Citations

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.

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.

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.

Mesenchymal stromal/stem cells (MSCs) belong to the endogenous cellular reparative system, and can be used exogenously in cell-based therapy. MSCs release extracellular vesicles (EVs), including exosomes and microvesicles, which mediate some of their therapeutic activity through intercellular communication. We have previously demonstrated that metabolic syndrome (MetS) modifies the cargo packed within swine EV, but whether it influences their phenotypical characteristics remains unclear. This study tested the hypothesis that MetS shifts the size distribution of MSC-derived EVs. Adipose tissue-derived MSC-EV subpopulations from Lean ( n = 6) and MetS ( n = 6) pigs were characterized for number and size using nanoparticle-tracking analysis, flow cytometry, and transmission electron microscopy. Expression of exosomal genes was determined using next-generation RNA-sequencing (RNA-seq). The number of EV released from Lean and MetS pig MSCs was similar, yet MetS-MSCs yielded a higher proportion of small-size EVs (202.4 ± 17.7 nm vs. 280.3 ± 15.1 nm), consistent with exosomes. RNA-seq showed that their EVs were enriched with exosomal markers. Lysosomal activity remained unaltered in MetS-MSCs. Therefore, MetS alters the size distribution of MSC-derived EVs in favor of exosome release. These observations may reflect MSC injury and membrane recycling in MetS or increased expulsion of waste products, and may have important implications for development of adequate cell-based treatments.

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.

Abstract Mesenchymal stem/stromal cells (MSCs) release extracellular vesicles (EVs), which shuttle proteins to recipient cells, promoting cellular repair. We hypothesized that cardiovascular risk factors may alter the pattern of proteins packed within MSC-derived EVs. To test this, we compared the protein cargo of EVs to their parent MSCs in pigs with metabolic syndrome (MetS) and Lean controls. Porcine MSCs were harvested from abdominal fat after 16 weeks of Lean- or MetS-diet (n = 5 each), and their EVs isolated. Following liquid chromatography mass spectrometry proteomic analysis, proteins were classified based on cellular component, molecular function, and protein class. Five candidate proteins were validated by Western blot. Clustering analysis was performed to identify primary functional categories of proteins enriched in or excluded from EVs. Proteomics analysis identified 6,690 and 6,790 distinct proteins in Lean- and MetS-EVs, respectively. Differential expression analysis revealed that 146 proteins were upregulated and 273 downregulated in Lean-EVs versus Lean-MSCs, whereas 787 proteins were upregulated and 185 downregulated in MetS-EVs versus MetS-MSCs. Proteins enriched in both Lean- and MetS-EVs participate in vesicle-mediated transport and cell-to-cell communication. Proteins enriched exclusively in Lean-EVs modulate pathways related to the MSC reparative capacity, including cell proliferation, differentiation, and activation, as well as transforming growth factor-β signaling. Contrarily, proteins enriched only in MetS-EVs are linked to proinflammatory pathways, including acute inflammatory response, leukocyte transendothelial migration, and cytokine production. Coculture with MetS-EVs increased renal tubular cell inflammation. MetS alters the protein cargo of porcine MSC-derived EVs, selectively packaging specific proinflammatory signatures that may impair their ability to repair damaged tissues. Stem Cells Translational Medicine 2019;8:430–440

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.

Background Ossabaw pigs are unique miniature swine with genetic predisposition to develop metabolic syndrome and coronary atherosclerosis after extended periods receiving atherogenic diets. We have hypothesized that transgenic Ossabaw swine expressing chimp PCSK9 (proprotein convertase subtilisin‐like/kexin type 9) containing the D374Y gain of function would develop familial hypercholesterolemia and coronary artery plaques more rapidly than Landrace swine with the same transgene. Methods and Results Ossabaw and Landrace PCSK9 gain‐of‐function founders were generated by Sleeping Beauty transposition and cloning. Histopathologic findings in the Ossabaw founder animal showed more advanced plaques and higher stenosis than in the Landrace founder, underscoring the Ossabaw genetic predisposition to atherosclerosis. We chose to further characterize the Ossabaw PCSK9 gain‐of‐function animals receiving standard or atherogenic diets in a 6‐month longitudinal study using computed tomography, magnetic resonance (MR) imaging, intravascular ultrasound, and optical coherence tomography, followed by pathological analysis of atherosclerosis focused on the coronary arteries. The Ossabaw model was consistently hypercholesterolemic, with or without dietary challenge, and by 6 months had consistent and diffuse fibrofatty or fibroatheromatous plaques with necrosis, overlying fibrous caps, and calcification in up to 10% of coronary plaques. Conclusions The Ossabaw PCSK9 gain‐of‐function model provides consistent and robust disease development in a time frame that is practical for use in preclinical therapeutic evaluation to drive innovation. Although no animal model perfectly mimics the human condition, this genetic large‐animal model is a novel tool for testing therapeutic interventions in the context of developing and advanced coronary artery disease.