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.     

Dexamethasone stimulates endothelin-1 gene expression in renal collecting duct cells

Aldosterone stimulates the endothelin-1 gene (Edn1) in renal collecting duct (CD) cells by a mechanism involving the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR). The goal of the present study was to determine if the synthetic glucocorticoid dexamethasone affected Edn1 gene expression and to characterize GR binding patterns to an element in the Edn1 promoter. Dexamethasone (1μM) induced a 4-fold increase in Edn1 mRNA in mIMCD-3 inner medullary CD cells. Similar results were obtained from cortical collecting duct-derived mpkCCD(c14) cells. RU486 inhibition of GR completely blocked dexamethasone action on Edn1. Similarly, 24h transfection of siRNA against GR reduced Edn1 expression by approximately 50%. However, blockade of MR with either spironolactone or siRNA had little effect on dexamethasone induction of Edn1. Cotransfection of MR and GR siRNAs together had no additive effect compared to GR-siRNA alone. The results indicate that dexamethasone acts on Edn1 exclusively through GR and not MR. DNA affinity purification studies revealed that either dexamethasone or aldosterone resulted in GR binding to the same hormone response element in the Edn1Edn1 promoter. The Edn1 hormone response element contains three important sequence segments. Mutational analysis revealed that one of these segments is particularly important for modulating MR and GR binding to the Edn1 hormone response element.     

Long term regulation of aquaporin-2 expression in vasopressin-responsive renal collecting duct principal cells.

Fine regulation of water reabsorption by the antidiuretic hormone [8-arginine]vasopressin (AVP) occurs in principal cells of the collecting duct and is largely dependent on regulation of the aquaporin-2 (AQP2) water channel. AVP-inducible long term AQP2 expression was investigated in immortalized mouse cortical collecting duct principal cells. Combined RNase protection assay, Western blot, and immunofluorescence analyses revealed that physiological concentrations of AVP added to the basal side, but not to the apical side, of cells grown on filters induced both AQP2 mRNA and apical protein expression. The stimulatory effect of AVP on AQP2 expression followed a V(2) receptor-dependent pathway because [deamino-8-d-arginine]vasopressin (dDAVP), a specific V(2) receptor agonist, produced the same effect as AVP, whereas the V(2) antagonist SR121463B antagonized action of both AVP and dDAVP. Moreover, forskolin and cyclic 8-bromo-AMP fully reproduced the effects of AVP on AQP2 expression. Analysis of protein degradation pathways showed that inhibition of proteasomal activity prevented synthesis of AVP-inducible AQP2 mRNA and protein. Once synthesized, AQP2 protein was quickly degraded, a process that involves both the proteasomal and lysosomal pathways. This is the first study that delineates induction and degradation mechanisms of AQP2 endogenously expressed by a renal collecting duct principal cell line.     

Dual influence of aldosterone on AQP2 expression in cultured renal collecting duct principal cells

In the renal collecting duct (CD) the major physiological role of aldosterone is to promote Na+ reabsorption. In addition, aldosterone may also influence CD water permeability elicited by vasopressin (AVP). We have previously shown that endogenous expression of the aquaporin-2 (AQP2) water channel in immortalized mouse cortical CD principal cells (mpkCCDC14) grown on filters is dramatically increased by administration of physiological concentrations of AVP. In the present study, we investigated the influence of aldosterone on AQP2 expression in mpkCCDC14 cells by RNase protection assay and Western blot analysis. Aldosterone reduced AQP2 mRNA and protein expression when administered together with AVP for short periods of time (< or =24 h). For longer periods of time, however, aldosterone increased AQP2 protein expression despite sustained low expression levels of AQP2 mRNA. Both events were dependent on mineralocorticoid receptor occupancy because they were both induced by a low concentration of aldosterone (10-9 m) and were abolished by the mineralocorticoid receptor antagonist canrenoate. Inhibition of lysosomal AQP2 protein degradation increased AQP2 protein expression in AVP-treated cells, an effect that was potentiated by aldosterone. Finally, both aldosterone and actinomycin D delayed AQP2 protein decay following AVP washout, but in a non-cumulative manner. Taken together, our data suggest that aldosterone tightly modulates AQP2 protein expression in cultured mpkCCDC14 cells by increasing AQP2 protein turnover while maintaining low levels of AQP2 mRNA expression.     

