| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Vol. 50, Issue 1, 107-142, March 1998
Departments of Pharmacology and Toxicology, Biochemistry, and Medicine, School of Medicine and Biomedical Sciences, State University of New York, Buffalo and V.A. Medical Center, New York
I. Introduction
A. History
B. Methodology
1. Clearance.
2. Sperber technique in chickens.
3. Isolated perfused kidney.
4. Tissue Preparations.
5. Molecular biology.
II. Renal Pathways for the Biotransformation of Drugs
A. Cytochrome P450 Dependent Mixed Function Oxidase System
1. Cytochrome P450 in the kidney.
2. Drugs that induce cytochrome P450 proteins.
3. Specific renal cytochrome P450 enzymes.
4. Nondrug factors that affect cytochrome P450 enzymes in the kidney that may modulate kidney drug metabolism.
B. N-Oxidation (Flavin-Containing Monooxygenases)
C. Alcohol Oxidation
D. Aldehyde Oxidation
E. Oxidative Deamination (Monoamine Oxidase)
F. Aldehyde and Ketone Reduction
1. Aldehyde reductase.
2. Ketone reductase.
3. Other.
G. Hydrolysis Mechanisms
1. Ester and amide hydrolysis/carboxylesterase and amidase.
2. Epoxide hydrolysis.
III. Phase II: Synthetic Conjugation Pathways
A. Glucuronidation
1. Isoforms.
2. Substrates and kinetics.
3. Induction of renal glucuronyl transferase.
4. Localization.
5. Relative contribution of renal glucuronidation.
B. Sulfation
C. Methylation
1. N-methylation.
2. O-methylation.
3. S-methylation.
D. Acetylation
E. Glutathione Conjugation
F. Mercapturic Acid Synthesis
G. Amino Acid Conjugation
1. Glycine conjugation.
2. Glutamine conjugation.
H. Cysteine Conjugate-Lyase
IV. Localization of Drug Metabolizing Enzymes in the Kidney
V. Effects of Renal Metabolism
Acknowledgments
References
 |
I. Introduction |
|---|
 Top NextReferences
|
|---|
A. History
The kidneys have important physiological functions including
maintenance of water and electrolyte balance, synthesis, metabolism and
secretion of hormones, and excretion of the waste products from
metabolism. In addition, the kidneys play a major role in the excretion
of drugs, hormones, and xenobiotics. Mechanisms involved in the
transport of drugs in the proximal tubule in the secretory direction
have been amply reviewed (Bessighir and Roch-Ramel, 1988
; Pritchard and
Miller, 1993). Reabsorptive transport for organic compounds,
particularly amino acids (Zelikovic and Chesney, 1989; Silbernagl,
1992) and choline (Acara and Rennick, 1973; Acara et al., 1979) also
have been studied. The concepts associated with pH dependence in the
nonionic passive back diffusion of drugs are well-described (Roch-Ramel
et al., 1992). However, the role of the kidney in the metabolism of
both endogenous and exogenous compounds has not received appropriate
attention.
Most of the current knowledge about drug metabolism is based on studies
in which the liver was the experimental organ. It is now clear that the
kidney actively metabolizes many drugs, hormones, and xenobiotics
(Anders, 1980; Bock et al., 1990). In some cases, certain
biotransformations occur at a faster rate in the kidney than in the
liver; e.g., glycination of benzoic acid (Poon and Pang, 1995). Bowsher
et al. (1983) found histamine N-methyltransferase activity to be higher
in concentration in rat renal tissue than in any other organ. Gamma
glutamyl transferase activity in mammalian tissues is at its highest in
the kidney (Goldbarg et al., 1960; see Section III.F.).
The heterogeneity of the kidney makes it important to define the
regional distribution of enzyme systems on a cellular and subcellular
level. The human kidney has two distinct regions: an outer cortical
region and the inner medullary region. The medulla is divided into
several pyramids, the base of which is at the corticomedullary junction
and the apex of which approaches the renal pelvis, forming a papilla.
This heterogeneity is caused by three successive excretory systems that
develop during embryonic development; and the latter two, the
mesonephros and metanephros, contribute to the formation of the kidney.
The ureteric bud, a specialized structure of the mesonephric duct,
gives rise to the collecting ducts, calyces, pelvis, and ureter. The
metanephros gives rise to the glomerulus, proximal, and distal tubules.
Whereas most studies have been performed either in whole kidney or in cortical tissue, biotransformations have also been identified in the
medullary region (Toback et al., 1977a; Lohr and Acara, 1990).
Information accumulated over the past 20 years demonstrates a large capacity for metabolism in the kidney, leading to activation or inactivation of numerous compounds and providing a major route for drug disposition. In addition, the metabolic products produced by the kidney may exert significant toxic effects. The pattern of blood flow through the kidney, the acidity of the urine, and the urinary concentrating mechanism provide an environment that facilitates the concentration of particular compounds in the medullary/papillary zone of the kidney, and sometimes even, their precipitation (e.g., uric acid) with resultant damage. Such reactions will be presented in a general way because the action of toxins on the kidney is beyond the scope of this review.
In this review, various methods will be described that have been used to study renal metabolism of drugs, xenobiotics, hormones, and endogenous compounds. The various types of metabolic reactions that occur in the kidney will be presented along with the compounds that occupy those particular routes. The contribution of the particular metabolic pathways to the direction of movement of metabolite, into blood or into urine, provides an interrelationship between transport and metabolism.
B. Methodology
Several different methods have been used to study the role of the kidney in the metabolism of drugs and xenobiotics. These vary from in vivo techniques, such as clearance and the Sperber chicken preparation, to in vitro studies of metabolism using organelles such as mitochondria and microsomes and molecular biology in which genes encoding specific enzymes of metabolism have been identified. Each technique has contributed different information regarding the way in which compounds are handled by the kidney.
1. Clearance.
Historically, the contribution of the kidney to
the elimination of a particular drug was measured as renal clearance
(Moller et al., 1928). The term "clearance" must be defined because
it is being used to describe an ever increasing number of functional equations. Renal clearance as described by Homer Smith (1956) is "the
volume of plasma required to supply the quantity X excreted in urine
each minute" or the volume of plasma completely cleared of that
substance in 1 minute time. However, these two definitions are not the
same because what is "cleared" may not necessarily appear in the
urine.
).
Clearance methods, developed to quantify renal function, were the first
methods used to study in vivo renal metabolism (Toretti and Weiner,
1976; Tucker, 1981). Metabolism may be detected during infusion of a
radiolabeled compound when the label in the urine is identified as
something other than the original compound (Quebbemann and Rennick,
1969; Acara and Rennick, 1972; Toback et al., 1977a,b).
Monitoring the fate of radiolabeled compounds after infusion into the
renal artery has provided some insight into the kidney's capacity to
metabolize. Diamond and Quebbemann (1981) demonstrated clearance of
radiolabeled metabolite during steady-state infusion of radiolabeled
metabolite (true clearance) and measurement of the clearance of
unlabeled metabolite during steady-state infusion of radiolabeled
metabolite and unlabeled precursor (apparent clearance). The difference
between the apparent clearance and true clearance is the renal
contribution to formation of urinary metabolite.
