The Study Of TIMP-1’s Regulation Of CCL2 And PAQRB In Mesangial Cells

Background:The mesangial proliferative glomerulonephritis (MesPGN) is a set of kidney diseases defined by pathological morphology, including IgA nephropathology and non-IgA nephrpathology.. Anti-Thyl nephritis model is the classical animal model of MesPGN with the characteristic of the self-limitedness in the proliferation of mesangial cells (MCs). It can be divided into three phases:the mesangial dissolved phase, the mesangial proliferative phase and the recovery phase. TIMPs are the endogenous inhibitors of matrix metalloproteinases (MMPs). TIMP-1 regulates many cytokines in the level of transcription and takes part in the process of inflammation. Our previous gene chip results showed that TIMP-1 and chemokine C-C ligand 2(CCL2) were increasing in the mesangial dissolved phase and peaked at the mesangial proliferative phase and finally backed to normal level in the last recovery phase.CCL2 is a chemokine known to recruit monocytes and macrophages to sites of inflammation.Nuclear factor kB (NF-kB) is an important transcription factor, it can promote the level of CCL2 in many different kinds of cells. Monocyte to macrophage differentiation-associated (MMD) is encoded by PAQRB gene, belonged to PAQR family, which induces monocyte differentiate into macrophage. We deduced thatin MesPGN, TIMP-1 could up-regulate the expression of CCL2 through NF-κB to promote the recruit of monocytes in the inflammation section, andby promoting PAQRB, induces monocyte differentiate into macrophage.Objective:Validate the mRNA level of TIMP-1 in anti-Thy-1 nephritis by Taqman technique;Test the regulation function of TIMP-1 in CCL2 and NF-kBby Taqman technique and Western blot;Validate the expression level of CCL2 after inhibiting NF-kBin over-expression cells; By RNA-Seq, we get the differential gene profiles in over expression TIMP-1 RMC; Detect the mRNA level of key gene PAQRB with Taqman probe technique in over-/low-expression TIMP-1 cell models.Method:(1) We established the anti-Thy-1 nephritis model, the animals were sacrificed at 0 day(control group),1 day,3 day,4day,5 day and 7 day (model groups) to collect the glomerular of kidney from each group for RNA isolation, mRNA level of TIMP-1 and MCP-1 were then detected at different time points of anti-Thy-1 nephritis by Taqman probe technique.(2) We established the TIMP-1 over-expression and low-expression rat mesangial cell model. About over-expression model, we transfect GFP-TIMP-llenti-virus into RMC and observe the transfection effect from day 3; TIMP-1 siRNA sequences were designed and the low expression model was created by liposome transfection technique. (3) Harvested TIMP-1 over expression cells of day 6 and the 48h cells of low-expression model. Then we detected their mRNA level and protein level of TIMP-1, MMP-2, MMP-9, NF-κB and CCL2 by Taqman probe technique and Western Blot.(4)We treated TIMP-1 over-expression cells with NF-κBinhibitor BAY11-7082(5μmol/L) at 4th day. After 48h-culture, we isolated the total RNA to obtain cDNA, then detected their mRNA level by Taqman probe technique. (5) By RNA-Seq technique, we found the differential gene profiles of TIMP-1 over-expression cells.GO-Analysis were then performed to identify the function of key gene PAQRB. (6) The expression level of PAQRB in TIMP-1 over-/low-expression models wasdetected by RT-PCR technique.Result:(1) In rat anti-Thy-1 nephritis model, the expression level of TIMP-1 and CCL2was increasing in the first place and then began to decrease. For TIMP-1, the increasing trend is obvious at the day of the 3rd,5th,7th day, and came to the peak at day 5, and the CCL2 came to the peak at day 2.(2) We established the TIMP-1 over-/low-expression model successfully, the results of fluorescent microscope revealed that the transfection efficiency was about 90%. (3) When TIMP-1 presented over-expression, the expression ofMMP-9, NF-κB and MCP-1 were up-regulated (P<0.05); on the contrast, when TIMP-1 presented low-expression, the expression of MMP-9, NF-κB and MCP-1 were down-regulated. (P<0.05)The change in expression of MMP2 was not obvious. (4) 48h after inhibition of NF-κB, the expression level of CCL2 in TIMP-1 over-expression cells was down-regulated (P<0.05).(5) TIMP-1 regulated many proteins related with cell proliferation/apoptosis, differentiation, inflammation, and signaltransduction, participated in the immune and inflammation reactions of MesPGN. The over-expression of TIMP-1 up-regulated the secretion of PAQRB, which promoted the differentiation of monocytes. (6) The expression level of PAQRB up-regulated when TIMP-1 presented over-expression(P<0.05), and down-regulated when TIMP-1 presented low-expression(P<0.05)Conclusion:TIMP-1 could participate in the immune and inflammation reactions of MesPGN by up-regulating the expression of CCL2 through NF-κB and promoting PAQRB in rat mesangial cells.