RESUMO
Magnesium (Mg2+) abnormalities during sepsis have been reported, but the underlying mechanisms during acute inflammation are poorly understood. We hypothesized that a decrease in GFR and/or changes in transporters or channels for Mg2+ could be responsible for the observed Mg2+ abnormalities. Therefore, we studied the metabolism of Mg2+ in a murine model of endotoxemia. LPS-induced hypermagnesemia was paralleled by a decrease in creatinine clearance and an increase in the fractional excretion of Mg2+. In agreement with an altered renal Mg2+ handling, endotoxemia decreased the renal expression of claudin (Cldn) 10b, Cldn16, Cldn19, parvalbumin, and of the solute carrier family (Slc) 41a3. Further, LPS increased the renal expression of Cldn14 and Slc41a1. The renal expression of the transient receptor potential melastin (Trpm) 6, Trpm7, and of cyclin M (Cnnm) 2 was unaltered in response to LPS. In vitro studies support a direct effect on the expression of Cldn10b, Cldn14, Cldn16, and Cldn19. Further, endotoxemia increased the fractional excretion of sodium, which was paralleled by a decrease of important renal sodium transporters. In the large intestine, the expression of Trpm7 was increased in response to LPS, whereas the expression of Trpm6 was decreased. Cnnm4 mRNA levels were unchanged in the large intestine. Further, Cldn12 and Na+-H+ exchanger 3 (Slc9a3) expressions were decreased in the small intestine in response to LPS. Our findings indicate that endotoxemia is associated with hypermagnesemia and a disturbed Mg2+ handling. It seems likely that LPS-induced hypermagnesemia is due to the decrease in renal function in response to LPS.
Assuntos
Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/metabolismo , Rim/metabolismo , Lipopolissacarídeos/farmacologia , Magnésio/metabolismo , Animais , Modelos Animais de Doenças , Endotoxemia/tratamento farmacológico , Endotoxemia/metabolismo , Inflamação/metabolismo , Masculino , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/metabolismo , Sódio/metabolismo , Trocador 3 de Sódio-Hidrogênio/metabolismoRESUMO
Fibroblast growth factor 23 (FGF23) levels are elevated in patients with acute kidney injury (AKI). The consequences on renal Ca2+, Mg2+, and Pi regulatory mechanisms are unknown. We hypothesized that renal ischemia-reperfusion (I/R) injury alters the expression of important renal Ca2+, Mg2+, and Pi transport proteins. I/R injury was induced in male C57BL/6 mice by clamping both renal arteries for 27 min. Mice were investigated 18 h later. The mRNA and protein levels of renal Ca2+, Mg2+, and Pi transport proteins were measured by RT-qPCR and western blot analysis. I/R injury-induced hyperphosphatemia and hypermagnesemia were paralleled by a decrease in glomerular filtration rate and an increase in the fractional excretion of Ca2+, Mg2+, and Pi. I/R injury affected the fibroblast growth factor 23 (FGF23)-klotho-vitamin D axis by increasing plasma levels of FGF23 and downregulation of renal klotho expression. Plasma levels of PTH and 1,25-dihydroxyvitamin D3 were unchanged. Further, downregulation of key genes for paracellular reabsorption of Ca2+ and Mg2+ (claudin (Cldn)2, Cldn10b, Cldn16, Cldn19) and for active transcellular transport of Ca2+, Mg2+, and Pi (calbindin-D28K, Ncx1, Pmca4, Cnnm2, Trpm7, NaPi-2a, and NaPi-2c) was observed. However, renal expression of Trpv5 and Trpv6 was increased. In vitro studies support a direct effect of proinflammatory cytokines on the mRNA expression of Cldn16, Cldn19, and Trpv6. Our findings indicate that renal I/R injury increases FGF23 blood levels independent of PTH and 1,25-dihydroxyvitamin D3. This increase is associated with hypermagnesemia, hyperphosphatemia, and increased or decreased expression of specific renal Ca2+, Mg2+, and Pi transporters, respectively.
Assuntos
Injúria Renal Aguda/metabolismo , Bombas de Íon/metabolismo , Traumatismo por Reperfusão/metabolismo , Animais , Cálcio/metabolismo , Fator de Crescimento de Fibroblastos 23 , Fatores de Crescimento de Fibroblastos/sangue , Glucuronidase/metabolismo , Proteínas Klotho , Magnésio/sangue , Masculino , Camundongos Endogâmicos C57BL , Fosfatos/sangue , Vitamina D/metabolismoRESUMO
Endotoxemia-related acute kidney injury (AKI) is associated with increased formation of prostaglandins, which may serve as a compensatory mechanism to maintain renal function. We hypothesized that an increase of renal EP2 or EP4 receptors and/or a downregulation of renal EP1 and EP3 receptors enhances PGE2-induced renal vasodilatation. Injection of lipopolysaccharide (LPS; 3 mg/kg i.p.) increased microsomal prostaglandin E synthase (mPGES)-1 and prostacyclin synthase expression, whereas mPGES-2 expression was unaltered. Further, LPS increased the mRNA abundance for the prostaglandin EP4 receptor, whereas the expressions of the EP1 and EP3 receptors were decreased. In isolated-perfused kidneys from control mice, PGE2 exerted a dual effect on renal vascular tone, inducing vasodilatation at lower concentrations and vasoconstriction at higher concentrations. In kidneys from endotoxemic mice, the vasodilatory component was more pronounced, whereas the vasoconstriction at higher PGE2 concentrations was absent. Similarly, prostacyclin (PGI2)-induced vasodilatation was more pronounced in endotoxemic kidneys. The enhanced vasodilatory effect was paralleled by an increase in renal vascular EP4 and prostacyclin IP receptor mRNA expression. Further, stimulation of renin secretion rate by PGE2 and PGI2 was enhanced in endotoxemic kidneys. Pretreatment with the cyclooxygenase (COX)-2 inhibitor SC-236 (10 mg/kg) did not alter the basal GFR, but augmented the LPS-induced decline in GFR, and attenuated the LPS-induced increase in plasma renin concentration in vivo. Our data suggest that an activation of the COX-2/mPGES-1 synthetic pathway is responsible for the increased renal formation of PGE2 in response to LPS and that the vasodilatory effect of PGE2 and PGI2 is enhanced during endotoxemia.