Tremaine, Diamond and Quebbemann (1984, 1985) developed another
radioisotope technique, termed the specific activity difference ratio
(SADRb) technique. This involves the infusion of
radiolabeled precursor as well as the infusion of unlabeled metabolites
if not present endogenously. The technique permits the quantification
of renal formation and excretion of several metabolites if they are
excreted in measurable amounts. Specific activity ratio technique is a standard isotope dilution method. This is performed by a constant infusion of radiolabeled metabolite along with a constant infusion of
unlabeled precursor. The specific radioactivities of metabolites in
plasma and urine are subsequently measured. The ratio of the specific
activity of metabolite in urine to that in plasma when subtracted from
one indicates the fraction of urinary metabolite formed in the kidney
and excreted; thus, determining the in vivo renal contribution to
formation of a compound.
2. Sperber technique in chickens.
Birds have a renal portal
circulation, accessible through a leg vein, which permits the
administration of substances to the ipsilateral kidney. Sperber (1946)
demonstrated that when substrates transported by organic excretory
transport carriers were infused into the leg vein, they were excreted
in excess in the urine from the ipsilateral kidney. Substances entering
the general circulation were excreted by both kidneys equally. Because
the chicken has no bladder, ureters from either side may be isolated
for urine collection (Campbell, 1960). Thus, when contralateral
excretion is subtracted from ipsilateral excretion and blood flow
through this system is considered, a value is obtained that describes the efficiency by which the compound is removed from the blood by the
kidney. In measuring the excretion of the metabolites of an infused
substance, those excreted in excess by the infused kidney represent the
results of intrarenal metabolism.
).
Whereas urinary metabolites clearly reflect active biochemical pathways
qualitatively, conversion rates are difficult to determine. The
technique does not account for the reabsorptive route of the metabolites but only for that route that ends with the appearance of
the metabolite in the urine.
3. Isolated perfused kidney.
The isolated perfused kidney
preparation permits the measurement of excretion, reabsorption, and
renal metabolism. (Nishiitsutsuji-Uwo, 1967; Bowman, 1978; Nizet,
1978). Because the kidney is removed from the animal, the influence of
other organs and tissues is not present. Renal clearance and urinary
clearance may be determined for a given compound. As previously
indicated, a large renal clearance associated with a low urinary
clearance suggests a metabolic component. Kidneys may be perfused for
up to 2 h and samples of perfusate and urine collected for
appropriate analyses.
). The conversion of enalapril, an inhibitor of angiotensin
converting enzyme, to its metabolite, enalaprilat, and the transport of
drug and metabolite across the basolateral and luminal membranes using
constant flow single pass and recirculating isolated perfused rat
kidney preparations provided an intrinsic clearance for renal drug
metabolism as well as identifying membrane transport steps (de Lannoy
et al., 1990).
Glutathione content of isolated perfused kidney is consistently lower
than that observed in vivo (Ross et al., 1980) and the maximum rates of
drug metabolism may not be observed. Functional shortcomings include
low glomerular filtration rate relative to perfusate flow and excessive
delivery of filtrate to the distal tubule with decreased concentrating
ability (Maack, 1980, 1986).
4. Tissue Preparations.
a. KIDNEY SLICES. Kidney slices have been used for the study of renal uptake and metabolism for decades (Forster, 1948).
Kidneys are removed quickly after onset of anesthesia and kept chilled
during slicing and until the start of incubation. Slices, thin enough
to permit oxygen to reach all of the tissue, may be incubated for up to 2 h. At the termination of the experiment, slices are blotted on
filter paper and weighed. Analyses of media and tissues for substrate
and metabolites can reveal accumulation of substrate against a
concentration gradient; as well as metabolic routes and intracellular
and extracellular amounts of substrate and metabolites. Phospholipid metabolism from (14C)-choline was
studied in mouse kidney slices during renal compensatory growth by
Toback et al. (1974). Inner cortical slices were found to have enhanced synthesis of choline containing lipids during kidney regeneration (Toback et al., 1977b). Relative specific activities of enzymes have
been studied in dissected cortex, outer medulla, and inner medulla.
Choline dehydrogenase activity was shown to increase in cortex and not
change in the inner medulla during hypernatremia (Grossman and Hebert,
1989). Addition of dimethylaminoethanol, an analogue of choline and an
inhibitor of choline oxidase, to isolated perfused kidneys as well as
dissected tissue regions decreased betaine production from
14C-choline (Lohr and Acara, 1990). Toback et al.
(1977b) studied the phosphorylation of choline into choline
phosphoglycerides in different kidney regions.However, information regarding the direction of movement, i.e.,
reabsorptive versus excretory, is not obtained using kidney slices.
Some controversy exists regarding whether or not tubules are collapsed.
Uniformity of slices is important because substrate and oxygen should
reach all cells. Sometimes the innermost cells of the slice are not
exposed to the same concentrations as the surface cells (Foulkes,
1996). The reproducibility and viability of tissue slices were greatly
improved by the more recent introduction of automated procedures to
produce "precision cut" kidney slices (Smith et al., 1985).
b. ISOLATED TUBULES.
Tubule segments may be obtained by
several methods. Microdissection of large numbers of tubule segments
may be performed after perfusion of the kidney with a collagenase
containing buffer and incubation with collagenase. The tubule segments
can then be used for metabolic studies in which the tubules are
typically incubated with a radiolabeled compound. After incubation, the
tubules can be separated from the medium either by centrifugation or
rapid filtration. Specific nephron segments can be obtained by this method but the tissue yield is low. Development of microassays has
permitted the study of enzyme activities in these segments (Endou,
1983a; Schlondorff, 1986). Relatively homogeneous proximal tubule suspensions can be obtained in
greater quantity by density gradient centrifugation, and, with
oxygenation, remain viable for approximately 2 h. Obtaining suspensions
of tubule segments other than proximal is more difficult because they
constitute lower percentages of the kidney mass.
c. CELL CULTURE.
Cultured cells have become a more
popular tool for metabolic studies. In addition to primary cultures,
there are now many continuous kidney cell lines (e.g., MDCK, LLC-PK1,
OK, A6, JTC-12, BSC1) available for study (Handler and Burg, 1992). The LLC-PK1, OK, and JTC-12 cell lines are of proximal tubular origin.
Of these the LLC-PK1 cells, derived from the Hampshire pig, have been
best characterized. They have characteristics such as Na-dependent
glucose uptake, Na-dependent phosphate uptake, and Na-dependent amino
acid uptake, and high activities of several brush border enzymes. The
MDCK cell line is most characteristic of distal tubule, and the A6 cell
line resembles the collecting duct.Primary cultures of proximal renal tubules may also be grown for use in
studies of renal drug metabolism. This may be accomplished with tubules
obtained by microdissection or by macro separation techniques using
rabbit, rat, dog or human kidney. Metabolic studies of cells in culture are generally performed on cells
which have reached confluence. The cells are rinsed with a buffer free
of the metabolic substrate to be studied and then incubated for an
appropriate time with substrate generally radiolabeled. The experiment
is ended by aspirating the medium, rinsing the cells, and disrupting
the cells with NaOH, scraping, or sonication. The cell fractions can
then be further analyzed. Fry and colleagues (1978) found that liver and kidney cells demonstrate
similar conjugated metabolite patterns. Jones et al. (1979) found that
the specific activities for formation of glucuronide and sulfate
derivatives in the kidney were approximately 5% of those for liver
cells, although formation of sulfhydryl derivatives was proportionately
higher in kidney cells with paracetamol as substrate.
d. SUBCELLULAR FRACTIONS.
