Interleukin-1 Receptor Activation Potentiates Salt Reabsorption in Angiotensin II-Induced Hypertension via the NKCC2 Co-transporter in the Nephron

Abstract

Hypertension is among the most prevalent and catastrophic chronic diseases worldwide. While the efficacy of renin angiotensin system (RAS) blockade in lowering blood pressure illustrates that the RAS is broadly activated in human hypertension, the frequent failure of RAS inhibition to prevent or reverse hypertensive organ damage highlights the need for novel therapies to combat RAS-dependent hypertension. We previously discovered elevated levels of the macrophage cytokine IL-1 in the kidney in a murine model of RAS-mediated hypertension. Here we report that IL-1 receptor (IL-1R1) deficiency or blockade limits blood pressure elevation in this model by mitigating sodium reabsorption via the NKCC2 co-transporter in the nephron. In this setting, IL-1R1 activation prevents intra-renal myeloid cells from maturing into Ly6C(+)Ly6G(-) macrophages that elaborate nitric oxide, a natriuretic hormone that suppresses NKCC2 activity. By revealing how the innate immune system regulates tubular sodium transport, these experiments should lead to new immunomodulatory anti-hypertensive therapies.

Figure 1

IL-1R1 deficiency or blockade limits the severity of angiotensin II-dependent hypertension. (A) Mean arterial pressures measured by radiotelemetry in the experimental groups at baseline (“pre”) and during chronic Ang II infusion. Wild-type (“WT”), circles. IL-1R1 KO (“KO”), open squares. n≥9 per group. (B) Mean arterial pressures measured by radiotelemetry in the anakinra-and vehicle-treated wild-type animals at baseline (“pre”), initiation of treatment (“In”), and during chronic Ang II infusion. n≥7 per group. (C) Ratio of heart weight/body weight (mg/g) in the WT and IL-1R1 KO (“KO”) groups after 28 days of Ang II infusion. (D) Ratio of heart weight/body weight (mg/g) in the anakinra-and vehicle-treated wild-type animals after 28 days of Ang II. Error bars represent SEM.

Figure 2

IL-1R1 stimulation augments angiotensin II-induced sodium retention by enhancing activity of NKCC2 sodium co-transporter. (A–B) Mice were placed into metabolic cages beginning 6 days prior to initiation of chronic angiotensin (Ang) II (“pump”) and continuing for 15 days of chronic Ang II infusion, (A) Cumulative sodium balances tabulated over 3-day periods in the WT and IL-1R1 KO (“KO”) groups, (B) Daily urine sodium excretion over same period, *P<0.05 vs. WT, n = 10 per group, (C) Mean arterial pressures measured by radiotelemetry at baseline (C1), followed by 5-day periods of 6% NaCl diet (HS) and restoration of normal diet (C2), n = 5 per group, *P=0.007 vs. WT C1, 0.03 vs. WT C2, ^#P=0.0002 vs. KO C1, 0.0006 vs. KO C2 by paired analysis, (D) Urine sodium/creatinine ratios measured on 3-hr urine samples from WT and IL-1R1 KO (“KO”) mice following IP injection of saline alone or with furosemide (“FURO”) are similar in naïve mice, (E) Results of furosemide challenge study repeated on day 10 of chronic Ang II infusion. IL-1R1 KOs have exaggerated natriuretic response to saline alone, but IL-1R1 KO natriuresis converges with that of WTs following NKCC2 blockade with furosemide. n = 6 per group, (F) Immunoblots for cortical (“c”) and medullary (“m”) NKCC and NKKC were performed with a constant amount of protein per lane. Quantitation in Figure S1D. Error bars represent SEM.

Figure 3

IL-1R1 activation potentiates RAS-mediated blood pressure elevation by limiting NOS2-dependent nitric oxide (NO) generation in the kidney. (A) Urinary excretion of nitric oxide metabolites in WT and IL-1R1 KO (“KO”) mice following 7 days of chronic Ang II infusion. (B) Urinary excretion of 8-isoprostane following chronic Ang II, n≥12 per group, (C) Mean arterial pressures measured by radiotelemetry in the groups at day 7 of Ang II prior to L-NAME treatment (“Pre-L-NAME”) and after 7 days of L-NAME ingestion (“Post-L-NAME”). (D–E) Renal mRNA expression of (D) NOS3 (eNOS) and (E) NOS2 (iNOS) during Ang II-dependent hypertension, n = 8 per group. Error bars represent SEM.

Figure 4

Activation of IL-1R1 constrains the accumulation of NO-producing myeloid cells in the hypertensive kidney. (A–C) Enhanced numbers of intra-renal F4/80⁺ macrophages in IL-1R1s compared to WT controls during Ang II-induced hypertension. Representative (A) WT and (B) IL-1R1 KO (“KO”) kidney sections stained for F4/80. (C) Blinded scoring of F4/80⁺ macrophages on WT and IL-1R1 KO kidney sections, (D) mRNA levels of inflammatory markers and cytokines IL-1β, IL-12b, TNF-a, CCL2, and Arg1 in CD11b⁺ macrophages isolated from kidneys of WT and IL-1R1 KO mice at day 7 of Ang II, n = 6 per group, (E–G) Enhanced production of nitric oxide by IL-1R1 KO activated macrophages infiltrating the kidney at day 7 of Ang II. Diaminofluorescein (DAF) staining for NO on intra-renal CD11b⁺ Ly6C⁺ myeloid cells from (E) WT and (F) IL-1R1 KO cohorts. (G) Proportions of DAF-positive activated macrophages as quantitated by FlowJo analysis. (H–J) Shift away from Ly6C⁺Ly6G⁺ double-positive immature myeloid populations toward single Ly6C⁺ phenotype among intra-renal IL-1R1 KO activated macrophages at day 7 of Ang II. Representative flow plots of Ly6C versus Ly6G staining among (H) WT and (I) IL-1R1 KO CD11b⁺ myeloid cells. (J) Summary data showing proportions of Ly6C⁺Ly6G⁻ and Ly6C⁺Ly6G⁺ macrophages in experimental groups. n = 8 per group. Error bars represent SEM.