RESUMEN
Leukocyte accumulation has been shown to be increased in sepsis. Moreover, in inducible nitric oxide synthase (iNOS) knockout mice, a further increase in leukocyte accumulation has been observed during sepsis, suggesting that nitric oxide (NO) may affect leukocyte/endothelial interaction. Accelerated peroxynitrite formation also occurs during sepsis. In the present study, the effect of peroxynitrite or NO on leukocyte adhesion to nitric oxide synthase (NOS)-inhibited or endotoxin-treated endothelium was examined. Bovine aortic endothelial cells were treated with either L-NAME or lipopolysaccharide (LPS) and interferon-gamma for 4 hr and subsequent leukocyte adhesion was measured. Both L-NAME and LPS treatment resulted in increased leukocyte adhesion compared with control. Neither a peroxynitrite donor, SIN-1, nor a direct NO donor, DETA-NO, had any effect on leukocyte adhesion to untreated endothelium. However, when the L-NAME or LPS-treated endothelial cells were treated simultaneously with either SIN-1 or DETA-NO, there was a significant reduction in leukocyte adhesion. Moreover, at the concentrations used in the present study, neither peroxynitrite nor NO showed harmful effects on normal cultured endothelial cells. These data demonstrating inhibition of leukocyte adhesion to endotoxin-treated endothelium suggest that peroxynitrite or NO may exert a beneficial effect during sepsis.
Asunto(s)
Endotelio Vascular/citología , Leucocitos/efectos de los fármacos , Nitratos/farmacología , Óxido Nítrico/farmacología , Animales , Bovinos , Adhesión Celular/efectos de los fármacos , Células Cultivadas , GMP Cíclico/análisis , Endotelio Vascular/química , Endotelio Vascular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Interferón gamma/farmacología , Leucocitos/citología , Lipopolisacáridos/farmacología , Molsidomina/análogos & derivados , Molsidomina/farmacología , NG-Nitroarginina Metil Éster/farmacología , Donantes de Óxido Nítrico/farmacología , Óxido Nítrico Sintasa/análisis , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo II , Óxido Nítrico Sintasa de Tipo III , Triazenos/farmacologíaRESUMEN
BACKGROUND: Renal failure is a frequent complication of sepsis with a high mortality. Tumor necrosis factor (TNF) has been suggested to be a factor in the acute renal failure in sepsis or endotoxemia. Recent studies also suggest involvement of nitric oxide (NO), generated by inducible NO synthase (iNOS), in the pathogenesis of endotoxin-induced renal failure. The present study tested the hypothesis that the role of TNF in endotoxic renal failure is mediated by iNOS-derived NO. METHODS: Renal function was evaluated in endotoxemic [Escherichia coli lipopolysaccharide (LPS), 5 mg/kg IP] wild-type and iNOS knockout mice. The effect of TNF neutralization on renal function during endotoxemia in mice was assessed by a TNF-soluble receptor (TNFsRp55). RESULTS: An injection of LPS to wild-type mice resulted in a 70% decrease in glomerular filtration rate (GFR) and in a 40% reduction in renal plasma flow (RPF) 16 hours after the injection. The results occurred independent of hypotension, morphological changes, apoptosis, and leukocyte accumulation. In mice pretreated with TNFsRp55, only a 30% decrease in GFR without a significant change in RPF in response to LPS, as compared with vehicle-treated mice, was observed. Also, the serum NO concentration was significantly lower in endotoxemic wild-type mice pretreated with TNFsRp55, as compared with untreated endotoxemic wild-type mice (260 +/- 52 vs. 673 +/- 112 micromol/L, P < 0.01). In LPS-injected iNOS knockout mice and wild-type mice treated with a selective iNOS inhibitor, 1400W, the development of renal failure was similar to that in wild-type mice. As in wild-type mice, TNFsRp55 significantly attenuated the decrease in GFR (a 33% decline, as compared with 75% without TNFsRp55) without a significant change in RPF in iNOS knockout mice given LPS. CONCLUSIONS: These results demonstrate a role of TNF in the early renal dysfunction (16 h) in a septic mouse model independent of iNOS, hypotension, apoptosis, leukocyte accumulation, and morphological alterations, thus suggesting renal hypoperfusion secondary to an imbalance between, as yet to be defined, renal vasoconstrictors and vasodilators.
