Urinary podocalyxin is an early marker for podocyte injury in patients with diabetes: establishment of a highly sensitive ELISA to detect urinary podocalyxin

Abstract

Aims/objective: Nephropathy, a major complication of diabetes, is the leading cause of end-stage renal disease. Recent studies have demonstrated that podocyte injury is involved in the onset of and progression to renal insufficiency. Here, we describe a novel, highly sensitive ELISA for detecting urinary podocalyxin, a glycoconjugate on the podocyte apical surface that indicates podocyte injury, particularly in the early phase of diabetic nephropathy.

Methods: Urine samples from patients with glomerular diseases (n = 142) and type 2 diabetes (n = 71) were used to quantify urinary podocalyxin by ELISA. Urine samples were obtained from 69 healthy controls for whom laboratory data were within normal values. Podocalyxin was detected in urine by immunofluorescence, immunoelectron microscopy and western blotting.

Results: Morphologically, urinary podocalyxin was present as a vesicular structure; western blotting showed it as a positive band at 165-170 kDa. Levels of urinary podocalyxin were elevated in patients with various glomerular diseases and patients with diabetes. In patients with diabetes, urinary podocalyxin was higher than the cut-off value in 53.8% patients at the normoalbuminuric stage, 64.7% at the microalbuminuric stage and 66.7% at the macroalbuminuric stage. Positive correlations were observed between urinary podocalyxin levels and HbA(1c), urinary β(2) microglobulin, α(1) microglobulin and urinary N-acetyl-β-D-glucosaminidase, although urinary podocalyxin levels were not correlated with other laboratory markers such as blood pressure, lipid level, serum creatinine, estimated GFR or proteinuria.

Conclusions/interpretation: Urinary podocalyxin may be a useful biomarker for detecting early podocyte injury in patients with diabetes.

Fig. 1

(a) Western blot analysis of monoclonal antibodies: 1) native PCX; 2) GST–Intra PC; 3) GST–PC-35; 4) GST–PC-46. (b) IF using cultured cells revealed positive staining with clone #45 when non-permeabilised cells were used (surface IF). When permeabilised cells were used (intracellular IF), clones #5 and no. 147 yielded positive staining. Clone #45 also showed positive staining on intracellular IF. (c) IF using monoclonal antibodies 22A4, #45, #5 and no. 147 and normal kidney sections. The antibodies showed bright glomerular staining, with faint staining of endothelial cells of the blood vessels

Fig. 2

(a) IF findings with urine precipitates after 453,000 g centrifugation of urine from a diabetic patient with normoalbuminuria. The anti-PCX monoclonal antibody 22A4 showed fine granular structures. Original magnification ×400. (b) IEM findings for the urine sample from a normoalbuminuric diabetic patient. Scale bar 100 nm. (c) The presence of PCX was confirmed by western blot analysis. The urine precipitate after centrifugation at 453,000 g of urine from two patients (patient 1 was a normoalbuminuric patient and patient 2 had IgA nephropathy) and glomerular lysate were used as samples with two anti-PCX monoclonal antibodies (22A4 and #5). Gl, glomerular lysate; M, molecular size marker; P1, patient 1; P2, patient 2

Fig. 3

u-PCX levels in various renal diseases (a) and diabetes (b). (a) Significantly higher levels of u-PCX were excreted by patients with IgA nephropathy, MCNS/FSGS and LN/MGN compared with controls (all ^*** p < 0.001). (b) Significantly higher levels of u-PCX were excreted by normoalbuminuric, microalbuminuric and macroalbuminuric patients with diabetes compared with healthy controls (all ^*** p < 0.001). A significant difference was noted between the normoalbuminuric and microalbuminuric groups (^† p < 0.05). Contr, controls; IgAN, IgA nephropathy; Macro, macroalbuminuric; Micro, microalbuminuric; Norm, normoalbuminuric