M2 macrophage infusion ameliorates diabetic glomerulopathy via the JAK2/STAT3 pathway in db/db mice.

Objectives: To explore the therapeutic effects of M2 macrophages in diabetic nephropathy (DN) and their mechanism.Methods: We infused M2 macrophages stimulated with IL-4 into 10-week-old db/db mice once a week for 4 weeks through the tail vein as M2 group. Then we investigated the role of M2 macrophages in alleviating the infammation of DN and explored the mechanism.Results: M2 macrophages hindered the progression of DN, reduced the levels of IL-1ß (DN group was 34%, M2 group was 13%, p < 0.01) and MCP-1 (DN group was 49%, M2 group was 16%, p < 0.01) in the glomeruli. It was also proven that M2 macrophages alleviate mesangial cell injury caused by a high glucose environment. M2 macrophage tracking showed that the infused M2 macrophages migrated to the kidney, and the number of M2 macrophages in the kidney reached a maximum on day 3. Moreover, the ratio of M2 to M1 macrophages was 2.3 in the M2 infusion group, while 0.4 in the DN group (p < 0.01). Mechanistically, M2 macrophages downregulated Janus kinase (JAK) 2 and signal transducer and activator of transcription (STAT) 3 in mesangial cells.Conclusions: Multiple infusions of M2 macrophages significantly alleviated inflammation in the kidney and hindered the progression of DN at least partially by abrogating the M1/M2 homeostasis disturbances and suppressing the JAK2/STAT3 pathway in glomerular mesangial cells. M2 macrophage infusion may be a new therapeutic strategy for DN treatment.

Astaxanthin attenuates diabetic kidney injury through upregulation of autophagy in podocytes and pathological crosstalk with mesangial cells.

Objectives: Astaxanthin (ATX) is a strong antioxidant drug. This study aimed to investigate the effects of ATX on podocytes in diabetic nephropathy and the underlying renal protective mechanism of ATX, which leads to pathological crosstalk with mesangial cells.Methods: In this study, diabetic rats treated with ATX exhibited reduced 24-h urinary protein excretion and decreased blood glucose and lipid levels compared to vehicle-treated rats. Glomerular mesangial matrix expansion and renal tubular epithelial cell injury were also attenuated in ATX-treated diabetic rats compared to control rats.Results: ATX treatment markedly reduced the α-SMA and collagen IV levels in the kidneys of diabetic rats. Additionally, ATX downregulated autophagy levels. In vitro, compared with normal glucose, high glucose inhibited LC3-II expression and increased p62 expression, whereas ATX treatment reversed these changes. ATX treatment also inhibited α-SMA and collagen IV expression in cultured podocytes. Secreted factors (vascular endothelial growth factor B and transforming growth factor-ß) generated by high glucose-induced podocytes downregulated autophagy in human mesangial cells (HMCs); however, this downregulation was upregulated when podocytes were treated with ATX.Conclusions: The current study revealed that ATX attenuates diabetes-induced kidney injury likely through the upregulation of autophagic activity in podocytes and its antifibrotic effects. Crosstalk between podocytes and HMCs can cause renal injury in diabetes, but ATX treatment reversed this phenomenon.

The pivotal role of glucose transporter 1 in diabetic kidney disease.

Diabetes mellitus (DM) is a significant public health problem. Diabetic kidney disease (DKD) is the most common complication of DM, and its incidence has been increasing with the increasing prevalence of DM. Given the association between DKD and mortality in patients with DM, DKD is a significant burden on public health resources. Despite its significance in DM progression, the pathogenesis of DKD remains unclear. Aberrant glucose uptake by cells is an important pathophysiological mechanism underlying DKD renal injury. Glucose is transported across the bilayer cell membrane by a glucose transporter (GLUT) located on the cell membrane. Multiple GLUT proteins have been identified in the kidney, and GLUT1 is one of the most abundantly expressed isoforms. GLUT1 is a crucial regulator of intracellular glucose metabolism and plays a key pathological role in the phenotypic changes in DKD mesangial cells. In an attempt to understand the pathogenesis of DKD better, we here present a review of studies on the role of GLUT1 in the development and progression of DKD.

