Developmental changes in organic osmolytes in prenatal and postnatal rat tissues

At high osmotic pressures, mammalian kidney medulla, heart, lens, and brain utilize organic osmolytes to regulate cell volume. However the types and proportions of these solutes vary among tissues in patterns and for non-osmotic roles not fully elucidated. To clarify these, we analyzed osmolyte-type solute contents in rat tissues at 7 and 2 days prenatal and at 0, 7, 14, 21 (weaning), 35 (juvenile) and 77 (adult) days postnatal. Placentas were dominated by betaine, taurine, and creatine, which decreased between the prenatal times. Fetuses were dominated by glutamate and taurine, which increased between the times. In cerebrum, hindbrain and diencephalon, taurine dominated at early stages, but dropped after postnatal day 7, while myo-inositol, glutamine, creatine and glutamate increased after birth, with the latter two dominating in adults. In olfactory bulb, taurine content declined gradually with age and was equal to glutamate in adults. In all brain regions, glycerophosphorylcholine (GPC) reached a peak in juveniles. In postnatal renal medulla, urea, sodium, GPC, betaine, and taurine increased sharply at day 21. Thereafter, most increased, but taurine decreased. In heart, taurine dominated, and increased with age along with creatine and glutamine, while glutamate decreased after postnatal day 7. In lens, taurine dominated and declined in adults. These patterns are discussed in light of hypotheses on non-osmotic and pathological roles of these solutes.     

Amino acids as well as polyols and methylamines accumulated in rat kidney during dehydration

Summary During antidiuresis cells in the renal inner medulla contain large amounts of sorbitol, myo-inositol, glycerophosphorylcholine and betaine to adjust the intracellular osmolality to the extracellular hyperosmolality. Although the accumulation of these four major organic osmolytes in the inner medulla of the dehydrated animal has been a consistent finding, the role of another class of organic osmolytes, amino acids, in osmoregulation in the kidney remains controversial. In the present study, renal responses of four major osmolytes and amino acids to dehydration were investigated using two HPLC systems. Taurine levels were significantly higher in the inner medulla of the dehydrated rats as compared with the control rats, and increased monotonically from the cortex to the inner medulla along the corticopapillary axis in the dehydrated rats. As for four major osmolytes, we confirm previously reported patterns in antidiuresis in greater detail. In conclusion, not only the four major osmolytes but taurine also plays a salient role in the osmoregulation in the kidney.     

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

Summary 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 mousePerognathus parvus (xeric), voleMicrotus montanus (mesic), and deer mousePeromyscus 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, withPerognathus 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 inMicrotus andPeromyscus subject to water diuresis followed by antidiuresis. In 102 h diuresis inMicrotus, 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. InPeromyscus, 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.Abbrevations GPC Glycero-3-phosphorylcholine - TCA trichloroacetic acid - M+P methylamines plus polyols     

Gap junctions favor normal rat kidney epithelial cell adaptation to chronic hypertonicity

Upon hypertonic stress most often resulting from high salinity, cells need to balance their osmotic pressure by accumulating neutral osmolytes called compatible osmolytes like betaine, myo-inositol, and taurine. However, the massive uptake of compatible osmolytes is a slow process compared with other defense mechanisms related to oxidative or heat stress. This is especially critical for cycling cells as they have to double their volume while keeping a hospitable intracellular environment for the molecular machineries. Here we propose that clustered cells can accelerate the supply of compatible osmolytes to cycling cells via the transit, mediated by gap junctions, of compatible osmolytes from arrested to cycling cells. Both experimental results in epithelial normal rat kidney cells and theoretical estimations show that gap junctions indeed play a key role in cell adaptation to chronic hypertonicity. These results can provide basis for a better understanding of the functions of gap junctions in osmoregulation not only for the kidney but also for many other epithelia. In addition to this, we suggest that cancer cells that do not communicate via gap junctions poorly cope with hypertonic environments thus explaining the rare occurrence of cancer coming from the kidney medulla.     

