Roles of sensory receptors in non-sensory organs: the kidney and beyond

Abstract

Olfactory receptors (ORs), taste receptors and opsins are well-known for their pivotal roles in mediating the senses of smell, taste and sight, respectively. However, in the past two decades, research has shown that these sensory receptors also regulate physiological processes in a variety of non-sensory tissues. Although ORs, taste receptors and opsins have all been shown to have physiological roles beyond their traditional locations, most work in the kidney has focused on ORs. To date, renal ORs have been shown to have roles in blood pressure regulation (OLFR78 and OLFR558) and glucose homeostasis (OLFR1393). However, sensory receptors remain drastically understudied outside of traditional sensory systems, in part because of inherent challenges in studying these receptors. Increased knowledge of the physiological and pathophysiological roles of sensory receptors has the potential to substantially improve understanding of the function of numerous organs and systems, including the kidney. In addition, most sensory receptors are G protein-coupled receptors, which are considered to be the most druggable class of proteins, and thus could potentially be exploited as future therapeutic targets.

Introduction

Olfactory receptors (ORs), taste receptors (TRs) and opsins (OPNs) are primarily known for their key roles as sensory receptors that mediate the senses of smell, taste and sight. Incredible diversity exists within the sensory systems, due in large part to their crucial roles in shaping how organisms perceive the outside world¹. ORs, TRs and OPNs are distinct receptor types with differential sensing and downstream signalling.

ORs were first identified in 1991 (ref. 2) and have since been shown to comprise >1,000 genes in rats and mice and ~350 genes in humans, making them the largest gene family in the genome³. All mammalian ORs are G protein-coupled receptors (GPCRs). In olfactory sensory neurons, OR-ligand binding activates the olfactory G protein (G_olf), which subsequently stimulates adenylate cyclase 3 (AC3) and increases cAMP levels. The influx of cAMP activates cyclic nucleotide-gated (CNG) channels, leading to an influx of ions and ultimately producing an action potential^4,5. Of note, mice cannot smell when either G_olf or AC3 is absent⁴.

Taste sensation can be divided into five basic taste qualities: sweet, bitter, salty, sour and umami⁶. GPCRs are involved in sensing sweet, umami and bitter tastes but not sour or salty taste⁷. Two different types of taste GPCRs have been identified: taste receptor type 1 (T1Rs) and taste receptor type 2 (T2Rs). T1Rs are class C GPCRs that function as obligate heterodimers between the T1R1, T1R2 and T1R3 subunits. The dimerization of T1R1 and T1R3 serves to detect umami, whereas the dimerization of T1R2 and T1R3 senses sweet taste^8,9. T2Rs consist of ~30 members in mammals and mediate sensing of bitter taste¹⁰. Ligand binding to a GPCR TR activates a heterotrimeric G protein, Gα-gustducin, and its βγ subunits, leading to the activation of phospholipase Cβ2 (PLCβ2), which converts the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) into the second messengers 1,4,5-inositol trisphosphate (IP3) and diacylglycerol (DAG). This signalling causes Ca²⁺ release from intracellular stores, activation of transient receptor potential channel M5 (TrpM5) and Na⁺ influx, ultimately leading to cell depolarization and generation of an action potential^11–13. In addition to the classic taste sensation, non-traditional taste modalities such as fatty taste have been described¹⁴. GPR120 is one of the most likely candidates for a fatty taste receptor¹⁴. A salt-sensitive GPCR, TAS2R7, that responds to divalent and trivalent salts such as zinc, calcium and copper, which are perceived as having a sour, bitter or metallic taste, has also been identified¹⁵.

OPNs also belong to the GPCR superfamily and are classified as either visual or non-visual photoreceptors¹⁶. In vertebrates, retinas have distinct photoreceptor cell types including rods and cones. Rod cells express rhodopsin (RH1) and function at low light levels, whereas cone cells express visual pigments and facilitate colour vision by responding to different wavelengths of light^17,18. Light absorption isomerizes retinal isoforms and activates the opsin, which in turn activates the heterotrimeric G protein transducin. The transducin-associated guanosine diphosphate (GDP) is exchanged for guanosine triphosphate (GTP) to dissociate into the GTP-bound α subunit and βγ complex. The G_α subunit then interacts with cyclic nucleotide phosphodiesterase (PDE), breaking down cGMP. The resulting decrease in cGMP levels causes CNG channels to close, preventing Ca²⁺ and Na⁺ influx and leading to hyperpolarization^19,20.

In addition to their roles in sensory neurons, sensory receptors are now recognized to have roles in non-sensory organs. ORs and TRs are expressed in multiple non-olfactory tissues, including the kidney, heart, eyes, muscle, prostate, small intestine and sperm^21–24, whereas OPNs are expressed in the bladder, skin, adipose tissue and cardiovascular systems^25–28. However, the functional roles of most of these sensory receptors in non-sensory organs remains to be elucidated. To date, functions have been identified for select sensory receptors expressed in the kidney and other tissues. These receptors have been found to respond to chemical stimuli, initiate signalling cascades and have important roles in cell biology and physiology.

In this Review, we discuss best practices for studying sensory receptors, highlight specific examples of sensory receptors with roles in non-sensory organs and illustrate the challenges and the great promise of uncovering the physiological functions of sensory receptors in the kidney and other tissues.

