Ontogeny of flow-stimulated potassium secretion in rabbit cortical collecting du - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-21 13:47 |只看该作者 |倒序浏览 |打印

作者：Craig B. Woda, Nobuyuki Miyawaki, Santhanam Ramalakshmi, Mohan Ramkumar, Raul Rojas, Beth Zavilowitz, Thomas R. Kleyman,  Lisa M. Satlin作者单位：1 Division of Pediatric Nephrology, Department ofPediatrics Mount Sinai School of Medicine, New York 10029; Division of Nephrology and Hypertension, Departmentof Medicine, Winthrop University Hospital, Mineola, New York 11501; and Renal-Electrolyte Division, Department of Medicine,University of Pitt
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      【摘要】, @( M9 L4 W! S0 J
   High urinary flow rates stimulate K secretion in the fully differentiated but not neonatal or weanling rabbit cortical collecting duct (CCD). Bothsmall-conductance secretory K and high-conductance Ca 2 /stretch-activated maxi-K channels have beenidentified in the apical membrane of the mature CCD by patch-clamp analysis. We reported that flow-stimulated net K secretion in the adult rabbit CCD is 1 ) blocked by TEA and charybdotoxin, inhibitors of intermediate- andhigh-conductance (maxi-K) Ca 2 -activated K channels, and 2 ) associated with increases in net Na absorption and intracellularCa 2 concentration([Ca 2 ] i ). The present study examined whetherthe absence of flow-stimulated K secretion early in life is due to a 1 ) limited flow-induced rise in net Na absorption and/or[Ca 2 ] i and/or 2 ) paucity of apicalmaxi-K channels. An approximately sixfold increase in tubular fluid flow ratein CCDs isolated from 4-wk-old rabbits and microperfused in vitro led to anincrease in net Na absorption and [Ca 2 ] i, similar in magnitude to the response observed in 6-wk-old tubules, but itfailed to generate an increase in net K secretion. By 5 wk of age, there was asmall, but significant, flow-stimulated rise in net K secretion that increasedfurther by 6 wk of life. Luminal perfusion with iberiotoxin blocked the flowstimulation of net K secretion in the adult CCD, confirming the identity ofthe maxi-K channel in this response. Maxi-K channel -subunit messagewas consistently detected in single CCDs from animals 4 wk of age byRT-PCR. Indirect immunofluorescence microscopy using antibodies directedagainst the -subunit revealed apical labeling of intercalated cells incryosections from animals 5 wk of age; principal cell labeling was generally intracellular and punctate. We speculate that the postnatalappearance of flow-dependent K secretion is determined by thetranscriptional/translational regulation of expression of maxi-K channels.Furthermore, our studies suggest a novel function for intercalated cells inmediating flow-stimulated K secretion. 1 ?7 i$ Z) M! }0 @9 x/ P
      【关键词】 maxiK channel iberiotoxin in vitro microperfusion intracellular calcium concentration mechanoregulation development! }! u8 h6 o* M5 z6 I
   NEWBORN HUMANS AND animals maintain a state of positive Kbalance, as is appropriate for growth, unlike their adult counterparts who arein net zero K balance ( 49 ). Itis now well established that the neonatal kidney contributes to this Kretention. Clearance studies provide abundant evidence for a low rate ofurinary K excretion early in life( 39, 49 ). In response to exogenous K loading, infants, like adults, can excrete K at a rate that exceeds itsfiltration ( 55 ), indicatingthe capacity for net tubular secretion. However, the rate of urinary Kexcretion in K-loaded infants and young animals is less than that observed inolder animals ( 27, 29 ).

Urinary K excretion is derived almost entirely from K secretion in theconnecting tubule and cortical collecting duct (CCD) ( 13 ). In contrast to the highrates of K secretion observed in CCDs isolated from adult animals andmicroperfused in vitro at physiological flow rates, segments isolated fromneonatal animals show no significant net K transport until after week 3 of postnatal life ( 38 ).Of note is that the rate of net Na absorption in the CCD at 2 wk of age is 60% of that measured in the adult( 38 ). By 6 wk of age, the rateof net K secretion in CCDs perfused at a flow rate of 1 nl ·min - 1 · mm - 1 iscomparable to that observed in the adult ( 38 ). These results indicatethat the low rates of urinary K excretion characteristic of the newborn kidneyare due, at least in part, to a low secretory capacity of the CCD.
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The CCD is comprised of two cell populations. Principal cells reabsorb Naand secrete K ( 24, 37, 47 ), whereas intercalated cells are thought to primarily function in acid-base homeostasis but canreabsorb K in response to dietary K restriction or metabolic acidosis( 44, 46, 63 ). Within the principalcell, Na reabsorbed across apical epithelial Na channels (ENaCs) is extrudedby the basolateral Na-K-ATPase in exchange for the uptake of K. Cell Kdiffuses down a favorable electrochemical gradient, established byelectrogenic Na absorption, into the tubular lumen through apical K-selectivechannels. Thus K secretion in the CCD requires Na absorption and the presenceof conducting apical K-selective channels.

Two types of apical K-selective channels have been functionally identifiedin the rabbit principal cell by patch-clamp analysis ( 41, 42 ). The prevalence of thelow-conductance secretory K (SK) channel and its high open probability at theresting membrane potential( 12, 42, 57 ) led to the premise that this channel mediates K secretion under baseline conditions. 100 pS) K channel, rarely open at physiological membranepotentials, exhibits kinetics similar to those of the maxi-K channel describedby others ( 18, 22, 35 ). In contrast to the SKchannel, detected within the CCD solely in principal cells, high-conductancemaxi-K channels, activated by membrane depolarization, stretch, elevation of intracellular calcium concentration([Ca 2 ] i ), or hypoosmotic stress( 11, 14, 21, 22, 35, 48, 50, 51 ), are present in bothprincipal and intercalated cells of the CCD( 35 ). In fact, the density ofmaxi-K channels in intercalated cells exceeds that detected in principal cells( 35 ).

