Renal Ischemia-reperfusion Injury Model Research Articles

Panax Notoginseng Saponins Inhibit Apoptosis and Alleviate Renal Ischemia-Reperfusion Injury Through the ROCK2/NF-κB Pathway.

Renal ischemia-reperfusion injury (RIRI) is a primary cause of acute kidney injury (AKI), frequently resulting in high mortality rates and progression to chronic kidney disease (CKD). This study aimed to investigate the therapeutic potential of total saponins from Panax notoginseng (PNS) in the context of RIRI. Utilizing a murine RIRI model, the efficacy of PNS was evaluated, demonstrating a significant reduction in renal inflammation and cellular pyroptosis. Furthermore, PNS was found to modulate the ROCK2/NF-κB signaling pathway, thereby attenuating the inflammatory response. Importantly, in vitro experiments with hypoxia/reoxygenation cell models corroborated these findings, showing that PNS inhibited pyroptosis and regulated the ROCK2/NF-κB pathway. This research underscores the therapeutic potential of PNS in the treatment of RIRI, providing a robust scientific basis for its consideration as a prospective clinical therapy.

An Experimental Insight Into the Role of Agomelatine in Renal Ischemia/Reperfusion Injury.

Acute kidney injury (AKI) is one of the leading causes of chronic kidney disease and accounts for 50%-75% of mortality following renal pathologies or organ transplantation. Ischemia‒reperfusion injury (IRI) involves an interrupted blood supply to organs and the kidney; IRI exacerbates AKI development. Owing to several pharmacological treatment methods, AKI still has a poor prognosis, and novel therapeutic options are needed. Agomelatine (AGM) is a melatonin receptor agonist (MT1 and MT2) with increased bioavailability and lipophilicity. In this study, we aimed to investigate the antioxidant and anti-inflammatory effects of AGM in experimental renal IRI via long-term and short-term applications. Sixty male Sprague-Dawley rats were randomly divided into six groups (n = 10): the control, I/R, AGM20S, AGM40S, AGM20L, and AGM40L groups. Following the establishment of the renal IRI model, the rats received agomelatine at 20 and 40 mg/kg orally, and agomelatine solvent (hydroxyethylcellulose) was used as a vehicle. At the end of the experiment, blood samples and renal tissues were harvested for histopathological and biochemical analysis. Urea, creatinine, tumor necrosis factor (TNF-α), and interleukin-1 beta (IL-1β) levels were measured in blood serum samples. Malondialdehyde (MDA) levels and increased superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSHPx), and total glutathione (GSH) levels were measured in renal tissue supernatants. Our biochemical results indicated that AGM reduced creatinine, TNF-α, IL-1β, and malondialdehyde levels and increased SOD, CAT, GSHPx, and total GSH levels. Agolematine reduced infiltration, intratubular hemorrhage, and intratubular cast formation histopathologically. Our results suggest that AGM could be a potential therapeutic adjuvant agent for ischemia‒reperfusion injury in the kidney and several other organs.

Sympathetic nerve promotes renal fibrosis by activating M2 macrophages through β2-AR-Gsa

Sympathetic nervous system overactivation is directly related to renal fibrosis. This study focused on the role of and mechanism by which sympathetic signaling regulates macrophage activation, as well as the contribution to renal fibrosis. Renal denervation alleviated tubular necrosis, tubulointerstitial fibrosis, and macrophage accumulation induced by unilateral ureteral obstruction and ischemia-reperfusion injury. In vitro, norepinephrine (NE) promoted macrophage alternative (M2) polarization by activating β2-adrenergic receptor (β2-AR) and heterotrimeric G stimulatory protein α-subunit (Gsa). The effects of NE-induced macrophage M2 polarization were blocked by a β2-AR selective antagonist and Gsa siRNA. Importantly, ablation of Gsa in macrophages alleviated tubulointerstitial fibrosis, macrophage accumulation, and M2 polarization in the renal ischemia-reperfusion injury model. Sympathetic nervous system overactivation regulates M2 polarization in macrophages as an important neuroimmune mechanism of renal fibrosis. The β2-AR-Gsa signaling pathway was responsible for NE-induced macrophage M2 polarization, which may be a therapeutic target for renal fibrosis.

Molecular mechanisms of ferroptosis in renal ischemia-reperfusion injury Investigated via bioinformatics analysis and animal experiments.

