RESUMO
BACKGROUND: Extracellular vesicles (EVs) have received considerable attention as ideal biomarkers for kidney diseases. Most reports have focused on urinary EVs, that are mainly derived from the cells in the urinary tract. However, the detection and the application of kidney-derived EVs in plasma remains uncertain. METHODS: We examined the kidney-derived small EVs (sEVs) in plasma that were supposedly released from renal mesangial and glomerular endothelial cells, using clinical samples from healthy controls and patients with kidney transplants. Plasma from healthy controls underwent ultracentrifugation, followed by on-bead flow cytometry, targeting α8 integrin, an antigen-specific to mesangial cells. To confirm the presence of kidney-derived sEVs in peripheral blood, plasma from ABO-incompatible kidney transplant recipients was ultracentrifuged, followed by western blotting for donor blood type antigens. RESULTS: Immunohistochemistry and immunoelectron microscopy confirmed α8 integrin expression in kidney mesangial cells and their sEVs. The CD9-α8 integrin double-positive sEVs were successfully detected using on-bead flow cytometry. Western blot analysis further revealed transplanted kidney-derived sEVs containing blood type B antigens in non-blood type B recipients, who had received kidneys from blood type B donors. Notably, a patient experiencing graft kidney loss exhibited diminished signals of sEVs containing donor blood type antigens. CONCLUSION: Our findings demonstrate the potential usefulness of kidney-derived sEVs in plasma in future research for kidney diseases.
Assuntos
Vesículas Extracelulares , Transplante de Rim , Humanos , Vesículas Extracelulares/metabolismo , Masculino , Feminino , Pessoa de Meia-Idade , Adulto , Estudos de Casos e Controles , Células Mesangiais/metabolismo , Biomarcadores/sangue , Sistema ABO de Grupos Sanguíneos , Tetraspanina 29/metabolismo , Citometria de Fluxo , Rim , Células Endoteliais/metabolismo , Incompatibilidade de Grupos SanguíneosRESUMO
A 16-year-old girl with fever that appeared after taking the second COVID-19 vaccine presented to the clinic with a serum creatinine of 0.89 mg/dL and C-reactive protein of 6.9 mg/dL. She had proteinuria and microscopic hematuria, with slowly worsening kidney function. Her kidney biopsy showed fibrocellular crescents in seven of nine glomeruli that were observed under light microscopy. Another glomerulus showed endocapillary hypercellularity and mesangial cell proliferation. Electron-dense deposits were significant in the mesangial area, with monoclonal IgG1-κ and C3 deposition by immunofluorescence. The patient was diagnosed with proliferative glomerulonephritis with monoclonal immunoglobulin deposits (PGNMID) and atypical pathological finding of diffuse crescent formation. The treatment regimen for PGNMID has not yet been established, and the appropriate duration of treatment is unknown. In our case, considering that rituximab acts by binding to CD20 on the surface of B cells through its crystallizable fragment, it was administered in addition to prednisolone, which successfully decreased the proteinuria over time.
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Vacinas contra COVID-19 , Glomerulonefrite , Feminino , Humanos , Adolescente , Rituximab/uso terapêutico , Anticorpos Monoclonais/metabolismo , Corticosteroides/uso terapêutico , Proteinúria/tratamento farmacológico , Proteinúria/etiologiaRESUMO
AIM: Irrespective of the cause, albumin/proteinuria induces tubulointerstitial damage and accelerates the progression of kidney diseases. Our series of studies demonstrated that proteinuria, an independent prognostic factor for chronic kidney disease (CKD), is correlated with urinary basigin/CD147 (Bsg) levels. We examined the morphology and origin of Bsg in the tubular lumen through the effects of filtered glucose and protein solutes on the tubules. METHODS: Diabetic kidney disease (DKD) patients (N = 50) were treated with spironolactone 25 mg for 4 weeks or by conservative treatment. The associations between urinary Bsg values and clinical indicators were examined. Primary-cultured proximal tubular epithelial cells (PTECs) from human adult kidneys were exposed to high glucose or bovine serum albumin (BSA). RESULTS: In patients with early phase DKD, urinary Bsg levels were closely correlated with proteinuria but not HbA1c. Full-length Bsg on extracellular vesicles (EVs) was investigated primarily in urine collected from DKD patients. EVs obtained from the urine of DKD patients included Bsg and SGLT2 proteins. Notably, spironolactone treatment concomitantly suppressed the release of Bsg-bearing EVs in correlation with decreased albuminuria. Exposure of PTECs to BSA (but not high glucose) enhanced the storage of supernatant Bsg in EVs despite the absence of exposure-specific changes in Bsg transcription. CONCLUSION: Proteinuria induces the release of Bsg-bearing EVs derived from PTECs into the tubular lumen.
