Renal Ischemia-reperfusion Injury Model Research Articles

Theaflavin pretreatment ameliorates renal ischemia/reperfusion injury by attenuating apoptosis and oxidative stress in vivo and in vitro

Oxidative stress-induced apoptosis is an important pathological process in renal ischemia/reperfusion injury (RIRI). Theaflavin (TF) is the main active pigment and polyphenol in black tea. It has been widely reported because of its biological activity that can reduce oxidative stress and protect against many diseases. Here, we explored the role of theaflavin in the pathological process of RIRI. In the present study, the RIRI model of 45 min ischemia and 24 h reperfusion was established in C57BL/6 J male mice, and theaflavin was used as an intervention. Compared with the RIRI group, the renal filtration function, renal tissue damage and antioxidant capacity of the theaflavin intervention group were significantly improved, while the level of apoptosis was reduced. TCMK-1 cells were incubated under hypoxia for 48 h and then reoxygenated for 6 h to simulate RIRI in vitro. The application of theaflavin significantly promoted the translocation of p53 from cytoplasm to nucleus, upregulated the expression of glutathione peroxidase 1 (GPx-1) in cells, and inhibited oxidative stress damage and apoptosis. Transfection with p53 siRNA can partially inhibit the effect of theaflavin. Thus, theaflavin exerted a protective effect against RIRI by inhibiting apoptosis and oxidative stress via regulating the p53/GPx-1 pathway. We conclude that theaflavin has the potential to become a candidate drug for the prevention and treatment of RIRI.

Lrg1 silencing attenuates ischemia-reperfusion renal injury by regulating autophagy and apoptosis through the TGFβ1- Smad1/5 signaling pathway

BackgroundDysfunction in the processes of autophagy and apoptosis within renal tubular epithelial cells (RTEc) contributes to renal ischemia-reperfusion injury (IRI). However, the factors influencing this dysfunction remain unclear. Leucine-rich alpha-2-glycoprotein 1 (Lrg1) plays a role in the progression of diabetic nephropathy and kidney fibrosis by modulating the activin receptor-like kinase 1 (ALK1)-Smad1/5/8 and TGF-β1/Smad3 pathways, respectively. Therefore, we aimed to investigate whether Lrg1 is involved in the pathological mechanisms of renal IRI and whether its effects are related to the dysregulation of autophagy and apoptosis in RTEc. MethodsWe conducted in vitro and in vivo experiments using CoCl2-induced hypoxic human kidney-2 (HK-2) cells and mice with renal IRI, respectively. Lrg1 was silenced using siRNA and lentiviral vectors in HK-2 cells and mouse kidneys. Rapamycin (Rapa) and methyladenine were applied to regulate autophagy in renal IRI models. ResultsIncreased Lrg1 expression was observed in hypoxic HK-2 cells and in the kidneys of mice with renal IRI. Silencing of Lrg1 through siRNA and lentiviral approaches restored autophagy and suppressed apoptosis in CoCl2-induced hypoxic HK-2 cells and renal IRI models. Additionally, reduced Lrg1 expression alleviated kidney damage caused by renal IRI. The downregulation of Lrg1 expression restrained the TGFβ-Smad1/5 signaling pathway in hypoxic-induced HK-2 cells and renal IRI by reducing ALK1 expression. Lastly, the enhancement of autophagy, achieved through Rapa treatment, provided protection against renal IRI in mice. ConclusionsOur findings suggest that Lrg1 silencing can be applied as a potential therapeutic target to inhibit the TGFβ1-Smad1/5 pathway, thereby enhancing autophagy and decreasing apoptosis in patients with acute kidney injury.

M6A methylase WTAP participates in renal ischemia-reperfusion injury by regulating FOXO1 expression

