Effect of electroacupuncture on renal function and expression of autophagy-related proteins in kidney of rats with diabetic nephropathy

Abstract

To observe the effect of electroacupuncture(EA)on renal function and autophagy-related protein expression in diabetic nephropathy (DN) rats，so as to explore its mechanisms underlying improvement of DN. Male Wistar rats were randomly divided into normal control，DN model and EA groups (n=8 rats in each group). The DN model was established by high-fat diet (for 4 weeks) + streptozotocin (STZ，35 mg/kg，i.p.). EA (2 Hz/15 Hz，1 mA) was applied to bilateral "Shenshu"(BL23)and "Zusanli"(ST36)for 20 min，5 times a week for 6 consecutive weeks. The urine volume in 24 h (24 h-UV) was recorded，the levels of urine protein in 24 h (24 h-UP)，blood urea nitrogen (BUN) and serum creatinine (Scr) were detected by using an automatic biochemical analyzer，and the fasting blood glucose (FBG) was detected using rapid blood glucose paper test. The histopathological changes of glomerulus of the left kidney were observed after periodic acid-Schiff stain (PAS) and the ultra-microstructural changes，the numbers of autophagosomes and autophagic vacuoles of the right kidney were observed by transmission electronic microscope (TEM). The expression and localization of podocyte marker protein Nephrin was detected by using immunofluorescence assay，and the expression levels of autophagy-related proteins microtubule associated protein 1 (LC3)，Beclin-1，p62 and Nephrin in the kidney tissue measured by Western blot. Before treatment,compared with the normal control group，the levels of 24 h-UV，24 h-UP and FBG were significantly increased (P<0.01) in the model and EA groups. After treatment, compared with the normal group，the levels of 24 h-UV，24 h-UP，FBG，BUN，Scr and p62 protein expression were signi-ficantly increased (P<0.01)，and the expression levels of LC3Ⅱ，Beclin-1 and Nephrin proteins and ratio of LC3Ⅱ/Ⅰ were significantly decreased (P<0.01) in the model group. Compared with the model group，the levels of 24 h-UV，24 h-UP，FBG，BUN and p62 were apparently decreased (P<0.01，P<0.05)，while the expression levels of LC3Ⅱ，Beclin-1 and Nephrin proteins and ratio of LC3Ⅱ/Ⅰ were obviously up-regulated (P<0.01，P<0.05) in the EA group. No significant changes were found in the level of Scr after EA (P>0.05) and in the expression levels of LC3 Ⅰ after modeling and EA (P>0.05). Outcomes of PAS showed blurred structure of the glomerulus with obvious proliferation of glomerular mesangial cells，increase of cell matrix，and thickening of basement membrane of the glomerulus and renal tubule，atrophy of epithelia cells of renal tubules and infiltration of inflammatory cells in the renal interstitum; and TEM revealed fusion and exfoliation of some foot processes，bareness or thickening of some basement membrane，and reduction of number of autophagosomes or autophagobubbles in podocytes in the model group，which was relatively milder in the EA group. EA can effectively alleviate kidney damage in DN rats，which may be related to its function in facilitating autophagy in the kidney.