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Published in final edited form as: Cell Metab. 2015 Dec 17;23(2):360–368. doi: 10.1016/j.cmet.2015.11.013

Interleukin-1 augments salt retention in angiotensin II-induced hypertension via nitric oxide-dependent regulation of the NKCC2 sodium co-transporter

Jiandong Zhang 1, Nathan P Rudemiller 1, Mehul B Patel 1, Norah S Karlovich 1, Min Wu 1, Alicia A McDonough 2, Robert Griffiths 1, Matthew A Sparks 1, Alexander D Jeffs 1, Steven D Crowley 1
PMCID: PMC4749461  NIHMSID: NIHMS741501  PMID: 26712462

Summary

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.

Graphical abstract

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Introduction

Hypertension is among the most prevalent chronic diseases, impacting over a billion adults worldwide (Lawes et al., 2008). The complications of uncontrolled hypertension such as stroke, heart failure, and kidney disease, are associated with substantial morbidity and mortality. However, the precise etiology of blood pressure elevation remains unclear in most affected individuals. Moreover, large numbers of hypertensive patients have blood pressure elevation that is resistant to existing treatment options (Egan et al., 2011), highlighting the urgent need for novel therapies.

A growing body of evidence has suggested that hypertension is an inflammatory disease. Reports of an inflammatory response during hypertension began to emerge several decades ago. Early biopsy studies revealed that immune cells figure prominently in the kidneys in patients with severe hypertension (Heptinstall, 1953). More recent epidemiological observations showed that low grade inflammation marked by increased C-reactive protein (CRP), a surrogate marker for interleukin 1 (IL-1) activity, precedes the onset of essential hypertension, suggesting that inflammation plays a role in the genesis of hypertension (Libby et al., 2002; Sesso et al., 2003). Moreover, linkage studies have demonstrated that polymorphisms in the genes encoding members of the IL-1 signaling pathway have been associated with essential hypertension (Fragoso et al., 2010; Khawaja et al., 2007).

IL-1 is a pro-inflammatory cytokine that plays a central role in both acute and chronic inflammation, acting as a primary inducer of the innate immune response. Both isoforms of IL-1, IL-1α and IL-1β, bind and signal via the type 1 IL-1 receptor (IL-1R1). We previously reported that expressions of both IL-1 isoforms are increased in the kidney during hypertension induced by activation of the renin angiotensin system (RAS) and correlates with the degree of blood pressure regulation (Crowley et al., 2010b). However, studies that directly address the role of IL-1R1 signaling in the pathogenesis of hypertension are lacking.

The present studies therefore test the hypothesis that activation of the IL-1R1 contributes to RAS-dependent hypertension. Herein, we elucidate a novel mechanism through which IL-1R1 stimulation potentiates blood pressure elevation by suppressing nitric oxide (NO)-dependent sodium excretion in the kidney. These experiments further identify IL-1R1 blockade as a potential strategy for treating hypertension.

Results

Genetic deficiency of IL-1R1 limits angiotensin II-induced blood pressure elevation

As IL-1α and β are both upregulated in the kidney during RAS-dependent hypertension (Crowley et al., 2010b) and bind to the type 1 IL-1 receptor (IL-1R1), we first examined the contribution of the IL-1 signaling pathway to hypertension by subjecting wild-type (WT) and IL-1R1-deficient (KO) mice to our angiotensin (Ang) II-dependent hypertension model. At baseline, WT and IL-1R1 KO mice had similar mean arterial blood pressures as measured by radiotelemetry (126±1 versus 132±3 mmHg; p=NS; Figure 1A). However, during chronic Ang II infusion, the IL-1R1 KO animals were partially protected from hypertension compared to the WT controls (165±6 versus 180±3 mmHg; p=0.048; Figure 1A). Consistent with their lower blood pressures, the Ang II-infused IL1R1 KOs had less cardiac hypertrophy following 4 weeks of hypertension (7.5±0.2versus 9.2±0.2 mg heart weight/g body weight, p<0.0001; Figure 1C).

Figure 1.

Figure 1

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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.