Pathways for organic osmolyte synthesis in rabbit renal papillary tissue, a metabolic study using 13C-labeled substrates

Renal papillary collecting duct cells have been postulated to adapt their intracellular osmolality to the large changes in interstitial osmolality by changing their content of 'non-perturbing' organic osmolytes such as sorbitol and myo-inositol. 13C-NMR was used in this study to elucidate the metabolic pathways leading to a synthesis of those compounds. Incubation of rabbit renal papillary tissue with [1-13C]glucose showed label scrambling mainly into sorbitol (C-1) and lactate (C-3). This result confirms activity of aldose reductase and glycolytic enzymes in renal papillary cells. Using [3-13C]alanine or [2-13C]pyruvate as carbon source, 13C-labeling of sorbitol and myo-inositol was observed, indicating that renal papillary tissue possesses, in addition, gluconeogenic activity. The latter assumption is supported by the result that in enzyme assays rabbit kidney papilla and isolated rat kidney papillary collecting duct cells show significant fructose-1,6-bisphosphatase activity.     

TMAO and other organic osmolytes in the muscles of amphipods (Crustacea) from shallow and deep water of Lake Baikal

Concentrations of trimethylamine oxide (TMAO) and other 'compatible' osmolytes were analyzed in the muscle tissue of Lake Baikal amphipods (Crustacea) in relation to water depth of the freshwater Lake Baikal. Using HPLC and mass spectrometry, glycerophosphoryl choline (GPC), betaine, S-methyl-cysteine, sarcosine, and taurine were detected for the first time in freshwater amphipods. These osmolytes were frequently found in the five species studied but mixtures were too complex to be quantified. The pattern of these osmolytes did not change with respect to water depth. The TMAO concentration, however, was significantly higher in the muscle tissue of amphipods living in deep water than of those living in shallow water, which supports the hypothesis that TMAO acts as a protective osmolyte at increased hydrostatic pressure. We propose that eurybathic amphipods, exposed to raised hydrostatic pressure in the extremely deep freshwater Lake Baikal, have elevated TMAO levels to counteract the adverse effect of high pressure on protein structure. The elevated intracellular osmotic pressure is balanced by upregulating the extracellular hemolymph NaCl concentration.     

Naturally occurring osmolytes modulate the nanomechanical properties of polycystic kidney disease domains

Polycystin-1 (PC1) is a large membrane protein that is expressed along the renal tubule and exposed to a wide range of concentrations of urea. Urea is known as a common denaturing osmolyte that affects protein function by destabilizing their structure. However, it is known that the native conformation of proteins can be stabilized by protecting osmolytes that are found in the mammalian kidney. PC1 has an unusually long ectodomain with a multimodular structure including 16 Ig-like polycystic kidney disease (PKD) domains. Here, we used single-molecule force spectroscopy to study directly the effects of several naturally occurring osmolytes on the mechanical properties of PKD domains. This experimental approach more closely mimics the conditions found in vivo. We show that upon increasing the concentration of urea there is a remarkable decrease in the mechanical stability of human PKD domains. We found that protecting osmolytes such as sorbitol and trimethylamine N-oxide can counteract the denaturing effect of urea. Moreover, we found that the refolding rate of a structurally homologous archaeal PKD domain is significantly slowed down in urea, and this effect was counteracted by sorbitol. Our results demonstrate that naturally occurring osmolytes can have profound effects on the mechanical unfolding and refolding pathways of PKD domains. Based on these findings, we hypothesize that osmolytes such as urea or sorbitol may modulate PC1 mechanical properties and may lead to changes in the activation of the associated polycystin-2 channel or other intracellular events mediated by PC1.     

Mammalian osmolytes and S-nitrosoglutathione promote Delta F508 cystic fibrosis transmembrane conductance regulator (CFTR) protein maturation and function

