Glomerular Mesangial Cell Proliferation Research Articles

Dapagliflozin can alleviate renal fibrosis in rats with streptozotocin‑induced type2 diabetes mellitus.

The aim of the present study was to explore the effects of Dapagliflozin on renal fibrosis in streptozotocin (STZ)-induced type 2 diabetes mellitus (T2DM) rats, and to determine the underlying mechanism of action. A total of 24 SPF male SD rats were randomly divided into 4 groups: A normal (Control) group, model group (STZ-induced T2DM rats), Dapagliflozin group (STZ-induced T2DM rats treated with 1 mg/kg Dapagliflozin), and a metformin group (STZ-induced T2DM rats treated with 200 mg/kg metformin), with 6 rats per a group. Peripheral blood and renal tissues were collected from these rats, and the renal indices of each group were examined. The fasting blood glucose (FBG), glycosylated hemoglobin (HbA1c), blood urea nitrogen (BUN), and serum creatinine (SCr) of rats were detected. After 24 h, the urine was collected and the urine protein levels were measured. Hematoxylin and eosin staining was used to detect histological changes in the rat kidney; Masson staining was used to observe the degree of fibrosis in rat renal tissues; and western blot was performed to determine the expression levels of α-smooth muscle actin (SMA), vimentin, E-cadherin, TGF-β1, Smad7, and p-Smad3 in rat renal tissues. Dapagliflozin effectively inhibited the increase in FBG and HbA1c levels in diabetic mice, reduced renal tissue damage, reduced the renal index values, reduced collagen deposition in the glomerulus and interstitial area, and reduced the proliferation of glomerular mesangial cells. In addition, Dapagliflozin significantly lowered the levels of BUN, SCr, and 24-h urine protein, decreased the protein expression of α-SMA, vimentin, TGF-β1, and p-Smad3, and increased the protein expression levels of E-cadherin and Smad7. Together, these results showed that Dapagliflozin alleviated renal fibrosis in STZ-induced T2DM rats, and its mechanism of action may be related to the inhibition of the TGF-β1/Smad pathway.

Qiteng Xiaozhuo granules medicated serum inhibits excessive proliferation and promotes apoptosis of human glomerular mesangial cells by targeting fat mass and obesity associated proteins.

To explore whether fat mass and obesity associated proteins (FTO) is an important target of Qiteng Xiaozhuo granules (QTXZG,) medicated serum in regulating proliferation and apoptosis of glomerular mesangial cells. Medicated serum was obtained from Sprague-Dawley (SD) rats administered intragastrically with QTXZG decoction. The optimal concentration and intervention time of medicated serum were selected with the cell counting kit 8 assay. Cell proliferation was assessed by 5-ethynyl-2'-deoxyuridine (EdU) and cell apoptosis was investigated using flow cytometry. The expression of FTO, Proliferating cell nuclear antigen, Cyclin D1, B-cell lymphoma 2 (Bcl2) and BCL2 assaciated X was detected by Western blot and Real-time quantitative polymerase chain reaction, respectively. Quantification of the m6A RNA methylation was utilized to determine the total level of m6A methylation modification. EdU and flow cytometry assays revealed that QTXZG medicated serum can remarkably inhibit proliferation and promote apoptosis of lipopolysaccharide (LPS)-induced human glomerular mesangial cells (HGMCs). The FTO overexpression plasmid could inhibit proliferation and promote apoptosis of LPS-induced HGMCs. The FTO inhibitor (FB23-2) can significantly attenuate the effect of QTZXG medicated serum on inhibiting excessive proliferation and promoting apoptosis. QTXZG medicated serum can significantly increase FTO expression and decrease the level of m6A methylation modification. FTO is a key target for QTXZG medicated serum in inhibiting excessive proliferation and promoting apoptosis of human glomerular mesangial cells.

Overexpression of FTO inhibits excessive proliferation and promotes the apoptosis of human glomerular mesangial cells by alleviating FOXO6 m6A modification via YTHDF3-dependent mechanisms.

