Human Endothelial Cell Seeding in Partially Decellularized Kidneys.

The excessive demand for organ transplants has promoted the development of strategies that increase the supply of immune compatible organs, such as xenotransplantation of genetically modified pig organs and the generation of bioartificial organs. We describe a method for the partial replacement of rat endothelial cells for human endothelial cells in a rat's kidney, obtaining as a final result a rat-human bioartificial kidney. First, in order to maintain parenchymal epithelial cells and selectively eliminate rat endothelial cells, three methods were evaluated in which different solutions were perfused through the renal artery: 0.1% sodium dodecyl sulfate (SDS), 0.01% SDS, and hyperosmolar solutions of sucrose. Then, partially decellularized kidneys were recellularized with human endothelial cells and finally transplanted in an anesthetized rat. The solution of 0.1% SDS achieved the highest vascular decellularization but with high degree of damage in the parenchyma side. On the contrary, 0.01% SDS and hyperosmolar solutions achieved a partial degree of endothelial decellularization. TUNEL assays reveal that hyperosmolar solutions maintained a better epithelial cell viability contrasting with 0.01% SDS. Partially decellularized kidneys were then recellularized with human endothelial cells. Histological analysis showed endothelial cells attached in almost all the vascular bed. Recellularized kidney was transplanted in an anesthetized rat. After surgery, recellularized kidney achieved complete perfusion, and urine was produced for at least 90 min posttransplant. Histological analysis showed endothelial cells attached in almost all the vascular bed. Therefore, endothelial decellularization of grafts and recellularization with human endothelial cells derived from transplant recipients can be a feasible method with the aim to reduce the damage of the grafts.

Squamousness gain defines pancreatic ductal adenocarcinoma hepatic metastases phenotype, and gemcitabine response.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a dismal disease with a survival rate of less than 7%, mainly due to the hepatic metastatic spread. Despite the importance of understanding PDAC metastases, central questions remain concerning their biology and chemosensitivity. Moreover, the transcriptomic divergence between primary tumor (PT) and hepatic metastases (HM) has been poorly studied and without a clear dissection of the confounding tumoral-surrounding tissue. METHODS: Here, to unravel key biological features not biased by the surrounding tissue, we implemented a blind source separation based on independent component analysis, ProDenICA, on a treatment-naïve cohort of PDAC paired samples and a cohort of 305 resectable patients. In addition, a time-lapse experiment was performed to assess the gemcitabine chemosensitivity profile between the PT and HM. RESULTS: We identified HM's specific transcriptomic characteristics related to the upregulation of cell cycle checkpoint, mitochondria activity, and extracellular matrix reorganization, which could be associated with metastatic niche adaptation mechanisms. Furthermore, squamous lineage emerged as a key feature linked with a downregulation in the epithelial-to-mesenchymal program that can stratifies PDAC HM independent of the classical/basal-like spectrum. Remarkably, we also demonstrated that gemcitabine response is influenced by the squamous profile, being the HM more refractory to the treatment than the PT. CONCLUSIONS: These results pointed out divergent HM aspects compared to PT and allowed their stratification through the squamous lineage. Moreover, we unravel a clinical actionable squamous signature that predicts the gemcitabine response.

Targeting REG3ß limits pancreatic ductal adenocarcinoma progression through CTGF downregulation.

The crosstalk between the transformed tumoral cells and their microenvironment is a key aspect for pancreatic ductal adenocarcinoma (PDAC) progression. This molecular dialog is intensively studied because it may result in an efficient therapeutic target. Contrary to this near microenvironment, the stromal portion in direct contact with the transformed cells, a far microenvironment, placed at the periphery of the tumor mass, produces factors signaling tumors. Among these factors, REG3ß, produced by this part of the pancreas, is an important factor in promoting tumor progression. This paper demonstrated that targeting REG3ß protein with specific antibodies limits the PDAC tumor growth in an orthotopic, syngeneic mice model induced by injection of Panc02 cells. Then, we showed that CTGF is over-expressed in response to REG3ß in PDAC-derived cells. Moreover, inactivation of REG3ß by treating tumors with anti-REG3ß antibodies results in a strong decrease of CTGF in PDAC tumors. Lastly, we demonstrated that forced expression of CTGF in xenografted Panc02 cells abolishes the therapeutic effect of the anti-REG3ß antibody treatment. Altogether, these results indicate that the effect of REG3ß in promoting PDAC progression is mediated by CTGF over-activation. Thus, REG3ß is a promising therapeutic target to treat PDAC with an original rationale. In conclusion, we demonstrated that the far microenvironment is essential for PDAC progression by producing active secretory factors, and some of them could be used as therapeutic targets.

