Apoptosis In HK-2 Cells Research Articles

Plasma exosomes may mediate the development of lupus nephritis in patients with systemic lupus erythematosus.

Lupus nephritis (LN) is the most serious complication of systemic lupus erythematosus (SLE), and plasma exosomes may serve as a bridge. MicroRNAs (miRNAs) are abundant in exosomes, so this study aimed to explore the role of exosome-derived miRNA in the development of LN. The publicly available data containing plasma exosomal miRNAs in SLE patients and healthy controls were researched, and differential expression and functional enrichment analysis of exosomal miRNA was conducted. Then, plasma exosomes from SLE patients were extracted, and the accuracy of differential expression and functional enrichment analysis was preliminarily verified. PKH26 dye was used to label exosomes to detect whether exosomes can enter HK2 cells. Evaluation of plasma exosomes impact on cell viability was done by utilizing CCK-8 assay. Flow cytometry was used to measure cell apoptosis. Plasma exosomes were successfully extracted and identified. Through differential expression analysis of the pulbilic data and subsequent qPCR validation, we observed that miR-20b-5p is overexpressed in plasma exosomes of SLE patients, whereas miR-181a-2-3p is downregulated. Then functional enrichment analysis revealed that these differential miRNAs primarily regulate processes such as apoptosis, autophagy, and inflammation. Then, flow cytometry analysis conducted after co-incubation of plasma exosomes and peripheral blood mononuclear cells confirmed that exosomes can indeed regulate apoptosis. And plasma exosomes can successfully enter HK2 cells without affecting cell activity. In addition, plasma exosomes promote HK2 cell apoptosis and autophagy. Overexpression of miR-181a-2-3p could inhibit HK2 cells apoptosis and upregulate the expression of bcl2, and beclin1. At the same time, a trend towards increased apoptosis rates was observed in HK2 overexpressed miR-20b-5p, although the difference did not reach statistical significance. And miR-20b-5p can enhance the expression of caspase3 and becin1 while suppressing the expression of bcl2 and LC3β. Our research indicates that the abundant presence of miR-20b-5p and the depletion of miR-181a-2-3p in plasma exosomes of SLE patients may mediate the promotion of apoptosis and autophagy in HK2 cells, thereby causing kidney damage and the development of LN.

CircTMCO3 alleviates sepsis-induced acute kidney injury via regulating miR-218-5p/ZEB2 axis.

Growing evidence has found the critical role of circular RNAs (circRNAs) in sepsis-induced acute kidney injury (S-AKI). CircTMCO3 has been found to be involved in tumor microenvironment changes of ovarian cancer. This study aimed to explore whether circTMCO3 functions in S-AKI, and if so, to elucidate the molecular mechanism. CircTMCO3 expression was analyzed in lipopolysaccharide (LPS)-induced HK-2 cells and in the kidney tissues of mice treated with cecal ligation and puncture (CLP), respectively. Furthermore, the effects of circTMCO3 on S-AKI and the related mechanisms were evaluated in both models through gain- and/or loss-of-function strategies. CircTMCO3 expression was suppressed in both S-AKI models. Upregulation of circTMCO3 mitigated LPS-induced apoptosis, oxidative stress and inflammation in HK-2 cells. In contrast, circTMCO3 downregulation exacerbated LPS-induced injuries in HK-2 cells. Intravenous injection of circTMCO3 lentivirus to increase circTMCO3 expression improved renal function and attenuated kidney injury in S-AKI mice, as evidenced by the decrease in serum creatinine and blood urea nitrogen concentrations, amelioration of tubular pathological injury, reduction of renal cell apoptosis, and mitigation of oxidative stress and proinflammatory cytokines (TNF-α, IL-1β, and IL-6). Moreover, circTMCO3 directly targeted miR-218-5p, and the mimic of which abolished the protective effect of circTMCO3 in cell models. ZEB2 was identified to be a target of miR-218-5p; its downregulation not only reversed the impacts of miR-218-5p inhibitor on S-AKI, but also mitigated the effects mediated by circTMCO3 upregulation in vitro. CircTMCO3 protects against S-AKI by regulating miR-218-5p/ZEB2 axis, thereby mediating anti-apoptotic, antioxidant and anti-inflammatory activities. This indicates that increasing circTMCO3 expression might be a future therapeutic method for S-AKI.

