Caspian Journal of Internal Medicine

Serum alpha–actinin antibody status in systemic

lupus erythematosus and its potential in the

diagnosis of lupus nephritis

Abstract

Background: In lupus nephritis (LN), deposition of pathogenic autoantibodies in the glomeruli is mediated via cross-reactivity with alpha-actinin. Association of serum alpha-actinin antibody (AαA) with LN has been shown in a few studies but the results are controversial.

Methods: Eighty patients into entered the study. The diagnosis of SLE was confirmed according to the American College of Rheumatology criteria and LN was diagnosed by proteinuria ≥ 500 mg/24 hour and kidney biopsy. Serum AαA was measured with ELISA method. Receiver operating characteristics curve (ROC) analysis was applied to determine an optimal cutoff value for AαA to discriminate patients with and without LN at the highest sensitivity and specificity. The association of AαA with LN was determined by logistic regression analysis with calculation of odds ratio (OR).

Results: Serum AαA was significantly lower in LN as compared with SLE patients without LN (P=0.001). Serum AαA at cutoff levels ≤ 59.5 pg/ml discriminated the two groups with sensitivity, specificity, positive predictive values of 60%. 90% and 85.7%, respectively. Serum AαA level ≤ 59.5 pg/ml was significantly associated with LN (OR=13.5, P=0.001) and the OR increased to 25.2 (P=0.003) after adjustment for age, sex, C3, C4, anti-ds-DNA, SLEDAI.

Conclusion: This study indicates that serum AαA decreases in LN and serum level ≤ 59.5 pg/ml is SLE and is predictive of nephritis.

Keywords: Systemic lupus erythematous, Nephritis, Anti-alpha-actinicn antibody, Diagnosis.

Citation:

Babaei M, Rezaieyazdi Z, Saadati R, et al. Serum alpha–actinin antibody status in systemic lupus erythematosus and its potential in the diagnosis of lupus nephritis. Caspian J Intern Med 2016; 7(4): 272-277.

Caspian J Intern Med 2016; 7(4):272-277

Systemic lupus erythematous (SLE) is associated with multiple organ involvement and high morbidity as well as mortality (1-4). Among the several manifestations of  SLE,  nephritis  is  of particular concern ,because lupus nephritis (LN)  is associated with excess risk  of death, malignancy and cardiovascular complications (3, 4). Alpha-actinin (α-actinin) is a ubiquitous cytoskeletal protein which belongs to the superfamily of filamentous actin (F-actin) crosslinking proteins. It is present in multiple subcellular regions of both muscle and non-muscle cells, including cell–cell and cell–matrix contact sites, cellular protrusions and stress fiber dense regions. It seems to bear multiple important roles in the cell by linking cytoskeleton to many different transmembrane proteins in a variety of junctions. Deposition of autoantibodies in the glomeruli seems to be crucial for development of LN (5).

In SLE, anti – alpha-actinin (AαA) is a major cross-reactive target for anti-dsDNA antibodies (6) and pathogenicity of some anti-DNA antibodies is mediated via cross-reactivity with alpha-actinin (7-9). Earlier studies have shown that renal pathogenicity of murine lupus antibodies are dependent on direct binding of antibodies to AαA (7-9). Active LN compared with SLE patients without nephritis displays greater AαA binding (6). It has been shown that pathogenic anti-ds DNA antibodies bind strongly to AαA and elevated levels of serum AαA antibodies are associated with a 2.5 -fold increase in the prevalence of nephritis (6). In one study, 10 out of 22 (45.1%) patients with AαA antibody had LN, while only 14 out of 78 (17.9%) SLE patients without AαA antibody had LN. This indicates a significant association between serum AαA antibody and LN (6). Nevertheless, SLE patients without nephritis and even patients without SLE may also have serum AαA antibody (8-10). Croqueted et al. compared the prevalence of AαA, between SLE and other rheumatic diseases versus healthy controls (9).

The results showed higher prevalence of AαA antibody in SLE compared with rheumatoid arthritis, Siogren syndrome, and healthy controls (22.3%, 3.92%, 3%, and 0.6% respectively). In Renaudineau et al,’s study (6), the prevalence rate of AαA antibody positivity was higher in anti-dsDNA positive versus anti-dsDNA negative SLE (33.8% vs 2.8%). Nevertheless, in a longitudinal study of 16 patients with new-onset-biopsy-proven LN, there was a positive association between measures of LN with anti-DNA and anti-nucleosome but not with AαA antibody (11).

