| Background and objectiveThe main clinical manifestation of mesangioproliferative glomerulonephritis,mesangial proliferative glomerulonephritis and the other kidney diseases (such as diabetic nephropathy, lupus nephritis)is nephrotic syndrome. The common feature of the patients is a lipid metabolism disorder with hyperlipidemia, associated with glomerular mesangial cells proliferation often, and this results in the occurrence of glomerular fibrosis and renal sclerosis in the clinical chronic progress course,although the incidence reasons of these diseases are different.They are the main reasons leading to the occurrence and development of chronic kidney disease(CKD).In recent years, end-stage renal disease(ESRD), which is advanced stage pathological changes of CKD, has been one of the major health problems of mankind,and the incidence of CKD and ESRD increased in the world year by year. Statistics from U.S. National Institutes of Health show that in 2004, the incidence of cancer mortality, the highest mortality rate in recent years, heart disease and the other types of diseases in the United States is gradually reduced, and incidence rate of CKD increased significantly. Studies have shown that the incidence of American adults with CKD is about 11% in United States, and the number of ESRD prevalence increased by 120 thousands at least in 2010, at the same time the U.S. National ESRD patients reached up to 650 thousands. The patients of CKD with heart and cerebrovascular diseases increased significantly, therefore ESRD and CKD increasingly became a major health problem that people need to face with the development of socioeconomics and the changes of the people's traditionary diet.Renal tissue fibrosis is the main pathology change of CKD, and some researchers believe that renal interstitial fibrosis caused by renal tubular lesions plays an important role in the genesis and development of chronic kidney disease, in particular,many patients of kidney disease such as diabetic nephropathy with hyperlipidemia are considered to be caused by the lipid-leading nephrotoxic effects, whose mechanism may involve the deposition of a large number of lipid in the renal tubules, the activation of renal tubular epithelial cells by low density lipoprotein and the release of some active cytokines. They also believe that the severity of kidney diseases consequences caused by renal tubular diseases is serious compared with kidney diseases caused by glomerular lesions, so the documents about kidney diseases concentrated on research of aspects of renal tubules disease in recent years. However,kidney diseases, especially glomerular lesions, often lead to kidney glomerular filtration rate as well as hemodynamics change,this would make blood flow reduction or result in interruption of blood supply by the afferent or efferent glomerular arteriole. Then a series of renal tubular pathology changes such as atrophy,degeneration or necrosis will appear, because of blood supply of renal tubule mainly from the glomerular efferent arterial. It can be considered that glomerular lesions are initiating factors of tubular diseases and trigger a lot of tubular diseases,and many tubular diseases are probably the secondary changes derived from glomerular lesions. Therefore, it is necessary to strengthen related research on the glomerular diseases. Discovered in recent years, glomerular mesangial cells, as the most important intrinsic cells in glomerulus, especially its transdifferentiation phenomenon by various damage factors, play an important role in ESRD and CKD process caused by glomerulonephritis. Attention to transdifferentiation phenomenon of mesangial cells increased recently.Mesangial cells (MsC) are one of three glomerular intrinsic cells whose function is most active in glomcrulus, and they are the main target cells of a variety of pathogenic factors in kidney disease.As glomerular intrinsic vascular pericytes,Histology and Embryology research confirmed that mesangial cells and vascular smooth muscle cells are homologous. Mesangial cells are similar to vascular smooth muscle cells which have different cell subtypes at different times or in different states.In the embryonic and larval development of glomcrulus glomerular mesangial cell may proliferate actively, and intracellular a-smooth muscle actin (a-SMA) expression is positive, so the cell type is proliferation/secretion one. With the gradual development of the glomerular body, mesangial cells are in a resting, inactive state with low metabolism, and the cells do not express a-SMA, then they are called static type cells. When mesangial cells are in a stationary state they only exercise phagocytosis function and maintain normal extracellular matrix metabolic function under physiological conditions, but transdifferentiation of mesangial cell can occur again and become proliferation/secretion cells stimulated by external factors under pathological conditions, at this time the cells are called myofibroblasts (MFB). Its shape changes, and a-SMA is the main cytoskeleton and accompanied by an increase in cell proliferation and mesangial extracellular matrix, thus it result in