Abstract 610: A Pepstatin-Sensitive Aspartyl Peptidase as an Early Urinary Marker of Renal Injury in the Diabetic Hypertensive mRen2.Lewis Female Rat

Abstract

We previously reported that induction of diabetes in estrogen-depleted, hypertensive mRen2.Lewis (mRen2) female rats leads to significant albuminuria, proteinuria and tubulointerstial oxidative stress at 28 days. The current study assessed the time course for the development of proteinuria and peptidase activities in the urine of the diabetic hypertensive mRen2 to begin to establish potential biomarkers of renal injury in this model. Heterozygous mRen2 underwent ovariectomy and diabetes was induced by a single administration of streptozotocin (65 mg/kg; STZ) at 15 weeks of age. Urinary samples were taken prior to (day 0) and 5 and 28 days following STZ. Aspartyl peptidases were assayed with the quenched fluorescent substrate Mca-GWPILFFRLW(Dnp)R in the presence or absence of the inhibitor pepstatin. Enzyme activities were expressed as nmols Mca per 30 min per mg creatinine. Pepstatin-sensitive activity [IC50 ∼ 0.4 nM] was detectable in the pre-diabetic urines [26 ± 9; n=8]; however, the diabetic rats exhibited a significant increase in urinary activity at days 5 and 28 [184 ± 25 and 204 ± 53, respectively; p<0.05 vs. day 0]. In contrast, pepstatin-insensitive activity was markedly lower and decreased between days 0 and 5 [22 ± 2 vs. 12 ± 4; p<0.05], but was unchanged at day 28 [34 ± 12]. Proteinuria was similar at days 0 and day 5, but significantly increased by day 28. Aspartyl peptidases in the kidney and urine that are sensitive to pepstatin include cathepsins D and E, as well as the enzyme napsin. However, immunoblots of concentrated urine samples using selective cathepsin and napsin antibodies failed to match protein expression to the increased activities in the diabetic rats. Finally, we assessed peptidase activity in isolated cortical slices from diabetic mRen2 to determine the extent of enzyme release. Aspartyl peptidase activity was readily detectable in the perfusate of the isolated kidney [6 ± 0.3 pmols Mca per min release, n=3] and completely inhibited by pepstatin suggesting that the urinary activity is of renal origin. We conclude that a pepstatin-sensitive aspartyl peptidase, potentially distinct from cathepsin D/E and napsin may constitute an early marker of renal injury in the diabetic hypertensive mRen2 strain.