中国生物工程杂志

Objective: To investigate the mechanism of steroid receptor coactivator 3 (SRC3) on renal dysfunction and podocyte injury in the diabetic kidney disease (DKD). Methods: Streptozotocin (STZ) was injected into C57BL/6 mice to induce DKD disease model. Fasting blood glucose (FBG), serum creatinine (Scr), blood urea nitrogen (BUN), 24-hour urine and urine albumin creatinine ratio (UACR) were detected to assess renal function. The glomerular pathology change and glomerular ultrastructure were observed by HE, PAS staining and transmission electron microscopy (TEM). Immunofluorescence was used to observe the relationship between SRC3 and glomerular injury in DKD patients and mice, and Western blot was used to detect the expression levels of SRC3, Podocin and WT1 in DKD glomeruli to verify the relationship between SRC3 and DKD mouse. SRC3^-/- DKD mouse model was induced by STZ injection, and the effect of SRC3 on kidney injury was investigated by renal function, pathological structure, and ultrastructural changes compared with WT DKD mice. Western blot and immunofluorescence were used to detect the levels of SRC3 and podocyte markers WT1, Podocin and Nephrin in SRC3^-/- DKD and WT DKD mice. Next, SRC3-silenced RNA and SRC3-overexpressed podocytes were constructed, and the negative control (NC), high glucose group (HG), high glucose+SRC3 silenced group (HG+si-SRC3), and overexpression of SRC3 group (SRC3^OE) were set. Western blot was used to detect the levels of SRC3, WT1, Podocin and apoptosis-related protein Caspase3, and immunofluorescence was used to detect the levels of SRC3 in podocytes under HG environment. To verify the effect of SRC3 on DKD glomerular injury under high glucose environment, all the groups including Normal, HG, HG+si-SRC3 and SRC3^OE were set to determine the relationship between SRC3 and the expression of inflammatory cytokines by detecting the levels of TNF-α and IL-1β of supernatant in both podocyte and mesangial cells. Results: Compared with WT group, FBG, BUN and Scr in WT+STZ group were significantly increased(P<0.001, P<0.001, P<0.001). The 24 h urine volume and UACR of WT DKD mice were the highest at 12 weeks(P<0.001, P<0.001). Glomerular injury shows glomerular sclerosis, mesangial hyperplasia and glomerular glycogen accumulation (P<0.01, P<0.01). The ultrastructure of the glomeruli showed that the podocyte process effaced obviously and the basement membrane thickness increased(P<0.001, P<0.001). The level of SRC3 in local glomeruli was significantly increased in DKD patients(P<0.001). Compared with WT mice, the levels of WT1 and Podocin in WT DKD mice were decreased (P<0.05, P<0.05), while the level of SRC3 was increased(P<0.05). Immunofluorescence also showed that SRC3 level was increased (P<0.001) while WT1 level was decreased (P<0.05). Compared with WT DKD mice, SRC3^-/- DKD mice had no significant changes in FBG, but Scr and BUN were decreased (P<0.01, P<0.05). The 24 h urine volume of SRC3^-/- DKD mice decreased significantly at 12 weeks (P<0.05). UACR was at a lower level in SRC3^-/- DKD mice at 12 weeks (P<0.001). Furthermore, the glomerular injury was alleviated in SRC3^-/- DKD mice. Western blot showed that SRC3^-/- DKD mice had almost no expression of SRC3(P<0.001), while levels of WT1 and Podocin were significantly increased (P<0.05, P<0.01). Immunofluorescence also showed a negative correlation between SRC3 and Nephrin (P<0.001, P<0.05). In vitro, within high glucose (HG), the levels of SRC3 and Caspase3 were increased (P<0.05, P<0.01), while the levels of WT1 and Podocin were decreased (P<0.01, P<0.05). The levels of SRC3 and Caspase3 were significantly decreased in group HG+si-SRC3 (P<0.01, P<0.05), while WT1 and Podocin were increased (P<0.05, P<0.05). The level of SRC3 in SRC3^OE was significantly increased (P<0.001), but there were no significant changes in podocyte markers and Caspase3 compared with NC group. Immunofluorescence also showed that SRC3 was significantly increased in podocytes in the high glucose environment(P<0.01). In podocytes and mesangial cells, the levels of TNF-α and IL-1β in HG group were significantly higher than those in Normal group (P<0.001, P<0.001). But all of them are decreased in HG+si-SRC3 (P<0.01, P<0.01). There was no significant difference between Normal and SRC3^OE. Conclusion: High expression levels of SRC3 were found in glomeruli of DKD patients and mice, and podocytes in the high glucose environment, confirming that SRC3 is positively correlated with glomerular injury in DKD. In addition in vitro experiments show that SRC3 could promote the production of inflammatory cytokines in podocytes and mesangial cells only under a high glucose environment.