The impact of hypoxia preconditioning on mesenchymal stem cells performance in hypertensive kidney disease.

BACKGROUND: Autologous mesenchymal stem cells (MSCs) have emerged as a therapeutic option for many diseases. Hypertensive kidney disease (HKD) might impair MSCs' reparative ability by altering the biomolecular properties, but the characteristics of this impairment are unclear. In our previous pre-clinical studies, we found hypoxic preconditioning (HPC) enhanced angiogenesis and suppressed senescence gene expression. Thus, we hypothesize that HPC would improve human MSCs by enhancing their functionality and angiogenesis, creating an anti-inflammatory and anti-senescence environment. METHODS: MSC samples (n = 12 each) were collected from the abdominal fat of healthy kidney donors (HC), hypertensive patients (HTN), and patients with hypertensive kidney disease (HKD). MSCs were harvested and cultured in Normoxic (20% O2) or Hypoxic (1% O2) conditions. MSC functionality was measured by proliferation assays and cytokine released in conditioned media. Senescence was evaluated by senescence-associated beta-galactosidase (SA-beta-gal) activity. Additionally, transcriptome analysis using RNA-sequencing and quantitative PCR (qPCR) were performed. RESULTS: At baseline, normoxic HTN-MSCs had higher proliferation capacity compared to HC. However, HPC augmented proliferation in HC. HPC did not affect the release of pro-angiogenic protein VEGF, but increased EGF in HC-MSC, and decreased HGF in HC and HKD MSCs. Under HPC, SA-ß-gal activity tended to decrease, particularly in HC group. HPC upregulated mostly the pro-angiogenic and inflammatory genes in HC and HKD and a few senescence genes in HKD. CONCLUSIONS: HPC has a more favorable functional effect on HC- than on HKD-MSC, reflected in increased proliferation and EGF release, and modest decrease in senescence, whereas it has little effect on HTN or HKD MSCs.

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.

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.

Evaluation of Renal Fibrosis Using Magnetization Transfer Imaging at 1.5T and 3T in a Porcine Model of Renal Artery Stenosis.

Renal fibrosis is an important marker in the progression of chronic kidney disease, and renal biopsy is the current reference standard for detecting its presence. Currently, non-invasive methods have only been partially successful in detecting renal fibrosis. Magnetization transfer imaging (MTI) allows estimates of renal fibrosis but may vary with scanning conditions. We hypothesized that MTI-derived renal fibrosis would be reproducible at 1.5T and 3T MRI and over time in fibrotic kidneys. Fifteen pigs with unilateral renal artery stenosis (RAS, n = 9) or age-matched sham controls (n = 6) underwent MTI-MRI at both 1.5T and 3T 6 weeks post-surgery and again 4 weeks later. Magnetization transfer ratio (MTR) measurements of fibrosis in both kidneys were compared between 1.5T and 3T, and the reproducibility of MTI at the two timepoints was evaluated at 1.5T and 3T. MTR at 3T with 600 Hz offset frequency successfully distinguished between normal, stenotic, and contralateral kidneys. There was excellent reproducibility of MTI at 1.5T and 3T over the two timepoints and no significant differences between MTR measurements at 1.5T and 3T. Therefore, MTI is a highly reproducible technique which is sensitive to detect changes in fibrotic compared to normal kidneys in the RAS porcine model at 3T.

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.

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.

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.

Hypoxic preconditioning induces epigenetic changes and modifies swine mesenchymal stem cell angiogenesis and senescence in experimental atherosclerotic renal artery stenosis.