An increase in tubular fluid flow rate in the fully differentiated CCDstimulates net K secretion ( 9, 15, 16, 28, 40 ). We recently reported thatflow-stimulated net K secretion is associated with increases in net Naabsorption, presumably due to stimulation of ENaC activity( 43 ), and increases in[Ca 2 ] i in both principal and intercalatedcells ( 58, 59 ). On the basis of thesensitivity of the response to TEA and charybdotoxin( 58 ) and theelectrophysiological evidence for apical SK and maxi-K channels in the rabbitCCD ( 41, 42 ), we proposed thatflow-dependent K secretion is mediated by the maxi-K channel, as had beensuggested by others ( 13, 35, 50 ). In contrast to the robustincrease in net K secretion elicited by flow in the adult rabbit CCD,flow-dependent net K secretion cannot be elicited until after week 4 of postnatal life ( 38 ). Yet,patch-clamp analysis reveals high-conductance (110 ± 6 pS) K channels,activated by membrane depolarization, in 10% of cell-attached patches ofthe apical membranes of rabbit principal cells at 2 wk of age( 41 ).4 U7 E/ r' u2 W7 ~# D% Z
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The purpose of the present study was to examine the cellular and molecularbasis for the delayed postnatal appearance of flow-stimulated K secretion inthe CCD, first observed 2 wk after baseline K secretion is established.Given our previous detection of high-conductance K channels in the neonatalrabbit principal cell by patch-clamp analysis, we initially speculated thatthe absence of flow-stimulated K secretion early in life was due to a limitedcapacity for flow to augment Na absorption and/or induce a rise in[Ca 2 ] i. However, once these flow-induced responses were shown not to be limiting, we used RT-PCR, Southern blotting,and indirect immunofluorescence to show that expression of maxi-K channel mRNAand protein is developmentally regulated.

MATERIALS AND METHODS
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Animals. Female adult New Zealand White rabbits and litters ofnewborns PA) and housed inthe Mount Sinai School of Medicine animal care facility. Newborns were allowedto remain with their mothers until weaning. Animals were fed standard rabbitchow and given free access to food and water. Rabbits were killed byintraperitoneal injection of pentobarbital sodium (100 mg/kg). All experiments were conducted in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.

Isolation of single tubules. The kidneys were removed via a midline incision and sliced into 2-mm coronal sections, and single tubuleswere dissected freehand in cold (4°C) Ringer solution containing (in mM)135 NaCl, 2.5 K 2 HPO 4, 2.0 CaCl 2, 1.2MgSO 4, 4.0 lactate, 6.0 L -alanine, 5.0 HEPES, and 5.5 D -glucose, pH 7.4, 290 ± 2 mosmol/kgH 2 O, aspreviously described ( 59 ). Asingle tubule was studied from each animal.

For in vitro microperfusion studies, each isolated tubule was immediatelytransferred to a temperature- and O 2 -CO 2 -controlled specimen chamber, assembled with a no. 1 glass coverslip (VWR Scientific,Media, PA) as its base. The CCD was mounted on concentric glass pipettes andcannulated. For measurement of [Ca 2 ] i, CCDswere affixed, basolateral membrane down, to coverslips previously painted witha 1-µl drop of Cell-Tak (Collaborative Biomedical Products, Bedford, MA),as previously described ( 59 ).Each tubule was perfused and bathed at 37°C with Burg's perfusatecontaining (in mM) 120 NaCl, 25 NaHCO 3, 2.5 K 2 HPO 4, 2.0 CaCl 2, 1.2 MgSO 4, 4.0Na lactate, 1.0 Na 3 citrate, 6.0 L -alanine, and 5.5 D -glucose, pH 7.4, 290 ± 2 mosmol/kgH 2 O ( 59 ). During the 60-minequilibration period and thereafter, the perfusion chamber was continuouslysuffused with a gas mixture of 95% O 2 -5% CO 2 to maintainpH of the Burg's solution at 7.4 at 37°C. The bathing solution wascontinuously exchanged at a rate of 10 ml/h using a peristaltic syringe pump(Razel, Stamford, CT).6 j  ~5 s5 C: Q: I
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For molecular studies, single tubules were dissected in cold PBS containing10 mM vanadyl ribonucleoside complex (Sigma, St. Louis, MO) to inhibit RNAdegradation, as previously described ( 3 ). On average, 15-18 mmtotal of CCDs or proximal tubules were pooled for each tubule sample. Tubuleswere rinsed three times in cold 1 x PBS and transferred to a 1.5-mlmicrocentrifuge tube for immediate RNA extraction. Dissection time was limited to 2 h to ensure RNA integrity.
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Measurement of cation fluxes. Isolated CCDs were microperfused invitro as previously described( 58 ). Transport measurements were performed in the absence of transepithelial osmotic gradients; thus watertransport was assumed to be zero. Samples of tubular fluid were collectedunder water-saturated light mineral oil by timed filling of a precalibrated 30-nl volumetric constriction pipette. The flow rate was varied from 1 to 6 nl · min - 1 ·mm - 1 by adjusting the height of the perfusate reservoir.
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For cation transport rates, three to four samples of tubular fluid werecollected at each flow rate and analyzed for K and Na concentrations. The Kand Na concentrations of perfusate and collected tubular fluid were determinedby helium glow photometry and the rates of net cation transport (in pmol· min - 1 ·mm - 1 tubular length) were calculated, aspreviously described ( 38 ). Todetermine the concentration of K and Na delivered to the tubular lumen,ouabain (100 µM) was added to the bath at the conclusion of each experiment to inhibit all active transport, and three to four samples of tubular fluidwere then obtained for analysis similar to that described above. Thecalculated ion fluxes were averaged to obtain a single mean rate of iontransport for the CCD at each flow rate. The sequence of flow rates wasrandomized within each group of tubules to minimize any bias induced bytime-dependent changes in ion transport.# `% u% h  q5 b8 q
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The transepithelial voltage (V te ) was measured betweensymmetrical calomel electrodes continuous with the perfusion pipette via a0.16 M NaCl agarose bridge and referenced to the bath, as previously described( 38 ). Voltages were monitoredusing a high-impedance electrometer (World Precision Instruments, New Haven,CT). Readings were taken at the midpoint of each timed collection andaveraged.
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A subset of CCDs, as indicated, was perfused with the scorpion venom toxiniberiotoxin (99% purity; Sigma), a selective inhibitor of the high-conductanceCa 2 -activated maxi-K channel( 5, 52 ). The final concentrationof 50 nM was prepared by diluting the toxin in the luminal perfusate., o$ B: J1 A! g9 D3 w! U

