ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 197, No. 1, October 1, pp. 277-284, 1979

Renal Cytochrome P-450’s-Electrophoretic and Electron Paramagnetic Resonance Studies H. J. ARMBRECHT, L. S. BIRNBAUM,” T. V. ZENSER, M. B. MATTAMMAL, AND B. B. DAVIS Geriatric Research,

Education and Clinical Center, Veterans’ Administration Medical Center, St. Louis, Missouri 63125, Departments ofBiochemistry and Medicine, St. Louis University, St. Louis, Missouri 63106 and *Masonic Medical Research Laboratory, Utica, New York, 13503

Received March 5, 1979; revised May 16, 1979 The cytochrome P-450’s of the microsomal mixed function oxidase systems from the rabbit renal cortex, outer medulla, inner medulla, and the liver were compared. Sodium dodecyl sulfate-(SDS) gel electrophoresis and electron paramagnetic resonance (EPR) studies detected cytochrome P-450 proteins in the liver, renal cortex, and outer medulla but not the inner medulla of normal animals. Two cytochrome P-450 peptides, which had molecular weights of 54,500 and 58,900 and which comigrated with known hepatic cytochrome P-450’s on SDS gels, were identified in the cortex and outer medulla. Treatment of animals with 3-methylcholanthrene (MC) enhanced the 54,500 and 58,900 peptides in the liver and cortex but produced little change in outer medulla. MC treatment induced faint cytochrome P-450 bands in the inner medulla. The EPR studies detected low spin heme iron absorption lines at g = 2.42, 2.26, and 1.92 in liver, cortex, and outer medulla from untreated animals. The amplitude of the low spin absorption lines was increased by ethanol, a reverse type I compound, and reduced by chloroform, a type I compound, in these tissues. MC treatment increased the amplitude of the heme absorption lines in these tissues, and it induced a barely detectable heme spectrum in the inner medulla. Differences in exogenous substrate binding between hepatic and renal microsomes from MC-treated animals were detected by EPR and optical difference spectroscopy. Acetone, 1-butanol, and 2-propanol gave evidence of binding to the hepatic cytochrome P-450’s but no evidence of binding to renal cortical microsomes. These results, along with previous enzymatic studies, suggest that the liver and each area of the kidney contain different substrate specificities and pathways for the metabolism of organic compounds.

The cytochrome P-450's function as the different pattern of mixed function oxidase terminal oxidase in the mixed function activity and enzyme induction by 3-methyloxidase system. This system oxidizes many cholanthrene (MC)’ (3). In the absence of compounds including carcinogens, fatty induction, cytochrome .P-450 could be deacids, and xenobiotics. The cytochrome P- tected spectroscopically only in the cortex. 450 system of the liver has been extensively The cortical and outer medullary microstudied, but the mixed function oxidases are somes from rabbits treated with MC exalso present in other tissues such as the hibited aryl hydrocarbon hydroxylase, amino kidney (1). The kidney is involved in the pyrene demethylase, and lauric acid hyconcentration and excretion of many drugs, droxylase activity. The inner medulla and the drug metabolizing capacity of the contained only the lauric acid hydroxylase kidney via the mixed function oxidase or activity, which was not inducable by MC other systems is therefore of great interest. and partially inhibited by carbon monoxide The kidney is composed of three anatomand metyrapone (3). ically and physiologically separate tissues-the cortex, outer medulla, and inner ’ Abbreviations used: MC, 3-methylcholanthrene; medulla (2). Each area demonstrates a SDS, sodium dodecyl sulfate. 277

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ARMBRECHT

The purpose of the present study is to further characterize the cytochrome P-450's in each area of the kidney in an effort to learn more about their drug-metabolizing potential. The cytochrome P-450 proteins of hepatic and renal microsomes were compared using sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis to separate proteins on the basis of their molecular weight. The distribution of P-450 in the kidney in the presence and absence of treatment with MC was studied by electron paramagnetic resonance (EPR). Finally, substrate binding by renal and hepatic microsomes was compared using optical difference and EPR spectroscopy. MATERIALS

