Landscape of GPCR expression along the mouse nephron

Abstract

Kidney transport and other renal functions are regulated by multiple G protein-coupled receptors (GPCRs) expressed along the renal tubule. The rapid, recent appearance of comprehensive unbiased gene expression data in the various renal tubule segments, chiefly RNA sequencing and protein mass spectrometry data, has provided a means of identifying patterns of GPCR expression along the renal tubule. To allow for comprehensive mapping, we first curated a comprehensive list of GPCRs in the genomes of mice, rats, and humans (https://hpcwebapps.cit.nih.gov/ESBL/Database/GPCRs/) using multiple online data sources. We used this list to mine segment-specific and cell type-specific expression data from RNA-sequencing studies in microdissected mouse tubule segments to identify GPCRs that are selectively expressed in discrete tubule segments. Comparisons of these mapped mouse GPCRs with other omics datasets as well as functional data from isolated perfused tubule and micropuncture studies confirmed patterns of expression for well-known receptors and identified poorly studied GPCRs that are likely to play roles in the regulation of renal tubule function. Thus, we provide data resources for GPCR expression across the renal tubule, highlighting both well-known GPCRs and understudied receptors to provide guidance for future studies.

INTRODUCTION

G protein-coupled receptor (GPCR) signaling plays a crucial role in renal function. Many key GPCRs expressed in renal tubule epithelia were initially identified through biochemical studies in isolated, microdissected renal tubules largely in the laboratory of Morel et al. (1, 2). Among other receptors, these studies localized the parathyroid hormone (PTH) receptor to the proximal tubule (PT) and thick ascending limb (TAL) (3), glucagon (4) and calcitonin receptors (5) to the TAL (6), and the vasopressin receptor to the collecting duct (CD) (1, 7). These and other studies of the same era defined an early pattern of GPCR expression along the renal tubule, geared chiefly to the regulation of transport, as demonstrated in isolated perfused tubule studies (8).

In general, all of these previously characterized receptors were identified through hypothesis-driven studies. Since the publication of various genome projects around 2,000, progress in identifying previously unsuspected gene products has been accelerated by the development of unbiased “-omics” approaches such as mass spectrometry-based proteomics and next-generation sequencing-based transcriptomics. Examples include transcriptomics in microdissected renal tubules (9–11), single-cell transcriptomics (10, 12–15), and proteomics in microdissected tubules (16). These studies have yielded a wealth of omics data, much of which is publicly accessible (see, for example, https://hpcwebapps.cit.nih.gov/ESBL/Database/) and available for hypothesis generation and analysis.

Insel et al. (17, 18) have proposed the use of unbiased comprehensive omics datasets to identify novel patterns of GPCR expression (GPCRomics) as tools for the discovery of new physiological functions that could lead to therapeutic drug targets. Although the work of several laboratories, including our own, has generated a wealth of omics data, these datasets have not been examined through a broad lens, particularly with respect to large gene families such as GPCRs. After creating a curated database of mouse GPCRs, we mapped the expression of 758 GPCRs, plus frizzled receptors, across 14 nephron segments using existing transcriptomic and proteomic data. From these data, we highlighted GPCRs with selective expression patterns in discrete tubule segments. The objective of this review is to integrate omics data and the existing literature to describe the landscape of GPCR expression among the segments of the renal tubule.

MAPPING GPCRs ACROSS THE NEPHRON

To map GPCR expression, we curated a complete list of mouse GPCRs (Fig. 1A) using multiple online databases. The resulting GPCR lists for the mouse, along with their rat and human orthologs, are provided at https://hpcwebapps.cit.nih.gov/ESBL/Database/GPCRs/. This curated GPCR list was mapped to RNA-sequencing (RNA-seq) data from 14 microdissected tubule segments and the glomerulus generated by Chen et al. (9) (Fig. 1, B and C). Expression levels for all these GPCRs are provided at https://hpcwebapps.cit.nih.gov/ESBL/Database/GPCRs/TubuleTPM.html. This yielded matches to 758 GPCRs across the renal tubule. From this extensive list, we ranked GPCR expression in each microdissected segment. The top 10 GPCRs expressed in each segment is shown in Table 1. From these data, we highlighted GPCRs that are selectively expressed in discrete tubule regions (Fig. 1D), defined as GPCR expression in one tubule region being at least 1.5 times greater than any other point along the renal tubule. This threshold was chosen to identify receptors with the potential for having functional roles in particular tubule segments. Several of the most highly expressed GPCRs in the renal tubule (Table 1), such as G protein-coupled receptor class C group 5 member C (Gprc5c) and adhesion G protein-coupled receptor G1 (Adgrg1), were expressed ubiquitously, suggesting nonspecific functions, i.e., housekeeping genes. This review will focus on GPCRs that were detected to be expressed in transcripts per kilobase million (TPM) value of 10 or greater, which corresponds to a rank of 105 of 498 GPCRs detected at ≥1.0 TPM. This criterion highlights the GPCRs most likely to be detected at the protein level. Additional details about how we cataloged and curated the GPCRs we present in this review, along with the cross-referenced datasets, are detailed in the following sections (see appendix).

Figure 1.

Creating a database of G protein-coupled receptors (GPCRs) and filtering for segment-specific expression. GPCR gene entries were combined from Mouse Genome Informatics (MGI), International Union of Basic and Clinical Pharmacology/British Pharmacological Society Guide to Pharmacology (IUPHAR), and Ensembl to create a complete GPCR database for mouse, rat, and human orthologs (A). This list was then mapped to RNA-sequencing (RNA-seq) data from 14 microdissected renal tubule segments (B) to get a complete picture of GPCR expression (C). From this mapped GPCR expression, segment-specific GPCRs were determined (D). These GPCRs were then compared with existing literature knowledge and cross referenced with other existing omics data sets. IMCD, inner medullary collecting duct; OMCD, outer medullary collecting duct; TPM, transcripts per kilobase million.

Table 1.

Most highly expressed G protein-coupled receptors in each tubule segment

Rank	PTS1	PTS2	PTS3	DTL1	DTL2	DTL3	ATL	MTAL	CTAL	DCT	CNT	CCD	OMCD	IMCD	Glom
1	Pth1r	Pth1r	Pth1r	Adgrg1	Adgrg1	Adgrg1	Adgrg1	Ptger3	Adgrg1	Pth1r	Adgrg1	Adgrg1	Adgrg1	Avpr2	Pth1r
	370	970	544	1,055	830	1,136	645	729	928	569	1,055	1,304	1,242	1,012	1,089
2	Gprc5c	Cckar	Gprc5c	Gprc5c	Ackr3	Gprc5c	Avpr2	Adgrg1	Pth1r	Adgrg1	Avpr2	Avpr2	Avpr2	Ptger1	F2r
	177	259	128	320	460	917	189	600	585	303	631	1,011	1,214	336	463
3	Adgrg1	Gprc5c	Tm2d1	Gprc5a	Gprc5c	Gprc5a	Adgre5	Gcgr	Ptger3	Ptgfr	Gprc5c	Gprc5c	Gprc5c	Adgrg1	Adgre5
	97	236	115	315	329	285	120	289	512	213	397	419	562	187	422
4	Lpar3	Tm2d1	Cckar	Adgrl4	Gprc5a	Adora1	Tm2d1	Avpr2	Gcgr	Gprc5c	Oxgr1	Avpr1a	Ptger1	Gprc5c	Agtr1a
	94	117	114	261	221	163	111	137	488	208	332	229	495	168	396
5	Tm2d1	Adgrg1	Adrb2	Ednrb	Adora1	F2rl1	F2r	Gprc5c	Casr	Oxgr1	Avpr1a	Oxgr1	Adgrf5	Tm2d1	Adgrf5
	82	63	46	217	190	144	92	130	225	174	301	206	406	162	231
6	Agtr1a	Adgra3	Adgrg1	F2rl1	Adgrl4	Tm2d1	Adora1	Tm2d1	Gprc5c	Gcgr	Adgrf5	Adgrf5	P2ry14	P2ry14	S1pr3
	52	58	36	115	174	125	63	127	195	160	155	186	237	146	219
7	Lgr4	Sucnr1	Gpr137	Adgrf5	Tm2d1	Lgr4	Ptger3	Fzd4	Tm2d1	Tm2d1	Tm2d1	Ptgfr	Tm2d1	Adgrf1	Calcrl
	44	54	35	111	162	107	36	58	124	126	133	146	153	122	188
8	Gpr160	Adgre5	Adra2b	Tm2d1	F2rl1	P2ry2	Gprc5c	P2ry2	Gpr160	Gpr160	Gcgr	Ackr3	Adora1	Adora1	Adgrl4
	41	52	32	102	106	102	36	54	108	104	129	136	150	96	159
9	Sucnr1	Adra2b	Smo	Fzd4	Fzd4	Gpr137	Adgra3	Gpr160	Fzd4	Fzd4	Ptgfr	Tm2d1	Ackr3	Gprc5b	Tbxa2r
	38	49	30	86	99	71	35	49	90	85	125	125	118	65	152
10	Adra2b	Adrb2	Adgra3	F2r	P2ry2	Adgrl4	Fzd4	Gpr137	Adgra3	Avpr1a	Gpr160	Gpr160	Fzd4	Gpr137	Ccrl2
	34	39	27	85	89	59	31	46	81	52	120	118	84	60	134

