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Saudi Journal of Kidney Diseases and Transplantation
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ORIGINAL ARTICLE  
Year : 2016  |  Volume : 27  |  Issue : 1  |  Page : 37-40
Assay of urinary protein-bound sialic acid can differentiate steroidsensitive nephrotic syndrome from steroid-resistant cases


1 Department of Biochemistry, Mahatma Gandhi Medical College and Research Institute (MGMC & RI), SBV, Pillaiyarkuppam, Puducherry, 607 402, India
2 Department of Biochemistry, Maulana Azad Medical College, New Delhi - 2, India
3 Maulana Azad Institute of Dental Sciences, New Delhi - 2, India
4 Jawaharlal Institute of Post-Graduate Medical Education and Research (JIPMER), Puduchery 6, India

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Date of Web Publication15-Jan-2016
 

   Abstract 

The protein selectivity index as measured from the ratio of urinary immunoglobulin to albumin failed to differentiate between steroid-sensitive (SS) and steroid-resistant (SR) cases of nephrotic syndrome (NS). Sialic acid contributes negative charges to many plasma proteins. The negative charge is a determinant of protein excretion rate. The prognostic significance of assay of urinary excretion of protein-bound sialic acid in NS has not been evaluated. Hence, the present study was designed to evaluate whether measurement of urinary protein bound sialic acid (UPBSA) can be used as a marker to differentiate SS from SR cases of NS. The urine samples of 70 (47 SS and 23 SR) pediatric NS children were assayed for UPBSA by Aminoff's method. The levels were compared and the receiver-operator curve was drawn to determine the optimum cutoff point to differentiate among the groups before starting the therapy. The excretion of UPBSA in SR cases of NS was significantly higher than that of SS cases (P<0.05). The optimum cutoff limit for UPBSA was 2.71 μg/mg of proteins with 75% sensitivity and 75.5% specificity for differentiating SS cases from SR cases (area under the plasma- concentration time curve = 0.814, P = 0.009). We conclude that UPBSA can differentiate SR cases from SS cases of NS in pediatric patients and may help in predicting the response to steroid therapy.

How to cite this article:
Gopal N, Koner BC, Bhattacharjee A, Bhat V. Assay of urinary protein-bound sialic acid can differentiate steroidsensitive nephrotic syndrome from steroid-resistant cases. Saudi J Kidney Dis Transpl 2016;27:37-40

How to cite this URL:
Gopal N, Koner BC, Bhattacharjee A, Bhat V. Assay of urinary protein-bound sialic acid can differentiate steroidsensitive nephrotic syndrome from steroid-resistant cases. Saudi J Kidney Dis Transpl [serial online] 2016 [cited 2019 Nov 14];27:37-40. Available from: http://www.sjkdt.org/text.asp?2016/27/1/37/174066

   Introduction Top


Nephrotic syndrome (NS) is primarily a pediatric disorder, being 15-times more common in this age group. The incidence is 2-6/100,000 per year. [1] Children with NS between the age group of one and eight years are presumed to have minimal lesion disease, which is steroid sensitive (SS). [2] However, children who continue to have proteinuria despite steroid therapy are considered steroid-resistant (SR) cases. The prognosis of the SRNS patients is not as good as the SSNS cases, and they often need other immunosuppressive drugs. [2]

The assessment of prognosis of NS depends on its response to corticosteroid therapy. The degree of proteinuria is not proportional to the severity of glomerular abnormality and hence cannot be used as a prognostic marker. [2],[3] Protein selectivity index (PSI), a measure of the immunoglobulin:albumin ratio, was found to be low in severe histological change in glomerulus, [3] and it did not provide much diagnostic or prognostic benefit in the evaluation of NS and is no longer performed routinely. [4] To the best of our knowledge, there is no dependable laboratory marker to predict the response to steroid therapy and prognosis of NS.

Sialic acid is a nine carbon containing negatively charged carbohydrate. Most of the plasma proteins (except albumin) are glycoproteins that contain sialic acid as the terminal moiety in their glycan units. Sialic acid adds a negative charge to these plasma proteins, which contributes to repulsion by glomerular filter bed. [5],[6] To the best of our knowledge, no study has evaluated the significance of appearance of sialo-glycoproteins in urine of NS cases.

