Gut Permeability and Glucose Absorption Are Affected at Early Stages of Graft Rejection in a Small Bowel Transplant Rat Model

Abstract

Background: Intestinal transplantation (ITx) faces many challenges due to the complexity of surgery and to the multiple immunological reactions that lead to the necessity of rigorous follow-up for early detection of acute cellular rejection (ACR). Our aim was to determine the kinetics of ACR using an experimental ITx model, with emphasis in the characterization of the process using different approaches, including the use of functional assays of absorptive and barrier function.

Methods: ITx in rats conducting serial sampling was performed. Clinical monitoring, graft histology, proinflammatory gene expression, and nitrosative stress determination were performed. Also, glucose absorption, barrier function using ovalbumin translocation, and contractile function were analyzed.

Results: The model used reproduced the different stages of ACR. Allogeneic ITx recipients showed signs of rejection from postoperative day (POD) 5, with increasing severity until 12 POD. Histological evaluation showed mild rejection in early sampling and severe rejection at late stages, with alterations in all graft layers. IL-6, CXCL 10, IFNg, and nitrite plasmas levels showed behavior coincident with histopathology. Remarkably, allogeneic grafts showed a marked alteration of glucose absorptive capacity from POD 5 that was sustained until endpoint. Coincidently, barrier function alteration was evidenced by luminal ovalbumin translocation to serum. Contractile function was progressively impaired along ACR.

Conclusions: Glucose absorption and barrier function are altered at early stages of ACR when histological alterations or gene expression changes were much subtle. This observation may provide simple evaluation tools that could be eventually translated to the clinics to contribute to early ACR diagnosis.

FIGURE 1

Clinical status of recipients and transplanted intestines are altered during ACR process. A, quantitative clinical score was determined for ALLO and ISO ITx recipients. **P < 0.01 (B) Worsening of ALLO group is also reflected in weight loss compared with ISO group at 10 to 12 days after transplantation. **P < 0.01 (C) Graft tightening (used as clinical rejection indicator) was evident in ALLO group in contrast with ISO ITx recipients. ****P < 0.001 (N = 10 in each group for each evaluation).

FIGURE 2

Functional features of ALLO intestinal grafts are altered at early stages of ACR. A, Glucose absorptive capacity was evaluated by glucose intraluminal administration and evaluation of glucose blood levels in both early (5 POD) and late (10-12 POD) evaluation times. Significant differences were observed between ALLO versus ISO and control groups (intraperitoneal and intestinal intraluminal administration in nontransplanted animals) 60 and 90 minutes after glucose administration. **P < 0.01. Mean and SD values of 6 different independent TX procedures are depicted. B, barrier function evaluation performed by plasma OVA determination by ELISA upon administration of OVA through graft proximal ostomy. Significant differences were observed in ALLO vs ISO and Sham groups (***P < 0.005). Mean and SD values of 6 different independent TX procedures are depicted. C, Maximum contractile strength evaluated by mecanotransduction. Significant differences were observed at late stages of ACR between ALLO vs ISO and control groups. *P < 0.05. Mean and SD values of 6 different independent TX procedures are depicted.

FIGURE 3

Histopathology analysis show evidences of the progression of the ACR process with increasing postoperative time. A, Histologic diagnosis of ACR and morphologic characteristics in ALLO and ISO groups after ITx (N = 10 in each group for each evaluation time). ALLO group showed histopathological sign of ACR from the fifth day after transplantation in contrast to ISO group. ***P < 0.005. B. Microscopic graft appearance in H/E stained intestinal samples of ISO and ALLO ITx groups. C, Apoptotic cell quantification and location in ALLO group evidence an increase in the number of apoptotic cells in late stages of ACR. **P < 0.01. D, Representative images of early and late stages of ACR in the ALLO group (20× magnification) by Tunel assay.

FIGURE 4

Proinflammatory gene expression analysis showed major differences at later stages of ACR process. A, Relative mRNA intestinal proinflammatory gene expression at 5 (early) and 10 to 12 (late) days after ITx in whole intestinal samples in ISO and ALLO groups (N = 6 in each group). Significant differences in terms of IL-6 and CXCL-1 expression were observed between later stages of ACR in ALLO group vs ISO group. *P < 0.05. B, Principal component analysis of gene expression profile in ISO and ALLO ITx groups at different times after ITX. The major difference is observed in the ALLO group during the later stages of ACR.

FIGURE 5

Proinflammatory gene expression was increased in different graft tissue layers upon ACR. Epithelial, muscular and serosa layers were obtained by laser microdissection and gene expression in each sample was determined by real-time quantitative polymerase chain reaction (N = 5 for each layer and group). Relative mRNA levels of IL22, IFNg, CXCL1, IDO, CXCL10, IL13 were evaluated.

FIGURE 6

Collagen type I/III increase considerably in ALLO group after 10 to 12 days after ITX compared with ALLO (N = 6) early and early and late ISO groups (N = 6). **P < 0.01. Also, representative microscopic images of different collagen types are shown.

FIGURE 7

A, Nitrites plasma levels increase from initial stages to late phases of ACR process. Significant differences were observed between ALLO (N = 6) vs ISO (N = 6) and basal nitrites levels. **P < 0.01. B, Plasma levels of protein carbonyls only increase significantly in later phases of ACR in ALLO group. *P < 0.05.