Tacrolimus Treatment Research Articles

B-222 Receptor-donor CYP3A5 and CYP3A4 genotype testing in liver transplant patients and its influence in tacrolimus blood levels: a retrospective study

Abstract Background Optimal immunosupression is crucial for transplant success. In orthohepatic transplants, blood target levels of tacrolimus, when coadministred with corticoids and mycophenolic acid, are between 6 and 10 ng/mL during the first three months post-transplant. Notwithstanding, achieving tacrolimus concentrations into this range may be tough because of factors such as liver function status, age, sex, drug-drug interactions, etc. In this way, pharmacogenetic testing of CYP3A5 and CYP3A4 genes have demonstrated to be useful in tacrolimus dosing optimization. Nonetheless, there is no evidence enough for pharmacogenetic based adjusting in liver transplant, specially if receptor and graft’s genotypes differ in these loci. The aim of the present study is to find out how the receptor and donor genotypes in CYP3A5 and CYP3A4 genes affect tacrolimus pharmacokinetics. Methods Genotypic profiling of 108 receptor-donor's pairs biopsies gathered from patients who were underwent hepatic transplant from 2012 to 2019 were carried out using Taqman® OpenArray™ PGx Express 120 panel (ThermoFisher™). We focused on CYP3A5*3, *6 and *7; and CYP3A4*1B, *3 and *22 alleles. Next to pharmacogenetic testing, each patient was classified as very poor, poor, intermediate, rapid or ultrarapid metabolizer according to their own genotype, graft’s genotype or both of them. Patient’s initial dose, at seven days after treatment start and after discharge of tacrolimus and their trough concentrations (C0) were collected from their electronic health records. Tacrolimus levels were measured by affinity chrome-mediated immunoassay. C0 was normalized by weight-adjusted dosage (C/D ratio); then, median C/D ratios between different metabolic profiles were compared through Kruskal-Wallis test. Results When patients were classified based on donor’s genotype isolated or combined with receptor’s genotype, lower C/D ratios were found as metabolizer ability increases (p=0.045 for donors and p=0.027 for combined genotypes, respectively). No statistically differences were found for C/D ratios at seven days and discharge, though that differences were almost significant for donor’s genotypes (p=0.06 in both cases). Conclusions These results show that previous information about both patient’s and specially donor’s CYP genes genotype may improve initial immunosupression and avoid tacrolimus induced toxicity. Pharmacogenetics has been currently proposed as a guide for tacrolimus treatment of some solid organ transplants as kidney transplanted patients. Better understanding of how receptor and donor’s CYP genotype affects its pharmacokinetics, along with liver’s damage markers and other genetic polymorphisms will allow clinicians, in the era of personalized medicine, to improve immunosupressive management.

Comparison of cyclosporine and tacrolimus after liver transplantation for primary biliary cholangitis: A propensity score–matched intention-to-treat registry study

The optimal calcineurin inhibitor after liver transplantation (LT) for primary biliary cholangitis (PBC) remains debated. We compared tacrolimus with cyclosporine in a propensity score–matched intention-to-treat analysis from the Scientific Registry of Transplant Recipients. We included adults with PBC who underwent primary LT from 1995 to 2022. Patients with initial cyclosporine treatment were 1:3 matched with those with initial tacrolimus treatment, ensuring exact calendar-period match. Primary outcomes were patient and graft survival. After matching, 579 patients with PBC and initial cyclosporine and 1348 with tacrolimus were well balanced for baseline characteristics. During a median follow-up of 11.1 years, 1044 (54%) deaths and 124 (6%) re-LTs occurred. In the overall matched sample, no significant survival difference emerged between cyclosporine and tacrolimus. However, tacrolimus conferred a survival advantage in some secondary analysis, such as LT after year 2000 and women, and in a 6-month landmark analysis excluding early postoperative events and calcineurin inhibitor switches. Cyclosporine did not reduce graft loss from PBC recurrence or affect laboratory markers of recurrence. In conclusion, we found no benefit of starting immunosuppression with cyclosporine after LT for PBC.

Pathogenic Th17 cells are a potential therapeutic target for tacrolimus in AChR-myasthenia gravis patients

In our study, we investigated the impact of tacrolimus (TAC) on CD4+ T cell subsets in 41 AChR-MG patients over 12 weeks. Twenty-seven patients were classified as the response group (RG) (improved myasthenia gravis composite scores ≥3), while 14 were non-response. We found that TAC treatment significantly reduced Th17 and pathogenic Th17 cells, along with IL-17 levels in RG, while Th1 and Tfh cells slightly decreased without affecting Th2 or Treg subsets. This indicates that TAC's clinical benefits may be due to its inhibitory effect on the Th17 response, enhancing our insight into its immunomodulatory mechanisms in MG management.

