Porcine Kidney Research Articles

Liquid Nitrogen-Based Cryoablation in In Vivo Porcine Tissue: A Pilot Study.

Introduction:Liquid nitrogen-based cryoablation induces freezing evenly throughout the probe tip surface, resulting in larger ablation volumes and faster treatment times. The purpose of this preliminary investigation is to determine the efficacy of the liquid nitrogen-based Visica2 Cryoablation System (Sanarus Technologies, Pleasanton, CA) in in vivo porcine kidney, liver, and fibro-fatty tissue. Methods:Ablations were performed under ultrasound guidance in 4 Yorkshire pigs. The target lesion cross-section width (W) and depth (D) were 1 cm for liver (n=8), kidney (n=4), and head-neck (n=5) and 2 cm for kidney (n=4). Expected axial length (L) of the resulting lesion is approximately 4 cm. After three-day survival, the ablated tissue was harvested and histologically analysed. The mean width and depth were compared with the target diameter using a one-sample t-test. Results:All animals survived the procedure. For the 1 cm target, mean dimensions (L x W x D) were 3.8±1.5 x 1.7±0.3 x 1.7±0.7 for liver, 3.0±0.5 x 2.0±0.4 x 1.7±0.6 for kidney, and 3.3±0.8 x 1.8±0.4 x 1.8±0.4 for head-neck. Mean width and depth were significantly greater than desired dimension. For the 2 cm target, mean dimensions were 3.2±0.5 x 3.1±0.8 x 1.9±0.7. Mean width and depth were not significantly different to desired target. Conclusion:Our preliminary results show that the Visica2 liquid nitrogen-based cryoablation system can efficiently and reproducibly create ablation volumes in liver, kidney, and fibro-fatty tissue within 4 minutes and 12 minutes for 1cm and 2cm targeted diameters, respectively. Further investigation is necessary to determine the optimal freeze-thaw-freeze protocol for larger ablation volumes.

Determining sensitivity of novel animal-derived cell cultures to clinical isolates of human enterovirus Echovirus 11 and Coxsackievirus B5

The cell culture laboratory of Ekaterinburg Research Institute of Viral Infections established and characterized diploid cell cultures of fetal porcine larynx, lungs, kidneys and muscles for the first time. Sensitivity of the novel animal-derived cell cultures to the priority clinical isolates of human enterovirus, Coxsackievirus B5 and Echovirus 11, was evaluated. Experimental studies with a Coxsackievirus B5 (CB5–8100) strain detected high sensitivity to the virus in the diploid laryngeal and renal cells and absence of such sensitivity in the cells of the lung and muscle tissues. We also documented the total absence of sensitivity to Echovirus 11 (111/RD) in all the cell cultures under study. The results will be used for further studies of animal-derived cell cultures. Certification of the novel fetal porcine renal and laryngeal cells will open up new opportunities for a wide variety of their uses in virology for studying the etiology of enteroviral infections caused by Coxsackievirus B5.

Treating Ischemically Damaged Porcine Kidneys with Human Bone Marrow- and Adipose Tissue-Derived Mesenchymal Stromal Cells During Ex Vivo Normothermic Machine Perfusion.

Pretransplant normothermic machine perfusion (NMP) of donor kidneys offers the unique opportunity to perform active interventions to an isolated renal graft before transplantation. There is increasing evidence that mesenchymal stromal cells (MSCs) could have a paracrine/endocrine regenerative effect on ischemia-reperfusion injury. The purpose of this study was to determine which cytokines are secreted by MSCs during NMP of a porcine kidney. Viable porcine kidneys and autologous whole blood were obtained from a slaughterhouse. Warm ischemia time was standardized at 20 min and subsequent hypothermic machine perfusion was performed during 2-3 h. Thereafter, kidneys were machine perfused at 37°C during 7 h. After 1 h of NMP, 0, 107 cultured human adipose tissue-derived MSCs, or 107 cultured bone marrow-derived MSCs were added (n = 5 per group). In a fourth experimental group, 7-h NMP was performed with 107 adipose tissue-derived MSCs, without a kidney in the circuit. Kidneys perfused with MSCs showed lower lactate dehydrogenase and neutrophil gelatinase-associated lipocalin levels in comparison with the control group. Also, elevated levels of human hepatocyte growth factor, interleukin (IL)-6, and IL-8 were found in the perfusate of the groups perfused with MSCs compared to the control groups. This study suggests that MSCs, in contact with an injured kidney during NMP, could lead to lower levels of injury markers and induce the release of immunomodulatory cytokines.

