OXIDATIVE STRESS AND HEMODYNAMIC MALADJUSTMENT IN CHRONIC RENAL DISEASE: A THERAPEUTIC IMPLICATION

Abstract

Hemodynamic maladjustment with predominant constriction at the efferent arteriole has been encountered in a variety of clinical settings of glomerulonephropathy. In essence, it induces not only intraglomerular hypertension but also exaggeratedly reduces the peritubular capillary flow, which supplies the tubulointerstitial compartment. The hemodynamic maladjustment is believed to reflect a glomerular endothelial cell dysfunction. In this regard, oxidative stress and antioxidant defect are likely responsible for the glomerular endothelial dysfunction. Improvement in renal function was accomplished following the correction of oxidant and antioxidant imbalance with antioxidant therapy and vasodilators. Following such therapy, there was a correction in hemodynamic maladjustment with a decline in intraglomerular hydrostatic pressure and an increase in renal perfusion with a subsequent increase in renal functions namely creatinine clearance, glomerular filtration rate and a decline in FEMg.

• Hemodynamic
• Chronic renal failure
• Intraglomerular hypertension
• Oxidant
• Antioxidant
• Vasodilators
• Glomerular endothelial dysfunction

INTRODUCTION

The pathogenetic mechanism of renal disease progression with particularly relevant to the development of tubulointerstitial fibrosis in chronic renal disease is complex which implicates both hemodynamic and non-hemodynamic (immunologic) factors.[[1]] Due to the unsuccessful attempt of immunosuppressant in preventing the progression of renal disease, the focus of interest has switched toward the issue of hemodynamic impact upon the mechanism of renal disease progression. Within this context, it has recently been demonstrated that there is a spatial relationship between renal perfusion and nephronal structure. A normal renal perfusion is usually associated with an intact nephronal structure with no tubulointerstitial disease such as that observed in steroid-sensitive, minimal change or mild mesangial proliferative nephrosis. In contrast, a reduction in renal perfusion such as peritubular capillary flow observed in nephrosis associated with focal segmental glomerulosclerosis generally encounters nephronal damage such as tubulointerstitial fibrosis.[[2]] Furthermore, Bohle and associates[[3]] also denoted that the degree of tubulointerstitial fibrosis correlated inversely with the intensity of postglomerular capillary patency. The cause-and-effect relationship between renal perfusion and nephronal structure has been implicated by the observation in steroid–resistant mesangial proliferative nephrosis that the reduction in peritubular capillary flow precedes the development of tubulointerstitial fibrosis.[[4]]

The reduction in renal perfusion is likely to be a reflection of glomerular endothelial dysfunction. The endothelial cell in nephrosis expresses procoagulant instead of anticoagulant surface and provasoconstrictive instead of vasodilating activity, thereby reducing the renal perfusion.[[2]] In this regard, an experimental study mimicking glomerular endothelial cell injury has recently been demonstrated that serum of nephrotic patient is capable of inducing endothelial cell cytotoxicity in vitro. That the greater incidence of endothelial cell cytotoxicity is derived from the serum of severe nephrosis associated with focal segmental glomerulosclerosis whereas the serum from mesangial proliferative nephrosis induces a low incidence of endothelial cell cytotoxicity. Furthermore, it is also denoted that the degree of in vitro endothelial cell cytotoxicity correlates with the magnitude of reduction in renal plasma flow in vivo determined by the intrarenal hemodynamic study.[[5]]

The preceding information has raised an interesting issue as to what would be the factor responsible for the induction of such endothelial cell cytotoxicity and dysfunction. Inasmuch as reactive oxygen species is capable of not only inducing proteinuria and nephronal damage in experimental model of puromycin aminonucleoside in animal,[[6]] but also participating in a variety of clinical setting of glomerulonephritides,[[7]], [[8]], [[9]], [[10]], [[11]] it is therefore of interest to perform in this study an assessment of oxidant and antioxidant status in nephrotic patients as well as in a group of patients associated with chronic renal failure who are likely to progress to an end-stage renal disease. If there would be any evidence of oxidative stress, it would also be interested to see whether a correction of oxidant/antioxidant imbalance would improve the renal function and retard the progression of renal disease in these patients.

