Background/Aim: Vascular endothelial growth factor (VEGF), required for renal development, is generated by alternative splicing of 8 exons to produce two families, pro-angiogenic VEGFxxx, formed by proximal splicing in exon 8 (exon 8a), and anti-angiogenic VEGFxxxb, generated by distal splicing in exon 8 (exon 8b). VEGF165b, the first described exon 8b-containing isoform, antagonises VEGF165 and is anti-angiogenic in vivo. Methods: Using VEGFxxxb-specific antibodies, we investigated its expression quantitatively and qualitatively in developing kidney, and measured the effect of VEGF165b on renal endothelial and epithelial cells. Results: VEGFxxxb formed 45% of total VEGF protein in adult renal cortex, and VEGF165b does not increase glomerular endothelial cell permeability, it inhibits migration, and is cytoprotective for podocytes. During renal development, VEGFxxxb was expressed in the condensed vesicles of the metanephros, epithelial cells of the comma-shaped bodies, invading endothelial cells and epithelial cells of the S-shaped body, and in the immature podocytes. Expression reduced as the glomerulus matured. Conclusion: These results show that the anti-angiogenic VEGFxxxb isoforms are highly expressed in adult and developing renal cortex, and suggest that the VEGFxxxb family plays a role in glomerular maturation and podocyte protection by regulating the pro-angiogenic pro-permeability properties of VEGFxxx isoforms.

Keywords:
Kidney development, Vascular endothelial growth factor 165b, Angiogenesis
1.
Carmeliet P: Angiogenesis in health and disease. Nat Med 2003;9:653–660.
2.
Robert B, Zhao X, Abrahamson DR: Coexpression of neuropilin-1, Flk1, and VEGF (164) in developing and mature mouse kidney glomeruli. Am J Physiol Renal Physiol 2000;279:F275–F282.
3.
Robert B, St John PL, Hyink DP, Abrahamson DR: Evidence that embryonic kidney cells expressing flk-1 are intrinsic, vasculogenic angioblasts. Am J Physiol 1996;271:F744–F753.
4.
Kitamoto Y, Tokunaga H, Tomita K: Vascular endothelial growth factor is an essential molecule for mouse kidney development: glomerulogenesis and nephrogenesis. J Clin Invest 1997;99:2351–2357.
5.
Kaipainen A, Korhonen J, Pajusola K, Aprelikova O, Persico MG, Terman BI, Alitalo K: The related FLT4, FLT1, and KDR receptor tyrosine kinases show distinct expression patterns in human fetal endothelial cells. J Exp Med 1993;178:2077–2088.
6.
Dumont DJ, Fong GH, Puri MC, Gradwohl G, Alitalo K, Breitman ML: Vascularization of the mouse embryo: a study of flk-1, tek, tie, and vascular endothelial growth factor expression during development. Dev Dyn 1995;203:80–92.
7.
Breier G, Albrecht U, Sterrer S, Risau W: Expression of vascular endothelial growth factor during embryonic angiogenesis and endothelial cell differentiation. Development 1992;114:521–532.
8.
Eremina V, Sood M, Haigh J, Nagy A, Lajoie G, Ferrara N, Gerber HP, Kikkawa Y, Miner JH, Quaggin SE: Glomerular-specific alterations of VEGF-A expression lead to distinct congenital and acquired renal diseases. J Clin Invest 2003;111:707–716.
9.
Eremina V, Quaggin SE: The role of VEGF-A in glomerular development and function. Curr Opin Nephrol Hypertens 2004;13:9–15.
10.
Gerber HP, Hillan KJ, Ryan AM, Kowalski J, Keller GA, Rangell L, Wright BD, Radtke F, Aguet M, Ferrara N: VEGF is required for growth and survival in neonatal mice. Development 1999;126:1149–1159.
11.
Schrijvers BF, Flyvbjerg A, De Vriese AS: The role of vascular endothelial growth factor (VEGF) in renal pathophysiology. Kidney Int 2004;65:2003–2017.
12.
Foster RR, Hole R, Anderson K, Satchell SC, Coward RJ, Mathieson PW, Gillatt DA, Saleem MA, Bates DO, Harper SJ: Functional evidence that vascular endothelial growth factor may act as an autocrine factor on human podocytes. Am J Physiol Renal Physiol 2003;284:F1263–F1273.
13.
Houck KA, Ferrara N, Winer J, Cachianes G, Li B, Leung DW: The vascular endothelial growth factor family: identification of a fourth molecular species and characterization of alternative splicing of RNA. Mol Endocrinol 1991;5:1806–1814.
14.
Bates DO, Cui TG, Doughty JM, Winkler M, Sugiono M, Shields JD, Peat D, Gillatt D, Harper SJ: VEGF165b, an inhibitory splice variant of vascular endothelial growth factor, is down-regulated in renal cell carcinoma. Cancer Res 2002;62:4123–4131.
