Adenosine A2A Receptor: A Target for Regulating Renal Interstitial Fibrosis in Obstructive Nephropathy

Renal interstitial fibrosis (RIF) is the common pathological process of chronic kidney diseases leading inevitably to renal function deterioration. RIF and its preceding epithelial-mesenchymal transition (EMT) are commonly triggered by an early occurring renal inflammation. However, an effective approach to prevent EMT and RIF is still lacking and of urgent need. Recently, the adenosine A2A receptor (A2AR) emerges as a novel inflammation regulator, therefore manipulation of A2AR may suppress the EMT process and as such protect against RIF. To test this hypothesis we applied a unilateral ureteral obstruction (UUO) model of RIF on A2AR knockout mice and their wild-type littermates, combined with the intervention of a selective A2AR agonist, CGS 21680. On days 3, 7 and 14 post-UUO we evaluated the effects of A2AR manipulation on the molecular pathological progresses of RIF, including the cellular component of interstitial infiltration, expression of profibrotic factors, cellular biomarkers of EMT, and collagen deposition of extracellular matrix. Our data demonstrated that activation of A2AR significantly suppressed the deposition of collagen types I and III, reduced the infiltration of CD4+ T lymphocytes, and attenuated the expression of TGF-β1 and ROCK1, which in turn inhibited and postponed the EMT progress. Conversely, genetic inactivation of A2AR exacerbated the aforementioned pathological processes of UUO-induced RIF. Together, activation of A2AR effectively alleviated EMT and RIF in mice, suggesting A2AR as a potential therapeutic target for the treatment of RIF.


Introduction
Regardless of the etiology, almost all forms of end-stage renal disease share the common pathological feature of progressive renal interstitial fibrosis (RIF) and tubular atrophy [1,2,3]. Renal inflammation after sustained injuries, e.g. IgA nephropathy and lupus nephritis, serves as a primer that sets up the fibrogenic stage and triggers tissue fibrogenesis [4]. During this pathological progress macrophage and lymphocyte play crucial roles. RIF is characterized by the myofibroblast activation and the accumulation of matrix proteins including collagen types I (Col I) and type III (Col III). While RIF is commonly triggered by inflammatory processes recent studies suggest that a succedent epithelialmesenchymal transition (EMT) may also play an important role in the progress of RIF [5]. Particularly, myofibroblast, with identified expression of a-SMA may contribute as a major source of increased production of matrix protein [6,7]. Nevertheless, an early initiated anti-inflammatory strategy is therefore of importance to prevent the progression of RIF. However, no therapeutic approach is currently available to achieve this goal [8,9]. Therefore, exploring new therapeutic target is in urgent need.
Recently, adenosine A 2A receptor (A 2A R) emerges as a novel inflammation regulator affecting the inflammation process and tissue repair. Pharmacology studies showed that A 2A R agonist, CGS21680 and ATL193, can effectively suppress inflammation [10,11]. Activation of A 2A R leads to attenuation of glomerulonephritis and renal injury [12,13,14]. Further, recent studies identified that A 2A R activation inhibits Rho/ROCK1 in hepatic stellate cells [15]. All of the above strongly suggest that A 2A R manipulation plays an important regulatory role in inflammation and may also affect EMT event. Therefore, we hypothesize that activation of A 2A R may suppress cellular infiltration, EMT event and profibrogenic factors, thereby preventing consequent pathology of RIF. Conversely, inactivation of A 2A R may lead exacerbation of RIF.
A unilateral ureteral obstruction (UUO) model has been refined to elucidate the pathogenesis and mechanisms responsible for RIF [16,17]. It has been shown that the infiltration of macrophages and T cells and lymphocyte dysfunction are two major mechan-isms contributing to the UUO-induced RIF model [18,19]. In this model, at the cellular level, tubular dilatation leads the tubular epithelia to lose their epithelial characteristics and acquire mesenchymal traits such as a-SMA expression and actin reorganization. At molecular level, TGF-b1 plays a key role in EMT via activation of its downstream Rho/ROCK signaling pathway [20].
Using the experimental UUO-induced RIF mouse model, the present study was aimed to evaluate the modulatory effect of A 2A R-based manipulation on several aspects of RIF progression, including interstitial lymphocyte infiltration, cellular biomarkers of EMT, expression of the profibrogenic factor TGF-b1 and its downstream Rho/ROCK1 pathway, as well as the consequent extracellular matrix accumulation.

