|Ahead of print publication
Serum levels of neutrophil gelatinase-associated lipocalin, kidney injury molecule-1, and cystatin-C in renal artery stenosis: A pilot study
Raghunandan Prasad1, Reeta Choudhary2, Anuradha Singh1, Surabhi Agarwal1, P Kaushik1, Kritika Singh3, Rajanikant R Yadav1, Amar Singh Yadav1, Dharmendra Singh Bhadauria4, Hira Lal1
1 Department of Radiodiagnosis, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India
2 Department of Biochemistry, Medanta – The Medicity, Gurugram, Haryana, India
3 Department of Clinical Immunology and Rheumatology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India
4 Department of Nephology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India
|Date of Submission||24-Jun-2020|
|Date of Acceptance||28-Jun-2020|
Department of Biochemistry, Medanta – The Medicity, Gurugram - 122 001, Haryana
Source of Support: None, Conflict of Interest: None
Background: The present pilot study evaluated the potential of newer renal biomarkers (neutrophil gelatinase-associated lipocalin [NGAL], cystatin-C [Cys-c], and kidney injury molecule-1 [KIM-1]) in estimating subclinical renal injury due to renal artery stenosis (RAS).
Materials and Methods: A total of 13 patients of magnetic resonance angiography confirmed RAS and 14 normotensive healthy controls were enrolled in the study after obtaining ethics approval and informed consent. Serum was collected from participants to check for serum levels of NGAL, KIM-1, and Cys-c by quantitative enzyme immunoassay. The data of RAS patients were compared and analyzed against the data of healthy controls.
Results: sNGAL, sKIM-1, and sCys-c values in RAS patients showed rising trend as compared to normal healthy control; however, the difference in their values was not statistically significant. This could be due to small and heterogeneous sample size. However, statistically significant difference was noted in the values of sNGAL and sCys-c between healthy controls and RAS patients with abnormal serum creatinine. This difference in the values of these biomarkers was also statistically significant between RAS patients with normal and elevated serum creatinine.
Conclusion: In RAS patients, sNGAL, sCys-c, and sKIM-1 seem to have potential as an early biomarker of kidney injury.
Keywords: Cystatin-C, kidney injury molecule-1, neutrophil gelatinase-associated lipocalin, renal artery stenosis, renal biomarkers
|How to cite this URL:|
Prasad R, Choudhary R, Singh A, Agarwal S, Kaushik P, Singh K, Yadav RR, Yadav AS, Bhadauria DS, Lal H. Serum levels of neutrophil gelatinase-associated lipocalin, kidney injury molecule-1, and cystatin-C in renal artery stenosis: A pilot study. Indian J Rheumatol [Epub ahead of print] [cited 2020 Oct 27]. Available from: https://www.indianjrheumatol.com/preprintarticle.asp?id=297400
| Introduction|| |
Renal artery stenosis (RAS) occurs when either of the renal arteries is narrowed at its origin or course. It is one of the most common causes of secondary hypertension and could be a result of many underlying diseases, including large-vessel vasculitis, atheromatous involvement of renal arteries, fibromuscular dysplasia, neurofibromatosis, extrinsic compression such as due to congenital bands, and following radiotherapy. RAS leads to the gradual reduction of intrarenal blood flow. This decrease in blood flow triggers renal autoregulation mechanism; however, beyond a critical level of stenosis, renal autoregulation fails (hemodynamically significant stenosis) and this results in intrarenal hypoxia and tissue loss, leading to gradual reduction of the glomerular filtration rate (GFR). This condition defined as ischemic nephropathy.,
Currently, serum creatinine and estimation of GFR are the two most commonly used markers of renal function; however, these markers are quite nonspecific and may identify advanced kidney injury at a stage when it is often irreversible. Moreover, serum creatinine is a marker of renal function and not of renal injury, and its measurement is quite insensitive. As per the current risk, injury, failure, loss-of-function to ESRD concept, acute and chronic kidney diseases exist in a continuum. Therefore, sensitive biomarkers that can identify kidney injury early before any degree of significant functional loss occurs are required, and there is also a need to assess its utility as a predictor of progression.,,
In recent years, several novel biomarkers of clinical and subclinical renal injury have been identified such as neutrophil gelatinase-associated lipocalin (NGAL), cystatin-C (Cys-c), and kidney injury molecule-1 (KIM-1). KIM-1 and NGAL are newly expressed in renal injury; hence, they are referred as de novo-synthesized biomarkers. Cyst-C referred as a surrogate tubular dysfunction biomarker is an established and promising biomarker of chronic kidney disease (CKD) progression. Such biomarkers, either synthesized de novo or as markers of surrogate tubular dysfunction, have been demonstrated to have a role in the evaluation of drug-related nephrotoxicity. In various studies, these markers have been found to correlate well with subclinical renal injury in very early stage before tissue damage becamwwwwwwwwwwwe apparent.
