Nox4 is involved in acute kidney injury associated to intravascular hemolysis.

Massive intravascular hemolysis occurs not unfrequently in many clinical conditions. Breakdown of erythrocytes promotes the accumulation of heme-derivates in the kidney, increasing oxidative stress and cell death, thus promoting acute kidney injury (AKI). NADPH oxidase 4 (Nox4) is a major source of reactive oxygen species (ROS) in the kidney, however it is unknown the role of Nox4 in hemolysis and whether inhibition of this enzyme may protect from heme-mediated injury. To answer these questions, we elicited intravascular hemolysis in wild type and Nox4 knockout mice. We also evaluated whether nephrotoxic effects of heme may be reduced by using Nox4 siRNA and pharmacologic inhibition with GKT137831, a Nox4 inhibitor, both in vivo and in cultured renal cells. Our results showed that induction of massive hemolysis elicited AKI characterized by loss of renal function, morphological alterations of the tubular epithelium and podocytes, oxidative stress, inflammation, mitochondrial dysfunction, blockade of autophagy and cell death. These pathological effects were significantly prevented in Nox4-deficient mice and in animals treated with GKT137831. In vitro studies showed that Nox4 disruption by specific siRNAs or Nox4 inhibitors declined heme-mediated ROS production and cell death. Our data identify Nox4 as a key enzyme involved in intravascular hemolysis-induced AKI. Thus, Nox4 inhibition may be a potential therapeutic approach to prevent renal damage in patients with severe hemolytic crisis.

Elevated plasma levels of TNF-R1 predict the development of acute ischemic events in coronary patients with diabetes. / Los niveles plasmáticos elevados de TNF-R1 predicen el desarrollo de eventos isquémicos agudos en pacientes coronarios con diabetes.

OBJECTIVES: To examine the relationship between inflammatory biomarkers and the occurrence of cardiovascular events in patients with type 2 diabetes mellitus (DM2) and stable coronary artery disease. METHODS: A total of 964 patients with stable coronary artery disease were included. Plasma levels of inflammatory markers, including tumour necrosis factor receptors 1 and 2 (TNF-R1 and TNF-R2), growth differentiation factor-15 (GDF-15), soluble suppression of tumorigenicity 2 (sST2), and high-sensitivity C-reactive protein (hsCRP) were measured. The primary endpoint was the development of acute ischaemic events (any type of acute coronary syndrome, stroke, or transient ischaemic attack). RESULTS: There were 232 diabetic patients and 732 non-diabetic patients. Patients with coronary artery disease and DM2 (232, 24%) had higher levels of TNF-R1, TNF-R2, GDF-15, sST2 (P<.001), and hsCRP compared to patients without DM2, indicating a higher inflammatory state. After a median follow-up of 5.39 (2.81-6.92) years, patients with DM2 more frequently developed the primary endpoint (15.9% vs 10.8%; P=.035). Plasma levels of TNF-R1 were independent predictors of the primary endpoint in patients with DM2, along with male gender, triglyceride levels, and the absence of treatment with angiotensin-converting enzyme inhibitors. None of these inflammatory markers predicted the development of this event in non-diabetic patients. CONCLUSIONS: Patients with stable coronary artery disease and DM2 exhibit elevated levels of the proinflammatory markers TNF-R1, TNF-R2, GDF-15, and sST2. Moreover, TNF-R1 is an independent predictor of acute ischaemic events only in diabetic patients.

Cardiac Rehabilitation Increases Plasma Klotho Levels.

Background: Mineral metabolism (MM), mainly fibroblast growth factor-23 (FGF-23) and klotho, has been linked to cardiovascular (CV) diseases. Cardiac rehabilitation (CR) has been demonstrated to reduce CV events, although its potential relationship with changes in MM is unknown. Methods: We performed a prospective, observational, case-control study, with acute coronary syndrome (ACS) patients who underwent CR and control patients (matched by age, gender, left ventricular ejection fraction, diabetes, and coronary artery bypass grafting), who did not. The inclusion dates were from August 2013 to November 2017 in CR group and from July 2006 to June 2014 in control group. Clinical, biochemical, and MM biomarkers were collected at discharge and six months later. Our objective was to evaluate differences in the modification pattern of MM in both groups. Results: We included 58 CR patients and 116 controls. The control group showed a higher prevalence of hypertension (50.9% vs. 34.5%), ST-elevated myocardial infarction (59.5% vs. 29.3%), and treatment with angiotensin-converting enzyme inhibitors (100% vs. 69%). P2Y12 inhibitors and beta-blockers were more frequently prescribed in the CR group (83.6% vs. 96.6% and 82.8% vs. 94.8%, respectively). After six months, klotho levels increased in CR patients whereas they were reduced in controls (+63 vs. -49 pg/mL; p < 0.001). FGF-23 was unchanged in the CR group and reduced in controls (+0.2 vs. -17.3 RU/dL; p < 0.003). After multivariate analysis, only the change in klotho levels was significantly different between groups (+124 pg/mL favoring CR group; IC 95% [+44 to +205]; p = 0.003). Conclusions: In our study, CR after ACS increases plasma klotho levels without significant changes in other components of MM. Further studies are needed to clarify whether this effect has a causal role in the clinical benefit of CR.

