Mol Cell Biochem (2008) 311:105–110 DOI 10.1007/s11010-008-9701-0
Oxidative stress in primary glomerular diseases: a comparative study Suchita Markan Æ Harbir Singh Kohli Æ Kamal Sud Æ Monica Ahuja Æ Tarunveer S. Ahluwalia Æ Vinay Sakhuja Æ Madhu Khullar
Received: 17 August 2007 / Accepted: 10 January 2008 / Published online: 25 January 2008 Ó Springer Science+Business Media, LLC. 2008
Abstract Objective To evaluate the status of oxidative stress in patients with different primary glomerular diseases (PGD) which have differential predisposition to renal failure. Methods Seventy-three patients with PGD and 50 controls were enrolled in the study. They were sub-grouped into non-proliferative glomerulonephritis (NPGN) and proliferative glomerulonephritis (PGN). Levels of serum malondialdehyde (MDA), reactive nitrogen intermediates (RNI), plasma total homocysteine (tHcy), urine 8-isoprostane (8-IP), RBC thiols, glutathione-S-transferase (GST) and serum superoxide dismutase (SOD) were measured spectrophotometrically. Results PGD patients showed a significant increase in MDA, RNI, tHcy, 8-IP levels (P \ 0.05) and decreased SOD, total thiols and protein bound thiol levels as compared to controls (P \ 0.05). Significantly higher levels of tHcy, MDA and 8-IP (P \ 0.05) and lower SOD enzyme activity (P \ 0.05) were observed in PGN group as compared to NPGN and control groups. These changes remained significant even after adjustment was made for creatinine. Conclusions Oxidative stress in PGN is significantly higher than NPGN, indicating higher oxidative stress in these patients, independent of degree of renal dysfunction.
S. Markan M. Ahuja M. Khullar (&) Department of Experimental Medicine and Biotechnology, Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh, India e-mail:
[email protected] H. S. Kohli K. Sud T. S. Ahluwalia V. Sakhuja Department of Nephrology, Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh, India
Keywords Non-proliferative glomerulonephritis Oxidative stress Proliferative glomerulonephritis Reactive oxygen species.
Introduction Primary glomerular diseases (PGD) constitute a heterogeneous group of diseases with different pathophysiology. They have different propensities for progression to renal failure; diseases with rapid proliferation of the parietal epithelial cells and infiltrating monocytes like crescentric glomerulonephritis are often associated with renal failure. However, the patients with minimal change disease (MCD) inherently do not progress to the end stage renal disease (ESRD) [1]. Reactive oxygen species (ROS) have been shown to play an important role in pathophysiology of renal diseases [2]. They have also been proposed to be the primary mediators in glomerulonephritis (GN), responsible for modification of glomerular permeability to proteins, development of morphologic lesions and alterations of glomerular haemodynamics [3, 4]. Increased endogenous generation of ROS such as superoxide anions, hydrogen peroxide and a decreased activity of antioxidant enzymes [superoxide dismutase (SOD), catalase and glutathione peroxidase] have been reported in the animal model of Anti-Thy 1.1 glomerulonephritis, indicating that glomeruli are vulnerable to oxidative injury in vivo [5]. Increased glomerular production of oxygen radicals has also been found in Mpv17 gene-inactivated mice, a model of steroidresistant nephrosis, similar to human focal segmental glomerulosclerosis (FSGS), and in rats with anti-Thy 1 nephritis [6]. There is limited human data that provides some proof of the concept that the observations in
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experimental models may be relevant to human diseases [7]. Evidence exists for the presence of increased oxidative stress in CKD [8–12] but there is limited correlative and cause–effect information about the role of oxidants in progressive kidney disease [7]. Recently, increased plasma malondialdehyde (MDA) levels have been reported in the patients with FSGS as compared to patients with MCD, which were associated with the degree of glomerulosclerosis, suggesting that oxidative stress occurs early and may play an important role in the pathogenesis of glomerulosclerosis [4]. An impaired antioxidative system has also been observed in patients with nephrotic syndrome, lupus nephritis and IgA nephropathy [13, 14]. However, Poelstra et al. reported no significant difference in glomerular antioxidative enzyme (AOE) expression in patients with non-proliferative primary glomerulopathies such as MCD, FSGS and membranous glomerulonephritis (MGN) [15]. Till now, only a few studies have examined oxidative stress in glomerular diseases such as FSGS or secondary glomerulonephritis; however, the status of oxidative stress in different PGD with differential predisposition for renal failure has not been evaluated. Although experimental studies have shown that oxidative stress may play an important role in the pathogenesis of glomerular diseases, there is limited human data that provides some proof of the concept that the observations in experimental models may be relevant to human diseases [7]. The present study was carried out to assess the status of oxidative stress in different glomerular diseases which have different propensities to develop renal failure.
