Rat Renal Proximal Tubular Cells Research Articles

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.

Mitochondrial transplantation attenuates toxicity in rat renal proximal tubular cells caused by Favipiravir.

Exogenous mitochondria transplantation or mitotherapy can be used to swap out unhealthy mitochondria for functioning ones. Treatment of mitochondrial diseases using this approach may be beneficial. In this study, we looked at the effect of transplanting newly isolated mitochondria on the toxicity that favipiravir (FAV) causes in renal proximal tubular cells (RPTCs). In this study, parameters such as lactate dehydrogenase (LDH) leakiness, reactive oxygen species (ROSs) production, damage to the lysosome membrane, reduced glutathione (GSH) content, extracellular oxidized glutathione (GSSG) content, GSH/GSSG ratio, ATP level, mitochondrial membrane potential (MMP) collapse, Bcl-2 content, and caspase-3 activity were used to assess the protective effects of mitochondrial transplantation against FAV-induced mitochondrial toxicity. The statistical analysis showed that the cytotoxicity, ROS production, MMP collapse, lysosomal damage, GSSG levels, and caspase-3 activity brought on by FAV in RPTCs were reduced by transplanting the healthy mitochondria. In addition, it led to an increase in ATP level, GSH content, Bcl-2 content, and GSH/GSSG ratio in RPTCs. A recent study found that mitochondrial transplantation is a powerful therapeutic approach for treating nephrotoxicity brought on by xenobiotics.

The Effect of Donor Rat Gender in Mitochondrial Transplantation Therapy of Cisplatin-Induced Toxicity on Rat Renal Proximal Tubular Cells.

Cisplatin-induced nephrotoxicity has been linked to a fundamental mechanism of mitochondrial dysfunction. A treatment called mitochondrial transplantation therapy can be used to replace damaged mitochondria with healthy mitochondria. Mitochondrial-related diseases may benefit from this approach. We investigated the effect of mitochondrial transplantation on cisplatin-induced nephrotoxicity using freshly isolated mitochondria obtained from renal proximal tubular cells (RPTCs). Based on our previous findings, we hypothesized that direct exposure of healthy mitochondria to cisplatin-affected RPTCs might improve cytotoxicity markers and restore mitochondrial function. Therefore, the primary objective of this study was to determine whether newly isolated mitochondrial transplantation protected RPTCs from cisplatin-induced cytotoxicity. The supply of exogenous rat kidney mitochondria to cisplatin-affected RPTCs was also a goal of this study to investigate the possibility of gender differences. After the addition of cisplatin (100 µM), rat RPTCs (106 cells/mL) were suspended in Earle's solution (pH = 7.4) at 37°C for two hours. Freshly isolated mitochondria were extracted at 4°C and diluted in 100 and 200 µg/mL mitochondrial protein. Statistical analysis revealed that transplantation of healthy mitochondria decreased ROS level, mitochondrial membrane potential (MMP) collapse, MDA level, glutathione depletion, lysosomal membrane damage, and caspase-3 activity induced by cisplatin in rat RPTCs. In addition, our results demonstrated that transplantation of female rat kidney mitochondria has higher protective activity at reducing toxicity parameters than male mitochondria. The findings reaffirmed that mitochondrial transplantation is a novel, potential, and promising therapeutic strategy for xenobiotic-induced nephrotoxicity.

Mitochondrial transplantation against gentamicin-induced toxicity on rat renal proximal tubular cells: the higher activity of female rat mitochondria.

Mitochondrial dysfunction is a fundamental mechanism leading to drug nephrotoxicity, such as gentamicin-induced nephrotoxicity. Mitochondrial therapy (mitotherapy) or exogenous mitochondria transplantation is a method that can be used to replace dysfunctional mitochondria with healthy mitochondria. This method can help in the treatment of diseases related to mitochondria. In this research, we studied the transplantation effect of freshly isolated mitochondria on the toxicity induced by gentamicin on renal proximal tubular cells (RPTCs). Furthermore, possible gender-related effects on supplying exogenous rat kidney mitochondria on gentamicin-induced RPTCs were investigated. At first, the normality and proper functioning of fresh mitochondria were assessed by measuring mitochondrial succinate dehydrogenase activity (SDH) and changes in mitochondrial membrane potential (MMP). Then, the protective effects of mitochondrial transplantation against gentamicin-induced mitochondrial toxicity were evaluated through parameters including lactate dehydrogenase (LDH) leakiness, reactive oxygen species (ROS) production, lipid peroxidation (LPO) content, reduced glutathione (GSH) level, extracellular oxidized glutathione (GSSG) level, ATP level, MMP collapse, and caspase-3 activity. According to the statistical analysis, transplanting the healthy mitochondria decreased the cytotoxicity, ROS production, MMP collapse, LPO content, GSSG levels, and caspase-3 activity caused by gentamicin in RPTCs. Also, it has caused an increase in the level of ATP and GSH in the RPTCs. Furthermore, higher preventive effects were observed for the female group. According to the current study, mitochondrial transplantation is a potent therapeutic method in xenobiotic-caused nephrotoxicity.