Evaluation of Clobetasol and Tacrolimus Treatments in an Imiquimod-Induced Psoriasis Rat Model.

Psoriasis is a chronic inflammatory skin disorder characterized by keratinocyte hyperproliferation, inflammation, and aberrant differentiation. Imiquimod-induced psoriasis in rodent models has been widely used to study the pathogenesis of the disease and evaluate potential therapeutic interventions. In this study, we investigated the efficacy of two commonly used treatments, Clobetasol and Tacrolimus, in ameliorating psoriatic symptoms in an Imiquimod-induced psoriasis Wistar rat model. Interestingly, rat models are poorly evaluated in the literature despite rats displaying several advantages in evaluating pharmacological substances. Psoriasis-like skin lesions were induced by topical application of Imiquimod cream on shaved dorsal skin for seven consecutive days. Following induction, rats in the treatment groups received either a Clobetasol or Tacrolimus ointment once daily for one week, while the control group did not receive any application. Disease severity was assessed using clinical scoring, histological examination, and measurement of proinflammatory cytokine levels. Both Clobetasol and Tacrolimus treatments significantly reduced psoriatic lesion severity compared to the control group. Clinical scoring revealed a decrease in erythema, scaling, transepidermal water loss, and thickness of skin lesions in both treatment groups with a more marked effect with Clobetasol. Histological analysis demonstrated reduced epidermal hyperplasia in treated animals compared to controls. Furthermore, Clobetasol led to a significant reduction in the expression levels of the interleukin-17 (IL-17a and IL-17f) proinflammatory cytokines in lesioned skin. Overall, our findings demonstrated the therapeutic efficacy of both Clobetasol and, in a modest manner, Tacrolimus in attenuating Imiquimod-induced psoriasis-like symptoms in a rat model. These results support the clinical use of these agents in the management of psoriasis and mitigating psoriatic inflammation. They also provide insights into the use of rats as a relevant species for the Imiquimod-induced psoriasis model.

Tacrolimus Treatment Improved Pregnancy Rate with Euploid Blastocyst in Women with Repeated Implantation Failures and Elevated T Helper 1/2 Cell Ratios

Tacrolimus Treatment Improved Pregnancy Rate with Euploid Blastocyst in Women with Repeated Implantation Failures and Elevated T Helper 1/2 Cell Ratios

Optimization of immunosuppression strategies for the establishment of chronic hepatitis E virus infection in rabbits.

Chronic hepatitis E mostly occurs in organ transplant recipients and can lead to rapid liver fibrosis and cirrhosis. Previous studies found that the development of chronic hepatitis E virus (HEV) infection is linked to the type of immunosuppressant used. Animal models are crucial for the study of pathogenesis of chronic hepatitis E. We previously established a stable chronic HEV infection rabbit model using cyclosporine A (CsA), a calcineurin inhibitor (CNI)-based immunosuppressant. However, the immunosuppression strategy and timing may be optimized, and how different types of immunosuppressants affect the establishment of chronic HEV infection in this model is still unknown. Here, we showed that chronic HEV infection can be established in 100% of rabbits when CsA treatment was started at HEV challenge or even 4 weeks after. Tacrolimus or prednisolone treatment alone also contributed to chronic HEV infection, resulting in 100% and 77.8% chronicity rates, respectively, while mycophenolate mofetil (MMF) only led to a 28.6% chronicity rate. Chronic HEV infection was accompanied with a persistent activation of innate immune response evidenced by transcriptome analysis. The suppressed adaptive immune response evidenced by low expression of genes related to cytotoxicity (like perforin and FasL) and low anti-HEV seroconversion rates may play important roles in causing chronic HEV infection. By analyzing HEV antigen concentrations with different infection outcomes, we also found that HEV antigen levels could indicate chronic HEV infection development. This study optimized the immunosuppression strategies for establishing chronic HEV infection in rabbits and highlighted the potential association between the development of chronic HEV infection and immunosuppressants.IMPORTANCEOrgan transplant recipients are at high risk of chronic hepatitis E and generally receive a CNI-based immunosuppression regimen containing CNI (tacrolimus or CsA), MMF, and/or corticosteroids. Previously, we established stable chronic HEV infection in a rabbit model by using CsA before HEV challenge. In this study, we further optimized the immunosuppression strategies for establishing chronic HEV infection in rabbits. Chronic HEV infection can also be established when CsA treatment was started at the same time or even 4 weeks after HEV challenge, clearly indicating the risk of progression to chronic infection under these circumstances and the necessity of HEV screening for both the recipient and the donor preoperatively. CsA, tacrolimus, or prednisolone instead of MMF significantly contributed to chronic HEV infection. HEV antigen in acute infection phase indicates the development of chronic infection. Our results have important implications for understanding the potential association between chronic HEV infection and immunosuppressants.

