RESUMO
The stress response was studied in suspensions of tubules from immature (IT) and mature (MT) rats after noninjury, heat, oxygen, and anoxia. Under all conditions, IT exhibited more exuberant activation of heat shock transcription factor (HSF) than MT. Characterization of activated HSF in immature cortex revealed HSF1. Also, 2 h after each condition, heat shock protein-72 (HSP-72) mRNA was twofold in IT. As the metabolic response to 45 min of anoxia, 20-min reoxygenation was assessed by measuring O2 consumption (O2C). Basal O2C was manipulated with ouabain, nystatin, and carbonylcyanide p-chloromethyoxyphenylhydrazone (CCCP). Basal O2C in IT were one-half the value of MT. After anoxia, basal O2C was reduced by a greater degree in MT. Ouabain reduced O2C to half the basal value in both noninjured and anoxic groups. Basal O2C was significantly stimulated by nystatin but not to the same level following anoxia in MT and IT. Basal O2C was also stimulated by CCCP, but after anoxia, CCCP O2C was significantly less in MT with no decrease in IT, suggesting mitochondria are better preserved in IT. Also, O2C devoted to nontransport activity was better maintained in IT.
Assuntos
Hipóxia Celular/fisiologia , Proteínas de Ligação a DNA/metabolismo , Túbulos Renais/metabolismo , Fatores Etários , Animais , Animais Recém-Nascidos , Northern Blotting , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , DNA Mitocondrial/metabolismo , Eletroforese , Proteínas de Choque Térmico HSP72 , Fatores de Transcrição de Choque Térmico , Proteínas de Choque Térmico/metabolismo , Temperatura Alta , Túbulos Renais/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Nistatina/farmacologia , Ouabaína/farmacologia , Consumo de Oxigênio , RNA Mensageiro/metabolismo , Ratos , Fatores de Transcrição/metabolismoRESUMO
The pattern of 72-kDa heat-shock protein (HSP-72) induction after renal ischemia suggests a role in restoring cell structure. HSP-72 activity in the repair and release from denatured and aggregated proteins requires ATP. Protein aggregates were purified from normal and ischemic rat renal cortex. The addition of ATP to cortical homogenates reduced HSP-72, Na(+)-K(+)-ATPase, and actin in aggregates subsequently isolated, suggesting that their interaction is ATP dependent. Altering ATP hydrolysis in the purified aggregates, however, had different effects. ATP released HSP-72 during reflow and preserved Na(+)-K(+)-ATPase association with aggregates at 2 h but had no effect in controls or at 6 h reflow and caused no change in actin. These results indicate that HSP-72 complexes with aggregated cellular proteins in an ATP-dependent manner and suggests that enhancing HSP-72 function after ischemic renal injury assists refolding and stabilization of Na(+)-K(+)-ATPase or aggregated elements of the cytoskeleton, allowing reassembly into a more organized state.
Assuntos
Trifosfato de Adenosina/farmacologia , Proteínas de Choque Térmico/metabolismo , Isquemia/metabolismo , Rim/irrigação sanguínea , Proteínas/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Actinas/análise , Trifosfato de Adenosina/metabolismo , Animais , Membrana Celular/enzimologia , Citoesqueleto/enzimologia , Proteínas de Choque Térmico HSP72 , Proteínas de Choque Térmico/análise , Hidrólise , Masculino , Proteínas/análise , Proteínas/química , Ratos , Ratos Sprague-Dawley , Reperfusão , ATPase Trocadora de Sódio-Potássio/análiseRESUMO
The small heat-shock proteins appear to have a regulatory role in actin dynamics. Since cytoskeletal disruption is integral to ischemic renal injury, we evaluated expression and intracellular distribution of heat-shock protein 25 (HSP-25) in rat renal cortex after 45 min of renal ischemia. HSP-25 was constitutively expressed and induced by ischemia with peak levels reached by 6 h reflow. Ischemia caused a shift of HSP-25 from the detergent-soluble into the insoluble cytoskeletal fraction. By 2 h reflow, the majority of HSP-25 had redistributed into the soluble fraction. HSP-25 was predominantly localized in a subapical distribution in control proximal tubules, a pattern intermediate between deoxyribonuclease (DNase)-reactive and filamentous actin. After ischemia, HSP-25 dispersed through the cytoplasm with small punctate accumulations similar to DNase-reactive actin. During later reflow, all three proteins were found in coarse intracytoplasmic accumulations; however, HSP-25 and DNase-reactive actin were in separate accumulations. HSP-25 and microfilamentous actin staining returned to the subapical domain. Thus the temporal and spatial patterns of HSP-25 induction and distribution suggest specific interactions between HSP-25 and actin during the early postischemic reorganization of the cytoskeleton. HSP-25 may have additional roles distinct from actin dynamics later in the course of postischemic recovery.
