| 【Objective】 China has more than 100 million people with diabetes currently. Diabetic nephropathy(DN), a microangiopathic complication of diabetes mellitus(DM), is one of the most important complications of DM, as well as the cause of end-stage renal disease(ESRD). Microalbuminuria is one of important indicators for the diagnosis and clinical treatment of DN. Effective drug treatment to reduce albuminuria is particularly important in the improvement of DN. Salidroside(SAL) is a phenylpropanoid glycoside isolated from the medicinal plant Rhodiola rosea. Recent studies have suggested that ethanol extract of Rhodiola rosea has beneficial effects on diabetic nephropathy and SAL inhibits high glucose(HG)-induced mesangial cell proliferation. The present study was to explore the underlying mechanism of SAL on alleviating diabetes-related albuminuria. The mechanism of proteinuria is seldom researched in the first barrier of glomerular filtration membrane—glomerular endothelial cells(GECs), and the main research interest is focused on the glycocalyx which covers on the surface of the GECs. GECs are a type of endothelial cell. Given the importance of caveolin-1(Cav-1) in transcytosis of albumin across endothelial barrier, proteinuria is not be observed in Cav-1-/- mice, and Cav-1 is expressed in the renal endothelium of human and mice. In view of the above-metioned facts, we guess that albumin is transcytosised across the GECs to produce proteinuria. In the present study, we used db/db mice, the most recognized DN animal model, to evaluate the effect of SAL on alleviating albuminuria. Meanwhile, we set up an in-vitro model of albumin to explore the underlying mechanism and the upstream signaling pathway. 【Methods】1. Establishment of an in-vitro albumin transcytosis modelIn this method, radioactive-labelled albumin was replaced by fluorescently labeled albumin to trace the process of albumin transcytosis across the cells. Considering that the process of albumin transcytosis across the endothelial cells is mediated by receptors and highly regulated, we set up the new in-vitro model of albumin transcytosis.2. Animal experimentsdb/db mice(male, 9-week old) were treated with metformin(Met, 200 mg/kg/day), salidroside(SAL, 25, 50, 100 mg/kg/day), Methl-β-cyclodextrin(MβCD, 100 mg/kg/week), N-ethylmaleimide(NEM, 1.7 mg/kg/week) or vehicle for 11 weeks(n = 12 ~ 15 per group). At the same time, age-matched male db/m mice were used as non-diabetic group(WT) and were gavaged the same amount of saline for 11 weeks. Postprandial blood glucose was measured with glucose meter. Blood samples and 24 h-urine samples were collected. After mice were sacrificed, kidneys were immediately excised and left kidney weight was measured. Serum and urine creatinine was measured using Creatinine Assay Kit(Nanjing Jiancheng Bioengineering Institute, China). Urinary albumin was determined using an ELISA assay kit from Exocell(Philadelphia, PA). The level of albuminuria was presented as the ratio of urinary albumin(μg) to urinary creatinine(mg). Coded sections were read by two independent observers unaware of the experimental protocol. Thirty glomeruli, cut through the vascular pole, were randomly selected from each animal. The PAS-positive material in the mesangial matrix and glomerular tuft area was identified using the computer image analysis.3. Celluar experimentsAn in-vitro model of albumin transcytosis was set up to explore the effects of SAL on the albumin transcytosis across GECs under normoglycemia and hyperglycemia condition. The phosphorylations of AMPK, Src, and Cav-1 were examined by western blot in GECs to identify the related pathways in regulation of albumin transcytosis. GECs were transfected with siRNA to lower the expression of AMPK or Cav-1 to confirm their role in albumin transcytosis and the relevance in the effects of SAL on the albumin transcytosis across GECs. To further investigate the relationship between the mitochondrial reactive oxygen species(mtROS) and the albumin transcytosis across GECs, mitochondria superoxide production and mitochondrial membrane potential were measured by fluorescent probes, MitoSOX and JC-1 respectively. The FITC-labeled albumin uptake was observed in GECs. 【Results】1. Establishment of an in-vitro albumin transcytosis model(1) FITC was used to label albumin and FITC-labelled albumin has stability and maneuverability.