In cystic fibrosis, the absence of functional CFTR results in thick mucous secretions in the lung and intestines, as well as pancreatic deficiency. Although expressed at high levels in the kidney, mutations in CFTR result in little or no apparent kidney dysfunction. In an effort to understand this phenomenon, we analyzed Delta F508 CFTR maturation and function in kidney cells under conditions that are common to the kidney, namely osmotic stress. Kidney cells were grown in culture and adapted to 250 mM NaCl and 250 mM urea. High performance liquid chromatography analysis of lysates from kidney cells adapted to these conditions identified an increase in the cellular osmolytes glycerophosphorylcholine, myo-inositol, sorbitol, and taurine. In contrast to isoosmotic conditions, hyperosmotic stress led to the proper folding and processing of Delta F508 CFTR. Furthermore, three of the cellular osmolytes, when added individually to cells, proved effective in promoting the proper folding and processing of the Delta F508 CFTR protein in both epithelial and fibroblast cells. Whole-cell patch clamping of osmolyte-treated cells showed that Delta F508 CFTR had trafficked to the plasma membrane and was activated by forskolin. Encouraged by these findings, we looked at other features common to the kidney that may impact Delta F508 maturation and function. Interestingly, a small molecule, S-nitrosoglutathione, which is a substrate for gamma glutamyltranspeptidase, an abundant enzyme in the kidney, likewise promoted Delta F508 CFTR maturation and function. S-Nitrosoglutathione-corrected Delta F508 CFTR exhibited a shorter half-life as compared with wild type CFTR. These results demonstrate the feasibility of a small molecule approach as a therapeutic treatment in promoting Delta F508 CFTR maturation and function and suggest that an additional treatment may be required to stabilize Delta F508 CFTR protein once present at the plasma membrane. Finally, our observations may help to explain why Delta F508 homozygous patients do not present with kidney dysfunction.     

Salting-in of neopentane in the aqueous solutions of urea and glycine-betaine

The effects of urea and glycine-betaine (GB) osmolytes on the hydrophobic interactions of neopentane in water have been studied using molecular dynamics simulations. From the study of the potentials of mean force, it is observed that both urea and GB decrease the association and solvation of neopentane. The calculated equilibrium constants show that urea and GB decrease the population of solvent-separated minima of neopentane. The hydrophobic association as well as solvation of neopentane molecules are stabilised by entropy and enthalpy in the mixtures. The radial distribution functions (RDFs) and running coordination numbers of water, urea and GB molecules show that neopentane shows salting-in behaviour in aqueous-GB, aqueous-urea and aqueous-urea-GB mixtures. Neopentane is preferentially solvated by GB in aqueous-GB and preferentially solvated by urea in aqueous-urea-GB solutions. The preferential solvation of neopentane by GB suggests that GB decreases the interaction between neopentane molecules i.e. salting-in of neopentane. The calculated solvation free energies and radial density profiles of neopentane also support the salting-in behaviour of neopentane in the mixtures of these osmolytes.     

The role of taurine in renal disorders

This article examines the actions of taurine on models of renal dysfunction, the potential mechanisms of taurine action and the possible clinical significance of these findings. Our laboratory has written previously on the role of taurine in renal function and we have focused upon the normal physiology of the kidney and on the mechanisms and regulation of the renal transport of taurine. This review is a distinct change of emphasis in that we describe a number of studies which have evaluated various aspects of renal dysfunction, including hypertension and proteinuria, specific glomerular and tubular disorders, acute and chronic renal conditions, urinary tract conditions including infection and nephrolithiasis, and diabetic nephropathy. The subject of chronic kidney disease and renal transplantation will also be examined relative to β amino acid. The studies evaluated will be mainly recent ones, recognizing that older reviews of the role of this taurine in the kidney are available.     

Cloning of the cDNa for a Na+/myo-inositol cotransporter, a hypertonicity stress protein.

Kidney medullary cells in situ, as well as kidney-derived Madin-Darby canine kidney (MDCK) cells accumulate nonperturbing, small organic solutes (osmolytes), including myo-inositol, when bathed in hypertonic media. Accumulation of osmolytes balances the osmolality of extracellular fluid without raising intracellular salts that would perturb cellular functions. In hypertonic media, increased myo-inositol accumulation is the result of increased activity of a Na+/myo-inositol cotransporter. We have isolated a cDNA encoding a Na+/myo-inositol cotransporter from MDCK cells using expression in Xenopus oocytes. The cDNA sequence predicts a protein of 718 amino acids with a significant amino acid sequence similarity to the Na+/D-glucose cotransporters of absorbing epithelia. Transporter mRNA is present in kidney and brain and is markedly induced in MDCK cells by medium hypertonicity, demonstrating that adaptation to hypertonic stress involves up-regulation of transporter mRNA accumulation.     

