Kidney Tubular Cells Research Articles

Exosomes derived from mesenchymal stem cells ameliorate renal fibrosis via delivery of miR-186-5p.

Evidence has shown that mesenchymal stem cells' (MSCs) therapy has potential application in treating chronic kidney disease (CKD). In addition, MSCs-derived exosomes can improve the renal function and prevent the progression of CKD. However, the mechanisms by which MSCs-derived exosomes (MSCs-Exo) ameliorate renal fibrosis in CKD remain largely unclear. To mimic an in vitro model of renal fibrosis, rat kidney tubular epithelial cells (NRK52E) were stimulated with transforming growth factor (TGF)-β1. In addition, we established an in vivo model of unilateral ureteric obstruction (UUO)-induced renal fibrosis. Meanwhile, we exploited exosomes derived from MSCs for delivering miR-186-5p agomir into NRK52E cells or kidneys in vitro and in vivo. In this study, we found that level of miR-186-5p was significantly downregulated in TGF-β1-stimulated NRK52E cells and the obstructed kidneys of UUO mice. In addition, miR-186-5p can be transferred from MSCs to NRK52E cells via exosomes. MSCs-delivered miR-186-5p markedly reduced the accumulation of extracellular matrix (ECM) protein, and inhibited epithelial-to-mesenchymal transition (EMT) and apoptosis in TGF-β1-stimulated NRK52E cells. Moreover, exosomal miR-186-5p from MSCs attenuated kidney injury and fibrosis in a UUO mouse model via inhibition of the ECM protein accumulation and EMT process. Meanwhile, dual-luciferase assay showed that miR-186-5p downregulated Smad5 expression via direct binding with the 3'-UTR of Smad5. Collectively then, these findings indicated that exosomal miR-186-5p derived from MSCs could attenuate renal fibrosis in vitro and in vivo by downregulation of Smad5. These findings may help to understand the role of MSCs' exosomes in alleviating renal fibrosis in CKD.

Kidney toxicity of the BRAF-kinase inhibitor vemurafenib is driven by off-target ferrochelatase inhibition

A multitude of disease and therapy related factors drive the frequent development of kidney disorders in cancer patients. Along with chemotherapy, the newer targeted therapeutics can also cause kidney dysfunction through on and off-target mechanisms. Interestingly, among the small molecule inhibitors approved for the treatment of cancers that harbor BRAF-kinase activating mutations, vemurafenib can trigger tubular damage and acute kidney injury. BRAF is a proto-oncogene involved in cell growth. To investigate the underlying mechanisms, we developed cell culture and mouse models of vemurafenib kidney toxicity. At clinically relevant concentrations vemurafenib induces cell-death in transformed and primary mouse and human kidney tubular epithelial cells. In mice, two weeks of daily vemurafenib treatment causes moderate acute kidney injury with histopathological characteristics of kidney tubular epithelial cells injury. Importantly, kidney tubular epithelial cell-specific BRAF gene deletion did not influence kidney function under normal conditions or alter the severity of vemurafenib-associated kidney impairment. Instead, we found that inhibition of ferrochelatase, an enzyme involved in heme biosynthesis contributes to vemurafenib kidney toxicity. Ferrochelatase overexpression protected kidney tubular epithelial cells and conversely ferrochelatase knockdown increased the sensitivity to vemurafenib-induced kidney toxicity. Thus, our studies suggest that vemurafenib-associated kidney tubular epithelial cell dysfunction and kidney toxicity is BRAF-independent and caused, in part, by off-target ferrochelatase inhibition.

Endoplasmic reticulum stress-mediated autophagy activation is involved in cadmium-induced ferroptosis of renal tubular epithelial cells

Acute cadmium (Cd) exposure is a significant risk factor for renal injury and lacks effective treatment strategies. Ferroptosis is a recently identified iron-dependent form of nonapoptotic cell death mediated by membrane damage resulting from lipid peroxidation, and it is implicated in many diseases. However, whether ferroptosis is involved in Cd-induced renal injury and, if so, how it operates. Here, we show that Cd can induce ferroptosis in kidney and renal tubular epithelial cells, as demonstrated by elevation of intracellular iron levels and lipid peroxidation, as well as impaired antioxidant production. Treatment with a ferroptosis inhibitor alleviated Cd-induced cell death. Intriguingly, we established that Cd-induced ferroptosis depended on endoplasmic reticulum (ER) stress, by demonstrating that Cd activated the PERK-eIF2α-ATF4-CHOP pathway and that inhibition of ER stress reduced ferroptosis caused by Cd. We further found that autophagy was required for Cd-induced ferroptosis because the inhibition of autophagy by chloroquine mitigated Cd-induced ferroptosis. Furthermore, we showed that iron dysregulation by ferritinophagy contributed to Cd-induced ferroptosis, by showing that the iron chelator desferrioxamine alleviated Cd-induced cell death and lipid peroxidation. In addition, ER stress is likely activated by MitoROS which trigger autophagy and ferroptosis. Collectively, our results indicate that ferroptosis is involved in Cd-induced renal toxicity and regulated by the MitoROS-ER stress-ferritinophagy axis.

