The Homocysteine Paradox in Diabetic Nephropathy: A Comprehensive Meta-Analysis Reveals Strong Biomarker Association but No Therapeutic Benefit from B-Vitamin Supplementation

The Homocysteine Paradox in Diabetic Nephropathy: A Comprehensive Meta-Analysis Reveals Strong Biomarker Association but No Therapeutic Benefit from B-Vitamin Supplementation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The Homocysteine Paradox in Diabetic Nephropathy: A Comprehensive Meta-Analysis Reveals Strong Biomarker Association but No Therapeutic Benefit from B-Vitamin Supplementation Hamid Benvidi, Saba Shafieizadegan, Alieh Gholaminejad This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7979615/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background The role of homocysteine (Hcy) in diabetic nephropathy (DN) remains paradoxical: while epidemiological studies consistently associate hyperhomocysteinemia with DN risk, interventional trials with B-vitamins have yielded conflicting results. This meta-analysis comprehensively integrates observational and interventional evidence to resolve this discrepancy and clarify the clinical utility of Hcy monitoring and B-vitamin therapy in DN. Methods We systematically searched PubMed, Scopus, and Web of Science through May 2024 for studies comparing Hcy, folate, and vitamin B12 levels between DN patients and diabetic controls, and randomized controlled trials (RCTs) evaluating B-vitamin supplementation. Random-effects models were used for meta-analysis, with subgroup analyses to explore heterogeneity. Results Across 45 studies (29 observational, 16 interventional), DN patients exhibited significantly higher Hcy levels (+ 2.81 µmol/L; 95% CI: 1.95–3.66) and lower folate levels (-2.09 nmol/L; 95% CI: -3.80 to -0.38) compared to diabetic controls without nephropathy. A clear dose-response relationship emerged: Hcy levels increased progressively from normoalbuminuria to microalbuminuria (+ 1.32 µmol/L) to macroalbuminuria (+ 3.58 µmol/L). Elevated Hcy was associated with 22% increased odds of DN (OR: 1.22; 95% CI: 1.13–1.32). Crucially, while B-vitamin supplementation effectively reduced Hcy levels (-3.35 µmol/L with folic acid), it conferred no renal benefit—showing no significant effects on eGFR (-1.91 mL/min/1.73m²) or creatinine levels. Conclusion This study establishes Hcy as a robust biomarker of DN severity but demonstrates that B-vitamin supplementation, despite effectively lowering Hcy, does not improve renal outcomes. These findings resolve the Hcy paradox in DN, suggesting that hyperhomocysteinemia may be a consequence rather than cause of renal impairment, and argue against routine B-vitamin supplementation for renal protection in DN patients. Further research is needed to explore potential benefits in specific patient subgroups. Systematic review registration: PROSPERO, CRD420251069868 Diabetic nephropathy Homocysteine Folic acid Vitamin B12 Vitamin B supplementation Meta-analysis Biomarkers Chronic kidney disease Type 2 diabetes Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Diabetic nephropathy (DN) is one of the most common microvascular complications of diabetes mellitus and a leading cause of chronic kidney disease (CKD) and end-stage renal disease (ESRD) worldwide. Although various biomarkers have been investigated to predict and manage DN progression, identification of modifiable metabolic markers, especially those associated with vascular and renal damage, is crucial for early intervention ( 1 – 4 ). Homocysteine (Hcy), a sulfur-containing amino acid derived from methionine metabolism, has been proposed as a potential biomarker and contributor to DN pathogenesis. Elevated levels of Hcy; known as hyperhomocysteinemia (Hcy) have been linked to endothelial dysfunction, oxidative stress, inflammation, and glomerular sclerosis, all of which are mechanisms implicated in renal microvascular damage and DN progression ( 5 – 7 ). Mechanistically, Hcy exerts direct endothelial toxicity by reducing nitric oxide bioavailability, promoting reactive oxygen species (ROS) generation, and inducing vascular smooth muscle proliferation, while also promoting inflammatory cytokine expression, collectively accelerating nephron damage ( 8 – 10 ). Despite these plausible mechanisms, epidemiological findings remain inconsistent. For instance, while Li et al. reported significantly elevated Hcy levels in DN patients compared to diabetics without nephropathy, some studies found no or less independent association after adjusting for glomerular filtration rate ( 11 – 13 ). One of the key metabolic pathways involved in homocysteine clearance is the methionine cycle, which is tightly interconnected with the folate cycle. As shown in the schematic (see graphical abstract), the enzyme methionine synthase (MS)—dependent on vitamin B12—remethylates Hcy into methionine, using 5-methyltetrahydrofolate (5-THF) derived from the folate cycle. Alternatively, Hcy can be catabolized to cysteine by transsulfuration pathway, which depends on vitamin B6 (pyridoxine) as a cofactor for cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE). Hence, deficiencies in vitamins B9 (folate), B12, and B6 can disrupt homocysteine metabolism, potentially exacerbating vascular and renal damage ( 14 , 15 ). This metabolic interdependence has led to increasing interest in B-vitamin supplementation as a possible intervention to modulate Hcy levels and reduce DN progression. Yet, interventional studies report conflicting outcomes: while some show that folate and B12 reduce Hcy and may improve renal outcomes, others report no significant benefit or even potential harm ( 16 – 19 ). While previous meta-analyses have examined homocysteine in broad CKD populations or B-vitamins in general diabetes management ( 20 , 21 )( 19 ), none have comprehensively addressed the critical disconnect between the strong observational associations and the null interventional effects specifically in diabetic nephropathy. This meta-analysis uniquely updates and integrates both strands of evidence to resolve the 'homocysteine paradox' in DN and provide definitive guidance for clinical practice. Therefore, we designed this meta-analysis to address the following objectives: ( 1 ) compare serum homocysteine levels between DN and DM patients; ( 2 ) assess serum folate and vitamin B12 levels in DN versus DM; and ( 3 ) evaluate the impact of B-vitamin supplementation on Hcy level as well as DN progression and renal outcomes. Methods Study Design This meta-analysis was conducted in accordance with PRISMA 2020 guidelines and included two distinct objectives: ( 1 ) To compare serum Hcy levels between patients with diabetic nephropathy (DN) and those with diabetes mellitus (DM) without nephropathy. ( 2 ) To assess folate and vitamin B12 levels in DN compare to DM patients, and the impact of B-vitamin supplementation on Hcy level and DN progression. Eligibility Criteria For part one, we included cross-sectional and cohort studies that compared serum homocysteine levels in DN and DM patients. For part two, we included (i) cross-sectional studies assessing folate and/or vitamin B12 levels in DN vs. DM, and (ii) randomized controlled trials (RCTs) investigating the effects of vitamin B supplementation on DN-related outcomes. Definition of DN DN was defined as type 1 or type 2 diabetes with either a glomerular filtration rate (GFR) 30 mg/g UAE > 30 mg/24h. DM control group included preserved renal function (GFR ≥ 60 mL/min/1.73m²) and normoalbuminuria. We excluded animal studies, case reports, reviews, editorials, and studies lacking adequate data or clear diagnostic criteria. Search Strategy A comprehensive literature search was conducted in PubMed, Scopus, and Web of Science up to May 2024, using keywords such as: “diabetic nephropathy,” “diabetic kidney disease,” “homocysteine,” “folic acid,” “vitamin B12,” and “B-vitamin supplementation.” We also manually reviewed reference lists. Duplicates were removed using EndNote. Only articles published in the English language were considered. Study Selection Two independent reviewers screened titles, abstracts, and full texts. Disagreements were resolved by discussion or consultation with a third reviewer. A machine learning–based tool (DeepSeek AI) was used to assist in full-text screening under expert supervision; details of its application and performance are provided in Supplementary Appendix 1, Table S0. Data Extraction and Quality Assessment Eligible studies were extracted using a standardized data form. The following variables were collected: First author, publication year, country, study design, sample size, participant demographics (age, sex, comorbidities), details of interventions or exposures (e.g., vitamin B dosage and duration), Outcome data including serum homocysteine, folate, and vitamin B12 levels; and clinical outcomes (e.g., changes in eGFR and albuminuria progression). For continuous outcomes, we extracted means ± standard deviations (SD) or mean changes ± SD. If outcomes were reported as medians with ranges or interquartile ranges (IQRs), we converted them to mean ± SD using the Meta-Converter tool ( 22 ). To calculate SD of change scores in pre-post studies, correlation coefficients (r) of 0.60 for homocysteine and 0.90 for creatinine were assumed based on prior literature ( Supplementary Appendix 1, Table S00 ). All data were extracted into Microsoft Excel and independently cross-checked by a second reviewer. Statistical Meta-analysis In total, meta-analyses were conducted using random-effects models (DerSimonian-Laird) and heterogeneity was assessed using the I² statistic, with values > 50% indicating moderate-to-high heterogeneity. All analyses were performed using STATA 17, with statistical significance set at p < 0.05. In Part 1, pooled mean differences (MD) in serum homocysteine levels between DN and DM groups were calculated using a random-effects model. Additionally, we performed separate meta-analyses based on the study design and reported effect measures; Odds ratios (ORs) from cross-sectional studies were pooled independently to assess the association between elevated homocysteine levels and DN. Hazard ratios (HRs) and incidence