Functional Profile of the Isolated Uremic Nephron

IMPAIRED WATERPERMEABILITY ANDADENYLATECYCLASE

RESPONSIVENESSOF THE CORTICALCOLLECTINGTUBULETO

VASOPRESSIN

LEONG. FINE, DETLEF SCHLONDORFF,WALTERTIuZNA, RICHARDM. GILBERT, andNEAL S. BRICKER, Department of Medicine, Division of Nephrology, Universityof Miami School of Medicine, Miami, Florida 33152, and Department ofMedicine, Division of Nephrology, Albert Einstein College of Medicine,Bronx, New York 10461

A B S T RA C T Resistance of the chronically diseasedkidney to vasopressin has been proposed as a possibleexplanation for the urinary concentrating defect ofuremia. The present studies examined the waterpermeability and adenylate cyclase responsiveness ofisolated cortical collecting tubules (CCT) from remnantkidneys of uremic rabbits to vasopressin. In the ab-sence of vasopressin the CCTs of both normal anduremic rabbits were impermeable to water. At thesame osmotic gradient, addition of a supramaximalconcentration of vasopressin to the peritubular bathingmedium led to a significantly lower net water fluxper unit length (and per unit luminal surface area) inuremic CCTs than in normal CCTs. Transepithelialosmotic water permeability coefficient, Pf, was 0.0232+0.0043 cm/s in normal CCTs and 0.0059+0.001 cm/sin uremic CCTs (P < 0.001). The impaired vasopressinresponsiveness of the uremic CCTs was observedwhether normal or uremic serum was present in thebath.

Basal adenylate cyclase activity per microgram pro-tein was comparable in normal and uremic CCTs.Stimulation by NaF led to equivalent levels of activityin both, whereas vasopressin-stimulated activity was50% lower in the uremic than in the normal CCTs(P < 0.025).

The cyclic AMP analogue, 8-bromo cyclic AMP,produced an increase in the Pf of normal CCTsclosely comparable to that observed with vasopressin.In contrast, the Pf of uremic CCTs was only minimally

Dr. Fine is the recipient of a Research Career DevelopmentAward from the National Institutes of Health.

Received for publication 8 August 1977 and in revisedform 13 January 1978.

increased by this analogue and was not further stimu-lated by theophylline.

These studies demonstrate an impaired responsive-ness of the uremic CCTto vasopressin. This functionaldefect appears to be a result, at least in part, of ablunted responsiveness of adenylate cyclase to vaso-pressin. The data further suggest that an additionaldefect in the cellular response to vasopressin may exist,involving a step (or steps) subse(luent to the formationof cyclic AMP.

A unifying concept of the urinary concentratinigdefect of uremia is proposed which incorporates a num-ber of hitherto unexplained observations on the con-centrating and diluting functions of the diseasedkidney.

INTRODUCTION

Amongthe several mechanisms proposed to explain theurinary concentrating defect of uremia is an impairedresponsiveness of the kidney to vasopressin (1, 2). Theevidence supporting this proposed mechanism is in-direct and has been derived from clearance-typestudies in man. Nevertheless, certain features of end-stage renal disease suggest that some aspect of theuremic state per se rather than a series of separatedefects, each specific for a different disease state, mayaccount for the failure of the uremic patient to produceurine which is maximally concentrated in response toan appropriate stimulus. A large variety of renaldiseases unrelated etiologically and vastly differentanatomically are characterized by a urinary concentrat-ing defect (1-5). Among these, disruption of the innermedullary structures (6, 7) or local alterations in medtul-lary blood flow (8) are of only occasional importance

J. Clin. Invest. © The American Society for Clinical Investigation, Inc., 0021-9738/78/0601-1519 $1.00 1519

as an explanation for the concentrating defect. Im-paired sodium chloride transport out of' the thick as-cending limb of Henle's loop does not appear to playan important role in this pathophysiological aberrationsince the ability to dilute the urine persists long afterthe concentrating defect becomes manifest (3). Simi-larly, the obligatory solute diuresis per nephron char-acteristic of most forms of chronic renal disease isunlikely to be the main underlying cause of the defectsince the maximal urine:plasma osmolality ratio isdiminished even when values are corrected for theeffects of increased solute excretion per nephron (3).

Recently we have demonstrated an impaired intra-renal recycling of urea and failure to concentratesolutes in the medullary interstitium in the pyelo-nephritic rat. This abnormality is associated with aninability to concentrate the urine normally with failureto conserve free-water maximallv (9). Amonig the pos-sible explanations for this phenomenon, anid one whichcould not be ruled out by the existing data was adiminished responsiveness to vasopression in the corti-cal collecting tubule. Because this segment of'the nephl-ron is essentially impermeable to urea but permeableto water in the presence of vasopressin (10), theremoval of water from this region serves to elevate theconcentration of urea within the tubular fluid. Hence,a high concentration of' urea is presented to the papil-lary collecting duct, a segmenit that is permeable tourea. By this mechanism, diff'usion of' urea into theinterstitium occurs. Impaired vasopressin responsive-ness would thus limit the ability of the kidney to con-centrate the urine by two separate mechanismiis: (a) im-paired diffusion of' water out of' the collecting tubule,and (b) failure to recycle urea anid accutmullate inter-stitial solute normally.

In the present studies, the vasopressin responsive-ness of the isolated cortical collecting tuibule of normaland uremic rabbits was studied in vitro.

