RESUMO
RATIONALE & OBJECTIVE: Posttransplant hyperparathyroidism is common, and treatment practices are poorly characterized. The goal of this study was to examine the incidence, associations, and outcomes of posttransplant parathyroidectomy and calcimimetic use in a cohort of Medicare-insured US kidney transplant recipients. STUDY DESIGN: Retrospective observational cohort study. SETTING & PARTICIPANTS: We used the US Renal Data System to extract demographic, clinical, and prescription data from Medicare Parts A, B, and D-insured patients who received their first kidney transplant in 2007-2013. We excluded patients with pretransplant parathyroidectomy. PREDICTORS: Calendar year of transplantation and pretransplant patient characteristics. OUTCOME: (1) Incidence of and secular trends in parathyroidectomy and cinacalcet use in the 3 years after transplant; (2) 90-day outcomes after posttransplant parathyroidectomy and cinacalcet initiation. ANALYTICAL APPROACH: Temporal trends and pretransplant correlates of parathyroidectomy and cinacalcet use were assessed using proportional hazards models and multivariable Poisson regression, respectively. RESULTS: The inclusion criteria were met by 30,127 patients, of whom 10,707 used cinacalcet before transplant, 551 underwent posttransplant parathyroidectomy, and 5,413 filled≥1 prescription for cinacalcet. The rate of posttransplant parathyroidectomy was stable over time. By contrast, cinacalcet use increased during the period studied. Long dialysis vintage and pretransplant cinacalcet use were strongly associated with posttransplant parathyroidectomy and cinacalcet use. Roughly 1 in 4 patients were hospitalized within 90 days of posttransplant parathyroidectomy, with hypocalcemia-related diagnoses being the most common complication. Parathyroidectomy (vs cinacalcet initiation) was not associated with an increase in acute kidney injury. LIMITATIONS: We lacked access to laboratory data to help assess the severity of secondary/tertiary hyperparathyroidism. The cohort was limited to Medicare beneficiaries. CONCLUSIONS: Almost one-fifth of our study cohort was treated with parathyroidectomy and/or cinacalcet. Further studies are needed to establish the optimal treatment for posttransplant hyperparathyroidism.
Assuntos
Hiperparatireoidismo Secundário , Falência Renal Crônica , Transplante de Rim , Humanos , Idoso , Estados Unidos , Cinacalcete/uso terapêutico , Calcimiméticos/uso terapêutico , Paratireoidectomia , Estudos Retrospectivos , Medicare , Hiperparatireoidismo Secundário/tratamento farmacológico , Hormônio Paratireóideo , Cálcio , Falência Renal Crônica/complicaçõesRESUMO
Solid organ transplant recipients are at risk for infectious complications due to chronic immunosuppression. The outbreak of coronavirus disease 2019 (COVID-19) in the United States has raised growing concerns for the transplant patient population. We seek to add to the current limited literature on COVID-19 in transplant recipients by describing the clinical course of two kidney transplant recipients with SARS-CoV-2 infection monitored by both RT-PCR and serology. Through careful adjustment of their immunosuppression regimen, both patients had excellent recovery with intact graft function and development of anti-SARS-CoV-2 antibodies.
Assuntos
Imunossupressores/administração & dosagem , Imunossupressores/uso terapêutico , Transplante de Rim , SARS-CoV-2 , Transplantados , Idoso , Feminino , Humanos , Hospedeiro Imunocomprometido , Pessoa de Meia-Idade , SoroconversãoRESUMO
Kidney transplant recipients may be at a high risk of developing critical coronavirus disease 2019 (COVID-19) illness due to chronic immunosuppression and comorbidities. We identified hospitalized adult kidney transplant recipients at 12 transplant centers in the United States, Italy, and Spain who tested positive for COVID-19. Clinical presentation, laboratory values, immunosuppression, and treatment strategies were reviewed, and predictors of poor clinical outcomes were determined through multivariable analyses. Among 9845 kidney transplant recipients across centers, 144 were hospitalized due to COVID-19 during the 9-week study period. Of the 144 patients, 66% were male with a mean age of 60 (±12) years, and 40% were Hispanic and 25% were African American. Prevalent comorbidities included hypertension (95%), diabetes (52%), obesity (49%), and heart (28%) and lung (19%) disease. Therapeutic management included antimetabolite withdrawal (68%), calcineurin inhibitor withdrawal (23%), hydroxychloroquine (71%), antibiotics (74%), tocilizumab (13%), and antivirals (14%). During a median follow-up period of 52 days (IQR: 16-66 days), acute kidney injury occurred in 52% cases, with respiratory failure requiring intubation in 29%, and the mortality rate was 32%. The 46 patients who died were older, had lower lymphocyte counts and estimated glomerular filtration rate levels, and had higher serum lactate dehydrogenase, procalcitonin, and interleukin-6 levels. In sum, hospitalized kidney transplant recipients with COVID-19 have higher rates of acute kidney injury and mortality.
