http://informahealthcare.com/rnf ISSN: 0886-022X (print), 1525-6049 (electronic) Ren Fail, Early Online: 1–5 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/0886022X.2014.964141
CLINICAL STUDY
The progress of inflammation and oxidative stress in patients with chronic kidney disease Gaosi Xu1, Kaiping Luo1,2, Huixin Liu1,2, Tianlun Huang1, Xiangdong Fang1, and Weiping Tu1 Department of Nephrology, Second Affiliated Hospital, Nanchang University, Nanchang, China and 2Medical Center of the Graduate School, Nanchang University, Nanchang, China
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1
Abstract
Keywords
Background: The variations and their correlation of inflammation and oxidative stress in chronic kidney disease (CKD) have not been thoroughly understood. Materials and methods: Biomarkers of inflammation and oxidative stress were measured in a cohort of 176 patients with CKD ranging from stage 1 to 5 and 67 healthy controls. Correlation analysis in levels between inflammation and oxidative stress was also performed with estimated glomerular filtration rate (eGFR) using the Modification of Diet in Renal Disease (MDRD) formula. Concentrations of serum creatinine (Scr), hs-CRP (hypersensitive C reactive protein) and MDA (malondialdehyde) of these participants were measured again after 12 month follow-up. Results: In the present study, with the development of CKD, serum levels of hs-CRP, interleukin-6 (IL-6) and MDA were significantly increased, and the serum levels of SOD (superoxide dismutase) and GSH-PX (glutathione peroxidase) were significantly decreased in these participants. eGFR was inversely associated with MDA and positively with SOD and GSH-PX when adjusting for age and hypertension therapy. IL-6 and hs-CRP were positively correlated with MDA, and negatively associated with SOD and GSH-PX. Notably, after 12-month follow-up, the increase in Scr was positively associated with the increase in hs-CRP (p50.01) and MDA (p50.05), respectively. Conclusions: Inflammation and oxidative stress interacted with each other and played pivotal roles in the development of CKD. Variation in eGFR was parallel with the changes of oxidative stress and inflammation when CKD developing.
Chronic kidney disease, inflammation, oxidative stress
Introduction A cross-sectional survey of a nationally representative sample of adult Chinese displayed that the overall prevalence of chronic kidney disease (CKD) is 10.8%.1 Therefore, the estimated number of patients with CKD in China is approximately 119.5 million, which exacerbate the burden of financial budget. Early detection and slowing progression to end-stage renal disease (ESRD) are two major aims for controlling the development of CKD. Oxidative stress appears to increase as CKD progresses and correlates significantly with the level of renal function. Oxidative stress and inflammation play pivotal roles in the pathogenesis and progression of CKD.2,3 Oxidative stress and inflammation are associated with higher mortality in patients receiving long-term hemodialysis.4 However, the precise prevalence of oxidative stress and inflammation among CKD patients remains undetermined due to the lack of population-based studies.5
History Received 1 May 2014 Revised 30 June 2014 Accepted 31 August 2014 Published online 6 November 2014
Cystatin C has a linear association with inflammatory biomarkers such as TNF-alpha in an ambulatory elderly cohort with estimated glomerular filtration rates (eGFR) not less than 60 mL/min/1.73 m2. 6 The latest published study indicated that cytokine-mediated inflammation was involved in early stages of impaired renal function.7 However, there has not correlation with eGFR between increased oxidative stress and inflammation in patients with CKD stage 3–5.8 None of the inflammatory cytokines, particularly CRP (C reactive protein), was affected by CKD stages.9 There have been no studies about the variance in serum creatinine (Scr) and the correlation between inflammatory cytokines and oxidative stress in patients with CKD. Therefore, the present work aims to investigate the relationship between inflammatory cytokines and oxidative stress in patients with CKD, and the association between the variance in Scr, inflammation and oxidative stress in these patients.
Materials and methods Study population Address correspondence to Gaosi Xu, Department of Nephrology, Second Affiliated Hospital, Nanchang University, No. 1, Minde Road, Nanchang 330006, P.R. China. Tel: +86-791-86270383; Fax: +86-79186270383; E-mail: [email protected]
The study protocol was proved by the Ethics Committee of Second Affiliated Hospital of Nanchang University. Informed consents were obtained from all the participated patients.
2
G. Xu et al.
Ren Fail, Early Online: 1–5
Table 1. General demographic characteristics of patients (n ¼ 176). Primary disease (n) CKD Control 1–2 3 4 5
Male/Female (n)
Age
eGFR
ACEI/ARB (n)
CCB (n)
CGN
DN
HN
Others
40/27 31/22 35/23 22/15 16/12
51.4 ± 15.5 52.8 ± 16.9 50.5 ± 14.1 48.2 ± 18.3 49.8 ± 17.6
118.6 ± 8.7 86.5 ± 8.4 48.4 ± 8.0 19.7 ± 4.3 9.5 ± 4.2
0 11 12 7 5
0 8 21 25 18
0 32 34 27 19
0 10 6 5 8
0 5 9 6 7
0 1 2 2 3
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Notes: CKD: chronic kidney disease, eGFR: estimated glomerular filtration rate (mL/min/1.73 m2), ACEI: angiotensin converting enzyme inhibitor, ARB: angiotensin receptor blocker, CCB: calcium channel blocker, CGN: chronic glomerular nephritis, DN: diabetic nephropathy, HN: hypertensive nephropathy, Others: including obstructive and uric acid nephropathy, etc.
