CRISPR-Cas9-Mediated Correction of SLC12A3 Gene Mutation Rescues the Gitelman's Disease Phenotype in a Patient-Derived Kidney Organoid System.

The aim of this study is to explore the possibility of modeling Gitelman's disease (GIT) with human-induced pluripotent stem cell (hiPSC)-derived kidney organoids and to test whether gene correction using CRISPR/Cas9 can rescue the disease phenotype of GIT. To model GIT, we used the hiPSC line CMCi002 (CMC-GIT-001), generated using PBMCs from GIT patients with SLC12A3 gene mutation. Using the CRISPR-Cas9 system, we corrected CMC-GIT-001 mutations and hence generated CMC-GIT-001corr. Both hiPSCs were differentiated into kidney organoids, and we analyzed the GIT phenotype. The number of matured kidney organoids from the CMC-GIT-001corr group was significantly higher, 3.3-fold, than that of the CMC-GIT-001 group (12.2 ± 0.7/cm2 vs. 3.7 ± 0.2/cm2, p < 0.05). In qRT-PCR, performed using harvested kidney organoids, relative sodium chloride cotransporter (NCCT) mRNA levels (normalized to each iPSC) were increased in the CMC-GIT-001corr group compared with the CMC-GIT-001 group (4.1 ± 0.8 vs. 2.5 ± 0.2, p < 0.05). Consistently, immunoblot analysis revealed increased levels of NCCT protein, in addition to other tubular proteins markers, such as LTL and ECAD, in the CMC-GIT-001corr group compared to the CMC-GIT-001 group. Furthermore, we found that increased immunoreactivity of NCCT in the CMC-GIT-001corr group was colocalized with ECAD (a distal tubule marker) using confocal microscopy. Kidney organoids from GIT patient-derived iPSC recapitulated the Gitelman's disease phenotype, and correction of SLC12A3 mutation utilizing CRISPR-Cas9 technology provided therapeutic insight.

Effect of dual inhibition of DPP4 and SGLT2 on tacrolimus-induced diabetes mellitus and nephrotoxicity in a rat model.

Sodium/glucose co-transporter-2 inhibitor (SGLT2i) or dipeptidyl peptidase IV inhibitor (DPP4i) is a newer anti-diabetic drug in type II diabetes mellitus (DM), but their use in tacrolimus (TAC)-induced DM is still undetermined. We performed this study to evaluate the effect of these two drugs in TAC-induced DM and nephrotoxicity in ex vivo and in vivo. In the experimental Sprague Dawley rat model of TAC-induced DM and nephrotoxicity, dual inhibition of DPP4 and SGLT2 significantly decreased blood glucose level, HbA1C and increased plasma insulin levels and pancreatic islet size compared with each drug. In the kidney, dual inhibition improved renal function decreased interstitial fibrosis and profibrotic cytokines compared with DPP4i and SGLT2i alone. Increased oxidative stress by TAC was remarkably decreased with DPP4i or SGLT2i in serum, pancreatic and renal tissues and this decrease was much more significant in the combination group. In in vitro study, TAC decreased the cell viability of human kidney-2(HK-2) cells and insulin-secreting beta-cell-derived line(INS-1) cells. SGLT2i protected TAC-induced cell death in HK-2 cells, but not in INS-1 cells. The addition of DPP4i to SGLT2i compensated for a lack of protective effect of SGLT2i on INS-1 cells. This finding provides the rationale for the combined treatment of SGLG2i and DPP4i in TAC-induced DM and nephrotoxicity.

Clinical significance of heart rate variability for the monitoring of cardiac autonomic neuropathy in end-stage renal disease patients.