Assuntos
Vesículas Extracelulares , Insuficiência Renal Crônica , Adulto , Humanos , Albuminúria/tratamento farmacológico , Albuminúria/metabolismo , Basigina/metabolismo , Espironolactona/metabolismo , Epitélio/metabolismo , Proteinúria , Insuficiência Renal Crônica/metabolismoRESUMO
Fibrillary glomerulonephritis (FGN), a rare disease is pathologically characterized by glomerular fibril accumulation ranging from 12 to 24 nm in diameter with negative Congo red staining. Recently, the identification of DnaJ homolog subfamily B member 9 (DNAJB9) as a highly sensitive and specific marker for FGN has revolutionized diagnosis of this disease. However, few recent studies have reported DNAJB9-negative glomerulonephritis with fibrillar deposits. As such, it remains unclear whether DNAJB9-negative cases can be considered equivalent to FGN. Here, we report the case of a 70-year-old woman who developed renal impairment and nephrotic-range proteinuria. Renal biopsy and pathological examination revealed focal glomerulonephritis with fibrocellular crescents. Immunofluorescence microscopy showed IgA-dominant deposition of polytypic IgG in the glomerulus. Electron microscopy revealed hump-like subepithelial electron dense deposits with fibrils of 15-25 nm in diameter. These findings were consistent with FGN; thus, Congo red and direct fast scarlet (DFS) staining, and immunohistochemistry for DNAJB9 were performed. In addition to negative Congo red/DFS/DNAJB9 staining, laser microdissection (LMD) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) resulted negative for DNAJB9, which is a highly sensitive and specific marker for FGN. The patient's renal function further declined, prompting administration of rituximab weekly for 2 weeks, similar to the treatment for FGN. This is a unique case of IgA-dominant glomerulonephritis with DNAJB9-negative fibrillar polytypic immunoglobulin deposits in the subepithelium, unlike previous DNAJB9-negative cases. Thus, DNAJB9-negative cases diagnosed based on accurate electron microscopic evaluation must be gathered, and LMD and LC-MS/MS must be used to analyze the organized fibrillar deposits to reveal the disease entity.
Assuntos
Glomerulonefrite por IGA , Glomerulonefrite , Feminino , Humanos , Idoso , Vermelho Congo , Cromatografia Líquida , Espectrometria de Massas em Tandem , Glomerulonefrite/patologia , Glomerulonefrite por IGA/diagnóstico , Imunoglobulina G , Imunoglobulina A , Proteínas de Membrana/análise , Chaperonas Moleculares/análise , Proteínas de Choque Térmico HSP40/análiseRESUMO
Introduction: Immunoglobulin A (IgA) nephropathy is a disease that presents with urinary symptoms such as glomerular hematuria and urinary protein positivity, with predominant deposition of IgA in the mesangial region of the glomerulus. Corticosteroids are mainly used for treatment; however, infection is a serious adverse event, and evidence regarding therapeutic efficacy is insufficient, thus new treatments are strongly desired. Mesenchymal stem cells (MSCs) contribute to the amelioration of inflammation and recovery of organ function in inflammatory environments by converting the character of leukocytes from inflammatory to anti-inflammatory and inducing the proliferation and differentiation of organ component cells, respectively. These properties of MSCs have led to their clinical application in various inflammatory diseases, but this study is the first clinical trial of MSCs for refractory glomerulonephritis in the world. This study is registered and assigned the number, jRCT2043200002 and NCT04342325. Methods: This will be a phase 1, open-label, multiple-center, dose-escalation study of adult patients with refractory IgA nephropathy resistant to or difficult to treat with existing therapies. ADR-001 will be administered intravenously to from three to six patients at a dose of 1 × 108 cells once in the first cohort and to six patients twice at 2-week intervals in the second cohort, and observation will continue until 52 weeks. The primary endpoint will be the evaluation of adverse events up to 6 weeks after the start of ADR-001 administration. Secondary endpoints will be the respective percentages of patients with adverse events, clinical remission, partial remission, remission of urine protein, remission of hematuria, time to remission, changes in urine protein, hematuria, and estimated glomerular filtration rate. Results: Following the administration of ADR-001 to patients with IgA nephropathy, the respective percentages of patients with adverse events, asymptomatic pulmonary emboli, clinical remission, partial remission, urine protein remission, hematuria remission, their time to remission, changes in urine protein, hematuria, and glomerular filtration rate will be determined. Conclusion: This study will evaluate the safety and tolerability of ADR-001 and confirm its therapeutic efficacy in adult patients with refractory IgA nephropathy.
