RESUMEN
BACKGROUND: SH2B3 (SH2B adaptor protein 3) is an adaptor protein that negatively regulates cytokine signaling and cell proliferation. A common missense single nucleotide polymorphism in SH2B3 (rs3184504) results in substitution of tryptophan (Trp) for arginine (Arg) at amino acid 262 and is a top association signal for hypertension in human genome-wide association studies. Whether this variant is causal for hypertension, and if so, the mechanism by which it impacts pathogenesis is unknown. METHODS: We used CRISPR-Cas9 technology to create mice homozygous for the major (Arg/Arg) and minor (Trp/Trp) alleles of this SH2B3 polymorphism. Mice underwent angiotensin II (Ang II) infusion to evaluate differences in blood pressure (BP) elevation and end-organ damage including albuminuria and renal fibrosis. Cytokine production and Stat4 phosphorylation was also assessed in Arg/Arg and Trp/Trp T cells. RESULTS: Trp/Trp mice exhibit 10 mmHg higher systolic BP during chronic Ang II infusion compared to Arg/Arg controls. Renal injury and perivascular fibrosis are exacerbated in Trp/Trp mice compared to Arg/Arg controls following Ang II infusion. Renal and ex vivo stimulated splenic CD8+ T cells from Ang II-infused Trp/Trp mice produce significantly more interferon gamma (IFNg) compared to Arg/Arg controls. Interleukin-12 (IL-12)-induced IFNg production is greater in Trp/Trp compared to Arg/Arg CD8+ T cells. In addition, IL-12 enhances Stat4 phosphorylation to a greater degree in Trp/Trp compared to Arg/Arg CD8+ T cells, suggesting that Trp-encoding SH2B3 exhibits less negative regulation of IL-12 signaling to promote IFNg production. Finally, we demonstrated that a multi-SNP model genetically predicting increased SH2B3 expression in lymphocytes is inversely associated with hypertension and hypertensive chronic kidney disease in humans.. CONCLUSIONS: Taken together, these results suggest that the Trp encoding allele of rs3184504 is causal for BP elevation and renal dysfunction, in part through loss of SH2B3-mediated repression of T cell IL-12 signaling leading to enhanced IFNg production.
Asunto(s)
Hipertensión Renal , Hipertensión , Proteínas Adaptadoras Transductoras de Señales , Angiotensina II/metabolismo , Angiotensina II/toxicidad , Animales , Arginina/efectos adversos , Arginina/metabolismo , Linfocitos T CD8-positivos/metabolismo , Fibrosis , Estudio de Asociación del Genoma Completo , Humanos , Hipertensión/metabolismo , Hipertensión Renal/metabolismo , Interferón gamma/metabolismo , Interleucina-12/efectos adversos , Interleucina-12/metabolismo , Riñón/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Polimorfismo de Nucleótido Simple , TriptófanoRESUMEN
The NOD, LRR, and pyrin domain-containing 3 (NLRP3) protein has been established as a central component of the inflammasome and regulates the inflammatory response to a myriad of environmental, microbial, and endogenous danger stimuli. Assembly of the NLRP3 inflammasome results in the cleavage and activation of caspase-1, in turn causing release of the pro-inflammatory interleukins 1-beta and 18. This activation response, while crucial to coordinated innate immune defense, can be aberrantly activated by the likes of cell-free DNA, and cause significant autoimmune pathology. Complications of autoimmunity induced by aberrant NLRP3 inflammasome activation have a great degree of mechanistic crossover with alloimmune injury in solid organ transplant, and stratagems to neutralize NLRP3 inflammasome activation may prove beneficial in solid organ transplant management. This article reviews NLRP3 inflammasome biology and the pathology associated with its hyperactivation, as well as the connections between NLRP3 inflammasome activation and allograft homeostasis.
