Proteomic analysis of rat livers in hepatic cold ischemia and reperfusion

CAMILA KNECHT; CECILIA BALABÁN; JOAQUÍN RODRÍGUEZ; EDGARDO GUIBERT; GERMÁN ROSANO

Rosario

Congreso; 8vo Congreso Latinoamericano de Organos Artificiales, Biomateriales e Ingeniería de Tejidos; 2014

Sociedad Latinoamericana de Biomateriales, Órganos Artificiales e Ingeniería de Tejidos

Ischemia and reperfusion (IR) injury constitutes a pivotal mechanism of tissue damage in pathological conditions such as stroke, myocardial infarction, vascular surgery, and organ transplant. In particular hepatic IR is a major cause of liver damage during transplant, leading to its functional deterioration and eventually to its rejection. Maintaining the viability of the organ during its ischemic transfer from donor to recipient is mainly based on hypothermia, which is applied to reduce hepatic metabolic activity. The present work deals with the setting of a rat model of liver donation to get samples for proteomic analysis. Livers were surgically removed and divided into four experimental groups (according to the stage of tissue sample collection): GI: Control - collection of sample immediately after surgical removal; GII: collection of sample after 90 min of ex vivo reperfusion using an isolated perfused rat liver model; GIII: collection of sample after 24 hs of preservation in HTK (histidine-tryptophan-ketoglutarate buffer) at 4ºC and GIV: collection of sample after 24 hs of preservation in HTK at 4ºC plus 90 min of ex vivo reperfusion. These groups were chosen as representative steps in a typicall liver transplantation process. To identify differentially expressed proteins in hepatic IR samples versus control, we examined pooled liver samples from each experimental group and subjected them to two-dimensional gel electrophoresis. The first dimension was run in a Multiphor II apparatus (GE) under denaturing conditions. The second dimension was run vertically in a Protean II system (BioRad). Proteins were detected by Coomassie staining. The gels were scanned and analyzed with the ImageMaster software (GE). Selected spots were subjected to MALDI-TOF for protein identification at the Analytical Biochemistry and Proteomics Unit of the Institute Pasteur in Montevideo, Uruguay. Several hepatic proteins whose expression levels were altered in livers subjected to the different treatments were identified. With respect to their relative abundance, 3 of the identified proteins remained unchanged after the entire procedure (spots: 280, 41 and 20), the levels of 4 of them increased after the treatments (spots 75, 237, 316 and 96) and 3 showed a down regulation in group IV with respect to I (Spots 52, 3 and 167). These data demonstrate the utility of proteomic applications in the search for novel sensitive and specific biomarkers of cellular response to IR for prospective clinical studies.