We then examined whether pharmacological blockade of IL-1R1 affords similar protection from hypertension by administering an IL-1R1 antagonist (anakinra) or vehicle to WT mice for 3 days prior to and during chronic Ang II infusion. As seen with IL-1R1 deficiency, anakinra treatment in WT mice did not influence baseline blood pressures compared to vehicle-treated controls (130±3 versus 130±1 mm Hg; p=NS; Figure 1B). However, IL-1R1 blockade with anakinra significantly attenuated the level of blood pressure elevation during chronic Ang II infusion (154±4 versus 167±3 mmHg; p=0.029; Figure 1B), leading to less cardiac hypertrophy after 4 weeks of hypertension (6.4±0.3 versus 7.1±0.2 mg heart weight/g body weight, p=0.05; Figure 1D). These data suggest that IL-1 receptor stimulation potentiates Ang II-mediated blood pressure elevation.

IL-1R1 signaling augments Ang II-induced sodium retention by enhancing NKCC2 co-transporter activity

To determine if the pro-hypertensive effects of IL-1 receptor activation were related to renal sodium retention, we placed WT and IL-1R1 KO mice into metabolic cages beginning 1 week prior to the initiation of chronic Ang II infusion and quantitated daily sodium ingestion and urinary sodium excretion to compute net sodium balances. Food ingestion remained similar in the 2 groups throughout the study (Figure S1A). Prior to Ang II, the 2 groups had similar net sodium balances (Figure 2A) and levels of urinary sodium excretion (Figure 2B). However, concurrent with the separation in blood pressures during the 2nd week of Ang II infusion, the WT animals excreted less sodium than their IL-1R1 KO counterparts (Figure 2B). Accordingly, over days 10–15 of Ang II, the WT mice sustained a positive sodium balance, whereas the IL-1R1 KO mice switched to a negative sodium balance (208.84±44.84 vs. −100.66±100.74 μmol/6days, p=0.013). Thus, IL-1 receptor deficiency limits Ang II-induced sodium reabsorption, mitigating the hypertensive response in our model. The actions of the IL-1 receptor to potentiate sodium retention appear to require RAS stimulation, as salt-loading at baseline had a similar impact to raise blood pressures in the WT and IL-1R1 KO cohorts (Figure 2C).

Figure 2.

Figure 2

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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.

To further examine the mechanism responsible for the attenuated hypertension and enhanced natriuresis in the IL-1R1 KO cohort, we measured the activity of 2 key renal sodium transporters, NKCC2 and NCC, either at baseline or following 10 days of chronic Ang II infusion when the difference in sodium excretion between the groups was most evident. To evaluate sodium transporter activity at these timepoints, we quantitated diuretic-induced sodium excretion during the 3 hours after an intraperitoneal saline infusion. At baseline prior to Ang II, there were no differences in sodium excretion between WT and IL1R1 KO mice either in response to saline control (90.3±16.9 vs. 62.3±12.9 mmol Na+/mmol Cr, p=NS) or furosemide (408.9±89.2 vs. 499.4±123.0 mmol Na+/mmol Cr, p=NS, Figure 2D).

By contrast, at day 10 of Ang II infusion, in response to IP saline injection alone, the IL-1R1 KO mice had exaggerated urine sodium to creatinine ratios, consistent with their enhanced natriuresis during hypertension in our original balance study (236.9±38.2 vs. 119.5±8.8 mmol/mmol Cr; p=0.016, Figure 2E). Blockade of NKCC2 with furosemide increased sodium excretion in both groups but abrogated the difference between the WT and IL-1R1 KOs (453.4±55.5 vs.480.5±103.1 mmol/mmol Cr; p=0.83, Figure 2E), suggesting that impaired NKCC2 activity in the Ang II-infused IL-1R1 KO mice accounts for their preserved capacity to excrete sodium during chronic Ang II infusion. Given the potential confounder of tubular creatinine secretion, we repeated the analysis without normalizing sodium excretion for creatinine and noted the same pattern (Figure S1B). By contrast, blockade of the NCC sodium transporter with hydrochlorthiazide did not attenuate the differences in sodium excretion between the Ang II-infused WT and IL-1R1 KOs (data not shown). At day 10 of Ang II, Western blot analysis revealed similar levels of total and phosphorylated NKCC expression in the renal cortex and medulla of the 2 groups (Figure 2F, Figure S1D), suggesting that IL-1 receptor activation modulates the function of NKCC without altering its translation or phosphorylation.

To determine if chronic in vivo NKCC2 blockade could prevent the separation in blood pressures between the Ang II-infused WT and IL-1R1 KO mice, we administered furosemide in the drinking water for a week beginning on day 5 of Ang II before the WT and IL-1R1 blood pressures diverged. Over the first 5 days of Ang II prior to furosemide treatment, blood pressures in the groups were similar. Oral furosemide significantly reduced blood pressures in both groups and prevented the separation in WT and IL-1R1 KO blood pressures (Figure S1C). Thus, IL-1R1 stimulation promotes Ang II-induced sodium reabsorption via the NKCC2 co-transporter.