Small (100 nm diameter)
closed vesicles which form when cells are disrupted by homogenization
and sediment at 100,000 × g are identified as
microsomes. They consist primarily of membranes of endoplasmic
reticulum and have proven to be a valuable tool in studying synthetic
and metabolic functions of the cell. Microsomes from kidney cortex have
been used to study drug metabolism. After homogenization and
centrifugation at low speed, the resulting supernatant is centrifuged
at high speed for 60 min. The microsomal fraction is the pellet that is
resuspended for use in drug metabolism studies. Substrates are added to
the microsomal incubation medium to study rates of conversion to
metabolites. Animals may be pretreated with various drugs and
xenobiotics, and the effects of these compounds on microsomal enzyme
activity determined in vitro. In addition to studying metabolism by use
of microsomes, the cytosolic fraction (i.e., the supernatant from the
microsomal fractionation) of cells can be isolated and used alone or in
conjunction with the microsomal fraction.
5. Molecular biology. The increased use of molecular biology techniques in the past 15 years has heavily impacted on how we classify and identify proteins. When available, IUBMB enzyme numbers are given for the enzymes discussed in this review. However, many enzyme activities have only been studied in membrane fractions or using nucleic acid probes, and IUBMB numbers are not available. A parallel system based on genetic information has arisen. The advances in molecular biology have allowed isolated proteins to be cloned and sequenced. In many cases, especially caused by the ease of analysis and amplification of small amounts of nucleic acid materials, it is easier to study the genetic material rather than the protein. Genes are isolated in different organs and species on the basis of homology to known genes whose enzymatic activities have been studied at the protein level. The transcriptional regulation of these genes can be studied and hypothetical protein sequences deduced. The explosion of molecular biology data has led to the same gene being isolated during studies of different phenotypes. To put some order into the system, there are organizations devoted to specific organisms: HUGO Gene Nomenclature Committee for human genes (accessed through http://gdb.org/gdb) and the Mouse Gene Nomenclature Committee for mouse genes (accessed through the Jackson Laboratory web site: http://www.informatics.jax.org). In addition, specific gene families have their own nomenclature organizations, such as the P450 Nomenclature Committee identified in the P450 section.
Molecular biology studies can never completely supersede the biochemical studies of isolated enzymes. The study of a gene isolated in the kidney as a homologue of a well-studied liver enzyme is extremely useful. However, it is not guaranteed that the enzyme encoded by that gene, even if it shows the same transcriptional regulation, has the same function in the kidney. The researcher is advised to check the gene banks for homologous genes, but to realize also that the strictly molecular biology studies do not indicate that the protein encoded by that gene has the implied function under physiological conditions in the organ of interest. With these caveats in mind, the authors of this review have attempted to use the most recent molecular biology designations of specific genes isolated in the kidney. Enzymatic activities of genes isolated in the kidney as homologues of liver genes are mentioned in the review, but they may be described more generally if their specific activity in the kidney has not been demonstrated.| |
II. Renal Pathways for the Biotransformation of Drugs |
|---|
|
TopPreviousNextReferences
|
|---|
The authors have organized the description of the specific pathways by first presenting an overview of the general reaction, then a discussion of the specific enzyme involved, and finally the role of this reaction in kidney drug metabolism.
A. Cytochrome P450 Dependent Mixed Function Oxidase System
The most well-studied drug metabolism reaction in the kidney (as well as in the liver) is the cytochrome P450 (CYP) mixed function oxidase (MFO) reaction, which catalyzes the hydroxylation of a diverse group of drugs as shown below:
|
(1) |
The localization of P450 MFO in the kidney has been known since the
early 1960s, and the early work in this area has been reviewed (Anders
et al., 1980i). Except for fatty acid hydroxylation (Oliw, 1994), which
is found to have greater activity in the kidney than in the liver, it
is clear that the renal metabolic contribution of the MFO system is
much less than that of the liver.
There are multiple components of the MFO, and different proteins are
described below. Cytochrome P450 is a heme containing enzyme that
serves as the terminal oxidase component of the electron transfer
system present in the endoplasmic reticulum. The usual second component
of the system is the flavoprotein nicotinamide adenine dinucleotide
phosphate (NADPH) dependent cytochrome P450 reductase that transfers
reducing equivalents from NADPH to cytochrome P450. In addition,
phospholipid is required for MFO activity. The lipid
phosphatidylcholine appears to be required for the coupling of the
cytochrome P450 to NADPH-dependent cytochrome P450 reductase. In
addition, cytochrome b5 and cytochrome
b5 reductase can also donate an electron
from nicotinamide adenine dinucleotide, reduced (NADH) to
cytochrome P450 (Guengerich, 1993).
In contrast to the wide range of cytochrome P450 proteins present in
the cell, there appears to be a limited number of NADPH cytochrome P450
reductases. This enzyme contains 1 mole of flavin adenine dinucleotide
(FAD) and 1 mole of flavin mononucleotide per mole of flavoprotein and
is found in close association with cytochrome P450 in the endoplasmic
reticulum. NADPH cytochrome P450 oxidoreductase
(NADPH:ferricytochromoxidoreductase, E.C. 1.6.2.4) has a Mr of 78.275 kDa and is found in close association with cytochrome P450 in the
endoplasmic reticulum (O'Leary et al., 1996).
The enzyme activity of NADPH-cytochrome c reductase has been determined
to be 34 and 77 nmol/mg protein/min in rabbit and mouse kidney,
respectively (Litterst et al., 1975). Human kidney was found to have
10.9 nmol reduced product/mg protein/min (Jakobsson et al., 1978).
These values range from 15 to 70% of that concentration found in the
liver of the respective species.
Microsomal NADPH cytochrome c reductase activity was found in
decreasing amounts from cortex to inner medulla (Zenser et al., 1978;
Endou, 1983a,b). When isolated tubules were examined, the activity was
greatest in the proximal tubule, although detectable in the glomerulus,
distal tubule, and collecting tubule (Endou, 1983a,b). Induction by
xylene and its isomers was observed in rat kidney by Toftgard and
Nilsen (1982).
1. Cytochrome P450 in the kidney.
The various cytochrome P450
proteins not only display different substrate activities, but they also
display different regional and stereo selectivities so that the fate of
a chemical in a tissue will be determined not only by the total
cytochrome P450 concentration but also by the form(s) present in that
tissue. There are a variety of oxidative reactions catalyzed by the
cytochrome P450 system. These include aliphatic hydroxylations,
aromatic oxidation, alkene epoxidation, nitrogen dealkylation,
oxidative deamination, oxygen dealkylation, nitrogen oxidation,
oxidative desulfurization, oxidative dehalogenation and oxidative
denitrification (Wislocki et al., 1980). Not all isoforms of cytochrome
P450 have been identified in the kidney.
, will be given
whenever possible. The reader is referred to this review for
information on enzyme functions and species location of the P450
families and subfamilies. The italicized root symbol (CYP for human)
will be followed by an Arabic number denoting the family, a letter
representing the subfamily, and an Arabic number representing the gene
within the subfamily. The gene products of the CYP genes, messenger
ribonucleic acid (mRNA), and proteins will be referred to using all
capital letters. Information from Nelson et al. (1993) was used to
provide the new name replacing the old common names for P450 enzymes.
However, much of the earlier work was done using enzyme assays or
immunological techniques. Because it is not possible to verify that the
P450 enzymes studied biochemically actually represent the enzymes
encoded by the isolated genes common names will appear in this review in addition to the current nomenclature of Nelson et al. (1996).
