Male SPF Wistar rats, weighing 200–250 g, were obtained from the Experimental Animals Center of the Hubei Academy of Medical Sciences (Wuhan, Hubei, China). The study was approved by the Ethics Committee of Wuhan University (Wuhan, Hubei, China). The experimental animal procedures were conducted in compliance with the EEC regulations (Official Journal of European Community L358 12/18/1986) and NIH standards (Guide for the Care and Use of Laboratory Animal, National Institutes of Health’s publication 85–23, revised 1996). The animals were housed in a controlled environment and provided with standard rodent chow and water.

The rats were divided into four experimental groups: (i) the SAP-vehicle group, in which rats were subjected to STC (Sigma-Aldrich, St. Louis, MO, USA) induced SAP (as described below) and administered an equivalent volume of the vehicle for TDZD-8 (10% DMSO solution, i.v.) at 30 min prior to the STC infusion (n=20); (ii) the SAP-TDZD-8 group, in which rats were subjected to STC-induced SAP (as described below) and administered TDZD-8 (no. T8325; 1 mg/kg, i.v.; Sigma-Aldrich) at 30 min prior to STC infusion (n=20); (iii) the sham-vehicle group, in which rats were treated with sham surgery (saline instead of STC) in combination with the administration of an equivalent volume of the vehicle for TDZD-8 (10% DMSO solution, i.v.) and observed for 12 h (n=20); (iv) the sham-TDZD-8 group, in which rats were treated with sham surgery (saline instead of STC) in combination with the administration of TDZD-8 (1 mg/kg, i.v.) and observed for 12 h (n=20).

All of the rats were sacrificed at 12 h following the induction of pancreatitis. The TDZD-8 dose used to reduce acute kidney injury was selected based on a previous study in which similar doses of TDZD-8 exerted protective effects in experimental models of the inflammatory response (14,17).

Prior to the study, the rats were deprived of food, but allowed access to water. The rats were anesthetised through an intraperitoneal injection of chloral hydrate (10%, 3 ml/kg). SAP was induced through the retrograde infusion of 5% STC solution (1 ml/kg) into the bile-pancreatic duct transduodenally using an angiocath with a standardised pressure-controlled infusion rate under laparotomy. Following infusion, the section of bile-pancreatic duct entering the duodenum was clipped using a non-invasive angiocath for 5 min. After assessing the bile leakage, the hole in the duodenum lateral wall was sutured. Subsequently, the abdomen was closed, and the rats were left to recover from the anaesthesia and allowed access to water.

The rats from each group were sacrificed through exsanguination at 12 h following the induction of pancreatitis. The blood samples were obtained through direct intracardiac puncture and the serum was stored at −20°C. Subsequently, the pancreas and kidney were immediately removed, harvested and fixed in 4% phosphate-buffered formaldehyde for histopathological observation. The remaining pancreatic and nephridial tissues were frozen in liquid nitrogen and stored at −80°C until further use.

Continuous sections of the paraffin-embedded tissue were obtained for pathological examination with hematoxylin-eosin staining. Two independent pathologists performed a blinded morphometric assessment under a light microscope (Olympus, Tokyo, Japan). The results were recorded on an evaluation form for pancreatic and kidney injury. The pancreatic histological assessment was determined by oedema, necrosis, haemorrhage and inflammation according to a previously described scale (18).

The histological assessment of kidney injury was performed using the point-counting method of Paller et al (19). For each kidney, 100 cortical tubules from at least 10 different areas were scored, and care was taken to avoid the repeated scoring of different convolutions of the same tubule. Higher scores represented more severe damage (maximum score per tubule was 10) and points were provided for the presence and extent of tubular epithelial cell flattening (1 point), brush border loss (1 point), cell membrane bleb formation (1 or 2 points), interstitial oedema (1 point), cytoplasmic vacuolisation (1 point), cell necrosis (1 or 2 points) and tubular lumen obstruction (1 or 2 points).

The serum samples were separated, and used for biochemical and cytokine measurements. Plasma amylase, lipase, Cr and BUN levels were measured with an automatic biochemistry analyser using standard techniques (Olympus).

IL-1β and IL-6 levels were examined in the plasma from SAP and sham-treated rats as previously described (20). The assay was performed using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (eBioscience, Vienna, Austria). The absorbance was measured using an automated microplate reader and the concentrations were calculated according to the standard curve run on each assay plate.

Neutrophil infiltration into the renal parenchyma was indirectly quantitated using an MPO assay, as previously described for neutrophil elicitation (21). Renal cortical samples were weighed and homogenised in 1:19 (wt/vol) in ice-cold homogenate buffer. MPO activity was spectrophotometrically determined using a commercial assay kit according to the manufacturer’s instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The absorbance was measured at 460 nm.

Pancreatic and renal tissue was placed in a decalcifying solution for 24 h and 4 μm sections were prepared from paraffin embedded tissues. Following deparaffinisation, 0.3% hydrogen peroxide was used to inactivate endogenous peroxidase activity. Non-specific adsorption was minimised following incubating the section in 5% normal goat serum in phosphate-buffered saline. Endogenous biotin and avidin binding sites were blocked using avidin and biotin, respectively. The sections were incubated overnight with a rabbit polyclonal anti-rat NF-κB p65 antibody (1:100 in PBS, v/v; Cell Signaling Technology, Inc., Danvers, MA, USA) in a moisture chamber. The sections were subsequently counterstained with hematoxylin. The negative control studies were performed in which PBS was used instead of the primary antibody.

