HG-induced HK-2 Cells Research Articles

MiR-1297 attenuates high glucose-induced injury in HK-2 cells via targeting COL1A2.

In this study, we aimed to explore whether COL1A2 and miR-1297 participated in the progression of diabetic nephropathy (DN) in vitro and classified the underlying mechanisms. d-Glucose (30 mM; high glucose, HG)-stimulated HK-2 cells were used to mimic DN condition. RNA and non-coding RNA profiles were from Gene Expression Omnibus (GEO) database. The interaction between miR-1297 and COL1A2 was measured by dual-luciferase reporter assay. Gene Set Enrichment Analysis (GSEA) method was conducted to analyse COL1A2-associated signalling pathways. The role of miR-1297/COL1A2 in biological behaviours of HG-induced HK-2 cells were analysed by cell counting kit-8 and apoptosis assays. Bioinformatics analysis revealed that COL1A2 was up-regulated in DN tissues. We predicted and verified miR-1297 as the regulatory miRNA of COL1A2, and the expression of miR-1297 was decreased in DN tissues and HG-stimulated HK-2 cells. Overexpression of miR-1297 could promote cell proliferation and inhibit apoptosis to protect HK-2 cells from HG-induced damage. And knockdown of COL1A2 enhanced the protective effects of miR-1297 on HG-stimulated HK-2 cells. GSEA results revealed that several inflammatory pathways were enriched in COL1A2 high-expression group. Meanwhile, transfection of miR-1297 reduced the phosphorylation of NFκB and expression of three important pro-inflammatory genes including cytokine CCL5, adhesion molecules ICAM1 and VCAM1 via targeting COL1A2. These results suggested that miR-1297 protected HG-treated HK-2 cells probably through suppressing inflammation via targeting COL1A2. This study sheds a light on the role miR-1297/COL1A2 in DN progression and provides a novel promising therapy strategy for suppressing DN progression.

CircHIPK3 Alleviates High Glucose Toxicity to Human Renal Tubular Epithelial HK-2 Cells Through Regulation of miR-326/miR-487a-3p/SIRT1.

BackgroundThe intervention of circular RNA HIPK3 (circHIPK3) in diabetes has drawn increasing attention in recent years. However, the underlying mechanism of circHIPK3 in diabetic nephropathy (DN) has not been fully elucidated. Thus, the current study aims to investigate the role of circHIPK3 in high glucose (HG)-induced toxicity to human renal tubular epithelial HK-2 cells.MethodsThe expression of circHIPK3 in HK-2 cells induced by HG was determined by qRT-PCR and Western blot. The regulatory effects of circHIPK3 and miR-326/miR-487a-3p on cells proliferative and apoptosis were evaluated by CCK-8 and flow cytometry. Dual-luciferase reporter assay was applied to predict the target genes of miR-326 or miR-487a-3p.ResultsExpression level of circHIPK3 in HK-2 cells was remarkably decreased after the treatment of HG. The overexpression of circHIPK3 effectively reversed the HG-induced HK-2 cell proliferation inhibition and apoptosis. Furthermore, SIRT1 was confirmed to be the target gene of miR-326 and miR-487a-3p, which were showed to be the downstream genes of circHIPK3. The silencing of miR-326 or miR-487a-3p was also proved to induce proliferation and reduce apoptosis in HG-induced HK-2 cells.ConclusionOur data suggest that overexpression of circHIPK3 can attenuate the proliferation inhibition of HK-2 induced by HG and inhibit apoptosis through sponging miR-326 or miR-487a-3p to regulate SIRT1.

Interference of S100A16 suppresses lipid accumulation and inflammation in high glucose-induced HK-2 cells.

Diabetic nephropathy (DN) is a major complication of diabetic mellitus and usually leads to the end-stage renal disease. Inflammation-induced lipid disorders have been proposed to play an important role in the pathogenesis of DN. S100A16 is a novel adipogenic factor, but has not been investigated in DN. This study aims to explore the role of S100A16 in high glucose (HG)-induced HK-2 cells. CCK-8 assay was used to detect cell viability. Cell transfection was performed to knockdown S100A16. Oil red staining was performed to assay lipid accumulation. qRT-PCR and western blotting were conducted to examine corresponding gene expression. Intracellular cholesterol was determined by enzymatic assay. Inflammatory cytokines production was measured using ELISA kits. The results exhibited lipid accumulation and upregulation of S100A16 in HG-induced HK-2 cells. S100A16 knockdown significantly reduced lipid droplets and cholesterol, and decreased the production of inflammatory cytokines induced by HG. Besides, S100A16 knockdown decreased the expression of SCAP, SREBP1, SCD1 and SCAP. However, the inhibitory effect in HG-induced HK-2 cells made by S100A16 was reversed by SREBP1 overexpression. These results suggested that S100A16 knockdown might protect against HG-induced lipid accumulation and inflammation in HK-2 cells through regulating SCAP/SREBP1 signaling.

