in the normal kidney , immunohistochemical expression of contractile protein alpha - smooth muscle actin ( asma ) is limited to the vascular smooth muscle cells . in pathological conditions , the expression of asma
the first experimental publication on this subject , by johnson et al , involved a series of clinical studies on the role of asma in diffuse renal diseases . in 1992
alpers and colleagues found higher expression of asma in proliferative types of glomerulonephritis gn . in iga nephropathy , asma expression in interstitium is related to damaged renal function and progression of disease .
the expression of asma in interstitial myofibroblasts helps to differentiate between minimal change disease and the early phase of idiopathic membranous gn . in patients with iga nephropathy ,
higher mesangial expression of asma predicts a progressive decline in renal function . in focal segmental glomerulosclerosis ,
, interest in the asma is increasing due to the fact that the asma in cytoplasm of the podocytes is associated with nephrin and thus is indirectly involved in the glomerular filtration barrier .
asma is analyzed in various types of gn together with markers such as metalloproteinase and nestin in investigation of tissue remodulation and epithelial - mesenchymal transition . to our best knowledge , this is the first morphometrical study of the correlation of asma expression in gn and renal function at the time of biopsy .
this retrospective study analyzed 142 patients  82 adults and 60 children  who underwent percutaneous renal biopsy in the department of internal medicine and department of pediatrics , university hospital , split , croatia in the period from 1994 to 2007 .
patients data were collected from the hospital records . due to the small numbers of each type of gn in the sample
, pathological diagnoses were classified into 2 categories  proliferative glomerulonephritis ( pg ) in 98 cases and non - proliferative glomerulonephritis ( npg ) in 44 cases . in the pg category
were : 29 iga nephropathy , 24 mesangioproliferative gn , 11 henoch - schenlein purpura , 9 focal segmental gn , 6 rapidly progressive gn , 6 endoproliferative gn and 6 lupus nephritis , 4 membranoproliferative gn and 1 hemolytic uremic syndrome , 1 alport syndrome and 1 churg - strauss syndrome . in the npg category were : 17 membranous gn , 15 focal segmental glomerulosclerosis , 5 minimal change disease , 2 fibrillar gn , 1 thin basement membrane disease , c1q nephropathy , igm nephropathy , amyloidosis and hereditary nephropathy .
absolute values of blood pressure ( bp ) , serum creatinine ( sc ) , creatinine clearance ( ccr ) and 24-hour urine protein , measured within 7 days before the biopsy , were collected from the hospital records . for adults ,
absolute values were categorized according to the common terminology criteria for adverse events ( ctcae ) . for children ,
bp was determined by body size and age using center for disease control and prevention growth charts and data from the national health and nutrition examination survey .
serum creatinine was categorized according to the pediatric reference ranges and divided into 3 categories : normal , high and low .
ccr was determined from sc , the patient s height and proportionality constant using the schwartz method .
the 24-hour urine protein was corrected for body surface area and categorized according to 95% confidence limits .
asma expression was analyzed by indirect immunohistochemistry ( envision / hrp system ( dako , denmark ) using mouse monoclonal anti - alpha smooth muscle antibody ( asma / hrp dako , denmark ) .
paraffin - embedded tissue sections of renal biopsies were deparaffinized in xylol and rehydrated in alcohol gradient .
tissue sections were incubated with primary asma antibody ( dilution 1:50 ) for 60 min and peroxidase - labeled secondary antibody for 20 min , followed by 10 min incubation with diaminobenzidine substrate - chromogen solution ( dako , denmark ) .
hematoxylin counter - staining was done ; slides were dehydrated in alcohol gradient , cleared in xylol and mounted with canada balsam .
positive control samples were normal renal tissues of 5 adult patients who underwent nephrectomy for renal cancer .
computer - assisted morphometric image analysis was used to measure glomerular and interstitial asma expression using ibm computer and digital camera ( olympus 4.1 zoom ) connected with olympus bx41 microscope ( olympus , japan ) .
a computer mouse was used to trace the perimeter of the area of interest on a computer monitor in successive sections , using  analysis  software ( analysis soft imaging system , usa ) .
all foci of asma expression in the interstitium ( including atrophic tubules ) , as well as the perimeter of histological slide , were measured in m at 100 magnification ; their ratio was calculated as a percentage of asma expression in interstitium .
area of asma expression in each glomerulus was measured in m at 400 magnification and added together for all glomeruli ( figure 1a ) .