the development of glomerulosclerosis, and this is an important step in the formation of kidney sclerosis. Therefore the role of mesangial cell transdifferentiation has important physiological and pathological significance.Hyperlipidemia, particularly LDL, is a major risk factor for atherosclerosis as known to all. LDL is very easily converted to oxidized low density lipoprotein(ox-LDL) due to various pathological factors such as oxidative stress in the body, and recent findings have shown that oxidized LDL has a stronger effect on atherosclerosis compared with LDL. Elevated plasma LDL concentrations in patients with abnormally lipid metabolism resulted by a variety of glomerulonephritis and diabetic nephropathy are more subject to researchers' attention. As early as 100 years ago,Virchow raised the view of lipid degeneration when describing Bright's kidney, later,Moorhead, the British scholar, raised the doctrine of kidney toxicity caused by plasma lipid metabolism in the 1980s. In recent years more and more experiments confirmed that hyperlipidemia are closely related to renal sclerosis, and there is a certain degree of similarity between the glomerular fibrosis and atherosclerosis in the morphological changes and the mechanisms, such as the proliferation of the cells, collagen and other extracellular matrix aggregation in the local tissue.Previous studies also have shown that phenotype adjustment of vascular smooth muscle cell plays an important role in atherosclerosis induced by ox-LDL, and it can be speculated that transdifferentiation of mesangial cells may also have important pathology significance in the development of glomerulonephritis and glomerular fibrosis under conditions of hyperlipidemia by oxidized low density lipoprotein stimulation as mesangial cells and vascular smooth muscle cells are of the same origin in the embryology. Therefore,depth research on regularity of mesangial cell proliferation and transdifferentiation in pathological conditions, such as ox-LDL, will help deepen understanding chronic progression mechanism of glomerulonephritis and glomerular fibrosis.In fact, the changing of mesangial cells and mesangial area are major pathological changes in the progression of kidney disease, but people pay more attention to the renal tubular diseases and focused more on the relationship between kidney interstitium and renal tubular epithelial cells, or some inflammatory mediators,cytokines on mesangial cells. However, there have been very few reports so far about the effects of ox-LDL inducing mesangial cells transdifferentiation directly and studies on ultrastructural stereological analysis of mesangial cells. This experiment intended to be used to investigate the role of ox-LDL on proliferation, cell cycle distribution, transdifferentiation, ultrastructure and function of mitochondria of rat mesangial cells cultured in vitro at the cellular level, and further study its effect on the glomerular fibrosis.Methods1. Cells culturing in vitro and toxicity effects of ox-LDL on mesangial cellsGlomerular mesangial cells were purchased from China Type Culture Collection located in Wuhan. The cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum in a humidified 5% CO2 incubator at 37?.The culture media were changed every 2 or 3 days. When cells reached con fluency,they were subcultured using the same incubation medium. Prior to experiments,subconfluent mesangial cells were serum starved and synchronized to quiescence by incubation in serum-free medium for 24h. Mesangial cells were then exposed to fresh culture medium containing final concentrations of ox-LDL (25, 50, 75, 100?g/ml) for the following experiments.Control mesangial cells were incubated in fresh serum-free medium with 0?g/ml ox-LDL. Then cells were collected after incubation for 72 hours and stained by trypan blue to count the quantitics of living cells.2. Mesangial cells proliferation level and cell cycle assayMTT was used to measure the proliferation level of mesangial cells in response to different concentration and different time; cell growth curve of seven days was used to observe mesangial cells growth trend; flow cytometry was used to detect cell cycle distribution and cells with proliferating cell nuclear antigen.3. Effects of ox-LDL on expression of ?-SMAmRNA, CTGFmRNA and protein, and FN, collagen ?Transmission electron microscopy and Western Blot were used to measure expression of ?-smooth muscle actin in mesangial cells stimulated by oxidized low density lipoprotein for 72 hours; RT-PCR was used to detect expression of ?