Human Kidney Spheroids and Monolayers Provide Insights into SARS-CoV-2 Renal Interactions.

Although coronavirus disease 2019 (COVID-19) causes significan t morbidity, mainly from pulmonary involvement, extrapulmonary symptoms are also major componen ts of the disease. Kidney disease, usually presenting as AKI, is particularly severe among patients with COVID-19. It is unknown, however, whether such injury results from direct kidney infection with COVID-19's causative virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), or from indirect mechanisms. Using ex vivo cell models, we sought to analyze SARS-CoV-2 interactions with kidney tubular cells and assess direct tubular injury. These models comprised primary human kidney epithelial cells (derived from nephrectomies) and grown as either proliferating monolayers or quiescent three-dimensional kidney spheroids. We demonstrated that viral entry molecules and high baseline levels of type 1 IFN-related molecules were present in monolayers and kidney spheroids. Although both models support viral infection and replication, they did not exhibit a cytopathic effect and cell death, outcomes that were strongly present in SARS-CoV-2-infected controls (African green monkey kidney clone E6 [Vero E6] cultures). A comparison of monolayer and spheroid cultures demonstrated higher infectivity and replication of SARS-CoV-2 in actively proliferating monolayers, although the spheroid cultures exhibited high er levels of ACE2. Monolayers exhibited elevation of some tubular injury molecules-including molecules related to fibrosis (COL1A1 and STAT6) and dedifferentiation (SNAI2)-and a loss of cell identity, evident by reduction in megalin (LRP2). The three-dimensional spheroids were less prone to such injury. SARS-CoV-2 can infect kidney cells without a cytopathic effect. AKI-induced cellular proliferation may potentially intensify infectivity and tubular damage by SARS-CoV-2, suggesting that early intervention in AKI is warranted to help minimize kidney infection.

Using intracellular metabolic profiling to identify novel biomarkers of cisplatin-induced acute kidney injury in NRK-52E cells

ABSTRACT The aim of this study was to investigate changes in the intracellular metabolism resulting from cisplatin (CDDP)-induced nephrotoxicity in normal kidney tubular epithelial NRK-52E cells. Cytotoxicity, cell cycle analysis, and apoptotic cell death were all evaluated in NRK-52E cells treated with CDDP. Subsequently, proton nuclear magnetic resonance (1H-NMR) spectroscopy was used to investigate cellular metabolic profiles. CDDP-induced nephrotoxicity was determined in vivo model. Cytotoxicity in the NRK-52E cells significantly rose following treatment with CDDP and these increases were found to be concentration-dependent. Both p53 and Bax protein expression was increased in CDDP-treated NRK-52E cells, correlating with enhanced cellular apoptosis. In addition, a number of metabolites were altered in both media and cell lysates in these cells. In cell lysates, citrate, creatinine, and acetate levels were dramatically reduced following treatment with 20 µM CDDP concentrations, while glutamate level was elevated. Lactate and acetate levels were significantly increased in culture media but citrate concentrations were reduced following high 20 µM CDDP concentrations incubation. In addition, excretion of clusterin, calbindin, neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1), selenium binding protein 1 (SBP1), and pyruvate kinase M2 (PKM2) into the culture media was significantly increased in CDDP-treated cells while expression of acetyl CoA synthetase 1 (AceCS1) was markedly reduced in these cells. These findings suggest that acetate-dependent metabolic pathway may be a reliable and useful biomarker for detecting CDDP-induced nephrotoxicity. Taken together, data demonstrate that the discovery of novel biomarkers by metabolite profiling in target cells may contribute to the detection of nephrotoxicity and new drug development.

Gambaran Histopatologi Ginjal Mencit (Mus musculus Balb/C) yang Diinfeksi dengan Plasmodium berghei

Background: P. berghei is a parasite that causes malaria in small rodents. This parasite is analogous to P. falciparum which causes severe malaria in humans with the highest risk of death compared to 4 other plasmodium species. Complications of falciparum malaria in renal microcirculation is AKI (Acute Kidney Injury). AKI is preceded by the incidence of ATN (Acute Tubular Necrosis) and MPGN (Mesangioproliferative Glomerulonephritis) with ATN being the highest prevalence.
 Objective: This study aims to determine the histopathological appearance of renal tubules of white mice Mus musculus Balb/C which are infected with P. berghei.
 Methods: Descriptive analysis method with quantitative approach is used in this study. The study was conducted at the Anatomical Pathology Laboratory of the Faculty of Medicine, Andalas University. The study was conducted since February to October 2020. The sample size was determined by the Federer formula (2008).
 Results: Percentage of renal tubular cell damage which were 8.42% for the control group, 4.52% for the pellet extract group, and 2.74% for supernatant extract group.
 Conclusion: The study concluded that highest percentage of kidney tubular cell damage was found in the kidney of mice immunization with the control group, and the lowest percentage of kidney tubular cell damage was found in the kidney of mice immunization with supernatant extract.

