Notch3-Mediated mTOR Signaling Pathway Is Involved in High Glucose-Induced Autophagy in Bovine Kidney Epithelial Cells

It is reported that Notch3 and mTOR signaling pathways are involved in autophagy, and both can be activated by high glucose (HG). However, the relationship between Notch3 and mTOR and how Notch3 affects mTOR to regulate HG-induced autophagy in bovine kidney epithelial cells is still unclear. The purpose of this study is to explore how Notch3 affects mTOR to modulate HG-induced autophagy in bovine kidney cells. Our results showed that HG treatment significantly decreased the cell viability of MDBK cells in a dose-dependent manner. HG treatment significantly increased the expression of LC3-II/I ratio and Beclin1 protein and significantly decreased the expression of p62 protein. Consistently, LC3 fluorescence signal formation was detected by immunofluorescence in both dose and time-dependent manners. In addition, HG treatment significantly increased the expression of Notch3 protein and decreased the expression of the p-mTOR protein in both dose and time-dependent manners. Inhibition of Notch3 upregulated the expression of p-mTOR and p62 protein, and downregulated the expression of LC3-II/I ratio and Beclin1 protein. Besides, the function of Notch3 was investigated. In this study, inhibition of Notch3 activity significantly increased the viability of HG-stimulated MDBK cells. In summary, our results revealed that the Notch3-mediated mTOR signaling pathway was involved in HG-induced autophagy in MDBK cells.


Introduction
Hyperglycemia can cause serious diabetic kidney disease or stress hyperglycemia in humans, mice, and rats [1][2][3]. The concentrations of glycemia in beef cattle significantly increase during transportation stress [4][5][6]. However, the cytological molecular mechanism of hyperglycemia in beef cattle injury remains unclear. The kidney is one of the organs most susceptible to glucose concentration [7]. HG is commonly used to simulate hyperglycemia in in vitro experiments. Previous studies usually focus on inflammation, apoptosis, and pyroptosis in HG-induced renal cells [8,9]. Moreover, studies have found that autophagy plays an important role in HG-induced cell injury [10] and can regulate inflammation, apoptosis, and pyroptosis [11][12][13]. For example, autophagy was activated by p53 deacetylation in diabetic mice and HG-treated HK-2 cells [14].
Notch receptors are the most conserved signaling molecules that are widely distributed on the surface of a variety of cells [15]. In mammals, there are four Notch receptors