This pilot study was designed to see how these novel and sensitive biomarkers of subclinical renal injury behave in RAS patients and to compare their levels with normal healthy controls.
| Design and Method|| |
This was a prospective study conducted at a tertiary care referral hospital in Northern India, after obtaining approval from the institutional ethics committee (IEC code: 2018-110-MD-EXP, Bioethics cell-Sanjay Gandhi Postgraduate Institute of Medical Sciences, Raebareli Road, Lucknow, date of approval-10/09/2018 and the document submission number to the said ethics committee-PGI/BE/424/2018. A total of 13 patients of renovascular hypertension with magnetic resonance angiography (MRA)-confirmed RAS were enrolled in the study after obtaining informed consent. Their demographic details and presence of risk factors for RAS (diabetes and coronary artery diseases) were recorded. Fourteen normotensive healthy controls with no history of any renal pathology were also included in the study after taking consent.
Serum samples were collected from all 27 subjects (14 healthy controls and 13 patients) to check for serum levels of NGAL, KIM-1, and Cys-c by quantitative enzyme immunoassay. Urine samples were also collected from all the patients to check for urine protein.
For all the patients with renovascular hypertension, their systolic and diastolic blood pressures were recorded. The presence of RAS was confirmed on MRA. Other findings such as kidney size, corticomedullary differentiation, and degree of RAS were also recorded on MRA. Differential GFR and enalapril scintigraphy results, if available, were also recorded and correlated.
Serum creatinine was measured using Jaffe rate reaction method. NGAL (catalog # DY1757), Cys-C (catalog # DY1196), and Human KIM-1 (catalog # DY1750) were measured in serum samples using sandwich enzyme-linked immunosorbent assay (ELISA, R&D Systems, Inc., USA, Canada). The lower detection limits of the kits were 78.1 pg/mL for NGAL, 62.5 pg/mL for Cys-C, and 15.6 pg/ml for KIM-1.
The data of healthy controls (n = 14) were labeled as Group 1 and that of RAS patients (n = 13) as Group 2. Both these groups were compared and analyzed using independent t-test [Table 1]. To further analyze, Group 2 patients' data were divided into Group 2A (RAS patients with normal serum creatinine [n = 6]) and Group 2B (RAS patients with elevated serum creatinine [n = 7]), and then both of these groups (Group 2A and Group 2B) were compared with healthy controls (Group 1) and with each other and analyzed using one-way ANOVA and Tukey's post hoc test [Table 2]. P ≤ 0.05/0.01 was considered statistically significant.
|Table 1: Comparison of serum levels of kidney injury biomarkers among control subjects (Group 1) and renal artery stenosis patients (Group 2)|
Click here to view
|Table 2: Comparison of serum levels of kidney injury biomarkers among control subjects (Group 1), renal artery stenosis patients with normal serum creatinine (Group 2A), and renal artery stenosis patients with elevated serum creatinine (Group 2B)|
Click here to view
| Results|| |
Total 27 subjects (14 healthy controls in Group 1 and 13 RAS patients in Group 2) were enrolled in the study. The mean age of patients in Group 2 (male = 9) was 38.46 years (range, 20–63 years), while for group 1, it was 35 years (range, 18–65 years). There was history of diabetes mellitus/coronary artery disease in four patients (risk factors for RAS), while in the remaining nine patients, there was no history of any risk factor for RAS.