Prostaglandin signaling in the renal collecting duct: release, reuptake, and oxidation in the same cell

Prostaglandins mediate autacrine and paracrine signaling over short distances. We used the renal collecting duct as a model system to test the hypothesis that local control of prostaglandin signaling is achieved by expressing inactivation in the same cell as synthesis. Immunocytochemical studies demonstrated that renal collecting ducts in situ express the prostaglandin (PG) synthesis enzyme, cyclooxygenase-1 (COX-1), as well as both components of prostaglandin metabolic inactivation, i.e. the prostaglandin uptake carrier prostaglandin transporter (PGT) and the enzyme 15-hydroxyprostaglandin dehydrogenase. We characterized this system further using the collecting duct cell line Madin-Darby canine kidney (MDCK), which retains COX-2 and prostaglandin dehydrogenase expression but which has lost PGT expression. When we reintroduced PGT, it was correctly sorted to the apical membrane where it altered the sidedness of prostaglandin E2 (PGE2) release, a process we call "vectorial release via sided reuptake." Importantly, although COX-2 and prostaglandin dehydrogenase are expressed in the same MDCK cell, they must be compartmentalized because even in the presence of excess dehydrogenase newly synthesized PGE2 is released largely un-oxidized. However, when PGE2 undergoes first release and then PGT-mediated reuptake, significant oxidation takes place, suggesting that PGT imports PGE2 into the prostaglandin dehydrogenase compartment. Our data are consistent with a new model that offers significant new mechanisms for the fine control of eicosanoid signaling.     

Spatial organisation of AKAP18 and PDE4 isoforms in renal collecting duct principal cells

A plethora of stimuli including hormones and neurotransmitters mediate a rise of the cellular level of cAMP and thereby activation of protein kinase A (PKA). PKA phosphorylates and thereby modulates the activity of a wide range of cellular targets. It is now appreciated that different stimuli induce the activation of PKA at specific sites where the kinase phosphorylates particular substrates in close proximity. The tethering of PKA to cellular compartments is facilitated by A kinase-anchoring proteins (AKAPs). The incorporation of phosphodiesterases (PDEs) into AKAP-based signalling complexes provides gradients of cAMP that regulate PKA activity locally. An example for a process depending on compartmentalised cAMP/PKA signalling is the arginine-vasopressin (AVP)-mediated water reabsorption in renal collecting duct principal cells. Upon activation through AVP, PKA phosphorylates the water channel aquaporin-2 (AQP-2) located on intracellular vesicles. The phosphorylation triggers the redistribution of AQP2 to the plasma membrane. AKAP-anchored PKA has been shown to be involved in AQP2 shuttling. Here, AKAP18 isoforms and members of the PDE4 family of PDEs are shown to be differentially localised in renal principal cells.     

Hypertonicity increases sodium transporters in cortical collecting duct cells independently of PGE2

Cyclooxygenase-2 (COX-2) expression is increased by hypertonicity. Therefore we hypothesized that hypertonicity increased PGE(2) can modulate the sodium transporters (Na(+)/K(+)-ATPase: NKA, epithelial sodium channel: ENaC, and sodium hydrogen exchanger: NHE) in M1 cortical collecting duct (CCD) cells. We demonstrated by immunoblotting a 2-fold increase in NKA expression and activity following hypertonic treatment. α-ENaC was also increased, however sgk1, an ENaC activator, decreased in response to hypertonicity. Other CCD sodium transporters (β-ENaC, NHE) were unchanged. Hypertonicity also increased PGE(2) but EP(4) receptor mRNA was unaltered. PGE(2) increased intracellular Na(+) and cAMP production in M1 cells, but PGE(2)-stimulated cAMP response was attenuated by hypertonicity. Overall, PGE(2) had no effect on sodium transporter levels. Since neither COX inhibition nor EP(4) siRNA altered the induction of NKA, we propose that sodium transporter regulation by hypertonicity is independent of PGE(2). Altogether, these data indicate that despite a concomitant increase in PGE(2) production and sodium transporter expression in hypertonicity, both pathways are acting independently of each other.     

