RESUMO
INTRODUCTION: Priming is the mechanism of protection induced by a previous exposition of a cell or organ to low or equal concentrations of a toxic substance. OBJECTIVE: To analyze the mechanism of priming induced by the previous exposition to gentamicin in human proximal tubular cells and nephrotoxic acute renal failure (ARF). METHODS: Wistar rats and tubular cells were exposed to gentamicin 2mM during 24h or 40 mg/kg during 3 days and after one rest week were exposed to the same concentration during 24h in cells and additional ten days in rats. The primed animals were compared to control rats receiving vehicle and GENTA animals treated with the gentamicin during the same period. Biochemical parameters were analyzed. The oxidative stress was analyzed by urinary hydroperoxides and carbonylated protein while antioxidant defense was studied by antioxidant activity of the plasma (FRAP), catalase, superoxide dismutase, heme-oxygenase 1 (HO-1) immunostaining and enzymatic activity in kidney. Necrosis, apoptosis, proliferation, endothelin 1 (ET-1) and HO-1 expression were studied in cells. RESULTS: Priming of the animals inhibited the increase in creatinine, urea, sodium excretion and urinary protein induced by gentamicin. Bosentan, ET-1 receptor antagonist, and hemin, HO-1 inducer, potentiate the inhibition. The mechanism of protection was mediated by induction of the antioxidant enzymes HO-1, catalase and SOD activity and oxidative stress reduction. Priming inhibited cell death and induced proliferation through ET-1 production. CONCLUSION: Priming is a persistent and multifactorial mechanism, the stimulation of the antioxidant defense could mimics partially the priming process and prevent the ARF.
Assuntos
Injúria Renal Aguda/induzido quimicamente , Antioxidantes/fisiologia , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/fisiologia , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/prevenção & controle , Animais , Células Cultivadas , Gentamicinas/administração & dosagem , Masculino , Estresse Oxidativo , Ratos , Ratos WistarRESUMO
RESUMO Introdução: Priming é um mecanismo de proteção induzida pela exposição anterior de uma célula ou órgão a baixas ou mesmas concentrações de uma substância tóxica. Objetivo: analisar o mecanismo de priming induzido pela exposição a gentamicina em células tubulares proximais humanas e na insuficiência renal aguda (IRA). Métodos: Células tubulares foram expostos a 2 mM de gentamicina durante 24 horas, enquanto ratos Wistar foram expostas a 40 mg/kg durante 3 dias. Depois de uma semana, as células foram expostas à mesma concentração durante 24h e os ratos durante dez dias. Os animais condicionados foram comparados com ratos controle e tratados com gentamicina durante 10 dias. Foram analisados parâmetros bioquímicos, o estresse oxidativo foi analisado por hidroperóxidos e proteínas carboniladas urinárias, enquanto a defesa antioxidante foi estudada pela atividade antioxidante do plasma e imunomarcação e atividade da catalase, superóxido dismutase, heme oxigenase-1 (HO-1) nos rins. Necrose, apoptose, proliferação e expressão da endotelina-1 (ET-1) e HO-1 foram estudadas em células. Resultados: o condicionamento dos animais inibiu o aumento da creatinina, ureia, excreção urinária de sódio e de proteína induzida por gentamicina. Bosentana, antagonista do receptor ET-1, e hemin, indutor de HO-1, potencializaram a inibição. O mecanismo de proteção foi mediado pela indução de enzimas antioxidantes HO-1, catalase e SOD atividade e redução do estresse oxidativo. O condicionamento inibiu a morte celular e induziu a proliferação via produção de ET-1. Conclusão: o mecanismo de condicionamento é persistente e multifactorial, o estímulo da defesa antioxidante poderia mimetizar o processo de condicionamento e impedir a IRA.