Asunto(s)
Lesión Renal Aguda/metabolismo , Endotoxemia/complicaciones , Óxido Nítrico Sintasa/genética , Factor de Necrosis Tumoral alfa/metabolismo , Lesión Renal Aguda/etiología , Animales , Antígenos CD/farmacología , Apoptosis , Presión Sanguínea , Tasa de Filtración Glomerular , Hipertensión Renal/etiología , Hipertensión Renal/metabolismo , Riñón/irrigación sanguínea , Riñón/enzimología , Leucocitos , Lipopolisacáridos/toxicidad , Masculino , Ratones , Ratones Noqueados , Óxido Nítrico/sangre , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo II , Receptores del Factor de Necrosis Tumoral , Receptores Tipo I de Factores de Necrosis Tumoral , Vasoconstricción/fisiologíaRESUMEN
BACKGROUND: In acute tubular necrosis (ATN), distal tubules are obstructed by casts formed by tubular debris, cells, and Tamm-Horsfall protein (THP). Since there are Arginine-Glycine-Aspartate (RGD) and Leucine-Aspartate-Valine (LDV) adhesive sequences in human THP, there may be direct integrin-mediated binding of tubular cells to THP. Alternatively, polymerization of THP may result in entrapment of the cells in its gel. METHODS: Adhesion of LLC-PK(1) cells to THP-coated wells was directly measured. THP concentrate was dissolved in solutions which mimic urine from ATN (ATN-S), distal convoluted tubule (DCT-S), collecting duct (CD-S), and monomeric buffer (M buffer). THP was also denatured by either boiling or N-glycanase digestion. Gel formation of THP was then measured. Inhibition of LLC-PK(1) cell adhesion to collagen type I was measured with each solution, as well as after the collagen was pretreated with either monomeric (mTHP) or polymeric (pTHP) THP. The effect of pTHP on the settling rate of LLC-PK(1) cells in suspension was also measured. RESULTS: LLC-PK(1) cells did not directly adhere to THP, a finding against integrin-mediated binding as a mechanism for in vivo tubular cell/THP cast formation. The high electrolyte concentration of ATN-S and CD-S, however, was associated with pTHP gel formation. Moreover, cells suspended in pTHP remained in suspension. In cell adhesion studies, mTHP attenuated cell adhesion by binding to the matrix, whereas pTHP attenuated cell adhesion by trapping cells in its gel. An active process was involved since both pTHP gel formation and attenuation of cell adhesion were abolished by boiling or oligosaccharide removal with N-glycanase digestion. CONCLUSIONS: With renal ischemia and proximal tubule cell shedding, ATN and collecting duct fluid composition enhance THP gel formation and thus favor tubular cast formation and obstruction. The present in vitro results indicate the importance of oligosaccharide residues in mediating the effect of the THP on gel formation and potential cast formation in ATN.
Asunto(s)
Mucoproteínas/fisiología , Animales , Líquidos Corporales/metabolismo , Adhesión Celular/efectos de los fármacos , Adhesión Celular/fisiología , Colágeno/fisiología , Electrólitos/metabolismo , Geles , Humanos , Túbulos Renales Proximales/metabolismo , Células LLC-PK1/citología , Células LLC-PK1/fisiología , Mucoproteínas/química , Mucoproteínas/farmacología , Oligosacáridos/fisiología , Polímeros/metabolismo , Porcinos , UromodulinaRESUMEN
Renal ischemia-reperfusion (I/R) injury was investigated in inducible nitric oxide synthase (iNOS) knockout mice. After a 26-min bilateral renal pedicle clamp, serum creatinine concentrations (in mg/dl) in wild-type mice after a 24-h reperfusion were 0.25 +/- 0.03 in sham-operated controls and 2.3 +/- 0.38 in ischemic mice (P < 0. 01); after 48 h, concentrations (in mg/dl) were 0.25 +/- 0.03 in controls and 2.0 +/- 0.18 in ischemic mice (P < 0.01). iNOS knockout mice demonstrated an attenuation of serum creatinine concentration after renal I/R injury. Serum creatinine concentrations (mg/dl) after a 24-h reperfusion were 2.3 +/- 0.22 in wild-type ischemic and 1.21 +/- 0.25 in iNOS knockout ischemic mice (P < 0.05); after 48 h, concentrations were 2.0 +/- 0.18 in wild-type ischemic and 0.96 +/- 0.25 in iNOS knockout ischemic mice (P < 0.01). Histological scoring of acute tubular necrosis in iNOS knockout mice was decreased compared with that in wild-type controls (0.88 +/- 0.2 vs. 3.3 +/- 0. 3, P < 0.05). iNOS protein in the renal cortex of wild-type mice subjected to renal I/R injury was undetectable up to 48 h. However, a strong upregulation of heat shock protein 72 expression was observed in renal cortex of iNOS knockout mice under basal conditions. In conclusion, kidneys of iNOS knockout mice were protected against ischemic acute renal failure. This protective effect may be related to a compensatory upregulation of heat shock protein 72.
