A salt-sensing kinase in T lymphocytes, SGK1, drives hypertension and hypertensive end-organ damage

Abstract

We previously showed that angiotensin II (Ang II) increases T cell production of IL-17A, and that mice deficient in IL-17A have blunted hypertension and attenuated renal and vascular dysfunction. It was recently shown that salt enhances IL-17A production from CD4+ T cells via a serum- and glucocorticoid-regulated kinase 1-dependent (SGK1-dependent) pathway. Thus, we tested the hypothesis that SGK1 signaling in T cells promotes hypertension and contributes to end-organ damage. We show that loss of T cell SGK1 results in a blunted hypertensive response to Ang II infusion by 25 mmHg. Importantly, renal and vascular inflammation is abrogated in these mice compared with control mice. Furthermore, mice lacking T cell SGK1 are protected from Ang II-induced endothelial dysfunction and renal injury. Loss of T cell SGK1 also blunts blood pressure and vascular inflammation in response to deoxycorticosterone acetate-salt (DOCA-salt) hypertension. Finally, we demonstrate that the Na+-K+-2Cl- cotransporter 1 (NKCC1) is upregulated in Th17 cells and is necessary for the salt-induced increase in SGK1 and the IL-23 receptor. These studies demonstrate that T cell SGK1 and NKCC1 may be novel therapeutic targets for the treatment of hypertension and identify a potentially new mechanism by which salt contributes to hypertension.

Keywords: Adaptive immunity; Cardiology; Cardiovascular disease; Hypertension; Inflammation.

Figure 1. T cell SGK1 deficiency attenuates angiotensin II–induced hypertension.

(A) Systolic blood pressures measured noninvasively using the tail-cuff method over 28 days of angiotensin II (Ang II) (490 ng/kg/min) or vehicle (sham) infusion in SGK1^fl/fl and SGK1^fl/fltg^CD4cre mice. **P < 0.01 between both Ang II–infused groups; regression analysis; n = 6–7 per group. (B) Systolic blood pressures, (C) mean arterial pressures, (D) diastolic blood pressures, and (E) heart rates measured invasively using carotid radiotelemetry over 28 days of Ang II infusion in SGK1^fl/fl and SGK1^fl/fltg^CD4cre mice. *P < 0.05, ***P < 0.001; regression analysis; n = 6–7 per group. All data are expressed as mean ± SEM.

Figure 2. T cell SGK1 deficiency prevents the chronic phase of angiotensin II–induced vascular inflammation.

(A) Representative flow cytometry dot plots showing gating strategy for total leukocytes (CD45⁺ cells), total T lymphocytes (CD45⁺CD3⁺ cells), and monocytes/macrophages (CD45⁺F4/80⁺ cells) in single-cell suspensions from the thoracic aorta of SGK1^fl/fl and SGK1^fl/fltg^CD4cre mice infused with angiotensin II (Ang II) or vehicle (sham) for 7 or 28 days. (B–D) Summary data of absolute numbers of indicated cell types per thoracic aorta. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; 2-way ANOVA/Holm-Sidak’s post-hoc test; n = 7–12 per group. All data are expressed as mean ± SEM.

Figure 3. T cell SGK1 deficiency prevents the chronic phase of angiotensin II–induced renal inflammation.

(A) Representative flow cytometry dot plots showing gating strategy for total leukocytes (CD45⁺ cells), total T lymphocytes (CD45⁺CD3⁺ cells), and monocytes/macrophages (CD45⁺F4/80⁺ cells) in single-cell suspensions from one kidney of SGK1^fl/fl and SGK1^fl/fltg^CD4cre mice infused with angiotensin II (Ang II) or vehicle (sham) for 7 or 28 days. (B–D) Summary data of absolute numbers of indicated cell types per kidney. **P < 0.01; 2-way ANOVA/Holm-Sidak’s post-hoc test; n = 6–10 per group. All data are expressed as mean ± SEM.

Figure 4. T cell SGK1 deficiency protects against angiotensin II–induced vascular and glomerular injury.