For all G protein-coupled receptor data, entries were sorted based on maximum expression (in transcripts per kilobase million) in each individual tubule segment. ATL, thin ascending limb of the loop of Henle; CCD, cortical collecting duct; CNT, connecting tubule; CTAL, cortical thick ascending limb of the loop of Henle; DCT, distal convoluted tubule; DTL1, short descending limb of the loop of Henle; DTL2, long descending limb of the loop of Henle in the outer medulla; DTL3, long descending limb of the loop of Henle in the inner medulla; Glom, glomerulus; IMCD, inner medullary collecting duct; MTAL, medullary thick ascending limb of the loop of Henle; PTS1, initial segment of the proximal convoluted tubule; PTS2, proximal straight tubule in cortical medullary rays; PTS3, last segment of the proximal straight tubule in the outer stripe of outer medulla; OMCD, outer medullary collecting duct.

These GPCRs were matched to known ligands, coupled G proteins, and known physiological roles in the segment in which it is selectively expressed. For ease of reference, principal pathways known to be activated by each G protein α-subunits are shown in Table 2. Although we primarily focused on the downstream effects of G α-subunits, it is worth noting that other heterotrimeric G protein subunits, Gβγ complexes, have been established to have downstream signaling effects, which have been the subject of detailed reviews (19, 20). In addition, β-arrestins have been implicated to have additional effects downstream of GPCR signaling (21). For example, established renal GPCRs such as PTH 1 receptor (Pth1r) and arginine vasopressin V2 receptor (Avpr2) have been found to have noncanonical signaling pathways via β-arrestins (22, 23). The focus of this review is on these selectively expressed GPCRs to identify understudied GPCRs with specific patterns of expression from which, given our current understanding of transport physiology, hypotheses and future studies could be proposed.

Table 2.

G protein α-subunits

G Protein α-Subunit	Downstream Effects
Gαs	Activate adenylyl cyclase → increase cAMP → activate PKA
Gαi/Gαo	Inhibit adenylyl cyclase → decrease cAMP
Gαq/Gα11	Activate phospholipase Cβ: IP3 generation → intracellular Ca2+ and diacylglycerol → protein kinase C
Gα12/Gα13	RhoGEF → RhoA → activate Rho kinase

Shown is a summary of G protein α-subunits that bind to G protein-coupled receptors and their general downstream effects. IP₃, inositol 1,4,5-trisphosphate; RhoGEF; rho-specific guanine nucleotide exchange.

GPCRs SELECTIVELY EXPRESSED ACROSS THE NEPHRON

Proximal Tubule

GPCRs that are selectively expressed in the PT, microdissected as three segments (PTS1, PTS2, and PTS3), are shown in Fig. 2. Known ligands, G proteins, and physiological roles for PT-selective GPCRs are shown in Table 3. This list includes multiple well-studied receptors that are known to localize to this segment, including Pth1r, angiotensin II (ANG II) receptor type 1a (Agtr1a), two α-adrenergic receptors (Adra1a and Adra2b), and succinate receptor 1 (Sucnr1).

Figure 2.

Proximal tubule-selective G protein-coupled receptors (GPCRs). GPCR expression data from microdissected renal tubules were filtered based on an individual genes’ maximum expression [in transcripts per kilobase million (TPM)] in the initial segment of the proximal convoluted tubule (PTS1), proximal straight tubule in cortical medullary rays (PTS2), or last segment of the proximal straight tubule in the outer stripe of outer medulla (PTS3) compared with its expression in any other tubule segment. PTS1, PTS2, and PTS3 expression that was 1.5-fold greater than any other tubule segment was considered selective. Entries are sorted starting with the greatest TPM ratio. Refer to Table 3 for additional information about each GPCR. ATL, thin ascending limb of the loop of Henle; CCD, cortical collecting duct; CNT, connecting tubule; CTAL, cortical thick ascending limb of the loop of Henle; DCT, distal convoluted tubule; DTL1, short descending limb of the loop of Henle; DTL2, long descending limb of the loop of Henle in the outer medulla; DTL3, long descending limb of the loop of Henle in the inner medulla; Glom, glomerulus; IMCD, inner medullary collecting duct; MTAL, medullary thick ascending limb of the loop of Henle; OMCD, outer medullary collecting duct.

Table 3.

Proximal tubule-selective G protein-coupled receptors

Gene Symbol	Annotation	Ligand	G Protein	Renal Physiological Process	Key Reference(s)
Lpar3	Lysophosphatidic acid receptor 3	Lysophosphatidic acid	Gαi/Gαq	Segment function not known. Blockade reduces ischemia-reperfusion injury	(24)
Cckar	Cholecystokinin A receptor	Cholecystokinin	Gαq/Gαs	Segment function not known. Proposed role in the natriuretic response	(25)
Sucnr1	Succinate receptor 1	Succinic Acid	Gαi/Gαq	Succinate binding induces renin release	(26, 27)
Agtr1a	angiotensin II receptor, type 1a	Angiotensin II	Gαi/Gαo/ Gαq	Na+/K+-ATPase activity, HCO3− HCO3− reabsorption, renin-angiotensin system activation	(28–31)
Adra2b	Adrenergic receptor, α2b	Epinephrine/norepinephrine	Gαi/Gαo	MAPK activation, Na+-K+-ATPase activity	(32–34)
Adgrg4	Adhesion G protein-coupled receptor G4	Orphan		Kidney function not known
Adra1a	Adrenergic receptor, α1a	Epinephrine/norepinephrine	Gαq/Gα11	Na+ absorption	(35, 36)
Lpar6	Lysophosphatidic acid receptor 6	Lysophosphatidic acid	Gαs/Gαi/ Gα12	Segment function not known
Pth1r	Parathyroid hormone 1 receptor	Parathyroid hormone	Gαs	Inhibits phosphate/bicarbonate absorption	(5, 37–39)

Shown are known ligands, coupled G protein α-subunits, and segment-specific physiological roles for G protein-coupled receptors selectively expressed in the proximal tubule.

Pth1r localizes to the PT (37, 40, 41). Classic isolated perfused tubule studies have demonstrated that PTH acting on the basolateral membrane strongly inhibits salt and water reabsorption (38, 42, 43) through effects to increase adenylyl cyclase activity (3) by activation of G_αs (39).