Measurement of intracellular Ca 2 concentration. After equilibration, microperfused tubules were loadedwith 20 µM of the acetoxymethyl ester of fura-2 (Calbiochem, La Jolla, CA)added to the bath for 20 min. With the use of a Nikon Eclipse TE300 inverted epifluorescence microscope linked to a cooled Pentamax CCD camera (PrincetonInstruments) interfaced with a digital imaging system (MetaFluor, UniversalImaging, Westchester, PA), intracellular Ca 2 concentration ([Ca 2 ] i ) was measured inindividually identified fura-2-loaded cells visualized using a Nikon S Fluor x 40 objective (numerical aperture 0.9, WD 0.3), as previously described( 59 ). We previously showedthat fura-2-loaded intercalated cells, identified by their selective apicalbinding of rhodamine PNA, appear more brightly fluorescent underepifluorescence illumination compared with principal cells, althoughsteady-state [Ca 2 ] i does not differ betweenthe two cell populations ( 59 ). Autofluorescence was not detectable at the camera gains used.: Z- R4 f: Z7 I# h. e: @$ u/ c

CCDs were alternately excited at 340 and 380 nm and images, acquired every2 to 10 s, were digitized for subsequent analysis. At the conclusion of eachexperiment, an intracellular calibration was performed, using 10 µM EGTA-AMin a Ca 2 -free bath and then a 2-mMCa 2 bath containing ionomycin (10 µM), as previouslydescribed ( 59 ). Standardequations were used to calculate experimental values of[Ca 2 ] i ( 17 ). Two to four principaland intercalated cells were analyzed in each CCD.
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RT-PCR and Southern blotting. RNA was extracted from tubules usinga modified acid guanidinium-phenol-chloroform RNA extraction method( 6 ). Briefly, each sample oftubules was placed in 50 µl of extraction buffer containing guanidiniumthiocyanate and allowed to sit on ice for 5 min. Five microliters of 2 M Naacetate, 50 µl of water-saturated phenol, and 20 µl ofchloroform-isoamyl alcohol were added to the buffer solution containing thetubules and gently mixed. The sample was then placed on ice for 15 min andcentrifuged at 14,000 rpm for 10 min at 4°C. The aqueous layer wasprecipitated with 100 µl of ice-cold 100% ethanol. To this, 0.6 µl ofthe coprecipitant linear acrylamide (Ambion, Austin, TX) were added andprecipitated for 1 h at -20°C. Segment-specific RNA was recovered bycentrifugation (30 min at 4°C) at 14,000 rpm. The pellet was resuspendedin 5 µl of diethyl pyrocarbonate-treated water. With the use of thestandard protocol provided by Invitrogen (Carlsbad, CA), the extracted RNA was treated with RNase-free DNase I and incubated at room temperature for 15 min.EDTA (25 mM) was added to inactivate the enzyme before heating to65°C.: L+ ^/ b: F- R- B, m

