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RETRACTED: Ontogeny of semaphorins 3A and 3F and their receptors neuropilins 1 and 2 in the kidney
Gene Expression Patterns 2 (2002) 151–155 www.elsevier.com/locate/modgep
Ontogeny of semaphorins 3A and 3F and their receptors neuropilins 1 and 2 in the kidney Guillermo Villegas, Alda Tufro* Department of Pediatrics/Nephrology, University of Virginia School of Medicine, Charlottesville, VA, USA Received 12 April 2002; received in revised form 13 June 2002; accepted 16 August 2002
Abstract Semaphorins 3A and 3F are axon guidance proteins during nervous system development. Their expression pattern and function outside the nervous system are unknown. Neuropilin 1 and 2 (NP-1, NP-2) are natural ligands for semaphorins 3A and 3F, respectively. NP-1 is also a coreceptor for vascular endothelial growth factor (VEGF) required for normal vascular development. We showed that VEGF is a direct chemoattractant for glomerular endothelial cells towards developing nephrons. To examine whether semaphorins could modulate VEGF endothelial cell guidance cues in the developing kidney, we studied the expression of semaphorin 3A and semaphorin 3F and their receptors NP-1 and NP-2 in the kidney during ontogeny using Northern blot analysis, in situ hybridization, Western blot analysis and immunohistochemistry. All four genes are developmentally regulated, with abundant expression during organogenesis and downregulation in the adult kidney. Semaphorin 3A and 3F are expressed by podocytes and tubules whereas their receptors NP-1 and NP-2 are localized to endothelial cells. In vitro, renal tubular epithelial cell lines (tsMPT, IRPT and MDCK) and glomerular endothelial cells express both semaphorins and their receptors, suggesting the presence of an autocrine system. The distribution of the receptors NP-1 and NP-2 in endothelial cells and developing vessels is complementary to that of the ligands in adjacent epithelial cells during kidney development. The sum of the guidance cues provided by VEGF and semaphorins 3A and 3F may be important determinants of the pattern of endothelial cell migration during kidney morphogenesis. q 2002 Elsevier Science B.V. All rights reserved. Keywords: Semaphorin 3A; Semaphorin 3F; Neuropilin 1; Neuropilin 2; Kidney development; Organogenesis; Guidance proteins; Podocytes; Renal epithelial cells; Endothelial cells
Semaphorins are secreted and transmembrane proteins that function as axon guidance proteins during nervous system development, causing axon repulsion in most cases, although they can provide chemoattractive cues (Raper, 2000; Polleux et al., 2000; Song et al., 1998; Chen et al., 1999). Semaphorins are expressed widely in the mouse during organogenesis; their distribution and function outside the nervous system are unknown (Behar et al., 1996; Raper, 2000). In the developing lung semaphorins are involved in branching morphogenesis (Kagoshima and Ito, 2001). Sema-3A and sema-3F are secreted proteins, the natural ligands for neuropilin 1 (NP-1) and neuropilin 2 (NP-2), that cause growth cone collapse and provide chemo-repulsive guidance for migrating axons (Kolodkin et al., 1997; Chen et al., 1997; Nakamura et al., 1998). Sema-3F competes with sema-3A for NP-1 binding (Chen et al., 1999). When sema* Corresponding author. Department of Pediatrics/Nephrology, University of Virginia School of Medicine, MR4 Bldg./Room 2017, Charlottesville, VA 22908, USA. E-mail address: [email protected] (A. Tufro).
