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.

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.

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.

Endovascular reversal of renovascular hypertension blunts cardiac dysfunction and deformation in swine.

OBJECTIVE: Renovascular hypertension (RVH) induces hemodynamic and humoral aberrations that may impair cardiac function, structure and mechanics, including cardiac twist and deformation. Revascularization of a stenotic renal artery can decrease blood pressure (BP), but its ability to restore cardiac mechanics in RVH remains unclear. We hypothesized that percutaneous transluminal renal angioplasty (PTRA) would improve cardiac function and left ventricular (LV) deformation in swine RVH. METHODS: Seventeen domestic pigs were studied for 16 weeks: RVH, RVH + PTRA and normal controls (n = 5-6 each). Global LV function was estimated by multidetector computed-tomography, and LV deformation by electrocardiographically triggered MRI tagging at the apical, mid, and basal LV levels. Cardiomyocyte hypertrophy, myocardial capillary density, and fibrosis were evaluated ex vivo. RESULTS: BP and wall thickness were elevated in RVH and decreased by PTRA, yet remained higher than in controls. LV myocardial muscle mass increased in RVH pigs, which also developed diastolic dysfunction, whereas cardiac output increased. Furthermore, both apical rotation and peak torsion angle increased in RVH compared with controls. Ex vivo, RVH induced myocardial fibrosis and vascular rarefaction. PTRA restored cardiac function and alleviated hypertrophy, vascular rarefaction, and fibrosis. PTRA also normalized apical rotation and peak torsion angle, and elevated basal peak radial strain and apical peak radial strain compared with RVH. CONCLUSION: In addition to cardiac LV adaptive hypertrophy and diastolic dysfunction, short-term RVH causes cardiac deformation. Despite only partial improvement in BP, PTRA effectively restored cardiac function and reversed abnormal mechanics. Hence, renal revascularization may be a useful strategy to preserve cardiac function in RVH.

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.

Mesenchymal Stem Cell-Derived Extracellular Vesicles Induce Regulatory T Cells to Ameliorate Chronic Kidney Injury.

Metabolic syndrome (MetS) profoundly changes the contents of mesenchymal stem cells and mesenchymal stem cells-derived extracellular vesicles (EVs). The anti-inflammatory TGF-ß (transforming growth factor-ß) is selectively enriched in EVs from Lean but not from MetS pigs, but the functional impact of this endowment remains unknown. We hypothesized that Lean-EVs more effectively induce regulatory T cells in injured kidneys. Five groups of pigs (n=7 each) were studied after 16 weeks of diet-induced MetS and unilateral renal artery stenosis (RAS; MetS+RAS). Two groups of MetS+RAS were treated 4 weeks earlier with an intrarenal injection of either Lean-EVs or MetS-EVs. MetS+RAS had lower renal volume, renal blood flow, and glomerular filtration rate than MetS pigs. Compared with Lean-EVs, MetS-EVs were less effective in improving renal function and decreasing tubular injury and fibrosis in MetS+RAS. Lean-EVs upregulated TGF-ß expression in stenotic kidney and increased regulatory T cells numbers more prominently. Furthermore, markedly upregulated anti-inflammatory M2 macrophages reduced proinflammatory M1 macrophages, and CD8+ T cells were detected in stenotic kidneys treated with Lean-EVs compared with MetS-EVs, and renal vein levels of interleukin-1ß were reduced. In vitro, coculture of Lean-EVs with activated T cells led to greater TGF-ß-dependent regulatory T cells induction than did MetS-EVs. Therefore, the beneficial effects of mesenchymal stem cells-derived EVs on injured kidneys might be partly mediated by their content of TGF-ß signaling components, which permitting increased Treg preponderance. Modulating EV cargo and transforming their functionality might be useful for renal repair.

Magnetization Transfer Imaging Is Unaffected by Decreases in Renal Perfusion in Swine.

OBJECTIVES: Multiparametric renal magnetic resonance imaging (MRI), including diffusion-weighted imaging, magnetic resonance elastography, and magnetization transfer imaging (MTI), is valuable in the noninvasive assessment of renal fibrosis. However, hemodynamic changes in diseased kidneys may impede their ability to measure renal fibrosis. Because MTI assesses directly tissue content of macromolecules, we test the hypothesis that MTI would be insensitive to renal hemodynamic changes in swine kidneys with acute graded ischemia. MATERIALS AND METHODS: Seven domestic pigs underwent placement of an inflatable silicone cuff around the right renal artery to induce graded renal ischemia. Multiparametric MRI was performed at baseline, 50%, 75%, and 100% renal artery stenosis as well as reperfusion. Measurements included regional perfusion, R2*, apparent diffusion coefficient (ADC), stiffness, and magnetization transfer ratio (MTR) using arterial spin-labeled MRI, blood oxygenation-dependent MRI, diffusion-weighted imaging, magnetic resonance elastography, and MTI, respectively. Histology was performed to rule out renal fibrosis. RESULTS: During graded ischemia, decreases in renal perfusion were accompanied with elevated R2*, decreased ADC, and stiffness, whereas no statistically significant changes were observed in the MTR. No fibrosis was detected by histology. After release of the obstruction, renal perfusion showed only partial recovery, associated with return of kidney R2*, ADC, and stiffness to baseline levels, whereas cortical MTR decreased slightly. CONCLUSIONS: Renal MTI is insensitive to decreases in renal perfusion and may offer reliable assessment of renal structural changes.

Improved renal outcomes after revascularization of the stenotic renal artery in pigs by prior treatment with low-energy extracorporeal shockwave therapy.

