| Renal fibrosis is a common pathologic lesion in the end stage of various progressive kidney diseases, and is characterized by interstitial inflammation, proliferation of myofibroblasts, and progressive accumulation of extracellular matrix(ECM). The JNK-associated leucine zipper protein (JLP) is a crucial scaffolding protein in signalling transduction and molecular trafficking. JLP has been found expressed in mouse tissues of brain, lung, spleen, and testis. We reported here, for the first time, the role of JLP deficiency in the progression of renal fibrosis in a mouse model of unilateral ureteral obstruction (UUO).Part I The role of scaffolding protein JLP deficiency in the progression of renal fibrosis in a mouse model of unilateral ureteral obstructionObjective:To observe the role of scaffolding protein JLP deficiency in the progression of renal interstitial fibrosis in a mouse model of unilateral ureteral obstruction, and to investigate the regulation mchanism of JLP in preventing renal interstitial fibrosis in obstructive nephropathy.Methods:jlp Wild type (jlp+/+) and jlp deficient (jlp-/-) mice were divided into four groups:jlp+/+-and jlp-/--sham-operated groups(jlp+/+-sham group and jlp-/--sham group), jlp+/+- and jlp-/--unilateral ureteral obstruction (UUO)-operated groups (jlp+/+-UUO group and jlp-/--UUO group). Mice were sacrificed 7 and 14 days later. Histological changes in renal tubular interstitium were observed with Masson taining. The expression of JLP was determined by immunofluorescence studies, immunohistochemistry staining and Western blotting in contralateral kidneys in jlp+/ mice. Immunohistochemical staining was used to detect the expression of a-smooth muscle actin (a-SMA), collagen Ⅰ (COL-Ⅰ) and collagen Ⅲ (COL-Ⅲ) in sham and UUO groups. Besides, the protein levels of a-SMA, COL-Ⅰ and COL-Ⅲ were also analyzed by Western blotting in each group.Results:Immunohistochemistry result showed that JLP was mainly distributed in the renal tubules in contralateral jlp+/+ mice. One or two weeks after surgery, more collagen deposition was observed in the renal interstitial area in jlp-/--UUO group than in jlp+/+-UUO group. Similar to that, the expression of α-SMA, COL-Ⅰ and COL-Ⅲ were significantly increased in the kidney cortices in jlp-/--UUO group. Meanwhile, Western blotting showed that protein levels of a-SMA, COL-Ⅰ and COL-Ⅲ were also obviously higher in jlp-/--UUO group than in jlp+/+-UUO group.Conclusions:Scaffolding protein JLP is critical in preventing renal fibrosis in obstructive nephropathy through the inhibition of myo-fibroblast induction.Part Ⅱ The effects of scaffolding protein JLP deficiency on the expression of inflammatory factors in renal interstitial fibrosis in a mouse model of unilateral ureteral obstructionObjective:To observe the effects of JLP deficiency on the infiltration of macrophages and the expression of inflammatory factors in renal interstitium in a mouse model of unilateral ureteral obstruction, and to investigate the role of JLP in preventing renal interstitial fibrosis in obstructive nephropathy.Methods:jlp Wild type (jlp+/+) and jlp deficient (jlp-/-) mice were divided into four groups:jlp+/+-and jlp-/--sham-operated groups(jlp+/+-sham group and jlp-1--sham group), ylp+/+-and ylp-/--unilateral ureteral obstruction (UUO)-operated groups (jlp+/+-UUO group and jlp-/--UUO group). Mice were sacrificed at 7 day to evaluate the expression of tumor necrosis factor(TNF-a) mRNA, interleukine-1β(IL-1β) mRNA and monocyte chemoattractant protein-1(MCP-1) mRNA by fluorescent quantitative Real-time PCR(Real-time PCR). Immunohistochemical staining was used to detect the expression of CD68, TNF-α, IL-1β and MCP-1 in sham and UUO groups. Besides, the protein levels of TNF-α, IL-1β and MCP-1 were also analyzed by Western blotting in four groups.Results:Real-time PCR showed that the expression of TNF-α mRNA, IL-1β mRNA and MCP-1 mRNA were significantly increased in the kidney cortices in jlp-/--UUO group than in jlp+/+-UUO group. Similar to that, the expression of CD68, TNF-a, IL-1β and MCP-1 were significantly increased in the kidney cortices in jlp-/--UUO group by Immunohistochemical staining. Meanwhile, Western blotting showed that protein levels of TNF-α, IL-1β and MCP-1 were obviously higher in jlp-/--UUO group than in jlp-/--UUO group.Conclusions:Scaffolding protein JLP is critical in preventing the infiltration of macrophages and the expression of inflammatory factors in renal interstitium in obstructive nephropathy.Part Ⅲ The Effects of scaffolding protein JLP deficiency on the expression of TGF-β1/Smad and apoptosis in renal interstitial fibrosis in a mouse model of unilateral ureteral obstructionObjective:To observe the effects of JLP deficiency on the expression of TGF-β1/Smad and apoptosis in renal interstitial fibrosis in a mouse model of unilateral ureteral obstruction.Methods:jlp Wild type (jlp+/+) and jlp deficient (jlp-/-) mice were divided into four groups:jlp+/+- and jlp-/--sham-operated groups(jlp+/+-sham group and jlp-/--sham group), jlp+/+- and jlp-/--unilateral ureteral obstruction (UUO)-operated groups (jlp+/+-UUO group and jlp-/--UUO group). Mice were sacrificed at the days of 7 and 14 to evaluate the expression of TGF-β1 mRNA by fluorescent quantitative Real-time PCR(Real-time PCR). Immunohistochemical staining was used to detect the expression of TGF-β1 in sham and UUO groups. The proteins of TGF-β1, p-Smad2, p-Smad3 and p-P65 were also analyzed by Western blotting in four groups. Besides, the renal cell apoptosis was evaluated by TUNEL-assay.Results:Real-time PCR showed that the expression of TGF-β1 mRNA was significantly increased in the kidney cortices in jlp-/--UUO group than in jlp+/+-UUO group. Similar to that, the expression of TGF-β1 were significantly increased in the kidney cortices in jlp-/--UUO group by Immunohistochemical staining. Meanwhile, Western blotting showed that the proteins of TGF-β1, p-Smad2, p-Smad3 and p-P65 were also obviously higher in jlp-/--UUO group compared to jlp+/+-UUO group. Moveover, the apoptosis rate was significantly increased in jlp-/-UUO group than in jlp+/+-UUO group.Conclusions:The function of scaffolding protein JLP is important in preventing renal fibrosis through the inhibition of TGF-β1/Smad, NF-κB expression and apoptosis induction in renal interstitial fibrosis. |
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