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.

podocytes results in foot process effacement and is associated with the pathogenesis of proteinuria, which is common among a spectrum of chronic kidney diseases. [3][4][5] Therefore, elucidating the mechanisms underlying podocyte apoptosis and F-actin cytoskeletal disruption is critical for the development of promising therapies for glomerular diseases.
Adriamycin (ADR)-induced nephrosis is a commonly used animal model of podocyte injury as ADR can induce apoptosis and F-actin cytoskeletal disaggregation in podocytes. We previously identified dozens of proteins that are differentially expressed between the isolated glomeruli of rats with ADR-induced nephropathy and those of normal control (NC) rats (data not shown here) in a proteome analysis via isobaric tags for relative and absolute quantification (iTRAQ).
Among these differentially expressed proteins, a cytoskeletal linker protein known as plectin caught our attention because of its versatility and significant suppression in the glomeruli of ADR-treated rats.
In addition to functioning as a linker, plectin plays a crucial role in cellular processes involving actin cytoskeleton dynamics 6,7 ; moreover, plectin can function as a scaffolding platform for signalling molecules, such as integrin α6β4 8,9 and focal adhesion kinase (FAK), 10,11 which is an intriguing new facet of its functional repertoire that has recently attracted attention from researchers. FAK is a key mediator of integrin signalling among different cellular functions in a variety of cells. Following activation by integrins, FAK undergoes autophosphorylation, forms a complex with other cellular proteins and triggers downstream signalling through its kinase activity or scaffolding function. 12,13 FAK and downstream p38 MAPK signalling have been shown to play vital roles in ADR-induced podocyte apoptosis and F-actin cytoskeletal remodelling. 4,14 Given that plectin is severely depleted in ADR-treated glomeruli, this study aimed to evaluate its effects in ADR-induced podocyte apoptosis and F-actin cytoskeletal rearrangement and explore its relationships with integrin α6β4 and FAK.

| Animals
Twenty male Sprague Dawley rats (200 g ± 20 g) were purchased from Shandong PengYue Laboratory Animals (Shandong, China). Rats were housed in a temperature-controlled room with a 12-hour light/ 12-hour dark cycle and given free access to food and water. These rats were randomly divided into two groups (n = 10 per group): the ADR group and NC group. ADR-induced nephropathy was achieved by a single injection of 7.5 mg/kg ADR (0.75 mg/mL in normal saline) via the tail vein. No rat died during the experiment. At the end of the 4th week after ADR injection, all rats were killed. Before death, body weight was recorded, and urine was collected from each rat in metabolic cages to determine 24-hour urine protein (UP) and 24-hour urine volume (UV).
Blood was collected from the abdominal aorta, and serum was pre-

| Cell culture and treatment
The mouse podocytes used in this study were conditionally immortal-

| Immunoblotting
The podocytes were washed with cold PBS and lysed with RIPA buffer containing a proteinase inhibitor cocktail (Roche) and phosphatase inhibitors. Total protein was extracted from frozen renal cortical tissue samples using RIPA AQ3 lysis buffer (Beyotime Institute of Biochemistry, Shanghai, China). The cell lysates were separated on 12%, 10% or 8% gels and probed with the indicated primary antibodies. The proteins were subsequently detected by enhanced chemiluminescence (Pierce: Waltham, MA). The blocking buffer used for phospho-immunoblotting contained 5% (w/v) BSA.

| Flow cytometry for apoptosis analysis
After treatment, the podocytes were stained with fluorescein-isothiocyanate-labelled Annexin V and propidium iodide according to the manufacturer′s instructions (BioLegend: San Diego, CA) and analysed by flow cytometry on an FACSAria III flow cytometer (Becton Dickinson, Franklin Lakes, NJ).

| Immunofluorescence staining
The cells were fixed in 4% paraformaldehyde for 30 minutes at room temperature and then incubated with 1% BSA (with 0.3% Triton X-100) for 60 minutes. The nuclei were stained with 4′,6-diamidino-2phenylindole (DAPI). For F-actin staining, the cells were stained with FITC-labelled phalloidin for 60 minutes at room temperature before staining with DAPI. Fluorescence was detected by laser scanning confocal microscopy (Leica, Wetzlar, Germany).

