Rat Glomerular Mesangial Cells Research Articles

Utilizing molecular docking and cell validation to explore the potential mechanisms of lupenone attenuating the inflammatory response via NF‐κB pathway

Diabetic nephropathy (DN), a common microvascular complicating disease of diabetes. Lupenone, a pentacyclic triterpenoid, has anti-inflammatory effects and can prevent type 2 diabetes mellitus and treat renal damage, however, the effects and mechanisms of lupenone in DN remain unclear. Thereby,the MTT method was used to investigate the antiproliferative effect of lupenoneon the cell line rat glomerular mesangial cells (HBZY-1). Molecular docking was used to investigate the combination of lupenone and MCP-1, IL-1β, TNF-α, IKKβ, IκBα, and NF-κB p65 proteins. The expression of mRNA of the pro-inflammatory cytokines (MCP-1, IL-1β and TNF-α) and the NF-κB signalling pathway in HBZY-1 cells were assessed by RT-PCR. The protein expressions of pro-inflammatory cytokines and NF-κB pathway were got by Western blot. Result showed that lupenone inhibited the proliferative activity of HBZY-1 cells at non-cytotoxic concentrations. Molecular docking results showed that lupenone combined well with the target proteins. Moreover, lupenone could significantly reduced the mRNA and protein expressions for pro-inflammatory cytokines and IKKβ, p-p65 and p-IκBα. Lupenone may play an anti-inflammatory role in DN treatment by inhibiting the NF-κB signalling pathway. These results provided a new understanding of the pharmacological mechanisms of lupenone in treatment of DN.

Red blood cell membrane-camouflaged prednisolone acetate-loaded PLGA nanoparticles for kidney-targeted drug delivery

The lack of renal targeting and adverse drug reactions are key issues in the treatment of glomerulonephritis. Here, we present the development of red blood cell membrane-camouflaged nanoparticles (RBC-PA-NPs) as a targeted drug delivery system for delivering prednisolone acetate (PA) to the glomerular mesangium. We successfully coated erythrocyte membranes onto PA-loaded PLGA nanoparticles (PA-NPs), and the resultant RBC-PA-NPs exhibited a well-defined core-shell structure with a favourable particle size of 104.20 ± 0.35 nm and a surface charge of −15.07 ± 2.13 mV. Toxicity was evaluated by cell viability assays using rat glomerular mesangial cells, and the RBC-PA-NPs showed good biocompatibility. RBC-PA-NPs exhibited reduced uptake by RAW 264.7 cells and greater uptake by rat glomerular mesangial cell lines than PA and PA-NPs. Biodistribution studies revealed that RBC-Cy5-NPs displayed the highest fluorescence intensity in renal tissue and effectively accumulated in the kidney, indicating their targeting effect. In summary, RBC-PA-NPs are a potential system for the kidney-targeted delivery of traditional drugs to improve their efficacy.

Retracted: Ganoderic Acid A Inhibits High Glucose-Induced Oxidative Stress and Extracellular Matrix Accumulation in Rat Glomerular Mesangial Cells.

[This retracts the article DOI: 10.1155/2022/5249910.].

Tetrandrine inhibits the proliferation of mesangial cells induced by enzymatically deglycosylated human IgA1 via IgA receptor/MAPK/NF-κB signaling pathway.

