Inhibition of ChREBP ubiquitination via the ROS/Akt-dependent downregulation of Smurf2 contributes to lysophosphatidic acid-induced fibrosis in renal mesangial cells.

BACKGROUND: Mesangial cell fibrosis, a typical symptom of diabetic nephropathy (DN), is a major contributor to glomerulosclerosis. We previously reported that the pharmacological blockade of lysophosphatidic acid (LPA) signaling improves DN. Although LPA signaling is implicated in diabetic renal fibrosis, the underlying molecular mechanisms remain unclear. Here, the role of carbohydrate-responsive element-binding protein (ChREBP) in LPA-induced renal fibrosis and the underlying mechanisms were investigated. METHODS: Eight-week-old wild-type and db/db mice were intraperitoneally injected with the vehicle or an LPAR1/3 antagonist, ki16425 (10 mg/kg), for 8 weeks on a daily basis, following which the mice were sacrificed and renal protein expression was analyzed. SV40 MES13 cells were treated with LPA in the presence or absence of ki16425, and the expression of ChREBP and fibrotic factors, including fibronectin, TGF-ß, and IL-1ß, was examined. The role of ChREBP in the LPA-induced fibrotic response was investigated by ChREBP overexpression or knockdown. The involvement of Smad ubiquitination regulatory factor-2 (Smurf2), an E3 ligase, in LPA-induced expression of ChREBP and fibrotic factors was investigated by Smurf2 overexpression or knockdown. To identify signaling molecules regulating Smurf2 expression by LPA, pharmacological inhibitors such as A6370 (Akt1/2 kinase inhibitor) and Ly 294002 (PI3K inhibitor) were used. RESULTS: The renal expression of ChREBP increased in diabetic db/db mice, and was reduced following treatment with the ki16425. Treatment with LPA induced the expression of ChREBP and fibrotic factors, including fibronectin, TGF-ß, and IL-1ß, in SV40 MES13 cells, which were positively correlated. The LPA-induced expression of fibrotic factors increased or decreased following ChREBP overexpression and knockdown, respectively. The production of reactive oxygen species (ROS) mediated the LPA-induced expression of ChREBP and fibrotic factors, and LPA decreased Smurf2 expression via Traf4-mediated ubiquitination. The LPA-induced expression of ubiquitinated-ChREBP increased or decreased following Smurf2 overexpression and knockdown, respectively. Additionally, Smurf2 knockdown significantly increased the expression of ChREBP and fibrotic factors. The pharmacological inhibition of Akt signaling suppressed the LPA-induced alterations in the expression of ChREBP and Smurf2. CONCLUSION: Collectively, the results demonstrated that the ROS/Akt-dependent downregulation of Smurf2 and the subsequent increase in ChREBP expression might be one of the mechanisms by which LPA induces mesangial cell fibrosis in DN.

Circular ribonucleic acid nucleoporin 98 knockdown alleviates high glucose-induced proliferation, fibrosis, inflammation and oxidative stress in human glomerular mesangial cells by regulating the microribonucleic acid-151-3p-high mobility group AT-hook 2 axis.

