Our study provides the first evidence showing that in patients with T1D or LADA, the peripheral total WBC count, neutrophil count, and NLR, even when within the normal range, are independently and significantly associated with DKD. The association between neutrophils and DKD persists even after controlling for conventional risk factors, including age, sex, smoking status, blood pressure, lipid profile, and glucose control, as well as obesity.
DKD is common to all types of diabetes; thus, hyperglycaemia is a major risk factor for DKD. However, hyperglycaemia does not account for all changes that are observed in the renal tissue.14 The pathogenesis of DKD is complex, including genetics, haemodynamic changes, disorders of glucose and lipid metabolism, effects of cytokines and growth factors, oxidative stress, and inflammatory responses. Several lines of evidence demonstrate inflammation as a cardinal pathogenic mechanism corresponding to the development and progression of DKD, in which several types of innate immune cells are actively involved.15 Neutrophils, as the most abundant and inflammation-related immune cell type in the circulation, might be involved in the pathogenesis of DKD.
The abnormal activation of blood neutrophils has been reported in diabetes patients.2, 16–18 Neutrophils from DKD patients exhibited faster exocytosis of primary granules than those from either normal subjects or patients without DKD. Neutrophils from DKD patients failed to remove CD11b (a subunit of Mac–1) from the cell membrane, and the adhesion molecule CD11b seems to persist at increased levels.16 The elevated expression of CD11b could play a role in directing neutrophil migration in inflamed renal tissue expressing upregulated levels of the cell adhesion molecule ICAM–1. In agreement with these data, spontaneous adhesion has been shown to be increased significantly in neutrophils from diabetes patients with proteinuria compared with those from patients with normoalbuminuria or healthy control subjects.17 The oxidation of serum albumin may cause neutrophil activation and further oxidation of albumin in diabetes, which are important to the severity and progression of DKD.18 In addition, the metabolic disturbances accompanying the impairment of diabetes control could also induce neutrophil adherence to foreign surfaces and superoxide anion production in diabetes patients.2
The exact molecular mechanism by which neutrophils are involved in the development of DKD is unclear. However, there is some evidence that neutrophils could play a role in this pathological process. The migration of neutrophils to the kidney is a critical step in the progression of DKD. The influx of neutrophils is associated with an acute reaction to inflammation or injury. Neutrophils secrete enzymes and oxidation products that can damage the local microenvironment and induce tissue injury.19 Recent studies revealed that neutrophils from T1D patients were primed to produce neutrophil extracellular traps (NETs) which are made of DNA, histones, and neutrophil proteins, and high glucose level can induce NETs in vitro.20,21 In addition, myeloperoxidase (MPO), a well-established marker of NET formation, was observed to increase in kidney of Streptozotocin-induced diabetic rats,22 suggesting neutrophil formed NETs might engage in the pathogenic mechanisms of DKD.
Furthermore, several epidemiological and clinical studies have evaluated the association of neutrophils or the NLR with DKD in T2D. Azab showed that the NLR could act as a predictor of worsening renal function in 338 American diabetic patients.4 Chung et al. demonstrated that peripheral neutrophil counts were independently and significantly associated with DKD in 1480 Chinese patients with T2D.3 In another study that recruited 253 Chinese subjects with T2D, an increased NLR was significantly associated with DKD, and the patients had a 2.088-fold increased risk of DKD for every unit increase in the NLR.5 Afsar et al. reported that the NLR was independently associated with the 24-hour urinary protein and urinary albumin excretion in 80 Turkish patients with newly diagnosed T2D.6 Ciray et al. also reported that the NLR was positively correlated with microalbuminuria but negatively correlated with the eGFR in 114 Turkish subjects with T2D.7 Akbas et al. showed that in 200 Turkish patients with T2D, the albuminuria levels increased as the NLR increased, and the NLR was found to be independently associated with albuminuria.8 Kahraman et al. also showed significant correlations among albuminuria, the glomerular filtration rate and the NLR in 112 patients with T2D.9 Additionally, NLR could predict renal function loss in 108 Pima Indians and 941 Europeans with T2D, as shown in a longitudinal study.10 In a recent study recruiting 247 patients with T2D and biopsy-confirmed DKD, the NLR was significantly associated with interstitial fibrosis, tubular atrophy and renal dysfunction.11
To the best our knowledge, our study provided the first clinical evidence addressing the relationship between neutrophil counts and DKD in T1D and LADA patients. We showed that the patients with DKD had higher neutrophil counts than the patients without DKD in the contexts of T1D and LADA. For all patients or those with normoalbuminuria, the neutrophil counts showed a significant decrease in patients with T1D compared to those with LADA, which is consistent with findings that reduced circulating neutrophil counts associated with T1D.23 However, the neutrophil counts were comparable among the albuminuria patients with T1D and LADA, indicating that the degrees of inflammation involved in DKD in different types of diabetes might be similar. Additionally, in T1D, hyperglycaemia usually starts in the first decades of life and is generally the only recognized cause of DKD. In contrast, in LADA which is also an autoimmune diabetes but frequently accompanied by metabolic syndrome,12 hyperglycaemia usually develops after 30 years of age when the kidneys begin suffering from the long-term consequences of ageing and other recognized promoters of chronic renal injury, such as arterial hypertension, obesity, dyslipidaemia, and smoking. However, the association between neutrophil counts and DKD persists in T1D and LADA after adjusting for age, sex and other risk factors, suggesting the independent influence of inflammatory markers such as neutrophils on the development of DKD.
Our study has several limitations. First, due to the relatively small sample size of LADA patients and the retrospectively cross-sectional design, future large-scale, longitudinal studies are needed to clarify the alterations and dynamic changes in the circulating neutrophil counts in the patients with different types of diabetes and to further investigate the concrete roles of the neutrophil counts in the pathogenesis of DKD in each type of diabetes. Second, this study did not provide serological data on neutrophil serine proteases, such as neutrophil elastase (NE), proteinase 3 (PR3), or cathepsin G (CG). Future studies focusing on whether neutrophils are activated to secrete the neutrophil serine proteases involved in the pathogenesis of DKD should be warranted in future.
In conclusion, our study suggests that the neutrophil counts reflect DKD in subjects with autoimmune diabetes. These findings support the roles of neutrophils in the pathogenesis of the kidney complications of diabetes and provide a possible perspective for using neutrophils as a potential biomarker for the early identification of individuals at high risk of developing DKD and as potential therapeutic targets for DKD.