The Effect of Cyclic AMPand Dibutyryl Cyclic AMPon

the Permeability Characteristics of the Renal Tubule

WILLIAM B. LORENIZ, JR.

From the Division of Pediatric Nephrology, Department of Pediatrics,University of Texas Medical Branch, Galveston, Texas 77550

A B S T R A C T The effect of adenosine-3',5'-cyclic mono-phosphate (cyclic AMP) and NM,02-dibutyryl adeno-sine-3',5'-cyclic monophosphate (dibutyryl cyclic AMP)on renal tubular permeability was studied by microin-jection techniques in anesthetized diuretic rats. Radio-active inulin and mannitol were microinjected simul-taneously into superficial proximal and distal convolu-tions and recovery of the isotopes was measured in theurine.

During control conditions, mannitol and inulin re-covery was essentially complete. However, during infu-sion of cyclic AMPor dibutyryl cyclic AMP, mannitolrecovery was significantly less than control after earlyproximal and late proximal microinjections, averaging79 and 85%, respectively. There was no loss of mannitolfrom the nephron after microinjection into distal con-volutions. Inulin recovery was complete after all mi-croinjections during cyclic AMP or dibutyryl cyclicAMPinfusion. Simultaneous clearances of mannitol andinulin as well as peritubular capillary microinjectionsstudies demonstrated bidirectional fluxes of mannitolacross the proximal tubular epithelium during infusionof cyclic AMPor dibutyryl cyclic AMP. Intratubularpressures were not different during control and ex-perimental periods.

These studies demonstrate a change in the permeabilitycharacteristic of the proximal convoluted tubule duringinfusion of cyclic AMPand dibutyryl cyclic AMP. Thischange in permeability of the proximal tubule could ac-count for the effects of cyclic AMPon proximal tubulartransport processes.

INTRODUCTIONIt is generally accepted that the effect of parathyroidhormone on the proximal tubule is mediated through

An abstract of this work appeared in 1973. Pediatr. Res.7: 409.

Received for publication 27 August 1973 and in revisedform 17 December 1974.

stimulation of adenyl cyclase (1-5) which subsequentlyincreases intracellular levels of adenosine-3',5'-cyclicmonophosphate (cyclic AMP).' Not only does para-thyroid hormone, through cyclic AMP, decrease netreabsorption of phosphate by the kidney, but it also de-creases the reabsorption of sodium, bicarbonate, andamino acids. Agus, Puschett, Senesky, and Goldberg (5)have shown that both parathormone and cyclic AMPin-hibit absolute and fractional reabsorption of phosphateand sodium in the proximal tubule of the dog. The workby Scriver and associates (6-7) in vitamin D deficiencyin infants as well as in the rat has demonstrated phos-phaturia and aminoaciduria in association with the sec-ondary hyperparathyroidism of vitamin D deficiency.Further, Winnberg and Bergstrom (8) have shown anacidification defect in untreated vitamin D deficiencyrickets secondary to decreased bicarbonate reabsorption.These studies indicate that parathormone, through stimu-lation of cyclic AMP, affects many aspects of proximaltubular transport. Although these effects are well docu-mented, there is very little information concerning theunderlying mechanism through which cyclic AMPmayinhibit net reabsorption in the proximal tubule. Theo-retically, cyclic AMP could inhibit specifically thesevarious active transport systems. However, it is equallypossible that cyclic AMP could interfere with mem-brane permeability and allow for greater passive backflux of these substances from the peritubular capillaryinto the tubular lumen. To evaluate the passive perme-ability characteristics of the proximal tubule during in-fusion of cyclic AMP, we have carried out experimentsutilizing the microinjection technique. These studies in-dicate that during infusion of cyclic AMPor dibutyrylcyclic AMP the proximal tubular epithelium remainsimpermeable to inulin but becomes permeable to man-

'Abbreviations used in this paper: ADH, antidiuretichormone; C, clearance; cyclic AMP, adenosine-3',5'-cyclicmonophosphate; dibutyryl cyclic AMP, N6,'O"-dibutyryladenosine-3',5'-cyclic monophosphate.

The Journal of Clinical Investigation Volume 53 May 1974*1250-125712.50

nitol to which the proximal tubule is normally imperme-able.

