Low-density granulocyte-driven NET formation is associated with poor prognosis in IgA nephropathy with thrombotic microangiopathy

Abstract

Introduction

Thrombotic microangiopathy (TMA) is a common pathological phenotype of immunoglobulin A nephropathy (IgAN). Low-density granulocytes (LDGs) exhibit potent pro-inflammatory properties and promote neutrophil extracellular trap (NET) formation, playing a pivotal role in thrombus development. This study aims to investigate the long-term prognosis of IgAN-TMA and explore the role of LDGs in its pathogenesis.

Methods

Sixty patients with IgAN-TMA and 249 high-risk IgAN controls were included. LDGs (CD14^lowCD15^high) were quantified via flow cytometry among 15 IgAN-TMA patients, 20 IgAN patients, and 15 healthy controls. Serum dsDNA and MPO–DNA complex levels were quantified via PicoGreen and ELISA, respectively. Serum-induced NET formation was assessed with fluorescence microscopy.

Results

Compared with controls, IgAN-TMA patients showed more adverse clinico-pathological profiles. TMA was identified as independently associated with adverse renal outcomes and poor renal prognosis in IgAN. Additionally, IgAN-TMA patients had a significantly higher proportion of circulating LDGs, correlating with disease severity. Furthermore, IgAN-TMA patients exhibited increased levels of circulating dsDNA and MPO–DNA complexes, serum from these patients significantly induced NET formation.

Conclusions

IgAN-TMA prognosis is worse than that of high-risk IgAN. LDGs are significantly elevated and correlate with the severity of the pathogenic process in IgAN-TMA, warranting further extensive investigation.

GRAPHICAL ABSTRACT

1. Introduction

Globally, immunoglobulin A nephropathy (IgAN) represents the predominant form of primary glomerulonephritis. It is the leading cause of renal failure among young adults. IgAN is characterized pathologically by the deposition of immune complexes, predominantly IgA subclass 1 (IgA1), within the mesangial region of renal glomeruli. Microangiopathic (MA) lesions of the renal arterioles are relatively common in IgAN. Approximately, 20% of patients with IgAN have varying degrees of small artery microvascular lesions. These microvascular lesions range from small artery wall thickening and translucency to microvascular lesions with or without thrombosis [1]. Among the numerous factors influencing the prognosis of IgAN, arterial and small artery lesions have attracted the attention of many researchers. Numerous investigations have demonstrated that microvascular lesions are linked to an unfavorable prognosis in patients with IgAN [1–3].

IgAN with microvascular lesions is primarily characterized by endothelial cell damage. If microthrombus formation is concurrently present, the condition is defined as thrombotic microangiopathy (TMA) lesion. TMA has been observed in 2–50% of IgAN patients, with prevalence varying across distinct study cohorts and diagnostic criteria [1,4–7]. However, the pathogenesis of TMA in IgAN remains unclear to date. Therefore, elucidating the specific molecular mechanism underlying microvascular lesions in IgAN is crucial for clarifying the pathogenesis – a specific pathological condition characterized by endothelial cell damage leading to thrombosis in glomerular capillaries, arterioles, interlobular arteries, and even arcuate arteries, along with the thickening of vessel walls and narrowing of vessel lumens [8]. Current research suggests the potential involvement of complement activation, inflammatory responses, and coagulation pathways in its disease mechanism [4,9,10]. Low-density granulocytes (LDGs) were first described in systemic lupus erythematosus (SLE), and these cells are enriched within the monocyte fraction following density gradient centrifugation [11–13]. Compared with normal-density neutrophils (NDGs), LDGs are more prone to play a critical role in vascular injury, thrombosis, and complement activation, owing to their potent pro-inflammatory effects, enhanced capacity to induce neutrophil extracellular trap formation (NETosis), and robust tissue-damaging activity [14]. Earlier investigations have indicated that LDGs contribute significantly to multiple autoimmune disorders, such as SLE, antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis, rheumatoid arthritis, and myasthenia gravis [15–20]. Recently, results from a study showed that NETosis is upregulated in IgAN and participates in its pathogenesis by promoting the release of inflammatory cytokines [21]. Furthermore, recent evidence has demonstrated that in patients with IgA vasculitis with nephritis (IgAV-N), IgA1 immune complexes induce FcαRI-mediated neutrophil activation, which elicits endothelial cell injury and thereby contributes to the pathogenesis of this disease [22]. Additionally, although our previous studies found that elevated neutrophil/lymphocyte ratios in patients with IgAN and IgAV-N were associated with disease severity and prognosis – suggesting a potential role for LDGs in IgAN [23,24] – LDGs have not yet been extensively studied in patients with IgAN-TMA.

In summary, we hypothesized that LDGs play an important role in patients with IgAN-TMA. In our study, we first analyzed the clinical case characteristics of patients with IgAN-TMA compared with those with IgAN alone and focused on the potential mechanism of LDGs in the pathogenesis of IgAN-TMA, with the aim of providing a theoretical basis and therapeutic direction for further elucidating the pathogenesis of patients with IgAN-TMA.

