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

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 (uACR), both of which reflect glomerular function or impairment. Tubular dysfunction, on the other hand, may also be associated with renal failure. 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.

© 2022 S. Karger AG, Basel

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].

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].

Fig. 1.

a 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-MEK-ERK 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.

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EGF promotes cellular proliferation, differentiation, and survival by binding to 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 injury 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 actually 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 immunoassays [24-26], enzyme-linked immunosorbent assays (ELISAs) [27-30], and ultramicroELISA assay [31]. Table 1 provides an overview of the features of the various techniques.

Table 1.

Overview of the characteristics of human EGF assays

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Because of serum protein matrix effects, the sensitivity of radioimmunoassays and radioreceptor assays for EGF analysis in 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. uEGF 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 had no effect on the observed uEGF concentrations. However, after 6 months, there was an increase in uEGF levels in both participants and quality control samples. The mean uEGF 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 uEGF declines [36]. So, age should always be taken into account when investigating uEGF concentration [37]. uEGF 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. uEGF/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 albuminuria), additional urine biomarkers of acute tubular injury have been identified [40]. However, only a few indicators reveal persistent tubular injury.

In population-level settings, uEGF could be a clinically applicable biomarker because it is stable over extended time 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). uEGF 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 (uACR). In adjusted models, uEGFR concentration had only a marginal relationship with mean eGFR reduction in the RENIS. In the PREVEND research, the youngest age group saw a very minor drop in 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 hyperfiltration. uEGF 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 PREVEND 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

/WebMaterial/ShowPic/1430152Kidney DiseasesLupus Nephritis

Lupus nephritis (LN) affects 40–60% of those with systemic lupus erythematosus (SLE). Late LN diagnosis is linked to an increased risk of kidney failure, with 10–15% of nephritis patients advancing to ESKD. There is an unmet need for a simple, noninvasive, accurate, and low-cost biomarker to help in the early identification and prediction of LN patients’ prognosis.

A proteomic-based method identified the uEGF/Cr ratio as a potentially relevant biomarker in an LN discovery cohort. LN patients’ urinary peptides matched the precursor proEGF protein. In two large multiethnic LN cohorts, active LN patients had significantly lower uEGF than patients with active nonrenal SLE, patients with inactive SLE, and kidney donors (Table 3). The histologic equivalents of chronic renal damage, such as interstitial fibrosis, tubular atrophy, and glomerular sclerosis, as well as the total of these components, known as the histologic chronicity index, were all linked with uEGF (r = −0.67, p value 0.001). A multivariate survival study revealed that uEGF was related to the time it took for serum creatinine to double, a sign of potential ESKD. Low uEGF/Cr at the start and/or over time was linked to progressive loss of kidney function, showing a greater risk of poor renal outcomes in people with LN. All patients who advanced to ESKD were identified by an uEGF/Cr threshold of 5.3 ng/mg. When paired with other indicators of kidney function during flare, such as proteinuria, eGFR, and the histologic chronicity index, uEGF/Cr levels at LN flare were independently linked with progressive loss of kidney function and improved the predictive value of these variables [19].

Table 3.

Relationship between uEGF and kidney function in patients with a kidney disease

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In another small study of 58 SLE patients and 30 healthy controls, uEGF values were considerably lower in the patient group. However, the urine protein/creatinine ratio (uPCR) did not correlate with the uEGF levels [42]. This could be due to a faster shift in uEGF than in uPCR, which is a glomerular disease activity measure. The reduced uEGF was connected to the histological findings on the renal biopsy. The mean uEGF concentration was higher in classes I, II, and III than in classes IV and V. There was a significant difference in uEGFR between classes II and III, which grew as the interclass comparison was widened (e.g., classes II and IV). At a cut-off value of 40.6, a receiver operating characteristic (ROC) analysis of uEGF as a predictor of LN revealed a sensitivity of 90.4% and a specificity of 83.3%.

A TaqMan low-density array examination of biomarkers in 54 archival formalin-fixed, paraffin-embedded renal biopsy tissues of patients with LN found an inverse association between mRNA EGF level and the severity of chronic damage and also renal function at the time of biopsy (unadjusted p < 0.01). EGF’s trophic and anti-apoptotic properties may block the activity of transforming growth factor-β (TGF-β), another major mediator of kidney injury in LN [52].

