Porcine Kidney Research Articles

Features of Reproduction of Tick-Borne Encephalitis Virus in a New Cell Line of the Siberian Bat Myotis sibiricus (Kastschenko, 1905)

Background. Tick-borne encephalitis virus (TBEV) in nature exists due to the constant circulation between vertebrate animals and tick viruses. To study the characteristics of reproduction of TBEV in the cells of vertebrate hosts of various species, it is necessary to simulate infection in the cell lines of both natural and accidental hosts of TBEV. Aim . To study the possibility of reproduction of TBEV in the cell line of the kidney of Siberian bat Myotis sibiricus (Kastschenko, 1905) – an accidental host of the virus. Materials and methods. The cell line of Siberian bat M. sibiricus was established by serial passages of primary culture of kidney cells. The SPEV line porcine kidney cells were used as reference. Cells were infected with a strain of TBEV of the Siberian subtype 92M and both cells and cell culture fluids were sampled every 2 hours during first 24 hours post infection. In addition, the samples were collected daily up to 5 days post infection. Evaluation of the amount of intracellular RNA of TBEV of positive polarity (+RNA) was performed using quantitative real-time PCR. The concentration of infectious TBEV was evaluated using the method of titration TBEV on plaque forming units. Results. The continuous cell line, designated as MdbK, was established from the kidney cell suspension of M. sibiricus and was used for experiments after 20 serial passages. MdbK cells were able to support the replication of TBEV RNA and production of infectious virus was also possible. The concentration of intracellular RNA had reached 9.1 lg copies/μl by day 3 post infection, whereas highest titer of infectious TBEV in cell culture fluid had comprised 5,5 PFU/ml and was detected by day 4. The concentration of intracellular RNA and the virus infectivity in MdbK cell line was significantly lower than in convenient SPEV line porcine kidney cell. Conclusions . The results suggest the low fitness of TBEV to the intracellular environment of an accidental host.

Improved Normothermic Machine Perfusion After Short Oxygenated Hypothermic Machine Perfusion of Ischemically Injured Porcine Kidneys.

In an era where global kidney shortage has pushed the field of transplantation towards using more marginal donors, modified kidney preservation techniques are currently being reviewed. Some techniques require further optimization before implementation in full scale transplantation studies. Using a porcine donation after circulatory death kidney model, we investigated whether initial kidney hemodynamics improved during normothermic machine perfusion if this was preceded by a short period of oxygenated hypothermic machine perfusion (oxHMP) rather than static cold storage (SCS). Kidneys subjected to 75 minutes of warm ischemia were randomly assigned to either SCS (n = 4) or SCS + oxHMP (n = 4), with a total cold storage time of 240 minutes. Cold preservation was followed by 120 minutes of normothermic machine perfusion with continuous measurement of hemodynamic parameters and renal function. oxHMP preserved kidneys maintained significantly lower renal resistance throughout the normothermic machine perfusion period compared to SCS kidneys (P < 0.001), reaching lowest levels at 60 minutes with means of 0.71 ± 0.35 mm Hg/mL/min/100 g (SCS) and 0.45 ± 0.15 mm Hg/mL/min/100 g (oxHMP). Accordingly, the oxHMP group had a higher mean renal blood flow versus SCS kidneys (P < 0.001). oxHMP kidneys had higher oxygen consumption during normothermic machine perfusion compared to SCS preserved kidneys (P < 0.001). Creatinine clearance remained similar between groups (P = 0.665). Preceding oxHMP significantly improved initial normothermic machine perfusion hemodynamics and increased total oxygen consumption. With the long period of warm ischemia, immediate kidney function was not observed, reflected by the findings of low creatinine clearance in both groups.

Effect of the drug cyclophosphamide on the activity of porcine kidney betaine aldehyde dehydrogenase.