Differentiation properties of renal collecting duct cells in culture

In the present study, we were particularly interested in distinguishing specific patterns of structural and functional proteins in the collecting duct system of neonatal and adult kidneys and in cultured renal collecting duct epithelia in order to ascertain the degree of differentiation in the cultures. We studied the distribution of specific renal collecting duct cell markers using morphological, immunohistochemical and biochemical procedures. Cultured renal collecting duct epithelium undergoes maturation in vitro. Examples of morphological differentiation include the appearance of cilia and microvilli at the apical cell pole, and a basement membrane at the basal aspect of the epithelium. Tight junctions with five to seven strands separate the wide intercellular spaces from the apical cell surface. Physiological maturation from a 'leaky' to a 'tight' epithelium is evident from the acquisition of the alpha-subunit of Na/K-ATPase and the development of a high transepithelial potential difference and resistance. Biochemical differentiation is revealed by the expression of specific proteins. The simple-epithelium cytokeratins, PKK1 and PKK2, which are typical intracellular-matrix proteins of mature collecting duct epithelium, maintain the same distribution in cell culture as in neonatal and adult kidneys. An indicator of maturation in vitro is the expression of the collecting duct-specific proteins, PCD2 and PCD3. Newly developed monoclonal antibodies against these antigens reacted similarly with cultured cells and cells of the mature collecting duct system, but they did not label the embryonic ampullae in the cortex of neonatal rabbit kidneys. In contrast, a third collecting duct-specific protein, PCD1, is not expressed by the cultured cells, which indicates the retention of an embryonic characteristic in vitro. Embryonic collecting duct ampullae of the neonatal kidney in situ contain laminin during their development. Laminin is, however, absent in cultured collecting duct epithelium. Biochemical stimulation of the adenylate cyclase system by arginine vasopressin resulted in a twofold stimulation of the enzyme activity. This degree of stimulation is similar to that found in maturing kidneys of neonatal rabbits and indicates another embryonic feature of the cultures.     

Prostaglandin E2 stimulates cystogenesis through EP4 receptor in IMCD-3 cells

Previously, we demonstrated that prostaglandin E(2) (PGE(2)) induced cAMP and cyst formation through PGE(2) receptor-2 (EP2) activity in human autosomal-dominant polycystic kidney disease (ADPKD) epithelial cells. In this study, we determined the role of EP2 and EP4 receptors in mediating PGE(2) stimulation of cAMP signaling and cystogenesis in mouse renal epithelial cells using the inner medullary collecting duct-3 (IMCD-3) cell line. In contrast to human ADPKD cells, using novel EP2 and EP4 antagonists, we found that IMCD-3 cells expressed functional EP4 but not EP2, which stimulated cAMP formation and led to cyst formation in 3D culture system. The involvement of EP4 receptors in IMCD-3 cells was further supported by the specific effect of EP4 siRNA that inhibited PGE(2)-induced cystogenesis. We also observed different cellular localization of EP2 or EP4 receptors in IMCD-3 transfected cells. Collectively, our results suggest an important role of different expression of EP2 or EP4 receptors in the regulation of cystogenesis.     

Differentiation properties of renal collecting duct cells in culture

In the present study, we were particularly interested in distinguishing specific patterns of structural and functional proteins in the collecting duct system of neonatal and adult kidneys and in cultured renal collecting duct epithelia in order to ascertain the degree of differentiation in the cultures. We studied the distribution of specific renal collecting duct cell markers using morphological, immuno-histochemical and biochemical procedures. Cultured renal collecting duct epithelium undergoes maturation in vitro. Examples of morphological differentiation include the appearance of cilia and microvilli at the apical cell pole, and a basement membrane at the basal aspect of the epithelium. Tight junctions with five to seven strands separate the wide intercellular spaces from the apical cell surface. Physiological maturation from a ‘leaky’ to a ‘tight’ epithelium is evident from the acquisition of the α-subunit of Na/K-ATPase and the development of a high transepithelial potential difference and resistance. Biochemical differentiation is revealed by the expression of specific proteins. The simple-epithelium cytokeratins. PKK1 and PKK2, which are typical intracellular-matrix proteins of mature collecting duct epithelium, maintain the same distribution in cell culture as in neonatal and adult kidneys. An indicator of maturation in vitro is the expression of the collecting duct-specific proteins, P^CD2 and P^CD3. Newly developed monoclonal antibodies against these antigens reacted similarly with cultured cells and cells of the mature collecting duct system, but they did not label the embryonic ampullae in the cortex of neonatal rabbit kidneys. In contrast, a third collecting duct-specific protein, PcDl, is not expressed by the cultured cells, which indicates the retention of an embryonic characteristic in vitro. Embryonic collecting duct ampullae of the neonatal kidney in situ contain laminin during their development. Laminin is. however, absent in cultured collecting duct epithelium. Biochemical stimulation of the adenylate cyclase system by arginine vasopressin resulted in a twofold stimulation of the enzyme activity. This degree of stimulation is similar to that found in maturing kidneys of neonatal rabbits and indicates another embryonic feature of the cultures.     