Assuntos
Injúria Renal Aguda/fisiopatologia , Endotoxemia/complicações , Rim/efeitos dos fármacos , Prostaglandinas/farmacologia , Vasodilatação , Vasodilatadores/farmacologia , Injúria Renal Aguda/etiologia , Injúria Renal Aguda/metabolismo , Animais , Rim/irrigação sanguínea , Rim/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Prostaglandina/genética , Receptores de Prostaglandina/metabolismo , Renina/metabolismoRESUMO
The protease renin is the key enzyme of the renin-angiotensin-aldosterone cascade, which is relevant under both physiological and pathophysiological settings. The kidney is the only organ capable of releasing enzymatically active renin. Although the characteristic juxtaglomerular position is the best known site of renin generation, renin-producing cells in the kidney can vary in number and localization. (Pro)renin gene transcription in these cells is controlled by a number of transcription factors, among which CREB is the best characterized. Pro-renin is stored in vesicles, activated to renin, and then released upon demand. The release of renin is under the control of the cAMP (stimulatory) and Ca(2+) (inhibitory) signaling pathways. Meanwhile, a great number of intrarenally generated or systemically acting factors have been identified that control the renin secretion directly at the level of renin-producing cells, by activating either of the signaling pathways mentioned above. The broad spectrum of biological actions of (pro)renin is mediated by receptors for (pro)renin, angiotensin II and angiotensin-(1-7).
Assuntos
Rim/metabolismo , Renina/metabolismo , Angiotensinas/genética , Angiotensinas/metabolismo , Animais , Regulação da Expressão Gênica , Humanos , Rim/citologia , Renina/genética , Transdução de SinaisRESUMO
INTRODUCTION: Mechanical and morphological ischemia and reperfusion (I/R) injury is reduced in septic hearts. The mechanism behind this "cardioprotection" is less well understood. As adenosine receptors play a major role for cardioprotection in non-septic hearts, we investigated the influence of adenosine receptors in a model of I/R in septic hearts. METHODS: SHAM operation or cecal ligation and puncture (CLP) was performed in adult male Wistar rats (n = 60). After 24 h of incubation, hearts were isolated and randomly assigned to a group with or without adenosine receptor (Ador) antagonists (SCH 58261 and MRS 1706) administered before reperfusion. Ischemia and reperfusion lasted for 40 min each. Cardiac function of the heart was determined by measuring left ventricular pressure (LVP). RESULTS: Before I/R, CLP hearts showed a significant mechanical left ventricular impairment (CLP: 63 ± 5 mmHg vs. SHAM: 104 ± 6 mmHg. After I/R, left ventricular function was significantly reduced in SHAM (24 ± 32 mmHg), but not in CLP hearts (65 ± 13 mmHg). mRNA expression for the AdorA2a and AdorA2b was significantly increased in CLP, but not in SHAM hearts. LVP of CLP hearts deteriorated when AdorA2a and AdorA2b were blocked. CONCLUSIONS: The morphological and functional I/R injury in septic animals is less pronounced compared to non-septic animals. By a combined blockade of AdorA2a and AdorA2b this "cardioprotective" effect is nearly abolished in septic hearts. This is the first study showing, that AdorA2a and AdorA2b may play an important role for a reduced functional I/R injury in the septic heart.
Assuntos
Traumatismo por Reperfusão Miocárdica/fisiopatologia , Receptor A2A de Adenosina/fisiologia , Receptor A2B de Adenosina/fisiologia , Sepse/fisiopatologia , Antagonistas do Receptor A2 de Adenosina/farmacologia , Antagonistas do Receptor A2 de Adenosina/uso terapêutico , Animais , Cardiotônicos/farmacologia , Cardiotônicos/uso terapêutico , Circulação Coronária/efeitos dos fármacos , Coração/efeitos dos fármacos , Coração/fisiologia , Masculino , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Traumatismo por Reperfusão Miocárdica/metabolismo , Miocárdio/metabolismo , RNA Mensageiro/metabolismo , Ratos Wistar , Receptor A2A de Adenosina/genética , Receptor A2B de Adenosina/genética , Sepse/tratamento farmacológico , Sepse/metabolismo , Função Ventricular Esquerda/efeitos dos fármacos , Pressão Ventricular/efeitos dos fármacosRESUMO
Thromboxane (Tx) A2 has been suggested to be involved in the development of sepsis-induced acute kidney injury (AKI). Therefore, we investigated the impact of cyclooxygenase (COX)-1 and COX-2 activity on lipopolysaccharide (LPS)-induced renal TxA2 formation, and on endotoxemia-induced AKI in mice. Injection of LPS (3 mg/kg ip) decreased glomerular filtration rate (GFR) and the amount of thrombocytes to â¼50% of basal values after 4 h. Plasma and renocortical tissue levels of TxB2 were increased â¼10- and 1.7-fold in response to LPS, respectively. The COX-1 inhibitor SC-560 attenuated the LPS-induced fall in GFR and in platelet count to â¼75% of basal levels. Furthermore, SC-560 abolished the increase in plasma and renocortical tissue levels of TxB2 in response to LPS. The COX-2 inhibitor SC-236 further enhanced the LPS-induced decrease in GFR to â¼40% of basal values. SC-236 did not alter thrombocyte levels nor the LPS-induced increase in plasma and renocortical tissue levels of TxB2. Pretreatment with clopidogrel inhibited the LPS-induced drop in thrombocyte count, but did not attenuate the LPS-induced decrease in GFR and the increase in plasma TxB2 levels. This study demonstrates that endotoxemia-induced TxA2 formation depends on the activity of COX-1. Our study further indicates that the COX-1 inhibitor SC-560 has a protective effect on the decrease in renal function in response to endotoxin. Therefore, our data support a role for TxA2 in the development of AKI in response to LPS.