ABSTRACT Introduction: Priming is the mechanism of protection induced by a previous exposition of a cell or organ to low or equal concentrations of a toxic substance. Objective: To analyze the mechanism of priming induced by the previous exposition to gentamicin in human proximal tubular cells and nephrotoxic acute renal failure (ARF). Methods: Wistar rats and tubular cells were exposed to gentamicin 2mM during 24h or 40 mg/kg during 3 days and after one rest week were exposed to the same concentration during 24h in cells and additional ten days in rats. The primed animals were compared to control rats receiving vehicle and GENTA animals treated with the gentamicin during the same period. Biochemical parameters were analyzed. The oxidative stress was analyzed by urinary hydroperoxides and carbonylated protein while antioxidant defense was studied by antioxidant activity of the plasma (FRAP), catalase, superoxide dismutase, heme-oxygenase 1 (HO-1) immunostaining and enzymatic activity in kidney. Necrosis, apoptosis, proliferation, endothelin 1 (ET-1) and HO-1 expression were studied in cells. Results: Priming of the animals inhibited the increase in creatinine, urea, sodium excretion and urinary protein induced by gentamicin. Bosentan, ET-1 receptor antagonist, and hemin, HO-1 inducer, potentiate the inhibition. The mechanism of protection was mediated by induction of the antioxidant enzymes HO-1, catalase and SOD activity and oxidative stress reduction. Priming inhibited cell death and induced proliferation through ET-1 production. Conclusion: Priming is a persistent and multifactorial mechanism, the stimulation of the antioxidant defense could mimics partially the priming process and prevent the ARF.
Assuntos
Animais , Masculino , Ratos , Injúria Renal Aguda/induzido quimicamente , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/fisiologia , Antioxidantes/fisiologia , Gentamicinas/administração & dosagem , Células Cultivadas , Ratos Wistar , Estresse Oxidativo , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/prevenção & controleRESUMO
BACKGROUND: Several studies have correlated perinatal malnutrition with diseases in adulthood, giving support to the programming hypothesis. In this study, the effects of maternal undernutrition during lactation on renal Na(+)-transporters and on the local angiotensin II (Ang II) signaling cascade in rats were investigated. METHODOLOGY/PRINCIPAL FINDINGS: Female rats received a hypoproteic diet (8% protein) throughout lactation. Control and programmed offspring consumed a diet containing 20% protein after weaning. Programming caused a decrease in the number of nephrons (35%), in the area of the Bowman's capsule (30%) and the capillary tuft (30%), and increased collagen deposition in the cortex and medulla (by 175% and 700%, respectively). In programmed rats the expression of (Na(+)+K(+))ATPase in proximal tubules increased by 40%, but its activity was doubled owing to a threefold increase in affinity for K(+). Programming doubled the ouabain-insensitive Na(+)-ATPase activity with loss of its physiological response to Ang II, increased the expression of AT(1) and decreased the expression of AT(2) receptors), and caused a pronounced inhibition (90%) of protein kinase C activity with decrease in the expression of the α (24%) and ε (13%) isoforms. Activity and expression of cyclic AMP-dependent protein kinase decreased in the same proportion as the AT(2) receptors (30%). In vivo studies at 60 days revealed an increased glomerular filtration rate (GFR) (70%), increased Na(+) excretion (80%) and intense proteinuria (increase of 400% in protein excretion). Programmed rats, which had normal arterial pressure at 60 days, became hypertensive by 150 days. CONCLUSIONS/SIGNIFICANCE: Maternal protein restriction during lactation results in alterations in GFR, renal Na(+) handling and in components of the Ang II-linked regulatory pathway of renal Na(+) reabsorption. At the molecular level, they provide a framework for understanding how metabolic programming of renal mechanisms contributes to the onset of hypertension in adulthood.
