Oxidized ATP Suppresses B Lymphocyte Activity to Attenuate Antibody-mediated Rejection of Kidney Allografts in Mice.

BACKGROUND: Antibody-mediated rejection (AMR) is a major cause of renal allograft dysfunction and loss. Targeting B cells and/or donor-specific antibody removal using plasma exchange and anti-CD20 antibodies are increasingly used in clinical practice, but the efficacy remains limited. Recent studies suggest that targeting purinergic P2X7 receptor/ATP axis can have profound immune regulatory effects in transplant models, but the mechanisms involved remain incompletely defined. METHODS: Purified B cells were isolated from the spleen of Balb/C mice and cultured with oxidized ATP at different concentrations. Proliferation and differentiation of B cells were examined. Effects of oxidized ATP were examined in a presensitized animal model where kidney allograft rejection mimics aspects of clinical AMR. Histopathology was assessed at the time of rejection or on day 5 after kidney transplantation. Infiltrating immune cells in renal allografts were detected by flow cytometry. RESULTS: Oxidized ATP inhibited B-cell activation and proliferation in vitro, significantly attenuated histological signs of graft injury and prolonged kidney allograft survival. Mechanistically, oxidized ATP inhibited antibody secretion by activated B cells in response to lipopolysaccharide stimulation and markedly suppressed the production of donor-specific antibody in kidney allograft recipients. Oxidized ATP also reduced graft infiltration by other inflammatory cells. CONCLUSIONS: These findings provide evidence for the involvement of the purinergic P2X7 receptor pathway in AMR and suggest that targeting this pathways may have important clinical implications.

Development of a Handheld Nano-centrifugal Device for Visual Virus Detection.

Gold nanoparticles (AuNPs) colorimetric assays based on distance-dependent optical characteristics have been widely employed for bioanalysis. However, this assay is not effective for visually detecting low-concentration targets due to the faint color change. Here, we developed a handheld nano-centrifugal device which could separate the crosslinked and non-crosslinked AuNPs. Results showed that the handheld nano-centrifugal device could easily reach more than 6000 r/min within 10 s simply by stretching and tightening the coiled rope in an appropriate rhythm. Further, combined with the CRISPR/Cas12a nucleic acids recognition system, a field-deployable colorimetric platform termed handheld nano-centrifugal device assisted CRISPR/Cas12a (Hand-CRISPR) has been validated. Moreover, clinical diagnostics applications for Epstein-Barr virus (EBV) and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) detection with high sensitivity and accuracy (100% consistency with reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) test results) have been demonstrated. Overall, the Hand-CRISPR platform showed great promise in point-of-care-test (POCT) application, expected to become a powerful supplement to the standard nucleic acid testing method in remote or poverty-stricken areas. Supplementary Information: The online version contains supplementary material available at 10.1007/s41664-022-00232-0.

Point-of-care non-invasive enzyme-cleavable nanosensors for acute transplant rejection detection.

Accurate and non-invasive monitoring of allograft posttransplant is essential for early detection of acute cellular rejection and determines the long-term survival of the graft. Clinically, tissue biopsy is the most effective approach for diagnosing transplant rejection. Nonetheless, the procedure is invasive and potentially triggers organ failure. This work aims to design and apply GzmB-responsive nanosensors (GBRNs) that can readily size-change in graft tissues. Subsequently, we investigate the activity of serine protease granzyme B by generating a direct colorimetric urinary readout for non-invasive detection of transplant rejection in under 1 h. In preclinical heart graft mice models of transplant rejection, GBRNs were cleaved by GzmB and excreted by the kidneys via accurate nanometre-size glomerular filtration. By exploiting the catalytic activity of ultrasmall gold nanoclusters, GBRNs urinalysis promotes ultrasensitive surveillance of rejection episodes with a receiver operator characteristic curve area under the curve of 0.896 as well as a 95% confidence interval of about 0.7701-1.000. Besides, the catalytic activity of gold nanoclusters in urine can be detected at point-of-care testing to predict the immunity responses in mice with insufficient immunosuppressive therapy. Therefore, this non-invasive, sensitive, and quantitative method is a robust and informative approach for rapid and routine monitoring of transplant allografts without invasive biopsy.

Photocontrolled crRNA activation enables robust CRISPR-Cas12a diagnostics.

CRISPR diagnostics based on nucleic acid amplification faces barriers to its commercial use, such as contamination risks and insufficient sensitivity. Here, we propose a robust solution involving optochemical control of CRISPR RNA (crRNA) activation in CRISPR detection. Based on this strategy, recombinase polymerase amplification (RPA) and CRISPR-Cas12a detection systems can be integrated into a completely closed test tube. crRNA can be designed to be temporarily inactivated so that RPA is not affected by Cas12a cleavage. After the RPA reaction is completed, the CRISPR-Cas12a detection system is activated under rapid light irradiation. This photocontrolled, fully closed CRISPR diagnostic system avoids contamination risks and exhibits a more than two orders of magnitude improvement in sensitivity compared with the conventional one-pot assay. This photocontrolled CRISPR method was applied to the clinical detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA, achieving detection sensitivity and specificity comparable to those of PCR. Furthermore, a compact and automatic photocontrolled CRISPR detection device was constructed.

