diosmin is a member of the citrus flavonoid family and is a modified form of hesperidin [ 1 - 4 ] . it is a semisynthetic drug that is used as an oral phlebotropic drug in the treatment of venous disease , i.e. , chronic venous insufficiency and hemorrhoidal disease . in some countries , daflon 500 mg ( which contains 90% diosmin+10% hesperidin ) is used as a prescription medication . diosmin prolongs the vasoconstrictor effect of norepinephrine on the vein wall , increases the venous tone , and therefore reduces venous capacitance , distensibility , and stasis . furthermore , it improves lymphatic drainage by increasing the frequency and intensity of lymphatic contractions and at the same time decreases the diameter of the lymphatic capillaries [ 1 - 4 ] . in addition to its use in vascular disease , diosmin has been found to be effective in mitigating hyperglycemia in diabetic rats . it is also speculated that diosmin may have potential in the treatment of neurodegenerative diseases , such as alzheimer 's disease . moreover , its anti - inflammatory and anti - apoptotic activity has been demonstrated in neuronal cells . it has been mentioned that the anticancer , cardiovascular , and anti - inflammatory activities are the properties of citrus flavonoids . until now , however , clinical studies of the effects of diosmin have been inconclusive . furthermore , only a limited number of articles have been published and few articles are available on the use of diosmin in renal diseases . kidney stones ( nephrolithiasis ) are a solid concretion or crystal aggregation formed in the kidneys from dietary minerals existing in the urine . the africo - asian stone - forming belt stretches from sudan , the arab republic of egypt , saudi arabia , the united arab emirates , the islamic republic of iran , pakistan , india , myanmar , thailand , and indonesia to the philippines . in this area of the world , the disease affects all age groups from less than 1 year old to more than 70 years old . because nephrolithiasis is a widespread disease , blocking the process of stone formation and finding new therapeutic options is an important area of study . the present study was therefore conducted to investigate the effects of diosmin in a rat model of calcium oxalate ( caox ) nephrolithiasis by use of stereological techniques . the quantitative data obtained showed the total volume of the deposited caox and structural changes to the kidney , including the total volume of the glomeruli , proximal and distal convoluted tubules ( pct and dct ) , loop of henle , collecting duct , interstitial tissue , and vessels . a rapid and simple stereological method was used to obtain the total quantities on one microscopic slide without the need for serial sectioning of the renal tissue . the animals were treated according to the standard directive as recommended and approved by the research authorities of shiraz university of medical sciences ( license no . the rats were randomly divided into three groups of 10 animals each , which is acceptable for stereological studies . the first group ( control ) did not receive any treatments . in groups two and three the second group received distilled water by gavage , whereas the third group received diosmin ( 80 mg / kg / d ) dissolved in a distilled water vehicle given by gavage for 21 days . to induce an experimental model of caox nephrolithiasis , the rats were orally administered 2.5% ( v / v ) ethylene glycol+2.5% ( w / v ) ammonium chloride ( 2 ml / d ) by gavage . in addition , the rats experienced a restriction on their intake of drinking water ( 20 ml / d ) for 21 days . after 21 days the primary volume of the kidney , vprimary , was measured by using the immersion method of sherle . estimation of the shrinkage and the length of the tubular structure of the kidney requires isotropic , uniform , random sections [ 13 - 15 ] . the estimated shrinkage was also used for the estimation of the final kidney volume to avoid the consecutive sectioning that is required for the cavalieri method . the final volume of the kidney ( the reference space ) was corrected by using the following formula : vfinal = vprimary(1-volume shrinkage ) . each sampled section was analyzed by using a video microscopy system that was made up of a microscope ( e-200 , nikon , tokyo , japan ) linked to a video camera , a computer , and a monitor to determine the parameters . between 8 and 12 microscopic fields were selected in a systematic random manner . afterwards , the stereological grids were superimposed on the images by means of the stereology software designed at histomorphometry & stereological research center , shiraz university of medical sciences , shiraz , iran . however , the volume density of each structure ( its volume per unit volume of the kidney ) was estimated by using the point - counting method [ 13 - 15 ] . 