Kidney transplantation is a pivotal treatment for end-stage renal disease. However, renal ischemia-reperfusion injury (IRI) during surgery significantly impacts graft function. Despite unclear molecular mechanisms, no specific therapies or preventative measures are available. Gene expression profiles from renal biopsies before and after IRI were downloaded from public databases. Differentially expressed genes were identified using the Wilcoxon rank-sum test and weighted gene co-expression network analysis. Ferroptosis-associated genes were screened using the FerrDb database. The genes with the highest connectivity were identified via the protein-protein interaction (PPI) network and upstream regulatory miRNAs were found through the gene-miRNA network. A mouse renal IRI model was constructed for transcriptome sequencing and quantitative real-time polymerase chain reaction (qRT-PCR) validation to elucidate the relationship between key ferroptosis genes and regulatory miRNAs in renal IRI. Differential analysis identified 15 ferroptosis-associated genes (TNFAIP3, IL6, KLF2, EGR1, JUN, ZFP36, GDF15, CDKN1A, HSPB1, BRD2, PDK4, DUSP1, SLC2A3, DDIT3, and CXCL2) involved in renal IRI regulation. In animal experiments, ferroptosis-related genes were also upregulated in the model group. Enrichment analysis and hematoxylin-eosin pathological staining suggested these genes are primarily involved in renal inflammatory responses. PPI network analysis revealed IL6 as the gene with the highest connectivity, and the gene-miRNA network indicated IL6 might be regulated by miR-let-7a. Animal experiments revealed decreased miR-let-7a and increased IL6 levels in the model group, identifying potential therapeutic targets. MiR-let-7a regulates ferroptosis in renal IRI by targeting IL6, highlighting IL6 as a crucial gene in the ferroptosis process of renal IRI.

Insights into Alkaline Phosphatase Anti-Inflammatory Mechanisms.

The endogenous ecto-enzyme and exogenously administered alkaline phosphatase (ALP) have been evidenced to significantly attenuate inflammatory conditions, including Toll-like receptor 4 (TLR4)-related signaling and cytokine overexpression, barrier tissue dysfunction and oxidative stress, and metabolic syndrome and insulin resistance, in experimental models of colitis, liver failure, and renal and cardiac ischemia-reperfusion injury. This suggests multiple mechanisms of ALP anti-inflammatory action that remain to be fully elucidated. Recent studies have contributed to a deeper comprehension of the role played by ALP in immune metabolism. This review outlines the established effects of ALP on lipopolysaccharide (LPS)-induced inflammation, including the neutralization of LPS and the modulation of purinergic signaling. The additional mechanisms of anti-inflammatory activity of ALP observed in different pathologies are proposed. The anti-inflammatory pathways of ALP may include a scavenger receptor (CD36)-mediated activation of β-oxidation and oxidative phosphorylation, caveolin-dependent endocytosis, and selective autophagy-dependent degradation.

Exploring Cuproptosis-Related Genes and Diagnostic Models in Renal Ischemia-Reperfusion Injury Using Bioinformatics, Machine Learning, and Experimental Validation.

Renal ischemia-reperfusion injury (RIRI) is a significant cause of acute kidney injury, complicating clinical interventions such as kidney transplants and partial nephrectomy. Recent research has indicated the role of cuproptosis, a copper-dependent cell death pathway, in various pathologies, but its specific involvement in RIRI remains insufficiently understood. This study aims to investigate the role of cuproptosis-related genes in RIRI and establish robust diagnostic models. We analyzed transcriptomic data from 203 RIRI and 188 control samples using bioinformatics tools to identify cuproptosis-related differentially expressed genes (CRDEGs). The relationship between CRDEGs and immune cells was explored using immune infiltration analysis and correlation analysis. Gene Set Enrichment Analysis (GSEA) was conducted to identify pathways associated with CRDEGs. Machine learning models, including Least Absolute Shrinkage and Selection Operator(LASSO) logistic regression, Support Vector Machine Recursive Feature Elimination (SVM-RFE), Clustering analysis, and weighted gene co-expression network analysis (WGCNA), were used to construct diagnostic gene models. The models were validated using independent datasets. Experimental validation was conducted in vivo using a mouse bilateral RIRI model and in vitro using an HK-2 cell hypoxia-reoxygenation (HR) model with copper chelation intervention. HE, PAS, and TUNEL staining, along with plasma creatinine and blood urea nitrogen (BUN) measurements, were used to evaluate the protective effect of the copper chelator D-Penicillamine (D-PCA) on RIRI in mice. JC-1 and TUNEL staining were employed to assess apoptosis in HK-2 cells under hypoxia-reoxygenation conditions. Immunofluorescence and Western blot (WB) techniques were used to verify the expression levels of the SDHB and NDUFB6 genes. A total of 18 CRDEGs were identified, many of which were significantly correlated with immune cell infiltration. GSEA revealed that these genes were involved in pathways related to oxidative phosphorylation and immune response regulation. Four key cuproptosis marker genes (LIPA, LIPT1, SDHB, and NDUFB6) were incorporated into a Cuproptosis Marker Gene Model(CMGM), achieving an area under the curve (AUC) of 0.741-0.834 in validation datasets. In addition, a five-hub-gene SVM model (MOAP1, PPP2CA, SYL2, ZZZ3, and SFRS2) was developed, demonstrating promising diagnostic performance. Clustering analysis revealed two RIRI subtypes (C1 and C2) with distinct molecular profiles and pathway activities, particularly in oxidative phosphorylation and immune responses. Experimental results showed that copper chelation alleviated renal damage and cuproptosis in both in vivo and in vitro models. Our study reveals that cuproptosis-related genes are significantly involved in RIRI, particularly influencing mitochondrial dysfunction and immune responses. The diagnostic models developed showed promising predictive performance across independent datasets. Copper chelation demonstrated potential therapeutic effects, suggesting that cuproptosis regulation may be a viable therapeutic strategy for RIRI. This work provides a foundation for further exploration of copper metabolism in renal injury contexts.

