Aquaporins are expressed in water transporting epithelia where they facilitate water transport across the cell membrane. Although much is known about the expression, regulation and physiological function of aquaporins in epithelia, thus far there have been only two indirect reports of aquaporin expression in cells of the skeletal system. Early developmental studies in the rat have demonstrated abundant AQP1 mRNA in the mesenchyme surrounding developing bone (Bondy et al. 1993). In addition AQP1 has been found in chondrocytes and periosteum of the guinea-pig inner ear (Stankovic et al. 1995). In this study we have examined the expression of AQP1, 2 and 3 in sections of porcine articular cartilage and freshly isolated porcine chondrocytes. Our previous experiments in human cartilage have confirmed AQP1 expression in human chondrocytes (Mobasheri et al. 2000; Trujillo et al. 2000).

Porcine articular cartilage was obtained at abattoir from the metacarpal-phalangeal joints of piglets within 4 h of slaughter. Full depth cartilage explants including subchondral bone were removed and fixed for 2 h in 4 % paraformaldehyde in phosphate-buffered saline (PBS). Explants were decalcified overnight in 10 % EDTA in PBS before paraffin embedding and sectioning (7 µm). Porcine chondrocytes were isolated by overnight digestion in Dulbecco’s modified Eagle’s medium supplemented with 5 % fetal calf serum, 1 % antibiotic/antimycotic solution and 0.05 % clostridial collagenase. Cell suspensions were fixed for 10 min in ice-cold methanol or 4 % paraformaldehyde in PBS before blocking in normal goat serum (10 % in PBS) to quench non-specific binding sites. The expression of AQP1, AQP2 and AQP3 in cartilage was compared with two water-transporting epithelia, kidney and prostate, by indirect immunofluorescence and immunohistochemistry using polyclonal antibodies to AQP and goat anti-rabbit IgG conjugated to FITC and alkaline phosphatase, respectively. Paraffin embedded sections of human (with Ethics Committee approval) and rat kidneys (animals were humanely killed) were used as a positive control for AQP expression in all experiments.

Immunohistochemical studies revealed moderate AQP1 expression in porcine chondrocytes, particularly in the superficial and calcified zones of articular cartilage. In addition to articular chondrocytes abundant expression of AQP1 was also observed in osteocytes, osteoblasts and bone marrow stromal cells. In kidney control sections AQP1 was localized to the apical and basolateral membranes of proximal convoluted tubules and descending thin limbs (Agre et al. 1993). Freshly isolated porcine chondrocytes were also found to express AQP1 and AQP3. The presence of AQP1 and AQP3 in chondrocytes supports a role for AQPs in water transport across the plasma membranes of these cells. AQP1 may be important in allowing chondrocytes to respond to changes in their ionic and osmotic environment. AQP3 may be involved in the transport of glycerol and related substances. Future studies will focus on the molecular identification and cellular localization of other aquaporins in cartilage.