2. Materials and methods

2.1. Participants

Our study was a single-center retrospective study that included patients diagnosed with primary IgAN combined with TMA by renal biopsy at the First Affiliated Hospital of Zhengzhou University between January 2019 and June 2024, as well as patients with IgAN at high risk of chronic kidney disease (CKD) progression, who served as disease controls.

This study ultimately included 60 patients with primary IgAN and biopsy-proven TMA, after excluding individuals with malignancies or with an insufficient number of glomeruli for Oxford classification (n = 28). In addition, 249 high-risk IgAN patients without TMA were enrolled as disease controls for subsequent analyses. The inclusion criteria for the high-risk IgAN without TMA group were as follows: (1) a pathological diagnosis of IgAN without TMA confirmed by renal biopsy; (2) age ≥ 18 years and a follow-up duration of >6 months; (3) with a range of change in 24 h proteinuria of >1.0 g/day and estimated glomerular filtration rate (eGFR) ≥30 mL/min/1.73 m² after 3 months of supportive therapy, in accordance with the KDIGO 2021 Clinical Practice Guideline criteria for high-risk progression [25].

To minimize potential confounding effects of therapeutic interventions on inflammatory status and neutrophil function, all patients included in the mechanistic investigations (flow cytometry and NET-related assays) were treatment-naïve at the time of sample collection. Specifically, none of these participants had received immunosuppressive therapy, including glucocorticoids or other immunomodulatory agents, prior to blood sampling. Furthermore, these individuals had not yet initiated standardized pharmacological treatment for IgAN at our center at the time of biopsy and sample acquisition.

Renal biopsy specimens from all patients were independently reviewed by two experienced renal pathologists who were blinded to the clinical and laboratory data. In the event of discrepancies, a consensus was reached through joint reassessment using a multi-headed microscope. IgAN was diagnosed based on the dominant deposition of IgA in the mesangial region, as detected by immunofluorescence or immunohistochemical staining, alongside electron-dense deposits in the same area observed via electron microscopy. Patients were excluded if they had Henoch–Schönlein purpura, liver cirrhosis, viral hepatitis, or other secondary causes of IgAN. TMA was diagnosed primarily based on pathological features, including intracapillary thrombosis in arterioles and/or glomeruli, frequently accompanied by fragmented erythrocyte accumulation within capillary lumens and focal ischemic or congested glomerular tufts [26]. We also excluded individuals with conditions recognized to induce TMA, such as thrombotic thrombocytopenic purpura, hemolytic-uremic syndrome, antiphospholipid syndrome, preeclampsia, and scleroderma renal crisis.

For the mechanistic investigations, 15 healthy controls (HCs) were recruited from members of our research team, clinical staff, and university volunteers. All HCs demonstrated normal renal function, defined by an eGFR > 90 mL/min/1.73 m², and tested negative for proteinuria via qualitative assessment of random urine samples at the time of enrollment. Detailed demographic and clinical characteristics of the HCs are provided in Table 1.

Table 1.

Baseline characteristics of enrolled participants for exploring the function of LDG.

Characteristic	IgAN-TMA (n = 15)	IgAN (n = 20)	p Valuea	HC (n = 15)
Baseline
Age (years)	52 (30–62)	37.5 (29–47)	0.029	31 (29–33)
Sex (male, %)	8 (53.3%)	13 (65%)	0.492	10 (66.6%)
SBP (mmHg)	138 (132–156)	135 (128.5–141)	0.394	129 (110–136)
DBP (mmHg)	85 (80–89)	86 (75–95)	0.828	82 (76–95)
eGFR (mL/min/1.73 m2)	53.1 (26.5–68.9)	70.5 (41.2–100.8)	0.042	119.5 (115–128)
24 h-UTP (g/d)	2.98 (1.30–4.49)	1.89 (1.41–4.21)	0.665	NA
Serum Alb (g/L)	35.0 (27.3–36.9)	39.0 (36.2–42.4)	0.003	42.2 (40.5–43.8)
WBC (×109)	5.30 (4.02–8.81)	6.95 (5.75–8.51)	0.194	5.65 (4.62–8.31)
Neutrophils	3.50 (2.14–5.90)	4.55 (3.63–5.82)	0.350	3.71 (2.96–5.69)
Platelet counts (×109)	197 (105–302)	259 (227.5–273.75)	0.138	278 (254–288)
Hemoglobin (g/L)	104 (87–115)	128 (113–143.75)	<0.001	157 (118–168)
Oxford classification (%)
M-1	7 (46.7)	6 (30.0)	0.320
E-1	9 (60.0)	10 (50.0)	0.562
S-1	12 (80.0)	10 (50.0)	0.073
T-1/T-2	6 (40.0)/3 (20.0)	3 (15.0)/3 (15.0)	0.129
C-1/C-2	7 (46.7)/0 (0.0)	9 (45.0)/4 (20.0)	0.128

SBP: systolic blood pressure; DBP: diastolic blood pressure; eGFR: estimated glomerular filtration rate; 24h-UTP: 24-h urine total protein; Alb: albumin; WBC: white blood cell; M: mesangial hypercellularity; E: endocapillary hypercellularity; S: segmental glomerulosclerosis; T: tubular atrophy/interstitial fibrosis; C: crescents; IgAN-TMA: IgA nephropathy with TMA; IgAN: IgA nephropathy; HCs: healthy controls.

