Kidney organoids derived from human pluripotent stem cells (hPSCs) represent a promising platform for modeling nephrogenesis and renal diseases. However, conventional differentiation protocols are continuous and time-sensitive, limiting their scalability and reproducibility. Here, we developed a method to pause and resume organoid formation through cryopreservation at an early differentiation stage using a chemically defined formulation that maintains high post-thaw viability and differentiation potential. We further found that synchronizing hPSCs in the G1 phase with PD-0332991 enhanced post-thaw organoid formation and transcriptional fidelity, while G2 phase enrichment with Ro-3306 promoted the development of SLC12A3-positive distal convoluted tubules. The post-thaw organoids exhibited well-organized nephron architecture and function comparable to uninterrupted cultured controls. This platform proved effective for modeling BK polyomavirus (BKV) infection, drug-induced nephrotoxicity, and renal fibrosis. Together, our cryopreservation and cell cycle synchronization strategy provides a flexible, practical framework to advance organoid-based research and translation. ### Competing Interest Statement Potential conflicts of interest include: Authors are named inventors on three patent applications covering aspects of the findings reported in this manuscript. These applications are currently pending review at the CNIPA. National Natural Science Foundation of China, 82471805, 82200846, 82470779, 82270789 Shanghai Municipal Key Clinical Specialty, shslczdzk05802 Shanghai Key Laboratory of Organ Transplantation, 09DZ2260300 Zhongshan Hospital, 2025XKPT32-5

Macrophage inflammatory plasticity is a central determinant of immune-mediated tissue injury, yet the intrinsic checkpoint mechanisms that restrain inflammatory macrophage programming remain incompletely defined. Here, using acute cardiac allograft rejection as an alloimmune tissue-injury model, we found that Vsir, encoding V-domain Ig suppressor of T-cell activation (VISTA), was preferentially expressed in graft macrophages but declined as macrophages acquired inflammatory and antigen-presenting states. Single-cell RNA sequencing and pseudotime analysis revealed that VISTA downregulation accompanied macrophage progression from reparative/resident-like states toward inflammatory programs. In vitro, VISTA overexpression intrinsically restrained pro-inflammatory macrophage polarization, reduced inflammatory cytokine expression, and limited the induction of antigen-presenting molecules including MHC-II and CD80, thereby attenuating macrophage-driven CD4+ T-cell proliferation. Integrative CUT&Tag and RNA-seq analyses further showed that VISTA overexpression was associated with reduced H3K4me3 -associated chromatin remodeling at inflammatory regulatory loci, including TRAF5 and CHDH, together with transcriptional repression of NF-κB-, TNF-, MAPK-, and IL-17-associated inflammatory programs. In vivo F4/80 promoter-directed Vsir restoration shifted intragraft macrophages away from inflammatory polarization, reduced T-cell accumulation, and attenuated early rejection-associated tissue injury. Together, these findings identify VISTA as an intrinsic regulator of macrophage inflammatory programming and point to H3K4me3-associated chromatin remodeling as a regulatory layer linked to VISTA-mediated inflammatory restraint.

Renal fibrosis, a progressive pathological feature of chronic kidney disease (CKD), is driven by impaired autophagic processes and persistent immune activation. The molecular mechanisms that interconnect these pathways remain inadequately understood. This study investigates the role of regulator of G-protein signaling 19 (RGS19), a novel autophagy-associated gene, in the pathogenesis of renal fibrosis. By analyzing transcriptomic data from the Gene Expression Omnibus (GEO) and applying machine learning algorithms, RGS19 was identified as a key fibrosis-related gene. In both in vitro and in vivo renal fibrosis models, we validated its functional role, focusing on autophagic flux and immune responses. We observed that RGS19 expression was elevated in fibrotic kidneys and correlated with increased CD8 + T cell infiltration. Knockdown of RGS19 using siRNA led to reduced p62 accumulation, suppressed rapamycin (p-mechanistic target of rapamycin (mTOR)) activity, and restored LC3B-II levels, reflecting enhanced autophagic flux. Additionally, the secretion of T cell chemoattractants, such as C-X-C motif chemokine ligand 9 (CXCL9) and C-X-C motif chemokine ligand 10 (CXCL10), was diminished. Notably, targeted delivery of RGS19 siRNA via RDYH58 nanoparticles effectively alleviated renal fibrosis in murine models by reducing collagen deposition and immune cell infiltration. These findings suggest that RGS19 plays a central role in linking autophagy dysfunction with immune activation in renal fibrosis and highlight its potential as a therapeutic target for CKD.

