Osmotically active organic solutes in the renal inner medulla

Kidney international, Vol. 31(1987), pp. 562—564

Osmotically active organic solutes in the renal inner medulla ROBERT S. BALABAN and MAURICE B. BURG Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung and Blood Institute, Bethesda, Maryland, USA

Several osmotically—active mammalian kidney organic so!utes have been identified in the medulla of mammalian kidney over the past 25 years, but only recently has their significance been clarified by the convergence of several otherwise unrelated lines of investigation, including electron probe and NMR

and sucrose), the methylamines (such as trimethylamine oxide and betaine), and the amino acids (such as glycine and proline). Urea is a special case. It is a perturbing solute which inhibits enzyme activity, yet it is employed by some species as a major

studies and comparative studies of marine organisms. The purpose of this review is to summarize those studies and to consider the likely roles of the osmotically—active organic solutes. Since the comparative studies provided much of the

apparent paradox is that in those species that employ urea as an osmolyte, it is generally accompanied by methylamines which counteract its harmful effects [1—3]. The ratio of methylamines

blood and intracellular osmolyte [1]. The resolution of this

to urea which is optimal for stabilizing enzymes against the

theoretical framework for this analysis, we will start with them. perturbing effects of urea is approximately 1:2, which is the It is generally accepted that cell membranes are incapable of ratio generally found in tissues containing high urea levels. sustaining any sizeable osmotic—pressure differences and that Despite this evidence in other classes (or phylums) of animals the osmotic pressure in cells is close to that of their surround- relatively little attention has been given to organic osmolytes in ings. Thus, the sum of the osmolalities of the intracellular mammals. The apparent reason is that most mammalian cells solutes equals that of the extracellular solutes. Comparative are not normally exposed to high salt concentrations with the studies recently reviewed by Yancey et al [1] demonstrate that exception of those of the renal inner medulla which contains cells of diverse organisms in high salt environments have high high extracellular concentrations of salt and urea. When metainternal solute concentrations, matching the high external bolic enzymes [1—3] or Na,K-ATPase [4], which serve imporosmolality. The point of special interest is that the concentra- tant functions in the inner medulla, were tested in broken cell tions of only particular types of solutes were found to be preparations, they were severely inhibited by high NaCI or urea elevated. In a wide variety of organisms the concentration of concentrations like those normally occurring in the inner me-

organic, rather than inorganic solutes (that is, Na, K, Cl), was increased to balance the osmolality of high external salt concentrations. The exception was certain Archaebacteria that contained high Na and K levels. Otherwise, plant, animal and bacterial cells in high salt environments all contained elevated concentrations of osmotically—active organic solutes, so—called

"osmolytes".

dulla. Nevertheless these enzymes apparently function ade-

quately in inner medullary cells. Based on comparative studies, we would therefore expect that intracellular concentrations of Na, K, and Cl are not elevated in the inner medulla, despite the high extracellular NaCl level, but that osmotically—active intracellular organic solutes should be present instead. Also, since urea presumably permeates the cells of the inner medulla, we

would expect that at least some of the osmolytes would be

The osmolytes mostly fall into three groups: polyols, methylamines. methylamines, and amino acids. Yancey et al [1] have proposed The intracellular Na, K, and Cl content of renal inner

a theoretical basis for understanding why cells employ these particular organic solutes as osmolytes. These authors distinguished between perturbing and nonperturbing solutes. High concentrations of perturbing solutes inhibit cellular enzymes, whereas even higher concentrations of nonperturbing solutes do not. The solutes that were perturbing included NaCl, KC1, and urea. The intracellular organic osmolytes were found to be nonperturbing. Concentrations of NaC1, KCI or urea high enough to balance osmotically the high salt concentration in the ocean were found to inhibit various cellular enzymes. The enzymes were not inhibited by comparable concentrations of the nonperturbing solutes, that is, the polyols (such as sorbitol

medullary cells has been investigated by two groups. Beck et al

[5] found that under antidiuretic conditions the extracellular concentration of NaCl was elevated in rat inner medullas, but the intracellular concentrations of Na, K and Cl were not. The

authors recognized that some unidentified solutes must be present in the cells to balance the extracellular hyperosmolality. The magnitude of the "osmotic gap" was 300 to 500 mOsm. In contradiction, Bulger, Beeuwkes and Sauberman [6] found high

intracellular NaC1 concentrations under apparently identical conditions to those used by Beck et al. Based on the studies

themselves, we cannot choose between these two results. Considering the comparative physiology results, however, we

are skeptical that renal inner medullary cells will join the Received for publication August 8, 1986

© 1987 by the International Society of Nephrology

Archaebacteria as exceptions to the rule that cells surviving in high salt environments do not have high internal salt concentrations. Taking the results of Bulger et al [6] at face value, 562

563

Inner medulla osmotically active solutes

there is still an osmotic gap between the intracellular and extracellular solute concentrations in rat inner medulla, but only 100 to 200 mOsm. Thus, there is agreement about the existence, if not the size, of the osmotic gap in the inner

Table 1. Approximate concentrations of non-urea organic osmolytes in the mammalian renal papilla

Content mmoles/kg

Osmolyte medulla. The question is what solutes fill the gap. Two methylamines have been identified in high concentration Sorbitol in the renal inner medulla, namely glycerolphosphorylcholine

