Mesangial cells possess irregular structures comprised of flattened cylinder-like cell bodies and contain actin, myosin, and α-actinin at both ends, granting them contractile features [1
]. The anchoring filaments from mesangial cells to the glomerular basement membrane can influence glomerular capillary blood flow [2
]. The structural support of mesangial cells in kidney glomeruli counteracts the expansible forces created by pressure gradients in capillary vessels and manipulates the capillary blood flow [4
]. Glomerular mesangial cells migrate in response to platelet-derived growth factor (PDGF) and angiotensin II, which is crucial for glomerulopathy and glomerular development [5
]. There is emerging evidence for the role of the abnormal migratory polarity of mesangial cells due to glomerular injury in the pathogenesis of proliferative glomerulonephritis [4
]. Glomerular injury-triggered molecular changes in mesangial cells in glomerulonephritis still remain elusive. In general, directional cell migration entails the establishment of cytoskeletal alterations that are essential for actin polarization and focal adhesion turnover [9
]. In the same context, the migratory process is controlled by coordinated actin dynamics and focal adhesion turnover at the peripheral ruffles in migrating mesangial cells [6
]. However, identifying the key molecular players in motility and clear-cut molecular functions remains challenging.
Glucose toxicity is a primary cause of glomerular injury in diabetic kidney diseases, and involves the production of advanced glycation end products (AGE) [11
]. Increased glucose levels result in glomerular oxidative stress via activation of metabolic pathways, which consequently produces and accumulates AGE, leading to glomerular injury [12
]. The activation of the AGE-receptor for advanced glycation end products (RAGE) influences endothelial actin cytoskeletal rearrangement and dysfunction [14
]. A recent report shows that AGE-induced oxidative stress stimulates proliferation and migration of vascular smooth muscle cells [16
]. Dynamics of focal adhesions containing vinculin and paxillin is entailed for cell polarization and motility, and extracellular matrix remodeling [17
]. Cell migration entails coordination between focal adhesion and actin cytoskeleton via F-actin-binding vinculin abundant in integrin-based focal adhesions [17
]. Exposure of retinal pericytes to AGE induces cell migration via phosphorylation of focal adhesion kinase and paxillin [19
]. Connective tissue growth factor prompts mesangial cell migration via disassembly of focal adhesion complexes and activation of cell polarization [20
]. On the other hand, autophagy is responsible for the degradation of AGE by the upregulation of lysosomal biogenesis and function in diabetic nephropathy (DN) [21
]. Accordingly, strategies aimed at enhancing the lysosomal function of autophagy-related proteins can hold promise for treating DN. One study shows that autophagy is likely to be a mechanism triggered to repair the reactive oxygen species-induced loss of the AGE-treated cells and thereby prompts cell survival [22
Numerous studies have demonstrated that natural compounds display renoprotective effects by diverse mechanisms [23
]. Polyphenolic flavonoids are known to attenuate hyperglycemia-induced renal endothelial barrier dysfunction, urinary albumin excretion, and glomerular hyperfiltration [23
]. Our previous studies showed that several natural compounds counteracted renal mesangial fibrosis and inflammation, renal tubulointerstitial fibrosis, glomerulosclerosis, and podocyte injury [26
]. Chrysin (5,7-dihydroxyflavone, Figure 1
A), a flavonoid abundant in edible plants such as passion flowers, mushrooms, honey, and bee propolis, is known to exhibit multiple biological effects, including anti-inflammatory, anti-atherogenic, and neuroprotective properties [29
]. In addition, growing evidence suggests that chrysin may display nephroprotective activities in rodents [32
]. Thus, the present study attempted to explore the renoprotective effects of chrysin on the formation of F-actin cytoskeleton and focal adhesion complex in AGE-exposed mesangial cells and diabetic mouse kidneys. Our recent study demonstrates that chrysin may inhibit glucose-mediated AGE-associated glomerulosclerosis and fibrosis [34
]. However, little is known about the renoprotective role of chrysin in actin cytoskeleton rearrangement, focal adhesion formation, and cell migration due to AGE. One investigation shows that the flavonoid myricetin suppresses retinal pericytes migration through suppressing activation of ERK1/2-focal adhesion kinase-paxillin by AGE [19
]. Whether the presence of chrysin modulated mesangial cell proliferation and motility by AGE was also examined. This study further elucidated how chrysin manipulated autophagy as a molecular player involved in the actin cytoskeleton, focal adhesion assembly, and motility.