| Renal histological investigation
Renal tissue specimens were fixed in a 4% formaldehyde solution for 48 hours, dehydrated in graded ethanol solutions, and then embedded in paraffin before being sectioned at a thickness of 5 μm. Morphological analysis of glomerular injury was conducted by light microscopy following haematoxylin and eosin staining.  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 timepoint. 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 ADRtreated 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 semiquantitative scoring system 15,16 to grade the intensity of the abovementioned immunoreactions. In each glomerulus, positively stained cells were scored according to their staining intensities, which were determined using the following scale: 0 (no staining), +1 (weak but detectable staining), +2 (moderate staining) and +3 (intense staining). Five areas in each slide were evaluated under a microscope at a magnification of 40×. An H-score was calculated for each tissue sample by multiplying the percentages of cells in each intensity category by the corresponding staining intensities and then adding these products together. The calculation was performed using the following formula: H-score = ∑(Pc × s), where s represents the intensity score, and Pc is the corresponding cell percentage. All immunohistochemical staining was independently assessed by two blinded pathologists.

| Electron microscopy
Kidney cortical tissue samples were separated into 1-μm-thick blocks on ice and then immediately placed in 3% glutaraldehyde for 2 hours at 48°C before being post-fixed with 1% osmium tetroxide for 1 hour, stained with 2% aqueous uranyl acetate, and dehydrated in graded ethanol solutions. After infiltration and polymerization, ultrathin sections were prepared, stained with uranyl acetate and lead citrate, and examined under an H-800 transmission electron microscope (Hitachi Electronic Instruments, Tokyo, Japan).

| Statistical analysis
Statistical analyses were performed with SPSS 13.0 (SPSS Inc., Chicago, IL). Data were analysed by Student's t test or one-way ANOVA followed by Student-Newman-Keuls post hoc tests. All statistical tests were two-sided and a P value < 0.05 was considered statistically significant for all tests.

| ADR down-regulated plectin expression and induced podocyte injury
We treated podocytes with 0.5 or 1 μg/mL ADR for 6, 12, or 24 hours and then assessed plectin expression by qPCR and Western blot. We found that ADR down-regulated plectin mRNA and protein expression in a concentration-and time-dependent manner in treated cells compared with control cells ( Figure 1A,B). However, there was no difference in plectin expression between the cells treated with ADR for 12 hours and those treated for 24 hours. We chose to treat podocytes with 0.5 μg/mL ADR for 12 hours in subsequent experiments (ADR-treated group) because treatment with ADR at the indicated concentration and for the indicated time induced typical podocyte injury as well as significant decreases in plectin expression.
In vitro studies have shown that the actin cytoskeletal changes, such as F-actin stress fibre loss, in cultured podocytes mimic the podocyte changes in vivo. 17 Furthermore, rescue of the cytoskeletal disaggregation by genetic or chemical means in vitro correlates with protection from proteinuria in vivo. 18 Therefore, we detected F-actin cytoskeleton organization by fluorescein-phalloidin staining to monitor podocyte injury. Immunofluorescence staining demonstrated that the F-actin cytoskeleton, which appears as a dynamic network of intracellular proteinaceous structural elements in NC podocytes, was disorganized, collapsed and peripherally located in ADR-treated cells ( Figure 1E).
The second marker of podocyte injury examined in this study was the podocyte apoptosis rate, which is a critical determinant of the progression of proteinuria and renal failure. Flow cytometry analysis showed that apoptosis was significantly increased in the ADR group compared with the NC group ( Figure 1C,D).
In this study, we also assessed the expression of several classical biomarkers that reflect podocyte injury. WT1 and synaptopodin are two specific podocyte markers whose expression is reduced when glomerular damage is present. Desmin has also been shown to be a sensitive marker of podocyte damage. WT1, synaptopodin and desmin protein levels were measured in podocytes by Western blot.
The results showed that WT1 and synaptopodin expression levels were significantly reduced and desmin expression levels were increased in cells treated with 0.5 μg/mL ADR for 12 hours compared with control cells ( Figure 1F,G).

| ADR down-regulated plectin expression through mitochondrial oxidative stress
Previous studies have shown that ADR caused mitochondrial dysfunction in podocytes, and the increased production of reactive oxygen species (ROS) by mitochondrial oxidative stress is one of the major manifestations. 19,20 In this study, we found that 0.5 μg/mL ADR treatment on podocytes for 1 hour caused significant increase F I G U R E 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.