Challenges associated with the analysis of sensory receptors

A number of challenges that are associated with the analysis of ORs and other sensory receptors must be considered when designing experiments and interpreting data. First, few validated antibodies are available. GPCRs, and particularly ORs, are poorly antigenic. Despite efforts to generate antibodies for this large gene family, only a few have been reported to be reliable²⁹. Numerous antibodies are sold commercially, but it is crucial to validate their fidelity. Whenever possible, antibodies should be validated by comparing the signal in wild-type and knockout mice. Unfortunately, in our experience, the majority of commercial antibodies for ORs lack fidelity.

When antibodies are not available, alternative approaches to localizing sensory receptors include using transgenic mice with reporter genes (such as GFP or LacZ) and/or evaluating RNA expression using PCR or in situ hybridization. As all mammalian ORs (and some taste receptors) have one coding exon, it is important to include controls for genomic DNA in reverse-transcriptase PCR (RT-PCR) experiments (given the lack of introns within the coding sequence, genomic DNA and RNA will yield the same size bands in a PCR reaction). In addition, given the large size of the OR gene family, PCR products should be sequenced to confirm their identity. As ORs have been reported to form chimeric transcripts in some tissues³⁰, a full-length PCR product (start to stop codon) should ideally be sequenced. Likewise, given the large size of the OR gene family, it is important to design in situ hybridization probes to minimize cross-reactivity with other ORs. In addition, high sensitivity is needed as the expression of ORs outside of the olfactory epithelium is fairly low. We have found that RNAscope probes have excellent specificity and sensitivity, helping to overcome these challenges. As for antibodies, the ideal control for RNAscope is the absence of signal in a knockout mouse.

Single-cell RNA-sequencing (scRNA-seq) databases are an excellent, ever-growing resource for localization of sensory receptors. Although positive data are exciting, negative data (that is, the absence of a receptor from a scRNA-seq database) may not be conclusive. As GPCRs have low copy numbers, sequencing depth may not always be sufficient for their identification and artefacts such as sample contamination can inhibit the ability to detect GPCRs³¹. In addition, sequencing reads that are short in length may not enable a unique genomic match for OR reads owing to the very large size of the OR gene family; thus, ORs may be omitted from databases if reads cannot be assigned to a specific transcript. One approach to investigating whether a cell type might functionally express ORs has been to look for the expression of G_olf³². However, such expression is not conclusive, particularly as ORs can couple to other G proteins^33,34.

Another challenge in working with ORs relates to identifying orthologues across species owing to the very large size of this gene family. Three ORs have clear orthologues across all placental mammals: human OR51E2 (murine OLFR78), human OR51E1 (murine OLFR558) and human OR6B1 (murine OLFR449)³⁵. (The OR nomenclature is different in humans, mice and rats). With the exception of these three well-conserved ORs, identification of orthologues is not easily accomplished using sequence similarity alone because of the large degree of sequence similarity within this very large gene family. Several common features may help to narrow down potential orthologues, including non-coding homology within the transcriptional unit³⁶. In addition, a database of predicted orthologues has been published³⁷. However, even when taking an evolutionary genomic approach, best practice remains to confirm that putative orthologues are in fact functional orthologues, that is, that they respond to the same ligand.

Detecting the response of an OR to a ligand is most often accomplished by transiently or stably transfecting the OR of interest into a cell line and then using a reporter assay to detect increases in a second messenger such as cAMP^38–40 (Box 1). Crucially, such assays require that the OR is expressed on the cell surface, which is a substantial hurdle given that exogenously expressed ORs are typically trapped in the endoplasmic reticulum^41–43. Chaperones and N-terminal tags that improve OR surface expression have been identified, including the chaperone receptor-transporting protein 1S (RTP1S) and the tags Rho and Lucy^38,44–46. Once surface expression has been experimentally confirmed, the receptor can be screened for responses to various ligands. Screening assays and approaches have been reviewed previously^38,47.

Box 1 |. Best practices for olfactory receptor ligand screening.

Currently, most olfactory receptors (ORs) remain orphan receptors with no known ligands. One of the major hurdles in identifying OR ligands is the inability to functionally express ORs in heterologous cell systems, which is a prerequisite for high-throughput ligand screens. Although compound libraries are commercially available, selecting the appropriate chemicals can be a challenge if data for the OR of interest are scarce. However, a number of steps can be taken to optimize the chances of successful de-orphanization.

Step 1: confirm surface expression of the OR in a heterologous cell system

Adding a tag (such as a Flag tag) to the N terminus of an OR can enable determination of whether the receptor is expressed on the plasma membrane via live-cell surface labelling or surface flow cytometry. Co-expression of the OR with chaperone proteins such as receptor-transporting protein 1S (RTP1S) and the inclusion of additional N-terminal tags including Rho and Lucy can help to facilitate proper OR folding and trafficking^{45,46,151–154}. Advances have also been made through codon optimization¹⁵⁵.

Step 2: determine an appropriate screening assay

ORs couple through Gαs signalling, which results in an increase in intracellular cAMP levels. Popular ligand screening assays include direct live-cell cAMP detection and a dual CREB luciferase reporter assay^156,157. ORs can also couple to other G proteins, including Gα15/16, when they are co-expressed, enabling the use of calcium reporter assays¹⁵⁸. In addition, yeast sensors have been used for de-orphanization studies¹⁵⁹ and for lower throughput assays, in vivo imaging of olfactory sensory neurons can be used to detect OR activation^160,161.

Step 3: identify appropriate chemicals for screening

ORs typically respond to volatile compounds and traditional screening assays have taken advantage of these odorant libraries to screen for activation. ORs are categorized into families and those within the same family (sibling ORs) often have ligands in common¹⁶². Thus, one strategy for ligand screening is to test an orphan OR using ligands that activate a sibling OR. In addition, advances in machine learning and the crystallization of the first OR have facilitated initial computer-based screening methods that can be confirmed experimentally^163,164.

