Primary Cilia of inv/inv Mouse Renal Epithelial Cells Sense Physiological Fluid Flow: Bending of Primary Cilia and Ca2+ Influx

Primary cilia sense environmental conditions, including osmolality, but whether cilia participate in the osmotic response in renal epithelial cells is not known. The transient receptor potential (TRP) channels TRPV4 and TRPM3 are osmoresponsive. TRPV4 localizes to cilia in certain cell types, while renal subcellular localization of TRPM3 is not known. We hypothesized that primary cilia are required for maximal activation of the osmotic response of renal epithelial cells and that ciliary TRPM3 and TRPV4 mediate that response. Ciliated [murine epithelial cells from the renal inner medullary collecting duct (mIMCD-3) and 176-5] and nonciliated (176-5Δ) renal cells expressed Trpv4 and Trpm3. Ciliary expression of TRPM3 was observed in mIMCD-3 and 176-5 cells and in wild-type mouse kidney tissue. TRPV4 was identified in cilia and apical membrane of mIMCD-3 cells by electrophysiology and in the cell body by immunofluorescence. Hyperosmolal stress at 500 mOsm/kg (via NaCl addition) induced the osmotic response genes betaine/GABA transporter ( Bgt1) and aldose reductase ( Akr1b3) in all ciliated cell lines. This induction was attenuated in nonciliated cells. A TRPV4 agonist abrogated Bgt1 and Akr1b3 induction in ciliated and nonciliated cells. A TRPM3 agonist attenuated Bgt1 and Akr1b3 induction in ciliated cells only. TRPM3 knockout attenuated Akr1b3 induction. Viability under osmotic stress was greater in ciliated than nonciliated cells. Akr1b3 induction was also less in nonciliated than ciliated cells when mannitol was used to induce hyperosmolal stress. These findings suggest that primary cilia are required for the maximal osmotic response in renal epithelial cells and that TRPM3 is involved in this mechanism. TRPV4 appears to modulate the osmotic response independent of cilia.

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Roles of dopamine receptor on chemosensory and mechanosensory primary cilia in renal epithelial cells

Frontiers in Physiology ◽

10.3389/fphys.2014.00072 ◽

2014 ◽

Vol 5 ◽

Cited By ~ 28

Author(s):

Viralkumar S. Upadhyay ◽

Brian S. Muntean ◽

Sarmed H. Kathem ◽

Jangyoun J. Hwang ◽

Wissam A. AbouAlaiwi ◽

...

Keyword(s):

Epithelial Cells ◽

Dopamine Receptor ◽

Primary Cilia ◽

Renal Epithelial Cells

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The renal inflammatory network of nephronophthisis.

10.1101/2021.01.07.425719 ◽

2021 ◽

Author(s):

Marceau Quatredeniers ◽

Frank Bienaime ◽

Giulia Ferri ◽

Pierre Isnard ◽

Esther Poree ◽

...

Keyword(s):

Epithelial Cells ◽

Primary Cilia ◽

Inflammatory Responses ◽

Renal Inflammation ◽

Renal Epithelial Cells ◽

Renal Deterioration ◽

Ccl2 Expression ◽

Genes Encoding ◽

Main Gene ◽

Autosomal Dominant Polycystic Kidney

Nephronophthisis (NPH) and autosomal dominant polycystic kidney disease (ADPKD) are caused by mutations in genes encoding primary cilia proteins. In ADPKD, altered cilia signaling promotes renal inflammation through the upregulation of the macrophage chemoattractant CCL2, which subsequently fuels disease progression. While inactivation of NPHP1, the main gene involved in NPH leads to increased CCL2 expression in cultured renal epithelial cells, little is known about renal inflammation in NPH. Here, we analyzed murine models of NPH as well as kidney tissues and urine derived renal epithelial cells (UREC) from NPH patients to dissect the renal inflammatory network involved in NPH. Similarly to ADPKD, NPH patients present kidney infiltration by macrophages, increased CCL2 expression by tubular cells and enhanced CCL2 urinary excretion. Yet, while tubule specific Ccl2 disruption dramatically reduced renal Ccl2 expression in a mouse model of NPH, this did not translate into reduced macrophage infiltration nor lessened renal deterioration. In further contrast to early ADPKD, infiltrating macrophages were accompanied by neutrophils and T cells in both human and murine NPH. Through analysis of transcriptome datasets from early diseased kidneys in two distinct mouse models of NPH, we identified a set of 17 soluble inflammatory mediators, which increased independently of CCL2. Among those, 8 were also significantly upregulated in UREC from NPH patients compared to controls. Collectively, these results unveil that mutations in ciliary proteins in NPH and ADPKD trigger divergent renal inflammatory responses. This work sheds light on the specific inflammatory network underlying NPH.

