Fig 1.
PAN-induced podocyte injury model.
(A) Podocytes treated with PAN (25, 50, 75, 100, or 125 μg/ml) for 24 h showed a dose-dependent decrease in viability. (B, C) The protein expression of nephrin, a marker of podocyte injury, was decreased following PAN treatment at concentrations ≥50 μg/ml, demonstrating that a PAN concentration ≥50 μg/ml was sufficient to induce podocyte injury. (D, E) Podocytes were examined by microscopy at 0 and 24 h. After 24 h, podocytes that were treated with 75 μg/ml PAN migrated faster than the controls, suggesting that the motility of PAN-treated podocytes was abnormal. (F) Phalloidin and DAPI staining of untreated control podocytes showed fine F-actin stress fibers, whereas podocytes exposed to 75 μg/ml PAN show a loss of stress fibers and the appearance of a disordered and thick cortical stress fiber distribution. (*p<0.05 vs. control; **p<0.01 vs. control; ns, no statistical significance; n = 3. Black bar = 200 μm; White bar = 40 μm. PAN: Puromycin aminonucleoside)
Fig 2.
Calcineurin and calpain were abnormal in PAN-treated podocytes.
(A) Enzyme activity of calcineurin in podocytes. Compared to untreated control podocytes, calcineurin activity was up-regulated in podocytes treated with various concentrations of PAN (25, 50, 75, 100, or 125 μg/ml) after 24 h. (B, C) Interestingly, the protein expression of full-length 60-kDa calcineurin was decreased in a dose-dependent manner in PAN-treated podocytes; this effect was the opposite of that observed on calcineurin activity. (D) Increased calpain activity was observed in PAN-treated podocytes. (E, F) Dose-dependent changes in the cleavage of non-erythroid α-spectrin after PAN exposure. The calpain-dependent truncation of α-spectrin from 250-kDa to 145-kDa and 150-kDa fragments suggests that calpain was activated. Increases in the abundance of the two smaller fragments indicated that calpain was abnormally activated in PAN-treated podocytes. (*p<0.05 vs. control; **p<0.01 vs. control; ns, no statistical significance; n = 3. PAN: Puromycin aminonucleoside)
Fig 3.
Calpain regulated both the expression and activity of calcineurin.
(A, B, G, H) After PAN treatment, the level of full-length 60 KDa calcineurin in podocytes was significantly decreased (lane 3). Calpain inhibition by ALLN or calpain knockdown restored the normal level of full-length calcineurin in PAN-treated podocytes (lane 4). (C, I) Calcineurin activity was abnormally enhanced after PAN exposure, whereas calpain inhibition with ALLN pre-treatment and calpain knockdown in PAN-treated podocytes suppressed the enhanced activity of calcineurin. (D, E) Screening of highly effective calpain siRNAs. Calpain protein expression decreased significantly after transfection with calpain siRNAs, especially siRNA #3, compared to control siRNA. In our study, calpain knockdown was performed using siRNA #3, as shown in 3G, 3H and 3I. (F) Phalloidin staining of untreated control and ALLN treated podocytes showed fine F-actin stress fibers, whereas podocytes exposed to 75 μg/ml PAN show a loss of stress fibers and the appearance of a disordered and thick cortical stress fiber distribution. After bloackade of calpain by using ALLN in PAN treated podocytes, the F-actin was present and recovered to fine but still a little disordered and thick cortical stress fiber distribution. (*p<0.05 vs. control or control siRNA; **p<0.01 vs. control; #p<0.05 vs. PAN or PAN+control siRNA; ns, no statistical significance; n = 3. PAN: Puromycin aminonucleoside)
Fig 4.
Calpain mediated the cleavage of calcineurin in PAN-treated podocytes.
(A, B) The cleavage pattern of calcineurin was examined in the presence of purified calpain 1 in vitro. Purified calpain 1 (1, 2, or 3 μg) was incubated with 1 mM Ca2+ at 37°C for 30 min. After the incubation, each sample was loaded onto a 10% SDS-PAGE gel, and the proteins were examined by western blotting. A 45-kDa fragment was detected, and the expression of this fragment was increased at the expense of 60-kDa calcineurin. (C, D) The expression of cleaved 45-kDa calcineurin was dependent on the increased concentration of PAN (25, 50, 75, 100, and 125 μg/ml) in podocytes and was associated with a dose-dependent decrease in 60-kDa calcineurin. (E, F) Inhibiting calpain with ALLN resulted in a reduced level of the 45-kDa calcineurin cleavage product (lane 4), whereas enhanced calcineurin cleavage (lane 3) was observed in the podocytes treated with PAN only. (G, H) Purified calpain 1 (2μg) was incubated with podocytes extracts in presence of 2 mM EGTA to chelate all the free calcium at 37°C for 30 min. Also, purified calpain 1 was also incubated with podocytes extracts at the same time. After incubation, the western blotting showed that the single calpain without calcium (lane 2) cannot increase the cleavage 45KDa calcineurin and calcineurin activity. (*p<0.05 vs. control or control siRNA; **p<0.01 vs. control; #p<0.05 vs. PAN; ns, no statistical significance; n = 3. PAN: Puromycin aminonucleoside)