RESUMO
Monocarboxylates, such as lactate and pyruvate, are precursors for biosynthetic pathways, including those for glucose, lipids, and amino acids via the tricarboxylic acid (TCA) cycle and adjacent metabolic networks. The transportation of monocarboxylates across the cellular membrane is performed primarily by monocarboxylate transporters (MCTs), the membrane localization and stabilization of which are facilitated by the transmembrane protein basigin (BSG). Here, we demonstrate that the MCT/BSG axis sits at a crucial intersection of cellular metabolism. Abolishment of MCT1 in the plasma membrane was achieved by Bsg depletion, which led to gluconeogenesis impairment via preventing the influx of lactate and pyruvate into the cell, consequently suppressing the TCA cycle. This net anaplerosis suppression was compensated in part by the increased utilization of glycogenic amino acids (e.g., alanine and glutamine) into the TCA cycle and by activated ketogenesis through fatty acid ß-oxidation. Complementary to these observations, hyperglycemia and hepatic steatosis induced by a high-fat diet were ameliorated in Bsg-deficient mice. Furthermore, Bsg deficiency significantly improved insulin resistance induced by a high-fat diet. Taken together, the plasma membrane-selective modulation of lactate and pyruvate transport through BSG inhibition could potentiate metabolic flexibility to treat metabolic diseases.
Assuntos
Basigina/deficiência , Fígado Gorduroso/genética , Resistência à Insulina/fisiologia , Animais , Humanos , CamundongosRESUMO
Lupus nephritis (LN) is a serious complication occurring in 50% of patients with systemic lupus erythematosus (SLE) for which there is a lack of biomarkers, a lack of specific medications, and a lack of a clear understanding of its pathogenesis. The expression of calcium/calmodulin kinase IV (CaMK4) is increased in podocytes of patients with LN and lupus-prone mice, and its podocyte-targeted inhibition averts the development of nephritis in mice. Nephrin is a key podocyte molecule essential for the maintenance of the glomerular slit diaphragm. Here, we show that the presence of fucose on N-glycans of IgG induces, whereas the presence of galactose ameliorates, podocyte injury through CaMK4 expression. Mechanistically, CaMK4 phosphorylates NF-κB, upregulates the transcriptional repressor SNAIL, and limits the expression of nephrin. In addition, we demonstrate that increased expression of CaMK4 in biopsy specimens and in urine podocytes from people with LN is linked to active kidney disease. Our data shed light on the role of IgG glycosylation in the development of podocyte injury and propose the development of "liquid kidney biopsy" approaches to diagnose LN.
Assuntos
Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina/metabolismo , Imunoglobulina G/metabolismo , Nefrite Lúpica/metabolismo , Podócitos/metabolismo , Adolescente , Adulto , Idoso , Animais , Linhagem Celular , Feminino , Fucose/metabolismo , Galactose/metabolismo , Humanos , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Pessoa de Meia-Idade , NF-kappa B/metabolismo , Fatores de Transcrição da Família Snail/metabolismo , Adulto JovemRESUMO
Transplant glomerulopathy (TG) is a major cause of late allograft loss. Increased urine podocin/creatinine ratio in TG signifies accelerated podocyte loss. The mechanisms that lead to podocyte injury in TG remain unclear. We report that IgG from kidney transplant recipients with TG, but not from those without TG, cause a reduction in the expression of nephrin, significant podocyte actin cytoskeleton, and motility changes. These changes are preceded by increased expression of calcium/calmodulin kinase IV (CAMK4). Mechanistically, we found that CAMK4 phosphorylates GSK3ß (glycogen synthase kinase 3 beta), activates the Wnt pathway and stabilizes the nephrin transcriptional repressor SNAIL. Silencing neonatal Fc Receptor (FcRn) or CAMK4 prevented the podocyte-damaging effects of IgG from patients with TG. Furthermore, we show that removal of N-linked glycosyl residues from these IgG did not interfere with its entry into the podocytes but eliminated its ability to upregulate CAMK4 and cause podocyte injury. The translational value of these findings is signified by the fact that CAMK4 is increased in podocytes of patients with TG but not in those without TG despite other forms of renal dysfunction. Our results offer novel considerations to limit podocyte injury in patients with kidney transplants, which may lead to eventual glomerular destabilization and transplant glomerulopathy.
Assuntos
Transplante de Rim , Podócitos , Cálcio , Proteínas Quinases Dependentes de Cálcio-Calmodulina , Humanos , Imunoglobulina G , Recém-Nascido , Transplante de Rim/efeitos adversosRESUMO
Hyaluronic acid (HA), a component of the extracellular matrix, is the ligand for CD44 and has been implicated in the pathogenesis of kidney inflammation in patients with systemic lupus erythematosus (SLE), but its direct role and mechanism of action have not been studied. Here we show that administration of hymecromone (4-Methylumbelliferone, 4-MU), an HA synthesis inhibitor, to lupus-prone mice suppressed dramatically lupus-related pathology. Interestingly, 4-MU stopped the appearance of disease when administered prior to its onset and inhibited the progression of disease when administered after its appearance. Inhibition of HA synthesis in vivo reduced tissue damage and the number of intrarenal lymphoid cell infiltrates including double negative CD3+CD4-CD8- T cells which are known to be involved in the pathogenesis of SLE. Exposure of human peripheral blood mononuclear cells to HA in vitro increased the generation of CD3+CD4-CD8- T cells through a mechanism involving Rho-associated kinase. Our results signify the importance of the HA-rich tissue microenvironment in the activation of lymphocytes to cause tissue damage in SLE and suggest the consideration of inhibition of HA synthesis to treat patients.
