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.

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.

Improving Postoperative Acute Kidney Injury Rates Following Primary Total Joint Arthroplasty.

BACKGROUND: Perioperative hip and knee arthroplasty complications remain a significant clinical and financial burden. Our institution has shifted to developing protocols to decrease these perioperative complications. This study focuses on acute kidney injury (AKI) rate status post primary total joint arthroplasty (TJA). Current literature demonstrates a 2%-15% incidence of AKI following TJA. However, there is a paucity of published literature on protocols that have effectively reduced AKI rates following TJA. The purpose of this study is to evaluate the effect that our institutionally developed perioperative renal protocol had on the postoperative AKI rates. METHODS: A retrospective cohort study was performed. Patient demographics, baseline creatinine, and postoperative creatinine values during the patient's hospitalization were collected and analyzed. The preintervention cohort data contained all patients at our institution who underwent a primary TJA from November 1, 2016 to January 1, 2018. The postintervention cohort included all primary TJA patients from July 1, 2018 to February 2, 2020. AKI was defined using the AKI Network classification system comparing baseline and postoperative creatinine values. A multivariate analysis was performed to determine the statistical significance of our results. RESULTS: Before intervention 1013 patients underwent a primary TJA with 68 patients developing an AKI postoperatively. After intervention 2169 patients underwent primary TJA with 90 patients developing an AKI (6.71% vs 4.15%; P = .0015, odds ratio = 0.59, 95% confidence interval = 0.42-0.82). CONCLUSION: This study demonstrated that implementation of a perioperative renal protocol can significantly reduce AKI rates. A reduction in AKI rates following TJA will result in improved outcomes and secondarily decrease the financial impact of postoperative complications seen following TJA.

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.

Transplanted senescent renal scattered tubular-like cells induce injury in the mouse kidney.

Cellular senescence, a permanent arrest of cell proliferation, is characterized by a senescence-associated secretory phenotype (SASP), which reinforces senescence and exerts noxious effects on adjacent cells. Recent studies have suggested that transplanting small numbers of senescent cells suffices to provoke tissue inflammation. We hypothesized that senescent cells can directly augment renal injury. Primary scattered tubular-like cells (STCs) acquired from pig kidneys were irradiated by 10 Gy of cesium radiation, and 3 wk later cells were characterized for levels of senescence and SASP markers. Control or senescent STCs were then prelabeled and injected (5 × 105 cells) into the aorta of C57BL/6J mice. Four weeks later, renal oxygenation was studied in vivo using 16.4-T magnetic resonance imaging and function by plasma creatinine level. Renal markers of SASP, fibrosis, and microvascular density were evaluated ex vivo. Per flow cytometry, irradiation induced senescence in 80-99% of STCs, which showed increased gene expression of senescence and SASP markers, senescence-associated ß-galactosidase staining, and cytokine levels (especially IL-6) secreted in conditioned medium. Four weeks after injection, cells were detected engrafted in the mouse kidneys with no evidence for rejection. Plasma creatinine and renal tissue hypoxia increased in senescent compared with control cells. Senescent kidneys were more fibrotic, with fewer CD31+ endothelial cells, and showed upregulation of IL-6 gene expression. Therefore, exogenously delivered senescent renal STCs directly injure healthy mouse kidneys. Additional studies are needed to determine the role of endogenous cellular senescence in the pathogenesis of kidney injury and evaluate the utility of senolytic therapy.

In a Phase 1a escalating clinical trial, autologous mesenchymal stem cell infusion for renovascular disease increases blood flow and the glomerular filtration rate while reducing inflammatory biomarkers and blood pressure.

Atherosclerotic renovascular disease (ARVD) reduces tissue perfusion and eventually leads to loss of kidney function with limited therapeutic options. Here we describe results of Phase 1a escalating dose clinical trial of autologous mesenchymal stem cell infusion for ARVD. Thirty-nine patients with ARVD were studied on two occasions separated by three months. Autologous adipose-derived mesenchymal stem cells were infused through the renal artery in 21 patients at three different dose levels (1, 2.5 and 5.0 × 105 cells/kg) in seven patients each. We measured renal blood flow, glomerular filtration rate (GFR) (iothalamate and estimated GFR), renal vein cytokine levels, blood pressure, and tissue oxygenation before and three months after stem cell delivery. These indices were compared to those of 18 patients with ARVD matched for age, kidney function and blood pressure receiving medical therapy alone that underwent an identical study protocol. Cultured mesenchymal stem cells were also studied in vitro. For the entire stem cell treated-cohort, mean renal blood flow in the treated stenotic kidney significantly increased after stem cell infusion from (164 to 190 ml/min). Hypoxia, renal vein inflammatory cytokines, and angiogenic biomarkers significantly decreased following stem cell infusion. Mean systolic blood pressure significantly fell (144 to 136 mmHg) and the mean two-kidney GFR (Iothalamate) modestly but significantly increased from (53 to 56 ml/min). Changes in GFR and blood pressure were largest in the high dose stem cell treated individuals. No such changes were observed in the cohort receiving medical treatment alone. Thus, our data demonstrate the potential for autologous mesenchymal stem cell to increase blood flow, GFR and attenuate inflammatory injury in post-stenotic kidneys. The observation that some effects are dose-dependent and related to in-vitro properties of mesenchymal stem cell may direct efforts to maximize potential therapeutic efficacy.

