ＭＺＴ（株）ゼブラフィッシュ創薬研究所

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In Vivo Assessment of Individual and Total Proteinuria in Zebrafish Larvae Using the Solvatochromic Compound ZMB741

To develop new fluorochromes for in vivo phenotypic screening, which is currently lacking globally, we reared transparent zebrafish (MieKomachi001) in a 96-well ZFplate and added one fluorochrome from a library of about 600 fluorochromes to the rearing water. A final concentration of 5 μM of each fluorescent dye was added to the rearing water, and image screening was performed 24 hours later. The results revealed that several fluorochromes selectively visualized blood vessels, with ZMB741 having the greatest fluorescence intensity. Subsequently, ZMB741 was found to bind to a number of plasma proteins, but not to the vessel wall, after noninvasive administration, leading to large-scale in vivo phenotypic screening. For example, it binds to most plasma proteins with slightly different affinities, such as apolipoprotein A-I (Kd=0.84 μM) and hemopexin (Kd=3.13 μM), which is the basis for its great utility in in vivo proteinuria quantification analysis. Furthermore, existing tracers such as fluorescent dextran, EB, and ICG can only be administered invasively, such as by cardiovascular injection, even in zebrafish. In addition, free ZMB741 in aqueous solution shows almost no fluorescence, but when bound to plasma proteins, its fluorescence quantum yield is highly enhanced, enabling more sensitive analysis of plasma protein dynamics than existing tracers, and thus enabling the analysis of medically important blood-brain barrier (BBB) breakdown (ACS Chem. Neurosci. 2013, 4, 1183-1193) and in vivo imaging of podocytopathy proteinuria.
Since the discovery of nephrin, one of the slit membrane component proteins of podocytes (glomerular epithelial cells), in 1998, our knowledge of podocytes has increased dramatically, and it has become clear that podocyte injury causes irreversible glomerulosclerosis and, eventually, chronic kidney disease (CKD) CKD. Kidney diseases in which podocyte injury is central to the pathogenesis are collectively considered podocytopathies, which typically include diseases of the primary nephrotic syndrome. Glucocorticoids and immunosuppressive drugs used in the treatment of these diseases have been reported to have direct podocytoprotective effects in addition to their immunological effects. However, at present, there is no therapeutic agent globally that has been established to strongly protect podocytes and inhibit the progression of CKD. It is hoped that the mechanism of podocytosis onset and progression will be elucidated, and specific treatment and prevention methods will be developed. Therefore, we created a zebrafish model of podocytosis by KO and KD of the nephrin gene and other genes, and constructed an in vivo proteinuria individual quantification system using ZMB741. This in vivo phenotypic screening has led to at least two therapeutic candidates with different mechanisms of action as a result of repositioning screening of existing drugs. We are currently analyzing the therapeutic mechanism of these new drug candidates for podocytosis and ultimately aiming for clinical application.

ＭＺＴ（株）ゼブラフィッシュ創薬研究所

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In Vivo Assessment of Individual and Total Proteinuria in Zebrafish Larvae Using the Solvatochromic Compound ZMB741

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