Renal Glomerular Endothelial Cells Research Articles

FBXO45 levels regulated ferroptosis renal tubular epithelial cells in a model of diabetic nephropathy by PLK1.

This research aims to investigate the role and underlying biological mechanism of FBXO45 in regulating ferroptosis of renal fibrocytes in a diabetic nephropathy (DN) model. C57BL/6 mice were fed with a high-fat diet and injected with streptozotocin to induce diabetes. Human renal glomerular endothelial cells stimulated with d-glucose. Serum FBXO45 mRNA expression was found to be down-regulated in patients with DN. There was a negative correlation between the expression of serum FBXO45 mRNA and serum α-SMA, Collagen I, and E-cadherin mRNA in patients with DN. Additionally, the expression of serum FBXO45 mRNA showed a negative correlation with blood sugar levels. Based on a 3D model prediction, it was observed that FBXO45 interacts with polo-like kinase 1 (PLK1) at GLY-271, ILE-226, GLY-166, LEU-165, ARG-245, and ASN-220, while PLK1 interacts with FBXO45 at TYR-417, ARG-516, HIS-489, TYR-485, GLN-536, and ARG-557. This interaction was confirmed through immunoprecipitation assay, which showed the interlinking of FBXO45 protein with PLK1 protein. These findings indicate that FBXO45 plays a role in mitigating ferroptosis in DN through the regulation of the PLK1/GPX4/SOX2 pathway. This highlights the potential of targeting FBXO45 as a therapeutic approach to ameliorate ferroptosis in DN.

#1071 The effect and mechanism of SGLT2 inhibitor on improving inflammatory injury of glomerular endothelial cells in diabetes

Abstract Background and Aims ATP-binding cassette transporter A1 (ABCA1), which reduces intracellular cholesterol accumulation, is highly expressed in the kidney. Studies have shown that sodium-glucose co-transporter 2 inhibitors (SGLT2) inhibitors promote ABCA1 expression in multiple tissues and organs. However, the effect of SGLT2 inhibitors on ABCA1 expression in diabetic kidney disease (DKD) is unclear. In this study, the effect of empagliflozin, an SGLT2 inhibitor, on ABCA1 in glomerular endothelial cells(GECs) of DKD and the possible mechanisms linking ABCA1 to GECs inflammatory injury were investigated. Method Kidney tissues were obtained from renal biopsies of patients with early and advanced DKD. db/db mice were used as animal models of type 2 diabetes and the effects of SGLT2 inhibitors on ABCA1 were analyzed by treatment with empagliflozin. The effects of ABCA1 overexpression on glomerular lipid deposition and kidney injury were examined in a type 2 diabetic mouse model with ABCA1 overexpression (db/db + ABCA1 mice), and human renal glomerular endothelial cells (HRGECs) cultured in high glucose and high cholesterol conditions, which simulated type 2 diabetes in vitro. Results The expression of ABCA1 in the glomeruli of DKD patients and db/db mice was significantly decreased. Enhancing ABCA1 expression in the kidney tissue of diabetic mice by adenovirus transduction improved renal cholesterol deposition, inhibited the overactivation of C3a/C5a, and improved renal inflammatory injury. In addition, in cultured HRGECs under high glucose and high cholesterol conditions, ABCA1 deficiency increased the deposit of cellular cholesterol, contributed to overactivation of C3a/C5a, and induced inflammatory injury. Overexpression of ABCA1 enhancing cholesterol efflux or inhibition of C3a/C5a activation in vitro significantly protected against glomerular endothelial injury stimulated by high glucose and high cholesterol. Moreover, SGLT2 inhibitor empagliflozin up-regulated the expression of ABCA1 and down-regulated the levels of cholesterol, C3a/C5a and inflammatory cytokines in diabetic mice and HRGECs. Conclusion SGLT2 inhibitor (empagliflozin) might play a renal protective role by up-regulating ABCA1-mediated cholesterol outflow, inhibiting the activation of C3a/C5a, and then ameliorating the inflammatory injury of GECs.

Azilsartan improves urinary albumin excretion in hypertension mice.

