Obesity Blunts the Effect of Mesenchymal Stem Cell-Derived Extracellular Vesicles.

Introduction: Mesenchymal stem/stromal cell-derived extracellular vesicles (MSC-EVs) are paracrine vectors with therapeutic functions comparable to their parent cells. However, it remains unclear if donor obesity affects their therapeutic functions. We tested the hypothesis that the curative effect of human adipose tissue-derived MSC-EVs (A-MSC-EVs) is blunted by obesity. Methods: MSC-EVs were isolated by ultracentrifugation from mesenchymal stem/stromal cells (MSCs) collected from abdominal subcutaneous fat of obese and lean human subjects (obese and lean-MSC-EVs, respectively) and injected into the aorta of mice 2 weeks after renal artery stenosis (RAS) induction. Magnetic resonance imaging studies were conducted 2 weeks after MSC-EVs delivery to determine renal function. The effect of MSC-EVs on tissue injury was assessed by histology and gene expression of inflammatory factors, including interleukin (IL)-1ß, IL-6, monocyte chemotactic protein-1 (MCP-1), and tumor necrosis factor alpha (TNF-α). Oxidative damage, macrophage infiltration, plasma renin, and hypoxia inducible factor-1α (HIF-1α) were also assessed. Results: Tracking showed that MSC-EVs localized in the kidney tissue, including glomeruli and tubules. All MSC-EVs decreased systolic blood pressure (SBP) and plasma renin and improved the poststenotic kidney (STK) volume, but obese-MSC-EVs were less effective than lean-MSC-EVs in improving medullary hypoxia, fibrosis, and tubular injury. Lean-MSC-EVs decreased inflammation, whereas obesity attenuated this effect. Only lean-MSC-EVs decreased STK cortical HIF-1α expression. Conclusion: Obesity attenuates the antihypoxia, antifibrosis, antiinflammation, and tubular repair functions of human MSC-EVs in chronic ischemic kidney disease. These observations may have implications for the self-repair potency of obese subjects and for the use of autologous MSC-EVs in regenerative medicine.

Renal ischemia alters the transcriptomic and epigenetic profile of inflammatory genes in swine scattered tubular-like cells.

BACKGROUND: Scattered tubular-like cells (STCs) are differentiated renal tubular cells that during recovery from ischemic injury dedifferentiate to repair other injured renal cells. Renal artery stenosis (RAS), often associated with chronic inflammatory injury, compromises the integrity and function of STCs, but the underlying mechanisms remain unknown. We hypothesized that RAS alters the transcriptomic and epigenetic profile of inflammatory genes in swine STCs. METHODS: STCs were harvested from pig kidneys after 10 weeks of RAS or sham (n=6 each). STC mRNA profiles of inflammatory genes were analyzed using high-throughput mRNA-sequencing (seq) and their DNA methylation (5mC) and hydroxymethylation (5hmC) profiles by DNA immunoprecipitation and next-generation sequencing (MeDIP-seq) (n=3 each), followed by an integrated (mRNA-seq/MeDIP-seq) analysis. STC protein expression of candidate differentially expressed (DE) genes and common proinflammatory proteins were subsequently assessed in vitro before and after epigenetic (Bobcat339) modulation. RESULTS: mRNA-seq identified 57 inflammatory genes up-regulated in RAS-STCs versus Normal-STCs (>1.4 or <0.7-fold, P<0.05), of which 14% exhibited lower 5mC and 5% higher 5hmC levels in RAS-STCs versus Normal-STCs, respectively. Inflammatory gene and protein expression was higher in RAS-STCs compared with Normal-STCs but normalized after epigenetic modulation. CONCLUSIONS: These observations highlight a novel modulatory mechanism of this renal endogenous repair system and support development of epigenetic or anti-inflammatory therapies to preserve the reparative capacity of STCs in individuals with RAS.

Obesity blunts amelioration of cardiac hypertrophy and fibrosis by human mesenchymal stem/stromal cell-derived extracellular vesicles.

