Delphine Dutheil

Protective roles of polyethylene glycol and trimetazidine against cold ischemia and reperfusion injuries of pig kidney graft.

Ischemia‐reperfusion injury (IRI) represents an allo‐independent risk factor which favors chronic allograft nephropathy (CAN). Here we analyzed the influence of preservation solutions on the function of autotransplanted pig kidneys over 1–16 weeks after surgery. Kidneys were cold‐flushed and cold‐stored for 24 or 48 h either in University of Wisconsin (UW), modified‐UW Hôpital Edouard Herriot, polyethylene glycol 20 kDa (PEG)‐supplemented preservation solutions with low K+ (ECPEG) or high K+ (ICPEG) content. Animals autotransplanted with kidneys cold‐stored for 24 h in ECPEG exhibited the greatest levels of creatinine clearance (Ccr: 161 ± 12 mL/min, n = 10) and the lowest levels of proteinuria (0.5 ± 0.03 mg/mL) 16 weeks after surgery as compared with pigs autotransplanted with kidneys cold‐stored in the other solutions tested (Ccr ranging from 80 and 140 mL/min). Similar differences, but with lower Ccr levels, were achieved after a prolonged period of cold‐storage(48 h). ECPEG better preserved the kidneys from monocytes/macrophages and CD4+ T cells infiltrations, VCAM‐1 and MHC class II overexpressions and occurrence of renal interstitial fibrosis (2%) as compared with the other preservation solutions (5%–20%). Adding the anti‐ischemic drug trimetazidine (TMZ) to the preservation solutions, particularly ECPEG, further improved the quality of the week‐16 post‐transplanted kidneys (Ccr: 182 ± 12 mL/min, n = 10). These findings demonstrated that adding PEG to extracellular‐like (with low K+ content) preservation solutions in combination with TMZ significantly improved the long‐term outcome of kidney grafts in this model of autotransplanted pig kidney.

Protective Effect of PEG 35 000 Da on Renal Cells: Paradoxical Activation of JNK Signaling Pathway During Cold Storage

Polyethylene glycol (PEG), a high‐molecular weight colloid, is added to preservation solutions in order to decrease cold‐ and ischemia‐induced injuries of the grafted organ. We evaluated on LLC‐PK1, a porcine proximal tubular epithelial cell line (1) the efficiency of several commercial preservation solutions (University of Wisconsin, Euro‐Collins, Celsior, SCOT, IGL‐1), and (2) whether adding PEG (400–35 000 Da) in a simple extracellular‐type buffer modified cell integrity and mitogen‐activated protein kinase (MAPK) signaling pathways. SCOT was the most efficient commercial solution. Moreover, only PEG 35 000 Da totally preserved cell viability, induced a decrease on reactive oxygen species production and a decrease on p38‐MAPK activation. Furthermore PEG 35 000 Da stimulated c‐Jun N‐terminal kinase (JNK). However, the inhibition of JNK pathway, with the specific SP600125 inhibitor, in the presence of PEG 35 000 Da did not affect cell survival. We also confirmed on whole pig kidney the protective effect of PEG 35 000 Da on cold‐induced tubular injuries. This study confirms PEG antioxidative properties, but we demonstrate that its effect on JNK signaling pathway had also a paradoxical effect on cell death. This sheds a new light on PEG effects during cell preservation, independently from the classical immuno‐camouflaging hypothesis.

Supplementation With a New Therapeutic Oxygen Carrier Reduces Chronic Fibrosis and Organ Dysfunction in Kidney Static Preservation

Static preservation is currently the most widely used organ preservation strategy; however, decreased donor organ quality is impacting outcome negatively. M101 is an O2 carrier with high‐oxygen affinity and the capacity to function at low temperatures. We tested the benefits of M101 both in vitro, on cold preserved LLC‐PK1, as well as in vivo, in a large white pig kidney autotransplantation model. In vitro, M101 supplementation reduced cold storage‐induced cell death. In vivo, early follow‐up demonstrated superiority of M101‐supplemented solutions, lowering the peak of serum creatinine and increasing the speed of function recovery. On the longer term, supplementation with M101 reduced kidney inflammation levels and maintained structural integrity, particularly with University of Wisconsin (UW). At the end of the 3‐month follow‐up, M101 supplementation proved beneficial in terms of survival and function, as well as slowing the advance of interstitial fibrosis. We show that addition of M101 to classic organ preservation protocols with UW and Histidine–Tryptophane–Ketoglutarate, the two most widely used solutions worldwide in kidney preservation, provides significant benefits to grafts, both on early function recovery and outcome. Simple supplementation of the solution with M101 is easily translatable to the clinic and shows promises in terms of outcome.

Evidence for a mitochondrial impact of trimetazidine during cold ischemia and reperfusion.

In organ transplantation, ischemia-reperfusion injury (IRI) has been implicated in delayed graft function (DGF) as well as in short- and long-term complications. Using an autotransplant pig kidney model, changes in renal function and morphology were determined after different periods of cold ischemia in kidneys preserved in the University of Wisconsin solution (UW), high-Na+ version of UW (HEH) or Celsior (CEL) a newly developed high-Na+ solution, with or without trimetazidine (TMZ). Kidney function was better preserved in CEL, UW and particularly HEH in combination with TMZ, particularly after 48 and 72 h. Mitochondria integrity was improved in TMZ-preserved groups. This study indicates that TMZ is efficiently protective against IRI even after prolonged preservation and in different preservation solutions.

Advancement in recombinant protein production using a marine oxygen carrier to enhance oxygen transfer in a CHO-S cell line

Abstract Recombinant proteins, particularly proteins used as therapeutics, are widely expressed for bioprocessing manufacturing processes. Mammalian cell lines represent the major host cells for bioproduction, according to their capacities of post-translational modifications and folding of secreted proteins. Many parameters can affect cell productivity, especially the rate of oxygen transfer. Dissolved oxygen, in high or low proportions, is a crucial parameter which can affect cell viability and thus productivity. HEMARINA has developed a new technology, commercially proposed as HEMOXCell®, to improve cell culture at a large production scale. HEMOXCell® is a marine oxygen carrier having properties of high oxygen sensitivity, to be used as an oxygen additive during cell culture manufacturing. In this study, we investigated the effects of HEMOXCell® on the culture of the commonly used CHO-S cell line. Two main objectives were pursued: 1) cell growth rate and viability during a batch mode process, and 2) the determination of the effect of this oxygen carrier on recombinant protein production from a CHO-transfected cell line. Our results show an increase of CHO-S cellular growth at a rate of more than four-fold in culture with HEMOXCell®. Moreover, an extension of the growth exponential phase and high cell viability were observed. All of these benefits seem to contribute to the improvement of recombinant protein production. This work underlines several applications using this marine-type oxygen carrier for large biomanufacturing. It is a promising cell culture additive according to the increasing demand for therapeutic products such as monoclonal antibodies.