Hypoglycaemic, antioxidative and nephroprotective effects of taurine in alloxan diabetic rabbits

The therapeutic potential of taurine was investigated under diabetic conditions. Alloxan diabetic rabbits were treated daily for three weeks with 1% taurine in drinking water. The following parameters were measured: 1) serum glucose, urea, creatinine and hydroxyl free radical (HFR) levels; 2) blood glutathione redox state; 3) urine albumin concentration; 4) hepatic and renal HFR levels, GSH/GSSG ratios and the activities of catalase, superoxide dismutase and the enzymes of glutathione metabolism; 5) renal NADPH oxidase activity; 6) the rates of renal and hepatic gluconeogenesis. Histological studies of kidneys were also performed. Taurine administration to diabetic rabbits resulted in 30% decrease in serum glucose level and the normalisation of diabetes-elevated rate of renal gluconeogenesis. It also decreased serum urea and creatinine concentrations, attenuated diabetes-evoked decline in GSH/GSSG ratio and abolished hydroxyl free radicals accumulation in serum, liver and kidney cortex. Animals treated with taurine exhibited elevated activities of hepatic gamma-glutamylcysteine syntetase and renal glutathione reductase and catalase. Moreover, taurine treatment evoked the normalisation of diabetes-stimulated activity of renal NADPH oxidase and attenuated both albuminuria and glomerulopathy characteristic of diabetes. In view of these data, it is concluded that: 1) diminished rate of renal gluconeogenesis seems to contribute to hypoglycaemic effect of taurine; 2) taurine-induced increase in the activities of catalase and the enzymes of glutathione metabolism is of importance for antioxidative action of this amino acid and 3) taurine nephroprotective properties might result from diminished renal NADPH oxidase activity. Thus, taurine seems to be beneficial for the therapy of both diabetes and diabetic nephropathy.     

Ultraviolet B radiation induces cell shrinkage and increases osmolyte transporter mRNA expression and osmolyte uptake in HaCaT keratinocytes

We have previously shown that compatible organic osmolytes, such as betaine, myo-inositol and taurine, are part of the stress response of normal human keratinocytes (NHKs) to ultraviolet B (UVB) radiation. In this regard, we tested human HaCaT keratinocytes as a surrogate cell line for NHK. HaCaT cells osmo-dependently express mRNA specific for transport proteins for betaine (BGT-1), myo-inositol (SMIT) and taurine (TAUT). Compared to normoosmotic (305 mosmol/l) controls, which strongly constitutively expressed BGT-1 mRNA, strong induction of SMIT and TAUT mRNA as well as low induction of BGT-1 mRNA expression was observed between 3 and 9 h after hyperosmotic exposure (405 mosmol/l). This expression correlated with an increased osmolyte uptake. Conversely, hypoosmotic (205 mosmol/l) stimulation led to a significant efflux of osmolytes. Exposure to UVB (290-315 nm) radiation induced cell shrinkage which was followed by an upregulation of osmolyte transporter mRNA levels and osmolyte uptake. These results demonstrate that human HaCaT keratinocytes possess an osmolyte strategy including UVB-induced cell shrinkage and following increased osmolyte uptake. However, several differences in osmolyte transporter expression and uptake were noted between NHK and HaCaT cells, indicating that the use of HaCaT cells as a surrogate cell line for NHK has limitations.     

Taurine Ameliorates Renal Oxidative Damage and Thyroid Dysfunction in Rats Chronically Exposed to Fluoride

Excessive exposure to fluoride poses several detrimental effects to human health particularly the kidney which is a major organ involved in its elimination from the body. The influence of taurine on fluoride-induced renal toxicity was investigated in a co-exposure paradigm for 45 days using five groups of eight rats each. Group I rats received normal drinking water alone, group II rats were exposed to sodium fluoride (NaF) in drinking water at 15 mg/L alone, group III received taurine alone at a dose of 200 mg/kg group IV rats were co-administered with NaF and taurine (100 mg/kg), while group V rats were co-administered with NaF and taurine (200 mg/kg). Administration of taurine significantly reversed the fluoride-mediated decrease in absolute weight and organo-somatic index of the kidney in the exposed rats. Taurine significantly prevented fluoride-induced elevation in plasma urea and creatinine levels in the exposed rats. Moreover, taurine restored fluoride-mediated decrease in the circulatory concentrations of triiodothyronine, thyroxine, and the ratio of triiodothyronine to thyroxine. Taurine ameliorated fluoride-mediated decrease in renal antioxidant status by significantly enhancing the antioxidant enzyme activities as well as glutathione level in the exposed rats. Additionally, taurine inhibited fluoride-induced renal oxidative damage by markedly decreasing the hydrogen peroxide and malondialdehyde levels as well as improved the kidney architecture in the treated rats. Collectively, taurine protected against fluoride-induced renal toxicity via enhancement of thyroid gland function, renal antioxidant status, and histology in rats.     