Renovascular hypertension (RVH) can induce cardiac damage that is reversible using adipose tissue-derived mesenchymal stromal/stem cells (A-MSCs). However, A-MSCs isolated from patients with obesity are less effective than lean-A-MSC in blunting hypertensive cardiomyopathy in mice with RVH. We tested the hypothesis that this impairment extends to their obese A-MSC-extracellular vesicles (EVs) progeny. MSCs were harvested from the subcutaneous fat of obese and lean human subjects, and their EVs were collected and injected into the aorta of mice 2 wk after renal artery stenosis or sham surgery. Cardiac left ventricular (LV) function was studied with MRI 2 wk later, and myocardial tissue ex vivo. Blood pressure, LV myocardial wall thickness, mass, and fibrosis that were elevated in RVH mice were suppressed only by lean EVs. Hence, human A-MSC-derived lean EVs are more effective than obese EVs in blunting hypertensive cardiac injury in RVH mice. These observations highlight impaired paracrine repair potency of endogenous MSCs in patients with obesity.NEW & NOTEWORTHY Injection of A-MSC-derived EVs harvested from patients who are lean can resolve myocardial injury in mice with experimental renovascular hypertension more effectively than A-MSC-derived EVs from patients with obesity. These observations underscore and might have important ramifications for the self-healing capacity of patients with obesity and for the use of autologous EVs as a regenerative tool.

Metabolic Syndrome Induces Epigenetic Alterations in Mitochondria-Related Genes in Swine Mesenchymal Stem Cells.

Autologous mesenchymal stem/stromal cells (MSCs) have demonstrated important therapeutic effects in several diseases. Cardiovascular risk factors may impair MSC mitochondrial structure and function, but the underlying mechanisms remain unknown. We hypothesized that metabolic syndrome (MetS) induces epigenetic alterations in mitochondria-related genes in swine MSCs. Pigs were fed a Lean or MetS diet (n = 6 each) for 16 weeks. MSCs were collected from subcutaneous abdominal fat, and DNA hydroxymethylation (5 hmC) profiles of mitochondria-related genes (MitoCarta-2.0) were analyzed by hydroxymethylated DNA immunoprecipitation and next-generation sequencing (hMeDIP-seq) in Lean- and MetS-MSCs untreated or treated with the epigenetic modulator vitamin (Vit)-C (n = 3 each). Functional analysis of genes with differential 5 hmC regions was performed using DAVID6.8. Mitochondrial structure (electron microscopy), oxidative stress, and membrane potential were assessed. hMeDIP-seq identified 172 peaks (associated with 103 mitochondrial genes) with higher and 416 peaks (associated with 165 mitochondrial genes) with lower 5 hmC levels in MetS-MSCs versus Lean-MSCs (≥2-fold, p < 0.05). Genes with higher 5 hmC levels in MetS + MSCs were primarily implicated in fatty acid metabolism, whereas those with lower 5 hmC levels were associated with electron transport chain activity. Vit-C increased 5 hmC levels in mitochondrial antioxidant genes, improved mitochondrial structure and membrane potential, and decreased oxidative stress. MetS alters 5 hmC levels of mitochondria-related genes in swine MSCs. Vit-C modulated 5 hmC levels in these genes and preserved mitochondrial structure and function in MetS-MSCs. These observations may contribute to development of strategies to overcome the deleterious effects of MetS on MSCs.

Obesity and dyslipidemia are associated with partially reversible modifications to DNA hydroxymethylation of apoptosis- and senescence-related genes in swine adipose-derived mesenchymal stem/stromal cells.

