Effect Of SOCS-1 On Diabetic Renal Injury In Vivo And In Vitro

Objectives: Diabetic nephropathy is one of the most common complications of diabetes, which is early characterized by glomerular hyperfiltration, glomerular and tubular epithelial hypertrophy, and the development of microalbuminuria, followed by the development of glomerular basement membrane thickening, the accumulation of mesangial matrix, and overt proteinuria, eventually leading to glomerulosclerosis and end stage renal disease (ESRD). Various growth factors and cytokines, including angiotensin II, monocyte chemoattractant protein-1(MCP-1), intercellular adhesion molecule-1 (ICAM-1), interleukin-6 (IL-6), and transforming growth factor (TGF)-β1 to be involved in the development of diabetic nephropathy. Janus kinase/signal transducers and activators of transcription (JAK/STAT) is an important signaling pathway, which is known to mediate the signaling of numerous cytokines and growth factors and is implicated in the regulation of a wide range of cellular processes, such as proliferation, differentiation, and apoptosis. Recent studies suggest that JAK/STAT signaling cascades may contribute to diabetic nephropathy. It was reported that the suppressors of cytokine signaling (SOCS) protein participate in the regulation to the injury of a series of diseases in many systems through negatively regulating the signaling pathway mediated by cytokines to affect the celluer basic biological behaviour, such as proliferation, differentiation and apoptosis. SOCS proteins have defined an important additional mechanism for the negative regulation of the JAK/STAT pathway. However, the role of SOCS gene on diabetic renal damage is barely understood. In this study, we used a streptozotocin (STZ)-induced diabetic model and human glomerular mesangial cells (HMCs) cultured in high glucose (HG) to observe the role of SOCS-1 overexpression on diabetic renal damage and its possible mechanism.Materials and Methods1 Induction of mice diabetes and SOCS-1 plasmid transfectionMale CD-1 mice were randomly divided into four groups: nondiabetic control mice (N); STZ-induced diabetic mice (DM); STZ-induced diabetic mice received empty expression plasmid vector (DM+V); STZ-induced diabetic mice received SOCS-1 plasmid (DM+S1). Mice received an intraperitoneal injection of STZ dissolved in 0.1mol/L sodium citrate (pH4.5) at a dose of 150 mg/kg body wt. The mice of control group only received an injection of the same volume of 0.1mol/L sodium citrate. The model of diabetes was considered to be successful when the blood glucose was≥16.7mmol/L and the glucose in urine was +++~++++ after 72 hours of the injection. Plasmid DNA was administrated into mice by rapid injection of a large volume of TransIT-EE Hydrodynamic Delivery Solution through the tail vein, according to the manufacturer's instruction. One group of diabetic mice received an injection of SOCS-1 plasmid at 1 mg/kg body wt at day 3 initially and every 7 days thereafter, and another group received an injection of vector in an identical manner. At 4 wk after STZ, six mice from each group were respectively sacrificed and partial renal tissures were fixed in 4% formaldehyde for light microscopic observation and imm- unohistochemical staining. Partial renal cortices were used to abstract total RNA and protein. The expression of F4/80, MCP-1, ICAM-1 and TGF-β1 protein were evaluated by immunohis- tochemistry and Western blot. The mRNA levels of MCP-1 and TGF-β1 were measured by reverse transcription and polymerase chain reaction (RT-PCR).2 Preparation of stable HMC cell lines overexpressing SOCS-1 and detection of JAK/STAT, TGF-β1 and fibronectinStable transfections of HMC with pCR3.1/vector and pCR3.1/SOCS-1 were performed with Lipofectamine 2000 according to the manufacturer's instructions. Subsequently, cells were cultured in selection medium containing 0.5mg/ml G418 for 4 weeks before single clones were isolated. HMCs were incubated with serum-free RPMI 1640 for 24 hours to synchronize the cell growth. Then cells were stimulated by NG(5.5mmol/L glucose);HG (30mmol/L glucose) and NG+M (24.5mmol/L mannitol). Cells were harvested to abstract total RNA and protein at 0-48h after incubation. The expression levels of SOCS-1, p-JAK2, p-STAT1, p-STAT3, JAK2, STAT1 and STAT3 proteins were examined by Western-blot. The mRNA level of SOCS-1 was examined by RT-PCR. The contents of TGF-β1 and fibronectin in the supernatants of the HMCs were measured by ELISA.Results:1 Effect of SOCS-1 overexpression on accumulation of monocytes/macrophages and expression of MCP-1, ICAM-1 and TGF-β1 in renal cortex of diabetic mice①Immunocytochemical staining showed that the expre- ssion of F4/80 positive cells, MCP-1, ICAM-1 and TGF-β1 proteins in DM and DM+V groups were obviously increased compared with control group. The expression of F4/80 positive cells, MCP-1, ICAM-1 and TGF-β1 proteins was inhibited by SOCS-1 overexpression in diabetic renal cortex.②Western blot analysis indicated that the expression of MCP-1, ICAM-1 and TGF-β1 at a higher level in DM and DM+V groups, and overexpression of SOCS-1 significantly decreased the expression of those proteins.③RT-PCR showed that the expression level of MCP-1 and TGF-β1 mRNA were upregulated compared with N group, and overexpression of SOCS-1 can downregulated the expression levels of MCP-1 and TGF-β1 mRNA in diabetic renal cortex.2 The activation of JAK/STAT and expressions of SOCS-1, TGF-β1 and fibronectin in HMCs under high glucose condition①Western blot showed that the expression level of SOCS-1 protein increased within 1h of HG stimulation, peaked at 4h, and gradually decreased to baseline at 24h. Compared with NG and NG plus mannitol, HG induced time-dependent tyrosine phosphorylation of JAK2, STAT1 and STAT3; the phosphory- lation levels were peaked at 12-24h, and remained at high levels until 48h. HG-induced activation of JAK2, STAT1 and STAT3 was inhibited by overexpression of SOCS-1 in the HMCs at 24h.②RT-PCR analysis showed that HG induced SOCS-1 mRNA expression increased within 1h, peaked at 4h, and gradually decreased to baseline at 24h. TGF-β1 mRNA level in HMCs in HG group is significant higher than those in NG group; and the transfection of pCR3.1/SOCS-1 significantly decreased the HG- induced overexpression of TGF-β1 mRNA in the HMCs at 48h.③The concentration of TGF-β1 and fibronectin in supernat- ants of the HMCs exposed to HG was higher than that in NG group at 48h and the transfection of pCR3.1/SOCS-1 significantly decreased the HG-induced high secretion of TGF-β1 and fibronectin.Conclusions:1 SOCS-1 overexpression in kidney ameliorated glomeru- lar hypertrophy, STAT1 activation, monocytes/macrophages accumulation, and expression of MCP-1, ICAM-1 and TGF-β1 in the early diabetic mice. So, we can speculate that the SOCS-1 can ameliorate inflammmation of kidney in early diabetic mice.2 SOCS-1 overexpression in HMCs inhibited HG-induced JAK/STAT signaling pathway activation, increased synthesis of TGF-β1 and fibronectin, and up-regulated TGF-β1 mRNA. These suggest that SOCS-1 inhibits HG-induced synthesis of TGF-β1 and fibronectin in HMCs, which may be partly via inhibiting activation of JAK/STAT signaling pathway.