Increase In Osmolality Research Articles

Seasonal Variation in Vascular Dehydration Risk: Insights from the KOBE Study

Abstract Objectives Dehydration, a risk factor for ischemic cerebrovascular diseases, is common in summer; however, the incidence of ischemic diseases is not necessarily higher in summer. The phenomenon may be influenced by the differences in the susceptibility to vascular dehydration based on the season. Therefore, this study aimed to distinguish types of dehydration in colder and warmer seasons by analyzing serum osmolarity, hematocrit, and daily non-alcohol drink (NAD) intake. Methods Participants in the Kobe Orthopedic and Biomedical Epidemiologic (KOBE) Study, consisting of healthy individuals, were categorized into two groups based on the examination month: the warmer and colder seasons. Multivariate analyses were conducted to examine disparities in serum osmolarity, hematocrit, and NAD intake between these two groups. Results Participant ratios by age group and the seasons (warmer season/colder season) were as follows: Women under 50 (35/62), 50-60 (77/126), 60-70 (123/170), over 70 (58/52); Men under 50 (14/17), 50-60 (22/36), 60-70 (57/71), over 70 (55/34). The colder season was found to be negatively correlated with serum osmolarity and NAD intake, but positively correlated with hematocrit, even after adjusting for relevant factors. Age was independently associated with serum osmolarity, but not with hematocrit and NAD intake. Conclusions This study highlights that vascular dehydration is more likely in the colder season despite lower serum osmolarity than the warmer season. Age-related increases in serum osmolarity without a corresponding rise in water intake may contribute to this. These findings emphasize the importance of addressing dehydration in the colder season, particularly in older adults. Key messages • The colder season showed a negative correlation with serum osmolarity and NAD intake but a positive correlation with hematocrit. • Raising awareness about the importance of hydration during winter may be essential.

Hyperosmotic Stress Induces the Expression of Organic Osmolyte Transporters in Porcine Intestinal Cells and Betaine Exerts a Protective Effect on the Barrier Function.

Background/objectives: The porcine intestinal epithelium plays a fundamental role as a defence interface against pathogens. Its alteration can cause severe inflammatory conditions and diseases. Hyperosmotic stress under physiological conditions and upon pathogen challenge can cause malabsorption. Different cell types counteract the osmolarity increase by accumulating organic osmolytes such as betaine, taurine, and myo-inositol through specific transporters. Betaine is known for protecting cells from hyperosmotic stress and has positive effects when fed to pigs. The aim of this study is to demonstrate the modulation of osmolyte transporters gene expression in IPEC-J2 during osmolarity changes and assess the effects of betaine. Methods: IPEC-J2 were seeded in transwells, where differentiate as a polarized monolayer. Epithelial cell integrity (TEER), oxidative stress (NO) and gene expression of osmolyte transporters, tight junction proteins (TJp) and pro-inflammatory cytokines were evaluated. Results: Cells treated with NaCl hyperosmolar medium (500 mOsm/L) showed a TEER decrease at 3 h and detachment within 24 h, associated with an osmolyte transporters reduction. IPEC-J2 treated with mannitol hyperosmolar medium (500 mOsm/L) upregulated taurine (TauT), myo-inositol (SMIT) and betaine (BGT1) transporters expression. A decrease in TJp expression was associated with a TEER decrease and an increase in TNFα, IL6, and IL8. Betaine could attenuate the hyperosmolarity-induced reduction in TEER and TJp expression, the NO increase and cytokines upregulation. Conclusions: This study demonstrates the expression of osmolyte transporters in IPEC-J2, which was upregulated upon hyperosmotic treatment. Betaine counteracts changes in intracellular osmolarity by contributing to maintaining the epithelial barrier function and reducing the inflammatory condition. Compatible osmolytes may provide beneficial effects in therapies for diseases characterized by inflammation and TJp-related dysfunctions.

Acute freshwater CO2 exposure does not impair seawater transfer in three different sizes of Atlantic salmon (Salmo salar) subjected to different photoperiod manipulations.

There is a growing interest in Atlantic salmon (Salmo salar) aquaculture to extend the time fish are reared in freshwater (FW) recirculating aquaculture systems (RAS), producing larger FW salmon that can then be induced to undergo smoltification before transfer into marine net pens for grow-out and harvest. Smolts can be produced by photoperiod (PT) manipulation in RASs, but little is known about how delaying smoltification to larger body sizes affects susceptibility to elevated CO2 levels (hypercapnia), which can occur at high stocking densities in FW RAS or during transport from FW RAS rearing facilities to marine net pens. To address this, Atlantic salmon were reared from hatch to one of three different sizes (~230, ~580, or ~1300 g) in FW (3 ppt) under continuous light (24:0, light:dark). Once fish reached the desired sizes, a group of salmon were maintained on continuous light 24L:0D to serve as a control salmon. A second group of salmon were exposed to 8 weeks of 12L:12D and then to 4 weeks of 24L:0D to serve as PT treatment salmon, which is the PT manipulation commonly used in Atlantic salmon aquaculture to induce smoltification. At the end of PT manipulation, both control and PT treatment salmon were exposed to 0% or 1.5% CO2 (30 mg/L) for 96 h in FW and then transferred to air-equilibrated seawater (SW, 35 ppt, normocapnia). Salmon were sampled at the end of the 96-h FW CO2 exposure and at 24 h and 7 days in SW for measurements of blood ion/acid-base status, muscle water content (MWC), and gill and kidney Na+/K+ ATPase (NKA) activity. Exposure to 96 h of CO2 in FW resulted in acid-base disturbances in fish from all three size classes, with decreases in blood pH and increases in blood PCO2 and plasma [HCO3 -] but no mortality. Despite these large acid-base disturbances in FW, after transfer to normocapnic SW, there were no significant effects of CO2 exposure on extracellular blood pH, intracellular red blood cell pH, or plasma osmoregulatory status for all three sizes of post-smolt salmon. In general, SW transfer was associated with significant increases in plasma ions and osmolality, as well as gill and kidney NKA activity after 24 h and 1 week in SW with no significant impacts between different sizes of salmon. Thus, exposure to 30 mg CO2/L that mimics levels experienced during transport from FW RAS to an SW transfer site may have minimal effects on Atlantic salmon smolts up to 1300 g.