Materials and methods The study was carried out in the Department of Experimental Medicine and Biotechnology in collaboration with the Department of Nephrology and Department of Pathology of the Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh. Histologically confirmed diagnosis of PGD as per light, immunofluorescence and electron microscopic examination of kidney biopsy were included in the study. Patients with secondary glomerulonephritis, or those on vitamin supplementation or with any previous history of steroids intake or immunosuppressant, were excluded from the study. Seventy-three patients of PGD (MCD: n = 10, FSGS: n = 25, MGN: n = 17, MPGN: n = 10, crescentic nephritis: n = 11) were enrolled in the study. The patients with MCD, FSGS and MGN were further grouped as non-proliferative glomerulonephritis (NPGN: n = 52) and patients of MPGN and RPGN as proliferative glomerulonephritis (PGN: n = 21). Fifty normotensive subjects without any history of renal or any other disease were enrolled as controls. The
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control subjects were not on any medications, vitamin supplementation or dietary restriction. Informed consents were obtained from all the subjects included in the study. The Indian Council of Medical Research (ICMR) approved the study protocol and the ethical clearance was obtained from the Institute ethics committee.
Materials Thiobarbituric acid (TBA), glutathione (GSH), 5,50 -dithiobis2-nitrobenzoic acid (DTNB), 1-chloro-2,4 dinitrobenzene (CDNB), butylated hydroxytoluene (BHT), hydroxylamine hydroquinone, nitrobluetetrazolium (NBT) were from Sigma–Aldrich Corporation (United States). Plasma homocysteine kit was from Axis Shield Diagnostics, UK, and urinary 8-Isoprostane was from Cayman chemicals, USA. All the other chemicals were of analytical grade.
Samples and methods Overnight fasting blood samples (5 ml) were collected in ethylenediamine tetra acetate (EDTA) coated and plain vials. The markers of oxidative stress were estimated within 6 h of the sample collection. Morning urine samples were used for the estimation of 8-isoprostane levels. Serum bilirubin, total proteins, albumin, calcium, urea and creatinine were measured spectrophotometrically using their respective kits (Erba Diagnostics Mannheim Gmbh, Germany). Blood Hb was measured spectrophotometrically using Drabkin’s solution. Serum MDA was measured by TBA method [16]. Reactive nitrogen intermediates (RNI) were assessed by measuring serum nitrite levels by reaction with Greiss reagent [17]; plasma total homocysteine (tHcy) and urinary 8-Isoprostane were measured by their respective ELISA kits. The glomerular filtration rate (GFR) was calculated by Cockcroft–Gault formula. Thiols were measured in whole blood lysate based on reactivity of thiol groups with DTNB forming a yellowcoloured complex with absorption maxima at 412 nm [18]. Glutathione-S-transferase (GST) activity was determined spectrophotometrically by measuring the conjugation of GSH and CDNB at 390 nm [19]. The SOD activity was measured using the enzymatic generation of superoxide by a microbial NADH diaphorase which reacted with hydroxylamine to yield nitrite which in turn formed colour in presence of sulphanilamide and naphthylethylene diamine with an absorption maxima at 540 nm [20]. The oxidative stress markers were correlated with serum creatinine, GFR, 24 h urinary proteins excretion and serum
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proteins to evaluate the possible association of these markers with parameters of oxidative stress.
