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Systemic lupus erythematosus (SLE) is an autoimmune disease, where the immune system loses the tolerance for endogenous nuclear materials like double strain DNA (ds-DNA) and the immune system also produces antibodies to against these endogenous nuclear materials. The intolerance of immune system will lead to systemic autoimmunity which will cause the damage of various organs and tissues. Lupus Nephritis (LN) is one of the most severe organ manifestations of autoimmune systemic lupus erythematosus (SLE), and LN is also a major cause of morbidity and mortality in patients with SLE. 1,2 It was shown that the overall survival of patients with SLE is 92% at 10 years after diagnosis, but the survival reduces to 88% when the Lupus Nephritis is developed, which means the presence of LN will significantly reduce the survival in patients with SLE. 3 The chance of SLE and developing lupus nephritis (LN) vary between different genders and ethnicities. SLE is more prevalent in women especially who are in the productive age than men in all ages and populations, and the black and Asian individuals tend to develop lupus nephritis than white and Hispanic.8 These differences might be attributed to genetic predisposition: the genotypes and antibodies which are closely related to the LN and SLE are more frequent in specific populations. Based on the accumulation of immune complex in glomeruli, the presence of mesangial orendocapillary proliferation, the overall extent of glomerular and glomeruli injury, lupus nephritis (LN) was classified into six classes by International Society of Nephrology/Renal Pathology Society (ISN/RPS) in 2003 4, and this classification gives a guidance to the treatment of LN. So far, many medications and treatments are clinically applied regarding different classes of LN. According to ISN/RPS, the patients who are classified to class Ⅲ or Ⅳ are highly recommended to use immunosuppressive agents and glucocorticoid(GC).3 Even though there are some novel therapies like calcineurin inhibitors (CNI) which can significantly reduce proteinuria and ameliorate the kidney failure, their potential nephrotoxicity associated with long-term administration remains a concern. So the corticosteroids combined with immunosuppressive agents like cyclophosphamide(CYC) or mycophenolate mofetil(MMF) azathioprine(AZA) are the current standard induction treatment regimens for patients with active severe LN, and low-dose corticosteroids plus either AZA or MMF are two commonly used maintenance regimens to maintain the disease in remission.1,5 However, there is an agreement that Long-term glucocorticoid administration is associated with damage and morbidity.4 The complications of GC treatment are extremely common, involving musculoskeletal, endocrinal, hematopoietic, and cardiovascular systems. The adverse effects which have particular medical importance include osteoporosis, glucose intolerance and diabetes, central obesity, muscle wasting, the risk of infection. 9 Even though the bone of patients who are treated with GC can be protected by bisphosphonates and teriparatide to prevent bone loss and fracture10,11, other adverse effects are hardly to be handled by specific and efficient medicine.9 Therefore developing agents which possess the intrinsic anti-inflammatory actions of glucocorticoids but do not have their detrimental effects was proposed, and this kind of agents were labeled selective glucocorticoid receptor agonists (SEGRAs). 12 It has been widely assumed that glucocorticoid exerts its effect by binding with glucocorticoid receptors (GRs) in the cytoplasm. This ligand binding will translocate the GRs to the nucleus, enabling them to bind with genes and alter the transcription. There are many mechanisms regarding glucocorticoid signaling pathways, but they can be broadly divided into transactivation and transrepression. These two mechanisms has not been fully understood, but it has long been hypothesized that auto-inflammatory actions of glucocorticoid are associated with transrepression since GC can bind with proinflammatory signaling molecules and prevent these factors from inducing expression of proinflammatory gene products. On the other hand, the adverse effects of long-term glucocorticoid use are typically assumed to be related to transactivation. Utilizing the difference between these two molecular pathways help researchers to develop the SEGRAs which have strong transrepressive capacity but reduced or absent transaction ability. 