Mechanisms and regulation of ion transport in the renal collecting duct

1. In the present paper the ion transport function of the renal mammalian collecting duct and its regulation is briefly reviewed. 2. The epithelium is characterized by different cell types: principal cells, intercalated cells, type A, and intercalated cells, type B. 3. Using microelectrodes and various microscopic techniques active Na+ absorption as well as K+ secretion has been localized to the principal cells, while Cl- absorption was found to proceed largely, though not exclusively, through the tight junctions between cells. 4. Intercalated cells of type A, which prevail in the outer medullary collecting duct, secrete H+ and intercalated cells of type B, which are most frequent in the late cortical collecting duct, secrete HCO3-. 5. This specialization of different cells in transporting individual ions provides the basis for the efficient adaptive regulation of urinary ion excretion.     

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.     

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.     

sgk is an aldosterone-induced kinase in the renal collecting duct. Effects on epithelial na+ channels.

The early phase of the stimulatory effect of aldosterone on sodium reabsorption in renal epithelia is thought to involve activation of apical sodium channels. However, the genes initiating this effect are unknown. We used a combination of polymerase chain reaction-based subtractive hybridization and differential display techniques to identify aldosterone-regulated immediate early genes in renal mineralocorticoid target cells. We report here that aldosterone rapidly increases mRNA levels of a putative Ser/Thr kinase, sgk (or serum- and glucocorticoid-regulated kinase), in its native target cells, i.e. in cortical collecting duct cells. The effect occurs within 30 min of the addition of aldosterone, is mediated through mineralocorticoid receptors, and does not require de novo protein synthesis. The full-length sequences of rabbit and mouse sgk cDNAs were determined. Both cDNAs show significant homology to rat and human sgk (88-94% at the nucleotide level, and 96-99% at the amino acid level). Coexpression of the mouse sgk in Xenopus oocytes with the three subunits of the epithelial Na+ channel results in a significantly enhanced Na+ current. These results suggest that sgk is an immediate early aldosterone-induced gene, and this protein kinase plays an important role in the early phase of aldosterone-stimulated Na+ transport.     

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.     

Calcium-sensing receptor regulation of renal mineral ion transport

Extracellular calcium has long been known to affect the rate and magnitude of renal calcium and phosphate recovery. In this review, we consider some of these findings in light of our present understanding of the tubular localization of the calcium-sensing receptor (CaSR). Experiments directly implicating the CaSR in regulating calcium and phosphate transport are described. These results point to an important role of the CaSR in regulating PTH-dependent calcium absorption by cortical thick ascending limbs and on PTH-sensitive proximal tubule phosphate transport. Possible avenues for further investigation are suggested.     

Mechanisms of vasopressin-induced intracellular Ca2+ oscillations in rat inner medullary collecting duct

Arginine vasopressin (AVP) causes increase in intracellular Ca(2+) concentration with an oscillatory pattern. Ca(2+) mobilization is required for AVP-stimulated apical exocytosis in inner medullary collecting duct (IMCD). The mechanistic basis of these Ca(2+) oscillations was investigated by confocal fluorescence microscopy and flash photolysis of caged molecules in perfused IMCD. Photorelease of caged cAMP and direct activation of ryanodine receptors (RyRs) by photorelease of caged cyclic ADP-ribose (cADPR) both mimicked the AVP-induced Ca(2+) oscillations. Preincubation of IMCD with 100 μM 8-bromo-cADPR (a competitive inhibitor of cADPR) delayed the onset and attenuated the magnitude of AVP-induced Ca(2+) oscillations. These observations indicate that the cADPR/RyR pathway is capable of supporting Ca(2+) oscillations and endogenous cADPR plays a major role in the AVP-induced Ca(2+) oscillations in IMCD. In contrast, photorelease of caged inositol 1,4,5-trisphosphate (IP(3)) induced Ca(2+) release but did not maintain sustained Ca(2+) oscillations. Removal of extracellular Ca(2+) halted ongoing AVP-mediated Ca(2+) oscillation, suggesting that it requires extracellular Ca(2+) entry. AVP-induced Ca(2+) oscillation was unaffected by nifedipine. Intracellular Ca(2+) store depletion induced by 20 μM thapsigargin in Ca(2+)-free medium triggered store-operated Ca(2+) entry (SOCE) in IMCD, which was attenuated by 1 μM GdCl(3) and 50 μM SKF-96365. After incubation of IMCD with 1 nM AVP in Ca(2+)-free medium, application of extracellular Ca(2+) also triggered Ca(2+) influx, which was sensitive to GdCl(3) and SKF-96365. In summary, our observations are consistent with the notion that AVP-induced Ca(2+) oscillations in IMCD are mediated by the interplay of Ca(2+) release from RyRs and a Ca(2+) influx mechanism involving nonselective cation channels that resembles SOCE.     