Animals
All animal experiments were conducted under approval of the Institutional Animal Care and Use Committee of Third Military Medical University (TMMU) and performed under the supervision of the facility veterinary staff. Mice used in the present study, i.e., genetic A 2A R knockout (KO) mice (A 2A R 2/2 ) and their wildtype (WT) controls (A 2A R +/+ ) were bred at the Animal Care Center of the Research Institute of Surgery of TMMU after being imported from Boston University School of Medicine as previously described [21]. Mice were maintained in a pathogen-free, humidity-and temperature-controlled environment with 12 h light-dark cycles and free access to food and drinking water. The A 2A R KO mice and their WT littermates were randomly designated into six experimental groups (see Table 1) according to the involvement of UUO procedure and CGS treatment. Ten mice from each experimental group were sacrificed at the designed experimental time-points, i.e., day 3, 7, and 14 after UUO. Mouse kidneys were harvested for the following imunohistochemistry evaluations.

Unilateral ureteral obstruction (UUO) model
Mice (20-25 g weight) were subjected to the UUO procedure under anesthesia as previously described [22] with modifications. All surgical procedures were performed under an operating microscope. Briefly, mice were first anesthetized with sodium pentobarbital (50 mg/kg, i.p.). After a left flank incision was taken, the left ureter was exposed, ligated with 6-0 silk sutures at two points, and cut between the two ligatures. Lastly, the peritoneal membrane and skin were sutured. Sham surgery was performed as control by following all steps of UUO-procedure except ligation and cut of ureter.

Reverse transcription quantitative real-time PCR (RT-qPCR)
Total RNA extraction of renal sample was conducted using a total RNA extraction kit (BioFlux, Cat# BSC52S1) and the reverse transcription reaction was performed using SYBR Premix Ex Taq kit (DRR041A, Dalian, China), according to the manufacturer's instructions. Then qPCR was performed to quantify the expression level of A 2A R, TGF-b1, and ROCK1 mRNAs using SYBR Premix Ex Taq kit (DRR041A, Dalian, China) and a qPCR reaction thermal cycle of 40 cycles of 95uC (30 s), 58uC (30 s), and 70uC (30 s). The glyceraldehyde 3phosphate dehydrogenase (GAPDH) was used as an internal control to normalize RT-qPCR readout. Gene mRNA expression levels were calculated relative to the expression level of GAPDH. All primers used (as shown in Table 2) were synthetized by Takara (Dalian, China).

Statistical analyses
The data are expressed as mean 6 SD. Statistical analysis was performed using one-way ANOVA followed by Bonferroni post hoc comparisons. P,0.05 was considered statistical significance.

A 2A R activation attenuated collagen deposition in matrix accumulation
To evaluate the effect of A 2A R on renal fibrosis, we applied the UUO model to mice combined with A 2A R agonist CGS21680 and genetic A 2A R inactivation (as aforementioned paradigm in Methods). Pathology assessment using H&E staining and immunohistostaining of Col I and Col III deposition were evaluated at day 3, 7, and 14 after UUO. Our H&E data demonstrated the successfulness of UUO modeling with featured pathological changes, e.g. progressively aggravated tubular dilatation and leukocytes infiltration (Figure 1 A). Our A 2A R immunochemistry data demonstrated that positive stained renal tubular epithelial cells were seen in WT mice (WT+Sham), but devoid in KO mice (KO+Sham) ( Figure 1B). Furthermore, we used RT-qPCR to detect the temporal changes of A 2A R mRNA expression in the progress of UUO-induced RIF. We showed that the mRNA level of A 2A R was significantly increased at day 3 through day 14 post-UUO, in a time-dependent manner. WT mice in WT+UUO+Veh group displayed an increase of 156%, 529% and 816% at day 3, 7 and 14, respectively, compared to non-UUO mice (F = 541.22, P,0.05, n = 10 per time point, Figure 1C). Conversely, A 2A R mRNA level in A 2A R KO (KO+UUO+Veh) mice remained under  Figure 1C).
Further, immunohistochemistry staining showed that starting at day 3, Col I and Col III progressively increased along with interstitial accumulation of extracellular matrix (ECM) in mouse kidneys from WT+UUO+Veh group (Figure 2 and 3), indicating the establishment of the UUO-induced RIF model. Importantly, quantitative morphometric analysis demonstrated that the depositions of Col I and Col III were both significantly reduced in the A 2A R agonist-treated WT+UUO+CGS group (a reduction of 40.6% and 55.9% at day 3, a reduction of 50.3% and 64.9% at day 7, respectively), compared to WT+UUO+Veh group (P,0.05, n = 10 per groups, Figure 2 and 3). Conversely, genetic inactivation of A 2A R significantly exacerbated collagen deposition in A 2A R KO (KO+UUO+Veh) mice, showing an increased Col I and Col III levels (by 39.6% and 57.1% at day 3, 29.5% and 31.7% at day 7, vs WT+UUO+Veh group, respectively, P,0.05, n = 10 per groups, Figure 2 and 3). Noteworthy, genetic A 2A R inactivation-induced exacerbation of collagen deposition was not affected by CGS treatment in KO+UUO+CGS group, showing significantly increased renal Col I and Col III levels, compared to WT+UUO+CGS group (P,0.05, n = 10 per groups, Figure 2

