Rat Renal Tubular Epithelial Cells Research Articles

HIF-1α participates in the regulation of S100A16-HRD1-GSK3β/CK1α pathway in renal hypoxia injury

S100 calcium-binding protein 16 (S100A16) is implicated in both chronic kidney disease (CKD) and acute kidney injury (AKI). Previous research has shown that S100A16 contributes to AKI by facilitating the ubiquitylation and degradation of glycogen synthase kinase 3β (GSK3β) and casein kinase 1α (CK1α) through the activation of HMG-CoA reductase degradation protein 1 (HRD1). However, the mechanisms governing S100A16-induced HRD1 activation and the upregulation of S100A16 expression in renal injury are not fully understood. In this study, we observed elevated expression of Hypoxia-inducible Factor 1-alpha (HIF-1α) in the kidneys of mice subjected to ischemia-reperfusion injury (IRI). S100A16 deletion attenuated the increased HIF-1α expression induced by IRI. Using a S100A16 knockout rat renal tubular epithelial cell line (NRK-52E cells), we found that S100A16 knockout effectively mitigated apoptosis during hypoxic reoxygenation (H/R) and cell injury induced by TGF-β1. Our results revealed that H/R injuries increased both protein and mRNA levels of HIF-1α and HRD1 in renal tubular cells. S100A16 knockout reversed the expressions of HIF-1α and HRD1 under H/R conditions. Conversely, S100A16 overexpression in NRK-52E cells elevated HIF-1α and HRD1 levels. HIF-1α overexpression increased HRD1 and β-catenin while decreasing GSK-3β. HIF-1α inhibition restored HRD1 and β-catenin upregulation and GSK-3β downregulation by cellular H/R injury. Notably, Chromatin immunoprecipitation (ChIP) and luciferase reporter assays demonstrated HIF-1α binding signals on the HRD1 promoter, and luciferase reporter gene assays confirmed HIF-1α‘s transcriptional regulation of HRD1. Additionally, we identified Transcription Factor AP-2 Beta (TFAP2B) as the upregulator of S100A16. ChIP and luciferase reporter assays confirmed TFAP2B as a transcription factor for S100A16. In summary, this study identifies TFAP2B as the transcription factor for S100A16 and demonstrates HIF-1α regulation of HRD1 transcription within the S100A16-HRD1-GSK3β/CK1α pathway during renal hypoxia injury. These findings provide crucial insights into the molecular mechanisms of kidney injury, offering potential avenues for therapeutic intervention.

SIRT1 alleviates Cd nephrotoxicity through NF-κB/p65 deacetylation–mediated pyroptosis in rat renal tubular epithelial cells

Cadmium (Cd) is a widely distributed environmental pollutant, primarily causing nephrotoxicity through renal proximal tubular cell impairment. Pyroptosis is an inflammation-related nucleotide-binding oligomerization segment-like receptor family 3 (NLRP3)-dependent pathway for programmed cell death. We previously reported that inappropriate inflammation caused by Cd is a major contributor to kidney injury. Therefore, research on Cd-induced inflammatory response and pyroptosis may clarify the mechanisms underlying Cd-induced nephrotoxicity. In this study, we observed that Cd-induced nephrotoxicity is associated with NLRP3 inflammasome activation, leading to an increase in proinflammatory cytokine expression and secretion, as well as pyroptosis-related gene upregulation, both in primary rat proximal tubular (rPT) cells and kidney tissue from Cd-treated rats. In vitro, these effects were significantly abrogated through siRNA-based Nlrp3 silencing; thus, Cd may trigger pyroptosis through an NLRP3 inflammasome–dependent pathway. Moreover, Cd exposure considerably elevated reactive oxygen species (ROS) content. N-acetyl-l-cysteine, an ROS scavenger, mitigated Cd-induced NLRP3 inflammasome activation and subsequent pyroptosis. Mechanistically, Cd hindered the expression and deacetylase activity of SIRT1, eventually leading to a decline in SIRT1–p65 interactions, followed by an elevation in acetylated p65 levels. The administration of resveratrol (a SIRT1 agonist) or overexpression of Sirt1 counteracted Cd-induced RELA/p65/NLRP3 pathway activation considerably, leading to pyroptosis. This is the first study to reveal significant contributions of SIRT1-triggered p65 deacetylation to pyroptosis and its protective effects against Cd-induced chronic kidney injury. Our results may aid in developing potential therapeutic strategies for preventing Cd-induced pyroptosis through SIRT1-mediated p65 deacetylation.

MicroRNA-630 alleviates inflammatory reactions in rats with diabetic kidney disease by targeting toll-like receptor 4.

