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 ()

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).

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 mm-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 no-denaturing conditions). For experimental details of these techniques, see the recent publications by BRZICA et al., 2009a and 2009b. The renal cortical basolateral and brush-border membranes were isolated from tissue homogenates by the established methods (BIBER et al., 1981; SCALERA et al., 1981).

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 mm.

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., 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

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