Asunto(s)
Inflamasomas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Animales , Autoinmunidad , ADN/inmunología , Humanos , Neutrófilos/inmunología , Neutrófilos/metabolismo , Neutrófilos/patología , Especificidad de Órganos/inmunología , Trasplante de Órganos , Procesamiento Proteico-PostraduccionalRESUMEN
The With-No-Lysine (K) (WNK) kinases play a critical role in blood pressure regulation and body fluid and electrolyte homeostasis. Herein, we introduce the first orally bioavailable pan-WNK-kinase inhibitor, WNK463, that exploits unique structural features of the WNK kinases for both affinity and kinase selectivity. In rodent models of hypertension, WNK463 affects blood pressure and body fluid and electro-lyte homeostasis, consistent with WNK-kinase-associated physiology and pathophysiology.
Asunto(s)
Sistema Cardiovascular/efectos de los fármacos , Imidazoles/farmacología , Riñón/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Pirrolidinas/farmacología , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Sistema Cardiovascular/metabolismo , Humanos , Imidazoles/química , Riñón/metabolismo , Pruebas de Función Renal , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Inhibidores de Proteínas Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Pirrolidinas/química , Ratas , Ratas Sprague-Dawley , Bibliotecas de Moléculas Pequeñas/químicaRESUMEN
RATIONALE: Accumulating evidence supports a role of adaptive immunity and particularly T cells in the pathogenesis of hypertension. Formation of memory T cells, which requires the costimulatory molecule CD70 on antigen-presenting cells, is a cardinal feature of adaptive immunity. OBJECTIVE: To test the hypothesis that CD70 and immunologic memory contribute to the blood pressure elevation and renal dysfunction mediated by repeated hypertensive challenges. METHODS AND RESULTS: We imposed repeated hypertensive challenges using either N(ω)-nitro-L-arginine methyl ester hydrochloride (L-NAME)/high salt or repeated angiotensin II stimulation in mice. During these challenges effector memory T cells (T(EM)) accumulated in the kidney and bone marrow. In the L-NAME/high-salt model, memory T cells of the kidney were predominant sources of interferon-γ and interleukin-17A, known to contribute to hypertension. L-NAME/high salt increased macrophage and dendritic cell surface expression of CD70 by 3- to 5-fold. Mice lacking CD70 did not accumulate T(EM) cells and did not develop hypertension to either high salt or the second angiotensin II challenge and were protected against renal damage. Bone marrow-residing T(EM) cells proliferated and redistributed to the kidney in response to repeated salt feeding. Adoptively transferred T(EM) cells from hypertensive mice homed to the bone marrow and spleen and expanded on salt feeding of the recipient mice. CONCLUSIONS: Our findings illustrate a previously undefined role of CD70 and long-lived T(EM) cells in the development of blood pressure elevation and end-organ damage that occur on delayed exposure to mild hypertensive stimuli. Interventions to prevent repeated hypertensive surges could attenuate formation of hypertension-specific T(EM) cells.
Asunto(s)
Presión Sanguínea/fisiología , Ligando CD27/deficiencia , Hipertensión/metabolismo , Enfermedades Renales/metabolismo , Cloruro de Sodio Dietético/efectos adversos , Animales , Presión Sanguínea/efectos de los fármacos , Hipertensión/inducido químicamente , Mediadores de Inflamación/metabolismo , Enfermedades Renales/inducido químicamente , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , NG-Nitroarginina Metil Éster/toxicidad , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismoRESUMEN
PURPOSE OF REVIEW: Hypertension is a leading cause of cardiovascular and renal morbidity, and mortality. Genome-wide association studies identified a single-nucleotide polymorphism in the gene SH2B3 encoding the lymphocyte adaptor protein, LNK, but, until recently, little was known about how LNK contributes to hypertension. This review summarizes recent work highlighting a central role for LNK in inflammation and hypertension. RECENT FINDINGS: Using a systems biology approach that integrates genomic data with whole blood transcriptomic data and network modeling, LNK/SH2B3 was identified as a key driver gene for hypertension in humans. LNK is an intracellular adaptor protein expressed predominantly in hematopoietic and endothelial cells that negatively regulates cell proliferation and cytokine signaling. Genetic animal models with deletion or mutation of LNK revealed an important role for LNK in renal and vascular inflammation, glomerular injury, oxidative stress, interferon-γ production, and hypertension. Bone marrow transplantation experiments revealed that LNK in hematopoietic cells is primarily responsible for blood pressure regulation. SUMMARY: LNK/SH2B3 is a key driver gene for human hypertension, and alteration of LNK in animal models has a profound effect on inflammation and hypertension. Thus, LNK is a potential therapeutic target for this disease and its devastating consequences.
