Development and translation of thiometallate sulfide donors using a porcine model of coronary occlusion and reperfusion.

Sulfide-releasing compounds reduce reperfusion injury by decreasing mitochondria-derived reactive oxygen species production. We previously characterised ammonium tetrathiomolybdate (ATTM), a clinically used copper chelator, as a sulfide donor in rodents. Here we assessed translation to large mammals prior to clinical testing. In healthy pigs an intravenous ATTM dose escalation revealed a reproducible pharmacokinetic/pharmacodynamic (PK/PD) relationship with minimal adverse clinical or biochemical events. In a myocardial infarction (1-h occlusion of the left anterior descending coronary artery)-reperfusion model, intravenous ATTM or saline was commenced just prior to reperfusion. ATTM protected the heart (24-h histological examination) in a drug-exposure-dependent manner (r2 = 0.58, p < 0.05). Blood troponin T levels were significantly (p < 0.05) lower in ATTM-treated animals while myocardial glutathione peroxidase activity, an antioxidant selenoprotein, was elevated (p < 0.05). Overall, our study represents a significant advance in the development of sulfides as therapeutics and underlines the potential of ATTM as a novel adjunct therapy for reperfusion injury. Mechanistically, our study suggests that modulating selenoprotein activity could represent an additional mode of action of sulfide-releasing drugs.

Reconfiguration and loss of peritubular capillaries in chronic kidney disease.

Functional and structural alterations of peritubular capillaries (PTCs) are a major determinant of chronic kidney disease (CKD). Using a software-based algorithm for semiautomatic segmentation and morphometric quantification, this study analyzes alterations of PTC shape associated with chronic tubulointerstitial injury in three mouse models and in human biopsies. In normal kidney tissue PTC shape was predominantly elongated, whereas the majority of PTCs associated with chronic tubulointerstitial injury had a rounder shape. This was reflected by significantly reduced PTC luminal area, perimeter and diameters as well as by significantly increased circularity and roundness. These morphological alterations were consistent in all mouse models and human kidney biopsies. The mean circularity of PTCs correlated significantly with categorized glomerular filtration rates and the degree of interstitial fibrosis and tubular atrophy (IFTA) and classified the presence of CKD or IFTA. 3D reconstruction of renal capillaries revealed not only a significant reduction, but more importantly a substantial simplification and reconfiguration of the renal microvasculature in mice with chronic tubulointerstitial injury. Computational modelling predicted that round PTCs can deliver oxygen more homogeneously to the surrounding tissue. Our findings indicate that alterations of PTC shape represent a common and uniform reaction to chronic tubulointerstitial injury independent of the underlying kidney disease.

Butyrate Supplementation Exacerbates Myocardial and Immune Cell Mitochondrial Dysfunction in a Rat Model of Faecal Peritonitis.

Mitochondrial dysfunction and immune cell dysfunction are commonplace in sepsis and are associated with increased mortality risk. The short chain fatty acid, butyrate, is known to have anti-inflammatory effects and promote mitochondrial biogenesis. We therefore explored the immunometabolic effects of butyrate in an animal model of sepsis. Isolated healthy human volunteer peripheral mononuclear cells were stimulated with LPS in the presence of absence of butyrate, and released cytokines measured. Male Wistar rats housed in metabolic cages received either intravenous butyrate infusion or placebo commencing 6 h following faecal peritonitis induction. At 24 h, splenocytes were isolated for high-resolution respirometry, and measurement of mitochondrial membrane potential (MMP), reactive oxygen species (mtROS), and intracellular cytokines (TNF alpha, IL-10) using flow cytometry. Isolated splenocytes from septic and septic butyrate treated rats were stimulated with LPS for 18 h and the effects of butyrate on cytokine release assessed. Ex vivo, butyrate (1.8 mM) reduced LPS-induced TNF alpha (p = 0.019) and IL-10 (p = 0.001) release by human PBMCs. In septic animals butyrate infusion reduced the respiratory exchange ratio (p < 0.001), consistent with increased fat metabolism. This was associated with a reduction in cardiac output (p = 0.001), and increased lactate (p = 0.031) compared to placebo-treated septic animals (p < 0.05). Butyrate treatment was associated with a reduction in splenocyte basal respiration (p = 0.077), proton leak (p = 0.022), and non-mitochondrial respiration (p = 0.055), and an increase in MMP (p = 0.007) and mtROS (p = 0.027) compared to untreated septic animals. Splenocyte intracellular cytokines were unaffected by butyrate, although LPS-induced IL-10 release was impaired (p = 0.039). In summary, butyrate supplementation exacerbates myocardial and immune cell mitochondrial dysfunction in a rat model of faecal peritonitis.

