Sayanthooran et al BMC Nephrology (2017) 18:31 DOI 10.1186/s12882-017-0440-x RESEARCH ARTICLE Open Access Potential diagnostic biomarkers for chronic kidney disease of unknown etiology (CKDu) in Sri Lanka: a pilot study Saravanabavan Sayanthooran1, Dhammika N Magana-Arachchi1*, Lishanthe Gunerathne2 and Tilak Abeysekera3 Abstract Background: In Sri Lanka, there exists chronic kidney disease of both known (CKD) and unknown etiologies (CKDu) Identification of novel biomarkers that are customized to the specific causative factors would lead to early diagnosis and clearer prognosis of the diseases This study aimed to find genetic biomarkers in blood to distinguish and identify CKDu from CKD as well as healthy populations from CKDu endemic and non-endemic areas of Sri Lanka Methods: The expression patterns of a selected panel of 12 potential genetic biomarkers were analyzed in blood using RT-qPCR Fold changes of gene expressions in early and late stages of CKD and CKDu patients, and an apparently healthy population of a CKDu endemic area, Girandurukotte (GH) were calculated relative to apparently healthy volunteers from a CKDu non-endemic area, Kandy (KH) of Sri Lanka, using the comparative CT method Results: Significant differences were observed between KH and early stage CKDu for both the insulin-like growth factor binding protein (IGFBP1; p = 0.012) and kidney injury molecule-1 (KIM1; p = 0.003) genes, and KH and late stage CKD and CKDu for the glutathione-S-transferase mu (GSTM1; p < 0.05) gene IGFBP1 and KIM1 genes showed significant difference between the early and late stage CKDu (p < 0.01) The glutamate cysteine ligase catalytic subunit (GCLC) gene had significantly different expression between KH and all the other study groups (p < 0.01) The GH group was significantly different from the KH group for the oxidative stress related genes, G6PD, GCLC and GSTM1 (p < 0.01), and also the KIM1 gene (p = 0.003) IGFBP1, insulin-like growth factor binding protein (IGFBP3), fibronectin (FN1) and KIM1 showed significant correlations with serum creatinine, and IGFBP1, KIM1 and kallikrein (KLK1) with eGFR (p < 0.05) Conclusion: A panel consisting of IGFBP1, KIM1, GCLC and GSTM1 genes could be used in combination for early screening of CKDu, whereas these genes in addition with FN1, IGFBP3 and KLK1 could be used to monitor progression of CKDu The regulation of these genes has to be studied on larger populations to validate their efficiency for further clinical use Keywords: Gene expression analysis, Kidney injury, Oxidative stress, RT-qPCR Background Chronic kidney disease (CKD) is increasing rapidly worldwide and is gaining much attention in both the developed as well as developing countries CKD is characterized by a reduced glomerular filtration rate (GFR) that is accompanied with structural or functional * Correspondence: dmaganaarachchi@gmail.com Cell Biology Group, National Institute of Fundamental Studies, Kandy, Sri Lanka Full list of author information is available at the end of the article abnormalities of the kidneys on urinalysis, biopsy and imaging [1] The concept of biomarker discovery in medicine is becoming increasingly important due to its potential for early screening, more effective treatment and a more personalized approach to medical care [2].“A biological marker (biomarker) is a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention.”[3] The biomarkers could be identified at any level along the © The Author(s) 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated Sayanthooran et al BMC Nephrology (2017) 18:31 genome – phenome continuum, and could be genomic biomarkers (DNA or RNA), proteomic biomarkers (proteins) or metabolic biomarker (metabolites) [3] Biomarkers in both acute kidney injury (AKI) and CKD look for similar effects of the diseases; decrease in nephron number, vascular insufficiency, and cell cycle disruption [1] The biomarkers of kidney disease should however be complemented with clinical assessments of patients with CKD and AKI and not be used in isolation [1] The intended biomarkers should provide rapid, noninvasive and specific measurements that correlate well with kidney tissue pathology [1] The most sensitive marker of CKD progression in clinical practice is proteinuria, especially when combined with the estimated glomerular filtration rate (eGFR) [2]; however, the process of kidney injury starts with the induction of molecular level changes, which therefore gives promise for identification of molecular markers for early diagnosis of disease process Biomarkers are needed in CKD to help estimate GFR, assess cardiovascular disease, determine metabolic abnormalities associated with CKD and differentiate inflammatory and fibrotic conditions of the kidney [1] The etiologies for CKD differ and therefore comorbidities exist in most CKD patients, hence a single biomarker would be incapable of satisfying all these needs [1, 2] In Sri Lanka, there exists CKD of known etiologies (with the majority being diabetes and cardiovascular diseases) and unknown etiology (CKDu) This CKDu is confined to certain geographical locations of the country, notably the dry zones, in the North Central Province and Uva Province where the majority of the population are involved in farming as the main occupation The majority of the hypotheses for the cause of the disease revolve around environmental stimuli based on the environmental location and occupation of the affected individuals [4–9] The main biomarkers for screening and identification of CKDu include dipstick proteinuria, serum creatinine and eGFR measurements, while excluding known etiologies of CKD such as diabetes, hypertension, and systemic lupus erythematosus [10] The presence of small, echogenic kidneys, and tubulointerstitial damage on renal biopsy further confirm diagnosis [11] As with CKD, the identification of diseased individuals takes place only in the later stages of the disease where the symptoms become apparent