Citations

Background: Scattered tubular-like cells (STCs) are dedifferentiated renal tubular cells endowed with progenitor-like characteristics to repair injured parenchymal cells. STCs may be damaged and rendered ineffective by renal artery stenosis (RAS), but the underlying processes remain unclear. We hypothesized that RAS alters the epigenetic landscape on DNA and the ensuing gene transcriptional profile of swine STCs. Methods: CD24+/CD133+ STCs were isolated from pig kidneys after 10 weeks of RAS or sham (n = 3 each) and their whole 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) profiles were examined by 5mC and 5hmC immunoprecipitation sequencing (MeDIP-/hMeDIP-seq, respectively). A subsequent integrated (MeDIP/hMeDIP-seq/mRNA-seq) analysis was performed by comparing all online available gene sets using Gene Set Enrichment Analysis. Apoptosis, proteolysis, and mitochondrial structure and function were subsequently evaluated in vitro. Results: Differential expression (DE) analysis revealed 239 genes with higher and 236 with lower 5mC levels and 275 genes with higher and 315 with lower 5hmC levels in RAS-STCs compared to Normal-STCs (fold change ≥1.4 or ≤0.7, p ≤ 0.05). Integrated MeDIP-/hMeDIP-seq/mRNA-seq analysis identified several overlapping (DE-5mC/mRNA and DE-5hmC/mRNA levels) genes primarily implicated in apoptosis, proteolysis, and mitochondrial functions. Furthermore, RAS-STCs exhibited decreased apoptosis, mitochondrial matrix density, and ATP production, and increased intracellular amino acid concentration and ubiquitin expression. Conclusions: Renal ischemia induces epigenetic changes in apoptosis-, proteolysis-, and mitochondria-related genes, which correlate with alterations in the transcriptomic profile and corresponding function of swine STCs. These observations may contribute to developing novel targeted interventions to preserve the reparative potency of STCs in renal disease.

Abstract BACKGROUND AND AIMS Patients with chronic kidney disease (CKD) have a higher risk to develop heart failure with preserved ejection fraction (HFpEF). We showed in a swine model of CKD with diastolic dysfunction and cardiac remodelling that these cardiac features are associated with blunted cardiac mRNA and protein expression of vascular endothelial growth factor (VEGF), a pro-angiogenic cytokine that plays important roles in cardiac vascular development, morphogenesis and contractility. Hence, we aim to identify potential underlying mechanisms of cardiac VEGF downregulation in CKD. METHOD CKD and normal pigs (n = 3 each) were studied for 14 weeks. Cardiac morphology and function (echocardiography) were quantified in vivo and microvascular density (3D micro-CT) ex vivo. Then, cardiac micro-RNA (miRNA) and 5-hydroxymethylcytosine (5-hmC) profiles of VEGFA gene were examined by hydroxymethylated DNA immunoprecipitation and miRNA sequencing (hMeDIP-seq and miRNA-seq, respectively) and validated by qPCR and Western blotting. RESULTS Blunted renal function in CKD pigs associated with left ventricular (LV) hypertrophy, and abnormal LV strain and diastolic dysfunction with pEF (Figure 1A) and reduced subendocardial microvascular density (Figure 1B) compared to normal. miRNA-seq showed that mir-183 was the only miRNA capable of targeting VEGFA and upregulated in CKD hearts (Figure 1C), accompanied by downregulation of VEGFA gene and VEGF protein (Figure 1D), whereas hMeDIP-seq revealed reduced VEGFA 5hmC levels in the hearts of CKD versus normal pigs (Figure 1E). CONCLUSION Our study suggests that miRNA-modulation and cardiac epigenetic changes in VEGF might contribute to cardiac damage and the development of HF in CKD. These observations may assist in developing novel therapies, like miRNA antagonists or epigenetic modulators, to preserve VEGF signalling and protect the heart in patients with CKD.

