Genome-Wide Association Study of Serum Creatinine Levels during Vancomycin Therapy

Vancomycin, a commonly used antibiotic, can be nephrotoxic. Known risk factors such as age, creatinine clearance, vancomycin dose / dosing interval, and concurrent nephrotoxic medications fail to accurately predict nephrotoxicity. To identify potential genomic risk factors, we performed a genome-wide association study (GWAS) of serum creatinine levels while on vancomycin in 489 European American individuals and validated findings in three independent cohorts totaling 439 European American individuals. In primary analyses, the chromosome 6q22.31 locus was associated with increased serum creatinine levels while on vancomycin therapy (most significant variant rs2789047, risk allele A, β = -0.06, p = 1.1 x 10^-7). SNPs in this region had consistent directions of effect in the validation cohorts, with a meta-p of 1.1 x 10^-7. Variation in this region on chromosome 6, which includes the genes TBC1D32/C6orf170 and GJA1 (encoding connexin43), may modulate risk of vancomycin-induced kidney injury.     

Single inward rectifier potassium channels in guinea pig ventricular myocytes. Effects of quinidine.

The effects of quinidine on single inward rectifier K channels were investigated in cell-attached patches with 4.5 mM pipette potassium concentrations. Under these conditions, the single-channel slope conductance of the predominant conductance level of the inward rectifier channels was 3.9 +/- 0.3 pS at membrane potentials between -75 and -150 mV. Quinidine reversibly decreased the likelihood of channel opening to the main conductance level without reducing the single-channel conductance, and also reduced the probability of channel opening to subconducting levels. Quinidine had no significant effects on the channel open times, and the inhibition of channel opening was only slightly voltage dependent over the range of membrane potentials investigated. Quinidine induced a complete cessation of channel openings for brief periods (up to 2 min), suggesting that quinidine promoted occupancy of a state from which opening was less likely. Occasional long periods (up to an hour) with an absence of channel activity were also observed but quinidine did not appear to promote this behavior. The data suggest that quinidine decreases the ability of the channel to enter both main and subconducting states. By binding to a particular closed conformation of the channel, quinidine could reduce the likelihood of channel opening. The main features of these observations could be accounted for using the three-state kinetic model proposed by Sakmann, B. and G. Trube (1984b. J. Physiol. [Lond.]. 347:659-683.) with quinidine binding to the middle closed state.     

Cytoplasmic Ca2+ inhibits the ryanodine receptor from cardiac muscle

Ca²⁺-dependent inhibition of native and isolated ryanodine receptor (RyR) calcium release channels from sheep heart and rabbit skeletal muscle was investigated using the lipid bilayer technique. We found that cytoplasmic Ca²⁺ inhibited cardiac RyRs with an average K _m = 15 mm, skeletal RyRs with K _m = 0.7 mm and with Hill coefficients of 2 in both isoforms. This is consistent with measurements of Ca²⁺ release from the sarcoplasmic reticulum (SR) in skinned fibers and with [³H]-ryanodine binding to SR vesicles, but is contrary to previous bilayer studies which were unable to demonstrate Ca²⁺-inhibition in cardiac RyRs (Chu, Fill, Stefani &; Entman (1993) J. Membrane Biol. 135, 49–59). Ryanodine prevented Ca²⁺ from inhibiting either cardiac or skeletal RyRs. Ca²⁺-inhibition in cardiac RyRs appeared to be the most fragile characteristic of channel function, being irreversibly disrupted by 500 mm Cs⁺, but not by 500 mm K⁺, in the cis bath or by solublization with the detergent CHAPS. These treatments had no effect on channel regulation by AMP-PNP, caffeine, ryanodine, ruthenium red, or Ca²⁺-activation. Ca²⁺-inhibition in skeletal RyRs was retained in the presence of 500 mm Cs⁺. Our results provide an explanation for previous findings in which cardiac RyRs in bilayers with 250 mm Cs⁺ in the solutions fail to demonstrate Ca²⁺-inhibition, while Ca²⁺-inhibition of Ca²⁺ release is observed in vesicle studies where K⁺ is the major cation. A comparison of open and closed probability distributions from individual RyRs suggested that the same gating mechanism mediates Ca²⁺-inhibition in skeletal RyRs and cardiac RyRs, with different Ca²⁺ affinities for inhibition. We conclude that differences in the Ca²⁺-inhibition in cardiac and skeletal channels depends on their Ca²⁺ binding properties.     

