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Metabolism of sulfolipids in isolated renal tubules from rat
Comparative Biochemistry and Physiology, Part B 140 (2005) 487 – 495 www.elsevier.com/locate/cbpb
Metabolism of sulfolipids in isolated renal tubules from rat Ken-ichi Nagaia, Keiko Tadano-Aritomia, Naoko Iida-Tanakaa,b, Hideki Yoshizawac, Ineo Ishizukaa,* a
Department of Biochemistry, Teikyo University School of Medicine, 2-11-1, Kaga, Itabashi, Tokyo 173-8605, Japan b Department of Food Science, Otsuma Woman’s University, 12, Sanbancho, Chiyoda, Tokyo 102–8357, Japan c Department of Biology, Matsumoto Dental College, 1780, Gobara Hirooka, Shiojiri, Nagano 399-0871, Japan Received 27 August 2004; received in revised form 16 November 2004; accepted 17 November 2004
Abstract Proximal-rich tubules were prepared from rat kidneys by using collagenase treatment. The isolated rat renal tubules were compared with the intact kidney on the following characteristics. (1) Composition of the sulfoglycolipid. (2) Sulfoglycolipid metabolism based on incorporation of [35S]sulfate or some properties of sulfoglycolipid metabolism, including the activities of anabolic and catabolic enzymes. The results indicated following characteristics of the isolated renal tubules in comparison to the kidney in vivo. (1) The sulfoglycolipid compositions are qualitatively similar, except that the content of glucosyl sulfatide, Gg3Cer II3-sulfate, and GM4 was slightly higher in the isolated tubules. (2) The apparent half-lives (15–55 min) of sulfoglycolipids in the isolated tubules could indicate the existence of a rapid turnover pool of these lipids. (3) The sulfotransferase and sulfatase activities related to sulfoamphiphiles in the renal tubule were similar to those reported for the whole kidney. Based on the above criteria, we conclude that the isolated rat renal tubule should be a useful metabolic system for clarification of the short-term physiological events, up to 90 min, of proximal tubular sulfoglycolipids. By using the present system, we showed that biosynthesis of the renal total sulfoglycolipid was significantly elevated in rats deprived of water for 24 h. D 2004 Elsevier Inc. All rights reserved. Keywords: Cholesterol sulfate; Collagenase; Dehydration; Half-life; LSIMS; Metabolism; Rat; Renal tubules; Sulfoglycolipids
1. Introduction Abbreviations: Chol, cholesterol; d18:1, 4-sphingenine; GalCer, galactosylceramide; GD3(NeuAc/NeuAc), II3-a-NeuAca8NeuAc-lactosylceramide; GM1a, II3-a-NeuAc-gangliotetraosylceramide; GM3(NeuAc), II3-a-NeuAc-lactosylceramide; GM4(NeuAc), I3-a-NeuAc-GalCer; HSO3Chol, cholesterol 3-sulfate; Lyso-SM4g, lyso seminolipid; NeuAc, N-acetylneuraminic acid; PAPS, 3V-phosphoadenylyl sulfate; SB1a, gangliotetraosylceramide II3,IV3-bis-sulfate; SB2, gangliotriaosylceramide II3,III3-bis-sulfate; SM2a, gangliotriaosylceramide II3-sulfate; SM3, lactosyl sulfatide, LacCer II3-sulfate; SM4s, galactosyl sulfatide, GalCer I3-sulfate; SM4s-Glc, glucosyl sulfatide, GlcCer I3-sulfate; SM4s-h, SM4s with hydroxy fatty acid; SM4s-nh, SM4s with nonhydroxy fatty acid; t18:0, 4-hydroxysphinganine. Abbreviations for lipids follow those of the IUPAC-IUB Joint Commission on Biochemical Nomenclature (IUPACIUB Joint Commission on Biochemical Nomenclature, 1999) and the symbols for sulfoglycolipids follow the system of Ishizuka (Ishizuka, 1997). * Corresponding author. Tel.: +81 3 3964 3749; fax: +81 3 5375 6366. E-mail address: [email protected] (I. Ishizuka). 1096-4959/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpc.2004.11.013
Sulfated amphiphiles, including sulfoglycolipids and cholesterol 3-sulfate (HSO3-Chol), are components of the cell membrane of the animals of deuterostome lineage, echinoderms to vertebrates (Ishizuka, 1997). Among various tissues of mammals, the kidney is one of the organs most enriched in these sulfolipids. Previous studies, based on the whole animal (Karlsson, 1982), whole kidney (Umeda et al., 1976), or renal epithelial cell lines (Niimura and Ishizuka, 1991), suggested that sulfated amphiphiles play specific roles in the maintenance of renal ionic homeostasis, although the operating biochemical mechanisms remain to be clarified (see Rev. Ishizuka, 1997). Wirthensohn and Guder (1990) reported that tubular suspensions prepared from rat kidneys by treatment with collagenase could be one of the systems suitable to study the renal metabolism. This system is not only
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convenient to process a large number of experiments simultaneously but also allows pretreatment of rats, in vivo, with hormones and drugs, or exposure to various environmental conditions, including salt-, water-load, and dehydration. The purpose of our present study is to establish a useful organ culture system for clarification of the functions of sulfolipids in renal tubules. To establish the validity of the present system, we compared the composition and metabolism of sulfolipids of isolated tubules with those of the whole rat kidney. Using this system, we showed that the synthesis of the total sulfoglycolipid was significantly elevated in the tubules, when the rats had been deprived of water for 24 h.
2. Materials and methods 2.1. Materials Male 6-week-old Wistar rats (Rattus norvegicus, 150– 180 g body mass) were purchased from SEASCO (Saitama, Japan) and fed with standard rat chow and tap water ad libitum. For in vivo dehydration experiment, rats were water-deprived for 24 h. The present study was carried out in accordance with the Teikyo University Guide for the Care and Use of Laboratory Animals, accredited by the Japanese Ministry of Education, Culture, Sports, Science and Technology. Every effort was taken to minimize any pain or discomfort of animals used in experiments. Reference gangliosides and sulfoglycolipids were prepared as previously described (Nagai et al., 1989). HSO3Chol, 2-hydroxypropyl-h-cyclodextrin, BSA essentially free of fatty acids, BSA-palmitic acid complex, 3V-phosphoadenylyl sulfate (PAPS), bovine brain galactosylceramide (GalCer), collagenase (type II from Clostridium histolyticum), 4-nitrocatechol, p-nitrocatechol sulfate (2-hydroxy-5nitrophenyl sulfate), 4-methylumbelliferone, and 4-methylumbelliferyl sulfate were purchased from Sigma-Aldrich Japan, Tokyo, Japan. Carrier-free H235SO4 and [35S]PAPS (59.2 GBq/mmol) were obtained from DuPont NEN Research Products, Wilmington, DE, USA. Sep-Pak Plus C18 cartridges (360 mg of sorbent, Waters, Tokyo, Japan) were prewashed sequentially with 10 mL portions of CHCl3/CH3OH/ H2O (30:60:8, by vol.), CH3OH, water, 0.1 M KCl; and Sep-Pak Plus NH2 (360 mg of sorbent) with 30 mL of water, 10 mL of CH3OH, and 10 mL of CHCl3/ CH3OH/H2O (30:60:8, by vol.). 2.2. Preparation of renal tubules from rat Tubules were isolated from rat kidneys according to the method of Guder (Guder et al., 1971; Guder, 1979) with slight modifications. Briefly, kidneys (1.2–1.4 g/animal) were removed from rats sacrificed under light ether anesthesia and the renal cortices cut out and minced with scissors. The tissue bbreiQ (0.8–1.0 g), prepared by forcing