Background: N6-methyladenosine (m6A) is a prevalent post-transcriptional modification presented in messenger RNA (mRNA) of eukaryotic organisms. Chronic glomerulonephritis (CGN) is characterised by excessive proliferation and insufficient apoptosis of human glomerular mesangial cells (HGMCs) but its underlying pathogenesis remains undefined. Moreover, the role of m6A in CGN is poorly understood. Methods: The total level of m6A modification was detected using the m6A quantification assay (Colorimetric). Cell proliferation was assessed by EdU cell proliferation assay, and cell apoptosis was detected by flow cytometry. RNA sequencing was performed to screen the downstream target of fat mass and obesity-associated protein (FTO). MeRIP-qPCR was conducted to detect the m6A level of forkhead box o6 (FOXO6) in HGMCs. RIP assay was utilized to indicate the targeting relationship between YTH domain family 3 (YTHDF3) and FOXO6. Actinomycin D assay was used to investigate the stability of FOXO6 in HGMCs. Results: The study found that the expression of FTO was significantly reduced in lipopolysaccharide (LPS)-induced HGMCs and renal biopsy samples of patients with CGN. Moreover, FTO overexpression and knockdown could regulate the proliferation and apoptosis of HGMCs. Furthermore, RNA sequencing and cellular experiments revealed FOXO6 as a downstream target of FTO in regulating the proliferation and apoptosis of HGMCs. Mechanistically, FTO overexpression decreases the level of FOXO6 m6A modification and reduces the stability of FOXO6 mRNA in a YTHDF3-dependent manner. Additionally, the decreased expression of FOXO6 inhibits the PI3K/AKT signaling pathway, thereby inhibiting the proliferation and promoting apoptosis of HGMCs. Conclusion: This study offers insights into the mechanism through which FTO regulates the proliferation and apoptosis of HGMCs by mediating m6A modification of FOXO6 mRNA. These findings also suggest FTO as a potential diagnostic marker and therapeutic target for CGN.

Sublytic C5b-9 induces TIMP3 expression by glomerular mesangial cells via TRAF6-dependent KLF5 K63-linked ubiquitination in rat Thy-1 nephritis

Rat Thy-1 nephritis (Thy-1N) is an experimental model for studying human mesangioproliferative glomerulonephritis (MsPGN), and its pathological features are glomerular mesangial cell (GMC) proliferation and extracellular matrix (ECM) accumulation. Although we have confirmed that renal lesions of Thy-1N rats are sublytic C5b-9-dependent, and ECM accumulation is related to tissue inhibitor of matrix metalloproteinase (TIMP) inhibiting matrix metalloproteinase (MMP) activity, whether sublytic C5b-9 can induce TIMP production by GMC in Thy-1N rat and the underlying mechanism remains unclear. In the study, we proved that the expressions of TIMP3, krϋppel-like transcription factor 5 (KLF5) and tumor necrosis factor receptor-associated factor 6 (TRAF6) were simultaneously up-regulated both in the renal tissues of Thy-1N rats (in vivo) and in the GMC exposed to sublytic C5b-9 (in vitro). Further mechanism exploration discovered that KLF5 and TRAF6 as two upstream molecules could induce TIMP3 gene transcription through binding to the same region i.e., −1801nt to −1554nt (GGGGAGGGGC) and −228nt to −46nt (GCCCCGCCCC) of TIMP3 promoter. In the process, TRAF6 mediated KLF5 K63-linked ubiquitination at K99 and K100 enhancing KLF5 nuclear localization and binding to TIMP3 promoter, augmenting its gene activation. Furthermore, the experiments in vivo exhibited that silencing KLF5, TRAF6 or TIMP3 gene could markedly lessen renal KLF5 K63-linked ubiquitination or TIMP3 induction, ECM accumulation and other pathological changes of Thy-1N rats. Besides, the positive expressions of above-mentioned these proteins and ECM accumulation and their correlation in the renal tissues of MsPGN patients were also demonstrated. Overall, our findings implicate that KLF5 and TRAF6 play a promoting role in sublytic C5b-9-triggered TIMP3 gene transcription and expression, which might provide a novel mechanistic insight into rat Thy-1N and human MsPGN.

