Pig Kidney Research Articles

Spatiotemporal immune atlas of a clinical-grade gene-edited pig-to-human kidney xenotransplant

Pig-to-human xenotransplantation is rapidly approaching the clinical arena; however, it is unclear which immunomodulatory regimens will effectively control human immune responses to pig xenografts. Here, we transplant a gene-edited pig kidney into a brain-dead human recipient on pharmacologic immunosuppression and study the human immune response to the xenograft using spatial transcriptomics and single-cell RNA sequencing. Human immune cells are uncommon in the porcine kidney cortex early after xenotransplantation and consist of primarily myeloid cells. Both the porcine resident macrophages and human infiltrating macrophages express genes consistent with an alternatively activated, anti-inflammatory phenotype. No significant infiltration of human B or T cells into the porcine kidney xenograft is detectable. Altogether, these findings provide proof of concept that conventional pharmacologic immunosuppression may be able to restrict infiltration of human immune cells into the xenograft early after compatible pig-to-human kidney xenotransplantation.

The importance of public engagement in clinical xenotransplantation

AbstractOver the past several decades, significant scientific progress in xenotransplantation has brought the field to the threshold of clinical trials. In the past 3 years in the United States, experimental pig kidney and heart xenotransplantation have been performed on human subjects recently declared dead by neurological criteria (decedents). In addition, two pig heart transplants have been carried out in living patients under the United States Food and Drug Administration's expanded access guidelines. However, though there has been a flurry of activity there remain unanswered questions regarding how the public views xenotransplantation, what concerns may exist, and how to address these concerns in a meaningful way. This paper aims to underscore the importance of public engagement in xenotransplantation, emphasizing the ongoing need for studies to assess public opinions. The current evidence on public engagement studies is reviewed and gaps in our understanding are identified. We propose practical steps to advance this field. Additional studies to determine the extent of racial/ethnic differences in attitudes to xenotransplantation should be conducted. Empirical and descriptive analysis of certain religious viewpoints—especially minority faiths—would be valuable. As public engagement is an important aspect of public acceptance of novel research that is accompanied by risk, we suggest that xenotransplantation biotechnology companies might consider leading the way in funding this research.

Neural radiance fields-based multi-view endoscopic scene reconstruction for surgical simulation.

In virtual surgery, the appearance of 3D models constructed from CT images lacks realism, leading to potential misunderstandings among residents. Therefore, it is crucial to reconstruct realistic endoscopic scene using multi-view images captured by an endoscope. We propose an Endoscope-NeRF network for implicit radiance fields reconstruction of endoscopic scene under non-fixed light source, and synthesize novel views using volume rendering. Endoscope-NeRF network with multiple MLP networks and a ray transformer network represents endoscopic scene as implicit field function with color and volume density at continuous 5D vectors (3D position and 2D direction). The final synthesized image is obtained by aggregating all sampling points on each ray of the target camera using volume rendering. Our method considers the effect of distance from the light source to the sampling point on the scene radiance. Our network is validated on the lung, liver, kidney and heart of pig collected by our device. The results show that the novel views of endoscopic scene synthesized by our method outperform existing methods (NeRF and IBRNet) in terms of PSNR, SSIM, and LPIPS metrics. Our network can effectively learn a radiance field function with generalization ability. Fine-tuning the pre-trained model on a new endoscopic scene to further optimize the neural radiance fields of the scene, which can provide more realistic, high-resolution rendered images for surgical simulation.

Human ST3Gal II and ST6GalNAc IV genes increase human serum-mediated cytotoxicity to xenogeneic cells.

