ecently, a new promising renal imaging radiopharmaceutical, technetium-99m mercaptoacetyltriglycine (MAG-3) has been introduced (1,2) and isundergoing clinical trials (3—10)as well as investigationsin animal experiments (1, 11—14).Most authors outlinethe similarity between MAG-3 and hippurate, sincerenal excretion of both is inhibited by probenecid andboth have higher renal clearance than glomerular markers, indicating secretion in the kidney. MAG-3 is thusrecommended as a substitute for hippurate for renalfunction studies. Some authors correct the MAG-3clearance by a simple factor to get renal plasma flow(15) and others use it as a measure of tubular excretionrate (4). However, several differences between these twosubstances have been reported (3,5, 10, 13, 14, 16, 17) andthe present study was performed to further characterize

ReceivedApr. 25, 1989;revision accepted July 21, 1989.For reprints contact: Roland Müller-Suur,Dept. of Clinical

Physiology,Danderyds Hospital, S-l82 88 Danderyd, Stockholm,Sweden.

the renal excretion mechanism for MAG-3 in comparison with hippurate. In the present study micropuncturetechnique was applied on the glomerular level for directmeasurement of glomerular filtration and on proximaland distal tubules to outline the secretory capacity alongthe nephron. Plasma protein binding measurementstogether with whole kidney clearances were also usedto estimate the filtered and secreted part of the totalexcretion.

METhODS

Male Wistar rats of a specialstrain with surfaceglomeruli(strain H, Versuchstierzucht, Hannover, FRG) were used withbodyweightsof 180—300g and freeaccessto normal rat chowand water. After anesthesia with thiobarbital (Inaktin, Byk,Constanz, FRG) 100—120mg/kg i.p. the trachea was cannulated and polyethylene catheters placed into the femoralartery for continuous blood pressure monitoring and arterialblood sampling. The leftjugular vein and/or the right femoralvein were also cannulated for infusion of radionuclide solutions and 0.9% Ringer solution to give a total infusion rate of

1986 MUIler-SuurandMUller-Suur The Journal of Nuclear Medicine

Glomerular Filtration and Tubular Secretionof MAG-3 in the Rat KidneyRoland MUller-Suur and Charlotte Müller-Suur

Karolinska Institute, Dept. ofClinical Physiology Danderyds Hospital, Stockholm and Dept. ofPhysiology and Medical Physics, Uppsala University, Uppsala Sweden

Technetium-99m mercaptoacetyltnglycine (MAG-3) has recently been introduced as a newradiopharmaceutical for dynamic renal scintigraphy. To elucidate the mechanism of renalexcretion,micropunctureexperimentswere performedin rat kidneysfor directmeasurementsof glomerular filtration and tubular secretory capacity. Fluid of Bowman space was collectedfrom superficial glomeruli and analyzed for its contents of [@“Tc]MAG-3,[125I]hippurateand[3H]inulinduring constant infusion of these compounds. The ratio of activity of ultrafiltrate tothat of arterial plasma was 0.23 for MAG-3, 0.68 for hippurate and 1.04 for inulin whichdemonstrates that the filtrated amount of MAG-3 is only 23% of that of inulin, presumablybecause of higher plasma protein binding which was also measured in vitro and found to be80 ±1.5% for MAG-3 and 32 ±2% for [125I]hippurate.Proximal and distal tubules were alsomicropunctured and their tubular fluid as well as the final urine analyzed for the activity ofhippurate and MAG-3. The tubular fluid to plasma ratio values along the nephron and in thefinal urine were all lower for MAG-3 than for hippurate, indicating a lower secretory capacity.From measurements of whole renal clearance, GFR and plasma protein binding the filteredamount of MAG-3 was 0.26 and of hippurate 0.87 ml/min.g kidney weight (p < 0.001) andthe secreted amount 2.01 and 2.38 mI/mm.g kidney weight (p < 0.05), respectively. Weconclude that MAG-3 is predominantly excreted by tubular secretion and that the lower renalclearance of MAG-3 as compared with that of hippurate is a result both of a substantiallydecreased glomerular filtration and of a lower tubular secretion.

