Apoptosis In NRK-52E Cells Research Articles

488-P: Isoliquiritigenin Protects Renal Tubular Epithelial Cells from Inflammatory and Oxidative Injuries via Directly Activating SIRT1 in Type 1 Diabetic Nephropathy

Emerging evidence shows that increasing fibrosis and apoptosis have been observed in the tubular epithelia in the progression of type 1 diabetic nephropathy (DN). Excessive inflammatory response and oxidative stress contribute to the pathogenesis of progression. Isoliquiritigenin (ISL), a natural chalcone compound, has been reported to exert anti-inflammatory and anti-oxidative properties. However, little is known about the effects of ISL on diabetic nephropathy and its mechanism. In this study, we investigated the potential protective effects and pharmacological mechanisms of ISL on DN in vivo and in vitro. We test beneficial activities of ISL on streptozotocin (STZ)-induced type 1 diabetes mellitus mice and renal tubular epithelial NRK-52E cells. The study showed that ISL attenuated diabetic nephropathy by decreasing both pro-fibrotic and pro-apoptotic biomarkers. Diabetes or high glucose (HG) induced inflammatory response and oxidative stress were eliminated by ISL administration. The molecular docking using the multiple ligand simultaneous docking (MLSD) software indicated that three molecules of ISL might bound to the binding site of SIRT1 and formation of a stable complex. We also found ISL could neutralize HG-mediated inactivation of SIRT1. Activation of SIRT1 could attenuate HG-induced inflammation, oxidative stress, fibrosis and apoptosis in NRK-52E cells, paralleled with the effects of ISL. While inhibition of SIRT1 could significantly abrogate the protective effects of ISL. Our study implicated that ISL might have great pharmacological actions on attenuating tubular damage of diabetic nephropathy. The beneficial effects of ISL are likely associated with inhibiting excessive inflammatory response and oxidative stress via directly activating SIRT1. Disclosure X. Chen: None. F. Shen: None. Funding Wenzhou Science and Technology Bureau (Y20190125); First Affiliated Hospital of Wenzhou Medical University (FHY2019015)

EZH2-inhibitor DZNep enhances apoptosis of renal tubular epithelial cells in presence and absence of cisplatin

BackgroundThe enhancer of zeste homolog 2 (EZH2) is a histone methyltransferase and induces the trimethylation of histone H3 lysine 27 (H3K27me3) in the promoter of many key genes; EZH2 acts as a transcriptional repressor and is an epigenetic regulator for several cancers. However, the role of EZH2 in nonneoplastic diseases, such as kidney diseases, is unknown and has been investigated.Materials and methodNRK-52E cells were treated with DZNep, a potent inhibitor of EZH2, with different concentrations and for different times to evaluate the apoptosis level of NRK-52E cells by Western blot and Flow cytometry analysis. The binding of EZH2 to the Deptor promoter was determined by ChIP assay.ResultsThe inhibition of EZH2 with 3-deazaneplanocin A (DZNep), a specific inhibitor of EZH2, led to the apoptosis of NRK-52E cells and the inhibition of mTORC1 and mTORC2 activity. A ChIP assay demonstrated that EZH2 bound the promoter region of Deptor, an endogenous inhibitor of mTORC1 and mTORC2, and regulated the transcription of Deptor by modulating H3K27me3 in its promoter region. Further experiments were performed to examine the effects of EZH2 inhibition on cisplatin-induced injured cells. Cisplatin induced the activation of mTORC1 and mTORC2 and apoptosis in NRK-52E cells, and DZNep inhibited mTORC1 and mTORC2 activity and aggravated cell apoptosis.ConclusionsThese data suggested that EZH2 inhibition increased the transcription of Deptor by modifying H3K27me3 in its promoter region, subsequently inhibited mTORC1 and mTORC2 activities, downregulated the expression of apoptosis suppressor genes, and finally led to apoptosis in renal tubular cells. The inhibition of EZH2 aggravated the cisplatin-induced injury in renal tubular cells by inactivating the mTOR complexes. The present study provides new insight into renal protection and suggests that EZH2 might be a target.

Selenium attenuates apoptosis and p-AMPK expressions in fluoride-induced NRK-52E cells.

