lupus nephritis ( ln ) is one of the most frequent manifestations of systemic lupus erythematosus ( sle ) and represents a major determinant of disease morbidity and mortality .
its clinical course is often characterized by flares of activity alternated with periods of quiescence , generally induced by therapy .
the identification of noninvasive biomarkers may help to predict the renal involvement at diagnosis and monitor relapses of ln during the follow - up .
many studies have tested the value of a number of autoantibodies for predicting or confirming the diagnosis of renal flares with contrasting results .
some [ 35 ] but not all studies   have demonstrated that anti - dsdna antibodies ( anti - dsdna ) and complement fractions may be useful in assessing the disease and the renal activity .
one paper   and a recent review   concluded that anti - nucleosome antibodies have high prevalence in severe ln but are of limited help in differentiating active from inactive ln .
a number of cross sectional studies found that antic1q antibodies ( antic1q ) have a significant association with renal involvement [ 915 ] . in our previous paper on a large cohort of sle patients evaluated prospectively for 6 years , we demonstrated that renal exacerbations seem to be quite improbable in the presence of normal values of c3 , c4 , anti - dsdna , anti - c1q , and that anti - c1q was slightly better than the other tests to confirm the clinical activity of ln .
noteworthy , in the vast majority of studies the diagnosis of ln flares relies on variable clinical definitions based on activity of urine sediment , amount of proteinuria , and deterioration of renal function , whilst the  gold standard  for the diagnosis of renal activity is represented by renal biopsy . in this prospective study
, serum samples at renal biopsy and after the induction therapy of 107 ln patients were tested for a panel of autoantibodies ( including anti - dsdna , anti - c1q , anti - nucleosome , anti - ribosome antibodies , and c3 and c4 complement fractions ) to investigate their association with the clinical and histological data .
one hundred and seven patients with sle , diagnosed according to the american college of rheumatology criteria   ( 94 females , 13 males ) at admission in two italian renal units ( fondazione ospedale maggiore and azienda ospedaliera ospedale san carlo borromeo , milano ) to undergo renal biopsy for assessment of ln , entered the study .
sera at renal biopsy were tested for a panel of auto antibodies including anti - dsdna and anti - c1q , anti - nucleosome , and anti - ribosome antibodies as well as c3 and c4 complement fractions .
we have obtained an informed consent to participate in the study from all the patients involved .
the aim of this study was to assess the performance of these tests in predicting : the histological classes of lupus nephritis , the activity and chronicity index at renal biopsy , the clinical feature of ln at renal biopsy , the response of lupus nephritis at 3 , 6 , and 12 months after the beginning of the induction therapy .
the histological classes of lupus nephritis , the activity and chronicity index at renal biopsy , the clinical feature of ln at renal biopsy , the response of lupus nephritis at 3 , 6 , and 12 months after the beginning of the induction therapy .
anti - dsdna antibodies were measured by a commercial quantitative elisa ( varelisa anti - dsdna antibodies , phadia gmbh , freiburg , germany ) and c3 and c4 plasma levels by nephelometry ( nephelometer analyser ii , behring , marburg gmbh , germany ) .
anti - c1q antibodies were detected using a home - made elisa as described by sinico et al . .
anti - nucleosome antibodies were measured by elisa according to manufacturer instructions using quanta lite chromatin assay ( inova diagnostics , inc . , san diego , ca , usa ) . .
anti - ribosome p antibodies were measured by elisa according to manufacturer instructions using quanta lite ribosomal p assay ( inova diagnostics , inc . , san diego , ca , usa ) .
at each clinical examination the activity of ln was classified as follows :  0 = complete renal remission : normal renal function for at least 6 months , proteinuria < 0.5  g/24  h , and urinary red blood cells < 5/hpf ;  1 = partial renal remission : for nephritic flare : improvement of at least 30% of serum creatinine but persistence of active urinary sediment ; for proteinuric flares improvement of 50% of proteinuria ;  2 = nephritic flare : increase of 30% of serum creatinine over the basal value and active urinary sediment ( > 10 red blood cells / hpf , cellular casts ) with or without an increase in proteinuria ;  3 = proteinuric flare : increase of proteinuria of at least 2  g / day in patients with non nephrotic syndrome or the doubling of nephrotic proteinuria with stable renal function ;  4 = persistent renal activity : the lack of achievement of remission after induction therapy . 0 = complete renal remission : normal renal function for at least 6 months , proteinuria < 0.5  g/24  h , and urinary red blood cells < 5/hpf ; 1 = partial renal remission : for nephritic flare : improvement of at least 30% of serum creatinine but persistence of active urinary sediment ; for proteinuric flares improvement of 50% of proteinuria ; 2 = nephritic flare : increase of 30% of serum creatinine over the basal value and active urinary sediment ( > 10 red blood cells / hpf , cellular casts ) with or without an increase in proteinuria ; 3 = proteinuric flare : increase of proteinuria of at least 2  g / day in patients with non nephrotic syndrome or the doubling of nephrotic proteinuria with stable renal function ; 4 = persistent renal activity : the lack of achievement of remission after induction therapy .
