Protein disulfide isomerase inhibition impairs Keap1/Nrf2 signaling and mitochondrial function and induces apoptosis in renal proximal tubular cells.

Renal proximal tubular apoptosis plays a critical role in kidney health and disease. However, cellular molecules that trigger renal apoptosis remain elusive. Here, we evaluated the effect of inhibiting protein disulfide isomerase (PDI), a critical thioredoxin chaperone protein, on apoptosis as well as the underlying mechanisms in human renal proximal tubular (HK2) cells. HK2 cells were transfected with PDI-specific siRNA in the absence and presence of an antioxidant, tempol. PDI siRNA transfection resulted in a decrease of ~70% in PDI protein expression and enzyme activity. PDI inhibition increased caspase-3 activity and induced profound cell apoptosis. Mitochondrial function, as assessed by mitochondrial cytochrome c levels, mitochondrial membrane potential, oxygen consumption, and ATP levels, was significantly reduced in PDI-inhibited cells. Also, PDI inhibition caused nuclear factor erythroid 2-related factor 2 (Nrf2; a redox-sensitive transcription factor) cytoplasmic sequestration, decreased superoxide dismutase and glutathione-S-transferase activities, and increased oxidative stress. In PDI-inhibited cells, tempol reduced apoptosis, caspase-3 activity, and oxidative stress and also restored Nrf2 nuclear translocation and mitochondrial function. Silencing Nrf2 in the cells abrogated the beneficial effect of tempol, whereas Kelch-like ECH-associated protein 1 (an Nrf2 regulatory protein) silencing protected cells from PDI inhibitory effects. Collectively, our data indicate that PDI inhibition diminishes Nrf2 nuclear translocation, causing oxidative stress that further triggers mitochondrial dysfunction and renal cell apoptosis. This study suggests an important role for PDI in renal cell apoptosis involving Nrf2 and mitochondrial dysfunction.

Simulated vehicle exhaust exposure (SVEE) in rats impairs renal mitochondrial function.

PURPOSE:  Vehicle exhaust emissions primarily comprise of nitrogen, oxygen, water, CO2, NO2, CO, hydrocarbons and particulate matter. While adverse effects of hydrocarbon and particulate matter on cardiovascular functions are known, the effect of pro-oxidants CO2, NO2 and CO are not clear. METHODS:  Here, using an animal model of a simulated mixture of pro-oxidants (0.04% CO2, 0.9 ppm NO2 and 3 ppm CO with air as a base), we examined the effect of simulated vehicle exhaust exposure (SVEE) on various cardiovascular parameters. Male Sprague-Dawley rats were exposed to SVEE or ambient air (Control: CON) for 30 min/day for 2 weeks. Thereafter, systolic and diastolic blood pressure, heart rate and glomerular filtration rate were measured. Later, rats were sacrificed, blood plasma and kidneys were collected. RESULTS:  The systolic and diastolic blood pressure, heart rate and glomerular filtration rate remained unchanged. Plasma corticosterone increased in SVEE rats when compared to CON group. Plasma 8-isoprostane, a systemic marker of oxidative stress, increased while total antioxidant capacity decreased in SVEE but not in CON. Kidney cortical tissue homogenates exhibited increase in superoxide, hydrogen peroxide and protein carbonylation in SVEE but not CON, all indicative of heightened oxidative stress. Renal cortical mitochondrial SOD activity was significantly reduced in SVEE than CON. CONCLUSION:  Significant decline in mitochondrial respiration and oxygen consumption was observed, in addition to low ATP, reduced ATP synthase and cytochrome C oxidase levels, as well as accelerated mitochondrial fission, and reduced fusion processes, were observed in SVEE than CON rats, all indicative of renal mitochondrial impairment.

ß2-Adrenoceptor signaling in airway epithelial cells promotes eosinophilic inflammation, mucous metaplasia, and airway contractility.

