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.

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.

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 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.

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.

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.

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.

Increased cellular senescence in the murine and human stenotic kidney: Effect of mesenchymal stem cells.

Cell stress may give rise to insuperable growth arrest, which is defined as cellular senescence. Stenotic kidney (STK) ischemia and injury induced by renal artery stenosis (RAS) may be associated with cellular senescence. Mesenchymal stem cells (MSCs) decrease some forms of STK injury, but their ability to reverse senescence in RAS remains unknown. We hypothesized that RAS evokes STK senescence, which would be ameliorated by MSCs. Mice were studied after 4 weeks of RAS, RAS treated with adipose tissue-derived MSCs 2 weeks earlier, or sham. STK senescence-associated ß-galactosidase (SA-ß-Gal) activity was measured. Protein and gene expression was used to assess senescence and the senescence-associated secretory phenotype (SASP), and staining for renal fibrosis, inflammation, and capillary density. In addition, senescence was assessed as p16+ and p21+ urinary exosomes in patients with renovascular hypertension (RVH) without or 3 months after autologous adipose tissue-derived MSC delivery, and in healthy volunteers (HV). In RAS mice, STK SA-ß-Gal activity increased, and senescence and SASP marker expression was markedly elevated. MSCs improved renal function, fibrosis, inflammation, and capillary density, and attenuated SA-ß-Gal activity, but most senescence and SASP levels remained unchanged. Congruently, in human RVH, p21+ urinary exosomes were elevated compared to HV, and only slightly improved by MSC, whereas p16+ exosomes remained unchanged. Therefore, RAS triggers renal senescence in both mice and human subjects. MSCs decrease renal injury, but only partly mitigate renal senescence. These observations support exploration of targeted senolytic therapy in RAS.

Percutaneous transluminal renal angioplasty attenuates poststenotic kidney mitochondrial damage in pigs with renal artery stenosis and metabolic syndrome.

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 H2 02 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.

Mesenchymal stem cells protect renal tubular cells via TSG-6 regulating macrophage function and phenotype switching.

Tumor necrosis factor (TNF)-α-induced gene/protein (TSG)-6 regulates the immunomodulatory properties of mesenchymal stem cells (MSCs), but its ability to protect the ischemic kidney is unknown. In a swine model of renal artery stenosis (RAS) and metabolic syndrome (MetS), we assessed the contribution of TSG-6 produced by MSCs to their immunomodulatory properties. Pigs were studied after 16 wk of diet-induced MetS and unilateral RAS and were either untreated or treated 4 wk earlier with intrarenal autologous adipose tissue-derived MSCs (n = 6 each). Lean, MetS, and RAS sham animals served as controls. We studied renal function in vivo (using computed tomography) and kidney histopathology and macrophage phenotype ex vivo. In vitro, TSG-6 levels were also measured in conditioned media of human MSCs incubated with TNF-α and levels of the tubular injury marker lactate dehydrogenase in conditioned media after coculturing macrophages with injured human kidney 2 (HK-2) cells with or without TSG-6. The effects of TSG-6 on macrophage phenotype (M1/M2), adhesion, and migration were also determined. MetS + RAS showed increased M1 macrophages and renal vein TNF-α levels. After MSC delivery, renal vein TSG-6 increased and TNF-α decreased, the M1-to-M2 ratio decreased, renal function improved, and fibrosis was alleviated. In vitro, TNF-α increased TSG-6 secretion by human MSCs. TSG-6 decreased lactate dehydrogenase release from injured HK-2 cells, increased expression of macrophage M2 markers, and reduced M1 macrophage adhesion and migration. Therefore, TSG-6 released from MSCs may decrease renal tubular cell injury, which is associated with regulating macrophage function and phenotype. These observations suggest that TSG-6 is endowed with renoprotective properties.NEW & NOTEWORTHY Tumor necrosis factor-α-induced gene/protein (TSG)-6 regulates the immunomodulatory properties of MSCs, but its ability to protect the ischemic kidney is unknown. In pigs with renal artery stenosis, we show that MSC delivery increased renal vein TSG-6, decreased kidney inflammatory macrophages, and improved renal function. In vitro, TSG-6 decreased inflammatory macrophages and tubular cell injury. Therefore, TSG-6 released from MSCs may decrease renal tubular cell injury, which is associated with regulating macrophage function and phenotype.

Renal ischemia alters expression of mitochondria-related genes and impairs mitochondrial structure and function in swine scattered tubular-like cells.

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-γ coactivator (PGC)-1α 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.

Selective intrarenal delivery of mesenchymal stem cell-derived extracellular vesicles attenuates myocardial injury in experimental metabolic renovascular disease.