Hypertension is one of the most important risk factors for chronic kidney diseases, leading to hypertensive nephrosclerosis, including excessive albuminuria. Azilsartan, an angiotensin II type 1 receptor blocker, has been widely used for the treatment of hypertension. However, the effects of Azilsartan on urinary albumin excretion in hypertension haven't been reported before. In this study, we investigated whether Azilsartan possesses a beneficial property against albuminuria in mice treated with angiotensin II and a high-salt diet (ANG/HS). Compared to the control group, the ANG/HS group had higher blood pressure, oxidative stress, and inflammatory response, all of which were rescued by Azilsartan dose-dependently. Importantly, the ANG/HS-induced increase in urinary albumin excretion and decrease in the expression of occludin were reversed by Azilsartan. Additionally, it was shown that increased fluorescence intensity of FITC-dextran, declined trans-endothelial electrical resistance (TEER) values, and reduction of occludin and krüppel-like factor 2 (KLF2) were observed in ANG/HS-treated human renal glomerular endothelial cells (HrGECs), then prevented by Azilsartan. Moreover, the regulatory effect of Azilsartan on endothelial monolayer permeability in ANG/HS-treated HrGECs was abolished by the knockdown of KLF2, indicating KLF2 is required for the effect of Azilsartan. We concluded that Azilsartan alleviated diabetic nephropathy-induced increase in Uterine artery embolization (UAE) mediated by the KLF2/occludin axis.

Detecting and exploring kidney-derived extracellular vesicles in plasma.

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. 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. 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. Our findings demonstrate the potential usefulness of kidney-derived sEVs in plasma in future research for kidney diseases.

Low dose cadmium inhibits syndecan-4 expression in glycocalyx of glomerular endothelial cells.

Cadmium (Cd) is one of the most polluting heavy metal in the environment. Cd exposure has been elucidated to cause dysfunction of the glomerular filtration barrier (GFB). However, the underlying mechanism remains unclear. C57BL/6J male mice were administered with 2.28 mg/kg cadmium chloride (CdCl2) dissolved in distilled water by oral gavage for 14 days. The expression of SDC4 in the kidney tissues was detected. Human renal glomerular endothelial cells (HRGECs) were exposed to varying concentrations of CdCl2 for 24 h. The mRNA levels of SDC4, along with matrix metalloproteinase (MMP)-2 and 9, were analyzed by quantitative PCR. Additionally, the protein expression levels of SDC4, MMP-2/9, and both total and phosphorylated forms of Smad2/3 (P-Smad2/3) were detected by western blot. The extravasation rate of fluorescein isothiocyanate-dextran through the Transwell was used to evaluate the permeability of HRGECs. SB431542 was used as an inhibitor of transforming growth factor (TGF)-β signaling pathway to further investigate the role of TGF-β. Cd reduced SDC4 expression in both mouse kidney tissues and HRGECs. In addition, Cd exposure increased permeability and upregulated P-Smad2/3 levels in HRGECs. SB431542 treatment inhibited the phosphorylation of Smad2/3, Cd-induced SDC4 downregulation, and hyperpermeability. MMP-2/9 levels increased by Cd exposure was also blocked by SB431542, demonstrating the involvement of TGF-β/Smad pathway in low-dose Cd-induced SDC4 reduction in HRGECs. Given that SDC4 is an essential component of glycocalyx, protection or repair of endothelial glycocalyx is a potential strategy for preventing or treating kidney diseases associated with environmental Cd exposure.

Clinical Significance of Apela in Acute Cardiorenal Insuffiency of Chronic Heart Failure

Introduction: Apela has a wide range of biological effects on the cardiovascular system, but the changes and significance of endogenous Apela in patients with chronic heart failure (CHF) and acute deterioration of cardiac and renal function are unclear. Methods: A total of 69 patients with stable CHF combined with well-preserved renal function were enrolled and followed for 12 months. The effects of Apela on human renal glomerular endothelial cells (hRGEC), human glomerular mesangial cells (hMC), and human renal tubular epithelial cells (HK-2) were observed. Results: Serum Apela concentration was positively correlated with NYHA class (r = 0.711) and N-terminal pro-brain natriuretic peptide (NT-proBNP) concentration (r = 0.303) but negatively correlated with left ventricular ejection fraction (LVEF) (r = −0.374) and 6-min walk distance (r = −0.860) in patients with stable CHF. Twenty-one patients experiencing deterioration of renal and cardiac function were diagnosed with cardiorenal syndrome (CRS) during the follow-up period. In addition, the serum Apela, as well as the difference in Apela between stable and worsening phases (ΔApela), was correlated with the estimated glomerular filtration rate (eGFR) and ΔeGFR in patients with CRS. Apela significantly inhibited the upregulated expression of MCP-1 and TNF-α induced by angiotensin II (AngII) in hRGEC, hMC, and HK-2 cells. Apela inhibited the adhesion of THP-1 cells to hRGEC and promoted the tubular formation of hRGEC. Moreover, Apela enhanced the expression of MMP-9 in hMC but inhibited the upregulated expression of α-SMA and vimentin in HK-2 cells by AngII. Conclusion: This study suggests that the level of Apela can be used to diagnose heart failure and assess the severity of cardiac dysfunction in patients with stable CHF, and its dynamic changes can be used to evaluate the damage to renal function in patients with CRS. Apela plays multiple protective effects on renal cells, highlighting its clinical application prospect in the prevention and treatment of CRS.