BACKGROUND: Atherosclerotic renal artery stenosis (ARAS) is a risk factor for ischemic and hypertensive kidney disease (HKD) for which autologous mesenchymal stem cell (MSC) appears to be a promising therapy. However, MSCs from ARAS patients exhibit impaired function, senescence, and DNA damage, possibly due to epigenetic mechanisms. Hypoxia preconditioning (HPC) exerts beneficial effects on cellular proliferation, differentiation, and gene and protein expression. We hypothesized that HPC could influence MSC function and senescence in ARAS by epigenetic mechanisms and modulating gene expression of chromatin-modifying enzymes. METHODS: Adipose-derived MSC harvested from healthy control (N = 8) and ARAS (N = 8) pigs were cultured under normoxia (20%O2) or hypoxia (1%O2) conditions. MSC function was assessed by migration, proliferation, and cytokine release in conditioned media. MSC senescence was evaluated by SA-ß-gal activity. Specific pro-angiogenic and senescence genes were assessed by reverse transcription polymerase chain reaction (RT-PCR). Dot blotting was used to measure global genome 5-hydroxymethylcytosine (5hmC) levels on DNA and Western blotting of modified histone 3 (H3) proteins to quantify tri-methylated lysine-4 (H3K4me3), lysine-9 (H3K9me3), and lysine-27 (H3K27me3) residues. RESULTS: Specific pro-angiogenic genes in ARAS assessed by RT-PCR were lower at baseline but increased under HPC, while pro-senescence genes were higher in ARAS at baseline as compared healthy MSCs. ARAS MSCs under basal conditions, displayed higher H3K4me3, H3K27me3, and 5hmC levels compared to healthy MSCs. During HPC, global 5hmC levels were decreased while no appreciable changes occurred in histone H3 tri-methylation. ARAS MSCs cultured under HPC had higher migratory and proliferative capacity as well as increased vascular endothelial growth factor and epidermal growth factor expression compared to normoxia, and SA-ß-gal activity decreased in both animal groups. CONCLUSIONS: These data demonstrate that swine ARAS MSCs have decreased angiogenesis and increased senescence compared to healthy MSCs and that HPC mitigates MSC dysfunction, senescence, and DNA hydroxymethylation in ARAS MSC. Thus, HPC for MSCs may be considered for their optimization to improve autologous cell therapy in patients with nephropathies.

Mesenchymal Stem/Stromal Cells and their Extracellular Vesicle Progeny Decrease Injury in Poststenotic Swine Kidney Through Different Mechanisms.

Novel therapies are needed to address the increasing prevalence of chronic kidney disease. Mesenchymal stem/stromal cells (MSCs) and MSC-derived extracellular vesicles (EVs) augment tissue repair. We tested the hypothesis that EVs are as effective as MSCs in protecting the stenotic kidney, but target different injury pathways. Pigs were studied after 16 weeks of renal injury achieved by diet-induced metabolic syndrome (MetS) and renal artery stenosis (RAS). Pigs were untreated or treated 4 weeks earlier with intrarenal delivery of autologous adipose tissue-derived MSCs (107) or their EVs (1011). Lean pigs and sham RAS served as controls (n = 6 each). Stenotic-kidney function was studied in vivo using computed tomography and magnetic resonance imaging. Histopathology and expression of necroptosis markers [receptor-interacting protein kinase (RIPK)-1 and RIPK-3], inflammatory, and growth factors (angiopoietin-1 and vascular endothelial growth factor) were studied ex vivo. Stenotic-kidney glomerular filtration rate and blood flow in MetS + RAS were both lower than Lean and increased in both MetS + RAS + MSC and MetS + RAS + EV. Both MSCs and EV improved renal function and decreased renal hypoxia, fibrosis, and apoptosis. MSCs were slightly more effective in preserving microvascular (0.02-0.2 mm diameters) density and prominently attenuated renal inflammation. However, EV more significantly upregulated growth factor expression and decreased necroptosis. In conclusion, adipose tissue-derived MSCs and their EV both improve stenotic kidney function and decrease tissue injury in MetS + RAS by slightly different mechanisms. MSCs more effectively preserved the microcirculation, while EV bestowed better preservation of renal cellular integrity. These findings encourage further exploration of this novel approach to attenuate renal injury.

Adjunctive mesenchymal stem/stromal cells augment microvascular function in poststenotic kidneys treated with low-energy shockwave therapy.