Hypoxia Activates FGF-23-ERK/MAPK Signaling Pathway in Ischemia-Reperfusion-Induced Acute Kidney Injury.

Both hypoxia and fibroblast growth factor-23 (FGF-23) are key factors in ischemia-reperfusion (I/R)-induced acute kidney injury (AKI). This study aimed to explore the relationship between hypoxia and FGF-23 in AKI. An I/R-AKI animal model was established using male BALB/c mice. HK-2 cells, a part of the human proximal tubular epithelial cell line, were subjected to hypoxia/reoxygenation (H/R). qPCR was used to measure FGF-23 and HIF1α, and ELISA was used to measure inflammatory and oxidative stress cytokines. Western blotting was used to measure the phosphorylation of extracellular signal-regulated kinase (ERK) level. In I/R mice, the levels of interleukin-6 (IL-6), tumor necrosis factor (TNF-α), malondialdehyde (MDA), and the phosphorylation of ERK (p-ERK) were increased, whereas the levels of interleukin-10 (IL-10), superoxide dismutase (SOD), glutathione peroxidase (GPx), and klotho were decreased, compared to the sham-operated mice. Silencing the FGF-23 expression in I/R mice normalized the levels of IL-6, IL-10, TNF-α, MDA, SOD, GPx, and p-ERK. In HK-2 cells, hypoxia-reperfusion (H/R) elevated the levels of IL-6, TNF-α, MDA, and p-ERK, but reduced IL-10, SOD, GPx, and klotho levels. Hypoxia induced apoptosis in HK-2 cells, but silencing of FGF-23 expression blocked the effects of hypoxia on cell apoptosis, pro-inflammatory factor levels, oxidative stress response, and p-ERK levels. FGF-23 is a key molecule in AKI, and hypoxia plays a crucial role in AKI by inducing cell apoptosis; however, its role is regulated by FGF-23. FGF-23 affects oxidative stress and the inflammatory response of kidney tissues by activating the ERK/mitogen-activated protein kinase (MAPK) signaling pathway.

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.

SMYD2 Promotes Calcium Oxalate-Induced Glycolysis in Renal Tubular Epithelial Cells via PTEN Methylation.

Background/Objectives: Damage to renal tubular cells (RTCs) represents a critical pathological manifestation in calcium oxalate (CaOx) stone disease, but the underlying mechanism remains elusive. Energy metabolism reprogramming is a vital influencer of RTC survival, and SMYD2 is a histone methylation transferase that has been extensively implicated in various metabolic disorders. Hence, this research aimed to identify whether SMYD2 induces the reprogramming of energy metabolism in RTCs exposed to CaOx nephrolithiasis. Methods: Kidney samples were obtained from patients who underwent laparoscopic nephrectomy for non-functioning kidneys caused by nephrolithiasis. The glyoxylate-induced CaOx stone mice model was established and treated with AZ505. The SMYD2-knockout HK-2 cell line was constructed. Histological changes were evaluated by HE, VK, Tunel, Masson stainings. The molecular mechanism was explored through co-immunoprecipitation and western blotting. Results: The results found that SMYD2 upregulation led to energy reprogramming to glycolysis in human kidney tissue samples and in mice with CaOx nephrolithiasis. We also identified the substantial involvement of glycolysis in the induction of apoptosis, inflammation, and epithelial-mesenchymal transition (EMT) in HK-2 cells caused by calcium oxalate monohydrate (COM). In vivo and in vitro results demonstrated that SMYD2 inhibition reduces glycolysis, kidney injury, and fibrosis. Mechanistically, SMYD2 was found to promote metabolic reprogramming of RTCs toward glycolysis by activating the AKT/mTOR pathway via methylated PTEN, which mediates CaOx-induced renal injury and fibrosis. Conclusions: Our findings reveal an epigenetic regulatory role of SMYD2 in metabolic reprogramming in CaOx nephrolithiasis and associated kidney injury, suggesting that targeting SMYD2 and glycolysis may represent a potential therapeutic strategy for CaOx-induced kidney injury and fibrosis.