METHODSMale Sprague-Dawley rats weighing 240-350 g were anes-thetized with sodium pentobarbital 50 mg/kg body wt in-traperitoneally and the left kidney was exposed for micro-puncture through a midline abdominal incision which wasextended laterally along the lower margin of the left ribcage. A metal retractor was used to immobilize the dia-phragm from above on the left and the kidney was packedwith cotton soaked in mineral oil. Throughout all ex-periments the animals were placed on a heated table andbody temperature was monitored continuously by meansof a rectal probe connected to a telethermometer (YellowSprings Instrument Co., Yellow Springs, Ohio). Body tem-perature was maintained at 37.5±10 C. A tracheotomy wasperformed and the right jugular vein was catheterized forinfusion of fluids and injections of lissamine green. A PE50 polyethylene catheter was placed in the left carotid ar-tery and threaded into the abdominal aorta to lie above thelevel of the renal arteries. The femoral artery was catheter-ized with a PE 50 polyethylene tubing for monitoring ofblood pressure. This tubing was connected to a Stathampressure transducer (Model P23 DC, Statham Instru-ments, Inc., Oxnard, Calif.) and blood pressure was re-corded on a Grass polygraph (Grass Instrument Co.,Quincy, Mass.). Both ureters were catheterized with PE50 polyethylene tubing.

For the microinjection experiments, the, kidney surfacewas illuminated with a fiberoptic light source (AmericanOptical Corp., Southbridge, Mass.). The kidney wasbathed with heated mineral oil delivered by a gravity sys-tem. Early proximal, late proximal, and distal convolu-tions were identified by the intravenous injection of 0.05ml of '5% lissamine green. The total amount of lissaminegreen administered to any animal did not exceed 0.5 mland no microinjections were performed until at least 30min had lapsed after the last injection of lissamine green.Intratubular pressures were measured by the method ofLandis as modified by Gottschalk and Mylle (9). Randomintratubular pressure measurements were made during bothcontrol and experimental periods. The animals were mademoderately diuretic by the infusion of 5%o mannitol in0.85% sodium chloride at a rate of 50 ul/min. Constrictionmicropipettes of known volume were used for microinjec-tion into superficial early and late proximal and distal con-volutions. A small volume (1-5 nl) of isotonic salinestained with nigrosine and containing trace amounts of[JH]inulin and [IC]mannitol was injected at an averagerate of 0.26±0.06 (SD) nl/s. The injection rate was con-trolled to avoid visible retrograde flow of the stained testsolution and dilatation of the tubular lumen. During ex-perimental periods either cyclic AMP or dibutyryl cyclicAMP was infused at a rate of 100 ,g/min through thecatheter placed in the abdominal aorta above the level ofthe renal arteries. After a 60-min equilibration period, aconvolution punctured during the control period was againentered through the original injection site and anothermicroinj ection performed.

[3H] Inulin and ['C] mannitol were obtained from theNew England Nuclear Corp., Boston, Mass. Cyclic AMPand dibutyryl cyclic AMPwere obtained from Calbiochem,La Jolla, Calif. Urine from both kidneys was collectedinto vials containing 10 ml of liquid scintillation fluid

(Aquasol, New England Nuclear Corp.) to which 2.5 mlof deionized H2Owas added.

Urine collection was started at the beginning of eachmicroinjection and three consecutive 3-min collections weremade after injections during both control and experi-mental conditions. Before each microinjection urine wascollected to measure residual radioactivity. Immediatelypreceding a microinjection, a timed urine collection of thesame duration as each sample after microinjection wastaken. A volume of test solution equal to that which wassubsequently injected was deposited in this sample andused as a reference standard. Significant quenching of thetritium counts without apparent cause was observed occa-sionally. Although there was little or no quenching of the"C counts, these results were discarded. Radioactivity wasmeasured in a Beckman 3 channel liquid scintillation spec-trometer (Beckman Instruments, Inc., Fullerton, Calif.).Each sample was counted to a preset error of ±3%o. Sam-ples with essentially background activity were counted fora total of 20 min. The recovery of injected radioactivesubstances was calculated as follows:

_ isotope in urine%recvin=~~xlOO%isotope in injectate

Clearance studies were performed in an additional 10 ratsmade similarly diuretic. A priming injection containing 15,uCi of ["C]mannitol and 40 gCi [8H]inulin was followedby the intravenous infusion of 20 pCi/h of ["C]mannitoland 60 ,tCi/h of [3H]inulin in 5% mannitol and 0.85%sodium chloride at a rate of 50 /l/min. After a 30-minequilibration period two 20-min control collections of urinewere made. Then an infusion of cyclic AMP or dibutyrylcyclic AMP at a rate of 100 ug/min was begun in theabdominal aorta. After a further 60-min equilibrationperiod, three 20-min experimental collections of urine wereperformed. Blood samples were withdrawn from the fe-moral artery at the midpoint of each clearance period.Radioactive levels and blood and urine were determinedas described above.

