HK-2 Cell Injury Research Articles

CD3D silencing alleviates diabetic nephropathy via inhibition of JAK/STAT pathway.

Diabetic nephropathy (DN) is a severe microvascular complication of diabetes that poses a significant burden to global health. This investigation aims to illustrate the functional role of CD3D and its relevant mechanisms in DN progression. The pivotal genes between the GSE47183 and GSE30528 datasets were identified using bioinformatics methods. The effects of CD3D silencing on renal damage, inflammatory response, and lipid metabolism were validated in DN mice. Furthermore, the impacts of CD3D knockdown on cell viability, apoptotic rate, inflammation, and lipid levels were investigated in HK-2 cells under high glucose (HG) conditions. Additionally, RO8191 was employed to investigate the role of CD3D in the JAK/STAT pathway in HG-treated cells. A total of 5 focal genes were identified through bioinformatics and were found to be upregulated in renal tissues from DN mice. CD3D silencing mitigated pathological damage to kidneys, reduced inflammatory response, and decreased lipid accumulation in DN mice. HG stimulation restrained viability, increased apoptosis, promoted the release of inflammatory cytokines, and affected expressions of hallmarks related to lipid metabolism in HG-treated cells; these changes were partially abolished by CD3D knockdown. Mechanistically, CD3D downregulation ameliorated HG-induced injury in HK-2 cells by blocking the JAK/STAT pathway. This study underscores that CD3D silencing has significant potential as a promising candidate in the treatment of DN.

Overexpression of miR-451a Aggravates Renal Ischemia–Reperfusion Injury by Targeting KLF1-ACSL4 to Promote Ferroptosis

Ischemia–reperfusion injury (IRI) is a predominant factor leading to delayed graft function (DGF) following kidney transplantation. MicroRNAs (miRNAs) play a pivotal role in the pathogenesis of renal IRI, with ferroptosis being a critical driving force throughout the process. In this study, we utilized bioinformatics methods to construct a network diagram of differentially expressed miRNAs, transcription factors (TFs), and ferroptosis-related genes. An I/R-induced renal injury model in mice and an in vitro H/R-induced HK-2 cell injury model were established. Quantitative real-time PCR (qRT-PCR) and Western blot analysis were used to measure the mRNA and miRNA levels in cells and tissues. The MDA concentration, iron levels, and GSH concentration were measured to evaluate the ferroptosis levels. CCK-8 assays were performed to assess cell viability. Luciferase reporter assays were conducted to validate the downstream targets of miRNA, and chromatin immunoprecipitation assays were performed to verify the interaction between TFs and mRNAs. Both the in vivo and in vitro results demonstrate that miR-451a was significantly enriched in the IRI renal tissues and cells, exacerbating ferroptosis. MiR-451a was found to reduce the expression of Kruppel-like factor 1 (KLF1) by directly binding to the 3′UTR of KLF1 mRNA. Additionally, KLF1 was identified as a negative transcription factor for acyl-CoA synthetase long-chain family member 4 (ACSL4). We demonstrated that IRI induced the upregulation of miR-451a, which reduced KLF1 expression, thereby promoting ferroptosis by upregulating ACSL4 expression, ultimately aggravating IRI-induced renal damage.

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.

HSPA12A stimulates "Smurf1-Hif1α-aerobic glycolysis" axis to promote proliferation of renal tubular epithelial cells after hypoxia/reoxygenation injury.

Proliferation of renal tubular epithelial cells (TECs) is critical for the recovery after kidney ischemia/reperfusion (KI/R). However, there is still a lack of ideal therapies for promoting TEC proliferation. Heat shock protein A12A (HSPA12A) shows abundant expression in kidney in our previous studies. To investigate the role of HSPA12A in TEC proliferation after KI/R, an in vitro KI/R model was simulated by hypoxia (12h) and reoxygenation (12h) in human kidney tubular epithelial HK-2 cells. We found that, when hypoxia/reoxygenation (H/R) triggered HK-2 cell injury, HSPA12A expression was downregulated, and extracellular lactate, the readout of glycolysis, was also decreased. Loss and gain of functional studies showed that HSPA12A did not change cell viability after hypoxia but increased cell proliferation as well as glycolytic flux of HK-2 cells after H/R. When blocking glycolysis by 2-deoxy-D-glucose or oxamate, the HSPA12A promoted HK-2 cell proliferation was also abolished. Further analysis revealed that HSPA12A overexpression increased hypoxia-inducible factor 1α (Hif1α) protein expression and nuclear localization in HK-2 cells in response to H/R, whereas HSPA12A knockdown showed the opposite effects. Notably, pharmacologicalinhibition of Hif1α with YC-1 reversed the HSPA12A-induced increases of both glycolytic flux and proliferation of H/R HK-2 cells. Moreover, the HSPA12A increased Hif1α protein expression was not via upregulating its transcription but through increasing its protein stability in a Smurf1-dependent manner. The findings indicate that HSPA12A might serve as a promising target for TEC proliferation to help recovery after KI/R.

