Effect of Qingshen granules on miR-23b and PINKl/Parkin pathway in rat NRK-52E cell transdifferentiation model / 中国药理学通报

Aim To investigate the targeting mechanism of miR-23b on PINKl/Parkin pathway in transdifferentiation of NRK-52E cellsinduced by TGF-β1, and to elucidate the intervention mechanism of Qingshen granules drug-containing serum on NRK-52E cell transdifferentiation. Methods Ultra-high performance liquid chromatography ( UPLC ) fingerprinting method was used to analyze Qingshen granules. The NRK-52E transdifferentiation model induced by TGF-β1 was constructed. The NRK-52E cells were divided into simulated no-load control group, miR-23b-5p simulated group, inhibitor no-load control group, and miR-23b-5p inhibitor group, after transfection with siRNA, and the effect of miR-23b-5p on PINK1 expression was ob-served. The NRK-52E cells were then divided into normal group, TGF-(31 group, Qingshen granule group, miR-23 b-mimic group, miR-23 b-mimic group, and miR-23b-mimic + Qingshen granule group. Western blot was used to detect the expression of Pinkl, Parkin, LC3 n, Beclin-1, P62 and a-SMA proteins, and RT- PCR was used to detect the expression of miR-23 b-5p, Pinkl, Parkin, Beclin-1 and a-SMA mRNA in NRK- 52E cells. Dual-Luciferase Reporter gene experiment was used to detect the targeting relationship between miR-23b-5p and PINKL Results UPLC fingerprinting method found 11 active components in Qingshen granules. After overexpression of miR-23b-5p, the expression of PINkl mRNA significantly increased (P 0. 05 ). The experimental results showed that the expressions of miR- 23b-5p, Pinkl, Parkin, Beclin-1, LC3 II and LC3 II/ I ratio in TGF-β1 group were significantly lower than those in normal group, but the expressions of P62 and a-SMA were significantly higher than those in normal group ( P <0.05). The expressions of miR-23 b-5 p, Pinkl, Parkin, Beclin-1, LC3 II and LC3 11/ I ratio in Qingshen granule group and miR-23 b-mimic group were significantly higher than those in TGF-β1 group, and the expressions of P62 and a-SMA were significantly lower than those in TGF-β1 group (P < 0. 05 ). The performance of miR-23 b-mimic + Qingshen granule group was better than that of miR-23 b-mimic group (P < 0. 05 ). Conclusions Qingshen granules can up- regulate the expression of miR-23b-5p in NRK-52E cellsand inhibit the transdifferentiation process of NRK- 52E cells by enhancing the mitochondrial autophagy activity mediated by PINKl/Parkin pathway.

Extracellular and intracellular productions of lysophosphatidic acids and cyclic phosphatidic acids by lysophospholipase D from exogenously added lysophosphatidylcholines to cultured NRK52E cells.

Lysophosphatidic acid (LPA) is a bioactive lysophospholipid that is a notable biomarker of kidney injury. However, it is not clear how LPA is produced in renal cells. In this study, we explored LPA generation and its enzymatic pathway in a rat kidney-derived cell, NRK52E cells. Culturing of NRK52E cells with acyl lysophosphatidylcholine (acyl LPC), or lyso-platelet activating factor (lysoPAF, alkyl LPC) was resulted in increased extracellular level of choline, co-product with LPA by lysophospholipase D (lysoPLD). Their activities were enhanced by addition of calcium ions to the cell culture medium, but failed to be inhibited by S32826, an autotaxin (ATX)-specific inhibitor. Liquid chromatography-tandem mass spectrometric analysis revealed the small, but significant extracellular production of acyl LPA/cyclic phosphatidic acid (cPA) and alkyl LPA/cPA. The mRNA expression of glycerophosphodiesterase (GDE) 7 with lysoPLD activity was elevated in confluent NRK52E cells cultured over 3 days. GDE7 plasmid-transfection of NRK52E cells augmented both extracellular and intracellular productions of LPAs (acyl and alkyl) as well as extracellular productions of cPAs (acyl and alkyl) from exogenous LPCs (acyl and alkyl). These results suggest that intact NRK52E cells are able to produce choline and LPA/cPA from exogenous LPCs through the enzymatic action of GDE7 that is located on the plasma membranes and intracellular membranes.