Cyclic AMP increases cell surface expression of functional Na,K-ATPase units in mammalian cortical collecting duct principal cells

Cyclic AMP (cAMP) stimulates the transport of Na(+) and Na,K-ATPase activity in the renal cortical collecting duct (CCD). The aim of this study was to investigate the mechanism whereby cAMP stimulates the Na,K-ATPase activity in microdissected rat CCDs and cultured mouse mpkCCD(c14) collecting duct cells. db-cAMP (10(-3) M) stimulated by 2-fold the activity of Na,K-ATPase from rat CCDs as well as the ouabain-sensitive component of (86)Rb(+) uptake by rat CCDs (1.7-fold) and cultured mouse CCD cells (1.5-fold). Pretreatment of rat CCDs with saponin increased the total Na,K-ATPase activity without further stimulation by db-cAMP. Western blotting performed after a biotinylation procedure revealed that db-cAMP increased the amount of Na,K-ATPase at the cell surface in both intact rat CCDs (1.7-fold) and cultured cells (1.3-fold), and that this increase was not related to changes in Na,K-ATPase internalization. Brefeldin A and low temperature (20 degrees C) prevented both the db-cAMP-dependent increase in cell surface expression and activity of Na,K-ATPase in both intact rat CCDs and cultured cells. Pretreatment with the intracellular Ca(2+) chelator bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid also blunted the increment in cell surface expression and activity of Na,K-ATPase caused by db-cAMP. In conclusion, these results strongly suggest that the cAMP-dependent stimulation of Na,K-ATPase activity in CCD results from the translocation of active pump units from an intracellular compartment to the plasma membrane.     

MAPK mediation of hypertonicity-stimulated cyclooxygenase-2 expression in renal medullary collecting duct cells

We have previously shown that hypertonicity stimulates cyclooxygenase-2 (COX-2) expression in cultured medullary epithelial cells. The aims of the present study were (i) to examine the role of cytoplasmic signaling through MAPK pathways in tonicity regulation of COX-2 expression in collecting duct cells and (ii) to assess the possible contribution of COX-2 to the survival of inner medullary collecting duct (IMCD) cells under hypertonic conditions. In mIMCD-K2 cells, a cell line derived from mouse IMCDs, hypertonicity induced a marked increase in COX-2 protein expression. The stimulation was reduced significantly by inhibition of MEK1 (PD-98059, 5-50 microm) and p38 (SB-203580, 5-100 microm) and was almost abolished by the combination of the two compounds. To study the role of JNK in tonicity-stimulated COX-2 expression, IMCD-3 cell lines stably transfected with dominant-negative mutants of three JNKs (JNK-1, -2, and -3) were used. Hypertonicity-stimulated COX-2 protein expression was significantly reduced in dominant-negative JNK-2-expressing cells and was unchanged in dominant-negative JNK-1- and JNK-3-expressing cells compared with controls. The reduction of COX-2 expression was associated with greatly reduced viability of dominant-negative JNK-2-expressing cells during hypertonicity treatment. 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) (2-8 microm), an inhibitor of Src kinases, reduced the tonicity-stimulated COX-2 expression in a dose-dependent manner, whereas PP3, an inactive analog of PP2, had no effect. Inhibition of COX-2 activity by NS-398 (30-90 microm) and SC-58236 (10-20 microm) significantly reduced viability of mIMCD-K2 cells subjected to prolonged hypertonic treatment. We conclude that 1) all three members of the MAPK family (ERK, JNK-2, and p38) as well as Src kinases are required for tonicity-stimulated COX-2 expression in mouse collecting duct cells and that 2) COX-2 may play a role in cell survival of medullary cells under hypertonic conditions.     

