3. ~. A. Bardakhch'yan, P. E. Povilaitite, and A. M. Polyak, Byul!. Eksp. Bioi .... !06, No. 6, 730-733 (1988).

4. B. V. Vtyurin and V. P. Tumanov, Byull. Eksp. Biol., 7_/2, No. i0, 108-110 (1971). 5. N. N. Karkishchenko, V. V. Solodilov, and ~. A. Bardakch'yan, Izv. Sev. Kavkaz. Tsentra

Vyssh, Shkoiy, No. 2, 122-127 (1987). 6. N. N. Karkishchenko, V. V. Solodilov, and ~. A. Bardakhch'yan, Izv. Sev. Kavkaz. Tsentra

Vyssh. Shkoiy, No. i, 121-125 (1988). 7. N. N. Karkishchenko and ~. A. Bardakhch'yan, Byull. ~ksp. Biol., 10._~9, No. i, 86-89

(1990). 8. P. E. Povilaitite and ~. A. Bardakhch'yan, Tsitol. Genet., 2__0, No. 5, 323-326 (1986). 9. ~. N. Popova, S. K. Lanin, and G. N. Krivitskaya, The Morphology of Adaptive Changes

in Nerve Structures [in Russian], Moscow (1976). A. D. Khor'kov, Arkh. Anat., 8__7, No. 12, 28-32 (1984). P. Schmidt and G. Tews, Human Physiology. Vo!. I: The Nervous System [Russian transla- tion], Moscow (1985).

i0. ii.

NEPHROTROPISM OF ~2SI-TRIOMBRAST AND GADOLINIUM-DTPA IN NORMAL RATS

AND RATS WITH EXPERIMENTAL GLYCEROL-INDUCED NEPHRITIS

E. N. Bolotova, V. S. Veksler, UDC 616.61-002.092.9-073.916 N. D. Mitrofanova, N. L. Shimanovskii, and P. V. Sergeev

The method of contrasting with roentgenoiogic contrast medium (RCM) containing iodine is widely used at the present time for the diagnosis of pathological changes in the urinary system. However, this method has various disadvantages: frequent side effects in patients, and lessening of the diagnostic value of the method when pathological changes have developed in the kidneys [2, 3].

Magnetic resonance tomography is a comparatively new method of medical diagnosis in the USSR, and because of its high informativeness, it is very promising [4]. An intensive search is currently in progress for paramagnetic agents which, on the one hand, will effectively change the relaxation time of tissue protons and, second, can be regarded as pharmacologic- ally inert, quickly eliminated from the body, and distinguished by high informativeness.

Among the paramagnetic agents known at present and belonging to the group of substances with unpaired electrons in their outer electron shell, trivalent gadolinium has optimal para- magnetic properties [9]. Its complexes with various chelating agents and, in particular, with diethyiaminopenta-acetate (DTPA), have begun to be widely used in recent years for con- trasting various organs and tissues [5-7]. The gadolinium-DTPA complex is readily soluble in water, stable in vivo, nontoxic in the doses used (50-100 mg/kg, intravenously), and is filtered sufficiently quickly by the kidneys [I0, ii].

The aim of this investigation was a comparative study of the nephrotropism of triombrast, a widely used Soviet RCM, and of gadolinium-DTPA in normal rats and rats with experimental glycerol-induced nephritis in the early stages of its development.

EXPERIMENTAL

Experiments were carried out on 450 noninbred male rats weighing 120-150 g, kept on the standard animal house diet. Acute renal failure was simulated by the method in [12], by giv- ing the animals a single intramuscular injection of a 50% solution of glycerol ("Fluka," Switzerland) at the rate of i0 ml/kg body weight.

A 76% solution of triombrast (from the M. V. Lomonosov Kiev Pharmaceutical Chemical Fac- tory) was used. 12Sl-labeled triombrast was obtained by the isotope exchange reaction by A. F. Volkov (Institute of Biophysics, Ministry of Health of the USSR).

Department of Molecular Pharmacology and Radiobiology, Medico-Biological Faculty, N. I. Pirogov Second Moscow Medical Institute. Chemical Faculty, M. V. Lomonosov Moscow State University. Translated from Khimiko-farmatsevitcheskii Zhurnal, Vol. 25, No. 4, pp. 13-15, April, 1991. Original article submitted July 12, 1990.

0091-150X/91/2504-0231512.50 �9 1992 Plenum Publishing Corporation 231

TABLE i. Concentration of 12Sl-Triom- brant in Rats' Kidneys and Blood after Injection in a Dose of 493 mg/kg (M • m)

Time, I min

5 15 30 60

control Nephritis blood I kidneys [blood I kidneys

1.08-+-0,08 5.0~0,5 2,745=0,05" 1,88___0,28" 0.31-4-0,09 1,37~0,2 2,23-+-0,1" 1,95-+-0,1" 0,16-4-0.02 1,16__0,1 2,05-+-0,06* 1.64-+-0,4 0,10-4-0,01 0.91-+-0,13 1.77-4-0,04" 1,55-4-0,4"

*P ~ 0.05.