6616 Copeptin Use To Reduce Hospital Stay In A Complicated DI Case

Abstract Disclosure: S.S. Kowsika: None. H. Moran: None. Introduction: While there is literature describing the use of copeptin for the diagnosis of polyuria-polydipsia syndrome, its utilization remains very limited in many hospitals. We present a case where the patient, initially misdiagnosed with central diabetes insipidus (DI), was appropriately diagnosed with partial nephrogenic DI through the use of copeptin. Case: A 48-year old female with a history of cardiac arrest, anoxic brain injury and diabetes insidious (DI) presented to the emergency department with hypernatremia (Na 154 mmol/L). The patient had been initiated on desmopresin (DDAVP) at an outside facility prior to her current admission. Despite receiving high doses of DDAVP, her sodium levels remained elevated, prompting further investigation. DDAVP administration was discontinued. A water deprivation test was subsequently performed, followed by IV administration of 2 mcg of DDAVP, which resulted in an increase in urine osmolality from 191 to 331 mosm/kg, which was less than the expected 50% increase observed in central DI. Additionally, a baseline copeptin level was obtained and noted to be 31 pmol/L, confirming the diagnosis of partial nephrogenic DI. The patient was started on Amiloride, leading to normalization of sodium levels. Discussion: Many patients with polyuria-polydipsia syndrome are frequently misdiagnosed and receive incorrect treatment, potentially worsening their clinical condition. Copeptin (Carboxy-Terminal-Pro-vasopressin) is the C-terminal peptide of pro-vasopressin, co-secreted with arginine vasopressin (AVP). It has been proposed as a direct test for diagnosing polyuria-polydipsia syndrome. Baseline Copeptin testing without stimulation can accurately diagnose nephrogenic DI if elevated (>21.4, pmol/L). To differentiate between central DI and chronic polyuria, prior thirsting with hypertonic saline or glucagon stimulation is necessary, with stimulated copeptin <4.9 pmol/L diagnosing central DI. Despite its diagnostic potential, Copeptin is underutilized in many settings. In this case, using Copeptin to accurately diagnose our patient led to appropriate treatment and the resolution of hypernatremia. Presentation: 6/1/2024

8155 Isolated Hypothalamic-Pituitary Langerhans' Cell Histiocytosis in Adult Patients

Abstract Disclosure: A. Vu: None. M. Aguilera: None. J. Shakil: None. R. Al-Ward: None. Introduction: Langerhans cell histiocytosis (LCH) is an infiltrative disease due to clonal proliferation of immature dendritic cells mainly affecting children and seen in adults in less than 30% of cases. It can present as localized or with systemic manifestations, with isolated hypothalamic-pituitary lesions being exceptionally rare. Our report adds to the limited published literature by presenting two cases of isolated hypothalamic-pituitary LCH diagnosed in adults. Furthermore, one of the cases reveals a rare association with papillary thyroid cancer (PTC). Case 1: 35-year-old woman with no past medical history presented with 4 year history of amenorrhea and weight gain and more recently, polyuria and polydipsia. Further investigations showed hypogonadotropic hypogonadism, central hypothyroidism, and mildly elevated prolactin. She couldn’t complete water deprivation test due to extreme thirst. A dedicated pituitary MRI showed marked infundibular enlargement, concerning for hypophysitis. An extensive workup including assessments for autoimmune etiology, sarcoidosis, infections, and low-grade tumors, did not identify a definitive etiology. A PET scan was obtained (to rule out LCH and Erdheim Chester disease), which showed increased uptake in the pituitary stalk. Due to otherwise negative workup, transsphenoidal biopsy of the pituitary lesion was done. Post-procedure, diabetes insipidus (DI) was diagnosed due to elevated sodium while fasting for the procedure. Tissue examination revealed increased histiocytes and lymphocytes, and a positive BRAF V600E mutation confirming LCH as the underlying diagnosis. Case 2: 26-year-old male with history of LCH limited to the skull, in remission for 8 years after chemotherapy, presented with 4-week history of polyuria, polydipsia and weight loss. A 24-hour urine collection resulted in 6.5L of urine. During water deprivation test, there was notable increase in serum sodium, serum osmolarity, and urine osmolarity. Initial pituitary MRI was normal, but subsequent imaging revealed the loss of the posterior pituitary bright spot. PET scan showed focal FDG avidity in the right lower thyroid lobe, confirmed on fine needle aspiration as PTC with BRAF mutation. He underwent total thyroidectomy and is currently receiving chemotherapy for LCH recurrence. Discussion: LCH is infrequent in adults, presenting with variable manifestations based on affected organs. In the multifocal form, the hypothalamic-pituitary axis is involved in 5-50% of cases. Our report describes two unique cases with only four other reported cases of isolated hypothalamic-pituitary axis LCH in adults. Central diabetes insipidus can be seen before the diagnosis or as the disease progresses. Pituitary biopsy, although invasive, should be considered when the diagnosis is not clear. The association of PTC with a BRAF mutation is extremely rare and has been reported. Presentation: 6/2/2024