15.
Perrin RM, Konopatskaya O, Qiu Y, Harper S, Bates DO, Churchill AJ: Diabetic retinopathy is associated with a switch in splicing from anti- to pro-angiogenic isoforms of vascular endothelial growth factor. Diabetologia 2005;48:2422–2427.
16.
Woolard J, Wang WY, Bevan HS, Qiu Y, Morbidelli L, Pritchard-Jones RO, Cui TG, Sugiono M, Waine E, Perrin R, Foster R, Digby-Bell J, Shields JD, Whittles CE, Mushens RE, Gillatt DA, Ziche M, Harper SJ, Bates DO: VEGF165b, an inhibitory vascular endothelial growth factor splice variant: mechanism of action, in vivo effect on angiogenesis and endogenous protein expression. Cancer Res 2004;64:7822–7835.
17.
Konopatskaya O, Churchill AJ, Harper SJ, Bates DO, Gardiner TA: VEGF165b, an endogenous C-terminal splice variant of VEGF, inhibits retinal neovascularisation in mice. Mol Vis 2006;12:626–632.
18.
Cebe Suarez S, Pieren M, Cariolato L, Arn S, Hoffmann U, Bogucki A, Manlius C, Wood J, Ballmer-Hofer K: A VEGF-A splice variant defective for heparan sulfate and neuropilin-1 binding shows attenuated signaling through VEGFR-2. Cell Mol Life Sci 2006;63:2067–2077.
19.
Glass CA, Harper SJ, Bates DO: The anti- angiogenic VEGF isoform VEGF165b transiently increases hydraulic conductivity, probably through VEGF receptor 1 in vivo. J Physiol 2006;572:243–257.
20.
Schumacher V, Jeruschke S, Eitner F, Becker J, Pitschke G, Ince C, Miner JH, Leuschner I, Engers R, Everding AS, Bulla M, Royer-Pokora B: Impaired glomerular maturation and lack of VEGF165b in Denys-Drash syndrome. J Am Soc Nephrol 2007;18:719–729.
21.
Varey AH, Rennel ES, Qiu Y, Bevan HS, Perrin RM, Raffy S, Dixon AR, Paraskeva C, Zaccheo O, Hassan AB, Harper SJ, Bates DO: VEGF165b, an antiangiogenic VEGF-A isoform, binds and inhibits bevacizumab treatment in experimental colorectal carcinoma: balance of pro- and antiangiogenic VEGF-A isoforms has implications for therapy. Br J Cancer 2008;98:1366–1379.
22.
Foster RR, Saleem MA, Mathieson PW, Bates DO, Harper SJ: Vascular endothelial growth factor and nephrin interact and reduce apoptosis in human podocytes. Am J Physiol Renal Physiol 2005;288:F48–F57.
23.
Rennel ES, H-Zadeh MA, Wheatley E, Schüler Y, Kelly SP, Cebe Suarez S, Ballmer-Hofer K, Stewart L, Bates DO, Harper SJ: Recombinant human VEGF165b protein is an effective anti-cancer agent in mice. Eur J Cancer 2008;44:1883–1894.
24.
Satchell SC, Anderson KL, Mathieson PW: Angiopoietin 1 and vascular endothelial growth factor modulate human glomerular endothelial cell barrier properties. J Am Soc Nephrol 2004;15:566–574.
25.
Bates DO, Macmillan PP, Manjaly JG, Qiu Y, Hudson SJ, Bevan HS, Hunter AJ, Soothill PW, Read M, Donaldson LF, Harper SJ: The endogenous anti-angiogenic family of splice variants of VEGF, VEGFxxxb, are down-regulated in pre-eclamptic placentae at term. Clin Sci (Lond) 2006;110:575–585.
26.
Cui TG, Foster RR, Saleem M, Mathieson PW, Gillatt DA, Bates DO, Harper SJ: Differentiated human podocytes endogenously express an inhibitory isoform of vascular endothelial growth factor (VEGF165b) mRNA and protein. Am J Physiol Renal Physiol 2004;286:F767–F773.
27.
Simon M, Rockl W, Hornig C, Grone EF, Theis H, Weich HA, Fuchs E, Yayon A, Grone HJ: Receptors of vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) in fetal and adult human kidney: localization and [125I]VEGF binding sites. J Am Soc Nephrol 1998;9:1032–1044.
28.
Tufro A, Norwood VF, Carey RM, Gomez RA: Vascular endothelial growth factor induces nephrogenesis and vasculogenesis. J Am Soc Nephrol 1999;10:2125–2134.
29.