Demographic details and MRI findings of the patients with RAS (Group 2) are shown in [Table 3]. Out of total 26 kidneys included in Group 2, corticomedullary differentiation was preserved only in 8 kidneys, while it was partially preserved in 12 kidneys and was totally lost in 6 kidneys. Out of 13 patients in Group 2, four patients were having right RAS, while two patients were having stenosis on left side. In 7 patients, renal arteries on both sides were stenosed. Average degree of stenosis on the right side was 56% ± 1.4%, while on the left side it was 64% ± 2.4% for Group 2A patients. For Group 2B patients, it was 69% ± 1.0% on the right side and 78% ± 1.0% on the left side. Out of 13 patients in Group 2, stenosis was > 70% in 8 patients. Differential GFR and enalapril scintigraphy results were available only for 6 and 5 patients, respectively. The values of serum biomarkers, S. creatinine, differential GFR, and enalapril scintigraphy in RAS patients with normal S. creatinine (Group 2A), and elevated S. creatinine (Group 2B) are depicted in [Table 4].
|Table 3: Demographic details and MRI findings in renal artery stenosis patients (Group 2)|
Click here to view
|Table 4: Serum biomarkers and serum creatinine values in renal artery stenosis patients with normal serum creatinine (Group 2A) and renal artery stenosis patients with elevated serum creatinine (Group 2B)|
Click here to view
The values of sNGAL, sCys-C, and sKim-1 were analyzed for Group 2, 2A, and 2B separately and compared with age-matched normal controls (Group 1).
The mean values of sNGAL, sCys-c, and sKIM-1 for normal healthy controls (Group 1) were 310.52 ± 179.356, 6.32 ± 2.81, and 40.64 ± 71.05, respectively, and were lower than the mean values of RAS patients (Group 2); however, no evident statistically significant difference was noted between the two groups [Table 1].
Further, the serum levels of sNGAL, sCys-c, and kidney injury biomarkers among control subjects (Group 1), RAS patients with normal S. creatinine (Group 2A), and RAS patients with elevated S. creatinine (Group 2B) were compared, which revealed statistically significant difference between Groups 1 and 2B and between Groups 2A and 2B. The mean values of sNGAL (253.67 ± 157.798 vs. 998.22 ± 676.422, P = 0.001) and sCys-c (3.79 ± 1.54 vs. 10.72 ± 2.31, P = 0.000) for RAS patients with normal S. creatinine (group 2A) were significantly lower than those of RAS patients with elevated S. creatinine (Group 2B) [Table 2].
| Discussion|| |
In this study, sNGAL, sKIM-1, and sCys-c values in RAS patients showed an increasing trend when compared to normal healthy control (Group 1); however, this difference in values did not came statistically significant. Possibly, this might be because of very heterogeneous and small sample size in our study. On further analyzing, sNGAL and sCys-c levels were high and statistically significant in Group 2B patients as compared to healthy controls. Statistically significant difference was also noted in the levels of sNGAL and sCys-c values, between Group 2A and 2B patients.
Various studies have documented increased expression of messenger ribonucleic acid (mRNA) of NGAL and NGAL protein in thick ascending limb of the loop of Henle and the intercalated cells of the collecting duct of kidney tubules, when exposed to ischemia, sepsis, or post-transplantation acute kidney injury (AKI). At present, it is also thought as a marker of ischemic renal injury as well.,,,
It is thought that NGAL upregulates heme oxygenase-1. This in turn preserves proximal tubule N-cadherin and inhibits cell death occurring thereafter by inhibiting apoptosis. Its expression is increased many fold in proximal tubules and can be detected in serum and urine within 3–6 h of ischemic or toxic kidney injury. Its level became high in various inflammatory conditions as well.,,,
Maatman et al. documented increased serum levels of NGAL within 2–6 h after cardiopulmonary bypass both in adults and children and after administration of intravenous contrast.,,
In a study, baseline NGAL level was compared to serum creatinine level in 157 patients of poisoning to predict AKI, in patients with poisoning within 24 h of event. It was reported by the authors that levels of NGAL at admission better predicted AKI than serum creatinine levels at a similar time point.
