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Based on the current guidelines in practice, a vast majority of the healthy Indian population is vitamin D deficient. Since the serum 25 hydroxycholecalciferol (25HCC) levels are affected by race and skin pigmentation, the normal range of vitamin D may differ in the Indians compared to the Western population. This study attempted to determine a population-specific threshold for 25 HCC levels associated with adequate bone health and calcium and phosphate homeostasis in healthy Indians. Subjects aged 20–50 years were included in the study. The exclusion criteria were obesity, chronic renal disease, liver failure, diabetes mellitus, thyroid disorders, a recent history of fracture, constant joint pain, and postmenopausal status. In addition, participants on prescribed medication such as glucocorticoids, anticonvulsants, or antifungals, as well as vitamin D and calcium supplementation, were also excluded. Blood samples were analyzed for serum calcium, phosphate, alkaline phosphatase, 25HCC, 1,25dihydroxycholecalciferol, parathyroid hormone (PTH), procollagen type-I N propeptide, and C-terminal telopeptide of type 1 collagen. Locally estimated smoothing scatter plot (LOESS) curves and Spearman correlation were utilized to study the correlation of all the biochemical parameters with 25 HCC to achieve thresholds. The study consisted of 270 healthy participants, out of which 97.8% were found to have vitamin D levels below 30 ng/ml. In addition, 8.8% had raised PTH, and 1.85% had hypocalcemia. Furthermore, 1.48% had raised serum alkaline phosphatase and hypophosphatemia, respectively. A weak inverse correlation was seen between 25 HCC and PTH (r s = −0.437, p < 0.001), as well as alkaline phosphatase (r s = −0.1475, p = 0.015), while a weak positive correlation was seen with serum phosphate (r s = 0.128, p = 0.047). For a healthy Indian population, the reference range of 25 HCC is much lower, and the lower limit of normal is approximately 13.5 ng/ml. This study indicates that vitamin D insufficiency in this population starts at 25 HCC values of 13.5 ng/ml and deficiency at 7 ng/ml. [ABSTRACT FROM AUTHOR]

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ahj.2006.02.021 Byline: David Charytan (a)(b), Laura Mauri (a)(c), Anupam Agarwal (c)(d), Steven Servoss (d), Benjamin Scirica (c), Richard E. Kuntz, (a)(c) Abstract: Dialysis patients have an excessive risk of cardiovascular death after myocardial infarction (MI). Underutilization of cardiac therapies may partially explain this risk, but whether patients on maintenance dialysis have differential rates of coronary angiography or revascularization during admission for MI compared with patients not on dialysis and whether these differences are explained by the presence of comorbid illness were uncertain. Author Affiliation: (a) Division of Clinical Biometrics, Department of Medicine, Brigham and Women's Hospital, Boston, MA (b) Division of Nephrology, Department of Medicine, Brigham and Women's Hospital, Boston, MA (c) Division of Cardiology, Department of Medicine, Brigham and Women's Hospital, Boston, MA (d) Division of Cardiology, Department of Medicine, Massachusetts General Hospital, Boston, MA Article History: Received 16 September 2005; Accepted 11 February 2006 Article Note: (footnote) This work was supported by NIH grant T32-DK07199-25 and was presented at the American Society of Nephrology, St. Louis, MO, in October 2004.

In present study aqueous extract of walnut husk was used for the simple, cost-effective synthesis of multiwalled carbon nanotubes (MWCNT) embedded with bimetallic iron-copper (Fe–Cu) nanoparticles, which exhibits adsorption capacity of 769.23 mg/g and 804.50 mg/g for Brilliant Blue (BB) dye and Direct Black (DB-19) dye respectively. The MWCNT@Fe–Cu were characterized by using FTIR, BET, FESEM, EDX, and XRD. The adsorption experiments were conducted by varying concentrations (50 ppm–200 ppm), pH (2.0–11.0), contact time (1 min–5 min), and temperature (30 °C–60 °C) to investigate their effect on adsorption. The adsorption data was best fit with the pseudo-second-order kinetics and Langmuir isotherm model, adsorption process was endothermic and spontaneous. The adsorption of each dye up to the 5th adsorption/desorption cycle was >95 %. From the 6th cycle to the 20th cycle the adsorption decreased from 97 % to 60.98 % and 95 % to56.76 % in the case of BB dye and DB-19 dye respectively. The unique feature of adsorbent is its pH independence as it showed more than 90 % removal efficiency in acidic as well basic medium. All findings underscore the effectiveness of synthesized MWCNT@Fe–Cu to be used as a potential adsorbent for organic dyes removal from industrial effluent. [Display omitted] • MWCNTs were synthesized at very low temperature 90°C using aqueous extract of walnut husk. • MWCNT@Fe–Cu was also fabricated by using aqueous extract of walnut husk. • Adsorption of DB-19 dye and BB dye was pH-independent. • Synthesized material MWCNT@Fe–Cu possess excellent adsorption capacity and regeneration ability. [ABSTRACT FROM AUTHOR]