Downregulation of Salusin-β protects renal tubular epithelial cells against high glucose-induced inflammation, oxidative stress, apoptosis and lipid accumulation via suppressing miR-155-5p

ABSTRACT Diabetic nephropathy (DN) is the main contributor to the excess mortality for patients suffering from diabetes. Here, C57BL/6 mice received 4 weeks of high-fat diet and intraperitoneal injection of STZ (100 mg/kg). Mice with random blood glucose level ≥16.7 mmol/L and positive urine protein were recognized as successful DN model. To construct an in vitro model, HK-2 cells were incubated with 30 mM glucose. RT-qPCR and western blot were employed to measure Salusin-β levels in kidney tissues of DN mice and HG-induced HK-2 cells. Meanwhile, RT-qPCR was performed to detect miR-155-5p level in kidney tissues of DN mice and HG-induced HK-2 cells. TNF-α, IL-6, IL-1β, ROS, SOD and CAT levels were assessed using commercial assay kits. Furthermore, apoptosis of HK-2 cells was assessed via flow cytometric analysis and TUNEL staining. In addition, intracellular lipid accumulation and total cholesterol levels were detected using Oil red O staining and TC ELISA kit. Herein, Salusin-β and miR-155-5p levels were distinctly upregulated in kidney tissues of DN mice and HG-induced HK-2 cells. Downregulation of Salusin-β reduced miR-155-5p expression. Salusin-β silencing dramatically relieved inflammatory and oxidative injury, suppressed apoptosis as well as lipid accumulation induced by HG in HK-2 cells. Besides, miR-155-5p elevation partially abrogated the alleviating effects Salusin-β silencing on HG-induced RTEC injury. In summary, downregulation of Salusin-β protected HK-2 cells against HG-induced inflammation, oxidative stress, apoptosis and ameliorated lipid accumulation through suppressing miR-155-5p, which indicated that Salusin-β could be a potential therapeutic drug for DN.

Hsa‐miR‐199b‐3p Prevents the Epithelial‐Mesenchymal Transition and Dysfunction of the Renal Tubule by Regulating E‐cadherin through Targeting KDM6A in Diabetic Nephropathy

Diabetic nephropathy (DN) is the leading cause of end-stage renal disease. The association between epithelial-mesenchymal transition (EMT) and fibrosis is quite ascertained, but its link to eventual tubule dysfunction is missing. Here, we show that human microRNA- (hsa-miR-) 199b-3p protects renal tubules from diabetic-induced injury by repressing KDM6A, a histone lysine demethylase regulating E-cadherin expression. Lower E-cadherin expression is related to a higher level of KDM6A, while E-cadherin is promoted upon treatment with the KDM6A inhibitor GSK-J4 in both high glucose- (HG-) induced HK2 cells and the kidneys from streptozotocin- (STZ-) induced type 1 diabetic mice. However, overexpression or RNA silencing of E-cadherin fails to alter KDM6A expression. We also show that the upregulation of KDM6A is associated with the increased methylation level of the E-cadherin promoter. Then, the target prediction results and a dual-luciferase assay show that hsa-miR-199b-3p is a new miRNA that targets KDM6A. Overexpression of hsa-miR-199b-3p increases E-cadherin expression and prevents EMT through repressing KDM6A expression in HG-induced HK2 cells. In contrast, inhibitor-induced hsa-miR-199b-3p knockdown has opposite effects, as it decreases E-cadherin level and worsens EMT, accompanied by increased levels of KDM6A. Besides, Mir199b-knockout mice without mmu-miR-119b-3p expression exhibit more renal tubule dysfunction and more serious kidney tissue damage upon treatment with STZ. These results demonstrate that hsa-miR-199b-3p improves E-cadherin expression and prevents the progression of DN through targeting KDM6A. miR-199b-3p could be a future biomarker or target for the diagnosis or treatment of DN.

