All animal experiments were approved by the Animal Care and Use Committee of Shanghai Jiao Tong University (Shanghai, China) and followed the Guidelines of the Care and Use of Laboratory Animals issued by the Chinese Council on Animal Research (project no. 2015DW001). Specifically, male Sprague-Dawley rats (200-250 g body weight) were purchased from the Chinese Academy of Sciences, bred, and maintained at the Animal Center Laboratory of Shanghai General Hospital. A total of 2 mice were housed per cage, under controlled temperature (22-25°C) and humidity (70%) conditions, along with a 12-h light/dark cycle, and the provision of free access to water. Rats were randomly assigned to either the 5/6 nephrectomized or the sham-operated control group (n=8 per group), and received surgical procedures according to a previous study (31). Briefly, the rats were anesthetized by an intraperitoneal injection of sodium pentobarbital (40 mg/kg) and received the 5/6 nephrectomy by surgical resection of the upper and lower thirds of the left kidney followed by right nephrectomy 7 days later. In the control group, only a sham operation was performed. During the experimental period, the animals were monitored two to three times per week for potential signs of suffering, mainly a weight loss >20% and significant changes in their behavior, mobility or body posture. In the case that the rats met one of these criteria, euthanasia would have been warranted on the same day in order to prevent further suffering. Animals were kept for 6 months. One CKD rat died in the late experimental period, and all control rats survived, similar to previous studies (32,33). Following adequate anesthesia with sodium pentobarbital (40 mg/kg) intraperitoneal injection, the bodyweight of each rat was recorded and blood samples were then collected through cardiac puncture, in order to measure creatinine, blood urea nitrogen (BUN), albumin (Alb), total cholesterol (TC) and triglyceride (TG) serum levels. Gastrocnemius muscle samples were collected and used to measure muscle wet weight, perform reverse transcription-quantitative PCR (RT-qPCR) and western blot analyses. Finally, the rats were sacrificed by decapitation immediately after the experimental procedure when the rats were still under deep anesthesia. Death was further confirmed by checking for the onset of rigor mortis (34).

Gastrocnemius muscle samples were fixed in 4% freshly made paraformaldehyde and processed for embedding in paraffin and sectioning. Tissue sections at a thickness of 4 µm were stained with H&E (Wuhan Servicebio Technology Co., Ltd.) at room temperature for 1-3 min, using standard procedures and observed under a light microscope (DFC550; Leica Microsystems, Inc.). Cross-sectional areas (CSAs) were analyzed using Image-Pro Plus 6.0 software (Media Cybernetics, Inc.) after counting at least 100 muscle fibers per muscle.

Rat L6 myoblasts were purchased from The Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (GNR 4), and were passaged according to previous studies (35,36). L6 cells were seeded in 6-well plates (1×10⁵ cells per well) in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.), supplemented with 10% fetal bovine serum [(FBS); (Gibco; Thermo Fisher Scientific, Inc.)] and 1% penicillin/streptomycin. Afterwards, the L6 myoblasts were incubated in a humidified incubator with 5% CO₂ at 37°C overnight. Then cell culture medium was replaced with DMEM containing only 2% horse serum to initiate differentiation for 48 h.

To mimic the conditions of CKD in rats and patients in vivo, L6 myoblasts were exposed to four different inflammatory agents [2 ng/ml IL-6; (400-06, Peprotech), 2 ng/ml TNF-α (400-14, Peprotech), 2 ng/ml INF-γ (400-20, Peprotech) and 10 ng/ml LPS (SMB00610, Sigma-Aldrich; Merck KGaA)] according to a previous study (30), and the CAPN6 expression levels were evaluated.

Plasmids carrying CAPN6 shRNA (hU6 -MCS-SV40-Neomycin-CAPN6 RNAi and CAPN6^−/−) and shRNA negative control were purchased from GeneChem, Inc. and the CAPN6 CRISPR activation plasmid (cat. no. sc-437325-ACT) and the control CRISPR activation plasmid (cat. no. sc-437275) were obtained from Santa Cruz Biotechnology, Inc. Plasmids carrying Raptor shRNA (phb-u6-mcs-pgk-puro-Raptor RNAi and Raptor^−/−) and shRNA negative control were purchased from Hanbio Biotechnology Co., Ltd. For cell transfection, L6 cells were grown overnight and transiently transfected at room temperature using OptiMEM medium with Lipofectamine 2000^® (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions. The final concentration of the plasmids was 2 ng/µl. Following 6 h of transfection at 37°C, treatments were applied to the cells.

Red and green fluorescent protein-tagged microtubule-associated protein 1 light chain 3 (RFP-GFP-LC3) adenoviral vectors (Hanbio Biotechnology Co., Ltd.) was used to transfect the L6 cells for the measurement of autophagy levels, using OptiMEM medium with Lipofectamine 2000^® (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The final concentration of the plasmids was 10 µl/ml. The transfection efficiency was inspected with a Leica Microsystems GmbH confocal microscope (Leica DM6000B). The RFP-GFP-LC3 levels in transfected cells was also visualized using a Leica Microsystems GmbH confocal microscope (Leica TCS SP8) and quantified using Image-Pro plus 6.0 (Media Cybernetics, Inc.).

