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Abstract: FR-PO616

Actinin-4 (ACTN4) Interacts with ALMS1 and NKCC2 in Thick Ascending Limbs (TAL) to Regulate NKCC2 Trafficking

Session Information

Category: Fluid and Electrolytes

  • 901 Fluid and Electrolytes: Basic

Authors

  • Maskey, Dipak, Henry Ford Hospital, Detroit, Michigan, United States
  • Ares, Gustavo R., Henry Ford Hospital, Detroit, Michigan, United States
  • Ortiz, Pablo A., Henry Ford Hospital, Detroit, Michigan, United States

Group or Team Name

  • Ortiz lab
Background

Loss-of-function mutations in the ALMS1 gene cause Alström syndrome, characterized by hypertension, early onset obesity, type 2 diabetes and progressive loss of renal function. Single nucleotide polymorphisms (SNPs) in the ALMS1 gene are associated with decreased renal function (lower GFR) and increased pulse pressure in the general population. However, the role of ALMS1 in the control of renal function is unclear. We recently found that ALMS1 physically interacts with the apical renal Na/K/2Cl cotransporter NKCC2 in the TAL, where it mediates its endocytosis. However, the molecular mechanisms by which ALMS1 mediates NKCC2 endocytosis are unclear.We hypothesized that ALMS1 is part of a protein complex that binds apical NKCC2 and promotes its endocytosis and recycling.

Methods

To begin studying these mechanisms we used a targeted proteomics screen to identify new binding partners for ALMS1 in the TAL as well as immuno-precipitation.

Results


GST-pull down with the C-terminus of ALMS1 identified several trafficking proteins. One of them, Actinin-4 (ACTN4), is involved in endocytosis and its mutation causes focal segmental glomerular disease. GST-ACTN4 (full length) pulled down both ALMS1 and NKCC2 from TALs. Immunoprecipitation of NKCC2 followed by mass spectrometry identified ACTN4. The role of ACTN4 in the nephron is unclear so we studied its localization. ACTN4 was abundant in glomeruli but also localized in cells along the nephron in a punctate vesicular pattern. ACTN4 was located in TALs (co-labeled with NKCC2) and co-localized with ALMS1 n the subapical space. To study the role of ALMS1 and ACTN4 we generated a mouse line with doxycycline (Dox) inducible nephron-specific deletion of ALMS1 (Dox-inducible-Pax8-Cre-ALMS1fl/f). 4 weeks after finishing Dox treatment, ALMS1 expression in medullary tubules was decreased by 78±14% (p<0.05). Interestingly, ACTN4 expression was also decreased by 45±9% (p<0.05) in nephron specific ALMS1 KO, compared to doxycycline treated controls (ALMS1fl/fl). In isolated mouse TALs, the surface to total NKCC2 ratio was increased by 55±19% (p<0.05), suggesting decreased retrieval from the surface.

Conclusion

We conclude that ACTN4 binds both ALMS1 and NKCC2 and ALMS1 is required for proper NKCC2 trafficking. Our data suggest that ACTN4 plays a role in tubular Na absorption in addition to its role in podocytes.

Funding

  • NIDDK Support