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Saudi Journal of Kidney Diseases and Transplantation
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Year : 2017  |  Volume : 28  |  Issue : 6  |  Page : 1256-1263
Neutrophil chemokines levels in different stages of nephrotic syndrome

1 Department of Physiology, College of Medicine, Al-Qassim University, Al-Qassim, Buraidah; Department of Physiology, King Saud University, Riyadh, Saudi Arabia
2 Department of Physiology, King Saud University, Riyadh, Saudi Arabia
3 Center of Excellence in Thrombosis and Hemostasis, College of Medicine, King Saud University, Riyadh, Saudi Arabia
4 Department of Medicine, College of Medicine, King Salman Chair for Kidney Disease Research, King Saud University, Riyadh, Saudi Arabia

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Date of Web Publication18-Dec-2017


Nephrotic syndrome (NS) is a disease of glomerular filtration barrier failure presenting with variable degrees of proteinuria, hypoalbuminemia, hyperlipidemia, and edema. Inflammation may contribute to the pathogenesis of NS. The aim of this study was to monitor the serum levels of three cytokines [i.e., granulocyte chemotactic protein-2 (GCP-2), growth-related oncogene-α (GRO-α), and interleukin-8 (IL-8)] in different stages of NS and to find out whether changes in the levels of these cytokines could be related to the severity of NS. This study included 125 patients who were divided into 40 patients with nephrotic range proteinuria (NRP), 45 patients with NS, and 40 patients who were in remission. This study also included 80 healthy participants as a control group. Enzyme-linked immunosorbent assay was used for the determination of the plasma levels of GRO-α, GCP-2, and IL-8. GCP-2 plasma levels were significantly higher in the NS and NRP groups when compared to the control group, whereas the GRO-α and IL-8 levels were significantly higher in all patient groups in comparison with the control group. All these chemokine levels were significantly decreased in remission as compared with the participants in the NS group (P <0.0001). There was a significant correlation between the cytokine levels and proteinuria and serum albumin in the NS group (P <0.0001). However, in the follow-up group, GCP-2 levels were significantly lower during remission as compared to those with active NS (P <0.0001). Our findings suggest that the pro-inflammatory cytokines GCP-2, GRO-α, and IL-8 could play a role in the pathogenesis of NS, particularly glomerular permeability.

How to cite this article:
Alsharidah AS, Alzogaibi MA, Bayoumy NM, Alghonaim M. Neutrophil chemokines levels in different stages of nephrotic syndrome. Saudi J Kidney Dis Transpl 2017;28:1256-63

How to cite this URL:
Alsharidah AS, Alzogaibi MA, Bayoumy NM, Alghonaim M. Neutrophil chemokines levels in different stages of nephrotic syndrome. Saudi J Kidney Dis Transpl [serial online] 2017 [cited 2021 Sep 22];28:1256-63. Available from: https://www.sjkdt.org/text.asp?2017/28/6/1256/220865

   Introduction Top

Nephrotic syndrome (NS) is a disease that results from the failure of the glomerular filtration barrier (GFB) and is characterized by proteinuria (>3–3.5 g/day in adults), hypoalbu-minemia, generalized edema, and hyperlipidemia.[1],[2],[3] Its incidence is about three new cases/100,000 each year in adults,[4] but it affects both children and adults.[5]

The pathogenesis of NS has not yet been clarified, but there is general agreement that the loss of selectivity of the GFB, which is the prime pathogenic feature of the disease, is the result of damage to the glomerular structures, mainly podocyte, glomerular basement membrane, or endothelial surface, which underlie the proteinuria.[6] The resulting proteinuria may potentiate pro-inflammatory responses such as the induction of cytokines that, in turn, result in inflammatory cell infiltration.[7],[8] On this basis, NS is considered as an immunoinflammatory disorder.[9]