Total cellular RNA was isolated from the tissues and cells using TRIzol^® reagent (cat. no. 15596-026; Invitrogen; Thermo Fisher Scientific, Inc.) and reverse transcribed into cDNA using the High Capacity cDNA Reverse Transcription kit (cat. no. K1622; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols. qPCR was performed using SYBR-Green (cat. no. F-415XL; Thermo Fisher Scientific, Inc.) in an ABI PRISM Sequence Detector System 7300 (Applied Biosystems; Thermo Fisher Scientific, Inc.). Conditions were set to initial 95°C for 10 min, then 40 cycles of 95°C for 15 sec and 60°C for 60 sec. Each experiment was performed in triplicate and repeated three times. The CAPN6 relative mRNA expression levels were calculated using the 2^−ΔΔCq method (37). The primers used were as follows: CAPN6 forward, 5′-TGT TTG GCT GTT CAG GAG TC-3′ and reverse, 5′-TGG GAA GCA AGT CGT CAA TC-3′; and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) forward, 5′-GTC GGT GTG AAC GGA TTT G-3′ and reverse, 5′-TCC CAT TCT CAG CCT TGA C-3′.

Tissue and cell samples were pulverized and lysed in the radioimmunoprecipitation assay (RIPA) buffer (Beyotime Institute of Biotechnology) at 4°C for 2 h. The lysates were centrifuged at 10,000 × g at 4°C for 10 min to collect the supernatant into separate tubes. The quality and concentration of protein were detected using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Inc.). Protein samples (20 µg) were separated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electro-transferred onto nitrocellulose membranes (EMD Millipore). The membranes were incubated in 5% skim milk in phosphate-buffered saline (PBS)-Tween 20 (PBS-T) at room temperature for 1 h, and subsequently with primary antibodies at 4°C overnight. The following primary antibodies used were purchased from Cell Signaling Technology, Inc.: anti-LC3 (1:1,000, cat. no. 2775), GAPDH (1:2,000, cat. no. 5174), phosphorylated (p-)eukaryotic translation initiation factor 4E binding protein 1 (4EBP-1; 1:1,000, cat. no. 2855), 4EPB-1 (1:1,000, cat. no. 9452); p-p70s6k1 (1:1,000, cat. no. 9234), p70s6k1 (1:1,000, cat. no. 9202) and (mTOR; 1:1,000, cat. no. 2983). Anti-CAPN6 (1:1,000, cat. no. ab76974) and Raptor (1:3,000, cat. no. ab40768) antibodies from Abcam were also used. GAPDH antibody was used as a loading control for the muscle tissues. Afterwards, the membranes were washed three times with PBS-T, then incubated with horseradish peroxidase-conjugated secondary anti-rabbit IgG (1:1.000, cat. no. A0208; Beyotime Institute of Biotechnology), or anti-mouse IgG (1:1.000, cat. no. A0216; Beyotime Institute of Biotechnology), for 1 h at room temperature. Positive protein bands were visualized using an enhanced chemiluminescence (ECL) substrate kit (cat. no. WBKLS0100; EMD Millipore), exposed to X-ray films and quantified using Tanon-5200 software (Tanon Science & Technology Co., Ltd.).

First, cell slides were washed with PBS (0.02 M) to remove the culture medium, and fixed with 4% formaldehyde for 30 min. Subsequently, the slides were washed three times for 3 min each with PBS (0.02 M) prior to permeabilization with Triton X-100 (0.5%) for 10 min. Consequently, the slides were washed three times with PBS (0.02 M), for 3 min each. Cells were blocked with 1% BSA for 1 h and washed with PBS (0.02 M) for 3 min, 3 times. Diluted primary antibodies were added drop by drop to each cell slide, and the slides were then placed in a wet box and incubated at 4°C overnight. Primary antibodies used in this experiment included Raptor (1:200, cat. no. ab5454; Abcam), mTOR (1:200, cat. no. 2983; Cell Signaling Technology, Inc.), lysosomal-associated membrane protein 1 (LAMP1; 1:500, cat. no. sc-20011; Santa Cruz Biotechnology, Inc.), α-tubulin (1:1,000, cat. no. 3873; Cell Signaling Technology, Inc.). A secondary antibody was then added in a dropwise manner, and the slides were placed into a wet box and incubated at 20°C for 1 h. The secondary antibodies used, included goat anti-rabbit IgG (FITC,1:1,000, ab6717) and goat anti-mouse IgG (TR,1:1000, ab5766), purchased from Abcam. DAPI (1:500, Beyotime Institute of Biotechnology) was used for nuclei staining at room temperature for 1 min. The slides were stored at −20°C until further analysis. Images were recorded using a fluorescence microscope (Olympus Corporation). A total of five randomly selected fields of each slide were selected and analyzed using Image-Pro plus 6.0 software (Media Cybernetics, Inc.).

