hes1-protein--human and Diabetic-Nephropathies

Diabetic nephropathy (DN) is a predominant cause of end‑stage renal disease. The impairment of the autophagy of human renal tubular epithelial cells (HK‑2 cells) is involved in the pathogenic mechanisms of DN. Sirtuin (Sirt)3 regulates the scavenging of damaged organelles and maintains energy balance. The present study aimed to examine the protective effects of Sirt3 on HK‑2 cells stimulated by high glucose (HG). HK‑2 cells were cultured in normal glucose (NG), HG or hyperosmotic medium. The viability of the HK‑2 cells was detected using a Cell Counting Kit‑8 assay. The expression and localization of Sirt3 were detected via immunofluorescence. Following transfection with an overexpression plasmid, the expression levels of key components in the Notch homolog 1 (Notch‑1)/hairy and enhancer of split‑1 (Hes‑1) pathway and those of the autophagy‑related proteins, Beclin‑1, LC‑3II and p62, were measured by western blot analysis and reverse transcription‑quantitative PCR (RT‑qPCR). As the Notch‑1/Hes‑1 pathway was inhibited, the expression levels of Beclin‑1, LC‑3II and p62 were also examined at transcriptional and translational level. It was found that prolonged culture in HG medium markedly reduced cell viability compared with the cells cultured in NG or in NG + mannitol, an effect that was aggravated with the increasing duration of culture. HG was capable of inhibiting the expression levels of Beclin‑1, LC‑3II and Sirt3, and upregulating p62 and the Notch‑1/Hes‑1 pathway, as verified by western blot analysis and RT‑qPCR. The results of immunofluorescence staining revealed that HG decreased Sirt3 expression. Sirt3 reversed the HG‑induced inhibition of the expression of Beclin‑1 and LC‑3II and the upregulation of p62. Moreover, Sirt3 reversed the HG‑induced inhibition of the Notch‑1/Hes‑1 signaling pathway. However, this autophagy‑promoting effect of Sirt3 was counteracted by the Notch‑1/Hes‑1 pathway activator. On the whole, the present study demonstrated that Sirt3 promoted the autophagy of HK‑2 cells, at least partly, via the downregulation of Notch‑1/Hes‑1.

Topics: Autophagy; Autophagy-Related Proteins; Beclin-1; Cell Line; Cell Survival; Diabetic Nephropathies; Down-Regulation; Epithelial Cells; Humans; Receptor, Notch1; Signal Transduction; Sirtuin 3; Transcription Factor HES-1

This study was purposed to figure out the contribution of Numb-induced Notch signaling to the development of diabetic nephropathy (DN).. Two hundred and twenty six DN patients were included, and human glomerular endothelial cells (RGEC) were cultured. MSCV-Numb-IRES-GFP, MSCV-Notch1-IRES-GFP and MSCV-Hes1-IRES-GFP were transfected to construct the recombinant retroviral vectors.. The over-expressed Numb and Notch1, as well as the under-expressed Hes-1 were correlated with the undesirable prognosis of DN patients (P < 0.05). Within the cell lines transfection with si-Numb would cut down E-cadherin and CD31 expressions (P < 0.05), yet elevated α-SMA and vimentin expressions (P < 0.05). The apoptotic rate of si-Numb cell lines underperformed ones categorized into the hyperglucose group (P < 0.05), whereas the lowly-expressed Notch1 and Hes1 were observably associated with inhibited proliferation of myofibroblasts (P < 0.05). Addition of ADPT caused under-expressed α-SMA and vimentin, along with the over-expressed E-cadherin and CD31 (P < 0.05). Silencing of Notch1 and Hes1 reversed the epithelial-mesenchymal transition (EMT) process that was triggered by high glucose (P < 0.05).. Numb negatively regulated Notch signaling pathway in EMT of DN, implying that they had great potentials to serve as therapeutic targets or diagnostic biomarkers for DN.

