ABSTRACT
Calpains are intracellular proteases that play a key role in inflammation/immunity. Rare studies show that they are partially externalized. However, the mechanism of this secretion and the functions of exteriorized calpains remain poorly understood. In this study, we found that mouse and human lymphocytes secreted calpains through an ABCA1-driven process. In turn, extracellular calpains inhibited IL-17A expression. We were able to attribute this function to a cleavage of the TLR2 extracellular domain, which prevented TLR2-induced transcription of molecules essential for IL-17A induction. Calpain exteriorization and TLR2 cleavage were critical for the control of IL-17A expression by low doses of IL-2. By using newly developed transgenic mice in which extracellular calpains are specifically inactivated, we provide evidence for the relevance of calpain externalization in vivo in regulating IL-17A expression and function in experimental sterile peritonitis and autoimmune arthritis, respectively. Thus, this study identifies calpain exteriorization as a potential target for immune modulation.
Subject(s)
ATP Binding Cassette Transporter 1/biosynthesis , Calpain/metabolism , Interleukin-17/biosynthesis , T-Lymphocytes/immunology , Toll-Like Receptor 2/metabolism , ATP Binding Cassette Transporter 1/genetics , Animals , Arthritis, Experimental , Cell Line , Cell Proliferation , Gene Expression Regulation , HEK293 Cells , Humans , Inflammation/immunology , Inflammation Mediators/immunology , Interleukin-17/genetics , Interleukin-2/therapeutic use , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Neutrophils/immunology , RNA Interference , RNA, Small Interfering , Spleen/cytologyABSTRACT
OBJECTIVE: Angiotensin II (AngII) infusion profoundly increases activity of calpains, calcium-dependent neutral cysteine proteases, in mice. Pharmacological inhibition of calpains attenuates AngII-induced aortic medial macrophage accumulation, atherosclerosis, and abdominal aortic aneurysm in mice. However, the precise functional contribution of leukocyte-derived calpains in AngII-induced vascular pathologies has not been determined. The purpose of this study was to determine whether calpains expressed in bone marrow (BM)-derived cells contribute to AngII-induced atherosclerosis and aortic aneurysms in hypercholesterolemic mice. APPROACH AND RESULTS: To study whether leukocyte calpains contributed to AngII-induced aortic pathologies, irradiated male low-density lipoprotein receptor(-/-) mice were repopulated with BM-derived cells that were either wild-type or overexpressed calpastatin, the endogenous inhibitor of calpains. Mice were fed a fat-enriched diet and infused with AngII (1000 ng/kg per minute) for 4 weeks. Overexpression of calpastatin in BM-derived cells significantly attenuated AngII-induced atherosclerotic lesion formation in aortic arches, but had no effect on aneurysm formation. Using either BM-derived cells from calpain-1-deficient mice or mice with leukocyte-specific calpain-2 deficiency generated using cre-loxP recombination technology, further studies demonstrated that independent deficiency of either calpain-1 or -2 in leukocytes modestly attenuated AngII-induced atherosclerosis. Calpastatin overexpression significantly attenuated AngII-induced inflammatory responses in macrophages and spleen. Furthermore, calpain inhibition suppressed migration and adhesion of macrophages to endothelial cells in vitro. Calpain inhibition also significantly decreased hypercholesterolemia-induced atherosclerosis in the absence of AngII. CONCLUSIONS: The present study demonstrates a pivotal role for BM-derived calpains in mediating AngII-induced atherosclerosis by influencing macrophage function.
Subject(s)
Angiotensin II , Aortic Aneurysm, Abdominal/prevention & control , Atherosclerosis/prevention & control , Calpain/deficiency , Inflammation/prevention & control , Leukocytes/enzymology , Animals , Aortic Aneurysm, Abdominal/chemically induced , Aortic Aneurysm, Abdominal/enzymology , Aortic Aneurysm, Abdominal/genetics , Atherosclerosis/chemically induced , Atherosclerosis/enzymology , Atherosclerosis/genetics , Bone Marrow Transplantation , Calcium-Binding Proteins/genetics , Calcium-Binding Proteins/metabolism , Calpain/genetics , Calpain/metabolism , Cell Adhesion/drug effects , Cell Movement/drug effects , Cells, Cultured , Coculture Techniques , Cysteine Proteinase Inhibitors/pharmacology , Diet, High-Fat , Disease Models, Animal , Endothelial Cells/drug effects , Endothelial Cells/enzymology , Genetic Predisposition to Disease , Inflammation/chemically induced , Inflammation/enzymology , Inflammation/genetics , Macrophages/drug effects , Macrophages/enzymology , Male , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Phenotype , Receptors, LDL/deficiency , Receptors, LDL/genetics , Whole-Body IrradiationABSTRACT
Excessive growth of pulmonary arterial (PA) smooth muscle cells (SMCs) is a major component of PA hypertension (PAH). The calcium-activated neutral cysteine proteases calpains 1 and 2, expressed by PASMCs, contribute to PH but are tightly controlled by a single specific inhibitor, calpastatin. Our objective was to investigate calpastatin during pulmonary hypertension (PH) progression and its potential role as an intracellular and/or extracellular effector. We assessed calpains and calpastatin in patients with idiopathic PAH and mice with hypoxic or spontaneous (SM22-5HTT(+) strain) PH. To assess intracellular and extracellular roles for calpastatin, we studied effects of the calpain inhibitor PD150606 on hypoxic PH in mice with calpastatin overexpression driven by the cytomegalovirus promoter (CMV-Cast) or C-reactive protein (CRP) promoter (CRP-Cast), inducing increased calpastatin production ubiquitously and in the liver, respectively. Chronically hypoxic and SM22-5HTT(+) mice exhibited increased lung calpastatin and calpain 1 and 2 protein levels and activity, both intracellularly and extracellularly. Prominent calpastatin and calpain immunostaining was found in PASMCs of remodeled vessels in mice and patients with PAH, who also exhibited increased plasma calpastatin levels. CMV-Cast and CRP-Cast mice showed similarly decreased PH severity compared with wild-type mice, with no additional effect of PD150606 treatment. In cultured PASMCs from wild-type and CMV-Cast mice, exogenous calpastatin decreased cell proliferation and migration with similar potency as PD150606 and suppressed fibronectin-induced potentiation. These results indicate that calpastatin limits PH severity via extracellular mechanisms. They suggest a new approach to the development of treatments for PH.
