Single-cell RNA transcriptomic reveal the mechanism of MSC derived small extracellular vesicles against DKD fibrosis.

Diabetic kidney disease (DKD), a chronic kidney disease, is characterized by progressive fibrosis caused due to persistent hyperglycemia. The development of fibrosis in DKD determines the patient prognosis, but no particularly effective treatment. Here, small extracellular vesicles derived from mesenchymal stem cells (MSC-sEV) have been used to treat DKD fibrosis. Single-cell RNA sequencing was used to analyze 27,424 cells of the kidney, we have found that a novel fibrosis-associated TGF-ß1+Arg1+ macrophage subpopulation, which expanded and polarized in DKD and was noted to be profibrogenic. Additionally, Actin+Col4a5+ mesangial cells in DKD differentiated into myofibroblasts. Multilineage ligand-receptor and cell-communication analysis showed that fibrosis-associated macrophages activated the TGF-ß1/Smad2/3/YAP signal axis, which promotes mesangial fibrosis-like change and accelerates renal fibrosis niche. Subsequently, the transcriptome sequencing and LC-MS/MS analysis indicated that MSC-sEV intervention could restore the levels of the kinase ubiquitin system in DKD and attenuate renal interstitial fibrosis via delivering CK1δ/ß-TRCP to mediate YAP ubiquitination degradation in mesangial cells. Our findings demonstrate the unique cellular and molecular mechanisms of MSC-sEV in treating the DKD fibrosis niche at a single-cell level and provide a novel therapeutic strategy for renal fibrosis.

LncRNA SNHG14 silencing attenuates the progression of diabetic nephropathy via the miR-30e-5p/SOX4 axis.

BACKGROUND: Diabetic nephropathy (DN) is a diabetic complication. LncRNAs are reported to participate in the pathophysiology of DN. Here, the function and mechanism of lncRNA small nucleolar RNA host gene 14 (SNHG14) in DN were explored. METHODS: Streptozotocin (STZ)-induced DN mouse models and high glucose (HG)-treated human mesangial cells (MCs) were used to detect SNHG14 expression. SNHG14 silencing plasmids were applied to examine the function of SNHG14 on proliferation and fibrosis in HG-treated MCs. Potential targets of SNHG14 were predicted using bioinformatics tools and verified by luciferase reporter, RNA pulldown, and northern blotting assays. The functional role of SNHG14 in DN in vivo was detected by injection with adenoviral vector carrying sh-SNHG14 into DN mice. Serum creatinine, blood urea nitrogen, blood glucose, 24-h proteinuria, relative kidney weight, and renal pathological changes were examined in DN mice. RESULTS: SNHG14 expression was elevated in the kidneys of DN mice and HG-treated MCs. SNHG14 silencing inhibited proliferation and fibrosis of HG-stimulated MCs. SNHG14 bound to miR-30e-5p to upregulate SOX4 expression. In rescue assays, SOX4 elevation diminished the effects of SNHG14 silencing in HG-treated MCs, and SOX4 silencing reversed the effects of SNHG14 overexpression. In in vivo studies, SNHG14 downregulation significantly ameliorated renal injuries and renal interstitial fibrosis in DN mice. CONCLUSIONS: SNHG14 silencing attenuates kidney injury in DN mice and reduces proliferation and fibrotic phenotype of HG-stimulated MCs via the miR-30e-5p/SOX4 axis.

Cross-species single-cell analysis uncovers the immunopathological mechanisms associated with IgA nephropathy progression.

IgA nephropathy (IgAN) represents the main cause of renal failure, while the precise pathogenetic mechanisms have not been fully determined. Herein, we conducted a cross-species single-cell survey on human IgAN and mouse and rat IgAN models to explore the pathogenic programs. Cross-species single-cell RNA sequencing (scRNA-Seq) revealed that the IgAN mesangial cells (MCs) expressed high levels of inflammatory signatures CXCL12, CCL2, CSF1, and IL-34 and specifically interacted with IgAN macrophages via the CXCL12/CXCR4, CSF1/IL-34/CSF1 receptor, and integrin subunit alpha X/integrin subunit alpha M/complement C3 (C3) axes. IgAN macrophages expressed high levels of CXCR4, PDGFB, triggering receptor expressed on myeloid cells 2, TNF, and C3, and the trajectory analysis suggested that these cells derived from the differentiation of infiltrating blood monocytes. Additionally, protein profiling of 21 progression and 28 nonprogression IgAN samples revealed that proteins CXCL12, C3, mannose receptor C-type 1, and CD163 were negatively correlated with estimated glomerular filtration rate (eGFR) value and poor prognosis (30% eGFR as composite end point). Last, a functional experiment revealed that specific blockade of the Cxcl12/Cxcr4 pathway substantially attenuated the glomerulus and tubule inflammatory injury, fibrosis, and renal function decline in the mouse IgAN model. This study provides insights into IgAN progression and may aid in the refinement of IgAN diagnosis and the optimization of treatment strategies.

