RESUMEN
BACKGROUND: Short-chain fatty acids derived from gut microbial fermentation of dietary fiber have been shown to suppress autoimmunity through mechanisms that include enhanced regulation by T regulatory cells (Tregs). METHODS: Using a murine kidney transplantation model, we examined the effects on alloimmunity of a high-fiber diet or supplementation with the short-chain fatty acid acetate. Kidney transplants were performed from BALB/c(H2d) to B6(H2b) mice as allografts in wild-type and recipient mice lacking the G protein-coupled receptor GPR43 (the metabolite-sensing receptor of acetate). Allograft mice received normal chow, a high-fiber diet, or normal chow supplemented with sodium acetate. We assessed rejection at days 14 (acute) and 100 (chronic), and used 16S rRNA sequencing to determine gut microbiota composition pretransplantation and post-transplantation. RESULTS: Wild-type mice fed normal chow exhibited dysbiosis after receiving a kidney allograft but not an isograft, despite the avoidance of antibiotics and immunosuppression for the latter. A high-fiber diet prevented dysbiosis in allograft recipients, who demonstrated prolonged survival and reduced evidence of rejection compared with mice fed normal chow. Allograft mice receiving supplemental sodium acetate exhibited similar protection from rejection, and subsequently demonstrated donor-specific tolerance. Depletion of CD25+ Tregs or absence of the short-chain fatty acid receptor GPR43 abolished this survival advantage. CONCLUSIONS: Manipulation of the microbiome by a high-fiber diet or supplementation with sodium acetate modified alloimmunity in a kidney transplant model, generating tolerance dependent on Tregs and GPR43. Diet-based therapy to induce changes in the gut microbiome can alter systemic alloimmunity in mice, in part through the production of short-chain fatty acids leading to Treg cell development, and merits study as a potential clinical strategy to facilitate transplant acceptance.
Asunto(s)
Fibras de la Dieta/administración & dosificación , Ácidos Grasos Volátiles/inmunología , Microbioma Gastrointestinal/inmunología , Rechazo de Injerto/prevención & control , Tolerancia Inmunológica/efectos de los fármacos , Linfocitos T Reguladores , Enfermedad Aguda , Aloinjertos/inmunología , Animales , Ácido Butírico/farmacología , Enfermedad Crónica , Suplementos Dietéticos , Disbiosis/etiología , Disbiosis/microbiología , Disbiosis/prevención & control , Microbioma Gastrointestinal/efectos de los fármacos , Rechazo de Injerto/patología , Rechazo de Injerto/fisiopatología , Supervivencia de Injerto/efectos de los fármacos , Supervivencia de Injerto/inmunología , Trasplante de Riñón/efectos adversos , Activación de Linfocitos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores Acoplados a Proteínas G/genética , Acetato de Sodio/farmacologíaRESUMEN
BACKGROUND: Studies have reported "dysbiotic" changes to gut microbiota, such as depletion of gut bacteria that produce short-chain fatty acids (SCFAs) through gut fermentation of fiber, in CKD and diabetes. Dietary fiber is associated with decreased inflammation and mortality in CKD, and SCFAs have been proposed to mediate this effect. METHODS: To explore dietary fiber's effect on development of experimental diabetic nephropathy, we used streptozotocin to induce diabetes in wild-type C57BL/6 and knockout mice lacking the genes encoding G protein-coupled receptors GPR43 or GPR109A. Diabetic mice were randomized to high-fiber, normal chow, or zero-fiber diets, or SCFAs in drinking water. We used proton nuclear magnetic resonance spectroscopy for metabolic profiling and 16S ribosomal RNA sequencing to assess the gut microbiome. RESULTS: Diabetic mice fed a high-fiber diet were significantly less likely to develop diabetic nephropathy, exhibiting less albuminuria, glomerular hypertrophy, podocyte injury, and interstitial fibrosis compared with diabetic controls fed normal chow or a zero-fiber diet. Fiber beneficially reshaped gut microbial ecology and improved dysbiosis, promoting expansion of SCFA-producing bacteria of the genera Prevotella and Bifidobacterium, which increased fecal and systemic SCFA concentrations. Fiber reduced expression of genes encoding inflammatory cytokines, chemokines, and fibrosis-promoting proteins in diabetic kidneys. SCFA-treated diabetic mice were protected from nephropathy, but not in the absence of GPR43 or GPR109A. In vitro, SCFAs modulated inflammation in renal tubular cells and podocytes under hyperglycemic conditions. CONCLUSIONS: Dietary fiber protects against diabetic nephropathy through modulation of the gut microbiota, enrichment of SCFA-producing bacteria, and increased SCFA production. GPR43 and GPR109A are critical to SCFA-mediated protection against this condition. Interventions targeting the gut microbiota warrant further investigation as a novel renoprotective therapy in diabetic nephropathy.