The following protein isoforms of the CYP genes have been found in the
kidney from studies of enzymes. CYP1A1 and CYP1A2 enzyme activities
were found (Ioannides and Parke, 1990). CYP4A1 (P450 LA
) was present in normal kidneys (Hardwick
et al, 1987.) Untreated kidney was shown to have low CYP1A enzyme
activity, which this was induced by polycyclic aromatic hydrocarbons,
-napthoflavone, and 2-acetylaminoflourine (Ioannides and Parke,
1990).
Molecular biology techniques have helped to find cytochrome P450 genes
in the kidney. Members of the CYP2 and
CYP4 gene families were among the earliest genes found
in the kidney (Gonzalez, 1989). CYP4A11 deoxyribonucleic
acid (DNA) was cloned from a human kidney complementary
deoxyribonucleic acid (cDNA) library independently by Palmer et al.
(1993) and Imaoka et al. (1993). Messenger ribonucleic acid (mRNA)
related to CYP4A11 was found in liver and kidney (with the highest abundance in the kidney) using Northern blot analysis and
ribonuclease protection assays (Palmer, 1993). The CYP4A5, A6, and A7 genes have been found in rabbit
kidney by Johnson et al. (1990), whereas the rat CYP4A2
was isolated by Kimura et al. (1989a,b). CYP4A3 is also
present in the kidney, and CYP3A4 is expressed in 80%
of human kidneys (Parkinson, 1996).
a. LOCALIZATION.
The localization of microsomal
cytochrome P450 in various regions of the kidney has been examined
(table 1). P450 is found in highest
concentration in the cortex, with smaller amounts in the outer medulla
and more in the inner medulla in rabbits (Zenser et al., 1978;
Armbrecht et al., 1979; Mohandas et al., 1981b). Using an isolated
tubule preparation, Endou (1983b) investigated the distribution of P450
in rat and rabbit kidney. P450 was found to be localized to the
proximal tubule, with the highest concentration in the S2 and S3
segments. At the same time using proximal and distal tubule suspensions
and spectrophotometric means cytochrome P450 was found to be present in
largest quantities in the proximal tubules (Cojocel et al., 1983).
Other investigators subsequently confirmed the localization of
cytochrome P450 to the proximal tubule (Foster et al., 1986).
Immunochemical analysis has shown that P450IIE1 is located primarily in
proximal tubules with some staining in distal tubules (Hu et al.,
1993).
|
).In rabbits, a five-fold increase in cortical renal cytochrome P450
content was seen after exposure to 3-MC, whereas the outer medullary
activity was only able to be identified after 3-MC treatment (Zenser et
al., 1978; Armbrecht et al., 1979). Likewise, 3-MC caused induction of
renal microsomal P450 in rats (Endou, 1983b; Funae et al., 1985; Wilson
et al., 1990). A 3-MC-inducible form of cytochrome P448 (CYP4A7), which
catalyzed the hydroxylation of BP, has been isolated from the cortex of
rabbit kidneys (Kusunose et al., 1989) and cytochrome P1450
(CYP1A1), but not Ps450, was induced by 3-MC (Tuteja et
al., 1985). In vivo hybridization of CYP4A8 to rat kidney sections
showed this DNA to be present in the outer stripe of the outer medulla
or proximal tubule (Stromstedt et al., 1990).
2. Drugs that induce cytochrome P450 proteins. The following sections describe systems in which P450 protein have been reported to be induced by various drugs. The researcher is directed to these references for more specific information.
a. ANALGESICS. Oxidation of acetaminophen by P450 in the kidney was reported by Mohandas et al. (1981a,b).
Immunohistochemical studies have been done that colocalized
acetaminophen and CYP2E1 protein in damaged kidney tissue after
administration of acetaminophen to mice (Hart et al., 1995).
Acetaminophen, a commonly used analgesic, induced nephrotoxicity in
males or females treated with testosterone, which corresponded with the
induction of renal CYP2E1 protein in mice as shown by Western blot
analysis and enzymatic activity (Hoivik et al., 1995). These results
are consistent with metabolism of acetaminophen by CYP2E1 protein
causing toxicity. Note that nephrotoxicity has also been hypothesized
to be related to prostaglandin synthesis activity in the kidney (cf
section in lipid metabolism).
b. ANESTHETICS.
In rats, ether anesthesia caused
lowering of total CYP protein, CYP1A, and CYP2B activities, whereas
increases in CYP2E1 activity were found. These changes were more
pronounced in fasted rats (Liu et al., 1993).
c. BARBITURATES.
Isozymes of renal P450 have been found
to be inducible by polycyclic aromatic compounds and barbiturates.
Phenobarbital (PB) induced CYP enzymes in cortical microsomes from
rabbit but not from rat (Kuo et al., 1982). Specifically CYP1A1 and A2
enzymes were induced by PB (Ioannides and Parke, 1990). Multiple forms of CYP2B and CYP4B1 were shown to be induced in the kidney by PB using
molecular biology studies (Ryan et al., 1993).
d. CHEMOTHERAPEUTIC AGENTS.
It has been known for a
while that cis-diaminedichloroplatinum caused an increase in CYP enzyme
activity as measured by spectral methods (Jollie and Maines, 1985).
Cis-platinum was found to specifically induce renal CYP2C23 (Ohishi et
al., 1994), and this immunological result was not correlated with any
increase in lauric acid 
-hydroxylation activity.
e. ALCOHOL.
The CYP2E1 gene is an ethanol inducible
form of P450 that metabolizes substrates such as ethanol, acetone,
diethyl ether, p-nitrophenol, halothane, benzene, and
N-nitrosodimethylamine, and is expressed in rabbit kidney (Khani et
al., 1988). The protein encoded by CYP2E1 is sometimes referred to a
P450 3a, P4502E1, or P450alc.Alcohol was shown to induce P450 isozyme 3a (CYP2E1) in the kidney
using antibodies to the liver protein. This increased expression accompanied a seven-fold increase in the isozyme 3a dependent rate of
aniline and butanol metabolism in kidney microsomes (Ding et al.,
1986). Ethanol has been found to induce specifically isozyme CYP2E1 in
the kidney by 50 to 80% (Ueng et al., 1987) and 500% (Ding et al.,
1986). Cyp2e-1 (the mouse ortholog of CYP2E1) is present in kidney and
induced by alcohol (Thomas et al., 1987). In addition to alcohol CYP2E1
protein was induced by bacterial lipopolysaccharide (LPS) irritants.
Bacterial LPS also induced CYP4A2 and 4A3 along with CYP2E1 (Sewer et
al., 1997). Inhalation of alcohol vapor was found to be the best way to induce the
protein encoded by CYP2E1, as detected by immunoblotting, in the
proximal convoluted tubule of the rat kidney. This result correlated
with induction of chlorzoxazone hydroxylation in rat kidney microsomes
(Zerilli et al., 1995). The half-life of immunoreactive CYP2E1 protein
in kidney was found to be approximately 6 h (Roberts et al.,
1994). The CYP2E1 protein and gene can also be induced by pyridine (Kim
et al., 1992).
f. IMMUNOSUPPRESSIVE AGENTS.
Total kidney cytochrome
P450 levels were increased after exposure to cyclosporin, whereas
hepatic P450 decreased (Mayer et al., 1989). Using Western blot
analysis, Yoshimura et al. (1989) showed that cyclosporin A induced a
protein in rat kidney that cross reacted to rabbit CYP2B protein. In
rat renal microsomes obtained after treatment of the animal with
cyclosporin A, immunoblotting with antibody to rabbit CYP2B4 protein
did not show any increase of this protein. In addition, levels of cross
reacting material to CYP4A5 (P450 kd) was also found after treatment
(Yoshimura et al., 1993b). Western blots of the membrane protein from
rat kidney obtained using antibodies to enzyme P450LM2, (CYP2B4) showed no induction of this protein by rapamycin. However, there was an
increase in aminopyrine N-demethylase activity after rapamycin treatment (Yoshimura et al., 1993a). Cyclosporin A has been shown to
induce CYP4A2 in rat kidney (Nakamura et al., 1994).