Assuntos
Angiotensina II/metabolismo , Rim/metabolismo , Lactação/metabolismo , Transdução de Sinais , Sódio/metabolismo , Angiotensina II/farmacologia , Animais , Transporte Biológico/efeitos dos fármacos , Pressão Sanguínea/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Colágeno/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Dieta com Restrição de Proteínas/efeitos adversos , Ingestão de Alimentos/efeitos dos fármacos , Feminino , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Taxa de Filtração Glomerular/efeitos dos fármacos , Rim/citologia , Rim/efeitos dos fármacos , Rim/fisiologia , Glomérulos Renais/citologia , Glomérulos Renais/efeitos dos fármacos , Glomérulos Renais/metabolismo , Glomérulos Renais/fisiologia , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/fisiologia , Masculino , Desnutrição/etiologia , Desnutrição/metabolismo , Desnutrição/patologia , Desnutrição/fisiopatologia , Mães , Gravidez , Proteína Quinase C/metabolismo , Proteinúria/etiologia , Proteinúria/metabolismo , Ratos , Receptores de Angiotensina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sódio/urina , ATPase Trocadora de Sódio-Potássio/metabolismo , Fatores de Tempo , DesmameRESUMO
BACKGROUND: Proximal tubule cells have specialized apical membranes with microvilli that provide an extensive surface area for unidirectional transport of solute from lumen to blood. The major structural solute component is F-actin, which interacts with transmembrane proteins, including ion transport molecules related to normal absorptive and secretory functions. Our study was to evaluate F-actin and fluid absorption (Jv) in proximal tubules after exposure to preservation solutions. METHODS: In vitro microperfusion technique and immunohistochemistry analysis. RESULTS: 1. Absorptions were similar in 1- and 24-hour-preserved tubules, as well as in fresh tubules. The exception was tubules for 24 hours in Euro-Collins solution, which did not show absorption, suggesting that it was affected. 2. Fluorescence intensity of actin tubules preserved for 1 hour in both solutions showed similar values to each other and to the control group; tubules preserved for 24 hours in both solutions were similar to each other, although statistically different than the control group and those preserved for 1 hour in Belzer (UW) solution. CONCLUSION: There were differences among groups in the distribution of F-actin; Jv values were different for 24-hour preservation in each solution, whereas fluorescence intensity was similar in both 24-hour solutions. Thus, actin cytoskeleton was not responsible for it, because 24-hour preservation in UW showed Jv results comparable to the control group.
Assuntos
Actinas/fisiologia , Citoesqueleto/fisiologia , Túbulos Renais Proximais/fisiologia , Soluções para Preservação de Órgãos/farmacologia , Preservação de Órgãos/métodos , Absorção , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/ultraestrutura , Actinas/ultraestrutura , Adenosina/farmacologia , Alopurinol/farmacologia , Animais , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/ultraestrutura , Glutationa/farmacologia , Insulina/farmacologia , Túbulos Renais Coletores/efeitos dos fármacos , Túbulos Renais Coletores/fisiologia , Túbulos Renais Coletores/ultraestrutura , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/ultraestrutura , Masculino , Microscopia Confocal/métodos , Microvilosidades/efeitos dos fármacos , Microvilosidades/ultraestrutura , Modelos Animais , Soluções para Preservação de Órgãos/farmacocinética , Perfusão/métodos , Coelhos , Rafinose/farmacologiaRESUMO
As consequence of glomerular filtration the viscosity of blood flowing through the efferent arteriole increases. Recently, we found that shear stress modulates proximal bicarbonate reabsorption and nitric oxide (NO.) was the chemical mediator of this effect. In the present work, we found that agonists of NO. production affected basolateral membrane potential (V (blm)) of the proximal convoluted tubule (PCT) epithelium. Using paired micropuncture experiments, we perfused peritubular capillaries with solutions with different viscosity while registering the V (blm). Our results showed that a 50% increment in the viscosity, or the addition of bradykinin (10(-5) M) to the peritubular perfusion solution, induced a significant and similar hyperpolarization of the V (blm) at the PCT epithelium of 6 +/- 0.7 mV (p < 0.05). Both hyperpolarizations were reverted by L-NAME (10(-4) M). Addition of 2,2'-(hydroxynitrosohydrazino) bis-ethanamine (NOC-18) 3 x 10(-4) M to the peritubular perfusion solution induced a hyperpolarization of the same magnitude of that high viscosity or bradykinin. These results strongly suggest the involvement of NO. in the effect of high viscosity solutions. This effect seems to be mediated by activation of K+(ATP) channels as glybenclamide (5 x 10(-5) M) added to peritubular solutions induced a larger depolarization of the V (blm) with high viscosity solutions. Acetazolamide (5 x 10(-5) M) added to high viscosity solutions induced a larger hyperpolarization (8 +/- 1 mV; p < 0.05), suggesting that depolarizing current due to HCO(-)3 exit across the basolateral membrane damps the hyperpolarizing effect of high viscosity. Considering that Na(+) and consequently water reabsorption is highly dependent on electrical gradient, the present data suggest that the endothelium of kidney vascular bed interacts in paracrine fashion with the epithelia, affecting V (blm) and thus modulating PCT reabsorption.
