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Mesenchymal stem/stromal cells (MSCs) facilitate repair in experimental diabetic kidney disease (DKD). However, the hyperglycemic and uremic milieu may diminish regenerative capacity of patient-derived therapy. We hypothesized that DKD reduces human MSC paracrine function. Adipose-derived MSC from 38 participants with DKD and 16 control subjects were assessed for cell surface markers, trilineage differentiation, RNA sequencing (RNA-seq), in vitro function (coculture or conditioned medium experiments with T cells and human kidney cells [HK-2]), secretome profile, and cellular senescence abundance. The direction of association between MSC function and patient characteristics were also tested. RNA-seq analysis identified 353 differentially expressed genes and downregulation of several immunomodulatory genes/pathways in DKD-MSC versus Control-MSC. DKD-MSC phenotype, differentiation, and tube formation capacity were preserved, but migration was reduced. DKD-MSC with and without interferon-γ priming inhibited T-cell proliferation greater than Control-MSC. DKD-MSC medium contained higher levels of anti-inflammatory cytokines (indoleamine 2,3-deoxygenase 1 and prostaglandin-E2) and prorepair factors (hepatocyte growth factor and stromal cell-derived factor 1) but lower IL-6 versus control-MSC medium. DKD-MSC medium protected high glucose plus transforming growth factor-β-exposed HK-2 cells by reducing apoptotic, fibrotic, and inflammatory marker expression. Few DKD-MSC functions were affected by patient characteristics, including age, sex, BMI, hemoglobin A1c, kidney function, and urine albumin excretion. However, senescence-associated β-galactosidase activity was lower in DKD-MSC from participants on metformin therapy. Therefore, while DKD altered the transcriptome and migratory function of culture-expanded MSCs, DKD-MSC functionality, trophic factor secretion, and immunomodulatory activities contributing to repair remained intact. These observations support testing of patient-derived MSC therapy and may inform preconditioning regimens in DKD clinical trials. [ABSTRACT FROM AUTHOR]

Obesity promotes dysfunction and impairs the reparative capacity of mesenchymal stem/stromal cells (MSCs), and alters their transcription, protein content, and paracrine function. Whether these adverse effects are mediated by chromatin-modifying epigenetic changes remains unclear. We tested the hypothesis that obesity imposes global DNA hydroxymethylation and histone tri-methylation alterations in obese swine abdominal adipose tissue-derived MSCs compared to lean pig MSCs. MSCs from female lean (n = 7) and high-fat-diet fed obese (n = 7) domestic pigs were assessed using global epigenetic assays, before and after in-vitro co-incubation with the epigenetic modulator vitamin-C (VIT-C) (50 μg/ml). Dot blotting was used to measure across the whole genome 5-hydroxyemthycytosine (5hmC) residues, and Western blotting to quantify in genomic histone-3 protein tri-methylated lysine-4 (H3K4me3), lysine-9 (H3K9me3), and lysine-27 (H3K27me3) residues. MSC migration and proliferation were studied in-vitro. Obese MSCs displayed reduced global 5hmC and H3K4m3 levels, but comparable H3K9me3 and H3K27me3, compared to lean MSCs. Global 5hmC, H3K4me3, and HK9me3 marks correlated with MSC migration and reduced proliferation, as well as clinical and metabolic characteristics of obesity. Co-incubation of obese MSCs with VIT-C enhanced 5hmC marks, and reduced their global levels of H3K9me3 and H3K27me3. Contrarily, VIT-C did not affect 5hmC, and decreased H3K4me3 in lean MSCs. Obesity induces global genomic epigenetic alterations in swine MSCs, involving primarily genomic transcriptional repression, which are associated with MSC function and clinical features of obesity. Some of these alterations might be reversible using the epigenetic modulator VIT-C, suggesting epigenetic modifications as therapeutic targets in obesity. [ABSTRACT FROM AUTHOR]

