Plectin protects podocytes from adriamycin-induced apoptosis and F-actin cytoskeletal disruption through the integrin α6β4/FAK/p38 MAPK pathway

Abstract

Podocyte injury is an early pathological change characteristic of various glomerular diseases, and apoptosis and F-actin cytoskeletal disruption are typical features of podocyte injury. In this study, we found that adriamycin (ADR) treatment resulted in typical podocyte injury and repressed plectin expression. Restoring plectin expression protected against ADR-induced podocyte injury whereas siRNA-mediated plectin silencing produced similar effects as ADR-induced podocyte injury, suggesting that plectin plays a key role in preventing podocyte injury. Further analysis showed that plectin repression induced significant integrin α6β4, focal adhesion kinase (FAK) and p38 MAPK phosphorylation. Mutating Y1494, a key tyrosine residue in the integrin β4 subunit, blocked FAK and p38 phosphorylation, thereby alleviating podocyte injury. Inhibitor studies demonstrated that FAK Y397 phosphorylation promoted p38 activation, resulting in podocyte apoptosis and F-actin cytoskeletal disruption. In vivo studies showed that administration of ADR to rats resulted in significantly increased 24-hour urine protein levels along with decreased plectin expression and activated integrin α6β4, FAK, and p38. Taken together, these findings indicated that plectin protects podocytes from ADR-induced apoptosis and F-actin cytoskeletal disruption by inhibiting integrin α6β4/FAK/p38 pathway activation and that plectin may be a therapeutic target for podocyte injury-related glomerular diseases.

Keywords: F-actin cytoskeleton; FAK; apoptosis; integrin α6β4; p38 MAPK pathway; plectin; podocyte.

Figure 1

ADR suppressed plectin expression and induced podocyte injury. A‐B, Changes in plectin mRNA and protein expression in podocyte treated with ADR for the indicated times and at the indicated doses. *P < 0.05 and **P < 0.01. N.S., not significant. #P < 0.05 1 μg/mL vs 0.5 μg/mL ADR‐treated podocyte at the same time‐point. C‐D, Flow cytometry analysis showed that apoptosis was significantly increased in ADR‐treated podocyte compared with NC podocyte. **P < 0.01 vs the NC group. E, Immunofluorescence staining with FITC‐labeled phalloidin showed that the F‐actin cytoskeleton was disorganized in the ADR group compared with the NC group. F‐G, Western blot analysis showed that WT1 and synaptopodin expression levels were significantly decreased and that desmin expression levels were increased in ADR‐treated podocyte compared with NC podocyte. **P < 0.01 vs the NC group. Data shown are representative of five independent experiments (n = 5). The ADR‐treated podocyte in C‐G were treated with 0.5 μg/mL ADR for 12 h. ADR, adriamycin; NC, normal control; FITC, fluorescein isothiocyanate; F‐actin, filamentous actin

Figure 2

ADR suppressed plectin expression through mitochondrial oxidative stress. A‐B, Flow cytometry analysis showed that 0.5 μg/mL ADR treatment on podocyte for the indicated times caused an increase in ROS production. **P < 0.01 vs NC group. C‐E, Changes in plectin mRNA and protein expression in podocyte treated with 0.5 μg/mL ADR for the indicated times. *P < 0.05, **P < 0.01 and N.S., not significant vs NC group. F‐G, Flow cytometry analysis showed that 0.5 μg/mL ADR treatment on podocyte for 4 h caused an increase in apoptosis rate. *P < 0.05, **P < 0.01 and N.S., not significant vs NC group. H, Flow cytometry analysis showed that mitochondrial protective agent MitoTEMPOL (10 μmol/L) significantly decreased ROS production in ADR‐treated podocyte. I‐J, Western blot analysis showed that plectin expression was significantly increased in ADR + MitoTEMPOL group compared with ADR group. Data shown are representative of five independent experiments (n = 5). NC, normal control podocyte; ADR, podocyte treated with 0.5 μg/mL adriamycin for 12 h; ADR + MitoTEMPOL, podocyte treated with 0.5 μg/mL adriamycin and 10 μmol/L MitoTEMPOL for 12 h

