Effects on rat renal osmolytes of extended treatment with an aldose reductase inhibitor

0306~4492/92 $5.00+ 0.00 Camp.Biachem.Physiol.Vol. 103C,No. 3, pp. 499-502, 1992 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHG 0 1992Pergamon Press Ltd Printed in Great Britain EFFECTS ON RAT RENAL OSMOLYTES OF EXTENDED TREATMENT WITH AN ALDOSE REDUCTASE INHIBITOR SCOTT EDWANDS and PAUL H. YANCEY Biology Department, Whitman College, Walla Walla, WA 99362, U.S.A. (Tel. 509 527-5498; Fax 509 527-5961) (Received 22 April 1992; accepted for publication 22 May 1992) Abstract-l. The mammalian renal medulla uses sorbitol, myo-jnositol, betaine and gIycerophosphorylcholine as intracellular osmotytes. 2. Sorbitol synthesis was inhibited by feeding male Wistar rats the aldose reductase inhibitor sorbinil at 40 mg/kg/day for 71 d, and renal inner medullas were extracted for analysis. 3. Aldose reductase activities and sorbitol contents were greatly reduced in sorbinil-treated animals, while betaine contents increased significantly (with no other osmolytes changing). 4. The betaine increase compensated for the sorbitol decrease such that the total organic osmolytes maintained the same ratio to sodium contents as controls. 5. These results are identical to the pattern previously reported for sorbinil treatment of rats for 10 d, but not for 21 d. INTRODUCTION In order to maintain cell volume, mammalian renal medulla ceils must be able to balance high external salt concentrations produced by the urine-concentrating mechanism of the kidney. Cells of a wide variety of organisms exposed to high external osmolalities use certain organic osmotic effecters termed “compatible osmolytes,” which do not exhibit the disruptive effects on macromol~ules that could result from increased cell NaCl or KC1 (Yancey et al., 1982). Compatible solutes include polyols, methylamines, and certain free amino acids; in renal medulla cells, the major osmolytes are sorbitol, myoinositol, glycerophosphorylcholine (GPC), betaine (N-t~methylglycine) (Bagnasco et al., 1986), and some amino acids (Nakanishi et ai., 1991), with excess NaCl thought to remain mainly outside the cells (Beck et ai., 1984). Many studies have confirmed the role of these solutes as osmolytes by altering renal osmotic conditions through variations in animal water or salt intake. In these experiments, total renal contents of these compounds are up- and down-regulated in antidiuretic and diuretic states respectively, such that cell volume appears to be maintained (Bagnasco et al., 1986; Gullans et al., 1988; Yancey, 1988; Yancey and Burg, 1989). Among the renal osmolytes, sorbitol, a polyol produced from glucose by aldose reductase (EC 1.1.121), is of particular interest because of its role in diabetes mellitus. During hyper~y~mia, some excess glucose is converted to sorbitol in certain tissues such as lens, neurons, and renal cortex and medulla (Beyer-Mears et al., 1984; Chauncey et al., 1988; Gaynes and Watkins, 1989), and damaging osmotic pressures may result (Burg and Kador, Abbr~~utio~: GPC, glycerophosphory~choline 1988). Drugs which inhibit aldose reductase reduce sorbitol ac~~ulation and can be beneficial to such tissues including the kidney (Beyer-Mears et al., 1984; Bank et al., 1989; Mauer et al., 1989; Kumari et al., 1991; McCaleb et al., 1991). However, sorbitol is used in normal renal osmoregulation, suggesting that aldose reductase inhibitors could impair this renal function. This possibility was supported by initial studies on cultured renal medulla cells (PAP-HT25). These cells rely mainly on sorbitol for cell volume maintenance when grown in normal growth medium with osmolality increased by NaCl. When exposed to aldose reductase inhibitors, sorbitol content is greatly decreased and cell survival is severely inhibited (Yancey et al., 1990a). This initial in vitro study has been followed up in three ways. First, in our laboratory, normal male Wistar rats were fed an aldose reductase inhibitor, sorbinil (Pfizer), for 10 and 21 d. In the 10-d treatment, sorbitol content of the inner medulla declined greatly, but in apparent compensation, betaine contents increased such that cell volume appeared to have been maintained. However, neither sorbitol