Insulin Resistant States Contributed Differentially to the Earlier Renal tubular Injury in Chinese Young Overweight Individuals with Hyperglucagonemia

Insulin Resistant States Contributed Differentially to the Earlier Renal tubular Injury in Chinese Young Overweight Individuals with Hyperglucagonemia | 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 Insulin Resistant States Contributed Differentially to the Earlier Renal tubular Injury in Chinese Young Overweight Individuals with Hyperglucagonemia Song Wen, Yanhong Huang, Lijiao Chen, Wu Wang, Liang Zeng, ChunXiang Fan, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7576426/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 6 You are reading this latest preprint version Abstract Background Hyperglucagonemia is associated to the metabolic dysregulation, but its early role in microvascular injury, especially with young and overweight state, remains unclear. Thus, this study investigates the association between hyperglucagonemia and renal tubular injury in Chinese young overweight with dysglycemic state, and compared the profile with Chinese aged type 2 diabetes (T2DM Aged). Methods The assessment was conducted on metabolic markers and renal tubular injury indicators. At 0, 30, and 120 minutes, the parameters of the oral glucose tolerance test (OGTT) were assessed. Analysis of relationships and predictors of renal impairment was conducted. Results Among young overweight patients, glucagon (GCG) levels were elevated in diabetic patients at both fasting and 30 minutes, with higher HOMA-IR and comparable HOMA-β. Elevated blood α1- and β2-MG were observed in newly diagnosed type 2 diabetic patients with hyperglucagonemia. However, when analyzed with T2DM aged (64.54 ± 10.91yrs), they exhibited elevated 30- and 120-minute GCG and reduced HOMA indices. In comparison to younger groups, aged patients exhibited profound elevated levels of α1MG. Correlational relationship analysis showed tubular markers in young patients significantly positively related to male gender, elevated FPG, HbA1c, insulin and GCG levels, as well as HOMA (P < 0.05), while ageing, elevated postprandial glucose, reduced insulin levels, postpone attenuating GCG in 30min and 120min, and decreased HOMA were significantly in T2DM Aged. The binary logistic regression analysis showed the risk of appearance of renal microvascular injury marker, UACR, in young patients was determined by 30-minute GCG (B = 0.105, OR = 1.111, P = 0.004) and increased eGFR (B = 0.050, OR = 1.052, P = 0.029), while it was associated with elevated HbA1c (B = 0.578, OR = 1.782, P = 0.002) and reduced eGFR (B=-0.039, OR = 0.962, P = 0.028) in the T2DM aged. Multilinear regression showed different tubular markers in young groups were differentially positively determined by FPG, insulin and C-peptide levels, 30minGCG and negatively with eGFR; for T2DM aged group, ageing, reduced eGFR and insulin levels, elevated postprandial GCG at 30min and 120min, reduced HOMA indices differentially contributed to the various elevated tubular markers. Conclusions Hyperglucagonemia and insulin resistance may serve as an early renal injury sign in the pathophysiology of overweight. While causes for microvascular and tubular injury were different between young overweight and aged T2DM patients which alert early effective bodyweight and metabolic management. Hyperglucagonemia Renal microvascular impairment Prediabetes Tubular injury Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction The recognition of glucagon (GCG) as a distinct hormone began in the early 20th century, marking a significant milestone in understanding metabolic regulation [ 1 ]. The story starts with the work of researchers exploring the pancreas, initially focused on insulin. In 1922, Banting and Best successfully isolated insulin from pancreatic extracts to treat diabetes, but they observed that crude pancreatic extracts sometimes caused hyperglycemia rather than hypoglycemia [ 2 ]. This paradoxical effect puzzled scientists and hinted at the presence of another factor. The breakthrough came in 1923 when Kimball and Murlin identified a hyperglycemic substance in pancreatic extracts distinct from insulin, naming it "glucagon," from the Greek "glukus" (sweet) and "agon" (leading), reflecting its glucose-elevating properties [ 3 ]. Initial studies suggested its production by pancreatic alpha cells, contrasting with insulin’s beta-cell origin [ 4 ]. However, its exact nature remained elusive due to limited purification techniques and the focus on insulin research at the time. Significant progress occurred in the 1950s when Staub, Behrens, and colleagues purified glucagon from porcine pancreas, confirming it as a 29-amino-acid peptide [ 5 ]. This enabled detailed physiological studies, revealing its role in counteracting insulin by stimulating hepatic gluconeogenesis and glycogenolysis [ 6 ]. By the 1960s and 1970s, radioimmunoassays and advanced biochemical techniques clarified its secretion patterns and receptor interactions, solidifying its status as a critical hormone in glucose homeostasis [ 7 ]. Hyperglucagonemia, characterized by elevated GCG levels, has since been linked to metabolic dysregulation in type 2 diabetes (T2DM) and prediabetes contributing to hyperglycemia [ 8 ]. Recent research has expanded its implications, suggesting a potential role in renal microvascular impairment, particularly in early disease stages, where the kidneys, especially renal tubules, are vulnerable to metabolic stress [ 9 ]. This is particularly relevant in young prediabetic (PreDM) individuals, where metabolic abnormalities may precede overt diabetes, and in aged patients with type 2 diabetes (T2DM Aged), where chronic disease processes dominate. Despite its metabolic significance, the specific contribution of hyperglucagonemia to renal injury across age groups remains underexplored. Young overweight individuals with prediabetes represent a critical population for early intervention to prevent chronic kidney disease (CKD), while aged T2DM patients often exhibit advanced renal complications, where glucagon’s role may differ due to age-related changes. Studies suggest that glucagon receptor (GCGR) activation in renal tubular cells may induce oxidative stress and inflammation, while chronic exposure could promote fibrosis [ 10 ], yet age-specific mechanisms are not well-defined. This study aims to investigate the association between hyperglucagonemia and renal tubular and microvascular injury in young individuals with PreDM, DM, and euglycemia in comparison to aged patients with T2DM. By analyzing metabolic markers, tubular and microvascular injury indicators, we seek to elucidate the role of GCG in renal microvascular impairment and identify age-tailored risk factors, providing insights for targeted preventive strategies. Understanding the mechanisms of early renal injury is a critical issue in light of the increasing global prevalence of obesity and diabetes. Methods and Materials Participant Recruitment and Obesity Diagnosis A total of 108 Chinese young overweight patients (age: 35.34 ± 8.66 yrs, BMI: 29.997 ± 5.32kg/m 2 ) were selected, categorized into normal glucose (Euglycemia, n = 46), prediabetes (PreDM, n = 45), and newly diagnosed diabetes (T2DM, n = 17) based on oral glucose tolerance test (OGTT) results, and compared with 131 long-standing aged type 2 diabetes patients (T2DM Aged; age: 64.54 ± 10.91yrs). All young participants with overweight had no prior renal replacement therapy and history for diabetes, and had a pre-contact on preparing for OGTT the day before consulting the clinic. The T2DM aged patients with completed OGTT results were recruited from the In-Patient history information system of Shanghai Pudong Hospital. This research is a component of our ongoing chronic disease weight management initiative, which is focused on overweight patients and was conducted from October 2024 to August 2025. Participants were selected from a cohort of overweight individuals who were diagnosed based on a body mass index (BMI) of ≥ 24 kg/m², in accordance with the most recent Chinese National Health Commission guideline, which were published in October 2024. This guideline suggests that the BMI thresholds for overweight be 24-27.9 kg/m² and for obesity be ≥ 28 kg/m². These thresholds are intended to reconcile the Asian population's higher percentage of visceral adipose tissue at lower BMIs than other populations. Secondary endocrine diseases, including adrenal gland tumor, pituitary tumor, hypothyroidism, and Cushing's syndrome, were excluded through a thorough medical history and physical examination after the diagnosis was confirmed. Imaging and laboratory investigations (e.g., thyroid function, cortisol levels) were implemented to the extent necessary for this purpose. The investigation highlighted primary obesity that was associated with metabolic and lifestyle factors, and patients with secondary obesity were excluded. Multidisciplinary Team (MDT) Evaluation The multidisciplinary team (MDT), guided by the 2024 Chinese National Health Commission guideline emphasizing clinical nutrition, behavioral intervention, exercise, and psychological support, conducted thorough evaluations. Medical interviews assessed dietary habits, physical activity, and psychological well-being, using tools like the PHQ-9 for depression screening. Comprehensive assessments included exercise physiology evaluations (e.g., VO2 max testing), nutritional analysis (e.g., 24-hour dietary recall), and tailored psychological support, reflecting the guidelines’ call for a balanced diet, physical activity, and family involvement. Measurement of Metabolic and Renal Markers Metabolic markers, including fasting and postprandial (0, 30, and 120 minutes) glucose, C-peptide, insulin, and glucagon (GCG), were