Cuff Anastomosis of Both Renal Artery and Vein to Minimize Thrombosis: A Novel Method of Kidney Transplantation in Mice.

OBJECTIVES: Anastomosis of renal artery and renal vein in mouse models of kidney transplantation is technically challenging. Conventional technique using suture may result in vascular thrombosis. We developed a simple cuff method to anastomose both renal artery and vein. MATERIALS AND METHODS: Briefly, the left renal artery was occluded at the junction with abdominal aorta using a small vessel clip, transected at the renal hilum, irrigated with heparinized saline, and passed through the lumen of a seamless tubing made of polyimide. The loose end of the artery was everted over the cuff and secured using an 8-0 silk suture. The cuffed artery was inserted into the donor renal artery and secured with an 8-0 suture. Anastomosis of the renal vein was performed similarly. Isograft transplantation was conducted using BALB/c mice as donor and recipient mice (n = 20). The total operative time was 77 ± 3 min, and the cold ischemic time of the graft kidney was minimized to 20 min. One animal was excluded due to anatomic variant vessels and another one died at three day after surgery without thrombosis. RESULTS: Serum creatinine increased insignificantly after transplantation and remained stable over 12 weeks posttransplant. Five recipient mice were sacrificed for histologic examination at 12 weeks after transplantation. No vascular thrombosis was observed at the site of anastomosis. The isografts showed no evidence of acute and chronic lesions such as extinctive ischemic sclerosis and interstitial fibrosis. CONCLUSION: In summary, cuff anastomosis can be used to eliminate thrombosis formation in the mouse model of kidney transplantation.

Curcumin attenuates renal ischemia reperfusion injury via JNK pathway with the involvement of p300/CBP-mediated histone acetylation.

Apoptosis is proved responsible for renal damage during ischemia/reperfusion. The regulation for renal apoptosis induced by ischemia/reperfusion injury (IRI) has still been unclearly characterized to date. In the present study, we investigated the regulation of histone acetylation on IRI-induced renal apoptosis and the molecular mechanisms in rats with the application of curcumin possessing a variety of biological activities involving inhibition of apoptosis. Sprague-Dawley rats were randomized into four experimental groups (SHAM, IRI, curcumin, SP600125). Results showed that curcumin significantly decreased renal apoptosis and caspase-3/-9 expression and enhanced renal function in IRI rats. Treatment with curcumin in IRI rats also led to the decrease in expression of p300/cyclic AMP response element-binding protein (CBP) and activity of histone acetyltransferases (HATs). Reduced histone H3 lysine 9 (H3K9) acetylation was found near the promoter region of caspase-3/-9 after curcumin treatment. In a similar way, SP600125, an inhibitor of c-Jun N-terminal kinase (JNK), also attenuated renal apoptosis and enhanced renal function in IRI rats. In addition, SP600125 suppressed the binding level of p300/CBP and H3K9 acetylation near the promoter region of caspase-3/-9, and curcumin could inhibit JNK phosphorylation like SP600125. These results indicate that curcumin could attenuate renal IRI via JNK/p300/CBP-mediated anti-apoptosis signaling.

NF45/NF90-mediated rDNA transcription provides a novel target for immunosuppressant development.

Herein, we demonstrate that NFAT, a key regulator of the immune response, translocates from cytoplasm to nucleolus and interacts with NF45/NF90 complex to collaboratively promote rDNA transcription via triggering the directly binding of NF45/NF90 to the ARRE2-like sequences in rDNA promoter upon T-cell activation in vitro. The elevated pre-rRNA level of T cells is also observed in both mouse heart or skin transplantation models and in kidney transplanted patients. Importantly, T-cell activation can be significantly suppressed by inhibiting NF45/NF90-dependent rDNA transcription. Amazingly, CX5461, a rDNA transcription-specific inhibitor, outperformed FK506, the most commonly used immunosuppressant, both in terms of potency and off-target activity (i.e., toxicity), as demonstrated by a series of skin and heart allograft models. Collectively, this reveals NF45/NF90-mediated rDNA transcription as a novel signaling pathway essential for T-cell activation and as a new target for the development of safe and effective immunosuppressants.

Dose- and Time-Dependent Effects of Human Mesenchymal Stromal Cell Infusion on Cardiac Allograft Rejection in Mice.