Assuntos
Actinas/metabolismo , Proteínas de Choque Térmico/biossíntese , Isquemia/metabolismo , Rim/irrigação sanguínea , Animais , Aorta Abdominal , Citoesqueleto/metabolismo , Citoesqueleto/patologia , Proteínas de Choque Térmico/metabolismo , Isquemia/patologia , Rim/metabolismo , Rim/patologia , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/patologia , Masculino , Ratos , Ratos Sprague-Dawley , Artéria RenalRESUMO
The tolerance of immature tissues to injury has been noted over the past several decades. Traditional teaching relates this tolerance to energy derived from anaerobic glycolysis. This mini-review describes investigations of the hypothesis that the immature kidney is less susceptible to oxygen deprivation than the mature kidney. Utilizing proximal tubule suspensions from immature and mature rats, studies assessing ATP levels as an index of cellular energy and lactate dehydrogenase (LDH) release as a determinant of plasma membrane damage demonstrate the developing kidney is resistant to prolonged anoxia. ATP is maintained at twofold higher levels during anoxia in the immature tubule compared with the mature tubule. The contribution of anaerobic glycolysis to the tolerance of the immature renal tubules is investigated by two inhibitors of the glycolytic pathway, L-glucose and iodoacetate. Following 70%-90% inhibition of glycolysis, ATP is decreased to similar levels in immature and mature tubules. However, immature tubules remain resistant to anoxic damage with no significant change in LDH release. Therefore, enhanced glycolytic activity does not play a dominant role in the tolerance of the developing kidney to anoxia, and this tolerance is not primarily dependent on preservation of cellular ATP.
Assuntos
Hipóxia/fisiopatologia , Túbulos Renais/crescimento & desenvolvimento , Túbulos Renais/fisiopatologia , Animais , Humanos , Túbulos Renais/metabolismo , RatosRESUMO
We have previously shown that the immature tubule is tolerant of prolonged anoxia. In addition, cellular ATP is maintained at 2-fold higher levels during anoxia in the immature tubules compared with the mature tubules. The purpose of this study was: 1) to determine whether anaerobic glycolysis contributes to the tolerance to anoxia and preservation of cellular ATP in immature tubules and 2) to evaluate whether the tolerance demonstrated by immature tubules is dependent on preservation of cellular ATP. Suspensions of proximal tubules from immature (8-10 d) and mature (8-10 wk) rats were subjected to 15 and 45 min of anoxia in a standard buffer and in buffers designed to inhibit glycolysis. Lactate dehydrogenase release was used to assess plasma membrane damage, ATP levels were determined as an index of cellular energy and total lactate production was measured to evaluate glycolytic activity. After 45 min of anoxia, total lactate production was less in immature tubules (101 +/- 48 micrograms of lactate/mg of DNA) compared with mature tubules (148 +/- 36 micrograms of lactate/mg of DNA). After inhibition of glycolytic metabolism, ATP decreased to similar levels in both immature and mature tubules. However, immature tubules remained resistant to anoxic damage (lactate dehydrogenase: mature tubules 38 +/- 4%, immature tubules 29 +/- 1.0%). Therefore, enhanced glycolytic activity does not play a dominant role in the tolerance of the developing kidney to anoxia, and this tolerance is not primarily dependent on preservation of cellular ATP.