3. Specific renal cytochrome P450 enzymes. The following sections discuss representative types of reactions attributed to kidney P450 enzymes (CYP) by purification and enzyme assay. These studies have found differences in activities in P450 enzymes isolated from kidneys of different species. If a specific P450 protein designation has been reported in the cited article the correct CYP nomenclature is given.
a. AROMATIC OXIDATION. This common reaction for drugs and xenobiotics that contain a benzene ring has been shown to occur in kidney microsomes. The hydroxylation of BP has been examined in rat (Mayer et al., 1989), rabbit (Zenser et al., 1978) hamster, and guinea
pig renal microsomes (Smith et al., 1986). Likewise, the hydroxylation
of aniline and biphenyl in various species was found to occur at low
levels of activity (Litterst et al., 1975).
b. N-DEALKYLATION.
N-dealkylation occurs in the
metabolism of drugs containing an alkyl group attached to an amine. In
all instances, its activity in the kidney is found to be equal to or
less than that of the liver (Orrhenius et al., 1973; Litterst et al.,
1975). The most commonly used substrate, aminopyrine (AP), undergoes
N-dealkylation in mouse, rat, rabbit, hamster, guinea pig, and human
kidney. Using AP as substrate, catalytic activity of human renal
cytochrome P450 was demonstrated in a reconstituted system using rat
NADPH-cytochrome P450 reductase and rat cytochrome b5
(Imaoka et al., 1990a). Zenser et al. (1978) examined the demethylation of AP in microsomes
from cortex, outer medulla, and inner medulla of rabbit kidney and
found activity only in the cortex, which was approximately one-third
that of liver. Pretreatment with 3-MC induced cortical activity and
caused appearance of activity in the outer medulla (Zenser et al.,
1978).N-demethylation of benzphetamine was demonstrated in rabbit renal
microsomes, but not in rat. The activity was induced three-fold by PB
treatment (Kuo et al., 1982). Likewise, benzphetamine demethylation was
induced by PB in renal microsomes from hamsters. Activity was 5 to 10%
that seen in liver (Smith et al., 1986).
O-DEALKYLATION.
Oxygen dealkylation occurs in the
metabolism of drugs containing an ether group. Renal cytochrome P450
2C23 (k2), A8(k-4), and 4A2(k-5) have been shown to catalyze the
O-deethylation of 7 ethoxycoumarin in a reconstituted system (Imaoka et
al., 1990a). Enzyme activity was demonstrated in microsomes from rat
and rabbit kidney. PB induced activity only in rabbits (Kuo et al.,
1982) and a two-fold induction was seen with ethanol (Ueng et al.,
1987). O-deethylation of 7-ethoxycoumarin and exthoxyresorufin in renal microsomes was approximately 15% that of liver in hamster, but much
less in guinea pig (Smith et al., 1986). No induction of ethoxycoumarin
deethylase by PB, polybrominated byphenyl or -napthoflavone (-NF)
occurred. Ethoxyresorufin deethylase activity in renal microsomes was
less than 10% that of liver in both hamster and guinea pig and
activity was induced by -NF (Smith et al., 1986). Rat kidney
activity was induced by xylene (Toftgard and Nilsen, 1982). Cytochrome
b5 independent O-dealkylation using 7-ethoxycoumarin was
found in rat and human kidney (Imaoka et al., 1990a). Cojocel et al.
(1983) reported O-deethylation of 7-ethoxycoumarin to be highest in the
proximal tubule fraction of the kidney. O-dealkylation of
ethoxyresorufin was shown in rat kidney microsomes exposed to the
polychlorinated biphenyl (PCB), Aroclor, by Beebe et al. (1995) and by
exposure to the pesticide Fenarimol (Paolini et al., 1996).
c. OXIDATIVE DEHALOGENATION.
Halogenated organic
compounds, such as general anesthetics, undergo oxidative
dehalogenation to the corresponding alcohol or acid. Renal microsomes
metabolize dichloromethane at a rate 5% that of liver microsomes
(Kubic and Anders, 1975).
4. Nondrug factors that affect cytochrome P450 enzymes in the kidney that may modulate kidney drug metabolism. In addition to being induced by drugs, various cytochrome P450s are induced by endogenous substrates, hormones, toxins, and various metabolic states. The presence of these additional effectors of P450 metabolism could affect the metabolism of drugs in the kidney. Below are representatives of these nondrug modulators on P450 function and expression in the rabbit kidney.
a. ENDOGENOUS LIPID METABOLISM. MFO and therefore P450 enzymes have been well-documented as playing a role in fatty acid and steroid metabolism in the liver. The kidney has been recently shown to be a site of these metabolic reactions. Expression of CYP3A, which catalyzes the 6-hydroxylation of endogenous steroids, has been found in amphibian (A6) renal cells and rat kidney and human kidney microsomes (Schuetz et al., 1992). In immunochemistry, studies using antibodies
raised to an adrenal cortex enzyme (steroid 21-hydroxylase),
immunoreactive protein was localized in the distal, cortical, and
medullary collecting tubules of the kidney. CYP21A1
(P450C21) enzymatic activity had previously been reported
in the bovine kidney (Sasano et al., 1988).The kidney has many P450s that are involved in fatty acid metabolism.
The general role of cytochrome P450 in the arachidonate cascade has
been reviewed by Capdevila and colleagues (1992). Zenser recognized in
1979 that there were at least two separate pathways for arachidonic
acid metabolism in the kidney. One pathway was the prostaglandin
cyclooxygenase pathway, whereas a second pathway involved the
NADPH-dependent mixed function oxidase P450 system. The action of the
CYP4A family (arachidonic acid /-1 oxygenase activity) metabolism
in the kidney has been related to hypertension (Laniado-Schwartzman et
al., 1996; Makita et al., 1996) and the action of CYP2A, the
arachidonic acid epoxygenase, in the kidney may also be related to
disease states (Makita et al., 1996). Thus, these target enzymes could
become the targets of drugs of the future.The renal cytochrome P450 arachidonic acid system has been reviewed by
Laniado-Schwartzman and Abrahams (1992). A gene encoding a putative rat
kidney arachidonic acid epoxygenase was isolated from a kidney cDNA
library. Overexpression of this gene in COS-1 cells produced a protein
that catalyzed the NADPH-dependent metabolism of arachidonic acid with
similar specificity to P450 2C23 (Karara et al., 1993).A P450 isozyme, responsible for the oxygenation of polyunsaturated
fatty acids, has been well-documented in the kidney (Oliw, 1994).
Cytochrome P450K (CYP2C6) protein, catalyzing the hydroxylation of
various saturated fatty acids to the corresponding - and -1 hydroxy derivatives has been studied in rat kidney cortex by spectral and enzymatic methods (Ellin et al., 1972). The CYP4 gene family, expressed in rat kidney tissue,
includes isoforms that catalyze the /(-1) hydroxylation of
prostaglandin A (CYP4A7) (Kusunose et al., 1989) and fatty acids
(Imaoka et al., 1990b). Members of this family of CYP4A proteins have
been shown to be induced in kidney by peroxisome proliferators such as
di-(2-ethylhexyl)phthalate (Okita et al., 1993). In the rat kidney
CYP4A1 mRNA was induced by methylclofenapate (Bell et al., 1992) and
CYP4A2 mRNA was induced by dehydroepiandrosterone, an adrenal steroid
that is a peroxisome proliferator at high dosages (Webb et al., 1996).
b. TOXIN BIOTRANSFORMATION.
