Vasopressin-dependent water permeability of the basolateral membrane of the kidney outer medullary collecting duct in postnatal ontogenesis in rats

Kidneys of new-born animals are resistant to arginine vasopressin (AVP). The ability of the hormone to regulate water permeability of the collecting duct can be seen from weaning period, probably due to the maturation of the intracellular signaling pathway. The purpose of the present work was to investigate the effect of V2 receptor agonist dDAVP on the water permeability of OMCD basolateral membrane in 10-, 22- and 60-day old Wistar rats. We also estimated ontogenetic gene expression of AQP2, AQP3, AQP4 and V2 receptor. Osmotic water permeability (Pf) of the basolateral membrane of microdissected OMCD was measured under control conditions and after incubation with the agonist V2 receptor desmopressin (dDAVP; 10(-7) M). Water permeability in 10- and 22-day old rats under control conditions were significantly higher than in adults. Desmopressin stimulated significant increase of this parameter in 22-day old pups (Pf = = 125 +/- 4.85; Pf = 174 +/- 8.2 microns/s, p < 0.001) and adult rats (Pf = 100.5 +/- 7.38; Pf = 178.8 +/- 9.54 microns/s, p < 0.001). Osmotic water permeability of the OMCD basolateral membrane in 10-day old rats does not depend on dDAVP (Pf = 172.5 +/- 23.8; Pf = 164.8 +/- 34 microns/s). With the RT-PCR, we observed a gradual increase of AQP2 and V2 receptor genes expression during postnatal ontogenesis. The gene expression of AQP3 and AQP4 remained unchanged during postnatal ontogenesis. In general, the water permeability of the OMCD basolateral membrane of rats can be stimulated by AVP since the 22nd day of postnatal life. The water permeability of the OMCD basolateral membrane under control conditions gradually decreased during postnatal development, while gene expression of AQP3 and AQP4 was unchanged. The mechanism of this decrease remains to be established.     

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.     

Regulation of water permeability of collecting ducts in mouse kidney during postnatal development

Water permeability of epithelial cells and response to vasopressin was studied on isolated fragments of collecting ducts in the kidney of C57BL/6J mice of three age groups (9, 18, and 60–90 days). The coefficient of osmotic water permeability P _f was evaluated from the rate of cell swelling after medium osmolality was changed from 300 to 200 mOsm/l. The P _f value proved to be significantly lower in mice at the age of 9 days than at the age of 18 days, i.e., after the transition to mixed feeding; although P _f at the age of 18 days does not reach the level of adult animals (58.6 ± 7.7, 94.5 ± 8.8, and 168.4 ± 11.8 μM/s, respectively). The antagonist of vasopressin V₂ receptors desmopressin at 1 nM significantly increased P _f in both 18-day-old and adult mice but induced no changes in 9-day-old animals. The inhibitor of protein kinase C Ro-31-8220 in the concentration of 100 nM inhibited the desmopressin effect on P _f in 18 day-old and adult mice but did not inhibit the effect of the analog of the vasopressin secondary messenger cAMP, N⁶,O²-dibutyryl cyclic monophosphate, on P _f of the plasma membrane in collecting duct cells. Thus, the response of collecting duct cells to vasopressin appears at the end of wining and correlates with the increase in unstimulated osmotic water permeability of the plasma membrane in collecting duct cells. The vasopressin signal transduction via V₂ receptors is proposed to require the activity of protein kinase C and calcium-dependent systems of intercellular mediators apart from the cAMP-mediated mechanism.     

Effect of dehydration and dDAVP on water permeability of basolateral membranes of epithelial cells in the kidney collecting tubules

Water permeability of the outer medullary collecting duct's (OMCD) basolateral membrane was determined in vitro in the tubules isolated from hyperhydrated or dehydrated Wistar rats. Oil was injected into the lumen to block apical membrane water permeability. OMCD fragments underwent a hypoosmic shock (600/300 mOsm) and epithelial cells volume increased ad recorded with a digital camera. The latter's rate was used to calculate apparent water permeability of the membrane (Pf). Treatment of the tubules with Hg2Cl2 suppressed the water permeability. Water deprivation and dDAVP induced an increase in the basolateral water permeability. The data obtained suggest that the water permeability of the OMCD basolateral membrane may be stimulated by vasopressin and water deprivation.     