Characterization of an in vitro system of human renal papillary collecting duct cells

Summary We have developed an in vitro model of human papillary collecting duct cells isolated from cadaver kidneys using methods similar to those we previously reported for the isolation of human proximal tubule cells. To date we have isolated papillary collecting duct cells from 100 normal human kidneys. Papillae were dissected and digested in Cellgro containing 400 U/ml collagenase. Cells were plated on fibronectin-coated culture flasks at a density of 10⁴ live cells/ml in Cellgro supplemented with insulin and 10% fetal bovine serum. Confluent monolayers, which were able to withstand 600 mOsm for 8 h, were obtained within 10 to 15 d. Cells of primary isolates and first passages exhibited epithelial cell ultrastructure including cell junctions, microvilli, and cilia. A dark-brown reaction product was observed in these cells when stained by the immunoperoxidase method with peroxidase-labeled peanut lectin (Arachis hypogaea), which binds specifically to human distal tubule and collecting duct cells. These cells were negative for Factor-VIII (a marker for endothelial cells) and γ-glutamyltransferase (a marker for proximal tubule cells). High activities of the glycolytic enzyme pyruvate kinase and arginine vasopressin-stimulated cAMP production in these cells are consistent with a distal nephron origin. The results indicate that human collecting duct cells can be isolated and cultured to provide an in vitro system to probe pathogenetic mechanisms of potential nephrotoxins. Part of this work was presented at a Symposium of the Center for Alternatives to Animal Testing, April 4–5, 1989, Johns Hopkins Medical Institutions, Baltimore, MD 21205. This work was supported in part by grants R01-AI24179, PO1-A804393 for the Public Health Service, U.S. Department of Health and Human Services, and by a grant from the National Kidney Foundation, Baltimore, MD affiliate.     

Oxygen regulation of the epithelial Na channel in the collecting duct

The PO(2) within the kidney changes dramatically from cortex to medulla. The present experiments examined the effect of changing PO(2) on epithelial Na channel (ENaC)-mediated Na transport in the collecting duct using the mpkCCD-c14 cell line. Decreasing ambient O(2) concentration from 20 to 8% decreased ENaC activity by 40%; increasing O(2) content to 40% increased ENaC activity by 50%. The O(2) effect required several hours to develop and was not mimicked by the acid pH that developed in monolayers incubated in low-O(2) medium. Corticosteroids increased ENaC activity at each O(2) concentration; there was no interaction. The pathways for O(2) and steroid regulation of ENaC are different since O(2) did not substantially affect Sgk1, α-ENaC, Gilz, or Usp2-45 mRNA levels, genes involved in steroid-mediated ENaC regulation. The regulation of ENaC activity by these levels of O(2) appears not to be mediated by changes in hypoxia-inducible factor-1α or -2α activity or a change in AMP kinase activity. Changes in O(2) concentration had minimal effect on α- or γ-ENaC mRNA and protein levels; there were moderate effects on β-ENaC levels. However, 40% O(2) induced substantially greater total β- and γ-ENaC on the apical surface compared with 8% O(2); both subunits demonstrated a greater increase in the mature forms. The α-ENaC subunit was difficult to detect on the apical surface, perhaps because our antibodies do not recognize the major mature form. These results identify a mechanism of ENaC regulation that may be important in different regions of the kidney and in responses to changes in dietary NaCl.     

Role of the Bacillus subtilis gsiA gene in regulation of early sporulation gene expression.

The Bacillus subtilis gsiA operon was induced rapidly, but transiently, as cells entered the stationary phase in nutrient broth medium. A mutation at the gsiC locus caused sporulation to be defective and expression of gsiA to be elevated and prolonged. The sporulation defect in this strain was apparently due to persistent expression of gsiA, since a gsiA null mutation restored sporulation to wild-type levels. Detailed mapping experiments revealed that the gsiC82 mutation lies within the kinA gene, which encodes the histidine protein kinase member of a two-component regulatory system. Since mutations in this gene caused a substantial blockage in expression of spoIIA, spoIIG, and spoIID genes, it seems that accumulation of a product of the gsiA operon interferes with sporulation by blocking the completion of stage II. It apparently does so by inhibiting or counteracting the activity of KinA.     

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.     

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.     

Augmentation of vasopressin-induced cAMP production by high potassium in rat renal papillary collecting tubule cells in culture.

