Shifting Paradigms for Suppressing Fibrosis in Kidney Transplants: Supplementing Perfusion Solutions With Anti-fibrotic Drugs

Great efforts have been made toward addressing the demand for donor kidneys. One of the most promising approaches is to use kidneys from donation after circulatory death donors. These kidneys, however, suffer from more severe ischemia and reperfusion injury than those obtained via donation after brain death and are thus more prone to develop interstitial fibrosis and tubular atrophy. Even though machine perfusion is increasingly used to reduce ischemia and reperfusion injury, there are no effective treatments available to ameliorate interstitial fibrosis and tubular atrophy, forcing patients to resume dialysis, undergo re-transplantation, or suffer from premature death. Safe and effective anti-fibrotic therapies are therefore greatly desired. We propose a new therapeutic approach in which machine perfusion solutions are supplemented with anti-fibrotic compounds. This allows the use of higher concentrations than those used in humans whilst eliminating side effects in other organs. To the authors' knowledge, no one has reviewed whether such an approach could reduce interstitial fibrosis and tubular atrophy; we therefore set out to explore its merit. In this review, we first provide background information on ischemia and reperfusion injury as well as interstitial fibrosis and tubular atrophy, after which we describe currently available approaches for preserving donor kidneys. We then present an evaluation of selected compounds. To identify promising compounds, we analyzed publications describing the effects of anti-fibrotic molecules in precision-cut kidneys slices, which are viable explants that can be cultured ex vivo for up to a few days whilst retaining functional and structural features. LY2109761, galunisertib, imatinib, nintedanib, and butaprost were shown to exert anti-fibrotic effects in slices within a relatively short timeframe (<48 h) and are therefore considered to be excellent candidates for follow-up ex vivo machine perfusion studies.

Quercetin Liposomal Nanoformulation for Ischemia and Reperfusion Injury Treatment

Ischemia and reperfusion injury (IRI) is a common complication caused by inflammation and oxidative stress resulting from liver surgery. Current therapeutic strategies do not present the desirable efficacy, and severe side effects can occur. To overcome these drawbacks, new therapeutic alternatives are necessary. Drug delivery nanosystems have been explored due to their capacity to improve the therapeutic index of conventional drugs. Within nanocarriers, liposomes are one of the most successful, with several formulations currently in the market. As improved therapeutic outcomes have been demonstrated by using liposomes as drug carriers, this nanosystem was used to deliver quercetin, a flavonoid with anti-inflammatory and antioxidant properties, in hepatic IRI treatment. In the present work, a stable quercetin liposomal formulation was developed and characterized. Additionally, an in vitro model of ischemia and reperfusion was developed with a hypoxia chamber, where the anti-inflammatory potential of liposomal quercetin was evaluated, revealing the downregulation of pro-inflammatory markers. The anti-inflammatory effect of quercetin liposomes was also assessed in vivo in a rat model of hepatic IRI, in which a decrease in inflammation markers and enhanced recovery were observed. These results demonstrate that quercetin liposomes may provide a significant tool for addressing the current bottlenecks in hepatic IRI treatment.

Translational Application of Fluorescent Molecular Probes for the Detection of Reactive Oxygen and Nitrogen Species Associated with Intestinal Reperfusion Injury

Acute mesenteric ischemia, caused by an abrupt interruption of blood flow in the mesenteric vessels, is associated with high mortality. When treated with surgical interventions or drugs to re-open the vascular lumen, the reperfusion process itself can inflict damage to the intestinal wall. Ischemia and reperfusion injury comprise complex mechanisms involving disarrangement of the splanchnic microcirculatory flow and impairment of the mitochondrial respiratory chain due to initial hypoxemia and subsequent oxidative stress during the reperfusion phase. This pathophysiologic process results in the production of large amounts of reactive oxygen (ROS) and nitrogen (RNS) species, which damage deoxyribonucleic acid, protein, lipids, and carbohydrates by autophagy, mitoptosis, necrosis, necroptosis, and apoptosis. Fluorescence-based systems using molecular probes have emerged as highly effective tools to monitor the concentrations and locations of these often short-lived ROS and RNS. The timely and accurate detection of both ROS and RNS by such an approach would help to identify early injury events associated with ischemia and reperfusion and increase overall clinical diagnostic sensitivity. This abstract describes the pathophysiology of intestinal ischemia and reperfusion and the early biological laboratory diagnosis using fluorescent molecular probes anticipating clinical decisions in the face of an extremely morbid disease.

Abstract 14580: Methodological Challenges Conducting In Vitro Ischemia and Reperfusion: A Fundamental Problem in Cell Viability Determination

Background: Oxygen & Glucose Deprivation (OGD) and Reperfusion (OGD-R) is considered one of the best models for studying ischemia and reperfusion injury (IRI) in vitro. Cell viability (CV) experimentation can aid in estimating cell survival after IRI insults and potential benefits from therapeutic attempts. However, the different metabolites used in various CV assays may compromise the usefulness and validity of various CV tools. We conducted two independent cell viability assays (WST-8 & ATP) after OGD and OGD-R on neurons (NE) and astrocytes (AS). Methods: NE (HT-22) and AS (C8-D1A) were subjected to 6h OGD only and 6h OGD followed by 20h Reperfusion (OGD-R). OGD is based on the combination of chemical ischemia (standard culture media depleted of glucose and other energy sources) and severe hypoxia (O2 ~1.4 to 1.8%) for 6h, while 20h reperfusion consists of readdition of standard culture media and conditions (21% O2, 5% CO2, 37°C). CV was determined by WST-8 and ATP “CellTiter-Glo 2.0” assays. Results: By WST-8 assay, OGD decreased CV in NE (Fig. 1A, P value = 0.0121), but increased CV in AS (Fig. 1A, P value = 0.0079). OGD-R decreased CV in NE and AS (Fig. 2A, P value = 0.0010, 0.0146, respectively). By ATP assay, OGD decreased CV in NE (Fig. 1B, P value <0.0001) and AS (Fig. 1C, P value <0.0001). OGD-R also decreased CV in NE (Fig. 2B, P value <0.0001) and AS (Fig. 2C, P value = 0.0001). Conclusions: The current data reveals opposite CV results after OGD in AS when comparing WST-8 and ATP assays; OGD caused CV decrease in NE but increased in AS, which constitutes a paradoxical response based on previous literature. However, OGD-R caused similar injury patterns in NE and AS from WST-8 and ATP assays. It is unknown why OGD increased CV in AS through WST-8 after OGD, however, it can potentially occur due to AS hypermetabolism leading to increased NADPH+ production, which is the main substrate in this CV estimation. We conclude that CV should be carefully assessed with multiple, independent CV assays.

Identification of ferroptosis-associated genes exhibiting altered expression in response to cardiopulmonary bypass during corrective surgery for pediatric tetralogy of fallot

Background Tetralogy of Fallot (ToF) is a life-threatening congenital cardiovascular disorder. Currently, the most effective therapeutic intervention for pediatric ToF remains corrective surgery with cardiopulmonary bypass (CPB). Ferroptosis is an iron-dependent form of regulated cell death, driven by an accumulation of lipid peroxides to levels sufficient to trigger cell death. Ferroptosis was recently linked to cardiac ischemia and reperfusion injury. However, few studies have examined CPB-associated ferroptosis. Method In the current study, pediatric ToF patient pre- and post-CPB atrial biopsy gene expression profiles were downloaded from a public database, and 117 differentially expressed genes (DEGs) were identified using the Wilcoxon rank-sum test and weighted gene correlation network analysis. These were screened for ferroptosis-associated genes using the FerrDb database, thereby identifying ten genes. Finally, the construction of gene-microRNA (miRNA) and gene-transcription factor (TF) networks, in conjunction with gene ontology and biological pathway enrichment analysis, were used to inform hypotheses regarding the molecular mechanisms underlying CPB-associated ferroptosis. Results Ten genes involved in CPB-associated ferroptosis(ATF3,TNFAIP3，CDKN1A, ZFP36, JUN，SLC2A3, IL6, CXCL2, PTGS2, and DDIT3). Ferroptosis-associated genes were largely involved in myocardial inflammatory responses and may be regulated by a number of identified miRNAs and TFs, thereby suggesting modulatable pathways potentially involved in CPB-associated ferroptosis. Conclusions Results suggest that CPB precipitates ferroptosis within cardiac tissue during corrective Surgery for Pediatric Tetralogy of Fallot. These findings may ultimately help improve outcomes of corrective surgery for pediatric ToF.

Relevance and Recommendations for the Application of Cardioplegic Solutions in Cardiopulmonary Bypass Surgery in Pigs

Cardioplegic solutions play a major role in cardiac surgery due to the fact that they create a silent operating field and protect the myocardium against ischemia and reperfusion injury. For studies on cardioplegic solutions, it is important to compare their effects and to have a valid platform for preclinical testing of new cardioplegic solutions and their additives. Due to the strong anatomical and physiological cardiovascular similarities between pigs and humans, porcine models are suitable for investigating the effects of cardioplegic solutions. This review provides an overview of the results of the application of cardioplegic solutions in adult or pediatric pig models over the past 25 years. The advantages, disadvantages, limitations, and refinement strategies of these models are discussed.

Evaluation of Combined Effects of Vitamin E and Methylprednisolone in Ischemia and Reperfusion Injury of the Testicle Post Torsion and Detorsion in West African Dwarf Goats

Background: To investigate the protective effects of vitamin E and methylprednisolone on ischemia and reperfusion testicular injury after testicular torsion and detorsion in West African dwarf (WAD) goats. Methods: Thirty-two male WAD goats were randomly allocated into four groups (n=8); group A was treated with vitamin E, group B with methylprednisolone, group C with combo-therapy of vitamin E and methylprednisolone and group D was the control. The levels of malondialdehyde (MDA), superoxide dismutase (SOD), glutathione (GSH), total tissue protein (TTP) and catalase (CAT) were assayed while tissue biopsies were harvested for evaluation of degeneration of germinal cells, desquamation in germinal cells, interstitial edema and haemorrhage. Result: Findings revealed that Vitamin E administered immediately post ligature application significantly (p less than 0.05) decreased MDA level in groups A and C. Vitamin E monotherapy and its combination with methylprednisolone respectively ameliorated more the cellular damage and histological alterations caused by ischaemia and reperfusion injury. This study, therefor, suggests that vitamin E monotherapy and its combination with methylprednisolone is more beneficial in the management of short and long term ischemia/reperfusion injury respectively.