METHODS

Experimental animalsExperiments were performed on isolated cortical collecting

tubules of white New Zealand femiiale rabbits 2-3 kg inweight. The animals vere maintain-ed on an ad libitum dietof standard rabbit chow anid tap water for 1-3 m11o beforethe study. The tubule segmenits studied were obtained from(a) the intact kidneys of normal rabbits and (b) the solitaryremnant kidneys of uremic rabbits. The surgical techni(qtue forthe creation of the remnant kidnev in the rabbit has leendescribed elsewhere (11).

Maximum urinary cotncentrating abilitylStudies were performed on seven normal and nine uiremic

rabbits. The animals were placed in metabolic cages anddeprived of food and water for 32 h. The urinie formed dturingthe first 24-h period wvas discarded. Each animilal theni re-

ceived five units of Pitressin tannate in oil (Parke, Davis &Co., Detroit, Mich.) and all the urine passed during theeinsuing 8 h wZas collected in a beaker under mineral oil. Urineosmolality wvas measured with a W7escor vapor pressure osmilo-mleter (Wescor Inc., Logan, Utah).

Prepa ratiotn of sera acnd perfusion solutionsUremiiie rabbit serumii was obtained from the femiioral artery of

uremie rabbits as dlescribedl elsewhere (11). Normal rabbitserumii was obtained commllercially (Microbiological As-sociates, Walkersville, Md.). Blood urea nitrogen (BUN)' wasmeasured on all sera using a Beckman BUNanalyzer (Beek-mani Instruments, Inc., Fullertoni, Calif.). Since the uiremicsera tencded to have higher osmolalities than the normalsera, all were adjusted to 330 mosmol/kg H20 by the ad-dition of the appropriate amiiounlt of urea. The perfisate usedin all experiments was comlposed of (in millimoles per liter);NaCl 60; K2HPO42.5, CaCl2 1.0 and MgSO4 1.2. (Osmolalitv130 mosmol/kg H20.) [carboxjy-'4C]Inulin (50 ,uCi/ml) wvasaddedl to the perfusate to act as ani impermiieant volumemarker. The pH of all solutions was adjusted to 7.4.

Perfusioni of cortical collecting tubulesCortical collecting tubultes (CCT) \vere dissected from nor-

mal or remiinant kidneys and( perflsed in vitro as describedpreviously from this laboratory (12, 13). CCTs from niniie nlor-mlal rabbits were studied; six were perfused in a bathi f norimlalserumii anid three in uLremic serrumil. CCTs from 13 uremiiie rab-bits were studied; 9 in uremiiic sera and 3 in normal sera.Tubule lengths varied fromii 0.71 to 3.18 1mm; there were nosignificanit differences in the m11ean1 lengths of' the segmenitsperfused betwveen the grouips.

Experimenits were conducted at 37°C at a transtubular osmo-tic gradient of 200 mosmiiol/kg H20. The bath was bubbledw ith 95%7 O2-5% CO., aind osm'olalitv was miaintained constanitby moniitorinig the addition of deionized water to the bath(12, 13). Transepithfelial poteintial difference (PD) wvas inleas-tired throuighouit the experimnenit as described previously (12,13). Both ends of' the tubule were instulated with Svlgard184 (Dow Corning C'orp., Midland, Nlich.). The imieain (±SE)perftusioni rate was 7.55± 1.03 rl/min in normiial tubuiles aind7.07 ±0.74 rl/min in turemiic- tuibuiles.

Influtiece of vasopressin. CCTs were generallvy mlouintedon the perfuisioni apparattus within 15-20 mni after the deathof thle ainimal. An equilibration period of 21/2 h was allowed,dluring which timle transepithelial PD hald stabilized aind thetubules hiad become impermeable to water (see Restults).

Four-five control collections of fltuid emerging from thettulbuile were *mdle. Vasopressin (Pitressin, Parke, Dakvis &Co.) 200 /AU/ml was the n added to the bath. Previousstudies have dlemono.strate(d that this is a stupraimaximiial closeof vasopressin (10, 14, 15). In five of the experiments onuremic tul)ules at close of' 2,000 AcU/ml was tused. After theadditioni of vasopressin. collections were made every 10min for 1 h. Samples f'romii each period were analyzed for14C activity and osmolality. The saimples for radioactivitywere pipetted directly into li(quiid scintillation fluid, whereasthose uised for osmolalitv measturements were depositeduinder oil aind theni transfesrred to the sample holder of a

'Abbreuiation.s i.sed in tl/is paper: 8-bromo-cAMP, 8-l)romoadenosine 3',53-cycJlic miionophosphoric acid; BUN,1)lood1 ireai nitrogen; CCT, cortical collecting tubule;Jv, niet water fluix; PD, potenitial clififerenee; Pf, tranisepitlhelialosmotic water permeability coefficient.

1520 L. G. Fine, D. Schlon(ldorff, WU. Triznia, R. Al. Gilbert, and N. S. Bricker

Clifton Nanioliter Osmometer (Cliftoni Techniical Physics,Hartford, N. Y.).