PAR2-Induced Tissue Factor Synthesis by Primary Cultures of Human Kidney Tubular Epithelial Cells Is Modified by Glucose Availability.

Coagulopathies common to patients with diabetes and chronic kidney disease (CKD) are not fully understood. Fibrin deposits in the kidney suggest the local presence of clotting factors including tissue factor (TF). In this study, we investigated the effect of glucose availability on the synthesis of TF by cultured human kidney tubular epithelial cells (HTECs) in response to activation of protease-activated receptor 2 (PAR2). PAR2 activation by peptide 2f-LIGRLO-NH2 (2F, 2 µM) enhanced the synthesis and secretion of active TF (~45 kDa) which was blocked by a PAR2 antagonist (I-191). Treatment with 2F also significantly increased the consumption of glucose from the cell medium and lactate secretion. Culturing HTECs in 25 mM glucose enhanced TF synthesis and secretion over 5 mM glucose, while addition of 5 mM 2-deoxyglucose (2DOG) significantly decreased TF synthesis and reduced its molecular weight (~40 kDa). Blocking glycosylation with tunicamycin also reduced 2F-induced TF synthesis while reducing its molecular weight (~36 kDa). In conclusion, PAR2-induced TF synthesis in HTECs is enhanced by culture in high concentrations of glucose and suppressed by inhibiting either PAR2 activation (I-191), glycolysis (2DOG) or glycosylation (tunicamycin). These results may help explain how elevated concentrations of glucose promote clotting abnormities in diabetic kidney disease. The application of PAR2 antagonists to treat CKD should be investigated further.

IFT88 deficiency in proximal tubular cells exaggerates cisplatin-induced injury by suppressing autophagy.

Primary cilia are widely regarded as specialized sensors in differentiated cells that have been implicated in the regulation of cell proliferation, differentiation, and viability. We have previously shown that shortening of primary cilia sensitizes cultured kidney tubular cells to cisplatin-induced apoptosis. Intraflagellar transport 88 (IFT88) is an essential component for ciliogenesis and maintenance. Here, we have further examined the effect of proximal tubule-specific IFT88 ablation on cisplatin-induced acute kidney injury (AKI). In this study, more severe AKI occurred in IFT88 knockout mice than age- and sex-matched wild-type mice. Mechanistically, cisplatin stimulated autophagy in kidney tubular cells as an intrinsic protective mechanism. However, renal autophagy was severely impaired in IFT88 knockout mice. In cultured HK-2 cells, cisplatin induced more apoptosis when IFT88 was knocked down. Tat-beclin 1 peptide, a specific autophagy activator, could partially prevent IFT88-associated cell death during cisplatin treatment, although cilium length was not improved significantly. Reexpression of IFT88 partially restored autophagy in IFT88 knockdown cells and suppressed apoptosis during cisplatin treatment. Taken together, these results indicate that defective autophagy in IFT88-deficient kidney cells and tissues contributes to the exaggerated AKI following cisplatin exposure.NEW & NOTEWORTHY Almost every cell has one hair-like, nonmotile antenna projecting from the cell surface, named the primary cilium. In kidney tubular cells, the primary cilium has a protective role, but the underlying mechanism is unclear. This study shows that a short cilium leads to the suppression of autophagy, which is responsible for the heightened injury sensitivity. These findings provide the clues of how to manipulate primary cilium and autophagy to save kidneys.

Annexin A1 Tripeptide Mimetic Increases Sirtuin-3 and Augments Mitochondrial Function to Limit Ischemic Kidney Injury.

