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Am J Physiol Renal Physiol 283: F1337-F1350, 2002. First published July 24, 2002; doi:10.1152/ajprenal.00165.2002
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Vol. 283, Issue 6, F1337-F1350, December 2002

Electrolyte and fluid secretion by cultured human inner medullary collecting duct cells

Darren P. Wallace1,2,3, Marcy Christensen1, Gail Reif1, Franck Belibi1, Brantley Thrasher4, Duke Herrell4, and Jared J. Grantham1,2,3

1 Kidney Institute and Departments of 2 Biochemistry and Molecular Biology, 3 Medicine, and 4 Urology, University of Kansas Medical Center, Kansas City, Kansas 66160


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Inner medullary collecting ducts (IMCD) are the final nephron segments through which urine flows. To investigate epithelial ion transport in human IMCD, we established primary cell cultures from initial (hIMCDi) and terminal (hIMCDt) inner medullary regions of human kidneys. AVP, PGE2, and forskolin increased cAMP in both hIMCDi and hIMCDt cells. The effects of AVP and PGE2 were greatest in hIMCDi; however, forskolin increased cAMP to the same extent in hIMCDi and hIMCDt. Basal short-circuit current (ISC) of hIMCDi monolayers was 1.4 ± 0.5 µA/cm2 and was inhibited by benzamil, a Na+ channel blocker. 8-Bromo-cAMP, AVP, PGE2, and forskolin increased ISC; the current was reduced by blocking PKA, apical Cl- channels, basolateral NKCC1 (a Na+-K+-2Cl- cotransporter), and basolateral Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchangers. In fluid transport studies, hIMCDi monolayers absorbed fluid in the basal state and forskolin reversed net fluid transport to secretion. In hIMCDt monolayers, basal current was not different from zero and cAMP had no effect on ISC. We conclude that AVP and PGE2 stimulate cAMP-dependent Cl- secretion by hIMCDi cells, but not hIMCDt cells, in vitro. We suggest that salt secretion at specialized sites along human collecting ducts may be important in the formation of the final urine.

kidney; chloride transport; salt secretion


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

INNER MEDULLARY COLLECTING DUCTS (IMCD), the last intrarenal sites in contact with the tubular fluid, are in a strategic location to have a significant impact on the composition of the urine. Proteins and mRNA for diverse electrolyte and fluid transport processes have been identified in primary cultures of rat IMCD cells (16, 48) and mIMCD-K2, a mouse IMCD cell line (52, 53). They include, but are not limited to, the epithelial Na+ channel (ENaC) and the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel, components that are critical to the regulation of salt and fluid transport by epithelia including the airway, intestine, and exocrine glands (2, 21). Whereas most studies of collecting duct function have focused on the absorptive pathways, recent studies demonstrated that collecting ducts have the capacity to secrete as well as absorb solutes and fluid (39, 54, 55, 57). Net salt and fluid transport is the sum of absorptive and secretory processes; consequently, changes in the rate of either salt absorption or secretion would determine the composition and the volume of the final urine.

The regulation of transport mechanisms has been studied in rat IMCD and cultured rat IMCD cells. Atrial natriuretic peptide (ANP) was shown to inhibit renal Na+ absorption (63) involving both ENaC phosphorylation and phosphorylation-independent mechanisms (47). Vandorpe et al. (53) characterized a cyclic nucleotide-gated (CNG) nonselective cation channel in IMCD cells that may also contribute to Na+ absorption. Although ENaC and CNG channels were shown to have distinctive characteristics, both were inhibited by benzamil (53). In many epithelia, cAMP agonists modulate net NaCl and fluid transport by stimulating Cl- secretion via CFTR, a cAMP-dependent Cl- channel, located in the apical membrane. Moreover, the activation of CFTR may diminish Na+ absorption through inhibition of ENaC (19, 44). In this regard, cAMP agonists may play an important role in the regulation of net solute and fluid transport within collecting ducts. To a large extent, our knowledge of electrolyte transport by IMCD has come from studies of isolated rat IMCD segments (39, 55, 57) and cultured rat and mouse IMCD cells (16, 18, 22-24, 48, 52, 53). Although rodent models are clearly important in defining electrolyte and fluid transport mechanisms in renal collecting ducts, there are major morphological and functional differences in collecting duct subsegments among the different species (5, 8, 27, 31). Thus mechanisms defined in rodent systems cannot be uniquely extrapolated to human collecting ducts. In the current study, we examined the functional contributions of classic modulators to electrolyte and fluid transport by initial (IMCDi) and terminal (IMCDt) IMCD cultured from human kidneys.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Human kidney tissue. Discarded human kidney tissue was obtained with the assistance of two surgeons in the urology section at the Kansas University Medical Center (KUMC) and the Midwest Transplant Network (MTN; Kansas City, KS), an organ retrieval agency. This protocol was approved by the KUMC Human Subjects Committee. Eleven kidney specimens were obtained from normal regions of kidneys removed for treatment of renal carcinomas. Renal tissue judged to be normal by histological examination was placed in sterile ice-cold PBS and brought to the lab for dissection. Four kidneys obtained from MTN had been perfused with an electrolyte preservation solution in preparation for transplantation. Kidneys that were deemed unsuitable for transplantation because of anomalous vasculature were maintained in ice-cold solution and delivered to the laboratory within 24 h from the time the renal vessels were clamped. We detected no difference in the quality or characteristics of the cultures obtained from the two sources.

Kidneys were cut sagittally to expose papillae protruding into the renal calyces. Papillae were isolated, and the adjoining cortical tissue was removed ~2 mm below the cortico-medullary boundary, leaving most of the medulla (Fig. 1) for study. In most cases, the terminal 4 mm of the papilla was removed and cultured separately, designated as hIMCDt. In kidneys perfused for transplantation, the boundary between the outer medulla (inner stripe) and the initial inner medulla was difficult to identify; therefore, the absolute distance from the papillary tip was used to define the initial inner medulla. A region of ~4 mm was removed from the upper portion of the inner medulla and designated as hIMCDi. Tissues used for morphological examination by electron microscopy were fixed in cold 2% glutaraldehyde in HEPES buffer. For lectin staining and immunolocalization of Na-K-ATPase, tissues were fixed in 4% paraformaldehyde (PFA) in PBS overnight at 4°C.