Assuntos
Endotélio Vascular/fisiologia , Células Epiteliais/fisiologia , Túbulos Renais Proximais/fisiologia , Acetazolamida/farmacologia , Adenosina Trifosfatases/antagonistas & inibidores , Animais , Bradicinina/farmacologia , Inibidores da Anidrase Carbônica/farmacologia , Proteínas de Transporte de Cátions/antagonistas & inibidores , Eletrofisiologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Inibidores Enzimáticos/farmacologia , Células Epiteliais/efeitos dos fármacos , Glibureto/farmacologia , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/efeitos dos fármacos , Masculino , Potenciais da Membrana/efeitos dos fármacos , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/metabolismo , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Compostos Nitrosos/farmacologia , Perfusão , Ratos , Ratos Wistar , Estresse MecânicoRESUMO
Albumin endocytosis in renal proximal tubule cells is a clathrin- and receptor-mediated mechanism that, in several pathophysiological conditions, is involved in initiating or promoting tubule-interstitial disease. Although much work has been done on this pathway, the regulation of albumin endocytosis in proximal tubule cells is not well understood. Here, we study the modulation by angiotensin II (Ang II) of albumin endocytosis in LLC-PK1, a model of proximal tubule cells. We observed that Ang II increases albumin endocytosis by approximately 100% at 10(-9) M. This effect is completely reversed by 10(-9) M PD123319, a specific AT(2) receptor antagonist, but not by losartan, a specific AT(1) receptor antagonist, at concentrations up to 10(-7) M. The Ang II effect on albumin endocytosis is also reversed by: phosphoinositide 3-kinase inhibitors LY294002 (2.5 x 10(-6) M) or wortmannin (10(-7) M), the protein kinase B inhibitor (2 x 10(-5) M), and staurosporine (2 x 10(-6) M), an inhibitor of 3'-phosphoinositide-dependent kinase 1. Ang II induced the selective phosphorylation of protein kinase B (PKB) at the Thr-308 residue without a change in Ser-473 phosphorylation, a combination that leads to an increase in PKB activity. These effects were completely abolished by 3 x 10(-6) M staurosporine or 10(-8) M PD123319. Our experiments also showed that PKB is present in the membrane fraction in overnight-starved LLC-PK1 cells. Taken together, these data show that Ang II increases albumin endocytosis through an AT(2) receptor mediated by activation of PKB in the plasma membrane, which depends on the basal activity of the phosphatidyl-inositol 3-kinase.