J Nuci Med 30:1986—1991,1989

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1 mi/h. 100 g BW [99mTc]MAG3 (kit prepared according toEnsing et al. (18) Mallinckrodt, Petten, Netherlands)and 125-1-hippurate (solution for ERPF measurements, AmershamInternational, New Hampshire, England) were diluted in thefinal solution to give —18MBqJh (500 foCi/hr) and 2.7 MBc@jhr(70 @zCi/hr),respectively. In two rats [3H]inulin (AmershamInternational as above) was infused solely at a rate of 2.7MBq/h (70 foCi/hr). A catheter was also placed into the leftureter for sampling ofunne. The left kidney was prepared freeofconnecting tissue and placed in a lucite chamber fixed withcotton tissue and superfused with prewarmed mineral oil. Thekidney surface was illuminated by a fiber optic light source.

Micropuncture was performed after a 60 mm equilibrationtime (Fig. I). Surface glomeruli appearing as clearly visiblecapillary balls at the surface of the kidney were puncturedwith sharpened glass pipets of 5—7@tmouter diameter filledwith sudan stained paraffin oil. Oil was injected into Bowmanspace and the proximal tubule and ultrafiltrate collected byapplying slight suction to the pipet, monitoring the meniscusof the oil block just at the glomerular neck of the proximaltubule. Ifby chance a glomerular capillary loop was puncturedand erythrocytes aspirated, the sample was discharged andanother glomerulum punctured. Late proximal tubules andearly distal tubules were identified by a single shot injectionof lissameen-green coloured Ringer solution into a randomlyselected proximal tubule by the aid of a micropuncture pipetwith a tip diameter of 1—2tim. They were then puncturedwith the collection pipet as above and tubular fluid wascollected in front ofan injected oil block (compare Fig. 1). Inthree nephrons, only collections at one site of the nephronwere successful. Glomeruiar collection and tubular collectionswere not performed in the same individual nephron.

Collection time was 2—5mm giving samples of 10 to 100ni. They were transferred to precalibrated parallel bore capillanes (Microcaps, Drummond, USA) to measure the volume.Aftertransferinto countingvialsfilledwith0.5 ml watertheirradioactivity was counted in a well type counter. The @mTcactivity was counted the same day and recounted after thedecay of 99mTcfor the 125!activity. The micro-samples of thetwo separate rats, where [3H]inulin was infused solely, werecounted in a scintillation counter (Beckman IS 3800, Fuller

ton, USA) after transferring them into 0.5 ml water and 3 mlscintillation cocktail (Picofluor 40, Packard Instruments,Groningen,Netherlands).Correspondingplasma samples(P)taken immediatelyafter each puncture and timed final urinesamples (U) were also analyzed for the radioactivity as above.The amount of filtrated MAG-3 and hippurate is given by theratio UF/P, where UF is the radioactivity of ultrafiltrate fromthe Bowmann space. The filtration rate of the single gbmerulum (SNGFR) equals the flowrate ofthe sample.

For measurement of plasma protein binding of MAG-3and hippurate plasma was centrifuged in special vials (Amicon, Centrifree, MA) at 3000 g. Radioactivity ofplasma beforecentrifugation and of the ultrafiltrate gave the amount ofprotein binding from the formula (l—F/P).100,where F isfiltrateactivityand P plasmaactivity.

For whole kidney clearances six separate rats were usedwith the same animal preparation as above. 1.8 MBq/hr[99mTcJMAG3, 360 KBuJhr [‘25I]hippurateand 320 K&iJhr51 Cr EDTA (Behringwerke, Marburg, FRG) were infusedsimultaneously at a rate of 1 ml/hr. 100 g BW. Plasma andurine aliquots were measured for their radioactivity in a welltype gamma counter with background, crosstalk and decaycorrection. MAG-3 and hippurate clearance and GFR, i.e.,EDTA clearancewerecalculated in the usual manner (i.e., U.V/P) corrected for kidney wet weight. Two to four samplingperiods of2O—30mm were averaged for every rat. The amountof filtrated and secreted MAG-3 or hippurate could be calcubatedfromthe protein freetracer fractiontimesGFR and totalclearance minus filtrated amount, respectively.