To evaluate the validity of the clearance studies, an ali-quot of urine from each clearance period was subjected todescending column chromatography utilizing a solvent sys-tem of three parts n-butanol, two parts pyridine, and onepart water by volume (10). After the chromatographyprocedure, the strips were analyzed for. radioactivity utiliz-ing a Beckman strip radiochromatograph scanner. Thestrips were then stained with the periodate reagent (11)to identify chemical inulin and mannitol.

Peritubular capillary microinjections were performed inanother group of five animals studied under identical con-trol and experimental conditions. Constriction micropipettesof 20-30 nl volume were utilized. A solution of isotonicsaline stained with lissamine green containing trace amountsof ['H]inulin and [C]mannitol was injected into peritubu-lar capillaries at an average rate of 31+11 (SD) nl/min.Urine from both kidneys was collected after microinjectionat 15-s intervals for the first 4 min, at 30-s intervals forthe next minute, and thereafter at 1-min intervals for an-other 4 min. Radioactivity levels in the urine were deter-mined as described above. For detection of precession onlythose counts exceeding background plus 2 SD of back-ground activity were considered significant. Recovery foreach sample was calculated as the percent of total radio-activity recovered during the 9-min collection period fol-lowing peritubular capillary injection.

Effect of Cyclic AMPon Renal Tubular Permeability 1251

l10r

30.

=0"I-'a

2

95

110

9go[Control Reinjection

C I

Control Ri

hOr

105

j1o

95

90

@00 @0 0000 00 0 0_ * ox00 of

° ;* *0° i t

A trol and reinjection periods. Inulin recovery averaged100.2±2.6% after control injections and 99.8±2.7%after reinjections. Mannitol recovery was 97.2±2.7%after control injections and averaged 98.2±2.5% afterreinjections. Neither of these was significantly different

---- from control. In Fig. 1B, mannitol and inulin recoveriesare shown as a function of time after the start of intra-venous infusion of 5% mannitol and 0.85% sodiumchloride at 50 Al/min. The results of these studies indi-cate that neither the reinjection technique or infusion of5% mannitol and 0.85% NaCi significantly affects the

Iniection recovery of inulin or mannitol after early proximalmicroinjection.

The recovery of mannitol and, inulin after microin-jection into early proximal convolutions during control

B and experimental conditions is shown in Fig. 2. Duringcontrol conditions mannitol recovery averaged 97.9+2.8%. However, during experimental periods there wasa significant loss of mannitol. Average mannitol recov-ery was 79.0±6.8%. This is significantly less than thecontrol (P <0.001). There was also a difference be-

0- -- tween recoveries after infusion of cyclic AMPor di-butyryl cyclic AMP. During infusion of cyclic AMP

0 mannitol recovery was 81.9±6.5%. Mannitol recoveryO during dibutyryl cyclic AMP infusion was 74.6±4.8%.

o- Inulin

TIme (nunutes)FIGURE 1 (A) Mannitol and inulin recoveries after simul-taneous early proximal microinjections and reinjections atthe same puncture site. The dashed horizontal line repre-sents 100% recovery of the injected substance. The solidlines connect reinjections pairs performed of the samepuncture site. (B) Mannitol and inulin recoveries aftersimultaneous early proximal microinjection as a functiontime from the start of intravenous infusion of 5%o mannitoland 0.85% NaCl at rate of 50 ul/min. The dashed hori-zontal line represents 100% recovery of injected substances.

Results are presented as means±1 SD. Student's t testwas used for evaluation of statistical significance.

RESULTS

A total of 186 satisfactory microinjections were per-formed in 27 animals. A microinjection was not con-sidered satisfactory if there was visible leakage at thepuncture site or retrograde flow. The results presentedare recoveries measured in the urine from the injectedkidney. All injections were performed with a test solu-tion which contained both inulin and mannitol.

The recovery of inulin and mannitol after early proxi-mal microinjection and reinjections during intravenousinfusion of 5% mannitol and 0.85% sodium chloride isshown in Fig. 1. Note in Fig. 1A that both inulin andmannitol recovery are essentially complete during con-

"Or

105 F

95 F

0

F-zz4%

at

90g

851-

80s-

751-

70 F

65

60'

105o

100

95

~9

D 85z

0so

75

70

*-cyclic AMPinfusedfor reinjections

o-dibutyryl cyclic AMPinfused for reinjectionso

Control Reinjection

65

60

ar-cyclic AMPinfusedfor reinjections

a-dibutyryl cyclic AMPinfused for reinjections

Control Reinjection

FIGURE 2 Mannitol and inulin recoveries after simultane-ous early proximal microinjections during control condi-tions and during infusion cyclic AMP or dibutyryl cyclicAMP. The dashed horizontal line represents 100% recoveryof injected substances. The solid lines connect reinjectionpairs performed at the same puncture site.

