Urea and methylamines have similar effects on aldose reductase activity

Maurice B. Burg and Eugenia M. Peters

Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung and Blood Institute, Bethesda, Maryland 20892

    ABSTRACT
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Abstract
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Methods
Results
Discussion
References

The concentration of urea in renal medullary cells is sufficiently high to inhibit activity of many enzymes, yet the cells survive and function. The generally accepted explanation is the counteracting osmolytes hypothesis, which holds that methylamines, such as glycerophosphorylcholine (GPC) and glycine betaine (betaine), found in the renal medulla stabilize biological macromolecules and oppose the effects of urea. The present study tests this hypothesis by determining the effects of urea and methylamines, singly and in combination, on the activity of aldose reductase, an enzyme that is important in renal medullas for catalyzing production of sorbitol from glucose. In apparent contradiction to the counteracting osmolytes hypothesis, urea (1.0 M) and three different methylamines (trimethylamine N-oxide, betaine, and GPC; 0.5 M) all have similar and partially additive inhibitory effects. They all decrease substantially both the Michaelis constant (Km) and the maximum velocity (Vmax). Also, a high concentration (0.5 M) of other organic osmolytes that are abundant in the renal medulla, namely inositol, sorbitol, or taurine, has a similar but lesser effect. KCl (0.3 M) causes a small increase in activity. We discuss the significance of these findings with regard to function of aldose reductase in the renal medulla and the counteracting osmolytes hypothesis.

glycerophosphorylcholine; betaine; sorbitol; taurine; inositol; counteracting organic osmolytes

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

HIGH CONCENTRATIONS OF UREA are present in the tissues of marine elasmobranchs and in the mammalian renal medulla. Urea generally destabilizes biological macromolecules, altering their structure and function. Such effects are expected to be deleterious. However, the urea-rich tissues also contain high concentrations of certain methylamine compounds, principally trimethylamine N-oxide (TMAO) in elasmobranchs (26) and glycine betaine (betaine) and glycerophosphorocholine (GPC) in mammals (1). These methylamines are believed to protect the tissues from urea by stabilizing macromolecules and thus counteracting the actions of urea (26). The two effects are independently additive (9, 26). When the ratio of methylamines to urea is appropriate (often 1:2), their opposing effects are reported to counteract, preserving macromolecular structure and function.

Numerous examples of counteraction of functional changes in enzymes have been reported. These include elevations of Michaelis constant (Km) by urea that are counteracted by TMAO [e.g., the Km for ADP of pyruvate kinase (3, 26) and creatine kinase (26), the Km for NADH of A4-lactate dehydrogenase, and the Km for glutamate of glutamate dehydrogenase (26)]. Similarly, TMAO counteracts urea-induced increase in the maximum velocity (Vmax) of A4-lactate dehydrogenase and urea-induced decrease in Vmax of creatine kinase and argininosuccinate lyase (26). Betaine increases the Vmax of porcine arginosuccinase, counteracting urea, which decreases the Vmax (22).

Effects of urea on protein structure are also counteracted by TMAO. These include effects on thermal stability of ribonuclease (8, 25), on the rate and extent of renaturation of lactate dehydrogenase after acid denaturation (25), and on reactivity of thiol groups in glutamate dehydrogenase (25).

In addition to these in vitro experiments, there also is direct evidence in living cells that methylamines and urea counteract each other. Since urea readily permeates Madin-Darby canine kidney (MDCK) cells and betaine is transported into them, their intracellular concentrations can be controlled by varying the concentrations in the medium. High intracellular concentration of either urea or betaine decreases colony-forming efficiency of MDCK cells, but when both solutes are added together, survival and growth of the cells is restored (23). Thus urea or betaine alone each apparently perturbs structure and/or function of critically regulated intracellular macromolecules in ways that are opposite. Since each solute upsets homeostasis, it is deleterious when it alone is present in high concentrations. However, when the two solutes are added simultaneously, they apparently counteract in a way that maintains the critical regulation and preserves cellular vitality.