This represents a subset of patients used for mechanistic assays. Data are presented as median (interquartile range).

^a p Value was used to indicate differences between the IgAN-TMA and IgAN groups. Bold values indicate statistical significance (p < 0.05).

This study protocol was approved by the Medical Ethics Committee of First Affiliated Hospital of Zhengzhou University. All patients provided written informed consent prior to participation in the study.

2.2. Clinical and pathological data

Clinical data for all participants were collected at the time of renal biopsy, encompassing age, sex, systolic blood pressure (SBP) and diastolic blood pressure (DBP), serum creatinine, baseline 24-h proteinuria, serum albumin, and complete blood counts. Hypertension was defined as a resting SBP of ≥140 mmHg and/or DBP of ≥90 mmHg, or a self-reported history of the condition. eGFR was computed using the Chronic Kidney Disease Epidemiology Collaboration formula. Pathological lesions in biopsy specimens with over eight glomeruli were assessed using the updated Oxford classification, MEST-C. The composite endpoint was defined as a 30% decline in eGFR, end-stage kidney disease (ESKD), or death, whichever occurred first.

2.3. Flow cytometry

Peripheral blood mononuclear cells (PBMCs) were extracted from the peripheral blood of patients with IgAN-TMA (n = 15), IgAN (n = 20), and HCs (n = 15) via density gradient centrifugation. Ten milliliters of peripheral blood was collected, centrifuged, and the serum obtained was stored at −80 °C. The blood samples were diluted using phosphate-buffered saline (PBS) before adding lymphocyte separation medium; and the sample was centrifuged for 30 min to isolate PBMCs.

The PBMCs were diluted once with PBS and lysed in 1× red blood cell lysis buffer (Solarbio, Beijing, China) for 10–15 min at room temperature. For the experiment, 100 μL of PBMCs were first pre-incubated with 5 µL of Human TruStain FcX™ (BioLegend Fc Receptor Blocking Solution, San Diego, CA) for 10 min at ambient temperature. Subsequently, the cells were stained with 5 μL of conjugated fluorescent antibodies – including FITC-CD15 and APC-CD14 (BioLegend, San Diego, CA) – under light protection for 30 min at room temperature. The cells were then washed three times with sterile cell staining buffer (BioLegend, San Diego, CA). Finally, the cells were resuspended in 500 μL of sterile cell staining buffer and immediately analyzed by flow cytometry. Spectral overlap was corrected using single stains, while fluorescence minus one staining was applied to the gate setting. FlowJo software was utilized for data analysis. Consistent with prior reports [11,20,27], LDGs were defined as CD14^low CD15^high cells and quantified as a percentage relative to total PBMCs.

2.4. Isolation and stimulation of human neutrophils

Neutrophils were isolated from 5 mL of ethylenediaminetetraacetic-anticoagulant venous blood from healthy donors by density gradient centrifugation, using a double gradient formed by layering 3 mL Histopaque-1077 over 3 mL Histopaque-1119 (Sigma, St. Louis, MO). Residual red blood cells were lysed at room temperature for 10 min. Neutrophils were suspended in phenol red-free Roswell Park Memorial Institute 1640 medium (Gibco, Waltham, MA) at a density of 2 × 10⁶ cells/mL and used for subsequent studies.

2.5. Quantification of extracellular deoxyribonucleic acid (DNA) release of NETs

Cell-free DNA levels in serum were quantified using the Quant-iT™ PicoGreen^® double-stranded DNA (dsDNA) Assay Kit (Thermo Fisher Scientific, Waltham, MA) following the manufacturer’s protocol. The serum was diluted at a ratio of 1:30. A 1 × DNA dye buffer was prepared and incubated with the samples, shielded from light. Following incubation, the fluorescence of samples was detected with a fluorescent microplate reader at standard fluorescein wavelengths (excitation ∼480 nm, emission ∼520 nm).