1252 W. B. Lorentz, Jr.

90

'IOr

. -0

"lOr

105-

-I0

I-jz-z4

le

100[

95F

85S

so8*-cyclic AMP

infused forr~einjection

o-dibutyryl cyclicAMPinfused forreinjection

ww 950u

IdI

D

"'85

so

75 -

70

a a -

Control Reinjection Control

FIGURE 3 Mannitol and inulin recoveries

o cyclic AMPinfusedfor reinjection

*-dibutyryl cyclicAMPinfused forreinjection

Reinjection

after simultane-ous late proximal microinj ections during control conditionsand infusion of cyclic AMPor dibutyryl cyclic AMP. Thedashed horizontal lines represent 100% recovery of injectedsubstances. The solid lines connect reinjection pairs per-formed at the same puncture site.

This difference is significant (P < 0.001). Inulin re-covery was essentially complete after both control andexperimental microinjections. Inulin recovery averaged99.4±2.7% after control injections and 99.1±1.7% afterexperimental microinjections. The transit time was sig-nificantly prolonged during experimental periods. Thetransit time for each microinjection is defined as thetime from beginning an injection until the first appear-ance of the nigrosine-stained test solution in the distalconvolution of the nephron under study. Transit timewas 34±14 s after control injections and 53±25 s afterexperimental injections (P < 0.001).

The recovery of inulin and mannitol after microinjec-tions into late proximal convolutions is shown in Fig.3. Mannitol recovery after late proximal injections dur-ing control periods was 98.9±2.6%. During infusion ofeither cyclic AMPor dibutyryl cyclic AMPmannitolrecovery averaged 89.4±2.0%. This difference is-highlysignificant (P < 0.001). Inulin recovery was again es-sentially complete. Inulin recovery averaged 99.3±2.5%during control periods and 98.8±2.1% during experi-mental periods. The transit time from the start of lateproximal injections until the injectate appeared in itsconvolution was 27±15 s during control microinjectionsand 32+26 during experimental injections. Althoughtransit time was prolonged after late proximal injec-tions this was not significant (0.05 < P < 0.1). Therewas no difference in recoveries after experimental in-

jections between cyclic AMPand dibutyryl cyclic AMP.Mannitol recovery during infusion of cyclic AMPav-eraged 89.7±1.7 and during dibutyryl cyclic AMPav-eraged 88.8±2.4%.

In contrast to the proximal convolution microinjec-tions, after microinjection into distal convolutions no lossof inulin or mannitol was demonstrable. Recovery afterdistal microinjections is shown in Fig. 4. Mannitol re-covery averaged 98.6±2.1% during control conditionsand during experimental periods averaged 98.7±2.6%.Inulin recovery was 100.1±2.4% during control periodsand averaged 99.4±1.2% during experimental periods.

Since a difference in mannitol recovery during infu-sion of cyclic AMPwas demonstrable, differences in theclearances between inulin and mannitol should also bepresent infusion of cyclic AMP. Simultaneous clearancesof inulin and mannitol were performed in ten animalswith two clearance periods performed during controlconditions followed by three clearance periods duringinfusion of cycylic AMP in five animals and dibutyrylcyclic AMPin five animals. The results are shown inTable I. Since the results were similar in both kidneys,they have been pooled for purposes of analysis. Therewas a significant decrease in urine flow and an increasein the urine/plasma (U/P) inulin ratios during theexperimental periods. There was a slight increase inmannitol clearance and a decrease in inulin clearanceduring the experimental periods, however, these resultsare not significantly different. In contrast, the ratios ofmannitol clearance to inulin clearance for each clearanceperiod were distinctly different. During control periodsmannitol clearance was slightly less than inulin clear-

105[LiJ

:

0u

I-zz4

90

851-

so8

0~~~~~~~~~~~~~~~~~~~ 1:

Idz-J

z

0

*-cyclic AMPinfused forreinjection

o-dibutyryl cyclicAMPinfused forreinjection

Contfol

90-

85

80

Reinjection Control

o -cyclic AMPinfused forreinjection

*-dibutyryl cyclicAMPinfused forreinjection

Reinjection

FIGuRE 4 Mannitol and inulin recoveries after simultane-ous distal convolution microinjections during control con-ditions and infusion of cyclic AMP or dibutyryl cyclicAMP. The dashed horizontal lines represent 100% recoveryof injected substances. The solid lines connect reinjectionpairs performed at the same puncture site.