BACKGROUND AND AIMS: The aim of this study is to determine whether the measurement of continuous heart rate variability (HRV) is useful in the evaluation of cardiac autonomic neuropathy (CAN) in end-stage renal disease (ESRD) patients. METHODS AND RESULTS: This cross-sectional study was performed at Seoul St. Mary's hospital between June 2017 and February 2018. Seventy-seven ESRD patients, and 29 healthy controls (HCs) were asked to wear a continuous ambulatory HRV monitor for 24 h. General cardiac function was evaluated using transthoracic echocardiogram (TTE), pulse wave velocity (PWV), coronary calcium scoring (CCS), and 24-h ambulatory blood pressure monitoring (ABPM). HRV parameters of ESRD patients and HCs, and the correlation of HRV parameters with cardiovascular screening methods were observed. All HRV parameters were significantly decreased in ESRD patients compared to HCs (P < 0.001). In the correlation analysis between TTE results and HRV parameters, 24-h standard deviation of all N-N intervals (24SDNN), 24-h standard deviation of sequential 5-min N-N interval means (24DANN) and Low Frequency Power/High Frequency Power (LF/HF) ratio showed negative correlations with E/e', LAVI and TR velocity which are representative indices for the diastolic function of the heart (P < 0.05). HRV parameters showed negative correlations with baPWV, CCS, and 24-h ABPM results as well (P < 0.05). Hemoglobin and serum albumin showed positive correlations with HRV parameters, and glucose, BUN, creatinine, and iPTH levels showed negative correlations (P < 0.05). CONCLUSION: Continuous HRV monitoring may be a useful tool for the evaluation of CAN in ESRD.

Effect of Klotho on autophagy clearance in tacrolimus-induced renal injury.

Recently, we showed that tacrolimus-induced renal injury was closely associated with impairment of autophagy clearance, and Klotho deficiency aggravated tacrolimus-induced renal injury. In this study, we evaluated the effect of Klotho treatment on autophagy clearance in tacrolimus-induced renal injury. We evaluated the effect of Klotho on tacrolimus-induced renal injury in an experimental mouse model and in vitro by treatment with tacrolimus and/or recombinant mouse Klotho. In vivo and in vitro studies showed that tacrolimus treatment impaired lysosomal acidification and decreased cathepsin B activity, expression of lysosome-associated membrane protein 2, and expression of transcription factor EB (TFEB), a master regulator for lysosomal biogenesis. These results were improved by Klotho treatment. Moreover, addition of bafilomycin A1, an inhibitor of lysosomal function, abolished the protective effect of Klotho, indicating that the protective effect of Klotho was closely associated with lysosome function. Klotho induced nuclear translocation of TFEB through inhibition of phosphorylation of glycogen synthase kinase 3ß (GSK3ß) by confirming using CHIR99021, a GSK3ß inhibitor. Collectively, our data suggest that Klotho improves autophagy clearance via activation of lysosomal function in tacrolimus-induced nephrotoxicity.-Lim, S. W., Shin, Y. J., Luo, K., Quan, Y., Ko, E. J., Chung, B. H., Yang, C. W. Effect of Klotho on autophagy clearance in tacrolimus-induced renal injury.

Host cell in vivo production of the synthetic drug anti-CD25/IL-10 using minicircle vector.

Synthetic biologic drugs are highly successful for induction therapy in transplantation, but the development of novel biologics is limited because of the high cost of synthesis and purification. In this study, we developed a novel strategy for the production of synthetic protein drugs in vivo by the host itself. We utilized minicircle (MC) technology, which can robustly express a target molecule and secrete it from cells, as an indirect method to produce a protein of interest in vivo. We designed an MC vector containing the sequences of basiliximab (anti-CD25 mAb) and IL-10. We verified the substantial production of the anti-CD25/IL-10 protein from the MC in vitro and in vivo. The therapeutic effect of MC-derived anti-CD25/IL-10 was evaluated in a skin allograft mouse model by single intravenous infusion. Mice treated with the MC encoding anti-CD25/IL-10 exhibited prolonged skin allograft survival times accompanied by improved histologic changes and immunologic regulation. These findings indicate that the anti-CD25/IL-10 protein drug obtained by MC technology is functionally active and relevant for reducing allograft rejection. This self-reproducible strategy for synthetic protein drugs using MCs is a promising tool for transplantation.-Lim, S. W., Shin, Y. J., Luo, K., Quan, Y., Ko, E. J., Chung, B. H., Yang, C. W. Host cell in vivo production of the synthetic drug anti-CD25/IL-10 using minicircle vector.

Coenzyme Q10 alleviates tacrolimus-induced mitochondrial dysfunction in kidney.