2. Materials and Methods
Fetal bovine serum (FBS), penicillin–streptomycin and trypsin–EDTA, were provided by BioWhittaker (San Diego, CA, USA). 3-(4, 5-Dimetylthiazol-yl)-diphenyl tetrazolium bromide (MTT) was obtained from DUCHEFA Biochemie (Haarlem, The Netherlands). Dulbecco’s modified Eagle media (DMEM), nutrient mixture F-12 Ham medium, mannitol, and D-glucose, were supplied by Sigma-Aldrich Chemical (St. Louis, MO, USA), as were all other reagents unless specifically stated otherwise. Antibodies of F-actin, α-smooth muscle actin (α-SMA), Arp2/3 antibody, mTOR, and phospho-mTOR were supplied by Abcam (Cambridge, UK). Antibodies of beclin-1, cortactin, Fascin1, and Ena/VASP (EVL) were provided by Santa Cruz Biotechnology (Dallas, TX, USA). Phospho-vinculin antibody was obtained from Biorbyt (Cambridge, UK). Atg7 antibody was purchased from Aviva system Biology (San Diego, CA, USA). Antibodies of Atg3 and phospho-paxillin were obtained from Cell Signaling Technology (Beverly, CA, USA). LC3 antibody was supplied by MBL International Corporation (Woburn, MA, USA). AGE-bovine serum albumin (AGE-BSA) was provided by Merck Millipore (Billerica, MA, USA). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG, goat anti-mouse, and donkey anti-goat IgG were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). SB03580 (MAP kinase inhibitor) was provided from Calbiochem (Billerica, MA, USA)
Chrysin (Sigma-Aldrich Chemical, St. Louis, MO, USA) was dissolved in dimethyl sulfoxide (DMSO) for live culture with cells; a final culture concentration of DMSO was <0.5%.
2.2. Culture of Human Renal Mesangial Cells (HRMC)
HRMC (Sciencell Research Laboratories, Carlsbad, CA, USA) were cultured at 37 °C humidified atmosphere of 5% CO2 in air. Routine culture of HRMC was performed in DMEM/F12 (7:1) media containing 15% FBS, 2 mM glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin. HRMC in 6-10th passage were sub-cultured at 80% confluence and used for further experiments. To mimic diabetic glomerular injury caused by chronic hyperglycemia, HRMC was incubated in 33 mM glucose- or 100 μg/mL AGE-BSA-supplemented DMEM containing 2% FBS and 2–8 μg/mL insulin for 3 days in the absence and presence of 1–20 μM chrysin. For osmotic control incubation, another set of HRMC was cultured in DMEM (5.5 mM) containing 2% FBS (+2 μg/mL insulin) and supplemented with 27.5 mM mannitol. Culture media was collected and stored at −20 °C.
After the 3-day incubation in 33 mM glucose and 100 μg/mL AGE-BSA, the MTT assay was routinely carried out for measuring cell proliferation. After the unconverted MTT was removed, cells were dissolved in isopropanol with gentle shaking. The absorbance of formazan dye was measured at λ
= 570 nm with background subtraction using λ
= 690 nm. There was no cytotoxicity of 1–20 μM chrysin per se observed [34
2.3. In Vivo Animal Experiments
Adult male db/db mice (C57BLKS/+Leprdb Iar; Jackson Laboratory, Sacramento, CA, USA) and their age-matched non-diabetic db/m littermates (C57BLKS/J; Jackson Laboratory, Sacramento, CA, USA) were used in the present study. Mice were kept on a 12 h light/12 h dark cycle at 23 ± 1 °C with 50 ± 10% relative humidity under specific pathogen-free conditions, fed a standard laboratory chow diet (CJ Feed, Seoul, Korea), and were provided with water ad libitum at the animal facility of Hallym University. This study included db/db mice at 7 weeks of age because they begin to develop diabetes (hyperglycemia) at the age of 7–8 weeks. The animals were allowed to acclimatize for a week before beginning the experiments. Mice were divided into three subgroups (n = 7–9 for each subgroup). The first group of mice was non-diabetic db/m control mice and db/db mice were divided into two groups. One group of db/db mice was daily supplemented 10 mg/kg BW chrysin via gavage for 10 weeks. Food intake, body weight, and drinking water intake of db/db mice increased, relative to those of db/m controls. However, the supplementation of chrysin led to a decline in water drinking after the 6–7th week. The measurement of fasting blood levels of glucose and glycated hemoglobin HbA1C were conducted every other week from mouse tail veins. Chrysin treatment diminished plasma levels of glucose and HbA1C elevated in db/db mice. The 24 h urine samples were collected in metabolic cages during the 10-week chrysin supplementation. The urine volume was reduced in chrysin-treated mice by ~50–60%, and diabetic proteinuria was alleviated.