Objective: Despite the use of renin-angiotensin system blockade and immunosuppressive drugs, including corticosteroids, the current treatment regimens for Immunoglobulins A nephropathy (IgAN) are severely limited. The proliferation of mesangial cell and deposition of deglycosylated human IgA1 immune complex are the most common pathologic features of IgAN. We examined the tetrandrine potential of suppressing the proliferation of mesangial cells and explored its underlying mechanisms with a focus on IgA receptor/MAPK/NF-κB signaling pathway. Methods: Standard human IgA (native IgA) were enzymatically desialylated (deS IgA) or further degalactosylated (deS/deGal IgA) using neuraminidase and β-galactosidase. Rat glomerular mesangial cells (HBZY-1) and human renal mesangial cells (HRMC) stimulated by IgA were used to observe the suppressive effect of tetrandrine. The MTT assay was used to detect the cell viability. The protein expression of IgA receptor/MAPK/NF-κB signaling pathway was examined by Western blot. Cell cycle analysis was measured by flow cytometer. Results: Native IgA and deS IgA showed limited stimulation effect on both HBZY-1 cells and HRMCs, whereas deS/deGal IgA significantly stimulated the proliferation of both HBZY-1 cells and HRMCs (p < 0.05). Compared with non-stimulation of deS/deGal IgA, 1-3μM of tetrandrine had stronger inhibitory effect on the proliferation of HBZY-1 cells and HRMCs with the stimulation of deS/deGal IgA (p < 0.05), suggesting that tetrandrine possibly inhibited the proliferation of mesangial cells induced by deglycosylated human IgA1 specifically. Molecular mechanism study revealed that tetrandrine decreased the expression of IgA1 receptor, CD71 and β4GALT1, and inhibited the activation of MAPK/NF-κB significantly (p < 0.05). Moreover, these inhibitory effect of tetrandrine caused cell cycle arrest and stopped the cell growth in the S phase companied with the upregulating of cyclin A2 and downregulating of cyclin D1. Conclusion: Taken together, tetrandrine inhibited the proliferation of mesangial cells induced by enzymatically deglycosylated human IgA1 via IgA receptor/MAPK/NF-κB signaling pathway. Based on these potential molecular mechanisms, tetrandrine would be an appealing therapeutic option for IgAN.

A proteoglycan isolated from Ganoderma lucidum attenuates diabetic kidney disease by inhibiting oxidative stress-induced renal fibrosis both in vitro and in vivo.

Ganoderma lucidum (G. lucidum) was regarded as "miraculous herb" by the Chinese and recorded detailly in the "Shen Nong Ben Cao Jing" as a tonic to improve health and prolong life. A proteoglycan (namely, FYGL) was extracted from Ganoderma lucidum, which was a water-soluble hyperbranched proteoglycan, and was found to be able to protect pancreatic tissue against oxidative stress damage. Diabetic kidney disease (DKD) is a complication of diabetes, but the effective treatment is still lack. Chronic hyperglycemia in diabetic patients induce the accumulation of ROS, which injure the renal tissue and lead to the renal dysfunction. In this work, the efficacy and target mechanics of FYGL on diabetic renal function were investigated. In the present study, the mechanism of the reno-protection of FYGL was analyzed on diabetic db/db mice and rat glomerular mesangial cells (HBZY-1) induced by high glucose (HG) with palmitate (PA) (HG/PA). In vitro, the levels of reactive oxygen species (ROS), malondialdehyde (MDA) and superoxide dismutase (SOD) were evaluated by commercial kits. the expressions of NOX1 and NOX4, phosphorylation of MAPK and NF-κB, and pro-fibrotic proteins were measured by Western blot. In vivo, diabetic db/db mice were gavaged with FYGL for 8 weeks, body weight and fasting blood glucose (FBG) were tested weekly. On 8th week, the serum, urine and renal tissue were collected for glucose tolerance test (OGTT), redox indicator (SOD, CAT, GSH and MDA), lipid metabolism (TC, TG, LDL and HDL), blood urea nitrogen (BUN), serum creatinine (Scr), uric acid (UA), 8-oxo-deoxyguanosine (8-OHdG), and the changes of histopathology and expression of collagen IV and AGEs. The results in vitro showed that FYGL significantly inhibited the HG/PA-induced HBZY-1cells proliferation, ROS generation, MDA production, promoted SOD activity, and suppressed NOX1, NOX4, MAPK, NF-κB, and pro-fibrotic proteins expression. In addition, FYGL markedly alleviated blood glucose, antioxidant activity and lipid metabolism, improved renal functions, and relieved renal histopathological abnormalities, especially renal fibrosis. The antioxidant activity of FYGL can reduce ROS caused by diabetes and protect renal from oxidative stress-induced dysfunction, thereby improving renal function. This study shows that FYGL has the potential to treat diabetic kidney disease.

Parthenolide Attenuates Sepsis-Induced Acute Kidney Injury in Rats by Reducing Inflammation.