Topics: Adult; Aged; Aged, 80 and over; Cells, Cultured; Diabetic Nephropathies; Epithelial-Mesenchymal Transition; Female; Gene Expression; Glucose; HEK293 Cells; Humans; Male; Membrane Proteins; Middle Aged; Nerve Tissue Proteins; Receptor, Notch1; Receptors, Notch; Signal Transduction; Transcription Factor HES-1

Several studies have provided compelling evidence implicating the Notch signalling pathway in diabetic nephropathy. Co-regulation of Notch signalling pathway genes with GREM1 has recently been demonstrated and several genes involved in the Notch pathway are differentially expressed in kidney biopsies from individuals with diabetic nephropathy. We assessed single-nucleotide polymorphisms (SNPs; n = 42) in four of these key genes (JAG1, HES1, NOTCH3 and ADAM10) for association with diabetic nephropathy using a case-control design.. Tag SNPs and potentially functional SNPs were genotyped using Sequenom or Taqman technologies in a total of 1371 individuals with type 1 diabetes (668 patients with nephropathy and 703 controls without nephropathy). Patients and controls were white and recruited from the UK and Ireland. Association analyses were performed using PLINK (http://pngu.mgh.harvard.edu/∼purcell/plink/) and haplotype frequencies in patients and controls were compared. Adjustment for multiple testing was performed by permutation testing.. In analyses stratified by centre, we identified six SNPs, rs8708 and rs11699674 (JAG1), rs10423702 and rs1548555 (NOTCH3), rs2054096 and rs8027998 (ADAM10) as being associated with diabetic nephropathy before, but not after, adjustment for multiple testing. Haplotype and subgroup analysis according to duration of diabetes also failed to find an association with diabetic nephropathy.. Our results suggest that common variants in JAG1, HES1, NOTCH3 and ADAM10 are not strongly associated with diabetic nephropathy in type 1 diabetes among white individuals. Our findings, however, cannot entirely exclude these genes from involvement in the pathogenesis of diabetic nephropathy.

Topics: ADAM Proteins; ADAM10 Protein; Adolescent; Adult; Amyloid Precursor Protein Secretases; Basic Helix-Loop-Helix Transcription Factors; Calcium-Binding Proteins; Child; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Epithelial-Mesenchymal Transition; Female; Genetic Predisposition to Disease; Genotype; Homeodomain Proteins; Humans; Intercellular Signaling Peptides and Proteins; Jagged-1 Protein; Male; Membrane Proteins; Polymorphism, Single Nucleotide; Receptor, Notch3; Receptors, Notch; Serrate-Jagged Proteins; Signal Transduction; Transcription Factor HES-1; Young Adult

The centrality of the transcriptional regulator Snail in epithelial-to-mesenchymal transformation (EMT), known to occur in models of diabetic nephropathy, has not been established. Transforming growth factor beta-1 (TGFbeta1) is induced in diabetic nephropathy and induces both Snail and EMT. Hypoxia inducible factors (HIFs) are known to induce Snail, independent of TGFbeta1. Notch induction is integral to Snail induction and EMT in tumour cells, but its role in the kidney is unknown. The present study was undertaken to determine the upstream regulators of Snail in the kidney in high glucose and hypoxic conditions. HK-2 cells were cultured in normoxic, hypoxic, high glucose and combined hypoxic/high glucose conditions. The expression of HIF1alpha, NotchIC, Snail, Lysyl oxidase-like 2 (Loxl2), and Hairy and Enhancer Split-1 (Hes1) were measured. We found that hypoxia increased HIF1alpha expression; however, concurrent exposure to high glucose blunted this effect. A similar pattern was observed in Lox12 expression, suggesting that Loxl2 was downstream of HIF1alpha, which was confirmed using siRNA techniques. Snail was upregulated by hypoxia and high glucose and in combination the effect was additive, suggesting independent upstream activation pathways by the two stimuli. Hes1 was upregulated by high glucose and to a lesser extent by hypoxia, but the effect of the combined stimuli was no greater than that observed with high glucose alone. NotchIC was downregulated by both hypoxia and high glucose, and in combination the effect was additive. Therefore, this study suggests that hypoxia and high glucose induce Snail expression through distinct pathways, independent of Notch signalling.

Topics: Amino Acid Oxidoreductases; Basic Helix-Loop-Helix Transcription Factors; Cell Line; Diabetic Nephropathies; Epithelial-Mesenchymal Transition; Homeodomain Proteins; Humans; Hyperglycemia; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Kidney Tubules, Proximal; Receptor, Notch1; RNA, Small Interfering; Signal Transduction; Snail Family Transcription Factors; Transcription Factor HES-1; Transcription Factors; Up-Regulation