To investigate the expression of WTAP, a m6A methylase, in a mouse model of renal ischemia-reperfusion (I/R) injury and the effect of WTAP knockdown on biological behavior of renal tubular epithelial cells exposed to I/R injury. Sixteen C57BL/6 mice with renal I/R injury or sham operation (n=8) were examined for blood urea nitrogen (BUN) and creatinine (Scr) levels to assess renal function, and renal pathologies were observed with HE staining. The expressions of WTAP and FOXO1 proteins in the kidneys of the mice were detected using immunohistochemistry. Human renal tubular epithelial cells (HK-2) were transfected with si-WTAP or si-NC followed by hypoxia-reoxygenation (H/R) exposure, Protein and mRNA expression were assessed by Western blot and qRT-PCR, and changes and changes in cell viability and apoptosis were assessed using CCK8 assay and TUNEL staining, respectively; LDH release level and caspase-3 activity of the cells were measured using commercial assay kits. FOXO1 m6A modification sites were predicted using SRAMP website (http://www.cuilab.cn/sramp/), and the interaction between WTAP and FOXO1 mRNA was analyzed with RIP experiment; the level of FOXO1 modified by m6A was detected by MeRIP-qPCR. Compared with sham-operated mice, the mice with renal I/R injury showed significantly increased Scr and BUN levels (P < 0.001) and renal expressions of WTAP mRNA and protein (P < 0.001). In cultured HK-2 cells, H/R exposure significantly decreased the cell viability (P < 0.001) and increased cellular LDH release (P < 0.001) and expressions of WTAP mRNA and protein (P < 0.001). WTAP knockdown obviously reduced the cell damage induced by I/R injury and significantly decreased the mRNA and protein levels of FOXO1 in the cells (P < 0.001). RIP experiment confirmed WTAP binding to FOXO1 mRNA, and inhibition of WTAP expression significantly reduced FOXO1 m6A level in HK-2 cells (P < 0.001). WTAP expression is up-regulated in the kidneys of mice with renal I/R injury and in HK-2 cells with H/R exposure. Inhibition of WTAP alleviates H/R-induced apoptotic damage in HK-2 cells possibly by inhibiting FOXO1 expression.

Molecular mechanism of NR4A1/MDM2/P53 signaling pathway regulation inducing ferroptosis in renal tubular epithelial cells involved in the progression of renal ischemia-reperfusion injury

Revealing the possible molecular mechanism of the NR4A1 (nuclear receptor subfamily 4 group A member 1)-MDM2 (MDM2 proto-oncogene)-P53 (tumor protein p53) signaling pathway that induces ferroptosis in renal tubular epithelial cells. Renal ischemia-reperfusion injury (RIRI) -related datasets were obtained from the GEO database. Differentially expressed genes in RIRI were analyzed using R language, intersected with RIRI-related genes in the GeneCard database, and retrieved from the literature to finally obtain differential ferroptosis-related genes. An in vitro cell model of RIRI was constructed using mouse renal cortical proximal tubule epithelial cells (mRTEC cells) treated with hypoxia-reoxygenation (H/R). Bioinformatic analysis showed that NR4A1 may be involved in RIRI through the induction of ferroptosis; in addition, we predicted through online databases that the downstream target gene of NR4A1, MDM2, could be targeted and regulated by ChIP and dual luciferase assays, and that NR4A1 could prevent MDM2 by inhibiting it, and NR4A1 was able to promote ferroptosis by inhibiting the ubiquitinated degradation of P53. NR4A1 expression was significantly increased in mRTEC cells in the hypoxia/reoxygenation model, and the expression of ferroptosis-related genes was increased in vitro experiments. NR4A1 reduces the ubiquitinated degradation of P53 by targeting the inhibition of MDM2 expression, thereby inducing ferroptosis and ultimately exacerbating RIRI by affecting the oxidative respiration process in mitochondria and producing oxidized lipids. This study presents a novel therapeutic approach for the clinical treatment of renal ischemia-reperfusion injury by developing drugs that inhibit NR4A1 to alleviate kidney damage caused by renal ischemia-reperfusion.

Targeted echogenic and anti-inflammatory polymeric prodrug nanoparticles for the management of renal ischemia/reperfusion injury

Ischemia/reperfusion (IR) injury is an inevitable pathological event occurring when blood is resupplied to the tissues after a period of ischemia. One of major causes of IR injury is the overproduction of reactive oxygen species (ROS) including hydrogen peroxide (H2O2), which mediates the expression of various inflammatory cytokines to exacerbate tissue damages. The overproduced H2O2 could therefore serve as a diagnostic and therapeutic biomarker of IR injury. In this study, poly(boronated methacrylate) (pBMA) nanoparticles were developed as nanotheranostic agents for renal IR injury, which not only generate CO2 bubbles to enhance the ultrasound contrast but also provide potent preventive effects in a H2O2-triggered manner. The surface of pBMA nanoparticles was decorated with taurodeoxycholic acid (TUDCA) that binds P-selectin overexpressed in inflamed tissues. In the mouse model of renal IR injury, TUDCA-coated pBMA (T-pBMA) nanoparticles preferentially accumulated in the injured kidney and markedly enhanced the ultrasound contrast. T-pBMA nanoparticles also effectively prevented renal IR injury by scavenging H2O2 and suppressing the expression of inflammatory cytokines. Treatment progress of IR injury could be also monitored by echogenic T-pBMA nanoparticles. Given their targeting ability, excellent H2O2-responsiveness, anti-inflammatory activity and H2O2-triggered echogenicity, T-pBMA nanoparticles have excellent translational potential for the management of various H2O2-related diseases including IR injury.

