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

Kidney fibrosis is a histological hallmark of chronic kidney disease and arises in large part through extracellular matrix deposition by activated fibroblasts. The signaling protein complex mTOR complex 2 (mTORC2) plays a critical role in fibroblast activation and kidney fibrosis. Protein kinase Cα (PKCα) is one of the major sub-pathways of mTORC2, but its role in fibroblast activation and kidney fibrosis remains to be determined. Here, we found that transforming growth factor β1 (TGFβ1) activates PKCα signaling in cultured NRK-49F cells in a time-dependent manner. Blocking PKCα signaling with the chemical inhibitor Go6976 or by transfection with PKCα siRNA largely reduced expression of the autophagy-associated protein lysosomal-associated membrane protein 2 (LAMP2) and also inhibited autophagosome–lysosome fusion and autophagic flux in the cells. Similarly to chloroquine, Go6976 treatment and PKCα siRNA transfection also markedly inhibited TGFβ1-induced fibroblast activation. In murine fibrotic kidneys with unilateral ureteral obstruction (UUO) nephropathy, PKCα signaling is activated in the interstitial myofibroblasts. Go6976 administration largely blocked autophagic flux in fibroblasts in the fibrotic kidneys and attenuated the UUO nephropathy. Together, our findings suggest that blocking PKCα activity may retard autophagic flux and thereby prevent fibroblast activation and kidney fibrosis.

Kidney fibrosis is a histological hallmark of chronic kidney disease and arises in large part through extracellular matrix deposition by activated fibroblasts. The signaling protein complex mTOR complex 2 (mTORC2) plays a critical role in fibroblast activation and kidney fibrosis. Protein kinase C␣ (PKC␣) is one of the major sub-pathways of mTORC2, but its role in fibroblast activation and kidney fibrosis remains to be determined. Here, we found that transforming growth factor ␤1 (TGF␤1) activates PKC␣ signaling in cultured NRK-49F cells in a time-dependent manner. Blocking PKC␣ signaling with the chemical inhibitor Go6976 or by transfection with PKC␣ siRNA largely reduced expression of the autophagy-associated protein lysosomalassociated membrane protein 2 (LAMP2) and also inhibited autophagosome-lysosome fusion and autophagic flux in the cells. Similarly to chloroquine, Go6976 treatment and PKC␣ siRNA transfection also markedly inhibited TGF␤1-induced fibroblast activation. In murine fibrotic kidneys with unilateral ureteral obstruction (UUO) nephropathy, PKC␣ signaling is activated in the interstitial myofibroblasts. Go6976 administration largely blocked autophagic flux in fibroblasts in the fibrotic kidneys and attenuated the UUO nephropathy. Together, our findings suggest that blocking PKC␣ activity may retard autophagic flux and thereby prevent fibroblast activation and kidney fibrosis.
Kidney fibrosis is one of the histological hallmarks of chronic kidney diseases (1,2). Although many cell types are involved in kidney fibrosis, fibroblast is the major cell type that contributes to interstitial myofibroblast accumulation as well as excessive extracellular matrix (ECM) 3 deposition (3). Deciphering the molecular mechanisms that regulate the conversion of fibroblast into ECM-producing myofibroblasts and exploring the efficient therapeutic strategy for retarding kidney fibrosis are necessary.
Autophagy is an important degradation system to maintain cellular homeostasis via the formation of autophagosomes followed by autolysosomes (4), which may be involved in regulating many cellular functions, including cell survival, differentiation, and metabolism (5,6). Microtubule-associated protein 1 light chain 3 (LC3) is the most widely monitored autophagyrelated protein that functions as a structural component in the formation of autophagosomes (7). LC3B is the best characterized form and the most widely used as an autophagic marker. The conversion of the cytosolic form of LC3B (LC3B-I) to the lipidated form (LC3B-II) indicates autophagosome formation. LC3B-binding protein SQSTM1/p62 (herein referred to as p62) regulates the formation of protein aggregates and is removed by autophagy, thus serving as an index of autophagic degradation (8,9). Lysosomal membrane proteins (LMPs) are involved in lysosomal acidification, cytoplasmic protein import, membrane fusion, and degraded product transportation to the cytoplasm (10). The best-known and most abundant LMPs are the lysosome associated proteins 1 (LAMP-1) and 2 (LAMP-2), which are both involved in maintaining lysosomal membrane integrity and phagolysosome formation (11,12).
Evidence is mounting that dysregulation of autophagy is implicated in the pathogenesis of various types of renal disease such as glomerulosclerosis, diabetic nephropathy, acute kidney injury, and kidney cystic disease (13). It has been well demonstrated that autophagy is induced in the obstructed renal tubule after UUO (14). Also, TGF␤1 may activate autophagy in cultured renal tubular and mesangial cells (15)(16)(17). Several studies found that activation of autophagy inhibits kidney fibrosis through promoting collagen degradation (16,18). Baisantry et al. (19) demonstrated that specific ablation of Atg5 from the proximal tubule exacerbated ischemic acute kidney injury at earlier time points but suppressed renal fibrosis during kidney recovery or repair. Furthermore, Livingston et al. (20) found that obstructive nephropathy was ameliorated in renal proxi-mal tubule Atg7-knockout mice, which was associated with the less production of pro-fibrotic cytokines regulated by autophagy (21). Autophagy is a highly dynamic, multistep process and is modulated at many levels. Among them, activation of mTORC1, a serine/threonine protein kinase, may suppress autophagy in many cell types (22,23). Our previous studies found that Rictor/mTORC2 promoted kidney tubular cell survival through induction of autophagy (24). However, whether PKC␣, one of the important downstream targets of mTORC2, can regulate autophagy in kidney fibroblast and its contribution to kidney fibrosis remains largely unknown.
In this study, we found that PKC␣ signaling activation mediates TGF␤1-induced fibroblast activation and contributes to kidney fibrosis by promoting autophagic flux. Go6976, a specific PKC␣ signaling inhibitor, may act as a novel therapeutic strategy for retarding kidney fibrosis.