An analysis of the molecular origin of osmolyte-dependent protein stability

Protein solvation is the key determinant for isothermal, concentration-dependent effects on protein equilibria, such as folding. The required solvation information can be extracted from experimental thermodynamic data using Kirkwood-Buff theory. Here we derive and discuss general properties of proteins and osmolytes that are pertinent to their biochemical behavior. We find that hydration depends very little on osmolyte concentration and type. Strong dependencies on both osmolyte concentration and type are found for osmolyte self-solvation and protein-osmolyte solvation changes upon unfolding. However, solvation in osmolyte solutions does not involve complex concentration dependencies as found in organic molecules that are not used as osmolytes in nature. It is argued that the simple solvation behavior of naturally occurring osmolytes is a prerequisite for their usefulness in osmotic regulation in vivo.     

Properties of a polarized primary culture from rat renal inner medullary collecting duct (IMCD) cells

Summary A primary culture from rat renal IMCD cells was established to investigate the permeability characteristics of the luminal and contraluminal plasma membranes of the papillary collecting duct in vitro. Freshly isolated IMCD cells were grown on filters in a special “epithelial cell” medium. Confluency was proved with an epithelial volt/ohm meter. After 7 d of culture the transepithelial resistance reached more than 1000 Ω×cm². A polarization of the cells with regard to a basolateral localization of a lactate efflux system, and an l-alanine transport system was achieved. The hypotonicity-activated release systems for the organic osmolytes sorbitol and betaine were also located basolaterally, whereas taurine, glycerophosphorylcholine, and myo-inositol left the cells at both cell poles but with different capacity. Morphological observations revealed also that the monolayer was well differentiated. Thus, a model of a renal collecting duct epithelium was established which can be used to analyze polarized and differentiated transport processes across the epithelial cells and their plasma membranes.     

Regulatory volume decrease after swelling induced by urea in fibroblasts: prominent role of organic osmolytes

Cell swelling, regulatory volume decrease (RVD), volume-sensitive Cl⁻ (Cl⁻ _swell) current and taurine efflux after exposure to high concentrations of urea were characterized in fibroblasts Swiss 3T3, and results compared to those elicited by hyposmotic (30%) swelling. Urea 70, 100, and 150 mM linearly increased cell volume (8.25%, 10.6%, and 15.7%), by a phloretin-inhibitable process. This was followed by RVD by which cells exposed to 70, 100, or 150 mM urea recovered 27.6%, 38.95, and 74.1% of their original volume, respectively. Hyposmolarity (30%) led to a volume increase of 25.9% and recovered volume in 32.5%. ³H-taurine efflux was increased by urea with a sigmoid pattern, as 9.5%, 18.9%, 71.5%, and 89% of the labeled taurine pool was released by 70, 100, 150, or 200 mM urea, respectively. Only about 11% of taurine was released by 30% hyposmolarity reduction in spite of the high increase in cell volume. Urea-induced taurine efflux was suppressed by NPPB (100 μM) and markedly reduced by the tyrosine kinase-general blocker AG18. The Cl⁻ _swell current was more rapidly activated and higher in amplitude in the hyposmotic than in the isosmotic/urea condition (urea 150 mM), but this was not sufficient to accomplish an efficient RVD. These results showed that at similar volume increase, cells swollen by urea showed higher taurine efflux, lower Cl⁻ _swell current and more efficient RVD, than in those swollen by hyposmolarity. The correlation found between RVD efficiency and taurine efflux suggest a prominent role for organic over ionic osmolytes for RVD evoked by urea in isosmotic conditions.