diabetes and diabetic nephropathy ( dn ) , a major and specific microvascular complication of diabetes , are increasingly prevalent and have become serious health problems . dn exhibits a high incidence rate in patients with diabetes and remains the main cause of end - stage renal disease . although the mechanisms by which diabetic patients develop dn are not completely understood , hyperglycemic exposure , hypertension , and other factors such as high levels of advanced glycation end products , oxidative stress , inflammation , and protein kinase c are generally considered to be risk factors for dn . the steno-2 study showed that strict metabolic control can effectively inhibit the progression of dn . the limited efficacy of current interventions , familial aggregation of dn , and clustering of dn within ethnic groups indicate that a genetic factor may contribute to susceptibility to dn . pentraxin 3 ( ptx3 ) , a form of long pentraxin , is a member of the acute - phase reactants superfamily and shares structural homology with short pentraxins , such as c - reactive protein ( crp ) and serum amyloid p component . ptx3 is produced by several cell types , most prominently by endothelial cells and monocytes , in response to inflammation . ptx3 is a sensitive biomarker of localized inflammatory reactions   and is associated with a variety of clinical diseases , including coronary artery disease , rheumatoid arthritis , sepsis , and chronic kidney diseases ( ckd ) . altered plasma levels of ptx3 are associated with development of dn ; plasma ptx3 levels are inversely associated with egfr , but positively associated with levels of albuminuria / proteinuria in patients with dn . single nucleotide polymorphisms ( snps ) in ptx3 have been found to influence circulating ptx3 levels [ 10,1215 ] and are associated with the risk of some diseases , including primary graft dysfunction after lung transplantation , pulmonary tuberculosis , and development of hepatocellular carcinoma in patients with chronic hepatitis c ( chc ) . however , no data are available on the association between ptx3 polymorphisms and dn in patients with diabetes . previous studies have shown that certain regions on chromosome 3q are associated with an increased susceptibility to dn , based on genome - wide scanning and linkage analysis . we hypothesized that variants of ptx3 may be associated with an increased risk of developing dn . the primary goal of the present study was to assess the relationship between ptx3 variants and dn in han chinese patients with type 2 diabetes . a total of 442 participants were recruited from the first affiliated hospital of wenzhou medical university between december 2015 and june 2016 for this case - control study . they included 290 patients known to have type 2 diabetes for longer than five years and 152 gender - matched normal controls ( nc ) . eligible patients with type 2 diabetes were categorized into two groups : the non - diabetic nephropathy ( ndn ) group ( n=155 ) and the diabetic nephropathy ( dn ) group ( n=135 ) . patients with type 2 diabetes were diagnosed according to the diagnostic criteria of the american diabetes association ( ada ) published in 2016 . albuminuria was determined according to the urine albumin - to - creatinine ratio ( acr ) suggested by the ada . estimation of the glomerular filtration rate ( egfr ) was performed using the chronic kidney disease epidemiology collaboration formula . dn was defined on the basis of persistent microalbuminuria detected during at least two examinations ( acr > 30 mg / g ) or overt albuminuria ( acr > 300 mg / g ) , with or without egfr < 60 ml / min/1.73 m and in the presence of diabetic retinopathy . patients with another kidney diseases , inflammatory diseases , pregnancy , or congestive heart disease were excluded from the study . patients with acr < 30 mg / g and egfr > 60 ml / min/1.73 m were considered ndn . all procedures were performed in accordance with the declaration of helsinki and informed consent was obtained from each individual . the study protocol was approved by the ethics committee of the first affiliated hospital of wenzhou medical university . genomic