Effect of Human Milk Fortifiers, Medium-chain Triglyceride Oil, Minerals, and Vitamin Supplements on the Osmolality of Pasteurized Donor Human Milk

Aim: We aimed to study the changes in osmolality of pasteurized donor human milk (PDHM) after the addition of four human milk fortifiers (HMFs), medium-chain triglyceride (MCT) oil, minerals, and vitamin supplements commonly used in neonates. Methods: The osmolality of 25 ml of PDHM was measured after the addition of 1 g each of HMF and 0.5 ml of MCT oil separately with a calibrated osmometer. The osmolality of 5 ml of PDHM was measured after the addition of minerals and vitamin supplements in therapeutic doses separately. All these measurements were carried out at 10 min, 30 min, and 2 h by a technician who was blinded to the fortifiers, minerals, and vitamin supplements used. Results: The osmolality of PDHM was 256 mOsm/kg (range = 256–259 mOsm/kg), which increased with all four HMFs tested, but remained below 450 mOsm/kg as recommended by the European Society for Paediatric Gastroenterology Hepatology and Nutrition (ESPGHAN). Proprietary fortifiers increased the osmolality of PDHM to a maximum of 429 mOsm/kg (range = 306–429 mOsm/kg). However, there was no change in the osmolality with MCT oil. A maximum increase in osmolality was observed with the addition of calcium phosphate syrup to PDHM (1181 mOsm/kg). The addition of multivitamins and Vitamin D3 in therapeutic doses increased osmolality (range = 552–753 mOsm/kg) beyond the safety limits of ESPGHAN recommendation. Conclusions: HMFs increased the osmolality of PDHM but were within the safety limits of ESPGHAN recommendation. MCT oil did not affect the osmolality of PDHM. Minerals and vitamin supplements added in therapeutic dosages significantly increased the osmolality of PDHM. Minerals and vitamin supplements need to be appropriately diluted to keep osmolality below 450 mOsm/kg to ensure safety. Future research should focus on manufacturing fortifiers and supplements with low osmolality to ensure safety.

Characterization of luminal contents from the fasted human proximal colon

To treat colonic diseases more effectively, improved therapies are urgently needed. In this respect, delivering drugs locally to the colon is a key strategy to achieve higher local drug concentrations while minimizing systemic side effects. Understanding the luminal environment is crucial to efficiently develop such targeted therapies and to predict drug disposition in the colon. In this clinical study, we collected colonic contents from an undisturbed fasted proximal colon via colonoscopy and characterized their composition with regard to drug disposition. Colonic pH, osmolality, protein content, bile salts, lipids, phospholipids and short-chain fatty acids were investigated in 10 healthy volunteers (8 male and 2 female, age 19–25). The unique environment of the proximal colon was reflected in the composition of the sampled luminal fluids and the effect of the microbiota could be observed on the pH (median 6.55), the composition of bile salts (majority deconjugated and secondary), and the abundance of short-chain fatty acids. At the same time, an increase in phospholipid concentration, osmolality and total protein content compared to reported ileal values was seen, likely resulting from desiccation. Lipids could only be found in low quantities and mainly in the form of cholesterol and free fatty acids, showing almost complete digestion and absorption by the time luminal contents reach the colon. All characteristics also displayed the considerable intersubject variability found in different regions of the gastrointestinal tract. This study contributes to an improved understanding of the luminal conditions in the proximal colon and facilitates the development of new predictive tools to study colonic drug absorption.

#1962 Development of triple-whammy induced pre-renal AKI is highly dependent on the hydration status