Preparation and characterization of cysteine-rich collagen peptide and its antagonistic effect on microplastic induced damage to HK-2 cells

Cysteine (Cys) plays a significant role in the stability and antioxidant capacity of peptide. This study was to prepare Cys-rich collagen peptide and evaluate its preventive effect on microparticle (MPs)-induced HK-2 cell damage. The results showed that the Cys-rich peptides (AMP-C) were successfully prepared from collagen hydrolysate by plastein reaction in the presence of exogenous Cys. The scavenging activities of 2,2-Diphenyl-1-picrylhydrazyl and hydroxyl radical of AMP-C were significantly higher than those of original peptides without Cys. The fraction with molecular weight < 3 kDa (AMP-CA) significantly improved MPs-induced damage and apoptosis of HK-2 cells. Compared with the same fractions lacking Cys (AMP-A), AMP-CA showed better performance in reducing reactive oxygen species and increasing the total superoxide dismutase and glutathione peroxidase levels, which may be related to its down-regulation of Kelch-like ECH-associated protein 1 (Keap1) expression. In addition, AMP-CA showed strong effects on inhibiting interleukin-6, tumor necrosis factor-α, monocyte chemoattractant protein-1, kidney injury molecule-1, and nuclear factor kappa-B levels. Finally, 330 Cys-containing peptides were isolated from AMP-CA, among which Leucine-Glycine-Asparagine-Glycine-Cysteine-Proline (LGNGCP) showed the lowest binding energy with Keap1 protein. In conclusion, the Cys-rich collagen peptide with higher antioxidant activity was successfully prepared by plastein reaction, which alleviated the HK-2 cells damage induced by MPs through antioxidant and anti-inflammatory. Our findings suggested that Cys-rich collagen peptide might be used as a functional food to mitigate health hazards associated with MPs accumulation.

KLF15 ATTENUATES LIPOPOLYSACCHARIDE-INDUCED APOPTOSIS AND INFLAMMATORY RESPONSE IN RENAL TUBULAR EPITHELIAL CELLS VIA PPARΔ.

Background: The kidney is the most commonly affected organ in sepsis patients, and Krüppel-like transcription factor 15 (KLF15) has a kidney-protective effect and is highly enriched in the kidneys. This study aims to explore the role of KLF15 in sepsis-related acute kidney injury. Methods: A septic injury model in HK2 cells was established through the administration of lipopolysaccharide (LPS), followed by the transfection of an overexpression plasmid for KLF15. Cell viability was assessed using Cell Counting Kit-8 assay, and apoptosis was measured via flow cytometry. The levels of inflammatory cytokines were detected using ELISA, and western blot assay was employed to assess the expression of KLF15, PPARδ, as well as inflammatory and apoptosis-related proteins. The interaction between KLF15 and PPARδ was confirmed through the utilization of online databases and immunoprecipitation experiments. The mechanism was further validated using PPARδ agonists and small interfering RNA. Results: LPS-induced HK2 cells showed downregulated expression of KLF15 and PPARδ, along with decreased viability, accompanied by increased levels of apoptosis, TNFα, IL-1β, and IL-6. Additionally, LPS upregulated the expression of Bax, cytoplasmic cytochrome C [Cytc (cyt)], Cox-2, and p-NF-κB-p65 in HK2 cells, while simultaneously downregulating the expression of Bcl2 and mitochondrial cytochrome c [Cytc (mit)]. immunoprecipitation experiment revealed a possible interaction between KLF15 and PPARδ in HK2 cells. Ov-KLF15, Ov-PPARδ, or administration of PPARδ agonists effectively alleviated the aforementioned alterations induced by LPS. However, interference with PPARδ significantly attenuated the protective effect of Ov-KLF15 on HK2 cells. Conclusion: KLF15 attenuates LPS-induced apoptosis and inflammatory responses in HK2 cells via PPARδ.

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.

LincRNA-p21/AIF-1/CMPK2/NLRP3 pathway promoted inflammation, autophagy and apoptosis of human tubular epithelial cell induced by urate via exosomes

