Berberine regulates mesangial cell proliferation and cell cycle to attenuate diabetic nephropathy through the PI3K/Akt/AS160/GLUT1 signalling pathway

Abstract High glucose (HG) is one of the basic factors of diabetic nephropathy (DN), which leads to high morbidity and disability. During DN, the expression of glomerular glucose transporter 1 (GLUT1) increases, but the relationship between HG and GLUT1 is unclear. Glomerular mesangial cells (GMCs) have multiple roles in HG‐induced DN. Here, we report prominent glomerular dysfunction, especially GMC abnormalities, in DN mice, which is closely related to GLUT1 alteration. In vivo studies have shown that BBR can alleviate pathological changes and abnormal renal function indicators of DN mice. In vitro, BBR (30, 60 and 90 μmol/L) not only increased the proportion of G1 phase cells but also reduced the proportion of S phase cells under HG conditions at different times. BBR (60 μmol/L) significantly reduced the expression of PI3K‐p85, p‐Akt, p‐AS160, membrane‐bound GLUT1 and cyclin D1, but had almost no effect on total protein. Furthermore, BBR significantly declined the glucose uptake and retarded cyclin D1‐mediated GMC cell cycle arrest in the G1 phase. This study demonstrated that BBR can inhibit the development of DN, which may be due to BBR inhibiting the PI3K/Akt/AS160/GLUT1 signalling pathway to regulate HG‐induced abnormal GMC proliferation and the cell cycle, supporting BBR as a potential therapeutic drug for DN.


| INTRODUC TI ON
Diabetic nephropathy (DN) is a common and serious chronic kidney disease that is the main reason for the high mortality of diabetes and its expanding global burden. 1 Current clinical treatment strategies can only delay its progression to a certain extent and cannot prevent its progression to end-stage renal disease. 2,3 Meanwhile, authoritative research believes that abnormal cell status will cause a series of cascading effects, promote pathological changes and damage to the kidney, and accelerate the progression of renal dysfunction and DN. 4 Among them, the abnormal proliferation of glomerular mesangial cells (GMCs) exists as early as the early stage of DN, and it plays an increasingly important role. 5 However, the exact mechanism is still unclear, and there is no ideal strategy to prevent abnormalities in GMCs and the resulting disease development. Therefore, the specific mechanisms and effective treatment strategies need to be studied in depth.
High glucose (HG) is the basic factor that accelerates the occurrence and development of DN. In recent years, several glucose transporters have been found in glomeruli and cultured glomerular cells, such as facilitating glucose transporters (GLUT1, 3 and 4) and sodiumglucose cotransporters (SGLTs). 6,7 The existing research mainly focuses on exploring the roles of SGLTs and promoting the clinical application of their inhibitors in the field of diabetes and its complications. 8,9 However, the role and mechanism of GLUT in the glomerulus are rarely mentioned. At the same time, multiple studies have found that hyperglycaemia can not only activate the abnormal proliferation of GMCs in DN animal models 10 but also has similar effects in in vitro studies, which is worthy of attention. 11 Moreover, abnormal expression of GLUT1 also appeared in the mesangial area stimulated by HG, but the specific mechanism has not been well elucidated. 12 Based on the above research, we aimed to explore the changes and corresponding mechanisms of GLUT1 in GMCs stimulated by HG and in DN.
The phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathway has been confirmed to be involved in many cellular processes, such as cell proliferation, differentiation, cell cycle progression and tumour growth. 13 In the liver cancer, the PI3K/Akt pathway can activate GLUT1 signalling to regulate insulin-dependent glucose metabolism. 14,15 During this process, some studies have found that AS160 (Akt substrate of 160 kDa/TbcId4), as a substrate of Akt, promotes GLUTs-mediated glucose metabolism by binding to GLUTs (GLUT1 or GLUT4) vesicles and the plasma membrane, which provides power to activate the cell cycle alterations and cell proliferation. 16,17 Our previous research found that HG can activate the PI3K/Akt signalling of podocytes to promote the development of DN. 18 In addition, preliminary research results indicate that HG conditions can cause abnormal changes in GMCs, and at this time the expression of GLUT1 is also significantly increased. In view of this, further in-depth research is needed to investigate the relationship among HG-induced abnormal GMC proliferation, the PI3K/Akt pathway and GLUT1 alterations in DN.
In recent years, traditional Chinese medicine has attracted increasing attention due to its outstanding performance in treating SARS-CoV-2-induced pneumonia, cardio-/cerebrovascular diseases and diabetes and its complications. [19][20][21] Among these, berberine (BBR), a famous herb extract generally extracted from rhizoma coptidis rhizomes and cortex phellodendri, has been reported to have potent renoprotective effects. 22 Multiple studies have shown that BBR can not only reduce hyperglycaemia, regulate dyslipidaemia and attenuate kidney inflammation but also improve insulin resistance and enhance insulin activity in an animal model of diabetes. 22,23 We previously reported that BBR can enhance autophagy by inhibiting PI3K/Akt signalling, thereby reducing glomerular podocyte injury in streptozocin (STZ)-induced DN. 18 However, the effect and underlying mechanism of BBR on GMCs in DN remain to be further studied.
Therefore, the present study aimed to evaluate the effect of BBR on abnormal GMC proliferation in DN and to further explore the underlying mechanism and relationship among BBR, abnormal GMC proliferation, the PI3K/Akt pathway and GLUT1 alterations, which will offer insights into controlling cellular responses to hyperglycaemia that initiate the progression of DN.