The effect of prolactin concentration on the efficacy of storing isolated porcine kidneys in a modified Biolasol solution

Abstract Introduction: Biolasol is a solution developed in Poland for flushing the kidneys, liver, heart and pancreas by simple hypothermia method prior to transplantation. The solution supports the cellular integrity of grafts during ischemia-reperfusion injury (IRI). The aim of the study was to evaluate the impact of the concentration of prolactin added to Biolasol on selected biochemical parameters of kidney injury. Materials and methods: Biolasol was modified by the addition of prolactin at 1 μg/L, 10 μg/L and 100 μg/L and by ascorbic acid at 0.5 mmol/L. After 2 h and 48 h of storage, the levels of alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase enzymes, sodium and potassium concentrations, pH and osmolarity parameters were assessed in the perfusates. Results: The addition of prolactin to Biolasol significantly improves the biochemical parameters of grafts in the models of rinsing, perfusion and reperfusion of isolated porcine kidneys. Conclusions: The study indicates the nephroprotective role of Biolasol with the addition of vitamin C and prolactin at a 100 μg/L.

Oral 2 - Comparison of Intrarenal Pressures Generated by Irrigation Devices During Ureterorenoscopy in a Porcine Kidney Model

Oral 2 - Comparison of Intrarenal Pressures Generated by Irrigation Devices During Ureterorenoscopy in a Porcine Kidney Model

SELECTED SEQUENCES OF PORCINE ELONGATION FACTOR 1 ALPHA PROMOTER LEAD TO SIGNIFICANT UPREGULATION OF TARGET GENE RESPONDING TO HUMAN SERUM INDUCTION IN PORCINE CELLS

Transgenic pig has been well known as a model and a potential source of cross-species organ transplantation. Fully and partially virus-originated promoters, CMV and CAG were predominantly used for generation of transgenic pigs. In this study we carry out to develop regulatory sequences originated from pig deriving efficient expression of target gene. We selected sequences according to analysis using AliBaba2.1 program, and synthesized the regulatory sequences equivalently to elongation factor 1 alpha (pEf1α) and actin (pAct) of pig, leading to construction of luciferase expression cassettes. Luciferase assay was showed that pEf1α promoter lead to significantly higher expression than CAG in ear skin fibroblasts (pEfb) and to similar expressions in porcine kidney cells pK15 and endothelial cells (pAec) compared with CAG. The pAct promoter was shown to be inefficient expression of luciferase compared with CAG and pEf1α in pEfb and pK15 cells. To inquire into that pEf1α promoters could spontaneously induce gene expression responding to rejection mechanisms by xenotransplantation, we treated normal human serum to pAec. Consequence was pEf1α promoter lead to significant upregulation of luciferase expression, while the level of endogenous pEf1α expression was not changed. We additionally constructed HLA-E and beta macroglobulin(b2m) expression cassettes in conjunction with pEf1α and CAG promoters, and transfected the cassettes into pEfb. We obtained stable cells by neomycin selection for 14 days, and subsequently analyzed HLA-E expression up to 30 days. We were able to observe that HLA-E expression by pEf1α promoter was consistently sustained for 30 days, but it by CAG promoter was dramatically downregulated from 18 days’ later selection. Taken together, we suggest that selected sequences of pEf1α gene in this study were able to regulate stably spatial and temporal expression of target gene in transgenic pig, and to induce distinct upregulation of the gene on rejection incurred in primate xeno-grafted of the pig. This work was carried out with the support of “Cooperative Research Program for Agriculture Science & Technology Development (Project No PJ01384202)” Rural Development Administration, Republic of Korea.

ASSOCIATION OF HAEMOGLOBIN LEVELS DURING NORMOTHERMIC MACHINE PERFUSION OF PORCINE DONATION AFTER CARDIAC DEATH KIDNEYS WITH RENAL METABOLISM AND FUNCTION