3 . the volume density ( vv ) of the renal cortex , the medulla , the glomeruli , the pct , the dct , the collecting ducts , the henle 's loop , the vessels , the connective tissue , and the caox deposition was obtained at the final magnification of 1,800 . the point - counting method used the following formula : vv ( structure , reference)=p(structure)/p(total ) where p(structure ) and p(total ) represent the sum of the number of the points hitting the structure and all sampled fields of the kidney , respectively . finally , the total volume of each structure was estimated by multiplying the fractional volume by the final volume of the kidney to prevent the " reference trap " [ 13 - 15 ] . the diameter of the capillaries and vessels sampled by the unbiased counting frame was measured . the diameter was measured perpendicular to the long axis where the vessel was widest [ 13 - 15 ] . the statistical comparisons between the group means were performed by analysis of variance and also mann - whitney u - test . the animals were treated according to the standard directive as recommended and approved by the research authorities of shiraz university of medical sciences ( license no . the rats were randomly divided into three groups of 10 animals each , which is acceptable for stereological studies . the first group ( control ) did not receive any treatments . in groups two and three the second group received distilled water by gavage , whereas the third group received diosmin ( 80 mg / kg / d ) dissolved in a distilled water vehicle given by gavage for 21 days . to induce an experimental model of caox nephrolithiasis , the rats were orally administered 2.5% ( v / v ) ethylene glycol+2.5% ( w / v ) ammonium chloride ( 2 ml / d ) by gavage . in addition , the rats experienced a restriction on their intake of drinking water ( 20 ml / d ) for 21 days . the primary volume of the kidney , vprimary , was measured by using the immersion method of sherle . 1 . the kidney was fixed in neutral buffered formaldehyde for 2 days . then the tissue shrinkage produced by fixation , processing , and staining was estimated . estimation of the shrinkage and the length of the tubular structure of the kidney requires isotropic , uniform , random sections [ 13 - 15 ] . the estimated shrinkage was also used for the estimation of the final kidney volume to avoid the consecutive sectioning that is required for the cavalieri method . the final volume of the kidney ( the reference space ) was corrected by using the following formula : vfinal = vprimary(1-volume shrinkage ) . each sampled section was analyzed by using a video microscopy system that was made up of a microscope ( e-200 , nikon , tokyo , japan ) linked to a video camera , a computer , and a monitor to determine the parameters . between 8 and 12 microscopic fields were selected in a systematic random manner . afterwards , the stereological grids were superimposed on the images by means of the stereology software designed at histomorphometry & stereological research center , shiraz university of medical sciences , shiraz , iran . however , the volume density of each structure ( its volume per unit volume of the kidney ) was estimated by using the point - counting method [ 13 - 15 ] . 3 . the volume density ( vv ) of the renal cortex , the medulla , the glomeruli , the pct , the dct , the collecting ducts , the henle 's loop , the vessels , the connective tissue , and the caox deposition was obtained at the final magnification of 1,800 . the point - counting method used the following formula : vv ( structure , reference)=p(structure)/p(total ) where p(structure ) and p(total ) represent the sum of the number of the points hitting the structure and all sampled fields of the kidney , respectively . finally , the total volume of each structure was estimated by multiplying the fractional volume by the final volume of the kidney to prevent the " reference trap " [ 13 - 15 ] . the diameter of the capillaries and vessels sampled by the unbiased counting frame was measured . the diameter was measured perpendicular to the long axis where the vessel was widest [ 13 - 15 ] . the statistical comparisons between the group means were performed by analysis of variance and also mann - whitney u - test . the mean and the standard deviation of the kidney volume and the volume of the cortex and medulla are presented in table 1 . the total volume of the glomeruli , pct , dct , henle 's loop , collecting duct , cortical and medullary degeneration , and fibrotic tissue in the different groups is shown in table 2 . however , after the induction of nephrolithiasis , the volume of the glomeruli , pct , dct , henle 's loop , collecting ducts , and vessels was reduced -57% , 34% , 35% , 58% , 54% , and 32% , respectively , in the rats ( p<0.001 ) . after treating the nephrolithiasis with diosmin , on average , -70% to 96% of the glomeruli , pct , of course , the degeneration of the cortical tissue was 5-fold that of the medulla . after treating the rats with nephrolithiasis with diosmin , the volume of caox deposition was -63% less in the rats with nephrolithiasis treated with diosmin ( p<0.01 ) . in the nephrolithiasis+diosmin rats , degeneration in the renal cortical tissue and medulla was reduced -70% and 44% compared with that in the rats with untreated nephrolithiasis ( p<0.01 ) . the diameter of the capillaries and vessels in the cortex was reduced -7% and -12% , respectively , in the rats with nephrolithiasis ( p<0.001 ) . after treating the nephrolithiasis with diosmin , the reduction of the diameter of the cortical capillaries and vessels was recovered ( table 3 ) . the mean and the standard