β₂-Adrenergic receptor agonism as a therapeutic strategy for kidney disease

Abstract

Approximately 14% of the general population suffer from chronic kidney disease that can lead to acute kidney injury (AKI), a condition with up to 50% mortality for which there is no effective treatment. Hypertension, diabetes, and cardiovascular disease are the main comorbidities, and more than 660,000 Americans have kidney failure. β₂-Adrenergic receptors (β₂ARs) have been extensively studied in association with lung and cardiovascular disease, but with limited scope in kidney and renal diseases. β₂ARs are expressed in multiple parts of the kidney including proximal and distal convoluted tubules, glomeruli, and podocytes. Classical and noncanonical β₂AR signaling pathways interface with other intracellular mechanisms in the kidney to regulate important cellular functions including renal blood flow, electrolyte balance and salt handling, and tubular function that in turn exert control over critical physiology and pathology such as blood pressure and inflammatory responses. Nephroprotection through activation of β₂ARs has surfaced as a promising field of investigation; however, there is limited data on the pharmacology and potential side effects of renal β₂AR modulation. Here, we provide updates on some of the major areas of preclinical kidney research involving β₂AR signaling that have advanced to describe molecular pathways and identify potential drug targets some of which are currently under clinical development for the treatment of kidney-related diseases.

INTRODUCTION

β-Adrenergic receptors (βARs) are a class of G protein-coupled receptors (GPCRs) that belong to the superfamily of seven-transmembrane cell-surface receptors. There are three subtypes of βARs including β₁, β₂, and β₃. In the classical pathway, β₂ARs couple with G_s proteins to activate adenylyl cyclase that converts ATP to the second messenger cAMP that in turn phosphorylates and activates protein kinase A (PKA) signaling (1–4). However, β₂ARs also couple with G_i proteins through a noncanonical pathway that signals via phosphoinositide 3-kinases (PI3Ks) and AKT (protein kinase B) (5). β₂ARs are known to be expressed in multiple parts of the kidney including proximal and distal convoluted tubules, glomeruli, and podocytes (6–9). Canonical and noncanonical β₂AR signaling pathways interface with other intracellular mechanisms in the kidney to regulate a range of critical physiological functions including renal blood flow, electrolyte balance and salt handling, glomerular filtration rate, and renin secretion from juxtaglomerular granular cells (10). Agents that selectively modulate β₂ARs in the kidney are being explored for nephroprotection; however, there is limited data on the pharmacology and potential side effects of such renal β₂AR modulation. Here, we review the current knowledge and implications of renal β₂ARs and their roles in physiology and disease. Key findings of the major studies reviewed here are presented in a schematic figure for each section and are collectively summarized in Table 1.

Table 1.

