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Saudi Journal of Kidney Diseases and Transplantation
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ORIGINAL ARTICLE  
Year : 2011  |  Volume : 22  |  Issue : 4  |  Page : 739-745
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of urinary protein in acute kidney injury


Department of Renal Medicine, Singapore General Hospital, Singapore

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Date of Web Publication9-Jul-2011
 

   Abstract 

Recent experimental and clinical studies have shown the importance of urinary proteomics in acute kidney injury (AKI). We analyzed the protein in urine of patients with clinical AKI using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for its diagnostic value, and followed them up for 40 months to evaluate prognosis. Urine from 31 consecutive cases of AKI was analyzed with SDS-PAGE to determine the low, middle and high molecular weight proteins. Fractional excretion of sodium (FENa) was estimated from serum and urine creatinine and sodium (Na). The cases were followed-up for 40 months from the end of the recruitment of study cases. Glomerular protein was higher in the hematuria group when compared with the non-hematuria group (P <0.04) and in the AKI group than in the acute on chronic renal failure (AKI-on-CRF) group (P <0.002). Tubular protein was higher in the AKI-on-CRF group (P <0.003) than in the AKI group. Tubular protein correlated with FENa in groups with diabetes mellitus (DM), AKI-on-CRF, and without hematuria (P <0.03, P <0.02 and P <0.004, respectively). Pattern of protein did not differ between groups with and without DM and clinical acute tubular necrosis (ATN). At the end of 40 months follow-up, category with predominantly glomerular protein progressed to chronic renal failure (CRF) or end-stage renal failure in higher proportion (P <0.05). In clinical AKI, we observed that glomerular protein dominated in cases with glomerular insult, as indicated by hematuria. Tubular protein was common in the study cases with CRF, DM and cases without hematuria. This indicates tubulo-interstitial injury for AKI in these cases. Patients with predominantly glomerular protein had an adverse outcome.

How to cite this article:
Suhail SM, Woo K T, Tan H K, Wong K S. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of urinary protein in acute kidney injury. Saudi J Kidney Dis Transpl 2011;22:739-45

How to cite this URL:
Suhail SM, Woo K T, Tan H K, Wong K S. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of urinary protein in acute kidney injury. Saudi J Kidney Dis Transpl [serial online] 2011 [cited 2019 Nov 13];22:739-45. Available from: http://www.sjkdt.org/text.asp?2011/22/4/739/82672

   Introduction Top


In a clinical set-up, acute kidney injury (AKI) has many causes. [1] We defined AKI as an increase in serum creatinine (SCr) of more than 0.5 mg/dL (44.2 μmol/L) over the baseline if less than 221 mol/L or an increase in SCr of more than 20%, if baseline value is higher. [2],[3],[4],[5],[6],[7] Pathologically, the injury may be inflammatory, ischemic or necrotic; clinically, these are not evident as biopsy is not done frequently. However, important changes in proteinuria can occur in addition to changes of urinary indices. [9],[10]

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is a sensitive method for the analysis of urinary proteins used in the evaluation of various renal diseases. [11],[12],[13] Tubular proteins (T) of low-molecular weight and glomerular proteins (G) of middle-and high-molecular weight appear in varying proportions depending on the pathologic mechanism of AKI. [8],[9],[10],[14]


   Aim Top


Our aim for this observational study was to analyze the distribution of glomerular and tubular protein in different types of clinical AKI to elucidate their etiologic and prognostic significance. We followed-up these cases for a period of 40 months to evaluate the importance of SDS-PAGE urinary protein analysis on the outcome of AKI.


   Materials and Methods Top


Thirty-one consecutive cases of clinical AKI as defined earlier were included in this observational study. Inclusion criteria were adult patients admitted with AKI or those who developed AKI during hospital stay, patients with chronic kidney disease (CKD) stage 1 to 4 with AKI and patients with non-renal sepsis with AKI. Patients admitted to the intensive care unit (ICU) with AKI were also included in this study. Patients who were treated with hemodialysis (HD), slow low-efficiency dialysis (SLED) and continuous veno-venous hemofiltration (CVVH) according to individual requirement, were also included. We excluded cases with CKD stage 5, end-stage renal failure (ESRF), urinary tract infection, urinary tract obstruction and cases with multi-organ failure. To avoid bias, we maintained blindness on data regarding pre-existing proteinuria. We performed renal biopsy in cases of AKI on the basis of clinical decision. Clinical acute tubular necrosis (ATN) was considered in patients with hypotension with or without sepsis in whom AKI did not reverse after restoration of normal blood pressure with rehydration. ATN was also considered in cases of nephrotoxicity with toxic substances and drugs other than non-steroidal anti-inflammatory drugs (NSAID). All cases of glomerulonephritis (GN) were biopsy-proven before enrollment for the study. Hematuria was defined as presence of dysmorphic red blood cells (RBC) in urine beyond the reference limit on phase contrast microscopy. Patients were stratified in paired groups [Table 1] as AKI and acute on chronic renal failure (AKI-on-CRF), with and without diabetes mellitus (DM and No DM), with and without hematuria (H and No H) and with and without clinical acute tubular necrosis (ATN and No ATN). We followed-up the cases for 40 months from the end of the recruitment of study cases.
Table 1: Patient's stratification with urinary parameters.

