The FSGS Protein Actinin-4 Interacts with NKCC2 to Regulate Thick Ascending Limb NaCl Reabsorption

Authors

Dipak Maskey, Henry Ford HealthFollow
Tang-Dong Liao, Henry Ford HealthFollow
D'Anna Potter, Henry Ford HealthFollow
Pablo A Ortiz, Henry Ford HealthFollow

Recommended Citation

Maskey D, Liao TD, Potter DL, and Ortiz PA. The FSGS protein actinin-4 (ACTN4) interacts with NKCC2 to regulate Thick Ascending Limb (TAL) NaCl reabsorption. Am J Physiol Renal Physiol 2024.

Document Type

Article

Publication Date

12-1-2024

Publication Title

American journal of physiology. Renal physiology

Abstract

In the kidney, the thick Ascending Limb (TAL) of the loop of Henle is crucial for NaCl homeostasis and blood pressure regulation. In animal models of salt-sensitive hypertension, NaCl reabsorption via the apical Na+/K+/2Cl cotransporter (NKCC2) is abnormally increased in the TAL. We showed that NaCl reabsorption is controlled by the presence of NKCC2 at the apical surface of TALs. However, the molecular mechanisms that maintain the steady-state levels of NKCC2 at the apical surface are not clearly understood. Here, we report that NKCC2 interacts with the F-actin cross-linking protein actinin-4 (ACTN4). We find that ACTN4 is expressed in TALs by Western blot and immunofluorescence microscopy. ACTN4 immunoprecipitated with NKCC2 and recombinant GST-ACTN4 pulled down NKCC2 from TAL lysates. ACTN4 is involved in endocytosis in other cells. Therefore, we hypothesized that ACTN4 binds apical NKCC2 and regulates its trafficking. To study this, we silenced ACTN4 in vivo via shRNA or CRISPR/Cas9 system to decrease ACTN4 expression in TALs. We observed that silencing ACTN4 in vivo via shRNA or CRISPR/Cas9 system increased the amount NKCC2 at the apical surface of TALs. Bumetanide-induced diuresis and natriuresis were enhanced by 35% after silencing of ACTN4 in vivo (AV-NKCC2-Cas9: 3841±709 vs AAV-gRNA-ACTN4: 5546±622 µmols Na/8h, n=5, p<0.05). We conclude that ACTN4, binds NKCC2 to regulate its surface expression. Selective depletion of ACTN4 in TALs using shRNA or CRISPR/Cas9 enhances surface NKCC2 and TAL NaCl reabsorption, indicating that regulation of the ACTN4-NKCC2 interaction is important for renal NaCl reabsorption and could be related to hypertension.

Medical Subject Headings

Solute Carrier Family 12, Member 1; Actinin; Loop of Henle; Animals; Humans; Sodium Chloride; Renal Reabsorption; Male

PubMed ID

39446130

ePublication

ePub ahead of print

Volume

327

Issue

6

First Page

1026

Last Page

1026