acute kidney injury is a common clinical syndrome that is characterized by a rapid decline in kidney function , often triggered by glomerular disease and/or tubulointerstitial disease and associated with high morbidity and mortality [ 14 ] . in addition to serum creatinine , 2-microglobulin is a biomarker that can be used to determine underlying causes of acute kidney injury .
serum 2-microglobulin derives from cellular membrane turnover , since 2-microglobulin forms the invariant light chain portion of major histocompatibility complex ( mhc ) class i in membranes of all cells [ 710 ] . as a single - chain small polypeptide ( mw = 11.8  kda ) ,
2-microglobulin is filtered almost completely through the glomeruli of the healthy kidney [ 11 , 12 ] and then reabsorbed by the renal proximal tubules .
only a small amount of 2-microglobulin can be detected in the urine under normal physiological conditions .
therefore , levels of serum and urinary 2-microglobulin reflect the functions of glomeruli and proximal tubules . in patients with acute kidney injury , an increase in serum creatinine , together with an increase in urinary 2-microglobulin , strongly suggests proximal tubule injury . on the other hand ,
an increase in serum creatinine with elevated serum 2-microglobulin is seen in patients with decrease in glomerular filtration rate .
elevated urinary 2-microglobulin was associated with tubular injury caused by viral infection , ischemia , and toxicity from medications or heavy metals [ 15 , 16 ] .
measurement of urinary 2-microglobulin is convenient and has been applied in routine clinical practice ( renalvysion bostwick laboratories , uniondale , ny ) to evaluate the tubular function . using immunohistochemical staining for cd133 in kidney biopsies , we demonstrated that urinary 2-microglobulin is a sensitive indicator for tubular injury .
however , a direct correlation between urinary 2-microglobulin and proximal tubule injury has not yet been fully established due to lack of a specific proximal tubule injury marker .
recently , kidney injury molecule-1 ( kim-1 ) was shown to be a biomarker for proximal tubule injury [ 1820 ] .
kim-1 is a type 1 cell membrane glycoprotein comprised of extracellular , transmembrane and intracellular domains [ 21 , 22 ] .
kim-1 is undetectable in the healthy kidney . with different forms of kidney injury , mrna and protein levels of kim-1
are upregulated and expressed [ 20 , 23 , 24 ] in injured tubules [ 23 , 2527 ] .
kim-1 binds to surface - specific epitopes on the apoptotic bodies and cellular debris derived from injured tubular cells , facilitating macrophages to engulf dying cells .
in addition , the extracellular domain of kim-1 can be cleaved by metalloproteinases into a soluble shedded part and short membrane bound fragments that are released into urine .
moreover , the expression of kim-1 in injured tubule cells is limited to the proximal tubular cells .
using immunohistochemistry , kim-1 staining in kidney tissue has been shown to accurately detect proximal tubular injury associated with ischemic and toxic type of tubular injury , allograft rejection in renal transplantation , and acute pyelonephritis [ 20 , 26 , 28 ] .
the present study was designed to examine the correlation between increase in urinary 2-microglobulin and proximal tubule injury assessed by kim-1 staining in kidney biopsies .
between january 2009 and december 2012 , a total of 5494 patients had urinary 2-microglobulin measurements by renalvysion analysis ( bostwick laboratories ) . among these ,
nine renal biopsies either contained less than 10 glomeruli per histological section or had insufficient tissue to perform immunohistochemical staining and were excluded from the present study .
the remaining forty - six biopsies met selection criteria and were included in the present study .
briefly , 10  ml fresh urine was collected from each patient and urine ph was adjusted to ph 68 with 1  m naoh .
the specimen was delivered to lab by overnight shipping and the level of 2-microglobulin was measured next day .
the majority of immunohistochemical staining was performed in archived unstained slides prepared at the time of renal biopsy . in those biopsies
in which unstained slides were not available , additional new slides were prepared from archival tissue from formalin - fixed paraffin - embedded blocks .