Sensory receptors in extrarenal non-sensory tissues

Sensory receptors are expressed throughout the human body. To date, ~97 different human ORs have been identified in ~12 tissues, including adipose, adrenal, brain, breast, colon, heart, liver, lung, ovary, testis, kidney and skeletal muscle^48,49. Some ORs are confined to a single tissue, whereas others are more widely expressed (Box 2). The testis seems to express the greatest number of ORs outside the olfactory epithelium and the most broadly expressed human OR is OR51E2 (refs. 48,49).

Box 2 |. When is an olfactory receptor not an olfactory receptor?

Olfactory receptors (ORs) were originally identified as a large G protein-coupled receptor (GPCR) family expressed in the olfactory epithelium and were named accordingly. However, the use of this nomenclature might lead researchers to conclude that ORs should not be expressed in other locations. Consequently, when an unbiased method (such as RNA sequencing or microarray) detects OR expression in a non-olfactory tissue, this result might be discounted by the researcher. ORs are perhaps better understood as chemosensory receptors that mediate olfaction and other processes. Notably, nearly the full repertoire of ORs is expressed in the olfactory epithelium, whereas most non-olfactory tissues express a much smaller subset of ORs.

Sensory receptors have diverse functions in non-sensory tissues^50,51. For example, ORs have been shown to govern cell migration and chemotaxis in keratinocytes and sperm, respectively^52–55; TRs have been linked to vasodilatory action in the lungs⁵⁶; and OPNs have been shown to modulate adiposity and lipolysis in adipocyte depots^28,57. As most sensory receptors are GPCRs, which are the most druggable class of proteins, they have important potential as therapeutic targets⁵⁸. For example, an OR agonist has been evaluated in a randomized control trial as a treatment for hair loss⁵⁹.

Olfactory receptors in the testis

One of the first reports of extra-nasal expression of ORs was in sperm in the human testis^52,54,60,61. More than 91 different transcripts corresponding to human ORs have been detected in spermatozoa but whether they all encode functional protein remains to be determined^48,62. Those that do encode proteins tend to be compartmentalized, suggesting that they govern different processes based on their subcellular localization⁶². One such function is linked to sperm motility and chemosensing. Several ORs, including OR51E2, OR17-4 and MOR23, have been shown to promote chemotaxis towards a ligand gradient^52–54. In addition, several known OR odorants have been detected in vaginal secretions (5a-androstat-16-en-3-one and 4-hydroxy-2,5-dimethyl-3(2H)-furanone) and the cervical mucus (short-chain fatty acids (SCFAs))^53,63. These findings suggest that ORs may help to guide sperm towards the oocyte. Decreased olfactory perception of some chemotactic chemicals is associated with idiopathic infertility, providing a possible direct link between OR sensation in the nose and the ability of sperm to fertilize an egg⁶⁴.

Olfactory receptors in the immune system

ORs have been found in cells of the immune system, including macrophages, at both the mRNA and protein levels^65,66. These ORs include murine Olfr2 and its human orthologue OR6A2 (refs. 66,67). Expression of Olfr2 increases upon consumption of a high-fat diet and activation of this receptor leads to inflammasome formation and IL-1β secretion, both of which contribute to atherosclerosis^66,67.

Murine Olfr78 is also expressed in bone-marrow-derived macrophages⁶⁸, with the highest expression in M2 macrophages, which are associated with anti-inflammatory properties⁶⁹. One of the reported OLFR78 ligands, lactate, has been shown to promote M2 macrophage differentiation and increase secretion of IL-4, IL-10 and transforming growth factor β (TGFβ)⁷⁰. This response was absent in macrophages isolated from Olfr78 knockout mice. Consistent with this finding, deletion of Olfr78 improved the survival of mice implanted with Lewis lung carcinoma cells⁶⁸. LPS and IFNγ decrease Olfr78 expression, suggesting opposite regulation of these macrophage-expressed ORs⁶⁸. In humans, OR2AT4 and OR1A2 are expressed in alveolar macrophages at the mRNA and protein levels⁷¹.

Studies suggest that macrophage-localized ORs rely on the canonical OR signalling cascade that exists in the nose. The genes that encode the OR signal transduction cascade (Adcy3, Cnga1, Cnga2, Cnga3, Cnga4 and Cngb1) were found to be expressed in macrophages isolated from Apoe-knockout mice, and stimulation with an OLFR2 ligand (octanal) led to an increase in cAMP levels and subsequent activation of CNG channels⁶⁷. This response was attenuated in heterozygous Acdy3 mice⁶⁷.

Although the functions of macrophage-expressed ORs are still being elucidated, evidence suggests that they may be involved in chemosensing and migration. Indeed, exposing macrophages to octanal led to increased cell migration and production of monocyte chemotactic protein 1 (ref. 66). Thus, it is tempting to speculate that ORs may sense changes in the inflammatory environment and facilitate rapid recruitment of macrophages to sites of injury or stress.

Olfactory receptors and taste receptors in lungs

Several bitter TR agonists, including saccharin, chloroquine and denatonium, have been shown to provoke a calcium response in cultured human airway smooth muscle cells⁵⁶. A screen for all bitter TRs found that these cells express upwards of 15 bitter TRs, with TAS2R10, TAS2R14 and TAS2R31 having the highest expression⁵⁶. Activation of these receptors promoted intracellular calcium release and subsequent vasodilation of smooth muscle cells.