To assess the cardiovascular safety of fenfluramine when used to treat children and young adults with Dravet syndrome. Patients with Dravet syndrome who completed one of three phase 3 clinical trials of fenfluramine could enroll in the open-label extension (OLE) study (NCT02823145). All patients started fenfluramine treatment at an oral dose of 0.2 mg/kg/day. The dose was titrated based on efficacy and tolerability to a maximum of 0.7 mg/kg/day (absolute maximum 26 mg/day) or 0.4 mg/kg/day (absolute maximum 17 mg/day) in patients concomitantly receiving stiripentol. Serial transthoracic echocardiography was performed using standardized methods and blinded readings at OLE entry, after 4–6 weeks, and every 3 months thereafter. Valvular heart disease (VHD) was defined as ≥ moderate mitral regurgitation or ≥ mild aortic regurgitation combined with physical signs or symptoms attributable to valve dysfunction. Pulmonary artery hypertension (PAH) was defined as systolic pulmonary artery pressure >35 mmHg. A total of 327 patients (median age, 9.0 years; range, 2–19 years) have enrolled in the OLE and received ≥1 dose of fenfluramine. The median duration of treatment was 23.9 months (range, 0.2–42.6 months) and the median dose of fenfluramine was 0.44 mg/kg/day. No patient demonstrated VHD or PAH at any time during the OLE. This study, which represents the largest, longest, and most rigorous examination of cardiovascular safety of fenfluramine yet reported, found no cases of VHD or PAH. These results, combined with fenfluramine's substantial antiseizure efficacy, support a strong positive benefit-risk profile for fenfluramine in the treatment of Dravet syndrome. • 327 Dravet syndrome patients were treated with fenfluramine for a median 23.9 months. • Serial echocardiograms were used to monitor cardiac valve function during treatment. • No cases of valvular heart disease were observed during the study. [ABSTRACT FROM AUTHOR]

Purpose: To assess the impact of fenfluramine (FFA) on the expected mortality incidence, including sudden unexpected death in epilepsy (SUDEP), in persons with Dravet syndrome (DS).Methods: In this pooled analysis, total time of exposure for persons with DS who were treated with FFA in phase 3 clinical trials, in United States and European Early Access Programs, and in two long-term open-label observational studies in Belgium was calculated. Literature was searched for reports of SUDEP mortality in DS, which were utilized as a comparison. Mortality rates were expressed per 1000 person-years.Results: A total of 732 persons with DS were treated with FFA, representing a total of 1185.3 person-years of exposure. Three deaths occurred, all in the phase 3 program: one during placebo treatment (probable SUDEP) and two during treatment with FFA (one probable SUDEP and one definite SUDEP). The all-cause and SUDEP mortality rates during treatment with FFA was 1.7 per 1000 person-years (95% CI, 0.4 to 6.7), a value lower than the all-cause estimate of 15.8 per 1000 person-years (95% CI, 9.9 to 25.4) and SUDEP estimate of 9.3 (95% CI, 5.0 to 17.3) reported by Cooper et al. (Epilepsy Res 2016;128:43-7) for persons with DS receiving standard-of-care.Conclusion: All-cause and SUDEP mortality rates in DS patients treated with FFA were substantially lower than in literature reports. Further studies are warranted to confirm that FFA reduces SUDEP risk in DS patients and to better understand the potential mechanism(s) by which FFA lowers SUDEP risk.Clinical Trial Registration: NCT02926898, NCT02682927, NCT02826863, NCT02823145, NCT03780127. [ABSTRACT FROM AUTHOR]

Iron is an essential metal involved in several major cellular processes required to maintain life. Because of iron's ability to cause oxidative damage, its transport, metabolism, and storage is strictly controlled in the body, especially in the small intestine, liver, and kidney. Iron plays a major role in acute kidney injury and has been a target for therapeutic intervention. However, the therapies that have been effective in animal models of acute kidney injury have not been successful in human beings. Targeting iron trafficking via ferritin, ferroportin, or hepcidin may offer new insights. This review focuses on the biology of iron, particularly in the kidney, and its implications in acute kidney injury. [ABSTRACT FROM AUTHOR]