Fluoride is widely distributed in the environment, and excessive fluoride intake can induce cytotoxicity, DNA damage, and cell cycle changes in many tissues and organs, including the kidney. Accumulating evidence demonstrates that selenium (Se) administration ameliorates sodium fluoride (NaF)-induced kidney damage. However, the potentially beneficial effects of Se against NaF-induced cytotoxicity of the kidney and the underlying molecular mechanisms of this protection are not fully understood. At present, in this study, the normal rat kidney cell (NRK-52E) was used to investigate the potentially protective mechanism of Se against NaF-induced apoptosis, by using the methods of pathology, colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometry, and Western blot. The experiment was designed with a control group, two NaF-treated groups (NaF, 5, 20mg/L), two sodium selenite-treated groups (Na2SeO3, 17.1, 34.2μg/L), and four Se + NaF-treated groups (Na2SeO3, 17.1, 34.2μg/L; NaF, 5, 20mg/L). The results indicate that selenium can attenuate apoptosis and AMPK phosphorylation in the NRK-52E cell induced with fluoride. These results imply that selenium is capable to modulate fluoride-induced NRK-52E cell apoptosis via regulating the expression levels of the proteins involved in mitochondrial pathway and changes in p-AMPK expressions may also be a key process in preventing fluorosis.

Telmisartan attenuates diabetic nephropathy progression by inhibiting the dimerization of angiotensin type-1 receptor and adiponectin receptor-1

AimsThe heterodimerization of angiotensin II receptors (AT1R and AT2R) with adiponectin receptor AdipoR1 and AdipoR2 may instigate high glucose (HG)-induced renal tubulointerstitial injury. This study examined the effect of telmisartan on diabetic nephropathy (DN) and its underlying mechanism. Main methodsDiabetes was induced in rats through a single intraperitoneal injection of streptozotocin. Diabetic rats treated with or without the intravenous injection of AdipoR1 siRNA were intragastrically administered with 5 mg/kg/d telmisartan or a vehicle for 12 weeks. The rat proximal tubular epithelial cell line NRK-52E was treated with HG (30 mmol/L) with or without telmisartan (10 μM) for 48 h. Key findingsIn streptozotocin-induced diabetic rats, telmisartan treatment could decrease the inulin clearance rate, restore the glomerular surface area and mesangial area, alleviate renal fibrosis, and decrease urinary albumin excretion. Furthermore, diabetic rats exhibited increased AT1R-AdipoR1 heterodimers in the renal tubular compartment, which could be attenuated by telmisartan treatment, accompanied by a decrease in the expression level of cytokines MIP-1α, ICAM-1 and MCP-1. In vitro, HG promoted the dimerization formation of AT1R-AdipoR1 in cultured NRK-52E cells, but this effect was not found in NRK-52E cells transfected with the AdipoR1-G269E,G273E mutant. Telmisartan could inhibit HG-induced AT1R-AdipoR1 dimerization, downregulate the expression levels of inflammatory cytokines, and alleviate cell apoptosis in NRK-52E cells. Furthermore, AdipoR1 knockdown could abate the renoprotective benefits of telmisartan. SignificanceThe heterodimerization of AT1R-AdipoR1 probably contributes to the renal injury of DN, and provides an additional mechanistic insight into how telmisartan prevents the development and progression of DN.

MiR-214 ameliorates acute kidney injury via targeting DKK3 and activating of Wnt/\u03b2-catenin signaling pathway

BackgroundmiR-214 was demonstrated to be upregulated in models of renal disease and promoted fibrosis in renal injury independent of TGF-β signaling in vivo. However, the detailed role of miR-214 in acute kidney injury (AKI) and its underlying mechanism are still largely unknown.MethodsIn this study, an I/R-induced rat AKI model and a hypoxia-induced NRK-52E cell model were used to study AKI. The concentrations of kidney injury markers serum creatinine, blood urea nitrogen, and kidney injury molecule-1 were measured. The expressions of miR-214, tumor necrosis factor-α, interleukin (IL)-1β, IL-6, were detected by RT-qPCR. The protein levels of Bcl-2, Bax, Dickkopf-related protein 3, β-catenin, c-myc, and cyclinD1 were determined by western blot. Cell apoptosis and caspase 3 activity were evaluated by flow cytometry analysis and caspase 3 activity assay, respectively. Luciferase reporter assay was used to confirm the interaction between miR-214 and Dkk3.ResultsmiR-214 expression was induced in ischemia–reperfusion (I/R)-induced AKI rat and hypoxic incubation of NRK-52E cells. Overexpression of miR-214 alleviated hypoxia-induced NRK-52E cell apoptosis while inhibition of miR-214 expression exerted the opposite effect. Dkk3 was identified as a target of miR-214. Anti-miR-214 abolished the inhibitory effects of DKK3 knockdown on hypoxia-induced NRK-52E cell apoptosis by inactivation of Wnt/β-catenin signaling. Moreover, miR-214 ameliorated AKI in vivo by inhibiting apoptosis and fibrosis through targeting Dkk3 and activating Wnt/β-catenin pathway.ConclusionmiR-214 ameliorates AKI by inhibiting apoptosis through targeting Dkk3 and activating Wnt/β-catenin signaling pathway, offering the possibility of miR-214 in the therapy of ischemic AKI.