Assuntos
COVID-19/epidemiologia , Rejeição de Enxerto/prevenção & controle , Terapia de Imunossupressão/métodos , Transplante de Rim/estatística & dados numéricos , Pandemias , SARS-CoV-2 , Transplantados , Idoso , Comorbidade , Europa (Continente)/epidemiologia , Feminino , Seguimentos , Rejeição de Enxerto/epidemiologia , Humanos , Hospedeiro Imunocomprometido , Imunossupressores/uso terapêutico , Masculino , Pessoa de Meia-Idade , América do Norte/epidemiologia , Estudos RetrospectivosRESUMO
The data on the outcomes of solid organ transplant recipients who have contracted coronavirus disease 2019 (COVID-19) are still emerging. Kidney transplant recipients are commonly prescribed renin-angiotensin-aldosterone system (AAS) inhibitors given the prevalence of hypertension, diabetes, and cardiovascular disease. As the angiotensin-converting enzyme 2 (ACE2) facilitates the entry of coronaviruses into target cells, there have been hypotheses that preexisting use of renin-angiotensin-aldosterone system (RAAS) inhibitors may increase the risk of developing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Given the common use of RAAS inhibitors among solid organ transplant recipients, we sought to review the RAAS cascade, the mechanism of SARS-CoV-2 entry, and pertinent data related to the effect of RAAS inhibitors on ACE2 to guide management of solid organ transplant recipients during the COVID-19 pandemic. At present, there is no clear evidence to support the discontinuation of RAAS inhibitors in solid organ transplant recipients during the COVID-19 pandemic.
Assuntos
Antagonistas de Receptores de Angiotensina/uso terapêutico , Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Betacoronavirus , Doenças Cardiovasculares/terapia , Infecções por Coronavirus/complicações , Transplante de Órgãos , Pneumonia Viral/complicações , COVID-19 , Doenças Cardiovasculares/complicações , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/terapia , Humanos , Pandemias , Pneumonia Viral/diagnóstico , Pneumonia Viral/terapia , Sistema Renina-Angiotensina/fisiologia , SARS-CoV-2RESUMO
Advances in solid organ transplantation have improved the survival of end-stage organ disease at the expense of an increased risk for opportunistic infections. Unusual clinical presentations and the possibility of concurrent infections make diagnosing invasive fungal infection (IFI) more difficult. Here, we present a case of simultaneous vertebral infection caused by Coccidioides immitis-posadasii and subcutaneous phaeohyphomycosis due to Nigrograna mackinnonii in a kidney transplant recipient. The diagnosis of both infections required invasive procedures to obtain tissue and a high index of suspicion that more than one IFI could be present. A multidisciplinary team approach for the management of immunocompromised patients with suspected or diagnosed IFI is warranted.
