| Diabetic nephropathy (DN) is micro-vascular complications of diabetic mellitus, and it is major cause of end stage of renal disease (ESRD). There are so many factors which led to increasing process of diabetic nephropathy, such as abnormal lipid metabolism, oxygen stress, disorder of renin-angiotensin system, inflammatory reaction, hemodynamic changes and gene polymorphism. The interference of the factors cannot block the development of diabetic nephropathy. It is important to explore the pathogenesis of diabetic nephropathy, finding new drug targets.Arrestins constitute a four-member family, Arrestinl (visual arrestin) and arrestin4 (cone arrestin) have restricted expression patterns and localized primarily in visual sensory tissue. β-arrestin-1 and β-arrestin-2 are extensively expressed in heart, brain, pituitary, spleen, liver, kidney, pancreas. The amino acid sequences of the two (3-arrestin isoforms are 78% identical. Structurally, these sequences comprise two reverse parallel (3 sandwiches, connected with a-helix through hinge. There are two domains in β-arrestins, N-terminal domain is responsible for the identification of active GPCRs, C-terminal domain is the identification of the second receptor. Several studies have suggested that β-arrestins are well known for negatively regulating G-protein-coupled receptors (GPCRs) signaling and that they participate in receptor desensitization and internalization.In recent decades, studies have shown that β-arrestins active many signaling pathways, such as proliferation, apoptosis, and immune response as scaffolding proteins. Many studies found β-arrestins are related to various diseases: cardiovascular disease, respiratory diseases, endocrine diseases, nerve system diseases, immune system diseases and tumor occurrence and development process. But the role of β-arrestins in diabetic nephropathy have not been published.Podocyte, renal capsule epithelial cell, attached to the outer edge of the glomerular basement membrane (GBM). Fenestrated glomerular endothelium, glomerular basement membrane and podocytes constitute filtration barrier. Podocyte damage happens in the early stage of diabetic nephropathy, podocyte apoptosis lead to glomerular filtration barrier damage, caused proteinuria, then lead to ESRD. There are multiple signaling pathways involved in podocyte injury in DN, the down regulation of autophagy in podocyte has an important role in kidney damage. But the process and mechanism of autophagy in the pathogenesis of DN is unclear. It is important to discuss the role of autophagy regulation in diabetic nephropathy.Objective1. Determine the expression pattern of β-arrestins and the role of them in diabetic nephropathy.2. Clarify the mechanism of P-arrestins in podocyte injury.3. Discuss the machenism of β-arrestins in the regulation of podocyte autophagy.MethodsPart 1:The expression and the role of β-arrestin-1/2 in diabetic nephropathy1.1 Tested the expression of β-arrestin-1 and β-arrestin-2 in human renal biopsy samples The renal section samples, including normal control, diabetic mellitus non-nephropathy (DM-NN) samples and diabetic nephropathy samples were collected. Immunohistochemical (IHC) and Real Time RT-PCR tested the protein and mRNA levels of β-arrestin-1 and P-arrestin-2, analysed the correlation of renal injury and the expression of P-arrestins.1.2 Animal studies1.2.1 The expression of β-arrestins in STZ-induced diabetic nephropathy Male C57BL/6J mice were randomized in two groups. To speed up the DN model, mice were uninephrectomized before intraperitoneal injection of streptozotocin (100 mg/kg body weight for three consecutive days). Fast blood glucose (>16mmol/l) and 24h urinary protein excretion were monitored to evaluate whether the diabetic nephropathy (DN) models were successful. Test the expression of β-arrestin-1/2 in renal cotex by real time RT-PCR and western blot (WB), which was further confirmed in paraffin-embedded sections of kidney tissues by in situ hybridization or immunohistochemical staining. Immunofluorescent to confirmed that both β-arrestin-1 and β-arrestin-2 expression levels in podocytes from DN mice. Then western blot tested the expression change of autophagy related proteins.1.2.2 Expression pattern of β-arrestin-1/2 in db/db miceThe expression of P-arrestin-1/2 in renal cotex of db/db mice were tested by WB.1.2.3 Changes of renal function in Arrbl and Arrb2 gene knockout diabeticnephropathy miceMale C57BL/6J mice were randomized in two groups. To speed up the DN