Rat Renal Tubular Epithelial NRK-52E Research Articles

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.

High sodium chloride affects BMP-7 and 1α-hydroxylase levels through NCC and CLC-5 in NRK-52E cells

A diet high in sodium chloride (NaCl) can affect renal function damage and increase urinary calcium excretion, leading to bone loss. in renal tubules, Na-Cl co-transporter (NCC) and chloride channel 5 (CLC-5) are involved in regulating urinary calcium excretion. In addition, some cytokines, such as Bone morphogenetic protein 7 (BMP-7) and 1α-hydroxylase, are synthesized by renal tubules, which target on bone and play important roles on bone metabolism. However, the specific mechanisms between NaCl and these ion channels or cytokines still need investigations from many aspects. This study, in culture normal rat renal tubular epithelial NRK-52E cells, showed that high concentrations of NaCl significantly inhibited the cell viability and increased the cell apoptosis. High concentration of NaCl reduce bone mineral density (BMD), as demonstrated by the significantly increased mRNA and protein levels of NCC and osteopontin (OPN), but decreased the levels of CLC-5, BMP-7, and 1α-hydroxylase. In addition, we found that ovariectomized (OVX) rats on a high-salt diet for 12 weeks had altered levels of these indices in the renal cortices. Moreover, the BMD in fourth and fifth lumbar vertebra (LV4 and 5) and femurs were significantly decreased and bone microstructure was destroyed of these rats. We also demonstrated that high concentration of NaCl enhanced the inhibition of these cytokines which is beneficial to increase BMD, induced by modulating ion channels NCC and CLC-5. In conclusion, our results indicate that high concentration of NaCl reduce BMD by regulating ion channels NCC and CLC-5.

Role of TGF-β1/ILK/FSP1 signaling pathway in cyclosporin A-induced epithelialmesenchymal transition in cultured renal tubular epithelial cells

To explore the role of transforming growth factor-β1/integrin-linked kinase/fibroblast-specific protein 1 (TGF- β1/ILK/FSP1) signaling pathway in cyclosporine A (CsA)-induced renal tubular epithelial cell transdifferentiation. Rat renal tubular epithelial NRK-52E cells were induced with 1 mg/L CsA, treated with TGF-β1 inhibitor (SB431542, 10 μmol/L), or transfected with the ILK-RNAi lentiviral expression vector (ILKshRNA) or a negative control vector before CsA induction. The expressions of TGF-β1, ILK and FSP-1 mRNAs and proteins in the cells were detected using real-time PCR and Western blotting. The positive cells for α-SMA expression were detected by immunohistochemistry. Compared with the blank control cells, the cells treated with CsA showed significantly increased levels of TGF-β1, ILK and FSP-1 mRNAs and proteins (P &lt; 0.05). The expressions of TGF-β1, ILK and FSP-1 were significantly lower in TGF-β1 inhibitor group than in CsA group (P &lt; 0.05). The levels of ILK and FSP-1 were significantly decreased after shRNA-mediated ILK silencing (P &lt; 0.05). The number of positive cells for α-SMA was significantly lower in cells treated with SB431542 and in cells with ILK silencing than in the cells treated with CsA alone (P &lt; 0.05). The activation of TGF-β1/ILK/FSP-1 signaling pathway is an important mechanism for CsA-induced transdifferentiation in rat renal tubular epithelial cells. ILK participates in CsA-induced epithelialmesenchymal transition of renal tubular epithelial cells.

1α,25(OH)2 D3 alleviates high glucose-induced lipid accumulation in rat renal tubular epithelial cells by inhibiting SREBPs.

Lipid accumulation is a vital event in the progression of diabetic nephropathy. 1,25-Dihydroxyvitamin D3 (1α,25(OH)2 D3 ) is considered to have a protective effect on diabetic nephropathy. However, it remains unclear whether 1α,25(OH)2 D3 can inhibit lipid accumulation, and the potential mechanisms responsible for lipid metabolism are incompletely understood. In this study, we evaluated the effects of 1α,25(OH)2 D3 on lipid metabolism in high glucose-exposed rat renal tubular epithelial NRK-52E cells. Results indicated that high glucose-enhanced lipid accumulation in NRK-52E cells and 1α,25(OH)2 D3 can remarkably decrease high glucose-induced lipid accumulation. Western blot showed that 1α,25(OH)2 D3 alleviated high glucose-induced upregulation of sterol regulatory element-binding protein-1c (SREBP-1c) and SREBP2, along with their established target genes fatty acid synthase (FASN) and hydroxymethylglutaryl CoA reductases (HMGCR). Overall, these findings suggest that 1α,25(OH)2 D3 downregulated the expressions of SREBPs to inhibit high glucose-induced lipid accumulation, which provides new sights into the protective effects of 1α,25(OH)2 D3 on diabetic nephropathy.

Transcriptome Identified lncRNAs Associated with Renal Fibrosis in UUO Rat Model.