In addition to its potential role as a biomarker of AKI, NGAL has emerged as a useful biomarker for CKD as well. In a study conducted by Vaidya et al. on 80 nondiabetic patients with CKD stages 2–4, serum NGAL was elevated in those patients, who were having most advanced CKD. Various other studies have also documented elevation of urinary and serum NGAL levels in a wide range of renal pathologies, such as diabetic nephropathy, IgA nephropathy, and autosomal polycystic kidney disease.,
The second biomarker of our study Cys-C is a 13-kD cysteine protease inhibitor and is produced by all nucleated cells at a constant rate. Its production in the body is genetically regulated and is not affected by environmental factors or renal diseases. It gets freely filtered by the renal glomerulus, followed by reabsorption (>95% of serum Cys-c get reabsorb) in the proximal tubule, with approximately 1/10th of the serum concentration being excreted in the urine.,,,
Functional impairment of proximal tubular epithelium results in increased levels of Cys-c in urine just like increase level of urine albumin in advanced CKD.,, In their study on diabetic nephropathy patients, it was found that serum Cys-c was significantly more sensitive as a marker of early Grade 2 diabetic nephropathy when compared with other markers of renal dysfunction. Similar observations were also true for the detection of early renal dysfunction, viz., stage 2 CKD. Molecular weight of Cystatin-c is nearly 120 times higher than Creatinine, resu lting in significant rise in serum cystatin-c concentration even in mild renal dysfunction.
The third marker of our study KIM-1 (also known as T cell immunoglobulin and mucin domain-containing protein-1 (TIM-1) and hepatitis A virus cellular receptor 1 (HAVCR-1), a de novo-synthesized biomarker in response to ischemia, is also showing rising trend like previous two markers, but with no evident statistically significant difference in the serum values between Group 1 and Group 2. Sustained KIM-1 expression has also been proposed to promote kidney fibrosis. KIM-1 is possibly one of the renal injury biomarkers with the highest sensitivity and specificity, whose levels increase in urine on the 1st day of even minor ischemic (10-min) or toxic kidney injuries in murine models. In another study in patients with type 1 diabetes and proteinuria, serum KIM-1 level at baseline strongly predicted the rate of estimated GFR loss and risk of developing ESRD during next 5–15 years of follow-up. Thus, it was concluded that KIM-1 could potentially play a role as a marker of both CKD as well as predict its progression. Sensitive assays to measure plasma and serum KIM-1 in mice, rats, and humans were developed and validated by Sabbisetti VS et al. In their study, they concluded that KIM-1 is a blood biomarker that specifically reflects acute and chronic kidney injury., In a cohort study of patients with CKD by Zhang and Parikh, urinary KIM-1, NGAL, and L-FABP levels predicted CKD progression.
RAS could be idiopathic as well as related to systemic diseases such as large-vessel vasculitis due to Takayasu arteritis and rarely variable vessel vasculitis such as Behcet's disease. It is possible that such subclinical renal injury could be occurring in individuals with large-vessel vasculitis who develop RAS, since many a time, such RAS requires interventions such as stent placement. This forms an area of future research.
We could not find any other study in the literature similar to ours, which compare baseline sNGAL, sCys-c, and Kim-1 levels in RAS patients and healthy controls. Our result indicates that NGAL, sCys-c, and Kim-1 can be considered as a sensitive biomarker for the detection of subclinical renal injury due to RAS. Determining the appropriate cutoff values in different grades of RAS appears to be next step in validating these biomarkers.
| Conclusions|| |
In RAS patients, sNGAL, sCys-c, and sKIM-1 seem to be potentially earlier and more sensitive biomarkers of kidney injury compared to traditionally used serum creatinine level; however, a large study with adequate homogenous sample size is required to validate these results further.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Textor SC, Wilcox CS. Renal artery stenosis: A common, treatable cause of renal failure? Ann Rev Med 2001;52:421-42.
