Abstract
Background. Rapamycin is known to induce autophagy, promote cell survival and inhibit the progression of diabetic nephropathy (DN).
Objectives. The aim of this study was to examine the role of autophagy in the treatment of DN with rapamycin to provide the basis for the DN treatment with rapamycin.
Materials and methods. Human mesangial cells (HMC) were cultured in a constant temperature incubator with 5% CO2, at 37°C and saturated humidity. Cells were divided into 5 groups and the 5-ethynyl-2-deoxyuridine (EdU) cell proliferation assay was used to determine cell proliferation. Flow cytometry was used to determine cell apoptosis, while GFP–RFP–LC3 showed autophagy flow. Western blot was employed to detect the expression of autophagy-related proteins LC3-II/LC3-I and P62. Enzyme-linked immunosorbent assay (ELISA) was used to determine the contents of type IV collagen fiber (Col4), hyaluronic acid (HA) and laminin (LA) in the extracellular matrix (ECM).
Results. Cell proliferation was the lowest in the hyperglycemic group. Additionally, the hyperglycemic group displayed the lowest number of autolysosomes compared to other groups. In contrast, the rapamycin group exhibited the highest number of autolysosomes. The LC3-II/LC3-I ratio was also the lowest in the hyperglycemic group, measuring 0.53 (0.50–0.58), while the expression level of P62 was significantly higher in that group at 0.98 (0.95–1.01) compared to other groups. Upon the introduction of rapamycin, the LC3-II/LC3-I ratio was significantly increased at 2.21 (1.95–2.21), and P62 was significantly decreased 0.38 (0.38–0.39) compared to the hyperglycemic group. Both changes were statistically significant, with p-values of 0.034 and 0.010, respectively. Enzyme-linked immunosorbent assay was employed to detect Col4, HA and LA content. The study findings demonstrated significantly higher levels of glucose in the hyperglycemic group in comparison to other groups. In contrast, the rapamycin group exhibited significantly lower levels of glucose than the hyperglycemic group, yet the difference was not statistically significant.
Conclusions. Hyperglycemic can inhibit the autophagic activity of HMC, promote cell apoptosis, enhance ECM accumulation, and facilitate the DN progression. In contrast, rapamycin can elicit autophagy, decrease mesangial matrix proliferation, and therefore impede DN progression.
Key words: diabetic nephropathy, autophagy, human mesangial cells
Background
At present, diabetic nephropathy (DN) has become the leading cause of end-stage renal disease (ESRD) in China. The treatment of DN is difficult, and there is no recognized specific drug that can cure DN. It is an urgent problem to further understand the pathogenesis of DN and seek effective treatment. Mesangial cells are extremely important target cells and effector cells in the development of DN, participating in the process of glomerular injury and repair, and promoting inflammation and the development of glomerular sclerosis together with extracellular matrix (ECM). At the same time, oxidative stress, inflammation and autophagy are also involved in various pathological processes of DN.
Autophagy refers to the formation of autophagosomes in the cytoplasm to degrade damaged organelles, proteins and other components to meet the metabolic needs of the cells themselves and help the cells survive. According to the different forms, autophagy can be divided into macroautophagy, microautophagy and chaperone-mediated autophagy, among which macroautophagy is the most common. Autophagy is a continuous dynamic process that involves 5 distinct stages: initiation, nucleation, expansion and elongation, closure and fusion, and cargo degradation The autophagy process is regulated by several autophagy-related genes (ATG). Autophagy is activated under stress, such as hypoxia, amino acid deficiency or low glucose, and forms autophagosomes after activation to remove damaged cell components and ensure cell survival. It is the process of fusing macromolecular proteins, mitochondria, ribosomes, and organelle fragments with autophagosomes to break them down into small molecules.1
Autophagy and proteasome pathways belong to 2 intracellular degradation pathways, but the contents degraded by the 2 are different. The latter specifically degrades ubiquitinated small protein molecules, while the denatured, misfolded large protein molecules and aging and deteriorated organelles are degraded by autophagy. Therefore, cells generally maintain some level of autophagy activity, and the level of autophagy varies depending on the tissue type. Under the condition of hyperglycemia, autophagy activity of mesangial cells is inhibited, but with the increase of intracellular advanced glycation end products (AGEs), a large number of proteins are inactivated, and organelle function is impaired. At this point, both protein degradation pathways may be activated. Although autophagy changes at different stages of DN are still a controversial issue, most evidence supports the fact that the process of renal autophagy is blocked in DN, promoting apoptosis and the occurrence of disease. As an inducer of autophagy, rapamycin enables cells to survive and resist external damage.