Diabetic kidney disease (DKD) is a major complication of diabetes mellitus. Renal tubular epithelial cell (TEC) damage, which is strongly associated with the inflammatory response and mesenchymal trans-differentiation, plays a significant role in DKD; However, the precise molecular mechanism is unknown. The recently identified microRNA-630 (miR-630) has been hypothesized to be closely associated with cell migration, apoptosis, and autophagy. However, the association between miR-630 and DKD and the underlying mechanism remain unknown. To investigate how miR-630 affects TEC injury and the inflammatory response in DKD rats. Streptozotocin was administered to six-week-old male rats to create a hyperglycemic diabetic model. In the second week of modeling, the rats were divided into control, DKD, negative control of lentivirus, and miR-630 overexpression groups. After 8 wk, urine and blood samples were collected for the kidney injury assays, and renal tissues were removed for further molecular assays. The target gene for miR-630 was predicted using bioinformatics, and the association between miR-630 and toll-like receptor 4 (TLR4) was confirmed using in vitro investigations and double luciferase reporter gene assays. Overexpression of miR-630 in DKD rats led to changes in body weight, renal weight index, basic blood parameters and histopathological changes. The expression level of miR-630 was reduced in the kidney tissue of rats with DKD (P < 0.05). The miR-630 and TLR4 expressions in rat renal TECs (NRK-52E) were measured using quantitative reverse transcription polymerase chain reaction. The mRNA expression level of miR-630 was significantly lower in the high-glucose (HG) and HG + mimic negative control (NC) groups than in the normal glucose (NG) group (P < 0.05). In contrast, the mRNA expression level of TLR4 was significantly higher in these groups (P < 0.05). However, miR-630 mRNA expression increased and TLR4 mRNA expression significantly decreased in the HG + miR-630 mimic group than in the HG + mimic NC group (P < 0.05). Furthermore, the levels of tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β), and IL-6 were significantly higher in the HG and HG + mimic NC groups than in NG group (P < 0.05). However, the levels of these cytokines were significantly lower in the HG + miR-630 mimic group than in the HG + mimic NC group (P < 0.05). Notably, changes in protein expression were observed. The HG and HG + mimic NC groups showed a significant decrease in E-cadherin protein expression, whereas TLR4, α-smooth muscle actin (SMA), and collagen IV protein expression increased (P < 0.05). Conversely, the HG + miR-630 mimic group exhibited a significant increase in E-cadherin protein expression and a notable decrease in TLR4, α-SMA, and collagen IV protein expression than in the HG + mimic NC group (P < 0.05). The miR-630 targets TLR4 gene expression. In vivo experiments demonstrated that DKD rats treated with miR-630 agomir exhibited significantly higher miR-630 mRNA expression than DKD rats injected with agomir NC. Additionally, rats treated with miR-630 agomir showed significant reductions in urinary albumin, blood glucose, TLR4, and proinflammatory markers (TNF-α, IL-1β, and IL-6) expression levels (P < 0.05). Moreover, these rats exhibited fewer kidney lesions and reduced infiltration of inflammatory cells. MiR-630 may inhibit the inflammatory reaction of DKD by targeting TLR4, and has a protective effect on DKD.

Observation of the Effects of Different Enzyme Digestion Time and Enzyme Digestion Methods on the Extraction of Primary Renal Tubular Epithelial Cells from Sprague-Dawley Suckling Mice.

Renal tubular epithelial cells are one of the essential functional cells in the kidney. Optimizing the isolation and culture method of primary renal tubular epithelial cells from SD mammary rats provides better experimental materials for renal tubule-related studies, which is essential for studying the pathogenesis of renal diseases, especially diabetic nephropathy and drug screening. SD rat renal tubular epithelial cells were isolated and purified by 2.5-mg/ml collagenase II or 2mg/ml trypsin + 2.5mg/ml collagenase II enzymatic digestion. The isolation and purification were observed at different time points (15min, 30min, 45min, and 60min) to determine the optimal extraction time for the enzymatic digestion method. After comparing the two enzymatic methods, it was determined that the trypsin + collagenase II enzymatic method was more effective. The primary renal tubular epithelial cells extracted by the trypsin + collagenase II digestion method were identified by the marker Cytokeratin 18 of renal tubular epithelial cells at 45min of digestion with high purity. We established a simple, efficient, and reproducible method for isolation and culture of renal tubular epithelial cells in SD mammary gland rats.

Evaluating the Effects of Kidney Preservation at 10 °C with Hemopure and Sodium Thiosulfate in a Rat Model of Syngeneic Orthotopic Kidney Transplantation.

