SAT-286 Analysis of Potassium and AngII-Regulated Phosphoproteome in mTORC2 Knockout Mice

Abstract Disclosure: R.J. Weber: None. E. Takagi: None. J. Demko: None. B. Saha: None. J. Oses-Prieto: None. A. Burlingame: None. D. Pearce: None. Background: Aldosterone is the primary mineralocorticoid in humans and its dysregulation is an important driver of hypertension. Aldosterone has two primary roles – augmenting potassium (K+) excretion and maintenance of blood pressure (BP). Much of this activity occurs in the distal nephron by ENaC translocation to the plasma membrane in response to mineralocorticoid receptor (MR) activation. However, this signaling pathway is modulated by mTORC2-dependent post-translational modifications. mTORC2 is a kinase complex that responds to both local electrolytes and circulating hormones, including angiotensin II (AngII) and insulin, to augment kaliuresis and sodium resorption by unleashing a kinase cascade that ultimately enhances translocation of ENaC and ROMK to the plasma membrane. Our lab has previously demonstrated that mice lacking mTORC2 are aldosterone resistant and has elucidated downstream pathways. However, there has not yet been a global evaluation of the interaction between mTORC2 and aldosterone. The majority of the known signaling changes are mediated by kinases; thus, we chose to perform phosphoproteomics. We hypothesized that by taking an unbiased approach, we may uncover novel mTORC2-dependent phosphorylation events secondary to stimulation by K+ and AngII. Methods: We utilized a transgenic mouse line with an inducible, nephron-specific mTORC2 knockout (TRKO) via a Pax-8 driven Cre and floxed Rictor, a necessary subunit of mTORC2. We used tail cuff blood pressure (BP) measurements and scanned a series of diets with varied K+ content. Wild type (WT) and TRKO mice were exposed to high or low K+ diets or injected subcutaneously with AngII. Subsequently, bilateral nephrectomy was performed, and slices of renal cortex were collected. We performed high throughput phosphoproteomic analysis of these slices using tandem mass tagged labeling. Results and Conclusions: We found that a 3% K+ diet decreased systolic BP in WT mice but paradoxically increased systolic BP in TRKOs. This correlated with a marked increase in serum aldosterone in TRKOs. We used this diet for phosphoproteomic analysis compared to a low K+ diet (0.5%) and compared to stimulation with AngII in both WT and TRKO mice. Phosphorylation of several well-established proteins directly downstream of mTORC2 was reduced in TRKO mice - AKT2, PKC, NEDD4-2, and Rictor itself. However, we also identified several ion channels and GPCRs not previously known to be downstream of mTORC2. Additionally, we used published kinase prediction tools to predict downstream kinases disrupted by mTORC2 deletion. Known downstream kinases were again confirmed and, in addition, we predict a family of CAMK kinases is linked to aldosterone signaling in the distal nephron which we are currently further exploring. Funding: Grants from NIH (T32- DK007418, R01-DK56695) and The James Hilton Manning and Emma Austin Manning Foundation. Presentation: Saturday, July 12, 2025