Apical sodium-dependent bile acid transporter upregulation is associated with necrotizing enterocolitis

Abstract

Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency of premature infants. Previously, we showed that luminal bile acids (BAs) are increased and correlated with disease development and that the apical sodium-dependent BA transporter (ASBT), which transports BAs from the ileal lumen into enterocytes, is upregulated in rats with NEC. We hypothesized that intraenterocyte, rather than luminal, BAs are associated with NEC and that upregulation of ASBT may be a mechanism by which this occurs. Neonatal rats with or without the ASBT inhibitor SC-435, mice in which ASBT was knocked out, and mice that overproduce BAs were subjected to the NEC protocol. Disease development, ASBT, and the farnesoid X receptor protein, along with luminal and intraenterocyte BA levels, were assessed. In addition, ileal sections from premature infants with and without NEC were examined for ASBT via immunohistology and real-time PCR. When BAs were not transported into enterocytes (rats given SC-435 and ASBT knockout mice), severity and incidence of NEC were reduced. In contrast, in mice that overproduce BAs, ASBT was elevated, intraenterocyte BAs were increased, and disease development was increased. ASBT staining was more intense on the apical membrane of ileal enterocytes from premature infants with NEC than premature infants with non-NEC diagnoses. In addition, ASBT mRNA levels were significantly higher in infants with NEC. These data show that accumulation of intraenterocyte BAs contributes to disease development, elevated ASBT increases disease severity in experimental models of NEC, and ASBT is elevated in human NEC. These data confirm that BAs and upregulation of ASBT play a crucial role in NEC pathogenesis and suggest that inhibition of ASBT could be utilized as a therapeutic modality against this disease.

Fig. 1.

Ileal structure in neonatal rat model of necrotizing enterocolitis (NEC). Representative histology shows areas of necrosis and damage to ileal architecture in the NEC group compared with NEC animals given SC-435 (NEC + SC-435).

Fig. 2.

Ileal structure in neonatal mouse models of NEC. Top: representative histology showing severe separation of lamina propria and moderate separation of submucosa in the apical sodium-dependent bile acid transporter (ASBT) wild-type (WT) NEC (ASBT WT NEC) group compared with the ASBT knockout (KO) NEC (ABST KO NEC) group. Bottom: representative histology showing areas of complete villous loss in the bile salt export pump (BSEP)-overexpressing (OE) group compared with the BSEP WT NEC group.

Fig. 3.

Representative Western blots and densitometry data from the rat NEC model. After normalization with β-actin, mean optical density (OD) for the dam-fed (DF) group (n = 5) was assigned a value of 1.0, and mean ODs for groups hand-fed with a cow's milk-based formula (NEC, n = 9) and NEC + SC-435 animals (n = 9) were determined relative to this number. ASBT was significantly elevated in the NEC and NEC + SC-435 groups compared with the DF group (*P ≤ 0.05 by ANOVA).

Fig. 4.

ASBT protein in neonatal mouse models of NEC. A: representative Western blots and densitometry data from ASBT WT DF (n = 5), ASBT WT NEC (n = 8), ASBT KO NEC (n = 8), and ASBT KO DF (n = 5) mice. After normalization with actin, mean OD for the ASBT WT DF group was assigned a value of 1.0, and mean ODs for all other groups were determined relative to this number. ASBT was significantly increased in the ASBT WT NEC group compared with ASBT WT DF and ASBT KO NEC groups (*P ≤ 0.05, #P ≤ 0.01). B: representative Western blots and densitometry data from BSEP WT DF (n = 5), BSEP WT NEC (n = 10), BSEP OE NEC (n = 10), and BSEP OE DF (n = 5) mice. After normalization with actin, mean OD for the BSEP WT DF group was assigned a value of 1.0, and mean ODs for all other groups were determined relative to this number. ASBT was significantly increased in BSEP WT NEC compared with BSEP WT DF animals (*P ≤ 0.05), BSEP OE NEC compared with BSEP WT NEC animals (*P ≤ 0.05), and BSEP OE NEC compared with BSEP OE DF animals (#P ≤ 0.01). Densitometry data are expressed as means ± SD. Statistical analyses were performed using ANOVA.

Fig. 5.

Farsenoid X receptor (FXR) protein in neonatal mouse models of NEC. A: representative Western blots and densitometry data from ASBT WT DF (n = 5), ASBT WT NEC (n = 8), ASBT KO NEC (n = 8), and ASBT KO DF (n = 5) mice. After normalization with actin, mean OD for the ASBT WT DF group was assigned a value of 1.0, and mean ODs for all other groups were determined relative to this number. FXR was significantly increased in ASBT WT NEC compared with all other groups (#P ≤ 0.01). B: representative Western blots and densitometry data from BSEP WT DF (n = 5), BSEP WT NEC (n = 10), BSEP OE NEC (n = 10), and BSEP OE DF (n = 5) mice. After normalization with actin, mean OD for the BSEP WT DF group was assigned a value of 1.0, and mean ODs for all other groups were determined relative to this number. FXR was significantly increased in the BSEP WT NEC compared with the BSEP WT DF group and in the BSEP OE NEC compared with the BSEP OE DF group (*P ≤ 0.05). Densitometry data are expressed as means ± SD. Statistical analyses were performed using ANOVA.

Fig. 6.

BAs in experimental NEC models. A: total luminal BAs. B: total intraenterocyte BAs. C: composition of intraenterocyte BAs. CA, cholic acid; CDCA, chenodeoxycholic acid; DCA, deoxycholic acid. Values are means ± SD. *P ≤ 0.05, #P ≤ 0.01. Statistical analyses were performed using ANOVA.

Fig. 7.

Correlation between total BA levels and ileal damage scores in rat (NEC and NEC + SC-435) and mouse (ASBT WT NEC, ASBT KO NEC, BSEP WT NEC, and BSEP OE NEC) models of NEC. Ileal luminal BA levels in rat (A) and mouse (B) models and intraenterocyte levels in rat (C) and mouse (D) models were compared with histological ileal damage scores. Rat luminal BAs (r = 0.320, P ≤ 0.05) and rat and mouse intraenterocyte BAs (r = 0.579 and 0.726, respectively; P ≤ 0.0001) are positively correlated with progression of ileal damage. Statistical analyses were performed using Spearman's rank correlation.

Fig. 8.

Representative ileal sections stained with anti-ASBT from premature infants diagnosed with NEC (A), non-NEC diagnoses (NND; B), and ileostomy-take down after NEC diagnosis (ITD after NEC; C). Note higher levels of ASBT on the apical membrane of ileal enterocytes from surgical specimens from NEC patients.

Fig. 9.

ASBT mRNA levels in NEC and NND infants. Mean steady-state mRNA levels for the NND group was assigned a value of 1.0, and mean mRNA levels for the NEC group were determined relative to this number. ASBT mRNA was significantly higher in the NEC than the NND group (#P ≤ 0.01). Values are means ± SD. Statistical analyses were performed using ANOVA.