TABLE 2. Parameters of Pharmacokinetics of 12Sl-Triombrast (493mg/kg) in Normal Rats and Rats with Renal Pathology (M -+ m)

Parameters _I Control ,I Nephritis

Total clearance of 1 2 51_ triombrast, ml/min 17,57-+-1,07 0,264~0,12"

Elimination constant, rain -I 0,092-+-0,008 0,002--+_0,0001" Half-elimination time, rain 29.69+1,1 144.4__.12,4"

*P ~ 0.05.

1251-triombrast was injected intravenously into the rats in a dose of 493 mg/kg (2"10 ~ Bq/kg). The animals were killed under superficial ether anesthesia 5, i0, 15, 20, 30, and 60 min after injection of the compound. Kidney slices were obtained and incubated with 12Sl- triombrast(10 -~ M) by the method described previously [i]. The quantity of triombrast in tissue samples was determined by measuring gamma-radiation on a Gamma-I spectrometer (USSR).

An aqueous solution of gadolinium-DTPA (40 mM) was injected intravenously into intact and sick animals in a dose of 0.I mmole/kg (57 mg/kg). The gadolinium content in the speci- mens was judged by calculating the relaxation amplification factor, reflecting a change in spin-lattice relaxation of protons:

( x ( L J experiment ~ )control.100 %. ET1 = . _ _

I

( ~ ) control

where TI denotes the spin-lattice relaxation time after injection of gadolinium (experiment) or physiological saline (control) into the animals.

In order to measure T1 of native tissues, blood samples and samples obtained separately from the renal cortex and medulla, weighing 100-200 mg, were taken from the animals immedi- ately after decapitation and transferred into the working ampule of the spectrometer not later than 3 h after preparation of the specimens. The value of TI was determined on a Minispek PCI20 instrument ("Brucker," West Germany, frequency 20 MHz, 40~ To measure TI of the native tissues, an 80 ~ - z - 90 ~ pulse sequence was used; to measure T2 (spin-spin relaxation time of protons) the Carr-Purcell-Meiboom-Gill (CPMG) sequence was used.

The experimental data were subjected to statistical analysis by Student's test, using the IMG 666B computer (Hungary).

RESULTS AND DISCUSSION

In early renal pathology (24 h after injection of glycerol) elimination of 12Sl-tri0m- blast from the blood was observed to be delayed and a fall in the ratio between the content of this compound in the kidney and blood to 0.7 was observed compared with 4.6 in the con- trol (Table i) 5 min after injection of triombrast. On the 2nd day the degree of neurotro- pism of the triombrast was reduced even more (Table 2). Whereas the intact animals excreted 76% of the triombrast in the course of 3 h, animals with glycerol-induced nephritis excreted only 53%, with a simultaneous increase in its blood level.

The results of the experiments in vitro showed that the kinetics of uptake and release of 12Sl-triombrast from kidney slices of rats with glycerol-induced nephritis (48 h) differs when compared with intact slices (Table 3). Reduction of the accumulation of this compound in the initial periods of incubation of the slices (5-10 min) and a decrease in its release from the kidney tissue starting with the 15th minute of incubation were noted. The velocity constant of uptake of the compound by kidney slices from rats with renal pathology was re- duced by 20-30% compared with the control.

The results explain the ~neffectiveness of the use of triombrast for contrasting the urinary system in acute renal failure.

Relaxation characteristics of kidney tissue protons were studied by proton magnetic re- laxation in the presence and in the absence of the paramagnetic agent gadolinium-DTPA.

232

TABLE 3. Kinetics of Assimilation and Release of 12Sl-Triombrast from Kidney Slices of Normal Rats and Rats with Renal Pathology [nmoles/(min.g), (M • m)]

Time, min

5 10 15 20 30

Assimilation Release Control I nephriti.r control I nephritis

23,8-4-0,8 18,3-4-1,1" 15,4-+-1,3 17,9+2,0 29,6• 23,6+1.6" 22,8• 19,6_---+-2,1 29,7+_ 1,7 23,7+_2,7 27,7-+- 1,2 19,8• 1,5" 34,5• 1,1 32,9• 28 ,5~ 1, I 20,4 + 1,9" 40,7:k2,3 41,5• 33,7+0,8 20,8/:2,0*

*P ~ 0.05.

TABLE 4. Spin-Lattice (TI) and Spin- Spin (T2) Relaxation Times (in sec) of Renal Tissue Protons from Intact Rats and Rats with Acute Renal Failure (M • m)

l

Parameters Cortex I Medulla J

T1 �9 I0 ~ Control 474_6 ,7 627__+ I0 Nephritis, ist day 522--+_11,5"* 677--+_13"

2nd ~ 530-+-20* 704-+-25* 3rd ~ 533• 709~15,5"

T2 Control 57,3• 85,6• 1,6 Nephritis, Ist day 66,7~1,9" 96,5--+-2,0"

>> 2nd ~ 70,6=+-5,6" 94,9-+-4,4 ~, 3rd ~ 63,0_0,07" 9 2 , 8 + 1,5"

*P < 0.05. **P < 0.01.