Background: Acute kidney injury (AKI) is one of the most common organ failures following surgery. We have developed a tripeptide mimetic (ANXA1sp) of the parent annexin A1 molecule that shows promise as an organ protectant limiting cellular stress; however, its potential as a kidney protective agent remains unexplored, and its mechanism of action is poorly understood. Our hypothesis was that ANXA1sp would limit kidney injury following surgical ischemic kidney injury.Methods: In a blinded fashion, wildtype mice were assigned to receive vehicle control or ANXA1sp one hour prior to and one hour after kidney vascular clamping. Our primary outcomes were markers of kidney injury and function as measured by serum creatinine and histologic injury scoring of kidney tissue sections. Immunofluorescence microscopy, real-time PCR, and Western blot were used to assess cell death, oxidative stress, and mitochondrial biomarkers. An in vitro model of oxygen-glucose deprivation in immortalized kidney tubule cells was used.Results: ANXA1sp given prior to and after ischemic kidney injury abrogated ischemic kidney injury. ANXA1sp limited cell death both in vivo and in vitro and abrogated oxidative stress following ischemia. ANXA1sp significantly increased the expression of markers associated with protective mitophagy and limited the expression of markers associated with detrimental mitochondrial fission. ANXA1sp upregulated the expression of the mitochondrial protectant sirtuin-3 (SIRT3) in the mitochondria of kidney tubular cells. Silencing of SIRT3 reversed ANXA1sp-mediated protection against hypoxic cell death.Conclusions: ANXA1sp limits kidney injury, upregulates SIRT3, and preserves mitochondrial integrity following ischemic kidney injury. ANXA1sp holds considerable promise as a perioperative kidney protectant prior to ischemia inducing surgery and kidney transplantation.

Ochratoxin A induces human kidney tubular epithelial cell apoptosis through regulating lipid raft/PTEN/AKT signaling pathway.

Ochratoxin A (OTA) is a fungal toxin that causes serious threat to human health. OTA could lead to the injury of various tissues, especially kidney injury. However, the toxic effects of OTA on human kidney tubular epithelial cell (HK-2) and the possible mechanism remains poorly understood. This study was to investigate the toxic effects of OTA on HK-2 and elucidate the molecular mechanism. HK-2 cells were treated OTA to evaluate the effect of OTA on cell viability and apoptosis. OTA inhibited the growth of HK-2 in a concentration-dependent manner. With the concentration increased, OTA significantly lead to the apoptosis of HK-2. OTA could increase the levels of reactive oxygen species (ROS) and Malondialdehyde (MDA). Superoxide dismutase (SOD) and glutathione (GSH) activities were decreased by OTA. Furthermore, OTA increased Caspase-3 and Bax expression and decreased BCL2 expression. Compared to the control group, the expression of PTEN was increased and the expression of PI3K and AKT were decreased in OTA treated groups. In addition, we found OTA could disrupt the formation of lipid raft by attenuating sphingomyelin and cholesterol levels. In conclusion, our results indicated that OTA induces apoptosis in HK-2 through regulating PTEN/AKT signaling pathway via disrupting lipid raft formation.

MO024THE ROLE AND MECHANISM OF DECOY RECEPTOR DCR2 IN EMBRYONIC KIDNEY DEVELOPMENT

Abstract Background and Aims Decoy receptor 2 (DcR2), a transmembrane receptor of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), has been regarded as a hallmark of cell senescence. This study aimed to explore the role and the potential mechanism of DcR2 in the embryonic kidney development. Method The 12.5d, 16.5d, 20.5d embryonic kidney and mature renal tissue (8w) form WT and DcR2-KO mice were obtained. The ureteric buds, S-shaped bodies, renal vesicles were observed by periodic Acid-Schiff staining. The expression of DcR2 were detected by IHC. The co-expression of DcR2 and proximal tubular markers (AQP-1, villin), distal tubular markers (AQP-2), senescent markers (P16, LaminB1), proliferation markers (Ki-67, PCNA, Edu) were analyzed by confocal. Results In WT kidneys, DcR2 were specifically expressed in the tubules and the percentage of DcR2 expression were higher than mature renal tissue. In addition, the expression of tubular DcR2 were significantly decreased in DcR2-KO kidney than WT. The number of ureteric buds, S-shaped bodies, renal vesicles were decreased in DcR2-KO kidney than WT. And the cortex of DcR2-KO kidney were thinner and medullar were enlarger than WT. DcR2 were co-expressed with proximal tubular markers AQP-1 and villin, but not with distal tubular markers AQP-2. Furthermore, DcR2 were co-expressed with senescent markers P16 and LaminB1, but not with proliferation markers Ki-67, PCNA, Edu. Conclusion DcR2 is specifically expressed in proximal tubular cells in embryonic kidney, and DcR2 is involved in early renal development. The potential mechanism is related to cell senescence participating in tissue remodeling and inhibiting cell proliferation.