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-ß1 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.

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

OBJECTIVE: 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 ß1(TGF-ß1)and the effect of Qingshen Granulesmedicated serum for alleviating transdifferentiation of NRK-52E cells. METHODS: NRK-52E cells with TGF-ß1-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. RESULTS: 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-ß1 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). CONCLUSION: Qingshen Granules-medicated serum reduces transdifferentiation of NRK-52E cells via miR-23b-5pmediated activation of the Nrf2 pathway.

[Numb activates the mTORC1 signaling pathway in proximal tubular epithelial cells by upregulating V1G1 expression].

OBJECTIVE: To investigate the role of Numb in regulating mammalian target of rapamycin (mTOR) complex 1 (mTORC1) signaling pathway. METHODS: Male BALB/C mouse models of acute kidney injury (AKI) were subjected to intravenous injections of Numb-siRNA or NC-siRNA with or without intraperitoneal cisplatin injections. After the treatments, the expressions and distribution of Numb and megalin in the renal tissues of the mice were detected with immunohistochemistry, and the renal expressions of Numb, S6, p-S6, S6K1, p-S6K1, 4EBP1 and p-4EBP1 were examined with Western blotting. The proximal renal tubular epithelial cells were isolated from the mice transfected with Numb-siRNA for in vitro culture. In NRK-52E cells, the effects of amino acid stimulation, Numb knockdown, and V1G1 overexpression, alone or in combination, on expressions of Numb, S6 and p-S6 were detected with Western blotting; the expressions of AMPK and p-AMPK were also detected in transfected NRK-52E cells, mouse kidneys and cultured mouse renal tubular epithelial cells. RESULTS: In BALB/C mice, injection of Numb-siRNA caused significant reductions of Numb and p-S6 expressions without affecting megalin expression in the renal proximal tubules (P < 0.05). Cisplatin treatment obviously upregulated p-S6K1 and p-4EBP1 expressions in the kidneys of the mice (P < 0.05), and this effect was significantly inhibited by treatment with Numb-siRNA (P < 0.05). In NRK-52E cells, amino acid stimulation significantly upregulated the expression of p-S6 (P < 0.05), which was strongly suppressed by transfection with Numb-siRNA (P < 0.05). Numb knockdown inhibited AMPK activation in NRK-52E cells, mouse kidneys and primary proximal tubular epithelial cells (P < 0.05). Numb knockdown significantly downregulated V1G1 expression in NRK-52E cells (P < 0.05), and V1G1 overexpression obviously reversed the inhibitory effect of Numb-siRNA on S6 phosphorylation (P < 0.05). CONCLUSION: Numb promotes the activation of mTORC1 signaling in proximal tubular epithelial cells by upregulating V1G1 expression.

Curcumin alleviates aristolochic acid nephropathy based on SIRT1/Nrf2/HO-1 signaling pathway.

