| BackgroundChronic kidney disease is a group of diseases based on the presence of kidney damage or decreased kidney function for 3 months or more, severely resulting in uremia. That the increased prevalence, high rate of mortality and disability and high treatment costs of chronic kidney disease has become an important "global public health problem ", seriously affecting people’s health and life. Early stages of disease are often asymptomatic, are detected during the assessment of comorbid disorders, and can be reversible. Rapidly progressive diseases can lead to kidney failure within months; however, most diseases evolve over decades and some patients do not progress during many years of follow-up. So it is necessary to make the diagnosis and treatment of chronic kidney disease at an early time. A large number of studies have shown that proteinuria is closely related to the development of kidney disease. Proteinuria is not only a nephrotic risk of the general population and signs in patients with chronic kidney disease, but also the main factor leading to nephropathy. The study found that the rates of deterioration in kidney disease and mortality in cardiovascular disease in numerous patients with albuminuria were about six times higher than patients with microalbuminuria or normal people. Especially macroalbuminuria (e.g. the total daily protein excretion is greater than 500 mg) was always associated with clinical proteinuria and deterioration of renal function. Proteinuria plays a pathological role in the further decline of renal function mainly from the following aspects:1. Ultrafiltration of various components in plasma-such as complement, growth factors, inflammatory mediators, alteration of the quantity and quality of protein can aggravate nephrotoxicity of proteinuria; 2. Macroalbuminuria can cause fluid retention, high blood pressure, dyslipidemia and endothelial dysfunction; 3. Excessive protein load in podocytes, will release transforming growth factor β (TGFβ) transforming mesangial cell to fibroblast, thereby increasing glomerular injury; 4. The increased urinary protein activates the complement cascade in kidney by forming membrane attack complex binding to specific receptors, thereby promoting the development of injury. Therefore, how to reduce or eliminate the leakage of urine protein is the key to treatment of kidney disease. Incidence of proteinuria is closely related to dysfunction of glomerular filtration membrane. That any layer of the glomerular filtration barrier has gene or acquired defects can cause damage to the glomerular filtration membrane, leading to proteinuria and renal disease. In all pathological glomerular proteinuria, it has appeared that macromolecules (mainly albumin) constantly filtered out from the barrier. There are many pathogens resulting in glomerular filtration membrane dysfunction:1. It has been shown that hyaluronidase and doxorubicin induced proteinuria by destruction of glycocalyx layer of endothelial cells; 2. Intrinsic charge layer on glomerular basement membrane is damaged, albumin with negative charge leaking easily; 3. In clinic, manifestation of various diseases in podocytes is proteinuria.Current pharmacological treatments for glomerular diseases are insufficient, with only a slowdown of progression for most diseases, mostly controlling systemic co-factors rather than the initiators or primary mediators. Glomerulopathies such as diabetic nephropathy are treated foremost by inhibiting the renin - angiotensin-aldosterone system (RAAS) with angiotensin-converting enzyme inhibitors (ACEi) or angiotensin II-receptor blockers (ARB), which have both antihypertensive and antiproteinuric effects. In addition, immune glomerulopathies, e.g. membranous nephropathy, are treated with immunemodulators such as glucocorticoids, calcineurin inhibitors (e.g. cyclosporine A) and cytotoxic drugs (e.g. cyclophosphamide), which are effective but have considerable side effects and easily result repeated condition. So seeking for the treatment of chronic kidney disease is undoubtedly one of the current strategic focuses in kidney disease.Self-assemble have recently gained intensive research interest owing to their dominant position of creating new function materials or use in medicine and therapeutics. And it is also one of most significant problem which requires immediately solution in 21th century. The initial concept of self-assembly is from the life sciences, refers to a single biomolecular interactions by weak bonds (van der Waals, electrostatic, hydrophobic interaction, π-π bonds, hydrogen bonds and coordination bonds), or voluntary organization, forming ordered process structure, such as aggregation of protein folding, biofilm