Temporal and sex‑dependent gene expression patterns in a renal ischemia–reperfusion injury and recovery pig model

Men are more prone to acute kidney injury (AKI) and chronic kidney disease (CKD), progressingto end-stage renal disease (ESRD) than women. Severity and capacity to regenerate after AKI are important determinants of CKD progression, and of patient morbidity and mortality in the hospital setting. To determine sex diferences during injury and recovery we have generated a female and male renal ischemia/reperfusion injury (IRI) pig model, which represents a major cause of AKI. This study was conducted using farm pigs, hybrids between Large White and Landrace. Five females and five males of four months old, free of specifc pathogens, between 30 and 40 kg of weight were included in this study. This age range was chosen due to the sexual maturity of the animal, allowing hormone efects. Using microarray assays, global transcriptomic analyses of kidney biopsies from this IRI pig model was conducted and revealed a sexual dimorphism in the temporal regulation of genes and pathways relevant for kidney injury and repair. Overall, this study constitutes an extensive characterization of the time and sex diferences occurring during renal IRI and recovery at gene expression level. On the frst day of surgery, expert urology surgeons from the team practiced a laparoscopic lef nephrectomy followed by 30 min of warm ischemia in the right kidney. The lef kidney was defned as control tissue at basal state (PR), clear form injury. By performing a lef nephrectomy, the compensation of the other kidney to maintain renal function during ischemia–reperfusion injury was prevented. Upon ischemia, the lower pole of the kidney was removed (approximately 3 cm of kidney parenchyma) and afer suturing, and 5 more minutes of arterial reperfusion, an upper polar biopsy was obtained (approximately 1 cm of renal parenchyma). Animals were housed until day 7-post surgery, when a laparoscopic nephrectomy of the right kidney was performed. Pigs were then euthanized at that point. Overall, three kidney biopsies were collected for each animal: prior to injury (PR), 5 min following 30 min of ischemia (PS) and one week afer ischemia (WL). RNA was extracted from the PR, PS and WL kidney biopsies from each animal. The extractions were performed starting from 50 mg of each biopsy performed with the NZyol Kit following manufacturer instructions (Nzytech genes & enzymes). Microarray hybridization was carried out at High Technology Unit (UAT) at the Vall d'Hebron Research Institute . RNA integrity was assessed by Agilent 2100 Bioanalyzer (Agilent, Palo Alto, Ca). Only samples with similar RNA integrity number were accepted for microarray analysis. Gene Titan Afymetrix microarray platform and the Genechip Porcine Gene 2.1 ST 16-Array plate were used for this experiment.