Successive histochemical differentiation steps during postnatal development of the collecting duct in rabbit kidney

Immunohistochemical experiments with monoclonal antibodies (mabs) on the kidney of neonatal rabbits revealed that the primary expression of collecting duct typic structures does not occur in a continuous and parallel, but in a subsequent developmental process. Only mabs RCT-30 A, and CD 4-V revealed immunoreactivity at the ontogenetically oldest parts of the collecting duct, the ampullae, while the other used markers (CD 1-3, CD 5-V, RCT-30 and RMCX) did not. In contrast, all of the tested antibodies showed positive reactions at the medullary and cortical collecting duct of the neonatal kidney as well as of the adult kidney. Additional incubations with wheat-germ agglutinin (WGA) a marker of terminal-differentiated collecting duct cells demonstrated weak-labelled ampulla cells beside intensively labelled ampullary neck and medullary collecting duct cells. With peanut agglutinin (PNA) labelling a 3 step transition could be illuminated: weak-labelled ampulla cells were found beside continuously bright labelled ampullary neck cells and finally a punctuate pattern downwards to the papilla. If the ampullary neck is the zone of proliferation, our findings of WGA- and PNA-co-labelling in this zone indicate, that in early developmental stages characteristic structures of different mature cells, probably principal and intercalated cells, are co-expressed within one single cell type. Thus intercalated cells might derive from principal cells.     

Successive histochemical differentiation steps during postnatal development of the collecting duct in rabbit kidney

Summary Immunohistochemical experiments with monoclonal antibodies (mabs) on the kidney of neonatal rabbits revealed that the primary expression of collecting duct typic structures does not occur in a continuous and parallel, but in a subsequent developmental process. Only mabs RCT-30 A, and CD 4-V revealed immunoreactivity at the ontogenetically oldest parts of the collecting duct, the ampullae, while the other used markers (CD 1–3, CD 5-V, RCT-30 and RMCX) did not. In contrast, all of the tested antibodies showed positive reactions at the medullary and cortical collecting duct of the neonatal kidney as well as of the adult kidney. Additional incubations with wheat-germ agglutinin (WGA) a marker of terminal-differentiated collecting duct cells demonstrated weak-labelled ampulla cells beside intensively labelled ampullary neck and medullary collecting duct cells. With peanut agglutinin (PNA) labelling a 3 step transition could be illuminated: weak-labelled ampulla cells were found beside continuously bright labelled ampullary neck cells and finally a punctuate pattern downwards to the papilla. If the ampullary neck is the zone of proliferation, our findings of WGA- and PNA-co-labelling in this zone indicate, that in early developmental stages characteristic structures of different mature cells, probably principal and intercalated cells, are co-expressed within one single cell type. Thus intercalated cells might derive from principal cells.     

Effect of sympathectomy on the heart pump function in rats during postnatal ontogenesis

The influence of guanetidine sympathectomy (30 mg/kg) on the heart pump function in rats during 3 weeks in postnatal ontogenesis has been investigated. Sympathectomy restrains age-dependent establishment of stroke volume, cardiac output and heart rate. The adaptation effects of regular physical training do not develop in the animals with sympathectomy, i.e. heart rate does not decrease and stroke volume does not increase. The initial stage of adaptation of the sympathectomized animals to physical training is accompanied by decrease in stroke volume and remarkable increase in heart rate which indicates the reduction of contractile activity in the myocardium.     