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Urine and blood samples were taken from patients at the time of diagnosis of AKI. The pattern of proteins was analyzed by SDS-PAGE, which was performed with the Phast-System (Pharmasia, Sweden) using PhastGel gradient 8-25 and PhastGel SDS buffer strips according to the manufacturer's instructions. [15] Albumin and transferrin bands were identified by the immunoblot technique. These two bands were considered middle molecular proteins. The relative distance from the middle molecular bands differentiated the low-and high-molecular weight proteins. Each of the three protein types was scored semi-quantitatively using the reference laboratory value. The middle-and high-molecular weight proteins were classified as glomerular protein (G) and low-molecular weight proteins as tubular protein (T). The tubular and glomerular protein ratio (TG) of each urine sample was determined. Tubular pattern was defined as TG ratio more than 1 (TG >1), while glomerular pattern was considered when TG ratio was not more than 1 (TG ≤1). FENa was calculated using the standard formula: (UnaXSCr) / (UCrXSNa) × 100.

At the end of follow-up, the cases were stratified into group with normal renal function (NRF) and group with chronic renal failure (CRF/ESRF). Patients in the NRF group maintained a normal renal function (SCr <141 μmol/L, according to reference laboratory value) after resolution of AKI. Patients of the CRF/ESRF group progressed to CRF or ESRF after the episode of AKI.


   Statistical Analysis Top


Statistical analysis was performed with SPSS software. Patterns of protein were analyzed for correlation with FENa within each stratified group using Pearson's correlation analysis with univariate model. Each type of protein was compared between groups using one sample "t" test. Categories of NRF and CRF/ESRF at the end of follow-up were analyzed using Yate's Chi-square test.


   Results Top


Thirty-one consecutive adult patients (male: female: 19:12), aged (mean ± SD) 59 ± 14 years, diagnosed as AKI during the hospital stay or on admission, were enrolled in this study. [Table 2] shows patient's demographics, admission diagnosis and causes of AKI. Fifteen patients (48%) had CRF (CKD stage 3 and 4) before admission. AKI resolved in 23 (74%) cases where SCr improved to baseline value; 11 patients (35%) required dialysis during the AKI phase. Three of them required CVVH in the ICU because of hypotension with sepsis. One patient died of overwhelming sepsis during the hospital stay (3% mortality).The mean score of glomerular (G) and tubular (T) protein, tubulo-glomerular proteinuria ratio (TG), SCr and FENa in different stratified groups are shown in [Table 1]. The mean urine protein-creatinine ratio (UPCR) was 2.6 ± 3.6 μg/mg (range 0.02-10.4).
Table 2: Patient's demographics and diagnosis at admission.

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Tubular pattern (TG >1) positively correlated with FENa in groups with DM, with CRF and the group without hematuria (P <0.03, P <0.004 and P <0.02, respectively; [Table 3]). Glomerular protein was significantly higher in the group with hematuria when compared with the group without it (P <0.04). Glomerular pattern and tubular pattern were significantly different in the AKI-on-CRF group when compared with the AKI group (P <0.002 and P <0.03). No significant difference was found between patients with and without DM and groups with and without clinical ATN [Table 4].
Table 3: Correlation of glomerular protein and tubular protein and TG ratio with FENa in different stratified groups.

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Table 4: Comparison of glomerular protein, tubular protein and TG ratio within each stratified pair.

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During the follow-up period of 40 months, 11 patients (36%) maintained normal renal function (NRF) and 19 cases (64%) progressed to CRF, of whom six patients developed ESRF, and were started on dialysis [Figure 1]. One patient died of myocardial infarction (MI) after reaching ESRF during the follow-up period. Thirteen patients of the CRF/ESRF category had glomerular pattern (TG ≤1) during their AKI phase, while this pattern was seen in only three patients in the NRF group (X2 7.929, df 3, P <0.04; [Figure 1]).
Figure 1: Number of patients at the end of follow-up in categories of chronic renal failure and normal renal function, with predominant pattern of proteinuria during their AKI phase.

