Synuclein alpha accumulation mediates podocyte injury in Fabry nephropathy

Current therapies for Fabry disease are based on reversing intra-cellular accumulation of globotriaosylceramide (Gb3) by enzyme replacement therapy (ERT) or chaperone-mediated stabilization of the defective enzyme, thereby alleviating lysosome dysfunction. However, their effect in the reversal of end-organ damage, like kidney injury and chronic kidney disease remains unclear. First, ultrastructural analysis of serial human kidney biopsies showed that long-term use of ERT reduced Gb3 accumulation in podocytes but did not reverse podocyte injury. Then, a CRISPR/CAS9-mediated alpha-Galactosidase knockout podocyte cell line confirmed ERT-mediated reversal of Gb3 accumulation without resolution of lysosomal dysfunction. Transcriptome-based connectivity mapping and SILAC-based quantitative proteomics identified alpha-synuclein (SNCA) accumulation as a key event mediating podocyte injury. Genetic and pharmacological inhibition of SNCA improved lysosomal structure and function in Fabry podocytes, exceeding the benefits of ERT. Together, this work reconceptualizes Fabry-associated cell injury beyond Gb3 accumulation, and introduces SNCA modulation as a potential intervention, especially for patients with Fabry nephropathy.

Fabry Nephropathy in a Young Female Patient Presenting with Only Urinary Mulberry Bodies Treated with Chaperone Therapy

Fabry disease (FD) is an X-linked disorder of the sphingolipid metabolism, caused by deficiency or decreased activity of α-galactosidase A. We report a rare case of Fabry nephropathy (FN) in a 21-year-old Japanese female patient presenting with only urinary mulberry bodies; she was treated with pharmacological chaperone therapy (PCT) after renal biopsy. The patient underwent a detailed examination because her mother was diagnosed with FD in the Division of Community Medicine of our hospital. She did not have renal dysfunction or proteinuria, and only mulberry bodies were detected in the urine. The activity of α-galactosidase A was low, and genetic analysis revealed the R301Q mutation. A percutaneous renal biopsy was performed, and the findings revealed enlargement and vacuolation of glomerular podocytes by light microscopy, and myelin and zebra bodies were detected in podocytes by electron microscopy. She was diagnosed with FN by renal biopsy and gene analysis. PCT was selected as the treatment to prevent cardiac events and renal dysfunction. The present case suggests that renal biopsy may be necessary even for young women with only mulberry bodies for the diagnosis of FN. It could be useful to evaluate the effect of treatment using the counts of mulberry bodies in the urine. In addition, due to its oral administration, PCT may be suitable for patients who are unable to visit the hospital frequently.

Urinary Extracellular Vesicles and Their miRNA Cargo in Patients with Fabry Nephropathy

Current biomarkers of Fabry nephropathy lack sensitivity in detecting early kidney damage and do not predict progression of nephropathy. Urinary extracellular vesicles (uEVs) and their molecular cargo could reflect early changes in renal impairment as they are secreted by the cells lining the urinary tract. We aimed to conduct a proof-of-concept study to investigate whether analysis of uEV characteristics and expression of uEV-derived microRNAs (miRNAs) could be applicable in studies to predict the development and progression of nephropathy in Fabry disease. A total of 20 Fabry patients were divided into two groups, depending on the presence of nephropathy. Chronological urine samples collected during 10-year follow-up were used for uEVs isolation with size exclusion chromatography. Nanoparticle tracking analysis was used to determine concentration and size of uEVs. We evaluated the expression of five uEV-derived miRNAs by qPCR (miR-23a-3p, miR-29a-3p, miR-30b-5p, miR-34a-5p, miR-200a-3p). There was no difference in the concentration and size of uEVs between patients with and without nephropathy at last follow-up or longitudinally. However, we found increased expression of miR-29a-3p and miR-200a-3p in uEVs isolated from chronological samples of patients with Fabry nephropathy. This may indicate an attempt by the organism to prevent the progression of renal damage leading to end-stage renal disease as previously reported in type 1 diabetes. In addition, we found an increased expression of miR-30b-5p in the 10-year period in uEVs of patients without renal dysfunction. miR-30b-5 was reported to have a protective role in podocyte injury and may possibly be important in Fabry nephropathy. These findings indicate that uEVs and their molecular cargo could be a promising target of studies focusing on elucidation of Fabry nephropathy. Nevertheless, total concentration and size of uEVs were neither indicative of the presence nor progression of Fabry nephropathy, while the role of the analyzed miRNAs in Fabry nephropathy progression was merely indicated and needs further in-depth studies.