Nephroprotective role of resveratrol in renal ischemia-reperfusion injury: a preclinical study in Sprague-Dawley rats.

Renal ischemia-reperfusion injury (IRI) is a significant contributor to renal dysfunction, acute kidney injury (AKI), and associated morbidity and mortality. Resveratrol, a polyphenol and phytoalexin, is known for its anti-inflammatory, antioxidant, and anti-cancer properties. This study investigates the nephroprotective potential of resveratrol in a rat model of renal IRI. Twenty-eight male Sprague-Dawley rats were divided into four groups: Sham, IRI, DMSO, and Resveratrol. The Sham group underwent identical procedures without renal pedicle clamping, while the IRI group experienced 30min of ischemia followed by 2h of reperfusion. The DMSO group received dimethyl sulfoxide (DMSO) intraperitoneally 30min before ischemia, and the Resveratrol group received 30mg/kg resveratrol intraperitoneally 30min before ischemia. Biochemical parameters (Urea, creatinine, IL-1β, NF-κβ, SOD, GSH, Bcl-2, and caspase-3) and histopathological changes were assessed. IRI caused a substantial increase in serum creatinine, Urea, IL-1β, NF-κβ, and caspase-3 levels, while simultaneously decreasing SOD, GSH, and Bcl-2 levels. Resveratrol treatment mitigated these effects by lowering inflammatory and apoptotic markers, enhancing antioxidant defenses, and improving histological outcomes. Resveratrol demonstrates significant nephroprotective effects in renal IRI, primarily through its antioxidant, anti-inflammatory, and anti-apoptotic properties.

Bidirectional Impact of Varying Severity of Acute Kidney Injury on Calcium Oxalate Stone Formation

Introduction: Acute kidney injury (AKI) is a prevalent renal disorder. The occurrence of AKI may promote the formation of renal calcium oxalate stones by exerting continuous effects on renal tubular epithelial cells (TECs). We aimed to delineate the molecular interplay between AKI and nephrolithiasis. Methods: A mild (20 min) and severe (30 min) renal ischemia-reperfusion injury model was established in mice. Seven days after injury, calcium oxalate stones were induced using glyoxylate (Gly) to evaluate the impact of AKI on the formation of kidney stones. Transcriptome sequencing was performed on TECs to elucidate the relationship between AKI severity and kidney stones. Key transcription factors (TFs) regulating differential gene transcription levels were identified using motif analysis, and pioglitazone, ginkgetin, and fludarabine were used for targeted therapy to validate key TFs as potential targets for kidney stone treatment. Results: Severe AKI led to increased deposition of calcium oxalate crystals in renal, impaired kidney function, and upregulation of kidney stone-related gene expression. In contrast, mild AKI was associated with decreased crystal deposition, preserved kidney function, and downregulation of similar gene expression. Transcriptomic analysis revealed that genes associated with inflammation and cell adhesion pathways were significantly upregulated after severe AKI, while genes related to energy metabolism pathways were significantly upregulated after mild AKI. An integrative bioinformatic analysis uncovered a TF regulatory network within TECs, pinpointing that PKNOX1 was involved in the upregulation of inflammation-related genes after severe AKI, and inhibiting PKNOX1 function with pioglitazone could simultaneously reduce the increase of calcium oxalate crystals after severe AKI in kidney. On the other hand, motif analysis also revealed the protective role of STAT1 in the kidneys after mild AKI, enhancing the function of STAT1 with ginkgetin could reduce kidney stone formation, while the specific inhibitor of STAT1, fludarabine, could eliminate the therapeutic effects of mild AKI on kidney stones. Conclusion: Inadequate repair of TECs after severe AKI increases the risk of kidney stone formation, with the upregulation of inflammation-related genes regulated by PKNOX1 playing a role in this process. Inhibiting PKNOX1 function can reduce kidney stone formation. Conversely, after mild AKI, effective cell repair through upregulation of STAT1 expression can protect TEC function and reduce stone formation, and activating STAT1 function can also achieve the goal of treating kidney stones.