BACKGROUND: Revascularization does not restore renal function in most patients with atherosclerotic renal artery stenosis (RAS), likely because of inflammation and fibrosis within the stenotic kidney. Low-energy shockwave therapy (LE-SWT) stimulates angiogenesis in the stenotic kidney, but its ability to improve renal function and structure after revascularization remains unexplored. We tested the hypothesis that a LE-SWT regimen before percutaneous transluminal renal angioplasty (PTRA) would enable PTRA to restore renal function in hypercholesterolemic pigs with RAS (HC+RAS), and that this would be associated with attenuation of renal inflammation and fibrosis. METHODS AND RESULTS: Twenty-six pigs were studied after 16 weeks of HC+RAS, HC+RAS treated with PTRA with or without a preceding LE-SWT regimen (bi-weekly for 3 weeks), and controls. Single-kidney renal blood flow (RBF), glomerular filtration rate (GFR), and oxygenation were assessed in vivo using imaging 4 weeks after PTRA, and then inflammation and fibrosis ex vivo.Four weeks after successful PTRA, blood pressure fell similarly in both revascularized groups. Yet, stenotic-kidney GFR remained lower in HC+RAS and HC+RAS+PTRA (P < 0.01 vs. normal), but was improved in HC+RAS+PTRA+SW (P > 0.05 vs. normal). Furthermore, reduced inflammation, medullary fibrosis, and cortical hypoxia were only shown in swine stenotic kidneys pretreated with LE-SWT before PTRA 4 weeks later. CONCLUSION: LE-SWT delivery before revascularization permitted PTRA to improve function and decrease cortical and medullary damage in the stenotic swine kidney. This study, therefore, supports the use of an adjunct SW pretreatment to enhance the success of PTRA in blunting loss of kidney function in experimental HC+RAS.

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.

Low-Energy Shockwave Therapy Improves Ischemic Kidney Microcirculation.

Microvascular rarefaction distal to renal artery stenosis is linked to renal dysfunction and poor outcomes. Low-energy shockwave therapy stimulates angiogenesis, but the effect on the kidney microvasculature is unknown. We hypothesized that low-energy shockwave therapy would restore the microcirculation and alleviate renal dysfunction in renovascular disease. Normal pigs and pigs subjected to 3 weeks of renal artery stenosis were treated with six sessions of low-energy shockwave (biweekly for 3 consecutive weeks) or left untreated. We assessed BP, urinary protein, stenotic renal blood flow, GFR, microvascular structure, and oxygenation in vivo 4 weeks after completion of treatment, and then, we assessed expression of angiogenic factors and mechanotransducers (focal adhesion kinase and ß1-integrin) ex vivo A 3-week low-energy shockwave regimen attenuated renovascular hypertension, normalized stenotic kidney microvascular density and oxygenation, stabilized function, and alleviated fibrosis in pigs subjected to renal artery stenosis. These effects associated with elevated renal expression of angiogenic factors and mechanotransducers, particularly in proximal tubular cells. In additional pigs with prolonged (6 weeks) renal artery stenosis, shockwave therapy also decreased BP and improved GFR, microvascular density, and oxygenation in the stenotic kidney. This shockwave regimen did not cause detectable kidney injury in normal pigs. In conclusion, low-energy shockwave therapy improves stenotic kidney function, likely in part by mechanotransduction-mediated expression of angiogenic factors in proximal tubular cells, and it may ameliorate renovascular hypertension. Low-energy shockwave therapy may serve as a novel noninvasive intervention in the management of renovascular disease.

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.

Assessment of renal artery stenosis using intravoxel incoherent motion diffusion-weighted magnetic resonance imaging analysis.

OBJECTIVES: Diffusion-weighted magnetic resonance imaging is a powerful tool to assess renal morphology. However, its quantitative index, apparent diffusion coefficient (ADC), derived from a conventional monoexponential model can vary with both functional and structural alterations as well as the choice of b values. In contrast, the intravoxel incoherent motion (IVIM) biexponential model provides independent parameters that may represent broader aspects of renal pathophysiology. We hypothesized that IVIM analysis is capable of detecting early morphological and functional changes in the swine kidney distal to renal artery stenosis (RAS). MATERIALS AND METHODS: Domestic pigs divided into 3 groups (n = 6-7 each) were studied for 16 weeks. Unilateral RAS was induced in 2 groups, of which 1 group was fed with a high-cholesterol diet to induce early atherosclerosis (ARAS), whereas the other (RAS) consumed regular diet. The third group included healthy pigs that served as control sham. Renal function, hemodynamics, tubular function, and morphology were assessed using multidetector computed tomography and histology. Diffusion-weighted magnetic resonance images were acquired at 3T and analyzed using monoexponential and biexponential models. Parameters of ADC and IVIM (diffusivity [D(t)], flow-dependent pseudodiffusivity [D(p)], and fluid fraction [f(p)]) were calculated in the cortex and medulla of the stenotic (STK) and contralateral kidneys (CLKs). Results were analyzed using analysis of variance, Student t test, and regression analysis. RESULTS: In both RAS and ARAS, the STK shrank and the CLK underwent hypertrophy. Glomerular filtration rate and renal blood flow declined in STKs, and CLKs manifested hyperfiltration. In addition, ARAS kidneys showed reduced mean transit time in distal tubular segments. Apparent diffusion coefficient and diffusivity both decreased in STK of RAS and ARAS. D(p) and f(p) were elevated in both the STK and CLK of RAS and more prominently in ARAS. The STK cortical ADC and D(t) correlated inversely with the degree of fibrosis and directly with glomerular filtration rate. Furthermore, D(p) correlated with tubular injury score in all kidneys. CONCLUSIONS: Apparent diffusion constant and D(t) both correlated with cortical and medullary fibrosis; however, IVIM-derived parameters can detect subtle functional and structural changes in the post-STK and may also serve as markers for tubular injury.