Introduction: The shortage of kidneys for transplantation remains. Therefore, kidneys of suboptimal quality are used more often. These derive from expanded criteria donors (ECD) and donation after circulatory death (DCD) donors. Normothermic machine perfusion (NMP) of these marginal donor kidneys is used to preserve and assess renal function. Although perfusion fluids are all based on red blood cells or alternative oxygen carriers, haemoglobin levels are mostly below 6 mmol/L. It is unknown to which extent oxygen carrying capacity is required to support kidney function during NMP. The aim of this study was to retrospectively evaluate the effect of haemoglobin levels on renal metabolism and function during NMP. Methods: Porcine kidneys obtained from a local slaughterhouse were considered eligible for this retrospective cohort study and were included when subjected to a warm ischemic time (WIT) ranging from 30 to 35 minutes and were subsequently preserved with oxygenated hypothermic machine perfusion (HMP) ranging from 3 to 24 hours. For all kidneys a 4 hours observation period with oxygenated NMP with an oxygen carrier was required. Kidneys were stratified according to haemoglobin concentrations (per 1 mmol/L increment). Variables measured were creatinine clearance, oxygen consumption, and fractional sodium reabsorption rates. Results: In total, 101 (84%) of 120 eligible kidneys were included. Preliminary results show that groups containing a haemoglobin level over 5 mmol/L or more result in higher oxygen consumption rates and increased reabsorption of sodium. Creatinine clearance seems to be improved in these groups as well.Conclusion: In this preliminary study, a minimum haemoglobin level of 5 mmol/l seems to be essential to effectively asses renal metabolism and function. It is unclear if lower haemoglobin levels result in renal injury.

GENOME-WIDE TRANSCRIPTOME ANALYSIS OF EXTREME MARGINAL RENAL GRAFTS INDICATES EARLIER REPAIR AND LESS DAMAGE FOLLOWING NORMOTHERMIC EX-VIVO KIDNEY PERFUSION PRESERVATION

Background: Normothermic ex-vivo kidney perfusion (NEVKP) was shown to improve outcomes of kidneys subjected to prolonged warm ischemia (WI) compared to static cold storage (SCS). To identify mechanisms for this finding, we compared the genomic profile of these grafts following storage to that of unmanipulated-naïve kidneys. Methods: Kidneys from 30kg-Yorkshire pigs were removed following 120min of WI and subjected to 8hrs of SCS or NEVKP. Kidney biopsies were collected on POD3 and gene expression was compared with naïve porcine kidneys utilizing the Affymetrix GeneChip® Porcine Gene 1.0 ST Array. Results: During NEVKP perfusion, 120min WI-grafts demonstrated favourable perfusion characteristics including lactate clearance (0hr:10.48±0.93mmol/L vs 7hr:1.48±0.85mmol/L,p<0.01,n=5), decreasing intra-renal resistance (0hr:2.26±0.9mmHg/mL/min vs 7hr:0.37±0.6mmHg/mL/min,p<0.01,n=5), and urine production. Post-transplantation, 120min WI-grafts with NEVKP, compared to SCS, demonstrated significantly decreased serum creatinine peak values (POD4:12.62±2.34mg/dl vs POD5:18.95±1.11mg/dL,p=0.001) and higher creatinine clearance (POD4:6.61±4.03mL/min vs 0.35±0.30mL/min,p=0.02). Comparison of POD3 transcript expression levels of 120min WI grafts subjected to NEVKP or SCS to naïve kidneys showed significant alterations in 5703 and 4699 genes, respectively (>±2-fold changes in expression over naïve,n=3,p<0.05, FDR<0.05). Genes related to regeneration and inflammation were among the most highly over expressed in SCS grafts with metabolism-associated genes highly downregulated (table 1). In contrast, genes related to regeneration were less elevated following NEVKP (table 2). Conclusions: Grafts damaged by prolonged WI highly over-express genes associated with regeneration. Lower levels of these genes in NEVKP with preserved metabolism-related genes likely correlates to earlier graft repair and/or less damage during storage.

Bestatin and bacitracin inhibit porcine kidney cortex dipeptidyl peptidase IV activity and reduce human melanoma MeWo cell viability