The major side effect of tacrolimus (Tac) is nephrotoxicity. We studied whether supplementation of coenzyme Q10, (CoQ10) a potent antioxidant, can reduce Tac-induced nephrotoxicity via improving mitochondrial function. In an in vitro study, CoQ10 reduced the production of Tac-induced mitochondrial reactive oxygen species and abolished the loss of mitochondrial membrane potential in proximal tubular cell line. Assessment of mitochondrial function revealed that CoQ10 decreased oxygen consumption and mitochondrial respiration rate increased by Tac, suggesting improvement of mitochondrial function to synthesize ATP with CoQ10 treatment. The effect of the CoQ10in vitro study was observed in an experimental model of chronic Tac-induced nephropathy. CoQ10 attenuated Tac-induced oxidative stress and was accompanied by function and histologic improvement. On electron microscopy, addition of CoQ10 increased not only the number but also the volume of mitochondria compared with Tac treatment only. Our data indicate that CoQ10 improves Tac-induced mitochondrial dysfunction in kidney. Supplementary CoQ10 treatment may be a promising approach to reduce Tac-induced nephrotoxicity.-Yu, J. H., Lim, S. W., Luo, K., Cui, S., Quan, Y., Shin, Y. J., Lee, K. E., Kim, H. L., Ko, E. J., Chung, B. H., Kim, J. H., Chung, S. J., Yang, C. W. Coenzyme Q10 alleviates tacrolimus-induced mitochondrial dysfunction in kidney.

The impact of cytomegalovirus infection on clinical severity and outcomes in kidney transplant recipients with Pneumocystis jirovecii pneumonia.

Cytomegalovirus (CMV) infection is associated with Pneumocystis jirovecii pneumonia (PJP) in kidney transplant recipients (KTRs), but its impact on clinical severity and outcomes in KTRs with PJP is unknown. We reviewed 1994 medical records of KTRs from January 1997 to March 2019. PJP or CMV infection was diagnosed by polymerase chain reaction or culturing using blood or respiratory specimens. We divided patients into PJP and PJP+CMV groups, and evaluated the clinical severity and outcomes. Fifty two patients had PJP (2.6%) in the whole study cohort. Among patients with PJP, 38 (73.1%) had PJP alone and 14 (26.9%) had combined PJP and CMV co-infection. The PJP+CMV group showed worse laboratory findings (serum albumin and C-reactive protein, P = 0.010 for both) and higher requirement of continuous renal replacement therapy than the PJP group (P = 0.050). The pneumonia severity was worse in the PJP+CMV group than in the PJP group (P < 0.05), and CMV infection was a high risk factor of pneumonia severity (odds ratio 16.0; P = 0.002). The graft function was worse in the PJP+CMV group (P < 0.001), and the incidence of graft failure was higher in the PJP+CMV group than in the PJP group (85.7% vs 36.8%; P < 0.001). Mortality was double in the PJP+CMV group than in the PJP group, but not statistically significant (21.4% vs 10.5%; P = 0.370). Our results show that approximately one in four patients with PJP after kidney transplantation develops CMV with increased clinical severity and risk of graft failure. The possibility of increased clinical severity and worse clinical outcomes by CMV co-infection should be considered in KTRs with PJP.

Moving closer to death: understanding psychosocial distress among older veterans with advanced cancers.

BACKGROUND: Early identification of psychosocial distress is important to address the needs of vulnerable populations and influence symptom management. Older veterans diagnosed with life-limiting cancers are particularly vulnerable because they often have unmet needs, experiencing psychological or emotional problems and gaps in healthcare communication, which extends suffering. Lack of emotional support, ongoing physical pain, and unresolved symptom control can further increase distress among older veterans, contributing to complexity of decision-making for end of life (EOL) care. OBJECTIVE: We explored older veterans' experiences and identification of psychosocial distress in cancer care to better understand how they describe distress while facing the end of life. METHODS: Guiding this study is a conceptual framework from psychosocial oncology with the multifactorial experience of distress indicated by NCCN guidelines for distress screening. We use a phenomenological approach to explore the experience of psychosocial distress among older veterans diagnosed with advanced cancers at risk for dying within a year. INCLUSION CRITERIA: Provider response of "no" to, "Would you be surprised if your patient died within a year?" and "yes", to the question, "Have you talked with your patient about the severity of their illness as being life-limiting, terminal?" RESULTS: Five themes emerged: (1) the meaning of distress: "It's hard to explain"; (2) severity of advanced cancer: "There's no stage five"; (3) distressing thoughts about the possibility of dying: "Either way, it's life limiting"; (4) coping: "Deal with it and hope for a better day"; and (5) personal factors: "I don't want to be anything but a man who can handle adversity." Findings suggest older veterans may have unique cancer experiences different from other populations. CONCLUSION: Older veterans in this study exhibited distressing symptoms which demonstrate they are at risk for declining health and in need of support for their distress. Healthcare providers are urged to understand the complexity of distress to provide the best possible treatment for older veterans.