Inhibition of PKC␣ reduces LAMP2 expression and induces lysosomal dysfunction in NRK-49F cells
To investigate the role of PKC␣ signaling in fibroblast activation, NRK-49F cells, a rat kidney interstitial fibroblast cell line, were treated with TGF␤1 (2 ng/ml) for different time points as indicated. As shown in Fig. 1A, the amount of p-PKC␣ and p-Akt (Ser-473) was increased at 4 h and maintained at a high level within 24 h after TGF␤1 treatment. NRK-49F cells were pretreated with the PKC␣ inhibitor Go6976 for 30 min, followed by TGF␤1 incubation, and harvested at 4 h and 8 h later. Western blotting analyses revealed that the abundance of p-PKC␣ 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 TGF␤1 treatment alone (Fig. 1B).
PKC␣ 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 M) 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 TGF␤1 treatment. The Western blotting analyses revealed that LAMP2 abundance was significantly decreased after Go6976 treatment (Fig. 1E). To further investigate the role of PKC␣ in regulating lysosomal function, NRK-49F cells transfected with scrambler or PKC␣ siRNA were treated with TGF␤1 for 4 h. PKC␣ siRNA transfection could markedly down-regulate PKC␣ mRNA expression, whereas the other isoforms of PKC remained unchanged. As shown in Fig. 1F, PKC␣ siRNA transfection markedly down-regulates LAMP2 protein expression. Immunofluorescent staining further confirmed Western blotting results (Fig. 1G). The lysosomal protease activity, measured by ␤-N-acetylglucosaminidase (NAG) assays, was not affected by Go6976 or PKC␣ 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 Lyso-Sensor (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 colocalization of the yellow/blue/autophagosome was inhibited in NRK-49F cells after CQ and Go6976 treatment (Fig. 1J).

Inhibition of PKC␣ 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 TGF␤1 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 PKC␣ signaling in regulating autophagic flux, NRK-49F cells were transfected with scramble and PKC␣ siRNA. Western blotting analyses revealed that PKC␣ 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 TGF␤1 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 TGF␤1 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 PKC␣ siRNA compared with those transfected with scrambler siRNA with or without TGF␤1 treatment. In NRK-49F cells, Go6976 but not CQ administration obviously inhibited the clearance of lipid droplets (Fig. 2H). Together, these results demonstrate that PKC␣ signaling is indispensable for maintaining autophagic flux in NRK-49F cells.