Renal GSK-3β, phospho-GSK-3β (Ser9), TNF-α, ICAM-1 and iNOS levels at 12 h following STC injection were determined by western blot analysis. The proteins were extracted using the Nuclear-Cytosol Extraction kit (Applygen Technologies Inc., Beijing, China). The protein concentrations in the samples were determined using the Bradford method with bovine serum albumin as a standard. Briefly, equal amounts of protein samples were electrophoresed using 8 or 10% sodium dodecyl sulphate polyacrylamide (SDS-PAGE) gels and subsequently transferred onto nitrocellulose membranes. The membrane was blocked with TBST buffer (TBS containing 5% non-fat dry milk and 0.1% Tween-20) at room temperature for 2 h and subsequently incubated with rabbit polyclonal anti-GSK-3β or anti-phospho-GSK-3β (Ser9) (Cell Signaling Technology, Inc.), anti-TNF-α (Abcam, Cambridge, MA, USA), anti-ICAM-1 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), or anti-iNOS (Cell Signaling Technology, Inc.) antibodies overnight at 4°C. Following extensive rinsing with TBST, the blots were incubated with a horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:5,000; Pierce Biotechnology, Inc., Rockford, IL, USA) at room temperature for 1–2 h, developed using an ECL reagent (Millipore, Bedford, MA, USA) and captured on a light-sensitive imaging film (Kodak, Shanghai, China). The protein bands were quantified through densitometry (Quantity One 4.5.0 software; Bio-Rad Laboratories, Inc., Hercules, CA, USA).

As demonstrated in Fig. 1A and B, significant hyperamylasemia and hyperlipasemia developed following the induction of pancreatitis, suggesting that establishing the SAP model was successful. TDZD-8 reduced amylase and lipase at 12 h following pancreatitis compared with the SAP-vehicle group (P<0.05). The rats subjected to SAP were associated with an increase in Cr and BUN levels, indicating that the rats experienced aggravated renal dysfunction. The changes in renal function induced by SAP were significantly improved following TDZD-8 treatment at 12 h after the STC infusion (P<0.05; Fig. 1C and D). The sham-vehicle and sham-TDZD-8-treated rats demonstrated no elevation in the serum levels of amylase, lipase, Cr and BUN levels.

Representative histological sections are demonstrated in Fig. 2A–D (pancreas) and Fig. 3A–D (kidney). STC-induced pancreatic damage was characterised by increased oedema, inflammatory cell infiltration, vacuolisation and necrosis. The sham rats demonstrated weak morphological evidence of pancreas injury, except for mild interstitial oedema. As revealed in Fig. 4A, there was a significant reduction in the pancreatic histological score in rats subjected to STC-induced pancreatitis and treated with TDZD-8 (P<0.05). In sham-vehicle and sham-TDZD-8-treated rats, the histological features of the pancreas were typical of a normal architecture.

Compared with sham-vehicle and sham-TDZD-8-treated rats (Fig. 3A and B), rats subjected to pancreatitis for 12 h exhibited the recognised features of kidney injury, including characteristic histological signs of tubular damage. Tubular epithelial cell flattening, brush border loss, cell membrane bleb formation, interstitial oedema, cytoplasmic vacuolisation, cell necrosis and tubular lumen obstruction were observed (P<0.05; Fig. 3C). The kidneys obtained from rats treated with TDZD-8 (Fig. 3D) demonstrated reduced histological features and pathological grading of kidney injury compared with the SAP-vehicle rats (P<0.05; Fig. 4B)

To determine whether GSK-3β inhibition modulates the inflammatory process through the regulation of IL-1β and IL-6, the plasma levels of IL-1β and IL-6 were analysed. A substantial increase in IL-1β and IL-6 formation was observed (Fig. 5A and B). By contrast, the plasma and pancreas levels of IL-1β and IL-6 were significantly reduced in the animals treated with TDZD-8 (P<0.05). No elevation in IL-1β and IL-6 plasma levels was observed in sham-vehicle and sham-TDZD-8 treated rats. The renal MPO activity following SAP was measured to examine the infiltration of neutrophils into the kidney. MPO activity was significantly elevated at 12 h following STC infusion in the vehicle-treated rats compared with the sham group (P<0.05). The MPO activity in the renal tissue from SAP rats treated with TDZD-8 was significantly reduced (P<0.05; Fig. 5C)

An immunohistochemical assay was used to assess the localisation of NF-κB p65 subunit expression. NF-κB p65 expression was primarily detected in the cytoplasm of cells from renal tissue. Immunostaining for NF-κB p65 (Fig. 6C) was markedly expressed in the nucleus following the administration of STC compared with that in the sham-control rats (Fig. 6A). Positive immunostaining for NF-κB p65 was significantly reduced in rats treated with TDZD-8 (Fig. 6D). As demonstrated in Fig. 6B, weak immunoreactivity was primarily observed in the cytoplasm in sham-TDZD-8-treated rats.

To obtain a mechanistic insight into the effects of the GSK-3β inhibitor used in the present study, a western blot analysis of the phosphorylation of GSK-3β (Ser9) was performed (Fig. 7A). As demonstrated in Fig. 7A, the phosphorylation of GSK-3β (Ser9) in renal tissue was significantly increased in the TDZD-8-treated SAP group than in the vehicle-treated SAP group, consistent with the inactivation of the kinase (P<0.05; Fig. 7B).

Taken together, these observations indicated that the degree of pancreatitis and associated kidney injury was gradually aggravated. Therefore, the effects of TDZD-8 treatment on TNF-α, ICAM-1 and iNOS expression in the kidney following 12 h were then examined. As demonstrated in Fig. 8, TNF-α, ICAM-1 and iNOS expression in renal tissue was significantly increased in the vehicle-treated SAP group (P<0.05). In addition, the inhibition of GSK-3β significantly decreased the activation of TNF-α, ICAM-1 and iNOS expression in the kidney following SAP (P<0.05).