Sorbitol Is a Severity Biomarker for PMM2-CDG with Therapeutic Implications.

OBJECTIVE: Epalrestat, an aldose reductase inhibitor increases phosphomannomutase (PMM) enzyme activity in a PMM2-congenital disorders of glycosylation (CDG) worm model. Epalrestat also decreases sorbitol level in diabetic neuropathy. We evaluated the genetic, biochemical, and clinical characteristics, including the Nijmegen Progression CDG Rating Scale (NPCRS), urine polyol levels and fibroblast glycoproteomics in patients with PMM2-CDG. METHODS: We performed PMM enzyme measurements, multiplexed proteomics, and glycoproteomics in PMM2-deficient fibroblasts before and after epalrestat treatment. Safety and efficacy of 0.8 mg/kg/day oral epalrestat were studied in a child with PMM2-CDG for 12 months. RESULTS: PMM enzyme activity increased post-epalrestat treatment. Compared with controls, 24% of glycopeptides had reduced abundance in PMM2-deficient fibroblasts, 46% of which improved upon treatment. Total protein N-glycosylation improved upon epalrestat treatment bringing overall glycosylation toward the control fibroblasts' glycosylation profile. Sorbitol levels were increased in the urine of 74% of patients with PMM2-CDG and correlated with the presence of peripheral neuropathy, and CDG severity rating scale. In the child with PMM2-CDG on epalrestat treatment, ataxia scores improved together with significant growth improvement. Urinary sorbitol levels nearly normalized in 3 months and blood transferrin glycosylation normalized in 6 months. INTERPRETATION: Epalrestat improved PMM enzyme activity, N-glycosylation, and glycosylation biomarkers in vitro. Leveraging cellular glycoproteome assessment, we provided a systems-level view of treatment efficacy and discovered potential novel biosignatures of therapy response. Epalrestat was well-tolerated and led to significant clinical improvements in the first pediatric patient with PMM2-CDG treated with epalrestat. We also propose urinary sorbitol as a novel biomarker for disease severity and treatment response in future clinical trials in PMM2-CDG. ANN NEUROL 20219999:n/a-n/a.

The aldose reductase inhibitor epalrestat exerts nephritic protection on diabetic nephropathy in db/db mice through metabolic modulation.

Epalrestat is an inhibitor of aldose reductase in the polyol pathway and is used for the management of diabetic neuropathy clinically. Our pilot experiments and accumulated evidences showed that epalrestat inhibited polyol pathway and reduced sorbitol production, and suggested the potential renal protection effects of epalrestat on diabetic nephropathy (DN). To evaluate the protective effect of epalrestat, the db/db mice were used and exposed to epalrestat for 8 weeks, both the physiopathological condition and function of kidney were examined. For the first time, we showed that epalrestat markedly reduced albuminuria and alleviated the podocyte foot process fusion and interstitial fibrosis of db/db mice. Metabolomics was employed, and metabolites in the plasma, renal cortex, and urine were profiled using a gas chromatography-mass spectrometry (GC/MS)-based metabolomic platform. We observed an elevation of sorbitol and fructose, and a decrease of myo-inositol in the renal cortex of db/db mice. Epalrestat reversed the renal accumulation of the polyol pathway metabolites of sorbitol and fructose, and increased myo-inositol level. Moreover, the upregulation of aldose reductase, fibronectin, collagen III, and TGF-ß1 in renal cortex of db/db mice was downregulated by epalrestat. The data suggested that epalrestat has protective effects on DN, and the inhibition of aldose reductase and the modulation of polyol pathway in nephritic cells be a potentially therapeutic strategy for DN.

Glycyrrhizic acid attenuated glycative stress in kidney of diabetic mice through enhancing glyoxalase pathway.