Macrophages communicate with mesangial cells through the CXCL12/DPP4 axis in lupus nephritis pathogenesis.

Lupus nephritis (LN) occurs in 50% of cases of systemic lupus erythematosus (SLE) and is one of the most serious complications that can occur during lupus progression. Mesangial cells (MCs) are intrinsic cells in the kidney that can regulate capillary blood flow, phagocytose apoptotic cells, and secrete vasoactive substances and growth factors. Previous studies have shown that various types of inflammatory cells can activate MCs for hyperproliferation, leading to disruption of the filtration barrier and impairment of renal function in LN. Here, we characterized the heterogeneity of kidney cells of LN mice by single-nucleus RNA sequencing (snRNA-seq) and revealed the interaction between macrophages and MCs through the CXC motif chemokine ligand 12 (CXCL12)/dipeptidyl peptidase 4 (DPP4) axis. In culture, macrophages modulated the proliferation and migration of MCs through this ligand-receptor interaction. In LN mice, treatment with linagliptin, a DPP4 inhibitor, effectively inhibited MC proliferation and reduced urinary protein levels. Together, our findings indicated that targeting the CXCL12/DPP4 axis with linagliptin treatment may serve as a novel strategy for the treatment of LN via the CXCL12/DPP4 axis.

Phillyrin alleviates high glucose-induced oxidative stress and inflammation in HBZY-1 cells through inhibition of the PI3K/Akt signaling pathway.

BACKGROUND: Phillyrin, the major lignin compound of Forsythia suspense (Thunb.) Vahl, has been shown the effects of anti-inflammatory and antioxidant. Our study was aimed to explore the protective effect of phillyrin on glomerular mesangial cells (HBZY-1) and the potential mechanism. METHODS: Cell viability, cytokine production, levels of reactive oxygen radicals (ROS), glutathione (GSH), malondialdehyde (MDA), and superoxide dismutase (SOD), as well as autophagy and apoptosis levels were determined to verify the mechanism of phillyrin on HBZY-1 cells. RESULTS: Our result indicated that phillyrin significantly inhibited HG-induced HBZY-1 proliferation by inhibiting Bcl-2 expression and upregulating Bad, cleaved caspase-3, and -9 expression. Also, phillyrin suppressed HG-induced mesangial extracellular matrix accumulation by inhibiting the expression of fibronectin and transforming growth factor-ß1. Further, phillyrin inhibited oxidative stress and inflammation by decreasing ROS, MDA, TNF-α, IL-1ß, and IL-6 contents and increasing SOD and GSH expression. Phillyrin also promoted autophagy by increasing LC3-II/LC3-I ratio and down-regulating p62 expression. Furthermore, WB assay showed that phillyrin inhibited oxidative stress caused by HG via activating Nrf2 signaling pathway, while attenuated proliferation and inflammation in HBZY-1 cells through inactivating PI3K/Akt/mTOR and NF-κB pathways. CONCLUSION: All results showed that phillyrin might be a promising therapeutic agent for the treatment of DN.

Detecting and exploring kidney-derived extracellular vesicles in plasma.

BACKGROUND: Extracellular vesicles (EVs) have received considerable attention as ideal biomarkers for kidney diseases. Most reports have focused on urinary EVs, that are mainly derived from the cells in the urinary tract. However, the detection and the application of kidney-derived EVs in plasma remains uncertain. METHODS: We examined the kidney-derived small EVs (sEVs) in plasma that were supposedly released from renal mesangial and glomerular endothelial cells, using clinical samples from healthy controls and patients with kidney transplants. Plasma from healthy controls underwent ultracentrifugation, followed by on-bead flow cytometry, targeting α8 integrin, an antigen-specific to mesangial cells. To confirm the presence of kidney-derived sEVs in peripheral blood, plasma from ABO-incompatible kidney transplant recipients was ultracentrifuged, followed by western blotting for donor blood type antigens. RESULTS: Immunohistochemistry and immunoelectron microscopy confirmed α8 integrin expression in kidney mesangial cells and their sEVs. The CD9-α8 integrin double-positive sEVs were successfully detected using on-bead flow cytometry. Western blot analysis further revealed transplanted kidney-derived sEVs containing blood type B antigens in non-blood type B recipients, who had received kidneys from blood type B donors. Notably, a patient experiencing graft kidney loss exhibited diminished signals of sEVs containing donor blood type antigens. CONCLUSION: Our findings demonstrate the potential usefulness of kidney-derived sEVs in plasma in future research for kidney diseases.