Asunto(s)
Nefropatías Diabéticas/prevención & control , Fibras de la Dieta/administración & dosificación , Ácidos Grasos Volátiles/fisiología , Receptores Acoplados a Proteínas G/fisiología , Albuminuria/prevención & control , Animales , Diabetes Mellitus Experimental/complicaciones , Disbiosis , Microbioma Gastrointestinal , Masculino , Ratones , Ratones Endogámicos C57BL , EstreptozocinaRESUMEN
BACKGROUND: We have previously demonstrated that absence of myeloid differentiation primary response gene 88 (MyD88) induced donor-specific kidney allograft tolerance. The upstream pathways of MyD88 that mediate this process, however, remain unclear. Toll-like receptor 4 (TLR4) is an innate immune receptor that is dependent upon MyD88 for activity of its dominant signaling pathway. Here, we investigated the role of TLR4 in kidney allograft rejection using a fully major histocompatibility complex-mismatched, life-sustaining, murine model of renal allograft rejection. METHODS: Donor (BALB/c) and recipient (C57BL/6) mice either both deficient or sufficient for TLR4 underwent heterotopic renal allograft transplantation, with an additional group of mice receiving renal isografts as controls. Survival was assessed up to 100 days posttransplantation. Animals were also sacrificed 14 days posttransplantation for assessment of the acute allograft rejection response. RESULTS: Both wild-type (WT) and TLR4 allografts showed inferior survival compared to isografts, with no difference in survival between the allograft groups. Serum creatinine was lower in TLR4 allografts at day 14 posttransplantation compared with WT allografts, but this was not sustained by day 100. At day 14 posttransplant, increased CD11c dendritic cell accumulation, expression of IL-2 and indoleamine 2,3-dioxygenase were evident in TLR4 compared with WT allografts, whereas expression of inducible nitric oxide synthase was decreased. CONCLUSIONS: Acute kidney allograft rejection was modestly attenuated in TLR4 mice; however, long-term allograft survival and function were not affected in our model. Protection against acute rejection may involve increased accumulation of CD11c cells and indoleamine 2,3-dioxygenase expression.
