Bioelectronic Approaches to Control Neuroimmune Interactions in Acute Kidney Injury

Abstract

Recent studies have shown renal protective effects of bioelectric approaches, including ultrasound treatment, electrical vagus nerve stimulation, and optogenetic brainstem C1 neuron stimulation. The renal protection acquired by all three modalities was lost in splenectomized mice and/or α7 subunit of the nicotinic acetylcholine receptor–deficient mice. C1 neuron-mediated renal protection was blocked by β2-adrenergic receptor antagonist. These findings indicate that all three methods commonly, at least partially, activate the cholinergic anti-inflammatory pathway, a well-studied neuroimmune pathway. In this article, we summarize the current understanding of neuroimmune axis-mediated kidney protection in preclinical models of acute kidney injury by these three modalities. Examination of the differences among these three modalities might lead to a further elucidation of the neuroimmune axis involved in renal protection and is of interest for developing new therapeutic approaches.

Acute kidney injury (AKI) is a common global health problem and it is associated with high morbidity and mortality. There are few pharmacological options to prevent and/or treat kidney injury; therefore, new approaches are needed. The evolving concept on neural control of inflammation provides a fresh look and new opportunity to intervene with nonpharmacological methods and bioelectronic approaches to reduce injury in AKI. Elucidation of mechanisms related to renal protection through neuroimmune system interactions is progressing rapidly, and the neuroimmune system-mediated kidney protection activated by three different modalities (pulsed ultrasound [US], vagus nerve stimulation [VNS], and brainstem C1 neuron stimulation) has been reported (Table 1). In this article, we show the current understanding of neuroimmune interactions in AKI and discuss the issues that remain to be investigated in the future.

Table 1.

Summary of bioelectronic and other approaches to control neuroimmune interactions in acute kidney injury

Modalities	Advantages	Disadvantages
Ultrasound	Noninvasive	Uncertain mechanism
Electrical stimulation	Technically easy	Invasive, nonselective
Optogenetic stimulation	Selective stimulation	Invasive, technically difficult
Drugs (nicotine, GTS-21)	Easy clinical application	Off-target effects

ACUTE KIDNEY INJURY

AKI is defined as a sudden (within 48 hours) reduction in kidney function based on elevated serum creatinine levels and decreased urinary output. The incidence of AKI has increased recently, both in the community and in hospital settings (Nash et al. 2002; Hsu et al. 2007). The estimated incidence of AKI is two to three cases per 1000 persons (Hoste and Schurgers 2008). AKI occurs in about 7% of hospitalized patients and about 67% of intensive care unit (ICU) patients, many of whom also have multiple organ dysfunction (Nash et al. 2002; Hoste et al. 2006). Recent advances in understanding AKI revealed that patients who completely recovered from AKI have a risk for developing chronic kidney disease (CKD) and end-stage renal disease (ESRD) as shown in animal models (for a review, see Venkatachalam et al. 2015 and Basile et al. 2016) and in humans (Jones et al. 2012). Around 10% of the population has CKD that might progress to ESRD that requires dialysis or kidney transplantation to survive and, indeed, more than two million patients worldwide require dialysis or kidney transplantation (Couser et al. 2011). Thus, treating and/or preventing AKI is important; however, few options exist despite extensive studies on the mechanisms of progression of kidney injury.

CAUSES OF AKI

Various factors such as infection, nephrotoxic drugs, and ischemia can cause AKI. The causes of AKI can be clinically divided into three categories despite difficulties in distinguishing them or multifactor causes: prerenal (caused by decreased renal perfusion such as dehydration, hypotension, and bleeding), intrinsic renal (caused by acute tubular necrosis, nephrotoxic drugs, sepsis, glomerulonephritis, interstitial nephritis, and cholesterol embolism, etc.), and postrenal (caused by urinary obstruction). In patients who already have underlying CKD, any of these factors may cause AKI in addition to the chronic impairment of renal function.