SGK1^fl/fl controls, tg^CD4cre controls, and SGK1^fl/fltg^CD4cre mice were infused with angiotensin II (Ang II) or vehicle (sham) for 28 days. Endothelium-dependent relaxation in response to increasing doses of acetylcholine (Ach) was measured in (A) SGK1^fl/fl mice and (B) SGK1^fl/fltg^CD4cre mice, and endothelium-independent relaxation in response to increasing doses of sodium nitroprusside (SNP) was measured in (C) SGK1^fl/fl mice and (D) SGK1^fl/fltg^CD4cre mice. ***P < 0.01; regression analysis; n = 4–6 per group. (E) Urinary albumin to creatinine ratio in the indicated groups. *P < 0.05, **P < 0.01, ***P < 0.001; 1-way ANOVA/Holm-Sidak’s post-hoc test; n = 8–14 per group. (F) Relative renal NGAL mRNA expression in the indicated groups. **P < 0.01; 2-way ANOVA/Holm-Sidak’s post-hoc test; n = 10 per group. All data are expressed as mean ± SEM. n.s., not significant.

Figure 5. T cell SGK1 deficiency abrogates angiotensin II–induced increase in splenic Th17 cells.

(A) Representative flow cytometry dot plots for CD4⁺IL-17A⁺ Th17 cells and CD8⁺IL-17A⁺ Tc17 cells in splenic single-cell suspensions from SGK1^fl/fl and SGK1^fl/fltg^CD4cre mice infused with angiotensin II (Ang II) or vehicle (sham) for 28 days. (B and C) Summary data of percentages of CD4⁺IL-17A⁺ Th17 cells out of total CD4⁺ cells and CD8⁺IL-17A⁺ Tc17 out of total CD8⁺ cells in the indicated groups. *P < 0.05; 1-way ANOVA/Holm-Sidak’s post-hoc test; n = 8–12 per group. All data are expressed as mean ± SEM.

Figure 6. T cell SGK1 deficiency attenuates hypertension and vascular inflammation in a DOCA-salt model of hypertension.

(A) Systolic blood pressures measured noninvasively using the tail-cuff method in SGK1^fl/fl and SGK1^fl/fltg^CD4cre mice in response to deoxycorticosterone acetate–salt (DOCA-salt) treatment over 21 days. *P < 0.05, **P < 0.01; regression analysis; n = 8–10 per group. (B–F) Summary flow cytometry data of absolute numbers of total leukocytes (CD45⁺ cells), total T lymphocytes (CD45⁺CD3⁺ cells), and monocytes/macrophages (CD45⁺F4/80⁺ cells) per thoracic aorta in SGK1^fl/fl and SGK1^fl/fltg^CD4cre mice after 21 days of DOCA-salt or vehicle (sham) treatment. *P < 0.05, **P < 0.01; 2-way ANOVA/Holm-Sidak’s post-hoc test; n = 8–14 per group. All data are expressed as mean ± SEM.

Figure 7. NKCC1 is upregulated in Th17 cells and mediates the salt-induced increase in SGK1 and IL-23R.

(A) Naive splenic CD4⁺ T cells were isolated from C57BL/6J WT mice and cultured for 72 hours on anti-CD3/anti-CD28–coated plates in the presence or absence of Th17-polarizing cytokines (Cytokines), an excess 40 mM NaCl (Salt), and cotreated with hydrochlorothiazide (HCTZ; A and E), spironolactone (B and F), furosemide (C and G), bumetanide (D and H), or corresponding vehicle (DMSO or ethanol). SGK1 expression (A–D) or IL-23 receptor (IL-23R) expression (E–H) was quantified by qRT-PCR and expressed as fold change relative to untreated cells (A–D) or relative to Cytokine-treated cells (E–H). (I) Naive splenic CD4⁺ T cells from WT mice were cultured as described above and sodium-potassium-2 chloride cotransporter 1 (NKCC1) expression was quantified by qRT-PCR and expressed as fold change relative to untreated cells. *P < 0.05, **P < 0.01, ***P < 0.001; ratio-paired t test was performed between the indicated groups with Bonferroni correction; n = 5–9 per group. NS, not significant.