Rabbit Renal Medulla Research Articles

P2A30 - Expression of CYP 3A isoform induced by dexamethasone in rat hepatocarcinoma cell line transfected with an episomal vector expressing the antisense IGF-I cDNA

Whole tissue reduced glutathione (GSH) concentration was found to be lowest in rabbit renal inner medulla and progressively higher in outer medulla and cortex. Activities of cytosolic glutathione reductase in inner medulla and outer medulla were similar, and each was only approximately 50% of that of cortex. Whole tissue and microsomal γ-glutamyl transpeptidase activities were high in cortex and outer medulla but were low in inner medulla. Cytosolic activity of selenium-dependent glutathione peroxidase (GPx-I) was similar in both outer medulla and inner medulla but was only 50% of that of cortex. Activity of cytosolic selenium-independent glutathione peroxidase (GPx-II) was highest in cortex and lowest in inner medulla (∼15% of cortex and ∼50% of outer medulla). Cytosolic glutathione S-transferase activity with l-chloro-2,4-dinitrobenzene as substrate was high in all three regions of kidney. With 1,2-dichloro-4-nitrobenzene and 1,2-epoxy-(4-nitrophenoxy)propane as substrates, cytosolic glutathione S-transferase activities were very low in cortex, outer medulla, and inner medulla. Microsomal activities of glutathione reductase, GPx-I, GPx-II and glutathione S-transferases were much lower than activities of corresponding cytosolic enzymes. Activities of the glutathione peroxidases in renal inner medulla would hence be expected to cause little interference to prostaglandin endoperoxide synthetase mediated cooxidative activation of paracetamol. It has been demonstrated that the paracetamol metabolite can react rapidly with GSH, forming not only glutathione conjugate but also paracetamol itself and oxidized glutathione. Low GSH concentrations, as well as low activities of glutathione reductase, GPx-I, GPx-II, and γ-glutamyl transpeptidase, may therefore render the inner medullary region of kidney particularly vulnerable to paracetamol-related analgesic nephropathy.

Cl- channels in basolateral renal medullary vesicles X. Cloning of a Cl- channel from rabbit outer medulla

These experiments were intended to identify candidate cDNAs which might encode basolateral membrane Cl- channels of the mTAL using a homology-based cloning strategy. We prepared a cDNA library using a 1.8 to 3.2 kb mRNA fraction from rabbit outer medulla that induces a Cl- conductance in cellular membranes of Xenopus laevis oocytes. The cDNA library was screened with two 32P-oligonucleotide probes corresponding to highly conserved sequences in other Cl- channels. We isolated two cDNAs: rbClC-Ka and rbClC-Kb. The protein sequences deduced from these two cDNAs had 99% homology. Using RT-PCR technology, cultured mouse mTAL cells were found to contain mRNA corresponding to those two cDNAs. Expression of the mRNAs corresponding to these two cDNAs was kidney-specific and was greater in rabbit renal medulla than rabbit renal cortex. Finally, by using RT-PCR technology in combination with microdissected glomeruli or tubule segments, we found mRNA for rbClC-Ka in glomeruli, proximal convoluted tubules, mTAL and cortical collecting tubules.

Hyperosmolality stimulates phospholipase A2 activity in rabbit renal medulla and in madin-darby canine kidney (MDCK) cells