MATERIAL AND METHODS

Fifteen patients associated with idiopathic nephrotic syndrome (8 patients with focal segmental glomerulosclerosis and 7 patients with mesangial proliferation) and 9 patients associated with chronic renal failure were included and subject to the following studies.

Oxidant and Antioxidant Study

Ten milliliters of blood was drawn from the vein. Blood was placed into heparinized tube and centrifuged at 1500 g for 15 min to separate plasma and RBCs. The RBCs were washed three times with cold saline, and the erythrocyte pellets were frozen at −20°C until further analysis.

Lipid Peroxidation (MDA)

MDA was assessed by thiobarbituric acid (TBA) colorimetric assay of hydroperoxides. The TBA assay was performed using a modification of the technique described by Askawa and Matsushita.[[12]]

Glutathione (GSH)

Determination of glutathione in the erythrocytes was made by colorimetric methods of Beutler et al.[[13]] using the glutathione disulfide reductase-DTNB (5-5′-dithiobis (2-nitrobenzoic acid)) to react with sulfhydryl compound and yield a stable yellow color. GSH concentration in the erythrocytes was expressed as µmol/g hemoglobin.[[14]] The hemoglobin concentration was assayed by using a cyanmethemoglobin technique.

Vitamin C

Ascorbic acid in serum and plasma was determined by specific enzymatic spectrophotometric method. Samples were analyzed indirectly by measuring the absorbance at 593 nm.[[15]]

Vitamin E

Vitamin E was assayed by the modified Emmeric and Engle's method.[[16]] The oxidation of xylene-extracted tocopherols from the blood sample by ferric chloride and the pink complex of ferrous ions with bathophenanthroline were measured colorimetrically at 536 nm.

Glutathione Peroxidase (GSH-Px)

GSH-Px activity was determined according to Gunzler et al.,[[17]] with modification by following oxidation of the reduced form of nicotinamide-adenine dinucleotide phosphate (NADPH) measured at 340 nm.

Renal Function Studies

Glomerular Function

A glomerular filtration rate was performed by measuring the 10-h endogenous creatinine clearance (CCr) or glomerular filtration rate (GFR) by the radioisotope technique using ^99mTc-labeled diethylene triamine pentaacetic acid (DTPA) and the value was converted to the body surface area of 1.73 m² by the method of calculation:

Intraglomerular pressure (PG mm Hg) was assessed by the method of calculation as previously described.[[18]]

Tubular Function

Tubular transport was assessed by a 10-h urinary collection during fasting. Blood drawn at the end of the test and urine were analyzed for creatinine, magnesium and protein. A reflection of tubular transport was derived from the determination of fractional excretion (FE) of filtered solute namely magnesium (Mg). The FE Mg was calculated through the formula.

Vascular Function

The renal plasma flow (RPF) value using ¹³¹I-labeled orthoiodohippuric acid (hippuran) and the renal afferent (RA) and efferent arteriolar resistance (RE) were determined by the previously described method.[[19]] A peritubular capillary flow (PTCF) is derived from the substraction of glomerular filtration rate from renal plasma flow.

Mode of Therapy

All of these 24 patients were treated with enhanced renal perfusion therapy, which consisted of (a) angiotensin converting enzyme inhibitor such as cilazapril 1.25–10 mg/day or enalapril (0.25–1 mg/kg/per day) or AII receptor antagonist 50–100 mg/day (b) calcium channel blocker isradipine 2.5–10 mg/day and (c) antiplatelet agent dipyridamole 3–5 mg/kg/day. In addition, all patients received antioxidants as a combination of vitamin E (800 units) and vitamin C (1000–3000 mg) daily. The nephrotic patients had been on a regular protein, non added salt diet and the patients in chronic renal failure group; in addition to the general supportive treatment such as correction of electrolyte and acid base imbalance, had been placed on a low protein, low cholesterol diet and adequate hydration.

STATISTICAL ANALYSIS

Values in text and tables are expressed as mean ± SEM. Non parametric Mann–Whitney was used to establish the significance of between group differences. The differences between pre- and post-treatment values between each treatment group was performed by Student's paired t-test. The difference was statistically significant when the p value was less than 0.05.