A common clinical dilemma is deciding whether to continue immunotherapy to avoid CKD or to discontinue immunotherapy and focus only on conservative therapies. Serum creatinine, for instance, has not always shown the best association with (decline in) renal function in LN patients [53]. The course of uEGF/Cr after LN treatment predicts the evolution of kidney failure in the future. This supports the role of uEGF/Cr as a tool of CKD, adding to the existing list of biomarkers. In the absence of any acute alterations that may occur during active disease, uEGF/Cr can be assessed during a LN flare and then following LN resolution to establish a baseline level. The uEGF/Cr level during a flare and its behavior following a flare may help identify people who are likely to regain kidney function, which may impact therapy choice, intensity, and persistence in patients with LN flare and acute renal damage. Before serum creatinine rises and eGFR declines, uEGF/Cr may be able to detect patients with CKD. After even one episode of aggressive LN, patients in remission often have persistent kidney damage that is not obvious from serum creatinine or eGFR. Serial uEGF/Cr monitoring in quiescent LN may provide a means of tracking silent histologic damage, alerting clinicians that such patients may require closer monitoring after exposure to nephrotoxic medications or may require dose adjustments of drugs excreted by the kidneys [19].

Diabetic Kidney DiseaseType 1 DM

Albuminuria is the characteristic first indication of DN, followed by a loss of kidney function and, eventually, ESKD [54]. Despite the fact that microalbuminuria is usually used to predict DN, only 30% of people advance to overt proteinuria. Some people who do not have microalbuminuria have severe renal pathology, indicating that microalbuminuria may not be the greatest early indicator of DKD [55]. To detect diabetic kidney deterioration early, more sensitive and specific indicators than microalbuminuria are necessary. Renal tubulointerstitial alterations, in addition to glomerular damage, are a common characteristic of DN and a strong predictor of renal failure [53]. Lower uEGF levels in children with type 1 DM (T1DM) may indicate a decreased kidney’s regenerative capacity, making uEGF a possible biomarker of early DN. Unlike microalbuminuria, which is a symptom of glomerular disease, a decrease in renal EGF production indicates tubulointerstitial kidney injury and increasing kidney injury. uEGF/Cr was considerably lower in T1DM when compared to age-matched healthy controls in a case-control study. Children with T1DM who had a decreased eGFR had significantly lower uEGF/Cr corrected for age. Only uEGF remained an independently linked parameter with renal function in a multivariate analysis [37].

Glomerular hyperfiltration precedes and even causes early tubulointerstitial kidney damage, and a combination of low microalbuminuria and low uEGF may indicate early renal function decrease due to tubulointerstitial fibrosis superimposed on glomerular injury. The screening potential of uEGF/Cr for DN in T1DM should be further explored in a long-term follow-up study [37].

Adding uEGF/creatinine to the standard of care regimen in T1DM patients will almost certainly result in a financial advantage. The earlier DN is identified, the sooner preventative medication can begin to halt the course of CKD. uEGF is a possible “new generation” biomarker linked to early DN symptoms in children and adolescents, and it deserves to be examined further in long-term follow-up research as a single biomarker or as part of a multi-biomarker panel for DN [37].

Type 2 DM

DKD is also a significant complication of T2DM, with up to 40% of the patients having renal failure [56]. In many cases, DKD does not follow the glomerular hyperfiltration pattern of microalbuminuria, resulting in rising levels of overt albuminuria and a decrease in eGFR [57]. The balance of protective growth factors and pro-inflammatory cytokines in DKD is thought to affect the extent of renal tissue damage and disease progression [46].