Aristolochic acid I (AA-I), presenting in a variety of natural medicinal plants, which could cause tubular epithelial cell injury. Curcumin (CUR), a polyphenolic substance isolated from turmeric, is a natural antioxidant. The aim of this experiment was to investigate whether CUR attenuated AA-I-induced renal injury in rats through the SIRT1/Nrf2/HO-1 signaling pathway. SD rats were treated with AA-I (10 mg/kg) or/and CUR (200 mg/kg) for 28 days to assess the protective effect of CUR on AA-I-induced renal injury in vivo. NRK-52E cells were treated with AA-I (40 µ M) or/and CUR (20 µ M) for 24 h in vitro. The intervention pathway of CUR against oxidative stress injury induced by AA-I was assessed by observing pathological changes, oxidative stress status, apoptosis and the expression of SIRT1/Nrf2/HO-1 signaling pathway-related factors. The results showed that AA-I exposure increased the contents of BUN, Cr, KIM-1, NGAL, ALT and AST in serum. It increased the content of MDA, decreased the activities of SOD, GST, GSH and the content of ATP in renal tissue. Pathological changes such as inflammatory cell infiltration and mitochondrial injury occurred in renal tissue. AA-I exposure resulted in a substantial rise in the levels of BAX, Ccaspase-9, Cleaved Caspase-9, Caspase-3, Cleaved Caspase-3 and a significant decrease in mRNA and protein expression levels of Bcl-2, SIRT1, Nrf2, NQO1, HO-1 and Keap1. However, these changes were reversed by CUR intervention. In summary, AA-I exposure caused mitochondrial dysfunction and triggered apoptosis through the oxidative stress pathway. However, CUR could reduce AA-I-induced renal injury by activating the SIRT1/Nrf2/HO-1 signaling pathway.

The overexpressed transcription factor RGPR-p117 suppresses the proliferation of normal rat kidney proximal tubular epithelial NRK-52E cells: Involvement of diverse signaling pathways.

AIMS: RGPR-p117 was originally discovered as a novel transcription factor, which specifically binds to a nuclear factor I (NFI) consensus motif TTGGC(N)6CC in the promoter region of the regucalcin gene. RGPR-p117 is also called as Lztr2 and SEC16B. The role of RGPR-p117 in cell regulation is poorly understood. This study was undertaken to determine whether the overexpression of RGPR-p117 impacts the proliferation of normal rat kidney proximal tubular epithelial NRK-52E cells in vitro. MAIN METHODS: The NRK-52E wild-type cells and RGPR-p117-overexpressing NRK-52E cells were cultured in DMEM containing fetal bovine serum. KEY FINDINGS: The overexpression of RGPR-p117 repressed colony formation and proliferation of NRK-52E cells. Interestingly, RGPR-p117 overexpression blocked cell proliferation promoted by culturing with Bay K 8644, a calcium-entry agonist, and phorbol 12-myristate 13-acetate, an activator of protein kinase C. The depressive effects of RGPR-p117 overexpression on cell proliferation were not occurred by culturing with various inhibitors of cell cycle and intracellular signaling processes. RGPR-p117 overexpression increased the translocation of RGPR-p117 into the nucleus of NRK-52E cells. Mechanistically, RGPR-p117 overexpression diminished the levels of Ras, PI3 kinase, Akt, mitogen-activated protein kinase, and mTOR, while it raised the levels of p53, Rb, p21, and regucalcin. Furthermore, RGPR-p117 overexpression protected cell death caused by apoptosis-inducing factors, suggesting that the suppressive effects of RGPR-p117 on cell growth are independent of cell death. SIGNIFICANCE: The present study demonstrates that the overexpressed transcription factor RGPR-p117 suppresses cell proliferation via targeting diverse signaling processes, suggesting a role of RGPR-p117 in cell regulation.

Metadherin silencing results in the inhibition of pyroptosis in lipopolysaccharide/adenosine triphosphate - stimulated renal tubular epithelial cells.

Pyroptosis is induced following inflammation via activation of the NLRP3 inflammasome. Lipopolysaccharide (LPS)-induced acute inflammation causes pyroptosis in renal tubular epithelial cells, which aggravates kidney damage and is involved in physiopathological processes in multiple renal diseases. Metadherin (Mtdh) induces inflammation by NLRP3 inflammasome activation. Specifically, it induces inflammatory injury in the kidney by activating the nuclear factor kappa B (NF-κB) signaling pathway, which is involved in NLRP3 inflammasome activation. However, the role of Mtdh in pyroptosis in renal tubular epithelial cells is unclear. Therefore, we investigated whether Mtdh participates in pyroptosis in LPS/adenosine triphosphate (ATP)-treated NRK-52E cells by activating the NLRP3 inflammasome and NF-κB signaling pathway. We induced pyroptosis in NRK-52E cells with LPS/ATP, after which Mtdh was silenced via transfection with small interfering RNA. LPS/ATP upregulated Mtdh expression and induced pyroptosis and NLRP3 inflammasome activation in NRK-52E cells. However, downregulation of Mtdh expression resulted in the alleviation of pyroptosis in LPS/ATP-treated NRK-52E cells. Additionally, activation of the NLRP3 inflammasome and NF-κB signaling pathway was inhibited. This demonstrates that downregulation of Mtdh expression results in the inhibition of pyroptosis in LPS/ATP-treated NRK-52E cells through the suppression of NLRP3 inflammasome activation, which occurs via inhibition of the NF-κB signaling pathway.

Caspase-11 promotes NLRP3 inflammasome activation via the cleavage of pannexin1 in acute kidney disease.

Ischemia/reperfusion (I/R) injury is a major cause of acute kidney injury (AKI) in clinic. The activation of NLRP3 inflammasome is associated with inflammation and renal injury in I/R-induced AKI. In the current study we explored the molecular and cellular mechanisms for NLRP3 inflammasome activation following renal I/R. Mice were subjected to I/R renal injury by clamping bilateral renal pedicles. We showed that I/R injury markedly increased caspase-11 expression and the cleavage of pannexin 1 (panx1) in the kidneys accompanied by NLRP3 inflammasome activation evidenced by the activation of caspase-1 and interlukin-1ß (IL-1ß) maturation. In Casp-11-/- mice, I/R-induced panx1 cleavage, NLRP3 inflammasome activation as well as renal functional deterioration and tubular morphological changes were significantly attenuated. In cultured primary tubular cells (PTCs) and NRK-52E cells, hypoxia/reoxygenation (H/R) markedly increased caspase-11 expression, NLRP3 inflammasome activation, IL-1ß maturation and panx1 cleavage. Knockdown of caspase-11 attenuated all those changes; similar effects were observed in PTCs isolated from Casp-11-/- mice. In NRK-52E cells, overexpression of caspase-11 promoted panx1 cleavage; pretreatment with panx1 inhibitor carbenoxolone or knockdown of panx1 significantly attenuated H/R-induced intracellular ATP reduction, extracellular ATP elevation and NLRP3 inflammasome activation without apparent influence on H/R-induced caspase-11 increase; pretreatment with P2X7 receptor inhibitor AZD9056 also attenuated NLRP3 inflammasome activation. The above results demonstrate that the cleavage of panx1 by upregulated caspase-11 is involved in facilitating ATP release and then NLRP3 inflammasome activation in I/R-induced AKI. This study provides new insight into the molecular mechanism of NLRP3 inflammasome activation in AKI.

Renoprotective activity of a new amide and a new hydroxycinnamic acid derivative from the fresh roots of Rehmannia glutinosa.

A new amide, named rehmagluamide (1), and a new hydroxycinnamic acid derivative, named nepetoidin F (2), together with six known compounds, 2'-O-methyluridine (3), puroglutamic acid (4), biliverdic acid (5), peterolactam (6), nicotinic acid (7), nicotinamide (8), were isolated from the fresh roots of Rehmannia glutinosa. All the structures of compounds were identified by the interpretation of their spectroscopic data and comparison with those reported in the literatures. The protective effects of compounds 1-7 on normal rat kidney tubule epithelioid (NRK-52e) cells injury induced by LPS were investigated. The results indicated that compounds 1, 2, and 7 exhibited protective effects against LPS-induced NRK 52e cells injury.

Burdock Fructooligosaccharide Attenuates High Glucose-Induced Apoptosis and Oxidative Stress Injury in Renal Tubular Epithelial Cells.