Percutaneous transluminal renal angioplasty (PTRA) has been used to treat renovascular disease (RVD), a chronic condition characterized by renal ischemia and metabolic abnormalities. Mitochondrial injury has been implicated as a central pathogenic mechanism in RVD, but whether it can be reversed by PTRA remains uncertain. We hypothesized that PTRA attenuates mitochondrial damage, renal injury, and dysfunction in pigs with coexisting renal artery stenosis (RAS) and metabolic syndrome (MetS). Four groups of pigs (n = 6 each) were studied after 16 weeks of diet‐induced MetS and RAS (MetS + RAS), MetS + RAS treated 4 weeks earlier with PTRA, and Lean and MetS Sham controls. Single‐kidney renal blood flow (RBF) and glomerular filtration rate (GFR) were assessed in vivo with multidetector computed tomography, and renal tubular mitochondrial structure and function and renal injury ex vivo. PTRA successfully restored renal artery patency, but mean arterial pressure remained unchanged. Stenotic kidney RBF and GFR, which fell in MetS + RAS compared to MetS, rose after PTRA. PTRA attenuated MetS + RAS‐induced mitochondrial structural abnormalities in tubular cells and peritubular capillary endothelial cells, decreased mitochondrial H202 production, and increased renal cytochrome‐c oxidase‐IV activity and ATP production. PTRA also improved cortical microvascular and peritubular capillary density and ameliorated tubular injury and tubulointerstitial fibrosis in the poststenotic kidney. Importantly, renal mitochondrial damage correlated with poststenotic injury and dysfunction. Renal revascularization attenuated mitochondrial injury and improved renal hemodynamics and function in swine poststenotic kidneys. This study suggests a novel mechanism by which PTRA might be relatively effective in ameliorating mitochondrial damage and improving renal function in coexisting MetS and RAS. [ABSTRACT FROM AUTHOR]

BACKGROUND Renovascular hypertension (RVH) often manifest with metabolic syndrome (MetS) as well. Coexisting MetS and hypertension increases cardiovascular morbidity and mortality, but the mechanisms underlying cardiac injury remain unknown. We hypothesized that superimposition of MetS induces myocardial mitochondrial damage, leading to cardiac injury and dysfunction in swine RVH. METHODS Pigs were studied after 16 weeks of diet-induced MetS with or without RVH (unilateral renal artery stenosis), and Lean controls (n = 6 each). Systolic and diastolic cardiac function were assessed by multidetector CT, and cardiac mitochondrial morphology (electron microscopy) and myocardial function in tissue and isolated mitochondria. RESULTS Body weight was similarly higher in MetS groups vs. Lean. RVH groups achieved significant stenosis and developed hypertension. Mitochondrial matrix density and adenosine triphosphate production were lower and H2O2 production higher in RVH groups vs. Lean and MetS. Lean + RVH (but not MetS + RVH) activated mitophagy, which was associated with decreased myocardial expression of mitophagy-related microRNAs. MetS groups exhibited higher numbers of intermitochondrial junctions, which could have prevented membrane depolarization/activation of mitophagy in MetS + RVH. Cardiac fibrosis, hypertrophy (increased left ventricular muscle mass), and diastolic function (decreased E/A ratio) were greater in MetS + RVH vs. Lean + RVH. CONCLUSIONS MetS+RVH induces myocardial mitochondrial damage and dysfunction. MetS + RVH failed to activate mitophagy, resulting in greater cardiac remodeling, fibrosis, and diastolic dysfunction. Mitochondrial injury and impaired mitophagy may constitute important mechanisms and therapeutic targets to ameliorate cardiac damage and dysfunction in patients with coexisting MetS and RVH. [ABSTRACT FROM AUTHOR]

