OBJECTIVES: Pyroptosis plays a critical role in tubulointerstitial lesions of diabetic kidney disease (DKD). Annexin A2 (ANXA2) is involved in cell proliferation, apoptosis, and adhesion and may be closely related to DKD, but its specific mechanism remains unclear. This study aims to investigate the role and molecular mechanism of ANXA2 in high glucose-induced pyroptosis of renal tubular epithelial cells, providing new targets for DKD prevention and treatment. METHODS: Human renal tubular epithelial HK-2 cells were divided into a normal glucose group (5.5 mmol/L), a high glucose group (30.0 mmol/L), and a osmotic control group (24.5 mmol/L mannitol+5.5 mmol/L glucose). ANXA2 expression was modulated by overexpression of plasmids and small interfering RNA (siRNA). Cell proliferation was measured by 5-ethynyl-2'-deoxyuridine (EdU) assay, apoptosis by flow cytometry, and ANXA2, p50, and p65 subcellular localization by immunofluorescence. Western blotting was employed to detect α-smooth muscle actin (α-SMA), fibronectin (FN), and collagen type IV (Col-IV). Real-time fluorescence quantitative PCR (RT-qPCR) and Western blotting were used to analyze nuclear factor-κB (NF-κB) subunits p50/p65 and the pyroptosis pathway factors NLR family Pyrin domain containing 3 (NLRP3), caspase-1, inferleukin (IL)-1β, and IL-18. Protein interactions between ANXA2 and p50/p65 were examined by co-immunoprecipitation, while chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays were used to examine NF-κB binding to the ANXA2 promoter. RESULTS: High glucose upregulated ANXA2 expression and promoted its nuclear translocation (P<0.01). High glucose reduced cell proliferation, increased apoptosis, and elevated α-SMA, FN, and Col-IV expression (all P<0.05); ANXA2 overexpression aggravated these effects (all P<0.05), while ANXA2 knockdown reversed them (all P<0.05). High glucose activated NF-κB and increased NLRP3, caspase-1, L-1β, and IL-18 mRNA and protein expression (all P<0.05); ANXA2 overexpression further enhanced this, whereas knockdown suppressed NF-κB activation and downstream factors (all P<0.05). Co-immunoprecipitation confirmed ANXA2 directly binds the NF-κB subunit p65. ChIP assays revealed p65 binds specifically to ANXA2 promoter regions (ChIP-2, ChIP-4, and ChIP-6), and luciferase activity in corresponding mutant constructs (M2, M4, and M6) was significantly increased versus controls (all P<0.05), confirming positive transcriptional regulation of ANXA2 by p65. CONCLUSIONS ANXA2 and NF-κB form a positive feedback loop that sustains NLRP3 inflammasome activation, promotes pyroptosis pathway activation, and aggravates high glucose-induced renal tubular epithelial cell injury. Targeting ANXA2 or blocking its interaction with p65 may be a novel strategy to slow DKD progression.
Humans ; Pyroptosis/drug effects* ; Annexin A2/physiology* ; Epithelial Cells/cytology* ; Kidney Tubules/cytology* ; Glucose/pharmacology* ; Diabetic Nephropathies/metabolism* ; NF-kappa B/metabolism* ; Cell Line ; Cell Proliferation ; Transcription Factor RelA/metabolism* ; Feedback, Physiological

OBJECTIVES: To investigate the effect of Didang Decoction-medicated serum on autophagy in high glucose (HG)-induced rat glomerular endothelial cells (RGECs) and explore the pathway that mediates its effect. METHODS: Primary RGECs were isolated and cultured using sequential sieving combined with collagenase digestion, followed by identification using immunofluorescence assay for factor VIII. High glucose medium was used to induce RGECs to simulate a diabetic environment, and the effects of Didang Decoction-medicated serum and 3-MA (an autophagy inhibitor), either alone or in combination, on autophagy of HG-exposed cells were evaluated by observing autophagic vacuoles using monodansylcadaverine (MDC) staining. RT-qPCR and Western blotting were employed to measure mRNA and protein expression levels of Beclin-1, p62, LC3B, p-PI3K, p-Akt, and p-mTOR. RESULTS: Compared with the control cells, the HG-exposed RGECs showed significantly reduced autophagic fluorescence intensity, decreased Beclin-1 mRNA expression, increased p62 mRNA expression, downregulated Beclin-1 protein and LC3-II/I ratio, and upregulated p62, p-PI3K, p-Akt, and p-mTOR protein levels. Didang Decoction-medicated serum significantly enhanced autophagic fluorescence intensity in HG-exposed cells, increased Beclin-1 mRNA expression, decreased p62 mRNA expression, upregulated Beclin-1 protein, and downregulated p62, p-PI3K, p-Akt, and p-mTOR protein levels. CONCLUSIONS Didang Decoction-medicated serum enhances autophagy in HG-exposed RGECs by regulating the PI3K/Akt/mTOR signaling pathway, which sheds light on a new therapeutic strategy for diabetic nephropathy.
Animals ; Autophagy/drug effects* ; Signal Transduction/drug effects* ; Rats ; TOR Serine-Threonine Kinases/metabolism* ; Drugs, Chinese Herbal/pharmacology* ; Proto-Oncogene Proteins c-akt/metabolism* ; Endothelial Cells/metabolism* ; Phosphatidylinositol 3-Kinases/metabolism* ; Glucose ; Cells, Cultured ; Kidney Glomerulus/cytology* ; Rats, Sprague-Dawley

