leupeptins and Diabetic-Nephropathies

Diabetic nephropathy (DN) is manifested as increased urinary protein level, decreased glomerular filtration rate, and final renal dysfunction. DN is the leading cause of end-stage renal disease worldwide and causes a huge societal healthcare burden. Since satisfied treatments are still limited, exploring new strategies for the treatment of this disease is urgently needed. Oxidative stress takes part in the initiation and development of DN. In addition, nuclear factor erythroid 2-related factor 2 (Nrf2) plays a key role in the cellular response to oxidative stress. Thus, activation of Nrf2 seems to be a new choice for the treatment of DN. In current review, we discussed and summarized the therapeutic effects of Nrf2 activation on DN from both basic and clinical studies.

Topics: Animals; Anticarcinogenic Agents; Antioxidant Response Elements; Antioxidants; Curcumin; Cysteine Proteinase Inhibitors; Diabetic Nephropathies; Enzyme Inhibitors; Humans; Isothiocyanates; Leupeptins; Molecular Targeted Therapy; NF-E2-Related Factor 2; Oxidative Stress; Resveratrol; Rutin; Signal Transduction; Stilbenes; Sulfoxides; Trace Elements; Zinc

Mesangial cells played a central role in the pathophysiology of diabetic nephropathy (DN). Our goal was to evaluate the molecular mechanism that regulates loss of BMP7 protein expression in DN. The mRNA and protein levels of BMP7 or UBE4A were detected using qRT-PCR and Western blot respectively. Mass spectrometry and co-immunoprecipitation were used to explore the E3 ligase which regulated BMP7 post-translationally. We initially confirmed that BMP7 protein, but not mRNA, is downregulated when cultured under high glucose mimicking DN conditions, which was rescued by MG-132 treatment. Proteomic analysis of NRK-52E cells ± MG-132 revealed a list of ubiquitin ligases associated with BMP7. Knockdown of the ubiquitin ligase UBE4A stabilized BMP7 expression in NRK-52E cells grown under high glucose conditions. Concurrent overexpression experiments confirmed that UBE4A is the ubiquitin ligase that degrades BMP7. Co-immunoprecipitation analysis confirmed that BMP7 and UBE4A interact. BMP7 expression in DN is regulated by post-translational mechanism.

Topics: Animals; Bone Morphogenetic Protein 7; Cells, Cultured; Cysteine Proteinase Inhibitors; Diabetic Nephropathies; Epithelial Cells; Gene Expression Regulation; Glucose; Kidney Tubules; Leupeptins; Rats; Sweetening Agents; Ubiquitin-Protein Ligases

Diabetic nephropathy (DN), the leading cause of end-stage renal disease (ESRD). To date, mounting evidence has shown that inflammation may contribute to the pathogenesis of DN. Recent reports have shown that proteasome inhibitors display cytoprotection by reducing the phosphorylation of Akt, a serine/threonine kinase, plays a critical role in cellular survival and metabolism and can crosstalk with inflammation. Therefore, we hypothesized that MG132, specific proteasome inhibitor, could provide renoprotection by suppressing Akt-mediated inflammation in DN. In vivo, male Sprague-Dawley rats were divided into normal control group (NC), diabetic nephropathy group (DN), DN model plus MG132 treatment group (MG132), and DN model plus deguelin treatment group (Deguelin)(deguelin, a specific inhibitor of Akt). In vitro, a human glomerular mesangial cell lines (HMCs) was exposed to 5.5 mmol/L glucose (CON), 30 mmol/L glucose (HG), 30 mmol/L glucose with 0.5 umol/L MG132 (MG132) and 30 mmol/L glucose with 5 umol/L deguelin (Deguelin). Compared with NC, DN showed a significant increase in the urinary protein excretion rate and inflammatory cytokines, as well as p-Akt. Compared with CON, HMCs co-cultured with HG was notably proliferated, which is in accord with α-smooth muscle actin (α-SMA) expression. These alterations were inhibited by administration of MG132 or deguelin. In conclusion, MG132 significantly inhibits the development of DN by regulating Akt phosphorylation-mediated inflammatory activation.