Transforming growth factor-β (TGF-β) is a multifunctional regulatory cytokine that is implicated in a variety of kidney diseases, including diabetic nephropathy and chronic transplant rejection, where it promotes stimulation of the extracellular matrix deposition, cell proliferation, and migration. TGF-β exerts its biological functions largely via its downstream complex signaling molecules, Smad proteins. Paradoxically, TGF-β also is essential for normal homeostasis and suppression of inflammation through mechanisms that are yet to be fully elucidated. One feasible mechanism by which TGF-β may exert its beneficial properties is through induction of heme oxygenase-1 (HO-1). Induction of this redox-sensitive enzyme is known to be cytoprotective through its potent antioxidant, anti-inflammatory, and anti-apoptotic properties in different conditions including several kidney diseases. In this overview, recent advances in our understanding of the role of TGF-β in kidney disease, its molecular regulation of HO-1 expression, and the potential role of HO-1 induction as a therapeutic modality in TGF-β-mediated kidney diseases are highlighted. [ABSTRACT FROM AUTHOR]

Kidney resident macrophages (KRMs) are involved in homeostasis, phagocytosis, defense against infectious agents, response to insults, inflammation, and tissue repair. They also play critical roles in the pathogenesis and recovery from many kidney diseases such as acute kidney injury. KRMs historically have been studied as one homogenous population, but the wide-ranging roles and phenotypes observed suggest that there is greater heterogeneity than previously understood. Advancements in RNA sequencing technologies (single-cell RNA sequencing and spatial transcriptomics) have identified specific subsets of KRMs that are molecularly, functionally, and spatially distinct with dynamic changes after kidney injury. Multiple studies have identified unique markers that represent these subpopulations, permitting further characterization of the function and roles they play in the kidney. Understanding the diversity of KRM subpopulations will be key in the development of novel therapies used in treating kidney diseases and promoting kidney health. [ABSTRACT FROM AUTHOR]

Hypertension is a complex genetic disorder caused by interplay between several ‘risk’ genes and environmental factors (genetic heritability ∼30%). Most genetic studies of hypertension use a candidate gene approach and two conclusions have been made: there is no association or linkage with the genes studied, or the hypertension phenotype is heterogeneous and subgroups with hypertension related to certain polymorphisms cannot be identified because of background noise. Studies using intermediate phenotypes suggest the latter is most likely. Another problem is the reliability of gene structure assessment: usually only one or two gene polymorphisms are assessed. The use of intermediate phenotypes and dense mapping of candidate genes would provide a better approach for identifying genotype–phenotype correlations, which might enable the use of genotypes to identify more-specific therapeutic and preventative measures for hypertensives. [Copyright &y& Elsevier]

Cardinal and colleagues describe the use of molecular hydrogen, the most abundant molecule in the universe, as a treatment for chronic allograft nephropathy (CAN) in a rat model of kidney transplantation. They demonstrate that the addition of hydrogen to the drinking water results in a decrease in the severity of CAN and increased graft survival, and they provide evidence that the mechanism of action could be due to a reduction in reactive oxygen species. [ABSTRACT FROM AUTHOR]

Awad and colleagues elucidate the spatiotemporal profile of neutrophil infiltration in the kidney following ischemia–reperfusion injury. Using elegant in vivo labeling techniques, they demonstrate increased neutrophil content in the kidney following ischemia–reperfusion, which is largely due to transmigration from the circulation into the interstitial compartment. The authors also provide mechanistic insights into this phenomenon and show that adenosine 2A receptor agonists reduce interstitial neutrophil infiltration and improve renal function.Kidney International (2009) 75, 674–676. doi:10.1038/ki.2008.689 [ABSTRACT FROM AUTHOR]

Summary Heme oxygenase-1 (HO-1) is a cytoprotective enzyme that controls inflammatory responses and redox homeostasis; however, its role during pulmonary tuberculosis (TB) remains unclear. Using freshly resected human TB lung tissue, we examined the role of HO-1 within the cellular and pathological spectrum of TB. Flow cytometry and histopathological analysis of human TB lung tissues showed that HO-1 is expressed primarily in myeloid cells and that HO-1 levels in these cells were directly proportional to cytoprotection. HO-1 mitigates TB pathophysiology by diminishing myeloid cell-mediated oxidative damage caused by reactive oxygen and/or nitrogen intermediates, which control granulocytic karyorrhexis to generate a zonal HO-1 response. Using whole-body or myeloid-specific HO-1-deficient mice, we demonstrate that HO-1 is required to control myeloid cell infiltration and inflammation to protect against TB progression. Overall, this study reveals that zonation of HO-1 in myeloid cells modulates free-radical-mediated stress, which regulates human TB immunopathology. Graphical Abstract Highlights • HO-1 is expressed in distinct microanatomic zones within the human TB lung • Macrophages and neutrophils are the major producers of HO-1 in human TB lungs • In diseased areas of human TB lungs, myeloid cells have low HO-1 and high ROS and RNS • Mice lacking HO-1 expression in myeloid cells are more susceptible to Mtb infection Heme oxygenase-1 (HO-1) is a cytoprotective enzyme that controls inflammation and redox homeostasis; however, its role in tuberculosis (TB) is unclear. Using freshly resected human lung tissue and HO-1-deficient mice, Chinta et al. demonstrate that HO-1 in myeloid cells is important for controlling inflammatory and free-radical-mediated tissue damage in TB. [ABSTRACT FROM AUTHOR]

