Cultured NRK-52E Cells Research Articles

Ezetimibe ketone protects against renal ischemia-reperfusion injury and attenuates oxidative stress via activation of the Nrf2/HO-1 signaling pathway.

Recently, ezetimibe (EZM) has been suggested to be a potent Nrf2 activator that is important for preventing oxidative stress. Interestingly, we found that its metabolite ezetimibe ketone (EZM-K) also has antioxidant effects. Thus, we investigated the role of EZM-K in preventing renal ischemia‒reperfusion injury (RIRI). Cultured NRK-52E cells were subjected to simulated IR with or without EZM-K. Rats were used to simulate in vivo experiments. EZM-K alleviated H2O2-induced apoptosis and reactive oxygen species (ROS) and upregulated Nrf2 and HO-1 levels in NRK-52E cells. A HO-1 and a Nrf2 inhibitor reversed the protective effects of EZM-K. In the rat RIRI model, pretreatment with EZM-K activated the Nrf2/HO-1 signaling pathway, suppressed tubular injury and inflammation, and improved renal function. EZM-K significantly prevented renal injury caused by ischemia‒reperfusion via the Nrf2/HO-1 signaling axis both in vivo and in vitro. The other metabolite of EZM, ezetimibe glucuronide (EZM-G) had no protective effects against ROS in RIRI. EZM-G also had no antioxidant effects and could not activate Nrf2/HO-1 signal pathway. Our findings also indicated the therapeutic potential of EZM-K in preventing RIRI.

Effect of Orthosiphon stamineus Extract on HIF-1Α, Endothelin-1, and VEGFR-2 Gene Expression in NRK-52E Renal Tubular Cells Subjected to Glucotixicity

This study aimed to investigate the impact of Orthosiphon stamineus extract on gene expression in NRK-52E cells under conditions of glucotoxicity. Gene expression analysis using RT-PCR was conducted following exposure of cultured NRK-52E cells to glucotoxic conditions and various concentrations of Orthosiphon stamineus extract. The results revealed a dose-dependent decrease in HIF-1α, ET-1 and VEGFR-2 gene expression levels upon treatment with Orthosiphon stamineus extract. The diverse array of phytochemicals found within Orthosiphon demonstrates a significant impact on the biochemical pathways implicated in the pathogenesis of the disease. Additionally, molecular docking studies suggested the potential inhibition of Rho-kinase as a mechanistic explanation for this observed effect. These findings suggest that Orthosiphon stamineus extract may possess renoprotective properties against glucotoxicity-induced cellular damage. This study contributes to understanding potential therapeutic interventions for managing renal complications associated with conditions such as diabetes.

Effect of Borreria hispida Extract on SIRT1, HIF-1α, ET-1 and VEGFR-2 Gene Expression in NRK-52E Cells Subjected to Glucotoxicity

This study aimed to investigate the impact of Borreria hispida extract on gene expression in NRK-52E cells under conditions of glucotoxicity. Gene expression analysis was conducted using RT-PCR following the exposure of cultured NRK-52E cells to glucotoxic conditions and varying concentrations of Borreria hispida extract. The results demonstrated a dose-dependent increase in SIRT1 gene expression and a concomitant decrease in HIF-1λ, ET-1, and VEGFR-2 gene expressions upon treatment with Borreria hispida extract. Additionally, molecular docking studies suggested the potential inhibition of Rho-kinase as a mechanistic explanation for these effects. Borreria hispida extract may confer renoprotective benefits against glucotoxicity-induced cellular damage. The potential therapeutic utility of Borreria hispida extracts in managing renal complications associated with conditions such as diabetes. Furthermore, our docking studies shed light on the potential molecular mechanisms underlying the observed effects, suggesting interactions between phytochemicals present in Borreria hispida extract and the JNK-1 protein. These interactions may contribute to the augmentation of SIRT1 activity, further bolstering the extract's therapeutic utility in DKD.

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.

Targeting lysine-specific demethylase 1A inhibits renal epithelial-mesenchymal transition and attenuates renal fibrosis.

