Ultrasonic Stimulation of the Cholinergic Anti-Inflammatory Pathway for Renal Protection

AKI encompasses a large spectrum of pathophysiological processes starting from the early reversible proximal tubular lesion to the definite loss of glomerular filtration and even death. Even discrete AKI (i.e., ≥0.3 mg/dl increase in serum creatinine in humans) has been reported to be associated with severe outcomes, such as an overall increased risk for longer length of hospital stay, ESRD, and mortality.¹ Renal ischemia-reperfusion injury (IRI) is a common process leading to AKI.² Renal IRI can develop after kidney transplantation and cardiopulmonary bypass, and it contributes to the main pathophysiological processes that occur during sepsis-, contrast- and rhabdomyolysis-induced renal injuries.

The past decade has seen remarkable progress in our understanding of the immunologic responses associated with renal IRI. In particular, innate immune responses mediated through infiltrating and resident immune effector cells (neutrophils, macrophages, and myeloid cells but also tubular cells) have been shown to play a major role very early in the course of renal IRI.³ In addition, converging evidence has suggested that successive AKI episodes can lead to interstitial fibrosis in experimental models, and successive episodes are now recognized as an independent risk factor for CKD, ESRD, and death.¹ Despite an overwhelming increase in AKI burden, neither prophylactic nor therapeutic strategies have proven effective. Therefore, the need to develop reliable strategies for prevention of AKI as early as possible is crucial.

Since the sixties, it has been reported that electrical stimulation of the vagus nerve enhances splenic release of acetylcholine (reviewed in reference 4). These seminal works have unveiled the concept of a brain–immune system route behind the so-called inflammatory reflex and the cholinergic anti-inflammatory pathway (CAP) endowed with important immunoregulatory properties. Along this regulatory pathway, acetylcholine is not released by the vagus nerve itself but by a particular CD4+ T cell subset that expresses β2-adrenergic receptors (β2-ARs) and choline acetyltransferase.⁵ Indeed, inflammatory cytokines and endogenous danger-associated molecular patterns (DAMPs), for example, after organ ischemia-reperfusion or exogenous pathogen-associated molecular patterns (PAMPs) trigger signals in the afferent vagus nerve that are transmitted to the brain (i.e., hypothalamus and the brain stem) before being redirected through the vagus efferent nerve into the celiac ganglion. From this last relay, adrenergic fibers extend to the spleen within neuro-immune synapse-like structures. Accordingly, on vagus nerve stimulation, adrenergic endings from the celiac ganglions stimulate the β2-AR⁺CD4⁺ T cells to secrete acetylcholine. In the splenic red pulp and the marginal zone, acetylcholine mainly acts on activated macrophages through cell surface homopentameric α-7 nicotinic acetylcholine receptors (nAchRs). The α7nAchRs are ionotropic cation channels, highly permeable to extracellular Ca2+, that rapidly open in the presence of acetylcholine or pharmacological agonists (i.e., nicotine, GTS-21 [3-(2,4-dimethoxybenzylidene)-anabaseine dihydrochloride], and cytisine). At the cellular level, α7nAchR activation suppresses NFκB translocation into the nucleus and the subsequent release of pro-inflammatory molecules (e.g., TNF-α, IL-1b, IL-6, IL-18, and high-mobility group box-1 [HMGB1]).⁶ The Jak2–Stat3 signaling pathway operates downstream of the α7nAchR, and IL-10 release seems unaffected.⁷

