Serum interleukin-35 as a prognostic biomarker in type 2 diabetes and diabetic nephropathy

Introduction

Diabetes mellitus is a chronic metabolic disorder characterized by persistent hyperglycemia. It is classified into many types such as type 1 diabetes (T1D), type 2 diabetes (T2D), other factors which causes special type of diabetes, and gestational diabetes mellitus (GDM) [1,2] . Diabetes can be caused by impaired β-cell pancreas due to the autoimmune-mediated destruction (insulin deficiency), impaired insulin receptor (resistance to the peripheral actions of insulin), or both [3] . Several complications may be caused for patients with T2D. The most common complications of T2D are nephropathy [4] . Diabetic nephropathy is a chronic inflammation of the kidney which is considered a serious complication of T2D causing proteinuria (albuminuria) and decline in renal functioning [5,6] . The average course of the disease is 10-20 years, though this might vary from patient to another based on a variety of factors, including glycemic management [7] . Previous studies explain that diabetic nephropathy may up to 30% in T1D patients and 40% in T2D patients, which means that 20-50% of individuals enrolled in renal failure programs have diabetes. It is the primary cause of end-stage renal disease (ESRD) prompting dialysis and kidney transplantation [8][9][10] . Actually, the pathobiology of the diabetic nephropathy is even more complicated, involving International Journal of Advanced Biochemistry Research https://www.biochemjournal.com modifications to kidney structure, function, and metabolism that are still not fully understood [11] . One of the many linked causes of disorder initiation is hyperglycemia, which triggers alternatively activated glucose pathways and their cytotoxic by products [12] . Oxidative stress [13] , immunological response and oxidized lipids all have affecting diabetic nephropathy [14] . All of these combined factors cause impaired to the kidney cells due to producing pro-inflammatory cytokines by DAMPs [15] . Although the body benefits from inflammation in the short term, prolonged inflammation can be highly harmful and lead to a variety of problems kidney [16] .

Inflammation is caused by an array of cytokines, and one of them is the IL-35. Il-35 is a novel heterodimeric cytokine in the IL-12 family [17] . An earlier study reported that IL-35 is a heterodimer of two subunits, EBI3 and P35 [18,19] . Cellspecific receptor subunit heterodimerization is required for IL-35 binding, i.e., GP130-GP130 (for T-cell), IL-12Rb2-IL12Rb2 and IL-12Rb2-GP130 (20, 21). Il-35 binds its while receptors then activate signaling pathway contain STAT1, STAT3 and STAT4 as well as JAK1 and JAK2 molecules [22,23] . IL-35, which is produced by regulatory T cell (Treg) and other lymphocytes, plays an immunosuppressive role through suppressing the generation of T-helper type-17 (Th17) cells and facilitating proliferation of Treg cells [24,25] . IL-35 has been associated with a variety of diseases from autoimmune diseases, cancer, to inflammatory response and sepsis [26,27] . In the past few years, researchers have created IL-35 transgenic mice with islet b-cell-specific expression of IL-35 and discovered that IL-35 has an inhibitory effect on type I diabetes [28] . The main mechanism for suppress of IL-35 is to inhibit the infiltration and proliferation of CD4 + T and CD8 + T [29] . Nevertheless, the expression of IL-35 in T2D and DN as well as its role in prognosis is largely unknown. Thus, in the present study, we investigated differences in patients with T2D and DN as well as whether serum Il-35 levels were associated with renal function hypothesizing that IL-35 be used as a potential biomarker for disease diagnosis and progression.

Materials and Methods

Study Design and Participants

Sample Collection and Biochemical Analysis

Fasting venous blood samples (5 mL) were collected from all participants using sterile vacutainer tubes. Samples were allowed to clot, centrifuged at 3,000 rpm for 10 minutes, and serum was separated and stored at-80 °C until analysis. Serum creatinine, blood urea nitrogen (BUN), and fasting glucose levels were measured using standard colorimetric methods.

Interleukin-35 Measurement

Serum levels of IL-35 were quantified using a commercially available ELISA kit (BT Lab Company, China) in accordance with the manufacturer's instructions. Optical densities were measured at 450 nm using a microplate reader (Model, Manufacturer). All samples were assayed in duplicate to ensure analytical consistency.