StatisticsAnalysis was performed using the students t-test for pairs

(MAG-3 versus hippurate in the same rats) or unpaired sampies (MAG-3 or hippurate versus inulin in different rats).Means±s.e.m.are given.Two-tailedP (2p)<0.05 wastakenas significant.

RESULTS

Table 1 gives the data from glomerular micropuncture and Table 2 those of micropuncture at early distal

AB

FIGURE 1Schematic drawing of the micropuncture technique for collection of ultrafiltrate from Bowman space (A), endproximalsite (B) or from the early distal tubule (C).

1987Volume 30 •Number 12 •December1989

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UF/PinulinRat

TABLE 1Glomerular Micropuncture Data

and 2.27 ±0.08 mb/mm .g kidney weight, respectively.The MAG-3 clearance was significantly lower than thatofOIH (2P < 0.00 1)and higher than that of EDTA (2P< 0.001). The amount offiltration calculated from GFR

and the protein free plasma fraction was 0.26 ±0.01ml/min.g kidney weight for MAG-3 and 0.87 ±0.04for OIH (2P < 0.001). The amount of secretion ofMAG-3 and OIH, i.e., the difference between totalclearance and filtrated amount was 2.00 ±0.10 and2.39 ±0.08 ml/min.g kidney weight (2p < 0.05, respectively.

DISCUSSION

Since MAG-3 has recently been introduced as a newradiopharmaceutical for renal dynamic scintigraphythere is much interest on the renal excretion mechanismfor this compound. Already in early studies, the similarity of MAG-3 to hippuran concerning its high renalclearance and the inhibitory effect of probenecid(1,2, 17,19)led to the conclusion that the same transportmechanism may be involved for the two substances.The high amount of binding to plasma proteins ofMAG-3 was taken as an argument, that very low filtration takes place in the gbomerulus and that tubularsecretion of MAG-3 is in the range of OIH to explainthe final clearance values (1,5,17). Both in animalexperiments (11—14)and all human clinical trials (2—6,8,10), the renal clearance ofMAG-3 was shown to beconsiderably less than that of OIH. There exists alsoexperimental evidence that the affinity of MAG-3 torat tubules is less than for OIH (13) and also in patientsa lower affinity to the tubular transport system compared with [‘31I]hippuratehas been shown (17). Thepresent study adds more evidence to these observations.We thus found that the amount of filtered MAG-3 isconsiderable less than that of inulin and that of OIH.Since it correlates strongly with the amount of proteinfree MAG-3 in arterial plasma the glomerular filtrationbarrier seem to permit all protein free MAG-3 to befiltered. The same conclusion is valid for the 125-I-hippurate preparation used in the present study. Itsprotein binding was 32% in rat plasma and the ultrafiltrated amount was 64% ofthe whole plasma activity.In human plasma and for other preparations of hippurate other values have been observed (10), MAG-3,however, showed in human studies similar high proteinbinding and consequently a restricted glomerular filtration. The present findings further demonstrate a lowersecretory capacity for MAG-3 than for hippurate at twotubular sites (compare Table 2 and Fig. 2) and also inthe final urine (compare Table 3 and Fig. 2). Fromwhole clearance and measurements of plasma proteinbinding in patients (9, 10) it is evident that similarconditions will exist also in the human kidney. Accord

SNGFRnI/mm

11.727.516.918.618.827.920.421.429.933.023.024.827.113.723.620.517.226.623.224.017.515.815.017.816.621.31.1

25

UF/PMAG-3

0.210.150.190.160.280.180.240.320.230.190.180.240.260.280.300.270.340.23

0.240.01

18

1.191.011.170.921.051.010.911.040.047

UF/POIH

0.620.430.470.760.860.570.760.870.620.480.560.480.770.580.660.630.680.70

0.640.03

180.001

2

3

4

5

6

Means.e.m.n2P<

. SNGFR = single nephron filtration rate, UF/P = ratio between

activityof the fluid in Bowmanspaceto that of arterialplasma.Valuesbelow1.0indicaterestrictedfiltrationand1.0freefiltration.

and late proximal site as well as of the activity in thefinal urine. The ratio of activity of ultrafiltrate in Bowman space to that ofarterial plasma was 1.04 for inulin,0.64 for OIH and 0.24 for MAG-3, which demonstrates,that only 23% of MAG-3 in the arterial plasma is freefiltrated, whereas 61 % of OIH and all inulin is freefiltrated.