Assuntos
Coccidioidomicose/diagnóstico , Coinfecção/diagnóstico , Coinfecção/microbiologia , Transplante de Rim/efeitos adversos , Feoifomicose/diagnóstico , Antifúngicos/uso terapêutico , Ascomicetos/isolamento & purificação , Biópsia/métodos , Coccidioides/isolamento & purificação , Coccidioidomicose/tratamento farmacológico , Coccidioidomicose/microbiologia , Coinfecção/tratamento farmacológico , Humanos , Hospedeiro Imunocomprometido , Imageamento por Ressonância Magnética/métodos , Masculino , Pessoa de Meia-Idade , Infecções Oportunistas/diagnóstico , Infecções Oportunistas/tratamento farmacológico , Infecções Oportunistas/microbiologia , Feoifomicose/tratamento farmacológico , Feoifomicose/microbiologia , Reação em Cadeia da Polimerase/métodos , Resultado do TratamentoRESUMO
Difficult behavior exhibited by dialysis patients is a spectrum that includes nonadherence, verbal or physical abuse, and threatening acts. Such behaviors may lead to harmful consequences to the patient, other patients, the facility, and staff and can culminate in involuntary discharge. It is important to recognize that these "difficult behaviors" may be due to underlying psychosocial or medical issues, which places an onus on care providers to explore further. According to the Conditions for Coverage (CfC) for dialysis facilities, it falls upon the medical director to coordinate and oversee policies for patient satisfaction, patient safety and rights, involuntary discharges, and adverse events and outcomes. Thus, medical directors are liable for their own actions, and their staff for which they have oversight, for harm or perceived harm to patients in response to difficult behaviors. Guidelines to deal with specific patient behavior scenarios have been published by the Decreasing Dialysis Patient Conflict National Task Force of the Forum of end-stage renal disease (ESRD) Networks. The common denominator for these difficult scenarios is impaired communication, and the majority of patient concerns involve issues with staff, policies, treatments, and diet. Involuntary discharge of a patient should always be viewed as a last resort, and there is a structured process described in the CfC that requires the involvement of the respective ESRD Network and the facility medical director. As physicians, we are bound by ethical and growing legal obligations to act in an appropriate, ethical, and fair manner to patients who are considered to be "difficult."
Assuntos
Adesão à Medicação , Segurança do Paciente , Satisfação do Paciente , Relações Médico-Paciente , Diálise Renal , Humanos , Guias de Prática Clínica como AssuntoRESUMO
AIMS/HYPOTHESIS: As insulin entry into muscle interstitium is rate-limiting for its overall peripheral action, defining the route and regulation of its entry is critical. Caveolin-1 is required for caveola formation in vascular endothelial cells (ECs) and for EC insulin uptake. Whether this requirement reflects simply the need for caveola availability or involves a more active role for caveolae/caveolin-1 is not known. Here, we examined the role of insulin-stimulated tyrosine 14 (Tyr(14))-caveolin-1 phosphorylation in mediating EC insulin uptake and the role of cellular Src-kinase (cSrc), TNF-α/IL-6 and high fat diet (HFD) in regulating this process. METHODS: Freshly isolated ECs from normal or HFD-fed rats and/or cultured ECs were treated with FITC-labelled or regular insulin with or without a Src or phosphotidylinositol-3-kinase inhibitor, TNF-α or IL-6, or transfecting FLAG-tagged wild-type (WT) or mutant (Y14F) caveolin-1. Tyr(14)-caveolin-1/Tyr(416) cSrc phosphorylation and FITC-insulin uptake were quantified by immunostaining and/or western blots. RESULTS: Insulin stimulated Tyr(14)-caveolin-1 phosphorylation during EC insulin uptake. Inhibiting cSrc, but not phosphotidylinositol-3-kinase, reduced insulin-stimulated caveolin-1 phosphorylation. Furthermore, inhibiting cSrc reduced FITC-insulin uptake by â¼50%. Overexpression of caveolin-1Y14F inhibited, while overexpression of WT caveolin-1 increased, FITC-insulin uptake. Exposure of ECs to TNF-α or IL-6, or to 1-week HFD feeding eliminated insulin-stimulated caveolin-1 phosphorylation and inhibited FITC-insulin uptake to a similar extent. CONCLUSIONS/INTERPRETATION: Insulin stimulation of its own uptake requires caveolin-1 phosphorylation and Src-kinase activity. HFD in vivo and proinflammatory cytokines in vitro both inhibit this process.