nor betaine levels were significantly altered in the 21-d treatment. We suggested that another type of compensation is occurring by 21 d of treatment, e.g., the kidney may up-regulate the production of sorbitol (Yancey et al., 1990b). Second, Bondy et al. (1990) showed that in young female Spragu~Dawley rats fed sorbinil for 10 d, renal papillary sorbitol contents were greatly reduced, but no other osmolyte (including betaine) changed. Cell volume may not have been maintained. Third, the results of our 10 d treatment in uivo (above) were confirmed with the renal cells (PAPHT25) in vitro: if betaine is added to the growth medium along with an aldose reductase inhibitor, the cells substitute betaine for sorbitol and their 499 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFE SCOTT EDMANDSand PAUL H. YANCEY 500 acid, NaOH) as a standard. The remaining supernatant was survival and growth is restored (Moriyama et al., neutral&d to pH 6.8-7.5 using 1 M KOH. The samples 1991). were passed through 0.45 nm HV filters (Millipore, Bedford, Thus, both zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA in vitro and in vivo, betaine and sorbitol MA) and Bond Elut C-18 cartridges (Varian, Harbor may serve as interchangeable compatible osmolytes City, CA). Overall tissue dilution was 18-fold. Peaks were during aldose reductase inhibition, but the in vivo identified using standards treated similarly. results are not consistent, with three different patUrine samples were analyzed for total osmotic pressure terns reported (reciprocal betaine-sorbitol changes, using a vapor pressure osmometer (model 5500, Wescor, no change in either, or reduced sorbitol with no Logan, UT), then were treated in the same manner as medullas, starting with perchloric acid treatment. Overall compensation). In an attempt to elucidate these urine dilution was IS-fold. patterns further, we have extended testing on Wistar Sodium in all samples was analyzed using flame photomrats to a much longer period (71 d), and include etry (model 2655-00. Cole-Parmer, Chicago, IL). Organic measurements of aldose reductase activities. MATERIALS AND METHODS Sorbinil was provided by Pfizer Central Research ton, CT). Other chemicals were purchased from Chemical Co. (St Louis. MO). (GroSigma Animals All animals used were male Wistar Rats initially 151-1758 (COBS, Charles River Laboratories, Wilmington, MA), housed under standard conditions and given Purina Rodent Laboratory Chow (5001, Purina Mills, St Louis, MO) and water ad Ii&turn for 14 d before the experiments were started. Two groups were fed ground chow, with one group as control and the second fed chow blended with sorbinil to yield an intake of 40 mg/kg/d. All animals were given water ad Ii&turn throughout, and body weights and, food consumption and sorbinil dosage were checked every 2-3 d. After 7ld animals were anaesthetized with CO, and killed by cervical dislocation. Urine samples, when present, were taken from the bladder with a syringe. One kidney from each animal was frozen in liquid nitrogen in a vial and stored at -7o’C for later osmolyte extraction. Aldose reductase assa? The second kidney was immediately dissected on a Petri plate on ice to obtain the inner medulla. The medulla was weighed to the nearest 0.1 mg. then homogenized in a glass homogenizer in 19 vol cold buffer (1OmM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES), 5 mM EDTA, 2mM EGTA. 2mM dithiothreitol, pH 7.0). The homogenates were centrifuged in a microcentrifuge 15 min at lO.OOOrpm. Supernatants were analyzed kinetically at 37-C for aldose reductase activity with 1OmM glyceraldehyde or xylose as substrate in 100 mM Na-phosphate buffer (pH 7.0) with 100 nM NADPH (Uchida et al., 1989). Osmolyte unaly sis The frozen kidneys were thawed in a cold room, and inner medullas immediately removed and weighed to the nearest 0.1 g, followed by homogenization in a glass homogenizer in 9.0 vol cold 7% perchloric acid. The homogenates were centrifuged for 15 min at 10,000 rpm in a microcentrifuge; 50 ~1 aliquots were removed from each homogenate for Na+ analysis (see below). The pellets were dissolved in 1 ml of 1 N NaOH, then analyzed for protein using the Bio-Rad protein assay (Bio-Rad