dna was extracted using a dna purification kit ( tiannamp blood dna kit ; tiangen biotech , china ) , according to the manufacturer s instructions . individual dna samples were genotyped using the abi prism snapshot method ( applied biosystems , ca , usa ) . eight snps ( rs2305619 , rs2120243 , rs1456099 , rs7634847 , rs1840680 , rs2316706 , rs2316709 , and rs7616177 ) in ptx3 were analyzed . the snapshot reactions were performed in a total reaction volume of 8 l containing snapshot multiplex reagent ( 5 l ) , extended primer mix ( 1 l ) , and templates ( 2 l ) , which consisted of purified multiplex polymerase chain reaction ( pcr ) products . the extension products were purified by incubation with 1 unit of shrimp alkaline phosphatase ( sap ) at 37c for 60 minutes and a subsequent incubation at 85c for 15 minutes for enzyme inactivation . the purified products ( 1 l ) , genescan-120 liz size standard ( applied biosystems , 0.5 l ) , and hi - di ( 8.5 l ) were mixed and identified using an abi 3730 sequencer ( applied biosystems ) . finally , the results were analyzed with genemapper 4.0 software ( applied biosystems co. , ltd . statistical analysis was performed using spss version 20.0 ( spss , chicago , il , usa ) . continuous variables with normal distribution are shown as mean  standard deviation ( sd ) . differences between groups were analyzed using by student s t - test and differences among groups were tested by one - way analysis of variance ( anova ) . the chi - square test was also used to evaluate whether genotype and allele frequencies were in hardy - weinberg equilibrium ( hwe ) . the association of each snp with the risk for dn was analyzed by multivariate logistic regression using the stepwise backward approach ; odds ratios ( or ) , and 95% confidence intervals ( 95% ci ) were also calculated . estimates of linkage disequilibrium between snps were determined by calculating pairwise d and r statistics using haploview ( version 4.2 ; www.broad.mit.edu/mpg/haploview/ ) ; haplotype analysis was also performed using this software . a total of 442 participants were recruited from the first affiliated hospital of wenzhou medical university between december 2015 and june 2016 for this case - control study . they included 290 patients known to have type 2 diabetes for longer than five years and 152 gender - matched normal controls ( nc ) . eligible patients with type 2 diabetes were categorized into two groups : the non - diabetic nephropathy ( ndn ) group ( n=155 ) and the diabetic nephropathy ( dn ) group ( n=135 ) . patients with type 2 diabetes were diagnosed according to the diagnostic criteria of the american diabetes association ( ada ) published in 2016 . albuminuria was determined according to the urine albumin - to - creatinine ratio ( acr ) suggested by the ada . estimation of the glomerular filtration rate ( egfr ) was performed using the chronic kidney disease epidemiology collaboration formula . dn was defined on the basis of persistent microalbuminuria detected during at least two examinations ( acr > 30 mg / g ) or overt albuminuria ( acr > 300 mg / g ) , with or without egfr < 60 ml / min/1.73 m and in the presence of diabetic retinopathy . patients with another kidney diseases , inflammatory diseases , pregnancy , or congestive heart disease were excluded from the study . patients with acr < 30 mg / g and egfr > 60 ml / min/1.73 m were considered ndn . all procedures were performed in accordance with the declaration of helsinki and informed consent was obtained from each individual . the study protocol was approved by the ethics committee of the first affiliated hospital of wenzhou medical university . genomic dna was extracted using a dna purification kit ( tiannamp blood dna kit ; tiangen biotech , china ) , according to the manufacturer s instructions . individual dna samples were genotyped using the abi prism snapshot method ( applied biosystems , ca , usa ) . eight snps ( rs2305619 , rs2120243 , rs1456099 , rs7634847 , rs1840680 , rs2316706 , rs2316709 , and rs7616177 ) in ptx3 were analyzed . the snapshot reactions were performed in a total reaction volume of 8 l containing snapshot multiplex