their ratio was calculated as a percentage of asma expression in glomeruli ( figure 1b ) . in 5 case controls , renal cortex in the highest distance from the tumor
the area measuring 0.14 mm was defined , and glomeruli and interstitium were analyzed in the same manner . in the control kidneys ,
nonparametric spearman s correlation , mann - whitney test , and analysis of variance ( anova , kruskal - wallis ) were made using graphpad prism statistical software ( graphpad software , inc .
this retrospective study analyzed 142 patients  82 adults and 60 children  who underwent percutaneous renal biopsy in the department of internal medicine and department of pediatrics , university hospital , split , croatia in the period from 1994 to 2007 .
patients data were collected from the hospital records . due to the small numbers of each type of gn in the sample
, pathological diagnoses were classified into 2 categories  proliferative glomerulonephritis ( pg ) in 98 cases and non - proliferative glomerulonephritis ( npg ) in 44 cases . in the pg category
were : 29 iga nephropathy , 24 mesangioproliferative gn , 11 henoch - schenlein purpura , 9 focal segmental gn , 6 rapidly progressive gn , 6 endoproliferative gn and 6 lupus nephritis , 4 membranoproliferative gn and 1 hemolytic uremic syndrome , 1 alport syndrome and 1 churg - strauss syndrome . in the npg category were : 17 membranous gn , 15 focal segmental glomerulosclerosis , 5 minimal change disease , 2 fibrillar gn , 1 thin basement membrane disease , c1q nephropathy , igm nephropathy , amyloidosis and hereditary nephropathy .
absolute values of blood pressure ( bp ) , serum creatinine ( sc ) , creatinine clearance ( ccr ) and 24-hour urine protein , measured within 7 days before the biopsy , were collected from the hospital records . for adults ,
absolute values were categorized according to the common terminology criteria for adverse events ( ctcae ) . for children ,
bp was determined by body size and age using center for disease control and prevention growth charts and data from the national health and nutrition examination survey .
serum creatinine was categorized according to the pediatric reference ranges and divided into 3 categories : normal , high and low .
ccr was determined from sc , the patient s height and proportionality constant using the schwartz method .
the 24-hour urine protein was corrected for body surface area and categorized according to 95% confidence limits .
asma expression was analyzed by indirect immunohistochemistry ( envision / hrp system ( dako , denmark ) using mouse monoclonal anti - alpha smooth muscle antibody ( asma / hrp dako , denmark ) .
paraffin - embedded tissue sections of renal biopsies were deparaffinized in xylol and rehydrated in alcohol gradient .
tissue sections were incubated with primary asma antibody ( dilution 1:50 ) for 60 min and peroxidase - labeled secondary antibody for 20 min , followed by 10 min incubation with diaminobenzidine substrate - chromogen solution ( dako , denmark ) .
hematoxylin counter - staining was done ; slides were dehydrated in alcohol gradient , cleared in xylol and mounted with canada balsam .
positive control samples were normal renal tissues of 5 adult patients who underwent nephrectomy for renal cancer .
computer - assisted morphometric image analysis was used to measure glomerular and interstitial asma expression using ibm computer and digital camera ( olympus 4.1 zoom ) connected with olympus bx41 microscope ( olympus , japan ) .
a computer mouse was used to trace the perimeter of the area of interest on a computer monitor in successive sections , using  analysis  software ( analysis soft imaging system , usa ) .
all foci of asma expression in the interstitium ( including atrophic tubules ) , as well as the perimeter of histological slide , were measured in m at 100 magnification ; their ratio was calculated as a percentage of asma expression in interstitium .
area of asma expression in each glomerulus was measured in m at 400 magnification and added together for all glomeruli ( figure 1a ) .
their ratio was calculated as a percentage of asma expression in glomeruli ( figure 1b ) . in 5 case controls , renal cortex in the highest distance from the tumor
the area measuring 0.14 mm was defined , and glomeruli and interstitium were analyzed in the same manner . in the control kidneys ,
nonparametric spearman s correlation , mann - whitney test , and analysis of variance ( anova , kruskal - wallis ) were made using graphpad prism statistical software ( graphpad software , inc .
the expression of asma in glomeruli and interstitium was not significantly different between pg and npg categories of gn in both children and adults ( table 1 ) .
the children with high blood pressure had greater expression of asma in interstitium compared to children with low blood pressure ( 10.79.4% vs. 3.74.3% , p=0.014 ) ( figure 2 ) .