AIMS/INTRODUCTION: This study aimed to investigate the role and mechanism of circular ribonucleic acid nucleoporin 98 (circNUP98) in diabetic nephropathy (DN). MATERIALS AND METHODS: Human glomerular mesangial cells (HMCs) were stimulated with high glucose (HG) to imitate the growth environment of cells under the DN condition. Levels of genes and proteins were tested by quantitative reverse transcription polymerase chain reaction and western blot. Cell proliferation, apoptosis and inflammatory response were analyzed by using cell counting kit-8, flow cytometry and enzyme-linked immunosorbent assay analysis, respectively. Oxidative stress and fibrosis were evaluated by detecting the activity of reactive oxygen species, malondialdehyde, superoxide dismutase, fibronectin and collagen IV. The binding interaction between microribonucleic acid (miR)-151-3p and high mobility group AT-hook 2 (HMGA2) or circNUP98 was confirmed using dual-luciferase reporter, pull-down and ribonucleic acid immunoprecipitation assays. Exosomes were isolated by ultracentrifugation, and qualified by transmission electron microscopy, nanoparticle tracking analysis and western blot. RESULTS: CircNUP98 expression was higher in the serum of DN patients and HG-stimulated HMCs. Functionally, circNUP98 knockdown alleviated HG-induced proliferation, fibrosis, inflammatory response and oxidative stress in HMCs. Mechanistically, circNUP98 directly sponged miR-151-3p, which targeted HMGA2. Rescue experiments showed that miR-151-3p reversed the inhibitory effects of circNUP98 knockdown on HG-induced HMC dysfunction. Furthermore, miR-151-3p re-expression also led to an inhibition of the aforementioned biological behaviors, which was attenuated by HMGA2 upregulation. Besides that, CircNUP98 was found to be packaged into exosomes of DN, and exosomal circNUP98 possessed diagnostic value for DN patients. CONCLUSION: CircNUP98 knockdown alleviates HG-induced proliferation, fibrosis inflammation and oxidative stress in HMCs by regulating the miR-151-3p-HMGA2 axis, which might provide a potential approach for DN therapeutics.

Astragalus mongholicus Bunge and Panax notoginseng formula (A&P) improves renal mesangial cell damage in diabetic nephropathy by inhibiting the inflammatory response of infiltrated macrophages.

BACKGROUND: Diabetic nephropathy (DN) is one of the main causes of end-stage renal disease with scantly effective treatment. Numerous evidences indicated that macrophages play an important role in the occurrence and pathogenesis of DN by secreting inflammatory cytokines. Mincle is mainly expressed in macrophages and promotes kidney inflammation and damage of acute kidney injury. However, the role of Mincle in DN is unclear. In this study, we aim to investigate the effect of Mincle-related macrophage inflammation on DN, and whether it can be identified as the therapeutic target for Astragalus mongholicus Bunge and Panax notoginseng Formula (A&P), a widely used Chinese herbal decoction for DN treatment. METHODS: In vivo experiments high-fat and high-sugar diet and streptozotocin was used to establish a diabetic nephropathy model, while in vitro experiments inflammation model was induced by high-glucose in mouse Bone Marrow-Derived Macrophages (BMDM) cells and mouse mesangial (MES) cells. Kidney pathological staining is used to detect kidney tissue damage and inflammation, Western blotting, Real-time PCR and ELISA are performed to detect Mincle signaling pathway related proteins and inflammatory cytokines. RESULTS: Mincle was mainly expressed in infiltrated macrophage of DN kidney, and was significant decreased after A&P administration. The in vitro experiments also proved that A&P effectively down-regulated the expression of Mincle in macrophage stimulated by high glucose. Meanwhile, the data demonstrated that A&P can reduce the activation of NFκB, and the expression and secretion of inflammatory cytokines in DN kidney or BMDM cells. Notably, we set up a co-culture system to conform that BMDM cells can aggravate the inflammatory response of mesangial (MES) cells under high glucose stimulation. Furthermore, we found that the anti-injury role of A&P in MES cells was dependent on inhibition of the Mincle in macrophage. CONCLUSION: In summary, our study found that A&P is effective in reducing renal pathological damage and improving renal function and inflammation in diabetic nephropathy by a mechanism mainly related to the inhibition of the Mincle/Card9/NFκB signaling pathway.

Discovery of caffeoylisocitric acid as a Keap1-dependent Nrf2 activator and its effects in mesangial cells under high glucose.