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.

Acidic preconditioning induced intracellular acid adaptation to protect renal injury via dynamic phosphorylation of focal adhesion kinase-dependent activation of sodium hydrogen exchanger 1

BackgroundDisruptions in intracellular pH (pHi) homeostasis, causing deviations from the physiological range, can damage renal epithelial cells. However, the existence of an adaptive mechanism to restore pHi to normalcy remains unclear. Early research identified H+ as a critical mediator of ischemic preconditioning (IPC), leading to the concept of acidic preconditioning (AP). This concept proposes that short-term, repetitive acidic stimulation can enhance a cell’s capacity to withstand subsequent adverse stress. While AP has demonstrated protective effects in various ischemia-reperfusion (I/R) injury models, its application in kidney injury remains largely unexplored.MethodsAn AP model was established in human kidney (HK2) cells by treating them with an acidic medium for 12 h, followed by a recovery period with a normal medium for 6 h. To induce hypoxia/reoxygenation (H/R) injury, HK2 cells were subjected to hypoxia for 24 h and reoxygenation for 1 h. In vivo, a mouse model of IPC was established by clamping the bilateral renal pedicles for 15 min, followed by reperfusion for 4 days. Conversely, the I/R model involved clamping the bilateral renal pedicles for 35 min and reperfusion for 24 h. Western blotting was employed to evaluate the expression levels of cleaved caspase 3, cleaved caspase 9, NHE1, KIM1, FAK, and NOX4. A pH-sensitive fluorescent probe was used to measure pHi, while a Hemin/CNF microelectrode monitored kidney tissue pH. Immunofluorescence staining was performed to visualize the localization of NHE1, NOX4, and FAK, along with the actin cytoskeleton structure in HK2 cells. Cell adhesion and scratch assays were conducted to assess cell motility.ResultsOur findings demonstrated that AP could effectively mitigate H/R injury in HK2 cells. This protective effect and the maintenance of pHi homeostasis by AP involved the upregulation of Na+/H+ exchanger 1 (NHE1) expression and activity. The activity of NHE1 was regulated by dynamic changes in pHi-dependent phosphorylation of Focal Adhesion Kinase (FAK) at Y397. This process was associated with NOX4-mediated reactive oxygen species (ROS) production. Furthermore, AP induced the co-localization of FAK, NOX4, and NHE1 in focal adhesions, promoting cytoskeletal remodeling and enhancing cell adhesion and migration capabilities.ConclusionsThis study provides compelling evidence that AP maintains pHi homeostasis and promotes cytoskeletal remodeling through FAK/NOX4/NHE1 signaling. This signaling pathway ultimately contributes to alleviated H/R injury in HK2 cells.

Knockdown of long non-coding RNA SBF2-AS1 inhibits calcium oxalate-induced HK-2 cell injury by regulating the miR-302e/NLRP3 pathway.