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Ácido Hialurônico/biossíntese , Rim/patologia , Leucócitos Mononucleares/metabolismo , Lúpus Eritematoso Sistêmico/metabolismo , Pele/patologia , Adulto , Animais , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/metabolismo , Feminino , Humanos , Receptores de Hialuronatos/metabolismo , Ácido Hialurônico/antagonistas & inibidores , Himecromona/farmacologia , Indicadores e Reagentes/farmacologia , Rim/efeitos dos fármacos , Rim/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Lúpus Eritematoso Sistêmico/patologia , Lúpus Eritematoso Sistêmico/prevenção & controle , Masculino , Camundongos Endogâmicos MRL lpr , Pele/efeitos dos fármacos , Quinases Associadas a rho/metabolismoRESUMO
Th1 and Th17 are important in the pathogenesis of autoimmune diseases and they depend on glycolysis as a source of energy. T cell antigen receptor signaling phosphorylates a serine/threonine kinase, calcium/calmodulin-dependent protein kinase IV (CaMK4), and promotes glycolysis. Based on these findings we hypothesized that CaMK4 promotes glycolysis. Camk4-deficient CD4+ T cells and cells treated with a CaMK4 inhibitor had less glycolysis compared with their counterparts. Pull-down of CaMK4 and mass spectrometry identified pyruvate kinase muscle isozyme (PKM), the final rate-limiting enzyme in glycolysis, as a binding partner. Coimmunoprecipitation and Western blotting showed that CaMK4 interacts directly with PKM2. Camk4-deficient CD4+ T cells displayed decreased pyruvate kinase activity. Silencing or pharmacological inhibition of PKM2 reduced glycolysis and in vitro differentiation to Th1 and Th17 cells, while PKM2 overexpression restored Th17 cell differentiation. Treatment with a PKM2 inhibitor ameliorated experimental autoimmune encephalomyelitis and CD4+ T cells treated with PKM2 inhibitor or Pkm2-shRNA caused limited disease activity in an adoptive cell transfer model of experimental autoimmune encephalomyelitis. Our data demonstrate that CaMK4 binds to PKM2 and promotes its activity, which is requisite for Th1 and Th17 differentiation in vitro and in vivo. PKM2 represents a therapeutic target for T cell-dependent autoimmune diseases.
Assuntos
Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina/metabolismo , Proteínas de Transporte/metabolismo , Linfopoese , Proteínas de Membrana/metabolismo , Células Th1/enzimologia , Células Th17/enzimologia , Hormônios Tireóideos/metabolismo , Animais , Proteínas de Transporte/antagonistas & inibidores , Dimetil Sulfóxido/farmacologia , Encefalomielite Autoimune Experimental/enzimologia , Encefalomielite Autoimune Experimental/imunologia , Inibidores Enzimáticos/farmacologia , Glicólise , Proteínas de Membrana/antagonistas & inibidores , Camundongos Endogâmicos C57BL , Naftoquinonas/farmacologia , Células Th1/efeitos dos fármacos , Células Th1/fisiologia , Células Th17/efeitos dos fármacos , Células Th17/fisiologia , Proteínas de Ligação a Hormônio da TireoideRESUMO
OBJECTIVE: Glutaminase 1 (Gls1) is the first enzyme in glutaminolysis. The selective Gls1 inhibitor bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES) suppresses Th17 development and ameliorates experimental autoimmune encephalomyelitis (EAE). The present study was undertaken to investigate whether inhibition of glutaminolysis is beneficial for the treatment of systemic lupus erythematosus (SLE), and the involved mechanisms. METHODS: MRL/lpr mice were treated with BPTES or vehicle control, and disease activity was examined. Then naive CD4+ T cells from patients with SLE were cultured under Th17-polarizing conditions with BPTES or vehicle. Furthermore, using newly generated Gls1 conditional-knockout mice, in vitro Th17 differentiation was examined, and EAE was induced in the mice. Glutaminolysis and glycolysis were measured with an extracellular flux analyzer. The expression of hypoxia-inducible factor 1α (HIF-1α) was examined by Western blotting. RESULTS: Treatment of MRL/lpr mice with BPTES improved autoimmune pathology in a Th17-dependent manner. T cells from patients with SLE treated with BPTES displayed decreased Th17 differentiation (P < 0.05). Using the conditional-knockout mice, we demonstrated that both in vitro Th17 differentiation (P < 0.05) and the development of EAE were dependent on Gls1. Gls1 inhibition reduced glycolysis and the expression of HIF-1α protein, which induces glycolysis. CONCLUSION: We demonstrated that inhibition of glutaminolysis represents a potential new treatment strategy for patients with SLE and Th17-related autoimmune diseases. Mechanistically, we have shown that inhibition of glutaminolysis affects the glycolysis pathway by reducing HIF-1α protein in Th17 cells.