IL-1R1 deficiency does not impair vascular responses to Ang II

IL-1 receptor stimulation could also regulate blood pressure and sodium excretion in our model by influencing vascular responses to Ang II (Vallejo et al., 2014). We therefore quantitated blood pressure elevation following an acute IV injection of Ang II into anesthetized animals (Figure S2A). By this measure, acute vascular responses of the IL-1R1 KO mice were intact. Moreover, renal vasoconstrictor responses as measured by reductions in renal blood flow following acute Ang II infusion were similar in anesthetized WT and IL-1R1 KO animals (Figure S2B). As an estimate of glomerular filtration rate (GFR), serum creatinines were comparable in the 2 groups at day 9 of chronic Ang II infusion (Figure S2C). These data would indicate that IL-1 receptor stimulation does not enhance Ang II-induced sodium retention by limiting delivery of sodium to the nephron.

IL1R1 stimulation promotes renal sodium reabsorption during RAS activation by limiting nitric oxide bioavailability in the kidney

Nitric oxide (NO) can facilitate renal sodium excretion by suppressing NKCC2 activity (Ortiz et al., 2001). We therefore examined 24-hour urinary excretion of nitric oxide metabolites as a marker of local NO production following 7 days of Ang II when the WT and IL-1R1 KO blood pressures diverged. As shown in Figure 3A, there was a 2.5-fold higher level of NO metabolites in the urines from the IL-1R1 KO cohort (152.5± 45.73 vs. 60.32 ±14.77nmol/24 h; p=0.05). Inversely, reactive oxygen species can limit NO bioavailability, and the Ang II-infused IL-1R1 group had significantly less urinary excretion of 8-isoprostane that marks renal oxidative stress (P=0.04; Figure 3B). These studies show that IL-1R1 deficiency preserves NO bioavailability during Ang II-dependent hypertension.

Figure 3.

Figure 3

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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.

To explore whether the enhanced NO bioavailability in the IL-1R1 KOs was responsible for their lower blood pressures in our hypertension model, we disrupted NO generation in both groups by administering L-NAME in the drinking water starting at day 7 of Ang II infusion. Similar to our initial hypertension experiment, IL1R1 KO mice had lower blood pressures than WT controls by day 7 of Ang II prior to L-NAME (165.4±3.2 vs. 179.3±3.2 mm Hg; p=0.006 Figure 3C). However, after L-NAME treatment, blood pressures in the groups converged (185.1±12.7 vs. 185.3±4.8 mmHg; p=NS Figure 3C). Thus, deprivation of NO bioavailability by L-NAME abrogated the protection from RAS-mediated blood pressure elevation in the IL1R1 KO mice, indicating that Ang II induces renal sodium retention and hypertension in part via IL-1R1-dependent suppression of NO generation.

Next, we sought to determine which of the NO synthases contributed to the exaggerated NO production in the Ang II-infused IL1R1 KO mice. Whole kidney mRNA expression of NOS3 (eNOS) was similar in the 2 groups (Figure 3D). By contrast, NOS2 (iNOS) expression was markedly upregulated in the IL-1R1 KO kidneys compared to WT controls (1.73±0.29 vs. 1±0.16 arbitrary units; p=0.045, Figure 3E). Thus, NOS2 rather than NOS3 appears to drive the exaggerated production of NO in the IL-1R1-deficient kidney during Ang II-dependent hypertension.

Enhanced accumulation of NO-producing macrophages in the IL-1R1-deficient kidney during RAS activation

As NOS2 catalyzes the generation of NO in macrophages infiltrating target organs and IL-1R1 can modulate macrophage function, we quantitated the number of F4/80+ macrophages infiltrating the kidney following chronic Ang II infusion. This analysis revealed 32% more intra-renal macrophages in the IL-1R1 KO kidney compared to WT controls (17.9 ± 0.8 versus 13.6 ± 1.7 per high power field; P=0.033, Figure 4A–C). However, CD11b+ macrophages isolated from the kidneys of IL-1R1 KO animals at day 7 of Ang II expressed lower mRNA levels of pro-inflammatory cytokines (Figure 4D) including IL-1β [0.68±0.06 vs. 1.00±0.05 arbitrary units (au); P=0.004] and tumor necrosis factor-α (TNF) (0.65±0.07 vs. 1.00±0.17 au; P=0.05). We therefore posited that the infiltrating macrophages in the IL-1R1 KO cohort were suppressing the hypertensive response through the generation of NO.