Renal medullary cells are able to accumulate glycerophosphocholine during adaptation to the high extracellular osmolality. The aim of this study was to investigate the effect of hyperosmolality on both phospholipase A2 activity and the rate of choline incorporation into glycerophosphocholine in rabbit renal medulla and Madin-Darby Canine Kidney cells. Phospholipase A2 activity was assayed in cellular subfractions isolated from both rabbit kidney medulla and Madin—Darby Canine Kidney cells in the presence of either 1-palmitoyl-2-[1- 14C]palmitoyl phosphatidylcholine or 1-stearoyl-2-[1- 14C]arachidonyl phosphatidylcholine as substrate. The rate of choline incorporation into glycerolphosphocholine was measured in Madin—Darby Canine Kidney cells growing in the presence of [methyl- 3H]choline in the growth medium. Water deprivation of rabbits resulted in an increase of phospholipase A2 activity from 2.7 ± 0.4 ( n = 5) and 5.7 ± 0.7 ( n = 5) to 5.0 ± 0.8 ( n = 5) and 10.8 ± 1.3 ( n = 5) pmol of fatty acid released/min per mg protein in mitochondrial and microsomal fractions, respectively, using dipalmitoyl phosphatidilcholine as substrate while the activity of cytosolic enzyme remained unchanged. Similarly, the addition of sodium chloride in order to increase growth medium osmolality (from 320 mOsm/kg to 520 mOsm/kg) resulted in an elevation of both mitochondrial (from 1.8 ± 0.1 to 4.9 ± 0.8 pmol of fatty acid released/min per mg protein, ( n = 4) and microsomal (from 8.7 ± 0.5 to 15.9 ± 1.7 pmol of fatty acid released/min per mg protein, n = 4) phospholipase A2 activities. An acceleration of phospholipase A2 activity in Madin—Darby Canine Kidney cells was accompanied by 1.5-fold stimulation of choline incorporation into intracellular glycerophosphocholine. In the presence of raffinose to increase the medium osmolality, phospholipase A2 was mainly activated in microsomal fraction of MDCK cells (up to 380% of the control value). The cytosolic PLA2 exhibited still low activity (1.3 ± 0.1 pmol of fatty acids released/min per mg protein) despite relatively high stimulation of this enzyme in MDCK cells growing in the raffinose containing, hyperosmotic medium. The data indicate that both animal antidiuresis in vivo as well as hyporosmolality of extracellular fluids in vitro result in an enhancement of phospholipase A2 activity in kidney medulla and renal cells in culture. It is concluded that activation of this enzyme is involved in the adaptation process of kidney to increased extracellular osmolality.

Cloning and characterization of the vasopressin-regulated urea transporter.

Urea is the principal end product of nitrogen metabolism in mammals. Movement of urea across cell membranes was originally thought to occur by lipid-phase permeation, but recent studies have revealed the existence of specialized transporters with a low affinity for urea (Km > 200 mM)2. Here we report the isolation of a complementary DNA from rabbit renal medulla that encodes a 397-amino-acid membrane glycoprotein, UT2, with the functional characteristics of the vasopressin-sensitive urea transporter previously described in in vitro-perfused inner medullary collecting ducts. UT2 is not homologous to any known protein and displays a unique pattern of hydrophobicity. Because of the central role of this transporter in fluid balance and nitrogen metabolism, the study of this protein will provide important insights into the urinary concentrating mechanism and nitrogen balance.

Inhibition of Na,K-ATPase by Cadmium: Different Mechanisms in Different Species

The mechanism of action of Cd on Na,K-ATPase was investigated in two "classical" model systems, the shark rectal gland and rabbit kidney outer medulla. In lyophilized plasma membranes from dogfish rectal gland Cd inhibited Na,K-ATPase activity after 30 min of preincubation with an I 50 of 1.3 × 10 −5 M. K-Dependent p-nitrophenylphosphatase (pNPPase) activity was inhibited 50% by Cd at 9.4 × 10 −6 M. Neither Na nor K altered the interaction of the enzyme with Cd. Decreasing the ATP concentration, however, lowered the apparent sensitivity of Na,K-ATPase to Cd. The inhibitory effect was also significantly reduced when the Mg concentration present during the preincubation was increased from 0.5 to 6.0 mM. The apparent Cd sensitivity of the K-dependent pNPPase is lower at 10 mM Mg than at 1 mM Mg. In initial rate experiments 4 × 10 −5 M Cd increased the apparent K m of the enzyme for Mg significantly from 0.88 ± 0.29 mM to 1.73 ± 0.3 mM whereas the V max (167 ± 32 μmol/min × mg protein compared to 140 ± 16 μmol/min × mg protein) remained essentially unchanged. In lyophilized rabbit kidney outer medulla, Cd was found to inhibit Na,K-ATPase activity with an I 50 of 1.9 × 10 −5 M. K-Dependent pNPPase was inhibited 50% under identical conditions by Cd at a nominal concentration of 2.1 × 10 −4 M. Increasing K concentrations protected the enzyme from the inhibitory action of Cd as indicated by a 10-fold decrease in sensitivity of pNPPase when the K concentration was increased from 1 to 20 mM. K, 20 mM, delayed also the onset of inhibition by about 15 min at 37°C. These studies suggest that the mode of action of Cd on Na,K-ATPase varies in different species. In rectal gland Cd competes with a Mg site (or sites) critically involved in ATP and pNPP hydrolysis, whereas in rabbit renal medulla Cd interacts with a potassium-binding site. Differences in the protein sequence, protein conformation, and/or in the kind of protein membrane-lipid interaction might contribute to this diversity observed in the inhibitory mechanisms.