SCOPE: Antiglycative effects of glycyrrhizic acid (GA) in kidney of diabetic mice were examined. METHODS AND RESULTS: GA at 0.05, 0.1, and 0.2% was supplied to diabetic mice for 9 wk. Results showed that GA intake increased its deposit in kidney, raised plasma insulin level, decreased plasma glucose and blood urine nitrogen levels, and improved creatinine clearance rate (p < 0.05). GA intake dose-dependently reduced renal carboxymethyllysine level, and at 0.1 and 0.2% decreased plasma HbA1c, urinary glycated albumin, and renal pentosidine levels (p < 0.05). Dietary GA intake declined renal aldose reductase activity and protein expression, as well as lowered renal fructose and sorbitol levels (p < 0.05). GA intake dose-dependently increased glyoxalase-1 activity and expression, and decreased renal methylglyoxal level (p < 0.05). This compound at 0.1 and 0.2% raised glyoxalase-2 activity and protein expression, and increased d-lactate formation (p < 0.05). GA intake dose-dependently suppressed renal expression of nuclear factor kappa B (NF-κB) p65 and p-p38, decreased reactive oxygen species production, and retained glutathione content (p < 0.05). This compound at 0.1 and 0.2% downregulated renal expression of NF-κB p50 and p-ERK1/2 (p < 0.05), and lowered renal level of monocyte chemoattractant protein-1 (MCP-1) and intercellular adhesion molecule-1 (ICAM-1). CONCLUSIONS: These findings suggest that glycyrrhizic acid is an antiglycative and renal-protective agent.

A novel urinary metabolite signature for diagnosing major depressive disorder.

Major depressive disorder (MDD) is a prevalent and debilitating mental disorder. Yet, there are no objective biomarkers available to support diagnostic laboratory testing for this disease. Here, gas chromatography-mass spectrometry was applied to urine metabolic profiling of 126 MDD and 134 control subjects. Orthogonal partial least-squares discriminant analysis (OPLS-DA) was used to identify the differential metabolites in MDD subjects relative to healthy controls. The OPLS-DA analysis of data from training samples (82 first-episode, drug-naïve MDD subjects and 82 well-matched healthy controls) showed that the depressed group was significantly distinguishable from the control group. Totally, 23 differential urinary metabolites responsible for the discrimination between the two groups were identified. Postanalysis, 6 of the 23 metabolites (sorbitol, uric acid, azelaic acid, quinolinic acid, hippuric acid, and tyrosine) were defined as candidate diagnostic biomarkers for MDD. Receiver operating characteristic analysis of combined levels of these six biomarkers yielded an area under the receiver operating characteristic curve (AUC) of 0.905 in distinguishing training samples; this simplified metabolite signature classified blinded test samples (44 MDD subjects and 52 healthy controls) with an AUC of 0.837. Furthermore, a composite panel by the addition of previously identified urine biomarker (N-methylnicotinamide) to this biomarker panel achieved a more satisfactory accuracy, yielding an AUC of 0.909 in the training samples and 0.917 in the test samples. Taken together, these results suggest this composite urinary metabolite signature should facilitate development of a urine-based diagnostic test for MDD.

Increased bone resorption may play a crucial role in the occurrence of osteopenia in patients with type 2 diabetes: Possible involvement of accelerated polyol pathway in its pathogenesis.

In order to investigate the underlying mechanism of alterations in bone mineral metabolism in patients with type 2 diabetes, we determined circulating levels of bone functional markers along with urinary excretion of sorbitol (SOR) and bone mineral density (BMD), and also examined their mutual interrelationship. A total of 151 male type 2 diabetic patients were examined in this study. Forty-eight age-matched male healthy subjects were also studied as the controls. A significant reduction of serum intact osteocalcin (i-OC) was found in the diabetic groups (p<0.01). On the other hand, circulating levels of tartrate resistant acid phosphatase (TRAP) in the diabetic patients were significantly higher than those in the controls (p<0.01). Interestingly, a significantly negative relationship was observed between BMD and serum TRAP (p<0.01), although no significant relationship was noted between BMD and serum i-OC in diabetic patients. Urinary excretion of SOR was significantly elevated in the diabetic patients when compared with the controls (p<0.01). In addition, a significantly positive correlation was observed between serum TRAP and urinary SOR (p<0.01), but not between serum i-OC and urinary SOR. Elevated serum TRAP in diabetes was reduced after the administration of aldose reductase inhibitor (p<0.05). It seems most likely that the increase in osteoclastic function probably due to accelerated polyol pathway plays a crucial role in the pathogenesis of decreased bone mineral content in male patients with type 2 diabetes.