In a few studies, the relationship between serum  AαA antibody and SLE disease activity index (SLDAI) or  anti-ds DNA  was assessed  and the results revealed  a negative correlation of AαA  with SLEDA and positive correlation with anti-dsDNA  (6, 9, 10, 12).

  Available data indicate that binding of pathogenic autoantibodies to AαA antibody is critical for the development of nephritis in SLE, suggesting a relationship between serum AαA antibody and LN. Nevertheless, the results of studies in this context are controversial (6, 10, 11, 12) which may be attributed to inadequate sample size, inappropriate study designs, patient selection or nonhomogeneous distribution of predisposing factors of LN across various studies. To overcome these shortcomings, the present case- control study was designed to compare SLE patients with and without nephritis regarding serum AαA antibody levels and to investigate the relationship between serum   AαA antibody and LN. The secondary purpose of this study was to determine a cutoff level of AαA antibody for the discrimination of SLE patients with and without nephritis.

Methods

Ninety patients with lupus erythematous  were recruited consecutively according to inclusion criteria among those who presented to rheumatology clinics of Mashhad University of Medical Sciences, Mashhad, Iran, Patient selection was performed over one year period from October 2011 to September 2012. 

The diagnosis of SLE was confirmed by the American College of Rheumatology criteria for systemic lupus erythematous (13) and the diagnosis of LN was confirmed in the presence of >  500 mg per 24 hours proteinuria for at least two occasions as well as kidney biopsy (13). The activity of SLE (SLEDAI) was assessed by a validated questionnaire for SLE disease activity (14). All patients with confirmed LN were included.

Exclusion criteria included SLE patients with diabetes, urinary tract infection or urinary nephrolithiasis, patients with overlap connective tissue disease, vasculitis syndrome, SLE patients with antiphospholipid syndrome, end- stage renal disease or patients on hemodialysis. SLE patients without proteinuria were considered as controls.

Sample size was estimated for detection of 30 % differences in proportion of AαA antibody positivity between SLE patients with and without nephritis. Based on an earlier prevalence of 15% AαA antibody   positivity in SLE patients without nephritis, (6) a sample size of 33 patients for each group was needed to detect such difference with 95% confidence interval (CI) and 80% power (15). However, we recruited additional patients to compensate the patients with missed data. All patients received appropriate treatment for SLE or LN to achieve remission. This study was confirmed by the Ethics Committee of the Mashhad University of Medical Sciences, Mashhad, Iran. Serum AαA antibody level was assessed with ELISA method according to manufacturer’s instruction using human alpha-actinin-4 kit (ACTN-4)  ELISA kit  CSB -E147 42h (96T) purchased from CUSABIO company.

Statistical analysis: Receiver operating characteristics ROC curve analysis was applied by plotting sensitivity against 1-specificity for various levels of serum AαA antibody .The optimal cutoff value that best distinguished patients with LN from those without LN was determined at maximum value for Youdens' index defined as   the difference between the true positive rate and the false positive rate [sensivitiy - (1-specificity)]. The overall diagnostic accuracy was estimated based on area under the ROC curve (AUC) expressed as mean ± SE.

 In additional analysis the levels of serum AαA antibody in patients with and without LN were compared with other predictive measures of nephritis like C3, C4, anti-ds DNA, serum creatinine (Cr). The status of distribution for all variables was examined by measures of skewness and kurtosis as well as using Kolmogrov -Smirnov test. Normality of distribution was assessed by Kolmogrov-Smirnov test. Parametric tests were used for the comparison of variables with normal distributions and nonparametric Mann-Whitney U test for comparison of skewed variables. Proportions were compared with chi -square or Fisher’s exact tests as appropriate. Association among categorical variables was determined by chi-square test with calculation of odds ratio (OR) and corresponding 95% confidence interval (95%CI). Correlations were performed using Spearman correlation coefficient.