The enzyme betaine aldehyde dehydrogenase (BADH EC 1.2.1.8) catalyzes the synthesis of glycine betaine (GB), an osmolyte and osmoprotectant. Also, it participates in several metabolic pathways in humans. All BADHs known have cysteine in the active site involved in the aldehyde binding, whereas the porcine kidney enzyme (pkBADH) also has a neighborhood cysteine, both sensitive to oxidation. The antineoplastic and immuno-suppressant pre-drug cyclophosphamide (CTX), and its bioactivation products, have two highly oxidating chlorine atoms. This work aimed to analyze the effect of CTX in the activity of porcine kidney betaine aldehyde dehydrogenase. PkBADH was incubated with varying CTX concentration (0 to 2.0 mM) at 25 °C and lost 50 % of its activity with 2.0 mM CTX. The presence of the coenzyme NAD+ (0.5 mM) decreased 95% the activity in 2.0 mM CTX. The substrate betaine aldehyde (0.05 and 0.4 mM, and the products NADH (0.1-0.5 mM) and GB (1 and 10 mM) did not have an effect on the enzyme inactivation by CTX. The reducing agents, dithiothreitol and β-mercaptoethanol, reverted the pkBADH inactivation, but reduced glutathione (GSH) was unable to restore the enzyme activity. Molecular docking showed that CTX could enter at the enzyme active site, where its chlorine atoms may interact with the catalytic and the neighboring cysteines. The results obtained show that CTX inactivates the pkBADH due to oxidation of the catalytic cysteine or because it oxidizes catalytic and neighborhood cysteine, forming a disulfide bridge with a concomitant decrease in the activity of the enzyme.

Techniques of orthotopic renal transplantation. II. Size-matched porcine grafts in monkey recipients.

ABSTRACTPurposeAs a classical xenotransplantation model, porcine kidneys have been transplanted into the lower abdomen of non-human primates. However, we have improved upon this model by using size-matched grafting in the orthotopic position. The beneficial aspects and surgical details of our method are reported herein.MethodsDonors were two newborn pigs (weighting 5 to 6 kg) and recipients were two cynomolgus monkeys (weighting, approximately, 7 kg). After bilateral nephrectomy, kidneys were cold-transported in Euro-Collins solution. The porcine kidney was transplanted to the site of a left nephrectomy and fixed to the peritoneum.ResultsKidneys transplanted to the lower abdomen by the conventional method were more susceptible to torsion of the renal vein (two cases). In contrast, early-stage blood flow insufficiency did not occur in orthotopic transplants of theleft kidney.ConclusionsSize-matched porcine-primate renal grafting using our method of transplanting tothe natural position of the kidneys contributes to stable post-transplant blood flow to the kidney.

Intrarenal temperature measurement associated with holmium laser intracorporeal lithotripsy in an ex vivo model.

IntroductionThe aim of this article was to quantify the effect of the use of holmium laser during intracorporeal lithotripsy in an ex vivo model.Material and methodsA simulated model for laser nephro-lithotripsy was designed. Two ex vivo porcine kidneys were used. Electronic thermometer electrodes were inserted on the upper calyx. Intracorporeal lithotripsy was simulated with a holmium laser. Intrarenal temperature was recorded both at the beginning and after one minute of laser use with delta temperature (DT) defined as the difference between them. Measurements were made at different irrigation heights (30, 40, and 50 cm H2O), frequency (Hz), and laser energy (J) in addition to the presence or absence of the access sheath. Analysis of factors associated with temperature change was performed.ResultsThirty-eight observations were recorded. The measurement without the use of access sheath showed an average DT of 4.9, 5.1, and 6.5°C for 5, 10, and 15 Hz, respectively; however, with a sheath, DTs were 0.2, 0.5, and 1.5°C. In terms of energy, mean DTs of 4.3, 6.1, 5.2, and 13.9°C for 0.5, 0.8, 1.0, and 1.5 J were recorded; in contrast, with a sheath, averages of 0.4, 0.5, 0.5, and 3.8°C, respectively were noted. In the adjusted model, energy, frequency, and use of sheath and water height were significant.ConclusionsThe configuration of the laser significantly modifies the intrarenal temperature and height of the bladder irrigation. The use of an access sheath provides lower intrarenal temperatures regardless of laser configuration and water height.

