Primary cilia‐regulated transcriptome in the renal collecting duct

Abstract

Renal tubular cells respond to mechanical stimuli generated by urinary flow to regulate the activity and transcript abundance of important genes for ion handling, cellular homeostasis, and proper renal development. The primary cilium, a mechanosensory organelle, is postulated to regulate this mRNA response. The aim of this study is to reveal the transcriptome changes of tubular epithelia in response to fluid flow and determine the role of primary cilia in this process. Inner-medullary collecting duct (CD) cells were subjected to either static or physiologically relevant fluid flow (∼0.6 dyn/cm). RNA-sequencing analysis of ciliated cells subjected to fluid flow showed up-regulation of 1379 genes and down-regulation of 1294 genes compared with static control cells. Strikingly, only 54 of these genes were identified as gene candidates sensitive to primary cilia sensing of fluid flow, of which 16 were linked to ion or water transport pathways in the CD. Validation by quantitative real-time PCR revealed that only the expression of transferrin receptor, which is involved in iron transport; and tribbles pseudokinase 3, which is involved in insulin signaling, were unequivocally regulated by primary cilia sensing of fluid flow. This study shows that the involvement of primary cilia in ion transport in the collecting duct is exceptionally specific.-Mohammed, S. G., Arjona, F. J., Verschuren, E. H. J., Bakey, Z., Alkema, W., van Hijum, S., Schmidts, M., Bindels, R. J. M., Hoenderop, J. G. J. Primary cilia-regulated transcriptome in the renal collecting duct.

Tubular flow activates magnesium transport in the distal convoluted tubule

“…Alternatively, the lack of IFT40 function could affect cell structure or differentiation, which indirectly modifies Mg 2+ transport due to potential altered channel localization or function. Our finding that primary cilia are not involved in FSS‐activated cellular Mg 2+ uptake in DCT is in line with previous studies showing no relation between flow sensing by primary cilia and CNT Ca 2+ transepithelial transport or the electrolyte transport transcriptome in the CD (21, 27). Possibly, the plethora of functions attributed to primary cilia sensing do not encompass electrolyte transport or it is limited to the ability to increase intracellular Ca 2+ levels, a function that is also under debate (53).…”

“…4). Conversely, the expression of Ptgs2 , encoding COX‐2, a known FSS‐sensitive gene (27, 38), was significantly up‐regulated ( P < 0.05) in FSS‐exposed cells.…”

“…However, in vivo , prourinary flow is of laminar nature (17, 54). In a more recent study (27) and in the present study, laminar flow was applied using the µ‐slide VI 0.4 channels. Therefore, the microfluidic model used in our study is of more physiologic relevance as compared with oscillatory models.…”

“…After experimentation, cells were washed with ice-cold PBS and fixed for 15 min in 10% (w/v) formalin in PBS followed by permeabilization in 0.3% (v/v) Triton X-100 in PBS and 0.1% (w/v) bovine serum albumin. Next, cells were incubated overnight at 4°C with rabbit anti-ADP ribosylation factor-like GTPase 13b (ARL13B, 1:200; Proteintech, Rosemont, IL, USA) in goat serum dilution buffer [16% v/v goat serum in PBS (pH 7.4)] (27). The following day, cells were washed with PBS and incubated for 1 h in the dark at room temperature with Alexa Fluor 594-conjugated anti-rabbit IgG (1:250; Molecular Probes, Eugene, OR, USA) in goat serum.…”

“…The variable urinary flow in DCT ranges from ∼0.3 to 1.2 dyn/cm 2 under physiologic conditions (26, 27). Primary cilia, expressed in almost all tubular epithelial cells along the nephron, act as mechanosensory organelles that sense changes in urinary flow (28, 29).…”