In vitro measurements showed that MAG-3 wasbound to proteins in rat plasma at 80 ±1.5% ascompared to 32 ±2% for OIH (2P < 0.001).

Table 2 shows the results ofmicropuncture at tubularsites and the activity in the final urine. Both the tubularfluid to plasma ratio, TF/P, at late proximal sites, earlydistal sites and in the final urine were lower for MAG3 than that for hippurate (2P < 0.001, 0.01 and 0.05,respectively). A considerable increase of the TF/P values along the nephron from the glomerulus to the finalurine was observed for both substances.

Table 3 gives the results of six separate rats wheresimultaneous measurements of gbomerular filtrationrate by 5 1-Cr-EDTA and of the OIH and MAG-3clearance provided means of 1.28 ±0.05, 3.25 ±0.08

1988 MUller-SuurandMUlIer-Suur The Journal of Nuclear Medicine

1

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TF/PproxRat Nephron MAG-3 OlHTF/P

distMAG-3 OlHU/P

finalMAG-3urine OIHClearanceMAG-3 OlH

1

. TF/P = ratio between activfty of tubular fluid to that of arterial plasma at proximal (TF/P@@) or distal (TF/P@.@) level. U/P = ratio of

activityin thefinalurineto that of arterialplasma.Clearancegivenin ml/min.gKW.

RatOlHLeft

renalclearancemI/mmg KW

MAG-3EDTASeermI/mm

OIHetedg KW

MAG-3FiltmI/mi

OIHeredn g KW

MAG-3A3.1712.3721.4922.1562.0741.015.298C3.3391.9291.2602.4821.677.857.252D3.1272.3101.1272.3612.085.766.225E3.5182.5931

.3002.6342.333.884.260F3.4582.0401.3362.5501.773.908.267G2.9332.3011.1632.1422.068.791.233Mean3.2582.2581

.2802.3882.0020.8700.256s.e.m.0.0900.0980.0540.0840.0970.0360.0112P<0.0010.050.001(MAG-3vs.

OIH)

17.1 32.8 589 858 2.413 3.518

TABLE 2Tubular Micropuncture and Final Urine Data

12I12112123

3.4 6.83.2 4.53.5 5.12.1 4.71.9 3.01.1 3.41.7 4.54.6 9.82.1 4.13.2 8.7

23

47

8

10.421.35889981.8853.20016.633.672015532.0904.5067.413.086920931

.6523.97816.237.11452661.9953.38110.819.25957992.1032.80717.822.89.112.313.1724.0158410942.0233.5651.473.36992610.1030.245886666

Mean2.685.46s.e.m.0.340.71n

1110102P<0.0010.01 0.05 0.01

ing to the tubular reabsorption or secretion the concentration of various substances changes along the lengthof the nephron (20). Inulin starts at the gbomeruluswith a concentration equal to that of the plasma waterand concentrates along the nephron based on the removal of water from the tubular fluid. The resultingprofile is shown in Figure 2 according to the data fromrats (21,22). P-aminohippuric acid, which is secretedactivily predominantly by the proximal tubulus (23)results in a profile higher than that of inulin (20,23).Fitting the present data into a graph of the concentration profile along the nephron (Fig. 2), it becomesobvious that MAG-3 places in between [‘251]hippurateand inulin, arguing for a lower tubular secretion rate ofMAG-3 than that of hippurate. For technical reasonswe were not able to simultaneously determine inulin inour micropuncture samples to correct directly in every

individual sample for the effect of water reabsorptionon the tubular concentration ratio. It is difficult to assay[3H]inulin in the presence of['25ljhippurate. The inulinconcentration profile along the nephron is, however,well known both from earlier experiments in otherlaboratories (21) and from our experiments (22). Separate experiments in two rats were, however, performedon the gbomerular bevel in the present study since wehad no earlier data. The observed inulin concentrationin Bowman space is in agreement with free filtration ofall inulin in the plasma water through the gbomerularifitration barrier whereas hippurate and MAG-3 arerestricted.