Extracellular vesicles (EVs) deliver genes and proteins to recipient cells, and mediate paracrine actions of their parent cells. Intrarenal delivery of mesenchymal stem cell (MSC)-derived EVs preserves stenotic-kidney function and reduces release of pro-inflammatory cytokines in a swine model of coexisting metabolic syndrome (MetS) and renal artery stenosis (RAS). We hypothesized that this approach is also capable of blunting cardiac injury and dysfunction. Five groups of pigs were studied after 16 weeks of diet-induced MetS and RAS (MetS + RAS), MetS and MetS + RAS treated 4 weeks earlier with a single intrarenal delivery of EVs-rich fraction harvested from autologous adipose tissue-derived MSCs, and lean and MetS Shams. Cardiac structure, function, and myocardial oxygenation were assessed in vivo using imaging, and cardiac inflammation, senescence, and fibrosis ex vivo. Inflammatory cytokine levels were measured in circulating and renal vein blood. Intrarenal EV delivery improved stenotic-kidney glomerular filtration rate and renal blood flow, and decreased renal release of monocyte-chemoattractant protein-1 and interleukin-6. Furthermore, despite unchanged systemic hemodynamics, intrarenal EV delivery in MetS + RAS normalized cardiac diastolic function, attenuated left ventricular remodeling, cellular senescence and inflammation, and improved myocardial oxygenation and capillary density in MetS + RAS. Intrarenal delivery of MSC-derived EVs blunts myocardial injury in experimental MetS + RAS, possibly related to improvement in renal function and systemic inflammatory profile. These observations underscore the central role of inflammation in the crosstalk between the kidney and heart, and the important contribution of renal function to cardiac structural and functional integrity in coexisting MetS and RAS.

Upregulated tumor necrosis factor-α transcriptome and proteome in adipose tissue-derived mesenchymal stem cells from pigs with metabolic syndrome.

INTRODUCTION: Mesenchymal stem cells (MSCs) have endogenous reparative properties, and may constitute an exogenous therapeutic intervention in patients with chronic kidney disease. The microenvironment of metabolic syndrome (MetS) induces fat inflammation, with abundant expression of tumor necrosis factor (TNF)-α. MetS may also alter the content of adipose tissue-derived MSCs, and we hypothesized that the inflammatory profile of MetS manifests via upregulating MSC mRNAs and proteins of the TNF-α pathway. METHODS: Domestic pigs were fed a 16-week Lean or MetS diet (n = 4 each). MSCs were harvested from abdominal subcutaneous fat, and their extracellular vesicles (EVs) isolated. Expression profiles of mRNAs and proteins in MSCs and EVs were obtained by high-throughput sequencing and proteomics. Nuclear translocation of the pro-inflammatory transcription factor (NF)-kB was evaluated in MSC and in pig renal tubular cells (TEC) co-incubated with EVs. RESULTS: We found 13 mRNAs and 4 proteins in the TNF-α pathway upregulated in MetS- vs. Lean-MSCs (fold-change > 1.4, p < 0.05), mostly via TNF-α receptor-1 (TNF-R1) signaling. Three mRNAs were upregulated in MetS-EVs. MetS-MSCs, as well as TECs co-incubated with MetS-EVs, showed increased nuclear translocation of NF-kB. Using qPCR, JUNB, MAP2K7 and TRAF2 genes followed the same direction of RNA-sequencing findings. CONCLUSIONS: MetS upregulates the TNF-α transcriptome and proteome in swine adipose tissue-derived MSCs, which are partly transmitted to their EV progeny, and are associated with activation of NF-kB in target cells. Hence, the MetS milieu may affect the profile of endogenous MSCs and their paracrine vectors and limit their use as an exogenous regenerative therapy. Anti-inflammatory strategies targeting the TNF-α pathway might be a novel strategy to restore MSC phenotype, and in turn function.

Renovascular disease induces mitochondrial damage in swine scattered tubular cells.

Scattered tubular-like cells (STCs) contribute to repair neighboring injured renal tubular cells. Mitochondria mediate STC biology and function but might be injured by the ambient milieu. We hypothesized that the microenviroment induced by the ischemic and metabolic components of renovascular disease impairs STC mitochondrial structure and function in swine, which can be attenuated with mitoprotection. CD24+/CD133+ STCs were quantified in pig kidneys after 16 wk of metabolic syndrome (MetS) or lean diet (Lean) with or without concurrent renal artery stenosis (RAS) (n = 6 each). Pig STCs were isolated and characterized, and mitochondrial structure, membrane potential, and oxidative stress were assessed in cells untreated or incubated with the mitoprotective drug elamipretide (1 nM for 6 h). STC-protective effects were assessed in vitro by their capacity to proliferate and improve viability of injured pig tubular epithelial cells. The percentage of STCs was higher in MetS, Lean + RAS, and MetS + RAS kidneys compared with Lean kidneys. STCs isolated from Lean + RAS and MetS + RAS pigs showed mitochondrial swelling and decreased matrix density, which were both restored by mitoprotection. In addition, mitochondrial membrane potential and ATP production were reduced and production of reactive oxygen species elevated in MetS, Lean + RAS, and MetS + RAS STCs. Importantly, mitoprotection improved mitochondrial structure and function as well as the capacity of MetS + RAS STCs to repair injured tubular cells in vitro. Renovascular disease in swine is associated with a higher prevalence of STCs but induces structural and functional alterations in STC mitochondria, which impair their reparative potency. These observations suggest a key role for mitochondria in the renal reparative capacity of STCs.