Weber-Mzell D, Kotanko P, Schumacher M, Klein W, Skrabal F. Coronary anatomy predicts presence or absence of renal artery stenosis. A prospective study in patients undergoing cardiac catheterization for suspected coronary artery disease. Eur Heart J 2002;23:1684-91.
May AG, Van de Berg L, DeWeese JA, Robb CG. Critical arterial stenosis. Surgery 1963;54:250-9.
Shukla A, Rai MK, Prasad N, Agarwal V. Short-term non-steroid anti-inflammatory drug use in spondyloarthritis patients induces subclinical acute kidney injury: Biomarkers study. Nephron 2017;135:277-86.
Vaidya VS, Ferguson MA, Bonventre JV. Bio-markers of acute kidney injury. Annu Rev Pharmacol Toxicol 2008;48:463-93.
Fassett RG, Venuthurupalli SK, Gobe GC, Coombes JS, Cooper MA, Hoy WE. Biomarkers in chronic kidney disease: A review. Kidney Int 2011;80:806-21.
Devarajan P. Neutrophil gelatinase-associated lipocalin (NGAL): A new marker of kidney disease. Scandinavian J Clin Lab Investig 2008;68:89-94.
Mishra J, Mori K, Ma Q, Kelly C, Yang J, Mitsnefes M, et al
. Amelioration of ischemic acute renal injury by neutrophil gelatinase-associated lipocalin. J Am Soc Nephrol 2004;15:3073-82.
Paragas N, Kulkarni R, Werth M, Schmidt-Ott KM, Forster C, Deng R, et al.
α-Intercalated cells defend the urinary system from bacterial infection. J Clin Invest 2014;124:2963-76.
Charlton JR, Portilla D, Okusa MD. A basic science view of acute kidney injury biomarkers. Nephrol Dial Transplant 2014;29:1301-11.
Schmidt-Ott KM, Mori K, Li JY, Kalandadze A, Cohen DJ, Devarajan P, et al
. Dual action of neutrophil gelatinase-associated lipocalin. J Am Soc Nephrol 2007;18:407-13.
Mishra J, Ma Q, Prada A, Mitsnefes M, Zahedi K, Yang J, et al
. Identification of neutrophil gelatinase-associated lipocalin as a novel early urinary biomarker for ischemic renal injury. J Am Soc Nephrol 2003;14:2534-43.
Mishra J, Mori K, Ma Q, Kelly C, Barasch J, Devarajan P. Neutrophil gelatinase-associated lipocalin: A novel early urinary biomarker for cisplatin nephrotoxicity. Am J Nephrol 2004;24:307-15.
Oikonomou KA, Kapsoritakis AN, Theodoridou C, Karangelis D, Germenis A, Stefanidis I, et al
. Neutrophil gelatinase-associated lipocalin (NGAL) in inflammatory bowel disease: Association with pathophysiology of inflammation, established markers, and disease activity. J Gastroenterol 2012;47:519-30.
Ichimura T, Asseldonk EJ, Humphreys BD, Gunaratnam L, Duffield JS, Bonventre JV. Kidney injury molecule-1 is a phosphatidylserine receptor that confers a phagocytic phenotype on epithelial cells. J Clin Invest 2008;118:1657-68.
Maatman RG, van de Westerlo EM, van Kuppevelt TH, Veerkamp JH. Molecular identification of the liver- and the heart-type fatty acid-binding proteins in human and rat kidney. Use of the reverse transcriptase polymerase chain reaction. Biochem J 1992;288(Pt 1):285-90.
Ahn JY, Lee MJ, Seo JS, Choi D, Park JB. Plasma neutrophil gelatinase-associated lipocalin as a predictive biomarker for the detection of acute kidney injury in adult poisoning. Clin Toxicol (Phila) 2016;54:127-33.