Diabetic nephropathy (DN) is one of the severe microvascular complications of diabetes mellitus. Oxidative stress resulting from aberrant metabolism of glucose mediates renal inflammation and fibrosis in the progression of DN. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor regulating the expression of antioxidant enzymes. Activating Nrf2 will give a promising therapy for DN. To discover novel Nrf2 activators, we have investigated caffeoylisocitric acid using mesangial cells under high glucose. The results showed at 10 µM, caffeoylisocitric acid significantly inhibited the self-limited proliferation of mesangial cells induced by high glucose. Further assessments have disclosed caffeoylisocitric acid mitigated oxidative stress, inflammation and accumulation of extracellular matrix resulting from high glucose via inactivating MAPK signalling. Meanwhile activation of Nrf2 was observed and involved in these effects through the interaction between Keap1 and caffeoylisocitric acid to disrupt Keap1-Nrf2 complex. Therefore, caffeoylisocitric acid is a promising Nrf2 activator targeting DN.

The DR1­CSE/H2S system inhibits renal fibrosis by downregulating the ERK1/2 signaling pathway in diabetic mice.

Glomerular mesangial cell (MC) proliferation and extracellular matrix deposition are the main pathological changes in diabetic nephropathy. Hydrogen sulfide (H2S) inhibits the proliferation of MCs. Dopamine 1 receptors (DR1) are expressed in MCs and serve important physiological roles. However, it is unclear whether DR1 activation inhibits MC proliferation by increasing endogenous H2S. The present study found that the production of H2S and the expression of DR1 and cystathionine­Î³­lyase (CSE) were decreased in the renal tissues of diabetic mice and high glucose (HG)­induced MCs. SKF38393 (a DR1 agonist) increased the production of H2S and the expression of DR1 and CSE and NaHS (an exogenous H2S donor) only increased H2S production and CSE expression but not DR1 expression. HG increased the thickness of the glomerular basement membrane, cell viability and proliferation, the expression of cyclin D1, PCNA, collagen 1 and α­smooth muscle actin and the activity of phosphorylated ERK1/2 and decreased the expression of P21 and MMP9. SKF38393 and NaHS reversed the effects of HG. PPG (a CSE inhibitor) abolished the beneficial effects of SKF38393. The beneficial effects of SKF38393 were similar to those of PD98059 (an ERK1/2 inhibitor). Taken together, the findings suggested that the DR1­CSE/H2S pathway activation attenuated diabetic MC proliferation and extracellular matrix deposition by downregulating the ERK1/2 signaling pathway.

Emodin alleviates high glucose-induced oxidative stress, inflammation and extracellular matrix accumulation of mesangial cells by the circ_0000064/miR-30c-5p/Lmp7 axis.

Emodin has been shown to exert a renoprotective effect in diabetic nephropathy (DN). In this paper, we investigated whether circular RNAs (circRNAs) might be involved in the renoprotective mechanism of emodin in DN. The levels of malondialdehyde (MDA), reactive oxygen species (ROS), superoxide dismutase (SOD), interleukin-1ß (IL-1ß), IL-6 and tumor necrosis factor-α (TNF-α) were measured using the corresponding assay kits. The expression levels of circ_0000064, microRNA (miR)-30c-5p, large multifunctional protease 7 (Lmp7), fibronectin (FN), and collagen type I (Col.1) were gauged by quantitative real-time polymerase chain reaction (qRT-PCR) or western blot. Subcellular localization assay was used to assess the cellular localization of circ_0000064. Targeted relationships among circ_0000064, miR-30c-5p and Lmp7 were confirmed by dual-luciferase reporter, RNA pull-down and RNA immunoprecipitation (RIP) assays. Our data showed the alleviative effect of emodin on HG-induced oxidative stress, inflammation and extracellular matrix (ECM) accumulation in SV-MES13 cells. Circ_0000064 was an importantly downstream effector of emodin function in HG-induced SV40-MES13 cells. Moreover, circ_0000064 directly targeted miR-30c-5p, and circ_0000064 modulated Lmp7 expression through miR-30c-5p. Circ_0000064 silencing alleviated HG-induced cell oxidative stress, inflammation and ECM accumulation via up-regulating miR-30c-5p. The enforced expression of miR-30c-5p attenuated HG-induced oxidative stress, inflammation and ECM accumulation in SV40-MES13 cells by targeting Lmp7. Our findings identified that emodin alleviated HG-induced oxidative stress, inflammation and ECM accumulation in SV40-MES13 cells at least partially by the regulation of the circ_0000064/miR-30c-5p/Lmp7 axis.