Lysine-specific histone demethylase 1 (LSD1) as the first identified histone/lysine demethylase regulates gene expression and protein functions in diverse diseases. In this study, we show that the expression of LSD1 is increased in mouse kidneys with unilateral ureteral obstruction (UUO) and in cultured NRK-52E cells undergoing TGF-β1-induced epithelial-mesenchymal transition (EMT). Inhibition of LSD1 with its specific inhibitor ORY1001 attenuated renal EMT and fibrosis, which was associated with decreased the deposition of extracellular matrix proteins and the expression of fibrotic markers, including α-smooth muscle actin (α-SMA) and fibronectin, and the recovery of E-cadherin expression and decrease of N-cadherin expression in UUO kidneys and in NRK-52E cells induced with TGF-β1. Targeting LSD1 also decreased the expression of Snail family transcriptional repressor 1 (Snail-1) and its interaction with LSD1 in UUO kidneys and in NRK-52E cells treated with TGF-β1. In addition, we identified a novel LSD1-14-3-3ζ-PKCα axis in the regulation of the activation of AKT and Stat3 and then the activation of fibroblasts. This study suggests that LSD1 plays a critical role in regulation of renal EMT and fibrosis through activation of diverse signaling pathways and places an emphasis that LSD1 has potential as a therapeutic target for the treatment of renal fibrosis.

MicroRNA-29b prevents renal fibrosis by attenuating renal tubular epithelial cell\u2013mesenchymal transition through targeting the PI3K/AKT pathway

PurposeThis 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.MethodsAdult 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).ResultsHistologic 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.ConclusionOur 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.

Fucoidan Ameliorates Renal Injury-Related Calcium-Phosphorus Metabolic Disorder and Bone Abnormality in the CKD-MBD Model Rats by Targeting FGF23-Klotho Signaling Axis.

Background: Recently, chronic kidney disease (CKD)-mineral and bone disorder (MBD) has become one of common complications occurring in CKD patients. Therefore, development of a new treatment for CKD–MBD is very important in the clinic. In China, Fucoidan (FPS), a natural compound of Laminaria japonica has been frequently used to improve renal dysfunction in CKD. However, it remains elusive whether FPS can ameliorate CKD–MBD. FGF23-Klotho signaling axis is reported to be useful for regulating mineral and bone metabolic disorder in CKD–MBD. This study thereby aimed to clarify therapeutic effects of FPS in the CKD–MBD model rats and its underlying mechanisms in vivo and in vitro, compared to Calcitriol (CTR). Methods: All male rats were divided into four groups: Sham, CKD–MBD, FPS and CTR. The CKD–MBD rat models were induced by adenine administration and uninephrectomy, and received either FPS or CTR or vehicle after induction of renal injury for 21 days. The changes in parameters related to renal dysfunction and renal tubulointerstitial damage, calcium-phosphorus metabolic disorder and bone lesion were analyzed, respectively. Furthermore, at sacrifice, the kidneys and bone were isolated for histomorphometry, immunohistochemistry and Western blot. In vitro, the murine NRK-52E cells were used to investigate regulative actions of FPS or CTR on FGF23-Klotho signaling axis, ERK1/2-SGK1-NHERF-1-NaPi-2a pathway and Klotho deficiency. Results: Using the modified CKD–MBD rat model and the cultured NRK-52E cells, we indicated that FPS and CTR alleviated renal dysfunction and renal tubulointerstitial damage, improved calcium-phosphorus metabolic disorder and bone lesion, and regulated FGF23-Klotho signaling axis and ERK1/2-SGK1-NHERF-1-NaPi-2a pathway in the kidney. In addition, using the shRNA-Klotho plasmid-transfected cells, we also detected, FPS accurately activated ERK1/2-SGK1-NHERF-1-NaPi-2a pathway through Klotho loss reversal. Conclusion: In this study, we emphatically demonstrated that FPS, a natural anti-renal dysfunction drug, similar to CTR, improves renal injury-related calcium-phosphorus metabolic disorder and bone abnormality in the CKD–MBD model rats. More importantly, we firstly found that beneficial effects in vivo and in vitro of FPS on phosphorus reabsorption are closely associated with regulation of FGF23-Klotho signaling axis and ERK1/2-SGK1-NHERF-1-NaPi-2a pathway in the kidney. This study provided pharmacological evidences that FPS directly contributes to the treatment of CKD–MBD.