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.

Quantitative Magnetization Transfer Detects Renal Fibrosis in Murine Kidneys With Renal Artery Stenosis.

BACKGROUND: Renal fibrosis is a common pathway in tubulointerstitial injury and a major determinant of renal insufficiency. Collagen deposition, a key feature of renal fibrosis, may serve as an imaging biomarker to differentiate scarred from healthy kidneys. PURPOSE: To test the feasibility of using quantitative magnetization transfer (qMT), which assesses tissue macromolecule content, to measure renal fibrosis. STUDY TYPE: Prospective. ANIMAL MODEL: Fifteen 129S1 mice were studied 4 weeks after either sham (n = 7) or unilateral renal artery stenosis (RAS, n = 8) surgeries. FIELD STRENGTH/SEQUENCE: Magnetization transfer (MT)-weighted images were acquired at 16.4T using an MT-prepared fast-low-angle-shot sequence. Renal B0, B1, and T1 maps were also acquired, using a dual-echo gradient echo, an actual flip angle, and inversion recovery method, respectively. ASSESSMENT: A two-pool model was used to estimate the bound water fraction (f) and other tissue imaging biomarkers. Masson's trichrome staining was subsequently performed ex vivo to evaluate renal fibrosis. STATISTICAL TESTS: Comparisons of renal parameters between sham and RAS were performed using independent samples t-tests. Pearson's correlation was conducted to investigate the relationship between renal fibrosis by histology and the qMT-derived bound pool fraction f. RESULTS: The two-pool model provided accurate fittings of measured MT signal. The qMT-derived f of RAS kidneys was significantly increased compared to sham in all kidney zones (renal cortex [CO], 7.6 ± 2.4% vs. 4.6 ± 0.6%; outer medulla [OM], 8.2 ± 4.2% vs. 4.2 ± 0.9%; inner medulla [IM] + P, 5.8 ± 1.6% vs. 2.9 ± 0.6%, all P < 0.05). Measured f correlated well with histological fibrosis in all kidney zones (CO, Pearson's correlation coefficient r = 0.95; OM, r = 0.93; IM + P, r = 0.94, all P < 0.05). DATA CONCLUSION: The bound pool fraction f can be quantified using qMT at 16.4T in murine kidneys, increases significantly in fibrotic RAS kidneys, and correlates well with fibrosis by histology. Therefore, qMT may constitute a valuable tool for measuring renal fibrosis in RAS. LEVEL OF EVIDENCE: 1 TECHNICAL EFFICACY STAGE: 3.

Tissue hypoxia, inflammation, and loss of glomerular filtration rate in human atherosclerotic renovascular disease.

The relationships between renal blood flow (RBF), tissue oxygenation, and inflammatory injury in atherosclerotic renovascular disease (ARVD) are poorly understood. We sought to correlate RBF and tissue hypoxia with glomerular filtration rate (GFR) in 48 kidneys from patients with ARVD stratified by single kidney iothalamate GFR (sGFR). Oxygenation was assessed by blood oxygenation level dependent magnetic resonance imaging (BOLD MRI), which provides an index for the levels of deoxyhemoglobin within a defined volume of tissue (R2*). sGFR correlated with RBF and with the severity of vascular stenosis as estimated by duplex velocities. Higher cortical R2* and fractional hypoxia and higher levels of renal vein neutrophil-gelatinase-associated-lipocalin (NGAL) and monocyte-chemoattractant protein-1 (MCP-1) were observed at lower GFR, with an abrupt inflection below 20 ml/min. Renal vein MCP-1 levels correlated with cortical R2* and with fractional hypoxia. Correlations between cortical R2* and RBF in the highest sGFR stratum (mean sGFR 51 ± 12 ml/min; R = -0.8) were degraded in the lowest sGFR stratum (mean sGFR 8 ± 3 ml/min; R = -0.1). Changes in fractional hypoxia after furosemide were also absent in the lowest sGFR stratum. These data demonstrate relative stability of renal oxygenation with moderate reductions in RBF and GFR but identify a transition to overt hypoxia and inflammatory cytokine release with severely reduced GFR. Tissue oxygenation and RBF were less correlated in the setting of reduced sGFR, consistent with variable oxygen consumption or a shift to alternative mechanisms of tissue injury. Identifying transitions in tissue oxygenation may facilitate targeted therapy in ARVD.