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  		Fig. 1.   A: Schematic of a cross section through a single renal papilla of a human kidney. The dimensions are estimated based on tissue obtained from adult male kidneys. The human medulla is much broader at the corticomedullary boundary than in the rat. The transitional zones of the subsegments of human kidneys have variable lengths, and the boundary between the outer medulla and the initial inner medulla is often difficult to distinguish. Thus, in the present study, ~11 mm of the inner medulla were removed from each papilla and separated into initial (IMCDi, upper 4 mm) and terminal (IMCDt, lower 4 mm) inner medulla collecting duct (IMCD). The transitional region between IMCDi and IMCDt was frequently discarded. B: schematic of a coronal section of a rat kidney through the single papilla for comparison to that of the human. The different regions of the medulla in the rat kidney are readily identifiable. The drawings are not drawn to scale. OMCD, outer medullary collecting duct. Anatomically, the rat IMCD can be subdivided into the outermost one-third (IMCD1), the middle one-third (IMCD2), and the terminal one-third (IMCD3).

Cell culture preparation. The method for preparing primary cultures was published previously (15, 56). Preparations of hIMCDt and hIMCDi cells were handled identically. The tissue was minced and digested in DME-F12 (1:1 vol/vol) mixture containing 220 IU/ml collagenase (type IV; Worthington Biochemical, Lakewood, NJ) and 100 IU/ml penicillin G-0.1 mg/ml streptomycin (P/S). The suspension of tissue was incubated at 37°C for at least 6 h or until the epithelial cells detached from the fibrous connective tissue. Collagenase digestion was stopped by adding FBS to a final concentration of 10%. The suspension was centrifuged, and the pellet was rinsed twice and resuspended in DME-F12 supplemented with 5% FBS, 5 µg/ml insulin, 5 µg/ml transferrin, and 5 ng/ml sodium selenite (ITS; Collaborative Biomedical Products, Bedford, MA) and P/S. The cells were transferred to a T75 culture flask and allowed to attach to the surface overnight. Unattached tissue and debris were removed the next morning, and fresh medium was added to the flask. After the cells had reached 70-80% confluence in the flask, they were lifted from the plastic with a trypsin-EDTA solution (Sigma) and counted with a hemocytometer.

Lectin staining. Normal human kidney tissue and cell monolayers grown either on eight-well chamber slides or permeable supports (Snapwell-Clear, 12-mm diameter; CoStar, Cambridge, MA) were examined with segment-specific lectins. Two lectins that bind selectively to collecting ducts, Dolichos biflorus agglutinin (DBA) and Arachis hypogaea agglutinin (peanut agglutinin; PNA) were used for lectin profiling of human IMCD cell cultures (14, 33). DBA and PNA were conjugated to horseradish peroxidase (HRP) and visualized with 3,3'-diaminobenzidine (DAB) (see Figs. 3 and 4). To confirm lectin-HRP staining, we used DBA and PNA conjugated to rhodamine (data not shown). The concentration of the lectins used in the study was 50 µg/ml. Specificity of lectin binding was determined by preincubating the lectins with the appropriate inhibiting sugars (14).

Preparation of monolayers on permeable supports. Primary cells were seeded at a density of 2.5 × 105 cells/1.13 cm2 on permeable supports (Snapwell-Tissue Culture Treated, 12-mm diameter; CoStar) as described previously (56). These supports have a lower inherent electrical resistance than the Snapwell-Clear. After 3 days in culture, the DME-F12, ITS, and P/S medium containing 5% FBS was changed to a medium containing 1% FBS. The reduced serum concentration increased and stabilized transepithelial electrical resistance (TER). Most experiments were performed 7-10 days after the cells were seeded; however, the monolayers responded to agonists after several weeks when maintained in the 1% FBS medium.

Electrical measurements. Snapwell supports containing confluent monolayers were inserted into modified Ussing chambers (Harvard Apparatus, Hollison, MA), and both surfaces of the cell monolayer were bathed in a HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-Ringer solution maintained at 37°C and equilibrated in 5% CO2-95% O2 (56). Two dual-voltage-clamp devices (Warner Instruments, Hamden, CT), each attached to four electrodes per chamber, were used to measure transepithelial potential difference (PD), short-circuit current (ISC), and TER in four monolayers simultaneously. IMCDi cells developed TER ranging from 100 to 500 Omega  · cm2; TER values of the IMCDt monolayers were lower (80-120 Omega  · cm2). Positive ISC reflects the active transport of cations (e.g., Na+) from the apical to basolateral media or the active transport of anions (e.g., Cl-) from the basolateral to apical media. ISC was continuously monitored and recorded with a chart recorder.

Fluid transport measurements. Dispersed hIMCDi cells were seeded (1 × 106 cells/4.52 cm2) on 24.5-mm-diameter cell culture supports (Transwell-Col; Costar) and incubated for 3 days in medium containing 5% FBS. After 3 days, the serum concentration was reduced to 1%. hIMCD cell monolayers developed a uniform, cuboidal appearance when grown on permeable supports (Fig. 2F). Rat IMCD cells grown in this manner retain many of the characteristics expressed in IMCD in situ (23).


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  		Fig. 2.   Morphology of human IMCD (hIMCD) in situ and cultured hIMCD cell monolayers grown on permeable supports. Electron microphotographs of initial hIMCD (hIMCDi; A), terminal hIMCD (hIMCDt; B), cultured hIMCDi cell monolayer (C), cultured hIMCDt cell monolayer (D), and apical junctional complex between two hIMCDi cells (E) are shown. Light microphotographs of a hIMCDi cell monolayer viewed with a phase microscope (F) and hematoxylin and eosin stain of a paraffin section of a hIMCDi cell monolayer (G) are also shown. In C, D, and F, the cells were grown on a tissue culture-treated Snapwell. The pores within the Snapwell are visible as parallel vertical lines. In F, cells were grown on a more transparent Transwell-Col.

The procedure for measuring fluid transport across epithelial cell monolayers was published previously (34, 59). Briefly, medium bathing the apical (upper) surface of the cell monolayer was removed and replaced with 200 µl of DME-F12 medium containing 1% FBS, ITS, and P/S. Sterile, water-saturated mineral oil was layered above the fluid to prevent fluid evaporation. Transwell supports containing the cell layer were placed in a six-well culture plate; each well contained 2.5 ml of basolateral medium. The fluid-oil mixture was collected after 24 h, and the volume of fluid was determined with calibrated microcapillary tubes (Drummond, Broomall, PA). The volume of fluid transported across the epithelium, expressed in microliters per hour per square centimeter, was determined from the change in volume over the 24-h period.

