Protein kinase Cα drives fibroblast activation and kidney fibrosis by stimulating autophagic flux

Inhibition of PKCα reduces LAMP2 expression and induces lysosomal dysfunction in NRK-49F cells

To investigate the role of PKCα signaling in fibroblast activation, NRK-49F cells, a rat kidney interstitial fibroblast cell line, were treated with TGFβ1 (2 ng/ml) for different time points as indicated. As shown in Fig. 1A, the amount of p-PKCα and p-Akt (Ser-473) was increased at 4 h and maintained at a high level within 24 h after TGFβ1 treatment. NRK-49F cells were pretreated with the PKCα inhibitor Go6976 for 30 min, followed by TGFβ1 incubation, and harvested at 4 h and 8 h later. Western blotting analyses revealed that the abundance of p-PKCα but not p-Akt (Ser-473) was diminished after Go6976 treatment (Fig. 1B). Immunofluorescent staining further confirmed Western blotting results (Fig. 1C). In addition, Smad3 phosphorylation was not markedly decreased in NRK-49F cells pretreated with Go6976 compared with TGFβ1 treatment alone (Fig. 1B).

PKCα was reported to play a key role for lysosome biogenesis (25). We then examined the intracellular intensity of lysosomes in cultured NRK-49F cells treated with Go6976 (5 μm) for a different time duration as indicated. As shown in Fig. 1D, LAMP2 expression was continuously down-regulated within 24 h after Go6976 treatment. We pretreated cells with Go6976 at different dosages as indicated for 30 min before TGFβ1 treatment. The Western blotting analyses revealed that LAMP2 abundance was significantly decreased after Go6976 treatment (Fig. 1E). To further investigate the role of PKCα in regulating lysosomal function, NRK-49F cells transfected with scrambler or PKCα siRNA were treated with TGFβ1 for 4 h. PKCα siRNA transfection could markedly down-regulate PKCα mRNA expression, whereas the other isoforms of PKC remained unchanged. As shown in Fig. 1F, PKCα siRNA transfection markedly down-regulates LAMP2 protein expression. Immunofluorescent staining further confirmed Western blotting results (Fig. 1G). The lysosomal protease activity, measured by β-N-acetylglucosaminidase (NAG) assays, was not affected by Go6976 or PKCα siRNA transfection in NRK-49F cells (Fig. 1H). Furthermore, we utilized the LysoSensor Yellow/Blue reagent, a pH-sensitive probe that emits predominantly yellow fluorescence in acidic organelles and blue fluorescence in less acidic organelles. NRK-49F cells were pre-transfected with RFP-LC3 expression plasmid and then incubated with LysoSensor (Fig. 1I). The blue/yellow ratio was not changed in the acidic vesicular organelles after Go6976 treatment, whereas chloroquine (CQ) administration significantly increased the blue/yellow ratio, reflecting an increase in pH values. The co-localization of the yellow/blue/autophagosome was inhibited in NRK-49F cells after CQ and Go6976 treatment (Fig. 1J).

Inhibition of PKCα retards autophagic flux in NRK-49F cells

Inspired by the above observations, we postulated that Go6976 may affect autophagosome–lysosome fusion and/or autophagy activity in NRK-49F cells. As shown in Fig. 2A, Western blotting analyses showed that Go6976 induced the accumulation of both MAP1LC3B-I (LC3B-I) and MAP1LC3B-II (LC3B-II) in a dose-dependent manner. Interestingly, SQSTM1/p62 protein abundance was also increased after Go6976 treatment in a dose-dependent manner at 4 h after TGFβ1 treatment, indicating the retardation of autophagic flux in NRK-49F cells. We then treated NRK-49F cells with CQ, and similar results were observed (Fig. 2B). To further explore the role of PKCα signaling in regulating autophagic flux, NRK-49F cells were transfected with scramble and PKCα siRNA. Western blotting analyses revealed that PKCα siRNA transfection significantly increased LC3B-II and SQSTM1/p62 abundance in NRK-49F cells (Fig. 2C). Electronic microscopic examination revealed an increased organized cytoplasmic structure typical of lysosomes and autophagosomes and/or autolysosomes and massive vacuolization containing undigested proteins and organelles in NRK-49F cells after Go6976 with or without TGFβ1 treatment (Fig. 2D).