Patients were recruited from outpatient and inpatient departments. Participants were all 418 years old and presence of CKD defined by glomerular filtration rate in agreement with the National Kidney Foundation.10 From September 2011 to January 2013, a cohort of 176 patients with CKD ranging from stage 1 to 5 and 67 healthy controls were included in the study. eGFR was calculated using the Modification of Diet in Renal Disease (MDRD) formula.11 Study participants underwent a detailed review of their disease history, and laboratory measurements consisting of biomarkers of serum oxidative stress and inflammatory cytokines at the time of enrollment and the end of the 12-month follow-up. Patients with infective diseases, active rheumatic diseases, tumor, trauma, cardio-cerebral break and severe hepatopathy were not included in this study. Participants who received corticosteroid, antioxidants and immunosuppressive agents within 3 months were also excluded from the present investigation. Blood sampling Fasting blood sample was obtained from all participants. Routine biochemical parameters were assayed in automated analyzer using commercial kits. All these measurements were carried out at the time of entry into the study, and partially Scr, hs-CRP (hypersensitive C reactive protein) and MDA (malondialdehyde) at the end of the 12-month follow-up. For serum screening of hs-CRP, samples were drawn into Vacutainer serum separator tubes containing clot activator. Tubes were kept at room temperature and centrifuged within 1 h after sample drawing. For serum IL-6 and biomarkers of oxidative stress detection, sample was drawn into Vacutainer tubes containing EDTA. Tubes were immediately placed on ice and centrifuged. Serum samples were kept at 4 C until frozen, stored at 70 C until analysis. Serum concentrations of hs-CRP were detected by latexenhanced immunoturbidimetry (Quantex hsCRP kit, BIOKIT, Barcelona, Spain), and levels of serum IL-6 were determined by a sandwich ELISA kit (Human IL-6 Quantikine HS, R&D Systems, GmbH, Wiesbaden, Germany). MDA, SOD (superoxide dismutase) and GSH-PX (glutathione peroxidase) were determined by the previously described method.12 Calculation of the variance ratios Calculation of the variance ratios (VRs) for Scr, hs-CRP and MDA was based on the following formula. For example, VRs for Scr ¼ (Scrafter 12 months Scrbaseline level)/Scrbaseline level%.
Statistical analysis Statistical analysis was performed using SPSS 17.0 software for windows. The results were expressed as mean ± standard deviation (SD). Demographic data and routine biochemical data were compared among the patient groups using one-way analysis of variance (ANOVA). Pearson correlation analysis was used to find the correlation of parameters between inflammation and oxidative stress. Spearman correlation coefficients were used to assess relationships among eGFR, inflammatory cytokines and oxidative stress. p50.05 was considered significant.
Results Patient characteristics The general characteristics of the study groups are displayed in Table 1. No significant differences obtained between groups in gender, age, receiving ACEI (angiotensin converting enzyme inhibitor) or ARBs (angiotensin receptor blockers) treatment and the primary diseases. The primary diseases include chronic glomerular nephritis, diabetic nephropathy, hypertensive nephropathy, obstructive and uric acid nephropathy, etc. Table 2 showed that the concentrations of inflammatory biomarkers hs-CRP (p ¼ 0.008) and IL-6 (p ¼ 0.006) were significantly higher in CKD patients compared with healthy controls. Similarly, the serum concentration of MDA (p ¼ 0.01) was significantly higher in CKD patients than in healthy participants. The serum levels of SOD (p ¼ 0.025) and GSHPX (p ¼ 0.031) were significantly lower in CKD participants than in healthy controls. Table 2 also showed the levels of nutritional markers in various study groups. Serum levels of hemoglobin and albumin were significantly decreased in CKD participants compared with healthy controls (p50.05, respectively). In Figure 1, differences in serum levels of hs-CRP, MDA, IL-6, SOD and GSH-Px in patients with different stage of CKD have been described in detail. To determine the potential relationship between the decrease in eGFR, inflammation and oxidative stress, the levels of eGFR, hs-CRP and IL-6 were compared with MDA, SOD and GSH-PX in 176 CKD participants. As shown in Table 3, when all the participants were analyzed together, there were positive correlations between inflammation and MDA and between eGFR and antioxidants (p50.05, respectively). However, negative associations were observed between eGFR and MDA, and between inflammation and SOD
Inflammation and oxidative stress in CKD
DOI: 10.3109/0886022X.2014.964141
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Table 2. Nutritional, inflammatory and oxidative stress variations in the study groups. CKD
N
Hb (g/L)
Alb (g/L)
hs-CRP (mg/L)
IL-6 (ng/L)
MDA (nmol/mL)
SOD (U/mL)
GSH-PX(U/L)
Control 1-2 3 4 5
67 53 58 37 28
127.6 ± 8.9 122.9 ± 25.5 98.4 ± 23.7 91.2 ± 19.3 75.6 ± 12.8
43.2 ± 4.4 38.5 ± 7.2 36.4 ± 6.3 35.6 ± 5.9 34.7 ± 6.8
1.16 ± 0.7 2.05 ± 1.21 3.68 ± 1.82 5.98 ± 1.92 7.31 ± 1.82
54.6 ± 13.9 95.2 ± 19.7 191.9 ± 40.4 274.8 ± 50.5 329.3 ± 64.4
3.21 ± 1.35 4.25 ± 1.89 5.18 ± 2.09 7.94 ± 3.45 12.14 ± 4.10
130.7 ± 31.5 92.8 ± 18.2 75.4 ± 15.3 70.4 ± 15.2 68.3 ± 17.6
276.4 ± 23.3 233.9 ± 11.8 180.4 ± 19.7 159.6 ± 18.2 142.4 ± 20.9
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Notes: CKD: chronic kidney disease, Hb: hemoglobin, Alb: albumin, hs-CRP: high-sensitive CRP, MDA: malondialdehyde, SOD: superoxide dismutase, GSH-PX: glutathione peroxidase.