Assuntos
Caveolina 1/metabolismo , Células Endoteliais/citologia , Resistência à Insulina , Quinases da Família src/metabolismo , Animais , Proteína Tirosina Quinase CSK , Dieta Hiperlipídica , Insulina/metabolismo , Interleucina-6/metabolismo , Masculino , Microscopia de Fluorescência , Fosforilação , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismo , Tirosina/químicaRESUMO
Background: Morbidity and mortality associated with coronavirus disease 2019 (COVID-19) infection in kidney transplant recipients are high and early outpatient interventions to prevent progression to severe disease are needed. SARS-CoV-2 neutralizing mAbs, including bamlanivimab and casirivimab-imdevimab, received emergency use authorization in the United States in November 2020 for treatment of mild to moderate COVID-19 disease. Methods: We performed a retrospective analysis of 27 kidney transplant recipients diagnosed with COVID-19 between July 2020 and February 2021 who were treated with bamlanivimab or casirivimab-imdevimab and immunosuppression reduction. We additionally identified 13 kidney transplant recipients with COVID-19 who had mild to moderate disease at presentation, who did not receive mAbs, and had SARS-CoV-2 serology testing available. Results: There were no deaths or graft failures in either group. Both infusions were well tolerated. Four of the 27 patients treated with mAbs required hospitalization due to COVID-19. Four of 13 patients who did not receive mAbs required hospitalization due to COVID-19. Patients who received mAbs demonstrated measurable anti-SARS-CoV-2 IgG with angiotensin-converting enzyme 2 (ACE2) receptor blocking activity at the highest level detectable at 90 days postinfusion, whereas ACE2 blocking activity acquired from natural immunity in the mAb-untreated group was weak. Conclusions: Bamlanivimab and casirivimab-imdevimab combined with immunosuppression reduction were well tolerated and associated with favorable clinical outcomes in kidney transplant recipients diagnosed with mild to moderate COVID-19.
Assuntos
COVID-19 , Transplante de Rim , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados , Anticorpos Neutralizantes , Humanos , Estudos Retrospectivos , SARS-CoV-2RESUMO
BACKGROUND: Humoral and cellular immune responses to SARS-CoV-2 vaccination among immunosuppressed patients remain poorly defined, as well as variables associated with poor response. METHODS: We performed a retrospective observational cohort study at a large Northern California healthcare system of infection-naïve individuals fully vaccinated against SARS-CoV-2 (mRNA-1273, BNT162b2, or Ad26.COV2.S) with clinical SARS-CoV-2 interferon gamma release assay (IGRA) ordered between January through November 2021. Humoral and cellular immune responses were measured by anti-SARS-CoV-2 S1 IgG ELISA (anti-S1 IgG) and IGRA, respectively, following primary and/or booster vaccination. RESULTS: 496 immunosuppressed patients (54% female; median age 50 years) were included. 62% (261/419) of patients had positive anti-S1 IgG and 71% (277/389) had positive IGRA after primary vaccination, with 20% of patients having a positive IGRA only. Following booster, 69% (81/118) had positive anti-S1 IgG and 73% (91/124) had positive IGRA. Factors associated with low humoral response rates after primary vaccination included anti-CD20 monoclonal antibodies (P < 0.001), sphingosine 1-phsophate (S1P) receptor modulators (P < 0.001), mycophenolate (P = 0.002), and B cell lymphoma (P = 0.004); those associated with low cellular response rates included S1P receptor modulators (P < 0.001) and mycophenolate (P < 0.001). Of patients who had poor humoral response to primary vaccination, 35% (18/52) developed a significantly higher response after the booster. Only 5% (2/42) of patients developed a significantly higher cellular response to the booster dose compared to primary vaccination. CONCLUSIONS: Humoral and cellular response rates to primary and booster SARS-CoV-2 vaccination differ among immunosuppressed patient groups. Clinical testing of cellular immunity is important in monitoring vaccine response in vulnerable populations.
Assuntos
COVID-19 , Vacinas Virais , Ad26COVS1 , Anticorpos Antivirais , Vacina BNT162 , COVID-19/prevenção & controle , Vacinas contra COVID-19 , Feminino , Humanos , Imunidade Humoral , Imunoglobulina G , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , SARS-CoV-2 , VacinaçãoRESUMO
As insulin's movement from plasma to muscle interstitium is rate limiting for its metabolic action, defining the regulation of this movement is critical. Here, we address whether caveolin-1 is required for the first step of insulin's transendothelial transport, its uptake by vascular endothelial cells (ECs), and whether IL-6 and TNFα affect insulin uptake or caveolin-1 expression. Uptake of FITC-labeled insulin was measured using confocal microscopy in control bovine aortic ECs (bAECs), in bAECs in which caveolin-1 was either knocked down or overexpressed, in murine ECs from caveolin-1(-/-) mice and in bAECs exposed to inflammatory cytokines. Knockdown of caveolin-1 expression in bAECs using specific caveolin-1 siRNA reduced caveolin-1 mRNA and protein expression by â¼ 70%, and reduced FITC-insulin uptake by 67% (P < 0.05 for each). Over-expression of caveolin-1 increased insulin uptake (P < 0.05). Caveolin-1-null mouse aortic ECs did not take up insulin and re-expression of caveolin-1 by transfecting these cells with FLAG-tagged caveolin-1 DNA rescued FITC-insulin uptake. Knockdown of caveolin-1 significantly reduced both insulin receptor protein level and insulin-stimulated Akt1 phosphorylation. Knockdown of caveolin-1 also inhibited insulin-induced caveolin-1 and IGF-1 receptor translocation to the plasma membrane. Compared with controls, IL-6 or TNFα (20 ng/ml for 24 h) inhibited FITC-insulin uptake as well as the expression of caveolin-1 mRNA and protein (P < 0.05 for each). IL-6 or TNFα also significantly reduced plasma membrane-associated caveolin-1. Thus, we conclude that insulin uptake by ECs requires expression of caveolin-1 supporting a role for caveolae mediating insulin uptake. Proinflammatory cytokines may inhibit insulin uptake, at least in part, by inhibiting caveolin-1 expression.