-SMA mRNA, CTGF mRNA; Western Blot was used to detect expression of a-SMA protein,CTGF protein; ELISA was used to detect expression of FN and collagen IV.4.Effects of ox-LDL on mitochondrial function and migration ability of mesangial cellActivity of the mitochondrial cytochrome C oxidase was used to measure mitochondrial function after mesangial cells stimulated by oxidized low density lipoprotein for 72 hours; Transwell migration was used to detect the migration ability of mesangial cells in vitro.5.Effects of ox-LDL on the ultrastructural and morphologic changes of mesangial cellsThe morphologic changes of mesangial cells were observed by using transmission electron microscope and inverted microscope; the ultrastructure morphological changes of mesangial cells stimulated by oxidized low density lipoprotein were tested by applications of the stereological principles and methods. Measuring the stereological parameters of the mitochondrial Vv(Volume Density), Sv(Surface Density), Nv(Numerical Density), S(Mean Surface ), V(Mean Volume ).6.Statistical AnalysisUsing SPSS 13.0 statistical software for quantitative analysis of the experimental results, calculating mean and standard deviation (expressed as (x±s) )in each group of measurement data. Factorial analysis of variance (two-factor) was used to analyze the results of ox-LDL effect on mesangial cell proliferation (the main and two-factor interactions effects)assayed by MTT method. The means between the various levels of the same factors was compared using one-way ANOVA, F value is adopted if homogeneity of variance, otherwise Welch value. SNK test was used for multiple comparisons when homogeneity of variance, and Tamhane's T2 method used if heterogeneity of variance. The test results of mesangial cell mitochondria stereological parameters analysed by using independent sample t test. Each comparison is considered statistically significant at P<0.05 level.Results1.Cytotoxic effect of ox-LDL on mesangial cells growthAfter exposure to different concentrations of ox-LDL for 72 hours, the results of trypan blue stain displayed that viable count of mesangial cells were more than 95%,(0.964±0.009),(0.961±0.006),(0.970±0.005),(0.969±0.013),(0.965±0.012) for each group respectively, the mean difference between groups was not statistically significant (F=0.641,P=0.642). The findings indicated that effects of all concentrations of ox-LDL in this study on proliferation, transdifferentiation and the other behavior of mesangial cells were involved in over 95% of living MsC.The experiment demonstrated that ox-LDL had no obvious cytotoxic effects in the range of 25?100?g/ml.2.Effects of ox-LDL on mesangial cells proliferation and cell cycle changesMesangial cell growth curve showed that oxidized low density lipoprotein can promot the growth of mesangial cells significantly.The culture growth was relatively slow from the first day to third, then it grew significantly faster from the fourth day to sixth, and thereafter the growth speed was relatively stable.Through the MTT method to detect the cell proliferation and the determination of absorbance of mesangial cells stimulated by oxidized low density lipoprotein of different concentrations. The cells proliferation rates after 24h of control group, and induction groups of ox-LDL were: (0.898±0.065),(1.645±0.086), (2.092±0.170),(2.309±0.168),(2.572±0.057); and after 48h were: (0.956±0.059),(1.939±0.058),(2.170±0.054),(2.524±0.168),(2.745± 0.055); while after 72h were: (1.281 ±0.053),(2.484±0.245),(2.662±0.087),(2.765±0.131 ),(2.912±0.079).Two-way factorial analysis of variance showed that mean differences caused by main effects of different concentrations of ox-LDL and processing time on mesangial cell proliferation was statistically significant (F=286.953, P<0.001; F=76.209, P<0.001), respectively.There was interaction effect between ox-LDL concentrations and processing time(F=2.735,P=0.022). The proliferative effects of ox-LDL on mesangial cells were concentration-dependent, time-dependent.Through flow cytometry method to detect the cell cycle of mesangial cells stimulated by oxidized low density at different concentrations for 72 hours. The cells cycle phase of control group and induction groups of ox-LDL were: (96.21 ±2.41)%,(93.66±0.77) %,(89.96±2.53)%,(86.92±1.68)%,(81.37±2.16)% in G0/G1 phase,and(3.62±1.66)%,(6.05±0.72)%,(9.17±1.58)%, (12.95±1.46)%,(17.43±2.41)% in S phase. The results showed that different concentrations of oxidized low density affected the cell cycle significantly in G0/G1 phase and S phase, and the mean differences between groups were statistically significant (F=33.321,P<0.001;F=44.048,P<0.001).Proliferating cell nuclear antigen of control group and induction groups of ox-LDL detected by flow cytometry were: (2.31±0.13), (6.82±0.27),(11.61 ±0.65),(18.44±0.18),(26.65±0.71). The mean difference between control group and induction groups of ox-LDL was statistically significant (F= 1764.774, P<0.001).3.Effects of ox-LDL on morphological changes of mesangial cellsThere were a lot of long and slim microvilli, intensively on the control mesangial cells surface, and no evident actin were found in cytoplasm. Following 72 hours of stimulation with different concentrations of ox-LDL, especially, at an ox-LDL concentration up to 100?g/ml, a significant change of mesangial cells ultrastructure was observed. The shape of mesangial cells appeared to be changed obviously.Length of the microvilli on the cell surface was shorter and became more thickness as well as confluence, and its quantity decreased visibly, while the quantity of rough endoplasmic reticulum and mitochondrion increased markedly. Actin-like architecture that were lined with cell. axis can be seen in cytoplasm, which indicated that transdifferentiation of mesangial cells had taken place, then the cells changed into myofibroblast and had its morphological characters such as shape,ultrastructure and so on. The results showed mesangial cells altered its cell phenotype stimulated by ox-LDL.4.Effects of ox-LDL on expression of a-SMAmRNA, CTGFmRNA and protein and expression of FN, collagen ?.Expression of a-SMAmRNA, CTGFmRNA of control group and induction groups of ox-LDL detected by RT-PCR were: a-SMAmRNA: (0.210±0.015)?