Semaglutide Improves Liver Steatosis and De Novo Lipogenesis Markers in Obese and Type-2-Diabetic Mice with Metabolic-Dysfunction-Associated Steatotic Liver Disease.

Metabolic-dysfunction-associated steatotic liver disease (MASLD) is a prevalent clinical condition associated with elevated morbidity and mortality rates. Patients with MASLD treated with semaglutide, a glucagon-like peptide-1 receptor agonist, demonstrate improvement in terms of liver damage. However, the mechanisms underlaying this beneficial effect are not yet fully elucidated. We investigated the efficacy of semaglutide in halting MASLD progression using a genetic mouse model of diabesity. Leptin-receptor-deficient mice with obesity and diabetes (BKS db/db) were either untreated or administered with semaglutide for 11 weeks. Changes in food and water intake, body weight and glycemia were monitored throughout the study. Body fat composition was assessed by dual-energy X-ray absorptiometry. Upon sacrifice, serum biochemical parameters, liver morphology, lipidomic profile and liver-lipid-related pathways were evaluated. The semaglutide-treated mice exhibited lower levels of glycemia, body weight, serum markers of liver dysfunction and total and percentage of fat mass compared to untreated db/db mice without a significant reduction in food intake. Histologically, semaglutide reduced hepatic steatosis, hepatocellular ballooning and intrahepatic triglycerides. Furthermore, the treatment ameliorated the hepatic expression of de novo lipogenesis markers and modified lipid composition by increasing the amount of polyunsaturated fatty acids. The administration of semaglutide to leptin-receptor-deficient, hyperphagic and diabetic mice resulted in the amelioration of MASLD, likely independently of daily caloric intake, suggesting a direct effect of semaglutide on the liver through modulation of the lipid profile.

Fibroblast growth factor 23 independently predicts adverse outcomes after an acute coronary syndrome.

AIMS: Abnormalities of mineral metabolism (MM) have been related to cardiovascular disorders. There are no reports on the prognostic role of MM after an acute coronary syndrome (ACS). We aim to assess the prognostic role of MM after an ACS. METHODS AND RESULTS: Plasma levels of components of MM [fibroblast growth factor 23 (FGF23), calcidiol, parathormone, klotho, and phosphate], high-sensitivity C-reactive protein, and N-terminal-pro-brain natriuretic peptide were measured in 1190 patients at discharge from an ACS. The primary outcome was a combination of acute ischaemic events, heart failure (HF) and death. Secondary outcomes were the separate components of the primary outcome. Age was 61.7 ± 12.2 years, and 77.1% were men. Median follow-up was 5.44 (3.03-7.46) years. Two hundred and ninety-four patients developed the primary outcome. At multivariable analysis FGF23 (hazard ratio, HR 1.18 [1.08-1.29], P < 0.001), calcidiol (HR 0.86 [0.74-1.00], P = 0.046), previous coronary or cerebrovascular disease, and hypertension were independent predictors of the primary outcome. The predictive power of FGF23 was homogeneous across different subgroups of population. FGF23 (HR 1.45 [1.28-1.65], P < 0.001) and parathormone (HR 1.06 1.01-1.12]; P = 0.032) resulted as independent predictors of HF. FGF23 (HR 1.21 [1.07-1.37], P = 0.002) and calcidiol (HR 0.72 [0.54-0.97), P = 0.028) were independent predictors of death. No biomarker predicted acute ischaemic events. FGF23 predicted independently the primary outcome in patients with estimated glomerular filtration rate > 60 mL/min/1.73 m2 . CONCLUSIONS: FGF23 and other components of MM are independent predictors of HF and death after an ACS. This effect is homogeneous across different subgroups of population, and it is not limited to patients with chronic kidney disease.

Tweak/Fn14 system is involved in rhabdomyolysis-induced acute kidney injury.