Blockade of PKC␣ signaling diminishes TGF␤1-induced fibroblast activation
We then treated NRK-49F cells with the PKC␣ inhibitor Go6976, followed 30 min later by TGF␤1 treatment for 24

PKC␣ activation promotes kidney fibrosis
and 48 h. Go6976 could remarkably inhibit TGF␤1-induced fibronectin (FN) and ␣-smooth muscle actin (␣-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 PKC␣ siRNAs as indicated. PKC␣ siRNA transfection could markedly down-regulate PKC␣ protein and mRNA expression (Fig. 3C). Down-regulation of PKC␣ could markedly reduce TGF␤1-induced FN and ␣-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 TGF␤1-induced FN and ␣-SMA expression (Fig. 3, E and F). Thus, it is clear that PKC␣ signaling mediates TGF␤1induced fibroblast activation through autophagy induction.

Blockade of PKC␣ inhibits autophagic flux in the interstitial myofibroblasts from UUO kidneys
Western blotting assays revealed that phosphorylated protein kinase C␣ (p-PKC␣) was increased at day 3 and reached peak at days 7 and 14 after UUO (Fig. 4A). Go6976 markedly decreased the abundance for p-PKC␣ at day 7 after UUO ( Fig.  4B). Immunofluorescent staining confirmed the results of Western blotting assay (Fig. 4D).

Figure 1. Inhibition of PKC␣ reduces LAMP2 expression and induces lysosomal dysfunction in NRK-49F cells.
NRK-49F cells were treated with TGF␤1 (2 ng/ml) with or without the PKC␣ inhibitor Go6976 for different times as indicated. A, Western blotting analyses show the induction of PKC␣ and Akt phosphorylation after TGF␤1 treatment in a time-dependent manner. Cell lysates were immunoblotted with Abs against p-Akt (Ser-473), p-PKC␣, and actin, respectively. B, Western blotting analyses revealing the remarkable reduction of PKC␣ but not Akt or Smad3 phosphorylation after TGF␤1 and Go6976 treatment compared with TGF␤1 treatment alone. Cell lysates were immunoblotted with Abs against p-PKC␣, p-Akt (Ser-473), Akt, p-Smad3, and actin, respectively. C, representative micrographs show the immunostaining for p-PKC␣ at 8 h after TGF␤1 (2 ng/ml) without Go6976 (5 M) treatment. Cells were co-stained with DAPI to visualize the nuclei; scale bar, 5 m. D, NRK-49F cells were treated with Go6976 for different time as indicated. Western blotting assay shows the down-regulation of LAMP2 expression after Go6976 treatment in a time-dependent manner. E, Western blotting analyses reveal the LAMP2 protein expression after TGF␤1 treatment and PKC␣ inhibitor Go6976 treated for different times or dosages as indicated. F, Western blotting analyses showing that knocking down PKC␣ reduced LAMP2 protein expression in NRK-49F cells. Cell lysates were immunoblotted with Abs against LAMP2 and actin, respectively. G, representative images showing the immunostaining for LAMP2 after various treatments as indicated. Cells were co-stained with DAPI to visualize the nuclei;

PKC␣ activation promotes kidney fibrosis
In NRK-49F cells, we found that PKC␣ 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 vehi- ; ###, p Ͻ 0.01 compared with Go6976-treated cells. F, representative images for SQSTM1/p62 and LAMP2 among different groups as indicated. Yellow dots in merged images represent colocalization of SQSTM1/ p62 and LAMP2. Cells were co-stained with DAPI to visualize the nuclei; scale bar, 5 m. Quantitative analysis for SQSTM1/p62 puncta/cell and merged dots/p62 in NRK-49F cells after various treatments is as indicated (lower part). *, p Ͻ 0.05; **, p Ͻ 0.01; ***, p Ͻ 0.001 compared with vehicle (Veh)-treated cells (n ϭ 5-7); #, p Ͻ 0.05 compared with cells treated with Go6976. G, representative images for p62 and LAMP2 among different groups as indicated. Yellow dots in merged images represent colocalization of p62 and LAMP2. Cells were co-stained with DAPI to visualize the nuclei; scale bar, 5 m. Quantitative analysis for p62 puncta/cell and merged dots/p62 in NRK-49F cells after various treatments as indicated (lower part). **, p Ͻ 0.01; ***, p Ͻ 0.001 compared with vehicle-treated cells (n ϭ 5-7). H, lipid droplet clearance assays were performed with vehicle, Go6976, or CQ. Representative images of BODIPY staining of lipid droplets in NRK-49F cells at 4 h post-treatment with vehicle, Go6976, or CQ; scale bar, 5 m. Quantification of BODIPY staining. n ϭ 3 independent experiments. ***, p Ͻ 0.001 compared with vehicle-treated cells (n ϭ 5-7); ###, p Ͻ 0.01 compared with Go6976-treated cells.