Urate nephropathy, a common complication of hyperuricemia, has garnered increasing attention worldwide. However, the exact pathogenesis of this condition remains unclear. Currently, inflammation is widely accepted as the key factor in urate nephropathy. Therefore, the aim of this study was to elucidate the interaction of lincRNA-p21/AIF-1/CMPK2/NLRP3 via exosomes in urate nephropathy. This study evaluated the effect of lincRNA-p21/AIF-1/CMPK2/NLRP3 using clinical data collected from patients with urate nephropathy and human renal tubular epithelial cells (HK2) cultured with different concentrations of urate. In clinical research section, the level of lincRNA-p21/AIF-1 in exosomes of urine in patients with hyperuricemia or urate nephropathy was found to be increased, particularly in patients with urate nephropathy. In vitro study section, the level of exosomes, inflammation, autophagy, and apoptosis was increased in HK2 cells induced by urate. Additionally, the expression of lincRNA-p21, AIF-1, CMPK2, and NLRP3 was upregulated in exosomes and HK2 cells. Furthermore, manipulating the activity of lincRNA-p21, AIF-1, CMPK2, and NLRP3 through overexpression or interference vectors regulated the level of inflammation, autophagy, and apoptosis in HK2 cells. In conclusion, the pathway of lincRNA-p21/AIF-1/CMPK2/NLRP3 contributed to inflammation, autophagy, and apoptosis of human renal tubular epithelial cell induced by urate via exosomes. Additionally, the specific exosomes in urine might serve as novel biomarkers for urate nephropathy.

Dapagliflozin restores autophagy and attenuates apoptosis via the AMPK/mTOR pathway in diabetic nephropathy rats and high glucose-induced HK-2 cells.

Diabetic nephropathy (DN) is a serious microvascular complication of diabetes mellitus. Significantly reduced levels of autophagy in diabetic kidneys play an important role in the development of DN. The present study investigated the effects of dapagliflozin (DAP) on renal autophagy and AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway in vivo and in vitro. We explored the effect of DAP in streptozotocin (STZ)-induced DN rats. The anti-DN effect of DAP was assessed by body weight, kidney weight/body weight ratio, blood and urine biochemical parameters, and pathological changes of kidney tissue. Number of autophagosomes in the kidney was investigated through Transmission electron microscopy. Besides, cell viability and apoptosis of DAP alone or combined with Compound C (CC, a selective AMPK inhibitor)-treated high glucose (HG)-induced HK-2 cells were detected by Cell Counting Kit-8 (CCK-8) and flow cytometry assays. Immunohistochemistry, Western blot, Enzyme-linked immunosorbent assay (ELISA), and immunofluorescence were employed to detect the expression levels of extracellular matrix (ECM) deposition, autophagy, apoptosis, and AMPK/mTOR pathway-associated targets in vivo and in vitro. The results showed that DAP ameliorated the body weight and decreased kidney weight, fasting blood glucose, and serum/urine biochemical parameters of renal damage, as well as renal pathological changes. Moreover, DAP significantly ameliorated HG-induced cell apoptosis and ECM deposition in HK-2 cells. However, these favorable effects of DAP could be abolished by co-treatment with CC in HG-induced HK-2 cells. Mechanistically, DAP can enhance autophagy in DN including increased LC3-II/I ratio, Beclin-1, p-AMPK protein levels, and decreased p62 and p-mTOR protein expressions, as well as inhibited renal fibrosis and apoptosis. In summary, DAP alleviated fibrosis, apoptosis, and autophagy in DN rats and HG-induced HK-2 cells by regulating the AMPK/mTOR pathway.

MicroRNA-223-3p Targeting SGK1 Regulates Apoptosis and Inflammation in Sepsis-Associated Acute Kidney Injury.

Sepsis and septic shock are significant contributors to the development of acute kidney injury (AKI) in critically ill patients. This study aimed to elucidate the role and mechanism of microRNA-223-3p in sepsis-associated AKI (SA-AKI). Bioinformatics methods were used to analyze the expression of microRNA-223-3p in sepsis patients, its correlation with inflammatory cytokines, and to predict the binding site of microRNA-223-3p with SGK1. The binding relationship between microRNA-223-3p and SGK1 was validated using a dual-luciferase reporter gene assay. The expression of microRNA-223-3p was assayed using qPCR in patient serum or lipopolysaccharide (LPS)-treated HK-2 cells. Cell apoptosis; expression of Bax, Bcl-2, cleaved caspase-3; and levels of TNF-α, IL-1β, and IL-6 were measured using TUNEL assay, Western blot (WB), and ELISA, respectively. SGK1 expression of HK-2 cells with different treatments was detected using qPCR and WB. The expression of microRNA-223-3p was found to be upregulated in sepsis patients and HK-2 cells treated with LPS. Furthermore, microRNA-223-3p promoted apoptosis and inflammation in LPS-induced HK-2 cells. This promotion was mediated by the negative regulation of SGK1 by microRNA-223-3p. The microRNA-223-3p was found to regulate SGK1 and promote apoptosis and inflammation in LPS-induced HK-2 cells. Our study has elucidated the mechanism of microRNA-223-3p in SA-AKI, providing a potential target for sepsis treatment.