Abstract Background and Aims The concomitant use of diuretics, non-steroidal anti-inflammatory drugs (NSAIDs), and angiotensin system inhibitors is known as Triple Whammy (TW) and significantly increases the risk of acute kidney injury (AKI) due to a reduction in the glomerular filtration rate. Maintenance of hydration contributes to correct kidney perfusion, and effective renal haemodynamic regulation allows the kidneys to cope with everyday stressors and challenges, such as TW therapy. Interference with these adaptive mechanisms frequently occurs due to extrinsic (environmental conditions, lifestyle, drugs) or intrinsic (comorbidities, ageing) factors and may lead to dehydration, a negative body liquid balance with a high impact on health. Identifying modifiable risk factors that pose individuals at high risk of AKI following TW therapy is key for the search and prescription of preventive strategies in a personalized manner. The aim of this work was to study the role of hydration status on the renal effect of TW. Method Two-month-old male Wistar rats were divided into 3 experimental groups: Blood and 24-hour urine samples were collected at basal state (B), two days after water deprivation (D0), and two days after TW therapy (D2). Body weight was monitored during the experiment. Haematocrit was assessed from blood samples, and osmolality, concentration of creatinine (Jaffe reaction), and urea (Jung method) were determined in plasma. Urinary flow was recorded, and specific gravity, osmolality and creatinine concentration were measured in urine samples. Results Dehydration was observed after 48 h water deprivation, evidenced by a 12% weight loss and significant increases in haematocrit and plasma osmolality. As expected, during this dehydrated state rats excreted a very reduced volume of a highly concentrated urine with elevated levels of specific gravity and osmolality. No changes in renal function were observed after 48 h water deprivation, nor after TW treatment in fully hydrated rats. Administration of TW triggered significant alterations of the renal function, indicative of AKI, only in rats previously deprived of water for 48 h. These alterations were markedly increased when dehydration was partially sustained during the TW treatment by a 60% water restriction. Conclusion TW-induced AKI in young Wistar rats is facilitated when the hydration status worsens. In this sense, assessing hydration and implementing (re)hydration strategies may help to reduce or prevent TW-induced AKI. This study was supported by Project PI21/01226, funded by Instituto de Salud Carlos III (ISCIII) and co-funded by the European Union, and a grant from the Conserjería de Educación, Junta de Castilla y León (IES160P20), Spain, co-funded by FEDER funds. Noelia Diaz-Morales is recipient of a Juan de la Cierva-Formación postdoctoral contract (FJC2020-043205-I) funded by MCIN/AEI/10.13039/501100011033 and European Union “NextGenerationEU/PRTR”.

#1408 Dehydration cannot substitute for any of the triple whammy drugs to cause acute kidney injury

Abstract Background and Aims Polymedication in patients with chronic diseases, such as hypertension, can lead to undesired drug interactions. The combination of angiotensin-converting enzyme inhibitors (ACEIs) or angiotensin II receptor blockers (ARBs) with diuretics and non-steroidal anti-inflammatory drugs (NSAIDs) can cause triple whammy (TW)-induced acute kidney injury (TW-AKI). Up to 9% of hypertensive patients used NSAIDs in combination with ACEIs / ARBs and diuretics. AKI has a mortality rate of 40% and TW increases the risk of AKI by 30% compared to the combination of only two drugs. In addition, TW-AKI is associated with other risk factors such as dehydration, which may exacerbate the injury. Consequently, the aim of our study was to determine whether dehydration combined with two of the TW drugs could induce AKI in an in vivo experimental model. Method Three months old male Spontaneously Hypertensive Rats (SHR) were divided into four experimental groups: control group (CT); TW group (TW), which received four days of double therapy with trandolapril and furosemide, followed by a triple therapy for two days with trandolapril, furosemide and ibuprofen; trandolapril and ibuprofen group (T+I), which received four days of trandolapril followed by two days of trandolapril and ibuprofen; furosemide and ibuprofen group (F+I), which received four days of furosemide followed by two days of furosemide and ibuprofen; and trandolapril and furosemide group (T+F), which received six days of trandolapril and furosemide. All groups were subjected to a 60-70% water restriction of basal intake (i.e. 15 mL/day) to generate mild dehydration. Blood and urine samples were collected at baseline (B), day 4 and day 6. Blood pressure was measured at baseline and day 4. Renal function was assessed by plasma creatinine, plasma urea and creatinine clearance, and hydration status was determined by plasma osmolarity and water balance (i.e. water intake minus urine flow). Results Under basal conditions, all rats were hypertensive. The combination of trandolapril and furosemide significantly reduced the mean blood pressure on day 4 (p = 0.0066), while trandolapril or furosemide alone did not achieve these decreases. After four days of water restriction to 15 mL/day, all groups showed mild dehydration with a decrease in water balance and an increase in plasma osmolarity by. Plasma creatinine and plasma urea values were only increased on day 6 in the TW group (p = 0.0009 and p = 0.02, respectively). Creatinine clearance was also clearly reduced in the TW group (p = 0.0004). F+I and T+F groups showed slight decreases in creatinine clearance at day 6, as well as slight increases in plasma urea. However, these changes were not as pronounced as those observed in the TW group. Conclusion In our in vivo model of TW-AKI, mild dehydration cannot replace any of the TW drugs, although it appears that combination of F+I and T+F in dehydrated rats slightly alter plasma urea and creatinine clearance values at day 6, suggesting that this condition maintained over time might lead to the development of AKI. Funding This research was funded by grants from Instituto de Salud Carlos III (ISCIII), Ministerio de Ciencia e Innovación (PI21/01226 and PI21/00548 co-funded by the European Union; and RICORS2040, RD21/0005/0004, co-funded by the European Union—NextGenerationEU, Mecanismo para la Recuperación y la Resiliencia (MRR)) and from Consejería de Educación, Junta de Castilla y León (IES160P20), co-funded by FEDER funds. Noelia Diaz-Morales is recipient of a Juan de la Cierva-Formación postdoctoral contract (FJC2020-043205-I) funded by MCIN/AEI/10.13039/501100011033 and the European Union “NextGenerationEU/PRTR”. Eva M. Baranda-Alonso is recipient of a predoctoral fellowship from the Junta de Castilla y Leon (Spain) and the European Social Fund from the European Commission.