MiR-30c-5p/ROCK2 axis regulates cell proliferation, apoptosis and EMT via the PI3K/AKT signaling pathway in HG-induced HK-2 cells.

Diabetic nephropathy (DN) is one of the most common complications of diabetes mellitus. Increasing evidence suggests that microRNA-30c-5p (miR-30c-5p) participates in the pathogenesis of DN, but the mechanism has not been clearly understood. Therefore, this study aimed to investigate the biological role of miR-30c-5p in human DN progression in vitro. Compared with the controls, DN tissues and high glucose-induced HK-2 cells had significantly reduced miR-30c-5p levels, while ROCK2 expression was prominently elevated. Additionally, the miR-30c-5p mimic distinctly facilitated cell proliferation and blocked cell apoptosis and epithelial–mesenchymal transition (EMT). However, ROCK2 was a target gene of miR-30c-5p, and the effects of miR-30c-5p mimic on cell proliferation, apoptosis and EMT were reversed by ROCK2 upregulation in vitro. Furthermore, the pathogenesis of DN was regulated by the miR-30c-5p/ROCK2 axis via the PI3K/AKT pathway. MiR-30c-5p regulating cell proliferation, apoptosis and EMT through targeting ROCK2 via the PI3K/AKT pathway provides the novel potential target for clinical treatment of DN.

Interference of Hsa_circ_0003928 Alleviates High Glucose-Induced Cell Apoptosis and Inflammation in HK-2 Cells via miR-151-3p/Anxa2.

BackgroundDiabetic nephropathy (DN) is a severe end-stage kidney disease developed from diabetes mellitus. The involvement of circular RNA (circRNAs) in the regulation of DN pathogenesis has been implied, but the underlying mechanism of DN is still lacking. This study aimed to investigate the effect of hsa_circ_0003928 on the inflammation and apoptosis of high glucose (HG)-induced renal tubular cells.MethodsThe expression of hsa_circ_0003928, miR-151-3p and Anxa2 in blood samples from DN patients and healthy controls was detected by RT-qPCR. Human renal epithelial cells HK-2 were incubated with D-glucose (30 mmol/l) to establish DN model in vitro. RT-qPCR analysis confirmed the transfection effects and detected the expressions of TNF-α, IL-6 and IL-1β. Western blotting analysis determined the protein expression of Anxa2, Bcl-2, Bax, cleaved caspase-3 and caspase-3. The production of ROS was detected by DCF-DA method and production of inflammatory cytokines was verified by ELISA assay. CCK-8 assay and TUNEL assay were performed to determine cell viability and apoptosis, respectively. Dual-luciferase reporter assay was performed to confirm the relationship between miR-151-3p and hsa_circ_0003928 or Anxa2.ResultsHsa_circ_0003928 and Anxa2 mRNA levels were increased, whereas miR-151-3p was decreased in both HG-induced HK-2 cells and patients with DN. Hsa_circ_0003928 knockdown could decrease cell viability loss and apoptosis, increase Bcl-2 expression, and decrease Bax and cleaved caspase-3 expression. Besides, hsa_circ_0003928 knockdown suppressed HG-induced overproduction of ROS, TNF-α, IL-6 and IL-1β. However, the effects made by miR-151-3p inhibition were opposite to those made by hsa_circ_0003928 knockdown. Furthermore, the binding sites between miR-151-3p and hsa_circ_0003928 or Anxa2 were predicted and verified. Protein expression of Anxa2 was suppressed by hsa_circ_0003928 knockdown, which was rescued by miR-151-3p inhibition.ConclusionThese results demonstrated that hsa_circ_0003928 could act as a sponge of miR-151-3p and regulate HG-induced inflammation and apoptosis partly through regulating Anxa2.

Potential Repressive Impact of microRNA-20a on Renal Tubular Damage in Diabetic Kidney Disease by Targeting C-X-C Motif Chemokine Ligand 6