Agtr1a also has established functions in the PT (30, 44, 45). Activation of this receptor by ANG II occupation results in acceleration of salt, glucose, and water reabsorption (29, 46, 47) through effects to decrease cAMP production (46, 47), consistent with its known coupling to G_αi (28). Notably, expression of Agtr1a is much lower in segments beyond the PT, a pattern confirmed in other single-cell RNA-seq studies (14).

The data shown in Fig. 2 also indicate that the PT selectively expresses two α-adrenergic receptors, α-1A (Adra1a) and α-2B (Adra2b), in agreement with the literature (32, 34, 36). Activation of α-adrenergic signaling increases Na⁺ and bicarbonate reabsorption in the PT by inhibiting cAMP (48–50). α-1A couples to G_αq/G_α11 (35), whereas α-2B couples to G_αi/G_αo (33). We note that the expression of multiple other adrenergic receptors across the nephron and will go into further detail on this GPCR subfamily later on.

Finally, Fig. 2 shows that Sucnr1 (also known as Gpr91) is selectively expressed in the PT, corroborating prior findings (26). Succinate receptor 1 activation leads to increasing Ca²⁺ mobilization and inhibition of cAMP via G_αi or G_αq coupling (26). A recent study has suggested that G_αi-induced Ca²⁺ signals are dependent on a separate G_αq-coupled signaling cascade (51). It’s possible that succinate receptor 1 signals in a similar fashion. Although normal physiological levels of extracellular succinate are insufficient to activate succinate receptor 1 (26, 27), plasma succinate is known to increase in animal disease models of diabetes (27), hypertension (52), and liver damage (53). The physiological role of succinate receptor 1 activation in the PT remains incompletely characterized.

In addition to GPCRs whose expression is consistent with prior studies, the unbiased nature of omics approaches reveal expression of receptors that have not been well explored in renal physiology. For example, the cholecystokinin (CCK) receptor (Cckar) is selectively expressed in the PT. CCK is a digestive regulatory hormone secreted by the proximal intestine, and Cckar is also highly expressed in the stomach, pancreas, and gastrointestinal smooth muscle (54). Cckar PT expression has been previously reported in mice and humans (25, 55, 56), but its function has not been fully explored. Of note, Takahashi-Iwanaga et al. (25) also reported the expression of CCK in renal medullary cells, which secrete CCK in response to body fluid volume expansion. This work suggests that CCKAR could be part of a feedback loop in response to Na⁺ and fluid balance, although these studies did not examine the effect of CCK on PT NaCl and water absorption. RNA-seq confirms that CCKAR ligand CCK is expressed in several medullary segments, namely, the descending thin limb of Henle’s loop (DTL), ascending thin limb of Henle’s loop (ATL), medullary TAL (MTAL), and inner medullary CD (IMCD) (9). Future studies involving treatment of isolated perfused PTs with CCK and measurement of transport would be a key experiment in uncovering the role of Cckar in kidney function.

Also present in the PT are adhesion G protein-coupled receptor G4 (Adgrg4), an orphan receptor, as well as lysophosphatidic acid (LPA) receptor 3/6 (Lpar3/6). Other receptors in these subfamilies are expressed in other nephron segments, and we go into more detail about trends in their expression in expression of gpcr families in the renal tubule.

There has also been increasing evidence in recent years of sex differences in kidney function, including transport regulation (57). In a single-cell RNA-seq study by Ransick et al. (14), clustering of renal tubule cells by gene expression revealed separation by sex, although only in the PT. Multiple GPCRs identified in our data as being selective for the PT also exhibited sex differences in expression, as shown in Fig. 3. Sucnr1 and Cckar, as previously described, were more highly expressed in males compared with females. Adra1a, one of multiple adrenergic receptors selectively expressed in the renal tubule, was more highly expressed in females. Future studies will be needed to determine if these GPCR sex differences directly translate to differences in tubule function and if these differences are relevant in human tissue.

Figure 3.

Sex differences in G protein-coupled receptor (GPCR) expression in the proximal tubule. Expression data from Ransick et al. (14) were compared between males and females in the initial segment of the proximal convoluted tubule (PTS1; top) and proximal straight tubule in cortical medullary rays (PTS2; bottom). Fold changes in gene expression [male/female (M/F)] for all detected GPCRs were averaged, and a 95% confidence interval was calculated. Shown here are all GPCRs outside this confidence interval. GPCRs preferentially expressed in males are shown in blue; GPCRs preferentially expressed in females are shown in red. [Adapted from data published in Ransick et al. (14).]

Thin limbs of Henle’s loop.

Functional studies in the isolated DTL and ATL are technically challenging, and so very few GPCRs have had their functions linked directly to this nephron region. GPCRs that are selectively expressed in the DTL (DTL1, DTL2, and DTL3) and ATL are provided in Supplemental Fig. S1 (all Supplemental Material is available at https://hpcwebapps.cit.nih.gov/ESBL/Database/GPCRSupplemental/). What is known about the ligands and physiological roles for receptors in the DTL and ATL is outlined in Supplemental Table S1.

TAL of Henle’s loop.

GPCRs that are selectively expressed in the TAL are shown in Fig. 4. Known ligands and physiological roles for these receptors are shown in Table 4. Four receptors were identified as being selectively expressed in the MTAL and/or cortical thick ascending limb, namely, the prostaglandin E₂ EP3 receptor (Ptger3) (58), Ca²⁺-sensing receptor (Casr) (61, 63), glucagon receptor (Gcgr) (6), and opsin 3 (Opn3). These receptors are also expressed at lower levels in the distal convoluted tubule (DCT), connecting tubule (CNT), and cortical CD (CCD). Ptger3, Casr, and Gcgr are all well-established TAL GPCRs. As elucidated by the classic studies by Morel and coworkers (3, 4, 64), Glucagon receptor along with the vasopressin and PTH receptor activate adenylyl cyclase in TAL cells consistent with signaling via G_αs (Table 4). Recent studies have corroborated these findings (60–62). Isolated perfused tubule studies have demonstrated that basolateral addition of vasopressin, PTH, and calcitonin increase NaCl absorption in the TAL (65–69). In contrast, prostaglandin E₂ inhibits NaCl absorption in the TAL, counteracting the stimulatory effect of vasopressin (67, 70). Activation of the EP3 receptor inhibits basolateral K⁺ channels, causing depolarization and thereby inhibiting NaCl reabsorption (59). Signaling by the EP3 receptor occurs via G_αi (Table 4). The Ca²⁺-sensing receptor is G_αi/G_αq coupled and inhibits voltage-dependent Ca²⁺ transport/reabsorption in the TAL (61).

Figure 4.

Thick ascending limb-selective G protein-coupled receptor (GPCRs). GPCR expression data from microdissected renal tubules were filtered based on an individual genes’ maximum expression [in transcripts per kilobase million (TPM)] in either the cortical thick ascending limb (CTAL) or medullary thick ascending limb (MTAL) compared with its expression in any other tubule segment. MTAL/CTAL expression that was 1.5-fold greater than any other tubule segment was considered selective. Entries are sorted starting with the greatest TPM ratio. Refer to Table 4 for additional information about each GPCR. ATL, thin ascending limb of the loop of Henle; CCD, cortical collecting duct; CNT, connecting tubule; DCT, distal convoluted tubule; DTL1, short descending limb of the loop of Henle; DTL2, long descending limb of the loop of Henle in the outer medulla; DTL3, long descending limb of the loop of Henle in the inner medulla; Glom, glomerulus; IMCD, inner medullary collecting duct; PTS1, initial segment of the proximal convoluted tubule; PTS2, proximal straight tubule in cortical medullary rays; PTS3, last segment of the proximal straight tubule in the outer stripe of outer medulla; OMCD, outer medullary collecting duct.