ANIMAL MODELS OF AKI

As shown above, the etiology for induction of kidney injury is multifold; therefore, a large number of animal models have been developed to mimic the clinical conditions of AKI. Ischemia-reperfusion injury (IRI) simulates the hemodynamic-induced changes in renal function that may occur in situations such as kidney transplantation and cardiac surgery. Drug-induced AKI, including aminoglycoside, cisplatin, and nonsteroidal anti-inflammatory drug (NSAID)-induced injury, mimics the AKI because of clinical administration of these respective drugs. Sepsis-induced AKI mimics the infection-induced AKI, and radiocontrast-induced AKI mimics renal failure in patients during use of radiocontrast media for various reasons such as cardiac catheterization and enhanced computerized tomography (CT) scan. Glycerol-induced kidney injury mimics rhabdomyolysis (Heyman et al. 2009; Singh et al. 2012). In this review, we focus on the IRI model, one of the most commonly used animal models in the AKI field.

THE ROLE OF IMMUNE CELLS IN AKI

Although IRI is a model of aseptic AKI, both innate and adaptive immune systems are directly involved in the pathogenesis of ischemic AKI. Renal ischemia-reperfusion mainly causes inflammation in the outer medulla, which is the part of the kidney with the lowest oxygen tension, leading to the highest degree of epithelial cell necrosis. Mechanical disruption of renal vascular endothelial integrity caused by IRI and the consequent increase in vascular permeability promotes immune cell infiltration into ischemic kidney after IRI. Damage-associated molecular patterns (DAMPs) released from dying cells, adhesion molecules, hypoxia-inducible factors (HIFs), and Toll-like receptors (TLRs) induce the recruitment of various immune cells during the early injury stage (Jang and Rabb 2015). Neutrophils, natural killer (NK) cells, and natural killer T (NKT) cells are recruited within hours of tissue injury, and the inflammatory cascade and release of proinflammatory molecules, such as interleukin (IL)-1, IL-6, and tumor necrosis factor (TNF)-α, are initiated by these immune cells. NK cells kill tubular epithelial cells and directly contribute to kidney damage after IRI. Complement system activation and proinflammatory cytokine and chemokine production enhances leukocyte infiltration into postischemic regions in the kidney (Jang and Rabb 2009). The number of renal dendritic cells increases in the injury site, and the dendritic cells mediate inflammation from the early to late injury phase. Macrophages also play roles in both innate and adaptive immune systems. Activated macrophages show strong phagocytic function and secrete several important cytokines. Inflammatory monocytes infiltrate damaged sites after infiltration of neutrophils and differentiate into macrophages. Then the differentiated macrophages are polarized into proinflammatory macrophages (M1) after the injury and enhance tissue damage. In the recovery phase, anti-inflammatory macrophages (M2) predominate in the damaged tissue and contribute to resolution of inflammation and tissue repair (Lee et al. 2011; Cao et al. 2015; Huen and Cantley 2015, 2017). Regulatory T cells (Tregs) exert renal protective function and play an important role in tubular regeneration to promote repair from IRI (Kinsey et al. 2013; Kinsey and Okusa 2014). B cells are activated during the injury stage, limiting tubular regeneration during the recovery phase and causing tubular atrophy (Jang et al. 2010).

PHARMACOLOGICAL THERAPY OF AKI

Regardless of advances in understanding the mechanism of AKI, limited treatment options for AKI are available and its management is primarily supportive. The key objective of management is assuring adequate renal perfusion by achieving and maintaining hemodynamic stability and avoiding hypovolemia. Monitoring electrolyte imbalances such as hyperkalemia and metabolic acidosis is important. All medications that may potentially damage the kidney, such as kidney toxic drugs or drugs that might reduce renal blood flow, should be discontinued if possible. Patients with rapidly progressive glomerulonephritis might need to be treated with pulse steroids and/or other immunosuppressive drugs often after confirmation of the diagnosis by kidney biopsy (Walters et al. 2015). In some patients, renal-replacement therapy such as hemodialysis will be required. Thus, new treatment options are greatly needed, and bioelectronics approaches are very attractive as nonpharmacological treatments.