All experiments were approved by the Committee on Animal Experimentation of Hallym University and performed in compliance with the University’s Guidelines for the Care and Use of Laboratory Animals (hallymR1 2016-10). No mice died and no apparent signs of exhaustion were observed during the experimental period.
2.4. Western Blot Analysis
Western blot analysis was conducted using whole-cell lysates and tissue extracts prepared from HRMC at a density of 3.0 × 105 cells. Whole-cell lysates and mouse renal tissue extracts were prepared in a lysis buffer containing 1 M β-glycerophosphate, 1% β-mercaptoethanol, 0.5 M NaF, 0.1 M Na3VO4, and protease inhibitor cocktail. Cell lysates and tissue extracts containing equal amounts of total proteins were electrophoresed on 6–15% SDS-PAGE and transferred onto a nitrocellulose membrane. Non-specific binding was blocked by soaking the membrane in a TBS-T buffer (50 mM Tris–HCl (pH 7.5), 150 mM NaCl, and 0.1% Tween 20) supplemented 3% BSA for 3 h. The membrane was incubated with an antibody to F-actin, α-SMA, phospho-paxillin, phospho-vinculin, cortactin, Arp2/3, fascin-1, EVL, beclin-1, LC3, Atg3, Atg7, mTOR, or phospho-mTOR. The membrane was then incubated with a secondary antibody of goat anti-rabbit IgG, goat anti-mouse IgG, or donkey anti-goat IgG conjugated to HRP. Each protein level was determined by using Supersignal West Pico Chemiluminescence detection reagents (Pierce Biotechnology, Rockford, IL, USA) and Konica X-ray film (Konica Co., Tokyo, Japan). Incubation with mouse anti-human β-actin was conducted for the comparative control.
2.5. Rhodamine-Phalloidin Staining of F-Actin
HRMC (7 × 104 cells) grown on 24 well glass chamber slides were exposed to 33 mM glucose or 100 μg/mL AGE-BSA in the absence and presence of 1–20 μM chrysin. Cells were fixed in 4% formaldehyde for 10 min and washed with pre-warmed phosphate-buffered saline (PBS). Subsequently, 10 units of the fluorescent dye rhodamine phalloidin were added to cells and incubated for 20 min. Nuclear staining was also conducted by using 4 mg/mL 4’,6-diamidino-2-phenylindole (DAPI). Each slide was mounted in VectaMount mounting medium (Vector Laboratories, Burlingame, CA, USA). Fluorescent images were taken with an Axiomager Optical fluorescence microscope (Zeiss, Oberkochen, Germany).
2.6. Mesangial Cell Motility
To assess the effects of glucose and AGE on mesangial cell motility in the absence and presence of chrysin, an in vitro scratch wound assay was employed. Briefly, mesangial cells were seeded onto a 12-well plate and incubated for 24 h in 10% FBS-containing media. Subsequently, confluent cells were scratched away horizontally on each well using a pipette tip. After scratching, injured cells were incubated for another 24 h in serum-free culture media containing 100 μg/mL AGE-BSA with and without 1–20 μM chrysin. Images of the scratches were photographed in the 2–3 microscopic fields per well using a microscope with CCD camera (Motic®, Wetzlar, Germany). A reduction of the scratched area indicates a sign of mesangial cell migration.
2.7. Immunocytochemical Staining
Immunofluorescent cytochemical staining was conducted to reveal the co-localization of Atg7 to F-actin fibers in mesangial cells grown on 24-well chamber slides. Cells were fixed with 4% formaldehyde for 20 min and permeated with 0.1% Triton X-100 for 10 min on ice. Cells were specifically blocked with 20% FBS for 1 h, and a primary antibody of Atg7 and a secondary antibody of FITC-conjugated IgG were applied to cells. Subsequently, the fluorescent dye rhodamine phalloidin was added to cells, and incubated for 20 min for F-actin staining. Nuclear counterstaining was performed with DAPI. Each slide was mounted in a mounting medium and images of each slide were taken using an optical Axiomager microscope (Zeiss, Oberkochen, Germany).