Statistical analysis The data was statistically analyzed and expressed as mean ± standard deviation (SD) of the mean. ANOVA and Tukey’s post-hoc tests were used to compute demographic and biochemical profile of the patients and to compare them between patients and control groups. Since most of the parameters of oxidative stress were not as per the Gaussian distribution, Kruskal–Wallis test and Mann– Whitney U test were used for the inter-group comparisons between patients and controls and PGN and NPGN groups. Pearson’s coefficient of correlation was calculated to look at the possible association of oxidative stress parameters with serum creatinine, GFR, 24 h urinary proteins excretion and serum proteins. Analysis of co-variance (ANCOVA) was performed to compare the parameters of oxidative stress after adjustment for creatinine. All the statistical analysis was performed using the SPSS (V10).
Results Demographic and biochemical profiles of the subjects enrolled in the study are shown in Tables 1 and 2, respectively. Patients with PGD had significantly increased levels of the serum MDA, RNI, urinary 8-Isoprostane and plasma homocysteine as compared to the normotensive controls (P \ 0.05). The RBC total thiols, protein bound thiols and SOD levels were significantly decreased in these patients (P \ 0.05) as compared to controls indicating decreased antioxidative activity. A slight increase in the RBC GST activity was observed in PGD cases with respect to controls (Table 3). However, this was statistically insignificant (P [ 0.05). Patients with PGD showed a
negative correlation between SOD (r = -0.296), GST activities (r = -0.304) and proteinuria (P \ 0.05). A positive association was seen between the serum total thiols and serum albumin levels (r = 0.314). Plasma homocysteine (tHcy) levels showed positive association with serum albumin (r = 0.311), creatinine (r = 0.308) and urea levels (r = 0.318) and were inversely associated with GFR (r = -0.380) (P \ 0.05). The levels of the serum MDA, RNI, urinary 8-Isoprostane and plasma homocysteine remained significantly elevated, and RBC total thiols, protein bound thiols and SOD levels significantly decreased in the patients with PGD as compared to controls after adjusting for creatinine using ANCOVA (P \ 0.05). Patients in PGN group had significantly higher levels of plasma homocysteine, GST activity, serum MDA and urine 8-Isoprostane (P \ 0.05) and significantly lower levels of SOD (P \ 0.05) as compared to the patients in the NPGN group (Table 4). No significant difference in the levels of the thiols and RNI levels was observed between PGN and NPGN patients. A positive correlation between SOD and GFR (r = 0.314), RBC total thiols and albumin (r = 0.315) and negative correlation between SOD (r = -0.296), GST (r = -0.349) and proteinuria was observed in the patients with PGN (P \ 0.05). Plasma homocysteine levels showed positive correlation with serum albumin (PGN: r = 0.314, NPGN: r = 0.314) and negative correlation with GFR (PGN: r = -0.298, NPGN: r = -0.380) in both (PGN and NPGN) groups of patients (P \ 0.05). The plasma homocysteine, serum MDA, urine 8-Isoprostane and GST enzyme activity remained significantly elevated after adjustment for creatinine levels with ANCOVA (P \ 0.05).