Table 4.

Thick ascending limb-selective G protein-coupled receptors

Gene Symbol	Annotation	Ligand	G Protein	Renal Physiological Process	Key References
Ptger3	Prostaglandin E receptor 3 (subtype EP3)	Prostaglandin E2	Gαi	Inhibits NaCl reabsorption	(58, 59)
Casr	Ca2+-sensing receptor	Ca2+	Gαi/Gαq	Inhibits Ca2+ reabsorption	(60, 61)
Gcgr	Glucagon receptor	Glucagon	Gαs	Stimulates adenylyl cyclase activity	(4, 6, 62)
Opn3	Opsin 3	Chromophore	Unknown	Kidney function not known

Shown are known ligands, coupled G protein α-subunits, and segment-specific physiological roles for G protein-coupled receptors selectively expressed in the thick ascending limb.

Opn3 is also expressed in the TAL and at lower levels in the DCT, CNT, and CCD. Opn3 has been characterized in brain, skin, and adipose tissue, but no functional studies have been conducted on Opn3 in the kidney (71–73). Studies have shown that opsin 3 can be light sensitive like rhodopsin, yet is expressed in tissues that are not exposed to light and has downstream functions that are not activated by light, suggesting light-independent mechanisms of opsin activation (71). These studies have yielded tools, such as a universal Opn3 reporter mouse, that could be valuable in kidney studies of this receptor.

Macula densa.

Macula densa cells are neuroepithelial-like cells that sense luminal NaCl concentration, providing a feedback signal that regulates renin secretion and tubuloglomerular feedback (74). The transcriptome of macula densa cells has recently been elucidated by single-cell RNA-seq studies in the mouse (75). These studies confirmed the expression of the arginine vasopressin 1a (V1a) receptor (76), where it mediates an indirect action to increase renin secretion. In addition, it identified two other GPCRs in macula densa cells, namely, adhesion G protein-coupled receptor F1 (Adgrf1) and thyroid stimulating hormone receptor (Tshr). The latter has previously been shown to be expressed in the kidney (77), although not specifically in the macula densa. Aside from thyroid stimulating hormone, it also acts as a receptor for the heterodimeric glycoprotein hormone GPHA2-GPHB5.

DCT and CNT.

GPCRs selectively expressed in the DCT and CNT are shown in Fig. 5. Known ligands and physiological roles for these receptors are shown in Table 5. Both the adenosine a2b receptor (Adora2b) and prostaglandin F receptor (Ptgfr) are selectively expressed in the DCT, although the latter has significant levels of expression extending into the CD. This pattern of expression is in agreement with other surveys of these receptors (79, 81).

Figure 5.

Distal convoluted tubule (DCT)- and connecting tubule (CNT)-selective G protein-coupled receptor (GPCRs). GPCR expression data from microdissected renal tubules were filtered based on an individual genes’ maximum expression [in transcripts per kilobase million (TPM)] in either the DCT or CNT compared with its expression in any other tubule segment. DCT/CNT expression that was 1.5-fold greater than any other tubule segment was considered selective. Entries are sorted starting with the greatest TPM ratio. Refer to Table 5 for additional information about each GPCR. ATL, thin ascending limb of the loop of Henle; CCD, cortical collecting duct; CTAL, cortical thick ascending limb of the loop of Henle; DTL1, short descending limb of the loop of Henle; DTL2, long descending limb of the loop of Henle in the outer medulla; DTL3, long descending limb of the loop of Henle in the inner medulla; Glom, glomerulus; IMCD, inner medullary collecting duct; MTAL, medullary thick ascending limb of the loop of Henle; PTS1, initial segment of the proximal convoluted tubule; PTS2, proximal straight tubule in cortical medullary rays; PTS3, last segment of the proximal straight tubule in the outer stripe of outer medulla; OMCD, outer medullary collecting duct.

Table 5.

Distal convoluted tubule- and connecting tubule-selective G protein-coupled receptors

Gene Symbol	Annotation	Ligand	G Protein	Cell Type	Renal Physiological Process	Key References
Distal convoluted tubule
Adora2b	Adenosine A2b receptor	Adenosine	Gαs		Induce H secretion via V-ATPase	(78–80)
Ptgfr	Prostaglandin F receptor	Prostaglandin F	Gαq/Gα11	Principal cells	Modulates K channels	(81–83)
Connecting tubule
Sctr	Secretin receptor	Secretin	Gαs	β-Intercalated cells > α-intercalated cells	Segment function not known	(84, 85)
Fzd7	Frizzled class receptor 7	Wnt	Gαs/Gαi		Also signals through Wnt, segment function not known
Oxgr1	oxoglutarate receptor 1	α-ketoglutarate	Gαq/Gα11	α-Intercalated cells > β-intercalated cells > principal cells	Segment function not known	(26, 86)

Known ligands, coupled G protein α-subunits, cell type-specific expression, and segment-specific physiological roles for G protein-coupled receptors selectively expressed in the distal convoluted tubule and connecting tubule are shown.

The effect of ADORA2B activation on DCT transport has not been studied as of this writing. However, activation of ADORA2B in medullary α-intercalated cells induced cAMP/PKA signaling to increase V-ATPase translocation, consistent with signaling through G_αs (78). In the context of hypertension, elevated levels of renal adenosine led to increased Adora2b expression, increasing hypoxia-inducible factor-1α and endothelin-1 (80), although these effects have not been linked to a particular tubule segment.

Prostaglandin F receptor has been shown to modulate K⁺ channels in the DCT (82). Its ligand, prostaglandin F_2α, is formed from prostaglandin H₂, catalyzed by the ubiquitous enzyme aldose reductase (AKR1B1/AKR1B3), which is also involved in osmotic regulation in the kidney (87). A recent study has shown that activation of PTGFR in the DCT stimulates Cl⁻ channel ClC-K2 (Clcknb) and NaCl cotransporter (NCC; Slc12a3) via protein kinase C and p38 activation (83). Ptgfr likely signals via G_αq (88, 89). Both CIC-K2 and NCC are also stimulated in models of salt-sensitive hypertension; the role of Ptgfr in this context may be of interest for future translational studies (31).

As shown in Fig. 5, the secretin receptor (Sctr) is also expressed in the CNT and to a lesser extent in the CCD. Single-cell RNA-seq studies (75) have shown expression of Sctr mRNA in both α- and β-intercalated cells but not in CNT cells, suggesting a role in acid-base transport. Similar single-cell RNA-seq studies of CD cell types also showed Sctr expression in intercalated cells but not in principal cells (12). Consistent with these results, early studies have shown that secretin can induce urinary alkalinization via increased ${\text{HCO}}_3^{-}$ excretion (90). In isolated perfused tubules, secretin treatment stimulated ${\text{HCO}}_3^{-}$ secretion via β-intercalated cells, which was significantly reduced in tubules from mice lacking cystic fibrosis transmembrane regulator (84). Secretin has also been proposed to increase water reabsorption in aquaporin-2-expressing segments, suggesting a role in CNT cells, principal cells, and/or IMCD cells (85), contrasting with the apparent absence of Sctr mRNA in aquaporin-2-expressing cells (12, 14, 75). Treatment with secretin alone or in conjunction with other compounds has been shown to ameliorate polyuria seen in X-linked nephrogenic diabetes insipidus mouse models (91). However, whether or not secretin can act as a true substitute for vasopressin by increasing water permeability remains an open question. Direct measurements of water permeability responses to secretin in isolated, perfused CDs or with in vivo perfusion of CNT segments will be required to determine whether observed effects of secretin on water excretion are direct or indirect.

