Alport syndrome (AS) is a hereditary nephritis related to a structural abnormality of the glomerular basement membrane (GBM) caused by pathogenic variants in genes of α3, α4, and α5 chains of type IV collagen (COL4A3, COL4A4, and COL4A5, respectively). The clinical manifestation of AS includes various degrees of hematuria, proteinuria, progressive renal insufficiency, and, eventually, end-stage kidney disease [1–3].

This article will focus on the pathological diagnosis of AS, reviewing the variations in pathological findings according to the different inheritance modes of AS and pathological differential diagnoses. Also, the recent advances in genetic tests will be briefly summarized.

Type IV collagen is present in all basement membranes of the human body. Genes named COL4A1 to COL4A6 are responsible for encoding α1 to α6 isoforms of type IV collagen. These isoforms assemble into three kinds of heterotrimers composed of α1-α1-α2, α3-α4-α5, and α5-α5-α6, and then assemble together to form a meshwork network. In the kidney, α3-α4-α5 type IV collagen is present in the GBM and distal tubular basement membrane (TBM), and α5-α5-α6 type IV collagen is present in Bowman’s capsule, while α1-α1-α2 collagen is present in all the basement membranes [4,5]. AS results from the mutation of genes producing α3, α4, and α5 chains. Therefore, the proportion of the α1-α1-α2 heterotrimer increases in the kidneys of AS patients [6]. The mechanisms of the subsequent structural and functional changes in the glomeruli of AS patients have not been fully elucidated; however, clues are obtained from in vitro and animal model studies. The GBM composed of α1-α1-α2 collagen is mechanically less stable than the GBM composed of α3-α4-α5 trimer. Podocytes interact with intact α3-α4-α5 GBM via discoidin domain receptor 1 and integrin α2β1 to maintain the function of the slit diaphragm, and this interaction is altered in AS. Moreover, podocytes recognizing mutated collagen upregulate the production of profibrotic factors, proteolytic enzymes, and chemokines such as transforming growth factor-β and matrix metalloproteinases, resulting in structural alteration of the GBM [6–9].

Light microscopic findings of AS are not specific to the disease. Glomeruli show no significant change in the early phase. With the progression of the disease, glomerular capillary loops become irregularly thick, and segmental or global glomerulosclerosis occurs. Mesangial hypercellularity may also be observed not infrequently [10]. Tubulointerstitium also shows tubular atrophy and interstitial fibrosis, which can be seen in other chronic kidney diseases. While interstitial foam cells can be observed in various glomerular diseases such as immunoglobulin A (IgA) nephropathy, membranous nephropathy, and idiopathic focal segmental glomerulosclerosis (FSGS), they are more frequently observed in AS patients [11], and they are not always associated with nephrotic range proteinuria (Fig. 1) [12]. Conventional immunofluorescence, including IgG, IgA, IgM, C3, C4, and C1q, does not show specific deposits. Since no specific findings are observed under light and immunofluorescent microscopies, and in some cases, the diagnosis of AS cannot be suspected by clinical history, the first clue of the diagnosis is obtained by electron microscopy [13]. The earliest electron microscopic finding of AS is segmental thinning of the lamina densa. As the disease progresses, GBM irregularity increases showing lamellation, splitting, and scalloping of the lamina densa. In severe cases, so-called “basket weaving” occurs. The podocytes show various degrees of foot process effacement. Microparticles or spherules can be observed within the GBM (Fig. 2) [4,12,13]. Similar findings are present in pediatric cases [14]. The above findings may present in other glomerular diseases; particularly, incomplete lamellation of the GBM can often be challenging to distinguish from nonspecific GBM damage or alteration. Therefore, it is not possible to confirm AS through conventional pathological examinations, and their significance lies in providing clues for performing specific diagnostic tests for AS. The confirmatory tests include type IV collagen subtype staining and recently emerging genetic tests.

Immunofluorescent staining of α chain subtypes reveals different staining patterns according to the inheritance pattern of AS [4,5]. Male X-linked AS patients who have mutations of COL4A5 show no staining of α5 and α3 chains in GBM, distal TBM, and Bowman’s capsule, while heterozygote females show mosaic pattern staining of α5 collagen in the GBM [15]. In autosomal recessive homozygote AS associated with COL4A3, α3 collagen and α5 collagen, which are dependent on α3, are decreased in the GBM and distal TBM. In contrast to X-linked AS, α5 collagen stain is preserved in Bowman’s capsule [12]. Autosomal dominant AS usually shows normal pattern expression of the α5 chain [16]. Therefore, the diagnosis requires further examination, such as next-generation sequencing (NGS). A commonly employed approach in clinical settings involves staining only α2 chain and α5 chain antibodies. The expression of α2 is considered as a control, and the diagnosis is determined by the expression of α5 (Fig. 3) [17]. However, immunofluorescent staining results are significantly influenced by the causative gene, the extent of abnormality, and the mode of inheritance. Especially, autosomal dominant AS cannot be detected by collagen staining. Recently, there has been an increasing number of reports on cases with pathologic variants in both COL4A3 and COL4A5 (“digenic” AS) [18], highlighting the limitations of collagen staining even more.

Due to the reasons mentioned above, genetic testing in diagnosing AS is becoming increasingly significant. The genetic mutations causing AS occur within COL4A3, COL4A4, and COL4A5, but due to the extensive diversity of these mutations, NGS has been recognized as the most suitable and widely used testing method. Consequently, this approach has led to the discovery of new types of previously unknown mutations [10,19,20]. For indications, strategies in using genetic tests, the clinical significance of specific mutations, interpretation of variants of uncertain significance, and other details on this topic, it is recommended to refer to specialized references that comprehensively cover these aspects [21].

The diagnosis of thin basement membrane disease (TBMD) requires diffuse thinning of the GBM without lamellation or basket weaving. The thickness of the GBM measured by electron microscopy varies depending on the tissue processing procedures, so each laboratory should establish its own standards. Generally, a 250 nm or less thickness in adults and in children, 180 nm or less may raise suspicion for TBMD [22]. The alternative term for TBMD, benign familial hematuria, is attributed to the perception of a favorable prognosis associated with TBMD. However, recent genetic studies have revealed that TBMD also harbors heterozygous mutations in COL4A3 or COL4A4, leading to its reclassification as part of the AS spectrum [23,24].

Some cases of IgA nephropathy are known to be associated with alterations in collagen type IV. In a familial IgA nephropathy pedigree, an association with the locus on chromosome 2q36, which includes COL4A3 and COL4A4, has been identified [25]. Subsequent studies have reported finding pathogenic mutations in COL4A3–5 in 20% of familial IgA nephropathy cases [26]. More recently, this phenomenon has also been reported in sporadic IgA nephropathy cases. Variants in COL4A3–5 genes were observed in 31.1% of IgA nephropathy cases with a thin GBM (<250 nm) [27]. Familial FSGS is another disorder where pathogenic variants in the same genes can be identified [28,29].

Since there are no specific light microscopy findings for AS, IgA nephropathy and FSGS could be considered as differential diagnoses of AS. On the other hand, when there is a family history or findings such as thin GBM in IgA nephropathy and FSGS, genetic testing for COL4A3–5 genes might also be considered.

When examined under light microscopy, AS patients’ kidney tissue may exhibit various changes such as glomerular FSGS, mesangial hypercellularity, and interstitial foam cell collection, but these changes are not diagnostic. Electron microscopy reveals alterations in the GBM thickness and features like basket weaving and lamellation. Efforts were made to confirm AS through collagen α chains staining; however, in recent times, the prevailing trend is to use genetic testing, including NGS.