Assuntos
Trifosfato de Adenosina/metabolismo , Glicólise/fisiologia , Hipóxia/fisiopatologia , Túbulos Renais/fisiologia , Anaerobiose/fisiologia , Animais , Estudos de Avaliação como Assunto , Túbulos Renais/citologia , L-Lactato Desidrogenase/metabolismo , Ácido Láctico/biossíntese , RatosRESUMO
Renal ischemia results in both a profound fall in cellular ATP and a rapid induction of the 70 kD heat-shock protein family, HSP-70. The present studies examined the relationship between cellular ATP and induction of the stress response in renal cortex. Cellular ATP, continuously monitored by in vivo 31P-NMR spectroscopy, was reduced and maintained at specific, stable levels in renal cortex by partial aortic occlusion for 45 min. Activation of heat-shock transcription factor (HSF) was detected by gel retardation assay and transcription was confirmed by Northern analysis. Activation of HSF was not present, and HSP-70 mRNA induction did not occur when ATP levels were maintained above 60% preocclusion (control) levels. Reduction in cortical ATP levels to 35-50% preocclusion values resulted in HSF activation and low-level expression of inducible HSP-70 mRNA. Cellular ATP of 20-25% control values resulted in a greater level of HSF activation and subsequent HSP-70 mRNA elaboration. HSF was activated at the end of 15 min of total occlusion. The studies indicate that a 50% reduction in cellular ATP in the renal cortex must occur before the stress response is detectable, that reduction of ATP below 25% control levels produces a more vigorous response, and that reperfusion is not required for initiation of a heat-shock response in the kidney. Cellular ATP, or the metabolic consequences associated with ATP depletion, may be a threshold factor for initiation of a stress response in the kidney.
Assuntos
Trifosfato de Adenosina/metabolismo , Proteínas de Choque Térmico HSP70/genética , Córtex Renal/metabolismo , Ativação Transcricional/fisiologia , Animais , Constrição Patológica , DNA/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Concentração de Íons de Hidrogênio , Isquemia , Córtex Renal/irrigação sanguínea , Masculino , Ligação Proteica , RNA Mensageiro/biossíntese , Ratos , Ratos Sprague-DawleyRESUMO
Very few data are available regarding the decreased susceptibility of the developing kidney to anoxia. Therefore, the purpose of this study was to develop an experimental system that would allow comparison of an anoxic insult in immature and mature proximal tubule segments and to investigate the hypothesis that the developing kidney is resistant to anoxia as compared with the mature kidney. Suspensions of proximal tubules from immature (age 8-10 d) and mature (8-10 wk) rats were obtained. The purity of the tubule suspension from the immature rats was documented by villin staining. A common buffer solution was developed to compare results from the immature and mature tubules. To study the response of the tubules to anoxia, we subjected the tubule suspension from both the immature and mature rats to 15, 30, 45, and 60 min of anoxia. Lactate dehydrogenase release was measured to assess plasma membrane damage, and ATP levels were determined as an index of cellular energy. After a short anoxic insult (15 or 30 min), the percentage of lactate dehydrogenase release was not significantly different from mature tubules. After prolonged anoxia (45 and 60 min) lactate dehydrogenase release continued to increase, whereas membrane integrity stabilized in the immature tubules. ATP levels decreased in both immature and mature tubules after anoxia, but the decline of ATP was greater in the mature tubules, with a plateau at 20% of basal ATP levels as compared with 40% in the immature tubules. Therefore, the developing kidney is resistant to prolonged anoxia.
Assuntos
Hipóxia/metabolismo , Túbulos Renais Proximais/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , DNA/metabolismo , Técnicas In Vitro , Túbulos Renais Proximais/crescimento & desenvolvimento , L-Lactato Desidrogenase/metabolismo , RatosRESUMO
Ischemia and reperfusion damage mitochondrial structure and impair respiratory function. In this study, 45 min of renal ischemia followed by varying periods of reflow profoundly depressed the activity of several respiratory complexes in mitochondria isolated from rat kidneys. The respiratory complexes are composed of subunits encoded by both the nuclear and mitochondrial genomes. To determine the role of mitochondrial gene expression in recovery of respiratory function, expression of mitochondrial RNA was examined during reperfusion. Both mature and incompletely processed cytochrome b mRNA levels were depressed after 45 min of ischemia and 15 min of reflow; levels rebounded to above normal after 2 h of reflow and then declined over the next 22 h. Another mitochondrial RNA showed a similar pattern; in contrast, the levels of a nuclear-encoded subunit mRNA for a respiratory enzyme and of 28S rRNA were unchanged. These data demonstrate that renal ischemia followed by reperfusion alters mitochondrial RNA expression. We speculate that mitochondrial RNA turnover is increased in response to continuing injury and that recovery is accompanied by enhanced RNA synthesis.