Mitoprotection attenuates myocardial vascular impairment in porcine metabolic syndrome.

Metabolic syndrome (MetS) leads to cardiac vascular injury, which may reflect in increased retention of endothelial progenitor cells (EPCs). Coronary endothelial cell (EC) mitochondria partly regulate vascular function and structure. We hypothesized that chronic mitoprotection would preserve EC mitochondria and attenuate coronary vascular injury and dysfunction in swine MetS. Pigs were studied after 16 wk of diet-induced MetS, MetS treated for the last 4 wk with the mitochondria-targeted peptide elamipretide (ELAM; 0.1 mg/kg sc once daily), and lean controls ( n = 6 each). Cardiac remodeling and function were assessed in vivo by multidetector-computed tomography (CT), and coronary artery and sinus blood samples were collected. EC mitochondrial density, apoptosis, oxidative stress, endothelial nitric oxide synthase immunoreactivity, myocardial microvascular density (three-dimensional microcomputed tomography), and coronary endothelial function (organ bath) were assessed ex vivo. The number and arteriovenous gradient of CD34+/KDR+ EPCs were calculated by FACS (a negative net gradient indicating EPC retention). MetS and MetS + ELAM pigs developed similar MetS (obesity, hyperlipidemia, insulin resistance, and hypertension). EC mitochondrial density decreased in MetS animals compared with lean animals but normalized in MetS + ELAM animals. ELAM also attenuated EC oxidative stress and apoptosis and improved subendocardial microvascular density. ELAM-induced vasculoprotection was reflected by decreased coronary retention of EPCs. ELAM also partly improved endothelial nitric oxide synthase immunoreactivity, coronary endothelial function, and vessel maturity, whereas myocardial perfusion was unaffected. Chronic mitoprotection improved coronary EC mitochondrial density and decreased vascular remodeling and dysfunction. However, additional mitochondria-independent mechanisms likely contribute to MetS-induced cardiac vascular injury. NEW & NOTEWORTHY The present study shows that chronic mitoprotection preserved coronary endothelial cell mitochondria and decreased vascular injury, subendocardial microvascular loss, coronary retention of endothelial progenitor cells, and release of markers of vascular injury. However, myocardial perfusion remained blunted, suggesting that additional mitochondria-independent mechanisms likely contribute to metabolic syndrome-induced cardiac vascular injury.

Mitoprotection preserves the renal vasculature in porcine metabolic syndrome.

NEW FINDINGS: What is the central question of this study? We hypothesized that chronic mitoprotection would decrease renal vascular remodelling and dysfunction in swine metabolic syndrome. What is the main finding and its importance? This study shows that experimental metabolic syndrome exerts renal microvascular and endothelial cell mitochondrial injury, which were attenuated by mitoprotection, underscoring the contribution of mitochondrial injury to the pathogenesis of metabolic syndrome-induced vascular damage. ABSTRACT: The metabolic syndrome (MetS) induces intrarenal microvascular disease, which may involve mitochondrial injury. The mitochondrial cardiolipin-targeting peptide elamipretide (ELAM) improves the microcirculation in post-stenotic kidneys, but its ability to attenuate MetS-induced renal vascular damage is unknown. We hypothesized that chronic treatment with ELAM would decrease renal vascular remodelling and function in swine MetS. Pigs were studied after 16 weeks of diet-induced MetS, MetS treated for the last 4 weeks with daily injections of ELAM (0.1 mg kg-1 ), and lean control (Lean) animals (n = 6 each). Single-kidney regional perfusion, blood flow and glomerular filtration rate were measured with multi-detector computed tomography (CT). Peritubular capillary (PTC) endothelial cell (EC) mitochondrial density and cardiolipin content were assessed in situ, as were PTC-EC apoptosis and oxidative stress. The spatial density of PTCs (Haematoxylin and Eosin staining) and renal microvessels (micro-CT), and renal artery endothelial function (organ bath) were characterized. Regional perfusion and serum creatinine were preserved in MetS pigs, but renal blood flow and glomerular filtration rate were higher compared with Lean. Mitochondrial density and cardiolipin content were diminished in MetS PTC-ECs, but improved in ELAM-treated pigs, as did PTC density. Elamipretide also attenuated PTC-EC oxidative stress and apoptosis. Furthermore, ELAM improved renal microvascular density, decreased microvascular remodelling and restored endothelial nitric oxide expression and endothelium-dependent relaxation of renal artery segments. In conclusion, MetS-induced mitochondrial alterations might contribute to renal PTC and microvascular loss and might impair renal artery endothelial function in pigs. Mitoprotection with ELAM preserved a hierarchy of renal vessels, underscoring its potential to ameliorate renal vascular injury in MetS.