Asunto(s)
Riñón/fisiopatología , Óxido Nítrico Sintasa/fisiología , Daño por Reperfusión/fisiopatología , Animales , Proteínas del Choque Térmico HSP72 , Proteínas de Choque Térmico/biosíntesis , Riñón/irrigación sanguínea , Riñón/patología , Ratones , Ratones Noqueados , Necrosis , Óxido Nítrico Sintasa de Tipo II , Daño por Reperfusión/patología , Regulación hacia ArribaRESUMEN
BACKGROUND: Nitric oxide (NO) and its metabolite, peroxynitrite (ONOO-), are involved in renal tubular cell injury. We postulated that if NO/ONOO- has an effect to reduce cell adhesion to the basement membrane, this may contribute to tubular obstruction and may be partially responsible for the harmful effect of NO on the tubular epithelium during acute renal failure (ARF). METHODS: We examined the effect of the NO donors (z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1- ium-1, 2-diolate (DETA/NO), spermine NONOate (SpNO), and the ONOO- donor 3-morpholinosydnonimine (SIN-1) on cell-matrix adhesion to collagen types I and IV and fibronectin using three renal tubular epithelial cell lines: LLC-PK1, BSC-1, and OK. RESULTS: In LLC-PK1 cells, DETA/NO (500 microM) had no effect, and SpNO (500 microM) had a modest effect on cell adhesion compared with controls. Exposure to SIN-1 caused a dose-dependent impairment in cell-matrix adhesion. Similar results were obtained in the different cell types and matrix proteins. The effect of SIN-1 (500 microM) on LLC-PK1 cell adhesion was not associated with either cell death or alteration of matrix protein and was attenuated by either the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, the superoxide scavenger superoxide dismutase, or the ONOO- scavenger uric acid in a dose-dependent manner. CONCLUSIONS: These results therefore support the possibility that ONOO- generated in the tubular epithelium during ischemia/reperfusion has the potential to impair the adhesion properties of tubular cells, which then may contribute to the tubular obstruction in ARF.
Asunto(s)
Túbulos Renales/efectos de los fármacos , Donantes de Óxido Nítrico/farmacología , Animales , Adhesión Celular/efectos de los fármacos , Línea Celular , Chlorocebus aethiops , Colágeno/metabolismo , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Fibronectinas/metabolismo , Depuradores de Radicales Libres/farmacología , Túbulos Renales/citología , Túbulos Renales/metabolismo , Células LLC-PK1 , Molsidomina/análogos & derivados , Molsidomina/farmacología , Nitratos/farmacología , Óxido Nítrico/farmacología , Zarigüeyas , Superóxidos/farmacología , PorcinosRESUMEN
Nitric oxide (NO) has been shown to be a mediator of hypoxic injury in rat renal proximal tubules (PT). However, the role of NO in hypoxic injury to mouse. PT has not been examined. The aim of the present study was to determine the effect of knockout of nitric oxide synthase (NOS) isoforms on hypoxic injury in mouse PT. Mouse PTs were isolated by collagenase digestion and Percoll centrifugation. The nonselective NOS inhibitor, N-nitro-L-arginine methyl ester (L-NAME, 10 mM), but not its inactive stereoisomer D-NAME, protected against hypoxic injury as assessed by LDH release. Carboxy-imidazolineoxyl N-oxide (carboxy-PTIO, 100 microM), a stable NO scavenger, also afforded cytoprotection against hypoxic injury. To determine the role of the different NOS isoforms in the hypoxic injury, we examined the effect of hypoxia on PT isolated from knockout mice in which either the inducible NOS (iNOS) endothelial NOS (eNOS) or neuronal NOS (nNOS) gene was lacking. PT isolated from iNOS knockout mice were resistant to hypoxic injury compared to wild-type controls. In contrast, PT isolated from both nNOS and eNOS knockout mice were not protected against hypoxic injury. In conclusion, the present study demonstrates that NO is a mediator of hypoxic PT injury in the mouse and that knockout of the iNOS gene is cytoprotective against this hypoxic PT injury.