Sevoflurane improves renal ischemia-reperfusion injury in rats through RISK signaling pathway

Purpose: To investigate the effect of sevoflurane on renal ischemia-reperfusion injury (IRI) in rats and its regulatory effect on reperfusion injury salvage kinase (RISK) signaling pathway.Methods: A total of thirty (30) Sprague-Dawley rats were randomly divided into sham, model and sevoflurane groups with 10 animals per group. Renal IRI models were created in model and sevoflurane groups, while sham group had no ligation. Renal injury was determined using hematoxylin and eosin (HE) staining. Blood urea nitrogen (BUN) and serum creatinine (Scr) levels were assayed while apoptosis was determined via terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining. Enzyme-linked immunosorbent assay (ELISA) was used to assess malonaldehyde (MDA) content and inflammatory factors in kidney tissues and peripheral blood, respectively. Reactive oxygen species (ROS) level was determined using 2,7-dichlorodi-hydro fluorescein diacetate (DCFHDA) while Western blotting was used to determine the expression of apoptosis- and RISK signaling pathway-related proteins in kidney tissues.Results: Compared to model group, renal injury in sevoflurane group rats was significantly alleviated (p &lt; 0.01). The levels of BUN and Scr in peripheral blood, apoptosis level in kidney tissues, MDA content and ROS level in kidney tissues, interleukin-1β (IL-1β), tumor necrosis factor-alpha (TNF-α) and IL-6 content, and content of caspase-3 protein in kidney tissues were significantly reduced (p &lt; 0.01), whereas IL-10 content, Bcl-2/Bax ratio and expression levels of p-ERK1/2, p-Akt and phosphorylated glycogen synthase kinase 3β (p-GSK-3β) were significantly increased (p &lt; 0.01) in the sevoflurane group.Conclusion: Sevoflurane represses the release of inflammatory factors, lowers ROS level and apoptosis of renal cells and improves renal function through activation of RISK signaling pathway in kidney tissues of rats with renal IRI. Thus, sevoflurane is a potential agent for the treatment of IRI.

Small extracellular vesicles delivering lncRNA WAC-AS1 aggravate renal allograft ischemia‒reperfusion injury by inducing ferroptosis propagation.

Ferroptosis is a predominant contributor to renal ischemia reperfusion injury (IRI) after kidney transplant, evoking delayed graft function and poorer long-term outcomes. The wide propagation of ferroptosis among cell populations in a wave-like manner, developing the "wave of ferroptosis" causes a larger area of tubular necrosis and accordingly aggravates renal allograft IRI. In this study, we decipher a whole new metabolic mechanism underlying ferroptosis and propose a novel spreading pathway of the "wave of ferroptosis" in the renal tissue microenvironment, in which renal IRI cell-secreted small extracellular vesicles (IRI-sEVs) delivering lncRNA WAC-AS1 reprogram glucose metabolism in adjacent renal tubular epithelial cell populations by inducing GFPT1 expression and increasing hexosamine biosynthesis pathway (HBP) flux, and consequently enhances O-GlcNAcylation. Additionally, BACH2 O-GlcNAcylation at threonine 389 in renal tubular epithelial cells prominently inhibits its degradation by ubiquitination and promotes importin α5-mediated nuclear translocation. We present the first evidence that intranuclear BACH2 suppresses SLC7A11 and GPX4 transcription by binding to their proximal promoters and decreases cellular anti-peroxidation capability, accordingly facilitating ferroptosis. Inhibition of sEV biogenesis and secretion by GW4869 and knockout of lncRNA WAC-AS1 in IRI-sEVs both unequivocally diminished the "wave of ferroptosis" propagation and protected against renal allograft IRI. The functional and mechanistic regulation of IRI-sEVs was further corroborated in an allograft kidney transplant model and an in situ renal IRI model. In summary, these findings suggest that inhibiting sEV-mediated lncRNA WAC-AS1 secretion and targeting HBP metabolism-induced BACH2 O-GlcNAcylation in renal tubular epithelial cells may serve as new strategies for protecting against graft IRI after kidney transplant.

GADD45A and GADD45B as Novel Biomarkers Associated with Chromatin Regulators in Renal Ischemia-Reperfusion Injury.