Esophageal Lichen Planus: The Efficacy and Safety of Tacrolimus in Reducing Inflammation and Need for Dilation.

Esophageal Lichen Planus (ELP) is a rare inflammatory disease most seen in middle-aged Caucasian women, manifested by sloughing mucosa, thick exudate, and proximal strictures. Most case reports and small series highlight using steroids and other immunosuppressants. To our knowledge, oral tablet tacrolimus has not been studied. We aimed to assess the change in ELP after oral tacrolimus treatment. The primary outcome was the efficacy of tacrolimus objectively through our scoring system, ELPSS. All consecutive adults with ELP who underwent more than one EGD by two esophagologists and being treated with tacrolimus or other treatment were eligible for inclusion in this retrospective cohort study. Inflammation and fibrostenotic disease were graded using the novel ELP Severity Score (ELPSS). Twenty two patients met the inclusion criteria. Half (11) received tacrolimus (dose 1-2 mg BID) and half (11) received other therapy (i.e. cyclosporine, topical steroids, or none). Mean ELPSS on first EGD, extraesophageal manifestations of disease, presenting symptoms, and baseline characteristics were similar between groups. Among patients on Tac vs No-Tac, there was a statistically significant improvement in ELPSS (mean difference 1.8 pts; 95% CI 0.25-3.38; P=0.02). Response rate was 89% with Tac vs 30% with No-Tac (P=0.025). All 22 patients underwent bougie dilation safely with a mean diameter of 16 mm achieved. Patients on Tac also required less frequent dilation. Oral tablet tacrolimus reduced the inflammatory and fibrostenotic components of ELP. Thus, low-dose oral tacrolimus is safe and should be considered in patients with more severe disease.

O-031 Tacrolimus treatment significantly improved IVF outcomes with euploid embryo transfer in women with repeated implantation failures (RIF) and elevated T helper (Th)1/Th2 cell ratios

Abstract Study question Dose tacrolimus improve implantation rate for RIF women showing elevated T helper (Th)1/Th2 cell ratios, particularly those undergoing IVF/ET with euploid blastocysts? Summary answer Tacrolimus improved the implantation rate in RIF women with elevated Th1/Th2 cell ratios undergoing IVF/ET using euploid blastocysts. What is known already The peripheral blood Th1 and Th2 cells determine immune inflammatory responses at the maternal-fetal interface. Previously, we reported that about 40% of women with RIF (≥4) had elevated Th1/Th2 cell ratios (≥10.3). The pregnancy rate was significantly improved when they were treated with tacrolimus, an immunosuppressive agent [AJRI 2015]. However, genetic embryonic concerns of RIF could not be ruled out since not all transferred embryos were genetically screened. Recently, we reported an adjusted cut-off value of the Th1/Th2 cell ratios (≥11.8) [AJRI 2021] for women with RIF based on the retrospective analysis. However, the prospective study has not been performed. Study design, size, duration A prospective cohort study was performed, including 569 infertile women with more than 4 times of RIF. The study participants received ET with euploid blastocysts from September 2020 to November 2021. All transferred blastocysts were confirmed as euploid or low-frequency mosaic by preimplantation genetic testing for aneuploidy, and the endometrial preparation for ET was made by using either hormone replacement cycle or natural ovulatory cycle. Participants/materials, setting, methods Before ET, all participants were measured for their peripheral blood Th1/Th2 (CD4+IFN-γ+/ CD4+IL-4+) cell ratios in the secretory phase. Women who had elevated Th1/Th2 cell ratios (≥10.3) received tacrolimus (2-4 mg daily). Tacrolimus was started 2 days before ET, and the hCG and gestational sac (GS) rates were calculated in women with or without tacrolimus. Main results and the role of chance Of 569 participants, 174 (30.6%) women showed elevated Th/Th2 cell ratios (≥10.3), whereas the remaining 395 women served as control (Control group). Among 174 women, 148 women received tacrolimus (Tac group), while 26 did not receive tacrolimus (no-Tac group). The hCG and GS rates of the Tac group were 73.0% and 64.2%, respectively, similar to the no-Tac group (69.2% and 57.7%, respectively, P=NS). Meanwhile the hCG and GS rates of the control group were 70.6% and 60.0%, respectively (P=NS). When the Th1/Th2 cell ratio cut-off value was set to ≥ 11.8, 123 women of the 569 participants (21.6%) had elevated Th1/Th2 cell ratios (≥11.8). Among them, 112 received tacrolimus (Tac2 group), and 11 did not tacrolimus (no-Tac2 group). The remaining 410 women were as a control group (Control2 group). The GS rate of the Tac2 group was 67.0%, which was significantly higher than that of the no-Tac group (36.3%, p < 0.05), while the GS rate of the Control2 group was 60.5% (P=NS, vs Control2 group). Tacrolimus dosing data was as follows; 11.8 or more but less than 15.8 of Th1/Th2 cell ratios were needed for 2 mg of tacrolimus daily, and 15.8 pr more was needed for 3 mg of tacrolimus. Limitations, reasons for caution The study was a controlled trial with a limited number of study population. Additionally, the endometrial changes or peripheral immune responses after tacrolimus were not evaluated thoroughly. Further study is needed for the tacrolimus effect on systematic immune responses and local endometrial immune milieu. Wider implications of the findings Before ET, screening for increased Th1/Th2 ratios (Cut-off value ≥11.8) may significantly improve IVF outcomes by selecting proper candidates for tacrolimus treatment and detecting the proper tacrolimus doses. Notably, a significant proportion of women with RIF have additional implantation failures, even with euploid embryos, when immune-inflammatory conditions were not controlled. Trial registration number not applicable