Effect of Cyclic AMPon Renal Tubular Permeability

qoF

75-

70-

"Or

1253

90

W. so-OC 80Dw> 7000 60w

c_50-> 401= EXPERIMENTAL-JD 20

D 100

2 4 6 8 10

1000 90 /w> 70M0U 06w

> 4CONTROL

-J-D 20.- MANNITOL

to 10- /INULIN

2 4 6 8 10

TIME (minutes)FIGURE 5 Cumulative mannitol and inulin recoveries fromthe experimental kidney after peritubular capillary injec-tion in a typical experiment. Note that mannitol recoveryprecedes inulin recovery during the experimental period inwhich -dibutyryl cyclic AMPwas infused.

ance. The ratio of mannitol clearance to inulin clearancefor each clearance period during control conditions was

TABLE IEffect of Cyclic AMPon Simultaneous Clearance

of Mannitol and Inulin

Control Experimental(n = 20) (n = 30) P

Urine flow 36.6411.2 21.045.7 <0.001(JA/min)

U/P inulin 107.4448.6 156.4438.3 <0.001

C mannitol 0.95 E0.33 0.98-40.30 >0.7(ml/min per 100 g body wt)

C inulin 0.992:0.31 0.892:0.29 >0.3(ml/min per 100 g body wt)

C mannitol 0.97 40.04* 1.122:0.08* <0.001C inulin

* Values represent mean24SD of ratios for each clearance period.

0.97±0.004. During experimental periods, however,mannitol clearance exceeded inulin clearance with anaverage ratio 1.12±0.08. This difference was significant(P < 0.001).

To check the validity of this clearance data, an aliquotof urine from each clearance period was subjected todescending column chromatography and scanning radio-chromatography. The results showed single peaks ofradioactivity which had the same mobility as [3H]inulinand ["C]mannitol from stock solutions of the radioiso-topes prepared for infusion. To further identify the ra-dioactive peaks, the strips were then stained with theperiodate reagent. The radioactive peaks were found tobe present in spots which stained identically with andhad the same chromatographic mobility as unlabeled in-ulin and mannitol. The chromatography studies showedthat both radioactive inulin and mannitol were ex-creted in the urine in an unaltered form.

The results of the microinjection studies and clearancestudies suggested that there was bidirectional movementof mannitol during infusion of cyclic AMPor dibutyrylcyclic AMP. To further investigate the possibility ofmovement of mannitol from the peritubular capillaryinto the tubular lumen, peritubular capillary microin-jection studies were performed in an additional five ani-mals. The animals were made diuretic as during themicroinjection and clearance studies by infusion of 5%mannitol in 0.85% saline at a rate of 50 il/min. Micro-injections into peritubular capillaries were made duringcontrol conditions and then a series of experimental mi-croinjections into peritubular capillaries was performedduring infusion of dibutyryl cyclic AMP.

During control periods inulin and mannitol were ex-creted in a similar fashion as is shown in the bottompanel of Fig. 5. However, during experimental periodsthere was precession of mannitol recovery over inulinrecovery in the urine of the injected kidney. This isshown in the top panel of Fig. 5. Precession of mannitolwas present in 14 of 15 microinjections during experi-mental periods. The percent recovery of mannitol at apoint in time when 50% of the inulin from the micro-injected kidney had been recovered averaged 47.0±1.2%during control periods and 58.5±3.0% during experi-mental periods (P < 0.001).

Random measurements of intratubular pressures inboth proximal and distal convolutions were made duringcontrol and experimental periods in all animals studied.Proximal intratubular pressure during control periodsaveraged 13.2±2.1 mmHg (n = 104). During experi-mental periods averaged proximal intratubular pressurewas 12.8±2.2 mmHg (n = 126). This difference wasnot significant. Distal intratubular pressures averaged6.4±1.5 mmHg (n = 52) during control periods and

1254 W. B. Lorentz, Jr.

during experimental periods averaged 6.1±1.6 mmHg(n = 60).

Mean arterial blood pressure during control periodsaveraged 110±13 mmHg (n = 112) and during ex-perimental periods averaged 102±11 mmHg (n = 124).This small difference was highly significant (P < 0.001).For purposes of analysis of continuous mean blood pres-sure recordings, the value immediately following a mi-croinjection or the midpoint of a clearance period waschosen as the value for analysis. Transient decreases inblood pressure following administration of pentobarbitalwere not considered for analysis.

DISCUSSION

The results of this study demonstrate that the tubularepithelium undergoes a change in its permeability char-acteristics during the infusion of cyclic AMP or di-butyryl cyclic AMP. It is interesting to note that thetubules remained impermeable to inulin during all con-ditions studied and reaffirms previous studies which indi-cate that inulin is the most satisfactory marker for glo-merular filtration during a variety of experimental con-ditions (12-16).