the above tissue pieces through a piece of nylon mesh (aperture 512 Am), was freed from tissue debris by washing three times with cold sulfate-free Krebs–Henseleit medium in which NaHCO3 and MgSO4 were replaced with triethanolamine–HCl buffer (Guder et al., 1971) and MgCl2, respectively. The pelleted bbreiQ was digested with collagenase (1700 U/g brei in 10 mL) in the above medium under pure oxygen gas phase with vigorous shaking (37 8C for 45 min). After the reaction was terminated by the addition of 2 vol. of the ice-cold medium, the supernatant transferred to another tube, and centrifuged at 10g for 3 min to separate the tubules from subcellular particles and cell debris. Finally, the tubules were washed twice in the centrifuge and resuspended in a fresh medium (4 mL/g brei, corresponding to 15–20 mg protein). This preparation was used immediately for extraction of lipids, [35S]sulfate incorporation experiments as below, or stored at 80 8C for enzyme assay. 2.3. Protein quantitation The protein concentrations were determined using a modified method of Wang and Smith (1975). To eliminate the interference of triethanolamine in the Krebs–Henseleit medium, the tubules were washed three times with 0.9% NaCl before protein assay (Peterson, 1983). The renal tubules together with control BSA was solubilized in 1 M NaOH by heating (80 8C, 1 h) prior to protein assay. 2.4. Incorporation of [35S]sulfate into renal tubular sulfolipids The tubular suspension (0.1 mL, up to 0.5 mg protein) was transferred to a 15 mL polypropylene tube with a cap and incubated with the sulfate-free Krebs–Henseleit medium (final vol., 1 mL) containing 370 kBq of carrier-free H235SO4 and 5 mM glucose at 37 8C with gentle shaking under an O2 gas phase. Incubations were stopped by chilling in ice water at the indicated time, and then 0.9 mL of the suspensions was transferred to a microcentrifuge tube and tubules collected by short centrifugation (10,000g for 5 s; Microfuge B, Beckman, Palo Alto, CA, USA). The extraction procedure for total lipids from the isolated tubules using mixtures of chloroform/methanol/water was similar to those described previously (Tadano and Ishizuka, 1982a; Nagai et al., 1984; Iida et al., 1989). In order to remove essentially all glycerophospholipids, the total lipid extract was treated with 0.2 M NaOH in methanol and neutralized. The fraction of total acidic lipids was prepared as follows. The crude alkali-resistant lipids were suspended in 1 mL of 0.1 M KCl by brief sonication and transferred to a C18 cartridge with the help of a reservoir. The residual lipids in the tube were washed with two 2 mL portions of 0.1 M KCl and applied to the cartridge (Nagai and Ishizuka, 1987). After eluting nonlipid compounds with 30 mL of
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water, the inlet of an NH2 cartridge was connected tandem to the outlet of the C18 cartridge. The lipids adsorbed on the C18 cartridge were eluted with 10 mL of CHCl3/CH3OH/ H2O (30:60:8, by vol.). Finally, the acidic lipids retained on the NH2 cartridge were procured by elution with 5 mL of CHCl3/CH3OH/3.6 M CH3COONH4 (30:60:8, by vol.) and concentrated to a syrup. After removing ammonium acetate by sublimation in vacuo, an aliquot of the alkali-resistant total acidic lipid was used to determine the radioactivity by a liquid scintillation counter. The rest was separated by high-performance TLC (Merck, Darmstadt, Germany) in a solvent system, CHCl3/CH3OH/CH3COCH3/CH3COOH/ H2O (7:2:4:2:1, by vol.) and the distribution of incorporated radioactivities analyzed using BAS-1500 Bioimaging Analyzer (Fuji Film, Tokyo, Japan). By addition of 35S-sulfated lipid mixture of known composition and radioactivity to the total lipid extract, the overall recovery (%) of individual sulfated lipids in this assay system was calculated as follows (meanFS.D., n=5): HSO3-Chol (56.8F7.3); glucosyl sulfatide (SM4s-Glc; 46.5F8.2); galactosyl sulfatide with nonhydroxy fatty acid (SM4s-nh; 43.8F5.9) and with hydroxy fatty acid (SM4s-h; 41.1F5.0); lactosyl sulfatide (SM3; 52.1F5.0); gangliotriaosylceramide II3-sulfate (SM2a; 39.1F6.1); gangliotriaosylceramide II3 ,III3 -bis-sulfate (SB2; 20.6F2.8); gangliotetraosylceramide II 3 ,IV 3 -bis -sulfate (SB1a; 17.5F2.3). The incorporation of 35S-sulfate into individual sulfolipids was corrected for the above recovery. 2.5. Pulse-chase experiment A batch of tubules (up to 15 mg tissue protein/tube) was incubated with [35S]sulfate for 60 min, washed three times with the nonradioactive, modified Krebs–Henseleit medium containing sulfate ion at 48C, then divided into smaller portions (0.5 mg protein/tube), and chase studies were done for 24, 36, and 48 min. The lipids extracted from the tubules were separated on TLC, and the radioactivity incorporated into the total lipid and individual sulfolipids was determined as described above. The half-lives were estimated based on the replot of the radioactivity. 2.6. Two-dimensional TLC of acidic lipids from rat whole kidneys and isolated renal tubules The extraction and isolation procedure for alkaliresistant acidic lipids of rat whole kidneys and isolated tubules was similar to those described (Tadano and Ishizuka, 1982a; Nagai et al., 1984; Iida et al., 1989). The 35S-labeled whole kidneys of 6-week male Wistar rats were obtained 16 h after intraperitoneal injection of carrierfree H235SO4 (26–37 MBq). The tubules from nonlabeled whole kidneys were also incubated with [35S]sulfate in a large scale. The 35S- and nonlabeled acidic lipids from the whole kidneys and tubules were separated by two-dimensional TLC using the solvent systems CHCl3/CH3OH/H2O
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(60:40:9, by vol., first direction) and CHCl3/CH3OH/ CH3COCH3/CH3COOH/H2O (7:2:4:2:1, by vol., second direction). Radioactive spots were analyzed using BAS1500, and nonradioactive glycolipids were stained by spraying orcinol-sulfuric acid reagent followed by heating at 110 8C. 2.7. Negative-ion liquid secondary ion mass spectrometry Each acidic lipid developed by two-dimensional TLC was transferred to a polyvinylidene difluoride membrane by iron-blotting (Far-Eastern blotting; Taki and Ishikawa, 1997), and the band on the membrane was excised and placed on a mass spectrometer probe tip with triethanolamine as the matrix (Tadano-Aritomi et al., 2000). Spectra were recorded at an accelerating voltage of 8 kV, with a scan rate of 5 s/decade, and at a resolution of 1000–2000 on a Concept IH mass spectrometer (Shimadzu-Kratos, Kyoto, Japan; Tadano-Aritomi et al., 1995). 2.8. Enzyme assay GalCer sulfotransferase activities were determined according to Tadano and Ishizuka (1979) with a slight modification (Okuda, 1995). Homogenates of the isolated tubules as the enzyme source were prepared after once of freezing and thawing by sonication for 30 s in ice water using Handy Sonic UR-20P (Tomy Seiko, Tokyo, Japan). The reaction mixture contained 140 AM GalCer, 0.4% Triton X-100, 40 AM [35S]PAPS (0.6 kBq/nmol), 100 mM imidazole–HCl (pH 6.8), 40 mM CaCl2, 4 mM ATP, and tubular homogenates (50–100 Ag protein) in a total volume of 200 AL. The mixture was incubated at 37 8C for 2 h, and the reaction was terminated by chilling on ice water followed by addition of 4 mL of CHCl3/CH3OH (2:1, by vol.). After partition in the Folch system (Ishizuka et al., 1978a; Tadano-Aritomi and Ishizuka, 1983), the organic phase was washed twice with the theoretical upper phase, and radioactivities of the final organic phase were measured in a liquid scintillation counter. The recovery of [35S]SM4s from the lower phase was 95.2F1.8% (meanFS.D., n=6). Cholesterol (Chol) sulfotransferase was assayed similarly to the procedure reported by Cui and Iwamori (1997). Briefly, the isolated tubules were diluted to 2.5 vol. with water and homogenized by sonication as described above. After centrifugation at 13,000 g for 15 min, the supernatant was used for the enzyme assay. The reaction mixture (200 AL) contained 0.25 mM Chol; 4.37 AM [35S]PAPS (7.4 kBq/nmol); 100 mM phosphate buffer (pH 7.5); 1.25% 2-hydroxypropyl-h-cyclodextrin; 0.5 mM dithiothreitol; and 100 Ag of tubular protein. After incubation at 378C for 60 min, the radioactivity of the product was assayed similarly to the procedure for GalCer sulfotransferase (see above). The recovery of H35SO3-Chol was 89.0F2.6% (meanFS.D., n=6).
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After removing cell debris by centrifugation at 600 g for 15 min, the whole homogenates of the isolated tubules obtained as above were centrifuged at 108,000 g for 60 min, and the resulting cytosolic and microsomal fractions were used for arylsulfatase A, B, and C assays (Momoeda et al., 1994), respectively. The activities of lactate dehydrogenase leaked into the incubation medium were taken as the indices of the stability of tubular cells (Vassault, 1983). Prior to and after incubation for 90 min at 0 or 37 8C, the tubules were separated from the medium by centrifugation (10,000 g for 5 s) and the supernatants were used for lactate dehydrogenase assay. Total tubular lactate dehydrogenase activities were determined after lysing the tubules in 0.1% Triton X-100 (by vol.). For all figures, means and standard deviations were calculated. Statistical comparison of two means was performed using unpaired Student’s t-test.
