Ultrastructural Alterations of the Glomerular Filtration Barrier in Fish Experimentally Exposed to Perfluorooctanoic Acid

The architecture of the glomerular filtration barrier of unexposed carp agrees with previous studies in eucoelomates and vertebrates, in general, and in European carp, in particular [1,55,56].

Perfluorooctanoic acid exposure was shown to affect the glomerular filtration barrier in all three of its main components (fenestrated endothelium, basement membrane, and podocyte) in carp experimentally exposed to 2 mg L⁻¹, promoting the outflow of proteinaceous material in the urinary space, hence confirming the glomerular origin of the protein leakage reported in a previous study on the same fish [22]. The pathophysiology of proteinuria (urine protein leakage) continues to intrigue researchers, focusing on the glomerular filtration barrier and/or proximal tubule integrity. With regard to the glomerular filtration barrier, all the constitutive components may be altered to a various degree and through possible reciprocal association due to the cross-talk between the main cellular components: fenestrated endothelial cells and podocytes [5,7,57,58,59,60]. As the first component of the glomerular filtration barrier, the glycocalyx is known to function as a negatively charged ion sieve, making it particularly efficient to prevent/reduce albumin leakage through the glomerular filtration barrier. As a consequence, its alteration is considered to be an incipient cause of proteinuria [4,5,57,61]. Unfortunately, glycocalyx requires dedicated processing/staining techniques to ensure its best evaluation under transmission electron microscopy [62,63], and because specimens were processed routinely for the present study, it was not possible to rule out possible alterations of glycocalyx related to PFOA exposure and its possible contribution to proteinuria. More recently, transcytosis by both the fenestrated endothelial cells and the podocytes has been proposed to be involved in the pathogenesis of albuminuria (urine albumin leakage), also without glycocalyx alteration, overtaking the conventional theory of “impairment of the size- and/or charge-selective filtration barrier” [60,64,65]. As a consequence, the possible role of glycocalyx and transcytosis should be addressed in further studies, with particular regard to the lowest, environmentally relevant PFOA concentration (200 ng L⁻¹), where no ultrastructural evidence of alteration of the glomerular filtration barrier was noted. Referring to the highest tested PFOA concentration (2 mg L⁻¹), the reported architecture disarrangement agrees with the known morphopathological evidences of glomerulonephrosis (i.e., the morphofunctional alteration of the glomerular filtration barrier), with particular regard to podocyte effacement [9,11,48,58,66,67]. Though PFOA and other PFAS can accumulate at high concentrations in the kidney and potentially affect renal function, no previous research has specifically addressed the effect of these pollutants on the glomerular filtration barrier [14,15,16,17,18,19,20,21,22,23,24], making it difficult to interpret and compare the observed alterations and speculate about the possible underlying pathophysiology. Interestingly, none of the lesions seen in other anatomical districts (namely mitochondrial cristolysis, vesiculation, swelling, and ballooning, autophagosomes occurrence, rough endoplasmic reticulum degranulation, disarrangement and enlargement in hepatocytes [54]; increased number and volume of cytoplasm vesiculations in cells of the first proximal tubular segment, mitochondrial focal vesiculation in cells of the distal tubular segment and of the collecting ducts in kidney [22]; rough endoplasmic reticulum enlargement and fragmentation, cytoplasm vacuolation, enhanced phagolysosomes formation in thyroid follicles [45]) were appreciated at the level of glomerular filtration barrier, suggesting a somewhat different pathogenesis compared to the previous tissues. Given the ultrastructural alterations documented during the present study, the plasma membrane, cytoskeleton, and adhesion molecules should be considered as possible targets of PFOA-induced damages. Indeed, PFOA and other PFAS have been shown to alter plasma membrane potential and to acidify cytosol in a human colon carcinoma HCT116 cell model due to their amphipatic structure, suggesting these alterations may precede reactive oxygen species production and mitochondrial transmembrane potential impairment [68]. Moreover, membrane potential dysregulation and alteration of the organization of membrane microdomains have been reported in boar spermatozoa experimentally exposed to PFOA and perfluorooctane sulfonate (PFOS) [69]. Recently, exposure of human HepaRG hepatoma cells to PFOA and PFOS has resulted in altered bile canalicular structure and bile flow impairment, caused by actin cytoskeleton disarrangement and to structural redistribution of the tight-junctional protein ZO-1 [70]. More information on the effects of PFOA and PFOS on F-actin, actin binding proteins, and adhesion molecules is available in Wang et al. [71]. Actin filament remodeling and an increase in endothelial permeability have been reported in human microvascular endothelial cells exposed to PFOS as a consequence of reactive oxygen species production [72]. The integrity of the plasma membrane, cytoskeleton, and adhesion molecules is of paramount importance in the maintenance of a proper and functional structure of the glomerular filtration barrier [5,10,11,61,73,74]. As a consequence, further studies should specifically address the topic with particular regard to the effect of PFOA and other PFAS on the fenestrated endothelium and on podocytes, both affected during the present study.

In the carp of the present study, the highest PFOA concentration was found in blood [51], the same as in other studies on fish and other vertebrates, though differences may arise according to the route of contamination, dosage/concentration, duration of exposure, and other biological parameters [18,20,23]. Worth noting is that PFAS bind to albumin and other blood proteins [23,75,76], so following the albumin route across the glomerular filtration barrier may contribute to shedding light on the underlying pathogenesis. Irrespective of how albumin can transit across the endothelial (e.g., transcytosis) and basement membrane barriers, it can be internalized by podocytes, partially bypassing the filtration slit diaphragm through transcytosis [77], allowing PFOA to enter the cells that are critical for maintenance of the morphofunctional integrity of the glomerular filtration barrier. Moreover, in humans, PFOA is known to be filtered freely in the glomerulus, actively excreted in the proximal tubule, and then readsorbed by organic anion transport (OAT) peptides [78,79]. Interestingly, in the mouse, kidney OAT-like peptides have been described in blood vessels, parietal epithelial cells, podocytes, distal convoluted tubules, connecting tubules, and collecting tubules [80]. The role of small molecule membrane transporters found in the mammalian podocyte in glomerular pathogenesis and as a possible therapeutic target has been discussed by Zennaro et al. (2014) [81]. OAT peptides are phylogenetically conserved and present in zebrafish, where marked variation according to tissue and sex has been reported [82,83]. As previously stressed by Manera et al. (2022), and differently from humans, where nephron segments are known to be differentially affected by toxicants, according to the implied transporter and its possible sex- and genetic-based modifications, there is a generalized lack of knowledge about nephron topographic toxicologic pathology for fish [22].

It is worth mentioning that pathology, as a discipline, relies on lesions to formulate a diagnosis, a lesion being the morphological evidence (at any integration level) of a disrupted function [88], providing precious information to elucidate the underlying pathogenesis. Furthermore, current nephropathological diagnostic guidelines, with particular regard to the glomerular filtration barrier, rely on qualitative detection and evaluation of codified ultrastructural alterations [11,48,49,50]. Nevertheless, and in spite of the paramount importance of electron microscopy to assess toxicity [89], further targeted studies are needed to elucidate at best the likely pathogenesis of PFOA at the glomerular filtration membrane level.