Unused warfare chemical agents, developed in World Wars I/II dumped in the ocean or buried at various sites across the world, pose significant environmental and human health risks. This review provides description of the neurotoxicity of arsenic-based warfare chemicals known as arsenicals. We specifically described the neuropathogenesis of diphenylarsinic acid (DPAA), a chemical warfare-related organoarsenicals and a degradation product of diphenylchloroarsine (DA), diphenylcyanoarsine (DC), also known as Clark I and Clark II respectively. These arsenicals are potent emetics, which were buried at a former naval base in the town of Kamisu, Japan. Several decades after burial, their environmental decay led to contamination of underground water table. Consumption of the contaminated water by the residents manifested a neurological syndrome, which was associated with damage to the cerebellum and brainstem as well as behavioral deficits. We summarized the chronology of this damage as recorded by monitoring the exposed population over time (∼15 years). Several simulating animal studies in primates and murine models demonstrate that DPAA caused this syndrome. [Display omitted] • Review on Clark-I/II, and DPAA formed under the aerial and aquatic environments. • Impact of Clark-I/II and their degradation arsenicals on human health. • DPAA caused developmental defects in children and neuropathology in adults. • Summarizes underlying molecular mechanisms employing both rodent and human models. [ABSTRACT FROM AUTHOR]

Osteoblastic differentiation of vascular smooth muscle cells (VSMCs) is involved in the pathogenesis of vascular calcification. Hydrogen sulfide (H2S) is a gas endogenously produced by cystathionine γ-lyase in VSMC. Here we determined whether H2S plays a role in phosphate-induced osteoblastic transformation and mineralization of VSMC. Hydrogen sulfide was found to inhibit calcium deposition in the extracellular matrix and to suppress the induction of the genes involved in osteoblastic transformation of VSMC: alkaline phosphatase, osteocalcin, and Cbfa1. Moreover, phosphate uptake and phosphate-triggered upregulation of the sodium-dependent phosphate cotransporter (Pit-1) were also prevented by H2S. Reduction of endogenous production of H2S by inhibition of cystathionine γ-lyase activity resulted in increased osteoblastic transformation and mineralization. Low plasma levels of H2S, associated with decreased cystathionine γ-lyase enzyme activity, were found in patients with chronic kidney disease receiving hemodialysis. Thus, H2S is a potent inhibitor of phosphate-induced calcification and osteoblastic differentiation of VSMC. This mechanism might contribute to accelerated vascular calcification in chronic kidney disease. [ABSTRACT FROM AUTHOR]

Plasma aldosterone concentration in the patient with diabetes mellitus. Background. Vascular injury at the microvascular and macrovascular levels plays a crucial role in the patient with diabetes mellitus. Evidence for renin-system activation in many patients with type 1 diabetes mellitus has raised the possibility that aldosterone—widely recognized as a contributor to vascular injury—could play a role. Methods. We examined the state of the renin-angiotensin-aldosterone system (RAAS) in 58 subjects with type 1 diabetes mellitus and 64 age-matched normal control subjects. All studies were performed on a fixed sodium (200 mmol/day) and potassium (100 mmol/day intake), and samples were drawn at 8:00 a.m. to avoid the influence of circadian rhythms. Results. The patient with diabetes mellitus showed an increase in plasma renin activity (PRA) ( P < 0.01), plasma angiotensin II concentration ( P < 0.01), and plasma aldosterone concentration ( P < 0.001). A striking influence of the angiotensin receptor blocker, candesartan, on plasma aldosterone concentration in the patients with diabetes mellitus suggested strongly that renin-system activation is responsible for the elevated plasma aldosterone concentration. Conclusion. Pharmacologic interruption of the effects of aldosterone at the tissue level could be especially useful in patients with diabetes mellitus. The dose of agents that block the renin-angiotensin system (RAS) should be adjusted to maximize the fall in plasma aldosterone concentration. [ABSTRACT FROM AUTHOR]