Hyperglycemia-induced apoptosis and oxidative stress injury are thought to play important roles in the pathogenesis of diabetic nephropathy (DN). Attenuating high glucose (HG)-induced renal tubular epithelial cell injury has become a potential approach to ameliorate DN. In recent years, burdock fructooligosaccharide (BFO), a water-soluble inulin-type fructooligosaccharide extracted from burdock root, has been shown to have a wide range of pharmacological activities, including antiviral, anti-inflammatory, and hypolipidemic activities. However, the role and mechanism of BFO in rat renal tubular epithelial cells (NRK-52E cells) have rarely been investigated. The present study investigated the protective effect of BFO on HG-induced damage in NRK-52E cells. BFO could protect NRK-52E cells against the reduced cell viability and significantly increased apoptosis rate induced by HG. These anti-oxidative stress effects of BFO were related to the significant inhibition of the production of reactive oxygen species, stabilization of mitochondrial membrane potential, and increased antioxidant (superoxide dismutase and catalase) activities. Furthermore, BFO increased the expression of Nrf2, HO-1, and Bcl-2 and decreased the expression of Bax. In conclusion, these findings suggest that BFO protects NRK-52E cells against HG-induced damage by inhibiting apoptosis and oxidative stress through the Nrf2/HO-1 signaling pathway.

Crocetin attenuating Urinary tract Infection and adherence of uropathogenic E. coli in NRK-52E cells via an inflammatory pathway.

BACKGROUND AND OBJECTIVE: Urinary tract infections (UTI) are commonly treated with broad-spectrum antibiotics, but treatment has limitation due to causes of nephrotoxicity in uroepithelial cells. Recently, the researcher focuses their research on alternative therapy for the treatment of UTI. This study evaluated the anti-infectious effect of crocetin against adherence of pathogenic [2-14 C]-acetate labeled Escherichia coli (MTCC-729) to rat proximal renal tubular cells (NRK-52E cells) and explores the possible mechanism of action. MATERIALS AND METHODS: In vitro cytotoxicity and radio acetate labeled tests were performed on NRK-52E cells. The rats were divided into five different groups as follows: normal control (NC), disease control (DC), and various doses of crocetin (1.25, 2.5, and 5 mg/kg) treated group rats. White blood cells in blood, urine, and bacterial colony counts were estimated at regular intervals. Pro-inflammatory cytokines, such as interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), interleukin-10 (IL-10), and interleukin-8 (IL-8), were also estimated. In the current study, we estimated the mRNA expression of toll-like receptor-4 (TLR-4) and toll-like receptor-2 (TLR-2) in the renal and bladder tissues. RESULTS: Crocetin significantly (p < .05) inhibited the adherence of E. Coli in NRK-52E cells. Crocetin suppresses the lipid peroxidation (LPO) 42% in cells treated with H2 O2 cells without crocetin. The white blood cells (WBC) count in blood and urine were augmented and crocetin treatment significantly (p < .05) reduced the WBC in urine and blood. The pro-inflammatory cytokines, such as IL-6, MCP-1, IL-10, and IL-8, significantly (p < .05) increased in the DC group and crocetin significantly (p < .05) reduced the pro-inflammatory cytokines. Dose-dependent treatment of crocetin significantly reduced the mRNA expression of TLR2 and TLR4 in the renal and bladder tissues. CONCLUSION: Crocetin considerably reduced the bacterial adherence to NRK-52E cells, attenuated the H2 O2 induced toxicity in NRK-52E cells and also improved the renal tubular function, and reduced the inflammatory response via altering the inflammatory and antioxidant markers. PRACTICAL APPLICATION: As we all know that urinary tract infection is the most common disease worldwide. In this study, we scrutinized the protective effect of crocetin against urinary tract infection. Crocetin treatment considerably reduced the zone of inhibition and improved radioactivity. Crocetin significantly reduced the levels of cytokines and inflammatory mediators. Crocetin can be used as a protective drug in the treatment of urinary tract infections.

Phenolic Compounds from Mori Cortex Ameliorate Sodium Oleate-Induced Epithelial-Mesenchymal Transition and Fibrosis in NRK-52e Cells through CD36.