Effect of dDAVP on basolateral cell surface water permeability in the outer medullary collecting duct

We report a novel approach for assessing the volume of living cells which allows quantitative, high-resolution characterization of dynamic changes in cell volume while retaining the cell functionality. The aim of this study was to evaluate the short-term effect of vasopressin on basolateral cell surface water permeability in the outer medullary collecting duct (OMCD). The permeability of the basolateral cell membrane was determined in the tubules where the apical membrane was blocked with oil injected into the lumen. The apparent coefficient of water permeability (P _f) was evaluated by measuring the cell swelling after the step from hypertonic to isotonic medium (600 mosm to 300 mosm). Desmopressin (dDAVP) induced an increase of the basolateral P _f from 113.7±8.5 μm/s in control cells to 186.6±11.4 μm/s in micro-dissected fragments of the OMCD incubated in vitro (10⁻⁷ M dDAVP, 30 min at 37 °C) (P<0.05). Mercury caused pronounced inhibition of basolateral water permeability (26.0±6.9 μm/s; P<0.05). The effect of mercury (1.0 mM HgCl₂) was reversible: after washing the fragments with PBS for 20 min, P _f values were restored to the control levels (125.0±9.5 μm/s). The results of the study indicate the existence of a mechanism controlling the osmotic water permeability of the basolateral cell membrane in the OMCD epithelium.     

FXYD5 (dysadherin) regulates the paracellular permeability in cultured kidney collecting duct cells

FXYD5 (dysadherin or RIC) is a member of the FXYD family of single-span transmembrane proteins associated with the Na(+)-K(+)-ATPase. Several studies have demonstrated enhanced expression of FXYD5 during metastasis and effects on cell adhesion and motility. The current study examines effects of FXYD5 on the paracellular permeability in the mouse kidney collecting duct cell line M1. Expressing FXYD5 in these cells leads to a large decrease in amiloride-insensitive transepithelial electrical resistance as well as increased permeability to 4-kDa dextran. Impairment of cell-cell contact was also demonstrated by staining cells for the tight and adherence junction markers zonula occludens-1 and β-catenin, respectively. This is further supported by large expansions of the interstitial spaces, visualized in electron microscope images. Expressing FXYD5 in M1 cells resulted in a decrease in N-glycosylation of β1 Na(+)-K(+)-ATPase, while silencing it in H1299 cells had an opposite effect. This may provide a mechanism for the above effects, since normal glycosylation of β1 plays an important role in cell-cell contact formation (Vagin O, Tokhtaeva E, Sachs G. J Biol Chem 281: 39573-39587, 2006).     

Cell membrane water permeability of rabbit cortical collecting duct

Summary The water permeability (P _osm) of the cell membranes of isolated perfused rabbit cortical collecting ducts was measured by quantitative light microscopy. Water permeability of the basolateral membrane, corrected for surface area, was 66 m·sec^–1 for principal cells and 62.3 m·sec^–1 for intercalated cells. Apical membraneP _osm values corrected for surface area, were 19.2 and 25 m·sec^–1 for principal and intercalated cells, respectively, in the absence of antidiuretic hormone (ADH). Principal and intercalated cells both responded to ADH by increasingP _osm of their apical membranes to 92.2 and 86.2 ·sec^–1 respectively. The ratio of the total basolateral cell membrane osmotic water permeability to that of the apical cell membrane was 271 in the absence of ADH and 71 in the presence of the hormone for both cell types. This asymmetry in water permeability is most likely due to the fact that basolateral membrane surface area is at least 7 to 8 times greater than that of the apical membrane. Both cell types exhibited volume regulatory decrease when exposed to dilute serosal bathing solutions. Upon exposure to a hyperosmotic serosal bath (390 mosm), pricipal cells did not volume regulate while two physiologically distinct groups of intercalated cells were observed. One group of intercalated cells failed to volume regulate; the second group showed almost complete volume regulatory increase behavior.     