Chromatin regulators (CRs) are essential upstream regulatory factors of epigenetic modification. The role of CRs in the pathogenesis of renal ischemia-reperfusion injury (IRI) remains unclear. We analyzed a bioinformatic analysis on the differentially expressed chromatin regulator genes in renal IRI patients using data from public domains. The hub CRs identified were used to develop a risk prediction model for renal IRI, and their expressions were also validated using Western blot, qRT-PCR, and immunohistochemistry in a murine renal IRI model. We also examined the relationships between hub CRs and infiltrating immune cells in renal IRI and used network analysis to explore drugs that target hub CRs and their relevant downstream microRNAs. The results of machine learning methods showed that five genes (DUSP1, GADD45A, GADD45B, GADD45G, HSPA1A) were upregulated in renal IRI, with key roles in the cell cycle, p38 MAPK signaling pathway, p53 signaling pathway, FoxO signaling pathway, and NF-κB signaling pathway. Two genes from the network, GADD45A and GADD45B (growth arrest and DNA damage-inducible protein 45 alpha and beta), were chosen for the renal IRI risk prediction model. They all showed good performance in the testing and validation cohorts. Mice with renal IRI showed significantly upregulated GADD45A and GADD45B expression within kidneys compared to sham-operated mice. GADD45A and GADD45B showed correlations with plasmacytoid dendritic cells (pDCs) in infiltrating immune cell analysis and enrichment in the MAPK pathway based on the weighted gene co-expression network analysis (WGCNA) method. Candidate drugs that target GADD45A and GADD45B include beta-escin, sertraline, primaquine, pimozide, and azacyclonol. The dysregulation of GADD45A and GADD45B is related to renal IRI and the infiltration of pDCs, and drugs that target GADD45A and GADD45B may have therapeutic potential for renal IRI.

Dissecting the Therapeutic Mechanisms of Sphingosine-1-Phosphate Receptor Agonism during Ischaemia and Reperfusion.

Sphingosine 1-phosphate (S1P) and S1P receptors (S1PR) regulate many cellular processes, including lymphocyte migration and endothelial barrier function. As neutrophils are major mediators of inflammation, their transendothelial migration may be the target of therapeutic approaches to inflammatory conditions such as ischaemia-reperfusion injury (IRI). The aim of this project was to assess whether these therapeutic effects are mediated by S1P acting on neutrophils directly or indirectly through the endothelial cells. First, our murine model of peritoneum cell recruitment demonstrated the ability of S1P to reduce CXCL8-mediated neutrophil recruitment. Mechanistic in vitro studies revealed that S1P signals in neutrophils mainly through the S1PR1 and 4 receptors and induces phosphorylation of ERK1/2; however, this had no effect on neutrophil transmigration and adhesion. S1P treatment of endothelial cells significantly reduced TNF-α-induced neutrophil adhesion under flow (p < 0.01) and transendothelial migration towards CXCL8 during in vitro chemotaxis assays (p < 0.05). S1PR1 agonist CYM5442 treatment of endothelial cells also reduced neutrophil transmigration (p < 0.01) and endothelial permeability (p < 0.005), as shown using in vitro permeability assays. S1PR3 agonist had no effects on chemotaxis or permeability. In an in vivo mouse model of renal IRI, S1PR agonism with CYM5442 reduced endothelial permeability as shown by reduced Evan's Blue dye extravasation. Western blot was used to assess phosphorylation at different sites on vascular endothelial (VE)-cadherin and showed that CYM5442 reduced VEGF-mediated phosphorylation. Taken together, the results of this study suggest that reductions in neutrophil infiltration during IRI in response to S1P are mediated primarily by S1PR1 signalling on endothelial cells, possibly by altering phosphorylation of VE-cadherin. The results also demonstrate the therapeutic potential of S1PR1 agonist during IRI.

