Chronic kidney disease (CKD); end stage kidney disease (ESKD); focal segmental glomerulosclerosis (FSGS); glomerular basement membrane (GBM); not otherwise specified (NOS); thin basement membrane nephropathy (TBMN).

Focal segmental glomerulosclerosis (FSGS) is a pathological diagnosis which underpins a variety of progressive renal diseases that commonly result in proteinuria, chronic kidney disease (CKD) and potential end stage kidney disease (ESKD) requiring renal replacement therapy ¹ . FSGS occurs in either a primary. secondary, or genetic form. Primary FSGS is thought to be largely immunological in nature perhaps driven by an elusive permeability factor and secondary FSGS caused by compensatory hyperfiltration due to a previous glomerular injury ² . In recent years there has been a greater appreciation of the contribution of underlying genetic causes of this histological pattern. Many of these genetic aetiologies for FSGS have potential clinical significance for at-risk family members, subsequent genetic counselling, future living related kidney transplantation and therapeutics including potential avoidance of immunosuppressive therapies otherwise used for primary immunologically-mediated FSGS. Although initial focus has been on genes that are involved in the maintenance of podocyte structure and function it has become apparent that abnormalities in genes responsible for the structural integrity of the glomerular basement membrane (GBM) may underpin a significant minority of cases of adult-onset FSGS ³ . Variants in COL4A3, COL4A4 and COL4A5 which encode the α3, α4 and α5 chains of type IV collagen respectively, the major constituent of the GBM previously linked to both Alport syndrome and thin basement membrane nephropathy (TBMN) may also underlie cases of FSGS ⁴ . This suggestion has biological plausibility. Podocytes, the glomerular epithelial cells responsible for maintenance of the filtration barrier of the glomerulus through which plasma is ultrafiltrated, are not only a source for the α-chains secreted to form the GBM but also are required to be anchored to this structure in order to maintain the aforementioned filtration barrier ^{2, 5} . It is not inconceivable then that inherited structural abnormalities of the GBM may lead to subsequent podocyte dysfunction and ultimately the lesion histologically characterised as FSGS.

The relationship between the three renal conditions intertwined around variants in the COL4A genes, Alport syndrome, TBMN, and FSGS is complex and incompletely understood. Whilst previously it was believed that patients heterozygous for variants in COL4A3 and COL4A4 would develop TBMN with persistent microscopic haematuria and an otherwise benign prognosis, this traditional thinking has been overturned by the discovery that some pedigrees with these variants will go on to develop significant proteinuria, FSGS lesions and the potential for progressive CKD or ESKD ^{6, 7} . More recently, targeted gene sequencing of adults thought to have primary FSGS or steroid resistant nephrotic syndrome (the paediatric equivalent) found that pathogenic or likely pathogenic variants in COL4A genes were present in up to 38% of families with familial FSGS, and 3% of those with sporadic FSGS ^{3, 8} . These findings highlight the importance of considering variants in these genes in the diagnosis and subsequent management of patients found to have FSGS histologically.

FSGS is a clinicopathological diagnosis that is made on the review of renal tissue obtained by percutaneous biopsy. This tissue is processed and subjected to several different staining techniques and methods of microscopy, including light, immunofluorescence and electron microscopy ⁹ . FSGS, suggested on light microscopy by the finding of focal, segmental lesions with sclerosis, and an increase in the glomerular matrix with obliteration of the capillary lumens only requires this finding in one glomerulus and light microscopy to diagnose ² . As such, some specimens may previously have not been sent for further processing and review with other techniques such as electron microscopy even though this histopathology has heterogenous causative aetiologies. The other potential renal lesions that may be caused by COL4A gene variants, Alport syndrome and TBMN, cause thinning, lamellation and fraying of the GBM and may also be associated with podocyte foot process effacement, all of which require electron microscopy to visualise ⁴ . Similar findings may also be present in FSGS cases associated with COL4A variants. One study of Chinese families with familial FSGS noted segmental thinning of the GBM similar to those which may be present with TBMN in four out of five families with a proven COL4A variant underlying their familial FSGS (electron microscopy in another family and a sporadic case with proven variants were both noted to be unremarkable) ¹⁰ . At present the sensitivity of these findings remain unknown. It should be noted that for Alport syndrome there are other techniques aside from electron microscopy available for identifying the diagnosis. Immunostaining of a renal biopsy specimen for type IV collagen may demonstrate the absence of alpha 3,4 or 5 chains in up to two thirds of patients, or a skin biopsy may show an absence of an alpha 5 chain ^{11, 12} , in addition to the aforementioned genetic testing. Such absence by immunostaining does not appear be present in FSGS ¹³ . Given that the pathological diagnosis of FSGS does not routinely require electron microscopy it is therefore conceivable that GBM lesions potentially associated with an underlying type IV collagen variant may have been missed. This represents an opportunity to reflect on our prior clinical behaviour to see if our multidisciplinary diagnostic practice may require improvement. We conducted a retrospective cohort analysis of prior renal biopsy results in two tertiary centres to see how many of those that had been given a histological diagnosis of FSGS were sent for subsequent electron microscopy.

