Biomarkers for the early diagnosis of Alport syndrome and associated kidney damage
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Abstract
Alport syndrome (AS) is a hereditary nephropathy characterized by progressive kidney damage that commonly leads to end-stage kidney disease. Early diagnosis is critical, as preemptive nephroprotective therapy, such as angiotensin-converting enzyme inhibitors, can significantly delay disease progression. However, the early diagnosis of AS remains challenging due to the lack of reliable preclinical or screening biomarkers, particularly before the onset of proteinuria. Although nonspecific microhematuria is often present, it is insufficient for definitive early detection. Recent studies have identified potential early cellular alterations as candidate biomarkers for the preclinical detection of AS, but none have been widely implemented in clinical practice. This review presents the current knowledge on early biomarkers of kidney damage for AS, highlights promising avenues for future research, and emphasizes the importance of developing effective diagnostic tools to enable timely intervention and improve patient outcomes.
Introduction
Alport syndrome (AS) is a rare hereditary disorder characterized by progressive kidney disease, sensorineural hearing loss, and ocular abnormalities [1]. It is caused by mutations in the COL4A3, COL4A4, and COL4A5 genes, which encode the α3, α4, and α5 chains of collagen type IV. This collagen is crucial for maintaining the structural integrity of basement membranes in the kidneys, cochlea, and eyes [2]. In the kidneys, the glomerular basement membrane (GBM) provides structural support between endothelial cells and podocytes, ensuring proper filtration and glomerular stability. Mutations in collagen type IV disrupt collagen assembly, leading to GBM abnormalities such as thinning, splitting, and irregular thickening. These defects compromise the GBM’s filtration barrier, resulting in podocyte effacement, proteinuria, and glomerular injury [3].
AS is inherited in four primary patterns: X-linked (XLAS), autosomal recessive (ARAS), autosomal dominant (ADAS), and digenic AS. Mutations in COL4A5 cause XLAS, whereas mutations in COL4A3 or COL4A4 lead to ARAS and ADAS [1,4]. Males with XLAS and individuals with ARAS experience a more severe disease course, with an estimated 100% progression to kidney failure, although the rate can vary depending on the specific mutation. In the past, females with XLAS were considered carriers, but they are now reclassified as having AS, with an estimated 25% progression to end-stage kidney disease (ESKD). Individuals with ADAS have nearly a 20% risk of progressing to ESKD if they have risk factors for disease progression, such as a family history of kidney failure, proteinuria, or findings on kidney biopsy like focal segmental glomerulosclerosis (FSGS), GBM thickening, or lamellation [5]. However, these subtypes are often underrecognized in the early stages of the disease. The most common clinical feature of AS is persistent microscopic hematuria, followed by microalbuminuria, which progresses to proteinuria during adolescence [4]. In the absence of treatment, males with XLAS have a high risk of progressing to ESKD: 50% by age 25, 80% by age 40, and nearly 100% by age 60 [6]. Early initiation of angiotensin-converting enzyme inhibitor therapy at the onset of microscopic hematuria or microalbuminuria has been shown to significantly slow disease progression in AS [7,8]. However, diagnosing AS can be challenging when there are no extrarenal manifestations or family history, particularly when proteinuria is the predominant symptom. Current diagnostic strategies rely on conventional biomarkers for chronic kidney disease (CKD), such as estimated glomerular filtration rate (eGFR), urinary protein excretion, and serum creatinine, but these biomarkers are typically more useful at later disease stages. While kidney biopsy remains a traditional diagnostic method, it has limitations, including bleeding risks and lower specificity in young children. Genetic testing is considered the gold standard for confirming AS, though it is expensive and not widely available, especially in resource-limited settings.
These challenges underscore the urgent need for reliable, noninvasive biomarkers to detect AS in its preclinical or early stages. Biomarkers that identify subtle kidney changes, differentiate AS from other conditions, and monitor disease progression could significantly improve early diagnosis and guide personalized treatment strategies. This review aims to discuss potential preclinical biomarkers and updates on markers of kidney damage in AS to enhance early detection, disease monitoring, and patient outcomes.