WNK1 is a central osmolality sensor for arginine vasopressin release and acts through the OSR1/SPAK kinase cascade

Background: Maintaining internal osmolality constancy and water homeostasis is essential for life. The circumventricular organs (CVOs) of the brain, including the organum vasculosum of the lamina terminalis (OVLT) and subfornical organ (SFO), lack a blood-brain barrier. Neurons in OVLT and SFO detect increases in serum osmolality that stimulate the production of vasopressin (AVP) in paraventricular nuclei (PVN) to be released in the posterior pituitary. We have recently reported that WNK1 in sensory neurons in CVOs functions as an osmolality sensor for AVP release (Jin et al., JCI, 2023). WNK1 activates Kv3.1 to increase action potential, traveling down to PVN to increase AVP synthesis for release from the posterior pituitary. Here, we propose that OSR1/SPAK acts as the downstream kinase for WNK1 in regulating AVP release. We further investigated the regulation of vasopressin release by the WNK1-OSR1/SPAK cascade in response to extracellular hyperosmolality. Methods: Cre recombinase-carrying retrograde AAV virus was stereotaxically injected into PVN nuclei from normal mice and SPAK-null mice with a homozygous OSR1-floxed allele (SAPK−/−;OSR1flox/flox). Metabolic cage studies were performed on mice. Urine output, water intake, serum osmolality, serum AVP, and copeptin levels were measured. K+ currents were measured from the OVLT neurons by whole-cell patch clamp. Results: Increased osmolality, either by water restriction or mannitol injection, activated OSR1/SPAK in OVLT, as evident by increased serine-373 phospho-SPAK and serine-325 phospho-OSR. Like the WNK1 deletion, the double deletion of OSR/SPAK in OVLT caused polyuria with decreased urine osmolality that persisted in water restriction. Circulating levels of AVP and copeptin were increased by water restriction in control mice. In contrast, water restriction failed to increase the levels in OSR/SPAK-deletion mice. Knockdown of the Kv3.1 channel in OVLT by shRNA showed the central diabetes insipidus (DI) phenotype as well. Hypertonicity increased K+ currents and WNKs inhibitor prevented the increase in OVLT neurons. Conclusions: Mice with neuronal-specific OSR1/SPAK deletion have water homeostasis defects consistent with partial central DI; Evidence supporting that OSR1/SPAK acts downstream of WNK1 to regulate AVP release; WNK1-OSR1/SPAK activates Kv3.1 to increase firing frequency of neurons projecting from CVOs to PVN. Our findings reveal that an intracellular protein acts as a sensor for extracellular tonicity and provide fresh insights into the mechanisms by which the body maintains osmolality constancy. Future studies will investigate how OSR1/SPAK regulates Kv3.1 channels. NIDDK. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Evaluation of therapeutic efficacy of Emustil drops for ocular discomfort and tear film osmolarity using different treatment management modes under dry environmental conditions

BackgroundWe aimed to check the efficacy of Emustil (oil in water emulsion) drops on tear film index and ocular surface dynamics in dry environments through protection and relief treatment modalities.MethodsThe subjects were exposed to a dry environment using a Controlled Environment Chamber (CEC) where the relative humidity (RH) was 5% and the temperature was 21 °C and screened for ocular symptoms, tear osmolarity, ocular surface temperature (OST) and tear production using ocular Surface Disease Index questionnaire (OSDI), OcuSense TearLab Osmometer, FLIR System ThermaCAM P620 and Schirmer strips/phenol red test respectively. Tear production was calculated by the Tear Function Index test (TFI).ResultsThe mean tear film osmolarity decreased significantly from 296.8 mOsm/l at 40% RH to 291 mOsm/l at 5%. (p = 0.01). Instillation of Emustil resulted in a significant increase in tear osmolarity in the relief method compared with osmolarity seen at 5% RH when no drop was used. The mean PRT value decreased from 26 ± 9 in normal conditions (40% RH) to 22 ± 4 mm in dry conditions (5% RH). Emustil drops did not induce any significant change in tear production in the PRT test. No significant change was found in OST following exposure to 5% RH. OST did not show a statistically significant change with the emulsion when used for relief (p > 0.05). The mean score of ocular discomfort observed was 70 at 5% RH. Still, the instillation of the oil-in-water emulsion (Emustil) resulted in a noticeable decrease in visual discomfort to 37 (p = 0.00) in protection and 59 in relief (p = 0.05). Emustil drops substantially improved tear film parameters under a desiccating environment, however, tear film parameters respond differently to the management modalities. In the protection method, tear film osmolarity was protected against a dry environment, while in the relief mode, tear production was improved.ConclusionCEC allows for a thorough evaluation of tear film parameters and dry eye treatment protocols in labs, providing greater confidence when applying them to patients. In addition, our study showed that Emustil not only provides protection and relief for dry eyes but also helps to maintain ocular homeostasis in desiccating environments. This indicates a promising potential for improving dry eye treatment protocols.

Immediate Impact on Tear Osmolarity Following the Application of Different Ocular Lubricants in Patients Experiencing Moderate Dry Eye Symptoms.

