scholarly journals Polycystin-2 is an essential ion channel subunit in the primary cilium of the renal collecting duct epithelium

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Xiaowen Liu ◽  
Thuy Vien ◽  
Jingjing Duan ◽  
Shu-Hsien Sheu ◽  
Paul G DeCaen ◽  
...  
Keyword(s):  
Ion Channel ◽  
Primary Cilium ◽  
Primary Cilia ◽  
Collecting Duct ◽  
Primary Cultures ◽  
Clear Understanding ◽  
Open Probability ◽  
Novel Method ◽  
Autosomal Dominant Polycystic Kidney ◽  
Collecting Duct Epithelium

Mutations in the polycystin genes, PKD1 or PKD2, results in Autosomal Dominant Polycystic Kidney Disease (ADPKD). Although a genetic basis of ADPKD is established, we lack a clear understanding of polycystin proteins’ functions as ion channels. This question remains unsolved largely because polycystins localize to the primary cilium – a tiny, antenna-like organelle. Using a new ADPKD mouse model, we observe primary cilia that are abnormally long in cells associated with cysts after conditional ablation of Pkd1 or Pkd2. Using primary cultures of collecting duct cells, we show that polycystin-2, but not polycystin-1, is a required subunit for the ion channel in the primary cilium. The polycystin-2 channel preferentially conducts K+ and Na+; intraciliary Ca2+, enhances its open probability. We introduce a novel method for measuring heterologous polycystin-2 channels in cilia, which will have utility in characterizing PKD2 variants that cause ADPKD.

scholarly journals PKD2 is an essential ion channel subunit in the primary cilium of the renal collecting duct epithelium

2017 ◽  
Author(s):  
Xiaowen Liu ◽  
Thuy Vien ◽  
Jingjing Duan ◽  
Shu-Hsien Sheu ◽  
Paul G. DeCaen ◽  
...  
Keyword(s):  
Ion Channel ◽  
Primary Cilia ◽  
Collecting Duct ◽  
Primary Cultures ◽  
Hek 293 ◽  
Duct Epithelium ◽  
293 Cells ◽  
Novel Method ◽  
Autosomal Dominant Polycystic Kidney ◽  
Collecting Duct Epithelium

ABSTRACTMutations in either Pkd1 or Pkd2 result in Autosomal Dominant Polycystic Kidney Disease (ADPKD). Although PKD2 is proposed to be an ion channel subunit, recordings of PKD2 ion channels conflict in their properties. Using a new ADPKD mouse model, we observe primary cilia are abnormally long in cells associated with cysts. Using primary cultures of collecting duct epithelial cells, we show that PKD2, but not PKD1, is a required subunit for primary cilia ion channel. The ciliary PKD2 channel conducts potassium and sodium ions, but little calcium. We also demonstrate that PKD2 is not constitutively active in the plasma membrane, but PKD2 channels are functional in primary cilia and are sensitized by high cilioplasmic [Ca2+]. We introduce a novel method for measuring PKD2 channels heterologously expressed in primary cilia of HEK-293 cells, which will have utility characterizing Pkd2 variants that cause ADPKD in their native ciliary membrane.

scholarly journals Primary cilia control the maturation of tubular lumen in renal collecting duct epithelium

2017 ◽  
Vol 313 (1) ◽  
pp. C94-C107 ◽  
Author(s):  
Thomas Ernandez ◽  
Olga Komarynets ◽  
Alexandra Chassot ◽  
Soushma Sougoumarin ◽  
Priscilla Soulié ◽  
...  
Keyword(s):  
Primary Cilium ◽  
Primary Cilia ◽  
Collecting Duct ◽  
Tubular Structures ◽  
Renal Epithelium ◽  
Tubular Lumen ◽  
Developing Kidney ◽  
Collecting Duct Cells ◽  
Collecting Duct Epithelium