C-X-C Motif Chemokine Ligand 6 (CXCL6) is an important chemokine. We attempt in this investigation to explore its role and possible mechanism in diabetic kidney disease (DKD). By intergrating GEO data, CXCL6 expression in DKD patients and normal controls was exhibited. miRWalk website and luciferase reporter assay were used to predict and verify the upstream miRNA of CXCL6. CCK-8 assay and flow cytometry were performed to detect proliferation and apoptosis capacities. The levels of inflammatory key factors (TNF-α, IL-6 and IL-8) were measured using ELISA analysis. Expression of CXCL6, miR-20a, and JAK/STAT3 pathway-related markers were detected by qRT-PCR or western blot assays. CXCL6 was increased in DKD. miR-20a was identified as an upstream regulatory miRNA of CXCL6, and its expression was decreased in DKD and HG-treated HK-2cells. miR-20a overexpression facilitated the proliferation of HG-treated HK-2cells, whereas miR-20a depletion exhibited the opposite phenomenon. The levels of TNF-α, IL-6 and IL-8 were increased by HG treatment in HK-2cells. CXCL6 antagonized the promoting impacts of miR-20a mimics on HG-exposed HK-2cell proliferation. The suppressive effect of miR-20a overexpression on apoptosis and inflammatory response of HG-induced HK-2cell was rescued by CXCL6 enhancement. The protein expression of p-JAK and p-STAT3 were reduced by miR-20a mimic while facilitated by CXCL6 overexpression in HG-stimulated HK-2cells. These consequences hinted that miR-20a might exert a repressive impact on DKD, possibly through targeting CXCL6 and mediating JAK/STAT3 pathway, which offer new targets for DKD treatment.

Circ_WBSCR17 aggravates inflammatory responses and fibrosis by targeting miR-185-5p/SOX6 regulatory axis in high glucose-induced human kidney tubular cells

BackgroundDiabetic nephropathy (DN), a severe microvascular complication of diabetes, has complex pathogenesis. Circular RNAs (circRNAs) exert broad biological functions on human diseases. This study intended to explore the role and mechanism of circ_WBSCR17 in DN. MethodsDN mice models were constructed using streptozotocin injection, and DN cell models were assembled using high glucose (HG) treatment in human kidney 2 cells (HK-2). The expression of circ_WBSCR17, miR-185-5p and SRY-Box Transcription Factor 6 (SOX6) was detected by quantitative real-time polymerase chain reaction (qRT-PCR). The protein levels of SOX6 and fibrosis markers were examined by western blot. The release of inflammatory cytokines, cell proliferation and apoptosis, were assessed by enzyme-linked immunosorbent assay (ELISA), cell counting kit-8 (CCK-8) assay and flow cytometry assay, respectively. The predicted interaction between miR-185-5p and circ_WBSCR17 or SOX6 was verified by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. ResultCirc_WBSCR17 was highly expressed in DN mice models and HG-induced HK-2 cells. Circ_WBSCR17 knockdown or SOX6 knockdown promoted cell proliferation and blocked cell apoptosis, inflammatory responses and fibrosis, while circ_WBSCR17 overexpression or SOX6 overexpression conveyed the opposite effects. MiR-185-5p was a target of circ_WBSCR17 and directly bound to SOX6. MiR-185-5p could reverse the role of circ_WBSCR17 or SOX6. Moreover, the expression of SOX6 was modulated by circ_WBSCR17 through intermediating miR-185-5p. ConclusionCirc_WBSCR17 triggered the dysfunction of HG-induced HK-2 cells, including inflammatory responses and fibrosis, which was accomplished via the miR-185-5p/SOX6 regulatory axis.

Psoralen alleviates high glucose-induced HK-2 cell injury by inhibition of Smad 2 signaling via upregulation of microRNA 874

BackgroundDiabetic nephropathy (DN) causes the vast proportion of excess mortality for patients with diabetes. Novel therapeutic approaches slowing down its incidence is still lacking. Psoralen is the major active ingredient of Psoralea corylifolia Linn. (PCL), which was used to treat a number of diseases. In this study, we aimed to investigate whether psoralen could alleviate DN using in vitro model.MethodsCell viability assay and immunofluorescence were used to evaluate the effect of psoralen on high glucose (HG)-stimulated human kidney HK-2 cells (48 h). RT-qPCR was used to detect the expressions of miRNA in cells. Cell transfection, apoptosis assay, inflammatory cytokines detection and Western blot were further performed to explore the underlying molecular mechanisms.ResultsHG-induced toxicity of HK-2 cells was alleviated by psoralen. Meanwhile, the secretion of inflammatory cytokines and extracellular matrix (ECM) accumulation induced by HG in HK-2 cells were also decreased by psoralen. In addition, the expression of miR-874 in HK-2 cells was significantly upregulated by psoralen. Western blot assays indicated that psoralen could reverse HG-induced increase of TLR-4/NF-κB and Smad2 via upregulation of miR-874.ConclusionThis study demonstrated that psoralen could significantly alleviate HG-induced HK-2 cell injury via upregulation of miR-874. In addition, HG-induced increase of TLR-4/NF-κB and Smad2 was revered by psoralen. Therefore, psoralen might serve as an agent for the treatment of DN.

