TREM1/3 Deficiency Impairs Tissue Repair After Acute Kidney Injury and Mitochondrial Metabolic Flexibility in Tubular Epithelial Cells.
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ABSTRACT:
SUBMITTER: Tammaro A
PROVIDER: S-EPMC6629955 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Macroautophagy/autophagy is a lysosomal degradation system which plays a protective role against kidney injury. RUBCN/Rubicon (RUN domain and cysteine-rich domain containing, Beclin 1-interacting protein) inhibits the fusion of autophagosomes and lysosomes. However, its physiological role in kidney proximal tubular epithelial cells (PTECs) remains uncertain. In the current study, we analyzed the phenotype of newly generated PTEC-specific rubcn-deficient (KO) mice. Additionally, we investigated the role of RUBCN in lipid metabolism using isolated rubcn-deficient PTECs. Although KO mice exhibited sustained high autophagic flux in PTECs, they were not protected from acute ischemic kidney injury. Unexpectedly, KO mice exhibited hallmark features of metabolic syndrome accompanied by expanded lysosomes containing multi-layered phospholipids in PTECs. RUBCN deficiency in cultured PTECs promoted the mobilization of phospholipids from cellular membranes to lysosomes via enhanced autophagy. Treatment of KO PTECs with oleic acid accelerated fatty acids transfer to mitochondria. Furthermore, KO PTECs promoted massive triglyceride accumulation in hepatocytes (BNL-CL2 cells) co-cultured in transwell, suggesting accelerated fatty acids efflux from the PTECs contributes to the metabolic syndrome in KO mice. This study shows that sustained high autophagic flux by RUBCN deficiency in PTECs leads to metabolic syndrome concomitantly with an accelerated mobilization of phospholipids from cellular membranes to lysosomes. Abbreviations: ABC: ATP binding cassette; ACADM: acyl-CoA dehydrogenase medium chain; ACTB: actin, beta; ATG: autophagy related; AUC: area under the curve; Baf: bafilomycin A1; BAT: brown adipose tissue; BODIPY: boron-dipyrromethene; BSA: bovine serum albumin; BW: body weight; CAT: chloramphenicol acetyltransferase; CM: complete medium; CPT1A: carnitine palmitoyltransferase 1a, liver; CQ: chloroquine; CTRL: control; EGFP: enhanced green fluorescent protein; CTSD: cathepsin D; EAT: epididymal adipose tissue; EGFR: epidermal growth factor receptor; EIF4EBP1: eukaryotic translation initiation factor 4E binding protein 1; FA: fatty acid; FBS: fetal bovine serum; GTT: glucose tolerance test; HE: hematoxylin and eosin; HFD: high-fat diet; I/R: ischemia-reperfusion; ITT: insulin tolerance test; KAP: kidney androgen regulated protein; KO: knockout; LAMP1: lysosomal associated membrane protein 1; LD: lipid droplet; LRP2: low density lipoprotein receptor related protein 2; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MAT: mesenteric adipose tissue; MS: mass spectrometry; MTOR: mechanistic target of rapamycin kinase; MTORC1: MTOR complex 1; NDRG1: N-myc downstream regulated 1; NDUFB5: NADH:ubiquinone oxidoreductase subunit B5; NEFA: non-esterified fatty acid; OA: oleic acid; OCT: optimal cutting temperature; ORO: Oil Red O; PAS: Periodic-acid Schiff; PFA: paraformaldehyde; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; PPARA: peroxisome proliferator activated receptor alpha; PPARGC1A: PPARG coactivator 1 alpha; PTEC: proximal tubular epithelial cell; RAB7A: RAB7A, member RAS oncogene family; RPS6: ribosomal protein S6; RPS6KB1: ribosomal protein S6 kinase B1; RT: reverse transcription; RUBCN: rubicon autophagy regulator; SAT: subcutaneous adipose tissue; SFC: supercritical fluid chromatography; SQSTM1: sequestosome 1; SREBF1: sterol regulatory element binding transcription factor 1; SV-40: simian virus-40; TFEB: transcription factor EB; TG: triglyceride; TS: tissue specific; TUNEL: terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling; UN: urea nitrogen; UQCRB: ubiquinol-cytochrome c reductase binding protein; UVRAG: UV radiation resistance associated; VPS: vacuolar protein sorting; WAT: white adipose tissue.