the difference in expression of asma in glomeruli was not statistically significant between children with high and low blood pressure ( 7.94.4% vs. 15.410.6% , p=0.051 ) . in adults ,
expression of asma in interstitium and in glomeruli were not significantly different between patients with high and normal blood pressure ( 10.714.9% vs. 9.912.6% , p=0.692 ) and ( 13.69.5% vs. 12.97.8% , p=1.00 ) , respectively . in children
, positive correlation was found between asma expression in interstitium and absolute value of sc ( r=0.45 , p=0.002 ) ( figure 3a ) . in categorized sc ,
the negative correlation of sc and expression of asma in glomeruli was not significant ( r=0.154 , p=0.291 ) . in adults ,
expression of asma in interstitium was correlated to sc ( r=0.528 , p<0.001 ) ( figure 3b ) .
the significance was confirmed in categorized sc , where grade iii had higher expression of asma in interstitium ( p=0.0009 ) .
significant negative correlation was found between expression of asma in glomeruli and sc ( r=0.395 , p=0.002 ) ( figure 3c ) . in children , absolute values of ccr
negatively correlated to expression of asma in interstitium ( r=0.375 , p=0.009 ) and positively to expression of asma in glomeruli ( r=1.00 , p<0.001 ) ( figure 4a , b ) . in adults
, there were no significant correlations between asma expression in glomeruli and ccr ( r=0.058 , p=0,643 ) .
children and adults with ccr grade ii and iii had higher expression of asma in interstitium ( p=0.0152 and p=0.0007 , respectively ) ( figures 5a , b ) .
no significant association between renal expression of asma and 24-hour urine protein was found , regardless of the patient s age .
the expression of asma in glomeruli and interstitium was not significantly different between pg and npg categories of gn in both children and adults ( table 1 ) .
the children with high blood pressure had greater expression of asma in interstitium compared to children with low blood pressure ( 10.79.4% vs. 3.74.3% , p=0.014 ) ( figure 2 ) .
the difference in expression of asma in glomeruli was not statistically significant between children with high and low blood pressure ( 7.94.4% vs. 15.410.6% , p=0.051 ) . in adults ,
expression of asma in interstitium and in glomeruli were not significantly different between patients with high and normal blood pressure ( 10.714.9% vs. 9.912.6% , p=0.692 ) and ( 13.69.5% vs. 12.97.8% , p=1.00 ) , respectively .
in children , positive correlation was found between asma expression in interstitium and absolute value of sc ( r=0.45 , p=0.002 ) ( figure 3a ) . in categorized sc ,
the negative correlation of sc and expression of asma in glomeruli was not significant ( r=0.154 , p=0.291 ) . in adults ,
expression of asma in interstitium was correlated to sc ( r=0.528 , p<0.001 ) ( figure 3b ) .
the significance was confirmed in categorized sc , where grade iii had higher expression of asma in interstitium ( p=0.0009 ) .
significant negative correlation was found between expression of asma in glomeruli and sc ( r=0.395 , p=0.002 ) ( figure 3c ) .
in children , absolute values of ccr negatively correlated to expression of asma in interstitium ( r=0.375 , p=0.009 ) and positively to expression of asma in glomeruli ( r=1.00 , p<0.001 ) ( figure 4a , b ) . in adults , there were no significant correlations between asma expression in glomeruli and ccr ( r=0.058 , p=0,643 ) .
children and adults with ccr grade ii and iii had higher expression of asma in interstitium ( p=0.0152 and p=0.0007 , respectively ) ( figures 5a , b ) .
no significant association between renal expression of asma and 24-hour urine protein was found , regardless of the patient s age .
studies of asma expression in renal parenchymal diseases began more than 2 decades ago , when it was noticed that a ) damaged glomerular mesangial cells change their immunophenotype expressing asma and b ) asma - positive myofibroblasts start interstitial fibrosis .
a number of papers were published about asma expression in different types of human gn , its connection to proliferation markers and prognostic impact .
most authors agree that asma expression in glomeruli was higher in proliferative gn and increases as the disease worsens , which makes asma expression a potential clinical prognostic factor .
some authors determined the relationship between iga nephropathy , lupus nephritis and other types of gn with expression of asma .
kim in 2001 found correlation between proliferation marker ki-67 and asma in different types of gn . in this study we focused on the connection of asma expression with impaired renal function in glomerulonephritis , measured at the time of biopsy .
we did not find correlation with a special type of gn nor proliferation , possibly because of heterogeneity of the sample and the arbitrary method of categorization into proliferative and non - proliferative gn .
increased expression of asma in the interstitium can be found in different diseases such as proliferative gn , diabetic nephropathy and renal transplant rejection .
the asma has prognostic value due to the association with interstitial fibrosis , urine protein and sc [ 8,2528 ] .
asma - positive myofibroblasts are responsible for the increased amount of extracellular matrix and renal fibrosis .
this study also found higher expression of asma in interstitium of all patients with higher sc .
greater expression of interstitial asma in children and adults was associated with higher grades of ccr and lower absolute values of ccr .
we confirm that elevated sc or lower values of ccr are associated with higher expression of asma in interstitium .
several studies have attempted to predict the development of progressive renal failure , measuring histomorphometric changes in the tubulointerstitial compartment .
the best correlating parameters of interstitial fibrosis with renal function are the ratio of the accumulation of tgf - beta-1 and its antagonist decorin , interstitial expression of asma , and accumulation of interstitial collagen . according to jiang et al .