Lipid deposition in the kidney can cause serious damage to the kidney, and there is an obvious epithelial-mesenchymal transition (EMT) and fibrosis in the late stage. To investigate the interventional effects and mechanisms of phenolic compounds from Mori Cortex on the EMT and fibrosis induced by sodium oleate-induced lipid deposition in renal tubular epithelial cells (NRK-52e cells), and the role played by CD36 in the adjustment process, NRK-52e cells induced by 200 µmol/L sodium oleate were given 10 µmoL/L moracin-P-2″-O-ß-d-glucopyranoside (Y-1), moracin-P-3'-O-ß-d-glucopyranoside (Y-2), moracin-P-3'-O-α-l-arabinopyranoside (Y-3), and moracin-P-3'-O-[ß-glucopyranoside-(1→2)arabinopyranoside] (Y-4), and Oil Red O staining was used to detect lipid deposition. A Western blot was used to detect lipid deposition-related protein CD36, inflammation-related protein (p-NF-κB-P65, NF-κB-P65, IL-1ß), oxidative stress-related protein (NOX1, Nrf2, Keap1), EMT-related proteins (CD31, α-SMA), and fibrosis-related proteins (TGF-ß, ZEB1, Snail1). A qRT-PCR test detected inflammation, EMT, and fibrosis-related gene mRNA levels. The TNF-α levels were detected by ELISA, and the colorimetric method was used to detects SOD and MDA levels. The ROS was measured by flow cytometry. A high-content imaging analysis system was applied to observe EMT and fibrosis-related proteins. At the same time, the experiment silenced CD36 and compared the difference between before and after drug treatment, then used molecular docking technology to predict the potential binding site of the active compounds with CD36. The research results show that sodium oleate can induce lipid deposition, inflammation, oxidative stress, and fibrosis in NRK-52e cells. Y-1 and Y-2 could significantly ameliorate the damage caused by sodium oleate, and Y-2 had a better ameliorating effect, while there was no significant change in Y-3 or Y-4. The amelioration effect of Y-1 and Y-2 disappeared after silencing CD36. Molecular docking technology showed that the Y-1 and Y-2 had hydrogen bonds to CD36 and that, compared with Y-1, Y-2 requires less binding energy. In summary, moracin-P-2″-O-ß-d-glucopyranoside and moracin-P-3'-O-ß-d-glucopyranoside from Mori Cortex ameliorated lipid deposition, EMT, and fibrosis induced by sodium oleate in NRK-52e cells through CD36.

Use of in vitro metabolomics in NRK cells to help predicting nephrotoxicity and differentiating the MoA of nephrotoxicants.

We describe a strategy using an in vitro metabolomics assay with tubular rat NRK-52E cells to investigate the Modes of Action (MoAs) of nephrotoxic compounds. Chemicals were selected according to their MoAs based on literature information: acetaminophen, 4-aminophenol and S-(trichlorovinyl-)L-cysteine (TCVC), (covalent protein binding); gentamycin, vancomycin, polymycin B and CdCl2 (lysosomal overload) and tenofovir and cidofovir (mitochondrial DNA-interaction). After treatment and harvesting of the cells, intracellular endogenous metabolites were quantified relative to vehicle control. Metabolite patterns were evaluated in a purely data-driven pattern generation process excluding published information. This strategy confirmed the assignment of the chemicals to the respective MoA except for TCVC and CdCl2. Finally, TCVC was defined as unidentified and CdCl2 was reclassified to the MoA "covalent protein binding". Hierarchical cluster analysis of 58 distinct metabolites from the patterns enabled a clear visual separation of chemicals in each MoA. The assay reproducibility was very good and metabolic responses were consistent. These results support the use of metabolome analysis in NRK-52E cells as a suitable tool for understanding and investigating the MoA of nephrotoxicants. This assay could enable the early identification of nephrotoxic compounds and finally reduce animal testing.

OM-85 in the prevention of respiratory infections: State-of-the-art and future perspectives in clinical practice.

Respiratory infections (RI) significantly burden patients, their families, and society. Respiratory infection recurrence (RRI) usually depends on a defect of the immune response, which can be more or less transient and/or selective. In particular, children, older people, heavy smokers, and patients with chronic diseases, characterized by an inadequate immune response, may be at risk of developing RRI. In this context, OM- 85 could represent a valuable option in the management of RRI. OM-85 is a bacterial lysate containing the extracts of some common pathogens, including Branhamella catarrhalis, Klebsiella pneumoniae, Klebsiella ozaenae, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus viridans, and Staphylococcus aureus. Methodologically rigorous studies have documented the mechanism of action, efficacy, and safety of OM-85. OM-85 enhances the natural and acquired immune response through multifaceted mechanisms. Substantial evidence has shown that OM-85 can prevent respiratory infections, reduce the number of COPD exacerbations, and shorten the disease duration at home or in hospital. OM-85 can enhance the effectiveness of the 'flu vaccination without affecting the vaccine tolerability. The preventive use of OM-85 can reduce the use of antibiotics, contributing to contrast antibiotic resistance and saving the high cost of chronic respiratory diseases. Further studies should define the ideal candidate to OM-85 treatment.

microRNA-29b prevents renal fibrosis by attenuating renal tubular epithelial cell-mesenchymal transition through targeting the PI3K/AKT pathway.

PURPOSE: This study aimed to investigate the effects of miR-29b on renal interstitial fibrosis in the obstructed kidney of mouse with unilateral ureteral obstruction (UUO) via inhibiting phosphatidylinositol 3-kinase/protein kinaseB (PI3K/AKT) signaling pathway. METHODS: Adult male CD-1 mice were intraperitoneally injected with vehicle or PI3K inhibitor LY294002 (3 mg/kg, 30 mg/kg) daily for 1 or 2 weeks after performing UUO or sham operation. The mice were sacrificed on days 7 and 14 after surgery. The rat proximal tubular epithelial cell (TEC) line NRK-52E was cultured in DMEM and treated with various concentrations angiotensin II (AngII). Obstructed and sham mouse kidneys were analyzed via HE, Masson and immunohistochemistry to assess the degree of renal fibrosis. Real-time quantitative polymerase chain reaction assays (RT-PCR) were performed to investigate changes in the levels of expression of miR-29b and Western blot was used to analyze the activation of PI3K/AKT signaling and expression of E-cadherin, α-smooth muscle actin (α-SMA). RESULTS: Histologic analyses of obstructed kidney revealed that LY294002 attenuated the degree of renal fibrosis. In this study, loss of miR-29b accompanied with increased epithelial-mesenchymal transition (EMT) was observed in renal tubules of mice after UUO and cultured NRK-52E cells exposed to AngII. LY294002 also prominently decreased phosphorylation of AKT in vivo and vitro. By RT-PCR and Western blot analysis, LY294002 blocked the PI3K/AKT-induced loss of E-cadherin expression and de novo increase of the expression of α-SMA in a time- and dose-dependent manner. The overexpression of miR-29b markedly reversed the phenotype induced by AngII in NRK-52E cells and the downregulation miR-29b expression with an miR-29b inhibitor resulted in enhanced EMT. In addition, the PI3K/AKT signaling pathway was found to be suppressed in the presence of overexpression of miR-29b by direct hybridization with 3'-untranslated region (3'-UTR) of PIK3R2. CONCLUSION: Our findings suggested that miR-29b significantly prevented tubulointerstitial injury in mouse model of UUO by attenuating renal tubular epithelial cell-mesenchymal transition via repressing PI3K/AKT signaling pathway.

Selenium Exerts Protective Effects Against Fluoride-Induced Apoptosis and Oxidative Stress and Altered the Expression of Bcl-2/Caspase Family.