Assuntos
Injúria Renal Aguda/prevenção & controle , Inibidores de Ciclo-Oxigenase 2/farmacologia , Endotoxemia/tratamento farmacológico , Taxa de Filtração Glomerular/efeitos dos fármacos , Rim/efeitos dos fármacos , Proteínas de Membrana/antagonistas & inibidores , Pirazóis/farmacologia , Tromboxano A2/metabolismo , Injúria Renal Aguda/enzimologia , Injúria Renal Aguda/etiologia , Injúria Renal Aguda/fisiopatologia , Animais , Plaquetas/efeitos dos fármacos , Plaquetas/enzimologia , Ciclo-Oxigenase 1/metabolismo , Modelos Animais de Doenças , Regulação para Baixo , Endotoxemia/induzido quimicamente , Endotoxemia/enzimologia , Endotoxemia/fisiopatologia , Endotoxinas , Rim/enzimologia , Rim/fisiopatologia , Masculino , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Inibidores da Agregação Plaquetária/farmacologia , Transdução de Sinais/efeitos dos fármacos , Fatores de TempoRESUMO
INTRODUCTION: Hypotension in septic patients results from hypovolemia, vasodilatation and hyporeactivity to vasoconstrictors, such as angiotensin II. The AT1 receptor-associated protein 1 (Arap1) is expressed in vascular smooth muscle cells and increases the surface expression of the AT1-receptor in vitro. We hypothesized that dysregulation of Arap1 may contribute to vascular hyporeactivity to angiotensin II during endotoxemia. METHODS: Arap1-deficient mice were used to assess the role of Arap1 in sepsis-induced hypotension. The isolated perfused kidney was used as an in vitro model to determine the relevance of Arap1 for vascular resistance and sensitivity to angiotensin II. RESULTS: During endotoxemia, mean arterial blood pressure (MAP) decreased in both genotypes, with the time course of sepsis-induced hypotension being markedly accelerated in Arap1-/- compared to +/+ mice. However, baseline MAP was similar in Arap1-/- and wildtype mice (102 ± 2 vs.103 ± 2 mmHg; telemetry measurements; n = 10; P = 0.66). Following lipopolysaccharide (LPS) injections (3 mg/kg), Arap1 expression was successively down-regulated in the wildtype mice, reaching levels below 10% of baseline expression. The endotoxemia-related decline in Arap1 expression could be recapitulated in cultured mesangial cells by incubation with pro-inflammatory cytokines, such as tumor necrosis factor α and interferon γ. Plasma renin concentration was increased in Arap1-/- mice compared to wildtype mice (66 ± 6 vs. 41 ± 4 ng AngI/ml/h; n = 23; P = 0.001), presumably contributing to preserved MAP under baseline conditions. The sensitivity of the vasculature to angiotensin II was reduced in Arap1-/- compared to +/+ mice, as determined in the isolated perfused kidney. CONCLUSIONS: Our data suggest that down-regulation of Arap1 expression during sepsis contributes to the development of hypotension by causing reduced vascular sensitivity to angiotensin II.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Hipotensão/etiologia , Hipotensão/metabolismo , Sepse/fisiopatologia , Animais , Pressão Sanguínea , Regulação para Baixo , Rim/metabolismo , Lipopolissacarídeos , Masculino , Camundongos , Renina/metabolismo , Sistema Renina-Angiotensina/fisiologia , Sepse/induzido quimicamenteRESUMO
Arap1 is a protein that interacts with angiotensin II type 1 (AT(1)) receptors and facilitates increased AT(1) receptor surface expression in vitro. In the present study, we assessed the tissue localization and regulation of Arap1 in vivo. Arap1 was found in various mouse organs, with the highest expression in the heart, kidney, aorta, and adrenal gland. Renal Arap1 protein was restricted to the vasculature and to glomerular mesangial cells and was absent from tubular epithelia. A similar localization was found in human kidneys. To test the hypothesis that angiotensin II may control renal Arap1 expression, mice were subjected to various conditions to alter the activity of the renin-angiotensin system. A high-salt diet (4% NaCl, 7 days) upregulated Arap1 expression in mice by 47% compared with controls (0.6% NaCl, P = 0.03). Renal artery stenosis (7 days) or water restriction (48 h) suppressed Arap1 levels compared with controls (-64 and -62% in the clipped and contralateral kidney, respectively; and -50% after water restriction, P < 0.01). Angiotensin II infusion (2 µg·kg(-1)·min(-1), 7 days) reduced Arap1 mRNA levels compared with vehicle by 29% (P < 0.01), whereas AT(1) antagonism (losartan, 30 mg·kg(-1)·day(-1), 7 days) enhanced Arap1 mRNA expression by 52% (P < 0.01); changes in mRNA were paralleled by Arap1 protein abundance. Experiments with hydralazine and epithelial nitric oxide synthase-/- mice further suggested that Arap1 expression changed in parallel with angiotensin II, rather than with blood pressure per se. Similar to in vivo, Arap1 mRNA and protein were suppressed by angiotensin II in a time- and dose-dependent manner in cultured mesangial cells. In summary, Arap1 is highly expressed in the renal vasculature, and its expression is suppressed by angiotensin II. Thus Arap1 may serve as a local modulator of vascular AT(1) receptor function in vivo.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Angiotensina II/farmacologia , Vasos Sanguíneos/fisiologia , Proteínas de Transporte/genética , Proteínas Ativadoras de GTPase/genética , Células Mesangiais/fisiologia , Circulação Renal/efeitos dos fármacos , Circulação Renal/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas de Transporte/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Receptor Tipo 1 de Angiotensina/metabolismo , Renina/metabolismo , Vasoconstritores/farmacologiaRESUMO