Assuntos
Albuminas/metabolismo , Angiotensina II/metabolismo , Endocitose/fisiologia , Túbulos Renais Proximais/fisiologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor Tipo 2 de Angiotensina/metabolismo , Albuminas/fisiologia , Androstadienos/farmacologia , Bloqueadores do Receptor Tipo 2 de Angiotensina II , Animais , Fracionamento Celular , Linhagem Celular , Cromonas/farmacologia , Endocitose/efeitos dos fármacos , Imidazóis/farmacologia , Túbulos Renais Proximais/citologia , Morfolinas/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação , Piridinas/farmacologia , Estaurosporina/farmacologia , Sus scrofa , WortmaninaRESUMO
ClC-2 is a CLC family member of chloride channels sensitive to changes in cell volume, pH and voltage. The ClC-2 is widely distributed along the nephron although in the kidney its role still not well understood. Aldosterone studies suggest that ClC-2 expression in the kidney may be hormonally regulated. To explore the possibility that estrogen control ClC-2 expression, we investigated whether its expression changed in the kidney of female Wistar rats subjected to ovariectomy with or without near-physiological or high doses of 17beta-estradiol benzoate treatment for 10 days. Total RNA isolated from rat kidney and dissected nephron segments was analyzed by ribonuclease protection assay and/or a semi-quantitative RT-PCR. The renal ClC-2 protein expression was analyzed by Western blot. The decreased renal expression of ClC-2 mRNA and protein observed in ovariectomized rats was restored to control levels after treatment with low doses of estradiol. Higher dose estradiol lead to an even greater increase in ClC-2 mRNA and protein expression. This change in overall expression was shown to be caused by the modulation of ClC-2 mRNA expression in the proximal tubule. These results suggest that ClC-2 may be involved in estrogen-induced Cl(-) transport in rat kidney.
Assuntos
Canais de Cloreto/genética , Estradiol/farmacologia , Túbulos Renais Proximais/fisiologia , Animais , Western Blotting , Canais de Cloro CLC-2 , Células Cultivadas , Estradiol/sangue , Feminino , Expressão Gênica/efeitos dos fármacos , Túbulos Renais Proximais/citologia , Néfrons/fisiologia , Ovariectomia , RNA Mensageiro/análise , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase ReversaAssuntos
Adenosina/farmacologia , Alopurinol/farmacologia , Glutationa/farmacologia , Soluções Hipertônicas/farmacologia , Insulina/farmacologia , Túbulos Renais Proximais , Soluções para Preservação de Órgãos/farmacologia , Rafinose/farmacologia , Animais , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/fisiologia , Masculino , Preservação de Órgãos/métodos , CoelhosRESUMO
BACKGROUND: The role of nitric oxide (NO) in acute renal failure (ARF) is not yet completely understood. L-Arginine (L-arg) is protective against different ARF models, while L-arg addition in isolated proximal tubules enhances hypoxia/reoxygenation (H/R) injury. The aim of this study was to evaluate the effects of L-arg on renal ischaemia. METHODS: In in vivo studies, Wistar rats were subjected to 60 min renal artery clamping, and renal function was evaluated 2 and 15 days after ischaemia. Four groups were studied: (1) control; (2) acute L-arg (50 mg/kg/bw i.v.); (3) L-nitro-arginine-methyl esther (L-NAME; 0.5 mg/kg/bw i.v.); and (4) chronic L-arg (L-arg 0.25% in drinking water/7 days). For the in vitro studies, proximal tubules (PTs), isolated by collagenase digestion and Percoll gradient, were studied from three groups: (1) untreated; (2) L-arg-treated (L-arg 0.25% in drinking water/7 days); and (3) L-NAME-treated rats (3 mg/kg in drinking water/7 days). PTs were kept oxygenated or subjected to 15 min hypoxia (H-15) and 35 min reoxygenation (R-35). In some experiments, additional doses of L-arg and L-NAME were administered. Cell injury was assessed by lactate dehydrogenase (LDH) release. NO production was evaluated by NO2-/NO3- measurement (Griess reaction) in both urine and isolation medium. RESULTS: After 2 days, L-arg infusion protected against ischaemia compared with control rats (0.4 vs 0.2 ml/min/100 g, P < 0.001), while neither L-NAME