Fluid shear stress increases transepithelial transport of Ca ²⁺ in ciliated distal convoluted and connecting tubule cells

Abstract

In kidney, transcellular transport of Ca is mediated by transient receptor potential vanilloid 5 and Na-Ca exchanger 1 proteins in distal convoluted and connecting tubules (DCTs and CNTs, respectively). It is not yet understood how DCT/CNT cells can adapt to differences in tubular flow rate and, consequently, Ca load. This study aims to elucidate the molecular mechanisms by which DCT/CNT cells sense fluid dynamics to control transepithelial Ca reabsorption and whether their primary cilia play an active role in this process. Mouse primary DCT/CNT cultures were subjected to a physiologic fluid shear stress (FSS) of 0.12 dyn/cm Transient receptor potential vanilloid 5 and Na-Ca exchanger 1 mRNA levels were significantly increased upon FSS exposure compared with static controls. Functional studies with Ca demonstrated a significant stimulation of transepithelial Ca transport under FSS compared with static conditions. Primary cilia removal decreased Ca transport in both static and FSS conditions, a finding that correlated with decreased expression of genes involved in transepithelial Ca transport; however, FSS-induced stimulation of Ca transport was still observed. These results indicate that nephron DCT and CNT segments translate FSS into a physiologic response that implicates an increased Ca reabsorption. Moreover, primary cilia influence transepithelial Ca transport in DCTs/CNTs, yet this process is not distinctly coupled to FSS sensing by these organelles.-Mohammed, S. G., Arjona, F. J., Latta, F., Bindels, R. J. M., Roepman, R., Hoenderop, J. G. J. Fluid shear stress increases transepithelial transport of Ca in ciliated distal convoluted and connecting tubule cells.

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).…”

“…Previous studies in primary DCT/CNT cultures using oscillatory flow have shown that transepithelial Ca 2+ transport is facilitated by an up‐regulation of the expression of calciotropic genes such as Trpv5 and Slc8a1 , which encode an apical Ca 2+ channel and a basolateral Na + ‐Ca 2+ exchanger, respectively (21). However, in vivo , prourinary flow is of laminar nature (17, 54).…”

“…After DNase treatment, cDNA synthesis was performed using Molony‐Murine Leukemia Virus‐Reverse Transcriptase (Thermo Fisher Scientific) as previously described (33). The cDNA was mixed with Power SYBR green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) and with primers (400 nM) as previously described (21). The expression of the following genes was assessed via real‐time quantitative PCR (qPCR) (7 min at 95°C, 40 cycles of 15 s at 95°C and 1 min at 60°C): Cnnm2, Kcna1, Proegf, Ptgs2, Trpm6 , and Trpm7 .…”

Primary cilia‐regulated transcriptome in the renal collecting duct

“…These effects of fluid flow on CD electrolyte handling lead to the conclusion that renal tubular cells respond to fluid flow to regulate the activity and abundance of electrolyte channels and transporters in the CD. In fact, this postulation has been demonstrated in the connecting tubule of the nephron, where fluid shear stress regulates the gene expression of transient receptor potential cation channel subfamily V (Trpv5) and solute carrier family 8 member a1 (Slc8a1), coding the proteins that allow transepithelial Ca 2+ transport in this segment of the nephron (14).…”

A perfusion chamber for monitoring transepithelial NaCl transport in an in vitro model of the renal tubule

“…An important mechanical stimulus for renal epithelial cells is FSS. In tubular epithelial cells cultured in vitro, physiological levels of FSS alters cytoskeletal organization and transport proteins resulting in enhanced epithelial cell phenotype (Duan, Weinstein, Weinbaum, & Wang, ; Mohammed et al, ; Raghavan, Rbaibi, Pastor‐Soler, Carattino, & Weisz, ). On the other hand, pathological levels of FSS may be responsible for losing of epithelial characteristics that may account for the progression of chronic kidney disease (Grabias & Konstantopoulos, ; Maggiorani et al, ).…”