Asunto(s)
Hipoxia/metabolismo , Túbulos Renales Proximales/metabolismo , Ratones Noqueados/genética , Ratones Noqueados/metabolismo , Óxido Nítrico Sintasa/genética , Animales , Benzoatos/farmacología , Inhibidores Enzimáticos/farmacología , Eliminación de Gen , Imidazoles/farmacología , Técnicas In Vitro , L-Lactato Deshidrogenasa/metabolismo , Ratones , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico Sintasa de Tipo I , Óxido Nítrico Sintasa de Tipo II , Óxido Nítrico Sintasa de Tipo IIIRESUMEN
The effect of glycine on hypoxia- and ionomycin-induced increases in calpain activity in rat proximal tubules was determined. Calpain activity was determined both in vitro and in the intact cell using the fluorescent substrate N-succinyl-Leu-Leu-Val-Tyr-7-amido-4-methyl coumarin (N-succinyl-Leu-Leu-Val-Tyr-AMC) and Western blotting for calpain-specific spectrin breakdown products (BDP), respectively. At 7.5 minutes of hypoxia (prelethal injury model) there was a significant (10-fold) increase in in vitro calpain activity that was not inhibited by glycine. At 15 minutes of hypoxia (postlethal injury model) there was a further increase in calpain activity that was inhibited by glycine. Normoxic tubules incubated with the calcium ionophore ionomycin (5 microM) for two minutes and 10 minutes had a significant increase in calpain activity that was not inhibited by glycine. After 15 minutes of hypoxia in the presence of glycine, there was an increase in calpain-specific spectrin breakdown products (BDP) in both Triton X-100 soluble and cytosolic extracts from proximal tubules. Glycine in concentrations up to 10 mM had no direct effect on the in vitro calpain activity of purified calpains. The present study demonstrates that: (1) prelethal increases in calpain activity stimulated by hypoxia and ionomycin treatment are not affected by glycine; (2) calpain-mediated spectrin breakdown during hypoxia occurs in the presence of glycine; (3) glycine does prevent the additional postlethal increase in calpain activity probably by maintaining membrane integrity to calcium influx.
Asunto(s)
Calpaína/metabolismo , Glicina/farmacología , Túbulos Renales Proximales/efectos de los fármacos , Animales , Calcio/metabolismo , Calpaína/antagonistas & inhibidores , Citoprotección , Ionomicina/farmacología , Túbulos Renales Proximales/enzimología , Masculino , Ratas , Ratas Sprague-Dawley , Espectrina/metabolismoRESUMEN
The effect of the newly developed, nonpeptide, calpain inhibitor, PD 150606, on hypoxia and ionomycin-induced increases in calpain activity in rat proximal tubules (PT) was determined. PD150606 inhibited both hypoxia and ionomycin-induced calpain activity as determined by the fluorescent substrate N-succinyl-Leu-Leu-Val-Tyr-7-amido-4-methyl coumarin (N-succinyl-Leu-Leu-Val-Tyr-AMC). This decrease in calpain activity was accompanied by dose-dependent cytoprotection against hypoxia and ionomycin-induced cell membrane damage. PD150606 had no effect on cathepsin B and L activity in PT as measured by the fluorescent substrate, benzyloxycarbonyl-L-phenylalanyl-L-arginine-7-amido-4-methyl coumarin (Z-Phe-Arg-AMC). The effects of low intracellular pH (pHi) or low free cytosolic calcium [Ca2+]i on this hypoxia-induced calpain activity were also determined. Both low pHi and low [Ca2+]i attenuated the hypoxia-induced increase in calpain activity. This attenuation of calpain activity was observed early before hypoxia-induced membrane damage and was associated with marked reduction in the typical pattern of hypoxia-induced cell membrane damage observed in this model. To identify the isoform of calpain activated in rat proximal tubules, normoxic, hypoxic and ionomycin treated tubules were fractionated by MONO-Q anion exchange chromatography and the fractions were assayed for calpain activity. A single peak of calpain activity characteristic of mu-calpain was found. The calcium dependency of the calpain activity was in the nanomolar range, further confirming that the activity was the low Ca(2+)-sensitive mu-calpain. The present study suggests that in rat proximal tubules: (1) PD 150606 is a specific inhibitor of calpain and not cathepsins B and L; (2) the cytoprotective effects of low pHi and low [Ca2+]i are mediated, at least in part, by inhibition of calpain activity; and (3) the predominant active form of calpain is the isoenzyme mu-calpain.
