CrossTalk opposing view: NKCC1 in the luminal membrane of choroid plexus is outwardly directed under basal conditions and contributes directly to cerebrospinal fluid secretion

Abstract

Our brain contains 80% water that is continuously renewed by de novo fluid secretion from the blood at a rate of approximately 500 ml day−1 in the adult human (Rubin et al. 1966). While cerebrospinal fluid (CSF) secretion has been acknowledged for centuries, the molecular mechanisms supporting this fluid movement remain controversial. Most of the CSF originates from the choroid plexus lining the fluid-filled ventricles of the brain (Welch, 1963; Pollay et al. 1985; Knuckey et al. 1991). These are mono-layered epithelial structures that feature characteristics common for secretory epithelia (Damkier et al. 2013). Each epithelial membrane (basolateral and luminal) hosts an array of specific membrane transporters (Fig. 1A), which mediate the directed transepithelial movement of ions and water and thereby generate the new CSF. An earlier view promoted CSF secretion as mainly relying on conventional osmotic water transport (Davson & Segal, 1970). This was proposed to occur analogously to the mammalian kidney collecting duct, in which aquaporin-mediated fluid reabsorption takes place in response to large osmotic gradients across the epithelium. While it seems appealing to extend this mechanism to the choroid plexus epithelium, several experimental observations suggest otherwise. Firstly, aquaporins are expressed on only one of the two epithelial membranes (Speake et al. 2003). Secondly, there is virtually no osmotic difference between the blood and the CSF (Hendry, 1962; Hochwald et al. 1974). Hence, there is no driving force to support the high rate of fluid secretion. Finally, it has been demonstrated that CSF secretion can occur independently of, and even against, a transepithelial osmotic gradient (Heisey et al. 1962; Hochwald et al. 1974). These three findings clearly suggest that CSF secretion cannot be solely based on osmotic forces, but must be realized through other means of water transport. In our recent work, we provided evidence for such an alternative mechanism (Steffensen et al. 2018). Instead of conventional water transport through aquaporins, we found that a substantial part of the CSF secretion in mice occurs by cotransport of water through the Na+–K+–2Cl− cotransporter, NKCC1. NKCC1 is expressed in the luminal membrane of the choroid plexus (Praetorius & Nielsen, 2006; Steffensen et al. 2018) and belongs to the expanding group of cotransporters sharing the ability to move water along their translocation pathway (Zeuthen & MacAulay, 2012). This water transport manifests itself by a fixed number of water molecules being translocated at the same time and in the same direction as the transported solutes – independently of the transmembrane osmotic gradient (Zeuthen, 2010). In line with this notion, we demonstrated that water can be moved independently of the osmotic gradient in acutely isolated choroid plexus in an NKCC1-dependent manner (Steffensen et al. 2018). The NKCC1-mediated water transport generated approximately 50% of the CSF in anaesthetized mice, as evidenced upon intraventricular pharmacological inhibition of this transporter with bumetanide (<50 μm following ventricular dilution of the infused bumetanide-containing artificial CSF (aCSF)), as also demonstrated in dogs (Javaheri & Wagner, 1993). A direct involvement of NKCC1 in CSF secretion via its ability to cotransport water across the luminal membrane of the choroid plexus necessitates a directed transport from inside the cells towards the ventricular cavity. In other words, NKCC1 activity must be outwardly directed. With an electroneutral transporter, such as NKCC1, the transport direction solely depends on the ion concentrations at both sides of the membrane. Our estimates of intra- and extracellular concentrations of the transported ions, obtained from choroid plexus rapidly extracted from mice, indeed suggested an outward direction. We verified this directionality in acutely extracted mouse choroid plexus by two different techniques (Steffensen et al. 2018). Firstly, choroid plexus was loaded with 86Rb+, which can replace K+ at the binding site and act as a tracer molecule for the transported K+ (and thereby illustrate the transport direction). The rate of 86Rb+ release thus mimics the K+ efflux from the ex vivo choroid plexus, and was found to be severely reduced in the presence of bumetanide (20 μm) (Keep et al. 1994; Steffensen et al. 2018). Such flux reduction can only be observed with NKCC1 operating in the outward transport direction. In a second approach, based on dynamic imaging in Hepes-based aCSF of acutely isolated choroid plexus with the Na+-sensitive fluorescent dye SBFI, bumetanide (20 μm) induced a reversible increase in intracellular [Na+] (Fig. 1B, upper trace). Such an increase is indicative of an outward operation of NKCC1 in choroid plexus. In contrast, in other preparations, i.e. Bergmann glia in acute brain slices, such an approach revealed a reversible decrease in [Na+]i and thus inwardly directed NKCC1 activity (Fig. 1B, lower trace) (Untiet et al. 2017). The recent study by Gregoriades