Hyperosmolality-induced damage to cells

高渗透压引起的细胞损伤

• 批准号: 8558068
• 负责人: MAURICE BENJAM BURG
• 金额: $ 48.94万
• 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/8558068
• 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; ChIP-seq; 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.

我们先前发现，在细胞培养和体内肾脏髓质中，高渗盐(高盐)都会引起DNA损伤和氧化应激。DNA损伤和氧化应激与细胞衰老有关，尤其是在衰老和癌症中。我们还发现，高盐在组织培养中会导致细胞衰老，在正常小鼠的肾脏髓质以及暴露在高盐下的线虫中，衰老细胞的年龄相关积累加速。因此，高渗透压不仅会造成DNA损伤和氧化应激，还会导致细胞衰老。 随着年龄的增长而导致的脱水是由于尿液集中能力下降和口渴。我们通过比较3个月、18个月和27个月大的小鼠的尿液浓度和水平衡进行了进一步的研究，这些小鼠摄入了等量的水。在限水期间，3月龄小鼠充分浓缩尿液以维持水分平衡(稳定体重)。18个月龄的小鼠也会浓缩尿液，但体重仍在下降(负水分平衡)。与18个月的小鼠相比，27个月的小鼠没有集中尿液，体重减轻得更多，这表明水的负平衡更大。老年小鼠的水负平衡伴随着加压素排泄的增加，这为脱水提供了进一步的证据。三组均保持水分平衡，同时只摄入水分含量为56%的凝胶食品中的水分。然而，两个年龄较大的组排出的尿量较少，渗透压较高，这表明更多的额外尿液水分丢失。由于它们的粪便也含有较少的水分，老年小鼠失去的多余水分显然是通过其他途径失去的，推测是通过呼吸道和皮肤失去知觉的。与尿液浓缩能力下降(27个月)相比，潜伏性水分丢失发生在更早的年龄(18个月)。我们认为，随着年龄的增长，通过皮肤和呼吸失去知觉的水分会增加，这是与衰老相关的脱水的主要原因。 Mre11在核DNA的维持、核DNA的修复、复制、减数分裂和端粒的维持等过程中起着重要的作用。线粒体DNA(MtDNA)的维持还没有得到很好的描述，Mre11是否参与也是未知的。我们先前发现，高盐导致一些mre11离开细胞核，但我们没有试图将其定位于细胞质中。在本研究中，我们从原代肾细胞分离的线粒体中发现了mre11，并且发现线粒体中mre11的含量随着细胞外盐浓度的升高而增加。此外，我们还通过共聚焦显微镜证实了mre11在细胞线粒体中的存在，并表明一些mre11与mtDNA共存。已知的破坏mtDNA的博莱霉素增加了mre11与mtDNA的共存。正常小鼠肾脏组织切片中也存在丰富的mre11，与近端小管线粒体和粗大的上肢细胞共存。为了探索Mre11的分布是否随细胞分化而变化，我们使用了一种通过在Matrigel基质中培养原代肾脏细胞来形成小管的实验模型。在未分化的细胞中，Mre11主要位于细胞核，但在细胞分化后，它主要变成细胞质。我们得出结论，mre11既存在于线粒体中，也存在于细胞核中，并且线粒体中的数量因细胞压力和分化而异。我们的结果表明，除了先前已知的维持核DNA的作用外，Mre11还在维持线粒体基因组完整性方面发挥了新的作用。 无论是在细胞培养中还是在体内，高浓度的氯化钠都会增加DNA断裂。只要氯化钠浓度保持在较高水平，断裂就会保持在较高水平，并在浓度降低时迅速修复。这些断裂的确切性质以及它们的位置尚不完全清楚，也不清楚细胞如何在肾内髓质等环境中存活、复制和保持基因组完整性，在这种环境中，额外的断裂持续存在，因为细胞不断暴露在高浓度的氯化钠中。在氯化钠被还原后，断裂的修复伴随着含有磷酸化H_2AX(GammaH_2AX)的焦点的形成。这种组蛋白修饰发生在DNA双链断裂周围，有助于它们的修复。我们发现(PNAS，在出版中)，在高盐诱导的DNA断裂修复过程中发生的GammaH2AX焦点在小鼠基因组中是非随机分布的。通过使用抗γ-H_AX抗体的染色质免疫沉淀和大规模平行测序(CHIP-SEQ)，我们发现在高盐诱导的双链断裂的修复过程中，γ-H_2AX主要定位于基因组中缺乏基因的区域(基因沙漠)，表明高盐诱导的双链断裂位于那里。基因沙漠的定位有助于解释为什么DNA断裂的危害性低于紫外线辐射、电离辐射和氧化剂等遗传毒性物质引起的随机断裂。我们认为，氯化钠在动物细胞周围的普遍存在直接影响了它们基因组结构的进化。