Secretory Leukocyte Proteinase Inhibitor Protects Acute Kidney Injury Through Immune and Non-Immune Pathways.

ABSTRACT: Acute kidney injury (AKI) is characterized by rapid loss of excretory function and is the clinical manifestation of several disorders affecting the kidney. The aim of the present study was to investigate the mechanism of action of Secretory Leukocyte Proteinase Inhibitor (SLPI) that protects the kidneys form AKI. In vivo and in vitro experiments were performed to assess the effect of SLPI on kidney injury. Animal models of kidney injury was generated by 40 min obstruction of kidney artery and vein (ischemia-reperfusion injury model) or daily administration of 60 mg/kg/day of gentamicine for 5 day (gentamicin-associated AKI model). For in vitro assessment, human renal epithelium HK-2 cells were cultured under serum starvation conditions or with tacrolimus. The administration of SLPI (250 µg/kg, i.p.) reduced elevated plasma creatinine and blood urea nitrogen levels, tissue myeloperoxidase content, and acute tubular necrosis induced by kidney damage. Furthermore, SLPI treatment reduced CD86, CD68, CD14, CCL2, TNFα, and IL-10 transcripts in kidney biopsies. To further analyze a direct effect of SLPI on renal epithelial cells, HK-2 cells from human renal epithelium were cultured under serum starvation conditions or with tacrolimus. Both conditions induced apoptosis of HK-2 cells which was reduced when SLPI was present in the culture medium. Furthermore, SLPI favored the proliferation and migration of HK-2 cells. An analysis of the gene profiles of HK-2 cells treated with calcineurin inhibitors affected inflammatory and non-inflammatory pathways that were reversed by SLPI. Among them, SLPI down modulated the expression of CCL2, SLC5A3, and BECN1 but up-regulated the expression of TLR4, ATF4, ATF6, HSP90B, BBC3 SLC2A1, and TNFRSF10B. Overall, these results suggest that SLPI, in addition to its activity on immune cells, may directly target tubular epithelial cells of the kidney to mediate the nephroprotective activity in AKI.

Estado actual del trasplanterenopancreático / Current status of the kidney-pancreas transplant

RESUMEN El trasplante de páncreas es un tratamiento alternativo para la diabetes. Sus modalidades e indicaciones son: 1) trasplante de páncreas simultáneo con riñón para pacientes con diabetes mellitus tipo 1 o con nefropatía diabética en estadio terminalen tratamiento sustitutivo o próximo al mismo; 2) trasplante de páncreas después de riñón parapacientes condiabetes mellitustipo 1 con un trasplante renal funcionante; 3) trasplante de páncreas aislado parapacientes con diabetes mellitustipo 1 con hipoglucemias aperceptivas que requieren internaciones o rescate de terceros. Algunos pacientes con diabetes mellitus tipo 2 seleccionados pueden ser candidatos a trasplante de páncreas. La selección de donantes es muy importante, el donante ideal es fallecido por traumatismo craneoencefálico, menor de 45 años, con un peso entre 30 y 90 kg, con un IMC menor a 30kg/m2, hemodinámicamente estable y sin antecedentes de paro cardiorespiratorio ni hipotensión sostenida. Hay varias estrategias de derivación de la función endócrina (sistémica y portal) y exócrina (entérica o vesical), la más utilizada es la derivación sistémica y entérica. En el manejo perioperatorio se destacan estrategias para mantener una buena presión de perfusión tisular, un control estricto de glucemia, para prevenir la trombosis del injerto debe implementarse un plan de antiagregación y anticoagulación, todo lo anterior junto a una profilaxis antibiótica, antifúngica y antiviral. Los esquemas clásicos de inmunosupresión incluyen una inducción con esteroides y anticuerpos deplecionantes de linfocitos T y un mantenimiento con un triple esquema con esteroides, tacrolimus y micofenolato. La clasificación de Banffdistingue rechazos celulares y humorales. La base del tratamiento del rechazo celular incluye pulsos de esteroides y anticuerpos deplecionantes de linfocitos T, mientras que los rechazos humorales requieren de plasmaféresis e inmunoglobulina endovenosa. Las principales complicaciones postoperatorias son el sangrado, la pancreatitis, la trombosis del injerto y las fístulas anastomóticas. En cuanto a los resultados, el trasplante de páncreas presenta, a cinco años, una supervivencia del paciente del 90% y un 77% del injerto pancreático. Las modalidades de trasplante solitario presentan menor supervivencia alejada del injerto. En Argentina hay una actividad de trasplante de páncreas de entre 60 y 80 trasplantes anuales. La reglamentación del INCUCAIprevé la inscripción anticipada en lista de espera de pacientes con nefropatía terminal con depuración de creatinina menor a 30ml/min.

