Renal Tubular Cell Injury Research Articles

Sirtuin1 Suppresses Calcium Oxalate Nephropathy via Inhibition of Renal Proximal Tubular Cell Ferroptosis Through PGC-1α-mediated Transcriptional Coactivation.

Calcium oxalate (CaOx) crystals induce renal tubular epithelial cell injury and subsequent nephropathy. However, the underlying mechanisms remain unclear. In the present study, single-cell transcriptome sequencing is performed on kidney samples from mice with CaOx nephrocalcinosis. Renal proximal tubular cells are identified as the most severely damaged cell population and are accompanied by elevated ferroptosis. Further studies demonstrated that sirtuin1 (Sirt1) effectively reduced ferroptosis and CaOx crystal-induced kidney injury in a glutathione peroxidase 4 (GPX4)-dependent manner. Mechanistically, Sirt1 relies on peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α) to promote resistance to ferroptosis in the tubular epithelium, and PGC-1α can recruit nuclear factor erythroid 2-related factor 2 (NRF2) to the promoter region of GPX4 and co-activate GPX4 transcription. This work provides new insight into the mechanism of CaOx crystal-induced kidney injury and identifies Sirt1 and PGC-1α as potential preventative and therapeutic targets for crystal nephropathies.

RBBP6-Mediated ERRα Degradation Contributes to Mitochondrial Injury in Renal Tubular Cells in Diabetic Kidney Disease.

Diabetic Kidney Disease (DKD), a major precursor to end-stage renal disease, involves mitochondrial dysfunction in proximal renal tubular cells (PTCs), contributing to its pathogenesis. Estrogen-related receptor α (ERRα) is essential for mitochondrial integrity in PTCs, yet its regulation in DKD is poorly understood. This study investigates ERRα expression and its regulatory mechanisms in DKD, assessing its therapeutic potential. Using genetic, biochemical, and cellular approaches, ERRα expression Was examined in human DKD specimens and DKD mouse models. We identified the E3 ubiquitin ligase retinoblastoma binding protein 6 (RBBP6) as a regulator of ERRα, promoting its degradation through K48-linked polyubiquitination at the K100 residue. This degradation pathway significantly contributed to mitochondrial injury in PTCs of DKD models. Notably, conditional ERRα overexpression or RBBP6 inhibition markedly reduced mitochondrial damage in diabetic mice, highlighting ERRα's protective role in maintaining mitochondrial integrity. The interaction between RBBP6 and ERRα opens new therapeutic avenues, suggesting that modulating RBBP6-ERRα interactions could be a strategy for preserving mitochondrial function and slowing DKD progression.

ADAR1 plays a protective role in proximal tubular cells under high glucose conditions by attenuating the PI3K/AKT/mTOR signaling pathway.

Adenosine deaminases acting on RNA 1 (ADAR1), an RNA editing enzyme, holds a role in cancer, inflammation, and immunity. However, its specific function in the nephropathy and high-glucose-induced human renal tubular epithelial cells (HK-2) injury in diabetic db/db mice is not clear. This study explored the expression characteristics of ADAR1 in proximal renal tubular cells of diabetic db/db mice, examining its function in the mechanism of high-glucose-induced HK-2 cell injury. Furthermore, it elucidated the molecular mechanism underlying the protective effect of ADAR1, the regulation of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB/Akt)/mammalian target of the rapamycin (mTOR) signaling. We observed a decrease in ADAR1 expression in proximal tubular cells of diabetic db/db mice, accompanied by an increase in the expression of inflammation-related markers (PI3K/AKT/mTOR). We constructed and validated ADAR1-overexpression plasmids and used an ADAR1 inhibitor (8-azaadenosine) to carry out cell experiments. The upregulation of ADAR1 expression alleviated high-glucose-induced endoplasmic reticulum stress, reduced HK-2 cell apoptosis, and reduced the expression of inflammation-related indicators (PI3K/AKT/mTOR). Taken together, the pivotal roles of ADAR1 in the progression of proximal renal tubulopathy and the mechanism of high-glucose-induced HK-2 injury in diabetic db/db mice suggest that ADAR1 may be a potential key factor in slowing the progression of diabetic kidney disease.