ADR-induced podocyte injury
To determine the role of plectin in ADR-induced podocyte injury, we restored plectin expression in ADR-treated podocytes via transfection with plasmids containing plectin cDNA and then measured the subsequent changes. We found that plectin expression levels were significantly increased in the ADR + plectin group compared with the ADR and ADR + Mock groups, as demonstrated by Western blot analysis in Figure 3C,D. Flow cytometry assays, the results of which are presented in Figure 3E,F, showed that podocyte apoptosis was decreased in the ADR + plectin group compared with the ADR and ADR + Mock groups. Restoring plectin expression also mitigated the above mentioned ADR-induced F-actin cytoskeletal disarrangements, a finding that was also supported by our immunofluorescence staining results ( Figure 3G). Western blot analysis showed that the expression levels of podocyte-specific markers WT1 and synaptopodin were significantly enhanced but the expression level of desmin, a marker of injury, was substantially decreased by plectin restoration (Figure 3H,I). The above results demonstrated that restoring plectin expression significantly attenuated ADR-induced podocyte injury, suggesting that plectin plays an important role in this pathological process.

| Plectin suppression produced similar effects as ADR-induced podocyte injury
To confirm the above findings, we disrupted plectin expression in normal podocytes using siRNA and observed the subsequent changes in podocyte injury. We screened three siRNAs and found that si-3 most effectively and significantly reduced plectin protein levels in the podocytes ( Figure 4A). Western blot analysis showed that plectin levels were significantly decreased in the NC + siPlectin group compared with the NC and NC + scramble groups ( Figure 5A

| Plectin suppression led to integrin α6β4, FAK and p38 activation
To elucidate the mechanism by which plectin suppression promotes podocyte injury, we monitored the activity of various signalling pathways in ADR-and siPlectin-treated podocytes. Plectin is known to be an important ligand for integrin α6β4. Phosphorylated integrin α6β4 can recruit and activate FAK, 21 an important upstream mediator of the p38 pathway. 12 Both FAK and p38 have been shown to be involved in nephropathy development. 12 Therefore, we specifically examined integrin α6β4, FAK and p38 expression and activity. Western blot analysis showed that integrin α6β4, FAK and p38 phosphorylation levels were significantly increased in the ADR-treated group compared with the NC group, whereas total protein levels were similar between the two groups ( Figure  Western blot analysis showed that the protein expression of plectin was significantly decreased after 48 hours siPlectin treatment and recovered after 120 hours siPlectin treatment ( Figure 4D). The activation of integrin α6β4 and FAK peaked at 72 and 120 hours, and decreased at 168 hours ( Figure 4E,F). Activities of p38 gradually increased after 72 hours siPlectin treatment and reached its peak at 168 hours ( Figure 4G). Flow cytometry revealed that the apoptosis rate of podocytes was significantly increased after 48 hours siPlectin treatment and further increased after 120 and 168 hours siPlectin treatment ( Figure 4C).

| Y1494 of integrin α6β4 contributed to plectin-dependent podocyte injury as well as FAK and p38 activation
The above experiments demonstrated that decreased plectin expression enhanced integrin α6β4, FAK and p38 phosphorylation levels.  Figure 5H,I), suggesting that FAK and p38 are downstream effectors of integrin α6β4 whose activation is controlled by plectin. In addition, we found that podocyte injury was mitigated in the NC + siPlectin + β4 mutant group compared to the NC + siPlectin and NC + siPlectin + Mock groups. This change manifested as decreased apoptosis ( Figure 5C,D), improved F-actin cytoskeletal organization ( Figure 5E), elevated WT1 and synaptopodin expression, and decreased desmin expression ( Figure 5F,G).

FAK promoted p38 activation
Activated integrin α6β4 has been shown to recruit FAK, thereby inducing Y397 autophosphorylation 21 and triggering the expression of downstream effectors, such as p38. 12  p38 activation by preventing FAK Y397 phosphorylation but had no effect on integrin α6β4 phosphorylation, suggesting that FAK is an upstream inducer of p38 and a downstream effector of integrin α6β4 ( Figure 6E,F). Western blot analysis also showed that WT1 and synaptopodin expression levels were higher, and desmin expression levels were lower in the NC + siPlectin + FAK inhibitor 14 group than in the NC + siPlectin and DMSO groups ( Figure 6B). Flow cytometry analysis and immunofluorescence staining also showed that podocyte injury was attenuated in the NC + siPlectin + FAK inhibitor 14 group compared with the NC + siPlectin and DMSO groups. These changes manifested as decreases in apoptosis ( Figure 6C) and improvements in F-actin cytoskeletal reorganization ( Figure 6D). Our findings suggested that blocking FAK Y397 phosphorylation inhibited p38 activation, thereby attenuating podocyte injury.

