Neutrophils harvest the intense green iron containing enzyme, Myeloperoxidase (MPO). MPO synthesis begins at the promyelocyte stage of neutrophil development and ends at the myeloid stage, during which the MPO containing azurophilic granules gets distributed to the daughter cells [1]. It is a highly cationic and glycosylated protein having a molecular weight of 144 kD [2]. They are released upon the activation of neutrophils, mostly during a state of inflammation. The peroxidase activity of MPO in the presence of H2O2 and chloride as co-substrates forms a major defense against myriad of bacteria, thus making it the key cellular defense mechanism in innate immunity [3].
Paradoxically, accumulating evidences implicated the role of MPO in initiation and progression of atherosclerosis [4]. MPO- H2O2-chloride triad results in the generation of many toxic end products like hypochlorite (HOCl), chloramines, hydroxyl radical, singlet oxygen and ozone. These agents when released out of the cell may damage the normal tissues. MPO has been found to be involved even in renal disease [5]. There are studies pointing to the presence of MPO and HOCl modified proteins in diseased renal tissues [6]. MPO is also linked to inflammation and oxidative stress due to its location in leucocytes and the role in catalyzing the formation of oxidative agents.
Nephrotic syndrome is a glomerular disease which causes intense proteinuria. Due to the tissue damage caused by numerous mechanisms, the porosity of the glomerular basement membrane is increased resulting in the leakage of protein eventually leading to an array of clinical symptoms. Role of MPO in the pathogenesis of Nephrotic syndrome has been a less researched subject. But there are substantial evidences available for neutrophils mediating glomerular injury in the nephritis models of rats [7]. Begenik et al., reported an increased MPO activity in adult Nephrotic syndrome patients [8]. Also there are studies which prove that MPO when bound to glomerular basement membrane can react with infused H2O2 in a chloride containing medium to induce glomerular injury [9]. Present study deals with the role of MPO and its association with other renal parameters in Nephrotic syndrome thus trying to elucidate a role of MPO in the prognosis of Nephrotic Syndrome.
Materials and Methods
The present study comprised of two phases. The first phase was a Case-Control study in which 40 newly diagnosed primary Nephrotic syndrome patients and 40 age and sex matched healthy subjects in the age group of 5-50 years were included. Control subjects were free from any clinical illness. Informed consent was obtained from all the subjects and the study was approved by the Institutional Ethics Committee. Fasting blood samples were collected in a sterile plain vacutainer from which the serum was separated and stored at -800C until analysis.
Myeloperoxidase was estimated using the method of Matheson et al., [10]. 100 μL serum was added to a mixture of 0.5 ml phosphate buffer (pH–6.1), 0.5 ml H2O2 and 0.5 ml 4-aminoantipyrine (substrate). Absorbance was recorded at one min intervals for 5 minutes at 512 nm in a UV-spectrophotometer. Average change in absorbance for one min was calculated. MPO activity was calculated using extinction coefficient of 13,900 M-1cm-1.
Serum urea, creatinine, total protein and albumin were measured in semi autoanalyser using commercially available kits. Blood Urea Nitrogen (BUN), BUN-Creatinine ratio (BUN/Cr), globulin and Albumin-Globulin ratio (A/G) were calculated using the standard formulae. Estimated Glomerular Filtration Rate (eGFR) was calculated using Modified Diet in Renal Disease (MDRD) formula [11]. Protein and creatinine was estimated in the 24 hour urine sample of Nephrotic syndrome patients using turbidimetric methods. Protein creatinine ratio was derived mathematically.
All the Nephrotic syndrome patients were treated with the recommended regime of steroids depending on the type of primary Nephrotic syndrome, following the KDIGO guidelines [12]. They were followed up during their regular check up in the hospital, for a period of ten months, in the second phase of the study. Out of 40 Nephrotic syndrome patients only 15 attained remission in this time period and they were included in the remission group. All the serum and urine analysis were repeated in these subjects.
