Several lines of evidence suggest that purinergic P2X7 receptors participate in the processes of various kidney diseases [14, 15, 16, 17]. In several previous studies, BBG, a selective P2X7R antagonist, was used as a potent inhibitor of P2X7R that reduces inflammation, immune cells activation, and fibrosis [10, 18, 19]. BBG was able to reduce renal injury in Dahl salt-sensitive hypertensive rats and in lupus nephritis model [6, 10] similarly to P2X7R null mice . Of note, BBG also antagonizes rat P2X4Rs, but its selectivity for P2X7R is 1000-fold greater [20, 21]. The present study addressed the effects of BBG on the initial process of renal interstitial inflammation, collagen deposition, renal cell apoptosis and proliferation, by using the model of unilateral ureteral obstruction. Our results showed that BBG attenuated renal damage, similar to those in P2X7R knockout mice  and in other disease models [10, 14, 18, 19, 22, 23].
Our previous study about the effects of P2X7 receptors used the same fibrogenic model of renal disease . In that study, we used knockout mice for P2X7R and we observed that these receptors could not be seen as constitutively expressed, but only in obstructed groups, probably localized on epithelial tubular cells. In addition, P2X7R were apparent only on day 7 of UUO and not on day 14. The present study was designed to observe the pathophysiological aspect of rat kidneys on day 3 of UUO, in which P2X7R was assumed to be expressed. In fact, P2X7R expression could be clearly seen by the immunohistochemical study, on the aspect of tubular epithelial cells, as well as in some interstitial cells. On the other hand, P2X7R were also expressed on sham-operated animals, although with significantly lower intensity. It is noteworthy that their expression was seen in BBG treated UUO group as significantly lower when compared with non-treated UUO group, an observation also mentioned by Marques et al  in another study using BBG. The explanation for the decreased expression of P2X7R on BBG treated group is not apparent at this moment, but one can argue about the possibility of receptor downregulation by BBG antagonism or allosteric modification of the epitope. Future studies are needed to elucidate this issue.
The results on the monocytes/macrophage recruitment after UUO clearly showed the expected increase of renal interstitial inflammation, which was decreased in BBG treated animals (Fig. 2). Nonetheless, perhaps the most specific feature associated with P2X7R activation and the development of tissue inflammation might be the secretion of inflammasome-related cytokines [24, 25]. In this regard, it has been consistently documented that the effect of P2X7R activation is closely linked to IL–1β secretion [24, 26]. In renal tissue, Deplano et al  and Jalilian et al  have previously demonstrated the role of ATP, as a damage-associated molecular pattern, on the activation of P2X7R to trigger the secretion of IL–1β. Our results showed that the increased IL–1β mRNA in UUO tissue kidneys was abrogated in BBG-treated animals (Fig. 2G).
A significant reduction in myofibroblast population (Fig. 3A-F) was observed in BBG treated group. Moreover, an effect on myofibroblast function was strongly suggested from the results of HSP47 (Fig. 3G-L). This chaperone is an endoplasmic reticulum (ER)-resident, stress inducible glycoprotein, collagen-specific heat-shock protein, which plays a key role in collagen biosynthesis and its structural assembly . It is also used as a biomarker to identify collagen-producing cells . A previous study using the model of UUO in mice clearly showed that HSP–47 was overexpressed in the renal interstitium of obstructed animals . In our study, BBG treated group showed decreased expression of the chaperone. Therefore, it is conceivable to suggest that the reduction of HSP–47 stained cells probably indicates a reduction of myofibroblasts function by the purinergic blockage.
The aspect of interstitial collagen deposition is a known striking histopathological feature of the UUO model. In this study, the expression of TGF-β, the fibrogenic cytokine associated with collagen deposition, was shown to be decreased in BBG treated rats (Fig. 5). Also, decreased collagen deposition, as expressed by picrosirius red staining, was seen in BBG treated group (Fig. 4A-F). Likewise, pro-collagen I, III and IV mRNA were shown to be increased in obstructed animals, with significant reduction in BBG treated rats (Fig. 4G-I).
Our previous study on P2X7 knockout mice clearly evidenced the implication of these receptors on the process of apoptosis of renal cells . In fact, the present study showed the decrease of apoptotic cells in kidneys from BBG treated group. Likewise, renal cells proliferation also increased in the group with BBG, and these results suggest that regenerative process after kidney damage by UUO can be up-regulated by P2X7R antagonism (Fig. 6). We have previously demonstrated the action of bone marrow-derived cells to induce and attenuate renal cell proliferation and apoptosis, respectively . Nevertheless, little is known about the involvement of P2X7 receptors activation in this setting. It is noteworthy, however, that Chen et al  have found an increase in P2X7R expression after derangement of retinal ganglion cells, which decreased in animals treated with human umbilical cord blood mesenchimal stem cells. This observation suggests a mechanistic antagonism between P2X7R expression and activation, and the action of progenitor cells to determine proliferative repair. In the present study, the putative BBG action to inhibit P2X7R activation, which was related to increased proliferation of renal cells, might suggest a modulatory role of this receptor on the mechanism of renal cell repair after epithelial cell injury.
In summary, the results from this study highlight the beneficial role of P2X7R antagonism, as can be accomplished by BBG, in the prevention of the early phase of inflammation and the ensuing fibrogenic process, even in the third day of UUO. As the previous studies using P2X7R antagonists in various disease models, one can suggest that targeting this receptor might be beneficial in selective conditions. In addition, this study also constitutes evidence that the blockage of purinergic P2X7 receptor may act in favor of renal cell proliferation and tissue regeneration, and the mechanism underlying this effect needs extensive investigation.