Influence of 8-bromoadetnosinie 3',5'-cyclic nionophos-phoric acid (8-bronmo-cAMP). Pre-ious studies on isolatedCCTs have demonstrated that only excessively high coni-centrations of cAMP (approximately 10 mMI) can mimicthe ,vater permeability response of vasopressin (10, 15). Inthe present studies the influeince of a more permeableanalogue, 8-bromo-cANIP (16), was evaluated in four normalanid five uremic CCTs. After the initial equilibration andconitrol periods 8-bromo-cA.MP was adde(d to the bathsolution in increasinig concenitrationis varying fromll 10 ,MNI to1 mM. At each bath conceentrationi of 8-broimo-cAMP, samipleswere collected everv 10 mill for 30 mnm anid analyzed for14C and osmolality as described above. To evaluate the pos-sibility that the decreased responise to exogenoIIs cAMPmight be due to increased phosphodiesterase activity, theuremic CCTs were studied durinig an addlitionial experimenitalperiod in which 1 m.M 8-bromo-cANIP plus 8 mnM theophyl-line were present in the bath.

CalctldationsPerfusion rate (\V) is calculated as 14C1J(Q4C)o t where '4CL

is the total amount of isotope collected, (14C)0 is the con-centration of isotope in the perfusate, and t is the dturationiof the collection. Net fluid reabsorption (net water flux), Jv,is e(qual to Vo - VL wshere 7L iS the collection rate. Jv isexpressed per millimeter length of tubule (nianioliters permillimeter per minutite) or per Unlit lumiinial surfice (nanio-liters per square centimeter per seconid). Tubthle lenigthl andinternal diameter were measured (luring perfuisioll with acalibrated reticle in the ocular of the microscope. Tralls-epithelial osmotic water permeability coefficient, Pf, (ceniti-meters per second) was computed accordinig to the expressionderived by Al-Zahid and co-xworkers (17):

- VOCO[CO - CL 1 (Cl -Cb) CO1Pi + lA Vw COCbCL (Cb)2 (C0 - CO)) Cl

where V, is the perfusion rate; Co, (b, and CI are theosmolalities of' the perftusate, bath, andl collecte(d flui(s, re-spectively; A is the luminal surface area; and( V\v is thepartial molar volume of water. This expressioni re(Iliires thatthe reflection coef'ficient of' the solute driv ing osmiioticflowv be unity.2

After the addition of vasopressin to the hath, net waterflux increased to maximal levels within 10 min and remainedstable for at least 30 min. All resuilts were thereforeexpressed as the mean of the values obtaine(d duriing thefirst three 10-min periods after the addition of vasopressin.

Assays for adenylate cyclase on isolatedcortical collecting tubulesAdenylate cyclase activity in isolate(d nephron segmiienits was

determined by a modification of the method of Imbert et al.

2 NaCI was the solute tused to genierate the osmoticgradient. 0NaC1= 1 in the normal rabbit CCT (18). aNaCwas measured in two experiments on uremic CCTs whereaNaCl is calculated as the ratio of the increment in netfluid movement (in the presence of vasopressin) caused by agiven osmotic gradient of NaCl to that resulting from an equalosmotic gradient or raffinose. Values for JNaCl of 0.96 and 0.94were obtained. These are regarded as being essentiallyeqjual to unity (a = reflection coefficienit).

(19) as previously described f'rom this laboratory (20). Afterremoval of the kidney, a cross section of cortex, 1-2 mmthick,was transferred to a dish of' modified Hank's solution whichserved as the dissection medium. In contrast to the techni(quedescribed by others (19, 21) no collagenase was used in thepreparation of tubules. All subsequent steps were carried outat 4°C.

Segments of' cortical collecting tubules were transferredinto each of f'our wvells of' a Terasaki microtest plate(Falcon Plastics, Div. of BioQuest, Oxford, Calif:) containinlg25 ,lA of dissection medium. Segments wvere pooled so that atotal length of approximately 5 mmwas used for each deter-mination of adenvlate cvelase under basal, NaF- and vaso-pressin-stimulated conditions.

The excess dissectioni solution was carefully aspirate(dunlder direct observation through an inverted microscopewith a fine capillary tube and 2 Al of hypotonic preincul)a-tion solution, containinig 8 mMTris HCI buffer, pH 7.4, with0.25 mnM EDTA, 1 mM M1gCl2 and 0.1% bovine serumilalbumin, added. Each w.ell wvas then photographed at x50through a calibrated eyepiece reticle and total thbule lengthwas determined from the photographs. For eaclh kidneystudied, three \vells were used for determination of basai,NaF- and vasopressin-stimulated adenylate eelase activityand a fourth well (containing a total length of' 10-15 mmllof collecting tubule segments) w as used f'or proteindetermination.

The wvells used for assay of NaF and vasopressin stimula-tion contained the same preincubation solution hut NaF andVP %vere added to result in respective concentrations of' 10mnMand 20 mU/ml in the final assay.3

The wells were sealed with coverslips and preincubated onice for 30 min. After this, the plates were frozen andthawed twice by applying them to a block of dry ice. (Thismaneuver together with the hypotonic preincubation solutionlcauses disruption of the cell membranes.) Assays for adenylatecyclase were carried out in a final volume of 10 ,l.

The reaction was initiated bv the addition of 8 Al of' areaction mixture containing 25 mMTris HCI, pH 7.71, 5 mNIMg C12, 1.4 mMEDTA, 1 mg/ml creatine phosphokinase(155 U/mg), 17 mMphosphocreatine, 1 mMcAMPand 0.125mNI [a32P]ATP (3-4 ,tCi/assav).

