Inflammatory macrophages in the kidney contribute to salt-sensitive hypertension

Abstract

This review will highlight recent studies that have investigated the relationship between Na⁺, renal macrophage polarization, and renal damage. A hyperosmotic environment drives the macrophage toward a proinflammatory phenotype and away from an anti-inflammatory phenotype. Animal models of salt-sensitive hypertension demonstrate a characteristic infiltration of macrophages into the kidney that is greatly reduced when blood pressure is lowered. Because general immunosuppression or macrophage depletion leads to a host of adverse side effects, more recent studies have modulated the interaction of specific signaling molecules, including NOD-like receptor family pyrin domain-containing 3, chemokine (C-X-C motif) ligand 16, and VEGF, to prevent the end-organ renal damage that accumulates in salt-sensitive disease.

INTRODUCTION

The progressive nature of hypertensive renal disease is in part due to the continued accumulation of immune cells in the kidney that appear to play a pathogenic role (24, 29). In particular, increases in dietary salt drive these immune cells toward a proinflammatory phenotype (32), which further contributes to renal injury. Understanding the early events by which the immune system is activated, specifically innate immune system activation, is vital to understanding the pathogenesis of hypertensive renal injury and also provides therapeutic opportunities to break the cycle between renal injury and immune system activation. This brief review will cover recent studies that illustrate the responsiveness of the macrophage, an integral component of innate immunity, to dietary salt and the resultant renal damage. We will initially present evidence that a hypertonic environment induces a proinflammatory phenotype in the macrophage (termed “M1”). We will then explore the relationship between dietary salt and macrophage activation, particularly in the kidney, through findings from two commonly used animal models of salt-sensitive hypertension, the Dahl salt-sensitive (SS) rat and the deoxycorticosterone acetate (DOCA)-salt model.

NaCl AND MACROPHAGE POLARIZATION

In vitro work has shown that the macrophage is a cell type that is sensitive to the osmolarity of its surroundings (14). Increasing the concentration of either glucose or NaCl in the media enhances the production of the inflammatory cytokines IL-1β, IL1α, and IL-6 in response to LPS stimulation, which appears to be facilitated through a caspase-1-dependent mechanism, which, in turn, activates the NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome (14). The osmotic balance in the body is tightly regulated in tissue compartments such as the plasma, heart, and brain; however, localized increases in Na⁺, particularly in the skin, have been reported in close proximity to sites of injury (16). This provides a condition where immune cells may be exposed to a hypertonic environment. In conjunction with these results, further in vitro work showed that a high salt environment increases macrophage activation, specifically the release of TNF and nitric oxide through p38/MAPK and nuclear factor of activated T cell 5 (NFAT5) signaling. This increased proinflammatory response results in an enhanced antimicrobial capacity and clearance of infection (16). A more recent study (11) has shown that in patients with end-stage renal disease, there is no difference in dermal Na⁺ concentration compared with healthy patient controls, but there is a notable increase in CD68⁺ macrophages and CD3⁺ T cells in the skin. These studies continue to show a relationship between renal injury and inflammatory activation in ectopic compartments in the body, although it is still unclear if the osmolarity of the dermal tissue is responsible for immune cell activation and whether those immune cells are responsible for the increased inflammation seen in the kidney and vasculature.

In general, macrophages exist on a spectrum ranging from a proinflammatory (M1) to an anti-inflammatory (M2) phenotype (28). In accordance with a proinflammatory shift, a hyperosmotic environment hinders the macrophage’s ability to adapt an M2 phenotype and to suppress T cell proliferation (6). This shift in polarization is not an artifact of the in vitro environment, as a high-salt diet in an experimental model of automimmune encephlomyelitis caused enhanced LPS-induced production of IL-6 and TNF-α as well as increased expression of inducible nitric oxide synthase in splenic macrophages (12). These macrophages from high-salt diet-fed animals also showed increased surface expression of major histocompatibility complex class II (MHC II), CD80, CD86, and CD40, which are classic markers of enhanced macrophage activation (12). At the same time, the high-salt diet resulted in a reduction in the expression of M2 anti-inflammatory genes such as arginase 1 and CD206. The enhanced proinflammatory polarization resulted in an increased ability for macrophages to induce T cell activation and proliferation (12). A recent study by Casper et al. (8) investigated an interesting phenomenon where kidney transplant recipients on loop diuretics exhibited increased urinary tract infections, leukocyturia, and bacteriuria. In these patients, depletion of the salt gradient in the kidney was associated with a decrease in chemokine (C-C motif) ligand 2 (CCL2) and a shift toward the M2 macrophage phenotype in the kidney and an inability to fight off infection. These results demonstrate that in human populations, Na⁺ concentration in the kidney drives macrophages toward a proinflammatory phenotype.