This study addresses the growing concern of Dry Eye Disease (DED), which has become increasingly prevalent due to modern lifestyles characterized by prolonged screen usage, global warming, polypharmacy, and extended life expectancy. Grounded in the Dry Eye Workshop II (DEWSII) diagnosis framework, the study focuses on DED as a multifactorial condition affecting the ocular surface's tear film homeostasis. The study evaluates the short-term impact of 5 commercially available ocular lubricants on disrupting the hyperosmolar environment and determine whether these lubricants can offer potential treatment benefits for DED. Conducted on 300 eyes (from 150 patients) with 5 preservative-free lubricants compared to a control group, the study reveals that all lubricants effectively reduced tear film osmolarity within 15 minutes of application. Notably, the control group exhibited an increase in average osmolarity (+0.98 mOsm/L) without lubricant use. Siccafluid demonstrated the most substantial osmolarity reduction after 15 minutes, with an average decrease of 11.54 mOsm/L. Statistical significance was observed for Siccafluid, Optive Fusion unique dose (UD), and Systane Ultra UD, while Hyabak and Freegen preservative free (PF) showed lower significance. Emphasizing the importance of disrupting the hyperosmolar environment to break the cycle of inflammation, the study concludes that ocular lubricants, at least as an immediate post-application effect, can interrupt this cycle and improve the hyperosmolar environment of the ocular surface.

A43 MECHANISMS OF UNIQUE COLONIZATION DYNAMICS AND IMMUNE RESPONSES TO MURIBACULACEAE

Abstract Background Commercially available probiotics have a limited ability to alter the gut microbiome and establish within a pre-existing microbial community. Increases in osmolality, a condition prevalent in intestinal diseases, depletes the highly abundant bacterial family Muribaculaceae (Mb) from the gut microbiome in a mouse model of short-term osmotic laxative use. Excitingly, despite microbial community re-equilibration in its absence, Mb supersedes other bacterial colonizers to its original abundance if reintroduced, suggesting unique colonization attributes and a potential privileged immune interaction. Aims We aimed to investigate whether and how Mb colonization affects intestinal innate and adaptive immune homeostasis, including the generation of Mb-specific antibodies. Methods To examine interactions between Mb and the immune system that allows for efficient colonization, we used the osmotic laxative polyethylene glycol (PEG) to clear Mb and then generated mice with complex microbiomes that either lack Mb completely (Mb naïve) or were introduced to 8 murine-derived Mb strains (Mb+). The same strains were then introduced to Mb naïve mice (primary exposure to the immune system). To model secondary exposure, Mb+ mice were treated with PEG, and Mb re-introduced 3 weeks later. Leukocytes from the small and large intestinal lamina propria and gut-draining mesenteric lymph nodes (mLNs) were profiled over 21 days to characterize colonization and recolonization dynamics. Results High-throughput 16SrRNA sequencing showed no significant difference between relative abundance of Mb isolates during colonization or recolonization, indicating that Mb is capable of establishing in mice that have never been colonized with it before, making up to 20% of the relative abundance. Broad immunophenotyping demonstrated robust CD11b-CD103+ dendritic cell and Germinal Centre B cell responses in the mLNs of mice seeing Mb for the first time, yet no subsequent plasmablast accumulation in the mLN or lamina propria. We also observed significantly increased type two innate lymphoid cells in the small intestine upon primary exposure to Mb. Recolonization elicited no detectable changes in the monitored populations. Conclusions The lack of immune reactivity upon reintroduction to Mb suggests Mb may evade immune detection upon repeat exposure and contribute to its robust colonization abilities. Determining mechanisms involved in Mb colonization and its impact on host immune responses could increase our understanding of colonization dynamics and highlights the potential of Mb as a model for microbiota reintroduction in therapeutic applications. Funding Agencies Weston Family Foundation

Characterizing the Robustness of Distinct Clinical Assessments in Identifying Dry Eye Condition of Animal Models

Purpose The study aims to characterize the robustness of distinct clinical assessments in identifying the underlying conditions of dry eye disease (DED), with a specific emphasis on the involvement of conjunctival goblet cells. Methods Seven rabbits receiving surgical removal of the lacrimal and Harderian glands were divided into two groups, one with ablation of conjunctival goblet cells by topical soaking of trichloroacetic acid (TCA) to the bulbar conjunctiva (n = 3) and one without (n = 4), and the conditions of DED were assessed weekly using Schirmer test, tear breakup time (TBUT), tear osmolarity, and National Eye Institute (NEI) fluorescein staining grading. After 8 weeks, the rabbits were sacrificed, and the eyes were enucleated for histopathological examination. Results Histopathological analysis revealed corneal epithelial thinning in both groups. While TCA soaking significantly decreased the density of conjunctival goblet cells, DED rabbits without TCA also showed a partial reduction in goblet cell density, potentially attributable to dacryoadenectomy. Both groups showed significant decreases in Schirmer test and TBUT, as well as an increase in tear osmolarity. In DED rabbits with TCA soaking, tear osmolarity increased markedly, suggesting that tear osmolarity is highly sensitive to loss and/or dysfunction of conjunctival goblet cells. Fluorescein staining was gradually and similarly increased in both groups, suggesting that fluorescein staining may not reveal an early disruption of the tear film until the prolonged progression of DED. Conclusion The Schirmer test, TBUT, tear osmolarity, and NEI fluorescein grading are distinct, yet complementary, clinical assessments for the evaluation of DED. By performing these assessments in definitive DED rabbit models, both with and without ablation of conjunctival goblet cells, the role of these cells in the homeostasis of tear osmolarity is highlighted. Characterizing the robustness of these assessments in identifying the underlying conditions of DED will guide a more appropriate management for patients with DED.