Discussion Although oxidative stress has been shown to be a major participant in the pathogenesis of renal diseases, its status
Table 1 Demographic profile of patients and controls NPGN (n = 52)
PGN (n = 21)
PGD (n = 73)
Controls (n = 50) 28.36 ± 8.88
Age (years)
31.55 ± 13.02
27.26 ± 9.79
30.59 ± 12.44
Male N (%)
38 (73.1)
7 (36.8)
44 (65.7)
26 (52)
Females N (%)
14 (26.9)
12 (63.2)
23 (34.3)
24 (48)
MAB (mmHg)
129.69 ± 15.68*
136.24 ± 18.49*
131.15 ± 16.43*
120.67 ± 5.67
BMI (kg/m2)
21.63 ± 4.63
19.98 ± 3.01*
21.26 ± 4.35
22.93 ± 3.33
Values are Mean ± SD; *P \ 0.05 between patients and controls ANOVA, independent sample t-test and Tukey’s post-hoc tests were used to compare parameters in between patients of different groups and to compare them between patients and control groups MAB: Mean arterial blood pressure, BMI: basal metabolic index, NPGN: non-proliferative glomerulonephritis, PGN: proliferative glomerulonephritis, PGD: primary glomerular disease patients (PGN + NPGN)
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Table 2 Biochemical profile of the subjects NPGN (n = 52)
PGN (n = 21)
PGD (n = 73)
Controls
Total proteins (g%)
4.91 ± 1.24*
6.09 ± 1.38
5.17 ± 1.35
6.04 ± 0.89
Albumin (g%)
3.26 ± 0.65
2.83 ± 0.87*
2.93 ± 0.84*
4.12 ± 0.25
Globulin (g%)
2.81 ± 0.61
3.07 ± 0.68*
2.87 ± 0.63
2.1 ± 0.43 0.89 ± 0.04
Bilirubin (g%)
0.70 ± 0.14
0.67 ± 0.01
0.69 ± 0.12
Creatinine (mg%)
1.44 ± 0.63*
5.94 ± 5.59*,**
3.07 ± 2.60*
65.82 ± 51.08*
104.60 ± 52.04*,**
74.50 ± 53.40*
Urea (mg%) Calcium (mg%) Sugar (mg%) 24 h Urine prot (g) GFR (ml/min) Hb
0.9 ± 0.08 21.21 ± 6.05
9.03 ± 4.98
8.74 ± 0.60
8.97 ± 4.39
9.67 ± 1.1
97.55 ± 27.16
96.08 ± 17.62
97.22 ± 25.22
92.3 ± 12.2
2.63 ± 1.60*
2.55 ± 1.49*
2.61 ± 1.57*
0.5 ± 0.08
63.46 ± 34.29* 12.0 ± 2.48
,
28.27 ± 23.47* ** 10.9 ± 2.2
54.36 ± 35.26* 11.8 ± 2.46
NA 11.67 ± 2.42
Values are Mean ± SD; independent sample t-test was used to compare parameters in between patients of different groups (PGN and NPGN) and to compare them between patients and control groups *P \ 0.05 between patients and controls; **P \ 0.05 between patients with PGN and NPGN PGN: Proliferative glomerulonephritis, PGD: primary glomerular disease patients (PGN + NPGN), NPGN: non-proliferative glomerulonephritis Table 3 Oxidative markers in patients with primary glomerular diseases and controls PGD (n = 73) SOD (U/mg prt) TT (U/Hb) NPT (U/Hb) PT (U/Hb)
Controls (n = 50)
5.00 (2.97–6.20)*
5.77 (5.08–6.97)
23.25 (20.32–28.19)*
55.31 (49.41–65.22)
0.45 (0.19–1.24) 22.21 (18.83–27.05)*
0.60 (0.48–0.80) 54.56 (48.72–64.64)
GST (U/Hb)
2.43 (2.01–3.07)
MDA (910-3 lmol/l)
0.46 (0.21–0.65)*
0.21 (0.12–0.30)
RNI (9103 lmol/l)
1.27 (0.47–2.36)*
0.73 (0.50–1.26)
tHcy (lmol/l) 8-IP (pg/mg)
16.50 (12.50–18.90)* 625.47 (261.17–1077.00)*
2.66 (2.31–3.14)
8.76 (6.59–10.64) 473.95 (617.07–330.83)
Values are Median (interquartile range); since most of the parameters of oxidative stress were not as per the Gaussian distribution, Kruskal– Wallis test and Mann–Whitney U test were used for the inter-group comparisons between patients with PGD and controls *P \ 0.05 between patients and controls PGD: Primary glomerular disease patients (PGN + NPGN), SOD: superoxide dismutase, TT: total thiols, NPT: non-protein thiols, PT: protein bound thiols, GST: glutathione-S-transferase, MDA: malondialdehyde levels, RNI: reactive nitrogen intermediates, tHcy: total homocysteine levels, 8-IP: urinary 8-isoprostane levels (pg/mg creatinine)