Subject(s)
Calcium-Binding Proteins/metabolism , Calpain/metabolism , Disease Progression , Extracellular Space/metabolism , Hypertension, Pulmonary/metabolism , Hypertension, Pulmonary/pathology , Acrylates/pharmacology , Acrylates/therapeutic use , Animals , Cell Movement/drug effects , Cell Proliferation/drug effects , Cytomegalovirus/genetics , Extracellular Space/drug effects , Heart Function Tests , Humans , Hypertension, Pulmonary/complications , Hypertension, Pulmonary/drug therapy , Hypoxia/complications , Hypoxia/metabolism , Hypoxia/physiopathology , Intracellular Space/drug effects , Intracellular Space/metabolism , Male , Mice, Inbred C57BL , Mice, Transgenic , Myocytes, Smooth Muscle/drug effects , Myocytes, Smooth Muscle/metabolism , Myocytes, Smooth Muscle/pathology , Promoter Regions, Genetic/genetics , Pulmonary Artery/pathologyABSTRACT
Vitamin D supplementation in humans should be accompanied by calcium administration to avoid bone demineralization through vitamin D receptor signaling. Here we analyzed whether long-term exposure of rats to vitamin D supplementation, with or without a calcium-rich diet, would promote kidney stone formation. Four groups of rats received vitamin D alone (100,000 UI/kg/3 weeks), a calcium-enriched diet alone, both vitamin D supplementation and calcium-enriched diet, or a standard diet (controls) for 6 months. Serum and urine parameters and crystalluria were monitored. Kidney stones were assessed by 3-dimensional micro-computed tomography, infrared spectroscopy, von Kossa/Yasue staining, and field emission scanning electron microscopy. Although serum calcium levels were similar in the 4 groups, rats receiving vitamin D had a progressive increase in urinary calcium excretion over time, especially those receiving both calcium and vitamin D. However, oral calcium supplementation alone did not increase urinary calcium excretion. At 6 months, rats exposed to both calcium and vitamin D, but not rats exposed to calcium or vitamin D alone, developed significant apatite kidney calcifications (mean volume, 0.121 mm(3)). Thus, coadministration of vitamin D and increased calcium intake had a synergistic role in tubular calcifications or kidney stone formation in this rat model. Hence, one should be cautious about the cumulative risk of kidney stone formation in humans when exposed to both vitamin D supplementation and high calcium intake.
Subject(s)
Calcium, Dietary/pharmacology , Dietary Supplements/adverse effects , Kidney Calculi/etiology , Vitamin D/pharmacology , Animals , Apatites/metabolism , Bone Demineralization, Pathologic/etiology , Calcium, Dietary/blood , Calcium, Dietary/urine , Disease Models, Animal , Drug Synergism , Kidney Calculi/blood , Kidney Calculi/chemistry , Kidney Calculi/urine , Male , Microscopy, Electron, Scanning , Rats , Rats, Sprague-Dawley , Receptors, Calcitriol/metabolism , Renal Elimination , Spectroscopy, Fourier Transform Infrared , X-Ray MicrotomographyABSTRACT
The activation of the calpain system is involved in the repair process following myocardial infarction (MI). However, the impact of the inhibition of calpain by calpastatin, its natural inhibitor, on scar healing and left ventricular (LV) remodeling is elusive. Male mice ubiquitously overexpressing calpastatin (TG) and wild-type (WT) controls were subjected to an anterior coronary artery ligation. Mortality at 6 wk was higher in TG mice (24% in WT vs. 44% in TG, P < 0.05) driven by a significantly higher incidence of cardiac rupture during the first week post-MI, despite comparable infarct size and LV dysfunction and dilatation. Calpain activation post-MI was blunted in TG myocardium. In TG mice, inflammatory cell infiltration and activation were reduced in the infarct zone (IZ), particularly affecting M2 macrophages and CD4(+) T cells, which are crucial for scar healing. To elucidate the role of calpastatin overexpression in macrophages, we stimulated peritoneal macrophages obtained from TG and WT mice in vitro with IL-4, yielding an abrogated M2 polarization in TG but not in WT cells. Lymphopenic Rag1(-/-) mice receiving TG splenocytes before MI demonstrated decreased T-cell recruitment and M2 macrophage activation in the IZ day 5 after MI compared with those receiving WT splenocytes. Calpastatin overexpression prevented the activation of the calpain system after MI. It also impaired scar healing, promoted LV rupture, and increased mortality. Defective scar formation was associated with blunted CD4(+) T-cell and M2-macrophage recruitment.