Comparative Effects of Fedratinib and Upadacitinib on Renal Ischemia-Reperfusion Injury in Rat Models

Renal ischemia-reperfusion injury (RIRI) is a severe condition that frequently occurs following kidney transplantation or surgical procedures affecting renal blood flow. Inflammatory responses, oxidative stress, and apoptotic pathways significantly contribute to RIRI. This study aimed to compare the renoprotective effects of Fedratinib, a JAK2 inhibitor, and Upadacitinib, a JAK1 inhibitor, in rat models of RIRI. Both inhibitors were administered one hour prior to ischemia induction, and the effects on renal function, inflammation, and cell death pathways were assessed. The findings revealed that both Fedratinib and Upadacitinib significantly reduced serum creatinine and urea levels, inflammatory cytokines (TNF-α, IL-6), and markers of apoptosis (BCL2/BAX) by suppressing the JAK/STAT signaling pathways. Histopathological analysis showed substantial reduction in renal tissue damage in the treated groups compared to controls. The study concludes that JAK inhibition by either drug provides significant renoprotection in RIRI, though with some differences in the degree of pathway inhibition and clinical implications.

APP-CD74 axis mediates endothelial cell-macrophage communication to promote kidney injury and fibrosis.

Kidney injuries often carry a grim prognosis, marked by fibrosis development, renal function loss, and macrophage involvement. Despite extensive research on macrophage polarization and its effects on other cells, like fibroblasts, limited attention has been paid to the influence of non-immune cells on macrophages. This study aims to address this gap by shedding light on the intricate dynamics and diversity of macrophages during renal injury and repair. During the initial research phase, the complexity of intercellular communication in the context of kidney injury was revealed using a publicly available single-cell RNA sequencing library of the unilateral ureteral obstruction (UUO) model. Subsequently, we confirmed our findings using an independent dataset from a renal ischemia-reperfusion injury (IRI) model. We treated two different types of endothelial cells with TGF-β and co-cultured their supernatants with macrophages, establishing an endothelial cell and macrophage co-culture system. We also established a UUO and an IRI mouse model. Western blot analysis, flow cytometry, immunohistochemistry and immunofluorescence staining were used to validate our results at multiple levels. Our analysis revealed significant changes in the heterogeneity of macrophage subsets during both injury processes. Amyloid β precursor protein (APP)-CD74 axis mediated endothelial-macrophage intercellular communication plays a dominant role. In the in vitro co-culture system, TGF-β triggers endothelial APP expression, which subsequently enhances CD74 expression in macrophages. Flow cytometry corroborated these findings. Additionally, APP and CD74 expression were significantly increased in the UUO and IRI mouse models. Immunofluorescence techniques demonstrated the co-localization of F4/80 and CD74 in vivo. Our study unravels a compelling molecular mechanism, elucidating how endothelium-mediated regulation shapes macrophage function during renal repair. The identified APP-CD74 signaling axis emerges as a promising target for optimizing renal recovery post-injury and preventing the progression of chronic kidney disease.

MicroRNA-223 alleviates inflammatory response in renal ischemia-reperfusion injury by targeting NLRP3.

We investigated the potential correlation between miR-223 and NAcHT, LRR, and PYd domain-containing protein 3 (NLRP3) in the context of renal ischemia-reperfusion injury (RIRI), which is a leading cause of acute renal failure with significant mortality rates. Additionally, miR-223 has been implicated in renal inflammation, further highlighting its relevance to this study. C57BL/6 male mice were used as RIRI models. After successful modeling, pathological examinations and serum creatinine and miR-223 levels were tested. Pro-inflammatory cytokine (IL-1β, IL-6, IL-8, NLPR3, TLR4) expression was detected in mice by western blot (kidney tissue) and enzyme-linked immunosorbent assay (serum). HK-2 cells were used for in vitro experiments. A hypoxia/reoxygenation (H/R) model was used, and miR-223 and pro-inflammatory cytokine levels were detected using PCR and western blot assays, respectively. A dual-luciferase reporter assay was conducted to confirm the binding of miR-223 to NLPR3. Next, NLRP3 was knocked down to determine whether the anti-inflammatory function of miR-223 is dependent on NLRP3. MiR-223 expression was lower in RIRI mice than in the sham operation group. The level of miR-223 negatively correlated with serum creatinine levels and the severity of tubule injury. Increased proinflammatory cytokine levels in RIRI mice were observed. In vitro, miR-223 alleviated the inflammatory response in H/R treated cells by inhibiting proinflammatory cytokines. Dual-luciferase reporter and western blot assays confirmed the binding of miR-223 to NLRP3. NLRP3 knockdown reversed the anti-inflammatory effects of miR-223 in HK-2 cells. MiR-223 plays an anti-inflammatory role in RIRI by targeting NLRP3 to repress pro-inflammatory factors.