Effective therapeutic strategies are needed to preserve renal function in patients with atherosclerotic renal artery stenosis (ARAS). Low-energy shockwave therapy (SW) and adipose tissue-derived mesenchymal stem/stromal cells (MSCs) both stimulate angiogenesis repair of stenotic kidney injury. This study tested the hypothesis that intrarenal delivery of adipose tissue-derived MSCs would enhance the capability of SW to preserve stenotic kidney function and structure. Twenty-two pigs were studied after 16 weeks of ARAS, ARAS treated with a SW regimen (bi-weekly for 3 weeks) with or without subsequent intrarenal delivery of adipose tissue-derived MSCs and controls. Four weeks after treatment, single-kidney renal blood flow (RBF) before and after infusion of acetylcholine, glomerular filtration rate (GFR), and oxygenation were assessed in vivo and the renal microcirculation, fibrosis, and oxidative stress ex vivo. Mean arterial pressure remained higher in ARAS, ARAS + SW, and ARAS + SW + MSC compared with normal. Both SW and SW + MSC similarly elevated the decreased stenotic kidney GFR and RBF observed in ARAS to normal levels. Yet, SW + MSC significantly improved RBF response to acetylcholine in ARAS, and attenuated capillary loss and oxidative stress more than SW alone. Density of larger microvessels was similarly increased by both interventions. Therefore, although significant changes in functional outcomes were not observed in a short period of time, adjunct MSCs enhanced pro-angiogenic effect of SW to improve renal microvascular outcomes, suggesting this as an effective stratege for long-term management of renovascular disease.

Alterations in genetic and protein content of swine adipose tissue-derived mesenchymal stem cells in the metabolic syndrome.

INTRODUCTION: Mesenchymal stem cells (MSCs) possess endogenous reparative properties and may serve as an exogenous therapeutic intervention in patients with chronic kidney disease. Cardiovascular risk factors clustering in the metabolic syndrome (MetS) might adversely affect cellular properties. To test the hypothesis that Mets interferes with MSC characteristics, we performed comprehensive comparison of the mRNA, microRNA, and protein content of MSCs isolated from Lean and MetS pigs. METHODS: Domestic pigs were fed a 16-week Lean or MetS diet (n = 4 each). Expression profiles of co-existing microRNAs, mRNAs, and proteins were obtained by high-throughput sequencing and liquid chromatography-mass spectrometry. TargetScan and ComiR were used to predict target genes of differentially expressed microRNAs, and DAVID 6.7 for functional annotation analysis to rank primary gene ontology categories for the microRNA target genes, mRNAs, and proteins. RESULTS: Differential expression analysis revealed 12 microRNAs upregulated in MetS-MSCs compared to Lean-MSCs (fold change>1.4, p < .05), which target 7728 genes, whereas 33 mRNAs and 78 proteins were downregulated (fold change<0.7, p < .05). Integrated analysis showed that targets of those microRNAs upregulated in MetS-MSCs overlap with at least half of mRNAs and proteins dysregulated in those cells. Functional analysis of overlapping mRNAs and proteins suggest that they are primarily involved in mitochondria, inflammation and transcription. MetS-MSCs also exhibited increased nuclear translocation of nuclear factor kappa-B, associated with increased SA-ß-Galactosidase and decreased cytochrome-c oxidase-IV activity. CONCLUSION: MetS alters the transcriptome and proteome of swine adipose tissue-derived MSCs particularly genes involved in mitochondria, inflammation and transcription regulation. These alterations might limit the reparative function of endogenous MSC and their use as an exogenous regenerative therapy.

Intrarenal Delivery of Mesenchymal Stem Cells and Endothelial Progenitor Cells Attenuates Hypertensive Cardiomyopathy in Experimental Renovascular Hypertension.

