Abstract
BackgroundIn clinical practice, there is a lack of markers for the non-invasive diagnosis and follow-up of kidney disease. Exosomes are membrane vesicles, which are secreted from their cells of origin into surrounding body fluids and contain proteins and mRNA which are protected from digestive enzymes by a cell membrane.MethodsToxic podocyte damage was induced by puromycin aminonucleoside in rats (PAN). Urinary exosomes were isolated by ultracentrifugation at different time points during the disease. Exosomal mRNA was isolated, amplified, and the mRNA species were globally assessed by gene array analysis. Tissue-specific gene and protein expression was assessed by RT-qPCR analysis and immunohistochemistry.ResultsGene array analysis of mRNA isolated from urinary exosomes revealed cystatin C mRNA as one of the most highly regulated genes. Its gene expression increased 7.5-fold by day 5 and remained high with a 1.9-fold increase until day 10. This was paralleled by a 2-fold increase in cystatin C mRNA expression in the renal cortex. Protein expression in the kidneys also dramatically increased with de novo expression of cystatin C in glomerular podocytes in parts of the proximal tubule and the renal medulla. Urinary excretion of cystatin C increased approximately 2-fold.ConclusionIn this proof-of-concept study, we could demonstrate that changes in urinary exosomal cystatin C mRNA expression are representative of changes in renal mRNA and protein expression. Because cells lining the urinary tract produce urinary exosomal cystatin C mRNA, it might be a more specific marker of renal damage than glomerular-filtered free cystatin C.
Highlights
An early and specific diagnosis and evaluation of disease activity are crucial elements for the choice of treatment modality in renal disease, i.e. immunosuppression versus conservative treatment
There is a multitude of basic science papers, none of these markers has been translated into clinical practice [1]
Exosomal gene expression Urine from rats at day 0, day 5 after induction of puromycin aminonucleoside nephrosis and on day 10 of the disease was collected in metabolic cages over a time frame of 16 hours
Summary
An early and specific diagnosis and evaluation of disease activity are crucial elements for the choice of treatment modality in renal disease, i.e. immunosuppression versus conservative treatment. The gold standard for diagnosis of renal disease is still a renal biopsy, an invasive diagnostic tool that is usually not suitable for follow-up diagnostics. There is a multitude of basic science papers, none of these markers has been translated into clinical practice [1] This might be due to the underlying problem that proteins in the urine usually exist in low quantities (e.g. nephrin, podocin), are often reabsorbed in the tubular system or are subjected to proteolytic digestion. We and others have not been able to simplify and standardize the method to allow for translation into clinical practice Another downside of podocyturia as a marker for glomerular disease might be the fact that only viable cells are being assessed, and it is probable that the larger proportion of apoptotic cells is being neglected. Exosomes are membrane vesicles, which are secreted from their cells of origin into surrounding body fluids and contain proteins and mRNA which are protected from digestive enzymes by a cell membrane
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