Cyclophosphamide Attenuates Fibrosis in Lupus Nephritis by Regulating Mesangial Cell Cycle Progression.

OBJECTIVES: Most patients with systemic lupus erythematosus (SLE) develop lupus nephritis (LN) with severe kidney manifestations. Renal fibrosis can be primarily attributed to overproliferation of mesangial cells (MCs), which are subject to drug treatment. Nevertheless, the detailed mechanisms remain elusive. We sought to identify the effect of cyclophosphamide (CTX), a drug commonly used for LN treatment, on MC proliferation and explore its underlying mechanisms. Material/Methods. Cell proliferation and fibrosis in mouse kidney tissues were determined by histopathology staining techniques. Flow cytometry was used for cell cycle analysis. Cell cycle regulators were examined in vitro following treatment of immortalized human MCs with platelet-derived growth factor subunit B (PDGF-B). Quantitative real-time PCR and western blot analyses were used to measure the mRNA and protein levels of candidate cell cycle regulators, respectively. RESULTS: CTX inhibited cell overproliferation induced by platelet-derived growth factor subunit B in vitro and in vivo. CTX (40 mg/l) was sufficient to induce G0/G1 phase cell cycle arrest. CTX treatment downregulated many critical cell cycle regulators including cyclins and cyclin-dependent kinases but upregulated cyclin-dependent kinase inhibitors. Additionally, CTX-treated samples showed significantly reduced fibrosis, as indicated by lower expression of interleukin-1ß and α-smooth muscle actin. CONCLUSION: CTX inhibits proliferation of MCs by modulating cell cycle regulator and therefore arresting them at G1 phase. CTX treatment significantly alleviates the severity of renal fibrosis. These findings provide novel insights into the mechanisms by which CTX affects LN.

Fucoxanthin regulates Nrf2 signaling to decrease oxidative stress and improves renal fibrosis depending on Sirt1 in HG-induced GMCs and STZ-induced diabetic rats.

Diabetic nephropathy (DN) is one of the major microvascular complications of diabetes. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a crucial cellular defense factor to cope with oxidative stress. Silent information regulator T1 (Sirt1) is a deacetylase with antioxidative stress activity. Fucoxanthin is a marine-derived carotenoid. This study was conducted to investigate whether fucoxanthin could alleviate oxidative stress by activating Sirt1/Nrf2 signaling to alleviate DN. In streptozotocin-induced diabetic rats, fucoxanthin treatment effectively improved renal function, alleviated glomerulosclerosis. Fucoxanthin reversed the decreased protein levels of Sirt1 and Nrf2 in the kidney of diabetic rats and glomerular mesangial cells cultured in high glucose. Conversely, EX527, a Sirt1 inhibitor, counteracted the effect of fucoxanthin on the expression of Nrf2. Furthermore, in vivo and vitro results showed that fucoxanthin treatment reversed the low expression and activity of superoxide dismutase and heme oxygenase 1, depending on Sirt1 activation. Our results suggest that fucoxanthin improves diabetic kidney function and renal fibrosis by activating Sirt1/Nrf2 signaling to reduce oxidative stress.

Melatonin Prevents T Lymphocyte Infiltration to the Kidneys of Hypertensive Rats, Induced by a High-Salt Diet, by Preventing the Expression of CXCR3 Ligand Chemokines.