Mitochondrial Transplantation Therapy against Ifosfamide Induced Toxicity on Rat Renal Proximal Tubular Cells.

Mitochondrial dysfunction is a basic mechanism leading to drug nephrotoxicity. Replacement of defective mitochondria with freshly isolated mitochondria is potentially a comprehensive tool to inhibit cytotoxicity induced by ifosfamide on renal proximal tubular cells (RPTCs). We hypothesize that the direct exposure of freshly isolated mitochondria into RPTCs affected by ifosfamide might restore mitochondrial function and reduce cytotoxicity. So, the aim of this study was to assess the protective effect of freshly isolated mitochondrial transplantation against ifosfamide-induced cytotoxicity in RPTCs. Therefore, the suspension of rat RPTCs (106 cells/ml) in Earle's solution with the pH of 7.4 at 37°C was incubated for 2 h after ifosfamide (4 mM) addition. Fresh mitochondria were isolated from the rat kidney and diluted to the needed concentrations at 4°C. The media containing suspended RPTCs was replaced with mitochondrial-supplemented media, which was exposed to cells for 4 hours in flasks-rotating in a water bath at 37°C. Statistical analysis demonstrated that mitochondrial administration reduced cytotoxicity, lipid peroxidation (LPO), reactive oxygen species (ROS) production, mitochondrial membrane potential (MMP) collapse, lysosomal membrane damage, extracellular oxidized glutathione (GSSG) level, and caspase-3 activity induced by ifosfamide in rat RPTCs. Moreover, mitochondrial transplantation increased the intracellular reduced glutathione (GSH) level in RPTCs affected by ifosfamide. According to the current study, mitochondrial transplantation is a promising therapeutic method in xenobiotic-caused nephrotoxicity pending successful complementary in vivo and clinical studies.

Anti-EMT and anti-fibrosis effects of protocatechuic aldehyde in renal proximal tubular cells and the unilateral ureteral obstruction animal model

Context Protocatechuic aldehyde (PCA) is a natural product that has various benefits for fibrosis. Objective This study evaluated the effects of PCA on renal fibrosis. Materials and methods Epithelial–mesenchymal transition (EMT) was induced by 20 ng/mL transforming growth factor-β1 (TGF-β1), followed by treatment with 1 and 5 μM PCA, in the rat renal proximal tubular cell line NRK-52E. Cell viability, protein expression, and scratch wound-healing assays were conducted. Sprague–Dawley (SD) rats underwent unilateral ureteral obstruction (UUO) surgery for renal fibrosis indication and were treated with 50 and 100 mg/kg PCA for 14 days. Results The IC50 of PCA was appropriately 13.75 ± 1.91 μM in NRK-52E cells, and no significant difference at concentrations less than 5 μM. PCA ameliorated TGF-β1-induced EMT, such as enhanced E-cadherin and decreased vimentin. Fibrotic markers collagen IV and α-smooth muscle actin (α-SMA) increased in TGF-β1-induced NRK-52E. Moreover, PCA reduced TGF-β1-induced migration in the wound-healing assay. Analysis of rat kidneys indicated that PCA reduced UUO-induced hydronephrosis (control: 15.11 ± 1.00%; UUO: 39.89 ± 1.91%; UUO + PCA50: 18.37 ± 1.61%; UUO + PCA100: 17.67 ± 1.39%). Protein level demonstrated that PCA not only decreased vimentin expression and enhanced E-cadherin expression, but inhibited UUO-induced collagen IV and α-SMA upregulation, indicating that it could mitigate EMT in a rat model of UUO-induced renal fibrosis. Discussion and conclusions This study suggested that PCA decreases TGF-β1-induced fibrosis and EMT in vitro and in vivo. These findings demonstrate pharmacological effects of PCA and might be a potential strategy for the prevention of organ fibrosis in clinics.

