Gastrin Attenuates Renal Ischemia/Reperfusion Injury by a PI3K/Akt/Bad-Mediated Anti-apoptosis Signaling

Abstract

Ischemic/reperfusion (I/R) injury is the primary cause of acute kidney injury (AKI). Gastrin, a gastrointestinal hormone, is involved in the regulation of kidney function of sodium excretion. However, whether gastrin has an effect on kidney I/R injury is unknown. Here we show that cholecystokinin B receptor (CCKBR), the gastrin receptor, was significantly up-regulated in I/R-injured mouse kidneys. While pre-administration of gastrin ameliorated I/R-induced renal pathological damage, as reflected by the levels of serum creatinine and blood urea nitrogen, hematoxylin and eosin staining and periodic acid-Schiff staining. The protective effect could be ascribed to the reduced apoptosis for gastrin reduced tubular cell apoptosis both in vivo and in vitro. In vitro studies also showed gastrin preserved the viability of hypoxia/reoxygenation (H/R)-treated human kidney 2 (HK-2) cells and reduced the lactate dehydrogenase release, which were blocked by CI-988, a specific CCKBR antagonist. Mechanistically, the PI3K/Akt/Bad pathway participates in the pathological process, because gastrin treatment increased phosphorylation of PI3K, Akt and Bad. While in the presence of wortmannin (1 μM), a PI3K inhibitor, the gastrin-induced phosphorylation of Akt after H/R treatment was blocked. Additionally, wortmannin and Akt inhibitor VIII blocked the protective effect of gastrin on viability of HK-2 cells subjected to H/R treatment. These studies reveals that gastrin attenuates kidney I/R injury via a PI3K/Akt/Bad-mediated anti-apoptosis signaling. Thus, gastrin can be considered as a promising drug candidate to prevent AKI.

Keywords: Akt; CCKBR; acute kidney injury; apoptosis; gastrin; ischemia-reperfusion injury.

FIGURE 1

Increased expression of CCKBR in the I/R-injured mouse kidneys. (A) Representative pictures showing pathological changes at the surface and cortico-medullary border of the I/R-injured kidneys (scale bar, 1 cm). (B) The mRNA level of CCKBR was significantly increased in the kidneys after I/R injury, compared with the sham kidneys (n = 3, *p < 0.05 vs. Sham). (C) A remarkable increase of CCKBR proteins in the kidneys after I/R injury (n = 3, *p < 0.05 vs. Sham). (D) Representative images of IHC staining indicating CCKBR was expressed in tubular cells and it was up-regulated after renal I/R injury (NC: negative control; scale bar, 50 μm; magnification of ×200 in the upper panel and ×400 in the lower panel).

FIGURE 2

Gastrin improves renal function and alleviates pathological damage after renal I/R injury. (A) Schematic illustration of the protocol used for I/R-injured mouse kidneys (NS: normal saline). (B) Representative pictures of mouse kidney specimens revealing that gastrin pre-treatment alleviates I/R-induced damage, while CI-988 blocked the protective effect. (C,D) Gastrin pre-treatment mitigated I/R-induced elevation of SCr and BUN, which was inhibited by the addition of CI-988 (n = 6, *p < 0.05 vs. Control-Sham; #p < 0.05 vs. Control-I/R; $p < 0.05 vs. Gastrin-I/R). (E) Gastrin alleviated renal pathological damage, revealed by the representative images of H&E staining and PAS staining, while CI-988 inhibited the beneficial effect (scale bar, 50 μm). (F) The semi-quantitative score of renal pathological damage (n = 6, *p < 0.05 vs. Control-Sham; #p < 0.05 vs. Control-I/R; $p < 0.05 vs. Gastrin-I/R). (G) The expression of KIM-1, a marker for tubule injury, was less elevated in I/R-injured kidneys pre-treated with gastrin, while the effect of gastrin was abolished in the presence of CI-988 (n = 3, *p < 0.05 vs. Control-Sham; #p < 0.05 vs. Control-I/R; $p < 0.05 vs. Gastrin-I/R).

FIGURE 3

Gastrin attenuates I/R-induced cell apoptosis in mouse kidneys. (A,B) TUNEL staining and statistical results showing gastrin pre-treatment reduced cell apoptosis induced by I/R injury (n = 6, *p < 0.05 vs. Control-Sham; #p < 0.05 vs. Control-I/R; scale bar, 50 μm). (C) Gastrin protected against the I/R-induced increase in caspase-3 activity, reflected by caspase-3 activity assay (n = 5, *p < 0.05 vs. Control-Sham; #p < 0.05 vs. Control-I/R). (D,E) Representative images and the statistic results showing that the phosphorylation of Akt was elevated after I/R injury and gastrin pre-treatment further increased the phosphorylation of Akt (p-: phosphorylated, n = 6 in Control and 8 in Gastrin, *p < 0.05 vs. Control-Sham; #p < 0.05 vs. Control-I/R).