ABSTRACT Pancreas transplantation is an alternative treatment for diabetes. Its modalities and indications are the following: 1) simultaneous pancreas and kidney transplantation: type 1 diabetes mellitus patients with end-stage diabetic nephropathy (in replacement treatment or close to it); 2) pancreas transplantation after kidney: type 1 diabetes mellitus patients with a functioning kidney transplant; 3) isolated pancreas transplantation: type 1 diabetes mellitus patients with unperceived hypoglycemia requiring hospitalization or rescue by third parties. Some of the screened type 2 diabetes mellitus patients may be pancreas transplantation candidates. Choosing a donor is very important: the ideal donor should be a deceased one who died due to intracranial injury, under 45 years of age, weighing between 30 and 90 kg, with a BMI below 30kg/m2, hemodynamically stable and having no history of cardiopulmonary arrest or sustained hypotension. There exist various strategies to divert the endocrine function (systemic and portal) and the exocrine function (vesical or enteric), systemic and enteric diversion being the most commonly used. Among the techniques which stand out during perioperative management, we could mention maintaining a good tissue perfusion, a strict glycemic control, an antiaggregation/anticoagulation plan to prevent graft thrombosis and antibiotic, antifungal and antiviral prophylactic treatment. Classic immunosuppression schemes consist of induction with T cell depleting steroids and antibodies and keeping a three-drug treatment including steroids, tacrolimus and mycophenolate. Banff classification draws a distinction between cellular and humoral rejection. The basis for cellular rejection treatment includes steroid-pulse therapy and T-cell depleting antibodies, while humoral rejection requires plasmapheresis and endovenous immunoglobulin. The main postoperative complications are bleeding, pancreatitis, graft thrombosis and anastomosis fistula. As for the results, the survival rate 5 years after pancreas transplantation is 90% for patients and 77% for pancreatic grafts. Isolated transplantation presents a lower long-term survival of the graft. In Argentina, between 60 and 80 pancreas transplants are performed every year. INCUCAI regulations provide for early registration on the waiting list for patients suffering from end-stage nephropathy with a creatinine clearance lower than 30 mL/min.

Evidencing a Pancreatic Ductal Adenocarcinoma Subpopulation Sensitive to the Proteasome Inhibitor Carfilzomib.

PURPOSE: Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer with a survival rate less than 5%. Multiple chemotherapeutic drugs have been tested to improve patient prognosis; however, the clinical efficacy of these treatments is low. One of the most controversial family of drugs are the proteasome inhibitors, which have displayed promising effects in preclinical studies, but low clinical performance. Here, we unravel a specific transcriptomic signature that discriminates a subgroup of patients sensitive to the proteasome inhibitor carfilzomib. EXPERIMENTAL DESIGN: First, we identified a subpopulation of PDAC-derived primary cells cultures (PDPCC) sensitive to the proteasome inhibitor carfilzomib. Then, we selected a transcriptomic signature that predicts carfilzomib chemosensitivity using independent component analysis on the transcriptome of PDPCC. Finally, we validated the signature in an independent cohort of PDAC biopsy-derived pancreatic organoids. RESULTS: Sensitive phenotype was characterized by a high expression of genes related with a cornified/squamous pathway and a downregulation of epithelial-mesenchymal transition genes. Interestingly, carfilzomib-sensitive transcriptomic profile did not show any association with the proteasome activity but strongly correlates with ATF4 and CHOP expression, which are key markers of the unfolded protein response and critical to trigger the cell death program. Concordantly, sensitive phenotype showed a high level of the de novo RNA and protein synthesis compared with the resistant one and, most important, cell death induced by carfilzomib is dependent of the translational activity. CONCLUSIONS: We demonstrate the existence of a carfilzomib-sensitive PDAC subgroup with a specific transcriptomic phenotype that could explain the biological reason for this responsiveness.

