Proteomic Analysis Identifies Distinct Glomerular Extracellular Matrix in Collapsing Focal Segmental Glomerulosclerosis.

BACKGROUND: The mechanisms leading to extracellular matrix (ECM) replacement of areas of glomerular capillaries in histologic variants of FSGS are unknown. This study used proteomics to test the hypothesis that glomerular ECM composition in collapsing FSGS (cFSGS) differs from that of other variants. METHODS: ECM proteins in glomeruli from biopsy specimens of patients with FSGS not otherwise specified (FSGS-NOS) or cFSGS and from normal controls were distinguished and quantified using mass spectrometry, verified and localized using immunohistochemistry (IHC) and confocal microscopy, and assessed for gene expression. The analysis also quantified urinary excretion of ECM proteins and peptides. RESULTS: Of 58 ECM proteins that differed in abundance between cFSGS and FSGS-NOS, 41 were more abundant in cFSGS and 17 in FSGS-NOS. IHC showed that glomerular tuft staining for cathepsin B, cathepsin C, and annexin A3 in cFSGS was significantly greater than in other FSGS variants, in minimal change disease, or in membranous nephropathy. Annexin A3 colocalized with cathepsin B and C, claudin-1, phosphorylated ERK1/2, and CD44, but not with synaptopodin, in parietal epithelial cells (PECs) infiltrating cFSGS glomeruli. Transcripts for cathepsins B and C were increased in FSGS glomeruli compared with normal controls, and urinary excretion of both cathepsins was significantly greater in cFSGS compared with FSGS-NOS. Urinary excretion of ECM-derived peptides was enhanced in cFSGS, although in silico analysis did not identify enhanced excretion of peptides derived from cathepsin B or C. CONCLUSIONS: ECM differences suggest that glomerular sclerosis in cFSGS differs from that in other FSGS variants. Infiltration of activated PECs may disrupt ECM remodeling in cFSGS. These cells and their cathepsins may be therapeutic targets.

Nephrotic Syndrome With Mutations in NPHS2: The Role of R229Q and Implications for Genetic Counseling.

Mutations in the NPHS2 gene, which encodes the podocyte slit diaphragm protein podocin, cause autosomal recessive steroid-resistant nephrotic syndrome (Online Mendelian Inheritance in Man [OMIM] #600995). Basic research and clinical studies have provided important insights about genotype-phenotype correlations. This knowledge allows personalized genetic (risk) counseling and should lead to changes in the advice given to patients. A patient who carries the R229Q variant (which has a high allele frequency of 3.7% in the European population) in combination with a pathogenic variant in exon 7 or 8 is at high risk for developing nephrotic syndrome that may not manifest before adulthood, whereas a patient with 2 pathogenic variants will develop congenital or childhood-onset nephrotic syndrome. In contrast, a patient who carries the R229Q variant in combination with a pathogenic variant in exons 1 to 6 is unlikely to develop nephrotic syndrome. In this article, we review the emerging knowledge about the NPHS2 gene and translate these findings from the bench to practical advice for the clinical bedside.

Increased expression of lysosome membrane protein 2 in glomeruli of patients with idiopathic membranous nephropathy.

Urinary microvesicles constitute a rich source of membrane-bound and intracellular proteins that may provide important clues of pathophysiological mechanisms in renal disease. In the current study, we analyzed and compared the proteome of urinary microvesicles from patients with idiopathic membranous nephropathy (iMN), idiopathic focal segmental glomerulosclerosis (iFSGS), and normal controls using an approach that combined both proteomics and pathology analysis. Lysosome membrane protein-2 (LIMP-2) was increased greater than twofold in urinary microvesicles obtained from patients with iMN compared to microvesicles of patients with iFSGS and normal controls. Immunofluorescence analysis of renal biopsies confirmed our proteomics findings that LIMP-2 was upregulated in glomeruli from patients with iMN but not in glomeruli of diseased patients (iFSGS, minimal change nephropathy, IgA nephropathy, membranoproliferative glomerulonephritis) and normal controls. Confocal laser microscopy showed co-localization of LIMP-2 with IgG along the glomerular basement membrane. Serum antibodies against LIMP-2 could not be detected. In conclusion, our data show the value of urinary microvesicles in biomarker discovery and provide evidence for de novo expression of LIMP-2 in glomeruli of patients with iMN.

Corrigendum: Kidney disease in adults with Prader-Willi syndrome: international cohort study and systematic literature review.