Development of End Stage Renal Disease after Long-Term Ingestion of Chaga Mushroom: Case Report and Review of Literature.

Chaga mushrooms are widely used in folk remedies and in alternative medicine. Contrary to many beneficial effects, its adverse effect is rarely reported. We here report a case of end-stage renal disease after long-term taking Chaga mushroom. A 49-year-old Korean man with end stage renal disease (ESRD) was transferred to our hospital. Review of kidney biopsy finding was consistent with chronic tubulointerstitial nephritis with oxalate crystal deposits and drug history revealed long-term exposure to Chaga mushroom powder due to intractable atopic dermatitis. We suspected the association between Chaga mushroom and oxalate nephropathy, and measured the oxalate content of remained Chaga mushroom. The Chaga mushroom had extremely high oxalate content (14.2/100 g). Estimated daily oxalate intake of our case was 2 times for four years and 5 times for one year higher than that of usual diet. Chaga mushroom is a potential risk factor of chronic kidney disease considering high oxalate content. Nephrologist should consider oxalate nephropathy in ESRD patients exposed to Chaga mushrooms.

Therapeutic Challenge of Minicircle Vector Encoding Klotho in Animal Model.

BACKGROUND: Klotho treatment is a promising approach against kidney injury, but its clinical application is still undetermined. We developed a novel strategy to allow self-production of Klotho protein, using minicircle (MC) technology, and evaluated its feasibility in therapeutic Klotho delivery. METHODS: We engineered MC vectors to carry cassette sequences of Klotho and verified the self-production of Klotho protein from in HEK293T cells. We evaluated the location and persistence of delivered MC in vivo, and the duration of Klotho protein production from MCs by serial measurement of Klotho protein in blood. We subsequently evaluated the therapeutic potential of Klotho-encoding MCs in experimental model of renal injury. RESULTS: We confirmed the production of Klotho from MC by its significant availability in cells transfected with the MC, as well as in its conditioned medium, compared to that in cells transfected with parent vector. MCs were delivered in vivo by hydrodynamic injection via tail vein. After a single injection of MCs, red fluorescence protein was detected until 30 days in liver, and Klotho protein was maintained until 10 days in the blood, suggesting the production of Klotho protein from MCs via protein synthesis machinery in liver. Therapeutic effect of MC was confirmed by functional and histological improvement seen in mouse model of acute ischemia-reperfusion injury and unilateral ureteral obstruction. CONCLUSION: Together, these findings implied that self-generated Klotho protein, using MC technology, is functionally active and relevant as a therapeutic approach in renal injury.

Clinical outcomes of ABO- and HLA-incompatible kidney transplantation: a nationwide cohort study.

This was a nationwide cohort study to investigate the impact of anti-A/B and donor-specific anti-HLA (HLA-DSA) antibodies on the clinical outcomes in kidney transplant recipients (KTRs). We classified a total of 1964 KTRs into four groups: transplants from ABO-incompatible donors (ABOi, n = 248); transplants in recipients with HLA-DSA (HLAi, n = 144); transplants from combined ABOi and HLAi donors (ABOi + HLAi, n = 31); and a control group for whom neither ABOi nor HLAi was applicable (CONT, n = 1541). We compared the incidence of biopsy-proven acute rejection (BPAR), allograft and patient survival rates. The incidence of BPAR was higher in the HLAi and ABOi + HLAi groups relative to the CONT group; in contrast, it was not higher in the ABOi group. Death-censored graft survival rates did not differ across the four groups. However, relative to the CONT group, patient survival rate was reduced in the ABOi and ABOi + HLAi groups, and with infection being the most common cause of death. Further, multivariable analysis revealed that desensitization therapy because of ABOi or HLAi was independent risk factors for patient mortality. HLAi was a more important risk factor for BPAR compared with ABOi. However, pretransplant desensitization therapy for either ABOi or HLAi significantly increased the risk of infection-related mortality.

A model predicting survival of patients with recurrent or progressive hepatocellular carcinoma: the MORE score.