Mesangial cell-derived CircRNAs in chronic glomerulonephritis: RNA sequencing and bioinformatics analysis.

BACKGROUND: Circular RNAs (circRNAs) have been shown to play critical roles in the initiation and progression of chronic glomerulonephritis (CGN), while their role from mesangial cells in contributing to the pathogenesis of CGN is rarely understood. Our study aims to explore the potential functions of mesangial cell-derived circRNAs using RNA sequencing (RNA-seq) and bioinformatics analysis. METHODS: Mouse mesangial cells (MMCs) were stimulated by lipopolysaccharide (LPS) to establish an in vitro model of CGN. Pro-inflammatory cytokines and cell cycle stages were detected by Enzyme-linked immunosorbent assay (ELISA) and Flow Cytometry experiment, respectively. Subsequently, differentially expressed circRNAs (DE-circRNAs) were identified by RNA-seq. GEO microarrays were used to identify differentially expressed mRNAs (DE-mRNAs) between CGN and healthy populations. Weighted co-expression network analysis (WGCNA) was utilized to explore clinically significant modules of CGN. CircRNA-associated CeRNA networks were constructed by bioinformatics analysis. The hub mRNAs from CeRNA network were identified using LASSO algorithms. Furthermore, utilizing protein-protein interaction (PPI), gene ontology (GO), pathway enrichment (KEGG), and GSEA analyses to explore the potential biological function of target genes from CeRNA network. In addition, we investigated the relationships between immune cells and hub mRNAs from CeRNA network using CIBERSORT. RESULTS: The expression of pro-inflammatory cytokines IL-1ß, IL-6, and TNF-α was drastically increased in LPS-induced MMCs. The number of cells decreased significantly in the G1 phase but increased significantly in the S/G2 phase. A total of 6 DE-mRNAs were determined by RNA-seq, including 4 up-regulated circRNAs and 2 down-regulated circRNAs. WGCNA analysis identified 1747 DE-mRNAs of the turquoise module from CGN people in the GEO database. Then, the CeRNA networks, including 6 circRNAs, 38 miRNAs, and 80 mRNAs, were successfully constructed. The results of GO and KEGG analyses revealed that the target mRNAs were mainly enriched in immune, infection, and inflammation-related pathways. Furthermore, three hub mRNAs (BOC, MLST8, and HMGCS2) from the CeRNA network were screened using LASSO algorithms. GSEA analysis revealed that hub mRNAs were implicated in a great deal of immune system responses and inflammatory pathways, including IL-5 production, MAPK signaling pathway, and JAK-STAT signaling pathway. Moreover, according to an evaluation of immune infiltration, hub mRNAs have statistical correlations with neutrophils, plasma cells, monocytes, and follicular helper T cells. CONCLUSIONS: Our findings provide fundamental and novel insights for further investigations into the role of mesangial cell-derived circRNAs in CGN pathogenesis.

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.

Ginsenoside Rg3 induces mesangial cells proliferation and attenuates apoptosis by miR-216a-5p/MAPK pathway in diabetic kidney disease.

BACKGROUND: Ginsenoside Rg3 is an active saponin isolated from ginseng, which can reduce renal inflammation. However, the role and mechanism of Rg3 in diabetic kidney disease (DKD) are far from being studied. METHODS: The effects of Rg3 and miR-216a-5p on the proliferation, apoptosis, and MAPK pathway in high glucose (HG)-induced SV40 MES 13 were monitored by CCK-8, TUNEL staining, and western blot. RESULTS: Rg3 treatment could accelerate proliferation and suppress apoptosis in HG-induced SV40 MES. Moreover, miR-216a-5p inhibition also could alleviate renal injury, prevent apoptosis, and activate the MAPK pathway in kidney tissues of diabetic model mice. CONCLUSION: Rg3 could attenuate DKD progression by downregulating miR-216a-5p, suggesting Rg3 and miR-216a-5p might be the potential drug and molecular targets for DKD therapy.

Polydatin attenuates diabetic renal inflammatory fibrosis via the inhibition of STING pathway.

