Anti-ANPEP monoclonal antibody, clone DWT20 [FITC]

Aminopeptidase N/CD13 (Anpep) is a membrane-bound protein that catalyzes the formation of natriuretic hexapeptide angiotensin IV (ANG IV) from ANG III. We previously reported that Anpep is more highly expressed in the kidneys of Dahl salt-resistant (SR/Jr) than salt-sensitive (SS/Jr) rats, Anpep maps to a quantitative trait locus for hypertension, and that the Dahl SR/Jr rat contains a functional polymorphism of the gene. This suggests that renal Anpep may be linked to salt sensitivity; however, its effect on renal Na handling has not been determined. Here, we examined regulation of basolateral Na+ -K+ -ATPase, a preeminent basolateral Na+ transporter in proximal tubule cells, by Anpep in LLC-PK1 cells. Treatment of the cells with Anpep siRNA increased total cellular Na+ -K+ -ATPase activity and basolateral Na+ -K+ -ATPase abundance by approximately twofold. Conversely, Anpep overexpression reduced Na+ -K+ -ATPase activity and basolateral abundance by similar to 50%. Similar effects were observed after treatment with ANG IV (10 nM, x30 min and 12 h). ANG IV receptor (AGTRIV) knockdown via specific siRNA relieved the decreases in basolateral Na+ -K+ -ATPase levels and activity induced by Anpep overexpression. In sum, these results demonstrate that Anpep reduces basolateral Na+ -K+ -ATPase levels via ANG IV/AGTRIV signaling. This novel pathway may be important in renal adaptation to high salt.

Background: Chemotherapy options in advanced urothelial carcinoma (UC) remain limited. Here we evaluated the peptide-based alkylating agent melphalan-flufenamide (mel-flufen) for UC. Methods: UC cell lines J82; RT4, TCCsup and 5637 were treated with mel-flufen, alone or combined with cisplatin, gemcitabine, dasatinib or bestatin. Cell viability (MTT assay), intracellular drug accumulation (liquid chromatography) apoptosis induction (apoptotic cell nuclei morphology, western blot analysis of PARP-1/caspase-9 cleavage and Bak/Bax activation) were evaluated. Kinome alterations were characterized by PathScan array and phospho-Src validated by western blotting. Aminopeptidase N (ANPEP) expression was evaluated in UC clinical specimens in relation to patient outcome. Results: In J82, RT4, TCCsup and 5637 UC cells, mel-flufen amplified the intracellular loading of melphalan in part via aminopeptidase N (ANPEP), resulting in increased cytotoxicity compared to melphalan alone. Mel-flufen induced apoptosis seen as activation of Bak/Bax, cleavage of caspase-9/PARP-1 and induction of apoptotic cell nuclei morphology. Combining mel-flufen with cisplatin or gemcitabine in J82 cells resulted in additive cytotoxic effects and for gemcitabine also increased apoptosis induction. Profiling of melflufen-induced kinome alterations in J82 cells revealed that mel-flufen alone did not inhibit Src phosphorylation. Accordingly, the Src inhibitor dasatinib sensitized for melflufen cytotoxicity. Immunohistochemical analysis of the putative mel-flufen biomarker ANPEP demonstrated prominent expression levels in tumours from 82 of 83 cystectomy patients. Significantly longer median overall survival was found in patients with high ANPEP expression (P = 0.02). Conclusion: Mel-flufen alone or in combination with cisplatin, gemcitabine or Src inhibition holds promise as a novel treatment for UC. (C) 2016 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.