Ischemia Reperfusion-induced Acute Kidney Injury Research Articles

Calycosin inhibited MIF-mediated inflammatory chemotaxis of macrophages to ameliorate ischemia reperfusion-induced acute kidney injury.

Inflammatory macrophage infiltration plays a critical role in acute kidney disease induced by ischemia-reperfusion (IRI-AKI). Calycosin is a natural flavone with multiple bioactivities. This study aimed to investigate the therapeutic role of calycosin in IRI-AKI and its underlying mechanism. The renoprotective and anti-inflammatory effects of calycosin were analyzed in C57BL/6 mice with IRI-AKI and lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. RNA-seq was used for mechanism investigation. The molecular target of calycosin was screened by in silico methods and validated by surface plasmon resonance (SPR). Macrophage chemotaxis was analyzed using Transwell and agarose gel spot assays. Calycosin treatment significantly reduced serum creatinine and urea nitrogen and attenuated tubular destruction in IRI-AKI mice. Additionally, calycosin markedly suppressed NF-κB signaling activation and the expression of inflammatory mediators IL-1β and TNF-α in IRI-AKI kidneys and LPS-stimulated RAW 264.7 cells. Interestingly, RNA-seq revealed calycosin remarkably downregulated chemotaxis-related pathways in RAW 264.7 cells. Among the differentially expressed genes, Ccl2/MCP-1, a critical chemokine mediating macrophage inflammatory chemotaxis, was downregulated in both LPS-stimulated RAW 264.7 cells and IRI-AKI kidneys. Consistently, calycosin treatment attenuated macrophage infiltration in the IRI-AKI kidneys. Importantly, in silico target prediction, molecular docking, and SPR assay demonstrated that calycosin directly binds to macrophage migration inhibitory factor (MIF). Functionally, calycosin abrogated MIF-stimulated NF-κB signaling activation and Ccl2 expression and MIF-mediated chemotaxis in RAW 264.7 cells. In summary, calycosin attenuates IRI-AKI by inhibiting MIF-mediated macrophage inflammatory chemotaxis, suggesting it could be a promising therapeutic agent for the treatment of IRI-AKI.

Protective effect and mechanism of Xiaoyu Xiezhuo decoction on ischemia–reperfusion induced acute kidney injury based on gut–kidney crosstalk

This study aimed to explore the mechanism of Xiaoyu Xiezhuo decoction (XXD) on ischemia–reperfusion-induced acute kidney injury (IRI-AKI) using network pharmacology methods and gut microbiota analysis. A total of 1778 AKI-related targets were obtained, including 140 targets possibly regulated by AKI in XXD, indicating that the core targets were mainly enriched in inflammatory-related pathways, such as the IL-17 signaling pathway and TNF signaling pathway. The unilateral IRI-AKI animal model was established and randomly divided into four groups: the sham group, the AKI group, the sham + XXD group, and the AKI + XXD group. Compared with the rats in the AKI group, XXD improved not only renal function, urinary enzymes, and biomarkers of renal damage such as Kim-1, cystatin C, and serum inflammatory factors such as IL-17, TNF-α, IL-6, and IL 1-β, but also intestinal metabolites including lipopolysaccharides, d-lactic acid, indoxyl sulfate, p-cresyl sulfate, and short-chain fatty acids. XXD ameliorated renal and colonic pathological injury as well as inflammation and chemokine gene abundance, such as IL-17, TNF-α, IL-6, IL-1β, ICAM-1, and MCP-1, in AKI rats via the TLR4/NF-κB/NLRP3 pathway, reducing the AKI score, renal pathological damage, and improving the intestinal mucosa’s inflammatory infiltration. It also repaired markers of the mucosal barrier, including claudin-1, occludin, and ZO-1. Compared with the rats in the AKI group, the α diversity was significantly increased, and the Chao1 index was significantly enhanced after XXD treatment in both the sham group and the AKI group. The treatment group significantly reversed this change in microbiota.