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Project description:Ischemia/reperfusion injury is a major cause of acute kidney injury. Improving renal repair would represent a therapeutic strategy to prevent renal dysfunction. The innate immune receptor Nlrp3 is involved in tissue injury, inflammation, and fibrosis; however, its role in repair after ischemia/reperfusion is unknown. We address the role of Nlrp3 in the repair phase of renal ischemia/reperfusion and investigate the relative contribution of leukocyte- versus renal-associated Nlrp3 by studying bone marrow chimeric mice. We found that Nlrp3 expression was most profound during the repair phase. Although Nlrp3 expression was primarily expressed by leukocytes, both leukocyte- and renal-associated Nlrp3 was detrimental to renal function after ischemia/reperfusion. The Nlrp3-dependent cytokine IL-1? remained unchanged in kidneys of all mice. Leukocyte-associated Nlrp3 negatively affected tubular apoptosis in mice that lacked Nlrp3 expression on leukocytes, which correlated with reduced macrophage influx. Nlrp3-deficient (Nlrp3KO) mice with wild-type bone marrow showed an improved repair response, as seen by a profound increase in proliferating tubular epithelium, which coincided with increased hepatocyte growth factor expression. In addition, Nlrp3KO tubular epithelial cells had an increased repair response in vitro, as seen by an increased ability of an epithelial monolayer to restore its structural integrity. In conclusion, Nlrp3 shows a tissue-specific role in which leukocyte-associated Nlrp3 is associated with tubular apoptosis, whereas renal-associated Nlrp3 impaired wound healing.
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Project description:IntroductionIntrauterine malnutrition impairs embryo kidney development and leads to kidney disease and hypertension in adulthood, yet the underlying mechanism remains unclear.MethodsWith a maternal protein restriction (MPR) rat model, we investigated the critical ciliogenesis factors and β-catenin pathway in FGR fetal kidneys and analyzed the impact of aberrant primary cilia on renal tubular epithelium.ResultsThe data showed decreased nephron number and renal tubular dysgenesis in FGR fetus. FGR fetus showed deregulated expression of ciliogenesis factors including upregulation of IFT88 and downregulation of DYNLT1, accompanied with cilia elongation in renal tubular epithelial cells. Wnt7b, the key ligand for Wnt/β-catenin signaling, was downregulated and nuclear translocation of β-catenin was decreased. The proapoptotic protein was upregulated. In vitro study with HK-2 cells showed that overexpression of IFT88 lengthened the cilia, inhibited β-catenin signaling. Besides, IFT88 overexpression suppressed cell proliferation, activated autophagy, and induced cell apoptosis. Inhibition of autophagy partly restored the cilia length and cell viability. Likewise, knockdown of DYNLT1 led to cilia elongation, suppressed cell proliferation, and promoted apoptosis in HK-2 cell. However, the cilia elongation induced by DYNLT1 knockdown was not autophagy-dependent, but associated with reactive oxygen species (ROS) accumulation.DiscussionWe elucidated that intrauterine protein malnutrition led to deregulation of ciliogenesis factors and cilia elongation in renal tubular epithelial, inhibited β-catenin signaling, and induced cell apoptosis and ultimately, compromised kidney development.
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Project description:Properdin, a positive regulator of complement alternative pathway, participates in renal ischemia-reperfusion (IR) injury and also acts as a pattern-recognition molecule affecting apoptotic T-cell clearance. However, the role of properdin in tubular epithelial cells (TECs) at the repair phase post IR injury is not well defined. This study revealed that properdin knockout (PKO) mice exhibited greater injury in renal function and histology than wild-type (WT) mice post 72-h IR, with more apoptotic cells and macrophages in tubular lumina, increased active caspase-3 and HMGB1, but better histological structure at 24 h. Raised erythropoietin receptor by IR was furthered by PKO and positively correlated with injury and repair markers. Properdin in WT kidneys was also upregulated by IR, while H2O2-increased properdin in TECs was reduced by its small-interfering RNA (siRNA), with raised HMGB1 and apoptosis. Moreover, the phagocytic ability of WT TECs, analyzed by pHrodo Escherichia coli bioparticles, was promoted by H2O2 but inhibited by PKO. These results were confirmed by counting phagocytosed H2O2-induced apoptotic TECs by in situ end labeling fragmented DNAs but not affected by additional serum with/without properdin. Taken together, PKO results in impaired phagocytosis at the repair phase post renal IR injury. Properdin locally produced by TECs plays crucial roles in optimizing damaged cells and regulating phagocytic ability of TECs to effectively clear apoptotic cells and reduce inflammation.
