ABSTRACT
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.
Subject(s)
Epithelial Cells/metabolism , Kidney Tubules, Collecting/metabolism , Osmoregulation , Osmotic Pressure , TRPM Cation Channels/metabolism , Animals , CRISPR-Cas Systems , Cell Line , Cilia/metabolism , Epithelial Cells/drug effects , GABA Plasma Membrane Transport Proteins/genetics , GABA Plasma Membrane Transport Proteins/metabolism , Gene Editing , Hydroxyprostaglandin Dehydrogenases/genetics , Hydroxyprostaglandin Dehydrogenases/metabolism , Kidney Tubules, Collecting/drug effects , Mice , Mice, Inbred C57BL , Mice, Transgenic , Osmoregulation/drug effects , Osmotic Pressure/drug effects , Saline Solution, Hypertonic/pharmacology , Signal Transduction , TRPM Cation Channels/genetics , TRPV Cation Channels/genetics , TRPV Cation Channels/metabolism , TransfectionABSTRACT
Primary cilia of renal epithelial cells express several members of the transient receptor potential (TRP) class of cation-conducting channel, including TRPC1, TRPM3, TRPM4, TRPP2, and TRPV4. Some cases of autosomal dominant polycystic kidney disease (ADPKD) are caused by defects in TRPP2 (also called polycystin-2, PC2, or PKD2). A large-conductance, TRPP2-dependent channel in renal cilia has been well described, but it is not known whether this channel includes any other protein subunits. To study this question, we investigated the pharmacology of the TRPP2-dependent channel through electrical recordings from the cilia of mIMCD-3 cells, a murine cell line of renal epithelial origin. The pharmacology was found to match that of TRPM3 channels. The ciliary TRPP2-dependent channel is known to be activated by depolarization and by increasing cytoplasmic Ca2+. This activation was greatly enhanced by external pregnenolone sulfate, an agonist of TRPM3 channels. Pregnenolone sulfate did not change the single-channel current-voltage relation. The channels were effectively blocked by isosakuranetin, a specific inhibitor of TRPM3 channels. Both pregnenolone sulfate and isosakuranetin were effective at concentrations as low as 1 µM. Knocking out TRPM3 by CRISPR/Cas9 genome editing eliminated the ciliary channel. Thus the channel is both TRPM3-dependent and TRPP2-dependent, suggesting that it may include both types of subunit. Knocking out TRPM3 did not change the level of TRPP2 protein in the cilia, so it is unlikely that the absence of functional ciliary channels results from a failure of trafficking.