Dual Actions of A2A and A3 Adenosine Receptor Ligand Prevents Obstruction-Induced Kidney Fibrosis in Mice

Kidney fibrosis is the final outcome of chronic kidney disease (CKD). Adenosine plays a significant role in protection against cellular damage by activating four subtypes of adenosine receptors (ARs), A1AR, A2AAR, A2BAR, and A3AR. A2AAR agonists protect against inflammation, and A3AR antagonists effectively inhibit the formation of fibrosis. Here, we showed for the first time that LJ-4459, a newly synthesized dual-acting ligand that is an A2AAR agonist and an A3AR antagonist, prevents the progression of tubulointerstitial fibrosis. Unilateral ureteral obstruction (UUO) surgery was performed on 6-week-old male C57BL/6 mice. LJ-4459 (1 and 10 mg/kg) was orally administered for 7 days, started at 1 day before UUO surgery. Pretreatment with LJ-4459 improved kidney morphology and prevented the progression of tubular injury as shown by decreases in urinary kidney injury molecular-1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL) excretion. Obstruction-induced tubulointerstitial fibrosis was attenuated by LJ-4459, as shown by a decrease in fibrotic protein expression in the kidney. LJ-4459 also inhibited inflammation and oxidative stress in the obstructed kidney, with reduced macrophage infiltration, reduced levels of pro-inflammatory cytokines, as well as reduced levels of reactive oxygen species (ROS). These data demonstrate that LJ-4459 has potential as a therapeutic agent against the progression of tubulointerstitial fibrosis.


Introduction
Kidney fibrosis is characterized by glomerulosclerosis, vascular sclerosis, and tubulointerstitial fibrosis and is considered the final outcome of chronic kidney disease (CKD) [1,2]. As the tubulointerstitial space occupies more than 90% of the kidney, tubulointerstitial fibrosis is an easily observed pathological process leading to end-stage kidney disease (ESKD) [3]. Tubulointerstitial fibrosis is accompanied by: (i) the infiltration of inflammatory cells, (ii) the activation of fibroblasts, (iii) the accumulation of extracellular matrix (ECM), and (iv) the production of tubular atrophy [4]. These events occur partially or together.
Adenosine is formed both intracellularly and extracellularly through catalytic enzymes that hydrolyze nucleotides [5]. Under distress conditions, including ischemia, hypoxia, and inflammation, extracellular ATP levels are increased and rapidly hydrolyzed to adenosine. Adenosine serves as a signaling molecule that initiates receptor-mediated functions. The adenosine receptors (ARs) are named adenosine receptor A 1 (A 1 AR), adenosine receptor A 2A (A 2A AR), adenosine receptor A 2B (A 2B AR), and adenosine receptor A 3 (A 3 AR) [6]. The four ARs belong to the family of G protein coupled receptors (GPCRs), and ARs signaling occurs not only through inhibition or stimulation of adenylyl cyclase (cAMP), but also through phospholipase C (PLC), Ca 2+ , and mitogen-activated protein kinases (MAPKs) [7]. 2 of 13 In the kidney, adenosine regulates the kidney physiological responses such as tubuloglomerular feedback (TGF), blood flow, glomerular filtration rate (GFR), renin release, and NaCl transport [8,9]. However, a chronically excessive kidney adenosine level causes tubulointerstitial fibrosis and kidney dysfunction [10,11].
Thus, the present study has determined the renoprotective effect of LJ-4459, a newly developed potent dual acting A 2A and A 3 AR ligand on UUO-induced tubulointerstitial fibrosis. LJ-4459 has been reported (i) to have high binding affinity to both hA 2A AR and hA 3 AR, (ii) to be a full A 2A AR agonist and a full competitive A 3 AR antagonist, (iii) to have 0.51 of log P, and (iv) to have similar anti-inflammatory potency as indomethacin in carrageenan-induced paw edema assay [31].