3. Results 3.1. Isolation of renal tubules Renal tubules corresponding to 15–20 mg protein were obtained from a rat with the body weight of 150–180 g. By microscopic observation, the renal tubular cells showed an irregular luminal surface, typical for the brush border of proximal tubules (data not shown) in agreement with Pfaller et al. (1984), who demonstrated from morphological criteria that more than 90% of the preparations obtained by their method consisted of proximal tubules. Since subtle variations of the metabolic conditions between individual tubular preparations are not avoidable, a direct comparison could be possible only within an identical batch of tubules. Lactate dehydrogenase-leakage rates of isolated tubules in the modified Krebs–Henseleit medium at 0 8C were
Fig. 1. TLC imaging of the alkali-stable acidic lipids from isolated renal tubules as compared with whole kidney. The alkali-stable acidic lipids from whole kidneys (A) and isolated renal tubules (B) corresponding to 8 mg of protein were separated on TLC first with solvent system CHCl3/CH3OH/H2O (60:40:9, by vol.) followed by CHCl3/CH3OH/CH3COCH3/CH3COOH/H2O (7:2:4:2:1, by vol.) to the vertical dimension and stained with orcinol-sulfuric acid reagent. The reference acidic glycolipids were obtained from rat brain and kidney. 35S-sulfolipids (1200 dpm) from rat whole kidneys labeled in vivo for 16 h (C) and tubules labeled in vitro for 90 min (D) were separated similarly to the nonradioactive lipids. The assignment of each lipid (Arabic numerals) is described in text. In panel (C), the migration of SB2 (9) and SB1a (10) was distorted due to the large amount of nonradioactive components. The faint spots, x comigrating with SB1a and y, and SB2 could not be characterized by iron-blot-mass spectrometry because of the limited amount of material and/or coexistence of other lipids.
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2.8F0.5% and 4.1F0.1% (meanFS.D., n=4) of the total cellular lactate dehydrogenase activities at zero and after 90 min, respectively, indicating the stability of the tubules on ice. In contrast, at higher temperature, i.e., at 37 8C for 90 min, the leakage increased to 21.7F0.4%, probably due to the substantial disruption of the integrity of tubular cell membranes. In spite of the cell membrane damage, the incorporation of [35S]sulfate into sulfolipids in the renal tubules continued to increase linearly up to 120 min (see below). 3.2. Acidic lipids from rat whole kidney and isolated tubules The total alkali-resistant acidic lipids from isolated tubules were analyzed by two-dimensional TLC (Fig. 1B). The band assignment below was confirmed by TLC blotting-negative-ion liquid secondary ion mass spectrometry (spectra not shown). The ceramide composition (fatty acid/sphingoid base) and most abundant molecule-related ions (m/z) are shown in parentheses. 1, HSO3-Chol ([M– H] , 465); 2, SM4s-Glc (24:0/4-hydroxysphinganine [t18:0]) ([M–H] , 908) (Iida et al., 1989); 3a, SM4s-nh (24:0/4-sphingenine [d18:1]) (Tadano and Ishizuka, 1982a) ([M–H] , 890); 3b, SM4s-h (Tadano and Ishizuka, 1982a) (24 h:0/d18:1) ([M–H] , 906); 4, SM3 (24:0/d18:1) ([M– H] , 1052); 5, I3-a-NeuAc-GalCer [GM4(NeuAc)] (24:0/ d18:1) ([M–H] , 1101); 6, SM2a (22:0/t18:0) (Tadano and Ishizuka, 1982a) ([M–H] , 1245) (Tadano-Aritomi et al., 1995); 7a, II3-a-NeuAc-lactosylceramide [GM3(NeuAc)] (24:0/d18:1) ([M–H] , 1263); 7b, GM3(NeuAc) (16:0/ d18:1) ([M–H] , 1151); 8, II3-a-NeuAca8NeuAc-lactosylceramide [GD3(NeuAc/NeuAc)] (24:0/d18:1) ([M– 2H+Na]d , 1576). Molecule-related ions for SB2 and SB1a could not be detected due to a large amount of the comigrating GM3(NeuAc) (7a,b in Fig. 1B). The acidic lipid components in isolated tubules and agematched whole kidneys were qualitatively identical with some quantitative difference of certain components. The relative intensity of the orcinol stain of SM4s-Glc, GM4(NeuAc), and SM2a of the proximal tubules (Fig. 1B), assessed by image analyses using BAS system (data not shown), was apparently stronger than that of the whole kidney (Fig. 1A). In contrast, SM4s-nh of the tubules was weaker as compared with that of whole kidneys. By the present method, all the major sulfolipids of rat kidney (Tadano and Ishizuka, 1982a,b; Tadano et al., 1982; Iida et al., 1989) could be detected also in the lipid extracts of isolated tubules. However, the less abundant (below 0.3 nmol/g) sulfoglycolipids, including gangliotriaosylceramide III3-sulfate (Tadano-Aritomi et al., 1996), isoglobotetraosylceramide IV3-sulfate and globotetraosylceramide IV3sulfate (Tadano-Aritomi et al., 1992), isoglobopentaosylceramide V3-sulfate (SMiGb5; Tadano-Aritomi et al., 1994), and II3-a-NeuAc-gangliotetraosylceramide (GM1a IV3)sulfate (Tadano-Aritomi et al., 1998), were below the detection limit.
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3.3. [35S]sulfate incorporation into renal tubular sulfolipids 35
S-labeling profiles of the alkali-resistant acidic lipid fraction from whole kidney (Fig. 1C) and isolated tubules (Fig. 1D) were essentially similar to those of orcinol staining (Fig. 1A,B). The incorporation into the total and individual sulfolipids increased linearly up to approximately 900 Ag (SM4s-Glc; up to 700 Ag) of tubular protein (Fig. 2) and 120 min of incubation (data not shown). The efficiency of sulfate incorporation was doubled ( pb0.01) when triethanolamine-HCl, instead of NaHCO3CO2, was selected as the buffer constituent of the Krebs– Henseleit medium. Among the potential energy substrates tested (Fig. 3), gluconeogenic substrates, glutamine and lactate, increased the total sulfate incorporation by three to four times (glutamine, pb0.05; lactate, pb0.0005), in which the major increase corresponded to HSO3-Chol. The incorporation into SM4s-Glc and SM4s-h was also increased, while that into longer chain sulfoglycolipids was markedly inhibited. It has been widely accepted that proximal tubular cells preferred fatty acids as a source of energy and palmitate (final 1 mM) complex of BSA is the substrate of choice (Wirthensohn et al., 1984). Unexpectedly, the sulfate incorporation into all sulfoglycolipids was significantly suppressed, not only by BSA-palmitate complex ( pb0.05), but also by fatty acid-free BSA ( pb0.005). This unheralded result might be explained by elimination of carrier-free [35S]sulfate from the reaction medium by BSA because even 40 Ag per tube of BSA, corresponding to 1/80 of BSA-palmitate, inhibited the sulfate incorporation into the total sulfated lipids to only 25% of the control (Nagai et al., unpublished results). Based on the above results, we chose glucose as the energy substrate in this assay system.
Fig. 2. Protein dependency of the incorporation of [35S]sulfate into the sulfolipids of the isolated tubules. Incubations were carried out for 90 min, and each point represents the means of triplicate determinations. The incorporation into HSO3-Chol should be read on the right ordinate. The results for the total sulfolipids are shown in the inset.
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Fig. 3. Effects of potential renal energy substrates on [35S]sulfate incorporation into the sulfolipids of the isolated tubules. Cont—control; Glc—glucose; Gln— l-glutamine; BSA—fatty acid-free BSA (3.15 mg/tube); BSA+C16:0—palmitic acid (1 mM) complexed to BSA (3.15 mg/tube). Incubations were performed for 90 min at 37 8C. Error bars indicate S.D. (n=4) of total incorporation.