Kidney transplantation is preferred for end-stage renal disease. The current gold standard for kidney preservation is static cold storage (SCS) at 4 °C. However, SCS contributes to renal graft damage through ischemia-reperfusion injury (IRI). We previously reported renal graft protection after SCS with a hydrogen sulfide donor, sodium thiosulfate (STS), at 4 °C. Therefore, this study aims to investigate whether SCS at 10 °C with STS and Hemopure (blood substitute), will provide similar protection. Using in vitro model of IRI, we subjected rat renal proximal tubular epithelial cells to hypoxia-reoxygenation for 24 h at 10 °C with or without STS and measured cell viability. In vivo, we preserved 36 donor kidneys of Lewis rats for 24 h in a preservation solution at 10 °C supplemented with STS, Hemopure, or both followed by transplantation. Tissue damage and recipient graft function parameters, including serum creatinine, blood urea nitrogen, urine osmolality, and glomerular filtration rate (GFR), were evaluated. STS-treated proximal tubular epithelial cells exhibited enhanced viability at 10 °C compared with untreated control cells (p < 0.05). Also, STS and Hemopure improved renal graft function compared with control grafts (p < 0.05) in the early time period after the transplant, but long-term function did not reach significance. Overall, renal graft preservation at 10 °C with STS and Hemopure supplementation has the potential to enhance graft function and reduce kidney damage, suggesting a novel approach to reducing IRI and post-transplant complications.

Qingshen Granules-medicated serum reduces transdifferentiation of NRK-52E cells by miR-23b-5p-mediated activation of the Nrf2 pathway

TTo investigate the targeted regulation of the Nrf2 pathway by miR-23b-5p in transdifferentiation of rat renal tubular epithelial NRK-52E cells induced by transforming growth factor β1(TGF-β1)and the effect of Qingshen Granulesmedicated serum for alleviating transdifferentiation of NRK-52E cells. NRK-52E cells with TGF-β1-induced transdifferentiation were transfected with miR-23b-5p mimic, miR-23b-5p inhibitor or the negative control(NC)siRNA and then treated with of Qingshen Granules-medicated serum.CCK8 assay was used to detectthe changes in viability of NRK-52E cells.The targeting relationship between miR-23b-5p and Nrf2 was verified using a dual luciferase reporter gene assay.The expressions of Nrf2, Keap1 and α-SMA mRNAs and proteins in the treated cells were detected with RT-qPCR and Western blotting, and ROS production in the cells was detected with flow cytometry. Transfection of NRK-52E cells with miR-23b-5p mimic significantly increased the expression of Nrf2 mRNA, while inhibition of miR-23b-5p obviously lowered Nrf2 mRNA in the cells.Rno-miR-23b-5p significantly down-regulated the luciferase activity of Rno-Nrf2-wt but not that of Rno-Nrf2-mu(P<0.05).Treatment with TGF-β1 significantly decreased the expressions of miR-23b-5p and Nrf2 and increased the expressions of Keap1, α-SMA and ROS in NRK-52E cells(P<0.05), and these changes were obviously ameliorated by treatment with 20% Qingshen Granules-medicated serum for 24 h.Transfection of the cells with miR-23b-mimic significantly decreased the expressions of Keap1, α-SMA and ROS(P<0.05), which were further decreased by treatment with the medicated serum(P<0.05). Qingshen Granules-medicated serum reduces transdifferentiation of NRK-52E cells via miR-23b-5pmediated activation of the Nrf2 pathway.

Astragalus polysaccharide promotes autophagy and alleviates diabetic nephropathy by targeting the lncRNA Gm41268/PRLR pathway

Background Astragalus polysaccharide (APS) is a major bioactive component of the Chinese herb astragalus, with well-established protective effects on the kidney. However, the effect of APS on diabetic nephropathy (DN) is unclear. Methods Long non-coding RNA (lncRNA) expression profiles in kidney samples from control, db/db, and APS-treated db/db mice were evaluated using RNA high-throughput sequencing techniques. Additionally, rat renal tubular epithelial (NRK-52E) cells were cultured in high glucose (HG) media. We inhibited the expression of Gm41268 and prolactin receptor (PRLR) by transfecting NRK-52E cells with Gm41268-targeting antisense oligonucleotides and PRLR siRNA. Results We found that APS treatment reduced 24-h urinary protein levels and fasting blood glucose and improved glucose intolerance and pathological renal damage in db/db mice. Furthermore, APS treatment enhanced autophagy and alleviated fibrosis in the db/db mice. We identified a novel lncRNA, Gm41268, which was differentially expressed in the three groups, and the cis-regulatory target gene PRLR. APS treatment induced autophagy by reducing p62 and p-mammalian target of rapamycin (mTOR) protein levels and increasing the LC3 II/I ratio. Furthermore, APS alleviated fibrosis by downregulating fibronectin (FN), transforming growth factor-β (TGF-β), and collagen IV levels. In addition, APS reversed the HG-induced overexpression of Gm41268 and PRLR. Reduction of Gm41268 decreased PRLR expression, restored autophagy, and ameliorated renal fibrosis in vitro. Inhibition of PRLR could enhance the protective effect of APS. Conclusions In summary, we demonstrated that the therapeutic effect of APS on DN is mediated via the Gm41268/PRLR pathway. This information contributes to the exploration of bioactive constituents in Chinese herbs as potential treatments for DN.

Increased Ca2 + transport across the mitochondria-associated membranes by Mfn2 inhibiting endoplasmic reticulum stress in ischemia/reperfusion kidney injury

Renal ischemia/reperfusion (I/R) injury, which leads to acute kidney injury (AKI), is a major cause of morbidity and mortality in a variety of clinical situations. This study aimed to investigate the protective role of Mfn2 during renal I/R injury. Overexpression of Mfn2 in NRK-52E rat renal tubular epithelial cells and rats, then we constructed hypoxia reoxygenation (H/R) cells and I/R rat model. Apoptosis, ROS, ATP, Ca2+ levels in cells and rats, as well as renal tissue and functional injury in rats were detected respectively. Endoplasmic reticulum (ER) stress was further examined in cells and rats. The morphological changes of mitochondria-associated ER membranes (MAMs) were also detected. Mfn2 expression is reduced in H/R-treated NRK-52E cells and renal tissue of I/R rats. At the cellular level, overexpression of Mfn2 promoted cell proliferation, inhibited cell apoptosis, attenuated mitochondrial damage and Ca2+ overload, and ER stress. In addition, Mfn2 also restored the MAMs structure. In vivo experiments found that overexpression of Mfn2 could improve renal function and alleviate tissue injury. Concomitant with elevated Mfn2 expression in the kidney, reduced renal cell apoptosis, restored mitochondrial function, and reduced calcium overload. Finally, ER stress in rat kidney tissue was alleviated after overexpression of Mfn2. These results reveal that Mfn2 contributes to ER stress, mitochondrial function, and cell death in I/R injury, which provides a novel therapeutic target for AKI.

The role of the SGK3/TOPK signaling pathway in the transition from acute kidney injury to chronic kidney disease.

Introduction: Profibrotic phenotype of renal tubular epithelial cells (TECs) featured with epithelial to mesenchymal transition (EMT) and profibrotic factors secretion, and aberrant accumulation of CD206+ M2 macrophages are the key points in the transition from acute kidney injury (AKI) to chronic kidney disease (CKD). Nevertheless, the underlying mechanisms involved remain incompletely understood. Serum and glucocorticoid-inducible kinase (SGK) is a serine/threonine protein kinase, required for intestinal nutrient transport and ion channels modulation. T-LAK-cell-originated protein kinase (TOPK) is a member of the mitogen activated protein kinase family, linked to cell cycle regulation. However, little is known about their roles in AKI-CKD transition. Methods: In this study, three models were constructed in C57BL/6 mice: low dose and multiple intraperitoneal injection of cisplatin, 5/6 nephrectomy and unilateral ureteral obstruction model. Rat renal tubular epithelial cells (NRK-52E) were dealt with cisplatin to induce profibrotic phenotype, while a mouse monocytic cell line (RAW264.7) were cultured with cisplatin or TGF-β1 to induce M1 or M2 macrophage polarization respectively. And co-cultured NRK-52E and RAW264.7 through transwell plate to explore the interaction between them. The expression of SGK3 and TOPK phosphorylation were detected by immunohistochemistry, immunofluorescence and western blot analysis. Results: In vivo, the expression of SGK3 and p-TOPK were gradually inhibited in TECs, but enhanced in CD206+ M2 macrophages. In vitro, SGK3 inhibition aggravated epithelial to mesenchymal transition through reducing the phosphorylation state of TOPK, and controlling TGF-β1 synthesis and secretion in TECs. However, SGK3/TOPK axis activation promoted CD206+ M2 macrophage polarization, which caused kidney fibrosis by mediating macrophage to myofibroblast transition (MMT). When co-cultured, the TGF-β1 from profibrotic TECs evoked CD206+ M2 macrophage polarization and MMT, which could be attenuated by SGK3/TOPK axis inhibition in macrophages. Conversely, SGK3/TOPK signaling pathway activation in TECs could reverse CD206+ M2 macrophages aggravated EMT. Discussion: We revealed for the first time that SGK3 regulated TOPK phosphorylation to mediate TECs profibrotic phenotype, macrophage plasticity and the crosstalk between TECs and macrophages during AKI-CKD transition. Our results demonstrated the inverse effect of SGK3/TOPK signaling pathway in profibrotic TECs and CD206+ M2 macrophages polarization during the AKI-CKD transition.

COX17 restricts renal fibrosis development by maintaining mitochondrial copper homeostasis and restoring complex IV activity.