In airway smooth muscle cells, TRs activated Gα-gustducin and the subsequent activation of PLCβ promoted the release of calcium from endoplasmic reticulum stores^56,72,73. Unlike global calcium release, which promotes contraction, this calcium release seemed to be more compartmentalized and may explain the relaxation phenotype⁷³. Concurrently, PLCβ can interact with the polarity protein, proteinase activated receptor 3 (Par3), which inhibits the serine kinase LIM domain kinase 1 (LIMK)⁷³. Phosphorylation of this protein activated the actin-severing protein, cofilin, ultimately resulting in increased smooth muscle relaxation⁷³.

Activation of bitter TRs might offer a unique treatment option for asthma. Indeed, bitter tastants were more effective than the β-adrenergic agonist, albuterol, in inducing tracheal and bronchial relaxation in a mouse model of allergic airway inflammation⁵⁶.

In addition to bitter TRs, human airway smooth muscle cells express several ORs (OR1J1, OR2A1, OR6A2 and OR51E2), which were confirmed through transcript detection and via the genotype-tissue expression dataset and RNA sequencing⁷⁴. These cells also express G_olf and AC3, indicating the ability to signal through the canonical pathway. SCFA-mediated activation of OR51E2 (using acetate or propionate) reduced cytoskeletal remodelling and proliferation of airway smooth muscle cells isolated from healthy and asthmatic lungs⁷⁴. OR2W3 expressed in airway smooth muscle cells has also been reported to mediate vasodilation, suggesting a potential pathway that could be leveraged to treat obstructive lung disease⁷⁵.

Sour taste receptors in the spinal cord

Most mammalian sensory receptors are GPCRs; however, sour taste is perceived through ion channels⁷⁶ such as polycystin-2-like protein 1 (PKD2L1)⁷⁷. This channel localizes to distinct regions of the tongue, where it participates in acid detection. In mice, loss of PKD2L1 expression led to a reduction in, but not complete elimination of, the detection of sour stimuli, whereas other taste perceptions were preserved^77,78.

In situ hybridization and confirmatory studies in reporter mice showed that Pkd2l1 is also expressed in distinct neurons lining the central canal of the spinal cord⁷⁷, which contains the cerebrospinal fluid that flows through the central nervous system. These PKD2L1-expressing neurons had the ability to detect a narrow range of acidic pH (pH 6.5–6.9)⁷⁷ and had a mechanosensory role in the detection of cerebrospinal fluid flow^79,80. The localization of PKD2L1-expressing neurons to the central canal is evolutionarily conserved across all bony vertebrate species and contributes to spinal curvature in zebrafish^80,81. PKD2L1 has also been localized to other neurons that surround the cerebrospinal fluid, including neurons in the ependymal cell layer that migrate away from the central canal during development⁸². Collectively, this research has identified a novel detection mechanism for pH changes and contributes to the increasing evidence that sensory receptors — both GPCRs and ion channels — contribute to the maintenance of homeostasis.

Opsins in adipose tissue

Photoreceptors within the retina detect light and colour. Opsins in rods and cones have a spectral sensitivity of 420–560 nm, enabling differentiation of colours along the visible spectrum. Non-visual opsins, also termed extraocular opsins, have functions that range from the regulation of the circadian clock to melanogenesis and include OPN3 (also known as encephalopsin or panopsin), OPN4 (melanopsin), OPN5 (neuropsin) and RRH (peropsin)^83–85. OPN3 and OPN4 can be activated by blue light and have been identified in adipocytes^28,57. Activation of these opsins by blue light has been shown to stimulate lipolysis, which has an important role in maintaining core body temperature under thermoneutral conditions. Opn3-knockout mice and mice that were housed without exposure to blue light had large adipocytes with few mitochondria and showed a reduction in core body temperature when housed under cold conditions⁵⁷. Similarly, chronic OPN4 activation by blue light led to a reduction in leptin and adiponectin secretion²⁸.

OPN5 is expressed in the hypothalamic preoptic area, specifically in neurons that synapse with and control the function of brown adipose tissue⁸⁶. These OPN5-expressing neurons can respond to violet light (380 nm) and their activation decreased core body temperature in mice, indicating a negative role of OPN5 in the maintenance of thermogenesis. Opn5-knockout mice and mice that were housed under conditions that lacked violet light showed improved maintenance of body temperature and thermogenesis upon cold exposure⁸⁶. Taken together, these findings highlight a clear connection between light sensitivity and adipose tissue function.

Opsins in the vasculature

Light can induce photo-relaxation of the vasculature, including the pulmonary arteries and aorta^87,88, and this phenomenon has been linked to the expression of OPNs. OPN3, OPN4 and a GPCR regulatory kinase, G protein coupled receptor kinase 2 (GRK2), were found to be expressed in rat and human pulmonary arterial smooth muscle cells and mouse tail arteries^27,89. Exposure to blue light (400–460 nm) induced relaxation of constricted arteries, but this response was attenuated in arteries from Opn4-knockout mice^27,89. The light-induced relaxation was cGMP dependent, suggesting a role of OPN signalling. OPN3 is also expressed in human airway smooth muscle, indicating conservation of function across species and translational potential⁹⁰. Blue light therapy has been successfully used to reduce arterial pressure in a rat model of pulmonary hypertension²⁷ and polymorphisms in OPN3 have been associated with an increased incidence of asthma⁹¹. Collectively, these data highlight an emerging area of interest in optogenetics and the clinical potential for phototherapy.