AND METHODS

Animals and treatment. New Zealand White male rabbits (60 days old) weighing 1.5-2.0 kg were purchased from Eldridge Laboratory Animals, Barnhart, Missouri, housed in a controlled environment, and allowed free access to food and water for 10 days before experimentation. Six to twelve rabbits were injected with either corn oil or 40 mg/kg 3-methycholanthrene in corn oil, intraperitoneally, once daily for 3 days. Preparation of microsomes. Rabbits were anesthetized with intravenous (iv) 20 mg/kg sodium thiopental. The kidneys and livers were quickly removed and placed in ice-cold 0.9% NaCl. Renal cortical, outer medullary, and inner medullary areas were separated by careful dissection. All subsequent steps were performed at 4°C. Tissue was minced, washed free of hemoglobin, and homogenized for 15 s with a Polytron homogenizer in 3 vol of 0.1 M phosphate buffer, pH 7.8, containing 20% glycerol and 10m4M dithiothreitol. The homogenate was centrifuged at 10,OOOg for 15 min and the subsequent supernatant at 100,OOOgfor 60 min. This pellet was layered with 1.15% KC1 and stored at -40°C. Under these storage conditions there were no detectable changes in either cytochrome P-450 spectra or mixed function oxidase enzymatic activities for up to 3 months (3). Before use, the microsomal pellets were resuspended in 1.15% KC1 by hand homogenization and recentrifuged for 30 min at 105,OOOg. Pellets were then resuspended in 0.1 M phosphate buffer, pH 7.8, with a PotterElvehjem Teflon glass homogenizer to give a final concentration of about 10 mg microsomal protein/ml. Aliquots of these suspensions were stored frozen at -40°C and they were stable for up to 2 weeks (3). Protein content was estimated by the method of Lowry using bovine serum albumin as a standard (4). Cytochrome P-450 content was measured from the carbon monoxide difference spectrum (19).

ET AL. Gel electropkoresis. Prior to electrophoresis the microsomes were washed with pyrophosphate as described by Birnbaum et al. (5). The samples were resuspended in 0.125 M Tris-HCI (pH 6.8)-20% glycerol and were divided into 100~~1 aliquots and stored frozen at -70°C until use. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) was performed using the procedures of Laemmli (6) with minor modifications. The polyacrylamide gels, which were either 0.75 or 3 mm thick and 10 cm long x 14 cm wide (Hoefer Scientific Co., SE 500), were preelectrophoresed for at least 1 h at 30 mA/gel. After heat denaturation of the sample in the presence of 2% SDS and 5% P-mercaptoethanol, the samples were applied and run at 20°C. The current was maintained at 15 mA/gel through the 3% stacking gel and raised to 30 m&gel during electrophoresis through the 7.5% separating gel. Electrophoresis was stopped when the bromphenol blue tracking dye had migrated to 1 cm from the bottom of the gel. Proteins were stained with Coomassie blue (7). The peroxidase activity of heme-containing peptides was detected by 3,3’,5,5’-tetramethylbenzidine (8). For this procedure the SDS concentration of the sample was dropped from 2.0 to O.l%, no reducing agent was added, and the denaturation step was eliminated. The gels were run at 5°C in the dark and the stain allowed to develop for at least 48 h. Protein standards were treated the same as the microsomal samples and were coelectrophoresed in order to determine the apparent molecular weight of the sample peptides (9). The standards used and their molecular weights were: phosphorylase a (94,000), bovine serum albumin (68,000), pyruvate kinase (57,000), egg albumin (43,000) aldolase (40,000), and lactic dehydrogenase (36,000). Eleetronparamagnetic resonance spectroscopy. The microsomal suspension was thawed, rehomogenized, and 0.4 ml pipeted into an EPR quartz sample tube (Varian Associates, Palo Alto, Calif.). The sample was frozen at -25°C and stored at this temperature until examination. EPR measurements were made within 2 h using a Varian E109E EPR spectrometer with lOO-kHz field modulation and operating at X-band frequency. The same experimental results were obtained when samples were frozen in liquid nitrogen and examined immediately. Samples were examined at 110°K using a Varian variable temperature accessory to maintain constant temperature. Microwave power was 3 mW, klystron frequency was 9100 MHz, and modulation amplitude was 40 G for all samples. To test the effect of different compounds on the microsomal EPR spectrum, the test compound was added directly to the microsomal suspension at room temperature. The sample was then vortexed and frozen for EPR study. The compounds butanol, acetone, 2propanol, ethanol, and aniline were added as liquids at

