Altered Vascular Homeostasis in Type 2 Diabetic Nephropathy

Abstract

Type 2 diabetic nephropathy is a primary cause of ESRD worldwide. Therapeutic strategy in patients with microalbuminuric or macroalbuminuric type 2 diabetic nephropathy usually fails to restore renal function but merely slows the renal disease progression. In contrast, a recent study implies that the restoration of renal function as well as renal perfusion can be accomplished in early stage of type 2 diabetic nephropathy (normoalbuminuria) by correcting the hemodynamic maladjustment in renal microcirculation with vasodilators. Therefore, we intend to study the mechanism of vascular homeostasis to explain why treatment in the late stage of diabetic nephropathy during microalbuminuria or macroalbuminuria fails to enhance renal perfusion or restore renal function. The results indicate that such therapeutic failure in late-stage type 2 diabetic nephropathy likely relates to multiple defects in vascular repair, namely deficiencies in angiogenic factors such as endothelial progenitor cell, angiopoietin-1, flt-1 receptor, as well as elevated levels of antiangiogenic factors such as angiopoietin-2 and KDR.

Keywords:

• type 2 diabetic nephropathy
• fractional excretion of magnesium
• vascular homeostasis
• angiopoietin
• VEGF receptors
• hemodynamic maladjustment

INTRODUCTION

The American Society of Nephrology Presidential Address has recently expressed concern over the continuous increase in number of CKD patients entering ESRD, of which type 2 diabetic nephropathy is the growing primary public health threat.[1] Because diagnosis of diabetic nephropathy is generally dependent upon the diagnostic marker microalbuminuria, such a diagnostic approach is likely to be insensitive to screen early-stage CKD associated with type 2 diabetes mellitus because the presence of microalbuminuria is usually apparent when the creatinine clearance drops to 50 percent of normal range.[2] The current therapeutic outcome under conventional therapy observed in type 2 diabetic nephropathy associated with microalbuminuria or macroalbuminuria is simply a slowing of renal disease progression, but it is unable to restore renal function.[3–6] Futrakul et al.[7] has recently demonstrated that treatment implementation at the early stage of type 2 diabetic nephropathy during normoalbuminuria by using fractional excretion of magnesium as a diagnostic marker for diabetic nephropathy can effectively enhance peritubular capillary flow as well as restore the renal function. The discrepancy between therapeutic response observed in early treatment during normoalbuminuria versus treatment in late stage associated with microalbuminuria or macroalbuminuria remains unclear. It is likely that the resistance to treatment observed in late stage of type 2 diabetic nephropathy, in which treatment is unable to either enhance renal perfusion or improve renal function, might reflect altered vascular homeostasis relevant to impaired vascular repair. Therefore, we intend to study the mechanism of vascular homeostasis in type 2 diabetic nephropathy.

MATERIALS AND METHODS

Fifty patients associated with type 2 diabetes mellitus were included in the study. The diagnosis of type 2 diabetic nephropathy was determined by the abnormally elevated level of fractional excretion of magnesium (FE Mg), which is in accordance with the previous study in renal disease demonstrating the direct correlation between FE Mg and the magnitude of tubulointerstitial fibrosis.[8] Ages of the patients were in the ranges between 40–60 years. Ten age-matched subjects without diabetes associated with normal value of FE Mg served as healthy controls. Diabetic subjects fulfilling the following inclusion criteria were included: no clinical evidence of heart disease, compliance to follow-up and investigative procedures, and no obvious renal disease. All individuals were subjects in the following studies.

Blood Collection and Preparation of Plasma Samples

One volume of 0.109 M trisodium citrate solution was mixed with nine volumes of blood, and the mixture was centrifuged at 2500 g for 10 min to collect plasma. The citrated plasma was stored at −20°C for the determination of plasma factors. Clotted blood was also collected for the determinations of serum creatinine and magnesium. Three milliliters of EDTA blood was collected for isolation of circulating endothelial cell (CEC).

Enumeration of Circulating Endothelial Cells

The numbers of CEC were examined using the buffy coat smear technique. Diluted blood was layered on a Ficoll-hypaque density gradient (Histopaque-1077, Sigma, USA) 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 buffet-coat 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 CEC/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 VEGF

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

Human Angiopoietin-1 or Angiopoietin-2 Immunoassay

This assay employs the quantitative sandwich enzyme immunoassay technique in a manner similar to that just described.

Human Tie-2, VEGF R2 Immunoassay

This assay also employs the quantitative sandwich enzyme immunoassay technique in a manner similar to that just described.

