Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, PC243
Vascular Endothelial Growth Factor-A (VEGF-A) overexpression in adult mouse glomeruli increases glomerular ultrafiltration coefficient due partly to the reduced coverage of the capillaries by the subpodocyte space (SPS).
C. R. Neal1, P. Patel1, T. Ahad1, S. Oltean1, C. Alsop1, T. Lee1, K. Sison2, S. E. Quaggin2, S. J. Harper1, A. H. Salmon1,3, D. Bates1
1. Microvascular Research Laboratories, School of Physiology and Pharmacology, University of Bristol, Bristol, United Kingdom. 2. The Samuel Lunenfeld Research Institute, Mt Sinai Hospital, Toronto, Ontario, United Kingdom. 3. Academic Renal Unit, Southmead Hospital, Bristol, United Kingdom.
Glomerular podocyte VEGF-A signals across the glomerular filtration barrier (GFB) to maintain permselectivity(1) and VEGF-A isoforms change in human nephropathy(2). We investigated the roles of the angiogenic mouse isoform VEGF-A164 in mature adult glomeruli in vivo using conditional inducible transgenic overexpression in mice. Procedures followed UK Home Office Legislation and had local ethics committee approval. podocin-rtTA mice and tetO-VEGF-A164 mice were crossed resulting in double-transgenic (podocin-rtTA x tetO-VEGF-A164: P+T+) mice. Podocyte-specific VEGF-A164 overexpression was induced in 8 to 22 week old mice by adding doxycycline to drinking water of adult P+T+ mice. Genotyping confirmed presence of the transgenes and kidney VEGF-A164 expression was measured by ELISA. The glomerular ultrafiltration coefficient (LPA) of P+T+ mice and wild type littermates (WT) was measured (3) and normalized to initial glomerular volume (LPA/Vi). Isolated glomeruli mounted on a pipette were bathed in 1% bovine serum albumin (BSA) in HEPES Ringer. Upon changing to 8% BSA, the efflux of water reduced glomerular volume. The initial (<0.1s) rate of glomerular volume reduction (JV) was used to calculate the glomerular ultrafiltration coefficient (LPA = Jv/-Δπ (nl.min-1mmHg-1)). Small portions (<1mm diameter) of P+T+ and WT kidney were fixed with glutaraldehyde, osmicated, dehydrated and embedded in resin. Glomerular sections were examined and electron micrographs used to estimate: glomerular basement membrane (GBM) thickness, podocyte filtration slit and endothelial fenestration frequency, coverage (%) of the GBM by the SPS(4), height of SPS, width of SPS exit pores and width of supporting anchor processes (5). P+T+ mice showed a 2 fold overexpression of VEGF-A164 over WT and LPA/Vi peaked by day 7 (WT: 0.78±0.13 (N=9); P+T+: 1.94±0.26 min-1.mmHg-1 (N=9); p<0.005). At day 7, overexpression of VEGF-A164 reduced subpodocyte space coverage of the glomerular capillary wall (65±4% WT; 43±6% P+T+ p<0.01). No other parameter differed between P+T+ and WT, indicating that the GFB was ultrastructurally unchanged. Using models developed by Neal et al(5), the SPS changes account for 63% of the increase in ultrafiltration coefficient. While the adult mouse GFB is relatively insensitive to induced VEGF-A164 over-expression, the podocyte appears to respond by reducing overall GFB resistance by increasing the fraction of GFB without SPS cover. This raises the ultrafiltration coefficient by allowing filtration through a greater area of the GFB that has lower resistance.
Where applicable, experiments conform with Society ethical requirements