The apparent half-lives (min) of sulfolipids were estimated by pulse-chase experiments as follows: HSO3Chol, 50; SM4s-Glc, 20; SM4s-nh, 35; SM4s-h, 45; SM3, 30; SM2a, 55; SB2, 20; and SB1a, 15. 3.4. Anabolic and catabolic enzyme activities of the sulfolipids in the isolated renal tubules Enzyme activities of tubular preparations related to synthesis and degradation of the sulfolipids are listed in Table 1. Cholesterol sulfotransferase activity (14.0 pmol/mg protein/h) of the rat-isolated tubules was three times higher than that reported for rabbit kidney (5.2; Cui and Iwamori, 1997). GalCer sulfotransferase activity (169.5 pmol/mg protein/h) was comparable to those of rat (180.0; Tennekoon et al., 1985) and human kidney (131.5; Sakakibara et al.,
Table 1 Activities of cholesterol sulfotransferase (Chol-ST), galactosylceramide sulfotransferase (CST), and arylsulfatases A, B, and C in isolated renal tubules Enzymea
Acceptor (+)
(pmol/mg protein/h) Chol-ST 19.9F3.7 CST 183.6F39.2 (nmol/mg protein/h) Arylsulfatase A Arylsulfatase B Arylsulfatase C
Acceptor ( ) 5.9F1.6 5.6F4.1
Endogenousb 14.0F4.7 169.5F37.2
492.5F35.0 1,956.0F201.8 15.7F0.3
The assay conditions were described in Materials and methods. a Enzyme activities (meanFS.D., n=4) were assayed using a single batch of tubule preparation. b The values of acceptor ( ) were subtracted from those of acceptor (+).
1991) but much higher than that of mouse kidney (approximately 0.5 pmol/mg protein/h; Hirahara et al., 2000). The total arylsulfatase activity (A, B plus C; 2500 nmol/ mg protein/h) was similar to that of rat cortical tubules (Velosa et al., 1981), while arylsulfatase A and B activities were comparable to those of rat whole kidney (Potter et al., 1972) and rat renal cortex (Leznicki and Rozanska, 1991). 3.5. Effects of dehydration on the metabolism of total sulfoglycolipids in the isolated renal tubules Previously, we showed that the renal tubular cell line, MDCK, adapts to the elevated osmolality of the culture medium by up-regulating the synthesis of sulfoglycolipids (Niimura and Ishizuka, 1991). To examine whether concentration of urine resulting from dehydration of the animal also up-regulate the synthesis of total sulfoglycolipids in the renal tubules, rats were exposed to absolute water-deprivation for 24 h. 35S-incorporation into the total sulfoglycolipid and GalCer sulfotransferase activity of the isolated renal tubules Table 2 Effects of dehydration on 35S-incorporation into the total sulfoglycolipid and the activity of galactosylceramide sulfotransferase (CST) of the isolated renal tubules Activitya
Control
24 h-water-deprived
35
2696.7F723.6
5760.0F1995.2**
S-incorporation (dpm/mg/90 min; n=8) CSTb (pmol/mg/h; n=4)
116.8F14.6
155.6F14.2*
The assay conditions were described in Materials and methods. a Values are meansFS.D. b Endogenous activity was subtracted (cf. Table 1). * Significantly different ( pb0.01) from the control. ** Significantly different ( pb0.005) from the control.
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from control and water-deprived rats were listed in Table 2. The 35S-incorporation of the water-deprived rats was 213% of the control rats, most probably due to the elevated activity of GalCer sulfotransferase (Table 2). The activity of the arylsulfatase A remained unchanged (data not shown).
4. Discussion In the present study, we attempted to establish a model system using isolated rat renal tubules for studying the metabolism of sulfoglycolipids in the kidney. We observed that the sulfolipid profiles of the rat whole kidney and isolated renal tubules were essentially similar, except for an apparently stronger staining of SM4s-Glc, GM4, and SM2a for the tubules. Trick et al. (1999) reported that SM2a is localized to brush border of the proximal tubule of rat kidney based on the immunohistological examination. As to SM2a, their observation coincides with our results. The apparent half-lives of the isolated tubular sulfolipids (15–55 min) were considerably shorter than those of a renal epithelial cell line, MDCK (50–64 h; Niimura and Ishizuka, 1991), derived from the collecting tubule (Ishizuka et al., 1978b), most probably because the cell line has lost its original metabolic properties. The turnover rate of SM4s observed for isolated tubules is much faster than that reported for whole kidney of rats (Green and Robinson, 1960) and mice (Ishizuka and Tadano, 1982). It has been described for rodent brain that the half-life of the metabolically more active, rapidly turning-over pool was 100-fold shorter than that of the more stable, slowly turningover sulfatides (Jungalwala, 1974; Burkart et al., 1981, 1982). Burkart et al. speculated that most of the newly synthesized sulfatides are transported by vesicles from their site of synthesis in the Golgi-endoplasmic reticulum region to their site of incorporation, the myelin membrane in the myelinating mouse brain. Parts of this lysosome-associated sulfatides remain in the lysosomes and are degraded later. Thus, lysosomes may play a major role not only in degrading sulfatides but also in their transport to the target site, the myelin membrane (Burkart et al., 1982). They also proposed that net sulfatide synthesis is partly regulated by lysosomal degradation during myelination of the brain (Burkart et al., 1981). In analogy, there might be two putative subcellular pools of sulfoglycolipids, fast turnover (lysosome-associated) and slow turnover (plasma membrane-associated) one, in renal cells, and it is most likely that the tubular cells are typically rich in the rapidly turningover pool. In our preliminary experiments in vitro, we found that a short-time hypertonic stress by addition of NaCl or mannitol inhibited sulfate incorporation into the tubular sulfoglycolipids to approximately 1/2 to 1/3, while submillimolar lactate, one of the renal gluconeogenetic substrates, enhanced the incorporation into some sulfoglycolipids by two- to threefold in a dose-dependent manner (Nagai et al., unpublished results). These observations also
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suggest that the rapid turnover pool of tubular sulfoglycolipids may be quickly regulated by environmental factors, and that the [35S]sulfate incorporation could serve as the good index for elucidation of the physiological functions of renal sulfoglycolipids if the labeling period is restricted to a short time such as our assay system. It has been reported that the concentration of HSO3-Chol in the kidney was highest among various rat tissues examined (Iwamori et al., 1976). Furthermore, [35S]sulfate incorporation rate into this lipid was also highest among all sulfated amphiphiles of the renal tubules similar to that observed for whole kidneys (Ishizuka and Tadano, 1982). These results enabled us to estimate catabolic and anabolic activities of HSO3-Chol of the isolated tubules. Cholesterol sulfotransferase activity (14.0 pmol/mg protein/h) of rat tubules was about threefold higher than that of rabbit kidney (5.2; Cui and Iwamori, 1997; Table 1). This velocity was 1/ 12 lower than that (170 pmol/mg protein/h) of GalCer sulfotransferase which synthesizes SM4s (Table 1), while the [35S]sulfate incorporation rate into HSO3-Chol was slightly higher than that into SM4s. This discrepancy could be explained by the relatively high anabolic enzyme (arylsulfatase A) activity for SM4s compared with that (arylsulfatase C) for HSO3-Chol (Table 1), although the anabolic enzyme activities in this study were measured with artificial substrates. It has been reported that biosynthesis of sulfoglycolipids, especially of SM4s, was activated by hyperosmotic stimuli on the whole animal (Karlsson, 1982), whole kidney (Umeda et al., 1976), or renal epithelial cell lines (Niimura and Ishizuka, 1991). These authors suggested that the sulfated amphiphiles may play specific roles in the maintenance of renal ionic homeostasis. In the present study, we also observed that concentration of urine brought about by dehydration of the animal elicited by 24 h-water-deprivation up-regulated the activity of GalCer sulfotransferase and, consequently, increased the biosynthesis of sulfoglycolipids in the isolated renal tubules (Table 2). The up-regulation of sulfotransferase and elevated synthesis of renal sulfoglycolipids, similar to the cultured renal cell line exposed more than 6 h in hypertonic media, was demonstrated for the first time with mammals. In conclusion, we propose that the renal tubule system is convenient to perform experiments not only by using anisosmotic medium, addition of renal substrates, drugs, hormones in vitro, but also using rats preacclimatized to anisosmotic or nutritional conditions (dehydration, salt- or water-loading, feed, starve, etc.) in vivo.
Acknowledgments We are grateful to Drs. M. Kasahara and T. Kasahara (Department of Physics, Teikyo University School of Medicine) for giving facility for usage of BAS-2000 Bioimaging Analyzer in the initial part of this study.
