Hyperosmolality-induced damage to cells

高渗透压引起的细胞损伤

• 批准号: 8344924
• 负责人: MAURICE BENJAM BURG
• 金额: $ 49.99万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8344924
• 关键词: Age; Aging; Animals; Antibodies; Apoptosis; Bleomycin; Caenorhabditis elegans; Cell Aging; Cell Culture Techniques; Cell Differentiation process; Cell Nucleus; Cell Proliferation; Cells; Cellular Stress; Confocal Microscopy; Cytoplasm; DNA; DNA Damage; DNA Double Strand Break; Dehydration; Double Strand Break Repair; Employee Strikes; Environment; Equilibrium; Evolution; Excretory function; Experimental Models; Feces; Food; Gel; Genes; Genome; Goals; Insensible Water Loss; Ionizing radiation; Junk DNA; Kidney; Kidney Concentrating Ability; Kidney Part; Left; Limb structure; Location; Maintenance; Malignant Neoplasms; Meiosis; Mitochondria; Mitochondrial DNA; Molecular; Mus; Mutagens; Nature; Nuclear; Osmolalities; Oxidants; Oxidative Stress; Participant; Proteins; Respiration; Respiratory System; Respiratory tract structure; Role; Route; Skin; Sodium Chloride; Stress; Structure; Telomere Maintenance; Thick; Thirst; Tissues; Ultraviolet Rays; Urea; Urine; Vasopressins; Water; Weight; age related; base; cell killing; chromatin immunoprecipitation; extracellular; histone modification; in vivo; kidney cell; kidney medulla; killings; matrigel; mouse genome; oxidation; premature; repaired; senescence; tissue culture; urinary

We previously found that hypertonicity (high NaCl) causes DNA damage and oxidative stress both in cell culture and in kidney medullas in vivo. DNA damage and oxidative stress are associated with cellular senescence, most striking in aging and in cancer. We also found that high salt causes cellular senescence in tissue culture and that age-associated accumulation of a senescent cells is accelerated in kidney medullas of normal mice, as well as in C. Elegans exposed to high salt. Thus, hyperosmolality not only causes DNA damage and oxidative stress, but also causes cellular senescence. Dehydration with aging is attributed to decreased urine concentrating ability and thirst. We further investigated by comparing urine concentration and water balance in 3, 18 and 27 months old mice, consuming equal amounts of water. During water restriction, 3 months mice concentrate their urine sufficiently to maintain water balance (stable weight). 18 months mice concentrate their urine as well, but still lose weight (negative water balance). 27 months mice do not concentrate their urine as well and lose even more weight than the 18 months mice, indicating a larger negative water balance. Negative water balance in older mice is accompanied by increased vasopressin excretion, providing further evidence of dehydration. All 3 groups maintain water balance while consuming only the water in gel food containing 56% water. However, both older groups excrete a smaller volume of urine of higher osmolality, indicating greater extra urinary water loss. Since their feces also contain less water, the excess water lost by the older mice apparently is through other routes, presumably insensible loss through the respiratory tract and skin. The greater insensible water loss occurs at an earlier age (18 months) than decreased urine concentrating ability (27 months). We propose that insensible water loss through skin and respiration increases with age, making a major contribution to aging related dehydration. Mre11 is a critical participant in upkeep of nuclear DNA, its repair, replication, meiosis and maintenance of telomeres. The upkeep of mitochondrial DNA (mtDNA) is less well characterized and whether Mre11 participates had been unknown. We previously found that high NaCl causes some of the Mre11 to leave the nucleus, but we did not then attempt to localize it within the cytoplasm. In the present studies we find Mre11 in mitochondria isolated from primary renal cells and show that the amount of Mre11 in mitochondria increases with elevation of extracellular NaCl. We additionally confirm the presence of Mre11 in the mitochondria of cells by confocal microscopy and show that some of the Mre11 colocalizes with mtDNA. Bleomycin, which is known to damage mtDNA, increases colocalization of Mre11 with mtDNA. Abundant Mre11 is also present in tissue sections from normal mouse kidneys, colocalized with mitochondria of proximal tubule and thick ascending limb cells. To explore whether distribution of Mre11 changes with cell differentiation we used an experimental model of tubule formation by culturing primary kidney cells in Matrigel matrix. In non-differentiated cells Mre11 is mostly in the nucleus, but it becomes mostly cytoplasmic upon cell differentiation. We conclude that Mre11 is present in mitochondria as well as in nuclei, and that the amount in mitochondria varies, depending on cellular stress and differentiation. Our results suggest a new role for Mre11 in the maintenance of genome integrity in mitochondria, in addition to previously known role in maintenance of nuclear DNA. High concentration of NaCl increases DNA breaks both in cell culture and in vivo. The breaks remain elevated as long as NaCl concentration remains high and are rapidly repaired when the concentration is lowered. The exact nature of the breaks, as well as their location, had not been entirely clear, nor had it been evident how cells survive, replicate and maintain genome integrity in environments like the renal inner medulla in which the additional breaks persist because the cells are constantly exposed to high NaCl concentration. Repair of the breaks after NaCl is reduced is accompanied by formation of foci containing phosphorylated H2AX (gammaH2AX). This histone modification, which occurs around DNA double-strand breaks, contributes to their repair. We find (PNAS, in press)that gammaH2AX foci that occur during repair of high NaCl-induced DNA breaks are non-randomly distributed in the mouse genome. By chromatin immunoprecipitation using anti-gamma H2AX antibody, followed by massive parallel sequencing (ChIP-Seq), we find that during repair of double strand breaks induced by high NaCl, gammaH2AX is predominantly localized to regions of the genome devoid of genes (gene deserts), indicating that the high NaCl-induced double-strand breaks are located there. Localization to gene deserts helps explain why the DNA breaks are less harmful than are the random breaks induced by genotoxic agents such as UV radiation, ionizing radiation and oxidants. We propose that the universal presence of NaCl around animal cells has directly influenced the evolution of the structure of their genomes.