#5449 CALYCOSIN ATTENUATES RENAL ISCHEMIA/REPERFUSION INJURY BY SUPPRESSING INFLAMMATION VIA PPARγ/EGR1 PATHWAY

Abstract Background and Aims Renal ischemia reperfusion injury (IRI) is a leading and common cause of AKI, and delayed recovery of IRI contributes to chronic kidney disease and even end-stage kidney disease. However, there is no effective prevention and treatment for IRI-AKI. Calycosin (CAL), an isoflavonoid phytoestrogen isolated from Radix astragali, has various pharmacological activities. However, whether CAL has a protective effect on renal IRI and its mechanism remains elusive. In this study, we aim to explore the effect and mechanism of CAL on IRI-AKI through bioinformatics and experiments in vitro and in vivo models, so as to provide potential candidate drugs for early intervention treatment of IRI-AKI. Method (1) In the first part, mice were intragastrically given CAL daily for 7 d, and subjected to bilateral renal artery occlusion for 35 min followed by reperfusion 24 h to establish the renal IRI model. HE staining was used to evaluate the renal pathological injury, and the renal function including serum creatinine (SCr) and blood urea nitrogen (BUN), was also examined. Meanwhile, inflammatory factors were detected. In cell experiments, HK-2 cells pretreated with 8,16 and 32 μM CAL were subjected with hypoxia/reoxygenation (H/R), and the effect of CAL on H/R induced cell injury were investigated by qRT-PCR and ELISA. (2) In the second part, the differentially expressed gene (DEGs) of IRI-AKI were obtained by bioinformatics analysis of GSE52004 dataset. Subsequently, the hub gene with the highest score was obtained by protein-protein interaction (PPI) analysis, and its upstream transcription factors were predicted by JASPAR database. In IRI-AKI mice, the predicted results of bioinformatics were verified by qRT-PCR, Western Blot and immunohistochemistry. In HK-2 cells, the regulatory relationship between the hub gene and its upstream transcription factor and the role of them in inflammatory process were verified by double luciferase reporter gene and siRNA. Furthermore, molecular docking was used to predict the interaction between CAL and the transcription factor, and the role of the target protein in the protection of H/R HK-2 cells by calycosin was verified by siRNA. Results (1) In the mice with renal IRI, CAL dose dependently alleviated SCr and BUN levels and renal tubular injury score. Meanwhile, the mRNA levels and concentrations of inflammatory cytokines were markedly inhibited by CAL pretreatment. (2) In the pre-experiment in vitro, the toxicity of CAL was negligible at concentrations of 32 μM or less for 24 h incubation. In H/R induced HK-2 cells, CAL incubation reduced the levels of hypoxia-inducible factor 1α (HIF-1α) and the inflammatory cytokines. (3) 58 DEGs were identified in the kidney of IRI mice. PPI analysis showed early growth response 1 (EGR1) is the hub gene the highest score. JASPAR database predicted that peroxisome proliferator-activated receptor γ (PPARγ) is the upstream transcription factor of EGR1. (4) In kidney after IRI, EGR1 was up-regulated and PPARγ was down-regulated, while the pretreatment of CAL decreased EGR1 and increased PPARγ in a dose-dependent manner. (5) In H/R induced HK-2 cells, after inhibited the expression of EGR1 by siRNA, the level of inflammatory factors decreased significantly, suggesting that EGR1 participated in and promoted the inflammatory process of tubular cells. In addition, dual luciferase reporter gene assay confirmed that PPARγ interact with EGR1 promoter, as EGR1 increased after EGR1 promoter plasmid transfection but decreased after PPARγ overexpression. (6) In mice after renal IRI, CAL promote PPARγ expression markedly. According to molecular docking, CAL, as a ligand, could be embedded into the orthosteric pocket of PPARγ with a binding energy of -6.46 kcal/mol. In CAL incubated cells, PPARγ increased and EGR1 decreased considerably. However, the protective effect of CAL on H/R induced inflammation was absent when PPARγ knockdown, indicated that CAL ameliorate renal IRI and inhibit inflammation via PPARγ/EGR1. Conclusion CAL could significantly modulate inflammation by targeting the PPARγ/EGR1 pathway, contributing to the alleviation of renal IRI.

NHWD-870 protects the kidney from ischemia/reperfusion injury by upregulating the PI3K/AKT signaling pathway (experimental study).

Renal ischemia-reperfusion injury is a critical clinical condition with a potentially fatal prognosis if not adequately managed. NHWD-870, a known Brd4 inhibitor with anti-cancer properties, exhibits additional attributes such as antioxidant, anti-inflammatory, and anti-apoptotic effects, suggesting its potential to preserve renal tissue and mitigate damage during ischemic insults. We aimed to assess the potential nephroprotective effect of NHWD-870 by investigating its anti-apoptotic, anti-inflammatory, and antioxidant properties in a rat model of renal ischemia-reperfusion injury. Male Wistar Albino rats (n=24) were randomly assigned to four groups: sham, control, vehicle, and NHWD-870. The control group experienced bilateral renal ischemia for 30 minutes, followed by 2 hours of reperfusion, while the sham group underwent a laparotomy without ischemia-reperfusion induction. The vehicle group received a DMSO injection, and the NHWD-870 group was administered 3mg/kg NHWD-870 orally 24 hours before repeating the control group protocol. Blood samples were collected after reperfusion for blood urea nitrogen (BUN) and serum creatinine (SCr) analysis. ELISA method was used to assess IL-1B, BCL-2, PGF-2, and PI3K/AKT signaling pathways in renal tissue. Tubular injury severity was evaluated through histopathological analysis. NHWD-870 treatment improved renal function and histological preservation compared to the control and vehicle groups. BUN, sCR, IL-1B, BCL-2, and PGF-2 levels in renal tissue were significantly improved in the NHWD-870 group (p<0.05). Furthermore, the PI3K/AKT signaling pathway was significantly upregulated (p<0.01), and tubular injury severity was reduced in the NHWD-870 group. NHWD-870 demonstrated substantial nephroprotective effects in reducing renal damage induced by ischemia-reperfusion injury in rats. These effects may be attributed to the anti-apoptotic properties, as indicated by increased levels of the anti-apoptotic protein Bcl-2, and the reduction in oxidative stress marker PGF-2 through upregulation of the PI3K/AKT signaling pathway, along with the decrease in the inflammatory marker IL-1B.