PKC␣ activation promotes kidney fibrosis
cle (Fig. 4D). These results suggest blocking PKC␣ 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 PKC␣ signaling ameliorates UUO nephropathy in mice
To further explore the role of PKC␣ signaling in kidney fibrosis in vivo, we created a mouse model with kidney fibrosis by UUO in male CD1 mice, and Go6976, a specific PKC␣ 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 ␣-SMA and FN expression by Western blotting analyses. FN and ␣-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 PKC␣ 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 TNF␣, 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, TNF␣, MCP1, and RANTES mRNA abundances were markedly up-regulated. Administration of Go6976 could significantly down-regulate RANTES, TNF␣, and MCP1 mRNA expression.

Discussion
In cultured NRK-49F cells, TGF␤1 treatment could induce both mTORC1 and mTORC2 signaling activation at a similar pattern, and blocking either one of them could partially interfere with TGF␤1-induced fibroblast activation (26). Our previous studies found that mTORC2/Akt signaling is involved in TGF␤1-induced fibroblast activation and kidney fibrosis (27). In addition to Akt, we demonstrated here that mTORC2/PKC␣ signaling activation may also contribute to TGF␤1-induced fibroblast activation and kidney fibrosis by promoting autophagic flux.
A growing body of evidence showed that autophagy induction may be an adaptive response against various stress stimuli and linked with the fibrotic diseases. Dysregulated autophagy has been implicated in disorders characterized by fibrosis in various tissues, including idiopathic pulmonary fibrosis, liver fibrosis, cardiac fibrosis, and renal fibrosis (28 -30). Autophagy may either promote kidney fibrosis via the induction of tubular atrophy and decomposition or attenuate kidney fibrosis via the intracellular degrading of excessive collagen. In the UUO rat model, autophagy was induced in a time-dependent manner, and inhibition of autophagy by 3-MA enhanced tubular cell

PKC␣ activation promotes kidney fibrosis
apoptosis and tubule-interstitial fibrosis in the obstructed kidney (14). Collagen deposition was increased in the kidneys of mice deficient in the autophagic protein beclin1 as compared with littermate mice. Genetic disruption of beclin1 led to increased type I collagen abundance in primary mouse mesan-gial cells (MMC) (16). Ding et al. (18) found that deletion of LC3B (LC3 Ϫ/Ϫ mice) resulted in increased collagen deposition and beclin1 heterozygous (beclin1 ϩ/Ϫ ) mice also displayed increased collagen deposition in the obstructed kidneys after UUO. However, other researchers found that autophagy induc-

PKC␣ activation promotes kidney fibrosis
tion may promote fibrogenesis. Livingston et al. (20) reported that persistent activation of autophagy in kidney proximal tubules promotes UUO nephropathy in mice. Baisantry et al. (19) found that lacking autophagy in proximal tubular S3 segments attenuates early survival mechanisms but prevents subsequent maladaptive repair and the development of chronic kidney diseases in mice. In addition, Hernandez-Gea et al. (29) found that animals with HSC-specific deletion of Atg7 display attenuated activation following liver injury, leading to reduced fibrosis in vivo. Consistently, in this study we found that the inhibition of autophagy with chloroquine could decrease NRK-49F cell activation after TGF␤1 treatment, suggesting a profibrotic role for autophagy induction in kidney fibroblasts.
The kinase mTOR is a critical regulator of autophagy. There is a tight, inverse coupling of autophagy induction and mTORC1 activation (31). Autophagy induction by genetic or pharmacologic inhibition of mTORC1 (TORC1 in yeast) was first demonstrated in yeast (32). In mammalian cells, mTORC1 and AMP-activated protein kinase regulate by inhibiting activation of UKL1, an autophagy-initiating UNC-5-like autophagy-activating kinase (ULK) complex (22). In addition, another layer of ULK1 regulation by mTORC1 has been suggested in which mTORC1 inhibits ULK1 stability by inhibitory phosphorylation of autophagy/beclin1 regulator 1 (AMBRA1) (33). Furthermore, mTORC1 also regulates autophagy at the transcriptional level by modulating localization of transcription factor EB (TFEB), a mas-

