Inducible Overexpression of sFlt-1 in Podocytes Ameliorates Glomerulopathy in Diabetic Mice

Abstract

Objective: Podocyte-specific, doxycycline (DOX)-inducible overexpression of the soluble VEGF receptor-1 (sFlt-1) in adult mice was used to investigate the role of VEGF-A/VEGF receptor (VEGFR) system in diabetic glomerulopathy.

Research Design and Methods: We studied non-diabetic and diabetic transgenic mice and wild-type controls, treated with vehicle (VEH) or DOX for 10-weeks. Glycemia was measured by a glucose-oxidase method and blood pressure by non-invasive technique. sFlt-1, VEGF-A, VEGFR2, and nephrin protein expression in renal cortex were determined by western immunoblotting; urine sFlt-1, urine free VEGF-A, and albuminuria by ELISA; glomerular ultrastructure by electron microscopy, VEGFR1 and VEGFR2 cellular localization with immunogold techniques.

Results: Non-diabetic DOX-treated transgenic mice showed a 2-fold increase in cortex sFlt-1 expression and 4-fold increase in sFlt-1 urine excretion (p<0.001). Urine free VEGF-A was decreased by 50% and cortex VEGF-A expression upregulated by 30% (p<0.04). VEGFR2 expression was unchanged while its activation reduced in DOX-treated transgenic mice (p<0.02). Albuminuria and glomerular morphology were similar among groups.

Results: DOX-treated transgenic diabetic mice showed a 60% increase in 24h urine sFlt-1 excretion and a ∼70% decrease in urine free VEGF-A when compared to VEH-treated diabetic mice (p<0.04), had lower urine albumin excretion at 10-weeks than VEH-treated diabetic mice (D-VEH vs D-DOX: 117.5[69-199] vs 43[26.8-69] μg/24h, geometric mean [95% CI], p=0.003). Diabetes-induced mesangial expansion, glomerular basement membrane thickening, podocyte foot-process fusion, and TGFβ1 expression were ameliorated in DOX-treated diabetic animals (p<0.05). Diabetes-induced VEGF-A and nephrin expression were not affected in DOX-treated mice.

Conclusions: Podocyte-specific sFlt-1 overexpression ameliorates diabetic glomerular injury, implicating VEGF-A in the pathogenesis of this complication.