rate ratios (IRRs) from prospective cohort studies were combined in a single meta-analysis to evaluate homocysteine as a temporal risk factor for DN onset or progression. The logarithmic values and standard errors of the effect estimates were used for pooling. Effect sizes were reported with 95% confidence intervals (CIs). In Part 2, MDs were used to compare folate and vitamin B12 levels. For RCTs, mean changes ± SD in clinical outcomes between intervention and control groups were pooled. In the next step, subgroup analyses for serum homocysteine levels were conducted to explore potential sources of heterogeneity across the included studies. Stratification was performed based on key study-level characteristics, including: Proportion of female participants (≥ 50% vs. <50%), Publication year (before vs. after median year), Geographic region (by continent), Reported central tendency (mean vs. median values), Mean participant age (< 58 vs. ≥58 years), Diagnostic criteria for diabetic nephropathy (eGFR + albuminuria vs. eGFR only vs. albuminuria only), Sample size (< 200 vs. ≥200 participants). In addition, subgroup analyses were pre-specified to baseline renal function (eGFR < 60 vs. ≥60 mL/min/1.73m²) and Albuminuria category (normoalbuminuria, microalbuminuria, macroalbuminuria) to examine the Hcy-DN relationship across different renal stages, addressing whether the association is independent of glomerular filtration rate or albuminuria. Risk of Bias Assessment Quality control of observational studies were assessed using the Newcastle-Ottawa Scale (NOS). RCTs were evaluated using the Cochrane Risk of Bias (RoB 2) tool. Sensitivity analyses were performed by excluding studies with high risk of bias to test the robustness of findings. Sensitivity analyses were performed using a leave-one-out approach, applying fixed-effect models, to evaluate the robustness of the pooled estimates. Publication bias was calculated using the Funnel plot and Egger's test (linear regression method), and a significant statistical publication bias was set at P < 0.05 for Egger's test. Finally, to evaluate the impact of the missing research in the presence of publication bias, the trim and fill approach was employed ( 23 ). Results Study Selection and Characteristics A total of 45 studies were included in this meta-analysis. 29 studies examined the relationship between homocysteine levels and diabetic nephropathy (DN), including 25 cross-sectional studies assessing mean differences and 8 studies reporting effect sizes (ORs, HRs and IRRs). Sixteen studies evaluated serum levels of folate and/or vitamin B12, or the effects of B-complex supplementation in DN patients. Figure 1 illustrates the PRISMA flow diagram. The baseline characteristics of included studies are summarized in Tables S1–S5 . Serum Homocysteine Levels in DN vs. DM Patients Across 25 cross-sectional studies (3,707 patients with DM and 3,179 with DN) ( Table S1 ) serum homocysteine levels were significantly higher in DN patients compared to DM controls. The pooled MD was + 2.81 µmol/L (95% CI: 1.95 to 3.66; I² = 91.9%) ( Fig. 2 A ) . No publication bias was observed (Egger’s p = 0.52), and funnel plots are available in Supplementary Figures S1. The sensitivity analysis indicated that the final estimated risk is not affected by a single study (Figures S2 ) . Subgroup analyses were performed according to sex, publication year, geographic region, central tendency index, age, sample size, HbA1c, eGFR status, and DN diagnostic criteria (Table S2 ) . Elevated Hcy levels were observed across all strata. The effect size was slightly higher in studies with < 50% female participants (MD = + 3.23 µmol/L) compared to ≥ 50% female (MD = + 2.52 µmol/L). Studies published after 2010 reported slightly greater mean differences (MD = + 3.14 µmol/L) than earlier studies (MD = + 2.28 µmol/L). The effect was more pronounced in Asian populations (MD = + 3.16 µmol/L) compared to other regions (MD = + 2.81 µmol/L). Studies reporting mean values yielded larger effects (MD = + 3.24 µmol/L) than those reporting medians (MD = + 1.21 µmol/L, non-significant). Younger cohorts (< 58 years) showed greater differences (MD = + 3.48 µmol/L) than older cohorts (MD = + 2.14 µmol/L). The association persisted across HbA1c strata, though slightly stronger in those with HbA1c < 8.5% (MD = + 3.83 µmol/L). Markedly higher effects were observed when DN was defined by combined eGFR and albuminuria criteria (MD = + 4.13 µmol/L) compared to albuminuria alone (MD = + 2.44 µmol/L). Despite stratification, high heterogeneity (I² >80%) remained in most subgroups, indicating that unmeasured factors may contribute to variability. Homocysteine levels rose progressively across DN stages ( Fig. 3 ) : Microalbuminuria vs. normoalbuminuria: +1.32 µmol/L (95% CI: 0.43 to 2.21), Macroalbuminuria vs. microalbuminuria: +2.38 µmol/L (1.28 to 3.47), Macroalbuminuria vs. normoalbuminuria: +3.58 µmol/L (2.10 to 5.05). Association of Homocysteine with DN Risk Six cross-sectional studies (n = 4,918) reporting odds ratios (ORs) were pooled ( Table S3 ). The meta-analysis yielded a pooled OR of 1.22 (95% CI: 1.13–1.32), indicating that elevated homocysteine levels are associated with 22% increased odds of having DN (Fig. 2 B). This association was statistically significant (p < 0.001) with low publication bias (Egger’s p = 0.27) ( Figures S1 ). Sensitivity analysis confirmed the robustness of the results (Figures S2 ) . Two cohort studies (n = 791) reported hazard ratios (HR) and incidence rate ratios (IRR) ( Table S3 ). The pooled effect size was 1.52 (95% CI: 0.98–2.34), suggesting a non-significant trend toward increased DN risk with higher homocysteine (Fig. 2 C). Folate Levels in DN vs. DM Patients Eight studies (DN: n = 1,051; DM: n = 1,596) reported folate concentrations ( Table S4 ). DN patients had significantly lower folate levels by − 2.09 nmol/L (95% CI: − 3.80 to − 0.38; I² = 69.8% ) ( Fig. 4 A ) . Egger’s test indicated publication bias ( p = 0.0001 ); trim-and-fill analysis imputed one missing study, which did not significantly alter the results (Figure S1 ). Vitamin B12 Levels in DN vs. DM Patients Eight cross-sectional studies reported serum vitamin B12 levels in DN (n = 1004) versus DM (n = 1590) patients ( Table S4 ). The pooled MD was − 27.36 pg/mL (95% CI: − 62.16 to 7.45), indicating a non-significant trend toward lower B12 in DN compared to DM patients (Fig. 4 B). No publication bias detected (Egger’s p = 0.196) and Sensitivity analysis confirmed the robustness of the results (Figure S1 -2). Effects of B-Vitamin Supplementation on Homocysteine and Renal Outcomes Ten RCTs were included, with a total of n = 2,005 participants ( Table S5 ). In 4 trials, the overall effect of B-vitamin supplementation (folate/B-complex) was a pooled reduction in homocysteine of − 5.30 µmol/L (95% CI: − 7.80 to − 2.80) ( Fig. 4 C ) . Among them, folic acid trials (n = 2 RCTs; n = 1,687) showed a significant reduction: − 3.35 µmol/L (95%CI: − 5.38 to–1.32) compared to placebo. However, these trials did not evaluate renal endpoints such as GFR or creatinine. Two RCTs (n = 493) on vitamin B6 in three trials showed no effect on serum creatinine: MD = − 0.01 mg/dL (95% CI: − 0.10 to + 0.08) ( Fig. 4 D ) . But in this group, the relationship between vitamin B and Hcy has not been investigated. Two RCTs (n = 318) on B-complex vitamins measured eGFR change and found no significant benefit: MD in eGFR = − 1.91 mL/min/1.73m² (95% CI: − 6.41 to + 2.58 ) ( Fig. 4 E ) . Discussion This meta-analysis provides comprehensive evidence that serum Hcy levels are significantly elevated in patients with DN compared to diabetic individuals without nephropathy. Moreover, he identification of a clear dose-response relationship between albuminuria severity and homocysteine levels represents a pivotal finding of our analysis. This graded association strongly suggests that homocysteine accumulation parallels diabetic nephropathy progression, providing compelling evidence for its role as a biomarker of renal injury severity. These findings are consistent with recent reports demonstrating elevated homocysteine in DN patients and its association with microvascular damage, glomerular dysfunction, and poor renal outcomes in diabetes ( 24 , 25 ). From a pathophysiological perspective, Hcy promotes oxidative stress, endothelial dysfunction, and pro-fibrotic signaling, particularly through the activation of transforming growth factor-β (TGF-β), which are all key contributors to renal injury in diabetes ( 14 , 26 ). Additionally, homocysteine interferes with methylation reactions and redox balance, impairing both vascular integrity and glomerular filtration. However, despite this plausible biological rationale, the causal link between homocysteine and DN remains debated. In our meta-analysis, cohort studies did not show a statistically significant association between baseline homocysteine levels and incident DN. This may reflect limited data, short follow-up duration, or confounding by reduced glomerular filtration rate (GFR) itself; a known determinant of homocysteine accumulation. This view is supported by Mendelian randomization studies, which suggest that the relationship between homocysteine and CKD is likely mediated by reduced renal clearance rather than a primary driver of nephropathy ( 27 ). Genetic susceptibility may further explain interindividual variation. A meta-analysis by Zhou et al. (2015) reported that the MTHFR C677T polymorphism, which impairs folate metabolism and elevates homocysteine, is associated with DN risk ( 28 ). Similarly, Huo et al. (2022) highlighted an association with the MTHFR A1298C polymorphism ( 29 ). These findings suggest that prolonged exposure to hyperhomocysteinemia, especially in genetically susceptible individuals, could contribute to DN pathogenesis. Regarding micronutrient status, our analysis showed significantly lower serum folate levels in DN patients, while vitamin B12 levels were not significantly different. These results align with previous studies reporting folate depletion in DN due to increased oxidative loss or reduced absorption, whereas B12 levels may be influenced