Nucleophosmin (NPM) Promotes Regulated Cell Death in Podocyte Injury.

Abstract

Background: The mechanism of regulated podocyte death during glomerulopathy is unknown, limiting therapeutic interventions to protect this essential kidney cell. We hypothesize that cytosolic nucleophosmin (NPM) accumulation promotes Bax-mediated podocyte injury during experimental and clinical glomerulopathy.

Methods: Cytosolic and total NPM were quantified in differentiated human podocytes subjected to mechanistically diverse stressors in vitro, in kidney tissue harvested from conditional, podocyte-specific integrin-linked kinase conditional knockout (ILK-cKO) mice, and in kidney tissue from patients with focal segmental glomerulosclerosis (FSGS), membranous nephropathy, or diabetic nephropathy using histologic and immunologic techniques. The effect of stress on NPM/Bax complex formation, mitochondrial Bax accumulation, and podocyte survival was assessed using an organelle-specific stain, NPM/Bax co-immunoprecipitation, and a colorimetric cell survival assay, respectively. To establish a potential role for NPM in regulated podocyte injury, NPM content was reduced and increased using molecular techniques, and the therapeutic effect of peptides designed to competitively inhibit NPM/Bax interaction was examined.

Results: Cytosolic NPM accumulation increased after transient exposure to Adriamycin or hydrogen peroxide. Hydrogen peroxide increased cytosolic NPM/Bax complex formation and increased mitochondrial Bax accumulation, early hallmarks of regulated cell death. siRNA-mediated NPM suppression significantly increased human podocyte survival, whereas NPM over-expression significantly reduced podocyte survival after acute stress. A novel TP10-fused peptide reduced NPM/Bax interaction and significantly increased podocyte survival after stress. In contrast to 2-day-old pups, increased NPM expression and cytosolic NPM accumulation were detected in podocytes of 4-week-old ILK-KO mice, an early FSGS model, as well as in the glomeruli of kidney tissue harvested from patients with diverse forms of clinical glomerulopathy.

Conclusions: Cytosolic NPM translocation accompanies experimental and clinical podocyte injury, promotes regulated podocyte death after stress, and is a promising target for protecting podocytes against glomerular injury.