Inflammation culminating in fibrosis contributes to progressive kidney disease. Cross-talk between the tubular epithelium and interstitial cells regulates inflammation by a coordinated release of cytokines and chemokines. Here we studied the role of heme oxygenase-1 (HO-1) and the heavy subunit of ferritin (FtH) in macrophage polarization and renal inflammation. Deficiency in HO-1 was associated with increased FtH expression, accumulation of macrophages with a dysregulated polarization profile, and increased fibrosis following unilateral ureteral obstruction in mice: a model of renal inflammation and fibrosis. Macrophage polarization in vitro was predominantly dependent on FtH expression in isolated bone marrow-derived mouse monocytes. Using transgenic mice with conditional deletion of FtH in the proximal tubules (FtHPT−/−) or myeloid cells (FtHLysM−/−), we found that myeloid FtH deficiency did not affect polarization or accumulation of macrophages in the injured kidney compared with wild-type (FtH+/+) controls. However, tubular FtH deletion led to a marked increase in proinflammatory macrophages. Furthermore, injured kidneys from FtHPT−/− mice expressed significantly higher levels of inflammatory chemokines and fibrosis compared with kidneys from FtH+/+ and FtHLysM−/− mice. Thus, there are differential effects of FtH in macrophages and epithelial cells, which underscore the critical role of FtH in tubular-macrophage cross-talk during kidney injury. [ABSTRACT FROM AUTHOR]

Although short-term outcomes in kidney transplantation have improved dramatically, long-term survival remains a major challenge. A key component of long-term, chronic allograft injury in solid organ transplants is arteriosclerosis characterized by vascular neointimal hyperplasia and inflammation. Establishing a model of this disorder would provide a unique tool not only to identify mechanisms of disease but also to test potential therapeutics for late graft injury. To this end, we utilized a mouse orthotopic renal transplant model in which C57BL/6J (H-2b) recipients were given either a kidney allograft from a completely mismatched Balb/cJ mouse (H-2d) or an isograft from a littermate. A unilateral nephrectomy was performed at the time of transplant followed by a contralateral nephrectomy on post-transplant day 7. Recipients were treated with daily cyclosporine subcutaneously for 14 days and then studied 8 and 12 weeks post transplantation. Renal function was significantly worse in allograft compared with isograft recipients. Moreover, the allografts had significantly more advanced tubulointerstitial fibrosis and profound vascular disease characterized by perivascular leukocytic infiltration and neointimal hyperplasia affecting the intrarenal blood vessels. Thus, we describe a feasible and reproducible murine model of intrarenal transplant arteriosclerosis that is useful to study allograft vasculopathy. [ABSTRACT FROM AUTHOR]

Heme oxygenase-1 (HO-1) catalyzes the rate-limiting step in heme degradation, producing equimolar amounts of carbon monoxide, iron, and biliverdin. Induction of HO-1 is a beneficial response to tissue injury in diverse animal models of diseases including acute kidney injury. In vitro analysis has shown that the human HO-1 gene is transcriptionally regulated by changes in chromatin conformation, but whether such control occurs in vivo is not known. To enable such an analysis, we generated transgenic mice, harboring an 87-kb bacterial artificial chromosome expressing human HO-1 mRNA and protein and bred these mice with HO-1 knockout mice to generate humanized BAC transgenic mice. This successfully rescued the phenotype of the knockout mice including reduced birth rates, tissue iron overload, splenomegaly, anemia, leukocytosis, dendritic cell abnormalities, and survival after acute kidney injury induced by rhabdomyolysis or cisplatin nephrotoxicity. Transcription factors such as USF1/2, JunB, Sp1, and CTCF were found to associate with regulatory regions of the human HO-1 gene in the kidney following rhabdomyolysis. Chromosome conformation capture and ChIP-loop assays confirmed this in the formation of chromatin looping in vivo. Thus, these bacterial artificial chromosome humanized HO-1 mice are a valuable model to study the human HO-1 gene, providing insight to the in vivo architecture of the gene in acute kidney injury and other diseases. [ABSTRACT FROM AUTHOR]

Renal injury induced by brain death is characterized by ischemia and inflammation, and limiting it is a therapeutic goal that could improve outcomes in kidney transplantation. Brain death resulted in decreased circulating nitrite levels and increased infiltrating inflammatory cell infiltration into the kidney. Since nitrite stimulates nitric oxide signaling in ischemic tissues, we tested whether nitrite therapy was beneficial in a rat model of brain death followed by kidney transplantation. Nitrite, administered over 2 h of brain death, blunted the increased inflammation without affecting brain death-induced alterations in hemodynamics. Kidneys were transplanted after 2 h of brain death and renal function followed over 7 days. Allografts collected from nitrite-treated brain-dead rats showed significant improvement in function over the first 2 to 4 days after transplantation compared with untreated brain-dead animals. Gene microarray analysis after 2 h of brain death without or with nitrite therapy showed that the latter significantly altered the expression of about 400 genes. Ingenuity Pathway Analysis indicated that multiple signaling pathways were affected by nitrite, including those related to hypoxia, transcription, and genes related to humoral immune responses. Thus, nitrite therapy attenuates brain death-induced renal injury by regulating responses to ischemia and inflammation, ultimately leading to better post-transplant kidney function. [ABSTRACT FROM AUTHOR]

