Intravital analysis of cilia function during injury in the kidney

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SUMMARY/ABSTRACT Primary cilia are present on most renal epithelial cells but their function is unknown. Recently, we and others found that cilia disruption alters the ability of the kidney to repair following acute kidney injury (AKI), eventually leading to cyst formation; but how cilia are connected with injury remains enigmatic. Based on in vitro studies, primary cilia were thought to be mechanosensors regulating a flow-induced Ca2+ signal requiring the cilia and cilia localized polycystin proteins (Pkd1 and Pkd2), two genes associated with human polycystic kidney disease (PKD). While in vitro data support a mechanosensor role, recent findings raise concerns with this model. First, cilia ablation in adult mice does not cause cysts for ~8 months and the cysts form focally, despite cilia loss on all tubule epithelium. Additionally, data from a cilia targeted Ca2+ biosensor indicate there is no Ca2+ change in the cilium when the axoneme is deflected in perfused tubules, nor is there a Pkd2 dependent Ca2+ current detected in cilia patch clamp studies. A potential limitation of this study is that it was not performed under conditions where cilia/Pkd1/Pkd2 are known to have critical roles in preventing rapid cyst formation (e.g. following injury). Thus, we propose that injury may induce a cilia response not present in non-injured states. We predict that understanding changes in cilia responses that occur under differing physiological conditions and following injury will be important for dissecting normal cilia function, mechanisms of mal-repair, and cyst formation in ciliopathies, such as PKD. To analyze in vivo roles of the cilium, we are using mouse lines with fluorescently tagged cilia, intravital fluorescence confocal microscopy, and a surgically implanted abdominal window approach to image cilia in intact nephrons. Our intravital imaging indicate that cilia are typically deflected in the direction of tubule flow under normal conditions. However, when flow is impaired, cilia behavior change and they begin to oscillate and can elongate or regress. Importantly, impaired tubule flow also occurs following injury. Thus, one objective of this project is to ascertain in vivo conditions that induce these changes in cilia behavior. We will analyze changes in cilia morphology, length, and number, determine whether there are changes in cilia Ca2+ signaling and transcriptional activity in the tubules associated with flow and altered cilia responses. The second objective is to measure changes in cilia responses during the injury and repair process. This will include testing if there is a cilia Ca2+ signal following injury and whether this is dependent on Pkd2. Finally, we will test the importance of cilia responses by disrupting cilia formation/function prior to and during the injury and repair process. In this way we can determine whether reassembly of the cilium and maintenance of proper cilia length (elongated or shortened) are important for the epithelium to return to a quiescent and differentiated state following injury. Applying our intravital imaging strategy to address these questions is innovative and is needed to understand renal cilia function and how the cilia respond to and regulate injury and repair mechanisms associated with cyst development.