Protective effects of Empagliflozin against Transforming growth factor‐beta‐induced fibrosis in renal proximal tubular cells

BackgroundChronic kidney disease (CKD) is the condition where the kidneys are unable to filter and clean the blood of waste properly. Hypertension, obesity, and diabetes are the leading risk factors for developing CKD. Fibrosis is regarded as a final common pathway leading to CKD. Sodium‐glucose co‐transporter 2 (SGLT‐2) inhibitors have shown to be renoprotective in type 2 diabetes patients. However, the mechanisms that underlie the renoprotective effects of SGLT‐2 inhibitors are unclear. Our study investigated whether empagliflozin (an SGLT‐2 inhibitor) treatment inhibits transforming growth factor β (TGF‐β)‐mediated fibrosis and profibrotic signaling in rat renal proximal tubular cells (NRK‐52E).MethodsNRK‐52E cells were cultured and treated with TGF‐β with or without empagliflozin for 24 and 48 hours. The expression of fibrosis markers ‐ alpha‐smooth muscle actin (α‐SMA) and vimentin, autophagy marker – LC3II, and TGF‐b‐mediated profibrotic signaling marker ‐ SMAD 2 – were measured by western blotting.ResultsTGF‐β treatment (at 5 ng/mL and 10 ng/mL) for 24 and 48 hours induced fibrosis and activated autophagy signaling in renal proximal tubular cells. For instance, 48 h TGF‐β treatment at 10 ng/mL induced the expression of α‐SMA by 1.43‐fold and LC3II by 2.6‐fold as compared to vehicle‐treated cells. Co‐treatment with empagliflozin (100 and 500 nM) reduced the expression of the fibrotic markers (α‐SMA, vimentin), autophagy marker (LC3II) and TGF‐β‐mediated phosphorylation of SMAD‐2 in renal proximal tubular cells.ConclusionsOur preliminary findings indicate that TGF‐β‐mediated induction of fibrosis, autophagy, and profibrotic signaling could be attenuated by empagliflozin treatment. Further studies are required to understand the molecular mechanisms that underpin the antifibrotic effects of empagliflozin against TGF‐β‐induced renal fibrosis.

SsDNA functionalized nanodiamonds for uranium decorporation

The hunt for agents that are suitable for actinide decorporation to reduce the whole-body load of actinide in accidental internal exposure is the ever-lasting goal in radiation protection and medical treatment in nuclear emergency. All current decorporation agents can be categorized as two groups, one is the molecular ligands, and the other is the nanoparticles decorated with molecular ligands. Here in this work, functional nanodiamonds (fNDs) with ssDNA (the endogenous biomacromolecule rich in phosphate groups) loaded on the NDs is reported, which poses good uranyl adsorption selectivity, high cellular uptake, fast excretion, and effective decorporation of uranyl from rat renal proximal tubular epithelial cells (NRK-52E). All those results corroborate that fNDs can potentially serve as a brand new family of chelators for actinide decorporation.

Blood glucose control contributes to protein stability of Ski-related novel protein N in a rat model of diabetes.

