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.
Solute carrier family 41 member A1 (SLC41A1) has been suggested to mediate magnesium (Mg 2+ ) transport by several in vitro studies. However, the physiological function of SLC41A1 remains to be elucidated. In this study, cellular Mg 2+ transport assays combined with zebrafish slc41a1 knockdown experiments were performed to disclose SLC41A1 function and its physiological relevance. The gene slc41a1 is ubiquitously expressed in zebrafish tissues and is regulated by water and dietary Mg 2+ availability. Knockdown of slc41a1 in zebrafish larvae grown in a Mg 2+ -free medium resulted in a unique phenotype characterized by a decrease in zebrafish Mg content. This decrease shows that SLC41A1 is required to maintain Mg 2+ balance and its dysfunction results in renal Mg 2+ wasting in zebrafish larvae. Importantly, the Mg content of the larvae is rescued when mouse SLC41A1 is expressed in slc41a1 -knockdown zebrafish. Conversely, expression of mammalian SLC41A1-p.Asp262Ala, harboring a mutation in the ion-conducting SLC41A1 pore, did not reverse the renal Mg 2+ wasting. 25 Mg 2+ transport assays in human embryonic kidney 293 (HEK293) cells overexpressing SLC41A1 demonstrated that SLC41A1 mediates cellular Mg 2+ extrusion independently of sodium (Na + ). In contrast, SLC41A1-p.Asp262Ala expressing HEK293 cells displayed similar Mg 2+ extrusion activities than control (mock) cells. In polarized Madin-Darby canine kidney cells, SLC41A1 localized to the basolateral cell membrane. Our results demonstrate that SLC41A1 facilitates renal Mg 2+ reabsorption in the zebrafish model. Furthermore, our data suggest that SLC41A1 mediates both Mg 2+ uptake and extrusion. Electronic supplementary material The online version of this article (10.1007/s00424-018-2234-9) contains supplementary material, which is available to authorized users.
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.
The PKD1 gene encodes polycystin-1 (PC1), a mechanosensor triggering intracellular responses upon urinary flow sensing in kidney tubular cells. Mutations in PKD1 lead to autosomal dominant polycystic kidney disease (ADPKD). The involvement of PC1 in renal electrolyte handling remains unknown since renal electrolyte physiology in ADPKD patients has only been characterized in cystic ADPKD. We thus studied the renal electrolyte handling in inducible kidney-specific Pkd1 knockout (iKsp- Pkd1) mice manifesting a precystic phenotype. Serum and urinary electrolyte determinations indicated that iKsp- Pkd1 mice display reduced serum levels of magnesium (Mg), calcium (Ca), sodium (Na), and phosphate (P) compared with control ( Pkd1) mice and renal Mg, Ca, and P wasting. In agreement with these electrolyte disturbances, downregulation of key genes for electrolyte reabsorption in the thick ascending limb of Henle's loop (TA;, Cldn16, Kcnj1, and Slc12a1), distal convoluted tubule (DCT; Trpm6 and Slc12a3) and connecting tubule (CNT; Calb1, Slc8a1, and Atp2b4) was observed in kidneys of iKsp- Pkd1 mice compared with controls. Similarly, decreased renal gene expression of markers for TAL ( Umod) and DCT ( Pvalb) was observed in iKsp- Pkd1 mice. Conversely, mRNA expression levels in kidney of genes encoding solute and water transporters in the proximal tubule ( Abcg2 and Slc34a1) and collecting duct ( Aqp2, Scnn1a, and Scnn1b) remained comparable between control and iKsp- Pkd1 mice, although a water reabsorption defect was observed in iKsp- Pkd1 mice. In conclusion, our data indicate that PC1 is involved in renal Mg, Ca, and water handling and its dysfunction, resulting in a systemic electrolyte imbalance characterized by low serum electrolyte concentrations.
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