Asunto(s)
Calpaína/metabolismo , Hipoxia/metabolismo , Túbulos Renales Proximales/enzimología , Acrilatos/farmacología , Análisis de Varianza , Animales , Calcio/fisiología , Calpaína/antagonistas & inhibidores , Calpaína/química , Catepsinas/análisis , Relación Dosis-Respuesta a Droga , Concentración de Iones de Hidrógeno , Técnicas In Vitro , L-Lactato Deshidrogenasa/análisis , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
In the present study, we directly monitored nitric oxide (NO) with an amperometric NO-sensor in suspensions of rat proximal tubules. Hypoxia-stimulated NO generation was characterized by an initial rise and a subsequent sustained increase which preceded cell membrane damage as assessed by lactic dehydrogenase (LDH) release. In contrast, the NO concentration remained unmeasurable in normoxic controls. Nitro-L-arginine-methyl ester (L-NAME) prevented the hypoxia-induced increase in NO in a dose dependent manner in parallel with incremental cytoprotection. The hypoxia-induced elevation in NO and the associated membrane injury were both markedly prevented by extracellular acidosis (pH 6.95). In vitro proximal tubular nitric oxide synthase (NOS) activity (3H-arginine to 3H-citrulline assay) was pH dependent with optimum activity at pH 8.0 and greatly reduced activity at acidic pH even in the presence of calcium and co-factors. However, glycine, a well recognized cytoprotective agent, did not attenuate the NO concentration during hypoxia. The present study therefore provides direct evidence that NO is generated by rat proximal tubules during hypoxia and demonstrates that the protective effect of low pH against hypoxic rat tubular injury is associated with an inhibition of this NO production.
Asunto(s)
Acidosis Tubular Renal/metabolismo , Hipoxia/metabolismo , Túbulos Renales Proximales/metabolismo , Óxido Nítrico/metabolismo , Animales , Arginina/análogos & derivados , Arginina/química , Arginina/farmacología , Glicina/farmacología , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Túbulos Renales Proximales/efectos de los fármacos , Cinética , L-Lactato Deshidrogenasa/metabolismo , Masculino , NG-Nitroarginina Metil Éster , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa/metabolismo , Ratas , Ratas Sprague-Dawley , EstereoisomerismoRESUMEN
Free fatty acids (FFA) and lysophospholipids accumulate during hypoxia (H) in rat proximal tubular epithelial cells partly as a result of increased phospholipase A2 (PLA2) activity. The role of FFA in mediating hypoxic injury and modulating PLA2 activity is not clear. In the present study, the effect of several FFA including arachidonic acid (AA, 20:4) on hypoxia-induced injury and PLA2 activity was assessed in freshly isolated rat proximal tubules. Hypoxia (H) was induced in the presence of either an unsaturated free fatty acid (uFFA) [AA or linoleic acid (LA, 18:2)] or a saturated FFA (sFFA) [palmitic (PA, 16:0) or myristic acid (MA, 14:0)]. Cell membrane injury was assessed by measuring lactate dehydrogenase release (LDH). AA markedly reduced LDH release during hypoxia in a dose dependent manner, while sFFA had no protective effect. LA showed similar protection to that observed with AA. AA did not affect buffer calcium concentration, buffer pH, intracellular pH or intracellular calcium concentration. Neither inhibiting the cyclooxygenase pathway with indomethacin, nor the lipoxygenase pathway with nordihydroguaiaretic acid (NDGA) had any effect on the AA observed cytoprotection. In vitro PLA2 activity in the control tubular extracts was compared to that following addition of AA or PA. PLA2 activity decreased significantly with AA but not with PA in a dose dependent manner. These data suggest that: (1) AA protects against hypoxic injury in rat proximal tubules. (2) This cytoprotection is not specific for AA and other uFFA have a similar effect. (3) AA significantly inhibits PLA2 activity, (4) AA induced cytoprotection may be related to a negative feedback inhibition of PLA2 activity.