model, mice were uninephrectomized before intraperitoneal injection of STZ (The method to induce diabetic nephropathy like the wild type mice).At the end of the study, urine was collected for 24 hours in a metabolic cage and urinary albumin excretion was measured using a mouse albumin enzyme-linked immunosorbent assay (ELISA)quantitation kit. Simultaneously, mice were killed under ketamine anesthesia. The fixed kidneys were paraffin-embedded, and sections were prepared and stained with periodic acid-schiff stain (PAS), and transmission electron microscope (TEM) were used to assess kidney injury. The GBM thickness, foot process width and the number of foot processes per μm of GBM were calculated using Image J as described.1.3 Studies in vitro1.3.1 Expression of β-arrestin-1/2 in podocyte under common detrimental factorsHuman podocyte were cultured in vitro. Effects of different stimuli on the expression of β-arrestin-1/2 in podocytes, such as high glucose (HG), advanced glycation end-product (AGE), transforming growth factor-β1 (TGF-β1) were carried out by WB.Part 2:The mechanism of β-arrestin-1/2 regulate autophagy in podocyte2.1 Role of β-arrestins in regulation of autophagy level in podocytePodocytes were transfected with shRNA-β-arrestin-1/2 by Lipofectamine 3000; the expression level of β-arrestin-1 and β-arrestin-2 were detected by WB. Detect the expression of LC3 under HG stimulation in podocyte with silence of P-arrestin-1 and β-arrestin-2. The transmission electron microscopy (TEM) was used to detect the number of typical autophagosomes; RFP-GFP-LC3 adenovirus construct was used to monitor progression from the autophagosome to autolysosome in this study.2.2 Role of β-arrestins in podocyteviability via autophagy To examine the role of autophagy in podocytes, autophagy inhibition by gene silencing of ATG-3 or 3-methyladenine (3-MA), one autophagy inhibitor, was used in this study.Furthermore, we examined whether P-arrestins regulate podocyte viability via autophagy, autophagy enhancer rapamycin and inhibitors (Chloroquine and Bafilomycin A1)were used.2.3 The effect of β-arreestin-1/2 on the formation of PI3K core complexCo-Immunoprecipitation (Co-IP) was carried out to assess the interaction between β-arrestinl/2 and beclin-1 or VPS34; Co-IP was carried out to assess the interaction between beclin-1 and VPS34 under β-arrestinl/2 silence.2.4 β-Arrestin-1 and β-arrestin-2 regulated ATG12-ATG5 conjugation systemCo-IP was carried out to assess the interaction between β-arrestinl/2 and ATG7. Then WB detected the expression of ATG12-ATG5.ResultsPart 1:The expression and the role of β-arrestin-1/2 in diabetic nephropathy1.1 Human renal biopsy samples studies:Expression patterns of β-arrestinl/2 in human diabetic renal tissues.Upregulation of β-arrestin-1 and β-arrestin-2 was observed in paraffin-embedded sections of human diabetic renal tissues by IHC, but weak staining in the kidney from normal controls or diabetic patients without nephropathy (DM-NN). Real time RT-PCR analysis further confirmed the changes of mRNA levels of β-arrestin-1 and β-arrestin-2 in renal biopsies from DN subjects. We further found that both β-arrestin-1 (Spearman r=-0.7471, P<0.01) and β-arrestin-2 (Spearman r=-0.7845, P<0.01) mRNA levels were negatively correlated with estimated glomerular filtration rate (eGFR) in all available subjects individually。1.2 Animal studies1.2.1 Expression patterns of β-arrestinl/2 in streptozotocin-induceddiabetic miceReal time RT-PCR and WB analyses showed that both β-arrestin-1 and β-arrestin-2 were upregulated in the kidney from diabetic mice, which was further confirmed in paraffin-embedded sections of kidney tissues byin situ hybridization (ISH)and IHC.1.2.2 Expression patterns of β-arrestinl/2 in db/db miceCompared to the sample from db/+ mice, we observed the enhanced renal expressions of β-arrestin-1 and P-arrestin-2 in the db/db mice.1.2.3 Gene knockout of Arrbl or Arrb2 ameliorated renal injury in DN mice In vivo gene knockout of Arrbl or Arrb2 ameliorated renal injury as evidenced by reduced UACR. PAS and TEM shown renal injury is ameliorated by the knockout of Arrbl or Arrb2.1.2.4 Gene knockout of Arrbl or Arrb2 recover the expression of autophagy related proteinsThe ATG12-ATG5 and LC3ⅡI/LC3Ⅰ ratio decrease in DN. Gene knockout of Arrb1 or Arrb2 increased the basal levels of ATG12-ATG5 and LC3Ⅱ/LC3Ⅰ ratio in the kidney.1.3 Studies in vitro1.3.1 Expression of β-arrestinl/2 were upregulated in podocyte under HGImmunofluorescent results further