The biotransformation of
many toxins by P450 enzymes has been shown to occur in the kidney.
Renal microsomal cytochrome P450 was induced by xylene in the rat
kidney and studied as the O-deethylation of 7-ethoxyresorufin (Toftgard
and Nilsen, 1982). Enzyme assays and immunoblots were used to
demonstrate that dietary exposure to Aroclor 1254, a PCB, induced P450
1A2 (Beebe et al., 1995) whereas exposure to the pesticide, Fenarimol,
induced CYP 1A1 (Paolini et al., 1996).Not all toxic agents stimulate kidney P450 enzymes. The mutagenic
compound, 3-chloro-4-(dichloromethyl)-5 hydroxy-2(5H)-furone (MX) has
been found in chlorinated drinking water and shown to inhibit kidney
EROD (7-ethoxyresorufin-O-deethylase) activity. However, note that MX
induced UDP glucuronosyltransferase activity in kidney (see
glucuronidation section). Studies have shown tissue specific expression of dioxin induced P450
enzymes. Form 6 (CYP1A1 protein in rabbit) was found to be induced by
dioxin (TCDD) in rat kidney, whereas form 4 (CYP1A2 in humans) was
found to be localized in the rabbit liver as measured by enzymatic
assay (Liem et al., 1980). Using more sensitive immunohistochemistry, it was found that P-450 isoforms 4 and 6 stained
proximal tubules and endothelium intensely after TCDD treatment. The aromatic hydrocarbon receptor is a small cytoplasmic protein
required for the induction of CYP1A1 at the transcriptional level. This
protein has the highest affinity for TCDD and is sometimes called the
Dioxin inhibitor. (Parkinson, 1996). The general action of the aromatic
hydrocarbon receptor and it's localization in the kidney has been
reported by Ioannides and Parke (1990). In rabbit kidney of untreated animals it was found that forms 2 and 3 were localized to proximal tubules (Dees et al., 1982). In addition,
the P450 gene family, P450 1A1 has been found in kidney tissue after
induction with TCDD (Gonzalez, 1989).
c. SEX HORMONES.
As in other organs, sex related
expression and metabolism of various xenobiotics and endogenous
substrates by P450 enzymes have been shown in the kidney (Hawke and
Welch, 1985; Hu et al., 1993). Renal P450 enzymes have been induced
both in males or testosterone treated females (Williams et al., 1986)
and by estrogen (Liehr et al., 1987). Inducers such as 3-MC, PB, and
purazole changed induction profiles of CYP2A by significantly
increasing 7 
-hydroxylase activity in male kidney microsomes,
whereas only PB increased activity in females (Hoivik et al., 1995;
Pelkonen et al., 1994). Male specific induction of the gene encoding P450 4A2 as tested by
Northern blot analysis after treatment with the peroxisome proliferator, clofibrate, has been reported in the rat kidney (Sundseth
and Waxman, 1992). Administration of androgens to female mice changed
the mouse kidney pattern of expression of P450 enzymes from female to
male in 8 days. Male mice missing the androgen receptor were not
responsive to testosterone and maintained the female P450 kidney
distribution. (Henderson and Wolf, 1991). CYP4A2, a P450 isozyme found
in greater abundance in female rat kidney, was also induced by growth
hormone under certain conditions (Imaoka et al., 1992).
d. FASTING, OBESITY, EXERCISE, AND DIABETES.
The P450
content/g of kidney was increased by 50% over that of controls in the
obese overfed rat (Corcoran and Salazar, 1988). Starvation has been
shown by enzyme assay, spectrophotometric methods, and Western blots
(Imaoka et al., 1990b) to increase activity of CYP4A2 (P450 K-5)
extending early work showing starvation increased amounts of P450 and
laurate--oxidation activity in rat kidney microsomes (Hasumura et
al., 1983; McMartin et al., 1981). In addition, exercise has been
demonstrated to increase renal microsomal P450 by up to 60% in male
rats and age to reduce it (Piatkowski et al., 1993).In rats with streptozotocin (STZ)-induced diabetes, the P450 content
was reduced by 50% (Del Villar et al., 1995). P450 2EI, 4A2, and 4A8
(K-4) in renal microsomes were induced by diabetes along with and
-1 hydroxylation activity (Shimojo et al., 1993). It is possible
that although total P450 content may decline, specific P450's may
increase in STZ-induced diabetes.
B. N-Oxidation (Flavin-Containing Monooxygenases)
Flavin containing monooxygenases (FMOs) are found in liver, kidney, and lung and can oxidize the nucleophilic nitrogen, sulfur, and phosphorus heteroatom of a variety of xenobiotics. They require NADPH and O2 and catalyze some of the same reactions as cytochrome P450. These are mostly detoxication reactions, and metabolites produced generally result from the chemical reaction between a peracid or peroxide. FMO plays a role in the N- and S-oxygenation of numerous xenobiotics.
FAD is reduced by NADPH and oxidized NADP+ remains bound to the enzyme, which then binds oxygen producing a relatively stable peroxide. During oxygenation, the 4a-hydroperoxyflavin is converted to 4a-hydroxyflavin and the flavin peroxide oxygen is transferred to the substrate.
cDNAs for five different FMOs (FMO1, FMO2, FMO3, FMO4, FMO5)
have been cloned and sequenced. Each of these genes has been mapped to
the long arm of chromosome 1. The open reading frames deduced from the
DNA sequence of FMO1, 2, 3, and 5 contain between 532 and
535 amino acid residues and the calculated molecular mass is
approximately 60 kDa. FMO 4 is believed to encode 25 more
amino acid residues. Each of these genes is expressed in a species and tissue specific manner in humans and other mammals. The kidney of
mouse, rat, and human contains relatively high levels of FMO1, and FMO3
is high in the mouse and rat but not in the human kidney (Dolphin et
al., 1991; Parkinson, 1996). The forms of FMO are distinct gene
products with different physical properties and substrate
specificities. Human FMOs 1 and 3 have been expressed in bacterial and
insect systems, and the proteins found to be functionally active in
catalyzing the N-oxidation of N-ethyl-N-methylaniline and pargyline
(Phillips et al., 1995).
Many basic drugs, such as benadryl, imipramine, chlorpromazine,
nicotine, morphine, methaqualone, methadone, and meperidine, form
N-oxides. The chicken kidney was found to produce 7.2 µmol/hr/g kidney of trimethylamine oxide from trimethyl amine in vivo (Acara et
al., 1977). The same metabolism in chicken liver homogenates occurred
at a rate of 9.3 µmol/hr/g liver (Baker et al., 1963). After
meperidine perfusion of the isolated rat kidney, meperidine N-oxide was
identified by GC/MS as the major renal metabolite (Acara et al., 1981).
Vickers et al. (1996) found that N-oxidation was the major renal
biotransformation pathway for the 5HT3
antagonist, tropisetron. Although the overall contribution to
tropisetron metabolism was very small, 2 to 12 pmol/hr/mg slice for
human rat and dog kidney were comparable to human and rat liver (but
not dog). In the kidney, the only metabolite formed of imipramine was
its N-oxide (Lemoine et al., 1990). The kinetic analysis indicated an
affinity of 7 mM for human liver microsomes versus 0.3 mM in kidney.