The wells wvere again sealed and incubations carried out at30°C in a water bath. For both normal and uremic collectingtubules, the reaction product increases linearly for at least30 min so that all incubations wvere carried out for 30 min.The reaction was terminated bv the addition 'of 100 Al of"stopping solution" which contained 10 mMcAMP, 40 mnMATP, 1% sodium dodecyl sulfate, and [3H]cANMP (approxi-mately 400,000 cpm) for calculation of recovery.

Each total incubate was transferred to a test tube and thevolume adjusted to 1 ml with water. The [32P]cAMP generatedwas then chromatographed on Dowex AG50W7-X8 (chlorideform) and neutral alumina columns and finally counted in 12ml of Aquasol liquid scintillation fluid (Newv England Nuclear,Boston, Mass.) wvith a Packard liquid scintillation counter(Packard Instruments, Inc., Dowvners Grove, Ill.). Recovery forthe [3H]cAMP was 40-60%.

In each experiment a triplicate incubation was carried outin the absence of tubule segments to serve as a blank. Themean of these blanks did not exceed 10% of the lowvest

3 In preliminary studies it \vas established for both normaland uremic tubules that maximal stimulation occurred at 200,uU/ml of vasopressin. Doses up to 20 mU/ml resulted in nofurther stimulation or inhibition of enzyme activity and thelatter dose was selected so that a comparison with studieson membrane preparations (20) could be made.

Vasopressin Responsiveness of Uremic Collecting Tubule 1521

experimental counts in any experiment. Results were cor-rected for their respective blank value and the percentrecovery was expressed as femtomoles cAMPgenerated permicrogram tubule protein (or per millimeter tubule) per30-min incubation. Protein content per millimeter tubule wasdetemined by the method of Lowry et al. (22). To minimizeinterassay variations, CCTs from normal and uremic animalswere generally studied together in the same assay.

MaterialscAMP, 8-bromo-cAMP (sodium salt), ATP, creatine phos-

phokinase, phosphocreatine, and neutral alumina were ob-tained from the Sigma Chemical Co., St. Louis, Mo. [3H]cAMP(20-40 Ci/nmol) and [a32P]ATP (10-30 Ci/nmol) were ob-tained from New England Nuclear. Dowex AG-50W-Zi wasobtained from Bio-Rad Laboratories, Richmond, Calif.

StatisticsThe statistical significance of the differences between groups

was assessed by Student t test.

RESULTS

Indices of renal function. Mean (+SE) BUN ofnormal rabbits was 8.8±0.9 mg/100 ml and of uremicrabbits was 62.5±10.8 mg/100 ml. Serum creatinineconcentration was 1.00±0.06 mg0100 ml in normal and3.13±0.38 mg/100 in uremic rabbits. Maximum urinaryosmolality was 1293 ±92 mosmol/kg H20 in normaland 659±49 mosmol/kg H20 in uremic rabbits (P< 0.005).

Morphology. CCTs from remnant kidneys ofuremic animals were identified according to the samecriteria as were used for normal CCTs (12, 13). Theremnant kidneys showed interstitial fibrosis makingthe dissection of intact CCTs relatively difficult. Only

one-third of the attempts to dissect an adequate lengthof CCT for perfusion were successful. The internaldiameters of normal and uremic CCTs are shown inTables I and II. The mean internal diameter of theuremic tubules was increased by approximately 50%(32.3 ,um).

Response of CCTs to vasopressin. In Fig. 1 themean (±SE) Jv of 9 normal and 13 uremic CCTs aredepicted for each of six 10-min periods after theaddition of a supramaximal concentration of vasopres-sin to the bath. Under the conditions employed (37°C,200 moSmol/kg gradient) there was a rapid increasein Jv followed by a gradual decline after 30 min in bothgroups of tubules. In almost all instances the maximumJv was observed either in the first or second 10-minperiod. The values for the first three 10-min periodswere not significantly different from each other andresults are expressed as the mean of these values.

In Table I the results of each experiment conductedon CCTs from nine normal rabbits are listed. TableII lists the results for 13 uremic CCTs. Perfusionrates were comparable in the two groups of animals.The mean transepithelial PD of normal CCTs was-22±6 mV and of stage III CCTs was -21±7 mV(lumen negative). These results are not significantlydifferent from each other. The influence of vasopressinon the transepithelial PD of normal and stage IIICCTs is depicted in Fig. 2. In accordance withprevious studies (14, 23, 24) vasopressin increased theluminal negativity of both normal CCTs (from -22±6to -26 22±7 mV; P < 0.02) and of uremic CCTs (from-21±7 to -25±7 mV; P < 0.02). During the 60 minof observation, there was no tendency of PDto decreaseto values below control after the initial increment.

TABLE IJv and Pf of CCTsfrom Normal Rabbit Kidneys

Tubule Jv JvPerfusion internal Control Pf

BUN rate diameter PD Control VP Control VP (Post-vasopressin)

mg/i00 ml ni/min Am mV ni/mm/min nl/cm2/s cm/s

11.2 11.65 23.0 -13 0.04 1.57 0.71 36.03 0.035712.3 5.75 21.0 -4 0.14 0.67 3.45 16.93 0.0168

5.8 3.98 15.5 -26 0.09 0.52 3.23 17.75 0.0271*5.7 7.64 22.2 -38 -0.05 0.52 -1.26 12.29

10.3 6.86 24.4 -14 -0.07 1.65 -1.54 33.87 0.0239*10.7 13.58 22.2 -15 0.29 1.18 5.89 28.17 0.043710.3 6.22 15.8 -61 0.23 0.32 7.67 9.19 0.0099

8.1 5.22 26.6 -11 -0.18 0.94 -3.37 18.71 0.0094*4.8 7.09 17.8 -10 -0.15 1.40 -4.51 41.83 0.0191

Mean 8.8 7.55 20.9 -22 0.03 0.97 1.14 23.86 0.0232+SE 0.9 1.03 1.3 6 0.05 0.17 1.40 3.82 0.0043

Tubules were studied in normal rabbit serum at 37°C with a 200-mosmol/kg H20 transepithelial osmotic gradient.* Indicates experiments with uremic rabbit serum in the bath. Vasopressin (VP) was added to the bath (200 j.U/ml).