Asunto(s)
Supervivencia de Injerto , Trasplante de Riñón , Complejo Mayor de Histocompatibilidad/inmunología , Receptor Toll-Like 4/deficiencia , Aloinjertos , Animales , Antígeno CD11c/metabolismo , Creatinina/sangre , Modelos Animales de Enfermedad , Rechazo de Injerto , Inmunidad Innata , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Interleucina-2/metabolismo , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Factor 88 de Diferenciación Mieloide/metabolismo , Transducción de Señal , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/metabolismoAsunto(s)
Fármacos Antiobesidad/efectos adversos , Dieta/efectos adversos , Hiperoxaluria/inducido químicamente , Intestinos/efectos de los fármacos , Enfermedades Renales/inducido químicamente , Riñón/efectos de los fármacos , Lactonas/efectos adversos , Ácido Oxálico/efectos adversos , Biopsia , Femenino , Humanos , Hiperoxaluria/diagnóstico , Hiperoxaluria/metabolismo , Hiperoxaluria/terapia , Mucosa Intestinal/metabolismo , Riñón/metabolismo , Riñón/patología , Enfermedades Renales/metabolismo , Enfermedades Renales/patología , Enfermedades Renales/terapia , Persona de Mediana Edad , Orlistat , Ácido Oxálico/metabolismo , Rheum/efectos adversos , Spinacia oleracea/efectos adversosRESUMEN
Toll-like receptors (TLRs) play a fundamental role in innate immunity and provide a link between innate and adaptive responses to an allograft; however, whether the development of acute and chronic allograft rejection requires TLR signaling is unknown. Here, we studied TLR signaling in a fully MHC-mismatched, life-sustaining murine model of kidney allograft rejection. Mice deficient in the TLR adaptor protein MyD88 developed donor antigen-specific tolerance, which protected them from both acute and chronic allograft rejection and increased their survival after transplantation compared with wild-type controls. Administration of an anti-CD25 antibody to MyD88-deficient recipients depleted CD4(+)CD25(+)FoxP3(+) cells and broke tolerance. In addition, defective development of Th17 immune responses to alloantigen both in vitro and in vivo occurred, resulting in an increased ratio of Tregs to Th17 effectors. Thus, MyD88 deficiency was associated with an altered balance of Tregs over Th17 cells, promoting tolerance instead of rejection. This study provides evidence that targeting innate immunity may be a clinically relevant strategy to facilitate transplantation tolerance.
Asunto(s)
Trasplante de Riñón/inmunología , Factor 88 de Diferenciación Mieloide/deficiencia , Animales , Citocinas/genética , Citocinas/metabolismo , Rechazo de Injerto/inmunología , Rechazo de Injerto/prevención & control , Supervivencia de Injerto/inmunología , Humanos , Tolerancia Inmunológica , Inmunidad Innata , Subunidad alfa del Receptor de Interleucina-2/antagonistas & inhibidores , Isoantígenos , Prueba de Cultivo Mixto de Linfocitos , Depleción Linfocítica , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/inmunología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal/inmunología , Linfocitos T Reguladores/inmunología , Células Th17/inmunología , Donantes de Tejidos , Trasplante HomólogoAsunto(s)
Electrólitos/uso terapéutico , Síndrome de Gitelman/terapia , Complicaciones del Embarazo/terapia , Resultado del Embarazo , Adulto , Ácido Aspártico/uso terapéutico , Cesárea , Diabetes Gestacional/dietoterapia , Femenino , Humanos , Magnesio/sangre , Potasio/sangre , Cloruro de Potasio/uso terapéutico , EmbarazoRESUMEN
Cumulative addition of atropine to the organ bath containing endothelium-intact (+E) rat aorta, which was precontracted with phenylephrine (PE, 1 microM) and subsequently relaxed with carbachol (1 microM), caused biphasic changes in the vascular contractility of +E rat aortic rings. Low concentrations of atropine (10 nM-1.0 microM) caused progressive restoration of contraction to PE; whereas at higher concentrations (1-100 microM), atropine caused progressive relaxation. Atropine-induced aortic relaxation was significantly inhibited upon endothelium removal by either rubbing or saponin treatment, but considerable relaxation still persisted in the range of 30-100 microM atropine. Similar findings were also obtained when the nitric oxide (NO) generation was inhibited with 300 microM NO synthase inhibitor, L-NAME. Atropine-induced relaxation was also observed when 5-hydroxytryptamine (5-HT) was used as the agonist and the atropine-relaxation was more potent at lower concentrations of PE and 5-HT. However, atropine had no effect on the contraction elicited by KCl or prostaglandin F(2 alpha). Also, atropine-induced relaxation was not affected by indomethacin (1-10 microM), nicotine (10-100 microM) or hexamethonium (30 microM). Pretreatment of +E aorta with tetraethylammonia (TEA, 3-10 mM) or 4-aminopyridine (4-AP, 1-3 mM) showed prominent inhibitory effect on atropine-induced relaxation; on the other hand, preincubation with glibenclamide (1-10 microM), BaCl(2) (1-30 microM) or 2 microM charybdotoxin and apamin, had little effect on the relaxation induced by atropine. When added to tissues after relaxation to atropine, TEA and 4-AP concentration-dependently reversed the relaxation in -E aorta, whereas in +E aorta, TEA up to 30 mM and 4-AP up to 10 mM only partially affected atropine-induced relaxation. Although TEA and 4-AP potentiated the PE-contraction, such potentiation is unlikely to contribute to the change in sensitivity to atropine-induced relaxation, since in the presence of 15 mM KCl, which also potentiated PE-contraction to a comparable extent, the atropine-relaxation remains unchanged. Scopolamine also acts like atropine, except that the effect of scopolamine was smaller than that of atropine and is primarily endothelium-dependent. Atropine-induced relaxation also occurs in medium artery (renal artery) and small muscular artery (mesenteric artery). In conclusion, atropine-relaxation is mediated in part via voltage-dependent K(+) channels in both smooth muscle and endothelium and forms the mechanistic basis for the observed vasodilation, reduced blood pressure and facial flushing following atropine overdose.
