Tear Fluid Progranulin as a Noninvasive Biomarker for the Monitoring of Corneal Innervation Changes in Patients With Type 2 Diabetes Mellitus

Abstract

Purpose: This study aimed to investigate the expression levels of progranulin (PGRN) in the tears of patients with diabetic retinopathy (DR) versus healthy controls. Additionally, we sought to explore the correlation between PGRN levels and the severity of ocular surface complications in patients with diabetes.

Methods: In this prospective, single-visit, cross-sectional study, patients with DR (n = 48) and age-matched healthy controls (n = 22) were included and underwent dry eye examinations. Tear fluid was collected, and its components were analyzed using the Luminex assay. The subbasal nerve plexus of all participants was evaluated by in vivo confocal microscopy.

Results: Patients with DR exhibited more severe dry eye symptoms, along with a reduction in nerve fiber density, length, and branch density within the subbasal nerve plexus, accompanied by an increase in the number of dendritic cells. Tear PGRN levels were also significantly lower in patients with diabetes than in normal controls, and the levels of some inflammatory factors (TNF-α, IL-6, and MMP-9) were higher in patients with DR. Remarkably, the PGRN level significantly correlated with nerve fiber density (R = 0.48, P < 0.001), nerve fiber length (R = 0.65, P < 0.001), and nerve branch density (R = 0.69, P < 0.001).

Conclusions: Tear PGRN levels might reflect morphological changes in the corneal nerve plexus under diabetic conditions, suggesting that PGRN itself is a reliable indicator for predicting the advancement of neurotrophic keratopathy in patients with diabetes.

Translational relevance: PGRN insufficiency on the ocular surface under diabetic conditions was found to be closely associated with nerve impairment, providing a novel perspective to discover the pathogenesis of diabetic complications, which could help in developing innovative therapeutic strategies.

Figure 1.

Representative IVCM images of the subbasal nerve plexus. (A) Confocal image of a healthy individual. (B) Confocal image of a patient with DR with fewer nerve fibers. Scale bar: 100 µm.

Figure 2.

Comparison of nerve morphological changes between the diabetic group and normal control group. (A) NFD was reduced in patients with diabetes (P < 0.001). (B) A significant reduction was observed in the NFL of patients with diabetes compared with that of normal controls (P < 0.001). (C) The NBD decreased in the diabetic group (P < 0.001). (D) The number of DCs increased among the patients with diabetes (P = 0.001).

Figure 3.

Level of different components in tears quantified using Luminex assay. (A) Tear PGRN levels were significantly reduced in patients with diabetes compared with that in normal controls (P < 0.001). (B–D) The levels of three of the pro-inflammatory cytokines increased in the DR group, including TNF-α (P = 0.055) (B), IL-6 (P < 0.001) (C), and MMP-9 (P = 0.007) (D). (E) No significant difference was observed in IL-1α levels between the two groups (P = 0.57).

Figure 4.

Scatter diagram with regression lines and 95% CI ranges indicating the relationship between tear PGRN level and dry eye parameters. (A) No significant correlation was observed between PGRN level and TBUT (R = 0.17; P = 0.16). (B) PGRN level was positively correlated with Schirmer's score with significance (R = 0.37; P = 0.0015). (C) No significant correlation was observed between PGRN level and staining scores (R = –0.18; P = 0.13).

Figure 5.

The correlation between tear PGRN level and subbasal nerve plexus changes. (A) The positive correlation between PGRN level and NFD was established (R = 0.48; P < 0.001). (B) PGRN level was positively correlated with NFL (R = 0.65; P < 0.001). (C) An excellent correlation was observed between PGRN level and NBD (R = 0.69; P < 0.001). (D) No strong correlation was detected between tear PGRN level and DC density (R = –0.17; P = 0.18).

Figure 6.

Further analysis for adjusting confounders (gender, age, and DR). In the scatter diagram, dots indicate female, triangles indicate male, and blue and red represent the DR and control groups, respectively. The size of the legend increases with age. (A) The correlation between tear PGRN level and Schirmer's test result was not significant considering the three covariates (P = 0.15). (B) There was a weak association between PGRN levels and NFD (P = 0.051). (C) PGRN level was weakly correlated with NFL (P = 0.069). (D) Comparison of tear PGRN level with NBD revealed significance (P = 0.0059).

Figure 7.

Graphical abstract of the study findings.