By 48 hpi, the yolk sac had continued

to darken and the edema increased to a moderate level. Severe pericardial edema and body curvature was observed in embryos at 72 hpi. Following documentation of live embryos, several zebrafish were selected for further analysis and processed through in situ hybridization with slc20a1a. The gene slc20a1a is a sodium dependent phosphate transporter that has previously been used to specifically distinguish the location of the proximal convoluted tubule (PCT) from the other segments in the zebrafish pronephros. 10 During EPZ5676 normal development, the expression of slc20a1a can be detected by 24 hpf in parallel tracks of the PCT ( Fig 4, B). 10 Between 24 and 20 hpf, slc20a1a transcripts continue to be highly expressed in the PCT, enabling its clear visualization. At approximately 48 hpf, the cells occupying the PCT begin morphogenesis from linear tubes into a compact coiled structure ( Fig 4, B).

Initially, the rostral-most PCT tubes display a lateral shift and form a characteristic selleck ‘Y’ shape, and then between 96 and 120 hpf undergo progressive coiling to form a tightly packed unit located rostral to the yolk sac at 120 hpf. The driving force behind the coiling of the PCT segment is fueled by a combination of cellular division within the distal segments, 10 and collective migration of distal segments. 80 and 81 However, gentamicin exposure obviates this process of nephron morphogenesis.

In our analysis, embryos fixed at three time points post-gentamicin injection (24, 48, and 72 hpi) and processed through whole mount in situ hybridization with slc20a1a revealed that gentamicin delayed the PCT coiling process ( Fig 4, B). In addition, spotted staining of cells within the tubule was noted. This could indicate PCT cells that should Bortezomib concentration have been stained with the marker had either undergone necrosis and sloughed off, or were too damaged for recognition by the slc20a1a RNA probe. To further analyze the effects of gentamicin exposure on tubular integrity and epithelial cell architecture, immunohistochemistry was performed on tissue cryosections of injected zebrafish at 24 and 48 hpi (Fig 5). The use of a transgenic line that stably expresses green fluorescent protein in larval zebrafish (Tg:enpep:eGFP) enabled the visualization of the pronephric duct and tubules. 82 In healthy rat kidneys, phalloidin has been characterized as having an affinity for the actin in the apical brush border microvilli of proximal tubule epithelial cells. 83 Tissue cryosections of healthy and injured embryos were stained with phalloidin at 24 and 48 hpi ( Fig 5). No disruption in tubule structure or epithelial polarity was noticeable in the healthy, uninjected control embryos at either time point; the lumen was clearly demarcated by a band of actin ( Fig 5, A).