Inhibition of STIM1 alleviates high glucose-induced proliferation and fibrosis by inducing autophagy in mesangial cells.

Diabetic nephropathy (DN) is a renal microvascular complication caused by diabetes mellitus. One of the most typical characteristics of DN is glomerular mesangial cells (GMCs) proliferation. Stromal interaction molecule 1 (STIM1), a Ca2+ channel, is involved in many diseases. In this study, we investigated the role of STIM1 in the proliferation and fibrosis in high glucose (HG)-induced HBZY-1 cells. We found that the expression of STIM1 was increased in renal tissues of diabetic rat and HBZY-1 cells stimulated by HG. Downregulation of STIM1-mediated SOCE suppressed hyperglycemic cell proliferation and fibrosis by activating autophagy. In addition, the inhibitory effect of downregulating STIM1 on cells was blocked by autophagy inhibitor Bafilomycin A1 (BafA1). Moreover, this experiment also showed that STIM1 regulated autophagy, cell proliferation and fibrosis via PI3K/AKT/mTOR signal pathway. These results clarify the role of STIM1 in HBZY-1 cells and its mechanism, and provide a new target for the treatment of DN.

Isoliquiritigenin attenuates high glucose-induced proliferation, inflammation, and extracellular matrix deposition in glomerular mesangial cells by suppressing JAK2/STAT3 pathway.

To investigate the effect of isoliquiritigenin (ISL) on high glucose (HG)-induced glomerular mesangial cells (GMCs) proliferation, extracellular matrix (ECM) deposition and inflammation, and the underlying mechanisms. Mouse GMCs (SV40-MES-13) were cultured in HG medium, with or without ISL. The proliferation of GMCs was determined by MTT assay. The production of proinflammatory cytokines was detected by qRT-PCR and ELISA. The expression of connective tissue growth factor (CTGF), TGF-β1, collagen IV, and fibronectin was measured by qRT-PCR and western blot. The phosphorylation of JAK2 and STAT3 was examined by western blot. Next, JAK2 inhibitor AG490 was applied to HG-exposed GMCs. The levels of JAK2/STAT3 phosphorylation and pro-fibrotic markers were analyzed by western blot, and the secretion of TNF-α and IL-1β was evaluated by ELISA. GMCs were treated with HG, HG plus ISL or HG plus ISL, and recombinant IL-6 (rIL-6) which is a JAK2 activator. The levels of JAK2/STAT3 activation, ECM formation, and proinflammatory cytokines secretion were determined by western blot and ELISA, respectively. In mouse GMCs, ISL successfully repressed HG-induced hyperproliferation; production of TNF-α and IL-1β; expression of CTGF, TGF-β1, collagen IV, and fibronectin; and activation of JAK2/STAT3. Similar to ISL, AG490 was able to reverse the inflammation and ECM generation caused by HG. Moreover, rIL-6 impeded the amelioration of ISL on HG-induced adverse effects. Our study demonstrated that ISL displayed preventive effects on HG-exposed GMCs through inhibiting JAK2/STAT3 pathway and provided an insight into the application of ISL for diabetic nephropathy (DN) treatment.

RNA sequencing reveals the mechanism of FTO in inhibiting inflammation and excessive proliferation of lipopolysaccharide-induced human glomerular mesangial cells.

Chronic glomerulonephritis (CGN) is a leading cause of end-stage renal disease in China; thus, there is an urgent need for effective therapeutic targets and strategies for CGN treatment. However, studies on CGN pathogenesis are limited. In this study, we found that the fat mass and obesity-associated protein (FTO) was significantly decreased in the lipopolysaccharide (LPS)-induced human glomerular mesangial cells (HGMCs) (P < 0.01) and kidney tissues of CGN patients (P < 0.05). Moreover, double-labeling immunofluorescence and flow cytometry assays demonstrated that the overexpression of FTO could inhibit inflammation and excessive proliferation of HGMCs. Furthermore, RNA-sequencing (RNA-seq) and real-time quantitative polymerase chain reaction (RT-qPCR) analyses revealed that FTO overexpression induced differential expression of 269 genes (absolute fold change ≥ 2 and P-value < 0.05), including 143 upregulated and 126 downregulated genes. Further functional analysis of these differentially expressed genes by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses suggested that FTO possibly mediates its inhibitory function by regulating the mammalian target of rapamycin (mTOR) signaling pathway and substance metabolism. Lastly, analysis of the PPI network and further identification of the top 10 hub genes (RPS15, RPS18, RPL18A, GNB2L1, RPL19, EEF1A1, RPS25, FAU, UBA52, and RPS6) indicated that FTO mediates its function by affecting the ribosomal proteins. Therefore, in this study, we elucidated the important role of FTO in the regulation of inflammation and excessive proliferation of HGMCs, suggesting FTO administration as a suitable therapeutic intervention for CGN.

