| 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 and glomerulosclerosis. 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 transducer 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 to be implicated in the regulation of a wide range of cellular processes such as proliferation, differentiation and apoptosis. It was reported that activation of JAK/STAT was induced by high glucose and angiotensin II in rat glomerular mesangial cells cultrued in vitro. There are few reports about the JAK/STAT signaling pathway in diabetic kidney. Experimental and clinical investigations indicated that antagonists of renin-angiotensin system, such as angiotensin-converting enzyme inhibitor (ACEI) and angiotensin recepter blockade (ARB), have renal protective effects. As classic and effective drugs that can reduce lipid, statins have been used widely in clinical renal diseases treatment. Recent investigations showed that statins have renal protective effects independent of the effect to reduce lipid. In this study, Wistar rats were used to induce experimental diabetes and rat glomerular mesangial cells were cultured in high glucose medium to investigate the expression of JAK/STAT signaling pathway and effects of AT1Ra and HMG-CoA reductase inhibitor. Methods 1. Induction of diabetes and detection of JAK/STAT 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 days 3,7,14,28 after STZ injection. Partial renal tissure was fixed in 4% formaldehydum and embeded with paraffin. Partial renal cortices were used to isolate glomeruli and abstract total RNA and protein. The levels of expression of JAK2,STAT1,p-STAT1,STAT3 and p-STAT3 were evaluated by immunohistochemistry and Western blot. The tyrosine phosphorylation of JAK2 was detected by immunoprecipitation and Western blot analysis. The expression of STAT1 mRNA and STAT3 mRNA was measured by reverse transcription and polymerase chain reaction (RT-PCR). 2. Effect of losartan on expression of JAK/STAT signaling pathway in diabetic glomeruli Male Wistar rats were used to induce diabetes by injection of STZ. Rats were divided into three groups: control group(A), diabetic group(B) and losartan treated group(C). The method to make diabetic model is the same as that of part 1. After the diabetic model was affirmed to be successful, the rats of group C were administered daily with losartan(40mg/kg) by gavage for 2 weeks. Blood, urine and kidney samples were collected at week 2. Partial renal tissure was fixed in 4% formaldehydum and embeded with paraffin. Partial renal cortices were used to isolate glomeruli and abstract total RNA and protein. Immunohistochemistry, Western blot and immunoprecipitation analysis were used to investigate the expression of JAK2, p-JAK2, STAT1, p-STAT1, STAT3 and p-STAT3. RT-PCR was used to detect the expression of STAT1 mRNA and STAT3 mRNA in diabetic glomeruli. 3. Cell culture and examination of JAK/STAT signaling pathwayGlomerular mesangial cells were separated from male Wistar rats. The fourth passage 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 measure the proliferation of mesangial cells at 12, 24, 48 and 72 hours, meanwhile to investigate the effect of AG490 on proliferation of mesangial cells in high glucose medium. Glomerular mesangial cells between the fifth and the tenth passages were randomly divided into four groups: LG group(5.5mmol/L glucose);LG+M group (5.5mmol/L glucose+24.5mmol/L mannitol);HG group(30mmol/L glucose)and HG+AG490 group(30mmol/L glucose+10μmol/L AG490). The cells were cultured in 24-pore plate and 25cm2 plastic culture flask. Mesangial cells were harvested to abstract total RNA and protein at 48 hours after incubation. The expressions of STAT1, STAT3, p-STAT1 and p-STAT3 were examined by immunohistochemistry and Western blot. The tyrosine phosphorylation of JAK2 was detected by immunoprecipitation and Western blot analysis. The expression of STAT1 mRNA and STAT3 mRNA was examined by reverse transcription and polymerase chain reaction (RT-PCR). The contents of laminin and type IV collagen in the supernatants of the GMCs were detected by radioimmunoassay. The contents of TGF-β1 and fibronectin in the supernatants of the GMCs were measured by Western blot. 4. Effects of valsartan and fluvastatin on expression of JAK/STAT signaling pathway in glomerular mesangial cells Glomerular mesangial cells between the fourth and the sixth passages 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 valsartan and fluvastatin on proliferation of mesangial cell in high glucose medium at different time and different drug concentration. Glomerular mesangial cells between the fifth and the tenth passages were cultured in 25cm2 plastic culture flask and divided into four groups: LG group(5.5mmol/L glucose); HG group(30mmol/L glucose);HG+Val group(30mmol/L glucose+10μmol/L valsartan)andHG+Flu group(30mmol/L glucose+1μmol/L fluvastatin).Total RNA and protein were abstacted at 48 hours after incubation. The expression of STAT1, STAT3, p-STAT1 and p-STAT3 was measured by Western blot. The tyrosine phosphorylation of JAK2 was detected by immunoprecipitation and Western blot analysis. TGFβ1mRNA was measured by reverse transcription and polymerase chain reaction (RT-PCR). The contents of laminin and type IV collagen in the supernatants of the GMCs were detected by radioimmunoassay. The contents of TGF-β1 and fibronectin in the supernatants of the GMCs were measured by Western blot. Results 1. Morphological changes and expression of JAK/STAT signaling proteins In vivo:①Compared