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Rab10 associates with primary cilia and the exocyst complex in renal epithelial cells

AJP Renal Physiology ◽

10.1152/ajprenal.00198.2010 ◽

2010 ◽

Vol 299 (3) ◽

pp. F495-F506 ◽

Cited By ~ 44

Author(s):

Clifford M. Babbey ◽

Robert L. Bacallao ◽

Kenneth W. Dunn

Keyword(s):

Epithelial Cells ◽

Membrane Transport ◽

Primary Cilia ◽

Glut4 Translocation ◽

Renal Epithelia ◽

Endocytic Recycling ◽

Renal Epithelial Cells ◽

Exocyst Complex ◽

Cultured Epithelia

Rab10, a mammalian homolog of the yeast Sec4p protein, has previously been associated with endocytic recycling and biosynthetic membrane transport in cultured epithelia and with Glut4 translocation in adipocytes. Here, we report that Rab10 associates with primary cilia in renal epithelia in culture and in vivo. In addition, we find that Rab10 also colocalizes with exocyst proteins at the base of nascent cilia, and physically interacts with the exocyst complex, as detected with anti-Sec8 antibodies. These data suggest that membrane transport to the primary cilum may be mediated by interactions between Rab10 and an exocyst complex located at the cilium base.

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

10.1101/2020.02.24.962860 ◽

2020 ◽

Cited By ~ 1

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.

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Vasopressin receptor-mediated functional signaling pathway in primary cilia of renal epithelial cells

AJP Renal Physiology ◽

10.1152/ajprenal.90509.2008 ◽

2009 ◽

Vol 296 (1) ◽

pp. F87-F97 ◽

Cited By ~ 43

Author(s):

Malay K. Raychowdhury ◽

Arnolt J. Ramos ◽

Peng Zhang ◽

Margaret McLaughin ◽

Xiao-Qing Dai ◽

...

Keyword(s):

Epithelial Cells ◽

Signaling Pathway ◽

Primary Cilium ◽

Primary Cilia ◽

Sensory Function ◽

Vasopressin Receptor ◽

Channel Activity ◽

Renal Epithelial Cells ◽

Selective Channel ◽

Cation Selective Channel

The primary cilium of renal epithelial cells is a nonmotile sensory organelle, implicated in mechanosensory transduction signals. Recent studies from our laboratory indicate that renal epithelial primary cilia display abundant channel activity; however, the presence and functional role of specific membrane receptors in this organelle are heretofore unknown. Here, we determined a functional signaling pathway associated with the type 2 vasopressin receptor (V2R) in primary cilia of renal epithelial cells. Besides their normal localization on basolateral membrane, V2R was expressed in primary cilia of LLC-PK1 renal epithelial cells. The presence of V2R in primary cilia was determined by spontaneous fluorescence of a V2R- gfp chimera and confirmed by immunocytochemical analysis of wild-type LLC-PK1 cells stained with anti-V2R antibodies and in LLC-PK1 cells overexpressing the V2R-Flag, with anti-Flag antibody. Ciliary V2R colocalized with adenylyl cyclase (AC) type V/VI in all cell types tested. Functional coupling of the receptors with AC was confirmed by measurement of cAMP production in isolated cilia and by testing AVP-induced cation-selective channel activity either in reconstituted lipid bilayers or subjected to membrane-attached patch clamping. Addition of either 10 μM AVP ( trans) or forskolin ( cis) in the presence but not the absence of ATP (1 mM, cis) stimulated cation-selective channel activity in ciliary membranes. This channel activity was reduced by addition of the PKA inhibitor PKI. The data provide the first demonstration for the presence of V2R in primary cilia of renal epithelial cells, and a functional cAMP-signaling pathway, which targets ciliary channel function and may help control the sensory function of the primary cilium.

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PKHD1 protein encoded by the gene for autosomal recessive polycystic kidney disease associates with basal bodies and primary cilia in renal epithelial cells

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0400073101 ◽

2004 ◽

Vol 101 (8) ◽

pp. 2311-2316 ◽

Cited By ~ 120

Author(s):

M.-Z. Zhang ◽

W. Mai ◽

C. Li ◽

S.-y. Cho ◽

C. Hao ◽

...