Figure 3

Restoring plectin expression prevented ADR‐induced podocyte injury. For the ADR group, podocyte was treated with 0.5 μg/mL ADR for 12 h. For the ADR + plectin group or ADR + MOCK group, podocyte was incubated with pEGFP‐N1‐plectin plasmids or empty vectors for 6 h and then cultured normally for 42 h, 0.5 μg/mL ADR was added 12 h prior to cell harvest. A‐B, Real‐time PCR and Western blot analysis showed that plectin expression was 2‐3 times of that in control group after transfecting pEGFP‐N1‐plectin plasmids into podocyte. C‐D, Western blot showed that plectin protein expression levels were significantly increased in the ADR + plectin group compared with the ADR and ADR + Mock groups. E‐F, Flow cytometry analysis showed that podocyte apoptosis was alleviated in the ADR + plectin group compared with the ADR and ADR + Mock groups. G, Immunofluorescence staining showed that restoring plectin expression in the ADR + plectin group ameliorated ADR‐induced F‐actin filament disruption. H‐I, Western blot showed that WT1 and synaptopodin protein expression levels were increased and that desmin protein expression levels were decreased in the ADR + plectin group compared with the ADR and ADR + Mock groups. J‐K, Western blot showed that integrin α6β4, FAK and p38 phosphorylation levels were higher in the ADR group than in the NC group. Restoring plectin expression in the ADR + plectin group suppressed integrin α6β4, FAK and p38 phosphorylation but had no effect on total integrin α6β4, FAK and p38 expression levels. Data shown are representative of three independent experiments (n = 3). **P < 0.01; N.S., not significant. ADR, adriamycin; F‐actin, filamentous actin; FAK, focal adhesion kinase; FITC, fluorescein isothiocyanate; NC, normal control

Figure 4

The time‐course effect of siPlectin on the phosphorylation of integrin α6β4, activation of FAK and p38. A‐B, Western blot analysis of plectin expression levels after siPlectin transfection. This experiment showed that siRNA #3 (si‐3) was the most efficient siRNA; therefore, this siRNA was used in subsequent experiments. C, Flow cytometry revealed that the apoptosis rate of podocyte was significantly increased after 48 h siPlectin treatment and further increased after 120 and 168 h siPlectin treatment. D, Western blot analysis showed that the protein expression of plectin was significantly decreased after 48 h siPlectin treatment and recovered after 120 h siPlectin treatment. E‐F, Western blot analysis showed that the phosphorylation of integrin α6β4 and FAK peaked at 72 and 120 h, and decreased at 168 h. G, Western blot analysis showed that the phosphorylation of p38 gradually increased after 72 h siPlectin treatment and reached its peak at 168 h

Figure 5

Plectin suppression produced similar effects as ADR‐induced podocyte injury by facilitating integrin α6β4‐mediated FAK and p38 activation. For the ADR group, podocyte was treated with 0.5 μg/mL ADR for 12 h. For the NC + siPlectin group and NC + Scramble group, podocyte was transiently transfected with siPlectin or scrambled RNA at a final concentration of 20 nmol/L for 4 h and then cultured normally for 68 h. A‐B, Western blot showed that plectin protein expression was significantly decreased in the NC + siPlectin group compared with the NC + Scramble groups. Mutating the Y1494 residue of integrin α6β4 in the NC + siPlectin + β4 mutant group had no effect on plectin protein expression. C‐D, Flow cytometry demonstrated increased podocyte apoptosis in the NC + siPlectin group compared with the NC + Scramble group. Mutating integrin α6β4 alleviated siPlectin transfection‐induced apoptosis. E, Immunofluorescence staining demonstrated the presence of significant F‐actin disruption in the NC + siPlectin group compared with the NC + Scramble group. Mutating integrin α6β4 reversed siPlectin transfection‐induced F‐actin disruption. F‐G, Western blot showed that WT1 and synaptopodin expression levels were significantly decreased and that desmin expression levels were significantly increased in the NC + siPlectin group compared with the NC + Scramble group. These abnormalities were partially reversed by integrin α6β4 mutation in NC + siPlectin + β4 mutant group. H‐I, Western blot showed that siPlectin transfection activated integrin α6β4, FAK and p38 phosphorylation, whereas mutating integrin α6β4 abolished the effects of siPlectin. Data shown are representative of three independent experiments (n = 3). **P < 0.01; N.S., not significant. ADR, adriamycin; F‐actin, filamentous actin; FAK, focal adhesion kinase; NC, normal control