Comparison of tacrolimus with or without prednisone therapy in primary membranous nephropathy: a retrospective clinical study

Previous studies showed tacrolimus monotherapy and dual therapy with tacrolimus and prednisone as effective treatment modalities in managing membranous nephropathy. However, few studies have compared these therapeutic regimens. The patients were divided into two groups based on the treatment regimen: (1) tacrolimus and prednisone dual therapy (T + P group, n = 67) treatment group; and (2) tacrolimus monotherapy (T group, n = 65) or the control group. Propensity matching method and subgroup analysis to eliminate the bias in the relationship between the treatment regimen and the outcomes. The mean remission times were 20.33 ± 2.75 weeks at T group and 9.50 ± 1.81 weeks at T + P group. The T group had a remission rates of 73.33, 76.66 and 66.66% at 12weeks, 24weeks and 48weeks, while the T + P group had a remission rate of 81.66, 86.66, 91.66%; At the follow-up of 48 weeks, the relapse rate for the T group was 21.66%, and that for the T + P group was 5%. The anti-PLA2R ab is positive and therapy may be the independent risk factors for predicting remission. Tacrolimus and low-dose prednisone dual therapy is efficacious in managing MN and lowers the recurrence rate in clinical practice.

Tacrolimus personalized therapy based on CYP3A5 genotype in Chinese patients with idiopathic inflammatory myopathies.

Idiopathic inflammatory myopathies (IIM) are a heterogeneous and life-threatening group of diseases; in particular, anti-melanoma differentiation-associated gene 5 antibody positive DM (MDA5+ DM) is reportedly strongly associated with high mortality rate. Tacrolimus (TAC) provides an excellent therapeutic option, but the trough concentration (Cmin)-outcome relationship remains unexplored. This study was undertaken to identify optimal Cmin and individualized dose based on CYP3A5 genotype for IIM patients. A total of 134 IIM patients with 467 Cmin were enrolled. We examined the relationship between TAC Cmin and relapses. The receiver operating characteristic analysis was used to confirm the optimal Cmin. Analyses of factors influencing Cmin were conducted. The dose requirement based on CYP3A5 genotype was confirmed. TAC Cmin is strongly associated with relapses. The optimal cutoff values were 5.30, 5.85, 4.85 and 5.35 ng/ml for acute, subacute, chronic and all-phase IIM patients (P = 0.001, 0.013, 0.002 and <0.001, respectively), as well as 5.35, 5.85, 5.55 and 5.85 ng/ml for acute, subacute, chronic and all-phase MDA5+ DM patients (P = 0.007, 0.001, 0.036 and <0.001, respectively). CYP3A5 genotype was one of the significant factors influencing TAC Cmin. CYP3A5 expressers required 0.059 mg/kg/day to attain the target Cmin, while nonexpressers required 0.046 mg/kg/day (P = 0.019). TAC treatment may elicit favorable outcome in patients with IIM and MDA5+ DM when Cmin exceeded 5.35 and 5.85 ng/ml, which is crucial to a lower relapse rate. The individualized dose based on the CYP3A5 genotype provides a reference for TAC personalized therapy in IIM.