The effect of high potassium, 60 mM KCl, on the cellular action of arginine vasopressin (AVP) was studied in rat renal papillary collecting tubule cells in culture. In the presence of 0.5 mM 3-isobutyl-1-methylxanthine AVP-induced cAMP production was enhanced by pretreatment of the cells with 60 mM KCl. Such an enhancement was not found in cells pretreated with Ca(2+)-free medium containing 1 mM EGTA or in Na(+)-free medium, which rather reduced AVP-induced cAMP production. Similar results were obtained with the blockers of cellular Ca2+ uptake, 1 x 10(-4) M verapamil and 1 x 10(-5) M nifedipine. The 60 mM KCl elevated the cellular sodium concentration ([Na+]i) from 15.1 to 18.8 mM, cellular pH (pHi) from 7.18 to 7.32, and basal cellular free calcium concentration ([Ca2+]i). These results indicate that high potassium promptly augments AVP-induced cAMP production in renal papillary collecting tubule cells. This effect is based on the alkalinized pHi and the increased [Ca2+]i.     

Regulation by vasopressin of NaCl absorption in the renal collecting duct

In the kidney, the fine control of NaCl absorption takes place in the distal nephron and is controlled by aldosterone and vasopressin. This review summarizes the effects of vasopressin on Na+ transport mediated by the amiloride-sensitive epithelial sodium channel (ENaC) and the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel in immortalized or primary cultured cortical collecting duct cells, expressing either the wild-type ENaC subunits, or mutations, or deletions of the PY domain of the beta- or gamma-ENaC subunits responsible for Liddle's syndrome, an inherited form of hypertension due to excessive salt absorption.     

Heterogeneity of tight junctions along the collecting duct in the renal medulla

Summary The tight junctions along the medullary collecting duct in the kidneys of the rat and the rabbit were studied with freeze-fracture electron microscopy and quantitated according to the number of strands and the apico-basal depth (nm) of the junctions.The most elaborate tight junctions were found in the inner stripe of the outer medulla; rat: 10.6±0.8 strands and 205±24nm; rabbit: 11.6±2.4 strands and 291±55 nm.The elaboration of the tight junctions decreased continuously towards the papillary tip. Inner zone I; rat: 9.3±2.6 strands and 186±38nm, rabbit: 9.5±2.3 strands and 247±59nm. Inner zone II; rat: 7.1±2.2 strands and 129±32nm, rabbit: 8.5±1.4 strands and 199±26nm. Inner zone III; rat: 6.0±1.6 strands and 111 + 19 nm, rabbit: 7.0±1.5 strands and 183±43 nm. In the inner zone III comprising the papillary tip tight junctions with only 1–3 strands were not infrequently seen. Preliminary findings in the kidney of the golden hamster indicate a similar decline of junctional tightness along the collecting duct.These morphological observations suggest that the permeability of the paracellular pathway of the medullary collecting duct increases towards the tip of the papilla, especially in the rat. The functional implications for the medullary recycling of urea and electrolytes, and for the urinary concentrating mechanism are discussed.In addition, the tight junctions of the papillary epithelium are described.     

Action of aldosterone on cultured renal collecting duct cells during the latent period.

The effects of aldosterone on protein synthesis in the latent period were investigated on cultured renal collecting duct cells from neonatal rabbit kidneys. Tissue was incubated with radioactively labelled uridine and amino acids and then precipitated with trichloroacetic acid in order to determine the intracellular precursor pool and identify new synthesis of RNA and protein. During the latent period, aldosterone increased the intracellular radioactive uridine pool and total radioactive RNA content already 20 and 60 min after its application; conversely 40 min after aldosterone introduction, no stimulation was found. Further experiments revealed that the intracellular radioactive amino acid pool was generally increased by aldosterone after 20, 40 and 60 min, while a distinct increased radioactive protein content was found to be induced by aldosterone only after 40 min. This indicates that aldosterone increases the uptake of RNA and protein precursors and the new synthesis of RNA and proteins. These events seem to to be regulated not continuously but intermittently. The induced proteins possibly take part in the mediation of the early hormone response. Experiments with the aldosterone antagonist, spironolactone, provide evidence for the specificity of the described hormone effects. The results after application of the Na+ channel blocker, amiloride, and the Na+/K(+)-ATPase inhibitor, G-strophanthin, indicate that the aldosterone effects are controlled by Na+ channels and Na+ pumps and therefore by the intracellular Na+ content. The inhibitory effect of cycloheximide on the aldosterone-induced protein synthesis indicates the role of these proteins on the hormone-stimulated Na+ transport.