The segmental recovery of mannitol after experi-mental microinjections is shown in Table II. When man-nitol recovery during infusion of either cyclic AMPordibutyryl cyclic AMPafter early proximal injections iscompared to mannitol recovery after late proximal in-jections there is a highly significant difference. Simi-larly when the results of late proximal injections anddistal microinjections are compared there is also ahighly significant difference.

This indicates that there is a change in the perme-ability of the proximal convoluted tubule to mannitolduring infusion of cyclic AMP. Since the entire proxi-mal convolution is not accessible to micropuncture, themicroinjections designated here as "late proximal" rep-resent approximately 70% of the total length of theentire proximal convoluted tubule (9). The loss ofmannitol after late proximal injection which has beendemonstrated in this study could, therefore, be from thepars recta of the proximal convoluted tubule or fromsome portion of the loop of Henle. No further distinc-tion can be made from this experimental design.

Previous work has shown that elevation of intratubu-lar pressures can lead to increased permeability of thenephron to mannitol (16). The intratubular pressuresduring both control and experimental periods in thisstudy were not different and were within the physio-logic range. They are well below the range of pressureswhich have been shown to be associated with mannitolloss from the nephron (16). Even though transit timeto the distal tubule was prolonged after experimentalinjections this should not have, in itself, caused loss of

mannitol. Aortic constriction which greatly prolongstransit time through the nephron, has been shown notto be associated with significant mannitol loss fromthe tubule (16).

The finding that mannitol clearance exceeded inulinclearance during infusion of cyclic AMP was unex-pected. There was a decrease in inulin clearance duringthe experimental periods. Cyclic AMP has been re-ported to cause vasodilatation of systemic arteries, butconversely constriction of the renal artery (17). Thedecrease in glomerular filtration rate could be due tochanges in systemic hemodynamics. However, unlessthere was transepithelial movement of mannitol thereshould have been a corresponding decrease in mannitolclearance. The results of the radiochromatography stud-ies and staining for identification of chemical inulinand mannitol indicate that carbon-14 and tritium wereexcreted intact in the urine on mannitol and inulin, re-spectively. Therefore, these results suggest that there isnet addition of mannitol to the urine. This, of course,does not contradict the results of the microinjectionstudies since only the unidirectional lumen to plasmaflux is studied by the microinjection technique.

The clearance studies were in accord with the resultsof the peritubular capillary microinjections. After in-jection into the peritubular capillaries during infusionof dibutyryl cyclic AMP, there was precession of man-nitol excretion in the urine from the experimental kid-ney. This indicates that there is movement of mannitolfrom the peritubular capillary lumen into the tubularlumen. If ,there were no movement of mannitol acrossthe tubular epithelium, mannitol appearing in the finalurine would have all had to be the result of glomerularfiltration. Mannitol excretion should have been sig-nificantly less than inulin excretion since the microin-jection studies indicate that during cyclic AMPinfusionthere is loss of mannitol from the proximal tubule.Therefore, all these studies indicate that during infusionof cyclic AMP or dibutyryl cyclic AMP there is abidirectional flux of mannitol across the epithelium of

TABLE I ISegmental Recovery of Mannitol During Infusion

of Cyclic AMP

Site %Recovery P

Early proximal (n = 23) 79.0-6.8<0.001*

Late proximal (n = 25) 89.4-2.0<0.001t

Distal (n = 29) 98.7-42.6

* Early proximal vs. late proximal.$ Late proximal vs. distal.

Effect of Cyclic AMPon Renal Tubular Permeability 1255

the proximal convoluted tubule and perhaps also someportion of the loop of Henle.

The results of these studies provide, of course, nodirect information concerning the structural alterationswhich may lead to changes in permeability or to theroute of movement of mannitol across the proximaltubular epithelium. There is a considerable body ofevidence that indicates the presence of low resistanceintercellular shunts which involves the tight junctionand lateral intercellular spaces (17-19). Boulpaep andSeely have shown an increased permeability of the nec-turus nephron to raffinose during conditions of volumeexpansion (20). Studies by Bulger, Lorentz, Colindres,and Gottschalk have shown changes in the tight junc-tion of the proximal tubule of the rat associated withincreased intratubular pressure due to elevation of ure-teral pressure or partial renal venous constriction (21).Associated with this there has been expansion of thelateral intercellular spaces. Therefore it would seemreasonable to propose that the increased permeabilityto mannitol induced by cyclic AMPis caused by changesin the permeability characteristics of the tight junctionwhich permits movement through the tight junctionand lateral . intercellular spaces.