In addition to DAMPs and PAMPs, inflammatory cytokines (e.g., IL-1b) also stimulate the vagus afferent nerve endings.⁶ Importantly, this inflammatory reflex is a key factor in the maintenance of immunologic homeostasis, preventing nonresolving inflammation after a variety of insults. Clearly, the cholinergic anti-inflammatory pathway depends on the expression of the α7nAchR. Indeed, α7nAChR^−/− mice display exquisite sensitivity to small doses of lipopolysaccharide compared with wild-type animals in experimental endotoxemia models.⁸ In addition, the protective effect of vagus nerve stimulation is lost during experimental endotoxemia in α7nAChR^−/− mice. Similarly, α7nAchR expression by macrophages is required to inhibit TNF synthesis by electrical stimulation of the vagus nerve.⁹ Biologically or anatomically, dysfunctional CAP is associated with chronic immune-mediated diseases, such as rheumatoid arthritis and inflammatory bowel disease.⁴ Accordingly, this homeostatic neuro-immune mechanism finely tunes the magnitude of many inflammatory responses and protects the host from tissue damage or life-threatening conditions, such as endotoxemia, peritonitis, colitis, and acute pancreatitis.¹⁰

Since the first description of α7nAChR expression by macrophages, additional cells have also been shown to be sensitive to acetylcholine. For instance, kidney cells (i.e., proximal tubular epithelial cells, glomerular endothelial cells, and mesangial cells) express several nAChR subunits.¹¹ In a mouse model of LPS-induced AKI, the administration of nicotinic agonists (i.e., nicotine or GTS-21) significantly decreased proinflammatory cytokine release (e.g., TNF-α, CCL-2 [Chemokine (C-C motif) ligand 2], and CXCL10 [C-X-C motif chemokine 10]) by renal cells. This nicotine-dependent regulation was associated with milder AKI and dependent on the inhibition of the kidney proteasome.¹¹ Interestingly, significant protection against experimental renal IRI can be achieved through either electric stimulation of the vagus nerve or administration of cholinergic agonists, such as nicotine and GTS-21.^12,13

In the current issue of JASN, Gigliotti et al.¹⁴ elegantly address the importance of CAP stimulation before renal IRI. In a simple but revealing first experiment, Gigliotti et al.¹⁴ show that a noninvasive ultrasound (US), which stimulates CAP through splenic afferences, efficiently prevents AKI induced by mild renal IRI in mice. The US stimulation of splenic nerves efficiently triggers the inflammatory reflex, which was shown by a significant reduction in leukocyte infiltrate (e.g., neutrophils and myeloid cells) and a reduction in IL-6 mRNA transcription. This result was also associated with a significant reduction in interstitial fibrosis, suggesting that this method may prevent the natural course of AKI to CKD and ESRD. This ultrasonic renal protection against IRI was strongly dependent on the presence of splenic CD4+ T cells and α7nAchR-expressing cells, confirming the involvement of CAP in the modulation of the course of AKI. Accordingly, splenectomy abrogated any protection against AKI, attesting to the important role played by the spleen in the CAP. The results by Gigliotti et al.¹⁴ are closely in line with previously published work from the same group and are supported by other works on IRI.^12,13

Clearly, it is important to consider what relevance this study may hold for human health. The US experimental strategy carried out by Gigliotti et al.¹⁴ in the mice is readily feasible in human subjects and does not involve the use of a pharmacological agent. Indeed, many cholinergic pharmacological agonists exhibit a narrow therapeutic index, limiting their clinical use. Importantly, the noninvasive US regimen relies on US settings within approved Food and Drug Administration guidelines. Opportunities arising from the work by Gigliotti et al.¹⁴ are numerous and promising, because many procedures that carry a very high risk of AKI, such as cardiac surgery with cardiopulmonary bypass, iodinated contrast intravenous injection, or renal conditioning, are planned before transplantation. In renal transplantation, enhanced IRI significantly contributes to delayed graft function, which affects the future of the transplanted organ.¹⁵ Moreover, brain death is associated with the termination of the CAP, which significantly contributes to premortem systemic inflammation and worsening of IRI in potential transplanted end organs.¹⁶ Other nonrenal diseases could be modulated through CAP stimulation (e.g., myocardial ischemia, hepatic injury, sepsis, and endotoxemia), significantly expanding the potential range of application of this method. Finally, in searching for novel approaches to prevent and even cure AKI, we believe that splenic US stimulation has a bright future ahead.

Disclosure

None.