John P. Leader

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.

Effects of anions on cellular volume and transepithelial Na+ transport across toad urinary bladder

SummaryThe effects of complete substitution of gluconate for mucosal and/or serosal medium Cl− on transepithelial Na+ transport have been studied using toad urinary bladder. With mucosal gluconate, transepithelial potential difference (VT) decreased rapidly, transepithelial resistance (RT) increased, and calculated short-circuit current (Isc) decreased. CalculatedENa was unaffected, indicating that the inhibition of Na+ transport was a consequence of a decreased apical membrane Na+ conductance. This conclusion was supported by the finding that a higher amiloride concentration was required to inhibit the residual transport. With serosal gluconateVT decreased,RT increased andIsc fell to a new steady-state value following an initial and variable transient increase in transport. Epithelial cells were shrunken markedly as judged histologically. CalculatedENa fell substantially (from 130 to 68 mV on average). Ba2+ (3mm) reduced calculatedENa in Cl− Ringers but not in gluconate Ringers. With replacement of serosal Cl− by acetate, transepithelial transport was stimulated, the decrease in cellular volume was prevented andENa did not fall. Replacement of serosal isosmotic Cl− medium by a hypo-osmotic gluconate medium (one-half normal) also prevented cell shrinkage and did not result in inhibition of Na+ transport. Thus the inhibition of Na+ transport can be correlated with changes in cell volume rather than with the change in Cl− per se. Nystatin virtually abolished the resistance of the apical plasma membrane as judged by measurement of tissue capacitance. With K+ gluconate mucosa, Na+ gluconate serosa, calculated basolateral membrane resistance was much greater, estimated basolateral emf was much lower, and the Na+/K+ basolateral permeability ratio was much higher than with acetate media. It is concluded the decrease in cellular volume associated with substitution of serosal gluconate for Cl− results in a loss of highly specific Ba2+-sensitive K+ conductance channels from the basolateral plasma membrane. It is possible that the number of Na+ pump sites in this membrane is also decreased.

Stabilization of supercooled fluids by thermal hysteresis proteins.

It has been reported that thermal hysteresis proteins found in many cold-hardy, freeze-avoiding arthropods stabilize their supercooled body fluids. We give evidence that fish antifreeze proteins, which also produce thermal hysteresis, bind to and reduce the efficiency of heterogenous nucleation sites, rather than binding to embryonic ice nuclei. We discuss both possible mechanisms for stabilization of supercooled body fluids and also describe a new method for measuring and defining the supercooling point of small volumes of liquid.

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.

Natural and Nanoengineered Chiral Reflectors: Structural Color of Manuka Beetles and Titania Coatings

A parallel study of natural and nanoengineered structurally chiral reflecting coatings is described. It is shown that the nanostructures are different in a minor way but are optically equivalent. Refractive index matching of nanoengineered chiral coatings on a plane substrate is shown to improve the saturation of structural color. Optical and electron microscopies reveal complexity in the multilayered chiral coatings that produce green metallic-like reflections from manuka (scarab) beetles. In particular, the reflectors are shown to have the form of small concave pits and troughs that are filled with contouring chiral material. Each chiral microreflector presents a range of pitch and tilt to an incident beam of light. Physical properties of the textured coatings are related to optical properties such as spectral reflectance, angle of spread, and perceived color, which has a high degree of saturation due to the filling of the pits. Observations of overlapping chiral mediums in beetle reflectors have inspired nanoengineering of related handed media such as Bragg reflectors for elliptically polarized light.

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.

Transepithelial transport of fluorescent p-glycoprotein and MRP2 substrates by insect Malpighian tubules: confocal microscopic analysis of secreted fluid droplets.

SUMMARY Transport of fluorescent substrates of p-glycoprotein (P-gp) and multidrug resistance-associated protein 2 (MRP2) by insect Malpighian tubules was examined using confocal laser scanning microscopy (CLSM). Isolated tubules of the cricket Teleogryllus commodus accumulated the MRP2 substrate Texas Red in the cells and lumen at concentrations up to 20 and 40 times, respectively, those in the bathing medium. Quantitative CLSM analysis of fluorochrome transport in some cricket tubules and all Drosophila tubules was not practical because of interfering effects of concretions in the cells and lumen. Samples of fluid secreted by tubules set up in Ramsay assays were therefore collected in hollow rectangle glass capillaries. Transepithelial dye flux was calculated as the product of fluid secretion rate (measured in the Ramsay assay) and dye concentration (measured by CLSM of the fluid samples). Dose–response curves for transport and the ratio of dye concentration in the secreted fluid to that in the bathing medium (S/M) were determined for Texas Red as well as for P-gp substrates (rhodamine 123, daunorubicin), the organic anion fluorescein and the organic cation quinacrine. Transepithelial transport of Texas Red was reduced by the MRP2 inhibitors MK571 and probenecid. Transport of daunorubicin was reduced by the P-gp inhibitors verapamil and quinacrine and also by the organic cation tetraethylammonium. The results indicate the presence of P-gp-like and MRP2-like transporters in the Malpighian tubules of both species.

Electrochemical characteristics of ion secretion in malpighian tubules of the New Zealand alpine weta (Hemideina maori).

Characteristics of ion and fluid secretion were investigated in isolated Malpighian tubules of the New Zealand Alpine Weta (Hemideina maori). Fluid secretion by tubules in iso-osmotic saline (500mOsm) occurred at a rate of 15+/-3nlh(-1) and was enriched in K(+) (approx. 125mmoll(-1)) relative to the saline (10mmoll(-1)). Maximal fluid secretion (112nlh(-1)) during simultaneous exposure to hypo-osmolality and dibutyryl cAMP resulted in an 8.8x increase in the quantity of K(+) secreted, compared to only a 2.4x increase in Na(+) secretion. Measurements of intracellular ion activities and membrane potentials indicated that Na(+) and K(+) were transported against a strong electrochemical gradient across the apical surface, regardless of saline osmolality. On the basolateral surface, there was a large driving force for Na(+) entry, while K(+) was distributed near its equilibrium potential. Neither bumetanide nor ouabain in the bathing saline had a significant effect on fluid secretion, but Ba(2+) and amiloride decreased fluid secretion by 79 and 57%, respectively. The effect of Ba(2+) on fluid secretion was consistent with a high basolateral permeability to K(+), relative to Na(+) and Cl(-). These results indicate that the characteristics of fluid secretion in this primitive insect are largely conserved with characteristics reported for other insects.

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.