SOCS-1 And SOCS-3 Gene Protect Diabetic Kidney Via JAK/STAT Signaling Pathway

Objectives: Diabetic nephropathy is one of the most common complications of diabetes, which is histologically characterized by hypertrophy of glumeruli and renal tubules, increased thickness of basement membrane, overaccumulation of extracellular matrix, glomerulosclerosis and interstitial fibrosis. The pathogenesis of diabetic nephropathy is very complicated. It is caused by multifactors, which can make kidney cell's signaling pathway change. Janus kinase/signal transducers and activators of transcription (JAK/STAT) is an important signaling pathway which can stimulate excessive proliferation and growth of glomerular mesangial cells, contributing 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. It was reported that SOCS protein participate in the regulation to the injury of a series of diseases in many systems. Our group has confirmed that JAK/STAT signaling pathway is involved in the glomerular damage associated with diabetes. However, the renal expression of SOCS gene, an important negative regulating factor of JAK/STAT signaling pathway is barely understood in diabetic kidney and renal cells. Aims of the present study are to examine the expression of SOCS-1/-3 in diabetic kidney, whether its possible protective role in diabetic nephropathy is mediated by inhibiting JAK/STAT signaling pathway, and further regulating cell proliferation, apoptosis, and extracellular matrix (ECM) production.In this study, Wistar rats were used to induce experimental diabetes model, human glomerular mesangial cells (HMC)and human kidney proximal tubular epithelial cells (HKC) were cultured in high glucose (HG) and advanced glycation end products (AGEs) in vitro to systemic investigate in early diabetic nephropathy and HMC or HKC induced by HG or AGEs from different levels in whole, cells and molecules. SOCS-3 expression vectors are transfected into renal cells (HMC/HKC) to construct the positive cell clones screened with G418 . we observed the HMC proliferation, apoptosis and extracellular matrix production (ECM) induced by HG and HKC transdifferentiation induced by AGEs in a state of SOCS-3 highexpression, in order to investigate the role of SOCS gene result in inhibition of cytokine-mediated signaling pathway. It is expected to provide a new thinking for the pathogenesis, the molecule mechanism and the prevention and cure of diabetic nephropathy.Methods1 SOCS-1/-3 expression in diabetic rat glomeruli Male Wistar rats were randomly divided into two groups: control group and diabetic group. The rats of diabetic group received a single intraperitoneal injection of STZ dissolved in 0.1mol/L sodium citrate (pH4.5) at a dose of 65mg/kg body weight. The rats 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 48 hours of the injection. Eight rats from each group were respectively sacrificed at weeks 1, 2, 4, 8 and 12 after STZ injection. Partial renal tissures were fixed in 4% formaldehyde and 2% glutaraldehyde for light microscopic and electron microscopic observation and immunohistochemical staining. Partial renal cortices were used to abstract total RNA and protein. The expression of p-JAK2, p-STAT1, SOCS-1 and SOCS-3 protein were evaluated by immunohistochemistry and Western blot. The mRNA levels of SOCS-1 and SOCS-3 were measured by reverse transcription and polymerase chain reaction (RT-PCR).2 Expression of SOCS-1, SOCS-3 in HMC induced by HG Human glomerular mesangial cells (HMC) were incubated with serum-free RPMI 1640 for 24 hours to synchronize the cell growth. The renal cells were then divided into three groups: NG group(5.5mmol/L glucose);HG group (30mmol/L glucose) ; HG+AG490 group (30mmol/L glucose+10μmol/L AG490). The cell morphology changes were observed by inverted microscope at 12, 24, 48 hours after incubation and the ultrastructure changes at 48 hours by electron microscope. Cells were harvested to abstract total RNA and protein at 2, 4, 6, 12, 24 and 48 hours after incubation. The