immunohistochemical staining for kim-1 was conducted on paraffin slides using dako autostainer ( dakocytomation , carpinteria , ca ) . to remove paraffin ,
sections were heated at 60c for 60  min , placed into xylene baths for 3  min , 3 times , followed by 100% alcohol , for 3  min , 3 times , and then washed by running water for 30  s. antigen retrieval was carried out using a tris - edta buffer at ph 8.0 for 20  min at 99c and cooled down for 20  min at room temperature , raised by tap water .
slides were placed into h2o2 for 15  min and then placed in tris buffer ( ph 7.6 ) .
finally , slides were placed into dako autostainer programmed by thermo scientific ultravision lp detection system ( kalamazoo , mi ) .
staining procedures included 5  min ultra v block , 30  min incubation with primary kim-1 monoclonal antibody ( akg7 clone at 1  :  10 dilution , kindly provided by dr .
joseph v. bonventre from renal division brigham and women 's hospital , boston ) , 8  min primary antibody enhancer , 10  min hrp polymer ( equivalent to secondary antibody ) , and 5  min of chromogen dab to achieve brown staining .
granular staining in the cytoplasm and membrane of proximal tubular epithelium was considered positive . staining intensity of kim-1
was scored from 0 to 3 + as follows : 0 , absence of staining ; 0.5 , focal weak fine granular staining ; 1 + , weak fine granular staining cytoplasm and/or membrane in entire tubular luminal surface ; 2 + , moderate granular staining ; and 3 + , strong coarse granular staining .
parameters related to tubular injury including serum creatinine , granular and cellular casts , and urine cytological findings were also recorded . staining with kim-1
was evaluated by two pathologists who were blinded to the clinical data , final pathological diagnosis and patient outcome .
linear regression analysis was used to correlate values of urinary 2-microglobulin and kim-1 staining scores .
odds ratio analysis was applied to express the association of urinary 2-microglobulin level and intensity of kim-1 staining .
data were also stratified according to urinary 2-microglobulin level as normal ( < 300  g / l ) versus increased to kim-1 staining ( positive or negative ) and evaluated by 2  2 contingency table ( table 2 ) .
the sensitivity , specificity , predictive value for positive result , and predictive value for negative result were calculated as follows :  sensitivity = true positive/(true positive + false negative ) ;  specificity = true negative/(true negative + false positive ) ;  predictive value for positive result = true positive/(true positive + false positive ) ;  predictive value for negative result = true negative/(true negative + false negative);the age of the patients was expressed as mean  sd . sensitivity = true positive/(true positive + false negative ) ; specificity = true negative/(true negative + false positive ) ; predictive value for positive result = true positive/(true positive + false positive ) ; predictive value for negative result = true negative/(true negative + false negative ) ;
among 5494 patients with urinary 2-microglobulin measurement , renal biopsy was obtained out in 56 patients ( 56/5494 , 1.0% ) . among these ,
thirty patients had increases in urinary 2-microglobulin ; it was in normal range in the remaining 16 patients .
the demographic details for 35 patients ( m  :  f = 22  :  13 ) with positive kim-1 staining in proximal tubules are shown in table 1 .
the age of patients at the time of biopsy was 54.9  10.7 years ranging from 16.0 to 87.0 years .
glomerular diseases included membranous nephropathy , antineutrophil cytoplasmic antibody- ( anca- ) associated glomerulonephritis , diabetic nephropathy , focal and segmental glomerulosclerosis , iga nephropathy , and lupus nephritis .
in addition to glomerular disease , five patients had tubular injury ( figures 1(c ) and 1(d ) ) , two of which were toxic type of injury with cytoplasmic vacuoles ( figure 1(d ) ) .
of the 26 patients with elevated urinary 2-microglobulin and positive kim-1 staining in proximal tubules , 18 patients had increased serum creatinine , ranging from 1.50  mg / dl to 6.00  mg / dl ( table 1 ) .
since the elevated urinary 2-microglobulin is associated with proximal tubular injury , in these 18 patients it was considered that the proximal tubular injury is the primary cause of increase in serum creatinine .
