|Year : 2016 | Volume
| Issue : 3 | Page : 539-545
|Serum sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of patients with membranous nephropathy and focal and segmental glomerulosclerosis
Pragya Pant1, RG Singh1, Usha2, Santosh K Singh3, Vijay P Singh1, Prodip K Doley1, M Sivasankar1
1 Department of Nephrology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India
2 Department of Pathology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India
3 Center for Experimental Medicine and Surgery, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India
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|Date of Web Publication||13-May-2016|
| Abstract|| |
Diagnosis of membranous nephropathy (MN) and focal and segmental glomerulo- sclerosis (FSGS) needs a renal biopsy, which is an invasive procedure with potentially serious complications. Proteomics may be applied for the development of a biomarker for these diseases which will obviate the need of biopsy. Serum sodium dodecyl sulfate-polyacrylamide gel electro-phoresis (SDS-PAGE) analysis gives an idea of the various proteins with different molecular weights (MWs) in a given sample. This study was conducted to analyze proteins with different MWs in patients with MN and FSGS and to compare the two groups with regard to their protein profile. This was a comparative, experimental study performed from June 2013 to July 2014 in the Department of Nephrology, Sir Sunderlal Hospital, Banaras Hindu University, Varanasi. Twenty-three histologically diagnosed cases of primary MN and 25 cases of FSGS were included in the study. Patients were categorized as having mild, moderate, and severe proteinuria with 24 h urinary protein levels of <4, 4- 8 and ≥8 g/24 h, respectively. SDS-PAGE analysis was performed by the method of Laemmli and revealed a significantly higher number of patients with FSGS (80%) having a protein corresponding to 29 kDa MW, than those with MN (39.1%) (P = 0.004). Protein of 5 kDa MW was present in a significantly higher number of patients with moderate (80%) and severe (100%) proteinuria than those with mild proteinuria (25%) (P <0.001). Thus, protein of MW 29 kDa may be a marker for FSGS and needs further characterization. Similarly, 5 kDa protein, present in patients with moderate and severe proteinuria, might be either contributing to or be a marker of severe illness.
|How to cite this article:|
Pant P, Singh R G, Usha, Singh SK, Singh VP, Doley PK, Sivasankar M. Serum sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of patients with membranous nephropathy and focal and segmental glomerulosclerosis. Saudi J Kidney Dis Transpl 2016;27:539-45
|How to cite this URL:|
Pant P, Singh R G, Usha, Singh SK, Singh VP, Doley PK, Sivasankar M. Serum sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of patients with membranous nephropathy and focal and segmental glomerulosclerosis. Saudi J Kidney Dis Transpl [serial online] 2016 [cited 2020 Dec 3];27:539-45. Available from: https://www.sjkdt.org/text.asp?2016/27/3/539/182393
| Introduction|| |
Membranous nephropathy (MN) and focal and segmental glomerulosclerosis (FSGS) are primarily pathological diagnoses. At present, they are diagnosed on histological examination of the renal biopsy specimen. Renal biopsy is an invasive procedure with potential complica- tions such as hemorrhage, infection, pneumo- thorax, hypotension, bleeding, gross hematuria, and adjacent organ injury or decrease in hemo- globin requiring blood transfusion. Compli- cation rates range between 0% and 30%.,Furthermore, in the presence of certain contra- indications like uncontrolled bleeding diathe- sis, solitary kidney, uncontrolled blood pres- sure, obesity and noncooperative patient, biopsy is deferred, and diagnosis remains unclear. Moreover, biopsy requires admission and mo- nitoring, which increases the financial burden of the patients as well as burden on the medi- cal infrastructure. Availability of a noninvasive marker to diagnose these conditions will ob- viate the need for renal biopsy and hence avoid the associated complications, and reduce the burden on hospitals and patients too.
During the past few years, proteomics has been extensively applied in several fields of medicine to identify novel biomarkers.Pro- teomics could be applied to patients with suspected glomerulopathies like MN and FSGS aiming for the development of a novel bio- marker for specific pathological lesions, which may obviate the need for a renal biopsy.,,Serum sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis gives an idea of the molecular weight (MW) of the proteins present in a sample; any protein if found to be of interest, may be further charac- terized by two-dimensional (2D)-electropho- resis and MALDI-TOF-MS.