Effectiveness of a machine perfusion preservation system, developed by industry-academic collaboration for warm ischemic porcine kidney

Effectiveness of a machine perfusion preservation system, developed by industry-academic collaboration for warm ischemic porcine kidney

Reaction Force Mapping by 3-Axis Tactile Sensing With Arbitrary Angles for Tissue Hard-Inclusion Localization.

Although robot-assisted diagnosis and minimally invasive surgery (MIS) brings distinct benefits, deficient multi-dimensional force feedback remains a noteworthy limitation and challenge in MIS. Aiming for a comprehensive high-fidelity perception of tissue-instrument interactions, we present a Fiber Bragg Grating (FBG)-based 3-axis tactile sensing for surface reaction force mapping, identification and localization of tissue hard-inclusion. The tactile sensing probe consists of five optical fibers inscribed with FBGs and a force-sensitive 3D printed deformable body. All fibers are suspended inside the deformable body in a parallel manner, leading to the direct compression or tension of each FBG. Such configuration can effectively avoid the chirping failure of FBG compared with the pasting FBG-based sensors. A linearized difference model is proposed to calibrate the 3-axis force detection and enhance the resistance to nonlinear interferences. Hard-inclusion identification experiments with varied hard-inclusion sizes and depths have been implemented through discrete palpation and dragging palpation modes. Results indicate that the probe can effectively identify the presence and location of these small hard-inclusions from the force mapping. Furthermore, lengthy vessels embedded in the phantom can be accurately identified through dragging palpation with an arbitrary contact angle. Another novelty of the probe is the reconstruction of the surface profile of a non-planar tissue, which further allows hard-inclusion identification and 3D localization. Ex-vivo tissue palpation on a porcine kidney further validates the effectiveness and feasibility of the probe to map surface reaction forces and localize the hard-inclusions intraoperatively.

Calculation of an Apical Efflux Ratio from P-Glycoprotein (P-gp) In Vitro Transport Experiments Shows an Improved Correlation with In Vivo Cerebrospinal Fluid Measurements in Rats: Impact on P-gp Screening and Compound Optimization.

P-glycoprotein (P-gp) is a major blood-brain barrier (BBB) efflux transporter. In vitro approaches, including bidirectional efflux ratio (ER), are used to measure P-gp-mediated transport, but findings can be inconsistent across models. We propose a novel, more physiologically relevant, in vitro model: unidirectional apical efflux ratio (AP-ER)-a ratio of permeability rates at the apical side of the BBB with and without P-gp inhibitor. To test our approach, ER and AP-ER were calculated for 3227 structurally diverse compounds in porcine kidney epithelial cells (LLC-PK1) overexpressing human or mouse P-gp and classified based on their passive transcellular P-gp permeability or charged properties. In vivo rat infusion studies were performed for selected compounds with high ER but low AP-ER. One-third of the 3227 compounds had bidirectional ER that was much higher than AP-ER; very few had AP-ER higher than ER. Compounds with a large difference between AP-ER and ER were typically basic compounds with low-to-medium passive permeability and high lipophilicity and/or amphiphilicity, leading to strong membrane binding. Outcomes in the human model were similar to those in mice, suggesting AP-ER/ER ratios may be conserved for at least two species. AP-ER predicted measured cerebrospinal fluid (CSF) concentration better than ER for the five compounds tested in our in vivo rat infusion studies. We report superior estimations of the CSF concentrations of the compounds when based on less resource-intensive AP-ER versus classic ER. Better understanding of the properties leading to high P-gp-mediated efflux in vivo could support more efficient brain-penetrant compound screening and optimization. SIGNIFICANCE STATEMENT: To address inconsistencies associated with the historical, bidirectional efflux ratio (ER) calculation of P-glycoprotein-mediated transport, we propose to use the novel, more physiologically relevant, unidirectional apical efflux ratio (AP-ER) model. In vitro experiments suggested that compounds with strong membrane binding showed the largest difference between AP-ER and ER, and in vivo infusion studies showed that AP-ER predicted cerebrospinal fluid concentrations of compounds better than ER; outcomes in the human model were similar to those in mice.

