Effect And Mechanism Of Periostin On Renal Glomeruli Lesions In Lupus Nephritis

Objective: Systemic lupus erythematosus(SLE) is characterized by the production of autoantibodies which drive immune-complex related inflammation and consequent tissue damage through binding to autoantigens in various tissues and organs including skin, joints, kidney and nervous system. Renal injury affects more than 60% patients with SLE, renal failure is a major contributor to morbidity and mortality. Currently, emerging evidence revealed that lupus nephritis is an important cause of chronic kidney disease(CKD). The morphological manifestation of lupus nephritis includes renal mesangial hypercellularity, mesangial matrix expansion, endocapillary proliferative lesions, glomerular basement membrane thickening, renal vein thrombosis and renal sclerosis. Among these renal lesions, the most typical features are mesangial cell proliferation and cumulative extracellular matrix.At present, increasing studies focus on a kind of extracellular matrix-related molecules, matricellular protein that is dynamically expressed non-structural protein and has regulatory role. Matricellular protein can modulate cell matrix interaction through binding cell surface receptors, as well as ECM structural proteins. The functions of matricellular protein are not only ECM remodeling, but also key regulations of matrix accumulation, fibrosis and cell-matrix interaction. Examples of matricellular proteins include thrombospondins(TSPs), osteopontin, CCN family, fibulin and secreted protein acidic rich in cysteine(SPARC) family.Osteoblast specific factor-2(periostin) is recently proven to be a kind of secretory, dissolved matricellular protein. It expresses mainly in such tissues abundant in collagen and has important physiological functions. Periostin is involved in the formation of extracellular matrix by binding to Collagen I, Collagen V, fibronectin, tendon raw protein-C and heparin, which mediates the adherence between cells and matrix and regulates cell physiological behavior such as cellular signal transduction, proliferation and differentiation. It was reported that periostin expressed in kidney of diabetic nephropathy, UUO mice, lupus nephritis and Ig A nephropathy. However, the exact function and mechanism of periostin in lupus nephritis is still not clear. Therefore, lupus mice and renal mesangial cells were chosen to determine the relationship and mechanism between periostin and renal ECM accumulation, cell hyperplasia.MRL/faslpr mice and MMC(mice mesangial cell) line were used in this study to investigate the role and possible mechanism of periostin on the proliferation and extracellular matrix accumulating of MMC, and to deternmine whether glomeruli proliferation level, glomerulus extracellular matrix accumulating and renal function of mice were inproved by knockdowning periostin expression in renal tissue of MRL/faslpr mice, which will provide an important basis for targeted therapy of lupus nephritis.Methods:1 The expression of periostin, PCNA, Fn and TGF-β1 in glomeruli of MRL/faslpr miceSix 24-week female MRL/MPJ mice and MRL/faslpr mice were respectively designed as control group and LN(lupus nephritis) group. 24 h urine, angular venous blood and renal tissue were collected for relevant detection. HE staining was used to observe the morphological structure of glomeruli, PAS staining was used to evaluate the deposition of glomeruli extracellular matrix. The expression of periostin, PCNA, Fn and TGF-β1 was detected by immunofluorescence, immunhistochemical and Western blot. 24 h Upro(urine protein), BUN(blood urea nitrogen) and Scr(serum creatinine) were detected by Rayto RT-9600 semiautomatic biochemistry analyzer.2 The effect and its mechanism of periostin on proliferation level and extracellular matrix accumulation of mice mesangial cell lineMice mesangial cell line(SV40 MES 130) were cultured in DMEM/F12(3:1) medium(Gibco BRL) supplemented with 10% fetal bovine serum(Gibco BRL). The cells were synchronised by culturing in serum-free medium for 24 h. ⑴To detect the effect of PDGF on periostin protein, cell proliferation and extracellular matrix accumulation. The cells were collected at 0, 2,4,6,12 h after stimulation with PDGF(10ng/ml) to detect the expression of periostin, PCNA, Fn and TGF-β1 by Western blot. ⑵To detect the effect of periostin on cell proliferation and extracellular matrix accumulating, transfections of cells with periostin vector or black vector were performed with Lipofectamine 2000 according to the manufacturer’s instructions. The cells were randomly divided into control group, black vector groups and periostin vector groups. Then the cells were collected to detect the expression of periostin, PCNA, Fn and TGF-β1 by Western blot and immunocytochemistry.