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K. Nagai et al. / Comparative Biochemistry and Physiology, Part B 140 (2005) 487–495 Umeda, T., Egawa, K., Nagai, Y., 1976. Enhancement of sulphatide metabolism in the hypertrophied kidney of C3H/He mouse with reference to [Na+, K+]-dependent ATPase. Jpn. J. Exp. Med. 46, 87 – 94. Vassault, A., 1983. Lactate dehydrogenase. In: Bergmeyer, H.U., Bergmeyer, J., Grassl, M. (Eds.), Methods of Enzymatic Analysis, 3rd edition, vol. 3. Verlag Chemie, Weinheim, pp. 118 – 126. Velosa, J.A., Shah, S.V., Ou, S.L., Abboud, H.E., Dousa, T.P., 1981. Activities of lysosomal enzymes in isolated glomeruli. Alterations in experimental nephrosis. Lab. Invest. 45, 522 – 526.
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Metabolism of sulfolipids in isolated renal tubules from rat Ken-ichi Nagaia, Keiko Tadano-Aritomia, Naoko Iida-Tanakaa,b, Hideki Yoshizawac, Ineo Ishizukaa,* a
Department of Biochemistry, Teikyo University School of Medicine, 2-11-1, Kaga, Itabashi, Tokyo 173-8605, Japan b Department of Food Science, Otsuma Woman’s University, 12, Sanbancho, Chiyoda, Tokyo 102–8357, Japan c Department of Biology, Matsumoto Dental College, 1780, Gobara Hirooka, Shiojiri, Nagano 399-0871, Japan Received 27 August 2004; received in revised form 16 November 2004; accepted 17 November 2004
Abstract Proximal-rich tubules were prepared from rat kidneys by using collagenase treatment. The isolated rat renal tubules were compared with the intact kidney on the following characteristics. (1) Composition of the sulfoglycolipid. (2) Sulfoglycolipid metabolism based on incorporation of [35S]sulfate or some properties of sulfoglycolipid metabolism, including the activities of anabolic and catabolic enzymes. The results indicated following characteristics of the isolated renal tubules in comparison to the kidney in vivo. (1) The sulfoglycolipid compositions are qualitatively similar, except that the content of glucosyl sulfatide, Gg3Cer II3-sulfate, and GM4 was slightly higher in the isolated tubules. (2) The apparent half-lives (15–55 min) of sulfoglycolipids in the isolated tubules could indicate the existence of a rapid turnover pool of these lipids. (3) The sulfotransferase and sulfatase activities related to sulfoamphiphiles in the renal tubule were similar to those reported for the whole kidney. Based on the above criteria, we conclude that the isolated rat renal tubule should be a useful metabolic system for clarification of the short-term physiological events, up to 90 min, of proximal tubular sulfoglycolipids. By using the present system, we showed that biosynthesis of the renal total sulfoglycolipid was significantly elevated in rats deprived of water for 24 h. D 2004 Elsevier Inc. All rights reserved. Keywords: Cholesterol sulfate; Collagenase; Dehydration; Half-life; LSIMS; Metabolism; Rat; Renal tubules; Sulfoglycolipids
1. Introduction Abbreviations: Chol, cholesterol; d18:1, 4-sphingenine; GalCer, galactosylceramide; GD3(NeuAc/NeuAc), II3-a-NeuAca8NeuAc-lactosylceramide; GM1a, II3-a-NeuAc-gangliotetraosylceramide; GM3(NeuAc), II3-a-NeuAc-lactosylceramide; GM4(NeuAc), I3-a-NeuAc-GalCer; HSO3Chol, cholesterol 3-sulfate; Lyso-SM4g, lyso seminolipid; NeuAc, N-acetylneuraminic acid; PAPS, 3V-phosphoadenylyl sulfate; SB1a, gangliotetraosylceramide II3,IV3-bis-sulfate; SB2, gangliotriaosylceramide II3,III3-bis-sulfate; SM2a, gangliotriaosylceramide II3-sulfate; SM3, lactosyl sulfatide, LacCer II3-sulfate; SM4s, galactosyl sulfatide, GalCer I3-sulfate; SM4s-Glc, glucosyl sulfatide, GlcCer I3-sulfate; SM4s-h, SM4s with hydroxy fatty acid; SM4s-nh, SM4s with nonhydroxy fatty acid; t18:0, 4-hydroxysphinganine. Abbreviations for lipids follow those of the IUPAC-IUB Joint Commission on Biochemical Nomenclature (IUPACIUB Joint Commission on Biochemical Nomenclature, 1999) and the symbols for sulfoglycolipids follow the system of Ishizuka (Ishizuka, 1997). * Corresponding author. Tel.: +81 3 3964 3749; fax: +81 3 5375 6366. E-mail address: [email protected] (I. Ishizuka). 1096-4959/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpc.2004.11.013
Sulfated amphiphiles, including sulfoglycolipids and cholesterol 3-sulfate (HSO3-Chol), are components of the cell membrane of the animals of deuterostome lineage, echinoderms to vertebrates (Ishizuka, 1997). Among various tissues of mammals, the kidney is one of the organs most enriched in these sulfolipids. Previous studies, based on the whole animal (Karlsson, 1982), whole kidney (Umeda et al., 1976), or renal epithelial cell lines (Niimura and Ishizuka, 1991), suggested that sulfated amphiphiles play specific roles in the maintenance of renal ionic homeostasis, although the operating biochemical mechanisms remain to be clarified (see Rev. Ishizuka, 1997). Wirthensohn and Guder (1990) reported that tubular suspensions prepared from rat kidneys by treatment with collagenase could be one of the systems suitable to study the renal metabolism. This system is not only
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convenient to process a large number of experiments simultaneously but also allows pretreatment of rats, in vivo, with hormones and drugs, or exposure to various environmental conditions, including salt-, water-load, and dehydration. The purpose of our present study is to establish a useful organ culture system for clarification of the functions of sulfolipids in renal tubules. To establish the validity of the present system, we compared the composition and metabolism of sulfolipids of isolated tubules with those of the whole rat kidney. Using this system, we showed that the synthesis of the total sulfoglycolipid was significantly elevated in the tubules, when the rats had been deprived of water for 24 h.
2. Materials and methods 2.1. Materials Male 6-week-old Wistar rats (Rattus norvegicus, 150– 180 g body mass) were purchased from SEASCO (Saitama, Japan) and fed with standard rat chow and tap water ad libitum. For in vivo dehydration experiment, rats were water-deprived for 24 h. The present study was carried out in accordance with the Teikyo University Guide for the Care and Use of Laboratory Animals, accredited by the Japanese Ministry of Education, Culture, Sports, Science and Technology. Every effort was taken to minimize any pain or discomfort of animals used in experiments. Reference gangliosides and sulfoglycolipids were prepared as previously described (Nagai et al., 1989). HSO3Chol, 2-hydroxypropyl-h-cyclodextrin, BSA essentially free of fatty acids, BSA-palmitic acid complex, 3V-phosphoadenylyl sulfate (PAPS), bovine brain galactosylceramide (GalCer), collagenase (type II from Clostridium histolyticum), 4-nitrocatechol, p-nitrocatechol sulfate (2-hydroxy-5nitrophenyl sulfate), 4-methylumbelliferone, and 4-methylumbelliferyl sulfate were purchased from Sigma-Aldrich Japan, Tokyo, Japan. Carrier-free H235SO4 and [35S]PAPS (59.2 GBq/mmol) were obtained from DuPont NEN Research Products, Wilmington, DE, USA. Sep-Pak Plus C18 cartridges (360 mg of sorbent, Waters, Tokyo, Japan) were prewashed sequentially with 10 mL portions of CHCl3/CH3OH/ H2O (30:60:8, by vol.), CH3OH, water, 0.1 M KCl; and Sep-Pak Plus NH2 (360 mg of sorbent) with 30 mL of water, 10 mL of CH3OH, and 10 mL of CHCl3/ CH3OH/H2O (30:60:8, by vol.). 2.2. Preparation of renal tubules from rat Tubules were isolated from rat kidneys according to the method of Guder (Guder et al., 1971; Guder, 1979) with slight modifications. Briefly, kidneys (1.2–1.4 g/animal) were removed from rats sacrificed under light ether anesthesia and the renal cortices cut out and minced with scissors. The tissue bbreiQ (0.8–1.0 g), prepared by forcing