(2) We successful set up an in-vitro new model of albumin transcytosis across GECs. Many factors must be considered, for example, the choice of aperture, the choice of fluorescent tracer, distinguishing albumin from transcytosis across the cell and from leakage, the effect of the osmotic pressure and the metabolism of albumin, etc.(3) We found that FITC-labeled albumin transcytosis was in both concentration- and time- dependent manner. The work provided a direct experimental basis for the occurrence of proteinuria. The model can also be used to screen drugs which can reduce albuminuria in-vitro. 2. SAL ameliorates diabetic nephropathy in db/db mice.(1) In animal experiments, compared with db/m group(WT), blood glucose, left kidney weight, serum creatinine, the ratio of albumin/creatinine, the glomerular surface area and mesangial surface area significantly increased in db/db mice(P < 0.05 or P < 0.01). It was in accordance with the characteristics of diabetic nephropathy model.(2) Left kidney weight, blood glucose, serum creatinine and the ratio of albumin/creatinine in db/db mice were significantly reduced after salidroside treatment(P < 0.05 or P < 0.01). Unexpectedly, Met reduced blood glucose, but did not alleviate albumin/creatinine in db/db mice. MβCD and NEM significantly reduced the ratio of albumin/creatinine in db/db mice.(3) SAL alleviated kidney morphrology change, but metformin,MβCD and NEM did not have the similar effect.3. Salidroside alleviates proteninuria by reducing albumin transcytossis across GECs(1) Using the established in-vitro albumin transcytosis model, we found that SAL decreased albumin transcytosis across GECs in normoglycemic or hyperglycemic conditions, whereas Met slightly increased the albumin transcytosis at normoglycemic conditions, and had no significant effect on the elevated albumin transcytosis induced by hyperglycemic condition.(2) We also examined the effects of AMPK activator, AICAR, and Src inhibitor, PP2 on albumin transport. Incubation with AICAR or PP2 inhibited the albumin transcytosis upregulated by high glucose but had no noticeable effect in normal glucose- treated GECs. The effect of SAL was partly blocked by the pharmacological inhibitor of AMPK, compound C, under hyperglycemic condition.(3) Under normoglycemic condition, SAL induced AMPK phosphorlation but inhibited the phosphorlations of Src and Cav-1 in a concentration-dependent manner. Knockdown of Cav-1 by specific siRNA resulted in significant reduction of albumin transcytosis.(4) Src/Cav-1 pathway and AMPK/Cav-1 pathway are responsible for the effect of SAL on albumin transcytosis:High glucose(HG) caused the inhibition of AMPK and the activation of Src, which both led to increased caveolin-1- mediated albumin transcytosis. SAL reversed the HG-induced alterations of AMPK and Src, as well as caveolin-1. GECs were transfected with AMPKα siRNA to specifically knockdown AMPKα expression. Both the phosphorylation of Cav-1 and albumin transcytosis across GECs was increased after AMPKα siRNA treatment. AMPKα siRNA partly inhibited SAL’s blocking effects on high glucose-upregulated Cav-1 phosphorylation in GECs, but did not affect SAL’s blocking effects on Src phosphorylation.(5) Met only reversed the decreased phosphorylation of AMPK, but had no effects on high glucose-stimulated phosphorylations of Src and Cav-1.(6) NAC, the ROS scavenger, abolished the high glucose-induced Src activation, as well as blocked the Cav-1 phosphorylation. SAL reduced mitochondrial superoxide anion in normal glucose condition, and reversed the rise of mitochondrial superoxide anion production stimulated by high glucose treatment. However, Met increased the mitochondrial superoxide production. SAL, Met, NAC, and CCCP caused a depolarization of mitochondrial membrane potential.(7) SAL result in a significant decrease of albumin uptake in GECs【Conclusions】 In conclusion, the present study demonstrates that salidroside alleviates albuminuria through reducing the albumin transcytosis across GECs. We find hyperglycemia exacerbates albumin transcytosis by upregulation of ROS/Src/Cav-1 pathway and downregulation of AMPK/Cav-1 pathway. SAL prevents hyperglycemia-induced albumin transcytosis by antioxidant activity and activation of AMPK. |
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