Saadiq, Ishran M.

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.

Tumor necrosis factor-α-induced gene/protein (TSG)-6 regulates the immunomodulatory properties of MSCs, but its ability to protect the ischemic kidney is unknown. In pigs with renal artery stenosis, we show that MSC delivery increased renal vein TSG-6, decreased kidney inflammatory macrophages, and improved renal function. In vitro, TSG-6 decreased inflammatory macrophages and tubular cell injury. Therefore, TSG-6 released from MSCs may decrease renal tubular cell injury, which is associated with regulating macrophage function and phenotype.

Abstract BACKGROUND Renovascular hypertension (RVH) often manifest with metabolic syndrome (MetS) as well. Coexisting MetS and hypertension increases cardiovascular morbidity and mortality, but the mechanisms underlying cardiac injury remain unknown. We hypothesized that superimposition of MetS induces myocardial mitochondrial damage, leading to cardiac injury and dysfunction in swine RVH. METHODS Pigs were studied after 16 weeks of diet-induced MetS with or without RVH (unilateral renal artery stenosis), and Lean controls (n = 6 each). Systolic and diastolic cardiac function were assessed by multidetector CT, and cardiac mitochondrial morphology (electron microscopy) and myocardial function in tissue and isolated mitochondria. RESULTS Body weight was similarly higher in MetS groups vs. Lean. RVH groups achieved significant stenosis and developed hypertension. Mitochondrial matrix density and adenosine triphosphate production were lower and H2O2 production higher in RVH groups vs. Lean and MetS. Lean + RVH (but not MetS + RVH) activated mitophagy, which was associated with decreased myocardial expression of mitophagy-related microRNAs. MetS groups exhibited higher numbers of intermitochondrial junctions, which could have prevented membrane depolarization/activation of mitophagy in MetS + RVH. Cardiac fibrosis, hypertrophy (increased left ventricular muscle mass), and diastolic function (decreased E/A ratio) were greater in MetS + RVH vs. Lean + RVH. CONCLUSIONS MetS+RVH induces myocardial mitochondrial damage and dysfunction. MetS + RVH failed to activate mitophagy, resulting in greater cardiac remodeling, fibrosis, and diastolic dysfunction. Mitochondrial injury and impaired mitophagy may constitute important mechanisms and therapeutic targets to ameliorate cardiac damage and dysfunction in patients with coexisting MetS and RVH.

AbstractPercutaneous 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 H202 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.

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.

Abstract BACKGROUND Mitochondria modulate endothelial cell (EC) function, but may be damaged during renal disease. We hypothesized that the ischemic and metabolic constituents of swine renovascular disease (RVD) induce mitochondrial damage and impair the function of renal artery ECs. METHODS Pigs were studied after 16 weeks of metabolic syndrome (MetS), renal artery stenosis (RAS), or MetS + RAS, and Lean pigs served as control (n = 6 each). Mitochondrial morphology, homeostasis, and function were measured in isolated primary stenotic-kidney artery ECs. EC functions were assessed in vitro, whereas vasoreactivity of renal artery segments was characterized in organ baths. RESULTS Lean + RAS and MetS + RAS ECs showed increased mitochondrial area and decreased matrix density. Mitochondrial biogenesis was impaired in MetS and MetS + RAS compared with their respective controls. Mitochondrial membrane potential similarly decreased in MetS, Lean + RAS, and MetS + RAS groups, whereas production of reactive oxygen species increased in MetS vs. Lean, but further increased in both RAS groups. EC tube formation was impaired in MetS, RAS, and MetS + RAS vs. Lean, but EC proliferation and endothelial-dependent relaxation of renal artery segments were blunted in MetS vs. Lean, but further attenuated in Lean + RAS and MetS + RAS. CONCLUSIONS MetS and RAS damage mitochondria in pig renal artery ECs, which may impair EC function. Coexisting MetS and RAS did not aggravate EC mitochondrial damage in the short time of our in vivo studies, suggesting that mitochondrial injury is associated with impaired renal artery EC function.

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