Subject(s)
CD4-Positive T-Lymphocytes/metabolism , Calcium-Binding Proteins/metabolism , Lymphocyte Activation , Macrophage Activation , Macrophages/metabolism , Myocardial Infarction/metabolism , Myocardium/metabolism , Ventricular Remodeling , Wound Healing , Animals , CD4-Positive T-Lymphocytes/immunology , Calcium-Binding Proteins/genetics , Calpain/metabolism , Chemotaxis, Leukocyte , Disease Models, Animal , Enzyme Activation , Genotype , Heart Rupture, Post-Infarction/metabolism , Heart Rupture, Post-Infarction/pathology , Heart Rupture, Post-Infarction/physiopathology , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Macrophages/immunology , Male , Mice, Inbred C57BL , Mice, Knockout , Myocardial Infarction/genetics , Myocardial Infarction/immunology , Myocardial Infarction/pathology , Myocardial Infarction/physiopathology , Myocardium/immunology , Myocardium/pathology , Phenotype , Time Factors , Up-Regulation , Ventricular Dysfunction, Left/metabolism , Ventricular Dysfunction, Left/pathology , Ventricular Dysfunction, Left/physiopathology , Ventricular Function, LeftABSTRACT
OBJECTIVE: Calpains, calcium-activated proteases, mediate the angiogenic signals of vascular endothelial growth factor. However, their involvement in vascular repair has not been investigated and the underlying mechanisms remain to be fully elucidated. METHODS AND RESULTS: A rapidly progressive form of glomerulonephritis in wild type and transgenic mice expressing high levels of calpastatin, a calpain-specific inhibitor, was studied. Calpastatin transgene expression prevented the repair of peritubular capillaries and the recovery of renal function, limiting mouse survival. In vitro analysis detected a significant reduction of both intracellular and extracellular calpain activities in transgene expressing cells, whereas Western blotting revealed that proangiogenic factors vascular endothelial growth factor and norepinephrine increased calpain exteriorization. In vitro, extracellular calpains increased endothelial cell proliferation, migration and capillary tube formation. In vivo, delivery of nonpermeable extracellular calpastatin was sufficient to blunt angiogenesis and vascular repair. Endothelial cell response to extracellular calpains was associated with fibronectin cleavage, generating fibronectin fragments with proangiogenic capacity. In vivo, fibronectin cleavage was limited in the kidney of calpastatin transgenic mice with nephritis. CONCLUSIONS: This study demonstrates that externalized calpains participate in angiogenesis and vascular repair, partly by promoting fibronectin cleavage and thereby amplifying vascular endothelial growth factor efficiency. Thus, manipulation of calpain externalization may have therapeutic implications to control angiogenesis.
Subject(s)
Calpain/physiology , Disease Progression , Glomerulonephritis/physiopathology , Neovascularization, Physiologic/physiology , Animals , Blood Vessels/growth & development , Calpain/genetics , Calpain/pharmacology , Cell Movement/drug effects , Cell Movement/physiology , Cell Proliferation/drug effects , Cells, Cultured , Disease Models, Animal , Endothelium, Vascular/cytology , Endothelium, Vascular/drug effects , Fibronectins/metabolism , Humans , Male , Mice , Mice, Inbred C57BL , Mice, TransgenicABSTRACT
RATIONALE: Sepsis, a leading cause of death worldwide, involves widespread activation of inflammation, massive activation of coagulation, and lymphocyte apoptosis. Calpains, calcium-activated cysteine proteases, have been shown to increase inflammatory reactions and lymphocyte apoptosis. Moreover, calpain plays an essential role in microparticle release. OBJECTIVES: We investigated the contribution of calpain in eliciting tissue damage during sepsis. METHODS: To test our hypothesis, we induced polymicrobial sepsis by cecal ligation and puncture in wild-type (WT) mice and transgenic mice expressing high levels of calpastatin, a calpain-specific inhibitor. MEASUREMENTS AND MAIN RESULTS: In WT mice, calpain activity increased transiently peaking at 6 hours after cecal ligation and puncture surgery. Calpastatin overexpression improved survival, organ dysfunction (including lung, kidney, and liver damage), and lymphocyte apoptosis. It decreased the sepsis-induced systemic proinflammatory response and disseminated intravascular coagulation, by reducing the number of procoagulant circulating microparticles and therefore delaying thrombin generation. The deleterious effect of microparticles in this model was confirmed by transferring microparticles from septic WT to septic transgenic mice, worsening their survival and coagulopathy. CONCLUSIONS: These results demonstrate an important role of the calpain/calpastatin system in coagulation/inflammation pathways during sepsis, because calpain inhibition is associated with less severe disseminated intravascular coagulation and better overall outcomes in sepsis.