As shown in Fig. 5, oxoglutarate receptor 1 (Oxgr1) is also highly expressed in the CNT and to a lesser extent in the DCT and CD, as seen in a prior study (26). Single-cell RNA-seq studies have indicated that Oxgr1 mRNA is expressed in intercalated cells, principal cells, and CNT cells, but at several-fold higher levels in intercalated cells (both α and β subtypes). In isolated perfused CCD experiments, treatment with oxoglutarate led to activation of OXGR1 to stimulate Cl⁻ reabsorption and bicarbonate secretion in a process mediated by the Cl⁻/bicarbonate exchanger pendrin (86, 92). Notably, these effects are mediated by apically localized OXGR1 in intercalated cells (92). The dependence of this effect on protein kinase C is consistent with OXGR1 signaling through G_αq (86). These effects are attributable to Oxgr1 in β-intercalated cells. Because Oxgr1 is also expressed in the other cell types of the CNT/CCD (12, 14), it seems possible that oxoglutarate has additional pendrin-independent functions in the CNT/CCD.

The Wnt receptor Frizzled class receptor 7 (Fzd7) is selectively expressed in the CNT and to a lesser extent in the DCT and CCD. Although Frizzled receptors do not typically couple to G proteins, FZD7 has been found to couple to G_αs or G_αi in different contexts (93). Canonical Wnt signaling is highly upregulated during kidney development, in particular as the ureteric bud differentiates into the CD system (94–96). Although this pathway is generally downregulated in adult tissue, upregulated Wnt signaling has been observed in chronic kidney disease and kidney injury (97–99). The role of Fzd7 in the adult kidney CNT is currently unknown (100).

Collecting duct.

GPCRs selectively expressed in the CD are shown in Fig. 6. This includes the CCD, outer medullary CD (OMCD), and IMCD. Known ligands and physiological roles for these receptors are shown in Table 6. GPCRs in this segment include the well-characterized receptors Avpr2 and prostaglandin E receptor 1 (Ptger1). In isolated perfused tubule studies, Grantham and Orloff (108) demonstrated that vasopressin increases osmotic water permeability in rabbit CCDs and that this action was mimicked by the addition of cAMP analogs and cyclic nucleotide phosphodiesterase inhibition. Later, Imbert et al. (7) demonstrated that vasopressin activates adenylyl cyclase in microdissected CDs. Although two vasopressin receptors are expressed in the CD, Avpr2 is the only one expressed in principal cells and IMCD cells, whereas the arginine vasopressin 1a receptor is abundant in α- and β-intercalated cells (12). Among the three vasopressin receptors (V1a, V1b, and V2), the V2 receptor is the only one coupled to G_αs (107). In recent years, extensive studies, including those in our laboratory, have demonstrated features of V2 receptor action in the CD. Aside from the well-studied effect of vasopressin action through the V2 receptor to speed water transport across the CD epithelium, vasopressin also increases urea transport via urea transporter (UT)-A1 and UT-A3 urea channels (111–113) and increases Na⁺ transport (114, 115) through regulation of β- and γ-epithelial Na⁺ channel subunits (116–119).

Figure 6.

Collecting duct (CD)-selective G protein-coupled receptor (GPCRs). GPCR expression data from microdissected renal tubules were filtered based on an individual genes’ maximum expression [in transcripts per kilobase million (TPM)] in either the cortical CD (CCD), outer medullary CD (OMCD), or inner medullary CD (IMCD) compared with its expression in any other tubule segment. CCD/OMCD/IMCD expression that was 1.5-fold greater than any other tubule segment was considered selective. Entries are sorted starting with the greatest TPM ratio. Refer to Table 6 for additional information about each GPCR. ATL, thin ascending limb of the loop of Henle; CNT, connecting tubule; CTAL, cortical thick ascending limb of the loop of Henle; DCT, distal convoluted tubule; DTL1, short descending limb of the loop of Henle; DTL2, long descending limb of the loop of Henle in the outer medulla; DTL3, long descending limb of the loop of Henle in the inner medulla; Glom, glomerulus; MTAL, medullary thick ascending limb of the loop of Henle; PTS1, initial segment of the proximal convoluted tubule; PTS2, proximal straight tubule in cortical medullary rays; PTS3, last segment of the proximal straight tubule in the outer stripe of outer medulla.

Table 6.

Collecting duct-selective G protein-coupled receptors

Gene Symbol	Annotation	Ligand	G Protein	Cell Type	Renal Physiological Process	Key Reference(s)
Adgrf1	Adhesion G protein-coupled receptor F1	Orphan			Kidney function not known
Ptger1	Prostaglandin E receptor 1	Prostaglandin E2	Gαq/Gα11	Principal cells	Inhibits Na+ absorption	(101, 102)
P2ry14	Purinergic receptor P2Y, G protein coupled, 14	UDP-glucose	Gαi	α-Intercalated cells	Inflammatory response	(103, 104)
Gprc5b	G protein-coupled receptor, family C, group 5, member B	Orphan		Principal cells	Kidney function not known
Adgrf5	Adhesion G protein-coupled receptor F5	Tethered agonist	Gαq/Gα11	α-Intercalated cells	V-ATPase trafficking	(105)
Npy6r	Neuropeptide Y receptor Y6	Neuropeptide Y/peptide YY/pancreatic peptide		Principal cells	Segment function not known, pseudogene in humans	(106)
Fzd1	Frizzled class receptor 1	Wnt	Gαi/Gαq	Principal cells	Also signals through Wnt
Npy1r	Neuropeptide Y receptor Y1	Neuropeptide Y/peptide YY	Gαi/Gαo		Kidney function not known	(106)
Avpr2	Arginine vasopressin receptor 2	Arginine vasopressin	Gαs	Principal cells	Water reabsorption	(7, 107, 108)
Celsr2	Cadherin, EGF LAG seven-pass G-type receptor 2	Orphan		Principal cells > β-intercalated cells > α-intercalated cells
Lpar1	Lysophosphatidic acid receptor 1	Lysophosphatidic acid	Gαi/Gαq/Gα12	Principal cells	Segment function not known
Gper1	G protein-coupled estrogen receptor 1	17β-Estradiol	Gαi/Gαo	Principal cells	Ca2+-H+-ATPase	(109)
S1pr3	Sphingosine-1-phosphate receptor 3	Sphingosine-1 phosphate	Gαi/Gαq	β-Intercalated cells	Kidney function not known	(110)

Known ligands, coupled G protein α-subunits, cell type-specific expression, and segment-specific physiological roles for G protein-coupled receptors selectively expressed in the collecting duct are shown.

As shown in Fig. 6, Ptger1, whose receptor is linked to G_αq or G_α11, is also expressed in CD cells (101, 102). Activation of this receptor inhibits water and NaCl reabsorption (102). In the CD, Ptger1 is expressed predominantly in principal cells rather than intercalated cells (12).

Frizzled class receptor 1 (Fzd1) is also selectively expressed in CD principal cells (12). A key component of the canonical Wnt signaling pathway, FZD1 binds Wnt along with coreceptors low-density lipoprotein receptor-related proteins 5 and 6 (LPR5 and LPR6, respectively). Single-cell RNA-seq studies have shown that Lpr6 is also selectively expressed in principal cells (120). As previously described, Wnt signaling plays key roles in kidney development; however, the role of Fzd1 in the adult kidney CD has not been established. In addition to its role in canonical Wnt signaling, FZD1 has also been reported to couple to either G_αi or G_αq in different contexts (93, 121, 122).

