hepatitis b virus ( hbv ) infection is an important public health problem worldwide , especially in developing countries , and hepatitis b virus - associated glomerulonephritis ( hbv - gn ) remains one of the most common secondary glomerular diseases .
ever since the association between hbv infection and glomerular diseases was first reported by combes et al . in 1971
the existence of hbv dna in the renal tissue of some nephritic syndrome patients led to the classification of hbv - gn , proposing a role for hbv in its pathogenesis .
the widely accepted view is that persistent viral infections could lead to immune complex - mediated nephritis .
hbv is a noncytopathic human hepadnavirus that causes acute and chronic hepatitis and hepatocellular carcinoma .
it contains a circular and partially double - stranded dna ( dsdna ) genome of approximately 3.2  kb that consists of four overlapping open reading frames  the c , s , p , and x regions . a key innate immune response to infection with microbial or viral pathogens and
.   showed that cytoplasmic dna triggers the formation of the absent melanoma 2 ( aim2 ) inflammasome by inducing aim2 oligomerization .
the inflammasome then activates caspase-1 , a cysteine protease that processes the inactive pro - interleukin-1b ( pro - il1 ) and pro - il18 to their respective active proinflammatory cytokines , il-1 and il-18 .
aim2 belongs to a family of hin-200 proteins that includes at least four members in humans 
hin-200 protein family members are interferon- ( ifn- ) inducible proteins with a 200-amino - acid repeat at the c - terminus , which is known as the hin domain , and an n - terminal pyrin domain ( pyd ) .
several studies have recently identified aim2 as a cytosolic sensor that binds dsdna through its c - terminal hin domain [ 6 , 812 ] .
the aim2 n - terminal pyd motif can recruit apoptosis - associated speck - like protein containing a card ( asc ) and thus caspase-1 , which can then lead to the formation and secretion of mature il-1 and il-18 , causing subsequent tissue damage .
although aim2 is known to be involved in the host defense against microbial invasion , its role in regulating the immune response to viruses , especially to hbv , has not been well understood .
given that hbv particles have been detected in the kidneys of hbv - gn patients , this cytoplasmic hbv dna could potentially be recognized by aim2 , leading to caspase-1 activation via the aim2 inflammasome and ultimately contributing to the renal damage seen in hbv - gn patients . in this study , we compared the expression of aim2 , caspase-1 , and il-1 in hbv - gn and chronic glomerulonephritis ( cgn ) patient kidney tissues .
the effects of aim2 expression status in primary human glomerular mesangial ( hgm ) cells transfected with hbv dna or vector control on caspase-1 , il-1 , and il-18 expression were also investigated .
our results showed that aim2 expression was higher in hbv - gn tissues than in cgn tissues and was correlated with renal inflammation associated with hbv - gn .
furthermore , sirna - mediated knockdown of aim2 decreased the expression of caspase-1 , il-1 , and il-18 in vitro .
thus , aim2 may play an important role in the development and progression of inflammation .
our retrospective study was approved by the ethics committee of jinan infectious disease hospital . a total of 79 patients diagnosed with chronic nephritis , identified between 2008 and 2011 at jinan infectious disease hospital and qilu hospital of shandong university shandong , china ,
the experimental group consisted of 54 hbv - gn patients , and the negative control group consisted of 25 cgn patients .
subjects received kidney puncture biopsy under ultrasound guidance to obtain tissue for diagnosis and subsequent research .
participation was dependent upon fulfillment of the following criteria : ( 1 ) patients must not have used an immune agent or antiviral agent in the past three months ; ( 2 ) patients must not have hav , hcv , hdv , hev , or hiv coinfection ; ( 3 ) patients must not have a history or current evidence of secondary glomerulonephritis ; and ( 4 ) consent for participation must have been obtained from those who participated . the diagnostic criteria used for cgn and hbv - gn were in accordance with the 2002 kidney disease outcome quality initiative ( k / doqi ) , edited by the national kidney foundation ( nkf ) .
frozen slices from biopsies of the 54 hbv - gn patients were kept in a low - temperature freezer .
monoclonal goat - anti - human hbsag and hbcag antibodies were purchased from dako ( denmark ) , and immunohistochemical staining for hbsag and hbcag in renal biopsies was used to confirm the diagnosis . for hbv - gn patients with undetectable hbsag or hbcag in the kidney tissue
tissue specimens were first fixed in 10% formalin , and then the tissue was cut , dehydrated , dipped in wax , embedded , and sectioned .
