrombin generation is enhanced during nephrosis, suggesting that thrombin may contribute to nephrotic progression. Moreover, nonspecific thrombin inhibition has been shown to decrease proteinuria in nephrotic animal models. We thus hypothesized that thrombin contributes to podocyte injury in a protease-activated receptor-specific manner during nephrosis. Here, we show that specific inhibition of thrombin with hirudin reduced proteinuria in two rat nephrosis models, and thrombin colocalized with a podocyte-specific marker in rat glomeruli. Furthermore, flow cytometry immunophenotyping revealed that rat podocytes express the protease-activated receptor family of coagulation receptors in vivo High-concentration thrombin directly injured conditionally immortalized human and rat podocytes. Using receptor-blocking antibodies and activation peptides, we determined that thrombin-mediated injury depended upon interactions between protease-activated receptor 3 and protease-activated receptor 4 in human podocytes, and between protease-activated receptor 1 and protease-activated receptor 4 in rat podocytes. Proximity ligation and coimmunoprecipitation assays confirmed thrombin-dependent interactions between human protease-activated receptor 3 and protease-activated receptor 4, and between rat protease-activated receptor 1 and protease-activated receptor 4 in cultured podocytes. Collectively, these data implicate thrombinuria as a contributor to podocyte injury during nephrosis, and suggest that thrombin and/or podocyte-expressed thrombin receptors may be novel therapeutic targets for nephrotic syndrome.
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Glomérulos Renales/metabolismo , Nefrosis/metabolismo , Podocitos/patología , Receptor PAR-1/metabolismo , Receptores de Trombina/metabolismo , Trombina/metabolismo , Animales , Antitrombinas/farmacología , Supervivencia Celular , Células Cultivadas , Modelos Animales de Enfermedad , Expresión Génica , Hirudinas/farmacología , Humanos , Inmunofenotipificación , Nefrosis/complicaciones , Nefrosis/patología , Nefrosis/orina , Podocitos/metabolismo , Proteinuria/etiología , Ratas , Receptor PAR-1/genética , Receptores de Trombina/genética , Transducción de Señal , Trombina/antagonistas & inhibidores , Trombina/farmacología , Trombina/orinaRESUMEN
Thrombotic disease, a major life-threatening complication of nephrotic syndrome, has been associated with proteinuria and hypoalbuminemia severity. However, it is not fully understood how disease severity correlates with severity of the acquired hypercoagulopathy of nephrotic syndrome. Without this knowledge, the utility of proteinuria and/or hypoalbuminemia as biomarkers of thrombotic risk remains limited. Here, we show that two well established ex vivo hypercoagulopathy assays, thrombin generation and rotational thromboelastometry, are highly correlated with proteinuria and hypoalbuminemia in the puromycin aminonucleoside and adriamycin rat models of nephrotic syndrome. Notably, in the puromycin aminonucleoside model, hyperfibrinogenemia and antithrombin deficiency were also correlated with proteinuria severity, consistent with reports in human nephrotic syndrome. Importantly, although coagulation was not spontaneously activated in vivo with increasing proteinuria, vascular injury induced a more robust thrombotic response in nephrotic animals. In conclusion, hypercoagulopathy is highly correlated with nephrotic disease severity, but overt thrombosis may require an initiating insult, such as vascular injury. Our results suggest that proteinuria and/or hypoalbuminemia could be developed as clinically meaningful surrogate biomarkers of hypercoagulopathy to identify patients with nephrotic syndrome at highest risk for thrombotic disease and potentially target them for anticoagulant pharmacoprophylaxis.