Immunostaining method. A monoclonal antibody to the alpha 1-subunit of Na-K-ATPase (clone M7-PB-E9; Affinity BioReagents, Golden, CO) was used to examine membrane polarity of hIMCDi cell monolayers grown on permeable supports. The immunostaining procedure was described previously (13). Briefly, hIMCDi cell monolayers were fixed in 4% PFA and permeabilized in 100% methanol at -20°C for 20 min. PBS containing 0.2% BSA, 5% goat serum, and 50 mM NH4Cl was used to block nonspecific binding sites. Endogenous biotin was blocked with an avidin-biotin blocking kit (Vector, Burlingame, CA). Monolayers were incubated in primary antibody (diluted 1:50 in 0.1% goat serum in PBS) for 2 h at room temperature. After several rinses, the secondary antibody (goat anti-mouse conjugated to biotin) in PBS plus 0.1% goat serum was added to the cell monolayers for 2 h at room temperature. Staining was visualized by incubating the monolayers in ExtrAvidin conjugated to peroxidase (Sigma) and developed with DAB (brown precipitate). Similar results were obtained with a fluorescein isothiocyanate-conjugated secondary goat anti-mouse antibody. We examined Na-K-ATPase localization in paraffin sections of fixed human initial inner medulla with a method similar to that described above.

Immunoblot method. Cell membrane extracts were prepared from fresh initial inner medullas from two normal human kidneys and hIMCDi cell monolayers grown in plastic petri dishes. Fresh inner medullas were isolated, rapidly frozen in liquid N2, and kept frozen at -80°C until cell membrane extracts were prepared. Crude cell lysates and membrane extracts were prepared at 4°C. Tissues were homogenized with a Polytron homogenizer (Brinkman, Westbury, NY) in lysate buffer containing 50 mM Tris (pH 7.4 with HCl), 10 mM imidazole, 0.3 M sucrose, and protease inhibitors [104 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 0.08 mM aprotinin, 2.1 mM leupeptin, 3.6 mM bestatin, 1.5 mM pepstatin A, 1 µg/ml antipain, 100 µM benzamidine, 1 µM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride], and cell lysates were further homogenized with a glass Dounce. The homogenates were centrifuged at 10,000 g for 15 min at 4°C to remove nuclei and debris. The supernatants were collected and centrifuged at 100,000 g for 90 min at 4°C. The cell membrane pellets were resuspended in 50 mM Tris (pH 7.4) containing 1% (vol/vol) Nonidet P-40, 0.1% SDS, and 0.5% sodium deoxycholate and protease inhibitors (same as above). Protein concentrations in each of the membrane lysates were determined by Pierce bicinchoninic acid protein assay (Rockford, IL).

Western blot analysis was used to determine the expression of NKCC1, the secretory form of Na+-K+-Cl- cotransporter, and AE2 anion exchanger, a member of the Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger family, in cell membrane fractions of human initial inner medullas and cultured hIMCDi cells. Immunoblotting for NKCC1 was performed with a mouse monoclonal antibody (T4, supernatant; Developmental Studies Hybridoma Bank, Iowa City, IA) directed against a 38-kDa fragment of the COOH terminus of NKCC1 from human colonic crypt (T84 cells). This antibody was described previously (10, 26). A T84 cell lysate was used as a positive control. These cells were originally purchased from American Type Culture Collection and maintained in culture as previously described (58). For AE2 immunoblotting, we used an antibody raised against the COOH terminus of mouse AE2 amino acids 1224-1237 (a generous gift from Dr. S. Alper, Harvard Medical School, Boston, MA). This antibody has been shown to stain rat and mouse IMCD (1, 49)

T84 and primary cultures of IMCDi cells were grown to confluence in petri dishes. Culture medium was removed, and the cells were washed three times in ice-cold PBS. Cells were scraped and collected and then homogenized with a glass Dounce in lysate buffer (same composition as used for tissue). Isolation of membrane proteins was carried out as described above.

A Mini-Protean III cell (Bio-Rad Laboratories) was used to resolve membrane proteins by 7.5% SDS-PAGE with a method described previously (61). Protein samples (10 µg for T84 and 20 µg for tissue and hIMCDi cells) were mixed with equal volume of 2× sample buffer (132 mM Tris · HCl pH 6.8, 4.2% SDS, 0.005% bromophenol blue, 21% glycerol, 0.72 M beta -mercaptoethanol, 20 mM dithiothreitol) and denatured at 37°C for 30 min. Proteins were transferred to a nitrocellulose membrane and blocked for 30 min at room temperature with blotting buffer containing 5% (wt/vol) nonfat powdered milk in TBS-T (20 mM Tris · HCl, pH 8.0, 137 mM NaCl, and 0.05% Tween 20). The nitrocellulose membranes were incubated overnight at 4°C in the blotting buffer containing antibody. The membranes were washed several times in TBS-T and incubated in anti-mouse antibody conjugated to HRP diluted in 5% nonfat milk in TBS-T for 60 min. The membranes were washed several times, and NKCC1 and AE2 proteins were visualized with an enhanced chemiluminescence system (ECL; Amersham Life Sciences, Arlington Heights, IL).

cAMP measurements. hIMCD cells were seeded (7.3 × 105 cells/0.33 cm2) on cell culture supports (Transwell-Col, 6.5-mm diameter; Costar) and grown as confluent monolayers under growth conditions similar to those used in the fluid transport experiments. Medium was changed to Ringer solution containing 10 µM benzamil in the apical reservoir for 15 min before the experiment (similar to ISC experiments). Basolateral medium was changed to a medium containing AVP, PGE2, or forskolin for 15 min. The medium was removed, and a 80% methanol-20% water mixture was used to extract cAMP from the cells. cAMP content was determined with an enzyme immunoassay system (Amersham Pharmacia Biotech, Little Chalfont, UK).

Statistics. Data are presented as means ± SE. Instat (GraphPad, San Diego, CA), a statistical package, was used for data analysis. Where appropriate, Student's t-test or one-way ANOVA and the Student-Newman-Keuls multiple-comparison posttest was used to determine statistical significance. Data groups containing heterogeneous variances indicated by Bartlett's test were analyzed with the nonparametric tests Mann-Whitney U-test or the Kruskal-Wallis nonparametric ANOVA and Dunn's posttest. P < 0.05 was taken to indicate statistical significance.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Morphological properties of hIMCDi and hIMCDt cell monolayers. The initial and terminal regions of the inner medulla of human kidneys were cultured separately to compare the functional response to cAMP agonists in hIMCDi and hIMCDt cells. Primary cultures of IMCD cells grown as confluent monolayers on permeable supports for 10 days developed a "cobblestone" appearance (Fig. 2, F and G). The cells appeared morphologically similar to rat IMCD cell monolayers described by Light et al. (23), and there were no measurable differences in the appearance of the hIMCDi and hIMCDt monolayers under low-magnitude light microscopy. Electron microscopy of the ultrastructure of hIMCDi and hIMCDt monolayers grown on Snapwell revealed the cell monolayers to be polarized, with basal surfaces of the cells attached to the filter supports (Fig. 2, C and D) and adjacent cells joined by apical junctional complexes (Fig. 2E). To a great extent, primary cultures of hIMCDi and hIMCDt cells appeared to have retained the basic cell appearance of collecting duct cells in situ (Fig. 2, A and B).

