diabetic patients , who consume vacor ( n-3-pyridylmethyl - n-p - nitrophenylurea , mw272 ) , experience microangiopathy causing retinopathy and nephropathy . the width of the capillary basement membrane in these patients thickens significantly compared to normal controls after 6 years of vacor ingestion , on average .
a number of animal models of diabetes have suggested that hyperglycemia can produce microangiopathic changes .
rodents made hyperglycemic with streptozotocin ( stz ) or alloxan developed glomerular changes that were similar to , but not strictly comparable to , those of humans .
vacor - induced diabetic rats developed acute glomerulopathy within a few months and the glomerulus revealed prominent thickening of the basement membrane .
the glomerular filtration barrier consists of complex matrix constituents interposed between the glomerular endothelial and epithelial cells , which constitute the lining of the glomerular capillaries .
they include collagen ( mostly type iv ) , laminin , polyanionic proteoglycans ( mostly heparan sulfate ) , fibronectin , entactin and several other glycoproteins .
these individual components of glomerular basement membrane ( gbm ) were immunohistochemically tagged using specific monoclonal antibodies and observed by light and electron microscopy .
changes of four major components of gbm , ( type iv collagen , laminin , fibronectin and proteoglycan ) , were studied in vacor - induced diabetic rats in this study .
adult male wistar rats ( n=42 ) , weight 200250 g , were used for this study .
a single dose of vacor , 80 mg / kg of body weight , was administered to the experimental animals by an orogastric canule , while distilled water was given to controls .
the experimental animals were sacrificed 0.5 , 1 , 3 , 7 , 14 , 28 and 56 days after the administration of vacor .
animals were perfused through the jugular vein with 300 ml of physiologic saline followed by an equal amount of 10% buffered formalin .
both kidneys were taken , and then routine formalin - fixed , paraffin - embedded tissue blocks were made for the immunohistochemical studies of glomerular basement membrane ( gbm ) components .
tissue sections , 4 m in thickness , were obtained , and stained with hematoxylin - eosin ( h&e ) and periodic acid schiff ( pas ) stain . for immunohistochemical studies of the gbm components , monoclonal antibodies for collagen type iv ( monosan , netherlands ) , laminin ( monosan , netherlands ) , fibronectin ( sigma bioscience , u.s.a . ) and proteogylcan chondroitin sulfate ( cspg , biogenesis , u.k . ) were used .
deparaffined sections were soaked in absolute methanol containing 0.3% hydrogen peroxide for 30 minutes at room temperature in order to block endogenous peroxidase activity .
after washing , the sections were incubated with 0.4% pepsin ( sigma ) in 0.01 n hci for 1 hour at 37c , then treated with 0.05% protease type vii ( sigma ) in phosphate - buffered saline ( pbs ) , ph 7.2 , for 15 minutes at 37c . with these enzyme pretreatments ,
after further washing in pbs , the sections were exposed to 1:20 diluted normal swine serum ( dako - immunoglobulins ltd . ,
. then sections were incubated with each primary antibody at a dilution of 1 : 500 overnight at 4c . after washing with pbs ,
treatment followed with anti - rabbit igg swine serum ( at a dilution of 1:20 , dako ) , and pap solution ( at a dilution of 1:80 , dako ) , for 30 minutes , respectively , at room temperature .
finally , the sections were soaked in 0.05 m tris - hci buffer , ph 7.6 , containing 3,3-diaminobenzidine hydrochloride ( 40 mg/100 ml ) and hydrogen peroxide ( 0.015% ) for 10 minutes , and counterstained with hematoxylin .
vascular walls and renal tubules in the specimens served as positive controls for the primary antibodies . as the negative control ,
the glomeruli in control animals revealed patent capillary loops demarcated by a thin , delicate glomerular basement membrane .
the basement membrane and mesangium were positively stained by pas stain . in the experimental groups ,
mild thickening of the gbm was evident at 7 days by h&e and pas stains .
increased thickening of the gbm and mesangial widening were noted at 14 to 56 days by pas and h&e stains ( fig .
the width of the gbm had more than doubled at 28 and 56 days compared with normal controls .
focal nodular proliferation of both glomerular endothelial and mesangial cells in the glomerulus was observed ( fig .
the glomeruli in control animals show thin linear staining along the gbm after immunohistochemical staining for collagen type iv and laminin ( fig .