The present study was conducted with the following aims and objectives:
- To study the proteins with different MW present in serum of patients with MN and FSGS
- To compare the findings of the two groups.
| Materials and Methods|| |
Data of this study were collected from June 2013 to July 2014 in the Department of Nephrology, Sir Sunderlal Hospital, Banaras Hindu University, Varanasi.
Patients with MN and FSGS who attended the nephrology outpatient department and/ or were admitted in the nephrology ward, SS Hospital were included in the study.
It is a comparative, experimental study design.
A total of 23 cases of primary MN (Group
1) and 25 cases of FSGS (Group 2) were included in the study.
Patients with lesions of MN and FSGS diag- nosed on light microscopy and immunofluo- resecence of renal biopsy specimens were in- cluded in the study.
The patients having the following features were excluded:
Informed consent was obtained from the sub- jects included in the study.
- Inability to obtain informed consent
- Co-existent septicemia
- Dialysis dependent state
- Secondary MN.
The patient's detailed history, demographic details, clinical findings, and laboratory re- ports were recorded on a predesigned, pre- tested, and structured performa.
Patients were categorized as having mild (Group A), moderate (Group B), and severe (Group C) proteinuria with 24 h urinary pro- tein levels of <4, 4-8 and ≥8 g/24 h, respec- tively.
Five mL of blood was collected and centri- fuged at 249.75 g for 5 min to separate serum. The serum sample was distributed into 0.5 mL aliquots and stored at −20°C until the proteo- mic experiment.
SDS-PAGE analysis of the samples was per- formed by the method of Laemlli. MWs of expressed proteins were noted down in all the patients. Patients with MN and FSGS were compared with respect to the proteins of spe- cific MWs expressed in the two groups.
Data analysis was performed by SPSS version 16.0 software (IBM, USA). For intergroup comparison, Chi-square test was applied. P <0.05 was considered significant.
| Results|| |
Group 1 included 23 subjects out of which 15 were males (65.2%) as compared to Group 2 which included 25 subjects with 20 males (80%) (P = 0.335). The mean age of the pa- tients was 41.6 ± 8.917 years in Group 1 versus 32.04 ± 10 years in Group 2 (P <0.001). [Table 1] shows baseline clinical and epidemio- logical characteristics of the study subjects.
|Table 1. Baseline parameters of patients in Group 1 (n=25) and Group 2 (n=23)|
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SDS-PAGE analysis of the study groups revealed a significantly higher number of pa- tients with FSGS (80%) having a protein corresponding to 29 kDa MW, than those with MN (39.1%) (P = 0.004). The protein with MW 18 kDa was also present in a higher num- ber of patients with FSGS than those with MN (80% vs. 56.5%), although the difference was not significant statistically (P = 0.074). The number of patients with bands at 4, 5, 15, and 43 kDa was similar in the two groups [Table 2].
|Table 2. Comparison of patients with membranous nephropathy (Group 1) and focal and segmental glomerulosclerosis (Group 2) with respect to the presence of proteins of different molecular weights|
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We also observed that protein of 5 kDa MW was present in significantly higher number of patients with moderate (4-8 g/day; 80%) and severe (≥8 g/day; 100%) proteinuria than those with mild proteinuria (<4 g/day; 25%) (P <0.001). The difference was not significant between those with moderate and severe pro- teinuria [Table 3].
|Table 3. Comparison of patients with mild (Group A), moderate (Group B) and severe (Group C) proteinuria with respect to the presence of proteins of different molecular weights|
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[Figure 1] and [Figure 2] depict the diagrammatic re- presentation of the data.
|Figure 1. Percentage positivity of bands corresponding to proteins with different molecular weight in Group 1 versus 2|
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|Figure 2. Percentage positivity of bands corresponding to proteins with different molecular weights in Groups A, B and C|
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| Discussion|| |
When blood flows through organs, each cell from the surrounding tissues sheds proteins or its fragments in it.Thus, it is likely that patho- logical changes in the kidneys result in a com- plex correlation with serum peptidome profiles.
Nowadays, the focus is on interrogating the 1% of plasma proteome that contains low abundance proteins, which may be a trove house of information.,SDS-PAGE can be an informative initial proteomic assay which allows low to moderate resolution of serum proteome. One-dimensional gel electrophoresis of proteins provides information about the molecular size, amount, and purity of a protein sample.
SDS (sodium dodecyl sulfate) is a detergent that dissolves hydrophobic molecules so that all proteins retain their primary structure only and also, covers all the proteins with a nega- tive charge. This means all proteins will mi- grate toward the positive pole when placed in an electric field with speeds determined only by their MWs.
