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Water permeability is a key feature of the cell plasma membranes, and it has seminal importance for several cell functions such as cell volume regulation, cell proliferation, cell migration, and angiogenesis to name a few. The transport of water occurs mainly through plasma membrane water channels, aquaporins. Aquaporins have very important function in physiological and pathophysiological states. Due to the above, the experimental assessment of the water permeability of cells and tissues is necessary. The development of new methodologies of measuring water permeability is a vibrant scientific field that constantly develops during the last three decades along with the advances in imaging mainly. In this chapter we describe and critically assess several methods that have been developed for the measurement of water permeability both in living cells and in tissues with a focus in the first category., (© 2023. The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd.)

In disease states, mesothelial cells are exposed to variable osmotic conditions, with high osmotic stress exerted by peritoneal dialysis (PD) fluids. They contain unphysiologically high concentrations of glucose and result in major peritoneal membrane transformation and PD function loss. The effects of isotonic entry of urea and myo-inositol in hypertonic (380 mOsm/kg) medium on the cell volume of primary cultures of rat peritoneal mesothelial cells and rat kidney outer medullary collecting duct (OMCD) principal cells were studied. In hypertonic medium, rat peritoneal mesothelial cells activated a different mechanism of cell volume regulation in the presence of isotonic urea (100 mM) in comparison to rat kidney OMCD principal cells. In kidney OMCD cells inflow of urea into the shrunken cell results in restoration of cell volume. In the shrunken peritoneal mesothelial cells, isotonic urea inflow caused a small volume increase and activated regulatory volume decrease (RVD). Isotonic myo-inositol activated RVD in hypertonic medium in both cell types. Isotonic application of both osmolytes caused a sharp increase of intracellular calcium both in peritoneal mesothelial cells and in kidney OMCD principal cells. In conclusion, peritoneal mesothelial cells exhibit RVD mechanisms when challenged with myo-inositol and urea under hyperosmolar isotonic switch from mannitol through involvement of calcium-dependent control. Myo-inositol effects were identical with the ones in OMCD principal cells whereas urea effects in OMCD principal cells led to no RVD induction.

Diseases of the cornea are a frequent cause of blindness worldwide. Keratoplasty is an efficient method for treating severely damaged cornea. The functional competence of corneal endothelial cells is crucial for successful grafting, which requires improving the media for the hypothermic cornea preservation, as well as developing the methods for the evaluation of the corneal functional properties. The transport of water and ions by the corneal endothelium is important for the viability and optic properties of the cornea. We studied the impact of SkQ1 on the equilibrium sodium concentration in the endothelial cells after hypothermic preservation of pig cornea at 4°C for 1, 5, and 10 days in standard Eusol-C solution. The intracellular sodium concentration in the endothelial cells was assayed using the fluorescent dye Sodium Green; the images were analyzed with the custom-designed CytoDynamics computer program. The concentrations of sodium in the pig corneal endothelium significantly increased after 10 days of hypothermic preservation, while addition of 1.0 nM SkQ1 to the preservation medium decreased the equilibrium concentration of intracellular sodium (at 37°C). After 10 days of hypothermic preservation, the permeability of the plasma membrane for sodium decreased in the control cells, but not in the cells preserved in the presence of 1 nM SkQ1. Therefore, SkQ1 increased the ability of endothelial cells to restore the intracellular sodium concentration, which makes SkQ1 a promising agent for facilitating retention of the functional competence of endothelial cells during cold preservation.

Background/aims: Cell volume regulation is a critical mechanism for cell homeostasis and depends on the osmotic water permeability (P f ) of the cell plasma membrane. The P f of human mesothelial cells is unknown although they contribute to serosal fluid turnover., Methods: In this study we measured the osmotic water permeability of benign human mesothelial cells (MeT-5A) and of epithelioid (M14K) and sarcomatoid (ZL34) malignant pleural mesothelioma (MPM) cells in response to acute hyperosmotic stress. We also assessed the changes in their P f after preconditioning with 4% glucose for 24 hours. In both cases we also assessed the role of AQP1 inhibition (0.1 mM HgCl 2 ) on the P f . Finally, we assessed corresponding changes in the AQP1 plasma membrane availability by immunofluorescence., Results: We report that MeT-5A cells have a significantly higher P f as compared to M14K and ZL34 MPM cells [4.85E-03±2.37E-03 cm/sec (n=17) versus 2.74E-03±0.74E-03 cm/sec (n=11) and 2.86E-03±0.11E-03 cm/sec (n=11)]. AQP1 inhibition significantly decreased the P f in all cells lines (p<0.001 in all cases). High glucose preconditioning for 24 hours significantly increased MeT-5A P f (p<0.001), did not influence M14K P f (p=0.19) and significantly reduced ZL34 P f (p=0.02). Comparing cell lines after high glucose preconditioning, MeT-5A P f was significantly higher than that of M14K and ZL34 MPM cells and the AQP1 inhibition effect was significant in MeT-5A and M14K cells. These results were corroborated by AQP1 immunofluorescence., Conclusion: We provide evidence for a differential regulation of P f in benign and MPM cells that require further mechanistic investigation., Competing Interests: The authors declare no conflicts of interest., (© Copyright by the Author(s). Published by Cell Physiol Biochem Press.)