Tritium labelling of amino sugars at C-2 by alkaline epimerization in tritiated water

N-Acetyl-D-[2-³H]glucosamine was synthesized from N-acetyl-D-mannosamineby alkaline 2-epimerization in pyridine containing ³H₂O andnickelous acetate. The reaction involves reversible formationof an enol intermediate and therefore also resulted in incorporationof tritium into N-acetylmannosamine. After completed reaction,the two N-acetylhexosamines were separated from other radioactiveproducts and Morgan-Elson chromogens by chromatography on acolumn of Sephadex G-10, which was eluted with 10% ethanol,and were then separated from each other by chromatography onSephadex G-15 in 0·27 M sodium borate (pH 7·8).The location of the incorporated tritium was established bytreatment of the N-acetylhexosamines with borate under the conditionsof the Morgan-Elson reaction, which converts the sugars to Kuhn'schromogen I with concomitant loss of the C-2 hydrogen. As expected,this treatment resulted in the formation of ³H₂O, indicatingthat the tritium was located at C-2. [2-³H]Glucosamine was preparedby acid hydrolysis of the labelled N-acetylglucosamine and wasconverted to [2-³H]glucosamine 6-phosphate by incubation withhexokinase and ATP. The sugar phosphate was used as a substratefor glucosamine 6-phosphate deaminase (isomerase, EC 5.3.1.10 [EC] )in a simple ³H₂O release assay. N-acetyl[2-³H]glucosamine N-acetyl[2-³H]mannosamine [2-³H]glucosamine glucosamine 6-phosphate deaminase [2-³H]mannosamine     

Metabolic response to anisoosmolarity of rat skeletal muscle in vitro.

Isolated rat hepatocytes exhibit an insulin-like anabolic response to hypoosmotic incubation and a glucagon-like catabolic response to hyperosmotic incubation. Recently, a distinct glycogenic response to hypoosmotic treatment was likewise reported for cultured rat myotubes. The present study examines the effects of anisoosmolar exposure on glucose metabolism in freshly isolated rat soleus muscle strips. Under the same experimental conditions as used for cultured myotubes, hypoosmolarity reduced net glycogen synthesis to 52%, while hyperosmolarity stimulated glycogen storage to 231% of isoosmolar control (nmol glucose incorporated into glycogen g(-1) x h(-1): hypoosmolar, 34+/-3; isoosmolar, 65+/-8; hyperosmolar, 150+/-11; p<0.01 each vs. isoosmolar). The responses of native skeletal muscle to anisoosmolarity are therefore in opposition to what has been described for hepatocytes and cultured myotubes. Further experiments on rat skeletal muscle revealed that the observed lack of a glycogenic response to hypoosmolarity persisted independent of medium composition, specifically with regard to prevailing glucose and K+ concentrations. In conclusion, hypoosmotic exposure inhibits glycogen synthesis in isolated rat soleus muscle, which clearly argues against the hypothesis that osmotic changes and cell swelling may be physiologically relevant stimulators of muscle glycogen synthesis.     

Perturbation of the molecular clockwork in the SCN of non-obese diabetic mice prior to diabetes onset

Circadian disruption is associated with the development of diabetes. Non-obese diabetic (NOD) mice show abnormal diurnal profiles in energy balance and locomotor activity suggesting circadian misalignment. Therefore, we analyzed cFos and mPER1 as markers for rhythmic neuronal activity within the suprachiasmatic nucleus (SCN) of wildtype (WT) and non-diabetic (nNOD) as well as acutely diabetic NOD (dNOD) mice. cFos levels show a day/night difference in both WT and nNOD but not in dNOD. mPER1 levels did not show a day/night difference in both nNOD and dNOD. This suggests that disruption of SCN rhythmicity in NOD mice precedes the actual onset of diabetes.     

Vanadium-induced Cl(-)-secretion in rabbit descending colon is mediated by prostaglandins.

Vanadium in the 4+ (vanadyl-ion) and 5+ (vanadate-ion) oxidation state stimulates furosemide-sensitive electrogenic Cl- secretion in isolated epithelia of rabbit descending colon. This effect is associated with an increased release of prostaglandin E2 from the tissue. Inhibitors of phospholipase A2 or cyclooxygenase abolish both vanadium-induced release of prostaglandin E2 and Cl- secretion. Neuronal mechanisms are not likely to be involved, as tetrodotoxin does not affect the vanadate induced Cl- secretion. Although vanadate is known to inhibit Na+,K(+)-ATPase activity, no inhibition of active Na+ transport was observed in intact colonic epithelia suggesting a rapid intracellular reduction of vanadate ions to vanadyl ions which have no inhibitory effect on the Na+,K(+)-ATPase. The present findings therefore indicate that vanadate stimulated colonic Cl- secretion involves intracellular conversion of vanadate to vanadyl and release of prostaglandin E2.