DPP8/9 inhibition attenuates the TGF-ß1-induced excessive deposition of extracellular matrix (ECM) in human mesangial cells via Smad and Akt signaling pathways.

The pathogenesis of glomerular diseases is strongly influenced by abnormal extracellular matrix (ECM) deposition in mesangial cells. Dipeptidyl peptidase IV (DPPIV) enzyme family contains DPP8 and DPP9, which are involved in multiple diseases. However, the pathogenic roles of DPP8 and DPP9 in mesangial cells ECM deposition remain unclear. In this study, we observed that DPP8 and DPP9 were significantly increased in glomerular mesangial cells and podocytes in CKD patients compared with healthy individuals, and DPP9 levels were higher in the urine of IgA nephropathy (IgAN) patients than in control urine. Therefore, we further explored the mechanism of DPP8 and DPP9 in mesangial cells and revealed a significant increase in the expression of DPP8 and DPP9 in human mesangial cells (HMCs) following TGF-ß1 stimulation. Silencing DPP8 and DPP9 by siRNAs alleviated the expression of ECM-related proteins including collagen Ⅲ, collagen Ⅳ, fibronectin, MMP2, in TGF-ß1-treated HMCs. Furthermore, DPP8 siRNA and DPP9 siRNA inhibited TGF-ß1-induced phosphorylation of Smad2 and Smad3, as well as the phosphorylation of Akt in HMCs. The findings suggested the inhibition of DPP8/9 may alleviate HMCs ECM deposition induced by TGF-ß1 via suppressing TGF-ß1/Smad and AKT signaling pathways.

Effect of M0 macrophage-derived exosome miR-181d-5p targeting BCL-2 to regulate NLRP3/caspase-1/GSDMD pathway on human renal mesangial cells pyroptosis.

BACKGROUND: Lupus nephritis (LN) is a type of autoimmune disease that impacts the kidneys. Exosomes are valuable for in-depth studies of the pathogenesis of LN. This study aimed to explore miR-181d-5p expression levels in M0 macrophage-derived exosomes and their role in human renal mesangial cells (HRMC) pyroptosis through binding to BCL-2. METHODS: Peripheral blood mononuclear cells (PBMCs) were collected from patients with lupus nephritis (LN) and healthy subjects. Monocytes isolated from these samples were induced into M0 macrophages using recombinant human granulocyte colony-stimulating factor (rhG-CSF). In a parallel process, THP-1 cells were induced into M0 macrophages using Phorbol Myristate Acetate (PMA). LPS- and ATP-stimulated HRMC were used to construct a cell pyroptosis model. We then introduced different miR-181d-5p mimic fragments into the M0 macrophages derived from the THP-1 cells. Subsequently, exosomes from these macrophages were co-cultured with HRMC. To evaluate the impact on HRMC, we conducted proliferation and apoptosis assessments using CellCountingKit-8assay and flow cytometry. The effect of exosomal miR-181d-5p on HRMC pyroptosis was assessed using western blot. The miR-181d-5p and BCL-2 targeting relationship was detected using real-time fluorescence quantitative PCR. IL-6, IL-1ß, and TNF-α levels in cell supernatants were detected using ELISA kits. RESULTS: In this study, we observed an increase in miR-181d-5p levels within exosomes secreted from M0 macrophages obtained by induction of monocytes from LN patients. It was found that miR-181d-5p can target binding to BCL-2. Exosomes with elevated levels of miR-181d-5p contributed to a significant increase in miR-181d-5p within HRMC, facilitating its proliferation and inhibiting apoptosis. Furthermore, exosomes expressing high levels of miR-181d-5p were observed to promote an inflammatory response and pyroptosis in HRMC. Notably, these effects were reversed when the levels of miR-181d-5p in the exosomes were reduced. CONCLUSION: Inhibition of miR-181d-5p, derived from M0 macrophage exosomes, effectively suppresses inflammation and pyroptosis in HRMC. This discovery indicates that miR-181d-5p holds the potential as a valuable target in the development of treatments for Lupus Nephritis (LN).