hIMCDi cells grown on glass stained intensely for Pan cytokeratin antibody (Sigma), indicating that the cells were of epithelial origin (Fig. 3A). Lectins bind to specific sugar residues that are uniquely distributed in different parts of the nephron (14). Fluorochrome- and HRP-labeled lectins were used as microscopic markers to characterize the hIMCDi and hIMCDt cells in culture. DBA, a lectin that binds preferentially to collecting duct cells in the inner medulla (20, 33), showed strong, distinct staining in hIMCDi cells (Fig. 3C). Nearly all cells were stained with DBA conjugated with HRP (visualized with DAB); however, the intensity of staining varied. Preincubation of DBA with 0.2 M N-acetylgalactosamine, a competing sugar that has a high affinity to DBA, inhibited staining (Fig. 3D). PNA, another collecting duct lectin, bound to the apical surface of hIMCD cells (Fig. 4A), but not proximal tubules or glomerular cells, within the cortex (Fig. 4B). PNA also showed strong, distinct staining of hIMCDi and hIMCDt cell monolayers grown on glass (Fig. 4, D and G) and a polarized hIMCDi monolayer grown on a Snapwell (Fig. 4J). Preincubation of the lectin with 0.2 M galactose, a competing sugar that has a high affinity to PNA, inhibited staining (Fig. 4, E, H, and K). These observations indicate that the cultures were highly enriched in IMCD cells. Similar results were obtained with DBA and PNA conjugated to rhodamine (not shown).


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  		Fig. 3.   Subconfluent culture of human IMCDi cells grown on glass microscope chamber slides. A: cells were stained with Pan anti-cytokeratin antibody (20 µg/ml) and visualized by a secondary antibody conjugated to FITC. B: no anti-cytokeratin antibody, but all other reagents were added. C: cells stained with 50 µM Dolichos biflorus agglutinin (DBA), a collecting duct-specific lectin. Most cells stained positive for DBA; however, the intensity of the staining was variable, suggesting that the cell culture was heterogeneous. Positive staining was observed also in confluent hIMCDi cell monolayers. D: mixture of 50 µM DBA and 200 µM N-acetylgalactosamine, a competing sugar specific for DBA.



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  		Fig. 4.   Arachis hypogaea agglutinin (peanut agglutinin; PNA) staining of a collecting duct within the initial region of a human inner medulla (A) and a distal tubule (red arrow) situated juxtaposed to a glomerulus within the renal cortex (B). In A, the red arrow denotes apical PNA staining of the collecting duct (CD) cells consistent with a previous report in rabbit CD (20). Confluent cultures of hIMCDi (C, D, E) and hIMCDt (F, G, H) cells grown on glass microscope chamber slides are also shown. hIMCDi and hIMCDt cells were incubated in the absence of lectin and the presence of all other reagents (C and F), 50 µM PNA (D and G), and a mixture of 50 µM PNA and 200 µM galactose, a competing sugar specific for PNA (E and H). Both cell types stained specifically for PNA (brown). A hIMCDi cell monolayer grown on a permeable support (Snapwell-Clear) also stained for PNA. The monolayer was divided into 3 sections and incubated in control medium (I), PNA (J), and PNA and galactose (K). Cultured cells were counterstained with Mayer's hematoxylin (blue of nuclei).

Asymmetric distribution of Na-K-ATPase between the apical and basolateral plasma membranes of polarized epithelial cells is necessary for the establishment of vectorial transport of ions. As in many transporting epithelia (2, 21, 31, 51, 52, 56, 57, 59), the Na-K-ATPase was found to be restricted to the basolateral membrane of human IMCDi in situ (Fig. 5A). Moreover, localization of Na-K-ATPase in lateral membrane of hIMCDi monolayers (Fig. 5B) indicates that hIMCDi cells establish an orthodox polarity when cultured on permeable supports.


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  		Fig. 5.   Immunolocalization of Na-K-ATPase in a human initial IMCD in situ (A) and a cultured hIMCDi cell monolayer grown on a Snapwell-Clear support (B). A monoclonal antibody directed against the alpha 1-isoform of Na-K-ATPase was visualized by 3,3'-diaminobenzidine (DAB; brown). Na-K-ATPase protein was located on the basolateral cell membranes of hIMCDi. Cellular localization of Na-K-ATPase to the lateral membranes of cells within the polarized monolayer validates the establishment of an orthodox polarity when hIMCDi cells are grown on permeable filters.

Comparison of effects of AVP and PGE2 on intracellular cAMP in hIMCDi and hIMCDt cells. We examined the effect of short-term incubation with media containing AVP, PGE2, or forskolin on total intracellular cAMP levels in confluent hIMCDi and hIMCDt cells grown on permeable supports. Intracellular cAMP was measured in four groups (n = 6 monolayers/group) treated for 15 min with control medium, 100 mU/ml AVP, 25 ng/ml PGE2, or 10 µM forskolin (Fig. 6). Benzamil (10 µM) was added to the apical media of all groups for 10 min before the experiment, to be consistent with the ISC experiments. There were qualitatively similar increases in intracellular cAMP levels in hIMCDi and hIMCDt monolayers after incubation in the presence of AVP, PGE2, and forskolin. These data indicate that cultured hIMCDi and hIMCDt cells have AVP and PGE2 receptors coupled to the cAMP pathway and that the capacity to generate cAMP by direct activation of adenylate cyclase with forskolin was not different between hIMCDi and hIMCDt cells.


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  		Fig. 6.   Effect of 100 mU/ml AVP, 25 ng/ml PGE2, and 10 µM forskolin, an activator of adenylyl cyclase, on intracellular cAMP levels in hIMCDi and hIMCDt cells grown on collagen-coated permeable supports; n = 6/group, 2 kidney preparations. Total cAMP (pmol/monolayer) was determined with an enzyme immunoassay system. Agonists were added 15 min before the cAMP was extracted with 80% methanol. *P < 0.05 compared with basal cAMP value.

