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.

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.

The soluble urokinase receptor is not a clinical marker for focal segmental glomerulosclerosis.

The soluble urokinase receptor (suPAR) promotes proteinuria and induces focal segmental glomerulosclerosis (FSGS)-like lesions in mice. A serum suPAR concentration cutoff of 3000 pg/ml has been proposed as a clinical biomarker for patients with FSGS. Interestingly, several studies in patients with glomerulopathy found an inverse correlation between the estimated glomerular filtration rate (eGFR) and suPAR. As patients with FSGS present at different eGFRs, we studied the relationship between eGFR and suPAR in a cohort of 476 non-FSGS patients and 54 patients with biopsy-proven idiopathic FSGS. In the non-FSGS patients, eGFR was the strongest significant determinant of suPAR. The proposed cutoff for suPAR in FSGS patients was exceeded in 17%, 39%, and 88% in patients with eGFRs of more than 60, 45-60, and 30-45 ml/min per 1.73 m(2), respectively. In patients with eGFR of <30 ml/min per 1.73 m(2), suPAR exceeded the cutoff in 95% of patients. Levels of suPAR in patients with idiopathic FSGS overlapped with non-FSGS controls and for any given eGFR did not discriminate FSGS cases from non-FSGS controls. In the overall cohort, there was a negative association between idiopathic FSGS and suPAR, and idiopathic FSGS was not an independent predictor of FSGS concentration over 3000 pg/ml. Thus, this study does not support an absolute, eGFR-independent, suPAR concentration cutoff as a biomarker for underlying FSGS pathology and questions the validity of relative, eGFR-dependent suPAR cutoff values.

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.

Pathological variants of focal segmental glomerulosclerosis in an adult Dutch population--epidemiology and outcome.

BACKGROUND: A working group has defined five subtypes of focal segmental glomerulosclerosis (FSGS) based on light microscopic assessment (Columbia classification). Limited information is available on the prognostic and therapeutic implications of this classification in a European population. We conducted a retrospective analysis in 93 adult patients with biopsy-proven FSGS to determine the clinical features and outcome of FSGS variants. METHODS: Renal biopsy specimens of adult patients (>16 years) diagnosed with FSGS between 1980 and 2003 were reviewed according to the Columbia classification without the knowledge of clinical outcome. The medical records were reviewed for clinical data. Primary outcomes were remission rate and renal survival. RESULTS: The frequencies of the FSGS variants were: 32% NOS (FSGS not otherwise specified), 37% tip, 26% perihilar and 5% collapsing. Cellular FSGS was not found in the biopsies. The nephrotic syndrome was less frequent in FSGS NOS (57%) and perihilar FSGS (25%) compared to the tip variant (97%). Renal function was significantly better in patients with the tip variant compared to FSGS NOS (P<0.05). Glomerular sclerosis and hyalinosis was most severe in patients with perihilar FSGS, intermediate in FSGS NOS and the least severe in patients with the tip variant. Patients with perihilar FSGS were less likely to receive immunosuppressive medication. Renal survival at 5 years was significantly better for patients with the tip variant (78% for tip vs 63% and 55% for FSGS NOS and perihilar FSGS; P=0.02). Type of FSGS and serum creatinine concentration were independent predictors of renal survival. Remission rate was higher in patients with the tip variant (P=0.1). CONCLUSION: The collapsing variant was rare in our population. Renal survival and remission rates were higher in patients with the tip variant. Use of the classification scheme for FSGS may be clinically useful.

Membranous nephropathy in the older adult: epidemiology, diagnosis and management.

Membranous nephropathy is the most important cause of the nephrotic syndrome in elderly patients (aged >65 years). The clinical presentation is similar in older and younger patients, although elderly patients more often present with renal failure. Notably, glomerular filtration rate (GFR) is usually lower in the elderly due to the physiological decline in GFR after the age of 30 years. Secondary causes, especially malignancies, are more common in older patients with membranous nephropathy. Therefore, elderly patients should undergo a thorough examination to exclude a secondary cause. The prognosis of elderly patients with idiopathic membranous nephropathy is not very different from that of younger patients. All elderly patients should receive symptomatic treatment aimed at reducing hypertension, oedema, proteinuria and hyperlipidaemia. It is recommended that elderly patients with a low serum albumin (<2 g/dL) receive prophylactic anticoagulation because of a high risk for thrombosis. Immunosuppressive therapy should be reserved for elderly patients at high risk of progression to end-stage renal disease because the elderly are particularly prone to the adverse effects and infectious complications of immunosuppressive therapy. High-risk elderly patients are characterised by renal insufficiency (GFR <45 mL/min/1.73m(2)), an increase in serum creatinine of >25% or a severe persistent nephrotic syndrome not responding to symptomatic treatment. In addition, elderly patients with a relatively normal GFR (>or=45 mL/min/1.73m(2)) and high urinary excretion of beta(2)-microglobulin and IgG are also at increased risk of developing end-stage renal disease; however, the deterioration in renal function is usually a slow process. Therefore, such patients benefit from immunosuppressive therapy only if their life expectancy is good. If immunosuppressive therapy is started, first-line treatment consists of prednisone and cyclophosphamide. If cyclophosphamide is contraindicated or fails to induce a remission, ciclosporin could be used. Treatment with ciclosporin should be limited to patients with a relatively normal renal function (GFR >60 mL/min/1.73m(2)) in view of its nephrotoxicity in patients with renal dysfunction.

Focal segmental glomerulosclerosis is not a sufficient predictor of renal outcome in patients with membranous nephropathy.

BACKGROUND: The course of idiopathic membranous nephropathy (iMN) is variable in untreated patients. Accurate prediction of renal outcome would allow optimal treatment decisions. We demonstrated that urinary beta2-microglobulin (beta2M) predicted prognosis in iMN with high sensitivity and specificity. It has been suggested that focal segmental glomerulosclerosis (FSGS) is a discriminative parameter with independent prognostic value. METHODS: We selected patients with iMN biopsied between 1988 and 2002. Biopsies were analysed for the presence of FSGS, interstitial fibrosis and vascular lesions. Serum creatinine, creatinine clearance, proteinuria and blood pressure were recorded at baseline. Outcome variables included remission of proteinuria, renal death (RD) defined as serum creatinine >135 micromol/l or increase of serum creatinine of >50%, or end-stage renal disease (ESRD). In a subgroup of patients, urinary beta2-microglobulin (beta2M) was measured. RESULTS: We included 53 patients (33M, 20F). Mean age was 51 years, serum creatinine 99 micromol/l, and proteinuria 7.0 g/10 mmol creatinine. FSGS was present in 22 patients. These patients were characterized by a higher serum creatinine at time of biopsy (P = 0.035), more severe interstitial fibrosis (P = 0.001) and higher stage of membranous nephropathy (P = 0.001). During follow-up 24 patients developed RD, almost equally distributed between patients with and without FSGS. Renal survival was numerically, but not significantly, lower in patients with FSGS. In Cox proportional hazard analysis, only serum creatinine at the time of biopsy was an independent predictor of RD or ESRD (P < 0.001). In patients with known urinary beta2M, there was no significant correlation with FSGS score (P = 0.174). CONCLUSION: FSGS is not an accurate prognostic marker in iMN. Histological scoring of FSGS is inferior to measurement of urinary proteins in predicting renal outcome in iMN.