formation, or the formation of a complex living organisms. Currently, the self-assembly of biomolecules has been widely used for the treatment and detection of cells, tissues and even organisms. Therefore, how to develop a self-assembled biofilm similar to glomerular filtration membrane for repairing renal function is a subject demanding urgent study.For the biofilm targeted to glomeruli, it has to find specific protein in glomerular filtration membrane. Numerous studies have demonstrated that Nephrin is the specific protein of glomerular filtration membrane. Therefore, we designed the following experiment:1. To prevent a large number of proteins filtering out from glomerular filtration barrier, based on the principles of self-assembly this thesis mainly focuses on the synthese of polymers with negatively charged, then assembles it into a porous membrane with suitable size and aperture, then self-assembles into Nephrin antibodies, referred as polymer self-assembly (PMb-anti-nephrin), the polymer self-assembly could specifically target to glomerular, reducing urinary protein.2. We explore toxicity range of polymer self-assembly (PMb-anti-nephrin) and examine its specific target to podocyte in vitro.3. We verify the impact of polymer self-assembly (PMb-anti-nephrin) on urinary protein and podocyte lesions and explore its mechanisms through establishment of different nephropathic model.Chapter one Self-assembly of PMb-anti-nephrinObjectiveBased on the principles of self-assembly, this thesis mainly focused on the synthese of polymers with negatively charged, then assembled it into a porous membrane with suitable size and aperture 。Methods1. Polymer was prepared.2. Modified the charge of the polymer.3. Self-assembled the porous film.4. Linked FITC to porous film.5. Modified anti-nephrin to porous film.Results1. The porous membrane with negative charges had been successfully synthesized by self-assembly method, and its size and aperture were adjustable.2. The anti-nephrin molecular was successfully connected to porous membrane, which could be targeted to the glomerular surface.SummaryThe prepared porous membrane with negative charges and full of aperture, it may simulate the glomerular filtration membrane and provide new ideas for the treatment of chronic kidney disease.Chapter two Podocyte-specific targeting of PMb-anti-nephrin and its effect on cell vitalityObjectiveAccording to the research, nephrin is kidney-specific molecules only expressed in podocytes, mainly gathering at the foot finger-like projections of the cell. Therefore, we observed the effect of self-assembly PMb-anti-nephrin on the podocyte-specific targeting and cell vitality by linking anti-nephrin to polymer PMb.MethodsImmortalized murine podocytes were grown in RPMI1640 medium containing 10% fetal bovine serum. To passage the cells, undifferentiated podocytes were grown under ’growth-permissive’ conditions, which involved growing cells at 33 ℃ in the presence of interferon-y (10 U/ml). For podocytes to acquire a differentiated phenotype, cells were grown under ’restrictive conditions’ at 37℃ in the absence of interferon-y for 10-14 days. Podocytes were grown adherently, passaged every 2 to 3 days. When 70-80% cells were sticking on the well, they were cultured for 6h in free serum medium. Cell vitality was assessed using the CCK-8, immunofluorescence staining tested whether self-assembly PMb-anti-nephrin could be joined to the podocytes.ResultsPMb-anti-nephrin had no effect on cell viability within a certain range of concentrations. CCK-8 assay indicated that high concentration (10 mg/ml) of PMb-anti-nephrin inhibited the proliferation of cells, cultured longer, more obvious toxicity, those viability was reduced nearly 20%, while the low concentration of PMb-anti-nephrin had little impact on cell viability, even a concentration of 0.1 mg/ml could promote cell proliferation and cultured longer, this effect was more pronounced (p= 0.005). Immunofluorescence confirmed that after the antibody nephrin occupyed the binding sites on the cell membrane, PMb-anti-nephrin followed up could not bind to the cells; Only PMb, unlinked to nephrin antibody, was also difficult to bind to the cells; but PMb-anti-nephrin added directly could successfully target to podocytes.Summary1. A high concentration of PMb-anti-nephrin could inhibit cell proliferation, while the low concentration of PMb-anti-nephrin could promote cell proliferation.2. PMb-anti-nephrin successfully targeted to podocytes through anti-nephrin.Chapter three Impact of PMb-anti-nephrin on proteinuria and podocyte lesions in proteinuria nephropathic ratsObjectiveWe verified the impact of PMb and PMb-anti-nephrin on proteinuria and podocyte lesions and explored