Role of the low-selective organic anion transport in regulation of osmotic balance of renal collecting duct principal cells under hypo-osmotic conditions.

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.

[Age-related changes of water transport by corneal endothelial cells in rats.]

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.

Regulation of V2 receptor by vasopressin is different in Brattleboro rats compared with Wistar.

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.

[The role of protein kinase C in the establishment of the mechanism of vasopressin antidiuretic action in the rat kidney during mammalian postnatal development].

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.

[Role of water channels in the regulation of the volume of principal cells of rat kidney collecting ducts in hypoosmotic medium].

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 pecularuties of aldosterone regulation of rat epithelial Na channel expression and functional activity].

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.

[G(i) proteins in regulation of water permeability of the rat kidney collecting tube epithelium cell membrane].

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.

[Calciumdependent mechanisms in vasopressin regulation of osmotic water permeability in the mouse kidney collecting duct cells].

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.

[Role of Ca2+ and aquaporin-2 in regulation of basolateral water permeability of collecting ducts in the rat kidney].

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.