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a concentration of 2.5%. This concentration has been shown to produce maximal EPR spectral changes in rat liver microsomes (10). Laurie acid was added as the sodium salt in water to give a final concentration of 0.4 InM. Optical difference spectroscopy. Optical difference spectra were recorded at room temperature using a dual beam Beckman Acta VI spectrophotometer with scale expander. The microsomal suspension was thawed and placed in both sample and reference cuvettes. The baseline was recorded and the test compound added to the sample cuvette. The cuvettes were allowed to stand for 1 min, and the difference spectrum was recorded. RESULTS

The multiple forms of cytochrome P-450 present in the kidney were studied using SDS-gel electrophoresis. Figure 1 is an electrophoretogram of rabbit and mouse microsomal proteins run in 2% SDS and stained with Coomassie blue. Protein standards were run in the outer wells (X, Y). Hepatic microsomes from 3-month-old, male C57BLKJ mice were included for comparison (M). Microsomes isolated from the rabbit liver (A, B), renal cortex (C, D), outer medulla (E, F) and inner medulla (G, H) were run in the center wells. Samples A, C, E, and G were from control rabbits while samples B, D, F, and H were from rabbits treated with MC. Figure 2 is a dia-

AND EPR STUDIES

279

_*_L1,XYiiABCOEFGH-MYX FIG. 1. Electrophoretogram of rabbit microsomal proteins. Microsomal samples containing 30 pg protein in 2% SDS and mercaphoethanol were heated and applied to 3-mm-thick 7.5% polyacrylamide slab gels. After electrophoresis, proteins were stained with Coomassie blue. KEY: X, protein standards: phosphorylase A, pyruvate kinase, and lactic acid dehydrogenase; Y, Protein standards: bovine serum albumin, egg albumin, aldolase; M, mouse hepatic microsomes; A and B, rabbit hepatic microsomes; C and D, rabbit cortical microsomes; E and F, rabbit outer medulla microsomes; G and H, rabbit inner medullar microsomes. Samples A, C, E, and G were from control rabbits. Samples B, D, F, and H were from rabbits treated with 3-methylcholanthrene.

grammatic representation of results obtained from four electrophoretograms of the type shown in Fig. 1. The same samples were also run under milder conditions and assayed for peroxidase activity (see Mate-

ABCDEFGH FIG. 2. Diagrammatic representation of rabbit hepatic and renal microsomal proteins in the 44,000 to 66,000 molecular weight region. The rabbit microsomal fractions A-H are as described in Fig. 1 and the apparent molecular weights of the protein bands are as in Table I. The asterisk indicates peroxidase activity as detected by staining with 3,3’,5,5’-tetramethylbenzidine.

ARMBRECHT TABLE MOLECULAR

Band No. 1 2 3 4 5 6

I

WEIGHTS OF MICROSOMAL PROTEIN BANDS

Apparent molecular weight” 62,600 58,900 54,500 49,800 48,200 46,700

+ k 2 2 f k

200 500 500 300 200 200

Previous nomenclature* LM7 LM4 LM2 LM 1 -

a Mean 2 standard deviation of three determinations. * Ref. (11).