A 2A R activation inhibited UUO-induced changes on Ecadherin and SMA
To evaluate A 2A R-mediated effects on the EMT process we detected the expression levels of a-SMA (the myofibroblast marker) and E-cadherin (the epithelial marker) that indicate the transdifferentiation status of epithelial to myofibroblast. Western blot assay showed that at day 3 no expression difference of a-SMA and E-cadherin was found between each of the UUO groups and sham groups (P.0.05, n = 5 per group, Figure 4), indicating the absence of EMT process. Notably, the expression level of a-SMA was enhanced by 58.6% at day 7, and 125.2% at day 14 in WT+UUO+Veh group compared to WT+Sham group (P ,0.05, n = 5 group, Figure 4). However, the expression level of Ecadherin was reduced by 35.4% at day 7 and 43.0% at day 14 in WT+UUO+Veh groups compared to WT+Sham group (P,0.05, n = 5 per group, Figure 4). Importantly, A 2A R agonist treatment reduced a-SMA level in WT+UUO+CGS group (by 21.7% at day 7 and 31.3% at day 14) compared to WT+UUO+Veh group, P,0.05, n = 5 per group, Figure 4). Meanwhile, A 2A R agonist treatment enhanced E-cadherin level in WT+UUO+CGS group (by 27.9% at day 7 and by 20.6% at day 14) compared to WT+UUO+Veh group (P,0.05, day 7 and day 14, n = 5 per group, Figure 4). Conversely, inactivation of A 2A R (KO+UUO+-Veh) led to an opposite effect on a-SMA and E-cadherin levels compared to A 2A R activation (WT+UUO+CGS) treatment. The expression of a-SMA was enhanced by 17.9% (day 7) and 54.2% (day 14), whereas the E-cadherin levels were decreased by 15.7% (day 7) and 39.6% (day 14), compared with WT+UUO+Veh group, (P,0.05, day 7 and day 14, n = 5 per group, Figure 4).
In addition, our immunochemistry data demonstrated that positive stained renal tubular epithelial cells were seen in vehicletreated WT mice (WT+UUO+Veh) and A 2A R KO mutants (KO+UUO+Veh), but devoid in WT mice which received CGS21680 treatment (WT+UUO+CGS), at day 7 post-UUO ( Figure 5). This immunohistology data is consistent with our Western blot evaluations of a-SMA. Together, A 2A R activationinduced reduction of a-SMA and the increase of E-cadherin suggest an inhibitory effect of A 2A R on the tubular EMT process.

A 2A R activation attenuated the expression of profibrotic mediators
To mechanistically evaluate the A 2A R modulation on RIF, we detected the mRNA expression of two crucial profibrotic mediators, TGF-b1 and ROCK1 using RT-qPCR. We showed that the expression level of TGF-b1 mRNA was significantly increased at day 3 through day 14 in WT+UUO+Veh group (an increase of 411%, 789% and 833% at day 3, 7 and 14 respectively) compared to WT+Sham control group (P,0.05, n = 10 per group, Figure 6). Importantly, A 2A R agonist treatment attenuated the increase of TGF-b1 mRNA expression in WT+UUO+CGS group, leading to a decrease of 60.9% (P,0.05) and 30.0% (P,0.05) at day 3 and day 7, respectively, vs. WT+UUO+Veh group (n = 10 per group). Conversely, genetic inactivation of A 2A R in KO+UUO+Veh group led to an additional enhancement in mRNA expression of TGF-b1, by 39.1% (day 3) and 37.5% (day 7) compared to WT+UUO+Veh group, correspondingly (P,0.05, n = 10 per group, Figure 6). Noteworthy, A 2A R activation-mediated inhibitory effect on TGF-b1 expression was blunt at 14 day post-UUO, with no difference compared to other UUO groups (P.0.05 Figure 6), suggesting that the A 2A R activation-induced suppression on TGF-b1 expression occurred at early but not later post-UUO stage.

