XB-ART-55535
Nat Commun
2018 Jun 04;91:2167. doi: 10.1038/s41467-018-04677-9.
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Cotransporter-mediated water transport underlying cerebrospinal fluid formation.
Steffensen AB
,
Oernbo EK
,
Stoica A
,
Gerkau NJ
,
Barbuskaite D
,
Tritsaris K
,
Rose CR
,
MacAulay N
.
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Cerebrospinal fluid (CSF) production occurs at a rate of 500 ml per day in the adult human. Conventional osmotic forces do not suffice to support such production rate and the molecular mechanisms underlying this fluid production remain elusive. Using ex vivo choroid plexus live imaging and isotope flux in combination with in vivo CSF production determination in mice, we identify a key component in the CSF production machinery. The Na+/K+/2Cl- cotransporter (NKCC1) expressed in the luminal membrane of choroid plexus contributes approximately half of the CSF production, via its unusual outward transport direction and its unique ability to directly couple water transport to ion translocation. We thereby establish the concept of cotransport of water as a missing link in the search for molecular pathways sustaining CSF production and redefine the current model of this pivotal physiological process. Our results provide a rational pharmacological target for pathologies involving disturbed brain fluid dynamics.
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Genes referenced: cps1 slc12a2 slc12a4 slc12a5 slc4a10
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Fig. 1. Cotransporter-mediated active water transport against an osmotic gradient. a Image of calcein fluorescence in mouse choroid plexus (CP, top) and the converted image (bottom) with indication of the region of interest (red box). Scale bar = 60 µm. b Volume changes of CPs challenged with a 100 mOsm gradient of mannitol (M), indicated by the extended bars (n = 5 CPs from five mice). c CPs challenged with a 100 mOsm gradient of first mannitol (M), then NaCl, and finally mannitol, indicated by the extended bars (n = 6 CPs from six mice). d CPs challenged with a 100 mOsm gradient of first mannitol (M), then KCl, and finally mannitol, indicated by the extended bars (n = 6 CPs from six mice). e Summary of the second volume change induced by identical osmotic gradients. f CPs challenged with 100 mOsm KCl alone (control, black symbols, data from d) or in the presence of 1 mM furosemide (purple symbols, n = 6 CPs from six mice). Inset magnifies the area of interest where furosemide blocks the KCl-induced swelling. g CPs challenged with 100 mOsm alone (control, black symbols, data from d) or in the presence of 10 µM bumetanide (blue symbols, n = 6 CPs from six mice). Inset magnifies the area of interest where bumetanide blocks the KCl-induced swelling. h CPs challenged with 100 mOsm KCl (control, black symbols, data from d) and in the absence of Na+ in the test solution (green symbols, n = 4 CPs from four mice). Inset magnifies the area of interest where lack of Na+ blocks the KCl-induced swelling. i Summary of the second volume change induced by KCl alone or with drug application/Na+ omission. Error bars represent standard error of the mean and statistical significance was tested with one-way ANOVA followed by Tukey’s multiple comparisons test. In panel e, the asterisks refer to a comparison to the mannitol-mediated shrinkage and in panel i, the asterisks refer to a comparison to the KCl-mediated swelling. **P < 0.01, ***P < 0.001, NS not significant | |
Fig. 2. NKCC1 and KCC1 are expressed in mouse choroid plexus. a mRNA expression levels of NKCC1 and the four KCC isoforms in mouse choroid plexuses. The five target genes were normalized to two reference genes, GAPDH and H2AFZT, and presented as relative expression; NKCC1 = 60.0 ± 3.9, KCC1 = 21.2 ± 1.8, KCC2 = 0.2 ± 0.1, KCC3 = 1.2 ± 0.1, and KCC4 = 3.2 ± 0.2 (n = 6 mice), error bars represent standard error of the mean. b Representative western blots to verify antibody specificity. NKCC1 and KCC1-4 were expressed in Xenopus laevis oocytes and purified membranes from these and uninjected oocytes were exposed to SDS-PAGE followed by western blot (n = 3 experiments). c−f Western blots of lysates from mouse choroid plexus demonstrated expression of NKCC1 (c) and KCC1 (d), while KCC2 (e) and KCC4 (f) were not detected. Beta-tubulin was employed as loading control (n = 3 experiments) | |
Fig. 3. NKCC1 is located at the luminal membrane facing the ventricles. a Immunostaining of mouse choroid plexus illustrated expression of NKCC1 in the membrane facing the lumen (green) with E-cadherin (red) as a basolateral marker and nuclei in blue. Scale bar = 10 µm. Previously unpublished image generously provided by Dr. Jeppe Praetorius. b Schematic illustration of the luminal surface biotinylation of mouse choroid plexus in situ. c Representative western blots showing expression of NKCC1, KCC1, and E-cadherin in total tissue fraction (Ft) and purified biotinylated (luminal) membrane protein fraction (Fl). Below is depicted the quantification of protein abundance represented as luminal membrane/total protein fraction (n = 4 mice). NKCC1 was expressed significantly higher in the purified luminal membrane fraction while KCC1 and E-cadherin were predominantly in the total fraction. Error bars represent standard error of the mean and statistical significance was determined with one-sample t test and comparison to equal distribution; Fl/Ft = 1 and the asterisks refer to a value significantly different from 1. **P < 0.01, ***P ≤ 0.001 | |
