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Diverse molecular species of sulfatide with differences in FA lengths, unsaturation degrees, and hydroxylation statuses are expressed in the kidneys. However, the physiological functions of specific sulfatide species in the kidneys are unclear. Here, we evaluated the distribution of specific sulfatide species in the kidneys and their physiological functions. Electron microscopic analysis of kidneys of Cst-deficient mice lacking sulfatide showed vacuolar accumulation in the cytoplasm of intercalated cells in the collecting duct, whereas the proximal and distal tubules were unchanged. Immunohistochemical analysis revealed that vacuolar H+-ATPase-positive vesicles were accumulated in intercalated cells in sulfatide-deficient kidneys. Seventeen sulfatide species were detected in the murine kidney by iMScope MALDI-MS analysis. The distribution of the specific sulfatide species was classified into four patterns. Although most sulfatide species were highly expressed in the outer medullary layer, two unique sulfatide species of m/z 896.6 (predicted ceramide structure: t18:0-C22:0h) and m/z 924.6 (predicted ceramide structure: t18:0-C24:0h) were dispersed along the collecting duct, implying expression in intercalated cells. In addition, the intercalated cell-enriched fraction was purified by fluorescence-activated cell sorting using the anti-vacuolar H+-ATPase subunit 6V0A4, which predominantly contained sulfatide species (m/z 896.6 and 924.6). The Degs2 and Fa2h genes, which are responsible for ceramide hydroxylation, were expressed in the purified intercalated cells. These results suggested that sulfatide molecular species with ceramide composed of phytosphingosine (t18:0) and 2-hydroxy FAs, which were characteristically expressed in intercalated cells, were involved in the excretion of NH3 and protons into the urine.

Summary: Elucidating fine architectures and functions of cellular and synaptic connections requires development of new flexible methods. Here, we created a concept called the “battle of transgenes,” based on which we generated strategies using genetically engineered battles of multiple recombinases. The strategies enabled split-tunable allocation of multiple transgenes. We demonstrated the versatility of these strategies and technologies in inducing strong and multi-sparse allocations of multiple transgenes. Furthermore, the combination of our transgenic strategy and expansion microscopy enabled three-dimensional high-resolution imaging of whole synaptic structures in the hippocampus with simultaneous visualizations of endogenous synaptic proteins. These strategies and technologies based on the battle of genes may accelerate the analysis of whole synaptic and cellular connections in diverse life science fields.