Tetrahydrocurcumin Attenuates Polymyxin B Sulfate-Induced HK-2 Cells Apoptosis by Inhibiting Endoplasmic Reticulum Stress-Mediated PERK/eIF2α/ATF4/CHOP Signaling Pathway Axis.

The clinical application of polymyxin B (PMB) is limited by its nephrotoxic effects, making the reduction of PMB-induced nephrotoxicity has become a pressing concern for clinicians. Tetrahydrocurcumin (THC), known for its beneficial characteristics in biological functions, presents an attractive option for intervention therapy to mitigate PMB-induced nephrotoxicity. However, the underlying mechanism of how THC mitigates PMB-induced nephrotoxicity is still poorly understood. Here, we first evaluated the potential of THC intervention therapy to mitigate PMB-induced nephrotoxicity in an in vitro model of PMB-induced cell injury. Moreover, we demonstrated that THC effectively protected HK-2 cells from PMB-induced apoptosis by using cell counting kit-8 and flow cytometry assay. THC could also suppress PMB-induced endoplasmic reticulum (ER) stress via PERK/eIF2α/ATF4/CHOP pathway. In addition, using PERK inhibitor GSK2606414 to inhibit ER stress also alleviated PMB-induced apoptosis. Taken together, these findings provide novel insights that THC possesses the ability to alleviate PMB-induced nephrotoxicity by inhibiting the ER stress-mediated PERK/eIF2α/ATF4/CHOP axis, which sheds light on the benefits of THC as an intervention strategy to reduce PMB-induced nephrotoxicity, thus providing a potential avenue for improved clinical outcomes in patients receiving PMB treatment.

LncRNA HILPDA promotes contrast-induced acute kidney injury by recruiting eIF4B to upregulate XPO1 expression.

Contrast-induced acute kidney injury (CI-AKI) is a serious and common complication following the use of iodinated contrast media, with a 20% fatality rate. The function of long non-coding RNA HILPDA (lnc-HILPDA) in CI-AKI development was investigated in this study. CI-AKI models were constructed by iopromide treatment. Kidney pathological changes were analyzed by HE staining. TUNEL labeling and flow cytometry were used to examine cell apoptosis. CCK-8 assay was used to determine cell viability. The interactions between lnc-HILPDA, eIF4B, and XPO1 were verified by RIP or Co-IP assay. Lnc-HILPDA was upregulated in CI-AKI, and its knockdown decreased contrast-trigged oxidative stress and apoptosis in HK-2 cells. Mechanically, lnc-HILPDA activated the NF-κB pathway by upregulating XPO1 through interacting with eIF4B. Moreover, the inhibitory effect of lnc-HILPDA downregulation on contrast-induced oxidative stress and apoptosis in HK-2 cells was weakened by XPO1 overexpression. Lnc-HILPDA accelerated CI-AKI progression by elevating XPO1 expression through eIF4B to activate NF-κB pathway.

Discovery of mmu-lncRNA129814/hsa-lncRNA582795 as a Potential Biomarker and Intervention Target for Ischemia Reperfusion Injury-Induced AKI.