Noninvasive Assessment of Renal Fibrosis with Magnetization Transfer MR Imaging: Validation and Evaluation in Murine Renal Artery Stenosis.

Purpose To test the utility of magnetization transfer imaging in detecting and monitoring the progression of renal fibrosis in mice with unilateral renal artery stenosis. Materials and Methods This prospective study was approved by the Institutional Animal Care and Use Committee. Renal artery stenosis surgery (n = 10) or sham surgery (n = 5) was performed, and the stenotic and contralateral kidneys were studied longitudinally in vivo at baseline and 2, 4, and 6 weeks after surgery. After a 16.4-T magnetic resonance imaging examination, magnetization transfer ratio was measured as an index of fibrosis (guided by parameters selected in preliminary phantom studies). In addition, renal volume, perfusion, blood flow, and oxygenation were assessed. Fibrosis was subsequently measured ex vivo by means of histologic analysis and hydroxyproline assay. The Wilcoxon rank sum or signed rank test was used for statistical comparisons between or within groups, and Pearson and Spearman rank correlation was used to compare fibrosis measured in vivo and ex vivo. Results In the stenotic kidney, the median magnetization transfer ratio showed progressive increases from baseline to 6 weeks after surgery (increases of 13.7% [P = .0006] and 21.3% [P = .0005] in cortex and medulla, respectively), which were accompanied by a progressive loss in renal volume, perfusion, blood flow, and oxygenation. The 6-week magnetization transfer ratio map showed good correlation with fibrosis measured ex vivo (Pearson r = 0.9038 and Spearman ρ = 0.8107 [P = .0002 vs trichrome staining]; r = 0.9540 and ρ = 0.8821 [P < .0001 vs Sirius red staining]; and r = 0.8429 and ρ = 0.7607 [P = .001 vs hydroxyproline assay]). Conclusion Magnetization transfer imaging was used successfully to measure and longitudinally monitor the progression of renal fibrosis in mice with unilateral renal artery stenosis. © RSNA, 2016 Online supplemental material is available for this article.

Efficacy of Human Embryonic Stem Cells Compared to Adipose Tissue-Derived Human Mesenchymal Stem/Stromal Cells for Repair of Murine Post-Stenotic Kidneys.

Clinical translation of mesenchymal stem/stromal cell (MSC) therapy has been impeded by the heterogenous nature and limited replicative potential of adult-derived MSCs. Human embryonic stem cell-derived MSCs (hESC-MSCs) that differentiate from immortal cell lines are phenotypically uniform and have shown promise in-vitro and in many disease models. Similarly, adipose tissue-derived MSCs (MSC(AT)) possess potent reparative properties. How these two cell types compare in efficacy, however, remains unknown. We randomly assigned mice to six groups (n = 7-8 each) that underwent unilateral RAS or a sham procedure (3 groups each). Two weeks post-operation, each mouse was administered either vehicle, MSC(AT)s, or hESC-MSCs (5 × 105 cells) into the aorta. Mice were scanned with micro-MRI to determine renal hemodynamics two weeks later and kidneys then harvested. hESC-MSCs and MSC(AT)s were similarly effective at lowering systolic blood pressure. However, MSC(AT)s more robustly increased renal perfusion, oxygenation, and glomerular filtration rate in the post-stenotic kidney, and more effectively mitigated tubular injury, fibrosis, and vascular remodeling. These observations suggest that MSC(AT) are more effective than hESC-MSC in ameliorating kidney dysfunction and tissue injury distal to RAS. Our findings highlight the importance of tissue source in selection of MSCs for therapeutic purposes and underscore the utility of cell-based therapy for kidney disease.

Human Obesity Attenuates Cardioprotection Conferred by Adipose Tissue-Derived Mesenchymal Stem/Stromal Cells.