Severe sepsis is often accompanied by acute renal failure with renal tubular dysfunction. Albuminuria is a common finding in septic patients and we studied whether it was due to an impairment of proximal tubular endocytosis of filtered albumin. We studied the regulation of megalin and cubilin, the two critical multiligand receptors responsible for albumin absorption, during severe experimental endotoxemia. Lipopolysaccharide (LPS) caused a time- and dose-dependent suppression of megalin and cubilin expression that was paralleled by a decrease in plasma albumin levels and an increase in the urine concentration of albumin in mice. Incubation of rat renal cortical slices with LPS also reduced the mRNA expression of megalin and cubilin. Further, LPS suppressed megalin and cubilin mRNA expression in murine primary proximal tubule cells and decreased the uptake of FITC albumin in these cells. In addition, the increase in urine levels of albumin in response to ischemia/reperfusion-induced acute renal failure was paralleled by a decrease in the expression of megalin and cubilin. Thus, our data indicate that the expression of megalin and cubilin is decreased during experimental endotoxemia and in response to renal ischemia/reperfusion injury. This downregulation may contribute, in part, to an increase in urine levels of albumin during acute renal failure.
Assuntos
Albuminúria/etiologia , Endotoxemia/complicações , Túbulos Renais Proximais/metabolismo , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Receptores de Superfície Celular/metabolismo , Injúria Renal Aguda/etiologia , Injúria Renal Aguda/genética , Injúria Renal Aguda/metabolismo , Albuminúria/sangue , Albuminúria/genética , Albuminúria/fisiopatologia , Albuminúria/urina , Animais , Biomarcadores/sangue , Células Cultivadas , Modelos Animais de Doenças , Regulação para Baixo , Endocitose , Endotoxemia/sangue , Endotoxemia/induzido quimicamente , Endotoxemia/genética , Endotoxemia/fisiopatologia , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/metabolismo , Capacidade de Concentração Renal , Túbulos Renais Proximais/fisiopatologia , Lipopolissacarídeos , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Superfície Celular/genética , Traumatismo por Reperfusão/complicações , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/metabolismo , Albumina Sérica/metabolismo , Fatores de TempoRESUMO
Acute renal failure (ARF) is frequently associated with polyuria and urine concentration defects and it is a severe complication of sepsis because it increases the mortality rate. Inhibition of NF-kappaB activation has been suggested to provide a useful strategy for the treatment of septic shock. However, the impact on sepsis-induced ARF is still unclear. Therefore, we examined the effect of pyrrolidine dithiocarbamate (PDTC) and of small interfering RNA (siRNA) silencing NF-kappaB p50/p105 on sepsis-induced downregulation of vasopressin V(2) receptors and aquaporin (AQP)-2 channels using a cecal ligation and puncture (CLP) mouse model. CLP caused a time-dependent downregulation of renal vasopressin V(2) receptor and of AQP2 expression without alterations in plasma vasopressin levels. Renal activation of NF-kappaB in response to CLP was attenuated by PDTC pretreatment, which also attenuated the downregulation of V(2) receptor and AQP2 expression. Furthermore, a strong nuclear staining for the NF-kappaB p50 subunit throughout the whole kidney in response to CLP was observed. siRNA against NF-kappaB p50 attenuated the CLP-induced nuclear translocation of the p50 subunit and the CLP-induced downregulation of V(2) receptor and AQP2 expression. Additionally, PDTC and siRNA pretreatment inhibited the CLP-induced increase in renal TNF-alpha and IL-1beta concentration and NOS-2 mRNA abundance. Moreover, PDTC and siRNA pretreatment ameliorated CLP-induced hypotension and ARF. Our findings suggest that NF-kappaB activation is of importance for the downregulation of AQP2 channel and vasopressin V(2) receptor expression during sepsis. In addition, our data indicate that NF-kappaB inhibition ameliorates sepsis-induced ARF.