HG Treatment Induced Autophagy Markers in MDBK Cells
To evaluate the effect of HG on MDBK cells viability, the cells were treated with 5.5, 10.5, 15.5, and 25.5 mM glucose for 24 h, and the cell proliferation was examined. The results showed that the MDBK cells viability of the 10.5, 15.5, and 25.5 mM glucose treatment groups decreased significantly (p < 0.05) compared with the 5.5 mM glucose treatment groups, respectively ( Figure 1A). Based on the above observations, glucose concentrations (5.5, 10.5, 15.5, and 25.5 mM) were chosen for subsequent experiments.
Next, to explore the effect of different concentrations of glucose (5.5, 10.5, 15.5, and 25.5 mM) on autophagy in MDBK cells, the protein and mRNA expression levels of LC3, p62, and Beclin1 in MDBK cells were detected by Western blot and qRT-PCR, and the intensity of red fluorescent labeled LC3 was detected by immunofluorescence assay. 10.5, 15.5 and 25.5 mM glucose treatment groups showed a significant increase (p < 0.01) in the protein levels of LC3-II/I and Beclin1 whereas the protein level of p62 was decreased significantly (p < 0.01) compared with 5.5 mM glucose treatment group ( Figure 1B,C). Consistent with the protein experiment results, relative to the 5.5 mM glucose treatment groups ( Figure 1D), the mRNA expression level of LC3B and Beclin1 was increased significantly (p < 0.01), and the mRNA expression level of p62 was decreased significantly (p < 0.01) in the 10.5, 15.5 and 25.5 mM glucose treatment groups. An immunofluorescence assay was used for measuring protein levels of LC3 in the MDBK cells in the present study. As shown in Figure 2, the 10.5, 15.5, and 25.5 mM glucose treatment groups showed a significant increase in the intensity of red fluorescent labeled LC3 compared with the 5.5 mM glucose treatment group. Moreover, the 25.5 mM glucose treatment group was the most significant among these groups.
Furthermore, to explore the effect of different HG treatment times on autophagy, MDBK cells were treated with 25.5 mM glucose for 0, 3, 6, 12, and 24 h. The protein and mRNA expression levels of LC3, p62, and Beclin1 in MDBK cells were detected by Western blot and qRT-PCR, and the intensity of red fluorescent labeled LC3 was detected by immunofluorescence assay. Figure 3A, B illustrated that the 3, 6, 12 and 24 h treatment groups showed a significant increase in the protein levels of LC3-II/I and Beclin1 (p < 0.01) whereas the protein level of p62 was decreased significantly (p < 0.01) compared with 0 h treatment group. Consistent with the protein experiment results, relative to the 0 h treatment groups ( Figure 3C), the mRNA expression level of LC3B and Beclin1 was increased significantly (p < 0.01), whereas the mRNA expression level of p62 was decreased significantly (p < 0.01) in the 3, 6, 12 and 24 h treatment groups. In addition, compared with other groups, the protein and mRNA expression levels of the 24 h treatment group were the most significant. As shown in Figure 4, the 3, 6, 12, and 24 h treatment groups showed a significant increase in the intensity of red fluorescent labeled LC3 compared with the 0 h treatment group. Moreover, the 24 h treatment group was the most significant among these groups.

HG Treatment Induced Autophagy Markers in MDBK Cells
To evaluate the effect of HG on MDBK cells viability, the cells were treated w 10.5, 15.5, and 25.5 mM glucose for 24 h, and the cell proliferation was examine results showed that the MDBK cells viability of the 10.5, 15.5, and 25.5 mM glucos ment groups decreased significantly (p < 0.05) compared with the 5.5 mM glucose ment groups, respectively ( Figure 1A). Based on the above observations, glucose c trations (5.5, 10.5, 15.5, and 25.5 mM) were chosen for subsequent experiments.  Next, to explore the effect of different concentrations of glucose (5.5, 10.5, 15.5, and 25.5 mM) on autophagy in MDBK cells, the protein and mRNA expression levels of LC3, p62, and Beclin1 in MDBK cells were detected by Western blot and qRT-PCR, and the intensity of red fluorescent labeled LC3 was detected by immunofluorescence assay. 10.5, 15.5 and 25.5 mM glucose treatment groups showed a significant increase (p < 0.01) in the protein levels of LC3-II/I and Beclin1 whereas the protein level of p62 was decreased significantly (p < 0.01) compared with 5.5 mM glucose treatment group ( Figure 1B,C). Consistent with the protein experiment results, relative to the 5.5 mM glucose treatment groups ( Figure 1D), the mRNA expression level of LC3B and Beclin1 was increased significantly (p < 0.01), and the mRNA expression level of p62 was decreased significantly (p < 0.01) in the 10.5, 15.5 and 25.5 mM glucose treatment groups. An immunofluorescence assay was used for measuring protein levels of LC3 in the MDBK cells in the present study. As shown in Figure 2, the 10.5, 15.5, and 25.5 mM glucose treatment groups showed a significant increase in the intensity of red fluorescent labeled LC3 compared with the 5.5 mM glucose treatment group. Moreover, the 25.5 mM glucose treatment group was the most significant among these groups. Furthermore, to explore the effect of different HG treatment times on autophagy, MDBK cells were treated with 25.5 mM glucose for 0, 3, 6, 12, and 24 h. The protein and mRNA expression levels of LC3, p62, and Beclin1 in MDBK cells were detected by West- To investigate whether HG induces autophagic flux in MDBK cells, chloroquine (CQ) was used. As shown in Figure S1 (Supplementary Materials), incubation of cells with CQ resulted in significantly increased LC3-II expression (p < 0.01), and treatment with HG and CQ revealed a further increase in LC3-II expression (p < 0.01).
In summary, these data indicated that HG induced autophagy in MDBK cells.   was used. As shown in Figure S1 (Supplementary Materials), incubation of cells with CQ resulted in significantly increased LC3-II expression (p < 0.01), and treatment with HG and CQ revealed a further increase in LC3-II expression (p < 0.01). In summary, these data indicated that HG induced autophagy in MDBK cells.   To investigate whether HG induces autophagic flux in MDBK cells, chloroquine (CQ) was used. As shown in Figure S1 (Supplementary Materials), incubation of cells with CQ resulted in significantly increased LC3-II expression (p < 0.01), and treatment with HG and CQ revealed a further increase in LC3-II expression (p < 0.01).
In summary, these data indicated that HG induced autophagy in MDBK cells.