Figure 4.

Figure 4

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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.

To test this possibility, we used a fluorescent cell sorting strategy to isolate CD45+ infiltrating mononuclear cells from the WT or IL-1R1 kidney at day 7 of Ang II and labeled these cells for CD11b and Ly6C to mark activated infiltrating macrophages. During the isolation procedure, we exposed these macrophages to diaminofluorescein (DAF-FM), which fluoresces green when conjugated to NO. Macrophages from the IL1R1 KO kidneys had nearly 2-fold greater DAF staining for NO than WT controls (4.1±0.6 vs. 2.1±0.5% of total infiltrating macrophages; P=0.039, Figure 4E–G), suggesting that IL-1R1 stimulation limits the accumulation of NO-producing macrophages in the hypertensive kidney.

Finally, as macrophages expressing the marker Ly6C but not the granulocyte marker Ly6G are capable of elaborating NO (Youn et al., 2008), we further stratified the CD45+ cells isolated from the hypertensive kidney based on their expression of these 2 markers to determine if activation of the IL-1R1 influences the maturation of renal myeloid-derived cells. Among the IL-1R1 KO macrophages, there were 50% more Ly6C+ Ly6G− cells (17.4±1.8 vs. 11.1±1.0%; p=0.002, Figure 4H–J) and 60% fewer immature myeloid cells marked by double positivity for both Ly6C and Ly6G (6.4±1.3 vs. 15.4±3.0%; P=0.015, Figure 4H–J). Moreover, upon re-stimulation of the renal infiltrating macrophages with LPS (Figure S3), the induction of NOS2 expression as measured by flow cytometry in the Ly6C+ Ly6G− cells was 2-fold greater in the IL-1R1 KOs than in WT controls (9.6±2.1 vs. 4.1±9.9 au; P<0.03). These data suggest that during RAS-dependent hypertension, IL-1R1 stimulation potentiates blood pressure elevation by suppressing the differentiation in the kidney of Ly6C+ Ly6G+ immature myeloid cells toward Ly6C+ Ly6G− myeloid cells that can elaborate NO and thereby constrain NKCC2-mediated sodium reabsorption.

Discussion

The pathogenesis of hypertension involves dysregulation of a complex circuit integrating inputs from the nervous system, the heart and vasculature, the kidney, and the immune system. Within the adaptive immune system, activated T lymphocytes can impart blood pressure elevation or reduction depending on the cytokines they elaborate (Barhoumi et al., 2011; Guzik et al., 2007; Madhur et al., 2010). Within the innate immune system, monocytes can increase blood pressure through effects on vascular remodeling (Wenzel et al., 2011), dendritic cells can promote hypertension by presenting antigens to T cells (Kirabo et al., 2014), and tissue macrophages can ameliorate salt-sensitive hypertension by facilitating the removal of excess sodium stores from the skin (Machnik et al., 2009; Wiig et al., 2013). In this context, the current experiments reveal an additional pathogenic immune mechanism in hypertension wherein IL-1 receptor stimulation blocks the accumulation in the kidney of NO-producing myeloid cells that can mitigate NKCC2-mediated sodium retention.

Several lines of evidence prompted us to focus on a possible role for IL-1 receptor activation in hypertension. Elevated serum levels of IL-1β have been documented in patients with essential hypertension (Dalekos et al., 1996) and in stroke-prone spontaneously hypertensive rats (Chiba et al., 2012). Also in rats, infusion of IL-1b into the intracerebral ventricles, the paraventricular nucleus, or the systemic venous circulation yielded an acute pressor response (Lu et al., 2009; Takahashi et al., 1992; Yamamoto et al., 1994). Finally, we previously detected elevated expression levels of IL-1α and β in the kidney during hypertension driven by activation of the renin angiotensin system (RAS) (Crowley et al., 2010b).

To capture signaling of both IL-1 isoforms, we have tested the effects of IL-1 receptor (IL-1R1) deficiency or blockade on the hypertensive response to chronic angiotensin (Ang) II infusion, the same model in which we previously detected IL-1 upregulation. We also favor this model as large percentages of patients treated with RAS blockade respond with some degree of blood pressure reduction, suggesting that the RAS is inappropriately activated in human hypertension (Matchar et al., 2008). Using both genetic and pharmacological approaches, we find that IL-1R1 signaling makes a striking and persistent contribution to Ang II-mediated blood pressure elevation.