Fructose overconsumption accelerates renal dysfunction with aberrant glomerular endothelial-mesangial cell interactions in db/db mice.

For the advancement of DKD treatment, identifying unrecognized residual risk factors is essential. We explored the impact of obesity diversity derived from different carbohydrate qualities, with an emphasis on the increasing trend of excessive fructose consumption and its effect on DKD progression. In this study, we utilized db/db mice to establish a novel diabetic model characterized by fructose overconsumption, aiming to uncover the underlying mechanisms of renal damage. Compared to the control diet group, the fructose-fed db/db mice exhibited more pronounced obesity yet demonstrated milder glucose intolerance. Plasma cystatin C levels were elevated in the fructose model compared to the control, and this elevation was accompanied by enhanced glomerular sclerosis, even though albuminuria levels and tubular lesions were comparable. Single-cell RNA sequencing of the whole kidney highlighted an increase in Lrg1 in glomerular endothelial cells (GECs) in the fructose model, which appeared to drive mesangial fibrosis through enhanced TGF-ß1 signaling. Our findings suggest that excessive fructose intake exacerbates diabetic kidney disease progression, mediated by aberrant Lrg1-driven crosstalk between GECs and mesangial cells.

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-ß1, 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-1ß 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-1ß; expression of CTGF, TGF-ß1, 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.

EIF2α/ATF4 pathway enhances proliferation of mesangial cell via cyclin D1 during endoplasmic reticulum stress in IgA nephropathy.

IgA nephropathy (IgAN) is an essential cause of kidney failure and end-stage kidney disease worldwide. Mesangial hypercellularity is an important characteristic of IgAN, but the underlying mechanism remains unclear. Endoplasmic reticulum (ER) stress is a series of stress responses to restore the function of endoplasmic reticulum. We aimed to explore how ER stress functioned in kidneys of IgAN. We first examined ER stress in IgAN kidneys in vivo and in vitro, by testing the levels of ER stress associated proteins (BIP, p-eIF2α and ATF4). Our results showed that ER stress was activated in IgAN patients, mice and cell model. ER stress activation was related to the distribution of IgA deposition and the degree of mesangial proliferation. To determine the role of ER stress in mesangial cell (MC) proliferation of IgAN, we then tested the levels of ER stress and MC proliferation (cyclin D1, cell viability and cell cycle) through inhibiting ER stress associated proteins. After inhibiting ER stress associated proteins, ER stress was inactivated and cell proliferation was inhibited in MCs. We also explored the correlation between ER stress in the glomerulus and the clinical outcomes of IgAN patients in a prospective study. Patients with lower expression of p-eIF2α or ATF4 had higher rates of hematuria remission, proteinuria remission and clinical remission. In summary, our work outlines that in IgAN, ER stress mediated by eIF2α/ATF4 pathway promotes MC proliferation via up-regulating the expression of cyclin D1. Furthermore, p-eIF2α and ATF4 in the glomerulus negatively correlate with the clinical remission of IgAN patients.

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

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: FTO is a key target for QTXZG medicated serum in inhibiting excessive proliferation and promoting apoptosis of human glomerular mesangial cells.

Cellular crosstalk of mesangial cells and tubular epithelial cells in diabetic kidney disease.

Diabetic kidney disease (DKD) is a major cause of end-stage renal disease and imposes a heavy global economic burden; however, little is known about its complicated pathophysiology. Investigating the cellular crosstalk involved in DKD is a promising avenue for gaining a better understanding of its pathogenesis. Nonetheless, the cellular crosstalk of podocytes and endothelial cells in DKD is better understood than that of mesangial cells (MCs) and renal tubular epithelial cells (TECs). As the significance of MCs and TECs in DKD pathophysiology has recently become more apparent, we reviewed the existing literature on the cellular crosstalk of MCs and TECs in the context of DKD to acquire a comprehensive understanding of their cellular communication. Insights into the complicated mechanisms underlying the pathophysiology of DKD would improve its early detection, care, and prognosis. Video Abstract.

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.

Macrophage-derived exosomes promote activation of NLRP3 inflammasome and autophagy deficiency of mesangial cells in diabetic nephropathy.