Ski-related novel protein N (SnoN) negatively regulates the transforming growth factor-β1 (TGF-β1)/Smads signaling pathway and is present at a low level during diabetic nephropathy (DN), but its underlying regulatory mechanism is currently unknown. The present study aimed to assess the effects of insulin-controlled blood glucose on renal SnoN expression and fibrosis in rats with diabetes mellitus (DM). Streptozotocin-induced DM rats were treated with insulin glargine (INS group) following successful model establishment. Blood samples were collected and centrifuged for biochemical indexes and the kidneys were collected for morphological analysis. In vitro, rat renal proximal tubular epithelial cells were treated with high-glucose medium for 24 h and transferred to normal glucose medium for 24 h. The expression levels of TGF-β1, SnoN, Smad ubiquitin regulatory factor 2 (Smurf2), Arkadia, Smads, E-cadherin, α-smooth muscle actin and collagen III were assessed by western blotting and immunohistochemistry. The ubiquitylation of SnoN was detected by immunoprecipitation, and the expression levels of SnoN mRNA were evaluated by reverse transcription-quantitative PCR. The biochemical parameters and morphology indicated that renal fibrosis was notable in the DM group and mitigated in the INS group. Compared with the control group, TGF-β1, phosphor (p)-Smad2, p-Smad3, Smurf2 and Arkadia levels were enhanced in the DM group, and the levels of SnoN protein were decreased, whereas the levels of SnoN mRNA and ubiquitylation were increased in renal tissues. Notably, treatment with insulin reversed this trend. Furthermore, changing the glucose levels in the medium from high to normal glucose suppressed the epithelial-mesenchymal transition of NRK-52E cells by restoring the SnoN protein levels, and this phenomenon was impaired by the knockout of SnoN. SnoN protein levels were likely reduced through a mechanism enhanced by the ubiquitin proteasome system, which reversed the transcriptional activation of SnoN during DN progression. In addition, controlling blood glucose may delay DN fibrosis by rescuing the protein stability of SnoN.

Overexpression of MnSOD Protects against Cold Storage-Induced Mitochondrial Injury but Not against OMA1-Dependent OPA1 Proteolytic Processing in Rat Renal Proximal Tubular Cells.

Kidneys from deceased donors undergo cold storage (CS) preservation before transplantation. Although CS is a clinical necessity for extending organ quality preservation, CS causes mitochondrial and renal injury. Specifically, many studies, including our own, have shown that the triggering event of CS-induced renal injury is mitochondrial reactive oxygen species (mROS). Here, we explored the role of OMA1-depedent OPA1 proteolytic processing in rat kidney proximal tubular epithelial (NRK) cells in an in vitro model of renal CS (18 h), followed by rewarming (6 h) (CS + RW). The involvement of mROS was evaluated by stably overexpressing manganese superoxide dismutase (MnSOD), an essential mitochondrial antioxidant enzyme, in NRK cells. Western blots detected rapid OPA1 proteolytic processing and a decrease in ATP-dependent cell viability in NRK cells subjected to CS + RW compared to control cells. Small interfering RNA (siRNA) knockdown of OMA1 reduced proteolytic processing of OPA1, suggesting that OMA1 is responsible for OPA1 proteolytic processing during CS + RW-induced renal injury. Overexpression of MnSOD during CS + RW reduced cell death, mitochondrial respiratory dysfunction, and ATP-dependent cell viability, but it did not prevent OMA1-dependent OPA1 processing. These data show for the first time that OMA1 is responsible for proteolytically cleaving OPA1 in a redox-independent manner during renal cell CS.

Antagonist targeting miR‑106b‑5p attenuates acute renal injury by regulating renal function, apoptosis and autophagy via the upregulation of TCF4.

Acute renal injury (ARI) is a life-threatening condition and a main contributor to end-stage renal disease, which is mainly caused by ischemia-reperfusion (I/R). miR-106b-5p is a kidney function-related miRNA; however, whether miR-106b-5p regulates the progression of ARI remains unclear. The present study thus aimed to examine the effects of miR-106b-5p antagonist on the regulation of ARI progression. It was found that miR-106b-5p expression was upregulated in the renal tissue of rats with I/R-induced ARI and in NRK-52E rat renal proximal tubular epithelial cells subjected to hypoxia-reoxygenation (H/R). In vitro, H/R induction suppressed the proliferation, and promoted the apoptosis and autophagy of NRK-52E cells, whereas miR-106b-5p antagonist (inhibition of miR-106b-5p) promoted the proliferation, and attenuated the apoptosis and autophagy of NRK-52E cells under the H/R condition. Dual luciferase reporter gene assay validated that transcription factor 4 (TCF4) was a target of miR-106b-5p. It was further found that TCF4 overexpression promoted the proliferation, and inhibited the apoptosis and autophagy of NRK-52E cells subjected to H/R. Moreover, the effects of miR-106b-5p antagonist on NRK-52E cell proliferation, apoptosis and autophagy were mediated through the regulation of TCF4. In vivo, miR-106b-5p antagonist reduced the severity of renal injury, decreased cell proliferation in renal tissues and lowered the serum creatinine (Scr) and blood urea nitrogen (BUN) levels in the blood samples from rats with I/R-induced ARI. On the whole, the findings presented herein demonstrate that miR-106b-5p antagonist attenuates ARI by promoting the proliferation, and suppressing the apoptosis and autophagy of renal cells via upregulating TCF4.