The mostly widely used bronchodilators in asthma therapy are ß2-adrenoreceptor (ß2AR) agonists, but their chronic use causes paradoxical adverse effects. We have previously determined that ß2AR activation is required for expression of the asthma phenotype in mice, but the cell types involved are unknown. We now demonstrate that ß2AR signaling in the airway epithelium is sufficient to mediate key features of the asthmatic responses to IL-13 in murine models. Our data show that inhibition of ß2AR signaling with an aerosolized antagonist attenuates airway hyperresponsiveness (AHR), eosinophilic inflammation, and mucus-production responses to IL-13, whereas treatment with an aerosolized agonist worsens these phenotypes, suggesting that ß2AR signaling on resident lung cells modulates the asthma phenotype. Labeling with a fluorescent ß2AR ligand shows the receptors are highly expressed in airway epithelium. In ß2AR-/- mice, transgenic expression of ß2ARs only in airway epithelium is sufficient to rescue IL-13-induced AHR, inflammation, and mucus production, and transgenic overexpression in WT mice exacerbates these phenotypes. Knockout of ß-arrestin-2 (ßarr-2-/-) attenuates the asthma phenotype as in ß2AR-/- mice. In contrast to eosinophilic inflammation, neutrophilic inflammation was not promoted by ß2AR signaling. Together, these results suggest ß2ARs on airway epithelial cells promote the asthma phenotype and that the proinflammatory pathway downstream of the ß2AR involves ßarr-2. These results identify ß2AR signaling in the airway epithelium as capable of controlling integrated responses to IL-13 and affecting the function of other cell types such as airway smooth muscle cells.

Superoxide via Sp3 mechanism increases renal renin activity, renal AT1 receptor function, and blood pressure in rats.

We tested a hypothesis that superoxide, by inducing Sp3, increases renal renin activity, renal angiotensin II type 1 receptor (AT1R) function, and blood pressure (BP) in rats. Group 1 rats were treated with vehicle, saline. Group 2 rats were treated with superoxide dismutase (SOD) inhibitor diethylthiocarbamate (DETC). Group 3 rats were treated with DETC and an SOD mimetic, tempol. Group 4 rats were treated with tempol only. All four groups of rats were treated for 2 wk then anesthetized, and BP was recorded. Thereafter, diuresis and natriuresis in response to AT1R blocker candesartan were determined. When compared with vehicle rats, BP increased in DETC rats. The increased BP in DETC rats decreased with tempol. Diuresis and natriuresis in response to candesartan increased in controls, and this further increased in DETC rats and decreased with tempol. A second set of four groups of rats underwent the same treatment as above and were anesthetized, and their kidneys were obtained for biochemical studies. The levels of superoxide but not hydrogen peroxide increased, whereas SOD activities decreased further in the renal cortical tissues of DETC rats than vehicle rats. These effects were attenuated with tempol in DETC rats. Moreover, tissue renin activity and abundance of membranous AT1R proteins increased more in DETC rats than vehicle rats, and decreased with tempol in DETC rats. Furthermore, the levels of lysine-acetylated, but not serine-phosphorylated, Sp3 increased more in the nuclei of DETC rats than vehicle rats. The increased levels of Sp3 lysine acetylation decreased in DETC rats with tempol. Taken together, our results suggest that superoxide activates renal Sp3 via lysine acetylation increasing renin activity, AT1R function, and BP in rats.

Age-related hypertension and salt sensitivity are associated with unique cortico-medullary distribution of D1R, AT1R, and NADPH-oxidase in FBN rats.

We examined effects of normal (NS) and high salt (HS) on blood pressure (BP) and cortico-medullary distribution of dopamine D1 receptor (D1R), angiotensin AT1 receptor (AT1R), NADPH oxidase-gp(91phox), and sodium transporters (NHE-3, Na, K ATPase) in adult and aged rats. Aged rats fed with NS diet had higher BP, which further increased with HS. HS increased D1R mRNA and protein levels in cortex and medulla of adult rats. NS or HS fed-aged rats had higher AT1R and gp(91phox) mRNA levels in cortex and medulla. Aged rats fed with NS diet had higher gp(91phox) protein levels in cortex. HS diet increased AT1R and gp(91phox) protein levels in medulla of aged rats. Aged rats fed with NS or HS diet had higher NHE-3 protein levels in medulla. HS increased Na, K ATPase protein levels in medulla of aged rats. HS increased urinary kidney injury molecule-1 (KIM-1) but not protein or albumin levels in aged rats. These results suggest that cortical gp(91phox) and medullary NHE-3 contribute to age-related hypertension. Whereas D1R (cortical and medullary) together with medullary AT1R, gp(91phox) and Na, K-ATPase contribute to salt sensitivity in aged rats. And, KIM-1 may be a better marker for kidney damage.