#1887 Comparison of the efficacy of preservation at 22°C versus 4°C in warm ischemic kidneys from MHC-inbred CLAWN miniature swine

Abstract Background and Aims Static cold preservation, the standard in transplantation, is primarily aimed at reducing metabolic and oxygen demand. Kidney transplantation from donors after cardiac death (DCD) addresses the shortage of donor organs, but these kidneys are susceptible to cold preservation. A previous study using small animal models demonstrated the potential benefits of preserving DCD kidneys at a warmer temperature of 22°C using extracellular-type solutions (Iwai S., PLoS One 2012). Based on this foundational research, our study aimed to evaluate the efficacy of preserving warm ischemic kidneys at temperatures of either 4°C or 22°C using preclinical large animal miniature swine models. Method After 2 hours of warm ischemia, the pig kidneys were preserved for 60 minutes at either 4°C or 22°C with extracellular-type solutions (ETK®). In Experiment 1, these preserved kidneys underwent ex-vivo perfusion for 120 minutes at 37°C with a mean arterial pressure of 85 mmHg. This perfusion used a Ringer's solution supplemented with red blood cells, aiming to determine the early functionality of kidneys subjected to 2-hour warm ischemia in transplantation and to assess whether preservation at 4°C or 22°C could maintain renal function during the acute postoperative period (n = 3 in each group). During this period, both physiological parameters (renal blood flow, intrarenal resistance, and urine output) and metabolic parameters (oxygen consumption) were evaluated. In Experiment 2, kidneys that underwent similar warm ischemia and preservation procedures were transplanted into MHC-matched recipients. These recipients received continuous tacrolimus treatment for 12 days to minimize rejection due to minor antigen differences. After transplantation, renal function was monitored and assessed by serum creatinine levels and renal biopsies for 14 days (n = 3 in each group). Results Experiment 1 demonstrated that kidneys subjected to 2 hours of warm ischemia and then preserved at 22°C had better physiological and metabolic indices during a subsequent 2-hour ex-vivo perfusion compared to those preserved at 4°C. The results were as follows: 22°C vs. 4°C—mean renal blood flow (27 ± 5 vs. 10 ± 0 ml/min), intrarenal resistance (3.5 ± 0.5 vs. 8.7 ± 0.1 mmHg/ml/min), total urine volume (14 ± 5 vs. 5 ± 4 ml), and oxygen consumption (224 ± 39 vs. 93 ± 16 ml/min/g). Importantly, there was no complete cessation of blood flow in either group, indicating the potential for long-term evaluation using a transplantation model. In Experiment 2, kidneys preserved at 22°C resulted in significantly lower peak post-transplant serum creatinine levels compared to those preserved at 4°C (3.9 ± 0.6 mg/dl vs 8.9 ± 0.3 mg/dl, p = 0.0015). There was a marked decrease in the area under the curve of creatinine from day 0 to day 14, with a 36% reduction from 61 mg/dL per day in the 4°C group to 39 mg/dL per day in the 22°C group. Renal biopsies performed on post-operative day 4 revealed extensive necrosis in the renal tubules of kidneys stored at 4°C. In contrast, kidneys preserved at 22°C showed more localized damage. In addition, PCNA staining indicated accelerated regeneration of the tubular epithelium in kidneys preserved at 22°C. Conclusion This study demonstrates that kidneys subjected to 2 hours of warm ischemia from MHC-inbred miniature swine are more effectively preserved at 22°C than at 4°C when using an extracellular-type solution. To our knowledge, this is the first report demonstrating the efficacy of organ preservation at 22°C in clinically relevant, large animal models. From the evaluation of ex-vivo perfusion of preserved kidneys, it was suggested that kidneys preserved at 22°C maintain better blood perfusion to severely ischemic kidneys immediately after transplantation, which leads to the rapid recovery of renal function of acute kidney injury. Future research will focus on elucidating the mechanisms underlying the superior outcomes associated with 22°C preservation. In addition, investigations will be conducted to determine whether similar benefits can be achieved using intracellular-type solutions, which are more commonly used in renal transplantation.

Tacrolimus-loaded Drug Delivery Systems in Vascularized Composite Allotransplantation: Lessons and Opportunities for Local Immunosuppression.

Long-term systemic immunosuppression is needed for vascularized composite allotransplantation (VCA). The high rate of acute rejection episodes in the first posttransplant year, the development of chronic rejection, and the adverse effects that come along with this treatment, currently prevent a wider clinical application of VCA. Opportunistic infections and metabolic disturbances are among the most observed side effects in VCA recipients. To overcome these challenges, local immunosuppression using biomaterial-based drug delivery systems (DDS) have been developed. The aim of these systems is to provide high local concentrations of immunosuppressive drugs while reducing their systemic load. This review provides a summary of recently investigated local DDS with different mechanisms of action such as on-demand, ultrasound-sensitive, or continuous drug delivery. In preclinical models, ranging from rodent to porcine and nonhuman primate models, this approach has been shown to reduce systemic tacrolimus (TAC) load and adverse effects, while prolonging graft survival. Localized immunosuppression using biomaterial-based DDS represents an encouraging approach to enhance graft survival and reduce toxic side effects of immunosuppressive drugs in VCA patients. Preclinical models using TAC-releasing DDS have demonstrated high local immunosuppressive effects with a low systemic burden. However, to reduce acute rejection events in translational animal models or in the clinical reality, the use of additional low-dose systemic TAC treatment may be envisaged. Patients may benefit through efficient graft immunosuppression and survival with negligible systemic adverse effects, resulting in better compliance and quality of life.