The key role of the primary cilium in developmental processes is illustrated by ciliopathies resulting from genetic defects of its components. Ciliopathies include a large variety of dysmorphic syndromes that share in common the presence of multiple kidney cysts. These observations suggest that primary cilia may control morphogenetic processes in the developing kidney. In this study, we assessed the role of primary cilium in branching tubulogenesis and/or lumen development using kidney collecting duct-derived mCCDN21 cells that display spontaneous tubulogenic properties when grown in collagen-Matrigel matrix. Tubulogenesis and branching were not altered when cilium body growth was inhibited by Kif3A or Ift88 silencing. In agreement with the absence of a morphogenetic effect, proliferation and wound-healing assay revealed that neither cell proliferation nor migration were altered by cilium body disruption. The absence of cilium following Kif3A or Ift88 silencing in mCCDN21 cells did not alter the initial stages of tubular lumen generation while lumen maturation and enlargement were delayed. This delay in tubular lumen maturation was not observed after Pkd1 knockdown in mCCDN21 cells. The delayed lumen maturation was explained by neither defective secretion or increased reabsorption of luminal fluid. Our results indicate that primary cilia do not control early morphogenetic processes in renal epithelium. Rather, primary cilia modulate tubular lumen maturation and enlargement resulting from luminal fluid accumulation in tubular structures derived from collecting duct cells.

Inward calcium current through the polycystin-2-dependent channels of renal primary cilia

2021 ◽  
Author(s):  
Steven J. Kleene ◽  
Nancy K. Kleene
Keyword(s):  
Primary Cilium ◽  
Calcium Current ◽  
Primary Cilia ◽  
Autosomal Dominant ◽  
Collecting Duct ◽  
Polycystic Kidney ◽  
Renal Epithelial Cells ◽  
Medullary Collecting Duct ◽  
The Mean ◽  
Autosomal Dominant Polycystic Kidney

In 15% of cases, autosomal dominant polycystic kidney disease (ADPKD) arises from defects in polycystin-2 (PC2). PC2 is a member of the TRPP subfamily of cation-conducting channels and is expressed in the endoplasmic reticulum and primary cilium of renal epithelial cells. PC2 opposes a pro-cystogenic influence of the cilium, and it has been proposed that this beneficial effect is mediated in part by a flow of Ca2+ through PC2 channels into the primary cilium. However, previous efforts to determine the permeability of PC2 channels to Ca2+ have yielded widely varying results. Here we report the mean macroscopic Ca2+ influx through native PC2 channels in the primary cilia of mIMCD-3 cells, which are derived from murine inner medullary collecting duct. Under conditions designed to isolate inward Ca2+ currents, a small inward Ca2+ current was detected in cilia with active PC2 channels, but not in cilia lacking those channels. The current was activated by addition of 10 µM internal Ca2+, which is known to activate ciliary PC2 channels. It was blocked by 10 µM isosakuranetin, which blocks the same channels. On average, the current amplitude was −1.8 pA at −190 mV; its conductance from −50 to −200 mV averaged 20 pS. Thus the native PC2 channels of renal primary cilium are able to conduct a small but detectable Ca2+ influx under the conditions tested. The possible consequences of this influx are discussed.

scholarly journals The native TRPP2-dependent channel of murine renal primary cilia

2017 ◽  
Vol 312 (1) ◽  
pp. F96-F108 ◽  
Author(s):  
Steven J. Kleene ◽  
Nancy K. Kleene
Keyword(s):  
Primary Cilium ◽  
Primary Cilia ◽  
Transient Receptor Potential ◽  
Single Channel ◽  
Receptor Potential ◽  
Open Probability ◽  
Electrophysiological Properties ◽  
Murine Cell Line ◽  
Autosomal Dominant Polycystic Kidney ◽  
Transient Receptor

Autosomal dominant polycystic kidney disease (ADPKD) is the most common life-threatening monogenic renal disease. ADPKD results from mutations in either of two proteins: polycystin-1 (also known as PC1 or PKD1) or transient receptor potential cation channel, subfamily P, member 2 (TRPP2, also known as polycystin-2, PC2, or PKD2). Each of these proteins is expressed in the primary cilium that extends from many renal epithelial cells. Existing evidence suggests that the cilium can promote renal cystogenesis, while PC1 and TRPP2 counter this cystogenic effect. To better understand the function of TRPP2, we investigated its electrophysiological properties in the native ciliary membrane. We recorded directly from the cilia of mIMCD-3 cells, a murine cell line of renal epithelial origin. In one-third of cilia examined, a large-conductance channel was observed. The channel was not permeable to Cl¯ but conducted cations with permeability ratios PK: PCa: PNa of 1:0.55:0.14. The single-channel conductance ranged from 97 pS in typical physiological solutions to 189 pS in symmetrical 145 mM KCl. Open probability of the channel was very sensitive to membrane depolarization or increasing cytoplasmic free Ca2+ in the low micromolar range, with the open probability increasing in either case. Knocking out TRPP2 by CRISPR/Cas9 genome editing eliminated the channel current, establishing it as TRPP2 dependent. Possible mechanisms for activating the TRPP2-dependent channel in the renal primary cilium are discussed.

