Loss of Polycystin-1 in Human Cyst-Lining Epithelia Leads to Ciliary Dysfunction Surya M. Nauli,* Sandro Rossetti, † Robert J. Kolb,* Francis J. Alenghat, ‡ Mark B. Consugar, † Peter C. Harris, † Donald E. Ingber, ‡ Mahmoud Loghman-Adham, § and Jing Zhou* *Renal Division, Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts; † Division of Nephrology, Mayo Clinic College of Medicine, Rochester, Minnesota; ‡ Vascular Biology Program, Departments of Pathology and Surgery, Children’s Hospital and Harvard Medical School, Boston, Massachusetts; and § Department of Pediatrics and Pediatrics Research Institute, St. Louis University School of Medicine, St. Louis, Missouri A “two-hit” hypothesis predicts a second somatic hit, in addition to the germline mutation, as a prerequisite to cystogenesis and has been proposed to explain the focal nature for renal cyst formation in autosomal dominant polycystic kidney disease (ADPKD). It was reported previously that Pkd1 null/null mouse kidney epithelial cells are unresponsive to flow stimulation. This report shows that Pkd1 +/null cells are capable of responding to mechanical flow stimulation by changing their intracellular calcium concentration in a manner similar to that of wild-type cells. This paper reports that human renal epithelia require a higher level of shear stress to evoke a cytosolic calcium increase than do mouse renal epithelia. Both immortalized and primary cultured renal epithelial cells that originate from normal and nondilated ADPKD human kidney tubules display normal ciliary expression of the polycystins and respond to fluid-flow shear stress with the typical change in cytosolic calcium. In contrast, immortalized and primary cultured cyst-lining epithelial cells from ADPKD patients with mutations in PKD1 or with abnormal ciliary expression of polycystin-1 or -2 were not responsive to fluid shear stress. These data support a two-hit hypothesis as a mechanism of cystogenesis. This report proposes that calcium response to fluid-flow shear stress can be used as a readout of polycystin function and that loss of mechanosensation in the renal tubular epithelia is a feature of PKD cysts. J Am Soc Nephrol 17: 1015–1025, 2006. doi: 10.1681/ASN.2005080830 A gene mutation can result in disease through direct or indirect mechanisms. For instance, in the gain-of- function mutation, a germline mutant allele confers new or enhanced protein activity with a pathologic function, whereas a dominant-negative mutation produces an aberrant protein that interferes with the function of the normal protein. In haploinsufficiency, a loss of 50% of normal protein as a result of a mutation in one of its alleles is sufficient to cause disease. In the two-hit mechanism, the disease results from a germline mutation in one allele, followed by the subsequent acquisition of a somatic mutation in the second normal allele with no remaining functional protein. Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary kidney disease. PKD1 and PKD2 are the genes that encode for the polycystin-1 (PC1) and poly- cystin-2 (PC2) proteins, respectively. Although patients with ADPKD carry heterozygous mutations in either PKD1 or PKD2 and present 100% penetrance of cystic kidney phenotypes, fewer than 5% of nephrons form cysts. These fluid-filled cysts are lined by a single layer of epithelial cells and can occur at any site along the nephron. The presence of renal cysts in ADPKD, despite the low number, results in a gradual decline in renal function. To explain the focal nature of renal cyst formation in ADPKD, Reeders (1) proposed a “two-hit” hypothesis suggest- ing that a second somatic alteration to the gene, in addition to a germline mutation, is a prerequisite to the disease phenotype. Although a mechanism based on haploinsufficiency has not been excluded, somatic mutations in either PKD1 or PKD2 indeed have been found in several ADPKD cyst-lining epithelia (2– 8), even though a somatic loss of other chromosomes or mutations in other loci also are found (2). These data provided hints that ADPKD is a recessive disease at the cellular level. The lack of a cellular assay for PC1 function has prevented an experimental demonstration of loss of function in cyst-lining epithelia in ADPKD. We and others have shown previously that PC1 and PC2 are localized to the primary cilia (9). The mechanosensation func- tion of polycystins can be assayed in cultured mouse kidney epithelial cells by monitoring changes in the intracellular cal- cium concentration in response to fluid-flow shear stress (10). To test the loss-of-function hypothesis in ADPKD with regard to mechanosensory ability, we used the flow assay to examine shear stress–induced calcium responses in cells that were de- rived from a heterozygous Pkd1 mouse model. Furthermore, we Received August 8, 2005. Accepted February 1, 2006. Published online ahead of print. Publication date available at www.jasn.org. Address correspondence to: Dr. Jing Zhou, Harvard Institutes of Medicine, Suite 520, 77 Avenue Louis Pasteur, Boston, MA 02115. Phone: 617-525-5860; Fax: 617-525-5861; E-mail: zhou@rics.bwh.harvard.edu Copyright © 2006 by the American Society of Nephrology ISSN: 1046-6673/1704-1015