Summary of renal β₂AR activity in the literature

No.	Cited Reference	Renal Condition/Study Model	β2AR Activity
1	Vandongen et al. (11)	Effects of β-AR modulation on renin secretion in isolated perfused rat kidney	Increased renin secretion
2	Grospietsch et al. (12)	Changes in hormones and salt handing following long-term infusion of β2-agonist fenoterol and calcium channel blocker verapamil in pregnant rabbits	Increased plasma renin and antidiuretic hormone, and reduced plasma aldosterone, urinary sodium, potassium excretion, and clearance of creatinine
3	Grospietsch et al. (13)	Changes in urine and electrolyte balance following a bolus injection of β2-agonist fenoterol in female rabbits	β2-Agonism had antidiuretic effect, increased urinary osmolarity, and reduced sodium, potassium, and urine excretion
4	Singh et al. (14)	Effects of β2 blocker ICI-118551 on tubular function in cultured rat proximal tubule epithelial cells	Increased the activity of Na-K-ATPase and apical sodium entry independent of the cAMP and β2-antagonism decreased it
5	Singh et al. (15)	The role of protein kinase C (PKC) in β2AR-induced Na+/K+-ATPase activity and sodium transport in cultured rat proximal tubule cells	PKC mediated the β2AR-induced activity of Na-K-ATPase and apical sodium entry
6	Nakamura et al. (16)	Dose-response study of β2-agonist terbutaline on lipopolysaccharide (LPS)-induced IL-6 production in primary rat renal macrophage cells	β2-Agonism decreased LPS-induced IL-6 production at low concentration via an independent-cAMP suppression of TNF-α and MAPK and increased it at high concentration via a PKA-cAMP pathway
7	Nakamura et al. (17)	Effects of terbutaline, a β2-AR agonist, on Shiga toxin (Stx)2-induced apoptosis in adenocarcinoma-derived (ACHN) cells, as a model of human renal tubular epithelial cells	β2-Agonism dose-dependently inhibited Shiga toxin (Stx)2-induced apoptosis through cAMP-PKA pathway and p38MAPK cascade
8	Nakamura et al. (18, 19)	The role of β2-ARs in a rat model of endotoxin-induced acute renal failure	Adenoviral delivery of β2-ARs increased GFR and sodium reabsorption
9	Nakamura et al. (20)	To determine the role of β2-AR in renal pathophysiology in Wistar rats over-expressing renal β2-AR and by analyzing intrarenal β2-AR expression in 34 children and the changes in serum creatinine levels of 99 children who received beta2-AR agonists	Treatment with terbutaline in rats overexpressing renal β2AR reduced glomerular function and systemic blood pressure to a greater extent and was associated with higher mortality. Clinically, renal β2AR expression slowly increases with age and was upregulated by steroid therapy and may increase the risk of renal side effects in children receiving β2-agonists.
10	Nakamura et al. (21)	The role of β2-inhibitor ICI118,551 in pathology of LPS-induced septic rats	The combination of LPS and the β2-antagonist increased renal TNFα, CD14, TLR4 and decreased Gsα and cAMP.
11	Nakamura et al. (22)	The effect of kidney-specific β2-AR gene delivery on kidney function in endotoxemic rat model of acute renal failure (ARF) induced by renal artery occlusion and subcutaneous injections of Escherichia coli	Renal-specific overexpression of β2-AR improved renal dysfunction and declined the reduction rate of creatinine clearance and decreased expression levels of cannabinoid-1 (CB-1) receptor, CD14, TLR4, and TNF-α protein in the kidney
12	Chen et al. (23)	Effect of β2-AR gene variants on glomerular filtration rate (GFR) decline rate in 580 African-American participants with progressive hypertensive nephropathy	Gly16Arg gene variant in haplotype-3 significantly correlated with the risk of end-stage renal disease
13	Mu et al. (24)	The role of β2-AR signaling on the pathology of salt-sensitive rat model of hypertension	β2-AR activation leads to Na+ retention and finally salt-dependent hypertension via downregulation of WNK4
14	Wills et al. (25)	Effects of β-agonists on mitochondrial biogenesis in primary renal proximal tubule cells (RPTC) isolated from female New Zealand white rabbits and primary adult feline cardiomyocytes (AFC)	β2-AR activation increased mitochondrial function in both cell lines
15	Wills et al. (25)	The in vivo changes of mitochondrial function following daily intraperitoneal injections of formoterol for 1 or 3 days	β2-AR agonism induced mitochondrial biogenesis, elevation in mtDNA copy number, and transcription of genes associated with mitochondrial electron transport chain in renal and cardiac tissues
16	Jesinkey et al. (26)	Effect of formoterol, on mitochondrial biogenesis and restoration of renal function in rats with ischemia-reperfusion induced acute kidney injury	β2-AR activation caused a rapid recovery of serum creatinine levels. It also reduced kidney injury molecule (KIM-1) levels and improved histological appearance and mitochondrial function
17	Li et al. (27)	The mechanism of cAMP-mediated protection in adriamycin (ADR) and puromycin aminonucleoside (PAN)-induced podocyte injury model	cAMP protected against PAN-induced podocyte loss via PKA. PKA signaling protected podocytes by inducing mitochondrial fusion. In vivo forskolin improved ADR-induced proteinuria and podocyte injury.
18	Wang et al. (28)	The role autoantibodies against β1, β2, and α1-AR in incidence of chronic cardiorenal syndrome (CRS) in 98 participants including 30 chronic cardiorenal syndrome CRS patients, 30 heart failure patients without kidney disease, and 38 healthy individuals	There is a strong correlation between the titer of auto-antibodies against β1, β2, and α1-AR and incidence of chronic cardiorenal syndrome (CRS)
19	Fujiu et al. (29)	The role of β2ARs in heart-brain-kidney interactions in response to pressure overload in a mouse model of transaortic constriction-induced heart failure	β2ARs in collecting duct cells are central to a proper hypertrophic response to pressure overload
20	Noh et al. (30)	Effects of β2AR agonists on TNF-α production in vitro (in phorbol myristate acetate-induced rat bone marrow macrophages and PBMCs isolated from streptozotocin-induced diabetic rats) and on diabetic nephropathy in vivo in Zucker diabetic fatty rats	β2-Agonism decreased TNF-a production in vitro and reduced monocyte activation, proinflammatory, and profibrotic responses in the kidneys in vivo
21	Cameron et al. (31)	Induction of mitochondrial biogenesis by formoterol	Unique structural features of formoterol allow it to interact with the β2-AR to activate the Gβγ-Akt-eNOS-sGC pathway to induce mitochondrial biogenesis
22	Stallaert et al. (32)	The influence of β2-AR on intracellular Ca2+ in HEK293S cells	Increased intracellular Ca2+ via activation of a IP3-dependent pathway through stimulating Gq-coupled P2Y purinergic receptors
23	Arif et al. (33)	The impact of formoterol, a long-acting β2-AR agonist on restoration of glomerular filtration in injured podocytes in acute nephrotoxic serum nephritis and chronic adriamycin glomerulopathy mouse models	Formoterol restored glomerular filtration via improving mitochondrial biogenesis in injured podocytes
24	Cameron et al. (34)	Investigating the cell type responsible for formoterol-induced recovery of renal function in mice after ischemia-reperfusion (IR) injury as a model of acute kidney injury (AKI) using mice with proximal tubule-specific knockout of the β2AR	Renal proximal tubule cells are required for formoterol-mediated protection against AKI
25	Ha et al. (35)	The prognostic value of β2AR gene (ADRB2) in patients with clear cell renal cell carcinoma (ccRCC)	Reduced ADRB2 expression is associated with poor prognosis in ccRCC patients
26	Zicha et al. (36)	Disputing the role of renal β2-adrenergic-WNK4-NCC pathway important in salt hypertension of Dahl rats	Propanolol failed to reduce hypertension in salt-sensitive rat suggesting no essential role for a renal βAR-WNK4-NCC pathway in pathogenesis and/or maintenance of salt hypertension in Dahl rats
27	Frame et al. (37)	β2-AR involvement in the sympathetic regulation of NCC in norepinephrine-evoked salt-sensitive hypertension in Sprague–Dawley rats	Norepinephrine-evoked hypertension is mostly mediated via β-adrenoceptor activation independently of the regulation of NCC.
28	Bhargava et al. (38)	The mechanism of formoterol-induced mitochondrial biogenesis in renal proximal tubule cells (RPTCs) isolated from female New Zealand White rabbit kidneys	β2AR agonist increased mitochondrial biogenesis (MB) through sGC/cGMP/PKG/p38/PGC-1 pathway
29	Cleveland et al. (39)	The role of formoterol on renal cortical mitochondrial function and energy production in diabetic db/db mice and in renal proximal tubule cells treated with high glucose	Formoterol restored expression of electron transport chain proteins in complexes I, II, III, and V and increased ATP levels in the renal cortex which were impaired in untreated db/db mice and glucose-exposed RPTC.

β₂AR, β₂ adrenergic receptors; cAMP, adenosine 3′,5′-cyclic monophosphate; CD, collecting duct; eNOS, endothelial nitric oxide synthase; GFR, glomerular filtration rate; IL-6, interleukin-6; MAPK, mitogen-activated protein kinase; NCC, sodium chloride cotransporter; PKA, protein kinase A; sGC, soluble guanylyl cyclase; TLR4, Toll-like receptor 4; TNF-α, tumor necrosis factor α; WNK4, with-no-lysine (K) kinase 4.