(GPC) and betaine. Ullrich [7] found approximately 125 mmoles/kg wet wt of GPC near the tip of dog renal inner medulla. Later, 31P nuclear magnetic resonance (NMR) dem-

onstrated 15 to 25 mmoles/kg wet wt GPC in rabbit inner medulla. GPC is produced by the action of phospholipases Al and A2 on phosphatidylcholine. High concentrations of these enzymes were found in the inner medulla [8], making local

Inositol Betaine Glycerolphosphorylcholine Glutamine Aspartate

Species

rabbit

rat rabbit dog rabbit rabbit dog

rat rat

wet wt

Reference

10 2 10 15 55 15 125 6

11

4

18 11

14 11 11

7

20 20

synthesis a likely source of the GPC. Betaine was found in whole kidneys by Martin and Finkelstein [9] and was subsequently localized to the inner medulla by '4N [10] and 'H [11] NMR and by mass spectrometry [11]. The betaine content of

dulla during antidiuresis. Since one role of osmolytes, presum-

aware of any studies indicating whether it is also present in the inner medulla. Two polyols, namely inositol and sorbitol, have been found in high concentrations in renal inner medulla. Schimassek, Kohl

concentrations of inositol [11, 14], betaine [11], sorbitol [11],

ably, is to balance the osmotic pressure of the extracellular rabbit inner medulla was 55 moles/kg wet wt). Betaine is NaCl, one might predict that the their concentration would vary synthesized in the kidney by choline oxidase [12]. This enzyme with the state of diuresis. The effect of hydration on inner was found in the proximal straight tubule [12], but we are not medullary osmolytes has been examined in two studies. The

and Bucher [13] found inositol in dog renal inner medulla. Later, chemical [14], autoradiographic [15], 'H NMR, and mass spectrometric [11] measurements demonstrated inositol in the

inner medulla of other species. Inner medullary inositol concentration ranged between 10 and 20 mmoles/kg wet wt. Since the medulla is a major site of inositol synthesis and breakdown in humans [16] and other mammals [17], local production is the likely source of the inositol. The sorbitol content of the renal inner medulla has been extensively studied, following observations of increased tissue sorbitol in renal failure and diabetes. Sorbitol was initially identified in rat inner medulla by Gabby

and GPC [11] were higher during antidiuresis than during diuresis. The changes were relatively small, however, compared to the concomitant changes in the level of urea. The sum of the concentrations of non-urea organic osmolytes

in whole renal inner medulla from antidiuretic rabbits was approximately 120 mmoleslkg wet wt (see Table 1). Assuming that organic osmolytes are entirely intracellular, the average concentration in the cells of the inner medulla will be higher

than in the whole tissue, and at the tip of the papilla the concentration in the cells should be at its highest. Thus, the cell

concentration of the known osmolytes may well equal the osmotic gap calculated from electron probe analysis of renal

inner medullas. The high concentration of methylamines in the inner medulla and O'Sullivan [18]. They found 2 mmoles/kg wet wt of sorbitol is especially interesting since these compounds are known to under normal conditions. In other studies 10 mmoles/kg wet wt offset the destabilizing effects of urea on metabolic enzymes. was found in rabbit renal inner medulla [11]. Sorbitol is pro- The optimal ratio for this effect was determined to be one part duced from glucose via aldose reductase. Aldose reductase of methylamine to two parts of urea [1]. The ratio of methylactivity has been identified in the kidney, and is concentrated in amines to urea is less than 1:2 in whole rabbit renal inner the renal inner medulla [19]. Therefore, local production is the medulla, however. Since the methylamines are probably conlikely source of sorbitol. fined to the cells while urea is present in the extracellular fluid There is no evidence of high free amino acid levels in the and urine, the ratio of methylamines to urea might well be 1:2 in

renal inner medulla, but few studies were devoted to that

the cells of the inner medulla, as it is in the cells of species subject. Chan et al [20] found 3.5 mmoles/kg aspartate and 6 adapted to high salt levels. Thus the methylamines might serve mmolesfkg wet wt glutamate in rat inner medulla, using chem- a similar protective role in both cases. Further studies are ical methods. Shalhoub et al [21] surveyed 19 amino acids in the needed, however, for a rigorous test of these speculations. In summary, the existence of significant amounts of non-urea dog papilla and found only glutamate, at 9 mmoles/kg wet wt, to be above 2 mmoles/kg wet wt. 'H NMR and mass spectrometry, organic osmolytes in the renal inner medulla has been clearly on the other hand, did not reveal any amino acid as a major demonstrated. The principal osmolytes are glycerolphosphorylosmolyte in rabbit or rat inner medulla [11]. choline, betaine, inositol, and sorbitol. Presumably they funcSince the inner medullary tissue that was analyzed included tion in osmotic regulation of the inner medullary cells and may extracellular fluid and urine, as well as cells, there is no direct protect the cellular enzymes from the potentially harmful efway to distinguish between intracellular and extracellular loca- fects of the hypertonic extracellular environment in the inner

tions of osmolytes. Since the levels of osmolytes in rabbit medulla. peripheral blood and urine were insignificant despite high levels in the inner medulla [11], however, the osmolytes in the inner medulla are most likely intracellular. Extracellular NaCI concentration increases in the inner me-

Reprint requests to Robert S. Balaban, Ph.D., Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung and Blood Institute, Bethesda, Maryland 20892, USA.

564

Balaban and Burg

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