Ex Vivo Porcine Model for Robot-Assisted Partial Nephrectomy Simulation at a High-Volume Tertiary Center: Resident Perception and Validation Assessment Using the Global Evaluative Assessment of Robotic Skills Tool.

Introduction: With increased demands on surgeon productivity and outcomes, residency robotics training increasingly relies on simulations. The objective of this study is to assess the validity and effectiveness of an ex vivo porcine training model as a useful tool to improve surgical skill and confidence with robot-assisted partial nephrectomy (RAPN) among urology residents. Methods: A 2.5 cm circular area of ex vivo porcine kidneys was marked as the area of the tumor. Tumor excision and renorrhaphy was performed by trainees using a da Vinci Si robot. All residents ranging from postgraduate year (PGY) 2 to 5 participated in four training sessions during the 2017 to 2018 academic year. Each session was videorecorded and scored using the global evaluative assessment of robotic skills (GEARS) by faculty members. Results: Twelve residents completed the program. Initial mean GEARS score was 16.7 and improved by +1.4 with each subsequent session (p = 0.008). Initial mean excision, renorrhaphy, and total times were 8.2, 13.9, and 22.1 minutes, which improved by 1.6, 2.0, and 3.6 minutes, respectively (all p < 0.001). Residents' confidence at performing RAPN and robotic surgery increased after completing the courses (p = 0.012 and p < 0.001, respectively). Overall, residents rated that this program has greatly contributed to their skill (4/5) and confidence (4.1/5) in robotic surgery. Conclusions: An ex vivo porcine simulation model for RAPN and robotic surgery provides measurable improvement in GEARS score and reduction in procedural time, although significant differences for all PGY levels need to be confirmed with larger study participation. Adoption of this simulation in a urology residency curriculum may improve residents' skill and confidence in robotic surgery.

Human recombinant IL-10 reduces xenogenic cytotoxicity via macrophage M2 polarization

Xenotransplantation has been considered an alternative to the moderate shortage of donor organs for transplantation. To achieve successful xenotransplatation, there is the need to overcome immune rejection. Although, hyperacute rejection has been overcome by α1,3-galactosyltransferase knockout pig, cellular immune rejection remains as a subsequent barrier. Interleukin-10 (IL-10) is known as an anti-inflammatory and immunomodulatory cytokine which has been shown to limit inflammatory responses by inhibiting macrophage activation in several animal experiments. To study the effect of human IL-10 (hIL-10) on pig-to-human xenotransplantation, porcine kidney epithelial cell line (PK(15)) expressing hIL-10 was established. The cytotoxicity of macrophages decreased by hIL-10 from transgenic cells. Furthermore, there is a decreased production of pro-inflammatory cytokines, tumor necrosis factor-α and interleukin-23, and increased anti-inflammatory cytokines like IL-10, but not transforming growth factor beta, in the presence of hIL-10. Also, macrophage polarization toward M2-like phenotype were induced by hIL-10 from transgenic PK(15) cells. Finally, we suggest that the cytotoxicity of human macrophages was reduced by hIL-10 from transgenic cells, inducing M2-like macrophage polarization. Therefore, these results show that hIL-10 transgenic pig can be used as a model to overcome acute immune rejection in pig-to-human xenotransplantation.