2.8. Data Analysis
The results are presented as mean ± SEM for each treatment group. Statistical analyses were performed using Statistical Analysis Systems statistical software package version 6.12 (SAS Institute Inc., Cary, NC, USA). Significance was determined by one-way ANOVA, followed by Duncan range test for multiple comparisons. Differences were considered significant at p < 0.05.
Ten major findings were observed in this study. (1) The enhanced mesangial F-actin induction and bundle formation by 33 mM glucose or 100 μg/mL AGE-BSA were attenuated by the presence of ≥10 μM chrysin in mesangial cells. (2) The mesangial induction of α-SMA by glucose and the tissue level of α-SMA in diabetic kidneys were reduced by chrysin, indicating its blockade of mesangial proliferation. (3) Treating chrysin inhibited the activation of vinculin and paxillin and the induction of cortactin, ARP2/3, fascin-1, and EVL in glucose- or AGE-exposed mesangial cells. (4) Oral treatment of 10 mg/kg chrysin diminished renal tissue levels of cortactin and fascin-1 elevated in diabetic mouse kidneys. (5) Mesangial cell motility was enhanced in glucose- or AGE-exposed mesangial cells, which was notably attenuated by adding chrysin. (6) Chrysin dampened the induction of autophagy-related genes of beclin-1, LC3 I/II, Atg3, and Atg7 in mesangial cells exposed to AGE. (7) Oral administration of chrysin lessened the tissue levels of beclin-1 and LC3 I/II enhanced in diabetic kidneys. (8) Chrysin reduced the mTOR activation in AGE-exposed mesangial cells and diabetic kidneys. (9) The presence of chrysin attenuated the induction of Atg7 localized to the F-actin cytoskeleton in mesangial cells. (10) The induction of mesangial F-actin, cortactin, and fascin-1 by AGE was near-completely abolished by the inhibition of autophagy. Therefore, chrysin may hamper diabetes-associated mesangial cell protrusion and migration through disturbing actin dynamics by interfering with autophagy via the blockade of beclin-1 and LC3 I/II.
The irregular mesangial structures in kidney glomeruli influence the blood capillary flow through manipulating expansible forces created by pressure gradients in glomerular vessels [4
]. A growing body of evidence shows that the aberrant proliferation and migratory polarity of mesangial cells plays a role in the pathogenesis of glomerulonephritis due to glomerular injury [4
]. However, the underlying molecular changes for mesangial aberrant proliferation and motility during glomerular injury remain elusive. The cell migratory process is controlled by well-coordinated actin dynamics and focal adhesion turnover at the peripheral ruffles in migrating cells [6
]. Numerous studies have shown that glomerular mesangial cells migrate in response to pathophysiological stimuli such as PDGF, angiotensin II, oxidants and inflammatory cytokines [5
]. Accordingly, alterations in induction and localization of cytoskeletal regulators trigger mesangial phenotypic alterations in glomerulonephritis [6
]. However, identifying the key molecular components steering motility and actin functions in mesangial cells remains challenging. This study investigated the mesangial dynamics of F-actin cytoskeleton and integrin-based focal adhesions in glomerular injury due to glucose and AGE implicated in the pathogenesis of diabetic complications. It was found that glucose and AGE prompted aberrant F-actin polymerization, bundling, and motility in mesangial cells. AGE induces alterations in cell morphology and function by affecting the cytoskeletal structure through activation of the receptor for AGE (RAGE) [14
Several studies have demonstrated that actin-targeting natural plant compounds influence actin dynamics, prompting either polymerization or depolymerization in signaling pathways through diverse mechanisms [42
]. A recent study shows that salvianolic acid A inhibits renal cytoskeletal dysfunction through inhibiting the AGE–RAGE signaling, thus effectively ameliorating early-stage DN [44
]. The polyphenol honokiol counteracts with cisplatin-induced cytoskeletal structure disruption in renal epithelial cells and preserves epithelial cell polarity and morphology [45
]. Our previous study found that chrysin, a naturally-occurring flavonoid, inhibited AGE-induced kidney fibrosis in renal mesangial cells and diabetic kidneys through inhibition of AGE–RAGE activation, contributing to blockade of glucose-mediated AGE-associated glomerulosclerosis and fibrosis [34
]. The current study examined whether chrysin inhibited glucose- and AGE-stimulated actin cytoskeleton rearrangement and focal adhesion dysfunction in renal mesangial cells. Herein, chrysin diminished the F-actin fiber formation highly enhanced by glucose and AGE. Chrysin attenuated the activation of F-actin-binding focal adhesion proteins of vinculin and paxillin in diabetic mesangial cells. This study also found that this compound encumbered the mesangial cell induction of cortactin, Arp2/3 complex, EVL, and fascin-1, all involved in actin polymerization and actin-crosslinking/bundling. The EVL proteins enhance in the cellular processes including axon guidance and cell migration as modulators of the actin cytoskeleton and cell migration [46
]. Unexpectedly, the renal tissue levels of F-actin, cortactin, and fascin-1 were reduced in diabetic mouse kidney, which was counteracted by chrysin. The kidney is composed of several different types of cells such as podocytes and glomerular vascular cells, and the mesangial cell population in the whole kidney is very small. Accordingly, the overall renal tissue levels of F-actin, cortactin, and fascin-1 might be lowered in diabetic kidneys comprised of diverse types of glomerular cells. Unlike mesangial cells, the induction of F-actin and cortactin was reduced in high glucose-exposed podocytes. Nevertheless, these findings shed light on a therapeutic role for chrysin in modulating aberrant actin remodeling and abnormal coordination between F-actin and focal adhesion in mesangial cells during glucose or AGE-induced glomerular injury.
