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1.  C-Peptide Reduces Mitochondrial Superoxide Generation by Restoring Complex I Activity in High Glucose-Exposed Renal Microvascular Endothelial Cells 
ISRN Endocrinology  2012;2012:162802.
Hyperglycemia-mediated microvascular damage has been proposed to originate from excessive generation of mitochondrial superoxide in endothelial cells and is the suggested mechanism by which the pathogenesis of diabetes-induced renal damage occurs. C-peptide has been shown to ameliorate diabetes-induced renal impairment. Yet, the mechanisms underlying this protective benefit remain unclear. The objective of this study was to determine whether C-peptide affords protection to renal microvascular endothelial cell mitochondria during hyperglycemia. Conditionally immortalized murine renal microvascular endothelial cells (MECs) were exposed to low (5.5 mM) or high glucose (25 mM) media with either C-peptide (6.6 nM) or its scrambled sequence control peptide for 24 or 48 hours. Respiratory control ratio, a measure of mitochondrial electrochemical coupling, was significantly higher in high glucose renal MECs treated with C-peptide than those of high glucose alone. C-peptide also restored high glucose-induced renal MEC mitochondrial membrane potential changes back to their basal low glucose state. Moreover, C-peptide prevented the excessive mitochondrial superoxide generation and concomitant reductions in mitochondrial complex I activity which are mediated by the exposure of the renal MECs to high glucose. Together, these data demonstrate that C-peptide protects against high glucose-induced generation of mitochondrial superoxide in renal MECs via restoration of basal mitochondrial function.
PMCID: PMC3388427  PMID: 22778984
2.  A remission spectroscopy system for in vivo monitoring of hemoglobin oxygen saturation in murine hepatic sinusoids, in early systemic inflammation 
During the early stages of systemic inflammation, the liver integrity is compromised by microcirculatory disturbances and subsequent hepatocellular injury. Little is known about the relationship between the hemoglobin oxygen saturation (HbsO2) in sinusoids and the hepatocellular mitochondrial redox state, in early systemic inflammation. In a murine model of early systemic inflammation, we have explored the association between the sinusoidal HbsO2 detected with a remission spectroscopy system and 1.) the NAD(P)H autofluorescence (an indicator of the intracellular mitochondrial redox state) and 2.) the markers of hepatocellular injury.
Animals submitted to 1 hour bilateral hindlimb ischemia (I) and 3 hours of reperfusion (R) (3.0 h I/R) exhibited lower HbsO2 values when compared with sham. Six hours I/R (1 hour bilateral hindlimb ischemia and 6 hours of reperfusion) and the continuous infusion of endothelin-1 (ET-1) further aggravated the hypoxia in HbsO2. The detected NAD(P)H autofluorescence correlated with the detected HbsO2 values and showed the same developing. Three hours I/R resulted in elevated NAD(P)H autofluorescence compared with sham animals. Animals after 6.0 h I/R and continuous infusion of ET-1 revealed higher NAD(P)H autofluorescence compared with 3.0 h I/R animals. Overall the analysed HbsO2 values correlated with all markers of hepatocellular injury.
During the early stages of systemic inflammation, there is a significant decrease in hepatic sinusoidal HbsO2. In parallel, we detected an increasing NAD(P)H autofluorescence representing an intracellular inadequate oxygen supply. Both changes are accompanied by increasing markers of liver cell injury. Therefore, remission spectroscopy in combination with NAD(P)H autofluorescence provides information on the oxygen distribution, the metabolic state and the mitochondrial redox potential, within the mouse liver.
PMCID: PMC545944  PMID: 15647116
3.  Hypercholesterolemia and microvascular dysfunction: interventional strategies 
Hypercholesterolemia is defined as excessively high plasma cholesterol levels, and is a strong risk factor for many negative cardiovascular events. Total cholesterol levels above 200 mg/dl have repeatedly been correlated as an independent risk factor for development of peripheral vascular (PVD) and coronary artery disease (CAD), and considerable attention has been directed toward evaluating mechanisms by which hypercholesterolemia may impact vascular outcomes; these include both results of direct cholesterol lowering therapies and alternative interventions for improving vascular function. With specific relevance to the microcirculation, it has been clearly demonstrated that evolution of hypercholesterolemia is associated with endothelial cell dysfunction, a near-complete abrogation in vascular nitric oxide bioavailability, elevated oxidant stress, and the creation of a strongly pro-inflammatory condition; symptoms which can culminate in profound impairments/alterations to vascular reactivity. Effective interventional treatments can be challenging as certain genetic risk factors simply cannot be ignored. However, some hypercholesterolemia treatment options that have become widely used, including pharmaceutical therapies which can decrease circulating cholesterol by preventing either its formation in the liver or its absorption in the intestine, also have pleiotropic effects with can directly improve peripheral vascular outcomes. While physical activity is known to decrease PVD/CAD risk factors, including obesity, psychological stress, impaired glycemic control, and hypertension, this will also increase circulating levels of high density lipoprotein and improving both cardiac and vascular function. This review will provide an overview of the mechanistic consequences of the predominant pharmaceutical interventions and chronic exercise to treat hypercholesterolemia through their impacts on chronic sub-acute inflammation, oxidative stress, and microvascular structure/function relationships.