MODELS OF SALT-SENSITIVE HYPERTENSION

Dahl SS Rats

The Dahl SS rat is a well-established preclinical model of salt-sensitive hypertension that recapitulates many of the phenotypes seen in human patients, including elevated blood pressure, renal damage, and renal immune cell infiltration (25). We have previously shown that macrophages accumulate around damaged glomeruli and medullary tubules when these animals are on a high-salt diet (27). More recently, using a flow cytometric technique, we demonstrated that the macrophage is the primary immune cell type to infiltrate into the kidneys in response to high salt, and these cells adopt a more M1-like, proinflammatory, phenotype (10). Wei et al. (33) recently performed an indepth time-course study to investigate the temporal pattern of renal damage in the Dahl SS rat in response to a high salt challenge. They demonstrated that alongside the progressive increase in blood pressure and histological renal injury, there was a continual increase in renal macrophages, clearly demonstrating sustained innate immune system activation in the kidney. A recent study by Ray et al. (31) demonstrated that the macrophage polarization in the kidneys of Dahl SS rats on a high-salt diet can be shifted from an M1 phenotype toward an M2 phenotype by oral sodium bicarbonate intake.

These results are similar to what is observed in salt-loaded spontaneously hypertensive rats, where lowering of blood pressure is associated with reduced renal macrophage infiltration and decreased proinflammatory cytokine production in the kidney (35). A similar result was recently observed in spontaneously hypertensive rats treated with nicotine, which further documented an attenuation in the rise of blood pressure and M1 macrophage infiltration by renal denervation and further demonstrated that ablation of this cell type, using an anti-CD161a⁺ antibody, from the body could also attenuate the elevation in blood pressure (30).

Various experimental manipulations to attenuate the salt-sensitive phenotype in the Dahl SS strain of animals have been associated with decreased renal macrophage infiltration. These manipulations include inhibition of the mammalian target of rapamycin complex 1 (mTorc1) pathway by rapamycin (20), treatment with hydrochlorothiazide (33), substitution of protein source in the diet (1), antagonizing chemerin chemokine-like receptor 1 via 2-(α-naphthoyl)ethyltrimethylammonium (1), treatment with an epoxyeicosatrienoic acid analog (13), sitagliptin treatment (7), or treatment with hydralazine, eplerenone, or tempol (17). Importantly, though, in high-salt diet-fed Dahl SS animals, when blood pressure is servo controlled to the left kidney, the number of infiltrating macrophages is similar to rats on a control diet (9). This indicates that the increase in dietary salt is not solely sufficient to induce immune cell infiltration but that increased renal perfusion pressure is necessary for renal immune cell infiltration and the associated renal injury. One of the more translational opportunities for future studies may be investigating how genetic factors may influence macrophages to adapt a proinflammatory phenotype. Dahl SS rat consomic and congenic panels may provide a valuable resource for teasing apart this very question (26).

DOCA Salt

Exogenous aldosterone administration induces a salt-sensitive phenotype with an increase in blood pressure accompanied by significant renal damage and sustained M1 macrophage infiltration, which is attenuated by spironolactone treatment (23). The aldosterone precursor DOCA can be used to stimulate Na⁺ and water reabsorption. When paired with uninephrectomy and a high-salt diet, this treatment causes an elevation in blood pressure and can be used as an alternative model of salt-sensitive hypertension. In rats, the renal damage observed in this model is associated with increased renal cytokines, including chemokine (C-X-C motif) ligand (CXCL)1, monocyte chemoattractant protein (MCP)-1, and IL-2 and robust renal macrophage infiltration (4). Investigators have demonstrated that renal denervation was able to prevent renal macrophage accumulation and resulted in a lower production of these proinflammatory cytokines. Additionally, they were able to detect the reduction in urinary cytokines after renal denervation (3). This reduction in blood pressure by renal denervation also seems to occur in the Dahl SS model but is not mediated by differential immune cell infiltration (2).