deviation of the kidney volume and the volume of the cortex and medulla are presented in table 1 . the total volume of the glomeruli , pct , dct , henle 's loop , collecting duct , cortical and medullary degeneration , and fibrotic tissue in the different groups is shown in table 2 . however , after the induction of nephrolithiasis , the volume of the glomeruli , pct , dct , henle 's loop , collecting ducts , and vessels was reduced -57% , 34% , 35% , 58% , 54% , and 32% , respectively , in the rats ( p<0.001 ) . after treating the nephrolithiasis with diosmin , on average , -70% to 96% of the glomeruli , pct , henle 's loop , collecting ducts , and vessels remained intact ( p<0.01 ) . of course , the degeneration of the cortical tissue was 5-fold that of the medulla . after treating the rats with nephrolithiasis with diosmin , the volume of caox deposition was -63% less in the rats with nephrolithiasis treated with diosmin ( p<0.01 ) . in the nephrolithiasis+diosmin rats , degeneration in the renal cortical tissue and medulla was reduced -70% and 44% compared with that in the rats with untreated nephrolithiasis ( p<0.01 ) . the diameter of the capillaries and vessels in the cortex was reduced -7% and -12% , respectively , in the rats with nephrolithiasis ( p<0.001 ) . after treating the nephrolithiasis with diosmin , the reduction of the diameter of the cortical capillaries and vessels was recovered ( table 3 ) . the results of the present investigation revealed that diosmin massively reduces caox formation and also protects the renal tissue from damage in a rat model of nephrolithiasis . formation of caox stones starts from the attachment of a crystal formed in the lumen of the renal tubules to the surface of the epithelial cells [ 16 - 23 ] . most of the crystals formed in the lumen of tubules are discharged in the urine . in healthy individuals , the crystals that attach to the surface of the epithelial cells are digested by macrophages or cellular lysosomes . however , in patients with hyperoxaluria or crystal urine , the renal tubular cells are injured , thus allowing crystals to easily attach to them . then , the crystals aggregate and grow , and finally a stone is formed [ 16 - 23 ] . several studies have provided evidence that oxalate - induced toxicity is related to superoxide and lipid peroxidation . the oxalate - induced increase in free radical production appears to be related to cell death in a concentration - dependent manner [ 16 - 23 ] . finding a therapy for preventing the caox deposition seems to be possible by controlling the above - mentioned mechanism , in particular controlling the tubular cell injury and the inflammation process . concluded that urinary glycosaminoglycans might play a critical role in preventing the adhesion of crystals by inhibiting the renal tubular cell injury . reported that the antioxidative effect of green tea decreases caox stone formation , osteopontin expression , and apoptosis and increases superoxide dismutase activity in the kidney tissues of rats . also , sarica et al .   reported a significant decrease in the mean level of apoptosis in the animals receiving vitamin e and allopurinol . in the present study , the protective role of diosmin on the renal structure diosmin has been used to prolong the vasoconstrictor effect of norepinephrine on the venous and lymphatic vessels . as a flavonoid , however , it also exhibits anti - inflammatory , free - radical scavenging , and antimutagenic properties . in the present research capillary hyperpermeability is one of the primary processes of the inflammation . at the microcirculation level , diosmin reduces capillary hyperpermeability and increases capillary resistance by protecting the microcirculation from the damaging processes [ 1 - 4 ] . in addition , diosmin reduces the expression of endothelial adhesion molecules and inhibits the adhesion , migration , and activation of leukocytes at the capillary level [ 1 - 4 ] . it has been reported that these effects lead to a reduction in the release of inflammatory mediators , principally oxygen free radicals and some prostaglandins . in a recent article , srinivasan and pari   showed the ameliorative effect of diosmin against the oxidative stress in the kidney of diabetic rats . reduction of the degenerative tissue as a result of using diosmin was another finding of the study . this finding might be explained by the reduction in tubular cell injury and inflammation after the treatment with diosmin . in addition , observation of more intact tubules after diosmin treatment in the present survey is an indicator of less tubular cell injury . another aspect of the present research was the reduction of the diameter of the capillaries and vessels in the cortex after nephrolithiasis and the recovery of this after diosmin treatment . as stated previously , diosmin prolongs the vasoconstrictor effect of norepinephrine on the vein . note the diameter reduction of the histological sections is a structural change that might be due to tissue fibrosis , because a significant reduction was seen in the cortex and not the medulla , which contains less fibrous tissue . diosmin reduces caox deposition and the degeneration of glomeruli and tubules in a rat model of nephrolithiasis .