Exploring the Knowledge, Attitude, and Practice of Sudanese Medical Doctors Towards Tacrolimus, at Ahmed Gasim Hospital, Kidney Transplant Center Unit, Khartoum, Sudan (2022 – 2023)

Tacrolimus is one of the most commonly prescribed immunosuppressive drugs that varies between individuals depending on different ethnic groups, the presence of different CYP3A5 genetic variants, and the duration of time since the transplantation, which makes targeting optimal tacrolimus concentration levels more challenging. This study aimed to explore the knowledge, attitude, and practice of Sudanese medical doctors towards Tacrolimus. An observational, cross-sectional study was conducted on the Sudanese medical doctors in the nephrology clinic at Ahmed Gasim Hospital, Kidney Transplant Center Unit, Khartoum, Sudan, who were in direct contact with kidney transplant patients under tacrolimus treatment. Data were collected using a questionnaire modified to record the pharmacogenomic impact. The data were analyzed using a statistical package for social sciences (SPSS). Total of 15 medical doctors, Female 8 (53.3%) and male 7 (46.7%) with mean ± SD age of 31.60 ± (3.906). All of them were currently practicing in governmental hospitals (n=15; 83.3%), private hospitals (n=2; 11.1%), and private clinics (n=1; 6.7%). There were 67% of the medical doctors had high knowledge about the genetic profile of the patient's impact on the tacrolimus trough concentrations, while 66.7% of the medical doctors had low attitude levels toward tacrolimus. The observations among the different Sudanese ethnic groups tacrolimus trough concentrations were found to be variable, as Arabs and Fulani were observed to have low trough concentrations (n=6; 40%), while Nuba and Beja were observed to have high trough concentrations (n=5; 33.3%), instead of fur observed to had fluctuations (n=2; 13.3%). A significant correlation between practice toward tacrolimus with the location of practice, doctor specialty, and years of experience was found (P value ≤ 0.05). The medical doctors exerted a high level of knowledge about the impact of the genetic profile (metabolic alleles) in the dosing and dose adjustment of tacrolimus.

The interplay between calcineurin inhibitors (CNIs) and WNK bodies

KS-WNK1 is a kidney-specific scaffolding isoform of the With-No-Lysine (WNK) kinase signaling pathway. It is the predominant WNK1 isoform in the kidney, lacks a functional kinase domain, and is highly expressed in the distal convoluted tubule (DCT) where it drives the formation of biomolecular condensates called WNK bodies. During hypokalemia, WNK bodies bring together KS-WNK1, L-WNK1, WNK4, and SPAK, facilitating activation of the kinase cascade. This leads to increased downstream phosphorylation of the SLC12 cotransporter NCC. Mechanisms governing the reversibility and size of WNK body condensate scaffolds are poorly understood. We propose that phosphatases regulate KS-WNK1 and WNK body formation--a likely candidate being the phosphatase calcineurin (PP3). Prior work suggests that calcineurin dephosphorylates and inhibits the WNK-SPAK-NCC pathway, whereas calcineurin inhibitors (CNIs) such as tacrolimus increase phosphorylation and activation of the pathway. Here, we tested the hypothesis that CNIs also increase the abundance of KS-WNK1, causing increased WNK body size and abundance, and activation of the WNK-SPAK-NCC pathway. To study the effect of endogenous WNK kinases on KS-WNK1 function, WT HEK293 cells or HEK293 L-WNK1 KO cells were transiently transfected with KS-WNK1-HA-mRuby, treated with tacrolimus and studied by immunofluorescence confocal microscopy and immunoblotting of supernatant and detergent-insoluble pellet fractions. In both WT and KO cells, tacrolimus significantly increased WNK body abundance and size. Consistent with its localization within WNK bodies, KS-WNK1 protein was enriched in the pellet fraction by 10-fold in both WT and KO cells. KS-WNK1 protein abundance was increased by tacrolimus in a dose- (0-400ng/ml) and time-dependent manner (0-72h), an effect that was independent of endogenous L-WNK1 expression. Consistent with previous studies, SPAK abundance and phosphorylation was increased with tacrolimus treatment in a dose and time-dependent manner. Interestingly, SPAK localization was shifted into the pellet fraction in WT cells expressing KS-WNK1, whereas it was localized in the supernatant in L-WNK1 KO cells. The tacrolimus-induced increase in KS-WNK1 and pSPAK correlated with increased SLC12 cotransporter phosphorylation in WT cells. These preliminary results suggest that tacrolimus promotes WNK body formation and activity via KS-WNK1, and that SPAK localization in HEK cell-reconstituted WNK bodies requires full length WNK1. There is growing evidence that biomolecular condensates concentrate biomolecules to optimize signaling pathways and enhance kinase specificity through spatiotemporal control. Here we propose a novel concept that WNK bodies are biomolecular condensates that are regulated by calcineurin inhibitors. NIH K08DK118211, U54DK137329, ASN Gottschalk Award, P30DK081943. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Does Uromodulin Play a Role in Tacrolimus-Mediated Upregulation of the Sodium Chloride Cotransporter?

