| Objective: Significant renal complications frequently occur during liver cirrhosis, such as water imbalance, sodium retention, and activation of intrarenal hormones. The causes of renal function impairment during liver cirrhosis are still unclear. The disturbance of renal hemodynamics caused by severity liver diseases may play an important role. The characters of renal hemodynamics during liver cirrhosis were as following: constriction of intrarenal blood vessels, redistribution of renal blood, diffluence of blood flowed from renal cortex to medulla, cortex ischemia, constriction of afferent glomerular arteriole and glomerular filtration rate (GFR) decreased. Despite elevated levels of vasodilatation factors in the systemic circulation for the reason that inactivation reduced or overproduction, activities of vasoconstriction factors were to gain advantage locally in the kidney. Consistent with the development of the liver cirrhosis, renal vessel constriction intensified, the resistance of renal vessel was increased. These may lead to a decrease of glomerular filtration rate and renal blood flow,resulting in proceeding uropenia, anuria, the concentrations of creatinine and urea nitrogen were increased, at last developing into hepatorenal syndrome. Recent studies found that carbon monoxide (CO), like nitric oxide (NO), is a gas messenger. Besides participating in the convection of signal in the central nervous system, it also plays an important role in vascular tone regulation. We therefore aimed to investigate whether CO may also play a pathogenic role in the regulation of renal vessel of liver cirrhotic rats. Heme oxygenase(HO)is a heme oxidizing enzyme,which can catalyze heme molecules and generate biliverdin, carbon monoxide and free iron. The present study was designed to investigate the role of HO-1 in the pathogenesis of renal dysfunction,and effects of treatment with the selective heme oxygenase inhibitor zinc protoporphyrin Ⅸ (ZnPP-Ⅸ) and activator hemin on renal hemodynamics of liver cirrhotic rats.Method: 1. Animal model and groups: Studies were performed in male Sprague-Dawley rats. Cirrhotic model was induced in rats by 40% carbon tetrachloride (CCl4) and 5% ethanol. All rats were divided into 4 groups at random: ⑴Control group; ⑵Cirrhotic group; ⑶Cirrhosis+ZnPP group; ⑷Cirrhosis+Hemin group. 2. Measurements of hemodynamics: A laparotomy was performed in animals under anesthesia with ketamine; portal vein, superior mesenteric artery and renal artery were isolated. An electromagnetic flowmeter (MFV-3200, Japan) was used to measurement of renal artery blood flow. Scalp acupunctures were placed into portal vein and superior mesenteric artery, and connected to pressure transducers (YP-100, Baoding) for measurement of portal venous pressure (PVP) and mean arterial pressure (MAP). 3.Histology analysis: (1) Immunohistochemical analysis: Tissue samples were fixed in 10% formaldehyde solution, dehydrated and embedded in paraffin, then cut into 5 μm-thick slices. After deparaffinization and dehydration, sections were pretreated with 3% hydrogen peroxidase in methanol to exhaust endogenous peroxidase activities. Antigen plerosis: hot antigen plerosis methods were used. The slices were put into a volume filled with citric acid buffer, kept at 92℃-94℃ for 40 min. Sections were preincubated with 10% goat serum for 40 min, then incubated with the HO-1 antibody (Boshide, Wuhan) at 1:50 dilution at 4℃ for 24h. Slides were washed and incubated with biotinylated secondary antibody at 37℃ for 30 min. Third antibody was incubated for 40 min. At last, antigens were visualized with the 3,3-diaminobenzidine systems. The positive expressions in cell were yellow color or filled with yellow particles. (2) Western blotting analysis: The kidney tissues were homogenized in lysis buffer containing PMSF. Samples were kept on ice for 1h, then centrifuged with 12,000 rpm at 4℃ for 15 min. The supernatant was removed into the other tube and the insoluble fraction was discarded. The protein concentrati...
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