Asunto(s)
Hipertensión/genética , Inflamación/genética , Interferón gamma/metabolismo , Proteínas/genética , Proteínas Adaptadoras Transductoras de Señales , Animales , Presión Sanguínea , Células Dendríticas/metabolismo , Humanos , Hipertensión/complicaciones , Hipertensión/metabolismo , Inflamación/complicaciones , Inflamación/metabolismo , Interferón gamma/biosíntesis , Péptidos y Proteínas de Señalización Intracelular , Riñón/fisiopatología , Proteínas/metabolismoRESUMEN
BACKGROUND: In cardiac transplant recipients, the development of antibodies to the endothelial intermediate filament protein vimentin (antivimentin antibodies, AVA) has been associated with rejection and poor outcomes. However, the incidence of these antibodies prior to transplantation and their association with early rejection has not been investigated. METHODS: Pre-transplant serum was analyzed from 50 patients who underwent de novo cardiac transplant at Johns Hopkins Hospital from 2004 to 2012. Demographic, one-yr rejection, and survival data were obtained from the transplant database. RESULTS: The incidence of pre-transplant AVA was 34%. AVA-positive patients were younger (p = 0.03), and there was an a trend toward incidence in females (p = 0.08). Demographic data were similar among both groups. AVA positivity did not predict rejection in the first year post-transplant. There was no difference in rejection-free graft survival (53 vs. 52%, p = 0.85) at one yr. Similarly, there was no difference in graft survival at one yr (82 vs. 88%, p = 0.56) or graft survival at a median follow-up of 23 and 26 months, respectively (76 vs. 85%, p = 0.41). CONCLUSIONS: AVA is common in the cardiac pre-transplant population with a higher incidence in the young. The presence of detectable AVA did not correlate with early post-transplant rejection or graft survival.
Asunto(s)
Autoanticuerpos/sangre , Rechazo de Injerto/etiología , Supervivencia de Injerto , Cardiopatías/cirugía , Trasplante de Corazón , Complicaciones Posoperatorias , Vimentina/inmunología , Adulto , Baltimore/epidemiología , Femenino , Estudios de Seguimiento , Rechazo de Injerto/epidemiología , Rechazo de Injerto/mortalidad , Cardiopatías/complicaciones , Humanos , Incidencia , Masculino , Pronóstico , Estudios Retrospectivos , Factores de Riesgo , Tasa de SupervivenciaRESUMEN
HLA matching in solid organ transplant is performed with the aim of assessing immunologic compatibility in order to avoid hyperacute rejection and assess the risk of future rejection events. Molecular mismatch algorithms are intended to improve granularity in histocompatibility assessment and risk stratification. PIRCHE-II uses HLA genotyping to predict indirectly presented mismatched donor HLA peptides, though most clinical validation studies rely on imputing high resolution (HR) genotypes from low resolution (LR) typing data. We hypothesized that use of bona fide HR typing could overcome limitations in imputation, improving accuracy and predictive ability for donor-specific antibody development and acute rejection. We performed a retrospective analysis of adult and pediatric kidney transplant donor/recipient pairs (N = 419) with HR typing and compared the use of imputed LR genotyping verses HR genotyping for PIRCHE-II analysis and outcomes. Imputation success was highly dependent on the reference population used, as using historic Caucasian reference populations resulted in 10 % of pairs with unsuccessful imputation while multiethnic reference populations improved successful imputation with only 1 % unable to be imputed. Comparing PIRCHE-II analysis with HR and LR genotyping produced notably different results, with 20 % of patients discrepantly classified to immunologic risk groups. These data emphasize the importance of using multiethnic reference panels when performing imputation and indicate HR HLA genotyping has clinically meaningful benefit for PIRCHE-II analysis compared to imputed LR typing.