Renal dopamine and angiotensin II receptor signaling in age-related hypertension.

Kidneys play a vital role in long-term regulation of blood pressure. This is achieved by actions of many renal and nonrenal factors acting on the kidney that help maintain the body's water and electrolyte balance and thus control blood pressure. Several endogenously formed or circulating hormones/peptides, by acting within the kidney, regulate fluid and water homeostasis and blood pressure. Dopamine and angiotensin II are the two key renal factors that, via acting on their receptors and counterregulating each other's function, maintain water and sodium balance. In this review, we provide recent advances in the signaling cascades of these renal receptors, especially at the level of their cross talk, and discuss their roles in blood pressure regulation in the aging process.

Human kidney-2 cells harbor functional dopamine D1 receptors that require Giα for Gq/11α signaling.

A recent study demonstrated that the dopamine D1 receptor (D1R) is nonfunctional in human kidney cells, HK2 cells, in terms of their inability to couple to Gs protein in response to the D1R agonist fenoldopam. Since D1R also couples to Gq protein, we tested whether D1R is functional in HK2 cells in terms of their ability to couple to Gq and produce downstream signaling. For comparison, we also studied another receptor, angiotensin II type 1 receptor (AT1R) known to couple to Gq. Protein kinase C (PKC) and (86)rubidium transport activities were determined as surrogate downstream signaling markers. Fenoldopam and angiotensin II increased PKC activity, which decreased in the presence of respective receptor antagonists (SCH23390 for D1R; candesartan for AT1R), PKC (chelerythrine chloride) and Gi protein (pertussis toxin) inhibitors and Gq/11α siRNA. Furthermore, fenoldopam and angiotensin II increased (35)S-GTPγS binding, an index of receptor-G protein coupling, which decreased with pertussis toxin and in Gq/11α-depleted cells. Also, fenoldopam-mediated inhibition of (86)rubidium transport (an index of Na-K-ATPase activity) was attenuated with SCH23390, chelerythrine chloride, pertussis toxin, and Gq/11α siRNA. Moreover, fenoldopam caused a decrease in cytosolic and increase in membranous abundance of Gq/11α. The immunoprecipitated levels of Gq/11α in the membranes were greater in fenoldopam-treated cells, and Giα coimmunoprecipitated with Gq/11α. Our results suggest that both D1R and AT1R are functional in HK2 cells, enabling Gq-mediated downstream signaling in a Gi dependent manner.

Waterpipe Tobacco Smoke Exposure during Lactation-Susceptibility of Reproductive Hormones and Oxidative Stress Parameters in Male Progeny Rats.

There is a growing evidence for the public health hazards associated with waterpipe tobacco smoking (WTS). While the adverse effects of WTS exposure during pregnancy on the offspring are widely reported, its impact during breastfeeding remains less understood. The effects of WTS exposure during lactation on the reproductive hormones and oxidative stress biomarkers of adult male progeny were examined. Lactating rats received either fresh air (controls) or mainstream WTS for 1 h twice/day from day 4 to day 21 of lactation. The offspring was then followed up until week 20. The data indicated that WTS exposure in the lactating animals reduced the levels of follicle-stimulating hormone (FSH), prolactin (P < 0.05), luteinizing hormone (LH) (P = 0.1146), and estradiol (P = 0.0773) in the blood in male progeny. While the activities of testicular superoxide dismutase (SOD), glutathione peroxidase (GPx) and the levels of thiobarbituric acid reactive substances (TBARS) and blood levels of testosterone (P >0.05) remained unaltered, the activity of catalase increased significantly indicating an increased oxidant load in the WTS exposed rats compared to the controls. WTS exposure during lactation impairs male reproductive hormonal profile, augments oxidative damage, and potentially affects male fertility in male offspring rats.