Cytokines are soluble low-molecular-weight (5–15 kDa) proteins[10] that are secreted by leukocytes and other cells. Some cytokines are chemoattractants, and these are responsible for leukocyte basal and inflammatory traffic control by chemotaxis.[11] Interleukin-8 (IL-8) was first discovered as a pro-inflammatory cytokine secreted by human blood monocytes and later redefined as a “chemokine” for its chemotactic capabilities.[12] Granulocyte chemotactic protein 2 (GCP-2) is expressed by macrophages and epithelial cells during inflammation.[13] Growth-related oncogene-alpha (GRO-alpha) is expressed by both epithelial cells and macrophages.[14] These three chemokines play a major role in chemoattraction of the neutrophils.[12],[15] It has recently been demonstrated that cytokines/chemokines are involved in the inflammatory process, in particular in the progression of chronic renal disease,[16] and this notion has been extended further by the suggestion that cytokines/chemokines might also act as biomarkers of renal disease progression.[17] In this context, the present study aimed to monitor the serum levels of the chemoattractive cytokines GRO-α, GCP-2, and IL-8 in different stages of NS. Furthermore, we also aimed to find out whether the levels of these cytokines could be related to proteinuria and serum albumin levels. To the best of our knowledge, this is the first study to look at the levels of GRO-α and GCP-2 in different stages of NS.

   Methods Top

This is a case–control, cross-sectional study of 125 patients in different stages of NS, stratified and divided into three patients groups [40 nephrotic range proteinuria (NRP), 45 NS, and 40 in remission]; 80 healthy individuals formed the control group. Patients were recruited at King Khalid University Hospital, Riyadh, Kingdom of Saudi Arabia. Ten patients were followed up from NS stage until remission. The healthy participants were recruited randomly from blood donors and academic staff and were age- and sex-matched with the patient groups.

This study protocol was approved by the Institutional Review Board of the College of Medicine, King Saud University, Riyadh. Written consent was obtained from all patients and controls. All participants underwent complete physical examination and routine biochemical blood testing. In all patients, the diagnosis was confirmed by renal biopsy [Table 1].
Table 1: Results of renal biopsy in patient groups.

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General exclusion criteria for all groups included secondary causes of NS such as diabetes mellitus, hepatitis, preeclampsia, HIV, and the use of nonsteroidal anti-inflammatory drugs.

Blood sample collection and processing

Five-milliliter blood was collected by venepuncture directly into sodium citrate tubes; proper mixing of blood and anticoagulant was attained by gentle inversion to give a final blood:citrate ratio of 9:1. Blood samples were transported without delay (within 1 h of collection) to the Physiology Laboratory, College of Medicine. Platelet-poor plasma (PPP) was prepared by centrifuging the citrated whole blood samples at 3000 rpm (1000 g) for 15 min. Using plastic pipettes, the PPP was separated and stored in aliquots at – 80°C, until analysis at a later date. All plasma specimens were thawed at 37°C for 15 min before assay.


The plasma levels of GRO-α, GCP-2, and IL-8 were measured by enzyme-linked immunosorbent assay kits (R&D Systems Inc., Minneapolis, MN, USA, Catalog Number DGR00, DGC00, DGR00, respectively).

   Statistical Analysis Top

Collected data were analyzed using the Statistical Package for the Social Sciences (SPSS) version 21.0 (SPSS Inc., Chicago, IL, USA). Data are expressed as median (25%–75%). The Kolmogorov–Smirnov test of normality was used to ensure the results followed normal distribution. However, we found that the obtained data were not normally distributed using the skew factor range from negative 0.09 to 3.6. The Kruskal–Wallis test was used for analysis of variance (ANOVA) and post-ANOVA pair-wise comparison between the groups. The Mann–Whitney test was applied to work out the significance of the difference between groups. Pearson’s correlation coefficient was used to determine the correlation between relevant variables. The P <0.05 was considered statistically significant.

   Results Top

The demographic data of patients and controls are shown in [Table 2]. Out of 45 NS patients, 26 male (57.8%) with a median age/year (25–75 percentile) of 30 (25–45) were evaluated in this study. The NRP group was made of 29 male (72.5%) with a median age of 42/year (30–52). Finally, in the 40 patients in the remission group, 23 were male (57.5%) and 17 were female (42.5%), with a median age of 35 years (23–49). A total of 80 healthy controls were evaluated, 50 males (62.5%) and 30 females (37.5%), with median age of 37 (29–42).
Table 2: Physical characteristics and biochemical parameters of control and study groups.