Long non-coding ribose nucleic acids (lncRNAs) have been implicated in the development of nephrolithiasis. The study aims to investigate the interplay of lncRNA SBF2-AS1 (SETbinding factor 2 antisense RNA 1) and NLR family pyrin domain containing 3 (NLRP3) in regulating the calcium oxalate monohydrate (COM)-induced human kidney HK-2 cell injury. HK-2 cells were treated with COM (100µg/mL) to create a cellular model of kidney injury. Gene and protein expression was assessed by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) and Western blot. Proliferation and apoptosis rates, as well as levels of malondialdehyde (MDA), lactate dehydrogenase (LDH), superoxide dismutase (SOD), tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 were measured. Additionally, potential miRNAs interacting with SBF2-AS1 and NLRP3 were predicted utilizing the starBase and TargetScan databases. The interference of SBF2-AS1 resulted in increased cell proliferation and SOD levels in HK-2 cells after COM induction. SBF2-AS1 silencing also reduced COM-induced cell death and inflammatory cytokine production by down-regulating NLRP3 protein expression. Conversely, forced upregulation of NLRP3 abrogated the effect of SBF2-AS1 interference. Notably, SBF2-AS1 interference on COM-induced oxidative stress and COM-induced cellular damage was rescued by antioxidant, indicating the involvement of oxidative burden in COM-induced damage. miR-302e acted as a mediator miRNA linking the functional association of SBF2-AS1 and NLRP3. Silencing SBF2-AS1 promoted miR-302e level and miR-302e reduced NLRP3 expression in HK-2 cells to protect against COM-induced damage. In summary, these findings suggest that downregulation of lncRNA SBF2-AS1 can potentially protect HK-2 cells from COM-induced injury by modulating the miR-302e/NLRP3 pathway.

Marrow mesenchymal stem cell mediates diabetic nephropathy progression via modulation of Smad2/3/WTAP/m6A/ENO1 axis.

Diabetic nephropathy (DN) is one of the common microvascular complications in diabetic patients. Marrow mesenchymal stem cells (MSCs) have attracted attention in DN therapy but the underlying mechanism remains unclear. Here, we show that MSC administration alleviates high glucose (HG)-induced human kidney tubular epithelial cell (HK-2 cell) injury and ameliorates renal injury in DN mice. We identify that Smad2/3 is responsible for MSCs-regulated DN progression. The activity of Smad2/3 was predominantly upregulated in HG-induced HK-2 cell and DN mice and suppressed with MSC administration. Activation of Smad2/3 via transforming growth factor-β1 (TGF-β1) administration abrogates the protective effect of MSCs on HG-induced HK-2 cell injury and renal injury of DN mice. Smad2/3 has been reported to interact with methyltransferase of N6-methyladenosine (m6A) complex and we found a methyltransferase, Wilms' tumor 1-associating protein (WTAP), is involved in MSCs-Smad2/3-regulated DN development. Moreover, WTAP overexpression abrogates the improvement of MSCs on HG-induced HK-2 cell injury and renal injury of DN mice. Subsequently, α-enolase (ENO1)is the downstream target of WTAP-mediated m6A modification and contributes to the MSCs-mediated regulation. Collectively, these findings reveal a molecular mechanism in DN progression and indicate that Smad2/3/WTAP/ENO1 may present a target for MSCs-mediated DN therapy.

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.

Wnt/β-catenin Pathway Aggravates Renal Fibrosis by Activating PUM2 Transcription to Repress YME1L-mediated Mitochondrial Homeostasis.

Chronic kidney disease (CKD) affects more than 10% of people worldwide and is a leading cause of death. However, the pathogenesis of CKD remains elusive. The oxidative stress and mitochondrial membrane potential were detected using Enzyme-linked immunosorbent assay and JC-1 assay. Co-immunoprecipitation, dual-luciferase assay, chromatin IP, RNA IP and RNA pull-down were used to validate the interactions among genes. Exploiting a H2O2-induced fibrosis model in vitro, PUM2 expression was upregulated in Human kidney 2 cell (HK-2) cells, along with reduced cell viability, enhanced oxidative stress, impaired mitochondrial potential, and upregulated expressions of fibrosis-associated proteins. While PUM2 knockdown reversed the H2O2-induced injury in HK-2 cells. Mechanically, Wnt/β-catenin pathway activated PUM2 transcription via TCF4. It was further identified that Wnt/β-catenin pathway inhibited YME1L expression through PUM2-mediated destabilizing of its mRNA. PUM2 aggravated H2O2-induced oxidative stress, mitochondrial dysfunction, and renal fibrosis in HK-2 cell via suppressing YME1L expression. Our study revealed that Wnt/β-catenin aggravated renal fibrosis by activating PUM2 transcription to repress YME1L-mediated mitochondrial homeostasis, providing novel insights and potential therapeutic targets for the treatment of kidney fibrosis.

CIRCVMA21-RELATED PATHWAY ALLEVIATES LIPOPOLYSACCHARIDE-INDUCED HK-2 CELL INJURY.