The finding that mannitol clearance exceeded inulinclearance during infusion of cyclic AMP or dibutyrylcyclic AMP indicates that there is net addition ofmannitol to the urine. Thus, the peritubular capillaryto lumen unidirectional flux must exceed the lumen toperitubular capillary unidirectional flux. Further, onewould have to assume that with water reabsorption inthe proximal tubule mannitol is moving against a con-centration gradient which would favor movement outof the tubular lumen. Since there is no known activetransport process for mannitol, another explanation forthis observed phenomena is necessary. Ussing andJohansen have demonstrated the phenomenon of anoma-lous solvent drag in the toad skin in which sucrose andurea were transported in a direction opposite to netwater movement (22). During sodium and water re-absorption from the proximal tubule, net water move-ment is from the lumen into the peritubular capillary.According to the standing osmotic gradient theory oftransport as proposed by Diamond (23), water move-ment is through the luminal border of the transportingepithelium into the cell and then into the lateral inter-cellular space. This pathway would remain impermeableto mannitol. However, under the influence of cyclicAMP the tight junction would become permeable forwater to back diffuse into the tubular lumen. Thus, netaddition of mannitol to the tubular lumen would beaccomplished by solvent drag through the now perme-able tight junction which would exceed movement of

mannitol out of the tubular lumen along its concentra-tion gradient.

This alteration of the passive permeability charac-teristics of the proximal tubule during infusion of cyclicAMPor dibutyryl cyclic AMPwould allow one to ex-plain the decreased net transport of such varied sub-stances as bicarbonate, phosphate, sodium, and aminoacids which have been directly observed (5) or in-ferred (6-8) with states of secondary hyperparathy-roidism. Agus et al. (5) have shown that the concen-tration of phosphate in the lumen of the proximal tubuleof dogs is less than that in an ultrafiltrate of plasmaafter active proximal tubular reabsorption. With achange in the permeability of the tight junction, therewould be movement of phosphate down a chemical con-centration gradient from the peritubular capillary intothe tubular lumen. There would likewise be the possi-bility of solvent drag if the proposed back diffusion ofwater does indeed occur from the lateral intercellularspace through the tight junction into the tubular lumen.Since the most recent electrophysiological studies ofthe rat proximal convoluted tubule indicate a small po-tential difference across the tubular epithelium withthe lumen positive, along most of its length, this move-ment would be down an electrical gradient (24, 25).This same potential difference would tend to inhibitback diffusion of sodium; however, one could assumethat solvent drag inward would overcome the effectsof the electrical gradient favoring outward movement.It is still possible that cyclic AMPcould, in addition,inhibit specific active transport processes taking placein the proximal convoluted tubule. However, this changein the passive permeability characteristics of the proxi-mal tubule allows one to advance a single hypothesiswhich would explain multiple effects.

Cyclic AMP and its dibutyryl derivative do notreadily cross cell membranes and very high blood con-centrations must be attained to produce physiologicalconcentrations within the cell (26). To consider theseresults a physiological effect one would have to assumethat none of the permeability changes were due to al-terations in systemic or renal hemodynamics and thatthe resultant intercellular levels were similar to thoseobtained after stimulation with parathyroid hormone.These questions cannot be answered from this study.

The finding that there was no change in the solutepermeability of the distal nephron was interesting whenone considers that current evidence suggests that theaction of antidiuretic hormone (ADH) to increasewater permeability of the collecting duct is mediatedthrough cyclic AMP (27, 28). Certainly the decreasein urine flow and increase in U/P inulin ratios in theclearance experiments during the experimental period

1256 W. B. Lorentz, Jr.

indicate there was an enhanced ADH effect present.Therefore, these results would be in accord with cur-rent theories concerning the activation of adenyl cyclasein the collecting duct by ADHand subsequent increasesin water permeability.

ACKNOWLEDGMENTSThe author expresses his appreciation to Dr. Gerald F.Powell for his assistance in performing the chromatogra-phy studies.

This work was supported in part by NIH GeneralResearch Support Grant RR 05427.

REFERENCES1. Liddle, G. W., and J. C. Hardman. 1971. Cyclic adeno-

sine monophosphate as a mediator of hormone action.N. Engl. J. Med. 285: 560.

2. Butlen, D., and S. Jard. 1972. Renal handling of 3'-5'-cyclic AMP in the rat. Pfluigers Arch. Eur. J. Physiol.331: 172.

3. Streeto, J. M. 1969. Renal cortical adenyl cyclase: ef-fect of parathyroid hormone and calcium. Metab. (Clin.Exp.). 18: 968.

4. Linarelli, L. B. 1972. Newborn urinary cyclic AMPand developmental renal responsiveness to parathyroidhormone. Pediatrics. 50: 14.