Predicting renal outcomes in diabetic individuals with normoalbuminuria is extremely difficult at the time. 642 participants from the Edinburgh Type 2 Diabetes Study, which were normoalbuminuric at baseline, were studied to see if uEGF could be used as a biomarker of progressive decline of renal function. At the outset of the study, there was a weakly positive correlation between eGFR and uEGF/Cr (r = 0.11; p < 0.01), and a higher proportion of patients in the highest tertile of uEGF/Cr had an uACR >0.5 mg/mmol. During the follow-up period, patients in the tertile with the lowest uEGF concentration had a substantially greater chance (p = 0.003) of developing an eGFR <60 mL/min/1.73 m2, despite the fact that uACR was not a meaningful prognostic factor. The unadjusted log (uEGF/Cr) odds ratios for incident eGFR of <60 mL/min/1.73 m2, fast worsening in renal function, or a combination of both end goals were 0.49 (95% confidence interval [CI]: 0.30–0.80)], 0.54 (95% CI: 0.35–0.84), and 0.45 (95% CI: 0.30–0.68), respectively. These correlations remained significant after correcting for baseline variables. So, regardless of other risk factors, a low uEGF/Cr ratio is linked to a rapid deterioration in renal function and the onset of CKD. The validity of employing uEGF as a biomarker for risk classification in diabetic individuals with normoalbuminuria should be tested in different populations [43].

In two Chinese population-based studies (the Cohort Research of Chronic Kidney Disease [C-STRIDE] and the Incident, Development, and Prognosis of DKD [INDEED] study), the relationships of uEGF with kidney involvement were studied in individuals at early and advanced stages of DKD. In the cross-sectional study of the INDEED cohort (early stage DKD), uEGF/Cr and uEGF/monocyte chemotactic peptide-1 (MCP-1) were found to have a negative correlation with the development of DKD. In the C-STRIDE cohort’s longitudinal investigation, uEGF/Cr, uMCP-1/Cr, and uEGF/MCP-1 were independently related with the composite outcome after controlling for relevant covariates. uMCP-1/Cr was found to be strongly linked to albuminuria, whilst uEGF/Cr was found to be more closely tied to eGFR. In advanced-stage DKD patients, uECF/Cr could be used as a predictive marker for kidney disease progression. When uMCP-1 levels were standardized and expressed as the uEGF:MCP-1 ratio, it not only remained significantly associated with the occurrence of DKD, but it also improved the ability to differentiate patients with kidney involvement from those with T2DM using clinical indicators such as eGFR and uACR. When compared to uEGF/Cr or uMCP-1/Cr alone, uEGF/MCP-1 exhibited a superior predictor of the renal composite outcome [44]. In terms of MCP-1’s underlying involvement in DKD pathogenesis, it may contribute to the course of the illness by recruiting and activating monocytes/macrophages and worsening tubulointerstitial inflammatory lesions [58]. As a result, it’s possible that the uEGF:MCP-1 ratio has such a significant relationship to kidney involvement in both the early and late phases of DKD because it is a better predictor of tubulointerstitial damage [44].

The pace of renal function decline varies widely across diabetic individuals [59-61], allowing researchers to look for indicators that predict the rate of decline and identify patients at risk many years before ESKD develops. Many criteria have been used to diagnose such decreases, but they all rely on serially recorded serum creatinine and an analysis of the trajectory of creatinine-based eGFR over time. The 2nd Joslin Kidney Study included 1,032 people with normal renal function who were assessed for factors of early progressive renal impairment in T2DM. Early renal deterioration was defined using eGFR slopes calculated from serial serum creatinine values throughout a 5- to 12-year period of follow-up. More specifically, the result was defined as a decrease in eGFR of more than 30% from baseline over 5 years. uEGF/Cr and uEGF/MCP-1 were substantially linked with the probability of early renal deterioration in univariate analysis (area under the curve (AUC) = 0.58, p = 0.003 and AUC = 0.75, p < 0.001). When the impacts of tumor necrosis factor receptor 1, kidney injury molecule-1, systolic blood pressure, uACR, and uEGF/MCP-1 ratio were combined, the latter had the most influence. When these indicators were merged into a multimarker prognosis test, the discriminatory performance of early renal deterioration risk prediction increased dramatically when compared to uACR and systolic blood pressure alone. However, the study only looked at T2DM individuals with mostly European ancestry, and the positive predictive value in albuminuric patients was only 50%. The suggested multimarker prognosis test represents a novel way of developing a prognostic test based on the findings of etiological research. The test has a low predictive value, and it cannot be recommended for clinical use at this time due to a lack of relevant indicators. To develop a better predictive test for such decline that may be employed in a clinical context, new and more informative makers are required [45].