scholarly journals Molecular dysregulation of ciliary polycystin-2 channels caused by variants in the TOP domain

2020 ◽  
Vol 117 (19) ◽  
pp. 10329-10338 ◽  
Author(s):  
Thuy N. Vien ◽  
Jinliang Wang ◽  
Leo C. T. Ng ◽  
Erhu Cao ◽  
Paul G. DeCaen
Keyword(s):  
Primary Cilia ◽  
Collecting Duct ◽  
Channel Structure ◽  
Superresolution Imaging ◽  
Channel Opening ◽  
Luminal Side ◽  
Autosomal Dominant Polycystic Kidney ◽  
Channel Assembly ◽  
A Site

Genetic variants in PKD2 which encodes for the polycystin-2 ion channel are responsible for many clinical cases of autosomal dominant polycystic kidney disease (ADPKD). Despite our strong understanding of the genetic basis of ADPKD, we do not know how most variants impact channel function. Polycystin-2 is found in organelle membranes, including the primary cilium—an antennae-like structure on the luminal side of the collecting duct. In this study, we focus on the structural and mechanistic regulation of polycystin-2 by its TOP domain—a site with unknown function that is commonly altered by missense variants. We use direct cilia electrophysiology, cryogenic electron microscopy, and superresolution imaging to determine that variants of the TOP domain finger 1 motif destabilizes the channel structure and impairs channel opening without altering cilia localization and channel assembly. Our findings support the channelopathy classification of PKD2 variants associated with ADPKD, where polycystin-2 channel dysregulation in the primary cilia may contribute to cystogenesis.

scholarly journals Exploring the Spectrum of Kidney Ciliopathies

Diagnostics ◽  
2020 ◽  
Vol 10 (12) ◽  
pp. 1099
Author(s):  
Matteo Santoni ◽  
Francesco Piva ◽  
Alessia Cimadamore ◽  
Matteo Giulietti ◽  
Nicola Battelli ◽  
...  
Keyword(s):  
Kidney Disease ◽  
Signaling Pathways ◽  
Polycystic Kidney Disease ◽  
Primary Cilium ◽  
Primary Cilia ◽  
Kidney Diseases ◽  
Ubiquitin Proteasome System ◽  
Polycystic Kidney ◽  
Ubiquitin Proteasome ◽  
Autosomal Dominant Polycystic Kidney

Ciliopathies are a group of multi-organ diseases caused by the disruption of the primary cilium. This event leads to a variety of kidney disorders, including nephronophthisis, renal cystic dysplasia, and renal cell carcinoma (RCC). Primary cilium contributes to the regulation of the cell cycle and protein homeostasis, that is, the balance between protein synthesis and degradation by acting on the ubiquitin-proteasome system, autophagy, and mTOR signaling. Many proteins are involved in renal ciliopathies. In particular, fibrocystin (PKHD1) is involved in autosomal recessive polycystic kidney disease (ARPKD), while polycystin-1 (PKD1) and polycystin-2 (PKD2) are implicated in autosomal dominant polycystic kidney disease (ADPKD). Moreover, primary cilia are associated with essential signaling pathways, such as Hedgehog, Wnt, and Platelet-Derived Growth Factor (PDGF). In this review, we focused on the ciliopathies associated with kidney diseases, exploring genes and signaling pathways associated with primary cilium and the potential role of cilia as therapeutic targets in renal disorders.

scholarly journals Polycystin-2 (TRPP2) Regulates Primary Cilium Length in LLC-PK1 Renal Epithelial Cells

2020 ◽  
Author(s):  
Paula L. Perez ◽  
Noelia Scarinci ◽  
Horacio F. Cantiello ◽  
María del Rocío Cantero
Keyword(s):  
Epithelial Cells ◽  
Gene Silencing ◽  
Primary Cilium ◽  
Primary Cilia ◽  
Cyst Formation ◽  
Channel Blockers ◽  
Wild Type ◽  
Polycystic Kidney ◽  
Renal Epithelial Cells ◽  
Autosomal Dominant Polycystic Kidney