Statistical Analysis
Sample size was calculated based on the statistical formula for paired sample analysis and it was found that 40 cases and 40 controls were adequate for the study. Sample size in remission group was taken according to the number of Nephrotic syndrome patients who attained remission in a time period of ten months. IBM SPSS, version 20.0 was used for the analysis. Quantitative data was expressed as mean ± standard deviation for normally distributed parameters and as median ± Inter Quartile Range (IQR) for non-normal distributions. All biochemical parameters which were normally distributed were analysed using paired students t-test, while those parameters which did not show normal distribution were subjected to Wilcoxon signed rank test. Pearsons and Spearmans correlation analysis was used for the parameters which follow normal and non-normal distribution respectively. p< 0.05 was considered statistically significant.
Results
[Table/Fig-1] shows the box plot of MPO levels in Nephrotic syndrome cases, controls and remission group. There was a significant increase in MPO levels in Nephrotic syndrome cases when compared with healthy controls. [Table/Fig-2] represents the values of renal function parameters in 40 cases and 40 control subjects. Urea, creatinine, BUN, BUN/Cr and eGFR values were higher in Nephrotic syndrome cases while total protein, albumin, globulin and A/G were less in them, when compared to the healthy controls.
Box plot showing MPO levels in healthy controls, cases and remission groups
Renal function parameters in Nephrotic syndrome cases and controls
| Sl No | Parameters | Cases (n=40) | Controls (n=40) | p-value |
|---|
| 1 | Urea | 38.52±26.26 | 22.70±6.51 | 0.001 |
| 2 | BUN | 18±12.27 | 10.60±3.04 | 0.001 |
| 3 | Creatinine | 1.63±0.98 | 0.86±0.19 | 0.025 |
| 4 | BUN/Creatinine | 22.79±18.48 | 12.81±4.40 | 0.001 |
| 5 | Total Protein | 4.24±1.07 | 7.69±0.79 | <0.001 |
| 6 | Albumin | 1.99±0.88 | 4.39±0.610 | <0.001 |
| 7 | Globulin | 2.25±0.45 | 3.28±1.08 | <0.001 |
| 8 | A/G | 0.91±0.42 | 1.54±0.71 | <0.001 |
| 9 | eGFR | 121.61±102.43 | 101.37±30.54 | 0.162 |
[Table/Fig-3] represents the comparison of MPO, renal function parameters and urine protein-creatinine ratio in 15 patients who attained remission, before and after steroid therapy. There was a significant decrease in MPO levels in the remission group when compared to their corresponding values before steroid therapy, while total protein, albumin and A/G ratio showed a significant increase [Table/Fig-3]. Correlation study of MPO showed a significant positive correlation with serum creatinine [Table/Fig-4] and urine protein-creatinine ratio [Table/Fig-5]. MPO also showed a weak positive correlation with serum urea and BUN. There was no correlation for MPO with eGFR and protein fractions.
Myeloperoxidase and renal function parameters in Nephrotic syndrome cases before and after steroid therapy
| Sl No | Parameters | Case (n=15) | Remission (n=15) | p-value |
|---|
| 1 | Myeloperoxidase | 91.5±45.3 | 44.5±21.6 | 0.003 |
| 2 | Urea | 42.92±29.17 | 30.07±8.88 | 0.057 |
| 3 | BUN | 20.06±13.63 | 14.05±4.15 | 0.056 |
| 4 | Creatinine | 1.11±0.67 | 0.77±0.18 | 0.078 |
| 5 | BUN/Creatinine | 20.1±12.43 | 18.54±5.05 | 0.659 |
| 6 | Total Protein | 4.29±1.10 | 6.78±1.41 | 0.001 |
| 7 | Albumin | 2.19±1.10 | 4.19±0.80 | <0.001 |
| 8 | Globulin | 2.10±0.42 | 2.59±0.91 | 0.097 |
| 9 | A/G | 1.11±0.62 | 1.89±1.15 | 0.046 |
| 10 | eGFR | 94.33±46.38 | 123.67±40.42 | 0.069 |
| 11 | Protein/Creatinine (urine) | 5.81±3.06 | 2.71±1.36 | 0.045 |
Correlation between MPO and Creatinine in Nephrotic syndrome cases
Correlation between Protein/Creatinine and MPO in Nephrotic syndrome cases
Discussion
Myeloperoxidase is a strongly cationic glycosylated protein with a molecular weight of 144 kD, consisting of two identical dimers linked by a disulfide bridge [13]. Even though it is important in enhancing the immune defense system, individuals possessing a promoter polymorphism and associated 2 fold reduction in MPO expression, was found to be cardio protected [14]. Generation of Reactive Oxygen Species via MPO catalyzed pathways may have a substantial impact on the promotion of inflammatory events. In this study, the MPO levels showed a marked increase in Nephrotic syndrome patients when compared to healthy controls. The primary reason for the increased MPO in Nephrotic syndrome may be as illustrated in [Table/Fig-6] [15,16].