NEUROIMMUNE INTERACTION THAT CONTROL INFLAMMATION

There are several known pathways linking the nervous and immune systems. The hypothalamic–pituitary–adrenal (HPA) axis is one of the well-known neuroimmune interactions; neuropeptides are secreted by nerve terminals and have both pro- and anti-inflammatory effects. Additionally, recent advances have shown that communication between the nervous and immune systems plays an important role in the regulation of immune function and inflammation. The inflammatory reflex is required for rapid homeostatic responses to inflammation, and the reflex mediates organ protection (Tracey 2002). The nervous system, including afferent and efferent pathways, is crucial for transmitting signals between neurons and from neurons to receptors and their effectors. Binding of chemical substances released in the inflammatory response to their receptors on sensory nerve terminals elicits transmission of neuronal signals to the brain via the afferent and somatosensory spinal nerve of the vagus nerve (Hosoi et al. 2005). The output signal is then transmitted to the effector via the efferent nervous system, which includes both the sympathetic and parasympathetic (e.g., vagus nerve) systems (Hosoi et al. 2005). This transmission is mediated by neurotransmitters such as acetylcholine (ACh) released by the vagus nerve and norepinephrine (NE) released by sympathetic nerves. As part of the inflammatory reflex, subsequent modulation of immune cell function underlies the regulation of inflammation (Tracey 2007).

Overview of the Cholinergic Anti-Inflammatory Pathway (CAP)

One of the well-studied neuroimmune pathways is the pathway called the cholinergic anti-inflammatory pathway (CAP), which is the efferent limb of the “inflammatory reflex pathway” mediated through the vagus nerve. This concept was first described by Tracey and co-workers using a lipopolysaccharide (LPS) model of inflammation (Borovikova et al. 2000; Tracey 2002, 2007, 2010; Huston et al. 2006; Pavlov and Tracey 2017). Receptors expressed on peripheral vagal afferent neurons bind bacterial products (pathogen-associated molecular patterns [PAMPs]), DAMPs, proinflammatory cytokines, immunoglobulins, and ATP (Niijima et al. 1995; Niijima 1996; Ek et al. 1998; Brouns et al. 2000; Page et al. 2000; Hermann and Rogers 2008; van der Kleij et al. 2010; Steinberg et al. 2016). Following detection of these inflammatory molecules at the receptors, signals from injured tissue and the immune cells are transmitted to a brain region called the nucleus tractus solitarius in the brainstem (Borovikova et al. 2000; Saeed et al. 2005). Through undefined mechanisms, the vagus efferent nerve is activated. Then the inflammatory reflex controls peripheral cytokine levels and inflammation through key cellular components such as macrophages and CD4⁺ T cells. Indeed, VNS shows anti-inflammatory and organ protective effects in various disorders, such as arthritis (McAllen et al. 2015), colitis (Goverse et al. 2016), ileus (Goverse et al. 2016), pancreatitis (van Westerloo et al. 2006), diabetes (Wang et al. 2014), hypertension (Carnevale et al. 2016), heart disease (Bernik et al. 2002), and kidney disease (Inoue et al. 2016).

Macrophages in the CAP

Macrophages were initially described as key components of the CAP. Tracey and coworkers found that direct electrical stimulation of the efferent vagus nerve significantly decreased the LPS-induced TNF-α expression in the serum (Borovikova et al. 2000). The TNF-mediated suppressive effect induced by VNS was abolished if the α7 subunit of the nicotinic acetylcholine receptor was deleted (α7nAChRKO). Although α7nAChR is predominantly expressed in neuronal tissues, it is also found on macrophages as shown histologically and functionally (Wang et al. 2003). ACh or nicotine, an agonist for the nicotinic receptor, suppressed LPS-induced TNF-α production in peritoneal macrophages, and the suppression was lost in α7nAChRKO-derived peritoneal macrophages (Wang et al. 2003). After activation of α7nAChR, inhibition of the nuclear translocation of nuclear factor (NF)-κB (Wang et al. 2004) and activation of the JAK2–STAT3 pathway (de Jonge et al. 2005) occur and ultimately reduce the production of inflammatory mediators.

CD4 T Cells in the CAP

CD4 T cells play important roles in the anti-inflammatory effect in the CAP. The role of β2-adrenergic receptors on CD4 T cells in the CAP was revealed by Vida et al. (2011). They showed that the β2 antagonist butoxamine efficiently abolished the VNS-mediated TNF-α suppression induced by LPS. The anti-inflammatory effect of VNS was lost in β2-adrenergic receptor-deficient mice, and the transfer of CD4⁺CD25⁻ cells (nonregulatory T cells), but not CD4⁺CD25⁺ cells (Tregs), from wild-type (WT) mice rescued the VNS-mediated TNF-α suppressive effect (Vida et al. 2011). These results suggest that β2-adrenergic receptors on CD4⁺CD25⁻ T cells contribute to the anti-inflammatory effect of the CAP.

Role of Spleen in the CAP

The spleen is a crucial organ in the CAP. Huston et al. (2006) revealed that the spleen is the major source of TNF-α after LPS exposure and the inhibition of systemic TNF-α production by VNS was lost in splenectomized animals. The splenic nerve, which is a component of the sympathetic (adrenergic) nervous system, releases NE, and the vagus nerve, which is a component of the parasympathetic (cholinergic) nervous system, releases ACh at nerve endings upon activation. In response to VNS, ACh levels in the spleen are up-regulated within 20 min (Rosas-Ballina et al. 2011) and plasma NE levels increase at 15 min (Vida et al. 2011) after stimulation. Splenic sympathetic nerves innervate spleen in rodents, but in the spleen little or no direct (cholinergic) innervation from the vagus was found (Bellinger et al. 1993; Nance and Sanders 2007). Rosas-Ballina et al. (2011) found that CD4⁺CD44^highCD62L^low cells express choline acetyltransferase ([ChAT], the ACh biosynthetic enzyme) and can release ACh in the spleen, and these cells can be the source of ACh for ACh receptor-positive macrophages during activation of the CAP. Catecholaminergic terminals of the splenic nerve in the white pulp of the spleen are located very close to lymphocytes, including ChAT-positive T cells. This anatomic relationship might permit the activation of ChAT⁺ CD4 T cells through splenic sympathetic nerve stimulation. Although the anti-inflammatory effects of VNS are lost in nude mice, which do not have functional T cells, reconstitution of nude mice with ChAT⁺CD4⁺ T cells partially restores the VNS-induced anti-inflammatory effect (Rosas-Ballina et al. 2011). The protective effect of VNS is lost in nude mice receiving spleen CD4⁺ T cells transfected with ChAT small interfering RNA (siRNA) (Rosas-Ballina et al. 2011). These studies provide a mechanism by which ACh levels increase in the spleen after VNS despite the lack of direct vagal innervation of the spleen. But what has not yet been determined is how NE is released in the spleen (or systemically) after VNS. In other words, the interaction between vagus (parasympathetic, cholinergic) and splenic (sympathetic, adrenergic) nerves in the CAP should be clarified in the future.

ULTRASOUND

US is a safe, noninvasive, and painless procedure widely and routinely used in clinical settings especially for imaging. US is based on the use of high-frequency sound waves ranging from 2 to 15 MHz. During US imaging, high-frequency sound waves are transmitted from a small transducer (probe) through a US gel applied directly to the skin. Sound waves that bounce back from insonated tissue are collected by the transducer, and the computer uses the sound waves to create an image. US images, which are captured in real time, can show the structure and movement of internal organs, as well as blood flow.