Bestatin and bacitracin are inhibitors of metallo aminopeptidases and bacterial proteases. However, their effects on other human peptidases, like dipeptidyl peptidase IV (DPP-IV, EC 3.4.14.5) are not established. Inhibitors of DPP-IV activity are used for treating type 2 diabetes mellitus, cancers and immune system diseases. Bacitracin and bestatin inhibited porcine membrane-bound DPP-IV (pDPP-IV) activity. Mechanisms were different, i.e. non-competitive with α > 1 (α = 3.9) and Ki value of 75 μM for bestatin, and competitive with Ki value of 630 μM for bacitracin. The binding mode in the tertiary complex enzyme:substrate:bestatin suggested the structural basis of the inhibitory effect and that bestatin is potentially selective for DPP-IV, ineffective vs. S9 family members dipeptidyl peptidase 8/9 and fibroblast activation protein. In the human melanoma MeWo cell line, bestatin and bacitracin inhibited aminopeptidase N (APN) and DPP-IV activities, reduced cell viability and increased DNA fragmentation, suggesting induction of apoptosis. Since bacitracin and bestatin are already marketed drugs, studying in depth the molecular mechanisms underlying their effects on melanoma cells is warranted. Additionally, bestatin emerges as a new lead compound for the development of DPP-IV inhibitors, and a promising dual APN/DPP-IV inhibitor for the treatment of pathologies in which both enzymes are upregulated.

Tick-Borne Encephalitis Virus Adaptation in Different Host Environments and Existence of Quasispecies.

A highly virulent strain (Hypr) of tick-borne encephalitis virus (TBEV) was serially subcultured in the mammalian porcine kidney stable (PS) and Ixodes ricinus tick (IRE/CTVM19) cell lines, producing three viral variants. These variants exhibited distinct plaque sizes and virulence in a mouse model. Comparing the full-genome sequences of all variants, several nucleotide changes were identified in different genomic regions. Furthermore, different sequential variants were revealed to co-exist within one sample as quasispecies. Interestingly, the above-mentioned nucleotide changes found within the whole genome sequences of the new variants were present alongside the nucleotide sequence of the parental strain, which was represented as a minority quasispecies. These observations further imply that TBEV exists as a heterogeneous population that contains virus variants pre-adapted to reproduction in different environments, probably enabling virus survival in ticks and mammals.

Abstract LB-285: Rapid needle biopsy assessment at point of care to advance personalized cancer therapy

Abstract Rationale: Although core needle biopsies (CNBs) are the preferred minimally invasive procedure for breast cancer diagnostics, the standard of care H&amp;E histopathologic analysis of core biopsies is both tissue destructive and labor intensive. Our novel millifluidic instrument, CoreView, is designed to evaluate fresh CNBs to make a real time cancer diagnosis or adequacy determination, conserve tissue for downstream diagnostics and assist in timely treatment planning. Technology and Methods: CoreView is designed to handle, stain, and transport CNBs with the use of millifluidics. The first stage of the CoreView removes biopsy procured tissue from side-cut and end-cut needle-type biopsy guns (14-16 gauge). The reliability of the novel low pressure fluidic process in removing intact CNBs was tested using freshly-excised (ex-vivo) porcine breast (N = 28), kidney (N = 21), liver (N = 42), lung (N = 25), and lymph node (N = 30) tissue. The shear rate, provided by the driving pressure of saline solution, was increased in increments until the CNB was removed and the resulting intactness was qualitatively analyzed by preserved overall shape and size. For optical imaging, a second stage streams fluorescence dye over the CNB for H&amp;E equivalent staining. High resolution imaging with 285 nm excitation is currently performed using microscopy with UV-surface excitation [MUSE, Levenson Group, UC Davis] for anatomical imaging. Previous studies have shown the optical sectioning thickness of MUSE is about 3x thicker than microtome-sectioned specimens. To improve the quality of images produced by MUSE within CoreView, we are limiting UV tissue penetration by steric hindrance of dye molecules using conjugated nanoparticles, such as 525-705 nm CdSe Qdots [ThermoFisher], as well as optical blocking dyes as counterstains. Results: The CNB removal stage was 3D-printed in a patent pending disposable design which is optically clear for monitoring the process. CNBs were fully released and structurally intact for analysis in 93% breast, 100% kidney, 100% liver, 84% lung, and 90% lymph node CNB samples. The average volume per time (µL/ms), an analog to shear rate, for each tissue type was 2.19 (σ;; = 1.04), 1.97 (σ;; =0.71), 1.74 (σ;; = 0.87), 2.05 (σ;; = 0.87), and 2.01 (σ;; = 0.87) for breast, kidney, liver, lung, and lymph node, respectively. The CNB tissue was removed in under 15 seconds, some as quickly as 2 seconds. Furthermore, the device's round channel, which holds a CNB with diameter 10-20% less than the channel, results in inherent structural preservation. Conclusions: The novel millifluidic biopsy removal device is an essential first stage to CoreView's automated biopsy handling and future MUSE imaging and adequacy analysis. The customizable CoreView system design allows optimization for specific CNB needle, tissue, and imaging procedure. We predict an adequacy check at nearly 100% reliability in CNB release and integrity within a few minutes at the point of care. Citation Format: David J. Cooper, Mark E. Fauver, Suzanne M. Dintzis, Eric J. Seibel. Rapid needle biopsy assessment at point of care to advance personalized cancer therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-285.