mean and standard deviation , together with median and interquartile ( iq ) range ( 2575 percentile ) were used as descriptive statistics . for continuous variables ,
the nonparametric wilcoxon test was used for assessing any difference between the two groups of patients , while the chi - square test was used for dichotomized variables .
multivariate logistic regression analysis has been used to find predictors of histological classes of lupus nephritis and for the predictors of complete renal response after the beginning of induction therapy .
odds ratios ( or ) and their 95% confidence interval ( ci ) for the covariates were derived as the antilogarithm of the regression coefficients .
the statistical package s - plus ( mathsoft inc . ) was used for all the analyses and plots .
the mean age at diagnosis of sle was 35.3  14.2 years , ( median 34 ) and that at renal biopsy was 36.4  13.9 years ( median 36 ) .
the mean time between the diagnosis of sle and that of renal involvement was 5.1  6.5 years , ( median 3 years ) . in 45 patients ,
considering that a preliminary analysis demonstrated no significant differences in the mean values of c3 , c4 , anti - dsdna , anti - c1q , anti - nucleosome , and anti - ribosome antibodies between class ii and class v and between class iii and class iv ln ( data not shown ) , the subsequent analysis was performed comparing class ii plus class v ( nonproliferative forms ; 26 patients ) versus class iii plus class iv ( proliferative forms ; 85 patients ) . at renal biopsy ,
high titers of anti - dsdna were present in 77.5% of cases , high titers of anti - c1q in 70.5% of cases , high titers of anti - nucleosome antibodies in 80.3% of cases , and high titers of anti- ribosome antibodies in 14% of cases ; c3 were low in 82% of cases and c4 in 74% of cases .  
table 2 reports the comparison at time of renal biopsy of clinical data and of the panel of autoantibodies between proliferative forms ( class iii plus class iv ) and nonproliferative forms ( class ii plus class v ) of ln . at univariate analysis , among the clinical parameters , proteinuria ( p = 0.02 ) and hemoglobin ( p = 0.0008 ) and among the immunological tests , c3 ( p = 0.02 ) and c4 ( p = 0.02 ) complement fractions , anti - dna ( p = 0.001 ) , anti - c1q ( p = 0.0005 ) , and high titers of anti - c1q antibodies or of anti - dsdna antibodies ( p = 0.0000 ) and anti - nucleosome antibodies ( p = 0.04 ) were able to differentiate proliferative from nonproliferative forms of ln . at multivariate analysis , hemoglobin ( p = 0.008 , or = 0.68 , ci : 0.520.9 ) and anti - c1q antibodies ( p = 0.03 , or = 1.004 , ci : 1.00031.007 ) were the independent predictors to discriminate between proliferative versus nonproliferative lupus nephritis .
excluding clinical parameters , at multivariate analysis , logarithm of erythrocyte sedimentation rate ( esr ) ( p = 0.03 , or = 1.9 , ci : 1.083.42 ) and high titers of anti - c1q antibodies or of anti - dsdna antibodies ( p = 0.005 , or = 8.67 , ci : 2.0337.3 ) are the independent predictors which are able to discriminate proliferative from nonproliferative lupus nephritis . among patients with proliferative forms of lupus nephritis ,
95% have high titers of anti c1q or of anti - dsdna ( 66.2% have high titers of both anti - c1q and of anti - dsdna ) while 5% have the results of both tests in a normal range . among patients with nonproliferative forms ,
64% have high titers of anti c1q or of anti dna while 36% have the results of both tests in a normal range ( p = 0.000 ) .
the correlations among the basal clinical and the immunological data and the activity and the chronicty index at renal biopsy are reported in table 3 .
all the clinical and immunological parameters evaluated with the exception for c reactive protein ( crp ) and anti - ribosome antibodies showed a significant correlation with activity index . at multivariate analysis proteinuria ( p = 0.0013 ) ,
low c4 ( p = 0.0010 ) , and high esr ( p = 0,037 ) were the independent predictors of the activity index . excluding the clinical variables , low c4 ( p = 0.0004 ) and high esr ( p = 0,0025 ) were the independent predictors of activity index .
