In Vivo Analysis of Cilia Function and Connections to Renal Disease and Physiology

Many cystic kidney diseases are caused by defects in proteins required for cilia assembly or activity. It has been proposed that cilia regulate cell proliferation, oriented cell division, responses to tissue injury, and as mechanosensors. The cilium is thought to mediate its effects by regulating the activity of several signaling proteins or channels including Polycystins 1 and 2, mTor, Stat3/6, Hedgehog, and purinergic receptors. Importantly, these signaling pathways are frequently altered when cilia are absent from cells and in cystic renal epithelium. However, the exact mechanisms connecting cilia dysfunction to cyst formation remains largely unknown. Recent data have raised concerns regarding the mechanosensory model for cystogenesis. Despite data from cell culture studies showing fluid flow mediated deflection of cilia results in changes in intracellular calcium levels, this has not been observed in vivo . Further, loss of cilia function in juvenile mice results in rapid cyst formation, but in adult mice cyst do not form for 6 to 8 months after cilia function is disrupted. This is not consistent with simple defects in mechanosensation. Our studies are trying to assess the functions of the cilium in vivo . We are addressing whether it has mechanosensory activities under different physiological conditions and how ciliary defects contribute to cyst formation. We are using conditional mutations in genes required for cilia formation and function along with a series of biosensors to analyze cilia function in live tissues. To begin addressing the in vivo roles of the cilium, we generated a Cilia GFP mouse allowing us to observe cilia in whole tissues of live animals. We found under normal conditions that cilia are consistently bent in the direction of fluid flow along the renal tubule. However, by altering physiological conditions, we show cilia oscillate or even reverse orientation, indicating that the behavior of cilia is dynamic and influenced by other systems in the body. We are determining how cilia react to changes in heart rate, blood pressure, glomerular filtration rates, restricted renal blood flow, during cyst formation, and after kidney injury. We will evaluate how changes in cilia behavior under different conditions influence Ca 2+ , Stat6, and mTor signaling activity and whether cilia morphology, length, or behavior is altered during cyst formation. Importantly, all of these studies will be performed in the kidney of a live mouse. Results will provide novel insights into how the cilium functions in the kidney and how defects in cilia are possibly involved in diseases. Support or Funding Information P30DK074038R01DK065655