A Simple Isolation Method for Basal-Lateral Plasma Membranes from Rat Kidney Cortex

Scalera, Vito; Huang, Yu-Kuo; Hildmann, Bruno; Murer, Heini

doi:10.3109/09687688109065422

Cited by 95 publications

(36 citation statements)

References 11 publications

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“…On the other hand, sulfate uptake (with inside HCO-= 50 mM) appeared to become saturated at about 10 mM so4, and half-maximal uptake occurred at 1-2 mM (Fig. 3B) (20 ,ug/mg of membrane protein). In the lower pair of curves (HCO-inside = outside), vesicles contained 100 mM mannitol/50 mM NaHCO3/10 mM Tris Hepes, pH 7.5, and were incubated with buffer containing 50 mM KCl, 50 mM NaHCO3, 10 mM Tris Hepes at pH 7.5, 0.5 mM CaCl2, and 50 ,1M 35SO2-with or without valinomycin.…”

Section: Methodsmentioning

confidence: 90%

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Renal sulfate transport at the basolateral membrane is mediated by anion exchange.

Pritchard¹,

Renfro²

1983

Proc. Natl. Acad. Sci. U.S.A.

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The transport of sulfate was studied in basolateral membrane (BLM) vesicles isolated from rat kidney cortex by centrifugation on a Percoll self-generating gradient. In contrast to sulfate transport at the luminal membrane, sulfate uptake by BLM vesicles was not sodium dependent. However, imposition of an inside greater than outside bicarbonate gradient stimulated BLM sulfate uptake nearly 10-fold and produced a transient overshoot of about 4-fold. This process appeared to become saturated at high concentrations of either bicarbonate or sulfate. Sulfate itself, thiosulfate, and hydroxyl, but not chloride or thiocyanate, were able to substitute for bicarbonate. None of these anions was as effective as bicarbonate. The sulfate/bicarbonate exchange was unaltered by manipulation of membrane potential, suggesting that it was electroneutral. Both bicarbonate stimulation and overshoot could be prevented by known inhibitors of anion transport, including mercuric chloride, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid, and phloretin. Bicarbonate-stimulated sulfate uptake also was inhibited by thiosulfate, probenecid, and acetazolamide. Thus, rat kidney BLM vesicles showed carrier-mediated anion exchange. Its properties indicate that this carrier may participate in both reabsorptive and secretory sulfate transport.

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Section: Methodsmentioning

confidence: 90%

“…BLM were isolated from the renal cortex of male Wistar rats (200-300 g) by density gradient centrifugation on self-orienting Percoll (Pharmacia) gradients essentially according to published methods (20,21). Briefly, rats were killed by decapitation and their kidneys were rapidly transferred to ice-cold saline.…”

Section: Methodsmentioning

confidence: 99%

Renal sulfate transport at the basolateral membrane is mediated by anion exchange.

Pritchard¹,

Renfro²

1983

Proc. Natl. Acad. Sci. U.S.A.

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“…BLM were isolated from the homogenates of various tissue zones by Percoll density gradient centrifugation (24), whereas BBM were isolated from the homogenates of cortical and outer stripe tissues of the rat kidney, or from the whole mouse kidney, by Mg 2ϩ /EGTA precipitation (4). The intestinal BBM were isolated from the rat jejunum, using the same method as for the renal membranes.…”

Section: Methodsmentioning

confidence: 99%

Rat renal glucose transporter SGLT1 exhibits zonal distribution and androgen-dependent gender differences