C. Alcohol Oxidation
The principle route of elimination of alcohol is by oxidation to the aldehyde and subsequently to the carboxylic acid. Alcohols can also be directly conjugated with glucuronic acid and metabolized by a microsomal P450 enzyme. Although 90% of ethanol metabolism occurs in the liver, the enzyme is ubiquitous and renal metabolism also plays a role in elimination.
Alcohol dehydrogenase (ADH) (E.C. 1.1.1.1) is a cytoplasmic
NAD+ dependent zinc metalloenzyme that catalyzes
the reaction oxidizing an alcohol to an aldehyde and reduces
NAD+ to NADH. At this time, the human ADH family
consists of seven genes, which have evolved from a common ancestral
gene. The genes encode proteins belonging to one of five classes of ADH
isoenzymes based on structural and kinetic features. Class I (ADH1,
ADH2, ADH3) has a low KM for ethanol
and is sensitive to inhibition by pyrazoles. Classes II (ADHP) and III
(ADH5) have a higher KM for ethanol,
greater affinity for long chain alcohols, and are insensitive to
pyrazole inhibition. Class IV (ADH7), isolated from rat stomach, has
enzyme characteristics of class II but substantial structural
differences (Pares et al., 1992). Class V (ADH6) has been described in
liver and stomach (Yasunami et al., 1991).
Kidney ADH has been studied in several species (Moser et al., 1968).
Five major isozymes were isolated from various tissues in baboons, with
the kidney extract showing the highest activity of Class I isozymes
termed ADH 1 and ADH 2 (Holmes et al., 1986). The specific activity of
ADH from kidney extract with 5 mM ethanol as substrate was
476 nmol/min/g tissue, roughly one-third that seen in liver extract.
ADH mRNA was found to be present in the inner cortex and medulla of
kidneys from female Wistar rats (Qulali et al., 1991). Treatment with
estradiol induced ADH mRNA resulting in a three-fold increase in ADH
activity. ADH activity of liver was 5 times that of kidney but showed
no induction with estradiol. Subsequent studies localized
estradiol-induced ADH mRNA only to kidney tubule cells and further
elucidated the role of hormones in the control of rat kidney ADH
(Qulali et al., 1993). Fasting, hyperthyroidism, and treatment of male
rats with estradiol increased ADH activity. Androgens were found to
induce ADH mRNA in mouse kidney (Felder et al., 1988; Tussey and
Felder, 1989; Watson and Paigen, 1990) and although androgen treatment
caused a difference in the transcription rate of mRNA in the kidney,
liver ADH level was controlled posttranscriptionally.
D. Aldehyde Oxidation
Aldehydes are produced as intermediates in many biological
reactions. The most common source of aldehyde in humans is acetaldehyde formed from the metabolism of ethanol. In addition, aldehyde formation may result from biogenic amine metabolism, amino acid metabolism, and
lipid peroxidation of polyunsaturated fatty acids (Ambruziak and
Pietruszko, 1993).
Aldehydes may be oxidized to their corresponding carboxylic acid by
enzymes such as aldehyde dehydrogenase (ALDH) (E.C. 1.2.1.3), aldehyde
oxidase, and xanthine oxidase. ALDH activity in the kidney was first
described by Deitrich (1966). The overall activity of ALDH in the rat
kidney varies from 20 to 80% of that in rat liver (Deitrich, 1966;
Vasilou and Marselos, 1989; Dipple and Crabb, 1993). Because proximal
tubule cells contain cytochrome P450 and ADH, the cells have the
potential to oxidize a variety of compounds to aldehydes that are
potential cytotoxins. The presence of ALDH in these cells is thus
beneficial.
Three major classes of ALDH (E.C.1.2.1.3) containing several isozymes
have been described. Class 1 are cytosolic ALDHs that have been
localized primarily in the liver and exhibit broad substrate specificity and a low KM with
acetaldehyde as a substrate. Class 2 ALDHs are mitochondrial enzymes
that are present at significant levels in human (Agarwal et al., 1989),
opossum (Holmes et al., 1991), rat (Dipple and Crabb, 1993), and mouse
(Ront et al., 1987) kidney, as well as the liver. Class 2 ALDHs also
show a low KM for acetaldehyde
and are active in aliphatic and biogenic amine metabolism. Class 3 ALDHs include the major corneal/stomach ALDH with a high
KM for acetaldehyde and have
not been described in the kidney.
The genomic structure of the gene encoding human class 1 ALDH protein
(Hsu et al., 1989) and class 2 ALDH protein (Hsu et al., 1988) have
been described. Each has approximately 50 kb and consists of 13 coding
exons separated by 12 introns. Human class 3 ALDH has also been cloned
and sequenced. Recently several additional human ALDH genes have been
identified but have not yet been assigned to gene classes. In
particular, ALDH7 cDNA was cloned from human kidney tissue
(Hsu et al., 1994). Information on ALDH genes may be obtained from
the Internet at the "Vasilou Laboratory Home Page"
(http://www.uchsc.edu/sp/sp/alcdbase/alcdbase.html).
An isozyme termed ALDH5 was found to be present in both cytosolic and
mitochondrial fractions of many opossum organs including the kidney
(Holmes et al., 1991). The isozyme ALDH4, a mitochondrial enzyme with a
high KM was found to have
significant activity in kidney (Agarwal et al., 1989; Holmes et al.,
1991). On purification, it was found to be identical with
glutamate-semi-ALDH (E.C. 1.5.1.2) (Forte-McRobbie and Pietruszko,
1986). Isozymes have been shown to vary in time of appearance during
development (Ront et al., 1987). A rat kidney ALDH isozyme that
catalyzes the oxidation of retinol to retinoic acid has been isolated
(Labrecque et al., 1995) and the cDNA encoding this protein has been
cloned (Bhat et al., 1995).
Subcellular fractionation of proximal tubule fragments from rabbit
kidney using propionaldehyde as a substrate showed ALDH activity to be
bimodally distributed in the mitochondrial and cytosolic fractions.
Kinetic characteristics suggested the presence of two isoenzymes
(Hjelle et al., 1983).
3-MC induced ALDH activity in rat kidney using benzaldehyde/NADP as
substrate but no change was seen after PB treatment (Vasilou and
Marselos, 1989). It appears that the pattern of ALDH induction is
dependent on the inducer.
E. Oxidative Deamination (Monoamine Oxidase)
Monoamine oxidase (MAO) amine:oxygen oxidoreductase (deaminating
flavin-containing E.C.1.4.3.4.) catalyzes the oxidative metabolism of
amines. MAO is tightly associated with the outer mitochondrial membrane. The reaction catalyzed by MAO consists of reductive and
oxidative half reactions. In the reductive half reaction, the amine
substrate is oxidized and the covalently attached FAD reduced to the
hydroxyquinolone. In the second half-reaction, the FAD is reoxidized by
oxygen with formation of
H2O2 and an aldehyde
(Weyler et al., 1990). The sum of the MAO partial reactions is:
|
(2) |
There are two isoforms of MAO, designated A and B, which are widely
distributed in mammalian tissues and function to breakdown not only
biogenic amines but a wide variety of amines including aliphatic,
aromatic, primary, secondary, and tertiary amines. The two forms were
originally demonstrated through selective inhibition by clorgyline
(MAO-A) (Johnston, 1968) and deprynel (MAO-B) (Knoll and Magyar, 1972).