1522 L. G. Fine, D. Schlondorff, W. Trizna, R. M. Gilbert, and N. S. Bricker

TABLE IIjv and Pf of CCTs from Remnant Kidneys of Uremic Rabbits

Tubule Jv JvPerfusion internal Control Pf

BUN rate diameter PD Control VP Control VP (Post-vasopressin)

mg/lOO ml nl/min /m mV nil/mm/min nl/cm's cm/s

58.7 5.23 23.0 -7 0.23 0.06 5.37 1.38 0.000935.1 10.06 22.2 -11 -0.02 0.59 -0.46 14.09 0.0063

121.2 5.61 31.1 -21 -0.22 0.14 -3.70 2.35 0.0069148.6 9.11 26.7 -15 -0.15 1.36 -0.95 27.07 0.0157

50.3 5.84 44.4 -13 -0.04 1.01 -0.52 12.16 0.0066*62.4 5.36 37.8 -12 -0.09 0.54 1.29 7.56 0.005727.9 4.96 24.4 -93 -0.14 0.15 -3.07 3.29 0.005024.5 9.723 33.3 -42 0.06 0.08 -0.95 1.27 0.006847.2 5.06 33.3 -6 0.09 0.13 1.38 2.13 0.003596.4 4.58 21.1 -4 -0.17 0.29 -4.19 7.35 0.0064*78.9 12.24 33.6 -25 0.27 0.27 4.28 10.53 0.007535.8 7.09 46.6 -26 0.24 0.24 2.73 3.74 0.0018*25.5 3.94 42.2 -3 0.13 0.13 1.72 6.34 0.0037

Mean 62.5 6.83 32.3 -21 0.02 0.38 0.23 7.64 0.0059+SE 10.8 0.72 2.4 -7 0.05 0.11 0.81 1.99 0.0010

Tubules were studied in uremic rabbit serum at 37°C with a 200-mosmol/kg H2O transepithelial osmotic gradient.* Indicates experiments with normal rabbit serum in the bath. Vasopressin (VP) was added to the bath (200-2,000 .LU/ml).

In the control periods (i.e., before the addition ofvasopressin) the CCTs of both groups of animals wereimpermeable to water and Jv was not significantlydifferent from zero in either of the groups (Tables I and11).4

After the addition of a supramaximal dose ofvasopression to the bath5 mean +SE Jv increased to 0.97+0.17 nl/mm per min in normal CCTs and to 0.38+0.11nl/mm per min in uremic CCTs (Tables I and II). Thusdespite the larger luminal surface area of the tubulesfrom the uremic rabbits, Jv was significantly lower thanin the normal animals (P <0.01). When results areexpressed per unit luminal surface area, the differencesare magnified even further, i.e., 23.86+±3.82 nl/cm2 per sin uremic CCTs (P < 0.001 vs. normal). The presence ofnormal or uremic rabbit serum in the bath did notinfluence the results which have consequently beenpooled.

Values for the transepithelial osmotic water per-meability coefficient, Pf, under the influence of vaso-pressin are listed in Tables I and II and depicted in Fig.3. Pf was significantly lower (P < 0.001) in the uremicCCTs than in the normals.

4Negative values for Jv reflect random variation of experi-mental results about zero.

5 In normal tubules a bath concentration of 200 /.uU/ml wasused; in uremic tubules 200 uU/ml was used in eightexperiments and 2,000 yU/ml in five experiments. Increasingthe concentrations by an order of magnitude did not influencethe results.

Adenylate cyclase responsiveness of isolated CCTs.Values for adenylate cyclase activity under basal con-ditions and NaF and vasopressin stimulation in normaland uremic CCTs are listed for each experiment inTable III and the mean results are depicted in Fig. 4.Mean protein content for normal CCTs was 0.264+0.023,ug/mm and for uremic CCTs 0.298+0.056 ,ug/mm.Basal activity (expressed per unit tubule length or permicrogram protein) in the uremic CCTs tended to behigher than in normal CCTs although this differencewas not statistically significant (Table III). NaF-stimulated adenylate cyclase activity was not signifi-cantly different between the two groups. Activity of theenzyme after vasopressin stimulation reached levelswhich were only 50%of those obtained in normal CCTs

VASOPRESSIN1.2 V

~0.8-E0.6 Normal

0.4-2 ,/ rs< Uremic0.2-

oI-10 0 20 40 60

MINUTES

FIGURE 1 Jv of normal and uremic CCTs after the additionof vasopressin (200-2,000 ,uU/ml) to the peritubular bathingmedium.

Vasopressin Responsiveness of Uremic Collecting Tubule 1523

Normal

VASOPRESSIN200 AU ml

-20 0 20 40 60 -20MINUTES

I UremicVASOPRESSIN

200-2000 pH ml

0 20 40 60

MINUTESFIGURE 2 Influence of vasopressin on the transepithelial PD of normal and uremic CCTs.

