SEX-RELATED EXPRESSION OF RENAL ORGANIC ANION AND CATION
TRANSPORTERS IN DIFFERENT SPECIES
Ivan Sabolić1, Marija Ljubojević1, Davorka Breljak1, Daniela Balen1, Hrvoje Brzica1,
Vedran Micek1, Nikola Radović2 and Ognjen Kraus3
1
Molecular Toxicology, Institute for Medical Research and Occupational Health,
Ksaverska cesta 2, 10001 Zagreb, Croatia. sabolic@imi.hr
2
Urology, Clinical Hospital “Dubrava”, Zagreb Croatia
3
Urology, Clinical Hospital “Sisters of Mercy”, Zagreb, Croatia
Abstract
In the mammalian kidney, transport of endogenous and xenobiotic organic compounds is
mediated by multispecific organic anion and cation transporters localized in the apical and
basolateral cell membrane domains of the specific nephron segments. These transporters
are also responsible for drug resistance, drug-drug interactions, and drug-induced
nephrotoxicity. In rat and mouse proximal tubules, a number of these transporters exhibit
sex differences in their expression. In comparison with those in rodents, in pig and human
proximal tubules some transporters are absent, some exhibit different localization in the
cell membrane domains, and none exhibits the sex-dependent expression. These
differences indicate that: a) the data on renal transporters in one species can not simply be
regarded as relevant for other species, and b) pigs may be better model then rodents for
drug-related studies relevant to humans.
Keywords: gender differences, human, kidney, mouse, organic compounds, pig, rat
1
Introduction
In the mammalian kidney, transport of organic anions (OA) and cations (OC) is
maintained by various, largely multispecific OA and OC transporters (OATs and OCTs,
respectively) which are localized in the apical (luminal) and basolateral membrane
domains in the cells along the nephron. These transporters use ATP or transmembrane ion
gradients to drive vectorial transport of their substrates in direction of secretion or
reabsorption (HEDIGER, 2004; STIEGER and HIGGINS, 2007). A polar distribution of
some of these transporters in the rat proximal tubule (PT), is illustrated in Fig. 1.
Fig. 1. Domain-specific localization of various OATs and OCTs in the rat PT. A. Definition of
various nephron segments in relation to specific tissue zones. CO, cortex; OS, outer stripe; IS,
inner stripe; IM, inner medulla; PAP, papilla; UR, ureter; CG, cortical glomerulus; JMG,
juxtamedullary glomerulus; PCT, proximal convoluted tubule; S3, PT straight segment; HL, Henle
loop; TALH, thick ascending limb of Henle; DT, distal tubule; CD, collecting duct. B.
Transmission electron micrograph of the PCT (cross section) in the cortex. BLM, basolateral
membrane; BBM, brush-border membrane; N, nucleus; M, mitochondria; V, vacuole). C.
Domain-specific distribution of various OATs and OCTs in the rat PT cell. Slc/SLC, ATPindependent solute carriers (Slc for animal and SLC for human secondary and tertiary active
carriers): NaDC1 and 3, sodium-dicarboxylate cotransporters; Oat1, 2, 3 and 5, organic anion
transporters; Oatp1 and 2, OA transporting polypeptides; Oct1, 2 and 3, organic cation
transporters; Octn1 and 2, zwitterion/cation transporters; Pept1 and 2, peptide transporters.
Abc/ABC, ATP-binding casette transporters (Abc for animal and ABC for human primary active
transporters). Mrp1-6, multidrug resistance associated proteins; Mdr/Pgp, multidrug resistance
protein/P-glycoprotein. GF, glomerular filtration.
2
Recent studies have shown that OATs and OCTs mediate: a) transport of various
endogenous organic compounds that are generated in normal metabolism, b) transport of
exogenous (xenobiotic) organic compounds, such as food constituents (flavonoids,
mycotoxins, pesticides) and drugs (antibiotics, chemotherapeutics, diuretics, antihypertensives, etc…), c) drug-drug interactions, d) drug resistance, e) development of
drug-induced nephrotoxicity, and f) specific diseases due to defect and/or malfunctional
transporters. Most of these studies have been performed in rodents (rats and mice); only a
limited amount of data is obtained from the studies in other animal species and humans.
Moreover, rats and mice are the most common animals used in testing pharmacology and
toxicology of various substances, including drugs to be used in human and veterinary
medicine. However, increasing evidence indicate that rodents may not be the best model
for such studies relevant to humans due to sex and species differences in various
properties.
Sex and species differences in renal transporters of organic anions and cations
Some observations in experimental animals and humans have indicated that renal
secretory and reabsorptive functions may be different in males (M) and females (F). These
differences may be related to sex hormone-regulated expression of specific transporters in
one of the membrane domains of (mainly) PT (reviewed in SABOLIC et al., 2007).