We also transfected NRK-49F cells with RFP-LC3 expression plasmid and then treated with Go6976 or CQ. As shown in Fig. 2E, Go6976 and CQ treatment could similarly increase RFP-LC3–positive and decrease LAMP2-positive dot numbers in NRK-49F cells. Interestingly, RFP-positive puncta were rarely co-localized with LAMP2 in NRK-49F cells treated with Go6976 or chloroquine. The accumulation of p62 represents the amount of substrate requiring degradation that accumulated in chloroquine and Go6976 administration cells. CQ and Go6976 treatment decreased the p62/LAMP2 merged dots/p62 puncta ratio in NRK-49F cells with or without TGFβ1 treatment (Fig. 2F). Furthermore, as shown in Fig. 2G, the number of p62 positive dots was increased, whereas the number of p62/LAMP2 merged dots/p62 dots ratio was decreased in cells transfected with PKCα siRNA compared with those transfected with scrambler siRNA with or without TGFβ1 treatment. In NRK-49F cells, Go6976 but not CQ administration obviously inhibited the clearance of lipid droplets (Fig. 2H). Together, these results demonstrate that PKCα signaling is indispensable for maintaining autophagic flux in NRK-49F cells.

Blockade of PKCα signaling diminishes TGFβ1-induced fibroblast activation

We then treated NRK-49F cells with the PKCα inhibitor Go6976, followed 30 min later by TGFβ1 treatment for 24 and 48 h. Go6976 could remarkably inhibit TGFβ1-induced fibronectin (FN) and α-smooth muscle actin (α-SMA) expression in a time- and dose-dependent manner (Fig. 3, A and B). Immunofluorescent staining further confirmed Western blotting results (Fig. 3B, right). NRK-49F cells were also transfected with PKCα siRNAs as indicated. PKCα siRNA transfection could markedly down-regulate PKCα protein and mRNA expression (Fig. 3C). Down-regulation of PKCα could markedly reduce TGFβ1-induced FN and α-SMA expression (Fig. 3D). To further clarify the role for autophagic flux retardation in fibroblast activation, we treated NRK-49F cells with CQ and 3-MA, and we found that CQ and 3-MA could remarkably inhibit TGFβ1-induced FN and α-SMA expression (Fig. 3, E and F). Thus, it is clear that PKCα signaling mediates TGFβ1-induced fibroblast activation through autophagy induction.

Blockade of PKCα inhibits autophagic flux in the interstitial myofibroblasts from UUO kidneys

Western blotting assays revealed that phosphorylated protein kinase Cα (p-PKCα) was increased at day 3 and reached peak at days 7 and 14 after UUO (Fig. 4A). Go6976 markedly decreased the abundance for p-PKCα at day 7 after UUO (Fig. 4B). Immunofluorescent staining confirmed the results of Western blotting assay (Fig. 4D).

In NRK-49F cells, we found that PKCα signaling activation is required for fibroblast activation through facilitating autophagic flux. We then investigated autophagic flux in kidneys with UUO nephropathy. On day 7 after UUO, Go6976 treatment increased the abundance of SQSTM1/p62 and LC3B-II, and it decreased LAMP2 abundance compared with the abundance from mice treated with vehicle (Fig. 4C). Immunofluorescent co-staining of anti-laminin and anti-SQSTM1/p62, or anti-laminin and anti-LC3B-II, or anti-laminin and anti-LAMP2, respectively, showed that the interstitial cells from UUO kidneys treated with Go6976 expressed more p62 and LC3B-II and less LAMP2 than from mice treated with vehicle (Fig. 4D). These results suggest blocking PKCα signaling inhibits autophagic flux and lysosome function in the interstitial myofibroblasts from kidneys with UUO nephropathy, which confirmed the results from NRK-49F cells.