Cells in the renal medulla contain two predominant methylamines, betaine and GPC (4). Although betaine evidently can counteract actions of urea in vitro and in renal cells in culture, the level of betaine in the renal medulla (13, 15, 21) and in tissue culture (11) does not correlate with that of urea, whereas the level of GPC does. In fact, betaine actually decreases with high urea. Thus renal cells apparently prefer GPC over betaine for counteracting the stress of urea. The reason for this is not clear. One intriguing hypothesis is that GPC counteracts the effect of molar levels of urea more effectively than does betaine (20). If so, this is not a general phenomenon, at least in vitro. GPC is no more effective than betaine at counteracting the increase in Km for ADP of pyruvate kinase caused by urea (3). It is important to remember, however, that this observation does not exclude the possibility that GPC counteracts some other effect of urea more effectively than does betaine.

The major goal of the present study was to compare the effects of urea, betaine, and GPC on another enzyme, aldose reductase. We chose aldose reductase because it has a major role in osmotic regulation in the renal medulla (4), and it functions in that tissue despite often high levels of urea. We find that 1) urea, TMAO, betaine, and GPC all decrease both the Km and Vmax of aldose reductase; 2) urea and the methylamines do not counteract each other; and 3) there is no substantial difference between betaine and GPC in these respects.

    METHODS
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Abstract
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Methods
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Discussion
References

Measurement of aldose reductase activity. Measurement of aldose reductase activity is based on a method previously described (6). Quartz cuvettes containing reaction mixture (complete, except for the substrate, which was either DL-glyceraldehyde or D-glucose) were prewarmed for 2 min to 37°C in the temperature-controlled 6-cell positioner (model CPS-240A) of a Shimadzu UV-1601 recording spectrophotometer. Then, the substrate, contained in 10% of the final volume, was added with mixing, and oxidation of NADPH was followed at 340 nm at 15-s intervals for a total of 90 s. The reaction slopes, which were linear through 90 s (data not shown), were recorded and calculated automatically by the spectrophotometer in its kinetics mode. In the experiments with DL-glyceraldehyde as substrate, kinetics were determined by adding a different amount to each cuvette, yielding from 0.0 to 2.0 mM final concentration, or activity was determined at a single high concentration (10 mM). In experiments with D-glucose, only 10 mM (final concentration) was used.

Recombinant rat lens aldose reductase (14), prepared fresh each month, was a generous gift of Drs. L. Rodriguez, P. F. Kador, and S. Sato. It was stored at 4°C until it was added to the reaction mixture immediately before each assay. The reaction mixture contained (final concentrations) ~50 mU/ml aldose reductase, 10 mM potassium phosphate buffer (pH 6.2), 0.104 mM NADPH, and particular organic osmolytes, as specified.

Km and Vmax were calculated by Eddie-Hofstee analysis (10) of each set of six reactions that used the different concentrations of DL-glyceraldehyde as substrate. The Eddie-Hofstee plots (not shown) were linear (mean R2 = 0.94). Vmax is expressed as absorbance units per minute (A/min).

Other reagents. Other reagents were TMAO (Sigma catalog no. T0514), betaine (Sigma catalog no. B2754), urea (ICN catalog no. 821527), sorbitol (Sigma catalog no. S1876), inositol (Sigma catalog no. I5125), taurine (Sigma catalog no. T0625), and GPC (1:1 cadmium chloride adduct; Sigma catalog no. G8005). Cadmium chloride was removed from the GPC before each experiment by shaking a dilute solution of the adduct for 1 h with a mixed bed ion-exchange resin (AG 501-X8, Bio-Rad). The GPC was then concentrated by lyophilizing the solution and dissolving the amorphous residue in a small volume of water. The final GPC concentration was originally confirmed both by direct analysis (7) and freezing-point depression (µOsmette, Precision Systems), following which each new batch was checked by freezing-point depression. Completeness of the removal of the cadmium was tested by flame atomic absorption spectroscopy (Allied Analytical Systems), using a cadmium lamp. No cadmium was detected (<2 µM cadmium in 70 mM GPC solution).

Statistics. Statistics were calculated with the GraphPad Instat program, using analysis of variance (ANOVA; Student-Newman-Keuls multiple comparison test). P < 0.05 was considered significant. Results are presented as means ± SE (n = number of measurements).