Renovascular hypertension (RVH) leads to left ventricular (LV) hypertrophy and diastolic dysfunction, associated with increased cardiovascular mortality. Intrarenal delivery of endothelial progenitor cells (EPCs) and mesenchymal stem cells (MSCs) improves kidney function in porcine RVH, and the potent anti-inflammatory properties of MSCs may serve to blunt inflammatory mediators in the cardiorenal axis. However, their relative efficacy in attenuating cardiac injury and dysfunction remains unknown. This study tested the hypothesis that the cardioprotective effect of EPCs and MSCs delivered into the stenotic kidney in experimental RVH are comparable. Pigs (n = 7 per group) were studied after 10 weeks of RVH or control untreated or treated with a single intrarenal infusion of autologous EPCs or MSCs 4 weeks earlier. Cardiac and renal function (fast CT) and stenotic kidney release of inflammatory mediators (ELISA) were assessed in vivo, and myocardial inflammation, remodeling, and fibrosis ex vivo. After 10 weeks of RVH, blood pressure was not altered in cell-treated groups, yet stenotic kidney glomerular filtration rate (GFR), blunted in RVH, improved in RVH + EPC, and normalized in RVH + MSCs. Stenotic kidney release of monocyte chemoattractant protein (MCP)-1 and its myocardial expression were elevated in RVH + EPC, but normalized only in RVH + MSC pigs. RVH-induced LV hypertrophy was normalized in both EPC- and MSC-treated pigs, while diastolic function (E/A ratio) was restored to normal levels exclusively in RVH + MSCs. RVH-induced myocardial fibrosis and collagen deposition decreased in RVH + EPCs but further decreased in RVH + MSC-treated pigs. Intrarenal delivery of EPCs or MSCs attenuates RVH-induced myocardial injury, yet MSCs restore diastolic function more effectively than EPCs, possibly by greater improvement in renal function or reduction of MCP-1 release from the stenotic kidney. These observations suggest a therapeutic potential for EPCs and MSCs in preserving the myocardium in chronic experimental RVH.

Obesity-metabolic derangement preserves hemodynamics but promotes intrarenal adiposity and macrophage infiltration in swine renovascular disease.

Obesity-metabolic disorders (ObM) often accompany renal artery stenosis (RAS). We hypothesized that the coexistence of ObM and RAS magnifies inflammation and microvascular remodeling in the stenotic kidney (STK) and aggravates renal scarring. Twenty-eight obesity-prone Ossabaw pigs were studied after 16 wk of a high-fat/high-fructose diet or standard chow including ObM-sham, ObM-RAS, Lean-RAS, or Lean-sham (normal control) groups. Single-kidney renal blood flow (RBF) and glomerular filtration rate (GFR) were assessed by multidetector computed tomography (CT), renal oxygenation and tubular transport capability by blood-oxygen-level-dependent MRI, and microcirculation by micro-CT for vessel density, and Western blotting for protein expressions of angiogenic factors (VEGF/FLK-1). Renal vein and inferior vena cava levels of inflammatory cytokines were measured to evaluate systemic and kidney inflammation. Macrophage (MØ) infiltration and subpopulations, fat deposition in the kidney, and inflammation in perirenal and abdominal fat were also examined. GFR and RBF were decreased in Lean-STK but relatively preserved in ObM-STK. However, ObM-STK showed impaired tubular transport function, suppressed microcirculation, and stimulated glomerulosclerosis. ObM diet interacted with RAS to blunt angiogenesis in the STK, facilitated the release of inflammatory cytokines, and led to greater oxidative stress than Lean-STK. The ObM diet also induced fat deposition in the kidney and infiltration of proinflammatory M1-MØ, as also in perirenal and abdominal fat. Coexistence of ObM and RAS amplifies renal inflammation, aggravates microvascular remodeling, and accelerates glomerulosclerosis. Increased adiposity and MØ-accentuated inflammation induced by an ObM diet may contribute to structural injury in the post-STK kidney.

Mesenchymal stem cells and endothelial progenitor cells decrease renal injury in experimental swine renal artery stenosis through different mechanisms.