Reverse transcription was performed using oligo dT under standard techniques with Superscript II (Invitrogen). Amplification of the slo pore region was conducted using a DNA Thermal Cycler 480 (Perkin Elmer,Norwalk, CT), using sequence-specific probes (sense:5'-GTTACGGGGACGTTTATGC-3'; antisense:5'-CCAACTTCAGCTCTGCAAG-3'), 1.5 mM MgCl 2, and Platinum Taq DNA Polymerase (Invitrogen), predicted to generate a product sizeof 608 bp. In total, 40 cycles of denaturation (94°C, 1 min),annealing (58°C, 1 min), and extension (72°C, 1 min) were conducted.Amplification of GAPDH was performed using a sense primer(5'-GCTGAACGGGAAACTCACTG-3') and an antisense primer(5'-TCCACCACCCTGTTGCTGTA-3'), expected to yield a product size of 307 bp. The PCR products were size-fractionated by electrophoresis on a2% agarose gel and visualized by UV fluorescence after ethidium bromidestaining to verify that the PCR products were of expected size. The sequencesof the PCR products were verified by direct sequencing (ABI Prism model 3700Sequencer).# Y2 |* W/ ?5 \# `
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In some cases, reverse transcriptase was omitted from the reaction as anegative control for amplification of genomic DNA. RNA extracted from wholekidney of adult rabbits was used as a positive control. Data from tubulesamples were omitted from further analysis if the sample failed to generate aGAPDH amplification product.& }% T. K/ o2 W' M5 X/ w4 f
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RT-PCR of single tubules was followed by Southern blotting with a labeledprobe (MegaPrime DNA Labeling System, Amersham, UK) encoding the slo pore region, generated using the primer sets identified above applied to RNAextracted from whole kidney of adult rabbit. PCR products were depurinatedwith 0.25 N HCl for 8 min. Alkaline denaturation of the gel was then performed twice with a solution containing 1.5 M NaCl and 0.5 M NaOH for 15 min,followed by neutralization with 1 M Tris and 1.5 M NaCl, pH 7.4, twice for 15min. With the use of a standard transfer technique with 10 x SSC buffer,the DNA was blotted onto a Hybond-N nylon membrane (Amersham). After overnight transfer, the membrane was baked for2hinan80°C vacuum oven andprehybridized at 42°C in Ultrahyb solution (Ambion) for 1 h. The labeledprobe was then added and the membrane was incubated overnight at 42°C. Theblot was washed twice with 2 x SSC and 0.1% SDS at 42°C for 5 min.Visualization was performed with a phosphorimager (Molecular Dynamics).$ d9 p: g" [) d8 D9 l/ J' }+ [4 M
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Western blotting of maxi-K channel protein in kidney. Rabbit kidneys were homogenized in a buffer containing 250 mM sucrose and 10 mMtriethanolamine, pH 7.6, supplemented with protease inhibitors (1:100 dilutionof Protease Inhibitor Cocktail Set III, Calbiochem, San Diego, CA). Aliquotsof the protein lysate (100 µg) were subject to 3-15% SDS-PAGE andtransferred to an Immobilon-NC membrane (Millipore, Waltham, MA). Blots wereblocked overnight with 5% nonfat dried milk in PBS (8 mM Na phosphate, 2 mM Kphosphate, 140 mM NaCl, 10 mM KCl, pH 7.4) plus 0.05% Tween 20 (PBS-Tween),probed with an affinity-purified antibody raised in chicken against thesequence CTANRPNRPKSRESRDKQN corresponding to a COOH-terminal region of mousemaxi-K -subunit (Aves Labs) at a concentration of 0.5 µg/ml inPBS-Tween 20 for 3 h at room temperature. Alternatively, blots were probed with the anti-maxi-K antibody that was preincubated overnight at 4°C withthe peptide immunogen at a concentration of 20 µg/ml. Bound antibody wasdetected following incubation with a 1:2,500 dilution of horseradishperoxidase-conjugated goat anti-chicken IgG (Kirkegaard and PerryLaboratories, Gaithersburg, MD) by chemiluminescence (Western BlotChemiluminescence Reagent Plus, New England Nuclear, Boston, MA).

Immunofluorescence localization of maxi-K channels in the kidney. Coronal sections of rabbit kidneys were fixed in 4% paraformaldehyde andsucrose and embedded in medium (Cryo-Gel, Instrumedics). Serial 4-µm-thicksections were cut on a cryostat (Leica CM1900) and collected on Superfrostmicroscopic slides (Fisher Scientific). Sections were hydrated and washed withPBS three times and quenched with PBS-0.02% Gly-1% BSA (PBS buffer solution). The tissue was then permeabilized with PBS buffer solution and 0.1% TritonX-100 for 10 min, blocked with PBS buffer solution for 30 min, and then with5% milk with 0.05% Tween 20 in PBS buffer solution for 60 min.9 `: b% c  X1 {
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Tissue sections were incubated with the anti-maxi-K antibody (6 µg/ml)for 1 h at room temperature. For peptide competition experiments, peptide wasadded to the primary antibody at a concentration of 80 µg/ml. Afterincubation with primary antibody, the sections were washed three times for 5min with PBS buffer solution with 0.05% Tween 20. The secondary antibody, aCy3-conjugated Affinipure F(ab') 2 fragment donkeyanti-chicken IgY, was applied at a 1:3,000 dilution (stock concentration of1.5 mg/ml) in PBS buffer with 0.05% Tween 20 for 45 min at room temperature;each section was colabeled with FITC-conjugated dolichos biflorus agglutinin(DBA; 5 µg/ml), a principal cell marker( 64 ). Sections were thenwashed three times with PBS buffer with 0.05% Tween 20 for 5 min followed bythree quick washes with PBS solution. All sections were mounted on coverslipswith phenylene diamine mounting media.! i) p5 T- J5 z6 E. H. s! N

Confocal microscopy was performed on a Leica TCS SL equipped with kryptonand green and red helium-neon lasers. Images were acquired with the use of a x 100 plan-apochromat objective (numerical aperture 1.4) and appropriatefilter combination [Kalman filter ( n = 4)]. Images were saved in atag-information-file format, and the contrast levels of the images wereadjusted in the Photoshop program (Adobe, Mountain View, CA) on Power PC G-4 Macintosh (Apple, Cupertino, CA). The contrast-corrected images were importedinto Freehand (Macromedia, San Francisco, CA) for viewing.$ x% W/ W* s" E/ C* c& J

