MO060: HIF1Α and Succinate Pathway: Role in Deceased Kidney Donors Inflammation

Abstract BACKGROUND AND AIMS The type of donor has an important impact on the renal prognosis of the recipient. Thus, it has been observed that the decline of renal function is higher in deceased donor (DD) than those observed in living donors (LD). These could be related with the pre-implantation inflammation state of the donor and the development of fibrotic processes. Own data indicate that the enzymatic complex succinate dehydrogenase (SDH) has a lower gene expression in DD grafts and is associated with inflammatory markers, macrophages and purinergic receptors and a worse renal function after transplantation. The aim of this study is to determine that in renal DD donation, ischemia/hypoxia can increase renal succinate levels, inducing infiltration of inflammatory cells. METHOD Preimplantation kidneys biopsies from 47 DD and 19 LD were collected in our institution. About 159 genes from these samples were analyzed by qRT–PCR with the TaqMan gene expression assay. Relative quantification of gene expression was performed with three internal controls. Genes with high positive correlation with HIF1α (Rho > 0.5) have been grouped in clusters using STRING database (http://string-db.org/). Succinate levels were measured in serum from 27 renal DD and in 10 healthy volunteers with the EnzyChromTM Succinate Assay Kit. All statistical data were analyzed using GraphPad Prism 5. HIF, SDH subunits and succinate levels in serum were analyzed with a Student's T-test (P-value of 0.001). Pearson correlation Test (Rho >0.5) was performed to analyze interdependence of HIF1α with the rest of genes. RESULTS To determine the hypoxia status of kidney donors, transcription levels of HIF1α and HIF2α were determined from renal tissue samples. In the graphic (Figure. 1A), a significant increase in HIF1α genic expression is observed in DD compared with LD (P < 0.001). The next figure (Figure. 2B) shows clusters in DD that are not present in LD and have a high correlation with HIF1a (Rho > 0.5). Twenty-four genes showed this strong correlation and can be grouped in three different clusters: Cluster 1: Transcription factors and metalloproteinases, Cluster 2: Extracellular Matrix (ECM) proteins and integrins and finally, Cluster 3: Inflammatory and Macrophages Markers. As is shown Figure 2A, we found reduced mRNA expression of all the SDH subunits (SDHA, SDHB, SDHC and SDHD) (P < 0.001) in DD samples compared with LD ones. As a consequence, succinate cannot be converted to fumarate in concordance with the greater succinate levels (P = 0.002) observed in sera of DD shown in the picture (Figure. 2B). CONCLUSION These results indicate that kidneys from DD undergo a greater hypoxia compared with LD, demonstrated by the increased transcription rate of HIF1α. Hypoxia not only increases HIF1α but also may disrupt the Krebs cycle, reducing the expression of SDH subunits, increasing extracellular levels of succinate that has a role in the maintenance of HIF1α levels, which could take part in a sustained inflammatory/anti-inflammatory process in kidney from DD.