Abstract BACKGROUND AND AIMS Chronic kidney disease (CKD) is an independent risk factor for heart failure with preserved ejection fraction (HFpEF), a progressive condition that affects ˃50% of patients in dialysis. We developed a swine model of CKD-HFpEF and showed that renal inflammatory signaling plays a key role in HFpEF pathophysiology via cardiac activation of nuclear factor-kappa B (NF-kB). However, the mechanisms of CKD-induced cardiac NF-κB activation remain unclear. We hypothesized that micro-RNA (miRNA)-mediated processes contribute to cardiac NF-kB activation in CKD-HFpEF. METHOD Using an in-silico approach (miRWalk3.0 and TargetScan 7.2), we identified all miRNAs capable of targeting the NFKB Inhibitor Alpha (NFKBIA) gene. We then identified those expressed in swine myocardium and measured their levels in normal and CKD pigs by qPCR. In a biomimetic heart culture system that mimics the cardiac CKD-HFpEF microenvironment, pig heart slices were exposed to plasma from normal or CKD pigs, and cardiac kinetics, NF-kB signaling, and miRNA expression levels were investigated in vitro. RESULTS We identified 5 miRNAs expressed in swine hearts capable of targeting NFKBIA, of which only miR-1271–3p showed higher expression in the myocardium of CKD-HFpEF pigs compared to normal pigs. miR-1271–3p upregulation was mirrored in pig heart slices exposed to plasma from CKD pigs, associated with blunted cardiac contraction/relaxation kinetics, downregulation of NFKBIA, and increased protein expression of NF-κB (Figure). CONCLUSION Our data support a novel pathophysiological mechanism for cardiac activation of NF-κB in CKD via post-transcriptional modulation of NFKBIA partly through miR-1271–3p. This miRNA might represent a novel therapeutic target to ameliorate the deleterious effects that renal inflammatory signaling in CKD imposes on the heart and may lead to HFpEF.

Background Chronic kidney disease (CKD) carries a higher cardiovascular mortality and is a prominent risk factor for development of HFpEF. However, the pathophysiology of HFpEF in CKD has not been established. We have recently shown that experimental CKD‐HFpEF is associated with increased renal release of inflammatory cytokines, particularly tumor necrosis factor (TNF)‐α and interleukin (IL)‐6, to the systemic circulation. Using a unique physiological biomimetic heart culture system, we test whether inflammatory cytokines of kidney origin contribute to the development of HFpEF in CKD. Methods Pigs (4 each) were assigned to sham or CKD (bilateral renovascular disease and dyslipidemia) groups and followed for 14 weeks. Circulating levels of TNF‐α and IL‐6 were quantified (ELISA). In a biomimetic heart culture system, pig heart slices were exposed to plasma from normal or CKD pigs, or from normal pigs enriched with TNF‐α and IL‐6 (matching CKD concentration) with or without cytokine neutralizing antibodies (NA). Contractility and relaxation kinetics in vitro were determined in a dose/time dependent manner. Results CKD‐HFpEF pigs exhibited elevated systemic levels of TNF‐α and IL‐6 (Figure 1A), associated with renal dysfunction, cardiac hypertrophy and fibrosis, and diastolic dysfunction. Plasma was collected from all animals. Then, pig heart slices were exposed to CKD‐HFpEF, normal, or normal plasma enriched with cytokines, showing impaired contractility and speed of relaxation that improved after TNF‐α and IL‐6 neutralization (Figure 1B). Conclusions Our observations implicate circulating inflammatory mediators released by dysfunctional kidneys in imposing damage to the remote myocardium, supporting a role of inflammatory cytokines of renal origin in renal‐cardio pathophysiology in CKD‐HFpEF.

Background Chronic kidney disease (CKD) is independently associated with incident heart failure with preserved ejection fraction (HFpEF), but the underlying mechanisms remain unknown. We recently developed a translational swine model of CKD‐HF that replicates many features of human HFpEF. This study tested the hypothesis that CKD‐induced HFpEF associates with increased pro‐inflammatory cytokine signaling of renal origin. Methods CKD and normal pigs (n=4 each) were studied for 14 weeks. Renal hemodynamics (multi‐detector CT) and cardiac morphology and function (echocardiography) were quantified in vivo . Tumor necrosis factor (TNF)‐ α and interleukin (IL)‐6 levels (ELISA) were measured in circulating, renal vein, and coronary sinus blood, and their renal and cardiac gradients quantified. Systemic TNF‐α and IL‐6 levels (Luminex) were also measured in patients with CKD and age/gender‐matched healthy volunteers (n=12 each) and correlated with biomarkers of heart failure. Results Pigs with CKD developed hypertension, renal failure (stage 3), left ventricular (LV) hypertrophy, and abnormal LV strain and diastolic dysfunction (E/A, E/e’ ratio) with pEF, accompanied by positive renal (renal release) and negative cardiac (cardiac retention) gradients of TNF‐α and IL‐6 (Fig. 1A). Circulating TNF‐α and IL‐6 were also higher in patients with CKD compared to normal subjects (Fig. 1B), which correlated directly with ANP and NT‐proBNP levels (Fig. 1C). Conclusions Our experimental and clinical data supports a crosstalk between the kidney and the heart in CKD, suggesting that inflammatory signaling led by TNF‐α and IL‐6 of renal origin may impose cardiac abnormalities towards development of HFpEF.