20-HETE As an Emerging Target to Improve Renal Health in Sickle Cell Disease

The kidney is one of the most severely affected organs in sickle cell disease (SCD), with glomerular hyperfiltration and microalbuminuria beginning in childhood, and progressing to chronic kidney disease (CKD) in majority of adults. Sickle individuals also suffer from acute kidney injury (AKI) often during characteristic vaso-occlusive crisis. Renal pathology in sickle cell nephropathy includes extensive microvascular congestions and thrombotic microangiopathy. The hypercoagulable and vasoconstrictive environment of SCD, in conjunction with medullary hypoperfusion and cortical hyperperfusion contributes to vaso-occlusive injury and deregulates intrarenal hemostasis leading to CKD development. Ischemic injury increases arachidonic acid hydroxylation producing 20-hydroxyeicosatetraenoic acid (20-HETE), which in turn triggers thrombosis and promotes vasoconstriction in the kidney. However, the role of 20-HETE in regulating renal hemostasis in SCD nephropathy has never been reported. We found that plasma 20-HETE is significantly elevated in SCD patients (n=9-11; p<0.01) compared to normal individuals. Similarly, transgenic SCD mice (SS), homozygous for human hemoglobin S, have increased plasma 20-HETE compared to control mice (AA), homozygous for human hemoglobin A (n=6; p<0.01). We have previously shown that elevated circulating heme mimicking hemolytic crisis triggers clinically relevant acute kidney injury (AKI) in SS mice (Blood (2020) 135(13): 1044-1048). We postulated that hemolytic stress may exacerbate 20-HETE production which in turn aggravates renal microvascular occlusion and vasoconstriction, leading to renal damage in SCD. Immunohistochemistry and biochemical experiments showed that the expression of CYP4A12a, a cytochrome 450-dependent enzyme responsible 20-HETE production in mice, is substantially elevated in the kidneys of SS mice compared to AA mice, associated with increased vascular congestion and renal thrombogenesis. Renal CYP4A12a and plasma 20-HETE were increased substantially following heme-induced AKI in SS mice, associated with a heightened expression of renal angiotensin II receptor 1 (ATIIR1), thus indicating induced vasoconstrictive activity. In vitro, we found that 20-HETE induced ATIIR1 expression in human renal glomerular endothelial cells (hRGEC), which was further exacerbated by low-concentration of heme challenge (5 mM for 24 hour). Nitro-oleic acid (NO 2-OA) is a nitrated fatty acid that binds ATIIR1 and inhibits angiotensin II induced vasoconstriction. Pretreatment with NO 2-OA reduced 20-HETE-dependent ATIIR1 expression in hRGEC, while NO 2-OA prophylaxis during hemin-induced AKI inhibited albuminuria, normalized plasma creatinine levels, and lowered the expression of ATIIR1 in SS mice kidneys. Overall, our data show that elevated 20-HETE levels during hemolytic stress may aggravate renal injury in SCD which can be prevented by NO 2-OA prophylaxis. Ongoing experiments will determine the direct effect of elevated 20-HETE in renal hemostasis and CKD development in SCD.

Insulin receptor expression to predict resistance to axitinib and elucidation of the underlying molecular mechanism in metastatic renal cell carcinoma.

The study aimed to examine the significance of insulin receptor (INSR) expression in predicting resistance to axitinib in clear cell renal cell carcinoma (ccRCC). Clinicopathological data were collected from 36 consecutive patients with metastatic RCC who received axitinib. Thirty-three primary tumours were obtained for immunohistochemistry. Patient-derived xenograft (PDX) models were created by transplanting primary tumours into immunodeficient mice, establishing axitinib-resistant PDX models. RCC cell lines were co-cultured with human renal glomerular endothelial cells (HGECs) treated with siRNA of INSR (HGEC-siINSR). Gene expression alteration was analysed using microarray. The patients with low INSR expression who received axitinib had a poorer outcome. Multivariate analysis showed that INSR expression was the independent predictor of progression-free survival. INSR expression decreased in axitinib-resistant PDX tumours. RCC cell lines showed upregulated interferon responses and highly increased interferon-β levels by co-culturing with HGEC-siINSR. HGECs showed decreased INSR and increased interferon-β after axitinib administration. RCC cell lines co-cultured with HGEC-siINSR showed high programmed death-ligand 1 (PD-L1) expression, which increased after interferon-β administration. Decreased INSR in RCC could be a biomarker to predict axitinib resistance. Regarding the resistant mechanism, vascular endothelial cells with decreased INSR in RCC may secrete interferon-β and induce PD-L1.

