| Objective: Up to date, the incidence of sepsis remains exceedingly high in intensive care unit (ICU), with a mortality rate of 18~29%. The main complication of sepsis is septic shock and mutiple organ dysfunction syndrome (MODS), which is the main cause of death in critical patients. In 19 to 51 percent septic patients, acute renal failure (ARF) is a most common complication, and the mortality rate is higher to 70%. Endothelin-1 (ET-1), a peptide of 21 amino acid residues, is the most potent vasoconstrictor substance as known. In pathological status, the concentration of ET-1 in plasma is high, and the binding of ET-1 to its cell surface coupled receptor induces constriction of vascular beds and hypoperfusion, which results in a high incidence of multiple organ dysfunction syndrome or multiple organ failure. Ulinastatin is a kind of ulinary trypsin inhibitor (UTI), which was extracted from men's urine. In recent studys, Ulinastatin was reported to have the effect of inhibiting the procession of systemic inflammatory response syndrome (SIRS) or MODS, which indicated its effective renal protection. In this study, in order to investigate the renal protective effects of ulinastatin and related mechanism, we investigate the level changes of ET-1 in plasma and ET-1 mRNA in renal specimen in septic rats which treated with Ulinastatin at same time and same dose (100,000 U/Kg). The histomorphological and ultrastructural changes of kidney were also observed to elucidate the results.Method: The model of cecal ligation and puncture (CLP) was used to duplicate severe sepsis model. Carotid artery catheterization was used to detect the blood pressure and heart rate. Nighty-six Sprague Dawley (SD) rats were randomly divided into three groups:①Sham-operation group (n=32, Rats received operation on abdomen, without giving CLP) ;②Saline group (n=32, Sodium Chloride was adminisrated into abdominal cavity after CLP );③Ulinastatin group (n = 32, Ulinastatin, at the dose of 100,000U/kg, was adminisrated into abdominal cavity after CLP). Every group was divided into four groups (n=8), which was preoperation, 3h, 6h and 12h subgroups. The blood in carotid artery were collected in each group, 1ml was enough. The plasma were preserved in -80℃and the renal specimen was preserved in nitrogen canister. The level of ET-1 in plasma was detected by Radioimmu-noassay (RIA). The expression of ET-1 mRNA in renal tissues was detected with reverse transcription-polymerase chain raction (RT-PCR) and was semi-quantitatively analyzed by Gel-pro Analyzer software. The histopathological morphologic changes in renal specimen were determined with HE staining under light microscopy (3h, 6h and 12h subgroups) and transmission electron microscopy technique (3h subgroup). SPSS11.5 software package was used for Statistical analysis. Data were expressed as mean±SD ( x±s), and the means of each group were analysed with ANOVA. A level of P<0.05 was considered statistically to be significant.Result1 The general status of rats in every group: The rats in Sham-operation group analepsied earlier than that in the other groups and acted actively. When the abdomen was opened, no abdominal dropsy and no evil smelling were found. The rats in Saline group analepsiaed later than that in Sham-operation group, and then depressed with inaction, piloerection, and tachypnea. When the abdomen was opened, bloody ascites, bowels edema and evil smelling could be found. The cecum was darkening in some rats. Compared with Saline group, the clinical manifestation in the rats of Ulinastatin groups were better.2 The histopathological changes in renal tissue were observed by light microscope: In sham-operation group, the structure of renal glomerulus and tubuli were normal, and we could see a few inflammatory cells in renal interstitium. In Saline group, renal glomerulus shrinked obviously, and renal proximal tubular epithelial cell degenerated, necrosised and shed. In addition, dilatation of renal capsular space and a lot of mononuclear leukocytic infiltration in renal interstitium could be found. Compared with Saline group, the changes of renal glomerulus and renal tubuli in Ulinastatin group were better.3 The ultrastructural changes in renal tissue were observed by electron microscope: In Sham-operation group, the glomerular filtration membrane was intact, and basement membrane thickness