Figure 1. Differences in serum levels of hs-CRP, MDA, IL-6, SOD and GSH-Px in patients with different stage of CKD.
Table 3. Spearman correlation analyses for inflammation and oxidative stress in patients with CKD. MDA
hs-CRP IL-6 eGFR
SOD
GSH-PX
r Value
p Value
r Value
p Value
r Value
p Value
0.896 0.861 0.904
50.01 50.05 50.01
0.901 0.893 0.963
50.01 50.01 50.01
0.885 0.869 0.872
50.01 50.05 50.05
(p50.01, respectively). Similarly, inflammation and GSHPX also correlated negatively with each other (p50.05, respectively). With the development of CKD, the associations of variance in parameters between Scr, inflammation and oxidative stress have not been previously investigated. Variance ratios for Scr, hs-CRP and MDA in 113 patients with CKD 2–4 after the 12-month follow-up were firstly analyzed in present study (Table 4). Significance of Pearson correlation analysis was performed between Scr and hs-CRP (p50.01), between Scr and MDA (p50.05), and between hs-CRP and MDA (p50.05), respectively.
Discussion The current leading cause of CKD in China is glomerular nephropathy, followed by diabetic nephropathy and hypertension.13 Inflammation and oxidative stress were common features of patients with advanced CKD, however, there were
Table 4. Variance ratios for hs-CRP and MDA in patients with CKD after 12 months of follow-up (%). CKD 2 3 4
Scr
hs-CRP
MDA
35.7 ± 8.1 59.2 ± 31.7 58.2 ± 20.9
41.5 ± 24.3 146.0 ± 84.8 93.4 ± 47.5
24.9 ± 23.6 65.5 ± 32.8 73.2 ± 37.4
Note: Significance of Pearson correlation analysis between Scr and hs-CRP (p50.01), between Scr and MDA (p50.05), and between hs-CRP and MDA (p50.05), respectively.
few studies examined the relationship between the biomarkers of inflammation and oxidative stress in these participants. The production of reactive oxygen species is usually in balance with the state of oxidative stress. There is a positive correlation between increasing oxidative stress and advancing stage of CKD.6,7,14 Several researches demonstrated the inverse relationship between eGFR and serum levels of inflammatory cytokines in advanced CKD patients.15,16 However, the extent to which eGFR is correlated with biomarkers of inflammatory cytokines is controversial.8,9 The most commonly used marker of lipid oxidation is MDA, which is formed naturally without any enzymatic activity.17 MDA is a kind of end-product generated by lipid peroxidation and has been considered as a biomarker of increased oxidative stress during CKD.18 Based on the abovementioned findings, we showed that there were significant differences in biomarkers of oxidative stress and inflammation
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G. Xu et al.
between CKD patients and healthy controls. Significant correlations were displayed between the levels of eGFR and the biomarkers of oxidative stress, between the levels of biomarkers for inflammation and oxidative stress. The most notable finding in this study was the first report of the close relationship in variance ratios between Scr, hs-CRP and MDA in patients with CKD after the 12-month follow-up. Therefore, we deduced that not only the serum levels of Scr, but also the variance extent of Scr are positively correlated with the concentrations of inflammatory cytokines in CKD participants. Our study put forward a significant positive correlation between hs-CRP, IL-6 and eGFR suggesting that these inflammatory cytokines seem to have a crucial role in the pathogenesis of CKD. ACE inhibitors or ARBs are frequently used in an attempt to delay the progression of CKD and to decrease proteinuria. Many of the effects of ACEI or ARBs signaling on target tissues are mediated via enhanced production of reactive oxygen species through activation of NADPH (nicotinamide adenine dinucleotide phosphate) oxidases.19 Several studies have also showed that the administration of ACEI or ARBs may have potential anti-inflammatory effects.20 However, there was no significant difference in recipient of ACEI or ARBs between the four CKD patient groups from stage 1 to 5. Increased levels of inflammatory mediators, i.e., hs-CRP and IL-6, may play independent as well as interdependent roles via several signaling pathways which lead to hyperglycemia-mediated augment in oxidative stress. However, increase in oxidative stress may reversely amplify inflammation, then setting up a vicious cycle by mechanisms including activation of the nuclear transcription factor kB (NF-kB), which contributes to the activation and recruitment of immune cells. Therefore, inflammatory cytokines associated with oxidative stress promote renal tissue damage by inflicting apoptosis, necrosis, fibrosis, and may be major mechanisms in the pathogenesis and progress of CKD.21 Although the exact mechanisms of inflammation and oxidative stress have not been accurately elucidated in CKD patients, a number of following described factors appear to be involved, including uremic toxins,3 rennin-angiotensin system,22 hypertension,23 underlying diseases (diabetes, autoimmune diseases, etc.),24 infection, iron overload,25 antioxidant deficiency,26 etc. Oxidative stress can provoke inflammation via activating NF-kB and consequent generation of pro-inflammatory cytokines.27 Production and release of reactive oxygen, nitrogen species and chlorine by the activated immune cells in turn promote the oxidative stress.28 The application of anti-oxidants for therapeutic intervention in patients with CKD may prove beneficial, but whether or not several anti-oxidants as a multi-drug therapy to target oxidant modifying pathways during the development of CKD remain unclear.29–32 In conclusion, our present study indicated that inflammation and oxidative stress interacted with each other and played pivotal roles in the development of CKD. Variation in eGFR was parallel with the changes of oxidative stress and inflammation when CKD developing. However, large-scale, multi-centered, controlled studies should be performed to reach the final conclusion.