Sepsis is a common complication of severe trauma, burns, infection, or major surgery. This disease-related end-organ dysfunction results from systemic inflammatory response syndrome (SIRS). Acute kidney damage (AKI), also known as acute renal failure, is one of the most frequent and serious sequelae of sepsis. Nuclear transcription factor-κB (NF-κB) regulates the transcription of inflammation-related genes and operates as a mediator in the immune system. While parthenolide (PTL) has been reported to prevent harmful inflammatory reactions, its effects on sepsis-associated AKI are unknown. The current study investigates the effects of PTL in sepsis-associated AKI using cell and cecal ligation and puncture (CLP) models. Lipopolysaccharide (LPS)-stimulated rat glomerular mesangial cells were treated with 10 μM PTL. Inflammatory mediators, including TNF-α, IL-6, and IL-1β, in the culture supernatants were measured by ELISA, and NF-κB levels were assessed by qPCR. After the generation of the septic CLP model, rats were intraperitoneally injected with 500 g/kg PTL and were euthanized after 72 h. Serum and kidney samples were analyzed. TNF-α, IL-1β, and IL-6 levels were elevated after LPS treatment of rat glomerular mesangial cells (p=0.004, p=0.002, and p=0.004, respectively) but were significantly reduced in the PTL treatment group (p ≤ 0.001, p=0.01, and p ≤ 0.001). NF-κB p65 levels were also increased after LPS treatment in this group and were reduced in the PTL treatment group. PTL treatment also reduced kidney damage after CLP induction, as shown by histological analysis and reductions in the levels of BUN, Cre, KIM-1, and NAGL. CLP-induced kidney inflammation together with increased levels of proinflammatory cytokines and inflammatory-related proteins. The elevated levels of renal TNF-α, IL-6, and IL-1β were downregulated after PTL treatment. The PTL treatment also reduced the CLP-induced activation of NF-κB p65 in the damaged kidneys. PTL reduced inflammation induced by CLP-induced AKI in rat models and LPS-induced damage to glomerular mesangial cells by suppressing NF-κB signaling.

Ganoderic Acid A Inhibits High Glucose-Induced Oxidative Stress and Extracellular Matrix Accumulation in Rat Glomerular Mesangial Cells.

Objective We aimed to investigate the role of ganoderic acid A (GAA) in glomerular mesangial cells (GMCs) under high glucose (HG). Methods GMCs were pretreated with GAA and then cultured under HG condition for 24 h. Cell proliferation was measured by CCK-8 assay. The production of intracellular ROS was determined using DCFH-DA. The activities of SOD and CAT were measured using ELISA kits. The expressions of NOX2, NOX4, fibronectin (FN), collagen IV (col IV), p38, and p-p38 were detected by western blot. Results GAA suppressed GMC proliferation in response to HG stimulation. GAA significantly attenuated HG-caused increase in ROS production and decreases in SOD and CAT activities in GMCs. In addition, the increased expressions of NOX2 and NOX4 and NOX activity in HG-induced GMCs were significantly decreased by GAA. Furthermore, GAA greatly inhibited the levels of FN and col IV in HG-stimulated GMCs. Mechanistic investigations showed that HG caused activation of p38 MAPK pathway, whereas the induction was mitigated by GAA. Notably, the specific agonist of p38 MAPK pathway (P79350) reversed the effects of GAA on GMCs. Conclusion GAA protected GMCs from HG-induced oxidative stress and ECM production, which was mediated by the inhibition of the p38 MAPK pathway.

Diphenyl Diselenide Alleviates Tert-Butyl Hydrogen Peroxide-Induced Oxidative Stress and Lipopolysaccharide-Induced Inflammation in Rat Glomerular Mesangial Cells.

Hyperglycemia, oxidative stress, and inflammation play key roles in the onset and development of diabetic complications such as diabetic nephropathy (DN). Diphenyl diselenide (DPDS) is a stable and simple organic selenium compound with anti-hyperglycemic, anti-inflammatory, and anti-oxidative activities. Nevertheless, in vitro, the role and molecular mechanism of DPDS on DN remains unknown. Therefore, we investigated the effects of DPDS on tert-butyl hydrogen peroxide (t-BHP)-induced oxidative stress and lipopolysaccharide (LPS)-induced inflammation in rat glomerular mesangial (HBZY-1) cells and explored the underlying mechanisms. DPDS attenuated t-BHP-induced cytotoxicity, concurrent with decreased intracellular ROS and MDA contents and increased SOD activity and GSH content. Moreover, DPDS augmented the protein and mRNA expression of Nrf2, HO-1, NQO1, and GCLC in t-BHP-stimulated HBZY-1 cells. In addition, DPDS suppressed LPS-induced elevations of intracellular content and mRNA expression of interleukin (IL)-6, IL-1β and TNF-α. Furthermore, LPS-induced NFκB activation and high phosphorylation of JNK and ERK1/2 were markedly suppressed by DPDS in HBZY-1 cells. In summary, these data demonstrated that DPDS improves t-BHP-induced oxidative stress by activating the Nrf2/Keap1 pathway, and also improves LPS-induced inflammation via inhibition of the NFκB/MAPK pathways in HBZY-1 cells, suggesting that DPDS has the potential to be developed as a candidate for the prevention and treatment of DN.