MO127CLEARED PODOCYTES AND NORMAL KIDNEY FUNCTION IN CLASSICAL FABRY MALES 15 YEARS AFTER START OF ENZYME REPLACEMENT THERAPY AT YOUNG AGE*

Background and Aims Fabry nephropathy may progress to kidney failure despite enzyme replacement therapy (ERT) when the treatment is initiated at a relatively late stage of the disease. This study evaluates long term effects of agalsidase in serial kidney biopsies and functional measurements in men with classical Fabry disease that commenced ERT at a young age. Method Six male Fabry patients with a median age of 20 years (range 7-30 years) at start of ERT were monitored over a median time of 15 years (range 14.5-17.0 years). The patients were treated with an agalsidase dose of 1.0 mg/kg/every other weak (eow) for 8.3 years (range 5-12 years) and with an agalsidase dose of 0.2-0.5 mg/kg/eow for 7.6 years (range 3-10 years). Kidney biopsies were evaluated with the scoring system of the International Study Group of Fabry Nephropathy (ISGFN) using both plastic embedded and paraffin embedded tissue with scoring of podocyte globotriaocylceramide (Gb3) in semithin toluidine blue sections (maximum score 4.0) and scoring of vacuolization in PAS-sections (maximum score 3.0). ISGFN composite score consists of both tissue scores giving a maximum score of 7.0. Renal function was evaluated with measurement of glomerular filtration rate (GFR) with iohexol clearance (mGFR) and urinary albumin creatinine ratio (uACR). Values are given in median (range). Results Kidney biopsies at baseline contained 24 (13-42) evaluable glomeruli and had an ISGFN composite score of 7.0 (6.9-7.0). After 15 years the ISGFN composite score had decreased to 0.56 (0-4.29), scored in 24 (9-52) evaluable glomeruli. At baseline mGFR was 106 (86-113) ml/min/1.73 m2 and after 15 years mGFR decreased to 97 (73-134) ml/min/1.73 m2. uACR was 5.6 (0.1-13.6) mg/mmol at baseline and had after 15 years increased to 10.0 (1.0-107) mg/mmol. The youngest patient had significant proteinuria with uACR 107 mg/mmol due to a chronic C3-glomerulonephritis superimposed on Fabry disease. Median uACR without this patient was at 15 years 5.8 (1.0-17.7) mg/mmol. The two youngest patients had no Gb3 visible (ISGFN composite score of 0) in their last biopsy and mGFR was normal in both. In all patients the reduction of podocyte-Gb3 was higher on an agalsidase dose of 1 mg/kg/eow; change composite score -4.66 (-7.9 to -1.8), compared to on an agalsidase dose of 0.2-0.5 mg/kg/eow; change composite score -0.15 (-5.1 to + 1.0). Conclusion Initiation of ERT at a relatively young age may clear the long living kidney cells of Gb3 and protect the kidneys from significant functional loss over a very long time period. The reduction of Gb3 in podocytes is higher on high dose compared to low dose of agalsidase.

A Case Study of Fabry Nephropathy and Its Progression: The First Reported Case in Malaysia

IntroductionFabry disease (FD) is a rare metabolic disorder caused by the genetic deficiency of the lysosomal hydrolase alpha-galactosidase A, causing common and serious kidney complications. We report the first case of Fabry disease detected in Malaysia. Clinical ScenarioA 35-year-old man had an incidental finding of proteinuria during routine health screening. His symptoms included frothy urine and intermittent lower limb swelling. Renal biopsy showed features of Fabry’s disease which was confirmed with genetic testing. He had classical hemizygous Fabry disease of c.610 C>T with Fabry nephropathy, cardiomyopathy, and cornea verticillata. He was then started on be Enzyme Replacement Therapy (ERT) with agalsidase till date. ConclusionProteinuria is the predominant factor in Fabry nephropathy predicting renal disease progression rate. Early Fabry Disease diagnosis could prevent kidney disease progression through the timely initiation of treatment which will help enhance the quality of life of the patient, as the disease progresses.

RIPK3 Contributes to Lyso-Gb3-Induced Podocyte Death

Fabry disease is a lysosomal storage disease with an X-linked heritage caused by absent or decreased activity of lysosomal enzymes named alpha-galactosidase A (α-gal A). Among the various manifestations of Fabry disease, Fabry nephropathy significantly affects patients’ morbidity and mortality. The cellular mechanisms of kidney damage have not been elusively described. Necroptosis is one of the programmed necrotic cell death pathways and is known to play many important roles in kidney injury. We investigated whether RIPK3, a protein phosphokinase with an important role in necroptosis, played a crucial role in the pathogenesis of Fabry nephropathy both in vitro and in vivo. The cell viability of podocytes decreased after lyso-Gb3 treatment in a dose-dependent manner, with increasing RIPK3 expression. Increased reactive oxygen species (ROS) generation after lyso-Gb3 treatment, which was alleviated by GSK’872 (a RIPK3 inhibitor), suggested a role of oxidative stress via a RIPK3-dependent pathway. Cytoskeleton rearrangement induced by lyso-Gb3 was normalized by the RIPK3 inhibitor. When mice were injected with lyso-Gb3, increased urine albuminuria, decreased podocyte counts in the glomeruli, and effaced foot processes were observed. Our results showed that lyso-Gb3 initiated albuminuria, a clinical manifestation of Fabry nephropathy, by podocyte loss and subsequent foot process effacement. These findings suggest a novel pathway in Fabry nephropathy.