Oxalate-upregulated annexin A6 promotes the formation of calcium oxalate kidney stones by exacerbating calcium release-mediated oxidative stress injury in renal tubular epithelial cells and crystal-cell adhesion

Oxalate-upregulated annexin A6 promotes the formation of calcium oxalate kidney stones by exacerbating calcium release-mediated oxidative stress injury in renal tubular epithelial cells and crystal-cell adhesion

Imperatorin ameliorates ferroptotic cell death, inflammation, and renal fibrosis in a unilateral ureteral obstruction mouse model

BackgroundImperatorin is a naturally occurring furocoumarin derivative found in traditional Chinese medicine Angelica dahurica for its anticancer, antihypertensive, and antidiabetic properties. Chronic kidney disease (CKD) is a global health issue, characterized by a high prevalence, significant morbidity and mortality, and a range of related complications. ObjectiveThis study aims to investigate the protective effects of imperatorin treatment and the specific underlying mechanisms in progressive CKD. MethodsImperatorin was orally administrated for 14 consecutive days to mice with unilateral ureteral obstruction (UUO) to investigate the renal pathological alternations, pro-inflammatory mediators, antioxidant response, and ferroptotic death signaling. Imperatorin was also tested in the erastin-induced injury of renal proximal tubular cells (NRK-52E). Cell viability, ferroptosis protein markers, erastin-induced oxidative stress, and lipid peroxidation were assessed. ResultsIn vivo, imperatorin treatment alleviated kidney histology alternations and attenuated the protein expression of fibrotic markers. Furthermore, imperatorin administration reduced inflammatory cell infiltration, and alleviated the oxidative stress burden by downregulating protein markers such as catalase, superoxide dismutase 2 (SOD-2), NADPH oxidase 4 (NOX-4), and thioredoxin reductase 1 (Trxr-1). It also mitigated ferroptosis markers such as glutathione peroxidase 4 (GPX4), solute carrier family 7 member 11/cystine transporter (SLC7A11/xCT), and transferrin receptor 1 (TFR-1), and attenuated renal cell apoptosis. In vitro, imperatorin treatment effectively decreased erastin-induced feroptotic cell death, restored the antioxidant enzyme levels, and mitigated lipid peroxidation as well as the expression of ferroptosis-related markers (XCT, GPX4, and p-p53) in a dose-dependent manner. ConclusionOur finding demonstrated for the first time, that imperatorin treatment holds therapeutic potential in a UUO mouse model of CKD and inhibits the erastin-induced oxidative stress, ferroptosis, and subsequent lipid peroxidation in vitro. This highlights the potential of imperatorin as a future therapeutic target for ferroptosis to improve the progression of CKD.

MiRNA-133a-3p Attenuates Renal Tubular Epithelial Cell Injury via Targeting MALM1 and Suppressing the Notch Signaling Pathway in Diabetic Nephropathy.

Diabetic nephropathy (DN) is a serious microvascular complication of diabetes characterized by structural and functional changes of kidneys. Human renal tubular epithelial (HK-2) cells are important for kidney recovery post injury and usually used for establishment of DN cell models. The study explored the role of microRNA (miR)-133a-3p in DN cell model and animal model. A cell model for DN was established via high glucose (HG) stimulation to HK-2 cells. Cell viability and apoptotic rate were measured by cell counting kit 8 and flow cytometry. Polymerase chain reaction was performed to quantify levels of miR-133a-3p and targets. Luciferase reporter assay was conducted to verify the binding of miR-133a-3p and MAML1. After establishment of a mouse model of DN, levels of renal function indicators were measured by biochemical analysis. Hematoxylin-eosin and periodic acid-schiff staining of kidney samples were performed to analyze histological changes. Western blotting was conducted to quantify levels of apoptotic markers, MAML1, and factors related to Notch signaling. Results showed that HG induced HK-2 cell apoptosis and the reduction of cell viability. MiR-133a-3p was lowly expressed in HG-stimulated HK-2 cells. Overexpressed miR-133a-3p improved HK-2 cell injury by increasing cell viability and hampering apoptosis under HG condition. In addition, miR-133a-3p directly targets MAML1 3'-untranslated region. MAML1 overexpression countervailed the repressive impact of miR-133a-3p on cell apoptosis in the context of HG. Moreover, miR-133a-3p inhibited the activity of Notch pathway by downregulating MAML1. MiR-133a-3p inhibits DN progression in mice, as evidenced by reduced fasting blood glucose level, improved levels of renal function parameters, and alleviation of kidney atrophy. In conclusion, miR-133a-3p improves HG-induced HK-2 cell injury and inhibits DN progression by targeting MAML1 and inactivating Notch signaling.

