Eirin, Alfonso

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.

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.

Background: The expansion of adipose mass requires adipocyte precursor cells that originate from multipotent mesenchymal stem cells (MSC). The inflammatory milieu in obesity often involves high levels of tumor necrosis factor (TNF)-α in adipose tissue, but whether this affects MSC function remains unknown. We tested the hypothesis that microenvironmental changes during the evolution of obesity affect MSC function, which may in turn promote obesity. Methods: Domestic pigs were fed with an adipogenic (n=7) or normal (n=7) diet for 16 weeks. Abdominal adipose tissue biopsies were collected at 8, 12, and 16 weeks for histology and MSC culture. Adipose tissue inflammation and MSC levels were evaluated in situ using immunohistochemistry staining for TNF-α, CD34, and Von Willebrand factor (vWF). The adipogenic propensity of MSC was tested in vitro using appropriate differentiation media, with or without supplementation of TNF-α or anti-TNF-α antibody for 3 weeks. Results: Obese pigs had increased body weight and abdominal fat mass (by CT) compared with lean pigs. TNF-α expression in adipose tissue (Figure 1A) was upregulated in obese pigs at 12 and 16 weeks, accompanied by increased numbers of MSC (CD34 + /vWF - ) at 16 weeks. In vitro, MSC derived from obese adipose tissue at 16weeks showed enhanced adipogenesis, which was abolished by anti-TNF-α treatment. Similarly, in lean MSC TNF-α treatment enhanced adipogenesis (Figure 1B). Conclusion: Obesity-induced adipose tissue inflammation may stimulate MSC differentiation into adipocytes, which further increase the adipose tissue mass. Our results support development of anti-inflammatory strategies for obesity management.

Abstract Chronic kidney disease (CKD) is a public health concern that affects over 200 million people worldwide and is associated with a tremendous economic burden. Therefore, deciphering the mechanisms underpinning CKD is crucial to decelerate its progression towards end-stage renal disease (ESRD). Renal tubular cells are populated with a high number of mitochondria, which produce cellular energy and modulate several important cellular processes, including generation of reactive oxygen species (ROS), calcium homeostasis, proliferation, and apoptosis. Over the past few years, increasing evidence has implicated renal mitochondrial damage in the pathogenesis of common etiologies of CKD, such as diabetes, hypertension, metabolic syndrome (MetS), chronic renal ischemia, and polycystic kidney disease (PKD). However, most compelling evidence is based on preclinical studies because renal biopsies are not routinely performed in many patients with CKD. Previous studies have shown that urinary mitochondrial DNA (mtDNA) copy numbers may serve as non-invasive biomarkers of renal mitochondrial dysfunction. Emerging data also suggest that CKD is associated with altered expression of mitochondria-related microRNAs (mitomiRs), which localize in mitochondria and regulate the expression of mtDNA and nucleus-encoded mitochondrial genes. This review summarizes relevant evidence regarding the involvement of renal mitochondrial injury and dysfunction in frequent forms of CKD. We further provide an overview of non-invasive biomarkers and potential mechanisms of renal mitochondrial damage, especially focusing on mtDNA and mitomiRs.

Abstract Renovascular disease (RVD) remains a common etiology of secondary hypertension. Recent clinical trials revealed unsatisfactory therapeutic outcomes of renal revascularization, leading to extensive investigation to unravel key pathophysiological mechanisms underlying irreversible functional loss and structural damage in the chronically ischemic kidney. Research studies identified complex interactions among various players, including inflammation, fibrosis, mitochondrial injury, cellular senescence, and microvascular remodeling. This interplay resulted in a shift of our understanding of RVD from a mere hemodynamic disorder to a pro-inflammatory and pro-fibrotic pathology strongly influenced by systemic diseases like metabolic syndrome (MetS), hypertension, diabetes mellitus, and hyperlipidemia. Novel diagnostic approaches have been tested for early detection and follow-up of RVD progression, using new imaging techniques and biochemical markers of renal injury and dysfunction. Therapies targeting some of the pathological pathways governing the development of RVD have shown promising results in animal models, and a few have moved from bench to clinical research. This review summarizes evolving understanding in chronic ischemic kidney injury.

Chronic kidney disease (CKD) is a progressive disorder with high cardiovascular morbidity and mortality. This work supports the role of inflammatory cytokines of renal origin in renal-cardio pathophysiology in CKD and that the heart may be a target. Furthermore, it supports the feasibility of a new strategy in a translational fashion, using targeted inhibition of renal NF-κB signaling to offset the development of cardiac injury in CKD.

Introduction: Morbidity and mortality attributable to hypertension are higher in African American (AAEH) compared to Caucasian essential hypertensive (EH) patients, possibly related to a differential effect on vascular injury and repair. While circulating endothelial progenitor cells (EPC) preserve endothelial integrity IEC detach from sites of injury and represent markers of vascular damage. We hypothesized that plasma levels of IEC and inflammatory markers would be higher in AAEH compared to EH patients. METHODS: Inferior vena cava levels of CD34+/KDR+ (EPC) and VAP-1+ (IEC) cells were measured by FACS in EH and AAEH under fixed sodium intake (150 mEq/d) and blockade of the renin-angiotensin-system, and compared to systemic levels in normotensive control subjects (n=19 each). Systemic levels of inflammatory cytokines and EPC homing factors were measured by Luminex. Results: Blood pressure, serum creatinine, lipids, antihypertensive medications, and EPC levels did not differ between EH and AAEH patients. Circulating IEC were higher in AAEH, and inversely correlated with EPC levels (Figure). Systemic levels of inflammatory cytokines and EPC homing factors were higher in AAEH compared to EH patients (Table), and correlated directly with IEC. Conclusion: Despite preserved kidney function and controlled BP, circulating inflammatory markers were elevated in AAEH and correlated with increased IEC and decreased EPC levels. Increased release of cytokines and IEC in AAEH may impair EPC reparative capacity and predispose to hypertensive vascular injury. This process may aggravate vascular damage and accelerate hypertension-related morbidity/mortality rates in AAEH.