Asunto(s)
Atropina/farmacología , Endotelio Vascular/fisiología , Antagonistas Muscarínicos/farmacología , Músculo Liso Vascular/efectos de los fármacos , Canales de Potasio/fisiología , Animales , Aorta Torácica/efectos de los fármacos , Aorta Torácica/fisiología , Endotelio Vascular/efectos de los fármacos , Técnicas In Vitro , Masculino , Arterias Mesentéricas/efectos de los fármacos , Arterias Mesentéricas/fisiología , Contracción Muscular/efectos de los fármacos , Relajación Muscular/efectos de los fármacos , Músculo Liso Vascular/fisiología , Óxido Nítrico/fisiología , Bloqueadores de los Canales de Potasio/farmacología , Ratas , Ratas Sprague-Dawley , Arteria Renal/efectos de los fármacos , Arteria Renal/fisiologíaRESUMEN
The effects of a newly discovered antifungal agent, pramanicin, on cytosolic Ca(2+) and cell viability of cultured bovine pulmonary artery endothelial cells and on endothelium-dependent relaxation of dog carotid arterial rings were investigated by digital dynamic fluorescence ratio imaging and morphological and contractility studies, respectively. Pramanicin 100 microM, previously shown to cause maximal endothelium-dependent and NO-mediated vascular relaxation, induced a small transient elevation of cytosolic Ca(2+) concentration in Ca(2+)-free medium; subsequent introduction of 1 mM Ca(2+) caused a steady, nonsaturating increase of Ca(2+), which could be brought down to the basal level by the addition of EGTA. At the single cell level, the elevation of cytosolic Ca(2+) initiates from the cell periphery and progresses toward the central region. When added to the plateau phase of phenylephrine-induced contraction, pramanicin induced a slow endothelium-dependent relaxation, which could be reversed with the NO synthase inhibitor, L-NOARG. When preincubated with vascular tissue, pramanicin resulted in an irreversible loss of endothelial function characterized by the lack of carbachol-induced relaxation. Pramanicin caused cell injury characterized by plasmalemmal bleb formation, leading to cell death characterized by Trypan blue staining of the nuclei in cultured vascular endothelial cells in a concentration- and time-dependent manner. Such pramanicin-induced cell death was not associated with Ca(2+)-mediated or NO-mediated mechanisms. The time course of Ca(2+) elevation corresponds with that of pramanicin-induced relaxation of precontracted arterial rings, whereas the time course of endothelial cell death corresponds to that of pramanicin-induced loss of endothelial function as assessed by carbachol-induced relaxation. The pramanicin analogue, PMC-A, a by-product of the biosynthesis of pramanicin, in which the epoxy group is replaced by a CC bond, caused little endothelial-dependent relaxation, but it was able to cause endothelial cell dysfunction, albeit to a lesser extent compared to pramanicin, suggesting a role of the epoxy group in pramanicin for its vasorelaxant effect.
