| Backgroud: Mesangial proliferative glomerulonephritis (MsPGN, includes IgAnephropathy and non-IgA mesangioproliferative glomerulonephritis), is the mostcommon cause of chronic kidney disease and end-stage renal disease in China. It’simportant to study the molecular mechanism of mesangial cell proliferation andexplore effective treatments. Our previous studies have shown that mesangialproliferation-related factors (including proliferation promoters and proliferationinhibitors) play an important role in MsPGN. The expression of MAX interactor1(Mxi1) decreases significantly during the proliferative period in anti-Thy1nephritismodel rats and then gradually rises as proliferation declines, thereby suggesting apossible role for Mxi1in mesangial proliferative glomerulonephritis.Objective: Regarding Mxi1as the breakthrough point, this thesis will research theeffects of Mxi1-knockout on mesangial cell proliferation and glomerular proteinexpression profile in vivo, observe the influences of Mxi1deficiency on mesangialproliferative nephritis model, and confirm its effects in vitro. The objective is todetect the effects of Mxi1on mesangial proliferation and the pathway Mxi1workthrough.Methods:12weeks old male Mxi1+/+mice, Mxi1+/-mice and Mxi1-/-mice (n=6)were used to collect renal tissue specimens. PAS staining and the number counting ofnucleated cells in the glomeruli were used to evaluate the renal biopsy pathology.PCNA immunohistochemistry and Western blot analyses were used to observe cellproliferation, and the expression of mesangial cell marker desmin was examed byWestern blot.2D-LC/MS was used to analysis the influence of Mxi1-knockout onprotein expression, and the expression of partial differentially expressed proteinswere validated by Western blot. Habu-snake venom (3.5mg/kg) was injected into tailvein of the12weeks old male mice (Mxi1+/+mice and Mxi1-/-mice) to makemesangial proliferative nephritis model. PAS staining and pathological evaluation were used to evaluate the renal biopsy pathology, PCNA immunohistochemistry wasused to observe cell proliferation, and the expressions of p-ERK1/2and ERK1/2(differentially expressed proteins detected by2D-LC/MS) were examed by Westernblot. In cultured mesangial cells, plasmid and siRNA transfection were used topositive-and negative-regulate the expression of Mxi1. The proliferation level ofmesangial cells was detected by CCK-8, and the cell cycle progression was testedusing flow cytometry. By Western blot, the expressions of ERK1/2pathwaymolecules were analyzed. And ERK1/2pathway was inhibited by the ERK1/2pathway inhibitor U0126to observe its influence on the proliferation regulatory effectof Mxi1.Results:(1) PAS staining showed that the renal tissue of Mxi1+/+mice had nosignificant pathological changes, but light-moderate mesangial cell proliferation werefound in some glomeruli of Mxi1-/-mice. Compared with Mxi1+/+mice, the numberof nucleated cells in glomeruli of Mxi1-/-mice was slightly increased, the glomerularPCNA expression levels in Mxi1-/-mice were significantly increased, as the desminprotein expression level.(2) By2D-LC/MS, a total number of129differentiallyexpressed proteins were detected, including65upregulated and64downregulatedproteins in Mxi1-/-mice. Some differentially expressed proteins which maybeassociated with cell proliferation including mitogen activated protein kinase1(MAPK1, also known as ERK2), mitogen activated protein kinase3(MAPK3, alsoknown as ERK1), apoptosis associated speck like protein containing a CARD(Pycard), histone H2B, catalase, sodium coupled monocarboxylate transporter(Slc5a8) and S-phase kinase-associated protein1(Skp1) were tested again by Westernblot, and the expression trends were basically in same pattern with the2D-LC/MSresults. The expressions of ERK1/2in Mxi1-/-mice were higher than Mxi1+/+mice.(3)After the model-making, the survival rate of Mxi1-/-mice was lower than Mxi1+/+mice. The renal pathology showed significant mesangial dissolution andcapillarectasia on1and3days after injection. On7days after injection, Mxi1+/+miceshowed increased mesangial cell proliferation and mesangial matrix, other lesionswere disappeared gradually, Mxi1-/-mice showed diffused severe mesangial proliferation, segmental sclerosis and balloon adhesion could be found in someglomeruli, pathological evaluation showed the lesion degree was more severecompared with Mxi1+/+mice. Immunohistochemical results showed that theexpression of PCNA of model groups in each time point were significantly highercompared with control group. On1and3days after injection of HSV, PCNAexpression of Mxi1-/-mice were significantly higher than Mxi1+/+mice. Western blotshowed the expressions of p-ERK1/2and ERK1/2of model groups in each time pointwere also obviously increased, and the expressions of p-ERK1/2in Mxi1-/-mice werehigher than Mxi1+/+mice.(4) In cultured mesangial cells, compared with the controltransfection group, upregulation of Mxi1expression via plasmid transfectionsignificantly reduced mesangial cell proliferation, decreased the percentage of Sphase cells, reduced the expressions of the molecules in ERK1/2pathway includingp-ERK1/2, ERK1/2, p-MEK, MEK, Cyclin D1, Cyclin E, CDK2and CDK6.Meanwhile inhibition of Mxi1expression by siRNA produced the opposite effects.U0126could antagonize siMxi1transfection-induced mesangial cell proliferation,cell proliferation level of siMxi1+U0126group was lower than siMxi1transfectiongroup, and higher than U0126group.Conclusion: In this study, we found that:(1) Mxi1-knockout could increase theproliferation level of mesangial cell;(2) Glomerular protein expression profile waschanged obviously in Mxi1-/-mice, the expressions of ERK1/2in Mxi1-/-mice werehigher than Mxi1+/+mice;(3) Compared with Mxi1+/+mice, mesangial cellproliferation and pathological injury were aggravated in the mesangial proliferativenephritis model which was making by Mxi1-/-mice;(4) In cultured mesangial cells,upregulation of Mxi1expression significantly reduced mesangial cell proliferationand the expressions of the molecules in ERK1/2pathway, downregulation of Mxi1produced the opposite effects. ERK1/2pathway inhibitor could antagonize siMxi1transfection-induced mesangial cell proliferation. In conclusion, experiments in vivoand in vitro confirmed that Mxi1is a mesangial proliferative inhibitory factor, it playsa role in inhibition of mesangial cell proliferation by inhibiting the ERK1/2pathway. |
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