FIGURE 7.

Podocyte VEGF^KD induces thiol-mediated mechanisms in diabetic and eNOS^−/−:VEGF^KD mice. (A) WB: Kidney GSNOR expression is not altered by diabetes or VEGF^KD , tubulin is shown as loading control. (B) GSNOR S-nitrosylation (SNO-GSNOR) detected by BST: VEGF^KD (+dox) decreases SNO-GSNOR in eNOS^−/−:VEGF^KD and diabetic kidneys; podocyte and kidney lysates are used as SNO positive and negative BST controls, respectively, input shows equal loading for BST. (C) Urine Cys thiol excretion (normalized to creatinine): podocyte VEGF^KD increases ∼2.5 fold Cys thiol excretion in eNOS^−/−:VEGF^KD + dox mice (red bar) (*, p = 0.013); diabetic mice (blue bars), irrespective of VEGF^KD , have ∼6-fold higher Cys thiol excretion than uninduced non-diabetic mice with intact eNOS (VEGF^KD − dox, white bar) (**, p = 0.004) or eNOS^−/−:VEGF^KD − dox (hatched red bar) (**, p = 0.004). (D) IHC: podocyte VEGF^KD (+ dox) increases S-nitrosylation of glomerular proteins in eNOS^−/−:VEGF^KD kidneys, SNO-Cys quantification is shown in (E), (****, p = 0.0003). (F) PLA: shows that podocyte VEGF^KD (+ dox) increases laminin S-nitrosylation (SNO-laminin) in eNOS^−/−:VEGF^KD kidneys, SNO-laminin PLA quantification is shown in (G) (*, p = 0.025). (H) PLA: shows β3-integrin S-nitrosylation (SNO-β3-integrin) in eNOS^−/−:VEGF^KD glomeruli, which is increased by podocyte VEGF^KD (+dox); SNO-β3-integrin quantification is shown in (I) (*, p = 0.036).