| Background:Diabetic kidney disease (DKD) is the most common microvascular complications of diabetes. Patients’renal function will progressive decline when accompanied with overt proteinuria. Due to the complexity of pathogenesis, more effective and safety treatments for DKD remain to be explored. As a medical practice based on syndrome differentiation and disease differentiation, Chinese medicine has many advantages in terms of DKD treatment. Previous clinical research showed that Tangshen Formula (TSF) provided a greater benefit in reducing urinary protein level, improving evaluated glomerular filtration rate (eGFR), increasing plasmic glutathione (GSH), and having renoprotective and antioxidant effect. It is now widely accepted that oxidative stress plays a key role in the development and progression of DKD. Reactive oxygen species (ROS) induces endothelial cells, mesangial cells, podocytes and tubulointerstitial injuries via activation of many signaling pathways, thereby exacerbates DKD. In recent years, myo-inositol oxygenase (MIOX), which mainly express in the renal tubular epithelial cells, received much attention due to its potential effects in the pathogenesis of DKD. Closely relate to oxidative stress, MIOX could regulate ROS generation and might be a new target for DKD treatment. In order to further observe and verify the long-term efficacy of TSF on DKD, and investigate its potential mechanisms, spontaneous type 2 DKD animal model, db/db mice will be employed. On this basis, the relationship between MIOX and oxidative stress will be explored in vitro. In addition, a multicenter, randomized, double-blind, placebo-controlled clinical trial investigating the efficacy and safety of TSF in type 2 DKD patients with overt proteinuria will be carried out, and oxidative stress biomarkers will be the secondary outcomes.Objectives:1. To investigate the renoprotective effect and mechanisms of TSF on db/db mice. Explore whether the mechanisms in part by suppressing oxidative stress, decreasing MIOX and improving podocyte injury.2. To explore the expression changes of MIOX in rat proximal tubular epithelial cell line (NRK52E) in high glucose ambience, and its role in oxidative stess in NRK52E.3. Since the multicenter, randomized, double-blind, placebo-controlled clinical trial is still ongoing, we summarized the critical process of quality control during the trial implemention.Methods:1. Study of renoprotective effect and mechanisms of TSF on db/db miceEight-week-old male C57BLKS/J db/m control and db/db mice were divided into 3 groups (db/m group, db/db group and db/db+TSFgroup), received TSF (2.4 g/kg-d) or distilled water for 12 weeks. General conditions were observed daily, body weights were measured weekly, blood glucose and 24h urine albumin levels were detected every four weeks. At week 12, mice were sacrificed after overnight fasting. Blood and tissues were collected for further analysis. Serum liver function indices, kidney function indices and lipid profiles were measured using automatic biochemical analyzer. Kidney tissues were separated and weighed. And part was prepared for histopathological analysis. Another part was isolated renal cortex for molecular biological detection. Paraffin sections were stained with periodic acid-Schiff (PAS), the degrees of glomerulosclerosis, defined as mesangial expansion were evaluated. Serum and 24h urine MDA levels were measured by TBA method. Western blot, Quantitative real-time PCR (qPCR) and immunohistochemistry methods were used to detected NADPH oxidase subunits, Nrf2 signaling, MIOX and podocyte biomarkers (nephrin, podocin and WT-1) expression.2. Role of MIOX in high glucose induced oxidative stress in renal tubular epithelial cellsUsing 5.5,10,20,30 mM glucose induced rat proximal tubular epithelial cells (NRK52E) for 48h,30 mM glucose induce NRK52E cells for 12,24,36,48 and 60h. Western blot method detected the expression changes of MIOX, Nox4 and Nrf2. Three siRNA Oligos targeting at MIOX gene sequence were designed and screened for higher efficiency of knocking down, and then were transfected into NRK52E cells for 6h. Thereafter cells were induced by 5.5 and 30 mM glucose for 48h. At last, cells were harvested to detect ROS generation, MIOX, Nox4, Nrf2 and NQO1 protein expression.3. Summarize the critical process of quality control during the multicenter, randomized, double-blind, placebo-controlled clinical trial.Results:1. TSF has renoprotective effect on db/db mice and the mechanism in part by lowering lipid profiles, suppressing oxidative stress and protecting podocyte injury.