US Activates the CAP and Protects the Kidney

US was described as the first bioelectronic approach to activate the CAP and reduce AKI. Gigliotti et al. (2013, 2015) found that prior US application (1 sec every 6 sec for 2 min focused on both kidneys and including spleen, mechanical index of 1.2) suppressed systemic and local (renal) inflammation such as IL-6 and TNF-α and attenuated AKI. US-mediated protection lasts up to 2 days before IRI (Gigliotti et al. 2013). A clinical US machine was employed in these mouse studies, as shown in Figure 1. Several findings resulted from studies examining the mechanism of US-mediated kidney protection. The crucial role of spleen in US-induced renal protection was proved by experiments with splenectomized mice (Gigliotti et al. 2013). US also protects kidneys in a cecal ligation and puncture-induced sepsis model (Gigliotti et al. 2015). The renal protective effect by prior US treatment was lost in Rag1-deficient mice (Rag1KO) that lack T and B lymphocytes. However, reconstitution of Rag1KO mice with CD4 T cells 10 days prior to US treatment restores the protective effect of US (Gigliotti et al. 2013). In addition, adoptive transfer of splenocytes from US-treated mice 24 h prior to IRI protected the kidney of the recipient mice from IRI (Gigliotti et al. 2015). These data suggest that US-mediated renal protection requires CD4 T cells in the spleen. The protective effects of US prior to IRI are mediated through α7nAChR signaling in the CAP, as the effect was lost in α7nAChRKO mice (Gigliotti et al. 2013). The importance of α7nAChRs on hematopoietic cells in US-related organ protection were further revealed by bone marrow chimera experiments (Gigliotti et al. 2015). Taken together, these findings indicate that the renal protective effect of US occurs through activation of the CAP. This also implies that there is a possibility that US might be useful for organ protection in humans such as kidney transplantation and cardiac surgeries. The mechanism underlying US-induced activation of the CAP is still unknown, but additional studies might accelerate our understanding and expedite bioelectronic medicine.

Figure 1.

Ultrasound (US) treatment in mice. US treatment is applied to the mice as shown in the left panel. After mice were anesthetized, fur was shaved and removed using a depilatory. Mice were then placed on a modified microscope stage, which was positioned under a US transducer held in place with a ring clamp. Prewarmed US gel was then placed on the depilated skin for US application. Mouse body temperature was monitored via rectal probe and maintained at 36.5°C with a heating pad and heat lamp. Top right panel shows the protocol for US treatment and renal ischemia-reperfusion injury (IRI). One day prior to IRI, US was applied to the mice, then IRI was performed 1 day after IRI. Bottom right panel shows the image where US was applied. This is an example of the left side of kidney. The enclosed area in red is the actual targeted region including kidney and spleen. US pulses (1 sec every 6 sec for 2 min) were applied to both kidneys including spleen. Control animals underwent the same preparation procedures but were not exposed to US pulses.

VAGUS NERVE STIMULATOR

For medically refractory epilepsy and treatment-resistant depression, more than 100,000 vagus nerve stimulators have been implanted, and the devices have been well-tolerated (Ben-Menachem et al. 2015). Although transvenous VNS failed to suppress systemic inflammation in healthy subjects (Kox et al. 2015), pilot studies revealed the efficacy of implanted vagus nerve stimulators in rheumatoid arthritis (Koopman et al. 2016) and Crohn’s disease (Bonaz et al. 2016) in humans. During the VNS treatment period, symptoms were significantly improved in 17 patients with rheumatoid arthritis, including patients in the early and late stages of the disease (Koopman et al. 2016). In seven patients with Crohn’s disease, VNS improved biological parameters evaluated at a 6-month follow-up (Bonaz et al. 2016). In addition, many other clinical trials are ongoing on a wide variety of disorders such as diabetes, hypertension, heart failure, and inflammation. Two noninvasive external devices to stimulate the vagus nerve through the skin have been developed (Ben-Menachem et al. 2015). In rats, the degree of tissue damage after cerebral ischemic injury was reduced with transcutaneous cervical VNS (Ay et al. 2016). In healthy human subjects, the release of inflammatory cytokines, such as IL-1β and TNF-α was down-regulated by VNS using a transcutaneous device on the neck (Lerman et al. 2016).