Although analyzing renal tissue samples would be most ideal as it is the affected tissue, the obtaining of biopsies is invasive, more expensive and not suitable for initial screening purposes [12] This brings about a need for other suitable tissues for the safe, reliable and inexpensive monitoring of biomarkers Body fluids like serum and urine are mainly targeted for the identification of biomarkers as they are the least Page of 12 invasive methods and replace the usage of tissue biopsies [13] In this pilot study, genes that have potential as screening and prognostic biomarkers of CKD, both known and unknown etiology, were selected from literature to cover more than one particular characteristic/function related to the disease The expression pattern of selected genes, namely kidney injury molecule-1 (KIM1), fibronectin (FN1), insulin like growth factor binding protein (IGFBP1), insulin like growth factor binding protein (IGFBP3), kallikrein (KLK1), glutathione S transferase mu (GSTM1), glutamate cysteine ligase catalytic subunit (GCLC), glucose-6-phosphate dehydrogenase (G6PD), cytochrome P450 enzyme 2D6 (CYP2D6) and 2C19 (CYP2C19) were analyzed in both chronic kidney disease patients of known and unknown etiology residing in Girandurukotte, a CKDu endemic area belonging to the dry zone of Sri Lanka The majority of cases in this region belong to the CKDu category Healthy individuals from the same area who are considered at risk and have been previously screened for CKDu and found negative, and healthy individuals from an area not endemic for CKDu, Kandy, located in the Central Province, wet zone of the country were selected as controls Among the genes selected, two were related to kidney injury and repair, KIM1 and FN1 KIM1 is a novel biomarker of kidney injury that is upregulated in dedifferentiated proximal tubule epithelial cells in kidney after ischemic or toxic injury [14] FN1 is a glycoprotein involved in cell adhesion and migration process including wound healing, blood coagulation and host defense It has also been linked to tissue scarring and fibrosis [15] Metabolic disorders such as diabetes and cardiovascular diseases are either causative factors or complications of chronic kidney diseases Therefore three genes related to cardiovascular complications and diabetes were selected to observe their use as possible prognostic biomarkers IGFBP1and IGFBP3 belong to a family of IGFBPs which has six proteins that specifically bind insulin like growth factors I (IGF-I) and II (IGF-II) The IGF system has been increasingly implicated in the development of cardiovascular diseases [16] They are multifunctional as they not only passively circulate transporters, but also play a variety of roles in the circulation, extracellular environment, and inside the cell [17] IGFBP3 has the major IGF transport function and is the most abundant circulating IGFBP It has been suggested to have a role of wound healing by binding to the IGF-1 and releasing them at the wound sites [17] KLK1 belongs to the kallikreins, a different group of proteins, serine proteases that have been related to human essential hypertension and associated complications Low levels of urinary kallikreins have been associated with hypertension and renal disease [18] Sayanthooran et al BMC Nephrology (2017) 18:31 There have been hypotheses suggesting the possible role of metal toxicity and environmental toxins as etiological factors of CKDu in Sri Lanka [19] We therefore selected genes related to metal toxicity and oxidative stress (GSTM1, GCLC and G6PD), and xenobiotic metabolism (CYP2D6 and CYP2C19) which could be possibly influenced by the environmental toxins, in turn acting as biomarkers of toxicity Glutathione (GSH) plays a crucial role in the antioxidant defense system and has a predominant role in the regulation of the intracellular redox state and protects cells from oxidative injury [20] Two genes related to GSH were selected to be studied; GCLC, the rate limiting enzyme in the production of GSH, and GSTM1, the enzyme that facilitates the detoxification action of GSH by providing a hydrophilic binding site for glutathione and a hydrophobic binding site for electrophilic substrates [21] The G6PD enzyme is another protein that enables cells to counterbalance the oxidative stress via the activation of the glutathione system by the production of nicotinamide adenine dinucleotide phosphate (NADPH) [22] Two genes belonging to the cytochrome P450 monoxygenase enzyme family, CYP2D6 and CYP2C19 were selected to study any differential expression influenced by drugs or xenobiotics CYP2D6 metabolizes 20–25% of the clinically used drugs [23], whereas CYP2C19 metabolizes 8% of all drugs [24] Such drug metabolizing enzymes are not only expressed in liver tissue, but also extra hepatic tissues like blood lymphocytes, kidney and intestine [25] Studies have shown that substrate metabolism and toxicity can be influenced by the xenobiotic modulation of the CYP genes and that they could hence be sensitive markers of chemical exposure [26, 27] The expression patterns of the genes were tested in the CKD, CKDu and apparently healthy, risk groups of the CKDu endemic area in comparison to apparently healthy individuals of a non-endemic area in order to identify possible early screening and prognostic markers Methods Patients The patients to be studied were recruited from June 2013 to November 2015 from the Renal Care & Research Centre, District Hospital, Girandurukotte, a region endemic for CKDu, located in the Uva Province, belonging to the dry zone of Sri Lanka The diagnosis of patients was carried out by the nephrologist attending the Renal Clinic of the hospital CKDu was labeled as having unknown etiology based on criteria set by the Ministry of Health, Sri Lanka with no past history of diabetes mellitus, chronic or severe hypertension, snake bite, glomerulonephritis or urological diseases being causes for the disease A normal Page of 12 HBA1C (