Evidence for the presence of a K-dependent acidifying adenosine triphosphatase in the rabbit renal medulla

To date direct evidence for the presence of a H-K-ATPase in the medulla comes from proton and potassium transport studies performed on K-restricted animals and K dependent ATP hydrolysis and Rb uptake in both normal and K-depleted animals. The present work examines K-dependent acidification in the medulla of rabbits on normal K diets. A membrane vesicle preparation was developed that was enriched for apical membranes derived from the renal medulla. Adenosine triphosphate (ATP)-dependent vesicular acidification was present and the extent of vesicular acidification was dependent on ambient K concentration. Moreover, ATP hydrolysis was dependent on ambient K concentration. K-dependent acidification was inhibited by the specific inhibitor of the gastric H-K-ATPase, SCH28080. However, significant acidification was observed in the absence of K that was not inhibited by SCH28080. The data suggest that an H-K-ATPase similar to the gastric H-K-ATPase is present in the renal medulla of rabbits on a normal K diet. The component of acidification and ATP hydrolysis that is independent of K concentration likely represents the previously characterized vacuolar H-ATPase.

A study on the neuronal and non-neuronal stores of dopamine in rat and rabbit kidney

The present study has examined the effects of 6-hydroxydopamine (6-OHDA) alone and in combination with pargyline, desipramine and GBR 12909 and denervation as induced by occlusion of the renal artery (RAO) on the endogenous dopamine (DA) and noradrenaline (NA) contents in rat and rabbit renal tissues; the effects of chemical denervation on catecholamine levels in the left ventricle were also studied. In rat and rabbit renal medulla and rat renal cortex, 6-OHDA and pargyline plus 6-OHDA selectively reduced NA (85-92% reduction) without a parallel decrease in DA tissue content (19-27% reduction). This 6-OHDA- and pargyline plus 6-OHDA-insensitive DA pool was found to be resistant to denervation as induced by RAO. The NA-depleting effect of 6-OHDA in these renal areas was found to be prevented by the previous administration of desipramine, but not with that of GBR 12909. In the rabbit renal cortex, 6-OHDA selectively reduced NA (90% reduction) without a parallel depletion of DA (20% reduction); previous treatment with pargyline abolished this selectivity. Again, only desipramine, but not GBR 12909, was found to prevent the NA and DA depleting effect of 6-OHDA in the rabbit renal cortex. Denervation induced by RAO was also found to produce a parallel depletion of DA and NA tissue levels in this renal area. In the left ventricle, 6-OHDA alone or in combination with pargyline produced a parallel depletion of DA and NA tissue levels (79-88% reduction) in both species. These results provide evidence against the presence of independent dopaminergic neurones in rat and rabbit kidney and suggest that in rat and rabbit renal medulla and rat renal cortex most of DA is stored in a non-neuronal compartment; in rabbit renal cortex some of the DA appears to be located in noradrenergic neurones, in a store different from that which contains NA.