Endothelial progenitor cells (EPC) and mesenchymal stem cells (MSC) augment tissue repair but possess slightly different properties. How the cellular phenotype affects the efficacy of this approach in renovascular disease is incompletely understood. This study tested the hypothesis that EPC and MSC protect the poststenotic kidney by blunting different disease pathways. Peripheral blood EPC and adipose-derived MSC were expanded and characterized by cell surface markers (e.g., CD34/kinase insert domain receptor, or CD44/CD90). Single-kidney hemodynamics and function were assessed in pigs after 10 weeks of renal artery stenosis (RAS) treated 4 weeks earlier with an intrarenal infusion of vehicle (n = 7), EPC (RAS+EPC) or MSC (RAS+MSC) (both 10 × 10(6), n = 6), and normal controls (n = 7). Kidney disease mechanisms were evaluated ex vivo. The ability of EPC and MSC to attenuate endoplasmic reticulum (ER) stress was also studied in isolated ER and in tubular cells cocultured with EPC and MSC. Glomerular filtration rate in RAS was lower than controls, increased in RAS+EPC, and further improved in RAS+MSC, although both improved renal blood flow similarly. EPC prominently enhanced renal growth factor expression and decreased oxidative stress, while MSC more significantly attenuated renal inflammation, ER stress, and apoptosis. Furthermore, MSC induced a greater decrease in caspase-3 and CHOP expression in cultured tubular cells through mechanisms involving cell contact. EPC and MSC achieve a comparable decrease of kidney injury in RAS by different mechanisms, although MSC elicited slightly superior improvement of renal function. These results support development of cell-based approaches for management of renovascular disease and suggest cell selection based on the underlying pathophysiology of kidney injury.

A mitochondrial permeability transition pore inhibitor improves renal outcomes after revascularization in experimental atherosclerotic renal artery stenosis.

Revascularization improves blood pressure but not renal function in most patients with atherosclerotic renal artery stenosis (ARAS), possibly related to injury incurred during renal reperfusion. Bendavia, a novel tetrapeptide that inhibits mitochondrial permeability transition pore opening, reduces apoptosis, oxidative stress, and ischemia-reperfusion injury in experimental models. However, its potential for improving renal response to revascularization of chronic ARAS is unknown. We hypothesized that adjunct Bendavia would improve renal structure and function after percutaneous transluminal renal angioplasty (PTRA). Pigs were treated after 6 weeks of ARAS or control with PTRA+stenting (or sham), adjunct continuous 4-hour infusion of Bendavia (0.05 mg/kg IV) or vehicle (n=7 each) during PTRA. Single-kidney renal blood flow and glomerular filtration rate were studied 4 weeks later and renal mitochondrial biogenesis, microvascular architecture, and injurious pathways evaluated ex vivo. Monocyte chemoattractant protein-1 levels rose after PTRA, suggesting inflammatory injury. Bendavia did not immediately affect inflammatory cytokine levels, yet 4 weeks later, stenotic kidney renal blood flow and glomerular filtration rate both improved (44.00 ± 0.21% and 36.40 ± 10.21%, respectively) in ARAS+PTRA+Bendavia compared with ARAS+PTRA+vehicle. Renal mitochondrial biogenesis was restored after PTRA+Bendavia, and microvascular rarefaction, apoptosis, oxidative stress, tubular injury, and fibrosis decreased. Infusion of Bendavia during PTRA preserved mitochondrial biogenesis, renal hemodynamics, and function, and attenuated tissue injury in swine ARAS. Thus, functional mitochondrial injury during renal reperfusion may sustain renal inflammatory injury and limit kidney recovery after PTRA. Potent antiapoptotic and antioxidant effects provide Bendavia a novel therapeutic potential for improving kidney outcomes after PTRA in experimental ARAS.

Adipose tissue-derived mesenchymal stem cells improve revascularization outcomes to restore renal function in swine atherosclerotic renal artery stenosis.

