Dataset Information

Screen-based identification and validation of four new ion channels as regulators of renal ciliogenesis.

ABSTRACT: To investigate the contribution of ion channels to ciliogenesis, we carried out a small interfering RNA (siRNA)-based reverse genetics screen of all ion channels in the mouse genome in murine inner medullary collecting duct kidney cells. This screen revealed four candidate ion channel genes: Kcnq1, Kcnj10, Kcnf1 and Clcn4. We show that these four ion channels localize to renal tubules, specifically to the base of primary cilia. We report that human KCNQ1 Long QT syndrome disease alleles regulate renal ciliogenesis; KCNQ1-p.R518X, -p.A178T and -p.K362R could not rescue ciliogenesis after Kcnq1-siRNA-mediated depletion in contrast to wild-type KCNQ1 and benign KCNQ1-p.R518Q, suggesting that the ion channel function of KCNQ1 regulates ciliogenesis. In contrast, we demonstrate that the ion channel function of KCNJ10 is independent of its effect on ciliogenesis. Our data suggest that these four ion channels regulate renal ciliogenesis through the periciliary diffusion barrier or the ciliary pocket, with potential implication as genetic contributors to ciliopathy pathophysiology. The new functional roles of a subset of ion channels provide new insights into the disease pathogenesis of channelopathies, which might suggest future therapeutic approaches.

SUBMITTER: Slaats GG

PROVIDER: S-EPMC4696498 | biostudies-literature | 2015 Dec

REPOSITORIES: biostudies-literature

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Screen-based identification and validation of four new ion channels as regulators of renal ciliogenesis.

Slaats Gisela G GG Wheway Gabrielle G Foletto Veronica V Szymanska Katarzyna K van Balkom Bas W M BW Logister Ive I Den Ouden Krista K Keijzer-Veen Mandy G MG Lilien Marc R MR Knoers Nine V NV Johnson Colin A CA Giles Rachel H RH

Journal of cell science 20151106 24

To investigate the contribution of ion channels to ciliogenesis, we carried out a small interfering RNA (siRNA)-based reverse genetics screen of all ion channels in the mouse genome in murine inner medullary collecting duct kidney cells. This screen revealed four candidate ion channel genes: Kcnq1, Kcnj10, Kcnf1 and Clcn4. We show that these four ion channels localize to renal tubules, specifically to the base of primary cilia. We report that human KCNQ1 Long QT syndrome disease alleles regulate ...[more]

PMID: 26546361

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Project description:Rationale: Renal cell carcinoma (RCC) accounts for about 2% of all adult cancers, and clear cell RCC (ccRCC) is the most common RCC histologic subtype. A hallmark of ccRCC is the loss of the primary cilium, a cellular antenna that senses a wide variety of signals. Loss of this key organelle in ccRCC is associated with the loss of the von Hippel-Lindau protein (VHL). However, not all mechanisms of ciliopathy have been clearly elucidated. Methods: By using RCC4 renal cancer cells and patient samples, we examined the regulation of ciliogenesis via the presence or absence of the hypoxic form of the voltage-dependent anion channel (VDAC1-?C) and its impact on tumor aggressiveness. Three independent cohorts were analyzed. Cohort A was from PREDIR and included 12 patients with hereditary pVHL mutations and 22 sporadic patients presenting tumors with wild-type pVHL or mutated pVHL; Cohort B included tissue samples from 43 patients with non-metastatic ccRCC who had undergone surgery; and Cohort C was composed of 375 non-metastatic ccRCC tumor samples from The Cancer Genome Atlas (TCGA) and was used for validation. The presence of VDAC1-?C and legumain was determined by immunoblot. Transcriptional regulation of IFT20/GLI1 expression was evaluated by qPCR. Ciliogenesis was detected using both mouse anti-acetylated ?-tubulin and rabbit polyclonal ARL13B antibodies for immunofluorescence. Results: Our study defines, for the first time, a group of ccRCC patients in which the hypoxia-cleaved form of VDAC1 (VDAC1-?C) induces resorption of the primary cilium in a Hypoxia-Inducible Factor-1 (HIF-1)-dependent manner. An additional novel group, in which the primary cilium is re-expressed or maintained, lacked VDAC1-?C yet maintained glycolysis, a signature of epithelial-mesenchymal transition (EMT) and more aggressive tumor progression, but was independent to VHL. Moreover, these patients were less sensitive to sunitinib, the first-line treatment for ccRCC, but were potentially suitable for immunotherapy, as indicated by the immunophenoscore and the presence of PDL1 expression. Conclusion: This study provides a new way to classify ccRCC patients and proposes potential therapeutic targets linked to metabolism and immunotherapy.

Dataset Information

Screen-based identification and validation of four new ion channels as regulators of renal ciliogenesis.

Publications

Screen-based identification and validation of four new ion channels as regulators of renal ciliogenesis.

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