HO-1 (heme oxygenase-1) is an inducible microsomal enzyme that catalyzes the degradation of pro-oxidant heme. The goal of this study was to characterize a minimal enhancer region within the human HO-1 gene and delineate its role in modulating HO-1 expression by participation with its promoter elements in renal epithelial cells. Deletion analysis and site-directed mutagenesis identified a 220-bp minimal enhancer in intron 1 of the HO-1 gene, which regulates hemin-mediated HO-1 gene expression. Small interfering RNA, decoy oligonucleotides, site-directed mutagenesis, and chromatin immunoprecipitation assays confirmed the functional interaction of Sp1 with a consensus binding sequence within the 220-bp region. Mutations of regulatory elements within the -4.5 kb promoter region (a cyclic AMP response and a downstream NF-E2/AP-1 element, both located at -4.0 kb, and/or an E-box sequence located at -44 bp) resulted in the loss of enhancer activity. A chromosome conformation capture assay performed in human renal epithelial (HK-2) cells demonstrated hemin-inducible chromatin looping between the intronic enhancer and the -4.0 kb promoter region in a time-dependent manner. Restriction digestion with ApaLI (which cleaves the 220-bp enhancer) led to a loss of stimulus-dependent chromatin looping. Sp1 small interfering RNA and mithramycin A, a Sp1 binding site inhibitor, resulted in loss of the loop formation between the intronic enhancer and the distal HO-1 promoter by the chromosome conformation capture assay. These results provide novel insight into the complex molecular interactions that underlie human HO-1 regulation in renal epithelial cells. [ABSTRACT FROM AUTHOR]

[Display omitted] Arsenicals belong to the class of chemical warfare agents known as vesicants, which are highly reactive, toxic and cause robust inflammatory response. Cutaneous exposure to arsenicals causes a wide range of systemic organ damage, beginning with cutaneous injuries, and later manifest multi-organ damage and death. Thus, the development of suitable antidotes that can effectively block injury following exposure to these agents is of great importance. Bromodomain 4 (BRD4), a member of the bromodomain and extra terminal domain (BET) family, plays crucial role in regulating transcription of inflammatory, proliferation and cell cycle genes. In this context, the development of potent small molecule inhibitors of BRD4 could serve as potential antidotes for arsenicals. Herein, we describe the synthesis and biological evaluation of a series of compounds. [ABSTRACT FROM AUTHOR]

Abstract: In vivo and in vitro studies revealed that nitroalkenes serve as protective mediators in the lung by inducing the cytoprotective enzyme heme oxygenase-1 (HO-1). Nitrolinoleic acid (LNO2) increased HO-1 mRNA, protein, and activity in cultured pulmonary epithelial cells treated with 5 to 50 μM LNO2 and in lungs of rats injected intraperitoneally with 2.6 mg/kg LNO2 twice daily for 20 days. Western blotting revealed that HO-1 protein increased significantly within 4 h of in vitro LNO2 addition and was preceded by an increase in HO-1 mRNA, consistent with transcriptional regulation of HO-1 expression by LNO2. LNO2 also dephosphorylated and activated eukaryotic initiation factor 2α, a key translational regulatory protein, indicating that increased translation may also contribute to LNO2-induced increases in HO-1. Exposure of cells to LNO2 activated ERK and JNK, as evidenced by increased phosphorylation. Downstream targets of ERK and JNK, Elk-1 and c-Jun, respectively, were also phosphorylated in response to LNO2 exposure. However, inhibitor studies revealed that only the ERK pathway is necessary for the LNO2-mediated increase in HO-1 mRNA and protein. These data reveal that LNO2 induces pulmonary epithelial HO-1 expression and downstream adaptive responses to inflammation via both transcriptional and translational regulatory mechanisms. [Copyright &y& Elsevier]

The mechanisms that allow Mycobacterium tuberculosis (Mtb) to persist in human tissue for decades and to then abruptly cause disease are not clearly understood. Regulatory elements thought to assist Mtb to enter such a state include the heme two-component sensor kinases DosS and DosT and the cognate response regulator DosR. We have demonstrated previously that O2, nitric oxide (NO), and carbon monoxide (CO) are regulatory ligands of DosS and DosT. Here, we show that in addition to O2 and NO, CO induces the complete Mtb dormancy (Dos) regulon. Notably, we demonstrate that CO is primarily sensed through DosS to induce the Dos regulon, whereas DosT plays a less prominent role. We also show that Mtb infection of macrophage cells significantly increases the expression, protein levels, and enzymatic activity of heme oxygenase-1 (HO-1, the enzyme that produces CO), in an NO-independent manner. Furthermore, exploiting HO-1+/+ and HO-1-/- bone marrow-derived macrophages, we demonstrate that physiologically relevant levels of CO induce the Dos regulon. Finally, we demonstrate that increased HO-1 mRNA and protein levels are produced in the lungs of Mtb-infected mice. Our data suggest that during infection, O2, NO, and CO are being sensed concurrently rather than independently via DosS and DosT. We conclude that CO, a previously unrecognized host factor, is a physiologically relevantMtb signal capable of inducing the Dos regulon, which introduces a new paradigm for understanding the molecular basis of Mtb persistence. [ABSTRACT FROM AUTHOR]