#2709 THE INVOLVEMENT OF NRF2-MEDIATED ANTIOXIDANT SYSTEM IN MYELOPEROXIDASE-POSITIVE ANTINEUTROPHIL CYTOPLASMIC ANTIBODY-ASSOCIATED VASCULITIS

Abstract Background and Aims Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a systemic autoimmune disease pathologically characterized by vascular necrosis with inflammation. The necrotic lesions consist of ANCA-mediated neutrophil extracellular traps (NETs). During AAV, activated neutrophils form NETs via reactive oxygen spices (ROS)s-mediated signaling pathway. The excessive NETs lead to endothelial vascular damage and serve as autoantigens against ANCAs in a vicious cycle. Although the role of NETs in AAV has been revealed, its treatment for targeting NETs remains unestablished. Since nuclear factor-erythroid 2-related factor 2 (Nrf2) regulates antioxidant proteins against oxidative stress and functions as a defense system, we investigated the role of Nrf2 in the development of AAV. Method In vitro, human neutrophils treated with the Nrf2 activator bardoxolone methyl (Bard) were stimulated with ANCA, and the effects on NETs formation and signaling pathways including intracellular ROS were evaluated by cell imaging and biochemical approaches. The effects of Nrf2 activators on NETs-induced cytotoxicity were examined using human renal glomerular endothelial cells (HRGECs). Nrf2 knockout or WT mouse-derived neutrophils were stimulated with anti-MPO antibody to induce NETs. In vivo studies, Bard was administered to ANCA-transfer AAV model or spontaneous AAV developing mice models (SCG/Kj). Renal function was assessed by biochemical analysis, and the expression of ROS and antioxidant protein NAD(P)H Quinone Dehydrogenase 1 (NQO1) in tissues was evaluated by fluorescent immunostaining and immunoblotting. The formation of NETs in the glomeruli was evaluated by immunostaining and glomerular vascular endothelial damage was assessed by the expression of CD31 and TUNEL positivity. To assess the role of Nrf2 in immunological abnormality during AAV, murine splenocytes were collected and flow cytometry were conducted using CD4, CD8, B220, MCHCII, CD11c, F4/80, Ly 6G, Ly6b, and CD11b antibodies. Moreover, to assess the genetic role of Nrf2 in AAV, we established SCG/Kj mice carrying a germ-line deletion of the Nrf2-genes. A spontaneous mouse model of vasculitis genetically deficient in Nrf2 was analyzed by the same parameters to evaluate the effect of Nrf2. N1 offspring mice were generated by crossing Nrf2-/- mice with SCG/Kj mice. These backcrossed mice were obtained by crossing Nrf2-/- mice to background SCG/Kj mice for three generations. The experimental group was mated to N4 heterozygous mice, and female mice in the experimental group were analyzed at 18 weeks. Results In vitro, pharmacological activation of Nrf2 suppressed ANCA-induced NETs via the upregulation of NQO1 and inhibition of ROS. While, Nrf2-deficienet neutrophils showed the massive NETs in response to ANCA, compared to WT neutrophils. Furthermore, Nrf2 activation directly protected endothelium from cellular injury caused by the exposure of ANCA-mediated NETs. In vivo, the pharmacological activation of Nrf2 ameliorated glomerulonephritis in two kinds of AAV models through the upregulation of antioxidant and inhibition of ROS-mediated NETs. In contrast, Nrf2 genetic deficiency exacerbated vasculitis in spontaneous AAV model. The activation of Nrf2 regulated the expansion of splenocytes in AAV mice. In particular, pan-T cells in spleen and the infiltration of Th17 cells in kidney were suppressed by Nrf2 activation. Conclusion During ANCA-stimulation in neutrophils, the activation of Nrf2 initiates antioxidant system and inhibits intracellular ROS, leading to the suppression of NETs formation. Nrf2 may be a novel therapeutic target for AAV.

Attenuation of Sepsis-Induced Acute Kidney Injury by Exogenous H2S via Inhibition of Ferroptosis.