β₂AR SIGNALING AND RENAL MITOCHONDRIAL FUNCTION

It is known that renal proximal tubular cells (RPTCs) contribute importantly to the pathogenesis of acute kidney injury (AKI) (40). Wills et al. (25) first demonstrated that β₂AR agonism increased mitochondrial biogenesis (MB) in the kidney. To investigate the effects of β₂AR signaling on MB in vitro, primary renal proximal tubule cells (RPTC) isolated from female New Zealand white rabbits and primary adult feline cardiomyocytes were treated with βAR agonists. Formoterol, a long-acting β₂-selective agonist, but neither isoproterenol (a nonspecific β-agonist) nor BRL 37244 (a selective β₃-agonist), significantly increased carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP)-uncoupled oxygen consumption rate (OCR), indicating augmented maximal electron transport chain (ETC) capacity and suggestive of higher ETC components. The enhancement of OCR was abrogated by selective β₂-antagonist ICI-118551 and nonselective β-blocker propranolol. Treatment of RPTCs with 30 nM formoterol increased mitochondrial DNA (mtDNA) copy number by 250% consistent with increased MB. Confirming these findings, daily intraperitoneal injections of mice with 100 μg/kg formoterol for 1–3 days, also increased mtDNA copy number, as well as the transcript levels of the master regulator of MB, peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), and several ETC genes in renal and cardiac tissues. The results supported pivotal roles for β₂-AR signaling in the regulation of MB in the heart and kidney (25).

To determine whether β₂ agonism restored acutely damaged kidney function by activating MB, Jesinkey et al. (26) subjected C57BL/6 mice to renal ischemia-reperfusion (IR) injury, followed by daily injections of formoterol (0.3 mg/kg) or vehicle (0.3% DMSO) delivered intraperitoneally for 5 days, and serially measured serum creatinine as an indicator of kidney function. Creatinine levels in the formoterol treatment group, but not control placebos were normalized after 5 days. Consistent with a reparative role for formoterol, levels of the kidney injury molecule (KIM-1) in cortical lysates from IR mice in the formoterol treatment group were significantly reduced relative to vehicle-treated mice, and formoterol treatment markedly reduced tubular necrosis as evidenced by histology at 5 days relative to the placebo IR group. State III respiration of renal mitochondria isolated after 5 days of treatment was also normalized in the IR-formoterol treatment group but remained depressed in the corresponding placebo group, suggesting reversal of mitochondrial dysfunction by formoterol. Levels of ETC proteins, including nuclear-encoded NDUFB8 and mitochondrial-encoded COX I, significantly reduced in renal mitochondria from 5-day IR placebo group, were normalized by formoterol treatment. The study concluded that formoterol administration after IR injury confers rapid renal repair of acute injury by mechanism(s) that include a central role for enhanced MB and augmented mitochondrial function.

The results of Jesinkey et al. (26) were confirmed and extended by Cameron et al. (34) who examined the role of RPTC β₂ARs in formoterol-induced recovery of renal and mitochondrial function after acute renal injury. Male C57Bl/6 mice with genetic deletion of proximal tubule-specific β₂AR and control wild-type (WT) mice underwent bilateral IR surgery, followed by daily intraperitoneal injections of 0.3 mg/kg formoterol beginning at 24 h postsurgery. Consistent with Jesinkey et al. (26), they found that serum creatinine and KIM-1 protein levels were normalized in WT mice after 5 days of treatment with formoterol, but not in the placebo group. Furthermore, whereas β₂AR knockout mice sustained similar levels of renal injury as WT mice, the reparative actions of formoterol treatment were eliminated in β₂AR knockout animals, as evidenced by sustained elevated levels of serum creatine, KIM-1, and tubular necrosis at 5 days. PCR and immunoblot assays were used to quantify mtDNA and proteins, respectively. Formoterol treatment was found to normalize the mtDNA content of renal mitochondria in the WT-IR-formoterol treatment group, whereas it remained depressed in the β₂AR knockout (KO)-IR-formoterol group. Similarly, IR β₂AR KO mice in the formoterol treatment group did not recover normal levels of ETC proteins NDUFS1 or COX1, markers of MB. Interestingly, the expression of NDUFS1 was increased in KO sham mice, a result that the authors interpreted to imply β₂AR-regulation of mitochondrial proteins in the physiological as well as in the injury state. However, such an interpretation seems at variance with the authors’ overall conclusion that β₂AR positively regulates renal MB. The authors went on to show that formoterol also restored mitochondrial fission and fusion proteins dynamin-related protein 1 (Drp1) and mitofusin (Mfn2), respectively, in WT but not in KO mice, results they suggest is consistent with formoterol-induced activation of PGC-1α (42–44). Electron microscopy confirmed that reductions of renal cortical mitochondrial number and mitochondrial area after IR were restored in the WT formoterol treatment group. Sham KO mice also had significantly fewer mitochondria, consistent with a positive role for β₂AR in physiological regulation of MB in RPTCs. Taken together with previous work by others and themselves, the authors conclude that formoterol drives recovery from kidney injury by stimulating RPTC β₂ARs and inducing MB through a pathway that includes activation of Akt and nitric oxide synthases (NOS) by Gβγ- and PI3K-dependent mechanism(s) (31). Although histological and protein studies suggested roles for β₂ARs in physiological mitochondrial homeostasis in RPTCs, the authors reported no difference in overall renal function or histopathology of sham-operated ADRB2Flox/Flox versus γGT-Cre:ADRB2Flox/Flox KO mice.

Cleveland et al. (45) extended the results to describe the effects of formoterol on the renal cortex and mitochondrial functions during diabetes. RPTCs exposed to high glucose exhibited increased ETC complex I, II, III, and V protein levels, increased maximal uncoupled mitochondrial respiration, and reduced ATP compared with controls. Similarly, renal cortex tissues from diabetic db/db versus nondiabetic db/m control mice also contained elevated levels of ETC I, III, and V and decreased ATP. In both cases, the effects were normalized by appropriate treatment with formoterol. The authors address this apparent paradox by suggesting that despite increased expression of ETC components, the diabetic environment creates dysfunctional mitochodnria, a state that is alleviated by formoterol perhaps by orchestrating a more organized and coordinated MB program. Diabetes or simulated hyperglycemia also conferred dysregulated expression and/or phosphorylation of the mitochondrial fission/fusion proteins Drp1 and Mfn1, effects that were also restored to control levels by formoterol. Mitochondrial homeostasis results from fusion and/or fission of pre-existing mitochondria, degradation of dysfunctional mitochondria via mitophagy or the ubiquitin proteasome system, and generation of new mitochondria from existing mitochondria (46–49). The authors conclude that exposure of RPTCs to a diabetic environment confers a pro-fission state wherein mitochondrial bioenergetic and dynamic profiles are adversely altered in ways that can be reversed by treatment with formoterol. They suggest possible therapeutic applications of formoterol for early-stage diabetic kidney disease with the caution that chronic administration could lead to impairment of cardiac relaxation, increased heart rate and blood pressure, as well as reduced mitochondrial protein synthesis and oxidative capacity (50).