Carbohydrate-antigens widely existed on glycoproteins and glycosphingolipids of all mammalian cells play a crucial role in self-defense and immunity. Xeno-reactive antibodies included in natural human sera play a protecting role in an acute phase-rejection of xenotransplantation. In this study, we investigated the effect of an alteration of glycosylation-pattern, caused by human sialyltransferases such as hST3Gal II or hST6GalNAc IV, on human serum mediated cytotoxicity in pig kidney PK15 cells. From LDH cytotoxicity assay, cytotoxicity to human serum was significantly increased in hST3Gal II and hST6GalNAc IV-transfected PK15 cells, as compared to the control. In the hST6Gal I-carrying cells, the cytotoxicity to human serum was rather decreased. Moreover, flow cytometry analysis revealed that an alteration of pig glycosylation-pattern by hST3Gal II or hST6GalNAc IV influences on a binding of human IgM or IgG, respectively, in pig kidney cells, regardless of Gal antigen alteration. Finally, we found that hST6GalNAc IV contributed to increase of terminal disialylated tetrasaccharide structure, disialyl T antigen, as evidenced by increase of the MAL II lectin binding capacity in the hST6GalNAc IV-transfected PK15 cells, compared with control. Therefore, our results suggest that carbohydrate antigens, such as disialyl T antigen, newly synthesized by the ST3Gal II- and ST6GalNAc IV are potentially believed to be new xeno-reactive elements.

Human PD-L1 overexpression decreases xenogeneic human T-cell immune responses towards porcine kidneys.

Xenotransplantation offers a promising alternative to circumvent the lack of donated human organs available for transplantation. Different attempts to improve the survival of xenografts led to the generation of transgenic pigs expressing various combinations of human protective genes or knocked out for specific antigens. Currently, testing the efficiency of porcine organs carrying different genetic modifications in preventing xenogeneic immune responses completely relies on in vitro assays, humanized mouse models, or non-human primate transplantation models. However, these tests are often associated with major concerns due to reproducibility and generation of insufficient data as well as they raise ethical, logistical, and economic issues. In this study, we investigated the feasibility of specifically assessing the strength of human T-cell responses towards the kidneys of wild-type (WT) or transgenic pigs overexpressing human programmed death-1 ligand 1 (hPD-L1) during ex vivo kidney perfusion (EVKP). Human T cells were shown to adhere to the endothelium and transmigrate into WT and hPD-L1 kidneys. However, transcript levels of TNF-a and IFN-y as well as cytotoxic molecules such as granzyme B and perforin secreted by human T cells were significantly decreased in the tissue of hPD-L1 kidneys in comparison to WT kidneys. These results were confirmed via in vitro assays using renal endothelial cells (ECs) isolated from WT and hPD-L1 transgenic pigs. Both CD4+ and CD8+ T cells showed significantly lower proliferation rates after exposure to hPD-L1 porcine renal ECs in comparison to WT ECs. In addition, the secretion of pro-inflammatory cytokines was significantly reduced in cultures using hPD-L1 ECs in comparison to WT ECs. Remarkably, hPD-L1 EC survival was significantly increased in cytotoxic assays. This study demonstrates the feasibility of evaluating the human response of specific immune subsets such as human T cells towards the whole xenograft during EVKP. This may represent a robust strategy to assess the potency of different genetic modifications to prevent xenogeneic immune responses and thereby predict the risk of immune rejection of new genetically engineered xenografts.

Porcine endogenous retroviruses in xenotransplantation.

Xenotransplantation using pig cells, tissues or organs is under development to alleviate the shortage of human donor organs. Meanwhile remarkably long survival times of pig organs in non-human primates were reported as well as the functionality of pig kidneys and hearts in brain-dead humans. Most importantly, two transplantations of pig hearts in patients were performed with survival times of the patients of 8 and 6 weeks. Xenotransplantation may be associated with the transmission of porcine microorganisms including viruses to the recipient. Porcine endogenous retroviruses (PERVs) are integrated in the genome of all pigs and cannot be eliminated like other viruses can. PERVs are able to infect certain human cells and pose therefore a risk for xenotransplantation. It is well known that retroviruses are able to induce tumors and immunodeficiencies. However, until now, PERV was not transmitted in all infection experiments using small animals and non-human primates, in all preclinical xenotransplantation trials in non-human primates and in all clinical trials in humans. In addition, several strategies including antiretrovirals, PERV-specific siRNA, vaccines and genome editing using CRISPR/Cas have been developed to prevent PERV transmission.

Plastinate Library: A Tool to Support Veterinary Anatomy Learning.