Background : It is reported that circVMA21 has an inhibition effect on sepsis-induced acute kidney injury (AKI). Therefore, the underlying molecular mechanisms of circVMA21 in AKI are worthy of further investigation. Material and Methods : Lipopolysaccharide (LPS) was used to induce HK2 cell injury. CircVMA21, miR-337-3p and ZEB2 expression was tested by qRT-PCR. Cell growth was detected by CCK8 assay, EdU assay, and flow cytometry. Protein levels were examined by western blot. The levels of inflammatory factors and oxidative stress markers were measured to evaluate cell inflammatory response and oxidative stress. RNA relationship as verified by dual-luciferase reporter assay, RIP assay, and RNA pull-down assay. Results : CircVMA21 had decreased expression in AKI patients. Overexpressed circVMA21 alleviated LPS-induced HK2 cell inflammation, apoptosis, and oxidative stress. Moreover, circVMA21 sponged miR-337-3p, and miR-337-3p targeted ZEB2. The inhibitory effect of circVMA21 on LPS-induced HK2 cell injury was reversed by miR-337-3p overexpression, and ZEB2 overexpression abolished the promotion effect of miR-337-3p on LPS-induced HK2 cell injury. Conclusions : CircVMA21 could inhibit LPS-induced HK2 cell injury via miR-337-3p/ZEB2 axis.

Knockdown of circ-Gatad1 alleviates LPS induced HK2 cell injury via targeting miR-22-3p/TRPM7 axis in septic acute kidney

BackgroundSepsis is a life-threatening, systemic inflammatory disease that can lead to a variety of conditions, including septic acute kidney injury (AKI). Recently, multiple circular Rnas (circRNAs) have been implicated in the development of this disease.MethodsIn this study, we aimed to elucidate the role of circ-Gatad1 in sepsis induced AKI and its potential mechanism of action. High-throughput sequencing was used to investigate abnormal expression of circRNA in AKI and healthy volunteer. Bioinformatics analysis and luciferase reporting analysis were used to clarify the interacted relationship among circRNA, miRNA and mRNA. HK2 cells were treated with lipopolysaccharide (LPS) to establish septic AKI cell model. HK2 cells were employ to analysis the ROS, inflammatory cytokines expression, proliferation and apoptosis under LPS condition.ResultsThe result show that the expression of circ-Gatad1 was increased in septic acute kidney patients. Downregulation circ-Gatad1 suppressed LPS-treated induced HK2 cells injury including apoptosis, proliferation ability, ROS and inflammatory cytokines level. Bioinformatics and luciferase report analysis confirmed that both miR-22-3p and TRPM7 were downstream targets of circ-Gatad1. Overexpression of TRPM7 or downregulation of miR-22-3p reversed the protective effect of si-circ-Gatad1 to HK2 after exposure to LPS (5 µg/ml) microenvironment.ConclusionIn conclusion, knockdown of circ-Gatad1 alleviates LPS induced HK2 cell injury via targeting miR-22-3p/TRPM7 axis in septic acute kidney.

Dual Immunoglobulin Domain-Containing Cell Adhesion Molecule Increases Early in Renal Tubular Cell Injury and Plays Anti-Inflammatory Role.

Dual immunoglobulin domain-containing cell adhesion molecule (DICAM) is a type I transmembrane protein that presents in various cells including renal tubular cells. This study evaluated the expression and protective role of DICAM in renal tubular cell injury. HK-2 cells were incubated and treated with lipopolysaccharide (LPS, 30 μg/mL) or hydrogen peroxide (H2O2, 100 μM) for 24 h. To investigate the effect of the gene silencing of DICAM, small interfering RNA of DICAM was used. Additionally, to explain its role in cellular response to injury, DICAM was overexpressed using an adenoviral vector. DICAM protein expression levels significantly increased following treatment with LPS or H2O2 in HK-2 cells. In response to oxidative stress, DICAM showed an earlier increase (2-4 h following treatment) than neutrophil gelatinase-associated lipocalin (NGAL) (24 h following treatment). DICAM gene silencing increased the protein expression of inflammation-related markers, including IL-1β, TNF-α, NOX4, integrin β1, and integrin β3, in H2O2-induced HK-2 cell injury. Likewise, in the LPS-induced HK-2 cell injury, DICAM knockdown led to a decrease in occludin levels and an increase in integrin β3, IL-1β, and IL-6 levels. Furthermore, DICAM overexpression followed by LPS-induced HK-2 cell injury resulted in an increase in occludin levels and a decrease in integrin β1, integrin β3, TNF-α, IL-1β, and IL-6 levels, suggesting an alleviating effect on inflammatory responses. DICAM was elevated in the early stage of regular tubular cell injury and may protect against renal tubular injury through its anti-inflammatory properties. DICAM has a potential as an early diagnostic marker and therapeutic target for renal cell injury.