BACKGROUND: Obesity dysregulates key biological processes underlying the functional homeostasis, fate decisions, and reparative potential of mesenchymal stem/stromal cells (MSCs). Mechanisms directing obesity-induced phenotypic alterations in MSCs remain unclear, but emerging drivers include dynamic modification of epigenetic marks, like 5-hydroxymethylcytosine (5hmC). We hypothesized that obesity and cardiovascular risk factors induce functionally relevant, locus-specific changes in 5hmC of swine adipose-derived MSCs and evaluated their reversibility using an epigenetic modulator, vitamin-C. METHODS: Female domestic pigs were fed a 16-week Lean or Obese diet (n = 6 each). MSCs were harvested from subcutaneous adipose tissue, and 5hmC profiles were examined through hydroxymethylated DNA immunoprecipitation sequencing (hMeDIP-seq) followed by an integrative (hMeDIP and mRNA sequencing) gene set enrichment analysis. For clinical context, we compared 5hmC profiles of adipose tissue-derived human MSCs harvested from patients with obesity and healthy controls. RESULTS: hMeDIP-seq revealed 467 hyper- (fold change ≥ 1.4; p-value ≤ 0.05) and 591 hypo- (fold change ≤ 0.7; p-value ≤ 0.05) hydroxymethylated loci in swine Obese- versus Lean-MSCs. Integrative hMeDIP-seq/mRNA-seq analysis identified overlapping dysregulated gene sets and discrete differentially hydroxymethylated loci with functions related to apoptosis, cell proliferation, and senescence. These 5hmC changes were associated with increased senescence in cultured MSCs (p16/CDKN2A immunoreactivity, senescence-associated ß-galactosidase [SA-ß-Gal] staining), were partly reversed in swine Obese-MSCs treated with vitamin-C, and shared common pathways with 5hmC changes in human Obese-MSCs. CONCLUSIONS: Obesity and dyslipidemia are associated with dysregulated DNA hydroxymethylation of apoptosis- and senescence-related genes in swine and human MSCs, potentially affecting cell vitality and regenerative functions. Vitamin-C may mediate reprogramming of this altered epigenomic landscape, providing a potential strategy to improve the success of autologous MSC transplantation in obese patients.

Effect of Hypoxia Preconditioning on the Regenerative Capacity of Adipose Tissue Derived Mesenchymal Stem Cells in a Model of Renal Artery Stenosis.

Atherosclerotic renal artery stenosis (ARAS) is associated with irreversible parenchymal renal disease and regenerative stem cell therapies may improve renal outcomes. Hypoxia preconditioning (HPC) may improve the regenerative functions of adipose tissue-derived mesenchymal stem cells (AMSC) by affecting DNA 5-hydroxymethylcytosine (5hmC) marks in angiogenic genes. Here, we investigated using a porcine ARAS model, whether growth of ARAS AMSCs in hypoxia (Hx) versus normoxia (Nx) would enhance renal tissue repair, and comprehensively analyze how HPC modifies DNA hydroxymethylation compared to untreated ARAS and healthy/normal pigs (n=5 each). ARAS pigs exhibited elevated serum cholesterol, serum creatinine and renal artery stenosis, with a concomitant decrease in renal blood flow (RBF) and increased blood pressure (BP) compared to healthy pigs. Renal artery injection of either autologous Nx or Hx AMSCs improved diastolic BP, reduced kidney tissue fibrosis, and inflammation (CD3+ T-cells) in ARAS pigs. In addition, renal medullary hypoxia significantly lowered with Nx but not Hx AMSC treatment. Mechanistically, levels of epigenetic 5hmC marks (which reflect gene activation) estimated using DNA immunoprecipitation technique were elevated in profibrotic and inflammatory genes in ARAS compared with normal AMSCs. HPC significantly reduced 5hmC levels in cholesterol biosynthesis and oxidative stress response pathways in ARAS AMSCs. Thus, autologous AMSCs improve key renovascular parameters and inflammation in ARAS pigs, with HPC mitigating pathological molecular effects on inflammatory and profibrotic genes which may play a role in augmenting regenerative capacity of AMSCs.

Autologous Extracellular Vesicles Attenuate Cardiac Injury in Experimental Atherosclerotic Renovascular Disease More Effectively Than Their Parent Mesenchymal Stem/Stromal Cells.

Atherosclerotic renovascular disease (RVD) leads to hypertension, chronic kidney disease (CKD), and heart disease. Intrarenal delivery of mesenchymal stem cells (MSCs) and MSC-derived extracellular vesicles (EVs) attenuate renal injury and suppress release of inflammatory cytokines in porcine RVD. We hypothesized that this strategy would also be useful for cardioprotection. Pigs with renovascular hypertension and metabolic syndrome were studied 4 weeks after treatment with a single intrarenal infusion of autologous MSCs, EVs, or vehicle. Cardiac structure and function were assessed in vivo, and myocardial remodeling and expression of the pro-fibrotic factor growth factor receptor-bound protein-2 (Grb2) were measured ex-vivo. Inflammatory cytokine levels were measured in the systemic circulation and myocardial tissue. Blood pressure was elevated in all RVD groups, but serum creatinine increased in RVD and decreased in both RVD + MSCs and RVD + EVs. RVD-induced diastolic dysfunction (lower E/A ratio) was normalized in both MSCs- and EVs- treated pigs. Intrarenal delivery of MSCs and EVs also attenuated RVD-induced myocardial fibrosis, collagen deposition, and Grb2 expression, yet EVs restored capillary density and inflammation more effectively than MSCs. These observations suggest that autologous EVs attenuate cardiac injury in experimental RVD more effectively than their parent MSCs.

