4-Hydroxynonenal is degraded to mercapturic acid conjugate in rat kidney

Free Radical Biology & Medicine, Vol. 19, No. 5, pp. 685-688, 1995 Copyright ~ 1995 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/95 $9.50 + .00

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Brief Communication 4-HYDROXYNONENAL

IS DEGRADED

CONJUGATE

IN

RAT

TO

MERCAPTURIC

ACID

KIDNEY

TOBIAS PETRAS,* WERNER G. SIEMS, t and TILMAN GRUNE* *Clinics of Physical Therapy and Rehabilitation, Medical Faculty (Charitr), Humboldt-University Berlin, Berlin, Germany; and tHerzog-Julius-Hospital for Rheumatology and Orthopaedics, Bad Harzburg, Germany (Received 23 February 1995; Revised 3 April 1995; Accepted 6 April 1995)

Abstract--The cytotoxic lipid peroxidation product 4-hydroxynonenal (HNE) was infused into rat kidney. During the first 2 min a rapid degradation of a 100 #M HNE solution was demonstrated. After 5 min the consumption rate of 4-HNE reached a steady state of about 75 nmoles/ml. The total HNE consumption rate was about 200 nmoles/g w.w./min. The excretion rate into urine was about 0.1% of total HNE consumption. It could be demonstrated that the HNE-mercapturic acid formation takes place in the kidney. The formation of the HNE-mercapturic acid contributes up to 6% to total HNE consumption. Within 10 min of perfusion 2% of the HNE-mercapturic acid were excreted into urine. The residual 98% flow back into the blood circulation. Keywords--Lipid peroxidation, 4-Hydroxynonenal, Mercapturic acid, Aldehyde metabolism, Kidney, Free radicals

INTRODUCTION

the organ of mercapturic acid formation. One has to favor the kidney as the dominant organ contributing to mercapturic acid formation. Therefore, we used the isolated perfused kidney of rats to measure the HNE-mercapturic acid formation in the venous effluent and the urine.

4-Hydroxynonenal ( H N E ) is an aldehyde formed by lipid peroxidation from omega-6-polyunsaturated fatty acids) Numerous biological effects like inhibition of DNA, RNA, and protein synthesis are connected with 4hydroxynonenal, which can also inhibit proliferation of cell lines and exerts various genotoxic effects. 2 The aldehyde easily reacts with the SH-groups of cysteine, glutathione, and proteins, 3 so it is able to inhibit the glycolytic enzymes glyceraldehyde phosphate dehydrogenase ( G A P D H ) and lactate dehydrogenase (LDH).4 Eucariotic cells rapidly metabolize HNE. The mean products of the H N E metabolism in hepatocytes and enterocytes were the H N E - G S H adduct, the 4-hydroxynonenoic acid, and 1,4-dihydroxynonene. 5 Experiments with injection of tritium-labeled 4-hydroxyhexenal ( H H E ) , another hydroxyalkenal, into the portal vein of rats have shown that a part of the radioactivity was excreted in the urine as the C-3 mercapturic acid conjugate. 6 Experiments with isolated hepatocytes demonstrated that there was no formation of the mercapturic acid conjugate of H N E in these cells] Therefore, it seems to be very important to investigate the metabolism of H N E with the goal to find

MATERIALS AND METHODS

All chemicals o f reagent grade were purchased from Merck, Darmstadt, Germany. 4-Hydroxynonenal and radioactively labeled 4-hydroxynonenal ( [ 2 - ~ H ] HNE; 68.2 m C i / m m o l ) were obtained from Prof. Esterbauer, Graz, Austria. Male Wistar rats weighing 340 _+ 50 g were used. The rats were anesthesized with diethyl ether. An incision from the symphysis to the sternum opened the abdomen. The left kidney, the aorta abdominalis, and the left ureter were used for the experiments. Vena cava inferior was separated from the aorta abdominalis, the vena renalis sinistra from the arteria renalis sinistra. Other minor vessels were thermocoagulated. Catheters were placed ( A ) via the aorta abdominalis into the arteria renalis sinistra to perfuse the kidney, ( B ) via the vena cava inferior into the vena renalis sinistra to collect the effluent, and ( C ) into the left ureter to measure the urine outflow of the kidney. First the kidney was perfused with a K r e b s - H e n s e l e i t buffer supple-

