Polycystic kidney disease (PKD) is characterized by the formation of renal cysts, cardiovascular abnormalities and hypertension. Various derangements including abnormal ion transport, altered cell polarity, accelerated cell growth and proliferation, protein mis-localization, and abnormal extracellular matrix composition have been demonstrated in various models of PKD. In models of autosomal recessive polycystic kidney disease (ARPKD), defects in cilia development or ciliary function have been proposed as a common pathogenic mechanism. Other work has shown that one function of cilia is to mediate flowdependent increases in cytosolic calcium concentration [Ca2+]i. The central hypothesis of this work is that cilia are tonic regulators of apical Ca2+ entry and that the absence of a functioning cilia in PKD leads to elevated and unregulated Ca2+ entry across the apical membrane. Studies will be performed in control collecting duct cell lines and in collecting duct cells from either the Oak Ridge Polycystic Kidney mouse model of ARPKD (orpk) in which cilia are absent, cilia(-), or from the cpk mouse which has a mutation in the ciliary protein, cystin.
The first aim will be to determine the role of cilia regulated apical Ca2+ entry by apical flow and store operated Ca2+ channels (SOCC). Studies will also determine if overall apical Ca2+ permeability is higher in the absence of a functioning cilia. Since transient receptor potential channels (TRP) are likely candidates for these two pathways, the second aim will identify which of the TRP channels are expressed in these cells and determine if there are differences in message and proteins levels in cilia+/- cells. siRNA or blocking antibodies will be used to establish which TRP isoforms are involved in flowdependent Ca2+ channels and SOCC activity and in overall Ca2+ permeability.
The third aim i s to define the role of cilia in the spatial organization of TRP channels at the apical membrane. The role of cilia cytoskeletal interactions in the regulating TRP location and activity will also be addressed.