2.6. Quantification of myeloperoxidase (MPO)–DNA complexes

MPO–DNA complex levels were determined using a modified capture enzyme-linked immunosorbent assay (ELISA) as reported previously [28,29]. Briefly, 96-well microtiter plates were coated with 5 µg/mL anti-MPO monoclonal antibody (Abcam, Cambridge, UK) overnight at 4 °C. After blocking with 3% bovine serum albumin (BSA), 50 µL of patient serum was added to each well, along with a peroxidase-labeled anti-DNA monoclonal antibody (Cell Death Detection ELISA kit component no. 2; Roche, Basel, Switzerland), following the manufacturer’s protocol. Samples were incubated for 2 h at room temperature on a shaker, washed with PBS, and 3,3′,5,5′-tetramethylbenzidine (TMB) substrate was added. Following a 15-min incubation at 37 °C in the dark, absorbance was measured at 450 nm using a microplate reader.

2.7. Visualization of NETs

A 200 μL suspension of neutrophils (5 × 10⁵ cells/mL) was added to 8 Lab-Tek Chamber Slides (Lab-Tek, Grand Rapids, MI). After incubation with serum or PBS at 37 °C in a 5% CO₂ incubator for 3 h, the cells were fixed with 4% paraformaldehyde for 30 min at room temperature. After blocking with 3% BSA/PBS for 1 h at room temperature, neutrophils were stained with primary anti-MPO antibody (dilution 1:200, Abcam, Cambridge, UK) overnight at 4 °C. Neutrophils were then stained with Cy3-conjugated goat anti-mouse IgG antibodies (1:500, Jackson, West Grove, PA) for 1 h at room temperature. Finally, the slides were mounted with a solution containing 4′,6-diamidino-2-phenylindole (Abcam, Cambridge, UK) for DNA staining. Randomly select three non-overlapping high-power fields (200×) from each well for counting. Then, we calculated the amount of NET formation by dividing the surface area of the obtained NETs by the surface area of neutrophils. The quantification was performed by two independent observers blinded to the clinical status of the participants.

2.8. Statistical analysis

Data analysis was performed using SPSS software (version 22.0; SPSS, Chicago, IL), and figures were generated with GraphPad Prism (version 8.0; GraphPad Software, San Diego, CA). Missing data were rigorously assessed for all variables prior to analysis. Patients with significant missing clinical or pathological data, such as baseline eGFR or Oxford classification scores, were excluded during the initial cohort selection. For the final cohort, as the missing rate for key clinical and pathological parameters was less than 5%, complete case analysis was employed, ensuring that only participants with a full set of required variables were included in the final evaluation. Continuous variables are presented as mean ± standard deviation (SD) or median (interquartile range, IQR), as appropriate. Between-group comparisons were performed using the independent samples t-test or the Kruskal–Wallis test. Categorical variables are expressed as percentages and compared using the Chi-square test. Correlations were assessed using Pearson’s correlation coefficient.

Renal prognosis was evaluated using Kaplan–Meier’s survival analysis with the log-rank test. Independent prognostic factors were examined using multivariable Cox proportional hazards models. To evaluate the independent contribution of TMA to renal outcomes, we employed a series of hierarchical models: model 1 adjusted for age and sex; model 2 further included baseline eGFR, proteinuria, and hypertension; and model 3 (the full model) additionally incorporated the Oxford MEST-C scores. The proportional hazards assumption was verified for all models using Schoenfeld residuals to ensure the validity of the hazard ratios. A two-sided p < 0.05 was considered statistically significant.

3. Results

3.1. Characteristics of IgAN with and without TMA lesions

Sixty patients with IgAN combined with TMA were included in this study, and 249 high-risk IgAN patients without TMA were included as disease controls. The baseline clinical and pathological data are shown in Table 2. As shown in Table 2, the median age of patients in TMA group was 38 years (IQR, 26–53), which was similar to that of the progressive IgAN group (37 years, IQR, 29–47). The proportion of males in both groups was comparable (42/70% vs. 145/58.2%; p = 0.094). Patients in the TMA group exhibited more severe symptoms, including elevated blood pressure: SBP was 150 mmHg (IQR 133–162) vs. 130 mmHg (IQR 120–140), p < 0.001; and DBP was 89 mmHg (IQR 82–100) vs. 80 mmHg (IQR 78–90), p = 0.001), increased 24-h urinary protein quantification (3.51 g [IQR 1.95–4.72] vs. 2.45 g [IQR, 1.63–4.13], p < 0.038), and lower serum albumin levels (36.8 g/L [IQR 32.2–41.8] vs. 39.0 g/L [IQR 36.6–42.5], p < 0.001). Patients in the TMA group also showed more severe Oxford pathological scores, including E1 [39 (65.0%) vs. 89 (35.7%), p = 0.001], S1 [54 (90.0%) vs. 164 (65.9%), p = 0.001], and T1/T2 [18 (30.0%)/32 (53.4%) vs. 91 (36.5%)/44 (17.7%), p < 0.001]. However, no significant differences were observed between the two groups for M1 [34 (56.7%) vs. 121 (48.6%), p = 0.262]. Additionally, patients in the TMA group had significantly lower platelet counts (205 [IQR 140–287] vs. 226 [IQR 194–271], p = 0.024) and hemoglobin levels (111 g/L [IQR 99–127] vs. 129 g/L [IQR 117–144], p < 0.001) than the IgAN group. However, no significant discrepancies were noted in the counts of white blood cells, lymphocytes, or neutrophils between the two groups.

