Cerebrospinal fluid formation is controlled by membrane transporters to modulate intracranial pressure

Disturbances in the brain fluid balance can lead to life-threatening elevation in the intracranial pressure (ICP), which represents a vast clinical challenge. Nevertheless, the molecular mechanisms governing cerebrospinal fluid (CSF) secretion are largely unresolved, thus preventing targeted and efficient pharmaceutical therapy of cerebral pathologies involving elevated ICP. Here, we employed experimental rats to demonstrate low osmotic water permeability of the choroid plexus, lack of an osmotic gradient across this tissue, and robust CSF secretion against osmotic gradients. Together, these results illustrate that CSF secretion occurs independently of conventional osmosis, which challenges the existing assumption that CSF production is driven entirely by bulk osmotic forces across the CSF-secreting choroid plexus. Instead, we reveal that the choroidal Na+/K+/Cl− cotransporter NKCC1, Na+/HCO3− cotransporter NBCe2, and Na+/K+-ATPase are actively involved in CSF production and propose a molecular mode of water transport supporting CSF production in this secretory tissue. Further, we demonstrate that inhibition of NKCC1 directly reduces the ICP, illustrating that altered CSF secretion may be employed as a strategy to modulate ICP. These insights identify new promising therapeutic targets against brain pathologies associated with elevated ICP.

Computational Modelling of Glucose Uptake by SGLT1 and Apical GLUT2 in the Enterocyte

It has been suggested that glucose absorption in the small intestine depends on both constitutively expressed SGLT1 and translocated GLUT2 in the brush border membrane, especially in the presence of high levels of luminal glucose. Here, we present a computational model of non-isotonic glucose uptake by small intestinal epithelial cells. The model incorporates apical uptake via SGLT1 and GLUT2, basolateral efflux into the blood via GLUT2, and cellular volume changes in response to non-isotonic conditions. The dependence of glucose absorption on luminal glucose, blood flow rate, and inlet blood glucose concentration is studied. Uptake via apical GLUT2 is found to be sensitive to all these factors. Under a range of conditions, the maximum apical GLUT2 flux is about half of the SGLT1 flux and is achieved at high luminal glucose (> 50 mM), high blood flow rates, and low inlet blood concentrations. In contrast, SGLT1 flux is less sensitive to these factors. When luminal glucose concentration is less than 10 mM, apical GLUT2 serves as an efflux pathway for glucose to move from the blood to the lumen. The model results indicate that translocation of GLUT2 from the basolateral to the apical membrane increases glucose uptake into the cell; however, the reduction of efflux capacity results in a decrease in net absorption. Recruitment of GLUT2 from a cytosolic pool elicits a 10–20% increase in absorption for luminal glucose levels in the a 20–100 mM range. Increased SGLT1 activity also leads to a roughly 20% increase in absorption. A concomitant increase in blood supply results in a larger increase in absorption. Increases in apical glucose transporter activity help to minimise cell volume changes by reducing the osmotic gradient between the cell and the lumen.

Efficient Screening Tests and Gene Mutation Spectrum for Hereditary Stomatocytosis in Japan

Background: Hereditary stomatocytosis (HSt) is a group of congenital hemolytic anemia caused by abnormally increased cation permeability of erythrocyte membranes. The most common subtype is dehydrated HSt (DHSt), and heterozygous mutations of the mechanosensitive calcium channel gene PIEZO1 have been associated with it most frequently. DHSt is suspected by screening tests such as erythrocyte morphology, cation concentration measurements inside and outside the erythrocyte membrane, or osmotic gradient ektacytometry; target-captured sequencing (TCS) is used for definitive diagnosis. We have shown that an increase in the residual red cells (%RRC) in a quantitative osmotic fragility test using a flow cytometer (FCM-OF) is useful as a screening test for DHSt. Purpose: We report the clinical findings and mutation spectrum of Japanese patients with DHSt confirmed by genetic testing. Methods: From April 2015 to June 2021, 27 patients who had a clinical diagnosis of DHSt and provided written consent were genetically tested. The clinical indications were hemolytic anemia with stomatocytes, accompanied by hemochromatosis, a family history, perinatal edema, and severe jaundice. Laboratory tests showed increased MCV, and subjects with an increased %RRC in FCM-OF were analyzed. TCS was performed using a hemolytic anemia-related gene panel. Results: Of the 27 patients, 14 had PIEZO1 variants, 3 had KCNN4 variants, and 2 had ABCB6 variants, for a total of 19 cases diagnosed as HSt. There was 1 SPTB mutation, 1 GCLC mutation, and 6 cases without mutations in genes known to be related to hemolytic anemia. There were 12 previously reported mutations (KCNN4: R352H, PIEZO1: V598M, T2014I, R2488Q, E2496ELE) and 5 novel mutations (KCNN4: P204R, A279T, PIEZO1: 427_428ins9AA, A1457V, K2323T).Notably, 5 E2496ELE mutations were found in unrelated individuals. There were no differences in age at diagnosis and severity of anemia between the E2496ELE mutation and other PIEZO1 mutations, but jaundice was more severe in patients with the E2496ELE mutation (p=0.007).The median age at diagnosis of the DHSt patients was 28 years (range: 2 months to 89 years); there were 6 men and 11 women. Three patients underwent splenectomy, and 2 patients with PIEZO1 mutations had postoperative thrombosis; 1 KCNN4 mutation had no complications, but no improvement in hemolytic anemia. Six patients had gallstones, 3 had fetal ascites, and 11 received red blood cell transfusions. Laboratory test results showed median Hb 10.4 g/dL (6.9-15.6), median MCV 99.7 fL (85-127.4), median MCHC 35.6% (33-39), and median T-Bil 3.4 mg/dL (0.5-37.9). The median ferritin level was 569.3 ng/mL (87.1-3895); of the 14 patients whose ferritin was measured at the time of diagnosis, 6 had already exceeded 1,000 ng/mL. The FCM-OF showed high values in all 16 cases tested. Discussion: Genetic testing was performed on cases in which DHSt was suspected based on clinical findings, smears, and FCM-OF; the diagnosis of DHSt was confirmed at a high percentage. As previously reported, the severity of hemolytic anemia was wide-ranging, and many cases of hemochromatosis were observed. The PIEZO1 mutation is the most common in the Japanese population, and the number of E2496ELE mutations is particularly conspicuous. Patients carrying E2496ELE mutations are reported to have a younger age at diagnosis and more severe hematological findings than other mutations; however, our results showed no significant differences in age at diagnosis or degree of anemia. Since the correlation between PIEZO1 gene mutation and phenotype has not yet been clarified, further research is considered necessary. Disclosures No relevant conflicts of interest to declare.