Proliferation of acid-secretory cells in the kidney during adaptive remodelling of the collecting duct

The renal collecting duct adapts to changes in acid-base metabolism by remodelling and altering the relative number of acid or alkali secreting cells, a phenomenon termed plasticity. Acid secretory A intercalated cells (A-IC) express apical H(+)-ATPases and basolateral bicarbonate exchanger AE1 whereas bicarbonate secretory B intercalated cells (B-IC) express basolateral (and apical) H(+)-ATPases and the apical bicarbonate exchanger pendrin. Intercalated cells were thought to be terminally differentiated and unable to proliferate. However, a recent report in mouse kidney suggested that intercalated cells may proliferate and that this process is in part dependent on GDF-15. Here we extend these observations to rat kidney and provide a detailed analysis of regional differences and demonstrate that differentiated A-IC proliferate massively during adaptation to systemic acidosis. We used markers of proliferation (PCNA, Ki67, BrdU incorporation) and cell-specific markers for A-IC (AE1) and B-IC (pendrin). Induction of remodelling in rats with metabolic acidosis (with NH(4)Cl for 12 hrs, 4 and 7 days) or treatment with acetazolamide for 10 days resulted in a larger fraction of AE1 positive cells in the cortical collecting duct. A large number of AE1 expressing A-IC was labelled with proliferative markers in the cortical and outer medullary collecting duct whereas no labeling was found in B-IC. In addition, chronic acidosis also increased the rate of proliferation of principal collecting duct cells. The fact that both NH(4)Cl as well as acetazolamide stimulated proliferation suggests that systemic but not urinary pH triggers this response. Thus, during chronic acidosis proliferation of AE1 containing acid-secretory cells occurs and may contribute to the remodelling of the collecting duct or replace A-IC due to a shortened life span under these conditions.     

Regulation of the vasopressin V2 receptor by vasopressin in polarized renal collecting duct cells

Binding of arginine-vasopressin (AVP) to its V2 receptor (V2R) in the basolateral membrane of principal cells induces Aquaporin-2-mediated water reabsorption in the kidney. To study the regulation of the V2R by dDAVP in a proper model, a polarized renal cell line stably-expressing V2R-GFP was generated. Labeled AVP-binding studies revealed an equal basolateral vs. apical membrane distribution for V2R-GFP and endogenous V2R. In these cells, GFP-V2R was expressed in its mature form and localized for 75% in the basolateral membrane and for 25% to late endosomes/lysosomes. dDAVP caused a dose- and time-dependent internalization of V2R-GFP, which was completed within 1 h with 100 nM dDAVP, was prevented by coincubation with a V2R antagonist, and which reduced its half-life from 11.5 to 2.8 h. Semiquantification of the V2R-GFP colocalization with E-cadherin (basolateral membrane), early endosomal antigen-1 (EEA-1) and lysosome-associated membrane protein-2 (LAMP-2) in time revealed that most dDAVP-bound V2R was internalized via early endosomes to late endosomes/lysosomes, where it was degraded. The dDAVP-internalized V2R did not recycle to the basolateral membrane. In conclusion, we established the itinerary of the V2R in a polarized cell model that likely resembles the in vivo V2R localization and regulation by AVP to a great extent.     

Cell type-specific glycoconjugates of collecting duct cells during maturation of the rat kidney

Summary The ontogeny of lectin-positive epithelial cell types and the maturation of polarized expression of the glycocalyx of the collecting ducts (CD) of the rat kidney were studied from samples of 18th-day fetal and neonatal kidneys of various ages. Lectins from Dolichos biflorus (DBA) and Vicia villosa (VVA), with preferential affinity to principal cells, stained virtually all CD cells of the fetal kidneys. However, within two days postnatally, the number of cells positive for DBA and VVA decreased to amounts found in the adult kidneys. Moreover, a characteristic change occurred rapidly after birth in the intracellular polarization of the reactive glycoconjugates, from a uniform plasmalemmal to a preferentially apical staining. In contrast, lectins from Arachis hypogaea (PNA), Maclura pomifera (MPA) and Lotus tetragonolobus (LTA), reacting indiscriminatively with principal and intercalated cells of adult kidneys, stained most CD cells in the fetal kidneys, and failed to show any postnatal change in the amount of positive cells or in the intracellular polarization.The immunocytochemical tests for (Na + K)-ATPase and carbonic anhydrase (CA II) revealed the characteristic postnatal decrease in the amount of principal cells and simultaneous increase in the amount of CA II rich intercalated cells. DBA and VVA reactive cells also decreased postnatally, paralleling the changes observed in the (Na + K)-ATPase positive principal cells. The present results suggest that the expression of the cell type-specific glycocalyx of principal and intercalated cells is developmentally regulated, undergoes profound changes during maturation, and is most likely associated with electrolyte transport phenomena.     