Autophagy serves as a vital mechanism to maintain kidney homeostasis, and its impairment is implicated in the pathogenesis of DN [47
]. One investigation shows that autophagy is involved in tumor cell motility and focal adhesion disassembly through targeted degradation of paxillin interacting with processed LC3 [48
]. The autophagy inhibition reduces tumor cell migration and invasion and attenuates metastasis [48
]. The present study demonstrated that glucose and AGE enhanced mesangial cell autophagy through inducing the beclin-1-LC3 I/II-Atg pathway, and that chrysin encumbered such diabetic induction of autophagy. This study further attempted to reveal that autophagy contributed to F-actin polymerization/bundling and focal adhesion dysfunction in the diabetic milieu. The presence of chrysin in mesangial cells reduced the induction of Atg7 localized to the F-actin cytoskeleton. Consistently, one study shows that a mouse Atg7 knockout inhibiting autophagosome formation displays severe defects in actin assembly owing to reduced expression of proteins involved in controlling actin dynamics [49
]. Additionally, the inhibition of autophagy deterred the induction of mesangial F-actin, cortactin, and fascin-1 by AGE, indicating that actin polymerization and bundling entailed autophagic activity. On the other hand, chrysin suppressed the mTOR activation in AGE-exposed mesangial cells and diabetic kidneys. Similarly, one study shows that paeoniflorin attenuates AGE-induced mesangial cell damage through inhibiting the RAGE/mTOR/autophagy pathway [50
]. Moreover, the blockade of mTOR activation highly attenuated the mesangial induction of F-actin, cortactin, and fascin-1. Growing evidence has suggested the interconnections of mTOR to autophagy in cellular signaling pathways in kidneys [51
]. Unfortunately, this study did not examine reciprocal interconnection of mTOR to autophagy in mesangial proliferation and motility due to AGE. Inhibition of mTOR attenuates the actin crosslinking protein filamin A-dependent focal adhesion formation and cell migration [52
This study elucidated that chrysin influenced glucose- or AGE-stimulated actin polymerization, focal adhesion disassembly and cell migration contributing to phenotypic alterations in mesangial cells. These diabetic stimuli induced mesangial cell proliferation, aberrant actin cytoskeleton, and focal adhesion formation. However, chrysin ameliorated dysregulation of mesangial actin assembly and bundling through blocking the induction of F-actin, cortactin, EVL, and fascin-1. In addition, this flavonoid hampered diabetic AGE-enhanced formation of focal adhesions involving paxillin and vinculin. Furthermore, the autophagy and mTOR pathways were involved in actin polymerization, focal adhesion disassembly and cell migration in AGE-stimulated mesangial cells. Therefore, chrysin may counteract diabetes-associated mesangial cell protrusion and migration through disturbing actin polymerization/bundling and focal adhesion formation by interfering with autophagy/mTOR activation. Since the mesangial cell population is very small in whole kidneys, the dissociation between the in vitro and in vivo results may take place. Accordingly, the immunohistochemical sequential double staining is needed to examine the mesangial level of F-actin, cortactin or fascin-1 in kidneys, along with mesangial α-SMA level. Western blotting with primary culture mesangial cells or glomeruli can be considered by using sieving methods at least cortex part of kidneys.