1999 European Regional Conference Proceeding
May 12 - 16, 1999
| Conference: | 1999 European Regional Conference |
|---|---|
| Title: | Modulation of aquaporin-2 expression in primary cultured rat inner medullary collecting duct (IMCD) cells |
| Authors: | Storm, Robert; Maric, Kenan; Klussmann, Enno; Rosenthal, Walter |
| Institutions: | Forschunginstitut fur Molekulare Pharmakologie, German Federal Ministry of Health and Social Security, Forschungsinstitut fur Molekulare Pharmakologie, Charite - Universitatsmedizin Berlin |
Our recently established primary IMCD cell culture model maintains long term aquaporin-2 (AQP-2) expression without genetic manipulation and thus provides an in vitro model to study the regulation of AQP-2. The aim of this study was to investigate conditions that modulate the expression of the water channel protein AQP-2.
Long term effects (i. e. on AQP-2 synthesis) were analysed by Western and Northern blot- techniques as well as by fluorescence microscopy. Short-term effects (AQP-2 protein shuttle) were monitored by fluorescence microscopy. When conducting long term experiments over six days we observed a modulating effect of extracellular acidification (media pH 6.4) on IMCD cell AQP-2 expression. Western blot analysis revealed a distinct increase in AQP-2 levels compared to controls. The increase on the protein level was accompanied by elevated AQP-2 mRNA levels as analysed by Northern blotting. Immunofluorescence microscopy showed increased signal intensity. The increase in the AQP-2 protein level was reversed by the non-selective sodium proton exchanger (NHE) inhibitor ethylisopropylamiloride (EIPA).
In contrast, cells cultured in media containing elevated calcium concentrations showed a decrease of AQP-2 on the protein level, as determined by Western blotting and immunofluorescence microscopy.
To investigate the influence of extracellularly lowered pH and elevated calcium concentration on the trafficking of AQP-2, short-term experiments with 30 minutes pretreatment prior to stimulation were performed with IMCD cells grown under control conditions. An effect of the above mentioned conditions on the trafficking of AQP-2 could not be detected.
Taken together these findings may establish a basis for the treatment of diuretic states, in particular of bthose caused by a decrease in AQP-2 expression.
The researchers used a cell culture model of primary cultured rat inner medullary collecting duct (IMCD) cells that was established by Maric et al. (1998). This model allows them to study the short-term regulation (minutes) of aquaporin-2 (AQP2) and, in addition to other cell culture models available, the long-term regulation (days and weeks) of AQP2. They observed that the expression (long-term regulation) of AQP2 could be easily modulated under cell-culture conditions.
The researchers noticed that when the extracellular environment of their model was acidic (pH 6.4), it resulted in a distinct increase in AQP2 expression. This increase could be reversed by a drug that inhibits sodium-proton exchangers present in IMCD cells. In contrast, cells cultured in an environment containing elevated calcium concentrations showed a decrease in AQP2. Both conditions, acidification and elevated calcium levels, seemed not to interfere with the AQP2s' ability to travel to the cell surface. The researchers suggest these findings may establish a basis for treatment of diuretic states, particularly those caused by a decrease in AQP2 expression.