Table 2.

Baseline characteristics of enrolled IgAN with and without TMA.

Characteristic	IgAN-TMA (n = 60)	IgAN (N = 249)	p Valuea
Baseline
Age (years)	38 (26–53)	37 (29–47)	0.097
Sex (male, %)	42 (70.0)	145 (58.2)	0.094
SBP (mmHg)	150 (133–162)	130 (120–140)	<0.001
DBP (mmHg)	89 (82–100)	80 (78–90)	0.001
eGFR (mL/min/1.73 m2)	27.5 (16–53)	63.2 (39.4–87.0)	<0.001
24 h-UTP (g/d)	3.51 (1.95–4.72)	2.45 (1.63–4.13)	0.038
Serum Alb (g/L)	36.8(32.2–41.8)	39.0(36.6–42.5)	<0.001
WBC (×109)	6.81 (5.31–8.46)	7.00 (6.05–8.29)	0.315
Neutrophils	4.45 (3.47–5.75)	4.21 (3.83–4.79)	0.885
Platelet counts (×109)	205 (140–287)	226 (194–271)	0.024
Hemoglobin (g/L)	111 (99–127)	129 (117–144)	<0.001
Oxford classification (%)
M-1	34 (56.7)	121 (48.6)	0.262
E-1	39 (65.0)	89 (35.7)	0.001
S-1	54 (90.0)	164 (65.9)	0.001
T-1/T-2	18 (30.0)/32 (53.4)	91(36.5)/44 (17.7)	<0.001
C-1/C-2	32 (53.3)/3 (5.0)	148 (44.0)/58 (17.3)	0.049

SBP: systolic blood pressure; DBP: diastolic blood pressure; eGFR: estimated glomerular filtration rate; 24 h-UTP: 24-h urine total protein; Alb: serum albumin; WBC: white blood cells; M: mesangial hypercellularity; E: endocapillary hypercellularity; S: segmental glomerulosclerosis; T: tubular atrophy/interstitial fibrosis; C: crescents; IgAN-TMA: IgA nephropathy combined with TMA; IgAN: IgA nephropathy.

Data are presented as median (interquartile range).

^a p Value was used to indicate the differences between the IgAN-TMA and IgAN groups. Bold values indicate statistical significance (p < 0.05).

3.2. A poorer prognosis for patients with IgAN-TMA

During a median follow-up period of 23.0 months (IQR, 16–28), 101 patients (40.6%) in the IgAN group and 28 patients (46.7%) in the IgAN-TMA group reached the composite endpoint. Kaplan–Meier’s analysis demonstrated significantly poorer renal survival in the IgAN-TMA cohort compared with the IgAN cohort (p < 0.001) (Figure 1).

Figure 1.

Kaplan–Meier’s survival curves showing renal outcome of IgA nephropathy with TMA patients and IgA nephropathy. IgAN-TMA patients had significantly lower renal survival rates than those in the progressive IgA nephropathy group (p < 0.001), and the composite end point was defined as a 30% estimated glomerular filtration rate (eGFR) decline or end-stage kidney disease (ESKD), or death. IgAN-TMA: IgA nephropathy with TMA patients.

Given the notable differences in baseline clinical and pathological features between the two groups, multivariable Cox proportional hazards models were applied to account for potential confounding. Prior to analysis, the proportional hazards assumption was tested and confirmed to be satisfied. To systematically evaluate the independent prognostic value of TMA, we constructed three progressively adjusted models (Table 3). Even after comprehensive adjustment for demographic factors, baseline clinical indicators (eGFR, proteinuria, and hypertension), and all components of the Oxford MEST-C score, the presence of TMA remained independently associated with an increased risk of adverse renal outcomes in patients with IgAN (HR, 1.782; 95% CI, 1.056–3.008; p = 0.030).

Table 3.

Multivariate Cox regression analysis verified that concurrent TMA is an independent risk factor for poor renal prognosis in IgA nephropathy.

Parameter	Unadjusted	Hazard ratio (95% confidence interval)
		Model 1a	Model 2b	Model 3c
TMA	2.060 (1.350–3.146)	2.760 (1.766–4.312)	2.428 (1.497–3.939)	1.782 (1.056–3.008)
	<0.001	<0.001	<0.001	0.030

TMA: thrombotic microangiopathy; IQR: interquartile range.

^a Model 1 adjusted for gender and age. Gender was analyzed as dichotomous data.

^b Model 2 adjusted for model 1 plus estimate glomerular filtration rate (eGFR), proteinuria, and hypertension. Hypertension was analyzed as categorical data.