#3261 SAR131675, A SELECTIVE VEGFR3 INHIBITOR, AMELIORATES RENAL INFLAMMATION AND LYMPHANGIOGENESIS IN THE MURINE LUPUS NEPHRITIS MODEL

Abstract Background and Aims Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by immune-complex deposits and inflammatory cell infiltrations in multiple organs, and approximately half of lupus patients have nephritis. Lymphangiogenesis is the proliferation of pre-existing lymphatic vessels (LVs), which regulate tissue fluid homeostasis and immune cell trafficking, responding to the tissue environment. In this study, we evaluated the therapeutic effect of SAR131672, a selective VEGFR3 inhibitor, on the murine lupus nephritis model by regulating inflammation and lymphangiogenesis. Method First, we reviewed medical records for the biopsy-proven lupus nephritis and performed an immunohistochemistry study for D2-40. For animal experiments, seven to eight-week-old male BALB/c mice were used. The back area's skin was shaved and treated topically three times per week, with 100 μg of resiquimod in 100 μl of acetone for eight weeks and concomitantly treatment of VEGFR3 inhibitor, SAR 131672 by oral gavage. We evaluated renal histology and immunofluorescent study for inflammatory cells and lymphatic vessels. We also evaluated inflammatory cytokines and chemokines, lymphangiogenic factors by qRT-PCR. Results In a human study, we found that the higher lupus activity index, the more D2-40(+) lymphatic vessels are expressed in the tubulointerstitial areas. In an animal study, eight weeks of topical treatment of resiquimod to Balb/c mice induces lupus-like symptoms such as weight loss, splenomegaly, and glomerular immune complexes deposits such as IgG, IgM, and C3 in immunofluorescent staining. Histologically, glomerular mesangial cell proliferation and increased inflammatory cells in tubulointerstitial areas were noted in the H&amp;E stain. Inhibition of VEGFR3 by oral SAR131672 treatment decreases glomerular and tubulointerstitial inflammation and decreases LYVE-1(+) lymphatic vessels. The pro-inflammatory cytokines and chemokines such as ICAM-1, VCAM-1, MCP-1, CCL19, CCL21, CCR7, CXCL13, and BAFF mRNA levels were increased compared with the vehicle-treated group. Treatment SAR131672 decreases pro-inflammatory cytokines and chemokine. Conclusion Inhibition of VEGFR3 by SAR131672 decreases the resiquimod-induced lupus nephritis model by regulating inflammation and lymphangiogenesis.

Astragaloside IV attenuates high glucose-induced NF-κB-mediated inflammation through activation of PI3K/AKT-ERK-dependent Nrf2/ARE signaling pathway in glomerular mesangial cells.