The toxicity of cisplatin derives from effects on renal organic ion transporters expression and serum endogenous substance levels

Acute kidney injury (AKI) is a worldwide public health problem with high morbidity and mortality. Cisplatin is a widely used chemotherapeutic agent for treating solid tumors, but the induction of AKI restricts its clinical application. In this study, the effect of cisplatin on the expression of organic ion transporters was investigated through in vivo and in vitro experiments. Targeted metabolomics techniques were used to measure the levels of selected endogenous substances in serum. Transmission electron microscopy was used to observe the microstructure of renal tubular epithelial cells. Our results show that the toxicity of cisplatin on HK-2 cells or HEK-293 cells was time- and dose-dependent. Administration of cisplatin decreased the expression of OAT1/3 and OCT2 and increased the expression of MRP2/4. Mitochondrial damage induced by cisplatin lead to renal tubular epithelial cell injury. In addition, administration of cisplatin resulted in significant changes in endogenous substance levels in serum, including amino acids, carnitine, and fatty acids. These serum amino acids and metabolites (α-aminobutyric acid, proline, and alanine), carnitines (tradecanoylcarnitine, hexanylcarnitine, octanoylcarnitine, 2-methylbutyroylcarnitine, palmitoylcarnitine, and linoleylcarnitine) and fatty acids (9E-tetradecenoic acid) represent endogenous substances with diagnostic potential for cisplatin-induced AKI.

Renogrit selectively protects against cisplatin-induced injury in human renal tubular cells and in Caenorhabditis elegans by harmonizing apoptosis and mitophagy

Cisplatin-induced nephrotoxicity restricts its clinical use against solid tumors. The present study elucidated the pharmacological effects of Renogrit, a plant-derived prescription medicine, using cisplatin-induced human renal proximal tubular (HK-2) cells and Caenorhabditis elegans. Quantification of phytochemicals in Renogrit was performed on HPTLC and UHPLC platforms. Renogrit was assessed in vitro in HK-2 cells post-exposure to clinically relevant concentration of cisplatin. It was observed that renoprotective properties of Renogrit against cisplatin-induced injury stem from its ability to regulate renal injury markers (KIM-1, NAG levels; NGAL mRNA expression), redox imbalance (ROS generation; GST levels), and mitochondrial dysfunction (mitochondrial membrane potential; SKN-1, HSP-60 expression). Renogrit was also found to modulate apoptosis (EGL-1 mRNA expression; protein levels of p-ERK, p-JNK, p-p38, c-PARP1), necroptosis (intracellular calcium accumulation; RIPK1, RIPK3, MLKL mRNA expression), mitophagy (lysosome population; mRNA expression of PINK1, PDR1; protein levels of p-PINK1, LC3B), and inflammation (IL-1β activity; protein levels of LXR-α). More importantly, Renogrit treatment did not hamper normal anti-proliferative effects of cisplatin as observed from cytotoxicity analysis on MCF-7, A549, SiHa, and T24 human cancer cells. Taken together, Renogrit could be a potential clinical candidate to mitigate cisplatin-induced nephrotoxicity without compromising the anti-neoplastic properties of cisplatin.

SET8 inhibition preserves PTEN to attenuate kidney cell apoptosis in cisplatin nephrotoxicity.

The aberrant expression of SET8, a histone methyltransferase that mediates H4 lysine 20 mono-methylation (H4K20me1), is implicated in the pathogenesis of various tumors, however, its role in acute kidney injury (AKI) is unknown. Here we showed that SET8 and H4K20me1 were upregulated in the murine kidney with AKI induced by cisplatin, along with increased renal tubular cell injury and apoptosis and decreased expression of E-cadherin and Phosphatase and Tensin Homolog (PTEN). Suppression of SET8 by UNC0379 improved renal function, attenuated tubule damage, and restored expression of PTEN, but not E-cadherin. UNC0379 was also effective in lessening cisplatin-induced DNA damage response (DDR) as indicated by reduced expression of γ-H2AX, p53, p21, and alleviating cisplatin-impaired autophagy as shown by retained expression of Atg5, Beclin-1, and CHMP2A and enhanced levels of LC3-II in the kidney. Consistently, inhibition of SET8 with either UNC0379 or siRNA mitigated apoptosis and DDR, and restored autophagy, along with PTEN preservation in cultured renal proximal tubular epithelial cell (TKPTs) exposed to cisplatin. Further studies showed that inhibition of PTEN with Bpv or siRNA potentiated cisplatin-induced apoptosis, DDR, and hindered autophagy, and conversely, alleviated by overexpression of PTEN in TKPTs. Finally, blocking PTEN largely abolished the inhibitory effect of UNC0379 on apoptosis. Taken together, these results suggest that SET8 inhibition protects against cisplatin-induced AKI and renal cell apoptosis through a mechanism associated with the preservation of PTEN, which in turn inhibits DDR and restores autophagy.