RAS activation causes chronic hypertension by driving sodium retention in the kidney (Crowley et al., 2006; Gonzalez-Villalobos et al., 2013). Moreover, we noted a gradual separation in WT and IL-1R1 KO blood pressures after a week of Ang II when prominent natriuresis can occur (Crowley et al., 2010a), leading us to explore whether IL-1R1 stimulation influences renal sodium handling during RAS activation. We find that IL-1R1 signaling exaggerates Ang II-induced sodium retention, preventing the natriuresis that would otherwise occur in response to substantial elevations in blood pressure. As the timings of the separations in blood pressure and sodium balances do not match precisely, we speculate that the metabolic cage conditions used for the balance studies may have attenuated eating and drinking behaviors and that mobilization of non-osmotic dermal sodium stores contributed to the net sodium balances in both groups (Machnik et al., 2009). Nevertheless, the higher sodium excretion coupled with lower blood pressures in the Ang II-infused IL-1R1 KOs suggests that their natriuresis caused rather than compensated for their blunted hypertensive response compared to WT controls.

The discovery that IL-1R1 activation potentiates salt reabsorption in our model is unexpected as acute IL-1 administration can inhibit sodium reabsorption in the collecting duct (Kohan et al., 1989; Sakairi et al., 1994). We speculate that the effects of IL-1 on renal sodium handling may be bi-phasic in which mild IL-1 elevations occurring in hypertension favor salt retention whereas fulminant IL-1 elevations during sepsis trigger natriuresis with a risk of circulatory collapse (Caverzasio et al., 1987). Such a phenomenon has been described with another pro-inflammatory cytokine, TNF (Ramseyer and Garvin, 2013). The apparent discrepancies in IL-1 actions could also be related to the source and distribution of IL-1 within the kidney as macrophages infiltrating the interstitium are likely a prominent source of intra-renal IL-1 in hypertension.

RAS activation regulates sodium transporters throughout the nephron (Gonzalez-Villalobos et al., 2013). Whereas Il-1 has previously been shown to inhibit sodium transport in the collecting tubule (Kohan et al., 1989), our diuretic challenge experiments indicate that IL-1R1 signaling potentiates the capacity of Ang II to activate NKCC2-mediated sodium transport in the medullary thick ascending limb (mTAL) of the nephron. By contrast, we did not find an effect of IL-1R1 activation on NCC activity in the distal convoluted tubule (data not shown). While there is precedent for IL-1β to upregulate NKCC in the epithelium of the middle ear (Kim et al., 2007), to our knowledge no previous study has demonstrated cooperative actions of Ang II and IL-1R1 activation to drive sodium reabsorption in the mTAL.

Nitric oxide (NO) is a potent driver of sodium excretion in the kidney that acts via cGMP and phosphodiesterase 2 to limit NKCC2 activity in the mTAL (Ares et al., 2008; Ortiz et al., 2001). Accordingly, we find that abrogating IL-1 signals enhances NO bioavailability in the kidney that, in turn, limits NKKC2-dependent sodium retention accruing from RAS activation. Our Western blot studies showing that NKKC protein is preserved in the IL-1R1 KO mice are consistent with prior reports that NO/cGMP regulate NKCC2 activity independently of its expression (Ares et al., 2008). Rather, we speculate that the alterations in NKKC2 function recorded in our experiments relate to effects on membrane trafficking of the transporter. While we have not examined transporter expression prior to the induction of hypertension, the similar blood pressures in the 2 groups at baseline would suggest that IL-1 receptor-deficiency does not alter expression levels in the absence of RAS activation. Our finding that IL-1 signaling limits renal NO generation in our hypertension model recapitulates a pattern seen with TNF in which both cytokines exaggerate Ang II-induced blood pressure elevation by reducing NO bioavailability and thereby enhancing NKKC2-mediated sodium transport (Ramseyer et al., 2012), highlighting a broad effect of these inflammatory cytokines to raise blood pressure by modulating NO-dependent actions in the nephron. Interestingly, TNF expression was also blunted in the renal infiltrating macrophages from our Ang II-infused IL-1R1 KO cohort.