Renal protective effects of helix B surface polypeptide in rats with puromycin aminonucleoside nephropathy

Background Recent studies have reported that helix B surface polypeptide (HBSP), an erythropoietin derivative, exhibits strong tissue protective effects, independent of erythropoietic effects, in a renal ischemia-reperfusion (IR) injury model. Meanwhile, the transforming growth factor-β (TGF-β) superfamily member glial cell line-derived neurotrophic factor (GDNF) demonstrated protective effect on podocytes in vitro. Using a rat puromycin aminonucleoside nephropathy (PAN) model, this study observed the renal protective effect of HBSP and investigated its renal protective effect on podocytes and mechanism related to GDNF. Methods Rats nephropathy model was induced by injection of 60 mg/kg of PAN via the tail vein. Rats in the PAN + HBSP group were injected intraperitoneally with HBSP (8 nmol/kg) 4 h before the model was induced, followed by intraperitoneal injections of HBSP once every 24 h for 7 consecutive days. The 24-hour urinary protein level was measured once every other day, and blood and renal tissue samples were collected on the 7th day for the examination of renal function, complete blood count, renal pathological changes and the expression levels of GDNF. Results Compared with the control group, the PAN nephropathy rat model showed a large amount of urinary protein. The pathological manifestations were mainly extensive fusion and disappearance of foot processes, along with vacuolar degeneration of podocytes and their separation from the glomerular basement membrane. GDNF expression was upregulated. Compared with the PAN + vehicle group, the PAN + HBSP group showed decreased urinary protein (p < 0.05). Pathological examination revealed ameliorated glomerular injury and vacuolar degeneration of podocytes. The expression of GDNF in the PAN nephropathy group was increased, when compared with the control group. The greatest expression of GDNF observed in the PAN + HBSP group (p < 0.05). Conclusions The expression of GDNF in the kidney of PAN rat model was increased. HBSP reduced urinary protein, ameliorated pathological changes in renal podocytes, increased the expression of GDNF in the PAN rat model. HBSP is likely to exert its protective effects on podocytes through upregulation of GDNF expression.

Ligustilide alleviates oxidative stress during renal ischemia-reperfusion injury through maintaining Sirt3-dependent mitochondrial homeostasis

BackgroundRenal ischemia-reperfusion (I/R) injury is an inevitable complication during renal transplantation and is closely related to patient prognosis. Mitochondrial damage induced oxidative stress is the core link of renal I/R injury. Ligustilide (LIG), a natural compound extracted from ligusticum chuanxiong hort and angelica sinensis, has exhibited the potential to protect mitochondrial function. However, whether LIG can ameliorate renal I/R injury requires further investigation. Delving deeper into the precise targets and mechanisms of LIG's effect on renal I/R injury is crucial. PurposeThis study aimed to elucidate the specific mechanism of LIG's protective effect on renal I/R injury. MethodsIn this study, an in vivo model of renal ischemia-reperfusion (I/R) injury was developed in mice, along with an in vitro model of hypoxia-reoxygenation (H/R) using human proximal renal tubular epithelial cells (HK-2). To assess the impact of LIG on renal injury, various methods were employed, including serum creatinine (Cr) and blood urea nitrogen (BUN) testing, hematoxylin and eosin (HE) staining, and immunohistochemistry (IHC) for kidney injury molecule-1 (KIM-1). The effects of LIG on oxidative stress were examined using fluorescent probes dihydroethidium (DHE) and dichlorodihydrofluorescein diacetate (DCFH-DA), TdT-mediated dUTP Nick-End Labeling (TUNEL) staining, and flow cytometry. Additionally, the influence of LIG on mitochondrial morphology and function was evaluated through transmission electron microscopy (TEM), Mito Tracker Red CMXRos staining, adenosine triphosphate (ATP) concentration assays, and JC-1 staining. The potential mechanism involving LIG and Sirt3 was explored by manipulating Sirt3 expression through cell transfection. ResultsThe results showed that LIG could provide protective function for mitochondria to alleviate oxidative stress induced by renal I/R. Further mechanistic studies indicated that LIG maintained mitochondrial homeostasis by targeting Sirt3. ConclusionOur findings demonstrated that LIG alleviated oxidative stress during renal I/R injury through maintaining Sirt3-dependent mitochondrial homeostasis. Overall, our data raised the possibility of LIG as a novel therapy for renal I/R injury.