Results

Eighty participants (95% females) achieved inclusion criteria that comprised 40 patients with LN with mean age of 29.9±19.7 years old  and 40  controls without LN with mean age of 30.7±10.7 years old  (P=0.63) (table 1). In the total number of patients, 54 (67.5%) patients were anti-ds DNA positive, 46 (57.5%) had low C3 levels, and 37 (46.3%) with low serum C4 levels. Distribution of  serum AαA antibody  in  the control group (patients without LN) was normal with mean  value of 124±56.2 pg/ml and median value of 121 pg/ml ,but in patients with LN , distribution  of  serum AαA antibody was skewed  to the  right  with  mean value of 78.2±56.9  pg.ml and median value of 50  pg/ml (P=0.001).

Based on the results of ROC curve analysis, serum AαA antibody level of ≤ 59.5 pg/ml yielded the highest Youden’s index value for discriminating patients with and without LN at sensitivity of 69% and specificity of 90%. At this level, serum AαA antibody exhibited a false positive rate of 10% and positive predictive value of 85.7% (95% confidence interval 66.4-95.3) and prevalence weighted likelihood ratio of 6 for diagnosis of LN (95% CI, 2.39-15).

 Serum AαA antibody at cutoff level of ≤ 59.5 pg/ml exhibited an AUC (±SE) value of 0.701±0.065   indicating 70.1%. The results of ROC curve analysis regarding other measures of SLE did not show significant ability in predicting LN (table 2). Association of AαA antibody with LN serum AαA antibody ≤ 59.5 pg/ml was significant by OR= 13.5 (95% CI, 4.05-45.3, P=0.001. After adjustment for age, sex, anti-ds DNA, C3,C4, creatinine, SLEDAI, serum AαA ≤ 59.5 pg, ml was independently associated with LN by adjusted odds ratio of 25.2 (95% CI, 3.02- 211.4, P=0.003). While the association of LN with anti-dsDNA, age, sex, C4, and creatinine did not reach to a statistically significant level. But serum C3 levels ≤ 12.5 U/ml were significantly associated with LN by adjusted odds ratio = 8.96 (95% CI, 1.114- 70.3, P=0.037).

Table 1: Characteristics of systemic lupus erythematous patients with and without lupus nephritis

^¥  Compared  by Mann-Whitney U test   ≠ Anti-alpha-actinin antibody

^҂ Systemic lupus erythematosus disease activity index

^€ Erythrocyte sedimentation rate

P value < 0.05 is significant.

Anti-ds DNA and LN: Twenty- nine patients with LN (53.7%) versus 25 controls without LN (46.3%) were anti-ds DNA positive (p=0.23). The levels of AαA antibody did not differ between the DNA negative and DNA positive groups (110±59.5 vs 96.8±61.4 pg/ml, p=0.36). In patients with LN serum AαA antibody was negatively correlated with SLEDAI (Spearman’s correlation coefficient= -0.352, P=0.05) but positively correlated with serum C3 level (r=0.419, P=0.014) as well as serum C4 level (r=0.335, P=0.05). Inasmuch as in the control group correlation between AαA antibody and SLEDA, C3 and C4 did not reach to statistically significant levels.

Table 2. Diagnostic performance of AαA antibody in the differentiation of SLE patients with and without nephritis in comparison to other conventional markers of lupus nephritis

^* Using receiver operating characteristics curve (ROC) analysis

Discussion

The findings of this study indicate significantly lower serum AαA antibody concentration in SLE with nephritis as compared to those without nephritis. In LN, low levels of serum AαA antibody correlated positively with serum C3, C4 and creatinine but negatively correlated with SLEDAI. The levels of AαA antibody levels ≤ 59.5 pg/ml distinguished SLE with and without nephritis with sensitivity of 60%, specificity of 90%, positive predictive value of 85.7 % with likelihood ratio of 6. In addition, serum AαA antibody ≤ 59.5 pg/ml was significantly associated with LN after adjustment for other associated risk factors such as anti-ds DNA positivity, low serum complement levels, sex, age, SLE activity by adjusted OR of 25.2.