Macrophages constitute a key source of NO generated via NOS2 and accumulate during RAS-dependent hypertension throughout the renal interstitium where they percolate among medullary epithelial cells (Ozawa et al., 2007). Accordingly, in our model we interrogated the phenotype of the infiltrating myeloid cells as it impacted their capacity for NO production. We find that IL-1R1 activation in this context hampers the differentiation of immature Ly6C+Ly6G+ myeloid cells toward an NO-producing Ly6C+Ly6G− macrophage phenotype (Youn et al., 2008). This phenomenon appears to be quite specific to the hypertensive kidney as IL-1 signaling in macrophages favors NO elaboration in response to Leishmania infection (Lima-Junior et al., 2013). Alternatively, it is conceivable that IL-1R1 activation on the kidney epithelial cells inhibits the differentiation of neighboring macrophages through a paracrine mechanism whose description may require a cell-specific gene targeting approach. Such “reverse” epithelial cell–macrophage cross-talk has been documented in the setting of acute kidney injury (Lee et al., 2011).

Lastly, we have focused on the actions of intra-renal macrophages to modulate sodium transport through NO production. However, we acknowledge that dermal macrophages make key contributions to sodium homeostasis (Machnik et al., 2009), that renal tubular cells can produce NOS2 (Markewitz et al., 1993), and that IL-1 has pleiotropic actions in other cell lineages that could also potentially contribute to its pro-hypertensive actions during RAS activation. For example, IL-1β can induce the pro-hypertensive cytokine IL-6 and also the differentiation of Th17 cells that produce IL-17 (Brands et al., 2010; Coccia et al., 2012; Madhur et al., 2010). Nevertheless, macrophages constitute major sources of IL-1 and NOS2, are the most abundant inflammatory cells that we can detect in the hypertensive kidney, and have therefore garnered our attention in the current investigation.

In sum we find that IL-1R1 stimulation accentuates the severity of RAS-dependent hypertension by promoting NKCC2-mediated sodium transport in the nephron. IL-1 enhances NKCC2 activity by releasing it from tonic NO-dependent inhibition, at least in part through effects on the maturation of myeloid cells infiltrating the kidney. Studies exploring the effects of IL-1R1 inhibition in other forms of experimental hypertension could further inform which human hypertensive patients would potentially derive benefit from IL-1R1 blockade. On the other hand, the safety profile of anakinra has been favorable in patients with rheumatic disease such that accelerated translational testing of IL-1R1 blockade could yield a novel immunomodulatory therapy to lower blood pressure and limit target organ damage in patients with resistant hypertension.

Experimental Procedures

(See also Supplemental Experimental Procedures)

Animals

IL1R1−/−(B6.129S7-Il1r1tm1Glm/J)mice on the C57/BL6 background were obtained from Jackson Laboratory and backcrossed for 6 generations to the 129/SvEv strain to enhance salt sensitivity. Then, IL1R1 heterozygotes were intercrossed to yield the IL1R1 KO and WT littermates for experiments. All of the animal studies were approved by the Durham Veterans Affairs Medical Center Institutional Animal Care and Use Committee and conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Model of Chronic Ang II-induced Hypertension

To render the animals more salt-sensitive, experimental mice (n≥9) underwent left nephrectomy followed one week later by implantation of a pressure-sensing catheter. To induce hypertension, an osmotic minipump was implanted to infuse Ang II continuously for 28 days.

Sodium Balance Study

Mice were placed into individual metabolic cages, and a gel diet containing nutrients, water and sodium (Nutra-Gel, Bio-serv, Frenchtown, NJ) was provided. Sodium ingestion and excretion were quantitated daily to allow quantitation of sodium balances.

Statistical Analysis

The values within a group are expressed as Mean±SEM. For comparisons between groups with normally distributed data, statistical significance was assessed using ANOVA followed by unpaired t-test. For comparisons within groups, normally distributed variables were analyzed by a paired t-test.

Supplementary Material

supplement

Acknowledgments

This work was supported by funding from (1) National Institutes of Health Grant DK087893 (SC) and DK083785 (AM), (2) the Department of Veterans Affairs, Veterans Health Administration, Office of Research and Development, Biomedical Laboratory Research and Development, Grant BX000893 and IK2BX002240, (3) the Edna and Fred L. Mandel Center for Hypertension and Atherosclerosis Research, and (4) a Grant-in-Aid and Postdoctoral Fellowship from the American Heart Association (12POST11910012).

Footnotes

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