IL-10 partly mediates the ability of MSC-derived extracellular vesicles to attenuate myocardial damage in experimental metabolic renovascular hypertension.

Extracellular vesicles (EVs) obtain properties of immunomodulation and tissue repair from their parental mesenchymal stem cells (MSCs), and upon delivery may be associated with fewer adverse events. EVs derived from adipose-tissue MSCs restored kidney function by attenuating kidney inflammation in a swine model of metabolic syndrome (MetS) and renal artery stenosis via anti-inflammatory pathways. EVs also ameliorated myocardial injury in renovascular hypertension (RVH) secondary to inflammation in cardiorenal disease, but the mechanisms regulating this effect are unknown. We hypothesize that the anti-inflammatory cytokine interleukin (IL)-10 mediates the reparative effects of EVs on cardiovascular complications in a preclinical swine model with coexisting MetS and RVH. Twenty-three pigs established as Lean controls or RVH models were observed for 16 weeks. At 12 weeks RVH subgroups received an intrarenal delivery of 1011 either wildtype (WT) EVs or EVs after IL-10 knockdown (KD) (RVH+WT-EVs or RVH+IL-10-KD-EVs, respectively). Cardiac and renal function were studied in-vivo and myocardial tissue injury in-vitro 4 weeks later. RVH pigs showed myocardial inflammation, fibrosis, and left ventricular diastolic dysfunction. WT-EVs attenuated these impairments, increased capillary density, and decreased myocardial inflammation in-vivo. In-vitro, co-incubation with IL-10-containing WT-EVs decreased activated T-cells proliferation and endothelial cells inflammation and promoted their migration. Contrarily, these cardioprotective effects were largely blunted using IL-10-KD-EVs. Thus, the anti-inflammatory and pro-angiogenic effects of EVs in RVH may be partly attributed to their cargo of anti-inflammatory IL-10. Early intervention of IL-10-containing EVs may be helpful to prevent cardiovascular complications of MetS concurrent with RVH.

Exogenous pericyte delivery protects the mouse kidney from chronic ischemic injury.

Pericytes are considered reparative mesenchymal stem cell-like cells, but their ability to ameliorate chronic ischemic kidney injury is unknown. We hypothesized that pericytes would exhibit renoprotective effects in murine renal artery stenosis (RAS). Porcine kidney-derived pericytes (5 × 105) or vehicle were injected into the carotid artery 2 wk after the induction of unilateral RAS in mice. The stenotic kidney glomerular filtration rate and tissue oxygenation were measured 2 wk later using magnetic resonance imaging. We subsequently compared kidney oxidative stress, inflammation, apoptosis, fibrosis, and systemic levels of oxidative and inflammatory cytokines. Treatment of xenogeneic pericytes ameliorated the RAS-induced loss of perfusion, glomerular filtration rate, and atrophy in stenotic kidneys and restored cortical and medullary oxygenation but did not blunt hypertension. Ex vivo, pericytes injection partially mitigated RAS-induced renal inflammation, fibrosis, oxidative stress, apoptosis, and senescence. Furthermore, coculture with pericytes in vitro protected pig kidney-1 tubular cells from injury. In conclusion, exogenous delivery of renal pericytes protects the poststenotic mouse kidney from ischemic injury, underscoring the therapeutic potential role of pericytes in subjects with ischemic kidney disease.NEW & NOTEWORTHY Our study demonstrates a novel pericyte-based therapy for the injured kidney. The beneficial effect of pericyte delivery appears to be mediated by ameliorating oxidative stress, inflammation, cellular apoptosis, and senescence in the stenotic kidney and improved tissue hypoxia, vascular loss, fibrosis, and tubular atrophy. Our data may form the basis for pericyte-based therapy, and additional research studies are needed to gain further insight into their role in improving renal function.

Effects of Elamipretide on Autophagy in Renal Cells of Pigs with Metabolic Syndrome.