Terpenoids from the Seeds of Toona sinensis and Their Ability to Attenuate High Glucose-Induced Oxidative Stress and Inflammation in Rat Glomerular Mesangial Cells.

Toona sinensis (A. Juss.) Roem is an edible medicinal plant that belongs to the genus Toona within the Meliaceae family. It has been confirmed to display a wide variety of biological activities. During our continuous search for active constituents from the seeds of T. sinensis, two new acyclic diterpenoids (1–2), together with five known limonoid-type triterpenoids (3–7), five known apotirucallane-type triterpenoids (8–12), and three known cycloartane-type triterpenoids (13–15), were isolated and characterized. Their structures were identified based on extensive spectroscopic experiments, including nuclear magnetic resonance (NMR), high-resolution electrospray ionization mass spectra (HR-ESI-MS), and electronic circular dichroism (ECD), as well as the comparison with those reported in the literature. We compared these findings to those reported in the literature. Compounds 5, 8, and 13–14 were isolated from the genus Toona, and compounds 11 and 15 were obtained from T. sinensis for the first time. The antidiabetic nephropathy effects of isolated compounds against high glucose-induced oxidative stress and inflammation in rat glomerular mesangial cells (GMCs) were assessed in vitro. The results showed that new compounds 1 and 2 could significantly increase the levels of Nrf-2/HO-1 and reduce the levels of NF-κB, TNF-α, and IL-6 at concentrations of 30 μM. These results suggest that compounds 1 and 2 might prevent the occurrence and development of diabetic nephropathy (DN) and facilitate the research and development of new antioxidant and anti-inflammatory drugs suitable for the prevention and treatment of DN.

Inhibitory Effect of Resveratrol on LPS-induced Glomerular Mesangial Cells Proliferation and TGF-β1 Expression via Sphingosine Kinase 1 Pathway.

To elucidate the renoprotective effect of resveratrol (RSV) on sphingosine kinase 1 (SphK1) signaling pathway and expression of its downstream molecules including activator protein 1 (AP-1) and transformation growth factor-β1 (TGF-β1) in lipopolysaccharide (LPS)-induced glomerular mesangial cells (GMCs). The rat GMCs line (HBZY-1) were cultured and randomly divided into 5 groups, including control, LPS (100 ng/mL), and 5, 10, 20 µmol/L RSV-treated groups. In addition, SphK1 inhibitor (SK-II) was used as positive control. GMCs were pretreated with RSV for 2 h and treated with LPS for another 24 h. GMCs proliferation was determined by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. The proteins expression of SphK1, p-c-Jun and TGF-β1 in GMCs were detected by Western blot, and DNA-binding activity of AP-1 was performed by electrophoretic mobility shift assay (EMSA). The binding activity between RSV and SphK1 protein was detected by AutoDock Vina and visualized by Discovery Studio 2016. LPS could obviously stimulate GMCs proliferation, elevate SphK1, p-c-Jun and TGF-β1 expression levels and increase the DNA-binding activity of AP-1 (P<0.05 or P<0.01), whereas these effects were significantly blocked by RSV pretreatment. It was also suggested that the effect of RSV was similar to SK-II (P>0.05). Moreover, RSV exhibited good binding affinity towards SphK1, with docking scores of -8.1 kcal/moL and formed hydrogen bonds with ASP-178 and LEU-268 in SphK1. RSV inhibited LPS-induced GMCs proliferation and TGF-β1 expression, which may be independent of its hypoglycemic effect on preventing the development of mesangial cell fibrosis and closely related to the direct inhibition of SphK1 pathway.

Modulation of IL-6 Expression by KLF4-Mediated Transactivation and PCAF-Mediated Acetylation in Sublytic C5b-9-Induced Rat Glomerular Mesangial Cells.