Glutathione depletion and rabbit renal medulla 6-ketoPGF 1α and TxB 2: Levels in vivo and following homogenate incubation in vitro

1. 1. The effect of glutathione (GSH) depletion on rabbit renal medullary homogenate 6-ketoPGF 1α and TxB 2 synthesizing capability was investigated. 2. 2. GSH depletion in vivo with diethyl maleate (DEM) produced higher ( P < 0.05) 6-ketoPGF 1α and TxB 2 renal medullary levels compared to controls. Homogenization and incubation lowered ( P < 0.05) GSH such that there were no differences in GSH between treatments after 5 min of incubation. By 30 min, GSH was lower ( P < 0.05) and 6-ketoPGF 1α higher ( P < 0.05) in homogenates from controls in comparison to those from DEM-treated rabbits. 3. 3. The results indicate GSH depletion increased 6-ketoPGF 1α levels in rabbit renal medulla in vivo but subsequent GSH catabolism prevented assessing the effect of this GSH depletion on prostanoid synthesizing capability.

Fibroblasts of rabbit kidney in culture. I. Characterization and identification of cell-specific markers

There is currently no information available as to whether different renal fibroblast subpopulation can be identified and whether they show differences in functional properties. We therefore compared the growth characteristics of interstitial fibroblasts derived from the rabbit renal cortex and inner medulla (papilla) and sought cell-specific markers for the two populations of cells. Analyses of the population dynamics revealed that the mitotic lifespan of papillary fibroblasts (PF) is approximately 50% longer than that of cortical fibroblasts (CF), with the former going through 20 cumulative population doublings (CPD) before transition into terminally differentiated postmitotic cells compared with 9 CPD in CF. PF and CF populations contained three types of mitotically active cells (MFI, MFII, MFIII) and three types of postmitotic cells (PMFIV, PMFV, PMFVI) differentiating along a terminal cell lineage from MFI through PMFVI. In both PF and CF cultures the percent of MF-type cells decreased and the percent of postmitotic cells increased with successive doublings. Two-dimensional polyacrylamide gel electrophoresis of uniform clonal populations of MFIII-type cells revealed two specific proteins for PF-MFIII-type cells, pf1 and pf2, and three specific proteins for CF-MFIII-type cells, cf1, cf2, and cf3. Additionally, a monoclonal antibody was raised that does not recognize CF in culture, but reacts strongly with PF. These studies demonstrate that rabbit renal PF have a pattern of growth in vitro that is distinct from that of CF and that they can be positively identified by specific immunological and protein markers in vitro.

In Vitro Effects of Synthetic Dyes on the Syntheses of Prostaglandin E2(PGE2) and 5-Hydroxyeicosatetraenoic acid(5-HETE).

The effects of thirty-one synthetic dyes (tar dyes in Japan), which are permitted in cosmetics, on three different enzymes were evaluated as an initial step in developing in vitro safety screens. The activities of the prostaglandin (PG)-synthetase from rabbit renal medulla and the lipoxygenases from potato tubers and guinea pig peritoneal polymorphonuclear leukocytes (PMN) were determined based on the biosyntheses of PGE2 and 5-hydroxyeicosatetraenoic acid (5-HETE), respectively. Xanthene dyes with halogen substituents exhibited marked inhibition on the PG-synthetase. Among the dyes which are permitted for any pharmaceutics and cosmetics, only the xanthene dyes exhibited inhibitory effects on the PG-synthetase at 2.5 mM. The xanthene dyes also showed inhibition and stimulation of potato lipoxygenase and PMN lipoxygenase, respectively.

The Effect of Verapamil on the Specific Activity of Na+-K+-activated Adenosine Triphosphatase in Rabbit Renal Medulla

The Effect of Verapamil on the Specific Activity of Na+-K+-activated Adenosine Triphosphatase in Rabbit Renal Medulla

Cl− transport in basolateral renal medullary vesicles: I. Cl− transport in intact vesicles

This paper provides the results of studies which characterized conductive 36Cl- flux in basolaterally enriched membrane vesicles prepared from rabbit renal outer medulla. Conductive 36Cl- uptake was studied under two different experimental conditions. In the first, 36Cl- flux was driven by an inside positive voltage created with oppositely directed Cl- and gluconate gradients. In the second, an inwardly direct K+ gradient was used to drive 36Cl- uptake. By these two methods, voltage-sensitive 36Cl- uptake was shown to comprise about 45 and 65%, respectively, of the initial rates of total 36Cl- flux. Separate paired studies demonstrated that the conductive 36Cl- uptake was inhibited by the Cl- channel blocker diphenylamine-2-carboxylate (DPC) with an IC50 for DPC of 154 microM. The voltage-dependent 36Cl- uptake had an activation energy of 6.4 kcal/mole. This 36Cl- conductance had an anion selectivity sequence of I- greater than Cl- greater than or equal to NO3- much greater than gluconate.