In this study, serum AαA antibody was not associated with anti-dsDNA which is in contrast with the results of Renaudineau et al. who have found a positive association between LN and anti-ds-DNA (6). Nonetheless, the association was only limited to anti-dsDNA positive nephritis (6). Similarly, Croquefer et al. found higher prevalence rate of AαA antibody positivity in SLE as compared with other rheumatic diseases as well as healthy controls regardless of nephritis (9). Similar to our study, Zhang et al, also reported an inverse relationship between serum AαA antibody and disease activity in SLE irrespective of LN (10). In another longitudinal study of 16 patients with LN, Manson et al. found higher baseline anti-dsDNA and anti-nucleosome but not AαA antibody in SLE than in the


healthy controls. In the latter study, serum AαA antibody had not been compared between patients with and without nephritis and the authors found no association between serum AαA antibody and associated factors of nephritis (16). In another case-control study by Becker- Merok et al. (12), serum AαA antibody was higher in anti-dsDNA positive SLE than other autoimmune rheumatic diseases and the serum AαA antibody was higher in renal flare and was independently correlated with anti-dsDNA. Notwithstanding, the association in this study was not SLE specifically because serum AαA antibody was not higher in other ANA positive autoimmune disease. Therefore, the observed association of serum AαA antibody and renal disease suggests cross-reactivity of AαA antibody with anti-dsDNA antibodies (12).

Cross-reactivity of anti-dsDNA and AαA antibody has been shown in a panel of 10 anti-dsDNA and/or AαA antibodies generated by Epstein Barr virus transformation of lymphocytes from patients with SLE. The results provided strong support for contribution of pathogenic cross-reactive anti-dsDNA/ AαA antibody in the development of LN (17).   In spite of many previously published studies regarding serum AαA antibody in SLE, yet the status of the serum AαA antibody in SLE patients with and without nephritis has not been addressed. The results of this study in consistent with similar reports (10, 18) present additional information to the existing data concerning the ability of this antibody in recognizing LN. LN is one of the most serious manifestations of SLE and a predictor of morbidity and mortality in these patients (3, 19). Early diagnosis and treatment of LN is of particular importance because treatment at earlier stage, prevents intractable kidney disease. Currently, the diagnosis of LN is based on clinical or laboratory findings which do not always correlate with pathologic abnormalities and thus, the diagnosis warrants certainty (20).

Although, renal biopsy is the gold standard method of diagnosis but it is an invasive procedure and the results of biopsy do not always provide additional benefits compared with clinical classification (21). Based on the findings of this study, serum AαA antibody ≤ 59.5 pg/ml provides supporting data in diagnosing LN with sensitivity of 60% and specificity of 90%. Diagnostic rate of LN in the clinical setting of the present study increased from the pre-test probability of 50% to post- test probability of 85.7%. Excellent likelihood ratio in this study indicates that the post-test probability is less subjected to sample bias.

Concerning the 50% prevalence of nephritis across various studies (22024), the population of this study should be considered the representative of SLE in general population. Several biomarkers were used for the diagnosis of nephritis in SLE, but none of them was validated in prospective studies and their performance may differ in various ethnic backgrounds (25, 26, 27).

The findings of this study should be considered with limitation since a number of SLE with asymptomatic LN may be missed because of lack of biopsy. Hence, the real number of LN may be underestimated. The strength of this study depends on the study population which was drawn from a homogenous population concerning ethnic and sociodemographic characteristics, treatment as well as diagnostic criteria. Another strength of this study is related to the study  design consisted of  two groups of SLE patients with similarity in many  baseline characteristics  including age, sex, renal function, serum complement levels and proportion of anti-dsDNA positivity. Adequate sample size and application of ROC curve analysis provides additional documents for validity. In conclusion, the findings of this study indicate that serum AαA antibody level is significantly higher in SLE with nephritis and at serum cutoff level ≤ 59.5 pg/ml differentiates SLE patients with and without nephritis with sensitivity of 60%, specificity of 90%. Serum AαA ≤ 59.5 ≤ 59.5 pg/ml is significantly associated with LN and yields a positive predictive value by 85.7%. The findings of this study require to be confirmed by longitudinal studies with biopsy-proven LN.

Acknowledgments

We thank the Clinical Research Development Unit Ayatollah Rouhani Hospital of Babol University of Medical Sciences.

Funding: The Research Council of Mashhad University of Medical Sciences, financially supported this project with grant number 900486.

Conflict of interests: The authors have no financial conflicts of interest to declare.

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Higher rate of microscopic hematuria in elderly patients who take regular doses of aspirin:

Result from AHAP Study

Abstract

Background: Aspirin is the most widely used drug in medicine for cardiovascular and as recently for its role in cancer prevention. Although the risk of bleeding events increased following regular use of aspirin, little is known about the association of aspirin and hematuria. The present study aimed to evaluate the association of regular aspirin use and microscopic hematuria in elderly.

Methods: In this study, we have extracted the data of elderly people who participated in Amirkola Health and Aging Project (AHAP) and taking regular doses of aspirin. The prevalence of microscopic hematuria was compared between the elderly who took aspirin regularly and those who did not take it.

Results: A total of 1243 individuals (54.22% males, 45.78% females) were entered in to the study. Two hundred and eighty-four (23%) elderly took regular doses of aspirin. Microscopic hematuria was seen in 305 (24.54%) elderly. The prevalence of microscopic hematuria was 27.27% in regular users of aspirin and 23.72% in non-users of aspirin (P=0.126). The prevalence of microscopic hematuria was significantly higher among the regular users of aspirin compared to non-users in multiple logistic regression analysis (P=0.035, OR=1.40, 95%CI: 1.02-1.92).

Conclusion: Taking regular doses of aspirin was accompanied with higher rate of microscopic hematuria in the elderly.

Keywords: Aspirin, Elderly, Hematuria, Prevalence

Citation:

Moudi E, Hosseini SR, Bijani A. Higher rate of microscopic hematuria in elderly patients who take regular doses of aspirin: Result from AHAP Study. Caspian J Intern Med 2016; 7(4): 278-282.

Caspian J Intern Med 2016; 7(4):278-282

Regular use of aspirin is one of the most common preventive strategies against cardiovascular diseases (1-5). More recently; its preventive effect in various types of cancers has emerged and therefore has supported its routine use in high risk populations (6-8). It is estimated that more than 35% of adults in the United States take daily doses of aspirin (5, 9). Despite useful effects of aspirin, certain complications such as gastrointestinal bleeding, hemorrhagic stroke and bronchospasm are prevalent among the regular users of aspirin (10). Aspirin interferes with the aggregation of platelets via inhibiting cyclooxygenase-1 and 2 (COX-1 and 2) enzymes. Inhibition of COX-1 and 2 enzymes leads to decrease formation of thromboxane -A₂ which plays a key role in platelet aggregation. Therefore, aspirin-users are at increased risk of bleeding events. However, the relationship between regular use of aspirin and hematuria is not well-studied yet. The present study aimed to compare the prevalence of microscopic hematuria in the elderly who took regular doses of aspirin and those who did not take it regularly.  

Methods

Study population and data collection: We extracted data on the aspirin use and prevalence of microscopic hematuria among elderly individuals who were recruited in Amirkola Health and Ageing Project (AHAP) (11). The protocol of the AHAP study has been fully described elsewhere. Briefly, health status of 1616 individuals aged 60 years or more were screened and their data were collected prospectively within a two-year period (2011-2012).

Several variables including demographic characteristics, body mass index (BMI), smoking status, chronic comorbidities (diabetes mellitus, hypertension, cardiovascular diseases etc.) and medicine use were evaluated and registered in AHAP database.

We have extracted the demographic data (age and gender), BMI, smoking history, history of diabetes, hematuria in urine analysis and the regular use of aspirin from the AHAP database using special checklists. The regular use of aspirin was defined as checking “yes” on the form of medication for daily use by the participant (self-reported).

Microscopic hematuria was defined as the presence of 5 red blood cells or more per high-power fieled in urine microscopic analysis. We excluded the elderly with self-reported history of urologic disorders (gross hematuria, urolithiasis, cancers of urogenital system), kidney disorders, taking anticoagulation and other antiplatelet medication and individuals with incomplete registry data from the study. Finally we have calculated the prevalence of microscopic hematuria among aspirin users and non-users applying multivariate statistical model.

Statistical Analysis: Data were analyzed using SPSS software Version 17 (IL, USA). T-test, chi-square, Fisher’s exact test were applied for comparing the frequency of microscopic hematuria among users and non-users of aspirin regarding their demographic characteristics, diabetes and smoking status. Multiple logistic regression analysis was used to adjust for potential confounding variables to isolate the association of aspirin use and the prevalence of microscopic hematuria. The p-value<0.05 was considered significant.

Results

Among the 1616 individuals who participated in AHAP, we excluded 373 persons who met the exclusion criteria. A total of 1243 elderly entered the final analysis of whom 674 (54.22%) were males and 569 (45.78%) were females. Two hundred and eighty four (23%) individuals reported regular use of aspirin and 957 (77%) did not report regular use of aspirin.

Demographic characteristics of the study population were shown in table 1.

Table 1. Demographic characteristics of elderly with and without regular use of aspirin (n=1243)

BMI, body mass index

Microscopic hematuria was seen in 305 (24.54%, 95%CI: 22.14%-26.93%) elderly.

The prevalence of microscopic hematuria was 27.27% (95%CI: 22.08%-32.47%) in regular users of aspirin and 23.72% (95% CI: 21.02%-26.42%) in non-users of aspirin (P=0.126).

The prevalence of microscopic hematuria was significantly higher among regular users of aspirin compared to non-users in multivariate analysis (P=0.035, OR=1.40, 95% CI: 1.02-1.92).

Multiple logistic regression analysis for determining the association of aspirin regular use and presence of microscopic hematuria was shown in table 2.

The prevalence of microscopic hematuria in the elderly with diabetes was 18.7%, 12.9% in the elderly with fasting blood glucose 180 milligram per deciliter and higher and 21.2% among elderly with FBS less than 180 (P=0.036).

Table 2. Multiple logistic regression analysis of the association of aspirin regular use and presence of microscopic hematuria in elderly (n=1243)

BMI, body mass index. DM, diabetes mellitus. *Chi-Square test

Discussion

Our study showed that the prevalence of microscopic hematuria is significantly higher in the elderly who took regular doses of aspirin compared to those who did not use aspirin. The effect of regular use of aspirin on the prevalence of microscopic hematuria was not associated with age, BMI, diabetes and smoking status of participants. Another finding of the present study was the lower rate of hematuria among diabetic participants.

 This was probably due to higher urinary volume in this group of participants that could dilute the urinary specimen. Higher rate of microscopic hematuria in DM patients with lower FBG levels may support this hypothesis. Microscopic hematuria is sometimes difficult to manage (12, 13). While, it could be an early sign of serious urological disorders in a group of patients, no specific disorders are detected at diagnostic investigations in another group (14, 15). In


patients with detected cause of
microscopic hematuria, urologic malignancies, urolithiasis, infections are the most common etiology. Our study revealed that taking regular doses of aspirin could be a potential cause of microscopic hematuria in the elderly who had no detectable cause for microscopic hematuria at initial work-up. Very few studies have investigated the association between aspirin use and the presence of microscopic hematuria in elderly population. Jeong et al. evaluated the risk of microscopic hematuria in aspirin users among 56632 adults who were screened in a health screening program. Microscopic hematuria was reported in 6.2% of their participants. The authors reported no significant association between low-dose aspirin and microscopic hematuria (6.1% in aspirin users versus 6.2% in non-users, P=0.71) (16). Another study with small population by Culclasure et al. also reported no significant association between aspirin users and microscopic hematuria(17). The first study assessed the adult population, whereas, the population of our study was old people which could be an explanation for the different findings. The latter study has only 69 aspirin-users in its analysis which can cause bias in their findings.

Our study was the first big one to evaluate the association of microscopic hematuria and the regular use of aspirin in elderly population. Based on our findings, regular use of aspirin is accompanied with greater risk of microscopic hematuria in elderly. It is a critical finding since microscopic hematuria may be a sign of benign condition such as urolithiasis or cancer in genitourinary system (18, 19).

Our study suggests that the clinician should think about aspirin causing microscopic hematuria in the elderly if the workup for microscopic hematuria showed no result. In conclusion, microscopic hematuria in elderly individuals with regular aspirin use is common, furthermore, the clinician should rule out other serious sources.

Acknowledgments

Thank you to Mrs Sakineh Kamali Ahangar of Clinical Research Center of Shahid Beheshti Hospital for her assistance during the study period.

Funding: The Vice-Chancellery for Research and Technology of Babol University of Medical Sciences financially supported the study.

Conflict of Interest: None declared.

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