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

Diabetologia, Nov 2012

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 HbA1c, urinary β2 microglobulin, α1 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.

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

M. Hara 0 2 3 K. Yamagata 0 2 3 Y. Tomino 0 2 3 A. Saito 0 2 3 Y. Hirayama 0 2 3 S. Ogasawara 0 2 3 H. Kurosawa 0 2 3 S. Sekine 0 2 3 K. Yan 0 2 3 0 Y. Tomino Department of Nephrology, Juntendo University , Tokyo, Japan 1 ) Department of Pediatrics, Yoshida Hospital , Yoshida 32-14, Tsubame City 959-0242, Niigata, Japan 2 K. Yan Department of Pediatrics, School of Medicine, Kyorin University , Mitaka, Tokyo, Japan 3 A. Saito Department of Medicine, Niigata University , Niigata, Japan 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. - Nephropathy is a major complication of diabetes and the leading cause of end-stage renal disease; it is clinically characterised by proteinuria and progressive renal insufficiency [1]. Human podocytes (Pods) have been demonstrated to be functionally and structurally injured in the natural history of diabetic nephropathy [2]. Recently, an increase in foot-process width has been identified in patients with type 1 diabetes and microalbuminuria, and foot-process width has been shown to correlate directly with the urinary albumin excretion rate [3]. Furthermore, the number and density of Pods have been reported to be markedly reduced (podocytopenia) in patients with diabetes [4]. Pods are located outside the glomerular basement membrane (GBM). Because of the proximity of the apical region of Pods to the urinary space, pathological events occurring in this region are expected to be more easily detectable in urine than those occurring in the basal or slit diaphragm regions of Pods [5, 6]. In this study, we report the establishment of a highly sensitive ELISA for the detection of urinary (u)-podocalyxin (PCX), one of the primary glycoconjugates on the Pod surface, for identifying Pod injury in various glomerular diseases, particularly in the early phase of diabetic nephropathies. Patients and samples Urine samples from patients with glomerular diseases (n 0 142) and type 2 diabetes (n 0 71) were used for u-PDX quantification by ELISA. Urine samples were obtained from 69 healthy controls with laboratory data within normal values. Urine samples were obtained from urine voided in the morning, and were stored at 70C within 2 h of collection until quantification by ELISA. The clinical characteristics of the patients and healthy controls are shown in Tables 1 and 2. This study was approved by the ethics committees of all the hospitals involved. Informed consent was obtained from patients. Monoclonal antibodies against PCX We used two series of monoclonal antibodies in this study: antibodies against native human PCX; and antibodies against recombinant PCX. Monoclonal antibodies against native human PCX were produced using, as the immunogen, native PCX prepared from isolated glomeruli. The glomeruli were isolated from the normal portion of the kidneys obtained during nephrectomy. Isolated glomeruli were extracted in 0.2% (vol./vol.) Triton X-100 in PBS containing protease inhibitors. The extract was then incubated with wheat germ agglutinin (WGA)Sepharosel; after washing, the sialic-acid-rich material that bound to the WGA column was removed with N-acetyl--glucopyranoside. BALB/c mice were immunised with 50 g WGAbound PCX. Spleen cells were fused according to standard procedures. Clones producing anti-PCX antibody were screened by indirect immunofluorescence (IF) on cryostat sections of human kidney and were further characterised by western blot analysis and immunoprecipitation. A number of positive clones were identified. Finally, three clones (22A4, 3H11 and 4D5) were obtained and confirmed as monoclonal antibodies against native human PCX [7]. The monoclonal antibodies against recombinant PCX were produced using purified recombinant human PCXglutathione transferase (GST) fusion protein, according to the method described by Kershaw et al [8] (electronic supplementary material [ESM]). BALB/c mice were immunised with purified recombinant human PCXGST fusion protein prepared by PCR amplification of the cDNA for base pairs 10041835 of the human PCX (named PC-46, and containing both intracellular and extracellular domains) by the standard method. The resulting hybridomas were cultured in 96 well plates and screened with an ELISA using polystyrene multi-well plates coated with a WGA-binding fraction in a Triton X-100 glomerular lysate. The PCXGST fusion proteins obtained using cDNA coding for parts of the extracellular and intracellular domains of human PCX (named PC-35 and Intra PC, respectively) were used to characterise the monoclonal antibodies. The monoclonal antibodies against native P (...truncated)


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M. Hara, K. Yamagata, Y. Tomino, A. Saito, Y. Hirayama, S. Ogasawara, H. Kurosawa, S. Sekine, K. Yan. Urinary podocalyxin is an early marker for podocyte injury in patients with diabetes: establishment of a highly sensitive ELISA to detect urinary podocalyxin, Diabetologia, 2012, pp. 2913-2919, Volume 55, Issue 11, DOI: 10.1007/s00125-012-2661-7