Animal experimentation is necessary to characterize human diseases and design adequate therapeutic interventions. In renal transplantation research, the limited number of in vitro models involves a crucial role for in vivo models and particularly for the porcine model. Pig and human kidneys are anatomically similar (characterized by multilobular structure in contrast to rodent and dog kidneys unilobular). The human proximity of porcine physiology and immune systems provides a basic knowledge of graft recovery and inflammatory physiopathology through in vivo studies. In addition, pig large body size allows surgical procedures similar to humans, repeated collections of peripheral blood or renal biopsies making pigs ideal for medical training and for the assessment of preclinical technologies. However, its size is also its main drawback implying expensive housing. Nevertheless, pig models are relevant alternatives to primate models, offering promising perspectives with developments of transgenic modulation and marginal donor models facilitating data extrapolation to human conditions.

We examined the effects of fetal exposure to a wide range of di-(2-ethylhexyl) phthalate (DEHP) doses on fetal, neonatal, and adult testosterone production. Pregnant rats were administered DEHP from Gestational Day (GD) 14 to the day of parturition (Postnatal Day 0). Exposure to between 234 and 1250 mg/kg/day of DEHP resulted in increases in the absolute volumes of Leydig cells per adult testis. Despite this, adult serum testosterone levels were reduced significantly compared to those of controls at all DEHP doses. Organ cultures of testes from GD20 rats exposed in utero to DEHP showed dose-dependent reductions in basal testosterone production. Surprisingly, however, no significant effect of DEHP was found on hCG-induced testosterone production by GD20 testes, suggesting that the inhibition of basal steroidogenesis resulted from the alteration of molecular events upstream of the steroidogenic enzymes. Reduced fetal and adult testosterone production in response to in utero DEHP exposure appeared to be unrelated to changes in testosterone metabolism. In view of the DEHP-induced reductions in adult testosterone levels, a decrease in the expression of steroidogenesis-related genes was anticipated. Surprisingly, however, significant increases were seen in the expression of Cyp11a1, Cy17a1, Star, and Tspo transcripts, suggesting that decreased testosterone production after birth could not be explained by decreases in steroidogenic enzymes as seen at GD20. These changes may reflect an increased number of Leydig cells in adult testes exposed in utero to DEHP rather than increased gene expression in individual Leydig cells, but this remains uncertain. Taken together, these results demonstrate that in utero DEHP exposure exerts both short-term and long-lasting effects on testicular steroidogenesis that might involve distinct molecular targets in fetal and adult Leydig cells.

Optimizing kidney preservation is a primary issue in transplantation, particularly in relation to new donor sources, such as expanded criteria donors (ECDs) and donation after cardiac death (DCD). Kidneys from these donors are highly sensitive to ischemia-reperfusion injuries--the emblematic lesions encountered during transplantation. Despite years of research, static cold storage, with solutions designed in the 1980s, remains the gold standard in kidney transplantation. This kind of preservation, however, is unable to fully protect an ECD or DCD kidney, highlighting the need for novel strategies to improve kidney preservation or promote kidney recovery. This Review provides an overview of the emerging strategies to prevent ischemia-reperfusion injuries in donor kidneys and describes strategies that are aimed at the donor, organ or recipient to improve graft outcome. These approaches include management of donors, preconditioning of the kidney, improvements in organ preservation solutions, postconditioning and regenerative therapies of the kidney graft following transplantation. In addition, machine perfusion provides an interesting opportunity to evaluate kidney graft quality before transplantation. Overall, a combination of therapeutic approaches seem to provide the best outcome, but preclinical studies using relevant models are needed before these approaches can be incorporated into clinical practice.

Chronic allograft nephropathy (CAN) is a leading cause of kidney graft failure following transplantation. Its causes are complex and include both immunological and nonimmunological factors. Here we have studied the development of CAN in a mouse model of kidney transplantation comparing isografts and allografts. Unlike the normal histology and normal serum creatinine of the uninephrectomized, nonrejecting isografted mice (0.219 ± 0.024 mg/dL), allografted mice demonstrated severe renal dysfunction (mean serum creatinine 0.519 ± 0.061 mg/dL; p < 0.005) with progressive inflammation and fibrosis of the kidney. These animals also showed an increased expression of connective tissue growth factor (CTGF), both systemically and within the graft. CTGF was highly expressed in tubuloepithelial cells of allografts, along with asmooth muscle actin, a marker of myofibroblasts, and transcriptionally associated with other markers of fibrosis. In vitro studies of tubular epithelium indicate that CTGF is capable of inducing EMT, independent of TGF-b . Finally, in human transplant recipients, serum and urine CTGF levels are significantly elevated compared to naïve individuals. Urinary levels correlated with the histological presence of CAN. These studies suggest a critical role of CTGF in graft fibrogenesis, for both mouse and man. Thus, CTGF has potential as a biomarker of CAN, and also a therapeutic target in managing graft fibrosis.