Bioelectrical properties of cultured hIMCDi and hIMCDt cells. We compared the electrical properties of hIMCDi and hIMCDt cell monolayers (3 kidney preparations). Pairs of hIMCDi and hIMCDt monolayers were mounted for measurement of TER, transepithelial PD, and ISC with two sets of chamber electrodes attached to a dual-voltage-clamp device. hIMCDi cell monolayers (n = 10) developed a TER of 258 ± 47 Omega  · cm2, a lumen-negative potential (PD; -0.3 ± 0.1 mV), and a positive ISC of 1.4 ± 0.5 µA/cm2. In contrast, the TER of hIMCDt monolayers (n = 10) was 152 ± 16 Omega  · cm2 and PD and ISC were not different from zero.

Evidence for Na+ absorption in human IMCDi. To investigate the contribution of Na+ absorption, through ENaC and CNG channels, to the baseline current in the hIMCDi cells, we examined the effect of adding ANP to the basolateral medium and benzamil to the apical medium. In hIMCDi monolayers (n = 13), ANP (10-1,000 nM) caused a small but significant reduction in baseline ISC (Delta ISC; 0.3 ± 0.1 µA/cm2, P < 0.05). The addition of benzamil (10 µM), a high-affinity inhibitor of ENaC and CNG (22, 53), to the apical fluid reduced baseline current from 1.9 ± 0.2 to 1.5 ± 0.2 µA/cm2 (n = 28 monolayers, 4 kidney preparations; P < 0.001). These data indicate that Na+ absorption via an ANP-sensitive apical Na+ conductance, possibly ENaC and CNG channels, contributes to only a small fraction of the basal current of cultured hIMCDi cell monolayers. In experiments in which we tested the effect of cAMP agonists on electrolyte secretion by IMCD monolayers, benzamil was included in the apical medium to reduce the contribution of these Na+ absorptive pathways to ISC.

Effect of cAMP agonists on anion transport by human IMCD cells. To investigate the effect of cAMP on anion secretion by human IMCD cells, we incubated IMCDi monolayers in the presence of apical benzamil and monitored ISC after the addition to apical and basolateral media of 200 µM 8-bromo-cAMP (8-BrcAMP), a permeant analog of cAMP. A typical response of IMCDi monolayers to benzamil and 8-BrcAMP is shown in Fig. 7. ISC increased after the addition of 8-BrcAMP from 0.7 to 5.9 µA/cm2. ISC reached a new steady state within 40 min. An apically negative transepithelial PD hyperpolarized from -0.1 to -1.2 mV. In a group of four hIMCDi monolayers, 8-BrcAMP increased benzamil-insensitive current from 2.2 ± 1.1 to 6.1 ± 0.7 µA/cm2 (P < 0.05; Fig. 8). In contrast, incubating human hIMCDt monolayers (prepared from the same kidney and grown under the same experimental conditions) in 8-BrcAMP did not significantly change ISC (Delta ISC = 0.0 ± 0.1 µA/cm2; n = 4).


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  		Fig. 7.   Effect of 8-bromo-cAMP (8-BrcAMP) and Cl- transport inhibitors on short-circuit current (ISC) across hIMCDi cell monolayer. Benzamil (10 µM) was added to the apical medium. 8-BrcAMP (200 µM) was added to both apical and basolateral media. DIDS (50 µM) and bumetanide (100 µM) were added to the basolateral medium.



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  		Fig. 8.   Effect of 8-BrcAMP on ISC in collecting duct cells cultured from IMCDi (A) and IMCDt (B); n = 4 monolayers/group. Benzamil (10 µM) was added to the apical media in both groups. 8-BrcAMP (200 µM) was added to both apical and basolateral media. *P < 0.01 compared with control ISC; #P < 0.001 comparison between cAMP-stimulated ISC in IMCDi and IMCDt.

Forskolin, which rapidly penetrates plasma membranes and elevates cAMP levels in hIMCDi cells (Fig. 6) by directly activating adenylyl cyclase, caused a peak increase in ISC in hIMCDi monolayers within 5-10 min that declined to a new steady-state current above baseline. Table 1 displays the changes in steady-state current for 50 monolayers cultured from six nephrectomy specimens. Forskolin stimulated an increase in benzamil-insensitive current in all hIMCDi cell monolayers (Delta ISC of all monolayers was 3.3 ± 0.2 µA/cm2); however, the magnitude of the response varied among the different preparations. Forskolin did not significantly increase ISC in the IMCDt monolayers (data not shown).

                              
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  		Table 1.   Effect of forskolin on ISC across individual preparations of human IMCDi cells

Effect of AVP and PGE2 on anion secretion in hIMCDi monolayers. The data in Fig. 6 indicated that AVP and PGE2 significantly increased cAMP in hIMCDi and hIMCDt cells; however, the response was much lower than that of a maximally effective concentration of forskolin. In cultured rat (16) and mouse (52) IMCD cells, the cAMP-stimulated ISC in the presence of benzamil was attributed to anion secretion involving apical CFTR Cl- channels. To determine whether AVP and PGE2 stimulated electrogenic anion secretion by the hIMCDi monolayers, we tested the effect of these agonists on ISC and PD (Table 2) after the addition of benzamil to the apical medium. In most monolayers, benzamil caused a small decline in baseline ISC. The addition of 100 mU/ml AVP to the basolateral medium significantly increased the current (Delta ISC = 1.0 ± 0.1 µA/cm2) and hyperpolarized the cell monolayers. PGE2, an important intrarenal autacoid involved in the regulation of blood pressure, was examined to determine whether it also affected electrolyte transport by hIMCD cells. PGE2 potently stimulated anion secretion by the hIMCDi cells. ISC increased by 3.6 ± 0.5 µA/cm2, and the monolayer hyperpolarized by -1.1 ± 0.1 mV. These observations indicate that AVP and PGE2 are important factors for promoting cAMP-dependent electrogenic Cl- secretion by hIMCDi cells.

                              
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  		Table 2.   Effect of AVP and PGE2 on ISC and potential difference in IMCDi monolayers

Effect of cAMP on fluid transport by hIMCDi cells. To determine whether the cAMP-induced anion transport was coupled to fluid secretion, we compared the direction and the rate of fluid transport in three groups of hIMCDi monolayers (n = 4 per group) incubated in 1) control medium, 2) apical medium containing 10 µM benzamil, or 3) apical medium containing benzamil and basolateral medium containing 10 µM forskolin. In Fig. 9, negative values represent fluid absorption and positive values indicate fluid secretion. In the control medium, human IMCDi monolayers absorbed fluid at a rate of -0.13 ± 0.03 µl · h-1 · cm-2 (P < 0.02; 1-sample t-test). Benzamil added to the apical medium had no effect on fluid absorption (-0.14 ± 0.02 µl · h-1 · cm-2), whereas the addition of apical benzamil and basolateral forskolin reversed the net flux of fluid to secretion (0.40 ± 0.05 µl · h-1 · cm-2; P < 0.001).