#2743 Impact of fatty acid-binding protein 4 on SUMOylation balance and renal tubular epithelial cell senescence in acute kidney injury

Abstract Background and Aims Dysregulated lipid metabolism and cellular senescence intricately interact in the context of acute kidney injury (AKI). Fatty acid-binding protein 4 (FABP4), a critical enzyme regulating lipid metabolism, potentially induces cell senescence through lipotoxicity in AKI. However, the specific mechanisms remain unclear. Method We established an ischemia-reperfusion-induced AKI (IRI-AKI) model in C57 mice and quantified and localized FABP4 expression in renal tissues using Western blotting (WB), qPCR, and immunofluorescence (IF). Oil Red O staining and beta-galactosidase staining were employed to assess lipid deposition and cellular senescence in tubular cells. The same measurements were repeated in IRI-AKI by knocking out Fabp4. Additionally, bulk mRNA sequencing and single-cell sequencing were conducted on renal tissues to analyze key molecules and related mechanisms. Results In vivo, we observed a significant decline in renal function, severe tubular epithelial damage, and abundant inflammation in the IRI-AKI group, which was noticeably alleviated in the Fabp4 knockout group (Fig. 1). Furthermore, we detected upregulation of FABP4 expression in the kidneys of IRI-AKI, primarily localized in the tubular epithelial cells. Fabp4 knockout effectively reduced tubular lipid accumulation and cellular senescence (Fig. 3). Through integrating mRNA and single-cell sequencing data, UBC9, an E2 enzyme involved in protein SUMOylation, emerged as a potential key regulator of cell senescence and AKI. WB and qPCR both confirmed the upregulation of UBC9 expression in IRI-AKI and its downregulation after Fabp4 knockout. Conversely, we observed that downregulated SUMOylation in IRI-AKI was upregulated in the Fabp4 knockout group (Fig. 4). Conclusion In conclusion, FABP4 modulates the expression of UBC9 and SUMOylation, thereby mediating cellular senescence and causing renal tubular cell damage in IRI-AKI. The mechanisms underlying FABP4’s regulation of UBC9 and the opposing trend between UBC9 and SUMOylation require further elucidation.

The role of PPP1R3G in promoting necroptosis in ischemia reperfusion induced acute kidney injury

Recent discoveries have revealed necroptosis as a major contributor to the pathogenesis of acute kidney injury (AKI). Protein phosphatase 1 regulatory subunit 3G (PPP1R3G) has been linked to the essentiality of RIPK1-dependent apoptosis and type I necroptosis. Nonetheless, the involvement of PPP1R3G in the regulation of necroptosis during ischemia reperfusion-induced acute kidney injury (IR-AKI) has yet to be elucidated. Here, we investigated the role of PPP1R3G in the regulation of necroptosis in IR using an in vitro cellular I/R model and in vivo IRI-AKI mouse model. Primary proximal tubular cells (PTCs) from C57BL/6 and Ppp1r3g−/− mice were cultured and cell I/R was induced through hypoxia/reoxygenation. Cell viability and necroptosis were analyzed using CellTiter-Glo assay and Annexin V-Cy3/ SYTOX green staining, respectively. The expression levels of necroptosis, oxidative stress, and inflammation factors were determined by real-time PCR, western blotting, immunofluorescence staining, and ELISA. In the in vivo IRI-AKI model, bilateral renal pedicles clamping was conducted in both C57BL/6 and Ppp1r3g−/− mice. The kidney injury and function were evaluated by plasma creatine, BUN, and KIM-1 levels and GFR values, respectively. Kidney morphological evidence of injury was assessed by PAS staining and cell death by immunofluorescent staining. We found that knockout of PPP1R3G in the primary tubular cells significantly preserved cell viability and attenuated tubular cell death. This protective effect was associated with the reduced expression and phosphorylation of necroptosis factors (RIP1, RIP3, and MLKL), and inflammatory factors (IL-6, IL-10 and TNF-α) in I/R injury cells. Deletion of PPP1R3G attenuated the I/R induced kidney injury indicated by 54% decrease in the plasma creatine (2.28±0.12mg/dL vs. 1.09±0.07mg/dL, p<0.0001, n=7) and 45% higher GFR values than WT mice at day 3 after reperfusion. Histology showed less diffuse renal tubular necrosis and casts comprising necrotic cells and debris in Ppp1r3g−/− mice, as well as 60% less cell death compared to WT mice. Moreover, the application of chemical compounds to prevent inhibitory phosphorylation of RIPK1 significantly reinstated cell death in Ppp1r3g−/− cells. In conclusion, our findings demonstrate, for the first time, that Ppp1r3g-mediated necroptosis significantly promotes IRI-AKI. Therefore, targeting Ppp1r3g to reduce necroptosis in the kidney holds promising potential for significant clinical benefits. National Institutes of Health Department of Molecular Pharmacology and Physiology, University of South Florida College of Medicine, Tampa, FL. 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.