[This corrects the article DOI: 10.3389/fendo.2023.1168648.].

Kidney Disease Associated With Mono-allelic COL4A3 and COL4A4 Variants: A Case Series of 17 Families.

Rationale & Objective: Mono-allelic variants in COL4A3 and COL4A4 (COL4A3/COL4A4) have been identified in a spectrum of glomerular basement membrane nephropathies, including thin basement membrane nephropathy and autosomal dominant Alport syndrome. With the increasing use of next generation sequencing, mono-allelic COL4A3/COL4A4 variants are detected more frequently, but phenotypic heterogeneity impedes counseling. We aimed to investigate the phenotypic spectrum, kidney biopsy results, and segregation patterns of patients with mono-allelic COL4A3/COL4A4 variants identified by whole exome sequencing. Study Design: Case series. Setting & Participants: We evaluated clinical and pathologic characteristics of 17 Dutch index patients with mono-allelic variants in COL4A3/COL4A4 detected by diagnostic whole exome sequencing and 25 affected family members with variants confirmed by Sanger sequencing. Results: Eight different mono-allelic COL4A3/COL4A4 variants were identified across members of 11 families, comprising 7 glycine substituted missense variants and 1 frameshift variant. All index patients had microscopic hematuria at clinical presentation (median age 43 years) and 14 had (micro)albuminuria/proteinuria. All family members showed co-segregation of the variant with at least hematuria. At end of follow-up of all 42 individuals (median age 54 years), 16/42 patients had kidney function impairment, of whom 6 had kidney failure. Reports of kidney biopsies of 14 patients described thin basement membrane nephropathy, focal segmental glomerulosclerosis, minimal change lesions, and Alport syndrome. Electron microscopy images of 7 patients showed a significantly thinner glomerular basement membrane compared with images of patients with idiopathic focal segmental glomerulosclerosis and other hereditary glomerular diseases. No genotype-phenotype correlations could be established. Limitations: Retrospective design, ascertainment bias toward severe kidney phenotypes, and familial hematuria. Conclusions: This study confirms the wide phenotypic spectrum associated with mono-allelic COL4A3/COL4A4 variants, extending from isolated microscopic hematuria to kidney failure with high intra- and interfamilial variability.

Kidney disease in adults with Prader-Willi syndrome: international cohort study and systematic literature review.

Background: Prader-Willi syndrome (PWS) is a rare, complex, genetic disorder characterized by hyperphagia, hypotonia, delayed psychomotor development, low muscle mass and hypothalamic dysfunction. Adults with PWS often have obesity, hypertension and type 2 diabetes mellitus (DM2), known risk factors for cardiovascular disease (CVD) and chronic kidney disease (CKD). Early symptoms of CVD and CKD may be masked by intellectual disability and inability to express physical complaints. Furthermore, kidney diseases are often asymptomatic. Therefore, renal and cardiovascular disease might be missed in patients with PWS. Microalbuminuria is an early sign of microvascular damage in the kidneys and other vascular beds. Therefore, we screened our adult PWS cohort for the presence of elevated urinary albumin and (micro)albuminuria. Methods: We retrospectively collected anthropometric measurements, blood pressure, medical history, medication use, urine dipstick and biochemical measurements form electronic patient files. In addition, we performed a systematic literature review on kidney disease in PWS. Results: We included 162 adults with genetically confirmed PWS (56% male, median age 28 years), of whom 44 (27%) had DM2. None had known CVD. All subjects had normal estimated glomerular filtration rate (eGFR) according to non-PWS reference intervals. Elevated urinary albumin or (micro)albuminuria was present in 28 (18%); 19 out of 75 (25%) had an increased urinary albumin-to-creatinine ratio (UACR) and 10 out of 57 (18%) had an increased urinary protein-to-creatinine ratio. Elevated urinary albumin was present at a young age (median age 26 (IQR 24-32) years) and was associated with an significantly higher BMI and LDL-cholesterol levels and higher prevalence of DM2, hypertension and dyslipidemia than those with normal UACR (p=0.027, p=0.019, p<0.001, p<0.001, p=0.011 and respectively). Conclusion: Upon screening, one in every five adults with PWS had increased urinary albumin or (micro)albuminuria, early signs of microvascular disease. All had normal eGFR, according to non-PWS reference intervals, and none had a formal diagnosis of CVD. As muscle mass is low in PWS, creatinine levels and eGFR may be spuriously normal. Urinalysis in this patient group can be used as a screening tool for microvascular (kidney) disease. We propose an algorithm for the detection and management of microvascular disease in adults with PWS.