Interleukin-6 (IL-6) overproduction has been considered to contribute to inflammatory damage of glomerular mesangial cells (GMCs) in human mesangial proliferative glomerulonephritis (MsPGN) and its rat model called Thy-1 nephritis (Thy-1N). However, the regulatory mechanisms of IL-6 expression in GMCs upon sublytic C5b-9 timulation remain poorly understood. We found that Krüppel-like factor 4 (KLF4) bound to the IL-6 promoter (−618 to −126 nt) and activated IL-6 gene transcription. Furthermore, lysine residue 224 of KLF4 was acetylated by p300/CBP-associated factor (PCAF), which was important for KLF4-mediated transactivation. Moreover, lysine residue 5 on histone H2B and lysine residue 9 on histone H3 at the IL-6 promoter were also acetylated by PCAF, which resulted in an increase in IL-6 transcription. Besides, NF-κB activation promoted IL-6 expression by elevating the expression of PCAF. Overall, these findings suggest that sublytic C5b-9-induced the expression of IL-6 involves KLF4-mediated transactivation, PCAF-mediated acetylation of KLF4 and histones, and NF-κB activation in GMCs.

Effects of Qijin granules on high glucose-induced proliferation, apoptosis and expression of nuclear factor- κB and MCP-1 in rat glomerular mesangial cells

Purpose: To investigate the effects of Qijin granules on high glucose-induced proliferation and apoptosis in rat glomerular mesangial cells (MC).Methods: MC cells from rats were passaged and cultured, and randomly divided into control group (CNG), high glucose group (HGG), Western medicine group (WMG, high glucose + Benazepril + Gliquidone), and Qijin granules 1/2/3 group (high glucose + different doses of Qijin granules). Mesangial cells proliferation was measured using MTT assay. The NF-κB, MCP-1 and inflammatory factors in supernatant were determined by ELISA. Apoptosis rate and cell cycle were assessed by flow cytometry. The apoptosis-related TGF-β1/Smad signaling pathway-related protein expressions were measured by Western blot.Results: The A-value and early apoptosis rate, apoptosis rate and S-phase percentage, and protein expressions of NF-κB, MCP-1, IL-6, IL-2, TNF-ɑ, Bax, Cyt-C, caspase-3, TGF-β1, and p-Smad3 of MC cells in the HGG at 12 h, 24 h and 48 h were higher than those in the CNG. The above indices were lower in the WMG, and Qijin granules 1/2/3 groups than in the HGG. The Bcl-2, Smad7 protein expression level and the percentage of G1 and G2/M phase were lower in the HGG than in the CNG, and the above indeices were higher in the WMG and Qijin granules 1/2/3 group than in HGG.Conclusion: Qijin granules can dose-dependently inhibit high glucose-induced proliferation and apoptosis in rat MC cells, block the cell cycle and reduce inflammatory responses. This may be related to the regulation of NF-κB, MCP-1 and TGF-β1/Smad signaling pathways. These findings provide theoretical and experimental basis for the clinical treatment of early diabetic nephropathy.

Improvement of E Se tea extracts on renal mesangial cell apoptosis and high-fat-diet/streptozotocin-induced diabetic nephropathy

E Se tea, the leaves of Malus toringoides (Rehd.) Hughes, is a kind of traditional beverage, which is often used to relieve hyperglycemia and hyperlipidemia and other symptoms, while its effect on diabetic nephropathy (DN) is still unclear. In this study, we evaluated the improvement of the ethanol extract of M. toringoides (EMT) on tert-butyl hydroperoxide (t-BHP)-induced rat glomerular mesangial cells (HBZY-1) apoptosis and high-fat-diet (HFD)/streptozotocin (STZ)-induced DN rats. The results showed that the EMT significantly reversed apoptosis, mitochondrial depolarization and cysteinyl aspartate specific proteinase (caspase) 3/9 activation in t-BHP (50 μM)-exposed HBZY-1 cells. Moreover, 8-weeks treatment of EMT improved blood glucose, proteinuria, serum creatinine and urea nitrogen, renal oxidative stress, proinflammatory mediators, histopathology and decreased activation of caspase 3. These results suggested that the E Se tea may therefore be a potential complementary therapy targeting DN patients.