Sensory receptors in the kidney

The role of one sensory receptor in the kidney, the epithelial sodium channel (ENaC), has been very well-studied. ENaC has a key role in sodium reabsorption in the distal nephron but is also thought to mediate a component of salt taste and thus can be considered a TR⁹². By contrast, the great majority of sensory receptors in the kidney have only begun to be examined. Several studies have used PCR-based approaches and/or whole kidney or single-cell RNA-seq analysis to identify OR and TR expression in the kidney (Table 1). In addition, a number of available databases can be used to search for GPCRs, including ORs, TRs and OPNs, across the murine and human nephron (Box 3).

Table 1 |.

Olfactory receptors and taste receptors identified in the kidney^a

Method	Genes		Expression	Refs.
	Olfactory receptors	Taste receptors
Mouse
PCR	Olfr78, Olfr90, Olfr1373, Olfr1392, Olfr1393	NA	Olfr78: renin cells and blood vessels; Olfr90: macula densa cell line; Olfr1393: proximal tubule	32,93,112
PCR	Olfr99, Olfr1426, Olfr31, Olfr545, Olfr691, Olfr693	Tas2r108, Tas2r119, Tas2r135, Tas2r137, Tas2r138, Tas2r140, Tas2r143, Tas1r1, Tas1r2, Tas1r3	NA	149
PCR	NA	Tas2r105, Tas2r106, Tas2r110, Tas2r113, Tas2r114, Tas2r134, Tas2r143	NA	150
RNA-seq	Olfr212, Olfr56, Olfr267, Olfr461, Olfr558, Olfr613, Olfr920, Olfr1033, Olfr1034, Olfr1396, Olfr1442, Olfr1443	NA	Olfr558: renin cells and vascular smooth muscle cells	101,103
RNA-seq	Olfr1016, Olfr1090, Olfr1112, Olfr127, Olfr1396, Olfr1406, Olfr23, Olfr273, Olfr373, Olfr39, Olfr432, Olfr50, Olfr554, Olfr568, Olfr617, Olfr691, Olfr735, Olfr745, Olfr801	NA	Olfr1016, Olfr1090, Olfr1112, Olfr127, Olfr1406, Olfr23, Olfr273, Olfr39, Olfr432, Olfr50, Olfr554, Olfr568, Olfr691, Olfr745, and Olfr801: DTL and glomerulus; Olfr1396: glomerulus; Olfr373: IMCD; Olfr617 and Olfr735: DTL	37
Human
RNA-seq	OR1L8, OR2A1/42, OR2A4/7, OR51E1, OR5K2, OR51E2, OR2W3, OR10A2/5, OR1C1, OR2L13, OR56B1	TAS1R1, TAS1R3, TAS2R1, TAS2R10, TAS2R14, TAS2R19, TAS2R20, TAS2R3, TAS2R30, TAS2R31, TAS2R4, TAS2R42, TAS2R43, TAS2R5, TAS2R50	OR51E1: human proximal tubule cells	48,102

^aThis table shows olfactory receptors and taste receptors that were reported by studies that focused on identifying sensory receptors. Many additional RNA-seq studies may have also reported expression of sensory receptors. The method listed in the first column was used for the initial identification of sensory receptor expression in the kidney; follow-up studies used additional methods to localize the expression of these receptors within the kidney. DTL, descending limb of the loop of Henle; IMCD, inner medullary collecting duct; NA, not available; RNA-seq, RNA sequencing.

Box 3 |. Transcriptomic databases for identifying sensory receptors in the nephron.

Following the advent of single-cell RNA sequencing, various studies have mapped the renal nephron for gene expression under healthy and pathophysiological states. These databases can be used to identify olfactory receptors, taste receptors and opsins that are expressed within the nephron.

• https://esbl.nhlbi.nih.gov/MRECA/Nephron/: a searchable database to identify gene expression across 14 microdissected tubule segments from C57BL/6 mice¹³⁵.

• https://humphreyslab.com/SingleCell/search.php: a searchable database to identify gene expression from >20 mouse and human RNA-seq studies.

• https://esbl.nhlbi.nih.gov/Databases/GPCRs/TubuleTPM.html: a searchable database to localize >700 G protein-coupled receptors expressed in the mouse kidney³⁷.

• https://susztaklab.com/index.php: a searchable database to identify gene expression across >20 mouse and human RNA-seq studies.

OLFR78 and renin regulation

The first OR that was identified as having a functional role in the kidney was mouse Olfr78 (ref. 93), which is one of only three ORs with a clear human orthologue (OR51E2). Studies using a lacZ reporter gene showed that Olfr78 localizes to renal blood vessels, including the renal afferent arteriole, which stores and secretes renin⁹³. Olfr78 is also expressed in blood vessels, primarily in smooth muscle cells, in a number of non-renal tissues^93,94 and has been reported to act as a hypoxia sensor in the carotid body⁹⁵ and to have roles in colon homeostasis and inflammation^96,97.

The ligands for OLFR78 and OR51E2 are the SCFAs acetate and propionate, which are mainly produced by gut microorganisms. Acetate and propionate can also be produced by the host in low quantities that probably only make a small contribution to circulating levels⁹⁸. Some studies have reported that OLFR78 can also be activated by lactate⁹⁵.

Studies using Olfr78-knockout mice demonstrated that OLFR78 activation increases renin release, which would be expected to increase blood pressure (Fig. 1). Initial studies in anaesthetized mice indicated that those with Olfr78 knockout had lower blood pressure than wild-type controls⁹³. However, subsequent studies using blood pressure telemetry did not show significant differences in baseline blood pressure in Olfr78-knockout and wild-type mice, but did report a lower percentage of glomeruli that were associated with renin protein staining in the knockout mice⁹⁹. In addition to effects on renin, propionate and acetate have been shown to induce acute decreases in blood pressure, probably by altering vascular resistance^93,100. Nevertheless, this effect is mediated primarily by a non-olfactory GPCR, GPR41, which has a similar ligand profile to OLFR78 (ref. 93).