By employing selective activators and inhibitors of β₂ARs and putative downstream targets, PKG, p38, and PGC-1 in primary rabbit kidney RPTCs, Bhargava et al. (38) proposed a signaling pathway for the soluble guanylyl cyclase-dependent induction of MB by formoterol. They reported that formoterol mediates the phosphorylation of p38 through cGMP-dependent stimulation of PKG that in turn phosphorylates PGC-1α on serine/threonine thereby conferring increased stability and nuclear translocation (38). The pathway is consistent with other work by this and other groups supporting central roles for cGMP and cAMP in driving β₂AR agonist-mediated MB, including the demonstration of enhanced MB in the renal cortex of mice driven by PDE3 inhibitors cilostamide and trequinsin and PDE5 inhibitor sildenafil (51). The proposed linear signaling pathway of sGC/cGMP/PKG/p38/PGC-1 does not necessarily preclude additional roles for ERK1/2, PI3K, GSK3, Akt or PKA/cAMP in the regulation of MB through PGC-1α mediated by β₂ agonists in the kidney.

Formoterol was also reported to restore glomerular filtration in mice by activating MB in injured podocytes after acute nephrotoxic serum nephritis and chronic adriamycin (ADR)-induced glomerulopathy (33). In these mouse models of focal and segmental glomerulosclerosis (FSGS) and podocytopathy, formoterol treatment enhanced podocyte MB as evidenced by increased mtDNA, PGC-1α, and ETC protein levels, restoration of the actin cytoskeleton, and normalization of the mitochondrial OCR. Formoterol-mediated repair of albuminuria-injured podocytes was also observed histologically. The authors conclude that the ability of formoterol to induce rapid recovery of damaged podocytes and glomerular function presents a novel therapeutic alternative for glomerular disease including FSGS where current treatments are of limited efficacy (52, 53). The results also suggest that MB via β₂AR agonism may be preferable to more direct augmentation of PGC-1α that can be detrimental to podocytes (54). The limited number of models tested so far means that further preclinical studies are required to determine whether the approach can be broadly applied to other glomerular diseases. In this study, the authors do not address or comment on the molecular pathway for the actions of formoterol on injured podocytes or whether the enhanced activity of PGC-1α involves cGMP and/or cAMP-dependent signaling.

Protein kinase A/CREB/cAMP signaling is implicated in protecting podocytes against injury by albuminuria induced by adriamycin (ADR) or puromycin aminonucleoside (PAN) exposure by enhancing MB (27, 55). In the former, ADR-increased urine excretion was normalized in mice by pretreatment with adenylate cyclase activator forskolin, in parallel with increased cAMP production. Histological analysis revealed that ADR-treated mice suffered effacement, retraction, and widening of podocyte foot processes that were reduced by forskolin treatment. Forskolin treatment mediated increased glomerular podocyte cell numbers and levels of podocyte-specific markers [Wilm’s tumor suppressor gene 1 (WT-1)] in ADR mice relative to placebo groups. PKA/cAMP agonism also prevented podocyte loss by puromycin aminonucleoside (PAN), an antibiotic that selectively confers damage to glomerular podocytes. Podocyte protection was mediated at least in part by PKA/cAMP-mediated activation of MB by pathways directed at PGC-1α and the mitochondrial fusion protein mitofusin (Mfn-1). Mfn-1 expression, reduced by PAN and ADR was normalized by forskolin or PKA agonist 8-(4-chlorophenylthio) adenosine 3′,5′-cyclic monophosphate-cAMP (pCPT-cAMP) treatment (27). In ADR-treated mice, pCPT-cAMP also enhanced mitochondrial function and augmented the levels of ETC proteins. By applying chromatin immunoprecipitation (Chip) assays, the authors demonstrated enrichment of CREB on PGC-1α and ND3 promoters, suggesting that these promoters are CREB targets (55).

MB is subjected to multiple and complex regulation through the family of PGC-1 proteins that may be stimulus- and tissue-specific [(42); reviewed by Popov (48) and Bhargava and Schnellmann (56)]. Different studies have identified components of several potential activation pathways that include cAMP/CREB (57, 58), sGC/cGMP/PKG/p38 (38, 51), AMPK/HAT1 (59, 60), SIRT1/3 (61–64), Gβγ/Akt/eNOS/sGC (31, 34), MEK1/2/Akt/FoxO3a (65, 66). Other studies have documented negative regulation by inflammatory mediators (tumor necrosis factor, TNF family) and TGF-β1, also acting at the level of PGC-1α (42). The cumulative evidence defines cell-specific pathways for β₂AR-regulation of MB initiated by cAMP and/or cGMP signaling in kidney cortex, RPTC, and podocytes with PGC-1α as the main target and downstream effects on mitochondrial ETC components, fusion/fission regulators transcription/translation machinery, DNA replication, and MB. β₂AR modulators and/or their downstream effectors clearly provide exciting possibilities for new pharmacological approaches to treat glomerular diseases. Figure 1 presents the summary of the role of β₂AR signaling in MB.

Figure 1.

Role of β₂ adrenergic receptor (β₂AR) signaling in mitochondrial biogenesis. β₂AR stimulation by formoterol leads to mitochondrial biogenesis via a myriad of pathways demonstrated in several animal models and primary renal cells (left). This is also achieved downstream in the β₂-adrenergic signaling pathway by the adenylate cyclase activator, forskolin as demonstrated (right). ADR: adriamycin; cGMP, guanosine 3′,5′-cyclic monophosphate; CREB, cAMP response element-binding protein; Drp-1, dynamin-related protein 1; ETC, electron transport chain; IR, ischemia reperfusion; KIM-1, kidney injury molecule-1; Mfn1, mitofusin 1; mtDNA #, mitochondrial DNA copy number; OCR, oxygen consumption rate; PAN, puromycin-aminonucleoside; PGC1-α, peroxisome proliferator-activated receptor γ coactivator 1-α; PI3K, phosphatidylinositol 3′-kinase; PKA, protein kinase A; PKG, protein kinase G; RPTCs, renal proximal tubular cells; sGC, soluble guanylyl cyclase.