The shortage of both time for anatomy courses in the new veterinary medicine curriculum and instructors prepared to teach biomedical sciences has raised a crisis in anatomical education. Often, students spend time out of their classes trying to learn not only concepts but also laboratory-wise content from 2D materials such as books and videos. In addition, since the global COVID-19 pandemic lockdown, studying and habits have been reviewed, with many people adopting an at-home style. The purpose of this study was to evaluate students' acceptance of taking plastinate anatomical specimens to study at home. Thirty-three students were divided into three groups. G1 took home a set of kidneys composed of equine, bovine, and swine materials; G2 took home a pig kidney; and G3 (control) did not take any plastinate specimen home. Acceptance was assessed using an anonymous survey and interview. The method had high acceptance by the students, who believed that having the plastinate library was advantageous from different perspectives, including aiding with learning the differences between kidneys from different species, time flexibility, no commute to study after hours of laboratory classes, and time redistribution to prioritize the laboratory topics. The plastinate library has the potential to be a supportive tool for anatomy students in the contemporaneous veterinary curriculum paradigm, considering that the G1 and G2 groups used the plastinate specimens at home to complement the time they spent in the laboratory.

A Comparison Between GalT-/-;hCD39;hCD55 and GalT-/-;hCD39;hCD46;hCD55;TBM Pig Kidneys Transplanted in Nonhuman Primates.

Because there is a shortage of donor kidneys, researchers are exploring the possibility of using genetically modified pig kidneys for transplantation. Approaches involving knockout of carbohydrate genes or knockin of protective proteins have been attempted to determine the best gene modifications. In this study, we utilized GalT-/-;hCD39;hCD55 and GalT-/-;hCD39;hCD46;hCD55;thrombomodulin (TBM) pigs for transplantation in nonhuman primates (NHPs). The NHPs survived for 4 weeks after kidney transplantation (4 WAT) from the GalT-/-;hCD39;hCD55 pig and for 6 WAT from the GalT-/-;hCD39;hCD46;hCD55;TBM pig. However, messenger RNA (mRNA) sequencing and immunohistochemistry analysis revealed that the 6 WAT kidney exhibited more severe apoptosis, inflammation, loss of renal function, and renal fibrosis than the 4 WAT kidney. These results indicate that additional knockin of complement regulator (hCD46) and coagulation regulator (TBM) is not enough to prevent renal damage, suggesting that improved immune suppression is needed for more prolonged survival.

A study of knowledge and acceptance of kidney xenotransplantation among Chinese kidney transplant recipients and candidates.

In recent years, the implementation of the first case of pig-to-human heart xenotransplantation and the report of three cases of pig-to-brain-dead human recipient kidney transplantation indicate that xenotransplantation is getting closer to clinical application. In the near future, China may also launch clinical trials of kidney xenotransplantation. Therefore, it is necessary to investigate the level of knowledge and acceptance of xenotransplantation among kidney transplant recipients and candidates in China. This study aims to investigate the level of comprehension and acceptance of kidney xenotransplantation in kidney transplant recipients and explore related factors, providing a reference for promoting the application and clinical trials of xenotransplantation in the near future. A questionnaire was completed by 211 kidney transplant recipients and 21 candidates. Answers to the questionnaires were self-administered by the respondents. Scores were compared using nonparametric tests, as well as using Chi-square test or Fisher's exact test to compare differences in answers. Respondents demonstrated a high score of 75 (out of 100) on knowledge and acceptance of kidney xenotransplantation. The sector "Knowledge and Attitude" received the overall highest score from respondents (85.0 out of 100), while "Risks and Concerns" received the lowest score (50 out of 100). Interestingly, respondents paid more attention to infection risks but showed less concern about rejection or unknown risks. Furthermore, 191 respondents (82.3%) expected that pig kidney xenografts could function for at least 5 years or more. The scores were statistically significant in terms of age, gender, level of education, level of knowledge on the case of xenotransplantation clinical trial, and willingness to donate organs. cognition CONCLUSIONS: The awareness rates of xenotransplantation are high among kidney transplant recipients and candidates, for which the majority showed a generally favorable attitude towards this procedure. Respondents did not have a comprehensive understanding of the specific knowledge of xenotransplantation and expressed more concern about the risk of infection compared to the risk of rejection and other unknown risks, while also expecting long-term survival similar to allograft transplantation for pig kidney xenografts.

Pigs in Transplantation Research and Their Potential as Sources of Organs in Clinical Xenotransplantation.