VNS Protects the Kidney from IRI by Activating the CAP

Inoue et al. (2016) revealed the protective role of electrical stimulation of the vagus nerve in the kidney field for the first time. The protection was not observed when the mice received VNS 10 min prior to IRI; however, when VNS was applied 24 h prior to renal ischemia and reperfusion, the kidneys were protected against IRI (Inoue et al. 2016). The protective effect was lost in α7nAChR-deficient mice and splenectomized mice. In addition, they found that adoptive transfer of splenocytes from VNS-treated α7nAChR^+/+ mice protected kidneys of recipient mice from IRI. On the other hand, the protection was lost in recipient mice receiving splenocytes from VNS-treated α7nAChR^−/− mice, thus demonstrating that protection requires α7nAChRs. They also observed a phenotypic change of macrophages in the kidney after VNS in α7nAChR^+/+ mice consistent with the observation that after IRI, inflammatory monocytes enter into the kidney and undergo a phenotypic change from M1 (early proinflammatory) to M2 (late “healing”) macrophage (Lee et al. 2011; Huen and Cantley 2017). Prior VNS suppressed the expression of Arg1 in macrophages in α7nAChR^+/+ mice, but the suppression was not observed in α7nAChR^−/− mice. Taken together, VNS protected the kidney through the phenotypic change of macrophages activated through the CAP.

Other CAP-Related New Findings in the Kidney Field

GTS-21 is a derivative of the natural product anabaseine, a selective α7nAChR agonist. This is a relatively safe drug candidate in humans and has been shown to significantly improve cognitive function and attention and is being investigated for the treatment of diseases such as Alzheimer’s disease and schizophrenia (Kitagawa et al. 2003). Very recently, the activation of the CAP by GTS-21 was reported to attenuate cisplatin-induced AKI in mice (Chatterjee et al. 2017). Cisplatin is a highly effective drug for cancers but is also nephrotoxic as mentioned above. This new finding with GTS-21 is important clinically, because AKI is observed in approximately 20%–30% of patients receiving cisplatin, thus requiring dose reduction or discontinuation (Miller et al. 2010), which compromises their chemotherapy. There is also one unique report about the direct role of α7nAChR on kidney cells. As renal IRI mainly induces damage in proximal tubules of the outer medulla, the protection of proximal tubular cells in the progression of AKI is essential. Activation of α7nAChR by GTS-21 was reported to attenuate ischemic AKI. Kim et al. (2018) found that heme oxygenase-1 (HO-1) expression is induced and inflammatory cytokine levels are decreased in proximal tubule by GTS-21. The degradation of heme to biliverdin, carbon monoxide, and free iron that are up-regulated under oxidative stress are catalyzed by HO-1 (Maines 1988). In myeloid cells and endothelial cells, HO-1 induction enhances cytoprotection against apoptotic cell death through its anti-inflammatory properties (Paine et al. 2010; Ryter and Choi 2016). In macrophages, HO-1 mediates a protective effect induced by α7nAChR stimulation (Tsoyi et al. 2011). Thus, HO-1 induction in proximal tubules through α7nAChR activation might play a critical protective role in kidney injury in addition to the importance of HO-1 in macrophages. In a very different approach, Ray et al. (2018) showed that oral NaHCO₃ administration changes the polarizing phenotype of macrophages from predominantly M1 (inflammatory) to M2 (regulatory), and FOXP3⁺CD4⁺ T-lymphocytes increased in the spleen, blood, and kidneys of rats. This change could be related with the CAP and oral NaHCO₃ might be used as an activator of the CAP.

Either Afferent or Efferent Vagus Nerve Stimulation Protects the Kidney

The findings mentioned above were obtained by stimulating the intact VNS (Inoue et al. 2016), but the vagus nerve bundle consists of both afferent and efferent fibers and it was unknown which fiber is important for the observed organ protection. To determine which component of vagus nerve (afferent or efferent) is essential for kidney protection, Inoue et al. performed selective electrical stimulation of either vagus afferents or efferents by cutting the vagus nerve and stimulating proximal or distal to the lesion (Fig. 2). Based on the accumulated evidence, efferent vagus nerve can activate the CAP and, indeed, stimulation of efferent vagus nerve protected the kidney from IRI. A little surprisingly, afferent stimulation also protected the kidney to the same extent as intact VNS (Inoue et al. 2016). This finding evoked further questions such as how afferent VNS shows organ protective effects. Based on the findings, there seem to be neuronal pathways in the brain that coordinate the CAP.

Figure 2.

