Expression Of PDGF-D And JAK/STAT And Effects Of Rapamycin In IgA Nephropathy

Objectives: IgA nephropathy (IgAN), a chronic and prosessive glomerular disease, is one of the major reasons of glomerulosclerosis and end-stage renal disease (ESRD), which induces a series of clinical and pathophysiological changes. IgAN is histologically characterized by overaccumulation of mensangial cell and extracellular matrix. With the development of renal tissue biopsy, diognosis ratio of children IgAN is increasing gradually. Janus kinase/signal transducer and activators of transcription (JAK/STAT) is an important signaling pathway, which may play roles as signal transducers and activators in transcription and was confirmed to mediate the signaling of numerous cytokines and growth factors and to be implicated in the regulation of a wide range of cellular processes. STAT3 mediated a series of cellular processes such as proliferation, differentiation and apoptosis. Suppressor of cytokine signaling (SOCS) proteins are classic inhibitors of JAK/STAT signaling pathway, and SOCS1 and SOCS3 are the two main inhibitors. Platelet-derived growth factor (PDGF) family is a major mitogen in vivo and in vitro, which has four members, A, B, C and D. A large number of experiments had verified the roles of PDGF-A and PDGF-B in various of renal diseases by inducing the activation of JAK/STAT. PDGF-D is a new member which was discovered in 2001. Few studies about PDGF-D and the JAK/STAT signaling pathway in IgAN were reported. Rapamycin is a new immunosuppressive agent which playes important roles in antiautoimmune and antiproliferation. Rapamycin is not yet used in clinical therapy and the precise mechanisms for rapamycin's effects were not fully understood. In the present study, we examined the expression of PDGF-D and JAK/STAT signaling pathway, investigated the dynamic change of PDGF-D, and observed the effects of rapamycin in IgA patients using Wistar rats model of experimental IgAN and human glomerular mesangial cells cultured in PDGF-D medium, as well as IgAN child patients.Methods:1. Detection of PDGF-D and JAK/STAT in IgAN childrenFifty-two children diagnosed as primary IgAN by renal biopsy at our hospital from January 2004 to October 2007 were involved in this study. There were 29 boys and 23 girls among them, whose ages were from 5 to 16 (10.7±3.1) -years old. These patients were divided into four groups according to their pathological changes: control group (n=16); mild proliferation group (MP, n=17); focal proliferation group (FP, n=18) and proliferaiorr sclerosis group (PS, n=17). Children in control group were all healthy and without any primary and secondary renal diseases. ELISA was used to detect the level of PDGF-D and PDGF-B in blood and urine. Immunohistochemistry was used to detect protein expressions of PDGF-D, PDGF-B, PDGFR-β, STAT3, p-STAT3, p-JAK2, SOCS1 and SOCS3 in renal tissue.2. Induction of IgAN and detection of PDGF-D and JAK/STAT in renal tussue of IgAN ratsMale Wistar rats were randomly divided into two groups: control group and IgAN group. The rats of IgAN group had been administered with bovine serum albumin by gavage at a dose of 400mg·kg-1 every other day for 6 weeks and injected 0.4ml castor oil plus 0.1ml CCl4 once a week for 9 weeks. Lipoplysaccharides were injected at weeks 6 and 8. These rats were observrd until 10th week. Renal tissue was cut into 3μm frozen secion. IgA accumulation in glomerular mensangial areas was examined under the microscope after stained with flurorescein-conjugated affinipure goat anti-rabbit IgG and rabbit anti-rat IgA by immunofluorescence. Proliferation of mensangial cell and overaccumulation of extracellular matrix were observed by PASM and Masson stains. These results indicated that the successful ratio of the IgAN model was 90%. Seven rats from each group were respectively sacrificed at weeks 1, 2, 4 and 8 after the models were established. Partial renal tissue was fixed in 4% formaldehydum and embeded with paraffin. Partial renal cortices were used to abstract total RNA and protein. The protein levels of PDGF-D, PDGF-B, PDGFR-β, p-STAT3, STAT3, p-JAK2, SOCS1 and SOCS3 were evaluated by immunohistochemistry and Western blot; The mRNA levels of PDGF-D, PDGF-B, STAT3, SOCS1 and SOCS3 were measured by reverse transcription and polymerase chain reaction (RT-PCR).3. Cell culture and examination of JAK/STAT signaling pathwayHuman glomerular mesangial cells were incubated in serum-free RPMI 1640 for 24 hours to synchronize the cell growth, and randomly divided into three groups: control group; PDGF-D group (20ng/ml PDGF-D) and