Therapeutic Potential of Selenium as a Component of Preservation Solutions for Kidney Transplantation

Selenium has strong antioxidant properties and diverse effects on the immune system. The aim of the study was to analyse the protective effect of selenium as a component of a kidney preservation solution on the prevention of ischemia-reperfusion injury of nephrons. The solution was modified by the addition of Se (1 µg/L), prolactin (0.1 µg/L) and Se with prolactin (1 µg/L Se + 0.1 µg/L PRL). The study used a model for storing isolated porcine kidneys in Biolasol® (modified Biolasol®), which minimizes ischemia-reperfusion injury of grafts. The introduction of Se4+ ions at a dose of 1 µg/L into the Biolasol® preservation solution in the form of Na2SeO3 caused an increase in the activity/concentration of the analysed biochemical parameters: aspartate transaminase, alanine transaminase, urea and protein. This suggests an adverse effect of Se4+ on nephron function during ischemia-reperfusion. The best graft protection was obtained by using Biolasol® modified with the addition of selenium (IV) at a dose of 1 µg/L and prolactin at a concentration of 0.1 µg/L. We proposed the mechanism of prolactin action in the metabolic reduction of selenite (SO32−) during ischemia/reperfusion.

Magnetization Transfer Imaging Predicts Porcine Kidney Recovery After Revascularization of Renal Artery Stenosis.

Stenotic kidney (STK) and contralateral kidney magnetization transfer ratios (MTRs; Mt/M0) were measured at 3.0-T magnetic resonance imaging, at offset frequencies of 600 and 1000 Hz, before and 1 month post-PTRA in 7 RVD pigs. Stenotic kidney MTR was correlated to renal perfusion, renal blood flow (RBF), and glomerular filtration rate (GFR), determined using multidetector computed tomography and with ex vivo renal fibrosis (trichrome staining). Untreated RVD (n = 6) and normal pigs (n = 7) served as controls. Renovascular disease induced hypertension and renal dysfunction. Blood pressure and renal perfusion were unchanged post-PTRA, but GFR and RBF increased. Baseline cortical STK-MTR predicted post-PTRA renal perfusion and RBF, and MTR changes associated inversely with changes in perfusion and normalized GFR. Stenotic kidney MTR at 600 Hz showed closer association with renal parameters, but both frequencies predicted post-PTRA cortical fibrosis. Renal STK-MTR, particularly at 600 Hz offset, is sensitive to hemodynamic changes after PTRA in swine RVD and capable of noninvasively predicting post-PTRA kidney perfusion, RBF, and fibrosis. Therefore, STK-MTR may be a valuable tool to predict renal hemodynamic and functional recovery, as well as residual kidney fibrosis after revascularization in RVD.

Increasing metformin concentrations and its excretion in both rat and porcine ex vivo normothermic kidney perfusion model

IntroductionMetformin can accumulate and cause lactic acidosis in patients with renal insufficiency. Metformin is known to inhibit mitochondria, while renal secretion of the drug by proximal tubules indirectly requires energy....

Metformin Preconditioning and Postconditioning to Reduce Ischemia Reperfusion Injury in an Isolated Ex Vivo Rat and Porcine Kidney Normothermic Machine Perfusion Model.

Metformin may act renoprotective prior to kidney transplantation by reducing ischemia‐reperfusion injury (IRI). This study examined whether metformin preconditioning and postconditioning during ex vivo normothermic machine perfusion (NMP) of rat and porcine kidneys affect IRI. In the rat study, saline or 300 mg/kg metformin was administered orally twice on the day before nephrectomy. After 15 minutes of warm ischemia, kidneys were preserved with static cold storage for 24 hours. Thereafter, 90 minutes of NMP was performed with the addition of saline or metformin (30 or 300 mg/L). In the porcine study, after 30 minutes of warm ischemia, kidneys were preserved for 3 hours with oxygenated hypothermic machine perfusion. Subsequently, increasing doses of metformin were added during 4 hours of NMP. Metformin preconditioning of rat kidneys led to decreased injury perfusate biomarkers and reduced proteinuria. Postconditioning of rat kidneys resulted, dose‐dependently, in less tubular cell necrosis and vacuolation. Heat shock protein 70 expression was increased in metformin‐treated porcine kidneys. In all studies, creatinine clearance was not affected. In conclusion, both metformin preconditioning and postconditioning can be done safely and improved rat and porcine kidney quality. Because the effects are minor, it is unknown which strategy might result in improved organ quality after transplantation.