Renal fibrosis relies on multiple proteins and cofactors in its gradual development. Copper is a cofactor of many enzymes involved in renal microenvironment homeostasis. We previously reported that intracellular copper imbalance occurred during renal fibrosis development and was correlated with fibrosis intensity. In this study, we investigated the molecular mechanisms of how copper affected renal fibrosis development. Unilateral ureteral obstruction (UUO) mice were used for in vivo study; rat renal tubular epithelial cells (NRK-52E) treated with TGF-β1 were adapted as an in vitro fibrotic model. We revealed that the accumulation of copper in mitochondria, rather than cytosol, was responsible for mitochondrial dysfunction, cell apoptosis and renal fibrosis in both in vivo and in vitro fibrotic models. Furthermore, we showed that mitochondrial copper overload directly disrupted the activity of respiratory chain complex IV (cytochrome c oxidase), but not complex I, II and III, which hampered respiratory chain and disrupted mitochondrial functions, eventually leading to fibrosis development. Meanwhile, we showed that COX17, the copper chaperone protein, was significantly upregulated in the mitochondria of fibrotic kidneys and NRK-52E cells. Knockdown of COX17 aggravated mitochondrial copper accumulation, inhibited complex IV activity, augmented mitochondrial dysfunction and led to cell apoptosis and renal fibrosis, whereas overexpression of COX17 could discharge copper from mitochondria and protect mitochondrial function, alleviating renal fibrosis. In conclusion, copper accumulation in mitochondria blocks complex IV activity and induces mitochondrial dysfunction. COX17 plays a pivotal role in maintaining mitochondrial copper homeostasis, restoring complex IV activity, and ameliorating renal fibrosis.

Emodin ameliorates renal injury and fibrosis via regulating the miR-490-3p/HMGA2 axis.

Renal fibrosis is a major pathological feature of chronic kidney disease (CKD). While emodin is reported to elicit anti-fibrotic effects on renal injury, little is known about its effects on microRNA (miRNA)-modulated mechanisms in renal fibrosis. In this study, we established a unilateral ureteral obstruction (UUO) model and a transforming growth factor (TGF)-β1-induced normal rat renal tubular epithelial cell line (NRK-52E) model to investigate the protective effects of emodin on renal fibrosis and its miRNA/target gene mechanisms. Dual-luciferase assay was performed to confirm the direct binding of miRNA and target genes in HEK293 cells. Results showed that oral administration of emodin significantly ameliorated the loss of body weight and the increase in physicochemical parameters, including serum uric acid, creatinine, and urea nitrogen in UUO mice. Inflammatory cytokines, including tumor necrosis factor-α, monocyte chemoattractant protein-1, and interleukin (IL)-1β, but not IL-6, were down-regulated by emodin administration. Emodin decreased the expression levels of TGF-β1 and fibrotic-related proteins, including alpha-smooth muscle actin, Collagen IV, and Fibronectin, and increased the expression of E-cadherin. Furthermore, miR-490-3p was decreased in UUO mice and negatively correlated with increased expression of high migration protein A2 (HMGA2). We further confirmed HMGA2 was the target of miR-490-3p. Transfection of miR-490-3p mimics decreased, while transfection of miR-490-3p inhibitors increased fibrotic-related proteins and HMGA2 expression levels in TGF-β1-induced NRK-52E cells. Furthermore, transfection of miR-490-3p mimics enhanced the anti-fibrotic effects of emodin, while transfection of miR-490-3p inhibitors abolished the protective effects of emodin. Thus, as a novel target of emodin that prevents renal fibrosis in the HMGA2-dependent signaling pathway, miR-490-3p has potential implications in CKD pathology.

Qinling liquid ameliorates renal immune inflammatory damage via activating autophagy through AMPK/Stat3 pathway in uric acid nephropathy.

Excessive deposition of uric acid (UA) is one of the risk factors for kidney damage. Qinling liquid (QL) has a certain therapeutic effect on uric acid nephropathy (UAN), but its regulation mechanism is still unclear. UAN rat models and UA induced rat renal tubular epithelial cells (NRK-52E) were constructed to evaluate the functional roles of QL. We firstly evaluated the kidney function and the degree of kidney damage in rats after QL treatment. Then, effects of QL on autophagy and NLRP3 inflammasome activation were assessed. Moreover, the regulation of QL in AMPK and Stat3 phosphorylation levels and the relationship among autophagy, AMPK/Stat3 pathway and NLRP3 inflammasomes were determined. QL could alleviate the inflammatory damage in UAN rats and promote the activation of autophagy. In addition, QL suppressed UA-induced activation of NLRP3 inflammasomes in rat renal tubular epithelial cells, which was partially reversed by autophagy inhibitor. Further, AMPK/Stat3 axis-mediated autophagy participated in the regulation of UA-induced NLRP3 inflammasome activation in NRK-52E cells. Finally, we confirmed that inhibiting AMPK/Stat3 pathway partly deteriorated the ameliorating effect of QL on renal immune inflammatory injury in UAN rats. Through in vivo and in vitro experiments, we found that QL promotes autophagy by activating the AMPK/Stat3 pathway, thereby improving renal immune inflammatory injury in UAN.