HG Induced Notch3 and mTOR Signaling Pathways in MDBK Cells
To investigate the role of Notch3 and mTOR in MDBK cells induced by HG, the protein and mRNA expression levels of Notch3 and mTOR in MDBK cells were detected by Western blot and qRT-PCR ( Figure 5A-C). Figure 5A showed that the protein level of Notch3 was increased significantly (p < 0.01) in the 10.5, 15.5, and 25.5 mM glucose treatment groups compared with the 5.5 mM glucose treatment group. Meanwhile, the protein level of p-mTOR was decreased significantly (p < 0.05) in the 10.5, 15.5, and 25.5 mM glucose treatment groups compared with the 5.5 mM glucose treatment group. Consistent with the protein experiment results, relative to the 5.5 mM glucose treatment groups ( Figure 5C), the mRNA expression level of Notch3 was increased significantly (p < 0.01), whereas the mRNA expression level of mTOR was decreased significantly (p < 0.01) in the 10.5, 15.5 and 25.5 mM glucose treatment groups. Moreover, compared with other groups, the protein and mRNA levels of Notch3 and p-mTOR were most significant in the 25.5 mM glucose treatment group.

HG Induced Notch3 and mTOR Signaling Pathways in MDBK Cells
To investigate the role of Notch3 and mTOR in MDBK cells induced by HG, the protein and mRNA expression levels of Notch3 and mTOR in MDBK cells were detected by Western blot and qRT-PCR ( Figure 5A-C). Figure 5A showed that the protein level of Notch3 was increased significantly (p < 0.01) in the 10.5, 15.5, and 25.5 mM glucose treatment groups compared with the 5.5 mM glucose treatment group. Meanwhile, the protein level of p-mTOR was decreased significantly (p < 0.05) in the 10.5, 15.5, and 25.5 mM glucose treatment groups compared with the 5.5 mM glucose treatment group. Consistent with the protein experiment results, relative to the 5.5 mM glucose treatment groups (Figure 5C), the mRNA expression level of Notch3 was increased significantly (p < 0.01), whereas the mRNA expression level of mTOR was decreased significantly (p < 0.01) in the 10.5, 15.5 and 25.5 mM glucose treatment groups. Moreover, compared with other groups, the protein and mRNA levels of Notch3 and p-mTOR were most significant in the 25.5 mM glucose treatment group. Furthermore, the protein levels of Notch3 showed a significant increase (p < 0.01) at 3, 6, 12, and 24 h in the treatment groups compared with the 0 h treatment group ( Figure  5B). The protein level of p-mTOR was decreased significantly (p < 0.01) in the 3, 6, 12, and Furthermore, the protein levels of Notch3 showed a significant increase (p < 0.01) at 3, 6, 12, and 24 h in the treatment groups compared with the 0 h treatment group ( Figure 5B). The protein level of p-mTOR was decreased significantly (p < 0.01) in the 3, 6, 12, and 24 h treatment groups compared with the 0 h treatment group ( Figure 5B). Similar to the protein experiment results, relative to the 0 h treatment groups ( Figure 5C), the mRNA expression level of Notch3 increased significantly (p < 0.01), whereas the mRNA expression level of mTOR decreased significantly (p < 0.01) in treatment groups at 3, 6, 12 and 24 h of treatment. Moreover, the protein and mRNA levels of Notch3 and mTOR were the most significant in the treatment group at 24 h compared with other groups.
These results revealed that HG induced Notch3 and mTOR signaling pathways in MDBK cells.