Anti-Hla donor-specific antibody monitoring in pancreas transplantation: Role of protocol biopsies.

In kidney transplantation, de novo donor-specific antibodies (DSA) correlate with poor graft survival, and Consensus Guidelines recommend a protocol biopsy. In pancreas transplantation, DSA are also associated with poor graft outcomes; however, there are no recommendations on protocol biopsies. We started an antibody screening protocol on pancreas transplant patients at 0, 3, 6, 12 months, and yearly. Patients with DSA or high MFI non-DSA were considered for protocol biopsies of both organs. Results: 143 pancreas recipients were screened. 84 patients had negative antibodies throughout the study, 11 patients were found to have antibodies at graft dysfunction, and 48 patients had positive antibodies at screening without acute organ dysfunction (study group). Among the 30 non-DSA patients, 9 had protocol simultaneous pancreas and kidney biopsies performed with negative results in all of them. In contrast, among the 18 DSA patients, 15 had these biopsies performed, and 47% presented with subclinical rejection of the kidney, the pancreas, or both. In addition, some of the DSA patients without a protocol biopsy presented with rejection during the first 15 months of follow-up. Conclusion: We conclude that protocol biopsies of both grafts may play a role in the follow-up of pancreas transplant patients with de novo DSA appearance.

Elucidating the mechanism of action of alpha-1-antitrypsin using retinal pigment epithelium cells exposed to high glucose. Potential use in diabetic retinopathy.

BACKGROUND: Alpha-1-antitrypsin is a protein involved in avoidance of different processes that are seen in diabetic retinopathy pathogenesis. These processes include apoptosis, extracellular matrix remodeling and damage of vessel walls and capillaries. Furthermore, because of its anti-inflammatory effects, alpha-1-antitrypsin has been proposed as a possible therapeutic approach for diabetic retinopathy. Our group tested alpha-1-antitrypsin in a type 1 diabetes mouse model and observed a reduction of inflammation and retinal neurodegeneration. Thus, shedding light on the mechanism of action of alpha-1-antitrypsin at molecular level may explain how it works in the diabetic retinopathy context and show its potential for use in other retinal diseases. METHODS: In this work, we evaluated alpha-1-antitrypsin in an ARPE-19 human cell line exposed to high glucose. We explored the expression of different mediators on signaling pathways related to pro-inflammatory cytokines production, glucose metabolism, epithelial-mesenchymal transition and other proteins involved in the normal function of retinal pigment epithelium by RT-qPCR and Western Blot. RESULTS: We obtained different expression patterns for evaluated mediators altered with high glucose exposure and corrected with the use of alpha-1-antitrypsin. CONCLUSIONS: The expression profile obtained in vitro for the evaluated proteins and mRNA allowed us to explain our previous results obtained on mouse models and to hypothesize how alpha-1-antitrypsin hinder diabetic retinopathy progression on a complex network between different signaling pathways. GENERAL SIGNIFICANCE: This network helps to understand the way alpha-1-antitrypsin works in diabetic retinopathy and its scope of action.

SLPI in the perfusion solution helps to identify graft quality in kidney transplants.

Aim: It is important to find biomarkers that identify the graft quality in kidney transplantation. Results & methodology: The level of SLPI in the cold preservation solution was used as a marker to predict early kidney graft function after transplantation. Before transplantation, kidneys were washed and SLPI was measured in the discarded solution. A retrospective analysis showed that patients with delayed graft function or rejection episodes in post-trasplant, had higher SLPI concentrations in the perfusion solution than patients without delayed graft function or rejections. Furthermore, SLPI could discriminate between patients with better or worse estimated glomerular filtration rate among low-risk patients (kidney donor profile index <80). Discussion & conclusion: These results suggest that the SLPI concentration in the perfusion solutions could be a predictor of short-term organ function and a complement to the kidney donor profile index score.