(P < 0.025) (Table III). Comparisons between normaland uremic CCTs under all three conditions are

depicted in Fig. 4.Response of CCTs to 8-bromo-cAMP. To establish

whether the decreased vasopressin responsiveness ofuremic tubules was explicable solely on the basis of thereduced adenylate cyclase activity (resulting in a de-creased production of cAMP), the effects of the cAMPanalogue, 8-bromo-cAMP were evaluated. The resultsfor four normal and five uremic CCTs are shown in Fig.5. In the normal CCTs, at a bath concentration of 0.1mM8-bromo-cAMP, Pf approached values obtainedwith vasopressin (i.e., 0.0202-+0.0026 cm/s). In contrast,at a bath concentration of 1 mM8-bromo-cAMO, the Pfof the uremic CCTs was only 0.007±0.0007 cm/s.Addition of 8 mMtheophylline to the bath did notincrease Pf further.

DISCUSSION

The present study provides documentation of the firstdirect observations on the function of the CCT inuremia. The experimental model in which isolated

400

-v 300E1-i

O 200O

QLI 100

0LNORMAL UREMIC

FIGURE 3 Pf of normal and uremic CCTs under the influenceof vasopressin.

segments of renal tubules from uremic animals are

perfused in vitro has been described elsewhere (11).The present observations indicate that CCTs derivedfrom remnant kidneys of uremic animals show a

marked hyporesponsiveness to vasopressin. Underbasal conditions, in the absence of vasopressin, thesenephron segments are essentially impermeable towater.

The CCTs studied showed evidence of compensa-

tory hypertrophy similar to that seen in the proximalstraight tubules studied in the same experimentalmodel (11). However, the degree of hypertrophy was

more variable and some tubules were of normal size.The mean luminal diameter of uremic CCTs perfusedat flow rates comparable to those used in normal

800_ C NORMAL

2 UREMIC

400

200r

BASAL NaF VASOPRESSIN

FIGURE 4 Adenylate cyclase activity of normal and uremicCCTs measured under basal, NaF-stimulated, and vaso-

pressin-stimulated conditions.

1524 L. G. Fine, D. Schlondorff, W. Trizna, R. M. Gilbert, and N. S. Bricker

-80

-60

E

b-

An

-21 0o :

X 1

TABLE IIIAdenylate Cyclase Activity of Isolated CCTsfrom Normal Kidneys and Uremic Remnant

Kidneys: Response to Sodium Fluoride and Vasopressin Stimulation

Basal NaF Vasopressin

fmols cAMPIug fmols cAMPIAg %basal fmols cAMPIug %basalproteinl30 min proteinl30 min proteinl30 min

Normal CCTs 96.2 726.0 754 893.8 92926.4 484.6 1,835 253.1 96060.6 843.0 1,391 1,048.8 1,73221.3 735.8 3,453 256.5 1,20466.8 593.5 888 444.3 665

142.8 903.4 632 616.3 431Mean+SE 68.9± 18.5 714.4+63.3 1,492+432 585.1±+135.2 986±184

Uremic CCTs 68.9 630.6 914 249.5 36253.7 128.1 238 197.3 38686.4 507.3 588 444.7 51613.7 379.2 2,767 105.5 777

395.7 1,577.5 398 338.5 85300.6 478.9 159 310.4 103

70.0 414.6 591 277.5 638297.3 1,052.7 354 256.4 86188.3 831.4 441 372.0 198

Mean±SE 163.8±45.5 666.7±144.5 722±266 283.0±33 352±86

tubules was increased by approximately 50%. How-ever, despite this increase in internal surface area, inthe presence of the same osmotic gradient and supra-maximal levels of vasopressin, Jv per unit length wassignificantly reduced in uremic vs. normal CCTs.When expressed per unit of luminal surface area, thedifference between normal and uremic CCTs ismagnified even further. The reduced osmotic waterpermeability coefficient of the uremic CCTs in re-sponse to vasopressin was an almost consistent finding.

To determine the possible biochemical mechanismsinvolved in this functional alteration, studies ofadenylate cyclase activity were performed on isolatedcollecting tubules of normal and uremic animals (19,

21,ZE-La

0-

15

10

50 ~ ~ ~~~~lrmago

50Ohmic

O 0.01mM 0.1mM mM mM+8 mmTheo

BATH CONCENTRATIONOF O-BROMO-cAMP

FIGURE 5 Pf of normal and uremic CCTs exposed to in-creasing bath concentrations of 8-bromo-cAMP. Uremictubules were also exposed to theophyline (Theo).

20). This technique offers the opportunity of correlatingfunctional measurements with biochemical or en-zymatic events in the same nephron segment.

Activation of membrane-bound adenylate cyclase byvasopressin at the peritubular surface of the nephronappears to be the initiating event in increasing thewater permeability of the luminal membrane of thecollecting tubule (25, 26). This leads to the genera-tion of increased amounts of intracellular cAMPwhich,through the activation of protein kinase, membraneprotein phosphorylation, and microtubule formation(25), ultimately effect a physical change in the luminalmembrane which increases its permeability to water.