Selective stimulation of vagus nerve with electricity. The left cervical vagus nerve was isolated via a midline cervical incision and placed on a bipolar silver wire electrode for stimulation. To perform intact vagus nerve stimulation (VNS), the nerve was left intact. To activate vagal afferents, the vagus nerve was cut and the central end was stimulated selectively. The peripheral end of the cut nerve was stimulated to activate vagal efferents.

OPTOGENETICS

Traditional methods for the functional analysis of neurons in the brain or peripheral nerves have relied on direct stimulation by tiny electrodes such as those used in electrical VNS shown above (Inoue et al. 2016). This method lacks precision in terms of temporal and spatial nerve stimulation. To probe the brain and reveal new circuits that regulate the CAP, optogenetics can be used to overcome the disadvantages of electrical stimulation. Optogenetics is a technique involving the use of light to control the activity of genetically modified neurons to express light-sensitive ion channels or pumps such as channelrhodopsin and halorhodopsin, respectively (Fig. 3) (Montgomery et al. 2016). These genetically encoded switches allow neurons to be turned on or off with the burst of light of a specific wavelength. The discovery of channelrhodopsin-1 (Nagel et al. 2002) and -2 (Nagel et al. 2003) (ChR1 and ChR2) from algae marked the genesis of optogenetics. Only 2 years after the discovery of ChR2, investigators introduced it in mammalian neurons, and they successfully activated ChR2-expressing neurons by blue light (Boyden et al. 2005). When ChR2 is stimulated by blue light (∼470 nm), the channel opens and functions as a nonselective cation channel that can depolarize the neuronal cell membrane and evoke an action potential. The utilization of halorhodopsin, an inhibitory channel, further expanded studies using optogenetics (Han and Boyden 2007). Yellow light (∼590 nm) is applied to neurons expressing halorhodopsin chloride ions to enter the cells, resulting in hyperpolarization and inhibition of neurons. By introducing an engineered viral vector containing the optogenetic gene into a specific Cre-expressing mouse or by crossing a mouse harboring the optogenetic gene with a Cre-expressing mouse, these excitatory or inhibitory proteins can be introduced into specific cell types. Thus, optogenetic techniques allow for targeted excitation or inhibition with cellular or projection specificity that cannot be achieved by electrical stimulation. This method was chosen as Method of the Year by Nature Methods in 2010 because it had a major impact on neuroscience research (Deisseroth 2011).

Figure 3.

Optogenetic neuron activation. channel rhodopsin, or halorhodopsin are introduced into specific neurons and used for optogenetic stimulation. When blue light is applied to neurons expressing channelrhodopsin (ChR2), the opsin functions as a nonselective cation channel, resulting in activation of the neurons. When yellow light is applied to halorhodopsin-expressing neurons, the chloride pump is activated, leading to inhibition of the neurons.

Recently, some interesting findings related with vagus nerve were reported using optogenetic methods (Chang et al. 2015; Williams et al. 2016; Nonomura et al. 2017). Specific markers identify several subgroups of vagal afferent sensory neurons, enabling selective introduction of ChR2 and optogenetic stimulation to dissect the role of these pathways in differentially modulating target organ function. Chang et al. (2015) found that selective activation of P2ry1-positive neurons in vagal afferents resulted in apnea, whereas the activation of Npy2r-positive neurons in vagal afferents caused rapid/shallow breathing. Williams et al. (2016) found that Gpr65-positive vagal sensory afferent neurons innervating intestinal villi detect nutrients and regulate gut motility, while Glp1r-positive vagal sensory neurons sense stomach and intestinal stretch. Nonomura et al. (2017) revealed the role of Peizo2 in vagal afferent fibers as an airway stretch sensor that is important for establishing efficient respiration. In a similar manner, future studies using optogenetic methods will likely facilitate elucidation of the selective neural circuits of the CAP.