MiR-182 enhances acute kidney injury by promoting apoptosis involving the targeting and regulation of TCF7L2/Wnt/β-catenins pathway

Acute kidney injury (AKI) is a sudden decay in renal function leading to increasing morbidity and mortality. miR-182 has been reported to be actively involved in kidney diseases. However, the function and molecular mechanism of miR-182 in AKI still need to be elucidated. The levels of serum creatinine (SCr), blood urea nitrogen (BUN), and urine Kim-1 in I/R-induced rat AKI model were detected by a Beckman Autoanalyzer. miR-182 and transcription factor 7-like-2 (TCF7L2) mRNA expression were measured by qRT-PCR. Flow cytometry and caspase-3 colorimetry analysis were performed to determine NRK-52E cell apoptosis. Bioinformatics and dual-luciferase reporter were used to identify the interaction between miR-182 and TCF7L2. miR-182 expression was increased in both I/R-induced rat models and hypoxia-treated NRK-52E cells, and miR-182 overexpression stimulated the apoptosis of hypoxia-induced NRK-52E cells. Dual-luciferase analysis disclosed that TCF7L2 was a target of miR-182. TCF7L2 suppressed hypoxia-induced apoptosis in NRK-52E cells, and the inhibitory effect of TCF7L2 on cell apoptosis could be reversed with miR-182 restoration. Moreover, the activity of Wnt/β-catenin signaling pathway was promoted following overexpression of TCF7L2 in NRK-52E cells with hypoxia treatment, and this effect was greatly attenuated by the increased miR-182 expression. Finally, in vivo experiment also validated the alleviation of miR-182 inhibitor on I/R-induced kidney injury and apoptosis via regulating TCF7L2/ Wnt/β-catenin pathway. miR-182 exacerbated AKI involving the targeting and regulation of TCF7L2/Wnt/β-catenin signaling, unveiling a novel regulatory pathway in ischemia-reperfusion injury and elucidating a potential biomarker for AKI treatment.

Fascin2 regulates cisplatin-induced apoptosis in NRK-52E cells

Previous studies have shown that the aging kidney has a marked loss of α(E)-catenin in proximal tubular epithelium. α-Catenin, a key regulator of the actin cytoskeleton, interacts with a variety of actin-binding proteins. Cisplatin-induced loss of fascin2, an actin bundling protein, was observed in cells with a stable knockdown of α(E)-catenin (C2 cells), as well as in aging (24 mon), but not young (4 mon), kidney. Fascin2 co-localized with α-catenin and the actin cytoskeleton in NRK-52E cells. Knockdown of fascin2 increased the susceptibility of tubular epithelial cells to cisplatin-induced injury. Overexpression of fascin2 in C2 cells restored actin stress fibers and attenuated the increased sensitivity of C2 cells to cisplatin-induced apoptosis. Interestingly, fascin2 overexpression attenuated cisplatin-induced mitochondrial dysfunction and oxidative stress in C2 cells. These data demonstrate that fascin2, a putative target of α(E)-catenin, may play important role in preventing cisplatin-induced acute kidney injury.