In the present studies basal adenylate cyclasespecific activity was higher in uremic than in normalCCTs although the difference did not achieve statisti-cal significance due to a large standard error in theformer values. Stimulation by NaF led to comparablelevels of absolute activity in both groups. However,under maximal vasopressin stimulation the uremictubules showed only 50% of the activity of normaltubules. Although it is possible that this defect is non-specific, it is of interest that parathyroid hormonestimulation of adenylate cyclase activity in uremicproximal convoluted tubules is not different from nor-mal6 suggesting that the present finding represents adecreased responsivity to vasopressin per se. It is pos-sible, however, that stimulation of renal adenylate

6 Fine, L. G., and D. Schlondorff. Unpublished ob-servations.

Vasopressin Responsiveness of Uremic Collecting Tubule

25

5

1525

cyclase by other hormones may be similarly impairedin uremia.

If the failure of vasopressin to stimulate adenylatecyclase activity normally were the only cellular defectunderlying the hyporesponsiveness of the uremic CCTto vasopressin (as measured by Jv and osmotic waterpermeability), it should be possible to correct thedefect with exogenous cAMP or one of' its analogues.cAMP itself' simulates the eff'ect of vasopressin on theisolated CCTonly in very high concentrations (10 mM)due to its low permeability across cell membranes (10).However, it has recently been shown that 8-(p-Cl-phenylthio)cAMP at a concentration of 10 ,uM mimicsthe ef'fect of'vasopressin on the hydraulic conductivityof'the isolated CCT(15). In the present study, 8-bromo-cAMP, another analogue of'cAMP (16), was uised. Whenadded to the peritubular bathing medium at a con-centration of' 0.1 mM, the Pf of' normal CCTs increasedto a level comparable to that which obtained withmaximal vasopressin stimulation. The same analogue ata concentration of 1 mMf'ailed to increase the Pf of'uremic CCTs. To exclude the possibility that increasedphosphodiesterase activity was responsible for thisblunted response, theophylline was added to the bathsolution in the presence of' 1 mM8-bromo-cAMP. Noadditional increase in Pf was observed. Although it isdifficult to interpret these results with certainty, f'ailureof the cAMP analogue to induce a normal waterpermeability response in uremic CCTs suggests thatmore than one biochemical alteration is responsible forthe observed functional difference between normaland uremic CCTs. Such an additional def'ect wouldhave to reside in a step or steps subse(luent to theformation of cAMP. These studies do not, however,exclude the possibility that 8-bromo-cAMP failed topermeate the cells of the uremic tubules to the sameextent as it does in normal tubules.

The mechlanisms underlying the functional and bio-chemical alterations described above are unelear. Itis possible that CCTs from remnant kidneys maydemonstrate "memory" effects of uremia in vitroinduced either by unidentified inhibitory substances(27) or by alterations in circulatinig hormone levels.7

The results of these studies may help to clarify anumber of hitherto unexplained aspects of the urinaryconcentrating defect of uremia. The clinical entity ofvasopressin resistant hyposthenuria has been de-scribed in a number of unrelated forms of renaldisease (1-4). The possibility that resistance to theeffect of vasopressin might be a more common eventthan was generally supposed was advanced by Hol-

7We have recently obtained plasma arginine vasopressin(pAVP) levels on six uremic and four normal rabbits.Radioimmunoassay of pAVP was performed by Dr. L. C. Keil,Ames Research Center, Calif pAVP was 5.7+0.4 pg/mi in thenormal and 23.6+6.9 pg/ml in the tiremic rabbits.

lidlay et al. (2) and Tannen et al. (1). Additionalstudlies by Schrier and Regal illustrated the unifornmimpairment of' renal conieentrating ability in patientswith renal disease, a significant proportion of whomhad vasopressin-resistant hyposthenuria (4). The dif:fereniee between an absolute inability to concentratethe uirine above the osmolality of plasma (hypo-sthenitiria) and the ability to raise the urinary osmola-lity to a value slightly greater than that of' plasma is a(uiaintitative one only. If' it is accepted that the kidneyin uremia can generate hypotonic tubular fluid (3, 9)the absolute amount of water renmoved by the distaltubule and collecting tubule will determine the finalurinie osmiiolality which mlay vary from hypo- to hyper-tonic. In the present study the resistanice to vasopressiiwas not complete.

One possible explanation for the resistance of uremicsubjects to vasopressiin is the existenice of a circulat-ing inhibitor to vasopressin (1). The present studiesprovidle no support for this. The impaired responsive-ness of' the urenmic collecting tubule was observedwlhen the tubules were studied in normal or uremicserumii and these observations suggest that the def'ectlies at the level of the tubular epithelial cell per se.They would also minimize the role of'other circulatinigionic or hormonal influences as important pathogeneticf'actors.

An additional observation of' importance relates tothe f'act that the uremic collecting tubules underbasal conditions (i.e., in the absence of vasopressin)were essentially impermeable to water. This findingprovides an important explanation for the observationthat the diluting ability of the diseased kidney is wellpreserved long after the concentrating defect becomesovertly manifest (3, 9). The ability of the diseasedkidney to dilute the urine requires that the generationof' hypotonic fluid by the ascending limb is intactand that little or no equilibration occurs betweenthis fluid and the hypertonic medullary interstitiumin the collecting tubule.