Assuntos
Isquemia/metabolismo , Mitocôndrias/metabolismo , RNA/metabolismo , Circulação Renal , Animais , Rim/metabolismo , Masculino , Oxirredução , Biossíntese de Proteínas , Ratos , Ratos Sprague-Dawley , Reperfusão , Fenômenos Fisiológicos Respiratórios , Sistema Respiratório/enzimologiaRESUMO
Previous studies from our laboratory have demonstrated that postischemic infusion of thyroxin (T4) will augment the restoration of cellular ATP and enhance the recovery of renal function. It has not been clear, however, whether T4 has a direct effect on mitochondrial ATP synthesis or an indirect effect by stabilization of the plasma membrane. To differentiate these putative effects, rats were subjected to 45 min of renal ischemia and given either normal saline (0.5 mL) or T4 (20 micrograms/100 g body weight) during the first 15 min of reflow. Cellular ATP levels were assessed by 31P-nuclear magnetic resonance spectroscopy, and release of lactate dehydrogenase (LDH) was used as an index of plasma membrane integrity at 30 and 120 min of reflow. In rats given normal saline, renal ATP had returned to only 57.9 +/- 1.4% of preischemic values at 30 min of reflow and 66.1 +/- 1.4% by 120 min. LDH release was 13 +/- 0.89% at 30 min and 14.6 +/- 1.6% at 120 min. In contrast, T4-treated animals had ATP levels of 70.2 +/- 2.0% at 30 min and 84.0 +/- 1.9% at 120 min, whereas LDH release was elevated to values similar to those in normal saline-treated rats, 14.9 +/- 1.5% and 14.4 +/- 0.5% at 30 min and 120 min, respectively (nonischemic LDH 8.8 +/- 0.8%). These data suggest that T4 stimulates the recovery of renal ATP by a direct effect on synthesis rather than an indirect effect related to global improvement in cellular integrity.
Assuntos
Trifosfato de Adenosina/metabolismo , Isquemia/metabolismo , Rim/irrigação sanguínea , Rim/metabolismo , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Isquemia/tratamento farmacológico , Rim/efeitos dos fármacos , L-Lactato Desidrogenase/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley , Tiroxina/farmacologiaRESUMO
The role of nucleoside uptake in the enhanced metabolic recovery seen with postischemic ATP.MgCl2 was assessed by determining the effect of S-(p-nitrobenzyl)-6-thioinosine (NBTI) on postischemic ATP recovery in rats given normal saline (NS), ATP.MgCl2, or adenosine after 45 min of bilateral renal ischemia. In NS-infused animals, postischemic administration of NBTI (250 nmol) had no significant effect on the pattern of ATP recovery. In animals given 50 mumol ATP.MgCl2, coinfusion of NBTI significantly reduced the renal ATP content 2 h after reperfusion but blocked only one-half of the enhancement in renal ATP content compared with animals given ATP.MgCl2 alone. In animals postischemically infused with [2,5,8-3H]ATP.MgCl2 (50 mumol) there was significant labeling of nucleotides, nucleosides, and bases after 2 h of reperfusion. The specific activity of the adenosine pool was consistent with significant label uptake in the form of adenosine. Coinfusion of NBTI led to a significant reduction in label incorporation into renal ATP and total adenine nucleotide pools. These data are consistent with an important role for an NBTI-sensitive nucleoside uptake mechanism in the enhanced metabolic and functional recovery observed in ischemically injured kidney treated by postischemic infusion of ATP.MgCl2.