expression of SOCS-1 and SOCS-3 proteins were examined by immunocytochemistry, Western-blot, and p-JAK2, p-STAT1 proteins by Western-blot. The mRNA levels of SOCS-1 and SOCS-3 were examined by RT-PCR.3 Expression of SOCS-1 and SOCS-3 in HKC induced by AGEsHuman kidney proximal tubular epithelial cells (HKC) were incubated with serum-free DMEM for 24 hours to synchronize the cell growth. The cells were divided into three groups: BSA group (50ug/ml); AGEs-BSA group (50ug/ml); AGEs+AG490 group (50ug/ml AGEs+10μmol/L AG490). The cell morphology changes were observed by inverted microscope at 12, 24, 48 hours after incubation and the ultrastructure changes at 48 hours by electron microscope. The renal cells were harvested to abstract total RNA and protein at 2, 4, 6, 12, 24, 48 hours after incubation. The expression of SOCS-1, SOCS-3 proteins were examined by immunocytochemistry, Western-blot and flow cytometry and p-JAK2, p-STAT1 proteins by Western-blot. The mRNA levels of SOCS-1 and SOCS-3 were examined by RT-PCR .4 Effects of SOCS-3 on proliferation, apoptosis and secretion of TGF-β,FN induced by HG in HMCPCR 3.1-SOCS-3 expression vector was constructed and transfected into the HMC with lipofectamine 2000 in vitro. The cells were divided into four groups: HG + PCR3.1- SOCS-3 transfection group (30mmol/L glucose, HG+PS); HG+ PCR 3.1-vector transfection group (30mmol/L glucose, HG+ PV); untransfection HG group (30mmol/L glucose, HG); blank control group (untransfected cells, CG). Cell morphological changes were observed by inverted microscope at 12, 24, 48 hours after HG stimulation. Immunoytochemistry were used to analyze the SOCS-3 tranfected HMC cell proliferation activity index (PI). Western-blot were used to detected the expression of p-JAK2, p-STAT1protein. The contents of TGF-βand fibronectin (FN) in the supernatants were measured by Western blot. DeadEndTM Colorimetric TUNEL System detection kit were used to analyze the cell apoptosis index (AI).5 Effects of SOCS-3 on the expression ofα-SMA, TGF-β, FN induced by AGEs in HKCPCR 3.1-SOCS-3 expression vector was constructed and transfected into the HKC with lipofectamine 2000 in vitro to construct the positive cell clones screened with G418. The cells are divided four groups: AGEs + PCR 3.1-SOCS-3 transfection group (50ug/ml BSA, A+S); AGEs + PCR 3.1-vector transfection group (50ug/mlAGEs-BSA, A+V); AGEs untransfection group (50ug/ml); blank control group (CG). The cell morphology changes were observed by inverted microscope at 12, 24, 48 hours induced by AGEs. Immunocytochemistry were used to analyze the expression ofα-SMA in HKC. Western-blot were used to detected the expression of p-JAK2, p-STAT1, TGF-β, FN protein.Results1 Morphological changes and the expressions of SOCS-1 and SOCS-3 protein and mRNA in diabetic rat glomeruli①No obvious morphological change of glomeruli at 1 and 2 weeks, whereas the diabetic rats showed slightly enlarged glomeruli at 4 weeks, and the number of cells was increased with glomeruli hypertrophy, tubular epithalial vacuolar degeneration, atrophy and thickening arterial at week 8 and week 12. Thickened glomerular basement membrane and accumulated mesangium matrix, the extensively effacement of foot process and decreased number of microvilli and vacuolar degeneration in proximal tubule epithelial cells were seen in DM group at 12 weeks.②Immunohistochemical positive staining of SOCS-1, SOCS-3 was observed in glomerular mesangial cells and renal tubular epithelial cytoplasim in both control group and diabetic group. Compared with those in control group, the expression levels of SOCS-1,SOCS-3 proteins was all increased in diabetic group at 1, 2, 4, 8 weeks. The expression of SOCS-1 peaked at week 2, whereas SOCS-3 peaked at week 4 (P<0.05).③From the results of Western blot analysis, the diabetic rats showed increased p-STAT1 expression in the kidney from week 1, and maintained at a higher level until week 8; the expression peaked at week 2, and started going down from week 4. The expression of JAK2 was increased from week 1, and gradually increased with duration of diabetes of diabetes.