Dysfunction of mesangial cells plays a significant role in the glomerular lesions and is implicated in the pathophysiology of diabetic nephropathy (DN). Macrophages infiltration is the main pathological feature of DN, which can ultimately lead to renal inflammation. Recent studies suggest that the crosstalk between kidney resident cells and inflammatory cells influences the development of DN, and that controlling this crosstalk may help treat DN. Here, we found that DN mice appeared renal pathological damage, including dilation of mesangial matrix and significant infiltration of macrophages, accompanied by increased inflammatory response, NLRP3 inflammasome activation and autophagy deficiency. Additionally, mesangial cells internalized exosomes from high glucose (HG) treated macrophage, resulting the activation of inflammatory cytokines and NLRP3 inflammasome and deficiency of autophagy in vitro and in vivo. Moreover, C57BL/6 mice injected HG-stimulated macrophages-derived exosomes exhibited renal dysfunction and mesangial matrix expansion. Taken together, the present study demonstrated that mesangial cells responded to HG treated macrophage-derived exosomes by promoting the activation of NLRP3 inflammasome and autophagy deficiency, thereby participating in the development of DN.

Expression of interferon-stimulated gene 20 (ISG20), an antiviral effector protein, in glomerular endothelial cells: possible involvement of ISG20 in lupus nephritis.

BACKGROUND: In addition to regulating the antiviral response, increased expression of Toll-like receptor 3 (TLR3) in resident renal cells plays a role in developing some forms of glomerulonephritis. TLR3 activation leads to type I interferon (IFN) production, which induces the expression of IFN-stimulated genes (ISGs). However, the role of ISG20 expression in resident renal cells remains unclear. METHODS: Cultured normal human glomerular endothelial cells (GECs) were treated with polyinosinic-polycytidylic acid (poly IC), Escherichia coli lipopolysaccharide (LPS), R848, and CpG (TLR3, TLR4, TLR7, and TLR9 agonists, respectively). The mRNA levels of ISG20, CX3CL1/fractalkine, and CXCL10/IP-10 were measured by quantitative reverse transcription-polymerase chain reaction. ISG20 protein expression was assessed by Western blotting. RNA interference was used to knockdown IFN-ß and ISG20 expression. CX3CL1 protein levels were assessed by enzyme-linked immunosorbent assay. We performed immunofluorescence to examine endothelial ISG20 expression in biopsy specimens from patients with lupus nephritis (LN). RESULTS: In GECs, the expression of ISG20 mRNA and protein was increased by polyIC, not by LPS, R848, or CpG treatment. Moreover, ISG20 knockdown prevented poly IC-induced CX3CL1 expression but had no effect on CXCL10 expression. Intense endothelial ISG20 immunoreactivity was observed in biopsy specimens obtained from patients with proliferative LN. CONCLUSION: In GECs, ISG20 was regulated via TLR3 but not via TLR4, TLR7, or TLR9 signaling. Moreover, ISG20 was involved in regulating CX3CL1 production. In addition to regulating antiviral innate immunity, ISG20 may act as a mediator of CX3CL1 production, thereby inducing glomerular inflammation, particularly in patients with LN.

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.

Hyperactivation of YAP/TAZ Drives Alterations in Mesangial Cells through Stabilization of N-Myc in Diabetic Nephropathy.