RESULTS

The initial assessment of oxidant revealed an elevated level of plasma MDA in nephrotic patients (3.4 ± 0.3 µM vs. 2.6 ± 1 µM of controls) as well as in patients with chronic renal failure (3.2 ± 0.6 µM). The initial level of erythrocyte MDA in both nephrotic (11.5 ± 2 nmol/L vs. 7.6 ± 0.9 nmol/L of control) and chronic renal failure patients (10.8 ± 1 nmol/L) were also elevated as depicted in Table 1. In respect to the antioxidant study, there was a significant depletion in GSH concentration in nephrotic (6.8 ± 1 µmol/g Hb vs. 9 ± 1 µmol/g Hb of control) and in chronic renal failure patients (6.3 ± 1 µmol/g Hb). The concentration of vitamin C was also found to be significantly depleted in nephrotic (1 ± 0.8 mg/L vs. 5.7 ± 4 mg/L of control) and in chronic renal failure patients (1 ± 1 mg/L). The level of vitamin E in plasma in both nephrotic and chronic renal failure patients were not significantly different from the controls. The level of glutathione peroxidase in nephrosis and chronic renal failure patients were 0.2 ± 0.2 nmol/10⁶ cells and 0.2 ± 0.18 nmol/10⁶ cells respectively which were not significantly different from the control value of 0.28 ± 0.4 nmol/10⁶ cells. The oxidant and antioxidant (OA) ratio in both groups of patients were significantly different from the control (Table 1).

Table 1. Oxidant and Antioxidant in Nephrotic and Chronic Renal Failure Patients

	NS	p Value	Control	p Value	CRF
Oxidant (O)
P MDA µM	3.4±0.3	<0.05	2.6±1	<0.05	3.2±0.6
R MDA nmol/L	11.5±2	<0.01	7.6±0.9	<0.01	10.8±1
Antioxidant (A)
GSH µmol/gHb	6.8±1	0.001	9±1	0.001	6.3±1
Vit C mg/L	1±0.8	<0.001	5.7±4	<0.001	1±1
Vit E mM	0.17±0.2	NS	0.17±0.1	NS	0.16±0.1
OA Ratio	1.9±0.5	<0.001	0.7±0.2	<0.001	1.9±0.5

The result of renal function study in the nephrosis group revealed a significant impairment. Of the glomerular function, the initial creatinine clearance (CCr) value was 44.8 ± 24 mL/min/1.73 m². The tubular function revealed a significant elevation of FE Mg (7.2 ± 2% vs. 2.2% of control). In the chronic renal failure (CRF) patients, the mean CCr was 17 ± 10 mL/min/1.73 m² and the FE Mg was 13 ± 6.3%. The intrarenal hemodynamic study revealed a significant reduction in renal plasma flow (RPF) 219 ± 84 mL/min/1.73 m² (normal 600 mL/min/1.73 m²), a low peritubular capillary flow (PTCF) 167 ± 67 mL/min/1.73 m² (normal 480 mL/min/1.73 m²), an elevated efferent arteriolar resistance (RE) 19442 ± 5206 dyne s cm⁻⁵ (normal 3000 dyne s cm⁻⁵) and an elevated intraglomerular hydrostatic pressure (PG) 56 ± 1 mm Hg vs. 53 mm Hg of control.

Following the therapy, there had been a significant improvement in oxidant and antioxidant status. The plasma and erythrocyte MDA declined to normal level whereas there had been a steady increase in the concentration of GSH, vitamin C and E as depicted in Table 2. In accordance with this improvement in antioxidant status, there was also a significant improvement in renal function. The creatinine clearance in nephrosis increased to 60 ± 34 mL/min/1.73 m² whereas the FE Mg declined to 5.7 ± 3% (Table 3). In the CRF patients, the CCr rose to 23 ± 12 mL/min/1.73 m² and the FE Mg declined to 11.4 ± 6%. The intrarenal hemodynamic study showed a significant improvement in RPF (391 ± 133 mL/min/1.73 m²), PTCF (314 ± 120 mL/min/1.73 m²), a reduction in RE (3918 ± 2040 dyne s cm⁻⁵) and PG (51 ± 0.8 mm Hg).