Indeed, as found in rat kidneys, NaDC3, Oat1 and Oat3 are localized in the BLM of
various nephron segments in the cortex, with M-dominant expression, whereas Oat2 and
Oat5 are localized in the BBM of (largely) S3 segments in the outer stripe and medullary
rays, with F-dominant expression (Fig. 2) (LJUBOJEVIC et al., 2004, 2007; SABOLIC et
al., 2007; and our unpublished data). As shown in castrated and sex hormone-treated rats,
sex differences in the expression of renal transporters are driven by stimulatory and/or
3
inhibitory actions of sex hormones at the level of mRNA and/or protein. A number of
other renal OATs and OCTs in rats, mice, and in few other animals also exhibit sex
differences in their expression at the level of protein and/or mRNA (reviewed in
SABOLIC et al., 2007).
Fig. 2. Sex differences in the expression of various OATs in the rat kidney. As found by
immunocytochemistry, OATs in the basolateral membrane (BLM; rNaDC3, rOat1 and rOat3)
exhibit strong M-dominant expression in various, mainly proximal convoluted tubules (PCT) in
the cortex (CO), whereas OATs in the brush-border membrane (BBM; rOat2 and rOat5) of
proximal tubule straight segments (S3) in the outer stripe (OS) exhibit strong F-dominant
expression. G, glomerulus; DT, distal tubule; CD, collecting duct. Bar = 20 m.
Accordingly, sex differences in the renal expression of membrane transporters can be
correlated with the equivalent differences in the excretion of various OA and OC in urine
(Table 1). In addition, prepubertal rats excrete some OA with much lower rate, and exhibit
sex-independent and much lower expression of renal OATs than adults (for references see
SABOLIC et al., 2007), whereas in mice, knock-out for Oat1 and Oat3, the urine
excretion of p-aminohippurate (PAH) and a few other OA is strongly impaired (ERALY
et al., 2006; SWEET et al., 2002).
4
Table 1. In rats and mice, sex differences (SD) in the expression of renal OATs and OCTs
correlate well with the excretion of some OA and OC in urine.
Transporter
Expression
Compound / Substrate
Excretion
(Oat/Oct)
(SD)
Oat1, Oat3
M>F
PAH (p-aminohyppurate), Furosemide
M>F
Oat3
M>F
Taurocholate
M>F
Oat2
M<F
PFOA (perfluorooctanoic acid)
M<F
Oct2
M>F
TEA (tetraethylammonium), Cis-platin,
M>F
(SD)
Amantadine
Contrary to the numerous cases of sex-dependent expression of renal transporters and
excretion of their substrates in rats and mice, in rabbits the expression of Oat1, Oat2,
Oat3, and Oct2 in the PT significantly increased after the puberty, but without exhibiting
sex differences in adult animals (GROVES at al., 2006). Accordingly, rabbits exhibit no
sex differences in urinary excretion of PAH and tetraethylammonium (TEA). Comparable
data were found in dogs. A few studies in humans have shown that the renal clearance of
some OA and OC is sex related, but the sex-related expression of relevant carriers in the
human nephron has not been reported (for references see SABOLIC et al., 2007). In
general, animals and humans exhibit similar, albeit not identical set of renal transporters.
However, recent immunochemical, mRNA and transport studies, performed by us and by
others, indicated that the presence, localization along the nephron, and the cell membrane
domain-specific localization of some OA and OC transporters are different in humans and
experimental animals.
5
Fig. 3. Localization of NaDC3 and Oat1/OAT1 in cortical PT of various species. In the
kidney cortex of indicated species, both OA transporters are localized in the BLM of
proximal tubules (arrows). G, glomerulus. Bar = 20 m.
In this respect, NaDC3 and Oat1/OAT1 proteins in the cortical PT of rats, mice, pigs and
humans were immunostained in the same membrane domain, e.g., BLM (Fig. 3). On the
other side, Oat2 in rats and mice was localized to the BBM of S3 segments in the outer
stripe and medullary rays, whereas in pigs and humans, the OAT2 protein was localized to
the BLM of cortical PT (Fig. 4). This means that Oat2 in rodents may have reabsorptive
function, whereas OAT2 in pigs and humans may have secretory function. Additional
experiments have shown that sex differences in the expression of NaDC3, Oat1, Oat2 and
Oat3 clearly exist in rats and mice, but not in pigs and humans (data not shown). This
further means that in rodents both the expression of various transporters and the transport
of relevant substrates are strongly influenced by sex hormones, whereas in humans (and
pigs), these hormones may play only a minor role in renal functions.
6
Fig. 4. Species-dependent localization of Oat2/OAT2 in different PT segments and cell membrane
domains. In rats and mice, Oat2 is localized to the BBM of S3 segments (arrowheads), whereas in
pigs and humans, the protein is localized to the BLM of cortical PCT (arrows). CO, cortex; OS,
outer stripe; G, glomerulus; PCT, proximal convoluted tubule; S3, proximal tubule straight
segment. Bar = 20 m.
Further studies have shown that: a) Oat5 is present in the BBM of S3 segments in rodents
(Fig. 3), but not in pigs and humans (data not shown), b) OAT4 in the human kidney was
localized to the PT BBM, whereas a similar transporter was absent from the rat nephron
[EKARATANAWONG et al., 2004], c) Oct1 was localized in the rat kidney largely to the
PT BLM in the cortex (KARBACH et al., 2000), whereas in the human kidney, OCT1
was not detected (GORBULEV et al., 1997), and d) Oct2 in the rat kidney was localized
predominantly to the BLM of PT S3 segment (KARBACH et al., 2000), whereas OCT2 in
the human kidney was localized to the BLM along the entire PT (MOTAHASHI et al.,
2002).