SIGNIFICANCE STATEMENT: Mesangial cells (MCs) in the kidney are essential to maintaining glomerular integrity, and their impairment leads to major glomerular diseases including diabetic nephropathy (DN). Although high blood glucose elicits abnormal alterations in MCs, the underlying mechanism is poorly understood. We show that YAP/TAZ are increased in MCs of patients with DN and two animal models of DN. High glucose directly induces activation of YAP/TAZ through the canonical Hippo pathway in cultured MCs. Hyperactivation of YAP/TAZ in mouse MCs recapitulates the hallmarks of DN. Activated YAP/TAZ bind and stabilize N-Myc, one of the Myc family. N-Myc stabilization leads to aberrant enhancement of its transcriptional activity and to MC impairments. Our findings shed light on how high blood glucose in diabetes mellitus leads to DN and support a rationale that lowering blood glucose in diabetes mellitus could delay DN pathogenesis. BACKGROUND: Mesangial cells (MCs) in the kidney are central to maintaining glomerular integrity, and their impairment leads to major glomerular diseases, including diabetic nephropathy (DN). Although high blood glucose elicits abnormal alterations in MCs, the underlying molecular mechanism is poorly understood. METHODS: Immunolocalization of YAP/TAZ and pathological features of PDGFRß + MCs were analyzed in the glomeruli of patients with DN, in Zucker diabetic fatty rats, and in Lats1/2i ΔPß mice. RiboTag bulk-RNA sequencing and transcriptomic analysis of gene expression profiles of the isolated MCs from control and Lats1/2iΔPß mice were performed. Immunoprecipitation analysis and protein stability of N-Myc were performed by the standard protocols. RESULTS: YAP and TAZ, the final effectors of the Hippo pathway, are highly increased in MCs of patients with DN and in Zucker diabetic fatty rats. Moreover, high glucose directly induces activation of YAP/TAZ through the canonical Hippo pathway in cultured MCs. Hyperactivation of YAP/TAZ in mouse model MCs recapitulates the hallmarks of DN, including excessive proliferation of MCs and extracellular matrix deposition, endothelial cell impairment, glomerular sclerosis, albuminuria, and reduced glomerular filtration rate. Mechanistically, activated YAP/TAZ bind and stabilize N-Myc protein, one of the Myc family of oncogenes. N-Myc stabilization leads to aberrant enhancement of its transcriptional activity and eventually to MC impairments and DN pathogenesis. CONCLUSIONS: Our findings shed light on how high blood glucose in diabetes mellitus leads to DN and support a rationale that lowering blood glucose in diabetes mellitus could delay DN pathogenesis.

Salviolone protects against high glucose-induced proliferation, oxidative stress, inflammation, and fibrosis of human renal mesangial cells by upregulating membrane metalloendopeptidase expression.

As one of complications of diabetes mellitus, diabetic nephropathy is related to renal dysfunction. Membrane metalloendopeptidase (MME) is associated with the pathogenesis of diabetic nephropathy and exerts a protective function in high glucose (HG)-treated podocytes. Salviolone, one of important bioactive components from Salvia miltiorrhiza, possesses an anti-inflammatory activity. However, the roles of salviolone in renal mesangial cell dysfunction under HG condition remain unknown. The targets of salviolone in diabetic nephropathy were predicted by bioinformatics analysis. Relative mRNA level of MME was detected by qPCR in HG-treated human renal mesangial cells (HRMCs). Cell viability was analyzed using CCK-8 assay. Cell proliferation was investigated by EdU staining. Oxidative stress was evaluated by detection of ROS generation and levels of oxidative stress-related biomarkers. The inflammatory cytokines and fibrosis-related biomarkers were examined by ELISA. Our results showed that MME expression was decreased in diabetic nephropathy and HG-treated HRMCs. Salviolone increased MME level in HG-treated HRMCs. Salviolone mitigated HG-induced HRMC proliferation by increasing MME expression. Salviolone attenuated HG-induced ROS generation, MDA level increase, and SOD activity decrease through upregulating MME expression. Moreover, salviolone suppressed HG-induced increase of levels of TNF-α, IL-1ß, IL-6, fibronectin, and collagen IV through upregulating MME expression. In conclusion, salviolone attenuates proliferation, oxidative stress, inflammation, and fibrosis in HG-treated HRMCs through upregulating MME expression.

Nanosized copper particles induced mesangial cell toxicity via the autophagy pathway.

Nanosized copper particles (nano Cu) have been incorporated into products in multiple industries, although studies have demonstrated that these particles are nephrotoxic. We investigated the cytotoxicity of nanosized copper particles on rat mesangial cells and measured rates of apoptosis, the expression of caspase-3, and generation of reactive oxygen species. We also measured autophagy through the acridine orange (AO) staining and expression of Beclin-1, microtubule-associated protein 1 light chain 3, and p62 to screen the underlying mechanism of toxicity. Nanosized copper particles inhibited mesangial cell viability, up-regulated the activity of caspase-3, and increased the rates of apoptosis and the generation of reactive oxygen species in a concentration-dependent manner. Exposure to nano Cu increased the formation of acidic vesicular organelles and the expression of Beclin-1, microtubule-associated protein 1 light chain 3, and p62, and treatment with an autophagy inhibitor reduced nephrotoxicity. This indicated that the autophagy pathway is involved in the toxicity induced by nanosized copper particles to mesangial cells. This finding can contribute to the development of safety guidelines for the evaluation of nanomaterials in the future.