Preclinical biomarkers for AS
Biomarkers for early disease diagnosis are measurable indicators that can detect a condition in its initial stages, often before symptoms become apparent. In AS, the earliest stage, referred to as stage 0, is characterized solely by microhematuria without microalbuminuria and can appear shortly after birth [7]. This stage precedes overt clinical manifestations such as albuminuria, proteinuria, or kidney dysfunction.
Biomarkers can be categorized into distinct types based on their timing and relevance to the progression of AS. Type A biomarkers are detectable preclinically and persist as the disease progresses. Type B biomarkers signal early molecular or structural changes before clinical symptoms emerge, while type C biomarkers appear when subtle clinical signs, such as microscopic hematuria, begin to manifest. Finally, type D biomarkers emerge in the later stages of the disease, such as with the onset of proteinuria, increasing serum creatinine, and low eGFR, reflecting advanced pathology in AS [9]. Understanding and identifying type A and type B biomarkers is crucial, as they hold the potential to detect preclinical alterations and enable early intervention, which could significantly alter the disease course.
For over a decade, studies by a research group in Germany using various AS models have provided valuable insights into preclinical biomarkers [9,10]. Building on findings from studies in mice, these researchers employed mass spectrometry to analyze samples from dogs with AS at stage 0, identifying over 70 potential biomarkers. These biomarkers were subsequently tested in two cohorts of children with AS, with some demonstrating significant elevation compared to healthy controls. Notably, collagen type XIII, hyaluronan-binding protein 2 (HABP2), and complement C4-binding protein (C4BP) in urine were identified as promising candidates for the early diagnosis of AS, with an area under the curve (AUC) of more than 80% [9]. Collagen type XIII, a transmembrane collagen, is involved in vascular endothelium and cell-matrix interactions. Normally absent in the vascular endothelium of healthy kidneys, collagen type XIII becomes abundant in the renal cortex of Alport mice. This aberrant expression promotes the selective migration of α1β1 integrin-positive monocytes, contributing to renal inflammation and fibrosis, central to AS pathophysiology [11]. Other potential biomarkers (HABP2 and C4BP) have shown promise, but their roles in AS patients remain understudied. HABP2, a serine protease involved in fibrinolysis and extracellular matrix (ECM) remodeling, regulates vascular homeostasis and inflammation [12], making it a potential early biomarker for monitoring AS progression. C4BP, a key regulator of the complement system, inhibits complement activation by binding to C4b [13]. Altered levels of C4BP may reflect disease activity and progression in AS, emphasizing its potential as a biomarker for both diagnosis and disease monitoring. Interestingly, the combination of collagen type XIII, HABP2, and C4BP in urine demonstrated superior diagnostic performance compared to individual biomarkers. This combination achieved a sensitivity of 65.2%, specificity of 99.5%, and an AUC of 81.9%, underscoring its potential as a promising diagnostic panel for assessing early AS status in clinical practice [9]. However, it is notable that the lack of comparisons with other glomerular diseases (e.g., immunoglobulin A nephropathy, FSGS) limits the ability to fully assess biomarker specificity for AS versus general kidney damage. Further research in larger and more diverse cohorts is necessary to validate the early diagnostic potential of these biomarkers for clinical use. Similarly, high mobility group box 1 (HMGB1), a protein involved in inflammation, cell proliferation, and tissue regeneration, has been found to be elevated in both serum and urine in the early stages of AS. Increased HMGB1 levels were correlated with a decrease in eGFR, supporting its potential as a serum biomarker for early kidney damage in AS [14].