In a small prospective cohort study [46] of T2DM with DKD at high risk for renal insufficiency development, the effectiveness of uEGF, uMCP-1, or their ratio in predicting fast decline of GFR was investigated. Patients with an annual eGFR reduction of >25% exhibited significantly higher baseline levels of uMCP-1 and uACR, as well as lower uEGF and uEGF/MCP-1 ratio. With ROC analysis, uMCP-1, uEGF, uEGF/MCP-1, and uACR all predicted primary outcomes. eGFR, uACR, uMCP-1, uEGF, uEGF/MCP-1, and blood pressure were all linked to rapid GFR reduction in univariate analysis. After multivariate analysis with conventional components, uMCP-1 and the uEGF/MCP-1 ratio exhibited considerable predictive potential. The uMCP-1 or uEGF/MCP-1 ratio were connected to rapid renal development in DKD, regardless of established risk factors. Previous research has indicated that MCP-1 expression in the kidneys is elevated in DKD. uMCP-1 levels have been connected to the degree of tubulointerstitial leukocyte infiltration [58, 62], but its significance in predicting renal prognosis in DKD remains uncertain. MCP-1-mediated macrophage accumulation and activation are critical processes in DN [63, 64]. High uMCP-1 levels were predictive of rapid renal function loss over a wide range of kidney function in Asian patients with stage 1–5 DKD, regardless of traditional variables. Low uEGF was a consistent predictor of quick renal deterioration over a wide range of GFR and albuminuria, including a subset of people with low albuminuria. In the context of more advanced DKD or noticeable proteinuria, the value of uEGF as a biomarker of rapid advancement over established markers may be lessened. Although a high uEGF/MCP-1 ratio was independent of fast GFR loss in DKD, the ratio’s overall performance was equal to that of uMCP-1 alone. As a result, given the higher expenditures, this research did not demonstrate the value of routine uEGF monitoring in addition to MCP-1. uEGF or uEGF/MCP-1 may be useful in the DKD subgroup with normo- or low-grade albuminuria and decreased eGFR. Larger studies are needed to determine the cost-benefit of uEGF testing in predicting adverse renal outcomes in this population [46].

Kidney Transplantation

EGF excretion has been assessed in the urine of living human kidney donors and recipients. After uninephrectomy, uEGF was approximately 65% of the preoperative value, reflecting a compensatory increase in uEGF excretion rate from the remaining kidney of approximately 30% [65]. This was in contrast to a 50% decrease in uEGF/Cr in five persons who underwent uninephrectomy due to renal malignancies [66]. GFR was also increased in response to compensatory changes in uEGF excretion rate, suggesting that compensatory changes in uEGF and eGFR are related, with both possibly being symptoms of nephron hypertrophy and hyperperfusion. The increased uEGF excretion of the recipients’ own kidneys may enhance the transplanted kidney’s EGF excretion. However, this impact is likely minimal owing to ESKD. GFR was consistently approximately 15% lower and uEGF excretion rate was consistently 40% lower in transplanted kidneys compared to identical kidneys remaining in donors. This could be due to ischemia and drug-induced damage, as well as the transplanted kidneys’ denervated status [65, 67].

Acute cellular rejection (ACR) of renal allografts is still a major cause of graft loss in human kidney transplantation. The kidney gene and protein expression, as well as the urine excretion rate of EGF, were examined in renal transplant recipients (16 with ACR and 13 with tubular damage/cyclosporine toxicity [ATD]). ACR patients had significantly lower EGF expression in the ascending limb of Henle’s loop and the distal convoluted tubule cells, as well as a prolonged positive IL-6 signal. The changed expression and distribution inside the allograft were closely paralleled in the urine pattern of IL-6 and EGF excretion. In ATD patients, EGF expression was significantly reduced. The IL-6/EGF ratio was considerably greater in ACR patients, but only modestly higher in ATD patients. As a result, kidney transplant patients with ACR or ATD had a specific pattern of IL-6 and EGF intragraft expression that was closely mirrored by their urinary excretion rate. EGF expression may be reduced as a result of tubular injury or as a reaction to an inhibitory signal. In individuals who recovered from ACR, uEGF levels returned to normal. To assess the therapeutic value and prognostic importance of EGF in each transplanted patient, more research is needed [68].