Heme oxygenase-1 is a highly inducible gene, the product of which catalyzes breakdown of the prooxidant heme. The purpose of this study was to investigate the regulation of the human heme oxygenase-1 gene in renal epithelial cells. DNase I hyper- sensitivity studies identified three distal sites (HS-2, -3, and -4) corresponding to approximately -4.0, -7.2, and -9.2 kb, respectively, of the heme oxygenase-1 promoter in addition to one proximal region, HS-1, which we have shown previously to be an E box. In vivo dimethyl sulfate footprinting of the HS-2 region revealed six individual protected guanines. Two mutations within HS-2 combined with a third mutation of the proximal E box abolished hemin- and cadmium-driven heme oxygenase-1 promoter activation, suggesting that these three sites synergized for maximal heme oxygenase-1 induction. Jun proteins bound to the antioxidant response element in the HS-2 region in vitro and associated with the heme oxygenase-1 promoter in vivo. JunB and JunD contribute opposing effects; JunB activated whereas JunD repressed heme oxygenase-1 expression in human renal epithelial cells, results that were corroborated in junB-/- and junD-/- cells. We propose that heme oxygenase-1 induction is controlled by a dynamic interplay of regulatory proteins, and we provide new insights into the molecular control of the human heme oxygenase-1 gene. [ABSTRACT FROM AUTHOR]

Nitroalkene derivatives of linoleic acid (LNO2) and oleic acid (OA-NO2) are present; however, their biological functions remain to be fully defined. Herein, we report that LNO2 and OA-NO2 inhibit lipopolysaccharide-induced secretion of proinflammatory cytokines in macrophages independent of nitric oxide formation, peroxisome proliferator-activated receptor-7 activation, or induction of heme oxygenase-1 expression. The electrophilic nature of fatty acid nitroalkene derivatives resulted in alkylation of recombinant NF-κB p65 protein in vitro and a similar reaction with p65 in intact macrophages. The nitroalkylation of p65 by fatty acid nitroalkene derivatives inhibited DNA binding activity and repressed NF-κB-dependent target gene expression. Moreover, nitroalkenes inhibited endothelial tumor necrosis factor-a-induced vascular cell adhesion molecule 1 expression and monocyte rolling and adhesion. These observations indicate that nitroalkenes such as LNO2 and OA-NO2, derived from reactions of unsaturated fatty acids and oxides of nitrogen, are a class of endogenous anti-inflammatory mediators. [ABSTRACT FROM AUTHOR]

Renal tubular cells elicit adaptive responses following expo- sure to nephrotoxins, such as cadmium. One response is the up-regulation of the 32-kDa redox-sensitive protein, heme oxygenase-1. Exposure of renal proximal tubular epithelial cells to 10 µM cadmium demonstrated induction (~20-fold) of heme oxygenase-1 mRNA and protein. Using a 4.5-kb human heme oxygenase-1 promoter construct, the importance of a previously identified cadmium response element (TGCTAGAT) in HeLa cells was verified in renal epithelial cells. Specific protein-DNA interaction with this sequence was demonstrated using nuclear extracts from cadmium-treated cells. Yeast one-hybrid screen of a human kidney cDNA library resulted in the identification of pescadillo, a unique nucleolar, developmental protein, as an interacting protein with the cadmium response element and was confirmed by chromatin immunoprecipitation in vivo and gel shift assays with purified glutathione S-transferase-pescadillo protein in vitro. The specificity of the DNA-protein interaction was verified by the absence of a binding complex when the core sequence of the cadmium response element was mutated or deleted. In addition, B23/nucleophosmin, another nucleolar protein, did not interact with the cadmium response sequence. Overexpression of pescadillo resulted in increased activity of the 4.5-kb human heme oxygenase-1 promoter construct but failed to activate this construct when the cadmium response sequence was mutated. The findings demonstrate the important and previously unrecognized role of pescadillo as a DNA-binding protein interacting specifically with the cadmium response element of the human heme oxygenase-1 gene. [ABSTRACT FROM AUTHOR]