④The mRNA levels of SOCS-1 and SOCS-3 in the kidney of diabetic rats increased at week 1, peaked week 2 and week 4 respectively, and maintained at higher levels until week 8.2 The expressions of SOCS-1, SOCS-3 protein and mRNA in HMCs①Under inverted microscope and electron microscope, the HMCs stimulated with HG revealed the decreased or shortened cell process, appeared many granula and vacular in cytoplasm; enlarged and retorted cell nucleli, less mitochondria and more rough endoplasmic reticulum, lysosome, in perticular, uncharachered granulues vacuole in cytoplasm.②Immunocytochemical staining showed that SOCS-1 and SOCS-3 proteins were mainly expressed in cytoplasm of HMC, positive signals were occasionally seen in nuclei. SOCS-1 is weakly expression. From the results of immunocytochemical, compared with normal glucose control group, the expressions of SOCS-1 and SOCS-3 in cytoplasm were increased and peaked at 4h and 6h respectively after HG stimulation.③Western blot analysis indicated that the expressions of P-JAK2, P-STAT1 were increased in HG group. The levels of expression of P-JAK2 and P-STAT1 in HG+AG490 were obviously lower than that in HG group. The same as the espression of SOCS-1 and SOCS-3. The nuclear translocation of SOCS-1 and SOCS-3 was also inhibited.④RT-PCR showed that the mRNA levels of SOCS-1 and SOCS-3 were weaked in control HMC, and up-regulated and the expression peaked at 4h and 6h respectively after stimulation by HG (P<0.01). The expression level of SOCS-1/-3mRNA was down-regulated by the treatment with AG490 (P<0.05), and higher when comparing to that of control group (P<0.05). 3 The expressions of SOCS-1 and SOCS-3 protein and mRNA in HKC①The HKC cell morphology changes were observed by inverted microscope and electron microscope: compared with control group, the cells grow spindle gradually with single cell rounding; karyopycnosis; the shortened cell process; appear many granula and vacular in cytoplasm; more microvillus on cell surface; less mitochondria and more rough endoplasmic reticulum, lysosome, vacuole in cytoplasm after AGEs stimulation.②Immunocytochemical staining showed that SOCS-1, SOCS-3 protein were expressed in cytoplasm of HKC, positive signals were occasionally seen in nuclei. SOCS-1 is weakly expressed. Compared with normal glucose control group, the expression of SOCS-1 and SOCS-3 in cytoplasm was increased by immunocytochemical and peaked at 6h and 12h respectively induced by AGEs.③Western blot analysis indicated that the expression of p-JAK2, p-STAT1 was increased in AGEs group. The levels of expression of p-JAK2 and p-STAT1 in HG+AG490 were obviously lower than that in AGEs group. The same as the espression of SOCS-1 and SOCS-3 .The nuclear translocation of SOCS-1 and SOCS-3 was inhibited.④RT-PCR showed that the expression level of SOCS-1 mRNA and SOCS-3 mRNA was weaked in control HKC , up-regulated and peaked at 6h and 12h respectively in AGEs group (P<0.01). The expression levels of SOCS-1/-3mRNA were down-regulated by the treatment with AG490 (P<0.05), but higher than control group (P<0.05).4 Transfection and over-expression of SOCS-3 in HMCs and HKCs.①PCR 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 SOCS-3 protein were expressed in more nuclei and cytoplasm of tranfected cells than control group, Which suggested a successful gene transfection and higher expression in protein level.③RT-PCR analysis indicated that SOCS-3 mRNA was up-regulated obviously in tranfected cells than control group. Which suggested a successful gene transfection and higher expression in mRNA level.5 Effects of SOCS-3 on HMC proliferation, apoptosis and secretion of TGF-β,FN induced by HG①The HMCs in HG+PS group and HG+PV group were observed very similar to those in HG group in morphology features, by inverted microscope, in compared with control group, whereas cells revealed spindle gradually with polygon shape, the shorter cell process and appeared many granula and vacular in cytoplasm.