BACKGROUND AND AIM: Most prognostic models for hepatocellular carcinoma are based on data at the time of diagnosis. However, the disease frequently recurs or progresses after initial treatment, with changes in tumor burden and clinical status. Therefore, we developed a risk score model to predict survival of hepatocellular carcinoma patients at the time of disease recurrence or progression. METHODS: Of 1972 patients newly diagnosed with hepatocellular carcinoma at the National Cancer Center, Korea, between January 2004 and December 2009, 1301 with recurrent or progressive disease were enrolled. They were randomly classified into a development (75%, n = 976) and a validation cohort (25%, n = 325). A survival prediction method was established in the development cohort using the multivariate Cox proportional hazards model, and its performance was evaluated on the validation cohort. RESULTS: A model predicting survival of patients with recurrent or progressive hepatocellular carcinoma was developed using some known independent prognostic factors for overall survival: age, albumin, Model for End-Stage Liver Disease score, tumor burden, serum alpha-fetoprotein level, and presence of ascites. In addition, initial treatment modality and best response after initial treatment were also independent prognostic factors and were incorporated in the model. The C-statistics and χ2 statistics of this novel score for the validation cohort were 0.808 (95% CI: 0.781-0.834) and 4.408 for 3-year survival. CONCLUSIONS: A new model to predict survival of patients with recurrent or progressive hepatocellular carcinoma was developed and validated. This model may be useful for planning subsequent treatments.

Comparative Study of Diabetes Knowledge, Attitudes, Family Support, Self-efficacy, and Self-management Behaviors Between Cancer Survivors With Diabetes and Diabetes Patients Without Cancer.

BACKGROUND: Studies have compared diabetes management quality indicators, focusing on physiological markers such as hemoglobin A1c, between cancer survivors with diabetes and general diabetes patients. However, research comparing differences in diabetes self-management behaviors and the factors influencing them between these groups is lacking. OBJECTIVES: This study aimed to compare self-management behaviors, guided by the information-motivation-behavior model, between cancer survivors with diabetes and general diabetes patients. In addition, we aimed to identify differences in factors such as diabetes knowledge, attitudes, family support, and self-efficacy that may influence diabetes self-management behaviors in both groups. METHODS: A total of 125 cancer survivors with diabetes and 126 general diabetes patients participated in this cross-sectional study. A structured questionnaire assessed demographics, diabetes knowledge, attitudes, self-efficacy, and self-management behaviors. RESULTS: Regarding diabetes education, 47.0% of cancer survivors and 61.6% of general diabetes patients received education. The cancer survivors had lower diabetes knowledge scores (10.30 ± 4.15, P < .001), a lower perceived value of strict blood glucose control (4.10 ± 0.56, P < .001), and less family support (15.50 ± 7.50, P = .019) than the patients without cancer (13.51 ± 3.84, 4.25 ± 0.65, and 17.57 ± 6.40, respectively). CONCLUSION: This study reveals significant differences in diabetes self-management between cancer survivors and general diabetes patients. Cancer survivors showed lower diabetes knowledge, glucose control perception, and family support. These findings highlight the need for tailored self-management programs for cancer survivors. IMPLICATIONS FOR PRACTICE: This study offers insights for developing tailored diabetes self-management programs and educational interventions for cancer survivors.

Prediction of diabetes mellitus after kidney transplantation using patient-specific induced pluripotent stem cells.

BACKGROUND: Multiple risk factors are involved in new-onset diabetes mellitus (DM) after organ transplantation; however, their ability to predict clinical prognosis remains unclear. Therefore, we investigated whether patient-specific induced pluripotent stem cells (iPSCs) could help predict DM development before performing kidney transplantation (KT). METHODS: We first performed whole transcriptome and functional enrichment analyses of KT patient-derived iPSCs. Our results revealed that insulin resistance, type 2 DM, and transforming growth factor beta signaling pathways are associated between the groups of DM and non-DM. We next determined whether the genetic background was associated with development of iPSCs into pancreatic progenitor (PP) cells. RESULTS: The levels of differentiation-related key markers of PP cells were significantly lower in the DM group than in the non-DM group. Moreover, the results of tacrolimus toxicity screening showed a significant decrease in the number of PP cells of the DM group compared with the non-DM group, suggesting that these cells are more susceptible to tacrolimus toxicity. CONCLUSION: Taken together, these results indicate that PP cells of the DM group showed low developmental potency accompanied by a significantly different genetic background compared with the non-DM group. Thus, genetic analysis can be used to predict the risk of DM before KT.

Validation of prediction model for successful discontinuation of continuous renal replacement therapy: a multicenter cohort study.