Assuntos
Injúria Renal Aguda/etiologia , Injúria Renal Aguda/metabolismo , Aquaporina 2/metabolismo , Regulação para Baixo/fisiologia , NF-kappa B/antagonistas & inibidores , Receptores de Vasopressinas/metabolismo , Sepse/complicações , Injúria Renal Aguda/prevenção & controle , Animais , Antioxidantes/farmacologia , Ceco/fisiopatologia , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Interleucina-1beta/metabolismo , Ligadura , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Pirrolidinas/farmacologia , Sepse/metabolismo , Tiocarbamatos/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , Vasopressinas/sangueRESUMO
In this prospective, randomized animal study, the role of proinflammatory cytokines in the pathogenesis sepsis-induced circulatory failure with downregulation of angiotensin-II-type-I-(AT(1))-receptors was investigated. Sepsis in wild-type mice and in mice with deficiencies for TNF-alpha, IL-1beta, IFN-gamma or IL-6 was induced by cecal ligation and puncture (CLP) and wild-type mice were injected with cytokines. Animals were treated with glucocorticoids or small interfering RNA (siRNA) targeting single or multiple cytokines or NF-kappaB. Vascular smooth muscle cells (VSMCs) were incubated with cytokines. CLP resulted in circulatory failure and a significant downregulation of AT(1)-receptors. Injection of single proinflammatory cytokines also strongly downregulated AT(1)-receptors paralleled by a markedly endogenous liberation of further cytokines, whereas, simultaneous blockade of these endogenously activated cytokines by dexamethasone prevented downregulation of AT(1)-receptors. Furthermore, inhibition of multiple but not single cytokines by treatment with siRNA against multiple cytokines or NF-kappaB significantly attenuated CLP-induced AT(1)-receptor downregulation and prevented septic circulatory failure. Our data demonstrate that downregulation of AT(1)-receptors during sepsis is due to multiple but not single cytokines and define a relevant role for NF-kappaB in the pathogenesis of septic shock.
Assuntos
Citocinas/antagonistas & inibidores , Regulação para Baixo , Receptor Tipo 1 de Angiotensina/metabolismo , Choque Séptico/metabolismo , Animais , Citocinas/genética , Citocinas/metabolismo , Dexametasona/metabolismo , Glucocorticoides/metabolismo , Hemodinâmica , Humanos , Rim/metabolismo , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , NF-kappa B/antagonistas & inibidores , NF-kappa B/genética , NF-kappa B/metabolismo , Estudos Prospectivos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Distribuição Aleatória , Receptor Tipo 1 de Angiotensina/genéticaRESUMO
Renal excretion of organic anions such as para-aminohippurate is reduced during severe sepsis and following ischemia/reperfusion injury. In order to better define the pathophysiology of sepsis-associated renal tubular dysfunction we measured the effect of lipopolysaccharide on renocortical organic anion transporter (OAT) expression in the rat. Prostaglandin E2 (PGE2) downregulates OATs in vitro, therefore, we also evaluated the effect of the cyclooxygenase (COX)-2 inhibitor parecoxib on this process. Endotoxemia caused a time- and dose-dependent decrease of OAT1 and OAT3 expression that paralleled increased renocortical COX-2 expression and PGE2 formation. Pretreatment with parecoxib decreased endotoxin-stimulated PGE(2) formation. Parecoxib attenuated OAT1 and OAT3 gene repression in the rat kidney following endotoxin treatment and during ischemia/reperfusion-induced acute renal injury. COX-2 inhibition improved the creatinine clearance in lipopolysaccharide-treated rats but not after ischemia/reperfusion-induced acute renal injury. The decreased clearance of para-aminohippurate in rats following endotoxin- or ischemia/reperfusion-induced renal injury was improved by parecoxib. Our findings show that COX-2 derived prostanoids downregulate OATs during lipopolysaccharide-induced acute renal injury.
Assuntos
Inibidores de Ciclo-Oxigenase 2/farmacologia , Ciclo-Oxigenase 2/fisiologia , Córtex Renal/metabolismo , Lipopolissacarídeos/farmacologia , Transportadores de Ânions Orgânicos/genética , Doença Aguda , Animais , Dinoprostona , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Isoxazóis/farmacologia , Nefropatias/induzido quimicamente , Masculino , Proteína 1 Transportadora de Ânions Orgânicos/genética , Transportadores de Ânions Orgânicos Sódio-Independentes/genética , Ratos , Ratos Sprague-DawleyRESUMO
Chronic heart failure is one of the most frequent causes of death in humans. Knockout of type 5 adenylyl cyclase (AC) in mice causes longevity and protection from cardiomyopathy, and an AC5 inhibitor reduces beta-adrenoceptor-stimulated Ca(2+) inward currents in isolated mouse cardiomyocytes. These data indicate that selective AC5 inhibitors may be beneficial in chronic heart failure. Therefore, we characterized AC in mouse heart membranes. Real-time polymerase chain reaction and immunoblot analysis suggested that AC5 is an important heart AC isoform. Enzyme kinetics of heart AC and recombinant AC5 in the presence of Mg(2+) were similar. Moreover, the inhibitory profile of eight 2'(3')-O-(N-methylanthraniloyl) (MANT)-nucleoside 5'-([gamma-thio])triphosphates on mouse heart in the presence of Mg(2+) was almost identical to that of AC5. MANT-ITP was the most potent inhibitor of heart AC and recombinant AC5, with K(i) values in the 15 to 25 nM range in the presence of Mg(2+) and in the 1 to 5 nM range in the presence of Mn(2+). However, in the presence of Mn(2+), we also noted differences between mouse heart AC and AC5 with respect to enzyme kinetics and forskolin analog effects. In conclusion, with regard to expression and kinetics and inhibition by MANT-nucleotides in the presence of Mg(2+), AC5 is an important AC isoform in heart, with MANT-ITP being an excellent starting point for the design of AC5-selective inhibitors. Unfortunately, a limitation of our study is the fact that immunologically and biochemically, AC5 and AC6 are quite similar, although they have different roles in heart. Moreover, lack of antibody specificity and Mn(2+) masking AC5 effects were problems.