Fluoride is widely distributed in nature, and at high concentrations, it targets the kidney and especially proximal tubule epithelial cells. Selenium is a typical trace element beneficial to humans, and the role of selenium in the prevention and treatment of fluoride-induced organ damage is an important research topic. The purpose of this study was to investigate the possible protective effects of selenium against fluoride-induced oxidative stress and apoptosis in rat renal tubular epithelial cells. We showed that the activity of antioxidant enzymes (superoxide dismutase and glutathione peroxidase) and total antioxidant capacity were significantly reduced in NaF-treated normal rat kidney cells (NRK-52E), whereas the levels of nitrogen monoxide (NO) and malondialdehyde (MDA) were significantly increased. Moreover, the number of apoptotic cells, mRNA expression of Bax, Bad, caspase-3, caspase-8, and caspase-9, and protein expression of Bax were elevated, while mitochondrial membrane potential and the protein expression of Bcl-2 were reduced. Compared with the NaF group, pretreatment with selenium enhanced the activity of antioxidant enzymes, mitochondrial membrane potential, and protein expression of Bcl-2, while the levels of NO and MDA, number of apoptotic cells, mRNA expression of Bax, Bad, caspase-3, caspase-8, and caspase-9, and protein expression of Bax were decreased. In conclusion, selenium exerted remarkable protective effect against NaF-induced oxidative stress and apoptosis and altered the expression of Bcl-2/caspase family.

Effect of cell cycle synchronization on cadmium-induced apoptosis and necrosis in NRK-52E cells.

Heavy metal pollution is a problem that cannot be ignored. Due to the prevalence of cadmium in the environment and its harmful effects on humans, cadmium pollution has become a research hotspot recently. The mechanism of cadmium-induced toxicity has also drawn much attention and most studies have been conducted using whole cells, but the toxicological mechanism of cadmium remains unclear. In this study, we aimed to obtain NRK-52E cells at different growth stages by various methods and analyze the differences in cadmium toxicity. The results show that the cadmium sensitivity of cells in each phase was different and the late apoptotic rate was increased significantly after 5 µM Cd treatment. In addition, cadmium easily induces apoptosis of G0- and S-phase cells, as well as necrosis of S- and M-phase cells, but has no significant effect on G1-phase cells. Overall, we first explored the differences in the effects of cadmium on NRK-52E cells at various growth phases. Besides, the findings of this study might provide a theoretical basis for further exploration of the toxicological mechanism of cadmium.Abbreviations Cd: cadmium; CDK: cyclin-dependent kinases; DAPI 2-(4-amidinophenyl)-1H-indole-6-carboxamidine; TBST: Tris-buffered saline with Tween-20; PI: propidium iodide; DMEM: Dulbecco's Modified Eagle Medium; BCA: bicinchoninic acid.

Global DNA Hypomethylation and Rassf1a and c-myc Promoter Hypermethylation in Rat Kidney Cells after Bisphenol A Exposure.

OBJECTIVES: Bisphenol A (BPA) is a synthetic monomer used in the production of polycarbonate and an environmental contaminant with endocrine disrupting properties. BPA release from plastic carriers is thought to cause high amounts of exposure, which result in high risk to human and environment health. MATERIALS AND METHODS: The study examined the possible changes in global DNA methylation, CpG promoter DNA methylation, and gene expressions of Rassf1a and c-myc after BPA exposure in rat kidney epithelial cells (NRK-52E). RESULTS: The IC50 values of BPA in NRK-52E cells were 133.42 and 101.74 µM in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and neutral red uptake tests, respectively. The cells were treated with BPA at 1 nM, 10 nM, 100 nM, 1 µM, and 10 µM concentrations for 24 h and at 100 nM concentration for 24, 48, 72, 96 h, and 6 days. Decreased global 5-methylcytosine levels were observed after 48, 72, 96 h, and 6 days at the concentration of 100 nM BPA. Changes in CpG promoter DNA methylation were detected in the genes of Rassf1a and c-myc in BPA-treated NRK-52E cells. Expression levels of Rassf1a and c-myc changed in response to BPA at the high concentrations after 24 h treatment, whereas 100 nM exposure to BPA altered gene expression after 48, 72, and 96 h. CONCLUSION: These results indicate that changes in global and gene-specific DNA methylation may play an important role in the mechanism of BPA toxicity in kidney cells.