This study will further verify the autophagy of cells under the condition of high glucose, whether high glucose has an effect on cell survival, and the effect of rapamycin on the autophagy activity of cells, providing evidence for the application of rapamycin in diabetic nephropathy.
Objectives
To study the role of autophagy in the treatment of DM with rapamycin and to provide the basis for the treatment of DN with rapamycin.
Materials and methods
Materials
Human mesangial cells (HMC) were purchased from the Shanghai Fuheng Biology (Shanghai, China; cat. No. FH0241) and cell culture flasks were purchased from FALCON (Chongqing, China; cat. No. 353014).
Culture and grouping of mesangial cells
Human mesangial cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 U/mL streptomycin in a constant temperature incubator with 5% CO2, at 37°C and saturated humidity. The HMC group was cultured in the original medium component without glucose. The hypoglycemic group was cultured in medium containing 5 mmol/L glucose. In the hyperglycemic group, HMCs were cultured in medium containing 30 mmol/L glucose. In isotonic mannitol group HMCs were cultured in medium containing 24.5 mmol/L mannitol and 5.5 mmol/L glucose. In the rapamycin group, HMCs were cultured in the medium containing 30 mmol/L glucose and 1 μM rapamycin.
Proliferation of 5-ethynyl-2-deoxyuridine (EdU) cells
The cells were treated with 2 × EdU working solution for 2 h, fixed with 4% paraformaldehyde and incubated for 30 min. The cells were cultured using conventional methods. After discarding the fixed solution in the culture medium, the cells were washed 3 times and incubated with a transparent solution at room temperature for 20 min. Following this, the cells were incubated at room temperature away from light for 30 min before being observed under a fluorescence microscope (Olympus IX71; Olympus Corp., Tokyo, Japan).
Apoptosis cells were determined with flow cytometry
After digesting the HMC with trypsin, they were collected by centrifugation at 2,000 rpm for 5–10 min and then washed again. Following this, 300 μL of buffer suspension cells were added, and 5 μL of Annexin V-FITC was mixed in. The resulting mixture was kept away from light. Before loading the sample, 5 μL of propidium iodide (PI) staining was added and allowed to bind for 5 min. Finally, after adding 200 μL of buffer, the results were observed.
GFP–RFP–LC3 labeling autophagy flow
The HMC were inoculated into 24-well plates, The amount of GFP–RFP–LC3 adenovirus in each group was within the range of multiplicity of infection (MOI) = 20. After 2 h, the medium was changed, and the cells were washed with culture medium or buffer. After the cleaning solution was carefully absorbed, the cells were fixed with a freshly prepared and pre-heated buffer containing 4% paraformaldehyde for 10 min at room temperature. The fixative was removed, and the cells were rinsed several times with an appropriate buffer. Nuclear staining was performed in a dark room to avoid light. Fluorescent dye was applied diluted (DAPI (4’,6-diamidino-2-phenylindole, 1:1,000)), washed cells were applied and incubate at room temperature for 7–10 min, and then wash 3 times for 5 min with phosphate-buffered saline (PBS); finally, the liquid was discarded. The film was sealed and observed with a confocal laser microscope (Leica TCS SP8; Leica Camera AG, Wetzlar, Germany).
Protein concentrations of LC3-II/LC3-I and P62 were detected with western blot
Cell pellets from each group were collected. A total of 150 μL of lysate was added to each sample, and gel plates were prepared in the following manner: 1. The polyvinylidene fluoride (PVDF) membrane was sealed with a sealing solution containing 5% skim milk powder at room temperature for 2 h. 2. The corresponding primary antibody (rabbit anti-human LC3, P62) was diluted with blocking solution. The PVDF membrane was then immersed in the primary antibody incubation solution and incubated overnight at 4°C. 3. Excess primary antibodies were removed by washing the PVDF membrane thoroughly in Tris-buffered saline with Tween (TBST) and repeating the process 3–4 times for 15 min each time. 4. The secondary antibody was applied (goat anti-rabbit IgG antibody) and the PVDF membrane was soaked in the solution for secondary antibody incubation and incubated at 37°C for 2 h. 5. Excess secondary antibodies were washed away by fully washing the PVDF membrane in TBST 3–4 times for 15 min each time. 6. Color exposure was performed.