(0.681 ±0.027 )?(0.984±0.015)?(1.387±0.026)?(1.938±0.029),CTGFmRNA:(0.500±0.013)?(0.940±0.064)?(1.210±0.011)?(1.271±0.043)?(1.473±0.035). The mean differences between control group and induction groups of ox-LDL were statistically significant (F=3241.339, P<0.001; F=377.897, P<0.001); a-SMA, CTGF of control group and induction groups of ox-LDL detected by Western Blot were:a-SMA: (0.097±0.042)?(0.247±0.019)?(0.452±0.068)?(0.562±0.018),(0.758±0.073), CTGF:(0.086±0.035)?(0.683±0.206)?(0.986±0.153)?(1.41±0.153)?(1.747±0.057). The mean differences between control group and induction groups of ox-LDL were statistically significant (F=108.708,P<0.001;F=88.634, P<0.001).Expression of FN and collagen IV of control group and induction groups of ox-LDL detected by ELISA were:FN: (3.03±0.41),(6.38±0.57),(10.44±0.35),(14.71±0.76),(19.23±0.59); collagen ?: (8.65±3.13),(16.01±1.89), (25.231±2.91),(32.95±3.30),(38.71±2.27).The ,mean differences between control group and induction groups of ox-LDL were statistically significant (F=539.784, P<0.001;F=78.799, P<0.001).5. Effects of ox-LDL on mitochondrial function of mesangial cellMitochondrial cytochrome C oxidase activity assay to detect the effect of ox-LDL on mitochondrial function of mesangial cells in vitro after 72 hours showed that the cytochrome C oxidase activity in the control group and ox-LDL-induced groups were(117.37 ± 51.81), (296.71±83.27), (477.85±150.53), (765.72±187.71),(953.28±176.13), and the mean difference between groups was statistically significant(F=23.435,P<0.001),indicating that ox-LDL enhanced mesangial mitochondrial function to provide more energy for cellular activities as needed.6. Effects of ox-LDL on the migration ability of mesangial cellTranswell migration assay to detect the effect of ox-LDL on migration of mesangial cells in vitro after 72 hours showed that the number of migrated mesangial cells in the control group and ox-LDL-induced groups were (10.25±3.50),(19.75±4.65), (30.00±6.00), (42.75±4.57),(66.25±5.56), and the mean difference between groups was statistically significant (F=78.064,P<0.001).7. Stereological measurements ultrastructure of mitochondria in mesangial cellsThe mitochondrial parameters of the control group and the induction group of ox-LDL whose concentration is 50?g/ml for 72 hours were as follows:control group: Volume Density(1.283±0.061); Surface Density(?m2/3),(0.113±0.015); Numerical Density(?m-3), (0.047±0.005); Mean Volume(?m3)(2.863±0.230); Mean Surface(?m2), (1.175±0.167);induction group: Volume Density (0.135±0.006); Surface Density(?m2/3),(1.171±0.005); Numerical Density(?m-3), (0.499±0.004); Mean Volume(?m3),(2.410±0.913); Mean Surface(?m2),(6.912±0.943).For these five parameters,the mean differences between two groups were statistically significant (Volume Density t=83.550, P<0.001 ; Surface Density t=-632.015,P<0.001; Numerical Density t=-311.759,P<0.001; Mean Volume t=6.749, P<0.001; Mean Surface t=-59.867, P<0.001).Conclusions1. Oxidized low density lipoprotein can promote mesangial cell proliferation, and the effect may be related to stimulate expression of proliferation cell nuclear antigen,and change cell cycle of mesangial cell;2. Oxidized low density lipoprotein can affect the morphology, ultrastructure of glomerular mesangial cells and promote mitochondria to supply more biological energy;3. Oxidized low density lipoprotein have the effect of inducing mesangial cells to transdifferent into myofibroblasts,and promote expressionof ?-SMA,FN,collagen? and CTGF;4. Oxidized low density lipoprotein is closed to relate with glomerular nephritis of mesangial cells proliferation and this probably lead to or aggravate glomerular fibrosis through the above-mentioned effects. Oxidized low density lipoprotein is a risk factor for glomerular fibrosis.New points1. The result has demonstrated that oxidized low density lipoprotein has promoting proliferation effect on mesangial cells cultured in vitro and the effect is connected with expression of proliferation cell nuclear antigen, and change of cell cycle of mesangial cells;2. The result has demonstrated for the first time that oxidized low density lipoprotein can affect morphology, ultrastructure of mesangial cell and induce them transdifferentiation into myofibroblast;3. The ultrastructure and morphological changes of the glomerular mesangial cells stimulated by oxidized low density lipoprotein were quantitatively analyzed with applications of the stereological principles and methods for the first time,and it provided support to reveal the transdifferentiation mechanism of mesangial cells. |
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