⑶The silencing effect of sh-periostin vector on periostin protein in mice mesangial cell was indentified by Western blot. Cells were randomly divided into control group, PDGF group, sh NC+PDGF group and sh-periostin+PDGF group to detect the role of periostin in PDGF-induced proliferation and extracellular matrix accumulation of mice mesangial cell. The proliferation level was measured by the expression of PCNA, and the accumulation of extracellular matrix was measured by the expression of Fn and TGF-β1.3 The effect of sh-periostin on glomeruli proliferation level, glomerulus extracellular matrix accumulating, and renal function of MRL/faslpr mice.Six 24-week female MRL/MPJ mice were designed as Control group, eighteen 24-week female MRL/faslpr mice were randomly divided into LN group, LN+sh NC group and LN+sh-periostin group. Electroporation technology was used to transfect vector in vivo in treatment group. Mice were sacrificed three weeks later after 24 h urine and angular venous blood were obtained, and renal cortex were collected for relevant detections. HE staining and PCNA detection were used to evaluate the proliferation of glomeruli in mice, PAS staining, Fn and TGF-β1 detection were used to evaluate the deposition of glomeruli extracellular matrix in mice. Expression of periostin was detected by immunofluorescence and Western blot, and expression of PCNA, Fn and TGF-β1 were observed by immunhistochemical staining and Western blot, 24 h urine protein, BUN and Scr was detected by Rayto RT-9600 semiautomatic biochemistry analyzer.4 The mechanism of PDGF mediated the periostin expression and the proliferation level and extracellular matrix accumulating of mice mesangial cell line.⑴Cells were randomly divided into six groups, and collected after stimulated by PDGF for 0, 30 min, 2h, 4h, 6h, 12 h. The expression of p-Akt-ser473,Akt protein was respectively evaluated by Western blot. ⑵LY294002, specific inhibitors of the PI3K/Akt pathway, was used to determine the role of PI3K/Akt pathway in PDGF-induced the expression of periostin, the proliferation level and extracellular matrix accumulating of MMC cells. Cells were randomly divided into four groups(control, PDGF, PDGF +LY294002, and PDGF +DMSO) to explore the effect of LY294002 on the expression of p-Akt-ser473, Akt, periostin, PCNA, Fn and TGF-β1. The cells in the PDGF group were pre-treated with LY294002(20 umol/L) for 1h. The cells were collected at 6h after stimulation with PDGF(10ng/ml), and the expression of periostin, p-Akt-ser473, Akt, PCNA, Fn and TGF-β1 were detected by Western blot.Results:1 The expression of periostin, PCNA, Fn and TGF-β1 were up-regulated in renal of MRL/faslpr mice.⑴HE staining showed that the volume of glomeruli and the number of glomeruli cells increased in the lupus nephritis group compared with control group. ⑵Result of immunofluorescence showed that periostin protein was mainly located in the cytoplasm of renal glomeruli cells, the expression of periostin protein was significantly higher in LN group than that in control group. No obviously PCNA, Fn and TGF-β1 expression was detected in glomeruli of control group mice. However, significantly increased PCNA, Fn, TGF-β1 were revealed in LN group by immunohistochemistry.⑶Western blot showed that the periostin, PCNA, Fn and TGF-β1 protein expression in renal cortex of LN group mice were up-regulated compared with control group. ELISA results showed that, in comparison with control group, the serum level of PDGF-BB in LN group increased significantly.2 periostin mediated PDGF-upregulated proliferation of MMC cells and extracellular matrix increase.⑴PDGF can elevate periostin protein expression in time dependent manner.Western blot result showed that in the PDGF-stimulated groups periostin protein expression was significantly higher than that in the control group. immunocytochemistry analysis revealed that the expression of periostin in the PDGF group was increased noticeably compared with the control group, The positive expression of periostin was located in the cytoplasm.⑵PDGF can increase mice mesangial cell proliferation and the expression of extracellular matrix. Western blot result showed that PCNA, Fn and TGF-β1 protein in the PDGF-stimulated groups increased at 6h compared with control group. ⑶The liposome-mediated gene transfer technology could successfully transfer periostin vector and black vector into mice mesangial cell line, and periostin vector significantly increased the expression of periostin protein in mice mesangial cells. Transfected periostin vector mesangial cells PCNA expression was enhanced significantly, and the expression of Fn and TGF-β1 protein increased significantly. Western blot and immunocytochemistry results showed that compared with black vector group, periostin vector group showed higher periostin, PCNA, Fn and TGF-β1.⑷Sh RNA vector aimed at periostin(sh-periostin vector) can partially reversed PDGF-induced mesangial cell proliferation and extracellular matrix expression. The liposome-mediated gene transfer technology could successfully transfer sh NC and sh-periostin vector into mice mesangial cell line, and sh-periostin vector significantly decreased the expression of periostin protein in mice mesangial cells. Transfected sh-periostin plasmid mesangial cells PCNA expression was attenuated greatly, and Fn and TGF-β1 generation decreased significantly. Periostin silence plasmid can partially reversed PDGF-induced mesangial cell proliferation and extracellular matrix accumulating.3 sh-periostin effectively decreases the glomeruli proliferation level, glomeruli extracellular matrix accumulating and 24 h urine protein of MRL/faslpr mice.⑴There was no obviously change in appearance, volume and weight of mice kidney among different groups, however, HE staining showed that the cell number of glomeruli decreased in sh-periostin group mice compared with LN group. PAS staining showed that the deposition of glomeruli extracellular matrix decreased in sh-periostin group mice compared with LN group. ⑶Immunofluorescence and Western blot showed that the expression of periostin protein decreased in glomeruli of mice of sh-periostin group compared with LN group. ⑷immunohistochemical staining and Western blot showed that the PCNA, Fn and TGF-β1 protein expression in renal cortex of sh-periostin group mice were down-regulated compared with LN group. The 24 h urine protein of mice in sh-periostin group decreased compared with LN group; and no significantly different was observed in BUN and Scr between two groups.4 PDGF mediated the periostin expression and the proliferation level and the generatin of extracellular matrix of mice mesangial cell line by PI3K/Akt signal pathway. ⑴Western blot results showed that in the PDGF-stimulated groups p-Aktser473(the phosphorylation of Akt ser473) reached peak at at 30 min, kept higher at 2h and 4h, began to decrease at 6 h and return to normal level at 12 h. ⑵Western blot results showed that the expression of periostin, p-Akt-ser473, PCNA, Fn and TGF-β1 protein was down-regulated in PDGF+ LY294002 group compared with PDGF+DMSO group.Conclusions:1 The expression level of periostin, PCNA, Fn and TGF-β1 were up-regulated in kidney of MRL/faslpr mice. knockdown of periostin expression could decrease glomeruli cell proliferation, glomeruli extracellular matrix accumulating in MRL/faslpr and improve proteinuria, which indicates that periostin might play an important role in regulating glomeruli proliferationand the deposition of glomeruli extracellular matrix of lupus nephritis.2 PDGF can enhance periostin protein expression and increase mice mesangial cell proliferation and extracellular matrix accumulation. periostin expression vector can increase periostin, PCNA, Fn and TGF-β1 protein in mice renal mesangial cells. sh RNA vector aimed at periostin can partially reversed PDGF-induced mesangial cell proliferation and extracellular matrix generation. PDGF mediated the periostin expression and the proliferation level and the expression of extracellular matrix of mice mesangial cell line by PI3K/Akt signal pathway.