Diabetic nephropathy (DN) is a complication of diabetes and is mainly characterized by renal fibrosis, which could be attributed to chronic kidney inflammation. Stimulator of interferon genes (STING), a linker between immunity and metabolism, could ameliorate various metabolic and inflammatory diseases. However, the regulatory role of STING in DN remains largely unexplored. In this study, knockdown of STING decreased extracellular matrix (ECM), pro-inflammatory, and fibrotic factors in high glucose (HG)-induced glomerular mesangial cells (GMCs), whereas overexpression of STING triggered the inflammatory fibrosis process, suggesting that STING was a potential target for DN. Polydatin (PD) is a glucoside of resveratrol and has been reported to ameliorate DN by inhibiting inflammatory responses. Nevertheless, whether PD improved DN via STING remains unclear. Here, transcriptomic profiling implied that the STING/NF-κB pathway might be an important target for PD. We further found that PD decreased the protein expression of STING, and subsequently suppressed the activation of downstream targets including TBK1 phosphorylation and NF-κB nuclear translocation, and eventually inhibited the production of ECM, pro-inflammatory and fibrotic factors in HG-induced GMCs. Notably, results of molecular docking, molecular dynamic simulations, surface plasmon resonance, cellular thermal shift assay and Co-immunoprecipitation assay indicated that PD directly bound to STING and restored the declined proteasome-mediated degradation of STING induced by HG. In diabetic mice, PD also inhibited the STING pathway and improved the pathological changes of renal inflammatory fibrosis. Our study elucidated the regulatory role of STING in DN, and the novel mechanism of PD treating DN via inhibiting STING expression.

Integrated network pharmacology and pharmacological investigations to discover the active compounds of Toona sinensis pericarps against diabetic nephropathy.

ETHNOPHARMACOLOGICAL RELEVANCE: Toona sinensis (A. Juss.) Roem. Is a deciduous woody plant native to Eastern and Southeastern Asia. Different parts of this plant have a long history of being applied as traditional medicines to treat various diseases. The fruits have been used for antidiabetic, antidiabetic nephropathy (anti-DN), antioxidant, anti-inflammatory, and other activities. AIM OF THE STUDY: The purpose of this study was to investigate the effects of EtOAc (PEAE) and n-BuOH extracts (PNBE) from T. sinensis pericarps (TSP) on kidney injury in high-fat and high-glucose diet (HFD)/streptozotocin (STZ)-induced DN mice by network pharmacology and pharmacological investigations, as well as to further discover active compounds that could ameliorate oxidative stress and inflammation, thereby delaying DN progression by regulating the Nrf2/NF-κB pathway in high glucose (HG)-induced glomerular mesangial cells (GMCs). MATERIALS AND METHODS: The targets of TSP 1-16 with DN were analyzed by network pharmacology. HFD/STZ-induced DN mouse models were established to evaluate the effects of PEAE and PNBE. Six groups were divided into normal, model, PEAE100, PEAE400, PNBE100, and PNBE400 groups. Fasting blood glucose (FBG) levels, organ indices, plasma MDA, SOD, TNF-α, and IL-6 levels, as well as renal tissue Nrf2, HO-1, NF-κB, TNF-α, and TGF-ß1 levels were determined, along with hematoxylin-eosin (H&E) and immunohistochemical (IHC) analysis of kidney sections. Furthermore, GMC activity screening combined with molecular docking was utilized to discover active compounds targeting HO-1, TNF-α, and IL-6. Moreover, western blotting assays were performed to validate the mechanism of Nrf2 and NF-κB in HG-induced GMCs. RESULTS: Network pharmacology predicted that the main targets of PEAE and PNBE in the treatment of DN include IL-6, INS, TNF, ALB, GAPDH, IL-1ß, TP53, EGFR, and CASP3. Additionally, major pathways include AGE-RAGE and IL-17. In vivo experiments, treatment with PEAE and PNBE effectively reduced FBG levels and organ indices, while plasma MDA, SOD, TNF-α, and IL-6 levels, renal tissue Nrf2, HO-1, NF-κB, TNF-α, and TGF-ß1 levels, and renal function were significantly improved. PEAE and PNBE significantly improved glomerular and tubule injury, and inhibited the development of DN by regulating the levels of oxidative stress and inflammation-related factors. In vitro experiments, compound 11 strongly activated HO-1 and inhibited TNF-α and IL-6. The molecular docking results revealed that compound 11 exhibited a high binding affinity towards the targets HO-1, TNF-α, and IL-6 (<-6 kcal/mol). Western blotting results showed compound 11 effectively regulated Nrf2 and NF-κB p65 protein levels, and significantly improved oxidative stress damage and inflammatory responses in HG-induced GMCs. CONCLUSION: PEAE, PNBE, and their compounds, especially compound 11, may have the potential to prevent and treat DN, and are promising natural nephroprotective agents.