FIGURE 4

Gastrin preserves the viability of H/R-treated HK-2 cells. (A) No obvious effect of gastrin on cell viability of HK-2 cells under normoxic condition, indicated by CCK-8 assay (Con: Control; n = 6). (B) Gastrin preserved the viability of H/R-treated HK-2 cells in a dose-dependent manner, reflected by CCK-8 assay (H/R: hypoxia/reoxygenation; n = 6, *p < 0.05 vs. Control; $p < 0.05 vs. H/R; #p < 0.05 vs. H/R). (C) Representative images showing gastrin (10⁻⁹ M) reduced the H/R-induced cell death, while CI-988 (10⁻⁵ M) blocked the protective effect of gastrin (G: Gastrin; CI: CI-988; scale bar, 50 μm). (D) Gastrin reduced the LDH release from HK-2 cells after H/R treatment, while CI-988 (10⁻⁵ M) abolished the protective effect of gastrin (10⁻⁹ M) (n = 6, *p < 0.05 vs. Control; #p < 0.05 vs. H/R; $ p < 0.05 vs. Gastrin + H/R). (E) CCKBR was significantly up-regulated in HK-2 cells after H/R treatment (n = 3, *p < 0.05 vs. Control).

FIGURE 5

Gastrin reduces HK-2 cell apoptosis and ROS production induced by H/R treatment. (A,B) Representative pictures and the statistical results of TUNEL assay illustrating that gastrin (10⁻⁹ M) reduced HK-2 cell apoptosis induced by H/R treatment, while CI-988 (10⁻⁵ M) abolished the anti-apoptotic effect of gastrin (Con: Control; H/R: hypoxia/reoxygenation; G: Gastrin; CI: CI-988; n = 6, *p < 0.05 vs. Control; #p < 0.05 vs. H/R; $ p < 0.05 vs. Gastrin + H/R; scale bar, 50 μm). (C) Gastrin (10⁻⁹ M) alleviated the H/R-induced increase in caspase-3 activity in HK-2 cells, while CI-988 (10⁻⁵ M) reduced the effect of gastrin, indicated by caspase-3 activity assay (n = 3, *p < 0.05 vs. Control; #p < 0.05 vs. H/R; $ p < 0.05 vs. Gastrin + H/R). (D,E) Representative images and statistical results of DHE staining indicating that gastrin (10⁻⁹ M) reduced the H/R-induced ROS production in HK-2 cells, while CI-988 (10⁻⁵ M) abolished the effect of gastrin (n = 3, *p < 0.05 vs. Control; #p < 0.05 vs. H/R; $ p < 0.05 vs. Gastrin + H/R; scale bar, 50 μm). (F) Gastrin (10⁻⁹ M) preserved the H/R-caused SOD activity reduction in HK-2 cells, while CI-988 (10⁻⁵ M) abolished the effect of gastrin (n = 6, *p < 0.05 vs. Control; #p < 0.05 vs. H/R; $ p < 0.05 vs. Gastrin + H/R). (G) Gastrin (10⁻⁹ M) ameliorated the H/R-induced increasing of MDA level in HK-2 cells, while CI-988 (10⁻⁵ M) blocked the effect of gastrin (n = 6, *p < 0.05 vs. Control; #p < 0.05 vs. H/R; $ p < 0.05 vs. Gastrin + H/R).

FIGURE 6

PI3K/Akt/Bad pathway is involved in the gastrin-mediated protective effect against H/R injury. (A) Gastrin (10⁻⁹ M) increased H/R-induced phosphorylation of PI3K, while CI-988 (10⁻⁵ M) blocked the effect of gastrin (p-: phosphorylated; Con: Control; H/R: hypoxia/reoxygenation; G: Gastrin; CI: CI-988; n = 3, *p < 0.05 vs. Control; #p < 0.05 vs. H/R; $p < 0.05 vs. Gastrin + H/R). (B) Gastrin (10⁻⁹ M) increased H/R-induced phosphorylation of Akt, while CI-988 (10⁻⁵ M) abolished the effect of gastrin (n = 3, *p < 0.05 vs. Control; #p < 0.05 vs. H/R; $p < 0.05 vs. Gastrin + H/R). (C) Gastrin (10⁻⁹ M) increased phosphorylation of Bad after H/R treatment, while CI-988 (10⁻⁵ M) reduced the effect of gastrin (n = 3, *p < 0.05 vs. Control; #p < 0.05 vs. H/R; $p < 0.05 vs. Gastrin + H/R). (D) Wortmannin (1 μM) blocked the gastrin-induced phosphorylation of Akt after H/R treatment (Wort: Wortmannin; n = 3, *p < 0.05 vs. Control). (E) Wortmannin (1 μM) and Akt inhibitor VIII (10 μM) reduced the protective effect of gastrin on viability of HK-2 cells subjected to H/R treatment, determined by CCK-8 assay (AKTI: AKT inhibitor VIII; n = 3, *p < 0.05 vs. the fifth group; #p < 0.05 vs. the sixth group).

FIGURE 7

A schematic representation of the protective effect of gastrin on renal I/R injury.