Proliferation, apoptosis and fluid secretion in cysts.
The formation and growth of cysts in PKD is accompanied by increased proliferation and apoptosis of cyst-lining epithelia, loss of epithelial polarity and de-differentiation, dysregulation of cell/matrix interactions and transformation of the absorptive epithelial phenotype to a secretory phenotype [54
]. Epidermal growth factor (EGF), transforming growth factor (TGF) alpha and EGF receptor (EGFR) play important roles in promoting cystic epithelial proliferation. In human PKDs and several animal models, EGFR is overexpressed and mislocalized to the apical membranes of cystic epithelial cells [57
]. Overexpression of TGF alpha in transgenic animals leads to renal cyst formation [58
]. Apoptosis is also essential for cystogenesis: deletion of the anti-apoptotic Bcl-2 and AP-2β genes and overexpression of the pro-apoptotic gene c-myc in mice results in renal cyst formation [59
]. Cystic fluid is derived from glomerular filtrate in the early stages of ADPKD, when cysts are still attached to the parent tubule [60
]. Cysts separate from the tubule of origin when they reach ~200 μm in diameter and continue to expand through a transepithelial chloride secretion mechanism mediated by cAMP [61
]. Chloride enters cells via
the basolateral Na-K-Cl cotransporter and accumulates in the cytoplasm. A chloride channel in the apical membrane, CFTR, mediates movement of chloride into the cystic lumen. Chloride secretion drives sodium into the cystic cavity through paracellular mechanisms; this causes movement of water through aguaporins [61
]. In contrast to ADPKD, cysts in ARPKD do not separate from affected collecting ducts. Therefore, proliferation but not transepithelial secretion is a major component causing cystic kidney volume enlargement in ARPKD.
Impaired cell-cell/matrix adhesion.
The overlapping expression and localization patterns of polycystin-1 and -2 support their role as a complex in regulating multiple processes in tubular epithelia [62
]. Both proteins are found in basolateral membranes and the primary cilium, where they may act together to regulate cellular adhesion and Ca2+
signaling. On the other hand, polycystin-2 is mainly expressed in endoplasmic reticulum, where it functions as a Ca2+
release channel [45
]. In addition, polycystin-1 is highly expressed during development, with significant down-regulation of its expression in adult tissues. In contrast, expression of polycystin-2 seems to persist into adult life [62
Experimental evidence from several groups has established an important role for polycystins in epithelial cell morphogenesis, including differentiation and maturation in vivo
]. Studies using in vitro
models of tubulogenesis and cystogenesis based on MDCK cells demonstrated that expression of polycystin-1 at cell-cell junctions at controlled levels is critical for proper tubular differentiation [65
]. It has been shown that polycystin-1 is directly involved in intercellular adhesion via
formation of strong homophilic interactions of its PKD (Ig-like) domains as shown in Fig. [36
]. A direct role for Ig-like domains in cell-cell adhesion was demonstrated by specific perturbation of intercellular adhesion using antibodies against Ig-like domains in cell cultures [36
]. Polycystin-1 was localized to the cell-cell adhesion complexes with adherens junctions and desmosomal junctions in epithelial cells of different origin [65
]. Because alterations in polycystin-1-mediated adhesion may cause the abnormal epithelial cell phenotype observed in ADPKD cells, including dedifferentiation and loss of epithelial polarity, several studies examined cell-cell adhesion junctions in primary cells derived from ADPKD kidneys [37
]. As shown in Fig. , abnormal adherens and desmosomal junctions were found in ADPKD: intracellular junctions were devoid of desmosomal cadherins and associated proteins, which were sequestered to the cytoplasmic pools, and adherens junctions appeared disrupted, accompanied by a great reduction of Ecadherin expression and partial compensatory expression of N-cadherin [68
Figure 2 Polycystin-1 in cell-cell/matrix adhesion in normal and ADPKD epithelia. (a) Polycystin-1 (PC-1) mediates cell-cell adhesion through homophilic interactions of its Ig-like domains. It localizes with desmosomal junctions (DJs) and adherens junctions (AJs). (more ...)
Interestingly, co-immunoprecipitation studies in ADPKD cells using an anti-polycystin-1 antibody showed that E-cadherin was lost from the complex, while beta-catenin remained associated with polycystin-1. Moreover, beta-catenin was still expressed at the plasma membrane despite the loss of E-cadherin, suggesting the presence of an alternative cadherin [68
]. N-cadherin, but not K-cadherin, was overexpressed in ADPKD cells and formed a complex with beta-catenin. In normal kidney, E-cadherin is expressed in distal segments of the nephron, while Ncadherin is expressed in proximal tubules [70
]. In the ADPKD kidney, N-cadherin is markedly increased in cysts of distal origin [68
]. However, the expression of N-cadherin in place of E-cadherin in ADPKD cells was not sufficient to maintain epithelial cell-cell adhesion [37
Polycystin-1 was shown to be indispensable in cellmatrix interactions (Fig. ) [54
]. Abnormalities in the basement-membrane composition and expression of matrix metalloproteases and their inhibitors were identified in PKD kidneys. Interestingly, the inactivation of tensin and insertional mutation in laminin alpha five result in cystogenesis [72
]. It has been shown that polycystin-1 interacts with focal adhesion complex molecules including α1β2 integrin, vinculin, paxillin, p130-cas, talin and focal adhesion kinase (FAK) [38
]. In ADPKD cells, the focal adhesion complex is disrupted due to loss of FAK (Fig. ).
PKD as a ciliopathy.