Simon M, Grone HJ, Johren O, Kullmer J, Plate KH, Risau W, Fuchs E: Expression of vascular endothelial growth factor and its receptors in human renal ontogenesis and in adult kidney. Am J Physiol 1995;268:F240–F250.
30.
Bailey E, Bottomley MJ, Westwell S, Pringle JH, Furness PN, Feehally J, Brenchley PE, Harper SJ: Vascular endothelial growth factor mRNA expression in minimal change, membranous, and diabetic nephropathy demonstrated by non-isotopic in situ hybridisation. J Clin Pathol 1999;52:735–738.
31.
Sadl V, Jin F, Yu J, Cui S, Holmyard D, Quaggin S, Barsh G, Cordes S: The mouse Kreisler (Krml1/MafB) segmentation gene is required for differentiation of glomerular visceral epithelial cells. Dev Biol 2002;249:16–29.
32.
Loughna S, Hardman P, Landels E, Jussila L, Alitalo K, Woolf AS: A molecular and genetic analysis of renal glomerular capillary development. Angiogenesis 1997;1:84–101.
33.
Takemoto M, He L, Norlin J, Patrakka J, Xiao Z, Petrova T, Bondjers C, Asp J, Wallgard E, Sun Y, Samuelsson T, Mostad P, Lundin S, Miura N, Sado Y, Alitalo K, Quaggin SE, Tryggvason K, Betsholtz C: Large-scale identification of genes implicated in kidney glomerulus development and function. EMBO J 2006;25:1160–1174.
34.
Wong MA, Cui S, Quaggin SE: Identification and characterization of a glomerular-specific promoter from the human nephrin gene. Am J Physiol Renal Physiol 2000;279:F1027–F1032.
35.
Robert B, Abrahamson DR: Control of glomerular capillary development by growth factor/receptor kinases. Pediatr Nephrol 2001;16:294–301.
36.
Dolan V, Hensey C, Brady HR: Diabetic nephropathy: renal development gone awry? Pediatr Nephrol 2003;18:75–84.
37.
Bernhardt WM, Schmitt R, Rosenberger C, Munchenhagen PM, Grone HJ, Frei U, Warnecke C, Bachmann S, Wiesener MS, Willam C, Eckardt KU: Expression of hypoxia-inducible transcription factors in developing human and rat kidneys. Kidney Int 2006;69:114–122.
38.
Carmeliet P, Ferreira V, Breier G, Pollefeyt S, Kieckens L, Gertsenstein M, Fahrig M, Vandenhoeck A, Harpal K, Eberhardt C, Declercq C, Pawling J, Moons L, Collen D, Risau W, Nagy A: Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature 1996;380:435–439.
39.
Ferrara N, Carver-Moore K, Chen H, Dowd M, Lu L, O’Shea KS, Powell-Braxton L, Hillan KJ, Moore MW: Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene. Nature 1996;380:439–442.
40.
Ferguson J, Qiu Y, Sage LM, Neal CR, Bates DO, Harper SJ, Salmon AH: Ultrafiltration co-efficient in isolated intact glomeruli from podocyte specific VEGF165B over-expressing transgenic mice. Microcirculation 2008, in press.
41.
Amin EM, Nowak DG, Saleem M, Bates D, Ladomery M: Relationship between WT1 and vascular endothelial growth factor (VEGF) splicing. Microcirculation 2008, in press.
42.
Holash J, Davis S, Papadopoulos N, Croll SD, Ho L, Russell M, Boland P, Leidich R, Hylton D, Burova E, Ioffe E, Huang T, Radziejewski C, Bailey K, Fandl JP, Daly T, Wiegand SJ, Yancopoulos GD, Rudge JS: VEGF-Trap: a VEGF blocker with potent antitumor effects. Proc Natl Acad Sci USA 2002;99:11393–11398.
43.
Kendall RL, Wang G, Thomas KA: Identification of a natural soluble form of the vascular endothelial growth factor receptor, FLT-1, and its heterodimerization with KDR. Biochem Biophys Res Commun 1996;226:324–328.
44.
Presta LG, Chen H, O’Connor SJ, Chisholm V, Meng YG, Krummen L, Winkler M, Ferrara N: Humanization of an anti-vascular endothelial growth factor monoclonal antibody for the therapy of solid tumors and other disorders. Cancer Res 1997;57:4593–4599.
45.
Belteki G, Haigh J, Kabacs N, Haigh K, Sison K, Costantini F, Whitsett J, Quaggin SE, Nagy A: Conditional and inducible transgene expression in mice through the combinatorial use of Cre-mediated recombination and tetracycline induction. Nucleic Acids Res 2005;33:e51.
46.
Quaggin SE: Transcriptional regulation of podocyte specification and differentiation. Microsc Res Tech 2002;57:208–211.
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