Subsequently, they have been distinguished by differences in substrate
preference (Lyles and Shaffer, 1979), tissue and cellular distribution
(Weyler et al., 1990), immunological properties (Denny et al., 1983),
and most recently determination of nucleotide sequences of cDNA (Bach
et al., 1988). cDNA cloning of human liver monoamine oxidase A and B
has determined that they are derived from separate genes. The MAO-A and
MAO-B human genes are located next to each other on the human X
chromosome. The A and B forms have subunits with molecular weights of
59.7 and 58.8 kDa, respectively with 70% sequence identity. The
obligatory cofactor FAD is covalently bound to cysteine in the same
pentapeptide sequence in both isoforms. The genes for monoamine oxidase
A and B have identical exon-intron organization, suggesting a common ancestral gene (Grimsby et al., 1991).
The kidney contains high concentrations of both MAO-A and -B. Most of
the activity has been shown to be in tubular cells and the enzyme is of
particular importance in the metabolism of amines filtered by the
kidney (Fernandes and Soares-da-Silva, 1990).
Table 2 shows the kinetic parameters of
MAO from homogenates of human and rat kidney. In human kidney, the
similar activities toward 5-hydroxytryptamine and phenylethylamine
suggest that differences in Vmax are caused by
differences in concentration of the enzyme (Fernandes and
Soares-da-Silva, 1992). The activity of MAO-A is similar in cortex and
medulla, whereas that of MAO-B is higher in the cortex. Studies in
rabbit renal proximal tubule using Western blot analysis and enzyme
assays show MAO-B to be the predominant isoform. This isoform holds the
I2 imidazoline binding site, a regulator of
MAO (Gargalidis-Mondanase et al., 1997). In rat tissue, MAO-A is the
predominant form. The MAO activity of human kidney is approximately
one-third that of liver (Vogel et al., 1983).
|
It has been demonstrated that in rat renal slices a substantial amount
of newly formed dopamine is deaminated to 34-dihydroxyphenylacetic acid
in renal tubular cells (Fernandes and Soares-da-Silva, 1990). The
effect of aging on MAO activity in the kidney has been studied, with
little change found in rabbits or mice (Feldman and Roche, 1978) but a
small decrease in MAO-A and MAO-B activity was seen in aging rats (Lai
et al., 1982).
F. Aldehyde and Ketone Reduction
Aldehydes and ketones are widely distributed and have several biological functions. In addition to ADH, there are several enzymes in the aldo-keto reductase family that may participate in the metabolism of aldehydes and ketones in the kidney. Aldose reductase (E.C. 1.1.1.21), aldehyde reductase (E.C. 1.1.1.2), and carbonyl reductase (E.C. 1.1.1.184), also referred to as ketone reductase, are members of this enzyme family that are found in relatively high amounts in the kidney. These enzymes reduce aldehydes and ketones to their corresponding alcohols. Aldose reductase has been found to metabolize only endogenous compounds in the kidney. Therefore it will not be discussed further in this review.
1. Aldehyde reductase.
The aldehyde reductases are members of
a superfamily of NADPH-dependent reductases that contribute to the
metabolism of certain carbonyl compounds (Bohren et al., 1989). The
enzymes are of monomeric structure and have broad substrate sensitivity
in reducing xenobiotics and naturally occurring carbonyl compounds. The
enzymes are widely distributed with kidney having the highest activity
in most species (Bosron and Prairie, 1973), and a concentration of up
to 0.5% of the total protein in tissue homogenates. Aldehyde reductase was first purified to homogeneity in pig kidney (Bosron and Prairie, 1972) and has been demonstrated to be active in the reduction of
p-nitrobenzaldehyde, indole-3-acetaldehyde,
DL-glyceraldehyde, and D-glucuronate (Flynn,
1986). The cDNA of human aldehyde reductase has been cloned and
sequenced and encodes a protein with a deduced Mr of 37 kDa.
). Aldehyde reductases are
characterized by inhibition by PB (Flynn, 1982).
2. Ketone reductase.
Ketone reductase (also known as carbonyl
reductase) is another member of the aldo-keto reductase superfamily
that may reduce certain unoxidized carbonyl substances. Aromatic,
acyclic, and unsaturated ketones may be reduced to free or conjugated
alcohols. The best substrates are quinones (Wermuth et al., 1986). The
various ketone reductases have common features such as ubiquitous
tissue distribution, primarily cytosolic localization, and preference for NADPH as a coenzyme.
). The structure and gene sequence of human carbonyl reductase
has now been identified and found to be a member of the short-chain
dehydrogenase family (Wermuth et al., 1988).
In the human kidney, carbonyl reductase was found to be localized to
the proximal convoluted and straight tubules (Wirth and Wermuth, 1992).
The enzyme was found to be identical with a prostaglandin 9-ketoreductase from human and pig kidney (Shieber et al., 1992).
The enzyme purified from rabbit kidney reduces several drugs with a
ketone group, such as acetohexamide, befunolol, metapyrone, levobunolol, daunorubicin, haloperidol, moperone, trifluperidol, and
loxoprofen, along with several other aldehydes and ketones (Imamura et
al., 1993; Higuchi et al., 1993). Dihydromorphinone reductase has been
isolated from chicken and rabbit kidney (Roerig et al., 1976), warfarin
reductase from human, rabbit, and rat kidney (Moreland and Hewick,
1975), and oxisuran, daunorubicin, and metapyrone reductase from rabbit
and human kidney (Ahmed et al., 1979). The specific activities of renal
reductase toward these substrates was generally high, second only to
liver in terms of tissue distribution (Ahmed et al., 1979). Although
generally found to be cytosolic enzymes, the microsomal fraction in
mouse, rat, and guinea pig kidney had significant metapyrone reductase activity (Opperman et al., 1991). The enzymes have been found to be
inhibited by quercetin (Felsted and Bachur, 1980) and by nonsteroidal
drugs such as indomethacin (Higuchi et al., 1994).
3. Other.
Other enzymes that have been shown to be involved in
the reduction of xenobiotic carbonyl compounds in the kidney are
dihydrodiol dehydrogenases and 11--hydroxysteroid dehydrogenase.
Dihydrodiol dehydrogenase (E.C. 1.3.1.20) catalyzes the reduction of
dicarbonyl compounds and some aldehydes in the presence of NADPH (Hara
et al., 1989) and plays a role in the detoxification of endogenous ketoaldehydes such as methylglyoxal and 3-deoxyglucosone. Dimeric dihydrodiol dehydrogenase has been isolated from monkey kidney (Nakagawa et al., 1989). 11--hydroxysteroid dehydrogenase has been
assayed in mouse kidney and displays xenobiotic carbonyl reductase
activity toward the drug metapyrone as well as endogenous glucocorticoid 11-B-oxidoreduction (Maser et al., 1994).
G. Hydrolysis Mechanisms
1. Ester and amide hydrolysis/carboxylesterase and amidase.
Carboxylesterases/amidases (E.C. 3.1.1.1) catalyze hydrolysis of
carboxylesters, carboxyamides, and carboxythioesters, as seen below in
equations 3 to 5, respectively (Heymann, 1980). The specificity of the
carboxylesterase/amidase action depends on the nature of R, R', R".
|
(3) |
|
|
(4) |
|
|
(5) |
|
).
Because proteins classified as carboxylesterases have also been shown
to be potential amidases, and vice versa, we will refer to this class
of enzymes simply as carboxylesterases. As with several other enzymes,
there are differences in classification schemes. In genetic
nomenclature, each esterase is termed "Es" </
