Loss of Polycystin-1 in Human Cyst-Lining Epithelia Leads to Ciliary Dysfunction

Abstract

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) A gene mutation can result in disease through direct or indirect mechanisms. For instance, in the gain-offunction 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 polycystin-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 suggesting 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 function of polycystins can be assayed in cultured mouse kidney epithelial cells by monitoring changes in the intracellular calcium 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 derived from a heterozygous Pkd1 mouse model. Furthermore, we

Human ADPKD primary cyst epithelial cells with a novel, single codon deletion in the PKD1 gene exhibit defective ciliary polycystin localization and loss of flow-induced Ca2+ signaling

“…5C). These data demonstrate localization of PC1 and PC2 to primary cilia in human NK cells, consistent with the recent observations of Nauli et al (31). In contrast, PKD cyst cells with normal PC1 abundance lack ciliary PC1, and PC2 is absent from most cilia of cyst cells.…”

“…However, accelerated decay of the flow-induced Ca 2ϩ signal in NK cells associated with overexpression of CD16.7-PKD1(115-226) requires integrity of that coiled-coil domain. These results confirm and extend the recent study by Nauli et al (31) published after completion of these experiments.…”

“…Our results thus show that ciliary localization of PC2 does not require immunocytochemically detectable levels of ciliary PC1, consistent with recent findings on ciliary targeting of PC2 (10). The continued ciliary expression of PC2 in 30% of cells with the novel PC1 ⌬L2433 mutation contrasts with the reported absence of ciliary PC2 in all SV40 large T-transformed, DBA-positive human 9 -12 cyst cells and in (genetically uncharacterized) primary human AD-PKD cyst epithelial cells (31). Ciliary localization of PC1 and PC2 was not described in a study of tsSV40LgT-immortalized human ADPKD cells with the E1537X germline mutation, which express normal PC2 levels with very low levels of PC1 (56).…”

“…The pkd1(Ϫ/Ϫ) mouse embryonic renal epithelial cells in which flow-induced signaling defects were observed completely lacked PC1 and PC2 polypeptides. A recent report appearing after completion of the work presented here extended this observation to human ADPKD cyst cells expressing little or no PC1 polypeptide (31). However, human ADPKD is almost always characterized by normal or increased renal levels of apparently full-length PC1 polypeptide (32,36,37), despite the significantly truncated proteins encoded by most PKD1 germline mutations (35).…”

“…PC1 and PC2 have been colocalized, along with other ciliary gene products linked to cystic kidney disease, to the primary cilium in mouse kidney, in primary cultured mouse kidney cells, and in several mammalian kidney cell lines (58,66,73). Human kidney cells in culture also exhibit primary cilia (40,31). Figure 3 shows the presence of cilia in both NK and PKD cyst cells as detected by immunostaining of the ciliary axoneme marker N-acetylated ␣-tubulin.…”