Characteristics of a 50S ribosomal subunit precursor particle as a substrate for ermE methyltransferase activity and erythromycin binding in Staphylococcus aureus.

Erythromycin is a macrolide antibiotic that inhibits not only mRNA translation but also 50S ribosomal subunit assembly in bacterial cells. An important mechanism of erythromycin resistance is the methylation of 23S rRNA by erm methyl transferase enzymes. A model for 50S ribosomal subunit formation suggests that the precursor particle which accumulates in erythromycin treated cells is the target for methyl transferase activity. Hybridization experiments identified the presence of 23S rRNA in the 50S precursor particle. The protein content of the 50S precursor particle was analyzed by MALDI-TOF mass spectrophotometry. These studies have identified 23 of 36 50S ribosomal proteins in the precursor. Methyltransferase assays demonstrated that the 50S precursor particle was a substrate for ermE methyltransferase. Competition experiments indicated that the enzyme could displace erythromycin from the 50S precursor particle and that the methyltransferase had a higher association constant for the precursor particle compared to that of erythromycin. Inhibition experiments showed that macrolide, lincosamide and streptogramin B compounds bound to the precursor particle with similar affinity and inhibited the ermE methyltransferase activity. These studies shed light on the interaction of ermE methyltransferase and erythromycin in this clinically important pathogen.

Grape Powder Improves Age-Related Decline in Mitochondrial and Kidney Functions in Fischer 344 Rats.

We examined the effects and mechanism of grape powder- (GP-) mediated improvement, if any, on aging kidney function. Adult (3-month) and aged (21-month) Fischer 344 rats were treated without (controls) and with GP (1.5% in drinking water) and kidney parameters were measured. Control aged rats showed higher levels of proteinuria and urinary kidney injury molecule-1 (KIM-1), which decreased with GP treatment in these rats. Renal protein carbonyls (protein oxidation) and gp (91phox) -NADPH oxidase levels were high in control aged rats, suggesting oxidative stress burden in these rats. GP treatment in aged rats restored these parameters to the levels of adult rats. Moreover, glomerular filtration rate and sodium excretion were low in control aged rats suggesting compromised kidney function, which improved with GP treatment in aged rats. Interestingly, low renal mitochondrial respiration and ATP levels in control aged rats were associated with reduced levels of mitochondrial biogenesis marker MtTFA. Also, Nrf2 proteins levels were reduced in control aged rats. GP treatment increased levels of MtTFA and Nrf2 in aged rats. These results suggest that GP by potentially regulating Nrf2 improves aging mitochondrial and kidney functions.

Superoxide increases angiotensin II AT1 receptor function in human kidney-2 cells.

The redox-sensitive Sp family transcription factor has been linked to the regulation of angiotensin II type 1 receptor (AT1R). However, the exact mechanism of AT1R regulation in renal cells is poorly understood. We tested the specificity of reactive oxygen species (ROS), superoxide vs. hydrogen peroxide (H2O2), and the specific role of Sp3 transcription factor, if any, in the regulation of AT1R in human kidney cells (HK2 cells). Superoxide dismutase (SOD) inhibitor diethyldithiocarbamate (DETC), but not H2O2 treatment, increased fluorescence levels of superoxide probe dihydroethidium (DHE). H2O2, but not DETC, treatment increased the fluorescence of the H2O2-sensitive probe dichloro-dihydro-fluorescein (DCFH). These data suggest that SOD inhibition by DETC increases the superoxide but not H2O2 and exogenously added H2O2 is not converted to superoxide in renal cells. Furthermore, DETC, but not H2O2, treatment increased nuclear accumulation of Sp3, which was attenuated with the superoxide dismutase (SOD)-mimetic tempol. DETC treatment also increased AT1R mRNA and protein levels that were attenuated with tempol, whereas H2O2 did not have any effects on AT1R mRNA. Moreover, Sp3 overexpression increased, while Sp3 depletion by siRNA decreased, protein levels of AT1R. In addition, Sp3 siRNA in the presence of DETC decreased AT1R protein expression. Furthermore, DETC treatment increased the levels of cell surface AT1R as measured by biotinylation and immunofluorescence studies. Angiotensin II increased PKC activity in vehicle-treated cells that further increased in DETC-treated cells, which was attenuated by AT1R blocker candesartan and SOD-mimetic tempol. Taken together, our results suggest that superoxide, but not H2O2, via Sp3 up-regulates AT1R expression and function in the renal cells.