Renal fibrosis represents a final common outcome of many renal diseases and has attracted a great deal of attention. To better understand whether lncRNAs could be a player in this process or be a biomarker for renal fibrosis diagnosis, we compared transcriptome sequencing data on renal tissues and urine respectively between UUO (unilateral ureteral obstruction) and shamed (Sham) rat model. Numerous genes including lncRNAs with significant changes in their expression were identified. 24 lncRNAs were up-regulated and 79 lncRNAs were down-regulated in the renal tissues of the UUO rats. 625 lncRNAs were up-regulated and 177 lncRNAs were down-regulated in urines of the UUO rats. Among the lncRNAs upregulated in renal tissue of UUO rats, 19 lncRNAs were predicted containing several conserved Smad3 binding motifs in the promoter. Among them, lncRNAs with putative promoter containing more than 4 conserved Smad3 binding motifs were demonstrated to be induced by TGF-β significantly in normal rat renal tubular epithelial NRK-52E cells. We further confirmed that lncRNA TCONS_00088786 and TCONS_01496394 were regulated by TGF-β stimulation and also can influence the expression of some fibrosis-related genes through a feedback loop. Based on transcriptome sequencing data, bioinformatics analysis and qRT-PCR detection, we also demonstrated lncRNA in urine are detectable and might be a novel biomarker of renal fibrosis. These results provide new information for the involvement of lncRNAs in renal fibrosis, indicating that they may serve as candidate biomarkers or therapeutic targets in the future.

Expression and cellular distribution of TLR4, MyD88, and NF-κB in diabetic renal tubulointerstitial fibrosis, in vitro and in vivo

AimInflammation and extracellular matrix hyperplasia are crucial in the pathogenesis of tubulointerstitial fibrosis (TIF) involved in diabetic nephropathy (DN). Macrophage accumulation plays a major role, but whether immune factors contribute to DN pathogenesis is not well understood. This study aimed to investigate TLR4-MyD88-NF-κB-dependent pathway's involvement in TIF pathogenesis. MethodsSTZ-induced diabetic rats and rat renal tubular epithelial NRK-52E cells cultured under high glucose conditions were used as in vivo and in vitro models, respectively. Real-time RT-PCR, western blot, immunohistochemistry and immunofluorescence were performed to examine the mRNA and protein levels of TLR4, MyD88, NF-κB, MCP-1, and α-SMA. ResultsCompared with 5.5mmol/L glucose, treatment of NRK-52E cells with 25 and 50mmol/L d-glucose resulted in significantly increased TLR4 and MyD88 mRNA and protein levels (P<0.05). TLR4 and MyD88 were detected in the cytoplasm of most NRK-52E cells cultured under high glucose. Pronounced damage in the renal tubulointerstitium was observed in diabetic rats (scores: 3.82±0.65 vs. 0.38±0.08, P<0.01). Compared with the normal controls, a sharp upregulation of TLR4, MyD88, NF-κB p65, MCP-1, and α-SMA mRNA and protein levels was observed in diabetic rat kidneys (P<0.05). In diabetic animals, TLR4 and MyD88 were strongly expressed in the cytoplasm, while NF-κB p65 was widely expressed in cytoplasm and nuclei of renal tubular epithelial cells. ConclusionThe inflammatory reaction and epithelial-mesenchymal transformation observed in renal tubulointerstitium may be the result of overactivation of the TLR4-MyD88-NF-κB-dependent innate immunity under high glucose, and may be involved in DN occurrence and progression.

Mitochondrial dysfunction contributes to the cytotoxicity induced by tentacle extract from the jellyfish Cyanea capillata in rat renal tubular epithelial NRK-52E cells

Our previous studies have shown that tentacle extract (TE) from the jellyfish Cyanea capillata could induce a delayed jellyfish envenomation syndrome with severe multiple organ dysfunctions, among which renal injury with tubular necrosis seemed to be most serious. So, in this study, we aimed to explore the toxic effect of TE on rat renal tubular epithelial NRK-52E cells. Based on the previous findings that TE could cause oxidative damage in erythrocytes, the effects of TE on cell oxidative stress conditions, including ROS production and lipid peroxidation, and mitochondrial dysfunction associated with cell death were investigated in NRK-52E cells. The results showed that TE caused cell morphological change and decreased cell viability through induction of apoptosis and necrosis in NRK-52E cells. Meanwhile, ROS overproduction and mitochondrial membrane potential decrease were found before the cell death occurred. It was concluded that TE could induce cytotoxicity, especially apoptosis and necrosis, in NRK-52E cells, and mitochondrial dysfunction and ROS overproduction might play important roles in the process of cell injury and death.