Five new phenylpropanoid glycosides from the flowers of Paulownia fortunei and their antioxidant activities

• Five new phenylpropanoid glycosides were isolated from the flowers of Paulownia fortunei. • Their structures were elucidated based on spectroscopic data analyses. • Compound 1 , 3 , 5 exhibited excellent antioxidant activities on NRK52e cell compared with a positive control. In this study, we focused on the antioxidant activities in NRK52e cells, a rat renal proximal tubular cell line, of five new phenylpropanoid glycosides, paulowninosides B–F (Compounds 1 – 5 ), which were isolated from the n -BuOH soluble part of the 50 % acetone-H 2 O extract of the flowers of Paulownia fortunei. The structures of the compounds were determined by extensive spectroscopic analyses (UV and IR spectroscopy, HR-ESI-MS, 1D-, 2D-NMR). The levels of antioxidant activities of the compounds were determined by real-time cell analysis (RTCA) of NRK52e cells that underwent H 2 O 2 -induced oxidative injury. Compounds 1 , 3 and 5 exhibited excellent antioxidant activities compared with control group.

NMR and LCMS analytical platforms exhibited the nephroprotective effect of Clinacanthus nutans in cisplatin-induced nephrotoxicity in the in vitro condition

BackgroundClinacanthus nutans (C. nutans) Lind. locally known as Belalai Gajah or Sabah snake grass is a medicinal plant belonging to Acanthaceae family. In Asia, this plant is traditionally used for treating skin rashes, insects and snake bites, diabetes mellitus, fever and for diuretic effect. C. nutans has been reported to possess biological activities including anti-oxidant, anti-inflammation, anti-cancer, anti-diabetic and anti-viral activities.MethodsProton Nuclear Magnetic Resonance (1H NMR) and Liquid Chromatography Mass Spectroscopy (LCMS) coupled with multivariate data analysis were employed to characterize the metabolic variations of intracellular metabolites and the compositional changes of the corresponding culture media in rat renal proximal tubular cells (NRK-52E).ResultsNMR and LCMS analysis highlighted choline, creatine, phosphocholine, valine, acetic acid, phenylalanine, leucine, glutamic acid, threonine, uridine and proline as the main metabolites which differentiated the cisplatin-induced group of NRK-52E from control cells extract. The corresponding media exhibited lactic acid, glutamine, glutamic acid and glucose-1-phosphate as the varied metabolites. The altered pathways perturbed by cisplatin nephrotoxic on NRK-52E cells included changes in amino acid metabolism, lipid metabolism and glycolysis.ConclusionThe C. nutans aqueous extract (1000 μg/mL) exhibited the most potential nephroprotective effect against cisplatin toxicity on NRK-52E cell lines at 89% of viability. The protective effect could be seen through the changes of the metabolites such as choline, alanine and valine in the C. nutans pre-treated samples with those of the cisplatin-induced group.

Oligo-Fucoidan Improves Diabetes-Induced Renal Fibrosis via Activation of Sirt-1, GLP-1R, and Nrf2/HO-1: An In Vitro and In Vivo Study.

Fucoidan extracted from brown algae has multiple beneficial functions. In this study, we investigated the effects of low-molecular-weight fucoidan (oligo-FO) on renal fibrosis under in vitro and in vivo diabetic conditions, and its molecular mechanisms. Advanced glycation product (AGE)-stimulated rat renal proximal tubular epithelial cells (NRK-52E) and diabetic mice induced by high-fat diet and intraperitoneal injection of streptozotocin and nicotinamide were used. Oligo-FO treatment significantly inhibited anti-high mobility group box 1 (HMGB1)/RAGE/ anti-nuclear factor-kappa B (NF-κB)/transforming growth factor-β1 (TGF-β1)/TGF-β1R/Smad 2/3/fibronectin signaling pathway and HIF-1α activation in AGE-stimulated NRK-52E cells. Conversely, the expression and activity of Sirt-1; the levels of ubiquitin-specific peptidase 22 (USP22), p-AMPK, glucagon-like peptide-1 receptor (GLP-1R), and heme oxygenase-1 (HO-1); and Nrf2 activation were remarkably increased by oligo-FO in AGE-stimulated cells. However, the above effects of oligo-FO were greatly diminished by inhibiting Sirt-1, HO-1, or GLP-1R activity. Similar changes of these pro-fibrotic genes in the kidney and a marked attenuation of renal injury and dysfunction were observed in oligo-FO-treated diabetic mice. These findings indicated that the inhibitory effects of the oligo-FO on diabetes-evoked renal fibrosis are mediated by suppressing TGF-β1-activated pro-fibrogenic processes via Sirt-1, HO-1, and GLP-1R dependence. Collectively, fucoidan-containing foods or supplements may be potential agents for ameliorating renal diseases due to excessive fibrosis.