3F binds both neuropilins the chemo-repulsive effect is abolished (Chen et al., 1999). Sema-3A also induces inhibition of endothelial cell migration and apoptosis (Miao et al., 1999). Sema-3A competes with vascular endothelial growth factor (VEGF) for NP-1 binding in endothelial cells (Miao et al., 1999). NP-1 is required for embryonic vessel formation (Kawasaki et al., 1999). Since VEGF induces endothelial cell migration via Flk-1 (Tufro, 2000; Ferrara, 2001) and this effect is potentiated by NP-1 acting as a co-receptor (Soker et al., 1998), sema-3A probably inhibits endothelial cell migration by displacing VEGF from the receptor complex. 1. Results and discussion We examined the expression level and localization of sema-3A and sema-3F and their receptors NP-1 and NP-2 in the developing mouse and rat kidney and determined which renal cell types express these proteins. Sema-3A and sema-3F mRNA levels are higher at E14 and newborn than in adult rat kidneys (Fig. 1). Sema-3A and sema-3F
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and arterioles during kidney vascularization (Fig. 4). In adult kidneys NP-1 and NP-2 expression is limited to peritubular capillaries and some glomerular capillaries (Fig. 4). This expression pattern is very similar to that of Flk-1, the major signaling VEGF receptor (Tufro et al., 1999). Our findings regarding NP-1 differ slightly from those of Robert et al. (2000) in that we found very little NP-1 expression in glomerular capillaries in adult kidneys. NP-2 expression, described here for the first time, overlaps with NP-1 localization. Thus, the localization of the ligands, sema-3A and sema-3F, and their respective receptors NP-1 and NP-2 is complementary throughout kidney development, suggesting paracrine interactions of these molecules during organogenesis. Interestingly, sema-3A and sema-3F transcripts co-
Fig. 1. Ontogeny of sema-3A and sema-3F in the kidney. Northern blots show mRNA levels in embryonic day 14 (E14), newborn (NB) and adult (A) rat kidneys. A total of 10 mg total RNA was resolved per lane. Transcript sizes are indicated in kb. (Top) Sema-3A mRNA; (middle) sema-3F mRNA; (bottom) GAPDH mRNA for loading control.
mRNAs are localized to renal tubular epithelial cells and podocytes (Fig. 2). Their expression is widespread at E12.5 in the mouse and E14 in the rat. Sema-3A and sema-3F mRNAs are detected in S-shaped bodies and ureteric buds at E15 and E17 in the mouse. At E17 in the mouse and E19 in the rat both semaphorin mRNAs are expressed in glomerular visceral and parietal epithelial cells as well as in the basolateral aspect of developing tubules. This peculiar mRNA distribution could be due to RNA transport. An artifact is unlikely since the tissue from all stages of development was processed identically. The co-localization of sema-3A and sema-3F during kidney organogenesis suggests overlapping or cooperative function. In adult kidneys mRNA expression of both semaphorins is limited to podocytes and the luminal aspect of collecting ducts and distal tubules. In embryonic kidneys NP-1 and NP-2 are several fold more abundant than in newborn and adult kidneys (Fig. 3). We detected two NP-1 isoforms of 180 and ,120 kDa in embryonic kidneys whereas in older kidneys NP-1 has the expected size (120 kDa) (Kolodkin et al., 1997). The larger NP-1 isoform could be due to complexing with another protein or dimerization of soluble NP-1 (90 kDa) (Gagnon et al., 2000) by thioether bonds resistant to standard denaturation procedures. NP-2 has the expected size (132 kDa) at all ages studied; duplet bands observed in newborn and adult kidneys may be due to different glycosylation (Chen et al., 1997). In developing kidneys NP-1 and NP-2 are detected in endothelial cells surrounding S-shaped bodies, in the vascular cleft and later on in glomerular capillaries
Fig. 2. Localization of sema-3A and sema-3F mRNAs in the kidney during development. Flk-1 (1/2) mouse kidneys (Shalaby et al., 1996) aged E12.5 to adult show semaphorin transcripts in S-shaped nephrons, visceral and parietal glomerular epithelial cells and tubular cells (purple-brown) and bgalactosidase stained (blue) endothelial cells. Sema-3A (left) and sema-3F (right) localization is very similar and does not overlap with endothelial cells. At E19 in the rat sema-3F localizes to the basolateral aspect of Sshaped nephrons and developing tubules; at all other ages the localization of both mRNAs in the rat kidney is identical to the mouse (data not shown). In adult kidneys sema-3A and sema-3F mRNAs are detected exclusively on the luminal side of collecting tubules and in podocytes. The original magnification is £ 400 in all pictures.