Viau A, El Karoui K, Laouari D, Burtin M, Nguyen C, Mori K, et al
. Lipocalin 2 is essential for chronic kidney disease progression in mice and humans. J Clin Invest 2010;120:4065-76.
Vaidya VS, Ford GM, Waikar SS, Wang Y, Clement MB, Ramirez V, et al
. A rapid urine test for early detection of kidney injury. Kidney Int 2009;76:108-14.
Ding H, He Y, Li K, Yang J, Li X, Lu R, et al
. Urinary neutrophil gelatinase-associated lipocalin (NGAL) is an early biomarker for renal tubulointerstitial injury in IgA nephropathy. Clin Immunol 2007;123:227-34.
Bolignano D, Coppolino G, Campo S, Aloisi C, Nicocia G, Frisina N, et al
. Neutrophil gelatinase-associated lipocalin in patients with autosomal-dominant polycystic kidney disease. Am J Nephrol 2007;27:373-8.
Newman DJ. Cystatin C. Ann Clin Biochem 2002;39:89-104.
Randers E, Erlandsen EJ. Serum cystatin C as an endogenous marker of the renal function –A review. Clin Chem Lab Med 1999;37:389-95.
Coll E, Botey A, Alvarez L, Poch E, Quintó L, Saurina A, et al
. Serum cystatin C as a new marker for noninvasive estimation of glomerular filtration rate and as a marker for early renal impairment. Am J Kidney Dis 2000;36:29-34.
Shimizu-Tokiwa A, Kobata M, Io H, Kobayashi N, Shou I, Funabiki K, et al
. Serum cystatin C is a more sensitive marker of glomerular function than serum creatinine. Nephron 2002;92:224-6.
Herget-Rosenthal S, van Wijk JA, Bröcker-Preuss M, Bökenkamp A. Increased urinary cystatin C reflects structural and functional renal tubular impairment independent of glomerular filtration rate. Clin Biochem 2007;40:946-51.
Russo LM, Sandoval RM, McKee M, Osicka TM, Collins AB, Brown D, et al
. The normal kidney filters nephrotic levels of albumin retrieved by proximal tubule cells: Retrieval is disrupted in nephrotic states. Kidney Int 2007;71:504-13.
Suzuki Y, Matsushita K, Seimiya M, Yoshida T, Sawabe Y, Ogawa M, et al
. Serum cystatin C as a marker for early detection of chronic kidney disease and grade 2 nephropathy in Japanese patients with type 2 diabetes. Clin Chem Lab Med 2012;50:1833-9.
Vaidya VS, Ramirez V, Ichimura T, Bobadilla NA, Bonventre JV. Urinary kidney injury molecule-1: A sensitive quantitative biomarker for early detection of kidney tubular injury. Am J Physiol Renal Physiol 2006;290:F517-29.
Humphreys BD, Xu F, Sabbisetti V, Grgic I, Naini SM, Wang N, et al
. Chronic epithelial kidney injury molecule-1 expression causes murine kidney fibrosis. J Clin Invest 2013;123:4023-35.
Sabbisetti VS, Waikar SS, Antoine DJ, Smiles A, Wang C, Ravisankar A, et al
. Blood kidney injury molecule-1 is a biomarker of acute and chronic kidney injury and predicts progression to ESRD in type I diabetes. J Am Soc Nephrol 2014;25:2177-86.
Zhang WR, Parikh CR. Biomarkers of acute and chronic kidney disease. Annu Rev Physiol 2019;81:309-33.
Misra DP, Naidu GSRSNK, Agarwal V, Sharma A. Vasculitis research: Current trends and future perspectives. Int J Rheum Dis 2019;22 Suppl 1:10-20.
Misra DP, Wakhlu A, Agarwal V, Danda D. Recent advances in the management of Takayasu arteritis. Int J Rheum Dis 2019;22 Suppl 1:60-8.
[Table 1], [Table 2], [Table 3], [Table 4]