This retrospective cohort analysis demonstrated that about two-thirds of native kidney biopsy samples across two institutions that were deemed to have primary FSGS underwent subsequent electron microscopy. Of those that did, two were reported to have characteristics that might be consistent with an underlying type IV collagen disorder. In both samples, electron microscopy revealed a diffusely thin GBM, with the second additionally identifying early focal splitting of the GBM. The first sample, in which the patient had the tip variant of FSGS, was suggested to be consistent with TBMN whereas there was no pathological comment made about the second from a patient with FSGS NOS. FSGS (NOS) was the most common lesion described in this study, consistent with prior reports ² . Notably, close to one in three cases of primary FSGS were not proceeding to electron microscopy despite an indication to do so and 1 in 20 cases within our cohort had structural changes that were consistent with an underlying type IV collagen variant. Whilst some samples were unable to undergo electron microscopy due to a lack of glomeruli in the biopsy core, in the majority of the others it is unclear why subsequent electron microscopy did not occur. The annualised rate of biopsy samples not subjected to electron microscopy varied by year, but on average around one in three samples were not subjected to electron microscopy despite receiving a histological diagnosis of FSGS.

Curiously, neither of the two patients in whom electron microscopy was suggestive of an underlying type IV collagen disorder was noted to have haematuria on their urinalysis at the time of presentation. Both of these samples were noted to have a thin basement membrane on electron microscopy, with focal splitting noted in one with an average thickness of 230.72 nm given. This is in keeping with a study of Chinese FSGS families in which four patients with a demonstrated mutation in COL4A and familial FSGS all had findings suggestive of TBMN on electron microscopy ¹⁰ . Unfortunately due to the retrospective nature of this study we were unable to send any samples for immunostaining of type IV collagen. Regrettably he authors also do not have any data as to whether the two patients in whom electron microscopy was suggestive of a type IV collagen disorder proceeded to subsequent genetic testing for COL4A mutations or whether they were subjected to immunosuppression and their subsequent clinical course. The authors suggest that sequencing of these electron microscopy variants when found could be an extension of current research to expand our knowledge in this field.

There is an increasing body of evidence indicating that inheritable variants in COL4A may underlie a proportion of cases of FSGS, with up to 12.5% cases of autosomal dominant FSGS attributable to COL4A3 in some cohorts ¹⁰ . Not subjecting these renal biopsy samples to electron microscopy represents a potential gap in the investigation and subsequent management of such patients given they are much less likely to respond to immunosuppressive therapy ¹⁶ which has otherwise been classically indicated ( Figure 2).

This study was designed as a retrospective cohort study looking at the number of samples sent for electron microscopy, as well as any potential changes which might be consistent with a type IV collagen glomerular basement membrane disorder. It is important to recognise that not all groups have found the characteristic changes associated with the type IV collagen disorders such as Alport’s Syndrome or TBMN on electron microscopy. One study described the typical pathological changes of FSGS but not the glomerular basement membrane abnormalities characterising Alport syndrome or TBMN in patients known to have variants in either COL4A3 or COL4A4 ⁷ . It is thus possible that a lack of classical findings for a type IV collagen glomerular basement membrane disorder may have accounted for the low number of those with GBM features on electron microscopy consistent with a type IV collagen disorder noted within our study. Indicating against this, however, another study suggested that biopsy samples from patients with the classical features of Alport Syndrome or TBMN showed podocyte detachment which might be expected and subsequently cause FSGS-type lesions ⁴ . Other studies which have looked at electron microscopy in FSGS cases have similarly found low numbers of abnormalities that may be consistent with an underlying collagen 4 disorder ^{3, 8} , which suggests the overall number of abnormalities to be found via electron microscopy may be low.

The process by which variants within the COL4A genes might cause FSGS remains unclear, particularly given their clear association with Alport Syndrome and TBMN. One proposal is that the ultrastructural changes induced by the type IV collagen variants, perhaps under the influence of modifier genes such as laminin, result in impaired podocyte attachment to the glomerular basement membrane which leads to accelerated podocyte detachment, subsequent foot process effacement as a response to the increased shear stress induced by the denuded basement membrane and at a critical level of podocyte loss collapse of the capillary network with the appearance of the classical segmental sclerotic lesion ^{2, 4, 19} . It also remains unclear as to whether the changes of FSGS are a secondary process occurring in those with TBMN or whether the type IV collagen variants are capable of causing primary FSGS ^{7, 20} . FSGS occurring as a secondary process to other basement membrane abnormalities may explain why immunosuppressive therapy has traditionally been less effective in inherited forms of FSGS, although there are case reports of the successful use of the calcineurin inhibitor cyclosporine for some patients harbouring inheritable type IV collagen disorders ⁶ .

In summary, this study has found that not all biopsy samples that had primary FSGS as a histological diagnosis were subjected to subsequent electron microscopy. This may have potentially led to inadvertently overlooking characteristic basement membrane abnormalities, which may suggest an underlying and heritable type IV collagen disorder. These findings reflect an opportunity to change practice in order to better investigate, counsel and provide clinical management to these and future patients.