5. Agus, Z. S., 3 B. Puschett, D. Senesky, and M.Goldberg. 1971. Mode of action of parathyroid hormoneand cyclic adenosine 3',5'-monophosphate on renal tubu-lar phosphate reabsorption in the dog. J. Clin. Invest.50: 617.

6. Fraser, D., S. W. Kooh, and C. P. Scriver. 1967. Hy-perparathyroidism as the cause of hyperaminoaciduriaand phosphaturia in human vitamin D deficiency. Pedi-atr. Res. 1: 425.

7. Grase, J. H., and C. P. Scriver. 1968. Parathyroid-dependent phosphaturia and aminoaciduria in the vita-min D-deficient rat. Am. J. Physiol. 214: 370.

8. Winberg, J., and T. Bergstrom. 1965. Renal acidifica-tion defect in infants with mild deficiency rickets. ActaPaediat. Scand. 54: 139.

9. Gottschalk, C. W., and M. Mylle. 1956. Micropuncturestudy ,of pressures in proximal tubules and peritubularcapillaries of the rat kidney and their relation to ure-teral and renal venous pressures. Am. J. Physiol. 185:430.

10. Smith, I. 1960. Chromatographic and ElectrophoreticTechniques. Interscience Publishers, Inc., New York.2nd edition. 248.

11. Smith, I. 1960. Chromatographic and ElectrophoreticTechniques. Interscience Publishers, Inc., New York.2nd edition. 252.

12. Smith, H. W. 1951. The Kidney, Structure and Func-tion in Health and Disease. Oxford University Press,Inc., New York. 39.

13. Gottschalk, C. W., F. Morel, and M. Mylle. 1965.Tracer microinjection studies of renal tubular perme-ability. Am. J. Physiol. 209: 173.

14. Gutman, Y., W. E. Lassiter, and C. W. Gottschalk.1965. Micropuncture study of inulin absorption in therat kidney. Science (Wash. D. C.). 147: 753.

15. Marsh, D., and C. Frasier. 1965. Reliability of inulinfor determining volume flow in rat renal cortical tubules.Am. J. Physiol. 209: 283.

16. Lorentz, W. B., Jr., W. E. Lassiter, and C. W. Gott-schalk. 1972. Renal tubular permeability during in-creased intrarenal pressure. J. Clin. Invest. 51: 484.

17. Windhager, E. E., E. L. Boulpaep, and G. Giebisch.1966. Electrophysiological studies on single nephrons.Proc. Int. Congr. Nephrol. 1: 35.

18. Giebisch, G. 1969. Functional organization of proximaland distal tubular electrolyte transport. Nephron. 6:260.

19. Boulpaep, E. L., and J. F. Seely. 1971. Electrophysiol-ogy of proximal and distal tubules in the autoperfuseddog kidney. Am. J. Physiol. 221: 1084.

20. Boulpaep, E. L. 1972. Permeability changes of the prox-imal tubule of necturus during saline loading. Am. J.Physiol. 222: 517.

21. Bulger, R. E., W. B. Lorentz, R. C. Colindres, andC. W. Gottschalk. 1974. Morphological changes in ratrenal proximal tubules and their tight junction withincreased intraluminal pressure. Lab. Invest. In press.

22. Ussing, H. H., and B. Johansen. 1969. Anamaloustransport of sucrose and urea in toad skin. Nephron. 6:317.

23. Diamond, J. M. 1962. The mechanism of solute trans-port by the gall bladder. J. Physiol. (Lond.). 161:474;

24. Barratt, L. J., F. C. Rector, Jr., J. P. Kokko, andD. W: Seldin. 1973. Variations in transepithelial po-tential difference (PD) along the rat proximal tubule(PCT). Clin. Res. 21: 675. (Abstr.).

25. Chirito, E., and J. F. Seely. 1973. Evidence for achange in the potential difference along the length ofthe rat proximal tubule in vivo: the role of chloride.Clin. Res. 21: 680. (Abstr.).

26. Henion, W. F., E. W. Sutherland, and T. Posterngk.1967. Effects of derivatives of adenosine 3',5'-phosphateon liver slices and intact animals. Biochim. Biophys.Acta. 148: 106.

27. Orloff, J., and J. S. Handler. 1962. The similarity ofeffects of vasopressin, adenosine-3',5'-monophosphate(cyclic AMP) and theophylline on the toad bladder.

J. Clin. Invest. 41: 702.28. Sutherland, E. W., G. A. Robinson, and R. W.

Butcher. 1968. Some aspects of the biological role ofadenosine 3',5'-monophosphate (cyclic AMP). Circula-tion. 37: 279.

Effect of Cyclic AMPon Renal Tubular Permeability 1257