In a previous study, we demonstrated that melatonin prevents kidney damage in a salt-induced hypertension model by decreasing oxidative stress. We hypothesized that this effect involves melatonin's immunomodulatory properties. In vivo Study-Dahl salt-sensitive (DSS) rats were fed normal chow, a high-salt diet (HSD), or a HSD and melatonin (30 mg/kg/day) in their water for eight weeks. Kidneys were harvested for immediate lymphocyte isolation and characterization by Flow cytometry (CD3+CD4+ and CD3+CD8+) and for lymphocyte chemoattractant (mainly CXCL chemokines) gene expression studies. In vitro study-rat mesangial cells (RMC) were cultured in a high-salt medium without and with melatonin. A HSD was associated with significant renal infiltration of CD4+ and CD8+ T lymphocytes compared to control. Melatonin significantly reduced renal lymphocyte infiltration. A HSD significantly increased mRNA expression of CXCL chemokines. Adding melatonin to the HSD abolished this effect. Treating RMC cells with salt increased the expression of CXCL10 and CXCL11 but not CXCL9. Adding melatonin to the culture media prevented this increase. Treating HSD-fed rats with melatonin decreased renal lymphocyte chemoattractant mRNA expression and is associated with significantly reducing renal T lymphocyte infiltration. Salt may have a direct effect on chemokine-producing renal cells, which is blunted by melatonin treatment.

Doxorubicin-Induced Fetal Mesangial Cell Death Occurs Independently of TRPC6 Channel Upregulation but Involves Mitochondrial Generation of Reactive Oxygen Species.

Doxorubicin (DOX), a category D pregnancy drug, is a chemotherapeutic agent that has been shown in animal studies to induce fetal toxicity, including renal abnormalities. Upregulation of the transient receptor potential cation (TRPC) 6 channel is involved in DOX-induced podocyte apoptosis. We have previously reported that TRPC6-mediated Ca2+ signaling promotes neonatal glomerular mesangial cell (GMC) death. However, it is unknown whether DOX alters mesangial TRPC expression or viability in the fetus. In this study, cell growth was tracked in control and DOX-treated primary GMCs derived from fetal pigs. Live-cell imaging demonstrated that exposure to DOX inhibited the proliferation of fetal pig GMCs and induced cell death. DOX did not alter the TRPC3 expression levels. By contrast, TRPC6 protein expression in the cells was markedly reduced by DOX. DOX treatment also attenuated the TRPC6-mediated intracellular Ca2+ elevation. DOX stimulated mitochondrial reactive oxygen species (mtROS) generation and mitophagy by the GMCs. The DOX-induced mtROS generation and apoptosis were reversed by the mitochondria-targeted antioxidant mitoquinone. These data suggest that DOX-induced fetal pig GMC apoptosis is independent of TRPC6 channel upregulation but requires mtROS production. The mtROS-dependent GMC death may contribute to DOX-induced fetal nephrotoxicity when administered prenatally.

Protective Effects of Ethanolic Extract from Rhizome of Polygoni avicularis against Renal Fibrosis and Inflammation in a Diabetic Nephropathy Model.

Progressive diabetic nephropathy (DN) in diabetes leads to major morbidity and mortality. The major pathological alterations of DN include mesangial expansion, extracellular matrix alterations, tubulointerstitial fibrosis, and glomerular sclerosis. Polygoni avicularis is widely used in traditional oriental medicine and has long been used as a diuretic, astringent, insecticide and antihypertensive. However, to the best of the authors' knowledge, the effects of the ethanolic extract from rhizome of Polygoni avicularis (ER-PA) on DN have not yet been assessed. The present study aimed to identify the effect of ER-PA on renal dysfunction, which has been implicated in DN in human renal mesangial cells and db/db mice and investigate its mechanism of action. The in vivo experiment was performed using Polygoni avicularis-ethanol soluble fraction (ER-PA) and was administrated to db/db mice at 10 and 50 mg/kg dose. For the in vitro experiments, the human renal mesangial cells were induced by high glucose (HG, 25 mM). The ER-PA group showed significant amelioration in oral glucose tolerance, and insulin resistance index. ER-PA significantly improved the albumin excretion and markedly reduced plasma creatinine, kidney injury molecule-1 and C-reactive protein. In addition, ER-PA significantly suppressed inflammatory cytokines. Histopathologically, ER-PA attenuated glomerular expansion and tubular fibrosis in db/db mice. Furthermore, ER-PA suppressed the expression of renal fibrosis biomarkers (TGF and Collagen IV). ER-PA also reduced the NLR family pyrin domain containing 3 inflammatory factor level. These results suggest that ER-PA has a protective effect against renal dysfunction through improved insulin resistance as well as the inhibition of nephritis and fibrosis in DN.