Astaxanthin attenuates diabetic kidney injury through upregulation of autophagy in podocytes and pathological crosstalk with mesangial cells

Objectives: Astaxanthin (ATX) is a strong antioxidant drug. This study aimed to investigate the effects of ATX on podocytes in diabetic nephropathy and the underlying renal protective mechanism of ATX, which leads to pathological crosstalk with mesangial cells.Methods: In this study, diabetic rats treated with ATX exhibited reduced 24-h urinary protein excretion and decreased blood glucose and lipid levels compared to vehicle-treated rats. Glomerular mesangial matrix expansion and renal tubular epithelial cell injury were also attenuated in ATX-treated diabetic rats compared to control rats.Results: ATX treatment markedly reduced the α-SMA and collagen IV levels in the kidneys of diabetic rats. Additionally, ATX downregulated autophagy levels. In vitro, compared with normal glucose, high glucose inhibited LC3-II expression and increased p62 expression, whereas ATX treatment reversed these changes. ATX treatment also inhibited α-SMA and collagen IV expression in cultured podocytes. Secreted factors (vascular endothelial growth factor B and transforming growth factor-β) generated by high glucose-induced podocytes downregulated autophagy in human mesangial cells (HMCs); however, this downregulation was upregulated when podocytes were treated with ATX.Conclusions: The current study revealed that ATX attenuates diabetes-induced kidney injury likely through the upregulation of autophagic activity in podocytes and its antifibrotic effects. Crosstalk between podocytes and HMCs can cause renal injury in diabetes, but ATX treatment reversed this phenomenon.

The role and mechanism of action of miR-483-3p in mediating the effects of IGF-1 on human renal tubular epithelial cells induced by high glucose

This study aimed to elucidate the influence of miR-483-3p on human renal tubular epithelial cells (HK-2) under high glucose conditions and to understand its mechanism. Human proximal tubular epithelial cells (HK-2) were exposed to 50 mmol/L glucose for 48 h to establish a renal tubular epithelial cell injury model, denoted as the high glucose group (HG group). Cells were also cultured for 48 h in a medium containing 5.5 mmol/L glucose, serving as the low glucose group. Transfection was performed in various groups: HK-2 + low glucose (control group), high glucose (50 mM) (HG group), high glucose + miR-483-3p mimics (HG + mimics group), high glucose +miR-483-3p inhibitor (HG + inhibitor group), and corresponding negative controls. Real-time quantitative polymerase chain reaction (qPCR) assessed the mRNA expression of miR-483-3p, bax, bcl-2, and caspase-3. Western blot determined the corresponding protein levels. Proliferation was assessed using the CCK-8 assay, and cell apoptosis was analyzed using the fluorescence TUNEL method. Western blot and Masson’s staining were conducted to observe alterations in cell fibrosis post miR-483-3p transfection. Furthermore, a dual-luciferase assay investigated the targeting relationship between miR-483-3p and IGF-1. The CCK8 assay demonstrated that the HG + mimics group inhibited HK-2 cell proliferation, while the fluorescent TUNEL method revealed induced cell apoptosis in this group. Conversely, the HG + inhibitor group promoted cell proliferation and suppressed cell apoptosis. The HG + mimics group upregulated mRNA and protein expression of pro-apoptotic markers (bax and caspase-3), while downregulating anti-apoptotic marker (bcl-2) expression. In contrast, the HG + inhibitor group showed opposite effects. Collagen I and FN protein levels were significantly elevated in the HG + mimics group compared to controls (P < 0.05). Conversely, in the HG + inhibitor group, the protein expression of Collagen I and FN was notably reduced compared to the HG group (P < 0.05). The dual luciferase reporter assay confirmed that miR-483-3p could inhibit the luciferase activity of IGF-1’s 3′-UTR region (P < 0.05). miR-483-3p exerts targeted regulation on IGF-1, promoting apoptosis and fibrosis in renal tubular epithelial cells induced by high glucose conditions.

Unveiling the role of mtDNA in Liver-Kidney Crosstalk: Insights from trichloroethylene hypersensitivity syndrome

In specific pathological conditions, addressing liver injury may yield favorable effects on renal function through the phenomenon of liver-kidney crosstalk. Mitochondrial DNA (mtDNA) possesses the capability to trigger downstream pathways of inflammatory cytokines, ultimately leading to immune-mediated organ damage. Consequently, understanding the intricate molecular mechanisms governing mtDNA involvement in diseases characterized by liver-kidney crosstalk is of paramount significance. This study seeks to elucidate the role of mtDNA in conditions marked by liver-kidney crosstalk. In previous clinical cases, it has been observed that patients with Trichloroethylene Hypersensitivity Syndrome (TCE-HS) who experience severe liver injury often also exhibit renal injury. In this study, patients diagnosed with trichloroethylene hypersensitivity syndrome were recruited from Shenzhen Occupational Disease Control Center. And Balb/c mice were treated with trichloroethylene. The correlation between liver and kidney injuries in patients with TCE-HS was assessed using Enzyme-Linked Immunosorbent Assay (ELISA). Alterations in mtDNA levels were examined in mouse hepatocytes, red blood cells (RBCs), and renal tubular epithelial cells utilizing immunofluorescence and PCR techniques. TCE-sensitized mice exhibited a significant increase in reactive oxygen species (ROS) and the opening of the mitochondrial permeability transition pore in hepatocytes, resulting in the release of mtDNA. Furthermore, heightened levels of mtDNA and Toll-like Receptor 9 (TLR9) expression were observed in RBCs. Additional experiments demonstrated elevated expression of TLR9 and its downstream mediator MyD88 in renal tubule epithelial cells of TCE-sensitized mice. In vitro investigations confirmed that mtDNA activates the TLR9 pathway in TCMK-1 cells. Collectively, these results suggest that mtDNA released from mitochondrial damage in hepatocytes is carried by RBCs to renal tubular epithelial cells and mediates inflammatory injury in renal tubular epithelial cells through activation of the TLR9 receptor.

Hepatic-derived BMP9 is involved in hepatic fibrosis-induced kidney injury through inhibition of renal VEGFA

Clinical observations suggest that acute kidney injury (AKI) occurs in approximately 20–50% of hospitalized cirrhotic patients, suggesting a link between the liver and kidney. Bone morphogenetic protein 9 (BMP9) is a protein produced primarily by the liver and can act on other tissues at circulating systemic levels. Previous studies have demonstrated that controlling abnormally elevated BMP9 in acute liver injury attenuates liver injury; however, reports on whether BMP9 plays a role in liver injury-induced AKI are lacking. By testing we found that liver injury in mice after bile duct ligation (BDL) was accompanied by a significant upregulation of the kidney injury marker kidney injury molecule (KIM-1). Interestingly, all these impairments were alleviated in the kidneys of hepatic BMP9 knockout (BMP9-KO) mice. Peritubular capillary injury is a key process leading to the progression of AKI, and previous studies have demonstrated that vascular endothelial growth factor A (VEGFA) plays a key role in maintaining the renal microvascular system. In animal experiments, we found that high levels of circulating BMP9 had an inhibitory effect on VEGFA expression, while renal tubular epithelial cell injury was effectively attenuated by VEGFA supplementation in the hypoxia-enriched-oxygen (H/R) constructs of the AKI cell model in both humans and mice. Overall, we found that elevated BMP9 in hepatic fibrosis can affect renal homeostasis by regulating VEGFA expression. Therefore, we believe that targeting BMP9 therapy may be a potential means to address the problem of clinical liver fibrosis combined with AKI.

Myricitrin inhibited ferritinophagy-mediated ferroptosis in cisplatin-induced human renal tubular epithelial cell injury.

Cisplatin-induced acute kidney injury (AKI) increases the patient mortality dramatically and results in an unfavorable prognosis. A strong correlation between AKI and ferroptosis, which is a notable type of programmed cell death, was found in recent studies. Myricitrin is a natural flavonoid compound with diverse pharmacological properties. To investigate the protective effect of myricitrin against cisplatin induced human tubular epithelium (HK-2) cell injury and the underlying anti-ferroptic mechanism by this study. Firstly, a pharmacology network analysis was proposed to explore the myricitrin's effect. HK-2 cells were employed for in vitro experiments. Ferroptosis was detected by cell viability, quantification of iron, malondialdehyde, glutathione, lipid peroxidation fluorescence, and glutathione peroxidase (GPX4) expression. Ferritinophagy was detected by related protein expression (NCOA4, FTH, LC3II/I, and SQSTM1). In our study, GO enrichment presented that myricitrin might be effective in eliminating ferroptosis. The phenomenon of ferroptosis regulated by ferritinophagy was observed in cisplatin-activated HK-2 cells. Meanwhile, pretreatment with myricitrin significantly rescued HK-2 cells from cell death, reduced iron overload and lipid peroxidation biomarkers, and improved GPX4 expression. In addition, myricitrin downregulated the expression of LC3II/LC3I and NCOA4 and elevated the expression of FTH and SQTM. Furthermore, myricitrin inhibited ROS production and preserved mitochondrial function with a lower percentage of green JC-1 monomers. However, the protection could be reserved by the inducer of ferritinophagy rapamycin. Mechanically, the Hub genes analysis reveals that AKT and NF-κB are indispensable mediators in the anti-ferroptic process. In conclusion, myricitrin ameliorates cisplatin induced HK-2 cells damage by attenuating ferritinophagy mediated ferroptosis.

Stevioside protects against acute kidney injury by inhibiting gasdermin D pathway

AbstractRecent studies indicate a significant upregulation of gasdermin D (GSDMD) in acute kidney injury (AKI), a severe medical condition characterized by high morbidity and mortality globally. In this study, we identified and validated the therapeutic effects of small molecule inhibitors targeting the GSDMD pathway for AKI treatment. Using a drug screening assay, we evaluated thousands of small molecules from DrugBank against Lipopolysaccharide (LPS) and Nigericin‐stimulated immortalized bone marrow‐derived macrophages (iBMDMs) to discern GSDMD pathway activators. We simulated AKI in primary renal tubular epithelial cells using hydrogen peroxide (H2O2) exposure. Furthermore, AKI in mouse models was induced via cisplatin and ischemia/reperfusion. Our findings highlight stevioside as a potent GSDMD activator exhibiting minimal toxicity. Experimental results, both in vitro and in vivo, demonstrate stevioside's significant potential in alleviating renal tubular epithelial cell injury and AKI histological damage. After stevioside treatment, a notable decrease in cleaved GSDMD‐N terminal levels was observed coupled with diminished inflammatory factor release. This observation was consistent in both cisplatin‐ and ischemia/reperfusion‐induced AKI mouse models. Collectively, our research suggests that stevioside could be a promising candidate for modulating GSDMD signaling in AKI treatment.

Exploring the Interplay between Calcium Oxalate Crystals and Renal Tubular Epithelial Cell Injury: Implications for the Formation and Prevention of Kidney Stones

Kidney stones are a prevalent and clinically significant disease that affects millions of individuals worldwide, which have emerged as a significant global public health concern. The majority of kidney stones are composed of calcium oxalate (CaOx). The mechanisms of stone formation and development are unclear, involving a complex interplay of physical and biochemical processes. The injury of tubular epithelial cells (TECs) represents a pivotal event in the pathogenesis of this condition, as it initiates oxidative stress and immune-inflammatory reactions. Macrophages play a pivotal role in the inflammatory process, interacting with a multitude of molecules and pathways, thereby influencing the stone formation. Furthermore, apoptosis and autophagy induce TECs injury and contribute to the pathogenesis of CaOx stones. The current treatment strategies mainly focus on the management of crystal-cell interactions and the protection of TECs, in conjunction with the application of antioxidants, anti-inflammatory agents, and inhibitors of apoptosis and autophagy. These strategies have demonstrated promising results. Future research will aim to modulate the immune-inflammatory response, offering hope for the effective prevention of stone recurrence.

WWP2 deletion aggravates acute kidney injury by targeting CDC20/autophagy axis

IntroductionAcute kidney injury (AKI) is associated with high morbidity and mortality rates. The molecular mechanisms underlying AKI are currently being extensively investigated. WWP2 is an E3 ligase that regulates cell proliferation and differentiation. Whether WWP2 plays a regulatory role in AKI remains to be elucidated. ObjectivesWe aimed to investigate the implication of WWP2 in AKI and its underlying mechanism in the present study. MethodsWe utilized renal tissues from patients with AKI and established AKI models in global or tubule-specific knockout (cKO) mice strains to study WWP2′s implication in AKI. We also systemically analyzed ubiquitylation omics and proteomics to decipher the underlying mechanism. ResultsIn the present study, we found that WWP2 expression significantly increased in the tubules of kidneys with AKI. Global or tubule-specific knockout of WWP2 significantly aggravated renal dysfunction and tubular injury in AKI kidneys, whereas WWP2 overexpression significantly protected tubular epithelial cells against cisplatin. WWP2 deficiency profoundly affected autophagy in AKI kidneys. Further analysis with ubiquitylation omics, quantitative proteomics and experimental validation suggested that WWP2 mediated poly-ubiquitylation of CDC20, a negative regulator of autophagy. CDC20 was significantly decreased in AKI kidneys, and selective inhibiting CDC20 with apcin profoundly alleviated renal dysfunction and tubular injury in the cisplatin model with or without WWP2 cKO, indicating that CDC20 may serve as a downstream target of WWP2 in AKI. Inhibiting autophagy with 3-methyladenine blocked apcin’s protection against cisplatin-induced renal tubular cell injury. Activating autophagy by rapamycin significantly protected against cisplatin-induced AKI in WWP2 cKO mice, whereas inhibiting autophagy by 3-methyladenine further aggravated apoptosis in cisplatin-exposed WWP2 KO cells. ConclusionTaken together, our data indicated that the WWP2/CDC20/autophagy may be an essential intrinsic protective mechanism against AKI. Further activating WWP2 or inhibiting CDC20 may be novel therapeutic strategies for AKI.

Protective Effect of CXCR7 Against Hypoxia/Reoxygenation Injury in Renal Tubular Epithelial Cells

Acute kidney injury (AKI) is a multifactorial syndrome with complex pathophysiology and prognosis. Ischaemia‒reperfusion injury (IRI) is a major cause of induced AKI. The aim of this study was to investigate the effect of upregulated CXCR7 expression on renal tubular epithelial cell apoptosis induced by hypoxia/reoxygenation (H/R). HK-2 cells were divided into three groups: control group (pcDNA3.1), hypoxia/reoxygenation + pcDNA3.1 group (H/R+pcDNA3.1) and CXCR7 overexpression + hypoxia/reoxygenation group (H/R+ Flag-CXCR7). Protein levels of renal tubular epithelial cell injury-, apoptosis- and autophagy-related markers were assessed by qRT‒PCR, Western blotting, flow cytometry (FCM), immunofluorescence and transmission electron microscopy (TEM). In addition, HK-2 cells were treated with the autophagy inhibitor 3-MA and divided into 3 groups: control group, 3-MA + pcDNA3.1 group, and 3-MA + Flag-CXCR7 group. Changes in autophagy and apoptosis in renal tubule epithelial cells were assessed by Western blotting and FCM. Compared with those in the control group, the protein and mRNA expression levels of CXCR7 in HK-2 cells were significantly lower under H/R conditions. Under H/R conditions, CXCR7 overexpression in HK-2 cells significantly downregulated the expression of NGAL. Moreover, CXCR7 overexpression significantly decreased H/R-induced cleaved PARP-1 and cleaved Caspase 3 levels, increased the level of the antiapoptotic protein BCL-2 and the autophagy-related molecules ATG5 and LC3B II, and significantly inhibited the expression of P62. Autophagy flow and TEM also showed that CXCR7 significantly promoted autophagy. CXCR7 significantly alleviated the 3-MA-induced inhibition of autophagy and increase in apoptosis. Upregulated CXCR7 expression can inhibit renal tubular epithelial cell apoptosis and damage by regulating autophagy. In conclusion, CXCR7 is a promising target for the prevention and/or treatment of AKI.

#921 Interferon regulatory factor 4 (IRF4)-specific deletion on dendritic cells protects against kidney inflammation and injury in IRI-induced AKI/AKD