Sepsis-associated acute kidney injury (SA-AKI) results in significant morbidity and mortality, and ferroptosis may play a role in its pathogenesis. Our aim was to examine the effect of exogenous H2S (GYY4137) on ferroptosis and AKI in in vivo and in vitro models of sepsis and explore the possible mechanism involved. Sepsis was induced by cecal ligation and puncture (CLP) in male C57BL/6 mice, which were randomly divided into the sham, CLP, and CLP + GYY4137 group. The indicators of SA-AKI were most prominent at 24 h after CLP, and analysis of the protein expression of ferroptosis indicators showed that ferroptosis was also exacerbated at 24 h after CLP. Moreover, the level of the endogenous H2S synthase CSE (Cystathionine-γ-lyase) and endogenous H2S significantly decreased after CLP. Treatment with GYY4137 reversed or attenuated all these changes. In the in vitro experiments, LPS was used to simulate SA-AKI in mouse renal glomerular endothelial cells (MRGECs). Measurement of ferroptosis-related markers and products of mitochondrial oxidative stress showed that GYY4137 could attenuate ferroptosis and regulate mitochondrial oxidative stress. These findings imply that GYY4137 alleviates SA-AKI by inhibiting ferroptosis triggered by excessive mitochondrial oxidative stress. Thus, GYY4137 may be an effective drug for the clinical treatment of SA-AKI.

Tanshinone IIA mediates protection from diabetes kidney disease by inhibiting oxidative stress induced pyroptosis

Ethnopharmacological relevanceSalvia miltiorrhiza is widely used traditional Chinese medicine in the treatment of diabetes kidney disease (DKD). Tanshinone IIA (Tan IIA) are one of the main components of the root of red-rooted Salvia miltiorrhiza Bunge. However, whether Tan IIA delay the progression of DKD and the underlying mechanisms are unknown. Aim of the studyClarify the mechanisms underlying the occurrence and progression of DKDs from a novel viewpoint and confirm the function and mechanism of Tan IIA. Materials and methodsWe experimented with models of DKD (db/db mice) and cultured human renal glomerular endothelial cells (HRGECs). We measured the biochemical indicators of mouse blood and urine to confirmed that Tan IIA exerted protective effects on the kidneys of db/db mice. Renal histopathology and immunohistochemical staining were used to determine the role of Tan IIA. High glucose-induced HRGECs pyroptosis based on the results of western blot, CCK-8 cell viability test, calcein/PI staining, ROS/superoxide anion generation and transmission electron microscope. We also confirmed that Tan IIA alleviated HRGEC pyroptosis through the same methods. The relationships between oxidative induction and regulation of NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome activation were investigated using western blot following the application of an NLRP3 inhibitor and oxidative stress inhibitor. ResultsTan IIA alleviated kidney injury and improved the levels of urine, blood indicators, the expression of NLRP3 and thioredoxin-interacting protein (Txnip) in db/db mice kidney. In vitro, high glucose inhibited HRGECs viability, increased ROS generation, enhanced the proportion of propidium iodide-stained cells. In addition, we discovered the expression of GSDMD-NT, NLRP3, cleaved IL-1β, cleaved caspase-1, and Txnip increased, but the expression of Trx1 decreased after treated by high glucose. These changes were partially ameliorated by Tan IIA. ConclusionHyperglycemia could induce pyroptosis in renal glomerular endothelial cells. However, Tan IIA could delay the progression of DKD by inhibiting pyroptosis by regulating the Txnip/NLRP3 inflammasome.

MiR-223-3p mediates the diabetic kidney disease progression by targeting IL6ST/STAT3 pathway.

Diabetic kidney disease (DKD), the most pervasive complication in diabetic patients, has become a major health threat to the aging population. Our previous miRNA profiling identified hsa-miR-223-3p as a dysregulated miRNA in the DKD samples, which may serve as a biomarker for DKD diagnosis. However, the specific mechanism of miR-223-3p in the pathogenesis of DKD remains to be elucidated. In this study, we first verified that miR-223-3p level was significantly decreased in the invitro cell model and invivo db/db DKD model, accompanied with endothelial cell damage. Importantly, inhibiting the expression of miR-223-3p exacerbated high-glucose induced damages in Human Umbilical Vein Endothelial Cells (HUVECs) and Human Renal Glomerular Endothelial Cells (HRGECs), while miR-223-3p overexpression showed the opposite effect. We further demonstrated that miR-223-3p associated with IL6T mRNA and attenuated the progression of DKD by suppressing the downstream STAT3 activation, indicative of the implication of miR-223-3p/IL6T/STAT3 axis in the pathogenesis of DKD.