In addition to human studies, several studies using AS animal models have been conducted to identify other promising biomarkers for early detection. In mice with the COL(IV)A3−/− mutation, three stages of AS were identified based on age: at 4.5 weeks (stage 0), 6 weeks (stage I), and 7.5 weeks (stage II) [10]. In the early stage (4.5 weeks), protein markers associated with ECM remodeling and cell damage, including transferrin, β2-glycoprotein 1, Apo A I, and vomeronasal 2 receptor 67, were found to be overexpressed. In the 6-week-old mice, when proteinuria developed, proteins such as haptoglobin, α1-antitrypsin 4, and complement factors H and I increased. Among these markers, gelsolin, which was detected in urine at 4.5 weeks and peaked at 6 weeks, was identified as a promising candidate for early kidney damage detection. Gelsolin has been studied in relation to other kidney conditions due to its role in cell movement and actin remodeling. Additionally, gelsolin plays a role in podocyte function [15], which becomes disrupted in AS. Another promising early biomarker identified in a mouse model of Alport-like disease (FVB/N Cd151−/−) is mindin, an ECM protein encoded by the SPON2 gene. Mindin is known for its role in immune responses, functioning as an integrin ligand, as well as mediating cell adhesion and tissue remodeling [16]. Naudin et al. [17] studied this model at 3 weeks of age, a stage characterized by the absence of glomerular inflammation or scarring but with abnormalities in GBM. They observed a significant increase in urinary mindin levels during early GBM damage. However, mindin was not detectable in the kidneys of 5-day-old mice, suggesting that while mindin may be a potential marker for disease progression, it is unlikely to reflect the initial events that trigger glomerular damage. Recently, a study conducted in 129 Sv Alport mice at 2 weeks of age revealed elevated endothelin 1 (ET-1) levels in kidney samples, highlighting its role in early glomerular damage. ET-1, a potent vasoactive peptide, mediates mesangial cell activation through the endothelin A receptor. This activation contributes to mesangial cell proliferation, excessive ECM deposition, and the secretion of inflammatory cytokines, initiating glomerular injury and disease progression [18]. Although ET-1 is involved in early pathogenic processes in AS, its utility as a diagnostic marker remains uncertain due to its lack of specificity, as it is also elevated in other glomerular diseases such as FSGS and hypertensive nephropathy [19]. Despite this limitation, ET-1 holds promise as a research biomarker, particularly in evaluating the efficacy of emerging therapies targeting AS.
In general, compared to other biomarkers mentioned in this section, urinary collagen type XIII, HABP2, and C4BP have shown significant promise based on studies conducted in various models, including animal models (mice and dogs) and validation in AS patients. Additional validation and ongoing research are required to identify and explore other potential biomarkers that could improve the early detection of AS and enhance patient outcomes. A summary of the biomarkers for early AS diagnosis is provided in Table 1.
Early biomarkers for diagnosing AS
Biomarkers of kidney damage in AS
Biomarkers indicating specific kidney cell damage in AS provide valuable insights into disease progression and pathophysiology (Fig. 1). Damage to key structures, such as podocytes, GBM, endothelial cells, and tubular cells, plays a central role in the pathophysiology of AS.
Biomarkers of kidney damage in Alport syndrome. PDlim2, PDZ and LIM domain 2; NOX4, NADPH oxidase 4; DDR1, discoidin domain receptor tyrosine kinase 1; MCP-1, monocyte chemoattractant protein-1; miR-21, microRNA-21; TNS1, tensin 1; CCND1, cyclin D1; GJA5, gap junction protein alpha-5; uEGF, urine epidermal growth factor.