②Comparing with those in CG group, HMCs in HG+PV group, HG group and HG+PS group showed increased numbers in PCNA positive cells and higher cell proliferation activity index (PI=68.24±8.34, PI=61.07±9.34); the high PI induced by HG was significantly retrieve by PCR 3.1-SOCS-3 plasmid treatment (PI =35.87±4.96 in HG+PS group).③Western blot analysis indicated: Compared with control group, the expression of p-JAK2 and p-STAT1 was increased (P<0.01) in transfected cells group (HG+PS and HG+PV) and HG group at 12, 24, 48 hours. However , the level in HG+PS group was lower than in HG+PV and HG group; the relative expression levels of TGF-βand fibronectin proteins were increased in HG+PS, HG+PV and HG group than those in CG group (P<0.05), and the over expressed proteins were downregulated by PCR 3.1-SOCS-3 transfer.④Comparing with that of CG group, cell apoptosis index (AI) was less increased in HG+PS group (AI=11.87±1.96) and more increased in HG+PV and HG group (AI=15.24±4.34, AI=13.93±5.34). whereas no significant difference was found between HG+PV and HG group groups.6 Effects of SOCS-3 gene on HKC transdifferentiation induced by AGEs①Compared with control group, The transfection HKC cell morphology changes were observed by inverted microscope, such as the spindled gradually with polygon shape, shortened cell process and many granula and vacular in cytoplasm in A+S, A+V and AGEs group.②Compring with that in BSA group, more expression ofα-SMA protein in cytoplasm was observed in A+S, A+V groups and AGEs group (P<0.05) by immunocytochemical staining, and the expression level in A+S group was lower than that in A+V group (P<0.05).The result was further confirmed by Westernblot analysis.③Compared with BSA control group, the expressions of p-JAK2 and p-STAT1 were increased (P<0.01) in transfected cells group (A+S and A+V) and AGEs group at 12, 24, 48 hours by Western blot. However, the level in A+S group was lower than in A+V and AGEs group. There is no significant difference between A+V and AGEs group.④Western blot analysis indicated the Expression levels of TGF-βand fibronectin proteins were increased in A+S, A+V and AGEs group after AGEs stimulation (P<0.05) , the higher expression in A+V and AGEs group.ConclusionsFrom above results we can draw the following conclusions:①SOCS-1 and SOCS-3 genes were expressed in normal rat kidney and cultures HMCs and HKCs at both protein and mRNA level; SOCS-1 showed a weak expression in cytoplasm, and occacionally in nuclei.②In the early stage of diabetes, along with the increased expression of p-JAK2 and p-STAT1 proteins, SOCS-1 and SOCS-3 genes were up-regulated at both mRNA and protein levels. HG/AGEs can induce high expressions of SOCS-1 and SOCS-3 genes in cultured HMCs/HKCs through JAK/STAT signaling pathway activation; AG490, specific inhibitor of JAK2, can inhibit activation of JAK/STAT signaling pathway and depressed phosphorylation of STAT1, meanwhile down-regulate the expression of SOCS-1 and SOCS-3 mRNA. could the results revealed that SOCS family member might participate in a negative regulation to JAK/STAT signaling pathway and could be involved in the glomerular pathological changes associated with diabetes.③SOCS-3 gene was successfully transfected into renal cells (HMCs/HKCs) with lipofectamine 2000 in vitro. RT-PCR and immunocytochemistry were used to analyze the higher expression of SOCS-3 mRNA and protein in transfected HMC/HKC.④HG could induce the activation of P-STAT1 and promote HMC proliferation, apoptosis and production of TGF-β, extracellular matrix production such as FN. SOCS-3 could prevent STAT1 from phosphorylating, to suppress HMC proliferation and production TGF-βextracellular matrix.⑤AGEs could induce the activation of P-STAT1 and promote HKC transdifferentiation. SOCS-3 might inhibit phosphorylation of STAT1 to suppress HMC transdifferentiation by feed-back to JAK/STAT signaling pathway, thereby to play a full role of protection in renal cells.