Ren Fail, Early Online: 1–5
Declaration of interest The research was supported by the National Natural Science Foundation of China (No. 81360122/H0518). No conflict of interests is declared.
References 1. Zhang L, Wang F, Wang L, et al. Prevalence of chronic kidney disease in China: A cross-sectional survey. Lancet. 2012; 379(9818):815–822. 2. Remuzzi G, Benigni A, Remuzzi A. Mechanisms of progression and regression of renal lesions of chronic nephropathies and diabetes. J Clin Invest. 2006;116(2):288–296. 3. Vaziri ND, Oveisi F, Ding Y. Role of increased oxygen free radical activity in the pathogenesis of uremic hypertension. Kidney Int. 1998;53(6):1748–1754. 4. Kotur-Stevuljevic J, Simic-Ogrizovic S, Dopsaj V, et al. A hazardous link between malnutrition, inflammation and oxidative stress in renal patients. Clin Biochem. 2012;45(15):1202–1205. 5. Massy ZA, Stenvinkel P, Drueke TB. The role of oxidative stress in chronic kidney disease. Semin Dial. 2009;22(4):405–408. 6. Keller CR, Odden MC, Fried LF, et al. Kidney function and markers of inflammation in elderly persons without chronic kidney disease: The health, aging, and body composition study. Kidney Int. 2007;71(3):239–244. 7. Nerpin E, Helmersson-Karlqvist J, Rise´rus U, et al. Inflammation, oxidative stress, glomerular filtration rate, and albuminuria in elderly men: A cross-sectional study. BMC Res Notes. 2012;5:537. 8. Oberg BP, McMenamin E, Lucas FL, et al. Increased prevalence of oxidant stress and inflammation in patients with moderate to severe chronic kidney disease. Kidney Int. 2004;65(3):1009–1016. 9. Morena M, Patrier L, Jaussent I, et al. Reduced glomerular filtration rate, inflammation and HDL cholesterol as main determinants of superoxide production in non-dialysis chronic kidney disease patients. Free Radic Res. 2011;45(6):735–745. 10. Ward RA, McLeish KR. Polymorphonuclear leukocyte oxidative burst is enhanced in patients with chronic renal insufficiency. J Am Soc Nephrol. 1995;5(9):1697–1702. 11. Levey AS, Bosch JP, Lewis JB, Greene T, Rogers N, Roth D. A more accurate method to estimate glomerular filtration rate from serum creatinine: A new prediction equation. Modification of Diet in Renal Disease Study Group. Ann Intern Med. 1999; 130(6):461–470. 12. Bartnicki P, Fijałkowski P, Majczyk M, Błaszczyk J, Banach M, Rysz J. Effect of methoxy polyethylene glycol-epoetin beta on oxidative stress in predialysis patients with chronic kidney disease. Med Sci Monit. 2013;19:954–959. 13. Liu ZH. Nephrology in China. Nat Rev Nephrol. 2013; 9(9):523–528. 14. Dounousi E, Papavasiliou E, Makedou A, et al. Oxidative stress is progressively enhanced with advancing stages of CKD. Am J Kidney Dis. 2006;48(5):752–760. 15. Stuveling EM, Hillege HL, Bakker SJ, Gans RO, De Jong PE, De Zeeuw D. C-reactive protein is associated with renal function abnormalities in a non-diabetic population. Kidney Int. 2003; 63(2):654661. 16. Pecoits-Filho R, Heimbu¨rger O, Ba´ra´ny P, et al. Associations between circulating inflammatory markers and residual renal function in CRF patients. Am J Kidney Dis. 2003;41(6):1212–1218. 17. Nielsen F, Mikkelsen BB, Nielsen JB, Andersen HR, Grandjean P. Serum malondialdehyde as biomarker for oxidative stress: Reference interval and effects of life-style factors. Clin Chem. 1997;43(7):1209–1214. 18. Atamer A, Kocyigit Y, Ecder SA, et al. Effect of oxidative stress on antioxidant enzyme activities, homocysteine and lipoproteins in chronic kidney disease. J Nephrol. 2008;21(6):924–930. 19. Wingler K, Wu¨nsch S, Kreutz R, Rothermund L, Paul M, Schmidt HH. Upregulation of the vascular NAD(P)H-oxidase isoforms Nox1 and Nox4 by the renin-angiotensin system in vitro and in vivo. Free Radic Biol Med. 2001;31(11):1456–1464. 20. Miyata T, van Ypersele de Strihou C, Ueda Y, et al. Angiotensin II receptor antagonists and angiotensin-converting enzyme inhibitors lower in vitro the formation of advanced glycation end