Fig. 4. NKCC1 is poised for outward transport in the luminal membrane. a Image of SBFI fluorescence of the choroid plexus taken with wide-field fluorescence microscope. Scale bar = 40 µm. b Representative sodium signals of choroid plexus during 10 min baseline, 10 min application of the NKCC1 inhibitor bumetanide (10 μM), and 20 min washout. Gray lines represent the SBFI ratio obtained from 40 single cells during one experiment, black line represents an average of these cells. c Average bumetanide-induced increase in SBFI ratio of choroid plexus cells, indicative of increased intracellular sodium concentration (n = 10 CPs from ten mice). d Representative images of calcein fluorescence of the choroid plexus either after 10 min in aCSF (top panel) or after 10 min of bumetanide treatment (bottom panel, time point marked as * in b). Scale bar = 40 µm. e Loss of 86Rb+ from choroid plexus as a function of time in control settings (black, n = 7 CPs from seven mice) or with treatment of either bumetanide (20 μM, blue, n = 5 CPs from five mice) or furosemide (1 mM, violet, n = 5 CPs from five mice). Y-axis is the natural logarithm of amount of 86Rb+ left in choroid plexus at time t (At) divided by the amount at time 0 (A0). f Efflux rate constants for 86Rb+ in control (0.041 ± 0.003 s−1, n = 7 CPs from seven mice), in the presence of bumetanide (0.008 ± 0.001 s−1, n = 5 CPs from five mice), or furosemide (0.005 ± 0.001 s−1, n = 5 CPs from five mice). Error bars represent standard error of the mean and statistical significance was tested with one-way ANOVA followed by Tukey’s multiple comparisons test and the asterisks above the bars indicate comparison to the control while the comparison between the two test solutions is indicated with a line above the relevant bars. ***P < 0.0001, NS not significant (P = 0.526) | |
Fig. 5. NKCC1 acts as a significant contributor to in vivo CSF production. a Schematic drawing of the infusion of aCSF containing dextran (dark red cannula) into the right lateral ventricle (RV) and collection of the diluted dextran (light red cannula) at cisterna magna (CM) of a ventilated mouse. b Representative time course of the fluorescence ratio of dextran (outflow/inflow) during a control ventriculo-cisternal perfusion. After stable baseline (60 min), the dextran/DMSO aCSF-solution was changed within 5–10 min. Inset depicts the average CSF production rate (n = 18 mice). c Summarized data from ventriculo-cisternal perfusion illustrating CSF production (percentage of baseline) as a function of time. Data normalized to the average of the two last samples before solution change. Control perfusion with the DMSO-vehicle is shown in black (n = 6 mice), treatment with bumetanide in blue (n = 6 mice), and furosemide (n = 6 mice) in purple. Inset illustrates the summarized CSF production rates after 60 min exposure to vehicle (black), bumetanide (blue), or furosemide (purple) normalized to own control. d Mid-sagittal section of a mouse brain after unilateral injection of Evans Blue revealed staining mainly in the injected, right lateral ventricle (top panel) compared to the contralateral ventricle (bottom image). Error bars represent standard error of the mean and statistical significance was tested with one-way ANOVA followed by Tukey’s multiple comparisons test with asterisks above the bars indicating comparison to control perfusion and comparison between test solutions indicated by lines above the respective bars. *P < 0.05, ***P < 0.001 | |
Fig. 6. Live imaging determines significant NKCC1 contribution to in vivo CSF production. a Illustration of pseudo-color fluorescence superimposed on a white light image from a white mouse after ventricular injection of 10 μM IRDye 800CW carboxylate dye. The dye content is quantified in the red box, placed in line with lambda. The intensity scale is arbitrary units and applies to all images. b Intensity of images were quantified and normalized to the first image (0 min) for control (vehicle, black), bumetanide (blue), or furosemide (purple) and plotted as a function of time. Inset illustrates summarized data at the 10-min time point; the fluorescence intensity of control mice reached 1.95 ± 0.12 (n = 8 mice), furosemide-treated 1.65 ± 0.05 (n = 6 mice), and bumetanide 1.63 ± 0.07 (n = 8 mice). c Unilateral injection of Evans Blue revealed staining mainly in the injected, right lateral ventricle (red oval) compared to the contralateral ventricle. d Representative images at 0, 5, and 10 min for control condition (top panels), bumetanide treatment (middle panels), and furosemide treatment (bottom panels). Error bars represent standard error of the mean and statistical significance was evaluated with two-way ANOVA (RM) followed by Tukey’s multiple comparisons test. Asterisks above the bars indicate comparison to control while comparisons between test solutions are indicated with a line above the respective bars. **P < 0.01, NS: not significant (P = 0.98) | |
Fig. 7. Schematic drawing of transporters in a choroid plexus epithelial cell. a The drawing depicts the selective expression of NKCC1 at the luminal membrane and indicates its unique outward transport direction, due to the high intracellular concentration of Na+ and Cl− in this tissue (ion concentrations given in mM). The NKCC1-mediated cotransport of water is indicated with a dashed arrow. The Na+/K+-ATPase and AQP114 are indicated on the luminal membrane of the choroid plexus epithelium (CPE), while KCC1 is localized to the basolateral membrane facing the vascular compartment (this study). b The drawing includes the many other coupled transporters localized to the choroidal epithelial membranes1. Note that NCBE may also be referred to as NBCn2 |
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