Podocyte biomarkers
Podocytes, which are specialized epithelial cells lining the outer surface of renal glomerular capillaries, play a crucial role in maintaining the structural and functional integrity of the glomerular filtration barrier [20]. Previous studies have indicated that podocyte depletion plays an important role in the mechanisms underlying the progression of glomerular disease, ultimately leading to ESKD [21]. A study by Ding et al. [22], involving 95 urine samples and 41 kidney biopsies from AS patients, demonstrated significantly elevated podocyte detachment in urine (through evaluating podocin messenger RNA levels compared to controls), which was consistent with biopsy findings. The loss of podocytes was strongly correlated with increased proteinuria, glomerulosclerosis, and declining eGFR. Notably, podocyte depletion progressed with age, with AS patients losing approximately 26 podocytes per glomerulus per year—more than ten times the rate observed in healthy kidneys (2.3 podocytes per glomerulus per year). Using a linear regression model, the authors proposed that urinary podocyte detachment could serve as a proxy for estimating glomerular podocyte numbers over time, highlighting its potential as an early marker for predicting AS progression [22]. Frank et al. [23] reported an elevated number of podocytes in the G1 phase of the cell cycle, along with increased cell hypertrophy, observed during disease progression in both male and female AS mice. This result was validated using in vitro experiments reflecting the same phenomenon. Particularly, the high expression of podocyte PDlim2 in the G1 phase of the cell cycle, derived from proteomic data in mice with AS, confirmed the same trend in an AS patient. Besides the loss of podocytes in AS, podocyte apoptosis was reported in COL4A3 mutations. In 2023, Tong et al. [24] found that NADPH oxidase 4 (NOX4) might induce podocyte apoptosis through the regulation of matrix metallopeptidase 2 (MMP-2) in patients with COL4A3 mutations. Following NOX4 inhibition, a decrease in podocyte apoptosis and low expression of MMP-2 were observed in both in vitro and in vivo studies.
GBM biomarkers
Discoidin domain receptor tyrosine kinase 1 (DDR1) is a receptor tyrosine kinase activated by collagens and plays a significant role in the progression of fibrotic kidney diseases. In 2021, Kim et al. [25] revealed a novel mechanism underlying the disruption of GBM integrity in an AS model. They demonstrated that the aberrant deposition of collagen type I triggers the activation of DDR1. This activation enhances the expression of CD36, a fatty acid transporter, which increases the uptake of free fatty acids and promotes lipid accumulation within podocytes. The resulting lipotoxicity contributes to podocyte dysfunction and accelerates kidney damage in AS. Additionally, the administration of ezetimibe, a cholesterol absorption inhibitor, has been found to interfere with the DDR1/CD36 interaction, thereby reducing podocyte lipotoxicity and improving renal outcomes in AS mouse models [25]. These findings suggest that DDR1 plays a crucial role in mediating kidney damage in AS through pathways involving collagen deposition and lipotoxicity. Recently, a study by Randles et al. [26] further elucidated these ECM changes in AS mouse models, such as Col4a3−/− and Col4a5−/− mice. This study demonstrated a reduction in essential GBM components, including collagen type IV, laminins, and collagen type XVIII, which are pivotal for maintaining GBM integrity. Simultaneously, there is a marked increase in interstitial ECM proteins, such as collagen types I, III, VI, and XV, as well as fibrinogens and nephronectin, reflecting a fibrotic response. These changes represent a shift from a specialized, filtration-supporting matrix to a fibrotic and dysfunctional ECM, a hallmark of AS pathology [26].
GEC biomarkers
In AS, glomerular endothelial cells (GEC) contribute to the dysfunction of the glomerular filtration barrier. Although the primary defect lies in the GBM due to mutations in collagen type IV, the altered GBM disrupts the interaction between endothelial cells and the basement membrane, leading to endothelial stress and dysfunction. A recent study in Col4a5 knockout AS mice identified apelin as a key molecule with pro-inflammatory properties, which is differentially expressed in a specific subpopulation of GEC. Transcriptomic analysis revealed elevated apelin expression in the brighttdT subpopulation, correlating with altered immune signaling pathways and endothelial dysfunction. These observations were further validated in human kidney biopsies from AS patients [27]. Apelin is an endogenous peptide expressed throughout the nephron in healthy kidneys. In CKD, plasma apelin levels are elevated and may serve as a potential biomarker for predicting the risk of kidney function decline [28]. Given its role in promoting inflammation and its consistent association with GEC injury, apelin shows promise as a biomarker for monitoring endothelial damage and disease progression in AS. However, further research is necessary to establish apelin as a definitive clinical biomarker for AS.
