Jennifer J. Bedford

ORE, a Eukaryotic Minimal Essential Osmotic Response Element THE ALDOSE REDUCTASE GENE IN HYPEROSMOTIC STRESS

Organisms, almost universally, adapt to hyperosmotic stress through increased accumulation of organic osmolytes but the molecular mechanisms have only begun to be addressed. Among mammalian tissues, renal medullary cells are uniquely exposed to extreme hyperosmotic stress. Sorbitol, synthesized through aldose reductase, is a predominant osmolyte induced under hyperosmotic conditions in renal cells. Using a rabbit renal cell line, we originally demonstrated that hyperosmotic stress induces transcription of the aldose reductase gene. Recently, we cloned the rabbit aldose reductase gene, characterized its structure, and found the first evidence of an osmotic response region in a eukaryotic gene. Now, we have progressively subdivided this 3221-base pair (bp) region into discrete fragments in reporter gene constructs. Thereby, we have functionally defined the smallest sequence able to confer hyperosmotic response on a downstream gene independent of other putative cis-elements, that is, a minimal essential osmotic response element (ORE). The sequence of the ORE is CGGAAAATCAC(C) (bp −1105/−1094). A 17-bp fragment (−1108/−1092) containing the ORE used as a probe in electrophoretic mobility shift assays suggests hyperosmotic induction of a slowly migrating band. Isolation of trans-acting factor(s) and characterization of their interaction with the ORE should elucidate the basic mechanisms for regulation of gene expression by hyperosmotic stress.

Lithium-induced Nephrogenic Diabetes Insipidus: Renal Effects of Amiloride

BACKGROUND AND OBJECTIVES Polyuria, polydipsia, and nephrogenic diabetes insipidus have been associated with use of psychotropic medications, especially lithium. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS The impact of psychotropic medications on urinary concentrating ability and urinary aquaporin 2 (AQP2) excretion was investigated after overnight fluid deprivation, and over 6 h after 40 microg of desmopressin (dDAVP), in patients on lithium (n = 45), compared with those on alternate psychotropic medications (n = 42). RESULTS Those not on lithium demonstrated normal urinary concentrating ability (958 +/- 51 mOsm/kg) and increased urinary excretion of AQP2 (98 +/- 21 fmol/micromol creatinine) and cAMP (410 +/- 15 pmol/micromol creatinine). Participants taking lithium were divided into tertiles according to urinary concentrating ability: normal, >750 mOsm/kg; partial nephrogenic diabetes insipidus (NDI), 750 to 300 mOsm/kg; full NDI, <300 mOsm/kg. Urinary AQP2 concentrations were 70.9 +/- 13.6 fmol/micromol creatinine (normal), 76.5 +/- 10.4 fmol/micromol creatinine (partial NDI), and 27.3 fmol/micromol creatinine (full NDI). Impaired urinary concentrating ability and reduced urinary AQP2, cAMP excretion correlated with duration of lithium therapy. Other psychotropic agents did not impair urinary concentrating ability. Eleven patients on lithium were enrolled in a randomized placebo-controlled crossover trial investigating the actions of amiloride (10 mg daily for 6 wk) on dDAVP-stimulated urinary concentrating ability and AQP2 excretion. Amiloride increased maximal urinary osmolality and AQP2 excretion. CONCLUSIONS By inference, amiloride-induced reduction of lithium uptake in the principal cells of the collecting duct improves responsiveness to AVP-stimulated translocation of AQP2 to the apical membrane of the principal cells.