MO082THE EFFECTS OF DIFFERENT MICROMOLAR CONCENTRATIONS OF CADMIUM IONS ON THE PERITUBULAR MEMBRANE POTENTIAL OF PROXIMAL TUBULAR CELLS IN PERFUSED FROG KIDNEYS

Abstract Background and Aims Cadmium (Cd2+) is toxic metal and environmental pollutant. Accumulation of cadmium in the kidney results initially in proximal tubule dysfunction. Although Cd2+ toxicity is well documented, all mechanisms that are involved in the early stages of nephrotoxicity, especially considering low micromolar concentrations of Cd2+ ions are still unknown. The Aim of this study was to investigate the effects acute exposure to different peritubular micromolar concentrations of cadmium (0.25, 0.50, 1.0, 2.0, 3.0, 5.0 μmol/L) on the peritubular cell membrane potential in proximal tubular cells of frog kidney. Method The experiments were performed on isolated, doubly perfused kidneys of Rana esculenta of both sexes. Aortic and portal vein were cannulated in order to perfusate luminal and peritubular cell membraine. In controled conditions, Ringer solution was simultaneously used to perfusate both cell membraines. Cadmium chloride (different concentrations: 0.25, 0.50, 1.0, 2.0, 3.0, 5.0 μmol/L) were added to the peritubular perfusate separately, by switching the peritubular perfusate from the control Ringer solution to Ringer solution with addition of cadmium ions. Peritubular cell membrane potentials (PD) were measured with conventional 3 mol/L KCl microelectrodes. Results The peritubular application of different micromolar Cd2+ concentrations led to a rapid, sustained, reversible hyperpolarization of the peritubular cell membrane: 0.25 µmol/L, by −3.3±0.4 mV (n=8, p<0.001); 0.50 µmol/L, by −3.0±0.5 mV (n=11, p<0.001); 1.0 µmol/L, by −2.9±0.6 mV (n=8, p<0.01); 2.0 µmol/L, by −4.2±0.4 mV (n=13, p<0.01); 3.0 µmol/L, by −3.4±0.3 mV (n=14, p<0.001); 5.0 µmol/L, by −3.0±0.4 mV (n=10, p<0.001). After switching the perfusion from Ringer solution with addition of cadmium ions to control Ringer, the peritubular membraine potential returned to the average values that were maintained before the peritubular Cd2+ application (p>0.05). Comparing the effect of different Cd2+ concentrations, there was no difference in the hyperpolarization of the peritubular cell membrane (p>0.05).Each cell served as its own control. Conclusion Different low micromolar concentrations of Cd2+ provoked rapid and sustained hyperpolarization of peritubular membrane potential that did not show concentration-dependent response.

MO307POLYCLONAL FREE LIGHT CHAINS AS A PREDICTOR OF CKD PROGRESSION IN NONPROLIFERATIVE GLOMERULOPATHIES

Abstract Background and Aims The mechanism of the epithelial-mesenchymal transition of kidney tubular cells leading to kidney fibrosis formation and CKD progression is well described for monoclonal free light chains (FLC) in patients with monoclonal gammopathies. As far as the interaction of FLC with megalin/cubulin receptors on proximal tubular epithelial cells is considered to be universal we hypothesize that polyclonal free light chains (pFLC) could have the same effect on tubulointerstitial compartment in patients with primary glomerulopathies. This retrospective study was performed to reveal the association of serum pFLC kappa (pFLC-κ) and lambda (pFLC-λ) assessed by Freelite® with clinical and morphological parameters and CKD progression in patients with nonproliferative glomerulopathies. Method 36 patients with morphologically proven diagnosis of nonproliferative glomerulopathies (minimal changed disease (n=11), membranous nephropathy (n=11) and focal segmental glomerulosclerosis (n=14)) were included. Serum levels of pFLC-κ and pFLC-λ were assessed by Freelite® (normal ranges: κ=3.3-19.4 mg/l; λ=5.7-26.3 mg/l; κ/λ ratio=0.26-1.65) at the time of kidney biopsy (KBx) in all cases. Patients with abnormal κ/λ-ratio due to monoclonal gammapathies were excluded. Apart demographical parameters, serum creatinine, estimated GFR (eGFR) by CKD-EPI, serum albumin and 24-hour proteinuria were measured. Morphological findings defined by light microscopy were measured semiquantitatively according to currently accepted criteria (0 - <10%, 1 - 10-25%, 2 – 26-50%, 3 - >50% of tissue involved). Data are presented as median and interquartile range (M (25%; 75%)) and mean and the standard error of mean (m±SEM) for semiquantitative parameters or %. Correlation between parameters was assessed by Spearman’s coefficient. Progression of CKD was determined as decline of eGFR >15% from the initial level at the end of follow-up. Cox proportional hazards regression was used to estimate the association of pFLC and other parameters with CKD progression. Differences were considered statistically significant at p <0.05. Median follow-up was 11 (1; 53) months. Results Demographic and clinical parameters at the time of KBx are shown in the Figure 1. Clinical and morphological parameters as well as correlation analysis data are presented in the Figure 2. Univariant Cox regression shows that pFLC-λ >N (Exp(β)=5.120; 95% CI: 1.011-25.924, p<0.05), both pFLC-κ and pFLC-λ >N (Exp(β)=6.646; 95% CI: 1.327-33.287, p=0.02), κ/λ ratio (Exp(β)=4.656; 95% CI: 1.411-15.362, p=0.01), as well as percent of sclerotic glomeruli (Exp(β)=1.039; 95% CI: 1.006-1.073, p=0.01), glomerular basement membrane segmental thickening (Exp(β)=3.129; 95% CI: 1.213-8.071, p<0.01), mesangial proliferation (Exp(β)=5.177; 95% CI: 1.146- 23.396, p=0.03), interstitial cell infiltration (Exp(β)=3.777; 95% CI: 1.258-11.340, p=0.02) and peritubular capillaritis (Exp(β)=5.177; 95% CI: 1.146- 23.396, p=0.03) were associated with CKD progression. Conclusion In nonproliferative glomerulopathies increased level of pFLC, either kappa or lambda, is associated with glomerular lesion, interstitial inflammation, tubular atrophy and interstitial fibrosis. Moreover, elevated levels of pFLC could be proposed as a predictor of CKD progression in studied patient cohort. The mechanisms of kidney injury by pFLC requires further investigation.