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  		Fig. 9.   Measurement of fluid transport rates across hIMCDi cell monolayers. Values are means ± SE; n = 4. Positive values indicate fluid secretion, and negative values indicate fluid absorption. Significance was determined by Kruskal-Wallis nonparametric analysis of variance and Dunn's test. *P < 0.05 compared with previous period.

Effect of Cl- channel blockers on cAMP-dependent transport. Mechanisms of cAMP-dependent Cl- secretion have been investigated in cultured rat and mouse IMCD cell monolayers (16, 18); however, cAMP-dependent anion secretion has not been previously characterized in hIMCD cells. Using pharmacological agents, we determined whether cAMP activated CFTR Cl- channels. We tested the effects of apical application of diphenylamine-2-carboxylate (DPC) and glybenclamide, known inhibitors of CFTR Cl- channels, on forskolin-stimulated anion secretion in hIMCDi monolayers. DPC inhibited 91 ± 6% of the forskolin-stimulated current, whereas glybenclamide, a weak inhibitor of CFTR, reduced the stimulated ISC by 48 ± 5% (Fig. 10). Niflumic acid, a potent inhibitor of epithelial Cl- channels including CFTR (4), also attenuated cAMP-stimulated current in hIMCDi monolayers (data not shown). DIDS is known to block Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange and some types of Cl- channels but is unable to block the Cl- conductance of CFTR (42). Apical application of DIDS did not significantly inhibit ISC (data not shown). The pharmacological profile of cAMP-induced anion secretion by hIMCDi to Cl- channel blockers is consistent with CFTR Cl- channels in the apical plasma membrane (42).


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  		Fig. 10.   Effect of Cl- channel inhibitors on forskolin-stimulated ISC in human IMCD cells. Currents were measured in the presence of benzamil (10 µM) added to the apical medium. Inhibitors were added after a steady state was reached in the presence of basolateral forskolin (10 µM). Diphenylamine-2-carboxylate (DPC) and glybenclamide were added to the apical medium. *P < 0.05 compared with the control value (benzamil alone); #P < 0.05 compared with the forskolin-stimulated current.

Mechanisms for Cl- uptake across basolateral membrane during cAMP-dependent Cl- secretion. A mouse monoclonal antibody (T4) to NKCC1, the secretory isoform of Na+-K+-Cl- cotransporter (26), has been localized within the basolateral membranes of a subfraction of rat collecting duct cells from the outer medulla (10). In the current study, we detected by Western blot abundant immunoreactivity of the T4 antibody to plasma membranes from fresh human initial inner medulla (Fig. 11, lane 1). There was a band at ~200 kDa and a broad band in the range of 150-170 kDa. NKCC1 has been reported to migrate during electrophoresis in a broad band of 145-205 kDa (10, 26). Multiple and broad bands for NKCC1 are often attributed to multiple states of glycosylation. When the supernatant of cultured hybridoma cells not expressing the relevant antibody was used as a control, no bands were observed. In confluent monolayers enriched in hIMCDi cells, we observed a major band at ~170 kDa in the membrane fraction (Fig. 11, lane 2). These results were confirmed with two additional hIMCDi preparations from other kidneys. These studies show that fresh human initial inner medulla and cultured hIMCDi cells express NKCC1 protein.


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  		Fig. 11.   Immunoblots for NKCC1, a secretory form of the Na+-K+-Cl- cotransporter, and AE2, an anion exchanger for HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and Cl-. Membrane proteins were isolated from a freshly dissected human initial inner medulla (lanes 1 and 3) and hIMCDi cells grown as confluent monolayers in petri dishes (lanes 2 and 4). A: human initial inner medulla and hIMCDi cells expressed a protein of 170 kDa that was detected by NKCC1 antibody. A higher-molecular-mass protein was also detected in the medullary tissue. B: an antibody that detects both AE1 and AE2 revealed a 165-kDa band in both medullary tissue and cultured cells, a protein consistent with AE2. A second band was also detected at ~205 kDa in the medullary tissue. Overexposure of the film revealed a weak band at ~ 100 kDa, the expected molecular mass of AE1 protein.

It is well established that AE anion exchangers, transporters that exchange HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and Cl-, are present in rodent IMCD (35). Measurements of mRNA and protein levels for the three known AE anion exchangers have suggested that AE2 is the most abundant form in renal cells (1). Recently, Alper and co-workers used an epitope-unmasking technique to show that AE2 immunolocalized to basolateral plasma membranes but not apical membranes of rat (1) and mouse (49) IMCD. To investigate whether human initial inner medulla and cultured hIMCDi cells express AE anion exchangers, we examined the protein expression of AE1 (~100 kDa) and AE2 (~165-180 kDa) by Western blot analysis with an antibody raised against the COOH terminus of mouse AE2 (amino acids 1224-1237). The COOH termini of AE2 and AE1 are sufficiently similar so that the antibody recognizes both proteins (49). In membrane fractions of human initial inner medulla (Fig. 11, lane 3) and cultured hIMCDi cells (Fig. 11, lane 4), the antibody detected a major band at ~165 kDa. This is consistent with the expression of AE2 protein. Overexposure of the film also revealed a band at ~100 kDa, indicating the presence of a less abundant AE1 anion exchanger.

To determine whether these Cl- transport mechanisms participate in cAMP-dependent anion secretion by hIMCDi cells, we tested the effect of basolateral application of bumetanide, an inhibitor of NKCC1, and DIDS, an inhibitor of AE2, on cAMP-stimulated ISC. In Fig. 7, we show a typical response to these inhibitors after steady-state stimulation by 200 µM 8-BrcAMP. In other experiments, hIMCDi cell monolayers (n = 6) incubated in medium containing benzamil exhibited a baseline ISC of 1.5 ± 0.6 µA/cm2 and forskolin increased this to a steady-state current of 6.0 ± 0.5 uA/cm2 (P < 0.001; Fig. 12). Basolateral addition of bumetanide (100 µM) reduced ISC to 3.8 ± 0.5 µA/cm2 (P < 0.01), and the subsequent addition of DIDS caused a further reduction of current to 2.0 ± 0.4 µA/cm2 (P < 0.001 compared with forskolin alone). After bumetanide and DIDS were combined, the current was not significantly different from the control current in benzamil alone. These data indicate that NKCC1 and AE2 are major pathways for Cl- entry across the basolateral membrane during cAMP-dependent Cl- secretion.