Asunto(s)
Genotipo , Rechazo de Injerto , Antígenos HLA , Prueba de Histocompatibilidad , Trasplante de Riñón , Humanos , Antígenos HLA/genética , Antígenos HLA/inmunología , Prueba de Histocompatibilidad/métodos , Rechazo de Injerto/genética , Rechazo de Injerto/inmunología , Estudios Retrospectivos , Adulto , Femenino , Masculino , Niño , Persona de Mediana Edad , Adolescente , Histocompatibilidad , Técnicas de Genotipaje/métodos , AlgoritmosRESUMEN
Evidence from nonhuman animal models demonstrates an important role for immune cells in hypertension, but immune cell changes in human hypertension are less clear. Using mass cytometry, we demonstrate novel and selective reductions in CCR10+ regulatory T cells (Tregs) and PD-1+CD57-CD8+ memory T cells. RNA sequencing reveals that CCR10+ Tregs exhibit gene expression changes consistent with enhanced immunosuppressive function. In addition, CITE-Seq demonstrates that PD-1+CD57-CD8+ memory T cells exhibit features of T-cell exhaustion. Taken together, these results provide novel evidence for decreases in anti-inflammatory and/or hypofunctional T-cell populations that may contribute to enhanced inflammation in human hypertension.
RESUMEN
BACKGROUND: Patients with end-stage kidney disease require complex and expensive medical management. Kidney transplantation remains the treatment of choice for end-stage kidney disease and is considered superior to all other modalities of renal replacement therapy or dialysis. However, access to kidney transplant is limited by critical supply and demand, making it extremely important to ensure longevity of transplanted kidneys. This is prevented through lifelong immunosuppression, with caution not to overly suppress the immune system, resulting in toxicity and harm. Transition of care to community nephrologists after initial kidney transplantation and monitoring at a transplant center is an important process to ensure delivery of effective and patient-centric care closer to home. Once transplanted, laborious surveillance of the immune system and monitoring for potential rejection and injury are undertaken through an armamentarium of screening modalities. Posttransplant surveillance for kidney function and injury remains key to follow-up care. While kidney function, quantified by estimated glomerular filtration rate and serum creatinine, and kidney injury, measured by proteinuria and hematuria, are standard biomarkers used to monitor injury and rejection posttransplant, they have recently been demonstrated to be inferior in performance to that of AlloSure (CareDx Inc, Brisbane, CA) circulating donor-derived, cell-free DNA (dd-cfDNA). OBJECTIVE: The outcomes and methods of monitoring renal transplant recipients posttransplant have remained stagnant over the past 15 years. The aim of this study is to consider intensive surveillance using AlloSure dd-cfDNA in an actively managed protocol, assessing whether it increases long-term allograft survival in kidney transplant recipients compared with current standard clinical care in community nephrology. METHODS: The study protocol will acquire data from a phase IV observational trial to assess a cohort of renal transplant patients managed using AlloSure dd-cfDNA and patient care managers versus 1000 propensity-matched historic controls using United Network for Organ Sharing U.S. Scientific Registry of Transplant Recipients data. Data will be managed in a centralized electronic data server. The primary outcome will be superior allograft survival, as a composite of return to dialysis, retransplant, death due to allograft failure, and death with a functional graft (infection, malignancy, and cardiovascular death). The secondary endpoints will assess improved kidney function through decline in estimated glomerular filtration rate and immune activity through development of donor-specific antibodies. RESULTS: The total sample is anticipated to be 3500 (2500 patients managed with AlloSure dd-cfDNA and 1000 propensity-matched controls). Active enrollment began in November 2020. CONCLUSIONS: Based on a significant literature base, we believe implementing the surveillance of dd-cfDNA in the kidney transplant population will have a positive impact on graft survival. Through early identification of rejection and facilitating timely intervention, prolongation of allograft survival versus those not managed by dd-cfDNA surveillance protocol should be superior. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): PRR1-10.2196/25941.
