Citations

Abstract Background and Aims Endothelial progenitor cells (EPCs) patrol the circulation and contribute to endothelial cell regeneration and tissue revascularization. Atherosclerotic renal artery stenosis (ARAS) induces microvascular loss in the stenotic kidney. A regimen of low–energy shockwave therapy (SW) induces angiogenesis and reduces chronic ischemia, but the mechanism remains unclear. This study tested the hypothesis that SW increases EPCs homing to the stenotic kidney and increases renal capillary regeneration in a unilateral ARAS swine model. Method Domestic pigs were randomized to normal control or unilateral ARAS (induced by a local irritant coil in the renal artery and a high-fat diet). ARAS pigs were treated with low-energy SW (0.1 mJ/mm2) or sham (n=6 each group), bi-weekly for 3 consecutive weeks, starting after 3 weeks of ARAS. Blood samples for EPCs (CD34+ and KDR+ by flow cytometry) and the homing factor SDF-1 were collected 4 weeks after completion of SW treatment from the inferior vena cava (IVC) and the stenotic kidney (STK) vein and artery. Urine was collected from the urinary bladder. Kidneys were studied ex vivo for morphology and expression of an endothelial cell marker (CD31) and the pro- angiogenic growth factor angiopoietin (Ang)-1. Results In ARAS, tubulointerstitial fibrosis, tubular score, urinary protein, serum creatinine and mean arterial pressure were significantly increased and capillary count (Fig. 1A) decreased (P<0.05 vs. control), but all markedly improved in ARAS+SW (P<0.05 vs. ARAS). EPC number was decreased in the IVC and renal artery of ARAS pigs (Fig. 1B), but improved in ARAS+SW. SDF-1 levels in the IVC, STK vein and artery, and Ang-1 expression in the kidney of ARAS+SW, were all increased (P<0.05 vs. ARAS). Conclusion Low-energy shockwave improves ischemic kidney capillary density, which is associated with and may be at least in part mediated by promoting EPC mobilization and homing to the stenotic kidney.

Abstract Background and Aims Tumor necrosis factor-α-induced gene/protein (TSG)-6 is a key factor influencing mesenchymal stem cells (MSCs) immunomodulatory properties, but its renoprotective efficacy is unknown. Using a novel swine model of renal artery stenosis (RAS) complicated by metabolic syndrome (Mets), we assessed the therapeutic effects of adipose tissue-derived MSCs-produced TSG-6 and mechanisms underlying the immunomodulatory properties of MSCs. Method Five groups of pigs (n=6 each) were studied after 16 weeks of diet-induced Mets and unilateral RAS (Mets+RAS), either untreated or treated 4 weeks earlier with a single intra-renal delivery of autologous posrcine adipose tissue-derived MSCs (pMSC). Lean, Mets, and RAS shams served as controls. We studied renal function in vivo (using CT imaging) and kidney histopathology and macrophage phenotype ex vivo. In vitro, TSG-6 levels were measured in conditioned media of human MSCs (hMSCs) incubated with or without TNF-α. Additionally, levels of the tubular injury marker LDH were measured in conditioned media after co-culturing macrophages with injured HK-2 cells (achieved by TNF-α and antimycin-A, AMA) with or without addition of TSG-6. The effects of TSG-6 on macrophage phenotype (M1/M2), adhesion, and migration capability were determined. Results Mets+RAS pigs showed increased renal M1 macrophages and renal vein TNF-α levels. After p-MSCs delivery, renal vein TSG-6 increased and TNF-α decreased, M1 macrophage switched to M2 (Fig. A),, renal function improved, and fibrosis alleviated. In vitro, TNF-α increased TSG-6 secretion by h-MSCs. TSG-6 decreased LDH release from injured HK-2, induced a macrophage phenotypic switch from M1 to M2 (Fig. B), and reduced M1 macrophage adhesion and migration (Fig. C). Conclusion TNF-α-induced TSG-6 release from MSCs in vivo and in vitro may decrease renal tubular cells injury, which is associated with and may be at least in part mediated by regulating macrophage function and phenotype.

Scattered tubular-like cells (STCs) are dedifferentiated surviving tubular epithelial cells that repair neighboring injured cells. Experimental renal artery stenosis (RAS) impairs STC reparative potency by inducing mitochondrial injury, but the exact mechanisms of mitochondrial damage remain unknown. We hypothesized that RAS alters expression of mitochondria-related genes, contributing to mitochondrial structural damage and dysfunction in swine STCs. CD24+/CD133+STCs were isolated from pig kidneys after 10 wk of RAS or sham ( n = 3 each). mRNA sequencing was performed, and nuclear DNA (nDNA)-encoded mitochondrial genes and mitochondrial DNA (mtDNA)-encoded genes were identified. Mitochondrial structure, ATP generation, biogenesis, and expression of mitochondria-associated microRNAs were also assessed. There were 96 nDNA-encoded mitochondrial genes upregulated and 12 mtDNA-encoded genes downregulated in RAS-STCs versus normal STCs. Functional analysis revealed that nDNA-encoded and mtDNA-encoded differentially expressed genes were primarily implicated in mitochondrial respiration and ATP synthesis. Mitochondria from RAS STCs were swollen and showed cristae remodeling and loss and decreased ATP production. Immunoreactivity of the mitochondrial biogenesis marker peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α and expression of the mitochondria-associated microRNAs miR-15a, miR-181a, miR-196a, and miR-296-3p, which target several mtDNA genes, were higher in RAS-STCs compared with normal STCs, suggesting a potential modulation of mitochondria-related gene expression. These results demonstrate that RAS induces an imbalance in mtDNA- and nDNA-mitochondrial gene expression, impairing mitochondrial structure and function in swine STCs. These observations support development of gene gain- and loss-of-function strategies to ameliorate mitochondrial damage and preserve the reparative potency of STCs in patients with renal ischemia.