ANGPT2/CAV1 regulates albumin transcytosis of glomerular endothelial cells under high glucose exposure and is impaired by losartan

BackgroundMicroalbuminuria is a common clinical symptom that manifests in the early stages of diabetic kidney disease (DKD) and is also the main feature of glomerular endothelial cells (GECs) injury. There is increasing evidence that the transcytosis of albumin across GECs is closely related to the formation of albuminuria. Our previous studies have shown that angiopoietin 2 (ANGPT2) can inhibit albumin transcytosis across renal tubular epithelial cells by activating caveolin 1 (CAV1) phosphorylation during high glucose (HG) exposure. The role of ANGPT2 in albumin transcytosis across GECs remains unclear. Losartan significantly reduces albuminuria, but the mechanism has not been clarified. MethodsWe established an in vitro albumin transcytosis model to investigate the change in albumin transcytosis across human renal glomerular endothelial cells (hrGECs) under normal glucose (NG), high glucose (HG) and losartan intervention. We knocked down ANGPT2 and CAV1 to evaluate their roles in albumin transcytosis across hrGECs and verified the relationship between them. In vivo, DKD mouse models were established and treated with different doses of losartan. Immunohistochemistry and Western blot were used to detect the expression of ANGPT2 and CAV1. ResultsIn vitro, the transcytosis of albumin across hrGECs was significantly increased under high glucose stimulation, and losartan inhibited this process. The expression of ANGPT2 and CAV1 were both increased in hrGECs under HG conditions and losartan intervention reduced the expression of them. Moreover, ANGPT2 downregulation reduced albumin transcytosis in hrGECs by regulating CAV1 expression. In vivo, the expression of ANGPT2 and CAV1 in the glomerulus was both increased significantly in DKD mice. Compared with DKD mice, losartan treatment reduced albuminuria and decreased the expression of ANGPT2 and CAV1 in a dose-dependent manner. ConclusionsANGPT2 exacerbated albumin transcytosis across GECs by increasing CAV1 expression during HG exposure, thereby increasing albuminuria. Losartan reduces albumin transcytosis and albuminuria formation in DKD by inhibiting the upregulation of ANGPT2 under HG conditions. Our findings suggest that ANGPT2 and CAV1 may be novel therapeutic targets for diabetic albuminuria. In addition, we provide new evidence to elaborate on the mechanism of losartan in the development of DKD.

Rutin alleviates EndMT by restoring autophagy through inhibiting HDAC1 via PI3K/AKT/mTOR pathway in diabetic kidney disease

BackgroundDiabetic kidney disease (DKD) is a primary microvascular complication of diabetes. However, a complete cure for DKD has not yet been found. Although there is evidence that Rutin can delay the onset of DKD, the underlying mechanism remains unclear. PurposeTo investigate the renoprotective effect of Rutin in the process of DKD and to explore its potential molecular mechanisms. MethodsDb/db mice and high glucose (HG)-induced human renal glomerular endothelial cells (GEnCs) were used as in vivo and in vitro models, respectively. Western blot (WB), Immunohistochemistry (IHC)and Immunofluorescence (IF) staining were used to identify the expression level of proteins associated with endothelial-to-mesenchymal transition (EndMT) and autophagy. Tandem Mass Tag (TMT)-based proteomics analysis was utilized to reveal the mechanism of Rutin in DKD. Transfection with small interfering RNA (siRNA) to reveal the role of histone deacetylase 1 (HDAC1) in HG-induced GEnCs. ResultsFollowing 8 weeks of Rutin administration, db/db mice's kidney function and structure significantly improved. In HG-induced GEnCs, activation of autophagy attenuates cellular EndMT. Rutin could alleviate EndMT and restore autophagy in vivo and in vitro models. Proteomics analysis results showed that HDAC1 significantly downregulated in the 200 mg/kg/d Rutin group compared with the db/db group. Transfection with si-HDAC1 in GEnCs partially blocked HG-induced EndMT and restored autophagy. Furthermore, Rutin inhibits the phosphorylation of the PI3K / AKT/ mTOR pathway. HDAC1 overexpression was suppressed in HG-induced GEnCs after using Rapamycin, a specific mTOR inhibitor, verifying the correlation between mTOR and HDAC1. ConclusionRutin alleviates EndMT by restoring autophagy through inhibiting HDAC1 via the PI3K/AKT/mTOR pathway in DKD.

Glomerular expression and urinary excretion of fatty acid-binding protein 4 in IgA nephropathy.

Fatty acid-binding protein 4 (FABP4) is secreted from adipocytes and macrophages in adipose tissue and acts as an adipokine. It has recently been reported that FABP4, but not liver-type FABP (L-FABP/FABP1), is also expressed in injured glomerular endothelial cells and infiltrating macrophages in the glomerulus and that urinary FABP4 (U-FABP4) is associated with proteinuria and kidney function impairment in nephrotic patients. However, the link between glomerular FABP4 and U-FABP4 has not been fully addressed in IgA nephropathy (IgAN). We investigated the involvement of FABP4 in human and mouse IgAN. In patients with IgAN (n = 23), the ratio of FABP4-positive area to total area within glomeruli (G-FABP4-Area) and U-FABP4 were positively correlated with proteinuria and were negatively correlated with eGFR. In 4-28-week-old male grouped ddY mice, a spontaneous IgAN-prone mouse model, FABP4 was detected in glomerular endothelial cells and macrophages, and G-FABP4-Area was positively correlated with urinary albumin-to-creatinine ratio (r = 0.957, P < 0.001). Endoplasmic reticulum stress markers were detected in glomeruli of human and mouse IgAN. In human renal glomerular endothelial cells, FABP4 was induced by treatment with vascular endothelial growth factor and was secreted from the cells. Treatment of human renal glomerular endothelial cells or mouse podocytes with palmitate-bound recombinant FABP4 significantly increased gene expression of inflammatory cytokines and endoplasmic reticulum stress markers, and the effects of FABP4 in podocytes were attenuated in the presence of an anti-FABP4 antibody. FABP4 in the glomerulus contributes to proteinuria in IgAN, and U-FABP4 level is a useful surrogate biomarker for glomerular damage in IgAN.

Retracted: Low‐Dose X‐Ray Increases Paracellular Permeability of Human Renal Glomerular Endothelial Cells

Retracted: Low‐Dose X‐Ray Increases Paracellular Permeability of Human Renal Glomerular Endothelial Cells

In vitro study of emodin-induced nephrotoxicity in human renal glomerular endothelial cells on a microfluidic chip

Emodin is an effective component of rhubarb with positive pharmacological effects on human health. However, it is also toxic to different cells or tissues to varying degrees. The effects of emodin on glomerular endothelial cells (GECs) remain to be tested, and the documented works were always performed in vitro and hardly reflect the real physiological situation. To study the effects of emodin on GECs in a biomimetic environment, we utilized a microfluidic chip to assess the physiological reaction of human renal glomerular endothelial cells to various concentrations of emodin in this work. The results showed that emodin caused cytotoxicity, impaired glomerular filtration barrier integrity to macromolecules, and increased barrier permeability in a dose-dependent manner. With the increase in emodin concentration, the concentration of the pro-inflammatory cytokine tumor necrosis factor-α, interleukin (IL)-6, transforming growth factor-β1, and monocyte chemoattractant protein (MCP-1) increased while the production of inflammatory cytokine IL-6 first increased and then decreased with the increase in emodin concentration. Our findings shed new light on emodin-induced nephrotoxicity and provide insights for the application of microfluidic chip devices to reveal drug-cell interactions.

Protective Effects of Xanthohumol against Diabetic Nephropathy in a Mouse Model

Introduction: Diabetic nephropathy (DN) is a long-term loss of renal function occurring in the diabetic patients, leading to 5 million deaths in 2015, and this number is dramatically growing annually. Due to unsatisfied outcome of current treatment, there is urgent need to develop more effective therapeutic drugs for DN. Methods: Approximately 150 kinds of natural small molecule drugs that have been used on the market or in the clinical trials in the presence of high glucose were tested individually on the same batch of human renal glomerular endothelial cells (GECs) and human kidney 2 (HK-2) cells with triplicated wells by using a robotic pipetting workstation to screen for the potential drug candidate. Cell viability and oxidative stress were examined in the GECs and HK-2 cells. DN mouse model was established and treated with 25 mg/kg xanthohumol. Results: By measuring cell viability, xanthohumol was selected as our predicted drug candidate for DN because it could mostly protect renal cells from high glucose with about 90% survived GECs and HK-2 cells, about 2.12- and 2.37-fold increase compared to glucose group which was with 42.78% and 37.69% survived GECs and HK-2 cells, respectively. Then, xanthohumol inhibited high glucose-induced oxidative stress through nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway in vitro. Moreover, xanthohumol (25 mg/kg) significantly decreased the levels of serum creatinine, blood urea nitrogen, urea protein, and kidney weight/body weight ratio in DN mice. In addition, the increase of reactive oxygen species production and the decrease of superoxide dismutase and catalase activities in DN mice were partially reversed by xanthohumol. mRNA levels of Nrf2, Hmox1, and Nqol genes were all decreased by xanthohumol DN mice. Conclusion: Xanthohumol could ameliorate DN-related impairments via Nrf2 signaling pathway, which might serve as a promising drug candidate for treatment of DN.