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   Discussion Top


Acute kidney injury results from many insults to renal structure and function. [16],[17] Sepsis, hypotension, disseminated intravascular coagulation, drug toxicity, contrast injury, cardiopulmonary bypass surgery and cardiopulmonary collapse are the most common factors responsible for glomerular and tubular dysfunction in the clinical set-up. Although reduction of urine output and rise of SCr are the hallmarks of AKI and AKI-on-CRF, there is only a minor degree of proteinuria. [18] Experimentally induced AKI in primates have consistently shown variation in the pattern of proteinuria. [19],[20] Many studies have demonstrated significant differences among the patterns of proteinuria in pediatric AKI. [18],[21] In clinical AKI, where there are various forms of injury to the kidney, the pattern of proteinuria has not been elucidated in detail elsewhere.

We used SDS-PAGE with Coumassie stain to qualify and quantify protein in urine. Proteins move according to their molecular weight in SDS-PAGE as the detergent in sodium dodecyl sulfate gel neutralizes the ionic charge. Hence, the proteins are separated according to their molecular weight. Several studies have shown that tubular proteins are of low molecular type and glomerular proteins are of middle and high molecular weight. [12],[22] The glomerular and tubular patterns were quantified using a semiquantitative scoring system. [9] Dominance of tubular protein was measured by the ratio of tubular and glomerular protein. A ratio greater than one was considered to be of tubular dominance in this study. Correlation of tubular pattern (TG >1) with FENa in groups with CRF, without hematuria and with DM indicated predominance of tubular injury in these groups of AKI as FENa is elevated in tubular dysfunction [Table 3]. Most studies that showed significance of tubular protein in AKI analyzed proteinuria by other methods. [21],[23] It is noted that when AKI cases were stratified with a clinical diagnosis of ATN, we could not demonstrate any differences in the pattern of proteinuria [Table 4].

Patients who had glomerular pattern (TG ≤1) during the AKI phase progressed to CRF or ESRF in a higher proportion than patients who maintained normal renal function (P <0.05, [Figure 1]). Very few studies have analyzed clinical AKI that showed varying patterns of proteinuria. Our study has revealed the presence of varying patterns of protein in urine using SDS-PAGE in different sub-groups of adult AKI. Study with long-term follow-up to see the prognosis of AKI has been performed previously. [24] But, to date, the long-term effect of pattern of proteinuria in the AKI phase has not been analyzed. This study has shown that SDS-PAGE analysis of urinary protein in clinical AKI cases has significant outcome effects.

In conclusion, we emphasize that in clinical AKI, the pattern of proteinuria, as analyzed by SDS-PAGE, might indicate diagnostic significance in terms of glomerular or tubular origin of AKI, and the pattern of proteinuria, particularly glomerular predominance, could predict progression to CRF/ESRF in the long run.

 
   References Top

1.Santos WJ, Zanetta DM, Pires AC, Lobo SM, Lima EQ, Burdmann EA. Patients with ischaemic, mixed and nephrotoxic acute tubular necrosis in the intensive care unit -a homogeneous population? Crit Care 2006;10(2): R68.  Back to cited text no. 1
    
2.Singri N, Ahja SN, Levin ML. Acute renal failure. JAMA 2003;289:747-51.  Back to cited text no. 2
    
3.Bellomo R, Kellum J, Ronco C. Acute renal failure: Time for consensus. Intensive Care Med 2001;27:1685-8.  Back to cited text no. 3
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4.Abosaif NY, Tolba YA, Heap M, Russell J, El Nahas AM. The outcome of acute renal failure in the intensive care unit according to RIFLE: model application, sensitivity, and predictability. Am J Kidney Dis 2005;46(6):1038-48.  Back to cited text no. 4
    
5.Kellum JA, Levin N, Bouman C, Lameire N. Developing a consensus classification system for acute renal failure. Curr Opin Crit Care 2002;8 : 509-14.  Back to cited text no. 5
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6.Mehta RL, Chertow GM. Acute renal failure definitions and classification: time for change? J Am Soc Nephrol 2003;14:2178-87.  Back to cited text no. 6
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7.Bellomo R, Ronco C, Kellum JA, Mehta R, Palevsky P; the ADQI workgroup. Acute renal failure-definition, outcome measures, animal models, fluid therapy and information technology needs: the Second International Consensus Coference of the Acute Dialysis Quality Initiative (ADQI) Group. Crit Care 2004;8: R204-12.  Back to cited text no. 7
    