Figure 6

Inhibiting FAK or p38 alleviated siPlectin‐induced podocyte injury. For the NC + siPlectin group and NC + Scramble group, podocyte was transiently transfected with siPlectin or scramble RNA at a final concentration of 20 nmol/L for 4 h and then cultured normally for 68 h. For inhibitor studies, podocyte was preincubated with the FAK inhibitor 14 (50 μmol/L), the p38 inhibitor SB203580 (5 μmol/L) or DMSO vehicle respectively for 1 h before siPlectin transfection and then incubated for an additional 72 h until podocyte collection. A, Western blot showed that siPlectin transfection successfully silenced plectin protein expression in the NC + siPlectin group. Inhibiting FAK in the NC + siPlectin + FAK inhibitor group or p38 in the NC + siPlectin + p38 inhibitor group had no effect on plectin expression. B, Western blot showed that FAK inhibition or p38 inhibition significantly attenuated the abnormalities in WT1, synaptopodin and desmin expression induced by siPlectin. C, Flow cytometry showed that FAK inhibition or p38 inhibition significantly alleviated siPlectin‐induced apoptosis. D, Immunofluorescence staining showed that FAK inhibition or p38 inhibition reversed siPlectin‐induced F‐actin disruption. E‐F, Western blot showed that integrin α6β4, FAK and p38 phosphorylation levels were elevated in the NC + siPlectin group. FAK inhibition at the Y397 site did not affect siPlectin‐induced integrin α6β4 phosphorylation levels but decreased p38 phosphorylation levels in the NC + siPlectin + FAK inhibitor group compared with the NC + siPlectin + DMSO group. p38 inhibition in the NC + siPlectin + p38 inhibitor group had no impact on integrin α6β4 and FAK phosphorylation levels. Data shown are representative of three independent experiments (n = 3). **P < 0.01; N.S., not significant. ADR, adriamycin; F‐actin, filamentous actin; FAK, focal adhesion kinase; NC, normal control

Figure 7

p38 induced podocyte apoptosis by activating Bax and caspase‐3. For the ADR group, podocyte was treated with 0.5 μg/mL ADR for 12 h. For the ADR + plectin group or ADR + MOCK group, podocyte was incubated with pEGFP‐N1‐plectin plasmids or empty vectors for 6 h and then cultured normally for 42 h, 0.5 μg/mL ADR was added 12 h prior to cell harvest. For the NC + siPlectin group and NC + Scramble group, podocyte was transfected with siPlectin or scramble RNA respectively at a final concentration of 20 nmol/L for 4 h and then cultured normally for 68 h. For p38‐MAPK inhibitor study, podocyte was preincubated with the p38 inhibitor SB203580 (5 μmol/L) or DMSO vehicle for 1 h before siPlectin transfection and then incubated for an additional 72 h until podocyte collection. A‐B, Western blot showed that Bax and cleaved caspase‐3 protein expression levels were elevated in ADR‐treated podocyte compared with NC podocyte and that recovering plectin expression inhibited the activation of these pro‐apoptotic proteins. C‐D, Western blot showed that siPlectin transfection increased Bax and cleaved caspase‐3 protein expression in the NC + siPlectin group compared with the NC + Scramble group, whereas p38 inhibition blocked the siPlectin‐induced activation of these proteins. Data shown are representative of three independent experiments (n = 3). *P < 0.05 and **P < 0.01

Figure 8

ADR contributed to the development of proteinuria and renal dysfunction by inhibiting plectin expression and activating the integrin α6β4/FAK/p38 pathway. ADR induced nephropathy was introduced by a single injection of 7.5 mg/kg ADR via the tail vein. All rats were killed at the end of the 4th week after ADR injection. A, Light microscopic examination revealed glomerular atrophy and disappearance as well as renal tubular swelling after ADR treatment. B, TEM examination of the NC group revealed the presence of normal glomerular ultrastructure. TEM examination of the ADR group revealed the presence of diffuse foot process effacement, GBM thickening, slit diaphragm loss and mesangial sclerosis. C‐D, Western blot showed that WT1 and synaptopodin protein expression levels were decreased and that desmin protein expression levels were increased in the ADR group compared with the NC group. E, Western blot showed that plectin expression was suppressed in the ADR‐treated group (n = 5) compared with the NC group (n = 5). F‐G, Immunohistochemical analysis showed that plectin expression in glomeruli was decreased in ADR treated kidney tissues (n = 5) compared with normal kidney tissues (n = 5). H‐I, Western blot showed that integrin α6β4, FAK and p38 were activated by ADR treatment. J‐K, Western blot showed that cleaved caspase‐3 and Bax protein expression was increased in the ADR group (n = 5) compared with the NC group (n = 5). **P < 0.01

Figure 9

Schematic representation of the mechanism by which plectin repression promotes apoptosis and F‐actin cytoskeletal disruption in podocyte