Pathological study of mitochondrial DNA deletion in circulatory diseases

为了探索线粒体DNA （mtDNA）缺失在体内形成的机制，我们在不同疾病模型的大鼠中检测了与年龄相关的4834bp mtDNA缺失。随着年龄的增长，这种缺失在大脑、心脏和肾脏皮层开始积累，但其在大脑和心脏的水平高于肾脏皮层，这支持了mtDNA缺失在有丝分裂后组织中积累的观点，增殖组织可以消除其积累。基于这些发现，我们将重点放在肾皮质mtDNA缺失上。幼龄大鼠和老年大鼠5/6肾切除，维持3个月。5/6肾切除大鼠均出现肾小球代偿性扩张和硬化，剩余组织出现小管扩张和上皮细胞萎缩。肾切除大鼠肾小管上皮细胞细胞色素氧化酶（COX）活性急剧下降，但幼鼠和老年大鼠之间无明显差异。无论是年轻的大鼠还是年老的大鼠，切除肾后的MtDNA缺失都没有增加，其水平几乎与未治疗的对照大鼠相当。接下来，我们使用了人类2型胰岛素依赖型糖尿病大鼠模型，即Otuska Long Evance Tokushima Fatty （OLETF）大鼠，发现肥胖、高血糖和高脂血症伴随着糖尿病肾病的发展。在衰老过程中，肾脏疾病进展，皮层也积累了4834bp的mtDNA缺失。此外，衰老相关的β-半乳糖苷酶（SA-β-gal）是细胞衰老超越增殖能力的标志，在一些小管上皮细胞中表达。最后，为了明确管状衰老与mtDNA缺失之间的关系，我们在体内和体外研究了镉中毒。Cd在体外初始干扰小管上皮细胞线粒体膜电位，强烈诱导细胞凋亡。经Cd毒性处理后存活的细胞培养超过100天，mtDNA不存在缺失。这可能与培养细胞具有高增殖能力的选择性消除机制有关。另一方面，长期低剂量Cd暴露大鼠肾脏功能障碍，肾小管上皮损伤，线粒体数量减少，氧化产物积累，SA-B-gal表达减少。此外，随着年龄相关mtDNA缺失的发生，肾皮质4834bp mtDNA缺失被大量积累。这些研究表明，在衰老过程中，当有毒刺激长期加载在组织上时，即使在增生性组织中，mtDNA缺失也会累积，导致功能恶化。这种现象在衰老相关疾病的发病机制中可能是重要的。少

To explore the mechanism of the formation of mitochondrial DNA (mtDNA) deletion in vivo, we examined age-related 4834bp mtDNA deletion using different disease models in rats. This deletion in the brain, heart and kidney cortex started to accumulate in accordance with increase of age, however its level was grater in the brain and heart than the kidney cortex, which supports the idea that mtDNA deletion accumulate in postmitotic tissues, and proliferative tissue -could eliminate its accumulation. Based on the findings, we focused on the mtDNA deletion in the kidney cortex. Young and old rat were 5/6-nephrecomized, and maintained for 3 months. Both of the rats with 5/6 nephrectomy revealed glomerular compensatory expansion and sclerosis, and tubular expansion with epithelial cell atrophy in remaining tissue. The activity of cytochrome oxidase (COX) in tubular epithelial cells decreased drastically in nephrectomized rats, while no different was however found between the young and old rats. … More MtDNA deletion was not accumulated by nephrectomy in either young or old rats, and the levels were almost equivalent to that of untreated control rats. We next used a rat model of human type 2, insulin-independent, diabetic mellitus, namely Otuska Long Evance Tokushima Fatty (OLETF) rats, which reveals obesity, hyperglycemia, and hyperlipidemia along with development of diabetic kidney diseases. During aging process, kidney disease was advanced, and 4834bp mtDNA deletion was also accumulated in the cortex. Moreover, senescent-associated beta-galactosidase (SA-β-gal), a marker of cell senescence beyond proliferative capacity, was expressed in some tubular epithelial cells. Finally, to specify the relationship between tubular senescence and mtDNA deletion, cadmium (Cd) intoxication was examined in vitro and in vivo. Cd in vitro initially perturbed mitochondrial membrane potential of tubular epithelial cells, and strongly induced apoptosis. When cells survived from Cd toxicity were cultured for more than 100 days, mtDNA deletion could not be induced. This may be due to the selective elimination mechanisms with high proliferation capacity in cultured cells. On the other hand, rats exposed for long time by low dose Cd induced kidney dysfunction and tubular epithelial damages along with decreased mitochondrial number, accumulation of oxidation products and expression of SA-B-gal. Moreover, 4834bp mtDNA deletion in kidney cortex was enormously accumulated over the occurrence of age-related mtDNA deletion. These studies indicated that when toxic stimuli are chronically loaded on tissues during senescence, mtDNA deletion even in proliferative tissue accumulates, results in functional deterioration. This phenomenon may be important in the pathogenesis, of the development of senescence-related diseases. Less