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Abstract: TH-PO330

CRISPR/Cas9-Mediated Suppression of the Focal Segmental Glomerulosclerosis Protein Actinin-4 (ACTN4) in Thick Ascending Limbs (TALs) Increases Sodium Chloride Reabsorption by Regulating NKCC2 Trafficking

Session Information

Category: Fluid‚ Electrolyte‚ and Acid-Base Disorders

  • 1001 Fluid‚ Electrolyte‚ and Acid-Base Disorders: Basic


  • Maskey, Dipak, Department of Physiology, Wayne State University,, Detroit, Michigan, United States
  • Ortiz, Pablo A., Department of Physiology, Wayne State University,, Detroit, Michigan, United States

Mutations in ACTN4 are known to cause focal segmental glomerulosclerosis. However, the role of ACTN4 in nephron ion transport has not been studied. We used a proteomics screen to find interacting proteins for the Na/K/2Cl cotransporter NKCC2 in the Thick Ascending Limb (TAL) and identified ACTN4. We showed that NKCC2 levels at the apical surface are controlled by endocytosis. ACTN4 has been involved in endocytosis in other cells. Therefore, we hypothesized that ACTN4 is a part of protein complex that binds apical NKCC2 and promotes its endocytosis.


Adeno-associated virus (AAV)-mediated CRISPR/Cas9 , Western blot, metabolic cages, Uni-nephrectomy, and Immunoprecipitation


To study the role of ACTN4 in NKCC2 endocytosis, we developed an adeno-associated virus (AAV)-mediated CRISPR/Cas9 approach to decrease ACTN4 expression in TALs. We tested 4 gRNAs targeting ACTN4 in neuroblastoma Neuro 2a/HF-Cas9-ROSA26 cells and selected the most effective guide, which decreased ACTN4 protein expression by 70-80% (n=3) and we packed this gRNA into AAV (AVV-U6-gRNA-ACTN4). To study the role of ACTN4 in TAL NaCl reabsorption, we uni-nephrectomized rats (right kidney removal), and a week later transduced the left kidneys with AV-pNKCC2Cas9 plus AAV-gRNA-ACTN4 or AV-pNKCC2-Cas9 alone (control). After 3 weeks, rats were placed in metabolic cages to measure bumetanide-induced natriuresis (4h) as an index of NKCC2-mediated NaCl absorption. We found that bumetanide induced UNa excretion was higher in rats transduced with Cas9-ACTN4, compared to rats transduced with Cas9 alone (Cas9 Control: 2880±381 vs Cas9-ACTN4: 4181±190 µmols Na/4h, n=5, p<0.05). Surface to total NKCC2 ratio was increased by 30±5% in TALs from rats transduced with Cas9-ACTN4 (p<0.05, n=3) whereas ACTN4 expression was 40±4% lower than control Cas9 rats (n=3, p<0.05). Recombinant ACTN4 (GST-ACTN4) or immunoprecipitation of ACTN4, pulled down NKCC2 from TAL lysates (n=4), suggesting that ACTN4 regulates surface NKCC2 involves protein-protein interactions.


We conclude that ACTN4 binds NKCC2 to regulate its surface expression. Selective depletion of ACTN4 in TALs using CRISPR/Cas9 enhances surface NKCC2 and TAL NaCl reabsorption, indicating that regulation of the ACTN4-NKCC2 interaction is important for renal NaCl reabsorption.


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