| Part One Establishment and identification of IgA nephropathy in rat modelObjective: To establish a rat model of IgA nephropathy by the oral immunization and observe the pathologic changes of the renal tissue for the study in the pathogenesis, prevention and treatment of IgA nephropathy.Methods: Twenty female sprague dawley (SD) rats were randomly divided into two groups (n=10), namely control group and IgA nephropathy group. Bovine serum albumin (BSA), endotoxin (LPS) and carbon tetrachloride (CC14) were jointly used to establish the rat model of IgA nephropathy and the specific implementation were as follows:BSA (400mg/kg, oral every other day) x6weeks+LPS (0.05mg, intravenous injection at the6th and8th weeks)+CC14(0.1ml dissolved in0.5ml castor oil, subcutaneous injection weekly) x9weeks. Animals were sacrificed in the tenth weekend, the glomerular deposition of IgA was detected by immunofluorescence, and the pathological changes of the kidney were observed by hematoxylin-eosin (HE) staining. Twenty-four-hour urine was collected to count the urinary red blood cells and to measure the24-hour urinary protein before animals were sacrificed.Results: In control group, no green fluorescence has been seen in the renal glomeruli, examination of kidney sections showed normal size of renal corpuscle, no significant proliferation of mesangial area, capillary loops were normal and no significant changes in the renal tubules and interstitial, microscopic hematuria has not been observed and the quantity of24-hour urine protein was normal. Compared with the control group, bright green fluorescence in a granular pattern in the mesangium has been seen in IgA nephropathy group. Examination of kidney sections from the IgA nephropathy group showed the structural lesions of glomeruli including glomerular hypertrophy, renal capsule dilation, renal glomerular smaller, glomerular vascular sparse, the mesangial proliferation, obvious fibrosis and the swelling of the renal tubule. Microscopic hematuria was observed and the quantity of24-hour urine protein increased in the IgA nephropathy group. Conclusion:Using bovine serum albumin (BSA)+endotoxin (LPS)+carbon tetrachloride (CC14) can successfully establish the rat model of IgA nephropathy. The success of establishment of rat IgA nephropathy model suggests that the intestinal mucosal barrier function damage and mucosal immune dysfunction may be involved in the pathogenesis of IgA nephropathy. Part Two The injury in intestinal mucosal barrier in the rat model of IgA nephropathy and its relationship with the pathogenesisObjective:To observe the pathologic changes in intestinal mucosal barrier in the rat model of IgA nephropathy, and to explore its relationship with the pathogenesis of IgA nephropathy.Fourteen female SD rats were randomly divided into two groups (n=7per group), namely control group and IgA nephropathy group. The pathologic changes in the intestinal mucosa were observed by hematoxylin-eosin staining, and combined with the image analysis system, the intestinal villus height, crypt depth, the ratio of them, volume density (Vv) and diameter of the central lacteals were measured. We observed the change of intestinal epithelial tight junctions of the rats using transmission electron microscope and the expression of intestinal epithelial tight junction proteins ZO-1and occludin using laser confocal microscope. The changes of the mRNA and protein expression of these proteins were measured by reverse transcription polymerase chain reaction (RT-PCR) and western blotting. The ratio of urinary lactulose/mannitol was measured by high performance liquid chromatography for assessing the intestinal permeability. The glomerular deposition of IgA was measured by immunofluorescence staining.Results:Compared with the control group, in IgA nephropathy group, intestinal epithelium sheded, intestinal villi became short and irregular, the number of intestinal villi reduced and their spacing widened. The congestive lamina propria, expanded lacteals and infiltration of inflammatory cells were observed in IgA nephropathy group. The results of image analysis showed that the intestinal villus height decreased, crypt depth deepened and the ratio decreased; Vv and diameter of the central lacteals increased; the density of tight junctions between intestinal epithelial cellls were reduced significantly; the mRNA and protein expression of intestinal epithelial tight junction protein ZO-1and occludin decreased; the ratio of urinary lactulose/mannitol increased; bright green fluorescence in a granular pattern in the mesangium was seen in IgA nephropathy group and the average optical density of glomerular IgA was positively correlated to the ratio of urinary lactulose/mannitol.Conclusions:In IgA nephropathy model group, the mucosal barriers were damaged, the permeability of the intestinal mucosa increased and the relative expression levels of the glomerular IgA was positively correlated to the permeability of the intestinal mucosa. These suggest that the mucosal barrier damage is closely related with the incidence and development of IgA nephropathy. Part Three Preventive and repairing effects of rhein on intestinal mucosal barrier injury in the rat model of IgA nephropathyObjective:To observe the preventive and repairing effects of rhein on intestinal mucosal barrier injury in the rat model of IgA nephropathy.Methods:Twenty-eight female SD rats were randomly divided into four groups (n=7per group), namely control group, IgA nephropathy group, rhein-treated group and rhein-prevented group. Using hematoxylin-eosin staining