reagent ( 5 l ) , extended primer mix ( 1 l ) , and templates ( 2 l ) , which consisted of purified multiplex polymerase chain reaction ( pcr ) products . the extension products were purified by incubation with 1 unit of shrimp alkaline phosphatase ( sap ) at 37c for 60 minutes and a subsequent incubation at 85c for 15 minutes for enzyme inactivation . the purified products ( 1 l ) , genescan-120 liz size standard ( applied biosystems , 0.5 l ) , and hi - di ( 8.5 l ) were mixed and identified using an abi 3730 sequencer ( applied biosystems ) . finally , the results were analyzed with genemapper 4.0 software ( applied biosystems co. , ltd . statistical analysis was performed using spss version 20.0 ( spss , chicago , il , usa ) . continuous variables with normal distribution are shown as mean  standard deviation ( sd ) . differences between groups were analyzed using by student s t - test and differences among groups were tested by one - way analysis of variance ( anova ) . the chi - square test was also used to evaluate whether genotype and allele frequencies were in hardy - weinberg equilibrium ( hwe ) . the association of each snp with the risk for dn was analyzed by multivariate logistic regression using the stepwise backward approach ; odds ratios ( or ) , and 95% confidence intervals ( 95% ci ) were also calculated . estimates of linkage disequilibrium between snps were determined by calculating pairwise d and r statistics using haploview ( version 4.2 ; www.broad.mit.edu/mpg/haploview/ ) ; haplotype analysis was also performed using this software . the baseline characteristics of the study participants in the dn , ndn , and nc groups are shown in table 1 . patients in the dn group were older than those in the ndn and nc groups ( p<0.05 ) . the proportion of patients with hypertension was significantly higher in the dn group than in the ndn and nc groups ( p<0.01 ) . compared with the ndn group , patients in the dn group had a longer duration of diabetes ( p=0.041 ) . the levels of fasting plasma glucose ( fpg ) and glycosylated hemoglobin ( hba1c ) in patients with diabetes were significantly higher than those of patients in the nc group ( all p<0.01 ) . egfr was lower in the dn group than in the nc group ( p<0.01 ) . as shown in table 2 , the genotype frequencies of rs2305619 differed significantly between the dn and the ndn groups ( p<0.05 ) , and the allele frequencies of rs2305619 differed significantly between the dn and the ndn groups ( p<0.05 ) . patients with the gg variant showed 4.078-fold higher susceptibility to dn than those with the aa variant ( or=4.078 , 95% ci=1.37012.135 , p=0.012 , table 3 ) . the genotype frequencies of rs2120243 differed significantly between the dn and the ndn groups ( p<0.05 , table 2 ) . we detected a lower risk of developing dn in individuals with the aa variant of rs2120243 ( or=0.213 , 95% ci=0.0550.826 , p=0.025 , table 3 ) . in addition , the a allele appeared to decrease the susceptibility to dn ( or=0.688 , 95% ci=0.4760.994 , p=0.046 , table 3 ) , which suggested that the a allele of rs2120243 may be a protective factor against dn in diabetic patients . although the genotype and allele frequencies of rs1840680 did not differ significantly between the dn and ndn groups ( p>0.05 , table 2 ) , we found that aa homozygous individuals showed a lower risk of dn than gg homozygous individuals ( or=0.370 , 95% ci=0.1400.977 , p=0.045 , table 3 ) . similarly , for rs2316706 , no differences in genotype or allele frequency were observed between the dn and ndn groups ( p>0.05 , table 2 ) ; however , the tt genotype exhibited a protective effect against dn ( or=0.345 , 95% ci=0.1320.903 , p=0.030 , table 3 ) . with respect to rs1456099 , rs7634847 , rs2316709 , and rs7616177 , no evidence of association with dn was found ( all p>0.05 , tables 2 , 3 ) . we quantified the linkage disequilibrium ( ld ) between the two ptx3 snps found to show a significant association with dn . we also investigated whether certain ptx3 haplotypes , or combinations of alleles at different loci , were associated with an increased risk of dn . we compared haplotype distributions between the dn and ndn groups , and observed that the caggg haplotype in block 1 was associated with dn ( p=0.0319 ) ( table 4 ) . as shown in table 5 , no difference in the allele frequencies of the eight snps was observed between the nc and t2 dm groups ( all p>0.05 ) . the baseline characteristics of the study participants in the dn , ndn , and nc groups are shown in table 1 . patients in the dn group were older than those in the ndn and nc groups ( p<0.05 ) . the proportion of patients with hypertension was significantly higher in the dn group than in the ndn and nc groups ( p<0.01 ) . compared with the ndn group , patients in the dn group had a longer duration of diabetes ( p=0.041 ) . the levels of fasting plasma glucose ( fpg ) and glycosylated hemoglobin ( hba1c ) in patients with diabetes were significantly higher than those of patients in the nc group ( all p<0.01 ) . egfr was lower in the dn group than in the nc group ( p<0.01 ) . as shown in table 2 , the genotype frequencies of rs2305619 differed significantly between the dn and the ndn groups ( p<0.05 ) , and the allele frequencies of rs2305619 differed significantly between the dn and the ndn groups ( p<0.05 ) . patients with the gg variant showed 4.078-fold higher susceptibility to dn than those with the aa variant ( or=4.078 , 95% ci=1.37012.135 , p=0.012 , table 3 ) . the genotype frequencies of rs2120243 differed significantly between the dn and the ndn groups ( p<0.05 , table 2 ) . we detected a lower risk of developing dn in individuals with the aa variant of rs2120243 ( or=0.213 , 95% ci=0.0550.826 , p=0.025 , table 3 ) . in addition , the a allele appeared to decrease the susceptibility to dn ( or=0.688 , 95% ci=0.4760.994 , p=0.046 , table 3 ) , which suggested that the a allele of rs2120243 may be a protective factor against dn in diabetic patients . although the genotype and allele frequencies of rs1840680 did not differ significantly between the dn and ndn groups ( p>0.05 , table 2 ) , we found that aa homozygous individuals showed a lower risk of dn than gg homozygous individuals ( or=0.370 , 95% ci=0.1400.977 , p=0.045 , table 3 ) . similarly , for rs2316706 , no differences in genotype or allele frequency were observed between the dn and ndn groups ( p>0.05 , table 2 ) ; however , the tt genotype exhibited a protective effect against dn ( or=0.345 , 95% ci=0.1320.903 , p=0.030 , table 3 ) . with respect to rs1456099 , rs7634847 , rs2316709 , and rs7616177 , no evidence of association with dn was found ( all p>0.05 , tables 2 , 3 ) . we quantified the linkage disequilibrium ( ld ) between the two ptx3 snps found to show a significant association with dn . we also investigated whether certain ptx3 haplotypes , or combinations of alleles at different loci , were associated with an increased risk of dn . we compared haplotype distributions between the dn and ndn groups , and observed that the caggg haplotype in block 1 was associated with dn ( p=0.0319 ) ( table 4 ) . as shown in table 5 , no difference in the allele frequencies of the eight snps was observed between the nc and t2 dm groups ( all p>0.05 ) . ptx3 is synthesized in extrahepatic tissues and cells , including atherosclerotic lesions , adipose tissue , vascular endothelial cells , and macrophages . a previous study in a mouse model showed that ptx3 is expressed in peritubular endothelial cells of the kidney . in humans , expression of ptx3 is also observed in primary proximal renal tubular epithelial cells ( ptecs ) , primary mesangial cells , and renal fibroblasts in kidney tissues . furthermore , increased plasma levels of ptx3 were observed in patients with ckd [ 1113 ] and dn . in addition , plasma ptx3 levels and urinary protein excretion can normalize though the treatment of the renin - angiotensin system , and/or with calcium channel blockers can normalize in patients with type 2 diabetes who show proteinuria . all the aforementioned evidence suggests that abnormal ptx3 expression may contribute to renal damage . here , we provide evidence of the association between ptx3 variants and dn in a population of han chinese patients with type 2 diabetes . we found that variants of rs2305619 and rs2120243 in ptx3 were