Textor, Stephen C.

Objective Activin A, an inflammatory mediator implicated in cellular senescence-induced adipose tissue dysfunction and profibrotic kidney injury, may become a new target for the treatment of diabetic kidney disease (DKD) and chronic kidney diseases. We tested the hypothesis that human DKD-related injury leads to upregulation of activin A in blood and urine and in a human kidney cell model. We further hypothesized that circulating activin A parallels kidney injury markers in DKD. Research design and methods In two adult diabetes cohorts and controls (Minnesota, USA; Galway, Ireland), the relationships between plasma (or urine) activin A, estimated glomerular filtration rate (eGFR) and DKD injury biomarkers were tested with logistic regression and correlation coefficients. Activin A, inflammatory, epithelial-mesenchymal-transition (EMT) and senescence markers were assayed in human kidney (HK-2) cells incubated in high glucose plus transforming growth factor-β1 or albumin. Results Plasma activin A levels were elevated in diabetes (n=206) compared with controls (n=76; 418.1 vs 259.3 pg/mL; p<0.001) and correlated inversely with eGFR (r s =−0.61; p<0.001; diabetes). After eGFR adjustment, only albuminuria (OR 1.56, 95% CI 1.16 to 2.09) and tumor necrosis factor receptor-1 (OR 6.40, 95% CI 1.08 to 38.00) associated with the highest activin tertile. Albuminuria also related to urinary activin (r s =0.65; p<0.001). Following in vitro HK-2 injury, activin, inflammatory, EMT genes and supernatant activin levels were increased. Conclusions Circulating activin A is increased in human DKD and correlates with reduced kidney function and kidney injury markers. DKD-injured human renal tubule cells develop a profibrotic and inflammatory phenotype with activin A upregulation. These findings underscore the role of inflammation and provide a basis for further exploration of activin A as a diagnostic marker and therapeutic target in DKD.

Background Hypertension may be associated with renal cellular injury. Cells in distress release extracellular vesicles ( EV s), and their numbers in urine may reflect renal injury. Cellular senescence, an irreversible growth arrest in response to a noxious milieu, is characterized by release of proinflammatory cytokines. We hypothesized that EV s released by senescent nephron cells can be identified in urine of patients with hypertension. Methods and Results We recruited patients with essential hypertension ( EH ) or renovascular hypertension and healthy volunteers (n=14 each). Renal oxygenation was assessed using magnetic resonance imaging and blood samples collected from both renal veins for cytokine‐level measurements. EV s isolated from urine samples were characterized by imaging flow cytometry based on specific markers, including p16 (senescence marker), calyxin (podocytes), urate transporter 1 (proximal tubules), uromodulin (ascending limb of Henle's loop), and prominin‐2 (distal tubules). Overall percentage of urinary p16+ EV s was elevated in EH and renovascular hypertension patients compared with healthy volunteers and correlated inversely with renal function and directly with renal vein cytokine levels. Urinary levels of p16 + /urate transporter 1 + were elevated in all hypertensive subjects compared with healthy volunteers, whereas p16 + /prominin‐2 + levels were elevated only in EH versus healthy volunteers and p16 + /uromodulin + in renovascular hypertension versus EH . Conclusions Levels of p16 + EV s are elevated in urine of hypertensive patients and may reflect increased proximal tubular cellular senescence. In EH , EV s originate also from distal tubules and in renovascular hypertension from Henle's loop. Hence, urinary EV s levels may be useful to identify intrarenal sites of cellular senescence.

Metabolic syndrome (MetS) is associated with nutrient surplus and kidney hyperfiltration, accelerating chronic renal failure. The potential involvement of podocyte damage in early MetS remains unclear. Mitochondrial dysfunction is an important determinant of renal damage, but whether it contributes to MetS-related podocyte injury remains unknown. Domestic pigs were studied after 16 wk of diet-induced MetS, MetS treated with the mitochondria-targeted peptide elamipretide (ELAM; 0.1 mg·kg−1·day−1 sc) for the last month of diet, and lean controls ( n = 6 pigs/group). Glomerular filtration rate (GFR) and renal blood flow (RBF) were measured using multidetector computed tomography, and podocyte and mitochondrial injury were measured by light and electron microscopy. Urinary levels of podocyte-derived extracellular vesicles (pEVs; nephrin positive/podocalyxin positive) were characterized by flow cytometry. Body weight, blood pressure, RBF, and GFR were elevated in MetS. Glomerular size and glomerular injury score were also elevated in MetS and decreased after ELAM treatment. Evidence of podocyte injury, impaired podocyte mitochondria, and foot process width were all increased in MetS but restored with ELAM. The urinary concentration of pEVs was elevated in MetS pigs and directly correlated with renal dysfunction, glomerular injury, and fibrosis and inversely correlated with glomerular nephrin expression. Additionally, pEV numbers were elevated in the urine of obese compared with lean human patients. Early MetS induces podocyte injury and mitochondrial damage, which can be blunted by mitoprotection. Urinary pEVs reflecting podocyte injury might represent early markers of MetS-related kidney disease and a novel therapeutic target.