and image analysis system, the intestinal villus height, crypt depth, the ratio of them, volume density (Vv) and diameter of the central lacteals were measured. We observed the change of intestinal epithelial tight junctions of the rats using transmission electron microscope and the expression of intestinal epithelial tight junction proteins ZO-1and occludin using laser confocal microscope. The changes of the mRNA and protein expression of these proteins were measured by reverse transcription polymerase chain reaction (RT- PCR) and western blotting. The ratio of urinary lactulose/mannitol was measured by high performance liquid chromatography for assessing the intestinal permeability.Results:In control group, intestinal villi arranged in neat rows, the tight junctions between epithelial cells located on the outer side of the cell membrane to the top, banded structure was dense, close fit between neighboring cells, no significant gap, tight junction protein ZO-1and occludin uniformly distributed in the intestinal epithelial cells at the top of the junction. Compared with the control group, in IgA nephropathy model group, the arrangement of intestinal villi was not structured, the intestinal villus height decreased, crypt depth deepened and the ratio of them decreased; Vv and diameter of the central lacteals increased; the density of tight junctions was reduced significantly and blur; the fluorescence of ZO-1and occludin were weaker, reduced and uneven; the mRNA and protein expression of intestinal epithelial tight junction protein ZO-1and occludin decreased; the ratio of urinary lactulose/mannitol increased. Compared with the IgA nephropathy group, the intestinal villus height increased, crypt depth shallow and the ratio of them increased, volume density (Vv) and diameter of the central lacteals decreased, the intestinal epithelial tight junctions were repaired with respect to structural integration, with dense connection and high electron density; the fluorescence of ZO-1and occludin were stronger; the mRNA and protein expression of ZO-1and occludin increased; the ratio of urinary lactulose/mannitol decreased in the rhein-prevented group and rhein-treated group.Conclusions:The RH can promote the growth of intestinal epithelium, increase the expression level of intestinal epithelial tight junction protein ZO-1and occludin, repair the damaged tight junctions, protect the intestinal mucosal and repair the intestinal mucosal injury. Part Four Preventive and repairing effects of rhein on renal tissue in the rat model of IgA nephropathyObjective: To observe the pathological changes of renal tissue in the rat model of IgA nephropathy and the preventive and repairing effects of rhein on renal tissue.Methods:Twenty-eight female SD rats were divided randomly into four groups, namely control group, IgA nephropathy group, rhein-treated group and rhein-prevented group. The pathologic changes in renal tissue were observed by hematoxylin-eosin staining, and combined with the image analysis system, volume density (Vv) of the renal corpuscle, renal capsule and renal glomerulus, the volume rate of the renal glomerulus and the renal capsule, number density (Nv) of the renal corpuscle, the long diameter of the renal corpuscle and glomerulus, the area of the lumen and the wall of the proximal renal tubule and the rate of them were measured. The glomerular deposition of IgA was measured by immunofluorescence staining. The amount of urinary red blood cells and24-hour urinary protein excretion were measured. Fibronectin (FN) and a-smooth muscle actin (α-SMA) expression on renal tissue were measured via immunohistochemistry.Results:Compared with the control group, glomerular hypertrophy, renal capsule dilation, renal glomerular smaller, glomerular vascular sparse, the mesangial proliferation, obvious fibrosis and the swelling of the renal tubule in the IgA nephropathy group. The image analysis results showed that, in IgA nephropathy group, Vv of the renal corpuscle increased, Vv of the renal capsule increased, while Vv of the renal glomerulus decreased, and the volume rate of the renal glomerulus and the renal capsule also decreased. The long diameter of the renal corpuscle increased and that of the renal glomerular decreased. The area of the lumen of the proximal renal tubule reduced, and the area of the wall of that enlarged and the rate of them decreased. Bright green fluorescence in a granular pattern in the mesangium has been seen, the amount of the urine red blood cells and24-hour urine protein increased, the FN and a-SMA expression level increased in the renal tissue in the IgA nephropathy group. Histological examination of kidney sections from both rhein-treated group and rhein-prevented group showed that glomerular hypertrophy, mesangial proliferation, renal capsule dilation and renal tubule narrow were markedly ameliorated compared with IgA nephropathy group. Moreover, rhein treatment significantly reduced IgA deposition in glomerular, the amount of urinary red blood cells and24-hour urinary protein excretion. More importantly, increased FN expression in IgA nephropathy was backed to normal level in rhein-treated and rhein-prevented group, which was along with the reduction of a-SMA expression in renal tissues.Conclusions:The renal tissue of IgA nephropathy modeling by BSA+LPS+CC14occur the swelling of the renal tubule, glomerular fibrosis and other pathological changes. RH has better control effect, its role may be mainly achieved through the protection of intestinal mucosal barrier. |
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