    RESULTS
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Abstract
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Methods
Results
Discussion
References

Urea (1.0 M) has a mixed effect on aldose reductase kinetics (Figs. 1-3). Although it inhibits enzyme activity at high substrate concentration (Vmax), it also reduces the concentration of substrate that produces half-maximal activation (Km). Combining the results in Figs. 1-3, Vmax is reduced by 59% (from 0.0747 ± 0.0026 to 0.0312 ± 0.0006 A/min), and Km is reduced by 42% (from 0.127 ± 0.009 to 0.074 ± 0.005 mM, n = 9). The decrease in Vmax is consistent with results of urea action on a number of other enzymes (26). TMAO (0.5 M) also decreases both Vmax (Fig. 1B) and Km (Fig. 1A) of aldose reductase. In fact, it decreases Km even more than does urea (Fig. 1A). The decrease in Km is consistent with results of TMAO action on a number of other enzymes (26).


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Fig. 1.   Effect of 0.5 M trimethylamine N-oxide (TMAO) and 1.0 M urea on aldose reductase. Substrate is glyceraldehyde. * P < 0.05 vs. control (n = 3). A: Michaelis constant (Km) for glyceraldehyde. P < 0.05 also for urea vs. TMAO and for urea vs. TMAO + urea. B: maximum velocity (Vmax). P < 0.05 also for TMAO vs. TMAO + urea and for urea vs. TMAO + urea.

When urea (1.0 M) and TMAO (0.5 M) are both added together, Vmax decreases more than with either added alone (Fig. 1B). Also, the combination decreases Km more than urea alone (but to the same extent as TMAO, alone) (Fig. 1A). Thus the effects of urea and TMAO are partially additive. In previous studies the effects of urea and TMAO were also found to be additive (3, 9, 26), but, since the effects of urea and TMAO were opposite in direction on Km, Vmax, or thermal transition temperature, the two effects counteracted each other, which evidently is not the case here.

The effects of betaine (0.5 M) on Vmax (Fig. 2B) and Km (Fig. 2A) of aldose reductase are similar to those of TMAO (Fig. 1).


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Fig. 2.   Effect of 0.5 M betaine and 1.0 M urea on aldose reductase. Substrate is glyceraldehyde. * P < 0.05 vs. control (n = 3). A: Km for glyceraldehyde. P < 0.05 also for urea vs. betaine and for urea vs. betaine + urea. B: Vmax. P < 0.05 also for urea vs. betaine and for urea vs. urea + betaine.

The effects of GPC (0.5 M) on Vmax (Fig. 3B) and Km (Fig. 3A) of aldose reductase are also similar to those of TMAO (Fig. 1), as well as to those of betaine (Fig. 2). These results do not rule out small differences between the effects of the different methylamines (TMAO, betaine, and GPC), but the similarities among the three are much more impressive than whatever small differences there may be.


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Fig. 3.   Effect of 0.5 M glycerophosphocholine (GPC) and 1.0 M urea on aldose reductase. Substrate is glyceraldehyde. * P < 0.05 vs. control (n = 3). A: Km for glyceraldehyde. P < 0.05 also for GPC vs. GPC + urea and for urea vs. GPC + urea. B: Vmax. P < 0.05 also for urea vs. GPC and for urea vs. urea + GPC.

Since urea and the methylamines decrease Km, as well as Vmax, their inhibition of aldose reductase activity might be less at low substrate concentrations than at concentrations that are high relative to the Km. To test this possibility, we chose to use as substrate D-glucose, for which the Km is high and which is an abundant natural substrate of the enzyme (Figs. 4 and 5). The Km for glucose of the recombinant rat lens aldose reductase that we used is 281 mM (18). We used 10 mM, which is well below the Km and is the lowest concentration that we were confident would allow accurate measurements. Using this relatively low concentration of glucose as substrate, we found that urea, TMAO, and betaine inhibit aldose reductase activity less than they do the Vmax of aldose reductase with DL-glyceraldehyde as substrate (Table 1). The exception is GPC, which inhibits the activity with 10 mM D-glucose as substrate as much as it inhibits the Vmax with DL-glyceraldehyde as substrate.


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Fig. 4.   Effect of urea (1.0) and other organic osmolytes (0.5 M) on aldose reductase. Substrate is 10 mM glucose. All differences are significant (P < 0.05), except for taurine vs. urea + betaine, urea vs. urea + betaine, taurine vs. urea, TMAO vs. betaine, and control vs. inositol.


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Fig. 5.   Effect of 0.5 M GPC and 1.0 M urea on aldose reductase. Substrate is 10 mM glucose. All differences are significant (P < 0.05), except for GPC vs. urea + GPC.