CD36 promotes NLRP3 inflammasome activation via the mtROS pathway in renal tubular epithelial cells of diabetic kidneys

Tubulointerstitial inflammation plays a key role in the pathogenesis of diabetic nephropathy (DN). Interleukin-1β (IL-1β) is the key proinflammatory cytokine associated with tubulointerstitial inflammation. The NLRP3 inflammasome regulates IL-1β activation and secretion. Reactive oxygen species (ROS) represents the main mediator of NLRP3 inflammasome activation. We previously reported that CD36, a class B scavenger receptor, mediates ROS production in DN. Here, we determined whether CD36 is involved in NLRP3 inflammasome activation and explored the underlying mechanisms. We observed that high glucose induced-NLRP3 inflammasome activation mediate IL-1β secretion, caspase-1 activation, and apoptosis in HK-2 cells. In addition, the levels of CD36, NLRP3, and IL-1β expression (protein and mRNA) were all significantly increased under high glucose conditions. CD36 knockdown resulted in decreased NLRP3 activation and IL-1β secretion. CD36 knockdown or the addition of MitoTempo significantly inhibited ROS production in HK-2 cells. CD36 overexpression enhanced NLRP3 activation, which was reduced by MitoTempo. High glucose levels induced a change in the metabolism of HK-2 cells from fatty acid oxidation (FAO) to glycolysis, which promoted mitochondrial ROS (mtROS) production after 72 h. CD36 knockdown increased the level of AMP-activated protein kinase (AMPK) activity and mitochondrial FAO, which was accompanied by the inhibition of NLRP3 and IL-1β. The in vivo experimental results indicate that an inhibition of CD36 could protect diabetic db/db mice from tubulointerstitial inflammation and tubular epithelial cell apoptosis. CD36 mediates mtROS production and NLRP3 inflammasome activation in db/db mice. CD36 inhibition upregulated the level of FAO-related enzymes and AMPK activity in db/db mice. These results suggest that NLRP3 inflammasome activation is mediated by CD36 in renal tubular epithelial cells in DN, which suppresses mitochondrial FAO and stimulates mtROS production.

Comprehensive metabolomic profiling in early IgA nephropathy patients reveals urine glycine as a prognostic biomarker.

Identification of a urinary metabolite biomarker with diagnostic or prognostic significance for early immunoglobulin A nephropathy (IgAN) is needed. We performed nuclear magnetic resonance‐based metabolomic profiling and identified 26 metabolites in urine samples. We collected urine samples from 201, 77, 47, 36 and 136 patients with IgAN, patients with membranous nephropathy, patients with minimal change disease, patients with lupus nephritis and healthy controls, respectively. We determined whether a metabolite level is associated with the prognosis of IgAN through Cox regression and continuous net reclassification improvement (cNRI). Finally, in vitro experiments with human kidney tubular epithelial cells (hTECs) were performed for experimental validation. As the results, the urinary glycine level was higher in the IgAN group than the control groups. A higher urinary glycine level was associated with lower risk of eGFR 30% decline in IgAN patients. The addition of glycine to a predictive model including clinicopathologic information significantly improved the predictive power for the prognosis of IgAN [cNRI 0.72 (0.28‐0.82)]. In hTECs, the addition of glycine ameliorated inflammatory signals induced by tumour necrosis factor‐α. Our study demonstrates that urinary glycine may have diagnostic and prognostic value for IgAN and indicates that urinary glycine is a protective biomarker for IgAN.

