<i>Pkd1</i> and <i>Nek8</i> mutations affect cell-cell adhesion and cilia in cysts formed in kidney organ cultures

Natoli, Thomas A.; Gareski, Tiffany C.; Dackowski, William; Smith, Laurie; Bukanov, Nikolay O.; Russo, Ryan J.; Husson, Hervé; Matthews, Douglas; Piepenhagen, Peter; Ibraghimov‐Beskrovnaya, Oxana

doi:10.1152/ajprenal.00362.2007

Cited by 42 publications

(42 citation statements)

References 55 publications

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“…A link between ciliary Ca 2+ signaling, the polycystin proteins, and cyst development has recently gained attention (42,(46)(47)(48)(49)(50)(51). The fact that the cilia in the InsP3R3 and PC2 knockdown cysts were still present, but absent in InsP3R1 knockdown cysts, by week 8, suggests that cilia may not be essential to the latter stages of cystogenesis and is broadly consistent with recent findings (52).…”

Section: Discussionsupporting

confidence: 81%

Cyst formation following disruption of intracellular calcium signaling

Kuo¹,

DesRochers

Kimmerling

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Mutations in polycystin 1 and 2 (PC1 and PC2) cause the common genetic kidney disorder autosomal dominant polycystic kidney disease (ADPKD). It is unknown how these mutations result in renal cysts, but dysregulation of calcium (Ca 2+ ) signaling is a known consequence of PC2 mutations. PC2 functions as a Ca 2+ -activated Ca 2+ channel of the endoplasmic reticulum. We hypothesize that Ca 2+ signaling through PC2, or other intracellular Ca 2+ channels such as the inositol 1,4,5-trisphosphate receptor (InsP3R), is necessary to maintain renal epithelial cell function and that disruption of the Ca 2+ signaling leads to renal cyst development. The cell line LLC-PK1 has traditionally been used for studying PKD-causing mutations and Ca 2+ signaling in 2D culture systems. We demonstrate that this cell line can be used in long-term (8 wk) 3D tissue culture systems. In 2D systems, knockdown of InsP3R results in decreased Ca 2+ transient signals that are rescued by overexpression of PC2. In 3D systems, knockdown of either PC2 or InsP3R leads to cyst formation, but knockdown of InsP3R type 1 (InsP3R1) generated the largest cysts. InsP3R1 and InsP3R3 are differentially localized in both mouse and human kidney, suggesting that regional disruption of Ca 2+ signaling contributes to cystogenesis. All cysts had intact cilia 2 wk after starting 3D culture, but the cells with InsP3R1 knockdown lost cilia as the cysts grew. Studies combining 2D and 3D cell culture systems will assist in understanding how mutations in PC2 that confer altered Ca 2+ signaling lead to ADPKD cysts. primary cilia | polycysin 2 | calcium release T he commonly occurring genetic kidney disorder, autosomal dominant polycystic kidney disease (ADPKD), is the result of mutations in polycystin 1 or 2 (PC1 or PC2). The progressive cyst formation within all segments of the nephron that defines the disorder leads to renal failure requiring treatment by dialysis and/or organ transplantation (1-3). Altered Ca 2+ signaling is one of several pathways that have been implicated in the disease (4, 5). A major limitation toward elucidating the role of Ca 2+ signaling in cyst formation has been the lack of easily manipulated, physiologically relevant experimental methodologies.In the past, ADPKD research has relied largely upon data from mouse models and cells maintained in 2D cell culture. Mouse models have played a significant role in understanding the biology of cyst formation but are unable to fully recapitulate the physiology of disease progression in humans due to the inherent differences between the species including life span, genetics, and environment. Two-dimensional cell culture has the ability to provide information on signaling pathways and response to therapies in a fast, high-throughput manner, but is incapable of replicating the inherent 3D nature of cyst formation. Advances in 3D tissue culture over the past 2 decades have improved the ability to model cyst development in vitro. However, previously published 3D tissue models of ADPKD have relied upon shortter...