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Fig. 3. Ontogeny of NP-1 and NP-2 expression in the kidney. Western blots showing NP-1 protein levels in E14, newborn and adult rat kidneys (100 mg/lane). (Top) In embryonic kidneys two NP-1 distinct bands of 120 and 180 kDa are detected. The smaller band is six fold more abundant in E14 and newborn (NB) than in adult kidneys. The larger band is barely visible in the newborn and absent in the adult. (Bottom) NP-2 (,130 kDa) is 13 fold more abundant in embryonic kidneys than in newborn or adult ones. Duplet bands may indicate developmental differences in glycosylation.
localize with VEGF whereas neuropilins co-localize with VEGF receptors Flk-1 and Flt-1 (Tufro et al., 1999), raising the possibility of combinatorial interactions among them. In vitro, VEGF binding to NP-1 potentiates Flk-1 signaling whereas sema-3A competes with VEGF for NP-1 binding and results in inhibition of endothelial cell migration and apoptosis (Soker et al., 1998; Miao et al., 1999). If sema-3A and sema-3F have similar functions during kidney organogenesis they could play a role in patterning endothelial cell migration as well as controlling endothelial cell number by modulating apoptosis (Bagnard et al., 2001). Further studies are in order to elucidate these potential functions of sema3A and sema-3F in kidney development. In contrast to intact tissue epithelial cells, proximal tubular epithelial cell lines IRPT, tsMPT and MDCK from rat, mouse and dog (Tang et al., 1995; Loghman-Adham et al., 1997; Gaush et al., 1966) as well as mouse glomerular endothelial cells (MGEC) (Tufro, 2000) express NP-1 and NP-2 protein in addition to sema-3A and sema-3F mRNA, as indicated by Western and Northern blot analysis (Figs. 5 and 6). In summary, sema-3A and sema-3F mRNAs are expressed in renal epithelial cells in a developmentally regulated manner, such that their abundance decreases as maturation proceeds and their localization becomes limited to podocytes and specific tubules where the transcripts shift from basolateral to luminal aspect of the cells. NP-1 and NP-2 are localized to endothelial cells throughout kidney development, their abundance also decreases and their localization is limited to peritubular capillaries in adult kidneys. Transformed tubular epithelial cell lines and primary glomerular endothelial cells express sema-3A and sema3F mRNA as well as both NP-1 and NP-2, suggesting that an autocrine system occurs in vitro.
Sema-3A and sema-3F mRNA levels were examined by Northern blot analysis as described previously (TufroMcReddie et al., 1997) using full-length sema-3A and sema-3F cDNAs (Chen et al., 1997; Behar et al., 1996). Sema-3A and sema-3F mRNA localization was examined in rat kidneys (E14 through adult) and Flk-1 (1/2) mouse kidneys (E12.5 through adult) by in situ hybridization in paraffin embedded tissue using DIG-labeled riboprobes (Roche Diagnostics) following the manufacturer’s instructions. DIG-labeled probes obtained from full-length sema3A and sema-3F cDNAs were hydrolyzed to obtain 300–500 bp cRNAs. Sections were counterstained with Neutral Fast Red (Vector). Kidneys from Flk-1 (1/2) mice were fixed in paraformaldehyde 1 glutaraldehyde and processed for bgalactosidase staining prior to paraffin embedding to identify endothelial cells (Shalaby et al., 1996). NP-1 and NP-2 protein expression levels were assessed by Western blot analysis in the kidney during ontogeny, in renal tubular epithelial cell lines IRPT, tsMPT and MDCK
Fig. 4. Localization of NP-1 and NP-2 in the kidney during development by fluorescent immunohistochemistry. (Left) NP-1 in E17, E19 and adult mouse kidneys. (Right) NP-2 in E15, E17 and adult mouse kidneys. Magnification is £ 400 except in NP1-E19 where the right side panel shows a glomerulus at £ 630. NP2-E17 shows NP-2 immunofluorescent labeling on the left and the corresponding bright light image with b-galactosidase staining of endothelial cells on the right. Both neuropilins are detected in developing glomeruli (G) and vessels (V) surrounding the forming nephrons whereas they are limited to peritubular capillaries (C) in adult kidneys.
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NP-1 (Gagnon et al., 2000). Negative controls for immunohistochemistry were absence of primary or secondary antibodies and substitution of primary antibody by rabbit serum. Acknowledgements We are grateful to Alan Kolodkin for providing us with the rat sema-3A and sema-3F cDNAs and the neuropilin antibodies, to Julie Ingelfinger for providing the IRPT cells and to Mahmoud Loghman-Adham for providing the tsMPT cells. This work was funded by NIH (K08-DK02421 and R01 DK59333). References
Fig. 5. Expression of sema-3A and sema-3F in renal epithelial cell lines. A total of 10 mg total RNA was loaded per lane. Transcript sizes are indicated in kb. (Top) Sema-3A mRNA levels; (bottom) sema-3F mRNA levels. Ethidium bromide staining and hybridization with a GAPDH probe verified equal loading (data not shown). Northern blots show that sema-3A and sema-3F mRNA levels in immortalized rat proximal tubular cells (IRPT), and immortalized mouse proximal tubular cells (tsMPT) are similar whereas they were lower in canine tubular cells (MDCK) and sema-3F expressed only the smallest transcript. This may be due to decreased species homology of the probe.