nine patients with normal urinary 2-microglobulin had positive kim-1 staining in proximal tubules , 6 of which had increase in serum creatinine , ranging from 1.10  mg / dl to 3.20  mg / dl .
granular casts were presented in 2 patients , who also had increase in both urinary 2-microglobulin and serum creatinine .
thirty - five of 46 biopsies showed positive kim-1 staining with a granular pattern in the cytoplasm and/or apical membrane of tubular epithelium , ranging from finely granular to coarsely granular ( figure 1(b ) ) .
the corresponding h&e slides of kidney biopsy showed flattening and simplification of tubular epithelium , ranging from focal to diffuse , overall focal tubular injury with varying degree , and mild tubular injury in the majority of the kidney biopsies . staining
intensity ranged from 0.5 + to 3 + with 1 + staining in 18 patients ( 18/35 , 51.4% ) .
the relation between value of urinary 2-microglobulin and kim-1 staining score is shown in figure 2 .
there was a significant correlation between levels of urinary 2-microglobulin and kim-1 staining ( r value = 0.293 , p < 0.05 ) ( figure 2 ) .
odds ratio was 7.229 , implying that levels of urinary 2-microglobulin are strongly associated with intensity of kim-1 staining .
confidence interval at the 95% range was 2.868 to 12.01 . among 35 biopsies with kim-1 positive staining ,
using positive kim-1 staining as the end point , sensitivity was 86.6% , specificity was 43.7% , positive predictive value was 74.2% , and negative predictive value was 63.6% .
we found that increased urinary 2-microglobulin was significantly correlated with kim-1 staining in proximal tubules , indicating that urinary 2-microglobulin is a sensitive ( sensitivity 86.6% ) assay for detecting tubular injury .
further , because kim-1 is a selective proximal tubule injury marker , the present study also demonstrated that measurement of urinary 2-microglobulin was a reliable assay to detect proximal tubule injury .
therefore , in patients with acute kidney injury with increase in serum creatinine , measurement of urinary 2-microglobulin is also helpful to identify the underlying tubular dysfunction , that is , proximal tubule injury .
acute tubular injury is a common clinical diagnosis . in a patient with classic clinical presentations such as hypotension together with a rapid rising of serum creatinine
, the diagnosis of severe tubular injury ( acute tubular necrosis ) can be made and does not require kidney biopsy to initiate treatment .
however , the diagnosis of glomerular disease accompanied by tubular injury is not easy to establish , as both may contribute to elevated serum creatinine .
tubular injury was the secondary diagnosis in 5/35 patients but other evidence of tubular injury such as tubular casts and an increase in tubular cells by urine analysis was found in 22/35 patients . moreover , 2 of 4 patients with secondary diagnosis of acute interstitial nephritis showed tubulitis , a feature associated with tubular injury .
these findings indicate that glomerular diseases associated with varying degree of tubular injury could be missed in renal biopsies due to sampling . in a prior study , using cd133 staining in injured tubular epithelium , it was demonstrated that urinary 2-microglobulin was a sensitive indicator for tubular injury . as a progenitor marker
in addition , cd133 positive cells are present not only in proximal tubular cells but also in distal tubular cells and/or atrophic tubules .
therefore , a careful morphological correlation is needed to exclude false positive staining . by comparison
, kim-1 is a selective proximal tubule injury marker that only presents in patients with proximal tubule injury [ 1820 ] .
therefore , kim-1 is a more specific marker than cd133 for detecting proximal tubular injury . in the present study ,
kim-1 was able to detect patients with mild , focal tubular injury evidenced by urine analysis as an increase in number of tubular cells ; some patients presented with normal levels of serum creatinine ( table 1 ) .
this was also supported with the finding in the present study that more than half of kim-1 staining score was weakly positive ( 1 + ) .
a good correlation of urinary 2-microglobulin and kim-1 staining ( r value of 0.293 ) indicates that measurement of urinary 2-microglobulin is a reliable assay for proximal tubule injury .
similar to cd133 staining , kim-1 was also able to identify two major types of tubular injury : ischemic and toxic type ( figures 1(c ) and 1(d ) ) .