The pig has long been used as a research animal and has now gained importance as a potential source of organs for clinical xenotransplantation. When an organ from a wild-type (i.e., genetically unmodified) pig is transplanted into an immunosuppressed nonhuman primate, a vigorous host immune response causes hyperacute rejection (within minutes or hours). This response has been largely overcome by 1) extensive gene editing of the organ-source pig and 2) the administration to the recipient of novel immunosuppressive therapy based on blockade of the CD40/CD154 T cell costimulation pathway. Gene editing has consisted of 1) deletion of expression of the 3 known carbohydrate xenoantigens against which humans have natural (preformed) antibodies and 2) the introduction of human 'protective' genes. The combination of gene editing and novel immunosuppressive therapy has extended life-supporting pig kidney graft survival to greater than 1 y and of pig heart survival to up to 9 mo. This review briefly describes the techniques of gene editing, the potential risks of transfer of porcine endogenous retroviruses with the organ, and the need for breeding and housing of donor pigs under biosecure conditions.

Enhanced grip force estimation in robotic surgery: A sparrow search algorithm-optimized backpropagation neural network approach.

The absence of an effective gripping force feedback mechanism in minimally invasive surgical robot systems impedes physicians' ability to accurately perceive the force between surgical instruments and human tissues during surgery, thereby increasing surgical risks. To address the challenge of integrating force sensors on minimally invasive surgical tools in existing systems, a clamping force prediction method based on mechanical clamp blade motion parameters is proposed. The interrelation between clamping force, displacement, compression speed, and the contact area of the clamp blade indenter was analyzed through compression experiments conducted on isolated pig kidney tissue. Subsequently, a prediction model was developed using a backpropagation (BP) neural network optimized by the Sparrow Search Algorithm (SSA). This model enables real-time prediction of clamping force, facilitating more accurate estimation of forces between instruments and tissues during surgery. The results indicate that the SSA-optimized model outperforms traditional BP networks and genetic algorithm-optimized (GA) BP models in terms of both accuracy and convergence speed. This study not only provides technical support for enhancing surgical safety and efficiency, but also offers a novel research direction for the design of force feedback systems in minimally invasive surgical robots in the future.

Dual-emissive Eu(III)-functionalized metal-organic frameworks for visual, rapid, and intelligent sensing of albendazole and albendazole sulfoxide in animal-origin food

Albendazole (ABZ), a benzimidazole-based anthelmintic, is widely used to treat helminth infections. The extensive and improper use of ABZ may cause drug residues in animal-origin food and anthelmintics resistance, which potentially threaten human health. Meanwhile, albendazole sulfoxide (ABZSO), a metabolite of ABZ, also exhibits toxic effects. Therefore, the detection of ABZ and ABZSO in animal-derived food is significantly necessary. Herein, a dual-emission europium fluorescent sensor (EuUHC-30) was rationally designed and constructed. EuUHC-30 exhibits high selectivity and sensitivity towards ABZ and ABZSO with a detection limit of 0.10 and 0.13 μM, respectively. Furthermore, EuUHC-30 was successfully applied for quantification of ABZ and ABZSO in milk and pig kidney, which were verified by HPLC analysis. Moreover, a smartphone-assisted EuUHC-30 fluorescent paper sensor was fabricated for the practical determination of ABZ and ABZSO in real food. Overall, this work provides a visual, rapid, and intelligent method for the detection of ABZ and ABZSO in animal-origin food.

Xenotransplantation experiments in brain-dead human subjects–A critical appraisal

Brain-dead human subjects (decedents) were recently introduced as a potential preclinical experimental model in xenotransplantation. Brain death is associated with major pathophysiological changes, eg, structural injury and cell infiltration in vital organs, and major hormonal, metabolic, inflammatory, and hemodynamic changes. In 2 of the 3 initial experiments, the design of the experiments resulted in little or no new information becoming available. In the third, the experiment was unfortunately unsuccessful as neither of the 2 pig kidneys transplanted into the decedent functioned adequately. Failure may well have been associated with the effects of brain death, but an immune/inflammatory response to the xenograft could not be excluded. Subsequently, 2 further pig kidney transplants and 2 pig heart transplants have been carried out in human decedents, but again the data obtained do not add much to what is already known. In view of the profound changes that take place during and after brain death, it may prove difficult to determine whether graft failure or dysfunction results from the effects of brain death or from an immune/inflammatory response to the xenograft. A major concern is that, if the results are confusing, they may impact decisions relating to the introduction of clinical xenotransplantation.