Genetic causes of focal segmental glomerulosclerosis: implications for clinical practice.

Focal segmental glomerulosclerosis (FSGS) is a common cause of steroid-resistant nephrotic syndrome in children and adults. Although FSGS is considered a podocyte disease, the aetiology is diverse. In recent years, many inheritable genetic forms of FSGS have been described, caused by mutations in proteins that are important for podocyte function. In the present commentary, we review these genetic causes of FSGS and describe their prevalence in familial and sporadic FSGS. In routine clinical practice, the decision to perform the costly DNA analysis should be based on the assessment if the results affect the care of the individual patient with respect to the evaluation of extra-renal manifestations, treatment decisions, transplantation and genetic counselling.

Later Response to Corticosteroids in Adults With Primary Focal Segmental Glomerular Sclerosis Is Associated With Favorable Outcomes.

INTRODUCTION: Guidelines advise initial therapy with corticosteroids (CSs) in patients with presumed primary focal segmental glomerular sclerosis (pFSGS). Many patients do not achieve complete remission (CR) after 8 or 16 weeks. Although these patients are considered steroid resistant, clinical outcomes are ill defined. METHODS: A retrospective cohort study of patients with pFSGS who were referred between January 1995 and December 2014. Data of clinical presentation until last follow-up were collected from patient records. RESULTS: A total of 51 patients (median age 47 years, 20 female/31 male) were included (median follow-up 7.1 years). There were 10 patients who achieved partial response (PR) at 8 weeks. High-dose CS monotherapy was continued for a median of 17 weeks (interquartile range [IQR] 11-21 weeks) (total duration 56 weeks [IQR 28-83 weeks]). With CSs, the cumulative incidence of CR + PR was 18% and 35%, respectively. Of 24 patients with persistent nephrotic-range proteinuria, 22 received additional immunosuppressive (IS) therapy, resulting in CR in 3 (14%) and PR in 11 patients (50%). A decrease of >20% of proteinuria at 8 weeks predicted response. In addition, 8 patients (36%) were considered primary nonresponders. A genetic cause was found in 2 patients. Proteinuria at end of follow-up was 1.2 g (IQR 0.4-3.0 g/24 hours or g/10 mmol creatinine). Renal survival at 3, 5, and 10 years was 92%, 87%, and 64%, respectively. CONCLUSION: Patients with presumed pFSGS often respond late to IS therapy. A decrease in proteinuria of >20% after 8 weeks of therapy is a predictor of responsiveness. Regardless of CR in some patients, improved biomarkers are needed to predict response/outcomes in patients with pFSGS.

Comparison of three methods for isolation of urinary microvesicles to identify biomarkers of nephrotic syndrome.

Urinary microvesicles, such as 40-100 nm exosomes and 100-1000 nm microparticles, contain many proteins that may serve as biomarkers of renal disease. Microvesicles have been isolated by ultracentrifugation or nanomembrane ultrafiltration from normal urine; however, little is known about the efficiency of these methods in isolating microvesicles from patients with nephrotic-range proteinuria. Here we compared three techniques to isolate microvesicles from nephrotic urine: nanomembrane ultrafiltration, ultracentrifugation, and ultracentrifugation followed by size-exclusion chromatography (UC-SEC). Highly abundant urinary proteins were still present in sufficient quantity after ultrafiltration or ultracentrifugation to blunt detection of less abundant microvesicular proteins by MALDI-TOF-TOF mass spectrometry. The microvesicular markers neprilysin, aquaporin-2, and podocalyxin were highly enriched following UC-SEC compared with preparations by ultrafiltration or ultracentrifugation alone. Electron microscopy of the UC-SEC fractions found microvesicles of varying size, compatible with the presence of both exosomes and microparticles. Thus, UC-SEC following ultracentrifugation to further enrich and purify microparticles facilitates the search for prognostic biomarkers that might be used to predict the clinical course of nephrotic syndrome.

NMR and MS urinary metabolic phenotyping in kidney diseases is fit-for-purpose in the presence of a protease inhibitor.

