VE-PTP promoter activity in adult mouse kidney.

<p><b>(A)</b> Whole mount X-gal staining of adult VE-PTP^tlacZ/+ mouse kidney. Evident β-galactosidase activity is observed in renal cortex (C), inner stripe (IS) of outer medulla, inner medulla (IM), and papilla (P). Right panel shows vascular distribution of β-galactosidase activity in renal cortex. C, cortex; OS, outer stripe; IS, inner stripe; IM, inner medulla; P, papilla. <b>(B)</b> β-galactosidase activity in adult VE-PTP^tlacZ/+ mouse kidney sections. In medulla, β-galactosidase activity is observed in vascular bundle (VB), subpopulations of medullary tubules (red arrows in panels c and d), and papillary cells (panel e). VB, vascular bundle. <b>(C)</b> β-galactosidase activity in cortical renal vasculature. RA, renal artery: RV, renal vein; AA, arcuate artery; AV, arcuate vein; IA, interlobular artery; IV, interlobular vein; Art, arteriole; G, glomerulus; af, afferent arteriole; ef, efferent arteriole. Note: VE-PTP promoter activity is limited in efferent arterioles, peritubular capillaries, and venous circulations. Scale bar, 200 μm in B-a and B-b; 100 μm in B-c, B-d, B-e, and C; 50 μm in an insert of C.</p

Expression of receptor-type protein tyrosine phosphatase in developing and adult renal vasculature

<div><p>Renal vascular development is a coordinated process that requires ordered endothelial cell proliferation, migration, intercellular adhesion, and morphogenesis. In recent decades, studies have defined the pivotal role of endothelial receptor tyrosine kinases (RPTKs) in the development and maintenance of renal vasculature. However, the expression and the role of receptor tyrosine phosphatases (RPTPs) in renal endothelium are poorly understood, though coupled and counterbalancing roles of RPTKs and RPTPs are well defined in other systems. In this study, we evaluated the promoter activity and immunolocalization of two endothelial RPTPs, VE-PTP and PTPμ, in developing and adult renal vasculature using the heterozygous LacZ knock-in mice and specific antibodies. In adult kidneys, both VE-PTP and PTPμ were expressed in the endothelium of arterial, glomerular, and medullary vessels, while their expression was highly limited in peritubular capillaries and venous endothelium. VE-PTP and PTPμ promoter activity was also observed in medullary tubular segments in adult kidneys. In embryonic (E12.5, E13.5, E15.5, E17.5) and postnatal (P0, P3, P7) kidneys, these RPTPs were expressed in ingrowing renal arteries, developing glomerular microvasculature (as early as the S-shaped stage), and medullary vessels. Their expression became more evident as the vasculatures matured. Peritubular capillary expression of VE-PTP was also noted in embryonic and postnatal kidneys. Compared to VE-PTP, PTPμ immunoreactivity was relatively limited in embryonic and neonatal renal vasculature and evident immunoreactivity was observed from the P3 stage. These findings indicate 1) VE-PTP and PTPμ are expressed in endothelium of arterial, glomerular, and medullary renal vasculature, 2) their expression increases as renal vascular development proceeds, suggesting that these RPTPs play a role in maturation and maintenance of these vasculatures, and 3) peritubular capillary VE-PTP expression is down-regulated in adult kidneys, suggesting a role of VE-PTP in the development of peritubular capillaries.</p></div

Anti-CD31 immunohistochemistry combined with β-galactosidase histochemistry of developing VE-PTP^tlacZ/+ and PTPμ^tlacZ/+ mice kidneys.

<p><b>(A through D)</b> Immunohistochemistry for CD31 was superimposed on β-galactosidase histochemistry in developing VE-PTP^tlacZ/+ (A, C) and PTPμ^tlacZ/+ (B, D) mice kidneys. Panels A and B show cortical area. Panels C and D display medullary region. In both mice, CD31 and β-galactosidase activity are colocalized in developing glomerular capillaries, including vascular clefts of S-shaped glomeruli (red arrows), and developing medullary vessels (black arrowheads in panels C and D). In VE-PTP^tlacZ/+ kidneys, β-galactosidase activity is also observed in endothelial cells forming peritubular capillaries (red arrowheads in panel A). Notes: β-galactosidase activity is observed in medullary tubular segments (black arrows) that are not labelled with CD31 in P0-stage VE-PTP^tlacZ/+ mouse kidney. Scale bar, 25 μm in A, C; 50 μm in B, D.</p

VE-PTP promoter activity in developing mouse kidneys.