Altered proximal tubule fatty acid utilization, mitophagy, fission and supercomplexes arrangement in experimental Fanconi syndrome are ameliorated by sulforaphane-induced mitochondrial biogenesis

The kidney proximal tubule function relies on oxidative phosphorylation (OXPHOS), thus mitochondrial dysfunction is characteristic of acute kidney injury (AKI). Maleic acid (MA) can induce an experimental model of Fanconi syndrome that is associated to oxidative stress and decreased oxygen consumption. Sulforaphane (SF) is an antioxidant known to protect against MA-induced AKI. The molecular basis by which SF maintains the bioenergetics in MA-induced AKI is not fully understood. To achieve it, rats were submitted to a protective scheme: SF (1 mg/kg/day i.p.) for four days and, at the fourth day, they received a single dose of MA (400 mg/kg i.p.), getting four main experimental groups: (1) control (CT), (2) MA-nephropathy (MA), (3) SF-protected and (4) SF-control (SF). Additionally, a similar protective schema was tested in cultured NRK-52E cells with different concentrations of SF and MA. In the animal model, SF prevented the MA-induced alterations: decrease in fatty acid-related oxygen consumption rate, OXPHOS capacity, mitochondrial membrane potential (Ψmt), and the activity of complex I (CI) as its monomeric and supercomplexes forms; the antioxidant also increased the activity of cytochrome c oxidase as well as mitochondrial biogenesis markers. Thus, SF prevented the MA-induced increase in fission, mitophagy and autophagy markers. In NRK-52E cells, we found that SF prevented the MA-induced cell death, increased mitochondrial mass and ameliorated the loss of Ψmt. We concluded that SF-induced biogenesis protects against mitochondrial dysfunction maintaining Ψmt, activities of mitochondrial complexes and supercomplexes, and prevents the extensive fission and mitophagy.

Fibroblast Growth Factor 2 Attenuates Renal Ischemia-Reperfusion Injury via Inhibition of Endoplasmic Reticulum Stress.

Acute kidney injury (AKI) is a serious clinical disease that is mainly caused by renal ischemia-reperfusion (I/R) injury, sepsis, and nephrotoxic drugs. The pathologic mechanism of AKI is very complex and may involve oxidative stress, inflammatory response, autophagy, apoptosis, and endoplasmic reticulum (ER) stress. The basic fibroblast growth factor (FGF2) is a canonic member of the FGF family that plays a crucial role in various cellular processes, including organ development, wound healing, and tissue regeneration. However, few studies have reported the potential therapeutic effect of FGF2 in the repair of renal ischemic injury in the past two decades. In the present study, we investigated the protective effect of FGF2 on renal I/R injury using Sprague-Dawley and NRK-52E cells. Our results showed that FGF2 significantly attenuates the apoptosis of kidney tissues after I/R injury through the inhibition of excessive ER stress. Moreover, FGF2 also alleviated the excessive ER stress and apoptosis in cultured NRK-52E cells injured by tert-Butyl hydroperoxide (TBHP). Significantly, phosphatidylinositol 3-kinase (PI3K)-selective inhibitor LY294002 and mitogen-activated protein kinase kinase (MEK)-selective inhibitor U0126 were utilized in the present study to examine the protective mechanism of FGF2. Our in vitro experimental results confirmed that both LY294002 and U0126 largely abolished the protective effect of FGF2. Taken together, the findings of the present study indicated that FGF2 attenuates I/R-induced renal epithelial apoptosis by suppressing excessive ER stress via the activation of the PI3K/AKT and MEK-ERK1/2 signaling pathways.

