Fig. 5.
Podocyte Vegf164 overexpression in diabetic mice induces semaphorin3a and VEGFR2 production. a Western blot and bar graph showing increased kidney semaphorin3a (SEMA3A) levels in diabetic +doxycycline (+dox) (black bar) and non-diabetic+doxycycline (light grey bar) mice overexpressing podocyte Vegf164. b NRP1 levels were increased in both groups of diabetic mice (black bar, dark grey bar) and in non-diabetic+doxycycline mice overexpressing podocyte Vegf164 (light grey bar). c VEGFR2 levels were increased in diabetic +doxycycline mice overexpressing Vegf164 (black bar). d Nephrin levels were decreased in diabetic+doxycycline and diabetic−doxycycline, and in non-diabetic+doxycycline (light grey bar) mice overexpressing podocyte Vegf164 compared with non-diabetic−doxycycline control (white bar). a–d Actin blots shown to document equal loading. Densitometric analysis shows mean±SEM fold change in arbitrary units as compared with controls, n≥4. *p<0.05 compared with control (−doxycycline) e Real-time PCR showing that relative mRNA expression of Vegfr2, Vegfr1 and soluble (s) Vegfr1 increased in diabetic+doxycycline mice overexpressing podocyte Vegf164. *p<0.05 compared with diabetic−doxycycline control. Nephrin, podocin, Wt1 and eNos mRNA levels were not significantly different. f Dual-immunostaining showed that podocyte SEMA3A level was increased in diabetic and non-diabetic glomeruli overexpressing podocyte Vegf164 (+doxycycline), whereas podocin was unchanged. Merge images demonstrate co-localisation. g Nephrin immunostaining shows decreased expression in both diabetic mouse groups (+doxycycline, −doxycycline) and in non-diabetic+doxycycline mice overexpressing podocyte Vegf164. Scale bars, 20 μm