Fig. 1 |. OLFR78 and renin release.

a, Binding of short-chain fatty acids (SCFAs), such as acetate and propionate, to OLFR78 expressed in renin-containing juxtaglomerular cells in the renal afferent arteriole increases renin release from these cells. The mechanism of OLFR78 signalling in renin-containing cells is not known but would be expected to involve elevations in cAMP. b, Renin is the ratelimiting enzyme in the renin–angiotensin–aldosterone system. Once released into the circulation, renin cleaves angiotensinogen into angiotensin I, which is subsequently converted into angiotensin II by angiotensin-converting enzyme (ACE). Angiotensin II stimulates aldosterone release from the adrenal cortex and acts as a vasoconstrictor, resulting in an increase in blood pressure.

OLFR558 and sex differences in blood pressure

Olfr558 also has a clear orthologue across placental mammals (OR51E1 in humans). Olfr558 has been reported to localize to vascular smooth muscle cells (but not endothelial cells) in numerous tissues, including the kidney and heart, and RNA-seq data indicate that the human orthologue, OR51E1, has a similar localization pattern¹⁰¹. OR51E1 is also expressed in human kidney cells and in the HK-2 human proximal tubule cell line¹⁰². In the mouse kidney, Olfr558 is expressed in the afferent arteriole where it colocalizes with renin¹⁰¹. Identified ligands of OLFR558 include 2-methylvaleric, 3-methylvaleric, 4-methylvaleric, butyric, cyclobutanecarboxylic and valeric acids. The most potent ligand for OLFR558 and OR51E1 is the SCFA butyric acid^103,104.

In humans and mice, the blood pressure of male individuals is ~10 mmHg higher than that of premenopausal female individuals^105–110. However, this sex difference in blood pressure is absent in Olfr558-knockout mice owing to increased blood pressure in females and reduced diastolic blood pressure in males¹⁰¹ (Fig. 2). Mechanistic studies indicated that female Olfr558-knockout mice had increased pulse wave velocity, indicating increased vascular stiffness, whereas male Olfr558-knockout mice had reduced renin expression and activity and an exaggerated relaxation response to sodium nitroprusside¹⁰¹.

Fig. 2 |. OLFR558 and blood pressure.

a, In the kidney, mouse Olfr558 and its human orthologue OR51E1 are expressed in vascular smooth muscle cells (VSMCs) and renin-expressing juxtaglomerular cells¹⁰¹. b, In wild-type mice, females have lower diastolic blood pressure (DBP) than males. However, this sex difference is absent in Olfr558-knockout mice owing to increased blood pressure in females and decreased blood pressure in males. Increased arterial stiffness probably contributes to increased blood pressure in female Olfr558-knockout mice, whereas reduced renin activity and altered vascular reactivity may explain decreased DBP in male Olfr558-knockout mice. Panel b modified with permission from ref. 101, AAAS.

Intriguingly, the OR51E1 locus has been found to be associated with diastolic blood pressure¹¹¹ and a rare OR51E1 missense variant (rs202113356, A156T) had a statistically significant sex interaction effect with diastolic blood pressure in humans¹⁰¹. These data indicate that OR51E1 has an evolutionarily conserved role in mediating sex differences in blood pressure. Although blood pressure is lower in premenopausal women than in men, a 2021 study reported that women also incur increases in cardiovascular risk at lower blood pressures than men¹¹⁰. Thus, uncovering how and why blood pressure is differentially regulated via OR51E1 in premenopausal women and men may provide novel insights into blood pressure regulation with potential clinical implications.

OLFR1393 and renal glucose handling

Olfr1393 is expressed in the renal proximal tubule and localizes to the apical plasma membrane in polarized Madin–Darby canine kidney (MDCK) cells¹¹². Ligands of Olfr1393 include small molecules with pre-constrained rings containing ketones or alcohols (cycloheptanone, cycloheptanone, cyclooctenone, cyclohexanone, 4,4 dimethylcyclohexanone, nopinone, norcamphor and 4-tertbutylcyclohexanone)¹¹². These ligands were shown to specifically activate OLFR1393 (and not OLFR1392, which differs by 15 amino acids), but the threshold of activation was in the millimolar range and thus the physiological relevance of these compounds is uncertain.

Although Olfr1393 has been reported to be expressed in various tissues outside of the kidney, the phenotype of Olfr1393-knockout mice is kidney specific¹¹². When knockout mice were fed a normal chow diet, they exhibited mild glycosuria (glucose wasting) despite maintaining euglycaemia, normal insulin levels and normal glomerular filtration rate¹¹². Moreover, when challenged with a glucose load, Olfr1393-knockout mice were able to clear glucose more efficiently and had improved glucose tolerance compared with wild-type controls, suggesting a specific renal impairment in glucose reabsorption. Examination of sodium-glucose co-transporter 1 (SGLT1) and SGLT2, which are expressed in the proximal tubule and mediate luminal uptake of all filtered glucose^113,114, revealed a reduction in luminal membrane localization of SGLT1, which is responsible for ~10% of all glucose reabsorption¹¹² (Fig. 3). Of note, SGLTs have emerged as important transporters in the diabetes field and beyond, and SGLT2 inhibitors (also known as gliflozins) can reduce hyperglycaemia and improve cardiometabolic phenotypes in patients with type 2 diabetes mellitus^115–120.