(ATCC, Bethesda, MD) and in MGEC; NP-1 and NP-2 distribution was examined by fluorescent immunohistochemistry using anti-rat NP-1 and NP-2 polyclonal antibodies (Kolodkin et al., 1997; Cloutier et al., 2002) as described (Tufro-McReddie et al., 1997). This anti-NP-1 antibody does not discriminate between NP-1 and soluble
Fig. 6. Expression of NP-1 and NP-2 in renal epithelial and endothelial cells. Western blots showing NP-1 protein levels in IRPT, tsMPT, MDCK and MGEC (100 mg/ lane). (Top) NP-1 protein levels are similar in all epithelial and endothelial cells; note that the NP-1 size is consistent across species and cell types; (bottom) NP-2 protein levels and size vary in epithelial cells from different origins, probably due to differences in cross-reactivity and glycosylation, respectively. Please note the marker indicating the size of the major NP-2 band in tsMPT cells.
Bagnard, D., Vaillant, C., Khuth, S., Dufay, N., Lohrum, M., Puschel, A., Belin, M.-F., Bolz, J., Thomasset, N., 2001. Semaphorin 3A-vascular endothelial growth factor-165 balance mediates migration and apoptosis of neural progenitor cells by the recruitment of shared receptor. J. Neurosci. 21, 3332–3341. Behar, O., Golden, J.A., Mashimo, H., Schoen, F.J., Fishman, M.C., 1996. Semaphorin III is needed for normal patterning and growth of nerves, bones and heart. Nature 383, 525–528. Chen, H., Chedotal, A., He, Z., Goodman, C., Tessier-Lavigne, M., 1997. Neuropilin-2, a novel member of the neuropilin family, is a high affinity receptor for the semaphorins sema E and sema IV but not sema III. Neuron 19, 547–559. Chen, H., He, Z., Tessier-Lavigne, M., 1999. Axon guidance mechanisms: semaphorins as simultaneous repellents and anti-repellents. Nat. Neurosci. 1, 436–439. Cloutier, J.-F., Giger, R., Koentges, G., Dulac, K., Kolodkin, A., Ginty, D., 2002. Neuropilin-2 mediates axonal fasciculation, zonal segregation, but not axonal convergence, of primary accessory olfactory neurons. Neuron 33, 877–892. Ferrara, N., 2001. Role of vascular endothelial growth factor in regulation of physiological angiogenesis. Am. J. Physiol. 280, C1358–C1366. Gagnon, M.L., Bielenberg, D.R., Gechtman, Z., Miao, H.-Q., Takashima, S., Soker, S., Klagsbrun, M., 2000. Identification of a natural soluble neuropilin-1 that binds vascular endothelial growth factor: in vivo expression and antitumor activity. Proc. Natl. Acad. Sci. USA 97, 2573–2578. Gaush, C.R., Hard, W.L., Smith, T.F., 1966. Characterization of an established line of canine kidney cells. Proc. Soc. Exp. Biol. Med. 122, 931– 935. Kagoshima, M., Ito, T., 2001. Diverse gene expression and function of semaphorins in developing lung: positive and negative regulatory roles of semaphorins in lung branching morphogenesis. Genes Cells 6, 559–571. Kawasaki, T., Kitsukawa, T., Bekku, Y., Matsuda, Y., Sanbo, M., Yagi, T., Fujisawa, H., 1999. A requirement for neuropilin-1 in embryonic vessel formation. Development 126, 4895–4902. Kolodkin, A., Levengood, D., Rowe, E., Tai, Y.-T., Giger, R., Ginty, D., 1997. Neuropilin is a semaphorin III receptor. Cell 90, 753–762. Loghman-Adham, M., Rohrwasser, A., Helin, C., Zhang, S., Terreros, D., Inoue, I., 1997. A conditionally immortalized cell line from murine proximal tubule. Kidney Int. 52, 229–239. Miao, H.-Q., Soker, S., Feiner, L., Alonso, J.-L., Raper, J.A., Klagsbrun, M., 1999. Neuropilin-1 mediates collapsin-1/semaphorin III inhibition of endothelial cell motility: functional competition of collapsin-1 and vascular endothelial growth factor-165. J. Cell Biol. 46, 233–241. Nakamura, F., Tanaka, M., Takahashi, T., Kalb, R., Strittmatter, S., 1998. Neuropilin-1 extracellular domains mediate semaphorin D/III-induced growth cone collapse. Neuron 21, 1093–1100.