Sabolić¹,

Škarica²,

Gorboulev³

et al. 2006

American Journal of Physiology-Renal Physiology

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Sabolić, Ivan, Mario Škarica, Valentin Gorboulev, Marija Ljubojević, Daniela Balen, Carol M. Herak-Kramberger, and Hermann Koepsell. Rat renal glucose transporter SGLT1 exhibits zonal distribution and androgen-dependent gender differences. Am J Physiol Renal Physiol 290: F913-F926, 2006. First published October 4, 2005 doi:10.1152/ajprenal.00270.2005.-SGLT1 (SLC5A1) mediates a part of glucose and galactose reabsorption in the mammalian proximal tubule (PT), but the detailed localization of the transporter along the tubule is still disputable. Here, we used several methods to localize rat SGLT1 (rSGLT1) in the kidneys of intact and variously treated male (M) and female (F) rats. In immunoblots of isolated cortical (C) and outer stripe (OS) brush-border membranes (BBM), a peptide-specific polyclonal antibody for rSGLT1 labeled a sharp inzone-, and genderdependent ϳ40-kDa protein and a broad ϳ75-kDa band that exhibited strong zonal (OS Ͼ C) and gender differences (F Ͼ M). In tissue cryosections, the antibody strongly stained BBM of the S3 PT segments in the OS and medullary rays (F Ͼ M) and smooth muscles of the blood vessels and renal capsule (F ϳ M) and weakly stained the apical domain of other PT segments in the C (F ϳ M). The phlorizinsensitive uptake of D-[ 3 H]galactose in BBM vesicles, as well as the tissue abundance of rSGLT1-specific mRNA, matched the immunoblotting data related to the 75-kDa protein and the immunostaining in S3, proving zonal and gender differences in the functional transporter. Ovariectomy had no effect, castration upregulated, whereas treatment of castrated rats with testosterone, but not with estradiol or progesterone, downregulated the 75-kDa protein and the immunostaining in S3. We conclude that in the rat kidney, the expression of SGLT1 is represented by a 75-kDa protein localized largely in the PT S3 segments, where it exhibits gender differences (F Ͼ M) at both the protein and mRNA levels that are caused by androgen inhibition. brush-border membrane; immunocytochemistry; kidney; sex differences; sodium-glucose cotransporters; steroid hormones THE EFFICIENT REABSORPTION of the filtered hexoses in the mammalian nephron is achieved by the concerted action of Na ϩ -dependent glucose cotransporters (SGLT1 and SGLT2), localized in the brush-border membranes (BBM), and Na ϩ -independent facilitated glucose transporters (GLUT1 and GLUT2), localized in the basolateral membranes (BLM) of proximal tubule cells (Refs. 11 and 34 and references therein). The two major brush-border glucose transporters in the proximal tubule, low-affinity/high-capacity SGLT2 and high-affinity/low-capacity SGLT1, differ in their affinity for glucose and Na ϩ (SGLT1 Ͼ SGLT2), their sensitivity to inhibitor phlorizin (SGLT1 Ͼ SGLT2), and their sugar selectivity (SGLT1 transports glucose and galactose equally well, whereas SGLT2 transports glucose at least 10 times better). At the protein level, both transporters have ϳ660 amino acid residues that share 59% homology and probably function as glycosylated monomers ...

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“…The kidneys were sagittally sliced; the cortices were manually separated and pooled, dispersed, and homogenized in the buffer described above for liver, and the basolateral membranes (BLM) were isolated by differential centrifugation and Percoll density gradient [43]. The isolated BLM were dispersed in the homogenizing buffer and stored at −70°C until use.…”

Section: Membrane Preparation and Western Blottingmentioning

confidence: 99%

“…The pellet with the liver TCM was resuspended in preloading buffer (300 mM mannitol, 10 mM HEPES-Tris, pH 7.4 with or without 10 mM oxalic acid) and stored in liquid nitrogen. Renal BLM vesicles from kidney cortex were prepared as described in [43] and preloaded with the same buffer, with or without 10 mM oxalic acid.…”

Section: Transport Of [mentioning

confidence: 99%

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

Brzica

Breljak

Krick

et al. 2008

Pflugers Arch - Eur J Physiol

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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.

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A Simple Isolation Method for Basal-Lateral Plasma Membranes from Rat Kidney Cortex

Cited by 95 publications

References 11 publications

Renal sulfate transport at the basolateral membrane is mediated by anion exchange.

Renal sulfate transport at the basolateral membrane is mediated by anion exchange.

Rat renal glucose transporter SGLT1 exhibits zonal distribution and androgen-dependent gender differences

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

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