Membranous translocation of murine double minute 2 promotes the increased renal tubular immunogenicity in ischemia-reperfusion-induced acute kidney injury.

Kidneys from donors with prolonged warm and cold ischemia are prone to posttransplant T cell-mediated rejection (TCMR) due to ischemia-reperfusion injury (IRI). However, the precise mechanisms still remain obscure. Renal tubular epithelial cells (TECs) are the main target during IRI. Meanwhile, we have previously reported that murine double minute 2 (MDM2) actively participates in TEC homeostasis during IRI. In this study, we established a murine model of renal IRI and a cell model of hypoxia-reoxygenation by culturing immortalized rat renal proximal tubule cells (NRK-52E) in a hypoxic environment for different time points followed by 24 h of reoxygenation and incubating NRK-52E cells in a chemical anoxia-recovery environment. We found that during renal IRI MDM2 expression increased on the membrane of TECs and aggregated mainly on the basolateral side. This process was accompanied by a reduction of a transmembrane protein, programmed death ligand 1 (PD-L1), a coinhibitory second signal for T cells in TECs. Using mutant plasmids of MDM2 to anchor MDM2 on the cell membrane or nuclei, we found that the upregulation of membrane MDM2 could promote the ubiquitination of PD-L1 and lead to its ubiquitination-proteasome degradation. Finally, we set up a coculture system of TECs and CD4+ T cells in vitro; our results revealed that the immunogenicity of TECs was enhanced during IRI. In conclusion, our findings suggest that the increased immunogenicity of TECs during IRI may be related to ubiquitinated degradation of PD-L1 by increased MDM2 on the cell membrane, which consequently results in T-cell activation and TCMR.NEW & NOTEWORTHY Ischemic acute kidney injury (AKI) donors can effectively shorten the waiting time for kidney transplantation but increase immune rejection, especially T cell-mediated rejection (TCMR), the mechanism of which remains to be elucidated. Our study demonstrates that during ischemia-reperfusion injury (IRI), the translocation of tubular murine double minute 2 leads to basolateral programmed death ligand 1 degradation, which ultimately results in the occurrence of TCMR, which may provide a new therapeutic strategy for preventing AKI donor-associated TCMR.

Ezetimibe ketone protects against renal ischemia-reperfusion injury and attenuates oxidative stress via activation of the Nrf2/HO-1 signaling pathway.

Recently, ezetimibe (EZM) has been suggested to be a potent Nrf2 activator that is important for preventing oxidative stress. Interestingly, we found that its metabolite ezetimibe ketone (EZM-K) also has antioxidant effects. Thus, we investigated the role of EZM-K in preventing renal ischemia‒reperfusion injury (RIRI). Cultured NRK-52E cells were subjected to simulated IR with or without EZM-K. Rats were used to simulate in vivo experiments. EZM-K alleviated H2O2-induced apoptosis and reactive oxygen species (ROS) and upregulated Nrf2 and HO-1 levels in NRK-52E cells. A HO-1 and a Nrf2 inhibitor reversed the protective effects of EZM-K. In the rat RIRI model, pretreatment with EZM-K activated the Nrf2/HO-1 signaling pathway, suppressed tubular injury and inflammation, and improved renal function. EZM-K significantly prevented renal injury caused by ischemia‒reperfusion via the Nrf2/HO-1 signaling axis both in vivo and in vitro. The other metabolite of EZM, ezetimibe glucuronide (EZM-G) had no protective effects against ROS in RIRI. EZM-G also had no antioxidant effects and could not activate Nrf2/HO-1 signal pathway. Our findings also indicated the therapeutic potential of EZM-K in preventing RIRI.