9,12,13 However, one of the major problems of developing such agents is that the negative effects of glucocorticoid are not only mediated by the transactivation but also the transrepression, so whether SEGRAs can completely eliminate the side effects of the GC is still questionable. Regarding the therapeutic effect and irreplaceable position of glucocorticoid (GC) in clinical treatment of Lupus Nephritis (LN) and its potential toxicity, Dr. Wang et al 6 proposed to use nanomedcine to address this challenge. They prepared a polyethylene glycol-based macromolecular prodrug (ZSJ-0228) of dexamethasone, which can self-assemble into micelles in aqueous media and the efficacy and toxicity of the ZSJ-0228 were also tested. Their research showed that ZSJ-0228 can be sequestrated in the inflamed kidney tissues based on the mechanism of “ELVIS”,14 which refers to Extravasation of nanomedicine through Leaky Vasculature and its subsequent Inflammatory cell-mediated Sequestration (ELVIS). Besides, the ZSJ-0228 showed a better therapeutic effect of ameliorating LN without apparent GC toxicities and complications compared with free dexamethasone. Based on their previous research, my project is to further explore the efficacy and toxicity of ZSJ-0228 in different dose. The significance of this project is that the biologic and side effects of the corticosteroids are significantly influenced by dosage and dose schedules. As mentioned before the adverse reactions of corticosteroids can vary from the mild and self-limited to major or life-threatening such as osteonecrosis or septic shock. Side effects are, in general, dose-related and thus the higher the initial and cumulative dose and the longer the course, the greater likelihood of significant side effects.16 Therefore to find an optimal dose and dose schedule for ZSJ-0228 is important for its further application. |
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Content |
The NZB/W_F1 mice which can develop glomerulonephritis spontaneously was used in this project, and all the mice are female because it was mentioned previously that the disease processes more rapidly in females, and lupus is about nine times more common in females than in males.7 All mice were randomized into 7 groups (every group has 13 mice) including 8 mg/kg/day Dex equivalent ZSJ0228 i.v. monthly (Very high dose group, VH), 3 mg/kg/day Dex equivalent ZSJ0228 i.v. monthly (High dose group, H), 1 mg/kg/day Dex equivalent ZSJ0228 i.v. monthly (Medium dose group, M), 0.5 mg/kg/day Dex equivalent ZSJ0228 i.v. monthly (Low dose group, L), 1 mg/kg/day Dex i.v. daily (Dex-D group) , 1 mg/kg/day Dex i.v. monthly (Dex-M group), and Saline i.v. monthly (Saline group). Only the mice which have established nephritis (proteinuria reading=2 in two consecutive days) are enrolled in this study. Mice that develop severe proteinuria (ge;2000 mg/dL) or showed any signs of distress (e.g., reduced mobility, weight loss gt;20%, edema, unkempt appearance) are sacrificed immediately. After 8 weeks, the surviving mice are euthanized by CO2 asphyxiation, and the kidney, formal bone and blood of the mice are isolated, weighed, and persevered in formalin solution. The kidneys are embedded and sectioned firstly, and then histological scoring, immune complex level and macrophage infiltration can be obtained via immunohistochemistry (IHC), immunofluorescence (IF). The blood needs to be centrifuged to obtain the serum, and then the analysis of total IgG and anti-dsDNA IgG can be implemented via enzyme-linked immunosorbent assay (ELISA). Femoral bone quality is analyzed using a Skyscan 1172 micro-CT system. The histological scoring will indicate the disease severity in each group. In the previous work, Dr. Wang et al 6proved that more than 40% of the saline and DEX-treated mice developed severe glomerulonephritis but only ~11% mice treated with ZSJ-0228 had severe glomeruli damage, which revealed the capability of ZSJ-0228 in treating Lupus Nephritis(LN). Systematic immunosuppression and osteopenia is associated with the toxicities of long-term exposure of GC, and in the ZSJ-0228 treatment for LN study, 6 the serum total IgG of animals which were treated with daily DEX administration was significantly dropped even until the end of the experiment and the bone mineral density (BMD) and trabecular thickness (tb.th) in the femoral trabecular bone of mice treated with DEX was lower. However, ZSJ-0228 treated mice did not have any alternations of their total serum IgG and the BMD and tb.th were much higher than DEX and saline treated group, which indicated this prodrug did not suppress the immune system and improved the bone quality of mice. Dr. Wang et al6 also found that ZSJ-0228 treatment significantly lowered the renal macrophage levels when compared with Dex treatment and the saline control, which suggested that this prodrug can exert its therapeutic effect partially by ameliorating macrophage infiltration to the kidneys. An intriguing thing was also observed in their study that ZSJ-0228 could significantly reduce renal immune complex without any decrease of serum anti-ds DNA IgG, which means immunosuppressive effect of ZSJ-0228 is not systemic but is rather restricted to the inflamed kidneys. Therefore the histological scoring, total serum IgG, bone quality, anti-ds DNA IgG, renal immune complex and macrophage infiltration is also tested via various methods and techniques in this project to further explore whether the different dose will have an impact on toxicities and therapeutic effect of ZSJ-0228. |
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Methodology |