Protection of Ndrg2 deficiency on renal ischemia-reperfusion injury via activating PINK1/Parkin-mediated mitophagy.

Renal ischemia-reperfusion (R-I/R) injury is the most prevalent cause of acute kidney injury, with high mortality and poor prognosis. However, the underlying pathological mechanisms are not yet fully understood. Therefore, this study aimed to investigate the role of N-myc downstream-regulated gene 2 (Ndrg2) in R-I/R injury. We examined the expression of Ndrg2 in the kidney under normal physiological conditions and after R-I/R injury by immunofluorescence staining, real-time polymerase chain reaction, and western blotting. We then detected R-I/R injury in Ndrg2-deficient (Ndrg2-/-) mice and wild type (Ndrg2+/+) littermates in vivo, and detected oxygen and glucose deprivation and reperfusion injury (OGD-R) in HK-2 cells. We further conducted transcriptomic sequencing to investigate the role of Ndrg2 in R-I/R injury and detected levels of oxidative stress and mitochondrial damage by dihydroethidium staining, biochemical assays, and western blot. Finally, we measured the levels of mitophagy in Ndrg2+/+ and Ndrg2-/- mice after R-I/R injury or HK-2 cells in OGD-R injury. We found that Ndrg2 was primarily expressed in renal proximal tubules and significantly decreased its expression 24h after R-I/R injury. Ndrg2-/- mice exhibited significantly attenuated R-I/R injury compared to Ndrg2+/+ mice. Transcriptomics profiling showed that Ndrg2 deficiency induced perturbations of multiple signaling pathways, downregulated inflammatory responses and oxidative stress, and increased autophagy following R-I/R injury. Further studies revealed that Ndrg2 deficiency reduced oxidative stress and mitochondrial damage. Notably, Ndrg2 deficiency significantly activated phosphatase and tensin homologue on chromosome ten-induced putative kinase 1 (PINK1)/Parkin-mediated mitophagy. The downregulation of NDRG2 expression significantly increased cell viability after OGD-R injury, increased the expression of heme oxygenase-1, decreased the expression of nicotinamide adenine dinucleotide phosphate oxidase 4, and increased the expression of the PINK1/Parkin pathway. Ndrg2 deficiency might become a therapy target for R-I/R injury by decreasing oxidative stress, maintaining mitochondrial homeostasis, and activating PINK1/Parkin-mediated mitophagy.

Role of TFEB-autophagy lysosomal pathway in palmitic acid induced renal tubular epithelial cell injury

Lysosomal dysfunction and impaired autophagic flux are involved in the pathogenesis of lipotoxicity in the kidney. Here, we investigated the role of transcription factor EB (TFEB), a master regulator of autophagy-lysosomal pathway, in palmitic acid induced renal tubular epithelial cells injury. We examined lipid accumulation, autophagic flux, expression of Ps211-TFEB, and nuclear translocation of TFEB in HK-2 cells overloaded with palmitic acid (PA). By utilizing immunohistochemistry, we detected TFEB expression in renal biopsy tissues from patients with diabetic nephropathy and normal renal tissue adjacent to surgically removed renal carcinoma (controls), as well as kidney tissues from rat fed with high-fat diet (HFD) and low-fat diet (LFD). We found significant lipid accumulation, increased apoptosis, accompanied with elevated Ps211-TFEB, decreased nuclear TFEB, reduced lysosome biogenesis and insufficient autophagy in HK-2 cells treated with PA. Kidney tissues from patients with diabetic nephropathy had lower nuclear and total levels of TFEB than that in control kidney tissues. Level of renal nuclear TFEB in HFD rats was also lower than that in LFD rats. Exogenous overexpression of TFEB increased the nuclear TFEB level in HK-2 cells treated with PA, promoted lysosomal biogenesis, improved autophagic flux, reduced lipid accumulation and apoptosis. Our results collectively indicate that PA is a strong inducer for TFEB phosphorylation modification at ser211 accompanied with lower nuclear translocation of TFEB. Impairment of TFEB-mediated lysosomal biogenesis and function by palmitic acid may lead to insufficient autophagy and promote HK-2 cells injury.

