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The Expression and Ubiquitination of SnoN Protein in Rats with Early Diabetic Nephropathy

Author: YangCheng
Tutor: XuYong
School: Luzhou Medical College
Course: Internal Medicine
Keywords: Diabetic nephropathy Ubiquitin- proteasome pathway TGF-β/Smad path SnoN protein
CLC: R692.9
Type: Master's thesis
Year: 2011
Downloads: 77
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Abstract

Objective: Diabetic Nephropathy (Diabetic Nephropathy, DN) is one of the most common microvascular disease of diabetes (diabetic mellitus, DM), its pathogenesis is unclear, the pathological features of early glomerular hypertrophy, mesangial proliferative later stage glomeruli, renal interstitial fibrosis is characterized. The present study: transforming growth factor-β (TGF-p) is closely related to the change in renal fibrosis is one of the key cytokines caused DN incidence. Mesenchymal transition in renal tubular epithelial cells (Epithelial-Mesenchymal Transition, EMT), TGF-β/Smad signal pathway activation, extracellular matrix synthesis, decreased degradation induced extracellular matrix continue to gather, and eventually cause renal fibrosis . SnoN protein corepressor of transcription in the nucleus. The study found that SnoN interacts with TGF-β downstream Smad proteins inhibit the cellular effects of TGF-β signaling generated SnoN protein degradation regulation by ubiquitination. Due to the inhibition of the transcriptional activity mediated by SnoN occur in the nucleus, and happens just before the target gene transcription in TGF-β, determines the SnoN in regulation TGF-β/smad signal path have an important role. In this study, a single intraperitoneal injection of streptozotocin (STZ) to establish early type 1 diabetic nephropathy rat model, specific proteasome inhibitor MG132 intraperitoneal injection therapy as an intervention factors discussed early nephropathy in diabetic rats transcription co-suppression factor SnoN protein expression and ubiquitination, and to explore the feasibility of a proteasome inhibitor treatment of diabetic nephropathy. Methods: 1, the model and the grouping of diabetic rats: Male Wistar rats were 30, were randomly divided into diabetic model group (20) and normal control group (Normal control, NC group, 10). Model group, a single intraperitoneal injection of streptozotocin STZ (60mg/kg), the control group received an equal volume of citrate buffer, after modeling were randomly divided into two groups: diabetic control group (Diabetes control DC group) and diabetes MG132 treatment group (Diabetes therapy, DT group). DT group 0.1mg/kg.d intraperitoneal injection of the proteasome inhibitor MG132, DC group and NC group given daily volume of 0.9% saline by intraperitoneal injection. Detection of 24-hour urinary albumin and collected rats urine 2,6 and 8 weeks; heart blood test fasting glucose, cystatin C (CysC). Pathological examination of the kidney tissue: Clean renal, embedded in paraffin, HE and Masson staining observed glomerular and tubular interstitial pathological changes and fibrosis change the situation. 4, immunohistochemistry and Western blot (Western-blot) detection SnoN protein expression in the kidney tissue. Real-time fluorescence quantitative RT-PCR was used to detect the groups kidney tissue of SnoN the mRNA expression. 6 Statistical analysis: application SPSS13.0 statistical software analysis of data, data expressed as mean ± standard deviation (x ± s), one-way ANOVA analysis and detection group differences between the two groups were compared using q test independent samples t test comparison group differences, α = 0.05 test, P lt; 0.05 indicates differences statistically significant. Results: 1, rats basic situation: ① weight: Compared with NC group, model group, body weight significantly reduced the DT group weight than DC group increased; difference is more pronounced in the eight weeks of each group (P lt; 0.05). ② blood sugar: NC group blood sugar at normal levels fluctuations, model group, blood glucose compared with NC group was significantly higher (P lt; 0.05), blood glucose differences between DT group and DC group had no statistical significance (P gt; 0.05); each group of 8 weeks showed the same trend, with six weeks blood sugar between the two was no significant difference (P gt; 0.05). ③ cystatin C (of CysC): the differences between groups and between eight weeks and six weeks, there was no significant statistical significance (P gt; 0.05). ④ 24 hour urinary protein: Compared with NC group, model group microalbuminuria significant DC group, the difference was statistically significant (P lt; 0.05); groups eight weeks and six weeks showed the same trend between the two significant difference. 2, HE, Masson staining: ① HE staining Show: compared with the NC group, DC group glomerular volume increases, tubular swelling DT group than in the DC group glomerular volume decreases; eight weeks compared with six weeks of glomerular volume The increased tubular swelling increased, and six weeks showed the same trend. ② Masson staining: Compared with NC group, DC group showed an increase in renal interstitial blue-positive material deposited DT group than in the DC group reduce; eight weeks compared with six weeks of blue-positive material deposited increased, with six weeks showed the same trend. SnoN protein immunohistochemical expression of kidney tissue: normal kidney tissue of the NC group visible SnoN protein, compared with the NC group, DC group SnoN protein expression is reduced DT group than in the DC group increased expression. The expression of eight weeks compared to 6 weeks reduced, same expression tendency six weeks. 4, the wester-blot detection of renal tissue SnoN protein: SnoN protein electrophoresis with the analysis results do gray value SnoN protein expression NC group detected a high level DC group than in the NC group SnoN protein expression was significantly reduced, MG132 treatment of DT group compared with the DC group SnoN protein expression was increased (P lt; 0.05); each group 8 weeks were reduced protein expression than six weeks, and six weeks showed the same trend (P lt; 0.05). Kidney tissue SnoN mRNA expression: among the groups SnoN mRNA expression was no significant difference between eight weeks and six weeks showed no significant difference (P lt; 0.05). Conclusion: The early diabetic nephropathy SnoN protein ubiquitination and degradation was low expression. SnoN protein, proteasome inhibitors can inhibit diabetic nephropathy Pan biotinylated degradation of DN has a therapeutic effect by inhibiting the activation of TGF-P pathway.

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