Also of interest is the purinergic receptor P2Y14 (P2rY14 gene; Fig. 6), which has been identified as being selectively expressed in α-intercalated cells (12, 103). Work by Azroyan et al. (103) has shown that activation of this receptor via UDP-glucose induces expression of proinflammatory cytokines, suggesting that purinergic receptors in α-intercalated cells are key to the regulation of inflammation in the CD. This was followed up in a study by Battistone et al. (104), who demonstrated that ablation of P2rY14 prevented the proinflammatory chemokine response after ischemia-reperfusion injury and reduced kidney damage, reinforcing this receptor’s role in the proinflammatory response. In vitro kidney cell models suggest that P2Y14 couples to G_αi, and activation leads to downstream ERK1/2 phosphorylation in human embryonic kidney and C6 cell lines, although this has not been verified in vivo (123).

As shown in Fig. 6, two neuropeptide Y (NPY) receptors, Npy1r and Npy6r, are selectively expressed in CD segments of the mouse, consistent with prior work (106). Npy6r is truncated in humans (124), where it is considered a pseudogene. NPY1R can bind a variety of ligands, including NPY, peptide YY (PYY), and pancreatic polypeptide (PP) (125, 126). Classically, NPY is secreted by renal adrenergic nerve fibers cosecreted with norepinephrine. PYY is secreted by the small intestine in response to feeding (127), and PP is secreted by γ-cells of pancreatic islets (128). Interestingly, expression of the NPY1R/NPY6R ligand NPY has been localized to the renal tubule (129), and our RNA-seq data indicate that NPY is expressed in both the DTL and OMCD, suggesting local activation of NPY receptors (9). NPY1R is coupled to G_αi, suggesting that it should inhibit cAMP production and decrease water permeability and active Na⁺ transport. Indeed, NPY was found to inhibit vasopressin-stimulated osmotic water permeability in isolated CDs (130). NPY signaling has been implicated in renal vasoconstriction and renin release, although these effects have not been linked to any NPY receptor activity in the CD (131).

Less well-established receptors selectively expressed in the CD (Fig. 6) include G protein-coupled estrogen receptor 1 (Gper1), sphingosine-1-phosphate receptor 3 (S1pr3), Adgrf1, adhesion G protein-coupled receptor F5 (Adgrf5), cadherin EGF Lag seven-pass G-type receptor 2 (Celsr2), and lysophosphatidic acid receptor 1 (Lpar1).

Most actions of estrogens are mediated by nuclear receptors that act as transcription factors, directly regulating transcription of target genes. However, a GPCR known as GPER1 or GPR30 has been identified that binds steroids, including estradiol (132, 133), and triggers intracellular Ca²⁺ mobilization and synthesis of phosphatidylinositol 3,4,5-trisphosphate (134). Prior studies have identified Gper1 in microdissected DCT, CNT, and CCD segments (109, 135), matching the distribution found by RNA-seq (Fig. 6). These tubule segments manifested 17β-estradiol-induced increases in Ca²⁺ concentration (109) consistent with GPER1 activation. Although the authors concluded that the site of Gper1 actions was in intercalated cells, recent single-cell RNA-seq analysis has identified Gper1 expression in CD principal cells, CNT cells, and DCT cells without a significant signal in intercalated cells (14). Consistent with a DCT cell site of action, GPER1 has been found to regulate thiazide-sensitive NCC of the DCT by binding aldosterone (136). Activation of GPER1 in the renal medulla has been found to cause natriuresis (selectively in female rats) via endothelin type A and B receptors, pointing to a physiological role of GPER1 in the regulation of Na⁺ and Cl⁻ excretion (137). The endothelin type B receptor is strongly expressed in the DCT and all CD segments (12).

S1pr3 is also expressed in the CD (Fig. 6). Although a function for this receptor has not been described in the CD, the functions of this receptor are well established in antigen-presenting cells in that kidney, which respond to sphingosine-1 phosphate to regulate inflammatory responses and immune cell migration (138). S1pr3 knockout mice have been described as being protected from kidney ischemia-reperfusion injury (110). S1pr3 is thought to be G_αi/G_αq coupled (139).

Of note, the most specific CD GPCR is Adgrf1, which has been shown to bind synaptamide (a DHA metabolite) in the fetal brain (140), although its function has not been explored in the kidney. In addition, Adgrf5, Celsr2, and Lpar1 are also expressed in the CD. These receptors belong to the adhesion class and lysophosphatidic acid families of GPCRs, which have multiple members expressed along the tubule and will be discussed in more detail later on. Finally, mRNA and protein expression of orphan receptor GPCR class C group 5 member B has been observed in the CD. Despite being highly expressed in the kidney CD, very little is known about this orphan receptor. Further studies are needed to uncover the function of these specific and highly expressed GPCRs.

EXPRESSION OF GPCR FAMILIES IN THE RENAL TUBULE

Adhesion GPCRs

Although we observe specific expression of well-characterized individual receptors, there are trends in multiple GPCR families expressed across the nephron. Expression of GPCR groups as well as specific localization can give us hints as to functional roles for these receptors. RNA-seq analysis has identified multiple members of the family adhesion GPCRs (aGPCRs) that are selectively expressed in tubule segments, including Adgrg4 in the PT, adhesion G protein-coupled receptors L3 and L4 (Adgrl3 and Adgrl4, respectively) in the DTL, adhesion G protein-coupled receptor E5 (Adgre5) in the ATL, and Adgrf1, Adgrf5, and Celsr2 in the CD (Fig. 7). Nine other members of this gene family are expressed more universally, including Adgrg1, by far the most highly expressed aGPCR in the nephron. Adgrg1 expression, along with Adgrf5 and adhesion G protein-coupled receptor L2 (Adgrl2), has been confirmed in rat proteomic studies (16). The role of aGPCRs in physiology and disease has been the subject of multiple reviews that provide a more detailed picture of our current understanding of this gene family (141–143).

Figure 7.

Expression of adhesion G protein-coupled receptors along the renal tubule. Colors indicate segment boundaries. CD, collecting duct; DCT, distal convoluted tubule; DTL, descending thin limb; Glom, glomerulus; PT, proximal tubule; TAL, thick ascending limb.

aGPCRs are characterized by their long extracellular domains, which contain a GPCR autoproteolysis-inducing (GAIN) domain, cleavage of which exposes a tethered ligand capable of activating the receptor (143, 144). However, aGPCRs are capable of being activated by multiple mechanisms, including exogenous ligands, mechanical stimuli, and cell-cell interactions with other external membrane proteins (145–148). ADGRG1, for example, can be activated by collagen type III (146), which is upregulated in renal fibrosis (149), and l-phenylalanine, although only at high levels (145).

To date, few aGPCR functions have been directly linked to the functions of specific kidney cell types, and many receptors remain orphans. Experimental progress has been challenged by a lack of validated antibodies and molecular tools (141). In addition, multiple knockout models for aGPCRs have been generated that have no overt renal phenotypes, suggesting redundant aGPCR functions (150, 151), although deeper analysis of renal function may yet identify functional roles. For example, mice lacking adhesion G protein-coupled receptor G3 (Adgrg3), which is expressed in the DTL and CD (9), were partially protected during acute kidney injury (152). The crystal structure of ADGRG3 as well as its binding to glucocorticoid ligands have also recently been published and may yield insights into the structure and binding properties of other aGPCRs (153). In addition, a recent study by Zaidman et al. (105) found a specific role for Adgrf5 (Gpr116) in α-intercalated cells of the CD, the cells normally responsible for urinary acidification through proton secretion (154). Adgrf5 knockout mice had acidified urine and metabolic alkalosis indicative of a static role of ADGRF5 to inhibit proton secretion, which appears to be mediated by regulation of trafficking of vacuolar H⁺-ATPase to the cell surface (105). The functions of many other aGPCRs expressed along the renal tubule are yet to be uncovered.