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Section: Discussionsupporting

confidence: 81%

Cyst formation following disruption of intracellular calcium signaling

Kuo¹,

DesRochers

Kimmerling

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…26 Similarly, we observed 2-and 3-fold higher cystogenic potentials in cultured jck/+ and jck/jck kidneys, compared with wt ( Figure 7A). Importantly, this increased cystogenesis was markedly reduced by treatment with Gant61 or Sant2.…”

Section: Small Molecule Hh Inhibitors Do Notsupporting

confidence: 69%

“…We further examined a direct role of Hh signaling in this and two other orthologous cystic kidney disease mouse mutants, in part by examining effects of Hh inhibitors on cystogenic potential of mutant kidneys cultured with cAMP, an assay proposed as a general model of cystic kidney disease 25 and useful for testing efficacy of potential pharmaceutical therapies. 26 …”

mentioning

confidence: 99%

Downregulating Hedgehog Signaling Reduces Renal Cystogenic Potential of Mouse Models

Tran

Talbott

Turbé-Doan

et al. 2014

Journal of the American Society of Nephrology

110

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Renal cystic diseases are a leading cause of renal failure. Mutations associated with renal cystic diseases reside in genes encoding proteins that localize to primary cilia. These cystoproteins can disrupt ciliary structure or cilia-mediated signaling, although molecular mechanisms connecting cilia function to renal cystogenesis remain unclear. The ciliary gene, Thm1(Ttc21b), negatively regulates Hedgehog signaling and is most commonly mutated in ciliopathies. We report that loss of murine Thm1 causes cystic kidney disease, with persistent proliferation of renal cells, elevated cAMP levels, and enhanced expression of Hedgehog signaling genes. Notably, the cAMP-mediated cystogenic potential of Thm1-null kidney explants was reduced by genetically deleting Gli2, a major transcriptional activator of the Hedgehog pathway, or by culturing with small molecule Hedgehog inhibitors. These Hedgehog inhibitors acted independently of protein kinase A and Wnt inhibitors. Furthermore, simultaneous deletion of Gli2 attenuated the renal cystic disease associated with deletion of Thm1. Finally, transcripts of Hedgehog target genes increased in cystic kidneys of two other orthologous mouse mutants, jck and Pkd1, and Hedgehog inhibitors reduced cystogenesis in jck and Pkd1 cultured kidneys. Thus, enhanced Hedgehog activity may have a general role in renal cystogenesis and thereby present a novel therapeutic target.

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“…46,47 Kidneys were removed from E14.5 embryos and cultured for 5 days with or without a cyst-inducing cAMP analog. In the presence of 8-Bromo-cAMP, jck/jck kidneys develop the greatest numbers of cysts, whereas Nek82/2 kidneys have the least cystogenic potential of all the genotypes ( Figure 5, E and F).…”

Section: Jck/nek82 Compound Heterozygotes Have Less Severe Cystic Dismentioning

confidence: 99%

Loss of the Ciliary Kinase Nek8 Causes Left-Right Asymmetry Defects

Manning¹,

Sergeev

Heesbeen

et al. 2013

Journal of the American Society of Nephrology

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A missense mutation in mouse Nek8, which encodes a ciliary kinase, produces the juvenile cystic kidneys (jck) model of polycystic kidney disease, but the functions of Nek8 are incompletely understood. Here, we generated a Nek8-null allele and found that homozygous mutant mice die at birth and exhibit randomization of left-right asymmetry, cardiac anomalies, and glomerular kidney cysts. The requirement for Nek8 in left-right patterning is conserved, as knockdown of the zebrafish ortholog caused randomized heart looping. Ciliogenesis was intact in Nek8-deficient embryos and cells, but we observed misexpression of left-sided marker genes early in development, suggesting that nodal ciliary signaling was perturbed. We also generated jck/Nek8 compound heterozygotes; these mutants developed less severe cystic disease than jck homozygotes and provided genetic evidence that the jck allele may encode a gain-of-function protein. Notably, NEK8 and polycystin-2 (PC2) proteins interact, and we found that Nek8 2/2 and Pkd2 2/2 embryonic phenotypes are strikingly similar. Nek8-deficient embryos and cells did express PC2 normally, which localized properly to the cilia. However, similar to cells lacking PC2, NEK8-depleted inner medullary collecting duct cells exhibited a defective response to fluid shear, suggesting that NEK8 may play a role in mediating PC2-dependent signaling.

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Pkd1 and Nek8 mutations affect cell-cell adhesion and cilia in cysts formed in kidney organ cultures

Cited by 42 publications

References 55 publications

Cyst formation following disruption of intracellular calcium signaling

Cyst formation following disruption of intracellular calcium signaling

Downregulating Hedgehog Signaling Reduces Renal Cystogenic Potential of Mouse Models

Loss of the Ciliary Kinase Nek8 Causes Left-Right Asymmetry Defects

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