Background: Tacrolimus, a calcineurin inhibitor (CNI), is a medication commonly prescribed to prevent organ transplant rejection. However, chronic CNI use has several adverse effects, including hypertension. Recent studies have revealed that the renal sodium chloride cotransporter (NCC) mediates tacrolimus-induced hypertension. However, the mechanisms underlying NCC upregulation are not fully understood. Previous studies indicate that uromodulin plays a key role in renal ion channel regulation. As such, we hypothesize that uromodulin mediates tacrolimus induced NCC upregulation. Experimental Design: To test this hypothesis, male C57BL/6 mice received tacrolimus (10 mg/kg/day) or vehicle (ethanol/DMSO/saline) for 21 consecutive days via intraperitoneal injection. The impact of tacrolimus on systolic blood pressure (BP) was monitored by tail-cuff plethysmography. To examine uromodulin and NCC expression, kidneys were harvested and processed for immunohistochemistry. Results: Compared to the BP of vehicle-treated mice (120 mm Hg ± 4.252), mice administered tacrolimus had significant increases in BP (170 mm Hg ± 3.528). Furthermore, tacrolimus treatment increased phosphorylated NCC (pNCC) and uromodulin expression. Conclusions: These results indicate that tacrolimus-induced hypertension is accompanied by pNCC and uromodulin upregulation in distal convoluted tubules. Significance: Overall, these findings highlight uromodulin as a possible regulator of NCC. Funding: R21 DK119879, R01 DK-133698, R25DK07838. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

CD8+ T cell-mediated rejection of allogenic human-induced pluripotent stem cell-derived cardiomyocyte sheets in human PBMC-transferred NOG MHC double knockout mice

BackgroundTransplantation of human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) has emerged as a promising therapy to treat end-stage heart failure. However, immunogenicity of hiPS-CMs in transplanted patients has not been fully elucidated. Thus, in vivo models are required to estimate immune responses against hiPS-CMs in transplant recipients. MethodsWe transferred human peripheral blood mononuclear cells (hPBMCs) into NOD/Shi-scid IL2rgnull (NOG) MHC class I/II double knockout (NOG-ΔMHC) mice, which were reported to accept hPBMCs without xenogeneic-graft-versus-host disease (xeno-GVHD). Then, hiPS-CM sheets generated from the hiPS cell line 201B7 harboring a luciferase transgene were transplanted into the subcutaneous space of NOG-ΔMHC mice. Graft survival was monitored by bioluminescent images using a Xenogen In Vivo Imaging System. ResultsThe human immune cells were engrafted for more than three months in NOG-ΔMHC mice without lethal xeno-GVHD. The hiPS-CMs expressed a moderate level of human leukocyte antigen (HLA)-class I, but not HLA-class II, molecules even after interferon-gamma (IFN-γ) stimulation. Consistently, the allogenic IFN-γ-treated hiPS-CMs induced weak CD8+ but not CD4+, T cell responses in vitro. hiPS-CM sheets disappeared approximately 17–24 days after transplantation in hPBMC-transferred NOG-ΔMHC mice, and CD8+ T cell depletion significantly prolonged graft survival, similar to what was observed following tacrolimus treatment. ConclusionhiPS-CMs are less immunogenic in vitro but induce sufficient CD8+ T cell-mediated immune responses for graft rejection in vivo.

The efficacy and safety of tacrolimus in treating refractory IgA vasculitis nephritis: a single-center retrospective study on 16 cases.

This study aimed to observe the efficacy and safety of tacrolimus in the treatment of refractory immunoglobulin A vasculitis nephritis (IgAVN). Sixteen patients with IgAVN who had been previously treated with cyclophosphamide shock therapy at least five times, some of whom had also received mycophenolate but still had persistent proteinuria, were enrolled. The clinical and pathological data were collected and analysed. The average (mean±standard deviation) age at the initial assessment for the group of 16 patients was 10±2.7years. Finally, at the end of their respective follow-up time point, 6 of the 16 patients achieved complete remission (37.5%), 5 achieved partial remission (31.2%), and 5 had no remission (31.2%). A significant difference was found in the median proteinuria before and after a 6-month course of tacrolimus treatment [19.2 (11.2, 31.9) vs 7.8 (4.3, 13.9) mg/kg/day] (P<.05). During the first 6months of tacrolimus treatment, all patients' estimated glomerular filtration rate levels remained normal. The mean tacrolimus blood concentration was 6.0±2.6ng/mL. The median prednisone dosage was decreased from 10mg/day to 5mg/day, and prednisone was eventually stopped in three individuals. No drug-related adverse effects were observed during treatment. Tacrolimus has demonstrated efficacy in increasing remission rates, significantly lowering urinary protein levels, and reducing steroid use in children with refractory IgAVN. Further research is required to investigate its optimal blood concentrations, long-term effects and renoprotective properties.