我们以前发现，高渗（高氯化钠）导致DNA损伤和氧化应激，在细胞培养和在体内肾髓质。DNA损伤和氧化应激与细胞衰老有关，在衰老和癌症中最引人注目。我们还发现，高盐会导致组织培养中的细胞衰老，在正常小鼠的肾髓质中，衰老细胞的年龄相关积累会加速，在C。暴露在高盐环境中的优雅。因此，高渗不仅导致DNA损伤和氧化应激，而且还导致细胞衰老。 衰老引起的脱水是由于尿液浓缩能力下降和口渴。我们通过比较3、18和27月龄小鼠的尿液浓度和水平衡进行了进一步研究，消耗等量的水。在限水期间，3个月的小鼠充分浓缩其尿液以维持水平衡（稳定的体重）。18个月的小鼠也会浓缩尿液，但体重仍然减轻（负水平衡）。27个月的小鼠也不会浓缩它们的尿液，并且比18个月的小鼠减轻更多的体重，这表明更大的负水平衡。老年小鼠的负水平衡伴随着加压素排泄增加，提供了脱水的进一步证据。 所有3组都保持水平衡，同时只消耗含有56%水的凝胶食品中的水。然而，两个老年组排出的尿量较少，渗透压较高，表明更多的额外尿水分流失。由于它们的粪便也含有较少的水，老年小鼠流失的多余水分显然是通过其他途径，可能是通过呼吸道和皮肤不知不觉地流失的。更大的无感觉水分丢失发生在较早的年龄（18个月）比降低尿液浓缩能力（27个月）。我们认为，随着年龄的增长，通过皮肤和呼吸的不知不觉的水分流失增加，使老化相关的脱水的主要贡献。 Mre 11是维持核DNA、其修复、复制、减数分裂和端粒维持的关键参与者。线粒体DNA（mtDNA）的维持不太好表征，Mre 11是否参与尚不清楚。我们以前发现，高NaCl导致一些Mre 11离开细胞核，但我们没有尝试将其定位在细胞质内。在目前的研究中，我们发现从原代肾细胞分离的线粒体中的Mre 11，并显示线粒体中的Mre 11的量随着细胞外NaCl的升高而增加。我们还确认了Mre 11的存在下，在线粒体的细胞通过共聚焦显微镜，并显示，一些Mre 11与mtDNA共定位。已知会损伤mtDNA的博来霉素增加了Mre 11与mtDNA的共定位。丰富的Mre 11也存在于正常小鼠肾脏的组织切片中，与近端小管和厚的升肢细胞的线粒体共定位。 为了探索Mre 11的分布是否随细胞分化而改变，我们使用了通过在Matrigel基质中培养原代肾细胞的小管形成的实验模型。在未分化的细胞中，Mre 11主要位于细胞核中，但在细胞分化后主要变为细胞质。我们的结论是，Mre 11存在于线粒体以及在细胞核中，线粒体中的量不同，这取决于细胞的压力和分化。我们的研究结果表明，除了先前已知的在维持核DNA中的作用之外，Mre 11在维持线粒体基因组完整性中的新作用。 高浓度的NaCl增加了细胞培养物和体内的DNA断裂。只要NaCl浓度保持高，断裂就保持升高，并且当浓度降低时迅速修复。 断裂的确切性质以及它们的位置还不完全清楚，也不清楚细胞如何在肾内髓等环境中存活、复制和保持基因组完整性，在肾内髓中，由于细胞不断暴露于高NaCl浓度，额外的断裂持续存在。在NaCl减少后，断裂的修复伴随着含有磷酸化H2 AX（γ H2 AX）的病灶的形成。这种发生在DNA双链断裂周围的组蛋白修饰有助于它们的修复。我们发现（PNAS，出版中），在修复高NaCl诱导的DNA断裂过程中发生的gammaH 2AX灶在小鼠基因组中是非随机分布的。通过使用抗γ H2 AX抗体进行染色质免疫沉淀，然后进行大规模平行测序（ChIP-Seq），我们发现在高NaCl诱导的双链断裂修复过程中，γ H2 AX主要定位于基因组中没有基因的区域（基因沙漠），表明高NaCl诱导的双链断裂位于那里。定位于基因沙漠有助于解释为什么DNA断裂比由遗传毒性剂（如紫外线辐射、电离辐射和氧化剂）诱导的随机断裂危害小。 我们认为，动物细胞周围普遍存在的NaCl直接影响了它们基因组结构的进化。