Subject(s)
Calcium-Binding Proteins/physiology , Sepsis/physiopathology , Animals , Apoptosis/physiology , Calpain/physiology , Cell-Derived Microparticles/physiology , Cytokines/physiology , Disease Models, Animal , Disseminated Intravascular Coagulation/physiopathology , Lymphocytes/physiology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Multiple Organ Failure/physiopathology , NF-kappa B/physiology , Sepsis/mortality , Thromboplastin/physiologyABSTRACT
The renal urothelium, the monolayered epithelium that covers the papilla, is the direct target of increased pressure during obstruction, yet most studies have mainly focused on tubules, fibroblasts, and inflammatory cells. We studied this epithelium in a unilateral ureteral obstruction mouse mode land found that it was disrupted and had broken tight junctions, enlarged intercellular space, with loss of apicaluroplakins, and marginal lumen desquamation. Shortly after obstruction these urothelial cells proliferated, peaking at day 2. By day 14, the renal urothelium was transformed into a multilayered barrier with newly synthesized uroplakins including the de novo induction of uroplakin II. This proliferation was found to be fibroblast growth factor (FGF)dependent. Renal urothelial cells constitutively express the FGF receptor 2, and obstruction activated the receptor by phosphorylation. Treatment with FGF receptor 2-antisense or vitamin A (an inhibitor of the MAP kinase in the FGFR2 pathway) decreased renal urothelial cell proliferation. Among known FGF receptor 2 ligands, only FGF7 was upregulated.Infusion of FGF7 into control mice caused the formation of a multilayered structure at 7 days, resembling the urothelium 14 days following obstruction. Thus, the pressure/stretching of renal monolayered urothelial cells is a very efficient trigger for proliferation, causing the formation of a bladder-like multistratified barrier with enhanced apical uroplakin plaques. Presumably, this ensures efficient barrier protection and repair.
Subject(s)
Cell Differentiation , Cell Proliferation , Cell Transdifferentiation , Kidney/pathology , Ureteral Obstruction/pathology , Urinary Bladder/pathology , Urothelium/pathology , Animals , Biomarkers/metabolism , Cell Differentiation/drug effects , Cell Proliferation/drug effects , Cell Transdifferentiation/drug effects , Disease Models, Animal , Female , Fibroblast Growth Factor 7/administration & dosage , Fibroblast Growth Factor 7/metabolism , Fibroblast Growth Factors/genetics , Fibroblast Growth Factors/metabolism , Kidney/drug effects , Kidney/metabolism , Mechanotransduction, Cellular , Mice , Mice, Inbred C57BL , Oligonucleotides, Antisense/metabolism , Phenotype , Phosphorylation , Pressure , Receptor, Fibroblast Growth Factor, Type 2/metabolism , Stress, Mechanical , Time Factors , Ureteral Obstruction/genetics , Ureteral Obstruction/metabolism , Urinary Bladder/drug effects , Urinary Bladder/metabolism , Uroplakins/metabolism , Urothelium/drug effects , Urothelium/metabolism , Vitamin A/pharmacologyABSTRACT
Rejection of solid organ allograft involves alloreactive T-cell expansion. The importance of NF-κB and NFAT in this process is underscored by the therapeutic efficacy of immunosuppressive agents, which target the two transcription factors. Since calpains, calcium-activated proteases, are involved in the activation of NF-κB and NFAT, we investigated the role of calpains in allograft rejection. In human transplant kidneys undergoing acute or chronic rejection, we show an increased expression of CAPN 1 gene encoding µ-calpain, associated with a marked expression of µ-calpain, mainly in infiltrating T cells. To address the role of calpain in rejection, we used a skin transplant model in transgenic mice expressing high levels of calpastatin, a calpain-specific inhibitor. We show that calpain inhibition extended skin allograft survival, from 11 to 20 days. This delay was associated with a limitation in allograft infiltration by T cells. In vitro, calpain inhibition by calpastatin transgene expression limited dramatically T-cell migration but, unexpectedly, increased slightly T-cell proliferation. Amplification of IL-2 signaling via the stabilization of IL-2R common γ-chain provided an explanation for the proliferation response. This is the first study establishing that calpain inhibition delays allograft rejection by slowing down T-cell migration rather than proliferation.