Role of Iberin as an anti-apoptotic agent on renal ischemia-reperfusion injury in rats.

Ischemia-reperfusion injury (IRI) is a major contributor to acute and chronic kidney failure, heart failure, and ischemic stroke. This study aimed to investigate the therapeutic potential of Iberin, known for its anti-inflammatory, antioxidant, and antiapoptotic properties, in a rat model of renal IRI. Twenty-four adult male rats were randomly divided into four groups: Group I (Sham group) underwent laparotomy without IRI induction; Group II (Control group) underwent laparotomy followed by renal artery clamping for 30 minutes to induce ischemia, followed by 2 hours of reperfusion; Group III (Iberin treatment group) received a pre-injection of Iberin (15 mg/kg) and underwent 30 minutes of ischemia followed by 2 hours of reperfusion; and Group IV (Vehicle-treated group) received the vehicle (ethanol) 1 hour prior to ischemia and reperfusion induction. Iberin was diluted with ethanol. Biomarkers associated with inflammation, oxidative stress, and apoptosis were measured using enzyme-linked immunosorbent assay. Iberin treatment significantly reduced levels of inflammatory cytokines interleukin-1β (IL-1β) and IL-6, Bcl-2-associated X protein (BAX), tumor necrosis factor α (TNF-α), nuclear factor kappa p56, high mobility group B1, and neutrophil gelatinase-associated lipocalin. Moreover, Iberin increased levels of heat shock protein and Bcl2 compared to the control and vehicle groups. Iberin treatment prolonged the ischemic tolerance of renal tissue, potentially preventing or delaying irreversible injuries. These findings highlight the potential of Iberin as a promising candidate for mitigating renal injury caused by ischemia-reperfusion, due to its ability to modulate inflammatory markers.

CC-4066 therapy delivered to kidneys during cold storage and assessed with normothermic reperfusion is feasible and safe.

Currently there is an urgent need to translate interventions that may be beneficial to marginal donor kidneys prior to transplant, to improve their quality from bench to bedside. This project investigated the effects of CC-4066, a potent dual inhibitor of cyclophilin proteins A and D, treatment during static cold storage (SCS) in a porcine model of renal ischemia-reperfusion injury (IRI) using Normothermic Reperfusion (NR). Porcine kidneys and autologous blood were retrieved in pairs from a local abattoir (n = 7). One kidney from each pair was randomly allocated to treatment and one allocated to control and flushed with preservation solution containing CC-4066 or vehicle. After 7 h of SCS kidneys underwent 3 h Normothermic Reperfusion (NR) with autologous whole blood while perfusion characteristics and samples were collected. Perfusion and metabolic parameters showed similar trends and no statistical differences were observed between the groups. IL-6 showed a significant increase over time but no significant difference between groups (p-value 0.009 and 0.14 respectively, two-way ANOVA). Oxygen consumption and lactate levels were similar between groups but there was increased vacuolation on histology in the control group. The addition of CC-4066 during SCS of kidneys is safe and feasible and has no adverse or detrimental effects on perfusion during assessment on NR. There was no difference in cytokine levels although there was a trend towards less vacuolation on histology in the treatment group.

PD31-06 THE EFFECT OF KIDNEY PRESERVATION AT 10°C WITH HEMOPURE AND HYDROGEN SULFIDE DONOR, SODIUM THIOSULFATE, IN A SYNGENEIC RAT RENAL TRANSPLANTATION MODEL