Up-regulation of miR-20a weakens inflammation and apoptosis in high-glucose-induced renal tubular cell mediating diabetic kidney disease by repressing CXCL8 expression

In our study, we determined the pattern of expression and biological roles of microRNA-20a (miR-20a) in diabetic kidney disease (DKD). The difference in the expression of miR-20a and proinflammatory genes (TNF-α, IL-6, and IL-1β) was measured across control, normal glucose (NG), and high glucose (HG) groups. Co-transfection miR-20a mimic and CXCL8 silence was used to assess the miR-20a/CXCL8 axis in the HG-induced HK-2 cell injury involved in DKD. miR-20a in HG group was significantly decreased, and a marked augmentation of inflammatory factor gene expression (TNF-α, IL-6, and IL-1β) in HK-2 cells was induced by HG. miR-20a over-expression enhanced cell proliferation, inhibited cell apoptosis, and suppressed the inflammatory response of HK-2 cells. CXCL8 knockdown strengthened the role of miR-20a. Our findings showed that miR-20a might be a significant regulator of HG-induced renal proximal tubular inflammatory injury mediating diabetic kidney disease through regulation of the expression of CXCL8 and the MEK/ERK pathway.

<p>Silencing of KCNQ1OT1 Decreases Oxidative Stress and Pyroptosis of Renal Tubular Epithelial Cells</p>

BackgroundLong noncoding RNAs (lncRNAs) can regulate the progression of DN. This research aimed to study the effect of lncRNA KCNQ1OT1 on the oxidative stress and pyroptosis of the renal tubular epithelial cells induced by high glucose (HG).MethodsRT-qPCR analysis detected the KCNQ1OT1 expression in serum with DN and HG-induced HK-2 cells, detect the expression of NLRP3, cleaved-caspase1, P-caspase1, IL-1β, p-IL-1β and GSDMD-N in HG-induced HK-2 cells, and confirm the transfection effects. The expression of NLRP3, cleaved-caspase1, P-caspase1, IL-1β, p-IL-1β and GSDMD-N in HG-induced HK-2 cells was also analyzed by Western blot analysis. ELISA assay detected the levels of TNF-α, IL-6 and MCP-1. The levels of ROS, MDA and SOD were determined by respective ELISA kits and ROS was also detected by the ROS assay kit (containing DCFH-DA).ResultsWe found that KCNQ1OT1 was increased in the plasma of patients with DN and HG-induced HK-2 cells and KCNQ1OT1 interference could decrease the inflammation, oxidative stress and pyroptosis of HG-induced HK-2 cells. In addition, KCNQ1OT1 directly targets miR-506-3p. MiR-506-3p was downregulated in the plasma of patients with DN and HG-induced HK-2 cells and KCNQ1OT1 interference promoted the expression of miR-506-3p. MiR-506-3p overexpression suppressed the inflammation, oxidative stress and pyroptosis of HG-induced HK-2 cells.ConclusionThis study demonstrated that downregulation of KCNQ1OT1 inhibited the inflammation, oxidative stress and pyroptosis of HG-induced HK-2 cells by up-regulating the expression of miR-506-3p, which provide new insights into the treatment of DN.

The Role of Akt2 in the Protective Effect of Fenofibrate against Diabetic Nephropathy.

