Tiziana Pacchiarotta1, Pieter van der Pol2, Hans de Fijter2, Cees van Kooten2, Oleg Mayboroda1
1LUMC Centre for Proteomics and Metabolomics 2Nephrology Department, LUMC
Ischemia/reperfusion injury (IRI) is an inevitable event after kidney transplantation, leading to extensive injury and poor graft survival. Current markers for the evaluation of IRI are considered insensitive and reliable markers are highly needed. Here we use a well-established rat model to document the protective role of MBL in renal IRI as target for therapeutic intervention in kidney transplantation and we show that the urinary metabolic pattern can be used to evaluate an in-tissue IRI event. Using a combination of statistical models, we link the metabolic trajectory to the recovery process under different antibody treatment. We demonstrate that “protective” urinary metabolic signature correlates with expression of GRP 78.
Kidney is by far the most transplanted organ worldwide (in the Netherlands in 2013 75% of transplanted organs were kidneys). Ischemia/reperfusion (IR) is an inevitable and often devastating event in organ transplantation. Ischemia is characterized by the reduction of oxygen and nutrient supply to the tissue leading to impaired energy metabolism at cellular level. Prolonged ischemia results in a malfunction or a failure of an affected organ. However, once the blood flow is restored the situation considerably aggravates because of the activation of a series oxidative stress and inflammation events by the immune system.
The mechanism and the exact sequence of the events that lead to mostly irreversible damage in ischemia reperfusion is poorly understood and the existing markers of the damage such as blood creatinine, blood urea nitrogen and creatinine clearance have long been considered unreliable . Another way to evaluate the kidney status is the estimated glomerular filtration, which suffers from interference with drugs. To this end, the only reliable source of information remains a renal biopsy, which is invasive, expensive and holds a risk of morbidity (bleeding complications). Post-transplant would benefit enormously from a proper way to assess the degree of IRI and an adequate insight into underlying mechanisms.
Here, we have used a rat model in which the renal pedicle was clamped for 45 minutes and groups of rats were sacrificed at early time points (2, 5 and 24 hrs post reperfusion). Along with classical reads-out, we present a metabolomics investigation of urinary profile applied to a well-established rat model. This may offer a solution to assess graft state and predict the outcome of transplantation because of its non-invasive character. In addition, this also offers the potential to elucidate the mechanism underlying ischemia/reperfusion
Histological analysis reveals that tubular alterations and loss of tight junctions already occurred at the cortico-medullary junction within 2 h upon reperfusion and further increased in time. Accordingly, this injury is accompanied by a significant enrichment of blood creatinine level within 24 h after reperfusion. Staining for 78 kDa glucose-regulated protein (GRP78) revealed that, tubular GRP78 protein expression is lost already 2 h after reperfusion. Altogether this points at an early induction of ER stress in this model of renal IR injury.
In addition, to assess the putative protective role of MBL in this process, we have therapeutically targeted MBL using the protective and blocking anti-MBL-A mAb (P7E4). Inhibition of MBL following IR significantly protected against renal dysfunction. Importantly, inhibition of MBL completely prevents the loss of tubular GRP78 early after reperfusion, whereas control-treated animals lose their GRP78 protein expression in the cortico-medullary tubular cells. This suggests that MBL might play a role in early tubular ER-stress following reperfusion.
We have addressed the question whether the effect of MBL inhibition on the occurrence of ischemia/reperfusion injury is reflected by changes of the urinary metabolome. To get better insight into the effect of time after the reperfusion and MBL inhibition, we have built a PLS-class regression models. Through these statistical models we have been able to link the urinary metabolic changes to the renal physiological status expressed as blood creatinine, blood urea, urine and blood neutrophil gelatinase-associated lipocalin (NGAL), interleukine-6 (IL-6) and 78 kDa glucose-regulated protein (GRP78) mRNA expression. Importantly, the protein staining for GRP78 is the only parameter reflecting the experimental condition and providing the possibility to investigate the different effect of the two antibodies (anti-MBL and control) on the metabolic space. We have closely analysed the discriminative feature of these models for the identification of molecular descriptors of the physiological status and we have compiled a list of metabolites which includes TCA cycle compounds as well as sugars and organic acids.
The signals of these compounds were integrated in order to provide semi-quantitative information to study the trend of the concentration level over time. These compounds all have shown an increase or decrease of multiple fold respect to the sham samples which obviously reflect the effect of the IRI damage. In addition, some of these molecules exhibited a different trend in rats treated with control or anti-MBL antibody (e.g. alpha-ketoglutaric acid).
Our results support a role of MBL in renal IRI. We have demonstrated that the inhibition of MBL in vivo protected tubular cells from loss of vital GRP78 and consequent tubular injury. This finding indicates that the therapeutic intervention at level of MBL is a promising target in kidney transplantation. In addition, we have shown that the urinary metabolic pattern can be used for the evaluation of ischemia/reperfusion injury event. Using a combination of statistical models, we could link the metabolic trajectory to the recovery process under different antibody treatment. Indeed, the protective effect of anti-rat MBL, expressed as GRP 78, has indicated a specific urinary metabolic signature.