In situ assessment of Mindin as a biomarker of podocyte lesions in diabetic nephropathy

Abstract

Diabetic nephropathy (DN) is the leading cause of chronic kidney disease and end-stage renal failure worldwide. Several mechanisms are involved in the pathogenesis of this disease, which culminate in morphological changes such as podocyte injury. Despite the complex diagnosis and pathogenesis, limited attempts have been made to establish new biomarkers for DN. The higher concentration of Mindin protein in the urine of patients with type 2 diabetes mellitus suggests that it plays a role in DN. Therefore, this study investigated whether in situ protein expression of Mindin can be considered a potential DN biomarker. Fifty renal biopsies from patients diagnosed with DN, 57 with nondiabetic glomerular diseases, including 17 with focal segmental glomerulosclerosis (FSGS), 14 with minimal lesion disease (MLD) and 27 with immunoglobulin A nephropathy (IgAN), and 23 adult kidney samples from autopsies (control group) were evaluated for Mindin expression by immunohistochemistry. Podocyte density was inferred by Wilms' tumor 1 (WT1) immunostaining, while foot process effacement was assessed by transmission electron microscopy. Receiver operative characteristic (ROC) analysis was performed to determine the biomarker sensitivity/specificity. Low podocyte density and increased Mindin expression were observed in all cases of DN, regardless of their class. In the DN group, Mindin expression was significantly higher than that in the FSGS, MCD, IgAN and control groups. Higher Mindin expression was significantly positively correlated with foot process effacement only in class III DN cases. Furthermore, Mindin protein presented high specificity in the biopsies of patients with DN (p < 0.0001). Our data suggest that Mindin may play a role in DN pathogenesis and is a promising biomarker of podocyte lesions.

Fig 1. Podocyte density and foot process effacement in renal biopsies of diabetic nephopathy.

(A) Significant reduction in podocyte density assessed by WT1 expression in DN cases compared to the control group. Mann‒Whitney test. (B) Representative biopsy of the control group showing high in situ WT1 expression in the glomerular compartment. (C) Representative biopsy of the DN group showing low WT1 expression in the glomerular compartment. (D) Foot process width in the control and DN groups. Mann‒Whitney test. (E) Foot process width in the control group and in different DN classes. Kruskal‒Wallis test followed by Dunn’s posttest. *p<0.05. (F) Normal pedicels under TEM in the control group. (G) Foot process effacement under TEM in the DN group.

Fig 2. Mindin in situ expression in the glomerular compartment.

(A) Comparison of Mindin in situ expression in DN cases, kidney biopsies from patients diagnosed with nondiabetic glomerular diseases and the control group. Kruskal‒Wallis test followed by Dunn’s posttest. p<0.05. (B) Low in situ Mindin expression in a representative biopsy of the control group. (C) Representative biopsy of the DN group showing high in situ Mindin expression. Representative biopsy of (D) FSGS, (E) MCD and (F) IgAN cases showing moderate in situ Mindin expression.

Fig 3. Mindin in situ expression and podocyte injury.

(A) Trend of positive correlation between Mindin expression and foot process width in the DN group. Spearman’s correlation (rS). (B) Significant positive correlation between Mindin and foot process width in the class III DN group. Spearman’s correlation (rS). (C) The Mindin ROC curve in the DN group revealed high specificity and moderate sensitivity, showing Mindin protein as a biomarker of podocyte injury in DN.

Fig 4. Mindin in situ expression and duration of DN.

(A) Comparison of Mindin in situ expression in the DN < 10 years and DN > 10 years groups. Mann‒Whitney test. The Mindin ROC curve in the (B) DN group < 10 years and (C) DN > 10 years group revealed high specificity and moderate sensitivity.