Scattered tubular-like cells (STCs) are dedifferentiated surviving tubular epithelial cells that repair neighboring injured cells. Experimental renal artery stenosis (RAS) impairs STC reparative potency by inducing mitochondrial injury, but the exact mechanisms of mitochondrial damage remain unknown. We hypothesized that RAS alters expression of mitochondria-related genes, contributing to mitochondrial structural damage and dysfunction in swine STCs. CD24+/CD133+ STCs were isolated from pig kidneys after 10 wk of RAS or sham (n = 3 each). mRNA sequencing was performed, and nuclear DNA (nDNA)-encoded mitochondrial genes and mitochondrial DNA (mtDNA)-encoded genes were identified. Mitochondrial structure, ATP generation, biogenesis, and expression of mitochondria-associated microRNAs were also assessed. There were 96 nDNA-encoded mitochondrial genes upregulated and 12 mtDNA-encoded genes downregulated in RAS-STCs versus normal STCs. Functional analysis revealed that nDNA-encoded and mtDNA-encoded differentially expressed genes were primarily implicated in mitochondrial respiration and ATP synthesis. Mitochondria from RAS STCs were swollen and showed cristae remodeling and loss and decreased ATP production. Immunoreactivity of the mitochondrial biogenesis marker peroxisome proliferator-activated receptor- coactivator (PGC)-1 and expression of the mitochondria-associated microRNAs miR-15a, miR-181a, miR-196a, and miR-296-3p, which target several mtDNA genes, were higher in RAS-STCs compared with normal STCs, suggesting a potential modulation of mitochondria-related gene expression. These results demonstrate that RAS induces an imbalance in mtDNA- and nDNA-mitochondrial gene expression, impairing mitochondrial structure and function in swine STCs. These observations support development of gene gain- and loss-of-function strategies to ameliorate mitochondrial damage and preserve the reparative potency of STCs in patients with renal ischemia. [ABSTRACT FROM AUTHOR]

Aims: The mechanisms responsible for cardiac damage in the early stages of metabolic syndrome (MetS) remain unknown. Mitochondria are intimately associated with cellular myofibrils, with the cytoskeleton functioning as a linkage coordinator, and closely associated to the calcium release sites of the sarcoplasmic reticulum (SR). We hypothesized that early MetS is characterized by mitochondria-related myocardial damage, associated with altered cytoskeletal-mitochondria-SR interaction. Methods And Results: Domestic pigs were studied after 16 weeks of diet-induced MetS, MetS treated for the last 4 weeks with the mitochondrial-targeted peptide elamipretide (ELAM; 0.1 mg/kg SC q.d), or Lean controls (n = 6/group). Cardiac remodeling and function were assessed by fast comuted tomography. Myocardial mitochondrial structure, SR-mitochondria interaction, calcium handling, cytoskeletal proteins, oxidative stress, and apoptosis were studied ex-vivo. MetS pigs developed hyperlipidemia, hypertension, and insulin resistance, yet cardiac function was preserved. MetS-induced mitochondrial disorganization, decreased (C18:2)4 cardiolipin, disrupted ATP/ADP balance, and decreased cytochrome-c oxidase (COX)-IV activity. MetS also increased mitochondrial hydrogen peroxide (H2O2) production, decreased nicotinamide adenine dinucleotide phosphate (NADPH)/NADP and GSH/GSSG, and decreased myocardial desmin and β2 tubulin immunoreactivity, and impaired SR-mitochondrial interaction and mitochondrial calcium handling, eliciting myocardial oxidative stress and apoptosis. ELAM improved mitochondrial organization and cardiolipin species profile, restored ATP/ADP ratio and COX-IV activity, decreased H202 production, and improved generation of NADPH and GSH. ELAM also improved cytoskeletal-mitochondria-SR interaction and mitochondrial calcium handling, attenuating oxidative stress, and apoptosis. Conclusions: Disorganization of cardiomyocyte cytoskeletal-mitochondria-SR network is associated with cardiac reversible changes in early MetS, preceding overt cardiac dysfunction. These findings may introduce novel therapeutic targets for blunting cardiac damage in early MetS. [ABSTRACT FROM AUTHOR]

