Khalid M. Elased (Advisor), Nadja Grobe (Committee Member), Mohammad Saklayen (Committee Member)
Master of Science (MS)
Chronic Kidney Disease (CKD) has reached epidemic proportions affecting more than 20 million adults in the US. CKD is routinely diagnosed and defined as reduced glomerular filtration rate (GFR), increased urinary albumin creatinine ratio (UACR) or both. Diabetes, hypertension and obesity share a major part in causing CKD. Angiotensin (1-7) (Ang (1-7)) is a vasodilator that plays an important renoprotective role in the renin angiotensin system, counteracting the vasoconstrictor and proliferative effects of Ang II. ACE2 and NEP which form Ang (1-7) by degrading Ang II and Ang I, respectively. A Disintegrin and Metalloproteinase (ADAM) 17, which is responsible for the ectodomain shedding of transmembrane proteins, sheds renal ACE2 and causes release of NEP from urinary exosomes. In this study, we aimed to investigate the levels of ACE2 and NEP in urine and to find their correlations with well-established markers of CKD in diabetic patients at different stages of albuminuria. Baseline UACR and estimated GFR (eGFR) were determined three months before initiation of the study in twenty nondiabetics (ND) and forty diabetic patients with normoalbuminuria, microalbuminuria, and macroalbuminuria. Based on their visits over the previous year, patients with history of microalbuminuria and whose UACR reversed to normal were considered as diabetic patients with normoalbuminuria. Metabolic and renal characteristics were measured in all groups. Enzyme activity of urinary ACE2 and NEP were measured using fluorogenic and mass spectrometric assays. We developed a sensitive and specific assay to measure ACE2 and NEP activities in human urine using matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). Significant inhibition of urinary ACE2 and NEP activities by MLN-4760 (specific ACE2 inhibitor) and thiorphan (specific NEP inhibitor) proved the specificity of the enzyme assays. Urinary ACE2 and NEP levels were significantly higher in diabetic patients compared to nondiabetic individuals, even before the onset of microalbuminuria (p<0.05). Immunoblotting confirmed increased urinary ACE2 and NEP expression in diabetic patients prior to the onset of microalbuminuria compared to nondiabetic patients. Unlike plasma ACE2 activity, which was not detectable, plasma NEP was increased in subjects with diabetes. Increase in urinary ADAM17 levels were observed in diabetic patients compared to nondiabetic individuals (p<0.05). Urinary ACE2 activity correlated with eGFR, blood glucose, glycated hemoglobin (HbA1C), and BUN (all p<0.05), but not UACR and urinary protein-to-creatinine ratio (UPCR). Regression analysis of urinary NEP concentration with various metabolic parameters showed correlations with blood glucose, BMI, HbA1C, and creatinine clearance. In conclusion, alterations in kidney function can be more precisely indicated by shedding of ACE2 and NEP in urine of diabetic patients prior to onset of microalbuminuria. These new findings provide novel insights for the possible role of urinary ACE2 and NEP as non-invasive biomarkers for diabetic kidney disease prior to the onset of microalbuminuria.
Department or Program
Department of Pharmacology and Toxicology
Year Degree Awarded
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