AbstractPolycystin-2 (PC2, TRPP2) is a Ca2+ permeable non-selective cation channel whose dysfunction generates autosomal dominant polycystic kidney disease (ADPKD). PC2 is present in different cell locations, including the primary cilium of renal epithelial cells. Little is known, however, as to whether PC2 contributes to the structure of the primary cilium. Here, we explored the effect(s) of external Ca2+, PC2 channel blockers, and PKD2 gene silencing on the length of primary cilia in wild type LLC-PK1 renal epithelial cells. To identify primary cilia and measure their length, confluent cell monolayers were fixed and immuno-labeled with an anti-acetylated α-tubulin antibody. Although primary cilia length measurements did not follow a Normal distribution, data were normalized by Box-Cox transformation rendering statistical difference under all experimental conditions. Cells exposed to high external Ca2+ (6.2 mM) decreased a 13.5% (p < 0.001) primary cilia length as compared to controls (1.2 mM Ca2+). In contrast, the PC2 inhibitors amiloride (200 μM) and LiCl (10 mM), both increased primary ciliary length by 33.2% (p < 0.001), and 17.4% (p < 0.001), respectively. PKD2 gene silencing by siRNA also elicited a statistically significant, 10.3% (p < 0.001) increase in primary cilia length, as compared to their respective scrambled RNA transfected cells. The data indicate that maneuvers that either regulate PC2 function or gene expression, modify the length of primary cilia in renal epithelial cells. Proper regulation of PC2 function in the primary cilium may be essential in the onset of mechanisms that trigger cyst formation in ADPKD.Significance StatementPolycystin-2 (PC2, TRPP2) is a Ca2+ permeable non-selective cation channel causing the autosomal dominant polycystic kidney disease (ADPKD). The importance of intact cilia and of fully functional polycystins in the onset of ADPKD cyst formation, point to yet unknown signaling mechanisms occurring within this organelle. We determined that the extracellular Ca2+ concentration, PC2 channel blockers, and PKD2 gene silencing, all contribute to the length of primary cilia in wild type LLC-PK1 renal epithelial cells. The data indicate that proper regulation of PC2 function in the primary cilium may be essential in the onset of mechanisms that trigger cyst formation in ADPKD.

The Primary Cilium of MDCK Cells is a Flow Sensor

2001 ◽  
Vol 7 (S2) ◽  
pp. 4-5
Author(s):  
H. A. Praetorius ◽  
K. R. Spring
Keyword(s):  
Primary Cilium ◽  
Mammalian Cells ◽  
Primary Cilia ◽  
Cultured Cells ◽  
Mdck Cells ◽  
Collecting Duct ◽  
Renal Tubules ◽  
Intercalated Cells ◽  
Principal Cells ◽  
Renal Tubular Cells

The primary cilium, a solitary non-motile structure projecting from the centriole of mammalian cells, has been a subject of interest of anatomists since 1898. Surprisingly, the function of the primary cilium in mammalian cells is completely unknown. Virtually all of the epithelial cells of the mammalian kidney, with the exception of the intercalated cells of the collecting duct, express a single primary cilium on their apical (lumenal) surface. These structures were ignored by physiologists until recently when the mechanical properties of the primary cilia of cultured cells of renal origin were measured and it was proposed that they could serve as flow sensors. The proposal stemmed from the observation that flow rates comparable to those observed in renal tubules resulted in a deflection of the cilium of a few microns and that the stiffness of the cilium was commensurate with mechanosensing.The primary cilia of renal tubular cells are generally about 2 - 3 μm long whereas those of cultured renal cells become much longer as the cells age and may extend 50 μm or more into the medium. MDCK cells, a cultured cell line derived from the collecting duct of the canine kidney, exhibit two cell types analogous to the principal and intercalated cells of the collecting duct. MDCK principal cells express a primary cilium that is visible by high-resolution DIC microscopy. About one week after splitting, the primary cilium of MDCK principal cells is about 8 μm long and is well suited for testing the hypothesis that the cilium serves as a mechanosensor of fluid flow.

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