AbstractAimsPatients with renovascular hypertension (RVH) exhibit elevated urinary mtDNA copy numbers, considered to constitute surrogate markers of renal mitochondrial injury. The modest success of percutaneous transluminal renal angioplasty (PTRA) in restoring renal function in RVH has been postulated to be partly attributable to acute reperfusion injury. We hypothesized that mitoprotection during revascularization would ameliorate PTRA–induced renal mitochondrial injury, reflected in elevated urinary mtDNA copy numbers and improve blood pressure and functional outcomes 3 months later.MethodsWe prospectively measured urinary copy number of the mtDNA genes COX3 and ND1 using qPCR in RVH patients before and 24 hrs after PTRA, performed during IV infusion of vehicle (n = 8) or the mitoprotective drug elamipretide (ELAM, 0.05 mg/kg/h, n = 6). Five healthy volunteers (HV) served as controls. Urinary mtDNA levels were also assessed in RVH and normal pigs (n = 7 each), in which renal mitochondrial structure and density were studied ex‐vivo.ResultsBaseline urinary mtDNA levels were elevated in all RVH patients vs HV and directly correlated with serum creatinine levels. An increase in urinary mtDNA 24 hours after PTRA was blunted in PTRA+ELAM vs PTRA+Placebo. Furthermore, 3‐months after PTRA, systolic blood pressure decreased and estimated glomerular filtration rate increased only in ELAM–treated subjects. In RVH pigs, mitochondrial damage was observed using electron microscopy in tubular cells and elevated urinary mtDNA levels correlated inversely with renal mitochondrial density.ConclusionsPTRA leads to an acute rise in urinary mtDNA, reflecting renal mitochondrial injury that in turn inhibits renal recovery. Mitoprotection might minimize PTRA–associated mitochondrial injury and improve renal outcomes after revascularization.

Hypertension, an important cause of chronic kidney disease, is characterized by peritubular capillary (PTC) loss. Circulating levels of endothelial microparticles (EMPs) reflect systemic endothelial injury. We hypothesized that systemic and urinary PTC-EMPs levels would reflect renal microvascular injury in hypertensive patients. We prospectively measured by flow cytometry renal vein, inferior vena cava, and urinary levels of EMPs in essential (n=14) and renovascular (RVH; n=24) hypertensive patients and compared them with peripheral blood and urinary levels in healthy volunteers (n=14). PTC-EMPs were identified as urinary exosomes positive for the PTC marker plasmalemmal-vesicle–associated protein. In 7 RVH patients, PTC and fibrosis were also quantified in renal biopsy, and in 18 RVH patients, PTC-EMPs were measured again 3 months after continued medical therapy with or without stenting (n=9 each). Renal vein and systemic PTC-EMPs levels were not different among the groups, whereas their urinary levels were elevated in both RVH and essential hypertension versus healthy volunteers (56.8%±12.7% and 62.8%±10.7% versus 34.0%±17.8%; both P ≤0.001). Urinary PTC-EMPs levels correlated directly with blood pressure and inversely with estimated glomerular filtration rate. Furthermore, in RVH, urinary PTC-EMPs levels correlated directly with stenotic kidney hypoxia, histological PTC count, and fibrosis and inversely with cortical perfusion. Three months after treatment, the change in urinary PTC-EMPs levels correlated inversely with a change in renal function ( r =−0.582; P =0.011). Therefore, urinary PTC-EMPs levels are increased in hypertensive patients and may reflect renal microcirculation injury, whereas systemic PTC-EMPs levels are unchanged. Urinary PTC-EMPs may be useful as novel biomarkers of intrarenal capillary loss.

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.

Background— Atherosclerotic renal artery stenosis reduces renal blood flow (RBF) and amplifies stenotic kidney hypoxia. Revascularization with percutaneous transluminal renal angioplasty (PTRA) and stenting often fails to recover renal function, possibly because of ischemia/reperfusion injury developing after PTRA. Elamipretide is a mitochondrial-targeted peptide that binds to cardiolipin and stabilizes mitochondrial function. We tested the hypothesis that elamipretide plus PTRA would improve renal function, oxygenation, and RBF in patients with atherosclerotic renal artery stenosis undergoing PTRA. Methods and Results— Inpatient studies were performed in patients with severe atherosclerotic renal artery stenosis scheduled for PTRA. Patients were treated before and during PTRA with elamipretide (0.05 mg/kg per hour intravenous infusion, n=6) or placebo (n=8). Stenotic kidney cortical/medullary perfusion and RBF were measured using contrast-enhanced multidetector CT, and renal oxygenation by 3-T blood oxygen level−dependent magnetic resonance imaging before and 3 months after PTRA. Age and basal glomerular filtration rate did not differ between groups. Blood oxygen level−dependent imaging demonstrated increased fractional hypoxia 24 hours after angiography and stenting in placebo (+47%) versus elamipretide (−6%). These were reverted to baseline 3 months later. Stenotic kidney RBF rose (202±29–262±115 mL/min; P =0.04) 3 months after PTRA in the elamipretide-treated group only. Over 3 months, systolic blood pressure decreased, and estimated glomerular filtration rate increased ( P =0.003) more in the elamipretide group than in the placebo group ( P =0.11). Conclusions— Adjunctive elamipretide during PTRA was associated with attenuated postprocedural hypoxia, increased RBF, and improved kidney function in this pilot trial. These data support a role for targeted mitochondrial protection to minimize procedure-associated ischemic injury and to improve outcomes of revascularization for human atherosclerotic renal artery stenosis. Clinical Trial Registration— URL: https://www.clinicaltrials.gov . Unique identifier: NCT01755858.