In the course of adaptation of the rat kidney collecting duct cells to hypo-osmotic medium, the organic anion transporter inhibitor probenecid reduced significantly the regulatory cell volume decrease in response to a hypotonic shock. Both probenecid and hypotonic shock delayed significantly the entry into a cell of the fluorescent dye calcein, which exists as anion at neutral pH. Thus, the organic osmolyte transport plays an important role in the regulatory decrease of the principal cell volume under the hypo-osmotic conditions.

Water permeability is a key feature of the cell plasma membranes and it has seminal importance for a number of cell functions such as cell volume regulation, cell proliferation, cell migration, and angiogenesis to name a few. The transport of water occurs mainly through plasma membrane water channels , the aquaporins, who have very important function in physiological and pathophysiological states. Due to the above the experimental assessment of the water permeability of cells and tissues is necessary. The development of new methodologies of measuring water permeability is a vibrant scientific field that constantly develops during the past three decades along with the advances in imaging mainly. In this chapter we describe and critically assess several methods that have been developed for the measurement of water permeability both in living cells as well as in tissues with a focus in the first category.

Senescence-associated alterations in structure and function of cornea make it more sensitive to traumas and disease. Trauma of cornea leads to edema and vision impairment and only corneal transplantation remains the effective for vision correction. Role of aquaporins for cornea endothelium function, as well as age-related changes of their activity, are not entirely understood. Herein, we studied changes with age the water permeability (Pf) of the plasma membranes of the corneal endothelial cells and the level of expression in them of the mRNA genes of the water channels of the aquaporins AQP1 and AQP3 in Wistar and senescence-accelerated OXYS rats. From the age of 3 to 18 months, Pf in Wistar rats increased, in OXYS - decreased and was twice lower than in Wistar rats. The expression of aqp1 mRNA (studied by RT-PCR) in the endothelium was the same in Wistar and OXYS rats at the age of 3 months. By the age of 18 months, it increased only in Wistar rats and became twice higher than in OXYS rats. The expression of aqp3 mRNA in the endothelium of 3-month-old OXYS rats was half that of Wistar rats and did not change with age, while in Wistar rats it decreased and at 18 months was 4 times lower than in 3 months. We supposed that increased water permeability of endothelial cells in Wistar rats is adaptive and compensates for the decrease in endothelial cell density with age, while the accelerated aging of OXYS rats abolishes this compensation.

Vasopressin (AVP) regulates the body salt-water balance. Brattleboro rats carry an AVP gene mutation resulting in a recessive form of central diabetes insipidus, being ideal for AVP deficiency studies. Herein, we studied the water permeability of the apical and basolateral sides of outer medullary collecting duct (OMCD) principal cells in response to dDAVP (a V2 receptor agonist) administration in Wistar and Brattleboro rats. Biophysical measurements of the water permeability (P f ) of isolated OMCD principal cells were performed with the calcein quenching method with/without dDAVP (10 -8  mol/L). mRNA transcripts and protein levels of AQP2, AQP3 and AQP4 were assessed by RT-PCR and western blot respectively. dDAVP increased the apical and basolateral P f of OMCD principal cells in Wistar rats, while in Brattleboro rats this effect was present basolaterally. Long-term dDAVP administration in both strains resulted in a significant increase in mRNA expression of all assessed AQP's while only the protein levels of AQP2 and AQP3 were significantly increased. Short-term (20 minutes) dDAVP treatment of isolated OMCD fragments resulted in significantly increased plasma membrane expression of AQP2 in Wistar rats and of AQP2 and AQP3 in Brattleboro rats. In summary, dDAVP induces different expression of AQP2, AQP3 and AQP4 in Wistar and Brattleboro rats during short- and long-term treatment. In Wistar rats dDAVP mainly increased AQP2 expression while in Brattleboro rats it increased functional water permeability mainly by AQP3 expression., (© 2016 John Wiley & Sons Australia, Ltd.)