in contrast , serum creatinine was the only parameter among those evaluated that showed a direct correlation with the chronicity index ( r : 0.4 , p = 0.0000 ) .
no correlation was found at time of renal biopsy between serum creatinine and the panel of autoantibodies , c3 and c4 complement fractions , and esr and crp . among these tests ,
anti - c1q only showed a significant direct correlation with the amount of proteinuria ( r = 0.2 , p = 0.03 ) .
in addition , patients with high titers of anti - c1q or of anti - dsdna had significant higher proteinuria ( median 2.7  g / day , iq 1.64.6 ) than those with both tests in normal range ( 1.8  g / day , iq 1.02.2 , p = 0.05 ) .
anti - c1q , anti - dsdna , and esr were inversely correlated with hemoglobin ( r-0.22 , p = 0.02 , r-0,24 , p = 0.01 , r-0.32 , p = 0.002 , resp . ) , while c3 and c4 were correlated with hemoglobin ( r 0.36 , p = 0.0002 and r 0.25 , p = 0.01 ) .
after renal biopsy and the beginning of induction therapy , 104 patients had a second evaluation between 3 and 12 months .  
table 4 reported the results of clinical and immunological tests in patients reevaluated at 3 months , at 6 months , and at 12 months . at 3 months , serum creatinine was unchanged and proteinuria did not show a significant improvement , while esr , c3 , anti - dna , anti - c1q , and anti - nucleosome antibodies showed a significant improvement in the median values . at 6 and at 12 months ,
proteinuria significantly improved together with all immunological tests with the exception of esr at 6 months and anti - ribosome antibodies at 12 months .
altogether , during the observation period , 39 patients ( 37.5% ) achieved and 65 ( 62.5% ) did not achieve complete renal remission ( 46 were in partial remission , 13 had persistent renal activity , and 6 had persistent nephrotic syndrome ) .
clinical and immunological tests at the time of renal biopsy have been tested as predictors of complete renal remission ( table 5 ) . at univariate
analysis , none of the immunological tests were predictive of complete remission . at multivariate analysis ,
proteinuria ( p = 0.015 , or : 0.76 ci 0.620.95 ) and the duration of therapy ( p = 0.03 or : 1.19 ci 1.0171.39 ) were the independent predictors of complete renal remission .
in this study , we have investigated the prevalence and the value of a panel of autoantibodies ( anti - dsdna , anti - c1q , anti - nucleosome , and anti - ribosome antibodies ) as well as c3 and c4 complement fractions in predicting the activity of ln at the time of renal biopsy .
the most important difference of our study compared to many previous studies is the timing of blood sampling in relation to renal activity . as a matter of fact , in the majority of the studies evaluating the predictive values of autoantibodies
[ 11 , 15 , 2123 ] , the renal activity of ln at the time of blood sampling was judged by clinical parameters but not confirmed by renal biopsy . to the best of our knowledge ,
only a few studies have evaluated the association of some autoantibodies with activity of ln at the time of renal biopsy [ 2427 ] .
reported that all but one out of 36 patients with proliferative lupus were positive for anti - c1q at the time of renal biopsy compared with 35% of patients with inactive ln . in 136 chinese patients , anti - c1q and anti - dsdna were more closely correlated with histological activity of ln at the time of renal biopsy than anti - extractable nuclear antigen antibodies , anti - c protein antibodies , anti cardiolipin , and anti beta2 glycoprotein antibodies .
the combination of anti - c1q and anti - dsdna indicates higher renal disease activity and predicts poor long term renal outcome .
another paper   investigated the clinical and pathological association of anti - c1q in ln and found a higher prevalence of the autoantibody in class iv than in the other histological classes . among the clinical variables
low haemoglobin was associated with anti - c1q positivity . in this paper , we have shown that there was a significant difference in the autoantibodies profile between proliferative forms ( class iii plus iv ) and the other forms of ln ( class v and class ll ) .
all the autoantibodies evaluated , with the exception of anti - ribosome antibodies , had significant higher prevalence and higher titres in proliferative than in nonproliferative forms of ln .
c3 and c4 complement fractions too were significantly lower in proliferative than in nonproliferative ln . at multivariate analysis , considering clinical and immunological tests , only low haemoglobin and high anti - c1q were the independent predictors of proliferative ln . excluding the clinical variables , high esr and positive anti - c1q or anti -
ninety - five percent of patients with proliferative ln had high titers of anti c1q or of anti - dsdna ( 66.2% had high titers of both tests ) while 4 patients only had the results of both tests in normal range .
the increasing power of the combination of anti - c1q or anti - dsdna positivity in predicting the activity of ln has been reported by other studies [ 25 , 28 ] .