Nonpeptidal compounds from the insect Polyphaga plancyi and their biological evaluation

Five new nitrogen-containing compounds (1–3, 5, and 6), two compounds which was firstly isolated from natural origin (7 and 10), along with six known ones, were isolated from the ethanol extract of the whole bodies of Polyphaga plancyi. The structures of the new compounds including their absolute configurations at stereogenic centers were assigned on the basis of spectroscopic analyses and computational methods. Racemic 10 was separated by chiral HPLC. Biological activities of these isolates against extracellular matrix components in rat renal proximal tubular cells, EV71, COX-2, ROCK2, JAK3, and tuberculosis were evaluated. Importantly, 8 was found to be a selective Smad3 phosphorylation inhibitor.

Neolignans and Norlignans from Insect Medicine Polyphaga plancyi and Their Biological Activities

Ten neolignans or norlignans (1–10) including eight new compounds were isolated from the whole bodies of Polyphaga plancyi Bolivar. Their structures were identified by spectroscopic data. Compounds 3, 4, 8, and 9 are racemates indicated by chiral HPLC analysis. Chiral separation followed by ECD calculations allowed to clarify the absolute configurations of all the antipodes. All the new compounds were evaluated for their biological properties toward extracellular matrix in rat renal proximal tubular cells, human cancer cells (K562, A549, and Huh7), EV71, ROCK2, JAK3, DDR1, and coagulation.Graphic

Micheliolide Attenuates Lipopolysaccharide-Induced Inflammation by Modulating the mROS/NF-κB/NLRP3 Axis in Renal Tubular Epithelial Cells.

Chronic kidney disease is a common disease closely related to renal tubular inflammation and oxidative stress, and no effective treatment is available. Activation of the nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome is an important factor in renal inflammation, but the mechanism remains unclear. Micheliolide (MCL), which is derived from parthenolide, is a new compound with antioxidative and anti-inflammatory effects and has multiple roles in tumors and inflammatory diseases. In this study, we investigated the effect of MCL on lipopolysaccharide- (LPS-) induced inflammation in renal tubular cells and the related mechanism. We found that MCL significantly suppressed the LPS-induced NF-κB signaling and inflammatory expression of cytokines, such as tumor necrosis factor-α and monocyte chemoattractant protein-1 in a rat renal proximal tubular cell line (NRK-52E). MCL also prevented LPS- and adenosine triphosphate-induced NLRP3 inflammasome activation in vitro, as evidenced by the inhibition of NLRP3 expression, caspase-1 cleavage, and interleukin-1β and interleukin-18 maturation and secretion. Additionally, MCL inhibited the reduction of mitochondrial membrane potential and decreases the release of reactive oxygen species (ROS). Moreover, MCL can prevent NLRP3 inflammasome activation induced by rotenone, a well-known mitochondrial ROS (mROS) agonist, indicating that the mechanism of MCL's anti-inflammatory effect may be closely related to the mROS. In conclusion, our study indicates that MCL can inhibit LPS-induced renal inflammation through suppressing the mROS/NF-κB/NLRP3 axis in tubular epithelial cells.