Changes in cellular composition of kidney collecting duct cells in rats with lithium-induced NDI

Lithium treatment for 4 wk caused severe polyuria, dramatic downregulation in aquaporin-2 (AQP-2) expression, and marked decrease in AQP-2 immunoreactivity with the appearance of a large number of cells without AQP-2 labeling in the collecting ducts after lithium treatment. Surprisingly, this was not all due to an increase in AQP-2-negative principal cells, because double immunolabeling revealed that the majority of the AQP-2-negative cells displayed [H⁺]ATPase labeling, which identified them as intercalated cells. Moreover, multiple [H⁺]ATPase-labeled cells were adjacent, which was never seen in control rats. Quantitation confirmed a significant decrease in the fraction of collecting duct cells that exhibited detectable AQP-2 labeling compared with control rats: in cortical collecting ducts, 40 ± 3.4 vs. 62 ± 1.8% of controls (P < 0.05; n = 4) and in inner medullary collecting ducts, 58 ± 1.6 vs. 81 ± 1.3% of controls (P < 0.05; n = 4). In parallel, a significant increase in the fraction of intercalated ([H⁺]ATPase-positive) cells was shown. Urine output, whole kidney AQP-2 expression, cellular organization, and the fractions of principal and intercalated cells in cortex and inner medulla returned to control levels after 4 wk on a lithium-free diet following 4 wk on a lithium-containing diet. In conclusion, lithium treatment not only decreased AQP-2 expression, but dramatically and reversibly reduced the fraction of principal cells and altered the cellular organization in collecting ducts. These effects are likely to be important in lithium-induced nephrogenic diabetes insipidus. nephrogenic diabetes insipidus; aquaporin; exchanger     

Effect of epicortical stimulation on depolarization of primary afferents in rats during early postnatal ontogenesis

Studies have been made on the parameters cord dorsum potentials (CDP) during stimulation of sensorimotor cortex in rats during first month of their postnatal life. First CDP were recorded from the 10th day, their latency being equal to about 80 msec, amplitude--65-70 microV, duration--more than 200 msec. During postnatal life of rats, the latent period decreases twice, the amplitude increases more than 3-fold, whereas the duration remains almost unchanged. These data indicate maturation of the descending pathways to the spinal cord, the increase in the propagation rate along these pathways and formation of segmentary mechanisms responsible for the generation of CDP. The effect of stimulation of the sensorimotor cortex on depolarization of primary afferent was also investigated. It was found that from the 2nd week of postnatal, life, formation of supraspinal control of afferent impulsation takes place.     

Vasopressin stimulates endocytosis in kidney collecting duct principal cells

In target epithelia, a vasopressin-induced water permeability increase is accompanied by the appearance of intramembranous particle (IMP) clusters, probably representing water-permeable patches, in the apical plasma membrane of responding cells. In the collecting duct principal cell, we have previously shown that these clusters are located in clathrin-coated pits. To determine whether vasopressin induces the endocytic uptake of these membrane domains in principal cells, we have examined the uptake of horseradish peroxidase (HRP) by principal cells of normal rats, vasopressin-deficient Brattleboro rats, and vasopressin-treated Brattleboro rats, following intravenous injection of HRP. By quantitative electron microscopy, principal cells of Brattleboro homozygous rats were found to take up much less HRP into cytoplasmic vesicles than normal rats, and HRP uptake was increased to normal levels in vasopressin-treated Brattleboro rats. Many invaginating coated pits at the cell surface were loaded with HRP reaction product, indicating their participation in the observed endocytosis of HRP. We conclude that vasopressin stimulates endocytosis in collecting duct principal cells. Since we have already shown that IMP clusters are found in coated pits at the cell surface, the endocytic removal of these putative water-permeable patches from the apical membrane seems to occur via a clathrin-mediated mechanism in this tissue.