DBP and BaP co-exposure induces kidney injury via promoting pyroptosis of renal tubular epithelial cells in rats

Dibutyl phthalate (DBP) and benzo(a)pyrene (BaP) are widespread environmental and foodborne contaminants that have detrimental effects on human health. Although people are often simultaneously exposed to DBP and BaP via the intake of polluted food and water, the combined effects on the kidney and potential mechanisms remain unclear. Hence, we treated rats with DBP and BaP for 90 days to investigate their effects on kidney histopathology and function. We also investigated the levels of paramount proteins and genes involved in pyroptosis and TLR4/NF-κB p65 signaling in the kidney. Our research showed that combined exposure to DBP and BaP triggered more severe histopathological and renal function abnormalities than in those exposed to DBP or BaP alone. Simultaneously, combined exposure to DBP and BaP enhanced the excretion of IL-1β and IL-18, along with the release of LDH in rat renal tubular epithelial cells (RTECs). Moreover, combined exposure to DBP and BaP increased the expression of pyroptosis marker molecules, including NLRP3, ASC, cleaved-Caspase-1, and GSDMD. Meanwhile, the combination of DBP and BaP activated TLR4/NF-κB signaling in the kidney. Taken together, the combined exposure to DBP and BaP causes more severe kidney injury than that caused by DBP or BaP exposure separately. In addition, pyroptosis of RTECs regulated by TLR4/NF-κB signaling may add to the kidney damage triggered by combined exposure to DBP and BaP.

N-acetylcysteine delayed cadmium-induced chronic kidney injury by activating the sirtuin 1–P53 signaling pathway

With the development of modern industrial civilization, cadmium (Cd), a known nephrotoxic metal, has become a growing public safety issue due to its ability to induce various types of kidney disease. Maladaptive proximal tubule repair is a significant cause of Cd-induced chronic kidney disease (CKD), which is characterized by premature senescence and pro-fibrosis. Previously, we demonstrated that cadmium causes DNA damage and cycle arrest in renal tubular epithelial cells, which may be relevant to premature senescence regulated by sirtuin 1 (SIRT1). In this study, in vivo and in vitro studies were conducted to elucidate the role of SIRT1-mediated premature renal senescence in Cd-induced CKD. As oxidative stress is a significant cause of aging, we evaluated whether N-acetylcysteine (NAC) would inhibit Cd-induced premature aging and dysfunction in rat renal tubular epithelial cells. Cadmium induced premature renal senescence and fibrosis, and NAC inhibited premature renal senescence and fibrosis through the SIRT1–P53 pathway and delayed CKD progression. Overall, the results suggested that the SIRT1–P53 pathway mediates oxidative stress, premature renal senescence, and renal fibrosis during cadmium exposure, which may be a potential therapeutic target for Cd-induced CKD.

Alpha lipoamide inhibits diabetic kidney fibrosis via improving mitochondrial function and regulating RXRα expression and activation.

Previous studies have shown mitochondrial dysfunction in various acute kidney injuries and chronic kidney diseases. Lipoic acid exerts potent effects on oxidant stress and modulation of mitochondrial function in damaged organ. In this study we investigated whether alpha lipoamide (ALM), a derivative of lipoic acid, exerted a renal protective effect in a type 2 diabetes mellitus mouse model. 9-week-old db/db mice were treated with ALM (50 mg·kg-1·d-1, i.g) for 8 weeks. We showed that ALM administration did not affect blood glucose levels in db/db mice, but restored renal function and significantly improved fibrosis of kidneys. We demonstrated that ALM administration significantly ameliorated mitochondrial dysfunction and tubulointerstitial fibrotic lesions, along with increased expression of CDX2 and CFTR and decreased expression of β-catenin and Snail in kidneys of db/db mice. Similar protective effects were observed in rat renal tubular epithelial cell line NRK-52E cultured in high-glucose medium following treatment with ALM (200 μM). The protective mechanisms of ALM in diabetic kidney disease (DKD) were further explored: Autodock Vina software predicted that ALM could activate RXRα protein by forming stable hydrogen bonds. PROMO Database predicted that RXRα could bind the promoter sequences of CDX2 gene. Knockdown of RXRα expression in NRK-52E cells under normal glucose condition suppressed CDX2 expression and promoted phenotypic changes in renal tubular epithelial cells. However, RXRα overexpression increased CDX2 expression which in turn inhibited high glucose-mediated renal tubular epithelial cell injury. Therefore, we reveal the protective effect of ALM on DKD and its possible potential targets: ALM ameliorates mitochondrial dysfunction and regulates the CDX2/CFTR/β-catenin signaling axis through upregulation and activation of RXRα. Schematic figure illustrating that ALM alleviates diabetic kidney disease by improving mitochondrial function and upregulation and activation of RXRα, which in turn upregulated CDX2 to exert an inhibitory effect on β-catenin activation and nuclear translocation. RTEC renal tubular epithelial cell. ROS Reactive oxygen species. RXRα Retinoid X receptor-α. Mfn1 Mitofusin 1. Drp1 dynamic-related protein 1. MDA malondialdehyde. 4-HNE 4-hydroxynonenal. T-SOD Total-superoxide dismutase. CDX2 Caudal-type homeobox transcription factor 2. CFTR Cystic fibrosis transmembrane conductance regulator. EMT epithelial mesenchymal transition. α-SMA Alpha-smooth muscle actin. ECM extracellular matrix. DKD diabetic kidney disease.Schematic figurewas drawn by Figdraw ( www.figdraw.com ).