Assuntos
Trifosfato de Adenosina/biossíntese , Isquemia/metabolismo , Rim/metabolismo , Nucleosídeos/farmacocinética , Circulação Renal , Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Sangue/metabolismo , Rim/irrigação sanguínea , Espectroscopia de Ressonância Magnética , Ratos , Tioinosina/análogos & derivados , Tioinosina/farmacologiaRESUMO
In order to elucidate the pattern of redistribution of cellular energy and the restoration of basic cellular metabolism following an ischemic renal insult, suspensions enriched in proximal tubule segments were studied after 45 min of bilateral artery occlusion and 15 min and 2 h of reflow from rats given either normal saline (control), ATP-MgCl2 (which enhances postischemic recovery of ATP), or alpha, beta-methyl adenosine diphosphate (AMPCP), which inhibits nucleotide degradation during ischemia. In non-ischemic control animals, approximately half of the energy is distributed to functional pump activity and half directed for non-transport purposes. When cellular ATP is reduced to 56% of control values, functional pump activity is significantly reduced to 61% of control, while energy delegated for non-transport purposes is decreased by a significantly smaller increment to only 78% of control at 15 min of reflow. In animals given ATP-MgCl2, the cellular and metabolic profile at 15 min of reflow was no different from ischemic control animals with cellular ATP levels similar at 58%. However, by 2 h, cellular ATP levels had increased significantly to 74%, and this was associated with a redistribution of cellular energy to functional pump activity (119% of control) with little change in non-transport function (76%). In animals treated with AMPCP, the cellular ATP levels were 74% of controls, similar to ATP-MgCl2-treated rats after 2 h of reflow. Despite the differences in reflow interval, the distribution of cellular energy was similar (functional pump activity 120% and non-transport activity 79%). By 2 h, cellular ATP was at 95% and both functional pump activity and non-transport activity were 100%.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Metabolismo Energético , Isquemia/metabolismo , Rim/irrigação sanguínea , Consumo de Oxigênio/fisiologia , Injúria Renal Aguda/metabolismo , Difosfato de Adenosina/análogos & derivados , Difosfato de Adenosina/farmacologia , Trifosfato de Adenosina/farmacologia , Animais , Metabolismo Energético/efeitos dos fármacos , Técnicas In Vitro , Rim/química , Rim/efeitos dos fármacos , Túbulos Renais/efeitos dos fármacos , Túbulos Renais/metabolismo , Microscopia Eletrônica , Consumo de Oxigênio/efeitos dos fármacos , Perfusão , Ratos , Ratos Endogâmicos , Distribuição TecidualRESUMO
A viable suspension of proximal tubules that had sustained an in vivo ischemic injury was harvested, and cellular integrity and viability were determined. The histopathological appearance of this preparation has characteristic features of an ischemic injury and ATP levels were comparable to those observed with nuclear magnetic resonance spectroscopy in vivo. Sprague-Dawley rats were subjected to 45 min of bilateral renal artery ischemia and the kidneys were allowed to reperfuse for either 15 min, 2 h, or 24 h before the harvest of the proximal tubule suspension. There was a decrease in base-line oxygen consumption from 34 +/- 0.8 nmol O2.min-1.mg protein-1 to 22 +/- 0.6 at 15 min of reflow. This decline in oxygen consumption persisted during the first 2 h of reflow and returned to control levels by 24 h. Residual respiration in the presence of ouabain was similar at all reflow intervals suggesting that the decrease in basal O2 consumption was related to decreased Na+-K+-ATPase in situ. In contrast, there was no significant difference in Na+-K+-ATPase activity when determined chemically under Vmax conditions in all experimental groups.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Isquemia/metabolismo , Túbulos Renais Proximais/metabolismo , Circulação Renal , Trifosfato de Adenosina/metabolismo , Animais , Técnicas Histológicas , Túbulos Renais Proximais/ultraestrutura , Nistatina/farmacologia , Ouabaína/farmacologia , Consumo de Oxigênio/efeitos dos fármacos , Ratos , Ratos Endogâmicos , ATPase Trocadora de Sódio-Potássio/metabolismo , Suspensões , Azul TripanoRESUMO
We have evaluated the impact of inhibiting adenine nucleotide dephosphorylation on the metabolic and functional consequences of renal ischemia. Intramuscular injection of the ADP-analogue adenosine alpha, beta-methylene diphosphate (AMP-CP) achieved a 70% reduction in 5'-nucleotidase activity, as measured in crude extracts of rat kidney. AMPCP-treated animals had an increased residual nucleotide pool at the end of 45 min of ischemia compared with untreated rats. Assessment of renal ATP by 31P-nuclear magnetic resonance (31P-NMR) in vivo during reflow demonstrates the following: 1) higher rapid initial recovery of ATP (69.3 +/- 1.2 vs. 50.0 +/- 0.5% control value, P less than 0.005), 2) accelerated rate of ATP restoration (0.20 +/- 0.02 vs. 0.11 +/- 0.01% control/min, P less than 0.005), and 3) significantly enhanced renal ATP content after 120 min (93.6 +/- 2.0 vs. 63.1 +/- 0.7% control, P less than 0.005). Kidney function, as measured by the rate of inulin clearance 24 h after the insult, was also significantly improved in AMPCP-treated rats (725 +/- 50 vs. 313 +/- 28 microliters.min-1.100 g body wt-1). Thus inhibition of 5'-nucleotidase results in enhanced metabolic and functional recovery from a renal ischemic insult.