measured using standardized enzymatic and immunoassays. Renal tubular injury indicators, such as blood retinol-binding protein (RBP), urinary N-acetyl-β-D-glucosaminidase (U-NAG), and α1-microglobulin (α1MG), were assessed via urine and serum samples collected during OGTT. Estimated glomerular filtration rate (eGFR) was calculated using the CKD-EPI equation. Oral Glucose Tolerance Test (OGTT) Procedure OGTT involved a 75g glucose load administered after an overnight fast, with blood samples drawn at 0, 30, and 120 minutes to assess dynamic metabolic responses, aligning with the NHC’s emphasis on monitoring glucose metabolism. Statistical Analysis Statistical analyses were conducted using Prism (GraphPad, version 10.0) and SPSS (IBM, version 26.0). Descriptive statistics summarized baseline characteristics, including age, BMI groups, HbA1c levels, reported as mean ± standard error of the mean (SEM) for continuous variables and frequencies/percentages for categorical variables. Chi-square tests assessed associations between categorical variables, such as age groups and gender, BMI groups and gender, and HbA1c groups and gender. Non-parametric Brown-Forsythe and Welch ANOVA tests, followed by post-hoc Dunn’s tests, compared age, BMI, HOMA-IR, UACR, OGTT parameters (glucose, C-peptide, insulin at 0, 30, and 120 minutes), and hormone levels (e.g., cortisol, GH, IGF-1) across the three glucose metabolism states. Spearman correlation analysis evaluated relationships between tubular proteins (α1MG, β2MG, RBP) and metabolic parameters, with significance at P < 0.05. Multilinear regression identified independent predictors of UACR, adjusting for age, gender, and metabolic factors. A significance threshold of P < 0.05 was applied for all analyses. Results 1. The glucose metabolic status of OVERWEIGHT youth as disclosed by OGTTs Newly diagnosed diabetic patients exhibited substantially higher fasting and postprandial glucose levels (at 30 and 120 minutes) than the normal glucose (Euglycemia) and prediabetic (PreDM) groups among young overweight patients (P<0.05). Conversely, PreDM patients exhibited elevated 120-minute postprandial glucose (P<0.05). Both DM and Prediabetic patients exhibited elevated 2-hour postprandial C-peptide and insulin levels in comparison to the normal glucose groups (P<0.05). In comparison to other groups, diabetic patients exhibited substantially elevated GCG levels at fasting and 30 minutes postprandial, while prediabetic and normal glucose groups exhibited comparable GCG concentrations. HOMA-β did not exhibit any significant differences among the three groups; however, the diabetic group exhibited a significant higher HOMA-IR ( Fig. 1 ). 2. The renal tubular injury markers in youth OVERWEIGHT patients were significantly different. Diabetic patients displayed higher blood α1-MG levels compared to normal glucose and prediabetic groups, and elevated β2MG compared to the Euglycemic group, indicating exacerbated tubular injury ( Fig.2 ). 3. The comparison of glucose metabolic status between aged and youth revealed significant C peptide and insulin levels, as well as retarded GCG response after 30min and 120min OGTT. Aged T2DM patients had higher HbA1c, fasting, 30min and 120-minute postprandial glucose levels compared to young Euglycemia and PreDM groups, but significantly reduced C-peptide and insulin levels (Fig.3 A-D). Fasting glucagon (GCG) was lower, while 30-minute and 120min postprandial GCG was higher than other groups. Interestingly, the HOMA-IR and HOMA-β were the lowest among all the groups ( Fig.3 ). 4. The comparison of renal tubular injury markers between youth and aged patients indicated different scale and spectrum of tubular impairment. Compared to young PreDM and Euglycemia, aged T2DM patients showed higher blood α1-microglobulin (α1MG) than Euglycemic groups, and urinary α1MG levels in young euglycemic, PreDM and DM groups; The level of blood β2MG was the highest among the groups; while we only observed increased urinary RBP in aged T2DM groups ( Fig.4 ). 5. The correlational relationships between the tubular injury markers and the state of glucose metabolism. We therefore performed the correlational relationships between tubular injury markers and OGTT parameters in young and aged patients, respectively. In youth, significant positive correlations were observed between tubular markers (e.g., b-α1MG, b-β2MG, b-RBP) and FPG, insulin, C-peptide, and GCG levels (P<0.05), indicating significant insulin resistance. We found gender’s different variation and ages, as well as HbA1c were significantly associated with the tubular markers ( Table.1 ). While in aged T2DM patients, the tubular markers were more frequently associated with the glycemic control, and reduced Insulin and C-peptide levels, as well as reduced HOMA-β and HOMA-IR, suggesting the association with the reduced insulin insufficiency ( Table.2 ). 6. The significant factors associated with the UACR elevation, which is an indicator of microvascular impairment in both youth and aged patients. In young patients, 30-minute glucagon (30min GCG) (OR=1.111, P=0.004), eGFR (OR=1.052, p=0.029) was strong independent risk for elevated urine albumin-to-creatinine ratio (UACR), which suggesting the insulin resistance, and early hyperfiltration of glomerular impairment was associated with the UACR increment; Higher postprandial GCG level was risk factor for elevation of UACR ( Table 3 ). In aged patients, however, HbA1c (OR=1.782, P=0.002 and eGFR(OR=0.962, P=0.028） were significant, indicating the blood glucose control, reduced renal glomerular infiltration function were responsible for elevation of UACR （ Table4 ） . 7. The determinants associated with the renal tubular markers’ alteration in both youth and aged, reflected by the analyses of multilinear regression models. The multilinear regression models provide insights into the determinants influencing alterations in renal tubular injury markers among both young and aged patients. In young patients, the analysis revealed a multifaceted set of predictors across different markers. For blood α1-microglobulin (α1MG), FPCP, estimated glomerular filtration rate (eGFR), and 30-minute glucagon (30min GCG) emerged as significant determinants, suggesting a complex interplay between metabolic control and renal filtration capacity ( Table 5 ). Blood β2-microglobulin (β2MG) was influenced by age, 30min GCG, eGFR and FPG, indicating that younger age, insulin resistance and reduced infiltration ability play critical roles in its elevation. For blood retinol-binding protein (RBP), a broader set of factors including gender, eGFR, FPCP, HOMA-β were identified as key contributors, highlighting the impact of both metabolic and renal function parameters. Urinary α1MG was notably associated with 30-minute postprandial C-peptide (30min PPCP), gender, 120minPPCP, underscoring the relevance of postprandial metabolic responses, and male gender in urinary marker changes. U-β2MG was associated with the HOMA-β and 30min postprandial insulin concentration (30min INS), while u-RBP was linked with 30minGCG, 120minPPCP (Table. 5). In aged patients, the determinants were more focused but equally significant. eGFR was a primary determinant for both α1MG and β2MG, reflecting the dominant role of renal filtration decline with age and disease progression (Table 6) . FPCP and 120min INS were significant indicative markers for elevated renal tubular markers, suggesting the mild reduced clearance circulation level of fasting C-peptide and insulin insufficiency both contributed the observed tubular changes. The levels of RBP were determined by FPCP, gender variation, reduced HOMA-IR, 120min GCG, as well as FPG. The urinary α1MG and NAG were associated with fasting Insulin, 30min GCG, and 30min PPG, respectively. Discussion The findings of this study underscore a significant association between hyperglucagonemia, as reflected by elevated glucagon (GCG) levels, and renal microvascular impairment, particularly in young prediabetic (PreDM) and diabetic (DM) individuals. The elevated postprandial GCG levels in young diabetic patients, coupled with increased tubular injury markers such as blood and urinary tubular markers, suggest that GCG may play a pivotal role in the early pathogenesis of renal damage. This is further supported by regression analyses, where 30-minute GCG emerged as an independent predictor of elevated urine albumin-to-creatinine ratio (UACR) in young patients, indicating a direct link between dysregulated prolonged glucagon exposure and glomerular dysfunction [ 11 ]. The consistency of these findings across multiple markers and time points reinforces the hypothesis that GCG dysregulation is a critical factor in the initiation of renal injury in this population ( Fig. 5 ) . In young overweight patients, the observed metabolic dysregulation—characterized by elevated postprandial glucose, C-peptide, and insulin in PreDM and DM groups—may exacerbate renal tubular injury. The significant correlation between GCG and tubular markers suggests that hyperglucagonemia could contribute to tubular stress through several mechanisms. These include increased gluconeogenesis, which elevates systemic glucose levels and places additional metabolic burden on the kidneys, as well as heightened oxidative stress and inflammation that impair renal tubular integrity [ 12 ]. The broader set of metabolic determinants identified in the multilinear regression models underscores the dynamic nature of renal injury in this population, potentially driven by acute metabolic fluctuations [ 13 ]. This dynamic response may reflect the heightened metabolic sensitivity of young individuals, where rapid changes in GCG levels could trigger early tubular damage before significant glomerular involvement [ 14 ]. In aged patients with type 