Role Of Heparanase In Septic Acute Kidney Injury

ObjectiveSepsis, which is a major cause of shock or death, is a severe systemic inflammatoryresponse triggered by a bacterial, viral or fungal infection. The mobidity rate for acutekidney injury is64%during the first twenty-four hours after sepsis occurs. The mortalityrate for sepsis doubles due to coincidence of acute kidney injury. The mobidity andmortality rates for septic acute kidney injury are high. To date, there has been mostlyanti-infectious and supportive therapies and specific therapy is absent because thepathogenesis of septic acute kidney injury is now still uncleared. Therefore, it ismeaningful to investigate the pathogenesis of the disease and consequently find a newtherapeutic target.Heparanase (HPSE) is the sole β-endo-glucuronidase that specifically cleavesheparan sulfate in the mammalian. Heparan sulfate is a major component of glycocalyxwhich is a linear proteoglycan compound coating luminal surface of the vascularendothelial cells. Some researches showed glycocalyx is involved in inflammatoryreactions. Vascular endothelial glycocalyx was found to be degraded and shedded during ainflammation which was correlated to leukocyte adhesion and vascular hyperpermeability.A recent experimetal research showed that heparanase protein activity of pulmonarymicrovascular endothelium was increased after the mice was administrated withlipopolysaccharide. The glycocalyx of the pulmonary microvascular endothelium waslosed and the consequent exposure of adhension molecules that were buried withinglycocalyx facilitated leukocyte adhesion which led to acute lung injury. Heparanaseinhibitors could alleviate sepsis-associtated acute lung injury. These results indicate thatheparanase might also play a role in septic acute kidney injury. This hypothesis has notbeen clarified until now.Accordingly, using animal and cellular models of sepsis, we observed the impact ofheparanase on kidney vascular endothelial glycocalyx in sepsis. We also observed the alterations of kidney leukocyte adhension and vascular permeability after interfering theactivity of heparanase to clarify if heparanase played a vital role in kidney inflammatoryinjury. Heparanase activity was interfered with agents in vivo and the impacts on septicacute kidney injury was observed. The aim was to clarify if heparanse inhibitors couldprotect kidney from septic insults. The current study provides a novel clue forpathogenesis of septic acute kidney injury and a new therapeutic target to septic acutekidney injury.MethodesFirstly, we observed the involvement of heparanase in development of septic acutekidney injury. A septic mouse model was constructed by a single-dose intraperitonealinjection of lipopolysaccharide. The kidneys were harvested. Heparananse protein levelswere detected by Western Blot and the protein distributions were identified byImmunohistochemistry. The aim of this part is to identify the action of heparanase in septicacute kidney injury and the possible influencing sites.Secondly, we explored the impact of heparanase on the capillary endothelialglycocalyx of renal interstitium and the role in leukocytes adhension during sepsis. Kidneyheparanase protein levels were detected at the different timepoints after sepsis occurred.The activated heparanase protein was detected after human umbilical vein endothelial cells(HUVEC) were stimulated with TNF-α. Then we evaluated the relationship of thechanging timepoints of heparanase and adhension molecules (ICAM-1and VCAM-1)protein expressions. The capillary endothelial glycocalyx of renal interstitium wasvisualized using lanthanum trace labeling with transmission electron microscopy. Then weevaluated the impact of heparanase on the capillary endothelial glycocalyx and the role ofTNF-αin this process. The changes of renal interstitial leukocyte infiltration wereobserved using hematoxylin-eosin staining after interfering the heparanase activity in vivo.The aim of this part was to investigate the role of heparanase in renal interstitialinflammation and the mechanism of this process.Thirdly, we explored the impacts of heparanase on the glomerular endothelial glycocalyx and proteinuria during sepsis. After incubation of TNF-α with human renalglomerular endothelial cells, heparanase protein levels of the cells were detected. The renalglomerular endothelial glycocalyx was visualized using lanthanum trace labeling withtransmission electron microscopy. Then we evaluated the impact of heparanase on the renalglomerular endothelial glycocalyx and the role of TNF-αin this process. The changes ofurinary albumin to creatinine ratios were evaluated after interfering the heparanase activityin vivo. The aim of this part was to investigate the role of heparanase in proteinuriadevelopment during sepsis and the mechanism of this process.Fourthly, we explored the protective effects of heparan sulfate mimetics on septicacute kidney injury. The lipopolysaccharide dosages of septic acute kidney injury modeland survival model were determined. Then we investigated the effects of heparan sulfatemimetics on kidney function, twenty-four-hour urine volume and survival rates.Results(1) Compared with that of normal mice, kidney heparanase protein level of sepsismice was increased, and the locations were renal interstitial vessles endothelial cell andrenal glomerular endothelial cell.(2) The timepoint of kidney heparanase protein level being upregulated was earlierthan the timepoints of adhension molecule (ICAM-1and VCAM-1). The activatedheparanase protein level was increased after HUVECs were stimulated with TNF-α.Administrating lipopolysaccharide or TNF-α diminished the mice renal interstitialcaplillary endothelial glycocalyx to dispeared. The glycocalyx recovered afteradministrating heparanase inhibitor to sepsis mice additionally. There were leukocyteinfiltration in the renal interstitium of sepsis mice. Administrating heparanase inhibitor tosepsis mice additionally attenuated renal interstitial leukocyte infiltration. Howerver,administrating heparin Ⅲ to degrade glycocalyx led to renal interstitial leukocyteinfiltration.(3) After renal glomerular endothelial cells was stimulated with TNF-α, the activatedheparanase protein level of the cells was significantly increased. Administrating lipopolysaccharide or TNF-αdiminished the mice renal glomerular endothelial glycocalyxto dispeared. The glycocalyx recovered after administrating heparanase inhibitor to sepsismice additionally. The urine albumin to creatinine ratio of sepsis mice was much higherthan that of normal mice. Administrating heparanase inhibitor to sepsis mice additionallylowered the urine albumin to creatinine ratio. Howerver, administrating heparin Ⅲ todegrade glycocalyx did not lowered the urine albumin to creatinine ratio.(4) The protocol of septic acute kidney injury mice model for observing kidneyfunctions was a single-dose intraperitoneal injection of lipopolysaccharide at10mg/kg fortwenty four hours. The mice model for observing survival rate was established by injectinglipopolysaccharide at a dose of20mg/kg intraperitoneally. After treatment of heparansulfate mimetics, the serum creatinine and urea nitrogen of sepsis mice decreased and thetwenty-four-hour urine volume increased than that of untreated sepsis mice. Heparansulfate mimetics improved survival rate of sepsis mice.ConclusionHeparanase plays a vital role in pathogenesis of septic acute kidney injury mice. Themechanism is as follow: Firstly, after sepsis occurs, TNF-αupregulates heparanase proteinexpression in renal capillary endothelium and consequently the glycocalyx is degradedwhich leads to leukocyte infiltration in renal interstitium. Secondly, TNF-α increasesheparanse protein expression in renal glomerular endothelium and consequently theglycocalyx is degraded which leads to destruction of glomerular filtration barrier andproteinuria. By inhibiting heparanase activity, Heparan sulfate mimetics can attenuate renaldysfunction in septic acute kidney injury and improve survival rate in sepsis.