Monoterpene indole alkaloids from the roots of Bousigonia mekongensis and their anti-diabetic nephropathy activity.

Four new monoterpene indole alkaloids (1-4) together with six known alkaloids (5-10) were isolated from the roots of Bousigonia mekongensis. Compounds 3 and 4 were the first examples of condylocarpan-adenine type alkaloids obtained from natural plant resource. Their structures were elucidated on the basis of spectroscopic data. All compounds were evaluated for their inhibiting glucose-induced mesanginal cell proliferation and protecting high glucose-evoked podocyte injury activities. (-)-demethoxycarbonyldihydrogambirtannine (5) can significantly antagonize glucose-induced podocyte injury with EC50 value of 6.5 ± 1.2 µM.

Transcriptional Suppression of Diabetic Nephropathy with Novel Gene Silencer Pyrrole-Imidazole Polyamides Preventing USF1 Binding to the TGF-ß1 Promoter.

Upstream stimulatory factor 1 (USF1) is a transcription factor that is increased in high-glucose conditions and activates the transforming growth factor (TGF)-ß1 promoter. We examined the effects of synthetic pyrrole-imidazole (PI) polyamides in preventing USF1 binding on the TGF-ß1 promoter in Wistar rats in which diabetic nephropathy was established by intravenous administration of streptozotocin (STZ). High glucose induced nuclear localization of USF1 in cultured mesangial cells (MCs). In MCs with high glucose, USF1 PI polyamide significantly inhibited increases in promoter activity of TGF-ß1 and expression of TGF-ß1 mRNA and protein, whereas it significantly decreased the expression of osteopontin and increased that of h-caldesmon mRNA. We also examined the effects of USF1 PI polyamide on diabetic nephropathy. Intraperitoneal injection of USF1 PI polyamide significantly suppressed urinary albumin excretion and decreased serum urea nitrogen in the STZ-diabetic rats. USF1 PI polyamide significantly decreased the glomerular injury score and tubular injury score in the STZ-diabetic rats. It also suppressed the immunostaining of TGF-ß1 in the glomerulus and proximal tubules and significantly decreased the expression of TGF-ß1 protein from kidney in these rats. These findings indicate that synthetic USF1 PI polyamide could potentially be a practical medicine for diabetic nephropathy.

The p70S6K/PI3K/MAPK feedback loop releases the inhibition effect of high-dose rapamycin on rat mesangial cell proliferation.