Heart transplantation is the final life-saving therapeutic strategy for many end-stage heart diseases. Long-term immunosuppressive regimens are needed to prevent allograft rejection. Mesenchymal stromal cells (MSCs) have been shown as immunomodulatory therapy for organ transplantation. However, the effect of dose and timing of MSC treatment on heart transplantation has not yet been examined. In this study, we infused three doses (1 × 106, 2 × 106, or 5 × 106 cells) of human MSCs (hMSCs) to the recipient BALB/c mice before (7 days or 24 h) or after (24 h) receiving C57BL/6 cardiac transplants. We found that infusion of high dose hMSCs (5 × 106) at 24 h post-transplantation significantly prolonged the survival time of cardiac grafts. To delineate the underlying mechanism, grafts, spleens, and draining lymph nodes were harvested for analysis. Dose-dependent effect of hMSC treatment was shown in: (1) alleviation of International Society of Heart and Lung Transplantation (ISHLT) score in grafts; (2) reduction of the population of CD4+ and CD8+ T cells; (3) increase of regulatory T (Treg) cells; (4) and decrease of serum levels of inflammatory cytokines and donor-specific antibodies. Taken together, we showed timing critical and dose-dependent immunomodulatory effects of hMSC treatment against acute allograft rejection in a mouse model of heart transplantation.

Human urine-derived stem cells protect against renal ischemia/reperfusion injury in a rat model via exosomal miR-146a-5p which targets IRAK1.

Rationale: Ischemia/reperfusion injury (IRI) is a major cause of acute kidney injury (AKI) that is associated with high morbidity and mortality, and for which specific treatments are lacking. In this study, we investigated the protective effect of human urine-derived stem cells (USCs) and their exosomes against IRI-induced AKI to explore the potential of these cells as a new therapeutic strategy. Methods: USCs were derived from fresh human urine. Cell surface marker expression was analyzed by flow cytometry to determine the characteristics of the stem cells. Adult male Sprague-Dawley rats were used to generate a lethal renal IRI model. One dose of USCs (2×106 cells/ml) or exosomes (20 µg/1 ml) in the experimental groups or saline (1 ml) in the control group was administered intravenously immediately after blood reperfusion. Blood was drawn every other day for measurement of serum creatinine (sCr) and blood urea nitrogen (BUN) levels. The kidneys were harvested for RNA and protein extraction to examine the levels of apoptosis and tubule injury. In vitro, the hypoxia-reoxygenation (H/R) model in human kidney cortex/proximal tubule cells (HK2) was used to analyze the protective ability of USC-derived exosomes (USC-Exo). Quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR), western blotting, superoxide dismutase activity, and malonaldehyde content analyses were used to evaluate oxidative stress in HK2 cells treated with USC-Exo after H/R. Exosomal microRNA sequencing techniques and bioinformatics analysis were used to search for enriched miRNAs in the exosomes and interacting genes. The interaction between miRNAs and the 3' untranslated region of the target gene was detected using a dual luciferase reporting system. The miRNA mimic and inhibitor were used to regulate the miRNA level in HK2 cells. Results: Treatment with USCs led to reductions in the levels of sCr, BUN, and renal tubular cell apoptosis; inhibited the infiltration of inflammatory cells; and protected renal function in the rat IRI model. Additionally, USC-derived exosomes protected against IRI-induced renal damage. miR-146a-5p was the most abundant miRNA in exosomes obtained from the conditioned medium (CM) of USCs. miR-146a-5p targeted and degraded the 3'UTR of interleukin-1 receptor-associated kinase 1 (IRAK1) mRNA, subsequently inhibited the activation of nuclear factor (NF)-κB signaling, and protected HK2 cells from H/R injury. USC transplantation also upregulated miR-146a-5p expression, downregulated IRAK1 expression and inhibited nuclear translocation of NF-κB p65 in the kidney of the rat IRI model. Conclusions: According to our experimental results, USCs could protect against renal IRI via exosomal miR-146a-5p, which could target the 3'UTR of IRAK1 and subsequently inhibit the activation of NF-κB signaling and infiltration of inflammatory cells to protect renal function. As a novel cell source, USCs represent a promising non-invasive approach for the treatment of IRI.

Ginkgo biloba extract EGb761 attenuates brain death-induced renal injury by inhibiting pro-inflammatory cytokines and the SAPK and JAK-STAT signalings.

This study aimed to investigate the protective effects of EGb761, a Ginkgo Biloba extract, against brain death-induced kidney injury. Sixty male Sprague Dawley rats were randomly divided into six groups: sham, brain-death (BD), BD + EGb b48h (48 hours before BD), BD + EGb 2 h (2 hours after BD), BD + EGb 1 h, and BD + EGb 0.5 h. Six hours after BD, serum sample and kidney tissues were collected for analyses. The levels of blood urea nitrogen (BUN) and serum creatinine significantly elevated in the BD group than in sham group. In all the EGb761-treated BD animals except for the BD + Gb 2 h group, the levels of BUN and serum creatinine significantly reduced (all P < 0.01). EGb761 attenuated tubular injury and lowered the histological score. In addition, the longer duration of drug treatment was, the better protective efficacy could be observed. EGb761 significantly reduced IL-1ß, IL-6, TNF-α, MCP-1, IP-10 mRNA expression and macrophage infiltration in the kidney. EGb761 treatment at 48 hour before brain death significantly attenuate the levels of p-JNK-MAPK, p-p38-MAPK, and p-STAT3 proteins (all P < 0.05, compared to BD group). In summary, our data showed that EGb761 treatment protected donor kidney from BD-induced damages by blocking SAPK and JAK-STAT signalings. Early administration of EGb761 can provide better protective efficacy.