Reno-protective strategies are needed to improve renal outcomes in patients with atherosclerotic renal artery stenosis (ARAS). Adipose tissue-derived mesenchymal stem cells (MSCs) can promote renal regeneration, but their potential for attenuating cellular injury and restoring kidney repair in ARAS has not been explored. We hypothesized that replenishment of MSC as an adjunct to percutaneous transluminal renal angioplasty (PTRA) would restore renal cellular integrity and improve renal function in ARAS pigs. Four groups of pigs (n = 7 each) were studied after 16 weeks of ARAS, ARAS 4 weeks after PTRA and stenting with or without adjunct intrarenal delivery of MSC (10 × 10(6) cells), and controls. Stenotic kidney blood flow (renal blood flow [RBF]) and glomerular filtration rate (GFR) were measured using multidetector computer tomography (CT). Renal microvascular architecture (micro-CT), fibrosis, inflammation, and oxidative stress were evaluated ex vivo. Four weeks after successful PTRA, mean arterial pressure fell to a similar level in all revascularized groups. Stenotic kidney GFR and RBF remained decreased in ARAS (p = .01 and p = .02) and ARAS + PTRA (p = .02 and p = .03) compared with normal but rose to normal levels in ARAS + PTRA + MSC (p = .34 and p = .46 vs. normal). Interstitial fibrosis, inflammation, microvascular rarefaction, and oxidative stress were attenuated only in PTRA + MSC-treated pigs. A single intrarenal delivery of MSC in conjunction with renal revascularization restored renal hemodynamics and function and decreased inflammation, apoptosis, oxidative stress, microvascular loss, and fibrosis. This study suggests a unique and novel therapeutic potential for MSC in restoring renal function when combined with PTRA in chronic experimental renovascular disease.

Enhanced endothelial progenitor cell angiogenic potency, present in early experimental renovascular hypertension, deteriorates with disease duration.

BACKGROUND: Endothelial progenitor cells (EPCs) augment neovascularization and repair of damaged tissues, but may undergo functional changes during exposure to cardiovascular risk factors. This study tested the hypothesis that early renovascular hypertension (RVH) modulates the temporal pattern of EPC function that deteriorates with disease duration. METHODS: RVH was induced in domestic pigs by unilateral renal artery stenosis. EPCs were cultured after 3, 6, and 12 weeks of RVH or normal control to evaluate EPC function, growth factor, and homing receptor expression. Plasma renin activity (PRA), vascular endothelial growth factor (VEGF), and its soluble receptor-1 (sFlt-1) were measured in plasma. EPCs (10 × 10) isolated from 3-week RVH or from normal pigs were also injected into control kidneys (n = 6-7, each group), and 4 weeks later single-kidney renal blood flow (RBF) and glomerular filtration rate (GFR) were evaluated. Microvascular density was studied ex vivo using microcomputed tomography. RESULTS: Blood pressure peaked at 3 weeks and remained higher than normal throughout the study. Systemic PRA also peaked after 3 weeks of RVH and declined thereafter, whereas sFlt-1 showed a reciprocal pattern. In vivo, only RVH but not normal EPCs increased RBF, GFR, and microvascular density. RVH-EPCs showed in vitro enhanced proliferation, tube formation, VEGF, and homing receptor expression that peaked at 3 weeks, which were abolished by valsartan and returned to baseline levels after 12 weeks of RVH. EPC number remained unchanged throughout the study. CONCLUSION: A transient enhancement of EPC function, mediated by angiotensin II, may contribute to compensatory vascular adaptation in early RVH, but is lost as hypertension persists.

Renal perfusion and hemodynamics: accurate in vivo determination at CT with a 10-fold decrease in radiation dose and HYPR noise reduction.

PURPOSE: To prospectively evaluate the accuracy of computed tomographic (CT) perfusion measurements of renal hemodynamics and function obtained by using images acquired with one-tenth the typical radiation dose and postprocessed with a highly constrained back-projection (HYPR)-local reconstruction (LR) noise-reduction technique. MATERIALS AND METHODS: This study was approved by the institutional Animal Care and Use Committee. Two consecutive CT perfusion acquisitions were performed in 10 anesthetized pigs over 180 seconds by using routine (80 kV, 160 mAs) and one-tenth (80 kV, 16 mAs) dose levels. Images obtained with each acquisition were reconstructed with identical parameters, and the one-tenth dose images were also processed with a HYPR-LR algorithm. Attenuation changes in kidneys were determined as a function of time to form time-attenuation curves (TACs). Extended gamma-variate curve-fitting was performed, and regional perfusion, glomerular filtration rate, and renal blood flow were calculated. Image quality was evaluated (in 10 pigs), and the agreement for renal perfusion and function between the routine dose and the one-tenth dose HYPR-LR images was determined (for 20 kidneys) by using statistical methods. Statistical analysis was performed by using the paired t test, linear regression, and Bland-Altman analysis. RESULTS: TACs obtained with the one-tenth dose were similar to those obtained with the routine dose. Statistical analysis showed that there were no significant differences between the routine dose and the one-tenth dose acquisitions in renal perfusion and hemodynamic values and that there were slight but statistically significant differences in some values with the one-tenth dose HYPR-LR-processed acquisition. The image quality of the one-tenth dose acquisition was improved by using the HYPR-LR algorithm. Linear regression and Bland-Altman plots showed agreement between the images acquired by using the routine dose and those acquired by using the one-tenth dose with HYPR-LR processing. CONCLUSION: A 10-fold dose reduction at renal perfusion CT imaging can be achieved in vivo, without loss of accuracy. The image quality of the one-tenth dose images could be improved to be near that of the routine dose images by using the HYPR-LR noise-reduction algorithm. SUPPLEMENTAL MATERIAL: http://radiology.rsna.org/lookup/suppl/doi:10.1148/radiol.2531081677/-/DC1.