| p38 activation triggered apoptosis and F-actin disruption
p38 is believed to be a critical mediator of cell apoptosis 28

| ADR treatment decreased plectin expression, activated integrin α6β4, FAK and p38, and induced renal injury in rats
The mean body weights of the two groups were similar before ADR treatment. Four weeks later, the body weight of the ADR-treated group was significantly lower than that of the NC group. The 24hour UP, BUN and SCr levels were significantly higher in the ADR group than in the NC group, and 24-hour UV was significantly lower in the ADR group than in the NC group. These results are shown in Table 2.
Light microscopic examination revealed the presence of significant histopathologic changes in the glomerular and tubulo-interstitial areas of the rat kidneys in the ADR group compared with those in the NC group ( Figure 8A).  Figure 6G). However, the staining intensity in the epithelial cells of the collecting ducts was not significantly different between the two groups (data not shown).
Integrin α6β4, FAK and p38 expression and activity were examined in vivo. Western blot assays showed that integrin α6β4, FAK and p38 phosphorylation levels were significantly increased in the ADR-treated group compared with the NC group, whereas the total protein levels were similar between the two groups ( Figure 8I,J).

Moreover, the expression levels of the podocyte apoptosis markers
Bax and cleaved caspase-3 were significantly up-regulated in the ADR-treated group compared with the NC group ( Figure 8K,L). 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 siPlectininduced apoptosis. D, Immunofluorescence staining showed that FAK inhibition or p38 inhibition reversed siPlectin-induced Factin 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 siPlectininduced integrin α6β4 phosphorylation levels but decreased p38 phosphorylation levels in the NC + siPlectin + FAK inhibitor group compared with the NC + siPlectin + DMSO group.   to podocyte injury and proteinuria. 36 Whereas the expression of integrin β1 in podocyte was considered negatively correlated with the severity of proteinuria. 37 Elevated expression of TGF-β1 or ROS induced decreased expression of integrin β1, resulting in the production of proteinuria. 38,39 In contrast to the other β subunits, the integrin β4 subunit contains an exceptionally large cytoplasmic domain comprising several major tyrosine phosphorylation sites and thus F I G U R E 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 regulates multiple intracellular signalling pathways. 40,41 The integrin  48 The results in this study showed that the Y1494 site of integrin β4 was phosphorylated when plectin expression was down-regulated.
According to the molecular spatial conformation that plectin binds to integrin β4, we speculate that, when plectin is bound to integrin β4, shown to protect podocytes from injury in vitro and ameliorate proteinuria in vivo. 29,53 A recent study showed that the FAK/p38 axis contributed to foot process effacement and proteinuria development in a rat model of short-term hypercholesterolaemia by modulating podocyte apoptosis and F-actin reorganization. 4  showed that EGFP was predominantly expressed in renal tubular epithelial cells with little expression in glomeruli. Western blot assay was also performed and the results showed that the protein expression of plectin in glomeruli of rats injected with Plectin-Ad-EGFP had no significant difference with that of the control rats. We adjusted the injection dose of Plectin-Ad-EGFP and the time-point of organ harvest, but the results remained the same (data not shown here).
These results suggest that adenovirus did not successfully enter the glomeruli, but selectively transfected into renal tubular epithelial cells. Therefore, the limitation for this part is the lack of the evidence showing in vivo function of plectin in protecting podocytes and regulating the downstream signalling pathways.

| CONCLUSION
In summary, our in vitro study demonstrated that ADR-or siRNA- This study has identified novel targets that may be used for the development of therapies for podocyte injury-related glomerulopathies.

ACKNOWLEDG EMENTS
This study was supported by grants from the National Natural Science Foundation of China 81600220; Natural Science Foundation of Shandong Province BS2014YY020; and Major Scientific Projects of Yankuang Group YK2015A017.

CONFLI CT OF INTEREST
All the co-authors declare that we have no conflict of interests in the submission of this manuscript.