Lopinavir and Nelfinavir Induce the Accumulation of Crystalloid Lipid Inclusions within the Reservosomes of Trypanosoma cruzi and Inhibit Both Aspartyl-Type Peptidase and Cruzipain Activities Detected in These Crucial Organelles

Several research groups have explored the repositioning of human immunodeficiency virus aspartyl peptidase inhibitors (HIV-PIs) on opportunistic infections caused by bacteria, fungi and protozoa. In Trypanosoma cruzi, HIV-PIs have a high impact on parasite viability, and one of the main alterations promoted by this treatment is the imbalance in the parasite’s lipid metabolism. However, the reasons behind this phenomenon are unknown. In the present work, we observed by transmission electron microscopy (TEM) that the treatment of T. cruzi epimastigotes with the HIV-PIs lopinavir and nelfinavir induced a huge accumulation of crystalloid-shaped lipids within the reservosomes, most of them deforming these key organelles. As previously reported, those structures are characteristic of lipid inclusions formed mostly of cholesterol and cholesterol-esters. The fractionation of nontreated epimastigotes generated two distinct fractions enriched in reservosomes: one mostly composed of lipid inclusion-containing reservosomes (Fraction B1) and one where lipid inclusions were much less abundant (Fraction B2). Interestingly, the extract of Fraction B2 presented enzymatic activity related to aspartyl-type peptidases 3.5 times higher than that found in the extract obtained from Fraction B1. The cleavage of cathepsin D substrate by this class of peptidases was strongly impaired by pepstatin A, a prototypical aspartyl PI, and the HIV-PIs lopinavir and nelfinavir. In addition, both HIV-PIs also inhibited (to a lesser extent) the cruzipain activity present in reservosomes. Finally, our work provides new evidence concerning the presence and supposed participation of aspartyl peptidases in T. cruzi, even as it adds new information about the mechanisms behind the alterations promoted by lopinavir and nelfinavir in the protozoan.

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

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.