Fig. 4

LipL32 induced pronephric malformation, inflammatory cell infiltration and translocation of NA-K-ATPase in zebrafish tubular epithelial cells.

(A) Quantification of normal, mild or severe deformities of pronephros between groups of wt1b:GFP larvae at 48 hpf (n = 30 to 35 in each group). Photos were collected by in vivo observation under fluorescence microscopy (dorsal view, anterior to the left). Pronephric kidneys show abnormalities in glomerular fusion, cystic changes, and deformities of pronephric tubules and ducts. Scale bar, 50 µm. (B) In situ hybridization shows that l-plastin positive cells (pseudo-colored by red) were increased in lipl32 mRNA-injected larvae compared to control. Arrows indicate a colocalization (orange) of GFP staining (green) and l-plastin in the pronephric tubule and duct. Scale bar, 50 µm. (C) Immunostaining for NA-K-ATPase shows the normal basolateral location of NA-K-ATPase in the pronephric ducts was disorganized in lipl32 mRNA injected larvae (scale bar, 10 µm). Arrows indicate the basolateral cell surface. Right panels are transverse sections on whole-mount stained larvae on the left (scale bar, 2 µm). A diagram illustrating the changes in the cellular location of NA-K-ATPase is shown. (D) Immunostaining for acetyl-tubulin shows no significant differences of pronephric cilia (arrows) between groups (scale bar, left panel, 10 µm, right panel, 2 µm). (E) The retention rates of 10-kDa rhodamine dextran as measured from the boxed region of the posterior pronephric ducts 6 hours after pericardial injection were significantly reduced in the lipl32 mRNA-injected larvae compared to myc-mRNA controls (**P < 0.01, n = 10 to 11 in each group, scale bar, 100 µm). Transverse sections demonstrate retention of rhodamine fluorescence in the lumen of pronephric ducts (circle) in lipl32 mRNA-injected larvae (scale bar, 5 µm).