| (Objective]Diabetic nephropathy is one of the most important causes of end-stage renal disease. Increasing evidence has shown that diabetic nephropathy in experimental animals, as well as in diabetic patients, develop fibrotic changes such as extracellular matrix (ECM) accumulation in glomeruli, which manifest as overt nephropathy. Transforming growth factors-β (TGF-β) plays a central role in the progression of glomerular fibrosis and transduces its signal through the type I activin receptor-like kinase (ALK)5, and then specifically induces expression of fibronectin (FN), the plasminogen activator inhibitor type1(PAI-1), and ECM accumulations. Several studies have shown that selective inhibit ALK5could be a potential therapy for chronic renal disease. Salt-inducible kinase1(SIKl), a serine/threonine protein kinase, is originally found in the adrenal glands of rats fed a high salt diet. SIK1belongs to a family of AMP-activated protein kinase (AMPK) due to the acid sequence closely related to catalytic a subunit of AMPK. Topical study also has shown that SIKl is identified as an inducible gene target of TGF-β/Smad signaling, and that SIK1forms complexes with the ALK5, the inhibitory Smad7and Smurf2to down-regulate ALK5as well as negative regulation of TGF-β signaling pathway. Although animal experiment reported that SIK mRNA is found in the highest levels in rat kidney, the specific role of SIK1in the kidney, especially in the glomeruli, of diabetic rats have not been evaluated yet, and there was limited knowledge concerning the specific rule of SIK1which involved in glucose metabolism at cellular level.Therefore, in the current study, we assessed the possible role of SIK1in glomeruli of Streptozotocin (STZ)-induced type1diabetic rat. We were also using rat glomerular mesangial cells line (HBZY-1cells) to examine the modulation of SIK1protein under the condition of high glucose. Furthermore, we have constructed two specific SIK1expression plasmid and transfected them into HBZY-1cells respectively to explore whether SIK1play a beneficial role during mesangial cell fibrogenesis by regulating ALK5signaling pathway.[Methods]1.The expression of SIK1in the kidney of STZ-induced diabetic rats.Thirty male Specific-pathogen-free (SPF) Wistar rats were housed in temperature (23±3℃)and humidity-controlled (60±5%) room with a12:12-hour light-dark cycle and fed with the standard diet. After1week adaptation, rats aged8weeks were randomized into2groups, ten rats were chosen randomly as normal group and the rest twenty rats as a model of diabetic mellitus. The STZ-induced rats were injected intraperitoneally with60mg/kg body weight STZ in50mM sodium citrate solution (pH4.5) to make type1diabetic model, meanwhile, rats in the normal group were injected with50mM sodium citrate solution. Type1diabetic were defined by fasting serum glucose level>16.7Mm after72h of STZ injection. All animals were given with equal volume distilled water every day. At the age of12,16weeks, diabetic rats were fasted for12h and anesthetized with intraperitoneal sodium pentobarbital (65mg/kg), its blood was then collected and its kidneys excised. After12weeks of administration, each last rat aged20was placed in a metabolic cage for24-h urine collection, then rats were weighed and sacrificed, the left kidney was removed and weighed. Glomeruli were isolated from rat kidneys by a modified procedure. Serum glucose was measured by an automatic biochemical analyzer. Urea and creatinine (Cr) were analyzed using enzymatic kits. Urinary microalbumin was measured by immunoturbidimetric assay using mALB kit. Albumin excretion rate (AER) was assessed as an index for kidney injury. Renal tissue samples for light microscopy were fixed in4%buffered paraformaldehyde,4-um-thick paraffin-embedded samples were used and Routine Hematoxylin-eosin (HE) staining and periodic acid Schiff (PAS) staining was performed, the glomerular tuft area and mesangial matrix index were measured. The lysates extracted from kidney and glomeruli tissues were used for Western blot and immunoprecipitation to examine the expression of SIK1and pT186-SIKl. The expression ALK5, FN and PAI-1expression were also examined of rats at the age of20weeks. Meanwhile, Kidney glomerular fibrosis is predominantly featured as the production of extracellular matrix (ECM) and Masson staining was used to observe the accumulation of ECM.2. High glucose significantly suppresses the expression and activity of SIK1, regulates the nuclear redistribution of SIK1and induces activation of the ALK5signaling pathway in HBZY-1cells, Overexpression of SIK1in HBZY-1cells resulted in downregulation of ALK5and its target gene FN and PAI-1, and reversed the high glucose-induced HBZY-1cellular overproliferation.HBZY-1cells, a rat glomerular mesangial cells line, were maintained at37℃in a humidified atmosphere of5%CO2and cultured in Dulbecco’s modified Eagle’s medium supplemented with10%fetal bovine serum,100U/mL penicillin and100μg/mL streptomycin. Cells were grown to-80%confluence and then incubated in serum-free