ABSTRACT
Objective To study the effect of Astragalus polysaccharide on the expressions of Nephrin and Desmin in high sugar stimulation mice podocytes. Methods Test one: the cultured mouse podocytes were divided into 5 groups: astragalus polysaccharide group [D－ glucose 30 mmol/L+astragalus polysaccharide (0.0 g/L, 0.1 g/L,0.2 g/L,0.4 g/L and 0.8 g/L) ]. Test two: In test one, we chose the concentration of astragalus polysaccharide which most affected both expression of Nephrin and Desmin in podocytes and named it as astragalus polysaccharide 0. X g/L. The cultured mouse podocytes were divided into 7 groups: astragalus polysaccharide group (D－ glucose 30 mmol/L+astragalus polysaccharide 0.X g/L) had been cultured for 0 h, 3 h, 6 h, 12 h,24 h,48 h and 72 h, respectively. Test three: Basted on test two, we chose the time which most affected both expression of Nephrin and Desmin in podocytes and named it as Yh. The cultured mouse podocytes were divided into 4 groups: the control group,mannitol hypertonic glucose control group, high sugar group,and astragalus polysaccharide group (high sugar group+ astragalus polysaccharide 0.X g/L) . Each group had been cultured for Yh. Nephrin and Desmin of each subgroup was detected by flow cytometry podocytes and real－time PCR. Results With the intervention of polysaccharide, Nephrin in podocyte cell surface gradually increased, Desmin gradually reduced in a concentration－dependent manner (P＜0.05) . With the intervention of astragalus polysaccharide, Nephrin gradually increase and Desmin expression gradually decreased in a concentration－dependent manner (P＜0.05) . Compared to the control group and the high sugar group,Nephrin mRNA increased and Desmin mRNA decreased in 0.8g/L astragalus polysaccharide co－cultured for 72h (P＜0.05) . Conclusion Astragalus polysaccharide intervention can increase Nephrin and reduce Desmin in podocytes, which are possible targets of Astragalus polysaccharide in treatment of DN proteinuria.
ABSTRACT
<p><b>BACKGROUND</b>Tubulointerstitial renal fibrosis is the common end point of progressive kidney diseases, and tubular epithelial-myofibroblast transdifferentiation (TEMT) plays a key role in the progress of tubulointerstitial renal fibrosis. Anaphylatoxin C3a and C5a are identified as novel profibrotic factors in renal disease and as potential new therapeutic targets. The aim of this study was to investigate whether C3a, C5a can regulate TEMT by transforming growth factor-β1 (TGF-β)/connective tissue growth factor (CTGF) signaling pathway and the effects of C3a and C5a receptor antagonists (C3aRA and C5aRA) on C3a- and C5a-induced TEMT.</p><p><b>METHODS</b>HK-2 cells were divided into C3a and C5a groups which were subdivided into four subgroups: control group, 10 ng/ml TGF-β1 group, 50 nmol/L C3a group, 50 nmol/L C3a plus 1 µmol/L C3aRA group; control group, 10 ng/ml TGF-β1 group, 50 nmol/L C5a group, 50 nmol/L C5a plus 2.5 µmol/L C5aRA group. TGF-β1 receptor antagonist (TGF-β1RA) 10 µg/ml was used to investigate the mechanism of C3a- and C5a-induced TEMT. Electron microscopy was used to observe the morphological changes. Immunocytochemistry staining, real-time PCR and Western blotting were used to detect the expressions of a smooth muscle actin (α-SMA), E-cadherin, Col-I, C3a receptor (C3aR), C5aR, CTGF and TGF-β1.</p><p><b>RESULTS</b>HK-2 cells cultured with C3a and C5a for 72 hours exhibited strong staining of α-SMA, lost the positive staining of E-cadherin, and showed a slightly spindle-like shape and loss of microvilli on the cell surface. The expressions of α-SMA, E-cadherin, Col-I, C3aR, C5aR, TGF-β1 and CTGF in C3a- and C5a-treated groups were higher than normal control group (P < 0.05). C3aRA and C5aRA inhibited the expressions of α-SMA, Col-I, C3aR, C5aR, and up-regulated the expression of E-cadherin (P < 0.05). TGF-β1 and CTGF mRNA expressions induced by C3a and C5a were partly blocked by TGF-β1RA (P < 0.05).</p><p><b>CONCLUSION</b>C3a and C5a can induce TEMT via the up-regulations of C3aR and C5aR mRNA and the activation of TGF-β1/CTGF signaling pathway in vitro.</p>