To explore the impact of obesity on reparative potency of adipose tissue-derived mesenchymal stromal/stem cells (A-MSC) in hypertensive cardiomyopathy, A-MSC were harvested from subcutaneous fat of obese and age-matched non-obese human subjects during bariatric or kidney donation surgeries, and then injected into mice 2 weeks after inducing renovascular hypertension (RVH) or sham surgery. Two weeks later, left ventricular (LV) function and deformation were estimated in vivo by micro-magnetic resonance imaging and myocardial damage ex vivo. Blood pressure and myocardial wall thickening were elevated in RVH + Vehicle and normalized only by lean-A-MSC. Both A-MSC types reduced LV mass and normalized the reduced LV peak strain radial in RVH, yet obese-A-MSC also impaired LV systolic function. A-MSC alleviated myocardial tissue damage in RVH, but lean-A-MSC decreased oxidative stress more effectively. Obese-A-MSC also showed increased cellular inflammation in vitro. Therefore, obese-A-MSC are less effective than lean-A-MSC in blunting hypertensive cardiomyopathy in mice with RVH.

Reliable Assessment of Swine Renal Fibrosis Using Quantitative Magnetization Transfer Imaging.

OBJECTIVES: Quantitative magnetization transfer (qMT) is useful for measurement of murine renal fibrosis at high and ultrahigh field strengths. However, its utility at clinical field strengths and in human-like kidneys remains unknown. We tested the hypothesis that qMT would successfully detect fibrosis in swine kidneys with unilateral renal artery stenosis (RAS) at 3.0 T. METHODS: The qMT protocol is composed of MT scans with variable flip angles and offset frequencies, and of B0, B1, and T1 mapping. Pigs were scanned 10 weeks after RAS or control. A 2-pool model was used to fit the bound pool fraction f of the renal cortex (CO) and outer medulla (OM). Then qMT-derived f in 5 normal and 10 RAS pigs was compared with histological fibrosis determined using Masson's trichrome staining and to renal perfusion assessed with computed tomography. RESULTS: The qMT 2-pool model provided accurate fittings of data collected on swine kidneys. Stenotic kidneys showed significantly elevated f in both the CO (9.8% ± 2.7% vs 6.4% ± 0.9%, P = 0.002) and OM (7.6% ± 2.2% vs 4.7% ± 1.1%, P = 0.002), as compared with normal kidneys. Histology-measured renal fibrosis and qMT-derived f correlated directly in both the cortex (Pearson correlation coefficient r = 0.93, P < 0.001) and OM (r = 0.84, P = 0.002), and inversely with stenotic kidney perfusion (r = 0.85, P = 0.002). CONCLUSIONS: This study demonstrates the feasibility of qMT for measuring fibrosis in human-like swine kidneys, and the association between tissue macromolecule content and renal perfusion. Therefore, qMT may be useful as a tool for noninvasive assessment of renal fibrosis in subjects with RAS at clinical field strengths.

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

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

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.

Magnetization Transfer Imaging Predicts Porcine Kidney Recovery After Revascularization of Renal Artery Stenosis.

MATERIALS AND METHODS: Stenotic kidney (STK) and contralateral kidney magnetization transfer ratios (MTRs; Mt/M0) were measured at 3.0-T magnetic resonance imaging, at offset frequencies of 600 and 1000 Hz, before and 1 month post-PTRA in 7 RVD pigs. Stenotic kidney MTR was correlated to renal perfusion, renal blood flow (RBF), and glomerular filtration rate (GFR), determined using multidetector computed tomography and with ex vivo renal fibrosis (trichrome staining). Untreated RVD (n = 6) and normal pigs (n = 7) served as controls. RESULTS: Renovascular disease induced hypertension and renal dysfunction. Blood pressure and renal perfusion were unchanged post-PTRA, but GFR and RBF increased. Baseline cortical STK-MTR predicted post-PTRA renal perfusion and RBF, and MTR changes associated inversely with changes in perfusion and normalized GFR. Stenotic kidney MTR at 600 Hz showed closer association with renal parameters, but both frequencies predicted post-PTRA cortical fibrosis. CONCLUSIONS: Renal STK-MTR, particularly at 600 Hz offset, is sensitive to hemodynamic changes after PTRA in swine RVD and capable of noninvasively predicting post-PTRA kidney perfusion, RBF, and fibrosis. Therefore, STK-MTR may be a valuable tool to predict renal hemodynamic and functional recovery, as well as residual kidney fibrosis after revascularization in RVD.

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

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