ET AL.

bands 1,2,3, and 6 in the inner medulla, but peroxidase activity was not detected in any of these bands. To study the cytochrome P-450 heme iron, microsomes from rabbit liver and kidney were examined by EPR. Figure 3 shows an EPR spectrum taken at X-band frequency at 110°K of cortical microsomes from MC-treated animals. The signals visible at g = 2.42, 2.26, and 1.92 are due to the cytochrome P-450 heme iron in the low spin form (12). The high spin form of the heme iron is not visible at this temperature (12). The addition of ethanol, a reverse type I compound, to the microsomal preparation caused an increase in the signal amplitude at g = 2.42, 2.26, and 1.92. The presence of chloroform, a type I compound, decreased the amplitudes of the same signals (Figure 3). The rest of the EPR microsomal spectrum was relatively unaffected by these additions. Since the line width of the g = 2.26 absorption line did not vary with tissue or the addition of substrate, the amplitude of this absorption line was used as a measure of low spin heme iron content of the microsomes. Table II shows the amplitude of the g = 2.26 EPR absorption line divided by the microsomal protein

rials and Methods). The presence of peroxidase activity in a region of the gel is indicated by an asterisk. Table I lists the molecular weights of the most prominent bands and the corresponding band nomenclature used by Haugen et al. (11). In the rabbit hepatic microsomal fraction there were at least six peptides with molecular weights between 46,000 and 63,000 which were detectable under these electrophoretic conditions. Only bands 2 and 3 had peroxidase activity. After treatment with MC, bands 2, 3, and 4 were induced and demonstrated peroxidase activity. Therefore, these peptides, with molecular weights of 58,900, 54,500, and 49,800 are probably cytochrome P-450 molecules. Peptides 1 and 5 were induced by MC, but no heme component was detected. Peptide 6 was not induced by MC, showed no peroxidase activity, and is probably not a cytochrome P-450 peptide. CPntrPl C~,orPfPrm Microsomes prepared from the renal cor----Ethmol tex and outer medulla showed prominent bands that corn&rated with bands 2, 3, and 6 of the liver. In the renal cortex, peptides 2 and 3 were induced by MC and showed 1.82 I’ 2.42 2.28 peroxidase activity. Peptide 6 did not apFIG. 3. Electron paramagnetic resonance first pear to be inducible in any area of the kidderivative absorption spectrum of cortical microsomes ney. In the outer medulla the major bands from 3-methylcholanthrene-treated animals. were 2, 3, and 6. Although bands 2 and 3 isolated Microsomes were examined at 110°K at X-band freexhibited peroxidase activity, these bands quency. The X axis is magnetic field with the g values did not appear to be induced by MC treatof the important absorption lines noted. The Y axis ment. Finally, the inner medullary microis the first derivative absorption in arbitrary units. somes from untreated animals showed only Chloroform and ethanol were added to control microband 6. MC-Treated animals showed faint some6 at 2.5% concentration.

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281

II

EPR SPECTRAL STUDIESOF HEPATICAND RENAL MICROSOMES P-450 content” (nmoI/mg protein)

EPR absorption ampIitudeb No added substrate

Chloroform

Ethanol

Liver Control MC”

1.00 1.90

8.49 12.5

3.61 9.70

9.46 21.9

Cortex Control MC

0.08 0.16

1.90 2.21

1.42 1.03

1.82 2.96

Outer medulla Control MC

N.D.d 0.01

0.61 0.88

0.41 0.52

0.89 1.14

Inner medulla Control MC

N.D. N.D.

N.D. Trace

-

Trace

o!Measured spectrophotometrically. b Numbers are peak-to-trough amplitude of g = 2.26 absorption line corrected for gain and divided by protein concentration. Substrate concentration was 2.5%. c 3-Methylcholanthrene-treated animals. d Not detected.

content for microsomes from different tissues. The P-450 content of the microsomal preparations as measured by optical difference spectroscopy is also included for comparison (Table II). In the microsomal preparations with no substrate added, the low spin iron content of the liver was greater than the cortex, which was in turn greater than outer medulla. MC treatment caused a modest increase in low spin iron in all microsomal preparations. A barely detectable EPR spectrum was seen in the inner medulla after MC treatment. In the liver microsomes, chloroform decreased the signal amplitude and ethanol increased it in both control and MC-treated animals. In the cortex, chloroform decreased the signal amplitude in both treated and untreated animals. Ethanol had little effect in the cortex from untreated animals, but it increased the signal amplitude in MC-treated animals. The EPR spectrum of outer medullary microsomes was decreased by chloroform and increased by ethanol in both types of animals. P-450 was detected in the liver and cortex using spectrophotometric methods, and the P-450 content of these tissues