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Trastornos de la Coagulación Sanguínea/sangre , Trastornos de la Coagulación Sanguínea/orina , Hipoalbuminemia/sangre , Síndrome Nefrótico/sangre , Proteinuria/sangre , Trombosis/etiología , Animales , Antitrombinas/sangre , Biomarcadores/sangre , Biomarcadores/orina , Coagulación Sanguínea/fisiología , Trastornos de la Coagulación Sanguínea/etiología , Modelos Animales de Enfermedad , Doxorrubicina , Elasticidad , Fibrinógeno/metabolismo , Hemostasis , Masculino , Síndrome Nefrótico/inducido químicamente , Síndrome Nefrótico/complicaciones , Puromicina Aminonucleósido , Ratas , Ratas Wistar , Albúmina Sérica/metabolismo , Índice de Severidad de la Enfermedad , Trombina/biosíntesisRESUMEN
Despite intensive research, the pathways that mediate calcium (Ca(2+))-stimulated glucose transport in striated muscle remain elusive. Since the sarcoplasmic reticulum calcium ATPase (SERCA) pump tightly regulates cytosolic [Ca(2+)], we investigated whether the SERCA pump is a major regulator of cardiac glucose transport. We used healthy and insulin-deficient diabetic transgenic (TG) mice expressing SERCA1a in the heart. Active cell surface glucose transporter (GLUT)-4 was measured by a biotinylated photolabeled assay in the intact perfused myocardium and isolated myocytes. In healthy TG mice, cardiac-specific SERCA1a expression increased active cell-surface GLUT4 and glucose uptake in the myocardium, as well as whole body glucose tolerance. Diabetes reduced active cell-surface GLUT4 content and glucose uptake in the heart of wild type mice, all of which were preserved in diabetic TG mice. Decreased basal AS160 and increased proportion of calmodulin-bound AS160 paralleled the increase in cell surface GLUT4 content in the heart of TG mice, suggesting that AS160 regulates GLUT trafficking by a Ca(2+)/calmodulin dependent pathway. In addition, cardiac-specific SERCA1a expression partially rescues hyperglycemia during diabetes. Collectively, these data suggested that the SERCA pump is a major regulator of cardiac glucose transport by an AS160 dependent mechanism during healthy and insulin-deficient state. Our data further indicated that cardiac-specific SERCA overexpression rescues diabetes induced-alterations in cardiac glucose transport and improves whole body glucose homeostasis. Therefore, findings from this study provide novel mechanistic insights linking upregulation of the SERCA pump in the heart as a potential therapeutic target to improve glucose metabolism during diabetes.
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Diabetes Mellitus Experimental/metabolismo , Transportador de Glucosa de Tipo 4/metabolismo , Glucosa/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Animales , Transporte Biológico , Western Blotting , Calcio/metabolismo , Calmodulina/metabolismo , Diabetes Mellitus Experimental/genética , Ecocardiografía , Fluorodesoxiglucosa F18 , Proteínas Activadoras de GTPasa/metabolismo , Homeostasis , Ratones Transgénicos , Miocardio/citología , Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Tomografía de Emisión de Positrones/métodos , Unión Proteica , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , Función Ventricular IzquierdaRESUMEN
Glucose uptake from the bloodstream is the rate-limiting step in whole body glucose utilization, and is regulated by a family of membrane proteins called glucose transporters (GLUTs). Although GLUT4 is the predominant isoform in insulin-sensitive tissues, there is recent evidence that GLUT12 could be a novel second insulin-sensitive GLUT. However, its physiological role in the heart is not elucidated and the regulation of insulin-stimulated myocardial GLUT12 translocation is unknown. In addition, the role of GLUT12 has not been investigated in the diabetic myocardium. Thus, we hypothesized that, as for GLUT4, insulin regulates GLUT12 translocation to the myocardial cell surface, which is impaired during diabetes. Active cell surface GLUT (-4 and -12) content was quantified (before and after insulin stimulation) by a biotinylated photolabeled assay in both intact perfused myocardium and isolated cardiac myocytes of healthy and type 1 diabetic rodents. GLUT localization was confirmed by immunofluorescent confocal microscopy, and total GLUT protein expression was measured by Western blotting. Insulin stimulation increased translocation of GLUT-4, but not -12, in the healthy myocardium. Total GLUT4 content of the heart was decreased during diabetes, while there was no difference in total GLUT12. Active cell surface GLUT12 content was increased in the diabetic myocardium, potentially as a compensatory mechanism for the observed downregulation of GLUT4. Collectively, our data suggest that, in contrast to GLUT4, insulin does not mediate GLUT12 translocation, which may function as a basal GLUT located primarily at the cell surface in the myocardium.