Schematic representation illustrating the possible cause for increased Myeloperoxidase level in Nephrotic syndrome
Vaso relaxant Nitric oxide (NO) is also a substrate for MPO and thus MPO acts as a catalytic sink for NO thereby reducing its bioavailability. This may lead to endothelial dysfunction [17]. MPO is known to mediate protein carbamylation which leads to post translational protein modification. This makes the resulting protein unsuitable for enzymatic digestion and results in peptides with unexpected retention time and masses in Mass Spectrometric experiments. Usually uremia secondary to chronic renal disease creates an optimum environment to initiate protein carbamylation, which has been hypothesized as a mechanism that causes repeated infections and constitutes one of the major causes for morbidity and mortality [18]. In this study, the values of urea, creatinine, BUN and BUN/Cr ratio increased in Nephrotic syndrome cases when compared to healthy controls. MPO levels showed a significant positive correlation with creatinine in Nephrotic syndrome cases. Even though not significant, there was a positive correlation for MPO with urea and BUN (r=0.246, p=0.126). Therefore both uremia and increased MPO levels may cause protein carbamylation of circulating protein, mainly the extracellular matrix (ECM) proteins of the glomerulus. Carbamylated ECM proteins induce adhesion of monocytes, which causes subsequent inflammation and tissue damage finally leading to proteinuria [18]. In line with this, the present result points to the presence of hypo albuminemia, hypo proteinemia and hypo globulinemia in Nephrotic syndrome.
MPO and HOCl modified proteins co-localize into the glomerular basement membranes and tubular epithelia which suggest that MPO is a main pathogenic factor in glomerular and tubulointerstitial diseases [5]. When MPO was perfused into the renal arteries of the rats it bound to glomerular capillary wall due to its cationic charge and lead to subsequent platelet influx and degranulation. Thus MPO mediated glomerular disease results in endothelial and mesangial cell injury and subsequent proliferative response. These morphological changes resemble those seen in several forms of inflammatory and proliferatory responses in man [19]. In this study MPO levels showed a significant positive correlation with urine protein-creatinine ratio which is continuously being used in clinical settings for the diagnosis and to assess the severity of the disease. Thus, MPO levels strongly associates with the severity of the disease in Nephrotic syndrome.
On the contrary, in a study conducted in Saudi, MPO levels were found significantly low in chronic renal failure patients. This might be due to the inhibition of uremic toxins [20]. In another study conducted on patients with end stage renal disease it was observed that process of haemodialysis itself resulted in a significant rise in plasma MPO levels [21].
Results obtained during remission showed that there was a significant decrease in MPO levels when compared to the disease state. The levels of urea, creatinine, BUN and BUN/Cr ratio came down, even though not very significant, during remission in Nephrotic syndrome. This might be due to the effect of steroids which reduces the inflammation and thus healing the glomerular basement membrane. Glucocorticoids inhibit the soluble mediators released by the T lymphocytes and also have a direct action on the glomerular podocytes [22]. In glomerulonephritis mouse model, steroid treatment significantly reduced the number of MPO specific plasma cells in the spleen [23].
Thus the components of MPO pathway can be used as attractive targets for the development of prognostic biomarkers and also can be implemented as an important therapeutic target for Nephrotic syndrome. MPO inhibition can be done at different levels either by active site blockage or by diversion from chlorination cycle. Oxidized inhibitors and hypochlorite scavengers can also be used as important therapeutic strategies [24].
Conclusion
Myeloperoxidase enzyme is found to be elevated and it strongly correlates with the severity of disease in Nephrotic syndrome and thus can be used as a prognostic marker. Further studies can be done to use MPO as therapeutic target in Nephrotic syndrome to ameliorate the symptoms.