β₂AR SIGNALING AND SALT HANDLING/ELECTROLYTE BALANCE

Direct roles for β-ARs in renin secretion have been recognized for almost 50 years. By applying a range of adrenergic receptor agonists and antagonists, and glucagon in a perfused kidney model, Vandongen et al. (11) first demonstrated an involvement of β-ARs in renal renin secretion. Shortly thereafter, Grospietsch et al. (12) reported that coadministration of β₂AR agonist fenoterol and calcium channel blocker verapamil during tocolytic therapy leads to markedly increased plasma renin and antidiuretic hormones and reduced plasma aldosterone. Coincidentally, urinary sodium and potassium excretion, and clearance of creatinine were significantly reduced. Studies from the same group reported that bolus fenoterol treatments of female rabbits markedly increased antidiuresis with elevated urinary osmolarity, and dose-dependent reduction of urinal sodium and potassium (13).

In parallel with earlier studies, Singh and Lina (14) reported that the activity of the sodium-potassium adenosine triphosphatase (Na⁺-K⁺-ATPase) in cultured proximal tubule epithelial cells was increased in a cAMP-independent manner by metaproterenol, a partially selective β₂AR agonist. It was prevented by ICI-118551, a selective β₂ blocker. The authors suggested that β₂AR-mediated regulation of sodium transport in proximal tubules involves protein kinase C signaling and serotonin receptors (14). Subsequent studies addressed the role of PKC in cultured rat proximal tubule cells (RPTC) exposed to phorbol 12-myristate 13-acetate (PMA)-tetradecanoylphorbol acetate (TPA), a PKC agonist. Exposure to PMA conferred a rapid, 100% increase of apical sodium influx and 30% decline of basal Na⁺-K⁺-ATPase activity (P < 0.05). Stimulation of RPTCs with metaproterenol conferred threefold enhanced PKC activity that was blocked by coincident ICI-118511 but not by metoprolol (a specific β₁AR inhibitor) confirming selectivity for β₂AR. To confirm the role of PKC in β₂AR-mediated transcellular sodium transport, Singh and Lina (15) further reported that staurosporine and calphostin C, selective PKC inhibitors, blocked metaproterenol-induced increases in Na uptake. These early studies established that PKC is responsible for the β₂AR-mediated increases in Na⁺-K⁺-ATPase activity in renal tubules.

The serine-threonine kinase, with-no-lysine (K) kinase 4 (WNK4), is a negative regulator of the thiazide-sensitive sodium chloride cotransporter (NCC) [reviewed by Furusho et al. (67)]. Mutations of the WNK genes including WNK1 and -4 have been linked with pseudohypoaldosteronism type II (PHAII), an inherited hypertensive disease, implicating WNKs with normal regulation of blood pressure (BP) and electrolyte balance. Under normal conditions, WNK4 inhibits NCC activity, reducing sodium reabsorption in the distal convoluted tubule to preserve normal BP. The expression of WNK kinases and thence NCC activity and BP are modulated by changes in dietary sodium. Roles for β₂AR signaling in the salt-sensitive regulation of WNK4 and NCC were first described by Mu et al. (24) and Fujita (68), using mouse models and salt-sensitive hypertension-prone (Dahl) rats. The authors reported that salt loading in DOCA-treated rats conferred increased sympathetic nervous system (SNS) activity and decreased WNK4 expression in the kidneys coincident with hypertension, effects that were reversed by renal denervation. They further found that WNK4 expression was downregulated in norepinephrine (NE)-infused mice coincident with upregulation of the NCC and salt-induced hypertension, effects that were reversed by treatment with propranolol, a β-AR blocker. The same effects were observed in wild-type and β1-KO mice infused with isoproterenol but not β₂AR-KO mice confirming a role for the β₂AR in activating the WNK4-NCC pathway. Mechanistically, the authors presented evidence that stimulation of the cAMP-PKA pathway by β₂AR-selective agonists conferred inhibition of histone deacetylase-8 (HDAC8), and increased acetylation of histones H3 and H4 at a negative glucocorticoid-responsive element (nGRE) in the promoter of the WNK4 gene. The result of such hyperacetylation was facilitated by binding of the glucocorticoid receptor to the nGRE and suppression of WNK4 transcription. Mu et al. (24) proposed that high salt-mediated hypertension in their models is driven by a pathway that includes β₂AR-WNK4-NCC.

Whereas the involvement of β₂AR pathways in the regulation of BP through activities of the Na⁺-K⁺-ATPase appears to be well established and may provide potential new targets for treating hypertension, subsequent work disputed the presence of a β₂AR-WNK4-NCC pathway in the regulation of BP. Uchida et al. (69) successfully reproduced the NE infusion models of Mu et al., but they did not observe any change of WNK4 mRNA or protein in the kidney caused by NE-infusion. More recently Zicha et al. (36) reported no effect of propranolol on hypertension induced by a high-salt diet on salt-sensitive rats and concluded that their results did not support an essential role for a renal βAR-WNK4-NCC pathway in pathogenesis and/or maintenance of salt hypertension in Dahl rats. Consistent with this, Frame et al. (37) and Puleo et al. (70) also used selective AR antagonism in NE-infused rats including the Dahl model to investigate the pathway of sympathetically mediated NCC regulation. These authors found that antagonism of α1- but not βARs restored dietary sodium-evoked suppression of the NCC and normalized BP by a pathway(s) that included WNK4, STE20/SPS1-related proline-alanine-rich kinase (SPAK), and oxidative stress response 1 (OSR1). The conflicting results particularly related to the mode of regulation of the NCC may reflect the use of different models, procedures, and reagents. In terms of drug development based on these pathways, recent research has focused on molecules that inhibit either the kinase or binding activities of WNKs, SPAK/OSR1, or MO25, an activator of SPAK/OSR1 [reviewed by Brown et al. (41)]. The schematic of the effects of β₂AR on renal electrolyte balance is demonstrated in Fig. 2.

Figure 2.

Effects of β₂ adrenergic receptor (β₂AR) signaling on salt handling in renal tubules. β₂AR signaling increases renal sodium reabsorption by: 1) Increasing the activity of Na-K-ATPase pump in proximal tubular epithelial cells in a protein kinase C (PKC)-dependent manner (left); 2) Blocking the inhibition of Na-Cl Co-transporter (NCC) by with-no-lysine (K) kinase 4 (WNK4) in distal renal tubular cells through a adenosine 3′,5′-cyclic monophosphate (cAMP)-protein kinase A (PKA)-mediated pathway (right). Image is from Servier Medical Art images (https://smart.servier.com).

β₂AR SIGNALING AND RENAL INFLAMMATION

Roles of β₂ARs in immunomodulation and inflammation have been recognized for more than 2 decades [reviewed by Kolmus et al. (71)]. β₂ARs are expressed on immune cells and participate in the adrenergic modulation of the actions of nuclear factor-κB (NF-κB), a master regulator of both innate and adaptive immune responses with important roles in susceptibility to disease such as sepsis and acute renal failure (ARF). Consistent with this, β₂AR agonists have been shown to suppress the cytokine cascade during the course of immunological responses, conferring regulation of tumor necrosis factor-α (TNF)-α and interleukin-6 (IL-6) in macrophages, and in renal glomerular mesangial and tubular epithelial cells, as well as brain astrocytes (22).

Terbutaline is a β₂ agonist with properties to modulate levels of pro- and anti-inflammatory cytokines including TNF-α, IL-10, and IL-6 in cancer and other cell types (72). It is currently used in the management of asthma symptoms and was previously used as a tocolytic to delay preterm labor (73, 74). Nakamura et al. (16, 75) first described biphasic pathways for the effects of terbutaline on lipopolysaccharide (LPS)-induced IL-6 production. The time course and dose-dependent effects of terbutaline on TNF-α release, MAPK, intracellular cAMP, and IL-6 expression were followed in LPS-stimulated primary rat renal macrophage cells. They showed that high dose (10⁻⁶ M) terbutaline significantly increased IL-6 expression, whereas it was similarly decreased at low-dose (10⁻⁸ M) terbutaline. TNF-α and MAPK were diminished, while there was a concentration-dependent increase of intracellular cAMP. The authors postulated that terbutaline-induced downregulation of IL-6 gene expression was mediated by an inhibitory effect of terbutaline on TNF-α, exerted through the MAPK and cAMP pathways, whereas the upregulation was caused by a direct action of intracellular cAMP. Consequently, the overall β₂AR-mediated regulation of IL-6 in renal macrophage cells is determined by the balance of the two pathways and regulated in a time and dose-dependent manner (16). Similar results and conclusions were reported for β₂AR regulation of TNF and IL-6 in primary rat renal resident macrophage cells (75). The findings were the first to suggest important renal immunomodulatory properties of β₂AR agonism.

Further studies by Nakamura et al. documented modulating roles for β₂ARs in the CD14-TLR4-TNFα signaling cascade in LPS-induced septic rats. It is noteworthy in this context that septic shock following surgery, trauma, burns, or severe infection is a common cause of acute renal failure (ARF), resulting in a high mortality rate (22). Seven-week-old Wistar rats were administered LPS (10 mg/kg ip) followed by β₂AR antagonist, ICI118,551 (3.14 μg/kg). After 24 h, a battery of inflammatory-related components including IL-6, IL-10, TNF, CRP, NO, norepinephrine, β₂-AR signaling proteins Giα, Gsα, cAMP as well as CD14, TLR4, and TNF-α proteins were quantified in the kidneys. The study showed that a combination of LPS and β₂-antagonist selectively increased renal TNF-α, CD14, TLR4, and decreased Gsα and cAMP. Accordingly, the authors proposed renal-specific β₂AR regulation of the CD14-TLR4-TNF-α signaling pathway, in which β₂ antagonism suppressed Gsα, decreased cellular cAMP and PKA activity, and increased renal TNF-α transcription. Therefore, by extrapolation, the studies predict decreased inflammation and subsequent tissue damage during sepsis-induced acute renal failure conferred by β₂ agonism (21).

Additional studies by Nakamura et al. (19) aimed to delineate the complex roles of β₂AR in renal physiology and determine whether tissue damage by endotoxin exposure could be rescued by overexpression of β₂AR in a gene therapy context. To test this, rat kidneys were injected intraperitoneally with 10⁹ viral particles of an adenoviral vector that contained human β₂AR cDNA. Transduced kidneys expressed approximately threefold higher levels of β₂ARs compared with sham controls. Two weeks after delivery, transduced mice had significantly increased glomerular filtration rate (GFR) and sodium reabsorption compared with controls. Administration of LPS to control rats conferred marked reductions in renal levels of GFR, β₂AR density, and cAMP, whereas TNF-α mRNA transcripts increased. In contrast, in rats overexpressing β₂AR, the effects of LPS on GFR and TNF-α mRNA were blocked. These results provide further support for a crucial role of β₂AR signaling in the pathophysiology of renal insufficiency.

Consistent with an anti-inflammatory role for β₂ agonism, kidney-specific β₂AR gene delivery by adenovirus, also protected against acute renal failure (ARF) in a rat model of systemic inflammation induced by renal artery occlusion and subcutaneous injection of Escherichia coli (22). In this model, renal-specific β₂AR overexpression ameliorated renal dysfunction as evidenced by increased rate of creatinine clearance and 40% improved survival. Although renal-specific overexpression of β₂AR did not affect serum levels of inflammatory cytokines, the renal levels of cannabinoid-1 (CB-1) receptor, CD14, toll-like receptor 4 (TLR4), and tumor necrosis factor (TNF)-α protein were reduced. Circulating serum levels of angiotensin II (ANG II) were decreased and nitric oxide (NO) increased in the treatment groups (22). These findings support positive therapeutic actions of renal-specific overexpression of β₂AR on renal function during septic stress, and β₂AR gene therapy presents a potential therapeutic option for acute renal failure secondary to sepsis.

