Figure 6.

Calpain-1 is activated by Transient Receptor Potential channel C6 (TRPC6)–dependent Ca²⁺ influx into the podocyte. Differentiated immortalized cultured podocytes were transfected with nontargeting scrambled (Scram) or calpain-1 (CPN-1^KD) siRNA. (A) Subsequently, they were treated with vehicle, adriamycin (ADRIA), or 1-oleoyl-2-acetyl-glycerol (OAG), and a calpain activity assay was performed. (B) In addition, medium was collected, and a calpain activity assay was performed to determine excretion of active calpain by podocytes in the culture medium. (C) Western blot analysis was performed to detect the downstream calpain target Talin-1 (Talin-1–to–β-actin ratio: 0.21 in ADRIA-treated cells versus 0.38 in ADRIA-treated Cpn-1^KD cells; 0.35 in OAG-treated cells versus 0.51 in OAG-treated Cpn-1^KD cells). To confirm that TRPC6 activates calpain-1, we used podocytes with an inducible stably transfected TRPC6 (TRPC6^KD) or scrambled siRNA construct (scram). (D) After activation of the constructs, we treated the cells with vehicle, ADRIA, or OAG and measured calpain activity. (E) Western blotting for Talin-1 was performed using TRPC6^KD or Scram cells treated with ADRIA or OAG (Talin-1–to–β-actin ratios: Veh-Scram, 0.20; ADRIA-scram, 0.02; ADRIA-TRPC6^KD, 0.24; OAG-scram, 0.18; OAG-TRPC6^KD, 0.23). (F) Subsequently, a calcineurin activity assay was performed. Results are representative of two experiments, with n=4 per group. 2-APB, 2-aminoethoxydiphenyl borate; siRNA, small interfering RNA. *P<0.05 compared with vehicle-treated scrambled cells; ^$P<0.05 compared with nontargeted transfected ADRIA-treated podocytes; ^†P<0.05 compared with scrambled siRNA transfected OAG-treated podocytes; ^‡P<0.05 compared with scrambled siRNA transfected vehicle-treated podocytes.