<p><b>(A through L)</b> β-galactosidase histochemistry of developing VE-PTP^tlacZ/+ mouse kidneys. In embryonic kidneys, evident VE-PTP activity is observed in penetrating and ingrowing renal arteries (black arrows in panels A, B, C, D, and J) and maturing glomeruli (G) (panels D and J). VE-PTP promoter activity is also observed in developing medullar vessels (arrowheads in panel G). In postnatal kidneys, VE-PTP promoter activity is observed in renal arterial vessels (panels E and F), maturing glomeruli (G) (panels E, F, and K), and medullary vessels (arrow heads in panels H and I) and tubular subpopulations (arrows in panels H and I). Note: VE-PTP promoter activity is observed in the cells intermittently distributed around developing glomeruli and tubules (red arrowheads in panel J) and in peri-tubular capillaries in postnatal kidneys (red arrows in panel K). The peritubular capillary VE-PTP promoter activity is down-regulated in 3-week old mouse kidney (panel L). RA, renal artery; G, glomerulus; IA, interlobular artery; V, venous vessel; T, developing tubules. Scale bar, 100 μm in A through I; 50 μm in J through L.</p

Co-immunostaining of VE-PTP and CD31 in developing and adult mice kidneys.

<p>Kidney sections from E16.5, P0 and adult mice were double immunolabeled for VE-PTP (red) and CD31 (green) as described in the “Materials & Methods”. In developing kidneys (E16.5 and P0), VE-PTP is expressed in endothelial cells in ingrowing arteries (A) and developing glomeruli (G), which are labeled with CD31. VE-PTP is also expressed in endothelial cells (yellow arrows) that distribute around the developing nephrons. In adult kidney, VE-PTP is expressed in endothelial cells in arterial and glomerular vasculature, while its expression is limited in peritubular capillaries. A, arterial vessel; IA, interlobular artery; G, glomerulus. Scale bar, 50 μm in E16.5 and P0 kidneys; 25 μm in adult kidney.</p

Immunolocalization of VE-PTP in developing and adult renal medulla.

<p><b>(A)</b> VE-PTP expression in E15.5, P0, and P7 kidneys was examined by immunohistochemistry using anti-VEPTP rat monoclonal antibody and ABC method as described in the “Materials & Methods”. VE-PTP-expressing cells (arrows) are dispersively distributed in medulla of E15.5 and P0 kidneys (left panels). Its connectivity suggests the formation of medullary vessels by these cells. In P7 kidney (middle and right panels), VE-PTP immunoreactivity is observed in medullary vessels (arrows) distributed along the tubules. Definite VE-PTP expression is not observed in tubular components. IS, inner stripe of outer medulla; IM, inner medulla; P, papilla; RA, renal artery. Scale bar, 100 μm in left and middle panels; 50 μm in right panels. <b>(B)</b> VE-PTP expression in adult mouse kidney was examined by immunohistochemistry as in (A). VE-PTP immunoreactivity is observed in vessels (arrows) distributed along the medullary tubules, while obvious VE-PTP expression is not observed in tubular components. Right panel shows the immunostaining without the 1^st antibody. IS, inner stripe of outer medulla; IM, inner medulla; P, papilla. Scale bar, 100 μm in left and right panels; 50 μm in middle panels.</p

Co-immunostaining of VE-PTP and CD31 in developing mouse glomeruli.