Although short-term graft survival has improved significantly in recent decades, chronic graft failure remains a key barrier to improving long-term outcomes in renal transplant recipients [69]. Chronic rejection, hypertension, oxidative stress, infection, calcineurin inhibitor toxicity, and proteinuria are all factors that contribute to graft failure, which leads to progressive fibrosis and loss of renal function [70]. The majority of biomarkers employed for follow-up are indicators of glomerular damage (uACR and uPCR) [71], which are indicative of the development of fibrosis and represent an early event in chronic rejection’s natural history [72]. Noninvasive biomarkers that can identify pathophysiological changes in renal tissue are being studied as prospective techniques for patient monitoring and early detection of persons at high risk of transplant failure [47].

In a prospective cohort research study [47], uEGF/Cr was measured in 649 stable renal transplant recipients with a working allograft of 1 year, no history of drug or alcohol addiction, or malignancy, and who were followed for 3 years. In individuals with impaired renal function, uEGF excretion was significantly lower. After adjusting for possible confounders, uEGF/Cr was negatively linked with the incidence of graft failure (p < 0.001). uEGF/Cr seems to be a better predictor of graft failure than albuminuria and urinary protein excretion. Although it is challenging to capture the complexities of graft failure with a single biomarker, fibrosis is a pivotal stage in the development of graft failure [73], and uEGF suppression is an early indicator of this phenomenon [17]. uEGF/Cr may be a marker for a specific pathological aspect of graft failure that begins prior to the development of glomerular injury [47]. The lack of a drop in uEGF with time following transplantation could be explained by a confusing impact of cyclosporin A (CsA) treatment, which lowers uEGF. It has been discovered that uEGF and the usage of calcineurin inhibitors have an inverse relationship [74, 75]. Besides the calcineurin inhibitors’ nephrotoxic impact, the relationship between uEGF/Cr and graft failure is probably mediated by other processes, including renal fibrosis [76]. Patients with stable graft function and proteinuria >500 mg/24 h had no significant differences in uEGF mRNA when compared to those with chronic allograft nephropathy [77]. More research and validation in larger and more diverse clinical trials are needed to establish the potential value of EGF directed treatment or the use of uEGF/Cr in clinical care of stable renal transplant patients [47].

Because of a renal Mg2+ leak, hypomagnesaemia has been linked to CsA therapy. As a result of CsA therapy, 60% of people develop hypomagnesaemia, with occurrences ranging from 1.5 to 100%. Confusion, muscular weakness, dysphagia, tetany, tremor, and general convulsions are some of the clinical symptoms of severe magnesium deficiency after CsA therapy [76, 78]. Animal studies have revealed a function for the Mg2+ channel Transient Receptor Potential Cation Channel Subfamily M Member 6, which is situated in the distal convoluted tubule and is activated by EGF [79, 80]. Transient Receptor Potential Cation Channel Subfamily M Member 6 mRNA was downregulated concurrently with EGF in an in vivo rat model of CsA nephrotoxicity, resulting in increased renal magnesium excretion [81]. Serum Mg2+ was considerably lower in the CsA group compared to the CKD group in a case-control study involving 55 renal transplant patients treated with CsA and 35 CKD patients, with significantly more CsA-treated patients developing hypomagnesaemia. Although the fractional Mg2+ excretion (FE Mg2+) did not differ substantially between the two groups, it was much higher in both when compared to normal reference values (FE Mg2+ <2%) [76, 82], as predicted given the degree of renal impairment [83, 84]. CsA-treated patients showed a substantially higher FE Mg2+ than CKD patients in a subanalysis of hypomagnesaemia patients (p = 0.05). The CsA group had a considerably lower uEGF excretion, which was a predictor of FE Mg2+ in both groups.

Renal magnesium reabsorption has been demonstrated to be enhanced in CKD patients in an attempt to maintain blood magnesium levels within normal ranges. However, in CsA-treated patients, the kidney is unable to augment magnesium reabsorption, and magnesium is lost in the urine. The CsA group had a more dramatic drop in EGF, which could be linked to FE Mg2+. Renal EGF production is connected to FE Mg2+, indicating that EGF regulates Mg2+ reabsorption in humans [74].

