The FSGS protein Actinin‐4 (ACTN4) is expressed in Thick Ascending Limbs and interacts with NKCC2 and ALMS1 to regulate NKCC2 trafficking

Abstract

NKCC2 is a Cl-dependent co-transporter that mediates all the NaCl absorption in the thick ascending limb (TAL). In humans, loss-of-function mutations in NKCC2 result in Bartter syndrome type 1, characterized by inability in concentrating urine, polyuria, and low blood pressure. Importantly, enhanced NKCC2 activity is associated with hypertension in humans and animal models. NKCC2 is present on the apical membrane and sub-apical space in the TAL and macula densa cells. We and others have shown that trafficking of NKCC2 to the apical membrane determines NKCC2-mediated NaCl absorption. These trafficking steps are controlled by signaling and protein-protein interactions. Very little is known about how NKCC2 endocytosis is controlled and only a handful of proteins that mediate NKCC2 internalization and trafficking are known. To identify new NKCC2 interacting partners we immunoprecipitated NKCC2 from TAL lysates using a C-terminus antibody and identified interacting proteins by mass spectrometry. We identified Actinin-4 (ACTN4), a protein studied in podocytes, that when mutated causes Focal segmental glomerulo-sclerosis (FSGS) in humans. We hypothesized that in addition to podocytes, ACTN4 is expressed along the nephron and is part of protein complex that binds apical NKCC2 and other proteins such as Alstrom Syndrome 1 (ALMS1) and promotes its endocytosis. To begin studying these mechanisms we used a targeted proteomics screen to identify new binding partners for ACTN4 in the TAL. We confirmed that glutathione-S-transferase (GST) ACTN4 (full length) pulled down NKCC2 and ALMS1 from TAL lysates identified by mass spectrometry (LC-MS) or Western blot. GST-ALMS1 also pulled down ACTN4, identified by LC-MS. Because there are no studies on the role of ACTN4 in NaCl reabsorption along the nephron, we studied its localization in the kidney. ACTN4 was abundant in glomeruli but also localized in cells along the nephron in a punctate vesicular pattern. ACTN4 was expressed in TALs and partially co-localized with NKCC2 the subapical space. To study if ACTN4 mediates NKCC2 trafficking, we generated adenoviruses expressing ACTN4 shRNA. In vivo transduction of the rat renal medulla decreased ACTN4 expression in TALs by 66±9% (p<0.04). Silencing ACTN4 in rat TALs increased the surface to total NKCC2 ratio by 49±16% (p<0.01) but did not affect total NKCC2, suggesting that ACTN4 regulates NKCC2 trafficking. To study the relationship of ACTN4 with ALMS1 and NKCC2, we generated nephron-specific ALMS1 knockout mice and measured surface NKCC2 and ACTN4. Deletion of ALMS1 increased surface to total NKCC2 ratio by 55±19% (p<0.05), but also decreased ACTN4 expression by 45±9% (p<0.05). We conclude that ACTN4 binds NKCC2 and ALMS1 and it is required for proper NKCC2 trafficking. It is likely that a large protein complex containing ACTN4 and ALMS1 mediates NKCC2 endocytosis to control renal salt reabsorption.