Elevated Levels of Urinary Extracellular Vesicle Fibroblast-Specific Protein 1 in Patients with Active Crescentic Glomerulonephritis

Abstract

Background/Aims: Extracellular vesicles (EVs), including exosomes, are present in various bodily fluids, including urine. We and others previously reported that cells expressing fibroblast-specific protein 1 (FSP1) accumulate within damaged glomeruli, and that urinary FSP1, as well as urinary soluble CD163, could potentially serve as a biomarker of ongoing glomerular injury. Methods: To test that idea, we collected urine samples from 37 patients with glomerular disease; purified the urinary EVs; characterized them using Nanosight, western blotting, and immunoelectron microscopy; and determined FSP1 and soluble CD163 levels using enzyme-linked immunosorbent assays. Results: Deemed to be mainly exosomes based on their size distribution, the EVs in urine contained FSP1, and a portion of the FSP1-positive vesicles was also positive for podocalyxin. FSP1 levels in urinary EVs were (1) positively correlated with rates of biopsy-proven cellular crescent formation (r = 0.562, p < 0.001) and total crescent formation (r = 0.448, p = 0.005) among total glomeruli; (2) significantly higher in patients with cellular crescents affecting 20% or more of their glomeruli than in those with fewer affected glomeruli (p = 0.003); and (3) significantly decreased after glucocorticoid and immunosuppressant therapy (p < 0.05). A positive correlation between FSP1 levels in urinary EVs and urinary soluble CD163 levels was confirmed (r = 0.367, p < 0.05). Conclusion: These data suggest that a portion of urinary FSP1 is secreted as EVs originating from podocytes, and that FSP1 levels reflect active and ongoing glomerular injury and disease activity, such as cellular crescent formation.

Urinary extracellular vesicles: A position paper by the Urine Task Force of the International Society for Extracellular Vesicles

“…EV‐based biomarkers in urine are currently investigated for an array of malignancies and other diseases such as polycystic kidney disease (Raimondo et al., 2016; Salih et al., 2016), cystinuria (Bourderioux et al., 2015), diabetes (Abe et al., 2018; Lytvyn et al., 2017; Zubiri et al., 2014), renal ischemia‐reperfusion injury (Sonoda et al., 2009), glomerulonephritis (Morikawa et al., 2018), renal interstitial fibrosis (Carreras‐Planella et al., 2020; Chun‐Yan et al., 2018), hypertension or lupus nephritis (Tangtanatakul et al., 2018) and in calcineurin inhibitor‐induced nephrotoxicity (Carreras‐Planella et al., 2020). However, many of the identified candidate biomarkers have not yet been validated in large independent cohorts or tested in more than one laboratory.…”

“…These results were the foundation for a prostate cancer diagnostic test that has been extensively validated in two prospective multi‐center US studies (McKiernan et al., 2016; McKiernan et al., 2018). Altogether, these promising results inspired the search for uEV‐based biomarkers for other urogenital tract pathologies such as polycystic kidney disease, cystinuria, diabetic nephropathy, acute kidney injury/ renal ischemia‐reperfusion injury, glomerulonephritis, renal interstitial fibrosis/ chronic kidney disease, lupus nephritis, nephronophthisis‐related ciliopathies, tubulopathies and primary and secondary hypertension (Abe et al., 2018; Bourderioux et al., 2015; Chun‐Yan et al., 2018; Corbetta et al., 2015; Gonzalez‐Calero et al., 2017; Kwon et al., 2017; La Salvia et al., 2020; Morikawa et al., 2018; Qi et al., 2016; Raimondo et al., 2016; Raimondo et al., 2020; Salih et al., 2016; Salih et al., 2018; Sonoda et al., 2009; Stokman et al., 2019; Tangtanatakul et al., 2018; van der Lubbe et al., 2012; Williams et al., 2020; Wolley et al., 2017; Zubiri et al., 2014). Many of the newly identified candidate biomarkers have not yet been validated in large independent cohorts or in additional laboratories, but nevertheless these examples highlight the enormous potential for uEV analyses as readouts for pathophysiological alterations within the urogenital and other systems.…”

Exosomes in Nephropathies: A Rich Source of Novel Biomarkers

“…Crescent formation, the hallmark of RPGN, has been associated with the presence of fibroblast-specific protein 1 (FSP1), a cytosolic protein expressed by increased number of renal cells in kidneys exhibiting ongoing injury [ 69 ]. In idiopathic membranous nephropathy, upregulation of blood and urinary MUC3A circular RNA (circRNA) and various small nucleolar RNAs (snoRNAs) such as SNORA51, SNORA31, SNORA70, SNORA75, and SNORD112 has been reported [ 70 ].…”

Roles for Exosome in Various Kidney Diseases and Disorders

“…In another study, level of fibroblast-specific protein 1 (FSP1) has been shown to increase in extracellular vesicles (including exosomes) derived from patients with active crescentic glomerulonephritis (Morikawa et al, 2019). Interestingly, its level was decreased after immunosuppressive therapy (Morikawa et al, 2019).…”

“…For other glomerular diseases, a previous study using a proteomics approach has shown that urinary exosomal aminopeptidase N, vasorin precursor, a-1-antitrypsin, and ceruloplasmin could differentiate immunoglobulin A (IgA) nephropathy from thin basement membrane nephropathy and healthy controls (Moon et al, 2011). In another study, level of fibroblast-specific protein 1 (FSP1) has been shown to increase in extracellular vesicles (including exosomes) derived from patients with active crescentic glomerulonephritis (Morikawa et al, 2019). Interestingly, its level was decreased after immunosuppressive therapy (Morikawa et al, 2019).…”