Methods to Measure Water Permeability.

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.

Comparison of Isotonic Activation of Cell Volume Regulation in Rat Peritoneal Mesothelial Cells and in Kidney Outer Medullary Collecting Duct Principal Cells.

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.

Mitochondrial Antioxidant SkQ1 Improves Hypothermic Preservation of the Cornea.

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.

Benign Pleural Mesothelial Cells Have Higher Osmotic Water Permeability than Malignant Pleural Mesothelioma Cells and Differentially Respond to Hyperosmolality.

BACKGROUND/AIMS: Cell volume regulation is a critical mechanism for cell homeostasis and depends on the osmotic water permeability (Pf) of the cell plasma membrane. The Pf 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 Pf after preconditioning with 4% glucose for 24 hours. In both cases we also assessed the role of AQP1 inhibition (0.1 mM HgCl2) on the Pf. Finally, we assessed corresponding changes in the AQP1 plasma membrane availability by immunofluorescence. RESULTS: We report that MeT-5A cells have a significantly higher Pf 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 Pf in all cells lines (p<0.001 in all cases). High glucose preconditioning for 24 hours significantly increased MeT-5A Pf (p<0.001), did not influence M14K Pf (p=0.19) and significantly reduced ZL34 Pf (p=0.02). Comparing cell lines after high glucose preconditioning, MeT-5A Pf 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 Pf in benign and MPM cells that require further mechanistic investigation.

Methods to Measure Water Permeability.

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.

Brattleboro rats have impaired apical membrane water permeability regulation in the outer medullary collecting duct principal cells.

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 (Pf ) 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 Pf 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.

Computational genomic analysis of PARK7 interactome reveals high BBS1 gene expression as a prognostic factor favoring survival in malignant pleural mesothelioma.

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.

The water permeability reduction after successive hypo-osmotic shocks in kidney principal cells is apically regulated.

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.

Quantitative estimation of transmembrane ion transport in rat renal collecting duct principal cells.

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.

Effect of vasopressin on the expression of genes for key enzymes of hyaluronan turnover in Wistar Albino Glaxo and Brattleboro rat kidneys.

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.

Regulatory volume decrease of rat kidney principal cells after successive hypo-osmotic shocks.

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.