Preparation, structural characterization, antioxidant activity and protection against cisplatin-induced acute kidney injury by polysaccharides from the lateral root of Aconitum carmichaelii.

Response surface methodology (RSM) and Box- Behnken design (BBD) based on one-way experiments were used to optimize the extraction parameters of the lateral root polysaccharides of Aconitum carmichaelii. The extracted polysaccharides were named as refined fucose polysaccharide. The optimal conditions included a water to raw material ratio of 43, an extraction time of 2h, and an extraction temperature of 90°C. The shape of RFP was shown by infrared spectroscopy (IR) and scanning electron microscopy (SEM) analysis. The monosaccharide composition and molecular weight of RFP was determined by high-performance liquid chromatography (HPLC). Furthermore, RFP exhibited moderate antioxidant activity by analyzing the scavenging rates of 2,2-diphenyl-1-picrylhydrazyl radical, superoxide anion radical, hydroxyl radical, and ABTS + radical. RFP exerted cytoprotective effects against hydrogen peroxide (H2O2)-induced injury in the rat renal tubular epithelial cell line rat renal tubular epithelial cells (NRK-52E) and inhibited apoptosis. In addition, researches found that RFP could alleviate cisplatin-induced acute kidney injury in mice by enhancing the levels of glutathione (GSH) and glutathione peroxidase-4 (GPX-4), decreasing the levels of malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE), reducing lipid peroxidation, and thus inhibiting ferroptosis. In conclusion, this study provides a good strategy for obtaining bioactive polysaccharides from Fuzi.

Melatonin Ameliorates Cisplatin-Induced Renal Tubular Epithelial Cell Damage through PPARα/FAO Regulation.

Previous studies revealed that melatonin ameliorated acute renal injury induced by cisplatin, but the mechanisms remain unclear. Peroxidase proliferative receptor α (PPARα) is considered the major regulator of fatty acid oxidation (FAO), which is an important source of energy for renal tubular epithelial cells. In this study, the aim was to investigate the role of melatonin in cisplatin-induced NRK-52E (rat renal tubular epithelial cell line) cell damage and the underlying mechanisms. We established a cisplatin-stimulated NRK-52E model in vitro. We assessed the levels of apoptotic proteins, including caspase-3, caspase-9, and B-cell lymphoma 2-associated X protein (Bax), as well as PPARα and FAO-related genes (Acadm, Acat1, Acsm2, Acsm3, PGC-1α, Pecr, Bdh2, and Echs1). Furthermore, we detected the effects of miR-21 and PPARα antagonist on the above indicators. We found that melatonin reduced the protein expression levels of caspase-3, caspase-9, and Bax, and increased the expression levels of the PPARα gene and protein and PPARα activity, as well as FAO-related genes, in NRK-52E cells. However, miR-21 mimics and PPARα antagonists partially antagonized the above effects of melatonin. Our data indicated that melatonin could alleviate cisplatin-induced cell damage through the upregulation of PPARα/FAO.

Deciphering the Pharmacological Mechanisms of Qidan Dihuang Decoction in Ameliorating Renal Fibrosis in Diabetic Nephropathy through Experimental Validation In Vitro and In Vivo

Objective QiDan DiHuang decoction (QDD) has been proven to have good efficacy in decreasing albuminuria levels, improving renal function, and inhibiting renal fibrosis in diabetic nephropathy (DN). However, the potential mechanism remains unclear. The purpose of this study was to explore the underlying mechanism of QDD for treating DN in vitro and in vivo. Methods Db/db mice were treated with QDD or saline intragastrically for 12 weeks. Non-diabetic db/m mice were used as controls. Rat renal tubular epithelial cells (NRK-52E) were cultured in high glucose conditions. ATF4 siRNA was transfected into NRK-52E cells. Different indicators were detected via UPLC, RT-PCR, western blotting, cell viability assays and apoptosis, transmission electron microscopy, histology, and immunofluorescence staining. Results Db/db mice experienced severe kidney damage and fibrosis, increased levels of PERK, eIF2α, and ATF4, and suppression of renal autophagy compared with db/m mice. The results showed a significant improvement in glucose intolerance, blood urea nitrogen, urine albumin, serum creatinine, and renal fibrosis in db/db mice with QDD treatment. Meanwhile, the application of QDD resulted in the downregulation of PERK, eIF2α, and ATF4 and the upregulation of autophagy in diabetic kidneys. In vitro, the exposure of NRK-52E cells to high glucose resulted in downregulation of the ratio of LC3-II/LC3-I and upregulation of P62, a reduction in the number of autophagosomes and upregulation of fibronectin (FN), collagen IV and TGF-β1 protein, which was reversed by QDD treatment through inhibiting ATF4 expression. Conclusions Taken together, our results suggest that QDD effectively alleviates diabetic renal injuries and fibrosis by inhibiting the PERK-eIF2α-ATF4 pathway and promoting autophagy in diabetic nephropathy.