Subject(s)
Calcium-Binding Proteins/metabolism , Calpain/metabolism , Graft Rejection/metabolism , Acrylates/therapeutic use , Adoptive Transfer , Animals , Calcium-Binding Proteins/genetics , Calpain/antagonists & inhibitors , Calpain/genetics , Cell Differentiation/genetics , Cell Differentiation/immunology , Cell Movement/genetics , Cell Movement/immunology , Cell Proliferation/drug effects , Gene Expression/genetics , Gene Expression/immunology , Graft Rejection/immunology , Graft Rejection/pathology , Graft Rejection/prevention & control , Homeodomain Proteins/genetics , Humans , Interleukin-2/metabolism , Kidney/immunology , Kidney/metabolism , Kidney Transplantation/immunology , Lymphocyte Activation/genetics , Lymphocyte Activation/immunology , Lymphocyte Culture Test, Mixed , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , NF-kappa B/metabolism , NFATC Transcription Factors/metabolism , Skin Transplantation/immunology , Skin Transplantation/pathology , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , T-Lymphocytes/pathology , T-Lymphocytes/transplantation , T-Lymphocytes, Cytotoxic/immunology , Th17 Cells/immunology , Th17 Cells/metabolism , Transplantation, HomologousABSTRACT
In hypertension, angiotensin (Ang) II is a critical mediator of cardiovascular remodeling, whose prominent features include myocardial and vascular media hypertrophy, perivascular inflammation, and fibrosis. The signaling pathways responsible for these alterations are not completely understood. Here, we investigated the importance of calpains, calcium-dependent cysteine proteases. We generated transgenic mice constitutively expressing high levels of calpastatin, a calpain-specific inhibitor. Chronic infusion of Ang II led to similar increases in systolic blood pressure in wild-type and transgenic mice. In contrast, compared with wild-type mice, transgenic mice displayed a marked blunting of Ang II-induced hypertrophy of left ventricle. Ang II-dependent vascular remodeling, ie, media hypertrophy and perivascular inflammation and fibrosis, was also limited in both large arteries (aorta) and small kidney arteries from transgenic mice as compared with wild type. In vitro experiments using vascular smooth muscle cells showed that calpastatin transgene expression blunted calpain activation by Ang II through epidermal growth factor receptor transactivation. In vivo and in vitro models of inflammation showed that impaired recruitment of mononuclear cells in transgenic mice was attributable to a decrease in both the release of and the chemotactic response to monocyte chemoattractant protein-1. Finally, results from collagen synthesis assay and zymography suggested that limited fibrogenesis was attributable to a decrease in collagen deposition rather than an increase in collagen degradation. These results indicate a critical role for calpains as downstream mediators in Ang II-induced cardiovascular remodeling and, thus, highlight an attractive therapeutic target.
Subject(s)
Calcium-Binding Proteins/metabolism , Calpain/metabolism , Cysteine Proteinase Inhibitors/metabolism , Genetic Therapy , Hypertension/complications , Hypertrophy, Left Ventricular/prevention & control , Ventricular Remodeling , Angiotensin II/administration & dosage , Animals , Aorta/enzymology , Aorta/pathology , Blood Pressure , Calcium-Binding Proteins/genetics , Calpain/antagonists & inhibitors , Cysteine Proteinase Inhibitors/genetics , Disease Models, Animal , Fibrosis , Genetic Therapy/methods , Hypertension/chemically induced , Hypertension/metabolism , Hypertension/physiopathology , Hypertension/therapy , Hypertrophy , Hypertrophy, Left Ventricular/etiology , Hypertrophy, Left Ventricular/metabolism , Hypertrophy, Left Ventricular/physiopathology , Inflammation/etiology , Inflammation/metabolism , Inflammation/physiopathology , Infusion Pumps, Implantable , Mice , Mice, Transgenic , Muscle, Smooth, Vascular/metabolism , Muscle, Smooth, Vascular/pathology , Myocardium/enzymology , Myocardium/pathology , NF-kappa B/metabolism , NFATC Transcription Factors/metabolism , Renal Artery/enzymology , Renal Artery/pathology , Time FactorsABSTRACT
Calpains, intracellular proteases specifically inhibited by calpastatin, play a major role in neoangiogenesis involved in tumor invasiveness and metastasis. They are partly exteriorized via the ATP-binding cassette transporter A1(ABCA1) transporter, but the importance of this process in tumor growth is still unknown. The aim of our study was to investigate the role of extracellular calpains in a model of melanoma by blocking their extracellular activity or exteriorization. In the first approach, a B16-F10 model of melanoma was developed in transgenic mice expressing high extracellular levels of calpastatin. In these mice, tumor growth was inhibited by â¼ 3-fold compared with wild-type animals. In vitro cytotoxicity assays and in vivo tumor studies have demonstrated that this protection was associated with a defect in tumor neoangiogenesis. Similarly, in wild-type animals given probenecid to blunt ABCA1 activity, melanoma tumor growth was inhibited by â¼ 3-fold. Again, this response was associated with a defect in neoangiogenesis. In vitro studies confirmed that probenecid limited endothelial cell migration and capillary formation from vascular explants. The observed reduction in fibronectin cleavage under these conditions is potentially involved in the response. Collectively, these studies demonstrate that probenecid, by blunting ABCA1 activity and thereby calpain exteriorization, limits melanoma tumor neoangiogenesis and invasiveness.
