Age-dependent sensitivity of the mouse kidney to chronic nicotine exposure.

BackgroundMany adolescents are exposed to nicotine via smoking, e-cigarette use, or second-hand smoke. Nicotine-induced renal oxidative stress and its long-term consequences may be higher in adolescents than in adults because of intrinsic factors in the adolescent kidney.MethodsAdolescent and adult male C57Bl/6J mice were subjected to 2 or 200 µg/ml nicotine, which closely emulates passive or active smoking, respectively, for 4 weeks. Extent of nicotine exposure (cotinine content), oxidative stress (HNE), renal function (creatinine), tubular injury (KIM-1), and pretreatment renal levels of select pro-oxidant (p66shc) and antioxidant (Nrf2/MnSOD) genes were determined. Impact of p66shc overexpression or Nrf2/MnSOD knockdown on low-/high-dose nicotine-induced oxidative stress was determined in cultured renal proximal tubule cells.ResultsDespite similar plasma/renal cotinine levels, renal HNE and KIM-1 contents were higher in adolescents compared with those in adults, whereas renal function was unaltered after passive or active smoking-equivalent nicotine exposure. Pretreatment levels of p66shc were higher, whereas Nrf2/MnSOD levels were lower in the adolescent kidney. In agreement with this, overexpression of p66shc or knockdown of Nrf2/MnSOD augmented nicotine-induced ROS production in renal proximal tubule cells.ConclusionChronic nicotine exposure incites higher oxidative stress in the adolescent than in adult kidney because of a pre-existent pro-oxidant milieu.

p66shc-mediated toxicity of high-dose alfa-tocopherol in renal proximal tubule cells

α-Tocopherol (TOC) is a widely used supplement known for its role as an antioxidant. Previously, we have shown that TOC elicits adaptive responses by upregulating the ERK/CREB/HO-1 pathway, which depends on its concentration in cultured renal proximal tubule cells (RPTCs). This suggests that high-dose TOC (hTOC) may elicit adverse effects via inflicting oxidative stress. Since the pro-oxidant p66shc is a major mediator of oxidant injury in various models of renal toxicants, we tested the hypothesis that hTOC elicits renal toxicity through activation of p66shc and consequent oxidative stress. RPTCs (NRK52E) were treated with high-dose TOC (hTOC; 400 nM) in cells where expression or mitochondrial cytochrome c-binding of p66shc was manipulated by genetic means. Intracellular production of reactive oxygen species (ROS), mitochondrial depolarization, and cell viability was also determined. Additionally, activation of the pro-survival ERK/CREB/HO-1 signaling and the p66shc promoter was determined via reporter luciferase assays. hTOC decreased cell viability via increasing ROS-dependent mitochondrial depolarization and suppressing the pro-survival ERK/CREB/HO-1 pathway via transcriptional activation of p66shc. Conversely, either knockdown of p66shc, mutation of its mitochondrial cytochrome c-binding site, or overexpression of ERK or HO-1 ameliorated adverse effects of hTOC and restored the pro-survival signaling. The pro-oxidant p66shc plays dual role in toxicity of high-dose TOC: it provokes oxidative stress and suppresses adaptive responses (AU)

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p66shc-mediated toxicity of high-dose α-tocopherol in renal proximal tubule cells.

α-Tocopherol (TOC) is a widely used supplement known for its role as an antioxidant. Previously, we have shown that TOC elicits adaptive responses by upregulating the ERK/CREB/HO-1 pathway, which depends on its concentration in cultured renal proximal tubule cells (RPTCs). This suggests that high-dose TOC (hTOC) may elicit adverse effects via inflicting oxidative stress. Since the pro-oxidant p66shc is a major mediator of oxidant injury in various models of renal toxicants, we tested the hypothesis that hTOC elicits renal toxicity through activation of p66shc and consequent oxidative stress. RPTCs (NRK52E) were treated with high-dose TOC (hTOC; 400 nM) in cells where expression or mitochondrial cytochrome c-binding of p66shc was manipulated by genetic means. Intracellular production of reactive oxygen species (ROS), mitochondrial depolarization, and cell viability was also determined. Additionally, activation of the pro-survival ERK/CREB/HO-1 signaling and the p66shc promoter was determined via reporter luciferase assays. hTOC decreased cell viability via increasing ROS-dependent mitochondrial depolarization and suppressing the pro-survival ERK/CREB/HO-1 pathway via transcriptional activation of p66shc. Conversely, either knockdown of p66shc, mutation of its mitochondrial cytochrome c-binding site, or overexpression of ERK or HO-1 ameliorated adverse effects of hTOC and restored the pro-survival signaling. The pro-oxidant p66shc plays dual role in toxicity of high-dose TOC: it provokes oxidative stress and suppresses adaptive responses.