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GCP-2 levels were significantly higher in NS (71.34 ng/mL) and NRP (49.31 ng/mL) groups when compared to the control group (39.42 ng/mL) (P <0.0001) [Figure 1]. Similarly, GRO-α and IL-8 median levels were significantly higher in NS (31.04 ng/mL, 9.9 ng/mL, respectively) and NRP (21.1 ng/mL, 6.1 ng/ mL, respectively) groups as compared to the control group (11.9 mg/mL, 4.6 ng/mL, respectively) (P <0.0001) [Figure 1]. However, levels of all three cytokines dropped significantly in remission (GCP-2 = 41.05 ng/mL, GRO-α = 16.17 ng/mL, IL-8 = 6.9 ng/mL) as compared to the NS group (GCP-2 = 71.34 ng/mL, GRO-α = 31.04 ng/mL, IL-8 = 9.9 ng/mL).
Figure 1: Cytokine levels in the control and patients groups.
GCP-2: Granulocyte chemotactic protein 2, GRO-α: Growth-related oncogene-α, IL-8: Interleukin-8, NS: Nephrotic syndrome, NRP: Nephrotic range proteinuria, ―: median, □: 25%–75%, Ι: Min-Max. Kruskal–Wallis test was used for ANOVA and post-ANOVA pair-wise to nonparametric statistical tests to compare between the groups.
*Statistically significant at P = 0.05.

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The levels of inflammatory cytokines GCP-2, GRO-α, and IL-8 correlated significantly with proteinuria and hypoalbuminemia in the NS group (P <0.0001) [Figure 2] and [Figure 3].
Figure 2: Correlation between cytokines and proteinuria.
GCP-2: Granulocyte chemotactic protein 2, GRO-α: Growth-related oncogene-α, IL-8: Interleukin-8. Pearson's correlation coefficient was used.

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Figure 3: Correlation between cytokines and albumin.
GCP-2: Granulocyte chemotactic protein 2, GRO-α: Growth-related oncogene-α, IL-8: interleukin-8. Pearson's correlation coefficient was used.

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In the follow-up group of patients, the levels of GCP-2 were significantly lower in the remission stage (39.42 ng/mL) as compared to the NS stage (85.02 ng/mL) (P <0.0001) [Figure 4].
Figure 4: Cytokine levels in the followed patients from NS until remission.
GCP-2: Granulocyte chemotactic protein 2, GRO-α: Growth-related oncogene-α, IL-8: Interleukin-8, NS: Nephrotic syndrome, : median; ―: 2%–75%; □: Min-Max. Mann–Whitney test was used to compare between two groups. *Statistically significant at P ≤0.05.

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   Discussion Top

The main findings results of the present study were the elevated levels of chemoattractant cytokines (GCP-2, GRO-α, and IL-8) in patients with NS and NRP. This indicates their possible pathogenic role in NS and NRP, especially in causing glomerular permeability. In addition, we found that the inflammatory process worsens with disease severity as the measured levels of inflammatory cytokines correlated with proteinuria and serum albumin.

IL-8 plays a regulatory role on neutrophil recruitment through binding to its two G-protein coupled receptors, C-X-C motif chemokine receptor 19 (CXCR1), and C-X-C CXCR type 2 (CXCR2).[12],[13] In the present study, the level of IL-8 was found to be substantially higher in all patient groups (NRP and NS) when compared to the control group (P <0.0001). The findings are similar to other studies, most of which reported a significant increase in IL-8 levels in pediatric patients with idiopathic NS.[18],[19],[20],[21],[22],[23] In contrast, Souto et al[24] in their study of children with idiopathic NS (8 patients with steroid-sensitive NS and 24 with steroid-resistant NS), investigated the possible relationship of the cytokine levels with disease activity (relapse and remission). They found no significant changes in the plasma levels of IL-8 between the groups. Our findings suggest that elevated levels of this chemokine in all groups of patients offers strong support to their possible involvement in the pathogenesis of adult NS by enhancing the glomerular inflammatory process.

The results obtained in the present study reveal that the levels of GCP2 were elevated significantly in both NRP and NS patient group as compared to the control group (P <0.0001). In addition, GRO-α plasma levels were significantly higher in all patient groups when compared to the control group (P <0.0001). However, all of these cytokines show a tendency toward a significant decrease in remission as compared with NS.

