Phenotypic analysis of Pod-Cre fVHL mice. A–D: PAS staining of paraffin sections of massively proteinuric Pod-Cre fVHL mice at four or 25 weeks. A: Entire kidney is fibrotic, and tubules and ducts are packed with proteinaceous material in 25-week-old Pod-Cre fVHL mice. B: Proteinuric Pod-Cre fVHL kidney at four weeks of age with dilated glomerular capillary lumen (arrow) and mesangial matrix expansion and hypercellularity (arrowhead). C: Proteinuric Pod-Cre fVHL at 4 weeks showing mesangial hypercellularity and glomerular crescent. D: Proteinuric Pod-Cre fVHL at 25 weeks showing a completely fibrotic glomerulus. E: PAS staining of paraffin sections of a four-week-old nonproteinuric Pod-Cre fVHL kidney shows occasional dilated tubules (arrows). Inset shows hypercellular mesangium with increased PAS+ matrix accumulation and prominent GBM. F: Kidney sections show labeling pattern of WT1 in podocytes (pink, arrows) and Ki-67 in mesangium (green, arrowheads) in nonproteinuric Pod-Cre fVHL mice. Slides were counterstained with 4′,6-diamidino-2-phenylindole. Numbers of Ki-67-positive (G) and WT1-positive cells (H) were counted in at least 10 glomeruli per animal (W = wild-type [n = 5], N = nonproteinuric [n = 5], and P = proteinuric [n = 2]). G: Significantly more Ki-67-positive cells are observed in 4-week-old proteinuric Pod-Cre fVHL glomeruli compared with wild-type (*P < 0.05). H: Conversely, fewer WT1-positive cells per glomerulus are observed in four-week-old Pod-Cre fVHL compared with four-week-old wild-type (**P < 0.01 proteinuric versus wild-type; *P < 0.05 nonproteinuric versus wild-type). I–N: Kidneys at eight weeks of age (I–K) or 41 weeks (L–N) were labeled with anti-synaptopodin antibody and counterstained with 4′,6-diamidino-2-phenylindole. In contrast to wild-type synaptopodin labeling (I and L), Pod-Cre fVHL glomeruli have decreased synaptopodin labeling in wide, central regions (J and M, arrow). Some Pod-Cre fVHL glomeruli completely lack synaptopodin labeling on one half of the glomerulus (K and N, dashed circles). O and P: Kidneys at eight weeks of age were labeled with anti-nephrin antibody. Pod-Cre fVHL glomeruli (P) have decreased nephrin labeling compared with wild-type (O). Original magnification: ×100 (A and E); ×200 (inset of E); ×400 (B, C, and D).