α2-Adrenoceptor agonist dexmedetomidine protects septic acute kidney injury through increasing BMP-7 and inhibiting HDAC2 and HDAC5

Bone morphogenetic protein (BMP)-7 protects sepsis-induced acute kidney injury (AKI). Dexmedetomidine (DEX), an α(2)-adrenoceptor (α(2)-AR) agonist, has anti-inflammatory effects. We investigated the protective effects of DEX on sepsis-induced AKI and the expression of BMP-7 and histone deacetylases (HDACs). In vitro, the effects of DEX or trichostatin A (TSA, an HDAC inhibitor) on TNF-α, monocyte chemotactic protein (MCP-1), BMP-7, and HDAC mRNA expression in LPS-stimulated rat renal tubular epithelial NRK52E cells, was determined using real-time PCR. In vivo, mice were intraperitoneally injected with DEX (25 μg/kg) or saline immediately and 12 h after cecal ligation and puncture (CLP) surgery. Twenty-four hours after CLP, we examined kidney injury and renal TNF-α, MCP-1, BMP-7, and HDAC expression. Survival was monitored for 120 h. LPS increased HDAC2, HDAC5, TNF-α, and MCP-1 expression, but decreased BMP-7 expression in NRK52E cells. DEX treatment decreased the HDAC2, HDAC5, TNF-α, and MCP-1 expression, but increased BMP-7 and acetyl histone H3 expression, whose effects were blocked by yohimbine, an α(2)-AR antagonist. With DEX treatment, the LPS-induced TNF-α expression and cell death were attenuated in scRNAi-NRK52E but not BMP-7 RNAi-NRK52E cells. In CLP mice, DEX treatment increased survival and attenuated AKI. The expression of HDAC2, HDAC5, TNF-α, and MCP-1 mRNA in the kidneys of CLP mice was increased, but BMP-7 was decreased. However, DEX treatment reduced those changes. DEX reduces sepsis-induced AKI by decreasing TNF-α and MCP-1 and increasing BMP-7, which is associated with decreasing HDAC2 and HDAC5, as well as increasing acetyl histone H3.

Role of NAD(P)H oxidase in transforming growth factor‐β1‐induced monocyte chemoattractant protein‐1 and interleukin‐6 expression in rat renal tubular epithelial cells

This study investigated the role of NAD(P)H oxidase in transforming growth factor-beta1 (TGF-beta1)-induced reactive oxygen species (ROS) generation, monocyte chemoattractant protein-1 (MCP-1), and interleukin-6 (IL-6) expression in rat renal tubular epithelial NRK-52E cells. The cells were treated with 10 ng/mL TGF-beta1, either in the presence or absence of the NAD(P)H oxidase inhibitor, diphenyleneiodonium (DPI), or short hairpin RNA (shRNA) suppressing p67phox expression. Expression of NAD(P)H oxidase subunits, MCP-1, and IL-6 at the mRNA levels was detected by reverse transcription polymerase chain reaction, while expression of NAD(P)H oxidase subunit p67phox protein was analyzed by western blot and MCP-1 by enzyme-linked immunosorbent assay. The cellular ROS generation was visualized using 2',7'-dichlorodihydrofluorescein diacetate by confocal microscopy. Compared to control, TGF-beta1 upregulated NAD(P)H oxidase subunit p67phox mRNA by 3.59-fold (P < 0.01), but had no effect on p22phox, gp91phox and p47phox NAD(P)H subunits. TGF-beta1 was also able to significantly increase intracellular ROS (P < 0.05), MCP-1 (P < 0.01) and IL-6 (P < 0.05) expression in NRK-52E cells. Further studies showed that generation of ROS and upregulation of MCP-1 and IL-6 by TGF-beta1 were significantly blocked by addition of DPI or shRNA-p67phox (P < 0.01), suggesting that these effects were NAD(P)H oxidase-dependent. TGF-beta1 differentially regulates the expression of NAD(P)H oxidase subunits and mediates MCP-1 and IL-6 expression in rat renal tubular cells via the NAD(P)H oxidase/p67phox-dependent mechanism.

Ceramide synthase is essential for endonuclease-mediated death of renal tubular epithelial cells induced by hypoxia-reoxygenation.

Ceramide is known to play a role in the cell signaling pathway involved in apoptosis. Most studies suggest that enhanced ceramide generation is the result of hydrolysis of sphingomyelin by sphingomyelinases. However, the role of ceramide synthase in enhanced ceramide generation has not been previously examined in hypoxia-reoxygenation injury. In the present study, we demonstrated that 60-min hypoxia of rat renal tubular epithelial NRK-52E cells in a gas chamber with 95% N2-5% CO2 with glucose deprivation resulted in a significant increase in ceramide generation. The ceramide level further increased after reoxygenation for 60 min. Exposure of cells to hypoxia-reoxygenation resulted in a significant increase in ceramide synthase activity without any significant change in acid or neutral sphingomyelinase. The hypoxia-reoxygenation of NRK-52E cells was also associated with the release of endonuclease G (EndoG) from mitochondria to cytoplasm measured by Western blot analysis and endonuclease activity assay. It further led to the fragmentation of DNA and cell death. A specific inhibitor of ceramide synthase, fumonisin B1 (50 microM), suppressed hypoxia-reoxygenation-induced ceramide generation and provided protection against hypoxia-reoxygenation-induced EndoG release, DNA fragmentation, and cell death. Taken together, our data suggest that hypoxia-reoxygenation results in an activation of ceramide synthase rather than sphingomyelinase and that ceramide synthase-dependent ceramide generation is a key modulator of EndoG-mediated cytotoxicity in hypoxia-reoxygenation injury to renal tubular epithelial cells.