| BackgroundSerious infection caused severe sepsis, shock and multiple organ dysfunction syndrome (MODS) remains a severe issues in the intensive care unit (ICU). Despite of advance development in the pathophysiology and treatment effort, morbidity and mortality are still unacceptablely high. Kidney is often the most vulnerable organ to be injuried according to the consensus of acute kidney injury (AKI) standard. Nearly8%of hospitalized patients and more than50%of ICU patients developed AKI. In addition, severe infection and sepsis shock are the main reason of AKI in critically ill patients: more than50%of AKI caused by sepsis. Despite of renal replacement therapy, AKI morbidity and mortality remain high, which might be related to its unclear underlying pathogenesis.Recent studies discovered that glycocalyx polysaccharide-protein complexes damage resulting in impaired microcirculation might be responsible for the AKI in sepsis. Growing bodies of stduies have shown the intravascular glycocalyx is important for cardiovascular disease. Glycocalyx is lining structure in the lumenar side of endothelial cell and glycocalyx is rich in polysaccharide, working as an important barrier between blood and tissue. Structural integrity of glycocalyx may maintain vascular permeability, suppress cell adhesion and provoke vascular protection. Once the integrity is destroyed, endothelium dysfunction and tissue edema happen, which may stimulate the neutrophils adhesion and thereafter inflammation. Previous studies have found that sepsis shock could cause glomerular capillary glycocalyx destruction following proteinuria. At the same time, the host produced a huge amount of TNF alpha. The exact mechanism for this phenomenon is still unclear: specifically what is TNF alpha’s role, the interaction between the neutrophils and glycocalyx, regulating mechanism and the damage mechanism.Ulinastatin (urinary trypsin inhibitor, UTI) was known for its potential anti-inflammation effects. There exist two forms of precursor molecules in the plasma, Intel-alpha inhibitor (I alpha I) and the pre-alpha inhibitor (P alpha I). UTI’s structure includes two Kunitz domains, O-sugar chain, chondroitin sulfate and N sugar chain. The C K area:inhibitevarious hydrolytic enzymes, the N K area:inhibit cathepsin G and elastase. O-:chondroitin sulfate sugar chain can combine with cells and calcium, which stablizes lysosomes, inhibits release of inflammatory cells. N-sugar chain:regulates the adhesion between endothelial cells and mononuclear cells, granulocytes and lymphocytes. With the two Kunitz domains structures, ulinastatin can restrain the activity of various enzymes, specifically decrease the activity of serine protease (including cathepsin, granulocyte elastase, fibrinolytic enzyme, stimulated peptide enzyme, trypsin and chymotrypsin, trypsin etc), reduce excessive activation of neutrophils, lymphocytes, macrophages and mast cells, inhibit fibrin degradation, slow excitation peptide generated, cause vasodilation, prevent protease activated receptor activation (PAR), and reduce the inflammatory cell activation. With all of these functions, inflammatory reaction will be localized. Ulinastatin can also work on precursor cells, reduce the secretion of cytokines, stabilize lysosomal membrane, reduce the release of the enzyme, achieve alleviate inflammation and improve microcirculation. The effect of ulinastatin on glycocalyx in AKI in sepsis remains unclear. Accordingly, in a lipopolysaccharide (LPS) induced rat shock model, the primary aim of the present study is to investigate the relationship between glycocalyx integrity and AKI developement, the role of glycocalyx in the sepsis induced AKI:only the target of inflammation or/and 《fire wood》 of inflammation. Secondly, whether current ulinastatin administration in sepsis could ameliorate AKI development in sepsis, if this is the case, what is the molecular mechanism for this beneficial effect. Objective:The aim of the present study was to investigate the protective effect and molecular mechanism of ulinastatin on AKI in a LPS induced rat septic shock model, furthermore, to provide therapeutic evidence through protecting endothelial cell glycocalyx integrity.Method:1) Septic model setup and serological marker detection Rats were anesthetized through the abdominal subcutaneous injection of chloral hydrate. Right carotid artery, right jugular vein and tail vein were catheterized. LPS was injected i.v (dose). Mean arterial pressure (MAP) was continuously monitored. Serum Cr and BUN were measured at time0,180and360minutes.2) Renal microcirculation assessment-laser Doppler Technology Laser Doppler velocimeter techinque was used to evaluate cortex microcirculation and perfusion.3) Kidney endothelium glycocalyx structural analysis Electron microscope was used to exam rat renal capillary endothelial glycocalyx structure and thickness. Renal venous heparan sulfate and syndecan-1levels were used indirectly to assess renal vascular endothelial glycocalyx damage.4) TNF-alpha synthesis from kidney endothelial cell related molecular signal pathwayImmunohistochemistry was used to detect p38MAPK and NF-kB expression in glomerular capillary endothelial cells. Circulating and renal venous TNF and other chemokines were measured with flow cyotmetry and CBA methods. Other MAPKs family moleculars and NF-kappa B expression in renal capillary endothelial cells were also measured.Results:1) After the injection of LPS (10ug/g), MAP decreased in the endotoxin group. While MAP increased stepwise to mornam in the saline intervention group and ulinastatin group with fluid resuscitation and vasopressors.. However, renal blood flow and cortex microcirculation showed the trend to decrease.2) Kidney endothelium glycocalyx layer in the sham group remains complete and continuous, about200nm thickness. In the sepis group, kidney capillary glycocalyx was not continuous with loose structure and became thinner compared to that of sham animals. Saline intervention group and ulinastatin drug intervention group rat kidney capillary glycocalyx structure showed non-continous, more looser and swollen compared to the sham group. Sulfate and syndecan-1concentrations in the circulation level and renal venous level started to increase even as early as one hour after sepsis, remained high and reached their peak at3hrs after LPS injection.3) TNF alpha started to increase at0.5hours after LPS injection, and remained high for3hrs thereafter followed by a rapid decline.The peak TNF alpha concentration in the circulation level was significantly lower than that of renal veinous blood. Compared to the sepsis group and saline interventaion group, ulinastatin treatment significantly decreased TNF alpha peak concentrations in both circulation level and renal venous level (P<0.05).4) The expression of NF-kB in the kidney capillary endothelium cell in the spsis group was significantly higher than that of sham group (P<0.01), ulinastatin treatment significantly decreased NF-kB expression compared to that of saline intervention group (P<0.05), and there was no significant difference among the rest groups.5) The expression of P38MAPK in the kidney capillary endothelium cell in the sepsis group was significantly increased than that of sham group (P<0.01), ulinastatin treatment significantly decreased the expression of P38MAPK compared to sepsis group (P<0.05), and there was no significant difference among the rest groups.Conclusion:Ulinastatin treatment amilorated the impaired microcirculation in kidney cortex in sepsis and decreased damage of renal vascular endothelial cell glycocalyx, thereafter raised kidney blood flow and decreased inflammatory response. Kidney endothelium glycocalyx damage might be responsible for the impaired microcirculation and AKI in sepsis. NF-kappa B/TNF-alpha molecular passway may be involved in the glycocalyx damage. By targeting glycocalyx, Ulinastatin may have potential therapeutic effect through improving impaired microcirculation and AKI in sepsis. |
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