Assuntos
Linfócitos T CD4-Positivos/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Encefalomielite Autoimune Experimental/genética , Glutaminase/antagonistas & inibidores , Glicólise/efeitos dos fármacos , Lúpus Eritematoso Sistêmico/genética , Sulfetos/farmacologia , Tiadiazóis/farmacologia , Adulto , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Diferenciação Celular/genética , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/metabolismo , Feminino , Glutaminase/genética , Glutamina/efeitos dos fármacos , Glutamina/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/efeitos dos fármacos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Técnicas In Vitro , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/metabolismo , Camundongos , Camundongos Endogâmicos MRL lpr , Camundongos Knockout , Pessoa de Meia-Idade , Células Th17/efeitos dos fármacos , Células Th17/imunologia , Células Th17/metabolismoRESUMO
Podocytes, which are susceptible to injury by various stimuli and stress, are critical regulators of proteinuric kidney diseases, regardless of the primary disease and pathogenesis. We further confirmed a significant correlation between urinary CD147/basigin (Bsg) levels and proteinuria in patients with focal segmental glomerulosclerosis. However, the molecular mechanism of podocyte injury involving Bsg is not fully understood. Here, the involvement of Bsg in the pathogenesis of podocyte injury was elucidated. Healthy podocytes rarely express Bsg protein. In two independent mouse models, including adriamycin-induced nephropathy and Nω-nitro-l-arginine methyl ester (l-name)-induced endothelial dysfunction, Bsg induction in injured podocytes caused podocyte effacement, which led to development of proteinuria. Bsg silencing in cultured podocytes exposed to transforming growth factor-ß suppressed focal adhesion rearrangement and cellular motility via the activation of ß1 integrin-focal adhesion kinase-matrix metallopeptidase signaling. In addition, induction of vascular endothelial growth factor and endothelin-1, which are implicated in podocyte-to-endothelial cross-communication, was lower in the supernatants of cultured Bsg-silenced podocytes stimulated with transforming growth factor-ß. In this setting, Bsg may be involved in a physiological positive feedback loop that accelerates podocyte cell motility and depolarization. The current study thus suggests that Bsg silencing via suppression of ß1 integrin-focal adhesion kinase-matrix metallopeptidase signaling may be an attractive therapeutic strategy for the maintenance of podocytes in patients with proteinuric kidney diseases.
Assuntos
Basigina/deficiência , Quinase 1 de Adesão Focal/metabolismo , Glomerulosclerose Segmentar e Focal/metabolismo , Podócitos/metabolismo , Proteinúria/metabolismo , Transdução de Sinais , Adulto , Animais , Modelos Animais de Doenças , Doxorrubicina/efeitos adversos , Doxorrubicina/farmacologia , Feminino , Glomerulosclerose Segmentar e Focal/induzido quimicamente , Glomerulosclerose Segmentar e Focal/patologia , Humanos , Masculino , Camundongos , Camundongos Knockout , NG-Nitroarginina Metil Éster/farmacologia , Podócitos/patologia , Proteinúria/induzido quimicamente , Proteinúria/patologiaRESUMO
Th17 cells favor glycolytic metabolism, and pyruvate dehydrogenase (PDH) is the key bifurcation enzyme, which in its active dephosphorylated form advances the oxidative phosphorylation from glycolytic pathway. The transcriptional factor, inducible cAMP early repressor/cAMP response element modulator (ICER/CREM), has been shown to be induced in Th17 cells and to be overexpressed in CD4+ T cells from the patients with systemic lupus erythematosus (SLE). We found that glycolysis and lactate production in in vitro Th17-polarized T cells was reduced and that the expression of pyruvate dehydrogenase phosphatase catalytic subunit 2 (PDP2), an enzyme that converts the inactive PDH to its active form, and PDH enzyme activity were increased in Th17 cells from ICER/CREM-deficient animals. ICER was found to bind to the Pdp2 promoter and suppress its expression. Furthermore, forced expression of PDP2 in CD4+ cells reduced the in vitro Th17 differentiation, whereas shRNA-based suppression of PDP2 expression increased in vitro Th17 differentiation and augmented experimental autoimmune encephalomyelitis. At the translational level, PDP2 expression was decreased in memory Th17 cells from patients with SLE and forced expression of PDP2 in CD4+ T cells from lupus-prone MRL/lpr mice and patients with SLE suppressed Th17 differentiation. These data demonstrate the direct control of energy production during Th17 differentiation in health and disease by the transcription factor ICER/CREM at the PDH metabolism bifurcation level.