Notch3-Mediated mTOR Signaling Pathway was Involved in HG-Induced Autophagy in MDBK Cells
To investigate the relationship between Notch3 and mTOR in autophagy induced by HG, MDBK cells were pretreated with 10 µM DAPT, a specific Notch inhibitor. Firstly, western blot results showed that Notch3, LC3-II/I, and Beclin1 proteins were increased significantly (p < 0.01), whereas the p-mTOR and p62 proteins were decreased significantly (p < 0.01) in the HG treatment group compared with the control group ( Figure 6A). Notch3, LC3-II/I, and Beclin1 proteins were decreased significantly (p < 0.01), whereas the p-mTOR and p62 proteins were increased significantly (p < 0.01) in the HG + DAPT treatment group compared with the HG treatment group ( Figure 6A). As shown in Figure 6C, compared with the control group, the mRNA levels of LC3B and Beclin1 increased significantly (p < 0.01), whereas the mRNA levels of mTOR and p62 decreased significantly (p < 0.01) in the HG treatment group. The mRNA levels of LC3B and Beclin1 decreased significantly (p < 0.01), whereas the mRNA levels of mTOR and p62 increased significantly (p < 0.01) in the HG + DAPT treatment group compared with the HG treatment group.
Besides, MDBK cell viability was examined by CCK-8 assay in the control group (5.5 mM glucose treatment group), HG group (25.5 mM glucose treatment group), and HG + DAPT group (25.5 mM glucose + 10 µM DAPT treatment group). The results showed that the MDBK cell viability of the HG treatment groups decreased significantly (p < 0.05) compared with the control groups ( Figure 6B). Moreover, compared with the HG group, the viability of the MDBK cells increased significantly in the HG + DAPT group (p < 0.01).
Furthermore, the autophagosomes also were detected by TEM in the current study. The results showed that compared with the control treatment group, the formation of autophagosomes was increased in the HG treatment group (Figure 7). Moreover, compared with the HG group, the formation of autophagosomes was decreased in the HG + DAPT treatment group (Figure 7).
Similar results were demonstrated in the immunofluorescence assay. As presented in Figure 8, compared with the control group, the intensity of red fluorescent labeled LC3 in the HG treatment group was increased. In addition, the intensity of red fluorescent labeled LC3 was decreased in the HG + DAPT treatment group compared with the HG treatment group.
In summary, these findings demonstrated that the Notch3-mediated mTOR signaling pathway was involved in HG-induced autophagy in MDBK cells. Besides, MDBK cell viability was examined by CCK-8 assay in the control group (5.5 mM glucose treatment group), HG group (25.5 mM glucose treatment group), and HG + DAPT group (25.5 mM glucose + 10 µM DAPT treatment group). The results showed that the MDBK cell viability of the HG treatment groups decreased significantly (p < 0.05) compared with the control groups ( Figure 6B). Moreover, compared with the HG group, the viability of the MDBK cells increased significantly in the HG + DAPT group (p < 0.01).
Furthermore, the autophagosomes also were detected by TEM in the current study. The results showed that compared with the control treatment group, the formation of autophagosomes was increased in the HG treatment group (Figure 7). M pared with the HG group, the formation of autophagosomes was decrease DAPT treatment group (Figure 7). Similar results were demonstrated in the immunofluorescence assay. in Figure 8, compared with the control group, the intensity of red fluorescen in the HG treatment group was increased. In addition, the intensity of re labeled LC3 was decreased in the HG + DAPT treatment group compared treatment group. In summary, these findings demonstrated that the Notch3-mediated mT pathway was involved in HG-induced autophagy in MDBK cells.  Similar results were demonstrated in the immunofluorescence assay. As prese in Figure 8, compared with the control group, the intensity of red fluorescent labeled in the HG treatment group was increased. In addition, the intensity of red fluore labeled LC3 was decreased in the HG + DAPT treatment group compared with the treatment group. In summary, these findings demonstrated that the Notch3-mediated mTOR sign pathway was involved in HG-induced autophagy in MDBK cells.