Susceptibility of immortalized porcine kidney macrophages to porcine reproductive and respiratory syndrome virus-2 infection

Porcine reproductive and respiratory syndrome virus (PRRSV) displays restricted tropism to porcine alveolar macrophages in nature. Meanwhile, non-porcine cell lines derived from African green monkey kidney cell lines are permissive to PRRSV, resulting in their widespread use in PRRSV research. Furthermore, genetically modified cell lines expressing receptors targeted by PRRSV have been established. We previously established porcine immortalized kidney-derived macrophages (IPKMs) that maintained typical macrophage function. In the present study, we demonstrated the advantages of IPKMs for PRRSV research. IPKMs expressed receptors for PRRSV such as CD163 and CD169. The efficiency of virus isolation from field biological samples was higher for IPKMs than for MARC-145 cells. Five different clusters of North American type PRRSV were propagated in IPKMs. Four field strains continuously produced progeny viruses during 10 continuous passages. The efficiency of virus isolation from field biological samples and continuous progeny virus production in the sequential passages using IPKMs indicated that these cells are good vessels for PRRSV research.

Evaluation of Temperature Distribution Around the Probe in Cryoablation of Lipiodol-Mixed-Tissue Phantom.

To determine whether lipiodol, which has low thermal conductivity, influences ice ball formation during cryoablation of a lipiodol-mixed-tissue phantom. Lipiodol-mixed-tissue phantoms were created by injecting lipiodol (4-6ml) into the renal arteries of ex vivo porcine kidneys (lipiodol group). A cryoprobe (CryoHit™ Needle S) with a holder that was set with thermocouples at various positions around the cryoprobe was inserted. After freezing for 300s, the followings were evaluated: ice ball size on CT, temperature distribution around the cryoprobe, and calculated distances at 0°C and -20°C. Each variable was compared between lipiodol group (n = 6) those obtained in a control group without lipiodol injection (n = 6). Mean ice ball diameter (width/length) on CT was 22.1 ± 2.3/22.9 ± 2.3mm in the lipiodol group and 21.6 ± 0.7/22.2 ± 1.3mm in the control group. Mean cryoprobe temperature was -118 ± 3.0°C in the lipiodol group and -117 ± 2.6°C in the control group. In both groups, temperature at the 3mm thermocouple reached approximately -50°C and was < 0°C within ~ 10mm of the cryoprobe. Temperature of 0/-20°C occurred at a mean distance from the cryoprobe of 11.1 ± 0.5/6.9 ± 0.4mm in the lipiodol group and 11.0 ± 0.2/6.9 ± 0.2mm in the control group. There was no significant difference in any variable between the groups. The inclusion of lipiodol in a tissue phantom had no negative effects on ice ball formation that were related to thermal conductivity.

The Impact of BPI Expression on Escherichia coli F18 Infection in Porcine Kidney Cells.

Simple SummaryEscherichia coli frequently causes bacterial diarrhea in piglets. Vaccine development and improved feeding and animal management strategies have reduced the incidence of bacterial diarrhea in piglets to some extent. However, current breeding strategies also have the potential to improve piglet resistance to diarrhea at a genetic level. This study sought to advance the current understanding of the functional and regulatory mechanisms whereby the candidate gene bactericidal/permeability-increasing protein (BPI) regulates piglet diarrhea at the cellular level.The efficacy and regulatory activity of bactericidal/permeability-increasing protein (BPI) as a mediator of Escherichia coli (E. coli) F18 resistance remains to be defined. In the present study, we evaluated lipopolysaccharide (LPS)-induced changes in BPI gene expression in porcine kidney (PK15) cells in response to E. coli F18 exposure. We additionally generated PK15 cells that overexpressed BPI to assess the impact of this gene on Toll-like receptor 4 (TLR4) signaling and glycosphingolipid biosynthesis-related genes. Through these analyses, we found that BPI expression rose significantly following LPS exposure in response to E. coli F18ac stimulation (p < 0.01). Colony count assays and qPCR analyses revealed that E. coli F18 adherence to PK15 cells was markedly suppressed following BPI overexpression (p < 0.01). BPI overexpression had no significant effect on the mRNA-level expression of genes associated with glycosphingolipid biosynthesis or TLR4 signaling. BPI overexpression suppressed the LPS-induced TLR4 signaling pathway-related expression of proinflammatory cytokines (IFN-α, IFN-β, MIP-1α, MIP-1β and IL-6). Overall, our study serves as an overview of the association between BPI and resistance to E. coli F18 at the cellular level, offering a framework for future investigations of the mechanisms whereby piglets are able to resist E. coli F18 infection.