Assuntos
Difosfato de Adenosina/análogos & derivados , Isquemia/prevenção & controle , Rim/irrigação sanguínea , Nucleotidases/antagonistas & inibidores , Circulação Renal/efeitos dos fármacos , 5'-Nucleotidase , Difosfato de Adenosina/farmacologia , Animais , Hipoxantina , Hipoxantinas/farmacologia , Cinética , Masculino , Nucleotidases/metabolismo , Ratos , Ratos EndogâmicosRESUMO
The concentrations of renal ATP have been measured by 31P-nuclear magnetic resonance (NMR) before, during, and after bilateral renal artery occlusion. Using in vivo NMR, the initial postischemic recovery of ATP increased with the magnitude of the residual nucleotide pool at the end of ischemia. ATP levels after 120 min of reflow correlated with functional recovery at 24 h. In the present study the effect of blocking the degradation of ATP during ischemia upon the postischemic restoration of ATP was investigated. Inhibition of adenosine deaminase by 80% with the tight-binding inhibitor 2'-deoxycoformycin led to a 20% increase in the residual adenine nucleotide pool. This increased the ATP initial recovery after 45 min of ischemia from 52% (in controls) to 62% (in the treated animals), as compared to the basal levels. The inhibition also caused an accelerated postischemic restoration of cellular ATP so that at 120 min it was 83% in treated rats vs. 63% in untreated animals. There was a corresponding improvement in the functional recovery from the insult (increase of 33% in inulin clearance 24 h after the injury). Inhibition of adenosine deaminase during ischemia results in a injury similar to that seen after a shorter period of insult.
Assuntos
Inibidores de Adenosina Desaminase , Rim/irrigação sanguínea , Nucleosídeo Desaminases/antagonistas & inibidores , Trifosfato de Adenosina/análise , Animais , Coformicina/análogos & derivados , Coformicina/farmacologia , Inulina/farmacocinética , Isquemia , Espectroscopia de Ressonância Magnética , Masculino , Pentostatina , Ratos , Ratos EndogâmicosRESUMO
To evaluate the effect of thyroxin (T4) on recovery from ischemic acute renal failure, rats were treated with T4 (10 or 20 micrograms/100 g body wt.) or normal saline (NS) either immediately prior to, immediately after or 24 h after 45 min of renal ischemia. Animals given T4 prior to ischemia had no significant increase in Inulin clearance (Cin) (377 +/- 40 microliters/min per 100 g body wt.) as compared with saline-treated ischemic controls (306 +/- 54). In contrast, animals treated immediately after ischemia with either dose of T4 demonstrated significantly better kidney function (Cin 515 +/- 59 microliters/min per 100 g body wt., Uosm 842 +/- 88 mosmol/kg, FENa 0.52% +/- 0.12% and Cin 543 +/- 71, Uosm 939 +/- 103, FENa 0.48 +/- 0.12, for 10 and 20 micrograms/100 g body wt., respectively). Moreover, the improvement in renal function was sustained and Cin was significantly better at day 3 (748 +/- 70) and day 7 (990 +/- 75) compared with saline controls (560 +/- 30 and 732 +/- 45, respectively). Animals which received T4 24 h after ischemia showed significantly higher Cin when compared with ischemic controls. To assess the impact of T4 on recovery of renal ATP, 31P-NMR was used. T4-treated rats demonstrated 90% +/- 5% recovery of renal ATP by 120 min of reflow, whereas NS animals had only 64% +/- 1%. In addition, cellular morphology was better preserved in T4 animals. These data indicate that animals treated postischemically with T4 showed accelerated and sustained recovery from acute renal failure. This beneficial effect appears to be related to cellular mechanisms which are essential for the restoration of sublethally injured cells.