Aquaporin Expression in Normal Human Kidney and in Renal Disease

Aquaporins (AQPs), membrane-inserted water channel proteins, play a highly important role in the reabsorption of water from the renal tubular fluid. Experimentally, both in rats and mice, failure to insert functional AQP molecules into renal tubular membranes leads to nephrogenic diabetes insipidus. In humans, most forms of renal disease lead to a reduction in the water handling capacity of the kidney. AQP distribution in various forms of human renal disease has not been documented. Immunohistochemical studies of biopsy samples from a wide range of renal diseases revealed a substantial and striking upregulation of AQP-1 in the glomeruli of most diseased kidneys. AQP-1 expression remained prominent in proximal tubules in all lesions. In contrast, there was judged qualitatively to be a reduction in the amounts of AQP-2 and AQP-3 expression, especially in lesions with substantial interstitial fibrosis and nephron loss, as compared with a healthy region of normal kidneys. The results were quantitatively confirmed by real-time reverse transcriptase-PCR. This is the first documentation of altered AQP expression in human renal disease. The significance of the increased AQP-1 expression requires further studies.

Freezing tolerance of the New Zealand alpine weta, Hemideina maori Hutton [Orthoptera; Stenopelmatidae]

Abstract 1. 1.|The New Zealand weta, Hemideina maori, is shown to withstand freezing, below its supercooling point, for at least 5 h at −9°C and for at least 8 h at −8°C. 2. 2.|The lower lethal temperature is about −10°C. 3. 3.|Insects collected in both summer and winter are freezing tolerant. 4. 4.|Haemolymph of “winter” insects has a much higher osmotic pressure (about 700 mOsm/kg) than “summer” forms, (about 450 mOsm/kg). 5. 5.|A large contribution to this high osmolality is made by unidentified compounds, which are non-ionic, and are not carbohydrate.

Amiloride restores renal medullary osmolytes in lithium-induced nephrogenic diabetes insipidus

In lithium-induced nephrogenic diabetes insipidus (NDI), alterations in renal medullary osmolyte concentrations have been assumed but never investigated. Amiloride can modify lithium-induced NDI, but the impact of amiloride in lithium-induced NDI on renal medullary osmolytes, aquaporins, and urea transporters is unknown and is the basis of this study. Rats fed lithium (60 mmol/kg dry food) over 4 wk developed NDI. Urine osmolality fell to 287 +/- 19 mosmol/kgH(2)O (controls 1,211 +/- 90 mosmol/kgH(2)O). Organic osmolytes in the renal medulla showed significant decreases compared with controls [inositol 221 +/- 35 to 85 +/- 10 mmol/kg protein; sorbitol 35 +/- 9 to 3 +/- 1 mmol/kg protein; glycerophosphorylcholine (GPC) 352 +/- 80 to 91 +/- 20 mmol/kg protein; and glycine betaine 69 +/- 11 to 38 +/- 38 mmol/kg protein]. Medullary urea content fell from 2,868 +/- 624 to 480 +/- 117 mmol/kg protein. Concurrent administration of amiloride (0.2 mmol/l) in the drinking water restored urine osmolality (1,132 +/- 154 mosmol/kgH(2)O), and reduced urine volume. Medullary osmolyte content were restored to control values (inositol, 232 +/- 12; sorbitol 32 +/- 6; GPC, 244 +/- 26; glycine betaine, 84 +/- 5 mmol/kg protein). Medullary urea rose to 2,122 +/- 305 mmol/kg protein. Reduced AQP2, AQP3, and urea transporter (UT-A1) expression was significantly reversed following amiloride therapy. Data presented here provide further understanding of how amiloride may substantially restore the lithium-induced impaired renal concentrating mechanism.

Hypotaurine, N‐Methyltaurine, Taurine, and Glycine Betaine as Dominant Osmolytes of Vestimentiferan Tubeworms from Hydrothermal Vents and Cold Seeps