BACKGROUND: Rhabdomyolysis is a severe clinical syndrome associated to acute kidney injury (AKI) and chronic kidney disease (CKD). TWEAK/Fn14 signaling axis regulates renal inflammation and tubular cell death. However, the functional role of TWEAK/Fn14 in rhabdomyolysis remains unknown. METHODS: Rhabdomyolysis was induced in wild-type, TWEAK- and Fn14-deficient mice or mice treated with TWEAK blocking antibody. Renal injury, inflammation, fibrosis and cell death were assessed. Additionally, we performed in vivo and in vitro studies to explore the possible signalling pathways involved in Fn14 regulation. FINDINGS: Fn14 renal expression was increased in mice with rhabdomyolysis, correlating with decline of renal function. Mechanistically, myoglobin (Mb) induced Fn14 expression via ERK and p38 pathway, whereas Nrf2 activation diminished Mb-mediated Fn14 upregulation in cultured renal cells. TWEAK or Fn14 genetic depletion ameliorated rhabdomyolysis-associated loss of renal function, histological damage, tubular cell death, inflammation, and expression of both tubular and endothelial injury markers. Deficiency of TWEAK or Fn14 also decreased long-term renal inflammation and fibrosis in mice with rhabdomyolysis. Finally, pharmacological treatment with a blocking TWEAK antibody diminished the expression of acute renal injury markers and cell death and lessened residual kidney fibrosis and chronic inflammation in rhabdomyolysis. INTERPRETATION: TWEAK/Fn14 axis participates in the pathogenesis of rhabdomyolysis-AKI and subsequent AKI-CKD transition. Blockade of this signaling pathway may represent a promising therapeutic strategy for reducing rhabdomyolysis-mediated renal injury. FUNDING: Spanish Ministry of Science and Innovation, ISCIII and Junta de Andalucía.

A mutual regulatory loop between miR-155 and SOCS1 influences renal inflammation and diabetic kidney disease.

Diabetic kidney disease (DKD) is a common microvascular complication of diabetes, a global health issue. Hyperglycemia, in concert with cytokines, activates the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway to induce inflammation and oxidative stress contributing to renal damage. There is evidence of microRNA-155 (miR-155) involvement in diabetes complications, but the underlying mechanisms are unclear. In this study, gain- and loss-of-function experiments were conducted to investigate the interplay between miR-155-5p and suppressor of cytokine signaling 1 (SOCS1) in the regulation of the JAK/STAT pathway during renal inflammation and DKD. In experimental models of mesangial injury and diabetes, miR-155-5p expression correlated inversely with SOCS1 and positively with albuminuria and expression levels of cytokines and prooxidant genes. In renal cells, miR-155-5p mimic downregulated SOCS1 and promoted STAT1/3 activation, cytokine expression, and cell proliferation and migration. Conversely, both miR-155-5p antagonism and SOCS1 overexpression protected cells from inflammation and hyperglycemia damage. In vivo, SOCS1 gene delivery decreased miR-155-5p and kidney injury in diabetic mice. Moreover, therapeutic inhibition of miR-155-5p suppressed STAT1/3 activation and alleviated albuminuria, mesangial damage, and renal expression of inflammatory and fibrotic genes. In conclusion, modulation of the miR-155/SOCS1 axis protects kidneys against diabetic damage, thus highlighting its potential as therapeutic target for DKD.

Intravascular hemolysis triggers NAFLD characterized by a deregulation of lipid metabolism and lipophagy blockade.

Intravascular hemolysis is a common feature of different clinical entities, including sickle cell disease and malaria. Chronic hemolytic disorders are associated with hepatic damage; however, it is unknown whether heme disturbs lipid metabolism and promotes liver steatosis, thereby favoring the progression to nonalcoholic fatty liver disease (NAFLD). Using an experimental model of acute intravascular hemolysis, we report here the presence of liver injury in association with microvesicular lipid droplet deposition. Hemolysis promoted serum hyperlipidemia and altered intrahepatic triglyceride fatty acid composition, with increments in oleic, palmitoleic, and palmitic acids. These findings were related to augmented expression of transporters involved in fatty acid uptake (CD36 and MSR1) and deregulation of LDL transport, as demonstrated by decreased levels of LDL receptor and increased PCSK9 expression. Hemolysis also upregulated hepatic enzymes associated with cholesterol biosynthesis (SREBP2, HMGC1, LCAT, SOAT1) and transcription factors regulating lipid metabolism (SREBP1). Increased LC3II/LC3I ratio and p62/SQSTM1 protein levels were reported in mice with intravascular hemolysis and hepatocytes stimulated with heme, indicating a blockade of lipophagy. In cultured hepatocytes, cell pretreatment with the autophagy inductor rapamycin diminished heme-mediated toxicity and accumulation of lipid droplets. In conclusion, intravascular hemolysis enhances liver damage by exacerbating lipid accumulation and blocking the lipophagy pathway, thereby promoting NAFLD. These new findings have a high translational potential as a novel NAFLD-promoting mechanism in individuals suffering from severe hemolysis episodes. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Potential Role of the mTORC1-PGC1α-PPARα Axis under Type-II Diabetes and Hypertension in the Human Heart.