Nephrotic syndrome with idiopathic membranous nephropathy as a major contributor, is characterized by proteinuria, hypoalbuminemia and oedema. Diagnosis is based on renal biopsy and the condition is treated using immunosuppressive drugs; however nephrotic syndrome treatment efficacy varies among patients. Multi-omic urine analyses can discover new markers of nephrotic syndrome that can be used to develop personalized treatments. For proteomics, a protease inhibitor (PI) is sometimes added at sample collection to conserve proteins but its impact on urine metabolic phenotyping needs to be evaluated. Urine from controls (n = 4) and idiopathic membranous nephropathy (iMN) patients (n = 6) were collected with and without PI addition and analysed using 1H NMR spectroscopy and UPLC-MS. PI-related data features were observed in the 1H NMR spectra but their removal followed by a median fold change normalisation, eliminated the PI contribution. PI-related metabolites in UPLC-MS data had limited effect on metabolic patterns specific to iMN. When using an appropriate data processing pipeline, PI-containing urine samples are appropriate for 1H NMR and MS metabolic profiling of patients with nephrotic syndrome.

Isolation and characterization of urinary extracellular vesicles: implications for biomarker discovery.

Urine is a valuable diagnostic medium and, with the discovery of urinary extracellular vesicles, is viewed as a dynamic bioactive fluid. Extracellular vesicles are lipid-enclosed structures that can be classified into three categories: exosomes, microvesicles (or ectosomes) and apoptotic bodies. This classification is based on the mechanisms by which membrane vesicles are formed: fusion of multivesicular bodies with the plasma membranes (exosomes), budding of vesicles directly from the plasma membrane (microvesicles) or those shed from dying cells (apoptotic bodies). During their formation, urinary extracellular vesicles incorporate various cell-specific components (proteins, lipids and nucleic acids) that can be transferred to target cells. The rigour needed for comparative studies has fueled the search for optimal approaches for their isolation, purification, and characterization. RNA, the newest extracellular vesicle component to be discovered, has received substantial attention as an extracellular vesicle therapeutic, and compelling evidence suggests that ex vivo manipulation of microRNA composition may have uses in the treatment of kidney disorders. The results of these studies are building the case that urinary extracellular vesicles act as mediators of renal pathophysiology. As the field of extracellular vesicle studies is burgeoning, this Review focuses on primary data obtained from studies of human urine rather than on data from studies of laboratory animals or cultured immortalized cells.

B cell suppression in primary glomerular disease.

Membranous nephropathy, focal segmental glomerulosclerosis (FSGS), and minimal change disease (MCD) are the most common causes of idiopathic nephrotic syndrome. For many years prednisone, alkylating agents, and calcineurin inhibitors have been the standard of therapy for these patients. More effective or better tolerated treatment modalities are needed. B cell targeted therapy was recently introduced in clinical practice. In this review, we briefly summarize the current standard therapy and discuss the efficacy of B cell targeted therapy in primary glomerular diseases. Observational, short-term studies suggest that rituximab is effective and comparable to standard therapy in maintaining remissions in patients with frequently relapsing or steroid-dependent MCD or FSGS. In contrast, response is limited in patients with steroid-resistant nephrotic syndrome. Rituximab also induces remissions in patients with membranous nephropathy. Controlled clinical trials on kidney endpoints are urgently needed to position B cell targeted therapy in clinical practice.

Microfiltration isolation of human urinary exosomes for characterization by MS.

PURPOSE: The purpose of this study was to address the hypothesis that small vesicular urinary particles known as exosomes could be selectively microfiltered using low protein-binding size exclusion filters, thereby simplifying their use in clinical biomarker discovery studies. EXPERIMENTAL DESIGN: We characterized a microfiltration approach using a low protein binding, hydrophilized polyvinylidene difluoride membrane to easily and efficiently isolate urinary exosomes from fresh, room temperature or 4°C urine, with a simultaneous depletion of abundant urinary proteins. Using LC-MS, immunoblot analysis, and electron microscopy methods, we demonstrate this method to isolate intact exosomes and thereby enrich for a low abundant urinary proteome. RESULTS: In comparison to other standard methods of exosome isolation including ultracentrifugation and nanofiltration, we demonstrate equivalent enrichment of the exosome proteome with reduced co-purification of abundant urinary proteins. CONCLUSION AND CLINICAL RELEVANCE: In conclusion, we demonstrate a microfiltration isolation method that preserves the exosome structure, reduces contamination from higher abundant urinary proteins, and can be easily implemented into mass spectrometry analysis for biomarker discovery efforts or incorporation into routine clinical laboratory applications to yield higher sample throughput.