Adrenergic Receptors

Renal sympathetic nerve activity plays key roles in blood pressure regulation in part through control of Na⁺ and water reabsorption along the renal tubule (155). These effects are mediated by binding of circulating epinephrine and norepinephrine to adrenergic receptors. Multiple members of the adrenergic receptor family are highly and selectively expressed in distinct renal tubule segments (Fig. 8). Adrenergic receptors can be divided into three groups, roughly in accord with the heterotrimeric G protein α-subunit that they signal through, viz. α₁-adrenergic receptors through G_αq/11, α₂-adrenergic receptors through G_αi, and β-adrenergic receptors through G_αs. PTs express all three, although in different distributions (Fig. 8). In isolated proximal convoluted tubules, norepinephrine was found to strongly increase NaCl and fluid absorption (156), seemingly consistent with a role in Na⁺ balance and blood pressure regulation. The finding that α₂-adrenergic receptors counter the increase in cAMP in PTs is consistent with a potential role of these receptors in stimulation of NaCl transport (50), which occurs through an increase in apical Na⁺/H⁺ exchange (157). RNA-seq results (Fig. 8) have identified Adra2b as the receptor most likely to mediate this effect. In contrast, proximal α₁-adrenergic receptors stimulate gluconeogenesis (158), and this action is likely to be restricted to the S-1 PT based on the RNA-seq finding that Adra1a is only expressed in S-1. In isolated perfused proximal S-2 segments, norepinephrine had no effect on NaCl and fluid absorption (156), perhaps because this segment expresses both β₂- and α_2b-adrenergic receptors (Fig. 8), which have opposing effects on cAMP production.

Figure 8.

Expression of adrenergic receptors along the renal tubule. Colors indicate segment boundaries. CD, collecting duct; DCT, distal convoluted tubule; Glom, glomerulus; DTL, descending thin limb; PT, proximal tubule; TAL, thick ascending limb.

Besides the PT, additional renal tubular sites of catecholamine-mediated regulation of salt and water transport are the CD and DCT. In the CCD, α₂-adrenergic receptor activation has been shown to oppose vasopressin action by decreasing cAMP (159, 160). Isolated perfused tubule experiments in CCDs showed that the α-adrenergic agonist phenylephrine markedly inhibited the ability of vasopressin to stimulate fluid absorption and osmotic water permeability (161). The RNA-seq results showed that the α-adrenergic receptor expressed in the CD is α_2a (Adra2a; Fig. 8), i.e., a different α₂-receptor than that expressed in the PT. Inhibition of Na⁺ and water absorption by adrenergic agents in the CD would be expected to decrease blood pressure and would tend to oppose α_2b-adrenergic receptor effects in the PT to increase Na⁺ and water absorption. As shown in Fig. 8, β-adrenergic receptors (Adrb2) are also expressed in CD segments. However, a single-cell RNA-seq study (12) has indicated that CD Adrb2 expression is exclusively in intercalated cells. Consistent with this, β-adrenergic agonists increased Cl⁻ absorption in isolated perfused CCDs without affecting net Na⁺ transport or water permeability (162).

β₂-Adrenergic receptors are also expressed in the DCT. Norepinephrine, acting through β-adrenergic receptors, has been demonstrated to increase phosphorylation of NCC of the DCT at a site demonstrated to be associated with increased transport activity (163). This effect occurs in part through regulation of the basolateral K⁺ channel Kcnj10 (164). Thus, β-adrenergic activation in the DCT may contribute to the blood pressure-raising effects associated with renal sympathetic nerve activation in the kidney. In addition to this, β₁-adrenergic receptors are present at low levels throughout the TAL and DCT. Activation of these receptors is well established to regulate renin secretion (155, 165).

Thus, multiple adrenergic receptors expressed in different renal tubule segments are expected to produce a complex aggregate effect on salt and water excretion and blood pressure regulation. Beyond this, adrenergic receptors in renin-secreting juxtaglomerular granular cells, vascular smooth muscle cells, and sympathetic neurons are all likely to make their own contributions to blood pressure regulation (155). The role of renal α-adrenergic receptors in hypertension and kidney function has been reviewed in greater detail by multiple groups (166–168).

Lysophosphatidic Acid Receptors

Lysophosphatidic acid receptors (LPARs) are also widely expressed, including Lpar3/6 in the PT and Lpar1 in the CD (Fig. 9). LPA is derived from membrane lipids and is present in most tissues, and its effect is regulated by the specific tissue and cellular distribution of LPARs (169). RNA and protein expression of Lpar1 and Lpar3 has been demonstrated in these segments in prior microdissection studies in rat nephrons (11, 16). Each Lpar is capable of binding to multiple G proteins: LPAR1 can couple to G_αi, G_αq, and G_α12, LPAR3 couples with G_αi and G_αq, and LPAR6 couples with G_αi and G_α12. The precise coupling of each Lpar in the kidney is still currently unknown.

Figure 9.

Expression of lysophosphatidic acid receptors (LPARs) along the renal tubule. Colors indicate segment boundaries. CD, collecting duct; DCT, distal convoluted tubule; Glom, glomerulus; DTL, descending thin limb; PT, proximal tubule; TAL, thick ascending limb.

LPARs have been shown to play roles in kidney disease models. In models of ischemia-reperfusion injury, blockade of LPAR3 reduced injury, whereas an LPAR3 agonist enhanced kidney injury (24). These results suggested that multiple Lpars with different affinities for LPA have functions in the ischemia-reperfusion injury response. Increased expression of Lpar 1/Lpar3 has also been observed in animal models of diabetic nephropathy (170, 171). In addition, elevated levels of LPA have been correlated with renal dysfunction (170).

To date, these effects have not been linked to a particular LPAR, nephron segment, or cell type. Treatment of microdissected tubules with LPA, in combination with pharmacological inhibitors and engineered in vivo knockout models, would be informative as to the direct effects of LPARs on transport across the nephron and could provide insights to the role of these receptors in disease states. In addition, LPAR-mediated signals interact with other pathways such as EGF or ANG II (172). The effects of LPAR activation in these discrete contexts remains an open question.

Olfactory Receptors Along the Nephron

Olfactory receptors (ORs) are the largest family of GPCRs, with over 1,109 mouse genes represented in our complete GPCR list. In recent years, ORs have been shown to have wide-ranging expression in nonolfactory tissues, where they have functional roles as chemosensors (173–175). Here, we show all ORs that have a TPM of 10 or greater at any point along the nephron, yielding 19 ORs (Supplemental Fig. S2). To our knowledge, these most highly expressed ORs have not been characterized in the kidney. In addition, 17 of 19 ORs with a maximum TPM greater than 10 are most highly expressed in DTL1, although the highest expressing OR found in our RNA-seq data set was Olfr373, an orphan receptor almost exclusively expressed in IMCD cells. The vast majority of ORs are still orphans with no known ligand, and creating specific antibodies is challenging due to a high level of homology between ORs (176). Despite this, multiple olfactory receptors have been identified and characterized to have functional roles in the kidney (177–181). The functional role of these orphan ORs represents a largely unanswered and potentially important physiological question.

RESOLVING DIFFERENT APPROACHES TO GPCR DETECTION

The expression of well-established receptors (Pth1r, Avpr2, Gcgr, etc.) has largely been confirmed by unbiased omics studies; however, discrepancies are inevitable when comparing results from different methodologies. One notable omission from our data is the expression of any of the dopamine receptors (Drd1–Drd5). Dopamine is a catecholamine that has been studied for decades as a vasodilator and has been shown to have natriuretic effects via interactions with dopamine receptors (182). However, we did not detect expression of any of the dopamine receptors in the renal tubule (9). These receptors have also not been reported in the tubule in other gene expression studies (14, 15). It is unknown why these receptors remain undetected in multiple omics datasets, despite well-established roles for dopamine in the kidney. It is possible that dopamine effects are mediated by adrenergic receptors, as cross talk has been observed in other tissues (183–185). In another example, we identified Agtr1a in the PT, but expression of Agtr1a was virtually zero outside of the PT. Nevertheless, multiple studies have identified ANG II actions in the TAL, DCT, and CD, where it has been implicated to play roles in salt and water transport (186–190). Although RNA-seq studies typically have a high degree of sensitivity, it is possible that transcript levels for these receptors are below the level of detection in these experiments while simultaneously producing enough protein to mediate receptor function. This could be true if Agtr1a protein had an exceptionally long half-life. As with dopamine, it is also possible that another receptor is responding to the same agonist that is yet to be identified. Resolving these discrepancies will require further functional studies to verify expression of these receptors and what their roles are.

