טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentSajrawi Clara
SubjectInvolvement of Heparanase in the Pathogenesis of Acute
Kidney Injury
DepartmentDepartment of Medicine
Supervisor Professor Zaid A. Abassi
Full Thesis textFull thesis text - English Version


Abstract

Acute kidney injury (AKI) is a common clinical problem characterized by high morbidity and mortality due to lack of effective treatment. The latter is largely attributed to poor understanding of the pathogenesis of AKI. Heparan sulfate proteoglycans, primary components at the interface between virtually every eukaryotic cell and its extracellular matrix, play important roles in ECM integrity, barrier function, and cell-ECM interactions. It is therefore not surprising that a HS degrading enzyme heparanase is critically involved in angiogenesis, tumor growth, metastasis, and inflammation. Although heparanase is recognized for its important role in progressive chronic kidney diseases, its involvement in the pathogenesis of AKI has been barely addressed.


We hypothesize that Heparanase is involved in the pathophysiology of ischemic AKI, and that heparanase inhibition by PG545, a potent heparanase inhibitor, could attenuate renal injury, and subsequently improve kidney function in ischemic AKI rat model. Therefore, the present research aimed to examine the alterations in renal heparanase following ischemia.


The present study includes 3 groups of Sprague Dawely rats:  Sham operated rats, rats  with renal injury induced by ischemia/reperfusion; and rats with renal ischemia pre-treated with PG545 2 days prior to the ischemia. Acute kidney injury was induced by clamping the renal artery of both kidneys for either 30, or 45 minutes, followed by reperfusion. Animals were sacrificed after 4, 24, 48 hours and 2 weeks. Measurement of renal function was determined by clearance studies in the animals that were sacrificed 4 hours post ischemia.  In Addition, blood samples were obtained from the 24, 48 hours and 2 weeks post ischemia sacrificed goups for measurement of serum creatinine (SCr) and blood urea nitrogen (BUN). Kidneys were harvested for histological analysis, heparanase activity, expression by means of RT-qPCR and western blot for inflammatory mediators involved in the kidney injury progression.

Ischemic kidney revealed remarkable cast formation, necrosis and congestion, a consistent pattern of acute tubular necrosis. AKI-Induced renal dysfunction and histological changes were associated with upregulation of renal Heparanase, as was evident by enhanced expression, immunoreactivity, and enzyme activity in the kidney . Moreover, Ischemic AKI of 24h and 48h, was associated with elevated SCr and BUN levels. Acute inhibition of heparanase by PG545 slightly improved GFR in rats with AKI that were sacrificed after 4h.  This beneficial effect of PG545 was associated with improved histological features of the ischemic kidney . Similarly, pretreatment with PG545 attenuated kidney dysfunction as was evident by improvement in GFR and moderate histological recovery in ischemia of 30 min after 48h. However, this encouraging effect was not prominent after 24h or when the ischemic insult lasted for 45 min. AKI was associated with enhanced expression and immunoreactivity of renal Cathepsin L, a key enzyme in the activation of Heparanase, and other pro-inflammatory mediators such as Vimentin, Akt, and TNFα.

Our study provides a better perspective of the pathogenesis of AKI, where the present results demonstrate that renal Heparanase expression/activity increases in early AKI and mediates the onset of renal dysfunction, and that its inhibition by PG545 exerted nephroprotective effect in injured kidney.