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  		Fig. 12.   Effect of basolateral addition of Cl- entry inhibitors on forskolin-stimulated anion current. Benzamil (10 µM) was added to the apical medium 10 min before control values were recorded. Forskolin, added to the basolateral medium, increased ISC in the 6 monolayers. All additions were made after the ISC reached a steady state after the prior addition. The combination of bumetanide, an inhibitor of Na+-K+-Cl- cotransport, and DIDS, an inhibitor of Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange, reduced ISC to near the control level. See text for mean ± SE values.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In the present study we used an in vitro method to explore the transport characteristics of primary cultures of cells enriched in hIMCDi or hIMCDt. The key observations of the study are as follows. 1) Benzamil caused a small decline in ISC in hIMCDi cells; however, it did not inhibit fluid absorption. 2) AVP, PGE2, and forskolin stimulated cAMP production in cells cultured from both initial and terminal regions of human IMCD. 3) cAMP stimulated electrogenic Cl- and fluid secretion in hIMCDi cell monolayers but not in hIMCDt monolayers. 4) NKCC1, a secretory form of Na+-K+-Cl- cotransporter, and AE2, a Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> anion exchanger, may represent parallel pathways for Cl- entry into hIMCDi cells across the basolateral membranes. 5) CFTR Cl- channels appear to mediate cAMP-regulated Cl- efflux across the apical membranes of hIMCDi cells.

Characteristics of hIMCDi and hIMCDt cell monolayers. The initial IMCD contain a heterogeneous population of mainly principal or "light" cells and alpha -type intercalated or "dark" cells, whereas the terminal IMCD contain a single cell type referred to as "the IMCD cell" (5, 27, 32). To compare the characteristics of hIMCDi and hIMCDt cells in culture, we isolated regions of the human initial and terminal inner medulla separately (Fig. 1) and prepared primary cultures with methods similar to those published previously (15, 16, 23, 56). Cells from both regions grew well in the presence of 5% FBS and formed confluent monolayers on permeable supports within 5-7 days. Adjacent cells in the monolayers formed apical junctional complexes (Fig. 2E) that were electrically tight as gauged by measurements of TER. The average resistance of hIMCDi cell monolayers was greater than that of hIMCDt monolayers. Moreover, the hIMCDi monolayers generated an apically negative PD and positive ISC, consistent with active ion transport in the basal state (Fig. 8, Table 2). In contrast, PD and ISC in IMCDt cells were not different from zero. Because hIMCDt cells generated cAMP in response to agonists as well as hIMCDi cells, the differences in transport between them are probably due to a lower abundance of solute transporters in hIMCDt. These results are in accord with measurements of PD in isolated, perfused rat collecting ducts (37, 40); however, we cannot exclude the possibility that the hIMCDt cells would have different basal transport characteristics in a medium with a composition comparable to the interstitial fluid of terminal inner medulla.

Morphological examination of cultured hIMCDi and hIMCDt monolayers showed a similar ultrastructure in the two cell populations (Fig. 2, C and D). In addition, assessment of the monolayers by electron microscopy showed a relatively homogeneous population of cells. Unfortunately, principal and intercalated cells could not be readily distinguished. hIMCDi and hIMCDt monolayers expressed cytokeratin and stained for collecting duct lectins, indicating that the cultures were derived from medullary collecting duct cells to a major extent (Figs. 3 and 4).

Absorption of Na+ by hIMCDi cells. Na+ absorption occurs at many loci along the nephron; however, the fine regulation of Na+ excretion is thought to occur in the collecting duct. ENaC has been shown to mediate Na+ absorption across the apical membrane of rat and mouse IMCD cells (23, 48, 62); however, its role in Na+ absorption by hIMCD cells has not been previously explored. In the present study, benzamil, a specific inhibitor of the ENaC and CNG channels (53), produced small but significant decreases in baseline ISC, consistent with the inhibition of Na+ absorption. On the other hand, benzamil failed to inhibit solute-coupled fluid absorption over a 24-h period (Fig. 9). Technical limitations in the method for measuring net fluid transport across cell monolayers may have prevented detection of a small inhibition of fluid absorption by benzamil. Moreover, our culture medium, which may lack critical hormones (i.e., aldosterone) involved in modulating Na+ absorption (62), may be inappropriate for investigating Na+ absorptive pathways. The results from this study, therefore, may underestimate the contribution of NaCl absorption by hIMCDi cells. Consequently, the molecular basis of fluid absorption by hIMCDi remains to be determined.

Salt secretion by IMCD cells. The role of electrolyte and fluid secretion by IMCD and its contribution to the composition and volume of urinary fluid have been debated for several decades. Sonnenberg (46) was the first to demonstrate, with a microcatheterization approach, net salt secretion by renal collecting ducts in acutely volume-expanded rats. Sonnenberg's data indicated that the IMCD secreted Na+ and water in amounts equal to ~10% of the filtered load (46). Recently, we confirmed (57) that, indeed, rat IMCD have an intrinsic capacity for cAMP-dependent salt and fluid secretion. Fluid secretion was inhibited by Cl- transport blockers, indicating that active Cl- secretion was coupled to the osmotic flow of water in IMCD. Husted and associates (16) showed that AVP and cAMP stimulated anion secretion by cultured rat IMCD cells and that this was mediated through CFTR Cl- channels present in the apical membrane. Thus the in situ observations by Sonnenberg (46) may be attributed to intrinsic salt and fluid secretion mechanisms in the initial region of the IMCD. Although there is mounting evidence that supports a role for cAMP-regulated salt secretion by the IMCD and, to a lesser extent, other regions of the collecting duct of rat, mouse and rabbit (Table 3), few studies have examined electrolyte transport by human collecting duct cells.

                              
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  		Table 3.   Mechanisms involved in salt-coupled fluid secretion by mammalian collecting ducts

We found that the cAMP agonists AVP and PGE2, and forskolin, increased the accumulation of intracellular cAMP in hIMCDi and hIMCDt cell monolayers (Fig. 5). Thus both cultured hIMCDi and hIMCDt cells have receptors for AVP and PGE2 that are coupled to the generation of cAMP. Furthermore, the capacity to maximally generate cAMP through the direct activation of adenylyl cyclase by forskolin was the same between the two cell lineages. On the other hand, the electrolyte transport response to cAMP was distinctly different. AVP, PGE2, forskolin, and 8-BrcAMP activated a benzamil-insensitive current in IMCDi but not in IMCDt cell monolayers. The current was inhibited by apical application of DPC, glybenclamide, and niflumic acid, drugs that block CFTR Cl- channels. Thus the evidence at hand indicates that transepithelial Cl- transport has a clear role in the secretion of fluid by hIMCDi but not by hIMCDt cells.

