| BackgroundDiabetic nephropathy (DN) involves serious diabetic microangiopathy complications. DN is the leading cause of disability and death in patients. Podocytes injury plays a critical role in the development and progression of DN. Therefore, the key to solve the problem is to explore the mechanism of podocyte injury and to find effective and feasible intervention drug. Recent investigations demonstrated that active vitamin D3 reduced albuminuria and improve renal function, which was associated with podocyte protection. The present study aimed to provide a mechanistic insight into the potential effects of 1,25-dihydroxyvitamin D3 (VD) in vivo and in vitro high glucose induced podocyte injury.MethodsDN rats were established by intraperitoneal injection with streptozocin. Rats were randomly divided into four groups:normal control (NC) group, calcitriol treatment (VD) group:calcitriol 0.1μg·kg-1·d-1, diabetic nephropathy (DN) group: streptozocin (STZ) 58mg·kg-1,DN treated with calcitriol (DN+VD) group:calcitriol 0.1μg·kg-1·d-1+STZ 58mg·kg-1. Rats were sacrificed 18 weeks after treatment. Blood glucose and body weight were assessed, and 24-hour urine was collected regularly. Blood and urine samples were taken for biochemical study, and kidney tissues were used for PAS and MASSON stains to assess histological changes. Ultramicro morphological changes in the podocyte foot processes were observed using electron microscopy. In in vitro study, an immortalized mouse podocytes were treated with 5mmol·L-1 glucose (NG) or 30mmol·L-1 glucose (HG), and then evaluated the effects of 1,25(OH)2 Vitamin D3, SKF-96365(a generic blocker of TRPC6 channels) and LY294002 (A specific and cell-permeable inhibitor of PI3K) on the cultured podocytes under the condition of HG Immunohistochenical staining was used to detect WT-1, Nephrin, Podocin, Desmin, VDR and p-Akt in the renal of SD rats. The Nephrin, Podocin, Desmin and TRPC6 in podocytes in vitro and TRPC6 in renal were examined by immunofluorescent staining. Western blotting was used to detect protein expression of Nephrin, Podocin, Desmin, VDR, TRPC6, PI3K, Akt and p-Akt.Results(1) Compared with the DN group, the DN+VD group exhibited significantly lower proteinuria by 36%, improved renal histology at the end of the experiment (P<0.05), and similar levels of blood glucose, serum urea nitrogen as well as body weight (P>0.05). There were also no differences in the serum concentrations of creatinine, calcium and phosphorus among the four groups (P>0.05). In DN rats, the expression levels of Nephrin, Podocin were significantly decreased (P>0.05) and the expression of Desmin was increased (P>0.05) compared to NC group. Calcitriol treatment could attenuate the above changes. The expression of WT-1 was no difference among the four groups. For in vitro studies, the expression of Nephrin and Podocin were decreased in MPC5 cells stimulated with 30mM glucose in a time-dependent manner. Meanwhile, the expression of Desmin was increased. The peak achieved at 24h after high glucose stimulation. There were up-regulated the expression of Nephrin and Podocin in MPC5 cells incubated with high glucose (30mM,24h) before the different doses 1,25(OH)2D3.1,25(OH)2D3 caused a decreased proportion of Desmin compared with the those untreated cells in a dose-dependent manner. 100nM 1,25(OH)2D3 gave the maximum response. The above results confirmed that podocyte injury is a typical characteristic of diabetic nephropathy, active vitamin D3 indeed protects podocytes to attenuate albuminuria in diabetic nephropathy. (2) We further explore the action of Nephrin-PI3K/p-Akt signaling pathway in aVitD3 ameliorating podocyte injury. The DN+VD group, the expression of PI3K-p85 and p-Akt were up-regulated compared with the DN group. The expression of Nephrin was positively correlated with either PI3K-p85 or p-Akt (r=0.736, P<0.05; r=0.855, P<0.05, respectively). For in vitro studies, the PI3K/p-Akt expression decreased in cultured MPC5 cells by high glucose exposure. Expectedly, 1,25(OH)2D3 reversed the decreased PI3K/p-Akt expression. Furthermore, LY294002, can abolished the protective effect of 1,25(OH)2D3 in podocytes by high glucose exposure. (3) The aim of this study is to investigate whether calcitriol up-regulates the PI3K/p-Akt signal pathway via inhabition of TRPC6 expression to ameliorate injury of podocyte in vivo and in vitro high glucose induced podocyte injury. In DN rats, the expression levels of VDR was significantly decreased (P>0.05) and the expression of TRPC6 was increased (P>0.05) compared to NC group. Calcitriol treatment could attenuate the above changes. Significant positive correlation was observed between TRPC6 and 24h urinary protein (r=0.739,P<0.05), Desmin (r=0.947, P<0.05) of podocyte injury maker. On the contrast, significant negative correlations were observed between TRPC6 protein and Nephrin expression (r=-0.752, P<0.05) and VDR (r=-0.685, P<0.05). For in vitro studies, the expression of VDR decreased in MPC5 cells stimulated with 30mM glucose at 24h. Meanwhile, the expression of TRPC6 was increased.1,25(OH)2D3 caused a decreased proportion of TRPC6 and a increased proportion of VDR. The expression of Nephrin and Podocin were increased in the presence of SKF-96365, a blocker of TRPC6. There were also no differences compared with 1,25(OH)2D3-treated cells. Expectedly, SKF-96365 reversed the decreased PI3K/p-Akt expression.Conclusion1) Podocyte injury was a typical pathological characteristic of diabetic nephropathy. 1,25(OH)2D3 ameliorated podocyte injury in vivo and in vitro high glucose induced podocyte injury.2) The PI3K/p-Akt signaling pathway was an important mechanism that high glucose induced podocyte impairment.1,25(OH)2D3 up-regulated the PI3K/p-Akt signaling pathway to ameliorate in vivo and in vitro high glucose induced podocyte injury.3) The expression of TRPC6 plays an important role in the pathogenesis of progressive podocyte injury in vivo and in vitro high glucose induced podocyte injury.1,25(OH)2D3 upregulates the PI3K/p-Akt signaling pathway via inhabition of TRPC6 expression to ameliorate podocyte injury in vivo and in vitro high glucose induced podocyte injury. |
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