Assuntos
Diferenciação Celular , Regulação Enzimológica da Expressão Gênica , Fosfoproteínas Fosfatases/biossíntese , Elementos de Resposta , Células Th17/enzimologia , Animais , Domínio Catalítico , Modulador de Elemento de Resposta do AMP Cíclico/genética , Modulador de Elemento de Resposta do AMP Cíclico/imunologia , Modulador de Elemento de Resposta do AMP Cíclico/metabolismo , Encefalomielite Autoimune Experimental/enzimologia , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Feminino , Humanos , Lúpus Eritematoso Sistêmico/enzimologia , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/patologia , Masculino , Camundongos , Camundongos Knockout , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/imunologia , Células Th17/imunologia , Células Th17/patologiaRESUMO
Podocyte malfunction occurs in autoimmune and nonautoimmune kidney disease. Calcium signaling is essential for podocyte injury, but the role of Ca2+/calmodulin-dependent kinase (CaMK) signaling in podocytes has not been fully explored. We report that podocytes from patients with lupus nephritis and focal segmental glomerulosclerosis and lupus-prone and lipopolysaccharide- or adriamycin-treated mice display increased expression of CaMK IV (CaMK4), but not CaMK2. Mechanistically, CaMK4 modulated podocyte motility by altering the expression of the GTPases Rac1 and RhoA and suppressed the expression of nephrin, synaptopodin, and actin fibers in podocytes. In addition, it phosphorylated the scaffold protein 14-3-3ß, which resulted in the release and degradation of synaptopodin. Targeted delivery of a CaMK4 inhibitor to podocytes preserved their ultrastructure, averted immune complex deposition and crescent formation, and suppressed proteinuria in lupus-prone mice and proteinuria in mice exposed to lipopolysaccharide-induced podocyte injury by preserving nephrin/synaptopodin expression. In animals exposed to adriamycin, podocyte-specific delivery of a CaMK4 inhibitor prevented and reversed podocyte injury and renal disease. We conclude that CaMK4 is pivotal in immune and nonimmune podocyte injury and that its targeted cell-specific inhibition preserves podocyte structure and function and should have therapeutic value in lupus nephritis and podocytopathies, including focal segmental glomerulosclerosis.
Assuntos
Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina/metabolismo , Glomerulosclerose Segmentar e Focal/enzimologia , Glomérulos Renais/enzimologia , Nefrite Lúpica/enzimologia , Transdução de Sinais , Animais , Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina/imunologia , Linhagem Celular Transformada , Feminino , Glomerulosclerose Segmentar e Focal/imunologia , Glomerulosclerose Segmentar e Focal/patologia , Humanos , Glomérulos Renais/imunologia , Glomérulos Renais/patologia , Nefrite Lúpica/imunologia , Nefrite Lúpica/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos MRL lpr , Camundongos Knockout , Proteinúria/enzimologia , Proteinúria/imunologia , Proteinúria/patologiaRESUMO
Glutaminolysis is a well-known source of energy for effector T cells but its contribution to each T cell subset and the mechanisms which are responsible for the control of involved metabolic enzymes are not fully understood. We report that Th17 but not Th1, Th2, or Treg cell induction in vitro depends on glutaminolysis and the up-regulation of glutaminase 1 (Gls1), the first enzyme in the glutaminolysis pathway. Both pharmacological and siRNA-based selective inhibition of Gls1 reduced in vitro Th17 differentiation and reduced the CD3/TCR-mediated increase of the mammalian target of rapamycin complex 1 activity. Treatment of mice with a Gls1 inhibitor ameliorated experimental autoimmune encephalomyelitis. Furthermore, RAG1-deficient mice that received Gls1-shRNA-transfected 2D2 T cells had reduced experimental autoimmune encephalomyelitis scores compared with those that received control-shRNA-treated cells. Next we found that T cells deficient in inducible cAMP early repressor (ICER), a transcriptional factor known to promote Th17 differentiation, display reduced activity of oxidative phosphorylation rates in the presence of glutamine and reduced Gls1 expression, both of which could be restored by ICER overexpression. Finally, we demonstrate that ICER binds to the gls1 promoter directly and increases its activity. These findings demonstrate the importance of glutaminolysis in the generation of Th17 and the direct control of Gls1 activity by the IL-17-promoting transcription factor ICER. Pharmaceutical modulation of the glutaminolysis pathway should be considered to control Th17-mediated pathology.