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Diabetes Mellitus Tipo 1/metabolismo , Proteínas Facilitadoras del Transporte de la Glucosa/metabolismo , Glucosa/metabolismo , Insulina/metabolismo , Miocardio/metabolismo , Animales , Transporte Biológico , Diabetes Mellitus Tipo 1/genética , Proteínas Facilitadoras del Transporte de la Glucosa/genética , Transportador de Glucosa de Tipo 4/genética , Transportador de Glucosa de Tipo 4/metabolismo , Humanos , RatonesRESUMEN
Oral acetate supplementation enhances glycogen synthesis in some mammals. However, while acetate is a significant energy source for skeletal muscle at rest in horses, its effects on glycogen resynthesis are unknown. We hypothesized that administration of an oral sodium acetate-acetic acid solution with a typical grain and hay meal after glycogen-depleting exercise would result in a rapid appearance of acetate in blood with rapid uptake by skeletal muscle. It was further hypothesized that acetate taken up by muscle would be converted to acetyl CoA (and acetylcarnitine), which would be metabolized to CO2 and water via the tricarboxylic acid cycle, generating ATP within the mitochondria and thereby allowing glucose taken up by muscle to be preferentially incorporated into glycogen. Gluteus medius biopsies and jugular venous blood were sampled from nine exercise-conditioned horses on two separate occasions, at rest and for 24 h following a competition exercise test (CET) designed to simulate the speed and endurance test of a 3 day event. After the CETs, horses were allowed water ad libitum and either 8 l of a hypertonic sodium acetate-acetic acid solution via nasogastric gavage followed by a typical hay-grain meal (acetate treatment) or a hay-grain meal alone (control treatment). The CET significantly decreased muscle glycogen concentration by 21 and 17% in the acetate and control treatments, respectively. Acetate supplementation resulted in a rapid and sustained increase in plasma [acetate]. Skeletal muscle [acetyl CoA] and [acetylcarnitine] were increased at 4 h of recovery in the acetate treatment, suggesting substantial tissue extraction of the supplemented acetate. Acetate supplementation also resulted in an enhanced rate of muscle glycogen resynthesis during the initial 4 h of the recovery period compared with the control treatment; however, by 24 h of recovery there was no difference in glycogen replenishment between trials. It is concluded that oral acetate could be an alternative energy source in the horse.
Asunto(s)
Ácido Acético/farmacología , Glucógeno/biosíntesis , Músculo Esquelético/metabolismo , Condicionamiento Físico Animal/fisiología , Acetato de Sodio/farmacología , Acetatos/sangre , Acetilcoenzima A/metabolismo , Acetilcarnitina/metabolismo , Animales , Glucemia/metabolismo , Prueba de Esfuerzo/veterinaria , Femenino , Glucógeno/metabolismo , Caballos , Masculino , Acetato de Sodio/metabolismoRESUMEN
We hypothesized that postexercise rehydration using a hypotonic electrolyte solution will increase the rate of recovery of whole body hydration, and that this is associated with increased muscle glycogen and electrolyte recovery in horses. Gluteus medius biopsies and jugular venous blood were sampled from six exercise-conditioned Standardbreds on two separate occasions, at rest and for 24 h following a competitive exercise test (CET) designed to simulate the speed and endurance test of a 3-day event. After the CETs, horses were given water ad libitum, and either a hypotonic commercial electrolyte solution (electrolyte) via nasogastric tube, followed by a typical hay/grain meal, or a hay/grain meal alone (control). The CET resulted in decreased total body water and muscle glycogen concentration of 8.4 +/- 0.3 liters and 22.6%, respectively, in the control treatment, and 8.2 +/- 0.4 liters and 21.9% in the electrolyte treatment. Electrolyte resulted in an enhanced rate of muscle glycogen resynthesis and faster restoration of hydration (as evidenced by faster recovery of plasma protein concentration, maintenance of plasma osmolality, and greater muscle intracellular fluid volume) during the recovery period compared with control. There were no differences in muscle Na, K, Cl, or Mg contents between the two treatments. It is concluded that oral administration of a hypotonic electrolyte solution after prolonged moderate-intensity exercise enhanced the rate of muscle glycogen resynthesis during the recovery period compared with control. It is speculated that postexercise dehydration may be one key contributor to the slow muscle glycogen replenishment in horses.