associated with susceptibility to dn in patients with diabetes . the snps rs2305619 and rs2120243 are located in the first intron and the 5 promoter region of ptx3 , respectively . these two snps may alter transcriptional regulation , resulting in abnormal splicing , or the translational dynamics of the ptx3 , as implied in previous studies that identified an association between rs2305619 and rs2120243 and plasma ptx3 levels [ 1315 ] . it is equally possible that these two snps may be in linkage disequilibrium with other functional loci for development of dn in patients with diabetes . further investigation is needed to clarify the role of these two snps in the pathogenesis of dn . although we identified a role of ptx3 in the pathogenesis of dn , the mechanism remains unclear . according to studies published to date , several mechanisms might be implicated in the process of renal function impairment in t2 dm , including endothelial dysfunction and/or chronic low - grade inflammation . endothelial dysfunction is an important contributor to the development of dn , as endothelial dysfunction influences mesangial cell and podocyte function and increases the permeability of glomerular basement membrane . indeed , ptx3 is known to be linked to endothelial dysfunction , and endothelial dysfunction has been reported to be ameliorated by suppression of ptx3 expression . given the strong links among ptx3 , endothelial dysfunction , and dn , ptx3 may play a role in renal damage through endothelial dysfunction . ptx3 is produced at sites of inflammation and regulated by stimulation with proinflammatory stimuli ( il-1 and tnf- )   and via the activation of toll - like receptors ( tlrs ) , and via several pathways , including the pi3k / akt axis and the nf-b pathway . moreover , these proinflammatory cytokines and signaling pathways are also known to be crucial for development of dn . on the basis of these findings , we speculate that ptx3 may act as an inflammatory mediator for the process of dn . we are interested in further investigation of the molecular mechanisms underlying the action of ptx3 in the development of dn . although plasma ptx3 level has previously been shown to be related to insulin resistance   and insulin secretion , we failed to detect a significant association between ptx3 polymorphisms and susceptibility to diabetes mellitus in this population . first , the sample size was relatively small , and only chinese individuals were included , which limits the power of our results . thus , further studies in a larger , ethnically diverse population are necessary to elucidate the role of ptx3 in development of dn . in conclusion , the present study found that certain variants of ptx3 predispose han chinese patients with type 2 diabetes to dn .
backgrounddiabetic nephropathy ( dn ) is a major microvascular complication of diabetes . 
 pentraxin 3 ( ptx3 ) is a member of the acute - phase reactants superfamily and altered plasma levels of ptx3 are associated with dn . 
 we performed a case - control study to analyze the relationship between single nucleotide polymorphisms ( snps ) in ptx3 and the risk for dn in patients with type 2 diabetes.material/methodsthe study included 135 dn patients , 155 non - diabetic nephropathy ( ndn ) patients , and 152 normal controls ( nc ) ( n=442 ) . 
 we genotyped eight ptx3 snps ( rs2305619 , rs2120243 , rs1456099 , rs7634847 , rs1840680 , rs2316706 , rs2316709 , and rs7616177 ) using the abi prism snapshot method.resultsthe genotype frequencies of rs2305619 and rs2120243 differed significantly between the dn and the ndn groups ( p=0.017 and p=0.033 , respectively ) . 
 patients with the gg variant of rs2305619 showed 4.078-fold higher susceptibility to dn than those with the aa variant ( or=4.078 , 95% ci=1.37012.135 , p=0.012 ) ; patients with the aa variant of rs2120243 had a lower risk of developing dn ( or=0.213 , 95% ci=0.0550.826 , p=0.025 ) . 
 haplotype analysis showed an association between the caggg haplotype in block 1 with dn ( p=0.0319).conclusionsour findings suggested that ptx3 polymorphisms were associated with an increased risk for dn in chinese patients with type 2 diabetes .