Statistical Analysis

Comparison of the sample mean of two quantitative variables was determined by the non-parametric method using the Mann-Whitney test. p values below 0.05 were considered to be significant.

RESULTS

As illustrated in Table 1, a significant increase in the number of circulating endothelial cells was observed in type 2 diabetic nephropathic patients. With respect to biomarkers of vascular repair, deficiencies in both angiopoietin-1 and flt-1 were documented in patients. VEGF did not significantly change in patients as compared to controls. With respect to antiangiogenic factors, both angiopoietin-2 and KDR were significantly elevated in patients.

Table 1 Vascular homeostasis in type 2 diabetic nephropathy

	Diabetic nephropathy	Normal	p value
Biomarker of vascular injury
Circulating endothelial cells	2010 ± 798	102 ± 112	< 0.001
Biomarkers of vascular repair, angiogenic factors
VEGF, pg/mL	219 ± 229	230 ± 220	NS
Angiopoietin-1, pg/mL	23721 ± 9731	52366 ± 32409	< 0.01
flt-1, pg/mL	33 ± 17	55 ± 11	< 0.01
Biomarkers of vascular repair, antiangiogenic factors
Angiopoietin-2, pg/mL	4588 ± 2225	1150 ± 1120	< 0.05
KDR, pg/mL	10414 ± 2198	7696 ± 1892	< 0.001

DISCUSSION

An increased number of circulating endothelial cells observed in type 2 diabetic nephropathy reflects the underlying macro, as well as microvascular disease. An increase in vascular injury usually triggers the vascular repairing process. Under normal circumstances, such vascular injury would recruit the endothelial progenitor cell to induce the proliferation of endothelial cells to replace the endothelial cell loss. In this process, it requires an adequate amount of vascular endothelial growth factor, which would activate the eNOS in order to enhancing the nitric oxide (NO) production from the arginine substrate.[9] To accomplish an adequate vascular repair or angiogenesis, it would require another angiogenic factor, namely angiopoietin-1, which would strengthen the repairable vessel (see Figure 1).[10] With respect to the study obtained from type 2 diabetic nephropathy, multiple defects in vascular repair were observed (see Figure 2). Endothelial progenitor cell was definitely reduced, and it also showed impaired migratory capacity compared with those of control.[11] This would impair the proliferation of endothelial cell to replace for the endothelial cell loss. With respect to VEGF, although we did not demonstrate a significant deficiency, Lindenmeyer et al.[12] have recently documented a decrease in VEGF–A expression, which correlated with a reduction in peritubular capillary densities in human diabetic nephropathy. Such a correlation implies the renal microvascular disease encountered in type 2 diabetic nephropathy. An increase in KDR receptor concurs with the experimental study in bovine aortic endothelial cells that increase KDR mRNA expression under high glucose environment.[13] In fact, KDR value was noted to be significantly increased in this study. In the presence of flt-1 deficiency, an activation of VEGF is likely to act through the KDR receptor, by which it would induce an uncoupling of eNOS; therefore, it is unable to enhance NO production. Thus, a deficient endothelial progenitor cell and impaired production of NO would impair the vascular repair or angiogenesis. In addition, a deficiency in angiopoietin-1 would also impair the vascular repair. The presence of an abnormally elevated level of angiopoietin-2, which induces the proliferation of vascular smooth muscle cells, would complicate the situation even further by inducing a progressive narrowing of vascular lumen, as well as a progressive decline in renal microvascular perfusion.[14] Taken together, these multiple defects in vascular repair would culminate in a default angiogenesis. The result of this study would likely explain the therapeutic failure in enhancing renal perfusion and restoring renal function if such treatment is initiated late in the course of diabetic nephropathy. This finding concurs with the evidence of the reduction of peritubular capillary densities in human diabetic nephropathy,[12] as well as the progressive reduction in peritubular capillary flow observed in late stage of type 2 diabetic nephropathy associated with albuminuria.[2] A successful restoration of renal function, observed in early treatment during normoalbuminuria in type 2 diabetic nephropathy by correcting the hemodynamic maladjustment in renal microcirculation with vasodilators, implies that the mechanism of vascular repair is likely to be adequately maintained in the early stage of diabetic nephropathy.[7] Thus, this study renders support that an effective therapeutic strategy aimed at restoring renal function should be implemented during normoalbuminuria in type 2 diabetic nephropathy.

Figure 1. Normal vascular homeostasis.

Figure 2. Impaired angiogenesis in type 2 diabetic nephropathy.

DECLARATION OF INTEREST

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

ACKNOWLEDGMENT

This study is supported by Thailand Research Fund and Thailand Research Council Fund.