RESUMEN
AIMS: Elevated serum immunoglobulins have been associated with experimental and human hypertension for decades but whether immunoglobulins and B cells play a causal role in hypertension pathology is unclear. In this study, we sought to determine the role of B cells and high-affinity class-switched immunoglobulins on hypertension and hypertensive end-organ damage to determine if they might represent viable therapeutic targets for this disease. METHODS AND RESULTS: We purified serum immunoglobulin G (IgG) from mice exposed to vehicle or angiotensin (Ang) II to induce hypertension and adoptively transferred these to wild type (WT) recipient mice receiving a subpressor dose of Ang II. We found that transfer of IgG from hypertensive animals does not affect blood pressure, endothelial function, renal inflammation, albuminuria, or T cell-derived cytokine production compared with transfer of IgG from vehicle infused animals. As an alternative approach to investigate the role of high-affinity, class-switched immunoglobulins, we studied mice with genetic deletion of activation-induced deaminase (Aicda-/-). These mice have elevated levels of IgM but virtual absence of class-switched immunoglobulins such as IgG subclasses and IgA. Neither male nor female Aicda-/- mice were protected from Ang II-induced hypertension and renal/vascular damage. To determine if IgM or non-immunoglobulin-dependent innate functions of B cells play a role in hypertension, we studied mice with severe global B-cell deficiency due to deletion of the membrane exon of the IgM heavy chain (µMT-/-). µMT-/- mice were also not protected from hypertension or end-organ damage induced by Ang II infusion or deoxycorticosterone acetate-salt treatment. CONCLUSIONS: These results suggest that B cells and serum immunoglobulins do not play a causal role in hypertension pathology.
Asunto(s)
Presión Sanguínea/inmunología , Hipertensión/inmunología , Cambio de Clase de Inmunoglobulina , Inmunoglobulina G/inmunología , Células B de Memoria/inmunología , Angiotensina II , Animales , Afinidad de Anticuerpos , Aorta/inmunología , Aorta/metabolismo , Aorta/patología , Células Cultivadas , Citidina Desaminasa/genética , Citidina Desaminasa/metabolismo , Acetato de Desoxicorticosterona , Modelos Animales de Enfermedad , Femenino , Hipertensión/sangre , Hipertensión/genética , Hipertensión/fisiopatología , Inmunoglobulina G/sangre , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/inmunología , Cadenas Pesadas de Inmunoglobulina/metabolismo , Inmunoglobulina M/genética , Inmunoglobulina M/inmunología , Inmunoglobulina M/metabolismo , Riñón/inmunología , Riñón/metabolismo , Riñón/patología , Masculino , Células B de Memoria/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Cloruro de Sodio DietéticoRESUMEN
LDL cholesterol (LDL-C) is cleared from plasma via cellular uptake and internalization processes that are largely mediated by the low-density lipoprotein cholesterol receptor (LDL-R). LDL-R is targeted for lysosomal degradation by association with proprotein convertase subtilisin-kexin type 9 (PCSK9). Gain of function mutations in PCSK9 can result in excessive loss of receptors and dyslipidemia. On the other hand, receptor-sparing phenomena, including loss-of-function mutations or inhibition of PCSK9, can lead to enhanced clearance of plasma lipids. We hypothesize that desolvation and resolvation processes, in many cases, constitute rate-determining steps for protein-ligand association and dissociation, respectively. To test this hypothesis, we analyzed and compared the predicted desolvation properties of wild-type versus gain-of-function mutant Asp374Tyr PCSK9 using WaterMap, a new in silico method for predicting the preferred locations and thermodynamic properties of water solvating proteins ("hydration sites"). We compared these results with binding kinetics data for PCSK9, full-length LDL-R ectodomain, and isolated EGF-A repeat. We propose that the fast k(on) and entropically driven thermodynamics observed for PCSK9-EGF-A binding stem from the functional replacement of water occupying stable PCSK9 hydration sites (i.e., exchange of PCSK9 H-bonds from water to polar EGF-A groups). We further propose that the relatively fast k(off) observed for EGF-A unbinding stems from the limited displacement of solvent occupying unstable hydration sites. Conversely, the slower k(off) observed for EGF-A and LDL-R unbinding from Asp374Tyr PCSK9 stems from the destabilizing effects of this mutation on PCSK9 hydration sites, with a concomitant increase in the persistence of the bound complex.