Statistics. All results are expressed as means ± SE; n equals the number of animal or tubule samples used for in vitromicroperfusion studies and RT-PCR. Multiple ( 3) blots and amplificationswere performed with different tubules or RNA samples that were isolated fromdifferent animals. CCDs from young animals were harvested from at least fivelitters of animals. Comparisons were made by paired and unpaired t -tests as appropriate. Significance was asserted if P$ ?$ @: W$ H! c
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Effect of age on flow-stimulated net K secretion. CCDs isolated from 4-wk-old rabbits showed baseline net K secretion (5.4 ± 0.3 pmol· min - 1 ·mm - 1, n = 5), albeit less than thatmeasured in the 6-wk-old animals (10.5 ± 1.4 pmol ·min - 1 · mm - 1, n = 5, P Fig.1 A ), when perfused at a flow rate of 1.3 ± 0.1 nl· min - 1 ·mm - 1. However, an increase in tubular fluid flowrate to 6.0 ± 0.1 nl · min - 1 · mm - 1 failed to elicit an increase in netK secretion in CCDs isolated from 4-wk-old animals( Fig. 1 A ), as wepreviously reported ( 38 ). At 5wk of age, an increase in tubular flow rate from 1.1 ± 0.1 to 6.3± 0.5 nl · min - 1 ·mm - 1 resulted in a small, but significant,increase in net K secretion (from 6.4 ± 0.5 to 11.4 ± 1.2 pmol· min - 1 ·mm - 1, n = 5, P increased further in CCDs isolated from 6-wk-old animals (from 10.5 ± 1.4 to 27.0 ± 1.9 pmol ·min - 1 · mm - 1 atflow rates of 1.3 ± 0.1 to 6.0 ± 0.2 nl ·min - 1 · mm - 1, n = 5, P Fig.1 A ). V te did not change significantly in anyage group in response to an increase in tubular fluid flow rate (4 wk:-6.4 ± 0.8 to -4.4 ± 0.7 mV, P = 0.24; 5wk: -7.4 ± 2.9 to -3.0 ± 1.6 mV, P = 0.15;6 wk: -8.1 ± 1.4 to -6.6 ± 2.1 mV, P =0.36).1 n$ k& N  b& g5 T2 ?5 a% h
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Fig. 1. Flow stimulation of net K secretion ( A ) and Na absorption( B ) in the maturing cortical collecting duct (CCD). Net transport wasmeasured at slow ( 1 nl · min - 1 · mm - 1 ) and fast ( 6 nl ·min - 1 · mm - 1 )flow rates in tubules isolated from 4 ( n = 5)-, 5 ( n = 5)-,and 6-wk-old ( n = 5) rabbits. Although a 6-fold increase in flow ratestimulated net Na absorption at 4 wk to levels comparable to those observed at6 wk of age, no flow-stimulated increase in net K secretion was detected at 1mo of life. The flow-stimulated increase in K secretion first became apparentat 5 wk of age. * P - 1 ·mm - 1.

Effect of age on flow-stimulated net Na absorption. To determine whether the absence of flow-stimulated K secretion in the 4-wk-old CCD was dueto a limited capacity of the tubule to augment net Na absorption with anincrease in flow rate, net Na absorption was measured in the same tubules asdescribed directly above. As shown in Fig.1 B, net Na absorption was similar in the three age groupsstudied at a flow rate of 1 nl · min - 1 · mm - 1. After an increase in tubular flowto 6 nl · min - 1 ·mm - 1, net Na absorption (in pmol ·min - 1 · mm - 1 )increased approximately fourfold (4 wk: 27.9 ± 2.1 to 106.8 ± 4.1; 5 wk: 26.2 ± 1.3 to 90.8 ± 5.4; 6 wk: 31.3 ± 2.2 to105.9 ± 10.2) in each age group studied ( P Na absorption areestablished before the onset of flow-stimulated K secretion. There was nosignificant difference ( P = not significant) noted between the ratesof net Na transport at the 6-nl · min - 1 · mm - 1 flow rate among the three age groups studied.' K6 h0 j$ z0 m& [: @8 K
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Effect of iberiotoxin on flow-stimulated net K secretion. To confirm that flow-stimulated net K secretion is mediated by a high-conductancemaxi-K channel, the effect of the specific channel inhibitor iberiotoxin wastested in three CCDs isolated from rabbits 6 wk of life. In the absence of iberiotoxin, net K secretion increased from 7.8 ± 2.1 to 23.0 ±1.1 pmol · min - 1 ·mm - 1 ( P ± 0.1 to 6.4 ± 0.2 nl ·min - 1 · mm - 1 ( Fig. 2 A ). Theflow-stimulated component of net K secretion was inhibited by addition of 50nM iberiotoxin to the luminal perfusate during a period of sustained high flow (6.9 ± 0.4pmol·min - 1 ·mm - 1, P ( Fig. 2 A ). Luminaliberiotoxin did not affect flow-stimulated net Na absorption, which averaged101.8 ± 6.6pmol·min - 1 ·mm - 1 in these same tubules ( P = not significant compared with transport rate in absence of iberotoxin) ( Fig.2 B ).2 P' o' |) I+ R# P
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Fig. 2. Effect of luminal iberiotoxin (IBX) on flow-dependent K secretion( A ) and Na absorption ( B ) in the adult CCD. Net transport(in pmol · min - 1 ·mm - 1 ) was measured in the absence of toxin at slowand then fast flow rates before 50 nM iberiotoxin was added to the luminalperfusate. Iberiotoxin inhibited the flow-stimulated increase in net Ksecretion but not Na absorption; n = 3. * P - 1 · mm - 1 inthe absence of iberiotoxin.