Type-2 diabetes (T2DM) and arterial hypertension (HTN) are major risk factors for heart failure. Importantly, these pathologies could induce synergetic alterations in the heart, and the discovery of key common molecular signaling may suggest new targets for therapy. Intraoperative cardiac biopsies were obtained from patients with coronary heart disease and preserved systolic function, with or without HTN and/or T2DM, who underwent coronary artery bypass grafting (CABG). Control (n = 5), HTN (n = 7), and HTN + T2DM (n = 7) samples were analysed by proteomics and bioinformatics. Additionally, cultured rat cardiomyocytes were used for the analysis (protein level and activation, mRNA expression, and bioenergetic performance) of key molecular mediators under stimulation of main components of HTN and T2DM (high glucose and/or fatty acids and angiotensin-II). As results, in cardiac biopsies, we found significant alterations of 677 proteins and after filtering for non-cardiac factors, 529 and 41 were changed in HTN-T2DM and in HTN subjects, respectively, against the control. Interestingly, 81% of proteins in HTN-T2DM were distinct from HTN, while 95% from HTN were common with HTN-T2DM. In addition, 78 factors were differentially expressed in HTN-T2DM against HTN, predominantly downregulated proteins of mitochondrial respiration and lipid oxidation. Bioinformatic analyses suggested the implication of mTOR signaling and reduction of AMPK and PPARα activation, and regulation of PGC1α, fatty acid oxidation, and oxidative phosphorylation. In cultured cardiomyocytes, an excess of the palmitate activated mTORC1 complex and subsequent attenuation of PGC1α-PPARα transcription of ß-oxidation and mitochondrial electron chain factors affect mitochondrial/glycolytic ATP synthesis. Silencing of PGC1α further reduced total ATP and both mitochondrial and glycolytic ATP. Thus, the coexistence of HTN and T2DM induced higher alterations in cardiac proteins than HTN. HTN-T2DM subjects exhibited a marked downregulation of mitochondrial respiration and lipid metabolism and the mTORC1-PGC1α-PPARα axis might account as a target for therapeutical strategies.

Reduction in the Risk of Peripheral Neuropathy and Lower Decrease in Kidney Function with Metformin, Linagliptin or Their Fixed-Dose Combination Compared to Placebo in Prediabetes: A Randomized Controlled Trial.

OBJECTIVE: To compare the effect of glucose-lowering drugs on peripheral nerve and kidney function in prediabetes. METHODS: Multicenter, randomized, placebo-controlled trial in 658 adults with prediabetes treated for 1 year with metformin, linagliptin, their combination or placebo. Endpoints are small fiber peripheral neuropathy (SFPN) risk estimated by foot electrochemical skin conductance (FESC < 70 µSiemens) and estimated glomerular filtration rate (eGFR). RESULTS: Compared to the placebo, the proportion of SFPN was reduced by 25.1% (95% CI:16.3-33.9) with metformin alone, by 17.3% (95% CI 7.4-27.2) with linagliptin alone, and by 19.5% (95% CI 10.1-29.0) with the combination linagliptin/metformin (p < 0.0001 for all comparisons). eGFR remained +3.3 mL/min (95% CI: 0.38-6.22) higher with the combination linagliptin/metformin than with the placebo (p = 0.03). Fasting plasma glucose (FPG) decreased more with metformin monotherapy -0.3 mmol/L (95%CI: -0.48; 0.12, p = 0.0009) and with the combination metformin/linagliptin -0.2 mmol/L (95% CI: -0.37; -0.03) than with the placebo (p = 0.0219). Body weight (BW) decreased by -2.0 kg (95% CI: -5.65; -1.65, p = 0.0006) with metformin monotherapy, and by -1.9 kg (95% CI: -3.02; -0.97) with the combination metformin/linagliptin as compared to the placebo (p = 0.0002). CONCLUSIONS: in people with prediabetes, a 1 year treatment with metformin and linagliptin, combined or in monotherapy, was associated with a lower risk of SFPN, and with a lower decrease in eGFR, than treatment with placebo.

Absence of High Lipoprotein(a) Levels Is an Independent Predictor of Acute Myocardial Infarction without Coronary Lesions.