Blockade of PKCα signaling ameliorates UUO nephropathy in mice

To further explore the role of PKCα signaling in kidney fibrosis in vivo, we created a mouse model with kidney fibrosis by UUO in male CD1 mice, and Go6976, a specific PKCα signaling inhibitor, was administered intraperitoneally once daily at 2 mg/kg/day from 2 days before the operation. The mice were sacrificed, and the kidneys were harvested at 1 and 2 weeks after UUO surgery. Periodic Acid-Schiff (PAS), Sirius red, and Masson staining showed that renal fibrotic lesions were significantly ameliorated, and interstitial matrix production was decreased in the Go6976-treated group at day 7 after UUO, compared with the vehicle-treated group (Fig. 5A). Kidney injury and fibrotic area were also assessed. Vehicle-treated UUO group exhibited a dramatic induction of kidney injury in the kidneys at 7 days after surgery, whereas Go6976 significantly ameliorated kidney injury. In addition, kidney fibrotic area in Go6976-treated group was also decreased compared with UUO control (Fig. 5B).

We then detected α-SMA and FN expression by Western blotting analyses. FN and α-SMA protein abundance were largely induced on days 7 and 14 in mice after UUO. Go6976 could markedly down-regulate their expression in the UUO kidneys (Fig. 6, A and B). Immunofluorescent staining confirmed the results of Western blotting analyses (Fig. 6C).

Previous studies revealed that inflammation contributes to the progression of kidney fibrosis. We also examined inflammatory cell accumulation and cytokine expression in kidney tissues. Macrophage, neutrophils, and T lymphocytes are identified by immunostaining of F4/80, ly6b, and CD3, respectively. A few F4/80-positive cells were detected in the sham kidneys from vehicle-treated group. In UUO kidneys, F4/80-positive cell number was largely increased compared with sham kidneys. Blocking PKCα signaling activation by Go6976 diminished the accumulation of F4/80-positive inflammatory cells in the UUO kidneys (Fig. 7, A and B). The ly6b-positive cell accumulation was also diminished in the Go6976-treated group compared with the vehicle-treated group, although the CD3-positive cell accumulation was not altered. We then determined the mRNA abundance of several proinflammatory cytokines, including TNFα, monocyte chemotactic protein-1 (MCP1), and RANTES (regulation on activation normal T cell expressed and secreted) in kidney tissues by real-time PCR assay. As shown in Fig. 7C, on day 14 after UUO, TNFα, MCP1, and RANTES mRNA abundances were markedly up-regulated. Administration of Go6976 could significantly down-regulate RANTES, TNFα, and MCP1 mRNA expression.

Mice

Male CD1 mice aged 6–8 weeks and weighing ∼18–20 g were acquired from the Specific Pathogen-Free Laboratory Animal Center of Nanjing Medical University and were maintained according to the guidelines of the Institutional Animal Care and Use Committee at Nanjing Medical University. UUO was performed as reported previously. The animals were divided into three groups: 1) sham control; 2) UUO mice treated with vehicle (5% DMSO); and 3) UUO treated with Go6976. Mice were treated with Go6976 (2 mg/kg·day) from 2 days before UUO surgery to 1 or 2 weeks after surgery. Go6976 (catalog no. S7119, Selleck, Boston) was prepared in a 5% DMSO solution and injected into mice intraperitoneally. The mice were euthanized on day 7 or 14 after UUO. The UUO as well as the contralateral kidneys were removed. One portion of the kidney was fixed in 10% phosphate-buffered formalin followed by paraffin embedding for histological and immunohistochemical staining. Another portion was immediately frozen in Tissue-Tek optimum cutting temperature compound (Sakura Finetek, Torrance, CA) for cryosection. The remaining kidney tissue was snap-frozen in liquid nitrogen and stored at −80 °C for extraction of RNA and protein. All experiments were performed in accordance with the approved guidelines and regulations of the Animal Experimentation Ethics Committee at Nanjing Medical University.