Glomerular Mesangial Cell pH Homeostasis Mediates Mineralocorticoid Receptor-Induced Cell Proliferation

Mineralocorticoids (e.g., aldosterone) support chronic inflammatory tissue damage, including glomerular mesangial injury leading to glomerulosclerosis. Furthermore, aldosterone leads to activation of the extracellular signal-regulated kinases (ERK1/2) in rat glomerular mesangial cells (GMC). Because ERK1/2 can affect cellular pH homeostasis via activation of Na+/H+-exchange (NHE) and the resulting cellular alkalinization may support proliferation, we tested the hypothesis that aldosterone affects pH homeostasis and thereby cell proliferation as well as collagen secretion also in primary rat GMC. Cytoplasmic pH and calcium were assessed by single-cell fluorescence ratio imaging, using the dyes BCECF or FURA2, respectively. Proliferation was determined by cell counting, thymidine incorporation and collagen secretion by collagenase-sensitive proline incorporation and ERK1/2-phosphorylation by Western blot. Nanomolar aldosterone induces a rapid cytosolic alkalinization which is prevented by NHE inhibition (10 µmol/L EIPA) and by blockade of the mineralocorticoid receptor (100 nmol/L spironolactone). pH changes were not affected by inhibition of HCO3− transporters and were not dependent on HCO3−. Aldosterone enhanced ERK1/2 phosphorylation and inhibition of ERK1/2-phosphorylation (10 µmol/L U0126) prevented aldosterone-induced alkalinization. Furthermore, aldosterone induced proliferation of GMC and collagen secretion, both of which were prevented by U0126 and EIPA. Cytosolic calcium was not involved in this aldosterone action. In conclusion, our data show that aldosterone can induce GMC proliferation via a MR and ERK1/2-mediated activation of NHE with subsequent cytosolic alkalinization. GMC proliferation leads to glomerular hypercellularity and dysfunction. This effect presents a possible mechanism contributing to mineralocorticoid receptor-induced pathogenesis of glomerular mesangial injury during chronic kidney disease.

388-P: GLP-1 Receptor Agonists Attenuates Secretion of Extracellular Matrix via Inhibiting HMGB1 Signaling in Rat Mesangial Cells

Background: GLP-1R agonists can exhibit a direct renoprotective effect in patients with diabetic nephropathy (DN) beyond the hypoglycemic effect, but the underlying mechanisms remain unknown. Recent studies have suggested that high mobility group box 1 (HMGB1) contributes to the development and progression of DN. In this study, we assessed whether GLP-1R agonists acts through HMGB1 and explored its downstream signaling pathways in DN. Methods: Rat glomerular mesangial cells (GMCs) were divided into four groups: normal control (NG), normal control with 10 nmol/L exendin-4 treatment (GLP-1 analog) (NGE), high glucose (HG), and high glucose with 10 nmol/L exendin-4 treatment (HGE) groups. GMCs were transfected with GLP-1R-siRNA using Lipofectamine TM2000 and divided as HG, HGE, HGE + GLP-1R-siRNA, or HGE + scrambled siRNA. FN and type IV collagen (COL-IV) were evaluated by enzyme-linked immunosorbent assay (ELISA). GLP-1R, HMGB1, TGF-β1, phosphorylated and total extracellular signal-regulated kinases (ERK), c-Jun NH2-terminal kinases (JNK), p38 mitogen-activated protein kinases (p38MAPK), and NF-κB p65 were measured by western blot analysis. Results: FN and COL-IV were higher in the HG group than NG group (P&amp;lt;0.05). Compared with NG group, the level of GLP-1R was decreased, HMGB1 and TGF-β1 were increased in the HG group (P&amp;lt;0.05). Additionally, NF-κB p65, the phosphorylation levels of ERK, JNK, and p38MAPK were all significantly elevated in the HG group (P&amp;lt;0.05). However, all these effects were reversed by Exendin-4 treatment (P&amp;lt;0.05), except phosphorylated p38MAPK (P&amp;gt;0.05). Moreover, the protective effects of Exendin-4 were abolished by GLP-1R knockdown (P&amp;lt;0.05). Conclusion: These results suggest that GLP-1 receptor agonists attenuates the ECM secretion of GMCs induced by high glucose. The potential mechanism involves it binding to and activating GLP-1R, which prevents ECM production by inhibiting HMGB1 and its signaling pathways. Disclosure W. Gu: None. M. Shi: None. H. Zhang: None. Funding National Natural Science Foundation of China (81200595, 81400807, 81700723)