Long noncoding RNA-GAS5 retards renal fibrosis through repressing miR-21 activity

Background/aimsLong noncoding RNA (lncRNA) is a large and diverse class of RNA molecules, and has received widespread attention for its role in the regulation of various biological processes, including stem cell transformation, neurological disease, and tumorigenesis. However, the role of lncRNA in renal fibrosis remains unclear. MethodsWe investigated the expression of lncRNA-GAS5 by quantitative real-time polymerase chain reaction (PCR) or fluorescence in situ hybridization in chronic kidney disease (CKD) by designing both in vivo and in vitro experiments. With over-expression of GAS5 or knockdown GAS5, miR-21 and its downstream target genes were tested using quantitative real-time PCR or western blots. Mutants of miR-21 were designed and transfected in cells. GAS5 in the plasma and urine of patients with CKD was measured by quantitative real-time PCR. ResultsIn normal rats, GAS5 was predominantly expressed in renal tubular epithelial cells. GAS5 induction was significantly reduced in obstructive kidneys at 7 days after unilateral ureteral obstruction. In vitro, GAS5 was inhibited in cultured normal rat renal proximal tubular cells (NRK-52E) after incubation with transforming growth factor β at 24 h. Ectopic over-expression of GAS5 repressed extracellular matrix (ECM) levels such as collagen type III and fibronectin 1. Conversely, knockdown GAS5 augmented ECM accumulation in NRK-52E cells. GAS5 suppressed miR-21 activity in a direct and mechanistic manner. It subsequently turned off the expression of miR-21 downstream target genes, matrix metallopeptidase 2 and 9, which resulted in excessive ECM synthesis and deposition. Of note, plasma GAS5 was positively correlated with estimated glomerular filtration rate levels in CKD patients with different etiologies while urine GAS5 was negatively correlated. ConclusionActivation of lncRNA-GAS5 attenuates kidney fibrosis by modulating miR-21 activity and may serve as a surrogate biomarker in monitoring CKD progression.

Alleviation of TGF-β1 induced tubular epithelial-mesenchymal transition via the δ-opioid receptor.

Renal fibrosis is a common pathological feature of progressive chronic kidney disease (CKD). It is indicated that transforming growth factor-β1 (TGF-β1) plays as a central mediator in renal fibrosis. The present study aimed to investigate the role of δ-opioid receptor (DOR) on renal fibrosis of the rat renal proximal tubular epithelial cell line (NRK-52E) induced by TGF-β1 and to elucidate its underlying mechanism, as well as its involvement in signaling pathways. Cells were treated with TGF-β1 (10ng·mL-1 ), along with a specific DOR agonist (UFP-512) or naltrindole (a DOR antagonist). Cell viability and morphology, as well as cell migration, were measured after drug administration. Western blotting was employed to examine the extracellular matrix (ECM) protein Fibronectin, and the tubular epithelial-mesenchymal transition (EMT) markers (E-cadherin and α-smooth muscle actin (α-SMA)), signal transducer (p-Smad3), and EMT-regulatory gene (Snail). The expression level of phosphorylated Akt and p38 was also examined. Our results showed that TGF-β1 induced fibroblastic appearance and increased the expression of Fibronectin, α-SMA, P-Smad3, and Snail, while it decreased the expression of E-cadherin in NRK-52E cells. Moreover, TGF-β1 induced the activation of Akt and p38 MAPK signaling pathways. DOR activation was found to efficiently block morphological changes and cell migration, as long as the expression changes of Fibronectin, E-cadherin, α-SMA, P-Smad3, Snail, P-Akt, and P-p38 were induced by TGF-β1. These findings suggest that DOR may serve as an antifibrotic factor for renal proximal tubule cells by inhibiting the fibrosis process via TGF-β/Smad, Akt, and p38 MAPK signaling pathways.

Sulfur and nitrogen-containing compounds from the whole bodies of Blaps japanensis

Pipajiains H–J (1–3), three new phenolic derivatives with an unusual sulfone group, pipajiamides A–C (4–6), three new amide derivatives, pipajiaine A (7), one new imidazole analogue, and pipajiaine B (8), a pair of new pyrrolidine derivatives, along with three known compounds were isolated from the insect Blaps japanensis. Their structures were identified by spectroscopic and computational methods. Chiral HPLC was used to separate the (−)- and (+)-antipodes of 4 and 8. Biological activities of all the new compounds against extracellular matrix in rat renal proximal tubular cells, human cancer cells (A549, Huh-7, and K562), COX-2, ROCK1, and JAK3 were evaluated. The results show that compounds 2, (+)-4, and (−)-4 are active against kidney fibrosis, whereas, compound 9 is active toward human cancer cells, inflammation, and JAK3 kinase.