medium for24h. To investigate the effects of glucose on protein expression of SIK1and pT186-SIK1, serum-deprived HBZY-1cells were cultured in DMEM containing30mM glucose (high glucose) for0,12,24and48h. At the end of each time, SIK1, pT186-SIK1, ALK5, FN and PAI-1expression were examined. In order to examine the direct effects of high glucose on the specific cellular localization of SIK1, HBZY-1cells were transfected with expression vectors for GFP-fused full-length SIK1(pEGFP-SIK1) and cultured with high glucose in glass-bottomed culture dishes for0and48h, and then observed and imaged by a Olympus confocal microscope. To examine SIK1negatively regulate ALK5signaling pathway, HBZY-1cells were randomly divided into three groups:untransfection normal group, empty vector control group and pCDFl-SIKl group. At48h after transfection, HBZY-1cells were collected and analyzed for SIK1expression, and were continuing cultured with high glucose for further48h to analyze SIK1, ALK5, FN and PAI-1expression by Western blot and immunocytochemistry analyses, MTT assays for HBZY-1cellular overproliferation analysis. Transient transfection of HBZY-1cells was carried out using Lipofectamine2000according to the manufacturer’s instruction. At48h after transfection, fluorescent microscopy was used to examine GFP expression and then the cells were collected for the extraction of protein.[Results]Part1(1). Twelve weeks after STZ injection, serum blood glucose level and systolic blood pressure were significantly higher in the type1diabetic group than in the normal group at the age of20weeks. The body weight of the rats in the diabetic groups was reduced in comparison with the normal group significantly. On the contrary, the ratio of kidney-to-body weight (KW/BW) was significantly higher in diabetic groups than in the normal group. The urine volume, AER, Cr, Ccr and Urea in the diabetic group were significantly increased compared with the normal group, indicating a damaged renal function in diabetic rats. Twelve wks after STZ injection, based on the results of HE and PAS staining, diabetic rats exhibited larger glomerular tuft area and an increased mesangial matrix index by two-fold compared with rats in normal group. Kidneys of the type1diabetic rats exhibited profound extracellular matrix deposition inside glomeruli. These indicating the pathological abnormalities were noteworthy in kidney of diabetic rats.(2). The expression of SIK1protein was investigated by immunohistochemistry and result showed that expression of the endogenous SIK1was detectable mainly in distal tubules and glomeruli but was weak in proximal tubules in the normal group. To our surprise, gradually decreased SIK1expression of diabetic rats was observed in glomerular tuft but not in distal tubules at the age of12,16and20weeks and lower than that of normal rats at age of20weeks. To further prove this finding and to investigate the activity changes of SIK1during diabetic nephropathy, we explored kidney SIK1protein expression and SIK1activity (Thr-182phosphorylation, pT182) by immunoprecipitation and Western blot. We found that there was no significant difference of the kidney SIK1expression among diabetic rats at the age of12,16and20weeks and normal rats at the age of20weeks. Nevertheless, SIK1activity (pT182) was significantly higher in kidney from normal rats at the age of20weeks when compared with diabetic rats at the age of12,16and20weeks. Activity of SIK1(pT182), with the diabetic state extending, was decrease rapidly in kidney from diabetic rats. Without any other process, Glomeruli-enriched tissues isolated from rat kidneys were lysed for Western blot analysis immediately. We found that the level of SIK1protein expression in the glomeruli isolated from kidneys of diabetic rat at the age of12,16and20weeks was significantly decreased than that of normal rats at the age of20weeks, glomerular SIK1expression of diabetic rat at the age of16and20weeks also markedly decreased than that of diabetic rat at the age of12weeks, but there was a negligible difference between diabetic rat at the age of16and20weeks.(3). To clarify the possible relationship between SIK1and ALK5signaling pathway under the state of diabetic glomerulopathy, Western blot analysis of ALK5, FN and PAI-1in glomeruli was performed. Of diabetes rats at the age of20weeks, as expected, glomerular expression of ALK5, FN and PAI-1were significantly higher than that of normal rats at the age of20weeks, in agreement with this, our further Masson staining showing the increased accumulation of glomerular ECM of diabetic rats compared with normal rats at the age of20weeks.Part2 (1). HBZY-1cells were stimulated with high glucose, and the activity resulting from Thr-182phosphorylation of the SIK1gene was monitored. The HBZY-1cells were cultured in media containing30mM glucose for0,12,24, or48h. Immunoprecipitation followed by Western blot analysis showed that SIK1activity decreased after glucose treatment in a time-dependent manner. The levels of Thr-182phosphorylation at12,24, and48h