Effects of protein association on the rates of photoinduced electron transfer from tryptophan residues to excited flavin in medium-chain acyl-Co A dehydrogenase. Molecular dynamics simulation

Rates of photoinduced electron transfer (ET) from tryptophan residues (Trps) to excited isoalloxazine (Iso*) of flavin adenine dinucleotide (FAD) in medium-chain acyl Co A dehydrogenase from porcine kidney (MCAD) have been studied using its entire tetrameric structures obtained by molecular dynamics simulation (MDS) and experimental ultrafast fluorescence decays reported. The ET rates were fastest from Trp166 to Iso* among the four Trps. The emission-wavelength dependent decays were elucidated by introducing emission-wavelength dependent electron affinity of Iso*. The ET rates in Sub C were much slower than those in other subunits, which was ascribed to non-equivalent electrostatic interactions among the subunits. Effect of the protein association on the ET rates and related physical quantities were examined comparing those in the tetramer and monomer reported. The donor-acceptor distances in the tetramer were shorter than those in the monomer. The ET rates from Trp166 in the tetramer were slower than that in the monomer. The ET rates and related physical quantities were examined at seven emission wavelengths. The values of standard free energy gap between the photo-products and reactants displayed to increase as the emission wavelength becomes longer at all subunits. The ET rates displayed to decrease in Sub A, Sub C and Sub D, as the emission wavelength becomes longer.

Ex vivo study of Ho:YAG and thulium fiber lasers for soft tissue surgery: which laser for which case?

The goal of this study was to assess the ablation, coagulation, and carbonization characteristics of the holmium:YAG (Ho:YAG) laser and thulium fiber lasers (TFL). The Ho:YAG laser (100W av.power), the quasi-continuous (QCW) TFL (120W av.power), and the SuperPulsed (SP) TFL (50W av.power) were compared on a non-frozen porcine kidney. To control the cutting speed (2 or 5mm/s), an XY translation stage was used. The Ho:YAG was tested using E = 1.5J and Pav = 40W or Pav = 70W settings. The TFL was tested using E = 1.5J and Pav = 30W or Pav = 60W settings. After ex vivo incision, histological analysis was performed in order to estimate thermal damage. At 40W, the Ho:YAG displayed a shallower cutting at 2 and 5mm/s (1.1 ± 0.2mm and 0.5 ± 0.2mm, respectively) with virtually zero coagulation. While at 70W, the minimal coagulation depth measured 0.1 ± 0.1mm. The incisions demonstrated zero carbonization. Both the QCW and SP TFL did show effective cutting at all speeds (2.1 ± 0.2mm and 1.3 ± 0.2mm, respectively, at 30W) with prominent coagulation (0.6 ± 0.1mm and 0.4 ± 0.1mm, respectively, at 70W) and carbonization. Our study introduced the TFL as a novel efficient alternative for soft tissue surgery to the Ho:YAG laser. The SP TFL offers a Ho:YAG-like incision, while QCW TFL allows for fast, deep, and precise cutting with increased carbonization.

A Novel Preservation Solution Containing Quercetin and Sucrose for Porcine Kidney Transplantation.