Keyword(s):

Kidney Disease ◽

Epithelial Cells ◽

Polycystic Kidney Disease ◽

Autosomal Recessive ◽

Primary Cilia ◽

Basal Bodies ◽

Polycystic Kidney ◽

Renal Epithelial Cells

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Characterization of Single Channel Currents from Primary Cilia of Renal Epithelial Cells

Journal of Biological Chemistry ◽

10.1074/jbc.m507793200 ◽

2005 ◽

Vol 280 (41) ◽

pp. 34718-34722 ◽

Cited By ~ 58

Author(s):

Malay K. Raychowdhury ◽

Margaret McLaughlin ◽

Arnolt J. Ramos ◽

Nicolás Montalbetti ◽

Richard Bouley ◽

...

Keyword(s):

Epithelial Cells ◽

Primary Cilia ◽

Single Channel ◽

Renal Epithelial Cells ◽

Single Channel Currents ◽

Channel Currents

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Polycystin-2 Cation Channel Function Is under the Control of Microtubular Structures in Primary Cilia of Renal Epithelial Cells

Journal of Biological Chemistry ◽

10.1074/jbc.m603643200 ◽

2006 ◽

Vol 281 (49) ◽

pp. 37566-37575 ◽

Cited By ~ 56

Author(s):

Qiang Li ◽

Nicolás Montalbetti ◽

Yuliang Wu ◽

Arnolt Ramos ◽

Malay K. Raychowdhury ◽

...

Keyword(s):

Epithelial Cells ◽

Primary Cilia ◽

Cation Channel ◽

Renal Epithelial Cells ◽

Channel Function ◽

Microtubular Structures

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Ca2+-Dependent Regulation by the Cyclic AMP Pathway of Primary Cilium Length in LLC-PK1 Renal Epithelial Cells

10.1101/2020.05.11.088336 ◽

2020 ◽

Author(s):

Noelia Scarinci ◽

Paula L. Perez ◽

María del Rocío Cantero ◽

Horacio F. Cantiello

Keyword(s):

Kidney Disease ◽

Epithelial Cells ◽

Primary Cilium ◽

Primary Cilia ◽

Autosomal Dominant ◽

Cyst Formation ◽

Polycystic Kidney ◽

Renal Epithelial Cells ◽

Autosomal Dominant Polycystic Kidney ◽

Cilium Length

AbstractThe primary cilium is a sensory organelle projecting from the apical surface of renal epithelial cells. Dysfunctional cilia have been linked to a number of genetic diseases known as ciliopathies, which include autosomal dominant polycystic kidney disease (ADPKD). Previous studies have determined that renal epithelial primary cilia express both the polycystin-2 (PC2, TRPP2) channel and the type-2 vasopressin receptor (V2R), coupled to local cAMP production. However, little is known as to how Ca2+ and cAMP signals lead to changes in the length of the primary cilium. Here, we explored how cAMP signals regulate the length of the primary cilium in wild type LLC-PK1 renal epithelial cells. Primary cilia length was determined by immunocytochemical labeling of the ciliary axoneme. Treatment of cells with the cAMP analog 8-Br-cAMP (1 mM) in normal external Ca2+ (1.2 mM) produced a 25.3% increase (p < 0.0001) in the length of the primary cilium, a phenomenon also observed in cells exposed to high external Ca2+ (6.2 mM). However, exposure of cells to vasopressin (AVP, 10 μM), which also increases cAMP in primary cilia of LLC-PK1 cells, mimicked the effect of 8-Br-cAMP in normal, but not in high Ca2+. Further, specific gene silencing of PC2 expression further increased primary cilium length after 8-Br-cAMP treatment, in normal, but not high Ca2+. The encompassed data indicate a crosstalk between the cAMP and Ca2+ signals to modulate the length of the primary cilium, in a phenomenon that implicates the expression of PC2.Significance StatementMorphological changes in primary cilia have been linked to genetic disorders, including autosomal dominant polycystic kidney disease (ADPKD), a major cause of kidney disease. Both cAMP and Ca2+ are universal second messengers that regulate polycystin-2 (PC2, TRPP2), a Ca2+ permeable non-selective cation channel implicated in ADPKD, and expressed in the primary cilium of renal epithelial cells. Despite current interest, little is known as to how second messenger systems and how aberrant regulation of PC2 may link primary cilium structure with cyst formation in ADPKD. Here we determined that both the cAMP analog 8-Br-cAMP and vasopressin increase the length of the primary cilium in renal epithelial cells. However, this phenomenon depends of external Ca2+ and PKD2 gene silencing. Proper cAMP signaling may be essential in the control of the primary cilium of renal epithelial cells, and the onset of cyst formation in ADPKD.

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