Identification and quantification of the osmodiuretic mannitol in urine for sports drug testing using gas chromatography-mass spectrometry.

The osmodiuretic mannitol can be potentially misused in sports, owing to its urine diluting effect and the possibility to decrease bodyweight. To reveal a doping offence, resulting urinary mannitol concentrations after a prohibited intravenous application and a permitted oral intake have to be differentiated. Therefore, a reliable gas chromatography-mass spectrometry (GC-MS) method was established based on peracetyl derivatives of the analytes. All possible hexitols (allitol, galactitol, iditol, altritols, sorbitol and mannitol) that can occur in human urine were separated and identified on a phenyl-methylpolysiloxane column (HP-5MS) within 10.75 min, and the method demonstrated its capability for quantification purposes. The lower limit of detection and lower limit of quantification were estimated at 0.9 microg mL(-1) and 2.4 microg mL(-1), respectively, and the assay was validated for mannitol and sorbitol regarding the parameters specificity, linearity, intra- (<10%) and inter-day precision (<15%) and accuracy (92-102%). To investigate urinary mannitol concentrations after oral intake the method was applied to an excretion study, providing a mean urinary excretion of mannitol of 19.5%. Comparison of theoretically expected urinary levels after a common therapeutic dose of mannitol and preliminary results on physiological urinary mannitol levels were promising, regarding a threshold level for mannitol that can be utilised for doping control purposes.

Inter- and intra-individual variations in normal urinary glycine betaine excretion.

OBJECTIVES: To establish whether normal human subjects excrete glycine betaine at a constant rate. DESIGN AND METHODS: Urine was collected from ten normal healthy male subjects for 14 days, during which fluid intake was systematically varied from <800 mL to >3 L per day. Glycine betaine, sorbitol and creatinine excretions were estimated per day and as millimole per mol creatinine. RESULTS: The intrasubject SD of urine glycine betaine was 3.5 mmol/mol creatinine, and the intersubject SD 5.8 mmol/mol creatinine. The intrasubject SD of plasma glycine betaine was 10.2 mol/L and the intersubject SD 14.2 mol/L. Water load had little effect on glycine betaine excretion and plasma glycine betaine. After 12 years, excretions and plasma concentrations tended to parallel the initial results. CONCLUSIONS: Normal subjects have consistent individual glycine betaine excretions that are not strongly influenced by urine volume. Abnormal excretions, or significant changes in excretion, can be interpreted as indicating a pathological process.

Analysis of polyols in urine by liquid chromatography-tandem mass spectrometry: a useful tool for recognition of inborn errors affecting polyol metabolism.

Several inborn errors of metabolism with abnormal polyol concentrations in body fluids are known to date. Most of these defects can be diagnosed by the assessment of urinary concentrations of polyols. We present two methods using tandem mass spectrometry for screening for inborn errors affecting polyol metabolism. Urine samples supplemented with internal standards ([13C4]erythritol, [13C2]arabitol and [2H3]sorbitol) were desalted by a mixed-bed ion-exchange resin. Separation was achieved by two different columns. Sugar isomers could not be separated using a Prevail Carbohydrate ES 54 column (method 1), whereas with the other column (Aminex HPX-87C) separation of the isomers was achieved (method 2). Multiple reaction monitoring polyol detection was achieved by tandem mass spectrometry with an electron ion-spray source operating in the negative mode. Age-related reference ranges of polyols (erythritol, treitol, arabitol, ribitol, xylitol, galactitol, mannitol, sorbitol, sedoheptitol and perseitol) in urine were established. The applicability of the method was demonstrated by the abnormal polyol concentrations observed in patients with transaldolase deficiency, ribose-5-phosphate isomerase deficiency and classical galactosaemia. This paper describes two methods for the analysis of urinary polyols by liquid chromatography-tandem mass spectrometry. Method 1 is a fast screening method with the quantification of total isomers and method 2 is a more selective method with the separate quantification of the polyols. Both methods can be used for diagnosing inborn errors of metabolism affecting polyol metabolism.