Background.In organ transplantation, the University of Wisconsin (UW) solution has been the gold standard for organ preservation. Quercetin (Que) has numerous antioxidant and anti-inflammatory activities, and sucrose (Suc) may be effective for cold storage (CS). This study aimed to investigate the in vitro protective effect of Que and Suc on cold injury to the kidney and to determine whether Que + Suc could improve ischemia-reperfusion injury during CS and hypothermic oxygenated perfusion (HOPE) in autologous transplantation models.Methods.BHK-21 cells were stored at 4°C for 3 days in UW solution for CS/machine perfusion (CS/MP-UW) with Que (33.1 μM, 3.3 μM, 0.33 μM) and Suc (0.1 M). In a porcine model of renal autologous transplantation, left kidney grafts were preserved under 3 conditions: group 1, CS preservation for 24 hours; group 2, CS preservation for 22 hours and HOPE with CS/MP-UW solution for 2 hours; and group 3, identical preservation as group 2, with Que and Suc added to the solution. Animals were euthanized on day 7 after autologous transplantation.Results.After 3 days of CS preservation, the CS/MP-UW solution with Que (33.1 μM, 3.3 μM) and Suc showed significant cell protection against cold injury. In the porcine model of renal autologous transplantation, the last blood Cre level and the blood lipid hydroperoxide on posttransplantation day 2 were significantly different between group 1 and group 3. Moreover, the total endothelial, glomerular, tubular, interstitial (EGTI) histology score in the kidney tissue was also significantly different. Regarding the change in renal resistance in HOPE, the decrease observed in group 3 was significantly larger than that in group 2.Conclusions.Our results suggest that the addition of Que and Suc to a UW solution can improve kidney preservation and could potentially enhance the outcome of kidney transplantation.

Evaluation of the Impact of Ureteroscope, Access Sheath, and Irrigation System Selection on Intrarenal Pressures in a Porcine Kidney Model.

Introduction: Elevated intrarenal pressure (IRP) during flexible ureterorenoscopy (FURS) is a predictor of postoperative complications. The aim of this study is to evaluate IRP during FURS in a porcine kidney model to determine the safest combination of irrigation device, ureteral access sheath (UAS), and ureteroscope. Methods: Urinary tracts were harvested from Landrace pigs slaughtered for the food chain. Two flexible ureteroscopes, 8.7F and 9.5F, were evaluated. Irrigation systems evaluated included the following: TraxerFlow™ (Rocamed, France), SAPS™ single action pumping system (Boston Scientific), Pathfinder Plus™ (Utah Medical), and a manual "bag squeeze." This experiment was conducted with no UAS, followed by an 11/13F UAS and then a 12/14F UAS. IRPs were measured in the prepared porcine kidney during all possible combinations of scope, UAS, and irrigation system. Results: Pressures were significantly reduced when using 12/14F UAS compared with 11/13F UAS (16.45 ± 5.3 cmH2O vs 32.73 ± 35.66 cmH2O, p = 0.006), and when using 11/13F UAS compared with no UAS (32.73 ± 35.66 cmH2O vs 49.5 ± 29.36 cmH2O, p = 0.02). Pressures were significantly reduced with the 8.7F scope compared with the 9.5F scope (24.1 ± 21.24 cmH2O vs 41.68 ± 34.5 cmH2O, p = 0.001). SAPS generates significantly greater IRP than TraxerFlow, Pathfinder Plus, and a "bag squeeze" (p < 0.05). The most dangerous combination was using the SAPS, no UAS, and larger ureteroscope leading to an IRP of 100.6 ± 16.1 cmH2O. The safest combination was using Pathfinder Plus with a 12/14F UAS and smaller ureteroscope giving an IRP of 11.6 ± 3.65 cmH2O. Conclusion: IRPs are reduced by selecting larger UAS and a small ureteroscope. The SAPS generates significantly higher IRPs than other irrigation systems. To maintain safe IRPs during FURS, urologists should use large UAS, narrow ureteroscopes, and be cautious in the selection of an irrigation device.

