EXPRESSION OF OXALATE TRANSPORTERS IN RAT RENAL PROXIMAL
TUBULES IS SEX-DEPENDENT
Hrvoje Brzica, Daniela Balen, Davorka Breljak and Ivan Sabolić
Molecular Toxicology, Institute for Medical Research and Occupational Health,
Ksaverska cesta 2, 10001 Zagreb, Croatia (hbrzica@imi.hr)
Oxalate is an end product of liver metabolism of glycolate generated through series of
enzyme-catalyzed reactions (POORE at al., 1997). Two transport proteins appear to play
major role in handling oxalate in, and its excretion from the organism. In liver, an anion
antiporter sat-1 (Slc26a1) in the hepatocyte basolateral (sinusoidal) membrane extrudes
intracellular oxalate in exchange for extracellular sulfate. Oxalate in blood is finally
removed from the body by secretion in kidneys, which occurs in two steps: 1) the sat-1
protein in the proximal tubule basolateral membrane exchanges oxalate for sulfate or some
other intracellular anion, such as chloride, formate or bicarbonate, and 2) the intracellular
oxalate is then extruded from the cell into the tubule lumen via the CFEX protein
(Slc26a6) in exchange for the filtered anions (Fig. 1) (ARONSON, 2006; JIANG et al.,
2002).
1
Fig. 1. Proposed function of sat-1 and CFEX proteins in transporting common substrates and
secreting oxalate in the mammalian proximal tubule. Chloride, formate, sulfate or bicarbonate are
reabsorbed from the glomerular filtrate in exchange for oxalate in concerted actions of the
basolateral sat-1 and apical CFEX. Oxalate is thus secreted and removed in urine. GF - glomerular
filtrate, BLM – basolateral membrane, BBM - brush border membrane.
Oxalate is known to be one of the major compounds of kidney stones giving rise to 79% of
all cases of urolithiasis in humans (COE at al., 2005). Men in the middle age (30–59 years)
have three-fold higher incidence of oxalate kidney stones compared to women. However
these differences are not present in young and elderly population (COSTA-BAUZA et al.,
2007). It is unclear whether the sex-dependent oxalate urolithiasis is related to the activity
and/or expression of oxalate transporters.
We recently started immunochemical, transport and molecular biology studies in rat
kidneys in order to discern possible sex differences in the expression of transporters
responsible for oxalate secretion in the mammalian kidney, e.g., sat-1 and CFEX (BRZICA
et al. 2009a). In these studies we used male and female Wistar rats from the breeding
colony of the Institute for Medical Research and Occupational Health in Zagreb.
Immunocytochemical studies were performed on 4 m-thick tissue cryosections, using the
optimal antigen retrieval methods and specific polyclonal antibodies. The detergent (SDS)treatment of cryosections was applied to retrieve sat-1, whereas heating of cryosections in
microwave oven in citrate buffer, pH 8, was applied to retrieve CFEX. Western blotting
was performed with the renal cortical basolateral membranes for sat-1 (membranes
prepared in Laemmli buffer at 65°C/15 min in denaturing conditions) or brush border
membranes for CFEX (membranes prepared in Laemmli buffer at 65°C/15 min in nodenaturing conditions). For experimental details of these techniques, see the recent
publications by BRZICA et al., 2009a and 2009b. The renal cortical basolateral and brushborder membranes were isolated from tissue homogenates by the established methods
(BIBER et al., 1981; SCALERA et al., 1981).
2
In the mammalian kidney, oxalate is secreted into the urine, and the main localization of
this secretory process seems to be proximal convoluted tubules (S1/S2 segments) in the
kidney cortex. Indeed, as shown by indirect immunofluorescence cytochemistry in
cryosections of the rat kidney (Fig. 1a), in the cortical proximal tubule cells the sat1
protein is localized in the basolateral membrane, where it mediates transport of oxalate
from blood into the cell. On the other side, CFEX is localized in the luminal (brush border
membrane) of the same cells, where it mediates transport of oxalate into the urine (Fig.
2b). Furthermore, the Western blot studies in isolated cortical basolateral and brush border
membranes revealed the presence of male-dominant sex differences in the abundance of
both transporters (Fig. 2c and d).
Fig. 2. Expression of sat-1 (a, c) and CFEX (b, d) proteins in immunocytochemical (a, b) and
Western blotting (c, d) studies in cryosections of the kidney cortex and isolated membranes,
respectively. The sat-1 antibody strongly stained the basolateral membrane of proximal tubule
S1/S2 segments (a, arrows), while the CFEX antibody labeled the apical (brush border) membrane
of the same tubules (b, arrows). In Western blots, sat-1 was labeled as a single protein band of ~85
kDa (c), whereas CFEX was labeled as a single ~100 kDa protein band (d). Both transporters
exhibited the male dominant sex differences in their expression. M, males; F, females. Bar = 20
m.
Localization of sat-1 protein in the proximal tubule cell basolateral membrane, as well as
in the hepatocyte sinusoidal membrane, was originally described by KARNISKI et al.,
3
1998. and QUANDAMATTEO et al., 2006, respectively. Earlier transport studies
indicated that formate and oxalate stimulate Cl- absorption in proximal tubules, and that
this anion exchange is mediated by CFEX in the proximal tubule brush-border membrane
(WANG et al., 1996). As shown in Fig. 2, we confirmed the apical localization of CFEX
by immunocytochemistry and by Western blotting (unpublished data). According to its
substrate specificity, CFEX has been assumed to contribute to development of oxalate
urolithiasis. However, recent studies in CFEX-knockout mice showed that lack of CFEX is
associated with higher incidence of oxalate urolithiasis, thereby revealing possible
preventing role of CFEX on the development of kidney stones (JIANG et al., 2006).