Continuous Renal Replacement Therapy During Long-term Normothermic Machine Perfusion of Human Donor Livers for up to 7 D.

Normothermic machine perfusion (NMP) is used to preserve and test donor livers before transplantation. During NMP, the liver is metabolically active and produces waste products, which are released into the perfusate. In this study, we describe our simplified and inexpensive setup that integrates continuous renal replacement therapy (CRRT) with NMP for up to 7 d. We also investigated if the ultrafiltrate could be used for monitoring perfusate concentrations of small molecules such as glucose and lactate. Perfusate composition (urea, osmolarity, sodium, potassium, chloride, calcium, magnesium, phosphate, glucose, and lactate) was analyzed from 56 human NMP procedures without CRRT. Next, in 6 discarded human donor livers, CRRT was performed during NMP by integrating a small dialysis filter (0.2 m2) into the circuit to achieve continuous ultrafiltration combined with continuous fluid substitution for up to 7 d. Within a few hours of NMP without CRRT, a linear increase in osmolarity and concentrations of urea and phosphate to supraphysiological levels was observed. After integration of CRRT into the NMP circuit, the composition of the perfusate was corrected to physiological values within 12 h, and this homeostasis was maintained during NMP for up to 7 d. Glucose and lactate levels, as measured in the CRRT ultrafiltrate, were strongly correlated with perfusate levels (r = 0.997, P < 0.001 and r = 0.999, P < 0.001, respectively). The integration of CRRT into the NMP system corrected the composition of the perfusate to near-physiological values, which could be maintained for up to 7 d. The ultrafiltrate can serve as an alternative to the perfusate to monitor concentrations of small molecules without potentially compromising sterility.

Osmoregulatory Plasticity in Oreochromis niloticus-Nile Tilapia, and it’s Possible Estuary Use for Rivers Dispersion

Nile tilapia is a widely farmed species globally, due to its resistance to environmental challenges. Additionally, it is a generalist consumer and a prolific breeder. In Brazil, this fish is recognized as an invasive species because of its high dispersion and establishment rates, which compromise native biota. Estuarine waters (0.06 - 32‰) are being used in tilapia aquaculture, which is a promisor fish farming for euryahalinity species. The tilapia’s euryhalinity is well-know, but most protocols are based on gradual exposure. This research showed the tolerance of Nile tilapia on abrupt exposure to 0‰ (FW), and 10, 20, 30, 35 or 40‰, over in 6, 12 and 24 hours. An abrupt exposure resistance demonstrate that this fish can use estuaries to disperse for other watersheds. After experimental design the blood sample was taken after anesthesia was administered. Subsequently, fish was euthanized by spinal section, for muscle and gills samples collections. All samples were keep freezing until ionic, dehydration and enzyme measurement. Nile tilapia survived 24h until 30‰, 12h at 35‰ and 9h at 40‰. Osmolality increase and muscle dehydration decrease both above 20‰. While sodium levels keep regulated in all salinities, chloride, potassium and magnesium levels also increased above 20‰. These results prove that tilapia can easily tolerate abrupt exposure until 20‰ without an osmotic stressor. Gill carbonic anhydrase (C.A.) activity decreased at 30‰, and NKA did not change along salinities and times. These results complements plasma and dehydration averages, showing the large tolerance of this specie to salinity challenges, even in abrupt exposure. Then, these results can be used for discuss about the estuary fish farming system for tilapia, as this species may use estuarine waters to nearby rivers and also can harm the native ecosystem.

Corneo‐conjunctival sensory neuron: Types, transduction channels and codification of nerve impulses

The cornea and the conjunctiva are innervated by the peripheral projections of a small number of primary sensory neurons whose somata are located at the ophthalmic region of the trigeminal ganglion. Most trigeminal neurons innervating the ocular surface are small and medium size, and contain neuropeptides such as SP and CGRP. Functionally, ocular surface neurons have been classified into five main neuronal types, based on their response to multiple modalities of stimuli (mechanical forces, heat, cold, H+, chemical substances, etc.). Polymodal nociceptors (activated by several modalities of stimulus, mechanical energy, heat, H+, and chemical irritants), mechanonociceptors (activated by mechanical energy), cold thermoreceptors (activated by temperature reductions and hyperosmolarity) are found in the cornea, the sclera and the conjunctiva. Besides, low‐threshold mechanoreceptors (activated by mechanical forces of low intensity) are found in the corneoscleral limbus and the lid border, and pruriceptors (activated by pruritogen substances) have been described in the tarsal conjunctiva, close to the lid margin.The ability to respond to the different modalities of stimulus lies in the types of transducing channels that each type of neuron expresses at its peripheral nerve endings. Sensory transduction involves the conversion of the physical or chemical stimuli acting on the nerve terminal into electrical energy, that is, into a local change of membrane potential that, if it is large enough, will generate action potentials that will travel along the axon to be processed by the CNS. So that, nerve terminals with TRPV1 channels are directly gated by heat, H+, and several chemical irritants such as capsaicin; nerve terminals with Piezo2 channels will be activated by mechanical forces; TRPM8 channels provide nerve terminals sensitivity to cold and osmolarity increases; et cetera.Ocular sensory neurons also express other ion channels that contribute to coding, that is to help the sensory neuron to establish a correspondence between the characteristics of the stimulus (intensity, temporal course, etc.) and specific parameters of the firing it induces. Voltage‐gated Ca2+, Na+ and K+ channels as well as cyclic nucleotide‐gated channels, among others, contribute to define the firing frequency and pattern to encode the traits of the stimulus. Thus, the ion channels present in the nerve terminals of each class of ocular sensory neuron provide the neurons the ability to detect and encode one or several modalities of stimulus, which constitutes the origin of the different sensations evoked on the ocular surface.(Supported by PID2020‐115934RB‐I00 from DOI: MCIN/AEI/10.13039/50110001103, and CIPROM/2021/48 from the Generalitat Valenciana, Spain)