Cell culture

NRK-49F cells were obtained from American Type Culture Collection (Manassas, VA). Cells were cultured in Dulbecco's modified Eagle's medium/F-12 medium supplemented with 10% fetal bovine serum (Invitrogen). The cells were seeded on 6-well culture plates to 60–70% confluence in complete medium containing 10% fetal bovine serum for 16 h and then changed to serum-free medium after washing twice with serum-free medium. Recombinant human TGFβ1 (catalog no. 100-B-010-CF, R&D Systems, Minneapolis, MN) was added to the serum-free medium for various periods of time. Go6976 (catalog no. ab141413, Abcam) or CQ (catalog no. C6628, Sigma) dissolved in DMSO or 3-MA (catalog no. s2767, Selleck) dissolved in double-distilled H₂O was added at 30 min before TGFβ1 stimulation. PKCα siRNA (GenePharma, Shanghai, China) and RFP-LC3 plasmid DNA was transfected into NRK-49F cells using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's instruction.

Histology and immunohistochemistry

Mouse kidney samples were fixed in 10% neutral formalin and embedded in paraffin. Three-μm thickness sections were used for PAS, Masson, and Sirius red staining. Kidney tubular injury was scored by percentage of morphological changes in the tubule, such as dilation, distortion of tubular basement membranes, and atrophy as follows: 0 = normal; 1 = <10%; 2 = 10–25%; 3 = 26–50%; 4 = 51–75%, and 5 = >75%. Ten random fields per section within the cortical area were selected for counting. For immunohistochemical staining, paraffin-embedded kidney sections were processed as per routine protocol. Sections were blocked with 10% normal donkey serum, followed by incubation with anti-Ly-6b (catalog no. MCA771G, AbD Serotec, Raleigh, NC) overnight at 4 °C. After incubation with secondary antibody for 1 h at room temperature, sections were incubated with ABC reagents for 1 h at room temperature before subjected to 3,3′-diaminobenzidine staining (Vector Laboratories, Burlingame, CA). Slides were viewed with a Nikon Eclipse 80i microscope equipped with a digital camera (DS-Ri1, Nikon, Shanghai, China).

Immunofluorescent staining

Three-μm thickness kidney cryosections were fixed for 15 min in 4% paraformaldehyde, followed by permeabilization with 0.2% Triton X-100 in 1× phosphate-buffered saline (PBS) for 5 min at room temperature. After blocking with 2% donkey serum for 60 min, the slides were immunostained with the following: anti-FN (catalog no. F3648, Sigma); anti-α-SMA (catalog no. A5228, Sigma); anti-p-PKCα (Thr-638/641) (catalog no. 9375, Cell Signaling Technology); anti-LAMP2 (catalog no. L0668, Sigma); anti-SQSTM1/p62 (catalog no. ab56416, Abcam); anti-LC3B (catalog no. L7543, Sigma); and anti-laminin (catalog no. ab44941, Abcam). Cells cultured on coverslips were washed twice with cold 1× PBS and fixed with cold methanol/acetone (1:1) for 10 min at −20 °C. After three times of extensive washing with 1× PBS, the cells were treated with 0.1% Triton X-100 for 5 min, blocked with 2% normal donkey serum in 1× PBS buffer for 40 min at room temperature, and incubated with antibodies against p-PKCα, FN, α-SMA, LAMP2, SQSTM1/p62, or LC3 followed by staining with FITC or tetramethylrhodamine-conjugated secondary antibody. Cells were also stained with 4′,6-diamidino-2-phenylindole to visualize the nuclei. Slides were viewed with a Nikon Eclipse 80i epi-fluorescence microscope equipped with a digital camera or Zeiss LSM710 (Zeiss).

Assessment of kidney fibrosis

Briefly, kidney sections (3 μm thickness) were stained with a Masson Trichrome kit (catalog no. HT15–1KT; Sigma) according to the manufacturer's instruction. Images were taken from 10 fields of one section under high magnification (×400) with a combination of cortex and medulla (including cortex and medulla with six and four fields selected, respectively). The percentage of interstitial fibrotic area relative to the selected field was analyzed with Image Pro Plus 6.0 software. An average percentage of kidney fibrotic area for each section was calculated.