Glomerular mesangial cell (MC) proliferation is one of the causative factors of glomerular diseases and one of their prominent pathological features. Rapamycin can inhibit MC proliferation and slow the progression to chronic renal fibrosis. The present study was designed to observe the role of rapamycin in MC proliferation and to explore the mechanism by which rapamycin acts on Akt and MAPK/ERK1/2 pathways in mesangial cells. MTT assay and flow cytometry were used to evaluate the proliferation and the cell cycle phase of glomerular mesangial cells respectively. The mRNA expression level of p70S6K was detected by RT-qPCR. Western blotting was performed to determine p70S6K, PI3K/Akt, and PI3K/MAPK protein expression. We found that rapamycin could reduce mesangial cell proliferation and arrest the cell cycle in the G1 phase, however the inhibition effect of 1000 nmol/L rapamycin was not higher than that in the 100 nmol/L group. The results of western blotting showed that 1000 nmol/L rapamycin more significantly inhibited the phosphorylation of p70S6K than 100 nmol/L, suggesting there should be another signaling pathway that activates the proliferation of MCs. Moreover, our results revealed that 1000 nmol/L rapamycin led to Raf1-MEK1/2-ERK pathway activation through a p70S6K-PI3K-mediated feedback loop in MCs. This study demonstrated that high-dose rapamycin leads to ERK1/2 activation through a p70S6K/PI3K/MAPK feedback loop in rat MCs, thus reducing the inhibitory effect of rapamycin on MC proliferation.

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

AIM: High glucose (HG) induces the production of transforming growth factor (TGF)-ß 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. METHODS: Rat glomerular mesangial cells were cultured in high glucose medium, with or without VD or VD receptor (VDR) siRNAs treatment. The levels of TGF-ß 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. RESULTS: In rat glomerular mesangial cells, VD treatment can repress the tyrosine phosphorylation of JAK2, STAT1 and STAT3. VD inhibited TGF-ß 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. CONCLUSION: Vitamin 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.

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.

Rosiglitazone attenuates high glucose-induced proliferation, inflammation, oxidative stress and extracellular matrix accumulation in mouse mesangial cells through the Gm26917/miR-185-5p pathway.

Rosiglitazone (RSG) is widely used to reduce the amount of sugar in the blood of patients with diabetes mellitus. Diabetic nephropathy is the most common microvascular complication of diabetes. The role of RSG in diabetic nephropathy is not fully understood. Diabetic nephropathy model was constructed in high glucose (HG)-treated mouse mesangial cells. The effects of RSG on cell viability and cell cycle were investigated using cell counting kit-8 (CCK-8) assay and flow cytometry assay. Oxidative stress was assessed according to ROS production and SOD activity in cells. Inflammatory responses were assessed according to the releases of inflammatory cytokines. Extracellular matrix (ECM) accumulation was determined by the levels of fibronectin and collagen IV using western blot. The expression of Gm26917 and microRNA-185-5p (miR-185-5p) was detected by quantitative real-time polymerase chain reaction (qPCR). The interaction between Gm26917 and miR-185-5p was validated by dual-luciferase reporter assay, RNA immunoprecipitation (RIP) assay and pull-down assay. RSG significantly inhibited HG-induced proliferation, oxidative stress, inflammatory responses and ECM accumulation in mouse mesangial cells. The expression of Gm26917 was induced by HG but weakened by RSG. Gm26917 knockdown alleviated HG-induced proliferation, oxidative stress, inflammatory responses and ECM accumulation in mouse mesangial cells, and Gm26917 overexpression partly abolished the effects of RSG. Moreover, miR-185-5p was a target of Gm26917, and miR-185-5p inhibition recovered proliferation, oxidative stress, inflammatory responses and ECM accumulation in mouse mesangial cells that were alleviated by Gm26917 knockdown. RSG ameliorated HG-induced mouse mesangial cell proliferation, oxidative stress, inflammation and ECM accumulation partially by governing the Gm26917/miR-185-5p pathway.

Activin A and Cell-Surface GRP78 Are Novel Targetable RhoA Activators for Diabetic Kidney Disease.