these sections were then placed on slides , baked , placed into xylene , cleared of the wax , rehydrated using graded ethanol , and immersed in 0.3% hydrogen peroxide for 5  min to reduce nonspecific background staining caused by endogenous peroxidase .
the slides were then washed with pbs buffer three times for five minutes each and placed in citrate buffer solution at a ph of 6.0 and then into a high temperature pressure pot to recover the tissue antigen . after being heated , the slides were cooled and restored at room temperature , washed three more times in pbs buffer , and incubated with aim2 rabbit anti - human polyclonal antibody ( ab93015 ; abcam , cambridge , uk ) , caspase-1 mouse anti - human polyclonal antibody ( sc-56063 ; santa cruz biotechnology inc .
, texas , usa ) , and il-1 rabbit anti - human polyclonal antibody ( ab2105 ; abcam , cambridge , uk ) .
the next day , the slides were washed with pbs buffer three times , each time lasting longer than 5  min , then incubated with the secondary antibody pv-9000 ( universal antibody ) at 37c for 10  min , and washed with pbs buffer , and dab staining was applied .
the stain was terminated using running water ; then the slides were washed with hydrochloric acid alcohol for differentiation .
lastly , the slides were washed with distilled water , cleared with xylene , and mounted . meanwhile , the aim2 , caspase-1 , and il-1 staining with pbs substituted primary antibody respectively in hbv - gn tissue , followed by dab , was shown as negative control . appearance of a tan stain in the cytoplasm signaled positive expression of the protein . after staining ,
scores were assigned based on stain intensity and percentage of positive cells as follows : for stain intensity , a score of 0 was given for no brown staining ( i.e. , no cells stained ) , 1 for light brown , 2 for brown , and 3 for dark brown ; for percentage of positive cells , a score of 0 was given for fewer than 5% positive cells , 1 for 5% to 30% , 2 for 30% to 60% , and 3 for greater than 60% .
scores for stain intensity and percent positive were then added together , and a negative sign (  ) was assigned for scores totaling 0 , mildly positive ( + ) for scores between 1 and 3 , moderately positive ( + + ) for scores between 4 and 6 , and strongly positive ( + + + ) for scores greater than 7 .
the human glomerular mesangial ( hgm ) cell line used in this study was purchased from sciencell research laboratories ( california , usa ) and isolated from human renal tissue .
hrmc are characterized by immunofluorescent methods with antibodies to fibronectin , thy-1 , and smooth muscle actin .
hrmc are negative for hiv-1 , hbv , hcv , mycoplasma , bacteria , yeast , and fungi .
cells were cultured in dmem ( gibco , california , usa ) , supplemented with 10% fetal bovine serum ( gibco , california , usa ) , 100  u / ml penicillin , and 100  g / ml streptomycin at 37c in a humidified atmosphere containing 5% co2 .
the 1.1-fold overlength hbv genome was cloned into the pcdna3 vector to generate pcdna3.0 - 1.1hbvdna , 1.1hbv as the expression gene and ampicillin resistance for antibiotic selection ( amresco , pennsylvania , usa ) .
aim2-sirna was synthesized accordingly : sense , 5-guc ccg cug aac auu auc att-3 ; antisense , 5-uga uaa ugu uca gcggga ctt-3 .
negative control sirna was synthesized accordingly : sense , 5-uuc ucc gaa cgu guc acg utt-3 ; antisense , 5-acg uga cac guu cgg aga att-3 .
transient transfection of sirna and plasmids was performed using lipofectamine 2000 ( invitrogen , california , usa ) . in brief
, hgm cells were seeded the day prior to transfection at a density of 3  10 cells / well in a 6-well plate with complete media .
for each well , 5  l sirna or plasmid ( 4  g ) was added into 250  l opti - mem ( gibco , california , usa ) medium , and then sirna or plasmids were mixed with lipofectamine 2000 .
the mixture was added to cells and incubated for 6  h before replacing the medium . protein and mrna analyses
total rna was isolated using trizol rna reagent ( invitrogen , california , usa ) .
quantitative real - time rt - pcr ( qrt - pcr ) was used to confirm the expression levels of mrnas .
reverse transcription was performed using the revertaid first stand cdna synthesis kit ( thermo fisher scientific , massachusetts , usa ) , according to the manufacturer 's instructions .