Reduction of Renal Preservation/Reperfusion Injury by Controlled Hyperthermia During Ex Vivo Machine Perfusion

The possible reno‐protective effect of a controlled brief heat‐shock treatment during isolated ex vivo machine perfusion of donor grafts prior to reperfusion should be investigated in a primary in vitro study. Porcine kidneys (n = 14) were retrieved after 20 minutes of cardiac standstill of the donor and subjected to 20 hours of static cold storage in University of Wisconsin solution. Prior to reperfusion, kidneys were subjected to 2 hours of reconditioning machine perfusion with gradual increase in perfusion temperature up to 35°C. In half of the kidneys (n = 7), a brief hyperthermic impulse (10 minutes perfusion at 42°C) was implemented in the machine perfusion period. Functional recovery of the grafts was observed upon normothermic reperfusion in vitro. Hyperthermic treatment resulted in a 50% increase of heat shock protein (HSP) 70 and HSP 27 mRNA and was accompanied by ~ 50% improvement of tubular re‐absorption of sodium and glucose upon reperfusion, compared with the controls. Furthermore, renal loss of aspartate aminotransferase was significantly reduced to one‐third of the controls as was urinary protein loss, evaluated by the albumin to creatinine ratio. It is concluded that ex vivo heat‐shock treatment seems to be an easily implementable and promising option to enhance renal self‐defense machinery against reperfusion injury after preservation that merits further investigation in preclinical models.

Foot-and-Mouth Disease Virus 3B Protein Interacts with Pattern Recognition Receptor RIG-I to Block RIG-I-Mediated Immune Signaling and Inhibit Host Antiviral Response.

Foot-and-mouth disease is a highly contagious disease of pigs, sheep, goats, bovine, and various wild cloven-hoofed animals caused by foot-and-mouth disease virus (FMDV) that has given rise to significant economic loss to global livestock industry. FMDV 3B protein is an important determinant of virulence of the virus. Modifications in 3B protein of FMDV considerably decrease virus yield. In the current study, we demonstrated the significant role of 3B protein in suppression of type I IFN production and host antiviral response in both human embryonic kidney HEK293T cells and porcine kidney PK-15 cells. We found that 3B protein interacted with the viral RNA sensor RIG-I to block RIG-I-mediated immune signaling. 3B protein did not affect the expression of RIG-I but interacted with RIG-I to block the interaction between RIG-I and the E3 ubiquitin ligase TRIM25, which prevented the TRIM25-mediated, Lys63-linked ubiquitination and activation of RIG-I. This inhibition of RIG-I-mediated immune signaling by 3B protein decreased IFN-β, IFN-stimulated genes, and proinflammatory cytokines expression, which in turn promoted FMDV replication. All of the three nonidentical copies of 3B could inhibit type I IFN production, and the aa 17A in each copy of 3B was involved in suppression of IFN-related antiviral response during FMDV infection in porcine cells. Together, our results indicate the role of 3B in suppression of host innate immune response and reveal a novel antagonistic mechanism of FMDV that is mediated by 3B protein.

Ex vivo delivery of Mirococept: A dose-finding study in pig kidney after showing a low dose is insufficient to reduce delayed graft function in human kidney.

The complement system plays a pivotal role in the pathogenesis of ischemia-reperfusion injury in solid organ transplantation. Mirococept is a potent membrane-localizing complement inhibitor that can be administered ex vivo to the donor kidney prior to transplantation. To evaluate the efficacy of Mirococept in reducing delayed graft function (DGF) in deceased donor renal transplantation, we undertook the efficacy of mirococept (APT070) for preventing ischaemia-reperfusion injury in the kidney allograft (EMPIRIKAL) trial (ISRCTN49958194). A dose range of 5-25mg would be tested, starting with 10mg in cohort 1. No significant difference between Mirococept at 10mg and control was detected; hence the study was stopped to enable a further dose saturation study in a porcine kidney model. The optimal dose of Mirococept in pig kidney was 80mg. This dose did not induce any additional histological damage compared to controls or after a subsequent 3hours of normothermic machine perfusion. The amount of unbound Mirococept postperfusion was found to be within the systemic dose range considered safe in the Phase I trial. The ex vivo administration of Mirococept is a safe and feasible approach to treat DGF in deceased donor kidney transplantation. The porcine kidney study identified an optimal dose of 80mg (equivalent to 120mg in human kidney) that provides a basis for further clinical development.