Inflammation is a key contributor to diabetic kidney disease pathogenesis, including reactive oxidation stress (ROS)-mediated nuclear factor-κB (NF-κB) signaling pathway. In this study, we examined the effect of Astragaloside IV (AS-IV) on anti-inflammatory and anti-oxidative properties under high glucose (HG) condition and the potential mechanism in glomerular mesangial cells (GMCs). We showed that AS-IV concentration-dependently reduced GMCs proliferation, restrained ROS release and hydrogen peroxide content, and suppressed pro-inflammatory cytokines as well as pro-fibrotic factors expression, which were associated with the inhibition of NF-κB and nuclear factor-erythroid 2-related factor 2 (Nrf2) signaling activation. Accordingly, both NF-κB overexpression by using RNA plasmid and Nrf2 gene silencing by using RNA interference weakened the ability of AS-IV to ameliorate HG-induced oxidative stress, inflammation, and cell proliferation. Furthermore, phosphatidylinositide 3-kinases (PI3K)/serine/threonine protein kinase (Akt) and extracellular regulated protein kinases (ERK) signaling pathway regulated the process of AS-IV-induced Nrf2 activation and antioxidant capacity, which evidenced by using PI3K inhibitor LY294002 or ERK inhibitor PD98059 that largely abolished the AS-IV efficacy. Taken together, these results indicated that AS-IV protected against HG-induced GMCs damage by inhibiting ROS/NF-kB-induced increases of inflammatory cytokines, fibrosis biomarkers, and cell proliferation via up-regulation of Nrf2-dependent antioxidant enzyme expression, which were mediated by PI3K/Akt and ERK signaling pathway activation.

Study of the shared gene signatures of polyarticular juvenile idiopathic arthritis and autoimmune uveitis.

To explore the shared gene signatures and potential molecular mechanisms of polyarticular juvenile idiopathic arthritis (pJIA) and autoimmune uveitis (AU). The microarray data of pJIA and AU from the Gene Expression Omnibus (GEO) database were downloaded and analyzed. The GEO2R tool was used to identify the shared differentially expressed genes (DEGs) and genes of extracellular proteins were identified among them. Then, weighted gene co-expression network analysis (WGCNA) was used to identify the shared immune-related genes (IRGs) related to pJIA and AU. Moreover, the shared transcription factors (TFs) and microRNAs (miRNAs) in pJIA and AU were acquired by comparing data from HumanTFDB, hTFtarget, GTRD, HMDD, and miRTarBase. Finally, Metascape and g: Profiler were used to carry out function enrichment analyses of previously identified gene sets. We found 40 up-regulated and 15 down-regulated shared DEGs via GEO2R. Then 24 shared IRGs in positivity-related modules, and 18 shared IRGs in negatively-related modules were found after WGCNA. After that, 3 shared TFs (ARID1A, SMARCC2, SON) were screened. And the constructed TFs-shared DEGs network indicates a central role of ARID1A. Furthermore, hsa-miR-146 was found important in both diseases. The gene sets enrichment analyses suggested up-regulated shared DEGs, TFs targeted shared DEGs, and IRGs positivity-correlated with both diseases mainly enriched in neutrophil degranulation process, IL-4, IL-13, and cytokine signaling pathways. The IRGs negatively correlated with pJIA and AU mainly influence functions of the natural killer cell, cytotoxicity, and glomerular mesangial cell proliferation. The down-regulated shared DEGs and TFs targeted shared DEGs did not show particular functional enrichment. Our study fully demonstrated the flexibility and complexity of the immune system disorders involved in pJIA and AU. Neutrophil degranulation may be considered the shared pathogenic mechanism, and the roles of ARID1A and MiR-146a are worthy of further in-depth study. Other than that, the importance of periodic inspection of kidney function is also noteworthy.

Alleviation of renal injury in rabbits by allisartan

The objective of this study was to determine the relationship between renal injury and inflammatory response induced by high-fat diet in rabbits and the interventional effect of allisartan. Fifteen 6-week-old healthy male rabbits were randomly divided into three groups: normal control (NC) group, high-lipid diet (HLD) group, high-lipid diet and allisartan (HLD+ALST) group. After allisartan treatment for 12 weeks, changes in total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), serum creatinine (Scr) and blood urea nitrogen (BUN) were measured enzymatically in the three groups. The left side of the kidney tissue was kept for paraffin section, and HE staining, periodic acid-Schiff (PAS) staining and Masson staining were used to observe the renal pathologic changes. TC, TG, LDL-C, Scr and BUN levels were all higher and HDL-C levels were lower in the HLD group compared with the NC group. Compared with the HLD group, Scr and BUN levels were significantly decreased in the HLD+ALST group. The results of HE staining showed that allisartan improved the changes of renal tissue morphology in rabbits on high-fat diet, reduced glomerular mesangial cell proliferation and improved glomerulosclerosis; PAS staining showed that glomerular glycogen deposition was reduced and glomerular red staining was significantly lighter; Masson staining showed that renal tubular blue-stained collagen fibers were reduced. In conclusion, hyperlipidemia can lead to aberrant expression of multiple cellular proteins and kidney tissue morphological damage in rabbits. On the other hand, allisartan attenuated renal injury and the mechanism may be related to the downregulation of the inflammatory response.