Suppression on T lymphocyte infiltration contributes to A 2A R-mediated renal protection against RIF
Infiltration of T lymphocyte, a key cellular inflammatory response, plays a crucial role in the initiation of EMT and RIF. Thus we examined renal T lymphocyte infiltration post-UUO  using immunostaining of T lymphocyte cell marker, CD3, CD4 and CD8. We observed that a prominent accumulation of CD3positive stained (CD3+) T lymphocyte was located around vessels in the kidneys from WT+UUO+Veh animals (data not shown). To identify the subtype of CD3+ T lymphocytes, we preformed immunohistostaining of CD4 and CD8. The data showed that the infiltrating T lymphocytes in UUO groups were identified as CD4positive stained (CD4+) (Figure 7), but not CD8 positive (CD8+) cells (data not shown). Further, quantitative morphometric analysis demonstrated that in WT+UUO+CGS mice there was less infiltration of CD4+ T lymphocyte, showing a reduction of 51.5% (day 3), 82.4% (day 7) and 89.9% (day 14) correspondently, compared with WT+UUO+Veh animals (P ,0.05, n = 10 per group, Figure 7). Conversely, infiltration of CD4+ T lymphocyte was exacerbated in UUO mice with genetic inactivation of A 2A R (KO+UUO+Veh), showing an increase of 37.8% (day 3), 57.5% (day 7), and 61.2% (day 14), respectively, vs WT+UUO+Veh group (P,0.05, n = 10 per group, Figure 7). In addition, immunohistostaining of CD11b (a marker for neutrophil granulocyte) as well as CD68 and F4/80 (markers for macrophage) were also performed to detect the involvement of other inflammatory cellular components. However, positive staining of CD68, F4/80, and CD11b were observed without significant difference (data not shown), suggesting a devoid of infiltration of macrophage or neutrophil granulocyte.
Lastly, we performed immunohistochemistry staining of Foxp3 (a marker of T cell), to evaluate the involvement of CD4+CD25+Foxp3+ regulatory (Treg) cells that are important inflammation regulators. We demonstrated the presence of Treg cells in all UUO groups at day 14 ( Figure 8). Further, the quantitative morphometric analysis showed that the ratio of Foxp3+ Treg cells to CD4+ T lymphocytes was enhanced 24.2% in WT+UUO+CGS animals (n = 10 per group), whereas genetic A 2A R inactivation significantly decreased this ratio by 54.8% in KO+UUO+Veh group, compared with WT+UUO+Veh group (P,0.05, n = 10 per group) at day 14 post-UUO. Together, these findings suggest that CD4+ T lymphocyte was the major component in the inflammatory infiltration after UUO while A 2A R-activation suppressed CD4+ T lymphocyte infiltration and enhanced the proportion of Treg cells.