Cadmium-induced heme-oxygenase-1 expression plays dual roles in autophagy and apoptosis and is regulated by both PKC-δ and PKB/Akt activation in NRK52E kidney cells

Heme oxygenase-1 (HO-1) protects cells against cadmium (Cd)-induced oxidative stress. However, the mechanism underlying this protection is not well understood. In this study, we elucidated the role of HO-1 in Cd-induced cytotoxicity. Exposure of NRK52E cells to Cd induced protein kinase B (PKB)/Akt, protein kinase C (PKC)-δ, and glycogen synthase kinase (GSK) 3αb phosphorylation, and eukaryotic initiation factor (eIF) 2α dephosphorylation. Pharmacological inhibition of Akt resulted in HO-1 suppression and eIF2α activation, which partially suppressed CHOP and PARP-1 cleavage, but promoted autophagy and decreased cell viability. Pharmacological inactivation of PKC-δ markedly suppressed Cd-induced phospho-serine (p-Ser) GSK3αβ, and HO-1, and partially inhibited PARP-1 cleavage, but massively induced autophagy and decreased cell viability. Pharmacological upregulation of p-Ser GSK3αβ enhanced Cd-induced HO-1, CHOP, and PARP-1 cleavage, but decreased autophagy. Genetic deficiency of GSK3β suppressed HO-1 and PARP-1 cleavage and increased autophagy. Genetic suppression of HO-1 reduced Cd-induced PARP-1 cleavage, but increased LC3-II. Cd exposure led to accumulation of p-PKC-δ, p-Ser GSK3αβ, and HO-1 in the nucleus and particulate fractions, suggesting that they have dual functions in response to Cd. N-acetylcysteine treatment suppressed Cd-induced activation of PKC-δ and Akt. These results indicate that HO-1 induced by Cd exposure is regulated by PKC-δ, p-Ser GSK3αβ, and PKB/Akt, which restrain autophagic cell death, but mildly induce apoptosis in NRK52E cells. Together, the results suggest that HO-1 expression in response to Cd maintains cellular homeostasis during oxidative stress.

The epoxyeicosatrienoic acid analog PVPA ameliorates cyclosporine-induced hypertension and renal injury in rats.

The introduction of calcineurin inhibitors (CNI) into clinical practice in the late 1970s transformed organ transplantation and led to significant improvement in acute rejection episodes. However, despite their significant clinical utility, the use of these agents is hampered by the development of hypertension and nephrotoxicity, which ultimately lead to end-stage kidney disease and overt cardiovascular outcomes. There are currently no effective agents to treat or prevent these complications. Importantly, CNI-free immunosuppressive regimens lack the overall efficacy of CNI-based treatments and put patients at risk of allograft rejection. Cytochrome P-450 epoxygenase metabolites of arachidonic acid, epoxyeicosatrienoic acids (EETs), have potent vasodilator and antihypertensive properties in addition to many cytoprotective effects, but their effects on CNI-induced nephrotoxicity have not been explored. Here, we show that PVPA, a novel, orally active analog of 14,15-EET, effectively prevents the development of hypertension and ameliorates kidney injury in cyclosporine-treated rats. PVPA treatment reduced proteinuria and renal dysfunction induced by cyclosporine. PVPA inhibited inflammatory cell infiltration into the kidney and decreased renal fibrosis. PVPA also reduced tubular epithelial cell apoptosis, attenuated the generation of reactive oxygen species, and modulated the unfolded protein response that is associated with endoplasmic reticulum stress. Consistent with the in vivo data, PVPA attenuated cyclosporine-induced apoptosis of NRK-52E cells in vitro. These data indicate that the cytochrome P-450/EET system offers a novel therapeutic strategy to treat or prevent CNI-induced nephrotoxicity.

Aldosterone induces NRK-52E cell apoptosis in acute kidney injury via rno-miR-203 hypermethylation and Kim-1 upregulation.

Acute kidney injury (AKI) is characterized by an acute reduction in kidney function as identified by an increase in serum creatinine levels and reduction in urine output. Kidney injury molecule-1 (Kim-1) is a hallmark of kidney diseases, since it is typically non-detectable in the non-injured kidney, but upregulated and excreted in the urine during AKI. Aldosterone (Aldo) is a mediator of the renin-angiotensin-Aldo system with a pivotal role in the regulation of salt and extracellular fluid metabolism. In the present study, mice subjected to renal ischemia/reperfusion-induced AKI were investigated. The mice exhibited elevated levels of Aldo and angiotensin II, together with increased Kim-1 expression levels in renal tissue. Treatment of the mice with the Aldo receptor antagonist spironolactone decreased Kim-1 expression levels. These results suggest that Aldo may be associated with the expression of Kim-1 during AKI. However, the molecular mechanism underlying the role of Aldo in Kim-1 expression is unclear, and thus was investigated using NRK-52E cells. Aldo was found to induce the apoptosis of NRK-52E cells via the hypermethylation of rno-microRNA (miR)-203 and upregulation of Kim-1. In addition, luciferase reporter assays demonstrated that Kim-1 was a target gene of rno-miR-203 in NRK-52E cells. Furthermore, Aldo-induced NRK-52E cell apoptosis was reduced by treatment with pre-miR-203 and spironolactone to a greater extent when compared with either alone. The results may provide a promising diagnostic marker or novel therapeutic target for AKI.

BMP-7 Attenuates TGF-β1-Induced Fibronectin Secretion and Apoptosis of NRK-52E Cells by the Suppression of miRNA-21.

Bone morphogenetic protein-7 (BMP-7) inhibited the pathogenesis of renal injury in response to a variety of stimuli. However, little is known about the molecular regulation and mechanism of endogenous BMP-7 and its renoprotective functions. This study examined the regulation of BMP-7 and its role in the fibronectin secretion and apoptosis of NRK-52E cells resulting from transforming growth factor-β1 (TGF-β1) in vitro. Results showed that TGF-β1 promoted factor-associated suicide (FAS), FAS ligand (FASL), fibronectin (FN), and miRNA-21 expression, while it downregulated phospho-Smad1 (pSmad1), pSmad5, and pSmad8 expressions in NRK-52E cells. In contrast, BMP-7 alleviated TGF-β1-induced cell apoptosis, inhibited TGF-β1-induced higher expression of miRNA-21 and FN, and enhanced TGF-β1-attenuated phosphorylation of Smad1, Smad5, and Smad8. Furthermore, a chemical inhibitor of miRNA-21 also negatively affected TGF-β1-induced apoptosis and FN secretion. On the other hand, overexpression of miRNA-21 counteracted the inhibitory effect of BMP-7 on TGF-β1-induced FN secretion and apoptosis. However, BMP-7 showed no effects on TGF-β1-induced FN secretion and apoptosis following knockdown of miRNA-21. Taken together, these findings demonstrated that BMP-7 might inhibit TGF-β1-induced FN secretion and apoptosis by the suppression of miRNA-21 in NRK-52E cells.

Induced Pluripotent Stem Cell-Derived Conditioned Medium Attenuates Acute Kidney Injury by Downregulating the Oxidative Stress-Related Pathway in Ischemia-Reperfusion Rats.

Teratoma-like formation addresses a critical safety concern for the potential utility of induced pluripotent stem cells (iPSCs). Therefore, therapy utilizing iPSC-derived conditioned medium (iPSC-CM) for acute kidney injury (AKI) has attracted substantial interest. A recent study showed that iPSC-CM effectively alleviated ventilator-induced lung injury in rats. It prompts us to assess the therapeutic effects of iPSC-CM on ischemic AKI. First, we assessed the changes in renal function and tubular cell apoptosis by intraperitoneal administration of iPSC-CM to ischemia-reperfusion (I/R) rats. Second, we explored the oxidative stress-related pathway in the apoptosis of renal tubular cells subjected to hypoxia-reoxygenation (H/R). Administration of iPSC-CM significantly improved renal function and protected tubular cells against apoptosis in rats with I/R-AKI, and the optimal effect was observed at the 50-fold concentrated iPSC-CM. iPSC-CM also mitigated the H/R-induced apoptosis of NRK-52E cells in vitro. Reactive oxygen species (ROS) production was augmented in kidneys following I/R and in NRK-52E cells subjected to H/R. Meanwhile, expressions of phosphorylated p38 MAPK, TNF-α, and cleaved caspase 3 and NF-κB activity were consistently increased in vivo and in vitro. Following administration of iPSC-CM, ROS production was abolished, and inflammatory cytokine expression was significantly suppressed. Annexin V-propidium iodide flow cytometry and in situ TUNEL assay further showed that iPSC-CM markedly attenuated H/R- or I/R-induced tubular cell apoptosis. Intriguingly, treatment with iPSC-CM significantly improved the survival of rats with I/R-induced AKI. iPSC-CM represents a favorable source of stem cell-based therapy and may serve as a potential therapeutic strategy for kidney repair in ischemic AKI.