Effects of NaCl, glucose, and aldose reductase inhibitors on cloning efficiency of renal medullary cells

To analyze the effects of sorbitol accumulation on the survival and growth of epithelial cells from rabbit renal inner medulla, cloning efficiency (an index of cell viability) was measured at normal and high glucose and NaCl concentrations and when sorbitol accumulation was prevented by Tolrestat and Sorbinil, which inhibit aldose reductase. With PAP-HT25 cells grown to near confluence, high NaCl increases aldose reductase activity, causing enough rise in cell sorbitol concentration to balance most of the increased osmolality of the high extracellular NaCl. Inhibition of aldose reductase prevents both the increased enzyme activity and sorbitol accumulation in a dose-related manner. Paralleling this, colony-forming efficiency is not affected by the inhibitors at a normal NaCl concentration but is greatly reduced when extracellular NaCl is high. On the other hand, high glucose levels, as occur in diabetes, increase sorbitol content well above the concentration required for osmotic balance and inhibit colony-forming efficiency. Under those conditions, aldose reductase inhibitors lower cell sorbitol and reverse (at 300-350 mosmol/kgH2O) or reduce (at 500-550 mosmol/kgH2O) the decrease in colony-forming efficiency caused by high glucose. Thus sorbitol accumulation is necessary for osmoregulation when induced by high osmolality but is harmful when induced by high glucose.

Ureteral obstruction decreases ATP dependent H+-pump activity of rabbit renal outer medulla.

Ureteral obstruction has been known to cause a renal acidification defect. To determine the underlying mechanisms of this defect, vacuolar-type H(+)-pump activity of the membrane vesicles taken from the rabbit renal outermedulla was measured in 24 hr obstructed and contralateral kidneys. H(+)-pump activity measured by acridine orange quenching method was resistant to oligomycin (10 micrograms/ml), vanadate (0.1 mM), and the absence of ambient K+, and it was highly sensitive to N-ethylmaleimide (0.5 mM) and dicyclohexylcarbodiimide (0.2 mM). H(+)-pump activity was 6.69 +/- 1.40 in the obstructed kidney, which was significantly lower than 10.32 +/- 1.88% AFU/mg.min in the contralateral kidney. In sham-operated rabbits, H(+)-pump activities were not different in both kidneys. Passive H+/OH- permeability measured by the rate of dissipation of the imposed pH gradient was not different in obstructed and contralateral kidneys. These results suggest that ureteral obstruction decreases vacuolar-type H(+)-pump activity of the kidney outermedulla, and this decrease may contribute to the distal acidification defect of obstructed uropathy.

Osmotic regulation of aldose reductase protein synthesis in renal medullary cells

Renal medullary cells are normally exposed to high extracellular NaCl as part of the urinary concentrating mechanism. They react to this stress by accumulating sorbitol and other organic osmolytes. PAP-HT25, a line of epithelial cells derived from rabbit renal inner medulla, expresses this response. In hypertonic medium, these cells accumulate large amounts of sorbitol. There is a large increase in the amount of aldose reductase, which catalyzes production of sorbitol from glucose. The purpose of the present study was to investigate whether the aldose reductase protein increases because of faster synthesis or slower degradation. We measured the rate of synthesis and degradation of aldose reductase protein by pulse-chase with [35S]methionine, followed by immunoprecipitation with specific antiserum and autoradiography. The protein synthesis rate was 6 times greater in cells grown in hypertonic (500 mosmol/kg) medium, than in those grown in normal (300 mosmol/kg) medium. When control cells were switched to hypertonic medium, the synthesis rate increased 15-fold by 24 h, then decreased to 11-fold after 48 h. In contrast, synthesis rate continued to increase past 24 h when accumulation of sorbitol was prevented by inhibiting aldose reductase activity with Tolrestat. Thus, there is a feedback mechanism by which cellular sorbitol accumulation inhibits aldose reductase protein synthesis. Degradation of aldose reductase protein was slow (only about 25% in 3 days) and was not affected by osmolality. Thus, the osmoregulatory increase in aldose reductase protein is due to an increase in its synthesis rate and not to any change in its degradation.