the primer pairs used to detect aim2 and the internal control genes are listed in table 1 .
qrt - pcr was performed using a bio - rad cycler cfx96 detection system ( bio - rad laboratories , california , usa ) and sybr premix ex taq ( takara bio , tokyo , japan ) , according to the manufacturer 's instructions .
briefly , 2  l cdna template was used for each reaction in a total volume of 20  l . all reactions were performed in triplicate .
the relative quantification of target gene expression was evaluated using the comparative cycle threshold ( ct ) method . before harvest ,
cells in 6-well plates were washed twice in pbs and lysed in ripa lysis buffer ( beyotime institute of biotechnology , nantong , china ) .
protein content of the samples was determined by the bradford assay using bovine serum albumin ( bsa ) as a standard .
proteins were separated by sds , polyacrylamide gel electrophoresis in tris / glycine buffer ( 25  mm tris and 250  mm glycine ) , and electroblotted onto polyvinylidene fluoride ( pvdf ) membranes ( 0.45  m , immobilon - p ; millipore , massachusetts , usa ) followed by blocking in tris - buffered saline with tween 20 ( tbst ) containing 5% w / v fat - free milk and 0.05% v / v tween 20 .
membranes were incubated with rabbit monoclonal antibody specific for human aim2 ( ab93015 ; abcam , cambridge , uk ) ( 1  :  200 diluted in primary antibody dilution buffer ) , rabbit monoclonal antibody specific for human caspase-1 ( 3345 - 1 ; epitomics , california , usa ) ( 1  :  1000 diluted in primary antibody dilution buffer ) , rabbit monoclonal antibodies specific for human il-1 ( ab2105 ; abcam , cambridge , uk ) ( 1  :  200 diluted in primary antibody dilution buffer ) and il-18 ( ab137664 ; abcam , cambridge , uk ) ( 1  :  500 diluted in primary antibody dilution buffer ) overnight at 4c , and il-6 rabbit anti - human polyclonal antibody ( ab6672 ; abcam , cambridge , uk ) ( 1  :  500 diluted in primary antibody dilution buffer ) . after washing in tbst
, the membranes were incubated with horseradish peroxidase- ( hrp- ) conjugated secondary antibody ( cwbio , beijing , china ) diluted 1  :  10,000 in tbst and incubated for 1  h at room temperature . after washing extensively in tbst
, membranes were immersed in ecl detection reagent ( beyotime institute biotechnology , nantong , china ) followed by exposure to chemidoc xrs + system ( bio - rad , california , usa ) . the spss program ( version 17.0 )
background factors were compared using student 's t - test ( numerical data ) or the chi - square test ( categorical data ) .
spearman 's two - tailed test was used for correlation analysis , and differences were regarded as significant if the p value was less than 0.05 on either side .
the expression of aim2 in biopsied kidney tissue from 54 hbv - gn and 25 cgn patients was determined by immunohistochemistry .
the results showed that aim2 expression was exclusive to the cellar cytoplasm of glomerular endothelial cells and mesangial cells in the tissue .
statistical analysis revealed that the positive expression rate of aim2 in hbv - gn patients was significantly higher than in cgn patients ( 81.4% versus 4.0% , p < 0.01 ) ( table 2 ) .
notably , aim2 expression was not affected by age ( p = 0.06 ) or gender ( p = 0.527 ) . to further clarify the factors affecting the expression of aim2 in hbv - gn tissue , we considered the potential influence of age , gender , and hbeag status in the serum .
as summarized in table 3 , the results showed that aim2 expression was not affected by age ( p = 0.937 ) or gender ( p = 0.627 ) . using elisa ,
serum hbeag was detected in 47 of the 54 hbv - gn patients , showing that aim2 expression was also not affected by hbeag status ( p = 0.614 ) .
lastly , as summarized in table 4 , aim2 expression was not affected by various hbv antigen types deposited in the kidney tissue ( p = 0.511 ) .
the correlation between the expression of aim2 , caspase-1 , and il-1 in patient tissues was analyzed .
statistical analysis revealed that the expression of aim2 was positively correlated with that of caspase-1 ( rs = 0.444 , p < 0.01 ) and il-1 ( rs = 0.379 , p < 0.01 ) , and the expression of caspase-1 was positively correlated with that of il-1 ( rs = 0.515 , p < 0.01 ) ( table 5 ) .