Sirt1 is essential for resveratrol enhancement of hypoxia-induced autophagy in the type 2 diabetic nephropathy rat

Type 2 diabetic nephropathy (DN) is a serious end-stage kidney disease worldwide. Multiple studies demonstrate that resveratrol (RSV) has a beneficial effect on DN. However, whether RSV-induced improvement in kidney function in diabetes is due to the regulation of autophagy remains unclear. Here, we investigated the mechanisms underlying RSV-mediated protection against DN in diabetic rats, with a special focus on the role of NAD-dependent deacetylase sirtuin 1 (Sirt1) in regulating autophagy. We found that long-term RSV treatment in rats promoted Sirt1 expression and improved related metabolic levels in the diabetic kidney. Our study showed that, in cultured NRK-52E cells, Sirt1 knockdown inhibited the autophagy levels of proteins Atg7, Atg5, and LC3 and impaired the RSV amelioration of dysfunctional autophagy under hypoxic condition. Furthermore, exposed to 1% O2 over time induced autophagy dysfunction and apoptosis in NRK-52E cells, which could be improved by RSV treatment. Our data highlight the role of the Sirt1-mediated pathway in the effects of RSV on autophagy in vivo and in vitro, suggesting RSV could be a potential new therapy for type 2 DN.

Biliverdin protects against cisplatin-induced apoptosis of renal tubular epithelial cells.

Biliverdin (BV) has long been thought to be a cytotoxic metabolic waste product. It has also been demonstrated to have important cytoprotective functions during oxidative stress. The present study aimed to examine the cytoprotective effect of BV on NRK-52E cells, a proximal tubular cell line derived from rat kidney. Cells were treated with 50 µmol/L cisplatin for 24 h (cisplatin group) or pre-treated with BV for 30 min, then with 50 µmol/L cisplatin for 24 h (cisplatin+BV group). Those given no treatment served as a control. Cell apoptosis was evaluated by flow cytometry and cell viability by Cell Counting Kit-8 (CCK-8). The protein expressions of cleaved caspase3, Bax and Bcl-2 were assessed by Western blotting. Reactive oxygen species (ROS) levels were measured using carboxydichlorodihydrofluorescein diacetate (H2DCF). The results showed that cisplatin induced the apoptosis of NRK-52E cells, decreased cell viability, and increased the formation of ROS by upregulating the expression of cleaved caspase3 and Bax and decreasing Bcl-2 protein expression. These effects could be significantly reversed by pretreatment with BV. It was concluded that BV can protect against cisplatin-induced cell apoptosis through the anti-oxidative effects.

Emodin ameliorates cisplatin-induced apoptosis of rat renal tubular cells in vitro by activating autophagy

A previous report shows that emodin extracted from the Chinese herbs rhubarb and giant knotweed rhizome can ameliorate the anticancer drug cisplatin-induced injury of HEK293 cells. In this study, we investigated whether and how emodin could protect renal tubular epithelial cells against cisplatin-induced nephrotoxicity in vitro. The viability and apoptosis of normal rat renal tubular epithelial cells (NRK-52E) were detected using formazan assay and flow cytometry analysis, respectively. The expression levels of cleaved caspase-3, autophagy maker LC3 I/II, and AMPK/mTOR signaling pathway-related proteins were measured with Western blot analysis. The changes of morphology and RFP-LC3 fluorescence were observed under microscopy. Cisplatin (10-50 μmol/L) dose-dependently induced cell damage and apoptosis in NRK-52E cells, whereas emodin (10 and 100 μmol/L) significantly ameliorated cisplatin-induced cell damage, apoptosis and caspase-3 cleavage. Emodin dose-dependently increased LC3-II levels and induced RFP-LC3-containing punctate structures in NRK-52E cells. Furthermore, the protective effects of emodin were abolished by bafilomycin A1 (10 nmol/L), and mimicked by rapamycin (100 nmol/L). Moreover, emodin increased the phosphorylation of AMPK and suppressed the phosphorylation of mTOR. The AMPK inhibitor compound C (10 μmol/L) not only abolished emodin-induced autophagy activation, but also emodin-induced anti-apoptotic effects. Emodin ameliorates cisplatin-induced apoptosis of rat renal tubular cells in vitro through modulating the AMPK/mTOR signaling pathways and activating autophagy. Emodin may have therapeutic potential for the prevention of cisplatin-induced nephrotoxicity.