Acute regulation of the predominant organic solutes of the rabbit renal inner medulla

High concentrations of organic solutes are present in the medulla of the antidiuretic kidney. However, their role in and response to acute changes in the diuretic state are unknown. In this study the organic solute content of the renal medulla was determined from extracts with the use of high-performance liquid chromatography following the acute dilution of the medullary interstitium during various forms of diuresis. After acute infusion of saline and furosemide, inner medullary urea, sodium, inositol, sorbitol, and betaine decrease significantly with no change in glycerophosphorylcholine (GPC) content. After diuresis, inner medullary urea and sodium contents eventually returned to control levels, although inositol, sorbitol, and betaine contents still remained low. Addition of antidiuretic hormone to the saline/furosemide infusion gave similar results. In contrast, induction of diuresis from mannitol infusion caused an acute decrease in all 4 organic solutes, whereas glucose infusion caused an acute decrease in all organic solutes except sorbitol. These data demonstrate that a decrease in all four organic solutes can accompany medullary dilution. However, GPC and sorbitol do not decrease when diuresis is induced by furosemide or glucose, respectively. In addition, the recovery of these compounds in a normally functioning kidney after diuresis is much slower than the regeneration of the sodium chloride and urea gradients.

Molecular cloning of cDNA coding for kidney aldose reductase: Regulation of specific mRNA accumulation by NaCl-mediated osmotic stress

Cells generally respond to long-term hyperosmotic stress by accumulating nonperturbing organic osmolytes. Unlike bacteria, in which molecular mechanisms involved in the increased accumulation of osmolytes have been identified, those in multicellular organisms are virtually unknown. In mammals, during antidiuresis, cells of the renal inner medulla are exposed to high and variable extracellular NaCl. Under these conditions, the cells contain a high level of sorbitol and other osmolytes which help balance the high extracellular osmolality. PAP-HT25 is a continuous line of cells derived from rabbit renal inner medulla. When medium osmolality is increased by raising the NaCl concentration, these cells accumulate sorbitol. The sorbitol is synthesized from glucose in a reaction catalyzed by aldose reductase. When the medium is made hyperosmotic, aldose reductase activity increases because of a larger increase in the amount of enzyme. This increase is produced by the accelerated rate of synthesis of aldose reductase protein. The purpose of the present studies was to examine the mechanism of this increase in aldose reductase protein by measuring the relative abundance of aldose reductase mRNA. A cDNA clone coding for rabbit kidney aldose reductase was isolated. Antisense RNA probes transcribed from this clone hybridized specifically with a 1.5-1.6 kilobase mRNA in Northern blots. Cells grown chronically in hyperosmotic medium had a relative abundance of this specific mRNA which was six times that of cells grown in isoosmotic medium. When cells grown in isoosmotic medium were switched to hyperosmotic medium, the level of aldose reductase mRNA peaked (18-fold) at 18-24 h. The induction of aldose reductase mRNA by osmotic stress was reversible. Our finding of increased abundance of a specific mRNA in direct response to hyperosmotic stress represents the first report of such an effect in animals.