You have accessJournal of UrologyCME1 Apr 2023PD31-06 THE EFFECT OF KIDNEY PRESERVATION AT 10°C WITH HEMOPURE AND HYDROGEN SULFIDE DONOR, SODIUM THIOSULFATE, IN A SYNGENEIC RAT RENAL TRANSPLANTATION MODEL Maria Abou Taka and Alp Sener Maria Abou TakaMaria Abou Taka More articles by this author and Alp SenerAlp Sener More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003324.06AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Renal transplantation is the preferred treatment for patients with end-stage renal disease. Yet, donor kidney demand outweighs supply. Static cold storage (SCS) at 4°C is the current gold standard for renal preservation. SCS contributes to kidney damage through renal ischemia-reperfusion injury (IRI) that involves cellular death and inflammation. In porcine models of renal transplantation, we added non-FDA-approved hydrogen sulfide (H2S) donor, AP39, to a bovine-based blood substitute, Hemopure, at 21°C and 37°C, which improved renal graft quality; though, the experimental setup was costly to meet renal metabolic demand. In rats, we investigated sodium thiosulfate (STS), an FDA-approved H2S donor, at 4°C and observed similar benefits. However, there is still a risk of 4°C cold IRI. Recent studies showed that 10°C human organ graft preservation enhanced patient survival without requiring extensive oxygen during preservation. Therefore, we hypothesize that preservation solutions with STS and Hemopure at 10°C will reduce renal IRI compared to SCS. METHODS: Using an in vitro model of rat renal IRI, we evaluated STS use at 4°C, 10°C, 21°C, and 37°C. We treated rat proximal tubular epithelial cells with 150 µM STS for 24 hours in hypoxic conditions to mimic ischemia, and 24 hours in normoxic conditions to mimic reperfusion. To assess cellular viability, we used flow cytometry and stained cells with FITC-Annexin-V and PerCP-Propidium Iodide to determine apoptosis and necrosis levels, respectively. RESULTS: STS treatment significantly enhanced cellular viability at 10°C compared to 4°C, 21°C, and 37°C, as determined by the percentage of cells negatively stained with FITC-Annexin-V and PerCP-Propidium Iodide. STS treatment also significantly decreased apoptotic and necrotic cells at 10°C compared to 4°C, 21°C, and 37°C, as shown by the percentage of cells positively stained with FITC-Annexin-V and PerCP-Propidium Iodide. CONCLUSIONS: Overall, 10°C STS treatment significantly protects rat proximal tubular epithelial cells from IRI, suggesting that 10°C kidney preservation may enhance renal graft survival, especially in a cost-effective manner, compared to other preservation temperatures currently used in transplantation. Source of Funding: Ontario Graduate Scholarship, Department of Surgery Internal Research Funds, Lawson Internal Research Fund © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e902 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Maria Abou Taka More articles by this author Alp Sener More articles by this author Expand All Advertisement PDF downloadLoading ...

Protective effect of IAXO-102 on renal ischemia-reperfusion injury in rats.

Ischemia/reperfusion injury (IRI) is a common cause of kidney damage, characterized by oxidative stress and inflammation. In this study, we investigated the potential protective effects of IAXO-102, a chemical compound, on experimentally induced IRI in male rats. The bilateral renal IRI model was used, with 24 adult male rats randomly divided into four groups (N=6): sham group (laparotomy without IRI induction), control group (laparotomy plus bilateral IRI for 30 minutes followed by 2 hours of reperfusion), vehicle group (same as control but pre-injected with the vehicle), and treatment group (similar to control but pre-injected with IAXO-102). We measured several biomarkers involved in IRI pathophysiology using enzyme-linked immunosorbent assay (ELISA), including High mobility group box1 (HMGB1), nuclear factor kappa b-p65 (NF-κB p65), interleukin beta-1 (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), 8-isoprostane, Bcl-2 associated X protein (BAX), heat shock protein 27 (HSP27), and Bcl-2. Statistical analysis was performed using one-way ANOVA and Tukey post hoc tests. Our results showed that IAXO-102 significantly improved kidney function, reduced histological alterations, and decreased the inflammatory response (IL-1, IL-6, and TNF) caused by IRI. IAXO-102 also decreased apoptosis by reducing pro-apoptotic Bax and increasing anti-apoptotic Bcl-2 without impacting HSP27. In conclusion, our findings suggest that IAXO-102 had a significant protective effect against IRI damage in the kidneys.

Research Highlights.

Research Highlights.

Effects of grape seed-derived proanthocyanidin B2 pretreatment on oxidative stress, endoplasmic reticulum stress and apoptosis of renal tubular epithelial cells in renal ischemia–reperfusion injury model of mice

PurposeTo investigate the effect of grape seed-derived proanthocyanidin B2 (GSPB2) pretreatment on acute renal ischemia–reperfusion injury model of mice.Methods50 mice were divided into 5 groups: Sham group: mice were treated with right nephrectomy. GSPB2 group: GSPB2 was injected intraperitoneally 45 min before right nephrectomy. IRI group: right kidney was resected and the left renal arteriovenous vessel was blocked for 45 min. GSPB2 + IRI group: GSPB2 was intraperitoneally injected 45 min before IRI established. GSPB2 + BRU + IRI group: GSPB2 and brusatol (BRU) were injected intraperitoneally 45 min before IRI established. Creatinine and urea nitrogen of mice were detected, and the kidney morphology and pathological changes of each group were detected by HE staining, PAS staining and transmission electron microscopy. Expressions of Nrf2, HO-1, GRP78, CHOP, and cleaved-caspase3 were detected by immunofluorescence staining and western blotting.ResultsMorphology and mitochondrial damages of kidney in GSPB2 + IRI group were significantly alleviated than those in IRI group. Expression levels of Nrf2 and HO-1 were significantly higher in GSPB2 + IRI group than those in IRI group. Expression levels of GRP78, CHOP and cleaved-caspase3 were significantly lower in GSPB2 + IRI group than those in IRI group. However, compared to GSPB2 + IRI group, protective effects of GSPB2 pretreatment were weakened in GSPB2 + BRU + IRI group.ConclusionsGSPB2 pretreatment could alleviate oxidative stress damage and reduce apoptosis of renal tubular epithelial cells, which might be related to activating the antioxidant system, up-regulating the expression of Nrf2 and HO-1, inhibiting the expressions of GRP78, CHOP and cleaved-caspase3. However, the protective effect could be reversed by brusatol.