The pathophysiological mechanisms underlying Myocardial Infarction with Non-Obstructive Coronary Artery Disease (MINOCA) are still under debate. Lipoprotein (a) [Lp(a)] has proinflammatory and prothrombotic actions and has been involved in the pathogenesis of atherosclerosis. However, no previous studies have linked Lp(a) levels with the probability of developing MINOCA. Moreover, the relationship between MINOCA and the plasma levels of other proatherogenic and proinflammatory molecules such as Interleukin-18 (IL18) and proprotein convertase subtilisin/kexin type 9 (PCSK9) has not been studied. We conducted a prospective, multicenter study involving 1042 patients with acute myocardial infarction (AMI). Seventy-six patients had no significant coronary lesions. All patients underwent plasma analysis on admission. MINOCA patients were younger (57 (47-68) vs. 61 (52-72) years; p = 0.010), more frequently female (44.7% vs. 21.0%; p < 0.001), and had lower rates of diabetes and of Lp(a) > 60 mg/dL (9.2% vs. 19.8%; p = 0.037) than those with coronary lesions; moreover, High Density Lipoprotein cholesterol (HDL-c) levels were higher in MINOCA patients. The absence of Lp(a) > 60 mg/dL and of diabetes were independent predictors of MINOCA, as well as female sex, high HDL-c levels, and younger age. IL-18 and PCSK9 levels were not predictors of MINOCA. During a follow-up of 5.23 (2.89, 7.37) years, the independent predictors of the primary outcome (acute ischemic events or death) in the whole sample were Lp(a) > 60 mg/dL, older age, low estimated Glomerular Filtration rate (eGFR), hypertension, previous heart failure (HF), coronary artery bypass graft, use of insulin, and no therapy with acetylsalicylic acid. In conclusion, in AMI patients, the absence of high Lp(a) levels, as well high HDL-c levels, were independent predictors of the inexistence of coronary artery disease. High Lp (a) levels were also an independent predictor of ischemic events or death.

The Synthetic Flavonoid Hidrosmin Improves Endothelial Dysfunction and Atherosclerotic Lesions in Diabetic Mice.

In diabetes, chronic hyperglycemia, dyslipidemia, inflammation and oxidative stress contribute to the progression of macro/microvascular complications. Recently, benefits of the use of flavonoids in these conditions have been established. This study investigates, in two different mouse models of diabetes, the vasculoprotective effects of the synthetic flavonoid hidrosmin on endothelial dysfunction and atherogenesis. In a type 2 diabetes model of leptin-receptor-deficient (db/db) mice, orally administered hidrosmin (600 mg/kg/day) for 16 weeks markedly improved vascular function in aorta and mesenteric arteries without affecting vascular structural properties, as assessed by wire and pressure myography. In streptozotocin-induced type 1 diabetic apolipoprotein E-deficient mice, hidrosmin treatment for 7 weeks reduced atherosclerotic plaque size and lipid content; increased markers of plaque stability; and decreased markers of inflammation, senescence and oxidative stress in aorta. Hidrosmin showed cardiovascular safety, as neither functional nor structural abnormalities were noted in diabetic hearts. Ex vivo, hidrosmin induced vascular relaxation that was blocked by nitric oxide synthase (NOS) inhibition. In vitro, hidrosmin stimulated endothelial NOS activity and NO production and downregulated hyperglycemia-induced inflammatory and oxidant genes in vascular smooth muscle cells. Our results highlight hidrosmin as a potential add-on therapy in the treatment of macrovascular complications of diabetes.

Role of Toll-like receptor 4 in intravascular hemolysis-mediated injury.

Massive intravascular hemolysis is a common characteristic of several pathologies. It is associated with the release of large quantities of heme into the circulation, promoting injury in vulnerable organs, mainly kidney, liver, and spleen. Heme activates Toll-like receptor 4 (TLR4), a key regulator of the inflammatory response; however, the role of TLR4 in hemolysis and whether inhibition of this receptor may protect from heme-mediated injury are unknown. We induced intravascular hemolysis by injection of phenylhydrazine in wildtype and Tlr4-knockout mice. In this model, we analyzed physiological parameters, histological damage, inflammation and cell death in kidney, liver, and spleen. We also evaluated whether heme-mediated-inflammatory effects were prevented by TLR4 inhibition with the compound TAK-242, both in vivo and in vitro. Induction of massive hemolysis elicited acute kidney injury characterized by loss of renal function, morphological alterations of the tubular epithelium, cell death, and inflammation. These pathological effects were significantly ameliorated in the TLR4-deficient mice and in wildtype mice treated with TAK-242. In vitro studies showed that TAK-242 pretreatment reduced heme-mediated inflammation by inhibiting the TLR4/NF-κB (nuclear factor kappa B) axis. However, analysis in liver and spleen indicated that TLR4 deficiency did not protect against the toxic accumulation of heme in these organs. In conclusion, TLR4 is a key molecule involved in the renal inflammatory response triggered by massive intravascular hemolysis. TLR4 inhibition may be a potential therapeutic approach to prevent renal damage in patients suffering from hemolysis. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

NT-proBNP Levels Influence the Prognostic Value of Mineral Metabolism Biomarkers in Coronary Artery Disease.

