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

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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.