this higher predictive value of anti c1q for proliferative ln confirmed our findings in a previous study in which we demonstrated that 80% of flares that developed in patients with proliferative forms were associated with high titres of anti - c1q in comparison to only 54% of those that occurred in the nonproliferative forms .
instead , other cohort studies did not show differences in the prevalence of antic1q between proliferative and nonproliferative lupus nephritis [ 2830 ] .
this discrepancy could be due to the fact that in these studies the diagnosis of renal activity was done on clinical grounds and not confirmed by renal biopsy .
again , anti - c1q , alone or associated with anti - dsdna , was the only test among the immunological parameters that significantly correlated with the amount of proteinuria . none of the immunological tests correlated with serum creatinine but the majority of our patients had normal renal function , and many tests correlated with hemoglobin , a manifestation not specific for ln but an expression of the general activity of sle .
low c4 and high esr were the independent predictors of a high activity index at multivariate analysis while none of the tests of the panel correlated with chronicity index .
, in our cohort , none of the immunological tests at the time of renal biopsy was predictive of the renal response , at least in the short term .
however , we have shown that , three months after the start of the induction therapy and prior to the improvement of proteinuria , a significant reduction of the mean value of anti - c1q , anti - dsdna , and anti - nucleosome antibody occurred .
the progressive and significant drop in autoantibodies titres continued at 6 and at 12 months together with a clinical improvement as reported in other studies [ 24 , 26 , 31 ] .
anti - c1q antibodies can be detected by different methods ( reviewed in [ 32 , 33 ] ) . in the early 1980s , a solid - phase assay using purified c1q , immobilized on plastic assay plates , was used for the detection of circulating immune complexes in sle patients . to differentiate between immune complexes and anti - c1q antibodies , high - salt concentrations ( 0.51.0  m sodium chloride )
the binding of the globular heads of c1q to immune complexes is prevented , whereas anti - c1q antibodies can still interact with the coated c1q .
subsequently , to eliminate the need to use high - ionic strength buffer , assays have been developed that utilize only the c1q collagen - like region .
however , additional exposed epitopes , by cleaving of the c1q molecule , might interfere with the results obtained with this assay .
more recently , peptides derived from c1q that have the properties to detect a major linear epitope in a high percentage of the patients in the absence of high - ionic strength buffer has been proposed . unfortunately , systematic studies comparing anti - c1q antibody detected by different assays are not available and different studies have used different methods . in our study , we have used the  classic  assay which has been used in the majority of published clinical studies because it is more readily available .
we found a significantly different autoantibodies profile between proliferative and nonproliferative forms of ln at the time of renal biopsy . among the panel of autoantibodies evaluated in this study ,
anti - c1q alone or in combination with anti - dsdna emerged as the most reliable in differentiating proliferative and nonproliferative ln and anti - c1q is the only test correlated with the clinical presentation of ln . after the beginning of therapy , the titer of the autoantibodies progressively and significantly reduced , but none of them was predictive of complete renal remission .
the results of this work , which outlines the role of autoantibodies and in particular of anti - c1q , in defining the activity of lupus nephritis at the time of renal biopsy , confirm their utility in diagnosing the acute exacerbations of ln made on clinical grounds only .
 few studies have correlated serum biomarkers with renal histology , the gold standard for renal activity , in lupus nephritis ( ln ) . 
 we tested a panel of autoantibodies and complement at the time of kidney biopsy and after treatment . 
 anti - dsdna , anti - nucleosome , anti - ribosome p , and anti - c1q antibodies and c3/c4 were measured in 107 patients with ln at the time of renal biopsy and after 612 months and were correlated with clinical / histological parameters . at multivariate analysis , 
 high titers of anti - c1q antibodies or of anti - dsdna antibodies ( p = 0.005 , or = 8.67 , ci : 2.0337.3 ) were the independent predictors that discriminate proliferative from nonproliferative ln . 
 all the immunological parameters , except anti - ribosome , showed a significant correlation with activity index but not with chronicity index . 
 only anti - c1q showed a significant correlation with the amount of proteinuria ( r = 0.2 , p = 0.03 ) . 
 none of the immunological parameters were predictive of remission at 6 and 12 months . 
 we found that anti - c1q alone or in combination with anti - dsdna emerged as the most reliable test in differentiating proliferative and nonproliferative ln . 
 anti - c1q was the only test correlated with the clinical presentation of ln . 
 after treatment , the titre of the autoantibodies was significantly reduced , but none was predictive of remission . 
1. Introduction
2. Patients and Methods
3. Statistical Analysis
4. Results
5. Discussion
6. Conclusions