Graft dysfunction in simultaneous pancreas kidney transplantation (SPK): Results of concurrent kidney and pancreas allograft biopsies.

Simultaneous pancreas and kidney transplants offer significant therapeutic advantages but present a diagnostic approach dilemma in the diagnosis of rejection. Because both organs are from the same donor, the kidney has been treated traditionally as the "sentinel" organ to biopsy, presumably representing the status of both allografts. Truly concurrent biopsy studies, however, are needed to confirm this hypothesis. We examined 101 concurrent biopsies from 70 patients with dysfunction in either or both organs. Results showed concurrent rejection in 23 of 57 (40%) of cases with rejection; 19 of 57 (33.5%) and 15 of 57 (26.5%) showed kidney or pancreas only rejection, respectively. The degree and type of rejection differed in the majority (13 of 23, 56.5%) of cases with concurrent rejection, with the pancreas more often showing higher rejection grade. Taking into account pancreas dysfunction, a positive kidney biopsy should correctly predict pancreas rejection in 86% of the instances. However, the lack of complete concordance between the 2 organs, the discrepancies in grade and type of rejection, and the tendency for higher rejection grades in concurrent or pancreas only rejections, all support the rationale for pancreas biopsies. The latter provide additional data on the overall status of the organ, as well as information on nonrejection-related pathologies.

Long-term apoptosis-related protein expression in the diabetic mouse ovary.

Emerging evidence has shown that oocytes from diabetic ovaries exhibit delayed maturation, mitochondrial dysfunction and meiotic defects, which are related increased apoptosis. The main objective of the present study was to analyze the apoptosis pathways activated during follicular loss at multiple time points in a diabetic mouse model. Twenty BALB/c mice were used in this study, and diabetes mellitus was induced by streptozotocin injection. Three diabetic and two control animals were sacrificed on days 15, 20, 70 and 80 posttreatment. The ovaries were then removed; one was used for follicular counting, TUNEL, immunohistochemistry and immunofluorescence, while the other was used for Western blot analysis. The proteins studied were BAX, BCL2, t-BID, FAS, FASL, active caspase 8, active caspase 9 and active caspase 3. Follicular apoptosis decreased over time, with the highest values observed at 15 days posttreatment. Granulosa cells were positive for active caspase 3, which showed constant expression levels at all time points. FAS, FASL, t-BID and active caspase 8 showed strong cytoplasmic immunostaining in the oocytes and granulosa cells of the diabetic mice, with significant increases observed at 15, 20 and 70 days posttreatment. BAX expression was slightly higher in the diabetic mouse ovaries than in the control ovaries at 15, 20 and 70 days posttreatment, whereas the highest active caspase 9 expression was at observed 20 days posttreatment. Low BCL2 protein levels were detected in the diabetic mouse ovaries at all time points. This study describes for the first time the behavior of apoptosis-related proteins in the diabetic mouse ovary and shows not only that the FAS/FASL pathway contributes to follicular loss but also that antral follicles are the most affected.

α-lipoic acid reduces postreperfusion syndrome in human liver transplantation - a pilot study.

A double-blind randomized controlled trial was performed to compare the safety and efficacy of α-lipoic acid (ALA) in liver transplantation (LT). The grafts were randomized to receive ALA or placebo before the cold ischemia time. Furthermore, patients transplanted with the ALA-perfused graft received 600 mg of intravenous ALA, while patients with the nonperfused graft received the placebo just before graft reperfusion. Hepatic biopsy was performed 2 h postreperfusion. Blood samples were collected before, during and 1 and 2 days after reperfusion. Quantitative polymerase chain reaction (qPCR) analysis was performed on biopsies to assess genes involved in the response to hypoxia, apoptosis, cell growth, survival and proliferation, cytokine production and tissue damage protection. Nine of 40 patients developed postreperfusion syndrome (PRS), but seven of them belonged to the control group. There was a decrease in PHD2 and an increase in alpha subunit of hypoxia-inducible factor-1 (HIF-1α) and baculoviral IAP repeat containing 2 (Birc2) transcript levels in the biopsies from the ALA-treated versus the control group of patients. Additionally, plasma levels of alarmins were lower in ALA-treated patients than control patients, which suggests that ALA-treated grafts are less inflammatory than untreated grafts. These results showed that ALA is safe for use in LT, induces gene changes that protect against hypoxia and oxidative stress and reduces the appearance of PRS.