, who analyzed asma production in peritoneal fibroblasts stimulated by tnf - beta-1 , hepatocyte growth factor could be important in blocking postoperative peritoneal adhesion .
analyzed 133 biopsies of various human renal diseases , and found tubular epithelial cells with mesenchymal phenotype ( vimentin and asma positive ) whose numbers have been associated with the level of sc and degree of interstitial damage .
analyzed 27 patients with iga nephropathy who had normal ccr at the time of biopsy , and found that expression of asma in mesangium predicts a progressive decline in renal function . in our study ,
a negative connection of asma expression in glomeruli and sc at the time of biopsy was found , statistically significant in adults and non - significant in children , but with the same trend .
it is well known that asma expression in the mesangial cell indicates its change to myofibroblastic immunophenotype .
our presumption is that asma - expressing mesangial cells are capable of higher contraction activity ; this leads to glomerular hyperfiltration as an early adaptive mechanism to glomerular damage .
in a recent experimental study of membranous glomerulonephritis in rats , osteopontin and asma are expressed together in myofibroblasts in the crescents of damaged glomeruli . in children , the absolute values of ccr
a possible explanation that children have a better ccr is due to phenotypic modulation of human mesangial cells to more contractile cells , which makes the glomerular function transitory normal or even increased .
we analyzed association of expression of asma and hypertension , because it is known that glomerular mesangial cells during hypertensive damage express myofibroblast phenotype and expression of asma increases .
the only significant association that we found was between expression of asma in interstitium and increased blood pressure in children .
we did not find significant correlation between expression of asma in glomeruli nor interstitium with 24-hour urine protein in our patients .
this result confirms previous findings that urine protein is primary linked to the changes in permeability of glomerular filtration barrier . nevertheless , according to some studies asma expression in interstitium
is linked to the degree of proteinuria and sc , and higher interstitial asma expression is an indicator of poor prognosis .
asma expression in interstitium is associated with sc and ccr and consecutively with decrease of renal function .
asma expression in glomeruli is associated with lower values of sc in adults , as well as with normal or higher ccr in children .
this correlation suggests that myofibroblastic phenotypic modulation of glomerular cells has a favorable impact on filtration . when calculated with precise computer - assisted quantitative morphometric technology , renal expression of asma
the disadvantage of this study is its small sample size with different types of gn ; the next survey should include a much narrower cohort of patients with a specific type of gn , as well as better phenotypic identification of asma - positive glomerular cells .
 summarybackgroundin a healthy kidney , contractile protein alpha - smooth muscle actin ( asma ) is immunohistochemically strongly expressed only in the blood vessels , while in pathological conditions it can be visualized in glomerular mesangial cells and interstitial myofibroblasts . 
 the aim of this study was to explore the possible correlation between expression of asma in glomerulonephritis ( gn ) and indicators of renal function.material/methodswe analyzed expression of asma in percutaneous renal biopsy of 142 adult and pediatric patients with gn and its correlation with blood pressure , serum creatinine , creatinine clearance and 24-hour urine protein at the time of biopsy . 
 immunoexpression of asma was analyzed quantitatively using computer - assisted morphometric analysis . 
 relative surface of asma expression in all glomeruli and interstitium was calculated for each patient.resultsin adults and children , greater expression of asma in interstitium was associated with higher serum creatinine and reduced creatinine clearance . 
 conversely , greater asma expression in glomeruli was associated with normal or decreased serum creatinine in adults and increased creatinine clearance in children . in children , correlation was found between high blood pressure and asma expression in interstitium.conclusionswe confirmed that interstitial expression of asma is associated with reduced renal function at time of biopsy . 
 the connection of asma expression in glomeruli with lower serum creatinine and normal or increased creatinine clearance suggests a favorable role of this phenotypic change in glomerular filtration rate ; further investigation is needed . 
Background
Material and Methods
Patients
Laboratory parameters
Immunohistochemistry
Morphometric analysis
Statistical analysis
Results
Renal ASMA expression
Blood pressure
Serum creatinine
Creatinine clearance
24-hour urine protein
Discussion
Conclusions