Altered immune cell phenotypes within chronically ischemic human kidneys distal to occlusive renal artery disease.

Renal artery stenosis (RAS) is a major cause of ischemic kidney disease, which is largely mediated by inflammation. Mapping the immune cell composition in ischemic kidneys might provide useful insight into the disease pathogenesis and uncover therapeutic targets. We used mass cytometry (CyTOF) to explore the single-cell composition in a unique data set of human kidneys nephrectomized due to chronic occlusive vascular disease (RAS, n = 3), relatively healthy donor kidneys (n = 6), and unaffected sections of kidneys with renal cell carcinoma (RCC, n = 3). Renal fibrosis and certain macrophage populations were also evaluated in renal sections. Cytobank analysis showed in RAS kidneys decreased cell populations expressing epithelial markers (CD45-/CD13+) and increased CD45+ inflammatory cells, whereas scattered tubular-progenitor-like cells (CD45-/CD133+/CD24+) increased compared with kidney donors. Macrophages switched to proinflammatory phenotypes in RAS, and the numbers of IL-10-producing dendritic cells (DC) were also lower. Compared with kidney donors, RAS kidneys had decreased overall DC populations but increased plasmacytoid DC. Furthermore, senescent active T cells (CD45+/CD28+/CD57+), aged neutrophils (CD45+/CD15+/CD24+/CD11c+), and regulatory B cells (CD45+/CD14-/CD24+/CD44+) were increased in RAS. RCC kidneys showed a distribution of cell phenotypes comparable with RAS but less pronounced, accompanied by an increase in CD34+, CD370+, CD103+, and CD11c+/CD103+ cells. Histologically, RAS kidneys showed significantly increased fibrosis and decreased CD163+/CD141+ cells. The single-cell platform CyTOF enables the detection of significant changes in renal cells, especially in subsets of immune cells in ischemic human kidneys. Endogenous pro-repair cell types in RAS warrant future study for potential immune therapy.NEW & NOTEWORTHY The single-cell platform mass cytometry (CyTOF) enables detection of significant changes in one million of renal cells, especially in subsets of immune cells in ischemic human kidneys distal to renal artery stenosis (RAS). We found that pro-repair cell types such as scattered tubular-progenitor-like cells, aged neutrophils, and regulatory B cells show a compensatory increase in RAS. Immune cell phenotype changes may reflect ongoing inflammation and impaired immune defense capability in the kidneys.

Clinical and pathological characteristics of acute kidney injury caused by diquat poisoning.

Introduction: Diquat poisoning leads to kidney injury, hepatotoxicity, rhabdomyolysis, gastrointestinal hemorrhage, and respiratory failure. Diquat has high mortality and no specific antidote. The pathology of acute kidney injury caused by diquat poisoning has been mainly investigated in animal studies and autopsies, and typically shows renal tubular necrosis. To our knowledge, antemortem renal biopsy has not been reported in humans.Case reports: Two males and one female presented following deliberate diquat self-poisoning. Their main clinical manifestations were abdominal pain, nausea, and emesis. All developed acute kidney injury. Kidney biopsy was performed in two cases which showed acute tubular necrosis with renal interstitial edema and multifocal inflammatory cell infiltration. Treatments given included gastric lavage, catharsis, early hemoperfusion combined with continuous kidney replacement therapy or hemodialysis, administration of glucocorticoids, and antioxidant therapy. All patients survived.Discussion: Despite potentially lethal ingestions three patients survived oral diquat poisoning with intensive supportive care. No clear relationship can be made between any of the therapies given and patient outcome.Conclusions: Kidney biopsy in these patients confirmed proximal renal tubular injury was the major pathological finding although interstitial injury was also present. The role of therapies that address renal pathology requires further study.

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.

Xenotransplantation: Current Challenges and Emerging Solutions.

To address the ongoing shortage of organs available for replacement, xenotransplantation of hearts, corneas, skin, and kidneys has been attempted. However, a major obstacle facing xenotransplants is rejection due to a cycle of immune reactions to the graft. Both adaptive and innate immune systems contribute to this cycle, in which natural killer cells, macrophages, and T-cells play a significant role. While advancements in the field of genetic editing can circumvent some of these obstacles, biomarkers to identify and predict xenograft rejection remain to be standardized. Several T-cell markers, such as CD3, CD4, and CD8, are useful in both the diagnosis and prediction of xenograft rejection. Furthermore, an increase in the levels of various circulating DNA markers and microRNAs is also predictive of xenograft rejection. In this review, we summarize recent findings on the advancements in xenotransplantation, with a focus on pig-to-human, the role of immunity in xenograft rejection, and its biomarkers.

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.

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.

Selective kidney targeting increases the efficacy of mesenchymal stromal/stem cells for alleviation of murine stenotic-kidney senescence and damage.

Chronic ischemia triggers senescence in renal tubules and at least partly mediates kidney dysfunction and damage through a p16Ink4a -related mechanism. We previously showed that mesenchymal stromal/stem cells (MSCs) delivered systemically do not effectively decrease cellular senescence in stenotic murine kidneys. We hypothesized that selective MSC targeting to injured kidneys using an anti-KIM1 antibody (KIM-MSC) coating would enhance their ability to abrogate cellular senescence in murine renal artery stenosis (RAS). KIM-MSC were injected into transgenic INK-ATTAC mice, which are amenable for selective eradication of p16Ink4a+ cells, 4 weeks after induction of unilateral RAS. To determine whether KIM-MSC abolish p16Ink4a -dependent cellular senescence, selective clearance of p16Ink4a+ cells was induced in a subgroup of RAS mice using AP20187 over 3 weeks prior to KIM-MSC injection. Two weeks after KIM-MSC aortic injection, renal senescence, function, and tissue damage were assessed. KIM-MSC delivery decreased gene expression of senescence and senescence-associated secretory phenotype factors, and improved micro-MRI-derived stenotic-kidney glomerular filtration rate and perfusion. Renal fibrosis and tubular injury also improved after KIM-MSC treatment. Yet, their efficacy was slightly augmented by prior elimination of p16Ink4a+ senescent cells. Therefore, selective targeting of MSC to the injured kidney markedly improves their senolytic potency in murine RAS, despite incomplete eradication of p16+ cells. KIM-MSC may constitute a useful therapeutic strategy in chronic renal ischemic injury.