Vascular Endothelium‐Derived ET‐1 Regulates The Renal T Cell‐Macrophage Interplay Under High Salt Diet

The vasoactive peptide endothelin-1 (ET-1) is a key contributor to high salt-induced elevations of blood pressure and end-organ damage. In addition, ET-1 is known to be pro-inflammatory in the high salt setting. Although ET-1 was first described as produced by the vascular endothelium, we now know that there are other cellular sources, like kidney tubular cells or immune cells. However, the exact role of endothelium-derived ET-1 in regulating immune cell populations during high salt consumption remains unclear. We hypothesized that endothelium-derived ET-1 is critical in regulating the kidney inflammation that is associated with high salt consumption. Male vascular endothelial cell ET-1 knockout (VEET KO) and control floxed ET-1 mice were placed on 4.0% NaCl diet (HSD) for 3 weeks. Urine was collected for assessment of kidney injury, and the immunological status was evaluated in kidneys and spleen by flow cytometry analysis of live, singlet cells. After 3 weeks of HSD, VEET KO mice presented reduced urinary protein excretion compared to floxed ET-1 mice (floxed ET-1 vs. VEET KO: 8.59 ± 1.90 vs. 3.56 ± 0.87, pg/day; p=0.03; n=6/group), suggesting that vascular ET-1 is critical for developing high salt-induced kidney injury. Lack of endothelium-derived ET-1 resulted in a 53.9% reduction in the frequency of kidney TH17 cells (IL-17A+ CD4+ T cells; floxed ET-1 vs.VEET KO: 1.650.17 vs. 0.770.10, % IL-17A of CD4+; p=0.001; n=5-6/group) and a 77.9% reduction in kidney absolute TH17 cell count (3.10.5 vs. 0.850.22, x102 cells; p=0.008). Interestingly, the intra-renal frequency of T regulatory (Treg) cells was similar between the genotypes (floxed ET-1 vs. VEET KO: 12.81.58 vs. 12.31.29, %; p=0.78), but the absolute Treg cell number in the kidney was reduced in VEET KO mice (2.260.25 vs. 1.210.30, x103 cells, p=0.02); these findings suggest a potential role for vascular ET-1 in the regulation of HSD-induced Treg expansion/proliferation in this organ. In addition, VEET KO mice also presented significantly blunted frequency and absolute numbers of the innate-like IL-17A+ +TCR+ T cell population in the kidney (floxed ET-1 vs. VEET KO; % IL-17A of IL-17A+ + TCR+: 36.61.92 vs. 21.22.63, p=0.0009; absolute numbers: 2.510.41 vs. 1.020.39, x102, p=0.03). Examination of the kidney myeloid cell populations under HSD also revealed a reduced frequency of activated resident macrophages (F4/80hi CD64+: 25.14.52 vs. 10.51.44, %, p=0.01; F4/80hi CD86+: 85.32.38 vs. 78.131.69 %, p=0.03), while no differences were found in the activation of infiltrating macrophages (F4/80lo CD64+ and F4/80lo CD86+) in the kidney. Lymphoid and myeloid immune cell populations in the spleen were similar between genotypes. These data indicate a critical role for intra-renal endothelium-derived ET-1 in the cellular response of immune cell populations under HSD. More specifically, our data demonstrate that endothelium-derived ET-1 regulates the immune landscape during HSD consumption by activating pro-inflammatory TH17 and IL-17A+ +TCR+ T cells as well as resident macrophages specifically in the kidney.

Mesenchymal Stem Cells Attenuate Renal Fibrosis via Exosomes-Mediated Delivery of microRNA Let-7i-5p Antagomir.

BackgroundRenal fibrosis is a chronic and progressive process affecting kidneys in chronic kidney disease (CKD). Mesenchymal stem cells-derived exosomes (MSCs-Exo) have been shown to alleviate renal fibrosis and injury, but the mechanism of MSCs-Exo-induced renal protection remains unknown.MethodsIn this study, MSCs were transfected with let-7i-5p antagomir (anti-let-7i-5p), and then exosomes were isolated from the transfected MSCs to deliver anti-let-7i-5p oligonucleotides to inhibit the level of let-7i-5p in kidney tubular epithelial cells (NRK-52E).ResultsIn both NRK-52E cells stimulated by TGF-β1 and the mouse kidneys after unilateral ureteral obstruction (UUO), we demonstrated increased level of let-7i-5p. In addition, MSCs-Exo can deliver anti-let-7i-5p to reduce the level of let-7i-5p in NRK-52E cells and increase the expression of its target gene TSC1. Moreover, exosomal anti-let-7i-5p reduced extracellular matrix (ECM) deposition and attenuated epithelial–mesenchymal transition (EMT) process in transforming growth factor beta 1 (TGF-β1)-stimulated NRK-52E cells and in the kidneys of UUO-treated mice. Meanwhile, mice received exosomal anti-let-7i-5p displayed reduced renal fibrosis and improved kidney function when challenged with UUO. Furthermore, exosomal anti-let-7i-5p promoted the activation the tuberous sclerosis complex subunit 1/mammalian target of rapamycin (TSC1/mTOR) signaling pathway in vivo and in vitro.ConclusionIn conclusion, exosomal anti-let-7i-5p from MSCs exerts anti-fibrotic effects in TGF-β1-induced fibrogenic responses in NRK52E cells in vitro as well as in UUO-induced renal fibrosis model in vivo. These results provided a novel perspective on improving renal fibrosis by MSCs-Exo.