                              
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Table 1.   Effect of organic osmolytes on aldose reductase activity

Sorbitol, inositol, and taurine also accumulate in the renal inner medulla when the interstitial NaCl concentration is high (4). Since all three are compatible organic osmolytes, they are not expected to affect enzyme activity very much (4, 24). We were interested to test their effect on aldose reductase activity, since aldose reductase is important for sorbitol accumulation under those conditions. With DL-glyceraldehyde as substrate, 0.5 M sorbitol, 0.5 M inositol, and 0.5 M taurine each significantly decrease both Vmax and Km (Fig. 6, A and B). However, the changes are smaller than with urea and the methylamines (Figs. 1-3). Taurine has a relatively small effect on Km, decreasing it by only 10% (Fig. 6A).


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Fig. 6.   Effect of 0.5 M inositol, sorbitol, or taurine on aldose reductase. Substrate is glyceraldehyde. * P < 0.05 vs. control (n = 3). A: Km for glyceraldehyde. P < 0.05 also for inositol vs. taurine and for sorbitol vs. taurine. B: Vmax. P < 0.05 also for inositol vs. taurine and for sorbitol vs. taurine.

Since the combined effect of the various organic osmolytes might differ from their individual effects, we measured aldose reductase activity with 10 mM DL-glyceraldehyde as substrate and a mixture of compatible organic osmolytes in a ratio similar to that found in rat renal medullary cells (13). The mixture contains 100 mM inositol, 50 mM betaine, 50 mM taurine, 50 mM sorbitol, and 250 mM GPC. The resultant aldose reductase activity (in A/min) is as follows: control, 0.0619 ± 0.0027; 1.0 M urea, 0.0231 ± 0.0004; mixture of osmolytes, 0.0158 ± 0.0007; and 1.0 M urea plus mixture of osmolytes, 0.0146 ± 0.0010 in A/min (n = 3). All of the differences are significant (P < 0.05) except that between urea and urea plus the mixture. Thus a combination of organic osmolytes similar to that present in renal medullary cells inhibits aldose reductase activity and does not counteract the effect of urea.

As already mentioned above, when both Km and Vmax are decreased, the inhibition of enzyme activity at low substrate concentration should be less than at concentrations greatly exceeding Km. Accordingly, 0.5 M sorbitol and 0.5 M inositol (which decrease Km substantially) do not inhibit aldose reductase activity with 10 mM D-glucose as substrate, and sorbitol and inositol evidently are compatible solutes in this respect. On the other hand, under the same conditions, 0.5 M taurine (which has relatively little effect on Km) inhibits the enzyme activity with 10 mM glucose as substrate by 39% (Fig. 4). Taurine was previously observed to perturb pyruvate kinase (3) and other enzymes.

Inorganic salts previously were shown to have little effect on aldose reductase activity in single tubules and in homogenate of renal cells (11, 17). To test this with the recombinant enzyme used in the present study, we measured aldose reductase activity with 10 mM DL-glyceraldehyde as substrate. The results are as follows: control, 0.067 ± 0.001 A/min; 0.3 M KCl added, 0.072 ± 0.001 A/min (n = 3). Although this small increase with KCl added is statistically significant (P < 0.05), we hesitate to generalize from it, because we also have found instances in which other inorganic salts decrease the activity (data not shown).

    DISCUSSION
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Methods
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Are studies of lens aldose reductase relevant to the kidney? The recombinant aldose reductase used for these studies was prepared using a cDNA derived from lens, but there is evidence that aldose reductase from kidney cells is also inhibited both by urea and by methylamines. Similar to the result with the recombinant lens enzyme, aldose reductase activity in homogenates of PAP-HT25 (rabbit renal medullary) cells is substantially inhibited by either 100-200 mM urea or by 100-200 mM betaine but not by 100 mM sorbitol or inositol (11). Therefore, the conclusions reached here by studying lens aldose reductase apparently apply also to the renal enzyme. In addition, we are unaware of any evidence for differences in aldose reductase protein between various tissues.

How can aldose reductase function adequately under the conditions in renal inner medullary cells, if it is inhibited by solutes normally present there? The present studies show that aldose reductase activity is inhibited by TMAO, betaine, GPC, and/or urea. The concentrations of these solutes in renal inner medullary cells generally are high. Nevertheless, the cells produce large quantities of sorbitol, catalyzed by aldose reductase. There are several explanations for the sufficiency of aldose reductase activity, despite the presence of these inhibitors.