Discussion
Notch3 and mTOR signaling pathways have been reported to be involved in aut agy, and both can be activated by HG. However, the relationship between Notch3

Discussion
Notch3 and mTOR signaling pathways have been reported to be involved in autophagy, and both can be activated by HG. However, the relationship between Notch3 and mTOR and whether HG induces autophagy in renal cells by Notch3/mTOR signaling pathway are still unclear. In the present study, we investigated how Notch3 influenced mTOR to regulate HG-induced autophagy in bovine renal cells.
Autophagy is an important process for the cell survival and maintenance of cells where organelles, proteins, and macromolecules in the cytoplasm and the circulation of breakdown products are degraded [29]. Moreover, autophagy plays an important regulatory role in HG-induced renal cell injury [30]. The glucose concentration in vitro is generally 5.5 mM~25.5 mM, which is used to simulate normal blood glucose and hyperglycemia respectively [31,32]. Furthermore, blood glucose levels in beef cattle subjected to transport stress increased from 5.5 mM to 15.2 mM [5]. As a result, the glucose concentration range for this test was set at 5.5 to 25.5 mM. In most studies, HG-induced autophagy in renal cells occurred within 24 h [33,34]. For example, Chen et al. [33] found that when human proximal tubular epithelial cells were treated with HG for 24 h, the LC3 protein expression increased, and the p62 protein expression decreased. Therefore, the action time range of HG is set at 0~24 h. Firstly, we examined whether HG treatment induces autophagy in MDBK cells. Our results showed a significant increase of LC3-II/LC3-I and Beclin1 whereas a remarkable decrease in p62 in both dose and time-dependent manners. This indicated that HG induced autophagy in MDBK cells. These results are consistent with previous findings [31][32][33]. Several evolutionarily-conserved genes (called autophagy-related genes, such as LC3, Beclin1, and p62) are involved in the process of autophagy [35][36][37]. Meanwhile, immunofluorescence results showed that the intensity of fluorescence increased in MDBK cells after HG treatment.
Next, we investigated the mechanism of autophagy induced by HG. mTOR is a significant negative regulator of autophagy, conscientious for nutrient consumption, low energy, or oxidative stress [38]. In this experiment, we observed a reduction in p-mTOR, indicating that the HG was capable of inhibiting mTOR activities. Consistent with our results, Zhao et al. [39] have also proposed that HG exposure induces autophagy in H9c2 cells by inhibiting mTOR. It has been demonstrated that highly active Notch3 is generally found to be related to kidney injuries [40][41][42]. In this research, an increase in Notch3 implied that the Notch3 signaling pathway was involved in autophagy induced by HG in MDBK cells. Similar results were also observed in the study by Huang et al. [19], which reported that the expression of Notch3 significantly increased in the renal tubular epithelial cells of 18 patients with obstructive nephropathy. Notch3 and mTOR are both involved in autophagy [20,26], but the relationship between Notch3 and mTOR is still unclear. Recent studies have found that the protein expression level of Notch3 was not affected when mTOR was inhibited [13], suggesting that Notch3 may be an upstream regulator of mTOR signaling. Furthermore, the relationship between Notch3 and mTOR was investigated in this study. From our study, co-treatment with the Notch3 inhibitor (DAPT) could reverse the reduction of p-mTOR protein expression, indicating that Notch3 was the major upstream regulator of mTOR. These results are consistent with previous findings by Ivanovska et al. [22]. Besides, the function of Notch3 also was studied. In this study, inhibition of Notch3 activity increased the viability of HG-stimulated MDBK cells, indicating that inhibition of Notch3 expression reduced the injury induced by HG in MDBK cells. Furthermore, results of ultrastructure observation and LC3 immunofluorescence staining showed that Notch3 inhibition could abolish the autophagy induced by HG. Taken together, these findings demonstrated that inhibition of Notch3-mediated mTOR signaling pathway alleviated HG-induced autophagy in MDBK cells.