Address correspondence to: Tilman Grune, Humboldt-University Berlin, Medical Faculty--Charit6, Clinics of Physical Therapy and Rehabilitation, Schumannstrafle 20-21, D-10098 Berlin, Germany. 685

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mented with 5 mM glucose, gased with 95% 02/5% CO2 at nearly 37°C for several minutes. The kidney was continuously infused intraarterially by use of a renal blood flow (RBF) of 3.5 ___ 0.3 ml/min. The urinary volume was about 20 #l/min without any pathologic excretion of glucose in the urine. The second solution was a Krebs-Henseleit buffer with a total concentration of 100 #M HNE and 5 mM glucose. In the case of tracerkinetic experiments we used radioactive labeled HNE with a final specific radioactivity of 40 #Ci/mmol. During the first 5 min of the experiment, every 30 s the effluent was collected from the vena renalis sinistra. The urinary volume we measured was 19.75 _ 2.75 #l/min. The wet weight of the kidneys were 1.68 + 0.17 g. To the perfusate was added an equal volume of acetonitrile/acetic acid (96:4; v / v ) . After centrifugation aliquots of the supernatant were eluted on TLC plates. To identify the HNE metabolites we used the method described by Grune et al.7 To determine the mercapturic acid we used an eluent of butanol/acetic acid/water (4:1:1; v / v / v ) . The separations on the TLC plates were measured by using of an automatic TLC linear analyser (Fa. Berthold, Wildbach, Germany). The extracts with acetonitrile/acetic acid were also used to determine HNE by an HPLCmethod. 7

RESULTS

By infusion of a 100 ~M HNE solution during the whole experiment via the arteria renalis sinistra we investigated the efflux rate of 4-hydroxynonenal in a perfused rat kidney (Table 1 ). During the first 2 min of perfusion the collected effluent from the vena renalis sinistra showed a rapid degradation rate of the exogenously added HNE; near 190 nmoles/g w.w./min. But already 5 min after perfusion the degradation of HNE approaches a steady state of about 160 nmoles/g w.w./ min (Table 1 ). So the HNE efflux rate of the venous effluent shows that during the first 2 min about 90% of the perfused HNE are metabolized in the kidney.

After 5 min the consumption rate of HNE reaches a steady state of about 75% (see HNE concentration difference; Table 1 ). The HNE excretion rate into urine is relatively constant, about 0.13 ___0.04 nmoles/ g w.w./min (Table 1 ). However, this is only 0.1% of the total HNE consumption rate. Simultaneously we observed a continuous increase of the HNE-mercapturic acid formation in the venous effluent and in urine (Fig. 1 ). During the first 2 min it reached a rate of about 4 nmoles/g w.w./min. (Table 2). After l0 min this value increased to a production rate of about 9 nmoles/g w.w./min. This means that about 6% of the total HNE consumption were used for the formation of mercapturic acid. The mercapturic acid rate of excretion into urine is about 0.1 nmoles/ g w.w./min (Table 2). So only about 2% of the total mercapturic acid production rate were found in the urine, whereas 98% were found in the venous effluent of the rat kidney. DISCUSSION

HNE-degradation in perfused rat kidney Experiments with addition of 4-hydroxyhexenal (HHE), another hydroxyalkenal, into the portal vein of rats have shown that the aldehyde did not accumulate to a large extent in the tissue of the rats. 6 These rats rapidly eliminated HHE mercapturic acid as a urinary metabolite (identified by tandem mass spectrometry). We were able to demonstrate a rapid degradation of the aldeyde 4-hydroxynonenal in the arterial perfused kidney of the rat. During the first 2 min the kidney was able to utilize about 150-200 nmoles/g w.w./min HNE. Isolated enterocytes and hepatocytes consumed about 25,000-28,000 nmoles/g w.w./min H N E . 14 So the ability of these isolated cells to degrade HNE during the first minutes seems to be more then 100-fold higher than in rat kidney. This difference may be due to a smaller metabolism of HNE in solid organs because of the diffusion distances. So an HNE consumption rate of about 60 nmoles/g w.w/min (after infusion

Table I. Balance of HNE Consumption in Perfused Rat Kidney Parameter

Dimension

HNE concentration of venous effluent HNE efflux rate of venous effluent HNE concentration difference (arterial influent-venous effluent) RBF-HNE consumption rate (arterial influent-venous effluent) HNE excretion into urine HNE excretion rate into urine Total HNE consumption rate

nmoles/ml nmolesTg w.w./min nmoles/ml nmoles/g w.w./min nmoles/ml nmoles/g w.w./min nmoles/g w.w./min