In the DOCA-salt model, the NLRP3 inflammasome is potently activated in the kidneys of mice and knockout of the NLPR3 complex protein ASC (18) as well as treatment with the complex inhibitor MCC950 (19) attenuates the salt-sensitive increases in blood pressure, renal damage, renal inflammatory cytokines, and macrophage recruitment. A study by Krishnan et al. (19) demonstrated that in contrast to the rat, DOCA-salt treatment in these C57BL/6J mice does not induce further M1 macrophage infiltration but rather leads to the recruitment of M2 macrophages into the kidney, even though M1 macrophages continue to outnumber M2 cells 2:1. CXCL16 is an additional cytokine upregulated in the kidney during DOCA-salt treatment, and CXCL16-deficient animals are protected from DOCA salt-induced renal damage and macrophage infiltration; interestingly, however, they are not protected from the increase in blood pressure (21). The NLRP3 complex and CXCL16 are two promising targets for preventing the end-organ renal damage associated with salt-sensitive hypertension. The studies described here demonstrate that, with respect to macrophage activation in the kidney, there appears to be stark mechanistic differences between the responses of mice and rats to DOCA salt. The presence of proinflammatory macrophages in the kidney of rats, which is absent in mice, may contribute to the observation that mice are more resistant to renal damage in various models of chronic kidney disease (34). This speaks to concerns in reproducibility of findings across experimental models, and there is a desperate need for confirmation in humans with salt-sensitive hypertension before consideration of any of these targets for therapeutic development.

DISCUSSION

This brief review has highlighted that an increased extracellular osmolarity drives the macrophage toward a proinflammatory state, increased renal perfusion pressure drives macrophages into the renal interstitium, and infiltrating macrophages increase renal damage through production of proinflammatory cytokines (Fig. 1). Unfortunately, all the presented animal experiments were performed exclusively in male animals, which raises an important caveat because there are distinct sexual dimorphisms in innate immune system activation (15). The association between renal macrophage infiltration and salt-sensitive hypertension and renal damage is undeniable. Because servo control of pressure prevents this infiltration even when salt-sensitive animals are on a high-salt diet, it appears that renal macrophages may not play a role in the initiation of hypertension but contribute to the progressive nature of the disease. The primary question moving forward is whether intervention, postinsult, can promote repair, removal of cellular debris, and restoration of homeostasis. In the salt loading of BALB/C mice, VEGF-C plays a protective role in reducing renal inflammation and macrophage recruitment, possibly due to its ability to induce substantial lymphangiogenesis (5). The role of the lymphatics in regulating the inflammatory state of renal tissue is becoming more evident. Kidney-specific VEGF-D-overexpressing mice demonstrate a dramatic augmentation of lymphatic density in the kidney, which not only reduces immune cell infiltration, including macrophages, but also completely eliminates the salt-induced increases in blood pressure after N-nitro-l-arginine methyl ester treatment (22). Although nonselective immunosuppression is ill advised, studies identifying specific therapeutic targets provide attractive opportunities to lessen disease burden and prevent progression of end-organ damage.

Fig. 1.

Increases in dietary salt can alter tissue osmolarity, which causes macrophages to shift to a more proinflammatory state. Additionally, dietary salt causes an increase in renal perfusion pressure and immune cell infiltration. These infiltrating macrophages contribute to renal damage and the production of proinflammatory cytokines. The altered fluid/electrolyte balance due to renal damage causes a further increase in blood pressure, creating a positive feedback loop and further macrophage infiltration. CCL2, chemokine (C-C motif) ligand 2; CXCL16, chemokine (C-X-C motif) ligand 16; MCP1, monocyte chemoattractant protein 1; MHC II, major histocompatibility complex class II.

GRANTS

This work was supported by National Heart, Lung, and Blood Institute Grants F31-HL-144084, HL-137748, and HL-116264 and by the Georgia Research Alliance.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

D.J.F. prepared figures; D.J.F. drafted manuscript; D.J.F. and D.L.M. edited and revised manuscript; D.J.F. and D.L.M. approved final version of manuscript.