Renal ischemia reperfusion (IR) injury is one of the major causes of acute kidney injury (AKI) and may contribute to the development of chronic kidney disease. However, the precise immunological mediators orchestrating these pathophysiological processes remain poorly defined. In the present study, we investigate the protective role of the V-domain Ig suppressor of T cell activation (VISTA), which is an immune checkpoint molecule and highly expressed in macrophages, in attenuating IR-induced renal injury. Using genetic deficiency animal models, we demonstrate that both VISTA deficient (Vsir−/−) mice and macrophages-specific VISTA knockout (Vsirfl/flLyz2Cre) mice exhibited significantly exacerbated renal dysfunction and histopathological damage post-IR injury. Mechanistically, hypoxia-inducible factor-1α (HIF-α), as a transcriptional regulator, induced VISTA expression in macrophages post IR injury. VISTA deficiency in macrophages reprogrammed these cells toward a pro-inflammatory phenotype via promoting NF-κB nuclear translocation, which amplified Th1 differentiation through IL-12 upregulation while simultaneously suppressing regulatory T cell expansion. Notably, neutralizing IL-12 activity rescued renal injury in VISTA-deficient mice, underscoring its role as a key effector in this pathway. Therapeutically, exogenous VISTA administration attenuated renal inflammation, fibrosis, and functional impairment, highlighting its direct renoprotective capacity. Therefore, VISTA emerges as a sentinel checkpoint protein that balances macrophage polarization and T cell immunity during AKI.

Objective To explore the correlation between allogeneic red blood cell(RBC)transfusion and healthcare-associated infections(HAIs)in patients undergoing coronary artery bypass grafting(CABG)during the perioperative peri-od.Methods A single-center retrospective cohort of 1,170 patients undergoing isolated CABG was analyzed.Multivariable logistic regression and restricted cubic splines(RCS)were employed to explore the nonlinear association between periopera-tive RBC transfusion(from intraoperative period to 72 hours postoperatively)and HAIs.Results Among the 1,170 CABG patients,109 patients(9.2%)received RBC transfusion during the operation or within 3 days after the operation.The risk of HAIs in those who received≥4 units of RBCs during and within 3 days after the operation was 6.89 times higher than that in the non-transfusion group(95%CI:3.65-17.20).Furthermore,there was a nonlinear threshold effect between the blood transfusion volume and postoperative HAIs(inflection point:7.8 units).When the transfusion volume was≤7.8 u-nits,the risk of HAIs increased by 61%for each additional unit transfused(OR=1.61,95%CI:1.21-2.15).Beyond this threshold,no statistically significant association was observed(P=0.289).Conclusion Perioperative RBC transfusion in CABG patients is associated with an increased incidence of HAIs.The perioperative blood transfusion volume has a curvilinear relationship with the risk of postoperative HAIs.When the blood transfusion volume is≤7.8 units,the blood transfusion vol-ume has a dose-dependent relationship with postoperative infection,with higher blood transfusion volumes correlating with greater postoperative infection risk.When the blood transfusion volume is＞7.8 units,the relationship between the two is not statistically significant.The preventive effect of reducing RBC transfusion on HAIs requires further validation in the future.

CD29/CD44/CD105-modified immunomagnetic liposomes (CD29/CD44/CD105-IMLs) were developed to isolate bone marrow mesenchymal stem cells (BMSCs) with distinct phenotypes. A key BMSC phenotype capable of alleviating chronic cyclosporine-induced nephropathy was identified through in vivo experiments in mice, demonstrating its potential as an effective adjunctive therapy for mitigating renal fibrosis-induced injury. Four weeks post-transplantation, mice receiving CD29+, CD44+, and CD105+ BMSC groups exhibited improved overall health compared to the control group. Western blot analysis of autophagy-related proteins LC3-II and P62 revealed significantly lower expression levels in the CD44+ BMSC group compared to the CD29+ and CD105+ BMSC groups. In vivo imaging further demonstrated enhanced chemotactic ability of CD44+ BMSCs in mice with chronic cyclosporine-induced nephropathy at various time points. These findings suggest that transplantation of CD29+/CD44+/CD105+ BMSCs can delay the progression of renal fibrosis in mice with cyclosporine-induced chronic kidney disease. Notably, CD44+ BMSCs exhibited superior efficacy in alleviating renal fibrosis compared to CD29+ and CD105+ BMSC groups.

Background The study aims to evaluate the preliminary efficacy and safety of basiliximab in treating acute T cellu2010mediated rejection (TCMR) after kidney transplantation, particularly in patients with poor responses to standard treatments. Methods We conducted a retrospective case series study involving eight kidney transplant recipients at Zhongshan Hospital, Fudan University, who received basiliximab for antiu2010TCMR treatment. Baseline characteristics, immunosuppression management data, and treatment outcomes were collected. Results Among the eight patients, two had biopsyu2010proven TCMR. Elevated serum soluble ILu20102R (sILu20102R) level were detected in all patients before basiliximab treatment. Following basiliximab therapy, all patients exhibited a rapid decrease in sILu20102R levels, and most cases showed a general downward trend in serum creatinine levels. Although two patients developed pulmonary infections posttreatment, no clear correlation was found between the use of basiliximab and infections. No other new infections or adverse reactions were reported. Conclusions Basiliximab therapy demonstrates efficacy and good tolerance in patients with TCMR.

Intraoperative cell salvage (IOCS) has been widely applied as an important blood conservation measure in surgical operations. However, there is currently a lack of clinical practice guidelines for the implementation of IOCS in patients with malignant tumors. This report aims to provide clinicians with recommendations on the use of IOCS in patients with malignant tumors based on the review and assessment of the existed evidence. Data were derived from databases such as PubMed, Embase, the Cochrane Library and Wanfang. The guideline development team formulated recommendations based on the quality of evidence, balance of benefits and harms, patient preferences, and health economic assessments. This study constructed seven major clinical questions. The main conclusions of this guideline are as follows: 1) Compared with no perioperative allogeneic blood transfusion (NPABT), perioperative allogeneic blood transfusion (PABT) leads to a more unfavorable prognosis in cancer patients (Recommended); 2) Compared with the transfusion of allogeneic blood or no transfusion, IOCS does not lead to a more unfavorable prognosis in cancer patients (Recommended); 3) The implementation of IOCS in cancer patients is economically feasible (Recommended); 4) Leukocyte depletion filters (LDF) should be used when implementing IOCS in cancer patients (Strongly Recommended); 5) Irradiation treatment of autologous blood to be reinfused can be used when implementing IOCS in cancer patients (Recommended); 6) A careful assessment of the condition of cancer patients (meeting indications and excluding contraindications) should be conducted before implementing IOCS (Strongly Recommended); 7) Informed consent from cancer patients should be obtained when implementing IOCS, with a thorough pre-assessment of the patient's condition and the likelihood of blood loss, adherence to standardized internally audited management procedures, meeting corresponding conditions, and obtaining corresponding qualifications (Recommended). In brief, current evidence indicates that IOCS can be implemented for some malignant tumor patients who need allogeneic blood transfusion after physician full evaluation, and LDF or irradiation should be used during the implementation process.