Asunto(s)
Simulación por Computador , Factor de Crecimiento Epidérmico/química , Conformación Proteica , Serina Endopeptidasas/química , Sitios de Unión , Línea Celular , Cristalografía por Rayos X , Factor de Crecimiento Epidérmico/genética , Humanos , Modelos Moleculares , Mutación , Proproteína Convertasa 9 , Proproteína Convertasas , Receptores de LDL/química , Serina Endopeptidasas/genética , Solventes/química , Relación Estructura-Actividad , Termodinámica , Agua/químicaRESUMEN
T and B cells have been implicated in hypertension, but the mechanisms by which they produce a coordinated response is unknown. T follicular helper (Tfh) cells that produce interleukin 21 (IL21) promote germinal center (GC) B cell responses leading to immunoglobulin (Ig) production. Here we investigate the role of IL21 and Tfh cells in hypertension. In response to angiotensin (Ang) II-induced hypertension, T cell IL21 production is increased, and Il21-/- mice develop blunted hypertension, attenuated vascular end-organ damage, and decreased interleukin 17A (IL17A) and interferon gamma production. Tfh-like cells and GC B cells accumulate in the aorta and plasma IgG1 is increased in hypertensive WT but not Il21-/-mice. Furthermore, Tfh cell deficient mice develop blunted hypertension and vascular hypertrophy in response to Ang II infusion. Importantly, IL21 neutralization reduces blood pressure (BP) and reverses endothelial dysfunction and vascular inflammation. Moreover, recombinant IL21 impairs endothelium-dependent relaxation ex vivo and decreases nitric oxide production from cultured endothelial cells. Finally, we show in humans that peripheral blood T cell production of IL21 correlates with systolic BP and IL17A production. These data suggest that IL21 may be a novel therapeutic target for the treatment of hypertension and its micro- and macrovascular complications.
Asunto(s)
Hipertensión/metabolismo , Interleucinas/genética , Interleucinas/metabolismo , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T Colaboradores-Inductores/metabolismo , Inmunidad Adaptativa , Adulto , Anciano , Anciano de 80 o más Años , Animales , Formación de Anticuerpos , Linfocitos B , Presión Sanguínea , Linfocitos T CD4-Positivos , Linfocitos T CD8-positivos , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Centro Germinal , Humanos , Hipertensión/genética , Hipertensión/patología , Inmunoglobulina G , Interleucina-17 , Ganglios Linfáticos/patología , Macrófagos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Proteínas RecombinantesRESUMEN
Aortic dissection (AD) is a life-threatening vascular disease with limited treatment strategies. Here, we show that loss of the GWAS-identified SH2B3 gene, encoding lymphocyte adaptor protein LNK, markedly increases susceptibility to acute AD and rupture in response to angiotensin (Ang) II infusion. As early as day 3 following Ang II infusion, prior to the development of AD, Lnk-/- aortas display altered mechanical properties, increased elastin breaks, collagen thinning, enhanced neutrophil accumulation, and increased MMP-9 activity compared with WT mice. Adoptive transfer of Lnk-/- leukocytes into Rag1-/- mice induces AD and rupture in response to Ang II, demonstrating that LNK deficiency in hematopoietic cells plays a key role in this disease. Interestingly, treatment with doxycycline prevents the early accumulation of aortic neutrophils and significantly reduces the incidence of AD and rupture. PrediXcan analysis in a biobank of more than 23,000 individuals reveals that decreased expression of SH2B3 is significantly associated with increased frequency of AD-related phenotypes (odds ratio 0.81). Thus, we identified a role for LNK in the pathology of AD in experimental animals and humans and describe a new model that can be used to inform both inherited and acquired forms of this disease.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/deficiencia , Proteínas Adaptadoras Transductoras de Señales/genética , Aorta/patología , Disección Aórtica/genética , Rotura de la Aorta/genética , Disección Aórtica/inducido químicamente , Disección Aórtica/patología , Angiotensina II/administración & dosificación , Animales , Aorta/efectos de los fármacos , Rotura de la Aorta/inducido químicamente , Rotura de la Aorta/patología , Modelos Animales de Enfermedad , Femenino , Predisposición Genética a la Enfermedad , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Masculino , Ratones , Ratones NoqueadosRESUMEN
