Urinary Epidermal Growth Factor: A Promising “Next Generation” Biomarker in Kidney Disease

Keywords Biomarker · Epidermal growth factor · Kidney disease · Urine

Abstract Background:

The epidermal growth factor (EGF) is a globular protein that is generated in the kidney, especially in the loop of Henle and the distal convoluted tubule. While EGF is nonexistent or hardly detectable in plasma, it is present in normal people's urine. Until now, risk stratification and chronic kidney disease (CKD) diagnosis have relied on estimated glomerular filtration rate (eGFR) and urine albumin/ creatinine ratio (ACR), both of which reflect glomerular function or impairment. Tubular dysfunction, on the other hand, may also be associated with renal failure. 

NEW HERBAL FORMULATION FOR CHRONIC KIDNEY DISEASE 

Summary: Because decreased urine EGF (uEGF) indicates tubular atrophy and interstitial fibrosis, this biomarker, together with eGFR and uACR, may be employed in the general population for risk assessment and diagnosis of CKD. uEGF levels have been shown to correlate with intrarenal EGF mRNA expression and have been found to decrease in a variety of glomerular and non-glomerular kidney disorders.

Key Message: uEGF, uEGF/creatinine, or uEGF/monocyte chemotactic peptide-1 are possible "new generation" biomarkers linked to a variety of kidney diseases that deserve further investigation as a single biomarker or as part of a multi-biomarker panel.

Introduction 

The epidermal growth factor (EGF) was first discovered by Stanley Cohen in the early 1950s [1]. A large molecular weight (MW) complex (MW: approximately 74,000 Da) was identified almost entirely in a crude homogenate of the male mouse's submaxillary gland, reversibly dissociable into EGF (MW: 6,045 Da) and an EGF-binding protein (MW: 29,000 Da) with arginyl-esterase activity [2]. Besides, EGF has been found in many human tissues and body fluids, including the kidneys (specifically the Henle's loop and distal convoluted tubules) and urine [3], saliva [4], parotid glands [5], milk, tears, and only in a minimal concentration in plasma [4]. Platelets are the principal source of blood EGF in humans, and the concentration steadily increases during coagulation following sample collection [6].

Fig. 1. an EGF is a 53-amino acid single-chain polypeptide with a relative MW of approximately 6,000 Da. b Chromosome 4 (q25–q27) includes the EGF gene, which is roughly 120 kb in size. c The structure of the EGFR, a member of the ErbB receptor family: the extracellular domain with four subdomains, the lipophilic transmembrane region, and an intracellular domain make up the EGFR. d Signaling pathways downstream of EGFR activation include the JAK-STAT pathway, the PI3K/Akt/ NF-B pathway, the PI3K/Akt/PTEN/ mTOR pathway, and the RAS-RAF-MEKERK MAPK network. EGFR, epidermal growth factor receptor; PI3K, phosphoinositide 3-kinase; JAK-STAT, Janus kinase-signal transducer and activator of transcription; ERK, extracellular signal-regulated kinase.

EGF is a nondialyzable, compact, heat-stable human polypeptide [7] of 53 amino acids with three intramolecular disulfide bonds (Fig. 1a) [8]. All the amino acids are present except phenylalanine, alanine, and lysine, and it has a C-terminal arginine residue [2]. The EGF gene has 24 exons and 23 introns and is found on chromosome 4q25–q27 (Fig. 1b) [9].

EGF promotes cellular proliferation, differentiation, and survival by binding 1:1 to the soluble extracellular domain of the EGF receptor, resulting in a complex of 2EGF:2 soluble extracellular domain of the EGF receptor (Fig. 1c) [10, 11]. This ligand-induced dimerization activates the receptor's intrinsic protein-tyrosine kinase activity (Fig. 1d), triggering a signaling cascade within the cell and resulting in a variety of biochemical changes. This eventually leads to DNA synthesis and cell proliferation [12]. In the kidney, EGF exerts several biological functions such as regulation of cellular metabolism and glomerular hemodynamics, modulation of cell growth, and renal repair after injury [13]. An experimental model of acute renal failure showed that EGF assisted with tubular jury recovery by activating regeneration pathways, resulting in reepithelialization of the injured tubules [14]. Through crosstalk with phosphoinositide 3-kinase, Janus kinase-signal transducer and activator of transcription, and extracellular signal-regulated kinase pathways [15, 16], higher urinary levels of EGF (uEGF) are thought to reflect functional tubular mass and regeneration potential [17]. In contrast, lower uEGF concentrations are associated with interstitial fibrosis and tubular atrophy (IFTA) [18]. Besides, the EGF protein detected in the urine of kidney disease patients and reported in several studies may be proEGF [19].

