Age-related changes of claudin expression in mouse liver, kidney, and pancreas

Abstract

Tight junctions (TJs) play crucial roles in tissue homeostasis and inflammation through their roles in the control of paracellular transport and barrier function. There is evidence that these functions are compromised in older organisms, but the exact mechanisms leading to TJ deterioration are not well understood. Claudin proteins are a family of membrane proteins that constitute the structural barrier elements of TJs and therefore play a major role in their formation and function. Using immunohistochemistry and immunoblotting, we have studied the expression of six different claudin proteins (claudin-1, -2, -3, -4, -5, and -7) in three tissues (liver, kidney, and pancreas) of aging male and female mice. In general, we find an age-dependent decrease in the expression of several claudin proteins in all three tissues observed, although the exact changes are tissue specific. Our findings provide a possible basis for the decrease in tissue barrier function in older organisms.

Figures

Figure 1.

Immunohistochemistry analysis of selected claudins in aging mouse liver. (A) Representative staining for claudin-3 from liver sections of 6- and 24-month-old male mice. (B) Effects of aging on the expression of the indicated claudins. Results shown were quantitated using the MCID image analysis software and represent the average of nine sections (three liver punches from three mice) (*p < .05, **p < .001; brackets show the significance between age groups (both male and female) and the absence of a bracket indicates significance for one gender within the age group). The staining data are shown as arbitrary units (A.U.). Results are shown for 6- and 24-month-old mice. Black bars represent males and gray bars females. Claudin-3 and claudin-4 were significantly decreased in both male and female mice. Claudin-1 was increased in both males and females. (C) The effect of age and gender on claudin expression in mouse liver. The expression of the indicated claudins was examined in the liver of 1-month-old (1M), 6-month-old (6M), 16-month-old (16M), and 24-month-old (24M) mice. Again, claudin-3 and claudin-4 showed a significant decrease in older mice.

Figure 2.

Immunohistochemistry analysis of selected claudins in aging mouse kidney. (A) Representative staining for claudin-3 from kidney sections of 6- and 24-month-old male mice. Claudin-3 shows a significant decrease in older mice. (B) Effects of aging on the expression of the indicated claudins. Results shown were quantitated using the MCID image analysis software and represent the average of nine sections (three kidney punches from three mice) (*p < .05, **p < .001; brackets show the significance between age groups (both male and female) and the absence of a bracket indicates significance for one gender within the age group). The staining data are shown as arbitrary units (A.U.). Results are shown for 6- and 24-month-old mice. Black bars represent males and gray bars females. Claudin-2, -3, -4, -5, and -7 were significantly decreased in both male and female mice. Claudin-1 was increased in both males and females. (C) The effect of age and gender on claudin expression in mouse liver. The expression of the indicated claudins was examined in the liver of 1-month-old (1M), 6-month-old (6M), 16-month-old (16M), and 24-month-old (24M) mice. The same trends were observed when these additional time points were studied.

Figure 3.

Immunohistochemistry analysis of selected claudins in aging mouse pancreas. (A) Representative staining for claudin-4 from pancreas sections of 6- and 24-month-old male mice. Claudin-4 is clearly reduced in older mice. (B) Effects of aging on the expression of the indicated claudins. Results shown were quantitated using the MCID image analysis software and represent the average of nine sections (three pancreas punches from three mice) (*p < .05, **p < .001; brackets show the significance between age groups (both male and female) and the absence of a bracket indicates significance for one gender within the age group). The staining data are shown as arbitrary units (A.U.). Results are shown for 6- and 24-month-old mice. Black bars represent males and gray bars females. Claudin-1, -3, -4, and -5, and claudin-4 were significantly decreased in both male and female mice. Claudin-2 and -7 were significantly decreased in females. (C) The effect of age and gender on claudin expression in mouse liver. The expression of the indicated claudins was examined in the liver of 1-month-old (1M), 6-month-old (6M), 16-month-old (16M), and 24-month-old (24M) mice. All the claudin examined tended to be decreased in the pancreas of older mice.

Figure 4.

Cellular fractionation and immunoblotting of aging mouse tissues. Kidney (A) and Liver (B) tissues were fractionated into cytoplasmic (c) and membrane (m) components before being run on sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Claudin-1 (Cldn1) and claudin-4 (Cldn4) expression patterns were observed. The lysates were also probed for glyceraldehyde 3-phosphate dehydrogenease (a cytoplasmic control) and E-cadherin (a membrane control). Claudin-1 is located predominantly in the membrane fraction and is increased in the kidney of older animals. Claudin-4, on the other hand, in mostly cytoplasmic and is decreased in both the kidney and liver of older mice.