ABCA1 deficiency-mediated glomerular cholesterol accumulation exacerbates glomerular endothelial injury and dysfunction in diabetic kidney disease

BackgroundHyperglycemia and dyslipidemia are two major characteristics of diabetes. In this study, the effects of glomerular cholesterol accumulation primarily due to ABCA1 deficiency on glomerular endothelial injury in diabetic kidney disease (DKD) and the possible mechanisms were investigated. MethodsThe effects of ABCA1 deficiency on glomerular lipid deposition and kidney injury were examined in a type 2 diabetic mouse model with ABCA1 deficiency in glomerular endothelial cells (DM-ABCA1−/− mice) and human renal glomerular endothelial cells (HRGECs) cultured in high glucose and high cholesterol conditions, which simulated type 2 diabetes in vitro. ResultsABCA1 deficiency in glomerular endothelial cells exacerbated renal lipid deposition and kidney injuries in type 2 diabetic mice and manifested as increased creatinine levels, more severe proteinuria, mesangial matrix expansion and fusion of foot processes, and more pronounced renal inflammatory injury and cell death. In HRGECs cultured under high glucose and high cholesterol conditions, ABCA1 deficiency increased the deposition of cellular cholesterol, contributed to inflammation and apoptosis, damaged the endothelial glycocalyx barrier, and induced endoplasmic reticulum stress (ERS). Conversely, ABCA1 overexpression enhancing cholesterol efflux or inhibition of ERS in vitro, significantly protected against glomerular endothelial injury stimulated by high glucose and high cholesterol. ConclusionsThese findings establish a pathogenic role of ABCA1 deficiency in glomerular endothelium injury and dysfunction and imply that ABCA1 may represent a potential effective therapeutic target for early diabetic kidney disease.

ANGPT2/CAV1 regulates albumin transcytosis of glomerular endothelial cells under high glucose exposure and is impaired by losartan

BackgroundMicroalbuminuria is a common clinical symptom that manifests in the early stages of diabetic kidney disease (DKD) and is also the main feature of glomerular endothelial cells (GECs) injury. There is increasing evidence that the transcytosis of albumin across GECs is closely related to the formation of albuminuria. Our previous studies have shown that angiopoietin 2 (ANGPT2) can inhibit albumin transcytosis across renal tubular epithelial cells by activating caveolin 1 (CAV1) phosphorylation during high glucose (HG) exposure. The role of ANGPT2 in albumin transcytosis across GECs remains unclear. Losartan significantly reduces albuminuria, but the mechanism has not been clarified. MethodsWe established an in vitro albumin transcytosis model to investigate the change in albumin transcytosis across human renal glomerular endothelial cells (hrGECs) under normal glucose (NG), high glucose (HG) and losartan intervention. We knocked down ANGPT2 and CAV1 to evaluate their roles in albumin transcytosis across hrGECs and verified the relationship between them. In vivo, DKD mouse models were established and treated with different doses of losartan. Immunohistochemistry and Western blot were used to detect the expression of ANGPT2 and CAV1. ResultsIn vitro, the transcytosis of albumin across hrGECs was significantly increased under high glucose stimulation, and losartan inhibited this process. The expression of ANGPT2 and CAV1 were both increased in hrGECs under HG conditions and losartan intervention reduced the expression of them. Moreover, ANGPT2 downregulation reduced albumin transcytosis in hrGECs by regulating CAV1 expression. In vivo, the expression of ANGPT2 and CAV1 in the glomerulus was both increased significantly in DKD mice. Compared with DKD mice, losartan treatment reduced albuminuria and decreased the expression of ANGPT2 and CAV1 in a dose-dependent manner. ConclusionsANGPT2 exacerbated albumin transcytosis across GECs by increasing CAV1 expression during HG exposure, thereby increasing albuminuria. Losartan reduces albumin transcytosis and albuminuria formation in DKD by inhibiting the upregulation of ANGPT2 under HG conditions. Our findings suggest that ANGPT2 and CAV1 may be novel therapeutic targets for diabetic albuminuria. In addition, we provide new evidence to elaborate on the mechanism of losartan in the development of DKD.