Fenofibrate (FF) protects against diabetic nephropathy (DN) in type 1 diabetic (T1D) mice by upregulating the expression of fibroblast growth factor 21 (FGF21), leading to the activation of the Akt-mediated Nrf2 antioxidant pathways. Here, we examined which isoforms of Akt contribute to FF activation of FGF21-mediated renal protection by examining the phosphorylation and expression of three isoforms, Akt1, Akt2, and Akt3. T1D induced by a single intraperitoneal dose of streptozotocin (STZ) resulted in reduced phosphorylation of one isoform, Akt2, but FF treatment increased renal Akt2 phosphorylation in these and normal mice, suggesting a potential and specific role for renal Akt2 in FF protection against T1D. This was further confirmed using in vitro cultured HK-2 human kidney tubule cells exposed to high glucose (HG) with siRNA silencing of the Akt2 gene and STZ-induced diabetic Akt2-knockout mice with and without 3-month FF treatment. In normal HK-2 cells exposed to HG for 24 hours, FF completely prevented cell death, reduced total Akt expression and glycogen synthase kinase (GSK)-3β phosphorylation, increased nuclear accumulation of Fyn, and reduced nuclear Nrf2 levels. These positive effects of FF were partially abolished by silencing Akt2 expression. Similarly, FF abolished T1D-induced renal oxidative stress, inflammation, and renal dysfunction in wild-type mice, but was only partially effective in Akt2-KO mice. Furthermore, FF treatment stimulated phosphorylation of AMPKα, an important lipid metabolism mediator, which in parallel with Akt2 plays an important role in FF protection against HG-induced HK-2 cells oxidative stress and damage. These results suggest that FF protects against DN through FGF21 to activate both Akt2/GSK-3β/Fyn/Nrf2 antioxidants and the AMPK pathway. Therefore, FF could be repurposed for the prevention of DN in T1D patients.

Empagliflozin reduces high glucose-induced oxidative stress and miR-21-dependent TRAF3IP2 induction and RECK suppression, and inhibits human renal proximal tubular epithelial cell migration and epithelial-to-mesenchymal transition

Proximal tubular epithelial cells (PTEC) in the S1 segment of the kidney abundantly express sodium-glucose co-transporters (SGLT) that play a critical role in whole body glucose homeostasis. We recently reported suppression of RECK (Reversion Inducing Cysteine Rich Protein with Kazal Motifs), a membrane anchored endogenous MMP inhibitor and anti-fibrotic mediator, in the kidneys of db/db mice, a model of diabetic kidney disease (DKD), as well as in high glucose (HG) treated human kidney proximal tubule cells (HK−2). We further demonstrated that empagliflozin (EMPA), an SGLT2 inhibitor, reversed these effects. Little is known regarding the mechanisms underlying RECK suppression under hyperglycemic conditions, and its rescue by EMPA. Consistent with our previous studies, HG (25 mM) suppressed RECK expression in HK-2 cells. Further mechanistic investigations revealed that HG induced superoxide and hydrogen peroxide generation, oxidative stress-dependent TRAF3IP2 upregulation, NF-κB and p38 MAPK activation, inflammatory cytokine expression (IL-1β, IL-6, TNF-α, and MCP-1), miR-21 induction, MMP2 activation, and RECK suppression. Moreover, RECK gain-of-function inhibited HG-induced MMP2 activation and HK-2 cell migration. Similar to HG, advanced glycation end products (AGE) induced TRAF3IP2 and suppressed RECK, effects that were inhibited by EMPA. Importantly, EMPA treatment ameliorated all of these deleterious effects, and inhibited epithelial-to-mesenchymal transition (EMT) and HK-2 cell migration. Collectively, these findings indicate that hyperglycemia and associated AGE suppress RECK expression via oxidative stress/TRAF3IP2/NF-κB and p38 MAPK/miR-21 induction. Furthermore, these results suggest that interventions aimed at restoring RECK or inhibiting SGLT2 have the potential to treat kidney inflammatory response/fibrosis and nephropathy under chronic hyperglycemic conditions, such as DKD.

LncRNA GAS5/miR-452-5p Reduces Oxidative Stress and Pyroptosis of High-Glucose-Stimulated Renal Tubular Cells.

BackgroundDiabetic nephropathy (DN) is the leading cause of end-stage renal failure worldwide. lncRNAs are demonstrated to improve the DN by changing the expression of miRNAs. This study was aimed to investigate the effect of lncRNA GAS5/miR-452-5p on the inflammation, oxidative stress and pyroptosis of high-glucose-induced renal tubular cells.MethodsHK-2 cells were induced by HG to simulate DN cells. RT-qPCR analysis confirmed the transfection effects and detected the expression of GAS5, NLRP3, caspase1, IL-1β, pro-caspase1, pro-IL-1β, GSDMD-N and miR-452-5p. Western blot analysis determined the protein expression of NLRP3, caspase1, IL-1β, pro-caspase1, pro-IL-1β and GSDMD-N. The expression of GSDMD-N was also verified by immunofluorescence. The levels of TNF-α, IL-6, MCP-1, ROS, MDA and SOD were measured by commercial assay kits, respectively. Dual-luciferase reporter assay indicated that GAS5 could combine with miR-452-5p.ResultsGAS5 expression was decreased in HG-induced HK-2 cells. GAS5 overexpression could decrease the levels of TNF-α, IL-6, MCP-1, ROS and MDA and increase the levels of SOD. Moreover, GAS5 overexpression suppressed the expression of NLRP3, caspase1, IL-1β and GSDMD-N, and the results of immunofluorescence verified the above results. miR-452-5p interference could cause the same changes as GAS5 overexpression for HG-induced HK-2 cells, and GAS5 inhibition could reverse the effect of miR-452-5p interference.ConclusionGAS5 overexpression inhibited the inflammation, oxidative stress and pyroptosis of HG-induced renal tubular cells by downregulating the expression of miR-452-5p.