Fig. 3 |. OLFR1393 and glucose handling.

OLFR1393 localizes to the apical membrane of renal proximal tubule cells, which is also the site of glucose and sodium reabsorption via sodium-glucose cotransporter 1 (SGLT1) and sodium-glucose cotransporter 2 (SGLT2). In the absence of OLFR1393 (for example in Olfr1393-knockout mice), luminal membrane expression of SGLT1 is reduced, resulting in reduced glucose reabsorption, mild glycosuria, and improved glucose tolerance. This results in an attenuation of diabetic phenotypes in the context of high-fat diet feeding or challenge with streptozotocin. The precise role of OLFR1393 ligand(s) in this process has not been elucidated.

Given the link between OLFR1393 and SGLT1, additional studies investigated whether Olfr1393-knockout mice have an altered diabetic phenotype. When these mice were challenged with either a 60% lard-based high-fat diet (to induce obesity and early stages of type 2 diabetes mellitus) or with streptozotocin (to induce type 1 diabetes mellitus), they exhibited generalized protection against the development of diabetes compared with wild-type controls^121,122. This protection was accompanied by a reduction in the luminal expression of SGLT2 in response to a high-fat diet and an increase in the total expression of SGLT1 in response to streptozotocin challenge^121,122.

Although several human ORs are expressed in the kidney¹²³, the functional human orthologue for OLFR1393 has not yet been identified. This missing link will be required to fully appreciate the translational potential of OLFR1393 and to determine whether this receptor contributes to the development or progression of diabetes. Interestingly, single nucleotide polymorphisms (SNPs) have been identified in human ORs from the gene family OR14 (ref. 124). These SNPs correlate with increased risk of diabetic complications including diabetic retinopathy and neuropathy. Further investigation into the role of ORs in renal glucose handling may ultimately lead to the development of novel treatment approaches and/or aid in the identification of people who are at a high risk of developing hyperglycaemia and insulin resistance.

Olfactory receptors in kidney fibrosis

Kidney fibrosis is characterized by the accumulation of extracellular matrix proteins and occurs as a result of nearly every type of chronic kidney disease, including lupus nephritis, glomerulonephritis, cystinosis and diabetic kidney disease¹²⁵. Deposition of extracellular matrix together with tissue scarring and severe inflammation leads to destruction of the renal tissue and kidney failure. A 2022 study highlighted a potential role of ORs in the development of kidney fibrosis¹²⁶. Time-course microarray datasets from mice with unilateral ureteral obstruction revealed nodes of ORs that were differentially expressed upon induction of fibrosis. Confirmatory studies found that the expression of four murine ORs, Olfr433, Olfr219, Olfr161 and Olfr622, was increased and remained elevated for up to 21 days after the onset of fibrosis. One of these ORs, Olfr433, was localized to macrophages, implying a potential role in the inflammatory response. In addition, the study showed that expression of Olfr1393 was reduced under conditions of kidney fibrosis¹²⁶. This finding suggests that in addition to the potential role of this OR in diabetes^121,122, it may have a general function in renal injury.

Taste receptors in podocytes

Tas2r108, which encodes taste receptor type 2 member 4 (TAS2R4), was shown to be expressed in mouse podocytes¹²⁷. In this study, chronic high glucose stimulation inactivated TAS2R4 and decreased downstream PLCβ2 expression in a mouse podocyte cell line. Quinine, an agonist of TAS2R4, increased TAS2R4 and PLCβ2 levels, podocyte cell viability and the levels of zonula occludens 1 (ZO-1) and nephrin in the high-glucose stimulated cell line. However, inhibition or knockdown of TAS2R4 abolished these effects. These findings suggest that TAS2R4 activation alleviates podocyte injury induced by chronic high glucose levels.

Opsins in the loop of Henle

Little is known about the potential roles of opsins in the kidney. Opn3 is selectively expressed in the thick ascending limb of the loop of Henle but its physiological function is unknown³⁷. Notably, Opn3 reporter mice¹²⁸ and Opn3-knockout mice¹²⁹ are viable and could be used to determine the role of this opsin in the kidney.

Proton-activated G protein-coupled receptors

Proton-sensing GPCRs belong to the class A GPCR subfamily and include GPR4, GPR65, GPR68 and GPR132, which act as cellular sensors of acidification¹³⁰. GPR4 expression is relatively abundant in the kidney, including in the renal cortex and medulla. Mice that lack GPR4 exhibit decreased renal acid secretion, leading to a non-gap metabolic acidosis¹³¹.

GPR68 is expressed in the glomerulus, proximal tubule and interstitium and GPR68-deficient mice had reduced calcium excretion during chronic metabolic acidosis¹³². Loss of this receptor was also shown to attenuate chronic kidney disease-induced cardiac impairment¹³³.

A study published in preprint form suggested that GPR65 may underlie some of the cardiovascular benefits of dietary fibre in a mouse model. This study reported that dietary fibre lowers intestinal pH, which leads to activation of GPR65, lowering both blood pressure and cardiovascular risk¹³⁴. The researchers suggested that GPR65 might mediate this cardioprotection primarily via immunosuppressive mechanisms, including changes in T cell populations in the kidney.

GPR132 has been detected in mouse kidney via RNA-seq¹³⁵. However, the role of this GPCR in the kidney has not yet been elucidated.