^c Model 3 adjusted for model 2 plus Oxford classification scores. Oxford classification: mesangial hypercellularity score (M1 > 0.5), the presence of endocapillary proliferation (E1: present), segmental glomerulosclerosis/adhesion (S1: present), severity of tubular atrophy/interstitial fibrosis (T1: 26–50%, T2 > 50%), and presence of crescent (C1: 1–25%, C2: 26–100%).

3.3. A correlation between the proportion of LDGs and the severity of the disease in patients

LDGs exhibit potent pro-inflammatory activity and a strong propensity for NET formation, contributing to vascular injury, thrombosis, and complement activation. Based on these pathogenic features, we sought to further elucidate the role of LDGs in patients with IgAN-TMA. The clinical characteristics of the 15 patients with IgAN-TMA, 20 patients with IgAN, and 15 HCs included in the analyses of circulating free NETs and serum-induced NETosis are summarized in Table 1. The proportion of LDGs (CD14^low CD15^high) was higher in patients with IgAN-TMA than in those with IgAN (p < 0.05) and HCs (p < 0.01) (Figure 2). However, no significant difference was observed between patients with IgAN and HCs. We further analyzed the correlation between the LDGs of patients in the TMA group and clinical and pathological indicators. The results showed that the proportion of LDGs was negatively correlated with eGFR (r = −0.56, p = 0.029) (Figure 3). In the previous section, we found that the numbers of E, S, T, and C lesions were higher in the IgAN-TMA than in the IgAN group. Therefore, we analyzed the correlation between LDGs and these pathological changes. The results showed that patients with more severe E, T, and S lesions had a higher proportion of LDGs, whereas no significant difference was observed in the proportion of circulating LDGs when comparing patients with crescents (C1/C2) to those without (C0) (Supplementary Figure 1). These results suggest that LDGs are related to the clinical and pathological severity of IgAN-TMA.

Figure 2.

Low density neutrophils significantly increase in peripheral blood of IgA nephropathy with TMA patients. (A–D) Gating strategies for LDGs in peripheral blood of HCs (A, B) and IgAN-TMA patients (C, D). LDGs were identified as the CD14^low CD15^high cell population in peripheral blood mononuclear cell (PBMC). (E) The percentage of LDGs in PBMCs was significantly higher in IgAN-TMA patients compared to HCs and progressive IgAN group. Data are expressed as mean ± SD. *p < 0.05, **p < 0.01.

Figure 3.

The percentage of LDGs is correlated with the clinical and pathological severity of patients with IgAN-TMA. (A) The proportion of LDGs is negatively correlated with eGFR. (B–D) The proportion of LDGs is correlated with the pathological severity of patients with IgAN-TMA. Patients with more severe pathology have a higher proportion of LDGs. LDGs: low-density granulocytes; eGFR: estimated glomerular filtration rate; S: segmental glomerulosclerosis; E: endocapillary hypercellularity; T: interstitial fibrosis/tubular atrophy. Data are expressed as mean ± SD. *p < 0.05, **p < 0.01.

3.4. Serum from patients with IgAN-TMA induces NETs

Given that LDGs have been shown to promote NET formation in previous studies, we next examined circulating NET levels in patients with IgAN-TMA. NETs are network-like fibrous structures released by neutrophils, primarily consisting of cellular DNA and histones. MPO is an important component of NETs. Therefore, dsDNA and MPO–DNA complexes were used to represent the expression levels of NETs in the serum. The results showed that, compared with HCs and patients with IgAN, patients with IgAN-TMA had significantly increased levels of dsDNA and MPO–DNA complexes, while no significant difference was observed between patients with IgAN and HC groups (Figure 4(A,B)). Additionally, neutrophils derived from HCs were stimulated with patient serum, further verifying that serum from patients in the IgAN-TMA group could induce neutrophil activation and NET formation (Figure 4(C,D)).

Figure 4.

The serum from patients with IgAN-TMA induces the formation of neutrophil extracellular traps (NETs). (A, B) Soluble markers of NETs are increased in serum from IgAN-TMA patients. Cell-free DNA and MPO–DNA complexes were quantified in the serum from IgAN (n = 20), IgAN-TMA (n = 15), and HC (n = 15). (C) Representative fluorescence microscopy images of NET formation in HCs neutrophils incubated with serum from HC, IgAN, and IgAN-TMA patients or PMA (positive control). (D) Quantification for serum-induced NET formation is shown for HC, IgAN, IgAN-TMA, and PMA (positive control). Data are expressed as mean ± SD. *p < 0.05, and ***p < 0.001. PMA: phorbol 12-myristate 13-acetate.