Background: Continuous renal replacement therapy (CRRT) has become the standard modality of renal replacement therapy (RRT) in critically ill patients. However, consensus is lacking regarding the criteria for discontinuing CRRT. Here we validated the usefulness of the prediction model for successful discontinuation of CRRT in a multicenter retrospective cohort. Methods: One temporal cohort and four external cohorts included 1,517 patients with acute kidney injury who underwent CRRT for >2 days in 2018 to 2020. The model was composed of four variables: urine output, blood urea nitrogen, serum potassium, and mean arterial pressure. Successful discontinuation of CRRT was defined as the absence of an RRT requirement for 7 days thereafter. Results: The area under the receiver operating characteristic curve (AUROC) was 0.74 (95% confidence interval, 0.71-0.76). The probabilities of successful discontinuation were approximately 17%, 35%, and 70% in the low-score, intermediate-score, and high-score groups, respectively. The model performance was good in four cohorts (AUROC, 0.73-0.75) but poor in one cohort (AUROC, 0.56). In one cohort with poor performance, attending physicians primarily controlled CRRT prescription and discontinuation, while in the other four cohorts, nephrologists determined all important steps in CRRT operation, including screening for CRRT discontinuation. Conclusion: The overall performance of our prediction model using four simple variables for successful discontinuation of CRRT was good, except for one cohort where nephrologists did not actively engage in CRRT operation. These results suggest the need for active engagement of nephrologists and protocolized management for CRRT discontinuation.

Mortality of elderly patients with acute kidney injury undergoing continuous renal replacement therapy: is age a risk factor?

Background: Whether advanced age is associated with poor outcomes of elderly patients with acute kidney injury (AKI) requiring continuous renal replacement therapy (CRRT) is controversial. This study aimed to evaluate age effect and predictors for mortality in elderly AKI patients undergoing CRRT. Methods: Data of 480 elderly AKI patients who underwent CRRT were retrospectively analyzed. Subjects were stratified into two groups according to age: younger-old (age, 65-74 years; n = 205) and older-old (age, ≥75 years; n = 275). Predictors for 28-day and 90-day mortality and age effects were analyzed using multivariable Cox regression analysis and propensity score matching. Results: Urine output at the start of CRRT (adjusted hazard ratio [aHR], 0.99; 95% confidence interval [CI], 0.99-1.00; p = 0.04), operation (aHR, 0.53; 95% CI, 0.30-0.93; p = 0.03), and use of an intra-aortic balloon pump (aHR, 3.60; 95% CI, 1.18-10.96; p = 0.02) were predictors for 28-day mortality. Ischemic heart disease (aHR, 1.74; 95% CI, 1.02-2.98; p = 0.04) and use of a ventilator (aHR, 0.56; 95% CI, 0.36-0.89; p = 0.01) were predictors for 90-day mortality. The older-old group did not exhibit a higher risk for 28-day or 90-day mortality than the younger-old group in multivariable or propensity score-matched models. Conclusion: Advanced age was not a risk factor for mortality among elderly AKI patients undergoing CRRT, suggesting that advanced age should not be considered for therapeutic decisions in critically ill elderly patients with AKI requiring CRRT.

Examining the Alterations in Metabolite Constituents and Antioxidant Properties in Mountain-Cultivated Ginseng (Panax ginseng C.A. Meyer) Organs during a Two-Month Maturation Period.

The current research was the first to prove the existence of fluctuations in the metabolite constituents and antioxidant properties in different organs (leaves, stems, and roots) of the mountain-cultivated ginseng (MCG) plant during a two-month maturation period. Four metabolites, including fatty acids, amino acids, ginsenosides, and phenolic phytochemicals, exhibited considerable differences in organs and maturation times with the following order: leaves > stems > roots. The predominant metabolite contents were found in leaves, with fatty acid (1057.9 mg/100 g) on 31 May, amino acid (1989.2 mg/100 g) on 13 July, ginsenosides (88.7 mg/g) on 31 May, and phenolic phytochemical (638.3 µg/g) on 31 May. Interestingly, ginsenoside content in leaves were highest, with 84.8 → 88.7 → 82.2 → 78.3 mg/g. Specifically, ginsenosides Re, Rd, and F2 showed abundant content ranging from 19.1 to 16.9 mg/g, 8.5 to 14.8 mg/g, and 9.5 to 13.1 mg/g, respectively. Phenolic phytochemicals exhibited remarkable differences in organs compared to maturation periods, with the highest total phenolic content and total flavonoid content recorded at 9.48 GAE and 1.30 RE mg/g in leaves on 31 May. The antioxidant capacities on radical, FRAP, and DNA protection differed significantly, with leaves on 31 May exhibiting the highest values: 88.4% (DPPH), 89.5% (ABTS), 0.84 OD593 nm (FRAP) at 500 µg/mL, and 100% DNA protection at 50 µg/mL. Furthermore, principal cluster analysis revealed metabolite variability as follows: ginsenoside (83.3%) > amino acid (71.8%) > phenolic phytochemical (61.1%) > fatty acid (58.8%). A clustering heatmap highlighted significant changes in metabolite components under the maturation times for each organ. Our findings suggest that MCG leaves on 31 May may be a potential source for developing nutraceuticals, offering highly beneficial components and strong antioxidants.