The content of Col4 HA, and LA of cells was determined by ELISA
Cells digested with trypsin were harvested and the resulting supernatant was collected. Biotinylated antibody working solution (100 μL) was added to each well, followed by incubation at 37°C for 1 h after covering the plate with film. Enzyme conjugate working solution (100 μL) was then added to each well, followed by substrate (tetramethylbenzidine) solution (90 μL). The enzyme plate was covered with film and incubated away from light. Stop solution (50 μL) was added to each well and the optical density (OD) was immediately measured at 450 nm using a microplate reader (Multiskan Mk3 Microplate Reader; Thermo Fisher Scientific, Waltham, USA).
Statistical analyses
IBM SPSS v. 26.0 (IBM Corp., Armonk, USA) software was utilized for statistical analysis. The experimental data were replicated 3 times for each group. All tested variables were expressed as medians with interquartile ranges (IQRs; 25th to 75th percentiles). We assessed statistical differences with the Kruskal–Wallis test, followed by Dunn’s test for multiple comparisons. We adjusted p-values using the Bonferroni correction. We considered a p-value below 0.05 statistically significant. Bootstrap medians and 95% confidence intervals (95% CIs) have been reported.
Results
EdU cell proliferation under different conditions
Hoechst 33342 stimulated the nuclei to emit blue fluorescence, while Azide 488 caused proliferation of cells resulting in green fluorescence. Figure 1 shows a significant number of cells in HMC group, hypoglycemic group and isotonic mannitol group, which were 948.5 (881.75–1008.5), 1019.5 (938.5–1055.5) and 920.5 (808.5–954.5), respectively. Additionally, there were a considerable number of proliferating cells: 463.0 (416.75–514.5), 512.5 (462.25–525.25 and 444.5 (411.75–513.25) were the measured values for total number of cells in hyperglycemic group. The group contained 912.0 (860.5–969.5) cells in total. Among them, only 141.0 (130.0–163.25) were found to be proliferating. The proliferation rate of cells in hyperglycemic group was statistically significant compared to HMC group (p-value 0.017), with a rate of 17.5% (13.75–19.75%). Table 1 displays the results, while Table 2 and Table 3 show the nonparametric test results and post hoc analysis, respectively. Hoechst 33342 induced blue fluorescence in all nuclei, while Azide 488 induced green fluorescence in proliferating cells. Figure 1A,B,D illustrates a significant number of cells: 948.5 (881.75–1008.5), 1019.5 (938.5–1055.5) and 920.5 (808.5–954.5), respectively. Furthermore, there was a considerable number of proliferating cells: the total cell count for hyperglycemic group was 912.0 (860.5–969.5) and the number of proliferating cells was 141.0 (130.0–163.25). The proliferation rate of cells in group C was 17.5% (13.75–19.75%), indicating significant statistical difference from HMC group with a p-value of 0.017.
Flow cytometry detection of cell apoptosis
Flow cytometry was utilized to evaluate cell apoptosis in various conditions, as depicted in Figure 2. The proportion of apoptotic cells in HMC group, hypoglycemic group and isotonic mannitol group were 2.6% (2.52–2.65%), 3.86% (3.77–3.87%) and 3.41% (3.37–3.53%), respectively, based on the data presented in Table 4. Notably, these rates were significantly lower than the 16.04% (16.03–16.55%) proportion in hyperglycemic group. The survival rates of cells in HMC group, hypoglycemic group and isotonic mannitol group were 93.0% (92.75–93.55%), 93.2% (92.4–93.25%) and 93.8% (93.25–94.0%), respectively. The survival rate of cells in group C was 79.0% (78.55–79.4%). The number of apoptotic cells in hyperglycemic group was statistically significant (p-value = 0.013) compared to HMC group. The nonparametric test results and post hoc analysis are shown in Table 5 and Table 6.
GFP-RFP-LC3 dual fluorescence labeling of autophagy flow
The red fluorescent protein (RFP) emitted red fluorescence, while the green fluorescent protein (GFP) emitted green fluorescence. Yellow spots represented autophagosomes, while red spots represented autolysosomes. As shown in Figure 3, hyperglycemic group had the lowest number of autolysosomes, while HMC group, hypoglycemic group and isotonic mannitol group had more autolysosomes than hyperglycemic group, with rapamycin group having the highest number of autolysosomes.