Alteration of urinary sorbitol excretion in WBN-kob diabetic rats - treatment with an aldose reductase inhibitor.

An accelerated polyol pathway in diabetes contributes to the development of diabetic complications. To elucidate diabetic nephropathy involving also renal tubular damage, we measured urinary sorbitol concentration concomitantly with urinary N-acetyl-D-glucosaminidase (NAG) excretion in WBN-kob diabetic rats.Twenty-four-hour urinary sorbitol concentrations increased in the diabetic rats in parallel with whole blood sorbitol concentrations. An increase in 24-h urinary NAG excretion coincided with the elevated urinary sorbitol levels in the diabetic rats. The administration of epalrestat, an aldose reductase inhibitor, reduced the increased whole blood and urinary sorbitol concentrations and urinary NAG excretion concomitantly with renal aldose reductase inhibition in the diabetic rats. These results indicate that diabetic nephropathy involves distorted cell function of renal tubules, and that treatment with epalrestat may prevent at least the progress of the nephropathy.

Urinary sorbitol measurement and the effect of an aldose reductase inhibitor on its concentration in the diabetic state.

The amounts of sorbitol (SOR) excreted in 24-h urine were determined on two groups, i.e., diabetic and nondiabetic patients, using an improved method in which ion exchange resin column processing was applied, and these levels were compared with SOR levels in whole blood. Urinary SOR concentration was also determined in diabetic and normal rats in the same manner and its relationship to aldose reductase (AR) activity in whole blood was investigated. Changes in SOR levels in urine and whole blood were compared in diabetic rats after administration of an AR inhibitor (ARI). Whole blood SOR levels and urinary SOR excretion were significantly higher in diabetic patients than in nondiabetic patients. The same results were obtained in the animal models. In diabetic rats, the urinary SOR excretion was about five times higher than that in control rats, and the AR activity in whole blood was also significantly higher. The increase in urinary SOR excretion and whole blood SOR levels, as well as AR activity, in blood in the diabetic state was inhibited by ARI administration. The influence of the diabetic state and the efficacy of the ARI were more marked in urinary SOR excretion than in whole blood SOR levels. These data indicate that determinations of urinary SOR excretion and AR activity are easily measurable and of benefit to assessing the diabetic condition.

Involvement of active transport systems in the mobilization of cadmium by dithiocarbamates in vivo.