Laboratories, Richmond, CA), using y-grobulin treated in the same manner (perchloric osmolytes were analyzed using high-performance liquid chromatography (HPLC) as previously described (Wolff et al., 1989) using a Sugarpak-I column (Waters, Milford, MA) at 84°C with peak detection by refractive index. The column separates inositol, sorbitol, GPC, urea, and betaine. A taurine peak was also detected but an unknown compound eluting at about the same time prevented accurate quantification; however the height and area of the peak containing taurine and the unknown solute did not change with sorbinil treatment. RESULTS In previous studies our laboratory reported osmolyte contents as mmol/kg wet wt (Yancey, 1988; Yancey and Burg, 1989; Yancey et al.. 1990b). In those studies, tissue protein contents did not differ among groups; however these did differ in the present study: the control group had 82.6 (f4.0 SD.) and the sorbinil group had 92.6 (k4.6 SD.) g protein/ kg wet wt (P = 0.002). Therefore, we report values on a per protein basis for solutes other than sodium and urea. After 71 d, sorbinil treatment greatly reduced renal aldose reductase activities (Table 1; P < 0.01). Assays for aldose reductase activity were also run separately with 10 mM xylose and 10 mM glyceraldehyde as substrates in order to distinguish between aldose and aldehyde reductase. Activity of rat kidney aldehyde reductase with 10 mM xylose is 0.7% of the activity with 10 mM glyceraldehyde, while that of rat lens aldose reductase is 26% (Terubayashi et al., 1989) and that of PAP (rabbit kidney) cells is 46% (Bedford et al., 1987). Velocities in the present study with xylose were 32% (in both groups) of the glyceraldehyde activity, suggesting that mainly aldose reductase was being measured. This aldose reductase inhibition resulted in a large decrease in sorbitol content (P = 0.003; Table 1; Fig. 1). Betaine content was elevated significantly (P = 0.03), with no other osmolytes showing differences from controls (Table 1; Fig. 1). Because of the reciprocal changes in sorbitol and betaine, sorbinil treatment did not change the ratio of renal sodium content to the total contents of organic osmolytes Table I. Contents of major osmolytes and aldose reductase activities in renal inner medullas of rats, treated in 2 groups for 71 d as described under Methods. Contents are in mmol/kg wet wt (k S.D.) for sodium and urea, and mmol/kg protein (*SD.) for the rest; aldose reductase activity (with glyceraldehyde as substrate) is in mmol substrate/min/kg protein (S.D.); “n” indicates number of animals. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPO Aldose reductase activitv GOlID Control Sorbinil (n = 7) (n = 6) *Significant difference 34. I f 4.5 9.4 f 1.5’ (P ~0.05) Sodium 202?29 210 f 21 from control Urea 215f70 264*50 Osmolyte GPC 217+43 195+ I5 content Inositol 146 & 22 160513 Betaine 62.4 k I I .2 85.1 f 20.4* Sorbitol 50.3 k 16. I 23.5 f 5.9’ Long-term sorbinil effects on renal osmolytes 501 assuming that the 40mg/kg/d sorbinil consumed is evenly distributed in the animal, the diluted extract in the cuvette would have at least 1 PM sorbinil. Sup porting this, Bondy et al. (1990) found no effect of dietary sorbinil on aldose reductase mRNA levels in renal medullas after 10d. Most of the sodium found in the renal medulla is probably extracellular (Beck et al., 1984) and urea is thought to equilibrate across cell membranes, while the (non-urea) organic osmolytes are thought to be largely intra~llular because of their low or no presence in blood and urine (this study; Bagnasco et al., 1986; Balaban and Burg, 1987). Thus an estimate of cell volume maintenance may be obtained by comparing contents of sodium and total methylamines Fig. 1. Contents of major osmolytes in renal inner plus polyols. As shown in Fig. 1, sorbinil treatment medullas of sorbinil-treated rats, using data from Table 1 for 71 d does not appear to impair volume regulation, but recalculated as percentages of control values. Total due to the reciprocal changes in sorbitol and betaine. osmolytes are the sum of methylamines and polyols calcuThis is also similar to in vitro studies with renal lated for each individual animal before applying statistics. medulla