The Na+-K+-Cl- cotransporter has been localized to the basolateral membranes of rat IMCD (6), and it has been projected to mediate Cl- secretion in rat IMCD (39, 57) and outer medullary collecting ducts (54). In the present study, NKCC1 protein (175 kDa) was found to be expressed in the membrane fraction of tissue isolated from human initial inner medulla and in cultured hIMCDi cell monolayers (Fig. 11). Bumetanide applied to the basolateral surface of the hIMCDi cells significantly reduced cAMP-stimulated ISC, pointing to a potential role for the NKCC1 transporter in Cl- entry during cAMP-dependent secretion.

AE2 anion exchanger, a transporter that exchanges HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and Cl-, has been immunolocalized to the basolateral, but not apical, membranes of rat and mouse IMCD cells (1, 49). Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchangers may contribute to the regulation of cell pH and cell volume. Two observations in the present study indicate that AE2 transporters may also contribute to transepithelial Cl- secretion in human IMCD cells during cAMP stimulation: 1) Cell membranes prepared from cell lysates of human initial inner medullary tissue and cultured hIMCDi cells contained abundant AE2 protein. 2) DIDS, an inhibitor of the AE2 transporter, reduced cAMP-stimulated anion secretion. Together, these findings indicate that NKCC1 and AE2 may operate in concert to mediate Cl- uptake across the basolateral membrane during cAMP-dependent Cl- secretion in hIMCDi.

Potential roles of collecting duct NaCl and fluid secretion. In the evolution of excretory organs, NaCl-coupled fluid secretion seems to be a conserved mechanism for regulating body fluid composition and volume (12). Marine birds and reptiles possess specialized salt glands that secrete a highly concentrated NaCl fluid (41), whereas marine teleosts, the bony fishes, secrete NaCl through "chloride cells" located in the gills (9) and elasmobranchs secrete NaCl via rectal glands (45). Beyenbach and Liu (3) demonstrated NaCl-coupled fluid secretion in the proximal tubules of aglomerular and glomerular fish. In the case of the aglomerular fish, urine formation was completely dependent on tubular salt and fluid secretion. Apical Cl- channels mediate Cl- secretion in many of the excretory organs of these nonmammalian species (3, 9, 12, 36, 41). In fact, stellar cells of the insect malpighian tubules secrete Cl- through CFTR-like channels (36).

Recent evidence of cAMP-dependent NaCl and fluid secretion in isolated intact rat IMCD (39, 54, 57) and cultured rat (16), mouse (18), and human IMCD cells demonstrates that this ancient secretory mechanism has been conserved in the renal collecting system of mammals. The mechanism for salt and fluid secretion is strategically located in the terminal portions of the renal tubule system. Were the NaCl secretion process situated in the proximal tubule exclusively, the contribution to urine formation of the relatively low magnitudes of net solute and fluid secretion might be nullified by reabsorption in more distal tubular segments. Previous studies may be interpreted to demonstrate a role for tubular secretion in the regulation of ECF. Luke (25) found in rats that withholding water for 24 h led to natriuresis and chloruresis, an effect on salt excretion that was mimicked by the administration of AVP. It was suggested that the increase in NaCl excretion might reflect a mechanism conserved in mammalian kidneys to maintain the tonicity of ECF during dehydration. Martinez-Maldonado et al. (28) showed similar natriuretic effects of AVP administered to dogs. It is conceivable that dehydration, mediated by AVP, unmasks a cAMP-regulated salt secretory mechanism in collecting ducts that blunts the elevation in plasma hypertonicity during desiccation.

It is also conceivable that NaCl-coupled fluid secretion by the renal tubule is important for maintaining patent lumens and propelling fluid out of the kidney during conditions of reduced or complete cessation of glomerular filtration, as may occur in acute renal failure, dehydration, and sudden blood volume loss caused by hemorrhage. This would enable the kidney to excrete to some extent potentially toxic products during periods in which glomerular filtration is insufficient. In this way, tubular secretion may serve as a "default" method of urine formation that has been in place since the era of aglomerular protovertebrates (12).

Role of NaCl and fluid secretion in renal cyst formation. The contribution of salt secretion to urine formation is difficult to assess clinically because massive quantities of salt and fluid are filtered by glomeruli and reabsorbed by many nephron segments. On the other hand, the intrinsic capacity to secrete NaCl is evident in renal cyst formation. In autosomal dominant polycystic kidney disease (ADPKD), cysts develop from the focal outgrowth of individual tubule cells and fluid accumulates within isolated sacs. The mechanisms of fluid secretion into ADPKD renal cysts were recently elucidated (50, 51, 56). cAMP agonists regulate fluid secretion by the mural epithelial cells through activation of apical CFTR and basolateral NKCC1 cotransporters (56). In the present study, cAMP-dependent NaCl and fluid secretion by renal cells from individuals without ADPKD supports the view that secretion is a normal physiological process within specific regions of the nephron. Thus the accumulation of NaCl and fluid within renal cysts could simply be a consequence of the fact that most cysts are isolated from their parent tubules, an anatomic outcome of the polycystin gene mutation. The lack of glomerular filtration into renal cysts may uncover a secretory process normally masked by absorption. The new discovery that cAMP agonists stimulate NaCl and fluid secretion in normal IMCD forces us to reconsider the origin of urinary fluid that has been frequently and most conveniently attributed to incomplete reabsorption of NaCl and water from the glomerular filtrate.


    ACKNOWLEDGEMENTS

We thank Dr. Tamio Yamaguchi for technical assistance and Dr. Larry Sullivan for reading the manuscript and for helpful discussions. The T4 NKCC1 monoclonal antibody developed by Lytle and Forbush was obtained from the Development Studies Hybridoma Bank developed under the auspices of the National Institute of Child Health and Human Development and maintained by the Department of Biological Sciences, University of Iowa (Iowa City, IA). We thank Dr. S. Alper for the generous gift of AE2 antibody.


    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants P01-DK-53763 and P50-DK-57301 (J. J. Grantham) and a National Research Service Award F32-DK-09929-01 (D. P. Wallace).

Portions of this study were published in abstract form (J Am Soc Nephrol 11: 38A, 2000).

Address for reprint requests and other correspondence: D. P. Wallace, Dept. of Medicine, Univ. of Kansas Medical Center, 3901 Rainbow Blvd, Kansas City, KS 66160-7382 (E-mail: dwallace{at}kumc.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

July 24, 2002;10.1152/ajprenal.00165.2002

Received 30 April 2002; accepted in final form 18 July 2002.


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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Am J Physiol Renal Fluid Electrolyte Physiol 283(6):F1337-F1350
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