Topics: Animals; Cell Line; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Disease Models, Animal; Glomerular Mesangium; Glucose; Humans; Inflammation; Inflammation Mediators; Kidney; Leupeptins; Male; Mesangial Cells; Proteasome Inhibitors; Protective Agents; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Rotenone; Signal Transduction

Smad7 is the main negative regulatory protein in the transforming growth factor-β (TGF-β) downstream signaling pathway, which plays an important role in diabetic nephropathy (DN) and may be related to the ubiquitin proteasome pathway (UPP).. We investigated the role of UPP in regulating TGF-β/SMAD signaling and explored the therapeutic effect of the ubiquitin proteasome inhibitor MG132 on DN.. Wistar rats were randomly divided into a diabetes group and a normal control group. Rats in the diabetes group were injected intraperitoneally with streptozotocin. Diabetic rats were then randomly divided into a diabetic nephropathy group (DN group), an MG132 high concentration (MH) group, and an MG132 low concentration (ML) group. After 8 weeks of treatment, 24-hour urinary microalbumin (UAlb), urinary protein/urinary creatinine (Up/Ucr) values, ALT, AST, Bcr, kidney damage, TGF-β, Smad7, fibronectin (FN), and Smurf2 were detected.. The body mass and Smad7 protein expression decreased in DN group, but kidney weight, kidney weight index, UAlb, Up/Ucr, FN and Smurf2 mRNA expression, and TGF-β protein expression increased. However, these changes diminished following treatment with MG132, and a more pronounced effect was evident in MH group compared to ML group.. MG132 alleviates kidney damage by inhibiting Smad7 ubiquitin degradation and TGF-β activation in DN.

Topics: Albuminuria; Animals; Diabetic Nephropathies; Dose-Response Relationship, Drug; Fibronectins; Gene Expression Regulation; Kidney; Leupeptins; Male; Organ Size; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Proteolysis; Random Allocation; Rats; Rats, Wistar; Signal Transduction; Smad7 Protein; Streptozocin; Transforming Growth Factor beta1; Ubiquitin-Protein Ligases; Ubiquitination

Transforming growth factor-β (TGF-β) has been shown to be involved in diabetic nephropathy (DN). The SnoN protein can regulate TGF-β signaling through interaction with Smad proteins. Recent studies have shown that SnoN is mainly degraded by the ubiquitin-proteasome pathway. However, the role of SnoN in the regulation of TGF- β/Smad signaling in DN is still unclear. In this study, diabetic rats were randomly divided into a diabetic control group (DC group) and a proteasome inhibitor (MG132) diabetes therapy group (DT group). Kidney damage parameters and the expression of SnoN, Smurf2, and TGF-β were observed. Simultaneously, we cultured rat glomerular mesangial cells (GMCs) stimulated with high glucose, and SnoN and Arkadia expression were measured. Results demonstrated that 24-hour urine protein, ACR, BUN, and the expression of Smurf2 and TGF- β were significantly increased (P < 0.05), whereas SnoN was significantly decreased in the DC group (P < 0.05). However, these changes diminished after treatment with MG132. SnoN expression in GMCs decreased significantly (P < 0.05), but Arkadia expression gradually increased due to high glucose stimulation (P < 0.05), which could be almost completely reversed by MG132 (P < 0.05). The present results support the hypothesis that MG132 may alleviate kidney damage by inhibiting SnoN degradation and TGF-β activation, suggesting that the ubiquitin-proteasome pathway may become a new therapeutic target for DN.