It is now well known that T lymphocytes play a critical role in the development of several cardiovascular diseases1,2,3,4,5. For example, studies from our group have shown that hypertension is associated with an excessive accumulation of T cells in the vessels and kidney during the development of experimental hypertension6. Once in these tissues, T cells produce several cytokines that affect both vascular and renal function leading to vasoconstriction and sodium and water retention1,2. To fully understand how T cells cause cardiovascular and renal diseases, it is important to be able to identify and quantify the specific T cell subsets present in these tissues. T cell subsets are defined by a combination of surface markers, the cytokines they secrete, and the transcription factors they express. The complexity of the T cell population makes flow cytometry and intracellular staining an invaluable technique to dissect the phenotypes of the lymphocytes present in tissues. Here, we provide a detailed protocol to identify the surface and intracellular markers (cytokines and transcription factors) in T cells isolated from murine kidney, aorta and aortic draining lymph nodes in a model of angiotensin II induced hypertension. The following steps are described in detail: isolation of the tissues, generation of the single cell suspensions, ex vivo stimulation, fixation, permeabilization and staining. In addition, several fundamental principles of flow cytometric analyses including choosing the proper controls and appropriate gating strategies are discussed.
Asunto(s)
Aorta/patología , Citometría de Flujo/métodos , Hipertensión/patología , Riñón/patología , Subgrupos de Linfocitos T/patología , Angiotensina II/efectos adversos , Animales , Citocinas/análisis , Citocinas/metabolismo , Modelos Animales de Enfermedad , Hipertensión/inducido químicamente , Riñón/metabolismo , Ganglios Linfáticos/metabolismo , Ganglios Linfáticos/patología , Ratones , Coloración y Etiquetado/métodos , Subgrupos de Linfocitos T/metabolismoRESUMEN
We previously showed that angiotensin II (Ang II) increases T cell production of IL-17A, and that mice deficient in IL-17A have blunted hypertension and attenuated renal and vascular dysfunction. It was recently shown that salt enhances IL-17A production from CD4+ T cells via a serum- and glucocorticoid-regulated kinase 1-dependent (SGK1-dependent) pathway. Thus, we tested the hypothesis that SGK1 signaling in T cells promotes hypertension and contributes to end-organ damage. We show that loss of T cell SGK1 results in a blunted hypertensive response to Ang II infusion by 25 mmHg. Importantly, renal and vascular inflammation is abrogated in these mice compared with control mice. Furthermore, mice lacking T cell SGK1 are protected from Ang II-induced endothelial dysfunction and renal injury. Loss of T cell SGK1 also blunts blood pressure and vascular inflammation in response to deoxycorticosterone acetate-salt (DOCA-salt) hypertension. Finally, we demonstrate that the Na+-K+-2Cl- cotransporter 1 (NKCC1) is upregulated in Th17 cells and is necessary for the salt-induced increase in SGK1 and the IL-23 receptor. These studies demonstrate that T cell SGK1 and NKCC1 may be novel therapeutic targets for the treatment of hypertension and identify a potentially new mechanism by which salt contributes to hypertension.