as shown in table 2 , tubular injury was an additional diagnosis included in 5 of 26 biopsies ( 25% ) ( table 1 ) , and all showed positive kim-1 staining in proximal tubules . using kim-1 staining as a proximal tubule injury marker in the present study ,
nine patients , rather than six patients with normal urinary 2-microglobulin , presented with positive kim-1 staining , which were considered false positive . with low specificity , clinical interpretation of elevated urinary 2-microglobulin should be closely correlated with clinical history , ( e.g. , history of hypotension , recent history of medications use , etc . ) and clinical presentation , including value of serum creatinine , as well as urine cytological findings .
the diagnosis of tubular injury with renalvysion was made not only based on the elevation of urinary 2-microglobulin and serum creatinine but also combined with morphological findings of tubular casts and the number of sloughed tubular epithelial cells in urine analysis .
renalvysion is a urine - based analysis that combines urine chemistry and cytology to detect glomerular disease and tubular injury .
elevation of urinary 2-microglobulin over 300  g / l was considered to be abnormal by renalvysion .
g / l and positive kim-1 staining in biopsies were considered to be false positive .
however , the actual values of urinary 2-microglobulin in 2 patients were marginally high in 280  g / l and 290  g / l .
the remaining 7 patients were found to have levels less than 210  g / l , which is the measurement threshold of the assay .
because the confidence interval at the 95% range in lower end was 2.868 in present study , the normal range of urinary 2-microglobulin applied in renalvysion should be reconsidered to be 2.868  g / l .
g / l should be considered as abnormal and belongs to true positive for kim-1 staining .
therefore , corrected sensitivity is 87.1% , specificity is 46.6% , positive predictive value is 77.1% , and negative predictive value is 63.6% .
our study has multiple limitations . only a small number of patients had follow - up renal biopsies after renalvysion ( 56/5494 , 1.0% ) , raising concern for selection and ascertainment biases .
as shown in table 1 , these patients had glomerular disease and/or tubular - interstitial disease .
the reason for this might be that renal biopsy is usually avoided in patients with a clear clinical diagnosis of severe tubular injury . a larger study cohort involving patients with severe tubular injury is needed to better correlate renal histopathology and immunohistochemistry . owing to limited sampling by biopsies ,
the retrospective nature of the study represents an additional limitation . in summary , measurement of urinary 2-microglobulin is a sensitive assay to screen patients with proximal tubule injury .
our data indicate a correlation of urinary 2-microglobulin with results of immunohistochemical staining of kim-1 in renal biopsy and consequently with proximal tubular injury .
 
 objective . after filtration through glomeruli , 2-microglobulin is reabsorbed in proximal tubules . increased urinary 2-microglobulin indicates proximal tubule injury and measurement of 2-microglobulin in urine is useful to determine the source of renal injury . 
 kidney injury molecule-1 ( kim-1 ) has been characterized as a selective proximal tubule injury marker . 
 this study was designed to evaluate the correlation of urinary 2-microglobulin concentration and kim-1 expression as evidence of proximal tubule injury 
 . methods . between 2009 and 2012 , 46 patients with urine 2-microglobulin ( renalvysion ) had follow - up kidney biopsy . 
 diagnoses included glomerular and tubule - interstitial disease . 
 immunohistochemical staining for kim-1 was performed and the intensity was graded from 0 to 3 + . 
 linear regression analysis was applied to correlate the values of urinary 2-microglobulin and kim-1 staining scores . 
 p < 0.05 was considered statistically significant . 
 results . 
 thirty patients had elevated urinary 2-microglobulin . 
 kim-1 staining was positive in 35 kidney biopsies . 
 there was a significant correlation between urinary 2-microglobulin and kim-1 staining ( p < 0.05 ) . 
 sensitivity was 86.6% , specificity was 43.7% , positive predictive value was 74.2% , and negative predictive value was 63.6% . conclusion . increased urinary 2-microglobulin is significantly correlated with kim-1 staining in injured proximal tubules . 
 measurement of urine 2-microglobulin is a sensitive assay for proximal tubule injury . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion