Does the primary cilium elongation play a role in urine concentration?
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Nearly every mammalian cell contains the primary cilium, a solitary, immotile cellular organelle, that operates as a mechanosensor and chemosensor [1]. Primary cilia are microtubule-based, hair-like organelles, projecting from the mammalian cells [2]. In the kidney, primary cilia are found largely in the epithelial cells of the parietal layer of Bowman’s capsule and throughout the renal tubular segments, including proximal and distal tubules and collecting ducts [2]. These primary cilia detect and convert extracellular signals from the tubular lumen into the cells, thereby activating intracellular signaling pathways. For example, Madin-Darby canine kidney cells with chloral hydrate-induced loss of primary cilia failed to exhibit fluid flow-induced intracellular Ca2+ increase [1]. Moreover, the appropriate functioning of primary cilia is essential for epithelial cell proliferation, differentiation, and kidney organogenesis [3]. Ciliopathies are a group of disorders caused by alterations in the structure and function of cilia [3]. Mutations in polycystin 1 (Pkd1), Pkd2, or polycystic kidney and hepatic disease 1 (Pkhd1) cause primary cilia dysfunction in the kidney, resulting in polycystic kidney disease, which leads to renal functional decline and end-stage renal disease [4]. However, how loss of cilia or cilia dysfunction leads to disease development remains elusive.
Interestingly, the length of the primary cilia in renal tubular cells was found to be dynamically altered under diverse conditions. A study done by Kong et al. [5] in this issue postulated that the alteration in primary cilium length in tubular epithelial cells is an important process for urine concentration. This may provide new insights into the relationship between urine concentration and cilia length alteration in renal tubular cells. Microtubules, the central elements of the primary cilium, undergo the processes of assembly and disassembly, which are linked to the lengthening and shortening of the primary cilia. Post-translational modifications regulate these processes. α-tubulin acetyltransferase 1 (αTAT1) adds acetyl groups to α-tubulins to make primary cilia longer, while histone deacetylase 6 (HDAC6) takes acetyl groups away from α-tubulins to make them shorter. Kong et al. [5] reported that high water intake (HWI) in mice induces elongation of the primary cilium in renal tubular cells through extracellular signal-regulated kinases 1 and 2 (ERK1/2) activation (phosphorylation at Thr202 and Tyr204) and increases in αTAT1 and Exoc5 (exocyst complex components 5) expressions. The expression and activity of HDAC6 were not affected by HWI. In contrast, ERK inhibition through inhibition of mitogen-activated kinase kinase (MEK) with an inhibitor (U0126) in mice blocks these HWI-induced changes, including α-tubulin acetylation and elongation of the primary cilium. Moreover, MEK inhibitor (U0126) treatment in mice inhibited the HWI-induced downregulation of aquaporin-2 (AQP2) and the kidney’s ability to produce diluted urine in response to HWI. Based on the results, the authors concluded that elongation of the primary cilium length in tubular epithelial cells via ERK1/2 activation is a required response to produce diluted urine under HWI conditions.
However, there are several complexities in the interpretation of the results. Previous studies have suggested that mitogen-activated protein kinases (MAPKs) are involved as a downstream signaling pathway of the vasopressin V2 receptor (V2R) and play a role in the regulation of AQP2 [6–8]. MAPKs, which are serine/threonine (Thr) kinases, convert extracellular stimuli into various cellular responses, including gene expression, metabolism, mitosis, and apoptosis. So far, five different groups of MAPKs have been characterized: ERK1/2; c-Jun amino-terminal kinases (JNKs) 1, 2, and 3; p38 isoforms α, β, γ, and δ; ERK 3 and 4; and ERK5. Among them, ERK1/2, JNKs, and p38 kinases are the most extensively studied. Each group of MAPKs comprises a set of three sequentially acting kinases: a MAPK kinase (MAPKK) kinase (MAPKKK), a MAPKK, and a MAPK. MAPKKK activation induces the phosphorylation and activation of MAPKKs [9], which in turn activate MAPK via phosphorylation on both Thr and tyrosine (Tyr) residues within the activation loop containing a conserved Thr-X-Tyr motif. MAPKs then phosphorylate their target substrate proteins on serine or threonine residues with proline at the +1 position [9]. Previous studies have demonstrated that vasopressin affects AQP2 phosphorylation at serine 261 (pS261-AQP2) through the regulation of MAPK activity. Nedvetsky et al. [6] demonstrated that the selective p38 MAPK inhibitor SB202190 decreases phosphorylation of p38 MAPK and pS261-AQP2 in primary cultured rat kidney inner medullary collecting duct (IMCD) cells. Pisitkun et al. [7] demonstrated that 1-deamino-8-D-arginine vasopressin, a selective V2R agonist, reduces ERK1/2 phosphorylation at Thr202 and Tyr204 in IMCD cells. In contrast, Cheung et al. [8] revealed that vasopressin dephosphorylates pS261-AQP2, but significantly increases phosphorylation of ERK1/2 at Thr202 and Tyr204 in LLC-PK1 cells stably expressing c-myc-tagged AQP2. These data indicated that the changes in MAPK expression after vasopressin stimulation and the roles of MAPKs in vasopressin signaling (i.e., the response to high or low water intake), including regulation of AQP2 and urine concentration, have not been clearly understood. A recent study demonstrated that tolvaptan, a V2R antagonist, increases ERK1/2 phosphorylation at Thr202 and Tyr204 in mpkCCD cells, which was dependent on protein kinase A (PKA) [10].
Furthermore, the authors did not provide a detailed explanation of how changes in the length of the primary cilium in tubular epithelial cells affect the expression and subcellular localization of AQP2 in the kidney collecting duct principal cells. Specifically, it is unclear whether downregulation of AQP2 protein abundance and decreased AQP2 in the membrane fractions are the direct results of the elongation in primary cilium length in the collecting duct principal cells in response to HWI. A previous study, however, demonstrated that Gd3+ (inhibiting intracellular Ca2+ entry) and forskolin treatment (increasing intracellular cyclic adenosine monophosphate [cAMP] levels), respectively, increased cilia length in the immortalized kidney collecting duct line (IMCD), and both the embryonically derived kidney epithelia and primary bone mesenchymal cells also showed similar findings [11]. In contrast, activation of Ca2+ signaling by triptolide or thapsigargin, as well as inhibition of PKA signaling (Rp-cAMPS, KT-5270, H-89), resulted in a reduction in primary cilium length [11]. AQP2 protein abundance and apical targeting in the collecting duct principal cells are largely dependent on an increase in intracellular cAMP levels and PKA activation, both of which, however, cause the elongation of primary cilia in IMCD cells [11]. As a result, this study demonstrating that HWI lengthens primary cilia while decreasing AQP2 expression contradicts previous findings.
Table 1 in the Kong et al.’s study [5] shows that ERK inhibition with U0126 in mice prevented the HWI-induced increase in urine volume. However, it seems difficult to determine exactly the changes in body water and sodium balance since the daily sodium intake and changes in plasma sodium levels and body weight are missing. Moreover, mice treated with the MEK inhibitor U0126 showed a dramatic decrease in urine sodium concentration in response to HWI compared to normal water intake (NWI), despite unchanged urine output, presumably due to altered food intake and a perturbation in daily sodium balance. Although it is not accurate to predict the solute-free water clearance (CH2O = V [1 − Uosm/Posm], where V means urine volume, Uosm means urine osmolality, and Posm means plasma osmolality) using the data presented in Table 1 [5], presumably the solute-free water clearance is different between NWI and HWI under the U0126 treatment, despite no difference in AQP2 expression levels. This issue needs to be clarified.
In addition, primary cilia have also been hypothesized to play a role in metabolic regulation, as they may be involved in detecting metabolic signals. Adipocyte development in adipose tissue is regulated by primary cilia through the transduction of the classical Hedgehog (HH) and Wnt signaling pathways. Moreover, noncanonical HH signaling controls glucose and energy metabolism in skeletal muscle and brown adipocytes [12]. The role of primary cilia in the metabolic processes of renal tubular epithelial cells remains unclear. In this study, mice were subjected to water containing 3% sucrose for a period of 2 days in order to induce HWI [5]. Metabolomics allows for the study of metabolic profiling of the kidney and urine [13]. Therefore, it is intriguing to investigate the alterations in the metabolism of the renal tubular epithelial cells, specifically in relation to the elongation of the primary cilia and the resulting modifications in signaling pathways. In summary, while this study is intriguing, it lacks mechanistic studies [5]. We should conduct more direct experiments to determine conclusively whether alterations in cilia length are associated with AQP2 and body water balance regulation.
Notes
Conflicts of interest
The author has no conflicts of interest to declare.
Funding
This work was supported by grants from the National Research Foundation of Korea (NRF) grant funded by the Korea Government (MIST) (2023R1A2C2005570).
Data sharing statement
The data presented in this study are available from the corresponding author upon reasonable request.