3 ) . fibronectin and proteoglycan were also identified along the gbm , but they stained more faintly than collagen type iv and laminin .
immunohistochemical staining for collagen type iv was the most sensitive way to demonstrate gbm thickening , evident 3 days after a single , large dose ( 80 mg / kg ) of vacor treatment .
the gbm was moderately thickened at 7 days , and was maximally thickened 28 to 56 days after the treatment by collagen type iv immunohistochemistry .
the pattern was initially focal and global from the gbm peripheral loop , and became subsequently diffuse ( fig .
3 ) . moderately thickened immunoreactivity with collagen type iv was noted after as many as 56 days .
laminin in the gbm also thickened after vacor treatment , and behaved similarly to collagen type iv ( fig .
4 ) . fibronectin in the gbm appeared faintly at 12 hours and 1 day after vacor treatment .
mild , focal and segmental thickening of fibronectin was evident at 3 days . from 7 days after vacor treatment , the fibronectin presence in the gbm began to resemble that of collagen type iv and laminin ( fig .
chondroitin sulfate proteoglycan ( cspg ) in the gbm did not change so prominently as type iv collagen , laminin and fibronectin .
only a mild focal thickening of cspg was noted along the gbm at 3 to 7 days after vacor treatment .
no identifiable changes of proteoglycan in the gbm were noted from 14 to 56 days after vacor treatment ( fig .
the glomeruli in control animals revealed patent capillary loops demarcated by a thin , delicate glomerular basement membrane .
the basement membrane and mesangium were positively stained by pas stain . in the experimental groups ,
mild thickening of the gbm was evident at 7 days by h&e and pas stains .
increased thickening of the gbm and mesangial widening were noted at 14 to 56 days by pas and h&e stains ( fig .
the width of the gbm had more than doubled at 28 and 56 days compared with normal controls .
focal nodular proliferation of both glomerular endothelial and mesangial cells in the glomerulus was observed ( fig .
the glomeruli in control animals show thin linear staining along the gbm after immunohistochemical staining for collagen type iv and laminin ( fig .
3 ) . fibronectin and proteoglycan were also identified along the gbm , but they stained more faintly than collagen type iv and laminin .
immunohistochemical staining for collagen type iv was the most sensitive way to demonstrate gbm thickening , evident 3 days after a single , large dose ( 80 mg / kg ) of vacor treatment .
the gbm was moderately thickened at 7 days , and was maximally thickened 28 to 56 days after the treatment by collagen type iv immunohistochemistry .
the pattern was initially focal and global from the gbm peripheral loop , and became subsequently diffuse ( fig .
3 ) . moderately thickened immunoreactivity with collagen type iv was noted after as many as 56 days .
laminin in the gbm also thickened after vacor treatment , and behaved similarly to collagen type iv ( fig .
4 ) . fibronectin in the gbm appeared faintly at 12 hours and 1 day after vacor treatment .
mild , focal and segmental thickening of fibronectin was evident at 3 days . from 7 days after vacor treatment , the fibronectin presence in the gbm began to resemble that of collagen type iv and laminin ( fig .
chondroitin sulfate proteoglycan ( cspg ) in the gbm did not change so prominently as type iv collagen , laminin and fibronectin .
only a mild focal thickening of cspg was noted along the gbm at 3 to 7 days after vacor treatment .
no identifiable changes of proteoglycan in the gbm were noted from 14 to 56 days after vacor treatment ( fig .
in humans , characteristic morphological changes of the glomeruli in diabetes mellitus include gbm thickening , diffuse glomerulosclerosis and nodular glomerulosclerosis .
diffuse thickening of the gbm occurs in virtually all diabetics , irrespective of the presence of proteinuria , and constitutes a part of diabetic microangiopathy .
careful morphometric studies demonstrate that this thickening begins as early as 2 years after the onset of type i diabetes and , by 5 years , develops to about a 30% increase
. diffuse and nodular glomerulosclerosis are late events for diabetics , and become pronounced after 10 to 20 years of diabetes . in vacor - treated rats , the glomeruli reveal prominent thickening of the gbm within a short period . the width of the gbm is more than twice that of normal controls , 28 and 56 days after vacor administration .
increased expressions of collagen iv , laminin , fibronectin and neutral mucopolysaccharide engendered the thickening of the gbm in this study .
proteoglycan immunoreactivity was not significantly different between vacor - treated and normal control groups . in a study of human diabetic diffuse glomeruloscleosis ,
the enlarged mesangial matrix also revealed increased staining reactions for collagen iv , v , laminin and fibronectin , whereas the staining pattern for heparan sulfate proteoglycan ( hspg ) was markedly reduced .