PDGF-D+AG490 group (20ng/ml PDGF-D +10μmol/L AG490). MTT assay was used to measure the proliferation of mesangial cells at 6, 12, 24 and 48 hours, meanwhile the effect of AG490 on proliferation of mesangial cells in PDGF-D medium was investigate. Mesangial cells were harvested to abstract total RNA and protein at 0, 30min, 60min, 120min and 240min after incubation. The expressions of STAT3, p-STAT3, p-JAK2, SOCS1 and SOCS3 proteins were examined by immunohistochemistry and Western blot. The levels of SOCS1 mRNA and SOCS3 mRNA were examined by RT-PCR.4. Effects of rapamycin on expression of JAK/STAT signaling pathway in human glomerular mesangial cells and renal tissues of rats with IgANMale Wistar rats were divided into five groups: control group, IgAN group and rapamycin treated group at three different doses (0.5mg·kg-1·d-1, 1mg·kg-1·d-1 and 2mg·kg-1·d-1). After the IgAN model was affirmed to be successful, the rats of rapamycin treated group were administered daily with rapamycin by gavage for 4 weeks. Blood, urine and kidney samples were collected at week 4. Partial renal tissure was fixed in 4% formaldehydum and embeded with paraffin. Partial renal cortices were used to abstract total RNA and protein. Immunohistochemistry and Western blot were used to investigate the expression of STAT3, p-STAT3, p-JAK2, SOCS1 and SOCS3 proteins. RT-PCR was used to detect the expression of STAT3, SOCS1 and SOCS3 mRNAs in the renal tissue of IgAN rats. Human glomerular mesangial cells were cultured in 96-pore plate. The cells were washed once with serum-free RPMI 1640 and further incubated for 24 hours to synchronize the cell growth. MTT assay was used to investigate the effect of rapamycin on proliferation of mesangial cell in PDGF-D medium at different time and different drug concentration. Human glomerular mesangial cells were cultured in 25cm2 plastic culture flask and divided into three groups: control group; PDGF-D group (20ng/ml PDGF-D); and PDGF-D+RPM group (20ng/ml PDGF-D +20μg/ml RPM). Total RNA and protein were abstacted 24 hours after incubation. The levels of STAT3, p-STAT3, p-STAT3, SOCS1 and SOCS3 proteins were measured by Western blot. SOCS1 mRNA and SOCS3 mRNA was measured by RT-PCR.Results:1. Expression of JAK/STAT signaling proteins in renal tissue of IgAN children, renal tissue of IgAN rats and human glomerular mensangial cell Immunohistochemical positive signals of STAT3 and p-STAT3 were increased in glomerular mensangial area and renal tubular epithelial area of the kidneys of patients with IgAN, and a correlation was found in the severity of glomerular injury and expression of these four proteins.Rats with IgAN:①No obvious morphological change was seen in glomeruli within 4 weeks and glomeruli was enlarged slightly at week 8, which hadn't statistical significance.②From immunohistochemical staining, the expression of p-STAT3, STAT3, SOCS1 and SOCS3 was positive in glomerular mensangial cells, renal tubular epithelial cells and interstitial cells of both control group and IgAN group, and the expression of p-STAT3, STAT3, SOCS1 and SOCS3 was increased in IgAN group.③Western blot analysis indicated that the expression of p-JAK2 was increased at week 1, and progressively increased with duration of IgAN. The expression levels of STAT3, p-STAT3 and SOCS1 were increased at week 1 in IgAN group, peaked at week 2, and slightly decreased at week 4 and week 8. SOCS3 was increased at week 2 in IgAN group, peaked at week 4, and slightly decreased at week 8.④RT-PCR analysis indicated that the mRNA levels of STAT3 and SOCS1 were increased at week 1 in IgAN group, peaked at week 2, and slightly decreased at week 4 and week 8. SOCS3 mRNA were increased at week 2 in IgAN group, peaked at week 4, and slightly decreased at week 8.In vitro:①STAT3 proteins were expressed in nuclei and cytoplasm of mesangial cells, p-STAT3 was expressed in nuclei, SOCS1 and SOCS3 were expressed in cytoplasm. Compared with those in control group, the expression levels of STAT3 and p-STAT3 were increased at 30min, peaked at 60min, and decreased at 120min and 240min after stimulation, whereas the levels of SOCS1 and SOCS3 were increased at 30min and 60min, peaked at 120min, and decreased at 240min. The expression levels of these four proteins in AG490 group were significantly lower than those of PDGF-D group.②Western blot analysis indicated that the expression levels of STAT3, p-STAT3 and SOCS1 were increased at 30min, peaked at 60min, and decreased at 120min and 240min after stimulation, whereas the levels of SOCS3 were increased at 30min and 60min, peaked at 120min, and decreased at 240min. The levels of p-JAK2 proteins were gradually increased. The levels of these five proteins were decreased in AG490 group comparing with those in PDGF-D group (P<0.05 or P<0.01).