in patients with different PGD is not well known. In the present study, we compared oxidative stress in PGD with differential propensity to progress to chronic renal failure (CRF), by measuring the levels of pro-oxidants (NO, tHcy), antioxidants (SOD, GST, Total thiols) and markers of oxidative stress (MDA, 8-isoprostane). A significant increase in the levels of the pro-oxidants and decreased levels of the antioxidants observed in the patients with PGD as compared to the controls indicate a state of oxidative stress in these diseases. The significant increase in levels of pro-oxidants observed in the present study indicates an increased production of ROS in these diseases. A similar increase in ROS levels has also been reported in MCD with relapse,
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lupus nephropathy, post-streptococcal glomerulonephritis and in the patients with IgA nephropathy [21]. Increased levels of ROS may be due to activation of neutrophils, monocytes and mesangial cells during metabolic processes in glomerular diseases and could induce severe oxidative and lipoperoxidative damage in these patients [3, 21]. The ROS have been shown to induce injury to proteins by oxidation of critical amino acids, resulting in loss of enzymatic activity or structural integrity, loss of membrane stability and integrity, which may lead to increased transepithelial permeability [2]. We also observed a significant increase in RNI which is an indirect measure of nitric oxide (NO). NO is known to react with ROS such as superoxide anions resulting in the formation of peroxynitrite, which
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Table 4 Oxidative markers in patients with proliferative and non-proliferative glomerulonephritis and controls NPGN (n = 52) SOD (U/mg) TT (U/Hb) NPT (U/Hb) PT (U/Hb)
5.35 (3.17–6.28) 23.04 (18.82–27.04)* 0.38 (0.19–1.10) 21.98 (17.94–26.53)*
PGN (n = 21) 3.25 (2.69–5.00)*,** 24.90 (20.61–24.90)* 0.74 (0.23–1.50) 23.40 (18.85–30.88)*
Controls (n = 50) 5.77 (5.08–6.97) 55.31 (49.41–5.22) 0.60 (0.48–0.80) 54.56 (48.72–64.64)
GST (U/Hb)
2.34 (1.93–2.77)
3.18 (2.79–6.24)*,**
RNI (9103 lmol/l)
1.24 (0.32–2.10)*
1.28 (0.50–2.83)*
0.73 (0.50–1.26)
MDA (910-3 lmol/l)
0.45 (0.21–0.58)*
0.65 (0.28–1.02)*,**
0.21 (0.12–0.30)
Hcy (lmol/l)
15.50 (12.12–17.50)*,**
8-IP (pg/mg)
356.59 (223.40–1015.00)**
18.90 (16–20)*,** 1056.00 (569.00–5196.00)
2.66 (2.31–3.14)
8.76 (6.59–10.64) 473.95 (617.07–330.83)
Values are Median (interquartile range); since most of the parameters of oxidative stress were not as per the Gaussian distribution, Kruskal– Wallis test and Mann–Whitney U test were used for the inter-group comparisons between patients and controls and PGN and NPGN groups *P \ 0.05 between patients and controls, **P \ 0.05 between patients with PGN and NPGN NPGN: non-proliferative glomerulonephritis, PGN: proliferative glomerulonephritis, GN: glomerulonephritis patients, SOD: superoxide dismutase, TT: total thiols, NPT: non-protein thiols, PT: protein bound thiols, GST: glutathione-S-transferase, MDA: malondialdehyde levels, RNI: reactive nitrogen intermediates, tHcy: total homocysteine levels, 8-IP: urinary 8-isoprostane levels (pg/mg creatinine)
can nitrosylate tyrosine residues, and thus cause tissue damage including renal damage [2]. Besides ROS, increased total homocysteine levels were also seen in patients with PGD. Total homocysteine has been proposed to induce cell injury/dysfunction through a mechanism involving oxidative stress [22]. Hyperhomocysteinaemia promotes production of hydroxyl radicals, which are known lipid peroxidation initiators, through tHcy autooxidation and thiolactone formation [22]. Thiol group of tHcy readily undergoes auto-oxidation in plasma to generate ROS. Moreover, total homocysteine has been shown to decrease the expression of a wide range of antioxidant enzymes, which further supports the hypothesis that tHcy may enhance the cytotoxic effects