Subject(s)
ATP Binding Cassette Transporter 1/antagonists & inhibitors , Calpain/metabolism , Melanoma, Experimental/drug therapy , Neovascularization, Pathologic/drug therapy , Probenecid/pharmacology , Skin Neoplasms/drug therapy , ATP Binding Cassette Transporter 1/genetics , ATP Binding Cassette Transporter 1/metabolism , Animals , Calcium-Binding Proteins/genetics , Calcium-Binding Proteins/metabolism , Cell Line, Tumor/transplantation , Cell Proliferation/drug effects , Humans , Male , Melanoma, Experimental/blood supply , Melanoma, Experimental/pathology , Mice , Mice, Transgenic , Neovascularization, Pathologic/pathology , Probenecid/therapeutic use , Skin Neoplasms/blood supply , Skin Neoplasms/pathologyABSTRACT
BACKGROUND: The specific mTor inhibitor sirolimus has been implicated in the pathogenesis of renal glomerular lesions and nephrotic syndrome appearance after transplantation. Podocyte injury and focal segmental glomerulosclerosis have been related to sirolimus therapy in some patients but the pathways underlying these lesions remain hypothetical. METHODS: To go further in the comprehension of these mechanisms, primary cultures of human podocytes were exposed to therapeutic-range concentrations of sirolimus. RESULTS: Cell viability was not affected after 2 days' exposure to the drug but changes in cell phenotype and cytoskeleton reorganization were observed. We also evidenced that vascular endothelial growth factor (VEGF) synthesis and Akt phosphorylation were decreased by sirolimus addition. We did not observe any loss of podocyte differentiation markers with the notable exception of WT1, a transcription factor essential for maintaining podocyte integrity. WT1 gene and protein expression in podocytes were decreased in a dose-dependent manner after incubation with sirolimus. CONCLUSION: Taken together, these data suggest that sirolimus could impair pathways essential for podocyte integrity and therefore predisposes to glomerular injury.
Subject(s)
Podocytes/drug effects , Podocytes/metabolism , Sirolimus/adverse effects , Base Sequence , Cell Differentiation/drug effects , Cell Survival/drug effects , Cells, Cultured , Cytoskeleton/drug effects , DNA Primers/genetics , Genes, Wilms Tumor/drug effects , Humans , Immunosuppressive Agents/adverse effects , Models, Biological , Phenotype , Podocytes/pathology , Polymerase Chain Reaction , Protein Kinases/drug effects , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction/drug effects , TOR Serine-Threonine Kinases , Vascular Endothelial Growth Factor A/biosynthesis , WT1 Proteins/genetics , WT1 Proteins/metabolismABSTRACT
Calpain 1 is a proinflammatory calcium-activated cysteine protease, which can be partly externalized. Extracellular calpains limit inflammatory processes and promote tissue repair, through cell proliferation and migration. Toll like receptor (TLR) 2 has been identified as a target of extracellular calpains in lymphocytes. The aim was to investigate the externalization of calpain 1 and the release of soluble TLR2 during tumor progression of pulmonary lepidic predominant adenocarcinoma (LPA). Extracellular calpain 1, soluble fragment of TLR2 and cytokines were analyzed by ELISA in bronchoalveolar lavage fluid (BALF) supernatants from patients with LPA (n=68). Source of calpain was analyzed by immunohistochemistry and soluble TLR2 by flow cytometry on polymorphonuclear neutrophils (PMN) and human lung cancer cell lines. Extracellular calpain 1, secreted by tumor cells, was associated to tumor progression, neutrophilic inflammation, with a poor prognostic factor on survival (P=0.003). TLR2 was expressed on PMN and tumor cells and decreased after calpain exposure. Soluble fragment of TLR2 in BALF supernatants was correlated to the extracellular calpain 1 concentration (r=0.624; P<0.001), and its high level was associated with tumor progression and a pro-inflammatory environment. Extracellular calpain 1 secreted by tumor cells, could participate in inflammatory microenvironment and tumor progression through TLR2 in LPA.
Subject(s)
Adenocarcinoma/metabolism , Bronchoalveolar Lavage Fluid/chemistry , Calpain/analysis , Lung Neoplasms/metabolism , Neoplasm Proteins/metabolism , Toll-Like Receptor 2/metabolism , Adenocarcinoma/mortality , Adenocarcinoma/pathology , Aged , Calpain/metabolism , Cell Line, Tumor , Disease Progression , Female , Humans , Inflammation/metabolism , Lung Neoplasms/mortality , Lung Neoplasms/pathology , Male , Neoplasm Proteins/analysis , Neutrophils/metabolism , PrognosisABSTRACT
Increased urinary oxalate excretion (hyperoxaluria) promotes the formation of calcium oxalate crystals. Monogenic diseases due to hepatic enzymes deficiency result in chronic hyperoxaluria, promoting end-stage renal disease in children and young adults. Ethylene glycol poisoning also results in hyperoxaluria promoting acute renal failure and frequently death. Stiripentol is an antiepileptic drug used to treat children affected by Dravet syndrome, possibly by inhibiting neuronal lactate dehydrogenase 5 isoenzyme. As this isoenzyme is also the last step of hepatic oxalate production, we hypothesized that Stiripentol would potentially reduce hepatic oxalate production and urine oxalate excretion. In vitro, Stiripentol decreased in a dose-dependent manner the synthesis of oxalate by hepatocytes. In vivo, Stiripentol oral administration reduced significantly urine oxalate excretion in rats. Stiripentol protected kidneys against calcium oxalate crystal deposits in acute ethylene glycol intoxication and chronic calcium oxalate nephropathy models. In both models, Stiripentol improved significantly renal function. Patients affected by Dravet syndrome and treated with Stiripentol had a lower urine oxalate excretion than control patients. A young girl affected by severe type I hyperoxaluria received Stiripentol for several weeks: urine oxalate excretion decreased by two-thirds. Stiripentol is a promising potential therapy against genetic hyperoxaluria and ethylene glycol poisoning.