2 Min 8.42 17.53 91.58 190.79 11.24 0.13 190.9

± 0.12 ___ 0.24 _ 0.12 _+ 0.2 __. 3.38 ± 0.04 ± 0.2

5 Min 26.42 55.06 73.58 153.29 11.71 0.14 153.4

± 3.36 _+ 7.0 __. 3.36 _ 7.0 ± 3.49 ± 0.04 ± 7.0

l0 Min 20.56 42.81 79.44 165.5 11.81 0.14 165.6

± 3.05 _+ 6.37 +_ 3.05 _+ 6.35 ± 3.44 ± 0.04 ± 6.35

HNE concentration of arterial influent was 100/zM. Mean wet weight of the kidney was 1.68 ± 0.7 g. Perfusion flow was 2.1 +_ 0.3 ml/ g w.w./min. Influent consists of Krebs-Henseleit buffer, pH 7.4. Temperature 37°C. Mean ± SD (n = 5).

Mercapturic acid formation in kidney

12

E 10 Urin

0

E =

8

<

6

k--

4

Venous effluent

2 ./

0

2

4 6 8 Time (min)

10

Fig. 1. Concentration of HNE-mercapturic acid conjugate in venous effluent and urine in perfused rat kidney. HNE concentration of the arterial influent was 100/zM. Wet weight of the kidney was 1.68 ___ 0.7 g. Perfusion flow was 2.1 _ 0.3 m l / g w.w./min. Rate of urine production 11.8 __. 1.6 # f i g w.w./min. Mean -L-_SEM (n = 5).

of 10 #M HNE) was reported for rat hearts, ~5 which is on the same order as those measured in this study. The qualitative difference between rat liver and rat kidney is the ability of renal cells to form the mercapturic acid of HNE.

Formation of HNE-mercapturic acid conjugate in rat kidney Mercapturic acids are S-substituted N-acetyl-L-cysteine derivates. 9 The first step of mercapturic acid biosynthesis is the intracellular conjugation with glutathione; the resultant glutathione S-conjugates leave the cells and are degraded in the extracellular compartment. ~6Acetylation of cysteine S-conjugates took place as well in superficial proximal convoluted tubules as

687

in the straight part of superficial proximal tubules, the capacity of acetylation, and the capacity of secretion of the mercapturic acid being almost twice as high in the straight part as in the convoluted part. s So it seems that the liver is no longer to be regarded as the primary site of acetylation of cysteine S-conjugates. Enzymes as glutamyl transpeptidase, cysteinyl-glycine dipeptidase and cysteine conjugate N-acetyltransferase are necessary to catalyze mercapturic acid formation, m In comparison to rat liver the activity of this enzyme in rat kidney is much higher. ~- ~3 So this seems to be the reason that until now no mercapturic acid of HNE has been detected in rat liver cells. 14 We investigated the HNE-mercapturic acid production in the rat kidney during the first 10 min of perfusion. So the total HNE-mercapturic acid production rate was initially about 2% of the whole HNE consumption during the first 2 min. After 10 min we observed an HNE-mercapturic acid production rate of about 5 - 6 % of the total HNE consumption. Although the highest concentration was measured in the urine, the largest flow of HNE-mercapturic acid was found in the venous effluent in comparison with the urine. The direction of mercapturic acid efflux from the kidney mainly depends on the different effluent volumes. So the predominant amount of the produced mercapturic acid flows via the efferent vessels of the kidney back into the blood circulation. Nevertheless, experiments with another labeled hydroxyalkenal [3H](HHE) could demonstrate that after 24 h about 80% of the aldehyde, injected into the portal vein of rats, distributed in the urine as HHE-mercapturic acid. 6 From the presented results one can assume that due to the concentration gradient of HNE-mercapturic acid from the urine to the perfusate the mercapturic acid as a product of HNE metabolism after multiple recirculations through the kidney will be excreted into the urine. Therefore, we could demonstrate for the first time that HNE-mercapturic acid formation takes place in the kidney. Although the initial mercapturic acid output via the urine seemed to be a limited process, it could be important due to the stability of mercapturic acid derivates. REFERENCES

I. Esterbauer, H. Aldehydic products of lipid peroxidation. In: McBrien, D. C.; Slater, T. F., eds. Free radicals, lipid peroxidation and cancer. New York: Academic Press: 101-128; 1982.