Signal for induction of aldose reductase in renal medullary cells by high external NaCl

GRB-PAP1 is a continuous line of epithelial cells derived from rabbit renal inner medulla. These cells accumulate large amounts of sorbitol when extracellular NaCl concentration is elevated. The accumulation involves an increase in aldose reductase, an enzyme that catalyzes production of sorbitol from glucose. The purpose of the present studies was to investigate the mechanism by which high NaCl triggers an increase in aldose reductase activity. When NaCl was added to increase medium osmolality from 300 to 500 mosmol/kg, enzyme activity began to increase after 12-24 h, was half-maximal in 2 days, and was maximal by 4 days. To test the effect of other solutes, medium osmolality was increased to 500 mosmol/kg by adding raffinose, urea, or glycerol. Raffinose increased aldose reductase activity as much as NaCl did. In contrast, neither urea nor glycerol affected the enzyme activity. NaCl and raffinose caused a sustained decrease in cell water content and an increase in cell sodium and potassium concentration, but urea did not. In some experiments, ouabain was added either with or without an increase in osmolality. Taking into account all of the experiments, with and without ouabain, we found that aldose reductase activity did not correlate with either cell sodium concentration or cell water content alone. It did correlate, however, with cell potassium concentration and even more strongly with the sum of cell sodium plus potassium concentration. We conclude that the signal by which hyperosmolality triggers an increase in aldose reductase activity most likely involves an increase in intracellular ionic strength.

Characterization and purification of a mammalian osmoregulatory protein, aldose reductase, induced in renal medullary cells by high extracellular NaCl.

GRB-PAP1 is a continuous line of epithelial cells derived from a rabbit renal inner medulla. Elevation of the NaCl concentration in the medium bathing these cells strongly induced the expression of a soluble protein with an apparent molecular mass of 39 kDa. The protein, purified by affinity chromatography with Amicon Matrex Gel Orange A, had enzyme activity characteristic of aldose reductase (alditol:NADPH+ oxidoreductase, EC 1.1.1.21). Goat antiserum against this purified aldose reductase selected the 39-kDa band from immunoblots of cells grown in a medium containing high NaCl. When the osmolality of the medium was increased by adding NaCl, the amount of aldose reductase protein and the aldose reductase activity increased together from very low to sustained high levels over several days. The aldose reductase protein was more than 10% of the soluble cell protein when cells were propagated in medium made hyperosmotic by adding NaCl to increase medium osmolality to 600 mosm.kg-1.

Proton NMR spectroscopy and imaging of the rabbit kidney, in vivo, at 4.7 tesla

Osmotically active organic solutes such as trimethylamines and polyols have been implicated in cell volume regulation in a variety of organisms, ranging from bacteria to mammals (I, 2). To avoid losing water in a hypertonic envionment (i.e., osmolality >300 mOsm/liter), animal cells must offset the osmotic pressure of their extracellular surroundings. Many cells have osmotically active organic solutes (“osmolytes”) to regulate their intracellular osmolality and hence their cell volume. The advantage of these organic solutes is that they do not increase intracell~ar ionic strength and thereby do not alter enzyme function; furthermore, studies have shown that these compounds protect enzyme function from the denaturing effects of urea which diffuses freely across cell membranes (I). Medullary cells of the mammalian kidney are exposed to a wide range of extracellular osmolalities, ranging from 50 to greater than 2000 mOsm/liter. To cope with this labile environment, renal medullary cells regulate the concentration of their osmolytes. The predominate osmolytes of the rabbit renal medulla include the trimethylamines betaine and glycerophosphorylcholine and the polyols sorbitol and inositol (2, 3). Averaged over the whole kidney, the concentration of these osmolytes is several millimolar; however, because these compounds are confined within the cells of the renal medulla, their local concentration may well exceed several hundred millimolar. Despite excellent sensitivity, in vivo ‘H NMR spectroscopy has lagged behind studies with other nuclei, such as 13C and 3’P, because of the dynamic range problem associated with a 110 A4 water signal and the limited spectral resolution afforded by a small chemical-shift range. These problems have been ameliorated by various techniques, such as binomial solvent suppression, soft pulse saturation, spin-echo relaxation, jump and-return-echo, and DANTE with homonuclear ‘H double-resonance editing sequences (4-10). Most of the in vivo ‘H NMR spectroscopy to date has been used to study brain metabolism (5, 7, 10). However, the sensitivity of the ‘H nucleus and the high concentration of osmolytes in the kidney provided good reason to try to use ‘H NMR spectroscopy to monitor osmoly-te concentrations. We have developed a kidney model in which ‘H NMR spectroscopy can monitor the regulation of these compounds, in vivo.