Contribution of TGF-Beta-Mediated NLRP3-HMGB1 Activation to Tubulointerstitial Fibrosis in Rat With Angiotensin II-Induced Chronic Kidney Disease.

Fibrosis is a common phenotype that often leads to the progression of blood pressure-induced chronic kidney disease (CKD). TGF-beta plays an important role in promoting pathogenesis, and NLRP3 is a critical mediator in the progression of blood pressure-induced CKD. However, the pathophysiological roles of the TGF-beta-mediated NLRP3 pathway in modulating fibrosis in blood pressure-induced CKD have not been elucidated. The present study aims to investigate the contribution of TGF-beta-mediated NLRP3 inflammasome to renal fibrosis in rats with high blood pressure. By treating rats with angiotensin II (Ang II) for 14 days, we observed the development of fibrosis, characterized by epithelial–mesenchymal transition (EMT) markers [alpha-smooth muscle actin (alpha-SMA), MMP-2, and MMP-9]. Immunohistochemical analysis further revealed that TGF-beta and NLRP3 inflammasome activation [high-mobility group box 1 (HMGB1), IL-1beta, and NLRP3] were significantly upregulated in the kidney of rats with Ang II-induced hypertension. Interestingly, we observed that Ang II could not increase the production of NLRP3 proteins, but TGF-beta could induce NLRP3 protein expression in cultured NRK-52E cells. Furthermore, we speculated that TGF-beta played a pathogenic role in Ang II-induced CKD because TGF-beta induced the activation of NLRP3 inflammasomes and Gasdermin D cleavage expression. We also proved that the pharmacological inhibition of NLRP3 by ISO caused a decrease in TGF-beta-induced NLRP3 inflammasome activation and the expression of EMT markers (alpha-SMA and CollagenI) and Gasdermin D cleavage. Collectively, these results suggest that TGF-beta-mediated NLRP3 inflammasome activation may cause the release of HMGB1 and an increase in Gasdermin D cleavage in NRK-52E, thereby contributing to renal fibrosis in Ang II-induced CKD. These findings provide novel insights into the pathogenic role of NLRP3 in CKD associated with high blood pressure.

FGF/FGFR2 Protects against Tubular Cell Death and Acute Kidney Injury Involving Erk1/2 Signaling Activation

Background: Fibroblast growth factors (FGFs) are heparin-binding proteins involved in a variety of biological processes, and part of them may act through binding with cell membrane receptor FGFR2. Objectives: To clarify the role and mechanisms of FGFR2 signaling in tubular cell survival and acute kidney injury (AKI). Method: In this study, kidney ischemia/reperfusion (IR) or cisplatin injection was used to induce AKI in mice. Results: In the kidneys after IR or cisplatin injection, the expression of FGFs and Erk1/2 phosphorylation were elevated. To investigate the role of FGFs in tubular cell survival and AKI, a mouse model with tubular cell specific FGFR2 gene disruption was generated. The knockouts were born normal. At 2 months of age, about one-third of the knockouts developed mild hydronephrosis. Ablation of FGFR2 in tubular cells aggravated acute kidney dysfunction as well as tubular cell apoptosis induced by IR or cisplatin. In addition, Erk1/2 phosphorylation was less in the knockout kidneys than in control littermates at day 1 after cisplatin injection. In cultured NRK-52E cells, recombinant FGF2 protein induced Erk1/2 phosphorylation and inhibited cisplatin-induced cell death. PD98059 abolished Erk1/2 phosphorylation and partly reversed the protective effect of FGF2 on cisplatin-induced cell death. Conclusions: This study indicates that FGF/FGFR2 signaling plays an important role in protecting against tubular cell death and AKI, which is partly through stimulating Erk1/2 activation.