et al. (2019) opposed this view by providing experimental evidence of an inward direction of NKCC1 in choroid plexus epithelial cells, thus questioning its direct role in choroidal fluid secretion. The authors based their conclusions on the choroid plexus epithelial cell volume and ion concentrations. These experiments were rigorously performed and the results convincingly demonstrate that under the chosen experimental paradigm, NKCC1 most likely functions in the inward transport mode. However, a few conditions set their experimental conditions and model preparation apart from those employed by Steffensen et al. (2018). In the latter study, most experiments were performed in acutely isolated intact choroid plexus (with limited access to the basolateral membrane) using bicarbonate-buffered, gas-equilibrated, and heated aCSF (Steffensen et al. 2018), while Gregoriades et al. (2019) conducted their experiments in bicarbonate-free, Hepes-based buffer. The absence of bicarbonate (CO2/HCO3−) will, however, essentially stop the transport activity of the elaborate array of bicarbonate transporters expressed in choroid plexus epithelium (i.e. AE2, NCBe2, NCBE and NCBn1; see Fig. 1A and Damkier et al. (2013)). Such functional elimination of a set of transporters, all coupled to Na+ or Cl−, will most likely affect their transport direction and alter the intracellular ion concentrations. Moreover, Gregoriades et al. (2019) performed their experiments on single choroid plexus epithelial cells, obtained by enzymatic treatment and mechanical isolation, which were subsequently cultured for hours (2–6 h) in cell culture medium with a K+ concentration much higher (8 mm, which is likely to promote inward NKCC1 transport) than that of the CSF (2.5–3 mm). Taken together, it is therefore highly likely that the inward NKCC1 transport direction arrived at by Gregoriades et al. (2019) originates, at least in part, from the mechanical/enzymatic/culturing treatment of cells prior to experimentation and the content of the test solutions, in which the experiments were conducted. The opposing results obtained in these two recent studies certainly add to the ongoing debate on the cellular mechanisms of CSF production. Future studies solving this issue may require novel, and more quantitative, intensity-independent fluorescence lifetime imaging of Na+ and Cl− (Untiet et al. 2017; Meyer et al. 2019). In conclusion, we propose that the high rate of CSF secretion simply cannot occur by conventional osmotic water transport given the lack of a transepithelial osmotic gradient, the low passive water permeability across the entire epithelium, and the ability of the choroid plexus to secrete fluid under conditions in which the osmotic gradient is either absent or unfavourable. Hence cotransport of water may serve as the missing link required to sustain a continuous fluid secretion across the choroid plexus epithelium. We suggest that NKCC1, via its outwardly directed transport of electrolytes and cotransported water, acts as a key contributor to the net CSF secretion across the luminal membrane of the choroid plexus. Readers are invited to give their views on this and the accompanying CrossTalk articles in this issue by submitting a brief (250 word) comment. Comments may be submitted up to 6 weeks after publication of the article, at which point the discussion will close and the CrossTalk authors will be invited to submit a ‘LastWord’. Please email your comment, including a title and a declaration of interest, to [email protected]. Comments will be moderated and accepted comments will be published online only as ‘supporting information’ to the original debate articles once discussion has closed. Nanna MacAulay, PhD, is Professor of Molecular Neurophysiology at Department of Neuroscience, University of Copenhagen. She trained in ion and water transport, and was, under the mentorship of Professor Thomas Zeuthen, amongst the pioneers discovering the concept of cotransport of water. Her research focuses on resolving the molecular mechanisms of water transport in various cell types of the mammalian brain and has demonstrated cotransport of water as a key component in choroid plexus, glia, and neurons. Christine R. Rose, PhD, is full Professor and Head of the Institute of Neurobiology at the Heinrich Heine University, Düsseldorf, Germany. She obtained a PhD in Zoology from the University of Kaiserslautern and worked as a Post-Doc at Yale School of Medicine (USA), at the Department of Physiology at Saarland University, and at the TU and LMU Munich. Her work is devoted to cellular ion regulation in the brain with a special focus on intracellular sodium homeostasis. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. None. Both authors have read and approved the final version of this manuscript and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed. This work was funded by Novo Nordic Foundation (NNF17OC0024718) to N.M., Thorbergs Foundation (53.734) to N.M., Læge Sofus Carl Emil Friis og hustru Olga Doris Friis'Legat to N.M., Carlsberg Foundation (CF15-0070) to N.M., Deutsche Forschungsgemeinschaft (DFG, SPP1757:Ro2327/8-2 and FOR2795:Ro2327/13-1) to C.R.R.

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