In this review, we will give an overview of the analytical aspects and the potential value of uEGF to evaluate kidney function in the general population as well as in adult patients with chronic kidney disease (CKD; diabetic kidney disease [DKD], primary glomerulonephritis, kidney transplantation, polycystic kidney disease). Besides, we highlight the potential unmet needs in this research field. 

Analytical Aspects 

Several methods have been developed for the measurement of the EGF concentration in human body fluids based on different principles such as radioimmunoassays [20–22], radioreceptor assays [23], enzyme immunoassaysays [24–26], enzyme-linked immunosorbent assays (ELISAs) [27–30], and ultramicro ELISA assay [31]. Table 1 provides an overview of the features of the various techniques

Because of serum protein matrix effects, the sensitivity of radioimmunoassays and radioreceptor assays for EGF analysis in the blood is insufficient without sample pretreatment (gel filtration or immunoaffinity chromatography) [33, 34]. Although no sample preparation is required for enzyme immunoassays, routine usage of these tests is problematic due to the fluorogenic substrates for the enzyme reaction [24–26]. ELISA is the gold standard for measuring EGF because of its great sensitivity and specificity. The effect of various storage conditions on uEGF stability, as well as other preanalytical techniques, has been investigated. VEGF concentrations were steady at 4°C for 48 h, as did variability as the standard deviation of mean uEGF values. Long-term storage at −80°C for 1 and 2 months did not affect the observed uEGF concentrations. However, after 6 months, there was an increase in VEGF levels in both participants and quality control samples. The mean VEGF levels were unaffected by repeated freeze-thaw cycles [35]. There is no diurnal variation in uEGF excretion, and its rate of excretion is similar when sampled on consecutive days, supporting its measurement in spot urine samples [32]. With age, the concentration of VEGF declines [36]. So, age should always be taken into account when investigating uEGF concentration [37]. EGF concentration is less likely than serum creatinine and albuminuria to be confounded by extrarenal factors, which may contribute to its evaluation and sensitivity as a biomarker for kidney failure [38]. In patients with very low or very high muscle mass, serum creatinine and estimated glomerular filtration rate (eGFR) do not correctly reflect renal function. EGF/Cr may provide a more realistic picture of the amount of chronic kidney impairment in such circumstances. When monitoring the uEGF/Cr ratio in the same individual over time, changes are considered significant when there is a difference of >30% compared to previous values [19].

General Population 

Tubular atrophy, interstitial fibrosis, and nephron loss define age-related glomerular filtration rate (GFR) reduction in seemingly healthy persons [39]. The amounts of mRNA EGF expressed by distal tubular epithelial cells are an indication of functional tubular mass and regeneration capability and are connected to GFR [17]. Aside from the traditional kidney damage indicators (eGFR and albuminutia), additional urine biomarkers of acute tubular injury have been identified [40]. However, only a few indicators reveal the persistent tubular injury. In population-level settings, uEGF could be a clinically applicable biomarker because it is stable over extended periods and can be reliably detected with just a few microliters of urine. In a prospective longitudinal study (follow-up of >5 years), the association between uEGF and rapid eGFR loss in subjects without diabetes mellitus (DM) or established CKD was investigated [38]. Because well-defined endpoints for CKD progression such as the incidence of end-stage kidney disease (ESKD), a doubling of serum creatinine, or a 30–40% decrease in eGFR are unlikely to occur in healthy subjects within a limited follow-up period [41], a fast eGFR decline was defined as a loss >3.0 mL/min/1.73 m2 per year. Lower uEGF was independently associated with rapid kidney function decline and incident CKD in two European population cohorts (the Renal Iohexol Clearance Survey [RENIS], which included 1,249 patients, and the Prevention of Renal and Vascular End-stage Disease [PREVEND], which included 4,534 patients). VEGF concentrations were highly associated with the occurrence of CKD, regardless of other risk factors (Table 2). The link remained (borderline) significant after controlling for age, gender, cohort, baseline eGFR, and urine albumin/creatinine ratio (ACR). In adjusted models, uEGFR concentration had only a marginal relationship with mean eGFR reduction in the RENIS. In the PREVENT research, the youngest age group saw a very minor drop in the eGFR change rate. The results might have been affected by a lower precision of eGFR in the normal range and, in certain cases, an abnormal elevation in eGFR in subpopulations due to prefiltration. VEGF may be a major contributing risk factor for CKD when combined with other genetic or environmental variables [38]. One SNP (rs11569017) in the EGF gene was found to be associated with ESKD in a Korean population [9]. Because the RENIS and PREVENT trials mostly involved white European participants, we believe that future research should evaluate the efficacy of uEGF in multiethnic population cohorts [38].

Table 2. Relationship between uEGF and kidney function in the general population