An SS31-rapamycin conjugate via RBC hitchhiking for reversing acute kidney injury

Mitochondrial dysfunction plays a major role in driving acute kidney injury (AKI) via alteration in energy and oxygen supply, which creates further ROS and inflammatory responses. However, mitochondrial targeting medicine in recovering AKI is challenging. Herein, we conjugated SS31, a mitochondria-targeted antioxidant tetrapeptide connecting a cleavable linker to rapamycin (Rapa), which provided specific interaction with FK506-binding protein (FKBP) in the RBCs. Once entering the bloodstream, SS31-Rapa could be directed to the intracellular space of RBCs, allowing the slow diffusion of the conjugate to tissues via the concentration gradient. The new RBC hitchhiking strategy enables the encapsulation of conjugate into RBC via a less traumatic and more natural and permissive manner, resulting in prolonging the t1/2 of SS31 by 6.9 folds. SS31-Rapa underwent the direct cellular uptake, instead of the lysosomal pathway, released SS31 in response to activated caspase-3 stimulation in apoptotic cells, favoring the mitochondrial accumulation of SS31. Combined with autophagy induction associated with Rapa, a single dose of SS31-Rapa can effectively reverse cisplatin and ischemia reperfusion-induced AKI. This work thus highlights a simple and effective RBC hitchhiking strategy and a clinically translatable platform technology to improve the outcome of other mitochondrial dysfunctional related diseases.

Neutrophil Membrane-Inspired Nanorobots Act as Antioxidants Ameliorate Ischemia Reperfusion-Induced Acute Kidney Injury.

Ischemia/reperfusion (I/R) injury causes excessive oxidative events and initiates destructive inflammatory responses, and it is an important promoter to the pathology of various pathema states. Ferroptosis is an iron-dependent type of nonapoptotic cell death accompanied by the accumulation of membrane lipid peroxide and consumption of polyunsaturated fatty acid, and it plays a key role in I/R injury diseases. Moreover, the excessive production of inflammatory cytokines contributes to the development of acute kidney injury. Here, we reported neutrophil membrane-coated copper-based nanoparticles (N-Cu5.4O@DFO NPs) for I/R kidney injury treatment. The highly biocompatible and stable N-Cu5.4O@DFO NPs showed excellent antioxidant and iron ion scavenging abilities in vitro. Our finding showed that the N-Cu5.4O@DFO NPs strategy could significantly accumulate in the inflammatory kidney, reduce oxidative damage events and inflammatory response, and finally achieve synergistic therapy against renal I/R injury. This work promotes the development of nanoantioxidant agents with multiple antioxidant properties for the therapy of other I/R injury diseases.

Casein Kinase 2 Alpha Inhibition Protects against Sepsis-Induced Acute Kidney Injury.

Sepsis-induced acute kidney injury (AKI) is a common complication in critically ill patients, often resulting in high rates of morbidity and mortality. Previous studies have demonstrated the effectiveness of casein kinase 2 alpha (CK2α) inhibition in ameliorating ischemia-reperfusion-induced AKI. In this study, our aim was to investigate the potential of the selective CK2α inhibitor, 4,5,6,7-tetrabromobenzotriazole (TBBt), in the context of sepsis-induced AKI. To assess this, we initially confirmed an upregulation of CK2α expression following a cecum ligation and puncture (CLP) procedure in mice. Subsequently, TBBt was administered to a group of mice prior to CLP, and their outcomes were compared to those of sham mice. The results revealed that, following CLP, the mice exhibited typical sepsis-associated patterns of AKI, characterized by reduced renal function (evidenced by elevated blood urea nitrogen and creatinine levels), renal damage, and inflammation (indicated by increased tubular injury score, pro-inflammatory cytokine levels, and apoptosis index). However, mice treated with TBBt demonstrated fewer of these changes, and their renal function and architecture remained comparable to that of the sham mice. The anti-inflammatory and anti-apoptotic properties of TBBt are believed to be associated with the inactivation of the mitogen-activated protein kinase (MAPK) and nuclear factor κB (NF-κB) signaling pathways. In conclusion, these findings suggest that inhibiting CK2α could be a promising therapeutic strategy for treating sepsis-induced AKI.