Evaluation of T Cell Response to SARS-CoV-2 in Kidney Transplant Recipients Receiving Monoclonal Antibody Prophylaxis and the Utility of a Bivalent mRNA Vaccine Booster Dose.

Monoclonal antibodies have been administered to kidney transplant recipients (KTRs) with a poor or non-responder status to SARS-CoV-2 vaccination. The cellular response to SARS-CoV-2 has been poorly studied in this context. We assessed the T cell response to SARS-CoV-2 in 97 patients on the day of the injection of tixagevimab/cilgavimab using an IFNγ enzyme-linked immunospot assay (ELISPOT). Among the 97 patients, 34 (35%) developed COVID-19 before the injection. Twenty-nine (85.3%) had an ELISPOT compatible with a SARS-CoV-2 infection. There was no difference between KTRs under belatacept or tacrolimus treatment. Sixty-three patients (64.9%) had no known COVID-19 prior to the ELISPOT, but nine (14.3%) had a positive ELISPOT. In 21 KTRs with a positive ELISPOT who received a booster dose of a bivalent mRNA vaccine, median antibody titers and spike-reactive T cells increased significantly in patients under tacrolimus but not belatacept. Our study emphasizes the potential usefulness of the exploration of immune cellular response to SARS-CoV-2 by ELISPOT. In KTRs with a positive ELISPOT and under CNI therapy, a booster dose of mRNA vaccine seems effective in inducing an immune response to SARS-CoV-2.

Age-Related Brain Atrophy and the Positive Effects of Behavioral Enrichment in Middle-Aged Beagles.

Aging dogs serve as a valuable preclinical model for Alzheimer's disease (AD) due to their natural age-related development of β-amyloid (Aβ) plaques, human-like metabolism, and large brains that are ideal for studying structural brain aging trajectories from serial neuroimaging. Here we examined the effects of chronic treatment with the calcineurin inhibitor (CNI) tacrolimus or the nuclear factor of activated T cells (NFAT)-inhibiting compound Q134R on age-related canine brain atrophy from a longitudinal study in middle-aged beagles (36 females, 7 males) undergoing behavioral enrichment. Annual MRI was analyzed using modern, automated techniques for region-of-interest-based and voxel-based volumetric assessments. We found that the frontal lobe showed accelerated atrophy with age, while the caudate nucleus remained relatively stable. Remarkably, the hippocampus increased in volume in all dogs. None of these changes were influenced by tacrolimus or Q134R treatment. Our results suggest that behavioral enrichment can prevent atrophy and increase the volume of the hippocampus but does not prevent aging-associated prefrontal cortex atrophy.

Hepatitis E virus infects human testicular tissue and Sertoli cells

Globally, hepatitis E virus (HEV) infections are prevalent. The finding of high viral loads and persistent viral shedding in ejaculate suggests that HEV replicates within the human male genital tract, but its target organ is unknown and appropriate models are lacking. We aimed to determine the HEV tropism in human testis and its potential influence on male reproductive health. We conducted ex vivo culture of human testis explants and in vitro culture of primary human Sertoli cells. Clinically derived HEV genotype 1 (HEV1) and HEV3 virions, as well as rat derived HEV-C1, were used for inoculation. Transcriptomic analysis was performed on testis tissues collected from tacrolimus-treated rabbits with chronic HEV3 infection. Our findings reveal that HEV3, but not HEV1 or HEV-C1, can replicate in human testis explants and primary human Sertoli cells. Tacrolimus treatment significantly enhanced the replication efficiency of HEV3 in testis explants and enabled successful HEV1 infection in Sertoli cells. HEV3 infection disrupted the secretion of several soluble factors and altered the cytokine microenvironment within primary human Sertoli cells. Finally, intratesticular transcriptomic analysis of immunocompromised rabbits with chronic HEV infection indicated a downregulation of genes associated with spermatogenesis. HEV can infect the human testicular tissues and Sertoli cells, with increased replication efficiency when exposed to tacrolimus treatment. These findings shed light on how HEV may persist in ejaculate of patients with chronic hepatitis E and provide valuable ex vivo tool for studying countermeasures.