Assuntos
Adenilil Ciclases/fisiologia , Miocárdio/enzimologia , Inibidores de Adenilil Ciclases , Adenilil Ciclases/química , Adenilil Ciclases/genética , Adenilil Ciclases/metabolismo , Animais , Catálise , Cátions Bivalentes/química , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Colforsina/análogos & derivados , Colforsina/farmacologia , Inibidores Enzimáticos/química , Feminino , Expressão Gênica/genética , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Guanosina Trifosfato/farmacologia , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Isoproterenol/farmacologia , Cinética , Metoprolol/farmacologia , Camundongos , Camundongos Endogâmicos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , SpodopteraRESUMO
AIM: Alterations in parathyroid hormone (PTH) and/or vitamin D signalling are frequently reported in patients with sepsis. The consequences on renal and intestinal Ca2+ and Pi regulatory mechanisms are still unclear. We hypothesized that endotoxaemia alters the expression of important renal and intestinal Ca2+ and Pi transport proteins. METHODS: Male C57BL/6 mice were treated with lipopolysaccharide (LPS; 3 mg/kg; i.p.). The mRNA and protein levels of renal and intestinal Ca2+ and Pi transport proteins were measured by RT-qPCR, immunohistochemistry and western blot analysis. RESULTS: Lipopolysaccharide-induced hypocalcaemia and hyperphosphataemia was paralleled by a decrease in glomerular filtration rate and urinary excretion of Ca2+ and Pi . Endotoxaemia augmented plasma levels of PTH and affected the fibroblast growth factor 23 (FGF23)-klotho-vitamin D axis by increasing plasma levels of FGF23 and downregulation of renal klotho expression. Renal expression of CYP27b1 and plasma levels of 1,25-dihydroxyvitamin D3 were increased in response to LPS. Endotoxaemia augmented the renal expression of TRPV5, TRPV6 and PiT1, whereas the renal expression of calbindin-D28K , NCX1, NaPi -2a and NaPi -2c were decreased. Incubation of primary distal tubule cells with LPS increased TRPV6 mRNA levels. Furthermore, LPS decreased the intestinal expression of TRPV6, calbindin-D9K and of NaPi -2b. CONCLUSION: Our findings indicate that endotoxaemia is associated with hypocalcaemia and hyperphosphataemia and a disturbed FGF23-klotho-vitamin D signaling. Further, LPS-induced acute kidney injury was accompanied by an increased or decreased expression of specific renal and intestinal Ca2+ and Pi transporters respectively. It seems unlikely that LPS-induced hypocalcaemia is due to renal loss of Ca2+ .
Assuntos
Cálcio/metabolismo , Endotoxemia/induzido quimicamente , Nefropatias/induzido quimicamente , Rim/metabolismo , Lipopolissacarídeos/toxicidade , Albuminúria , Animais , Transporte Biológico , Cálcio/urina , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Creatinina/urina , Regulação para Baixo , Endotoxemia/patologia , Fator de Crescimento de Fibroblastos 23 , Regulação Enzimológica da Expressão Gênica , Glucuronidase/genética , Glucuronidase/metabolismo , Hiperfosfatemia/induzido quimicamente , Hipocalcemia/induzido quimicamente , Proteínas Klotho , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase em Tempo Real/métodos , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismo , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , Regulação para CimaRESUMO
It has been reported that the serine protease kallikrein stimulates and that aprotinin, a protease inhibitor, inhibits renal renin secretion. Since direct stimulation of the protease-activated receptor (PAR) 2 increases renin secretion in isolated perfused mouse kidneys, we hypothesized that activation of PAR2 receptors by serine proteases could be involved in the synthesis and secretion of renin in vivo. We therefore determined the response of plasma renin concentration (PRC) to acute intraperitoneal administration of the PAR2 agonist SLIGRL, isoproterenol, hydralazine, furosemide, losartan, or lipopolysaccharide in conscious wild-type (WT) and Par2-deficient mice. Basal PRC was not different in Par2-deficient mice compared with WT mice. All six acute treatments caused significant increases of PRC in both WT and Par2-deficient mice. The response was significantly lower only in endotoxin-treated Par2-deficient mice. Chronic treatment with losartan, low salt intake, the combination of both, or furosemide caused an increase of PRC and renin mRNA in WT mice, whereas a high salt intake caused a decrease. Alterations in PRC and renal renin mRNA expression were not different between WT and Par2 -/- mice in response to chronic treatments. Par2-deficiency did not alter furosemide-induced diuresis and natriuresis. Systolic blood pressure responses to chronic treatments were not different between WT and Par2 -/- mice. In conclusion, deficiency of Par2 receptors does not alter renin secretion and renin gene expression modulated by a variety of typical maneuvers. However, activation of Par2 receptors by serine proteases seems to be of importance for renin secretion in the context of inflammation.