AbstractEffective 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.

Cell-derived extracellular vesicles (EVs) can be isolated from various body fluids, including urine. Urinary EVs have gained important recognition as potential diagnostic biomarkers in renal disease since their cargo includes nucleic acids, proteins, and other cellular components, which likely mirror the physiological and possibly pathophysiological state of cells along the nephron. Accumulating evidence highlights the feasibility of using EVs as biomarkers for diagnostic, prognostic, and therapeutic purposes in several forms of renal disease, such as acute kidney injury, glomerulonephritis, and renal transplantation. Additionally, exogenous delivery of EVs released in vitro by cells in culture may have salutary benefits for renal diseases. In this review, we introduce recent studies that attempt to identify urinary EVs as candidate biomarkers for human kidney diseases and consider their potential implication as a therapeutic option in key kidney diseases.

Background Peripheral microvascular endothelial dysfunction (PMED) has been linked to an increased risk of cardiovascular events, but there is a lack of information characterizing the predictive value of PMED for future risk of ischemic stroke (IS). Methods and Results This retrospective observational cohort study enrolled 637 patients who underwent non‐invasive microvascular endothelial function assessment using reactive hyperemia peripheral arterial tonometry. Reactive hyperemia peripheral arterial tonometry index ≤2 was defined as PMED. Of 280 patients with PMED, 12 (4.3%) patients developed IS, compared with only 4 (1.1%) of 357 patients without PMED during a median follow‐up of 5.3 years. Patients with PMED had lower IS‐free survival compared with patients without PMED (log‐rank P =0.03). Cox proportional hazard ratio (HR) analyses showed that PMED predicted the incidence of IS, with a HR of 3.43, 95% CI, 1.10–10.63 ( P =0.03); adjusted HR of 3.70, 95% CI, 1.18–11.59 ( P =0.02) after adjusting for sex, smoking history, and atrial fibrillation; adjusted HR of 3.45, 95% CI, 1.11–10.72 ( P =0.03) after adjusting for CHA 2 DS 2 ‐VASc score; adjusted HR of 5.70, 95% CI, 1.40–23.29 ( P =0.02) after adjusting for revised Framingham Stroke Risk Score. Reactive hyperemia peripheral arterial tonometry index improved discrimination of risk for IS after adding reactive hyperemia peripheral arterial tonometry index to CHA 2 DS 2 ‐VASc score and revised Framingham Stroke Risk Score. Conclusions PMED was associated with a >3‐fold increased risk of IS. These findings underscore the concept of the systemic nature of endothelial dysfunction, which could act as a potential marker to predict future risk of IS.

Background Coronary artery disease risk factors are associated with atrial fibrillation ( AF ) and coronary endothelial dysfunction ( CED ). We hypothesized that CED is associated with increased risk of incident AF among patients with chest pain and nonobstructive coronary artery disease. Methods and Results Three hundred patients with chest pain, nonobstructive coronary artery disease, and no history of AF underwent intracoronary acetylcholine infusion for evaluation of baseline epicardial (decrease in mid–left anterior descending coronary artery diameter in response to acetylcholine) and microvascular (<50% increase in coronary blood flow in response to acetylcholine) CED . Primary outcome was incident AF over a mean follow‐up period of 10.5±5.5 years. Mean age was 53.3±10.8 years, and 70% were women. Baseline clinical and echocardiographic characteristics were similar between patients with CED (n=256) and those with normal endothelial function (n=44). Overall, 35 of 300 (12%) patients developed AF , among whom 34 of 35 (97%) had CED at baseline. Compared with normal endothelial function, the presence of CED was associated with 11% increased absolute risk and 5.8‐fold increased relative risk of incident AF . Moreover, CED ( odds ratio , 3.87; 95% CI , 1.27–47.0) and increased (>34 mL/m 2 ) left atrial volume index (odds ratio, 3.87; 95% CI , 1.60–9.11) were independent predictors of incident AF . Conclusions Patients with normal coronary endothelial function, as compared with those with CED and similar AF risk factors, have significantly lower incidence of AF on long‐term follow‐up. The potential mechanistic link between vascular dysfunction and AF development warrants further investigation.

Endothelial progenitor cells (EPCs) patrols the circulation and contributes to endothelial cell regeneration. Atherosclerotic renal artery stenosis (ARAS) induces microvascular loss in the stenotic kidney (STK). Low-energy shockwave therapy (SW) can induce angiogenesis and restore the STK microcirculation, but the underlying mechanism remains unclear. We tested the hypothesis that SW increases EPC homing to the swine STK, associated with capillary regeneration. Normal pigs and pigs after 3 wk of renal artery stenosis were treated with six sessions of low-energy SW (biweekly for three consecutive weeks) or left untreated. Four weeks after completion of treatment, we assessed EPC (CD34+/KDR+) numbers and levels of the homing-factor stromal cell-derived factor (SDF)-1 in the inferior vena cava and the STK vein and artery, as well as urinary levels of vascular endothelial growth factor (VEGF) and integrin-1β. Subsequently, we assessed STK morphology, capillary count, and expression of the proangiogenic growth factors angiopoietin-1, VEGF, and endothelial nitric oxide synthase ex vivo. A 3-wk low-energy SW regimen improved STK structure, capillary count, and function in ARAS+SW, and EPC numbers and gradients across the STK decreased. Plasma SDF-1 and renal expression of angiogenic factors were increased in ARAS+SW, and urinary levels of VEGF and integrin-1β tended to rise during the SW regimen. In conclusion, SW improves ischemic kidney capillary density, which is associated with, and may be at least in part mediated by, promoting EPCs mobilization and homing to the stenotic 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 β-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.