MiR-34b Alleviates High Glucose-Induced Inflammation and Apoptosis in Human HK-2 Cells via IL-6R/JAK2/STAT3 Signaling Pathway.

BackgroundIt is well established that inflammation and apoptosis of renal tubular epithelial cells caused by hyperglycemia contribute to the development of diabetic nephropathy (DN). Although microRNAs (miRNAs) are known to have roles in inflammation-related disorders, the exact role of miR-34b in DN has not been defined, and the regulatory mechanism has been unclear. This study aimed to clarify the role of miR-34b in DN pathogenesis.Material/MethodsExpression of miR-34b, IL-6R, and other key factors of inflammation, apoptosis (TNF-α, IL-1β, IL-6, caspase-3) in high glucose (HG)-induced HK-2 cells were measured by real-time PCR, Western blot, and flow cytometric cell apoptosis assays. We used luciferase reporter assay to detect the target of miR-34b. Moreover, the targeting gene of miR-34b and its downstream JAK2/STAT3 signaling pathway were explored.ResultsIt was demonstrated that miR-34b overexpression inhibited apoptosis and expression levels of TNF-α, IL-1β, IL-6, and caspase-3 in HG-treated HK-2 cells. We also found that IL-6R is a direct target of miR-34b, which could rescue inflammation and apoptosis in HG-treated HK-2 cells transfected with miR-34b mimic. Furthermore, we showed that overexpression of miR-34b inhibited the IL-6R/JAK2/STAT3 signaling pathway in HG-treated HK-2 cells.ConclusionsOur data suggest that overexpression of miR-34b improves inflammation and ameliorates apoptosis in HG-induced HK-2 cells via the IL-6R/JAK2/STAT3 pathway, indicating that miR-34b could be a promising therapeutic target in DN.

Long-chain non-coding RNA UCA1 inhibits renal tubular epithelial cell apoptosis by targeting microRNA-206 in diabetic nephropathy

In this study, the inhibitory effect of long-chain non-coding RNA urothelial carcinoma associated 1 (UCA1) on renal tubular epithelial cell apoptosis by targeting microRNA (miRNA)-206 in diabetic nephropathy (DN) was investigated through DN rat model. The results showed that UCA1 expression was significantly reduced in diabetic renal tubular epithelial tissues and HG-induced HK-2 cells. UCA1 significantly inhibited HG-induced apoptosis and inflammation of renal tubular epithelial cells in HK-2 cells. In addition, UCA1 can directly act as an anti-pro-cytokine by inhibiting the expression of miR-206, and finally inhibit the apoptosis and inflammation of renal tubular epithelial cells. We conclude that UCA1 inhibits renal tubular epithelial cell apoptosis by targeting miRNA-206 in DN and can be used as a potential therapeutic target for DN.

Cyclocarya paliurus triterpenic acids fraction attenuates kidney injury via AMPK-mTOR-regulated autophagy pathway in diabetic rats

BackgroudDiabetic nephropathy is the most serious complication of diabetes. Cyclocarya paliurus (CP), an herbal plant in China, has been reported the biological activity of anti-hyperglycemia. However, its effects on the diabetic nephropathy (DN) remain unclear. PurposeWe aimed to investigate the potential role of CP and its underlying mechanisms on DN. Study designIn this study, the effects of triterpenic acids-enriched fraction from CP (CPT) on DN was evaluated in streptozotocin (STZ)-induced rats and high glucose (HG)-induced HK-2 cells models. MethodsAfter oral administration with or without CPT for 10 weeks, body weight, glucose, microalbumin, serum creatinine and blood urea in STZ-induced rats were detected. Histological analysis was performed to evaluate renal function of mice. Moreover, the level of autophagy was detected by western blot or immunostaining. In vitro, HG-induced HK-2 cell was conducted to evaluate the renal protection and mechanism of CPT. ResultsCPT dramatically decreased the levels of microalbumin, serum creatinine and blood urea nitrogen and ameliorated increased mesangial matrix and glomerular fibrosis. In addition, we found the CPT prevented renal damage and cell apoptosis through the autophagy. Furthermore, CPT could increase the phosphorylation of AMPK and decrease its downstream effector phosphorylation of mTOR. Besides, the expression of LC3-II were locked by AMPK inhibitor dorsomorphin dihydrochloride (compound C), implying that the autophagy may be regulated with AMPK activation. ConclusionThese findings suggested that CPT might be a desired candidate against diabetes, potentially through AMPK-mTOR-regulated autophagy pathway.