Early podocyte injury and elevated levels of urinary podocyte-derived extracellular vesicles in swine with metabolic syndrome: role of podocyte mitochondria.

Metabolic syndrome (MetS) is associated with nutrient surplus and kidney hyperfiltration, accelerating chronic renal failure. The potential involvement of podocyte damage in early MetS remains unclear. Mitochondrial dysfunction is an important determinant of renal damage, but whether it contributes to MetS-related podocyte injury remains unknown. Domestic pigs were studied after 16 wk of diet-induced MetS, MetS treated with the mitochondria-targeted peptide elamipretide (ELAM; 0.1 mg·kg-1·day-1 sc) for the last month of diet, and lean controls (n = 6 pigs/group). Glomerular filtration rate (GFR) and renal blood flow (RBF) were measured using multidetector computed tomography, and podocyte and mitochondrial injury were measured by light and electron microscopy. Urinary levels of podocyte-derived extracellular vesicles (pEVs; nephrin positive/podocalyxin positive) were characterized by flow cytometry. Body weight, blood pressure, RBF, and GFR were elevated in MetS. Glomerular size and glomerular injury score were also elevated in MetS and decreased after ELAM treatment. Evidence of podocyte injury, impaired podocyte mitochondria, and foot process width were all increased in MetS but restored with ELAM. The urinary concentration of pEVs was elevated in MetS pigs and directly correlated with renal dysfunction, glomerular injury, and fibrosis and inversely correlated with glomerular nephrin expression. Additionally, pEV numbers were elevated in the urine of obese compared with lean human patients. Early MetS induces podocyte injury and mitochondrial damage, which can be blunted by mitoprotection. Urinary pEVs reflecting podocyte injury might represent early markers of MetS-related kidney disease and a novel therapeutic target.

Hypertension with Hyperhomocysteinemia Increases the Risk of Early Cognitive Impairment after First-Ever Ischemic Stroke.

BACKGROUND: Hypertension and hyperhomocysteinemia (HHcy) are independent risk factors of stroke and are associated with each other. Although evidence suggests that they are related to cognitive impairment, the relationship between hypertension accompanied with HHcy and poststroke cognitive impairment (PSCI) is unclear. OBJECTIVE: To define the relationship between hypertension with HHcy and early cognitive impairment after acute cerebral infarction. MATERIALS AND METHODS: Our study enrolled 232 patients with acute first-ever ischemic stroke. Patients were assigned to 3 groups by blood pressure and homocysteine (Hcy) levels: hypertension with HHcy, simple hypertension, or control. Cognition was assessed by the Montreal cognitive assessment at admission and at 3- and 6-month follow-ups. RESULTS: The hypertension with HHcy group exhibited the highest incidence of early cognitive impairment (simple hypertension: p = 0.000; control: p = 0.000). This group also had lower visual space/executive scores than the simple hypertension group (p = 0.000) and lower delayed recall scores than the control group (p = 0.011). Multivariate analysis showed that hypertension with HHcy (OR 7.797; 95% CI 2.917-20.843; p = 0.000), the level of serum Hcy (OR 1.063; 95% CI 1.109-1.109; p = 0.005), education years (OR 0.797; 95% CI 0.722-0.880; p = 0.000), and Fazekas scale of leukoaraiosis (OR 1.648; 95% CI 1.239-2.191; p = 0.001) were independent influencing factors of early PSCI; however, simple hypertension (OR 1.183, 95% CI 0.208-6.737; p = 0.850) and simple HHcy (OR 1.112, 95% CI 0.181-6.810; p = 0.909) were not. CONCLUSION: Patients with both hypertension and HHcy are at an increased risk of early cognitive impairment after acute first-ever ischemic stroke.

Renal Artery Stenosis Alters Gene Expression in Swine Scattered Tubular-Like Cells.

BACKGROUND: Scattered tubular-like cells (STCs) proliferate and differentiate to support neighboring injured renal tubular cells during recovery from insults. Renal artery stenosis (RAS) induces renal ischemia and hypertension and leads to loss of kidney function, but whether RAS alters renal endogenous repair mechanisms, such as STCs, remains unknown. We hypothesize that RAS in swine modifies the messenger RNA (mRNA) profile of STCs, blunting their in vitro reparative capacity. METHODS: CD24+/CD133+ STCs were isolated from pig kidneys after 10-weeks of RAS or sham (n = 3 each) and their gene cargo analyzed using high-throughput mRNAseq. Expression profiles for upregulated and downregulated mRNAs in RAS-STCs were functionally interpreted by gene ontology analysis. STC activation was assessed by counting the total number of STCs in pig kidney sections using flow cytometry, whereas cell proliferation was assessed in vitro. RESULTS: Of all expressed genes, 1430 genes were upregulated and 315 downregulated in RAS- versus Normal-STCs. Expression of selected candidate genes followed the same fold change directions as the mRNAseq findings. Genes upregulated in RAS-STCs were involved in cell adhesion, extracellular matrix remodeling, and kidney development, whereas those downregulated in RAS-STCs are related to cell cycle and cytoskeleton. The percentage of STCs from dissociated kidney cells was higher in RAS versus Normal pigs, but their proliferation rate was blunted. CONCLUSIONS: Renal ischemia and hypertension in swine induce changes in the mRNA profile of STCs, associated with increased STC activation and impaired proliferation. These observations suggest that RAS may alter the reparative capacity of STCs.

Obesity-induced mitochondrial dysfunction in porcine adipose tissue-derived mesenchymal stem cells.

Transplantation of autologous mesenchymal stem cells (MSCs) may be a viable option for treatment of several diseases. MSCs efficacy depends on adequate function of their mitochondria, which might be impaired in a noxious milieu. We hypothesized that obesity compromises MSCs mitochondrial structure and function, possibly via micro-RNA (miRNA)-based mechanisms. MSCs were collected from swine abdominal adipose tissue after 16 weeks of Lean or Obese diet (n = 7 each). Mitochondrial structure was assessed by electron microscopy and function by membrane potential and cytochrome-c oxidase (COX)-IV activity. Oxidative stress was assessed by Mito-SOX and dihydroethidium staining. Next-generation sequencing (RNA-seq) was performed to identify miRNAs expression in MSCs, and predicted mitochondrial target genes were then identified (MitoCarta). Compared to Lean-MSCs, mitochondria from Obese-MSCs were smaller and showed cristae remodeling and loss. Mitochondrial membrane potential and COX-IV activity decreased in Obese-MSCs, associated with increased mitochondrial oxidative stress. RNA-seq generated reads for 413 miRNAs, of which 5 miRNAs were upregulated in Obese-MSCs (fold change >2, p < 0.05) and found to target 43 specific mitochondrial genes. Obesity impairs MSC mitochondrial structure and function, possibly mediated partly through miRNA-induced mitochondrial gene regulation, leading to increased oxidative stress. Importantly, these alterations may limit the therapeutic use of autologous MSCs in subjects with obesity.

The metabolic syndrome alters the miRNA signature of porcine adipose tissue-derived mesenchymal stem cells.

Autologous transplantation of mesenchymal stem cells (MSCs) is a viable option for the treatment of several diseases. Evidence indicates that MSCs release extracellular vesicles (EVs) and that EVs shuttle miRNAs to damaged parenchymal cells to activate an endogenous repair program. We hypothesize that comorbidities may interfere with the packaging of cargo in MSC-derived EVs. Therefore, we examined whether metabolic syndrome (MetS) modulates the miRNA content packed within MSC-derived EVs. MSCs were collected from swine abdominal adipose tissue after 16 weeks of lean or obese diet (n = 7 each). Next-generation RNA sequencing of miRNAs (miRNA-seq) was performed to identify miRNAs enriched in MSC-derived EVs and their predicted target genes. Functional pathway analysis of the top 50 target genes of the top 4 miRNAs enriched in each group was performed using gene ontology analysis. Lean- and MetS-EVs were enriched in, respectively, 14 and 8 distinct miRNAs. Target genes of miRNAs enriched in MetS-EVs were implicated in the development of MetS and its complications, including diabetes-related pathways, validated transcriptional targets of AP1 family members Fra1 and Fra2, Class A/1 (Rhodopsin-like receptors), and Peptide ligand-binding receptors. In contrast, miRNAs enriched in Lean EVs target primarily EphrinA-EPHA and the Rho family of GTPases. MetS alters the miRNA content of EVs derived from porcine adipose tissue MSCs. These alterations could impair the efficacy and limit the therapeutic use of autologous MSCs in subjects with MetS. Our findings may assist in developing adequate regenerative strategies to preserve the reparative potency of MSCs in individuals with MetS. © 2017 International Society for Advancement of Cytometry.