these data suggest that vacor - induced glomerulopathy in rats can provide a useful experimental model to study human diabetic glomerulopathy .
expression of type iv collagen and its mrna in glomeruli and the interstitium of diabetic rats increased at 3 , 7 and 14 days after streptozotocin ( stz ) administration .
the expression was associated with up - regulation of transforming growth factor ( tgf)-. administration of tgf- increased collagen synthesis in normal rat glomeruli in a dose - dependent manner up to 5 ng / ml . these data indicate a regulatory role for tgf- in renal glomerular collagen synthesis in normal and diabetic rats .
inhibition of tgf- and type iv collagen expression by insulin treatment suggests that they may be useful structural markers for determining the efficacy of therapeutic intervention during early diabetic nephropathy .
concomitant increased staining patterns for laminin and type iv collagen have been reported in human diabetic glomerular lesions and experimental diabetic nephropathy . over - expression of tgf- was also partly associated with the up - regulation of glomerular laminin gene expression in diabetic mice .
the occurrence of collagen type iii in late diffuse glomerulosclerosis could be interpreted as an irreversible change in glomerular structure .
fibronectin also increases in the gbm and mesangial matrix in human diabetic nephropathy and stz - induced diabetic rats .
fibronectin is a multifunctional matrix protein important in wound healing , and is markedly increased in glomerular crescents .
there are two phases of fibronectin metabolism in anti - gbm models of rapidly progressive glomerulonephritis ( rpgn ) in the rabbit .
phase i is associated with increased glomerular fibronectin content from plasma , and phase ii with increased fibronectin mrna in glomeruli at days 7 to 14 .
this finding suggests that fibronectin synthesis may take place in glomeruli and that it may also be a molecule regulating glomerular cell proliferation and fibrosis .
two types of proteoglycan , hspg and basement membrane - specific cspg , have been studied in human diabetes and diabetic rats .
the expression of hspg decreases in the gbm and other capillaries in diabetes , while cspg increases in stz - diabetic rats .
a mildly increased expression of cspg was noted only after 3 to 7 days of vacor administration in this experiment .
other studies suggest that changes of cspg displace the capillary endothelial cells from the gbm causing loss of fenestrae , while loss of the hspg results in alteration of gbm charge - selectivity .
the abnormal levels of type iv collagen , laminin , fibronectin and proteoglycan may , in part , be the cause of diabetic nephropathy induced by vacor administration .
to elucidate the biochemical component changes in the gbm in vacor - induced diabetic glomerulopathy , immunohistochemical analyses of type iv collagen , laminin , fibronectin and cspg were performed in adult male wistar rats .
mild thickening of the gbm was evident 7 days after vacor administration , and the width of the gbm was more than twice that of normal controls at 28 and 56 days . significantly increased expressions of type iv collagen , laminin , fibronectin and neutral polysaccharide in the thickened gbm was noted 14 to 56 days after the administration , and the mildly increased expression of cspg was present after 3 to 7 days .
these abnormal levels of gbm components might , in part , be the cause of diabetic nephropathy induced by vacor administration .
 objectivesthe thickening of the glomerular basement membrane in rats after vacor ingestion was examined by electron microscopy . 
 this study was performed to elucidate which biochemical components changed in the glomerular basement membrane after vacor - induced diabetic glomerulopathy.methodsimmunohistochemical analyses of type iv collagen , laminin , fibronectin and chondroitin sulfate proteoglycan were performed . 
 a single dose of vacor ( molecular weight 272 ) , 80 mg / kg , was administered to adult male wistar rats by orogastric canule , and the animals were sacrificed at 0.5 , 1 , 3 , 7 , 14 , 28 and 56 days after administration.resultsmild thickening of the glomerular basement membrane was evident 7 days after vacor administration , and the width of the glomerular basement membrane was more than twice that of normal controls at 28 and 56 days . 
 significantly increased expressions of type iv collagen , laminin , fibronectin and neutral polysaccharide in the thickened glomerular basement membrane were noted 14 to 56 days after administration , and a mildly increased expression of chondroitin sulfate proteoglycan appeared between 3 to 7 days.conclusionthese abnormally increased glomerular basement membrane components might be part of what causes diabetic nephropathy after vacor administration 
INTRODUCTION
MATERIALS AND METHODS
RESULTS
Glomerular changes examined by H&E and PAS stains
Changes of GBM components by immunohistochemical stains
DISCUSSION
CONCLUSION