its mechanism through establishing proteinuria nephropathic model.MethodsAlbumin overload nephropathy model:40 normal female Wistar-Kyoto rats (initial weight 100-150 g), purchased from the Sourthern Medical University Animal Experiment Center, were subjected either to unilateral nephrectomy (UNX) or to sham operation (sham). One week after the operation, the UNX rats were received daily intraperitoneal injection of bovine serum albumin dissolved in saline (pH 7.4) at 5 g/kg/d, six days a week. Three days later, the UNX rats were randomized into 3 groups and received the following treatment:intravenously injection of PMb dissolved in saline (pH 7.4) at 30 mg/kg, intravenously injection of PMb-anti-nephrin at 30 mg/kg, or vehicle (saline, pH 7.4) alone, four days one injection. Animals were sacrificed on 32nd after BSA injection.PAN nephropathy model:24 normal male SD rats were received a single intraperitoneal injection of PA (the PA powder solute with sterile saline,20 mg/ml), 150 mg/kg. Three days after injection,16 randomly selected PAN nephropathy rats,8 rats were given intravenously 3 mg/kg PMb-anti-nephrin, others given intravenously normal saline of equal volume. Ten days after injection of PA, rats were killed.ADR nephropathy model:24 normal male BABL/c mice were received a single tail intravenous injection of adriamycin (the AD powder solute with sterile saline,2 mg/ml),10 mg/kg. Three weeks after injection,16 randomly selected ADR nephropathy mice,8 mice were given intravenously 30 mg/kg PMb-anti-nephrin, others given intravenously normal saline of equal volume. Five weeks after injection of AD, mice were killed.During the experiment,24 h urine or 12 h urine was collected for urinary protein, which was detected using Coomassie blue method. Blood was collected for serum creatinine. After stimulation, mice were anesthetized with 3% pentobarbital sodium (35 mg/kg, i.p.), heart, liver, lung, kidney and other tissues were collected for HE or Masson staining. Immunofluorescence staining was carried out to assess the expression of Podocin. Transmission electron microscopy was carried out to detect podocyte lesions.ResultsTrends of 24 h urinary protein in albumin overload nephropathy rats indicated that PMb could reduce urinary protein excretion up to 300 mg (P<0.05); while PMb-anti-nephrin reduced urinary protein excretion up to 500 mg (P<0.05), but neither was able to recover urinary protein to normal level. When stoping interfering during 35-41 days, the level of urinary protein began to rise again.24h urinary protein in PAN rats showed that PMb-anti-nephrin could significantly decrease urinary protein excretion (P<0.05), but it was not able to recover urinary protein to normal level. Urinary protein in ADR mice indicated that in the third week after giving PMb-anti-nephrin intervention, the level of urinary protein decreased significantly compared with the same period of the ADR group, only about half of which in the ADR group; elevated serum creatinine was significantly decreased after the intervention of two weeks, while the low glomerular filtration rate also increased significantly. As could be seen from pathological evaluation of various vital organs, PMb polymer self-assembly was safe and non-toxic. Histology stained with HE detected that PMb and PMb-anti-nephrin could make renal interstitial inflammatory cells significantly reduced, a large number of protein casts disappeared and renal tubular cell morphology restored. Immunofluorescence demonstrated that after PMb and PMb-anti-nephrin reduced urinary protein excretion, shape of Podocin was changed from a discontinuous distribution of fine particles into a kind of line-like distribution along the glomerular capillary loops, indicating that low protein expression of Podocin became significantly higher (P<0.05); Similar findings were also confirmed by transmission electron microscopy (TEM), in proteinuria nephritic group, we observed distortion of foot process architecture resulting in foot process effacement (FPE) or flattening at varying degrees; there was obvious vacuole degeneration in highly swollen cell body; foot process were widespread fused even loss, actin microfilaments segment got together to form plaque-like structures within the diffuse foot process fusion. After giving intervention of PMb and PMb-anti-nephrin, these lesions were significantly improved, only detecting segmental fusion of foot process.Summary1. PMb-anti-nephrin could significantly reduce proteinuria, which might be due to the porous biofilm simulating the filtration membrane and having negative charges.2. PMb-anti-nephrin could improve renal pathological alteration and prevent further damage of podocytes. |
PDF Full Text Request