Metabolite-sensing G protein-coupled receptors

Under aerobic conditions, glucose can be fully metabolized to produce adenosine triphosphate (ATP) and carbon dioxide. A large part of this process occurs via the tricarboxylic acid cycle, which oxidizes acetyl CoA through a series of intermediate steps. Two of the intermediate metabolites in this cycle, succinate and α-ketoglutarate (αKG), activate the chemosensory GPCRs GPR91 (also known as SUCNR1) and GPR99 (also known as OXGR1), respectively. These GPCRs share 33% amino acid identity and are predominantly, but not exclusively, expressed in the kidney¹³⁶. mRNA localization of GPR91 was reported in the proximal and distal tubules¹³⁶ and protein localization confirmed expression of the protein in the afferent arteriole and glomerulus as well in the apical membranes of the macula densa, thick ascending limb of the loop of Henle and the intermedullary collecting duct^137–139. GPR99 was found to be confined to the distal tubule with antibody localization confirming its expression on the apical membranes of intercalated cells of the collecting duct, particularly on non-A, non-B cells that express the chloride/bicarbonate exchanger, pendrin^136,140,141.

GPR91 is selectively activated by succinate, leading to the induction of Gi and Gq signalling cascades. Increased levels of succinate promote renin release and afferent arteriole vasodilation¹³⁶. Increased succinate (and renin) are associated with excess levels of glucose (hyperglycaemia)¹³⁸ and circulating levels of succinate are increased under diabetic conditions. Loss of GPR91 prevents succinate- and glucose-stimulated prorenin expression, renin release and prostaglandin production^136,138. Synthetic agonists for GPR91 have been developed that have the potential to modulate the GPR91–succinate–hypertension pathway and provide therapeutic intervention¹⁴¹.

αKG also has biological functions outside of the tricarboxylic acid cycle, most notably through activation of GPR99 and modulation of acid–base balance. In mice, GPR99 was shown to increase chloride and bicarbonate exchange and promote NaCl reabsorption when activated by αKG^136,142. Gpr99-knockout mice exhibit a decreased urinary pH with a concomitant increase in urinary titratable acid and αKG levels, suggesting that GPR99 aids in the regulation of acid–base balance¹⁴².

Non-olfactory receptor short-chain fatty acid G protein-coupled receptors

SCFAs are metabolic byproducts of gut microbial metabolism that can affect numerous host signalling pathways. As mentioned above, OLFR78 and OLFR558 are receptors for SCFAs. At least three other non-OR SCFA receptors are expressed in the kidney: GPR41, GPR43 and GPR109A (which is also a receptor for niacin). GPR41 and GPR43 have been localized to renal blood vessels⁹³ and GPR41 has been localized to the aortic vascular endothelium¹⁴³. In addition, all three receptors are expressed in immune cells^144,145. GPR109A has also been localized to podocytes, where it helps to mediate a protective effect of the SCFA butyrate¹⁴⁶. Other studies have reported that SCFAs protect against the progression of diabetic kidney disease via GPR109A and GPR43 and that SCFAs can protect mesangial cells from oxidative stress and inflammation via GPR43 (ref. 147). In addition, reduced levels of GPR41 and GPR43 have been associated with increased arterial stiffness in humans¹⁴⁴.

Conclusions

Olfactory, taste and opsin receptors are not only found in sensory organs but are also widely expressed in a variety of non-sensory tissues throughout the body. The roles of sensory receptors in these tissues are only beginning to be uncovered, in part because of the challenges inherent in studying these receptors. However, the available evidence clearly indicates that sensory receptors act to help to protect homeostasis in many different cell types and influence a variety of processes, from sensing the pH of cerebrospinal fluid to modulating macrophage function.

The kidney, which is sometimes described as the master regulator of homeostasis, is a particularly promising location for the identification of sensory receptors given its crucial role in maintaining homeostasis. Although only a small number of renal sensory receptors have been examined in detail, the roles uncovered to date are varied and important. Thus, the numerous renal sensory receptors that have yet to be studied represent great promise for advancing our understanding of kidney function in health and disease.

Studies of sensory receptors may also lead to the development of novel therapies¹⁴⁸. As the kidney is the master regulator of a large number of parameters, studies of novel renal receptors could potentially lead to insights into a wide variety of diseases, including electrolyte disorders, uraemia, hypertension, oedema, acidosis, bone diseases and anaemia. To realize this potential, future studies should rigorously investigate the localization and ligand profiles of sensory receptors in the kidney and leverage animal models (such as knockouts) to uncover their functional roles. In addition, it will be important to develop small molecular agonists and antagonists that can modulate sensory receptors. These small molecules could serve as key tools for interrogating the physiological roles of sensory receptors and as potential lead compounds for pharmaceutical targeting of these receptors.

Key points.

• Olfactory receptors (ORs), taste receptors and opsins have functional roles in non-sensory organs and tissues, including in the kidney, lungs, testis, spinal cord, adipose tissue, vasculature and immune system.

• Studies of ORs are challenging but use of best practices in the field enable these important receptors to be interrogated in a rigorous fashion.

• ORs have roles in sperm chemotaxis, macrophage chemosensation and migration, and lung vasodilation; taste receptors modulate lung vasodilation and sense pH changes in cerebrospinal fluid; and opsins have been reported to have roles in adipose tissue function and thermogenesis.

• In the kidney, Olfr78 is expressed in blood vessels; activation of this OR in the renal afferent arteriole increases renin release.

• Olfr558 localizes to blood vessels, including the renal afferent arteriole, and is required for sex differences in blood pressure.

• Olfr1393 is expressed in the renal proximal tubule, where it modulates sodium–glucose cotransporters, resulting in effects on glucose tolerance.