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† Both the authors have equally contributed to this work.Static preservation is currently the most widely used organ preservation strategy; however, decreased donor organ quality is impacting outcome negatively. M101 is an O 2 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 followup, 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.Key words: Graft preservation, ischemia reperfusion injury, kidney transplantation, oxygen, oxygen transporters Abbreviations: ATP, adenosine 5 -triphosphate; CS, cold storage; HBL-Hb, hexagonal-bilayer hemoglobin; HBOC, hemoglobin-based oxygen carrier; HTK, histidine tryptophane ketoglutarate; IFTA, interstitial fibrosis and tubular atrophy; IRI, ischemia reperfusion injury; LDH, lactate dehydrogenase; M101, hemarina-M101; NBT, nitroblue tetrazolium; PFCs, perfluorocarbons; SOD, superoxide dismutase; UW, University of Wisconsin.

†Authors contributed equally to this paper. Ischemia reperfusion injury (IRI) is pivotal for renal fibrosis development via peritubular capillaries injury.Coagulation represents a key mechanism involved in this process. Melagatran R (M), a thrombin inhibitor, was evaluated in an autotransplanted kidney model, using Large White pigs. To mimic deceased after cardiac death donor conditions, kidneys underwent warm ischemia (WI) for 60 min before cold preservation for 24 h in University of Wisconsin solution. Treatment with M before WI and/or in the preservation solution drastically improved survival at 3 months, reduced renal dysfunction related to a critical reduction in interstitial fibrosis, measured by Sirius Red staining. Tissue analysis revealed reduced expression of transforming growth factor-b (TGF-b ) and activation level of its effectors phospho-Smad3, Smad4 and connective tissue growth factor (CTGF) after M treatment. Fibrinolysis activation was also observed, evidenced by downregulation of PAI-1 protein and gene expression. In addition, M reduced S100A4 expression and vimentin staining, which are markers for epithelial mesenchymal transition, a major pathway to chronic kidney fibrosis. Finally, expression of oxidative stress markers Nox2 and iNOS was reduced. We conclude that inhibition of thrombin is an effective therapy against IRI that reduces chronic graft fibrosis, with a significantly positive effect on survival.

We previously found that platelet-derived growth factor (PDGF) and 17beta-estradiol stimulate gonocyte proliferation in a dose-dependent, nonadditive manner. In the present study, we report that gonocytes express RAF1, MAP2K1, and MAPK1/3. Inhibition of RAF1 and MAP2K1/2, but not phosphoinositide-3-kinase, blocked PDGF-induced proliferation. AG-370, an inhibitor of PDGF receptor kinase activity, suppressed not only PDGF-induced proliferation but also that induced by 17beta-estradiol. In addition, RAF1 and MAP2K1/2 inhibitors blocked 17beta-estradiol-activated proliferation. The estrogen receptor antagonist ICI 182780 inhibited both the effects of 17beta-estradiol and PDGF. PDGF lost its stimulatory effect when steroid-depleted serum or no serum was used. Similarly, 17beta-estradiol did not induce gonocyte proliferation in the absence of PDGF. The xenoestrogens genistein, bisphenol A, and DES, but not coumestrol, stimulated gonocyte proliferation in a dose-dependent and PDGF-dependent manner similarly to 17beta-estradiol. Their effects were blocked by ICI 182780, suggesting that they act via the estrogen receptor. AG-370 blocked genistein and bisphenol A effects, demonstrating their requirement of PDGF receptor activation in a manner similar to 17beta-estradiol. These results demonstrate the interdependence of PDGF and estrogen pathways in stimulating in vitro gonocyte proliferation, suggesting that this critical step in gonocyte development might be regulated in vivo by the coordinated action of PDGF and estrogen. Thus, the inappropriate exposure of gonocytes to xenoestrogens might disrupt the crosstalk between the two pathways and potentially interfere with gonocyte development.