Background. Mineral metabolism (MM) system and N-terminal pro-brain natriuretic peptide (NT-ProBNP) have been shown to add prognostic value in patients with stable coronary artery disease (SCAD). However, the influence of NT-ProBNP on the prognostic role of MM in patients with SCAD has not been shown yet. The objective of this study is to assess the influence of NT-ProBNP on the prognostic role of MM markers in patients with SCAD. Methods: We analyzed the prognostic value of MM markers (parathormone (PTH), klotho, phosphate, calcidiol (25-hydroxyvitamin D3), and fibroblast growth factor-23) in 964 patients with SCAD and NT-ProBNP > 125 pg/mL vs. patient with NT-ProBNP ≤ 125 pg/mL included in five hospitals in Spain. The main outcome was the combination of death, heart failure, and ischemic events (any acute coronary syndrome, ischemic stroke, or transient ischemic attack). Results: A total of 622 patients had NT-proBNP > 125 pg/mL and 342 patients had NT-ProBNP ≤ 125 pg/mL. The median follow-up was 5.1 years. In the group of NT-proBNP > 125 pg/mL, the patients were older, and there were more females and smokers than in the group of patients with normal NT-proBNP. Additionally, the proportion of patients with hypertension, atrial fibrillation, ejection fraction < 40%, cerebrovascular attack, or prior coronary artery bypass graft was higher in the high NT-proBNP group. In the high NT-proBNP patients, the predictors of poor prognosis were PTH (HR = 1.06 (1.01−1.10), p < 0.001) and NT-proBNP (HR = 1.02 (1.01−1.03), p = 0.011), along with age (HR = 1.039 (1.02−1.06), p < 0.001), prior coronary artery bypass graft (HR = 1.624 (1.02−2.59), p = 0.041), treatment with statins (HR = 0.32 (0.19−0.53), p < 0.001), insulin (HR = 2.49 (1.59−4.09), p < 0.001), angiotensin receptor blockers (HR = 1.73 (1.16−2.56), p = 0.007), nitrates (HR = 1.65 (1.10−2.45), p = 0.014), and proton pump inhibitors (HR = 2.75 (1.74−4.36), p < 0.001). In the NT-proBNP ≤ 125 pg/mL subgroup, poor prognosis predictors were plasma levels of non-high-density lipoprotein (non-HDL) cholesterol (HR = 1.01 (1.00−1.02), p = 0.014) and calcidiol (HR = 0.96 (0.92−0.99), p = 0.045), as well as treatment with verapamil (HR = 11.28 (2.54−50.00), p = 0.001), and dihydropyridines (HR = 3.16 (1.63−6.13), p = 0.001). Conclusion: In patients with SCAD and NT-ProBNP > 125 pg/mL, PTH and NT-ProBNP, which are markers related to ventricular damage, are predictors of poor outcome. In the subgroup of patients with NT-ProBNP ≤ 125 pgm/L, calcidiol and non-HDL cholesterol, which are more related to vascular damage, are the independent predictors of poor outcome. Then, in patients with SCAD, baseline NT-ProBNP may influence the type of biomarker that is effective in risk prediction.

Kidney microRNA Expression Pattern in Type 2 Diabetic Nephropathy in BTBR Ob/Ob Mice.

Diabetic nephropathy (DN) is the main leading cause of chronic kidney disease worldwide. Although remarkable therapeutic advances have been made during the last few years, there still exists a high residual risk of disease progression to end-stage renal failure. To further understand the pathogenesis of tissue injury in this disease, by means of the Next-Generation Sequencing, we have studied the microRNA (miRNA) differential expression pattern in kidneys of Black and Tan Brachyury (BTBR) ob/ob (leptin deficiency mutation) mouse. This experimental model of type 2 diabetes and obesity recapitulates the key histopathological features described in advanced human DN and therefore can provide potential useful translational information. The miRNA-seq analysis, performed in the renal cortex of 22-week-old BTBR ob/ob mice, pointed out a set of 99 miRNAs significantly increased compared to non-diabetic, non-obese control mice of the same age, whereas no miRNAs were significantly decreased. Among them, miR-802, miR-34a, miR-132, miR-101a, and mir-379 were the most upregulated ones in diabetic kidneys. The in silico prediction of potential targets for the 99 miRNAs highlighted inflammatory and immune processes, as the most relevant pathways, emphasizing the importance of inflammation in the pathogenesis of kidney damage associated to diabetes. Other identified top canonical pathways were adipogenesis (related with ectopic fatty accumulation), necroptosis (an inflammatory and regulated form of cell death), and epithelial-to-mesenchymal transition, the latter supporting the importance of tubular cell phenotype changes in the pathogenesis of DN. These findings could facilitate a better understanding of this complex disease and potentially open new avenues for the design of novel therapeutic approaches to DN.

Meta-Inflammation and De Novo Lipogenesis Markers Are Involved in Metabolic Associated Fatty Liver Disease Progression in BTBR ob/ob Mice.