A highly efficient liquid chromatography–tandem mass spectrometry (LC–MS/MS) assay for etomidate and etomidate acid in urine, liver and kidney

Etomidate (ETO) is a highly-efficient drug that can induce anesthesia with increasing doses, thus subject to strict regulation. However, an accurate and efficient method for ETO intake detection is currently lacking. Therefore, this study developed a straightforward sample preparation method using LC-MS/MS to analyze ETO and its primary metabolite, etomidate acid (ETA), in urine, liver, and kidney samples. Snap frozen pig liver and kidney samples were ground into a fine powder. Then, all the biological samples, including human urine, pig liver and kidney tissues, were deproteinized using acetonitrile and filtered for analysis. The separation was achieved in 9.01 min with gradient elution. The calibration curves ranged from 0.5 to 50 ng/mL for ETO in urine and 0.5 to 50 ng/g in liver and kidney, while the curves ranged from 1 to 100 ng/mL for ETA in urine and 1 to 100 ng/g in liver and kidney. The correlation coefficients (R2) were greater than 0.9957. The Limit of detection (LOD) and limit of quantitation (LOQ) for ETO were 0.2 and 0.5 ng/mL in urine samples and 0.2 and 0.5 ng/g in liver and kidney samples, respectively. For ETA, the LOD and LOQ were 0.5 and 1 ng/mL in urine samples and 0.5 and 1 ng/g in liver and kidney samples. This method was assessed by validation parameters, including selectivity, intra- and inter-day precision and accuracy, recovery, matrix effect, dilution integrity and stability. It was successfully applied to a practical case, revealing ETO and ETA concentrations in urine of 1.01 and 5.58 μg/mL, in liver samples of 12.30 and 1.13 μg/g, and in kidney samples of 6.95 and 4.23 μg/g. This suggests that the method is suitable for routine forensic detection of illicit ETO abuse.

In vitro characterization, evaluation of enzymatic activity and capacity of intestinal absorption of daogest (Dao extract from pig kidney)

In vitro characterization, evaluation of enzymatic activity and capacity of intestinal absorption of daogest (Dao extract from pig kidney)

Transcriptomic analysis of the coexisting nonhuman primate kidney with a transgenic pig kidney

Transcriptomic analysis of the coexisting nonhuman primate kidney with a transgenic pig kidney

Preliminary investigation of organ-protective effects of fullerenol in vivo in acute toxicity during doxorubicin therapy in healthy pigs

Abstract Objectives The potential protective effect of fullerenol [C60(OH)24, Frl], a strong antioxidant, was investigated at a single dose of 25 mg/kg, administered orally (per os; p.o.) and intraperitoneally (i.p.), in the liver, lung, kidney, heart and spleen of healthy pigs, prior to doxorubicin (Dox)-induced toxicity at a single i.p. dose of 10 mg/kg. Methods We used the F1 generation of an in-vivo pig model (whose parents were Swedish Landrace and Large Yorkshire) to explore whether Frl, administered 6 h for p.o. and 30 min for i.p. prior to Dox treatment, could protect organs against damage caused by oxidative stress. Key findings According to the macroscopic, haematological, biochemical and physiological results, Frl exerted a potential protective effect on all investigated organs (heart, liver, lung, spleen and kidney) in pigs after i.p. administration. However, the results from Frl p.o. administration were inconclusive, therefore warranting further investigation. Conclusions Frl at a low dose of 25 mg/kg demonstrated satisfactory organ protection against Dox-induced toxicity in healthy pigs after i.p. injection, However, additional follow-up studies in pig models of cancer and with longer Dox and Frl treatment periods would be required to further delineate the optimal and clinically relevant conditions for Frl administration.

Sodium Hydrosulfide Treatment During Porcine Kidney Ex Vivo Perfusion and Transplantation.