Selective mitochondrial antioxidant MitoTEMPO reduces renal dysfunction and systemic inflammation in experimental sepsis in rats.

BACKGROUND: Excess mitochondrial reactive oxygen species (mROS) in sepsis is associated with organ failure, in part by generating inflammation through the NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome. We determined the impact of a mitochondrial-targeted antioxidant (MitoTEMPO) on mitochondrial dysfunction in renal proximal tubular epithelial cells, peritoneal immune cell function ex vivo, and organ dysfunction in a rat model of sepsis. METHODS: The effects of MitoTEMPO were assessed ex vivo using adenosine triphosphate and lipopolysaccharide-stimulated rat peritoneal immune cells and fresh rat kidney slices exposed to serum from septic rats. We assessed mROS production and phagocytotic capacity (flow cytometry), mitochondrial functionality (multiphoton imaging, respirometry), and NLRP3 inflammasome activation in cell culture. The effect of MitoTEMPO on organ dysfunction was evaluated in a rat model of faecal peritonitis. RESULTS: MitoTEMPO decreased septic serum-induced mROS (P<0.001) and maintained normal reduced nicotinamide adenine dinucleotide redox state (P=0.02) and mitochondrial membrane potential (P<0.001) in renal proximal tubular epithelial cells ex vivo. In lipopolysaccharide-stimulated peritoneal immune cells, MitoTEMPO abrogated the increase in mROS (P=0.006) and interleukin-1ß (IL-1ß) (P=0.03) without affecting non-mitochondrial oxygen consumption or the phagocytotic-induced respiratory burst (P>0.05). In vivo, compared with untreated septic animals, MitoTEMPO reduced systemic IL-1ß (P=0.01), reduced renal oxidative stress as determined by urine isoprostane levels (P=0.04), and ameliorated renal dysfunction (reduced serum urea (P<0.001) and creatinine (P=0.05). CONCLUSIONS: Reduction of mROS by a mitochondria-targeted antioxidant reduced IL-1ß, and protected mitochondrial, cellular, and organ functionality after septic insults.

DNA Nanodevices with Selective Immune Cell Interaction and Function.

DNA nanotechnology produces precision nanostructures of defined chemistry. Expanding their use in biomedicine requires designed biomolecular interaction and function. Of topical interest are DNA nanostructures that function as vaccines with potential advantages over nonstructured nucleic acids in terms of serum stability and selective interaction with human immune cells. Here, we describe how compact DNA nanobarrels bind with a 400-fold selectivity via membrane anchors to white blood immune cells over erythrocytes, without affecting cell viability. The selectivity is based on the preference of the cholesterol lipid anchor for the more fluid immune cell membranes compared to the lower membrane fluidity of erythrocytes. Compacting DNA into the nanostructures gives rise to increased serum stability. The DNA barrels furthermore functionally modulate white blood cells by suppressing the immune response to pro-inflammatory endotoxin lipopolysaccharide. This is likely due to electrostatic or steric blocking of toll-like receptors on white blood cells. Our findings on immune cell-specific DNA nanostructures may be applied for vaccine development, immunomodulatory therapy to suppress septic shock, or the targeting of bioactive substances to immune cells.