Neuronal Swelling: A Non-osmotic Consequence of Spreading Depolarization

An acute reduction in plasma osmolality causes rapid uptake of water by astrocytes but not by neurons, whereas both cell types swell as a consequence of lost blood flow (ischemia). Either hypoosmolality or ischemia can displace the brain downwards, potentially causing death. However, these disorders are fundamentally different at the cellular level. Astrocytes osmotically swell or shrink because they express functional water channels (aquaporins), whereas neurons lack functional aquaporins and thus maintain their volume. Yet both neurons and astrocytes immediately swell when blood flow to the brain is compromised (cytotoxic edema) as following stroke onset, sudden cardiac arrest, or traumatic brain injury. In each situation, neuronal swelling is the direct result of spreading depolarization (SD) generated when the ATP-dependent sodium/potassium ATPase (the Na+/K+ pump) is compromised. The simple, and incorrect, textbook explanation for neuronal swelling is that increased Na+ influx passively draws Cl− into the cell, with water following by osmosis via some unknown conduit. We first review the strong evidence that mammalian neurons resist volume change during acute osmotic stress. We then contrast this with their dramatic swelling during ischemia. Counter-intuitively, recent research argues that ischemic swelling of neurons is non-osmotic, involving ion/water cotransporters as well as at least one known amino acid water pump. While incompletely understood, these mechanisms argue against the dogma that neuronal swelling involves water uptake driven by an osmotic gradient with aquaporins as the conduit. Promoting clinical recovery from neuronal cytotoxic edema evoked by spreading depolarizations requires a far better understanding of molecular water pumps and ion/water cotransporters that act to rebalance water shifts during brain ischemia.

Acquired Decline in Ultrafiltration in Peritoneal Dialysis: The Role of Glucose

Ultrafiltration is essential in peritoneal dialysis (PD) for maintenance of euvolemia, making ultrafiltration insufficiency preferably called ultrafiltration failure—an important complication. The mechanisms of ultrafiltration and ultrafiltration failure are more complex than generally assumed, especially after long-term treatment. Initially, ultrafiltration failure is mainly explained by a large number of perfused peritoneal microvessels, leading to a rapid decline of the crystalloid osmotic gradient, thereby decreasing aquaporin-mediated free water transport. The contribution of peritoneal interstitial tissue to ultrafiltration failure is limited during the first few years of PD, but becomes more important in long-term PD due to the development of interstitial fibrosis, which mainly consists of myofibroblasts. A dual hypothesis has been developed to explain why the continuous exposure of peritoneal tissues to the extremely high dialysate glucose concentrations causes progressive ultrafiltration decline. First, glucose absorption causes an increase of the intracellular NADH/NAD+ ratio, also called pseudohypoxia. Intracellular hypoxia stimulates myofibroblasts to produce profibrotic and angiogenetic factors, as well as the glucose transporter GLUT-1. Second, the increased GLUT-1 expression by myofibroblasts increases glucose uptake in these cells, leading to a reduction of the osmotic gradient for ultrafiltration. Reduction of peritoneal glucose exposure to prevent this vicious circle is essential for high-quality long-term PD.