Objective Hyperinsulinemia elicits an increase sympathetic nervous system activity directed toward skeletal muscle (muscle sympathetic nerve activity, MSNA). The insulin‐mediated increase in MSNA is primarily attributed to its effect within the central nervous system. In addition to direct central effects, insulin also elicits peripheral vasodilation, which may further increase MSNA via arterial baroreflex‐mediated mechanisms in order to maintain blood pressure (BP). Herein we examined the contribution of insulin‐induced peripheral vasodilation and role for the arterial baroreflex in the MSNA response to hyperinsulinemia. We hypothesized that rescuing peripheral resistance with co‐infusion of the vasoconstrictor phenylephrine would attenuate the MSNA response to hyperinsulinemia. We further hypothesized the insulin‐mediated increase in MSNA would be recapitulated with another vasodilator (sodium nitroprusside, SNP). Methods In 33 healthy adults (28M/5F, 28±1 yrs, BMI 25.0±0.5 kg/m 2 ) MSNA (microneurography) and BP (Finometer/brachial catheter) were measured, and total peripheral resistance (TPR, ModelFlow) was calculated at rest and during intravenous insulin (n=20) or SNP (n=13) infusion. A subset of participants receiving insulin (n=7) were co‐infused with phenylephrine. Spontaneous arterial baroreflex sensitivity was determined over 5‐min at baseline and during pharmacological intervention(s). Results Insulin infusion increased MSNA (p=0.05) and decreased TPR (p<0.05), with no effects on arterial baroreflex sensitivity or BP (p>0.05). Co‐infusion with phenylephrine returned MSNA and TPR to baseline, with no effect on baroreflex sensitivity (p>0.05). Similar to insulin, SNP increased MSNA (p<0.01) and decreased TPR (p<0.05), with no effect on baroreflex sensitivity (p>0.05). Conclusions Present findings reveal that acute hyperinsulinemia increases MSNA without a change in arterial baroreflex sensitivity in healthy men and women. Insulin‐mediated increases in MSNA are lost with co‐infusion of phenylephrine and can be recapitulated with infusion of SNP. Results support a dynamic interplay between central and peripheral mechanisms during hyperinsulinemia to increase MSNA and maintain BP.

Background Prior research has established some risk factors for an increased risk of severe disease and mortality from coronavirus disease 2019 (COVID-19). However, the impact of HIV infection on SARS-CoV-2 susceptibility and severity is a significant gap in the literature. In the same way, not many studies across the globe have analyzed the degree of vaccination willingness among people living with HIV/AIDS (PLWHA) and considerations regarding prioritizing this population during vaccination plans, particularly in developing countries. Methods A descriptive-analytical cross-sectional study was conducted. Self-completed electronic surveys directed to PLWHA were performed via Twitter in February 2021, using accounts of HIV activists. Results 460 (87.1%) participants were willing to be vaccinated with any COVID-19 vaccine. The reasons for that were listed as 1) the belief that vaccination prevents both the COVID-19 infection (81.3%) as well as being a spreader (52.2%); 2) having a high occupational risk of becoming infected with COVID-19 (22%); and 3) the belief that they would be at high risk of death because of COVID-19 (21.3%). Only 56 (10.6%) participants expressed hesitancy toward vaccination, and 12 (2.2%) stated they did not want to get vaccinated. Conclusions Our results may support the prioritization of people living with HIV during the implementation of vaccination plans in developing countries. New strategies should be adopted to overcome the hesitancy and unwillingness toward the COVID-19 vaccination, especially in populations with risk factors for severe disease.