4. Discussion

Microangiopathy is relatively common in patients with IgAN. It has been estimated that approximately 20% of patients with IgAN have varying degrees of microangiopathy in small arteries. Prior research has indicated an association between microangiopathies and hypertension. However, in recent years, multiple studies have confirmed that microangiopathy in IgAN is not always secondary to hypertension. Moreover, hypertension is not the sole cause, and simply controlling blood pressure may be insufficient to curb disease progression [1,5,30]. Studies in China, Netherlands, and France have confirmed that microangiopathy is a pathological injury contributing to disease progression in IgAN. It is an independent risk factor for poor long-term renal prognosis in patients with IgAN. TMA is a complex disease involving endothelial damage and microvascular thrombosis. Complement, coagulation, and genetics are involved in its development and progression [10]. When IgAN is combined with TMA, patients present not only with the clinical manifestations of IgAN but also with TMA-related symptoms, such as worsening anemia, thrombocytopenia, and rapid deterioration of renal function. Compared to patients with isolated IgAN, those with concurrent TMA have a poorer prognosis, are more likely to progress to ESRD, and have a higher mortality rate. Multiple studies have shown that the renal survival rate of patients with IgAN-TMA is significantly lower than that of patients with IgAN without TMA. Early identification and intervention are crucial to improving the prognosis of these patients [1,5,31–33]. This study conducted a retrospective analysis comparing the clinical disease characteristics and prognosis of patients with IgAN-TMA at our center with those of patients with high-risk CKD progression in IgAN. Simultaneously, the mechanism underlying LDGs in patients with IgAN-TMA was preliminarily explored.

In this study, the patients with IgAN-TMA showed a significantly higher proportion of males compared to females, and most had a history of uncontrolled hypertension, whether malignant or not. Compared to patients in the IgAN group, those in the IgAN-TMA group had a more severe clinicopathological phenotype during renal biopsy, mainly manifesting as massive proteinuria, lower eGFR, hemoglobin, and platelet count. Additionally, patients in the IgAN-TMA group had more severe pathological phenotypes, including capillary endothelial cell hyperplasia (E), segmental glomerular sclerosis (S), interstitial fibrosis/renal tubular atrophy (T), and crescentic lesions (C). Furthermore, the survival analysis indicated that, compared to patients with IgAN at a high risk of CKD progression, those with IgAN-TMA had a poorer prognosis, warranting utmost attention from clinicians. Prior studies have suggested that the complement system, inflammation, and tumors may be related to the pathogenesis of TMA. However, the pathogenesis of microvascular lesions has not been fully elucidated. Some studies have indicated that complement activation may play an important role in IgAN with microvascular lesions. Recent studies have also shown that, in the Chinese population, over 90% of patients with IgAN and microvascular lesions have deposits of C3d, C5b-9, and C4d in their arteries, further confirming that complement activation may be involved in the occurrence of microvascular lesions. Furthermore, whole-exome sequencing revealed that, in addition to the complement system, the coagulation pathway may also be involved in the occurrence and development of this lesion. In our study, the platelet count of patients with IgAN-TMA was significantly decreased, and their clinicopathological phenotypes were poor, resulting in a worse overall prognosis, consistent with previous literatures [2].

LDGs are a unique subset of neutrophils that exhibit strong proinflammatory activity and promote NET formation. They are critical for regulating innate and adaptive immune responses in multiple inflammation-associated diseases. Meanwhile, growing evidence suggests that LDGs contribute substantially to the pathogenesis and progression of diverse conditions such as infections, autoimmune diseases, and malignancies. LDGs have been extensively studied in autoimmune diseases, such as rheumatoid arthritis, SLE, ANCA-associated vasculitis, and idiopathic myositis. In this study, we found that the proportion of LDGs in the circulation was significantly increased in patients with IgAN-TMA and was correlated with disease severity, suggesting that LDGs may play an important role in the pathogenesis of TMA. Studies have shown that, during inflammation or a state of stress, the body releases various cytokines and chemokines that promote LDG production. Therefore, the increased proportion of LDGs in the circulation of patients with IgAN-TMA may be related to the immune-inflammatory components. Although systemic immune inflammation in IgAN has received less attention, the recognized role of mucosal immunity in its pathogenesis and the role of glucocorticoids in treatment have prompted further exploration of circulating inflammatory mechanisms responses in these patients.

Excessive activation of neutrophils can lead to the formation of NETs, which are widely regarded as inducers of atherosclerosis and venous thromboembolism [10,34,35]. More importantly, NETs contribute to endothelial damage in diseases such as diabetic nephropathy, ANCA-associated vasculitis, and SLE [36–38]. Also, circulating levels of NETs are significantly elevated in CKD patients, are associated with thrombotic events, and constitute a risk factor for poor long-term prognosis [39].