In another model of human renal toxicity, Nakamura et al. showed that terbutaline conferred a dose-dependent inhibitory effect on apoptosis of adenocarcinoma-derived (ACHN) cells exposed to Shiga (Stx)2 toxin, a toxin commonly produced by bacteria Shigella dysenteriae and some serotypes of E. coli. The anti-apoptotic effect of terbutaline was indicated by reduced DNA degradation, lower activities of caspases 3 and 7, and annexin V and enhanced mitochondrial membrane potential, following treatment with Stx2 and terbutaline. Treatments with cAMP-PKA inhibitors (H-89, KT5720) and β₂AR antagonist (ICI118,551) blocked protection by terbutaline, whereas SB203580, a p38MAPK but not PD098059, a p42/p44MAPK inhibitor, further decreased caspase-3 activity. The authors concluded that β₂AR agonism by terbutaline suppressed caspase-3 activation through a PKA-cAMP pathway with parallel inhibition of p38MAPK (17). The study is consistent with global protection against renal toxicity by β₂-agonism.

Further studies support antifibrosis as well as anti-inflammation roles for β₂AR agonists in the kidney. Salbutamol treatment of Zucker diabetic fat (ZDF) rats reduced the expression of fibronectin and collagen types I and IV in the kidneys and markedly attenuated collagen deposition in renal cortices (30). The authors attributed the changes to reductions of monocyte activation and macrophage influx into renal tissue, mediated by β₂AR-mediated inhibition of the β-arrestin2-NF-kB pathway in THP-1 cells thereby suppressing the inherent high-glucose-induced proinflammatory responses (30). The study highlights the potential use of β₂AR antagonists in the prevention of diabetic kidney disease. The role of β₂AR and its downstream effects in renal inflammation are recapped in Fig. 3.

Figure 3.

Role of β₂ adrenergic receptor (β₂AR) signaling in renal inflammation. β₂AR agonism by various agents is shown to reduce the proinflammatory response and renal apoptosis and fibrosis in ex vivo and in vitro models (left). β₂AR inhibition in lipopolysaccharide (LPS)-induced sepsis in rats increases renal inflammation by inducing cluster of differentiation 14 (CD14), Toll-like receptor 4 (TLR4), and tumor necrosis factor α (TNFα) mediated by suppressed adenosine 3′,5′-cyclic monophosphate (cAMP)/ protein kinase A (PKA) activity (middle). Overexpression of β₂ARs via gene delivery improves renal function [increased creatinine clearance and glomerular filtration rate (GFR)] in rat models of endotoxin-induced acute renal failure (ARF) and of systemic inflammation (right). ACHN, adenocarcinoma renal cell line; ANG II, angiotensin II; MAPK, mitogen-activated protein kinase; NF-kB, nuclear factor κ B; NO, nitric oxide; PKC, protein kinase C; Stx2, Shiga toxin-2; ZDF, Zucker diabetic fatty.

β₂ARS IN HEART-BRAIN-KIDNEY CROSS TALK

Fujiu et al. (29) investigated the roles for β₂AR cross talk between heart, brain, and kidney in a mouse model of pressure overload and heart failure by transaortic constriction (TAC). The study revealed that sympathetic nerves activated by cardiac pressure overload confer remote stimulation of collecting duct (CD) epithelial cells in the kidney via a β₂AR pathway. Activated duct cells respond by incorporating renal macrophages that in turn cause renal endothelial cell (EC) activation. Activated-renal ECs release colony-stimulating factor 2 (CSF2) and stimulate cardiac Ly6C^lo macrophages to produce amphiregulin, a major cardioprotective mediator. Confirming these findings, the renal response to the cardiac pressure overload was abolished by renal sympathetic nerve ablation, β₂AR inhibition by butoxamine or CD-cell-specific deficiency of KLF5 (Krüppel-like factor 5), a transcription factor known to influence the accumulation of inflammatory monocytes and macrophages during renal inflammation in response to injury, as well as in hypertrophic cardiac responses. The pathway implicates β₂ARs in cross talk between cardiac macrophages and renal ECs and inflammatory mediators in a pathway that supports adaptive/protective myocardial hypertrophy in the setting of myocardial pressure overload (29).

The predicted cardioprotective effects of such an enhanced β₂-receptor-mediated renal response driven by TAC supported by recent studies have demonstrated cardioprotective actions of β₂-receptor agonists in human and animal models of heart failure (HF) (76). For example, combined therapy with a β₂-receptor agonist and a β₁-blocker in a rat model of postmyocardial-infarction-dilated cardiomyopathy showed a long-term survival benefit over β₁-blocker monotherapy (77). The authors conclude that pharmacological modulation of β₂-receptor signaling would affect the heart-brain-kidney network, and their study highlights a need to re-evaluate the effects of pharmacological adrenergic receptor intervention at the multisystem and multiorgan levels (29). Figure 4 summarizes the implications of β₂AR signaling in the brain-heart-kidney cross talk.

Figure 4.

Role of β₂ adrenergic receptor (β₂AR) signaling in heart-brain-kidney cross-talk during myocardial adaptation to pressure overload. Upon induction of cardiac pressure overload in a mouse model of transaortic constriction, sympathetic nerves are activated and stimulate collecting duct (CD) epithelial cells in the kidney via a β₂AR-dependent pathway. Activated duct cells respond by incorporating renal macrophages that in turn cause endothelial cell (EC) activation. Activated ECs release colony-stimulating factor 2 (CSF2) and stimulate cardiac Ly6C^lo macrophages to produce the cardioprotective mediator amphiregulin. Image is from Servier Medical Art images (https://smart.servier.com).

RENAL β₂AR IN CLINICAL STUDIES

A clinical study analyzed the effect of β₂AR gene variants on the GFR in 580 African-American patients with hypertensive nephrosclerosis (23). GFR decline rates associated with progressive hypertensive nephropathy were calculated. The study reported a more rapid GFR decline in individuals with haplotype-3 (–804G→173T→16Arg→27Gln), whereas participants with haplotype-1 (–804G→173T→16Gly→27GIn) showed the lowest GFR decline rate. A Gly16Arg variant that exists in haplotype-3 was also significantly correlated with elevated risk of end-stage renal disease in the cohort population studied. Therefore, genetic variation within the β₂AR locus contributes to risk and progression of chronic kidney disease (23).