Western blot analysis

Cultural NRK-49F cells were lysed in 1× SDS sample buffer. The kidneys were lysed with radioimmunoprecipitation assay solution containing 1% Nonidet P-40, 0.1% SDS, 100 mg/ml phenylmethanesulfonyl fluoride, 1% protease inhibitor mixture, and 1% phosphatase I and II inhibitor mixture (Sigma) on ice. The supernatants were collected after centrifugation at 13,000 × g at 4 °C for 30 min. The primary antibodies were as follows: anti-p-PKCα (Thr-638/641) and anti-PKCα (catalog no. 2056, Cell Signaling Technology); anti-p-Akt (Ser-473) (catalog no. 3868, Cell Signaling Technology); anti-Akt (catalog no. 4691, Cell Signaling Technology); anti-β-actin (C4) (catalog no. sc-47778, Santa Cruz Biotechnology, Dallas, TX); and anti-LAMP2, anti-SQSTM1/p62, anti-LC3B, anti-FN, anti-α-SMA, and anti-tubulin (catalog no. sc53646, Santa Cruz Biotechnology). Quantification was performed by measuring the intensity of the signals with the aid of ImageJ software package (National Institutes of Health).

Lipid droplet clearance assay

NRK-49F cells grown on coverslips of 6-well culture plates to 60–70% confluence and then changed to serum-free medium with Go6976 or CQ for 4 h. Lipid droplets were stained with BODIPY493/503 (2 μg/ml) for 30 min prior to the time of examination. Slides were viewed with a Nikon Eclipse 80i epi-fluorescence microscope equipped with a digital camera.

NAG assay

NAG assays were performed using a kit from Nanjing Jiancheng Bioengineering Institute (catalog no. A031, Nanjing, China), based on the principle that NAG hydrolyzes the substrate to generate free p-nitrophenol that can be measured colorimetrically at 400 nm following ionization at basic pH. Briefly, NRK-49F cells treated with Go6976 (5 μm) or CQ (50 μm) for 4 h or transfected with PKCα or Scramble siRNA for 24 h were lysed in RIPA buffer (100 μl). Ten micrograms of cell lysates from each sample were normalized to equal volume and measured in quadruplicate for NAG activity following the protocol provided by the supplier.

EM assay

Lysosomes and autophagosomes and/or autolysosome structures were evaluated by EM. NRK-49F cells were seeded in 6-well plates. Go6976 (5 μm) was added to the wells and then treated with or without TGFβ1. After 4 h, the cells were collected and fixed with pentanediol and observed by transmission electron microscopy (Tecnai G2 Spirit Bio TWIN).

RNA isolation and real-time quantitative reverse transcriptase-PCR

Total RNA was extracted using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. cDNA was synthesized with 1 μg of total RNA, ReverTra Ace (Vazyme, Nanjing, China), and oligo(dT)_12–18 primers. Gene expression was measured by real-time PCR assay (Vazyme) and the 7300 real-time PCR system (Applied Biosystems, Foster City, CA). The relative amount of mRNA or gene to internal control was calculated using the equation 2ΔCT, in which ΔCT = CT_gene − CT_control.

Assessment of lysosome acidification

To assess lysosome acidification, we used LysoSensor Yellow/Blue DND-160 (PDMPO) (Invitrogen). We diluted the 1 mm probe stock solution in the 1 μm working concentration in the growth medium. When NRK-49F cells reached the desired confluence, we removed the medium from the dish and added the pre-warmed (37 °C) probe-containing medium. We incubated the cells for 30 min under growth conditions and then replaced the loading solution with fresh medium, and the fluorescence images were collected at the wavelength range from 510 to 641 nm (yellow) and at the wavelength range from 404 to 456 nm (blue) with the Zeiss LSM710 (Zeiss). The mean fluorescence intensity in the fluorescence-positive area was measured using ImageJ, for yellow and blue fluorescence, respectively. The blue/yellow ratio was calculated by division process in each section. The average blue/yellow ratio of a given sample was calculated from five sections. Intensity was measured using at least three independent experiments.

Statistical analysis

All data examined are presented as mean ± S.E. Statistical analyses of the data were performed using the SigmaStat software (Jandel Scientific Software, San Rafael, CA). Comparison between groups was made using one-way analysis of variance, followed by the Student-Newman-Keuls test. p < 0.05 was considered statistically significant.