A brief harvesting-freezing delay significantly alters the kidney metabolome and leads to false positive and negative results

Abnormalities in distinct metabolic pathways have been associated with the pathogenesis and progression of many forms of kidney disease. Metabolomics analyses can be used to determine organ-specific metabolic fingerprints and, ideally, should represent the metabolic state of the organ at the exact moment the sample is harvested. However, conventional harvesting methods depend on posteuthanasia tissue harvest, which results in ischemia conditions and metabolome changes that could potentially introduce artifacts into the final studies. We recently optimized a modified clamp-freezing technique for rodent kidney harvesting and freezing, significantly reducing ischemia and freezing times and granting a closer snapshot of in vivo metabolism. In this study, we characterized and compared the metabolome of kidneys harvested using our modified approach versus traditional techniques to determine which metabolites are preferentially affected by a brief lapse of ischemia and freezing delay and which are more stable. We used Sprague-Dawley rats as a model of wild-type (WT) kidneys and PCK [polycystic kidney disease (PKD)] rats as a model of chronic kidney disease kidneys. Finally, we compared the metabolic profile of clamp-frozen and delayed WT and PKD kidneys to determine which metabolic changes are most likely observed in vivo in PKD and which could be subjected to false positive or negative results. Our data indicate that a short harvesting-freezing delay is sufficient to impart profound metabolic changes in WT and PKD kidneys, leading to false positive and negative differences when comparing these genotypes. In addition, we identified a group of metabolites that were more stable. Interestingly, while the delay had a similar effect between WT and PKD, there were notable differences. The data obtained indicate that the quick clamp-freezing technique for kidney metabolomics provides a more accurate interpretation of the in vivo metabolic changes associated with the disease state. <b>NEW & NOTEWORTHY</b> Our study shows that a brief harvesting-freezing delay associated with organ collection and freezing can significantly alter the kidney metabolic profile of both male and female wild-type and a genetic model of chronic kidney disease. Importantly, given that the effect of this delay differs among genotypes, it is not safe to assume that equally delaying harvesting-freezing in wild-type and polycystic kidney disease kidneys adequately controls this effect, ultimately leading to false positive and negative results among different renal diseases.