Asunto(s)
Ácido Araquidónico/farmacología , Hipoxia/prevención & control , Túbulos Renales Proximales/efectos de los fármacos , Fosfolipasas A/metabolismo , Animales , Calcio/metabolismo , Inhibidores de la Ciclooxigenasa/farmacología , Activación Enzimática , Concentración de Iones de Hidrógeno , Hipoxia/metabolismo , Hipoxia/patología , Líquido Intracelular/efectos de los fármacos , Líquido Intracelular/metabolismo , Túbulos Renales Proximales/metabolismo , Túbulos Renales Proximales/patología , L-Lactato Deshidrogenasa/metabolismo , Masculino , Ácido Palmítico , Ácidos Palmíticos/farmacología , Fosfolipasas A/efectos de los fármacos , Fosfolipasas A2 , Potasio/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Increased free fatty release during hypoxia is believed to contribute to cell injury. This phenomenon is likely to be mediated through activation of specific isoforms of phospholipase A2 (PLA2). In this study, PLA2 enzymatic activity was measured in cell-free extracts prepared from rat renal proximal tubules. Both soluble and membrane-associated PLA2 activity were detected. All PLA2 activity detected during normoxia was Ca2+ dependent. Fractionation of cytosolic extracts by gel filtration revealed three peaks of PLA2 activity. Exposure of tubules to hypoxia resulted in stable activation of soluble PLA2 activity, which correlated with disappearance of the highest molecular mass form (> 100 kDa) and appearance of a low-molecular-mass form (approximately 15 kDa) of PLA2. Hypoxia also resulted in release of a low-molecular-mass form of PLA2 into the extracellular medium. Pretreatment of tubules with glycine before hypoxia blocked this release of PLA2 but not activation of soluble PLA2 activity. These studies provide direct evidence for PLA2 activation during hypoxia and suggest that multiple mechanisms regulate free fatty acid release associated with hypoxia injury.
Asunto(s)
Hipoxia/enzimología , Túbulos Renales Proximales/enzimología , Fosfolipasas A/metabolismo , Animales , Sistema Libre de Células , Fraccionamiento Químico , Glicina/farmacología , Isoenzimas/metabolismo , Túbulos Renales Proximales/efectos de los fármacos , Fosfolipasas A2 , Ratas , Ratas Sprague-Dawley , Valores de ReferenciaRESUMEN
The role of the lysosomal proteases cathepsins B and L and the calcium-dependent cytosolic protease calpain in hypoxia-induced renal proximal tubular injury was investigated. As compared to normoxic tubules, cathepsin B and L activity, evaluated by the specific fluorescent substrate benzyloxycarbonyl-L-phenylalanyl-L-arginine-7-amido-4-methylcoumarin, was not increased in hypoxic tubules or the medium used for incubation of hypoxic tubules in spite of high lactate dehydrogenase (LDH) release into the medium during hypoxia. These data in rat proximal tubules suggest that cathepsins are not released from lysosomes and do not gain access to the medium during hypoxia. An assay for calpain activity in isolated proximal tubules using the fluorescent substrate N-succinyl-Leu-Tyr-7-amido-4-methylcoumarin was developed. The calcium ionophore ionomycin induced a dose-dependent increase in calpain activity. This increase in calpain activity occurred prior to cell membrane damage as assessed by LDH release. Tubular calpain activity increased significantly by 7.5 min of hypoxia, before there was significant LDH release, and further increased during 20 min of hypoxia. The cysteine protease inhibitor N-benzyloxycarbonyl-Val-Phe methyl ester (CBZ) markedly decreased LDH release after 20 min of hypoxia and completely prevented the increase in calpain activity during hypoxia. The increase in calpain activity during hypoxia and the inhibitor studies with CBZ therefore supported a role for calpain as a mediator of hypoxia-induced proximal tubular injury.