Alpha-1-antitrypsin ameliorates inflammation and neurodegeneration in the diabetic mouse retina.

Diabetic retinopathy (DR) is the most common cause of blindness in the working age population. Early events of DR are accompanied by neurodegeneration of the inner retina resulting in ganglion cell loss. These findings together with reduced retinal thickness are observed within the first weeks of experimental DR. Besides, an inflammatory process is triggered in DR in which the innate immune response plays a relevant role. Alpha 1 antitrypsin (AAT), an inhibitor of serine proteases, has shown anti-inflammatory properties in several diseases. We aimed at evaluating the use of AAT to prevent the early changes induced by DR. Diabetic AAT-treated mice showed a delay on ganglion cell loss and retinal thinning. These animals showed a markedly reduced inflammatory status. AAT was able to preserve systemic and retinal TNF-α level similar to that of control mice. Furthermore, retinal macrophages found in the AAT-treated diabetic mouse exhibited M2 profile (F4/80+CD206+) together with an anti-inflammatory microenvironment. We thus demonstrated that AAT-treated mice show less retinal neurodegenerative changes and have reduced levels of systemic and retinal TNF-α. Our results contribute to shed light on the use of AAT as a possible therapeutic option in DR.

Cementoin-SLPI fusion protein binds to human monocytes and epithelial cells and shows higher biological activity than SLPI.

Secretory Leukocyte Proteinase Inhibitor (SLPI) is an antiinflammatory peptide that blocks the activity of serine proteases, primarily the neutrophil elastase. In an attempt to direct the activity of SLPI on inflamed sites, a chimera consisting of the transglutaminase II substrate domain of trappin 2 (cementoin), and the mature SLPI protein was constructed. Cell attachment and biological activity were compared between SLPI and this chimera. By using whole cell ELISA, fluorescence microscopy and flow cytometry assays we observed that the cementoin-SLPI fusion protein (FP) but not SLPI attached to a human lung epithelial cell line and monocytes. A maximum attachment was achieved 15 min after FP was added to the cell cultures. In an elastase activity assay, we observed that FP retained its antiprotease activity and that at equimolar amount of proteins, FP was more efficient than SLPI in the inhibition. Both, FP and SLPI inhibits IL-2-induced lymphocyte proliferation, however, lower amounts of FP were required to achieve this inhibition. Furthermore, FP binds to mycobacteria and maintained the bactericidal activity observed for SLPI. Overall, these results show that this new chimera is able to attach to the cell surfaces retaining and improving some biological activities described for SLPI.

Alpha Lipoic Acid: A Therapeutic Strategy that Tend to Limit the Action of Free Radicals in Transplantation.

Organ replacement is an option to mitigate irreversible organ damage. This procedure has achieved a considerable degree of acceptance. However, several factors significantly limit its effectiveness. Among them, the initial inflammatory graft reaction due to ischemia-reperfusion injury (IRI) has a fundamental influence on the short and long term organ function. The reactive oxygen species (ROS) produced during the IRI actively participates in these adverse events. Therapeutic strategies that tend to limit the action of free radicals could result in beneficial effects in transplantation outcome. Accordingly, the anti-oxidant α-lipoic acid (ALA) have been proved to be protective in several animal experimental models and humans. In a clinical trial, ALA was found to decrease hepatic IRI after hepatic occlusion and resection. Furthermore, the treatment of cadaveric donor and recipient with ALA had a protective effect in the short-term outcome in simultaneous kidney and pancreas transplanted patients. These studies support ALA as a drug to mitigate the damage caused by IRI and reinforce the knowledge about the deleterious consequences of ROS on graft injury in transplantation. The goal of this review is to overview the current knowledge about ROS in transplantation and the use of ALA to mitigate it.