Assuntos
Rim/metabolismo , Receptor PAR-2/fisiologia , Renina/biossíntese , Animais , Relação Dose-Resposta a Droga , Furosemida/farmacologia , Expressão Gênica/efeitos dos fármacos , Hidralazina/farmacologia , Isoproterenol/farmacologia , Rim/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Losartan/farmacologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oligopeptídeos/farmacologia , Receptor PAR-2/agonistas , Receptor PAR-2/genética , Renina/sangue , Renina/genética , Cloreto de Sódio/farmacologiaRESUMO
OBJECTIVE: Here we characterize the impact of nuclear factor-kappaB and cytokines on cecal ligation and puncture-induced circulatory failure and regulation of vasopressin V1A-receptors during inflammation. DESIGN: Prospective animal trial. SETTING: Laboratory of the Department of Anesthesiology. SUBJECTS: Male C57/BL6 mice. INTERVENTIONS: The effects of cecal ligation and puncture on hemodynamic parameters and V1A-receptor expression were measured in cytokine knock-out mice, in mice with/without treatment with glucocorticoids or NF-kappaB-inhibitors, in mice pretreated with small interfering RNA silencing NF-kappaB and in mice treated with V1 receptor agonists. Furthermore, the effects of cytokines on V1A-receptor expression were determined. MEASUREMENTS AND MAIN RESULTS: Cecal ligation and puncture resulted in a hyperdynamic circulatory failure with diminished blood pressor dose response to V1 receptor agonists and down-regulation of V1A-receptors. Dexamethasone inhibited proinflammatory cytokine production and attenuated cecal ligation and puncture-induced cardiovascular failure in parallel with attenuated down-regulation of V1A-receptor expression. Tumor necrosis factor-alpha, interleukin-1beta, interferon-gamma or interleukin-6 dose-dependently decreased V1A-receptor expression, whereas cecal ligation and puncture-induced down-regulation of V1A-receptors was not affected in cytokine knock-out mice. In contrast, inhibition of NF-kappaB strongly reduced induction of cytokines, prevented septic circulatory failure and down-regulation of V1A-receptor gene expression and improved survival of septic animals. CONCLUSIONS: Our data demonstrate that down-regulation of V1A-receptor expression during sepsis may be due to proinflammatory cytokines. Our findings explain the failure of therapeutic strategies targeting single cytokines as well as the success of glucocorticoid therapy and define a critical role for NF-kappaB in the pathogenesis of septic shock.
Assuntos
Citocinas/biossíntese , NF-kappa B/fisiologia , Receptores de Vasopressinas/fisiologia , Sepse/metabolismo , Choque/metabolismo , Animais , Ceco/cirurgia , Citocinas/fisiologia , Regulação para Baixo/fisiologia , Ligadura , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Choque/etiologiaRESUMO
Based on recent evidence that renal cyclooxygenase-2 (COX-2) gene expression is suppressed by immunosuppressive agents such as cyclosporin A (CsA), tacrolimus and dexamethasone, this study aimed to characterize the effect of the new immunosuppressant everolimus on COX-2 expression in the rat kidney. Oral application of everolimus (3 mg kg(-1) day(-1)) to male Sprague-Dawley rats (175-200 g; n=8) for 7 days lowered COX-2 expression in the rat renal cortex and outer medulla, while COX-2 expression in the inner medulla as well as COX-1 expression remained unaltered. Furthermore, everolimus decreased renocortical prostaglandin (PG) E(2) concentration. Everolimus also attenuated the stimulation of renocortical COX-2 expression by furosemide (12 mg day(-1) for 7 days; s.c. via osmotic minipumps), by low salt intake (0.02% NaCl, wt wt(-1)) or by a combination of low salt intake with the AT(1)-receptor antagonist valsartan (30 mg kg(-1) day(-1); oral). In line with these findings, everolimus decreased renocortical PGE(2) concentration during these treatment maneuvers. Everolimus moderately increased natriuresis and diuresis, while the urinary excretion of PGE(2), 6-keto PGF(1alpha) and thromboxane B(2) was decreased. These findings suggest that everolimus inhibits basal and also stimulated expression of renocortical COX-2 and of tissue prostanoid formation. Since inhibition of renal prostanoid formation by everolimus was associated by an increased rather than decreased natriuresis and diuresis, it appears as if everolimus also inhibits tubular salt and water resorption.
Assuntos
Regulação para Baixo , Imunossupressores/farmacologia , Rim/efeitos dos fármacos , Sirolimo/análogos & derivados , Animais , Creatinina/sangue , Creatinina/urina , Everolimo , Immunoblotting , Rim/enzimologia , Masculino , Potássio/urina , Prostaglandinas/urina , RNA Mensageiro/biossíntese , Ratos , Ratos Sprague-Dawley , Sirolimo/farmacologia , Sódio/urina , Cloreto de Sódio na Dieta/administração & dosagemRESUMO
1 It is known that nonselective cyclo-oxygenase (COX) inhibitors have small but significant effects on blood pressure (BP), most notably in hypertensive patients on antihypertensive medication. Whether selective COX-2 inhibitors also interfere with BP regulation is not well understood. Therefore, we aimed to examine the effect of chronic treatment with a selective COX-2 inhibitor (rofecoxib) on systolic blood pressure (sBP) in normotensive Wistar-Kyoto rats (WKY) and on the developmental changes of sBP in young spontaneously hypertensive rats (SHR). In addition, we investigated a possible influence of salt intake on the effects of COX-2 inhibition on BP in these two rat strains. 2 Rofecoxib dose dependently increased sBP and decreased plasma levels of 6-keto prostaglandin (PG)F(1alpha) in WKY rats fed a normal salt diet (0.6% NaCl, wt wt(-1)), without affecting serum thromboxane (TX)B(2) levels. 3 Rofecoxib significantly elevated sBP in both rat strains fed normal salt or high salt diet (8% NaCl, wt wt(-1)), but not in rats on low salt intake (0.02% NaCl, wt wt(-1)). 4 Rofecoxib significantly decreased plasma levels of 6-keto PGF(1alpha) in both rat strains fed normal or high salt diet, but not in rats during low salt intake. 5 Rofecoxib exerted no influence on the changes of body weight nor on water intake. Plasma renin activity (PRA) and renocortical renin mRNA abundance were not changed by rofecoxib, but plasma aldosterone concentration (PAC) was significantly reduced. 6 These results suggest that chronic inhibition of COX-2 causes an increase of blood pressure that depends on prostacyclin synthesis. Furthermore, this increase is independent on genetic predisposition and can be prevented by salt deprivation. Since water intake and body weight gain were not changed by rofecoxib, fluid retention appears not to be a major reason for the development of hypertension. Similarly, an activation of the renin-angiotensin-aldosterone axis appears to be an unlikely candidate mechanism.