<p><b>(A)</b> Kidney sections from E16.5 and P0 mice were double immunolabeled for VE-PTP (red) and CD31 (green). <b>(B)</b> VE-PTP expression in developing glomeruli was also examined by immunohistochemistry with ABC method as described in the “Materials & Methods”. VE-PTP is expressed in glomerular endothelial cells (yellow arrows in panel A, black arrows in panel B) that are distributed in vascular clefts of S-shaped glomeruli and within capillary loop stage and maturing glomeruli. VE-PTP immunoreactivity is increased as glomerular capillary development proceeds. No VE-PTP immunoreactivity is observed in developing podocytes (P) and mesangial cells (M) (panel B). VE-PTP expression is also observed in the cells dispersively distributed around developing glomeruli (white arrowheads in panel A, black arrowheads in panel B). G, developing glomerulus; VC, vascular cleft. Scale bar, 50 μm in A; 20 μm in B.</p

VE-PTP promoter activity in adult mouse kidney.

<p><b>(A)</b> Whole mount X-gal staining of adult VE-PTP^tlacZ/+ mouse kidney. Evident β-galactosidase activity is observed in renal cortex (C), inner stripe (IS) of outer medulla, inner medulla (IM), and papilla (P). Right panel shows vascular distribution of β-galactosidase activity in renal cortex. C, cortex; OS, outer stripe; IS, inner stripe; IM, inner medulla; P, papilla. <b>(B)</b> β-galactosidase activity in adult VE-PTP^tlacZ/+ mouse kidney sections. In medulla, β-galactosidase activity is observed in vascular bundle (VB), subpopulations of medullary tubules (red arrows in panels c and d), and papillary cells (panel e). VB, vascular bundle. <b>(C)</b> β-galactosidase activity in cortical renal vasculature. RA, renal artery: RV, renal vein; AA, arcuate artery; AV, arcuate vein; IA, interlobular artery; IV, interlobular vein; Art, arteriole; G, glomerulus; af, afferent arteriole; ef, efferent arteriole. Note: VE-PTP promoter activity is limited in efferent arterioles, peritubular capillaries, and venous circulations. Scale bar, 200 μm in B-a and B-b; 100 μm in B-c, B-d, B-e, and C; 50 μm in an insert of C.</p

VE-PTP expression in developing and adult mouse renal vasculature.

<p><b>(A through G)</b> Immunolocalization of VE-PTP was assessed by immunofluorescence staining (A though D) or immunohistochemistry with ABC method (E through G) as described in the “Materials & Methods”. At E15.5 (panel A) and E17.5 (panel E), VE-PTP expression is observed in ingrowing renal arteries (A), glomeruli (G) in juxta-medullary region, and the cells (arrows in panel E) surrounding developing glomeruli and tubules. At postnatal day 0 (panel B) and day 7 (panels C and F), VE-PTP is expressed in renal arterial vessels (A), maturing glomeruli (G), and peritubular capillaries (PTC). In adult kidney (panels D and G), VE-PTP is expressed in renal arterial vasculature and glomeruli, while its expression is limited in peritubular capillaries (PTC) and venous circulation (V). Asterisks in panels F and G indicate non-specific tubular staining. RA, renal artery; A, arterial vessel; Art, arteriole; G, glomerulus; PTC, peritubular capillary; V, vein. Scale bar, 50 μm in A, B, C, D, F, and G; 40 μm in E.</p

Anti-CD31 immunohistochemistry combined with β-galactosidase histochemistry of adult VE-PTP^tlacZ/+ and PTPμ^tlacZ/+ mice kidneys.

<p><b>(A through F)</b> Immunohistochemistry for CD31 was superimposed on β-galactosidase histochemistry of adult VE-PTP^tlacZ/+ (A, C, E) and PTPμ^tlacZ/+ (B, D, F) mice kidneys. CD31 is co-localized with β-galactosidase activity in glomeruli and arterioles (panels A and B) and medullary vessels (red arrows) in inner stripe of outer medulla (ISOM) and papilla (panels C through F) in VE-PTP^tlacZ/+ and PTPμ^tlacZ/+ mice kidneys. Glom, glomerulus; Art, arteriole; IA, interlobular artery. Notes: β-galactosidase activity is absent in podocytes (black arrows in A and B). β-galactosidase activity is also observed in tubular segments (red arrowheads) that are not labelled with CD31. Scale bar, 25 μm in A and B; 50 μm in C through F.</p