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Project description:The repair of the kidney after ischemia/reperfusion injury involves proliferation of proximal tubular epithelial cells as well as cell migration and differentiation. Immediately after reperfusion, expression of hypertension-related calcium-regulated gene (HCaRG/COMMD5) decreases, but its expression increases even higher than baseline during repair. HCaRG inhibits proliferation and accelerates wound healing and differentiation in cultured cells, but whether HCaRG can stimulate renal repair after ischemia/reperfusion injury is unknown. Here, transgenic mice overexpressing human HCaRG survived longer and recovered renal function faster than littermate controls after ischemia/reperfusion (64% versus 25% survival at 7 days). Proliferation of proximal tubular epithelial cells stopped earlier after ischemia/reperfusion injury, E-cadherin levels recovered more rapidly, and vimentin induction abated faster in transgenic mice. HCaRG overexpression also reduced macrophage infiltration and inflammation after injury. Taken together, these data suggest that HCaRG accelerates repair of renal proximal tubules by modulating cell proliferation of resident tubular epithelial cells and by facilitating redifferentiation.
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Project description:Peritubular capillaries (PTCs) are closely related to renal tubules in structure and function, and both are pivotal regulators in the development and progression of acute kidney injury (AKI). However, the mechanisms that underlie the interaction between PTCs and tubules during AKI remain unclear. Here we explored a new mode of tubulovascular crosstalk mediated by small extracellular vesicles (sEV) after AKI. In response to renal ischemia/reperfusion (I/R) injury, endothelial proliferation of PTCs and tubular expression of vascular endothelial growth factor-A (VEGF-A) were increased, accompanied by a remarkable redistribution of cytoplasmic VEGF-A to the basolateral side of tubular cells. Meanwhile, the secretion mode of VEGF-A was converted in the injured tubular cells, which showed a much greater tendency to secrete VEGF-A via sEV other than the free form. Interestingly, tubular cell-derived VEGF-A-enriched sEV (sEV-VEGF-A) turned out to promote endothelial proliferation which was regulated by VEGF receptors 1 and 2. Furthermore, inhibition of renal sEV secretion by Rab27a knockdown resulted in a significant decrease in the proliferation of peritubular endothelial cells in vivo. Importantly, taking advantage of the newly recognized endogenous repair response of PTCs, exogenous supplementation of VEGF-A + sEV efficiently recused PTC rarefaction, improved renal perfusion, and halted the AKI to CKD transition. Taken together, our study uncovered a novel intrinsic repair response after AKI through renal tubule-PTC crosstalk via sEV-VEGF-A, which could be exploited as a promising therapeutic angiogenesis strategy in diseases with ischemia.
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Project description:Coagulation abnormalities and increased risk of atherothrombosis are common in patients with chronic kidney diseases (CKD). Mechanisms that alter renal hemostasis and lead to thrombotic events are not fully understood. Here we show that activation of protease activated receptor-2 (PAR2) on human kidney tubular epithelial cells (HTECs), induces tissue factor (TF) synthesis and secretion that enhances blood clotting. PAR-activating coagulation-associated protease (thrombin), as well as specific PAR2 activators (matriptase, trypsin, or synthetic agonist 2f-LIGRLO-NH2 (2F), induced TF synthesis and secretion that were potently inhibited by PAR2 antagonist, I-191. Thrombin-induced TF was also inhibited by a PAR1 antagonist, Vorapaxar. Peptide activators of PAR1, PAR3, and PAR4 failed to induce TF synthesis. Differential centrifugation of the 2F-conditoned medium sedimented the secreted TF, together with the exosome marker ALG-2 interacting protein X (ALIX), indicating that secreted TF was associated with extracellular vesicles. 2F-treated HTEC conditioned medium significantly enhanced blood clotting, which was prevented by pre-incubating this medium with an antibody for TF. In summary, activation of PAR2 on HTEC stimulates synthesis and secretion of TF that induces blood clotting, and this is attenuated by PAR2 antagonism. Thrombin-induced TF synthesis is at least partly mediated by PAR1 transactivation of PAR2. These findings reveal how underlying hemostatic imbalances might increase thrombosis risk and subsequent chronic fibrin deposition in the kidneys of patients with CKD and suggest PAR2 antagonism as a potential therapeutic strategy for intervening in CKD progression.