ABSTRACT Background Renal artery stenosis (RAS) is an important cause of chronic kidney disease and secondary hypertension. In animal models, renal ischemia leads to downregulation of growth factor expression and loss of intrarenal microcirculation. However, little is known about the sequelae of large-vessel occlusive disease on the microcirculation within human kidneys. Method This study included five patients who underwent nephrectomy due to renovascular occlusion and seven nonstenotic discarded donor kidneys (four deceased donors). Micro-computed tomography was performed to assess microvascular spatial densities and tortuosity, an index of microvascular immaturity. Renal protein expression, gene expression and histology were studied in vitro using immunoblotting, polymerase chain reaction and staining. Results RAS demonstrated a loss of medium-sized vessels (0.2–0.3 mm) compared with donor kidneys (P = 0.037) and increased microvascular tortuosity. RAS kidneys had greater protein expression of angiopoietin-1, hypoxia-inducible factor-1α and thrombospondin-1 but lower protein expression of vascular endothelial growth factor (VEGF) than donor kidneys. Renal fibrosis, loss of peritubular capillaries (PTCs) and pericyte detachment were greater in RAS, yet they had more newly formed PTCs than donor kidneys. Therefore, our study quantified significant microvascular remodeling in the poststenotic human kidney. RAS induced renal microvascular loss, vascular remodeling and fibrosis. Despite downregulated VEGF, stenotic kidneys upregulated compensatory angiogenic pathways related to angiopoietin-1. Conclusions These observations underscore the nature of human RAS as a microvascular disease distal to main vessel stenosis and support therapeutic strategies directly targeting the poststenotic kidney microcirculation in patients with RAS.

Introduction: The metabolic syndrome (MetS) leads to cardiac vascular injury, which may reflect in increased retention of endothelial progenitor cells (EPC). Coronary endothelial cell (EC) mitochondria partly regulate vascular function and structure. We hypothesized that chronic mitoprotection would preserve EC mitochondria and attenuate coronary vascular injury and dysfunction in swine MetS. Methods: Pigs were studied after 16 weeks of diet-induced MetS, MetS treated for the last 4 weeks with the mitochondria-targeted peptide elamipretide (ELAM, 0.1mg/kg SC q.d), and lean controls (n=6 each). Coronary artery and sinus blood samples were collected. EC mitochondrial density (electron microscopy), endothelial nitric oxide (eNOS) immunoreactivity (staining), myocardial microvascular density (3D micro-CT), and coronary endothelial function (organ bath) were assessed ex-vivo. The number and arteriovenous gradient of CD34+/KDR+ EPC was calculated by FACS (a negative gradient indicating EPC retention). Results: MetS and MetS+ELAM pigs developed similar MetS (obesity, hyperlipidemia, insulin resistance, and hypertension). EC mitochondrial density decreased in MetS compared to lean, but normalized in MetS+ELAM. ELAM also improved eNOS immunoreactivity, subendocardial microvascular density, and coronary endothelial function. ELAM-induced vasculoprotection was reflected in decreased coronary retention of EPCs. Conclusions: These observations underscore the role of mitochondria in regulating the cardiac circulation in experimental MetS, and the benefits of mitoprotection to preserve EC myocardial microvascular integrity.

Background: Atherosclerotic renal artery stenosis (ARAS) reduces renal blood flow (RBF) and amplifies regional kidney hypoxia, but their relationship to tissue inflammation and injury markers is poorly understood. We tested the hypothesis that renal injury, reflected by renal vein levels of neutrophil gelatinase associated lipocalin (NGAL) and inflammatory cytokines monocyte chemoattractant protein-1 (MCP-1) would be magnified with severe occlusive vascular disease in humans with ARAS. Methods: Inpatient studies performed in patients with ARAS (n=40, 71.3±11% occlusion by CT angiography) or essential hypertension (EH) (n=30), during fixed Na+ intake and ACE/ARB Rx. Single-kidney cortical and medullary perfusion and RBF were measured using multidetector CT, and glomerular filtration rate (GFR) by iothalamate clearance. Tissue deoxyhemoglobin levels (R2*) and fractional kidney hypoxia (% of axial area with R2*>30/s) were measured by BOLD-MRI at 3T. Results: Single kidney RBF, cortical and medullary perfusion were reduced in the post-stenotic kidney, as was GFR. Renal vein NGAL, MCP-1 and fractional hypoxia were higher in patients with ARAS than EH. NGAL and MCP-1 levels correlated directly with cortical R2* and fractional hypoxia (r = 0.52, P <0 .0001 and r = 0.4, P = 0.0006) and with the degree of artey stenosis (r = 0.37, P = 0.04 and r = 0.35, P = 0.05). GFR correlated inversely with NGAL (r= - 0.5, P <0.0001). Conclusions: Our results demonstrate that elevated renal venous markers of kidney inflammation (MCP-1) and injury (NGAL) in ARAS are correlated with the severity of tissue hypoxia as measured by BOLD MR, suggesting a possible link between kidney inflammation and hypoxia.