Abstract: TH-PO0285
Interferon-Gamma (IFNg) Induces Sodium-Potassium-2 Chloride Cotransporter (NKCC2) Phosphorylation in the Thick Ascending Limbs of Hypertensive Rats, but Not Normotensive Rats: Role of SPAK
Session Information
- Hypertension and CVD: Mechanisms
November 06, 2025 | Location: Exhibit Hall, Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Hypertension and CVD
- 1601 Hypertension and CVD: Basic
Authors
- Granados Pineda, Jessica, Henry Ford Health System, Detroit, Michigan, United States
- Mendez, Mariela, Henry Ford Health System, Detroit, Michigan, United States
- Ortiz, Pablo A., Henry Ford Health System, Detroit, Michigan, United States
Background
Inflammation has a pathogenic contribution on the development and maintenance of elevated blood pressure. Mice deficient in the innate immunity cytokine IFNg, have a blunted blood pressure increase to Angiotensin II infusion and a greater ability to excrete sodium. However, it is not clear if IFNg modulates sodium cotransporters, the nephron segments where it acts, or its downstream signaling in the nephron. We hypothesize IFNg, acting directly on thick ascending limbs (TALs), increases the phosphorylation of the apical Na/K/2Cl cotransporter (NKCC2) at Thr96,101, in part due to the activation of SPAK (STK39) kinase.
Methods
TALs were freshly isolated from the outer medulla of Sprague Dawley, Dahl Salt Sensitive (Dahl SS) rats or SPAK knock-out Dahl SS rats. Isolated TALs were treated (30min) with IFNg (30 or 300 ng/ml) and then analyzed for expression of NKCC2 and SPAK (total and phosphorylated forms) by immunoblotting.
Results
In TALs from Sprague Dawley rats, IFNg did not significantly change NKCC2 phosphorylation (p<0.05, n=4). In TALs from Dahl SS rats on normal salt diet (0.4% NaCl), IFNg increased NKCC2 phosphorylation and the SPAK inhibitor ZT-1a (10 μM) abrogated this stimulatory effect (baseline= 100, IFNg = 221 ± 48, ZT-1a+IFNg = 11.0 ± 4.6 % of baseline, p<0.05, n=4). SPAK phosphorylation was increased by IFNg and ZT-1a abrogated this effect (IFNg= 156 ± 34 vs ZT-1a+IFNg = 42.0 ± 9.2% of baseline, n=3). Then, we fed DSS rats with a high salt diet (4% NaCl) for 1 week to induce hypertension. IFNg didn’t further increase NKCC2 or SPAK phosphorylation, while ZT-1a decreased baseline and IFNg-induced phosphorylation of NKCC2 (p<0.05, n=4). Finally, in TALs from SPAK knock-out Dahl SS rats, IFNg didn’t significantly increase NKCC2 phosphorylation (p<0.05, n=4) and SPAK was completely absent.
Conclusion
Our data show that IFNg stimulates SPAK and NKCC2 phosphorylation in TALs from genetically hypertensive rats, but not in normal rats, suggesting that additional signaling or pro-inflammatory conditions are required for IFNg to stimulate NKCC2 phosphorylation. Our data link inflammatory mediators with increased tubular NaCl reabsorption in hypertension and kidney damage during chronic inflammation.
Funding
- Other NIH Support