Acute kidney injury (AKI) is associated with higher perioperative mortality and morbidity, as well as increased medical expenses. The molecular mechanisms underlying ischemia-reperfusion (I/R)-induced AKI remain unclear. We applied an RT-qPCR assay to measure the expression of mmu-lncRNA129814, hsa-lncRNA582795, and miRNA-494-5p, immunoblotting to detect IL-1α and cleaved caspase-3 expression, and TUNEL staining and flow cytometry (FCM) to evaluate apoptosis. The experiments were conducted using BUMPT and HK-2 cells, as well as C57BL/6J mice. Mechanistically, mmu-lncRNA129814 could sponge miRNA-494-5p and upregulate IL-1α expression to promote cell apoptosis. Furthermore, knockdown of mmu-lncRNA129814 ameliorated I/R-induced progression of AKI by targeting the miRNA-494-5p/IL-1α pathways. Interestingly, hsa-lncRNA582795, a homolog of mmu-lncRNA129814, also promoted I/R-stimulated HK-2 cell apoptosis and AKI progression by regulating the miRNA-494-5p/IL-1α axis. Finally, we found that patients with I/R-induced AKI exhibited significantly elevated plasma and urinary levels of hsa-lncRNA582795 compared to those who underwent ischemia-reperfusion without developing AKI. Spearman's test demonstrated a significant correlation between serum creatinine and plasma hsa-lncRNA582795 in I/R patients. Plasma hsa-lncRNA582795 showed high sensitivity but low specificity (86.7%) compared to urinary hsa-lncRNA582795. The mmu-lncRNA129814/hsa-lncRNA582795/miRNA-494-5p/IL-1α axis was found to modulate the progression of ischemic AKI, and hsa-lncRNA582795 could act as a diagnosis biomarker and potential therapy target of I/R-induced AKI.

Chlorogenic Acid Alleviates High Glucose-induced HK-2 Cell Oxidative Damage through Activation of KEAP1/NRF2/ARE Signaling Pathway.

To investigate the alleviating effect of chlorogenic acid (CGA) on oxidative damage in high glucose (HG)-induced HK-2 cells and to explore its potential mechanisms. We cultured the human proximal tubular cell line HK-2 and divided them into the control group and different concentrations of CGA groups (0, 5, 10, 25, 50, 100, 200 μM). The trypan blue dye test was used to detect CGA's potential cytotoxicity on HK-2 cells. Then, we treated HK-2 with HG and CGA; the Cell Counting Kit-8 (CCK-8) method was used to detect the cell viability of HK-2 cells in each group. Flow cytometry was employed to measure the apoptosis rate of cells. Western blot was performed to detect the expression of apoptosis proteins B-cell lymphoma-2 (BCL-2), BCL-2-associated X protein (BAX), cysteinyl aspartate specific proteinase (CASPASE)-9, and CASPASE-3. In addition, enzymatic activities, including superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), and lipid peroxide (LPO), were measured with the corresponding detection kits. 2',7'-Dichlorodihydrofluorescein diacetate (DCFH-DA) assay and flow cytometry were performed to detect reactive oxygen species (ROS) production. Western blot analysis and Reverse Transcription-Polymerase Chain Reaction (RT-PCR) were conducted to evaluate protein and mRNA expressions of the Kelch-like ECH-associated protein-1 (KEAP1)/Nuclear factor erythroid 2-related factor 2 (NRF2)/Antioxidant Response Elements (ARE) signaling pathway. The outcomes showed that, in a dose-dependent way, CGA dramatically increased the vitality of HK-2 induced by HG. Furthermore, CGA significantly reduced the HG-stimulated HK-2 cell apoptosis, which may be linked to the promotion of BCL-2 and the suppression of BAX, cleaved-CASPASE-3, and cleaved-CASPASE-9 expression. In HK-2 cells, CGA reduced the formation of ROS generated by HG levels and markedly boosted the activity of the antioxidant enzymes SOD, GSH-Px, and CAT. Furthermore, compared with the HG group, CGA significantly raised NRF2 nuclear expression and downregulated NRF2 cytosolic expression and increased the mRNA expression of NRF2 and its target genes, heme oxygenase-1 (HO-1), KEAP1, and NAD(P)H dehydrogenase quinone 1 (NQO1). These results show that CGA might be useful in managing oxidative damage in HG-induced HK-2 cells.

Overexpression of CISD2 alleviates septic acute kidney injury via activating Sonic Hedgehog signaling pathway.