with the control group, there was no obvious morphological change of glomeruli at day 3 and 7, but the number of cells in each glomerular cross section was increased and glomeruli was enlarged at day 14 and 28. ②Immunohistochemical staining showed that the expression of STAT1, STAT3, p-STAT1 and p-STAT3 was positive in glomeruli of both control group and diabetic group. The expression of p-STAT1 and p-STAT3 was increased in diabetic glomeruli, compared with control group. ③Compared with control group, the level of tyrosine phosphorylation of JAK2 was increased at day 3, 7, 14 and 28. ④Western blot analysis indicated that the expression of STAT1, STAT3, p-STAT1 and p-STAT3 was increased at day 3 in DM group, peaked at day 14, and a slight decrease was observed at day 28. The expression of JAK2 was increased at day 7, and progressively increased with duration of diabetes. ⑤RT-PCR analysis indicated that the levels of exprssion of STAT1 mRNA and STAT3 mRNA were increased at day 3 in DM group, peaked at day 14, and slightly decreased at day 28. In vitro: ①Immunocytochemical staining showed that four STATs proteins were expressed in nuclei and cytoplasm of mesangial cells. Compared with low glucose control group, the expression of p-STAT1 and p-STAT3 was increased in high glucose group,and STAT1 and STAT3 were observedmainly in nuclei. The levels of expression of p-STAT1 and p-STAT3 in AG490 group were obviously lower than that of the high glucose control group. The nuclear translocation of STAT1 and STAT3 was inhibited. ②Compared with low glucose control group, phosphorylation level of JAK2 was increased in high glucose group at 48 hours(IOD: low glucose 132.2±21.9;high glucose 1339.2±96.7,p<0.01)and phosphorylation of JAK2 was inhibited by AG490 (IOD: 687.8±79.5,p<0.01)。③Compared with low glucose control group, the expression of p-STAT1 and p-STAT3 was increased (p<0.01)in high glucose group at 48 hours. The expression of p-STAT1 and p-STAT3 in AG490 group was decreased compared with high glucose group. ④RT-PCR showed that the relative expression level of TGF-β1 mRNA was increased in high glucose medium. After treatment with AG490, the relative expression level of TGF-β1 mRNA was decreased. ⑤The concentration of TGF-β1, fibronectin, laminin and type IV collagen in supernatants of the GMCs exposed to high glucose was higher than that in low glucose group at 48 hours. After treament with AG490, the secretion of TGF-β1, fibronectin, laminin and type IV collagen was decreased. 2. The effect of losartan on the expression of JAK/STAT signaling pathway Results from STZ-induced diabetic rats treated with losartan for 14 days are as follows: ①The phosphorylation level of JAK2 was decreased after losartan treatment (DM group: 3.12±0.45, losartan treatment group: 1.86±0.21, p<0.05). ②The expression of p-STAT1 and p-STAT3 was down-regulated in losartan treatment group( p-STAT1: DM group 2.93±0.34, losartan treatment group 1.76±0.19; p-STAT3: DM group 3.14±0.59, losartan treatment group 2.16 ±0.42 ). Losartan could not affect the expression of JAK2, STAT1, and STAT3. ③The expression of STAT1 and STAT3 mRNA was up-regulated in DM group, and losartan could not affect the expression of STAT1 and STAT3 mRNA. 3. Effects of valsartan and fluvastatin on expression of JAK/STAT signaling pathway in glomerular mesangial cellsCompared with low glucose, mannitol had no effect on proliferation of mesangial cells, while high concentration of glucose could promote the proliferation of mesangial cells. 1, 10 and 100μmol/L valsartan could suppress the proliferation of mesangial cells in high glucose. In fluvastatin treated group, there was little suppressing effect on the proliferation of mesangial cells at dose of 0.1umol/L, and the suppressing effect became apparent at a dose of 1umol/L. The suppressing effect was over at a dose of 10umol/L. Immunoprecipitation and Western blot analysis indicated that phosphorylation level of JAK2 was increased in high glucose group(IOD: low glucose 235.8±29.7;high glucose 2673.1±267.9,p<0.01), and was decreased in valsartan group and fluvastatin group (IOD: valsartan 987.6±112.3;fluvastatin 1259.7±164.5 p<0.01). Western blot analysis showed that high glucose stimulation could activate phosphorylation of STAT1 and STAT3 at 48 hours, valsartan and fluvastatin could reduce the level of phosphorylation of STAT1 and STAT3 in mesangial cells cultured in high glucose medium. The expression of STAT1 and STAT3 showed no significant difference among different groups. RT-PCR analysis indicated that the expression TGF-β1 mRNA was higher in high glucose group than in low glucose control group, while valsartan and fluvastatin could down-regulate the expression TGF-β1 mRNA in mesangial cells cultured in high glucose medium. The secretion of TGF-β1, fibronectin, laminin and type IV collagen in mesangial cells cultured in high glucose medium could be inhibited by valsartan and fluvastatin. Conclusions From above results we can draw the following conclusions: ①In the early stage of diabetes, the expression of JAK2, STAT1, STAT3, p-JAK2, p-STAT1 and p-STAT3 was up-regulated in glomeruli,with increase of STAT1 and STAT3 mRNA. High glucose can activate JAK/STAT signaling pathway in rat glomerular mesangial cells (enhance phosphorylation of JAK2, STAT1 and STAT3). AG490, specific inhibitor of JAK2, can inhibit activation of JAK/STAT signaling pathway and secretion of TGF-β1 and ECM in glomerular mesangial cells under high glucose medium, meanwhile...
|
PDF Full Text Request