Mitochondria play a critical role in the cardiomyocyte physiology by generating majority of the ATP required for the contraction/relaxation through oxidative phosphorylation (OXPHOS). Aging is a major risk factor for cardiovascular diseases (CVD) and mitochondrial dysfunction has been proposed as potential cause of aging. Recent technological innovations in Seahorse XFe24 Analyzer enhanced the detection sensitivity of oxygen consumption rate and proton flux to advance our ability study mitochondrial function. Studies of the respiratory function tests in the isolated mitochondria have the advantages to detect specific defects in the mitochondrial protein function and evaluate the direct mitochondrial effects of therapeutic/pharmacological agents. Here, we provide the protocols for studying the respiratory function of isolated murine cardiac mitochondria by measuring oxygen consumption rate using Seahorse XFe24 Analyzer. In addition, we provide details about experimental design, measurement of various respiratory parameters along with interpretation and analysis of data.

The presence of receptors for angiotensin II (AII) on the luminal membranes of various nephron segments has been well established for many decades.1,2 Originally their function remained unclear because tubular fluid AII concentrations were thought to be quite low due to the presence of various degrading enzymes on the brush border of proximal tubular cells. However, a series of reports in the 1990s demonstrated that the proximal tubular concentrations of AI and AII are in the nanomolar range and much higher than can be explained by tubular fluid reabsorption or equilibration with the circulating levels. 3,4 These findings led to a paradigm shift in our concepts regarding the role of luminal AII receptors in various nephron segments, and it is now well accepted that intraluminal AII and other angiotensin peptides exert various actions on transport systems in essentially all nephron segments predominantly through activation of AT1 receptors. 5-10In further studies, proximal tubular fluid samples were incubated with excess renin to determine substrate availability. The resultant AI concentrations demonstrated that the proximal tubular fluid angiotensinogen (AGT) concentrations are also very high 11 and greater than the circulating concentrations, indicating that it is unlikely the tubular AGT concentrations are derived from filtered AGT, in particular considering the limited permeability of the AGT because of its large size.11 Using in situ perfusion of proximal tubules with artificial tubular fluid with the delivery of filtrate blocked, Braam et al.4 collected the tubular fluid from downstream segments and found these also had elevated AII concentrations in the nanomolar range, supporting tubular secretion of AII or its precursors. Studies on isolated perfused tubules from S2 segments indicated that AII is produced intracellularly and secreted preformed into the tubular lumen, supporting the presence of intact AGT in isolated proximal tubule segments.12 These findings, along with clear evidence for the presence of AGT mRNA and protein in proximal tubular cells, [13][14][15] provided the foundation for the concept that the intratubular AII concentrations are derived primarily from locally synthesized substrate. The local production of AGT in the proximal tubule was supported by Terada et al., 14 who demonstrated the presence of a large signal for AGTmRNA in microdissected proximal convoluted and straight tubules. In vitro studies have also consistently demonstrated mRNA encoding AGT in proximal tubular cell lines extracted from proximal tubule segments.15,16 However, Pohl et al. 17 recently reported predominant localization of mRNA encoding AGT to S3 segments.The findings of kidney tubular mRNA encoding AGT notwithstanding, the kidney's ability to produce AGT is dwarfed by that of the liver, the organ primarily responsible for the maintenance of circulating AGT concentrations. In addition, it is well recognized that filtered AGT can be taken up by proximal tubule scavenger receptors such as megalin and cubilin, ...