1) The mixed kinetics of the aldose reductase inhibition reduce its impact. The inhibition is characterized by large decreases in Km, as well as Vmax. Therefore, for substrate concentrations well below Km, the reduction in reaction rate is relatively less than the reduction in Vmax. That applies specifically to glucose, which is the natural substrate in renal medullary cells. Intracellular glucose concentration certainly is much less than the Km for glucose, which is >100 mM. Using 10 mM glucose in our assay, we find that inhibition of aldose reductase activity by the three methylamines and by urea is considerably less than the inhibition of Vmax with DL-glyceraldehyde as substrate (Table 1).

2) GPC substitutes for sorbitol when urea is high, which reduces the requirement for sorbitol. Sorbitol is only one of five predominant "compatible" organic osmolytes in the renal medulla (4). The total concentration of all five, not the concentration of any individual one, is important for their function as compatible osmolytes (12). High urea specifically induces accumulation of GPC, which in effect partially replaces the other compatible organic osmolytes (13, 15, 21). Thus, under hypertonic conditions, less, rather than more, sorbitol is needed when urea increases, and, accordingly, the level of sorbitol either does not change or decreases (11, 13).

3) Aldose reductase is remarkably abundant in renal cells exposed to hypertonicity. Thus aldose reductase may compensate in bulk for any lack of catalytic efficiency. In PAP-HT25 cells stressed by high NaCl, aldose reductase comprises 10% of the soluble cell protein (2).

Failure of methylamines to counteract inhibition by urea is not unique to aldose reductase. As detailed in the introduction, there are numerous examples of counteraction by methylamines of the actions of urea on specific macromolecules in vitro. Also, betaine counteracts the decrease in survival and growth of renal cells caused by high urea in tissue culture. Based on these and other observations, the general importance of counteraction has been appropriately stressed (19, 24). From this point of view, the failure of methylamines to counteract the inhibition of aldose reductase by urea might seem surprising. However, there are several other instances in which counteraction was not observed. Those exceptions include that 1) TMAO does not counteract the perturbing effects of urea on enzyme-substrate interactions of A4-lactate dehydrogenase with pyruvate or of glyceraldehyde-3-phosphate dehydrogenase with glyceraldehyde 3-phosphate (26), 2) TMAO does not counteract urea effects on phosphofructokinase activity and structure (5), and 3) betaine does not counteract Km effects of urea on uricase (22).

These findings raise an apparent paradox. Although counteracting osmolytes evidently protect cells from harmful effects of urea (23), these osmolytes do not counteract the effects of urea on all enzymes tested in vitro. It has been emphasized that maintaining Km values within a narrow range is essential for optimal rates and regulation of catalysis (26). If so, how can counteracting osmolytes protect adequately from the effects of high urea if they do not protect all enzymes? Perhaps, fine tuning is more important for some enzymes than for others, and cells can get along adequately even if not all of their enzymes are functioning completely normally.

That leaves the questions of which enzymes are critical and what distinguishes them. At present it is difficult to estimate how many enzymes in renal medullary cells are affected by urea and which of the effects of urea are counteracted by methylamines. The levels of urea found in renal inner medullas inhibit many but certainly not all enzymes. Of diverse enzymes that were tested, 10 were competitively inhibited by urea (i.e., the Km was elevated), 3 were uncompetitively inhibited (i.e., the Vmax was reduced), and 5 were not inhibited at all by urea concentrations up to 2 M (16). Competitive inhibition by urea is often counteracted by methylamines, as discussed above. Nevertheless, even this mode of counteraction is not found universally. Urea competitively inhibits uricase (16), which is not counteracted by betaine (22).

Inhibition of aldose reductase by organic osmolytes and urea cannot be attributed solely to high osmolality. High concentrations of the various methylamines and urea all similarly inhibit aldose reductase activity, which raises the suspicion that the effect is an osmotic one, related simply to osmolality of the cosolvents and independent of their specific identity. However, the observations that 0.3 M KCl slightly increases activity and that 0.5 M sorbitol and inositol do not inhibit (Fig. 4) or have a substantially smaller effect are not consistent with such a simple explanation.

    FOOTNOTES

Address for reprint requests: M. B. Burg, Bldg. 9, Rm. 1N105, National Institutes of Health, Bethesda, MD 20892-0951.

Received 12 May 1997; accepted in final form 7 August 1997.

    REFERENCES
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Abstract
Introduction
Methods
Results
Discussion
References

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