the above tissue pieces through a piece of nylon mesh (aperture 512 Am), was freed from tissue debris by washing three times with cold sulfate-free Krebs–Henseleit medium in which NaHCO3 and MgSO4 were replaced with triethanolamine–HCl buffer (Guder et al., 1971) and MgCl2, respectively. The pelleted bbreiQ was digested with collagenase (1700 U/g brei in 10 mL) in the above medium under pure oxygen gas phase with vigorous shaking (37 8C for 45 min). After the reaction was terminated by the addition of 2 vol. of the ice-cold medium, the supernatant transferred to another tube, and centrifuged at 10g for 3 min to separate the tubules from subcellular particles and cell debris. Finally, the tubules were washed twice in the centrifuge and resuspended in a fresh medium (4 mL/g brei, corresponding to 15–20 mg protein). This preparation was used immediately for extraction of lipids, [35S]sulfate incorporation experiments as below, or stored at 80 8C for enzyme assay. 2.3. Protein quantitation The protein concentrations were determined using a modified method of Wang and Smith (1975). To eliminate the interference of triethanolamine in the Krebs–Henseleit medium, the tubules were washed three times with 0.9% NaCl before protein assay (Peterson, 1983). The renal tubules together with control BSA was solubilized in 1 M NaOH by heating (80 8C, 1 h) prior to protein assay. 2.4. Incorporation of [35S]sulfate into renal tubular sulfolipids The tubular suspension (0.1 mL, up to 0.5 mg protein) was transferred to a 15 mL polypropylene tube with a cap and incubated with the sulfate-free Krebs–Henseleit medium (final vol., 1 mL) containing 370 kBq of carrier-free H235SO4 and 5 mM glucose at 37 8C with gentle shaking under an O2 gas phase. Incubations were stopped by chilling in ice water at the indicated time, and then 0.9 mL of the suspensions was transferred to a microcentrifuge tube and tubules collected by short centrifugation (10,000g for 5 s; Microfuge B, Beckman, Palo Alto, CA, USA). The extraction procedure for total lipids from the isolated tubules using mixtures of chloroform/methanol/water was similar to those described previously (Tadano and Ishizuka, 1982a; Nagai et al., 1984; Iida et al., 1989). In order to remove essentially all glycerophospholipids, the total lipid extract was treated with 0.2 M NaOH in methanol and neutralized. The fraction of total acidic lipids was prepared as follows. The crude alkali-resistant lipids were suspended in 1 mL of 0.1 M KCl by brief sonication and transferred to a C18 cartridge with the help of a reservoir. The residual lipids in the tube were washed with two 2 mL portions of 0.1 M KCl and applied to the cartridge (Nagai and Ishizuka, 1987). After eluting nonlipid compounds with 30 mL of
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water, the inlet of an NH2 cartridge was connected tandem to the outlet of the C18 cartridge. The lipids adsorbed on the C18 cartridge were eluted with 10 mL of CHCl3/CH3OH/ H2O (30:60:8, by vol.). Finally, the acidic lipids retained on the NH2 cartridge were procured by elution with 5 mL of CHCl3/CH3OH/3.6 M CH3COONH4 (30:60:8, by vol.) and concentrated to a syrup. After removing ammonium acetate by sublimation in vacuo, an aliquot of the alkali-resistant total acidic lipid was used to determine the radioactivity by a liquid scintillation counter. The rest was separated by high-performance TLC (Merck, Darmstadt, Germany) in a solvent system, CHCl3/CH3OH/CH3COCH3/CH3COOH/ H2O (7:2:4:2:1, by vol.) and the distribution of incorporated radioactivities analyzed using BAS-1500 Bioimaging Analyzer (Fuji Film, Tokyo, Japan). By addition of 35S-sulfated lipid mixture of known composition and radioactivity to the total lipid extract, the overall recovery (%) of individual sulfated lipids in this assay system was calculated as follows (meanFS.D., n=5): HSO3-Chol (56.8F7.3); glucosyl sulfatide (SM4s-Glc; 46.5F8.2); galactosyl sulfatide with nonhydroxy fatty acid (SM4s-nh; 43.8F5.9) and with hydroxy fatty acid (SM4s-h; 41.1F5.0); lactosyl sulfatide (SM3; 52.1F5.0); gangliotriaosylceramide II3-sulfate (SM2a; 39.1F6.1); gangliotriaosylceramide II3 ,III3 -bis-sulfate (SB2; 20.6F2.8); gangliotetraosylceramide II 3 ,IV 3 -bis -sulfate (SB1a; 17.5F2.3). The incorporation of 35S-sulfate into individual sulfolipids was corrected for the above recovery. 2.5. Pulse-chase experiment A batch of tubules (up to 15 mg tissue protein/tube) was incubated with [35S]sulfate for 60 min, washed three times with the nonradioactive, modified Krebs–Henseleit medium containing sulfate ion at 48C, then divided into smaller portions (0.5 mg protein/tube), and chase studies were done for 24, 36, and 48 min. The lipids extracted from the tubules were separated on TLC, and the radioactivity incorporated into the total lipid and individual sulfolipids was determined as described above. The half-lives were estimated based on the replot of the radioactivity. 2.6. Two-dimensional TLC of acidic lipids from rat whole kidneys and isolated renal tubules The extraction and isolation procedure for alkaliresistant acidic lipids of rat whole kidneys and isolated tubules was similar to those described (Tadano and Ishizuka, 1982a; Nagai et al., 1984; Iida et al., 1989). The 35S-labeled whole kidneys of 6-week male Wistar rats were obtained 16 h after intraperitoneal injection of carrierfree H235SO4 (26–37 MBq). The tubules from nonlabeled whole kidneys were also incubated with [35S]sulfate in a large scale. The 35S- and nonlabeled acidic lipids from the whole kidneys and tubules were separated by two-dimensional TLC using the solvent systems CHCl3/CH3OH/H2O
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(60:40:9, by vol., first direction) and CHCl3/CH3OH/ CH3COCH3/CH3COOH/H2O (7:2:4:2:1, by vol., second direction). Radioactive spots were analyzed using BAS1500, and nonradioactive glycolipids were stained by spraying orcinol-sulfuric acid reagent followed by heating at 110 8C. 2.7. Negative-ion liquid secondary ion mass spectrometry Each acidic lipid developed by two-dimensional TLC was transferred to a polyvinylidene difluoride membrane by iron-blotting (Far-Eastern blotting; Taki and Ishikawa, 1997), and the band on the membrane was excised and placed on a mass spectrometer probe tip with triethanolamine as the matrix (Tadano-Aritomi et al., 2000). Spectra were recorded at an accelerating voltage of 8 kV, with a scan rate of 5 s/decade, and at a resolution of 1000–2000 on a Concept IH mass spectrometer (Shimadzu-Kratos, Kyoto, Japan; Tadano-Aritomi et al., 1995). 2.8. Enzyme assay GalCer sulfotransferase activities were determined according to Tadano and Ishizuka (1979) with a slight modification (Okuda, 1995). Homogenates of the isolated tubules as the enzyme source were prepared after once of freezing and thawing by sonication for 30 s in ice water using Handy Sonic UR-20P (Tomy Seiko, Tokyo, Japan). The reaction mixture contained 140 AM GalCer, 0.4% Triton X-100, 40 AM [35S]PAPS (0.6 kBq/nmol), 100 mM imidazole–HCl (pH 6.8), 40 mM CaCl2, 4 mM ATP, and tubular homogenates (50–100 Ag protein) in a total volume of 200 AL. The mixture was incubated at 37 8C for 2 h, and the reaction was terminated by chilling on ice water followed by addition of 4 mL of CHCl3/CH3OH (2:1, by vol.). After partition in the Folch system (Ishizuka et al., 1978a; Tadano-Aritomi and Ishizuka, 1983), the organic phase was washed twice with the theoretical upper phase, and radioactivities of the final organic phase were measured in a liquid scintillation counter. The recovery of [35S]SM4s from the lower phase was 95.2F1.8% (meanFS.D., n=6). Cholesterol (Chol) sulfotransferase was assayed similarly to the procedure reported by Cui and Iwamori (1997). Briefly, the isolated tubules were diluted to 2.5 vol. with water and homogenized by sonication as described above. After centrifugation at 13,000 g for 15 min, the supernatant was used for the enzyme assay. The reaction mixture (200 AL) contained 0.25 mM Chol; 4.37 AM [35S]PAPS (7.4 kBq/nmol); 100 mM phosphate buffer (pH 7.5); 1.25% 2-hydroxypropyl-h-cyclodextrin; 0.5 mM dithiothreitol; and 100 Ag of tubular protein. After incubation at 378C for 60 min, the radioactivity of the product was assayed similarly to the procedure for GalCer sulfotransferase (see above). The recovery of H35SO3-Chol was 89.0F2.6% (meanFS.D., n=6).