Retracted: Repression of microRNA-382 inhibits glomerular mesangial cell proliferation and extracellular matrix accumulation via FoxO1 in mice with diabetic nephropathy.

Retracted: Repression of microRNA-382 inhibits glomerular mesangial cell proliferation and extracellular matrix accumulation via FoxO1 in mice with diabetic nephropathy.

Alleviation of renal injury in rabbits by allisartan

The objective of this study was to determine the relationship between renal injury and inflammatory response induced by high-fat diet in rabbits and the interventional effect of allisartan. Fifteen 6-week-old...

FADD phosphorylation contributes to development of renal fibrosis by accelerating epithelial-mesenchymal transition

ABSTRACT FADD, a classical apoptotic signaling adaptor, has recently been reported to exhibit a series of non-apoptotic functions. Here, we report that FADD may play a critical role in the development of renal fibrosis. Neutrophil infiltration in the renal interstitial part, glomerular mesangial cell proliferation, and base-membrane thickening were observed in FADD-D mice by H&E, PAS, and PASM staining. Immunofluorescence analysis revealed that macrophage infiltration was significantly enhanced in FADD-D mice. Renal fibrosis might be induced by IgA nephritis in FADD-D mice as evidenced by increased Ki67 and type IV collagen. Additionally, the levels of α-SMA, Fibronectin, and Vimentin were also found to be elevated. Mechanism study indicated that the TLR4/myD88/NF-κB signaling pathway was activated in FADD-D mice. Moreover, FADD phosphorylation activated the mTOR and TGF-β/Smad pathway and accelerated the process of epithelial mesenchymal transition. Further studies indicated that the TGF-β1 pathway was also activated and the process of EMT was accelerated in both FADD-disrupted HEK293 cells and FADD-deficient MES cells. Thus, we concluded that FADD phosphorylation could lead to IgA nephritis and eventually result in renal fibrosis. Taken together, our study provides evidence, for the first time, that FADD, especially in its phosphorylated form, has an effect on the development of renal fibrosis. Abbreviations: FADD: FAS-associated protein with death domain; DED: death effector domain; DD: death domain; CKD: chronic kidney disease; ECM: extracellular matrix; ESRD: end-stage renal disease; RRT: renal replacement therapy; H&E: hematoxylin and eosin; PASM: periodic acid silver methenamine

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.

Exosomal miR-7002-5p derived from highglucose-induced macrophages suppresses autophagy in tubular epithelial cells by targeting Atg9b.

Macrophage infiltration plays an important role in the progression of diabetic nephropathy (DN). Previously, we demonstrated that highglucose-stimulated macrophage-derived exosomes (HG-exo) induces proliferation and extracellular matrix accumulation in glomerular mesangial cells, but its effect on tubular cells is unclear. This study aimed to explore the role of HG-exo on renal tubular injury in DN. The results show that HG-exo could induce dysfunction, autophagy inhibition, and inflammation in mouse tubular epithelial cell (mTEC) and C57 mouse kidney. Moreover, miR-7002-5p was differentially expressed in HG-exo based on miRNAs sequencing and bioinformatics analysis. A dual-luciferase reporter assay confirmed that Atg9b was the direct target gene of miR-7002-5p. Further experimentation showed that miR-7002-5p inhibition in vivo and vitro reserves HG-exo effects. These results demonstrated that HG-exo carries excessive miR-7002-5p and inhibits autophagy through targeting Atg9b; this process then induces renal tubular dysfunction and inflammation. In conclusion, our study clarifies the important role of macrophage-derived exosomes in DN and is expected to provide new insight on DN prevention and treatment.

