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Fig. 2. Resorption of macromolecules by the proximal segment. The blood of embryos was labeled by cardiac injection of fluor-tagged dextrans, LDL particles, or proteins. Embryos were photographed 15 min post injection for live images, or allowed to resorb for 20 h then fixed and counterstained for Na+K+ATPase expression via FISH. (A) Dextrans cross the pronephric glomeral filtration barrier are excreted. Fluorescent 70 kDa dextran can be seen in the lumen of proximal tubules (blue arrow), distal tubule, and pronephric duct (white arrow) 15 min post injection in live embryo. (B) LDL particles cross the glomeral filtration barrier and are excreted. Fifteen minutes post injection, live embryo. Both the lumen and the epithelium of the proximal segment are fluorescent, while only the lumen of the distal segment and duct is labeled. (C) Serum albumin in the lumen of pronephric tubules 15 min post injection, live embryo. (D) Fluorescent urine (yellow arrow) from filtered 70 kDa dextran flowing from the cloaca. A real-time animation of protein excretion in urine is also available in the supplemental materials (BSA_proteinuria.mov). (E) Example of acetylated LDL resorption out-competing excretion. Little or no LDL is present in the lumen or urine. Red arrows indicate labeled endothelial cells. (F) Resorption of serum albumin by proximal tubules of a living tadpole. (G) Localization of 70 kDa FITC-dextran resorption, 20 h post injection. (H) Counterstain of G with Na+K+ATPase and a Cy3-tyramide substrate. Both FITC and Cy3 channels are shown. (I) Localization of Alexa488-LDL resorption domain. Tadpole was fixed 2 h post LDL injection. (J) Counterstain of I with Na+K+ATPase and a Cy3- tyramide substrate. Both Alexa-488 and Cy3 channels are shown. (K) Resorption of serum albumin. Tadpole was fixed 2 h post serum albumin injection. (L) Counterstain of the sample shown in K with Na+K+ATPase using an FITC-tyramide. The distal tubule (dt) has no serum albumin, nor does the duct. Both Texas Red (serum albumin) and FITC channels are shown. Scale bars represent 200 Am. Unlabeled blue arrows, proximal tubule. Unlabeled white arrows, pronephric duct. Unlabeled red arrows, endothelial cells. |