Limited overlap between plasma, renal, and urinary microRNAs altered by ischemia reperfusion-induced Acute Kidney Injury (AKI)

Over 13 million people worldwide are affected by acute kidney injury (AKI), or the rapid reduction of renal function over a short period of time. Causes of AKI include genetics, nephrotoxic drugs, and medical interventions, when combined with lack of prevention and treatment interventions results in a death rate of 300,000/year. MicroRNAs (miRs) are thought to regulate apoptotic and inflammatory processes involved with AKI primarily through suppressing gene expression. Thus, miR expression profiles could serve as viable AKI biomarkers. Our goal was to identify miRs that are altered in kidney cortex tissue, urine, and plasma samples following renal ischemia-reperfusion injury (IRI). We hypothesized that in the 24 hours following IRI, alterations in miR abundance in the kidney would be reflected by similar or inverse changes in clinically accessible measured bio-fluids (urine and plasma), and that miRs altered in those bio-fluid samples would overlap. AKI was induced in 10-wk-old male Sprague Dawley rats by bilaterally clamping both the renal artery and vein for 30 mins, followed by 24 hrs of reperfusion (n=7). Sham surgeries were performed without vessel occlusion (n=5). Upon surgical recovery, rats were placed in metabolic cages for 24-hour urine collection. Blood, urine, and kidneys were collected and processed for blood assays, small RNA library preparation and sequencing following the 24-hour period. Small RNA sequencing results were analyzed using EdgeR2 to evaluate differential expression (DE) based on Benjamini-Hochberg adjusted p-value (p.adj) ≤ 0.05. An extensive literary search was performed to identify DE miRs involvement in AKI based on renal IRI models.Blood panel results show SCr (IR: 3.8 vs sham: 0.32mg/dL) and BUN (IR: 96.57 vs sham: 17.60mg/dL) were elevated in renal IRI male rats (p< 0.05). The miRNA seq data assayed 528 miRs, 174 were differentially altered in the cortical, urinary, and plasma samples (p.adj ≤ 0.05) following renal IRI. DE miRs in only 1 sample: 53 cortex-specific (10↑, 43↓); 50 plasma-specific (20↑, 30↓) and 27 urine-specific (16↑, 11↓). In total, 19 DE miRs were common to 2 sample types: 5 in plasma and urine samples, 8 in cortex and plasma samples; 6 in cortex and urine samples; 2 miRs in all three. Of the 21 overlapped DE miRs, 9 are associated with AKI pathology in published literature.AKI presents economic and clinical burdens that call for novel biomarkers of disease pathology that facilitate earlier treatment interventions. Using a clinically relevant AKI IR rat model, we observed that 11% of total DE miRs identified were common to 2 sample types and only 1% were common to all three. Future directions call for understanding miR abundance levels in circulation while also determining miR origin. This project highlights the need for more comprehensive analysis of miRs that are proposed as clinically relevant biomarkers of disease pathology that are representative of both the primary insult and circulation. The project described was supported by Award Number 2T32HL007852. The content is solely the responsibilities of the authors and does not necessarily represent the official views of the NIH. This is the full abstract presented at the American Physiology Summit 2023 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.

USP25-PKM2-glycolysis axis contributes to ischemia reperfusion-induced acute kidney injury by promoting M1-like macrophage polarization and proinflammatory response

M1-like macrophages have been reported to play critical roles in acute kidney injury (AKI). Here, we elucidated the role of ubiquitin-specific protease 25 (USP25) in M1-like macrophages polarization and AKI. High USP25 expression was correlated with a decline in renal function in patients with acute kidney tubular injury and in mice with AKI. In contrast, USP25 knockout reduced M1-like macrophage infiltration, suppressed M1-like polarization, and improved AKI in mice, indicating that USP25 was necessary for M1-like polarization and proinflammatory response. Immunoprecipitation assay and liquid chromatography-tandem mass spectrometry showed that the M2 isoform of pyruvate kinase, muscle (PKM2) was a target substrate of USP25. Kyoto Encyclopedia of Genes and Genomes pathway analysis indicated the USP25 regulated aerobic glycolysis and lactate production during M1-like polarization via PKM2. Further analysis showed that the USP25-PKM2-aerobic glycolysis axis positively regulated M1-like polarization and exacerbated AKI in mice, providing potential therapeutic targets for AKI treatment.

NIR-II Photoacoustic-Active DNA Origami Nanoantenna for Early Diagnosis and Smart Therapy of Acute Kidney Injury.