Pretreatment of LJ-4459 Improves Kidney Function and Attenuates Kidney Tubular Injury in the Obstructed Kidney
Our previous studies have established an obstruction-induced tubulointerstitial fibrosis model in rat and mice [12,32]. Based on these analyses, mice were administered LJ-4459 to start 1 d prior to UUO surgery and all mice were euthanized after 7 d of treatment ( Figure 1A). The urinary adenosine excretion was significantly increased after UUO surgery (data not shown), which was not affected by LJ-4459 treatment (Supplementary Figure S1). We examined the effect of LJ-4459 on kidney dysfunction and kidney tubular injury in the obstructed kidneys. Plasma creatinine and blood urea nitrogen (BUN), markers of kidney injury, were significantly increased in UUO mice [32]. Plasma creatinine was effectively reduced by 10 mg/kg LJ-4459 treatment ( Figure 1B). BUN tended to be decreased by LJ-4459 treatment, but it did not reach statistical significance ( Figure 1C). In addition, kidney tubular injury markers such as urinary KIM-1 and NGAL were significantly reduced by 10 and 1 mg/kg treatment with LJ-4459, respectively (Figure 2A,B). The KIM-1 mRNA levels increased in the obstructed kidneys, which were reduced by LJ-4459 treatment ( Figure 2C). The NGAL mRNA and protein expression levels increased after UUO surgery and were decreased by LJ-4459 treatment ( Figure 2D-H). The results of PAS staining showed morphology changes as indicated by tubular atrophy after UUO surgery, which was attenuated by LJ-4459 treatment ( Figure 2I). Tubular cell apoptosis indicated by TUNEL staining was increased in the obstructed kidneys, which was decreased by LJ-4459 treatment ( Figure 2J). To examine the toxicity of LJ-4459, we performed the MTT assay using mouse proximal tubular epithelial (mProx) cells. LJ-4459 did not affect cell viability up to 100 µM (Supplementary Figure S2), suggesting lack of toxicity. tion of ECM in the obstructed kidneys. The excessive accumulation of ECM was reduced after LJ-4459 treatment ( Figure 4A,B). The collagen I staining results showed that positively stained areas were increased in the obstructed kidneys and were decreased in LJ-4459-treated obstructed kidneys ( Figure 4C,D). Consistently, the collagen I, collagen IV, fibronectin, and α-SMA protein expression levels were significantly increased in obstructive kidneys. These ECM proteins and α-SMA expression levels were inhibited by LJ-4459 treatment ( Figure 4E-I).

Pretreatment of LJ-4459 Decreases Kidney Inflammation in the Obstructed Kidney
We evaluated the anti-inflammatory effect of LJ-4459 in the obstructed kidneys. As expected, the obstructed kidneys led to an increase of inflammatory cytokines such as ICAM-1, iNOS, and IL-6, which were reduced in response to LJ-4459 treatment ( Figure 3A-C). Macrophage infiltration, as indicated by F4/80-positive staining, was increased in the obstructed kidneys, whereas LJ-4459-treated obstructed kidneys had decreases in these effects ( Figure 3D,E). In parallel, the protein expression levels of ICAM-1 and iNOS were increased in the obstructed kidneys, which were reduced by LJ-4459 treatment ( Figure 3F-H).

Pretreatment with LJ-4459 Inhibits Tubulointerstitial Fibrosis in the Obstructed Kidney
In order to confirm whether LJ-4459 has an anti-fibrotic effect, we detected the ECM proteins, such as collagen I, collagen IV, and fibronectin, and α-smooth muscle actin (α-SMA). Picrosirius red staining indicated that there was a lot of UUO-induced accumulation of ECM in the obstructed kidneys. The excessive accumulation of ECM was reduced after LJ-4459 treatment ( Figure 4A,B). The collagen I staining results showed that positively stained areas were increased in the obstructed kidneys and were decreased in LJ-4459-treated obstructed kidneys ( Figure 4C,D). Consistently, the collagen I, collagen IV, fibronectin, and α-SMA protein expression levels were significantly increased in obstructive kidneys. These ECM proteins and α-SMA expression levels were inhibited by LJ-4459 treatment ( Figure 4E-I).

Pretreatment with LJ-4459 Reduces Oxidative Stress in the Obstructed Kidney
We evaluated the state of oxidative stress in the obstructed kidneys. The mRNA expression levels of NOX1, NOX2, and NOX3 were increased in the obstructed kidneys. These mRNA expression levels were reduced by LJ-4459 treatment ( Figure 5A-C). The 8-oxo-dG staining results showed that positively stained areas were increased in obstructed kidneys. These positively stained areas were reduced in LJ-4459-treated obstructed kidneys ( Figure 5D,E). The 4-hydroxynonenal (4-HNE) staining, which indicates oxidative stress, was increased in the obstructed kidneys, whereas LJ-4459-treated obstructed kidneys had decreases in these effects ( Figure 5F,G).