roughly doubled in MC-treated animals. P450 was detected spectrophotometrically in the outer medulla only after MC pretreatment, and it was not detected at all in the inner medulla. The ability of renal cortex and hepatic microsomes to form complexes with various compounds was tested using optical difference and EPR spectroscopic methods. Microsomes from MC-treated animals were mixed with the test compound and the optical difference spectrum and the EPR spectrum recorded (Table III). In the liver butanol, acetone, 2-propanol, and ethanol all gave reverse type I optical spectra with a maximum at 420 nm and a minimum at about 390 nm. These compounds also increased the amplitude of the g = 2.26 EPR absorption line in liver. However, the cortical microsomes at the same protein concentration gave no evidence of forming complexes with butanol, acetone, and 2-propanol. These compounds elicited no optical difference spectra and produced little or no increase in the EPR signal. Aniline behaved as a type II compound, maximum at about 430 nm and minimum at 395 nm, in both

282

ARMBRECHT

cortex and liver, although two other type II compounds, dimethylaniline and n-octylamine, gave evidence of complex formation only in the liver (data not shown). Laurie acid gave a type I optical spectrum, maximum at 380 nm and minimum at 410 nm, and also caused a decrease in the EPR spectrum, characteristic of a type I compound, in both cortex and liver. The optical difference spectra of cortical and hepatic microsomes in the presence of ethanol and 2-propanol are shown in Fig. 4. In these studies the microsomal concentration was adjusted to give equal P-450 concentrations in both cortical and hepatic preparations. Ethanol gave the characteristic reverse type I spectrum with cortical and hepatic microsomes while 2-propanol produced no observable spectrum in the cortical microsomes. DISCUSSION

These studies of the renal microsomal cytochrome P-450 oxidases demonstrate TABLE

ET AL.

differences in the distribution and induction of the cytochrome P-450 mixed function oxidase system in each area of the kidney. They also demonstrate differences in substrate binding by the hepatic and cortical microsomes. This is consistent with a previous study which showed differences in microsomal enzyme activity between the liver and the three regions of the kidney (3). The gel electrophoresis and EPR studies indicated that the P-450 was present in the liver, cortex, and outer medulla of both control and MC-treated animals (Fig. 2 and Table III). There was no evidence of P450 in the inner medulla of control animals. After MC treatment some faint bands were detected on SDS-gels of inner medullary microsomes, and a barely detectable P-450 EPR spectrum was observed. In our laboratory both gel electrophoresis and EPR were more sensitive detectors of the presence of P-450 than spectral measurements, which did not detect P-450 in the control outer medulla (Table II). III

EFFECT OF EXOGENOUS COMPOUNDS ON OPTICAL AND EPR MICROSOMAL SPECTRA Compound”

Tissue*

Type of spectrum

Optical differenceC WD)

EPR spectrum amplituded (% of control)

I-Butanol

Liver Cortex

Rev I -

0.195 N.S.’

59 -11

Acetone

Liver Cortex

Rev I -

0.086 N.S.

85 11

2-Propanol

Liver Cortex

Rev I -

0.211 N.S.

108 8

Ethanol

Liver Cortex

Rev I Rev I

0.131 0.009

75 34

Aniline

Liver Cortex

II II

0.084 0.015

42 43

Laurie acid

Liver Cortex

I I

N.M.” N.M.

-30 -14

0 All compounds were added as liquids to a final concentration of 2.5%, except for lauric acid which was added in water to a final concentration of 0.4 mM. * Microsomes were prepared from 3-methylcholanthrene-treated animals. Protein concentration was 2.04 mg/ml for both tissues. c Measured at absorption maximum. d Measured at 9 = 2.26. e Abbreviations: N.S., no observed spectrum; N.M., not measured.

RENAL 0.02-

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P-450’s: ELECTROPHORETIC

AND EPR STUDIES

a

283

2-PROPANOL ETHANOL

-0.02

I 400 Wavelength

I 500

I 450 [nm]

-0.027

I 400

I 500

I 450

Wavelength

Inml

FIG. 4. Optical difference spectra of cortical and hepatic microsomes. Microsomes were suspended in 0.1 M phosphate buffer (pH 7.4) to give a final P-450 concentration of 0.4 nmol/ml for each microsomal preparation. Microsomal suspension was placed in both sample and reference cuvettes and the baseline recorded. Ethanol or 2propanol(2.5% final concentration) was added to sample cuvette, and after 1 min the difference spectrum was recorded.