Flow-induced [Ca 2 ] i transients. We previously reported that an increase in tubular flow ratein the adult rabbit CCD is associated with an increase in[Ca 2 ] i ( 59 ). To determine whetherflow induces comparable increases in [Ca 2 ] i in maturing CCDs, CCDs from 1- to 5-wk-old rabbits were loaded with fura-2 and[Ca 2 ] i was measured. Baseline[Ca 2 ] i was lower in principal cells at 1 wkof age (53.4 ± 14.3 nM, P with older agegroups (97.8 ± 26.9, 115.8 ± 42.2, 99.1 ± 14.0, and 77.1± 15.6 nM at 2, 3, 4, and 5 wk, respectively, n = 3 in allgroups except for 5-wk-old subjects, where n = 4)( Fig. 3 ). Resting[Ca 2 ] i in intercalated cells in 1-wk-oldCCDs (70.4 ± 11.5 nM) was not significantly different from thatmeasured at 2 (86.6 ± 26.0 nM) and 3 (125.2 ± 43.8 nM) wk ofage, but it was less than that detected in the older age groups (100.7± 4.7 and 120.7 ± 13.3 nM at 4 and 5 wk, respectively)( Fig. 3 ). An acute increase intubular fluid flow rate, sufficient to increase tubular diameter by 25.3± 3.4%, increased [Ca 2 ] i in bothprincipal and intercalated cells at all ages ( Fig. 3 ). However, the peak[Ca 2 ] i elicited by high flow was significantly lower in 1-compared with 5-wk-old principal ( P P
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Fig. 3. Effect of high flow on intracellular Ca 2 concentration ([Ca 2 ] i ). CCDs isolated from1- to 5-wk-old rabbits exhibited a significant increase in[Ca 2 ] i in both principal (PC) andintercalated (IC) cells following an acute increase in tubular fluid flow rate(associated with an increase in luminal diameter of 20-25%) ( n = 3 CCDs at each age, except 5 wk old, where n = 4, * P 2 ] i ).5 F) b) A$ X, A  `
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Developmental expression of slo message in single tubules. To examine the developmental expression of slo mRNA in the CCD, RT-PCRwas performed on single CCDs ( n = 8 to 20 samples/age group) andproximal tubules ( n = 7 to 11 samples/age group) using primersdirected against the pore region. Slo transcripts were notconsistently detected until week 4 of life in the CCD (Figs. 4 and 5 ). CCD samples were split forselective amplification of slo and GAPDH; a single band ofappropriate size for GAPDH was detected at all age groups( Fig. 4 ), indicating that theabsence of slo transcripts early in life was not due to RNAdegradation. In the absence of reverse transcriptase, bands were not detected.The sequence of the amplified product showed a 99.5% homology to the publishedrabbit slo channel at both the nucleotide and predicted amino acidlevel.0 a5 S$ o% L( W4 \! |8 z: x

Fig. 4. Maxi-K channel ( -subunit) mRNA expression in CCDs. RT-PCR of CCDs( top ), using primer pairs designed to amplify the pore region of the -subunit, revealed no sequence-specific signal until week 4 oflife. Southern blot ( middle ) analysis of the PCR products, using apore-specific probe, revealed transcripts in CCDs isolated from the 2-wk-oldanimal only on this blot (out of 4 in total). GAPDH-specific PCR products( bottom ) were detected at each age confirming that degradation of theRNA did not occur. KDN, adult whole kidney.( }  ]1 J  V# M( F2 b7 x8 E0 n$ Q6 m

Fig. 5. Incidence of maxi-K transcripts in the CCD during postnatal maturation.Numbers in parentheses indicate the total number of GAPDH-positive CCD samplesobtained at each week of postnatal life. At 1 wk of age, maxi-K message wasnot detected in any of 4 CCD sets sampled. By week 2 4 wkold.- q) Y6 G/ j$ o! r: u
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Southern blotting of the RT-PCR gels, using a channel pore region-specific probe, revealed a signal in only one of four CCD samples harvested from2-wk-old kidneys (Figs. 4 and 5 ). Transcripts were never detected in any CCD samples isolated from 1- and 3-wk-old rabbits ( Fig. 5 ), whereas all CCDsdissected from animals 4 wk of life showed slo transcripts. Slo transcripts were also not detected in any proximal tubule segmentat any age, although all tubule samples expressed GAPDH( Fig. 6 ).! q& w/ S4 v9 Y* X7 G  Z7 p
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Fig. 6. Comparison of maxi-K channel ( -subunit) mRNA expression in CCDs andproximal tubules during postnatal life: representative Southern blot. RT-PCRof proximal tubules (P; all samples positive for GAPDH message) failed togenerate maxi-K channel sequence-specific transcript at any age, even whenprobed on a Southern blot. Similar to the results shown in Fig. 4, Southern blot analysisof CCD PCR products (C) revealed transcripts in samples from 5- and 6-wk-oldanimals. A, whole kidney from an adult rabbit.

Western analysis of maxi-K channel protein in kidney homogenate. An immunoblot of whole rabbit kidney lysate was performed to characterize theanti-maxi-K channel antibody. Polypeptides with apparent molecular weights of 200 and 90 kDa were recognized by the antibody but not by antibodypreincubated with the immunizing peptide( Fig. 7 ). The 90-kDapolypeptide has a lower apparent molecular mass than was reported for therabbit -subunit isoform rbSlo1 expressed in HEK293 cells (146 kDa)( 56 ) and may represent asplice variant of rbSlo1. The 200-kDa polypeptide may represent an -subunit dimer.+ ?, V% [& R+ Z3 U' E: q( F
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Fig. 7. Immunoblot of rabbit kidney lysate probed with chicken antibody directedagainst the -subunit of the maxi-K channel ( lane 1 ) orantibody incubated with the immunizing peptide ( lane 2 ). One-hundredmicrograms of protein were loaded in each lane. Polypeptides with apparentmolecular weights of 200 and 90 kDa were specifically recognized by theanti-maxi-K channel antibody.