Statistical Analysis

Data analysis was performed using SPSS version 26.0 (IBM, USA). Continuous variables were expressed as mean ± standard deviation. Comparisons between groups were made using one-way ANOVA followed by Tukey's post hoc test. Pearson's correlation was used to assess associations between IL-35 levels and renal function markers. ROC curve analysis was employed to evaluate the diagnostic performance of IL-35. A p-value of<0.05 was considered statistically significant.

Experiments Experimental Groups

 Group 1 (DKD): Patients with confirmed diabetic kidney disease (n = 30)  Group 2 (DM): Diabetic patients without nephropathy (n = 30)  Group 3 (HC): Healthy, age-and sex-matched individuals (n = 30)

Primary Outcome

The primary experimental outcome was the comparison of serum IL-35 levels among the three groups.

Secondary Outcomes  Correlation between IL-35 and serum creatinine, BUN, and glucose levels  Diagnostic accuracy of IL-35 as assessed by ROC curve analysis

Experimental Procedure

All laboratory procedures were conducted under standardized conditions. The ELISA assay was validated for sensitivity and specificity, and calibration curves were generated with each run. Assay variability was monitored by including internal controls in every batch.

Results

The general characteristics of the enrolled participants is presented in table 1, including control subjects, patients with diabetes mellitus (DM), and those with diabetic nephropathy (DN). Several statistically significant differences were observed among the groups. Patients with DN were significantly older than both control (p = 0.001) and DM groups (p = 0.026). Body mass index (BMI) showed a marked decline across groups, being highest in controls and lowest in DN patients (p<0.001 for all comparisons). Fasting serum glucose (FSG) levels were significantly elevated in both DM and DN groups compared to controls (p<0.001), while no difference was observed between the DM and DN groups (p = 0.736). Fasting insulin (FI) levels were significantly lower in both DM and DN groups compared to controls (p<0.001), with a borderline difference between DM and DN (p = 0.050). Similarly, HOMA-IR and HOMA-β were significantly altered between control and disease groups (p<0.05), though the difference between DM and DN was not statistically significant for HOMA-β (p = 0.699). Triglycerides (TG) and very low-density lipoprotein cholesterol (VLDL-C) levels were significantly higher in controls than in both DM and DN patients (p<0.05), with borderline differences between DM and DN (p = 0.059). In contrast, total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) levels were significantly elevated in the DN group compared to both DM and control groups (p<0.05). High-density lipoprotein cholesterol (HDL-C) was markedly reduced in both DM and DN groups relative to controls (p<0.001). Notably, serum IL-35 levels were significantly decreased across the groups, with the lowest levels observed in DN patients. IL-35 levels were significantly lower in both DM and DN groups compared to controls (p<0.001), and further significantly decreased in DN compared to DM patients (p = 0.001). While several parameters such as waist circumference (WS), hip circumference (HIP), and%HbA1c showed expected trends, their comparisons between certain groups did not reach statistical significance. suggesting that IL-35 may reflect immune-mediated renal damage rather than glycemic dysregulation.

Diagnostic Performance of Serum IL-35 in Discriminating Disease Groups

The receiver operating characteristic (ROC) curve analysis is performed to assess the diagnostic potential of serum ILIn a recent study, IL-35 levels were found to be significantly decreased in diabetic patients with renal involvement compared to those without nephropathy, implicating its role in modulating renal inflammation [30] . Though most prior research has focused on autoimmune renal diseases like lupus nephritis, IL-35's diagnostic potential extends to broader renal pathologies. Nassif et al. reported that IL-35 levels in both serum and urine were significantly lower in lupus nephritis patients, suggesting its utility as a noninvasive biomarker of renal inflammation [31] .

The results align with experimental models in which IL-35 administration ameliorated renal inflammation. For instance, Yao et al. observed that IL-35 levels decreased in septic mice with acute kidney injury, and IL-35 supplementation mitigated renal damage through suppression of inflammatory cytokines and oxidative stress [32] . These findings suggest that diminished IL-35 may contribute to unchecked inflammation in chronic diabetic states, accelerating renal injury. Similarly, in a recent rodent model, IL-35 pretreatment significantly alleviated lipopolysaccharide-induced kidney injury by inhibiting nuclear factor-kappa B (NF-κB) activation, a key regulator of inflammatory gene expression [33] . While our study is cross-sectional, the inverse correlation between IL-35 levels and disease severity reinforces IL-35's proposed nephroprotective role. Interestingly, our data showed no significant correlation between IL-35 and glycemic markers (fasting serum glucose, HbA1c, HOMA indices), suggesting that IL-35 may reflect immune-mediated damage more than metabolic dysregulation. This echoes findings in type 1 diabetes models where IL-35 was shown to modulate immune responses independent of glycemic control, offering protection against β-cell destruction [34] . Moreover, Neubauer-Geryk et al. identified IL-35 alterations in young patients with type 1 diabetes, correlating the cytokine's levels with cutaneous microvascular dysfunction a complication also tied to systemic inflammation [35] . Such results support the role of IL-35 as a sensitive marker for inflammatory microangiopathic changes, a hallmark of diabetic complications.