Species differences in the expression and/or cellular localization of the indicated
transporters may influence the secretory and/or reabsorptive direction of renal transport of
their substrates and thus may affect their urinary excretion. They further indicate that the
findings regarding sex differences and effects of sex (and, possibly, other) hormones upon
7
renal transporters in one species can not simply be regarded as relevant for other species.
In humans, the tested renal OA and OC transporters exhibit no sex differences in their
expression, whereas their localization in specific nephron segments and cell membrane
domains are similar to those in pigs and not in rats and mice. Therefore, pigs may be a
plausible model to study drug transport, pharmacotherapy, drug-drug interactions, and
drug-induced nephrotoxicity relevant to humans.
References
EKARATANAWONG, S., N. ANZAI, P. JATUBHA, H. MIYAZAKI, R. NOSHIRO, M.
TAKEDA, Y. KANAI, S. SOPHASAN, H. ENDOU (2004): Human organic anion
transporter 4 is a renal apical organic anion/dicarboxylate exchanger in the proximal
tubules. J. Pharmacol. Sci. 94, 297-304.
ERALY, S. A., V. VALLON, D. A. VAUGHAN, J. A. GANGOITI, K. RICHTER, M.
NAGLE, J.C. MONTE, T. RIEG, D. M. TRUONG, J. M. LONG, B. A. BARSHOP, G.
KALER, S. K. NIGAM (2006): Decreased renal organic anion secretion and plasma
accumulation of endogenous organic anions in OAT1 knock-out mice. J. Biol. Chem. 281,
5072-5083.
GORBOULEV, V., J. C. ULZHEIMER, A. AKHOUNDOVA, I. ULZHEIMERTEUBER, U. KARBACH, S. QUESTER, C. BAUMANN, F. LANG, A. E. BUSCH, H.
KOEPSELL (1997): Cloning and characterization of two human polyspecific organic
cation transporters. DNA Cell Biol. 16, 871-881.
GROVES, C. E., W. B. SUHRE, N. J. CHERRINGTON, S. H. WRIGHT (2006): Sex
differences in the mRNA, protein, and functional expression of organic anion transporter
(Oat) 1, OAT3, and organic cation transporter (OCT) 2 in rabbit renal proximal tubules. J.
Pharmacol. Exp. Therap. 316, 743-752.
8
HEDIGER, M. A. (Guest Editor) (2004) The ABCs of solute carriers: physiological,
pathological, and therapeutic implications of human membrane transport proteins.
Pflügers Arch. Eur. J. Physiol. Vol. 447, No. 5.
KARBACH, U., J. KRICKE, F. MEYER-WENTRUP, V. GORBOULEV, C. VOLK, D.
LOFFING-CUENI, B. KAISSLING, S. BACHMANN, H. KOEPSELL (2000):
Localization of organic cation transporters OCT1 and OCT2 in rat kidney. Am. J. Physiol.
Renal Physiol. 279, F679-F687.
LJUBOJEVIC, M., C. M. HERAK-KRAMBERGER, Y. HAGOS, A. BAHN, H.
ENDOU, G. BURCKHARDT, I. SABOLIC (2004): Rat renal cortical OAT1 and OAT3
exhibit gender differences determined by both androgen stimulation and estrogen
inhibition. Am. J. Physiol. Renal Physiol. 287, F124-F138.
LJUBOJEVIC, M., D, BALEN, D. BRELJAK, M. KUSAN, N. ANZAI, A. BAHN, G.
BURCKHARDT, I. SABOLIC (2007): Renal expression of organic anion transporter
OAT2 in rats and mice is regulated by sex hormones. Am. J. Physiol. Renal Physiol. 292,
F361-F372.
MOTOHASHI, H., Y. SAKURAI, H. SAITO, S. MASUDA, Y. URAKAMI, M. GOTO,
A. FUKATSU, O. OGAWA, K. I. INUI (2002): Gene expression levels and
immunolocalization of organic ion transporters in the human kidney. J. Am. Soc. Nephrol.
13, 866-874.
SABOLIC, I., A. ASIF., W. BUDACH, C. WANKE, A. BAHN, G. BURCKHARDT,
(2007): Gender differences in kidney function. Pflugers Arch. Eur. J. Physiol. 455, 397429.
STIEGER, B., C. F. HIGGINS (Guest Editors) (2007): Twenty years of ABC transporters.
Pflügers Arch. Eur. J. Physiol. Vol. 453, No. 5.
9
SWEET, D. H., D. S. MILLER, J. B. PRITCHARD, Y. FUJIWARA, D. R. BEIER, S. K.
NIGAM (2002): Impaired organic anion transport in kidney and choroid plexus of organic
anion transporter 3 (Oat3 (Slc22a8)) knockout mice. J. Biol. Chem. 277, 26934-26943.
10