We recently showed by transport studies in isolated membrane vesicles that basolateral
membranes from the rat kidney cortex, as well as the total cell membranes from the rat
liver, have higher sulfate uptake when preloaded with oxalate, and that this uptake is much
more apparent in vesicles derived from male then from female animals. In the same study
we measured ~80% higher oxalate levels in the male plasma, and twofold higher oxalate
concentration in the male urine (BRZICA et al., 2009a). These male-dominant sex
differences in the rat plasma and urine oxalate concentrations, as well as the comparable
sex differences in the rate of oxalate/sulfate exchange in membrane vesicles from the
kidney cortex, are well correlated with the comparable, sex-dependent abundance (male >
female) of the sat-1 and CFEX proteins in the respective membranes. A higher abundance
of oxalate transporters in the kidney membranes from male rats, as shown in our studies,
may contribute to formation and higher incidence of oxalate urolithiasis in male rats, and
possibly humans, but the nature and extent of this contribution for each transporter should
be further studied.
References
4
ARONSON, P. S. (2006): Essential roles of CFEX-mediated Cl− oxalate exchange in
proximal tubule NaCl transport and prevention of urolithiasis. Kidney Int. 70, 1207–1213.
BIBER, J., B. STIEGER, W. HAASE, H. MURER (1981): A high yield preparation for rat
kidney brush-border membranes: different behavior of lysosomal markers. Biochim.
Biophys. Acta 647, 169-176.
BRZICA, H., D. BRELJAK, W. KRICK, M. LOVRIĆ, G. BURCKHARDT, B.C.
BURCKHARDT, I. SABOLIĆ (2009a): The liver and kidney expression of sulfate anion
transporter sat-1 in rats exhibits male-dominant gender differences. Eur. J. Physiol. 457,
1381-1392.
BRZICA, H., D. BRELJAK, M. LJUBOJEVIĆ, D. BALEN, V. MICEK, N. ANZAI, I.
SABOLIĆ (2009b): Optimal methods of antigen retrival for organic anion transporters in
cryosections of rat kidney. Arh. Hig. Rada Toksikol. 60, 7-17.
COE, F. L., A. EVAN, E. WORCESTER (2005): Kidney stone disease. J. Clin. Invest.
115, 2598–2608.
COSTA-BAUZÀ, A., M. RAMIS, V. MOSTESINOS, A. CONTE, P. PIZÀ, E. PIERAS,
F. GARSES (2007): Type of renal calculi: variation with age and sex. World J. Urol. 25,
415–421.
JIANG, Z, I. I. GRICHTCHENKO, W. B. BORON, P. S. ARONSON (2002): Specificity
of anion exchange mediated by mouse Slc26a6. J. Biol. Chem. 277, 33963–33967.
JIANG, Z., J. R. ASPLIN, A. P. EVAN, V. M. RAJENDRAN, H. VELAZQUEZ, T. P.
NOTTOLI, H. J. BINDER, P. S. ARONSON (2006): Calcium oxalate urolithiasis in mice
lacking anion transporter Slc26a6. Nature Gen. 38, 474-478.
5
LEE, Y. H., W. C. HUANG, J. K. HUANG, L. S. CHANG (1996): Testosterone enhances
whereas estrogen inhibits calcium oxalate stone formation in ethylene glycol treated rats. J.
Urol. 156(2 Pt 1), 502-505.
KARNISKI, L. P., M. LÖTSCHER, M. FUCENTESE, H. HILFIKER, J. BIBER, H.
MURER (1998): Immunolocalization of sat-1 sulfate/oxalate/bicarbonate anion exchanger
in the rat kidney. Am. J. Physiol. Renal Physiol. 275, F79–F87.
POORE, R. E., C. H. HURST, D. G. ASSIMOS, R. P. HOLMES (1997): Pathways of
hepatic oxalate synthesis. Am. J. Physiol. Cell Physiol. 272, C289–C294.
QUONDAMATTEO, F., W. KRICK, Y. HAGOS, M. H. KRÜGER, K. NEUBAUERSAILE, R. HERKEN, G. RAMADORI, G. BURCKHARDT, B. C. BURCKHARDT
(2006): Localization of the sulfate/anion exchanger in the rat liver. Am. J. Physiol.
Gastrointest. Liver Physiol. 290, G1075–G1081.
SCALERA, V., Y. K. HUANG, B. HILDMAN, H. MURER (1981): A simple isolation
method for basal-lateral plasma membranes from rat kidney cortex. Membr. Biochem. 4,
49-64.
WANG, T., A. L. EGBERT, T. ABBIATI, P. S. ARONSON, G. GIEBISCH (1996):
Mechanisms of stimulation of proximal tubule chloride transport by formate and oxalate.
Am. J. Physiol. Renal Fluid Electrllyte Physiol. 40, F446 – F450
YOSHIHARA, H, S. YAMAGUCHI, S. YACHIKU (1999): Effect of sex hormones on
oxalate-synthesizing enzymes in male and female rat livers. J. Urol. 161, 668–673
6