C1 Neuron Stimulation Protects the Kidney from IRI

By using optogenetic stimulation (Fig. 4), a new role of brainstem C1 neurons in activating the CAP was revealed (Abe et al. 2017). C1 neurons are located in the medulla oblongata and innervate many regions of the brain, including the dorsal motor nucleus of the vagus nerve, the paraventricular nucleus of the hypothalamus, other brainstem regions, and sympathetic and parasympathetic preganglionic neurons in the periphery (Guyenet et al. 2013). Physiological roles of C1 neurons include regulation of reproduction (I’Anson et al. 2003), HPA-mediated stress responses (Fuzesi et al. 2007), food consumption (Verberne and Sartor 2010), body temperature (Madden et al. 2013), breathing (Burke et al. 2014; Wenker et al. 2017), and glucose homeostasis (Zhao et al. 2017). These neurons, when confronted with major acute stresses such as hypoxia, acute infection, blood loss, and hypotension, elicit responses to promote organism survival (Guyenet et al. 2013). As circulating IL-1 and LPS strongly activate subsets of C1 neurons (Li et al. 1996), C1 neurons were thought to be involved in the inflammatory reflex.

Figure 4.

Optogenetic C1 neuron stimulation. Initially, virus carrying ChR2 (AAV2-ChR2-mCherry) was injected into a specific region (left rostral ventrolateral medulla, location of C1 neurons) of the mouse (dopamine β-hydroxylase-cre mice; targeting adrenergic C1 neurons) and at the same time an optical fiber was implanted close to the target region. Five to 6 weeks later, the virus-injected target region (C1 neurons) expressed ChR2. After the implanted optical fiber and the laser were connected, light pulses (470 nm, 10 msec duration, 5 Hz) were delivered for 10 min.

Abe et al. (2017) showed that optogenetic C1 neuron stimulation by blue laser protects kidneys from IRI and that the spleen, β2-adrenergic receptors, and α7nAChRs are necessary for the protective effect. This suggests that the CAP is involved in the organ-protective effect induced by C1 neuron activation. Given the role of C1 neurons in stress-mediated responses, it was interesting to find that restraint stress for a period of only 10 min also protects the kidney from IRI. The protective effect by restraint stress is lost when C1 neurons are selectively inhibited by the Gi-coupled designer receptors exclusively activated by designer drugs (DREADDs) system or destroyed by caspase, meaning that C1 neurons are required for renal protection induced by restraint stress. Although ganglionic blockade abolished the protection by C1 neuron stimulation, subdiaphragmatic vagotomy or corticosterone receptor blockade did not affect C1 neuron-mediated renal protection. These findings indicate that C1 neurons activate the CAP through a sympathetic (adrenergic) route, but not through a vagal route.

CONCLUDING REMARKS

We summarized the current understanding of bioelectronic approaches to control neuroimmune interactions mainly in AKI (Fig. 5) (Inoue et al. 2017). Neuroimmune interactions play very important roles in various disorders including kidney diseases. Although the mechanisms linking these two different systems are very complex and incompletely understood, bioelectronic stimulations such as US treatment, electrical VNS, and optogenetic C1 neuron stimulation by laser showed a significant organ protection. These studies might lead to a powerful nonpharmacological approach to block inflammation and preserve organ function. Additional studies with advanced tools such as optogenetics will provide a further molecular understanding of neuroimmunomodulatory mechanisms that control inflammation and organ injury.

Figure 5.

Current understanding of the cholinergic anti-inflammatory pathway (CAP) in acute kidney injury. The activated cholinergic anti-inflammatory pathway links the nervous system and immune system, then protects the kidney from injury. The activity of afferent vagus nerve fibers is stimulated by cytokines and pathogen-associated molecular patterns (PAMPs). The signal activates efferent vagus nerve fibers through the nucleus tractus solitarius (NTS) and dorsal motor nucleus of the vagus (DMV) in the brain. The efferent vagus nerve (cholinergic) stimulates CD4 T cells in spleen via the splenic sympathetic (adrenergic) nerve. Release of norepinephrine (NE) binds to β2-adrenergic receptors (β2ARs) on CD4 T cells, which then elicits release of acetylcholine (ACh). ACh binding to α7 nicotinic acetylcholine receptors (α7nAChRs) on macrophages produces an anti-inflammatory response, such as tumor necrosis factor (TNF)-α suppression. Ultrasound and C1 neuron stimulation also activate the pathway, at least partially, not directly through vagus nerve and produce organ protective effects (Abe et al. 2017).