Metabolic consequences of lactate dehydrogenase inhibition by oxamate in hyperglycemic proximal tubular cells

Diabetic kidney disease (DKD) is associated with altered metabolic patterns, leading to increased lactate production even in the presence of sufficient oxygen supply. Studies have shown hyperglycemia to be an important factor in determining development of DKD.Here we explore the metabolic consequences of lactate dehydrogenase (LDH) inhibition exerted by the LDH inhibitor, oxamate, in the isolated rat renal proximal tubular cells (NRK-52E) under hyperglycemic conditions.Cells treated with oxamate (100 mM) for 24 h, with or without high D-glucose (25 mM) load, were investigated with hyperpolarized [1-13C]pyruvate in a 1T NMR system. Respiratory measurements using an oxygen microsensor system was conducted.Oxamate treatment of cells with or without the presences of high D-glucose, reduced the lactate production/accumulation with 36.5% or 22.5% respectively. Reduced proliferation, hypertrophic effects, as well as elevated vascular endothelial growth factor (VEGF) expression in the NRK-52E cells were found. The increased glycolytic flux in high D-glucose cultured NRK-52E cells resulted in an upregulation of the cellular oxygen consumption rate upon treatment with oxamate.Our findings suggested that in vitro cultured NRK-52E cells exposed to hyperglycemic conditions, could redirect the glycolytic flux towards oxidative phosphorylation by LDH inhibition. This link between aerobic and anaerobic metabolism may be determined by the redox balance (NAD+/NADH ratio). In conclusion, hyperglycemic conditions and oxamate treatment alters the metabolic phenotype of NRK-52E cells towards increased oxygen utilization mediated by a decreased NAD+/NADH ratio, which in turn decreases cell proliferation/survival.

Ferulic Acid Protects Hyperglycemia-Induced Kidney Damage by Regulating Oxidative Insult, Inflammation and Autophagy.

Oxidative insult, inflammation, apoptosis and autophagy play a pivotal role in the etiology of diabetic nephropathy, a global health concern. Ferulic acid, a phytochemical, is reported to protect against varied diseased conditions. However, the ameliorative role and mechanisms of ferulic acid in averting STZ-mediated nephrotoxicity largely remains unknown. For in vivo study, a single intraperitoneal injection of streptozotocin (50 mg kg-1 body wt.) was administered in experimental rats to induce diabetes. The diabetic rats exhibited a rise in blood glucose level as well as kidney to body weight ratio, a decrease in serum insulin level, severe kidney tissue damage and dysfunction. Elevation of intracellular ROS level, altered mitochondrial membrane potential and cellular redox balance impairment shown the participation of oxidative stress in hyperglycemia-triggered renal injury. Treatment with ferulic acid (50 mg kg-1 body wt., orally for 8 weeks), post-diabetic induction, could markedly ameliorate kidney injury, renal cell apoptosis, inflammation and defective autophagy in the kidneys. The underlying mechanism for such protection involved the modulation of AGEs, MAPKs (p38, JNK, and ERK 1/2), NF-κB mediated inflammatory pathways, mitochondria-dependent and -independent apoptosis as well as autophagy induction. In cultured NRK-52E cells, ferulic acid (at an optimum dose of 75 μM) could counter excessive ROS generation, induce autophagy and inhibit apoptotic death of cells under high glucose environment. Blockade of autophagy could significantly eradicate the protective effect of ferulic acid in high glucose-mediated cell death. Together, the study confirmed that ferulic acid, exhibiting hypoglycemic, antioxidant, anti-inflammatory, anti-apoptotic activities and role in autophagy, could circumvent oxidative stress-mediated renal cell damage.

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.

Combined Ischemic Postconditioning and Ozone Postconditioning Provides Synergistic Protection Against Renal Ischemia and Reperfusion Injury Through Inhibiting Pyroptosis