were24.8%,58%, and58.4%, respectively, compared to0h. The level of phospho-Thr-182is important to maintain the SIK1protein level, which was also true in the HBZY-1cells treated with high glucose. The density ratios of SIKl/β-actin at12,24, and48h were18.2%,61.2%, and72%, respectively, of that at0h. Further semi-quantitative RT-PCR analysis also demonstrated equivalent results at the transcriptional level under high glucose conditions at each time point. These results showed that high glucose concentrations induced the decreased activity and expression of SIK1. Because the intracellular distribution of SIK1reflects its functional activity we decided to examine the localization of SIK1protein in HBZY-1cells, immuno-positive signals for SIK1were detected in the cytoplasm and nucleus of unstimulated HBZY-1cells. After stimulation with high glucose, the SIK1signals gradually redistributed into the nucleus with decreased intensity.To confirm our results from immunocytochemical analysis, green fluorescence protein (GFP)-tagged SIK1protein was expressed in HBZY-1cells and the subcellular translocation of GFP signals was monitored before and after the addition of high glucose. In unstimulated cells, the GFP-SIK1signal was observed in both the nuclear and cytoplasmic compartments. When the cells were treated with high glucose, the green fluorescence signal was subsequently translocated into the nuclei from the cytoplasm. These results indicated that high glucose induced the nuclear import of SIK1, which might be a result from the reduction in SIK1kinase activity. (2). Western blot analysis using HBZY-1cells also showed that the level of ALK5was upregulated by high glucose with1.82-and2.92-fold inductions at24and48h, respectively (normalized with β-actin), whereas no significant difference in the ALK5level was observed between0and12h. In contrast to ALK5, protein levels for FN and PAI-1, downstream targets of ALK5, were also increased by high glucose in a time-dependent manner. Immunocytochemical analysis of FN and PAI-1protein in HBZY-1cells also provided consistent results at48h, suggesting that high glucose induces the activation of the ALK5signaling pathway, which may be attributed to the downregulation of SIK1.(3). To examine the involvement of the reduced levels of SIK1in the upregulation of ALK5signaling in glomerular mesangial cells, we decided to perform paradoxical assays through forced expression of SIK1in HBZY-1cells. The transformation efficiency, monitored by the control copGFP-expression, was approximately60%. When the SIK1expression plasmid was transformed into HBZY-1cells, the levels of SIK1protein and mRNA increased approximately1.8-and1.9-fold, respectively, compared to non-or mock-transfected cells. After stimulation of the cells with high glucose for48h, a negative correlation between the levels of SIK1protein and ALK5, FN, and PAI-1proteins was observed. When the SIK1expression plasmid was transformed into HBZY-1cells, the ALK5protein expression decreased to approximately38%in pCDFl-SIK1transfection cells compared to control cells. Immunocytochemical analysis of FN and PAI-I also supported the negative correlation between SIK1and ALK5signaling. A significant reduction of proliferation of HBZY-1cells from overexpression of SIK1was also observed.[Conclusions](1).STZ-induced type1diabetes rat model to demonstrate the possible role of SIK1in early diabetic nephropathy. Twelve weeks later, diabetic rats developed diabetic nephropathy showing typical disorder features which developed severe hyperglycemia, albuminuria and renal pathological changes such as the expansion of mesangial matrix and the accumulation of ECM, all of those are characteristic of early diabetic nephropathy, and demonstrated that an animal model of diabetic nephropathy was successfully established。(2). Endogenous SIKl was detectable mainly in distal tubules and glomeruli but was weak in proximal tubules in the normal group. Gradually decreased SIK1expression of diabetic rats was observed in glomerular tuft but not in distal tubules at the age of12,16and20weeks and lower than that of normal rats at age of20weeks. SIK1activity (pT182) was significantly higher in kidney from normal rats at the age of20weeks when compared with diabetic rats at the age of12,16and20weeks. Of diabetes rats at the age of20weeks, glomerular expression of ALK5, FN and PAI-1were significantly higher than that of normal rats at the age of20weeks. Our results indicated that decreasing expression and activity of SIK1in glomerular cells of diabetic rats may play an important role associated with abnormal fibrosis processing.(3). High glucose induces the depression of the activity and expression of SIK, the activation of the ALK5signaling pathway in HBZY-1cells and regulates the specific localization and distribution of SIKl in HBZY-1cells. But, forced expression of SIK1induces the degradation of ALK5and reduces the ECM production and reversed the high glucose-induced overproliferation in HBZY-1cells. |
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