A network pharmacology-based study on the mechanism of astragaloside IV alleviating renal fibrosis through the AKT1/GSK-3β pathway

Ethnopharmacological revelvanceAstragaloside IV, a glycoside derived from Astragalus membranaceus, has anti-renal fibrosis effects. However, its mechanism of action has not yet been fully elucidated. Aim of the studyThe purpose of this study was to investigate the anti-fibrotic effect of AS-IV and to clarify its underlying mechanism. Materials and methodsThe network pharmacology method, molecular docking and surface plasmon resonance (SPR) was used to identify potential targets and pathways of AS-IV. A unilateral ischemia-reperfusion injury (UIRI) animal model, as well as TGF-β1-induced rat renal tubular epithelial cells (NRK-52E) and renal fibroblasts (NRK-49F) were used to investigate and validate the anti-fibrotic activity and pharmacological mechanism of AS-IV. Network pharmacology was performed to construct a drug-target-pathway network. The anti-fibrosis effect of AS-IV was determined using hematoxylin-eosin (H&E) and MASSON staining, as well as immunostaining methods. qRT-PCR and western blotting were used to elucidate and validate the mechanism of AS-IV. ResultsNetwork pharmacology revealed that the PI3K/AKT pathway is an important pathway in AS-IV. AS-IV inhibited the expression of α-SMA, collagen I, and fibronectin in NRK-52E, NRK-49F, and UIRI rats, and reduced serum creatinine and blood urea nitrogen levels in UIRI rats. AS-IV inhibited AKT phosphorylation, blocked GSK-3β phosphorylation, and restored GSK-3β activity, which contributed to the degradation of β-catenin, thereby preventing epithelial-mesenchymal transition (EMT). ConclusionAstragaloside IV alleviated renal fibrosis through the AKT1/GSK-3β pathway. In addition, our findings indicate that the network pharmacology method is a powerful tool for exploring the pharmacological mechanisms of drugs.

Isorhapontigenin Modulates SOX9/TOLLIP Expression to Attenuate Cell Apoptosis and Oxidative Stress in Paraquat-Induced Acute Kidney Injury.

Paraquat (PQ) is a widely used herbicide but can be lethal to humans. The kidney is vital for PQ elimination; therefore, explorations for therapeutic approaches for PQ-induced acute kidney injury (AKI) are of great significance. Here, the effects of a natural bioactive polyphenol isorhapontigenin (ISO) on PQ-AKI were investigated. In vitro experiments carried out in PQ-intoxicated rat renal tubular epithelial cells (NRK-52E) showed that ISO treatment inhibited PQ-induced cell apoptosis and oxidative stress, which was evidenced by the decreased proapoptotic proteins [cleaved caspase 3/9 and poly (ADP-ribose) polymerase (PARP)], the reduced oxidative stress indicators [reactive oxygen species (ROS), malondialdehyde (MDA), and lactate dehydrogenase (LDH) leakage], and the increased antioxidants [superoxide dismutase (SOD), nuclear factor E2-related factor 2 (NRF2), and oxygenase-1 (HO-1)]. Furthermore, 50 mg/kg ISO pretreatment before PQ administration significantly attenuated PQ-AKI in rats, as manifested by the improved renal tubule damage, the reduced serum and urine markers of kidney injury, and the inhibited cell apoptosis and oxidative stress in the renal cortex. Furthermore, expression of sex-determining region Y box 9 (SOX9) and Toll-interacting protein (TOLLIP) in NRK-52E cells and the renal cortex was significantly upregulated after ISO treatment. Overexpression of SOX9 increased TOLLIP transcription and attenuated PQ-induced apoptosis and oxidative stress, whereas knockdown of SOX9 impaired the protective effects of ISO on NRK-52E cells against PQ toxicity. In conclusion, the present study demonstrated that ISO modulated SOX9/TOLLIP expression to attenuate cell apoptosis and oxidative stress in PQ-AKI, suggesting the potential of ISO in treating PQ-poisoned patients.