Evaluation of Cell Lines for the Isolation of Foot-and-Mouth Disease Virus and Other Viruses Causing Vesicular Disease.

The most sensitive cell culture system for the isolation of foot-and-mouth disease virus (FMDV) is primary bovine thyroid (BTY) cells. However, BTY cells are seldom used because of the challenges associated with sourcing thyroids from FMDV-negative calves (particularly in FMD endemic countries), and the costs and time required to regularly prepare batches of cells. Two continuous cell lines, a fetal goat tongue cell line (ZZ-R 127) and a fetal porcine kidney cell line (LFBK-αVβ6), have been shown to be highly sensitive to FMDV. Here, we assessed the sensitivity of ZZ-R 127 and LFBK-αVβ6 cells relative to primary BTY cells by titrating a range of FMDV original samples and isolates. Both the ZZ-R 127 and LFBK-αVβ6 cells were susceptible to FMDV for >100 passages, and there were no significant differences in sensitivity relative to primary BTY cells. Notably, the LFBK-αVβ6 cell line was highly sensitive to the O/CATHAY porcine-adapted FMDV strain. These results support the use of ZZ-R 127 and LFBK-αVβ6 as sensitive alternatives to BTY cells for the isolation of FMDV, and highlight the use of LFBK-αVβ6 cells as an additional tool for the isolation of porcinophilic viruses.

Novel Porcine Kidney-Based Microsurgery Training Model for Developing Basic to Advanced Microsurgical Skills.

Microsurgery training is critical to the practice of microvascular procedures in many surgical areas. However, even simple procedures require different levels of complex skills. Therefore, simulation-based surgical training, mainly in the area of vascular anastomosis, is of great importance. In this paper, we present a new microsurgery training model for the development of basic to advanced microsurgical skills. Porcine kidneys were purchased from a legal butchery slaughterhouse. First, kidneys were washed with water to remove blood and clots inside vessels. Then, dissection was performed throughout the vascular pedicle from the renal arteries to the segmentary branches. Finally, the longitudinal sectioning of the kidney parenchyma was performed to expose the vessels necessary for training. Sixty end-to-end anastomoses were performed. Specific instruments and materials were used to perform anastomoses and dissections with magnification by a video system. We evaluated the diameter of vessels, time to perform anastomosis, and patency of anastomosis. There was no great anatomical variation among the porcine kidneys. The total length for dissection training was 25.80 ± 7.44 cm using the arterial and venous vessel. The average time to perform arterial anastomoses was 23.79 ± 4.55 minutes. For vessel diameters of ≤ 3, 4 to 6, and 7 to 10 mm, the average procedure times were 27.68 ± 3.39, 22.92 ± 4.12, and 20.77 ± 3.44 minutes, respectively. Regarding venous anastomosis, the average duration of the procedure was 26.17 ± 4.80 minutes, including durations of 31.61 ± 3.86, 25.66 ± 4.19, and 21.24 ± 3.79 minutes for vessel diameters of ≤ 7, 8 to 10, and >10 mm, respectively. Positive patency was achieved in all surgeries. The porcine kidney provides an inexpensive and convenient biological model for modeling microanastomosis with high fidelity to vascular structures.

Gentamicin-Induced Acute Kidney Injury in an Animal Model Involves Programmed Necrosis of the Collecting Duct.