The vasopressin unresponsiveness of'the uremic col-lecting tubule provides a unifying framework withinwhich a number of observations on the concentratingand diluting functions of the diseased kidney can beexplained. First, as pointed out above, it explains howdiluting functions can be maintained in the absence of'normal concentrating ability. Second, it explains theinability of uremic subjects to respond to maximaldoses of vasopressin and since the defect is intrinsicto the renal epithelial cell rather than to a circulatinginhibitor, accounts for the reported failure of hemo-dialysis to correct this abnormality (1). Failure ofwater removal from the collecting tubule under theinfluence of vasopressin not only directly limits theability of the kidney to concentrate the urine butindirectly influences its ability to generate a hypertonicmedullary interstitium via an influence on urea re-

1526 L. G. Fine, D. Schlondorff, W. Trizna, R. M. Gilbert, and N. S. Bricker

cycling. In the presence or absence of vasopressin,the CCT is impermeable to urea (10). Water abstractionfrom the CCT under the influence of vasopressinconsequently leads to a progressive increase in ureaconcentration of the fluid coursing through the col-lecting tubules leading to a high tubular fluid ureaconcentration in the terminal papillary collectingtubules. The relative high urea permeability of theselatter nephron segments (28) allows urea to diffuseinto the papillary interstitiuim down its concentra-tion gradient and thereby facilitates concentration ofother solutes in the interstitium according to the"passive theory" of countercurrent multiplication (29).We have recently described a disturbance of suchurea recycling and failure to accumulate interstitialsoluite in rats with chronic pyelonephritis (9). If vaso-pressin unresponsiveness is a component of the uremicstate, it is highly likely that failure to abstract waterfrom the cortical and outer medullary collectingtubules plays an important role in the failure of thediseased kidney to concentrate the urine normally.

ACKNOWLEDGMENTS

These studies were supported by grant 7 RO1 AM 19822from the National Institutes of' Health and by grant HRC211from the New York Health Research Council.

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7. Lubowitz, H., M. L. Purkerson, and N. S. Bricker. 1966.Investigation of single nephrons in the chronicallydiseased (pyelonephritic) kidney of the rat using micro-punieture techni(qtues. Nephron. 3: 73-83.

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Bricker. 1976. On the influience of' the natriuretic f'actorfrom patients with chronic uremia on the bioelectricproperties and sodium transport of' the isolated mam-malian collecting tubule.J. Clin. Invest. 58: 590-597.

13. Fine, L. G., and W. Trizna. 1977. Influeniee of'prostaglan-dins on sodium transport of' isolated medullary nephronsegments. Am. J. Physiol. 232: F383-F390.

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15. Hall, D. A., L. D. Barnes, and T. P. Douisa. 1977.Cyclic AMPin action of'antidiuretic hormone: eff'ects of'exogenous cyclic AMP and its newv analogue. Anm. J.Physiol. 1: F368-F376.

16. Meyer R. B., and J. P. Miller. 1974. Analogs of eyelicAMP and cyclic GMP: General methods of' synthesisand the relationship of' strtuctture to enzvmie activity.Life Sci. 14: 1019-1040.

17. Al-Zahid, G., J. A. Schafer, S. L. Trouitmclan, andl T. E.Andreoli. 1977. Ef'fect of' antidiuretic hormonie on vaterand solute permeation, and the activation eniergies f)rthese processes in mammalian cortical collectinig tubules.

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antidiuretic hormone on solute flows in mammaliancollecting tubules.J. CliGu. Inxvest. 51: 1279-1286.

19. Imbert, M., D. Chabardes, M. Montegut, A Cli(que, anidF. Morel. 1975. Adenylate cyclase activity along the rabbitnephron as measured in single isolated segments.Pfluegers Arch. Eur. J. Physiol. 354: 213-228.

20. Schlondorff, D., H. Weber, W. Trizna, and L. G. Fine.1977. Vasopressin responsiveness of' renal adenylatecyclase in newborn rats and rabbits. Am. J. Physiol.234: F16-F21.

21. Chabardes, D., M. Imbert, A. Cli(que, M. Montegut, andF. Morel. 1975. PTH senisitive adenyl cyclase activityin dif'ferent segments of' the rabbit nephron. PfluegersArch. Eur. J. Physiol. 354: 229-239.

22. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, anid R. J.Randall. 1951. Protein measturement with the Foliophenol reagent.J. Biol. Chetmi. 193: 265-275.

23. Helman, S. I., J. J. Grantham, and M. B. Buirg. 1971.Effect of vasopressini on electrical resi stance of renal corti-cal collecting tubuiles. Am. J. Phtysiol. 220: 1825-1832.

24. Frindt, G., and M. B. Bturg. 1972. Eff'ect of' vasopressiion sodium transport in renial cortical collectinig tubules.Kidney IJut. 1: 224-231.

25. Hays, R. M., and S. D. Levine. 1974. Vasopressin.Kidney Int. 6: 307-322.

26. Dousa, T., and H. Valtin. 1976. Cellular actionis of vaso-pressin in the mammaliani kidney. Kidtey IJut. 10:46-63.

27. Webster, S. K., and N. Beek. 1976. Impairmiienit of vaso-pressin-dependent cyclic AMPand urinary concentratingability in uremia. Clin. Res. 24: 415A. (Abstr.)

28. Rocha, A. S., and J. P. Kokko. 1974. Permeability ofmedullary nephron segmiienits to urea and vater: Eflfectof vasopressin. Kidney Int. 6: 379-397.

29. Kokko, J. P., and F. C. Rector, Jr. 1972. Countereurrentmultiplication system without active transport in innermedulla: New model. Kidntey IJut. 2: 214-224.

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