To investigate whether ischemic postconditioning (IPO) and ozone postconditioning (OP) could synergistically attenuate renal ischemia-reperfusion (I/R) injury and its possible mechanism. An in vivo rat model of renal I/R injury was established, and the serum and kidneys were harvested after reperfusion to assess renal function and histologic changes. For the in vitro study, the cultured NRK-52E cells were subjected to 3 hours of hypoxia (5% CO2, 1% O2, and 94% N2) followed by 24 hours of reoxygenation (5% CO2, 21% O2, and 74% N2). The mRNA expression levels were analyzed by real-time polymerase chain reaction, and the protein expression levels were analyzed by using Western blot, immunofluorescence staining and enzyme-linked immunosorbent assay. Kidneys undergone I/R showed characteristic renal dysfunction and pyroptosis. IPO or OP could prevent the elevated blood urea nitrogen and creatinine, renal damage, as well as pyroptosis, however, the combined application of them had more obvious protection. Oxidative stress and pyroptosis were increased in hypoxia and reoxygenation (H/R) model using NRK-52E cells. The combination of hypoxic postconditioning and OP had more protective effects on oxidative abnormalities and pyroptosis compared with the single application of hypoxic postconditioning or OP. Our in vivo and in vitro studies show the combination of IPO and OP synergistically prote-cted the kidney from I/R by attenuating pyroptosis in kidney cells.

HDAC6 Inhibition Promotes Transcription Factor EB Activation and Is Protective in Experimental Kidney Disease.

To contend with the deleterious effects of accumulating misfolded protein aggregates or damaged organelles cells rely on a system of quality control processes, among them the autophagy-lysosome pathway. This pathway is itself controlled by a master regulator transcription factor termed transcription factor EB (TFEB). When TFEB localizes to the cell nucleus it promotes the expression of a number of genes involved in protein clearance. Here, we set out to determine (1) whether TFEB expression is altered in chronic kidney disease (CKD); (2) whether inhibition of the cytosolic deacetylase histone deacetylase 6 (HDAC6) affects TFEB acetylation and nuclear localization; and (3) whether HDAC6 inhibition, in turn, alters the natural history of experimental CKD. TFEB mRNA and protein levels were observed to be diminished in the kidneys of humans with diabetic kidney disease, accompanied by accumulation of the protein aggregate adaptor protein p62 in tubule epithelial cells. In cultured NRK-52E cells, HDAC6 inhibition with the small molecule inhibitor Tubastatin A acetylated TFEB, increasing TFEB localization to the nucleus and attenuating cell death. In a rat model of CKD, Tubastatin A prevented the accumulation of misfolded protein aggregates in tubule epithelial cells, attenuated proteinuria progression, limited tubule cell death and diminished tubulointerstitial collagenous matrix deposition. These findings point to the common occurrence of dysregulated quality control processes in CKD and they suggest that TFEB downregulation may contribute to tubule injury in CKD. They also identify a regulatory relationship between HDAC6 and TFEB. HDAC6 inhibitors and TFEB activators both warrant further investigation as treatments for CKD.

Anti-inflammatory effects of Lefty-1 in renal tubulointerstitial inflammation via regulation of the NF-κB pathway.

Renal tubulointerstitial inflammation has an important role in fibrosis, which is the main pathogenetic alteration associated with chronic kidney disease (CKD). The left-right determination factor 1 (Lefty-1) gene pleiotropically and biologically regulates transforming growth factor, mitogen-activated protein kinase and other signaling pathways, and is considered to have a potential anti-inflammatory function. However, its role in renal tubulointerstitial inflammation, which is often a long-term consequence of renal fibrosis, is currently unknown. In the present study, the effects of adenovirus-mediated overexpression of Lefty-1 (Ad-Lefty-1-flag) on renal tubulointerstitial inflammation were determined using a mouse model of unilateral ureteral obstruction (UUO) and a rat renal tubular duct epithelial cell line (NRK-52E), which was treated with lipopolysaccharide (LPS). In vivo results indicated that the inflammatory response was increased in UUO mice, as evidenced by the increase in inflammatory cytokines and chemokines. Conversely, Lefty-1 significantly reversed the effects of UUO. Furthermore, the results of the in vitro study demonstrated that Lefty-1 significantly inhibited LPS-induced inflammatory marker expression in cultured NRK-52E cells via the nuclear factor (NF)-κB signaling pathway. These results suggested that Lefty-1 may ameliorate renal tubulointerstitial inflammation by suppressing NF-κB signaling. In conclusion, the findings of the present study indicated that Lefty-1 may be considered a potential novel therapeutic agent for inhibiting renal tubulointerstitial inflammation or even reversing the CKD process.

Hyperhomocysteinemia Exacerbates Cisplatin-induced Acute Kidney Injury.

Hyperhomocysteinemia (HHcy) has been linked to several clinical manifestations including chronic kidney disease. However, it is not known whether HHcy has a role in the development of acute kidney injury (AKI). In the present study, we reported that HHcy mice developed more severe renal injury after cisplatin injection and ischemia-reperfusion injury shown as more severe renal tubular damage and higher serum creatinine. In response to cisplatin, HHcy mice showed more prevalent tubular cell apoptosis and decreased tubular cell proliferation. Mechanistically, a heightened ER stress and a reduced Akt activity were observed in kidney tissues of HHcy mice after cisplatin injection. Stimulating cultured NRK-52E cells with Hcy significantly increased the fraction of cells in G2/M phase and cell apoptosis together with decreased Akt kinase activity. Akt agonist IGF-1 rescued HHcy-induced cell cycle arrest and cell apoptosis. In conclusion, the present study provides evidence that HHcy increases the sensitivity and severity of AKI.

Synchronous Kikuchi-Fujimoto disease and gastric adenocarcinoma mimicking malignant lymphoma on 18F-FDG PET/CT

The Notch signaling pathway consists of several receptors and their ligands Delta and Jagged and is important for embryogenesis, cellular differentiation and proliferation. Activation of Notch receptors causes their cleavage yielding cytoplastic domains that translocate into the nucleus to induce target proteins such as the basic-loop-helix proteins Hes and Hey. Here we sought to clarify the significance of the Notch signaling pathway in acute kidney injury using a rat ischemia-reperfusion injury model and cultured NRK-52E cells. Analysis of the whole kidney after injury showed increased expression of Delta-1 and Hes-1 mRNA and protein along with processed Notch-2. Confocal microscopy, using specific antibodies, showed that Delta-1, cleaved Notch-2 and Hes-1 colocalized in the same segments of the injured renal proximal tubules. Recombinant Delta-1 significantly stimulated NRK-52E cell proliferation. Our study suggests that the Delta-1/Notch-2/Hes-1 signaling pathway may regulate the regeneration and proliferation of renal tubules during acute kidney injury.

CDK5 promotes renal tubulointerstitial fibrosis in diabetic nephropathy via ERK1/2/PPARγ pathway.

Cyclin-dependent kinase 5 (CDK5) has been documented in podocyte injuries in diabetic nephropathy (DN), however its role in renal tubular epithelial cells has not been elucidated. We report here that CDK5 is detrimental and promotes tubulointerstitial fibrosis (TIF) via the extracellular signal-regulated kinase 1/2 (ERK1/2)/peroxisome proliferator-activated receptor gamma (PPRAγ) pathway in DN. In high glucose cultured NRK52E cells, blocking CDK5 activity inhibited epithelial-to-mesenchymal transition (EMT) and fibrosis via ERK1/2/PPARγ pathway. In diabetic rats, CDK5 inhibitor roscovitine decreased renal fibrosis and improved renal function as demonstrated by a decrease in levels of blood urine nitrogen (BUN), serum creatinine and β2-microglobulin. Further studies revealed that improved renal fibrosis and function in diabetic rats were associated with inactivation of ERK1/2 and PPARγ signaling pathways. In late staged DN patients, the upregulation of CDK5 and p35 activated phosphorylated ERK1/2 and PPARγ, leading to decreased levels of E-cadherin but increased Vimentin and Collagen IV. Accordingly, renal fibrosis and function were worsened as revealed by decreased estimated glomerular filtration rate (eGFR) and increased serum BUN, creatinine, β2-microglobulin, 24-hour proteinuria and urine albumin to creatinine ratio (UACR). These findings demonstrate a novel mechanism that CDK5 increases tubulointerstitial fibrosis by activating the ERK1/2/PPARγ pathway and EMT in DN. CDK5 might have therapeutic potential in diabetic nephropathy.