Abstract: Xanthine oxidoreductase (XOR) activity has been previously noted to be responsive to changes in O2 tension. While prior studies have focused on the extremes (0–3% and 95–100%) of O2 tensions, we report the influence of 10% O2 on endothelial cell XOR, a concentration resembling modest arterial hypoxia commonly found in patients with chronic cardiopulmonary diseases. Exposure of bovine aortic endothelial cells to 10% O2 increased XOR mRNA and protein abundance by 50%. Concomitantly, there was a 3-fold increase in XOR activity, XOR-dependent reactive oxygen species production, and cellular export of active enzyme. Although increases in mRNA and immunoreactive protein levels were observed, inhibition of transcription, translation, or protein degradation did not significantly alter cellular XOR specific activity, suggesting only modest contributions to 10% O2-induced effects. Exposure to 10% O2 did not increase cellular HIF-1α protein levels and hypoxia mimics did not alter XOR activity. Treatment of control cells with adenosine resulted in increased XOR activity similar to hypoxia. Exposure to the adenosine receptor agonist NECA increased enzymatic activity 4-fold while 8SPT, an adenosine receptor antagonist, reduced hypoxic induction of XOR activity ∼50%. Combined, these data reveal that moderate hypoxia significantly enhances endothelial XOR specific activity, release, and XOR-derived reactive oxygen species generation. These effects appear to be mediated in part via adenosine-dependent processes. [Copyright &y& Elsevier]

Mn-SOD serves as the primary cellular defense against oxidative damage by converting superoxide radicals (O2...) to O2 and H2O2. A unique characteristic of this mitochondrial anti-oxidant enzyme is the conservation from bacteria to man of a rapidly formed product inhibited state. Using site-directed mutagenesis, we have generated an active site mutant (H30N) of human Mn-SOD, which exhibits significantly reduced product inhibition and increased enzymatic efficiency. Overexpression of the H30N enzyme causes anti-proliferative effects in vitro and anti-tumor effects in vivo. Our results provide a teleological basis for the phylogenetically invariant nature of position His-30 and the evolutionary conservation of product inhibition. These data also provide more direct intracellular evidence for the signaling role associated with H2O2. [ABSTRACT FROM AUTHOR]

Hydrogen peroxide mediates FK506-induced cytotoxicity in renal cells. Background. The nephrotoxicity induced by immunosuppressant FK506 remains a serious clinical problem, and the underlying mechanism has not been completely understood. The present study was undertaken to determine the role of hydrogen peroxide in FK506-mediated cytotoxicity in a porcine renal proximal tubular cell line, LLC-PK1 cells, and human embryonic kidney (HEK293) cells. Methods. Cytotoxicity was estimated by crystal violet and lactate dehydrogenase release assays. The activity of reactive oxygen species (ROS) was detected by flow cytometry. FK506-induced cell death was examined in the presence of the hydrogen peroxide scavenger, catalase, or a scavenger of hydroxyl radicals, sodium benzoate. As a control, FK506-induced cell death was also measured in the presence of superoxide anion inhibitor, 4,5-dihydroxy-1,2-benzene disulfonic acid (Tiron), TEMPO, or overexpressed human manganese superoxide dismutase (MnSOD). Catalase was also used in tumor necrosis factor-α (TNF-α)-induced cell injury to determine whether the enzyme specifically protected cells against FK506-mediated cytotoxicity. Results. FK506 induced cell death in a dose-dependent manner and coincided with a dose-dependent increase in ROS activity. Abrogation of FK506-mediated ROS by catalase and N-acetylcysteine blunted FK506-induced cell death. Furthermore, overexpression of catalase, sodium benzoate, and deferoxamine inhibited the cytotoxic effect of FK506. In contrast, Tiron, TEMPO, or overexpression of human MnSOD failed to show cytoprotection. In fact, TEMPO or expression of MnSOD enhanced the effect of FK506. Catalase did not significantly affect TNF-α–induced cell injury. Conclusion. Catalase is uniquely required in cellular protection against FK506 cytotoxicity, which suggests an important role for hydrogen peroxide in the cellular actions of FK506. [ABSTRACT FROM AUTHOR]

Recent reports indicate successful transduction of pancreatic islets using recombinant adeno-associated viral (rAAV) vectors. This advance offers new possibilities in rendering islets resistant to rejection and recurrence of autoimmune destruction in the setting of islet transplantation as treatment of type 1 diabetes. Most gene delivery approaches using islets have thus far involved transduction with a single gene. However, the concomitant delivery of more than one gene encoding cytoprotective and/or immunoregulatory molecules may offer superior clinical utility. Here, we have generated a bicistronic rAAV (serotype 2) vector incorporating a viral internal ribosome entry site (IRES), derived from polio virus type 1, to allow for translation of two coupled cDNAs from a single mRNA transcript. Our study demonstrates the ability of this vector to produce significant expression of two reporter proteins in human and mouse islets in vitro. This expression did not interfere with β-cell function. Transduction was maintained in vivo following transplantation of mouse islets. These data are the first report of efficient islet cell transduction with two genes using a single bicistronic rAAV vector and have direct implications for strategies aimed at enhancing islet transplant survival. [Copyright &y& Elsevier]