Impact of Low-Level Donor-Specific Anti-HLA Antibody on Posttransplant Clinical Outcomes in Kidney Transplant Recipients.

Background: The clinical significance of low-level donor-specific anti-HLA antibody (low-DSA) remains controversial. We investigated the impact of low-DSA on posttransplant clinical outcomes in kidney transplant (KT) recipients. Methods: We retrospectively reviewed 1,027 KT recipients, namely, 629 living donor KT (LDKT) recipients and 398 deceased donor KT (DDKT) recipients, in Seoul St. Mary's Hospital (Seoul, Korea) between 2010 and 2018. Low-DSA was defined as a positive anti-HLA-DSA result in the Luminex single antigen assay (LABScreen single antigen HLA class I - combi and class II - group 1 kits; One Lambda, Canoga Park, CA, USA) but a negative result in a crossmatch test. We compared the incidence of biopsy-proven allograft rejection (BPAR), changes in allograft function, allograft survival, patient survival, and posttransplant infections between subgroups according to pretransplant low-DSA. Results: The incidence of overall BPAR and T cell-mediated rejection did not differ between the subgroups. However, antibody-mediated rejection (ABMR) developed more frequently in patients with low-DSA than in those without low-DSA in the total cohort and the LDKT and DDKT subgroups. In multivariate analysis, low-DSA was identified as a risk factor for ABMR development. Its impact was more pronounced in DDKT (odds ratio [OR]: 9.60, 95% confidence interval [CI]: 1.79-51.56) than in LDKT (OR: 3.76, 95% CI: 0.99-14.26) recipients. There were no significant differences in other outcomes according to pretransplant low-DSA. Conclusions: Pretransplant low-DSA has a significant impact on the development of ABMR, and more so in DDKT recipients than in LDKT recipients, but not on long-term outcomes.

Discovery of cellular and genetic signatures of immune tolerance in kidney transplant recipients through single cell RNA sequencing analysis.

The objective of this study was to uncover distinct cellular and genetic signatures of transplant operational tolerance (TOT) in kidney transplant recipients (KTRs) through single cell RNA sequencing (scRNA-seq) using peripheral blood mononuclear cells (PBMCs). PBMCs were isolated from 12 KTRs, including those with TOT (TOT, n = 4), stable allograft function on maintenance immunosuppression (STA, n = 4) and biopsy-proven allograft rejection (BPAR, n = 4). ScRNA-seq of PBMCs was analyzed using 20 cell surface marker antibody sequencing to annotate clusters and 399 immune response panel to identify gene expression. Differences in cellular distribution and gene expression were compared among the three groups. Heatmap hierarchical clustering showed that overall cellular distribution pattern was distinct in TOT in comparison with those in the other two groups, with the proportion of B cells being higher in TOT, attributed to immature B cell fraction (TOT vs. STA vs. BPAR: 4.61% vs. 1.27% vs. 2.53%, p = 0.01). Transcript analysis of B cells revealed that genes involved in allo-immune pathway were downregulated in TOT. In T cell subset analysis, the proportion of naïve T cells and regulatory T cells (Tregs) was increased. In transcript analysis, genes associated with inflammation were decreased, while expression levels of CCR6 in Tregs were increased in TOT. Proportions of NKT and NK cells were increased in TOT than in the other two groups. This study showed that TOT has distinct cellular and genetic signatures such as increases of immature B cells, naïve T cells and Tregs and high expression levels of CCR6 in Tregs.