Previously, we reported that the action of cadmium (Cd) complexing dithiocarbamates, such as N-benzyl-D-glucamine dithiocarbamate (BGD) and N-p-hydroxymethylbenzyl-D-glucamine dithiocarbamate (HBGD), in removing Cd from the kidney involves a probenecid-sensitive organic anion transport system. However, other mechanisms responsible for Cd mobilizing effects of BGD and HBGD are still unclear. Therefore, in the present study we examined the effects of phloretin (an inhibitor of plasma membrane glucose carrier), phloridzin (an inhibitor of Na(+)-dependent active hexose transport) and alpha-aminoisobutyric acid (AIB, an inhibitor of amino acid transport) on the excretion and distribution of the chelating agents and Cd in mice. Phloretin pretreatment markedly decreased the biliary and urinary excretions of BGD and HBGD. Phloridzin pretreatment also decreased the biliary and urinary excretions of HBGD, but had no effect on the BGD. AIB pretreatment had no effect on the excretions of either BGD or HBGD. Phloretin pretreatment increased the hepatic and renal contents of BGD and HBGD. Contrary to this, phloridzin pretreatment decreased the hepatic content of BGD and hepatic and renal contents of HBGD, while AIB pretreatment decreased the renal contents of BGD and HBGD. The mobilizing effects of BGD and HBGD on the hepatic and renal Cd was also investigated using Cd-exposed mice. Phloretin or phloridzin pretreatment decreased the mobilizing effect of BGD and HBGD on the hepatic Cd, but had no effect on the renal Cd. These results suggest that BGD and HBGD are taken up into the liver and kidney by phloridzin- and phloretin-sensitive transport system, respectively; that Cd-BGD and Cd-HBGD complexes formed in the hepatic cells are secreted to the bile by phloridzin- and phloretin-sensitive transport systems; and that free BGD and HBGD secreted from these organ to the bile and urine might have occurred, at least in part, by different mechanisms.

Evaluation of hepatic function in liver cirrhosis: clinical utility of galactose elimination capacity, hepatic clearance of D-sorbitol, and laboratory investigations.

Assessment of hepatic function is based on both liver blood tests and functional tests, the extensive application of which is still controversial. The aim of this study was to evaluate the clinical utility of a few selected tests as discriminatory and prognostic indexes: serum albumin, pseudocholinesterase, prothrombin time, as well as galactose elimination capacity and hepatic sorbitol clearance. Two separate studies were performed: Study I to investigate how well these tests assessed severity, and Study II to evaluate their prognostic value. A total of 128 consecutive cirrhotic patients classified according to the Child-Pugh score were included in Study I; Study II was carried out on 47 of these 128 during a two-year follow-up period. Pairwise correlations between all tests and Child-Pugh score yielded higher significant values for liver blood tests than for the functional ones. In Study I functional tests such as galactose elimination capacity and hepatic sorbitol clearance did not appear to be better than conventional biochemical tests in discriminating clinical severity of cirrhotic patients, as defined by Child-Pugh classification. Results of Study II confirmed that in severe liver cirrhosis Child-Pugh score remains the best method for medium- and long-term prognosis and for planning liver transplantation. Functional tests should be reserved for defining the residual functioning liver mass or for studies about functional liver plasma flow.

Intrarenal distribution of organic osmolytes in human kidney.

The distribution pattern of renal organic osmolytes in surgically explanted human kidneys was investigated and compared with that of untreated Sprague-Dawley rats (Uosmo = 1186 mosmol/kg H2O). Sorbitol, myo-inositol, glycerophosphorylcholine and betaine were measured by liquid chromatography (HPLC) in homogenates of five different kidney zones from the cortex towards the papillary tip. All four organic osmolytes were detected as in human as in rat kidney. The expected increase from the inner medulla to the papillary tip for sorbitol and betaine, and from the outer medulla to the papilla for glycerophosphorylcholine, was observed in rats, but not in explanted human kidney. An inverse distribution pattern was observed with decreasing tissue contents from the inner and outer medulla to the papillary tip for all organic osmolytes and urea. This intrarenal osmolyte profile is in accordance with that observed during water and lithium diuresis in rats. Therefore it can be assumed that a loss of renal medullary osmolytes during the intra-operative treatment of patients led to the observed osmolyte pattern. We conclude that organic osmolytes are involved in renal medullary osmoadaptation of humans.

Short-term response of nonurea organic osmolytes in human kidney to a water load and water deprivation.