Chemical Profile Assessment and Potential Bioactive Component Screening of a Chinese Patent Herbal Drug Yi-Shen-Hua-Shi Granule

Yi-Shen-Hua-Shi (YSHS) Granule is a well-known patented herbal drug for treating chronic glomerulonephritis (CGN) in China. Its chemical compositions and anti-CGN components are not fully understood. This study aimed to establish the chemical profile and to identify the anti-CGN components of YSHS Granule. For these purposes, a HPLC-Q-TOF-MS/MS method was developed. A total of 105 peaks were detected in the mass spectrum of the Granule. Of these, 99 compounds were tentatively identified as terpenoids, flavonoids, coumarins, alkaloids, phenols, and other types of compounds, and 15 were further identified with reference substances. To screen bioactive compounds, a cell membrane immobilized chromatography (CMIC) method was used. Lipopolysaccharide (LPS)-challenged rat glomerular mesangial cells (HBZY-1) were incubated with YSHS Granule (100 μg/mL), and the binding components to the cell membrane were extracted and analyzed using the established HPLC-Q-TOF-MS/MS method. Seven potential bioactive components that bound to HBZY-1 cell membranes were detected and identified as calycosin-7- O-β-D-glucoside, 6-gingerol, naringin, ginsenoside Re, poncirin, liquiritigenin, and isoliquiritigenin. The outcomes of the present study provide a chemical basis for clinical use of the Granule in managing CGN, and provide quality control markers for the Granule’s production and use.

Systematic investigation of the pharmacological mechanism for renal protection by the leaves of Eucommia ulmoides Oliver using UPLC-Q-TOF/MS combined with network pharmacology analysis

Bark is the traditional medicinal component of Eucommia ulmoides Oliver (E. ulmoides). However, the demand for E. ulmoides medicinal materials seriously limits their sustainability. To alleviate resource constraints, the bioactivity of E. ulmoides leaves and its pharmacodynamic basis were investigated. In the present study, extracts of E. ulmoides leaves were found to display potential renal protective properties in rat glomerular mesangial (HBZY-1) cells treated with high levels of glucose, suggesting that they possess potential factors capable of treating diabetic nephropathy. Ultra-performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) was used to comprehensively characterize the chemical components of E. ulmoides leaves. A total of 83 possible chemical components, including 12 iridoids, 13 flavonoids, 14 lignans, 20 phenylpropanoids, 14 phenolic acids, and 10 additional components, were identified in E. ulmoides leaves. Network pharmacology was used for a preliminary exploration of the potential mechanism of action of renal protection afforded by E. ulmoides leaves towards diabetic nephropathy. The network pharmacology results were verified using a series of biological experiments. The present study provided the basis for the comprehensive development and utilization of E. ulmoides leaves and the discovery of potential drugs.

Vitamin D protects glomerular mesangial cells from high glucose-induced injury by repressing JAK/STAT signaling

AimHigh glucose (HG) induces the production of transforming growth factor (TGF)-β and reactive oxygen species, which further activates JAK/STAT signaling and promotes the synthesis of matrix proteins, contributes to the pathophysiological processes of diabetic nephropathy. This study aims to investigate the protection role of vitamin D (VD) in the kidney in high glucose condition.MethodsRat glomerular mesangial cells were cultured in high glucose medium, with or without VD or VD receptor (VDR) siRNAs treatment. The levels of TGF-β and fibronectin were detected by qRT-PCR, immunoblotting and enzyme-linked immunosorbent assay (ELISA). The levels of phosphorylated JAK2, STAT1 and STAT3, and JAK/STAT signaling downstream genes were examined by immunoblotting and qRT-PCR.ResultsIn rat glomerular mesangial cells, VD treatment can repress the tyrosine phosphorylation of JAK2, STAT1 and STAT3. VD inhibited TGF-β and fibronectin expression which was rescued by vitamin d receptor (VDR) siRNA and STATs inhibitor perficitinib. The JAK/STAT signaling downstream protein coding genes including SOCS1, SOCS3 and type IV collagen were repressed by VD. Meanwhile, the expression of non-coding RNAs such as miR-181a, miR-181b, was repressed by VD, and the expression of miR-34a and Let-7b was upregulated by VD.ConclusionVitamin D (VD) treatment inhibits the function of HG on fibronectin production through regulating JAK/STAT pathway. These results provide direct evidences that VD protects glomerular mesangial cells from high glucose-induced injury through repressing JAK/STAT signaling, which has the potential for clinical DN treatment.