Subject(s)
Dioxolanes/pharmacology , Ethylene Glycol/poisoning , Hyperoxaluria, Primary/prevention & control , Nephrolithiasis/prevention & control , Animals , Calcium Oxalate/metabolism , Epilepsies, Myoclonic/drug therapy , Epilepsies, Myoclonic/metabolism , Epilepsies, Myoclonic/pathology , Female , Hepatocytes/metabolism , Hepatocytes/pathology , Humans , Hyperoxaluria, Primary/metabolism , Hyperoxaluria, Primary/pathology , Kidney/metabolism , Kidney/pathology , Male , Nephrolithiasis/metabolism , Rats , Rats, Sprague-DawleyABSTRACT
The National Institute of Health and Medical Research (Inserm), the Society of Nephrology, and the French Kidney Foundation recognized the need to create a National Research Program for kidney and urinary tract diseases. They organized a conference gathering 80 researchers to discuss the state-of-the art and evaluate the strengths and weaknesses of kidney and urinary tract disease research in France, and to identify research priorities. From these priorities emerged 11 of common interest: 1) conducting epidemiologic studies; 2) conducting large multicenter cohorts of well-phenotyped patients with blood, urine and biopsy biobanks; 3) developing large scale approach: transcriptomics, proteomics, metabolomics; 4) developing human and animal functional imaging techniques; 5) strengthening the expertise in renal pathology and electrophysiology; 6) developing animal models of kidney injury; 7) identifying nontraumatic diagnostic and prognostic biomarkers; 8) increasing research on the fetal programming of adult kidney diseases; 9) encouraging translational research from bench to bedside and to population; 10) creating centers grouping basic and clinical research workforces with critical mass and adequate logistic support; 11) integrating and developing european research programs.
Subject(s)
Kidney Diseases , Research/trends , Urologic Diseases , Foundations , France/epidemiology , Humans , Incidence , Kidney Diseases/classification , Kidney Diseases/epidemiology , Kidney Transplantation/statistics & numerical data , Urologic Diseases/classification , Urologic Diseases/epidemiologyABSTRACT
Calpains are ubiquitous pro-inflammatory proteases, whose activity is controlled by calpastatin, their specific inhibitor. Transgenic mice over-expressing rabbit calpastatin (CalpTG) are protected against vascular remodelling and angiotensin II-dependent inflammation. We hypothesized that specific calpain inhibition would protect against aging-related lesions in arteries and kidneys. We analysed tissues from 2-months and 2-years-old CalpTG and wild-type mice and performed high throughput RNA-Sequencing of kidney tissue in aged mice. In addition, we analysed inflammatory response in the kidney of aged CalpTG and wild-type mice, and in both in vivo (monosodium urate peritonitis) and in vitro models of inflammation. At two years, CalpTG mice had preserved kidney tissue, less vascular remodelling and less markers of senescence than wild-type mice. Nevertheless, CalpTG mice lifespan was not extended, due to the development of lethal spleen tumors. Inflammatory pathways were less expressed in aged CalpTG mice, especially cytokines related to NF-κB and NLRP3 inflammasome activation. CalpTG mice had reduced macrophage infiltration with aging and CalpTG mice produced less IL-1α and IL-1ß in vivo in response to inflammasome activators. In vitro, macrophages from CalpTG mice produced less IL-1α in response to particulate activators of inflammasome. Calpains inhibition protects against inflammaging, limiting kidney and vascular lesions related to aging.
Subject(s)
Aging/drug effects , Calcium-Binding Proteins/pharmacology , Calpain/antagonists & inhibitors , Cysteine Proteinase Inhibitors/pharmacology , Peritonitis/drug therapy , Animals , Arteries/drug effects , Arteries/growth & development , Calcium-Binding Proteins/therapeutic use , Calpain/metabolism , Cells, Cultured , Cysteine Proteinase Inhibitors/therapeutic use , Cytokines/metabolism , Inflammasomes/metabolism , Kidney/drug effects , Kidney/growth & development , Mice , Mice, Inbred C57BL , NF-kappa B/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , RabbitsABSTRACT
Repair of inflammatory and/or ischemic renal injury involves endothelial, mesangial and epithelial regeneration. These structures may be rebuilt by resident progenitor cells and bone marrow-derived stem cells. Resident progenitor cells in adult kidney have not yet been conclusively identified. They are likely to be slowly cycling cells located mainly in the outer medulla and renal papilla. In glomerulonephritis with mesangiolysis, mesangial regenera- tion involves progenitor cells migrating from the juxtaglomerular apparatus and also bone marrow-derived cells. In acute ischemic renal failure, epithelial regeneration of proximal tubules results from the migration, proliferation and differentiation of resident progenitor cells; bone marrow-derived cells may play an accessory role. Molecular mechanisms underlying these repair processes could be targets for new therapeutic approaches.