The cells of the inner medulla of the mammalian kidney accumulate high concentrations of nonurea organic osmolytes. The organic osmolytes found in the kidney include glycine betaine and sorbitol. This study was designed to measure changes in the urinary excretion of glycine betaine and sorbitol and the plasma concentration of glycine betaine in response to an acute water load (20 ml/kg) or acute water deprivation in young healthy males. In response to a water load the urinary excretion of glycine betaine and sorbitol increased parallel with or shortly after urinary urea excretion. The increase in urinary urea and sorbitol excretions preceded maximum minute volume, whereas peak glycine betaine excretion was closely related to maximum urine minute volume. Subsequently, urea, sorbitol, and glycine betaine excretion rates returned to baseline. In contrast, during water deprivation no change in glycine betaine, sorbitol, and urea urinary excretions occurred during the study period. Plasma glycine betaine concentration was stable during both diuresis and antidiuresis. We conclude that the organic osmolytes glycine betaine and sorbitol are components of a physiological and dynamic system in response to an acute water diuresis.

Abnormal glycine betaine content of the blood and urine of diabetic and renal patients.

In normal human plasma the concentrations of the renal osmolyte, glycine betaine, are usually between 20 and 70 mumol/l, in adult males (median 44 mumol/l) higher than in females (34 mumol/l). Concentrations are lower in renal disease (median 28 mumol/l) and normal in diabetes. Urinary excretion of glycine betaine shows no sex difference and is frequently elevated both in renal disease and in diabetes (medians: normal, 6.2, renal 12.3 and diabetes, 39.7 mmol/mol creatinine). The elevation in diabetes does not strongly correlate with known renal disease, nor with either urinary microalbumin or plasma creatinine. There is no correlation with glycated haemoglobin. The positive correlation with the excretions of another renal osmolyte, sorbitol, was highly significant in diabetic subjects. In the diabetic group there was also a significant negative correlation between plasma glycine betaine and urine microalbumin.

Dose dependency of fermentation and the extent of renal excretion of palatinit (isomalt) in rats with respect to its energy value.

The impact of dose-dependent caloric salvage by microbial fermentation processes in the lower gut and the extent of renal excretion for the overall energetic availability of the alternative bulk sweetener Palatinit were investigated in rats. To evaluate the extent of dose-dependent fermentation a conventional and a germ-free rat model were used and fecal excretions of Palatinit after intragastric application were compared. Because of the lack of bacterial colonization in the gastrointestinal tract in germ-free rat the difference in fecal excretion of Palatinit between germ-free and conventional rat is mainly due to bacterial fermentation. To determine the amount of renal excretion of Palatinit the urine was collected. The experiments were conducted using different amounts of Palatinit (300 and 1,200 mg/kg body weight = mg/kg b.w.). Fecal excretions of Palatinit and its monomers (sorbitol and mannitol) were measured by high-performance liquid chromatography (HPLC) and for the determination of renal excretions a gas chromatography system was used. After the application of 300 mg/kg b.w. Palatinit only the breakdown product sorbitol could be recovered in the feces of germ-free rats (29% of the applied dose). No intact Palatinit could be found. In contrast, neither Palatinit nor the breakdown products sorbitol or mannitol could be detected in the feces of conventional rats after application of the same dose. After the application of the higher dose only small amounts of intact Palatinit were found in the feces of germ-free rats (average 12%). There was no intact measurable Palatinit in the feces of conventional rats. The fecal excretions of sorbitol and mannitol in the feces of the germ-free rats were 55% and 39%; in conventional rats only 21% sorbitol was excreted. Only traces of Palatinit, sorbitol or mannitol were found in the urine of conventional and germ-free rats after application of the low as well as the high dose. In conclusion, this study clearly shows the dose dependency of fermentation and therefore the dose dependency of the energetic (i.e., caloric) availability of this disaccharide sugar alcohol. In the calculation of the energy value of Palatinit the renal excretion of Palatinit and its monomers can be neglected.

Sorbitol-accumulating pyrimidine derivatives.