Metabolic associated fatty liver disease (MAFLD) is a hepatic manifestation of metabolic syndrome and usually associated with obesity and diabetes. Our aim is to characterize the pathophysiological mechanism involved in MAFLD development in Black Tan and brachyuric (BTBR) insulin-resistant mice in combination with leptin deficiency (ob/ob). We studied liver morphology and biochemistry on our diabetic and obese mice model (BTBR ob/ob) as well as a diabetic non-obese control (BTBR + streptozotocin) and non-diabetic control mice (BTBR wild type) from 4-22 weeks. Lipid composition was assessed, and lipid related pathways were studied at transcriptional and protein level. Microvesicular steatosis was evident in BTBR ob/ob from week 6, progressing to macrovesicular in the following weeks. At 12th week, inflammatory clusters, activation of STAT3 and Nrf2 signaling pathways, and hepatocellular ballooning. At 22 weeks, the histopathological features previously observed were maintained and no signs of fibrosis were detected. Lipidomic analysis showed profiles associated with de novo lipogenesis (DNL). BTBR ob/ob mice develop MAFLD profile that resemble pathological features observed in humans, with overactivation of inflammatory response, oxidative stress and DNL signaling pathways. Therefore, BTBR ob/ob mouse is an excellent model for the study of the steatosis to steatohepatitis transition.

CCN2 (Cellular Communication Network Factor 2) Deletion Alters Vascular Integrity and Function Predisposing to Aneurysm Formation.

BACKGROUND: CCN2 (cellular communication network factor 2) is a matricellular protein involved in cell communication and microenvironmental signaling responses. CCN2 is known to be overexpressed in several cardiovascular diseases, but its role is not completely understood. METHODS: Here, CCN2 involvement in aortic wall homeostasis and response to vascular injury was investigated in inducible <i>Ccn2</i>-deficient mice, with induction of vascular damage by infusion of Ang II (angiotensin II; 15 days), which is known to upregulate CCN2 expression in the aorta. RESULTS: Ang II infusion in CCN2-silenced mice lead to 60% mortality within 10 days due to rapid development and rupture of aortic aneurysms, as evidenced by magnetic resonance imaging, echography, and histological examination. <i>Ccn2</i> deletion decreased systolic blood pressure and caused aortic structural and functional changes, including elastin layer disruption, smooth muscle cell alterations, augmented distensibility, and increased metalloproteinase activity, which were aggravated by Ang II administration. Gene ontology analysis of RNA sequencing data identified aldosterone biosynthesis as one of the most enriched terms in CCN2-deficient aortas. Consistently, treatment with the mineralocorticoid receptor antagonist spironolactone before and during Ang II infusion reduced aneurysm formation and mortality, underscoring the importance of the aldosterone pathway in Ang II-induced aorta pathology. CONCLUSIONS: CCN2 is critically involved in the functional and structural homeostasis of the aorta and in maintenance of its integrity under Ang II-induced stress, at least, in part, by disruption of the aldosterone pathway. Thus, this study opens new avenues to future studies in disorders associated to vascular pathologies.

Impact of renal function on the prognostic value of mineral metabolism in patients with chronic ischaemic heart disease patients with chronic ischaemic heart disease. / Impacto de la función renal en el valor pronóstico del metabolismo mineral en pacientes con cardiopatía isquémica crónica.

BACKGROUND: Parathormone (PTH) is a component of the Mineral Metabolism (MM) system that has been shown recently to add prognostic value in pts. with stable coronary artery disease (SCAD) and average renal function. However, the influence of renal function on the prognostic role of PTH in pts. with SCAD has not been shown yet. PURPOSE: To assess the influence of estimated glomerular filtration rate (eGFR) on the prognostic role of PTH and other MM markers in pts. with SCAD. METHODS: We analyzed the prognostic value of MM markers (PTH, klotho, phosphate, calcidiol [25-hydroxyvitamin D], and fibroblast growth factor-23 [FGF23]) in 964 pts. with SCAD and eGFR<60ml/min/1.73 m2 (LGFR) vs pts. with eGFR≥60ml/min/1.73 m2 (HGFR) included in five hospitals of Madrid. The main outcome was the combination of death with ischemic events (any acute coronary syndrome, ischemic stroke or transient ischemic attack). eGFR was calculated by the Chronic Kidney Disease Epidemiology Collaboration equation (CKD-EPI). RESULTS: Age was 60.0 (52.0-72.0) years, 76.2% of patients were men, and eGFR was 80.4 (65.3-93.1) ml/min/1,73 m2. Median follow-up was 5.39 (2.81-6.92) years. There were 790 pts. with HGFR and 174 with LGFR. In HGFR pts., predictors of ischemic events or death were plasma levels of calcidiol [HR=0.023 (0.94-0.99) p=0.023], FGF23 [HR=1.00 (1.00-1.003) p=0.036], non-HDL cholesterol [HR=1.01 (1.00-1.01) p=0.026] and high sensitivity troponin I [HR=5.12 (1.67-15.59) p=0.004], along with age [HR=1.03 (1.01-1.05) p=0.01], treatment with statins [HR=0.36 (0.19-0.68) p=0.002], nitrates [HR=1.13 (1.07-2.79) p=0.027], dihydropyridines [HR=1.71 (1.05-2.77) p=0.032], verapamil [HR=5.71 (1.35-24.1) p=0.018], and proton-pump inhibitors [HR=2.23 (1.36-3.68) p= 0.002]. In the LGFR subgroup, predictors of death or ischemic events were PTH plasma levels, [HR=1.01 (1.00-1.01) p=0.005], eGFR [HR=0.96 (0.94-0.99) p=0.004], age [HR=1.06 (1.02-1.10) p=0.003], caucasian race [HR=0.04 (0.004-0.380) p=0.005], and treatment with insulin [HR=2.6 (1.20-5.63) p=0.015]. CONCLUSIONS: In pts. with SCAD, PTH is an independent predictor of poor outcomes only in those with eGFR<60ml/min/1.73 m2, while in pts. with eGFR≥60ml/min/1.73 m2 calcidiol and FGF23 become the only components of MM that may predict prognosis. Then, renal function influences the predictive power of MM markers in pts. with SCAD.