Assuntos
Caveolina 1/fisiologia , Endotélio Vascular/metabolismo , Insulina/metabolismo , Animais , Aorta/citologia , Aorta/metabolismo , Transporte Biológico , Bovinos , Caveolina 1/genética , Membrana Celular/metabolismo , Células Cultivadas , Endotélio Vascular/citologia , Expressão Gênica , Mediadores da Inflamação/metabolismo , Camundongos , Camundongos Knockout , Fosforilação , Transporte Proteico , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/metabolismo , RNA Interferente Pequeno , Receptor de Insulina/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND AND OBJECTIVES: In patients with kidney failure due to IgA nephropathy, IgA deposits can recur in a subsequent kidney transplant. The incidence, effect, and risk factors of IgA nephropathy recurrence is unclear, because most studies have been single center and sample sizes are relatively small. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: We performed a multicenter, international, retrospective study to determine the incidence, risk factors, and treatment response of recurrent IgA nephropathy after kidney transplantation. Data were collected from all consecutive patients with biopsy-proven IgA nephropathy transplanted between 2005 and 2015, across 16 "The Post-Transplant Glomerular Disease" study centers in Europe, North America, and South America. RESULTS: Out of 504 transplant recipients with IgA nephropathy, recurrent IgA deposits were identified by kidney biopsy in 82 patients; cumulative incidence of recurrence was 23% at 15 years (95% confidence interval, 14 to 34). Multivariable Cox regression revealed a higher risk for recurrence of IgA deposits in patients with a pre-emptive kidney transplant (hazard ratio, 3.45; 95% confidence interval, 1.31 to 9.17) and in patients with preformed donor-specific antibodies (hazard ratio, 2.59; 95% confidence interval, 1.09 to 6.19). After kidney transplantation, development of de novo donor-specific antibodies was associated with subsequent higher risk of recurrence of IgA nephropathy (hazard ratio, 6.65; 95% confidence interval, 3.33 to 13.27). Immunosuppressive regimen was not associated with recurrent IgA nephropathy in multivariable analysis, including steroid use. Graft loss was higher in patients with recurrence of IgA nephropathy compared with patients without (hazard ratio, 3.69; 95% confidence interval, 2.04 to 6.66), resulting in 32% (95% confidence interval, 50 to 82) graft loss at 8 years after diagnosis of recurrence. CONCLUSIONS: In our international cohort, cumulative risk of IgA nephropathy recurrence increased after transplant and was associated with a 3.7-fold greater risk of graft loss.