G. Villegas, A. Tufro / Gene Expression Patterns 2 (2002) 151–155 Polleux, F., Morrow, T., Ghosh, A., 2000. Semaphorin 3A is a chemoattractant for cortical apical dendrites. Nature 404, 567–573. Raper, J.A., 2000. Semaphorins and their receptors in vertebrates and invertebrates. Curr. Opin. Neurobiol. 10, 88–94. Robert, B., Zhao, X., Abrahamson, D.R., 2000. Coexpression of neuropilin1, Fk-1 and VEGF164 in developing and mature mouse kidney glomeruli. Am. J. Physiol. 279, F275–F282. Shalaby, F., Rossant, J., Yamaguchi, T., Gertsenstein, M., Wu, X., Breitman, M., Schuh, A., 1996. Failure of blood-island formation and vasculogenesis in Flk-1-deficient mice. Nature 376, 62–66. Soker, S., Takashima, S., Miao, H.-Q., Neufeld, G., Klagsbrun, M., 1998. Neuropilin-1 is expressed by endothelial and tumor cells as an isoformspecific receptor for vascular endothelial growth factor. Cell 92, 745. Song, H.-J., Ming, G.-L., He, Z., Lehman, M., McKerracher, L., TessierLavigne, M., Poo, M.-M., 1998. Conversion of neuronal growth cone
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responses from repulsion to attraction by cyclic nucleotides. Science 281, 1515–1518. Tang, S.S., Jung, F., Diamant, D., Brown, D., Bachinsky, D., Hellman, P., Ingelfinger, J., 1995. Temperature-sensitive SV40 immortalized rat proximal tubule cell line has functional renin-angiotensin system. Am. J. Physiol. 268, F435–F446. Tufro, A., 2000. VEGF spatially directs angiogenesis during metanephric development in vitro. Dev. Biol. 227, 558–566. Tufro, A., Norwood, V.F., Carey, R.M., Gomez, R.A., 1999. Vascular endothelial growth factor induces nephrogenesis and vasculogenesis. J. Am. Soc. Nephrol. 10, 2125–2134. Tufro-McReddie, A., Norwood, V.F., Aylor, K.W., Botkin, S.J., Carey, R.M., Gomez, R.A., 1997. Oxygen regulates vascular endothelial growth factor-mediated vasculogenesis and tubulogenesis. Dev. Biol. 183, 139–149.