Tissue processing, fixation and sectioning: Before paraffin embedding, kidneys should be soaked in ethanol with graded concentration and Histo-Clear II to clear and dehydrate. The dehydrated kidneys are processed into paraffin which are preheated to 60℃. The kidney and paraffin attached to the cassette has formed a block which supports the kidneys structure and enables very thin sections to be cut and mounted onto microscope slides for analysis. All embedded kidneys are cut at a thickness of 5mu;m for further histological evaluation. Histological Evaluation: Paraffin sections of the mouse kidneys were deparaffinized, rehydrated, washed with water, and stained for evidence of glomerulonephritis, Glomerulonephritis was assessed by a pathologist using a semiquantitative 0-4 scale by light microscope. Scores of 1 and 2 represent mild and moderate focal disease respectively. Scores of 3 and 4 refer to diffuse disease and as such were classified as lsquo;severe glomerulonephritis.15 Immunofluorescence (IF) which combines the use of antibodies with fluorescence imaging technique to visualize the biomolecules in tissue sample. In our case, we use IF to obtain the graphical and quantifiable data of the macrophage infiltration in the glomeruli. When performing IF, macrophage can be detected using primary antibody covalently conjugated fluorophores. The concise procedure of kidney macrophage infiltration immunofluorescence includes Deparaffinization and rehydration of the kidney sections through Histo-Clear II and graded alcohol series; Retrieval of antigen which is to expose antigenic sites and allow antibodies to bind; Antibody incubation using 5ug/ml rat anti-mouse F4/80 (eBioscience, 41-4801-82) for 20 hours at 4℃; Coverslip mounting with Prolong Antifade Mounting Medium with DAPI; Imaging under fluorescence microscope. The number of macrophages in 25 glomeruli in every kidney section is counted, and then the average macrophages number of each sample is calculated. Immune complex level is analyzed by Immunohistochemistry (IHC), which also uses antibodies to detect the location of proteins and other antigens in tissue sections. The mechanism and protocol of IHC is quite similar with IF, but in IHC the antibody-antigen interaction is visualized using chromogenic detection with a colored enzyme substrate. After deparaffinization and rehydration, a rabbit-specific HRP/DAB (ABC) detection IHC kit (Abcam) was applied. Briefly, quenching endogenous peroxidase activity is performed after the antigen retrieval to avoid the negative effects of endogenous enzymes on chromogenic detection, and the antibody binding is visualized by the DAB staining. The staining intensity (represented as arbitrary gray units) of 50 glomeruli per mouse was quantified using Zeiss AxioVision software (version 4.6.3.0). Analysis of Serum Immunoglobulin and Autoantibody Levels: Enzyme-linked immunosorbent assay (ELISA) is used to quantify anti-dsDNA IgG and total serum IgG, which can indicate the serum immunoglobulin and autoantibody levels. The Mouse anti-dsDNA IgG ELISA Kit (Alpha Diagnostic, San Antonio, TX, USA) and Mouse IgG Antigen ELISA Kit(Innovation Research, Novi, MI, USA) are used to analyze the anti-dsDNA IgG and total serum immunoglobulin G respectively. The principle of the test is that the mouse IgG and anti-dsDNA IgG in the sample can bind with the affinity purified capture antibody coated on the microtiter plate. After a washing step horseradish peroxidase (HRP) labeled polyclonal anti-mouse IgG antibody binds to the captured protein. Excess antibody is washed away and chromogenic substrate (TMB) is added and color is developed by the enzymatic reaction of HRP on the substrate and then the absorbance at 450nm is measured by microwell reader, which is directly proportional to the amount of IgG and anti-dsDNA IgG present in the sample. A standard calibration curve is prepared along with the samples to be measured using dilutions of mouse IgG. The quality of Bone Skyscan 1172 Micro-CT system is used to observe the formal bone quality of the mice, in which attenuation coefficient (AC) is used to determine the density in Micro-CT image. AC is determined both by mass density and elemental composition of the material. However, when the X-ray absorption of a material is dominated by one specific material, then we can relate AC to the mass density of that material. The dominated material of X-ray absorption in bone is calcium hydroxyapatite (CaHA), the formula of which is Ca5(PO4)3OH. Based on this mechanism, the formal bones of each mouse are scanned under the parameters as below: Voltage: 55KV, Current: 181mu;A, Columns: 2000times;1336, Pixel size: 8.85 (9), Al 0.5mm Filter. After the scanning, three dimensional reconstructions were obtained using the NRecon and DataViewer software (Bruker micro-CT). A consistent polygonal region of interest of trabecular bone at the distal femur, from 20 slices (0.25 mm) to 100 slices proximal (1.25 mm) to the growth plate, was selected for bone quality analysis. The bone mineral density (BMD), bone volume/tissue volume (Bv/Tv), trabecular number (Tb.N), trabecular spacing (Tb.Sp), and trabecular thickness (Tb.Th) were quantified using the Bruker CTAn software. |
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Schedule |
Week 1-2: bone analysis. Week 3-4: kidney histological scoring; Week 5-6: kidney macrophage infiltration analysis; Week 7-9: kidney immune complex level analysis; Week 10-12: serum anti-dsDNA IgG analysis; Week 13-14: serum total IgG analysis; |
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Reference |
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