Nephroprotective Effects of Synthetic Flavonoid Hidrosmin in Experimental Diabetic Nephropathy.

Diabetes mellitus (DM) is a high-impact disease commonly characterized by hyperglycemia, inflammation, and oxidative stress. Diabetic nephropathy (DN) is a common diabetic microvascular complication and the leading cause of chronic kidney disease worldwide. This study investigates the protective effects of the synthetic flavonoid hidrosmin (5-O-(beta-hydroxyethyl) diosmin) in experimental DN induced by streptozotocin injection in apolipoprotein E deficient mice. Oral administration of hidrosmin (300 mg/kg/day, n = 11) to diabetic mice for 7 weeks markedly reduced albuminuria (albumin-to-creatinine ratio: 47 ± 11% vs. control) and ameliorated renal pathological damage and expression of kidney injury markers. Kidneys of hidrosmin-treated mice exhibited lower content of macrophages and T cells, reduced expression of cytokines and chemokines, and attenuated inflammatory signaling pathways. Hidrosmin treatment improved the redox balance by reducing prooxidant enzymes and enhancing antioxidant genes, and also decreased senescence markers in diabetic kidneys. In vitro, hidrosmin dose-dependently reduced the expression of inflammatory and oxidative genes in tubuloepithelial cells exposed to either high-glucose or cytokines, with no evidence of cytotoxicity at effective concentrations. In conclusion, the synthetic flavonoid hidrosmin exerts a beneficial effect against DN by reducing inflammation, oxidative stress, and senescence pathways. Hidrosmin could have a potential role as a coadjutant therapy for the chronic complications of DM.

N-Terminal Pro-Brain Natriuretic Peptide Plasma Levels Are Associated with Intermediate-Term Follow-Up Cancer in Coronary Patients.

N-terminal pro-brain natriuretic peptide (NT-proBNP) plasma levels are increased in patients with cancer. In this paper, we test whether NT-proBNP may identify patients who are going to receive a future cancer diagnosis (CD) in the intermediate-term follow-up. We studied 962 patients with stable coronary artery disease and free of cancer and heart failure at baseline. This sample represents a re-analysis of a previous work expanding the sample size and the follow-up. NT-proBNP, galectin-3, monocyte chemoattractant protein-1, high-sensitivity C-reactive protein, high-sensitivity cardiac troponin I (hsTnI), and calcidiol (vitamin D) plasma levels were assessed. The primary outcome was new CD. After 5.40 (2.81-6.94) years of follow-up, 59 patients received a CD. NT-proBNP [HR 1.036 CI (1.015-1.056) per increase in 100 pg/mL; p = 0.001], previous atrial fibrillation (HR 3.140 CI (1.196-8.243); p = 0.020), and absence of previous heart failure (HR 0.067 CI (0.006-0.802); p = 0.033) were independent predictors of receiving a CD in the first three years of follow-up. None of the variables analyzed predicted a CD beyond this time. The number of patients developing heart failure during follow-up was 0 (0.0%) in patients receiving CD in the first three years of follow-up, 2 (6.9%) in those receiving a CD diagnosis beyond this time, and 40 (4.4%) in patients not developing cancer (p = 0.216). These numbers suggest that future heart failure was not a confounding factor. In patients with coronary artery disease, NT-proBNP was an independent predictor of CD in the first three years of follow-up but not later, suggesting that it could be detecting subclinical undiagnosed cancers.