Subject(s)
Kidney/blood supply , Reperfusion Injury/therapy , Stem Cells/physiology , Humans , Kidney/cytology , Kidney/physiopathology , Regeneration/physiologyABSTRACT
Antidiuretic hormone or arginine vasopressin (AVP) increases water reabsorption in the collecting ducts of the kidney. Three decades ago, experimental models have shown that AVP may increase calcium reabsorption in rat kidney. The objective of this study was to assess whether AVP modulates renal calcium excretion in humans. We analyzed calcium, potassium, and sodium fractional excretion in eight patients affected by insipidus diabetes (nephrogenic or central) under acute vasopressin receptor agonist action and in 10 patients undergoing oral water load test affected or not by inappropriate antidiuretic hormone secretion (SIADH). Synthetic V2 receptor agonist (dDAVP) reduced significantly calcium fractional excretion from 1.71% to 0.58% (P < 0.05) in patients with central diabetes insipidus. In patients with nephrogenic diabetes insipidus (resistant to AVP), calcium fractional excretion did not change significantly after injection (0.48-0.68%, P = NS). In normal subjects undergoing oral water load test, calcium fractional excretion increased significantly from 1.02% to 2.54% (P < 0.05). Patients affected by SIADH had a high calcium fractional excretion at baseline that remained stable during test from 3.30% to 3.33% (P = NS), possibly resulting from a reduced calcium absorption in renal proximal tubule. In both groups, there was a significant correlation between urine output and calcium renal excretion. In humans, dDAVP decreases calcium fractional excretion in the short term. Conversely, water intake, which lowers AVP concentration, increases calcium fractional excretion. The correlation between urine output and calcium excretion suggests that AVP-related antidiuresis increases calcium reabsorption in collecting ducts.
ABSTRACT
Renal stone incidence has progressively increased in industrialized countries, but the implication of Randall plaque in this epidemic remains unknown. Our objectives were to determine whether the prevalence of Randall plaque-related stones increased during the past decades after having analyzed 30,149 intact stones containing mainly calcium oxalate since 1989 (cross-sectional study), and to identify determinants associated with Randall plaque-related stones in patients (case-control study). The proportion of Randall plaque-related stones was assessed over 3 time periods: 1989-1991, 1999-2001, and 2009-2011. Moreover, we analyzed clinical and biochemical parameters of 105 patients affected by calcium oxalate stones, with or without plaque. Of 30,149 calcium oxalate stones, 10,282 harbored Randall plaque residues (34.1%). The prevalence of Randall plaque-related stones increased dramatically during the past years. In young women, 17% of calcium oxalate stones were associated with Randall plaque during the 1989-1991 period, but the proportion rose to 59% 20 years later (Pâ<â0.001). Patients with plaques experienced their first stone-related event earlier in life as compared with those without plaque (median age 26 vs 34 years, Pâ=â0.02), had increased ionized serum calcium levels (Pâ=â0.04), and increased serum osteocalcin (Pâ=â0.001) but similar 25-hydroxyvitamin D levels. The logistic regression analysis showed that age (odds ratio [OR] 0.96, confidence interval [CI] 0.926-0.994, Pâ=â0.02), weight (OR 0.97, CI 0.934-0.997, Pâ=â0.03), and osteocalcin serum levels (OR 1.12, CI 1.020-1.234, Pâ=â0.02) were independently associated with Randall plaque. The prevalence of the FokI f vitamin D receptor polymorphism was higher in patients with plaque (Pâ=â0.047). In conclusion, these findings point to an epidemic of Randall plaque-associated renal stones in young patients, and suggest a possible implication of altered vitamin D response.
Subject(s)
Calcium Oxalate/metabolism , Calcium Phosphates/metabolism , Kidney Calculi/chemistry , Kidney Calculi/epidemiology , Adult , Aged , Female , France/epidemiology , Humans , Male , Middle Aged , Risk FactorsABSTRACT
Somatostatin (SRIF) exerts anti-inflammatory effects, in part by deactivating monocytes/macrophages. Thus, the objective of this study was to characterize specific receptors for SRIF on these cells. Macrophages isolated from mouse peritoneal cells bound [125I]Tyr(0), D-Trp(8) SRIF(14) specifically. Scatchard analysis of saturation binding data revealed two classes of binding sites with an affinity of 0.44+/-0.13 and 2.58+/-0.56 nM, respectively. By sensitive and specific RT-PCR, the mRNAs for the five SRIF receptors (SSTR1 to SSTR5) could be detected. Evidence for the involvement of SSTR1 and SSTR2 in the binding of SRIF to the high and low affinity sites, respectively, was obtained by the demonstration that (1) only SSTR1 and SSTR2 subtype-specific agonists were active in competing for [125I]Tyr(0), D-Trp(8) SRIF(14) binding to high and low affinity sites, respectively, and (2) [125I]Tyr(0), D-Trp(8) SRIF(14) bound to high but not low affinity sites on macrophages isolated from SSTR2 knock-out mice. In conclusion, we have identified and characterized two different SRIF receptor subtypes in murine macrophages.