Ontogeny of semaphorins 3A and 3F and their receptors neuropilins 1 and 2 in the kidney Guillermo Villegas, Alda Tufro* Department of Pediatrics/Nephrology, University of Virginia School of Medicine, Charlottesville, VA, USA Received 12 April 2002; received in revised form 13 June 2002; accepted 16 August 2002
Abstract Semaphorins 3A and 3F are axon guidance proteins during nervous system development. Their expression pattern and function outside the nervous system are unknown. Neuropilin 1 and 2 (NP-1, NP-2) are natural ligands for semaphorins 3A and 3F, respectively. NP-1 is also a coreceptor for vascular endothelial growth factor (VEGF) required for normal vascular development. We showed that VEGF is a direct chemoattractant for glomerular endothelial cells towards developing nephrons. To examine whether semaphorins could modulate VEGF endothelial cell guidance cues in the developing kidney, we studied the expression of semaphorin 3A and semaphorin 3F and their receptors NP-1 and NP-2 in the kidney during ontogeny using Northern blot analysis, in situ hybridization, Western blot analysis and immunohistochemistry. All four genes are developmentally regulated, with abundant expression during organogenesis and downregulation in the adult kidney. Semaphorin 3A and 3F are expressed by podocytes and tubules whereas their receptors NP-1 and NP-2 are localized to endothelial cells. In vitro, renal tubular epithelial cell lines (tsMPT, IRPT and MDCK) and glomerular endothelial cells express both semaphorins and their receptors, suggesting the presence of an autocrine system. The distribution of the receptors NP-1 and NP-2 in endothelial cells and developing vessels is complementary to that of the ligands in adjacent epithelial cells during kidney development. The sum of the guidance cues provided by VEGF and semaphorins 3A and 3F may be important determinants of the pattern of endothelial cell migration during kidney morphogenesis. q 2002 Elsevier Science B.V. All rights reserved. Keywords: Semaphorin 3A; Semaphorin 3F; Neuropilin 1; Neuropilin 2; Kidney development; Organogenesis; Guidance proteins; Podocytes; Renal epithelial cells; Endothelial cells
Semaphorins are secreted and transmembrane proteins that function as axon guidance proteins during nervous system development, causing axon repulsion in most cases, although they can provide chemoattractive cues (Raper, 2000; Polleux et al., 2000; Song et al., 1998; Chen et al., 1999). Semaphorins are expressed widely in the mouse during organogenesis; their distribution and function outside the nervous system are unknown (Behar et al., 1996; Raper, 2000). In the developing lung semaphorins are involved in branching morphogenesis (Kagoshima and Ito, 2001). Sema-3A and sema-3F are secreted proteins, the natural ligands for neuropilin 1 (NP-1) and neuropilin 2 (NP-2), that cause growth cone collapse and provide chemo-repulsive guidance for migrating axons (Kolodkin et al., 1997; Chen et al., 1997; Nakamura et al., 1998). Sema-3F competes with sema-3A for NP-1 binding (Chen et al., 1999). When sema* Corresponding author. Department of Pediatrics/Nephrology, University of Virginia School of Medicine, MR4 Bldg./Room 2017, Charlottesville, VA 22908, USA. E-mail address: [email protected] (A. Tufro).
3F binds both neuropilins the chemo-repulsive effect is abolished (Chen et al., 1999). Sema-3A also induces inhibition of endothelial cell migration and apoptosis (Miao et al., 1999). Sema-3A competes with vascular endothelial growth factor (VEGF) for NP-1 binding in endothelial cells (Miao et al., 1999). NP-1 is required for embryonic vessel formation (Kawasaki et al., 1999). Since VEGF induces endothelial cell migration via Flk-1 (Tufro, 2000; Ferrara, 2001) and this effect is potentiated by NP-1 acting as a co-receptor (Soker et al., 1998), sema-3A probably inhibits endothelial cell migration by displacing VEGF from the receptor complex. 1. Results and discussion We examined the expression level and localization of sema-3A and sema-3F and their receptors NP-1 and NP-2 in the developing mouse and rat kidney and determined which renal cell types express these proteins. Sema-3A and sema-3F mRNA levels are higher at E14 and newborn than in adult rat kidneys (Fig. 1). Sema-3A and sema-3F
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and arterioles during kidney vascularization (Fig. 4). In adult kidneys NP-1 and NP-2 expression is limited to peritubular capillaries and some glomerular capillaries (Fig. 4). This expression pattern is very similar to that of Flk-1, the major signaling VEGF receptor (Tufro et al., 1999). Our findings regarding NP-1 differ slightly from those of Robert et al. (2000) in that we found very little NP-1 expression in glomerular capillaries in adult kidneys. NP-2 expression, described here for the first time, overlaps with NP-1 localization. Thus, the localization of the ligands, sema-3A and sema-3F, and their respective receptors NP-1 and NP-2 is complementary throughout kidney development, suggesting paracrine interactions of these molecules during organogenesis. Interestingly, sema-3A and sema-3F transcripts co-
Fig. 1. Ontogeny of sema-3A and sema-3F in the kidney. Northern blots show mRNA levels in embryonic day 14 (E14), newborn (NB) and adult (A) rat kidneys. A total of 10 mg total RNA was resolved per lane. Transcript sizes are indicated in kb. (Top) Sema-3A mRNA; (middle) sema-3F mRNA; (bottom) GAPDH mRNA for loading control.