Assuntos
Anticorpos/sangue , Glomerulonefrite por IGA/epidemiologia , Falência Renal Crônica/etiologia , Falência Renal Crônica/cirurgia , Adulto , Aloenxertos/imunologia , Aloenxertos/patologia , Biópsia , Brasil/epidemiologia , Europa (Continente)/epidemiologia , Feminino , Glomerulonefrite por IGA/complicações , Glomerulonefrite por IGA/tratamento farmacológico , Glomerulonefrite por IGA/patologia , Sobrevivência de Enxerto , Humanos , Incidência , Rim/patologia , Transplante de Rim , Masculino , Pessoa de Meia-Idade , Recidiva , Estudos Retrospectivos , Fatores de Risco , Estados Unidos/epidemiologiaRESUMO
Insulin resistance strongly associates with decreased nitric oxide (NO) bioavailability and endothelial dysfunction. In the vasculature, NO mediates multiple processes that affect insulin delivery, including dilating both resistance and terminal arterioles in skeletal muscle in vivo. However, whether NO directly regulates vascular endothelial cell (EC) insulin uptake and its transendothelial transport (TET) is unknown. We report in this article that L-N(G)-nitro-L-arginine methyl ester (L-NAME) pretreatment blocked, whereas L-arginine and sodium nitroprusside (SNP) each enhanced, EC uptake of fluorescein isothiocyanate (FITC)-labeled insulin. SNP also partly or fully reversed the inhibition of EC insulin uptake caused by L-NAME, wortmannin, the Src inhibitor PP1, and tumor necrosis factor-α. In addition, SNP promoted [(125)I]Tyr(A14)insulin TET by ~40%. Treatment with insulin with and without SNP did not affect EC cyclic guanosine monophosphate (cGMP) levels, and the cGMP analog 8-bromo-cGMP did not affect FITC-insulin uptake. In contrast, treatment with insulin and SNP significantly increased EC protein S-nitrosylation, the colocalization of S-nitrosothiol (S-NO) and protein-tyrosine phosphatase 1B (PTP1B), and Akt phosphorylation at Ser(473) and inhibited PTP1B activity. Moreover, a high-fat diet significantly inhibited EC insulin-stimulated Akt phosphorylation and FITC-insulin uptake that was partially reversed by SNP in rats. Finally, inhibition of S-nitrosylation by knockdown of thioredoxin-interacting protein completely eliminated SNP-enhanced FITC-insulin uptake. We conclude that NO directly promotes EC insulin transport by enhancing protein S-nitrosylation. NO also inhibits PTP1B activity, thereby enhancing insulin signaling.
Assuntos
Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Insulina/metabolismo , Óxido Nítrico/metabolismo , Animais , Aorta/citologia , Aorta/efeitos dos fármacos , Aorta/metabolismo , Arginina/farmacologia , Bovinos , Células Cultivadas , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Masculino , NG-Nitroarginina Metil Éster/farmacologia , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Nitroprussiato/farmacologia , Fosforilação/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Insulin's trans-endothelial transport (TET) is critical for its metabolic action on muscle and involves trafficking of insulin bound to its receptor (or at high insulin concentrations, the IGF-I receptor) via caveolae. However, whether caveolae-mediated insulin TET involves actin cytoskeleton organization is unknown. Here we address whether insulin regulates actin filament organization in bovine aortic endothelial cells (bAEC) and whether this affects insulin uptake and TET. We found that insulin induced extensive cortical actin filament remodeling within 5 min. This remodeling was inhibited not only by disruption of actin microfilament organization but also by inhibition of phosphatidylinositol 3-kinase (PI3K) or by disruption of lipid rafts using respective specific inhibitors. Knockdown of either caveolin-1 or Akt using specific small interfering RNA also eliminated the insulin-induced cortical actin filament remodeling. Blocking either actin microfilament organization or PI3K pathway signaling inhibited both insulin uptake and TET. Disruption of actin microfilament organization also reduced the caveolin-1, insulin receptor, and IGF-I receptor located at the plasma membrane. Exposing bAEC for 6 h to either TNFα or IL-6 blocked insulin-induced cortical actin remodeling. Extended exposure (24 h) also inhibited actin expression at both mRNA and protein levels. We conclude that insulin-induced cortical actin filament remodeling in bAEC is required for insulin's TET in a PI3K/Akt and plasma membrane lipid rafts/caveolae-dependent fashion, and proinflammatory cytokines TNFα and IL-6 block this process.