Tubular damage biomarkers
Tubular damage occurs as a consequence of glomerular dysfunction, leading to tubular atrophy, interstitial fibrosis, and progressive kidney injury in AS. Epidermal growth factor (EGF) is predominantly produced in the renal tubules and plays a crucial role in cell proliferation, tissue repair, and the maintenance of tubular integrity. Low expression of EGF is associated with CKD [29]. Currently, a study involving 117 children with AS and 146 healthy controls has been exploring the relationship between the urinary EGF (uEGF)/creatinine ratio and eGFR, as well as its decline with age in children with AS. A lower uEGF/creatinine ratio was significantly associated with a lower eGFR during long-term follow-up. Notably, the uEGF/creatinine ratio decreased by approximately 60% in ARAS patients and by 73% in AS patients who progressed to CKD, compared to the healthy group [30]. This ratio demonstrated a strong predictive value for progression to late-stage AS, with the highest AUC of 88% compared to other factors such as 24-hour proteinuria and eGFR. Therefore, the uEGF/creatinine ratio may serve as a promising marker for tubular damage and interstitial fibrosis, aiding in the prediction of AS progression in children.
Other biomarkers related to AS progression
The monocyte chemoattractant protein-1 (MCP-1) is a chemokine that plays a critical role in recruiting monocytes and macrophages to sites of tissue injury or inflammation. It is produced by various kidney cell types, including podocytes, mesangial cells, and renal tubular cells. In CKD, elevated MCP-1 levels contribute to monocyte infiltration in the kidney, thereby aggravating inflammation and tissue damage [31]. A 5-year follow-up study involving 28 patients with AS revealed higher urinary MCP-1/creatinine levels in AS patients compared to control groups. This increased ratio may correlate with a faster decline in kidney function, particularly in male patients with AS [32].
MicroRNAs, which are small noncoding RNAs, are closely associated with modulating tissue repair responses, including inflammation, fibrosis, and regeneration in kidney injury. A study involving 27 AS patients found that microRNA-21 (miR-21) levels were significantly elevated in kidney biopsy samples, correlating with worsened proteinuria, blood urea nitrogen, and eGFR compared to control groups [33]. Furthermore, inhibiting miR-21 can attenuate fibrosis and improve renal outcomes, emphasizing its potential as a therapeutic target. These findings highlight the pivotal role of miR-21 in AS pathogenesis and its promise for developing novel treatment strategies.
Spatial transcriptomics, which maps gene expression data to tissue architecture, provides additional insights into the spatial relationships between healthy and diseased cells in the kidney [34]. In 2024, Clair et al. [35] used digital spatial profiling to investigate the mechanisms underlying kidney disease progression in three patients with AS, two with FSGS, two with membranous nephropathy, and three non-disease controls. Focusing on the two young AS patients, functional annotation of the top differentially expressed genes compared to control groups revealed enrichment in pathways associated with AS, including ECM organization, cell adhesion, and collagen activation. Gap junction protein alpha-5, tensin 1, and cyclin D1 were associated with glomerular injury progression in AS patients [35]. These genes represent potential candidate markers for AS; however, further studies involving larger cohorts of AS patients are needed to validate their clinical relevance.
Conclusion
Early diagnosis plays a pivotal role in the effective management and prognosis of AS. This review updated the potential of preclinical biomarkers such as collagen type XIII, HABP2, and C4BP as valuable noninvasive candidates for detecting AS. While numerous biomarkers associated with kidney damage in AS continue to be investigated, several candidates show promise for monitoring disease progression, including podocin (specific to podocytes), DDR1 (associated with the GBM), apelin (for GEC), and the uEGF/creatinine ratio (indicative of tubular cells). However, long-term and large-scale studies are essential to confirm their roles in early diagnosis and disease prediction, as well as to ensure their applicability across diverse patient populations. Overall, these findings hold significant promise for improving diagnosis, monitoring disease progression, and facilitating early therapeutic interventions.
Notes
Conflicts of interest
No potential conflict of interest relevant to this article was reported.
Funding
None.
Author contributions
All the work was done by HDTN and MHC.