CircTAOK1 regulates high glucose induced inflammation, oxidative stress, ECM accumulation, and apoptosis in diabetic nephropathy via targeting miR-142-3p/SOX6 axis.

Diabetic nephropathy (DN) is a complication caused by diabetes. Circular RNAs (circRNAs) are a kind of RNA with a closed circular structure, which has high stability and is involved in many disease-related processes. The mechanism of circRNA TAO kinase 1 (circTAOK1) in the pathogenesis and development of DN is unclear. CircTAOK1, microRNA (miR)-142-3p, and sex-determining region Y-box transcription factor 6 (SOX6) mRNA levels were analyzed by real-time quantitative polymerase chain reaction (RT-qPCR). Cell counting kit-8 (CCK8) and 5-ethynyl-2'-deoxyuridine (EdU) assays were used to analyze cell proliferation. Cell cycle distribution was detected by flow cytometry. Western blot assay was performed to test B-cell lymphoma 2 (Bcl-2), Bcl-2 associated X (Bax), cleaved-caspase 3, and fibronectin (FN), collagen I (Col I), and collagen IV (Col IV) protein levels. ELISA assay was used to measure interleukin 1β (IL-1β), interleukin 6 (IL-6), and tumor necrosis factor (TNF-α) levels. The reactive oxygen species (ROS) and malondialdehyde (MDA) levels and the superoxide dismutase (SOD) activity were assessed by the corresponding kits. And the correlation between miR-142-3p and circTAOK1 or SOX6 was confirmed by dual luciferase reporter assay, RNA immunoprecipitation assay and RNA pull down assay. CircTAOK1 and SOX6 expression levels were up-regulated, while miR-142-3p expression was down-regulated in DN serum and HG-treated HK-2 cells. Knockdown of circTAOK1 could inhibit cell injury of HG-induced HK-2 cells. The inhibitory effect of circTAOK1 knockdown on HG-induced HK-2 cell injury was restored by miR-142-3p downregulation. CircTAOK1 acted as a sponge for miR-142-3p, and SOX6 was targeted by miR-142-3p. The overexpression of SOX6 could recover the effect of miR-142-3p overexpression on HG-induced HK-2 cell injury. CircTAOK1 regulated the expression of SOX6 by targeting miR-142-3p. CircTAOK1 knockdown inhibited HG-induced HK-2 cell damage in DN by the miR-142-3p/SOX6 axis.

Circ_0006944 aggravates LPS-induced HK2 cell injury via modulating miR-205-5p/UBL4A pathway

Circular RNAs (circRNAs) has been manifested to be involved in the development of human diseases, including sepsis-associated acute kidney injury (SA-AKI). However, the function and mechanism of circ_0006944 in SA-AKI has not been validated. Lipopolysaccharide (LPS) was utilised to induce AKI cell model. Levels of genes and proteins were monitored by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. Cell counting kit 8 assay, EdU assay and flow cytometry were exploited to estimate cell proliferation and apoptosis. The concentrations of inflammation factors were measured via using ELISA assay. The levels of MDA and SOD were tested by the corresponding kits. The relationship between miR-205-5p and circ_0006944 or UBL4A was verified by dual-luciferase reporter assay and RIP assay. Circ_0006944 was overexpressed in SA-AKI patients, and interference of circ_0006944 restrained LPS-stimulated HK2 cell proliferation repression, apoptosis, inflammation and oxidative stress. Mechanistically, circ_0006944 could sponge miR-205-5p, and miR-205-5p interference counteracted circ_0006944 inhibition-mediated impact on the biological functions in LPS-induced HK2 cell. Additionally, UBL4A was targeted by miR-205-5p, and UBL4A overexpression also partially abolished the repressive impacts of miR-205-5p on LPS-triggered HK2 cell damage. Importantly, circ_0006944 sponged miR-205-5p to mediate the expression of UBL4A. Our outcomes identified that circ_0006944 exacerbated SA-AKI development via miR-205-5p/UBL4A axis, which might be a potential treatment and diagnosis biomarker for SA-AKI.

Sweroside plays a role in mitigating high glucose-induced damage in human renal tubular epithelial HK-2 cells by regulating the SIRT1/NF-κB signaling pathway.