The aim of our study was to assess the differential gene expression of Parkinson protein 7 (PARK7) interactome in malignant pleural mesothelioma (MPM) using data mining techniques to identify novel candidate genes that may play a role in the pathogenicity of MPM. We constructed the PARK7 interactome using the ConsensusPathDB database. We then interrogated the Oncomine Cancer Microarray database using the Gordon Mesothelioma Study, for differential gene expression of the PARK7 interactome. In ConsensusPathDB, 38 protein interactors of PARK7 were identified. In the Gordon Mesothelioma Study, 34 of them were assessed out of which SUMO1, UBC3, KIAA0101, HDAC2, DAXX, RBBP4, BBS1, NONO, RBBP7, HTRA2, and STUB1 were significantly overexpressed whereas TRAF6 and MTA2 were significantly underexpressed in MPM patients (network 2). Furthermore, Kaplan-Meier analysis revealed that MPM patients with high BBS1 expression had a median overall survival of 16.5 vs. 8.7 mo of those that had low expression. For validation purposes, we performed a meta-analysis in Oncomine database in five sarcoma datasets. Eight network 2 genes (KIAA0101, HDAC2, SUMO1, RBBP4, NONO, RBBP7, HTRA2, and MTA2) were significantly differentially expressed in an array of 18 different sarcoma types. Finally, Gene Ontology annotation enrichment analysis revealed significant roles of the PARK7 interactome in NuRD, CHD, and SWI/SNF protein complexes. In conclusion, we identified 13 novel genes differentially expressed in MPM, never reported before. Among them, BBS1 emerged as a novel predictor of overall survival in MPM. Finally, we identified that PARK7 interactome is involved in novel pathways pertinent in MPM disease., (Copyright © 2015 the American Physiological Society.)

This paper presents the new priority data on the effect of genetically determined deficiency of endogenous vasopressin on the functioning of V2 receptor in the plasma membrane of the principal cells of the collection ducts of Brattleboro rats. It was found that 2-day desmopressin administration results in the activation of V2 receptor gene expression in Brattleboro rat, whereas in Wistar rats the content of mRNA did not change. In addition, short-term exposure of the collection ducts of the kidneys to the hormone led to V2 receptor internalization from the plasma membrane in Wistar rats, but had no effect on the protein content in Brattleboro rats.

Background/aims: Renal principal cells maintain their intracellular water and electrolyte content despite significant fluctuations of the extracellular water and salt concentrations. Their water permeability decreases rapidly (within a few seconds) after successive hypo-osmotic shocks. Our aim was to investigate the contribution of the apical and basolateral surface to this effect and the potential influence of fast reduction in AQP-2, -3 or -4 plasma membrane content., Methods: Rat principal cells of kidney collecting duct fragments underwent hypo-osmotic challenge applied apically or basolaterally and the regulatory volume decrease (RVD) was measured by the calcein quenching method. The AQP -2, -3 and -4 content of the plasma membrane fraction was quantified by Western blotting., Results: The hypo-osmotic shock applied apically causes rapid swelling with high apparent water permeability and fast RVD. An identical successive shock after 15-20 sec causes significantly lower swelling rate with 3-fold reduction in apparent water permeability. This reaction is accompanied by AQP2 decrease in the plasma membrane while AQP3 and AQP4 are unaffected. The contribution of the basolateral cell surface to RVD is significantly lower than the apical., Conclusion: These results indicate that in principal cells the effective mechanism of RVD is mainly regulated by the apical cell plasma membrane., (© 2014 S. Karger AG, Basel.)

Kidney collecting duct principal cells play a key role in regulated tubular reabsorption of water and sodium and secretion of potassium. The importance of this function for the maintenance of the osmotic homeostasis of the whole organism motivates extensive study of the ion transport properties of collecting duct principal cells. We performed experimental measurements of cell volume and intracellular sodium concentration in rat renal collecting duct principal cells from the outer medulla (OMCD) and used a mathematical model describing transmembrane ion fluxes to analyze the experimental data. The sodium and chloride concentrations ([Na+]in = 37.3 ± 3.3 mM, [Cl-]in = 32.2 ± 4.0 mM) in OMCD cells were quantitatively estimated. Correspondence between the experimentally measured cell physiological characteristics and the values of model permeability parameters was established. Plasma membrane permeabilities and the rates of transmembrane fluxes for sodium, potassium and chloride ions were estimated on the basis of ion substitution experiments and model predictions. In particular, calculated sodium (PNa), potassium (PK) and chloride (PCl) permeabilities were equal to 3.2 × 10-6 cm/s, 1.0 × 10-5 cm/s and 3.0 × 10-6 cm/s, respectively. This approach sets grounds for utilization of experimental measurements of intracellular sodium concentration and cell volume to quantify the ion permeabilities of OMCD principal cells and aids us in understanding the physiology of the adjustment of renal sodium and potassium excretion.

