1. Loss of Claudins 2 and 15 From Mice Causes Defects in Paracellular Na+ Flow and Nutrient Transport in Gut and Leads to Death from Malnutrition 
Masami Wada, Atsushi Tamura, Nobuyuki Takahashi, Sachiko Tsukita 
Gastroenterology 
Volume 144, Issue 2, Pages (February 2013)
DOI: /j.gastro
Copyright © 2013 AGA Institute Terms and Conditions

2. Figure 1
Infant death phenotype of DKO (Cldn2−/−Cldn15−/−) mice. (A) Growth curves for WT, DHet (Cldn2+/−Cldn15+/−), and DKO (Cldn2−/−Cldn15−/−) mice (n = 5–7/group). Representative data are shown. (B) Survival curves for WT, DHet, and DKO mice (n = 11–14/group). (C) H&E staining and thin-section electron micrographs of the jejunum. Scale bar: 200 μm and 200 nm, respectively. (D) Fluorescent staining for claudin-2 or -15 (green), with counterstaining for occludin (red) and DAPI (blue) in the jejunum. Scale bar: 10 μm. (E) qRT-PCR for claudin-2, -3, -4, -7, -15, -23, and -25 in the WT and DKO jejunum (n = 6/group). (F) Electron micrographs of freeze-fracture replicas of the jejunum of WT and DKO mice. Scale bar: 200 nm.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2013 AGA Institute Terms and Conditions

3. Figure 2
Glucose malabsorption in DKO (Cldn2−/−Cldn15−/−) mice intestine. (A) The concentration of glucose and total ketone bodies in the serum of WT and DKO mice under nonfasted conditions or 4 hours of fasting (n = 4–6/group). (B) The 1.5 g/kg body weight OGTT curves in the DKO mice after fasting for 4 hours indicated glucose malabsorption (n = 3/group). (C) Immunofluorescence images, qRT-PCR (n = 5–6/group), and immunoblotting for SGLT1 in the jejunum of WT and DKO mice under nonfasted conditions. The magnified image shows an area of the apical membrane. Scale bar: 100 μm. (D) Immunofluorescence images of GLUT2 in WT and DKO mice given 1.5 g/kg body weight glucose or mannitol after fasting for 4 hours. Scale bar: 100 μm. The magnification shows an area of the apical membrane of the small intestine after glucose administration. Scale bar: 50 μm. Arrowheads show GLUT2 localization in the apical membrane. qRT-PCR for GLUT2 and GLUT5 in the jejunum of WT and DKO mice under nonfasted conditions (n = 5–6/group).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2013 AGA Institute Terms and Conditions

4. Figure 3
Ion homeostasis in the DKO (Cldn2−/−Cldn15−/−) mouse intestine. (A) Na+ concentration in the luminal contents of the small intestine and colon under normal feeding (left panel). Luminal Na+ concentration in individual segments of the small intestine under fasted conditions (right panel) (n = 3–5/group). (B) K+ concentration in the luminal contents of the small intestine and colon under normal feeding (left panel). Luminal K+ concentration in individual segments of the small intestine under fasted conditions (right panel) (n = 3–5/group). (C) The 1.5 g/kg body weight OGTT curves in DKO mice with 200 mmol/L NaCl or LiCl after fasting for 4 hours (n = 3/group). (D) Electrophysiological analyses of the ion conductance and Na+ permeability in the jejunum of WT and DKO mice (n = 3/group).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2013 AGA Institute Terms and Conditions

5. Figure 4
Amino acid malabsorption in DKO (Cldn2−/−Cldn15−/−) mouse intestine. (A) Time-course of changes in the concentration of various amino acids in the serum of WT and DKO mice after the oral administration of a mixture of 20 different amino acids (300 mg/kg total) after fasting for 4 hours (n = 5–6/group). (B) Time-course of the change in amino acid concentrations in the serum of DKO mice after the administration of 20 kinds of amino acids (300 mg/kg total) with 200 mmol/L NaCl or 200 mmol/L LiCl (n = 4/group). (C) qRT-PCR for amino acid transporters in the jejunum of WT and DKO mice under nonfasted conditions (n = 5–6/group).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2013 AGA Institute Terms and Conditions

6. Figure 5
Fat malabsorption in the DKO (Cldn2−/−Cldn15−/−) mouse intestine. (A) Time-course of the concentration change of TGs in the serum of WT and DKO mice. Mice were given 9.5 g/kg body weight of olive oil orally after fasting for 4 hours (n = 4/group). (B) Measurement of unabsorbed FFAs and TGs in the colonic contents of WT and DKO mice with controlled intake (n = 4–6/group). (C) BODIPY-FA uptake in the small intestine of WT and DKO mice after fasting for 4 hours. The relative fluorescence intensity was determined from the intensity of BODIPY-FA in the cytoplasm of epithelial cells in DKO mice compared with WT mice (n = 15/group). (D) qRT-PCR for FATP4 and FAT/CD36 in the jejunum of WT and DKO mice under nonfasted conditions (n = 5/group).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2013 AGA Institute Terms and Conditions

7. Figure 6
Bile acid malabsorption in the DKO (Cldn2−/−Cldn15−/−) mouse intestine. (A) Bile acid absorption in WT and DKO mice. Serum concentration of bile acids in the portal vein was measured at 0 minutes (left panel); concentration changes of bile acids in the portal vein were measured 10 minutes after administration of cholic acid (CA) in mice fasted for 4 hours (right panel) (n = 3–4/group). (B) Bile acid pool size in the WT and DKO mice (n = 3/group). (C) qRT-PCR for ASBT in the ileum of WT and DKO mice (n = 3/group). (D) Change in bile acid concentration in the portal vein 10 minutes after the administration of CA with Na+ or Li+ in mice fasted for 4 hours (right panel) (n = 3–4/group). (E) Schematic drawing of the claudin-2/-15–based nutrient absorption in the WT intestine and the malabsorption in the DKO intestine owing to the loss of paracellular Na+ permeability.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2013 AGA Institute Terms and Conditions

8. Figure 7
Malabsorption of 3 major nutrients causes infant death. (A) Na+-dependent absorption of glucose, leucine, and cholic acid as determined by ΔIsc. By using Ussing chambers, the apical sides of the jejunum or ileum of either WT or DKO mice were bathed in buffer containing Na+ and K+ in concentrations similar to the luminal side of the WT or DKO jejunum/ileum in vivo (n = 3–4/group). (B) Schematic drawing of the absorption of 3 major nutrients in the small intestine. In WT mice, glucose and amino acids are mainly absorbed via a Na+-dependent transporter. In fat absorption, bile acids absorbed Na+ dependently in the terminal ileum are circulated, and then the stored bile acids are secreted into the lumen of the upper small intestine to digest TGs into FFAs. The absorption of these 3 major nutrients is related closely to the luminal [Na+] regulated by claudin-2 and claudin-15. In contrast, DKO mice show malabsorption of the 3 major nutrients caused by glucose, amino acid, and bile acid transporter dysfunction owing to low luminal [Na+].
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2013 AGA Institute Terms and Conditions