BACKGROUND:Myeloid-derived suppressor cells (MDSCs) comprise monocytic MDSCs (M-MDSCs) and granulocytic MDSCs (G-MDSCs), both of which are effective for controlling T cell responses. Although Nrf2 participates in the expansion of MDSCs and MDSC-mediated immunosuppression, the underlying mechanisms of Nrf2 in MDSC differentiation remain poorly understood. METHODS:In this study, G-MDSCs or M-MDSCs were induced and sorted from the bone marrow (BM) of wild-type (WT) or Nrf2-/- mice using flow cytometry in vitro, with or without GW9662 treatment. Mouse models of tumorigenesis, acute kidney injury (AKI), and chronic kidney disease (CKD) were used to evaluate the immunosuppressive function of WT or Nrf2-/- M-MDSCs treated with or without GW9662. Histological analysis was performed to evaluate tumor angiogenesis or kidney injury. Immunohistochemical staining was used to evaluate lymphocyte infiltration in kidney. Masson's trichrome and Sirius red staining were performed to evaluate kidney fibrosis. Additionally, RNA sequencing (RNA-seq) was conducted to identify gene expression difference between WT and Nrf2-/- M-MDSCs, with real-time PCR or western blot used to validate key findings. RESULTS:The immunosuppressive function of M-MDSCs generated from BM of Nrf2-/- mice and sorted from Nrf2-/- tumor-bearing mice was dramatically enhanced compared to the G-MDSC counterparts. Moreover, M-MDSCs with an Nrf2 deficiency could effectively ameliorate the inflammatory disorders in mice with AKI or CKD. Intriguingly, the efficacy of Nrf2-/- M-MDSCs could be attributed to increased expression of inducible nitric oxide synthase but decreased levels of arginase 1, indicating an unconventional mechanism of activation. Further mechanistic studies using RNA-seq and bioinformatics analyses identified an essential role of peroxisome proliferator-activated receptor-γ (PPARγ) in the differentiation and suppressive ability of Nrf2-/- M-MDSCs with which the effect could be markedly blocked with GW9662, a specific PPARγ inhibitor. CONCLUSIONS:Our findings elucidate an immunoregulatory mechanism of Nrf2 deficiency related to M-MDSC function and provides a foundation for the application of Nrf2-/- M-MDSCs in inflammatory diseases.

Background Kidney transplantation stands out as the most effective renal replacement therapy for patients grappling with end-stage renal disease. However, post-transplant renal fibrosis is a prevalent and irreversible consequence, imposing a substantial clinical burden. Unfortunately, the clinical landscape remains devoid of reliable biological markers for diagnosing post-transplant renal interstitial fibrosis. Methods We obtained transcriptome and single-cell sequencing datasets of patients with renal fibrosis from NCBI Gene Expression Omnibus (GEO). Subsequently, we employed Weighted Gene Co-Expression Network Analysis (WGCNA) to identify potential genes by integrating core modules and differential genes. Functional enrichment analysis was conducted to unveil the involvement of potential pathways. To identify key biomarkers for renal fibrosis, we utilized logistic analysis, a LASSO-based tenfold cross-validation approach, and gene topological analysis within Cytoscape. Furthermore, histological staining, Western blotting (WB), and quantitative PCR (qPCR) experiments were performed in a murine model of renal fibrosis to verify the identified hub genes. Moreover, molecular docking and molecular dynamics simulations were conducted to explore possible effective drugs. Results Through WGCNA, the intersection of core modules and differential genes yielded a compendium of 92 potential genes. Logistic analysis, LASSO-based tenfold cross-validation, and gene topological analysis within Cytoscape identified four core genes (CD3G, CORO1A, FCGR2A, and GZMH) associated with renal fibrosis. The expression of these core genes was confirmed through single-cell data analysis and validated using various machine learning methods. Wet experiments also verified the upregulation of these core genes in the murine model of renal fibrosis. A positive correlation was observed between the core genes and immune cells, suggesting their potential role in bolstering immune system activity. Moreover, four potentially effective small molecules (ZINC000003830276-Tessalon, ZINC000003944422-Norvir, ZINC000008214629-Nonoxynol-9, and ZINC000085537014-Cobicistat) were identified through molecular docking and molecular dynamics simulations. Conclusion Four potential hub biomarkers most associated with post-transplant renal fibrosis, as well as four potentially effective small molecules, were identified, providing valuable insights for studying the molecular mechanisms underlying post-transplant renal fibrosis and exploring new targets.