Hyaluronan (HA), the major glycosaminoglycan of the interstitial matrix, is heterogeneously distributed within the kidney. Using real-time RT-PCR, we tested the assumption that renal HA may be involved in the long-term effect of vasopressin on water reabsorption. The expression of the genes encoding hyaluronan synthase-2 (Has2), hyaluronidase-1 and hyaluronidase-2 (Hyal1 and Hyal2) was studied in the kidneys of Wistar Albino Glaxo (WAG) and homozygous vasopressin-deficient Brattleboro rats treated with the V2 receptor-selective vasopressin analogue dDAVP (100 μg (kg body wt)(-1), i.p., twice a day for 2 days). The Has2 mRNA content was the highest in the kidney papilla of the hydrated WAG and control Brattleboro rats, devoid of vasopressin. In WAG rats, dDAVP induced a considerable decrease in Has2 mRNA content in the papilla, with less pronounced changes in the cortex. The changes elicited by dDAVP in Brattleboro rats tended to be the same as in WAG rats, but weaker. In contrast to Has2, dDAVP treatment caused a significant increase in the Hyal1 and Hyal2 mRNA content in the renal papilla of WAG and Brattleboro rats. In rats of both strains, there was a good fit between Hyal1 and Hyal2 transcriptional levels and changes in hyaluronidase activity in the renal tissue. It is suggested that vasopressin is able to inhibit the synthesis of HA and concomitantly promote its degradation in the interstitium of the renal papilla, thereby facilitating water flow between elements of the renal countercurrent system. The implications for this effect are discussed in the context of the data in the literature.

Outer Medullary Collecting Duct (OMCD) principal cells are exposed to significant changes of the extracellular osmolarity and thus the analysis of their regulatory volume decrease (RVD) function is of great importance in order to avoid cell membrane rupture and subsequent death. In this paper we provide a sub-second temporal analysis of RVD events occurring after two successive hypo-osmotic challenges in rat kidney OMCD principal cells. We performed experimental cell volume measurements and created a mathematical model based on our experimental results. As a consequence of RVD the cell expels part of intracellular osmolytes and reduces the permeability of the plasma membrane to water. The next osmotic challenge does not cause significant RVD if it occurs within a minute after the primary shock. In such a case the cell reacts as an ideal osmometer. Through our model we provide the basis for further detailed studies on RVD dynamical modeling., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The kidney of immaturely born mammals in early postnatal development is insensitive to the effect of the antidiuretic hormone, vasopressin. It has been demonstrated that water permeability of the epithelial cells in the collecting ducts of a rat kidney increases during development; in this process, the response to desmopressin, an agonist of vasopressin V2 receptors, appears at the age of 20 days. The observed increase in water permeability is connected with an increased content of the water channel's proteins aquaporins AQP2 and AQP3 in the plasma membrane. The calcium-dependent protein kinase C isoforms are the likely components of the vasopressin signal's transduction and are possibly involved in the mechanisms underlying the maturation of sensitivity to this hormone. The contents of three protein kinase C isoforms (alpha, delta, and zeta) in rats at different periods of their postnatal development were estimated using Western blot hybridization. It has been shown that the contents of protein kinase C isoforms alpha and delta increase with development, whereas the content of isoform zeta remains constant. The most likely participant of the mechanism providing for maturation of the cell's hormonal competence for vasopressin is the calcium-dependent protein kinase Ca, because it's content in the plasma membrane is maximal on days 20-24, which coincides with the time when the vasopressin action appears.

The effect of plasma membrane water permeability on the rate of changes in the volume of principal cells of collecting ducts of the outer substantia medullaris under conditions of hypoosmotic shock has been studied. Changes in cell volume were studied by the fluorescent method. It was shown that the hypotonic shock induced a rapid increase in the cell volume with the characteristic time that depended on plasma membrane water permeability. The decrease in volume occurred much more slowly, and the rate of volume decrease directly correlated with the rate of swelling. The inhibition of potassium transport by barium chloride decreased the rate of volume restoration, without affecting substantially the duration of the swelling phase. The inhibition of mercury-sensitive water channels by mercury caused a significant increase in the time of both cell swelling and volume restoration. It was concluded that the state of water channels largely determines the rate of the regulatory response of epithelial cells of collecting ducts to hypoosmotic shock and affects the exchange of cell osmolites.