FUNCTIONAL SIGNIFICANCE

Our conclusions are primarily drawn from RNA-seq data, which can provide a more accurate representation of GPCR expression along the renal tubule than can be achieved by proteomic methods due to greater sensitivity. Hypotheses from transcriptomic and proteomic studies ultimately will require follow-up functional studies to learn the true role of the newly identified receptors in the kidney. We therefore cross referenced our findings with other omics datasets including single-cell RNA-seq studies (14, 75), which are useful for identifying GPCR expression patterns in cellular subpopulations. By conducting differential gene expression analysis using kidney cell type-specific markers (12), localization as well as cell type-specific GPCR expression can be confirmed. We have also cross referenced this data with prior proteomics data from microdissected rat kidneys (16). Proteomics approaches to detecting GPCR expression are limited, and a lower level of GPCR expression than proteomic detection thresholds may be sufficient to affect physiological function. In addition, a general lack of specific validated antibodies can make detection with traditional biochemical approaches such as Western blot analysis difficult. Ultimately, neither mRNA nor protein expression necessarily indicate functional activity. Additional factors such as downstream signaling targets are also yet to be uncovered for many of these receptors. For many GPCRs, further experimental validation in animal models is needed to accurately determine receptor function.

CONCLUSIONS

In this review, we provide a new resource for the study of GPCRs in the renal tubule. Using a recently published transcriptome for mouse renal tubule segments, we created a comprehensive list of GPCRs and ORs expressed in the mouse and where they are expressed. This GPCR expression set will help to form new hypotheses for future independent studies to validate GPCR expression and function in cellular and animal models. Given that GPCRs are the most targeted gene group for therapeutics, we hope this will encourage study of lesser known GPCRs that will lead to new discoveries.

SUPPLEMENTAL DATA

Supplemental Figs. S1–S3 and Supplemental Table S1: https://hpcwebapps.cit.nih.gov/ESBL/Database/GPCRSupplemental/.

GRANTS

This work was primarily funded by National Heart, Lung, and Blood Institute Grants ZIAHL001285 and ZIAHL006129 (to M.A.K.). This work was also supported through a National Institutes of Health Bench-to-Bedside award from the National Institutes of Health Office of Clinical Research.

DISCLOSURES

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

AUTHOR CONTRIBUTIONS

B.G.P., L.C., and M.A.K. conceived and designed research; B.G.P., L.C., C-L.C., and M.A.K. analyzed data; B.G.P. and M.A.K. prepared figures; B.G.P., L.C., C-L.C., V.R., and M.A.K. drafted manuscript; B.G.P., L.C., C-L.C., V.R., and M.A.K. edited and revised manuscript; B.G.P., L.C., C-L.C., V.R., and M.A.K. approved final version of manuscript.

APPENDIX

Curation of a Comprehensive List of GPCR Genes in the Mouse, Human, and Rat Genomes

We curated a complete list of mouse GPCRs using multiple online databases. This list includes data from the International Union of Basic and Clinical Pharmacology/British Pharmacological Society (IUPHAR/BPS) (https://www.guidetopharmacology.org/GRAC/GPCRListForward?class=A), Mouse Genome Informatics (MGI; http://www.informatics.jax.org/), and Ensembl Biomart (https://m.ensembl.org/info/data/biomart/index.html). Human and rat orthologs were mapped from data obtained from Ensembl Biomart and the Rat Genome Database (RGD; https://rgd.mcw.edu/). The resulting GPCR lists for the mouse, rat, and human genomes are provided at https://hpcwebapps.cit.nih.gov/ESBL/Database/GPCRs/. This list contains 758 mouse GPCRs and their human and rat orthologs (Fig. 1A).

This list does not include ORs, which we provide as a separate list. For mouse ORs, we obtained a gene list searching for “olfactory receptors” from MGI (http://www.informatics.jax.org/searchtool/Search.do?query=Olfactory+receptor&submit=Quick%0D%0ASearch), yielding 1,109 gene symbols after we filtered out the pseudogenes. We mapped the mouse ORs to human and rat ORs using tools at the Human Genome Organization Gene Nomenclature Committee website (https://www.genenames.org/data/genegroup/#!/group/139) and RGD. This curated list is provided at https://hpcwebapps.cit.nih.gov/ESBL/Database/GPCRs/MouseHumanRatORs.html. We used the comprehensive GPCR and OR lists to extract expression data from our omics datasets, thus providing a complete picture of GPCR expression.

Sorting for GPCRs Selectively Expressed in Renal Tubule Segments

The curated lists of GPCRs and ORs were mapped to RNA-seq data from microdissected renal tubule segments from the mouse generated by Chen et al. (9) (Fig. 1B). This yielded matches to 758 GPCRs and 1,109 ORs across 14 tubule segments as well as the glomerulus. Expression levels for all GPCRs can be found at https://hpcwebapps.cit.nih.gov/ESBL/Database/GPCRs/TubuleTPM.html. GPCRs with a maximum TPM of 10 or less were filtered out to eliminate low-expressing receptors. Only 104 GPCRs and 19 ORs had TPM values of greater than 10 (Fig. 1C). From this, GPCR expression was ranked in each tubule segment (top 10 GPCRs for each segment shown in Table 1). Several top expressed GPCRs were present ubiquitously across all segments, suggesting that these receptors are less responsible for individual segment functions. For this review, we wanted to highlight understudied receptors that have distinct patterns of expression that suggest functional roles. To determine nephron segment-selective expression of GPCRs, we identified the TPM values for each gene in a particular nephron segment group. Groups of nephron segments were combined and analyzed by “regions” as follows: the PT, which includes PTS1, PTS2, and PTS3; DTL, which includes DTL1, DLT2, and DTL3; ATL; MTAL and cortical TAL; DCT; CNT; and CD, which includes the CCD, OMCD, and IMCD. To identify GPCRs selectively expressed in these regions, we compared maximum TPM values for segments within the region with maximum TPM values for segments outside the region (Fig. 1D). A ratio of these two values of 1.5 or greater was considered to be selective for a particular region. These GPCRs are presented based on the segment in which they are selectively expressed (Figs. 2, 3, 4, 5, 6).

Note that glomerular GPCRs were not covered in this review. GPCRs selectively expressed in the glomerulus are provided in Supplemental Fig. S3. Multiple transcriptomic studies have been published recently focusing on the glomerulus, which provide a more complete picture of the glomerular expression landscape (191, 192).

Selectively expressed GPCRs were matched to known ligands, coupled G proteins, and known physiological roles in the corresponding nephron segment. Ligand and G protein coupling data were obtained from the IUPHAR/BPS database (https://www.guidetopharmacology.org) and the GPCRdb database (193) (see Tables 3–6 and Supplemental Table S1). The curated GPCR list was also mapped to proteomics data obtained from microdissected nephron segments of Sprague–Dawley rats, published in Ref. 16 and available at https://esbl.nhlbi.nih.gov/KTEA/, which yielded 27 GPCRs. The curated GPCR list was also mapped to single-cell RNA-seq data from intercalated and principal cells from mouse CDs, published in Ref. 12 and available at https://hpcwebapps.cit.nih.gov/ESBL/Database/scRNA-Seq/, which yielded 40 GPCRs.