Multi-parametric MRI-based machine learning model for prediction of pathological grade of renal injury in a rat kidney cold ischemia-reperfusion injury model

BackgroundRenal cold ischemia-reperfusion injury (CIRI), a pathological process during kidney transplantation, may result in delayed graft function and negatively impact graft survival and function. There is a lack of an accurate and non-invasive tool for evaluating the degree of CIRI. Multi-parametric MRI has been widely used to detect and evaluate kidney injury. The machine learning algorithms introduced the opportunity to combine biomarkers from different MRI metrics into a single classifier.ObjectiveTo evaluate the performance of multi-parametric magnetic resonance imaging for grading renal injury in a rat model of renal cold ischemia-reperfusion injury using a machine learning approach.MethodsEighty male SD rats were selected to establish a renal cold ischemia -reperfusion model, and all performed multiparametric MRI scans (DWI, IVIM, DKI, BOLD, T1mapping and ASL), followed by pathological analysis. A total of 25 parameters of renal cortex and medulla were analyzed as features. The pathology scores were divided into 3 groups using K-means clustering method. Lasso regression was applied for the initial selecting of features. The optimal features and the best techniques for pathological grading were obtained. Multiple classifiers were used to construct models to evaluate the predictive value for pathology grading.ResultsAll rats were categorized into mild, moderate, and severe injury group according the pathologic scores. The 8 features that correlated better with the pathologic classification were medullary and cortical Dp, cortical T2*, cortical Fp, medullary T2*, ∆T1, cortical RBF, medullary T1. The accuracy(0.83, 0.850, 0.81, respectively) and AUC (0.95, 0.93, 0.90, respectively) for pathologic classification of the logistic regression, SVM, and RF are significantly higher than other classifiers. For the logistic model and combining logistic, RF and SVM model of different techniques for pathology grading, the stable and perform are both well. Based on logistic regression, IVIM has the highest AUC (0.93) for pathological grading, followed by BOLD(0.90).ConclusionThe multi-parametric MRI-based machine learning model could be valuable for noninvasive assessment of the degree of renal injury.

The use of ultrasound for noninvasive monitoring of anesthetic effects in renal ischemia-reperfusion injury.

Compared with the use of ultrasound for noninvasive monitoring of the anesthetic sodium pentobarbital versus tribromoethanol in an animal model of renal ischemia-reperfusion injury in rats. Adult rats were randomly assigned to a renal ischemia-reperfusion injury model, and preoperative anesthetics were administered as either sodium pentobarbital or tribromoethanol. Color Doppler ultrasound and spectral Doppler ultrasound were used to detect changes in respiratory rate and heart rate during and after the surgery, as well as measure renal hemodynamic parameters including peak systolic velocity, end-diastolic velocity, and resistance index. The frequency of changes in respiration and heart rate was significantly higher in the sodium pentobarbital anesthesia group compared to the tribromoethanol anesthesia group. The peak systolic velocity and end-diastolic velocity values in the sodium pentobarbital anesthesia group were significantly lower than those in the tribromoethanol group. However, the resistance index in the sodium pentobarbital group was higher than that in the tribromoethanol group. Ultrasound can be used to dynamically monitor the effects of anesthesia during the experiment, including changes in respiratory rate and heart rate, as well as semi-quantitatively monitor hemodynamic changes in the kidneys, which indirectly reflects whole-body hemodynamic changes in rats.

#348 Neutrophil membrane-derived nanovesicles deliver IL-37 to repair renal endothelial cells in ischemia‒reperfusion injury

Abstract Background and Aims Renal ischemia‒reperfusion injury (IRI) is one of the most important causes of acute kidney injury (AKI). Interleukin (IL)-37 is a novel anti-inflammatory factor, but its application is still limited by its low stability and delivery efficiency. In recent years, engineered extracellular vesicles (EVs) have attracted substantial attention as an emerging drug delivery system. The aim of this study was to investigate the reparative effect of neutrophil membranes-derived nanovesicle (N-MV)-delivered IL-37 on renal IRI and its mechanism of action. Method The density gradient centrifugation method was used to extract peripheral blood neutrophils from rats, and the differential centrifugation method was used to extract neutrophil membranes. Cell membranes and IL-37 were then extruded with polycarbonate porous membranes with different pore sizes using a mini extruder to prepare IL-37 that was coated with neutrophil membrane (N-MV@IL-37). Subsequently, the physical and chemical properties and in vitro biological functions were identified. The in vivo distribution of N-MV@IL-37 as well as the renal function and inflammatory response in rats were further evaluated in a rat renal IRI model, and the targeting and anti-inflammatory mechanisms of N-MV@IL-37 were further explored. Results N-MV@IL-37 not only enhanced the stability of IL-37 but also targeted endothelial cells via P-selectin glycoprotein ligand-1 (PSGL-1) on neutrophil membrane surfaces. In vitro experiments showed that N-MV@IL-37 inhibited endothelial cell apoptosis and inflammatory factor production and promoted endothelial cell viability and angiogenesis. In the rat renal IRI model, N-MV@IL-37 inhibited renal cell apoptosis and alleviated inflammatory responses, thereby improving renal function in IRI rats. Conclusion N-MVs provide an effective method of delivering IL-37, thereby alleviating the inflammatory response due to renal IRI and repairing renal endothelial cells; these vesicles could be a potential system for the treatment of renal IRI. Meanwhile, N-MVs also provided a drug delivery platform to provide a new strategy for the treatment of other IRI-like diseases.