Assuntos
Anti-Inflamatórios não Esteroides/efeitos adversos , Pressão Sanguínea/efeitos dos fármacos , Inibidores de Ciclo-Oxigenase/efeitos adversos , Lactonas/efeitos adversos , 6-Cetoprostaglandina F1 alfa/sangue , Actinas/metabolismo , Aldosterona/sangue , Animais , Peso Corporal/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ingestão de Líquidos/efeitos dos fármacos , Frequência Cardíaca/efeitos dos fármacos , Hematócrito , Córtex Renal/efeitos dos fármacos , Córtex Renal/metabolismo , Masculino , Potássio/sangue , RNA Mensageiro/metabolismo , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Renina/sangue , Renina/genética , Renina/metabolismo , Sódio na Dieta/administração & dosagem , SulfonasRESUMO
Proteinase-activated receptors (PARs) 1 to 4 are highly expressed in the kidney and are involved in the regulation of renal hemodynamics and tubular function. Since intravascular infusion of the proteinase thrombin, which activates PARs, has been shown to decrease plasma renin activity in rats, we investigated the effects of the respective PAR subtypes on renin release using the isolated perfused mouse kidney model. Thrombin dose-dependently reduced perfusate flow and inhibited renin secretion rates (RSRs) that had been prestimulated by the ß-adrenoreceptor agonist isoproterenol. The suppression of RSRs was prevented by the selective PAR1 inhibitor SCH79797, and direct activation of PAR1 by TFLLR mimicked the effects of thrombin on RSRs and vascular tone. Moreover, TFLLR suppressed the stimulations of RSRs in response to the loop diuretic bumetanide, to prostaglandin E(2), or to a decrease in renal perfusion pressure but not in response to a reduction in extracellular calcium. The PAR2-activating peptide SLIGRL concentration dependently increased RSR and perfusate flow. The stimulation of RSRs by SLIGRL was markedly attenuated by N(G)-nitro-L-arginine methyl ester, suggesting an NO-dependent mechanism. Activation of PAR4 by AYPGKF did not modulate RSRs or perfusate flow. PAR1 and PAR2 immunoreactivity were detected in the juxtaglomerular region and were colocalized with renin immunoreactivity. Our data provide evidence that PAR1 activation inhibits renal renin secretion and induces renal vasoconstriction, whereas PAR2 activation stimulates renin release and induces vasodilation mainly via the release of NO.
Assuntos
Sistema Justaglomerular/metabolismo , Rim/metabolismo , Receptor PAR-1/metabolismo , Receptor PAR-2/metabolismo , Renina/metabolismo , Agonistas Adrenérgicos beta/farmacologia , Animais , Relação Dose-Resposta a Droga , Isoproterenol/farmacologia , Sistema Justaglomerular/efeitos dos fármacos , Rim/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Óxido Nítrico/metabolismo , Oligopeptídeos/farmacologia , Pirróis/farmacologia , Quinazolinas/farmacologia , Receptor PAR-1/antagonistas & inibidores , Receptor PAR-1/efeitos dos fármacos , Receptor PAR-2/efeitos dos fármacos , Trombina/farmacologiaRESUMO
(3'-5')-Cyclic diguanylate (c-di-GMP) is a bacterial second messenger with immunomodulatory activities in mice suggesting potential applications as a vaccine adjuvant and as a therapeutic agent. Clinical studies in larger animals or humans will require larger doses that are difficult and expensive to generate by currently available chemical or enzymatic synthesis and purification methods. Here we report the production of c-di-GMP at the multi-gram scale from the economical precursors guanosine monophosphate (GMP) and adenosine triphosphate by a "one-pot" three enzyme cascade consisting of GMP kinase, nucleoside diphosphate kinase, and a mutated form of diguanylate cyclase engineered to lack product inhibition. The c-di-GMP was purified to apparent homogeneity by a combination of anion exchange chromatography and solvent precipitation and was characterized by reversed phase high performance liquid chormatography and mass spectrometry, nuclear magnetic resonance spectroscopy, and further compositional analyses. The immunomodulatory activity of the c-di-GMP preparation was confirmed by its potentiating effect on the lipopolysaccharide-induced interleukin 1ß, tumor necrosis factor α, and interleukin 6 messenger RNA expression in J774A.1 mouse macrophages.