Impact of organ preservation using HTK for graft flush and subsequent storage in UW in rat kidney transplantation.

BACKGROUND: In kidney transplantation, preservation has a significant influence on organ function. Since previous reports have indicated a benefit of combining histidine-tryptophan-ketoglutarate (HTK) and University of Wisconsin (UW) solution, we evaluated the effects of initial flush with low viscosity HTK, followed by storage in UW. MATERIAL AND METHODS: Kidneys from inbred Lewis rats were procured using HTK or UW for initially perfusion and re-flushed after 30 min with either solution. In a third group, after perfusion with HTK, organs were re-flushed with UW. Organs were stored for 16-24 h (4 degrees C). Study parameters were high-energy phosphates, histology, apoptosis, recipient survival and urine excretion of 15-F2t -isoprostanes (oxidative stress marker). RESULTS: Prior to transplantation, tissue ATP/ADP concentrations were: HTK/UW > UW-only > HTK-only. In transplanted kidneys, histological damage was highest after preservation in HTK-only. Twenty-four hours after transplantation (24 h cold ischemia time - CIT), cleaved-PARP was most abundant using UW-only. 16 h of CIT resulted in higher urine concentrations of isoprostanes in the order HTK-only (368 +/- 308) > UW-only (157 +/- 105) > HTK/UW (67 +/- 26), and was lower in HTK/UW after 24 h of CIT (146 +/- 38) vs. UW-only (507 +/- 33 pg/mg creatinine). Survival (24 h CIT) was significantly reduced, and percentage of initial non-functioning (INF) kidneys highest in HTK-only (2.6 +/- 0.3 days, 100%), compared to UW-only (13 +/- 4.4 days, 75%) and HTK/UW (18.5 +/- 4.6 days, 33%). CONCLUSIONS: In long-term preservation, UW is superior over HTK. However, our results indicate that perfusion with HTK prior to storage in UW may improve the results of UW alone which is reflected by better survival, lower rate of INF, higher cellular energy conservation and a decrease of free radicals.

Greater free plasma VEGF and lower soluble VEGF receptor-1 in acute mountain sickness.

Vascular endothelial growth factor (VEGF) is a hypoxia-induced protein that produces vascular permeability, and limited evidence suggests a possible role for VEGF in the pathophysiology of acute mountain sickness (AMS) and/or high-altitude cerebral edema (HACE). Previous studies demonstrated that plasma VEGF alone does not correlate with AMS; however, soluble VEGF receptor (sFlt-1), not accounted for in previous studies, can bind VEGF in the circulation, reducing VEGF activity. In the present study, we hypothesized that free VEGF is greater and sFlt-1 less in subjects with AMS compared with well individuals at high altitude. Subjects were exposed to 4,300 m for 19-20 h (baseline 1,600 m). The incidence of AMS was determined by using a modified Lake Louise symptom score and the Environmental Symptoms Questionnaire for cerebral effects. Plasma was collected at low altitude and after 24 h at high altitude, or at time of illness, and then analyzed by ELISA for VEGF and for soluble VEGF receptor, sFlt-1. AMS subjects had lower sFlt-1 at both low and high altitude compared with well subjects and a significant rise in free plasma VEGF on ascent to altitude compared with well subjects. We conclude that increased free plasma VEGF on ascent to altitude is associated with AMS and may play a role in pathophysiology of AMS.

Domains of Pit-1 required for transcriptional synergy with GATA-2 on the TSH beta gene.

Previous studies showed that Pit-1 functionally cooperates with GATA-2 to stimulate transcription of the TSH beta gene. Pit-1 and GATA-2 are uniquely coexpressed in pituitary thyrotropes and activate transcription by binding to a composite promoter element. To define the domains of Pit-1 important for functional cooperativity with GATA-2, we cotransfected a set of Pit-1 deletions with an mTSH beta-luciferase reporter. Plasmids were titrated to express equivalent amounts of protein. A mutant containing a deletion of the hinge region between the POU and homeodomains retained the ability to fully synergize with GATA-2. In contrast, mutants containing deletions of amino acids 2-80 or 72-125 demonstrated 56 or 34% of the synergy found with the full-length protein, suggesting that these regions contributed to cooperativity. Mutants with deletions of the POU-specific or homeodomain further reduced the effect signifying the requirement for DNA binding. GST interaction studies demonstrated that only the homeodomain of Pit-1 interacted with GATA-2. Finally, several mutations between the Pit-1 and GATA-2 sites on the TSH beta promoter reduced binding for each factor and greatly reduced ternary complex formation. Thus multiple domains of Pit-1 are required for full synergy with GATA-2 and sequences between the two binding sites contribute to co-occupancy with both factors on the proximal TSH beta promoter.