Role Of SOCS-1 And SOCS-3 In The Inflammatory Pathogenesis Of Diabetic Nephropathy

Objectives:Diabetic nephropathy (DN) is one of the most common complications of diabetes. Now DN has become the most frequent cause of end-stage renal disease. The pathogenesis of diabetic nephropathy is very complicated. From a pathophysiological point of view, the critical importance of metabolic and haemodynamic factors for the risk of developing ND is clear. However, the intimate mechanisms leading from chronic hyperglycaemia to the development of renal injury are complex and not yet fully unravelled. Although DN is traditionally considered as a nonimmune disease, many evidence has indicated that immunologic and inflammatory mechanisms play a significant role in its development and progression. A mumber of experimental and clinical studies have demonstrated that inflammation, more specifically inflammatory cytokines, plays a determinant role in the development of DN. Inflammatory cytokines are produced locally by resident and infiltrating cells. These molecules, including chemokines, adhesion molecules, and proinflammmatory cytkines, exhibit pleiotropic effects on the setting of DN. The first suggestion, that inflammatory cytokine could participate in the development of DN, was already made by Hasegawa et al. In addition, the recent studies have shown that patients with type 2 diabetes and overt nephropathy exhibit high levels of diverse acute-phase markers of inflammation. More important, these acute phase markers are associated with the thickness of GBM, a crucial lesion of DN. In addition, in db/db mice, a model of type 2 diabetes and DN, exhibited an increased expression of intracellular adhesion molecule-1 (ICAM-1) and a marked increase in macrophage infiltration. Kelly and colleafues demonstrated that in the model of diabetes, albuminuria was reduced and renal function was preserved treated with ruboxistaurin. All these studies illustrate that inflammation plays a significant role in the pathogenesis of DN.DN is histologically characterized by hypertrophy of glumeruli and renal tubules in the early stage and glomerulosclerosis and tubular interstitial fibrosis in the late. Tubular interstitial fibrosis is behaved to be the common final pathway leading to end-stage renal failure and is the strongest predictor of the degree and progression of chronic renal failure. The accumulation of myofibroblasts play a key role in this progression. These cells, a subset of fibroblasts, express the mesenchymal marker a-smooth muscle actin (a-SMA) and are a major source of extracellular matrix proteins in tubular interstitial fibrosis. There isn't myofibroblast in normal nephridial tissue, but it appeares in chronic renal disease. It has been suggested that myofibroblast derive from the differentiation of a resident interstitial fibroblast or migration of vascular smooth muscle cells into the interstitium. Mountaining evidence has shown that a proportion of interstitial myofibroblast detected during tubular interstitial fibrosis originated from tubular epithelial cells by a process called tubular epithelial-myofibroblast transdifferentiation. The transdifferentiation can be induced by multiple factors. Nowdays it was suggested that interactions between inflammatory infiltrates and resident tubular epithelial cells may play important roles in the development of tubular interstitial fibrosis by promoting the tubular epithelial-myofibroblast transdifferentiation.IL-1βhas been shown to exert profibrotic activity in a number of disease model. Recent studies have identified that IL-1βcan induce tubular epithelial-myfibroblast transdifferentiation, by which IL-1βinduces renal fibrosis. In additional experiments, it was identified that OSM is a novel inducer of tubular epithelial-myfibroblast transdifferentiation and is likely to be one of several cytokines that contribute to tubulointerstitial fibrosis. Therefor both IL-1βand OSM can induce