The aim of our study was to evaluate the urinary protein-bound sialic acid (UPBSA) levels in pediatric NS cases to determine their clinical and prognostic significance in NS.


   Patients and Methods Top


This study was conducted at the Department of Biochemistry of the Jawaharlal Institute of Post-graduate Medical Education and Research (JIPMER), Puducherry, India, in collaboration with the Department of Pediatrics. This study was approved by the Institute Human Ethics Committee of JIPMER.

A total of 70 children with first-onset NS were included in this study. Among them, 47 children were found to be SS and 23 were detected to be either SRNS as assessed from the response to prednisone therapy and renal histopathology. All cases were between one and eight years of age, and they were not suffering from any other major illnesses. Their parents/guardians were informed about our study and written consent was obtained from them to include their children in our study. Assents were also obtained from the children.

After confirmation of the diagnosis of NS, about 100 mL of casual urine samples were collected from each newly diagnosed case in the pediatric Outpatient Department, nephrotic clinic or the children's wards before starting steroid therapy. The diagnosis of SR was confirmed from the children's histopathology reports and presence of chronic renal failure was ruled out at the time of sample collection. The protein from the collected urine was precipitated by centrifuging with trichloroacetic acid. The protein precipitate was stored at -50°C. From the precipitate, UPBSA was estimated by Aminoff's method. [7] All the cases were followed-up at least for 18 weeks. Children with first-onset NS who responded to steroid therapy with trace or nil proteinuria had been grouped SSNS and children who continued to have proteinuria (2+ or greater) even after eight weeks of steroid therapy were labeled SRNS. A subset of patients who had a relapse while they were on alternate day steroid therapy or within eight weeks of stopping prednisone were treated with oral prednisolone (60 mg/m 2 /day) and responded well; they were also included as SSNS.


   Statistical Analysis Top


The data are presented as the mean with standard deviation. For comparison of the means, unpaired Student t test was used. A receiver- operating curve (ROC) was drawn to determine the optimum cutoff for predicting SSNS from SRNS cases. A P-value <0.05 was considered as significant for all the statistical tests.


   Results Top


Of 70 NS cases who received steroids, 47 responded with trace/nil proteinuria and were included in the SSNS group. There were 23 cases in the SRNS group. The mean UPBSA levels were compared between these groups. The UPBSA levels were significantly high in the SRNS cases compared with the SSNS cases [Table 1].
Table 1: Comparison of demographic characteristics and baseline biochemical parameters among steroid sensitive and steroid resistant cases of nephrotic syndrome.

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An ROC curve was drawn and the optimum cutoff value of UPBSA was 2.71 μg/mg of protein having sensitivity of 75% and specificity of 75.5% to differentiate SSNS cases from SRNS cases [Figure 1]. The area under the plasma-concentration time curve (AUC) of the ROC was 0.814.
Figure 1: ROC curve showing sensitivity and specificity at different cutoff values of UPBSA for differentiating steroid-resistant from steroidsensitive cases of nephrotic syndrome (area under the plasma concentration–time curve: 0.814) (P-value = 0.009).

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   Discussion Top


Our study found higher levels of UPBSA in SRNS in comparison with SSNS, and may be used to differentiate between them clinically.

The protein selectivity index, which was once thought to be a useful marker, is no more used because of large overlap, [3],[4] and it did not have optimum sensitivity and specificity to be a clinically useful prognostic marker. [4] The measurement of excretion of immunoglobulin or albumin failed to differentiate between SRNS and SSNS.

Although the derangement in the glomerular basement membrane architecture determines the extent of proteinuria, loss of negative charge on GBM is another important determinant of proteinuria. The extent of loss of this negative charge might have some prognostic significance. The GBM architecture may be studied by microscopy, but there is no direct method to assay for the negative charge on it. The negatively charged moieties on the plasma proteins are repelled by the negatively charged carbohydrate moieties on the healthy glomerular filter bed.