Grape powder treatment prevents anxiety-like behavior in a rat model of aging.

Earlier, we have reported that grape powder (GP) treatment prevented pharmacologic and psychological stress-induced anxiety-like behavior and memory impairment in rats. Protective effects of GP were attributed to its antioxidant effects. In this study, we tested the hypothesis that age-associated behavioral and cognitive deficits such as anxiety and memory impairment will be ameliorated with GP treatment. Using a National Institute of Aging recommended rodent model of aging, we examined a potentially protective role of antioxidant-rich GP in age-associated anxiety-like behavior and memory impairment. Male Fischer 344 rats were randomly assigned into 4 groups: young rats (3 months old) provided with tap water or with 15 g/L GP dissolved in tap water for 3 weeks, aged rats (21 months old) provided with tap water or with GP-treated tap water for 3 weeks (AG-GP). Anxiety-like behavior was significantly greater in aged rats compared with young rats, GP-treated young rats, or aged control rats (P < .05). Also, GP treatment prevented age-induced anxiety-like behavior in AG-GP rats (P < .05). Neither short-term nor long-term age-associated memory deficits improved with GP treatment in AG-GP rats. Furthermore, aged rats showed increased level of physiological stress (corticosterone) and increased oxidative stress in the plasma (8-isoprostane) as well as in selected brain areas (protein carbonylation). Grape powder treatment prevented age-induced increase in corticosterone levels and plasma 8-isoprostane levels in aged rats (P < .05), whereas protein carbonylation was recovered in the amygdala region only (P < .05). Grape powder by regulating oxidative stress ameliorates age-induced anxiety-like behavior in rats, whereas age-associated memory deficits seem unaffected with GP treatment.

Epinephrine Activation of the ß2-Adrenoceptor Is Required for IL-13-Induced Mucin Production in Human Bronchial Epithelial Cells.

Mucus hypersecretion by airway epithelium is a hallmark of inflammation in allergic asthma and results in airway narrowing and obstruction. Others have shown that administration a TH2 cytokine, IL-13 is sufficient to cause mucus hypersecretion in vivo and in vitro. Asthma therapy often utilizes ß2-adrenoceptor (ß2AR) agonists, which are effective acutely as bronchodilators, however chronic use may lead to a worsening of asthma symptoms. In this study, we asked whether ß2AR signaling in normal human airway epithelial (NHBE) cells affected mucin production in response to IL-13. This cytokine markedly increased mucin production, but only in the presence of epinephrine. Mucin production was blocked by ICI-118,551, a preferential ß2AR antagonist, but not by CGP-20712A, a preferential ß1AR antagonist. Constitutive ß2AR activity was not sufficient for IL-13 induced mucin production and ß-agonist-induced signaling is required. A clinically important long-acting ß-agonist, formoterol, was as effective as epinephrine in potentiating IL-13 induced MUC5AC transcription. IL-13 induced mucin production in the presence of epinephrine was significantly reduced by treatment with selective inhibitors of ERK1/2 (FR180204), p38 (SB203580) and JNK (SP600125). Replacement of epinephrine with forskolin + IBMX resulted in a marked increase in mucin production in NHBE cells in response to IL-13, and treatment with the inhibitory cAMP analogue Rp-cAMPS decreased mucin levels induced by epinephrine + IL-13. Our findings suggest that ß2AR signaling is required for mucin production in response to IL-13, and that mitogen activated protein kinases and cAMP are necessary for this effect. These data lend support to the notion that ß2AR-agonists may contribute to asthma exacerbations by increasing mucin production via activation of ß2ARs on epithelial cells.