Data are presented as the mean ± standard deviation (SD) and were statistically analyzed using SPSS 17.0 statistical software (SPSS, Inc.). In particular, data with a normal distribution were analyzed using one-way analysis of variance (ANOVA), followed by pairwise comparisons with the Tukey's test. Data with unequal and non-parametric features were analyzed using ANOVA on ranks followed by the Dunn's method for pairwise comparisons. Comparisons between two groups were analyzed with an independent Student's t-test. P≤0.05 was considered to indicate a statistically significant difference.

To explore the involvement of CAPN6 in the regulation of myoblast autophagy, the expression of the autophagy-related genes, LC3 and p62, in rat L6 myoblast cells were examined. First, CAPN6 overexpression and knockdown was induced in L6 cells (Fig. S1). CAPN6 shRNA decreased CAPN6 protein levels by 55.75% and increased the ratio of LC3II/I by 124.815%, whereas CAPN6 cDNA increased the CAPN6 levels by 23.6% and decreased the ratio of LC3II/I by 45.03% (Fig. 1). This was also confirmed by p62, a cargo protein degraded inside autolysosomes, protein level detection. The level of p62 decreased when CAPN6 expression decreased (Fig. 1).

To examine the autophagic flux status, L6 myoblasts were transfected with RFP-GFP-LC3 plasmid, according to a previous study (38). The green fluorescent protein (GFP) level is quenched in the lysosomal environment and the red fluorescent protein (RFP) signal is more stable in the acidic environment according to a previous study (39). Thus, the autophagosomes and autolysosomes were labeled with a yellow (green and red) or red color, respectively. Through the detection and the analysis of the two different fluorescent signals, the autophagic flux was monitored. After culturing the myoblasts for 48 h with CAPN6 cDNA, decreased red fluorescence was observed in the CAPN6 cDNA groups (CAPN6 group), in comparison with the CRISPR-transfected groups (Control-CRISPR group). This indicated that CAPN6 may decrease autophagy (Fig. 1).

Furthermore, the mechanism responsible for the effects of CAPN6 on autophagy was explored. It was observed that the co-localization of mTOR and LAMP1 proteins (a lysosomal marker) was increased in the CAPN6 overexpression group (CAPN6 group), indicating an increased combination of mTOR and lysosomal degradation. Additionally, the co-localization of mTOR, Raptor and α-tubulin (a marker of microtubules) in the CA PN6 group indicated an increased combination of mTORC1 and microtubules. These results indicated that a high CAPN6 expression was positively related to mTORC1 combined with lysosomal and microtubules, and this may be a mechanism through which CAPN6 inhibits autophagy (Fig. 2).

Following a decrease in mTORC1 through transfection with Raptor shRNA, it was revealed that the co-localization of mTOR and LAMP1 proteins, mTOR and α-tubulin, Raptor and α-tubulin decreased in the CAPN6 overexpression + Raptor shRNA-transfected group (CAPN6 + Raptor shRNA group). In addition, an increase in red fluorescence was observed in the CAPN6 + Raptor shRNA group, as compared with the CAPN6 group. This indicated that mTORC1 combined with lysosomal or microtubules plays an important role in CAPN6 inhibiting autophagy flux (Fig. 2).

L6 myoblasts were exposed to 2 ng/ml IL-6, 2 ng/ml TNF-α, 2 ng/ml INF-γ and 10 ng/ml LPS separately and it was demonstrated that the protein expression of CAPN6 increased, particularly in the IFN-γ-treated L6 myoblasts (Fig. 3). The mRNA levels also increased when the cells were exposed to the four inflammatory agents simultaneously and in a time-dependent manner. The expression levels increased following 6 h of treatment and were higher at 24 h (Fig. 3).

Subsequently, the effects of CAPN6 on autophagy in myoblasts exposed to the four inflammatory agents were assessed. CAPN6 expression significantly increased in the cytokine mixture-treated L6 myoblasts (M group), while the level of LC3II/I decreased significantly. LC3II/I expression increased following CAPN6 knockdown using shRNA in the cytokine mixture-treated L6 myoblasts (M + shRNA group; Fig. 4). The autophagic was also examined with fluorescence. It was observed that the fluorescence was not significantly altered in the M group; however, fluorescence increased in the M + shRNA group, suggesting an increase in the autophagic flux (Fig. 4C).

Finally, the expression and activity of mTORC1 in L6 myoblasts following treatment with the control or cytokine mixture (M group) was evaluated and an increase in mTOR, p-p70s6k1/p70s6k1 and p-4EBP1/4EBP1 expression was observed. Additionally, CAPN6 knockdown (M + shRNA group) culminated in a decrease in protein expression (Fig. 5). However, Raptor expression was not significantly altered in the three groups.

In the in vivo experiments, a rat model of CKD was established. The rats exhibited a decrease in body weight, and reduced muscle mass and muscle CSA (Fig. 6). Blood chemical levels were similar to those similar to those of CKD patients (9), including an increase in BUN, serum creatinine and TC levels, and lower TG and Alb levels (Table I). CAPN6 mRNA and protein expression levels in the gastrocnemius muscles of rats with CKD were also examined, and it was revealed that there was a non-significant increase only (Fig. 7).