Asunto(s)
Antifúngicos/farmacología , Calcio/metabolismo , Citosol/efectos de los fármacos , Citosol/metabolismo , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Compuestos Epoxi/farmacología , Lactamas/farmacología , Animales , Bovinos , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Células Cultivadas , Perros , Endotelio Vascular/metabolismo , Femenino , MasculinoRESUMEN
To investigate their potentially toxic effects on mammalian vascular smooth muscle, pentane extracts of papaya seeds and the chief active ingredient in the extracts, benzyl isothiocyanate (BITC), were tested for their effects on the contraction of strips of dog carotid artery. BITC and the papaya seed extract caused relaxation when added to tissue strips that had been pre-contracted with phenylephrine (PE). Incubation of the tissue with papaya seed extract or BITC caused inhibition of contraction when the strips were subsequently contracted with KCl or PE. This relaxation and inhibition of contraction did not appear to be endothelium-dependent, as endothelium-denuded rings showed the same degree of relaxation or inhibition of contraction in response to the preparations/drugs as those with the endothelium intact. The effects of both BITC and the extract were irreversible, i.e., the tissue did not recover to normal contractile ability after extensive washing. Exposure of the tissue to the papaya seed extract caused slower relaxation of the tissue, compared to controls, both after contraction with PE and subsequent addition of carbachol (CCh), and after contraction with KCl and then washing. Calcium imaging studies using cultured endothelial cells showed strong influxes of Ca2+ into the cells in response to addition of the papaya seed extract. We conclude that these extracts, when present in high concentration, are cytotoxic by increasing the membrane permeability to Ca2+, and that the vascular effects of papaya seed extracts are consistent with the notion that BITC is the chief bio-active ingredient.
Asunto(s)
Carica/química , Isotiocianatos/farmacología , Contracción Muscular/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Semillas/química , Vasoconstricción/efectos de los fármacos , Animales , Calcio/metabolismo , Carbacol/farmacología , Cardiotónicos/farmacología , Arterias Carótidas/efectos de los fármacos , Bovinos , Células Cultivadas , Perros , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Humanos , Técnicas In Vitro , Contracción Muscular/fisiología , Músculo Liso Vascular/citología , Músculo Liso Vascular/fisiología , Fenilefrina/farmacología , Extractos Vegetales/farmacología , Cloruro de Potasio/farmacología , Vasoconstrictores/farmacologíaRESUMEN
Changes in endothelial cell (EC) shape result in inter-EC gap formation and subsequently regulate transendothelial passage. In this work, we investigated the effects of protein phosphorylation (induced by inhibition of protein phosphatases) on EC shape changes. Treatment of bovine pulmonary artery endothelial cells (BPAEC) with calyculin A (100 nM, an inhibitor of protein Ser/Thr phosphatases 1 and 2A) resulted in cell retraction, surface bleb formation and cell rounding. Trypan blue and electrophysiological experiments suggested that the plasma membrane of these rounded cells maintained functional integrity. Calyculin A-induced morphological changes were strongly inhibited by staurosporine, but not affected by specific inhibitors of the myosin light chain (MLC) kinase, protein kinases A, C and G, and tyrosine kinases. The calyculin A effects were not mimicked by phorbol myristate acetate, dibutyryl cAMP, 8-bromo-cGMP or ionomycin. Cytochalasin B (an inhibitor of actin polymerization) almost completely abolished such shape changes while colchicine (an inhibitor of microtubule polymerization) had no inhibitory effect at all. Ca(2+) imaging experiments showed that the morphological changes were not associated with any global or local cytosolic Ca(2+) concentration ([Ca(2+)](i)) elevation. The results suggest that calyculin A unmasked the basal activities of some protein Ser/Thr kinases other than MLC kinase and protein kinases A, C and G; these unknown kinases might cause BPAEC shape changes by a mechanism involving actin polymerization but not [Ca(2+)](i) elevation.