In the correlation analysis, age was the only variable that showed a statistically significant positive relationship with IL-35 levels in DN patients. This finding contrasts with expectations, as aging typically promotes a proinflammatory milieu ("inflammaging"). One plausible explanation could be a compensatory immune-regulatory mechanism in older individuals, particularly in chronic disease states, although more data are required to confirm this hypothesis. The lack of association between IL-35 and lipid parameters, BMI, or waist-hip ratio indicates that IL-35 may be a more specific marker of immunologic and renal perturbation than general metabolic status. This study reinforces the potential of serum interleukinAlthough focused on periodontal inflammation, they noted that IL-35 had a diagnostic accuracy of 82.2% using ROC analysis in diabetes-related immune dysregulation, suggesting systemic immunoinflammatory alterations in diabetic patients influence IL-35 levels broadly [36] . Also, comparable diagnostic performance of IL-35 has been observed in other immune-mediated renal conditions. For instance, in patients with lupus nephritis, Nassif (2021) reported an AUC of 0.914 for serum IL-35, distinguishing nephritic from non-nephritic SLE patients, reinforcing its diagnostic relevance in inflammatory renal conditions [31] . Similarly, Zhang et al. evaluated IL-35 in idiopathic membranous nephropathy and found that serum IL-35 levels could predict remission duration, highlighting its prognostic as well as diagnostic potential [37] . Additionally, Farahat and Hassan (2024) demonstrated elevated diagnostic value of IL-35 in lupus nephritis with strong ROC metrics, reinforcing our conclusion that IL-35 performs robustly as a non-invasive biomarker across a spectrum of renal inflammatory diseases [38] .

Although IL-35 has not been widely studied in the context of diabetic nephropathy, its role as an immunomodulatory cytokine positions it well for such clinical utility. Its ability to suppress pro-inflammatory signaling-especially pathways involving IL-6, IL-17, and TNF-α-may explain its depletion in advanced disease states, where inflammatory responses are heightened. Given its impressive AUC, sensitivity, and specificity in our study, IL-35 may aid in early detection of nephropathy in diabetic patients, potentially informing timely therapeutic interventions. These findings, in conjunction with our results, suggest that IL-35 could serve as an early and reliable marker for detecting the onset and progression of DN in diabetic individuals. Its immunosuppressive role in modulating inflammatory cytokines such as IL-6 and TNF-α likely underpins this diagnostic utility, reflecting the level of immune activation across disease states. However, further prospective studies are warranted to validate its clinical application and assess its performance in larger, diverse populations.

The study has several limitations that should be considered. Firstly, the cross-sectional design limits our ability to infer causality between serum IL-35 levels and diabetic kidney disease (DKD) progression, and longitudinal studies are needed to assess the temporal relationship. Secondly, the relatively small sample size of 30 participants per group may affect the generalizability of the findings, necessitating larger, multi-center studies to confirm these results.

Additionally, the lack of data on renal tissue expression of IL-35 prevents a full understanding of its role in kidney inflammation, and future studies should incorporate tissue analysis. Furthermore, potential confounding factors, such as medication use, diabetes duration, and comorbidities, were not fully controlled, which could influence inflammatory markers. Finally, while several clinical and biochemical variables were assessed, the study did not evaluate other relevant inflammatory mediators that might interact with IL-35, suggesting that broader cytokine profiling could provide more comprehensive insights into its regulatory role in DKD.

Conclusion

In summary, the findings confirm that serum IL-35 levels are significantly reduced in diabetic nephropathy and inversely associated with disease progression. The cytokine's immune-modulatory properties, coupled with consistent findings in experimental and human studies, underscore its potential as a biomarker for DKD.

Prospective and mechanistic studies are needed to validate IL-35's diagnostic and therapeutic utility in diabetesassociated renal disease.

Declarations

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