figures 1(a ) , 1(b ) , and 1(c ) illustrate the positive expression of aim2 , caspase-1 , and il-1 , respectively , in these patients , and figure 1(d ) shows the negative expression of aim2 , caspase-1 , and il-1 with pbs substituted primary antibody in hbv - gn tissue .
these results suggested that the expression of aim2 was specifically correlated with inflammation in hbv - gn tissue and that elevation of aim2 corresponding to hbv infection or replication may contribute to the inflammatory damage associated with the development of hbv - gn .
hgm cells were divided into three groups : aim2-sirna and hbv dna cotransfected group ( group a ) , hbv dna - transfected group without aim2-sirna ( group b ) , and aim2-sirna and empty plasmids cotransfected group ( group c ) .
three aim2-sirnas were designed and used for this experiment . as shown in figure 2 , the expression of aim2 mrna and protein was tested in transiently transfected hgm cells .
aim2-sirna#2 was identified as the most effective silencer , resulting in a 3.8-fold inhibition , and was therefore selected as a positive control for the following experiments .
the expression of caspase-1 ( figure 3 ) , il-1 ( figure 4 ) , and il-18 ( figure 5 ) genes was measured at the posttranscriptional level by qrt - pcr and normalized to -actin mrna ( mean  se ,
n = 3 ) . compared to group b , sirna - mediated knockdown of aim2 in group a resulted in a 1.9-fold decrease of caspase-1 , a 2.8-fold decrease of il-1 , and a 1.8-fold decrease of il-18 mrna levels . compared to group c , the expression of caspase-1 , il-1 , and il-18 in group a was downregulated 2.4- , 3.3- , and 2.7-fold , respectively .
these changes were also noted at the translational level , as measured by western bolt .
overall , the expression of caspase-1 , il-1 , and il-18 was reduced in group a compared to the controls at both the posttranscriptional and translational levels . with aim2 knockdown , expression of all genes studied including caspase-1 , il-1 , and il-18 was decreased . in order to exclude the changes caused by aim2-sirna degrading all mrna , we detected the mrna and protein of il-6 after sirna transfection as control , and the results showed that the expression of il-6 in group a detected by qrt - pcr had not changed compared with group b and group c(a ) .
these similar changes were also noted at the translational level , as measured by western blot ( figure 6 ) . taken together , these results suggested that downregulation of aim2 could inhibit inflammation in hbv - gn .
increasing epidemiological , clinical , and immunological evidence has suggested a relationship between hbv infection and the development of nephropathy .
hbv is currently viewed as a noncytopathic virus , and hbv - associated renal damage is thought to be the consequence of a long lasting cytolytic immune response against infected renal tissue .
both innate and adaptive arms of the immune system are generally involved in responding to viral infection , with innate responses being important for control of viral replication and dissemination very early after infection , as well as for timely orchestration of virus - specific adaptive responses .
activation of inflammasome complexes is a key innate immune response against infection with microbial or viral pathogens and tissue damage , and the aim2 inflammasome appears to be an important mediator of this process in hbv - gn .
aim2 was first reported to act as a putative tumor suppressor in malignant melanoma , but the aim2 inflammasome complex is more widely known as an essential mediator of host defense against cytosolic bacteria and dna viruses . the aim2 inflammasome is a cytosolic nucleic acid sensor that detects microbial dna in the cytoplasm  a compartment in which host dna is usually not present .
dna is physiologically stored in the nucleus and in the mitochondria , and this compartmentalization of nucleic acids within subcellular organelles enables the host to distinguish self from nonself .
binding of multiple aim2 proteins to a single molecule of dsdna could lead to a process mimicking nlr core structure formation .
recent research has demonstrated that aim2 senses potentially dangerous cytoplasmic dsdna and forms the inflammasome with asc , which then activates caspase-1 and the subsequent release of mature il-1 and il-18 , ultimately leading to tissue damage . as a nonspecific receptor for cytoplasmic dna , aim2 can be activated by and bind to plasmid dna , dna from the bacterium l. monocytogenes and even synthetic dsdna .
aim2 has also been detected in the small intestine , spleen , peripheral white blood cells , and testis .
this study describes the expression of aim2 in renal tissue and the exclusive expression of aim2 in glomerular endothelial cell and mesangial cell cytoplasm in kidney tissue .