Assuntos
Injúria Renal Aguda/tratamento farmacológico , Isquemia/tratamento farmacológico , Rim/irrigação sanguínea , Tiroxina/uso terapêutico , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/patologia , Trifosfato de Adenosina/metabolismo , Animais , Isquemia/metabolismo , Isquemia/patologia , Rim/metabolismo , Rim/patologia , Masculino , Ratos , Ratos Endogâmicos , ATPase Trocadora de Sódio-Potássio/metabolismo , Fatores de TempoRESUMO
Renal vasoconstriction is an important pathophysiological component of an ischemic acute renal injury. The postischemic infusion of ATP-MgCl2 enhances recovery of glomerular and tubular function, accelerates regeneration of sublethally injured tubular cells, and augments resynthesis of cellular nucleotides. Since both ATP and MgCl2 are vasoactive compounds, postischemic enhancement of renal blood flow (RBF) by a pharmacological agent, dopamine, was examined to study the possible contribution of vasodilation. At 2 h, the infusion of dopamine resulted in RBF (1.70 +/- 0.09 ml X min-1 X 100 g body wt-1 X kidney-1) and inulin clearance (CIn, 400 +/- 44 microliter X min-1 X 100 g body wt-1) similar to rats treated with ATP-MgCl2 (1.73 +/- 0.27 RBF, 404 +/- 38 CIn) and significantly (P less than 0.01) greater than saline-treated rats (0.80 +/- 0.04 RBF, 78 +/- 19 CIn; P less than 0.01). However, by 24 h CIn in dopamine animals had not continued to improve (460 +/- 25 microliter X min-1 X 100 g body wt-1) and was similar to normal saline rats (388 +/- 40). In contrast, CIn in ATP-MgCl2 animals showed sustained recovery (676 +/- 28 microliter X min-1 X 100 g body wt-1, P less than 0.01). These differences resulted from improved integrity of tubular epithelium as reflected by decreased cell swelling and necrosis. Moreover, the recovery of renal ATP levels, as assessed by 31P nuclear magnetic resonance, in animals given saline (63 +/- 3%) or dopamine (66 +/- 5%) was slow and incomplete by 120 min after ischemia.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Trifosfato de Adenosina/metabolismo , Isquemia/fisiopatologia , Rim/metabolismo , Circulação Renal , Trifosfato de Adenosina/farmacologia , Animais , Dopamina/farmacologia , Hemodinâmica , Isquemia/metabolismo , Isquemia/patologia , Masculino , Ratos , Ratos Endogâmicos , Circulação Renal/efeitos dos fármacosRESUMO
Renal energy metabolism was investigated before, during, and after ischemic insults of varying durations with in vivo 31P NMR spectroscopy. The postischemic recovery of renal ATP was found to be a biphasic process regardless of the length of the ischemia. This two-stage recovery consisted of a quick initial component immediately upon reflow followed by a slower, more gradual return toward preischemic levels. To characterize the source of each phase of the recovery, kidneys were extracted with perchloric acid at the end of the different periods of ischemia (before reflow). Concentrations of adenine nucleotides and breakdown products adenosine, inosine, and hypoxanthine were determined by 1H NMR spectroscopy. Excellent correlation was found between the residual nucleotide pool and the magnitude of the initial phase of ATP recovery. Additionally, the renal ATP content after 120 min of reflow was shown to have a strong correlation with subsequent functional recovery. These experiments show that in vivo 31P NMR can provide new and dynamic information concerning the biochemical recovery from ischemia. Furthermore, this data has the potential to predict the eventual functional recovery of the organ.