2 diabetes (T2DM Aged), a distinctly different profile of renal impairment emerges. Despite lower fasting GCG and reduced C-peptide/insulin levels, the elevated 30- and 120-minute GCG and higher HbA1c suggest a chronic hyperglycemic state that may shift the injury pattern toward both glomerular and tubular decline [ 15 ]. The prominence of eGFR as a determinant for α1MG and β2MG, and GCG, indicates that renal filtration capacity and sustained glucagon elevation are key factors in aged renal damage [ 16 ]. This shift may reflect age-related declines in renal reserve and compensatory mechanisms, where increased ratio of postprandial GCG relative to insulin exposure could amplify existing glomerular damage rather than initiate it [ 17 ]. The insulin insufficiency in aged patients further complicates this picture, potentially leading to an unopposed GCG effect that exacerbates renal fibrosis and vascular stiffness over time [ 18 , 19 ]. The differential contribution of GCG to renal injury between young and aged populations may stem from age-specific physiological responses. In young patients, the acute elevation of GCG, particularly postprandially, may act as an early trigger for tubular injury, potentially through glucagon receptor-mediated pathways that increase sodium reabsorption and oxidative stress in the proximal tubule [ 20 – 22 ]. This acute effect is supported by the significant associations with u-α1MG and UACR, suggesting that GCG acts as an early stressor in metabolically active young kidneys [ 23 ]. In aged patients, the chronic elevation of GCG, combined with reduced insulin secretion, may exacerbate pre-existing glomerular sclerosis and tubular atrophy. This chronicity could lead to a more pronounced glomerular injury pattern, as sustained GCG levels may contribute to hypertension and endothelial dysfunction, key drivers of glomerulosclerosis in aging populations [ 24 – 26 ]. Recent research provides mechanistic insights into GCG’s role in chronic kidney disease (CKD) and early renal injury [ 27 , 28 ], offering a framework to interpret these findings. This GCG, traditionally viewed as a counter-regulatory hormone to insulin, contributes to hyperglycemia by stimulating hepatic gluconeogenesis and glycogenolysis [ 29 , 30 ]. In the context of renal health, elevated GCG levels may exacerbate CKD progression by promoting inflammation and fibrosis, as evidenced by increased expression of pro-inflammatory cytokines (e.g., TNF-α, IL-6) and extracellular matrix proteins (e.g., collagen IV) in animal models of diabetic nephropathy [ 31 – 33 ]. It also notes that GCG resistance may develop in advanced CKD due to downregulated glucagon receptor (GCGR) expression, yet persistent hyperglucagonemia still drives systemic inflammation and glomerular hypertension, accelerating glomerulosclerosis [ 34 ]. Specifically, GCG’s role in early renal injury may involve activation of the GCGR in renal tubular cells, supraphysiological concentration leading to increased cyclic AMP (cAMP) signaling [ 35 , 36 ]. This pathway can enhance sodium and water reabsorption, increase tubular workload and generate reactive oxygen species (ROS) that contribute to oxidative stress and cellular damage in the proximal tubule [ 37 ]. Over time, this chronic stress may transition into fibrosis, as GCG-induced inflammation recruits macrophages and activates transforming growth factor-beta (TGF-β) signaling, a well-established pathway in renal scarring [ 38 , 39 ]. In advanced CKD stages, the review suggests that GCG’s effects may shift toward systemic metabolic dysregulation, where reduced GCGR sensitivity in the kidney amplifies its hepatic gluconeogenic impact, further worsening glycemic control and renal perfusion pressure [ 40 ]. In this study, the acute GCG elevation in young patients aligns with its role in early tubular stress, supported by the significant association with α1MG and UACR. The dynamic metabolic environment in young patients, as reflected by multiple OGTT time points, may amplify this effect, making GCG a potential therapeutic target to prevent progression to CKD [ 41 ]. In aged patients, the chronic GCG elevation and its link to urinary tubular markers suggest a transition to glomerular damage, consistent with CKD progression. The significant HOMA index across groups further emphasizes GCG’s independent contribution, beyond insulin resistance, highlighting its dual role as both a metabolic and renal stressor. Recent hotspots, such as glucagon receptor antagonists (e.g., REMD-477) reducing albuminuria in diabetic models, support targeting GCG in young prediabetic individuals to prevent early renal impairment. For aged patients, addressing chronic glomerular protection alongside GCG modulation may be necessary, given the interplay of filtration decline, fibrosis, and sustained glucagon levels [ 42 ]. The multilinear regression models indicate that gender significantly influences renal tubular injury markers in young patients, with males showing higher tubular makers’ levels compared to females. This suggests sex-specific modulation of renal damage [ 43 ]. Hormonal factors, such as estrogen’s reno-protective effects in females (e.g., anti-inflammatory and antioxidant properties) versus testosterone’s potential to promote sodium retention in males, may explain these differences [ 44 ]. Additionally, females’ higher visceral fat in obesity could amplify tubular stress, correlating with HbA1c and β2MG, while males’ muscle mass might buffer damage [ 45 ]. Pubertal hormonal surges may further accentuate these effects in youth. In aged patients, gender’s lack of significance (Table 6 ) suggests that age-related renal decline and chronic hyperglucagonemia overshadow sex-specific influences. Future studies should explore hormonal profiling to tailor interventions for gender-specific renal risks. The comparison of renal injury patterns reveals that young patients exhibit a predominance of tubular damage linked to metabolic dynamism, whereas aged patients show a mixed glomerular-tubular injury pattern driven by chronicity and filtration decline. This age-dependent divergence suggests that GCG’s contribution varies by disease stage and renal reserve, with acute effects dominating in youth and chronic effects prevailing in the aged. Future research should validate these mechanisms through longitudinal studies and explore GCG-targeted therapies tailored to age-specific renal pathology, potentially integrating GCGR antagonists with anti-fibrotic agents to address the full spectrum of GCG-mediated renal injury. Limitations This study has several limitations that warrant consideration. The cross-sectional design precludes establishing causality between hyperglucagonemia and renal tubular injury, necessitating longitudinal studies to confirm temporal relationships. The study’s focus on overweight young patients and aged T2DM may limit generalizability to non-overweight or other age demographics. Conclusion This study provides compelling evidence that hyperglucagonemia may contribute to renal microvascular impairment through tubular dysfunction, particularly in young prediabetic individuals, where 30-min and 120-minute GCG contributed to the early glomerular and tubular injury. The findings highlight GCG as a potential early marker of renal risk in this population, contrasting with aged T2DM patients, where chronic renal filtration decline and sustained GCG elevation dominate. Further longitudinal research is essential to validate these findings and refine therapeutic approaches. Declarations Ethical Approval and consent to participate Human Ethics and Consent to Participate declarations This study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of Shanghai Pudong Hospital (Approval No. AZ-010). The study was a retrospective analysis that strictly adhered to the ethical principles outlined in the Declaration of Helsinki. The study protocol was reviewed and approved by the Ethics Committee of Shanghai Pudong Hospital (Approval No. AZ-010). As a retrospective study, informed consent was waived, and all data were anonymized to protect patient confidentiality. Patient data were anonymized by removing personal identifiers (e.g., name, ID number) and stored securely on an encrypted server accessible only to authorized personnel involved in the study. The Ethics Committee of Shanghai Pudong Hospital waived the requirement for consent to participate (No. AZ-010). Consent to Publication Not applicable. Clinical trial Number: Not applicable. Availability of Data and Materials The data that support the findings of this study are not publicly available due to privacy reasons but are available from the corresponding author upon request. Competing interests The authors declare that there is no conflict of interest. Funding This work was supported by Integrated Traditional Chinese and Western Medicine (YC-2023-0404),Fudan Zhangjiang Clinical Medicine Innovation Fund Project (KP0202118), Fudan Good Practice Program of Teaching and Learning (FD2023A227), Project of Key Medical Discipline of Pudong Hospital of Fudan University (Zdxk2020-11), Project of Key Medical Specialty and Treatment Center of Pudong Hospital of Fudan University (Zdzk2020-24), Pudong New Area Clinical Plateau Discipline Project (PWYgy-2021-03), the Natural Science Foundation of China (21675034), Pudong New Area Clinical Characteristic Discipline Project (PWYts2021-11)，Pudong New Area Clinical Characteristic Discipline Project (PWYts2021-01), Pudong Research Project (PWRl2023-08, YC-2023-0128; YC-2023-0129; YC-2023-0202; YC-2023-0607). Shanghai Pudong New Area Health Commission Science and Technology Project (PW2024A-05);Shanghai Pudong New Area Health Science and Technology Project (PW2024A-05); Shanghai Pudong Hospital Talent Introduction Project (YJYJJRC202306); Fudan University Affiliated Pudong Hospital Key Specialty (Zdzk2024-04)，AI Health grant (RZ-CYAI-01-24-0258, 202401065), Integrated Large Model Platform Project of Clinical and Scientific Research for Chronic Disease Management (Grant No. 2024-GZL-RGZN-02012), AI-Driven Causal Analytics for Multimodal Disease Data in Healthcare Management (Grant No. 202401065). Authors' contributions Dr. Song Wen, and Dr. Yanhong Huang contributed equally to drafting this work. Dr. Yanhong Huang and Lijiao Chen were responsible for data collection; Dr. Wu Wang, Liang Zeng were responsible for project administration; Dr. Jiyu Li, Dr. Ligang Zhou, Dr, Chunxiang Fan and Dr. Dan Liu contributed equally to the conception and study design. Dr. Jiyu Li and Dr. Ligang Zhou were responsible for funding acquisition. All authors gave final approval of the version to be published, agreed on the journal to which the article was submitted, and accept responsibility for all aspects of the work. Acknowledgements We would like to express our sincere gratitude to all the patients who actively participated in this study, as well as to all the staff, especially the nursing team in the Department of Endocrinology at Shanghai Pudong Hospital, to whom we have made significant contributions. References A century of glucagon . Nature Reviews Endocrinology 2023, 19 (6):311-311. Best CH: The internal secretion of the pancreas . Can Med Assoc J 1962, 87 (20):1046-1051. Murlin JR, Clough HD, Gibbs CBF, Stokes AM: AQUEOUS EXTRACTS OF PANCREAS III. SOME PRECIPITATION REACTIONS OF INSULIN . Journal of Biological Chemistry 1923, 58 :337-346. 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Bomholt AB, Johansen CD, Galsgaard KD, Elmelund E, Winther-Sørensen M, Holst JJ, Wewer Albrechtsen NJ, Sørensen CM: Glucagon receptor activation contributes to the development of kidney injury . Am J Physiol Renal Physiol 2024, 327 (5):F712-f724. Liu JJ, Liu S, Gurung RL, Chan C, Ang K, Tang WE, Tavintharan S, Sum CF, Lim SC: Relationship Between Fasting Plasma Glucagon Level and Renal Function-A Cross-Sectional Study in Individuals With Type 2 Diabetes . J Endocr Soc 2019, 3 (1):273-283. Kajani S, Laker RC, Ratkova E, Will S, Rhodes CJ: Hepatic glucagon action: beyond glucose mobilization . Physiol Rev 2024, 104 (3):1021-1060. Yamashita K, Yamashita S, Yasuda H, Oka Y, Ogata E: A decreased response of cyclic adenosine monophosphate concentrations to glucagon in liver slices from streptozotocin-induced diabetic rats . Diabetes 1980, 29 (3):188-192. Chen W, Cui W, Wu J, Zheng W, Sun X, Zhang J, Shang H, Yuan Y, Li X, Wang J et al : Blocking IL-6 signaling improves glucose tolerance via SLC39A5-mediated suppression of glucagon secretion . Metabolism 2023, 146 :155641. Li XC, Zhuo JL: Targeting glucagon receptor signalling in treating metabolic syndrome and renal injury in Type 2 diabetes: theory versus promise . Clin Sci (Lond) 2007, 113 (4):183-193. McFarlin BE, Duffin KL, Konkar A: Incretin and glucagon receptor polypharmacology in chronic kidney disease . Am J Physiol Endocrinol Metab 2024, 326 (6):E747-e766. Bilbrey GL, Faloona GR, White MG, Knochel JP: Hyperglucagonemia of renal failure . J Clin Invest 1974, 53 (3):841-847. Rodgers RL: Glucagon and cyclic AMP: time to turn the page? Curr Diabetes Rev 2012, 8 (5):362-381. Rodgers RL: A reappraisal of the role of cyclic AMP in the physiological action of glucagon . Peptides 2023, 159 :170906. Kolanowski J, Salvador G, Desmecht P, Henquin JC, Crabbé J: Influence of glucagon on natriuresis and glucose-induced sodium retention in the fasting OVERWEIGHT subject . Eur J Clin Invest 1977, 7 (3):167-175. Sakamoto K, Butera MA, Zhou C, Maurizi G, Chen B, Ling L, Shawkat A, Patlolla L, Thakker K, Calle V et al : Overnutrition causes insulin resistance and metabolic disorder through increased sympathetic nervous system activity . Cell Metab 2025, 37 (1):121-137.e126. Ye J: Mechanism of insulin resistance in obesity: a role of ATP . Front Med 2021, 15 (3):372-382. Grøndahl MFG, Lange AH, Suppli MP, Bagger JI, Thing M, Gluud LL, Kofod DH, Hornum M, van Hall G, Trammell SAJ et al : Glucagon Clearance Is Decreased in Chronic Kidney Disease but Preserved in Liver Cirrhosis . Diabetes 2024, 73 (10):1641-1647. Parving HH, Noer J, Kehlet H, Mogensen CE, Svendsen PA, Heding L: The effect of short-term glucagon infusion on kidney function in normal man . Diabetologia 1977, 13 (4):323-325. Sherwin RS, Fisher M, Hendler R, Felig P: Hyperglucagonemia and blood glucose regulation in normal, OVERWEIGHT and diabetic subjects . N Engl J Med 1976, 294 (9):455-461. Huang W, Xie C, Jones KL, Horowitz M, Rayner CK, Wu T: Sex differences in the plasma glucagon responses to a high carbohydrate meal and a glucose drink in type 2 diabetes . Diabetes Res Clin Pract 2024, 214 :111769. Napit PR, Ali MH, Shakya M, Mandal SK, Bheemanapally K, Mahmood A, Ibrahim MMH, Briski KP: Hindbrain Estrogen Receptor Regulation of Ventromedial Hypothalamic Glycogen Metabolism and Glucoregulatory Transmitter Expression in the Hypoglycemic Female Rat . Neuroscience 2019, 411 :211-221. Kim JY, Bacha F, Tfayli H, Michaliszyn SF, Yousuf S, Arslanian S: Adipose Tissue Insulin Resistance in Youth on the Spectrum From Normal Weight to OVERWEIGHT and From Normal Glucose Tolerance to Impaired Glucose Tolerance to Type 2 Diabetes . Diabetes Care 2019, 42 (2):265-272. Tables Tables 1 to 6 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Tables.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 17 Oct, 2025 Reviewers invited by journal 10 Oct, 2025 Editor invited by journal 15 Sep, 2025 Editor assigned by journal 11 Sep, 2025 Submission checks completed at journal 11 Sep, 2025 First submitted to journal 09 Sep, 2025 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. 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1","display":"","copyAsset":false,"role":"figure","size":138166,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThis figure illustrates the HbA1c (A), dynamic of blood glucose (B), C-peptide (C), insulin (D), and glucagon (GCG) (E), as well as HOMA-IR (F), HOMA-β (G) among the three glucose upgradation state groups in youth, which are represented as euglycemia, prediabetes and diabetes.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNote: *: p\u0026lt;0.05; **: p\u0026lt;0.01; ***: p\u0026lt;0.001; ****: p\u0026lt;0.0001; HbA1c: Glycated Hemoglobin A1c; BG: blood glucose; GCG: glucagon; HOMA: homeostatic models of assessment.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7576426/v1/f69bec987e146f317353b3a8.png"},{"id":94224097,"identity":"4877ff35-55d0-48dc-8a48-00d3130889a7","added_by":"auto","created_at":"2025-10-23 19:15:50","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":70135,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe comparison of tubular injury markers among three groups represented by Euglycemia, preDM, DM.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNote: *：p\u0026lt;0.05; α1MG：α1 microglobin; β2MG: β2 microglobin；RBP：retinol-binding protein; NAG: N-acetyl-β-D-glucosaminidase\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7576426/v1/e94b961a14115bc22b67ede0.png"},{"id":94224791,"identity":"3d1814ba-a530-42e0-ae72-a1f204639719","added_by":"auto","created_at":"2025-10-23 19:23:50","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":206588,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe comparison between aged and youth patients in reference to the HbA1c (A), dynamic of blood glucose (B), C-peptide (C), insulin (D), and glucagon (GCG) (E), HOMA-IR (F), HOMA-β(G) among the three degrade of glucose state groups in youth represented as Euglycemia, prediabetes, diabetes.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNote: *: p\u0026lt;0.05; **: p\u0026lt;0.01; ***: p\u0026lt;0.001; ****: p\u0026lt;0.0001; HbA1c: Glycated Hemoglobin A1c; BG: blood glucose; GCG: glucagon; HOMA: homeostatic models of assessment.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7576426/v1/3bc97841a75b9fa4bfd4954b.png"},{"id":94224793,"identity":"2904c089-a5b8-41a9-9ed9-f5edd6348e9b","added_by":"auto","created_at":"2025-10-23 19:23:50","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":189791,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe comparison of tubular injury markers between the youth and aged T2DM patients in relation to the renal tubular injury markers, including blood α1MG (A), β2MG(B)，RBP(C)，urinary α1MG(D), β2MG (E)，RBP(F).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNote: *：p\u0026lt;0.05; ***: p\u0026lt;0.001; ****: p\u0026lt;0.0001; \u003cstrong\u003eα\u003c/strong\u003e1MG：α1 microglobin; β2MG: β2 microglobin；RBP：retinol-binding protein; NAG: N-acetyl-β-D-glucosaminidase\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7576426/v1/ce653212796ab94812183917.png"},{"id":94225639,"identity":"62d36040-0a52-461c-82a0-e62005fdb0cf","added_by":"auto","created_at":"2025-10-23 19:31:50","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":107763,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe diverse causal agents for renal microvascular and tubular impairment among young overweight patients and aged T2DM patients. \u003c/strong\u003eHyperglucagonemia is a primary cause of renal microvascular and tubular injury in young overweight individuals; However, multiple factors contributing to renal microvascular injury in aged diabetic patients.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7576426/v1/fd42b32c8f1d8c3a1366f1a1.png"},{"id":94226417,"identity":"e7c1fb2f-2900-4bba-9fa4-5ac350383c1c","added_by":"auto","created_at":"2025-10-23 19:47:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3743907,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7576426/v1/51e720f7-3a88-4027-b7c5-ae28785ba69a.pdf"},{"id":94224096,"identity":"4ab6ce96-e6da-43ac-aa18-4c29ab10a94e","added_by":"auto","created_at":"2025-10-23 19:15:50","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":46143,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-7576426/v1/92688c6a25a8fca050a07362.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Insulin Resistant States Contributed Differentially to the Earlier Renal tubular Injury in Chinese Young Overweight Individuals with Hyperglucagonemia","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe recognition of glucagon (GCG) as a distinct hormone began in the early 20th century, marking a significant milestone in understanding metabolic regulation [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The story starts with the work of researchers exploring the pancreas, initially focused on insulin. In 1922, Banting and Best successfully isolated insulin from pancreatic extracts to treat diabetes, but they observed that crude pancreatic extracts sometimes caused hyperglycemia rather than hypoglycemia [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. This paradoxical effect puzzled scientists and hinted at the presence of another factor. The breakthrough came in 1923 when Kimball and Murlin identified a hyperglycemic substance in pancreatic extracts distinct from insulin, naming it \"glucagon,\" from the Greek \"glukus\" (sweet) and \"agon\" (leading), reflecting its glucose-elevating properties [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Initial studies suggested its production by pancreatic alpha cells, contrasting with insulin\u0026rsquo;s beta-cell origin [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. However, its exact nature remained elusive due to limited purification techniques and the focus on insulin research at the time. Significant progress occurred in the 1950s when Staub, Behrens, and colleagues purified glucagon from porcine pancreas, confirming it as a 29-amino-acid peptide [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. This enabled detailed physiological studies, revealing its role in counteracting insulin by stimulating hepatic gluconeogenesis and glycogenolysis [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. By the 1960s and 1970s, radioimmunoassays and advanced biochemical techniques clarified its secretion patterns and receptor interactions, solidifying its status as a critical hormone in glucose homeostasis [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eHyperglucagonemia, characterized by elevated GCG levels, has since been linked to metabolic dysregulation in type 2 diabetes (T2DM) and prediabetes contributing to hyperglycemia [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Recent research has expanded its implications, suggesting a potential role in renal microvascular impairment, particularly in early disease stages, where the kidneys, especially renal tubules, are vulnerable to metabolic stress [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. This is particularly relevant in young prediabetic (PreDM) individuals, where metabolic abnormalities may precede overt diabetes, and in aged patients with type 2 diabetes (T2DM Aged), where chronic disease processes dominate. Despite its metabolic significance, the specific contribution of hyperglucagonemia to renal injury across age groups remains underexplored. Young overweight individuals with prediabetes represent a critical population for early intervention to prevent chronic kidney disease (CKD), while aged T2DM patients often exhibit advanced renal complications, where glucagon\u0026rsquo;s role may differ due to age-related changes. Studies suggest that glucagon receptor (GCGR) activation in renal tubular cells may induce oxidative stress and inflammation, while chronic exposure could promote fibrosis [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], yet age-specific mechanisms are not well-defined.\u003c/p\u003e\u003cp\u003eThis study aims to investigate the association between hyperglucagonemia and renal tubular and microvascular injury in young individuals with PreDM, DM, and euglycemia in comparison to aged patients with T2DM. By analyzing metabolic markers, tubular and microvascular injury indicators, we seek to elucidate the role of GCG in renal microvascular impairment and identify age-tailored risk factors, providing insights for targeted preventive strategies. Understanding the mechanisms of early renal injury is a critical issue in light of the increasing global prevalence of obesity and diabetes.\u003c/p\u003e"},{"header":"Methods and Materials","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eParticipant Recruitment and Obesity Diagnosis\u003c/h2\u003e\u003cp\u003eA total of 108 Chinese young overweight patients (age: 35.34\u0026thinsp;\u0026plusmn;\u0026thinsp;8.66 yrs, BMI: 29.997\u0026thinsp;\u0026plusmn;\u0026thinsp;5.32kg/m\u003csup\u003e2\u003c/sup\u003e) were selected, categorized into normal glucose (Euglycemia, n\u0026thinsp;=\u0026thinsp;46), prediabetes (PreDM, n\u0026thinsp;=\u0026thinsp;45), and newly diagnosed diabetes (T2DM, n\u0026thinsp;=\u0026thinsp;17) based on oral glucose tolerance test (OGTT) results, and compared with 131 long-standing aged type 2 diabetes patients (T2DM Aged; age: 64.54\u0026thinsp;\u0026plusmn;\u0026thinsp;10.91yrs). All young participants with overweight had no prior renal replacement therapy and history for diabetes, and had a pre-contact on preparing for OGTT the day before consulting the clinic. The T2DM aged patients with completed OGTT results were recruited from the In-Patient history information system of Shanghai Pudong Hospital.\u003c/p\u003e\u003cp\u003eThis research is a component of our ongoing chronic disease weight management initiative, which is focused on overweight patients and was conducted from October 2024 to August 2025. Participants were selected from a cohort of overweight individuals who were diagnosed based on a body mass index (BMI) of \u0026ge;\u0026thinsp;24 kg/m\u0026sup2;, in accordance with the most recent Chinese National Health Commission guideline, which were published in October 2024. This guideline suggests that the BMI thresholds for overweight be 24-27.9 kg/m\u0026sup2; and for obesity be \u0026ge;\u0026thinsp;28 kg/m\u0026sup2;. These thresholds are intended to reconcile the Asian population's higher percentage of visceral adipose tissue at lower BMIs than other populations. Secondary endocrine diseases, including adrenal gland tumor, pituitary tumor, hypothyroidism, and Cushing's syndrome, were excluded through a thorough medical history and physical examination after the diagnosis was confirmed. Imaging and laboratory investigations (e.g., thyroid function, cortisol levels) were implemented to the extent necessary for this purpose. The investigation highlighted primary obesity that was associated with metabolic and lifestyle factors, and patients with secondary obesity were excluded.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eMultidisciplinary Team (MDT) Evaluation\u003c/h3\u003e\n\u003cp\u003e The multidisciplinary team (MDT), guided by the 2024 Chinese National Health Commission guideline emphasizing clinical nutrition, behavioral intervention, exercise, and psychological support, conducted thorough evaluations. Medical interviews assessed dietary habits, physical activity, and psychological well-being, using tools like the PHQ-9 for depression screening. Comprehensive assessments included exercise physiology evaluations (e.g., VO2 max testing), nutritional analysis (e.g., 24-hour dietary recall), and tailored psychological support, reflecting the guidelines\u0026rsquo; call for a balanced diet, physical activity, and family involvement.\u003c/p\u003e\n\u003ch3\u003eMeasurement of Metabolic and Renal Markers\u003c/h3\u003e\n\u003cp\u003eMetabolic markers, including fasting and postprandial (0, 30, and 120 minutes) glucose, C-peptide, insulin, and glucagon (GCG), were measured using standardized enzymatic and immunoassays. Renal tubular injury indicators, such as blood retinol-binding protein (RBP), urinary N-acetyl-β-D-glucosaminidase (U-NAG), and α1-microglobulin (α1MG), were assessed via urine and serum samples collected during OGTT. Estimated glomerular filtration rate (eGFR) was calculated using the CKD-EPI equation.\u003c/p\u003e\n\u003ch3\u003eOral Glucose Tolerance Test (OGTT) Procedure\u003c/h3\u003e\n\u003cp\u003eOGTT involved a 75g glucose load administered after an overnight fast, with blood samples drawn at 0, 30, and 120 minutes to assess dynamic metabolic responses, aligning with the NHC\u0026rsquo;s emphasis on monitoring glucose metabolism.\u003c/p\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eStatistical analyses were conducted using Prism (GraphPad, version 10.0) and SPSS (IBM, version 26.0). Descriptive statistics summarized baseline characteristics, including age, BMI groups, HbA1c levels, reported as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM) for continuous variables and frequencies/percentages for categorical variables. Chi-square tests assessed associations between categorical variables, such as age groups and gender, BMI groups and gender, and HbA1c groups and gender. Non-parametric Brown-Forsythe and Welch ANOVA tests, followed by post-hoc Dunn\u0026rsquo;s tests, compared age, BMI, HOMA-IR, UACR, OGTT parameters (glucose, C-peptide, insulin at 0, 30, and 120 minutes), and hormone levels (e.g., cortisol, GH, IGF-1) across the three glucose metabolism states. Spearman correlation analysis evaluated relationships between tubular proteins (α1MG, β2MG, RBP) and metabolic parameters, with significance at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Multilinear regression identified independent predictors of UACR, adjusting for age, gender, and metabolic factors. A significance threshold of P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was applied for all analyses.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e1. The glucose metabolic status of OVERWEIGHT youth as disclosed by OGTTs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNewly diagnosed diabetic patients exhibited substantially higher fasting and postprandial glucose levels (at 30 and 120 minutes) than the normal glucose (Euglycemia) and prediabetic (PreDM) groups among young overweight patients (P\u0026lt;0.05). Conversely, PreDM patients exhibited elevated 120-minute postprandial glucose (P\u0026lt;0.05). Both DM and Prediabetic patients exhibited elevated 2-hour postprandial C-peptide and insulin levels in comparison to the normal glucose groups (P\u0026lt;0.05). In comparison to other groups, diabetic patients exhibited substantially elevated GCG levels at fasting and 30 minutes postprandial, while prediabetic and normal glucose groups exhibited comparable GCG concentrations. HOMA-β did not exhibit any significant differences among the three groups; however, the diabetic group exhibited a significant higher HOMA-IR (\u003cstrong\u003eFig. 1\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2. The renal tubular injury markers in youth OVERWEIGHT patients were significantly different. \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDiabetic patients displayed higher blood α1-MG levels compared to normal glucose and prediabetic groups, and elevated β2MG compared to the Euglycemic group, indicating exacerbated tubular injury (\u003cstrong\u003eFig.2\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3. The comparison of glucose metabolic status between aged and youth revealed significant C peptide and insulin levels, as well as retarded GCG response after 30min and 120min OGTT.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAged T2DM patients had higher HbA1c, fasting, 30min and 120-minute postprandial glucose levels compared to young Euglycemia and PreDM groups, but significantly reduced C-peptide and insulin levels (Fig.3 A-D). Fasting glucagon (GCG) was lower, while 30-minute and 120min postprandial GCG was higher than other groups. Interestingly, the HOMA-IR and HOMA-β were the lowest among all the groups (\u003cstrong\u003eFig.3\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4. The comparison of renal tubular injury markers between youth and aged patients indicated different scale and spectrum of tubular impairment. \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCompared to young PreDM and Euglycemia, aged T2DM patients showed higher blood α1-microglobulin (α1MG) than Euglycemic groups, and urinary α1MG levels in young euglycemic, PreDM and DM groups; The level of blood β2MG was the highest among the groups; while we only observed increased urinary RBP in aged T2DM groups (\u003cstrong\u003eFig.4\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5. The correlational relationships between the tubular injury markers and the state of glucose metabolism.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe therefore performed the correlational relationships between tubular injury markers and OGTT parameters in young and aged patients, respectively. In youth, significant positive correlations were observed between tubular markers (e.g., b-α1MG, b-β2MG, b-RBP) and FPG, insulin, C-peptide, and GCG levels (P\u0026lt;0.05), indicating significant insulin resistance. We found gender’s different variation and ages, as well as HbA1c were significantly associated with the tubular markers (\u003cstrong\u003eTable.1\u003c/strong\u003e). While in aged T2DM patients, the tubular markers were more frequently associated with the glycemic control, and reduced Insulin and C-peptide levels, as well as reduced HOMA-β and HOMA-IR, suggesting the association with the reduced insulin insufficiency (\u003cstrong\u003eTable.2\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e6. The significant factors associated with the UACR elevation, which is an indicator of microvascular impairment in both youth and aged patients.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn young patients, 30-minute glucagon (30min GCG) (OR=1.111, P=0.004), eGFR (OR=1.052, p=0.029) was strong independent risk for elevated urine albumin-to-creatinine ratio (UACR), which suggesting the insulin resistance, and early hyperfiltration of glomerular impairment was associated with the UACR increment; Higher postprandial GCG level was risk factor for elevation of UACR (\u003cstrong\u003eTable 3\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn aged patients, however, HbA1c (OR=1.782, P=0.002 and eGFR(OR=0.962, P=0.028）\u0026nbsp;were significant, indicating the blood glucose control, reduced renal glomerular infiltration function were responsible for elevation of UACR\u003cstrong\u003e（\u003c/strong\u003e\u003cstrong\u003eTable4\u003c/strong\u003e\u003cstrong\u003e）\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e7. The determinants associated with the renal tubular markers’ alteration in both youth and aged, reflected by the analyses of multilinear regression models.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe multilinear regression models provide insights into the determinants influencing alterations in renal tubular injury markers among both young and aged patients. In young patients, the analysis revealed a multifaceted set of predictors across different markers. For blood α1-microglobulin (α1MG), FPCP, estimated glomerular filtration rate (eGFR), and 30-minute glucagon (30min GCG) emerged as significant determinants, suggesting a complex interplay between metabolic control and renal filtration capacity (\u003cstrong\u003eTable 5\u003c/strong\u003e). Blood β2-microglobulin (β2MG) was influenced by age, 30min GCG, eGFR and FPG, indicating that younger age, insulin resistance and reduced infiltration ability play critical roles in its elevation. For blood retinol-binding protein (RBP), a broader set of factors including gender, eGFR, FPCP, HOMA-β were identified as key contributors, highlighting the impact of both metabolic and renal function parameters. Urinary α1MG was notably associated with 30-minute postprandial C-peptide (30min PPCP), gender, 120minPPCP, underscoring the relevance of postprandial metabolic responses, and male gender in urinary marker changes. U-β2MG was associated with the HOMA-β and 30min postprandial insulin concentration (30min INS), while u-RBP was linked with 30minGCG, 120minPPCP (Table. 5).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn aged patients, the determinants were more focused but equally significant. eGFR was a primary determinant for both α1MG and β2MG, reflecting the dominant role of renal filtration decline with age and disease progression \u003cstrong\u003e(Table 6)\u003c/strong\u003e. FPCP and 120min INS were significant indicative markers for elevated renal tubular markers, suggesting the mild reduced clearance circulation level of fasting C-peptide and insulin insufficiency both contributed the observed tubular changes. The levels of RBP were determined by FPCP, gender variation, reduced HOMA-IR, 120min GCG, as well as FPG. The urinary α1MG and NAG were associated with fasting Insulin, 30min GCG, and 30min PPG, respectively.\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe findings of this study underscore a significant association between hyperglucagonemia, as reflected by elevated glucagon (GCG) levels, and renal microvascular impairment, particularly in young prediabetic (PreDM) and diabetic (DM) individuals. The elevated postprandial GCG levels in young diabetic patients, coupled with increased tubular injury markers such as blood and urinary tubular markers, suggest that GCG may play a pivotal role in the early pathogenesis of renal damage. This is further supported by regression analyses, where 30-minute GCG emerged as an independent predictor of elevated urine albumin-to-creatinine ratio (UACR) in young patients, indicating a direct link between dysregulated prolonged glucagon exposure and glomerular dysfunction [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The consistency of these findings across multiple markers and time points reinforces the hypothesis that GCG dysregulation is a critical factor in the initiation of renal injury in this population \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn young overweight patients, the observed metabolic dysregulation\u0026mdash;characterized by elevated postprandial glucose, C-peptide, and insulin in PreDM and DM groups\u0026mdash;may exacerbate renal tubular injury. The significant correlation between GCG and tubular markers suggests that hyperglucagonemia could contribute to tubular stress through several mechanisms. These include increased gluconeogenesis, which elevates systemic glucose levels and places additional metabolic burden on the kidneys, as well as heightened oxidative stress and inflammation that impair renal tubular integrity [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The broader set of metabolic determinants identified in the multilinear regression models underscores the dynamic nature of renal injury in this population, potentially driven by acute metabolic fluctuations [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. This dynamic response may reflect the heightened metabolic sensitivity of young individuals, where rapid changes in GCG levels could trigger early tubular damage before significant glomerular involvement [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn aged patients with type 2 diabetes (T2DM Aged), a distinctly different profile of renal impairment emerges. Despite lower fasting GCG and reduced C-peptide/insulin levels, the elevated 30- and 120-minute GCG and higher HbA1c suggest a chronic hyperglycemic state that may shift the injury pattern toward both glomerular and tubular decline [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The prominence of eGFR as a determinant for α1MG and β2MG, and GCG, indicates that renal filtration capacity and sustained glucagon elevation are key factors in aged renal damage [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. This shift may reflect age-related declines in renal reserve and compensatory mechanisms, where increased ratio of postprandial GCG relative to insulin exposure could amplify existing glomerular damage rather than initiate it [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The insulin insufficiency in aged patients further complicates this picture, potentially leading to an unopposed GCG effect that exacerbates renal fibrosis and vascular stiffness over time [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe differential contribution of GCG to renal injury between young and aged populations may stem from age-specific physiological responses. In young patients, the acute elevation of GCG, particularly postprandially, may act as an early trigger for tubular injury, potentially through glucagon receptor-mediated pathways that increase sodium reabsorption and oxidative stress in the proximal tubule [\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. This acute effect is supported by the significant associations with u-α1MG and UACR, suggesting that GCG acts as an early stressor in metabolically active young kidneys [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In aged patients, the chronic elevation of GCG, combined with reduced insulin secretion, may exacerbate pre-existing glomerular sclerosis and tubular atrophy. This chronicity could lead to a more pronounced glomerular injury pattern, as sustained GCG levels may contribute to hypertension and endothelial dysfunction, key drivers of glomerulosclerosis in aging populations [\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eRecent research provides mechanistic insights into GCG\u0026rsquo;s role in chronic kidney disease (CKD) and early renal injury [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], offering a framework to interpret these findings. This GCG, traditionally viewed as a counter-regulatory hormone to insulin, contributes to hyperglycemia by stimulating hepatic gluconeogenesis and glycogenolysis [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In the context of renal health, elevated GCG levels may exacerbate CKD progression by promoting inflammation and fibrosis, as evidenced by increased expression of pro-inflammatory cytokines (e.g., TNF-α, IL-6) and extracellular matrix proteins (e.g., collagen IV) in animal models of diabetic nephropathy [\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. It also notes that GCG resistance may develop in advanced CKD due to downregulated glucagon receptor (GCGR) expression, yet persistent hyperglucagonemia still drives systemic inflammation and glomerular hypertension, accelerating glomerulosclerosis [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eSpecifically, GCG\u0026rsquo;s role in early renal injury may involve activation of the GCGR in renal tubular cells, supraphysiological concentration leading to increased cyclic AMP (cAMP) signaling [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. This pathway can enhance sodium and water reabsorption, increase tubular workload and generate reactive oxygen species (ROS) that contribute to oxidative stress and cellular damage in the proximal tubule [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Over time, this chronic stress may transition into fibrosis, as GCG-induced inflammation recruits macrophages and activates transforming growth factor-beta (TGF-β) signaling, a well-established pathway in renal scarring [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. In advanced CKD stages, the review suggests that GCG\u0026rsquo;s effects may shift toward systemic metabolic dysregulation, where reduced GCGR sensitivity in the kidney amplifies its hepatic gluconeogenic impact, further worsening glycemic control and renal perfusion pressure [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn this study, the acute GCG elevation in young patients aligns with its role in early tubular stress, supported by the significant association with α1MG and UACR. The dynamic metabolic environment in young patients, as reflected by multiple OGTT time points, may amplify this effect, making GCG a potential therapeutic target to prevent progression to CKD [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. In aged patients, the chronic GCG elevation and its link to urinary tubular markers suggest a transition to glomerular damage, consistent with CKD progression. The significant HOMA index across groups further emphasizes GCG\u0026rsquo;s independent contribution, beyond insulin resistance, highlighting its dual role as both a metabolic and renal stressor. Recent hotspots, such as glucagon receptor antagonists (e.g., REMD-477) reducing albuminuria in diabetic models, support targeting GCG in young prediabetic individuals to prevent early renal impairment. For aged patients, addressing chronic glomerular protection alongside GCG modulation may be necessary, given the interplay of filtration decline, fibrosis, and sustained glucagon levels [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe multilinear regression models indicate that gender significantly influences renal tubular injury markers in young patients, with males showing higher tubular makers\u0026rsquo; levels compared to females. This suggests sex-specific modulation of renal damage [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Hormonal factors, such as estrogen\u0026rsquo;s reno-protective effects in females (e.g., anti-inflammatory and antioxidant properties) versus testosterone\u0026rsquo;s potential to promote sodium retention in males, may explain these differences [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Additionally, females\u0026rsquo; higher visceral fat in obesity could amplify tubular stress, correlating with HbA1c and β2MG, while males\u0026rsquo; muscle mass might buffer damage [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Pubertal hormonal surges may further accentuate these effects in youth. In aged patients, gender\u0026rsquo;s lack of significance (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e6\u003c/span\u003e) suggests that age-related renal decline and chronic hyperglucagonemia overshadow sex-specific influences. Future studies should explore hormonal profiling to tailor interventions for gender-specific renal risks.\u003c/p\u003e\u003cp\u003eThe comparison of renal injury patterns reveals that young patients exhibit a predominance of tubular damage linked to metabolic dynamism, whereas aged patients show a mixed glomerular-tubular injury pattern driven by chronicity and filtration decline. This age-dependent divergence suggests that GCG\u0026rsquo;s contribution varies by disease stage and renal reserve, with acute effects dominating in youth and chronic effects prevailing in the aged. Future research should validate these mechanisms through longitudinal studies and explore GCG-targeted therapies tailored to age-specific renal pathology, potentially integrating GCGR antagonists with anti-fibrotic agents to address the full spectrum of GCG-mediated renal injury.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eLimitations\u003c/h2\u003e\u003cp\u003eThis study has several limitations that warrant consideration. The cross-sectional design precludes establishing causality between hyperglucagonemia and renal tubular injury, necessitating longitudinal studies to confirm temporal relationships. The study\u0026rsquo;s focus on overweight young patients and aged T2DM may limit generalizability to non-overweight or other age demographics.\u003c/p\u003e\u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study provides compelling evidence that hyperglucagonemia may contribute to renal microvascular impairment through tubular dysfunction, particularly in young prediabetic individuals, where 30-min and 120-minute GCG contributed to the early glomerular and tubular injury. The findings highlight GCG as a potential early marker of renal risk in this population, contrasting with aged T2DM patients, where chronic renal filtration decline and sustained GCG elevation dominate. \u0026nbsp;Further longitudinal research is essential to validate these findings and refine therapeutic approaches.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical Approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHuman Ethics and Consent to Participate declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of Shanghai Pudong Hospital (Approval No. AZ-010). The study was a retrospective analysis that strictly adhered to the ethical principles outlined in the Declaration of Helsinki. The study protocol was reviewed and approved by the Ethics Committee of Shanghai Pudong Hospital (Approval No. AZ-010). As a retrospective study, informed consent was waived, and all data were anonymized to protect patient confidentiality. Patient data were anonymized by removing personal identifiers (e.g., name, ID number) and stored securely on an encrypted server accessible only to authorized personnel involved in the study. The Ethics Committee of Shanghai Pudong Hospital waived the requirement for consent to participate (No. AZ-010).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial Number:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of Data and Materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are not publicly available due to privacy reasons but are available from the corresponding author upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there is no conflict of interest. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Integrated Traditional Chinese and Western Medicine (YC-2023-0404),Fudan Zhangjiang Clinical Medicine Innovation Fund Project (KP0202118), Fudan Good Practice Program of Teaching and Learning (FD2023A227), Project of Key Medical Discipline of Pudong Hospital of Fudan University (Zdxk2020-11), Project of Key Medical Specialty and Treatment Center of Pudong Hospital of Fudan University (Zdzk2020-24), Pudong New Area Clinical Plateau Discipline Project (PWYgy-2021-03), the Natural Science Foundation of China (21675034), Pudong New Area Clinical Characteristic Discipline Project (PWYts2021-11)，Pudong New Area Clinical Characteristic Discipline Project (PWYts2021-01), Pudong Research Project (PWRl2023-08, YC-2023-0128; YC-2023-0129; YC-2023-0202; YC-2023-0607). Shanghai Pudong New Area Health Commission Science and Technology Project (PW2024A-05);Shanghai Pudong New Area Health Science and Technology Project (PW2024A-05); Shanghai Pudong Hospital Talent Introduction Project (YJYJJRC202306); Fudan University Affiliated Pudong Hospital Key Specialty (Zdzk2024-04)，AI Health grant (RZ-CYAI-01-24-0258, 202401065), Integrated Large Model Platform Project of Clinical and Scientific Research for Chronic Disease Management (Grant No. 2024-GZL-RGZN-02012), AI-Driven Causal Analytics for Multimodal Disease Data in Healthcare Management (Grant No. 202401065).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDr. Song Wen, and Dr. Yanhong Huang contributed equally to drafting this work. Dr.\u0026nbsp;Yanhong Huang\u0026nbsp;and Lijiao Chen were responsible for data collection; Dr. Wu Wang, Liang Zeng were responsible for project administration; Dr. Jiyu Li, Dr. Ligang Zhou, Dr, Chunxiang Fan and Dr. Dan Liu contributed equally to the conception and study design. Dr. Jiyu Li and Dr. Ligang Zhou were responsible for funding acquisition. All authors gave final approval of the version to be published, agreed on the journal to which the article was submitted, and accept responsibility for all aspects of the work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to express our sincere gratitude to all the patients who actively participated in this study, as well as to all the staff, especially the nursing team in the Department of Endocrinology at Shanghai Pudong Hospital, to whom we have made significant contributions.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003e\u003cstrong\u003eA century of glucagon\u003c/strong\u003e. \u003cem\u003eNature Reviews Endocrinology \u003c/em\u003e2023, \u003cstrong\u003e19\u003c/strong\u003e(6):311-311.\u003c/li\u003e\n\u003cli\u003eBest CH: \u003cstrong\u003eThe internal secretion of the pancreas\u003c/strong\u003e. \u003cem\u003eCan Med Assoc J \u003c/em\u003e1962, \u003cstrong\u003e87\u003c/strong\u003e(20):1046-1051.\u003c/li\u003e\n\u003cli\u003eMurlin JR, Clough HD, Gibbs CBF, Stokes AM: \u003cstrong\u003eAQUEOUS EXTRACTS OF PANCREAS III. 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subjects\u003c/strong\u003e. \u003cem\u003eN Engl J Med \u003c/em\u003e1976, \u003cstrong\u003e294\u003c/strong\u003e(9):455-461.\u003c/li\u003e\n\u003cli\u003eHuang W, Xie C, Jones KL, Horowitz M, Rayner CK, Wu T: \u003cstrong\u003eSex differences in the plasma glucagon responses to a high carbohydrate meal and a glucose drink in type 2 diabetes\u003c/strong\u003e. \u003cem\u003eDiabetes Res Clin Pract \u003c/em\u003e2024, \u003cstrong\u003e214\u003c/strong\u003e:111769.\u003c/li\u003e\n\u003cli\u003eNapit PR, Ali MH, Shakya M, Mandal SK, Bheemanapally K, Mahmood A, Ibrahim MMH, Briski KP: \u003cstrong\u003eHindbrain Estrogen Receptor Regulation of Ventromedial Hypothalamic Glycogen Metabolism and Glucoregulatory Transmitter Expression in the Hypoglycemic Female Rat\u003c/strong\u003e. \u003cem\u003eNeuroscience \u003c/em\u003e2019, \u003cstrong\u003e411\u003c/strong\u003e:211-221.\u003c/li\u003e\n\u003cli\u003eKim JY, Bacha F, Tfayli H, Michaliszyn SF, Yousuf S, Arslanian S: \u003cstrong\u003eAdipose Tissue Insulin Resistance in Youth on the Spectrum From Normal Weight to OVERWEIGHT and From Normal Glucose Tolerance to Impaired Glucose Tolerance to Type 2 Diabetes\u003c/strong\u003e. \u003cem\u003eDiabetes Care \u003c/em\u003e2019, \u003cstrong\u003e42\u003c/strong\u003e(2):265-272.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 6 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"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":"bmc-endocrine-disorders","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bend","sideBox":"Learn more about [BMC Endocrine Disorders](http://bmcendocrdisord.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bend/default.aspx","title":"BMC Endocrine Disorders","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Hyperglucagonemia, Renal microvascular impairment, Prediabetes, Tubular injury","lastPublishedDoi":"10.21203/rs.3.rs-7576426/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7576426/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003eBackground\u003c/b\u003e\u003c/p\u003e\u003cp\u003eHyperglucagonemia is associated to the metabolic dysregulation, but its early role in microvascular injury, especially with young and overweight state, remains unclear. Thus, this study investigates the association between hyperglucagonemia and renal tubular injury in Chinese young overweight with dysglycemic state, and compared the profile with Chinese aged type 2 diabetes (T2DM Aged).\u003c/p\u003e\u003cp\u003e\u003cb\u003eMethods\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe assessment was conducted on metabolic markers and renal tubular injury indicators. At 0, 30, and 120 minutes, the parameters of the oral glucose tolerance test (OGTT) were assessed. Analysis of relationships and predictors of renal impairment was conducted.\u003c/p\u003e\u003cp\u003e\u003cb\u003eResults\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAmong young overweight patients, glucagon (GCG) levels were elevated in diabetic patients at both fasting and 30 minutes, with higher HOMA-IR and comparable HOMA-β. Elevated blood α1- and β2-MG were observed in newly diagnosed type 2 diabetic patients with hyperglucagonemia. However, when analyzed with T2DM aged (64.54\u0026thinsp;\u0026plusmn;\u0026thinsp;10.91yrs), they exhibited elevated 30- and 120-minute GCG and reduced HOMA indices. In comparison to younger groups, aged patients exhibited profound elevated levels of α1MG. Correlational relationship analysis showed tubular markers in young patients significantly positively related to male gender, elevated FPG, HbA1c, insulin and GCG levels, as well as HOMA (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while ageing, elevated postprandial glucose, reduced insulin levels, postpone attenuating GCG in 30min and 120min, and decreased HOMA were significantly in T2DM Aged. The binary logistic regression analysis showed the risk of appearance of renal microvascular injury marker, UACR, in young patients was determined by 30-minute GCG (B\u0026thinsp;=\u0026thinsp;0.105, OR\u0026thinsp;=\u0026thinsp;1.111, P\u0026thinsp;=\u0026thinsp;0.004) and increased eGFR (B\u0026thinsp;=\u0026thinsp;0.050, OR\u0026thinsp;=\u0026thinsp;1.052, P\u0026thinsp;=\u0026thinsp;0.029), while it was associated with elevated HbA1c (B\u0026thinsp;=\u0026thinsp;0.578, OR\u0026thinsp;=\u0026thinsp;1.782, P\u0026thinsp;=\u0026thinsp;0.002) and reduced eGFR (B=-0.039, OR\u0026thinsp;=\u0026thinsp;0.962, P\u0026thinsp;=\u0026thinsp;0.028) in the T2DM aged. Multilinear regression showed different tubular markers in young groups were differentially positively determined by FPG, insulin and C-peptide levels, 30minGCG and negatively with eGFR; for T2DM aged group, ageing, reduced eGFR and insulin levels, elevated postprandial GCG at 30min and 120min, reduced HOMA indices differentially contributed to the various elevated tubular markers.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConclusions\u003c/b\u003e\u003c/p\u003e\u003cp\u003eHyperglucagonemia and insulin resistance may serve as an early renal injury sign in the pathophysiology of overweight. While causes for microvascular and tubular injury were different between young overweight and aged T2DM patients which alert early effective bodyweight and metabolic management.\u003c/p\u003e","manuscriptTitle":"Insulin Resistant States Contributed Differentially to the Earlier Renal tubular Injury in Chinese Young Overweight Individuals with Hyperglucagonemia","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-23 19:15:46","doi":"10.21203/rs.3.rs-7576426/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"165998904472304087568114027101441858312","date":"2025-10-17T12:37:22+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-10T05:00:35+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-09-15T09:02:53+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-11T07:46:08+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-11T07:45:41+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Endocrine Disorders","date":"2025-09-09T18:15:26+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-endocrine-disorders","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bend","sideBox":"Learn more about [BMC Endocrine Disorders](http://bmcendocrdisord.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bend/default.aspx","title":"BMC Endocrine Disorders","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0ac32000-bea7-4f62-9a37-594cbbb5dca6","owner":[],"postedDate":"October 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-10-23T19:15:46+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-23 19:15:46","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7576426","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7576426","identity":"rs-7576426","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}