transdifferentiation, but the mechanisms by which they operate in DN are poorly understood.Recently, a new family of negative regulators called suppressors of cytokine signaling (SOCS) has been isolated. It was reported that SOCS protein can negatively regulat the signaling pathway mediated by cytokines to affect the celluer basic biological behaviour. Our group has confirmed that SOCS-1 and SOCS-3 are involved in the DN. However, the relationship among the SOCS-1/SOCS-3, inflammation of DN and tubular epithelial-myofibroblast transdifferentiation is barely understood. In this study, many methods have been used to examine the infiltration of macrophage and the expression of IL-1βand OSM in diabetic kidney, the effect of IL-1βand OSM on the tubular epithelial-myofibroblast transdifferentiation. Besides, SOCS-1 and SOCS-3 gene transfection has been used to explore the role of SOCS-1 and SOCS-3 overexpression on the inflammatory pathogenesis of DN and further to provide a new thinking for the molecule mechanism and the prevention and cure of diabetic nephropathy.Methods:1 Determination of CK18,α-SMA, p-STAT1, SOCS-1, SOCS-3, CD68, L-1βand OSM in control and STZ-induced diabetic miceMale CD-1 mice were randomly divided into two groups:control group (N), diabetic group (DM). The mice of diabetic group received a single intraperitoneal injection of STZ dissolved in 0.1mol/L sodium citrate (pH4.5) at a dose of 150mg/kg body weight. 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. Six mice from contrlo and diabetic group were respectively sacrificed at weeks 4,8 and 12 after STZ injection, Partial renal tissures were fixed in 4% formaldehyde and 4% glutaraldehyde for light microscopic and electron microscopic observation and immunohistochemical staining. Partial renal cortices were freezed at-80℃for Western blot, reverse transcription and polymerase chain reaction (RT-PCR) and enzyme linked immunosorbent assay (ELISA). Partial renal cortices were fixed in 70% alcohol for flow cytometry. The expression of CK18,α-SMA, p-STAT1, SOCS-1, SOCS-3 protein and mRNA was respectively evaluated by immunohistochemistry, Western blot and RT-PCR. The expression of CD68, the marker of macrophage, was evaluated by flow cytometry. The level of IL-1βand OSM were evaluated by ELISA.2 Determination of CK18,α-SMA, SOCS-1, SOCS-3, FN and Col I in HKC induced by IL-1βor OSMHKC were maintained at 37℃in a humidified atmosphere of 5% CO2 in Dulbecco's modified Eagle's medium (DMEM) containing penicillin/streptomycin (100 U/ml and 100μg/ml, respectively) and 10% fetal bovine serum (FBS). Prior to use, cells were incubated in serum-free DMEM for 24 hours to synchronize the cell growth. The cells were divided into five groups:control group (N), IL-1β(10ng/ml) group (IL), IL-1β+AG490 (10μmol/L) group (IL+A), OSM (10ng/ml) group (OSM), OSM+AG490 (10μmol/L) group (O+A). The cells ultrastructure changes at 72 hours were observed by electron microscope. The cells were harvested to abstract total RNA and protein at 12,24,48 hours after incubation. The medium was collected for detecting the level of FN and Col I. The expression of CK18, a-SMA, SOCS-1, SOCS-3 proteins and mRNA was examined by immunocytochemistry, Western blot and RT-PCR. The p-STAT1 protein was examined by Western blot. The FN and Col I proteins were examined by ELISA.3 Determination of CK18,α-SMA, FN and Col I in HKC overexpressing SOCS-1, SOCS-3PCR 3.1-SOCS-1,SOCS-3 expression vector was constructed and transfected into HKC with lipofectamine 2000 in vitro to construct the positive cell clones screened with G418. The cells are divided nine groups:control group (N), IL-1β(lOng/ml) group (IL), IL-1β+PCR 3.1-SOCS-1 transfection group (IL+PS1), IL-1P+PCR 3.1-SOCS-3 transfection group (IL+PS3) and IL-1β+PCR 3.1-vector transfection group (IL+PV), OSM(10ng/ml) group (OSM), OSM+PCR 3.1-SOCS-1 transfection group (OSM+PS1), OSM+PCR 3.1-SOCS-3 transfection group (OSM+PS3) and OSM+PCR 