Hepatitis B Virus X Protein Modulates Apoptosis in NRK-52E Cells and Activates Fas/FasL Through the MLK3-MKK7-JNK3 Signaling Pathway

Background/Aims: The hepatitis B virus X protein (HBx) contributes to HBV-induced injury of renal tubular cells and induces apoptosis via Fas/FasL up-regulation. However, the mechanism of Fas/FasL activation is unknown. Recent studies indicated that HBx induction of apoptosis in hepatic cells depends on activating the MLK3-MKK7-JNKs signaling module, which then up-regulates FasL expression. In this study, we used NRK-52E cells transfected an HBx expression vector to examine the role of the MLK3-MKK7-JNKs signaling pathway on HBx-induced renal tubular cell injury. Methods: NRK-52E cells were transfected with pc-DNA3.1(+)-HBx to establish an HBx over-expression model, and with pc-DNA3.1(+)-HBx and pSilencer3.1-shHBx to establish an HBx low expression model. One control group was not transfected and another control group was transfected with an empty plasmid. Cell proliferation was determined by the formazan dye method (Cell Counting Kit-8) and apoptosis was measured by flow cytometry and fluorescence microscopy. Western blotting was used to measure the expression of Fas, FasL, and MLK3-MKK7-JNKs signaling pathway-related proteins. The activity of caspase-8 was measured by spectrophotometry. Results: Transfection of NRK-52E cells with pc-DNA3.1(+)-HBx inhibited cell proliferation and increased apoptosis and caspase-8 activity. The expression of Fas, FasL, and MLK3-MKK7-JNKs signaling pathway-related proteins were also greater in the pc-DNA3.1(+)-HBx group, but lower in RNAi group. Furthermore, the activity of MLK3-MKK7-JNKs signaling pathway, expression of Fas/FasL, and apoptosis were significantly lower in the pc-DNA3.1(+)-HBx group when treated with K252a, a known inhibitor of MLK3. Conclusions: Our results show that HBx induces apoptosis in NRK-52E cells and activates Fas/FasL via the MLK3-MKK7-JNK3-c-Jun signaling pathway.

Peroxiredoxin 1 inhibits the oxidative stress induced apoptosis in renal tubulointerstitial fibrosis.

Apoptosis is one of the most important mechanisms underlying renal tubulointerstitial fibrosis. We identified a role of protein Peroxiredoxin 1 (Prx1) in protecting apoptosis occurred in tubular epithelial cells of the rat and human kidney. Immunohistochemistry (IHC) staining was used to detect Prx1 expression in kidney derived from unilateral-ureteral obstruction (UUO) rats or patients with obstructive nephropathy. Modulation of Prx1 expression by transfecting siRNA and overexpression plasmid approach were carried out in NRK-52E (rat kidney tubular epithelial cell line) cells. UUO-induced apoptosis was determined using TUNEL assay. Immunohistochemistry staining showed that Prx1 expressed in the cytoplasm of renal tubular epithelial cells, in the kidneys of UUO rats. The reduction was confirmed by both IHC and real-time polymerase chain reaction following a course of renal tubulointerstitial fibrosis in UUO rats and a decrease of Prx1 occurred concomitantly with an elevation of TUNEL-positive cells. Fluorofenidone (AKF-PD), a new anti-tubulointerstitial fibrotic agent, attenuated Prx1 reduction in UUO rats. Furthermore, hydrogen peroxide (H2 O2 )-derived oxidative stress activated p38 MAPK, and induced apoptosis in NRK-52E cells; knockdown of Prx1 sensitized both events in NRK-52E cells, and overexpression of Prx1 diminished the apoptosis and the phosphorylation of p38 CONCLUSION: Downregulation of Prx1 occurred in renal tubular epithelial cells of UUO rats and patients with obstructive nephropathy. Prx1 may alleviate the pathogenesis by inhibiting H2 O2 -induced apoptosis via inhibiting the p38 MAPK pathway. Prx1 may represent a useful target for a protective therapy towards renal tubulointerstitial fibrosis.