The gel electrophoresis studies showed that the renal microsomes contain two cytochrome P-450 species (bands 2 and 3) which are identical to hepatic species on SDS-gel electrophoresis. Another hepatic P-450 (band 4) was not detected in any region of the kidney. These electrophoretic studies are in good agreement with results obtained with rabbit hepatic microsomes (11, 13-15) and on renal microsomes isolated from the whole kidney (1). In the latter renal microsome study, a band was detected which had a molecular weight of approximately 57,000 and was inducible by MC. This probably corresponds to the band 3 seen in this study. However, this whole kidney study did not note any induction of the 54,000 molecular weight component (band 2). In the present study, this component was found to be increased in the cortex but not in the outer medulla after MC treatment. The EPR studies of renal microsomal preparations detected absorption lines at g = 2.42, 2.26, and 1.92 due to the heme iron of the P-450 proteins (Fig. 3). At the temperature of these EPR studies, only the low spin form of the heme iron was observable, but it has been estimated that the low spin iron form accounts for twothirds of the P-450 heme iron in hepatic

microsomes from control rabbits (10). Induction by MC produced modest increases in the amplitude of the low spin P-450 iron spectrum observable by EPR in liver, cortex, and outer medulla (Table III). Induction by MC has been shown to increase the high spin form of the heme iron preferentially and raise the fraction of heme iron in the high spin form to one-half (10). Thus, MC caused a larger percentage increase in the total microsomal P-450 content, as measured by spectral methods, than in the low spin P-450 iron content with no substrate added, as detected by EPR (Table II). The studies of substrate binding by hepatic and cortical microsomes showed significant differences between these two tissues even at equal P-450 concentrations. EPR and optical difference spectroscopy demonstrated interaction of ethanol, aniline, and lauric acid with cortical and hepatic microsomes. However, in contrast to liver, no interaction of 1-butanol, acetone, or 2propanol with cortical microsomes was observed (Table III and Fig. 4). These results demonstrate a difference in specificity or affinity between hepatic and renal P-450 proteins. They may be related to the complete absence of certain P-450 proteins in the kidney, such as band 4. In summary, these results show that the

ARMBRECHT

284

renal cortex and outer medulla contain multiple cytochrome P-450 oxidases and that two of the P-450 peptides are very similar to those found in liver. On the other hand, the substrate binding studies and previously reported hydroxylase activities (3) show that there are significant differences between the P-450's of the kidney and liver and probably between cortex and outer medulla. Although the total P-450 content of the liver is greater than that of the kidney, the importance of renal oxidative pathways must be evaluated in terms of the kidney’s anatomical environment and physiological function. The cortex receives 20% of the cardiac output, but it is only 0.4% of the body weight. This may result in the exposure of this tissue to relatively high levels of toxic materials. Certain drugs with nephrotoxic potential reach high concentrations in the renal inner medulla (16, 17). In this regard, it is important that little or no cytochrome P-450 was detected in the inner medulla. It has recently been shown that the compound l,&diphenylisobenzofuran is metabolized in the inner medulla via cooxygenation with arachidonic acid by prostaglandin cyclooxygenase (18). In the cortex, metabolism of this compound was almost exclusively via the cytochrome P-450 mixed function oxidase pathway. Thus, each area of the kidney may contain separate pathways for the metabolism of organic compounds, with non-P-450 pathways being of great importance in the inner medulla. ACKNOWLEDGMENTS The authors gratefully acknowledge the excellent technical assistance of Mr. John Zongrone (Masonic Medical Research Laboratory) and the secretarial assistance of Mrs. Sandy Melliere (St. Louis VA Medical Center). This research was supported in part by the Veterans Administration. L. S. B. was the recipient of NRSA Postdoctoral Fellowship CA 05536.

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