Immunolocalization of maxi-K channels in the kidney. Cryosections of adult rabbit kidney were labeled with antibodies directed against the -subunit of the maxi-K channel. Principal cells were labeled with DBA.Labeling with both markers was observed in discrete groups of tubules,presumably collecting ducts ( Fig.8 ). Apical localization of maxi-K -subunits in DBA-positive tubules was observed in intercalated (i.e., DBA negative) cells ( Fig. 8, arrows). Intracellularlocalization of maxi-K channels in CCDs was observed in both DBA-positiveprincipal ( Fig. 8, arrowheads)as well as DBA-negative intercalated cells. These results suggest that apicalmembrane expression of maxi-K channels is restricted mainly to intercalatedcells and are in agreement with the previous electrophysiological finding thatthe density of conducting maxi-K channels in CCDs is greater in intercalated than principal cells ( 35 ).+ l  [9 U4 o4 h/ D* o0 {( W

Fig. 8. Immunolocalization of maxi-K -subunits in the adult rabbit kidney.Indirect immunofluorescence labeling of the -subunit of maxi-K channelswas performed in adult rabbit kidney as described under MATERIALS AND METHODS. Sections were also labeled with FITC-conjugated dolicosbifloris agglutinin (DBA), a principal cell marker. A, D,and G : DBA localization (green). B and E : maxi-K -subunit localization (red). C and F : localization ofboth DBA (green) and maxi-K -subunit (red). The maxi-K channel antibodyexhibited a heterogeneous staining pattern in tubular profiles that coexpressDBA (i.e., collecting ducts). Maxi-K channel protein was detected along theapical membranes of DBA-negative cells (intercalated cells, arrows) in CCDs.Intracellular staining was observed in DBA-positive cells (arrowheads) as wellas in DBA-negative cells (not shown). H and I : section ofcortex labeled with antibody in the presence of excess immunizing peptideshowed no maxi-K staining.

We also examined the expression of maxi-K channels in the developing kidneyof the rabbit. No significant immunostaining with the anti-maxi-K antibody wasobserved until week 5 of postnatal life, at which time staining wasobserved in both DBA-negative intercalated cells as well as DBA-positiveprincipal cells ( Fig. 9 ). As inadults, apical membrane expression of maxi-K channels was limited toDBA-negative cells ( Fig. 9,arrows), and intracellular staining was observed in both DBA-positive ( Fig. 9, arrowheads) andDBA-negative cells ( Fig. 9 ).These results correlate well with developmental appearance of flow-dependent Ksecretion and with message encoding the maxi-K -subunit.
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Fig. 9. Immunolocalization of maxi-K channel in the maturing rabbit kidney.Sections of kidney were labeled as described in the legend for Fig. 8. Maxi-K -subunit(red) was not detected in collecting ducts [DBA-positive (green) tubularprofiles] until week 5 of life. Sections of 5- and 6-wk-old kidneysshowed both apical (arrows) and intracellular immunodetectable channels inCCDs. Apical membrane localization of maxi-K -subunits in CCDs wasobserved in DBA-negative (i.e., intercalated) cells. Intracellularlocalization of maxi-K -subunits was observed in both DBA-positive(arrowheads) and DBA-negative cells./ |* I% g" j8 x6 j

DISCUSSION6 z0 N8 A' F* @9 x/ u7 M& c9 o) l
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The results of the present study demonstrate that flow-stimulated net Ksecretion is not detected in the maturing rabbit CCD until 5 wk of age( Fig. 1 A ) and followsthe developmental appearance of baseline net K secretion( 38 ) that is presumed to bemediated by the SK channel (i.e., ROMK). SK channel activity increasesprogressively after the first week of postnatal life ( 42 ), a developmental processthat closely parallels increases in ROMK mRNA levels and expression ofimmunodetectable ROMK protein at the apical membrane of CCD principal cells( 3, 64 ). The absence offlow-stimulated net K secretion early in life is not limited by the capacityof the CCD to respond to an increase in flow with augmented Na absorption or arise in [Ca 2 ] i (Figs. 1 B and 3 ). In fact, the transport andsignaling pathways mediating these mechano-induced responses appear to beestablished at an early age.- J/ k7 k* ~, f  r0 D
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We recently proposed that flow-stimulated K secretion is mediated by theCa 2 /stretch-activated high-conductance maxi-K channel ( 58 ) previously identified byelectrophysiological analysis of the apical membrane of rat and rabbit CCDs( 35, 41 ). This notion is supportedby the present observation that iberiotoxin, a scorpion venom toxin known toblock the maxi-K channel with high affinity( 5, 52 ), prevents flow-stimulatedK secretion without affecting baseline K secretion( Fig. 2 A ).) K) G  `* i5 J  R/ l# }

Two subunits of the maxi-K channel have been cloned( 2, 8, 23 ): a pore-forming -subunit, a member of the slo family of K channels originallycloned from Drosophila, and a regulatory -subunit. Coexpressionof both subunits in Xenopus laevis oocytes enhances the voltage,Ca 2  , and charybdotoxin sensitivity of the channelcompared with expression of the -subunit alone ( 20, 30 ). All slo genesgenerate multiple transcripts via alternative splicing( 1, 4, 25, 33 ). Heterologous expression of the unique variants reveals differences in their voltage, Ca 2  , and hormonal sensitivity( 54, 61 ) and, as suggested in morerecent studies, their subcellular localization and association withinteracting proteins ( 32 ).8 _& p3 `! `$ D& S" I$ |7 e3 w7 Y# k
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Two alternatively spliced transcripts of the maxi-K channel -subunithave been cloned from the rabbit medullary thick ascending limb (MTAL), asegment that expresses conducting channels ( 33 ). Rbslo1 containsa 174-bp insert immediately following the predicted S8 transmembrane domain( 33 ). Although the amino acidsequence of rbslo1 is highly homologous to slo channels cloned from mouse, human, and chicken( 33 ), the rabbit subunit exhibits enhanced Ca 2 and voltage sensitivitiescompared with the other cloned isoforms due to the COOH-terminus insertion sequence ( 19 ). Rbslo2 has a 104-bp deletion between the S9 and S10 regions( 33 ). Rbslo1 and 2 are expressed in glomeruli, thin and thick limbs of Henle, andcortical and medullary collecting ducts, but they are rarely detected inproximal tubule ( 33 ). Functional studies provide evidence for the differential expression of rbslo transcripts in unique segments and cell types in the rabbitkidney. For example, stretch activation (application of pipette suction) ofmaxi-K channels in cell-attached or inside-out patches on rabbit CCDintercalated cells persists even after chelation of freeCa 2 with EGTA in the pipette or the bath solutions,implying that stretch activation of these channels is not mediated byincreased Ca 2 entry into the cell and is notCa 2 sensitive( 35 ). In contrast, the rabbitconnecting tubule maxi-K channel is not stretch activated in the absence ofambient Ca 2 ( 50 ).# Y# `8 _5 u. h/ F0 [. b- K& P/ ?
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We considered it theoretically possible that the differential expression ofunique slo splice variants during maturation could account, in part,for our early (2 wk after birth) electrophysiological detection ofhigh-conductance K channels, activated by depolarization ( 41 ), in 10% of principalcells, and relatively late appearance (5 wk of age; Fig. 1 A ) offlow-stimulated K secretion in the rabbit CCD. Given that our primer pairswere designed to amplify the highly conserved pore region of the -subunit common to all splice variants, our failure to detect any -subunit-specific transcripts in CCDs isolated from animals 4 and 5 ) likely reflects either that 1 ) levels of expression are below the threshold for detection by ourmolecular analyses or 2 ) high-conductance K channels detected in theneonatal principal cell are encoded by a gene distinct from slo. It should be stated that the high-conductance K channels were not rigorouslycharacterized in the neonatal CCD but were assumed to be maxi-K channels basedon their conductance and similarity in kinetics to the well-described ratchannel ( 35 ). Future effortsshould be directed at a thorough electrophysiological analysis of thesechannels in the maturing CCD.

