New Treatment Strategy On Diabetic Kidney Disease By Targeting To Inflammation

Background:Diabetic kidney disease(DKD) was one of the most serious complications in Diabetes Mellitus (DM).DKD could affect the prognosis.The treatments of DKD so far were limited. More and more evidence indicated that the micro-inflammation in kidney in local tissue were important pathogenic factors of Type2DM and DKD, whose molecular mechanism was unclear. It was reported that inflammasome, microRNA might have an great influence on the inflammation state of DKD. Parthenolide(PTN), known as the inhibitor of NF-KB and NLRP3, could inhibit the systemic inflammation state. Our previous research showed PTN could relief the clinical and pathologic condition of DKD in db/db mice. And it could also improve the local IR and decrease the inflammatory factors, as MCP-1and IL-6. The purpose of this research was to confirm whether there was local inflammation in DKD patients which showed changes of CD68, NF-Kb and NLRP3. And we tested the changes of NF-Kb pathway and NLRP3Pathway in the db/db mice to indicat how PTN inhibited the local inflammation and delayed the progress of DKD.Objectives:to reveal the phenomenon of The recruitment of macrophages, activation of NF-K B and NLRP3in DKD patients. And we studied the changes of NLRP3pathway and NF-κB pathway in db/db mice model. And besides the next generation sequencing was used to investigate the changes of microRNA expression profiling in mice. And the aim of this research was also to explain how PTN delay the DKD through suppression of local micro-inflammation.Methods:(1) Retrospective analysis the clinical and examine data of CG patient from January2011to January2014, there were37patients diagnosed as DKD by confirm of renal biopsy. There were7patients with shorter diabetic kidney disease history and with pathology of glomerular hypertrophy were encoded as early stage group (DKD1). Meanwhile, the other30patients were encoded as middle and end stage group (DKD2). And we picked10patients diagnosed minimal change disease and15patents diagnosised membranous nephropathy as control groups (CTL1and CTL2). Immunohistochemistry was used to test CD68, NF-K B and NLRP3in the tissue of kidney to confirm local inflammation state.(2) Three groups of mice was used:1) db/m mice treated with normal saline (NS) as control;2) db/db mice treated with NS as DKD model;3) db/db mice treated with Parthenolide (PTN) as treatment group. The mice were sacrificed at TO (week8), T4(weekl2), T8(weekl6) and8Tusse from T8(2Control,2DKD model and4PTN group) were sent to sequence. Real time PCR was used to test the expression in different time of T0, T4, T8in db/m+NS, db/db+NS and db/db+PTN.(3)Real-time PCR, western blotting, inmmunohistochemistry and Enzyme Linked Immunosorbent Assay were used to test the expression of IRAK1, TRAF6, IK B, NF-K B, NLRP3, Caspase-1and IL-1in db/db mice.Results:It was observed that there was positive CD68in cytoplasm of glomerular and tubulointerstitial cells. With the exacerbation of pathology, the positive CD68increased yet. In the kidney of minimal change disease and membranous nephropathy as control, the CD68positive area was extremely limited,. In pathology of the DKD patients in early stage (DKD1) which only showed glomerular hypertrophy, the positive CD68area(OD0.0069±0.0067)in glomerulus and tubulointerstitium increased than control groups. The difference was statistically significant. And meanwhile, it showed plenty of positive CD68in the tissue of middle and end stage(DKD2) than both control groups and DKD1group. There were obvious statistical significant differences. In accordance with CD68, the NF-K B and NLRP3also tent to be more serious in the wake of DKD aggravation. The OD of NLRP3among DKD1group and DKD2group were0.158±0.077、0.186±0.077respectively. DKD2group was statistical different with the control groups and DKD1group. And The OD of NF-K B among DKD1group and DKD2group were0.054±0.018、0.127±0.080respectively. DKD1and DKD2were statistical different with the CTL groups. In the contrast of DKD and control group, the numbers of up-regulation and down-regulation microRNA were206and91respectively. And compare with DKD and PTN group, the the numbers of up-regulation and down-regulation microRNA were101and76respectively. If the filter criteria was that on the premise of some amount of expression, the expression in DKD group was down-regulated than control, meanwhile it was the most significant up-regulation from DKD group to PTN group. There were68microRNA meets the condition. The top five were microRNA146a-5p, microRNA223-3p, microRNA16-5p, microRNA126-3p, microRNA195-5p. And on the contrary,72microRNA meets the condition of up-regulation in DKD gtoup and down-regulation in PTN group on the premier of some amount expression. They were microRNA204-5p, microRNA7d-3p, microRNA2137, microRNA5115and microRNA193b-3p. most of above microRNA were relative weth inflammation, tumor, revascularization and so on. microRNA146a-5p meets the condition of down-regulation statistical significantly in DKD group and up-regulation most in PTN group. Though tested in real time PCR, We found microRNA146a-5p maintained stable expression in db/m T0, T4, T8amd db/db+NS T0, db/db+PTN TO groups. As the mice aging, the expression of db/db+NS group reduced gradually, until T8group reach the statistical significant. The Δ OD of db/db+NS T4and T8were0.42±0.38,0.34±0.12respectively. After treatment by PTN, the expression of microRNA146a-5p in db/db+PTN group up-regulated both in T4and T8. TheΔ OD were1.07±0.96,0.72±0.53respectively. The expressions were double fold than db/db+NS group with same age, but without statistical significance.The expression of IRAKI,TRAF6, IK B, NF-K B, NLRP3, Caspase-1and IL-1were stabilized in a low level in control groups(db/m+NS).Comparison with DKD group and control group,the premier one was higher statistical significantly. And contract on DKD group, the expression of PTN group decreased. IK B as the inhibitor of NF-K B, showed positive on the proximal renal tubule in control group. And with the ages, the IK B faded away. But After treatment of PTN, the IK B showed up again. On the contrary of IK B, NF-K B showed limited in the control group. With the mice ages, the inmmunohistochemistry positive area of db/db+NS group increased. And after PTN treatment, it turned to decrease. And contract on DKD group, the expression of PTN group decreased. Pro-IL-1was not affected by PTN. And with the ages, the expression of NLRP3, caspase-1and IL-1increased in db/db+NS group..Conclusion:there were chronic micro-inflammation in diabetic nephropathy patients and db/db diabetic nephropathy mice. There were activation of NF-K B pathway and NLRP3pathway in DKD patients and db/db DKD model. And changes of microRNA were also important factors in DKD. Especially microRNA146a-5p were inhibited statistical significantly, which could induced the activation NF-KB and NLRP3.And with treatment of PTN, the expression of microRNA146a-5p elevated double. On conclusion, PTN might take path to release the microRNA146a-5p and inhibit NF-KB, NLRP3and then DKD local micro-inflammation, and suspending DKD.