Gentamicin is a potent aminoglycoside antibiotic that targets gram-negative bacteria, but nephrotoxicity limits its clinical application. The cause of gentamicin-induced AKI has been attributed mainly to apoptosis of the proximal tubule cells. However, blocking apoptosis only partially attenuates gentamicin-induced AKI in animals. Mice treated with gentamicin for 7 days developed AKI, and programmed cell death pathways were examined using pharmacologic inhibitors and in RIPK3-deficient mice. Effects in porcine and murine kidney cell lines were also examined. Gentamicin caused a low level of apoptosis in the proximal tubules and significant ultrastructural alterations consistent with necroptosis, occurring predominantly in the collecting ducts (CDs), including cell and organelle swelling and rupture of the cell membrane. Upregulation of the key necroptotic signaling molecules, mixed lineage kinase domain-like pseudokinase (MLKL) and receptor-interacting serine/threonine-protein kinase 3 (RIPK3), was detected in gentamicin-treated mice and in cultured renal tubule cells. In addition, gentamicin induced apical accumulation of total and phosphorylated MLKL (pMLKL) in CDs in mouse kidney. Inhibiting a necroptotic protein, RIPK1, with necrostatin-1 (Nec-1), attenuated gentamicin-induced necrosis and upregulation of MLKL and RIPK3 in mice and cultured cells. Nec-1 also alleviated kidney inflammation and fibrosis, and significantly improved gentamicin-induced renal dysfunction in mice. Furthermore, deletion of RIPK3 in the Ripk3-/- mice significantly attenuated gentamicin-induced AKI. A previously unrecognized role of programmed necrosis in collecting ducts in gentamicin-induced kidney injury presents a potential new therapeutic strategy to alleviate gentamicin-induced AKI through inhibiting necroptosis.

BIOLOGICAL ACTIVITY OF NICKEL NANOPARTICLES

Objective. Study the biological activity of nickel nanoparticles (Ni NPs) against pathogenic viruses and fungi, pathogens of farm animals and plants, investigate their bactericidal and growthregulating properties. Methods. Virological, microbiological and statistical. Virucidal activity of NiNPs was evaluated by the difference of virus titres in the control and under the action of NPs, bactericidal — by the difference of bacterial titres, fungicidal and fungistatic — by the difference of diameters of fungal colonies, growth-regulating — by the difference in leaf blade length and roots ofgerms of winter wheat. The antiviral activity of NPs was determined in the culture of versenalisedembryonic porcine kidney cells. Bactericidal activity was investigated in a liquid digest medium.Fungistatic activity was determined on wort agar. Wheat germs were germinated in Petri dishesaccording to generally accepted methods. The viral titre was calculated by Reed-Muench method.The bacterial titre was determined by seeding the suspensions on agar medium. Statistical processing was performed in Microsoft Office Excel and statsoft STATISTICA 12, using Student's t-test,post-hoc tests: Fisher’s least significant difference test and Duncan’s new multiple range test.Results. It was found that Ni NPs show high virucidal activity against Teschovirus A strainDniprovskyi-34, reducing the virus titre by 2.46 lg TCD50/cm3, and have a chemotherapeutic indexof 4. Ni NPs have low fungistatic activity against Acremonium cucurbitacearum 502, reducing theaverage diameter of colonies by 6.58 to 20.22 %. Regarding Acremonium strictum 048 and Fusarium sp. 072, Ni NPs have a slight stimulating activity, increasing the average diameter of the colonies by 10.18 to 12.44 % and 14.84 to 22.18 %, respectively. Under the action of Ni NPs, the titre ofBacillus subtilis ІМВ В-7023 and Pseudomonas chlororaphis subsp. aureofaciens UKM B-306(2687) upon the cultivation of bacteria in meat-peptone broth significantly (by 80.96 to 82.77 %and 15.67 to 36.39 %, respectively) increases. Ni NPs exhibits growth-promoting properties onwheat germs, increasing the average length of leaf blades and roots by 41.79 and 36.76 %, respectively. Conclusion. According to the study findings, Ni NPs cannot be recommended for the creation of antifungal preparations. Further research is needed to develop Ni NP-based antiviral preparations, virucidal and disinfecting agents, growth- regulating products for cultivated plants, and toincrease the titre of beneficial soil bacteria.

An orally delivered microbial cocktail for the removal of nitrogenous metabolic waste in animal models of kidney failure.

Patients with kidney failure commonly require dialysis to remove nitrogenous wastes and to reduce burden to the kidney. Here, we show that a bacterial cocktail orally delivered in animals with kidney injury can metabolize blood nitrogenous waste products before they diffuse through the intestinal mucosal barrier. The microbial cocktail consists of three strains of bacteria isolated from faecal microbiota that metabolize urea and creatinine into amino acids, and is encapsulated in calcium alginate microspheres coated with a polydopamine layer that is selectively permeable to small-molecule nitrogenous wastes. In murine models of acute kidney injury and chronic kidney failure, and in porcine kidney failure models, the encapsulated microbial cocktail significantly reduced urea and creatinine concentrations in blood, and did not lead to any adverse effects.