Expression levels of autophagy markers LC3 and P62 were detected with western blotting
The results indicate that in hyperglycemic group, there was a significant increase in the expression of P62 and an apparent decrease in the expression of autophagy marker LC3-II, while the expression of LC3-I was apparently increased, as compared to HMC group, hypoglycemic group and isotonic mannitol group. These results are presented in Figure 4 and Figure 5. When rapamycin was added to the sample (rapamycin group), the expression level of LC3-II increased significantly, while the expression levels of LC3-I and P62 decreased significantly. The LC3-II/LC3-I ratio was 2.21 (1.95–2.21), which was significantly higher than that of the other groups. Also, the expression level of P62 was 0.38 (0.375–0.39), which was significantly lower than that of the other groups. The levels of P62 expression and the ration of LC3-II/LC3-I in hyperglycemic group significantly differed from those in rapamycin group, with p-values of 0.01 and 0.03, respectively, as displayed in Table 7. Table 8 and Table 9 illustrate the nonparametric test results and post hoc analysis.
The contents of Col4, HA and LA in cells were detected with ELISA
The main elements of ECM were detected using ELISA and displayed in Figure 6. In the hyperglycemic group, the levels of IV collagen fiber (Col4), hyaluronic acid (HA) and laminin (LA) were significantly higher compared to those in the HMC, hypoglycemic and mannitol isotonic groups. The levels were as follows: 18.65 (17.04–19.43) ng/mL, 89.17 (87.73–93.23) ng/mL and 56.71 (47.76–74.76) ng/mL, respectively. Compared to the HMC group, there was a significant difference in Col4 and LA levels with p-values of 0.035 and 0.012, respectively. Upon the addition of rapamycin, the hyperglycemic group demonstrated a significant decrease in the levels of Col4, HA and LA, although no statistical significance was observed, as indicated in Table 10. The nonparametric test results and post hoc analysis are presented in Table 11 and Table 12. Under hyperglycemic conditions, the concentrations of Col4, HA and LA were significantly elevated in comparison to the other groups (represented by the hyperglycemic group as compared to HMC; p < 0.05). Although there was no significant difference between the rapamycin group and the hyperglycemic group, the concentrations of Col4, HA and LA were significantly reduced in the rapamycin group.
Discussion
Diabetic nephropathy has become the predominant cause of chronic kidney disease (CKD) in China.2 Under high glucose conditions, mesangial cells experience abnormal proliferation, resulting in the secretion and deposition of a considerable amount of ECM in the mesangial region.3 Furthermore, high-glucose conditions also impair autophagy activity in glomerular and renal tubular cells.4, 5, 6 An inhibition of such activity reduces ECM degradation and facilitates DN progression. In many studies on the mechanism of mesangial cells and matrix proliferation, autophagy in mesangial cells has received little consideration.7
Autophagy is an intracellular self-degradation process that degrades and recycles misfolded proteins and damaged organelles to maintain cell homeostasis.8, 9 This process is realized through the transport of cytoskeletal microtubule network system, and its molecular mechanism involves essential proteins, such as LAMP1, LAMP2, Rab7, and UVRAG.10 The mTOR signaling pathway is the main regulatory pathway of autophagy.11 It contains at least 2 protein complexes with different functions: mTORC 1 and mTORC 2.12, 13 Of these, mTORC 1, that is a rapamycin sensitive protein complex, is one of the main regulatory factors controlling the activity of ULK1 complex. It can modulate autophagy by phosphorylating and dephosphorylating related components within the ULK1 complex, specifically ULK1 and Atg13.
This study aimed to elucidate the impact of high-glucose condition on HMC. The 5-ethynyl-2-deoxyuridine is a nucleoside analogue of thymine (T) and an effective replacement for T in replicating DNA during cell proliferation. The green fluorescence of newly proliferated cells is observed under a fluorescence microscope after staining with Azide 488, with excitation wavelength falling between 495–519 nm. After performing DNA staining with Hoechst 33342, all cells emit blue fluorescence at an excitation wavelength of 346–460 nm, including newly proliferated cells and previously undivided proliferated cells. The results of EdU cell proliferation experiment showed that the number of nuclei and cell proliferation in hyperglycemic group were significantly reduced compared with the other 3 groups (912.0 (860.5–969.5) and 141.0 (130–163.25, respectively), and the proliferation rate was the lowest: 17.5 (13.75–19.75).