Pretreatment with LJ-4459 Reduces Oxidative Stress in the Obstructed Kidney
We evaluated the state of oxidative stress in the obstructed kidneys. The mRNA pression levels of NOX1, NOX2, and NOX3 were increased in the obstructed kidn These mRNA expression levels were reduced by LJ-4459 treatment ( Figure 5A-C). Th oxo-dG staining results showed that positively stained areas were increased in obstru kidneys. These positively stained areas were reduced in LJ-4459-treated obstructed neys ( Figure 5D,E). The 4-hydroxynonenal (4-HNE) staining, which indicates oxida

Pretreatment with LJ-4459 Suppresses ERK and NF-κB Phosphorylation in the Obstructed Kidney
To dissect the mechanisms involved in the renoprotective effect of LJ-4459, we dete mined that the expression levels of ERK and NF-κB. ERK [12] and NF-kB [17] have be shown to mediate A3AR-induced kidney injury. The obstructed kidneys were marked increased in total (t-NF-κB) expression levels and the phosphorylation of NF-κB (p-N κB). UUO-induced increases in total and phosphorylation of NF-κB were suppressed LJ-4459 treatment ( Figure 6A-C). In addition, phosphorylation of ERK (p-ERK) expressi levels was upregulated in obstructed kidneys, which was decreased by LJ-4459 treatme ( Figure 6D,E).

Pretreatment with LJ-4459 Suppresses ERK and NF-κB Phosphorylation in the Obstructed Kidney
To dissect the mechanisms involved in the renoprotective effect of LJ-4459, we determined that the expression levels of ERK and NF-κB. ERK [12] and NF-kB [17] have been shown to mediate A 3 AR-induced kidney injury. The obstructed kidneys were markedly increased in total (t-NF-κB) expression levels and the phosphorylation of NF-κB (p-NF-κB). UUO-induced increases in total and phosphorylation of NF-κB were suppressed by LJ-4459 treatment ( Figure 6A-C). In addition, phosphorylation of ERK (p-ERK) expression levels was upregulated in obstructed kidneys, which was decreased by LJ-4459 treatment ( Figure 6D,E).

Discussion
The present data demonstrated that LJ-4459, a newly developed dual-acting ligand acting as both an A2AAR agonist and an A3AR antagonist [31], attenuated the progression of tubulointerstitial fibrosis in UUO mice. In addition, kidney injuries including kidney dysfunction and inflammation were improved by LJ-4459 treatment.
Kidney fibrosis is a consequence of multiple mechanisms, including the infiltration of inflammatory cells, the production of fibrotic cytokines and growth factors, and the deposition of ECM [4,33]. In addition, ROS regulate various signaling pathways, leading to inflammation and fibrosis [34]. UUO has been used as a tubulointerstitial fibrosis model characterized by the excessive accumulation of matrix protein, degradation of the proximal tubular mass, and increased tubular cell death [35,36]. In our experimental condition, we confirmed that the UUO operation leads to kidney injury, including tubulointerstitial fibrosis (Figure 4), tubular injury ( Figure 2), inflammation (Figure 3), and oxidative stress ( Figure 5).
ARs are a family of GPCRs widely distributed in almost all organs, playing roles in physiological and pathological functions by mediating downstream signaling [37]. All ARs affect cAMP levels, and the Gi-coupled A1AR and A3AR inhibit AC activity. On the other hand, the Gs-coupled A2AAR and A2BAR stimulate AC activity. Furthermore, this leads to activation of cAMP-dependent protein kinase A (PKA), MAPKs, phospholipase C (PLC), and calcium-dependent protein kinases (PKC) pathways [6,7].

Discussion
The present data demonstrated that LJ-4459, a newly developed dual-acting ligand acting as both an A 2A AR agonist and an A 3 AR antagonist [31], attenuated the progression of tubulointerstitial fibrosis in UUO mice. In addition, kidney injuries including kidney dysfunction and inflammation were improved by LJ-4459 treatment.
Kidney fibrosis is a consequence of multiple mechanisms, including the infiltration of inflammatory cells, the production of fibrotic cytokines and growth factors, and the deposition of ECM [4,33]. In addition, ROS regulate various signaling pathways, leading to inflammation and fibrosis [34]. UUO has been used as a tubulointerstitial fibrosis model characterized by the excessive accumulation of matrix protein, degradation of the proximal tubular mass, and increased tubular cell death [35,36]. In our experimental condition, we confirmed that the UUO operation leads to kidney injury, including tubulointerstitial fibrosis (Figure 4), tubular injury ( Figure 2), inflammation (Figure 3), and oxidative stress ( Figure 5).
ARs are a family of GPCRs widely distributed in almost all organs, playing roles in physiological and pathological functions by mediating downstream signaling [37]. All ARs affect cAMP levels, and the Gi-coupled A 1 AR and A 3 AR inhibit AC activity. On the other hand, the Gs-coupled A 2A AR and A 2B AR stimulate AC activity. Furthermore, this leads to activation of cAMP-dependent protein kinase A (PKA), MAPKs, phospholipase C (PLC), and calcium-dependent protein kinases (PKC) pathways [6,7].
In our present study, we showed that phosphorylation of ERK and NF-κB were increased in obstructed kidneys, and this was attenuated by LJ-4459 treatment ( Figure 6). Previous studies have shown that NF-κB is a target gene of ERK signaling [38] and it contributes to kidney fibrosis [39]. In line with our data, A 2A AR agonists have antiinflammatory effects through the decreased phosphorylation of ERK [40,41] and NF-κB [41] in neutrophils and T cells. In addition, an A 3 AR antagonist blocked fibrosis via decreased phosphorylation of ERK [12] and NF-κB [17]. Therefore, these results suggest that the renoprotective effects of LJ-4459 are associated with the ERK and NF-κB signaling pathways.
In contrast, A 2A AR agonists increased phosphorylation of ERK in liver [42] and brain [43] tissue. In addition, A 3 AR antagonists increased phosphorylation of NF-κB in microglial cells [44] and mesothelial cells [45]. More importantly, an A 3 AR agonist has been shown to protect against sepsis kidney injury [46]. These controversial effects suggest that ARs mediate different roles in tissue and cell types. Moreover, the ARs have been affected by acute versus chronic diseases [9,47]. The detailed mechanism of the effect of LJ-4459 on A 2A AR and A 3 AR in CKD is not clear yet.
In summary, LJ-4459, a new dual-acting agent that acts as both an A 2A AR agonist and an A 3 AR antagonist, effectively prevented obstruction-induced kidney dysfunction, inflammation, tubulointerstitial fibrosis, and oxidative stress.