Proteinase-activated receptor 2 stimulates Na,K-ATPase and sodium reabsorption in native kidney epithelium

Proteinase-activated receptors 2 (PAR2) are expressed in kidney, but their function is mostly unknown. Since PAR2 control ion transport in several epithelia, we searched for an effect on sodium transport in the cortical thick ascending limb of Henle's loop, a nephron segment that avidly reabsorbs NaCl, and for its signaling. Activation of PAR2, by either trypsin or a specific agonist peptide, increased the maximal activity of Na,K-ATPase, its apparent affinity for sodium, the sodium permeability of the paracellular pathway, and the lumen-positive transepithelial voltage, featuring increased NaCl reabsorption. PAR2 activation induced calcium signaling and phosphorylation of ERK1,2. PAR2-induced stimulation of Na,K-ATPase Vmax was fully prevented by inhibition of phospholipase C, of changes in intracellular concentration of calcium, of classical protein kinases C, and of ERK1,2 phosphorylation. PAR2-induced increase in paracellular sodium permeability was mediated by the same signaling cascade. In contrast, increase in the apparent affinity of Na,K-ATPase for sodium, although dependent on phospholipase C, was independent of calcium signaling, was insensitive to inhibitors of classical protein kinases C and of ERK1,2 phosphorylation, but was fully prevented by the nonspecific protein kinase inhibitor staurosporine, as was the increase in transepithelial voltage. In conclusion, PAR2 increases sodium reabsorption in rat thick ascending limb of Henle's loop along both the transcellular and the paracellular pathway. PAR2 effects are mediated in part by a phospholipase C/protein kinase C/ERK1,2 cascade, which increases Na,K-ATPase maximal activity and the paracellular sodium permeability, and by a different phospholipase C-dependent, staurosporine-sensitive cascade that controls the sodium affinity of Na,K-ATPase.     

Angiotensin II stimulates epithelial sodium channels in the cortical collecting duct of the rat kidney

We examined the effect of angiotensin II (ANG II) on epithelial Na(+) channel (ENaC) in the rat cortical collecting duct (CCD) with single-channel and the perforated whole cell patch-clamp recording. Application of 50 nM ANG II increased ENaC activity, defined by NP(o) (a product of channel numbers and open probability), and the amiloride-sensitive whole cell Na currents by twofold. The stimulatory effect of ANG II on ENaC was absent in the presence of losartan, suggesting that the effect of ANG II on ENaC was mediated by ANG II type 1 receptor. Moreover, depletion of intracellular Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM failed to abolish the stimulatory effect of ANG II on ENaC but inhibiting protein kinase C (PKC) abolished the effect of ANG II, suggesting that the effect of ANG II was the result of stimulating Ca(2+)-independent PKC. This notion was also suggested by the experiments in which stimulation of PKC with phorbol ester derivative mimicked the effect of ANG II and increased amiloride-sensitive Na currents in the principal cell, an effect that was not abolished by treatment of the CCD with BAPTA-AM. Also, inhibition of NADPH oxidase (NOX) with diphenyleneiodonium chloride abolished the stimulatory effect of ANG II on ENaC and application of superoxide donors, pyrogallol or xanthine and xanthine oxidase, significantly increased ENaC activity. Moreover, addition of ANG II or H(2)O(2) diminished the arachidonic acid (AA)-induced inhibition of ENaC in the CCD. We conclude that ANG II stimulates ENaC in the CCD through a Ca(2+)-independent PKC pathway that activates NOX thereby increasing superoxide generation. The stimulatory effect of ANG II on ENaC may be partially the result of blocking AA-induced inhibition of ENaC.     

Prostaglandin E2 induced changes in renal blood flow, renal interstitial hydrostatic pressure and sodium excretion in the rat

Prostaglandin E2, when infused into the renal artery of the dog, is a vasodilator and increases both renal interstitial hydrostatic pressure and sodium excretion. Similar studies in the rat, however, have been inconclusive. The present study examined the effect of prostaglandin E2 infusion into the renal interstitium, by means of a chronically implanted matrix, on renal blood flow, renal interstitial hydrostatic pressure and sodium excretion in the rat. Prostaglandin E2 was continuously infused directly into the kidney interstitium to mimic endogenous prostaglandin E2 production by renal cells. The maximum change in each of these parameters occurred when 10(-5) M PGE2 was infused. Renal blood flow increased from 4.70 +/- 0.91 to 5.45 +/- 0.35 ml/min (p less than 0.05) while renal interstitial hydrostatic pressure decreased from 3.9 +/- 0.4 to 2.6 +/- 0.5 mmHg (p less than 0.05) and fractional excretion of sodium decreased from 1.02 +/- 0.20 to 0.61 +/- 0.12% (p less than 0.05). Thus, the present study demonstrates that renal interstitial infusion of prostaglandin E2 increases total renal blood flow but decreases both renal interstitial hydrostatic pressure and urinary sodium excretion in the rat.