Primary Glomerulonephritis

Primary glomerulonephritis (GN) is a group of illnesses that together account for one of the leading causes of ESKD [85]. Regardless of the underlying disease, glomerular injury activates similar profibrotic pathways such as tubular shrinkage and interstitial fibrosis, resulting in progressive nephron loss [86, 87]. Renal function degrades at varying rates in GN and is more strongly related to the degree of tubulointerstitial disease than to glomerular lesions [86-88]. Patients with extensive tubulointerstitial involvement at risk of more rapid development cannot be properly identified using traditional clinical indicators such as proteinuria, hypertension, or a lowered GFR. Currently, only a kidney biopsy can establish the extent of IFTA, which is a major prognostic factor in GN. The severity of IFTA is likely determined by an imbalance between pro-inflammatory cytokines like MCP-1 and protective cytokines like EGF. A link was discovered between IFTA and uEGF, uMCP-1, and the uEGF/MCP-1 ratio in a cross-sectional study. eGFR, uEGF, and uEGF/MCP-1 ratio were all linked to IFTA in univariate analysis. In multivariate analysis, only the uEGF/MCP-1 ratio was independently associated with IFTA. The uEGF/MCP-1 ratio had an IFTA sensitivity of 88% and a specificity of 74%. uEGF/MCP-1 discriminated well against IFTA (AUC = 0.85). However, there was no significant difference between uEGF alone and uEGF/MCP-1. Because of their opposing fibrotic effects, the balance of uEGF and uMCP-1 may be a better predictor of IFTA severity and renal disease progression than either cytokine alone. In univariate analysis, the eGFR, uEGF, and uEGF/MCP-1 ratio were all shown to be significant. uEGF may be a good candidate biomarker for moderate to severe IFTA, while it may not be as beneficial in detecting persons with early tubuloin­terstitial damage. More research with larger study populations is needed to thoroughly examine the function of uEGF/MCP-1 as a biomarker in predicting long-term prognosis or response to therapy [48].

IgAN is the most common primary GN, and many people develop ESKD within 10–20 years after initial renal biopsy. The presence of impaired renal function, arterial hypertension, and the severity of histologic lesions, as well as the degree of proteinuria at the time of renal biopsy, are all well-known prognostic indicators for the development of ESKD in IgAN patients [89]. Evidence suggests that the degree of tubulointerstitial alterations, such as tubular atrophy, interstitial cell infiltration, and fibrosis, is closely linked to the loss of renal function in the course of renal injury [90]. The relationships between urine levels of IL-6, EGF, MCP-1, and TGF-β1 and clinical, biochemical, and histological aspects were evaluated in 58 individuals with primary IgAN, as well as their ability to predict the degree of glomerular and/or interstitial sclerosis lesions. uIL-6, uTGF-β1, and uMCP-1 were associated with focused glomerulosclerosis and interstitial fibrosis extension, but not with mesangial, extracapillary, or endocapillary proliferation. uEGF had a negative connection with interstitial fibrosis. Individuals with T1 and T2 scores exhibited significantly higher levels of uIL-6, uMCP-1, uTGF-β1, and significantly lower uEGF levels than patients with T0 scores, according to the Oxford classification. uMCP-1, uIL-6, and uEGF concentrations were revealed to be independent predictors of the fibrosis surface in multiple regression and logistic regression studies after age and eGFR were corrected [49].

Cytokines generated by both resident and nonresident renal cells play a significant role in the advancement of renal damage during IgAN. Subjects with uEGF/MCP-1 ratios in the lowest tertile showed a significant worsening in renal survival at 48 and 84 months of follow-up in a cohort of 132 patients with biopsy-proven IgAN, whereas individuals in the highest tertile achieved 100% renal survival. In a multivariate Cox’s regression analysis, the uEGF/MCP-1 ratio was revealed to be an independent predictive factor and to be indirectly related to the combined outcome. The AUC was also used to compute the predictive value. The AUC of the uEGF/MCP-1 ratio was significantly greater than that of uEGF or uMCP-1 alone, histologic grade, creatinine clearance, or proteinuria. As a result, the uEGF/MCP-1 ratio could be employed as a predictive diagnostic for ESRD in IgAN patients [50].