Alpha-Tocopherol protects renal cells from nicotine- or oleic acid-provoked oxidative stress via inducing heme oxygenase-1

Smoking and obesity increases renal oxidative stress via nicotine (NIC) or free fatty acid such as oleic acid (OA) but decreases levels of the vitamin E-derivative alpha-tocopherol (TOC), which has shown to stimulate the antioxidant system such as heme oxygenase-1 (HO-1). Hence, we hypothesized that supplementation of TOC may protect renal proximal tubules from NIC- or OA-mediated oxidative stress by upregulating the HO-1 gene. NIC- or OA-dependent production of reactive oxygen species (ROS) was determined in the presence or absence of various pharmacologic or genetic inhibitors that modulate HO-1 activation and enhancer elements in the HO-1 promoter such as the antioxidant response element (ARE) and the cAMP-response element (CRE) in renal proximal tubule cells (NRK52E). Activity of the HO-1 promoter, the ARE and the CRE was determined in luciferase assays. We found that pre- or posttreatment with TOC attenuated NIC- or OA-dependent ROS production that required HO-1 activation. TOC activated the HO-1 promoter via the CRE but not the ARE enhancer through the extracellular signal-regulated kinase (ERK) and protein kinase A (PKA). Consequently, inhibitors of ERK, PKA, or CRE activation mitigated beneficial effects of TOC on NIC- or OA-mediated ROS production. Hence, vitamin E supplementation-via induction of the cytoprotective HO-1-may help to reduce renal oxidative stress imposed by smoking or obesity

α-Tocopherol protects renal cells from nicotine- or oleic acid-provoked oxidative stress via inducing heme oxygenase-1.

Smoking and obesity increases renal oxidative stress via nicotine (NIC) or free fatty acid such as oleic acid (OA) but decreases levels of the vitamin E-derivative α-tocopherol (TOC), which has shown to stimulate the antioxidant system such as heme oxygenase-1 (HO-1). Hence, we hypothesized that supplementation of TOC may protect renal proximal tubules from NIC- or OA-mediated oxidative stress by upregulating the HO-1 gene. NIC- or OA-dependent production of reactive oxygen species (ROS) was determined in the presence or absence of various pharmacologic or genetic inhibitors that modulate HO-1 activation and enhancer elements in the HO-1 promoter such as the antioxidant response element (ARE) and the cAMP-response element (CRE) in renal proximal tubule cells (NRK52E). Activity of the HO-1 promoter, the ARE and the CRE was determined in luciferase assays. We found that pre- or posttreatment with TOC attenuated NIC- or OA-dependent ROS production that required HO-1 activation. TOC activated the HO-1 promoter via the CRE but not the ARE enhancer through the extracellular signal-regulated kinase (ERK) and protein kinase A (PKA). Consequently, inhibitors of ERK, PKA, or CRE activation mitigated beneficial effects of TOC on NIC- or OA-mediated ROS production. Hence, vitamin E supplementation-via induction of the cytoprotective HO-1-may help to reduce renal oxidative stress imposed by smoking or obesity.

The plant-derived natural compound Flavin 7 attenuates oxidative stress in cultured renal proximal tubule cells.

BACKGROUND: Cancer therapies and cancer progression can increase oxidative stress that might account for renal toxicity in cancer patients. Flavin 7 (F7) is a natural polyphenol-containing dietary supplement with potential antioxidant activity. Therefore, it might help to attenuate renal toxicity of chemotherapeutics. MATERIALS AND METHODS: Cultured mouse renal proximal tubule cells were subjected to H(2)O(2)-mediated oxidative stress. Potential antioxidant effects of F7 were assessed by measuring the production of reactive oxygen species (ROS), mitochondrial depolarization and injury (lactate dehydrogenase release as well as trypan blue exclusion) in cells that were pretreated with F7 prior to treatment with H(2)O(2). RESULTS: F7 pretreatment significantly attenuated H(2)O(2)-induced ROS production, mitochondrial depolarization and consequent injury in renal proximal tubule cells. CONCLUSION: F7 supplementation might be beneficial for cancer patients in order to prevent renal toxicity of anticancer drug- or cancer progression-related oxidative stress.

Effect of a plant-derived natural compound, Flavin7, on the ERK signaling pathway in immortalized mouse proximal tubule cells.