3.1-vector transfection group (OSM+PV). The cells were harvested to abstract total RNA and protein at 12,24,48 hours after incubation. The medium was collected for detecting the level of FN and Col I. The expression of CK18,α-SMA, SOCS-1, SOCS-3 proteins and mRNA was examined by immunocytochemistry, Western blot and RT-PCR. The p-STAT1 protein was examined by Western blot. The FN and Col I proteins were examined by ELISA.4 Determination of CK18,α-SMA, p-STAT1, SOCS-1, SOCS-3, CD68, L-1βand OSM in mice before and after transfectionMale CD-1 mice were randomly divided into five groups:control group (N), diabetic group (DM), PCR3.1-Vector group (P+V), PCR3.1-SOCS-1 group (P+S1) and PCR3.1-SOCS-3 group (P+S3). 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. Two groups of diabetic mice received an injection of SOCS-1 or SOCS-3 plasmid at 1mg/kg body weight at week 8 initially and every 7 days thereafter, and another group received an injection of vector in an identical manner. Six mice from every group were respectively sacrificed at weeks 16 after STZ injection. Partial renal tissures were fixed in 4% formaldehyde for light microscopic observation and immunohistochemical staining. Partial renal cortices were freezed at-80℃for Western blot, reverse transcription and polymerase chain reaction (RT-PCR) and enzyme linked immunosorbent assay (ELISA). Partial renal cortices were fixed in 70% alcohol for flow cytometry. The expression of CK18, a-SMA, p-STAT1, SOCS-1, SOCS-3 protein and mRNA was respectively evaluated by immunohistochemistry, Western blot and RT-PCR. The expression of CD68, the marker of macrophage, was evaluated by flow cytometry. The expression of IL-1βand OSM was evaluated by ELISA.Results;1 The inflammatory infiltration and tubular epithelial-myofibroblast transdifferentiation in DN①The diabetic mice showed slightly glomeruli hypertrophy, mesangium matrix increase, partial tubular epithalial vacuolar degeneration from week 4 and tubule atrophy, interstitial widening at week 16 by light microscope. Ultrastructure changes, including thickened glomerular basement membrane, the extensively efacement of foot process and vacuolar degeneration in proximal tubule epithelial cells, were detected by electron microscope.②Immunohistochemical positive staining of SOCS-1, SOCS-3 was observed in cytoplasim of glomerulu and tubule cells. Compared with that in control group, the expression level of SOCS-1, SOCS-3 protein was increased in diabetic group.③From the results of immunohistochemistry and Western blot analysis, the diabetic mice showed gradually decreased expression of CK18 and increased expression of a-SMA. The expression of CK18 and a-SMA mRNA was the same with the expression of protein.④The expression of p-STAT1 in DM group was higher than that in control group.⑤The level of CD68 in DM was higher than that in control group. The result of ELISA showed that the level of IL-1βand OSM in DM group was higher than that in control group.2 The effects of IL-βand OSM on the expression of SOCS-1, SOCS-3, CK18 and a-SMA in HKC①The morphology changes of HKC were observed by electron microscope. Compared with control group, the cells stimulated by IL-1βor OSM displayed expansion of rough endoplasmic reticulum, less mitochondria and loss of mitochondrial crista. Actin filaments appeared in HKC when stimulated 72 hours.②Immunocytochemical staining showed that CK18,α-SMA and SOCS-1, SOCS-3 protein were all expressed in cytoplasm of HKC. Compared with control group, the expression ofα-SMA occurred and the expression of CK18 decreased in HKC induced by IL-1βor OSM. Besides, the expression of SOCS-1, SOCS-3 was increased in IL-1βor OSM group. The changes of these protien in HKC induced by IL-1βor OSM were partly reversed by AG490.③Western blot analysis also indicated that the expression of a-SMA gradually increased while the expression of CK18 decreased in IL-1βor OSM group. The expression of SOCS-1, SOCS-3 and p-STAT1 was also increased in IL-1βor OSM group, but they peaked at 24h. The changes of these protien in HKC induced by IL-1βor OSM were partly reversed when co-stimulated with AG490.