Network pharmacology and molecular docking technology-based predictive study of the active ingredients and potential targets of rhubarb for the treatment of diabetic nephropathy

Diabetic nephropathy (DN) is one of the most serious complications of diabetes and the main cause of end-stage renal failure. Rhubarb is a widely used traditional Chinese herb, and it has exhibited efficacy in reducing proteinuria, lowering blood sugar levels and improving kidney function in patients with DN. However, the exact pharmacological mechanism by rhubarb improves DN remain unclear due to the complexity of its ingredients. Hence, we systematically explored the underlying mechanisms of rhubarb in the treatment of DN. We adopted a network pharmacology approach, focusing on the identification of active ingredients, drug target prediction, gene collection, Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes enrichment. Molecular docking technology was used to verify the binding ability between the main active compounds and central therapeutic targets, and screen out the core active ingredients in rhubarb for the treatment of DN. Finally, molecular dynamics simulation was performed for the optimal core protein-ligand obtained by molecular docking using GROMACS software. The network analysis identified 16 active compounds in rhubarb that were linked to 37 possible therapeutic targets related to DN. Through protein–protein interaction analysis, TP53, CASP8, CASP3, MYC, JUN and PTGS2 were identified as the key therapeutic targets. By validation of molecular docking, finding that the central therapeutic targets have good affinities with the main active compounds of rhubarb, and rhein, beta-sitosterol and aloe-emodin were identified as the core active ingredients in rhubarb for the treatment of DN. Results from molecular dynamics simulations showed that TP53 and aloe-emodin bound very stably with a binding free energy of − 26.98 kcal/mol between the two. The results of the gene enrichment analysis revealed that the PI3K-Akt signalling pathway, p53 signalling pathway, AGE-RAGE signalling pathway and MAPK signalling pathway might be the key pathways for the treatment of DN, and these pathways were involved in podocyte apoptosis, glomerular mesangial cell proliferation, inflammation and renal fibrosis. Based on the network pharmacology approach and molecular docking technology, we successfully predicted the active compounds and their respective targets. In addition, we illustrated the molecular mechanisms that mediate the therapeutic effects of rhubarb against DN. These findings provided an important scientific basis for further research of the mechanism of rhubarb in the treatment of DN.

Circ_0114428 promotes proliferation, fibrosis and EMT process of high glucose-induced glomerular mesangial cells through regulating the miR-185-5p/SMAD3 axis

Circular RNA (circRNA) has been confirmed to be the key regulators of diabetic nephropathy (DN) progression. However, the role of circ_0114428 in the DN progression remains unclear. Glomerular mesangial cells (GMCs) were treated with high glucose (HG) to mimic DN cell models in vitro. The expression levels of circ_0114428, microRNA (miR)-185-5p, and SMAD3 mRNA were examined by quantitative real-time PCR. Cell proliferation ability was detected by MTT assay, EdU staining and flow cytometry. The protein levels of proliferation marker, fibrosis markers, epithelial-mesenchymal transition (EMT) markers and SMAD3 were measured by western blot assay. The interaction between miR-185-5p and circ_0114428 or SMAD3 was confirmed via dual-luciferase reporter assay, RIP assay and RNA pull-down assay. Our data showed that circ_0114428 was upregulated in HG-induced GMCs. Circ_0114428 overexpression could aggravate the promotion effect of HG on the proliferation, fibrosis and EMT process of GMCs, while its knockdown had an opposite effect. In the terms of mechanisms, circ_0114428 could sponge miR-185-5p to regulate SMAD3. MiR-185-5p inhibitor could reverse the suppressive effect of circ_0114428 knockdown on the proliferation, fibrosis and EMT process in HG-induced GMCs. Also, SMAD3 overexpression abolished the inhibition of miR-185-5p on the proliferation, fibrosis and EMT process in HG-induced GMCs. Taken together, our data suggested that circ_0114428 might promote DN progression by regulating the miR-185-5p/SMAD3 axis.

Sestrin2 attenuates renal damage by regulating Hippo pathway in diabetic nephropathy.

