Isoflurane activates intestinal sphingosine kinase to protect against renal ischemia-reperfusion-induced liver and intestine injury

Abstract

Background: Renal ischemia-reperfusion injury (IRI) is a major cause of acute kidney injury and often leads to multiorgan dysfunction and systemic inflammation. Volatile anesthetics have potent antiinflammatory effects. We aimed to determine whether the representative volatile anesthetic isoflurane protects against acute kidney injury-induced liver and intestinal injury and to determine the mechanisms involved in this protection.

Methods: Mice were anesthetized with pentobarbital and subjected to 30 min of left renal ischemia after right nephrectomy, followed by exposure to 4 h of equianesthetic doses of pentobarbital or isoflurane. Five hours after renal IRI, plasma creatinine and alanine aminotransferase concentrations were measured. Liver and intestine tissues were analyzed for proinflammatory messenger RNA (mRNA) concentrations, histologic features, sphingosine kinase-1 (SK1) immunoblotting, SK1 activity, and sphingosine-1-phosphate concentrations.

Results: Renal IRI with pentobarbital led to severe renal, hepatic, and intestinal injury with focused periportal hepatocyte vacuolization; small-intestinal apoptosis; and proinflammatory mRNA up-regulation. Isoflurane protected against renal IRI and reduced hepatic and intestinal injury via induction of small-intestinal crypt SK1 mRNA, protein and enzyme activity, and increased sphingosine-1-phosphate. We confirmed the importance of SK1 because mice treated with a selective SK inhibitor or mice deficient in the SK1 enzyme were not protected against hepatic and intestinal dysfunction with isoflurane.

Conclusions: Isoflurane protects against multiorgan injury after renal IRI via induction of the SK1/sphingosine-1-phosphate pathway. Our findings may help to unravel the cellular signaling pathways of volatile anesthetic-mediated hepatic and intestinal protection and may lead to new therapeutic applications of volatile anesthetics during the perioperative period.

Fig. 1

Plasma alanine aminotransferase (ALT, U/L) after renal ischemia–reperfusion injury (IRI). (A) Plasma ALT was measured in C57BL/6 mice exposed to 4 h of pentobarbital (PB) or 1.2% isoflurane (Iso) after sham operation (N = 7 for each group) or renal IRI (N = 8 for each group). (B) Plasma ALT from C57BL/6 mice treated with the sphingosine kinase (SK) inhibitor SKI-II or from SK1 knockout (SK1KO) mice after sham operation (PB anesthesia; N = 4 for each group) or renal IRI (N = 6 for each group) followed by exposure to 4 h of PB or Iso. In all cases (A and B), plasma ALT was measured 24 h after renal IRI. # P less than 0.05 versus. sham mice. * P less than 0.05 versus. PB Renal IRI group. Data presented as mean ± SEM.

Fig. 2

Isoflurane protects against liver injury after renal ischemia–reperfusion injury (IRI). Representative photomicrographs of liver from 4 experiments (hematoxylin and eosin staining, magnification of 400×) of mice subjected to sham operation (A) or to renal IRI followed by 4 h of pentobarbital (B) or 1.2% isoflurane (C). Tissues were collected 24 h after renal IRI. Arrows point to areas of significant hepatocyte vacuolization.

Fig. 3

Isoflurane protects against small intestinal injury after renal ischemia–reperfusion injury (IRI). Representative photomicrographs of small intestine from 4 experiments (hematoxylin and eosin staining, magnifications of 200× and 600×). Sham-operated mice show normal intestinal morphology (A-B). The intestines of mice exposed to 4 h of pentobarbital after renal IRI demonstrate marked epithelial villous swelling (C, swollen villi highlighted by [lsqb]*[rsqb]), and an enlarged image of a single, swollen villous (D) shows numerous apoptotic bodies (circled). In contrast, the intestines of mice exposed to 4 h of 1.2% isoflurane after renal IRI were protected from severe injury (E-F). Tissues were collected 24 h after renal IRI.

Fig. 4

Isoflurane protects against intestinal apoptosis after renal ischemia–reperfusion injury (IRI). Representative fluorescence photomicrographs (of 4 experiments) illustrating apoptotic nuclei (TUNEL fluorescence staining, green) in the small intestine. The left side of each panel depicts a 100× fluorescence photomicrograph with a highlighted area (white box) enlarged to 400× on the right side the panel. Mice were exposed to 4 h of pentobarbital after sham operation (A) or renal IRI (C) or to 4 h of 1.2% isoflurane after sham operation (B) or renal IRI (D). Tissues were collected 24 h after renal IRI.

Fig. 5

Isoflurane protects against renal ischemia–reperfusion injury (IRI) mediated hepatic and intestinal pro-inflammatory messenger RNA (mRNA) upregulation. Mice were subjected to renal IRI followed by exposure to 4 h of pentobarbital (open bars) or 1.2% isoflurane (closed bars). Liver and small intestine tissues were collected 24 h after renal IRI. Densitometric quantifications of band intensities relative to GAPDH from RT-PCR reactions. N = 4 per group. * P less than 0.05 versus. appropriate pentobarbital group. Data presented as mean ± SEM.

Fig. 6

Isoflurane reduces vascular permeability after renal ischemia–reperfusion injury (IRI). Quantification of Evans blue dye (EBD) extravasation as an index of vascular permeability of liver, jejunum, and ileum tissues in mice 24 h after sham operation (sham, pentobarbital (PB) anesthesia) or renal IRI followed by exposure to 4 h of PB or 1.2% isoflurane (Iso). N = 3 per group. Data are presented as means ± SEM # P less than 0.05 versus. PB sham group. * P less than 0.05 versus. PB renal IRI group.

Fig. 7

Isoflurane activates intestinal sphingosine kinase (SK) 1, but not SK2, messenger RNA (mRNA) expression. (A) Representative gel images of RT-PCR (of 4 experiments) of SK1, SK2, and GAPDH from the small intestines of sham mice exposed to 4 h of pentobarbital or 1.2% isoflurane. (B) Densitometric quantifications of band intensities relative to GAPDH from RTPCR reactions. * P less than 0.05 versus. Pentobarbital sham group. Data presented as mean ± SEM.

Fig. 8

Isoflurane increases intestinal sphingosine kinase (SK) 1, but not SK2, protein expression. (A) Representative immunoblot images (of 4 experiments) of SK1, SK2, and β-actin from the small intestines of sham mice exposed to 4 h of pentobarbital or 1.2% isoflurane. (B) Densitometric quantifications of band intensities relative to β-actin from immunoblot images. * P less than 0.05 versus. Pentobarbital sham group. Data presented as mean ± SEM.

Fig. 9

Isoflurane increases sphingosine kinase (SK) 1 in small intestinal crypts. Representative immunofluorescence images (of 4 experiments) for SK1 (red) and nuclear staining (blue) from sham mice exposed to 4 h of pentobarbital (A) or 1.2% isoflurane (B). SM designates smooth muscle layer. Arrows point to small intestinal crypts.

Fig. 10

Isoflurane exposure increases sphingosine kinase (SK) 1 activity and sphingosine-1-phosphate (S1P) formation. (A) Relative SK1 activity (fold over pentobarbital group) from intestines of sham mice exposed to 4 h of pentobarbital or isoflurane (N = 6 per group). (B) Formation of S1P (fold over pentobarbital group) from the intestines of sham mice exposed to 4 h of pentobarbital or isoflurane (N = 4 per group). * P less than 0.05 versus. Pentobarbital group. Data presented as mean ± SEM.