Abstract Background and Aims Ischemia-reperfusion injury (IRI) is a frequent clinical cause of acute kidney injury and disease (AKI/AKD) which can lead to acute tubular necrosis and irreversible kidney function loss. Mononuclear phagocytes, including dendritic cells (DCs), Monocytes, and macrophages, play important roles in various phases of injury and repair processes. Previous research has indicated that conventional dendritic cells (cDCs) are subdivided into cDC1s and cDC2s, and these two subsets could have opposing functions. We also demonstrated that cDC1s have a protective role as deficiency of cDC1s results in aggravated inflammation and injury in the IRI-induced AKI/AKD model in our previous study. Based on these researches, we aim to investigate the function of cDC2s in the IRI-induced AKI/AKD model. Method We used interferon regulatory factor 4 (IRF4)-deficient mice (IRF4fl/flClec9acre) to specifically delete IRF4 on DC lineage cells as our experimental group, and used wide-type mice as our control group. After unilateral IRI (UIRI) surgery, we measured the GFR and plasma creatinine of the mice on day 1 (D1) and day 5 (D5), then collected the injured kidney issue after sacrificing mice. We compared the differences between IRF4fl/flClec9acre mice and wild-type mice in terms of DC subpopulation changes, neutrophil accumulation, T cell differentiation, intensity of inflammatory responses, and tissue repair capabilities in damaged kidney tissue, by using techniques such as immunohistochemistry, immunofluorescence, flow cytometry, qRT-PCR, etc. Results ([KOMean-WTMean] ± SEM): IRF4fl/flClec9acre mice showed a lower percentage of cDC2s without affecting cDC1s in the kidney compared with wide-type mice (D1: −0.04467 ± 0.01356, P = 0.0301; D5: −1.108 ± 0.2248, P = 0.0004). During post-ischemic AKI/AKD, IRF4 deficiency in cDCs had several beneficial effects compared to wild-type mice, including reducing renal tubular cell injury (PAS score D1: −0.9375 ± 0.1523, P &amp;lt; 0.0001; PAS score D5: −1.119 ± 0.1445, P &amp;lt; 0.0001), decreasing loss of kidney function (GFR D1: 8.983 ± 4.091, P = 0.1041; GFR D5: 14.9 ± 5.01, P = 0.0172), and promoting kidney function recovery. This was associated with increased expression of anti-inflammatory cytokines (IL-4 D1: 4.486 ± 1.363, P = 0.0079; IL-4 D5: 3.403 ± 1.447, P = 0.0366; IL-10 D1: 2.103 ± 0.7342, P = 0.0299; IL-10 D5: 2.654 ± 0.8859, P = 0.0166), decreased expression of pro-inflammatory cytokines(TNF-α D1: −1.418 ± 0.5467, P = 0.0433; TNF-α D5: −2.434 ± 1.919, P = 0.0074; IL-23a D1: −1.320 ± 0.3432, P = 0.0061; IL-23a D5: −16.12 ± 3.587, P = 0.0004) and acute tubular cell death, reduced neutrophil chemoattractant expression (CXCL-2) (Fig. 6) and recruitment of neutrophils (−6.107 ± 1.664, P = 0.0214), and differentiation of Th2 (−0.1867 ± 0.03333, P = 0.005) and Th17 cells (−0.1767 ± 0.01333, P = 0.0002). Conclusion In conclusion, our data demonstrate a critical role of IRF4 in the kidney cDC2 subset and highlight that deficiency of cDC2s can protect against kidney inflammation, reduce kidney injury, and promote kidney recovery in the IRI-induced AKI/AKD model. This suggests that cDC2s have an immunoregulatory role in AKI/AKD and could serve as a potential therapeutic target for kidney inflammation.

Baicalein alleviates cisplatin-induced acute kidney injury by inhibiting ALOX12-dependent ferroptosis

BackgroundIn acute kidney injury (AKI), ferroptosis is the main mechanism of cell death in the renal tubular epithelium. Baicalein, a traditional Chinese medicine monomer, plays a protective role in various kidney diseases; however, the effect of baicalein on ferroptosis in AKI still needs further exploration. PurposeIn this study, we explored the role of baicalein and its specific mechanism in mediating ferroptosis in cisplatin-induced AKI. MethodsWe used a cisplatin-induced AKI model to study the effects of baicalein on renal tissue and tubular epithelial cell injury. The effects of baicalein on tubular epithelial cell ferroptosis were detected in cisplatin-induced AKI and further verified by folic acid-induced AKI. The Swiss Target Prediction online database was used to predict the possible mechanism by which baicalein regulates ferroptosis, and the specific target proteins were further verified. Molecular docking and SPR were used to further determine the binding potential of baicalein to the target protein. Finally, RNA interference (RNAi) technology and enzymatic inhibition were used to determine whether baicalein regulates ferroptosis through target proteins. ResultsBaicalein alleviated cisplatin- and folic acid-induced renal dysfunction and pathological damage and improved cisplatin-induced HK2 cell injury. Mechanistically, baicalein reduced the expression of 12-lipoxygenase (ALOX12), which inhibits phospholipid peroxidation and ferroptosis in AKI. Molecular docking and SPR demonstrated direct binding between baicalein and ALOX12. Finally, we found that silencing ALOX12 had a regulatory effect similar to that of baicalein. Comparable results were also obtained with the ALOX12 inhibitor ML355. ConclusionThis was the first study to confirm that baicalein regulates ferroptosis both in vitro and in vivo in cisplatin-induced AKI and to verify the regulatory effect of baicalein in folic acid-induced AKI. Our results reveal the critical role of ALOX12 in kidney damage and ferroptosis caused by cisplatin and emphasize the regulatory effect of baicalein on renal tubular epithelial cell ferroptosis mediated by ALOX12. Baicalein is an effective drug for treating AKI, and ALOX12 is a potential drug target.