| Animals model and experimental design
Male C57BL/6 mice (6-8 weeks) were purchased from the Experimental Animal Centre of Anhui Medical University (Hefei, China) and provided with adaptable feeding (free access to standard food and water, 22 ± 2°C and humidity of 60 ± 5%) for 7 days. The

| Biochemical analyses
Collected blood was centrifuged for testing of blood urea nitrogen (BUN), serum creatinine (Scr) and the ratio of urine total protein (UTP) using an automated biochemistry analyser (Model 7600 Series Automatic Analyzer, Hitachi Corporation) as described previously. 24

| Kidney histopathology
The fixed kidney tissues were embedded in paraffin and crosssectioned (4 μm) for histological examination. Haematoxylin and eosin and periodic acid-Schiff (PAS) staining were performed as described previously. 25 Finally, the mesangial expansion index and tubular-interstitial injury index of renal tissue were evaluated from six randomly selected fields. 26

| Transmission electron microscopy
Kidney tissue was fixed in 2.5% glutaraldehyde and 1% osmic acid at 4°C. Sections were with 1% uranyl acetate and embedded in epoxy resin (EPON) polymerized in gelatin capsules at 60°C for 48 h. 26 The sections were observed using a transmission electron microscope (JEM1400, Hitachi).

| Immunohistochemistry and immunohistofluorescence
Renal sections were heated in a microwave at 60°C for 2 h and incubated with 3% hydrogen peroxide. The paraffin tissue sections were treated with anti-GLUT1 (1:250) and anti-Cyclin D1 (1:200) antibodies for 24 h at 4°C, followed by secondary antibodies (goat anti-rabbit IgG/H&L for 30 min and rabbit immunoglobulin G/Cy3 for 60 min) at 37°C. After staining with DAB for 5 min and DAPI for 5-8 min, the slides were observed under a microscope (Zeiss Spot, Carl Zeiss).

| Flow cytometry
Glomerular mesangial cells were washed twice with cold phosphate buffer solution (PBS) and fixed in cold 70% ethanol overnight at 4°C. Fixed cells were washed with cooled PBS and stained using a cell cycle assay kit. The experiment was repeated three times. Finally, samples were analysed on a Beckman Coulter instrument, and data were collected for 10,000 single-cell events.
The percentage of cells in the G1, S and G2 phases of the cell cycle was determined by CytExpert software. Finally, data are presented as histograms.

| Fluorescent EdU
Glomerular mesangial cells at a density of 1 × 10 4 cells/well were cultured in 24-well plates for 24 h, and then the EdU assay was performed using fluorescence microscopy (DM2000, Leica) in different groups. Briefly, the cells were fixed with 4% paraformaldehyde (PFA) for 15 min at room temperature, and then permeabilized with 0.3% Triton X-100 for 15 min. Then, the click reaction solution was added and samples were incubated for 30 min in the dark. After staining with Hoechst solution for 10 min, images were obtained with a microscope and were analysed with Image-Pro Plus. The EdU incorporation rate was calculated as the ratio of EdU-positive cells (green cells) to total Hoechst-positive cells (blue cells). (1:1000), anti-GLUT1 (1:1000) and secondary antibody. All of the immunoblots were detected with HRP western blotting detection reagents (Millipore Corporation). Equal loading was confirmed using anti-GAPDH or antiβ-actin antibody (1:1000).

| RT-qPCR analysis
To detect the relative mRNA level, a Transcription First Strand cDNA Synthesis kit (Vazyme Biotech) was used to perform the reverse transcription. The reaction conditions were as follows: 50℃ for 15 min and 85℃ for 5 s. A PCR reaction system was prepared using SYBR ® -Green Real-Time PCR Master mix (Thermo Fisher Science, ABI7500). The PCR reaction conditions were as follows: 95℃ for 10 s, followed by 40 cycles of 10 s at 95℃ and 30 s at 60℃. GAPDH served as an internal control to normalize the relative expression of GAPDH, PI3K, Akt, GLUT1 and AS160. All data were quantified using the 2 −ΔΔC t method and run-in triplicate for each sample. The primer sequences used in RT-qPCR are listed in Table 1.

| 2-NBDG assay for glucose uptake
Briefly, GMCs were seeded at a concentration of 1 × 10 5 cells/ml/ well in duplicate in a six well plate and incubated overnight at 37℃.
The cells were washed twice with cold KBR after HG treatment and then cultured with 2-NBDG for 30 min at 37°C. Finally, the cells were washed twice with cold KBR buffer to halt the glucose uptake and then resuspended in KBR for fluorescence detection using a flow cytometer at a fluorescence excitation wavelength of 488 nm and emission wavelength of 520 nm.