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products: Biochemical mechanisms. J Am Soc Nephrol. 2002; 13(10):2478–2487. Gupta S, Gambhir JK, Kalra O, et al. Association of biomarkers of inflammation and oxidative stress with the risk of chronic kidney disease in Type 2 diabetes mellitus in North Indian population. J Diabetes Complications. 2013;27(6):548–552. Kim CH, Vaziri ND. Hypertension promotes integrin expression and reactive oxygen species generation by circulating leukocytes. Kidney Int. 2005;67(4):1462–1470. Manning Jr RD, Tian N, Meng S. Oxidative stress and antioxidant treatment in hypertension and the associated renal damage. Am J Nephrol. 2005;25(4):311–317. Usharani P, Fatima N, Muralidhar N. Effects of Phyllanthus emblica extract on endothelial dysfunction and biomarkers of oxidative stress in patients with type 2 diabetes mellitus: A randomized, double-blind, controlled study. Diabetes Metab Syndr Obes. 2013;6:275–284. Lim CS, Vaziri ND. Iron and oxidative stress in renal insufficiency. Am J Nephrol. 2004;24(6):569–575. Galli F, Ghibelli L, Buoncristiani U, et al. Mononuclear leukocyte apoptosis in haemodialysis patients: The role of cell thiols and vitamin E. Nephrol Dial Transplant. 2003;18(8):1592–600.
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27. Stenvinkel P, Ketteler M, Johnson RJ, et al. IL-10, IL-6, and TNFalpha: Central factors in the altered cytokine network of uremia— The good, the bad, and the ugly. Kidney Int. 2005;67(4):1216–1233. 28. Sela S, Shurtz-Swirski R, Cohen-Mazor M, et al. Primed peripheral polymorphonuclear leukocyte: A culprit underlying chronic lowgrade inflammation and systemic oxidative stress in chronic kidney disease. J Am Soc Nephrol. 2005;16(8):2431–2438. 29. Small DM, Coombes JS, Bennett N, Johnson DW, Gobe GC. Oxidative stress, anti-oxidant therapies and chronic kidney disease. Nephrology (Carlton). 2012;17(4):311–321. 30. Boaz M, Smetana S, Weinstein T, et al. Secondary prevention with antioxidants of cardiovascular disease in endstage renal disease (SPACE): Randomised placebo-controlled trial. Lancet. 2000; 356(9237):1213–1218. 31. Yusuf S, Dagenais G, Pogue J, Bosch J, Sleight P. Vitamin E supplementation and cardiovascular events in high-risk patients. The Heart Outcomes Prevention Evaluation Study Investigators. N Engl J Med. 2000;342(3):154–160. 32. Xu G, Yan W, Li J. An update for the controversies and hypotheses of regulating nonthyroidal illness syndrome in chronic kidney diseases. Clin Exp Nephrol. 2014 Apr 22. [Epub ahead of print]. doi:10.1007/s10157-014-0974-1.
CLINICAL STUDY
The progress of inflammation and oxidative stress in patients with chronic kidney disease Gaosi Xu1, Kaiping Luo1,2, Huixin Liu1,2, Tianlun Huang1, Xiangdong Fang1, and Weiping Tu1 Department of Nephrology, Second Affiliated Hospital, Nanchang University, Nanchang, China and 2Medical Center of the Graduate School, Nanchang University, Nanchang, China
Ren Fail Downloaded from informahealthcare.com by Universiteit Twente on 12/09/14 For personal use only.
1
Abstract
Keywords
Background: The variations and their correlation of inflammation and oxidative stress in chronic kidney disease (CKD) have not been thoroughly understood. Materials and methods: Biomarkers of inflammation and oxidative stress were measured in a cohort of 176 patients with CKD ranging from stage 1 to 5 and 67 healthy controls. Correlation analysis in levels between inflammation and oxidative stress was also performed with estimated glomerular filtration rate (eGFR) using the Modification of Diet in Renal Disease (MDRD) formula. Concentrations of serum creatinine (Scr), hs-CRP (hypersensitive C reactive protein) and MDA (malondialdehyde) of these participants were measured again after 12 month follow-up. Results: In the present study, with the development of CKD, serum levels of hs-CRP, interleukin-6 (IL-6) and MDA were significantly increased, and the serum levels of SOD (superoxide dismutase) and GSH-PX (glutathione peroxidase) were significantly decreased in these participants. eGFR was inversely associated with MDA and positively with SOD and GSH-PX when adjusting for age and hypertension therapy. IL-6 and hs-CRP were positively correlated with MDA, and negatively associated with SOD and GSH-PX. Notably, after 12-month follow-up, the increase in Scr was positively associated with the increase in hs-CRP (p50.01) and MDA (p50.05), respectively. Conclusions: Inflammation and oxidative stress interacted with each other and played pivotal roles in the development of CKD. Variation in eGFR was parallel with the changes of oxidative stress and inflammation when CKD developing.