RESUMEN
Angiotensin II-induced hypertension is associated with an increase in T-cell production of interleukin-17A (IL-17A). Recently, we reported that IL-17A(-/-) mice exhibit blunted hypertension, preserved natriuresis in response to a saline challenge, and decreased renal sodium hydrogen exchanger 3 expression after 2 weeks of angiotensin II infusion compared with wild-type mice. In the current study, we performed renal transporter profiling in mice deficient in IL-17A or the related isoform, IL-17F, after 4 weeks of Ang II infusion, the time when the blood pressure reduction in IL-17A(-/-) mice is most prominent. Deficiency of IL-17A abolished the activation of distal tubule transporters, specifically the sodium-chloride cotransporter and the epithelial sodium channel and protected mice from glomerular and tubular injury. In human proximal tubule (HK-2) cells, IL-17A increased sodium hydrogen exchanger 3 expression through a serum and glucocorticoid-regulated kinase 1-dependent pathway. In mouse distal convoluted tubule cells, IL-17A increased sodium-chloride cotransporter activity in a serum and glucocorticoid-regulated kinase 1/Nedd4-2-dependent pathway. In both cell types, acute treatment with IL-17A induced phosphorylation of serum and glucocorticoid-regulated kinase 1 at serine 78, and treatment with a serum and glucocorticoid-regulated kinase 1 inhibitor blocked the effects of IL-17A on sodium hydrogen exchanger 3 and sodium-chloride cotransporter. Interestingly, both HK-2 and mouse distal convoluted tubule 15 cells produce endogenous IL-17A. IL17F had little or no effect on blood pressure or renal sodium transporter abundance. These studies provide a mechanistic link by which IL-17A modulates renal sodium transport and suggest that IL-17A inhibition may improve renal function in hypertension and other autoimmune disorders.
Asunto(s)
Lesión Renal Aguda/metabolismo , Angiotensina II/farmacología , Hipertensión/metabolismo , Interleucina-17/metabolismo , Túbulos Renales Proximales/metabolismo , Simportadores del Cloruro de Sodio/metabolismo , Lesión Renal Aguda/fisiopatología , Análisis de Varianza , Animales , Determinación de la Presión Sanguínea , Células Cultivadas , Modelos Animales de Enfermedad , Hipertensión/fisiopatología , Immunoblotting , Masculino , Ratones , Ratones Endogámicos C57BL , Distribución Aleatoria , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Sensibilidad y Especificidad , Miembro 3 de la Familia de Transportadores de Soluto 12/metabolismoRESUMEN
Type 2 diabetes is a polygenic disease which afflicts nearly 200 million people worldwide and is expected to increase to near epidemic levels over the next 10-15 years. Glucokinase (GK) activators are currently under investigation by a number of pharmaceutical companies with only a few reaching early clinical evaluation. A GK activator has the promise of potentially affecting both the beta-cells of the pancreas, by improving glucose sensitive insulin secretion, as well as the liver, by reducing uncontrolled glucose output and restoring post-prandial glucose uptake and storage as glycogen. Herein, we report our efforts on a sulfonamide chemotype with the aim to generate liver selective GK activators which culminated in the discovery of 3-cyclopentyl-N-(5-methoxy-thiazolo[5,4-b]pyridin-2-yl)-2-[4-(4-methyl-piperazine-1-sulfonyl)-phenyl]-propionamide (17c). This compound activated the GK enzyme (alphaK(a) = 39 nM) in vitro at low nanomolar concentrations and significantly reduced glucose levels during an oral glucose tolerance test in normal mice.