confirmed the increase in podocyte P-arrestin-1 and (3-arrestin-2 expression in the kidney from STZ-induced diabetic mice in vivo. We found that HG, AGE, or TGF-β1 increased β-arrestin-1 and β-arrestin-2expression in a concentration dependent manner in podocytes.Part 2:The mechanism of β-arrestin-1/2 regulate autophagy in podocyte2.1 β-arrestinl/2 contributed to HG-inhibited basal autophagy in podocytesThe silence of shRNA-β-arrestin-1 and shRNA-p-arrestin-2 is 50% downregulated. The typical autophagosomes with double membranes weresignificantly increased in shRNA-β-arrestin-1 and shRNA-β-arrestin-2 transfected podocytes compared with scramble transfected podocytes under HG condition by TEM. Gene silencing of β-arrestin-1 and P-arrestin-2 restored the ratio of autophagy-related proteins LC3Ⅱ/LC3Ⅰ by WB analysis. We utilizedthe tandem RFP-GFP-LC3 adenovirusconstruct to further confirm autophagy induction byform punctate that represent autophagosome formation. After infection with the tandem construct, we evaluated the successful introduction of this adenovirus showing both fluorescent proteins. In addition to accumulation of LC3, there were morered and yellow puncta in shRNA-β-arrestin-1 and shRNA-p-arrestin-2 transfected podocytes than in control cells under HG condition.2.2 Gene silencing of Arrbl/2 reduced apoptosis in podocytes with HG treatmentThe apoptosis of podocytes is increase under the condition of the autophagy inhibitor 3-MA. Also when we silence ATG3 with shRNA-ATG3, the apoptosis of potocyte increased compared with scramble. So we know the autophagy can protect podocyte from apoptosis.To further investigate the role of β-arrestinl/2 on podocyte function, gene silencing of Arrbl or Arrb2 by shRNA-β-arrestin-1 and shRNA-β-arrestin-2 or overexpression of β-arrestin-1 and β-arrestin-2 by pCMV6-p-arrestin-1 and pCMV6-β-arrestin-2 transfection were used in this study. We found that HG-enhanced the apoptosis of podocyte was decreased by Arrbl and Arrb2 knockdown by flow cytometry.2.3 β-arrestin-1/2 had no effects on the formation of PI3K core complex in podocytes under HG condition.Co-Immunoprecipitation (Co-IP) assay showed that β-arrestin-1 interacted with VPS34 and beclin-1 and the interaction between VPS34 and β-arrestin-1 was significantly enhanced in podocytes with HG treatment. Similarly, an increased tendency was also observed for the interaction between VPS34 and β-arrestin-2. However, there was no interaction change between belcin-1 and VPS34 in podocytes with HG treatment as well as gene silencing of β-arrestin-1 and β-arrestin-2. These results indicated that neither β-arrestin-1 nor β-arrestin-2 had no effects on the formation of PI3K core complex under HG condition in podocytes.2.4 β-arrestin-1/2 negatively regulated ATG12-ATG5 conjugation system in podocytes with HG treatment.Co-IP assay showed that β-arrestin-1 interacted with ATG7 and the interaction between ATG7 and β-arrestin-1 was significantly enhanced in podocytes with HG treatment. Similarly to β-arrestin-2. Gene silencing of Arrbl and Arrb2 restored the levels of autophagy-related proteins ATG12-ATG5 and the ratio of LC3Ⅱ/LC3Ⅰ by WB analysis. Overexpression of β-arrestin-1 and β-arrestin-2, the levels of autophagy-related proteins ATG12-ATG5 and the ratio of LC3Ⅱ/LC3Ⅰ decreased by WB analysis.The enhanced interaction between P-arrestin-1/2 and ATG7 may block ATG7 to freely access and activate glycine residue of ATG12, thereby reducing ATG12-ATG5 conjugation and suppressing autophagy.Conclusion and innovation1. β-arrestin-1/2 play an important role in podocytes dysfunction in diabetic nephropathy. The apoptosis of podocyte increased with overexpression of β-arrestin-1/2 under HG condition, and the autophagy is inhibited. Knockout of Arrbl or Arrb2 alleviate renal damage of diabetic nephropathy.2. The machenism of β-arrestin-1/2 inhibit autophagy in podocyte:The enhanced interaction between P-arrestin-1/2 and ATG7 may block ATG7 to freely access and activate glycine residue of ATG12, thereby reducing ATG12-ATG5 conjugation and suppressing autophagy.3. In conclusion, this study for the first time explores the role of P-arrestins in DN indicating that β-arrestins are critical components of signal transduction pathways that link renal injury to reduced autophagy and increase apoptosis in diabetic nephropathy. Modulation of these pathways may be an innovative therapeutic strategy for treating patients with diabetic nephropathy. |
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