The liver and kidney expression of sulfate anion transporter sat-1 in rats exhibits male-dominant gender differences

Abstract

The sulfate anion transporter (sat-1, Slc26a1) has been cloned from rat liver, functionally characterized, and localized to the sinusoidal membrane in hepatocytes and basolateral membrane (BLM) in proximal tubules (PT). Here, we confirm previously described localization of sat-1 protein in rat liver and kidneys and report on gender differences (GD) in its expression by immunochemical, transport, and excretion studies in rats. The ∼85-kDa sat-1 protein was localized to the sinusoidal membrane in hepatocytes and BLM in renal cortical PT, with the maledominant expression. However, the real-time reversetranscription polymerase chain reaction data indicated no GD at the level of sat-1 mRNA. In agreement with the protein data, isolated membranes from both organs exhibited the male-dominant exchange of radiolabeled sulfate for oxalate, whereas higher oxalate in plasma and 24-h urine indicated higher oxalate production and excretion in male rats. Furthermore, the expression of liver, but not renal, sat-1 protein was: unaffected by castration, upregulated by ovariectomy, and downregulated by estrogen or progesterone treatment in males. Therefore, GD (males > females) in the expression of sat-1 protein in rat liver (and, possibly, kidneys) are caused by the female sexhormone-driven inhibition at the posttranscriptional level. The male-dominant abundance of sat-1 protein in liver may conform to elevated uptake of sulfate and extrusion of oxalate, causing higher plasma oxalate in males. Oxalate is then excreted by the kidneys via the basolateral sat-1 (males > females) and the apical CFEX (Slc26a6; GD unknown) in PT and eliminated in the urine (males > females), where it may contribute to the male-prevailing development of oxalate urolithiasis.

Oxalate: From the Environment to Kidney Stones

“…The SO 4 2-/anion exchange system that transports various anions including oxalate was demonstrated in the sinusoidal (114,115,121 membrane of rat hepatocytes (129). In support of these fi ndings, Sat-1, an exchanger of SO 4 2-and an oxalate, was localized in rats to the hepatocyte sinusoidal membrane (116,117,130). Recent transport studies on Sat-1 expressing oocytes demonstrated that Sat-1 can exchange SO 4 2-or HCO 3 -for oxalate in both directions, but the affi nity for oxalate is lower than for counter anions.…”

“…As shown in Fig. 4, in rat kidneys the Sat-1 protein was localized by immunochemical methods to the proximal tubule BLM (117,118).…”

“…However, real time RT-PCR data in the rat liver and kidney tissues showed an absence of signifi cant sex differences in Sat-1 mRNA expression, while Sat-1 protein expression and its activity, shown by Western blotting, immunocytochemistry, and transport studies in the same organs, were male-dominant, indicating a posttranscriptional regulation for this transporter. Additional experiments showed that, at least in the rat kidneys, the male-dominant sex differences in Sat-1 protein expression were driven by the inhibitory effects of estrogens and progesterone (117). In mice, sex hormones may also infl uence the expression of DRA in the large intestine; the expression of DRA mRNA in the colon of adult animals was 134-fold higher than in prepubertal mice, possibly due to the up-regulating effects of sex hormones after the onset of puberty (121).…”

“…Sat-1 (Slc26a1) is usually associated with kidneys and liver, but recently its mRNA expression was also detected in mouse cecum (112) and human small intestine and colon (113). However, although it is presumed that Sat-1 is localized in the BLM of intestinal epithelial cells (114,115), in rats an anti-Sat-1 antibody clearly labeled the Sat-1 protein in specifi c membrane domains of hepatocytes and proximal tubules (116)(117)(118), whereas in the intestine, the same antibody stained intracellular organelles, possibly mitochondria, with (11,14).…”

Role of microwave heating in antigen retrieval in cryosections of the formalin-fixed mammalian tissues

“…However, the treatment of tissue cryosections with sodium dodecyl sulfate (SDS), without heating, has been efficiently used to enhance immunostaining with some antibodies (Brown et al, 1996;Sabolić et al, 1999), thus indicating that unmasking techniques may be beneficial for revealing the antibody binding epitopes also in cryosections. Furthermore, we have recently described that heating cryosections of the PFAfixed rat and mouse organs in a microwave oven can be used to enhance immunostaining with specific antibodies for various transporters of organic anions (Bahn et al, 2005;Breljak et al, 2010;Brzica et al, 2009b;Ljubojević et al, 2004Ljubojević et al, & 2007Yokoyama et al, 2008), glucose (Balen et al, 2008;Sabolić et al, 2006), and sulfate (Brzica et al, 2009a). In order to demonstrate the importance of various unmasking protocols for immunocytochemical studies in tissue cryosections, we have used cryosections of the rat kidney and liver tissues that had been fixed with 4% PFA in vivo, treated them with various antigen retrieval protocols without and with the use of microwave heating, and tested for the intensity of immunostaining of several representative proteins known to reside in the cell membrane (cell adhesion molecule 105 (CAM105), megalin (GP 330 ), Na/K-ATPase, aquaporin 1 (AQP1)), cytoplasm (metallothionein), cell membrane and intracellular organelles (vacuolar H + -ATPase (V-ATPase)), and cytoskeleton (actin, tubulin).…”