Dihydromyricetin ameliorates diabetic renal fibrosis via regulating SphK1 to suppress the activation of NF-κB pathway.

Dihydromyricetin (DHM) is a flavonoid from vine tea with broad pharmacological benefits, which improve inflammation by blocking the NF-κB pathway. A growing body of research indicates that chronic kidney inflammation is vital to the pathogenesis of diabetic renal fibrosis. Sphingosine kinase-1 (SphK1) is a key regulator of diabetic renal inflammation, which triggers the NF-κB pathway. Hence, we evaluated whether DHM regulates diabetic renal inflammatory fibrosis by acting on SphK1. Here, we demonstrated that DHM effectively suppressed the synthesis of fibrotic and inflammatory adhesion factors like ICAM-1, and VCAM-1 in streptozotocin-treated high-fat diet-induced diabetic mice and HG-induced glomerular mesangial cells (GMCs). Moreover, DHM significantly suppressed NF-κB pathway activation and reduced SphK1 activity and protein expression under diabetic conditions. Mechanistically, the results of molecular docking, molecular dynamics simulation, and cellular thermal shift assay revealed that DHM stably bound to the binding pocket of SphK1, thereby reducing sphingosine-1-phosphate content and SphK1 enzymatic activity, which ultimately inhibited NF-κB DNA binding, transcriptional activity, and nuclear translocation. In conclusion, our data suggested that DHM inhibited SphK1 phosphorylation to prevent NF-κB activation thus ameliorating diabetic renal fibrosis. This supported the clinical use and further drug development of DHM as a potential candidate for treating diabetic renal fibrosis.

Non-voltage-gated Ca2+ channel signaling in glomerular cells in kidney health and disease.

Positioned at the head of the nephron, the renal corpuscle generates a plasma ultrafiltrate to initiate urine formation. Three major cell types within the renal corpuscle, the glomerular mesangial cells, podocytes, and glomerular capillary endothelial cells, communicate via endocrine- and paracrine-signaling mechanisms to maintain the structure and function of the glomerular capillary network and filtration barrier. Ca2+ signaling mediated by several distinct plasma membrane Ca2+ channels impacts the functions of all three cell types. The past two decades have witnessed pivotal advances in understanding of non-voltage-gated Ca2+ channel function and regulation in the renal corpuscle in health and renal disease. This review summarizes the current knowledge of the physiological and pathological impact of non-voltage-gated Ca2+ channel signaling in mesangial cells, podocytes and glomerular capillary endothelium. The main focus is on transient receptor potential and store-operated Ca2+ channels, but ionotropic N-methyl-d-aspartate receptors and purinergic receptors also are discussed. This update of Ca2+ channel functions and their cellular signaling cascades in the renal corpuscle is intended to inform the development of therapeutic strategies targeting these channels to treat kidney diseases, particularly diabetic nephropathy.

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.

Trapping mechanism by di-d-psicose anhydride with methylglyoxal for prevention of diabetic nephropathy.