by factors such as metformin use, dietary intake, or assay variability ( 26 ). Our findings also indicate that B-vitamin supplementation was shown to reduce homocysteine levels substantially. However, this biochemical improvement did not translate into renal protection. For instance, the landmark RCT by House et al. (2010) reported that high-dose folic acid, B6, and B12 led to a greater decline in GFR and increased vascular events in DN patients ( 18 ). Similarly, Schneider et al. (2014) observed no improvement in proteinuria after 4 weeks of high-dose folate therapy ( 30 ). These observations reinforce the idea that biomarker modulation does not necessarily lead to clinical benefit. Recent meta-analyses such as Mokgalaboni et al. (2024) confirmed the ability of folic acid to reduce homocysteine and inflammatory markers in patients with type 2 diabetes. Our meta-analysis fills this gap by evaluating clinical and biochemical endpoints in parallel, providing a more comprehensive picture of the utility and limitations of B-vitamin therapy in DN ( 19 ). Our findings are in line with the Cochrane review by Raval et al. (2015), which concluded that while B-vitamin supplementation lowers Hcy levels, it does not significantly improve renal function. Our study extends these findings by including evidence from both observational and cross-sectional evidence on B9/B12 status ( 17 ). Importantly, the possibility of adverse effects from high-dose B-vitamin supplementation such as accumulation of unmetabolized folic acid or unbalanced methylation cannot be ignored ( 31 , 32 ). Therefore, routine supplementation in DN patients should be approached with caution and individualized based on genetic, biochemical, and clinical profiles. In other meta-analyses similar to ours, Zhu et al. also conducted a meta-analysis limited to patients with type 2 diabetes, which reported a significant association between elevated homocysteine and diabetic nephropathy risk. Our findings are broadly consistent, but our analysis included both type 1 and type 2 diabetes, offering broader generalizability across diabetic populations ( 21 ). Several limitations should be acknowledged. First, substantial heterogeneity was observed across the included studies, potentially due to differences in patient demographics, assay methods, DN diagnostic criteria, and regional dietary factors. Although subgroup analyses were performed to explore these sources of variability, residual confounding may persist. Second, most studies were observational and cross-sectional in design, limiting causal inference. Third, only a few randomized controlled trials (RCTs) reported hard clinical outcomes, and most were of short duration with varied dosing strategies, reducing the generalizability of their findings. Additionally, evidence from Mendelian randomization and longitudinal data supports the notion that elevated Hcy levels may largely reflect declining renal function, rather than directly causing DN. Our findings resolve a long-standing paradox in diabetic nephropathy management: while homocysteine demonstrates strong, graded associations with DN severity consistent with a potential pathogenic role, therapeutic lowering of Hcy with B-vitamins fails to translate into renal protection. This discrepancy suggests several mechanistic interpretations: ( 1 ) Hcy may primarily be a marker rather than mediator of renal damage; ( 2 ) the timing of intervention may be critical—once DN is established, Hcy-lowering may be too late; or ( 3 ) Hcy might interact with other uremic toxins in established CKD, limiting the efficacy of isolated B-vitamin supplementation. Thus, further high-quality, long-term, and genetically stratified RCTs are needed to elucidate whether targeting homocysteine confers renal benefit in specific patient populations. Conclusion This meta-analysis provides the most comprehensive evidence to date that while homocysteine serves as an excellent biomarker for DN risk stratification, therapeutic targeting of Hcy with B-vitamins is ineffective for renal protection. Clinicians should utilize Hcy measurements for prognostic information but not as a therapeutic target. Future research should focus on earlier intervention in prediabetic or normoalbuminuric stages, and consider combinatorial approaches addressing the broader metabolic milieu of DN. Abbreviations ACR albumin-to-creatinine ratio CI confidence interval CKD chronic kidney disease DM diabetes mellitus DN diabetic nephropathy ESRD end-stage renal disease eGFR Estimated glomerular filtration rate HR hazard ratio HHcy hyperhomocysteinemia IRR incidence rate ratio I² I-squared statistic MD mean difference NOS Newcastle-Ottawa Scale OR odds ratio PLP pyridoxal-5′-phosphate, RCT:randomized controlled trial RoB 2 Risk of Bias 2 tool SD standard deviation UAE urinary albumin excretion Declarations Competing interests The authors declare no competing interests. Funding The authors received no funds for this study. Author contributions HB and AG participated in the design, data searching and collection, analysis, data interpretation, and drafting of the manuscript. SS participated in the data interpretation, and drafting of the manuscript. All the authors have reviewed the final version of the manuscript. References Wani ZA, Ahmed S, Saleh A, Anna VR, Fahelelbom KM, Raju SK, et al. Biomarkers in diabetic nephropathy: A comprehensive review of their role in early detection and disease progression monitoring. Diabetes Res Clin Pract. 2025;226:112292. Gholaminejad A, Moein S, Roointan A, Mortazavi M, Nouri R, Mansourian M et al. Circulating β2 and α1 microglobulins predict progression of nephropathy in diabetic patients: a meta-analysis of prospective cohort studies. Acta Diabetol. 2022. Roointan A, Shafieizadegan S, Ghaeidamini M, Gheisari Y, Hudkins KL, Gholaminejad A. The Potential of Cardiac Biomarkers, NT-ProBNP and Troponin T, in Predicting the Progression of Nephropathy in Diabetic Patients: A Meta-Analysis of Prospective Cohort Studies. Diabetes Res Clin Pract. 2023:110900. Roointan A, Gheisari Y, Hudkins KL, Gholaminejad A. Non-invasive metabolic biomarkers for early diagnosis of diabetic nephropathy: Meta-analysis of profiling metabolomics studies. Nutr Metabolism Cardiovasc Dis. 2021;31(8):2253–72. Park S, Lee S, Kim Y, Cho S, Kim K, Kim YC, et al. Causal Effects of Homocysteine, Folate, and Cobalamin on Kidney Function: A Mendelian Randomization Study. Nutrients. 2021;13(3):906. Wu HHL, McDonnell T, Chinnadurai R. Physiological Associations between Vitamin B Deficiency and Diabetic Kidney Disease. Biomedicines. 2023;11(4):1153. Esse R, Barroso M, Tavares de Almeida I, Castro R. The Contribution of Homocysteine Metabolism Disruption to Endothelial Dysfunction: State-of-the-Art. Int J Mol Sci. 2019;20(4):867. Chermiti R, Burtey S, Dou L. Role of Uremic Toxins in Vascular Inflammation Associated with Chronic Kidney Disease. J Clin Med. 2024;13(23):7149. Yi F, Li P-L. Mechanisms of Homocysteine-Induced Glomerular Injury and Sclerosis. Am J Nephrol. 2007;28(2):254–64. FRIEDMAN AN, BOSTOM AG, SELHUB J, LEVEY AS, ROSENBERG IH. The Kidney and Homocysteine Metabolism. J Am Soc Nephrol. 2001;12(10):2181–9. Li H, Liu C, Zhang J, Wang W, Cheng W, Yang R, et al. The association of homocysteine level with the risk of diabetic nephropathy and diabetic retinopathy in NHANES. Acta Diabetol. 2023;60(7):907–16. Wang B, Li H, Wang N, Li Y, Song Z, Chen Y, et al. The impact of homocysteine on patients with diabetic nephropathy: a mendelian randomization study. Acta Diabetol. 2025;62(1):123–30. Hovind P, Tarnow L, Rossing P, Teerlink T, Stehouwer CD, Emeis JJ, et al. Progression of diabetic nephropathy: role of plasma homocysteine and plasminogen activator inhibitor-1. Am J Kidney Dis. 2001;38(6):1376–80. Cappuccilli M, Bergamini C, Giacomelli FA, Cianciolo G, Donati G, Conte D, et al. Vitamin B Supplementation and Nutritional Intake of Methyl Donors in Patients with Chronic Kidney Disease: A Critical Review of the Impact on Epigenetic Machinery. Nutrients. 2020;12(5):1234. Friedman AN, Bostom AG, Selhub J, Levey AS, Rosenberg IH. The Kidney and Homocysteine Metabolism. J Am Soc Nephrol. 2001;12(10). Righetti M, Serbelloni P, Milani S, Ferrario G. Homocysteine-Lowering Vitamin B Treatment Decreases Cardiovascular Events in Hemodialysis Patients. Blood Purif. 2006;24(4):379–86. Raval AD, Thakker D, Rangoonwala AN, Gor D, Walia R. Vitamin B and its derivatives for diabetic kidney disease. Cochrane Database Syst Rev. 2015;1(1):Cd009403. House AA, Eliasziw M, Cattran DC, Churchill DN, Oliver MJ, Fine A, et al. Effect of B-vitamin therapy on progression of diabetic nephropathy: a randomized controlled trial. JAMA. 2010;303(16):1603–9. Mokgalaboni K, Mashaba GR, Phoswa WN, Lebelo SL. Folic acid supplementation on inflammation and homocysteine in type 2 diabetes mellitus: systematic review and meta-analysis of randomized controlled trials. Nutr Diabetes. 2024;14(1):22. Zhou X, Shi A, Zhou X. A meta-analysis of serum Hcy in diagnosis of diabetic nephropathy. Pteridines. 2020;31(1):1–8. Zhu M, Fan Y. Association of Circulating Homocysteine Level with the Risk of Nephropathy in Type 2 Diabetes Mellitus: A Meta-Analysis. Horm Metab Res. 2025;57(02):117–23. Abbas A, Hefnawy MT, Negida A. Meta-analysis accelerator: a comprehensive tool for statistical data conversion in systematic reviews with meta-analysis. BMC Med Res Methodol. 2024;24(1):243. Duval S, Tweedie R. Trim and fill: a simple funnel-plot–based method of testing and adjusting for publication bias in meta‐analysis. Biometrics. 2000;56(2):455–63. Zheng X, Liu Q, Liu Z. Serum homocysteine concentration as a marker for advanced diabetic nephropathy in a cohort of elderly patients. BMC Endocr Disorders. 2023;23(1):114. Ye B, Zhu X, Zeng Z, Ji X, Ji M. Clinical significance of serum homocysteine as a biomarker for early diagnosis of diabetic nephropathy in type 2 diabetes mellitus patients. Pteridines. 2021;32(1):11–6. Schleicher E, Didangelos T, Kotzakioulafi E, Cegan A, Peter A, Kantartzis K. Clinical Pathobiochemistry of Vitamin B12 Deficiency: Improving Our Understanding by Exploring Novel Mechanisms with a Focus on Diabetic Neuropathy. Nutrients. 2023;15(11):2597. Park S, Lee S, Kim Y, Cho S, Kim K, Kim YC, et al. Causal effects of homocysteine, folate, and cobalamin on kidney function: a Mendelian randomization study. Nutrients. 2021;13(3):906. Zhou T-B, Drummen GP, Jiang Z-P, Li H-Y. Methylenetetrahydrofolate reductase (MTHFR) C677T gene polymorphism and diabetic nephropathy susceptibility in patients with type 2 diabetes mellitus. Ren Fail. 2015;37(8):1247–59. Huo Y, Zhang W, Zhang X, Su L, Li H, Wang F, et al. The Association of Methylenetetrahydrofolate Reductase (MTHFR) A1298C Gene Polymorphism with Susceptibility to Diabetic Nephropathy: A Meta-Analysis. Horm Metab Res. 2022;54(12):845–51. Schneider MP, Schneider A, Jumar A, Kistner I, Ott C, Schmieder RE. Effects of folic acid on renal endothelial function in patients with diabetic nephropathy: results from a randomized trial. Clin Sci. 2014;127(7):499–505. Bailey SW, Ayling JE. The extremely slow and variable activity of dihydrofolate reductase in human liver and its implications for high folic acid intake. Proceedings of the National Academy of Sciences. 2009;106(36):15424–9. Obeid R, Kasoha M, Kirsch SH, Munz W, Herrmann W. Concentrations of unmetabolized folic acid and primary folate forms in pregnant women at delivery and in umbilical cord blood. Am J Clin Nutr. 2010;92(6):1416–22. Supplementary Files graphicalabstract.png Graphical Abstract.Graphical summary of systematic review and meta-analysis SupplementalAppendix1.docx SupplementalAppendix2.docx SupplementalAppendix3.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7979615","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":544972871,"identity":"25f6ead8-775b-4d28-9f81-a0c4c8213096","order_by":0,"name":"Hamid Benvidi","email":"","orcid":"","institution":"Isfahan University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Hamid","middleName":"","lastName":"Benvidi","suffix":""},{"id":544972872,"identity":"753f9f1d-ef79-44ff-87b8-4fd98d26e380","order_by":1,"name":"Saba Shafieizadegan","email":"","orcid":"","institution":"Isfahan University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Saba","middleName":"","lastName":"Shafieizadegan","suffix":""},{"id":544972873,"identity":"36f88576-08e3-4e7d-bc2d-8e1660f6178f","order_by":2,"name":"Alieh Gholaminejad","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3UlEQVRIiWNgGAWjYDACdgYGZgYGCyDJfICBsYEYLcxgJAEk2RJI1cLAY0CcFv5mBubXBRUS8rrtPN8kfu6wkWNgP3x0Az4tEocZ2KxnnJEw3HaYd5tk75k0YwaetLQbeK0BajHmbZNgBGmR4G07nNggwWOGV4s8VIv9tsM8zyT/EqPF4DAD82OglkSgFjZpomwxBNrCzHNGInnbYTZja9m2NGM2Qn6RO97A/JmnwsZ22/nDD2++bbOR42c/fAy/9xn4v0lAWSxgBht+5WDA/AGdMQpGwSgYBaMABQAAJklDEeO2/K0AAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0003-0329-2937","institution":"Isfahan University of Medical Sciences","correspondingAuthor":true,"prefix":"","firstName":"Alieh","middleName":"","lastName":"Gholaminejad","suffix":""}],"badges":[],"createdAt":"2025-10-29 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07:05:05","extension":"html","order_by":25,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":95140,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7979615/v1/633d583abca9617a85b1e629.html"},{"id":96792647,"identity":"0089af88-ddb0-4dbd-9b22-e331cfe747d4","added_by":"auto","created_at":"2025-11-26 07:05:04","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":982849,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePRISMA 2020 flow diagram illustrating the study selection process for both parts of the meta-analysis.\u003c/strong\u003e A total of 551 records were screened for Part 1 (homocysteine in DN patients compare to DM patients), resulting in 29 included studies. For Part 2 (vitamin B12, folate, and intervention studies), 16 studies were included after full-text assessment. Reasons for exclusion at each stage are detailed.\u003c/p\u003e","description":"","filename":"Picture1.png","url":"https://assets-eu.researchsquare.com/files/rs-7979615/v1/82b7f5f4a57a3358fb71b390.png"},{"id":96916603,"identity":"612f88e0-0740-415b-aaee-e5e657a93daf","added_by":"auto","created_at":"2025-11-27 14:08:46","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":443393,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMeta-analysis of homocysteine levels in patients with diabetic nephropathy (DN) compared to diabetes mellitus (DM) without nephropathy.\u003c/strong\u003e (A) Pooled mean difference across cross-sectional studies. (B) Pooled odds ratio (OR) for DN in patients with elevated homocysteine. (D) Longitudinal risk estimates (hazard ratio/incidence rate ratio) from cohort studies.\u003c/p\u003e","description":"","filename":"Picture2.png","url":"https://assets-eu.researchsquare.com/files/rs-7979615/v1/68c110c83a0e407baee962a7.png"},{"id":96916752,"identity":"3d6a372b-c03b-47be-b02c-d2e27e4a406a","added_by":"auto","created_at":"2025-11-27 14:08:52","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":285933,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMeta-analysis of homocysteine levels in diabetes patients with different level of albuminuria. \u003c/strong\u003e(A) micro albuminuria versus normal albuminuria. (B) macro albuminuria versus micro albuminuria. (C) macro albuminuria versus normal albuminuria.\u003c/p\u003e","description":"","filename":"Picture3.png","url":"https://assets-eu.researchsquare.com/files/rs-7979615/v1/35a332530a2f0709028d77be.png"},{"id":96792646,"identity":"97d01f70-7bbd-422f-85a9-0af6c6ba65e9","added_by":"auto","created_at":"2025-11-26 07:05:04","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":750035,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFolate and vitamin B12 in DN vs. DM and Effects of B-vitamin supplementation in patients with diabetic nephropathy. \u003c/strong\u003e(A) Folate is decreased in DN vs. DM. (B) Vitamin B12 showed no significant changes in DN vs DM. (C) Vitamin B group and Folate significantly reduced serum homocysteine. (D) Vitamin B6 supplementation showed no significant effect on serum creatinine levels. (E) Vitamin B supplementation showed no significant change in eGFR.\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7979615/v1/d1983ff1afc6bafceaff6156.jpg"},{"id":98421881,"identity":"17cd900d-2030-47a4-bbf5-8eeef202bd24","added_by":"auto","created_at":"2025-12-17 16:29:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3373203,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7979615/v1/05a5cf18-86b1-4601-85ce-aa1f303f87b3.pdf"},{"id":96916753,"identity":"9c91bf73-2ece-4c75-8d2e-7febf2ccecab","added_by":"auto","created_at":"2025-11-27 14:08:52","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":469220,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical Abstract.\u003c/strong\u003eGraphical summary of systematic review and meta-analysis\u003c/p\u003e","description":"","filename":"graphicalabstract.png","url":"https://assets-eu.researchsquare.com/files/rs-7979615/v1/07429c249a3b1261c1f8cf96.png"},{"id":96792644,"identity":"9292b3f1-28bb-4206-a4fa-5601976e5431","added_by":"auto","created_at":"2025-11-26 07:05:04","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":26141,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalAppendix1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7979615/v1/3d79ca7c0289c8c75593e9a8.docx"},{"id":96915542,"identity":"776f6a82-2793-4820-a808-6d7764736d1f","added_by":"auto","created_at":"2025-11-27 14:07:21","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":118655,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalAppendix2.docx","url":"https://assets-eu.researchsquare.com/files/rs-7979615/v1/14b304ec5ea24752e03174af.docx"},{"id":96792656,"identity":"8fa2bb05-afd9-456a-ac6e-3707cd5360f6","added_by":"auto","created_at":"2025-11-26 07:05:04","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":720115,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalAppendix3.docx","url":"https://assets-eu.researchsquare.com/files/rs-7979615/v1/b146f12085dcbeeed3c36b8a.docx"}],"financialInterests":"","formattedTitle":"The Homocysteine Paradox in Diabetic Nephropathy: A Comprehensive Meta-Analysis Reveals Strong Biomarker Association but No Therapeutic Benefit from B-Vitamin Supplementation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDiabetic nephropathy (DN) is one of the most common microvascular complications of diabetes mellitus and a leading cause of chronic kidney disease (CKD) and end-stage renal disease (ESRD) worldwide. Although various biomarkers have been investigated to predict and manage DN progression, identification of modifiable metabolic markers, especially those associated with vascular and renal damage, is crucial for early intervention (\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eHomocysteine (Hcy), a sulfur-containing amino acid derived from methionine metabolism, has been proposed as a potential biomarker and contributor to DN pathogenesis. Elevated levels of Hcy; known as hyperhomocysteinemia (Hcy) have been linked to endothelial dysfunction, oxidative stress, inflammation, and glomerular sclerosis, all of which are mechanisms implicated in renal microvascular damage and DN progression (\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Mechanistically, Hcy exerts direct endothelial toxicity by reducing nitric oxide bioavailability, promoting reactive oxygen species (ROS) generation, and inducing vascular smooth muscle proliferation, while also promoting inflammatory cytokine expression, collectively accelerating nephron damage (\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Despite these plausible mechanisms, epidemiological findings remain inconsistent. For instance, while \u003cem\u003eLi et al.\u003c/em\u003e reported significantly elevated Hcy levels in DN patients compared to diabetics without nephropathy, some studies found no or less independent association after adjusting for glomerular filtration rate (\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eOne of the key metabolic pathways involved in homocysteine clearance is the methionine cycle, which is tightly interconnected with the folate cycle. As shown in the schematic (see graphical abstract), the enzyme methionine synthase (MS)\u0026mdash;dependent on vitamin B12\u0026mdash;remethylates Hcy into methionine, using 5-methyltetrahydrofolate (5-THF) derived from the folate cycle. Alternatively, Hcy can be catabolized to cysteine by transsulfuration pathway, which depends on vitamin B6 (pyridoxine) as a cofactor for cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE). Hence, deficiencies in vitamins B9 (folate), B12, and B6 can disrupt homocysteine metabolism, potentially exacerbating vascular and renal damage (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThis metabolic interdependence has led to increasing interest in B-vitamin supplementation as a possible intervention to modulate Hcy levels and reduce DN progression. Yet, interventional studies report conflicting outcomes: while some show that folate and B12 reduce Hcy and may improve renal outcomes, others report no significant benefit or even potential harm (\u003cspan additionalcitationids=\"CR17 CR18\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eWhile previous meta-analyses have examined homocysteine in broad CKD populations or B-vitamins in general diabetes management (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e)(\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e), none have comprehensively addressed the critical disconnect between the strong observational associations and the null interventional effects specifically in diabetic nephropathy. This meta-analysis uniquely updates and integrates both strands of evidence to resolve the 'homocysteine paradox' in DN and provide definitive guidance for clinical practice. Therefore, we designed this meta-analysis to address the following objectives: (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) compare serum homocysteine levels between DN and DM patients; (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) assess serum folate and vitamin B12 levels in DN versus DM; and (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) evaluate the impact of B-vitamin supplementation on Hcy level as well as DN progression and renal outcomes.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStudy Design\u003c/h2\u003e\u003cp\u003eThis meta-analysis was conducted in accordance with PRISMA 2020 guidelines and included two distinct objectives:\u003c/p\u003e\u003cp\u003e(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) To compare serum Hcy levels between patients with diabetic nephropathy (DN) and those with diabetes mellitus (DM) without nephropathy.\u003c/p\u003e\u003cp\u003e(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) To assess folate and vitamin B12 levels in DN compare to DM patients, and the impact of B-vitamin supplementation on Hcy level and DN progression.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eEligibility Criteria\u003c/h3\u003e\n\u003cp\u003eFor part one, we included cross-sectional and cohort studies that compared serum homocysteine levels in DN and DM patients. For part two, we included (i) cross-sectional studies assessing folate and/or vitamin B12 levels in DN vs. DM, and (ii) randomized controlled trials (RCTs) investigating the effects of vitamin B supplementation on DN-related outcomes.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eDefinition of DN\u003c/strong\u003e\u003cp\u003eDN was defined as type 1 or type 2 diabetes with either a glomerular filtration rate (GFR)\u0026thinsp;\u0026lt;\u0026thinsp;60 mL/min/1.73m\u0026sup2; or albuminuria (UACR\u0026thinsp;\u0026gt;\u0026thinsp;30 mg/g UAE\u0026thinsp;\u0026gt;\u0026thinsp;30 mg/24h. DM control group included preserved renal function (GFR\u0026thinsp;\u0026ge;\u0026thinsp;60 mL/min/1.73m\u0026sup2;) and normoalbuminuria. We excluded animal studies, case reports, reviews, editorials, and studies lacking adequate data or clear diagnostic criteria.\u003c/p\u003e\u003c/p\u003e\n\u003ch3\u003eSearch Strategy\u003c/h3\u003e\n\u003cp\u003eA comprehensive literature search was conducted in PubMed, Scopus, and Web of Science up to May 2024, using keywords such as: \u0026ldquo;diabetic nephropathy,\u0026rdquo; \u0026ldquo;diabetic kidney disease,\u0026rdquo; \u0026ldquo;homocysteine,\u0026rdquo; \u0026ldquo;folic acid,\u0026rdquo; \u0026ldquo;vitamin B12,\u0026rdquo; and \u0026ldquo;B-vitamin supplementation.\u0026rdquo; We also manually reviewed reference lists. Duplicates were removed using EndNote. Only articles published in the English language were considered.\u003c/p\u003e\n\u003ch3\u003eStudy Selection\u003c/h3\u003e\n\u003cp\u003eTwo independent reviewers screened titles, abstracts, and full texts. Disagreements were resolved by discussion or consultation with a third reviewer. A machine learning\u0026ndash;based tool (DeepSeek AI) was used to assist in full-text screening under expert supervision; details of its application and performance are provided in \u003cb\u003eSupplementary Appendix 1, Table S0.\u003c/b\u003e\u003c/p\u003e\n\u003ch3\u003eData Extraction and Quality Assessment\u003c/h3\u003e\n\u003cp\u003eEligible studies were extracted using a standardized data form. The following variables were collected: First author, publication year, country, study design, sample size, participant demographics (age, sex, comorbidities), details of interventions or exposures (e.g., vitamin B dosage and duration), Outcome data including serum homocysteine, folate, and vitamin B12 levels; and clinical outcomes (e.g., changes in eGFR and albuminuria progression).\u003c/p\u003e\u003cp\u003eFor continuous outcomes, we extracted means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviations (SD) or mean changes\u0026thinsp;\u0026plusmn;\u0026thinsp;SD.\u003c/p\u003e\u003cp\u003eIf outcomes were reported as medians with ranges or interquartile ranges (IQRs), we converted them to mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD using the Meta-Converter tool (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). To calculate SD of change scores in pre-post studies, correlation coefficients (r) of 0.60 for homocysteine and 0.90 for creatinine were assumed based on prior literature (\u003cb\u003eSupplementary Appendix 1, Table S00\u003c/b\u003e). All data were extracted into Microsoft Excel and independently cross-checked by a second reviewer.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Meta-analysis\u003c/h2\u003e\u003cp\u003eIn total, meta-analyses were conducted using random-effects models (DerSimonian-Laird) and heterogeneity was assessed using the I\u0026sup2; statistic, with values\u0026thinsp;\u0026gt;\u0026thinsp;50% indicating moderate-to-high heterogeneity. All analyses were performed using STATA 17, with statistical significance set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003cp\u003eIn Part 1, pooled mean differences (MD) in serum homocysteine levels between DN and DM groups were calculated using a random-effects model. Additionally, we performed separate meta-analyses based on the study design and reported effect measures; Odds ratios (ORs) from cross-sectional studies were pooled independently to assess the association between elevated homocysteine levels and DN. Hazard ratios (HRs) and incidence rate ratios (IRRs) from prospective cohort studies were combined in a single meta-analysis to evaluate homocysteine as a temporal risk factor for DN onset or progression. The logarithmic values and standard errors of the effect estimates were used for pooling. Effect sizes were reported with 95% confidence intervals (CIs). In Part 2, MDs were used to compare folate and vitamin B12 levels. For RCTs, mean changes\u0026thinsp;\u0026plusmn;\u0026thinsp;SD in clinical outcomes between intervention and control groups were pooled.\u003c/p\u003e\u003cp\u003eIn the next step, subgroup analyses for serum homocysteine levels were conducted to explore potential sources of heterogeneity across the included studies. Stratification was performed based on key study-level characteristics, including: Proportion of female participants (\u0026ge;\u0026thinsp;50% vs. \u0026lt;50%), Publication year (before vs. after median year), Geographic region (by continent), Reported central tendency (mean vs. median values), Mean participant age (\u0026lt;\u0026thinsp;58 vs. \u0026ge;58 years), Diagnostic criteria for diabetic nephropathy (eGFR\u0026thinsp;+\u0026thinsp;albuminuria vs. eGFR only vs. albuminuria only), Sample size (\u0026lt;\u0026thinsp;200 vs. \u0026ge;200 participants). In addition, subgroup analyses were pre-specified to baseline renal function (eGFR\u0026thinsp;\u0026lt;\u0026thinsp;60 vs. \u0026ge;60 mL/min/1.73m\u0026sup2;) and Albuminuria category (normoalbuminuria, microalbuminuria, macroalbuminuria) to examine the Hcy-DN relationship across different renal stages, addressing whether the association is independent of glomerular filtration rate or albuminuria.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eRisk of Bias Assessment\u003c/h3\u003e\n\u003cp\u003eQuality control of observational studies were assessed using the Newcastle-Ottawa Scale (NOS). RCTs were evaluated using the Cochrane Risk of Bias (RoB 2) tool.\u003c/p\u003e\u003cp\u003eSensitivity analyses were performed by excluding studies with high risk of bias to test the robustness of findings. Sensitivity analyses were performed using a leave-one-out approach, applying fixed-effect models, to evaluate the robustness of the pooled estimates.\u003c/p\u003e\u003cp\u003ePublication bias was calculated using the Funnel plot and Egger's test (linear regression method), and a significant statistical publication bias was set at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 for Egger's test. Finally, to evaluate the impact of the missing research in the presence of publication bias, the trim and fill approach was employed (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e).\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eStudy Selection and Characteristics\u003c/h2\u003e\u003cp\u003eA total of 45 studies were included in this meta-analysis. 29 studies examined the relationship between homocysteine levels and diabetic nephropathy (DN), including 25 cross-sectional studies assessing mean differences and 8 studies reporting effect sizes (ORs, HRs and IRRs). Sixteen studies evaluated serum levels of folate and/or vitamin B12, or the effects of B-complex supplementation in DN patients. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e illustrates the PRISMA flow diagram. The baseline characteristics of included studies are summarized in \u003cb\u003eTables S1\u0026ndash;S5\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eSerum Homocysteine Levels in DN vs. DM Patients\u003c/h2\u003e\u003cp\u003eAcross 25 cross-sectional studies (3,707 patients with DM and 3,179 with DN) (\u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e) serum homocysteine levels were significantly higher in DN patients compared to DM controls. The pooled MD was +\u0026thinsp;2.81 \u0026micro;mol/L (95% CI: 1.95 to 3.66; \u003cem\u003eI\u0026sup2; =\u003c/em\u003e 91.9%) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. No publication bias was observed (Egger\u0026rsquo;s p\u0026thinsp;=\u0026thinsp;0.52), and funnel plots are available in Supplementary \u003cb\u003eFigures S1.\u003c/b\u003e The sensitivity analysis indicated that the final estimated risk is not affected by a single study \u003cb\u003e(Figures \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eSubgroup analyses were performed according to sex, publication year, geographic region, central tendency index, age, sample size, HbA1c, eGFR status, and DN diagnostic criteria \u003cb\u003e(Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e)\u003c/b\u003e. Elevated Hcy levels were observed across all strata. The effect size was slightly higher in studies with \u0026lt;\u0026thinsp;50% female participants (MD\u0026thinsp;=\u0026thinsp;+\u0026thinsp;3.23 \u0026micro;mol/L) compared to \u0026ge;\u0026thinsp;50% female (MD\u0026thinsp;=\u0026thinsp;+\u0026thinsp;2.52 \u0026micro;mol/L). Studies published after 2010 reported slightly greater mean differences (MD\u0026thinsp;=\u0026thinsp;+\u0026thinsp;3.14 \u0026micro;mol/L) than earlier studies (MD\u0026thinsp;=\u0026thinsp;+\u0026thinsp;2.28 \u0026micro;mol/L). The effect was more pronounced in Asian populations (MD\u0026thinsp;=\u0026thinsp;+\u0026thinsp;3.16 \u0026micro;mol/L) compared to other regions (MD\u0026thinsp;=\u0026thinsp;+\u0026thinsp;2.81 \u0026micro;mol/L). Studies reporting mean values yielded larger effects (MD\u0026thinsp;=\u0026thinsp;+\u0026thinsp;3.24 \u0026micro;mol/L) than those reporting medians (MD\u0026thinsp;=\u0026thinsp;+\u0026thinsp;1.21 \u0026micro;mol/L, non-significant). Younger cohorts (\u0026lt;\u0026thinsp;58 years) showed greater differences (MD\u0026thinsp;=\u0026thinsp;+\u0026thinsp;3.48 \u0026micro;mol/L) than older cohorts (MD\u0026thinsp;=\u0026thinsp;+\u0026thinsp;2.14 \u0026micro;mol/L). The association persisted across HbA1c strata, though slightly stronger in those with HbA1c\u0026thinsp;\u0026lt;\u0026thinsp;8.5% (MD\u0026thinsp;=\u0026thinsp;+\u0026thinsp;3.83 \u0026micro;mol/L). Markedly higher effects were observed when DN was defined by combined eGFR and albuminuria criteria (MD\u0026thinsp;=\u0026thinsp;+\u0026thinsp;4.13 \u0026micro;mol/L) compared to albuminuria alone (MD\u0026thinsp;=\u0026thinsp;+\u0026thinsp;2.44 \u0026micro;mol/L). Despite stratification, high heterogeneity (I\u0026sup2; \u0026gt;80%) remained in most subgroups, indicating that unmeasured factors may contribute to variability.\u003c/p\u003e\u003cp\u003eHomocysteine levels rose progressively across DN stages \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e: Microalbuminuria vs. normoalbuminuria: +1.32 \u0026micro;mol/L (95% CI: 0.43 to 2.21), Macroalbuminuria vs. microalbuminuria: +2.38 \u0026micro;mol/L (1.28 to 3.47), Macroalbuminuria vs. normoalbuminuria: +3.58 \u0026micro;mol/L (2.10 to 5.05).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eAssociation of Homocysteine with DN Risk\u003c/h2\u003e\u003cp\u003eSix cross-sectional studies (n\u0026thinsp;=\u0026thinsp;4,918) reporting odds ratios (ORs) were pooled (\u003cb\u003eTable \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e\u003c/b\u003e). The meta-analysis yielded a pooled OR of 1.22 (95% CI: 1.13\u0026ndash;1.32), indicating that elevated homocysteine levels are associated with 22% increased odds of having DN (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). This association was statistically significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) with low publication bias (Egger\u0026rsquo;s p\u0026thinsp;=\u0026thinsp;0.27) (\u003cb\u003eFigures \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/b\u003e Sensitivity analysis confirmed the robustness of the results \u003cb\u003e(Figures \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003eTwo cohort studies (n\u0026thinsp;=\u0026thinsp;791) reported hazard ratios (HR) and incidence rate ratios (IRR) (\u003cb\u003eTable \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e\u003c/b\u003e). The pooled effect size was 1.52 (95% CI: 0.98\u0026ndash;2.34), suggesting a non-significant trend toward increased DN risk with higher homocysteine (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eFolate Levels in DN vs. DM Patients\u003c/h2\u003e\u003cp\u003eEight studies (DN: n\u0026thinsp;=\u0026thinsp;1,051; DM: n\u0026thinsp;=\u0026thinsp;1,596) reported folate concentrations (\u003cb\u003eTable S4\u003c/b\u003e). DN patients had significantly lower folate levels by \u0026minus;\u0026thinsp;2.09 nmol/L (95% CI: \u0026minus;\u0026thinsp;3.80 to \u0026minus;\u0026thinsp;0.38; \u003cem\u003eI\u0026sup2; = 69.8%\u003c/em\u003e) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. Egger\u0026rsquo;s test indicated publication bias (\u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.0001\u003c/em\u003e); trim-and-fill analysis imputed one missing study, which did not significantly alter the results \u003cb\u003e(Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eVitamin B12 Levels in DN vs. DM Patients\u003c/h2\u003e\u003cp\u003eEight cross-sectional studies reported serum vitamin B12 levels in DN (n\u0026thinsp;=\u0026thinsp;1004) versus DM (n\u0026thinsp;=\u0026thinsp;1590) patients (\u003cb\u003eTable S4\u003c/b\u003e). The pooled MD was \u0026minus;\u0026thinsp;27.36 pg/mL (95% CI: \u0026minus;\u0026thinsp;62.16 to 7.45), indicating a non-significant trend toward lower B12 in DN compared to DM patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). No publication bias detected (Egger\u0026rsquo;s p\u0026thinsp;=\u0026thinsp;0.196) and Sensitivity analysis confirmed the robustness of the results \u003cb\u003e(Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e-2).\u003c/b\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eEffects of B-Vitamin Supplementation on Homocysteine and Renal Outcomes\u003c/h2\u003e\u003cp\u003eTen RCTs were included, with a total of n\u0026thinsp;=\u0026thinsp;2,005 participants (\u003cb\u003eTable S5\u003c/b\u003e). In 4 trials, the overall effect of B-vitamin supplementation (folate/B-complex) was a pooled reduction in homocysteine of \u0026minus;\u0026thinsp;5.30 \u0026micro;mol/L (95% CI: \u0026minus;\u0026thinsp;7.80 to \u0026minus;\u0026thinsp;2.80) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e. Among them, folic acid trials (n\u0026thinsp;=\u0026thinsp;2 RCTs; n\u0026thinsp;=\u0026thinsp;1,687) showed a significant reduction: \u0026minus;\u0026thinsp;3.35 \u0026micro;mol/L (95%CI: \u0026minus;\u0026thinsp;5.38 to\u0026ndash;1.32) compared to placebo. However, these trials did not evaluate renal endpoints such as GFR or creatinine.\u003c/p\u003e\u003cp\u003eTwo RCTs (n\u0026thinsp;=\u0026thinsp;493) on vitamin B6 in three trials showed no effect on serum creatinine: MD = \u0026minus;\u0026thinsp;0.01 mg/dL (95% CI: \u0026minus;\u0026thinsp;0.10 to +\u0026thinsp;0.08) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e. But in this group, the relationship between vitamin B and Hcy has not been investigated.\u003c/p\u003e\u003cp\u003eTwo RCTs (n\u0026thinsp;=\u0026thinsp;318) on B-complex vitamins measured eGFR change and found no significant benefit: MD in eGFR = \u0026minus;\u0026thinsp;1.91 mL/min/1.73m\u0026sup2; (95% CI: \u0026minus;\u0026thinsp;6.41 to +\u0026thinsp;2.58\u003cb\u003e) (\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis meta-analysis provides comprehensive evidence that serum Hcy levels are significantly elevated in patients with DN compared to diabetic individuals without nephropathy. Moreover, he identification of a clear dose-response relationship between albuminuria severity and homocysteine levels represents a pivotal finding of our analysis. This graded association strongly suggests that homocysteine accumulation parallels diabetic nephropathy progression, providing compelling evidence for its role as a biomarker of renal injury severity. These findings are consistent with recent reports demonstrating elevated homocysteine in DN patients and its association with microvascular damage, glomerular dysfunction, and poor renal outcomes in diabetes (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). From a pathophysiological perspective, Hcy promotes oxidative stress, endothelial dysfunction, and pro-fibrotic signaling, particularly through the activation of transforming growth factor-β (TGF-β), which are all key contributors to renal injury in diabetes (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Additionally, homocysteine interferes with methylation reactions and redox balance, impairing both vascular integrity and glomerular filtration. However, despite this plausible biological rationale, the causal link between homocysteine and DN remains debated.\u003c/p\u003e\u003cp\u003eIn our meta-analysis, cohort studies did not show a statistically significant association between baseline homocysteine levels and incident DN. This may reflect limited data, short follow-up duration, or confounding by reduced glomerular filtration rate (GFR) itself; a known determinant of homocysteine accumulation. This view is supported by Mendelian randomization studies, which suggest that the relationship between homocysteine and CKD is likely mediated by reduced renal clearance rather than a primary driver of nephropathy (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eGenetic susceptibility may further explain interindividual variation. A meta-analysis by Zhou et al. (2015) reported that the MTHFR C677T polymorphism, which impairs folate metabolism and elevates homocysteine, is associated with DN risk (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Similarly, Huo et al. (2022) highlighted an association with the MTHFR A1298C polymorphism (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). These findings suggest that prolonged exposure to hyperhomocysteinemia, especially in genetically susceptible individuals, could contribute to DN pathogenesis.\u003c/p\u003e\u003cp\u003eRegarding micronutrient status, our analysis showed significantly lower serum folate levels in DN patients, while vitamin B12 levels were not significantly different. These results align with previous studies reporting folate depletion in DN due to increased oxidative loss or reduced absorption, whereas B12 levels may be influenced by factors such as metformin use, dietary intake, or assay variability (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eOur findings also indicate that B-vitamin supplementation was shown to reduce homocysteine levels substantially. However, this biochemical improvement did not translate into renal protection. For instance, the landmark RCT by House et al. (2010) reported that high-dose folic acid, B6, and B12 led to a greater decline in GFR and increased vascular events in DN patients (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Similarly, Schneider et al. (2014) observed no improvement in proteinuria after 4 weeks of high-dose folate therapy (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). These observations reinforce the idea that biomarker modulation does not necessarily lead to clinical benefit.\u003c/p\u003e\u003cp\u003eRecent meta-analyses such as Mokgalaboni et al. (2024) confirmed the ability of folic acid to reduce homocysteine and inflammatory markers in patients with type 2 diabetes. Our meta-analysis fills this gap by evaluating clinical and biochemical endpoints in parallel, providing a more comprehensive picture of the utility and limitations of B-vitamin therapy in DN (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Our findings are in line with the Cochrane review by Raval et al. (2015), which concluded that while B-vitamin supplementation lowers Hcy levels, it does not significantly improve renal function. Our study extends these findings by including evidence from both observational and cross-sectional evidence on B9/B12 status (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Importantly, the possibility of adverse effects from high-dose B-vitamin supplementation such as accumulation of unmetabolized folic acid or unbalanced methylation cannot be ignored (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Therefore, routine supplementation in DN patients should be approached with caution and individualized based on genetic, biochemical, and clinical profiles.\u003c/p\u003e\u003cp\u003eIn other meta-analyses similar to ours, Zhu et al. also conducted a meta-analysis limited to patients with type 2 diabetes, which reported a significant association between elevated homocysteine and diabetic nephropathy risk. Our findings are broadly consistent, but our analysis included both type 1 and type 2 diabetes, offering broader generalizability across diabetic populations (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eSeveral limitations should be acknowledged. First, substantial heterogeneity was observed across the included studies, potentially due to differences in patient demographics, assay methods, DN diagnostic criteria, and regional dietary factors. Although subgroup analyses were performed to explore these sources of variability, residual confounding may persist. Second, most studies were observational and cross-sectional in design, limiting causal inference. Third, only a few randomized controlled trials (RCTs) reported hard clinical outcomes, and most were of short duration with varied dosing strategies, reducing the generalizability of their findings. Additionally, evidence from Mendelian randomization and longitudinal data supports the notion that elevated Hcy levels may largely reflect declining renal function, rather than directly causing DN.\u003c/p\u003e\u003cp\u003eOur findings resolve a long-standing paradox in diabetic nephropathy management: while homocysteine demonstrates strong, graded associations with DN severity consistent with a potential pathogenic role, therapeutic lowering of Hcy with B-vitamins fails to translate into renal protection. This discrepancy suggests several mechanistic interpretations: (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) Hcy may primarily be a marker rather than mediator of renal damage; (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) the timing of intervention may be critical\u0026mdash;once DN is established, Hcy-lowering may be too late; or (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) Hcy might interact with other uremic toxins in established CKD, limiting the efficacy of isolated B-vitamin supplementation. Thus, further high-quality, long-term, and genetically stratified RCTs are needed to elucidate whether targeting homocysteine confers renal benefit in specific patient populations.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis meta-analysis provides the most comprehensive evidence to date that while homocysteine serves as an excellent biomarker for DN risk stratification, therapeutic targeting of Hcy with B-vitamins is ineffective for renal protection. Clinicians should utilize Hcy measurements for prognostic information but not as a therapeutic target. Future research should focus on earlier intervention in prediabetic or normoalbuminuric stages, and consider combinatorial approaches addressing the broader metabolic milieu of DN.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eACR\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ealbumin-to-creatinine ratio\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCI\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003econfidence interval\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCKD\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003echronic kidney disease\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eDM\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ediabetes mellitus\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eDN\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ediabetic nephropathy\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eESRD\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eend-stage renal disease\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eeGFR\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eEstimated glomerular filtration rate\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eHR\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ehazard ratio\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eHHcy\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ehyperhomocysteinemia\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eIRR\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eincidence rate ratio\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eI\u0026sup2;\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eI-squared statistic\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eMD\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003emean difference\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eNOS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eNewcastle-Ottawa Scale\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eOR\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eodds ratio\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePLP\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003epyridoxal-5\u0026prime;-phosphate, RCT:randomized controlled trial\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eRoB 2\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eRisk of Bias 2 tool\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eSD\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003estandard deviation\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eUAE\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eurinary albumin excretion\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCompeting interests\u003c/h2\u003e\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThe authors received no funds for this study.\u003c/p\u003e\u003ch2\u003eAuthor contributions\u003c/h2\u003e\u003cp\u003eHB and AG participated in the design, data searching and collection, analysis, data interpretation, and drafting of the manuscript. SS participated in the data interpretation, and drafting of the manuscript. All the authors have reviewed the final version of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWani ZA, Ahmed S, Saleh A, Anna VR, Fahelelbom KM, Raju SK, et al. Biomarkers in diabetic nephropathy: A comprehensive review of their role in early detection and disease progression monitoring. Diabetes Res Clin Pract. 2025;226:112292.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGholaminejad A, Moein S, Roointan A, Mortazavi M, Nouri R, Mansourian M et al. Circulating β2 and α1 microglobulins predict progression of nephropathy in diabetic patients: a meta-analysis of prospective cohort studies. Acta Diabetol. 2022.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRoointan A, Shafieizadegan S, Ghaeidamini M, Gheisari Y, Hudkins KL, Gholaminejad A. The Potential of Cardiac Biomarkers, NT-ProBNP and Troponin T, in Predicting the Progression of Nephropathy in Diabetic Patients: A Meta-Analysis of Prospective Cohort Studies. Diabetes Res Clin Pract. 2023:110900.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRoointan A, Gheisari Y, Hudkins KL, Gholaminejad A. Non-invasive metabolic biomarkers for early diagnosis of diabetic nephropathy: Meta-analysis of profiling metabolomics studies. Nutr Metabolism Cardiovasc Dis. 2021;31(8):2253\u0026ndash;72.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePark S, Lee S, Kim Y, Cho S, Kim K, Kim YC, et al. Causal Effects of Homocysteine, Folate, and Cobalamin on Kidney Function: A Mendelian Randomization Study. Nutrients. 2021;13(3):906.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWu HHL, McDonnell T, Chinnadurai R. Physiological Associations between Vitamin B Deficiency and Diabetic Kidney Disease. Biomedicines. 2023;11(4):1153.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEsse R, Barroso M, Tavares de Almeida I, Castro R. The Contribution of Homocysteine Metabolism Disruption to Endothelial Dysfunction: State-of-the-Art. Int J Mol Sci. 2019;20(4):867.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChermiti R, Burtey S, Dou L. Role of Uremic Toxins in Vascular Inflammation Associated with Chronic Kidney Disease. J Clin Med. 2024;13(23):7149.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYi F, Li P-L. Mechanisms of Homocysteine-Induced Glomerular Injury and Sclerosis. Am J Nephrol. 2007;28(2):254\u0026ndash;64.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFRIEDMAN AN, BOSTOM AG, SELHUB J, LEVEY AS, ROSENBERG IH. The Kidney and Homocysteine Metabolism. J Am Soc Nephrol. 2001;12(10):2181\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi H, Liu C, Zhang J, Wang W, Cheng W, Yang R, et al. The association of homocysteine level with the risk of diabetic nephropathy and diabetic retinopathy in NHANES. Acta Diabetol. 2023;60(7):907\u0026ndash;16.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang B, Li H, Wang N, Li Y, Song Z, Chen Y, et al. The impact of homocysteine on patients with diabetic nephropathy: a mendelian randomization study. Acta Diabetol. 2025;62(1):123\u0026ndash;30.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHovind P, Tarnow L, Rossing P, Teerlink T, Stehouwer CD, Emeis JJ, et al. Progression of diabetic nephropathy: role of plasma homocysteine and plasminogen activator inhibitor-1. Am J Kidney Dis. 2001;38(6):1376\u0026ndash;80.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCappuccilli M, Bergamini C, Giacomelli FA, Cianciolo G, Donati G, Conte D, et al. Vitamin B Supplementation and Nutritional Intake of Methyl Donors in Patients with Chronic Kidney Disease: A Critical Review of the Impact on Epigenetic Machinery. Nutrients. 2020;12(5):1234.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFriedman AN, Bostom AG, Selhub J, Levey AS, Rosenberg IH. The Kidney and Homocysteine Metabolism. J Am Soc Nephrol. 2001;12(10).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRighetti M, Serbelloni P, Milani S, Ferrario G. Homocysteine-Lowering Vitamin B Treatment Decreases Cardiovascular Events in Hemodialysis Patients. Blood Purif. 2006;24(4):379\u0026ndash;86.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRaval AD, Thakker D, Rangoonwala AN, Gor D, Walia R. Vitamin B and its derivatives for diabetic kidney disease. Cochrane Database Syst Rev. 2015;1(1):Cd009403.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHouse AA, Eliasziw M, Cattran DC, Churchill DN, Oliver MJ, Fine A, et al. Effect of B-vitamin therapy on progression of diabetic nephropathy: a randomized controlled trial. JAMA. 2010;303(16):1603\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMokgalaboni K, Mashaba GR, Phoswa WN, Lebelo SL. Folic acid supplementation on inflammation and homocysteine in type 2 diabetes mellitus: systematic review and meta-analysis of randomized controlled trials. Nutr Diabetes. 2024;14(1):22.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhou X, Shi A, Zhou X. A meta-analysis of serum Hcy in diagnosis of diabetic nephropathy. Pteridines. 2020;31(1):1\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhu M, Fan Y. Association of Circulating Homocysteine Level with the Risk of Nephropathy in Type 2 Diabetes Mellitus: A Meta-Analysis. Horm Metab Res. 2025;57(02):117\u0026ndash;23.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAbbas A, Hefnawy MT, Negida A. Meta-analysis accelerator: a comprehensive tool for statistical data conversion in systematic reviews with meta-analysis. BMC Med Res Methodol. 2024;24(1):243.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDuval S, Tweedie R. Trim and fill: a simple funnel-plot\u0026ndash;based method of testing and adjusting for publication bias in meta‐analysis. Biometrics. 2000;56(2):455\u0026ndash;63.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZheng X, Liu Q, Liu Z. Serum homocysteine concentration as a marker for advanced diabetic nephropathy in a cohort of elderly patients. BMC Endocr Disorders. 2023;23(1):114.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYe B, Zhu X, Zeng Z, Ji X, Ji M. Clinical significance of serum homocysteine as a biomarker for early diagnosis of diabetic nephropathy in type 2 diabetes mellitus patients. Pteridines. 2021;32(1):11\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSchleicher E, Didangelos T, Kotzakioulafi E, Cegan A, Peter A, Kantartzis K. Clinical Pathobiochemistry of Vitamin B12 Deficiency: Improving Our Understanding by Exploring Novel Mechanisms with a Focus on Diabetic Neuropathy. Nutrients. 2023;15(11):2597.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePark S, Lee S, Kim Y, Cho S, Kim K, Kim YC, et al. Causal effects of homocysteine, folate, and cobalamin on kidney function: a Mendelian randomization study. Nutrients. 2021;13(3):906.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhou T-B, Drummen GP, Jiang Z-P, Li H-Y. Methylenetetrahydrofolate reductase (MTHFR) C677T gene polymorphism and diabetic nephropathy susceptibility in patients with type 2 diabetes mellitus. Ren Fail. 2015;37(8):1247\u0026ndash;59.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHuo Y, Zhang W, Zhang X, Su L, Li H, Wang F, et al. The Association of Methylenetetrahydrofolate Reductase (MTHFR) A1298C Gene Polymorphism with Susceptibility to Diabetic Nephropathy: A Meta-Analysis. Horm Metab Res. 2022;54(12):845\u0026ndash;51.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSchneider MP, Schneider A, Jumar A, Kistner I, Ott C, Schmieder RE. Effects of folic acid on renal endothelial function in patients with diabetic nephropathy: results from a randomized trial. Clin Sci. 2014;127(7):499\u0026ndash;505.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBailey SW, Ayling JE. The extremely slow and variable activity of dihydrofolate reductase in human liver and its implications for high folic acid intake. Proceedings of the National Academy of Sciences. 2009;106(36):15424\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eObeid R, Kasoha M, Kirsch SH, Munz W, Herrmann W. Concentrations of unmetabolized folic acid and primary folate forms in pregnant women at delivery and in umbilical cord blood. Am J Clin Nutr. 2010;92(6):1416\u0026ndash;22.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Diabetic nephropathy, Homocysteine, Folic acid, Vitamin B12, Vitamin B supplementation, Meta-analysis, Biomarkers, Chronic kidney disease, Type 2 diabetes","lastPublishedDoi":"10.21203/rs.3.rs-7979615/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7979615/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eThe role of homocysteine (Hcy) in diabetic nephropathy (DN) remains paradoxical: while epidemiological studies consistently associate hyperhomocysteinemia with DN risk, interventional trials with B-vitamins have yielded conflicting results. This meta-analysis comprehensively integrates observational and interventional evidence to resolve this discrepancy and clarify the clinical utility of Hcy monitoring and B-vitamin therapy in DN.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eWe systematically searched PubMed, Scopus, and Web of Science through May 2024 for studies comparing Hcy, folate, and vitamin B12 levels between DN patients and diabetic controls, and randomized controlled trials (RCTs) evaluating B-vitamin supplementation. Random-effects models were used for meta-analysis, with subgroup analyses to explore heterogeneity.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eAcross 45 studies (29 observational, 16 interventional), DN patients exhibited significantly higher Hcy levels (+\u0026thinsp;2.81 \u0026micro;mol/L; 95% CI: 1.95\u0026ndash;3.66) and lower folate levels (-2.09 nmol/L; 95% CI: -3.80 to -0.38) compared to diabetic controls without nephropathy. A clear dose-response relationship emerged: Hcy levels increased progressively from normoalbuminuria to microalbuminuria (+\u0026thinsp;1.32 \u0026micro;mol/L) to macroalbuminuria (+\u0026thinsp;3.58 \u0026micro;mol/L). Elevated Hcy was associated with 22% increased odds of DN (OR: 1.22; 95% CI: 1.13\u0026ndash;1.32). Crucially, while B-vitamin supplementation effectively reduced Hcy levels (-3.35 \u0026micro;mol/L with folic acid), it conferred no renal benefit\u0026mdash;showing no significant effects on eGFR (-1.91 mL/min/1.73m\u0026sup2;) or creatinine levels.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eThis study establishes Hcy as a robust biomarker of DN severity but demonstrates that B-vitamin supplementation, despite effectively lowering Hcy, does not improve renal outcomes. These findings resolve the Hcy paradox in DN, suggesting that hyperhomocysteinemia may be a consequence rather than cause of renal impairment, and argue against routine B-vitamin supplementation for renal protection in DN patients. Further research is needed to explore potential benefits in specific patient subgroups.\u003c/p\u003e\u003ch2\u003eSystematic review registration:\u003c/h2\u003e\u003cp\u003ePROSPERO, CRD420251069868\u003c/p\u003e","manuscriptTitle":"The Homocysteine Paradox in Diabetic Nephropathy: A Comprehensive Meta-Analysis Reveals Strong Biomarker Association but No Therapeutic Benefit from B-Vitamin Supplementation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-26 07:05:00","doi":"10.21203/rs.3.rs-7979615/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"17b77564-de52-4deb-b0d1-fe47c75aa4b7","owner":[],"postedDate":"November 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-11T00:15:48+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-26 07:05:00","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7979615","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7979615","identity":"rs-7979615","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}