A retrospective clinical study investigated possible implications of renal β₂AR overexpression on renal dysfunction in children secondary to β₂AR activation (20). β₂ Agonists used for non-renal ailments of children, most commonly asthma, are eliminated intact by the kidney and are therefore likely to activate nephron β₂ARs. The results showed no statistically significant overall difference in serum creatinine between children receiving therapeutic β₂AR agonists and control groups. Distributions of β₂ARs were assayed in renal biopsy tissue of children, aged 2–15 yr olds who were admitted to hospital with either proteinuria and/or hematuria. Control groups of 23 children with normal creatinine clearance as well as children with nephrotic syndrome, receiving prednisolone therapy (steroid group) were included. Immunohistochemical detection of β₂AR expression revealed that children under steroid therapy overexpressed β₂AR in distal tubules and glomeruli. The results suggest that steroid therapy may upregulate β₂AR in immature kidneys, with possible adverse side effects on kidney function in these subjects. Another possible side effect relates to tolerance that can develop with chronic β₂AR stimulation secondary to primary treatments with medications such as steroids or salbutamol. Tolerance is driven by phosphorylation of the receptors by GPCR kinases (GRK) that facilitate complexing with arrestin family proteins. There are four arrestin proteins that function as cytosolic adaptors and mediate receptor internalization and degradation thereby reducing cell surface numbers (78). Such desensitization has wide implications for cell biology and physiology but few studies have investigated the clinical repercussion.

Wang et al. measured autoantibodies in 98 participants including 30 patients with chronic cardiorenal syndrome (CRS), 30 patients with heart failure without kidney disease, and 38 healthy controls. They reported strong correlations between titers of autoantibodies against β₁, β₂, and α₁-ARs, and elevated incidence of chronic cardiorenal syndrome (CRS). Therefore, in this cohort of patients, higher frequencies and levels of antibodies against β₁-, β₂-, and α₁ARs were significantly associated with predisposal to chronic CRS relative to healthy control and heart failure groups. Cause and effect of the correlations were not established but the results corroborate preclinical studies that have linked dysregulated adrenergic signaling with cardiorenal syndrome (28). In support of this, a recent study reported that reduced β₂AR levels were associated with poor prognosis of renal cell carcinoma implying a prognostic significance of renal β₂AR expression in this indication (35). The highlights of these clinical studies are shown in Fig. 5.

Figure 5.

Renal β₂ adrenergic receptor (β₂AR) in clinical studies. Summary of clinical studies investigating β₂AR-related effects renal disease. ccRCC, clear cell renal cell carcinoma; CRE, creatinine; CRS, cardiorenal syndrome; ESRD, end-stage renal disease; GFR, glomerular filtration rate; HF, heart failure.

DISCUSSION

In this review we outline the progress, timeline, and clinical relevance of β₂AR signaling in the kidney with a focus on renal pathophysiology, pharmacological modulation, and potential for therapy. As delineated in the timeline (Table 1), early studies in this area were focused primarily on characterizing the effects of β₂AR signaling on kidney physiology as it relates to the renin-angiotensin-aldosterone system. There was then a shift in focus toward the pharmacological effects of β₂ agonists and antagonists on kidney function and related pathophysiology. The clinical utility of such β₂AR modulation by a variety of selective ligands was already well established for other indications including pulmonary (asthma and chronic obstructive pulmonary disease) and cardiovascular (heart failure, coronary artery disease, hypertension). Because kidney dysfunction is an eventual sequela of many common chronic systemic diseases and represent a major unmet clinical need, it will be important to fully characterize the effects of targeted β₂AR modulation on normal renal function as well as the possible side effects of acute and chronic drug administration. Perhaps the most significant work with potential for clinical translation involves studies that investigated the roles of β₂AR signaling in the restoration of renal function and reversal of kidney injury. Central roles of β₂AR signaling in regulating blood pressure, mitochondrial biogenesis, inflammation, and apoptosis in the kidney in a range of pathological settings suggests exciting directions for selective pharmacological and molecular modulation of β₂ARs to combat renal disease. Thus far, negative effects of chronic β₂AR agonism have been described for cardiovascular indications (reviewed in Ref. 79). For example, chronic exposure can lead to increased Na⁺ and water retention and elevated blood pressure, effects that would be counter indicated for hypertensive patients. β₂AR desensitization is also a well-established cardiovascular side effect of chronic agonism that may also have widespread negative implications for renal function and physiology. However, few studies have investigated the clinical repercussions of chronic β₂AR agonism on kidney function.

Perspectives and Significance

Although our knowledge of β₂AR signaling in the kidney has increased in recent years, substantial gaps remain particularly related to the precise molecular signaling pathways and (patho)physiological cross-reacivities between other organs and parallel signaling pathways. In this review, we provide both a historic perspective and current state-of-the-art of the subject matter available. Cumulative integration of our present knowledge of the extensive and varied contributions of β₂AR signaling to renal physiological and pathological pathways combined with the well-established pharmacology for other indications is predicted to facilitate an efficient development of novel therapeutic strategies. In most cases, further preclinical studies are required to identify the optimal candidates and signaling intermediates, extrapolate promising findings to large animal and/or nonhuman primate models that more closely mimic the human disease, define appropriate dosing and routes of administration, and implement standard preclinical efficacy/safety/toxicity profiles of candidate reagents before clinical trials. From this literature review, the programs that appear to be most advanced from the translational perspective include targeting of PGC-1α via cGMP/PKA/cAMP/CREB to induce reparative mitochondrial biogenesis in RPTC and podocytes, and salt handling/electrolyte balance for hypertension via regulators of WNK, SPAK, and OSR1 kinases.

GRANTS

This work was supported by National Institutes of Health (NIH) Grant 1R01HL140468 (to L.A. Shehadeh) and the Miami Heart Research Institute (to L.A. Shehadeh), and NIH Diversity Supplement Award 3R01HL140468-02S1 (to J.C. Dunkley). K. Yousefi is a recipient of American Heart Association Predoctoral Fellowship 18PRE33960070.

DISCLOSURES

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

AUTHOR CONTRIBUTIONS

A.K., K.Y., J.C.D., K.A.W., and L.A.S. drafted manuscript; A.K., K.Y., J.C.D., K.A.W., and L.A.S. edited and revised manuscript; A.K., K.Y., J.C.D., K.A.W., and L.A.S. approved final version of manuscript.