AbstractNumerous myofibroblasts are arisen from endothelial cells (ECs) through endothelial to mesenchymal transition (EndMT) triggered by TGF-β. However, the mechanism of ECs transforms to a different subtype, or whether there exists an intermediate state of ECs remains unclear. In present study, we demonstrate Midkine (MDK) mainly expressed by CD31 + ACTA2+ECs going through partial EndMT contribute greatly to myofibroblasts by spatial and single-cell transcriptomics. MDK is induced in TGF-β treated ECs, which upregulates C/EBPβ and increases EndMT genes, and these effects could be reversed by siMDK. Mechanistically, MDK promotes the binding ability of C/EBPβ with ACTA2 promoter by stabilizing the C/EBPβ protein. In vivo, knockout of Mdk or conditional knockout of Mdk in ECs reduces EndMT markers and significantly reverses fibrogenesis. In conclusion, our study provides a mechanistic link between the induction of EndMT by TGF-β and MDK, which suggests that blocking MDK provides potential therapeutic strategies for renal fibrosis.

Background: Blood transfusion is an indispensable supportive therapy. It plays a pivotal role in the perioperative management of cardiac surgery. The aim of this study was to develop a model for predicting the transfusion volume in isolated mitral valve surgery. Methods: We gathered data from 677 patients undergoing isolated mitral valve surgery with and without simultaneous tricuspid valve operation. The dataset was partitioned into a training dataset (70%) and a testing dataset (30%). We evaluated 18 machine-learning algorithms, incorporating inputs from 36 demographic and perioperative features. Additionally, the performance of multiple linear regressions was compared with machine-learning algorithms. CatBoost was selected for further analysis, and Shapley additive explanation (SHAP) values were employed to evaluate feature importance. Finally, we explored the impact of various features on the accuracy of CatBoost by analyzing the reasons for misjudgment. Results: CatBoost outperformed all 18 machine learning algorithms with an R-squared value of 0.420, mean absolute error of 0.702, mean squared error of 1.208, and root mean squared error of 1.090, surpassing multiple linear regression. The analysis of the testing group achieved 72.5% accuracy. SHAP identified 20 pertinent features influencing transfusion volume. No significant differences were observed between correctly and incorrectly predicted groups in tricuspid valve repair, American Society of Anesthesiologists classification, or platelet count. Conclusion: CatBoost effectively predicts the intraoperative transfusion volume in mitral valve surgery, aiding clinicians in transfusion decision-making and enhancing patient care.

Renal fibrosis is a pathological endpoint of maladaptation after ischemia-reperfusion injury (IRI), and despite many attempts, no good treatment has been achieved so far. At the core of renal fibrosis is the differentiation of various types of cells into myofibroblasts. MSCs were once thought to play a protective role after renal IRI. However, growing evidence suggests that MSCs have a two-sided nature. In spite of their protective role, in maladaptive situations, MSCs start to differentiate towards myofibroblasts, increasing the myofibroblast pool and promoting renal fibrosis. Following renal IRI, it has been observed that Bone Marrow-Derived Mesenchymal Stem Cells (BM-MSCs) and Renal Resident Mesenchymal Stem Cells (RR-MSCs) play important roles. This review presents evidence supporting their involvement, discusses their potential mechanisms of action, and suggests several new targets for future research.