Nephronectin (NPNT) is a novel extracellular matrix protein and a new ligand of integrin α8β1. Recent studies showed that NPNT is highly expressed in kidney, lung, thyroid, etc, and it may play an important role in many pathological conditions. NPNT is involved in the process of kidney development and acute kidney injury, regulates proliferation and differentiation of osteoblast, and induces the vasculogenesis in vitro. NPNT may play a key role in pathological osteoporosis and therefore be a new therapeutic target of bone diseases. NPNT gene variants are not only associated with lung function, but also potentially implicated in chronic airway diseases development. Moreover, NPNT is also an important factor that mediates pathology of cardiac, epidermis, breast, liver and teeth diseases. In this paper, we reviewed some research progresses on the structure, distribution, physiological and pathophysiological functions of NPNT.
Cell Differentiation ; Cell Proliferation ; Extracellular Matrix Proteins ; physiology ; Humans ; Kidney ; physiology ; Osteoblasts ; cytology ; Osteoporosis

Objective To induce adipose-derived stem cells (ADSCs) to differentiate into intermediate mesoderm (IM)-like cells ,with IM-like cells for recellularizing kidney scaffolds,and then to obtain a tissue-engineering kidney with renal structures and functions through co-culture.Methods After inguinal fat pads of Wistar rats were surgically harvested,the primary ADSCs were isolated,induced,and cultured for stem cell identification. ADSCs were inducted to differentiate into IM-like cells by adding glycogen synthase kinase-3 inhibitor (CHIR99021) and fibroblast growth factor 9 (FGF9) at different stages. Seven days later,the IM-like cells were identified. The induced IM-like cells and well-prepared kidney decellularized scaffolds were co-cultured for 10 days to obtain recellularized tissue-engineered kidneys and their differentiation was identified.Results The ADSCs harvested had osteogenic and adipogenic abilities and could express the stem cell surface markers. After 7 days of induction,the positive expressions of odd-skipped related 1 and paired-box 2 were observed in IM-like cells by immunofluorescence technique. After 10 days of co-culture with kidney decellularized scaffolds,the positive expressions of Wilms'tumor 1,GATA-binding protein-3,and E-cadherin were observed by immunofluorescence technique.Conclusion ADSCs can be induced into IM-like cells,and renal cell differentiation can be observed through combining the induced IM-like cells with kidney decellularized scaffolds.
Adipose Tissue ; Animals ; Cell Differentiation ; Cells, Cultured ; Kidney ; growth & development ; Mesoderm ; cytology ; Rats ; Rats, Wistar ; Regeneration ; Stem Cells ; cytology ; Tissue Engineering ; Tissue Scaffolds

OBJECTIVETo study the clinical value of red blood cell distribution width (RDW) in the early prediction of acute kidney injury (AKI) in children with sepsis.

METHODSA total of 126 children with sepsis were divided into an AKI group (n=66) and a non-AKI group (n=60) according to the presence or absence of AKI. These patients were also classified into high-RDW and low-RDW groups according to the mean RDW. The groups were compared in terms of age, male-to-female ratio, body mass index (BMI), Acute Physiology and Chronic Health Evaluation II (APACHE II) score, Sequential Organ Failure Assessment (SOFA) score, serum blood urea nitrogen (BUN), creatinine (Cr), uric acid (UA), serum C-reactive protein (CRP), and routine blood test results. Independent factors associated with RDW were analyzed by multiple linear regression.

RESULTSAge, male-to-female ratio, BMI, CRP, SOFA score, and APACHE II score did not differ significantly between the AKI and non-AKI groups (P>0.05), but the AKI group had significantly higher BUN, Cr, UA, and RDW levels than the non-AKI group (P<0.05). Age, male-to-female ratio, and BMI did not differ significantly between the high-RDW and low-RDW groups (P>0.05), but the high-RDW group had significantly higher BUN, Cr, UA, CRP, SOFA score, APACHE II score, Hb, and mean corpuscular volume (MCV) than the low-RDW group (P<0.05). The multiple linear regression analysis showed that age, sex, APACHE II score, Cr, Hb, and MCV were independent factors associated with RDW.