mRNAs are localized to renal tubular epithelial cells and podocytes (Fig. 2). Their expression is widespread at E12.5 in the mouse and E14 in the rat. Sema-3A and sema-3F mRNAs are detected in S-shaped bodies and ureteric buds at E15 and E17 in the mouse. At E17 in the mouse and E19 in the rat both semaphorin mRNAs are expressed in glomerular visceral and parietal epithelial cells as well as in the basolateral aspect of developing tubules. This peculiar mRNA distribution could be due to RNA transport. An artifact is unlikely since the tissue from all stages of development was processed identically. The co-localization of sema-3A and sema-3F during kidney organogenesis suggests overlapping or cooperative function. In adult kidneys mRNA expression of both semaphorins is limited to podocytes and the luminal aspect of collecting ducts and distal tubules. In embryonic kidneys NP-1 and NP-2 are several fold more abundant than in newborn and adult kidneys (Fig. 3). We detected two NP-1 isoforms of 180 and ,120 kDa in embryonic kidneys whereas in older kidneys NP-1 has the expected size (120 kDa) (Kolodkin et al., 1997). The larger NP-1 isoform could be due to complexing with another protein or dimerization of soluble NP-1 (90 kDa) (Gagnon et al., 2000) by thioether bonds resistant to standard denaturation procedures. NP-2 has the expected size (132 kDa) at all ages studied; duplet bands observed in newborn and adult kidneys may be due to different glycosylation (Chen et al., 1997). In developing kidneys NP-1 and NP-2 are detected in endothelial cells surrounding S-shaped bodies, in the vascular cleft and later on in glomerular capillaries
Fig. 2. Localization of sema-3A and sema-3F mRNAs in the kidney during development. Flk-1 (1/2) mouse kidneys (Shalaby et al., 1996) aged E12.5 to adult show semaphorin transcripts in S-shaped nephrons, visceral and parietal glomerular epithelial cells and tubular cells (purple-brown) and bgalactosidase stained (blue) endothelial cells. Sema-3A (left) and sema-3F (right) localization is very similar and does not overlap with endothelial cells. At E19 in the rat sema-3F localizes to the basolateral aspect of Sshaped nephrons and developing tubules; at all other ages the localization of both mRNAs in the rat kidney is identical to the mouse (data not shown). In adult kidneys sema-3A and sema-3F mRNAs are detected exclusively on the luminal side of collecting tubules and in podocytes. The original magnification is £ 400 in all pictures.
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Fig. 3. Ontogeny of NP-1 and NP-2 expression in the kidney. Western blots showing NP-1 protein levels in E14, newborn and adult rat kidneys (100 mg/lane). (Top) In embryonic kidneys two NP-1 distinct bands of 120 and 180 kDa are detected. The smaller band is six fold more abundant in E14 and newborn (NB) than in adult kidneys. The larger band is barely visible in the newborn and absent in the adult. (Bottom) NP-2 (,130 kDa) is 13 fold more abundant in embryonic kidneys than in newborn or adult ones. Duplet bands may indicate developmental differences in glycosylation.
localize with VEGF whereas neuropilins co-localize with VEGF receptors Flk-1 and Flt-1 (Tufro et al., 1999), raising the possibility of combinatorial interactions among them. In vitro, VEGF binding to NP-1 potentiates Flk-1 signaling whereas sema-3A competes with VEGF for NP-1 binding and results in inhibition of endothelial cell migration and apoptosis (Soker et al., 1998; Miao et al., 1999). If sema-3A and sema-3F have similar functions during kidney organogenesis they could play a role in patterning endothelial cell migration as well as controlling endothelial cell number by modulating apoptosis (Bagnard et al., 2001). Further studies are in order to elucidate these potential functions of sema3A and sema-3F in kidney development. In contrast to intact tissue epithelial cells, proximal tubular epithelial cell lines IRPT, tsMPT and MDCK from rat, mouse and dog (Tang et al., 1995; Loghman-Adham et al., 1997; Gaush et al., 1966) as well as mouse glomerular endothelial cells (MGEC) (Tufro, 2000) express NP-1 and NP-2 protein in addition to sema-3A and sema-3F mRNA, as indicated by Western and Northern blot analysis (Figs. 5 and 6). In summary, sema-3A and sema-3F mRNAs are expressed in renal epithelial cells in a developmentally regulated manner, such that their abundance decreases as maturation proceeds and their localization becomes limited to podocytes and specific tubules where the transcripts shift from basolateral to luminal aspect of the cells. NP-1 and NP-2 are localized to endothelial cells throughout kidney development, their abundance also decreases and their localization is limited to peritubular capillaries in adult kidneys. Transformed tubular epithelial cell lines and primary glomerular endothelial cells express sema-3A and sema3F mRNA as well as both NP-1 and NP-2, suggesting that an autocrine system occurs in vitro.