Sublytic C5b-9 induces glomerular mesangial cell proliferation via ERK1/2-dependent SOX9 phosphorylation and acetylation by enhancing Cyclin D1 in rat Thy-1 nephritis

Glomerular mesangial cell (GMC) proliferation is a histopathological alteration in human mesangioproliferative glomerulonephritis (MsPGN) or in animal models of MsPGN, e.g., the rat Thy‐1 nephritis (Thy-1N) model. Although sublytic C5b-9 assembly on the GMC membrane can trigger cell proliferation, the mechanisms are still undefined. We found that sublytic C5b-9-induced rat GMC proliferation was driven by extracellular signal‐regulated kinase 1/2 (ERK1/2), sry-related HMG-box 9 (SOX9), and Cyclin D1. Here, ERK1/2 phosphorylation was a result of the calcium influx-PKC-α-Raf-MEK1/2 axis activated by sublytic C5b-9, and Cyclin D1 gene transcription was enhanced by ERK1/2-dependent SOX9 binding to the Cyclin D1 promoter (−582 to −238 nt). In addition, ERK1/2 not only interacted with SOX9 in the cell nucleus to mediate its phosphorylation at serine residues 64 (a new site identified by mass spectrometry) and 181 (a known site), but also indirectly induced SOX9 acetylation by elevating the expression of general control non-repressed protein 5 (GCN5), which together resulted in Cyclin D1 synthesis and GMC proliferation. Moreover, our in vivo experiments confirmed that silencing these genes ameliorated the lesions of Thy‐1N rats and reduced SOX9 phosphorylation, acetylation and Cyclin D1 expression. Furthermore, the renal tissue sections of MsPGN patients also showed higher phosphorylation or expression of ERK1/2, SOX9, and Cyclin D1. In summary, these findings suggest that sublytic C5b-9-induced GMC proliferation in rat Thy-1N requires SOX9 phosphorylation and acetylation via enhanced Cyclin D1 gene transcription, which may provide a new insight into human MsPGN pathogenesis.

Mesenchymal Stem Cell-Derived Exosomes Carry MicroRNA-125a to Protect Against Diabetic Nephropathy by Targeting Histone Deacetylase 1 and Downregulating Endothelin-1

BackgroundMesenchymal stem cell (MSC)-derived exosomes have seen great advances in human disease control in a minimally invasive manner. This research aimed to explore the function of MSC-derived exosomes in diabetic nephropathy (DN) progression and the molecules involved.MethodsA rat model with DN and rat glomerular mesangial cell (GMC) models treated with high glucose (HG) were established, which were treated with exosomes from adipose-derived-MSCs (adMSCs). The levels of blood glucose, serum creatinine, and urinary protein, the urine albumin-to-creatinine ratio (UACR), kidney weight/body weight, and mesangial hyperplasia and kidney fibrosis in rats were determined. The expression of interleukin-6 (IL-6), collagen I (Col. I), fibronectin (FN), Bax and Bcl-2 in HG-treated GMCs was assessed. The microRNA (miRNA) carried by adMSC-exosomes was identified, and the implicated down-stream molecules were analyzed.ResultsadMSC-derived exosomes decreased levels of blood glucose, serum creatinine, 24-h urinary protein, UACR and kidney weight/body weight, and they suppressed mesangial hyperplasia and kidney fibrosis in DN rats. The exosomes also suppressed levels of IL6, Col. I and FN in HG-treated GMCs and promoted cell apoptosis. miR-125a was at least partially responsible for the above protective events mediated by adMSC-exosomes. miR-125a directly bound to histone deacetylase 1 (HDAC1), while HDAC1 further regulated endothelin-1 (ET-1) activation. Up-regulation of HDAC1 blocked the functions of adMSC-exosomal miR-125a.ConclusionThis study suggested that adMSC-derived exosomes inhibit DN progression and alleviate the symptoms by carrying miR-125a, during which HDAC1 and ET-1 were inhibited. This study may provide novel effects into DN treatment.