was uniform, and endothelial cell foot process arranged in order. The shape of endothelial cells was normal, and we could see the window. The brush border in proximal convoluted tubule endothelial cell bloomed and lined up in order. The chromatin distribute were uniformly, and the structure of mitochondria was normal. In Saline group, glomerular basement membrane was thicken and humped, some foot process cells conjugated, and some vascular endothelia cells hyperplasied and became thicken. In liberation face of proximal convoluted tubule, the quantity of microvillus was decreased and arranged irregularly, and some crista mitochondriales could be seen, and mitochondria membrane fusion decreased or disappeared. Rough endoplasmic reticulum stretched lightly, and the phenomenon of degranulation was obviously. Compared with Saline group, ultrastructure changes of renal glomerulus and proximal convoluted tubule in Ulinastatin group were better.4 The change of ET-1 level in plasma: In Sham-operation group , the level of ET-1 in plasma of 3h, 6h and 12h subgroups were higher than that of pre-operation obviously (29.065±43.731 vs 82.768±3.599pg/ml, P<0.01; 387.635±14.598 vs 82.768±3.599 pg/ml, P<0.01; 391.783±15.673 vs 82.768±3.599pg/ml, P < 0.01, respectively). In Saline group, the level of ET-1 in plasma of 3h, 6h and 12h subgroups were higher than that of Sham-operation group obviously (456.363±24.048 vs 329.065±43.731pg/ml, P<0.05; 583.054±48.971 vs 387.635±14.598 pg/ml, P<0.05; 476.613±24.322 vs 391.783±15.673 pg/ml, P<0.05, respectively). The level of ET-1 in plasma of septic rats was highest at six hours after CLP, and 3h, 12h subgroups were both lower than that of 6h subgroup obviously(456.363±24.048 vs 583.054±48.971 pg/ml, P<0.05; 476.613±24.322 vs 583.054±48.971 pg/ml, P<0.05, respectively). In Ulinastatin group, the level of ET-1 in plasma of 3h, 6h subgroups were lower than that of Saline group obviously (336.828±22.330 vs 456.363±24.048 pg/ml, P<0.05; 458.868±46.269 vs 583.054±48.971 pg/ml, P <0.05, respectively), but no significant difference was found in 12h subgroup(P>0.05).5 The expression of ET-1 mRNA in renal tissue: In Sham- operation group, the level of ET-1 mRNA in renal tissue of 3h, 6h and 12h subgroups were higher than that of pre-operation obviously(0.562±0.045 vs 0.140±0.005, P<0.01; 0.477±0.048 vs 0.140±0.005, P<0.01; 0.052±0.010 vs 0.140±0.005, P<0.05, respectively). In Saline group, the level of ET-1 mRNA in renal tissue of 3h, 6h, and 12h subgroups were higher than that of Sham-operation group obviously (0.770±0.039 vs 0.562±0.045, P<0.05; 0.694±0.064 vs 0.477±0.048, P<0.05; 0.881±0.028 vs 0.052±0.010, P<0.01, respectively). The level of ET-1 mRNA in renal tissue of septic rats was increased at post-CLP three hours and decreased at six hours, which increased at twelve hours again, but there was no significant difference between 3h and 6h subgroup (P>0.05). In 12h subgroup, The level of ET-1 mRNA was lower than that of 6h subgroup obviously (0.881±0.028 vs 0.694±0.064, P<0.05). In Ulinastatin group, the level of ET-1 mRNA in renal tissue of 3h, 6h and 12h subgroups were lower than that of Saline group obviously (0.437±0.046 vs 0.770±0.039, P<0.01; 0.382±0.051 vs 0.694±0.064, P<0.01; 0.553±0.084 vs 0.881±0.028, P<0.05, respectively).Conclusions1 In this study, the animal model of sepsis was duplicated by cecal ligation and puncture (CLP) successfully.2 The high level of ET-1 in plasma was caused by trauma and infection, the same as the high expression of ET-1 mRNA in renal tissue. The level of ET-1 in plasma in septic rats was highest at six hours after CLP. The expression of ET-1 mRNA in renal of septic rats was increased at three hours after CLP, decreased at six hours, and increase at twelve hours again. The histopathological and ultrastructural changes in renal tissue were observed at three hours after CLP.3 Ulinastatin possesses the renal protective effects in septic rats, which may be related to the synthesis and release of ET-1. Ulinastatin could improve the clinical manifestation and physical sign of septic rats. In early stage of sepsis, Ulinastatin could reduce the level of ET-1 in plasma, furthmore, it regulates the expression of ET-1 mRNA in renal on gene level and lessen the histopathological and ultrastructural changes in renal. |
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