The novel compounds SDI 157 (2-methyl-4(4-N,N-dimethylaminosulfonyl-1-piperazino)pyrimid ine, CAS 131816-54-1) and SDI 158 (2-hydroxymethyl-4-(4-N,N-dimethylaminosulfonyl-1- piperazino) pyrimidine, CAS 140687-51-0) have been found to be inhibitors of sorbitol dehydrogenase. In normal and diabetic animals both compounds induced a dose-dependent increase of tissue sorbitol, especially in the peripheral nerve, without alteration of the blood glucose. In contrast to SDI 158, SDI 157 does not act in vitro. However, in the isolated perfused rat liver SDI 157 induced a high sorbitol release and plasma samples of animals treated with SDI 157 induced a sorbitol accumulation in vitro in erythrocytes like SDI 158. SDI 157 seems to be a prodrug. In accordance with the polyol theory, the chronic administration of SDI 157 to diabetic rats accelerated the cataract development and depleted the lens of total and oxidized glutathione. Surprisingly, however, it accelerated the motor nerve conduction velocity in normal and diabetic rats, normalized the glomerular filtration rate in diabetic rats and did not induce retinal capillary lesions in normal rats or aggravate those in diabetic rats. At single oral doses up to 100 mg/kg, SDI 157, was well tolerated by diabetic and normal rats. Except for a reduction of drinking water consumption, the chronic administration of SDI 157 in drinking water at doses up to 100 mg/kg per day had no side effects in normal, diabetic and galactoselfructose-rich diet rats.

Methylamines and polyols in kidney, urinary bladder, urine, liver, brain, and plasma. An analysis using 1H nuclear magnetic resonance spectroscopy.

Methylamines and polyols are known to behave as organic osmolytes in the adaptation of many cells to hyperosmolar conditions. Using 1H nuclear magnetic resonance spectroscopy to analyze perchloric acid extracts we have examined several tissues in the rat for the presence of these compounds. Methylamines such as glycerophosphorylcholine, choline and betaine were observed in the renal inner medulla, urinary bladder, urine, liver, brain, and plasma. Myoinositol was relatively abundant in the renal inner medulla and brain whereas sorbitol was detected only in the inner medulla. A variety of unidentified compounds was also detected in each tissue. Although these methylamines and polyols are known to respond to osmotic changes in the renal inner medulla, their responses in other tissues remain to be investigated.

Osmotic effectors in kidneys of xeric and mesic rodents: corticomedullary distributions and changes with water availability.

Urea, sodium, the methylamines glycine betaine and glycerophosphorylcholine (GPC), and the polyols sorbitol and myo-inositol are reported to be the major osmolytes in kidneys of laboratory mammals. These were measured (millimoles per kilogram wet weight) in kidney regions and urines of three species of wild rodents with different dehydration tolerances: the pocket mouse Perognathus parvus (xeric), vole Microtus montanus (mesic), and deer mouse Peromyscus m. gambeli (intermediate). In animals kept without water for 4-6 days, sodium, urea, betaine and GPC + choline were found in gradients increasing from cortex to outer to inner medulla in all species, with Perognathus having the highest levels. Sorbitol was high in the inner medulla but low in the cortex and outer medulla; inositol was highest in the outer medulla. Totals of methylamines and methylamines plus polyols in the medulla showed high linear correlations (positive) with urea and with sodium values. Whole medullae were analyzed at several time points in Microtus and Peromyscus subject to water diuresis followed by antidiuresis. In 102 h diuresis in Microtus, all osmolytes decreased except inositol; however, only urea, sodium and sorbitol reached new steady states within 24 h. Urea returned to initial values in 18 h antidiuresis, while other osmolytes required up to 90 h. In Peromyscus, all osmolytes except the polyols declined in diuresis (max. 78 h test period). During antidiuresis, urea and GPC + choline rose to initial values in 18 h, with sodium and betaine requiring more time. In plots of both species combined, total methylamines + polyols correlated linearly (positive) with sodium, and GPC + choline with urea. Estimates of tissue concentrations suggest that total methylamines + polyols can account for intracellular osmotic balance in all species in antidiuresis and that sufficient concentrations of methylamines may be present to counteract perturbing effects of urea on proteins.