8.Cappi SB, Sakr Y, Vincent JL. Daily evaluation of organ function during renal replacement therapy in intensive care unit patients with acute renal failure. J Crit Care 2006;21 (2):179-83.  Back to cited text no. 8
    
9.Woo KT, Lau YK, Lee GS, Wei SS, Lim CH. Pattern of proteinuria in IgA nephritis by SDS-PAGE: Clinical significance. Clin Nephrol 1991;38:6-11.  Back to cited text no. 9
    
10.Coimbra TM, Furtado MR, Carvalho IF. Crossed immuno-electrophoresis analysis of the urinary excretion of alpha 1-acid glycol-protein, alpha 1-antitrypsin, albumin, and transferrin in normal subjects and in patients with renal disease. Braz J Med Biol Res 1984;17(1):35-41.  Back to cited text no. 10
    
11.Lau YK, Woo KT. SDS-PAGE is underutilised as a tool for investigating renal patients. Nephron 2002;90:227-9.  Back to cited text no. 11
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12.Boesken WH. Diagnostic significance of SDS-PAA-electrophoresis of urinary proteins: different forms of proteinuria and their correlation to renal diseases. Curr Prob Clin Biochem 1979;9:235-48.  Back to cited text no. 12
    
13.Grillenberger A, Weninger M, Lubec G. Determination of urinary low molecular weight proteins for the diagnosis of tubular damage. Padiatr Pathol 1987;22(3):229-34.  Back to cited text no. 13
    
14.Pires MT, Da Cunha AS, Virella G, Simoes J. Analytical characterization of urinary proteins by sodium dodecyl sulphate-polyacrylamide gel electrophoresis in renal disease. Clinical and histopathological correlations. Nephron 1975;14(5):361-72.  Back to cited text no. 14
    
15.Weber MH, Verwiebe R, Neuhoff V, Scheler F. Analysis of proteinuria by Micro-PAGE electrophoresis. Contr Nephrol 1988;68:179-87.  Back to cited text no. 15
    
16.Uchino S, Bellomo R, Goldsmith D, Bates S, Ronco C. An assessment of the RIFLE criteria for acute renal failure in hospitalized patients. Crit Care Med 2006;34(7):1913-7.  Back to cited text no. 16
    
17.Gursel G, Demir N. Incidence and risk factors for the development of acute renal failure in patients with ventilator-associated pneumonia. Nephrology (Carlton) 2006;11(3):159-64.  Back to cited text no. 17
    
18.Murakami T, Itagaki A, Takahashi H. A change of urinary proteins from a glomerular pattern to a tubular pattern during a late diuretic phase of acute renal failure. Child Nephrol Urol 1988-89;9(3):160-2.  Back to cited text no. 18
    
19.De Tata V, Bombara M, Novelli M, Pingitore R, Bergamini E. Glycated plasma proteins in experimentally induced acute toxic renal failure by dichromate injection: evidence for loss with urine and decreased plasma levels. Int J Exp Pathol 1998;79(3):141-9.  Back to cited text no. 19
    
20.Furuhama K, Onodera T. Studies on experimental renal damage in rats, II, beta 2-microglobulin: purification, radioimmunoassay, and urine and serum levels in tubular nephropathy. Nippon Yakurigaku Zasshi 1982;79(5): 409-19.  Back to cited text no. 20
    
21.Tomlinson PA, Dalton RN, Hartley B, Haycock GB, Chantler C. Low molecular weight protein excretion in glomerular disease: a comparative analysis. Pediatr Nephrol 1997;11(3):285-90.  Back to cited text no. 21
    
22.Lubec D, Grillenberger A, Weninger M, Lubec G. Value of SDS-polyacrylamide gel electrophoresis, small molecular weight proteins and alpha-1-acid glycoprotein for the diagnosis of tubular damage. Padiatr Padol 1987;22(4):319-24.  Back to cited text no. 22
    
23.Tomlinson PA, Dalton RN, Turner C, Chantler C. Measurement of beta 2-microglobulin, retinol-binding protein, alpha 1-microglobulin and urine protein 1 in healthy children using enzyme-linked immunosorbent assay. Clin Chim Acta 1990;192(2):99-106.  Back to cited text no. 23
    
24.Gentric A, Cledes J. Immediate and long term prognosis in acute renal failure in elderly. Nephrol Dial Transplant 1991;6(2):86-90.  Back to cited text no. 24
    

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Correspondence Address:
Sufi M Suhail
Department of Renal Medicine, Singapore General Hospital, Outram Road
Singapore
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PMID: 21743220

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