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After removing cell debris by centrifugation at 600 g for 15 min, the whole homogenates of the isolated tubules obtained as above were centrifuged at 108,000 g for 60 min, and the resulting cytosolic and microsomal fractions were used for arylsulfatase A, B, and C assays (Momoeda et al., 1994), respectively. The activities of lactate dehydrogenase leaked into the incubation medium were taken as the indices of the stability of tubular cells (Vassault, 1983). Prior to and after incubation for 90 min at 0 or 37 8C, the tubules were separated from the medium by centrifugation (10,000 g for 5 s) and the supernatants were used for lactate dehydrogenase assay. Total tubular lactate dehydrogenase activities were determined after lysing the tubules in 0.1% Triton X-100 (by vol.). For all figures, means and standard deviations were calculated. Statistical comparison of two means was performed using unpaired Student’s t-test.
3. Results 3.1. Isolation of renal tubules Renal tubules corresponding to 15–20 mg protein were obtained from a rat with the body weight of 150–180 g. By microscopic observation, the renal tubular cells showed an irregular luminal surface, typical for the brush border of proximal tubules (data not shown) in agreement with Pfaller et al. (1984), who demonstrated from morphological criteria that more than 90% of the preparations obtained by their method consisted of proximal tubules. Since subtle variations of the metabolic conditions between individual tubular preparations are not avoidable, a direct comparison could be possible only within an identical batch of tubules. Lactate dehydrogenase-leakage rates of isolated tubules in the modified Krebs–Henseleit medium at 0 8C were
Fig. 1. TLC imaging of the alkali-stable acidic lipids from isolated renal tubules as compared with whole kidney. The alkali-stable acidic lipids from whole kidneys (A) and isolated renal tubules (B) corresponding to 8 mg of protein were separated on TLC first with solvent system CHCl3/CH3OH/H2O (60:40:9, by vol.) followed by CHCl3/CH3OH/CH3COCH3/CH3COOH/H2O (7:2:4:2:1, by vol.) to the vertical dimension and stained with orcinol-sulfuric acid reagent. The reference acidic glycolipids were obtained from rat brain and kidney. 35S-sulfolipids (1200 dpm) from rat whole kidneys labeled in vivo for 16 h (C) and tubules labeled in vitro for 90 min (D) were separated similarly to the nonradioactive lipids. The assignment of each lipid (Arabic numerals) is described in text. In panel (C), the migration of SB2 (9) and SB1a (10) was distorted due to the large amount of nonradioactive components. The faint spots, x comigrating with SB1a and y, and SB2 could not be characterized by iron-blot-mass spectrometry because of the limited amount of material and/or coexistence of other lipids.
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2.8F0.5% and 4.1F0.1% (meanFS.D., n=4) of the total cellular lactate dehydrogenase activities at zero and after 90 min, respectively, indicating the stability of the tubules on ice. In contrast, at higher temperature, i.e., at 37 8C for 90 min, the leakage increased to 21.7F0.4%, probably due to the substantial disruption of the integrity of tubular cell membranes. In spite of the cell membrane damage, the incorporation of [35S]sulfate into sulfolipids in the renal tubules continued to increase linearly up to 120 min (see below). 3.2. Acidic lipids from rat whole kidney and isolated tubules The total alkali-resistant acidic lipids from isolated tubules were analyzed by two-dimensional TLC (Fig. 1B). The band assignment below was confirmed by TLC blotting-negative-ion liquid secondary ion mass spectrometry (spectra not shown). The ceramide composition (fatty acid/sphingoid base) and most abundant molecule-related ions (m/z) are shown in parentheses. 1, HSO3-Chol ([M– H] , 465); 2, SM4s-Glc (24:0/4-hydroxysphinganine [t18:0]) ([M–H] , 908) (Iida et al., 1989); 3a, SM4s-nh (24:0/4-sphingenine [d18:1]) (Tadano and Ishizuka, 1982a) ([M–H] , 890); 3b, SM4s-h (Tadano and Ishizuka, 1982a) (24 h:0/d18:1) ([M–H] , 906); 4, SM3 (24:0/d18:1) ([M– H] , 1052); 5, I3-a-NeuAc-GalCer [GM4(NeuAc)] (24:0/ d18:1) ([M–H] , 1101); 6, SM2a (22:0/t18:0) (Tadano and Ishizuka, 1982a) ([M–H] , 1245) (Tadano-Aritomi et al., 1995); 7a, II3-a-NeuAc-lactosylceramide [GM3(NeuAc)] (24:0/d18:1) ([M–H] , 1263); 7b, GM3(NeuAc) (16:0/ d18:1) ([M–H] , 1151); 8, II3-a-NeuAca8NeuAc-lactosylceramide [GD3(NeuAc/NeuAc)] (24:0/d18:1) ([M– 2H+Na]d , 1576). Molecule-related ions for SB2 and SB1a could not be detected due to a large amount of the comigrating GM3(NeuAc) (7a,b in Fig. 1B). The acidic lipid components in isolated tubules and agematched whole kidneys were qualitatively identical with some quantitative difference of certain components. The relative intensity of the orcinol stain of SM4s-Glc, GM4(NeuAc), and SM2a of the proximal tubules (Fig. 1B), assessed by image analyses using BAS system (data not shown), was apparently stronger than that of the whole kidney (Fig. 1A). In contrast, SM4s-nh of the tubules was weaker as compared with that of whole kidneys. By the present method, all the major sulfolipids of rat kidney (Tadano and Ishizuka, 1982a,b; Tadano et al., 1982; Iida et al., 1989) could be detected also in the lipid extracts of isolated tubules. However, the less abundant (below 0.3 nmol/g) sulfoglycolipids, including gangliotriaosylceramide III3-sulfate (Tadano-Aritomi et al., 1996), isoglobotetraosylceramide IV3-sulfate and globotetraosylceramide IV3sulfate (Tadano-Aritomi et al., 1992), isoglobopentaosylceramide V3-sulfate (SMiGb5; Tadano-Aritomi et al., 1994), and II3-a-NeuAc-gangliotetraosylceramide (GM1a IV3)sulfate (Tadano-Aritomi et al., 1998), were below the detection limit.
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3.3. [35S]sulfate incorporation into renal tubular sulfolipids 35
S-labeling profiles of the alkali-resistant acidic lipid fraction from whole kidney (Fig. 1C) and isolated tubules (Fig. 1D) were essentially similar to those of orcinol staining (Fig. 1A,B). The incorporation into the total and individual sulfolipids increased linearly up to approximately 900 Ag (SM4s-Glc; up to 700 Ag) of tubular protein (Fig. 2) and 120 min of incubation (data not shown). The efficiency of sulfate incorporation was doubled ( pb0.01) when triethanolamine-HCl, instead of NaHCO3CO2, was selected as the buffer constituent of the Krebs– Henseleit medium. Among the potential energy substrates tested (Fig. 3), gluconeogenic substrates, glutamine and lactate, increased the total sulfate incorporation by three to four times (glutamine, pb0.05; lactate, pb0.0005), in which the major increase corresponded to HSO3-Chol. The incorporation into SM4s-Glc and SM4s-h was also increased, while that into longer chain sulfoglycolipids was markedly inhibited. It has been widely accepted that proximal tubular cells preferred fatty acids as a source of energy and palmitate (final 1 mM) complex of BSA is the substrate of choice (Wirthensohn et al., 1984). Unexpectedly, the sulfate incorporation into all sulfoglycolipids was significantly suppressed, not only by BSA-palmitate complex ( pb0.05), but also by fatty acid-free BSA ( pb0.005). This unheralded result might be explained by elimination of carrier-free [35S]sulfate from the reaction medium by BSA because even 40 Ag per tube of BSA, corresponding to 1/80 of BSA-palmitate, inhibited the sulfate incorporation into the total sulfated lipids to only 25% of the control (Nagai et al., unpublished results). Based on the above results, we chose glucose as the energy substrate in this assay system.
Fig. 2. Protein dependency of the incorporation of [35S]sulfate into the sulfolipids of the isolated tubules. Incubations were carried out for 90 min, and each point represents the means of triplicate determinations. The incorporation into HSO3-Chol should be read on the right ordinate. The results for the total sulfolipids are shown in the inset.
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Fig. 3. Effects of potential renal energy substrates on [35S]sulfate incorporation into the sulfolipids of the isolated tubules. Cont—control; Glc—glucose; Gln— l-glutamine; BSA—fatty acid-free BSA (3.15 mg/tube); BSA+C16:0—palmitic acid (1 mM) complexed to BSA (3.15 mg/tube). Incubations were performed for 90 min at 37 8C. Error bars indicate S.D. (n=4) of total incorporation.