③RT-PCR showed that the mRNA levels of SOCS1 were increased at 30min, peaked at 60min, and decreased at 120min and 240min after stimulation, whereas the mRNA levels of SOCS3 were increased at 30min and 60min, peaked at 120min, and decreased at 240min. The increased expressions were inhibited by the treatment with AG490.2. The expression of PDGF-D, PDGF-B and PDGFR-βin the kidneys of patients and animal models with IgANClinical experiments:①The levels of PDGF-D and PDGF-B were progressively increased in blood and urine of IgAN children with the severity of glomerular injury.②Immunocytochemical staining showed that the expressions of PDGF-D, PDGF-B and PDGFR-βwere colocalized and increased in renal tissues of IgAN children.③Relevant analyze showed that the levels of PDGF-D had positive correlation with 24h urine protein excretion rate in blood and urine of IgAN children (r=0.546, P<0.05; r=0.76, P<0.01). There was negative correlation between the levels of PDGF-D and serum albumin (r=-0.649, P<0.01; r=-0.528, P<0.05); the levels of PDGF-B had positive correlation with 24h urine protein excretion rate in blood and urine of IgAN children (r=0.634, P<0.01; r=0.577, P<0.05). There was negative correlation between the levels of PDGF-B and serum albumin (r=-0.613, P<0.01; r=-0.531, P<0.05).Animal experiments: From the results of immunocytochemical staining, the expressions of PDGF-D and PDGF-B were increased at week 1 in IgAN group, and progressively increased with duration of IgAN.②Western blot analysis indicated that the expression of PDGF-B was increased at week 1 in IgAN group, and progressively increased with duration of IgAN. The expressions of PDGF-D and PDGFR-βwas increased at week 2 in IgAN group, and progressively increased with duration of IgAN.③RT-PCR showed the mRNA levels of PDGF-B were increased at week 1 in IgAN group, peaked at week 2, and slightly decreased at week 4 and week 8. PDGF-D mRNA were increased at week 2 in IgAN group, peaked at week 4, and slightly decreased at week 8.3. Effects of rapamycin on expression of JAK/STAT signaling pathway in the kidney of IgAN rats and human glomerular mesangial cellsIn vivo:①Immunocytochemical staining showed that the expression levels of p-STAT3, STAT3, SOCS1 and SOCS3 proteins were increased significantly in IgAN group, and the increased expressions were down-regulated in rapamycin treatment group.②Western blot showed the expression levels of p-JAK2, STAT3, p-STAT3, SOCS1 and SOCS3 were increased in IgAN group.③The mRNA levels of STAT3, SOCS1 and SOCS3 were up-regulated in IgAN group, and down-regulated by rapamycin treatment.In vitro:①Compared with control group, PDGF-D could stimulate the proliferation of mesangial cells, rapamycin at a concentration of 4, 20 and 100μg/ml significantly inhibited the increased proliferation of mesangial cells in PDGF-D . In rapamycin treated group, the suppressing effect became apparent at a dose of 20μg/ml after 12h and 24h.②Western blot analysis indicated that levels of p-JAK2, STAT3, p-STAT3, SOCS1 and SOCS3 proteins were increased in PDGF-D group, and decreased in rapamycin treated group (P<0.05 or P<0.01).③RT-PCR analysis indicated that the expression of SOCS1 mRNA and SOCS3 mRNA was higher in PDGF-D group than in control group, while rapamycin down-regulated the expression of SOCS1 mRNA and SOCS3 mRNA in mesangial cells cultured in PDGF-D medium.Conclusions:From above results we can draw the following conclusions:①In IgAN, the expression levels of p-JAK2, STAT3, p-STAT3, SOCS1, SOCS3 and PDGF-D were up-regulated in renal tissue of the patients with IgAN. PDGF-D and JAK/STAT signaling pathway may be of pathogenic significance in IgAN.②PDGF-D could activate JAK/STAT signaling pathway in human glomerular mesangial cells. PDGF-D enhanced phosphorylation of JAK2/STAT3 and increased the expression of SOCS1 and SOCS3. AG490, specific inhibitor of JAK2, could inhibit activation of JAK/STAT signaling pathway in glomerular mesangial cells under PDGF-D stimulation, which suggested PDGF-D may play an important role in the pathogenesis of IgAN through JAK/STAT signaling pathway.③Rapamycin could inhibit phosphorylation of JAK2 and STAT3 in IgAN, which may be responsible for the renal protective effects of rapamycin in rats with IgAN. Rapamycin may be a promising new drug in the treatment of IgAN.④PDGF-D may have therapeutic effects on IgAN. JAK/STAT inhibitors may be new target of treatment of IgAN.