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Project description:Corneal transparency, dependent on the integrity of epithelial cells, is essential for vision. Corneal epithelial damage is one of the most commonly observed ocular conditions and proper wound healing is necessary for corneal transparency. Sirt6, a histone deacetylase, has been shown to regulate many cellular events including aging and inflammation. However, its specific role in corneal epithelial wound healing remains unknown. Here we demonstrated that Sirt6 was expressed in corneal epithelial cells and its expression decreased with age. In an in vivo corneal epithelial wound healing model, Sirt6 deficiency resulted in delayed and incomplete wound healing and was associated excessive inflammation in the corneal stroma and dysfunction of Notch signaling, leading to keratinization of the corneal epithelium and corneal opacity. Aging Sirt6-deficient mice spontaneously developed corneal keratitis with extensive infiltration of inflammatory cells into the cornea. In vitro experiments demonstrated that primary corneal epithelial cells with Sirt6 downregulation expressed increased basal levels of inflammatory genes and exhibited hyper-inflammatory reactivity to IL-1β and TNFα treatment. These results provide compelling evidence that Sirt6 is a critical regulator of inflammation in the cornea, and is responsible for corneal epithelial wound healing, thus contributing to the maintenance of epithelial integrity and corneal transparency.
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Project description:CALHM1 is a cell surface calcium channel expressed in cerebral neurons. CALHM1 function in the brain remains unknown, but recent results showed that neuronal CALHM1 controls intracellular calcium signaling and cell excitability, two mechanisms required for synaptic function. Here, we describe the generation of Calhm1 knockout (Calhm1(-/-)) mice and investigate CALHM1 role in neuronal and cognitive functions. Structural analysis revealed that Calhm1(-/-) brains had normal regional and cellular architecture, and showed no evidence of neuronal or synaptic loss, indicating that CALHM1 deficiency does not affect brain development or brain integrity in adulthood. However, Calhm1(-/-) mice showed a severe impairment in memory flexibility, assessed in the Morris water maze, and a significant disruption of long-term potentiation without alteration of long-term depression, measured in ex vivo hippocampal slices. Importantly, in primary neurons and hippocampal slices, CALHM1 activation facilitated the phosphorylation of NMDA and AMPA receptors by protein kinase A. Furthermore, neuronal CALHM1 activation potentiated the effect of glutamate on the expression of c-Fos and C/EBPβ, two immediate-early gene markers of neuronal activity. Thus, CALHM1 controls synaptic activity in cerebral neurons and is required for the flexible processing of memory in mice. These results shed light on CALHM1 physiology in the mammalian brain.
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Project description:Metabolic flexibility is the capacity for skeletal muscle to shift reliance between lipids and glucose during fasting or in response to insulin. We hypothesized that body fat, adipose tissue characteristics, e.g. larger adipocytes, presence of inflammatory gene markers and impaired suppression of non-esterified fatty acids (NEFAs) during insulin infusion might be related to metabolic flexibility. We measured changes in respiratory quotient (DeltaRQ) before and during euglycemic-hyperinsulinemic clamp in healthy young males. Body fat by DXA, laboratory measurements, abdominal subcutaneous adipose tissue biopsies and fat cell size (FCS) were obtained after an overnight fast. Gene expression for 17 adipose tissue genes related to lipid synthesis, uptake, oxidation and storage, lipolysis and inflammation were measured. Reduced metabolic flexibility was associated with higher body fat, larger FCS and impaired insulin suppression of NEFAs. Metabolic flexibility was associated with higher serum adiponectin levels. Lower adipose tissue gene expression for inflammation markers was associated with greater NEFA suppression by insulin and metabolic flexibility. Combined, these results indicate that body fat, larger adipocytes, failure of insulin to suppress NEFAs, decreased adiponectin levels and inflammation markers in adipose tissue are associated with decreased insulin-stimulated glucose uptake and oxidation, which is an important component of reduced metabolic flexibility.
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