Glomerular mesangial cell proliferation and extracellular matrix accumulation contribute to the progression of diabetic nephropathy (DN). As a conserved stress-inducible protein, sestrin2 (Sesn2) plays critical role in the regulation of oxidative stress, inflammation, autophagy, metabolism, and endoplasmic reticulum stress. In this study, we investigated the role of Sesn2 on renal damage in diabetic kidney using transgenic mice overexpressing Sesn2 and the effect of Sesn2 on mesangial cell proliferation and extracellular matrix accumulation in diabetic conditions and the possible molecular mechanisms involved. Sesn2 overexpression improved renal function and decreased glomerular hypertrophy, albuminuria, mesangial expansion, extracellular matrix accumulation, and TGF-β1 expression, as well as oxidative stress in diabetic mice. In vitro experiments, using human mesangial cells (HMCs), revealed that Sesn2 overexpression inhibited high glucose (HG)-induced proliferation, fibronectin and collagen IV production, and ROS generation. Meanwhile, Sesn2 overexpression restored phosphorylation levels of Lats1 and YAP and inhibited TEAD1 expression. Inhibition of Lats1 accelerated HG-induced proliferation and expression of fibronectin and collagen IV. Verteporfin, an inhibitor of YAP, suppressed HG-induced proliferation and expression of fibronectin and collagen IV. However, Sesn2 overexpression reversed Lats1 deficiency-induced Lats1 and YAP phosphorylation, nuclear expression levels of YAP and TEAD1, and proliferation and fibronectin and collagen IV expressions in HMCs exposed to HG. In addition, antioxidant NAC or tempol treatment promoted phosphorylation of Lats1 and YAP and inhibited TEAD1 expression, proliferation, and fibronectin and collagen IV accumulation in HG-treated HMCs. Taken together, Sesn2 overexpression inhibited mesangial cell proliferation and fibrosis via regulating Hippo pathway in diabetic nephropathy. Induction of Sesn2 may be a potential therapeutic target in diabetic nephropathy.

The involvement of branched-chain amino acids (BCAAs) in aromatic trihalogenated DBP exposure-induced kidney damage in mice

Disinfection by-products (DBPs) are inevitably generated in the process of disinfection. Among them, aromatic halogenated DBPs, such as 2,4,6-trichlorophenol (TCP), 2,4,6-tribromophenol (TBP) and 2,4,6-triiodophenol (TIP), have attracted considerable interest for their high toxicity. A systematic nephrotoxicity evaluation of 2,4,6-trihalophenols is still lacking. In this study, mice were exposed to TCP, TBP and TIP ranging from environmental-related low concentration to high concentration that commonly used in animal study (0.5–200 μg/L). Kidney histopathology, urine protein detection and urine metabolomics were performed. Remarkable changes including kidney damage, proteinuria and glomerular mesangial cell proliferation were observed after three 2,4,6-trihalophenol exposure, even at low concentration of 0.5 μg/L. The nephrotoxicity rank order was TIP > TBP > TCP. Additionally, in vivo exposure to 2,4,6-trihalophenols also led to apparent changes in urinary metabolic profiles. Biosynthesis pathways of branched-chain amino acids (BCAAs, containing valine, leucine and isoleucine) were disturbed even at the early stage of exposure (4 weeks). Intriguingly, it has been reported that BCAAs could promote the proliferation of glomerular mesangial cells. Thus, in vitro cell experiments were further performed on mouse glomerular mesangial cell line MES-13. Consistently with in vivo results, cell proliferation was observed in MES-13 cells after exposure to 2,4,6-trihalophenols, especially to TBP and TIP. Meanwhile, TCP at high concentration, TBP and TIP at not only high concentration but also low concentration, induced BCAAs accumulation in glomerular mesangial cells, which was completely commensurate to that observed in cell proliferation assay. Then the proliferation of MES-13 cells induced by 2,4,6-trihalophenols was remarkably inhibited after BCAAs interference. Here we provide direct link between disturbed BCAAs and the nephrotoxicity of 2,4,6-trihalophenols. 2,4,6-trihalophenols could induce excess BCAAs, which further led to proliferation of glomerular mesangial cells and renal injury. This study revealed the nephrotoxicity of aromatic trihalogenated DBPs and provided new insights into the potential toxic mechanisms.