PMCID: PMC2996379  PMID: 21087503
4.  Alteration of renal respiratory Complex-III during experimental type-1 diabetes 
Diabetes has become the single most common cause for end-stage renal disease in the United States. It has been established that mitochondrial damage occurs during diabetes; however, little is known about what initiates mitochondrial injury and oxidant production during the early stages of diabetes. Inactivation of mitochondrial respiratory complexes or alteration of their critical subunits can lead to generation of mitochondrial oxidants, mitochondrial damage, and organ injury. Thus, one goal of this study was to determine the status of mitochondrial respiratory complexes in the rat kidney during the early stages of diabetes (5-weeks post streptozotocin injection).
Mitochondrial complex activity assays, blue native gel electrophoresis (BN-PAGE), Complex III immunoprecipitation, and an ATP assay were performed to examine the effects of diabetes on the status of respiratory complexes and energy levels in renal mitochondria. Creatinine clearance and urine albumin excretion were measured to assess the status of renal function in our model.
Interestingly, of all four respiratory complexes only cytochrome c reductase (Complex-III) activity was significantly decreased, whereas two Complex III subunits, Core 2 protein and Rieske protein, were up regulated in the diabetic renal mitochondria. The BN-PAGE data suggested that Complex III failed to assemble correctly, which could also explain the compensatory upregulation of specific Complex III subunits. In addition, the renal F0F1-ATPase activity and ATP levels were increased during diabetes.
In summary, these findings show for the first time that early (and selective) inactivation of Complex-III may contribute to the mitochondrial oxidant production which occurs in the early stages of diabetes.
PMCID: PMC2636815  PMID: 19166612
5.  Liver dysfunction after lung recruitment manoeuvres during pressure-controlled ventilation in experimental acute respiratory distress 
Critical Care  2007;11(1):R13.
Consequences of lung recruitment with prolonged high positive end-expiratory pressure (PEEP) ventilation for liver function are unclear. We therefore investigated liver dysfunction during two different ventilation treatment regimens of experimental acute respiratory distress syndrome.
Sixteen anaesthetised juvenile pietrain pigs were ventilated in the pressure-controlled mode (PCV) with an inspiratory fraction of oxygen (FiO2) of 1.0, a respiratory frequency of 30 per minute, a tidal volume of 6 ml/kg, and a PEEP of 5 cm H2O. After lung injury was induced by repeated pulmonary lavage with normal saline, animals were randomly assigned into two groups (n = 8 each) for a 24-hour trial: PCV (unchanged ventilation) and PCV with recruitment (PCV+R) (starting with a sustained inflation of 50 cm H2O for 1 minute, the ventilation was continued while increasing PEEP in increments of 3 cm H2O every 15 minutes as long as arterial oxygen tension [PaO2] improved). After recruitment, FiO2 was reduced to 0.4 and the PEEP was lowered every 15 minutes until PaO2 decreased to 12.0 to 14.7 kPa (90 to 110 torr). Serum levels of hyaluronic acid (HA), routine liver serum markers, and plasma disappearance rate of indocyanine green (ICG) were tested before and after lung injury, and 6 and 18 hours after randomisation. Liver serum markers were also tested at 24 hours. Paraffin sections of liver tissue stained by haematoxylin and eosin were made after euthanisation.
The PCV+R group exhibited more polymorphonuclear neutrophils and lymphocytes in the liver sinusoids: median score (interquartile range) of 1.5 (1.4 to 1.5) compared to 0.9 (0.7 to 1.1) (p = 0.01). Elevation of bilirubin, aspartate aminotransferase, and lactate dehydrogenase was more prominent in the PCV+R group. Plasma disappearance rate of ICG indicated no liver dysfunction. HA levels in the PCV+R group gradually increased and were significantly higher (p < 0.001) at 6 and 18 hours with 59 (57 to 64) and 75 (66 to 84) ng/ml, respectively, than in the PCV group with 34 (32 to 48) and 41 (38 to 42) ng/ml, respectively.
The PCV+R group showed a more prominent inflammatory reaction in their liver sinusoids accompanied by increased serum levels of liver enzymes and HA. Therefore, recruitment with higher PEEP levels for treatment of respiratory failure might lead to liver dysfunction.
PMCID: PMC2151872  PMID: 17261192

Results 1-5 (5)