MiR-21 inhibits autophagy by targeting Rab11a in renal ischemia/reperfusion

Renal ischemia–reperfusion (I/R) is one of the main causes of the acute kidney injury (AKI) that usually occurs during clinical surgery. Autophagy plays an important role in recovery from acute ischemic kidney injury. MicroRNA-21 (miR-21) was reported to inhibit autophagy in several diseases. However, the molecular mechanism of miR-21 on autophagy during renal I/R is still unclear. For the in vitro study, NRK-52E cells were transfected with miR-21 mimics and subjected to I/R. Results showed that miR-21 mimics inhibited cell viability and induced cell apoptosis in NRK-52E cells. Expression of beclin-1 and LC3-II was induced, and p62 was decreased by I/R. miR-21 mimics inhibited this induction. RT-PCR and western blotting assay showed that miR-21 mimics inhibited the protein level of Rab11a, but not the mRNA level. A luciferase activity assay showed that miR-21 directly targeted Rab11a 3′-UTR. Rab11a overexpression attenuated the effect of miR-21 mimics and I/R on cell viability and cell apoptosis. The expression of beclin-1 and LC3-II was increased, and p62 was decreased by Rab11a overexpression. For the in vivo assay in a rat I/R model, the miR-21 level was increased during renal I/R injury. Pre-treatment with miR-21 inhibitor injection attenuated the renal injury, and enhanced expression of LC3-II and beclin-1. The results showed that miR-21 inhibited autophagy by targeting Rab11a in renal I/R, indicating that Rab11a might be a new medical target for the treatment of renal I/R.

Dexmedetomidine protects against apoptosis induced by hypoxia/reoxygenation through the inhibition of gap junctions in NRK-52E cells

AimsThe α2-adrenoceptor inducer dexmedetomidine (Dex) provides renoprotection against ischemia/reperfusion (I/R) injury, but the mechanism of this effect is largely unknown. The present study investigated the effect of Dex on apoptosis induced by hypoxia/reoxygenation (H/R) and the relationship between this effect and gap junction intercellular communication (GJIC). Main methodsIn vitro, two cell lines of normal rat kidney proximal tubular cells (NRK-52E) and HeLa cells that were transfected with a connexin 32 (Cx32) plasmid were exposed to H/R. The role of Dex in the modulation of H/R-induced apoptosis was explored by the manipulation of connexin expression, and hence gap junction (GJ) function, using a GJIC inhibitor, heptanol, and a GJIC inducer, retinoic acid. GJ function and the Cx32 protein level were determined by the parachute dye-coupling assay and Western blotting, respectively. Key findingsDex and heptanol significantly reduced H/R-induced apoptosis in NRK-52E cells. The anti-apoptosis effect of Dex was exhibited only in Cx32-expressing HeLa cells. One hour Dex exposure inhibited GJ function mainly via a decrease in Cx32 protein levels in NRK-52E cells. SignificanceOur data suggest that Dex reduced H/R-induced apoptosis through the inhibition of GJ activity by reducing Cx32 protein levels.

Role of Reactive Oxygen Species-Mediated Endoplasmic Reticulum Stress in Contrast-Induced Renal Tubular Cell Apoptosis

Background: Renal tubular cell apoptosis is a key mechanism of contrast-induced acute kidney injury. It has been reported that endoplasmic reticulum (ER) stress is the underlying mechanism of high osmolar contrast-induced renal tubular cell apoptosis. Whether ER stress is involved in low osmolar contrast-induced renal tubular cell injury remains unclear. In the present study, the roles of ER stress in iopromide-induced (a low osmolar contrast) renal tubular cell apoptosis and the effects of N-acetylcysteine (NAC) on ER stress were investigated. Methods: NRK-52E cells were exposed to different concentrations of iopromide [50, 100 and 150 mg iodine (I)/ml] for 4 h. In a separate experiment, NRK-52E cells were exposed to iopromide (100 mg I/ml, 4 h) with or without NAC (10 mmol/l). NAC was added 1 h before incubation with iopromide. Apoptosis was determined by Hoechst staining and flow cytometry. The intracellular formation of reactive oxygen species (ROS) was detected by confocal microscopy with fluorescent probe CM-H2DCFDA. The expression of glucose-regulated protein 78 (GRP78) and CAAT/enhancer-binding protein homologous protein (CHOP) was determined by Western blot. Results: Iopromide induced NRK-52E cell apoptosis in a concentration-dependent manner. The intracellular ROS production increased significantly following iopromide exposure in the NRK-52E cells. Significantly increased expressions of GRP78 and CHOP were observed in the NRK-52E cells exposed to iopromide for 4 h; NAC attenuated iopromide-induced NRK-52E cell apoptosis by inhibiting the overproduction of intracellular ROS and subsequently suppressing the overexpression of GRP78 and CHOP. Conclusion: ROS-mediated ER stress is involved in contrast-induced renal tubular cell apoptosis.