BACKGROUND: Since MAP kinases represent an important pathway of transducing external stimuli to internal signals in cells, determining their possible role in cancer cells may offer a promising way for the treatment and prognosis of malignant diseases. Our previous experiments have shown that a flavonoid-rich solution, Flavin7, was able to diminish kidney tumor growth in vivo. MATERIALS AND METHODS: Effects of Flavin 7 on the MAPK signaling pathway were determined in immortalized mouse proximal tubule cells by determining cell viability, flow cytometric analysis, luciferase assays and Western blots. RESULTS: At a nontoxic dose, Flavin7 markedly reduced phosphorylation of ERK and inhibited activity of its downstream targets such as Elk1 and CREB via inhibition of the ERK-kinase MEK1. CONCLUSION: Because of its ability to temporarily inhibit kidney tumor growth and activation of the MEK1/ERK pathway in vitro, further in vivo investigations may determine the potential role of Flavin7 in the treatment of malignancies.

All-trans retinoic acid (ATRA) suppresses transcription of human papillomavirus type 16 (HPV16) in a dose-dependent manner.

Earlier we found that SiHa cervical squamous carcinoma cells that harbor HPV type 16 respond to ATRA treatment in a dose-dependent manner: high-dose (10(-5)-10(-4) M) but not low-dose (10(-7)-10(-6) M) ATRA induced growth arrest. Growth of HPV-infected cells is highly dependent on the expression of the viral E6/E7 proteins. Thus, targeting expression of the viral E6/E7 genes might influence growth properties of HPV-infected cells. Here, we demonstrated that high-dose ATRA inhibited expression of HPV16 E7 through suppression of the HPV16 promoter (p97) activity. Gelshift assay (EMSA) revealed that binding of the AP-1 transcription factor to an oligonucleotide originated from the HPV type 16 promoter was diminished after high-dose, but not low-dose ATRA treatment. This suggests that high-dose ATRA suppresses HPV 16 promoter activity, at least in part, via a decreased AP-1 binding. Our data might be useful in treatment of cervical dysplasias and/or carcinomas.

All-trans-retinoic acid activates caspase-1 in a dose-dependent manner in cervical squamous carcinoma cells.

Earlier we observed that all-trans-retinoic acid (ATRA) dose-dependently suppressed the growth of cervical carcinoma cells. Suppression of growth required sustained activation of interferon regulatory factor 1 (IRF-1), which was achieved by high-dose (10(-4) M), but not low-dose (10(-6) M), ATRA treatment. In this paper we examine the role of IRF-1 in cell death that accompanied the growth suppression in high-dose ATRA-treated cells. We found that high-dose, but not low-dose, ATRA treatment activated caspase-1 in those cervical carcinoma cells. Transient transfection of an antisense-IRF-1 construct diminished high-dose ATRA-mediated caspase-1 activation. On the other hand, ATRA was not able to induce caspase-1 expression in a STAT1 (signal transducer and activator of transcription 1) knockout cell line, but transient transfection of STAT1 restored it. These results suggested the importance of both IRF-1 and STAT1 in high-dose ATRA-induced activation of caspase-1. Our results might be useful in the treatment of retinoid-resistant cervical neoplasias.

Suppression of growth by all-trans retinoic acid requires prolonged induction of interferon regulatory factor 1 in cervical squamous carcinoma (SiHa) cells.

All-trans retinoic acid (ATRA) suppresses growth of cervical dysplasias in vivo, although the sensitivity to retinoids is frequently lost during cervical carcinogenesis. It has been suggested that prolonged treatment or use of higher doses of retinoids might offer favorable response rates. We found SiHa cervical squamous carcinoma cells that were virtually resistant to ATRA-induced growth-inhibitory effects at physiological doses (10(-7 to) 10(-6) M) to be more responsive at pharmacological doses (10(-5 to) 10(-4) M). The growth inhibition by high-dose (10(-4) M) ATRA was associated with a sustained activation of interferon regulatory factor 1 (IRF-1), while a low dose (10(-6) M) of ATRA activated IRF-1 only transiently. Antisense IRF-1 inhibited the high-dose (10(-4) M), ATRA-mediated growth arrest; forced expression of IRF-1 caused a significant reduction in cell growth. High-dose (10(-4) M) ATRA increased binding of NF-kappaB and STAT1 proteins to sequences that originated from the IRF-1 promoter region, while low-dose (10(-6) M) ATRA induced only NF-kappaB binding. A delayed tyrosine phosphorylation of the signal transducer and activator of transcription-1 (STAT1) was observed after high-dose (10(-4) M) but not low-dose (10(-6) M) ATRA treatment. In agreement with this, induction of IRF-1 mRNA by ATRA was only modest and transient in a STAT1 knockout cell line, suggesting the importance of STAT1 in sustained IRF-1 expression. Our data showed that ATRA is capable of inducing dose-dependent cellular changes, which might be appropriate to overcome resistance to retinoids that frequently develops during cervical carcinogenesis.