④RT-PCR showed that the expression of CK18,α-SMA and SOCS-1 and SOCS-3 mRNA was the same as the protein.⑤The secretion level of ColⅠand FN was higher in IL-1βor OSM group than that in control group. AG490 could inhibit the secretion of ColⅠand FN induced by IL-1βor OSM.3 Effect of SOCS-1 and SOCS-3 overexpression on tubular epithelial-myofibroblast transdifferentiation induced by IL-1βor OSM in HKC①PCR 3.1-SOCS-1 and PCR 3.1-SOCS-3 plasmid amplified in DH5a and examined by the agarose gel electrophoresis, the effects showed the same strap as the standard plasmid DNA.②Immunocytochemical staining showed that the expression level of SOCS-1 protein was significant higher in PCR 3.1-SOCS-1 transfection group and SOCS-3 protein was significant higher in PCR 3.1-SOCS-3 transfection group than that in control group, which suggested a successful gene transfection and higher expression in protein level.③RT-PCR analysis indicated that SOCS-1, SOCS-3 mRNA was up-regulated obviously in tranfected cells than that in control group, which also suggested a successful gene transfection and higher expression in mRNA level. The overexpression of SOCS-1, SOCS-3 could partly restore the expression of CK18 and decrease the expression ofα-SMA, p-STAT1 and the secretion of ColⅠand FN induced by IL-1βor OSM.4 Effect of SOCS-1, SOCS-3 on inflammation and tubular epithelial-myofibroblast transdifferentiation in DN①Compared with DM and DM+V group, the expression level of SOCS-1 in DM+S1 group and SOCS-3 in DM+S3 group was significant increased. The expression of mRNA was consistent with protein. These results suggest that the gene transfection is successful.②From the results of immunohistochemistry and Western blot analysis, the diabetic mice showed gradually decreased expression of CK18 and increased expression ofα-SMA. Overexpression of SOCS-1, SOCS-3 by gene transfection could reverse the expression of those in bubular epithelial cells in DM. The expression of CK18 andα-SMA mRNA in various groups was the same with the expression of protein.③The expression level of CD68 in DM was higher than that in control group. Overexpression of SOCS-1, SOCS-3 could inhibit the expression of CD68.④The result of ELISA showed that the expression level of IL-1βand OSM in DM group was higher than that in control group. SOCS-1, SOCS-3 gene transfection could inhibit the expression of IL-1βand OSM.Conclusions:①There are infiltration of macrophage and increasing expression of IL-1β, OSM and p-STAT1 in diabetic kidney, along with the transdifferentiation of bubular epithelial cells to myofibroblast. These findings suggest that inflammation, tubular epithelial-myofibroblast transdifferentiation and activation of JAK/STAT signaling pathway are involved in DN.②IL-1βand OSM could induce the expression ofα-SMA and p-STAT1 and depress the expression of CK18 in HKC. While AG490 can partly reverse the expression of them, Which suggest that IL-1βand OSM could induce the tubular epithelial-myofibroblast transdifferentiation via activating JAK/STAT signaling pathway.③SOCS-1, SOCS-3 overexpression could depress the expression ofα-SMA and p-STAT1 and partly restore the expression of CK18 induced by IL-1βand OSM in HKC. These results suggest that overexpression of SOCS-1, SOCS-3 could inhibit the tubular epithelial-myofibroblast transdifferentiation induced by IL-1βor OSM, which maybe partly via inhibiting activation of JAK/STAT signaling pathway.④Overexpression of SOCS-1, SOCS-3 could ameliorate macrophage infiltration, the expression of IL-1βand OSM and the transdifferention of bubular epithelial cells to myofibroblast in kidney. From these results we can conclude that SOCS-1 and SOCS-3 could inhibit the tubular epithelial-myofibroblast transdifferentiation partly by ameliorating inflammation of DN.