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PATHOLOGY Kidney Disease Genes Tied to Flow Sensing Gene Products May Be Part of Mechanical Response to Fluid Flow Across Kidney Cell Cilium Although the genetic defect that causes polycystic kidney disease (PKD) is known, how it leads kidneys to enlarge and choke with fluid-filled cysts has been frustratingly elusive. A new line of evidence points to a cellular appendage called the primary cilium, which may act as a mechanical sensor in cells lining kidney ducts. The latest study, led by Jing Zhou and published online in the Jan. 6 Nature Genetics, shows that the two genes involved in PKD help sense fluid flow across the primary cilium of kidney cells. Although still preliminary, the work is bringing a new focus in PKD research and turning attention to the role of mechanical sensation in cells. In this model, the primary cilium of a mouse kidney epithelial cell acts as an antenna sensing fluid flow. The shear stress from the bending cilium activates PC1, which signals PC2, a calcium channel, to allow calcium into the cell. This influx activates intracellular ryanodine receptors, releasing additional calcium from pools in the cell. The amplified signal regulates still unknown processes related to tissue development. (Image courtesy of Jing Zhou) PKD, the most common life-threatening genetic disease, is caused by mutations in one of two genes. Their activity has been unclear, but a clue surfaced with the discovery that the products of both genes, polycystin-1 (PC1) and polycystin-2 (PC2), were concentrated in the primary cilium of kidney epithelial cells. In this model, the primary cilium acts like a lever amplifying force. Other observations have supported the link between the primary cilium and kidney dysfunction. A gene found to control the formation of cilia in the alga Chlamydomonas turned out to be a homologue of a mouse gene whose defect leads to PKD in mice. These mice have shortened or absent primary cilia in their kidneys. Another breakthrough came when a group showed that bending the primary cilium on kidney cells caused a rise in cellular calcium, suggesting the cilium conveys a mechanical signal. Going with the Flow The laboratory of Zhou, HMS associate professor of medicine at Brigham and Women's Hospital, had been studying the polycystin family of proteins for years. Based on the recent findings, she speculated on a role for polycystins in ciliary signaling. A chance encounter she had with Donald Ingber while traveling led the two researchers to team up. An HMS professor of pathology at Children's Hospital, Ingber suggested that ciliary polycystins may be acting as mechanical sensors, since disrupting mechanical signals could lead to abnormal tissue development. Genes implicated in polycystic kidney disease help sense fluid flow in kidney cells, according to a study by (l to r) Ying Luo, Francis Alenghat, Surya Nauli, Donald Ingber, and Jing Zhou. (Photo by Graham Ramsay) The two labs collaborated to run flow studies, led by postdoctoral fellow Surya Nauli and HST student Francis Alenghat. They found that when a layer of mouse kidney cells was exposed to an abrupt onset of fluid flow, calcium levels spiked. "The cells don't respond to all kinds of flow," Zhou said, but the level of flow that did activate them was similar to fluid speeds in renal collecting ducts. But in cells lacking PC1, this influx of calcium was inhibited, and the same was true in normal cells given antibodies to PC1 and PC2. The response not only depended on the expression of these proteins but on the structure of the primary cilium; Nauli and Ying Luo, another postdoctoral fellow in Zhou's lab, also grew cells under conditions that yielded short or absent cilia, and these did not respond to flow. Researchers Rediscover Sense Organ of the Cell Look at a cross-section of a kidney cell and you will likely miss the primary cilium, but when the surface of the kidney epithelium is viewed under a scanning electron microscope, the cilia can be seen as lone spikes that poke prominently from each cell. In fact, this structure sticks like a tail out of most cell types, though the typical cell diagram fails to include it. What is this overlooked appendage? The primary cilium surfaces after mitosis from one of the cell's centrioles. Most primary cilia were thought to have little function--perhaps were even an evolutionary artifact from a unicellular ancestor, like a tailbone that never quite disappeared. But in the past two or three years, research has begun to implicate this structure in important sensory functions, suggesting it may serve as a kind of cellular antenna. Unlike the motile cilia of the respiratory tract, the primary cilium is usually stiff and can act as a lever to convey mechanical signals. Specialized sensory cells like olfactory neurons and photoreceptors use modified cilia to detect environmental cues, and "that has driven the idea that the primary cilium is a sensory organelle," said Gregory Pazour, assistant professor of molecular medicine at the University of Massachusetts Medical School. Pazour was studying ciliated algae when he discovered that a gene implicated in kidney disease is required for ciliary assembly. "This was not surprising since the cilium is a very highly conserved structure," he said. "But what was surprising was the fact that a ciliary defect could cause polycystic kidney disease." Ciliary defects also lead to problems in left-right symmetry, and research in mice has shown that flow across cilia influences embryonic left-right patterning. In the kidney, it remains to be seen what the calcium rise induced by cilia means, but Jing Zhou said the latest study "tells us that, indeed, when urine is flowing across cells, the cilia are not doing nothing; they're providing signals." Based on what is already known about the functions of PC1 and PC2, the team proposed a model of how the proteins might cooperate to transmit signals. Zhou and her colleagues previously showed that PC1 behaves like a G-protein-coupled receptor, while "PC2 looks like and acts like an ion channel," Zhou said. The two membrane proteins have been shown to interact at their interior tails. Zhou believes that PC1 senses the flow of fluid from shear stress along the bent cilium and then transmits a signal to PC2, which allows calcium into the cell. The response they saw depended on the availability of calcium both outside the cells and in intracellular pools. The researchers believe the initial intake of calcium may signal to the intracellular storehouses to release further calcium, amplifying the response. In this model, the primary cilium acts like a lever amplifying force. Although there is precedence for such a mechanism in specialized structures like auditory hair cells, so far the primary cilia of other cell types have not been thought to have such a function (see sidebar). Ingber points out that because of the research already conducted on PC1 and PC2, "We know more about the early steps of mechanotransduction in this cell than any other cell that's not a specialized mechanotransducer cell." However, the downstream steps are still anybody's guess. The Disease Connection Though this new line of research is promising, it remains to be seen whether ciliary dysfunction causes human PKD. M. Amin Arnaout, HMS professor of medicine and chief of the renal unit at Massachusetts General Hospital, said the pathology of PKD is characterized by three main problems: a failure in the terminal differentiation of epithelial cells, cell proliferation that leads to cyst formation, and an increase in fluid secretion that causes cysts to expand and the kidneys to eventually fail. "The question," Arnaout said, "is how are these three changes tied to PC1 and PC2?" A defect in ciliary sensing offers a plausible--but still unproven--scenario. In any case, he said, "It will certainly stimulate a lot of studies of the role of the primary cilium in cell function." Damage to kidneys is the most ravaging symptom of PKD, but patients can develop cysts in the liver and pancreas, as well as aneurysms and heart valve defects. "It's a systemic disease," said Zhou, so any pathological mechanism may have implications for other cell types. --Courtney Humphries