Autophagy eliminates excessive nutrients and maintains homeostasis. Obesity and metabolic syndrome (MetS) dysregulate autophagy, possibly partly due to mitochondria injury and inflammation. Elamipretide (ELAM) improves mitochondrial function. We hypothesized that MetS blunts kidney autophagy, which ELAM would restore. Domestic pigs were fed a control or MetS-inducing diet for 16 weeks. During the 4 last weeks, MetS pigs received subcutaneous injections of ELAM (0.1 mg/kg/day, MetS + ELAM) or vehicle (MetS), and kidneys were then harvested to measure protein expression of autophagy mediators and apoptosis. Systemic and renal venous levels of inflammatory cytokines were measured to calculate renal release. The function of isolated mitochondria was assessed by oxidative stress, energy production, and pro-apoptotic activity. MetS slightly downregulated renal expression of autophagy mediators including p62, ATG5-12, mTOR, and AMPK vs. control. Increased mitochondrial H2O2 production accompanied decreased ATP production, elevated apoptosis, and renal fibrosis. In MetS + ELAM, mito-protection restored autophagic protein expression, improved mitochondrial energetics, and blunted renal cytokine release and fibrosis. In vitro, mitoprotection restored mitochondrial membrane potential and reduced oxidative stress in injured proximal tubular epithelial cells. Our study suggests that swine MetS mildly affects renal autophagy, possibly secondary to mitochondrial damage, and may contribute to kidney structural damage in MetS.

Renal Ischemia Induces Epigenetic Changes in Apoptotic, Proteolytic, and Mitochondrial Genes in Swine Scattered Tubular-like Cells.

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.

Microvascular remodeling and altered angiogenic signaling in human kidneys distal to occlusive atherosclerotic renal artery stenosis.

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.

Effects of obesity on reparative function of human adipose tissue-derived mesenchymal stem cells on ischemic murine kidneys.

INTRODUCTION: Obesity is a health burden that impairs cellular processes. Mesenchymal stem/stromal cells (MSCs) are endowed with reparative properties and can ameliorate renal injury. Obesity impairs human MSC function in-vitro, but its effect on their in-vivo reparative potency remains unknown. SUBJECTS AND METHODS: Abdominal adipose tissue-derived MSC were harvested from patients without ('lean') or with obesity ('obese') (body mass index <30 or ≥30 kg/m2, respectively) during kidney donation or bariatric surgery, respectively. MSC (5 × 105/200 µL) or vehicle were then injected into 129S1 mice 2 weeks after renal artery stenosis (RAS) or sham surgery (n = 8/group). Two weeks later, mice underwent magnetic resonance imaging to assess renal perfusion and oxygenation in-vivo, and kidneys then harvested for ex-vivo studies. RESULTS: Similar numbers of lean and obese-MSCs engrafted in stenotic mouse kidneys. Vehicle-treated RAS mice had reduced stenotic-kidney cortical and medullary perfusion and oxygenation. Lean (but not obese) MSC normalized ischemic kidney cortical perfusion, whereas both effectively mitigated renal hypoxia. Serum creatinine and blood pressure were elevated in RAS mice and lowered only by lean-MSC. Both types of MSCs alleviated stenotic-kidney fibrosis, but lean-MSC more effectively than obese-MSC. MSC senescence-associated beta-gal activity, and gene expression of p16, p21, and vascular endothelial growth factor correlated with recipient kidney perfusion and tissue injury, linking MSC characteristics with their in-vivo reparative capacity. DISCUSSION: Human obesity impairs the reparative properties of adipose-tissue-derived MSCs, possibly by inducing cellular senescence. Dysfunction and senescence of the endogenous MSC repair system in patients with obesity may warrant targeting interventions to restore MSC vitality.

Renal Revascularization Attenuates Myocardial Mitochondrial Damage and Improves Diastolic Function in Pigs with Metabolic Syndrome and Renovascular Hypertension.