Diabetic kidney disease (DKD) is the leading cause of kidney failure. RhoA/Rho-associated protein kinase (ROCK) signaling is a recognized mediator of its pathogenesis, largely through mediating the profibrotic response. While RhoA activation is not feasible due to the central role it plays in normal physiology, ROCK inhibition has been found to be effective in attenuating DKD in preclinical models. However, this has not been evaluated in clinical studies as of yet. Alternate means of inhibiting RhoA/ROCK signaling involve the identification of disease-specific activators. This report presents evidence showing the activation of RhoA/ROCK signaling both in vitro in glomerular mesangial cells and in vivo in diabetic kidneys by two recently described novel pathogenic mediators of fibrosis in DKD, activins and cell-surface GRP78. Neither are present in normal kidneys. Activin inhibition with follistatin and neutralization of cell-surface GRP78 using a specific antibody blocked RhoA activation in mesangial cells and in diabetic kidneys. These data identify two novel RhoA/ROCK activators in diabetic kidneys that can be evaluated for their efficacy in inhibiting the progression of DKD.

Targeted Delivery of Soluble Guanylate Cyclase (sGC) Activator Cinaciguat to Renal Mesangial Cells via Virus-Mimetic Nanoparticles Potentiates Anti-Fibrotic Effects by cGMP-Mediated Suppression of the TGF-ß Pathway.

Diabetic nephropathy (DN) ranks among the most detrimental long-term effects of diabetes, affecting more than 30% of all patients. Within the diseased kidney, intraglomerular mesangial cells play a key role in facilitating the pro-fibrotic turnover of extracellular matrix components and a progredient glomerular hyperproliferation. These pathological effects are in part caused by an impaired functionality of soluble guanylate cyclase (sGC) and a consequentially reduced synthesis of anti-fibrotic messenger 3',5'-cyclic guanosine monophosphate (cGMP). Bay 58-2667 (cinaciguat) is able to re-activate defective sGC; however, the drug suffers from poor bioavailability and its systemic administration is linked to adverse events such as severe hypotension, which can hamper the therapeutic effect. In this study, cinaciguat was therefore efficiently encapsulated into virus-mimetic nanoparticles (NPs) that are able to specifically target renal mesangial cells and therefore increase the intracellular drug accumulation. NP-assisted drug delivery thereby increased in vitro potency of cinaciguat-induced sGC stabilization and activation, as well as the related downstream signaling 4- to 5-fold. Additionally, administration of drug-loaded NPs provided a considerable suppression of the non-canonical transforming growth factor ß (TGF-ß) signaling pathway and the resulting pro-fibrotic remodeling by 50-100%, making the system a promising tool for a more refined therapy of DN and other related kidney pathologies.

Methylglyoxal-Lysine Dimer, an Advanced Glycation End Product, Induces Inflammation via Interaction with RAGE in Mesangial Cells.

INTRODUCTION: Advanced glycation end products (AGEs) and receptor of advanced glycation end products (RAGE) mediate renal function during diabetic and non-diabetic nephropathy development. Methylglyoxal-lysine dimer (MOLD), a typical toxic advanced glycation end product (TAGE), contributes to inflammatory responses during renal diseases. This study determines the effect of MOLD on inflammatory responses in mouse mesangial cells. METHODS AND RESULTS: The murine mesangial cell line SV40 MES 13 is used to assess nuclear factor-kappa B (NF-κB) expression, reactive oxygen species (ROS) production, and mitochondria labeling. The interaction model between RAGE and MOLD is also determined. MOLD treatment of mesangial cells markedly increases RAGE expression and the linkage with V-type Ig domain of RAGE. MOLD induces ROS production and mitochondrial dysfunction. MOLD activates phosphatidylinositol 3-kinase-protein kinase B (PI3KB) and NF-κB signaling pathways. It is confirmed that these changes are reversed when ROS is suppressed. These effects may be regulated through mitogen-activated protein kinases and pro-inflammatory cytokines in circulatory inflammation responses. CONCLUSION: MOLD plays a major role in nephropathy via ROS production and mitochondrial dysfunction through direct association with RAGE. Further, the NF-kB and PI3K/AKT signaling pathways triggered by ROS mediate the inflammatory response to exacerbate MOLD-induced damages in inflammation-related diabetic and non-diabetic renal diseases.