nor chronic L-arg supplementation ameliorated renal function. After 15 days, both acute and chronic L-arg groups showed a higher glomerular filtration rate (0.6 and 0.75 ml/min/100 g) compared with control rats (0.3 ml/min/100 g, P < 0.05) and L-NAME-treated rats (0.2 ml/min/100 g, P < 0.05). Despite similar recovery in both L-arg groups, the mortality rate was 25% in the chronic L-arg group. Tubular function was also better preserved in the acute L-arg group. PTs isolated from L-arg-treated rats were more sensitive to isolation injury. L-Arg addition enhanced H/R injury (44.9 vs 51.8%, P < 0.05), whereas L-NAME addition protected (44.9 vs 24%, P < 0.001) in untreated rats. In L-arg-treated rats, addition of L-arg did not enhance H/R injury (49.6 vs 53.5%, NS) and L-NAME was still protective (49.6 vs 32.3%, P < 0.001). In PTs from L-NAME-treated rats, L-arg addition also did not enhance H/R injury (50 vs 54%, NS) whereas L-NAME was protective (50 vs 27%, P < 0.001). NO2-/NO3- production paralleled L-arg and L-NAME supplementation. CONCLUSION: It was demonstrated that acute L-arg infusion was beneficial in in vivo renal ischaemia while it was harmful in isolated H/R tubules. In contrast, chronic L-arg supplementation was deleterious both in in vivo and in vitro renal ischaemia, suggesting that injurious effects had overcome the beneficial effects during excess NO exposure.
Assuntos
Arginina/farmacologia , Arginina/toxicidade , Isquemia/tratamento farmacológico , Rim/irrigação sanguínea , Rim/efeitos dos fármacos , Injúria Renal Aguda/etiologia , Injúria Renal Aguda/fisiopatologia , Injúria Renal Aguda/prevenção & controle , Animais , Arginina/administração & dosagem , Inibidores Enzimáticos/farmacologia , Técnicas In Vitro , Isquemia/fisiopatologia , Rim/lesões , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/fisiologia , Masculino , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/fisiologia , Óxido Nítrico Sintase/antagonistas & inibidores , Ratos , Ratos Wistar , Traumatismo por Reperfusão/etiologia , Traumatismo por Reperfusão/fisiopatologia , Traumatismo por Reperfusão/prevenção & controleRESUMO
Crotalus durissus cascavella (C.d.c) is a snake usually found in scrubland of Brazilian Northeast and its bite constitutes an important public health problem. Isolated kidneys from wistar rats, weighing 240 to 280 g, were perfused with Krebs Henseleit solution containing 6 g% of previously dialysed bovine serum albumin. The effects of C.d.c venom were studied on the perfusion pressure (PP), urinary flow (UF), glomerular filtration rate (GFR), percent of sodium tubular transport (%TNa+) and percent of proximal tubule sodium transport (%pTNa+). All experiments were preceded by a 30 min internal control period and an external control group. The infusion of C.d.c (10 microg ml(-1)) increased the PP, UF at 60 and 90min of perfusion, and decreased the GFR, %TNa+ and %pTNa+ at 120 min of perfusion. The proximal renal tubule was the major site for this toxic effect. In the group treated with the venom we found hyalin cylinders inside all tubules and proteinaceous material, alternating from moderate to intense presence in urinary space. Dexamethasone (Dexa 20 microg ml(-1)) protected against the increase in PP, UF, and against the decrease in GFR, it produced the reversion of the effect also in %TNa+ and %pTNa+. Indomethacin (Indo 10 microg ml(-1)) antagonized the effect observed in PP and UF, but was not able to reverse the changes in GFR, %TNa+ and %pTNa+. Nifedipine (Nif 10 microg ml(-1)) promoted a reversion of almost all functional changes, except the %pTNa+ was not reversed. We conclude that these alterations may be caused by a direct action of the venom on the kidneys and indirectly by the release of mediators from endothelial cells. Dexa protected against renal lesions caused by the venom, perhaps by inhibiting phospholipase A2 a toxic component of the venom. The reversion partially induced by indo may be due to cyclooxygenase inhibition that will inhibit the formation of prostaglandins. Nif blocked the renal alterations that may involve cell calcium influx that resulted from the venom aggression.