In IgAN patients, it is occasionally essential to repeat the kidney biopsy, but because this is an intrusive procedure, a cost-benefit analysis is required. In these circumstances, measuring the EGF/MCP-1 ratio could help us foresee the ongoing loss of renal function, avoiding more intrusive procedures when they are not absolutely necessary. These findings could be used to identify high-risk patients who would benefit from more frequent monitoring and vigorous treatment. To back up this idea, more prospective trials with a bigger patient group are needed [50].

Antineutrophil Cytoplasmic Antibody Vasculitis

Antineutrophil cytoplasmic antibody-associated vasculitis (AAV) is a collection of potentially fatal autoimmune diseases that includes granulomatosis with polyangiitis, microscopic polyangiitis, and eosinophilic granulomatosis with polyangiitis [91]. Approximately 25% of AAV patients are still resistant to induction therapy, and 20–25% of AAV patients develop ESKD [92]. Finding noninvasive biomarkers that can predict renal response to therapy and renal function decline in AAV patients is crucial.

The relationship between uEGF/Cr and renal disease severity and outcomes was studied in a cohort of 173 patients with active AAV and 143 patients in remission [51]. Intrarenal EGF mRNA expression was found to be highly related to log2eGFR at biopsy in AAV patients (β = 0.63, p < 0.001). It’s not unexpected that the quantity of uEGF/Cr was linked to eGFR because uEGF levels are strongly tied to EGF mRNA levels. The quantity of uEGF/Cr in patients in remission was substantially higher than in individuals with ongoing disease, both in patients with sequential readings and in cross-sectional comparison. The quantity of uEGF/Cr at the time of sampling was favorably related to log2eGFR in both the active and remission periods (p < 0.001, p < 0.001, respectively). Patients with resistive renal illness had significantly lower uEGF/Cr levels than responders. uEGF/Cr was also linked with a composite outcome of ESKD or a 30% reduction in eGFR after controlling for other potential predictors [93, 94].

Lower uEGF/Cr ratios in AAV patients are associated with more severe renal impairment, treatment resistance, and a higher chance of progression to the composite outcome. The strong link between a lower uEGF/Cr and a worse long-term outcome in AAV patients could be a reflection of the degree of tubular damage occurring. This could be due to the severity of an ongoing autoimmune disease or to immunological injury to the tubular compartment in the past. Studies have demonstrated that high uEGF levels diminish renal epithelial sodium channel activity, potentially lowering glomerular and renal tubular damage [93, 94]. However, more research into EGF’s protective role in AAV development is needed. In individuals with AAV, the amount of uEGF/Cr may be a valuable noninvasive biomarker for evaluating tubular damage and the possibility of advancing to chronic progressive kidney disease, but it has to be verified in a larger, multicenter cohort research [51].

Polycystic Kidney Disease

EGF and EGF-reactive peptide species are formed in the apical media of cultured autosomal dominant polycystic kidney disease (ADPKD) epithelia, and cyst fluids from ADPKD patients contain large, possibly mitogenic amounts [95, 96]. EGF and other growth factors play a role in the regulation of numerous epithelial ion channels in the kidney. EGF, for example, increases the number of store-operated Ca2+ channels in human mesangial cells via an intracellular signaling cascade that includes tyrosine kinase and protein kinase C [97]. EGF activates store-operated Ca2+ channels via phospholipase C, but not inositol triphosphate receptors [98].

uEGF levels have been measured in patients with ADPKD with or without CKD, as well as their relatives and 20 healthy subjects. uEGF was significantly lower in ADPKD patients with CKD than in those without CKD (p = 0.004). There was no statistically significant difference between ADPKD patients who did not have CKD, their relatives, or healthy controls, respectively. Only a small rise in uEGF was observed in the ADPKD group without CKD, suggesting that EGF production is linked to kidney mass in the early stages of renal function impairment [99]. Before uEGF may be utilized as a prognostic indicator in ADPKD patients to anticipate the onset of renal function impairment, more research is needed [99].