Assuntos
Modulador de Elemento de Resposta do AMP Cíclico , Glutaminase , Células Th17 , Animais , Autoimunidade , Modulador de Elemento de Resposta do AMP Cíclico/genética , Modulador de Elemento de Resposta do AMP Cíclico/metabolismo , Encefalomielite Autoimune Experimental/imunologia , Glutaminase/antagonistas & inibidores , Glutaminase/metabolismo , Glutamina/metabolismo , Camundongos , Camundongos Transgênicos , Células Th17/citologia , Células Th17/imunologia , Células Th17/metabolismoRESUMO
BACKGROUND: Precise understanding of kidney disease activity is needed to design therapeutic strategies. CD147/basigin is involved in the pathogenesis of acute kidney injury and renal fibrosis through inflammatory cell infiltration. The present study examined the clinical relevance of CD147 in biopsy-proven kidney diseases that lead to the progression of chronic kidney disease. METHODS: Kidney biopsy specimens and plasma and urine samples were obtained from patients with kidney diseases, including IgA nephropathy (IgAN), Henoch-Schönlein purpura nephritis (HSPN), diabetic kidney disease (DKD), focal segmental glomerulosclerosis (FSGS), and membranous nephropathy (MN), who underwent renal biopsy between 2011 and 2014. Plasma and urinary CD147 levels were measured and evaluated for their ability to reflect histological features. Disease activity of IgAN tissues was evaluated according to the Oxford classification and the Japanese histological grading system. RESULTS: In biopsy tissues, CD147 induction was detected in injured lesions representing renal inflammation. Plasma CD147 values correlated with eGFR in patients with inflammation-related kidney diseases such as IgAN, HSPN, and DKD. Particularly in IgAN patients, plasma CD147 levels were correlated with injured regions comprising more than 50% of glomeruli or with tubular atrophy/interstitial injury in biopsy tissues. Proteinuria showed a closer correlation with urinary values of CD147 and L-FABP. Of note, plasma and urinary CD147 levels showed a strong correlation with eGFR or proteinuria, respectively, only in DKD patients. CONCLUSION: Evaluation of plasma and urinary CD147 levels might provide key insights for the understanding of the activity of various kidney diseases.
Assuntos
Basigina/sangue , Nefropatias/sangue , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biópsia , Estudos Transversais , Feminino , Glomerulonefrite por IGA , Humanos , Rim , Nefropatias/diagnóstico , Nefropatias/patologia , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
Activated T cells play crucial roles in the pathogenesis of autoimmune diseases, including lupus nephritis (LN). The activation of calcineurin/nuclear factor of activated T cells (NFAT) and STAT4 signaling is essential for T cells to perform various effector functions. Here, we identified the growth factor midkine (MK; gene name, Mdk) as a novel regulator in the pathogenesis of 2,6,10,14-tetramethylpentadecane-induced LN via activation of NFAT and IL-12/STAT4 signaling. Wild-type (Mdk+/+) mice showed more severe glomerular injury than MK-deficient (Mdk-/-) mice, as demonstrated by mesangial hypercellularity and matrix expansion, and glomerular capillary loops with immune-complex deposition. Compared with Mdk-/- mice, the frequency of splenic CD69+ T cells and T helper (Th) 1 cells, but not of regulatory T cells, was augmented in Mdk+/+ mice in proportion to LN disease activity, and was accompanied by skewed cytokine production. MK expression was also enhanced in activated CD4+ T cells in vivo and in vitro. MK induced activated CD4+ T cells expressing CD69 through nuclear activation of NFAT transcription and selectively increased in vitro differentiation of naive CD4+ T cells into Th1 cells by promoting IL-12/STAT4 signaling. These results suggest that MK serves an indispensable role in the NFAT-regulated activation of CD4+ T cells and Th1 cell differentiation, eventually leading to the exacerbation of LN.
Assuntos
Diferenciação Celular , Citocinas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Nefrite Lúpica/patologia , Ativação Linfocitária , Fatores de Transcrição NFATC/metabolismo , Transdução de Sinais , Células Th1/citologia , Animais , Citocinas/deficiência , Inflamação/patologia , Glomérulos Renais/lesões , Glomérulos Renais/patologia , Glomérulos Renais/ultraestrutura , Nefrite Lúpica/imunologia , Camundongos , Midkina , Modelos Biológicos , Baço/patologia , Linfócitos T/imunologiaRESUMO
OBJECTIVE: Interleukin-17 (IL-17)-producing T cells (Th17 cells) play critical roles in the pathogenesis of immune-related diseases, including systemic lupus erythematosus. However, the fundamental mechanism regulating Th17 cell differentiation is not fully understood. Recently, we demonstrated that plasma levels of CD147/basigin (Bsg) in patients with lupus nephritis (LN) were closely associated with disease activity. but the molecular mechanism involving Bsg has been elusive. Here, we addressed the role of Bsg in the pathogenesis of LN. METHODS: Injections of pristane (2,6,10,14-tetramethylpentadecane [TMPD]) were administered to Bsg(-/-) or Bsg(+/+) mice to induce LN. The mice were killed 6 months after being injected, for histologic and biochemical analyses of the kidneys and spleens. RESULTS: Pristane induced LN more strikingly in Bsg(-/-) mice than in Bsg(+/+) mice, even though humoral autoimmunity was similarly increased in both genotypes. The increased number of Th17, but not Th1, Treg cells, was augmented in Bsg(-/-) mice. The expression of IL-17 was also increased in the kidneys of Bsg(-/-) mice, in proportion to LN disease activity. Furthermore, treatment with anti-IL-17 antibody reduced LN disease activity in Bsg(-/-) mice. Complementary to these phenotypes of Bsg(-/-) mice, Bsg expression was enhanced in activated CD4+ T cells in vivo and in vitro. Bsg deficiency selectively augmented in vitro differentiation of naive CD4+ T cells to Th17 cells and STAT-3 phosphorylation during this differentiation. Moreover, STAT-3 phosphorylation was suppressed by crosslinking of Bsg with its antibody. CONCLUSION: Bsg plays an indispensable role in Th17 cell differentiation as a negative regulator by suppressing the IL-6/STAT-3 pathway.