Angiotensin II (AngII) is a critical physiologic regulator of volume homeostasis and mean arterial pressure (MAP), yet it also is known to induce immune mechanisms that contribute to hypertension. This study determined the role of interleukin-6 (IL-6) in the physiologic effect of AngII to maintain normal MAP during low-salt (LS) intake, and whether hypertension induced by plasma AngII concentrations measured during LS diet required IL-6. IL-6 knockout (KO) and wild-type (WT) mice were placed on LS diet for 7 days, and MAP was measured 19 h/day with telemetry. MAP was not affected by LS in either group, averaging 101 ± 4 and 100 ± 4 mmHg in WT and KO mice, respectively, over the last 3 days. Seven days of ACEI decreased MAP ∼25 mmHg in both groups. In other KO and WT mice, AngII was infused at 200 ng/kg per minute to approximate plasma AngII levels during LS. Surgical reduction of kidney mass and high-salt diet were used to amplify the blood pressure effect. The increase in MAP after 7 days was not different, averaging 20 ± 5 and 22 ± 6 mmHg in WT and KO mice, respectively. Janus Kinase 2 (JAK2)/signal transducer of activated transcription (STAT3) phosphorylation were not affected by LS, but were increased by AngII infusion at 200 and 800 ng/kg per minute. These data suggest that physiologic levels of AngII do not activate or require IL-6 to affect blood pressure significantly, whether AngII is maintaining blood pressure on LS diet or causing blood pressure to increase. JAK2/STAT3 activation, however, is tightly associated with AngII hypertension, even when caused by physiologic levels of AngII.

Growing evidence indicates that prorenin receptor (PRR) is upregulated in collecting duct (CD) of diabetic kidney. Prorenin is secreted by the principal CD cells, and is the natural ligand of the PRR. PRR activation stimulates fibrotic factors, including fibronectin, collagen, and transforming growth factor-β (TGF-β) contributing to tubular fibrosis. However, whether high glucose (HG) contributes to this effect is unknown. We tested the hypothesis that HG increases the abundance of PRR at the plasma membrane of the CD cells, thus contributing to the stimulation of downstream fibrotic factors, including TGF-β, collagen I, and fibronectin. We used streptozotocin (STZ) male Sprague–Dawley rats to induce hyperglycemia for 7 days. At the end of the study, STZ-induced rats showed increased prorenin, renin, and angiotensin (Ang) II in the renal inner medulla and urine, along with augmented downstream fibrotic factors TGF-β, collagen I, and fibronectin. STZ rats showed upregulation of PRR in the renal medulla and preferential distribution of PRR on the apical aspect of the CD cells. Cultured CD M-1 cells treated with HG (25 mM for 1 h) showed increased PRR in plasma membrane fractions compared to cells treated with normal glucose (5 mM). Increased apical PRR was accompanied by upregulation of TGF-β, collagen I, and fibronectin, while PRR knockdown prevented these effects. Fluorescence resonance energy transfer experiments in M-1 cells demonstrated augmented prorenin activity during HG conditions. The data indicate HG stimulates profibrotic factors by inducing PRR translocation to the plasma membrane in CD cells, which in perspective, might be a novel mechanism underlying the development of tubulointerstitial fibrosis in diabetes mellitus.

Introduction Muscle, fat and bone mass may play some roles to keep physical activity and favorable outcome in patients with cardiovascular diseases. However, there is a paucity of data regarding the effects on the prognosis of skeletal muscle, fat, and bone mass in patients with ST-segment elevation myocardial infarction (STEMI). Purpose Our purpose was to examine whether skeletal muscle, fat, and bone mass each affect the prognosis after STEMI. Methods A total of 354 male patients with STEMI were enrolled in this study. Dual-energy X-ray absorptiometry scan was performed before discharge. All patients were followed up for the primary composite outcome of all-cause death, nonfatal myocardial infarction, nonfatal ischemic stroke, hospitalization for congestive heart failure, and unplanned revascularization. Results During a median follow-up of 32 months, 57 patients experienced primary composite outcome. Each of skeletal muscle, fat, and bone mass were indexed by height squared (kg/m2) and divided into two groups using the cut-off value obtained from the maximum Youden index to predict the primary composite outcome. The event rate was significantly higher in patients with low appendicular skeletal muscle mass index (ASMI) (29.2% vs 11.7%, p<0.001), low fat mass index (FMI) (22.9% vs 13.3%, p=0.030), and low bone mass index (23.8% vs 11.6%, p=0.002). After adjustment for age, renal function, diabetes mellitus, infarct size, Killip classification, and body mass index, low ASMI but not FMI (p=0.150) and bone mass index (p=0.159) was independently and significantly associated with the primary composite outcome (adjusted hazard ratio 2.12, 95%-confidence interval 1.05–4.31, p=0.035). Conclusions Index about muscle mass rather than fat and bone mass have prognostic impact in male patients with STEMI.