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Fibroblasts from patients with Tangier disease carrying ATP-binding cassette A1 (ABCA1) loss-of-function mutations are characterized by cardiolipin accumulation, a mitochondrial-specific phospholipid. Suppression of ABCA1 expression occurs in glomeruli from patients with diabetic kidney disease (DKD) and in human podocytes exposed to DKD sera collected prior to the development of DKD. We demonstrated that siRNA ABCA1 knockdown in podocytes led to reduced oxygen consumption capabilities associated with alterations in the oxidative phosphorylation (OXPHOS) complexes and with cardiolipin accumulation. Podocyte-specific deletion of Abca1 ([Abca1.sup.fl/fl]) rendered mice susceptible to DKD, and pharmacological induction of ABCA1 improved established DKD. This was not mediated by free cholesterol, as genetic deletion of sterol-oacyltransferase-1 (SOAT1) in [Abca1.sup.fl/fl] mice was sufficient to cause free cholesterol accumulation but did not cause glomerular injury. Instead, cardiolipin mediates ABCA1-dependent susceptibility to podocyte injury, as inhibition of cardiolipin peroxidation with elamipretide improved DKD in vivo and prevented ABCA1-dependent podocyte injury in vitro and in vivo. Collectively, we describe a pathway definitively linking ABCA1 deficiency to cardiolipin-driven mitochondrial dysfunction. We demonstrated that this pathway is relevant to DKD and that ABCA1 inducers or inhibitors of cardiolipin peroxidation may each represent therapeutic strategies for the treatment of established DKD., Introduction Diabetic kidney disease (DKD) remains the most common cause of chronic kidney disease (CKD), comprising about 40% of patients with CKD (1). However, not all patients with diabetes develop [...]

High dietary fat intake leads to insulin resistance in skeletal muscle, and this represents a major risk factor for type 2 diabetes and cardiovascular disease. Mitochondrial dysfunction and oxidative stress have been implicated in the disease process, but the underlying mechanisms are still unknown. Here we show that in skeletal muscle of both rodents and humans, a diet high in fat increases the [H.sub.2][O.sub.2]-emitting potential of mitochondria, shifts the cellular redox environment to a more oxidized state, and decreases the redox-buffering capacity in the absence of any change in mitochondrial respiratory function. Furthermore, we show that attenuating mitochondrial [H.sub.2][O.sub.2] emission, either by treating rats with a mitochondrial-targeted antioxidant or by genetically engineering the overexpression of catalase in mitochondria of muscle in mice, completely preserves insulin sensitivity despite a high-fat diet. These findings place the etiology of insulin resistance in the context of mitochondrial bioenergetics by demonstrating that mitochondrial [H.sub.2][O.sub.2] emission serves as both a gauge of energy balance and a regulator of cellular redox environment, linking intracellular metabolic balance to the control of insulin sensitivity., Introduction Obesity has become a worldwide epidemic, the consequences of which represent a major health care challenge in the 21st century. A decrease in the sensitivity of skeletal muscle to [...]

Unilateral ureteral obstruction (UUO) is characterized by decreases in renal function, increased interstitial fibrosis, tubular apoptosis, and cellular infiltration. It has been suggested that inhibition of tubular apoptosis may protect against renal damage in obstruction. We have recently developed a series of peptides which are concentrated in the inner mitochondrial membrane and prevent cell death. These peptides are also active in vivo, in myocardial infraction, ischemic brain injury, and amyotrophic lateral sclerosis models. We therefore used SS-31, a prototype of these peptides, and assessed its effects on renal damage and oxidative stress in a 14-day obstruction model. SS-31 (1 or 3 mg/kg) or saline was given 1 day before and throughout the 14 days of obstruction. Kidneys were harvested and assessed for apoptosis (terminal transferase-dUTP-nick-end labeling, caspase 3 expression), fibrosis (trichrome staining), macrophage infiltration, fibroblast expression (immunoperoxidase), and oxidative damage (8-OH deoxyguanosine and heme oxygenase-1 expression), cytokines, and signaling pathways (transforming growth factor-[beta], CCR-1, p38-MAPK, NF-[kappa]B). SS-31 significantly attenuated the effects of obstruction on all aspects of renal damage which were examined, with both the 1 and 3 mg/kg doses showing efficacy. We noted increased oxidative stress in obstruction, which was also attenuated by SS-31 treatment. Signaling via NF-[kappa]B and p38 MAPK pathways were both affected by SS-31 treatment. This study provides a proof of concept that peptides which protect mitochondria in vitro can provide protection from renal damage in a UUO model. The mechanism by which protection is afforded requires further studies both in vitro and in vivo. mitochondria; fibrosis; peptides

Opiates such as morphine are the choice analgesic in the treatment of chronic pain. However their long-term use is limited because of the development of tolerance and dependence. Due to its importance in therapy, different strategies have been considered for making opiates such as morphine more effective, while curbing its liability to be abused. One such strategy has been to use a combination of drugs to improve the effectiveness of morphine. In particular. [delta] opioid receptor ligands have been useful in enhancing morphine's potency. The underlying molecular basis for these observations is not understood. We propose the modulation of receptor function by physical association between [micro] and [delta] opioid receptors as a potential mechanism. In support of this hypothesis, we show that [micro]-[delta] interacting complexes exist in live cells and native membranes and that the occupancy of [delta] receptors (by antagonists) is sufficient to enhance [micro] opioid receptor binding and signaling activity. Furthermore, [delta] receptor antagonists enhance morphine-mediated intrathecal analgesia. Thus, heterodimeric associations between [micro]-[delta] opioid receptors can be used as a model for the development of novel combination therapies for the treatment of chronic pain and other pathologies.