CONCLUSIONSRDW has a certain clinical value in the early prediction of AKI in children with sepsis.

APACHE ; Acute Kidney Injury ; blood ; diagnosis ; Adolescent ; Child ; Child, Preschool ; Creatinine ; blood ; Erythrocyte Indices ; Erythrocytes ; cytology ; Female ; Humans ; Infant ; Male ; Retrospective Studies ; Sepsis ; blood ; diagnosis

Objective To identify and verify the distribution of Telocytes derived from heterogeneous interstitial cells in the vital organs of ApoE mice.Methods Heart,kidney,and liver tissues were harvested from ApoE adult mice. Immunohistochemical assays were performed by using different immunobiological markers.Results Telocytes were found in these vital organs. The expressions of immunobiological markers differed among different organs. CD34,CD117,and CD28 were positively expressed in Telocytes in cardiac tissue;CD117 and plateled-derived growth factor-Α were negatively expressed in Telocytes in renal tissue;and CD117 and plateled-derived growth factor receptor-Α had negative expression in Telocytes in hepatic tissue. Furthermore,the distribution of Telocytes also differed in the same organ.Conclusions Telocytes exist in the vital organs of ApoE mice,as demonstrated by immunohistochemisty assay. The expressions of immunobiological markers differ among Telocytes in different organs.
Animals ; Antigens, CD34 ; metabolism ; CD28 Antigens ; metabolism ; Kidney ; cytology ; Liver ; cytology ; Mice ; Mice, Knockout, ApoE ; Myocardium ; cytology ; Proto-Oncogene Proteins c-kit ; metabolism ; Telocytes ; cytology

In the kidney, pericyte is the major source of myofibroblast (MyoF) in renal interstitium. It is reported that pericyte-myofibroblast transition（PMT）is one of the important pathomechanisms of renal interstitial fibrosis（RIF）. Among them, the main reasons for promoting RIF formation include pericyte recruitment, activation and isolation, as well as the lack of pericyte-derived erythropoietin. During the PMT startup process, pericyte activation and its separation from microvessels are controlled by multiple signal transduction pathways, such as transforming growth factor-β（TGF-β）pathway, vascular endothelial growth factor receptor (VEGFR) pathway and platelet derived growth factor receptor (PDGFR) pathway;Blocking of these signaling pathways can not only inhibit PMT, but also suppress renal capillaries reduction and further alleviate RIF. In clinic, many traditional Chinese medicine compound prescriptions, single traditional Chinese herbal medicine (CHM) and their extracts have the clear effects in alleviating RIF, and some of their intervention actions may be related to pericyte and its PMT. Therefore, the studies on PMT and its drug intervention will become the main development direction in the research field of anti-organ fibrosis by CHM.
Drugs, Chinese Herbal ; pharmacology ; Fibrosis ; Humans ; Kidney ; cytology ; drug effects ; pathology ; Myofibroblasts ; cytology ; Pericytes ; cytology ; Receptors, Platelet-Derived Growth Factor ; metabolism ; Signal Transduction ; Vascular Endothelial Growth Factor A ; metabolism

This study was aimed to investigate the effect and mechanism of Zhenwu Tang on AVP-V2R-AQP2 pathway in NRK-52E cells . Forty eight male SD rats were randomly divided into eight groups with 6 animals in each group. Distilled water or 22.68 g·kg⁻¹·d⁻¹ Zhenwu Tang(calculated by raw drug dosage meter) was given by gavage. Blood samples were collected by cardiac puncture， and the medicated serum was centrifuged from the blood by 3 000 r·min⁻¹. NRK-52E cells were treated with different medicated serum or dDAVP. The condition of cell proliferation was detected by RTCA. The distribution of V2R and AQP2 in cells were detected by immunofluorescence. The expression of V2R， PKA and AQP2 were detected by Western blot and AQP2 mRNA level was detected by real-time PCR. Results showed that the level of AQP2 mRNA(<0.01) and protein expression of V2R， PKA and AQP2(<0.05， <0.01， <0.05) of Z7d group which was treated with Zhenwu Tang medicated serum for 24 h were significantly higher than that of normal rat serum group. And the expression level of V2R， p-AQP2 and AQP2(<0.01， <0.05， <0.01) of Z7d+dDAVP group were significantly increased comparing to normal rat serum group. The results indicate that the applying of Zhenwu Tang medicated serum could increase the expression level of V2R， PKA and AQP2 which exist in AVP-V2R-AQP2 pathway in NRK-52E， and there is synergistic effect between Zhenwu Tang medicated serum and dDAVP. So the pathway of AVP-V2R-AQP2 may be one of the mechanism for which Zhenwu Tang regulate balance of water transportation.
Animals ; Aquaporin 2 ; metabolism ; Cell Line ; Cyclic AMP-Dependent Protein Kinases ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Kidney ; cytology ; Male ; RNA, Messenger ; Rats ; Rats, Sprague-Dawley ; Receptors, Vasopressin ; metabolism ; Signal Transduction

BACKGROUNDSurfactant protein-A (SP-A) contributes to the regulation of sepsis-induced acute kidney injury. In a previous study, we demonstrated that the expression of SP-A in the human renal tubular epithelial (HK-2) cells can be stimulated by lipopolysaccharide (LPS). The present study evaluated the possible signal-transducing mechanisms of LPS-induced SP-A biosynthesis in the HK-2 cells.