Probably when the glomerular filter bed loses the negative charges beyond a certain point, there remain hardly any barriers to prevent small-sized albumin from being excreted. It probably becomes a freely filterable substance not affected by the variation in the glomerular charge that occurs between SRNS and SSNS. On the other hand, immunoglobulins are so big that they encounter a high mechanical glomerular barrier. For large proteins such as immunoglobulins, the mechanical barrier becomes a major determinant of the rate of filtration. Hence, in spite of some variation in glomerular charges between SRNS and SSNS, the excretion rate of immunoglobulins is minimally altered to discriminate SRNS from SSNS cases by the PSI.

Hence, the appearance of proteins having more negatively charged moieties in urine, because of binding to sialic acid, is surmised to be an index of the loss of the negative charges on the GBM.

The large AUC of the ROC and high sensitivity and specificity of cutoff to differentiate SRNS from SSNS cases indicates that this parameter has great potential to be included as a prognostic marker in NS. The measurement of UPBSA levels reflects the collective sialic acid contributed by different sialo-glycoproteins excreted by kidney from plasma. They are diverse in size, shape and other characters. In the present study, this collective measurement of excretion of these proteins in the form of UPBSA was able to differentiate the cases of SRNS from SSNS. Similarly, by computer simulation with diverse lipid molecules, BarEvan et al, demonstrated that at a molecular level also, the mixture of diverse molecules generates a newer collective property that is not present in any of these individual molecules. [8]

We conclude that SRNS patients excrete more sialo-glyproteins in urine. Assay of UPBSA may help in differentiating these cases from SSNS cases. It can predict the response to steroid therapy in NS and a different plan of action may be initiated for such cases from the very beginning instead of waiting for the result of the steroid trial. Larger longitudinal studies are required to confirm our results.

Conflict of Interest: None declared.

 
   References Top

1.
Alhassan A, Mohamed WZ, Alhaymed M. Patterns of childhood nephrotic syndrome in Aljouf region, Saudi Arabia. Saudi J Kidney Dis Transpl 2013;24:1050-4.  Back to cited text no. 1
[PUBMED]  Medknow Journal  
2.
Ding WY, Koziell A, McCarthy HJ, et al. Initial steroid sensitivity in children with steroid-resistant nephrotic syndrome predicts post-transplant recurrence. J Am Soc Nephrol 2014;25:1342-8.  Back to cited text no. 2
    
3.
Saleem MA, Tizard J, Dudley J, Inward C, Caward R, McGrow M. Disorders of the urinary system. In: McIntosh N, Helms P, Smyth R, Logan S, eds. Forfar Et Arneil's Text Book of Pediatrics. 7th ed. New York: Churchill Livingstone/Elsevier; 2008. p. 545-84.  Back to cited text no. 3
    
4.
Wang HH, Fu LW, Yang LY, Chen WP, Tsai SJ, Lin CY. A study of the relationship between IgG subclass/IgM and idiopathic nephrotic syndrome. Zhonghua Min Guo Xiao Er Ke Yi Xue Hui Za Zhi 1997;38:21-7.  Back to cited text no. 4
    
5.
Ito M, Sugihara K, Asaka T, et al. Glycoprotein hyposialylation gives rise to a nephrotic-like syndrome that is prevented by sialic acid administration in GNE V572L point-mutant mice. PLoS One 2012;7:e29873.  Back to cited text no. 5
    
6.
Schauer R. Achievements and challenges of sialic acid research. Glycoconj J 2000;17:485-99.  Back to cited text no. 6
    
7.
Aminoff D. Methods for the quantitative estimation of N-acetylneuraminic acid and their application to hydrolysates of sialomucoids. Biochem J 1961;81:384-92.  Back to cited text no. 7
    
8.
Bar-Even A, Shenhav B, Kafri R, Lancet DJ. The lipid world: From catalytic and informational head groups to micelle replication and evolution without nucleic acids. In: Seckbach J, ed. Life in the Universe. The Netherlands: Kluwer Academic Publishers; 2005. p. 111-4.  Back to cited text no. 8
    

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Correspondence Address:
Niranjan Gopal
Department of Biochemistry, Mahatma Gandhi Medical College and Research Institute, SBV, Pillaiyarkuppam, Puducherry - 607 402
India
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DOI: 10.4103/1319-2442.174066

PMID: 26787564

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