The apparent half-lives (min) of sulfolipids were estimated by pulse-chase experiments as follows: HSO3Chol, 50; SM4s-Glc, 20; SM4s-nh, 35; SM4s-h, 45; SM3, 30; SM2a, 55; SB2, 20; and SB1a, 15. 3.4. Anabolic and catabolic enzyme activities of the sulfolipids in the isolated renal tubules Enzyme activities of tubular preparations related to synthesis and degradation of the sulfolipids are listed in Table 1. Cholesterol sulfotransferase activity (14.0 pmol/mg protein/h) of the rat-isolated tubules was three times higher than that reported for rabbit kidney (5.2; Cui and Iwamori, 1997). GalCer sulfotransferase activity (169.5 pmol/mg protein/h) was comparable to those of rat (180.0; Tennekoon et al., 1985) and human kidney (131.5; Sakakibara et al.,
Table 1 Activities of cholesterol sulfotransferase (Chol-ST), galactosylceramide sulfotransferase (CST), and arylsulfatases A, B, and C in isolated renal tubules Enzymea
Acceptor (+)
(pmol/mg protein/h) Chol-ST 19.9F3.7 CST 183.6F39.2 (nmol/mg protein/h) Arylsulfatase A Arylsulfatase B Arylsulfatase C
Acceptor ( ) 5.9F1.6 5.6F4.1
Endogenousb 14.0F4.7 169.5F37.2
492.5F35.0 1,956.0F201.8 15.7F0.3
The assay conditions were described in Materials and methods. a Enzyme activities (meanFS.D., n=4) were assayed using a single batch of tubule preparation. b The values of acceptor ( ) were subtracted from those of acceptor (+).
1991) but much higher than that of mouse kidney (approximately 0.5 pmol/mg protein/h; Hirahara et al., 2000). The total arylsulfatase activity (A, B plus C; 2500 nmol/ mg protein/h) was similar to that of rat cortical tubules (Velosa et al., 1981), while arylsulfatase A and B activities were comparable to those of rat whole kidney (Potter et al., 1972) and rat renal cortex (Leznicki and Rozanska, 1991). 3.5. Effects of dehydration on the metabolism of total sulfoglycolipids in the isolated renal tubules Previously, we showed that the renal tubular cell line, MDCK, adapts to the elevated osmolality of the culture medium by up-regulating the synthesis of sulfoglycolipids (Niimura and Ishizuka, 1991). To examine whether concentration of urine resulting from dehydration of the animal also up-regulate the synthesis of total sulfoglycolipids in the renal tubules, rats were exposed to absolute water-deprivation for 24 h. 35S-incorporation into the total sulfoglycolipid and GalCer sulfotransferase activity of the isolated renal tubules Table 2 Effects of dehydration on 35S-incorporation into the total sulfoglycolipid and the activity of galactosylceramide sulfotransferase (CST) of the isolated renal tubules Activitya
Control
24 h-water-deprived
35
2696.7F723.6
5760.0F1995.2**
S-incorporation (dpm/mg/90 min; n=8) CSTb (pmol/mg/h; n=4)
116.8F14.6
155.6F14.2*
The assay conditions were described in Materials and methods. a Values are meansFS.D. b Endogenous activity was subtracted (cf. Table 1). * Significantly different ( pb0.01) from the control. ** Significantly different ( pb0.005) from the control.
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from control and water-deprived rats were listed in Table 2. The 35S-incorporation of the water-deprived rats was 213% of the control rats, most probably due to the elevated activity of GalCer sulfotransferase (Table 2). The activity of the arylsulfatase A remained unchanged (data not shown).
4. Discussion In the present study, we attempted to establish a model system using isolated rat renal tubules for studying the metabolism of sulfoglycolipids in the kidney. We observed that the sulfolipid profiles of the rat whole kidney and isolated renal tubules were essentially similar, except for an apparently stronger staining of SM4s-Glc, GM4, and SM2a for the tubules. Trick et al. (1999) reported that SM2a is localized to brush border of the proximal tubule of rat kidney based on the immunohistological examination. As to SM2a, their observation coincides with our results. The apparent half-lives of the isolated tubular sulfolipids (15–55 min) were considerably shorter than those of a renal epithelial cell line, MDCK (50–64 h; Niimura and Ishizuka, 1991), derived from the collecting tubule (Ishizuka et al., 1978b), most probably because the cell line has lost its original metabolic properties. The turnover rate of SM4s observed for isolated tubules is much faster than that reported for whole kidney of rats (Green and Robinson, 1960) and mice (Ishizuka and Tadano, 1982). It has been described for rodent brain that the half-life of the metabolically more active, rapidly turning-over pool was 100-fold shorter than that of the more stable, slowly turningover sulfatides (Jungalwala, 1974; Burkart et al., 1981, 1982). Burkart et al. speculated that most of the newly synthesized sulfatides are transported by vesicles from their site of synthesis in the Golgi-endoplasmic reticulum region to their site of incorporation, the myelin membrane in the myelinating mouse brain. Parts of this lysosome-associated sulfatides remain in the lysosomes and are degraded later. Thus, lysosomes may play a major role not only in degrading sulfatides but also in their transport to the target site, the myelin membrane (Burkart et al., 1982). They also proposed that net sulfatide synthesis is partly regulated by lysosomal degradation during myelination of the brain (Burkart et al., 1981). In analogy, there might be two putative subcellular pools of sulfoglycolipids, fast turnover (lysosome-associated) and slow turnover (plasma membrane-associated) one, in renal cells, and it is most likely that the tubular cells are typically rich in the rapidly turningover pool. In our preliminary experiments in vitro, we found that a short-time hypertonic stress by addition of NaCl or mannitol inhibited sulfate incorporation into the tubular sulfoglycolipids to approximately 1/2 to 1/3, while submillimolar lactate, one of the renal gluconeogenetic substrates, enhanced the incorporation into some sulfoglycolipids by two- to threefold in a dose-dependent manner (Nagai et al., unpublished results). These observations also
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suggest that the rapid turnover pool of tubular sulfoglycolipids may be quickly regulated by environmental factors, and that the [35S]sulfate incorporation could serve as the good index for elucidation of the physiological functions of renal sulfoglycolipids if the labeling period is restricted to a short time such as our assay system. It has been reported that the concentration of HSO3-Chol in the kidney was highest among various rat tissues examined (Iwamori et al., 1976). Furthermore, [35S]sulfate incorporation rate into this lipid was also highest among all sulfated amphiphiles of the renal tubules similar to that observed for whole kidneys (Ishizuka and Tadano, 1982). These results enabled us to estimate catabolic and anabolic activities of HSO3-Chol of the isolated tubules. Cholesterol sulfotransferase activity (14.0 pmol/mg protein/h) of rat tubules was about threefold higher than that of rabbit kidney (5.2; Cui and Iwamori, 1997; Table 1). This velocity was 1/ 12 lower than that (170 pmol/mg protein/h) of GalCer sulfotransferase which synthesizes SM4s (Table 1), while the [35S]sulfate incorporation rate into HSO3-Chol was slightly higher than that into SM4s. This discrepancy could be explained by the relatively high anabolic enzyme (arylsulfatase A) activity for SM4s compared with that (arylsulfatase C) for HSO3-Chol (Table 1), although the anabolic enzyme activities in this study were measured with artificial substrates. It has been reported that biosynthesis of sulfoglycolipids, especially of SM4s, was activated by hyperosmotic stimuli on the whole animal (Karlsson, 1982), whole kidney (Umeda et al., 1976), or renal epithelial cell lines (Niimura and Ishizuka, 1991). These authors suggested that the sulfated amphiphiles may play specific roles in the maintenance of renal ionic homeostasis. In the present study, we also observed that concentration of urine brought about by dehydration of the animal elicited by 24 h-water-deprivation up-regulated the activity of GalCer sulfotransferase and, consequently, increased the biosynthesis of sulfoglycolipids in the isolated renal tubules (Table 2). The up-regulation of sulfotransferase and elevated synthesis of renal sulfoglycolipids, similar to the cultured renal cell line exposed more than 6 h in hypertonic media, was demonstrated for the first time with mammals. In conclusion, we propose that the renal tubule system is convenient to perform experiments not only by using anisosmotic medium, addition of renal substrates, drugs, hormones in vitro, but also using rats preacclimatized to anisosmotic or nutritional conditions (dehydration, salt- or water-loading, feed, starve, etc.) in vivo.
Acknowledgments We are grateful to Drs. M. Kasahara and T. Kasahara (Department of Physics, Teikyo University School of Medicine) for giving facility for usage of BAS-2000 Bioimaging Analyzer in the initial part of this study.
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