Table 2. Balance of Mercaptudc Acid Formation in Perfused Rat Kidney Parameter RBF-mercapturic acid production rate Mercapturic acid rate of excretion into urine Total mercapturic acid production rate Mercapturic acid production as % of HNE consumption rate

Dimension

2 Min

5 Min

10 Min

nmoles/g w.w./min nmolesdg w.w./min nmoles/g w.w./min %

4.22 _+ 0.17 0.07 _+ 0.01 4.29 _+ 0.17 2.2

9.29 +_ 1.83 0.12 _+ 0.03 9.41 +_ 1.83 6.1

9,48 _+ 1.72 0.13 _+ 0.03 9,61 + 1.72 5.8

Conditions as in Table 1. Rate of urine production was 11.8 _+ 1.6/zl/g w.w./min. Mean _+ SD (n = 5).

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2. Esterbauer, H.; Schauer, R. J.; Zollner, H. Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes. Free Radic. BioL Med. 11:81-128; 1991. 3. Esterbauer, H.; Zollner, H. Methods of determination of aldehydic lipid peroxidation products. Free Radic. Biol. Med. 7(2):197-203; 1989. 4. Schauenstein, E.; Esterbauer, H.; Zollner, H.; Lagnado, J. R., eds. Aldehydes in biological systems. London: Pion Limited; 1977. 5. Siems, G. W.; Zollner, H.; Grune, T.; Esterbauer, H. Qualitative and quantitative determination of metabolites of the lipid peroxidation product 4-hydroxynonenal from hepatocytes, enterocytes and tumor cells. Fresenius J. Anal. Chem. 343:75-76; 1992. 6. Winter, C. K.; Segall, H. J.; Jones, A. D. Distribution of trans4-hydroxy-2-hexenal and tandem mass spectrometic detection of its urinary mercapturic acid in the rat. Drug Metab. Dispos. 15(5) :608-612; 1987. 7. Grune, T.; Siems, W.; Kowalewski, J.; Zoilner, H.; Esterbauer, H. Identification of metabolic pathways of the lipid peroxidation product 4-hydroxynonenal by enterocytes of rat small intestine. Biochem. Int. 25:963-971; 1991. 8. Kraus, P.; Kloft, H. D. The activity of glutathione-S-transferases in various organs of the rat. Enzyme 25:158-160; 1980. 9. Vermeulen, N. P. E. Analysis of mercapturic acids as a tool in biotransformation, biomonitoring and toxicological studies. TiPS 10:177-181; 1989. 10. Sausen, P. J.; Elfarra, A. A. Cysteine conjugate S-oxidase. J. Biol. Chem. 265( 11 ):6139-6145; 1990. 11. Green, R. M.; Elce, J. S. Acetylation of S-sustituted cysteines

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by a rat liver and kidney microsomal N-acetyltransferase. Biochem. J. 147:283-290; 1975. Anderson, M. E.; Meister, A. Inhibition of gamma-glutamyl transpeptidase and induction of glutathionuria by gamma-glutamyl amino acids. Proc. Natl. Acad. Sci. USA 83:5029-5032; 1986. Meister, A., Anderson, M. E. Glutathione. Annu. Rev. Biochem. 52:711-760; 1983. Siems, W. G.; Grune, T.; Zollner, H.; Esterbauer, H. Formation and metabolism of the lipid peroxidation product 4-hydroxynonenal in liver and small intestine. In: Poli, G.; Albano, E.; Dianzani, M. U., eds. Free radicals: From basis science to medicine. Basel/Switzerland: Birkhaeuser Vedag; 1993: 89-101. Grune, T.; Schrnheit, K.; Blasig, I.; Siems, W. Reduced 4hydroxynonenal degradation in hearts of spontaneously hypertensive rats during normoxia and postischemic reperfusion. Cell. Biochem. Func. 12:143-147; 1994. Heuner, A.; Dekant, W.; Schwegler, J. S.; Silbemagl, S. Localization and capacity of the last step of mercapturic acid biosynthesis and the reabsorption and acetylation of cysteine S-conjugate in the rat kidney. Eur. J. Physiol. 417:523-527; 1991. ABBREVIATIONS

HNE--4-hydroxy-2,3-trans-nonenal nenal ) HPLC--high-performance

(4-hydroxyno-

liquid chromatography

RBF--renal blood flow TLC--thin layer chromatography