In rodents, hydrogen sulfide (H2S) reduces ischemia-reperfusion injury and improves renal graft function after transplantation. Here, we hypothesized that the benefits of H2S are conserved in pigs, a more clinically relevant model. Adult porcine kidneys retrieved immediately or after 60 min of warm ischemia (WI) were exposed to 100 µM sodium hydrosulfide (NaHS) (1) during the hypothermic ex vivo perfusion only, (2) during WI only, and (3) during both WI and ex vivo perfusion. Kidney perfusion was evaluated with dynamic contrast-enhanced MRI. MRI spectroscopy was further employed to assess energy metabolites including ATP. Renal biopsies were collected at various time points for histopathological analysis. Perfusion for 4 h pig kidneys with Belzer MPS UW + NaHS resulted in similar renal perfusion and ATP levels than perfusion with UW alone. Similarly, no difference was observed when NaHS was administered in the renal artery before ischemia. After autotransplantation, no improvement in histologic lesions or cortical/medullary kidney perfusion was observed upon H2S administration. In addition, AMP and ATP levels were identical in both groups. In conclusion, treatment of porcine kidney grafts using NaHS did not result in a significant reduction of ischemia-reperfusion injury or improvement of kidney metabolism. Future studies will need to define the benefits of H2S in human, possibly using other molecules as H2S donors.

Ischemia-Induced Metabolic Patterns Associate With Kidney Function During Normothermic Kidney Perfusion, a Preclinical Study.

To investigate if ischemia alters donor kidney metabolism and whether these changes associate with organ function. An unmet need in kidney transplantation is the ability to predict post-transplant organ function before transplantation. Key to such viability testing is a profound understanding of the organ's complex biochemistry and how ischemia, inevitable during the transplantation process, influences this. First, metabolic changes in glucose, lactate and 20 amino acids induced by no, 1h of warm, or 22h of cold ischemia were investigated during 4h perfusion of pig kidneys with autologous whole blood (n=6/group), simulating the ischemia-reperfusion phase of transplantation. Next, we confirmed similar metabolic changes during normothermic preservation of pig (n=3/group; n=4 for cold ischemia) and discarded human kidneys (n=6) perfused with a red-blood cell based perfusate. At 2h of perfusion with autologous whole blood, abundances of 17/20 amino acids were significantly different between groups, reflecting the type of ischemia. Amino acid changes at 15 min and 2h of perfusion correlated with future kidney function during perfusion. Similar metabolic patterns were observed during perfusion preservation of pig and discarded human donor kidneys, suggesting an opportunity to assess kidney viability before transplantation. Perfusate metabolite changes during normothermic kidney perfusion represent a unique non-invasive opportunity to assess graft viability. These findings now need validation in transplant studies.

Sustained Pituitary T3 Production Explains the T4-mediated TSH Feedback Mechanism.

The regulation of thyroid activity and thyroid hormone (TH) secretion is based on feedback mechanisms that involve the anterior pituitary TSH and medial basal hypothalamus TSH-releasing hormone. Plasma T3 levels can be "sensed" directly by the anterior pituitary and medial basal hypothalamus; plasma T4 levels require local conversion of T4 to T3, which is mediated by the type 2 deiodinase (D2). To study D2-mediated T4 to T3 conversion and T3 production in the anterior pituitary gland, we used mouse pituitary explants incubated with 125I-T4 for 48 hours to measure T3 production at different concentrations of free T4. The results were compared with cultures of D1- or D2-expressing cells, as well as freshly isolated mouse tissue. These studies revealed a unique regulation of the D2 pathway in the anterior pituitary gland, distinct from that observed in nonpituitary tissues. In the anterior pituitary, increasing T4 levels reduced D2 activity slightly but caused a direct increase in T3 production. However, the same changes in T4 levels decreased T3 production in human HSkM cells and murine C2C12 cells (both skeletal muscle) and mouse bone marrow tissue, which reached zero at 50 pM free T4. In contrast, the increase in T4 levels caused the pig kidney LLC-PK1 cells and kidney fragments to proportionally increase T3 production. These findings have important implications for both physiology and clinical practice because they clarify the mechanism by which fluctuations in plasma T4 levels are transduced in the anterior pituitary gland to mediate the TSH feedback mechanism.