Biomarkers of Endothelial Injury in Focal Segmental Glomerulosclerotic Nephrosis

Abstract

Enhanced circulating endothelial cells, elevated transforming growth factor beta, and depleted vascular endothelial growth factor were observed in nephrosis associated with focal segmental glomerulosclerosis (FSGS). Increased endothelial cell loss may be due to the elevated transforming growth factor beta, which can induce apoptosis of podocyte as well as tubular epithelium. Such injury may explain the depletion of vascular endothelial growth factor and increased endothelial cell loss in these patients. There biomarkers may have relevance to the altered intrarenal hemodynamics commonly observed in FSGS nephrosis.

• circulating endothelial cell
• hemodynamics
• transforming growth factor beta
• vascular endothelial growth factor
• nephrosis

Introduction

Glomerular endothelial cell dysfunction and renal microvascular injury have previously been noted in nephrosis associated with focal segmental glomerulosclerosis (FSGS). These are reflected by an enhanced endothelial cell cytotoxicity induced in vitro by a patient's own serum[1] and by a decreased endothelial factor VIII staining in the renal microcirculation of FSGS nephrosis,[2] respectively. Such glomerular endothelial cell dysfunction can be assessed by intrarenal hemodynamic study that usually reveals a reduction in renal plasma flow and peritubular capillary flow which supplies the tubulointerstitium.[3&4] The reduction in peritubular capillary flow has therapeutic implications, because peritubular capillary flow reduction is relevant to the pathogenesis of tubulointerstitial fibrosis and renal disease progression.[5] Early detection of glomerular endothelial cell injury or dysfunction would have better impact on the clinical response to therapy than the late recognition of the vascular disease. In this regard, determination of intrarenal hemodynamic study is generally not available due to limited resource, therefore, a search for an alternative biomarker that would reflect endothelial cell injury would have relevance to the therapeutic strategy. It is the purpose of this study to perform biomarkers that would reflect endothelial cell injury, such as circulating endothelial cell, transforming growth factor beta, and vascular endothelial growth factor in nephrotic patients associated with FSGS.

Material and Method

Fifteen nephrotic patients associated with FSGS were enrolled in the study. Ten age-match subjects served as healthy controls.

Enumeration of Circulating Endothelial Cells

The number of circulating endothelial cells was examined using the buffy coat smear technique as described by Solovey.[6] Diluted blood was layered on a Ficoll-Hypaque density gradient (Histopaque-1077, Sigma) and sedimented for 30 min at 250 g. The pooled supernatant and interface were centrifuged for 5 min at 1200 g, and a smear was made of buffy-coated pellet. After being air dried, the smear was fixed with 4% paraformaldehyde for 10 min and stained with FITC-conjugated sheep antihuman von Willebrand factor (Seroter, UK). The number of circulating endothelial cells per 1 ml of whole blood was examined using fluorescence microscopy. Negative controls were provided by the white cells on the smears and by parallel slides prepared with control negative antibody.

ELISA for Vascular Endothelial Growth Factor (VEGF)

This assay employed the quantitative sandwich enzyme immunoassay technique. Standards and samples were pipetted into the wells, and any VEGF present was bound by the immobilized antibody. After washing away any unbound substances, an enzyme-linked polyclonal antibody specific for VEGF was added to the wells. Following a wash to remove any unbound antibody enzyme reagent, a substance solution was added to the wells, and color developed in proportion to the amount of VEGF bound in the initial step. The color development was stopped, and the intensity of the color was measured.

ELISA for Transforming Growth Beta (TGF-β)

TGF-β-soluble receptor type II, which bound TGF-β1, had been precoated onto a microplate. Standards and samples were pipetted into the wells, and any TGF-β1 present was bound by the immobilized receptor. After washing away any unbound substances, an enzyme-linked polyclonal antibody specific for TGF-β1 was added to the wells to sandwich the TGF-β1 immobilized during the first incubation. Following a wash to remove any unbound-enzyme reagent, a substrate solution was added to the wells and color developed in proportion to the amount of TGF-β1 bound in the initial step. The color development was stopped, and the intensity of the color was measured.

Statistical Analysis

Comparison of the sample mean of two quantitative variables was determined by the nonparametric method using the Mann–Whitney test. The difference between groups was performed by Student's unpaired t-test. The p values below 0.05 were considered to be significant.

Results

Table 1 showed a significantly elevated number of circulating endothelial cells, transforming growth factor beta, and depleted level of vascular endothelial growth factor.

Table 1. Biomarkers of endothelial injury in FSGS nephrosis

	Circulating endothelial cell (cell/mL)	TGF β (pg/mL)	VEGF (pg/mL)
I Control	46 ± 67	3224 ± 712	299 ± 150
p value	< 0.001	< 0.001	< 0.05
II FSG nephrosis	2528 ± 585	7796 ± 2400	64 ± 50

Discussion

By virtue of its location facing the circulating blood and toxic elements such as oxidative stress, proinflammatory cytokine, and toxins, the glomerular endothelial cell is likely to be injured by such substances and is generally the primary site where kidney inflammation takes place.[7] The result of our study tends to support this conceptual view. An increased number of circulating endothelial cells was noted in FSGS nephrosis. This finding implies enhanced endothelial cell loss. Glomerular endothelial cell dysfunction,[8] namely, charges selective proteinuria, a defective release of endothelium-dependent vasodilators, which has previously been substantiated in nephrotic syndrome, concurs with the increased endothelial cell loss. The increased production of TGF-β observed in these nephrotic patients may be relevant to the mechanism of endothelial cell loss and suppression of VEGF production, because TGF-β can induce apoptosis of podocyte[9] as well as tubular epithelium,[10] which are the main sources of VEGF production. In addition, depleted VEGF production may be due to enhanced detachment of podocyte from the basement membrane secondary to hemodynamic maladjustment.[3] VEGF production was noted to be depleted (Table 1). Because VEGF is a crucial growth factor that is essential to the survival and regeneration of endothelial cells, depletion of VEGF may be relevant to enhanced endothelial cell loss.

In accordance with the preceding information, an increased number of circulating endothelial cells may be a suitable marker that reflects renal microvascular disease. Accumulating evidence indicates that renal microvascular disease is crucial to the development of nephronal damage.[2], [11-13] Early detection of renal microvascular disease or glomerular endothelial cell injury by such a marker would be beneficial to the preventive strategy of progressive renal disease.

Acknowledgment

This study is supported by the Thailand Research Fund.