| Statistical analysis
Data were assessed using SPSS 23.0 (IBM Corporation). Significant differences were evaluated using one-way ANOVA with a post hoc Bonferroni correction (GraphPad Prism 5.0; GraphPad Software). A two-sided p-value <0.05 was considered significant. The data are presented as the mean ± SEM.

| BBR ameliorates renal pathological changes in a DN mouse model
To test whether berberine provides renal protection in the high-  Figure 1D).

| BBR changes the PI3K/Akt pathway in DN mice
As shown in Figure

| BBR regulates cell cycle redistribution of GMCs
In this section, we used flow cytometry to explore the effect of HG and BBR on regulating the cell cycle redistribution of GMCs

| BBR affects GLUT1-mediated glucose uptake, cell cycle redistribution and abnormal GMCs proliferation
Based on the above results, BBR can inhibit the signal transduction of the PI3K/Akt/AS160/GLUT1 pathway to reduce the level of membrane GLUT1. In Figure 6A, a 2-NBDG assay was used to measure the glucose uptake, and the results indicated that HG significantly in-

| DISCUSS ION
The present study explored the renoprotective role of BBR in mouse  Figure 4B; *p < 0.05, vs. HG-12h in Figure 4C,4D. HG, high glucose abnormal GMC proliferation, and BBR showed an ameliorating effect in these mice at 12 weeks. Moreover, excessive glycogen deposition demonstrated excessive glucose metabolism in the glomerular area compared with that of control mice, and BBR reduced glycogen deposition. Transmission electron microscopy results also show that berberine can significantly reduce the GBM thickening and foot process fusion. Given all this, hyperglycaemia may induce additional glucose uptake, which contributes to dysfunction of GMCs, eventually causing glomerular damage and abnormal kidney function. 28 When treated with BBR, these abnormal changes can be significantly improved, and the effect is similar to metformin, which indicates that BBR is likely to exert its renoprotective effect through its glucose regulation function.
Some studies have demonstrated that the GLUT1-modulated glucose metabolism can be activated and regulated by the PI3K/ Akt pathway. [29][30][31] During this process, the Akt 160 kDa/TbcId4 substrate, named AS160, can bind to GLUT1 vesicles and the plasma membrane to regulate glucose metabolism and provide power to activate cell cycle alterations and cell proliferation. 32 In the cur- This supports our hypothesis that major pathological responses are generated from HG-induced abnormal GMC proliferation, and HG most likely stimulates GMCs to enter S phase, and increasing the total proportion of G1 phase cells by BBR treatment could exert a renoprotective effect. Therefore, it may be possible to interfere with the G1 and S phase distribution to ameliorate abnormal GMC proliferation by BBR treatment.
Studies have shown that GLUT1 is a vital regulator glucose homeostasis throughout the body, and its membrane recruitment is essential for the glucose uptake process. 34,35 In addition, a study indicated that the PI3K/Akt pathway functions as an upstream signalling pathway to stimulate the GLUT1 translocation to the cell surface in multiple tumour cells, thereby promoting glucose uptake. 31,36 During this process, GLUT1 translocation is mediated by AS160, which is activated through Akt phosphorylation. 29 In the present study, BBR played a similar role to the PI3K inhibitor LY294002, which indicated that BBR can significantly inhibit HG-induced PI3K-p85 expression. Subsequently, the elevated expression levels of p-Akt, p-AS160 and membrane GLUT1 were significantly decreased after treatment with BBR, while their total protein expression changed undistinguishably. These results indicate that BBR reduces the membrane transport of GLUT1 protein when inhibiting PI3K/Akt/AS160 signal transduction rather than changing the level of total GLUT1 protein. This is consistent with the mechanism of action of GLUTs reported by authoritative research. Meanwhile, we found that mRNA had the same changing trend as proteins. These original observations provide the important knowledge that BBR inhibits the PI3K/Akt/AS160 signalling pathway to reduce the overexpression and membrane transport of GLUT1, as well as the abnormal cell cycle and GMC proliferation, eventually relieving the corresponding pathological changes.
In conclusion, the present study demonstrated that BBR could ameliorate DN progression to a certain degree. The mechanism may involve BBR inhibiting the activation of the PI3K/Akt/AS160/ GLUT1 signalling pathway to prevent the HG-induced abnormal GMC proliferation by retarding the cell cycle to remain at G1 phase.
Therefore, regulating the cell cycle of GMCs is a potential therapeutic strategy, and BBR can also be considered as a promising therapeutic drug in the treatment of DN. Despite the advantages of BBR, its clinical application still has certain limitations, the most important of which is its low bioavailability due to its poor water solubility and gastrointestinal absorption. In view of this, in the future research, we will pay more attention to the structural modification and bioavailability of BBR while exploring its renoprotective mechanism.

CO N FLI C T O F I NTE R E S T
The authors declare no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.