PKC␣ activation promotes kidney fibrosis
ter transcriptional regulator of lysosomal and autophagy genes (34). However, how mTORC2 regulates autophagy is not fully understood. MTORC2 phosphorylates AKT (Ser-473), which can lead to the activation of the AKT/mTORC1 signaling axis (35). Therefore, mTORC2 may indirectly suppress autophagy by activating mTORC1. Our previous study found that Rictor/mTORC2 has a critical role in autophagy induction in kidney tubular cells (22).
Recent studies reported conflicting regulation of autophagy by protein kinase C (PKC) signaling. It has been shown that activation of PKC suppressed starvation-or rapamycin-induced autophagy, whereas PKC inhibitors dramatically induced autophagy (36). Deleting the Prkc␣ gene, which encodes PKC␣, reverses diabetes-induced autophagy impairment, and PKC␣ increases the expression of miR-129-2, which is a negative regulator of autophagy and mediates the inhibitory effect of maternal diabetes on autophagy (37). Other researchers found that PKC activation induced the Epstein-Barr virus lytic cycle through the activation of p38 MAPK and autophagy induction, which resulted in a prosurvival effect, as indicated by p38 or ATG5 knockdown experiments (38). Tan et al. (39) found that inhibition of classical PKC isoforms (PKC␣) was able to effectively suppress palmitic acid-induced autophagy, which found to be independent of mTOR regulation. In agreement with the positive regulation of PKC␣ on autophagy, our results showed that the PKC␣-signaling pathway may promote autophagy flux in kidney fibroblast.
Lysosomes are degradation and signaling centers that coordinate cellular metabolism with clearance (34,40). Activated PKC␣ and PKC␦ cause inactivation of GSK3␤, which in turn causes TFEB nuclear translocation and then promotes lysosome biogenesis (25). In agreement with the positive effect of PKC␣ on lysosome, our findings in cultured cells demonstrated that blocking PKC␣ activity decreased the abundance of LAMP-2, a principal lysosomal membrane protein. Lysosomal dysfunction leads to lower autophagic clearance, resulting in autophagosome accumulation (41). In this study, the impaired autophagosome-lysosome fusion after PKC␣ inhibition may due to the lysosomal dysfunction. It was previously shown that mutation of LAMP-2 causes X-linked vacuolar cardiomyopathy and myopathy (also called Danon disease) (42). In our study, transmission electron microscopy showed that lysosomal accumulation was observed after PKC␣ inhibitor treatment. However, how PKC␣ regulates LAMP2 expression remains to be further determined.
In conclusion, this study demonstrated that PKC␣ signaling contributes to TGF␤1-induced fibroblast activation and development of kidney fibrosis by promoting autophagy. Targeting this signaling pathway may inhibit kidney fibrosis in patients with chronic kidney diseases.

Mice
Male CD1 mice aged 6 -8 weeks and weighing ϳ18 -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/kg⅐day) 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 Ϫ80°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 TGF␤1 (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 H 2 O was added at 30 min before TGF␤1 stimulation. PKC␣ 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-m 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 ϭ normal; 1 ϭ Ͻ10%; 2 ϭ 10 -25%; 3 ϭ 26 -50%; 4 ϭ 51-75%, and 5 ϭ Ͼ75%. 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 3,3Ј-diaminobenzidine staining (Vector Laboratories, Burlingame, CA). Slides were viewed with a

Assessment of kidney fibrosis
Briefly, kidney sections (3 m 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 (ϫ400) 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.

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 g/ml) for 30 min prior to the time of examination. Slides were viewed with a Nikon Eclipse 80i epifluorescence 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 M) or CQ (50 M) for 4 h or transfected with PKC␣ or Scramble siRNA for 24 h were lysed in RIPA buffer (100 l). 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 M) was added to the wells and then treated with or without TGF␤1. 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 g of total RNA, ReverTra Ace (Vazyme, Nanjing, China), and oligo(dT) [12][13][14][15][16][17][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 2⌬CT, in which ⌬CT ϭ CT gene Ϫ 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 M 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 Ϯ 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 Ͻ 0.05 was considered statistically significant.