Assuntos
Basigina/imunologia , Diferenciação Celular/imunologia , Interleucina-6/imunologia , Nefrite Lúpica/imunologia , Fator de Transcrição STAT3/imunologia , Células Th17/imunologia , Animais , Basigina/genética , Interleucina-6/metabolismo , Rim/metabolismo , Rim/patologia , Nefrite Lúpica/genética , Nefrite Lúpica/patologia , Camundongos , Camundongos Knockout , Transdução de Sinais , Células Th17/metabolismoRESUMO
The effects of endothelium-derived hyperpolarizing factors have been attributed to cytochrome P450-derived epoxyeicosatrienoic acids (EETs), but the regulation and role of EETs in endothelial dysfunction remain largely unexplored. Hypertension is a primary risk factor for renal dysfunction, which is frequently accompanied by various systemic diseases induced by endothelial dysfunction in the microcirculation. We previously reported that the endothelial growth factor midkine (MK) enhances hypertension in a model of CKD. Here, we investigated the hypothesis that MK regulates EET activity and thereby BP. MK gene-deleted mice were resistant to hypertension and developed less glomerulosclerosis and proteinuria after administration of a nitric oxide synthase (NOS) inhibitor in the setting of uninephrectomy. The hypertension observed in uninephrectomized wild-type mice after NOS inhibition was ameliorated by anti-MK antibody. MK-deficient mice produced higher amounts of EETs, and EETs dominantly regulated BP in these mice. Furthermore, MK administration to MK-deficient mice recapitulated the BP control observed in wild-type mice. EETs also dominantly regulated renal blood flow, which may influence renal function, in MK-deficient mice. Taken together, these results suggest that the MK/EET pathway is physiologically engaged in BP control and could be a target for the treatment of hypertension complicated by endothelial dysfunction.
Assuntos
Pressão Sanguínea , Citocinas/metabolismo , Eicosanoides/metabolismo , Endotélio Vascular/metabolismo , Compostos de Epóxi/metabolismo , Animais , Fatores Biológicos/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Hipertensão/etiologia , Masculino , Camundongos , Midkina , Óxido Nítrico Sintase/antagonistas & inibidores , Distribuição Aleatória , Circulação Renal , Insuficiência Renal/etiologia , Sistema Renina-Angiotensina , Sistema Nervoso Simpático/metabolismoRESUMO
The glycosylated transmembrane protein CD147/basigin, also known as extracellular matrix metalloproteinase (MMP) inducer (EMMPRIN), contributes to cell survival, migration and cancer invasion. In normal kidneys, high expression of CD147 is detected only in the basolateral side of tubular epithelial cells (TECs). The pathophysiological roles of CD147 in the kidneys are diverse, ranging from involvement in the occurrence of acute kidney injury (AKI) that is frequently accompanied by ischemia, inflammation and a loss of self-tolerance to the progression of chronic kidney disease (CKD) that is caused by an imbalance in extracellular matrix protein turnover. In AKI induced by ischemia, it is the CD147 on neutrophils, rather than that on TECs, that coordinately participates in massive neutrophil recruitment via acting as a physiological ligand for E-selectin, which is specifically enhanced in the endothelium upon inflammatory stimulation. In the CKD that follows AKI, a molecular circuit involving CD147, MMPs and transforming growth factor-ß may be involved in the pathogenesis of progressive fibrosis through hyaluronan production and macrophage infiltration. Whereas CD147 thus plays deleterious roles in ischemic and fibrotic kidney injuries, CD147 expression on lymphocytes might decrease the disease activity of lupus nephritis (LN) by functioning as a potential negative regulator of the extraordinary proliferation of lymphocytes that occurs in this disease. In line with these basic studies, our clinical data indicate the potential of plasma CD147 to function as a critical biomarker for both ischemic AKI and LN. CD147 is also involved in crosstalk between the kidneys and distant organs, which may be mediated by chemotactic cytokines that are derived from circulating inflammatory cells and damaged organs. Disruption of such a vicious chain reaction involving CD147 would therefore be required in order to overcome kidney diseases. Multidisciplinary research regarding CD147 functions may open a new avenue for targeting therapeutics for kidney diseases.