The Immunodiagnostic Utility of Antinuclear Antibody Patterns: A Prediction for Renal Involvement in Systemic Lupus Erythematosus Patients in the Western Region of Saudi Arabia

Objectives Previous studies have noted associations between the immunofluorescence patterns of antinuclear autoantibodies (ANA) and the autoimmune responses seen in systemic lupus erythematosus (SLE). In this study, the authors tested the hypothesis of whether ANA patterns predict renal involvement in SLE patients. Method A retrospective study was carried out on consecutive SLE patients who had ANA staining pattern data and who were screened for renal involvement defined as all-stage proteinuria or chronic kidney disease (CKD) at a referral tertiary center in western Saudi Arabia from December 2021 to February 2022. Demographic data and levels of lupus immune markers including ANA titers, anti-double-stranded deoxyribonucleic acid antibodies (anti-dsDNA), complements C3 and C4, anticardiolipin (aCL) immunoglobulin (Ig) G and IgM, anti-β2 glycoprotein (β2-IgM and β2-IgG), and lupus anticoagulant (LA) antibodies were collected. Result Among 243 patients included, 25.1% had renal involvement (95% confidence interval {CI}=19.8-31.0). A mixed ANA pattern was associated with a higher prevalence of renal involvement (46.2%), followed by homogenous (26.5%) and speckled (25.6%) patterns, compared with 4.5% for the other patterns (p=0.044). No further association of renal involvement was observed with other biological markers. Adjusted logistic regression showed age (odds ratio {OR}=0.95; 95% CI=0.92-0.97) and mixed ANA pattern (OR=26.66; 95% CI=2.53-281.11) to be independently associated with renal involvement, explaining 12.6% of the variance. Conclusion A mixed homogenous/speckled ANA staining pattern is associated with an increased risk of renal involvement, independent of ANA titer or other lupus immune markers. The potential clinical applications of the ANA staining pattern in SLE should be explored in various subtypes of SLE and patient groups.


Introduction
Systemic lupus erythematosus (SLE) is a complex autoimmune disorder with multiorgan involvement, resulting from the interplay of genetic susceptibility, environmental factors, and immune/hormonal triggers [1,2]. It imposes significant health and economic challenges, including increased morbidity, cardiovascular complications, and reduced health-related quality of life (HRQoL), with variable epidemiological figures across ethnicities [3][4][5][6][7][8][9][10]. Recent research has deepened our understanding of SLE's immunopathogenesis, highlighting an aberrant immune response against nuclear autoantigens and the production of antinuclear autoantibodies (ANA). This involves disruptions in myeloid and lymphoid immunity, increased activation of autoreactive T and B lymphocytes, impaired clearance of immune complexes, and enhanced interferon pathways, all culminating in systemic autoimmunity [2,11,12]. Accordingly, SLE management focuses on mitigating disease activity, preventing organ damage, and enhancing survival and HRQoL [2].
Renal involvement, or lupus nephritis, represents a common and severe complication of SLE, observed between 7% and 50% of patients, depending on the stage and duration of the disease, as well as the ethnic group [13,14]. The prevalence of biopsy-proven lupus nephritis was estimated to be between 20% and 40% [15,16]. Lupus nephritis may be present at the time of SLE diagnosis or develop a few months to several years after diagnosis [17]. It may occur in flares with no clinical renal manifestation [18] as it may manifest as overt chronic kidney disease (CKD), the risk of which is potentiated by other factors such as a history of cardiovascular disease, atherosclerosis, or sepsis [14,19]. Therefore, the early diagnosis and timely management of lupus nephritis are paramount, since a delayed diagnosis increases the risk of end-stage renal disease (ESRD) [20]. An international meta-analysis study that included 187 articles and 18,300 patients showed that lupus nephritis was associated with a five-year risk of ESRD of about 10%-12% [21].
Several immunopathological mechanisms are intricately involved in lupus nephritis. These include systemic autoantibodies targeting different kidney structures, leading to the persistent presence of nuclear material in the extracellular space, which triggers further autoantigen-specific adaptive immune response locally and leads to the fixation of immune memory. Renal injury results from the local deposition of the immune complex and the activation of the complement, which leads to intrarenal inflammation [22]. The recent understanding of the correlations between immunological and histological disorders enabled defining several markers to enhance the early diagnosis of renal involvement in SLE. Thus, the occurrence of lupus nephritis is associated with higher autoimmune profile, indicated by higher anti-double-stranded deoxyribonucleic acid antibodies (anti-dsDNA) and greater consumption of complement indicated by low C3 [18,23]. Another marker of renal involvement is anti-C1q antibodies, which are more accurate in ruling out the diagnosis with a negative predictive value of 100%, besides being strong positive markers for lupus nephritis flares and proteinuria [24][25][26].
While monitoring these markers may enhance early diagnosis, certain genotypes have been identified to increase the risk or severity of lupus nephritis [27]. However, due to the impracticality of genotyping studies in routine practice, it remains of interest to prospectively characterize the patients' profiles associated with greater risk for developing lupus nephritis. This would enable a better appraisal of the renal prognosis and adaptation or renal monitoring in high-risk patients. In a previous work, the author evidenced a correlation of autoimmune activity in SLE patients with the immunofluorescence pattern of ANA [28]. This study tests the hypothesis of whether ANA patterns were also predictive for renal involvement in SLE patients.