Topics: Animals; Diabetic Nephropathies; Down-Regulation; Glucose; Kidney Function Tests; Kidney Glomerulus; Leupeptins; Male; Mesangial Cells; Nerve Tissue Proteins; Proteasome Inhibitors; Proteinuria; Proteolysis; Rats, Wistar; Streptozocin; Transcription Factors; Transforming Growth Factor beta

Oxidative stress is a major cause of diabetic nephropathy. Upregulation of the key antioxidative transcription factor, nuclear factor-erythroid 2-related factor 2 (Nrf2), was found to prevent the development of diabetic nephropathy. The present study was designed to explore the therapeutic effect of Nrf2 induced by proteasomal inhibitor MG132 at a low dose (10 μg/kg) on diabetic nephropathy. Transgenic type 1 diabetic (OVE26) mice displayed renal dysfunction with albuminuria by 3 mo of age, at which time MG132 treatment was started. After 3-mo treatment with MG132, renal function, morphology, and biochemical changes were examined with real-time PCR, Western blotting, and immunohistochemical examination. Compared with age-matched, nontreated diabetic mice, MG132-treated diabetic mice showed significant improvements in terms of renal structural and functional alterations. These therapeutic effects were associated with increased Nrf2 expression and transcriptional upregulation of Nrf2-regulated antioxidants. Mechanistic study using human renal tubular HK11 cells confirmed the role of Nrf2, as silencing the Nrf2 gene with its specific siRNA abolished MG132 prevention of high-glucose-induced profibrotic response. Furthermore, diabetes was found to significantly increase proteasomal activity in the kidney, an effect that was significantly attenuated by 3 mo of treatment with MG132. These results suggest that MG132 upregulates Nrf2 function via inhibition of diabetes-increased proteasomal activity, which can provide the basis for the therapeutic effect of MG132 on the kidney against diabetes-induced oxidative damage, inflammation, fibrosis, and eventual dysfunction.

Topics: Animals; Cells, Cultured; Cysteine Proteinase Inhibitors; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Female; Gene Expression Regulation; Kidney; Leupeptins; Mice; Mice, Transgenic; NF-E2-Related Factor 2; RNA, Small Interfering; Streptozocin; Transcriptional Activation

Oxidative stress may play an important role in the pathogenesis of diabetic nephropathy (DN). Recent studies have shown that the ubiquitin-proteasome pathway (UPP) and oxidative stress have interaction. We aimed to investigate whether inhibiting the proteasome has a preventive effect on DN through suppression of renal oxidative stress.. Male Sprague-Dawley rats were randomly divided into three groups: a normal control (NC) group, a streptozotocin-induced DN model group, and a DN plus MG132 (10 μg/kg) treatment group.. Increased 24-h urinary protein excretion rate (UPER) and renal pathological changes were all improved after MG132 administration. Furthermore, enhanced renal 26S proteasome activity and concentration in DN rats were effectively reduced after MG132 administration. Increased p47phox and nitrotyrosine (NT) expressions in kidneys of DN rats were decreased after MG132 treatment. Renal mRNA and protein expressions of NF-E2 related factor 2 (Nrf2) were up-regulated by MG132 in comparison to DN alone. Decreased renal mRNA expression of superoxide dismutase 1 (SOD1), catalase (CAT) and glutathione peroxidase (GPx) in DN rats was heightened after MG132 intervention. Depressed activities of renal SOD, CAT and GPx in DN rats were also improved by MG132 treatment. Increased renal nuclear factor κB (NF-κB) activity was inhibited after MG132 administration in DN rats at the end of 12 weeks.. Our present data suggest that inhibition of the proteasome by low-dose MG132 has a preventive effect on DN development and progression in rats through the up-regulation of antioxidant genes.

Topics: Animals; Antioxidants; Blotting, Western; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Electrophoretic Mobility Shift Assay; Enzyme-Linked Immunosorbent Assay; Kidney; Leupeptins; Male; Oxidative Stress; Protease Inhibitors; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Proteinuria; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction

Chronic hypoxia has been recognized as a key regulator in renal tubulointerstitial fibrosis, as seen in diabetic nephropathy, which is associated with the activation of hypoxia-inducible factor (HIF)-1α. We assess here the effects of the biguanide, metformin, on the expression of HIF-1α in diabetic nephropathy using renal proximal tubular cells and type 2 diabetic rats.. We explored the effects of metformin on the expression of HIF-1α using human renal proximal tubular epithelial cells (HRPTECs). Male Zucker diabetic fatty (ZDF; Gmi-fa/fa) rats were treated from 9 to 39 weeks with metformin (250 mg ⋅ kg(-1) ⋅ day(-1)) or insulin.. Metformin inhibited hypoxia-induced HIF-1α accumulation and the expression of HIF-1-targeted genes in HRPTECs. Although metformin activated the downstream pathways of AMP-activated protein kinase (AMPK), neither the AMPK activator, AICAR, nor the mTOR inhibitor, rapamycin, suppressed hypoxia-induced HIF-1α expression. In addition, knockdown of AMPK-α did not abolish the inhibitory effects of metformin on HIF-1α expression. The proteasome inhibitor, MG-132, completely eradicated the suppression of hypoxia-induced HIF-1α accumulation by metformin. The inhibitors of mitochondrial respiration similarly suppressed hypoxia-induced HIF-1α expression. Metformin significantly decreased ATP production and oxygen consumption rates, which subsequently led to increased cellular oxygen tension. Finally, metformin, but not insulin, attenuated tubular HIF-1α expression and pimonidazole staining and ameliorated tubular injury in ZDF rats.. Our data suggest that hypoxia-induced HIF-1α accumulation in diabetic nephropathy could be suppressed by the antidiabetes drug, metformin, through the repression of oxygen consumption.

Topics: Adenylate Kinase; Analysis of Variance; Animals; Cell Line; Cysteine Proteinase Inhibitors; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Epithelial Cells; Humans; Hypoglycemic Agents; Hypoxia-Inducible Factor 1, alpha Subunit; Immunohistochemistry; Insulin; Kidney Tubules, Proximal; Leupeptins; Male; Metformin; Oxygen Consumption; Rats; Rats, Zucker; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction

Cell loss by apoptosis occurs in renal injury such as diabetic nephropathy. TNF-alpha is a cytokine that induces apoptosis and has been implicated in the pathogenesis of diabetic nephropathy. The aim was to investigate whether C-peptide or insulin could modulate TNF-alpha-mediated cell death in opossum kidney proximal tubular cells and to examine the mechanism(s) of any effects observed. C-peptide and insulin protect against TNF-alpha-induced proximal tubular cell toxicity and apoptosis. Cell viability was analyzed by methylthiazoletetrazolium assay; cell viability was reduced to 60.8 +/- 2.7% of control after stimulation with 300 ng/ml TNF-alpha. Compromised cell viability was reversed by pretreatment with 5 nM C-peptide or 100 nM insulin. TNF-alpha-induced apoptosis was detected by DNA nick-end labeling and by measuring histone associated DNA fragments using ELISA. By ELISA assay, 300 ng/ml TNF-alpha increased apoptosis by 145.8 +/- 4.9% compared with controls, whereas 5 nM C-peptide and 100 nM insulin reduced apoptosis to 81.6 +/- 4.8 and 77.4 +/- 3.1% of control, respectively. The protective effects of C-peptide and insulin were associated with activation of NF-kappaB. Activation of NF-kappaB by C-peptide was pertussis toxin sensitive and dependent on activation of Galpha(i). Phosphatidylinositol 3-kinase but not extracellular signal regulated mitogen-activated protein kinase mediated C-peptide and insulin activation of NF-kappaB. The cytoprotective effects of both C-peptide and insulin were related to increased expression of TNF receptor-associated factor 2, the product of an NF-kappaB-dependent survival gene. These data suggest that C-peptide and/or insulin activation of NF-kappaB-regulated survival genes protects against TNF-alpha-induced renal tubular injury in diabetes. The data further support the concept of C-peptide as a peptide hormone in its own right and suggest a potential therapeutic role for C-peptide.

Topics: Animals; Apoptosis; C-Peptide; Cell Line; Diabetic Nephropathies; GTP-Binding Protein alpha Subunits, Gi-Go; Humans; Insulin; Kidney Tubules, Proximal; Leupeptins; NF-kappa B; Opossums; Signal Transduction; TNF Receptor-Associated Factor 2; Tumor Necrosis Factor-alpha