Arbutin protects HK-2 cells against high glucose-induced apoptosis and autophagy by up-regulating microRNA-27a

Arbutin (ARB) has been widely used in skin pigmentation disorders. Nevertheless, the involvements of ARB in diabetic nephropathy (DN) are still unknown. We investigated the functions of ARB in high glucose (HG)-induced cell apoptosis and autophagy in HK-2 cells. Cell viability was examined through CCK-8 in HK-2 cells after disposal with 45 mM glucose and ARB (10-50 μM). Flow cytometry and western blot tested cell apoptosis and the related protein levels in HK-2 cells after 45 mM glucose and 50 μM ARB administration. RT-qPCR delved microRNA (miR)-27a expression in HG and ARB co-treated HK-2 cells. Effect of miR-27a on ARB affected cell apoptosis and autophagy was investigated after miR-27a inhibitor transfection. JNK and mTOR pathways were finally assessed by western blot. ARB alleviated HG-induced cell apoptosis, autophagy and regulated the related protein levels in HK-2 cells. MiR-27a expression was reduced in HG-treated cells, but was accelerated in HG and ARB co-treated HK-2 cells with the increased concentration. Inhibition of miR-27a apparently abolished the outcomes of ARB in HG-induced HK-2 cells apoptosis and autophagy. Besides, ARB blocked JNK and mTOR pathways by regulating miR-27a. The findings demonstrated that ARB alleviated apoptosis and autophagy in HG-treated HK-2 cells by regulating miR-27a/JNK/mTOR axis.

RETRACTED: Protective effect of DMDD, isolated from the root of Averrhoa carambola L., on high glucose induced EMT in HK-2 cells by inhibiting the TLR4-BAMBI-Smad2/3 signaling pathway

Hyperglycemia stimulated epithelial-mesenchymal transition (EMT) plays a critical role in initiating and progressing renal fibrosis in diabetic kidney disease (DKD). It is crucial to explore novel renal protective drugs for the treatment of DKD. The present study is to confirm our hypothesis and to accumulate the information for the application of DMDD (2-Dodecyl-6-methoxycyclohexa-2,5-diene-1,4-dione) as a novel therapeutic agent to potentially inhibit renal fibrogenesis and EMT in the DKD. High glucose induced renal proximal tubular epithelial cell line (HK-2 cells) was cultured and treated with DMDD. The cell viability and DMDD cytotoxicity were assessed by CCK8. Immunofluorescence was used for detection of TLR4 and downstream protein in normal and high glucose induced HK-2 cells. HK-2 cells were transfected with lentivirus codifying for BAMBI (BMP and activin membrane bound inhibitor) and interfering RNA for determination of the effect of BAMBI over-expression and silencing, respectively. TLR4-BAMBI-Smad2/3 pathway was analyzed by means of RT-PCR and western blot. A high concentration (60mM) of glucose induced significant EMT process and TLR4 expression was increased obviously in this circumstance. DMDD inhibited high expressions of TLR4 and Smad2/3 in HG induced cells and decreased the expression of BAMBI. In addition, the effects of decreased BAMBI expression and increased Smad2/3 expression in HG cultured cells were reversed in the cells of TAK-242 (TLR4 signaling inhibitor) intervention. BAMBI gene silencing dramatically increased EMT process and the over-expression of BAMBI was opposite in HK-2 cells with HG condition. These observations of EMT were ameliorated when the HK-2 cells were pre-treated with DMDD. Our study demonstrates that DMDD treatment improves EMT in the HG induced HK-2 cells. In addition, DMDD significantly inhibits EMT by TLR4-BAMBI-Smad2/3 pathway, which hints that DMDD may be an alternative approach in diabetic renal injury.