Chronic kidney disease, inflammation, oxidative stress
Introduction A cross-sectional survey of a nationally representative sample of adult Chinese displayed that the overall prevalence of chronic kidney disease (CKD) is 10.8%.1 Therefore, the estimated number of patients with CKD in China is approximately 119.5 million, which exacerbate the burden of financial budget. Early detection and slowing progression to end-stage renal disease (ESRD) are two major aims for controlling the development of CKD. Oxidative stress appears to increase as CKD progresses and correlates significantly with the level of renal function. Oxidative stress and inflammation play pivotal roles in the pathogenesis and progression of CKD.2,3 Oxidative stress and inflammation are associated with higher mortality in patients receiving long-term hemodialysis.4 However, the precise prevalence of oxidative stress and inflammation among CKD patients remains undetermined due to the lack of population-based studies.5
History Received 1 May 2014 Revised 30 June 2014 Accepted 31 August 2014 Published online 6 November 2014
Cystatin C has a linear association with inflammatory biomarkers such as TNF-alpha in an ambulatory elderly cohort with estimated glomerular filtration rates (eGFR) not less than 60 mL/min/1.73 m2. 6 The latest published study indicated that cytokine-mediated inflammation was involved in early stages of impaired renal function.7 However, there has not correlation with eGFR between increased oxidative stress and inflammation in patients with CKD stage 3–5.8 None of the inflammatory cytokines, particularly CRP (C reactive protein), was affected by CKD stages.9 There have been no studies about the variance in serum creatinine (Scr) and the correlation between inflammatory cytokines and oxidative stress in patients with CKD. Therefore, the present work aims to investigate the relationship between inflammatory cytokines and oxidative stress in patients with CKD, and the association between the variance in Scr, inflammation and oxidative stress in these patients.
Materials and methods Study population Address correspondence to Gaosi Xu, Department of Nephrology, Second Affiliated Hospital, Nanchang University, No. 1, Minde Road, Nanchang 330006, P.R. China. Tel: +86-791-86270383; Fax: +86-79186270383; E-mail: [email protected]
The study protocol was proved by the Ethics Committee of Second Affiliated Hospital of Nanchang University. Informed consents were obtained from all the participated patients.
2
G. Xu et al.
Ren Fail, Early Online: 1–5
Table 1. General demographic characteristics of patients (n ¼ 176). Primary disease (n) CKD Control 1–2 3 4 5
Male/Female (n)
Age
eGFR
ACEI/ARB (n)
CCB (n)
CGN
DN
HN
Others
40/27 31/22 35/23 22/15 16/12
51.4 ± 15.5 52.8 ± 16.9 50.5 ± 14.1 48.2 ± 18.3 49.8 ± 17.6
118.6 ± 8.7 86.5 ± 8.4 48.4 ± 8.0 19.7 ± 4.3 9.5 ± 4.2
0 11 12 7 5
0 8 21 25 18
0 32 34 27 19
0 10 6 5 8
0 5 9 6 7
0 1 2 2 3
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Notes: CKD: chronic kidney disease, eGFR: estimated glomerular filtration rate (mL/min/1.73 m2), ACEI: angiotensin converting enzyme inhibitor, ARB: angiotensin receptor blocker, CCB: calcium channel blocker, CGN: chronic glomerular nephritis, DN: diabetic nephropathy, HN: hypertensive nephropathy, Others: including obstructive and uric acid nephropathy, etc.
Patients were recruited from outpatient and inpatient departments. Participants were all 418 years old and presence of CKD defined by glomerular filtration rate in agreement with the National Kidney Foundation.10 From September 2011 to January 2013, a cohort of 176 patients with CKD ranging from stage 1 to 5 and 67 healthy controls were included in the study. eGFR was calculated using the Modification of Diet in Renal Disease (MDRD) formula.11 Study participants underwent a detailed review of their disease history, and laboratory measurements consisting of biomarkers of serum oxidative stress and inflammatory cytokines at the time of enrollment and the end of the 12-month follow-up. Patients with infective diseases, active rheumatic diseases, tumor, trauma, cardio-cerebral break and severe hepatopathy were not included in this study. Participants who received corticosteroid, antioxidants and immunosuppressive agents within 3 months were also excluded from the present investigation. Blood sampling Fasting blood sample was obtained from all participants. Routine biochemical parameters were assayed in automated analyzer using commercial kits. All these measurements were carried out at the time of entry into the study, and partially Scr, hs-CRP (hypersensitive C reactive protein) and MDA (malondialdehyde) at the end of the 12-month follow-up. For serum screening of hs-CRP, samples were drawn into Vacutainer serum separator tubes containing clot activator. Tubes were kept at room temperature and centrifuged within 1 h after sample drawing. For serum IL-6 and biomarkers of oxidative stress detection, sample was drawn into Vacutainer tubes containing EDTA. Tubes were immediately placed on ice and centrifuged. Serum samples were kept at 4 C until frozen, stored at 70 C until analysis. Serum concentrations of hs-CRP were detected by latexenhanced immunoturbidimetry (Quantex hsCRP kit, BIOKIT, Barcelona, Spain), and levels of serum IL-6 were determined by a sandwich ELISA kit (Human IL-6 Quantikine HS, R&D Systems, GmbH, Wiesbaden, Germany). MDA, SOD (superoxide dismutase) and GSH-PX (glutathione peroxidase) were determined by the previously described method.12 Calculation of the variance ratios Calculation of the variance ratios (VRs) for Scr, hs-CRP and MDA was based on the following formula. For example, VRs for Scr ¼ (Scrafter 12 months Scrbaseline level)/Scrbaseline level%.