The flow cytometry results indicated that the percentage of mesangial cell apoptosis was 16.04% (16.03–16.55%) under high-glucose conditions, surpassing that of the other groups. The high-glucose conditions significantly affected cell survival rates, while the proliferation rate and apoptosis number of cells in the mannitol isotonic group did not differ significantly from those in the HMC group. This finding further demonstrates that high-glucose conditions or its glucose metabolites can impede cell proliferation and promote cell apoptosis, but this effect is unaffected by high osmotic state. Second, this study confirmed that high-glucose conditions inhibited HMC proliferation and autophagy activity. Autophagy flow detection can determine autophagy state. Changes in autophagy flow are primarily monitored using GFP–RFP–LC3 tandem fluorescent protein labeling, which has sensitivity different to lysosomal acidic microenvironment.14
The strength of autophagy can be assessed through red and yellow fluorescence intensity. This study utilized a GFP–RFP–LC3-labeled autophagy flow experiment to demonstrate that the hyperglycemic group had significantly diminished autolysosome count compared to the HMC group, indicating subdued autophagy activity. Previous research has demonstrated that inhibition of autophagy exacerbates oxidative damage in cells. Damaged and aging organelles and macromolecules within cells may not be eliminated in a timely manner, resulting in their accumulation and worsening of cellular damage, aging, and eventual promotion of cell apoptosis. This process could potentially be one of the mechanisms behind the development of DN.15, 16, 17
LC3 and P62 are proteins commonly used as markers for autophagy, reflecting both its expression and intensity. LC3 is found in 2 forms, LC3-I and LC3-II. Initially, Atg4 cleaves the precursor LC3 to produce LC3-I, which later, under the activation of Atg7, generates its membrane binding form (LC3-II), which locates at autophagosome and autolysosome membranes18. The changes in LC3-II protein content can offer insight into the changes in autophagic structures, such as autophagosomes and autolysosome.19
P62, a protein that binds to ubiquitin, is essential for autophagy and serves as a selective autophagy receptor that links LC3 to the ubiquitinated substrate targeted for degradation.20 Western blotting analysis revealed that the expression of LC3-II was significantly lower in the hyperglycemic group compared to the HMC group. Additionally, the LC3-II/LC3-I ratio was reduced, while P62 expression levels were elevated, strongly indicating that hyperglycemia significantly inhibits cellular autophagy activity.
Under high-glucose conditions, inhibition of autophagy decreases ECM degradation, resulting in an ECM synthesis and degradation imbalance. This leads to the accumulation of ECM components such as Col4, HA and LA in the mesangial region of the kidney. Significant increases in Col4, HA and LA contents were observed in the hyperglycemic group compared to other groups.
Rapamycin is a known inducer of autophagy. The study revealed that the addition of rapamycin into the HMC of the hyperglycemic group resulted in a significant increase in the expression of LC3-II compared to other groups. Additionally, the expression of LC3-I and P62 decreased significantly, indicating that rapamycin could activate inhibited autophagy. The levels of Col4, HA and LA of ECM components were reduced in the rapamycin group, as confirmed with ELISA. This indicates that after autophagy was activated, the accumulation of ECM decreased significantly, leading to an improvement in the condition. Nonetheless, no statistical difference was observed between the rapamycin group and the hyperglycemic group, which may be due to the small sample size.
Limitations
The study had a small sample size and an imperfect test grouping. At the outset, the rapamycin group did not undergo cell proliferation or apoptosis tests, and the ELISA test did not yield statistically significant differences in HA, resulting in poor persuasiveness. Additionally, no experiments were conducted to verify rapamycin’s autophagy induction effect.
Conclusions
Autophagy activity of glomerular mesangial cells is significantly inhibited under high-glucose conditions, but rapamycin can induce cell autophagy, improve cellular metabolic processes, and reduce mesangial cell proliferation and matrix hyperplasia, thereby inhibiting the progression of DN. This provides a theoretical basis for the clinical application of rapamycin in DN treatment.
Data availability
The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.
Consent for publication
Not applicable.