Myocardial microvascular function during acute coronary artery stenosis: effect of hypertension and hypercholesterolaemia.

AIMS: Coronary collateral arteries (CCA) reduce cardiovascular events. We tested the hypothesis that new microvessels that proliferate in early atherosclerosis may be associated with myocardial protection during acute subtotal coronary artery obstruction (CAO). METHODS AND RESULTS: Acute left anterior descending CAO was induced by a balloon catheter in pigs after 12 weeks of high-cholesterol (HC) diet, renovascular hypertension (HTN), or normal control. Cardiac structure, myocardial perfusion, and functional response to iv adenosine and CAO were studied in vivo using electron beam computed tomography (CT). The intra-myocardial microvessels were subsequently evaluated ex vivo using micro-CT, and myocardial expression of growth factors using immunoblotting. Basal myocardial perfusion and microvascular permeability were similar among the groups, whereas their responses to adenosine were attenuated in HC and HTN. A significant decline in myocardial perfusion in normal pigs during acute CAO was attenuated in HC and abolished in HTN. CAO also elicited an increase in normal anterior wall microvascular permeability (+202 +/- 59%, P < 0.05), which was attenuated in HC and HTN (+55 +/- 9 and +31 +/- 8%, respectively, P < 0.05 vs. normal). Microvascular (<200 microm) spatial density was significantly elevated in HC and HTN, accompanied by increased myocardial growth factor expression. CONCLUSION: This study demonstrates that early exposure to the cardiovascular risk factors HC and HTN protects the heart from decreases in myocardial perfusion during acute subtotal CAO. This protective effect is associated with and potentially mediated by pre-emptive development of intra-myocardial microvessels that might serve as recruitable CCA.

Endothelial progenitor cells restore renal function in chronic experimental renovascular disease.

BACKGROUND: Endothelial progenitor cells (EPCs) promote neovascularization and endothelial repair. Renal artery stenosis (RAS) may impair renal function by inducing intrarenal microvascular injury and remodeling. We investigated whether replenishment with EPCs would protect the renal microcirculation in chronic experimental renovascular disease. METHODS AND RESULTS: Single-kidney hemodynamics and function were assessed with the use of multidetector computed tomography in vivo in pigs with RAS, pigs with RAS 4 weeks after intrarenal infusion of autologous EPCs, and controls. Renal microvascular remodeling and angiogenic pathways were investigated ex vivo with the use of micro-computed tomography, histology, and Western blotting. EPCs increased renal expression of angiogenic factors, stimulated proliferation and maturation of new vessels, and attenuated renal microvascular remodeling and fibrosis in RAS. Furthermore, EPCs normalized the blunted renal microvascular and filtration function. CONCLUSIONS: The present study shows that a single intrarenal infusion of autologous EPCs preserved microvascular architecture and function and decreased microvascular remodeling in experimental chronic RAS. It is likely that restoration of the angiogenic cascade by autologous EPCs involved not only generation of new vessels but also acceleration of their maturation and stabilization. This contributed to preserving the blood supply, hemodynamics, and function of the RAS kidney, supporting EPCs as a promising therapeutic intervention for preserving the kidney in renovascular disease.