Table 2. Initial and Post-treatment Values of Oxidant and Antioxidant in Nephrotic and CRF Patients

	Initial	Post-treatment	p Value
GSH µmol/gHb	6.8±1.5	8.2±1.7	<0.001
Vit C mg/L	1.2±1.4	4.3±1.9	<0.001
Vit E mM	0.17±0.1	0.29±0.1	<0.001
P-MDA µM	3.2±0.9	1.5±0.9	<0.001
R-MDA nmol/L	00.2±3	5.6±3	<0.001

Table 3. Initial and Post-treatment Values of Renal Function in Nephrosis

	Initial	Post-treatment	p Values
CCr mL/min/1.73 m^3	44.8 ± 24	60.6 ± 34	<0.05
FE Mg%	7.2 ± 2	5.7 ± 3	<0.05

DISCUSSION

Reactive oxygen species are implicated in cell signaling, gene transcription, mitosis, apoptosis and vasoconstriction.[[20]], [[21]] The cellular sources of reactive oxygen species are multiple namely NADPH oxidase, lipoxygenase, cyclooxygenase from plasma membrane, electron transport system from mitochondria, xanthine oxidase, hemoglobin, transition metals (Fe^2+/3+, Cu1^+/2+) from cytosol and cytochrome P-450 from endoplasmic reticulum.[[22]] Increased cellular metabolism with enhanced production of reactive oxygen species have been delineated in a variety of glomerulonephropathy and renal failure.[[23]], [[24]] This study indicates that both increased reactive oxygen species and decreased antioxidant defense have been substantiated in our renal patients. Increased plasma MDA and erythrocyte MDA in conjunction with a depleted plasma vitamin C and a reverse ratio of oxidant and antioxidant imply that there is an oxidant/antioxidant imbalanced state. Similar observation has also been reported.[[23]], [[24]], [[25]] In excess, reactive oxygen species and their byproducts are capable of causing oxidative damage and cytotoxicity to cells. These result in increased oxidized LDL, advanced glycation end products of carbohydrates, fat and protein.[[26]] In the presence of antioxidant imbalance, the defective antioxidant would allow the excessively generated reactive oxygen species to induce a sustained oxidative damage to cells in particular the endothelial cells which is optimally situated at the interface between the circulating blood and the vessel wall to serve as a sensor and transducer of signals within the circulatory microenvironment. The oxidant and antioxidant imbalance observed in this study is likely to explain the in vitro increased endothelial cell cytotoxicity induced by sera of nephrotic patients.[[5]] Increased oxidative stress to the glomerular endothelial cell would induce dysfunctioning of the endothelial cell and in excessive amount incriminate in endothelial cell death. In response to such oxidative injury, the endothelial cell would increase productions of vasoconstrictive substances namely angiotensin II, endothelin and thromboxaneA₂ whereas the production of endothelium dependent vasodilator such as nitric oxide is defective as well as being neutralized by the excessive amount of reactive oxygen species. Such a provasoconstrictive state would induce a hemodynamic maladjustment with a predominant vasoconstriction at the efferent arteriole. The preponderant vasoconstriction at the efferent arteriole not only increases the intraglomerular hydrostatic pressure but also exaggeratedly reduces the peritubular capillary flow, which supplies the tubulointerstitial compartment. The hemodynamic study indicates that there is indeed an increase in intraglomerular hydrostatic pressure to 56 ± 1 mm Hg (normal ≤53 mm Hg) implicating an intraglomerular hypertension. The presence of intraglomerular hypertension in conjunction with the reduction in renal plasma flow (mean 219 ± 84 mL/min/1.73 m²) and with the hemorheologic alteration secondary to the additive effect of oxidative stress to the endothelial cell inducing vascular inflammatory gene expression such as vascular adhesion molecule, mononuclear cell infiltration and procoagulant surface expression; would culminate in the injury to the glomerular cell inducing glomerulosclerosis.[[27]], [[28]], [[29]], [[30]]