METHODSTetrazolium salt colorimetry (MTT) assay was used to detect cell viability of HK-2 cells after LPS stimulation on different time points. HK-2 cells were stimulated with 100 ng/ml of LPS for different durations to determine the effects of LPS on SP-A and toll-like receptor 4 (TLR4) messenger RNA (mRNA) expression, as well as phosphorylation of mitogen-activated/extracellular signal-regulated kinase (MEK) 1, extracellular signal-regulated kinase 1/2 (ERK1/2), p38 mitogen-activated protein kinase (p38MAPK), and nuclear factor-kappa B (NF-κB) inhibitor-alpha (IkB-α). Then, HK-2 cells were pretreated with CLI-095, a TLR4 inhibitor, to analyze mRNA and protein levels of SP-A and TLR4 and expression of NF-κB in the cytoplasm and nucleus of HK-2 before LPS exposure.

RESULTSHK-2 cells exposed to 100 ng/ml of LPS for 1, 6, and 24 h did not affect cell viability which showed no toxic effect of 100 ng/ml LPS on cells (P = 0.16); however, the biosynthesis of SP-A mRNA and protein in HK-2 cells was significantly increased (P = 0.02). As to the mechanism, LPS enhanced transmembrane receptor TLR4 protein expression. Sequentially, LPS time dependently augmented phosphorylation of MEK1, ERK1/2, and p38MAPK. In addition, levels of phosphorylated IκB-α and nuclear NF-κB were augmented with LPS exposure for 2 h. LPS-induced SP-A and TLR4 mRNA as well as NF-κB expression were significantly inhibited by pretreatment with CLI-095.

CONCLUSIONSThe present study exhibited that LPS can increase SP-A synthesis in human renal epithelial cells through sequentially activating the TLR4-related MEK1-ERK1/2-NF-κB-dependent pathway.

Cell Line ; Cell Survival ; drug effects ; physiology ; Colorimetry ; Humans ; Kidney ; cytology ; metabolism ; Lipopolysaccharides ; toxicity ; Mitogen-Activated Protein Kinase 1 ; metabolism ; Mitogen-Activated Protein Kinase 3 ; metabolism ; NF-kappa B ; metabolism ; Pulmonary Surfactant-Associated Protein A ; metabolism ; Sulfonamides ; pharmacology ; Tetrazolium Salts ; chemistry ; Toll-Like Receptor 4 ; antagonists & inhibitors ; metabolism

OBJECTIVETo investigate the effect of arctiin on advanced oxidation protein product (AOPP)-induced epithelial-to-mesenchymal transition (EMT) in tubular cells and explore the mechanisms underlying this effect.

METHODSHuman proximal tubular cells (HK-2 cells) were treated with bovine serum albumin (BSA) or AOPPs in the presence or absence of arctiin. The expressions of E-cadherin, vimentin, and GRP78 at the protein and mRNA levels in the cells were examined using Western blotting and quantitative real-time PCR. The level of reactive oxygen species (ROS) was measured by flow cytometry with DCFH-DA as the fluorescent probe.

RESULTSCompared with BSA-treated cells, the cells treated with AOPPs showed decreased expression of epithelial cell marker E-cadherin and overexpression of mesenchymal marker vimentin and endoplasmic reticulum stress marker GRP78 with an increased ROS level. These changes induced by AOPPs were partly inhibited by arctiin.

CONCLUSIONArctiin can ameliorate AOPP-induced EMT in tubular cells by inhibiting endoplasmic reticulum stress, and oxidative stress response may participate in this process.

Advanced Oxidation Protein Products ; adverse effects ; Cadherins ; metabolism ; Cell Line ; Endoplasmic Reticulum Stress ; Epithelial Cells ; cytology ; drug effects ; Epithelial-Mesenchymal Transition ; Furans ; pharmacology ; Glucosides ; pharmacology ; Heat-Shock Proteins ; metabolism ; Humans ; Kidney Tubules ; cytology ; drug effects ; Oxidative Stress ; Reactive Oxygen Species ; metabolism ; Vimentin ; metabolism