Sema-3A and sema-3F mRNA levels were examined by Northern blot analysis as described previously (TufroMcReddie et al., 1997) using full-length sema-3A and sema-3F cDNAs (Chen et al., 1997; Behar et al., 1996). Sema-3A and sema-3F mRNA localization was examined in rat kidneys (E14 through adult) and Flk-1 (1/2) mouse kidneys (E12.5 through adult) by in situ hybridization in paraffin embedded tissue using DIG-labeled riboprobes (Roche Diagnostics) following the manufacturer’s instructions. DIG-labeled probes obtained from full-length sema3A and sema-3F cDNAs were hydrolyzed to obtain 300–500 bp cRNAs. Sections were counterstained with Neutral Fast Red (Vector). Kidneys from Flk-1 (1/2) mice were fixed in paraformaldehyde 1 glutaraldehyde and processed for bgalactosidase staining prior to paraffin embedding to identify endothelial cells (Shalaby et al., 1996). NP-1 and NP-2 protein expression levels were assessed by Western blot analysis in the kidney during ontogeny, in renal tubular epithelial cell lines IRPT, tsMPT and MDCK
Fig. 4. Localization of NP-1 and NP-2 in the kidney during development by fluorescent immunohistochemistry. (Left) NP-1 in E17, E19 and adult mouse kidneys. (Right) NP-2 in E15, E17 and adult mouse kidneys. Magnification is £ 400 except in NP1-E19 where the right side panel shows a glomerulus at £ 630. NP2-E17 shows NP-2 immunofluorescent labeling on the left and the corresponding bright light image with b-galactosidase staining of endothelial cells on the right. Both neuropilins are detected in developing glomeruli (G) and vessels (V) surrounding the forming nephrons whereas they are limited to peritubular capillaries (C) in adult kidneys.
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NP-1 (Gagnon et al., 2000). Negative controls for immunohistochemistry were absence of primary or secondary antibodies and substitution of primary antibody by rabbit serum. Acknowledgements We are grateful to Alan Kolodkin for providing us with the rat sema-3A and sema-3F cDNAs and the neuropilin antibodies, to Julie Ingelfinger for providing the IRPT cells and to Mahmoud Loghman-Adham for providing the tsMPT cells. This work was funded by NIH (K08-DK02421 and R01 DK59333). References
Fig. 5. Expression of sema-3A and sema-3F in renal epithelial cell lines. A total of 10 mg total RNA was loaded per lane. Transcript sizes are indicated in kb. (Top) Sema-3A mRNA levels; (bottom) sema-3F mRNA levels. Ethidium bromide staining and hybridization with a GAPDH probe verified equal loading (data not shown). Northern blots show that sema-3A and sema-3F mRNA levels in immortalized rat proximal tubular cells (IRPT), and immortalized mouse proximal tubular cells (tsMPT) are similar whereas they were lower in canine tubular cells (MDCK) and sema-3F expressed only the smallest transcript. This may be due to decreased species homology of the probe.
(ATCC, Bethesda, MD) and in MGEC; NP-1 and NP-2 distribution was examined by fluorescent immunohistochemistry using anti-rat NP-1 and NP-2 polyclonal antibodies (Kolodkin et al., 1997; Cloutier et al., 2002) as described (Tufro-McReddie et al., 1997). This anti-NP-1 antibody does not discriminate between NP-1 and soluble
Fig. 6. Expression of NP-1 and NP-2 in renal epithelial and endothelial cells. Western blots showing NP-1 protein levels in IRPT, tsMPT, MDCK and MGEC (100 mg/ lane). (Top) NP-1 protein levels are similar in all epithelial and endothelial cells; note that the NP-1 size is consistent across species and cell types; (bottom) NP-2 protein levels and size vary in epithelial cells from different origins, probably due to differences in cross-reactivity and glycosylation, respectively. Please note the marker indicating the size of the major NP-2 band in tsMPT cells.
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