Organic osmolytes, solutes that regulate cell volume, occur at high levels in marine invertebrates. These are mostly free amino acids such as taurine, which are “compatible” with cell macromolecules, and methylamines such as trimethylamine oxide, which may have a nonosmotic role as a protein stabilizer, and which is higher in many deep‐sea animals. To better understand nonosmotic roles of osmolytes, we used high‐performance liquid chromatography and 1H‐nuclear magnetic resonance (NMR) to analyze vestimentiferans (vestimentum tissue) from unusual marine habitats. Species from deep hydrothermal vents were Riftia pachyptila of the East Pacific Rise (2,636 m) and Ridgeia piscesae of the Juan de Fuca Ridge (2,200 m). Species from cold hydrocarbon seeps were Lamellibrachia sp. and an unnamed escarpid species from subtidal sediment seeps (540 m) off Louisiana and Lamellibrachia barhami from bathyal tectonic seeps (1,800–2,000 m) off Oregon. Riftia were dominated by hypotaurine (152 mmol/kg wet wt), an antioxidant, and an unidentified solute with an NMR spectrum consistent with a methylamine. Ridgeia were dominated by betaine (N‐trimethylglycine; 109 mmol/kg), hypotaurine (64 mmol/kg), and taurine (61 mmol/kg). The escarpids were dominated by taurine (138 mmol/kg) and hypotaurine (69 mmol/kg). Both Lamellibrachia populations were dominated by N‐methyltaurine (209–252 mmol/kg), not previously reported as a major osmolyte, which may be involved in methane and sulfate metabolism. Trunk and plume tissue of the Oregon Lamellibrachia were nearly identical to vestimentum in osmolyte composition. The methylamines may also stabilize proteins against pressure; they were significantly higher in the three deeper‐dwelling groups.

The carbohydrate levels of insect haemolymph

Abstract 1. 1. Several species of insects and terrestrial arthropods were analysed for levels of carbohydrates in their haemolymph. 2. 2. Trehalose in all terrestrial arthropods and primitive insects analysed was either absent or present in very small amounts. 3. 3. Glucose showed a similar pattern to trehalose, but in the more primitive animals was generally present in moderate amounts. 4. 4. In the higher insects, high levels of trehalose and moderate amounts of glucose were detected in the haemolymph.

The composition of the haemolymph and muscle tissue of the shore crab, Hemigrapsus edwardsi, exposed to different salinities

Abstract 1. 1. Analysis showed that this crab was euryhaline and only imperfectly capable of regulation of the concentration of sodium and chloride in the haemolymph. 2. 2. Three different assumptions were used to derive intracellular concentrations of ions of muscle tissue. All calculations indicated the same general trends. 3. 3. Although the concentration of sodium chloride in the haemolymph fell by >40% over the survival range, muscle fibre water rose by only 25%, and the amounts of intracellular ions showed no change over the same range. 4. 4. Free amino acid concentration in the muscle tissue fell more than would be expected on adaptation to lower salinities. 5. 5. It was concluded that adaptation of Hemigrapsus in low salinities involves reduction of the concentration of free amino acids in the muscle tissues.

Betaine is the principal counteracting osmolyte in tissues of the elephant fish, Callorhincus millii (Elasmobranchii, Holocephali)

Abstract Analysis of the organic constituents of tissues of the elephant fish, Callorhincus millii , using both NMR and HPLC, revealed that trimethylamine oxide (TMAO) was present in only low concentrations in all tissues. The principal counteracting osmolyte in the elephant fish was the methylamine glycine betaine, which occurred at a concentration of 50–70 mmol kg −1 fresh weight in those tissues analysed. The compatible osmolyte taurine was found to be in high concentration (39 mmol kg −1 fresh weight) in heart muscle, and significant amounts of serine were found in both heart and brain.

The composition of the fluid compartments of two chondrichthyans, Callorhyncus millii and Squalus acanthias

1. 1. The major cations and anions were measured in the plasma, red blood cells and muscle tissue of the elasmobranch, Squalus acanthias, and the holocephalan, Callorhyncus millii. 2. 2. These were found to be similar to those reported from other marine elasmobranchs. 3. 3. The organic fraction of these tissues was also determined in these two fish: urea, trimethylamine oxide and free amino acids. 4. 4. The most interesting feature of this was the large amounts of the amino acids, taurine (up to 90 mmol), glycine (up to 90 mmol) and proline (up to 25 mmol) found in the muscle tissue and more especially the red blood cells. The concentrations of the free amino acids in the plasma of both fish were low.