POS-337 ASPARAGINYL ENDOPEPTIDASE PROTECTS AGAINST PODOCYTE INJURY IN DIABETIC NEPHROPATHY THROUGH CLEAVING COFILIN-1

Abstract

Asparaginyl endopeptidase (AEP) is a lysosomal cysteine protease that specifically cleaves its substrates after asparagine residues. Physiologically, AEP is most abundant in kidney proximal tubule and is required for normal kidney physiology and homeostasis. Recent studies reported that AEP exerts anti-fibrotic effect through degrading of matrix fibronection during unilateral ureteral obstruction (UUO) animal model. However, its role and mechanism in podocyte injury during diabetic nephropathy (DN) remains unclear. Diabetic mice model was established by intraperitoneal injection of streptozotocin (STZ) and oral administration of AEP inhibitor was adopted to inhibit AEP activity, and also, AEP knockout mice were employed. In vitro study, human podocyte cell line was employed, and subjected to different treatments after differentiation. RNA interfere and adenovirus expression vector were utilized to knockdown AEP or overexpression of AEP and cofilin 1 fragments respectively. Moreover, AEP recombinase was used to detect its substrate---cofilin 1 cleavage activity in vitro. In the present study, AEP was highly upregulated in diabetic glomeruli and hyperglycemic stimuli treated-podocytes; however, AEP gene knockout and its compound inhibitor treatment accelerated DN in streptozotocin-induced diabetic mice, whereas specific induction of AEP in glomerular cells attenuated podocyte injury and renal function deterioration. In vitro, elevated AEP was involved in actin cytoskeleton maintenance and anti-apoptosis effects. Mechanistically, we found that AEP directly cleaved the actin-binding protein cofilin-1 after the asparagine 138 (N138) site. The protein levels of endogenous cofilin-1 1-138 fragments were upregulated in diabetic podocytes, consistent with the changes in AEP levels. Importantly, we found that cofilin-1 1-138 fragments were remarkably unphosphorylated than full-length cofilin-1, indicating the enhanced cytoskeleton maintenance activity of cofilin-1 1-138. Then we validated cofilin-1 1-138 could rescue podocytes from cytoskeleton disarrangement and injury in diabetic conditions. Taken together, our data suggests a protective role of elevated AEP in podocyte injury during DN progression through cleaving cofilin-1 to maintain podocyte cytoskeleton dynamics and defend damage.