(1) Renoprotective effect:Compared with the db/m mice, the db/db mice showed higher body weights during the experiment (P<0.001), the db/db+TSF mice showed weight loss began at the sixth week (16 weeks old) and continued to the end of the study (P < 0.05). TSF intervention decreased db/db mice kidney weights (P< 0.01). During the intervention period, the blood glucose levels of the db/m mice maintained stable, while both in the db/db and db/db+TSF mice increased (P<0.01) with no difference between these two groups. Compared with the db/m mice, the AST and ALT levels of the db/db mice increased (P<0.01, P< 0.001), and TSF treatment could improve the abnormal liver function (P< 0.05, P<0.05). The contents of TC, FFA and LDL-C in the db/db mice were higher than that in the db/m mice (P< 0.001, P<0.05, P< 0.001), and in the db/db+TSF mice, the dyslipidemia were ameliorated (P<0.001, P<0.05, P<0.001). During the experiment, the db/db mice showed high levels of the 24h urine albumin than db/m mice (P< 0.001), the db/db+TSF mice showed declined trend from the eighth week (P<0.01), and more close to the db/m mice at week 12 (P<0.05). Morphologically, staining with PAS revealed that the db/db mice without treatment developed glomerulosclerosis and extracellular matrix deposition (P<0.01), in contrast, treatment with TSF significantly inhibited the histological damages (P<0.01).(2) Anti oxidative stress mechanism:Results from TBA assay showed that compared with the db/db mice, serum and 24h urine MDA increased in the db/db mice (P<0.01, P< 0.001), TSF intervention could reduce their levels (P<0.05, P<0.05). According to the results of qPCR, compared with the db/m mice, renal oxidative stress largely increased in the db/db mice, including a marked upregulation of NADPH oxidase (Nox2, Nox4, p22phox) mRNA levels (P< 0.01, P< 0.05, P< 0.01), TSF could downregulate their contents (P< 0.001, P< 0.05, P< 0.01). While for p47phox, there was no significant difference between the three groups (P> 0.05). Western blot and immunohistochemistry demonstrated that the protein expression of Nox2 and Nox4 were also increased in the db/db mice (P<0.05, P<0.001;P<0.05, P<0.001) and decreased by TSF significantly (P <0.05,P<0.001; P<0.05, P<0.001). For Nrf2 signaling, Nrf2 and NQO1 mRNA levels were increased in the db/db mice (P<0.05, P<0.01) and decreased in the db/db+TSF mice significantly (P<0.05, P<0.05). In contrast, HO-1 showed opposite changes (P<0.05, P < 0.05). Western blot and immunohistochemistry revealed that TSF could attenuate the increasing Nrf2 and NQO1 protein levels in the db/db mice (P<0.05, P<0.001; P<0.05, P< 0.001). Furthermore, Immunohistochemical results showed that MIOX express in proximal renal tubule. Western blot and immunohistochemistry indicated that compared with the db/m mice, the db/db mice showed higher MIOX protein expression (P<0.01, P< 0.001), TSF could reduce its level (P<0.05, P<0.001).(3) Podocyte protective mechanism:According to the results of qPCR, as compared with the db/m mice, the nephrin, podocin and WT-1 levels decreased in the db/db mice (P< 0.05, P<0.05, P<0.05), TSF could significantly improve their expression (P<0.01,P< 0.01, P< 0.05). Immunohistochemistry results also confirm that TSF could increase the expression of nephrin and podocin in the renal cortex of db/db mice (P<0.01, P<0.01).2. MIOX mediated high glucose induced oxidative stress in renal tubular epithelial cells(1) The effect of high glucose on MIOX expression:With the increasing concentration of glucose in the medium, the expression of MIOX and Nox4 increased, Nrf2 decreased. After NRK52E induced by 30 mM glucose for 0,12,24,36,48 and 60 hours, the expression of MIOX, Nox4 and Nrf2 increased at first and then decreased. Nrf2 reached to the peak at 12 hour, while for MIOX and Nox4, the peak was 48 hour.(2) The effect of MIOX knock down on oxidative stress:One pair of siRNA sequences targeting at MIOX showed 72.1%knockdown rate, and was transfected into NRK52E cells for 6 hours, followed by adding the final concentration of 30 mM glucose medium for another 48 hours. Increased ROS generation, MIOX, Nox4 and NQO1 expression were found in 30mM glucose cells, but Nrf2 reduced. While after MIOX gene was knocked down, NRK52E cells in 30 mM glucose ambience showed decreasing of ROS generation and downregulating of Nox4, NQO1, and Nrf2 showed further reduction.3. Subjects recruitment, data management, monitoring, and compliance of investigator and subjects were the key processes of quality control during the clinical trial.Conclusion:1. TSF can reduce the body weights, kidney weights, urinary protein excretion; ameliorate dyslipidemia and renal pathological damages in the db/db mice, which indicte that TSF has a renoprotective effect on type 2 DKD. And the mechanism in part by ameliorating dyslipidemia, suppressing oxidative stress via reducing the expression of NADPH oxidase and MIOX, protecting podocyte injuries.2. MIOX promote oxidative stress in renal tubular, the effect in part by increading the expression of Nox4, which provide a new target in exploring Chinese medicine intervention. |
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