Polycystins, fibrocystin and numerous other proteins or cystoproteins such as nephrocystin-1, -3, -4, -5, inversin, ALMS1, OFD1, BBS1-8, cystin, polaris and Nek8, which cause PKD in humans and animals, were recently discovered in a distinct subcellular compartment, the primary cilium [10
]. Cilia are microtubule-containing organelles that project from the surface of most eukaryotic cells [76
]. The primary (non-motile) cilia are known to transduce sensory stimuli such as concentrations of growth factors, osmolarity and fluid flow [76
]. They primary cilia are formed in fully differentiated cells during interphase and grow out of the basal bodies (Fig. ). The ciliary basal body is formed by the centrosomes, more specifically by the mother centriole that moves to the membrane where the axoneme, the structural core, is formed [77
]. Construction and maintenance of the axoneme requires a bidirectional intraflagellar transport system (IFT) to deliver structural components from the cell body to the tip of the cilium.
Figure 3 Ciliary functions of cystoproteins: mechanosensation and cell cycle control. Polycystin-1 (PC-1), polycystin-2 (PC-2) and other cystoproteins (not shown) are expressed in primary cilia, basal bodies or centrosomes. The primary cilium forms in fully differentiated (more ...)
It was recently discovered that defects in ciliary structure or function underlie multiple human diseases with diverse phenotypes, including retinal degeneration, neural tube defects, obesity, polydactyly and PKD. Initial linkage between cilia and PKD came from mating behavior studies in Caenorhabditis elegans
]. Mutations in the lov-1 and PKD-2 genes of C. elegans
, which are closely related to human polycystins, were associated with mechanosensation defects of ciliated sensory neurons. Direct evidence linking defects in ciliogenesis and PKD came from the study of renal tissue-specific inactivation of KIF3A, a subunit of kinesin-II essential for cilia formation [79
]: inactivation of KIF3A in renal tubular epithelial cells resulted in development of PKD. Several studies have shown that defects in ciliary assembly are associated with PKD. It was demonstrated that the primary cilia in the cystic kidney of mice with a mutation in the Tg737 gene (homologous to the IFT gene of Chlamydomonas
, IFT88) are shorter than normal [80
]. On the other hand, the primary cilia of cystic epithelial cells in jck mice were found to be significantly longer than the cilia in wild-type mice [75
]; this study suggested that ciliary protein Nek8, which is mutated in the jck mice, may play a role in control of ciliary length. Taken together, these data established a link between ciliary dysfunction and PKD.
Cilia and mechanosensation in PKD.
A number of studies support a role for the primary cilium as a mechanosensor in kidney tubular epithelia. Praetorius et al. reported that the primary cilia of renal epithelial MDCK cells can serve as a flow sensor [81
]. Bending the cilium resulted in intracellular Ca2+
influx followed by calcium release from IP3
-sensitive stores. The calcium signal was spread as a wave through gap junctions of cells [81
]. The authors concluded that the primary cilium in MDCK cells is mechanically sensitive and responds to flow by increasing intracellular calcium. A subsequent study demonstrated that polycystins can serve as flow-sensitive ciliary mechanosensors in kidney epithelia [46
]. More specifically, ciliary polycystin-1 and polycystin-2 function together with ryanodine receptors to mediate mechanotransduction into the intracellular Ca2+
signaling response (Fig. ). The influx of Ca2+
across the plasma membrane constitutes the initial response to mechanical stimulation, and downstream signaling is mediated through intracellular Ca2+
]. It is possible that polycystin-1 functions as a sensor of ciliary bending, while polycystin-2 transduces themechanical signal into a calcium response. Changes in intracellular Ca2+
concentration are known to regulate multiple cellular functions including gene expression, cell cycle, differentiation and apoptosis. Cells with mutated polycystin-1 fail to respond to the fluid flow: it was shown that in ADPKD-derived cells, the ciliary mechanosensation of fluid-flow shear stress by poly-cystins is absent [46
Cilia and cell cycle. There is an intimate link between cilia and the cell cycle. The basal bodies/centrosomes of the cilia act as organizers of the mitotic spindle poles during cell division, directly connecting ciliogenesis with cell cycle regulation, and cilia are resorbed when cells enter the cell cycle (Fig. ). As we discussed previously, a number of cystoproteins causing PKD in humans and animals are expressed, at least partially, in cilia or the basal body of the cilia. Polycystins and other cystoproteins may play an important role in connecting the mechanosensory function of the cilia to the centrosome and thus influence cell cycle control. Disruption of cystoproteins associated with cilia or basal bodies could, therefore, lead to dysregulation of the cell cycle and proliferation, resulting in cystic disease.
Several lines of evidence support this hypothesis: Overexpression of exogenous polycystin-1 in cultured cells resulted in growth arrest in G0/G1 phase of the cell cycle (Fig. ) [82
]. Further analysis has shown that polycystin-1 expression inhibits Cdk2 and induces p21waf1
. Polycystin-1 activates the JAK-STAT pathway, thereby up-regulating p21waf1 and inducing cell cycle arrest in G0/G1. Functional polycystin-2 was shown to be essential for this process [82
]. Other cystoproteins localized to cilia have also been recently shown to directly regulate the cell cycle. For example, the IFT88/polaris protein, a component of the IFT, was shown to be tightly associated with the centrosome during cell cycle transitions [83
]. Similar to polycystin-1, overexpression of polaris resulted in cell cycle arrest at the G0/G1 stage (Fig. ). On the other hand, down-regulation of polaris mRNA promoted progression of the cell cycle. Polycystin-2 has been shown to participate in cell cycle regulation as well: it can associate with the helix-loop-helix protein Id2 and block its translocation to the nucleus, preventing proliferation [84
]. The translocation of Id2 in cells with mutated polycystins is associated with downregulation of p21 expression, leading to an increase in CDK2 activity and cell cycle progression (Fig. ). Thus, evidence of a direct link between cystoproteins, ciliary dysfunction and cell cycle dysregulation continues to accumulate.