Sweroside is a natural monoterpene derived from Swertia pseudochinensis Hara. Recently, studies have shown that sweroside exhibits a variety of biological activities, such as anti-inflammatory, antioxidant, and hypoglycemic effects. However, its role and mechanisms in high glucose (HG)-induced renal injury remain unclear. Herein, we established a renal injury model in vitro by inducing human renal tubular epithelial cell (HK-2 cells) injury by HG. Then, the effects of sweroside on HK-2 cell activity, inflammation, reactive oxygen species (ROS) production, and epithelial mesenchymal transition (EMT) were observed. As a result, sweroside treatment ameliorated the viability, inhibited the secretion of inflammatory cytokines (TNF-α, IL-1β, and VCAM-1), reduced the generation of ROS, and inhibited EMT in HK-2 cells. Moreover, the protein expression of SIRT1 was increased and the acetylation of p65 NF-kB was decreased in HK-2 cells with sweroside treatment. More importantly, EX527, an inhibitor of SIRT1, that inactivated SIRT1, abolished the improvement effects of sweroside on HK-2 cells. Our findings suggested that sweroside may mitigate HG-caused injury in HK-2 cells by promoting SIRT1-mediated deacetylation of p65 NF-kB.

Paeoniflorin alleviates hypoxia/reoxygenation injury in HK-2 cells by inhibiting apoptosis and repressing oxidative damage via Keap1/Nrf2/HO-1 pathway

Acute kidney injury (AKI) is a serious disorder associated with significant morbidity and mortality. AKI and ischemia/reperfusion (hypoxia/reoxygenation, H/R) injury can be induced due to several reasons. Paeoniflorin (PF) is a traditional herbal medicine derived from Paeonia lactiflora Pall. It exerts diverse therapeutic effects, including anti-inflammatory, antioxidative, antiapoptotic, and immunomodulatory properties; thus, it is considered valuable for treating several diseases. However, the effects of PF on H/R injury-induced AKI remain unknown. In this study, we established an in vitro H/R model using COCL2 and investigated the functions and underlying mechanisms of PF on H/R injury in HK-2 cells. The cell vitality was evaluated using the cell count kit-8 assay. The DCFH-DA fluorescence probe was used to measure the levels of reactive oxygen species (ROS). Oxidative damage was detected using superoxide dismutase (SOD) and malondialdehyde (MDA) assay kits. Apoptotic relative protein and Keap1/Nrf2/HO-1 signaling were evaluated by Western blotting. Our results indicated that PF increased cell viability and SOD activity and decreased the ROS and MDA levels in HK-2 cells with H/R injury. PF inhibits apoptosis by increasing Bcl-2 and decreasing Bax. Furthermore, PF significantly upregulated the expression of HO-1 and Nrf2, but downregulated the expression of HIF-1α and Keap1. PF considerably increased Nrf2 nuclear translocation and unregulated the HO-1 expression. The Nrf2 inhibitor (ML385) could reverse the abovementioned protective effects of PF, suggesting that Nrf2 can be a critical target of PF. To conclude, we found that PF attenuates H/R injury-induced AKI by decreasing the oxidative damage via the Nrf2/HO-1 pathway and inhibiting apoptosis.

Carnosine reduces serum uric acid in hyperuricemia rats via restoring hepatorenal dysfunction and enhancing uric acid excretion by inhibiting inflammation

Carnosine is a new type of food supplement with various bioactive functions, which has been widely used in fields such as health products, functional beverages, cosmetology, etc. However, there are few studies and application of carnosine in hyperuricemia. In the present study, the hyperuricemia rat model and the HK-2 cell injury model were used for investigating the anti-hyperuricemia effect and mechanism of carnosine. The results shown that, after carnosine administration, the content of serum uric acid(SUA) was decreased by 40.9%, the hepatic oxidative injury and the renal inflammatory injury were inhibited, the gene expressions of renal urate transporter 1(URAT1) and glucose transporter type 9(GLUT9) were remarkably downregulated. In the HK-2 cell injury model, the protein expressions of p-p65, p-JNK, NLRP3, caspase-1, URAT1 and GLUT9 were inhibited by carnosine. Furthermore, the protein expressions of URAT1 and GLUT9 were significantly decreased after the inhibition of p-p65 and p-JNK by QNZ and SP600125.