Analysis of thioredoxin 1 levels in a mouse model of unilateral renal ischemia-reperfusion injury

Kidney disease, acute and chronic, is associated with high morbidity and mortality. Currently, a major obstacle in the field of nephrology is the lack of novel and more sensitive biomarkers that would allow an early diagnose and a proper monitoring of disease progression. Oxidative stress and the increased production of reactive oxygen species (ROS) have been implicated in the pathophysiology of a variety of renal diseases. Proteins of the thioredoxin (Trx) family, among others thioredoxins (Trxs) and glutaredoxins (Grxs), represent one of the most important protective systems in the defense against ROS as well as in the repair of their intracellular effects. Thioredoxin 1 (Trx1) is a cytosolic redox protein that catalyzes the reduction of protein disulfides using the thiol groups of two cysteine residues on its CGPC active site. Trx1 is abundantly expressed in different segments of the nephron and has been shown to be released from cells under conditions of oxidative stress in vitro and in vivo. We hypothesized that renal IRI induces the release of Trx1 from cells into extracellular fluids such as serum and tubular fluid/urine and that Trx1 increase may correlate with the degree of damage in the kidney. We established an effective mouse model of unilateral renal ischemia-reperfusion injury (IRI) and studied Trx1 levels in the serum, urine, as well as in various nephron segments of mice following IRI or sham surgery. We compared different reperfusion times (4h, 8h, 16h, 24h, 48h, 96h, and 168h) to track Trx1 levels fluctuation over time. Serum levels of Trx1 increased 4h after renal IRI, while other markers such as blood urea nitrogen and creatinine remained low. Serum Trx1 showed a positive correlation with tubular injury score helping predict intrarenal damage over time. Additionally, immunohistochemistry studies revealed an increase in Trx1 signals in the proximal tubules and collecting ducts as well as a strong nuclear localization in the same nephron segments that became more intense by 96h, a time point characterized by an intense mitotic activity in the tubules. Between 16 and 24h, Trx1 appeared in the lumen of proximal tubules, however urinary levels of Trx1 did not significantly increase. Based on these findings we conclude that serum Trx1 may represent a novel biomarker for assessing renal damage. In addition to this, we hypothesize that intrarenal changes in Trx1 expression and localization might be related to the activation of regeneration mechanisms in the renal tubular epithelium. Further studies are needed to understand the role of Trx1 in the regeneration process after renal IRI. This work was funded by the Center of Integrative Mammalian Research of Ross University School of Veterinary Medicine and by a Startup funding from Long Island University. 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.

Inhibition of Usp14 ameliorates renal ischemia-reperfusion injury by reducing Tfap2a stabilization and facilitating mitophagy

Mitochondrial dysfunction is recognized as a pivotal contributor to the pathogenesis of renal ischemia-reperfusion (IR) injury. Mitophagy, the process responsible for removing damaged protein aggregates, stands as a critical mechanism safeguarding cells against IR injury. Currently, the role of deubiquitination in regulating mitophagy still needs to be completely elucidated. This study aimed to evaluate the impact of ubiquitin-specific peptidase 14 (Usp14), a deubiquitinase, in IR injury by influencing mitophagy. Utilizing a murine model of renal IR injury, Usp14 silencing was found to ameliorate kidney injury, leading to decreased levels of serum creatinine and blood urea nitrogen, alongside diminished oxidative stress and inflammation. In renal epithelial cells subjected to hypoxia/reoxygenation (H/R), Usp14 knockdown increased cell viability and reduced apoptosis. Further mechanistic studies revealed that Usp14 interacted with and deubiquitinated transcription factor AP-2 alpha (Tfap2a), thereby suppressing its downstream target gene, TANK binding kinase 1 (Tbk1), to influence mitophagy. Tfap2a overexpression or Tbk1 inhibition reversed the protective effects of Usp14 silencing on renal tubular cell injury and its facilitation of mitophagy. In summary, our study demonstrated the renoprotective role of Usp14 knockdown in mitigating renal IR injury by promoting Tfap2a-mediated Tbk1 upregulation and mitophagy. These findings advocate for exploring Usp14 inhibition as a promising therapeutic avenue for mitigating IR injury, primarily by enhancing the clearance of damaged mitochondria through augmented mitophagy.