Assuntos
Citoesqueleto de Actina/metabolismo , Células Endoteliais/metabolismo , Insulina/fisiologia , Actinas/antagonistas & inibidores , Actinas/metabolismo , Animais , Aorta/citologia , Transporte Biológico , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Bovinos , Caveolina 1/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Endotélio Vascular/citologia , Insulina/metabolismo , Interleucina-6/farmacologia , Interleucina-6/fisiologia , Microdomínios da Membrana/metabolismo , Microscopia de Fluorescência , Receptor IGF Tipo 1/metabolismo , Receptor de Insulina/metabolismo , Tiazolidinas/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Fator de Necrose Tumoral alfa/fisiologiaRESUMO
Endothelial nitric oxide synthase (eNOS) activity is tightly regulated by posttranscriptional modification and its subcellular localization. Here we examined whether insulin modulates nitric oxide (NO) production by regulating eNOS subcellular localization. We used confocal microscopy and immunoblots to examine the time course for 1) subcellular targeting/association of eNOS and caveolin-1 (CAV-1); 2) eNOS Ser(1179) phosphorylation; and 3) NO production in cultured bovine aorta endothelial cells. Serum starvation increased eNOS/CAV-1 localization to the perinuclear region. Adding insulin provoked their prompt translocation to and association at the plasma membrane (PM). Specific monoclonal antibodies against either CAV-1 or eNOS coimmunoprecipitated the other from bovine aorta endothelial cell membrane extracts, and insulin increased this interaction. Insulin stimulated NO production transiently despite a persistent eNOS Ser(1179) phosphorylation. The decline of NO production correlated temporally to insulin-induced translocation of eNOS and CAV-1 to PM. Knockdown of CAV-1 expression with a specific small interfering RNA duplex resulted in eNOS redistributing to the perinuclear region and nearly doubled insulin-induced NO production. Inhibition of phosphatidylinositol 3-kinase activity with wortmannin not only significantly inhibited insulin-induced translocation of eNOS and CAV-1 to PM but also blocked insulin-induced interaction of CAV-1 with eNOS at PM. Insulin increased incorporation of [(3)H]palmitic acid into eNOS immunoprecipitates by approximately 140%. Insulin-induced translocation of eNOS and CAV-1 to PM was palmitoylation dependent. Inhibiting eNOS and CAV-1 palmitoylation enhanced the NO production while blocking the translocation of eNOS and CAV-1 to PM induced by insulin. These data show that insulin acutely regulates eNOS and CAV-1 trafficking to PM of vascular endothelial cells where their interaction can regulate eNOS activity.
Assuntos
Caveolina 1/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/enzimologia , Insulina/farmacologia , Óxido Nítrico Sintase Tipo III/metabolismo , Óxido Nítrico/biossíntese , Aciltransferases/metabolismo , Androstadienos/farmacologia , Animais , Bovinos , Caveolina 1/antagonistas & inibidores , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Células Endoteliais/citologia , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/enzimologia , Lipoilação/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Fosfosserina/metabolismo , Ligação Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , RNA Interferente Pequeno/metabolismo , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/enzimologia , WortmaninaRESUMO
OBJECTIVE: In vivo evidence suggests that insulin entry into skeletal muscle is rate limiting for its overall metabolic action. Although there has been controversy regarding whether insulin crosses the endothelium by a passive (transcellular or paracellular) or mediated process, accumulating data favor the latter. Here, we addressed whether insulin signaling within the endothelial cell is required for the first step of transendothelial insulin transport: its uptake by the endothelial cell. RESEARCH DESIGN AND METHODS: Bovine aortic endothelial cells (bAECs) were incubated in serum-free medium for 6 h before addition of 50 nmol/l fluoroisothiocyanate (FITC)-labeled insulin for 30 min, and uptake of FITC insulin was quantified by confocal immunocytochemistry. RESULTS: Cellular insulin uptake was temperature dependent, being greater at 37 vs. 4 degrees C (P < 0.05). Inhibiting phosphatidylinositol 3-kinase (PI 3-kinase) (wortmannin), mitogen-activated protein kinase kinase (MEK) (PD98059), the cSrc-family tyrosine kinase (PP1), or the insulin receptor tyrosine kinase (genistein) markedly diminished FITC insulin uptake (P < 0.05 for each). In contrast, inhibiting the phosphotyrosine phosphatase protein tyrosine phosphatase 1B further stimulated insulin uptake (P < 0.05). Addition of the inflammatory cytokine 5 ng/ml tumor necrosis factor-alpha (TNF-alpha) for 6 h before adding 50 nmol/l FITC insulin diminished insulin uptake significantly (P < 0.05). This inhibitory effect of TNF-alpha could be partially reversed by a specific p38 MAPK inhibitor (SB203580). CONCLUSIONS: Insulin uptake by bAECs requires intact insulin signaling via both the PI 3-kinase and MEK signaling cascades and the cSrc-family tyrosine kinases, and endothelial cell insulin uptake is sensitive to cytokine-induced insulin resistance.