Assuntos
Trifosfato de Adenosina/metabolismo , Metabolismo Energético , Isquemia/metabolismo , Rim/metabolismo , Circulação Renal , Difosfato de Adenosina/metabolismo , Monofosfato de Adenosina/metabolismo , Animais , Cinética , Espectroscopia de Ressonância Magnética , Masculino , Ratos , Ratos Endogâmicos , Fatores de TempoRESUMO
Postischemic administration of ATP-MgCl2 is known to enhance recovery of renal function and accelerate the restitution of cellular ATP levels. To differentiate between a direct and indirect effect of the exogenous nucleotide, rats were subjected to 45 min of bilateral renal ischemia and were infused with either ATP-MgCl2, AMP-MgCl2, or normal saline. The immediate recovery of the cellular ATP was similar in all three groups of animals, whereas the subsequent recovery was accelerated by the infusion of either nucleotide. Since ATP-MgCl2 and AMP-MgCl2 produced similar results, this study provides evidence that exogenous ATP may act by providing precursors for the resynthesis of the cellular adenine nucleotide pool rather than being a direct source of energy.
Assuntos
Trifosfato de Adenosina/metabolismo , Isquemia/metabolismo , Rim/metabolismo , Circulação Renal , Animais , Rim/patologia , Masculino , Ratos , Ratos EndogâmicosRESUMO
To determine whether a preformed basement membrane contributes to the maintenance of morphology and function of type II pneumocytes, we cultured isolated adult rat type II pneumocytes on the basement membrane and stromal surfaces of an acellular human amnionic membrane and on plastic. The presence of lamellar bodies on transmission electron microscopy and epithelial morphology in culture and a characteristic phospholipid profile after incubation with 3H-acetate identified the cells as type II. When type II cells were cultured on a preexisting basement membrane, they formed a well-organized monolayer with polarity, centrally located surface microvilli, and more basally located nuclei. Individual cells maintained a cuboidal morphology for 8-10 days. Intracellularly, there were numerous mitochondria, endoplasmic reticulum (ER), and lamellar bodies. The cells secreted a new basal lamina of their own. When cultured on the stromal side of the amnion, the cells became flattened within 48-60 hours, formed small lamellar bodies, and had scanty surface microvilli; they formed clumps and appeared less ordered. These cells did not secrete a visible basement membrane, and the majority detached from the stromal surface after 7-8 days in culture. In addition, culture on the basement membrane aspect of the amnion prevented the rapid decline in the percentage of 3H-acetate label incorporated in phosphatidylcholine after 72 hours of culture. We conclude that a preformed basement membrane influences the function and morphology of type II pneumocytes, organizes them into a monolayer in culture, and influences deposition of a visible basal lamina. Thus, the acellular human amnion provides an excellent model for the systematic study of basement membrane influence on the biology and pathology of these cells.
Assuntos
Membrana Basal/fisiologia , Pulmão/citologia , Âmnio/fisiologia , Células Cultivadas , Colágeno/metabolismo , Meios de Cultura , Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Humanos , Laminina/metabolismo , Microscopia Eletrônica , Microscopia Eletrônica de Varredura , Modelos Biológicos , Surfactantes Pulmonares/metabolismoRESUMO
Glucose oxidation by developing rat intestine changed dramatically during the period of suckling and weaning. After weaning, glucose oxidation to CO2 by intestinal slices increased over 3-fold This was associated with an increase in lactate production from glucose and an increase in the rate of pyruvate decarboxylation. Active pyruvate dehydrogenase in intestine of developing rats also increases in activity at the time of weaning, suggesting that the suppression of glucose oxidation during the suckling period is controlled by pyruvate dehydrogenase. Glucose oxidation to CO2 and pyruvate decarboxylation to CO2 by intestinal slices of postweaned animals was inhibited by exogenous 3-hydroxybutyrate. But exogenous 3-hydroxybutyrate did not inhibit glucose and pyruvate oxidation in intestine of suckling animals which have higher levels of endogenous 3-hydroxybutyrate than intestine of postweaned rats. Palmitate, in contrast, inhibited glucose and pyruvate oxidation by both pre- and postweaned intestine.