Platelets, on activation by endothelial damage, release ADP, ATP, serotonin, epinephrine, and norepinephrine. Although ATP is known to augment the action of norepinephrine in cardiovascular and endocrine systems, the possible interaction between ATP and catecholamines in regulation of platelet reactivity has not been reported. The addition of ATP (1-5 [micro]M) to human platelet-rich plasma did not induce platelet aggregation; however, it selectively augmented the aggregatory response to norepinephrine and epinephrine, but not to serotonin. This potentiating action of ATP was dose dependent and was not due to contamination by, or hydrolysis to, ADP. The action of ATP was blocked by 10 [micro]M of adenosine 3'-phosphate 5'-phosphosulfate, a selective [P.sub.2][Y.sub.1] receptor antagonist. ATP alone did not cause release of intracellular [Ca.sup.2+], but produced a significant [Ca.sup.2+] response in the presence of norepinephrine. In contrast, the [P.sub.2][X.sub.1] receptor agonists [P.sup.1],[P.sup.6]-diadenosine-5' hexophosphate and [alpha],[beta]-methylene-ATP had no effect on norepinephrine-induced platelet aggregation even when added at 100 [micro]M. This synergistic interaction between ATP and norepinephrine in stimulating platelet aggregation may have significant clinical implications and suggests a prothrombotic role for ATP in stress. norepinephrine; synergism

We recently discovered an opioid peptide analgesic, 2',6'-dimethyltyrosine (Dmt)-D-Arg-Phe-Lys-N[H.sub.2] ([[Dmt.sup.1]]DALDA), that can protect against ischemia-induced myocardial stunning. In buffer-perfused hearts, 30-min global ischemia followed by reperfusion resulted in a significant increase in norepinephrine (NE) overflow immediately upon reperfusion and significant decline in contractile force (45%). Pretreatment with [[Dmt.sup.1]]DALDA before ischemia completely abolished myocardial stunning and significantly reduced NE overflow (68%). In contrast, pretreatment with morphine before ischemia only provided brief protection against myocardial stunning and no reduction in NE overflow. [[Dmt.sup.1]]DALDA inhibited [[sup.3]H]NE uptake into cardiac synaptosomes in vitro (I[C.sub.50] = 3.9 [micro] M), whereas morphine had no effect. Surprisingly, protection against myocardial stunning was apparent even when hearts were perfused with [[Dmt.sup.1]]DALDA only upon reperfusion, whereas reperfusion with morphine had no effect. Binding studies with [[sup.3]H][[Dmt.sup.1]]DALDA revealed no high-affinity specific binding in cardiac membranes, suggesting that the cardioprotective actions of [[Dmt.sup.1]]DALDA are not mediated via opioid receptors. These findings suggest that [[Dmt.sup.1]]DALDA is a potent analgesic that may be useful for myocardial stunning resulting from cardiac interventions or myocardial ischemia. myocardial ischemia; norepinephrine uptake; [mu]-opioid; cardiac contractility; norepinephrine transporter

Dynorphin A (Dyn A) stimulates the release of ACTH in fetal sheep, a response that involves N-methyl-D-aspartate (NMDA) receptors but not the secretogogues corticotropin-releasing hormone or arginine vasopressin. We now find that neither Dyn A-(1-13) (0.5 mg/kg, iv) nor NMDA (4 mg/kg, iv) elicits ACTH release in postnatal lambs. This led us to hypothesize that Dyn A-(1-13) and NMDA might act to release placental ACTH. However, the ability of Dyn A-(1-13), NMDA, and the [kappa]-opioid receptor agonist U-50488H (1 mg/kg, iv) to release ACTH was lost after either fetal hypophysectomy (n = 4) or hypothalamo-pituitary disconnection (n = 4). These results indicate that neither the placenta nor the fetal pituitary is the site of action for these agonists and suggest a hypothalamic or suprahypothalamic site of action. Furthermore, the release of ACTH by Dyn A-(1-13) and NMDA was abolished after pretreatment with indomethacin, suggesting that they might cause the release of a prostanoid, possibly from the placenta, that subsequently acts at the hypothalamus or serves as a permissive factor in the action of Dyn A-(1-13) and NMDA at the hypothalamus. [kappa]-opioid receptor; hypothalamo-pituitary-adrenal axis; excitatory amino acid; prostanoid; placenta