Histological alterations in the corpus region of the stomach. (A) Representative H&E staining of a transverse section of stomach from mice at one year of age. In WT mice, progenitor cells are located in the isthmus (asterisk), PCs (basic staining) stain magenta with H&E and are located in the neck (black arrow). ECL cells and zymogenic cells (acid staining) stain dark purple with H&E and are located at the base of oxyntic glands (gray arrow). In KI/KI mice, cellular disorganization and increased thickness of the stomach wall were observed, characterized by the presence of proliferating cells (at isthmus in WT mice) and ECL-cells (at base in WT mice) within the neck region of gastric corpus glands (asterisk). In addition, high numbers of epidermoid cysts surrounded by a single layer of cuboidal epithelium (gray arrowhead), vacuolization of gastric mucosal cells (white arrow) and widely opened lumen of the oxyntic glands (white arrowhead) were seen in KI/KI mice. In the enlarged image at the far right, increased number of grouped cells from the isthmus region (black arrowhead), dysplastic cytoplasms of PCs (black arrow) and hyalinosis (light gray arrow), indicating a premalignant condition, are shown. (B) IHC of gastric corpus stained with anti-Ki67 antibody. In WT mice, the staining is limited to the isthmus region, which is the proliferative zone. Increased Ki67 staining is observed in the KI/KI stomach isthmus, but is also seen in the neck region (black arrow). (C) IHC of gastric corpus stained with anti-chromogranin A antibody. In WT mice, the staining is limited to the base of the oxyntic glands whereas in KI/KI stomachs it is also seen in the entire neck region (black arrows). (D) Alcian Blue-PAS staining of the corpus region of the stomach of WT (left) and KI/KI (right) mice. White arrows show areas of glandular metaplasia in the KI/KI stomach.

Hypoacidity, anemia and hypergastrinemia in KI/KI mice. (A) Gastric pH values determined by direct measurement with colorimetric paper at necropsy. Measurements of two different animals are shown for each genotype; gastric pH values were ∼1, ∼4 and ∼7 for WT, KI/+ and KI/KI mice, respectively. (B) Ferritin concentrations in plasma (mM) of WT, KI/+ and KI/KI male (left) and female (right) mice. (C) Gastrin concentrations in serum (pM) of WT, KI/+ and KI/KI mice at ages indicated. (D) Representative IHC images of the antrum region of the stomach stained with anti-gastrin antibody in WT and KI/KI mice at 350 days of age. In both genotypes, the anti-gastrin antibody labels G-cells at the base of pyloric glands (black arrows). (E) IHC with anti-Ki67 antibody of the gastric antrum from representative WT and KI/KI mice. In both genotypes, progenitor cells are located at the base of pyloric glands (asterisks). (F) Gastrin concentrations in serum (pM) of WT, KI/+ and KI/KI mice. n=6 per genotype. ns, not significant. Data in B,C,F are represented as mean±s.d.

Phenotype prevention by treatment with HCl water. (A) Representative macroscopic external (left) and internal (right) images of stomachs are shown for HCl water­-treated and untreated WT and KI/KI mice at >350 days of age. No lesions or injuries were observed in WT mice following this treatment. Gastric hyperplasia in treated KI/KI mice (pH 4) is drastically reduced compared with KI/KI mice without treatment (pH 7). Scale bar: 1 cm. (B) Representative H&E staining images of KI/KI stomachs from treated mice compared with WT and non-treated KI/KI mice at 350 days of age. Similar architecture consisting of progenitor neck mucosa cells located in the isthmus (asterisks), PCs in the neck (black arrows), and ECL and zymogenic cells in the base of oxyntic glands (dark gray arrows) were observed in WT and treated KI/KI stomachs. Widely opened oxyntic glands (white arrowhead), grouped nuclei from the isthmus region (black arrowhead) and hyalinosis (light gray arrow), observed in untreated KI/KI stomachs, were not detected in treated mice. (C) Serum gastrin concentrations (pM) in WT, KI/+ and KI/KI mice treated for the times indicated. (D) Ferritin concentrations in plasma (µM) of treated WT, KI/+ and KI/KI males (top) and females (borrom). (E) IHC of corpus region of a representative treated KI/KI stomach with anti-Ki67 (left) and anti-chromogranin A (right) antibodies. Staining is limited to the isthmus and the base of the oxyntic glands, respectively, as observed in WT mice (Fig. 2B). (F) Alcian Blue-PAS staining of the corpus region of a representative KI/KI stomach from a 350-day-old mouse. n=6 per genotype. ns, not significant. Data in C,D are represented as mean±s.d.