Zhou X and Vize PD (2004), Proximo-distal specialization of epithelial tra...

XB-ART-3354

Dev Biol 2004 Jul 15;2712:322-38. doi: 10.1016/j.ydbio.2004.03.036.

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Proximo-distal specialization of epithelial transport processes within the Xenopus pronephric kidney tubules.

Zhou X , Vize PD .

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The embryonic kidneys of larval aquatic vertebrates such as fish and frogs serve as excellent model systems for exploring the early development of nephric organs. These experimental systems can easily be manipulated by microsurgery, microinjection, genetics, or combinations of these approaches. However, little is known about how physiologically similar these simple kidneys are to the more complex mammalian adult kidneys. In addition, almost nothing is known about proximo-distal patterning of nephrons in any organism. In order begin to explore the physiological specialization of the pronephric tubules along the proximo-distal axis, a combination of uptake assays using fluorescently tagged proteins, LDL particles and dextrans, and an informatics-targeted in situ screen for transport proteins have been performed on embryos of the frog, Xenopus laevis. Genes identified to be expressed within unique subdomains of the pronephric tubules include an ABC transporter, two amino acid cotransporters, two sodium bicarbonate cotransporters, a novel sodium glucose cotransporter, a sodium potassium chloride cotransporter (NKCC2), a sodium chloride organic solute cotransporter (ROSIT), and a zinc transporter. A novel combination of colorimetric and fluorescent whole-mount in situ hybridization (FCIS) was used to precisely map the expression domain of each gene within the pronephros. These data indicate specialized physiological function and define multiple novel segments of the pronephric tubules, which contain at least six distinct transport domains. Uptake studies identified functional transport domains and also demonstrated that early glomeral leakage can allow visualization of protein movement into the pronephric tubules and thus establish a system for investigating experimentally induced proteinuria and glomerulonephritis.

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Species referenced: Xenopus laevis
Genes referenced: abcf2 adm atp1a1 slc12a1 slc30a1 slc4a11 slc4a4 slc5a9 slc6a14 slc6a14.2 slc6a19 slc7a8

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	Fig. 3. Expression of XSGLT1k examined by colorimetric in situ, FISH and FCIS methodologies. (A and B) XSGLT1k expression in stage 28/29 pronephros (red arrow). (C and D) XSGLT1k expression in stage 37 pronephros. (E) XSGLT1K expression in stage 37 embryo detected via FISH with an FITC-tyramide substrate. (F) Na+K+ATPase expression in the same embryo as panel F, detected with a Cy3-tyramide substrate. (G) Overlay of panels E and F. The proximal domain expressing XSGLT1k appears green. (H) XSGLT1k expression in a stage 30 embryo developed with a purple substrate. (I) The same embryo as shown in panel H but developed for Na+K+ATPase expression with an FITC-tyramide substrate. (J) FCIS overlay of panels H and I performed via the protocol described in Methods. The XSGLT1k domain appears blue, while the pronephric tubules in which it is not expressed appear orange.
	Fig. 4. Localization of XAA1 expression. Embryo stages are shown on the lower left of each panel. Panels A–H illustrate colorimetric in situs, and panels K and N FCIS with a Na+K+ATPase counterstain. XAA1 is strongly expressed in the late proximal segment from stages 32 onwards. Very low-level expression is also observed in the distal tubule and pronephric duct at stage 38.
	Fig. 5. XAA2 expression in the early proximal segment. In stage 28 embryos, low-level expression of XAA2 is observed in the future distal segment. This expression declines and is not detectable by stage 32. At stage 32, strong expression is activated in the early proximal tubule (C, D) and remains restricted to this segment until at least stage 40. Embryonic stages are shown at the lower left of panels A–L, M and P. The blue pronephric region in the FCIS panels shows the site of high level expression.
	Fig. 6. Pronephric expression of an ABC transporter. Embryo stages are shown on the lower left of each panel. XABC expression in the pronephros is first detected at stage 26 (A and B), red arrow. The XABC1 gene is expressed in both early and late proximal segments (A–L). Other expressing tissues include the somites, brachial arches, and head. By stage 35, expression of XABC in nonpronephric tissues increases, necessitating shorter development times, hence the relatively weak signal in later panels. Panels M–R illustrates FCIS staining. Purple development of the transporter is shown in the left column, FITC-tyramide development of the Na+K+ATPase counterstain shown in the middle, and FCIS overlay shown on the right.
	Fig. 7. XNBC1 expression in the developing kidney. XNBC1 is expressed in multiple pronephric regions. Expression in the early and late proximal domains begins at stage 29. At stage 33, a new domain of expression is activated in the late distal segment. Once active, this segment expresses much higher levels of XNBC1 than do the proximal tubules. Proximal expression declines over time and is barely detectable by stage 40, at which time late distal expression remains robust. Red arrow, proximal tubule expression. Blue arrow, late distal segment expression. Black arrow, pink coloration in proximal tubules representing low-level expression.
	Fig. 8. XNBC2 expression. XNBC2 is expressed in the pronephros from stage 28. Up until stage 35, expression is present in both the early proximal and early distal segments, but at only very low levels in the intervening late proximal segment (see panel F, black arrow). The early distal expression decreases over time and cannot be detected by stage 35. From stage 35 onwards, strong expression is observed in the early proximal segment and the weak expression in the late proximal domain is also retained.
	Fig. 9. XNKCC2 is a pronephric specific marker of the early distal segment. XNKCC2 expression is first detected at stage 29 when strong expression commences in the future early proximal segment. This pattern is maintained until at least stage 40.
	Fig. 10. Expression of XROSIT, an organic solute cotransporter. XROSIT is expressed in a small pronephric domain from stage 28 onwards. Staining in the dorsal fin (black arrow, panel E) is background and was also observed in controls. In conventional staining (panels A–H), precise localization within the pronephros is difficult, but the FCIS overlap (panel K) clearly shows that expression is restricted to the early proximal domain (pink region, panel K).
	Fig. 11. Expression of a zinc transporter, XZnSLC30, in the embryonic kidney tubules. XZnSLC30 (M–R) is expressed in the tips of the dorsal branches at commencing at stage 28. By stage 35, this expression has expanded slightly into the early proximal segment (panels F and N) but expression remains highest in the dorsal branches. As the pronephric tubules coil, the staining is still present but is less obvious as the dorsal branches lie proximal to the lateral coiled tubules (H).
	solute carrier family 5 (sodium/glucose cotransporter), member 9 (slc5a9) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 39, lateral view, anterior left, dorsal up.
	solute carrier family 6 (amino acid transporter), member 14, gene 2 (slc6a14.2) gene expression in Xenopus laevis embryo, (blue) examined by double in situ hybridization with atp1a1 (NaK ATPase). Expression is restricted to the late proximal segment (S3) of the pronephric nephron.
	Xenopus solute carrier family 7 (cationic amino acid transporter, y+ system), member 8 (slc7a8) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 34/35, lateral view, anterior left, dorsal up.
	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.