Sirtuin 3 deficiency promotes acute kidney injury induced by sepsis via mitochondrial dysfunction and apoptosis.

Objective(s):To explore the regulation mechanism of Sirtuin 3 (SIRT3) on the mitochondrial function and apoptosis of acute kidney injury (AKI) in septic mice.Materials and Methods:The sepsis-induced AKI model was constructed in the wild-type and SIRT3 knockout (KO) mice, and the levels of serum creatinine (Scr) and plasma kidney injury molecule 1 (pKIM-1) in mice were detected by ELISA. The mitochondrial damage of kidney tubular epithelial cells (KTEC) was observed by electron microscopy, the apoptosis of KTEC was detected by TUNEL assay, and the mRNA levels of SIRT3, Bax, Caspase-3, and Bcl-2 were detected by RT-qPCR.Results:SIRT3 KO caused increased expression of Scr, pKIM-1, and inducible nitric oxide synthase protein in the kidneys of septic mice, and decreased the levels of superoxide dismutase, catalase, and mitochondrial complex enzymes I/II/III/IV. SIRT3 deficiency exacerbated histopathological and mitochondrial damage to the proximal tubules of the kidney. In addition, SIRT3 KO resulted in a significantly increased apoptosis of KTEC, increased the mRNA levels of Bax and Caspase-3, and decreased the mRNA levels of Bcl-2. Conclusion:Our study suggests that SIRT3 deficiency promotes sepsis-induced AKI via increasing oxidative stress, mitochondrial dysfunction, and inducing apoptosis.

Enhanced Ultrasound Contrast of Renal-Clearable Luminescent Gold Nanoparticles.

Renal-clearable nanoparticles are typically fast eliminated through the free glomerular filtration, which show weak interaction with the renal compartments and negligible ultrasound signals, raising challenges in direct imaging of kidney diseases. Here, we report the ultrasmall renal-clearable luminescent gold nanoparticles (AuNPs) with both pH-induced charge reversal and aggregation properties, and discover that enhanced ultrasound contrast could be facilely acquired through the increased tubular reabsorption and in situ aggregation of AuNPs in renal tubule cells in injured kidneys. The tuning elimination pathway of the renal-clearable luminescent AuNPs is further demonstrated to provide a synergistical fluorescence and ultrasound imaging strategy for diagnosing early kidney injury with precise anatomical information.

Therapeutic Effect of Endothelin-Converting Enzyme Inhibitor on Chronic Kidney Disease through the Inhibition of Endoplasmic Reticulum Stress and the NLRP3 Inflammasome.

Chronic inflammation and oxidative stress significantly contribute to the development and progression of chronic kidney disease (CKD). The NOD-like receptor family pyrin containing domain-3 (NLRP3) inflammasome plays a key role in the inflammatory response. The renal endothelin (ET) system is activated in all cases of CKD. Furthermore, ET-1 promotes renal cellular injury, inflammation, fibrosis and proteinuria. Endothelin-converting enzymes (ECEs) facilitate the final processing step of ET synthesis. However, the roles of ECEs in CKD are not clear. In this study, we investigated the effects of ETs and ECEs on kidney cells. We found that ET-1 and ET-2 expression was significantly upregulated in the renal tissues of CKD patients. ET-1 and ET-2 showed no cytotoxicity on human kidney tubular epithelial cells. However, ET-1 and ET-2 caused endoplasmic reticulum (ER) stress and NLRP3 inflammasome activation in tubular epithelial cells. The ECE inhibitor phosphoramidon induced autophagy. Furthermore, phosphoramidon inhibited ER stress and the NLRP3 inflammasome in tubular epithelial cells. In an adenine diet-induced CKD mouse model, phosphoramidon attenuated the progression of CKD by regulating autophagy, the NLRP3 inflammasome and ER stress. In summary, these findings showed a new strategy to delay CKD progression by inhibiting ECEs through autophagy activation and restraining ER stress and the NLRP3 inflammasome.