Developmental changes of mRNA alfa-subunit ENaC abundance in renal cortex of 10-day old and in adult rats and aldosterone (10 nM) influence on it were studied. The mRNA level was lower in young rat renal cortex and there was no aldosterone effect on it within 5 hours and 30 minutes, in contrast to adult rats. Intracellular sodium concentration [Na+ i] measured by fluorescent dye Na+ Green in CCD fragments micro-dissected from the kidneys was lower in fragments from immature kidneys, whereas fast nongenomic of aldosterone (10 nM) was the same at both ages. Aldosterone significantly raised [Na+ i] by 40 and 50% in conditions of low sodium concentration (14 mM) in outer medium in epithelial cells of both type of CCD fragments from 10-day old and adult animals, respectively (p < 0.05). We assume that heterochrony of fast and long time genomic effect takes place in aldosterone molecular mechanism maturation. Fast nongenomic effect on the [Na+ i] appeared earlier compared to delayed genomic effect of aldosterone mediated through the changes of the mRNA alpha-subunit ENaC abundance.

Principal mechanism of the transepithelial water permeability increase in the kidney collecting ducts in response to vasopressin involves insertion of aquaporin 2 (AQP2) into the apical membrane. Previously we have shown that water permeability of the basolateral membrane also may be increased with stimulation of V2-receptors. It is known that inhibition of G(i)-proteins with pertussis toxin blocks redistribution of AQP2 into the apical membrane following the application of vasopressin or forskolin. The aim of the present study was to investigate potential involvement of G(i)-proteins in regulation of basolateral membrane water permeability. Effect of pertussis toxin on the ability of desmopressin to increase the basolateral membrane osmotic water permeability was investigated, and the expression of Galpha(i)2 and Galpha(i)3 genes under normal conditions and after 2 days of water deprivation were evaluated. We demonstrated that dehydration leds to a 30% increase of Galpha(i)3 mRNA content while the Galpha(i)2 mRNA level remains unchanged. In control experiments, basolateral membrane water permeability increased in response to desmopressin from 59.2 +/- 6.61 to 70.6 +/- 9.2 microm/s (p < 0.05, paired t-test). Pertussis toxin completely blocked this reaction (53.5 +/- 5.18 vs 50.1 +/- 6.50 microm/s, respectively). We conclude that G(i)-proteins participate in the mechanism of the basolateral membrane water permeability increase in response to stimulation of V2-receptors. Clarification of the G(i)-proteins role in this process requires further investigation, but most likely they are involved in regulation of aquaporin transport and insertion into the cell membrane.

The role of AQP2,3 and intracellular calcium in vasopressin-induced increase in the water permeability of the basolateral cell membrane in microdissected rat kidney OMCD was studied. It was shown that increase in the water permeability of the basolateral membranes correlated with increase in the content of AQP2 and AQP3 in the membrane fraction isolated from outer kidney medulla. Preliminary loading of cells with BAPTA-AM which binds intracellular Ca2+ abolished the increase in the water permeability and prevented the rise of the AQP2 content in response to dDAVP. BAPTA was ineffective to block the enhancement of AQP2 content in membrane fraction in presence of dDAVP. These results suggest that the increase in intracellular calcium activity and the enhanced content of AQP2 in plasma membrane are important for the antidiuretic effect of dDAVP.

In the study, the role of PKC and Ca++ in vasopressin regulation of the plasma membrane water permeability was studied in the cells of the mouse kidney collecting duct. Coefficient of osmotic water permeability of total cell surface (Pf) was calculated from the initial rate of cell swelling following the osmotic shock caused by changing the medium osmolarity from isotonic to hypotonic (300 mOsm to 200 mOsm). Desmopressin (dDAVP 1 nM) increased the Pf in hydrated mice from 168.4 +/- 11.8 microm/s up to 231.3 +/- 14.7 microm/s. The Ca++ chelator BAPTA prevented the desmopressin-induced increase in water permeability. Inhibition of PKC (Ro-31-8220 0.1 microM) also abolished the desmopressin-stimulated increase of plasma membrane water permeability, whereas inhibitor of PKC alone did not suppress the stimulation of the water permeability by db-cAMP. The PKC activity and calciumdependent second messengers seem to be important for regulation of water permeability by vasopressin.