Discussion
Our study demonstrate, for the first time, that A 2A R activation can protect and postpone RIF in experimental UUO animals by the following findings: (i) A 2A R activation significantly attenuated UUO-induced pathology consequence and collagen deposition at Regulation of the infiltration of T lymphocytes is the principal mechanism of A 2A R manipulation in UUO-induced RIF in mice. The infiltration of lymphocyte, as a macrophage-independent response, plays an important role in the process of RIF and nephritis [19,25,26,27,28]. T cell infiltration was observed in the kidneys of patient with chronic kidney disease [29] and in the models of UUO [30,31,32]. Furthermore, there is reduced lymphocyte infiltration and fibrosis in the kidney after UUO when CC-chemokine receptor-1 mediated migration of lymphocytes into inflamed tissue is blocked [31,33]. Activation of A 2A R, as a Gs coupling protein receptor, can significantly increase cAMP level in immune cells, and in turn, alter immune responses including antigen presentation, T cell activation, clonal expansion, and the survival of immune memory [34,35]. This study shows that activation of A 2A R significantly reduced the CD4+ T cell infiltration whereas genetic inactivation of A 2A R exerted the opposite effect. Noteworthy, while macrophages played an important role in renal fibrosis in a model of immune-associated chronic inflammation [13] and in aristolochic acid-induced RIF [36], in the presented UUO model no significant amount of macrophage (with CD68+ and F4/80+ staining) was observed participating in leukocyte infiltration around vessels post-UUO.
Treg cells are critical to maintain immune-cell homeostasis by enforcing a dominant negative regulation on other immune cells. Thus, Tregs are of great interest due to its immunosuppressive effect and inhibitory effect against fibrosis [37]. Most recent, it was reported that Tregs' negative regulation on immune cells was mediated by A 2A R activation whereas deletion of A 2A R abolished Tregs' regulatory effect [38]. These reports support our findings that A 2A R activation by CGS21680 significantly increased the ratio of Tregs to CD4+ T lymphocytes, whereas this ratio was significantly decreased in A 2A R KO mutants post-UUO. Thus, regulation of Tregs recruitment and (CD4+) T lymphocyte infiltration acts as underlying mechanism of A 2A R-mediated effects against RIF.
Another important finding in this study is that A 2A R could affect EMT-related changes in E-cadherin and SMA. While more direct evidence and evaluations are needed in human studies, EMT is recently proposed as a crucial mechanism in RIF [5]. During the EMT process, renal tubular epithelial cell lost the E-cadherin phenotype and acquire the myofibroblast phenotype a-SMA. Our findings demonstrated that activation of A 2A R restored expression level of a-SMA and E-cadherin to a basal level in sham animals ( Figure 4). Though indirectly based on Western blot reflecting total renal tissue rather than TECs-specific on-site changes, this finding indicates that A 2A R activation maintained intrinsic phenotypes of epithelia and myofibroblast, i.e., inhibited the process of EMT. To find the mechanism by which A 2A R affects EMT, we demonstrated that activation of A 2A R significantly reduced the expression of TGF-b1, a key profibrotic mediator in EMT, along with ROCK1, the regulatory protein in the TGF-b1 downstream pathway Rho/ROCK signaling. In UUO, the enhanced TGF-b1 may (i) act as a mitogenic factor to affect collagen synthesis and (ii) facilitate the EMT process [6,39]. Importantly, activation of A 2A R restored both aforementioned consequences of TGF-b1 post-UUO. Furthermore, this study showed that TGF-b1-mediated EMT is regulated by the Rho/ROCK-dependent signaling pathway [20], and the ROCK pathways play an important role in RIF and phenotypic modulation of epithelial cells [40,41]. While ROCK has two types (ROCK1 and ROCK2), the ROCK1 is predominantly expressed in the kidney and regulates cell adhesion, chemotaxis and contraction, as well as epithelial differentiation [42]. Meanwhile, E-cadherin, not only as a marker, is also the most important component for maintaining the integrity and polarity of epithelial cells [43]. The loss of E-cadherin expression in the renal tubular epithelial cells will lead to a loss of cell-cell adhesion facilitating the renal tubular epithelial cells enter the renal interstitium. Studies showed that E-cadherin is regulated by ROCK1 [42,44], moreover, a-SMA as the important structure protein of the myofibroblast, is also influenced by the Rho/ROCK signaling pathway [45]. Importantly, the Rho/ROCK-1 pathway is closely linked to adenosine activity [15]. Thus activation of A 2A R may, via ROCK1, regulate cell adhesion of tubular epithelial cells and the EMT process. Further evidence is needed to address this potential mechanism.
The increase of A 2A R after UUO may account for a compensatory protective mechanism. In line with our finding, Lee et al also demonstrated this phenomena [46]. However, it is still unclear whether the increased A 2A R mRNA attribute to inherent renal cells or immigrated cells, e.g., bone marrow-derived cells [47], via inflammatory processes post-UUO. Our study suggested that the effect on T lymphocyte infiltration contribute to A 2A R-mediated protection against RIF. Noteworthy, some of the A 2A R activation resulting effects on post-UUO animals were blunt in the late stage after UUO with unknown reason. This may be due to the severity of phenotypes in late post-UUO stage and the progressive aggravation in pathology unless the pathogenic factors of tubular obstruction might have been removed. The severe pathology changes in late post-UUO stage might be irreversible; however, we also noted the suppressive effect of A 2A R activation on expression of TGF-b1 was devoid at day 14 after UUO. This may be due to a redundant mechanism between ROCK1 and TGF-b1, for instance a down-regulation of ROCK1 by A 2A R agonist may result in an increased expression of TGF-b1 [48]. To clarify the noticed phenomenon, using a model with mild severity or slow progress may help on this point in the future.
In summary, this study for the first time demonstrates the beneficial effect of A 2A R activation in preventing the progression of RIF in the UUO animal model. This provides a novel therapeutic strategy against renal interstitial fibrosis by targeting the adenosine A 2A receptor.