Alport Syndrome

Alport syndrome is a rare inherited kidney disease that results in kidney failure. In recent decades, it has become well-known as a kind of glomerular disease with the usual pathological alterations of the glomerular basement membrane. In terms of illness development, Alport syndrome patients have a wide range of symptoms. The necessity for identifying patients at high risk of fast kidney function decline remains unfulfilled [100]. Patients with Alport syndrome have been found to have tubulointerstitial fibrosis, a sign of renal disease development [101, 102].

In a cross-sectional study of 117 pediatric patients with Alport syndrome and 146 healthy children, uEGF/Cr dropped much quicker in pediatric patients with Alport syndrome than in healthy children of the same age group. After adjusting for age, uEGF/Cr was strongly linked with eGFR (r = 0.75, p < 0.001), but not with 24-h proteinuria. In 38 patients with long-term follow-up, there was a substantial correlation between uEGF/Cr and the eGFR slope (r = 0.58, p < 0.001). Patients with a lower uEGF/Cr level had a higher chance of progressing to a later stage of CKD [100]. The regenerative activity of EGF is likely to explain the link between uEGF/Cr and progression [15]. Lower uEGF/Cr indicates that the tubular cells’ regenerative capacity is hindered, which is linked to progression. Even while eGFRs were still above 60 mL/min/1.73 m2, uEGF/Cr had an AUC of 0.88 for distinguishing progressors from nonprogressors, compared to 0.77 for eGFR and 0.81 for 24-h urine protein [100].

Further research into whether and how EGF is involved in tubular damage healing and regeneration in Alport syndrome is needed. To validate the predictive usefulness of uEGF/Cr in children with Alport syndrome, a prospective study with more patients and a longer follow-up is needed [100].

Conclusion and Future Perspectives

The present review article shows that the majority of studies have looked at uEGF, uEGF/Cr, or uEGF/MCP-1 as a standalone biomarker. As a result, its performance and connection with other tubule function biomarkers are unknown, and more research is needed. Despite this, the significant signals seen with uEGF, which are independent of baseline eGFR, albuminuria, and other risk variables, make it a promising new addition to the list of tubule cell dysfunction markers [103]. As there are currently no uEGR, uEGF/Cr, or uEGF/MCP-1 reference values, it is necessary to use standardized uEGF tests with calculated reference values [47]. The next step would be to establish an age-dependent normal uEGF/Cr and uEGF/MCP-1 range [100].

The widespread clinical standardization of uEGF by creatinine as a biomarker of renal tissue damage would have several advantages: (1) it is tissue specific, making it resistant to extrarenal events that may damage the accuracy of other nonspecific biomarkers; and (2) it is well proven that the kidney function has a considerable influence on uEGF/Cr, even in the normal creatinine range [104]. uEGF/Cr has only a weak relationship with glomerular damage indicators such as urine protein excretion, indicating that this ratio is a representation of a distinct independent pathophysiologic process [105-107] and could be used in conjunction with these other parameters.

The precise place of uEGF, uEGF/Cr, and uEGF/MCP-1 measurement to indicate rapid renal function decrease is still unknown [38]. Because uEGF is a possible indicator of intrarenal health, integrating it into a risk assessment system may help predict which patients at high risk of CKD progression will actually progress. uEGR determination may also be useful in assessing kidney function beyond serum creatinine and proteinuria, as well as in selecting subjects for kidney transplantation and before prescribing medications known to cause toxicity to kidney tubular cells, such as certain antibiotics and anti-HIV medications [38, 108, 109]. Changes in uEGF/Cr can only be regarded as significant when they vary by at least 30% when monitoring repeated samples in the same person. In contrast to increasing serum creatinine, which can indicate either acute renal injury or chronic kidney damage, reduced uEGF/Cr levels may indicate an increased disease activity and is related to the severity of chronic kidney impairment. Low uEGF/Cr levels during a flare, as well as declining levels over time, are linked to poor long-term kidney outcomes [19].

Conflict of Interest Statement

The authors have no conflicts of interest to declare.

Funding Sources

No funding has been received for this study.

Author Contributions

C.C. and M.M.S. wrote the first draft and edited the manuscript. R.S. and J.R.D. were responsible for conceptualization, critical review, and editing of the manuscript. All the authors have read and approved the final manuscript.

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