We propose to examine the novel K channel blocker, clotrimazole (CLT, classically used as an antifungal agent), as a treatment for ADPKD which will act on the secretory, proliferative, and perhaps also signalling phenotypes of the disease. Our previous studies document the properties of CLT which form the basis for this proposal. CLT blocks calcium-gated and some voltage-gated K channels. In sickle erythrocytes, in a mouse model of sickle cell disease, and in sickle cell patients this property prevents K efflux from and dehydration of red cells. CLT blockade of epithelial K recycling at the basolateral membrane can also lead to inhibition of secretagogue-induced short-circuit current in T84 colon carcinoma cells, a model for transepithelial chloride secretion. Lastly, CLT also inhibits cell proliferation in concert with depletion of intracellular Ca stores. Despite these diverse effects, clotrimazole at high oral doses is very well tolerated in mice and normal humans, and well tolerated in humans with sickle cell disease. CLT is off-patent and available at low cost. We will test the following hypothesis: the combined antisecretory and antiproliferative properties of CLT will display characteristics in vitro and in mouse models of PKD which will recommend its consideration for clinical trials in PKD patients who have not yet developed severe renal insufficiency.
The specific aims which will subject this hypothesis to experimental test are to: 1. Characterize the ability of CLT to block transepithelial Cl secretion by monolayers and cysts of Type I MDCK, mIMCD-K2, immortalized cpk kidney cells, and ADPKD monolayer in comparison with monolayers and cysts of T84 cells. 2. Characterize the ability of CLT to inhibit proliferation of type I MDCK, mIMCD.K2, cpk, ADPKD, and T84 cells. 3. Characterize the ability of CLT to deplete intracellular Ca stores in Type I MDCK, mIMCD-K2, and cpk cells in comparison with T84 cells. 4. Characterize the ability of CLT to block K channels in Type I MDCK cells, mIMCD-K2 cells, cpk cells, ADPKD cells, and T84 cells. Clone CLT- sensitive K channel(s) from T84, MDCK, and ADPKD cells. 5. Test the ability of oral CLT administration to retard the onset of or to attenuate the severity of polycystic kidney disease in the mouse models jck, cpk, and newly bred strains carrying these genotypes on alternate genetic backgrounds.