It is of interest to note that, in the follow-up group, GCP-2 levels dropped during remission, indicating a tendency of the return of the levels of this cytokine back toward normal. However, no significant differences in the levels of GRO-α and IL-8 were found in the follow-up group from NS till remission; although their levels started to decrease and their tendency to normalize was quite clear, they did not reach the significant level. There was a positive correlation to the levels of the cytokines GCP-2, GRO-α, and IL-8 and the degree of proteinuria and serum albumin. This would suggest a role of these cytokines in a pathogenic factor in the development to glomerular permeability, in NS. There have been reports of elevated levels of IL-8 in children with NS and significant correlation between IL-8 and proteinuria.[24],[25] IL-8, in particular, has been shown to have effects on glomerular permeability by increasing the catabolism of heparan sulfate on the glomerular basement membrane and subsequently decreasing the negative charge of heparan sulfate.[26],[27]

One limitation of our study is the inclusion of the patient with the heterogeneous cause of NS subgroups and also the small number of patients [Table 1]. The findings of this study need to be confirmed in a larger sample size.

   Conclusion Top

In summary, patients with NS and NRP had higher levels of GCP-2, GRO-α, and IL-8, which could play a role in the pathogenesis of NS, particularly glomerular permeability. Patients in remission had significantly lower levels of all three of these chemokines, indicating that their levels started to drop to reach normal levels. Finally, the GCP-2, GRO-α, and IL-8 levels were correlated with the levels of proteinuria and serum albumin, indicating that inflammatory process worsens with disease severity.

   Acknowledgments Top

The authors would like to thank Mr. Alwaleed Alhmmad for his technical assistance and the financial support of Al-Qassim University is sincerely acknowledged.

Conflict of interest: None declared.

   References Top

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Zachwieja J, Bobkowski W, Zaniew M, et al. Intracellular synthesis of cytokines as the index of function of peripheral blood lymphocytes and monocytes in children with first relapse of nephrotic syndrome. Pol Merkur Lekarski 2003;14:289-94.  Back to cited text no. 18
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Cho MH, Lee HS, Choe BH, et al. Interleukin-8 and tumor necrosis factor-alpha are increased in minimal change disease but do not alter albumin permeability. Am J Nephrol 2003; 23:260-6.  Back to cited text no. 20
Ece A, Atamer Y, Gürkan F, Bilici M, Koçyigit Y. Anti-oxidant status in relation to lipoproteins, leptin and pro-inflammatory cytokines in children with steroid-sensitive nephrotic syndrome. Nephrology (Carlton) 2004;9:366-73.  Back to cited text no. 21
Kanai T, Yamagata T, Momoi MY. Macrophage inflammatory protein-1beta and interleukin-8 associated with idiopathic steroid-sensitive nephrotic syndrome. Pediatr Int 2009;51:443-7.  Back to cited text no. 22
Araya C, Diaz L, Wasserfall C, et al. T regulatory cell function in idiopathic minimal lesion nephrotic syndrome. Pediatr Nephrol 2009;24:1691-8.  Back to cited text no. 23
Souto MF, Teixeira AL, Russo RC, et al. Immune mediators in idiopathic nephrotic syndrome: Evidence for a relation between interleukin 8 and proteinuria. Pediatr Res 2008;64:637-42.  Back to cited text no. 24
Garin EH, Laflam P, Chandler L. Antiinterleukin 8 antibody abolishes effects of lipoid nephrosis cytokine. Pediatr Nephrol 1998; 12:381-5.  Back to cited text no. 25
Garin EH, West L, Zheng W. Interleukin-8 alters glomerular heparan sulfate glycosaminoglycan chain size and charge in rats. Pediatr Nephrol 2000;14:284-7.  Back to cited text no. 26
Araya CE, Wasserfall CH, Brusko TM, et al. A case of unfulfilled expectations. Cytokines in idiopathic minimal lesion nephrotic syndrome. Pediatr Nephrol 2006;21:603-10.  Back to cited text no. 27

Correspondence Address:
Ashwag S Alsharidah
Physiology Department, College of Medicine, Alqassim University, Al-Qassim, Buraidah 51491
Saudi Arabia
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DOI: 10.4103/1319-2442.220865

PMID: 29265036

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4]

  [Table 1], [Table 2]


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