Functional evidence for high-conductance,Ca 2 -activated K channels has been demonstrated not onlyin the MTAL, connecting tubule, and collecting duct, but also in proximaltubule ( 31, 33, 52 ). However, neither we( Fig. 6 ) nor others( 33 ) identified slo transcripts in proximal tubules of mammalian kidneys, raising uncertainty tothe molecular identity of the proximal tubule channel. The functional maxi-Kchannel detected in this segment may be encoded by another gene.
: N9 Y' E, E& H5 _' {
An unexpected finding of the present investigation was the immunolocalization of the -subunit of the maxi-K channel predominantly to the apical surface of intercalated cells. Whereas principal cells alsoexhibited anti-maxi-K channel antibody labeling, the signal appeared to belocalized primarily within the cell, with little apical label detected. Theseresults, although in good agreement with the previous electrophysiologicalfindings of a higher density of maxi-K channels in intercalated than principalcells ( 35 ), suggest that theflow-activated component of net K secretion may be mediated by intercalatedcells, a cell population not traditionally considered to participate in net Ksecretion. Of note is that these cells possess an apical H-K-ATPase( 7, 45 ). Others( 45, 60 ) have suggested thepresence of an apical K channel in intercalated cells that allows for K thatis absorbed via the H-K-ATPase to recycle back into the tubular fluid, toensure a continuous supply of luminal K for the apical pump. Perhaps thisfunction is accomplished by the maxi-K channel.; w- q2 z) T3 e3 e  ~3 O
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Our finding that a rapid increase in tubular flow rate leads to an increasein both principal and intercalated cell[Ca 2 ] i in the differentiating CCD isconsistent with our recent observations in the fully differentiated CCD( 26, 59 ). Emerging evidence suggests that the apical cilium present in principal but not intercalatedcells is a flow sensor ( 34, 36 ). We recently proposed thatflow-induced shear or hydrodynamic impulses at the cilium or apical membraneof the fully differentiated CCD stimulate release ofCa 2 from inositol-1,4,5-trisphosphate (IP 3 )-sensitive internal stores and influx from the extracellular space ( 26 ). The maturationalincrease in the magnitude of the flow-induced increase in[Ca 2 ] i in CCD cells( Fig. 3 ) is thus of particularinterest. The modest response of the neonatal principal cell to flow cannot beexplained by the absence of an apical central cilium; indeed, scanningelectron microscopic analyses reveal that the principal cell cilia at birthare longer than those in the adult( 10 ). Functional expression ofCa 2 -conducting channels in the collecting duct may be low in early renal development( 53 ). Studies in developing neurons suggest that IP 3 receptors and Ca 2 channels appear to be present early in life but are not assembled to allow IP 3 -assisted, Ca 2 -inducedCa 2 release( 62 ).
4 ], H3 t( J; R4 h. m! u  I& I
In summary, our data suggest that the absence of flow-dependent K secretionearly in life is due to the developmental regulation of expression of maxi-Kchannel -subunit message and protein. The temporal association betweenappearance of flow-stimulated K secretion in the maturing CCD( Fig. 1 A ) anddetection of message encoding slo (Figs. 4 and 5 ) and the immunodetectable protein ( Fig. 9 ) lends furthersupport to the premise that the maxi-K channel mediates flow-stimulated Ksecretion. The expression of maxi-K channels in the apical membrane ofpredominantly intercalated cells rather than principal cells suggests that intercalated cells mediate, at least in part, flow-stimulated K secretion.

DISCLOSURES( ^$ j8 V* n6 v
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This work was supported by National Institutes of Health (NIH) GrantsDK-38470 (to L. M. Satlin) and DK-51391 (to T. R. Kleyman). C. B. Woda wassupported by NIH Grant T32 HD-07537 (Training Grant in Developmental Biologyof Membrane Transport; L. Satlin, PI) and N. B. Miyawaki by NIH Grant T32DK-07757 (Renal Medicine Training Grant; P. Klotman, PI). Abstracts of thiswork were presented at the Annual Meetings of the American Society of Nephrology in 2001 (San Francisco, CA) and 2002 (Philadelphia, PA)., ?5 ]6 b# }+ I$ q' V2 N

ACKNOWLEDGMENTS
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The authors thank J. Bruns for technical support.
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