Angulin-1 (LSR) Affects Paracellular Water Transport, However Only in Tight Epithelial Cells

Water transport in epithelia occurs transcellularly (aquaporins) and paracellularly (claudin-2, claudin-15). Recently, we showed that downregulated tricellulin, a protein of the tricellular tight junction (tTJ, the site where three epithelial cells meet), increased transepithelial water flux. We now check the hypothesis that another tTJ-associated protein, angulin-1 (alias lipolysis-stimulated lipoprotein receptor, LSR) is a direct negative actuator of tTJ water permeability depending on the tightness of the epithelium. For this, a tight and an intermediate-tight epithelial cell line, MDCK C7 and HT-29/B6, were stably transfected with CRISPR/Cas9 and single-guide RNA targeting angulin-1 and morphologically and functionally characterized. Water flux induced by an osmotic gradient using 4-kDa dextran caused water flux to increase in angulin-1 KO clones in MDCK C7 cells, but not in HT-29/B6 cells. In addition, we found that water permeability in HT-29/B6 cells was not modified after either angulin-1 knockout or tricellulin knockdown, which may be related to the presence of other pathways, which reduce the impact of the tTJ pathway. In conclusion, modulation of the tTJ by knockout or knockdown of tTJ proteins affects ion and macromolecule permeability in tight and intermediate-tight epithelial cell lines, while the transepithelial water permeability was affected only in tight cell lines.

Stored water in the inner bark and sapwood: Atypical patterns of daily discharge and recharge, radial osmotic gradient and freezing resistance

Stored water in inner tissues can affect plant water balance and its freezing resistance. We studied the water storages in the inner bark and sapwood of Araucaria araucana, a species with thick inner bark. Specifically, we analyzed its daily behavior, the driving force to radial water movement and its freezing resistance. The whole-stem water content and diameter and sap flow increased in the morning and decreased in the afternoon. An osmotic gradient between stem tissues was involved in the morning water storage recharge. There were no lags in the onset of sap flow between different stem positions, however sap flow at 6m height was higher than basal sap flow in the afternoon, at the time that sapwood water content started to decline followed by the inner bark. Extracellular freezing was delayed down to -6˚C in the inner bark and to -8˚C in the leaves. The unusual diurnal pattern of internal water use may enhance freezing resistance as a consequence of the lower water content and higher osmotic potential when the lowest temperatures occur. The contribution of stem tissues to daily water use and the pattern of ice nucleation observed make this species less susceptible to drought and very low temperatures.

Deletion of Cdh-16 Ksp-cadherin leads to a developmental delay in the ability to maximally concentrate urine in mouse

Ksp-cadherin (Cadherin-16) is an atypical member of the cadherin superfamily of cell adhesion molecules that is ubiquitously expressed on the basolateral membrane of epithelial cells lining the nephron and the collecting system of the mammalian kidney. The principal aim of the present study was to determine if Ksp-cadherin played a critical role in the development and maintenance of the adult mammalian kidney by generating and evaluating a mouse line deficient in Ksp-cadherin. Ksp-null mutant animals were viable and fertile, and kidneys from both neonates and adults showed no evidence of structural abnormalities. Immunolocalization and Western analyses of Na/K-ATPase and E-cadherin indicated that Ksp-cadherin is not essential for either the genesis or maintenance of the polarized tubular epithelial phenotype. Moreover, E-cadherin expression was not altered to compensate for Ksp-cadherin loss. Plasma electrolytes, total CO2, BUN, and creatinine levels were also unaffected by Ksp-cadherin deficiency. However, a subtle but significant developmental delay in the ability to maximally concentrate urine was detected in Ksp-null mice. Expression analysis of the principal proteins involved in the generation of the cortico-medullary osmotic gradient and the resultant movement of water identified misexpression of Aquaporin-2 in the inner medullary collecting duct as the possible cause for the inability of young adult Ksp-cadherin deficient animals to maximally concentrate their urine. In conclusion, Ksp-cadherin is not required for normal kidney development but its absence leads to a developmental delay in maximal urinary concentrating ability.

Aquaporin water channels as regulators of cell-cell adhesion proteins

Aquaporin (AQP) water channels facilitate passive transport of water across cellular membranes following an osmotic gradient. AQPs are expressed in a multitude of epithelia, endothelia, and other cell types where they play important roles in physiology, especially in the regulation of body water homeostasis, skin hydration, and fat metabolism. AQP dysregulation is associated with many pathophysiological conditions, including nephrogenic diabetes insipidus, chronic kidney disease, and congestive heart failure. Moreover, AQPs have emerged as major players in a multitude of cancers where high expression correlates with metastasis and poor prognosis. Besides water transport, AQPs have been shown to be involved in cellular signaling, cell migration, cell proliferation, and in regulation of junctional proteins involved in cell-cell adhesion; all cellular processes which are dysregulated in cancer. This Mini-Review focuses on AQPs as regulators of junctional proteins involved in cell-cell adhesion.