cells (Moriyama ei al., 1991; see Introduc*Statistically different from control group. Error bars tion). Thus, betaine and sorbitol can apparently serve indicate S.D. fUpper level of bar results from eliminating as interchangeable compatible osmolytes in medulla one sorbinil-treated animal which had distinctly lower cells during both short and long periods of aldose betaine and higher sorbitoi than any other in its group (see Results). reductase inhibition. The signal for this compensation, apparently for maintaining cell volume, is not known. (methylamines plus polyoIs). This is shown in the However, it is important to note that sorbitol and right-hand side of Fig. 1. betaine may not have identical functions within the Only one animal in the sorbinil-treated group had cell. Betaine, but not sorbitol, has been found to be a low betaine content (53.5 mmol/kg protein) overa counteracting osmolyte, in that it can counteract lapping the range of the control group, but interthe inhibitory effects of high urea contents on cell estingly it also had the highest sorbitol content growth in vitro (Yancey and Burg, 1990). Therefore (33 mmol/kg protein) of the sorbinil group. If the the two compounds may not be functionally equivalanimals data are eliminated, the mean values of ent, and further studies need to be done to examine the sorbinil group (N = 5) become 91.4 3_ 14.9 for the effect of substituting betaine for sorbitol. In the betaine (P = 0.003) and 21.7 rt: 4.5 for sorbitol 71 d treatment there was no obvious damage to the (P = 0.002). kidney, but we did note a 12% increase in medullary Body weights (not shown) and urine osmo~alities protein content in the sorbinil group (see Results), (2218 k 357 SD. for controls, 1924 + 214 SD. for perhaps due to a lower water content. sorbinil-treated) were not significantly affected by the Two inconsistencies remain unexplained. First, in drug. Urines did not differ in their concentrations of our previous study (Yancey et al., 1990b) the same sodium (114 mM + S.D. for controls, 111 It 52 S.D. type of rats (200 g male Wistar) showed no changes for sorbinil-treated) or urea (444 mM + 95 S.D. for in renal sorbitol or betaine after an intermediate controls, 526 f 70 S.D. for sorbinil-treated). No period of sorbinil treatment (21d). Thus cell volume betaine, GPC, or inositol was detected, but one maintenance was apparently maintained, as it was sample in each group had about 1mM sorbitol. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFED after 10 d and ‘71d but by a different mechanism. We note that around 21 d in our experiment the rats were undergoing sexual maturation with possibly DI SCU SSI ON different metabolisms from younger and older aniAnimals used in these and previous experiments mals. Second, the study on small (< 100 g) female from our laboratory (Yancey et al., 1990b) were male Sprague-Dawley rats (Bondy et ai., 1990) under 10 d Wistar rats at about 20&23Og body weight at the of sorbinil treatment gave a third pattern of reduced start of the experiment. Therefore results of the two renal sorbitol with no apparent compensation by studies should be directly comparable. The apparent other osmolytes. The authors concluded that cell use of increased renal betaine content to compensate volume regulation might have been impaired. for reduced sorbitol content is a pattern virtually Whether this represents an age or strain difference identical to that we previously found for 1Od sorbinil needs to be investigated. treatment (see Fig. 1 in Yancey et al., 1990b). Thus, In summary, in male Wistar rats, the renal inner it seems clear that dietary sorbinil maintains a similar medulla appears to be able to maintain cell volume level of effectiveness across short and long periods. by substituting betaine for sorbitol during short-and long-term aldose reductase treatment, and by some Although the enzyme rates in Table 1 are measured unknown mechanism of sorbitol maintenance during in a lOO-fold dilution of the medulla (in the assay an intermediate treatment coinciding with sexual cuvette), they probably reflect inhibition by sorbinil in the extract rather than any down-regulation of maturation. 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