Kidney preservation at subzero temperatures using a novel storage solution and insect ice-binding proteins.

BACKGROUND: Contemporary kidney preservation methods involve storing at 4 degree C up to 24 h prior to transplantation. By decreasing the storage temperature to below 0 degree C, we hypothesized that the safe storage time could be significantly lengthened. OBJECTIVE: The efficacy of a proprietary CryoStasis (CrS) storage solution for the subzero preservation of kidneys was tested, with or without addition of a hyperactive insect antifreeze protein (TmAFP). MATERIALS AND METHODS: Rat kidneys were stored in either University of Wisconsin (UW) solution (4 degree C, 24 h), CrS (-2 degree C, 48 h), or CrS with 61.5 µM TmAFP (-4.4 degree C, 72 h). Following storage, viability was assessed with MTT reduction assays and live vs. dead cell (FDA/PI) staining. Markers of ischemic damage were analyzed using fluormetric substrates for caspase-3 and calpain activity. RESULTS: Kidneys stored in CrS for 48 h and CrS with TmAFP for 72 h displayed similar levels of enzymatic activity compared to 24 h UW controls. CONCLUSION: This methodology shows promise to prolong the safe storage time of kidneys and offers the potential of increased organ availability for renal transplants.

Metabolic opportunists: feeding and temperature influence the rate and pattern of respiration in the high arctic woollybear caterpillar gynaephora groenlandica (Lymantriidae)

Arctic woollybear caterpillars, Gynaephora groenlandica, had the capacity to rapidly and dramatically increase respiration rates up to fourfold within 12-24 h of feeding and exhibited similar decreases in respiration of 60-85 % in as little as 12 h of starvation. At the peak of their feeding season, the respiration rates of caterpillars also increased significantly with temperature from 0.5 to 22 degreesC for both fed and starved caterpillars (Q10=1-5). Indicative of diapause, late season caterpillars had depressed respiration rates which were less sensitive to temperature changes (Q10 approximately 1.5), while respiration rates for caterpillars that had spun hibernacula were even lower. G. groenlandica did not appear to demonstrate metabolic cold adaptation compared with other temperate lepidopteran larvae. The seasonal capacity to adjust metabolic rate rapidly in response to food consumption and temperature (which can be elevated by basking) may promote the efficient acquisition of energy during the brief (1 month) summer growing and feeding season, while conserving energy by entering diapause when conditions are less favorable. These adaptations, along with their long 15-20 year life cycle and the retention of freeze tolerance year-round, promote the survival of G. groenlandica in this harsh polar environment.

Glycerol metabolism in a freeze-tolerant arctic insect: an in vivo 13C NMR study.

Freeze-tolerance in larvae of Gynaephora groenlandica is enhanced by the accumulation of glycerol in the winter. Since summer larvae remain freeze-tolerant despite the lack of glycerol, we investigated glycerol metabolism as a function of acclimation and body temperature using noninvasive 13C NMR spectroscopy. Major constituents of hemolymph isolated from cold- and warm-acclimated larvae were identified with the aid of standard NMR spectra and confirmed by TLC and GLC. Spectra obtained on live, warm-acclimated larvae showed the presence of lipids, glycogen, glucose, trehalose and amino acids. Similar spectra of cold-acclimated or previously frozen larvae showed the additional presence of glycerol. In vitro time-lapse 13C spectra of D-[1-13C]glucose added separately to hemolymph or extracted fat body tissue showed that glycerol is synthesized from glucose in the fat body tissue and distributed to the peripheral tissue via hemolymph. In vivo time-lapse 13C spectra of cold- and warm-acclimated larvae were obtained after injection with D-[1-13C]glucose to monitor the production of labeled metabolic intermediates and end-products. [13C]Glycerol was produced between -30 degrees C and 30 degrees C but accumulated only below 5 degrees C. Above 5 degrees C glycerol was degraded and the 13C label incorporated mainly into glycogen. The mechanism underlying temperature control of glycerol biosynthesis and degradation may provide a clue to the role of glycerol in enhancing freeze-tolerance in these insects.

Cold-induced mitochondrial degradation and cryoprotectant synthesis in freeze-tolerant arctic caterpillars.

The larvae of Gynaephora groenlandica, a long-lived moth endemic to the high arctic, are perennially freeze-tolerant and able to increase their freeze-tolerance by synthesizing glycerol. Cold-induced mitochondrial changes were correlated (using electron microscopy, DNA staining, cytochrome c assay, and oxygen uptake) with glycerol production (using NMR spectroscopy) in larvae under different acclimations and in the field. Hypometabolism in summer- or warm-acclimated larvae led to glycerol accumulation. Extended exposure to near-zero or freezing temperatures caused mitochondrial degradation and glycerol accumulation. Rapid freezing of warm-acclimated larvae did not result in mitochondrial breakdown. Mitochondrial reconstitution upon warm-acclimation occurred much more rapidly (less than 1 week) than did degradation (greater than 2 months). Concomitant with mitochondrial breakdown was reduced oxidative metabolism, but the cytochrome c concentration remained independent of acclimation temperature. The adaptive response to cold by mitochondrial degradation and glycerol accumulation by G. groenlandica may be linked to diapause in other species of ectotherms.