Previous studies have demonstrated that complement and NETs play critical roles in the pathogenesis of diverse inflammatory disorders, including autoimmune and infectious diseases [40]. Accumulating evidence has further revealed a complex bidirectional crosstalk between the complement cascade and NETosis, which is critically involved in the development of multiple conditions such as anti-neutrophil cytoplasmic antibody-associated vasculitis, SLE, and antiphospholipid syndrome [40,41]. The interaction between complement activation and NETs carries important implications for therapeutic strategies. In the recent years, novel agents targeting NETs degradation (recombinant DNase I [42] and anti-DNase antibody [43]) and complement component inhibitors (anti-C5 humanized monoclonal antibody, C5aR inhibitor) have emerged and demonstrated promising efficacy in autoimmune diseases. Extensive evidence indicates that NETs amplify complement activation, whereas complement-derived factors promote NET formation, thereby establishing a vicious cycle of inflammation and tissue injury. This bidirectional crosstalk suggests that simultaneous targeting of complement components and NETs may exert synergistic effects, mitigate immune-mediated tissue damage, and improve long-term disease prognosis. Complement activation has emerged as a key focus in IgAN research, with numerous complement-targeted agents currently under investigation [44]. Previous studies have shown that the complement system is involved in the interaction between NETs and is related to the occurrence and development of TMA [9,40]. Furthermore, studies have shown that complement C5a promotes NETs formation, thereby facilitating the formation of arterial thrombi [45]. Therefore, targeting C5a may reduce the formation of NETs and thrombosis, thereby contributing to the treatment of related autoimmune diseases. In this study, we found that the stimulation of neutrophils from healthy individuals with serum from IgAN-TMA significantly induced NET formation. This confirms that LDGs and NETs formation may be involved in the pathogenesis and inflammatory response to IgAN-TMA.

Based on current understanding, we speculate that pathogenic IgA1 molecules may trigger inflammatory responses and complement activation. This, in turn, promotes neutrophil activation, release of inflammatory mediators, endothelial injury, and activation of the coagulation system, ultimately contributing to microvascular lesions and thrombosis (Figure 5). These mechanisms warrant further investigation and may provide potential targets for future therapeutic strategies. One promising approach involves the direct degradation or inhibition of NETs. Recombinant human DNase I, which has established efficacy in treating cystic fibrosis, could potentially be repurposed to digest the DNA scaffold of NETs, thereby reducing microvascular occlusion and endothelial exposure to cytotoxic histones. Additionally, inhibitors of peptidylarginine deiminase 4, the enzyme responsible for histone citrullination and subsequent chromatin decondensation, are currently under investigation in various autoimmune conditions and could potentially prevent NETosis at its source.

Figure 5.

Proposed model: crosstalk between neutrophil extracellular traps (NETs) and the complement system may contribute to IgAN pathogenesis. In IgAN patients, inflammatory factors activate neutrophils to induce NETs formation, which further triggers the coagulation and complement pathways, forming a proinflammatory amplification loop that ultimately leads to vascular endothelial injury and thrombosis.

To our knowledge, this is the first study to demonstrate that the proportion of LDGs is significantly elevated in patients with IgAN-TMA and is associated with disease severity. Moreover, we observed that serum from these patients can stimulate NET formation, suggesting a potential role of NETs in the pathogenesis of IgAN-TMA.

Despite providing novel insights, our study has several limitations. First, this single-center retrospective study utilized a specific ‘high-risk’ IgAN control group; while this intentionally minimized confounding from baseline disease severity, it limits the generalizability of our findings to milder IgAN populations. Second, the mechanistic assays were conducted on a small convenience subset due to the strict requirement for freshly isolated cells. This resulted in baseline imbalances (e.g., age and eGFR) that could independently influence neutrophil biology. Furthermore, our cross-sectional design precludes definitive causal inference, leaving it unclear whether the LDG-NET axis primarily drives TMA or is a secondary consequence of the pro-inflammatory microthrombotic environment. Third, the NET surrogate markers evaluated (dsDNA and MPO–DNA complexes) are semi-quantitative and lack absolute specificity, as they may also be elevated during necrosis or apoptosis. Finally, the absence of longitudinal data to monitor post-treatment biomarker dynamics and the undefined downstream molecular pathways highlight the need for future prospective, multi-center investigations.

In summary, our research findings indicate that, compared to patients with IgAN who are at high risk of CKD progression, those with IgAN-TMA have a poorer prognosis. The proportion of LDGs in patients with IgAN-TMA was significantly higher in the peripheral circulation and correlated with disease severity. NET formation is also significantly increased in patients with IgAN-TMA. These results indicate that LDGs might exert a notable role in the pathogenesis of IgAN-TMA, warranting further in-depth investigation.

Funding Statement

This study was supported by the National Natural Science Foundation of China (82300801 to Qianqian Li), the Henan Province Science and Technology Innovation Talent Program (LHGJ20230241 to Qianqian Li), and Open Project of Henan Key Laboratory of Imaging and Intelligent Processing (HKLIIP2023-A01 to Genyang Cheng).

Ethical approval

This study was approved by the Medical Ethics Committee of First Affiliated Hospital of Zhengzhou University (Approval No. 2023-KY-0591). All methods were conducted in accordance with relevant guidelines and regulations, including the Declaration of Helsinki.

Consent form

Written informed consent was obtained from all participants and/or their legal guardians prior to their participation in the study.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.