The mechanism for the low secretion of MAG-3 isof interest. In our first experimental study on MAG-3,we showed less penetration of MAG-3 into blood cellsthan was found for hippurate (11). The same has been

TABLE 3Data on ClearanceAs WellAs Filtratedand SecretedAmounts

Volume 30 •Number 12 •December1989 1989

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REFERENCES1. Fritzberg AR, Kasina 5, Eshima D, Johnson DL.

Synthesis and biologic evaluation of technetium-99m-MAG3 as a hippuran replacement. J Nucl Med 1986;27:111—116.

2. Taylor A, Eshima D, Frizberg AR, Christian PE, Kasina S. Comparison of I-l31-OIH and Technetium99m-MAG3renal imaging in volunteers. J Nucl Med1986; 27:795—803.

3. Taylor A, Ziffer JA, Stevens A, et al. Clinical comparison of 1-131 orthoiodohippurate and the kit formulation of Tc-99m mercaptoacetyltriglycine.Radiology1989;170:721—725

4. Bubeck B, Brandau W, Steinbaecher M, et al. Technetium-99-m labeled renal functioning and imagingagents: II. Clinical evaluation of 99m Tc-MAG3(99mTc..mercapto-acetylglycine).NucI Med Biol 1988;15:109—b18.

5. Jafri AR, Britton KE, Nimmon CE, et al. Technetium99m-MAG3, a comparison with iodine-l23 an diodine-l3l-orthoiodohippurate in patients with renal disorders.JNuclMed 1988;29:147—158.

6. RussellCD, Thorstad B,Yester MV, et al. Comparisonof Tc-99m-MAB3with I-l3l-hippuran by a simultaneous dual channel technique. J Nucl Med 1988;29:1189—b193.

7. Thorstad BL, Russell CD, Dubovsky EV, et al. Abnormal Captopril Renogram with a Technetium-99-M-Labeled Hippuran Analog. J Nuci Med 1988;29:1730—1737.

8. Martin WG, Wolf IH, Kempken K, et al. Tc99m-MAG3 versus 1123 OIH; comparison of quantitativeparameters in renal function. In: Schmidt HEA, Buraggi GL eds. Nuclear medicine. Stuttgart-New York:Sehattauer Verlag; 1989; 413—416.

9. Al-Nahhas AA, Jafri RA, Bntton KE, et al. Clinicalexperience with 99mTcMAG mercaptoacetyltriglycine and a comparison with 99mTc..DTPA.Eur J NuclMed 1988; 14:453—462.

10. Mübler-SuurR, Bois-Svensson I, Mesko L. A comparative study of renal scintigraphy and clearance with99m-Tc-MAG3 and 123-I-hippurate in patients withrenal disorders. [Abstract]. J Nucl Med I989; 30(4):2Ab.

I 1. MUller-Suur R, Müller-SuurC. Renal and extrarenalhandling of a new imaging compound (99m-TcMAG3) in the rat. Eur J NuclMed 1986; 12:438—442.

12. Coveney JR, Robbins MS. Comparison of technetium-99m MAG3kit with HPLC-purified technetium99m MAG3 and OIH in rats. J Nucl Med 1987;12:1881—1887.

13. Coveney JR. Pilcher GD, Robbins MS. Tc-99m-MAG3 and I-b3 1 o-iodohippurate (OIH) transportkinetics in rat kidney slices. Eur J Nucl Med 1988;14:256.

14. de Jong R, Nielsen J, van Steenbergen J, et al. Resultsof ERBF and ERPF measurements in healthy dogswith two new radiopharmaceutical principles. ContrNephrol 1987; 56:49—52.