An electric field is applied across the gel. After the set amount of time, the biomolecules will migrate toward the positive electrode by different distances determined by their weight. Following electrophoresis, the gel may be stained for proteins, most commonly with Coomassie Brilliant Blue, allowing visua- lization of the separated proteins. Molecules are run in a separate lane in the gel to calibrate the gel and determine the approximate mole- cular mass of unknown biomolecules by comparing the distance traveled relative to the marker.
SDS-PAGE analysis has been used earlier as a method to investigate biomarkers for various glomerulopathies, especially FSGS, as there is lot of clinical and experimental evidence to suggest that FSGS is caused by a permeability factor.,,,,
A pivotal work by Sharma et al showed that the permeability factor resides in a 30-50 kDa plasma fraction, but this information did not lead to identification of the causal factor.
In a meticulous study by Sharma et al, it was found that under nondenaturing conditions, electrophoresis of the FSGS 70% supernatant showed a prominent low MW band that was not evident in the 70% supernatant from normal plasma.Injection of the 70% FSGS supernatant into rats caused a 3-fold increase in urine protein in collections from 6 to 24 h after injection. No increase in proteinuria occurred in rats injected with 70% supernatant from normal individuals. They concluded that the FSGS factor is a low MW protein with the potential to cause proteinuria in vivo.
Le Berre et al observed in their study that on SDS-PAGE of 70% ammonium sulfate (AS) supernatant from plasma of patients with FSGS, a 23 kDa protein was much more concentrated than in the non-FSGS plasma and was identified as apolipoprotein A.After sequential ultrafiltration of 70% AS super- natant on 30 and 50 kDa cutoff membranes, a second band of 43 kDa was found to be present in much higher concentration in the FSGS sample than in the non-FSGS nephrotics and healthy people.
Ramjee et al compared the reliability of the conventional selectivity index (SI) of serum and urinary transferrin and immunoglobulin G (IgG) with other tests of urinary proteins, such as SDS-PAGE and isoelectric focusing (IEF).Thirty-one children with steroid-resistant nephrotic syndrome (SRNS) were compared with 26 who had biopsy-proven FSGS and who were steroid resistant. SDS-PAGE and IEF revealed excretion of albumin and trans- ferrin only, with homogeneous anionic charge, respectively, in SRNS but unrestricted excre- tion of additional proteins IgG, beta 2- microglobulin, and lysozyme with heterogeneity of electrical charge in FSGS. With SDS-PAGE and IEF, they were able to predict all children who had SRNS and FSGS; the SI test pre- dicted all steroid-resistant patients with FSGS but was able to predict only 41.7% of the patients with SRNS.
In our study, the mean age of patients was significantly higher in the MN group as compared to the FSGS group. This is in concordance with the existing knowledge that MN is commoner in adults aged above 40 years.
In our study, we found significantly higher number of patients with FSGS (80%) having a protein of MW 29 kDa than those with MN (39.1%) (P = 0.004); this could be due to the elusive soluble urokinase-type plasminogen activator receptor (suPAR) or its fragments or due to apolipoprotein A. suPAR has an MW of 20-50 kDa.Few patients with MN also had this band (9/23). Indeed, in a study by Wei et al, it was observed that 4/11 patients with MN had elevated suPAR levels.In our study, the mean serum creatinine level did not differ significantly between the patients with FSGS and MN. This implies that variations in eGFR between the two groups did not confound the results.
We also observed a protein of 5 kDa mole- cular weight to be present in significantly higher number of patients with moderate (4-8 g/day; 80%) and severe (≥8 g/day; 100%) proteinuria than those with mild proteinuria (<4 g/day; 25%) (P <0.001). The difference was not significant between those with mode- rate and severe proteinuria. This protein might be either contributing to the severity of illness or it might be indicative of severe illness. No literature reporting the presence of a similar serum protein in severe nephrotic illnesses could be found, although it is known that low MW proteinuria corresponds to tubular protei- nuria and may be predictive of future deve- lopment of chronic renal failure.,,
The exact characterization of these proteins (5 and 29 kDa) will need further analysis with 2D-electrophoresis and MALDI-TOF. The study may provide a background on which further research may be done regarding the proteomic profile of patients with MN and FSGS.
Conflict of interest: None declared.
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Department of Nephrology, Institute of Medical Sciences, Banaras Hindu University, Varanasi - 221 005, Uttar Pradesh
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]
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