Tripterygium glycoside suppresses epithelial­to­mesenchymal transition of diabetic kidney disease podocytes by targeting autophagy through the mTOR/Twist1 pathway.

Tripterygium glycoside (TG) is a traditional Chinese medicine extract with immunosuppressive, anti­inflammatory and anti­renal fibrosis effects. Epithelial­mesenchymal transition (EMT) and cell apoptosis are considered to be the major cause of podocyte injury in diabetic kidney disease (DKD). However, it remains unknown as to whether TG is able to alleviate podocyte injury to prevent DKD progression. Therefore, the present study aimed to clarify the podocyte protective effects of TG on DKD. TG, Twist1 small interfering RNA (siRNA) and Twist1 overexpression vector were added to DKD mouse serum­induced podocytes in vitro. Autophagic and EMT activities were evaluated by immunofluorescence staining and western blot analysis. Apoptotic activity was evaluated by Annexin V­FITC/PI flow cytometric analysis. The results revealed that after treatment with DKD mouse serum, autophagy was decreased, whereas EMT and apoptotic rate were increased, in podocytes. In addition, Twist1 expression was increased in DKD­induced podocytes. Furthermore, following Twist1­small interfering RNA transfection, the DKD­induced podocyte EMT and apoptotic rate were markedly reduced, indicating that Twist1 may be a promising therapeutic target for DKD. The present results also revealed that overexpression of Twist1 increased podocyte apoptosis, although this was decreased after TG treatment, indicating that TG may exhibit a protective effect on podocytes by inhibiting the Twist1 signaling pathway. After the addition of 3­benzyl­5­((2­nitrophenoxy) methyl)­dihydrofuran­2(3H)­one, an activator of mTORC1, the effects of TG on podocyte EMT, apoptosis and the autophagy were reversed. These findings indicated that TG may alleviate EMT and apoptosis by upregulating autophagy through the mTOR/Twist1 signaling pathway in DKD.

The association between time-mean serum uric acid levels and the incidence of chronic kidney disease in the general population: a retrospective study.

BACKGROUND: Investigations on the role of the time-mean serum uric acid (SUA) value in determining the risk of chronic kidney disease (CKD) are limited. We investigated whether the time-mean SUA value indicates the risk of CKD, and explored associations of the baseline and time-mean SUA levels with kidney function decline and incident CKD in a healthy population. METHODS: We initiated an inhabitant-based cohort study between January 2011 and December 2016. All participants completed a yearly medical check-up at the Zhejiang Province People's Hospital and had baseline estimated glomerular filtration rates (eGFR) > 60 ml/min/1.73m2. The SUA level and eGFR were assessed every year in the follow-up period. A multivariate adjusted binary logistic regression analysis and Cox proportional hazards models were used to evaluate the risk of newly-developed CKD among different stratified groups. RESULTS: During the 6-year follow-up period, 227 (4.4%) participants developed CKD. In multivariable-adjusted analyses, the odds ratio (OR) for new-onset CKD increased, with higher time-mean SUA levels than at baseline (OR: 1.00 [reference], 2.709 [95% confidence interval: 1.836-5.293], 3.754 [1.898-7.428], and 7.462 [3.694-15.073]). After adjustment for potential cofounders, a multivariate Cox proportional hazard model showed that a higher SUA increased the risk of developing CKD (the adjusted hazard ratios of the highest and lowest quartiles for the baseline and time-mean SUA levels were 1.689 [1.058-2.696] and 6.320 [3.285-12.159], respectively). CONCLUSION: An increased time-mean and single SUA value were independently associated with an increased likelihood of eGFR decline and development of new-onset CKD in the general population.

Rapamycin Reduces Podocyte Apoptosis and is Involved in Autophagy and mTOR/ P70S6K/4EBP1 Signaling.

BACKGROUND/AIMS: The purpose of this study was to investigate the impact of rapamycin (RAP) on autophagy in podocytes and the therapeutic effects of RAP on idiopathic membranous nephropathy (IMN). METHODS: We established an in vitro model of IMN by preconditioning mouse podocytes with puromycin aminonucleoside (PAN). A Cell Counting Kit-8 was used to detect the proliferation of each group of podocytes. Podocyte apoptosis was analyzed by flow cytometry via annexin V/propidium iodide dual staining. Subsequently, we observed the number of autophagosomes by transmission electron microscopy. Western blotting was used to detect the levels of LC3, mTOR, p-mTOR, 4EBP1, p-4EBP1, P70S6K, and p-P70S6K in each group. RESULTS: The number of podocytes in the PAN + 100 ng/mL RAP group, PAN + 200 ng/mL RAP group, and PAN + 300 ng/mL RAP group was significantly increased (P < 0.01). The apoptotic rate of podocytes was significantly different between the PAN group and the PAN + RAP group (P < 0.001). There were fewer autophagic corpuscles in the PAN group and more autophagosomes were observed in the PAN + RAP group. LC3 protein expression was down-regulated in the PAN group, while its expression was up-regulated in the PAN + RAP group. In the PAN group, the levels of phosphorylated mTOR, 4EBP1, and P70S6K were increased, while in the PAN + RAP group, protein phosphorylation was reduced. CONCLUSIONS: RAP can effectively inhibit the mTOR/P70S6K/4EBP1 signaling pathway, and activate podocyte autophagy, consequently reducing podocyte apoptosis. Therefore, RAP could be used for the treatment of idiopathic membranous nephropathy.

Effect of autophagy and stromal interaction molecule 1 on podocyte epithelial-mesenchymal transition in diabetic nephropathy.

AIM: We aimed to assess the effect of autophagy and stromal interaction molecule 1 (STIM1) on podocyte epithelial-mesenchymal transition in diabetic nephropathy. METHODS: The sera of 8-week-old db/db and C57BL/KsJ rats were used to culture MPC5 cells. The experiment was divided into 4 groups: MPC5 + siRNA-Scr + 10% C57BL/KsJ (Group A), MPC5 + siRNA-STIM1 + 10% C57BL/KsJ (Group B), MPC5 + siRNA-Scr + 10% db/db (Group C), and MPC5 + siRNA-STIM1 + 10% db/db (Group D). Podocyte autophagy was evaluated via immunofluorescence staining for LC3II and P62, and via Western blotting for P62 and LC3 (LC3II/LC3I). Western blotting was also used to assess the expression of TRPC6, Orai1, Beclin-1, Bcl-2, Caspase3, E-cadherin, fibronectin, and α-SMA protein. Furthermore, podocyte apoptosis was assessed via flow cytometry. RESULTS: We found that, in podocytes cultured in the serum of diabetic nephrotic rats, the autophagy level decreased, whereas the apoptosis level increased, and EMT can be advanced. However, after silencing STIM1 with siRNA, a converse outcome was noted. Furthermore, in diabetic nephropathy rats, the up-regulated expression of podocyte STIM1 can activate TRPC6 and Orai1 channels, which results in Ca2+ entry. CONCLUSIONS: We found that, in podocytes cultured in the serum of diabetic nephrotic rats, the autophagy level increased, whereas the apoptosis level decreased, and EMT can be inhibited by silencing STIM1 with siRNA.