Di-d-psicose anhydride (DPA), derived from functional rare saccharide as d-psicose, is investigated for its strong chelating ability. Methylglyoxal (MGO), an important precursor of advanced glycation end-products (AGEs), promotes obesity, and causes complications such as diabetic nephropathy. On mesangial cells, DPA can substantially reduce the negative effects of MGO. DPA effectively trapping MGO in mesangial cells. The bonding properties of the DPA-MGO adduct were discussed by mass spectrometry and nuclear magnetic resonance (NMR). The NMR spectra of the DPA-MGO adduct provide evidence for chelation bonding. The inhibition of AGE formation and the mass spectrometry results of the DPA-MGO adduct indicate that DPA can scavenge MGO at a molar ratio of 1:1. DPA suppressed 330 % of the up-regulated receptor for an AGEs protein expression to a normal level and restored the suppressed glyoxalase 1 level to 86 % of the normal group. This research provides important evidence and theoretical basis for the development of AGE inhibitors derived from rare saccharide.

Ursolic acid alleviates lupus nephritis by suppressing SUMO1-mediated stabilization of NLRP3.

BACKGROUND: Systemic lupus erythematosus (SLE) is a multi-system autoimmune disease that affects multiple organs and cause a wide range of severe clinical manifestations, including lupus nephritis (LN), which is a major risk factor for morbidity and mortality in individual with SLE. Ursolic acid (UA) is a natural compound with favorable anti-inflammatory properties and has been employed to treat multiple disease, including inflammatory diseases, diabetes, and Parkinson's disease. However, its therapeutic potential on LN and the underlying mechanisms remains unclear. PURPOSE: This aim of this study was to investigate the impact of UA on LN and its underlying mechanism. METHODS: MRL/lpr lupus-prone mouse model was used and UA was administered orally for 8 weeks. Dexamethasone was used as a positive control. After 8 weeks of administration, the spleen-to-body-weight ratio, renal function, urine albumin excretion, cytokines levels, and the deposition of immune complex were measured. The primary mouse glomerular mesangial cells (GMCs) and SV40-MES-13 were stimulated by lipopolysaccharide (LPS), either alone or in combination with nigericin, to establish an in vitro model. The activation of NLRP3 inflammasome were investigated both in vivo and in vitro using qRT-PCR, immunoblotting, and immunofluorescence. RESULTS: Our results revealed that UA prominently alleviated LN in MRL/lpr lupus-prone mice, leading to a significant reduction in proteinuria production, infiltration of immune cells infiltration, and histopathological damage in the renal tissue. In addition, UA exerted inhibitory effects on the secretion of IL-1ß, IL-18, and caspase-1, pyroptosis, and ASC speck formation in primary mouse GMCs and SV40-MES-13 cells. Furthermore, UA facilitated the degradation of NLRP3 by suppressing SUMO1-mediated SUMOylation of NLRP3. CONCLUSION: UA possess a therapeutical effect on LN in MRL/lpr mice by enhancing the degradation of NLRP3 through inhibition of SUMO1-mediated SUMOylation of NLRP3. Our findings provide a basis for proposing UA as a potential candidate for the treatment of LN.

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.

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.

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.

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.

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.

Crosstalk among podocytes, glomerular endothelial cells and mesangial cells in diabetic kidney disease: an updated review.

Diabetic kidney disease (DKD) is a long-term and serious complication of diabetes that affects millions of people worldwide. It is characterized by proteinuria, glomerular damage, and renal fibrosis, leading to end-stage renal disease, and the pathogenesis is complex and involves multiple cellular and molecular mechanisms. Among three kinds of intraglomerular cells including podocytes, glomerular endothelial cells (GECs) and mesangial cells (MCs), the alterations in one cell type can produce changes in the others. The cell-to-cell crosstalk plays a crucial role in maintaining the glomerular filtration barrier (GFB) and homeostasis. In this review, we summarized the recent advances in understanding the pathological changes and interactions of these three types of cells in DKD and then focused on the signaling pathways and factors that mediate the crosstalk, such as angiopoietins, vascular endothelial growth factors, transforming growth factor-ß, Krüppel-like factors, retinoic acid receptor response protein 1 and exosomes, etc. Furthermore, we also simply introduce the application of the latest technologies in studying cell interactions within glomerular cells and new promising mediators for cell crosstalk in DKD. In conclusion, this review provides a comprehensive and updated overview of the glomerular crosstalk in DKD and highlights its importance for the development of novel intervention approaches.

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).