Design and participants
This retrospective study was carried out at the Diagnostic Immunology Division, Department of Laboratory Medicine, King Abdulaziz University Hospital (KAUH), Jeddah, Saudi Arabia, from December 2021 to February 2022. KAUH is a referral immunodiagnostic center in the western region of Saudi Arabia. The study protocol was reviewed and ethically approved by the institutional review board of KAUH (reference number: 130-21).

Participants
Consecutive patients who were diagnosed with SLE and treated in KAUH from January 2018 to December 2020 and who had sufficient data to test the primary objective of the study were included. Hence, patients who had no results for ANA pattern or renal involvement were not included. SLE diagnosis and classification were carried out according to the 2019 European League Against Rheumatism/American College of Rheumatology (EULAR/ACR) criteria [29]. A non-probability, convenience sampling was used to include all eligible patients.

Diagnostic immunology assays
All immunodiagnostic analyses were carried out at the same laboratory and using the same methods for all patients and in compliance with the respective manufacturers' guidelines. ANA test was performed by indirect immunofluorescence (IIF) technique utilizing human epithelial cells (human epithelial type 2 {HEp-2}) fixed on glass slides using Aesku kits (Aesku Diagnostics, Windlesham, Germany). ANA patterns were categorized into peripheral, speckled, homogenous, nucleolar, and centromere.
Anti-dsDNA were analyzed by the enzyme-linked immunosorbent assay (ELISA) technique, using the INOVA System Quanta Lite™ Ds-DNA Kit (San Diego, CA).
In addition, CRP level, leucocyte count, hemoglobin level, PT, and PTT were analyzed in accordance with the standard laboratory methods (Diagnostica Stago, Asnières-sur-Seine, France).

Dependent Variable
The primary outcome was renal involvement, indicated by the presence at diagnosis or occurrence during the follow-up period of proteinuria of >500 mg/24 hours, [30] or the diagnosis of a chronic kidney disease (CKD), defined as chronic reduction of eGFR of <60 mL/minute/1.73 m 2 [14].

Independent Variables
Independent variables included ANA pattern and titer, demographic data, and other specific and nonspecific biological markers.

Statistical methods
The Excel database was checked for critical data missing, duplicates, and outliers and then transferred to Statistical Package for Social Sciences (SPSS) version 21 (IBM SPSS Statistics, Armonk, NY) for Windows for data analysis. Descriptive statistics were carried out to present frequencies and percentages for categorical variables and mean and standard deviation (SD) and/or median and interquartile range (IQR) for numerical variables. The association of renal involvement with categorical variables was analyzed using chi-square or Fisher's exact test, as applicable. The association of renal involvement with numerical variables used independent t-test or Mann-Whitney test, as applicable. A p-value of <0.05 was considered statistically significant.

Participants' characteristics
The study included 243 patients, with a mean (SD) age of 39.36 (15.72) years, and 86.4% were female. The majority were from an Arabian ethnic group (69.5%) ( Table 1).

Specific and nonspecific biological markers
The biological profiles of the patients are presented in Table 2

Renal involvement and its association with ANA titers and patters
Of the total patients, 25.1% had renal involvement (95% CI=19.8-31.0). While no significant association was observed between renal involvement and ANA titer (p=0.374), mixed pattern was associated with higher prevalence of renal involvement (46.2%), followed by homogenous (26.5%) and speckled (25.6%) patterns, while the "other" patterns had low prevalence of renal involvement (4.5%), and the result was statistically significant (p=0.044) (

Association of biological markers with renal involvement
Patients with renal involvement had more frequently positive CRP (40.7% versus 30.6%); however, this was not statistically significant (p=0.157). No further association of renal involvement was observed with other specific and nonspecific biological markers (

Context and summary
Renal involvement in SLE and the consequent CKD and ESRD are associated with impaired HRQoL, high morbidity, and up to 26-fold risk of mortality compared to the general population, which entails a substantial health and economic burden [4,31]. This study retrospectively analyzed the association of renal involvement with ANA pattern and titer, in addition to other lupus markers. The findings support that mixed ANA pattern is independently associated with a 27-fold risk of renal involvement, independent of ANA titers and the levels of other lupus markers. Additionally, the risk of lupus nephritis decreases by 5% with every year of the patient's age. Furthermore, male patients and those from African or Asian ethnic groups had relatively greater risk; however, this was not statistically significant.

Prevalence of renal involvement in SLE patients
The prevalence of renal involvement was approximately 25%, which is consistent with data from the literature. Various case definitions and estimation methods for renal involvement are reported. By considering the time of SLE onset, the prevalence of renal symptoms varies between 25% and 50%, whereas the life incidences of lupus nephritis in adult SLE patients may reach 60% [22]. By considering case definition, cases diagnosed with biopsy were reported at a comparable prevalence of 20%-40% [15,16]. Kidney biopsy represents the golden standard in the diagnosis of lupus nephritis. It enables excluding differentials, appraising renal damage, and classifying the level of severity of nephritis [32]. The International Society of Nephrology/Renal Pathology Society (ISN/RPS) criteria and the National Institutes of Health (NIH) activity and chronicity indices are based on the histological features found in kidney biopsy [33]. Additionally, a repeat kidney biopsy is recommended to monitor the treatment efficacy, in combination with clinical and biological signs including serum creatinine concentration, proteinuria, or eGFR [34,35]. In this retrospective study, renal involvement was doomed based on clinical and biological data, and no biopsy results were available for the patients.

Renal involvement and associated demographic factors
The authors observed that patients with renal involvement were 10 years younger, on average with reference to their counterparts; every additional year of the patient's age is associated with 5% decrease in the risk of renal involvement. This may be due to younger patients having more likely a juvenile-onset SLE (JO-SLE). Data from studies comparing adults with JO-SLE showed that lupus nephritis is significantly more prevalent in juvenile forms (up to 82%) compared with adult-onset forms (up to 53%) [16,36,37]. The younger age of SLE patients who develop lupus nephritis represents an additional factor in the health and economic burden of the disease.
Regarding gender, males were likely at higher risk, although the results were not statistically significant. The literature reports frequent male preponderance of lupus nephritis, with a greater renal damage and a higher prevalence ranging from 27% to 75%, compared with 16% to 52% in females [38][39][40].
Another observation is the relatively high prevalence of renal involvement among South Asian and Southeast Asian ethnic groups (40.0%), followed by African descents (33.3%), although the differences were not statistically significant (p=0.093). The differential risk of lupus nephritis across ethnic groups stands for the genetic backgrounds of SLE. A multiethnic study by Lanata et al., involving 1,244 SLE patients, found that the prevalence of lupus nephritis was highest among Asian patients (62%), followed by African-Americans (55.2%) and Hispanics (52.1%), while it was lowest among Europeans (≤40%) [41]. This is in line with the findings in this study, which showed Asians and Africans having the highest risks. The same study by Lanata et al. carried out further gene-based analyses and showed several genotypes to be associated with lupus nephritis in certain ethnic groups [41]. Comparable observations have been reported in a cohort of US patients with Medicaid coverage (2000)(2001)(2002)(2003)(2004), which showed a significantly greater risk for lupus nephritis among Hispanic, African-American, and Asian patients, and this risk is associated with a younger age at diagnosis [42]. This is also in line with an international cohort study by Hanly et al., showing African, Asian, and Hispanic ethnic groups to be at a greater risk for having lupus nephritis at inception (≤15 months of SLE diagnosis) [16].

Renal involvement and ANA pattern
The findings from this study demonstrate that mixed ANA pattern is independently associated with a 27-fold risk of lupus nephritis with reference to ANA patterns other than speckled and homogenous. Although the regression model, which also included age variable, explained only 12.6% of the outcome, such a finding indicates the potential utility of ANA immunofluorescence staining pattern in predicting the renal prognosis. Studies that analyzed the association of renal involvement with ANA pattern are scarce. A Swedish study by Frodlund et al. found that homogenous pattern was significantly associated with proliferative lupus nephritis, follow by speckled pattern, whereas mixed homogenous/speckled pattern was more frequently observed in low-class lupus nephritis [43]. This observation is not in contradiction with the findings from this study, as the severity of renal involvement was not assessed in this study. Furthermore, both homogenous and speckled patterns were observed to have relatively high risk of lupus nephritis, although this was not statistically significant. Conversely, Novianti et al. observed no significant correlation between ANA pattern and renal involvement, as indicated by proteinuria, in a group of 89 newly diagnosed, pediatric SLE patients [44]. Another intriguing case of a 34-year-old male was reported in Japan. This person had SLE cutaneous vasculitis with a mixed speckled/homogenous ANA pattern that progressed into lupus nephritis with the conversion of ANA pattern into discrete speckled type [45]. These observations indicate the potential interest of ANA pattern in predicting the organ damage in SLE.
In a previous study, the author demonstrated that ANA pattern had a significant immunodiagnostic value for disease activity as it showed several correlations with different SLE immune markers. Among these observations, mixed pattern was associated with remarkably higher levels of lupus anticoagulant [28]. This is consistent with a case-control study by Alba et al., which showed that lupus nephritis was associated with positive lupus anticoagulant with an odds ratio of 1.85 and 1.98 in unadjusted and adjusted analysis, respectively. Lupus nephritis was further associated with anti-dsDNA (OR=2.35) and anti-Smith autoantibody (OR=3.3) [46]. Another study by Farrugia et al. compared SLE patients with positive and negative lupus anticoagulants and showed that lupus anticoagulant was associated with a significant threefold risk of thrombosis and nonstatistically significant increase in serum creatinine levels. Histological examination showed no difference between the two groups in terms of activity or chronicity index [47]. Furthermore, lupus anticoagulant was demonstrated to be associated with adverse outcome in pregnant females with SLE, including lupus nephritis, preeclampsia, and proteinuria [22].
Other data showed that renal involvement was associated with other immune markers, notably low levels of C3 and C4, indicating high complement consumption and deposition [48]. Additionally, fluctuations in C3 and C4 levels were also demonstrated to be predictive for lupus nephritis [49]. The present study failed to demonstrate the correlation of renal involvement with any of the investigated immune markers.

Limitations
This study bears certain intrinsic limitations that merit acknowledgment. Primarily, given our reliance on retrospective data from the hospital's electronic databases, we faced challenges in procuring comprehensive clinical details. Specifically, the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) score and certain treatment data, which might offer crucial insights into the relation between renal involvement and ANA patterns in SLE patients, were not uniformly available for all participants. This limitation could potentially affect the depth of our findings. Additionally, the single-center nature of the study imposes constraints on the generalizability of our results. The observations derived from our cohort, based in one institution, might not be representative of SLE patients in other clinical settings or among different ethnic groups. Variations in patient demographics, clinical management, and disease manifestations could influence renal complications and ANA patterns in SLE. Therefore, caution should be exercised when extrapolating our findings to broader populations. Multicenter investigations with diverse patient samples are advocated to derive a more holistic understanding of the association between renal involvement and ANA patterns in SLE.

Conclusions
The staining pattern of ANA in immunofluorescence assays provides valuable indications on the severity and prognosis of SLE and organ damage. A mixed homogenous/speckled staining pattern is associated with a substantial risk of renal involvement, independent of ANA titer or other lupus immune markers. Other patterns that are likely to be associated with renal involvement include speckled and homogenous. Furthermore, younger patients are at higher risk of renal nephritis and should benefit from stringent renal monitoring. The potential clinical applications of ANA staining patterns in SLE should be explored in various subtypes of SLE and ethnic groups, considering the multifaceted nature of the disease and the multiple prognostic factors.

Additional Information Disclosures
Human subjects: Consent was obtained or waived by all participants in this study. The institutional review board of King Abdulaziz University Hospital (KAUH) issued approval 130-21. The study protocol was reviewed and ethically approved by the institutional review board of KAUH (reference number: 130-21). Animal subjects: All authors have confirmed that this study did not involve animal subjects or tissue.

Conflicts of interest:
In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: The author thanks the Deanship of Scientific Research of King Abdulaziz University, Jeddah, Saudi Arabia, for funding this research (grant number: KEP-9-140-42). Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work.