Patients with sepsis are often complicated by acute kidney injury (AKI), which greatly increases mortality. In this study, our purpose was to explore the expression and function of CDGSH iron sulfur domain 2 (CISD2) in septic AKI, and the underlying molecular mechanism. Western blot and quantitative real-time polymerase chain reaction (RT-PCR) were employed to detect protein and mRNA levels in cells. The inflammation level of cells was evaluated by detecting the content of inflammatory factors (TNF-α, IL-1β, IL-6). Apoptosis of cells was evaluated by Caspase-3 activity assay, flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP Nick-End Labeling (TUNEL) staining. CISD2 was down-regulated in HK-2 cells treated with lipopolysaccharide (LPS). LPS treatment increased the level of inflammatory factors, the activity of Caspase-3, and the rate of apoptosis in HK-2 cells. However, overexpression of CISD2 significantly suppressed these effects. Moreover, overexpression of CISD2 activated the Sonic Hedgehog (SHH) signaling pathway. The use of cyclopamine (Cyc), a SHH signaling pathway inhibitor, eliminated the effect of overexpressing CISD2, that is, inhibiting LPS-induced inflammation and apoptosis of HK-2 cells. LPS treatment down-regulated CISD2 in HK-2 cells, and overexpression of CISD2 could inhibit LPS-induced inflammation and apoptosis of HK-2 cells by activating the SHH signaling pathway.

Zn2+ improves sepsis-induced acute kidney injury by upregulating SIRT7-mediated Parkin acetylation.

Zn2+ levels are reported to be correlated with kidney function. We explored the significance of Zn2+ in sepsis-induced acute kidney injury (SI-AKI) through the regulation of sirtuin 7 (SIRT7) activity. The sepsis rat model was established by cecal ligation and perforation (CLP) and intraperitoneally injected with ZnSO4 or SIRT7 inhibitor 97491 (SIRT7i), with renal tubular injury assessed by hematoxylin and eosin staining. In vitro, human renal tubular epithelial cells (HK-2) were induced with lipopolysaccharide to obtain a renal injury cell model, followed by ZnSO4 or SIRT7i and autophagy inhibitor (3-methyladenine) treatment. Interleukin (IL)-1β, IL-18, reactive oxygen species (ROS), Parkin acetylation level, kidney injury molecule-1 (KIM-1), and neutrophil gelatinase-associated lipocalin (NGAL) expression levels were determined. The renal tubule injury, inflammation condition, and pyroptosis-related and autophagy-related protein levels were assessed. The pyroptosis in kidney tissues and autophagosome formation were observed by transmission electron microscopy. Zn2+ alleviated renal injury in CLP rats and inhibited pyroptosis and its related protein levels by inhibiting SIRT7 activity in septic rat renal tissues. In vitro, Zn2+ increased HK-2 cell viability and reduced KIM-1, NGAL, IL-1β, IL-18, NLRP3 inflammasome, cleaved caspase-1, gasdermin D-N levels, and pyroptotic cell number. Zn2+ increased autophagosome number and LC3BII/LC3BI ratio and decreased TOM20, TIM23, P62, and mitochondrial ROS levels. Zn2+ increased Parkin acetylation by repressing SIRT7 activity. Inhibiting mitophagy partially averted Zn2+-inhibited NLRP3 inflammasome activation and apoptosis in HK-2 cells. Zn2+ upregulated Parkin acetylation by repressing SIRT7 activity to promote mitophagy and inhibit NLRP3 inflammasome activation and pyroptosis, thus improving SI-AKI.NEW & NOTEWORTHY Zn2+ upregulated Parkin acetylation by repressing sirtuin 7 activity to promote mitophagy and inhibit NLRP3 inflammasome activation and pyroptosis, thus improving sepsis-induced acute kidney injury.

Quercetin prevented diabetic nephropathy by inhibiting renal tubular epithelial cell apoptosis via the PI3K/AKT pathway.

Diabetic nephropathy (DN) is the most common and serious complication of diabetes, posing a significant threat to human health. Currently, safe and effective preventive strategies for DN are lacking. The study aimed to explore the preventive effect and the underlying mechanism of quercetin against DN. In the in vivo experiments, we established a mouse model of type 2 diabetes mellitus (T2DM) induced by a combination of high-fat diet (HFD) and streptozotocin (STZ) to explore the preventive effect of quercetin on DN and its protective role against renal tubular epithelial cell apoptosis. Subsequently, in vitro experiments using human tubular epithelial cells (HK-2 cells) were conducted to further validate the protective effects of quercetin on renal tubular epithelial cell apoptosis. Additionally, we employed RNA sequencing analysis (RNA-seq) and network pharmacology analysis to comprehensively elucidate the molecular mechanisms involved. In vivo, we observed a significant increase in the ratio of urinary microalbumin to creatinine in diabetic mice compared to control mice, accompanied by the activation of renal tubular epithelial cell apoptosis. Remarkably, all of these changes were reversed after quercetin treatment. In vitro, high-glucose-induced apoptosis in HK-2 cells was significantly attenuated by quercetin. Subsequent RNA sequencing analysis and network pharmacology analysis revealed that quercetin was most likely to inhibit high-glucose-induced HK-2 cell apoptosis through the PI3K/AKT signaling pathway. Western Blotting results further demonstrated that quercetin could inhibit the activation of the PI3K/AKT signaling pathway in HK-2 cells induced by high glucose. Our results supported that quercetin could prevent DN by inhibiting tubular epithelial cell apoptosis via the PI3K/AKT pathway. Quercetin might be a promising candidate for the prevention of DN.

