P2X7 receptor inhibition protects against ischemic acute kidney injury in mice

Abstract

Activation of the purinergic P2X7 receptor (P2X7R) has been associated with the development of experimental nephritis and diabetic and hypertensive nephropathy. However, its role in acute kidney injury (AKI) remains unknown. In this study, we examined the effects of P2X7R inhibition in a murine model of ischemia-reperfusion (I/R)-induced AKI using A438079, a selective inhibitor of P2X7R. At 24 h after I/R, mice developed renal dysfunction and renal tubular damage, which was accompanied by elevated expression of P2X7R. Early administration of A438079 immediately or 6 h after the onset of reperfusion protected against renal dysfunction and attenuated kidney damage whereas delayed administration of A438079 at 24 h after restoration of perfusion had no protective effects. The protective actions of A438079 were associated with inhibition of renal tubule injury and cell death and suppression of renal expression of monocyte chemotactic protein-1 and regulated upon expression normal T cell expressed and secreted (RANTES). Moreover, I/R injury led to an increase in phosphorylation (activation) of extracellular signal-regulated kinases 1/2 in the kidney; treatment with A438079 diminished this response. Collectively, these results indicate that early P2X7R inhibition is effective against renal tubule injury and proinflammatory response after I/R injury and suggest that targeting P2X7R may be a promising therapeutic strategy for treatment of AKI.

Keywords: acute kidney injury; apoptosis; purinergic receptors; renal tubular cells.

Fig. 1.

Experimental design. Experiments were designed to evaluate the effect of A438079 on acute kidney injury (AKI) when it was given at 0 (I–IV), 6 (V–VIII), and 24 (IX–XII) h after onset of reperfusion. I/R, ischemia-reperfusion.

Fig. 2.

Inhibition of P2X7 by A438079 meliorates renal dysfunction in I/R-induced AKI in mice. Mice were subject to ischemia for 30 min followed by administration of A438079 or vehicle immediately (0), 6, or 24 h after onset of reperfusion. After treatments for an additional 24 h, blood was collected for measuring serum creatinine (A, C, and D) and blood urea nitrogen (BUN; B, D, and F). Data are represented as the mean ± SE (n = 6). Means with different superscript letters are significantly different from one another (P < 0.05).

Fig. 3.

A438079 protects against renal morphological damage in the murine model of I/R-induced AKI. After I/R and A43079 treatments as indicated in Fig. 2, the kidneys underwent periodic acid-Schiff (PAS) staining (A). Morphological changes were scored based on the scale described in materials and methods (B, C, and D). Data are represented as the means ± SE (n = 6). Means with different superscript letters are significantly different from one another (P < 0.05).

Fig. 4.

A438079 inhibits I/R-induced expression of P2X7 receptor (P2X7R) in renal tubular cells. Mice were subject to ischemia for 30 min followed by administration of A438079 immediately at onset of reperfusion. 24 h after treatments, kidneys were harvested for immunofluorescent staining of P2X7R (A) and the whole kidney tissue lysates were subjected to immunoblot analysis with specific antibodies against P2X7R and α-tubulin (B). Representative immunoblots are 2 samples from 6 animals in each group. The expression level of P2X7R was quantified by densitometry and normalized with α-tubulin. Means with different symbols are significantly different from one another (P < 0.05; C).

Fig. 5.

A438079 inhibits I/R-induced expression of neutrophil gelatinase-associated lipocalin (NAGL) in renal tubular cells. Mice were subject to ischemia for 30 min followed by administration of A438079 immediately at onset of reperfusion. Twenty-four hours after treatments, kidneys were harvested for immunofluorescent staining of NGAL (A) and tubules expressing NGAL were counted in 3 fields (1 × 100) of each kidney from 3 kidneys (B). The whole kidney tissue lysates were subjected to immunoblot analysis with specific antibodies against NGAL and α-tubulin (C). Representative immunoblots are 3 samples from 6 animals in each group. The expression level of NGAL was quantified by densitometry and normalized with α-tubulin (D). Means with different symbols are significantly different from one another (P < 0.05). The section of I/R-injured kidney was costained with antibodies to Tamm-Horsfall protein (THP) and NGAL (E). Tubular cells with positive staining for both THP and NGAL, or THP, or NGAL alone were counted in 5 high-power fields and expressed as means ± SE (F).

Fig. 6.

A438079 inhibits I/R-induced renal epithelial cell death and cleavage of poly(ADP-ribose)polymerase-1 (PARP-1). Mice were subject to ischemia for 30 min followed by administration of A438079 immediately at onset of reperfusion. Twenty-four hours after treatments, kidneys were harvested for terminal deoxynucleotidyl transferased UTP-mediated nick-end labeling (TUNEL) staining (A). Positive TUNEL staining cells were counted in 10 high-power fields and expressed as means ± SE (B). The whole kidney tissue lysates were subjected to immunoblot analysis with specific antibodies against the PARP-1 fragment at 55 kDa and α-tubulin (C and D). The expression level of cleaved PARP-1 was quantified by densitometry and normalized with α-tubulin. Data are means ± SE (n = 6). Representative immunoblots are 2 samples from 6 animals in each group. Means with different symbols are significantly different from one another (P < 0.05).

Fig. 7.

A438079 inhibits I/R-induced expression of monocyte chemotactic protein-1 (MCP-1) and regulated upon expression normal T cell expressed and secreted (RANTES) in the kidney. Mice were subject to ischemia for 30 min followed by administration of A438079 immediately at onset of reperfusion. At 24 h after treatments, kidneys were harvested for histochemical staining of MCP-1 and RANTES (A). The expression level of MCP-1 and RANTES was quantified by densitometry. Data are represented as the means ± SE (n = 6). Means with different symbols are significantly different from one another (P < 0.05; B and C).

Fig. 8.

A438079 administration inhibits I/R-induced phosphorylation of ERK1/2 in the kidney. Mice were subject to ischemia for 30 min followed by administration of A438079 immediately at onset of reperfusion. At 24 h after treatments, kidneys were harvested and the whole kidney tissue lyates were used for immunoblot analysis with specific antibodies against p-ERK1/2 and ERK1/2 (A). Representative immunoblots are 3 samples from 6 animals in each group. The expression level of ERK1/2 was quantified by densitometry and normalized with β-actin. Means with different symbols are significantly different from one another (P < 0.0; B).