Background: An increased number of podocyte-derived extracellular vesicles (pEVs) may reflect podocyte injury in renal disease. Elevated glomerular pressure and other insults may injure podocytes, yet it remains unclear whether the numbers of pEVs are altered in hypertensive patients. We tested the hypothesis that urinary pEV levels would be elevated in patients with renovascular hypertension (RVH) compared with essential hypertension (EH) or healthy volunteers (HVs). Methods: We prospectively enrolled patients with EH (n = 30) or RVH (n = 31) to study renal blood flow (RBF) and cortical perfusion using multidetector computed tomography under controlled condition (regulated sodium intake and renin-- angiotensin blockade). After isolation fromurine samples, pEVs (nephrin and podocalyxin positive) were characterized by flow cytometry. Fourteen RVH patients were studied again 3 months after stenting or continued medical therapy. HVs (n = 15) served as controls. Results: The fraction of pEV among urinary EVs was elevated in RVH compared with HVs and EH (11.4± 6.4, 6.8 ± 3.4 and 6.3 ± 3.7%, respectively; P < 0.001) and remained unchanged after 3 additional months of therapy and after controlling for clinical parameters. However, eGFR- and age-adjusted pEV levels did not correlate with any clinical or renal parameters. Conclusions: In hypertensive patients under controlled conditions, urinary pEV levels are elevated in patients with RVH and low eGFR compared with patients with EH and relatively preserved renal function. These pEVs may reflect podocyte injury secondary to kidney damage, and their levels might represent a novel therapeutic target. [ABSTRACT FROM AUTHOR]

BACKGROUND: Left ventricular (LV) hypertrophy (LVH) plays an important role in hypertensive heart disease, and may be accompanied by myocardial autophagy. However, the pattern of autophagy during evolution of LVH is unclear. We hypothesized that autophagy activation indicates advancing cardiac LVH with tissue remodeling. METHODS: Ten domestic pigs with a 10-week unilateral renovascular hypertension (HTN) were classified as mild or moderate HTN (n = 5 each group) based on the degree of renal artery stenosis (above or below 75%). Seven normal pigs served as controls. Left ventricular remodeling, function, and microvascular density were assessed using multi-detector- and micro-computed tomography and histology. Markers of myocardial autophagic and endoplasmic reticulum (ER) stress-related unfolded protein response (UPR), apoptosis, and fibrosis were examined ex vivo. RESULTS: Both HTN groups had increased myocyte cross-sectional area, but it was greater in moderate HTN, accompanied by elevated LV muscle-mass. Moderate, but not mild HTN, also showed impaired microvascular density and impaired myocardial perfusion. Autophagy mediators were unaltered in mild HTN but UPR markers were increased, while in moderate HTN they were all upregulated, whereas UPR markers were suppressed. Myocardial apoptosis and fibrosis were also greater in moderate HTN. Autophagic proteins were correlated with LVH and fibrosis. CONCLUSIONS: Autophagic activity is stimulated during the exacerbation of LVH, following a transient early increase in ER stress, and may be involved in the progression of cardiac remodeling in renovascular hypertensive heart disease. [ABSTRACT FROM AUTHOR]

Objective To establish and characterize a novel domestic porcine model of obesity. Methods Fourteen domestic pigs were fed normal (lean, n = 7) or high-fat/high-fructose diet (obese, n = 7) for 16 weeks. Subcutaneous abdominal adipose tissue biopsies were obtained after 8, 12, and 16 weeks of diet, and pericardial adipose tissue after 16 weeks, for assessments of adipocyte size, fibrosis, and inflammation. Adipose tissue volume and cardiac function were studied with multidetector computed tomography, and oxygenation was studied with magnetic resonance imaging. Plasma lipids profile, insulin resistance, and markers of inflammation were evaluated. Results Compared with lean pigs, obese pigs had elevated cholesterol and triglyceride levels, blood pressure, and insulin resistance. Both abdominal and pericardial fat volume increased in obese pigs after 16 weeks. In abdominal subcutaneous adipose tissue, adipocyte size and both tumor necrosis factor (TNF)-α expression progressively increased. Macrophage infiltration showed in both abdominal and pericardial adipose tissues. Circulating TNF-α increased in obese pigs only at 16 weeks. Compared with lean pigs, obese pigs had similar global cardiac function, but myocardial perfusion and oxygenation were significantly impaired. Conclusions A high-fat/high-fructose diet induces in domestic pigs many characteristics of metabolic syndrome, which is useful for investigating the effects of the obesity. [ABSTRACT FROM AUTHOR]