Elucidation of the new role of nitric oxide in the cortical collecting duct that regulates and acid base balance

肾皮质集管（COD）由主细胞和插层细胞组成，在酸碱平衡中起重要作用。CCD包含两种插层细胞：a插层细胞分泌H， b插层细胞分泌HCO3（-）。另一方面，一氧化氮（NO）几乎在所有组织和器官中产生，在生理和病理条件下都发挥着多种生物作用。一氧化氮由三种不同的一氧化氮合成酶（NOS）亚型合成：神经元型、诱导型和内皮型NOS。已知CCD可表达各种异构体，NO在CCD中起重要作用。但是对于一氧化氮如何影响CCD中酸碱平衡的维持，我们所知甚少。首先，我们研究了COD对代谢性酸中毒或碱中毒的适应是否需要NO的产生。我们从正常小鼠肾脏中取出CCD，用no敏感荧光染料二氨基荧光素（DAF）对其进行孵育。改变酸或碱孵育液后，在CCD中测量其荧光。只有当pH变低时，一氧化氮才会立即在部分细胞处产生并在CCD内扩散。在这些实验之后，为了确定哪些细胞产生NO，我们尝试用AE1（主要细胞的标记）、PNA（6个细胞的标记）或band 3（1个细胞的标记）标记相同的CCD。但我们无法识别这些细胞。接下来，我们分别用NOS单敲除小鼠和NOS三敲除小鼠来研究NO在CCD中调节酸碱平衡的作用。我们报道了NOS三重基因敲除小鼠在出生后后期引起肾源性尿崩症，CCD肾小管凋亡参与了肾源性尿崩症的发病机制。因此，我们需要在出生后的早期阶段进行CCD检查。我们正计划用它做实验。

The cortical collecting duct (COD) of the kidney plays an important role in acid base homeostasis and is comprised of principal cells and intercalated cells. The CCD contains two types of intercalated cells: A-intercalated cells secrete H and B-intercalated cells secrete HCO3 (-). On the other hand, Nitric oxide (NO) is produced in almost all tissues and organs, exerting multiple biological actions under both physiological and pathological conditions. NO is synthesized by three different isoforms of NO synthase (NOS): neuronal, inducible, and endothelial NOSs. The CCD is known to express each isoform and NO has important actions in the CCD. But less is known about how NO affects the maintenance of acid-base homeostasis in the CCD. At first, we examined whether NO production was required for adaptation of the COD to metabolic acidosis or alkalosis. We incubated the dissected CCD from normal mouse kidney and loaded it with NO-sensitive fluorescent dyes, diaminofluoresceins (DAF). We measured its fluorescence in the CCD after changing acid or alkali incubation solution. Only When it changed into low pH, NO was produced immediately at, the some cells and spread in the CCD. After these experiments, to identify which cells produce NO, we tried to label the same CCD with either AE1 (the marker of Principal cells), PNA (the marker of 6 cells), or band 3 (the marker for a cells). But we were not able to identify the cells. Next, we used each single NOS knockout and triple NOS knockout mice to investigate the role of NO in regulating the acid-base balance in the CCD. We have reported that triple NOS knockout mice cause nephrogenic diabetes insipidus at the late stage after birth, and that renal tubular apoptosis in the CCD is involved in the pathogenesis of nephrogenic diabetes insipidus. Therefore we need the CCD at the early stage after birth. We are planning to experiment using it.