15. Jafri RA, Nimmon CC, Britton KE, et al. Tc-99mmercapto acetyl-tnglycine MAG3, a comparison with131-I-and 1-123orthoiodohippurate for routine renalwork [Abstract].J NucIMed 1987;28:647.

16. Schaap GH, de Jong RBJ, Alferink THR, et ab.99mTc-MAG3 is not suitable for determination of theeffectiverenal plasma flow.Kidney Int 1987; 32:775.

17. Brandau W, Bubeck B, Berger J, et al. Excretion

1000

500

200-

I 00

50

20

10

5

2.0

1.0

0.5

0.2

0I-

z0I-

I-zwC.)z0C.)

C,)

—I

0I-wz

0@

0@

bPROXIM. LOOP OF@DISTAL @OLLEC1bFINAL@TUBULE HENLE@ TUBULE iTUBULE@ URINE

+GLOMERULuS

shown also in patients (6,10). That observation mayalso be true for penetration into tubular secretory cellswhich may be responsible for lower outward transportcapacity into tubular lumen. The high protein bindingof MAG-3 may be involved in such a hypotheticalchain of events. However, other substances predominantly protein bound have not shown an increasedclearance when their protein binding is reduced (24).No such experiments have been performed until nowwith MAG-3. Thus, the reason for a lower transportcapacity for MAG-3 in the kidney is still unclear andfurther experiments are necessary.

To summarize, we have demonstrated that the lowerclearance of MAG-3 is partly explained by a boweramount of filtration, due to a higher plasma proteinbinding but also by a lower tubular secretion rate bothat the proximal and the distal tubular level.

ACKNOWLEDGMENTS

This study was partly supported by research grants fromthe Karolinska Institute and from Malhinckrodt, Petten, Netherlands. The typing of the manuscript by Karin Hultberg isgratefully acknowledged. The authors especially thank Ingeborg Bois-Svensson who prepared the MAG3 solution.

1990 MUller-SuurandMUlIer-Suur The Journal of Nuclear Medicine

p

125-l-HIPP4― /

,‘çIMAG-3

/NULIN

FIGURE 2Change of concentrationof inulin,hippurateand MAG3along the nephron. Observe the logarithmic scale. Filledand open squares give mean values of the present study.The profile for inulin is taken from the literature (20—22).

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characteristics of Tc-99m-MAG3 in patients. In:Schmidt HEA, Buraggi GL eds. Nudear medicine.Stuttgart-New York: Schattauer Verlag; 1989; 409—412.

18. Ensing GJ, Nielsen J, Panek-Finda H, Panek K. Dcvelopment of a kit formulation for the labelling ofmercaptoacetyl tnglycine (MAG3) with 99mTc. EurJNuclMed 1988;14:303.

19. Müller-SuurR, Müller-SuurC. Renal and extrarenalexcretion of a new renal scanning agent: 99mTcMAG-3 in comparison to 125-I-hippurate (OIH) and5bCr-EDTA. Abstract Europ NuclMed Congr Gosbar,FRG 1986.

20. Guyton AC. Textbook ofMedicalPhysiology. VII Ed.

Philadelphia: W. B. Saunders, 1986.21. Giebisch 0, Windhager EE. Renal tubular transfer of

sodium, chloride and potassium. Am J Med 1964;36:643—649.

22. Mulber-Suur R, Norlen BJ, Persson AEG. Resetting oftububoglomerubarfeedback in rat kidneys after unibatcrab nephrectomy. Kidney Int 1980; 18:48—57.

23. Shimomura A, Chonko AM, Grantham JJ. Basis forheterogeneity of para-aminohippurate secretion inrabbit proximal tubulus. Am J Physiol 1981;240:F430—F436.

24. Ochwadt BK, Pitts RF. Disparity between phenol redand Diodrast clearances in the dog. Am J Physiol1956; 187:318—322.

1991Volume 30 •Number 12 •December1989

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1989;30:1986-1991.J Nucl Med.   Roland Müller-Suur and Charlotte Müller-Suur  Glomerular Filtration and Tubular Secretion of MAG-3 in the Rat Kidney

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