G. S. Baturina

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.

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.

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

•  What is the central question of this study? The interstitium of the renal inner medulla contains abundant linear negatively charged glycosaminoglycan hyaluronan (HA), which affects the water flow depending on their polymeric state. It remains an open question whether HA is involved in the long‐term effect of vasopressin on water reabsorption via the regulation of the expression of genes for key enzymes of HA turnover. •  What is the main finding and its importance? Using real‐time RT‐PCR, we show that in rats vasopressin inhibits the synthesis of HA and concomitantly promotes its degradation in the renal interstitium through its effect on genes for key enzymes of HA turnover (hyaluronan synthase‐2 and hyaluronidase‐2). Analysis of the long‐term effect of vasopressin on the transcription of these genes demonstrated a new mechanism in hormonal regulation of the renal concentrating function.

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.

Effect of hypoosmotic shock on the volume of renal collecting duct epithelial cells of brattleboro rats with hereditarily defective vasopressin synthesis.

102 The problem of maintaining a constant volume under conditions of extracellular and intracellular osmotic pressure fluctuations is particularly relevant for transport epithelium cells, such as renal collecting duct cells. The mechanisms regulating the cell volume in osmotic shock are specific for hypotensive and hypertensive effects [1, 2]. Recent studies have shown that the swelling rate of the cell in a hypoosmotic envii ronment and the effectiveness of regulatory volume decrease largely depend on the water permeability of the plasma membrane. The lipid bilayer of the cell membrane is highly permeable for water molecules due to the presence of specific proteins that form water channels (aquaporins, AQPs) [3]. Thus, the regulation of the water permeability of the cell membrane affects the parameters of the regulatory volume decrease (RVD). The transepithelial water permeability of renal collecting ducts increases as a result of AQP2 insertion into the apical membrane of cells in response to the neurohypophysial hormone vasopressin [4]. In the absence of the hormone or in the case of its inability to bind to receptors, the urine output and, respectively, the bodys fluid demands increase tenfold. In Brattlee boro rats, derived from LonggEvans rats [5], a single deletion in the vasopressin gene was found [6], which leads to disruption of the hormone synthesis [5, 7, 8]. Homozygous Brattleboro rats are characterized by a sharp decrease in the concentrating ability of the kidd neys, polydipsia, and polyuria. In this study, we invess tigated the effect of the absence of endogenous vasoo pressin in Brattleboro rats in response to changes in the volume of the principal cells of renal collecting ducts exposed hypoosmotic environment. The experiments were performed on Brattleboro and Wistar rats of both sexes. All animals were obtained from the laboratory of experimental animals, Russian Academy of Sciences (Novosibirsk). Experii ments were performed with adult 600dayold animals of the corresponding line, weighing 200–250 g. On the day of the experiment, animals were placed into mett abolic cages. The osmolarity of their spontaneously excreted urine samples was determined cryoscopically using an OSMETTE microosmometer (Precision Syss tems Inc., United States). Before the experiment, Brattleboro rats were kept under standard conditions and received dry food and water ad libitum. In experii ments we used the rats that drank a water amount that exceeded 50% of their body weight per day, which indii cated that they were homozygotes. The osmolality of spontaneously excreted urine was …

Study of the reaction of kidney collecting duct principal cells to hypotonic shock. Experiment and mathematical model

The reaction of rat kidney collecting duct principal cells to hypotonic shock was studied. The changes in cell relative volume were measured using fluorescent dye calcein, and a mathematical model based on our experimental results was developed. It was shown that regulatory volume decrease is mainly provided by significant release of osmolytes from the cell and decrease of the plasma membrane water permeability. Using our model, we calculated the membrane water permeability and found it to decrease from 2 · 10−1 to 2 · 10−2 cm/s. We conclude that for effective RVD to occur, a dramatic increase in the membrane permeability to K+, Cl− and organic anions is necessary.

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.

The effect of vasopressin on the expression of genes of key enzymes of the interstitial hyaluronan turnover and concentration ability in WAG rat kidneys

In mammals arginine-vasopressin (AVP) is a major hormone involved in the regulation of renal water reabsorption, acting via the increase of the osmotic permeability of the epithelium of the collecting duct. The AVP-induced intracellular events include, as a core step, the trafficking of the vesicles containing the water channels, aquaporin-2, to the apical plasma membrane of the collecting duct’s principal cells. The interstitium of the renal inner medulla contains abundant linear negatively charged glycosaminoglycan, hyaluronan (HA), which affects the water flow between structures of the concentrating mechanism, depending on its polymeric state. Using real-time RT-PCR, we tested the assumption that the renal hyaluronan may be involved in the long-term vasopressin effect on water reabsorption. The expression of the genes encoding hyaluronan synthase-2 (Has2), hyaluronidase-1, and hyaluronidase-2 (Hyal1 and Hyal2) in the kidneys of Wistar Albino Glaxo (WAG) was studied. The Has2 mRNA content was the highest in the papilla of the kidneys of the hydrated rats. The V2 receptor-selective vasopressin analog dDAVP (100 μg/kg of body weight, twice a day intraperitoneally for 2 days) induced a considerable decrease in the Has2 mRNA content in the papilla with less pronounced changes in the cortex. In contrast to Has2, the dDAVP treatment caused a significant increase in the Hyal1 and Hyal2 mRNA content in the renal papilla. There was a good fit between the Hyal1 and Hyal2 transcriptional levels and changes in hyaluronidase activity in the renal tissue. It was suggested that vasopressin is able to inhibit the synthesis of hyaluronan and concomitantly promotes its degradation in the renal papilla interstitium, thereby facilitating water flow between the elements of the renal countercurrent system. The implications for this effect are discussed in the context of the data in the literature.

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.

Age-Related Changes in Water Transport by Corneal Endothelial Cells in Rats

Senescence-associated alterations in the structure and function of the cornea make it more sensitive to such external agents as surgery, traumas, and disease, resulting in edema and vision impairment that can be corrected only by cornea transplantation. The role of aquaporins for cornea endothelium functioning, as well as age-related changes in their activity, is not entirely understood. We have studied age-related changes in the water permeability (Pf) of corneal endothelium plasma membranes and the mRNA expression levels of aquaporins aqp1 and aqp3 genes in Wistar and senescence-accelerated OXYS rats. At the age of 3 to 18 months, Pf increased in Wistar rats and decreased in OXYS rats, becoming two times lower than in the Wistar line. The expression of AQP1 mRNA (studied by real-time 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 two times 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 it decreased in Wistar rats and at 18 months was four times lower than at 3 months. We propose that the 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 eliminates this compensation.