Statistical analysis Statistical analysis was performed using SPSS 17.0 software for windows. The results were expressed as mean ± standard deviation (SD). Demographic data and routine biochemical data were compared among the patient groups using one-way analysis of variance (ANOVA). Pearson correlation analysis was used to find the correlation of parameters between inflammation and oxidative stress. Spearman correlation coefficients were used to assess relationships among eGFR, inflammatory cytokines and oxidative stress. p50.05 was considered significant.
Results Patient characteristics The general characteristics of the study groups are displayed in Table 1. No significant differences obtained between groups in gender, age, receiving ACEI (angiotensin converting enzyme inhibitor) or ARBs (angiotensin receptor blockers) treatment and the primary diseases. The primary diseases include chronic glomerular nephritis, diabetic nephropathy, hypertensive nephropathy, obstructive and uric acid nephropathy, etc. Table 2 showed that the concentrations of inflammatory biomarkers hs-CRP (p ¼ 0.008) and IL-6 (p ¼ 0.006) were significantly higher in CKD patients compared with healthy controls. Similarly, the serum concentration of MDA (p ¼ 0.01) was significantly higher in CKD patients than in healthy participants. The serum levels of SOD (p ¼ 0.025) and GSHPX (p ¼ 0.031) were significantly lower in CKD participants than in healthy controls. Table 2 also showed the levels of nutritional markers in various study groups. Serum levels of hemoglobin and albumin were significantly decreased in CKD participants compared with healthy controls (p50.05, respectively). In Figure 1, differences in serum levels of hs-CRP, MDA, IL-6, SOD and GSH-Px in patients with different stage of CKD have been described in detail. To determine the potential relationship between the decrease in eGFR, inflammation and oxidative stress, the levels of eGFR, hs-CRP and IL-6 were compared with MDA, SOD and GSH-PX in 176 CKD participants. As shown in Table 3, when all the participants were analyzed together, there were positive correlations between inflammation and MDA and between eGFR and antioxidants (p50.05, respectively). However, negative associations were observed between eGFR and MDA, and between inflammation and SOD
Inflammation and oxidative stress in CKD
DOI: 10.3109/0886022X.2014.964141
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Table 2. Nutritional, inflammatory and oxidative stress variations in the study groups. CKD
N
Hb (g/L)
Alb (g/L)
hs-CRP (mg/L)
IL-6 (ng/L)
MDA (nmol/mL)
SOD (U/mL)
GSH-PX(U/L)
Control 1-2 3 4 5
67 53 58 37 28
127.6 ± 8.9 122.9 ± 25.5 98.4 ± 23.7 91.2 ± 19.3 75.6 ± 12.8
43.2 ± 4.4 38.5 ± 7.2 36.4 ± 6.3 35.6 ± 5.9 34.7 ± 6.8
1.16 ± 0.7 2.05 ± 1.21 3.68 ± 1.82 5.98 ± 1.92 7.31 ± 1.82
54.6 ± 13.9 95.2 ± 19.7 191.9 ± 40.4 274.8 ± 50.5 329.3 ± 64.4
3.21 ± 1.35 4.25 ± 1.89 5.18 ± 2.09 7.94 ± 3.45 12.14 ± 4.10
130.7 ± 31.5 92.8 ± 18.2 75.4 ± 15.3 70.4 ± 15.2 68.3 ± 17.6
276.4 ± 23.3 233.9 ± 11.8 180.4 ± 19.7 159.6 ± 18.2 142.4 ± 20.9
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Notes: CKD: chronic kidney disease, Hb: hemoglobin, Alb: albumin, hs-CRP: high-sensitive CRP, MDA: malondialdehyde, SOD: superoxide dismutase, GSH-PX: glutathione peroxidase.
Figure 1. Differences in serum levels of hs-CRP, MDA, IL-6, SOD and GSH-Px in patients with different stage of CKD.