Asunto(s)
Calpaína/metabolismo , Catepsina B/metabolismo , Catepsinas/metabolismo , Cisteína Endopeptidasas/metabolismo , Endopeptidasas , Hipoxia , Corteza Renal/fisiopatología , Túbulos Renales Proximales/fisiopatología , Animales , Catepsina L , Dipéptidos/farmacología , Técnicas In Vitro , Ionomicina/farmacología , Corteza Renal/efectos de los fármacos , Corteza Renal/patología , Túbulos Renales Proximales/efectos de los fármacos , Túbulos Renales Proximales/patología , Cinética , Masculino , Ratas , Ratas Sprague-Dawley , Valores de Referencia , Inhibidores de Serina Proteinasa/farmacologíaRESUMEN
A protective effect of verapamil against hypoxic renal proximal tubule injury has been demonstrated in vitro. In contrast to other cytoprotective agents such as glycine or alanine, the protective effect of verapamil is associated with better maintenance of cellular ATP and potassium levels. These findings suggested a possible direct effect of verapamil on the mitochondria in addition to known effects of verapamil on membrane Ca channels. In the present study, the direct effects of verapamil on Ca-induced swelling, respiratory dysfunction, Ca uptake rate and phospholipase activity of renal cortical mitochondria were determined. Verapamil (100 microM) significantly inhibited Ca-induced mitochondrial swelling and partially prevented the associated reduction in respiratory control ratio (State 3/State 4: Ca + verapamil: 2.8 +/- 0.1 vs. Ca alone, 2.0 +/- 0.2; P < .01). A phospholipase A2 inhibitor, dibucaine (100 microM), significantly inhibited Ca-induced mitochondrial swelling and attenuated the decrease in respiratory control ratio (Ca + dibucaine: 2.9 +/- 0.1 vs. Ca alone, 2.0 +/- 0.2; P < .001). Neither agent, either alone or combined, completely prevented the respiratory dysfunction. Either verapamil or dibucaine attenuated the mitochondrial Ca uptake rate and reduced the rate of Ca-stimulated polyunsaturated free fatty acid accumulation; verapamil treatment also was associated with diminished net release of saturated and monounsaturated free fatty acids. These findings demonstrate that verapamil exerts a protective effect against Ca-induced mitochondrial damage which may be mediated in part by its effect to suppress mitochondrial Ca uptake and mitochondrial phospholipase activity.
Asunto(s)
Calcio/farmacología , Dilatación Mitocondrial/efectos de los fármacos , Consumo de Oxígeno/efectos de los fármacos , Verapamilo/farmacología , Animales , Calcio/metabolismo , Masculino , Fosfolipasas/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
The role of cytosolic free Ca2+ ([Ca2+]i) in hypoxic injury was investigated in rat proximal tubules. [Ca2+]i was measured using fura-2 and cell injury was estimated with propidium iodide (PI) in individual tubules using video imaging fluorescence microscopy. [Ca2+]i increased from approximately 170 to approximately 390 nM during 5 min of hypoxia. This increase preceded detectable cell injury as assessed by PI and was reversible with reoxygenation. 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA; 100 microM) reduced [Ca2+]i under basal conditions (approximately 80 nM) and during hypoxia (approximately 120 nM) and significantly attenuated hypoxic injury. When [Ca2+]i and hypoxic cell injury were studied concurrently in the same individual tubules, the 10 min [Ca2+]i rise correlated significantly with subsequent cell damage observed at 20 min. 2 mM glycine did not block the rise in [Ca2+]i, yet protected the tubules from hypoxic injury. These results indicate that in rat proximal tubules, hypoxia induces an increase of [Ca2+]i which occurs before cell damage. The protective effect of BAPTA supports a role for [Ca2+]i in the initiation of hypoxic proximal tubule injury. The glycine results, however, implicate calcium-independent mechanisms of injury and/or blockade of calcium-mediated processes of injury such as activation of phospholipases or proteases.
Asunto(s)
Calcio/metabolismo , Hipoxia de la Célula , Citosol/metabolismo , Túbulos Renales Proximales/metabolismo , Adenosina Trifosfato/análisis , Animales , Citosol/efectos de los fármacos , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Glicina/farmacología , Técnicas In Vitro , Túbulos Renales Proximales/efectos de los fármacos , Masculino , Microscopía Fluorescente , Potasio/análisis , Ratas , Ratas Sprague-Dawley , Verapamilo/farmacología , Grabación en VideoRESUMEN
Nitric oxide (NO), among several other functions, may play a role in hypoxia and reoxygenation injury due to its free radical nature and high reactivity with the superoxide radical to yield peroxynitrite, an oxidant molecule. The present study was undertaken to evaluate a potential role for NO, either endogenous or exogenous, in a model of hypoxia/reoxygenation (H/R) in freshly isolated rat proximal tubules. NO synthase activity, as assessed by conversion of L-[3H]arginine to L-[3H]citrulline, was detected in normoxic tubules. This activity could be inhibited by N-nitro-L-arginine methyl ester (L-NAME), a NO synthase inhibitor, and was stimulated by 15 min of hypoxia. The injury in proximal tubules caused by 15 min of hypoxia followed by 35 min of reoxygenation was completely prevented by L-NAME as assessed by release of lactate dehydrogenase, whereas D-NAME, which does not inhibit NO synthase, had no effect. In contrast, L-arginine (NO substrate) enhanced the H/R injury. These effects were paralleled by nitrite/nitrate production. In separate experiments, the addition of sodium nitroprusside, a NO donor, to proximal tubules enhanced the H/R injury; this effect could be blocked by hemoglobin, a NO scavenger. Also, addition of nitroprusside reversed L-NAME protection against H/R injury. These results demonstrate that NO is synthesized in rat proximal tubules and participates as one of the mediators in rat tubular H/R injury.