the expression of aim2 in the hbv - gn group was significantly higher than in the cgn group .
as neither age nor gender was statistically different between the two groups , this suggests that there is indeed a relationship between chronic hbv infection and aim2 elevation .
we also considered the potential influence of hbeag status in serum and tissue , which may have contributed to mutation of the hbv dna - p - bcp ( basal core promoter ) or pre - c region , affecting activation and binding of aim2 to hbv dna .
importantly , the expression of aim2 was not significantly different between the hbeag positive and negative groups ; the same results were shown for other hbv - associated antigens deposited in renal tissue groups , collectively indicating that aim2 activation and binding to hbv dna are not influenced by serum hbeag status or hbv - associated antigen deposits .
il-1 is an important intracellular cytokine which binds to receptors and activates the downstream nf-b signaling pathway , releasing inflammatory factors that cause tissue damage .
il-1 governs the recruitment of inflammatory cells such as neutrophils to the site of infection and is important in the generation of optimal adaptive immunity .
il-1 is not only essential to innate immune defense , but is also an important mediator of adaptive immune response to viral infections .
detailed array analyses of immediate response genes triggered by hbv identify il-1 as the central activator of further adaptive immune responses . in this study ,
expression of il-1 and il-18 from hgm cells was increased by hbv dna . il-1 and il-18 play an important role in local immunity and in the activation of lymphocytes and macrophages in the antiviral response [ 21 , 22 ] , while il-18 is mainly involved in coordinating ifn- production from nk cells and t cells at the early and late phases of infection , respectively . in this study ,
the expression of aim2 was positively correlated with the expression of caspase-1 , il-1 , and il-18 in hbv - gn .
indeed , immunohistochemistry analysis showed positive staining of aim2 , caspase-1 , and il-1 in glomerular endothelial cells and mesangial cells in hbv - gn tissue but negative staining in cgn tissue . within the experimental group ,
the expression level of aim2 was positively correlated with the level of caspase-1 , and the level of caspase-1 was positively correlated with the levels of il-1 and il-18 , suggesting that the aim2 inflammasome - caspase-1 inflammation signal transfer pathway may be active in hbv - gn .
the data further suggest that downregulation of aim2 could directly inhibit the expression of caspase-1 mrna and subsequently inhibit the expression of il-1 and il-18 at both the transcriptional and protein levels in hbv - infected hgm cells , evident by the results of sirna - mediated knockdown of aim2 in hbv - infected cells . in summary
, we found the expression of aim2 , a cytosolic dna sensor , to be significantly higher in hbv - gn patient kidney tissues than in cgn tissues .
the likely binding of hbv dna to aim2 appears to trigger activation of caspase-1 and subsequent release of il-1 and il-18 , leading to renal damage .
our findings may help provide a new therapeutic target for hbv - gn and a new avenue for researching its pathogenesis and therapeutic options .
 
 background & aims . 
 aim2 plays an important role in innate immunity , but its role in regulating the immune response to hepatitis b virus ( hbv ) is unknown . 
 we hypothesized that aim2 expression is positively correlated with hbv - mediated inflammation in patients with hbv - associated glomerulonephritis ( hbv - gn ) , potentiating inflammation and leading to renal damage . 
 we therefore analyzed the expression of aim2 and inflammatory factors in hbv - gn tissues and cell lines relative to the inflammatory response to hbv infection and hbv status . 
 methods . 
 seventy - nine patients with chronic nephritis ( cn ) were included : 54 with hbv - gn and 24 with chronic glomerulonephritis ( cgn ) . 
 expression of aim2 , caspase-1 , and il-1 was detected by immunohistochemistry in renal biopsies from each patient . 
 following sirna - mediated knockdown of aim2 in hbv - infected and hbv - uninfected human glomerular mesangial ( hgm ) cells , expression of caspase-1 , il-1 , and il-18 was detected by qrt - pcr and western blot . 
 results . 
 aim2 expression in hbv - gn biopsies ( 81.4% ) was significantly higher than in cgn ( 4.0% ) and positively correlated with caspase-1 and il-1 expression in hbv - gn . in vitro , aim2 knockdown reduced caspase-1 , il-1 , and il-18 expression in hbv - infected and hbv - uninfected hgm cells . 
 conclusion . 
 aim2 elevation during hbv infection or replication may contribute to inflammatory damage , thus providing a putative therapeutic target for hbv - gn . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion