Impact of Antiglycemic & Immunosuppressive Therapies on NETosis in Diabetes & Kidney Disease (NETs - Neutrophil Traps)

Recruiting

• Conditions: Diabetes Mellitus; Kidney Disease

• Registration Number: NCT06821919
• Lead Sponsor: Western Galilee Hospital-Nahariya

Brief Summary

This study aims to investigate whether new glucose-lowering medications, such as SGLT2 inhibitors (e.g., Forxiga/Jardiance) and GLP-1 receptor agonists (e.g., Ozempic), can reduce NETosis in diabetic patients, thereby mitigating secondary complications such as cardiovascular disease and kidney damage. By targeting dysregulated NET formation, the study seeks to establish a link between reduced NETosis and improved clinical outcomes in diabetes.

Additionally, the study will evaluate the effects of immunosuppressive therapies on NETosis in patients with immune-mediated kidney diseases, such as ANCA-associated vasculitis. By correlating NETosis activity with disease progression and treatment response, this research will assess whether reducing NETosis contributes to better management of inflammation and secondary morbidity in these conditions.

Through these evaluations, the study aims to identify potential therapeutic strategies to improve outcomes in both diabetic and chronic kidney disease populations.

Detailed Description

Neutrophil extracellular traps (NETs) are an essential component of the innate immune system, designed to trap and neutralize invading pathogens. NETosis, the process by which neutrophils release decondensed chromatin (DNA and histones) decorated with antimicrobial proteins such as myeloperoxidase (MPO) and neutrophil elastase (NE), is a critical mechanism in host defense. This process is induced by stimuli such as PMA, antibodies, cytokines, chemokines, and sterile triggers, including high glucose, cholesterol, and hypoxia. This stimulation activates the Raf-MEK-ERK pathway and NADPH oxidase-dependent production of reactive oxygen species.1,2 An increase in cytosolic calcium cations activates NADPH oxidases and acts as a cofactor for peptidylarginine deiminase 4 (PAD4). PAD4 catalyzes citrullinated histone H3 (Cit-H3) inducing chromatin decondensation, resulting in a cell extrusion of mixture of DNA and bactericidal proteins, including MPO and NE, which all serves as a specific markers of NETosis.1,2 While beneficial in infection settings, excessive or dysregulated NET formation can cause significant tissue damage and organ dysfunction. NETs have been implicated in the pathogenesis of various acute and chronic inflammatory diseases, including myocardial infarction, atherosclerosis, autoimmune diseases, diabetes, and chronic kidney disease (CKD). Elevated NETosis markers, including cell-free DNA (cfDNA) and Cit-H3, are commonly observed in these conditions, underscoring their role in disease progression.3 Given the dual nature of NETosis, understanding how different factors influence this process is critical for developing targeted therapies. This study focuses on two key aspects: evaluating the effects of antiglycemic medications on NETosis and investigating NETosis across different stages of immune-mediated kidney disease before and after immunosuppressive therapy.

(The hemodialysis part of this trial is not included in the current protocol)

Part 1: Evaluating the Effects of Antiglycemic Medications on NETosis Dysregulated NETosis in diabetic and CKD patients contributes to systemic inflammation and organ damage. Studies have shown elevated levels of NETosis markers, such as cfDNA and Cit-H3, in these populations. While metformin has demonstrated the ability to reduce NET formation in diabetic patients, the effects of newer glucose-lowering agents, including SGLT2 inhibitors and GLP-1 receptor agonists, remain unexplored.4 Both SGLT2 inhibitors and GLP-1 receptor agonists have demonstrated robust cardiovascular and renal protective effects.5 This study aims to evaluate their impact on basal and stimulated NETosis in diabetic and CKD patients with various etiologies. By analyzing NETosis markers before and after treatment, this research will provide insights into whether these agents can modulate NETosis, thereby offering additional anti-inflammatory benefits and reducing disease-associated complications.

Part 2: Evaluating NETosis at Different Stages of Immune-Mediated Kidney Disease and After Immunosuppressive Therapy Immune-mediated kidney diseases, such as ANCA-associated vasculitis, involve heightened NETosis that contributes to kidney injury and systemic inflammation.2 Chronic kidney disease resulting from these conditions often exhibits elevated NET formation, exacerbating disease progression. This study seeks to investigate the dynamics of NETosis at various stages of CKD caused by immune-mediated diseases.

In addition, the effects of immunosuppressive therapy on NETosis will be assessed. Immunosuppressive medications, a cornerstone in the treatment of diseases like ANCA-associated vasculitis, can influence neutrophil activity. By monitoring NETosis markers, including cfDNA, Cit-H3, MPO, and NE, before and after initiating immunosuppressive therapy, the study aims to identify patterns of response and the potential therapeutic modulation of NETosis.

Conclusion This comprehensive investigation into the effects of antiglycemic medications and immunosuppressive therapy on NETosis will provide critical insights into the interplay between treatment, inflammation, and disease progression in diabetic and CKD patients. These findings may help pave the way for targeted interventions aimed at modulating NETosis and improving outcomes in high-risk populations.

Study Timeline

• Study Start: 2024-05-13
• Study First Submitted: 2025-01-26
• Status Verified: 2025-02
• Study First Submitted QC: 2025-02-10
• Last Update Submitted: 2025-02-10
• Study First Posted: 2025-02-12
• Last Update Posted: 2025-02-12
• Primary Completion: 2030-12-30
• Study Completion: 2031-12-30

Recruitment & Eligibility

• Status: RECRUITING
• Sex: All
• Target Recruitment: 70

Inclusion Criteria

DM therapy study: - Diabetic patients aged 18 years or older (men and women).

• Patients who have not previously received SGLT2 inhibitors or GLP-1 receptor agonists.

  -Chronic kidney disease (CKD) patients :50 CKD patients with various etiologies.

  • Focus:

• This part of the study will specifically evaluate immune-mediated kidney disease, such as ANCA-associated vasculitis, and the effects of immunosuppressive therapy on NETosis.

  •

Exclusion Criteria

• Patients with acute infections, hematologic or oncologic diseases, or positive for HIV or Hepatitis B/C

Study & Design

• Study Type: OBSERVATIONAL
• Study Design: Not specified

Primary Outcome Measures

Name	Time	Method
NETosis marker- citrullinated histone H3 (citH3)	Before treatment, 1,2 , 3, 6 and 12 months after starting anti-diabetic drugs or immunosuppression	* NETosis markers: NET formation will be assessed in sera blood samples across all groups.; * Focus on Naïve Patients: Emphasis will be placed on evaluating the initial effects of antiglycemic medications in diabetic patients and the impact of immunosuppressive therapy in CKD patients with immune-mediated diseases.; * Samples will be collected at identical intervals and compared among diabetic andCKD, groups to assess differences in NETosis levels over time (controls- only baseline NETosis markers) and in response to treatment.; All serum samples will be diluted 1:2 and citrullinated histone H3 (citH3) will be quantified using the Citrullinated Histone H3 ELISA Kit: O.D. (501620; Cayman Chemical, Ann Arbor, USA).
NETosis marker- Myeloperoxidase	Before treatment, 1,2,3,6 and12 moths after treatment.	Samples will be collected at identical intervals and compared among diabetic andCKD, groups to assess differences in NETosis levels over time (controls- only baseline NETosis markers) and in response to treatment.; All serum samples will be diluted 1:2 . MPO measurement will be conducted using Myeloperoxidase ELISA Kit , ng/ml (501410; Cayman Chemical).
NETosis marker- Neutrophil elastase (NE)	Before treatment, 1,2 , 3, 6 and 12 months after starting anti-diabetic drugs or immunosuppression	Samples will be collected at identical intervals and compared among diabetic andCKD, groups to assess differences in NETosis levels over time (controls- only baseline NETosis markers) and in response to treatment.; All serum samples will be diluted 1:2 , and NE measurement will be conducted using Human Neutrophil Elastase ELISA Kit, ng/ml (ab204730; Abcam
NETosis marker: Peptidylarginine deiminase 4-PAD4	Before treatment, 1,2 , 3, 6 and 12 months after starting anti-diabetic drugs or immunosuppression	Samples will be collected at identical intervals and compared among diabetic andCKD, groups to assess differences in NETosis levels over time (controls- only baseline NETosis markers) and in response to treatment.; All serum samples will be diluted 1:2 and PAD4 measurement will be conducted using (PADI4 ELISA Kit, ng/ml (ELH-PADI4, RayBiotech) according to the manufacturer's instructions

Secondary Outcome Measures

Name	Time	Method
Kidney function	Before treatment, 1,2 , 3, 6 and 12 months, and each year (during 5 years) after starting anti-diabetic drugs or immunosuppression therapy.	1. Kidney Function; * Estimated glomerular filtration rate (eGFR, ml/min) will be measured using CKD-EPI creatinine) equation.; * These measures will monitor changes in kidney function over time and will be correlated with NETosis markers assessed prior to and during treatment.
All cause mortality	1,2 , 3, 6 and 12 months, and each year (during 5 years) after starting anti-diabetic drugs or immunosuppression therapy	All-cause mortality: Survival outcomes will be tracked through yearly analyses.; * Correlations will be drawn between NETosis markers and mortality rates to understand their predictive value and treatment effects. These secondary outcomes will highlight the association between NETosis and long-term renal and cardiovascular outcomes
Urine protein/ creatinine ratio	Before treatment, 1,2 , 3, 6 and 12 months, and each year (during 5 years) after starting anti-diabetic drugs or immunosuppression therapy	Urine protein/ creatinine ratio w (mg/gr) will be measured.; o These measures will monitor changes in kidney function over time and will be correlated with NETosis markers assessed prior to and during treatment
Major adverse cardiovascular events (MACE)	1,2 , 3, 6 and 12 months, and each year (during 5 years) after starting anti-diabetic drugs or immunosuppression therapy	Major adverse cardiovascular events (MACE): Including myocardial infarction, stroke, heart failure and cardiovascular death (present/absent).; * Yearly analyses will evaluate the occurrence of these events in relation to NETosis markers

Trial Locations

Locations (1)

Galilee Medical Center; 🇮🇱 Nahariya, Israel