Percutaneous transluminal renal angioplasty (PTRA) may improve cardiac function in renovascular hypertension (RVH), but its effect on the biological mechanisms implicated in cardiac damage remains unknown. We hypothesized that restoration of kidney function by PTRA ameliorates myocardial mitochondrial damage and preserves cardiac function in pigs with metabolic syndrome (MetS) and RVH. Pigs were studied after 16 weeks of MetS+RVH, MetS+RVH treated 4 weeks earlier with PTRA, and Lean and MetS Sham controls (n=6 each). Cardiac function was assessed by multi-detector CT, whereas cardiac mitochondrial morphology and function, microvascular remodeling, and injury pathways were assessed ex vivo. PTRA attenuated myocardial mitochondrial damage, improved capillary and microvascular maturity, and ameliorated oxidative stress and fibrosis, in association with attenuation of left ventricular remodeling and diastolic dysfunction. Myocardial mitochondrial damage correlated with myocardial injury and renal dysfunction. Preservation of myocardial mitochondria with PTRA can enhance cardiac recovery, underscoring its therapeutic potential in experimental MetS+RVH.

Superimposition of metabolic syndrome magnifies post-stenotic kidney injury in dyslipidemic pigs.

BACKGROUND: Dyslipidemia aggravates kidney injury distal to atherosclerotic renal artery stenosis (ARAS). Besides dyslipidemia, metabolic syndrome (MetS) also involves development of obesity and insulin-resistance (IR). We hypothesized that concurrent obesity and IR magnify swine stenotic-kidney damage beyond dyslipidemia. METHODS: Pigs with unilateral RAS were studied after 16 weeks of atherogenic diets without (ARAS) or with (MetS + RAS) development of obesity/IR (n=6 each). Additional pigs on normal diet served as normal or non-dyslipidemic RAS controls (n=6 each). Stenotic-kidney renal blood flow (RBF), glomerular filtration rate (GFR), and microvascular architecture were studied using CT, and oxygenation was studied using blood oxygen level-dependent magnetic-resonance-imaging. We further compared kidney adiposity, oxidative stress, inflammation, apoptosis, fibrosis, and systemic levels of oxidative and inflammatory cytokines. RESULTS: ARAS and MetS + RAS developed hypertension and dyslipidemia, and MetS + RAS also developed obesity and IR. RBF and GFR were similarly decreased in all post-stenotic pig kidneys compared to normal pig kidneys, yet MetS + RAS aggravated and expanded medullary hypoxia and microvascular loss. RAS and ARAS increased systemic levels of tumor necrosis factor (TNF)-α, which were further elevated in MetS + RAS. Renal oxidative stress and TNF-α expression increased in ARAS and further in MetS + RAS, which also upregulated expression of anti-angiogenic angiostatin, and magnified apoptosis, tubular injury, and fibrosis. CONCLUSION: Beyond dyslipidemia, obesity and insulin-resistance aggravate damage in the post-stenotic kidney in MetS, despite relative hyperfiltration-related preservation of renal function. These observations underscore the need to control systemic metabolic disturbances in order to curb renal damage in subjects with ischemic kidney disease.

Impaired immunomodulatory capacity in adipose tissue-derived mesenchymal stem/stromal cells isolated from obese patients.

Immune-modulatory properties of adipose tissue-derived mesenchymal stem/stromal cells (MSCs) might be susceptible to metabolic disturbances. We hypothesized that the immune-modulatory function of MSCs might be blunted in obese human subjects. MSCs were collected from abdominal subcutaneous fat of obese and lean subjects during bariatric or kidney donation surgeries, respectively. MSCs were co-cultured in vitro for 24 h with M1 macrophages, which were determined as M1or M2 phenotypes by flow cytometry, and cytokines measured in conditioned media. In vivo, lean or obese MSCs (5 × 105 ), or PBS, were injected into mice two weeks after unilateral renal artery stenosis (RAS) or sham surgeries (n = 6 each). Fourteen days later, kidneys were harvested and stained with M1 or M2 markers. Lean MSCs decreased macrophages M1 marker intensity, which remained elevated in macrophages co-cultured with obese MSCs. TNF-α levels were four-fold higher in conditioned media collected from obese than from lean MSCs. RAS mouse kidneys were shrunk and showed increased M1 macrophage numbers and inflammatory cytokine expression compared with normal kidneys. Lean MSCs decreased M1 macrophages, M1/M2 ratio and inflammation in RAS kidneys, whereas obese MSCs did not. MSCs isolated from lean human subjects decrease inflammatory M1 macrophages both in vivo and in vitro, an immune-modulatory function which is blunted in MSCs isolated from obese subjects.

Carotid Plaques From Symptomatic Patients Are Characterized by Local Increase in Xanthine Oxidase Expression.

Background and Purpose: XO (xanthine oxidase) is a key enzyme of uric acid metabolism and is thought to contribute to oxidative pathways that promote atherosclerotic plaque progression, yet its role in plaque destabilization is not well elucidated. We hypothesized that XO is expressed in carotid plaque from symptomatic patients in association with cardiovascular risk factors. Methods: Patients were stratified by symptoms, defined as presentation with an ipsilateral cerebral ischemic event. Carotid atherosclerotic plaques were obtained from 44 patients with symptomatic plaque and 44 patients without ischemic cerebral events. Protein expression of XO was evaluated by immunohistochemical staining and the percentage of cells expressing XO and CD68 (macrophage marker) compared between the groups. Biochemical and demographic cardiometabolic risk factors of study participants also were measured. Results: Carotid atherosclerotic plaques from symptomatic patients were associated with significantly higher XO expression versus asymptomatic plaque (median [interquartile range]: 1.24 [2.09] versus 0.16 [0.34]; P<0.001) and with significantly higher circulating uric acid levels (mean±SD: 7.36±2.10 versus 5.37±1.79 mg/dL; P<0.001, respectively). In addition, XO expression in atherosclerotic carotid plaque was inversely associated with serum high-density lipoproteins cholesterol levels (P=0.010, r=−0.30) and directly with circulating uric acid levels (P<0.001, r=0.45). The average percentage of macrophages that expressed XO was significantly higher in symptomatic versus asymptomatic plaques (median [interquartile range]: 93.37% [25] versus 46.15% [21], respectively; P<0.001). Conclusions: XO overexpression in macrophages is associated with increased serum uric acid and low high-density lipoproteins cholesterol levels and may potentially have a mechanistic role in carotid plaque destabilization. The current study supports a potential role for uric acid synthesis pathway as a target for management of carotid atherosclerosis in humans.

The Micro-RNA Cargo of Extracellular Vesicles Released by Human Adipose Tissue-Derived Mesenchymal Stem Cells Is Modified by Obesity.

Obesity is a chronic disease that interferes with normal repair processes, including adipose mesenchymal stem/stromal cells (ASCs) function. ASCs produce extracellular vesicles (EVs) that activate a repair program in recipient cells partly via their micro-RNA (miRNA) cargo. We hypothesized that obesity alters the miRNA expression profile of human ASC-derived EVs, limiting their capacity to repair injured cells. Human ASCs were harvested from obese and age- and gender-matched non-obese (lean) subjects during bariatric or cosmetic surgeries, respectively (n = 5 each), and their EVs isolated. Following high-throughput sequencing analysis, differentially expressed miRNAs were identified and their gene targets classified based on cellular component, molecular function, and biological process. The capacity of human lean- and obese-EVs to modulate inflammation, apoptosis, as well as mitogen-activated protein kinase (MAPK) and Wnt signaling in injured human proximal tubular epithelial (HK2) cells was evaluated in vitro. The number of EVs released from lean- and obese-ASCs was similar, but obese-EVs were smaller compared to lean-EVs. Differential expression analysis revealed 8 miRNAs upregulated (fold change > 1.4, p < 0.05) and 75 downregulated (fold change < 0.7, p < 0.05) in obese-EVs vs. lean-EVs. miRNAs upregulated in obese-EVs participate in regulation of NFk-B and MAPK signaling, cytoskeleton organization, and apoptosis, whereas those downregulated in obese-EVs are implicated in cell cycle, angiogenesis, and Wnt and MAPK signaling. Treatment of injured HK2 cells with obese-EVs failed to decrease inflammation, and they decreased apoptosis and MAPK signaling significantly less effectively than their lean counterparts. Obesity alters the size and miRNA cargo of human ASC-derived EVs, as well as their ability to modulate important injury pathways in recipient cells. These observations may guide development of novel strategies to improve healing and repair in obese individuals.

Diabetic Kidney Disease Alters the Transcriptome and Function of Human Adipose-Derived Mesenchymal Stromal Cells but Maintains Immunomodulatory and Paracrine Activities Important for Renal Repair.

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-ß-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 ß-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.