of agents or conditions known to generate ROS [22]. Thus, hyperhomocysteinaemia seen in patients with PGD in the present study could significantly contribute to renal injury in these patients. An association between hyperhomocysteinaemia and renal dysfunction has been reported earlier also [23]. We observed significantly lower levels of reduced thiols which are known to have potent antioxidant activity. The significantly lower levels of the thiols in patients with PGD could be due to hypoalbuminemia in these patients. Massive albuminuria has been incriminated as the cause of decreased thiols [24] as most of serum thiols are albumin bound. The positive association of the RBC thiols with serum albumin levels observed in our patients further supports this. Decrease in endogenous antioxidative activities was further confirmed by decreased SOD levels in PGD patients. The SOD is an important antioxidant enzyme which neutralizes the superoxide anions. Decrease in SOD activity in patients with PGD may result from inactivation of the enzyme by ROS. The negative association between SOD and the urinary proteins seen in the
present study further reflects loss of the enzymes due to massive proteinuria in these patients. Since PGD patients had significantly higher creatinine levels, indicative of renal insufficiency, which could also result in oxidative stress [25], therefore, we carried out ANCOVA to eliminate the effect of serum creatinine levels on oxidative stress. We observed that even after adjusting the oxidative stress parameters for serum creatinine, these parameters remained significantly altered which suggests that ROS play a role in causing renal injury but does not rule out the possibility that renal injury also causes increased oxidative stress. The MPGN and crescentic glomerulonephritis are accompanied by proliferation of mesangial cells and extracellular matrix protein accumulation, whereas MCD, MGN and FSGS do not show significant proliferation of the resident cells. Patients with PGN experience rapid progression to CRF as compared to the patients with NPGN [1]. A comparison of levels of oxidative stress biomarkers in these two groups showed a significantly greater oxidative stress as indicated by increased levels of MDA and 8isoprostane and decreased activity of AOEs in patients with PGN as compared to the patients with NPGN. The increased oxidative stress in PGN might be due to the rapid proliferation of the glomerular cells (mesangial cells, endothelial cells and podocytes) which are a source of ROS or could be due to infiltration of the macrophage and the neutrophils in the patients with PGN. We observed significantly increased GST enzyme activity in the patients with PGN as compared to the patients with NPGN which may be a compensatory mechanism to increase the glomerular antioxidative defense [26]. The levels of MDA, 8isoprostane and GST enzyme activity were found to be significantly higher (P \ 0.05) in the PGN as compared to
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NPGN group even after adjustment for creatinine using ANCOVA, indicating that oxidative stress may contribute to the rapid progression of the PGN patients to renal failure. Thus, our results show that the presence of oxidative stress in PGD in general may contribute to the pathophysiology of glomerulonephritis. Moreover, PGN appears to be associated with severe oxidative stress and could be a contributing factor to progressive renal failure in these patients. In conclusion, our data provides evidence of oxidative stress in PGD, which is more pronounced in PGN as compared to NPGN, independent of degree of renal dysfunction.
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