DKK3 promotes renal fibrosis by increasing MFF-mediated mitochondrial dysfunction in Wnt/β-catenin pathway-dependent manner

Background Chronic kidney disease (CKD) lacks effective treatments and renal fibrosis (RF) is one of CKD’s outcomes. Dickkopf 3 (DKK3) has been identified as an agonist in CKD. However, the underlying mechanisms of DKK3 in CKD are not fully understood. Methods H2O2-treated HK-2 cells and ureteric obstruction (UUO) mice were used as RF models. Biomarkers, Masson staining, PAS staining, and TUNEL were used to assess kidney function and apoptosis. Oxidative stress and mitochondria function were also evaluated. CCK-8 and flow cytometry were utilized to assess cell viability and apoptosis. Western blotting, IHC, and qRT-PCR were performed to detect molecular expression levels. Immunofluorescence was applied to determine the subcellular localization. Dual luciferase assay, MeRIP, RIP, and ChIP were used to validate the m6A level and the molecule interaction. Results DKK3 was upregulated in UUO mouse kidney tissue and H2O2-treated HK-2 cells. Knockdown of DKK3 inhibited oxidative stress, maintained mitochondrial homeostasis, and alleviated kidney damage and RF in UUO mice. Furthermore, DKK3 silencing suppressed HK-2 cell apoptosis, oxidative stress, and mitochondria fission. Mechanistically, DKK3 upregulation was related to the high m6A level regulated by METTL3. DKK3 activated TCF4/β-catenin and enhanced MFF transcriptional expression by binding to its promoter. Overexpression of MFF reversed in the inhibitory effect of DKK3 knockdown on cell damage. Conclusion Upregulation of DKK3 caused by m6A modification activated the Wnt/β-catenin pathway to increase MFF transcriptional expression, leading to mitochondrial dysfunction and oxidative stress, thereby promoting RF progression.

Activation of the Nrf2/ARE signaling pathway ameliorates hyperlipidemia-induced renal tubular epithelial cell injury by inhibiting mtROS-mediated NLRP3 inflammasome activation.

Dyslipidemia is the most prevalent independent risk factor for patients with chronic kidney disease (CKD). Lipid-induced NLRP3 inflammasome activation in kidney-resident cells exacerbates renal injury by causing sterile inflammation. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that modulates the cellular redox balance; however, the exact role of Nrf2 signaling and its regulation of the NLRP3 inflammasome in hyperlipidemia-induced kidney injury are poorly understood. In this study, we demonstrated that activation of the mtROS-NLRP3 inflammasome pathway is a critical contributor to renal tubular epithelial cell (RTEC) apoptosis under hyperlipidemia. In addition, the Nrf2/ARE signaling pathway is activated in renal tubular epithelial cells under hyperlipidemia conditions both in vivo and in vitro, and Nrf2 silencing accelerated palmitic acid (PA)-induced mtROS production, mitochondrial injury, and NLRP3 inflammasome activation. However, the activation of Nrf2 with tBHQ ameliorated mtROS production, mitochondrial injury, NLRP3 inflammasome activation, and cell apoptosis in PA-induced HK-2 cells and in the kidneys of HFD-induced obese rats. Furthermore, mechanistic studies showed that the potential mechanism of Nrf2-induced NLRP3 inflammasome inhibition involved reducing mtROS generation. Taken together, our results demonstrate that the Nrf2/ARE signaling pathway attenuates hyperlipidemia-induced renal injury through its antioxidative and anti-inflammatory effects through the downregulation of mtROS-mediated NLRP3 inflammasome activation.