Contro 1

I

o I l I I o a 5 lO

Ex eriment (ist day)

Experiment (3rd day)

I I I I o,,l I i Io a .5"

Fig. I. Kinetics of accumulation of gad- olinium-DTPA in renal medulla under normal and pathological conditions (M • mt). Here and in Fig. 2: abscissa, time after injec- tion of gadolinium-DTPA, min; ordinate, re- laxation enhancement factor, %.

The proton magnetic relaxation time is a biophysical characteristic of the tissues and can be used as an indicator of their state under normal conditions and in disease. In cells and tissues, which contain up to 80% of water, the relaxation time mainly characterizes mo- bility of the protons of water, and it is a sensitive indicator of the water balance of the tissues and depends on the content of water and of paramagnetic centers (the iron of hemo- globin) in the tissues and on mobility of nonaqueous molecules.

In acute renal insufficiency the spin-lattice relaxation time of the protons (TI) changes in the cortical and medullary layers of the kidneys (Table 4). Only 24 h after injection of glycerol into the animals the values of T1 increased significantly by 10% (for the renal cor- tex) and by 8% (for the renal medulla) compared with intact animals. By the 3rd day of de- velopment of nephritis the tendency for TI to increase was preserved, with values of 12 and 13% respectively. In all probability the increase in the spin-lattice relaxation time is connected with the development of edema of the kidney tissue in this pathology.

The spin-spin relaxation time (T2) of the kidney tissue protons also was changed (Table 4): on the ist day of development of the disease it rose by 16% in the cortex and by 13% in the medulla compared with the control.

Injection of gadolinium-DTPA into the animals was shown to reduce the proton relaxation time of the renal cortex and medulla by 2-3 times, confirming the high contrasting power of this paramagnetic agent.

To analyze the data we used the relaxation enhancement factor, the value of which is directly proportional to the concentration of the paramagnetic agent in the tissue. We found that the kinetics of accumulation of gadolinium-DTPA in the kidneys of rats with acute

233

Contro 1

800

1 0 0

0 i i L 0 6"

k

i

I0

Experiment Experiment (lss day) (3rd day)

t L I,, ,Y ~ / 0

L I [ I

Fig. 2. Kinetics of accumulation of gado- linium-DTPA in renal cortex under normal and pathological conditions (M • mt).

renal failure differed sharply from the control (Pigs. i and 2). These differences were par- ticularly marked in the early stage of injection of gadolinium. Only 24 h after the develop- ment of the pathological state the content of gadolinium in the renal medulla i min after its injection into the animals was 2.2 times less than in the control, and on the 3rd day of development of nephritis it was 2.7 times less (Fig. i). Similar changes in the kinetics of gadolinium accumulation were observed in the cortex (Fig. 2). Whereas in the intact kid- ney gadolinium accumulated to the greatest degree 1 min after its intravenous injection, in rats with glycerol-induced nephritis, peak accumulation of gadolinium in the cortex and me- dulla was shifted to 3-5 min after injection, after which its concentration in both cortex and medulla gradually fell. Under these circumstances the kinetics of elimination of gado- linium-DTPA from the blood was virtually unchanged; the maximal level of increase of proton relaxation in kidney tissue did not differ significantly from the control.

Thus to sum up data in the literature and our own findings, it can be concluded that by contrast with angiourographic roentgenologic contrast media, magnetic resonance contrast media of the gadolinium-DTPA type preserve their nephrotropism in renal pathology, and a change in the kinetics of their accumulation or elimination by the kidneys can serve as a diagnostic test of disturbance of functional activity of the urinary system.

LITERATURE CITED

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gation of the Kidneys and Urinary Tract [in Russian], Moscow (1987). 3. P. V. Sergeev, N. K. Sviridov, and N. L. Shimanovskii, Radiocontrast Media [in Russian],

Moscow (1980). 4. P. V. Sergeev, N. L. Shimanovskii, and V. O. Panov, Khim.-farm. Zh., No. 5, 540 (1989). 5. D. N. Carr, S. Brown, G. M. Bydder, et al., Am. J. Radiol., 157, 44 (1985). 6. W. L. Curati, M. Graif, D. P. Kingsley, et el., Radiology, I~8, 447 (1986). 7. M. Graif, G. M. Bydder, R. E. Steiner, et al., Radiology, 157, 125 (1985). 8. L. Kaufman et al., (eds.), Nuclear Magnetic Resonance in Medicine, New York (1982),

pp. 46-54. 9. V. M. Runge, S. A. Clanton, C. M. Lukehart, et al., Am. J. Roentgenol., 141, 1209 (1983).

i0. V. Tauber, H.-J. Wemman, M. Pancer, et al., Arzneimittel-Forsch., 36, 1089 (1986). ii. H. S. Weinman, R. C. Brasch, W. R. Press, et al., Lancet, ~, 484 (1984). 12. M. Yates, C. Bowmer, and L. En,nerson, Biochem. Pharmacol., 33, 1695 (1984).

234