Robbins Mike E, Zhao Weiling, Davis Charles S, Toyokuni Shinya, Bonsib Stephen M
Free Radical and Radiation Biology Program, Department of Radiology, The University of Iowa, B-180 ML, Iowa City, IA 52242, USA.
Micron. 2002;33(2):133-41. doi: 10.1016/s0968-4328(01)00006-3.
Kidney irradiation clearly leads to a progressive reduction in function associated with concomitant glomerulosclerosis and/or tubulointerstitial fibrosis. However, the particular cell types, mediators and/or mechanisms involved in the development and progression of radiation nephropathy remain ill defined. Angiotensin II (Ang II) plays a major pathogenic role; administration of Ang II blockers markedly abrogates the severity of radiation nephropathy in experimental models. Both ionizing radiation and Ang II signal via generation of reactive oxygen species (ROS). Thus, we hypothesized that localized kidney irradiation might lead to a chronic oxidative stress. In view of the difficulty in measuring ROS in vivo we adopted an indirect immunohistochemical approach in which we used a monoclonal antibody specific for 8-hydroxy-2'-deoxyguanosine (8-OHdG), one of the most commonly used markers of DNA oxidation. The right kidney of 7-8 week-old male Sprague-Dawley rats was removed. Five to 6 weeks later the remaining hypertrophied kidney was irradiated with single doses of 0-20.0 Gy X-rays. Groups of rats, three per dose, were killed at 4, 8, 16 and 24 weeks post-irradiation, their kidneys fixed, and sections stained with the 8-OHdG-specific antibody N45.1. For quantitation of glomerular DNA oxidation with the N45.1 antibody stained sections, 50 glomeruli/animal were counted. The presence of any intensely stained nuclei within the glomerular tuft was scored as positive. Quantitation of tubular DNA oxidation employed a 10 x 10 point ocular grid. Sections were examined at 400 magnification; 250 tubular profiles were counted. All tubules with any nuclear staining were scored as positive.Sham-irradiated kidneys showed little evidence of DNA oxidation over the experimental period. In contrast, localized kidney irradiation led to a marked, dose-independent increase in glomerular and tubular cell nuclear DNA oxidation. This increase was evident at the first time point studied, i.e. 4 weeks after irradiation, and persisted for up to 24 weeks postirradiation. DNA oxidation in the irradiated kidney was only seen in apparently viable glomerular and tubular cells. Thus, while from 16 to 24 weeks post-irradiation structural alterations had progressed to glomerular sclerosis and tubular atrophy, positive staining for 8-OHdG was not observed in severely atrophic tubules. Similarly, fewer positive staining cells were noted in glomeruli undergoing sclerosis, while none were seen in totally sclerotic glomeruli. These data support the hypothesis that renal irradiation is associated with a chronic and persistent oxidative stress.
肾脏照射显然会导致功能逐渐减退,并伴有肾小球硬化和/或肾小管间质纤维化。然而,辐射性肾病发生和发展过程中涉及的特定细胞类型、介质和/或机制仍不明确。血管紧张素II(Ang II)起主要致病作用;在实验模型中,给予Ang II阻滞剂可显著减轻辐射性肾病的严重程度。电离辐射和Ang II均通过产生活性氧(ROS)来传递信号。因此,我们推测局部肾脏照射可能会导致慢性氧化应激。鉴于体内测量ROS存在困难,我们采用了一种间接免疫组织化学方法,使用一种对8-羟基-2'-脱氧鸟苷(8-OHdG)特异的单克隆抗体,8-OHdG是最常用的DNA氧化标志物之一。切除7-8周龄雄性Sprague-Dawley大鼠的右肾。5至6周后,对剩余的肥大肾脏进行单次剂量为0-20.0 Gy的X射线照射。每组三只大鼠,在照射后4、8、16和24周处死,取其肾脏固定,切片用8-OHdG特异性抗体N45.1染色。对于用N45.1抗体染色的切片进行肾小球DNA氧化定量分析,每只动物计数50个肾小球。肾小球丛中任何强染色的细胞核记为阳性。肾小管DNA氧化定量采用10×10点的目镜网格。切片在400倍放大倍数下检查;计数250个肾小管轮廓。所有有核染色的肾小管记为阳性。假照射的肾脏在实验期间几乎没有DNA氧化的迹象。相比之下,局部肾脏照射导致肾小球和肾小管细胞核DNA氧化显著增加,且与剂量无关。这种增加在研究的第一个时间点即照射后4周就很明显,并持续到照射后24周。照射肾脏中的DNA氧化仅见于明显存活的肾小球和肾小管细胞。因此,虽然在照射后16至24周结构改变已发展为肾小球硬化和肾小管萎缩,但在严重萎缩的肾小管中未观察到8-OHdG阳性染色。同样,在正在发生硬化的肾小球中阳性染色细胞较少,而在完全硬化的肾小球中未观察到阳性染色细胞。这些数据支持了肾脏照射与慢性持续性氧化应激相关的假说。