Reagents
All chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise specified.

Animal Experiments
All experimental animals were approved by the Institutional Animal Care and Use Committee at Ewha Womans University (IACUC No. 18-007, 9 March 2018). The 6-weekold male C57BL/6J mice were purchased from the Ewha Laboratory Animal Genomic center (Seoul, Korea). Unilateral ureteral obstruction (UUO) surgery was performed as described in our previous study [12,32]. Mice were housed in a room maintained at 22 ± 2 • C with a 12 h dark/12 h light cycle and were randomly divided into three groups: (i) UUO without LJ-4459 treatment (0 mg/kg), (ii) UUO+LJ-4459 1 mg/kg, and (iii) UUO+LJ-4459 10 mg/kg. Briefly, to creative the kidney tubulointerstitial fibrosis model, the left ureter was ligated at two points with silk (4-0; Ailee Co., Ltd., Busan, Korea) and was cut between the two ligation points. The UUO mice were administered 0.25% carboxymethyl cellulose (CMC) or LJ-4459 (1 or 10 mg/kg) for 7 d by oral gavage. Drug administration was start at 1 d before the UUO surgery, and all mice were euthanized after 7 d of treatment. Contralateral kidney of UUO without LJ-4459 treatment was used as a sham kidney.

Measurements of Blood Parameters
Blood was collected from the jugular vein before sacrifice and centrifuged at 3000 rpm for 15 min at 4 • C to collect the serum from the supernatant. Plasma creatinine (Arbor Assays, Ann Arbor, MI, USA) and blood urea nitrogen (BUN, Arbor Assays, Ann Arbor, MI, USA) were measured by using ELISA kits.

Measurements of Urine Parameters
Urine was collected in metabolic cage for 24 h and centrifuged at 3000 rpm for 15 min at 4 • C. Urinary kidney injury molecular-1 (KIM-1, MKM100, R&D Systems, Minneapolis, MN, USA) and urinary neutrophil gelatinase-associated lipocalin (NGAL, Immunology Consultants Laboratory, Inc., Portland, OR, USA) were measured by using ELISA kits.

Terminal Transferase-dUTP-Nick-End Labeling (TUNEL) Assay
Apoptosis was measured using the TUNEL assay according to the manufacturer's protocol (Roche Diagnostics, Mannheim, Germany). Briefly, after deparaffinization and rehydration, kidney sections were incubated with TUNEL reaction mixture for 60 min at 37 • C in a humidified dark chamber. Images were taken using a Zeiss ApoTome Axiovert 200M microscope (Carl Zeiss Microscopy).

Quantitative Real Time Reverse Transcriptase Polymerase Chain Reaction
Total cellular RNA was extracted with TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Expression of mRNAs were measured by real-time PCR using a 20 µL reaction volume consisting of cDNA transcripts, primer pairs, and SYBR Green PCR Master Mix (Applied Biosystems, Carlsbad, CA, USA) with the StepOne TM (Applied Biosystems). 18S was used as an internal control to normalize the genes. The primer sequences are shown in Table 1.

Statistical Analyses
All results are expressed as the mean ± standard error (SE). Analysis of variance (ANOVA) was used to assess the differences between multiple groups, followed by Fisher's least significant difference (LSD) test. The level of statistical significance was set at p-values less than 0.05.