Extracellular Vesicles as Theranostic Tools in Kidney Disease.

Extracellular vesicles are important vectors for cell-cell communication and show potential value for diagnosis and treatment of kidney diseases. The pathologic diagnosis of kidney diseases relies on kidney biopsy, whereas collection of extracellular vesicles from urine or circulating blood may constitute a less invasive diagnostic tool. In particular, urinary extracellular vesicles released mainly from resident kidney cells might provide an alternative tool for detection of kidney injury. Because extracellular vesicles mirror many features of their parent cells, cargoes of several populations of urinary extracellular vesicles are promising biomarkers for disease processes, like diabetic kidney disease, kidney transplant, and lupus nephritis. Contrarily, extracellular vesicles derived from reparative cells, such as mesenchymal stem cells, tubular epithelial progenitor cells, and human umbilical cord blood represent promising regenerative tools for treatment of kidney diseases. Furthermore, induced pluripotent stem cells-derived and engineered extracellular vesicles are being developed for specific applications for the kidney. Nevertheless, some assumptions regarding the specificity and immunogenicity of extracellular vesicles remain to be established. This review focuses on the utility of extracellular vesicles as therapeutic and diagnostic (theranostic) tools in kidney diseases and future directions for studies.

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.

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.

Progressive Cellular Senescence Mediates Renal Dysfunction in Ischemic Nephropathy.

BACKGROUND: Peripheral vascular diseases may induce chronic ischemia and cellular injury distal to the arterial obstruction. Cellular senescence involves proliferation arrest in response to stress, which can damage neighboring cells. Renal artery stenosis (RAS) induces stenotic-kidney dysfunction and injury, but whether these arise from cellular senescenceand their temporal pattern remain unknown. METHODS: Chronic renal ischemia was induced in transgenic INK-ATTAC and wild type C57BL/6 mice by unilateral RAS, and kidney function (in vivo micro-MRI) and tissue damage were assessed. Mouse healthy and stenotic kidneys were analyzed using unbiased single-cell RNA-sequencing. To demonstrate translational relevance, cellular senescence was studied in human stenotic kidneys. RESULTS: Using intraperitoneal AP20187 injections starting 1, 2, or 4 weeks after RAS, selective clearance of cells highly expressing p16Ink4a attenuated cellular senescence and improved stenotic-kidney function; however, starting treatment immediately after RAS induction was unsuccessful. Broader clearance of senescent cells, using the oral senolytic combination dasatinib and quercetin, in C57BL/6 RAS mice was more effective in clearing cells positive for p21 (Cdkn1a) and alleviating renal dysfunction and damage. Unbiased, single-cell RNA sequencing in freshly dissociated cells from healthy and stenotic mouse kidneys identified stenotic-kidney epithelial cells undergoing both mesenchymal transition and senescence. As in mice, injured human stenotic kidneys exhibited cellular senescence, suggesting this process is conserved. CONCLUSIONS: Maladaptive tubular cell senescence, involving upregulated p16 (Cdkn2a), p19 (Cdkn2d), and p21 (Cdkn1a) expression, is associated with renal dysfunction and injury in chronic ischemia. These findings support development of senolytic strategies to delay chronic ischemic renal injury.

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.

Mesenchymal Stem/Stromal Cell-Derived Extracellular Vesicles Elicit Better Preservation of the Intra-Renal Microvasculature Than Renal Revascularization in Pigs with Renovascular Disease.

BACKGROUND: Percutaneous transluminal renal angioplasty (PTRA) confers clinical and mortality benefits in select 'high-risk' patients with renovascular disease (RVD). Intra-renal-delivered extracellular vesicles (EVs) released from mesenchymal stem/stromal cells (MSCs) protect the kidney in experimental RVD, but have not been compared side-by-side to clinically applied interventions, such as PTRA. We hypothesized that MSC-derived EVs can comparably protect the post-stenotic kidney via direct tissue effects. METHODS: Five groups of pigs (n = 6 each) were studied after 16 weeks of RVD, RVD treated 4 weeks earlier with either PTRA or MSC-derived EVs, and normal controls. Single-kidney renal blood flow (RBF) and glomerular filtration rate (GFR) were assessed in vivo with multi-detector CT, and renal microvascular architecture (3D micro CT) and injury pathways ex vivo. RESULTS: Despite sustained hypertension, EVs conferred greater improvement of intra-renal microvascular and peritubular capillary density compared to PTRA, associated with attenuation of renal inflammation, oxidative stress, and tubulo-interstitial fibrosis. Nevertheless, stenotic kidney RBF and GFR similarly rose in both PTRA- and EV-treated pigs compared RVD + Sham. mRNA sequencing reveled that EVs were enriched with pro-angiogenic, anti-inflammatory, and antioxidants genes. CONCLUSION: MSC-derived EVs elicit a better preservation of the stenotic kidney microvasculature and greater attenuation of renal injury and fibrosis compared to PTRA, possibly partly attributed to their cargo of vasculo-protective genes. Yet, both strategies similarly improve renal hemodynamics and function. These observations shed light on diverse mechanisms implicated in improvement of post-stenotic kidney function and position EVs as a promising therapeutic intervention in RVD.