Cell, Chemical and Reagents
MDBK cells, an immortalized bovine kidney epithelial cell line, were purchased from China Center for Type Culture Collection, Wuhan, China. DMEM Low Glucose medium, DMEM High Glucose medium and DMEM/F12(HAM) medium were purchased from Biological Industries, Kibbutz Beit Haemek, Israel. Notch3 inhibitor (DAPT) was purchased from Selleck, Houston, TX, USA.

Cell Counting Kit-8 (CCK-8) Assay for Cell Growth
Trypsinized MDBK cells (2 × 10 5 cells) were resuspended in complete DMEM/F12(HAM) medium and seeded in a 96-well plate and then incubated with 5.5, 10.5, 15.5, and 25.5 mM glucose in the previously mentioned DMEM Low Glucose medium under 37 • C, 5% CO 2 . Cell proliferation was evaluated by CCK-8 assay following the manufacturer's instructions (Solarbio) after 24 h. At least triplicate repeats were completed in all the experiments.

Quantitative Real-Time PCR (qRT-PCR) Analysis
Primers for qRT-PCR were designed by Primer Premier Software 5.0 (Premier Biosoft International, San Francisco, CA, USA). The sequences are shown in Table 1

Transmission Electron Microscopy (TEM) Analysis
MDBK cells were cultured as described above. MDBK cells were washed with PBS three times and collected by a cell scraper. Then, the cells were centrifuged at 1000 g for 10 min. Precipitated MDBK cells were fixed with ice-cold 3% glutaraldehyde (Solarbio) and then post-fixed in 1% osmium tetroxide. The cell clumps were cut into ultrathin sections after they were embedded in Epon epoxy resin (Chengdu Rongshengke Biotechnology Co., Ltd., Chengdu, China). The ultrathin sections were stained with 0.1% lead citrate (Chengdu Rongshengke Biotechnology Co., Ltd.) and 10% uranyl acetate (Chengdu Rongshengke Biotechnology Co., Ltd.) and were observed with a transmission electron microscope (JEOL, Beijing, China, JEM-1400PLUS).

Statistical Analyses
All the experiments were performed in triplicates. The data were presented as the mean ± SD and were analyzed using variance SPSS 24.0. The differences in the means were determined by one-way analysis of variance (ANOVA) for multiple comparisons followed by the least significant difference (LSD) test for two-group comparisons among the multiple comparisons. p-value of less than 0.05 was considered statistically significant (# or * p < 0.05) and p-value of less than 0.05 was considered extremely significant (## or ** p < 0.01).

Conclusions
In conclusion, this study demonstrated that the Notch3-mediated mTOR signaling pathway was involved in HG-induced autophagy in MDBK cells, as summarized in Figure 9. This study may provide a certain theoretical basis for the pathogenesis and treatment of nephropathy induced by hyperglycemia, such as diabetic nephropathy and stress hyperglycemia. However, the role of autophagy in HG-induced renal injury needs to be further explored.
In conclusion, this study demonstrated that the Notch3-mediated mTOR sign pathway was involved in HG-induced autophagy in MDBK cells, as summarized in ure 9. This study may provide a certain theoretical basis for the pathogenesis and t ment of nephropathy induced by hyperglycemia, such as diabetic nephropathy and s hyperglycemia. However, the role of autophagy in HG-induced renal injury needs further explored.  Institutional Review Board Statement: Ethical approval is not applicable, because the research not contain any studies with human or animal subjects.

Data Availability Statement:
The data presented in this study are available on request from corresponding author.  Institutional Review Board Statement: Ethical approval is not applicable, because the research does not contain any studies with human or animal subjects.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.