Table 3. Spearman correlation analyses for inflammation and oxidative stress in patients with CKD. MDA
hs-CRP IL-6 eGFR
SOD
GSH-PX
r Value
p Value
r Value
p Value
r Value
p Value
0.896 0.861 0.904
50.01 50.05 50.01
0.901 0.893 0.963
50.01 50.01 50.01
0.885 0.869 0.872
50.01 50.05 50.05
(p50.01, respectively). Similarly, inflammation and GSHPX also correlated negatively with each other (p50.05, respectively). With the development of CKD, the associations of variance in parameters between Scr, inflammation and oxidative stress have not been previously investigated. Variance ratios for Scr, hs-CRP and MDA in 113 patients with CKD 2–4 after the 12-month follow-up were firstly analyzed in present study (Table 4). Significance of Pearson correlation analysis was performed between Scr and hs-CRP (p50.01), between Scr and MDA (p50.05), and between hs-CRP and MDA (p50.05), respectively.
Discussion The current leading cause of CKD in China is glomerular nephropathy, followed by diabetic nephropathy and hypertension.13 Inflammation and oxidative stress were common features of patients with advanced CKD, however, there were
Table 4. Variance ratios for hs-CRP and MDA in patients with CKD after 12 months of follow-up (%). CKD 2 3 4
Scr
hs-CRP
MDA
35.7 ± 8.1 59.2 ± 31.7 58.2 ± 20.9
41.5 ± 24.3 146.0 ± 84.8 93.4 ± 47.5
24.9 ± 23.6 65.5 ± 32.8 73.2 ± 37.4
Note: Significance of Pearson correlation analysis between Scr and hs-CRP (p50.01), between Scr and MDA (p50.05), and between hs-CRP and MDA (p50.05), respectively.
few studies examined the relationship between the biomarkers of inflammation and oxidative stress in these participants. The production of reactive oxygen species is usually in balance with the state of oxidative stress. There is a positive correlation between increasing oxidative stress and advancing stage of CKD.6,7,14 Several researches demonstrated the inverse relationship between eGFR and serum levels of inflammatory cytokines in advanced CKD patients.15,16 However, the extent to which eGFR is correlated with biomarkers of inflammatory cytokines is controversial.8,9 The most commonly used marker of lipid oxidation is MDA, which is formed naturally without any enzymatic activity.17 MDA is a kind of end-product generated by lipid peroxidation and has been considered as a biomarker of increased oxidative stress during CKD.18 Based on the abovementioned findings, we showed that there were significant differences in biomarkers of oxidative stress and inflammation
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G. Xu et al.
between CKD patients and healthy controls. Significant correlations were displayed between the levels of eGFR and the biomarkers of oxidative stress, between the levels of biomarkers for inflammation and oxidative stress. The most notable finding in this study was the first report of the close relationship in variance ratios between Scr, hs-CRP and MDA in patients with CKD after the 12-month follow-up. Therefore, we deduced that not only the serum levels of Scr, but also the variance extent of Scr are positively correlated with the concentrations of inflammatory cytokines in CKD participants. Our study put forward a significant positive correlation between hs-CRP, IL-6 and eGFR suggesting that these inflammatory cytokines seem to have a crucial role in the pathogenesis of CKD. ACE inhibitors or ARBs are frequently used in an attempt to delay the progression of CKD and to decrease proteinuria. Many of the effects of ACEI or ARBs signaling on target tissues are mediated via enhanced production of reactive oxygen species through activation of NADPH (nicotinamide adenine dinucleotide phosphate) oxidases.19 Several studies have also showed that the administration of ACEI or ARBs may have potential anti-inflammatory effects.20 However, there was no significant difference in recipient of ACEI or ARBs between the four CKD patient groups from stage 1 to 5. Increased levels of inflammatory mediators, i.e., hs-CRP and IL-6, may play independent as well as interdependent roles via several signaling pathways which lead to hyperglycemia-mediated augment in oxidative stress. However, increase in oxidative stress may reversely amplify inflammation, then setting up a vicious cycle by mechanisms including activation of the nuclear transcription factor kB (NF-kB), which contributes to the activation and recruitment of immune cells. Therefore, inflammatory cytokines associated with oxidative stress promote renal tissue damage by inflicting apoptosis, necrosis, fibrosis, and may be major mechanisms in the pathogenesis and progress of CKD.21 Although the exact mechanisms of inflammation and oxidative stress have not been accurately elucidated in CKD patients, a number of following described factors appear to be involved, including uremic toxins,3 rennin-angiotensin system,22 hypertension,23 underlying diseases (diabetes, autoimmune diseases, etc.),24 infection, iron overload,25 antioxidant deficiency,26 etc. Oxidative stress can provoke inflammation via activating NF-kB and consequent generation of pro-inflammatory cytokines.27 Production and release of reactive oxygen, nitrogen species and chlorine by the activated immune cells in turn promote the oxidative stress.28 The application of anti-oxidants for therapeutic intervention in patients with CKD may prove beneficial, but whether or not several anti-oxidants as a multi-drug therapy to target oxidant modifying pathways during the development of CKD remain unclear.29–32 In conclusion, our present study indicated that inflammation and oxidative stress interacted with each other and played pivotal roles in the development of CKD. Variation in eGFR was parallel with the changes of oxidative stress and inflammation when CKD developing. However, large-scale, multi-centered, controlled studies should be performed to reach the final conclusion.
Ren Fail, Early Online: 1–5
Declaration of interest The research was supported by the National Natural Science Foundation of China (No. 81360122/H0518). No conflict of interests is declared.
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