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PMID:22832925

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Citation

Ghosh, AK, Hurd, T and Hildebrandt, F (2012) 3D spheroid defects in NPHP knockdown cells are rescued by the somatostatin receptor agonist octreotide. Am. J. Physiol. Renal Physiol. 303:F1225-9

Abstract

Ciliopathies are a heterogeneous group of diseases that exhibit broad clinical phenotypes, including renal cysts, retinal degeneration, and cerebellar vermis aplasia. Nephronophthisis (NPHP) is a renal ciliopathy that causes chronic kidney disease and is characterized by kidney cysts at the cortico-medullary border. Among the 10 different disease-causing genes (NPHP1-NPHP10), mutations in NPHP3, NPHP6, or NPHP8 cause the most severe ciliopathy variants of NPHP, Joubert syndrome, and Meckel Syndrome. In this study, we tested the hypothesis that loss of function of these three most severe disease-associated genes leads to morphological defects in a three-dimensional (3D) renal cell culture [murine (m) inner medullary collecting duct (IMCD) 3] model by either lack of cilia formation and/or cell polarity defects. Stable knockdown cell lines were examined in 3D spheroid culture followed by rhodamine-phalloidin staining to assess spheroid architecture. We observed significantly higher percentages of abnormal spheroids for all three stable cell lines compared with control short-hairpin RNA cells. In addition, stable knockdown of Nphp3, Nphp6, and Nphp8 results in reduced cilia numbers and elevated cAMP levels in mIMCD3 cells. We demonstrate that, following gene knockdown of Nphp3, Nphp6, or Nphp8, treatment with the somatostatin agonist octreotide (2 μM) reduces the percentage of abnormal spheroids compared with control. This study reveals that the loss of Nphp3, Nphp6, or Nphp8 leads to cilia abnormalities and cell polarity defects, resulting in spheroid abnormalities, which can be rescued by inhibiting cAMP levels with octreotide treatment.

Links

PubMed PMC3469674 Online version:10.1152/ajprenal.00135.2012

Keywords

Animals; Cell Culture Techniques; Cell Line; Cilia/drug effects; Cilia/genetics; Cilia/metabolism; Cilia/pathology; Kidney Diseases, Cystic/congenital; Kidney Diseases, Cystic/genetics; Kidney Diseases, Cystic/metabolism; Kidney Diseases, Cystic/pathology; Kidney Tubules, Collecting/drug effects; Kidney Tubules, Collecting/metabolism; Kidney Tubules, Collecting/pathology; Mice; Octreotide/pharmacology; Receptors, Somatostatin/genetics; Receptors, Somatostatin/metabolism

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

MOUSE:SSR3

located_in

GO:0097730: non-motile cilium

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

MOUSE:CE290

acts_upstream_of_or_within

GO:0007163: establishment or maintenance of cell polarity

ECO:0000315: mutant phenotype evidence used in manual assertion

P

  • occurs_in:(CL:1000546)

Seeded From UniProt

complete

MOUSE:CE290

acts_upstream_of_or_within

GO:1905515: non-motile cilium assembly

ECO:0000315: mutant phenotype evidence used in manual assertion

P

  • occurs_in:(CL:1000546)

Seeded From UniProt

complete

MOUSE:NPHP3

acts_upstream_of_or_within

GO:0007163: establishment or maintenance of cell polarity

ECO:0000315: mutant phenotype evidence used in manual assertion

P

  • occurs_in:(CL:1000546)

Seeded From UniProt

complete

MOUSE:NPHP3

acts_upstream_of_or_within

GO:1905515: non-motile cilium assembly

ECO:0000315: mutant phenotype evidence used in manual assertion

P

  • occurs_in:(CL:1000546)

Seeded From UniProt

complete

MOUSE:FTM

acts_upstream_of_or_within

GO:0007163: establishment or maintenance of cell polarity

ECO:0000315: mutant phenotype evidence used in manual assertion

P

  • occurs_in:(CL:1000546)

Seeded From UniProt

complete

MOUSE:FTM

acts_upstream_of_or_within

GO:1905515: non-motile cilium assembly

ECO:0000315: mutant phenotype evidence used in manual assertion

P

  • occurs_in:(CL:1000546)

Seeded From UniProt

complete

MOUSE:NPHP3

acts_upstream_of_or_within

GO:0007163: establishment or maintenance of cell polarity

ECO:0000315: mutant phenotype evidence used in manual assertion

P

occurs_in:(CL:1000546)

Seeded From UniProt

complete

MOUSE:NPHP3

acts_upstream_of_or_within

GO:1905515: non-motile cilium assembly

ECO:0000315: mutant phenotype evidence used in manual assertion

P

occurs_in:(CL:1000546)

Seeded From UniProt

complete

MOUSE:FTM

acts_upstream_of_or_within

GO:0007163: establishment or maintenance of cell polarity

ECO:0000315: mutant phenotype evidence used in manual assertion

P

occurs_in:(CL:1000546)

Seeded From UniProt

complete

MOUSE:FTM

acts_upstream_of_or_within

GO:1905515: non-motile cilium assembly

ECO:0000315: mutant phenotype evidence used in manual assertion

P

occurs_in:(CL:1000546)

Seeded From UniProt

complete

MOUSE:CE290

acts_upstream_of_or_within

GO:1905515: non-motile cilium assembly

ECO:0000315: mutant phenotype evidence used in manual assertion

P

occurs_in:(CL:1000546)

Seeded From UniProt

complete

MOUSE:CE290

acts_upstream_of_or_within

GO:0007163: establishment or maintenance of cell polarity

ECO:0000315: mutant phenotype evidence used in manual assertion

P

occurs_in:(CL:1000546)

Seeded From UniProt

complete


See also

References

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