Seasonal variation in vascular dehydration risk: insights from the Kobe Orthopedic and Biomedical Epidemiologic (KOBE) study.

Dehydration, a risk factor for ischemic cerebrovascular diseases, is common in summer; however, the incidence of ischemic diseases is not necessarily higher in summer. Therefore, this study aimed to clarify the relationships between serum osmolarity, hematocrit, daily non-alcohol drink (NAD) intake and factors such as season and age as risk factors for dehydration. Participants (703 women and 306 men) in the follow-up survey, in 2012 and 2013, of the Kobe Orthopedic and Biomedical Epidemiologic (KOBE) Study, consisting of healthy individuals living in Kobe, Japan, were categorized into two groups based on the examination month: the warmer and colder seasons. Multivariate analyses were conducted to examine disparities in serum osmolarity, hematocrit, and NAD intake between these two groups. The colder season was found to be negatively correlated with serum osmolarity and NAD intake, but positively correlated with hematocrit, even after adjusting for relevant factors. Age was independently associated with serum osmolarity, but not with hematocrit and NAD intake. This study suggests that intra-vascular volume depletion is more likely in the colder season despite lower serum osmolarity compared to the warmer season. Age-related increases in serum osmolarity without a corresponding rise in water intake may contribute to this. These findings support the importance of addressing dehydration in the colder season, particularly in older adults.

Metabolites, ions, and the mechanisms behind seasonal cold hardening of Pyrochroa coccinea (Pyrochroidae) larvae

The larvae of the black headed cardinal beetle Pyrochroa coccinea, overwinters under the bark of dead logs in northern European dioecious forests, and are thus exposed to temperatures below the melting point of their bodily fluids. Here we explore the mechanisms behind their seasonal cold hardening by characterising field samples collected monthly throughout the year. Both the lower lethal temperature and supercooling point dropped as much as 10℃ in the second half of November, reaching values around –15℃ by the beginning of December. This change was accompanied by a 320 mosmol/kg increase in hemolymph osmolality, which is a doubling compared to the summer levels. We used NMR metabolomics to identify and measure the absolute concentrations of the responsible cryoprotective C-H containing metabolites in the hemolymph. The largest increase was found to be in either glucose or trehalose, with an average total increase of 120 mM. Proline, alanine, and choline concentrations were found to increase by around 10 mM each. Contrarily, phosphocholine and phosphoethanolamine were halved, resulting in a total decrease of around 50 mM. These measurements were complemented with ion exchange chromatography measurements. This allowed us to account for all the osmotic pressure in the summer hemolymph, and the measured concentration changes explained as much as 40 % of the observed osmolality increase upon cold hardening. Preliminary results indicate that the remainder may be explained by non-colligative protein contributions.

Osmolar Modulation Drives Reversible Cell Cycle Exit and Human Pluripotent Cell Differentiation via NF-κВ and WNT Signaling.

Terminally differentiated cells are commonly regarded as the most stable cell state in adult organisms, characterized by growth arrest while fulfilling their specialized functions. A better understanding of the mechanisms involved in promoting cell cycle exit will improve the ability to differentiate pluripotent cells into mature tissues for both pharmacological and therapeutic use. Here, it demonstrates that a hyperosmolar environment enforces a protective p53-independent quiescent state in immature hepatoma cells and in pluripotent stem cell-derived models of human hepatocytes and endothelial cells. Prolonged culture in hyperosmolar conditions stimulates changes in gene expression promoting functional cell maturation. Interestingly, hyperosmolar conditions do not only trigger growth arrest and cellular maturation but are also necessary to maintain this maturated state, as switching back to plasma osmolarity reverses the changes in expression of maturation and proliferative markers. Transcriptome analysis revealed sequential stages of osmolarity-regulated growth arrest followed by cell maturation, mediated by activation of NF-κВ, and repression of WNT signaling, respectively. This study reveals that a modulated increase in osmolarity serves as a biochemical signal to promote long-term growth arrest and cellular maturation into different lineages, providing a practical method to generate differentiated hiPSCs that resemble their mature counterpart more closely.