Asunto(s)
Túbulos Renales/lesiones , Túbulos Renales/metabolismo , Óxido Nítrico/metabolismo , Daño por Reperfusión/etiología , Aminoácido Oxidorreductasas/antagonistas & inhibidores , Aminoácido Oxidorreductasas/metabolismo , Animales , Arginina/análogos & derivados , Arginina/farmacología , Túbulos Renales/efectos de los fármacos , L-Lactato Deshidrogenasa/metabolismo , Masculino , NG-Nitroarginina Metil Éster , Óxido Nítrico Sintasa , Nitroprusiato/farmacología , Ratas , Ratas Sprague-Dawley , Daño por Reperfusión/metabolismo , Daño por Reperfusión/prevención & controlRESUMEN
In freshly isolated hypoxic rat proximal tubules, Ca2+ uptake rate increases promptly, within 1 min, and remains significantly elevated throughout a 20-min period of hypoxia. Lactate dehydrogenase (LDH) release, a sign of membrane injury, increases only after 5 min of hypoxia and thereafter rises progressively. The potential effect of increased Ca2+ uptake rate to activate phospholipases, which would then initiate membrane injury, was evaluated by treating hypoxic tubules with three dissimilar phospholipase inhibitors, i.e., mepacrine, dibucaine, or p-bromophenacyl bromide (PBPB). LDH release averaged 11.9 and 13.8% after 10 and 20 min of normoxia, respectively. With 10 or 20 min of hypoxia LDH release increased to 46.0 and 65.2%, respectively (P < 0.01), and Ca2+ uptake rate increased from 2.56 in normoxia to 4.71 nmol.mg-1 x min-1 at 10 min of hypoxia (P < 0.01) and from 2.82 in normoxia to 3.76 nmol/mg at 20 min of hypoxia (P < 0.05). In a separate series of tubules, after 10 min of hypoxia LDH release was reduced by pretreatment with 50 microM mepacrine (66.1 to 47.3%, P < 0.01) or 50 microM dibucaine (53.1 to 38.5%, P < 0.02). The increase in Ca2+ uptake rate also was significantly reduced. After 20 min of hypoxia neither mepacrine nor dibucaine reduced Ca2+ uptake rate; LDH release was modestly reduced by dibucaine but not mepacrine. Higher doses of mepacrine (500 microM) and dibucaine (250 microM) also reduced cell injury at 10 min of hypoxia as assessed by LDH release.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Calcio/metabolismo , Dibucaína/farmacología , Ácidos Grasos no Esterificados/metabolismo , Hipoxia/metabolismo , Túbulos Renales Proximales/metabolismo , L-Lactato Deshidrogenasa/análisis , Quinacrina/farmacología , Animales , Transporte Biológico/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Técnicas In Vitro , Túbulos Renales Proximales/efectos de los fármacos , Cinética , Ratas , Factores de TiempoRESUMEN
We studied the effects of glycine (2 mM) on hypoxia-induced changes in phospholipids and fatty acids in isolated rat proximal tubules. In this preparation, 25 min of hypoxia caused cell injury, as reflected by the release of lactate dehydrogenase (LDH) (13.1 +/- 0.8 vs. 43.5 +/- 3.2%; P < 0.01). Hypoxia caused increases in fatty acids and in lysophospholipids. Glycine prevented the hypoxia-induced cell injury (LDH 13.1 +/- 0.8 vs. 11 +/- 0.7%; not significant) but did not attenuate the increases in fatty acids or lysophospholipids. In additional experiments, the effects of glycine on phospholipid changes and cell injury induced by exogenous phospholipase A2 (PLA2) were studied. PLA2 caused dramatic increases in fatty acids and lysophospholipids and mild cell injury; these effects were not influenced by glycine. In contrast, glycine attenuated increases in LDH release induced by exposing the tubules to exogenous arachidonic acid. In conclusion, glycine does not prevent the phospholipid degradation induced by either exogenous PLA2 or hypoxia in isolated proximal tubules and yet affords protection against hypoxia and exogenous arachidonic acid.