Herein, we designed and synthesized a novel microRNA (miR)-responsive nanoantenna capable of early diagnosis and smart treatment of acute kidney injury (AKI). The nanoantenna was made of two miniature gold nanorods (AuNRs) (e.g., length: ∼48 nm; width: ∼9 nm) linked together by a rectangular DNA origami nanostructure (rDONs) scaffold (e.g., length: ∼90 nm; width: ∼60 nm) (rDONs@AuNR dimer). The surface plasmon resonance peak of the constructed nanoantenna is located within the NIR-II window (e.g., ∼1060 nm), thus guaranteeing photoacoustic (PA) imaging of the nanoantenna in deep tissues. Intriguingly, the nanoantenna displayed exclusive kidney retention in both healthy mice and ischemia reperfusion-induced AKI mice by leveraging the kidney-targeting ability of rDONs. Distinguished from the stable signals in the healthy mice, the PA signals of the nanoantenna would turn down in the AKI mice due to the AuNR detached from rDONs upon interaction with miR-21, which were up-expressed in AKI mice. The limit of detection toward miR-21 was down to 2.8 nM, enabling diagnosis of AKI as early as 10 min post-treatment with ischemia reperfusion, around 2 orders of magnitude earlier than most established probes. Moreover, the naked rDON scaffold generated by AKI could capture more reactive oxygen species (e.g., 1.5-fold more than rDONs@AuNR dimer), alleviating ischemic AKI. This strategy provided a new avenue for early diagnosis and smart treatment of AKI.

Identification of resident progenitors labeled with Top2a responsible for proximal tubular regeneration in ischemia reperfusion-induced acute kidney injury

BackgroundAcute kidney injury is a common fatal disease with complex etiology and limited treatment methods. Proximal tubules (PTs) are the most vulnerable segment. Not only in injured kidneys but also in normal kidneys, shedding of PTs often happens. However, the source cells and mechanism of their regeneration remain unclear. MethodsScRNA and snRNA sequencing data of acute injured or normal kidney were downloaded from GEO database to identify the candidate biomarker of progenitor of proximal tubules. SLICE algorithm and CytoTRACE analyses were employed to evaluate the stemness of progenitors. Then the repairing trajectory was constructed through pseudotime analyses. SCENIC algorithm was used to detect cell-type-specific regulon. With spatial transcriptome data, the location of progenitors was simulated. Neonatal/ adult/ aged mice and preconditioning AKI mice model and deconvolution of 2 RNA-seq data were employed for validation. ResultsThrough cluster identification, PT cluster expressed Top2a specifically was identified to increase significantly during AKI. With relatively strong stemness, the Top2a-labeled PT cluster tended to be the origin of the repairing trajectory. Moreover, the cluster was regulated by Pbx3-based regulon and possessed great segmental heterogeneity. Changes of Top2a between neonatal and aged mice and among AKI models validated the progenitor role of Top2a-labeled cluster. ConclusionsOur study provided transcriptomic evidence that resident proximal tubular progenitors labeled with Top2a participated in regeneration. Considering the segmental heterogeneity, we find that there is a group of reserve progenitor cells in each tubular segment. When AKI occurs, the reserve progenitors of each tubular segment proliferate and replenish first, and PT-progenitors, a cluster with no obvious PT markers replenish each subpopulation of the reserve cells.

Kidney targeting of formoterol containing polymeric nanoparticles improves recovery from ischemia reperfusion-induced acute kidney injury in mice

The β2 adrenergic receptor agonist, formoterol, is an inducer of mitochondrial biogenesis and restorer of mitochondrial and kidney function in acute and chronic models of kidney injury. Unfortunately, systemic administration of formoterol has the potential for adverse cardiovascular effects, increased heart rate, and decreased blood pressure. To minimize these effects, we developed biodegradable and biocompatible polymeric nanoparticles containing formoterol that target the kidney, thereby decreasing the effective dose, and lessen cardiovascular effects while restoring kidney function after injury. Male C57Bl/6 mice, treated with these nanoparticles daily, had reduced ischemia-reperfusion-induced serum creatinine and kidney cortex kidney injury molecule-1 levels by 78% and 73% respectively, compared to control mice six days after injury. With nanoparticle therapy, kidney cortical mitochondrial number and proteins reduced by ischemic injury, recovered to levels of sham-operated mice. Tubular necrosis was reduced 69% with nanoparticles treatment. Nanoparticles improved kidney recovery even when the dosing frequency was reduced from daily to two days per week. Finally, compared to treatment with formoterol-free drug alone, these nanoparticles did not increase heart rate nor decrease blood pressure. Thus, targeted kidney delivery of formoterol-containing nanoparticles is an improvement in standard formoterol therapy for ischemia-reperfusion-induced acute kidney injuries by decreasing the dose, dosing frequency, and cardiac side effects.

Role of tRNA derived fragments in renal ischemia–reperfusion injury

Background Ischemia–reperfusion injury (IRI) is one of the major causes of acute kidney injury (AKI). tRNA derived fragments (tRFs/tiRNAs) are groups of small noncoding RNAs derived from tRNAs. To date, the role of tRFs/tiRNAs in renal IRI has not been reported. Herein, we aimed to investigate the involvement of tRFs/tiRNAs in the occurrence and development of ischemia–reperfusion-induced AKI. Methods Moderate/severe renal IRI mouse models were established by bilateral renal pedicle clamping. The tRF/tiRNA profiles of healthy controls and moderate/severe IRI-stressed kidney tissues were sequenced by Illumina NextSeq 500. Candidate differentially expressed tiRNAs were further verified by RT-qPCR. Biological analysis was also performed. Results Overall, 152 tRFs/tiRNAs were differentially expressed in the moderate ischemic injury group compared with the normal control group (FC > 2, p < 0.05), of which 47 were upregulated and 105 were downregulated; in the severe ischemic injury group, 285 tRFs/tiRNAs were differentially expressed (FC > 2, p < 0.05), of which 157 were upregulated, and 128 were downregulated. RT-qPCR determination of eight abundantly expressed tiRNAs was consistent with the sequencing results. Gene Ontology analysis for target genes of the tRFs/tiRNAs showed that the most enriched cell components, molecular functions and biological processes were Golgi apparatus, cytoplasmic vesicles, protein binding, cellular protein localization and multicellular organism development. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that these target genes were mainly involved in the natural killer cell mediated cytotoxicity pathway, citrate cycle, and regulation of actin cytoskeleton signaling pathway. Conclusion Our results indicated that tRFs/tiRNAs were involved in renal IRI. These tRFs/tiRNAs may be effective partly via regulation of renal immunity, inflammation and metabolism processes. Candidate genes, including tiRNA-Gly-GCC-003, tiRNA-Lys-CTT-003, and tiRNA-His-GTG-002, might be potential biomarkers and therapeutic targets of ischemia–reperfusion injury-induced acute kidney injury.

The Role of the Superior Cervical Sympathetic Ganglion in Ischemia Reperfusion-Induced Acute Kidney Injury in Rats.

Acute kidney injury (AKI) has been found to be a serious clinical problem with high morbidity and mortality, and is associated with acute inflammatory response and sympathetic activation that subsequently play an important role in the development of AKI. It is well known that the sympathetic nervous system (SNS) and immune system intensely interact and mutually control each other in order to maintain homeostasis in response to stress or injury. Evidence has shown that the superior cervical sympathetic ganglion (SCG) participates in the bidirectional network between the immune and the SNS, and that the superior cervical ganglionectomy has protective effect on myocardial infarction, however, the role of the SCG in the setting of renal ischemic reperfusion injury has not been studied. Here, we sought to determine whether or not the SCG modulates renal ischemic reperfusion (IR) injury in rats. Our results showed that bilateral superior cervical ganglionectomy (SCGx) 14 days before IR injury markedly reduced the norepinephrine (NE) in plasma, and down-regulated the increased expression of tyrosine hydroxylase (TH) in the kidney and hypothalamus. Sympathetic denervation by SCGx in the AKI group increased the level of blood urea nitrogen (BUN) and kidney injury molecule-1 (KIM-1), and exacerbated renal pathological damage. Sympathetic denervation by SCGx in the AKI group enhanced the expression of pro-inflammatory cytokines in plasma, kidney and hypothalamus, and increased levels of Bax in denervated rats with IR injury. In addition, the levels of purinergic receptors, P2X3R and P2X7R, in the spinal cord were up-regulated in the denervated rats of the IR group. In conclusion, these results demonstrate that the sympathetic denervation by SCGx aggravated IR-induced AKI in rats via enhancing the inflammatory response, thus, the activated purinergic signaling in the spinal cord might be the potential mechanism in the aggravated renal injury.

Study of Renal Accumulation of Targeted Polycations in Acute Kidney Injury.

Acute kidney injury (AKI) is a global healthcare burden characterized by rapid loss of renal function and high morbidity and mortality. Chemokine receptor CXCR4 participates in the renal infiltration of immune cells following injury and in local inflammatory enhancement. Injured renal tubule cells overexpress CXCR4, which could be used as a target for improved drug delivery in AKI. Plerixafor is a small-molecule CXCR4 antagonist that has shown beneficial effects against AKI and has been previously developed into a polymeric analog (polymeric plerixafor, PP). With the goal of gaining a better understanding of how overall charge and hydrophilicity affect renal accumulation of PP, we have synthesized PP copolymers containing hydroxyl, carboxyl, primary amine, and alkyl moieties using Michael-type addition copolymerization. All synthesized copolymers showed excellent CXCR4-binding and inhibiting ability in vitro and improved cellular uptake in hypoxia-reoxygenation stimulated mouse tubule cells. Analysis of serum protein binding revealed that polymers with hydroxyl group modification showed the least amount of protein binding. Biodistribution of the polymers was tested in a unilateral ischemia reperfusion-induced AKI mouse model. The results showed significant differences in accumulation in the injured kidneys depending on the net charge and hydrophilicity of the polymers. The findings of this study will guide the development of polymeric drug carriers for targeted delivery to injured kidneys for better AKI therapy.

SIRT3 mediates mitofusin 2 ubiquitination and degradation to suppress ischemia reperfusion-induced acute kidney injury

Ischemia reperfusion-induced acute kidney injury (IR-induced AKI) is a life-threatening disease with many complications. Mitofusin 2 (Mfn2) ubiquitination is related to AKI. But the underlying molecular mechanisms remain unknown. This study aimed to probe the mechanism of Mfn2 ubiquitination in IR-induced AKI development. In IR-induced AKI mouse models, orbital blood and urine were collected for assessing kidney function. The kidney injury, ultrastructure of mitochondria, and histopathology in mice were evaluated after injection of G5, an ubiquitination inhibitor. Oxygen glucose deprivation/reoxygenation (OGD/R) models were established in HK-2 cells, and the mitochondria were extracted. Cell viability, apoptosis, oxidative stress, inflammatory reaction, mitochondrial membrane potential, and ATP production were measured. Mfn2 ubiquitination in mouse and cell models was evaluated. si-SIRT3 and pcDNA3.1-SIRT3 were transfected into cell models. Consequently, kidney function in mice was impaired by IR-induced AKI. Mfn2 ubiquitination and degradation promoted IR-induced AKI. OGD/R induced renal tubular epithelial cell injury and disrupted mitochondrial dynamics and functions through promoting Mfn2 ubiquitination. SIRT3 knockdown led to Mfn2 ubiquitination by binding to UBC; while its overexpression alleviated tubular epithelial cell injury. Briefly, SIRT3 mediates Mfn2 ubiquitination to relieve IR-induced AKI. This investigation may offer new insights for the treatment of IR-induced AKI injury.

Hyaluronic acid coated bilirubin nanoparticles attenuate ischemia reperfusion-induced acute kidney injury

Acute kidney injury (AKI) is a common pathological process that is globally associated with a high morbidity and mortality rate. The underlying AKI mechanisms include over-produced reactive oxygen species (ROS), inflammatory cell infiltration, and high levels of inflammatory mediators. Bilirubin is an endogenous compound with antioxidant, anti-inflammatory and anti-apoptotic properties, and could, therefore, be a promising therapeutic candidate. Nanotechnology-mediated therapy has emerged as a novel drug delivery strategy for AKI treatment. In this study, we report a hyaluronic acid (HA) coated ε-polylysine-bilirubin conjugate (PLBR) nanoparticle (nHA/PLBR) that can selectively accumulate in injured kidneys and alleviate the oxidative/inflammatory-induced damage. The in vitro study revealed that nHA/PLBR has good stability, biocompatibility, and exhibited higher antioxidant as well as anti-apoptotic effects when compared to nPLBR or bilirubin. The in vivo study showed that nHA/PLBR could target and accumulate in the injured kidney, effectively relieve oxidative stress and inflammatory reactions, protect the structure and function of the mitochondria, and more importantly, inhibit the apoptosis of tubular cells in an ischemia/reperfusion-induced AKI rat model. Therefore, nHA/PLBR has the capacity to enhance specific biodistribution and delivery efficiency of bilirubin, thereby providing better treatment for AKI in the future.

Identification of Hub Genes Associated with the Development of Acute Kidney Injury by Weighted Gene Co-Expression Network Analysis

Background: Acute kidney injury (AKI) is a severe clinical syndrome, causing a profound medical and socioeconomic burden worldwide. This study aimed to explore underlying molecular targets related to the progression of AKI. Methods: A public database originated from the NCBI GEO database (serial number: GSE121190) and a well-established and unbiased method of weighted gene co-expression network analysis (WGCNA) to identify hub genes and potential pathways were used. Furthermore, the unbiased hub genes were validated in 2 classic models of AKI in a rodent model: chemically established AKI by cisplatin- and ischemia reperfusion-induced AKI. Results: A total of 17 modules were finally obtained by the unbiased method of WGCNA, where the genes in turquoise module displayed strong correlation with the development of AKI. In addition, the results of gene ontology revealed that the genes in turquoise module were involved in renal injury and renal fibrosis. Thus, the hub genes were further validated by experimental methods and primarily obtained Rplp1 and Lgals1 as key candidate genes related to the progression of AKI by the advantage of quantitative PCR, Western blotting, and in situ tissue fluorescence. Importantly, the expression of Rplp1 and Lgals1 at the protein level showed positive correlation with renal function, including serum Cr and BUN. Conclusions: By the advantage of unbiased bioinformatic method and consequent experimental verification, this study lays the foundation basis for the pathogenesis and therapeutic agent development of AKI.

Fenofibrate attenuates ischemia reperfusion-induced acute kidney injury and associated liver dysfunction in rats.

Ischemia/reperfusion (I/R) is one of the common reasons for acute kidney injury (AKI) and we need to develop effective therapies for treating AKI. We investigated the role of fenofibrate against I/R-induced AKI and associated hepatic dysfunction in rats. In male wistar albino rats, renal pedicle occlusion for 40 min and 24 h reperfusion resulted in AKI. I/R-induced AKI was demonstrated by measuring serum creatinine, creatinine clearance, urea, uric acid, potassium, fractional excretion of sodium and urinary microproteins. Oxidative stress in rat kidneys was quantified by assaying superoxide anion generation, thiobarbituric acid reactive substances, and reduced glutathione levels. AKI-induced hepatic damage was quantified by assaying serum aminotransferases, alkaline phosphatase and bilirubin levels. Moreover, serum cholesterol, high density lipoprotein and triglycerides were quantified. Hematoxylin-eosin staining of renal and hepatic tissues was done and the kidney and liver injury scores were determined. Immunohistology of endothelial nitric oxide synthase (eNOS) was done in rat kidneys. Fenofibrate was administered for 1 week before subjecting rats to AKI. In separate group, the nitric oxide synthase inhibitor, L-nitroarginine methyl ester (L-NAME) was administered prior to fenofibrate treatment. In I/R group, significant alteration in the serum/urine parameters indicated AKI and hepatic dysfunction along with marked increase in kidney and liver injury scores. Treatment with fenofibrate attenuated AKI and associated hepatic dysfunction. Moreover, I/R-induced decrease in renal eNOS expression was abrogated by fenofibrate. Pre-treatment with L-NAME abolished fenofibrate mediated reno- and hepato-protective effects. In conclusion, fenofibrate attenuates I/R-induced AKI and associated hepatic dysfunction putatively through modulation of eNOS expression.

MiR-20a-5p is enriched in hypoxia-derived tubular exosomes and protects against acute tubular injury.

Exosomes have been shown to effectively regulate the biological functions of target cells. Here, we investigated the protective effect and mechanism of hypoxia-induced renal tubular epithelial cells (TECs)-derived exosomes on acute tubular injury. We found that in vitro hypoxia-induced tubular exosomes (Hy-EXOs) were protective in acute tubular injury by promoting TECs proliferation and improving mitochondrial functions. By using exosome miRNA sequencing, we identified miR-20a-5p was abundant and was a key mechanism for the protective effect of Hy-EXOs on tubular injury as up-regulation of miR-20a-5p enhanced but down-regulation of miR-20a-5p inhibited the protective effect of Hy-EXOs on tubular injury under hypoxia conditions. Further study in a mouse model of ischemia-reperfusion-induced acute kidney injury (IRI-AKI) also confirmed this notion as pre-treating mice with the miR-20a-5p agomir 48 h prior to AKI induction was capable of inhibiting IRI-AKI by lowering serum levels of creatinine and urea nitrogen, and attenuating the severity of tubular necrosis, F4/80(+) macrophages infiltration and vascular rarefaction. Mechanistically, the protective effect of miR-20a-5p on acute kidney injury (AKI) was associated with inhibition of TECs mitochondrial injury and apoptosis in vitro and in vivo. In conclusion, miR-20a-5p is enriched in hypoxia-derived tubular exosomes and protects against acute tubular injury. Results from the present study also reveal that miR-20a-5p may represent as a novel therapy for AKI.