The reduction in peritubular capillary flow secondary to the hemodynamic maladjustment at the efferent arteriole exerts a significant hemodynamic impact upon the tubulointerstitial structure. It is of notion that a simulated ischemia in flow-adapted endothelial cells leads to generation of reactive species and cell signaling through the NADPH oxidase pathway. This is followed by an increased production of nuclear factor-kappa B which then upregulates the inflammatory gene expressions namely cytokines, growth factors and adhesion molecules.[[27]], [[28]] A sustained reduction in peritubular capillary flow in conjunction with the oxidative stress would therefore, induce an ischemic injury to the tubulointerstitial structure and the development of tubulointerstitial fibrosis. In this regard, increased production of nuclear factor-kappa B was detected in nephrosis associated with focal glomerulosclerosis[[31]] and increased chemokine expression was also demonstrated in puromycin aminonucleoside nephrosis.[[32]] In this regard, it has recently been demonstrated that there is an inverse correlation between the peritubular capillary flow and the incidence of tubulointerstitial fibrosis.[[4]] That the progressive reduction in peritubular capillary flow as the disease severity progresses inversely increases the magnitude of tubulointerstitial fibrosis. In addition, we have recently demonstrated that there is also a correlation between the renal perfusion and FE Mg.[[33]]

The preceding information renders a supportive view that the oxidative stress and antioxidant defect is likely to be responsible for the endothelial cell cytotoxicity and a spontaneous endothelial cell dysfunction by which it induces a progressive reduction in renal perfusion as the disease severity progresses. In accordance with the therapeutic strategy, a correction of antioxidant defect in conjunction with the administration of vasodilators to correct the hemodynamic maladjustment would likely improve the renal perfusion and prevent the progression of renal disease. Based upon this therapeutically strategic approach, the administration of antioxidants namely vitamin C and vitamin E restores the antioxidant status toward normal. It has been a general consensus that vitamin C is capable of neutralizing super oxide anion, reactive nitrogen species such as peroxynitrite, nitrogen dioxide[[34]] and also acts as a coantioxidant by regenerating ∝-tocopherol (vitamin E) from the ∝-tocopherol radical.[[35]], [[36]], [[37]] Vitamin C has also been shown to regenerate glutathione and B-carotene in vitro from their respective one-election oxidation product.[[34]], [[38]] Another major property that makes vitamin C such an effective antioxidant is the stability and low reactivity of the ascorbyl radical formed when ascorbate scavenges a reactive oxygen or nitrogen species.[[39]] The impaired endothelium-dependent vasodilation was markedly improved by vitamin C in essential hypertension.[[40]]

In respect to vitamin E, a combined vitamin E and selenium or glutathione deficiency leads to pronounced and progressive oxidative damage to renal structure and function.[[41]], [[42]], [[43]] Increasing dietary vitamin E level significantly attenuates renal oxidative damage in the puromycin nephrotoxicity model of FSGS in the rat.[[44]], [[45]] Therefore, both vitamins C and E administration would assist in neutralizing the reactive oxygen species and thereby minimizing the tissue damage by oxidative stress. Such an event would spare the vasodilating status of nitric oxide (NO). Increased available NO would exert a cellular protection to the nephronal structure as well as to the glomerular endothelial cell. A decreased endothelial cell cytotoxicity was demonstrated following the therapeutic administration of antioxidants and vasodilators.

In respect to the renal function, the therapeutic regimen reduced the renal arteriolar resistance. The relaxation of efferent arteriole not only reduced the intraglomerular hydrostatic pressure (PG 51 ± 0.8 mm Hg), but also enhanced the peritubular capillary flow. The peritubular capillary flow increased from 167 ± 67 mL/min/1.73 m² to 314 ± 120 mL/min/1.73 m² following treatment. The improvement in renal perfusion correlated with the glomerular function as well as the tubular function. The creatinine clearance in nephrosis increased from 44.8 ± 24 mL/min/1.73 m² to 60.6 ± 34 mL/min/1.73 m², the glomerular filtration rate increased from 52 ± 21 mL/min/1.73 m² to 76 ± 22 mL/min/1.73 m² and the FE Mg significantly reduced from 7 ± 2% to 5.7 ± 3% following treatment, p < 0.05. In chronic renal failure, the CCr rose from 17 mL/min/1.73 m² to 23 ± 12 mL/min/1.73 m².

Thus the preceding information renders a supportive view that the oxidant and antioxidant imbalance is likely to induce the glomerular endothelial dysfunction with subsequent hemodynamic maladjustment by which the correction of such disorders by antioxidant therapy and vasodilators can improve the renal function and prevent the renal disease progression.

Acknowledgment

This work was supported by the National Research Council of Thailand.