OGG1 aggravates renal ischemia-reperfusion injury by repressing PINK1-mediated mitophagy.

Renal ischemia-reperfusion injury (IRI) is mainly responsible for acute kidney injury for which there is no effective therapy. Accumulating evidence has indicated the important role of mitophagy in mitochondrial homeostasis under stress. OGG1 (8-oxoguanine DNA glycosylase) is known for functions in excision repair of nuclear and mitochondrial DNA. However, the role of OGG1 in renal IRI remains unclear. Herein, we identified OGG1, induced during IRI, as a key factor mediating hypoxia-reoxygenation-induced apoptosis in vitro and renal tissue damage in a renal IRI model. We demonstrated that OGG1 expression during IRI negatively regulates mitophagy by suppressing the PINK1/Parkin pathway, thereby aggravating renal ischemic injury. OGG1 knockout and pharmacological inhibition attenuated renal IRI, in part by activating mitophagy. Our results elucidated the damaging role of OGG1 activation in renal IRI, which is associated with the regulatory role of the PINK1/Parkin pathway in mitophagy.

Single-cell transcriptome analysis of a rat model of bilateral renal ischemia-reperfusion injury

Ischemia-reperfusion injury (IRI) causes massive tissue damage. Renal IRI is the most common type of acute renal injury, and the defects caused by it may progress to chronic kidney disease (CKD). Rodent models of renal IRI, with various patterns, have been used to study the treatment of human kidney injury. A rat model of bilateral IRI, in which the bilateral kidney blood vessels are clamped for 60 min, is widely used, inducing both acute and chronic kidney disease. However, the molecular mechanisms underlying the effects of bilateral IRI on kidney cells have not yet been fully elucidated. This study aimed to perform a whole-transcriptome analysis of the IRI kidney using single-cell RNA sequencing. We found renal parenchymal cells, including those from the proximal tubule, the loop of Henle, and distal tubules, to be damaged by IRI. In addition, we observed significant changes in macrophage population. Our study delineated the detailed cellular and molecular changes that occur in the rat model of bilateral IRI. Collectively, our data and analyses provided a foundation for understanding IRI-related kidney diseases in rat models.

Renal Ischemia-reperfusion Injury Attenuated by Exosomes Extracted From Splenic Ischemic Preconditioning Models.

To investigate the protective effects of exosomes extracted from splenic ischemic preconditioning (sIPC) models on renal ischemia-reperfusion injury (IRI). sIPC was conducted on mice before renal IRI, and exosomes derived from sIPC mice were infused into a mouse model of renal IRI. The kidney tissue and serum were collected 24 h later. The morphological changes, inflammation and apoptosis in IR kidneys were determined by hematoxylin-eosin (HE), terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL), and Ki-67 immunohistochemical staining. In addition, the proinflammatory cytokines in serum and cell supernatant were measured with enzyme-linked immunosorbent assays (ELISAs). Then, we administered exosomes to mouse renal epithelial cells. TUNEL assays and flow cytometry were used to evaluate cell apoptosis. Bax and Bcl-2 levels were measured via Western blotting. HE staining showed that the renal IRI was attenuated after sIPC. TUNEL results showed that renal tissue apoptosis was greatly reduced after sIPC or injection of exosomes. ELISAs showed that the serum creatinine (sCr), tumor necrosis factor alpha, and interleukin-1 β levels induced by IRI decreased with sIPC. In vitro, exosomes extracted from the hypoxia/reoxygenation (H/R) splenic fibroblast model had the same protective effect. TUNEL and flow cytometry results showed that the exosomes reduced apoptosis. ELISAs showed that tumor necrosis factor alpha and interleukin-1 β were significantly increased in the H/R group but decreased due to the exosomes treated with starvation. WB results showed that Bax expression was increased and Bcl-2 expression was decreased in the H/R group. However, exosomes decreased the Bax level and increased the Bcl-2 level. Exosomes extracted from sIPC models exerted a protective effect to attenuate renal IRI.